Camptothecin derivative combined with DDX5 protein and prodrug thereof

Abstract

The present invention relates to: an active camptothecin derivative (a) represented by Formula 1 designed so as to bind to a DDX5 protein and an E3 ligase; a prodrug (b) thereof, preferably an antibody-drug conjugate (ADC) thereof, designed so as to release the active camptothecin derivative (a) at a target site in vivo; the active camptothecin derivative (a) or the FL118 compound shown in the formula 2 is connected with a complex (c) containing a target DDX5 protein ligand through a joint, so that the 1-type topoisomerase inhibition capability in the active camptothecin derivative (a) or the FL118 compound shown in the formula 2 is inactivated. The active camptothecin derivative designed according to the present invention can bind to DDX5 protein in cells and induce cell death by degradation of the DDX5 protein.

Description

Technical Field

[0001] The present invention relates to: an active camptothecin derivative (a) represented by the following formula 1, which is designed in a manner of binding to a DDX5 protein and an E3 ligase; a prodrug (b) thereof, which is designed in a manner of releasing the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug-conjugate (CDC), in particular an antibody-drug conjugate (ADC); or a complex (c) containing a ligand targeting a DDX5 protein, which is connected by a linker to inactivate the type 1 topoisomerase inhibitory ability in the active camptothecin derivative (a) or the FL118 compound of formula 2.

[0002] The active camptothecin derivatives designed according to the present invention can bind to the DDX5 protein in cells and induce cell death by degrading the DDX5 protein. Background Art

[0003] Cancer genes are genes that originally exist to control the normal functions of cells, but if they mutate or become abnormally regulated, they can lead to cancer. One of the normal cell functions is apoptosis, and related to this, there are apoptosis-promoting factors and apoptosis-inhibiting factors.

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

[0005] Cancer is a group of diseases in which the cell cycle of growth and division is dysregulated. Fig.30 As shown, cancer occurs when the programmed cell death (e.g., apoptosis) machinery is impaired.

[0006] Cancer is caused by mutations in genes whose protein products are involved in cell cycle regulation. Many cancers are associated with the overexpression of specific genes or the abnormal activity of their mutant protein products (Oncogenes).

[0007] A single mutation does not usually cause cancer. Often, several genes that regulate cell growth mutate before a cell becomes cancerous.

[0008] HER2-positive breast cancer, which has a high abnormal expression of HER2 in breast epithelial cells, accounts for about 1 / 4 of all breast cancer patients. Compared with female hormone-related breast cancer, it has a higher recurrence rate during chemotherapy and a poorer prognosis. Herceptin (ingredient name: Trastuzumab), a HER2-targeted therapy drug, treats breast cancer by reducing the amount of HER2 on the cell surface and helping immune cells kill HER2-positive breast cancer cells.

[0009] HER2-positive breast cancer is a breast cancer that retains amplification or overexpression of the HER2 (ErbB2) tumor gene, a member of the ErbB receptor. Trastuzumab (Herceptin) has improved the treatment of HER2-positive breast cancer, but compared with other breast cancers, HER2-positive breast cancer is more aggressive, and more than half of patients develop resistance to existing targeted therapies or develop resistance during treatment.

[0010] As ligands of ErbB receptors attached to the cell membrane, NRG1 and HB-EGF activate ErbB ligands to bind to their receptors (EGFR, HER3) and catalyze phosphorylation (phosphorylation, P) through the binding between receptors (EGFR-HER2, HER2-HER3) to induce intracellular growth and proliferation signals. Excessive activation of these ErbB receptors can lead to resistance to anti-HER2 therapeutic drugs.

[0011] On the other hand, dysregulated transcription factors can reduce the sensitivity of cancer cells to chemotherapy or radiotherapy. Drug resistance in cancer treatment can be either intrinsic or acquired. In cancer treatment, intrinsic drug resistance may be caused by abnormally expressed transcription factors, which are key regulators of cell proliferation and apoptosis. Abnormal overexpression of NF-κB, STAT3, HIF-1, AR or ER and inactivation of p53 and FOXO3a in tumor tissues can protect cancer cells from apoptosis caused by anticancer drugs, resulting in intrinsic drug resistance.

[0012] However, chemotherapy or radiotherapy also induces acquired drug resistance through the activation of dysregulated transcription factors. Studies have shown that ionizing radiation can stimulate the activation and induction of several different transcription factors, including STAT3, NF-κB, and HIF-1. The upregulation of NF-κB and STAT3 induces the transcription of anti-apoptotic genes in cancer cells and promotes cancer cells to enter the cell cycle and proliferate, resulting in radioresistance. In addition, the upregulation of transcription factors NF-κB and HIF-1 by radiation can induce genes responsible for maintaining EMT cells and cancer stem cells that are resistant to cancer treatment. In addition, the activation of the transcription factor STAT3 also promotes resistance to cancer treatment. Feedback activation of STAT3 by chemotherapy or radiotherapy has been shown to be a molecular mechanism of resistance to cancer treatment. Therefore, co-targeting dysregulated transcription factors and their main targets may be a promising approach to overcome resistance in cancer treatment.

[0013] The STAT (abbreviation for signal transducer and activator of transcription) transcription factor family includes STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6. When stimulated by growth factors, cytokines, interferons or tumor genes, JAK kinase activates STAT proteins to regulate interferon signaling. The DNA binding domain of STAT is an immunoglobulin-like structure that mediates the binding of specific DNA target sequences. During activation, phosphorylation of STAT3 by JAK kinase activates dimerization. The dimer then moves to the nuclear compartment and binds to the sequence of the target promoter to induce the expression of the target gene. STAT3 target genes include survivin, bcl-xl, mcl-1, Cyclin D1, MMP2, MMP9, VEGF, Myc, Sox2, etc. STAT proteins regulate many biological processes, including cell growth, apoptosis, differentiation and immunity. Among the STAT family members, STAT3 and STAT5 are most strongly associated with cancer progression.

[0014] Dysregulated STAT3 and activated STAT5 are considered tumor genes that increase angiogenesis and improve the survival of cancer cells. In a study using a large number of samples from the cBioPortal database, it was found that STAT3 was more frequently overexpressed in lung cancer, ovarian cancer, gastric cancer, hematopoietic cancer, and brain cancer compared with normal tissues, and STAT3 overexpression was associated with poor overall survival. Phosphorylation of STAT3 by JAK kinase is the first step in STAT3 activation. In another study of 90 glioblastoma patients, high p-STAT3 values ​​were found to be significantly associated with reduced progression-free survival and overall survival. Multivariate survival analysis showed that high p-STAT3 levels can have an important prognostic indicator role for poor progression-free survival and overall survival.

[0015] Type 1 topoisomerase I inhibitors (irinotecan, topotecan, etc.) are anticancer mechanisms whose efficacy and safety have been clinically verified, and their excellent anticancer efficacy against various refractory solid cancers such as colon cancer, lung cancer, breast cancer, and ovarian cancer has been verified in clinical trials.

[0016] In this regard, camptothecin derivatives are known as low-molecular compounds that exhibit antitumor effects by inhibiting type 1 topoisomerase (topoisomeras e-1).

[0017] Camptothecin (CPT) has low solubility in water, and in preparation for clinical trials, the National Cancer Institute (NCI) of the United States prepared a water-soluble sodium salt (NSC100880). Because the compound exhibits high toxicity (hemorrhagic cystitis, gastrointestinal toxicity such as nausea, vomiting, diarrhea, and bone marrow suppression, especially leukopenia and thrombocytopenia), Phase 1 and Phase 2 clinical trials have not been completed.

[0018] Subsequently, many camptothecin (CPT) analogs were synthesized to obtain less toxic and more water-soluble compounds. Two drugs are irinotecan (CPT-11) and topotecan.

[0019] Irinotecan (CPT-11), jointly developed by Dallchi and Yakult of Japan, is the world's first camptothecin anticancer drug. In 1994, it was proven to be effective against lung cancer (small cell and non-small cell lung cancer) and was launched in Europe and Japan. In 1995, its effectiveness against colon cancer and breast cancer was confirmed. In addition, topotecan developed by GlaxoSmith Kline was approved by the US FDA for the treatment of metastatic ovarian cancer in April 1995 and was launched on the market. CKD-602 (belot ecan) is a new camptothecin derivative recently developed in South Korea. It has a strong inhibitory effect on type 1 topoisomerase and is water-soluble, successfully overcoming the existing toxicity caused by poor solubility.

[0020] All camptothecin derivatives identified so far contain a five-ring parent structure that is required for cytotoxicity ( Figure 1 ). In terms of molecular structure, the E ring, A ring, and B ring sites are confirmed as important sites. Camptothecin contains a five-ring lactone structure in the E ring required for cytotoxicity. Among them, the lactone group and α hydroxyl group at the C20 position of the E ring are important for the stability of type 1 topoisomerase-DNA byproducts, and it has been confirmed that modifications of the A ring and the B ring can increase water solubility and activity. Modifications on the first ring have been confirmed to increase solubility in water and receive greater tolerance, for example, in the case of the drugs mentioned above.

[0021] CKD-602 also attempts to replace the B-ring site of carbon 7 to increase water solubility and anticancer effects. Lee et al. reported that CKD-602 has excellent anticancer effects in a variety of cancer cell lines compared to camptothecin and topotecan. In addition, it was confirmed to be a relatively safe drug in the L1210 leukemia nude mouse model, with a maximum tolerated dose (MTD) of 25 mg / kg. Generally, the side effects of known camptothecin drugs can be roughly divided into hematological side effects and non-hematological side effects. As hematological side effects, there are neutropenia, sepsis, bleeding, etc. accompanied by fever, and as non-hematological side effects, skin side effects such as nausea, vomiting, alopecia, etc., as well as toxicity to the gastrointestinal tract, kidneys, and nervous systems can be cited. In the CKD-602 animal study, Kim et al. found that among the side effects of these camptothecin drugs, no abnormal drug reactions other than increased gastric secretion were found in the case of high-volume administration more than 10 times higher than the clinically applicable dose. In addition, its relative safety has been demonstrated in recent clinical studies in South Korea, and only reversible and controllable side effects of neutropenia and leukopenia have been reported, with no reports of severe systemic toxicity. However, so far, its main indications are patients who have failed or cannot undergo standardized therapy, that is, patients who have relapsed or worsened after standard therapy and are judged to be difficult to achieve the effect of further anticancer chemotherapy or surgery, and the treatment of refractory or recurrent ovarian cancer and colon cancer, resistant or recurrent limited-stage small cell lung cancer that has failed initial chemotherapy, and extensive-stage small cell lung cancer, and its use is restricted.

[0022] SN-38 has an IC in the nanomolar range in various cell lines 50 It is a potent topoisomerase-I inhibitor with high value. It is the active form of the prodrug irinotecan used for the treatment of colorectal cancer, and is also active in lung cancer, breast cancer, and brain cancer. Trop-2-SN-38ADC has been successfully developed in various cancer types such as TNBC, bladder cancer, and gastric cancer, but there are still drug resistance problems caused by overexpression of drug efflux transporters, epigenetic silencing of Top1, and increase in anti-apoptotic proteins.

[0023] On the other hand, Daiichi Sankyo Co., Ltd. The development of HER2 used a cytotoxic drug DXD (exatecan class) that is approximately 10 times more active in cancer cells than SN-38. DXD has good solubility, is relatively safe, and has a good bystander effect, so it has advantages in the treatment of heterogeneous tumors. However, the half-life that can reduce off-target effects is very short. DXD was bioconjugated to the cysteine ​​residues of the anti-HER2 antibody via a maleimide linker, and the homogeneous DAR value reached 8. Despite the high DAR value, DXD is very stable, with only 2.1% released into plasma within 21 days (Ogitani et al., 2016). HER2 was approved by the US Food and Drug Administration (USFDA) in 2019, and the target patients are adult patients with unresectable metastatic Her2-positive breast cancer who have received at least two HER2-targeted therapies in the past.

[0024] Derivatives of camptothecins, such as SN-38 and Dxd (a derivative of the clinical-stage topoisomerase I inhibitor exotecan), have recently been successfully developed as novel payloads for antibody-drug conjugates (ADCs). As a result, two ADCs using camptothecin payloads (Trodelvy (trade name, Chinese name: Tuo Dawei, generic name: Sacituzumab Govitecan) with SN-38 payload and Enhertu (trade name, Chinese name: You He De, generic name: Trastuzumab Deruxtecan) with Dxd payload) have recently been approved by the FDA. Compared with ADCs with existing ultra-toxic payloads such as MMAE and MMAF (DAR=2 or 4), these camptothecin payloads have moderate cytotoxicity, but due to their excellent safety profile, higher drug-antibody ratios (DAR), such as 8, can be used. In addition, with these camptothecin payloads, linker systems with faster drug release characteristics, such as CL-2A or GGFG linker systems, can be used. By combining these components, new ADCs with a wider range of therapeutics have been developed.

[0025] Antibody-drug conjugates (ADCs) such as Trodelvy and Enhertu that successfully use camptothecin derivatives as payloads can selectively deliver camptothecin compounds that show strong anti-cancer efficacy only to cancer cells, and can also bring excellent anti-cancer efficacy without serious systemic side effects, although there are still many disadvantages, the most representative example is the drug resistance caused by overexpression of the ABCG2 drug efflux pump. Many camptothecin derivatives are rapidly exported to the extracellular space due to ABCG2, so compared with cancer cells that do not express ABCG2 or express it at a low normal level, cancer cells that overexpress ABCG2 show a problem of severely reduced therapeutic efficacy. Overexpression of these ABC G2s is not only the main reason for the reduced therapeutic efficacy of low-molecular-weight camptothecin compounds such as irinotecan and topotecan, but also the main reason for the reduced therapeutic efficacy of ADCs such as Trodelvy and Enhertu that use camptothecin compounds as payloads. Summary of the invention

[0026] Technical issues

[0027] The object of the present invention is to provide a camptothecin derivative designed to bind to the DDX5 protein and the E3 ligase as a candidate drug for causing apoptosis of target cells.

[0028] The present inventors synthesized the FL118 compound ( Figure 2 ), the FL118 compound is a camptothecin drug with excellent type 1 topoisomerase inhibition ability and dual MoA of degrading oncoprotein DDX5 (p68), and various studies on its physicochemical properties continue. However, due to the limitation of physicochemical properties, the FL118 compound is limited in dosage form development.

[0029] In this regard, based on the structure of FL118, which is different from the SN38 drug, the inventors designed and synthesized a camptothecin derivative with a new structure, which exerts a dual mechanism of action (dual MoA) in terms of type 1 topoisomerase inhibition and DDX5 degradation, which are the advantages of FL118.

[0030] Therefore, the object of the present invention is to design and provide new active camptothecin derivatives and / or their prodrugs, i.e., carrier-drug conjugates (CDCs), in particular antibody-drug conjugates (ADCs), wherein the active camptothecin derivatives have dual mechanisms of action (dual MoA) of both type 1 topoisomerase inhibition and degradation of oncoprotein DDX5, and the prodrugs are designed to release the active camptothecin derivatives in vivo.

[0031] In addition, the purpose of the present invention is to provide an anti-cancer mechanism, which is a new form of killing cancer cells and / or surrounding cells before intrinsic or acquired drug resistance, so that active camptothecin derivatives bind to DDX5 protein in cells together with the bystander effect and induce cell apoptosis (cell death) through degradation of DDX5 protein.

[0032] Solutions to the problem

[0033] The first embodiment of the present invention provides the following: an active camptothecin derivative (a) represented by the following formula 1 is designed in a manner to bind to the DDX5 protein and the E3 ligase; or a prodrug thereof (b) is designed in a manner to release the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC); or a complex (c) containing a ligand targeting the DDX5 protein, wherein the type 1 topoisomerase inhibitory ability of the active camptothecin derivative (a) or the FL118 compound of formula 2 is inactivated by a linker connection,

[0034]

[0035] X 1 and X 3 are independently carbon, oxygen, nitrogen or sulfur, X 1 and X 3 Can be the same or different,

[0036] X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond,

[0037] X 1 、(X 2 )n and X 3 It can form a hexagonal ring or a 7-corner ring (n=1 to 2),

[0038] Y 1 , Y 2 and Y 3 They may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0039] In the complex containing a ligand targeting DDX5 protein, the ligand targeting DDX5 protein may be a ligand that binds to the DDX5 protein.

[0040] Active camptothecin derivatives can be designed as follows: In Formula 1, X 1 、(X 2 )n、X 3 , Y 1 , Y 2 and / or Y 3 By modifying the protein, its hydrophobicity or cell membrane permeability can be adjusted as needed to exert a bystander effect in tumor tissue or control the extent of its effect.

[0041] Preferably, the active camptothecin derivative (a) designed in a manner to bind to the DDX5 protein and the E3 ligase may have the function of a molecular glue degrader having a ligand (binder) that binds to the E3 ligase; and / or, may cause apoptosis of target cells expressing the DDX5 protein through a molecular glue degrader mechanism (MoA); and / or, may have the ability to inhibit type 1 topoisomerase while also having a mechanism of action (MoA) for decomposing the oncoprotein DDX5.

[0042] The second embodiment of the present invention provides a pharmaceutical composition or cancer diagnostic composition for preventing or treating cancer, wherein the composition comprises: the active camptothecin derivative (a) of the first embodiment; its prodrug (b), preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC); or a complex (c) containing a ligand targeting the DDX5 protein.

[0043] For example, it is administered for the treatment of HER2-positive cancer, breast cancer, lung cancer, and colon cancer.

[0044] Preferably, the pharmaceutical composition for preventing or treating cancer can be used as a first-line treatment drug after cancer diagnosis or can be administered to an individual who (over)expresses DDX5 in cancer tissue.

[0045] The third embodiment of the present invention provides a preparation method, which is used to prepare: an active camptothecin derivative (a) designed in a manner to bind to the DDX5 protein and the E3 ligase; a prodrug (b) thereof designed in a manner to release the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC); or a complex (c) containing a ligand targeting the DDX5 protein, which is connected by a linker to inactivate the type 1 topoisomerase inhibitory ability in the active camptothecin derivative (a) or the FL118 compound of Formula 2, characterized in that the preparation method comprises the following steps: designing the active camptothecin derivative (a) in a manner that includes the camptothecin skeleton represented by Formula 1 as a parent core, or selecting a compound designed in this manner, or synthesizing a compound designed in this manner.

[0046] For example, the step of designing in such a way that the active camptothecin derivative (a) comprises the camptothecin skeleton represented by Formula 1 as a core, or selecting the compound so designed may be:

[0047] (1) selecting an active camptothecin derivative designed to have a mechanism of action (MoA) for inhibiting type 1 topoisomerase and / or a mechanism of action (MoA) for degrading oncoprotein DDX5 from a library of compounds comprising a camptothecin skeleton represented by Formula 1 as a core, and / or,

[0048] (2) Whether the compound comprising the camptothecin skeleton represented by Formula 1 as a core inhibits type 1 topoisomerase and / or degrades oncoprotein DDX5 is confirmed by in vitro experiments and / or in vivo experiments.

[0049] The fourth embodiment of the present invention provides a preparation method, which is used to prepare: an active camptothecin derivative (a) that binds to DDX5, which acts as an oncoprotein in cells, and induces cell apoptosis (cell death) through degradation of DDX5 protein; a prodrug (b) thereof, which is designed in a manner to release the active camptothecin derivative (a) in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC); or a complex (c) containing a ligand targeting DDX5 protein, in which the type 1 topoisomerase inhibitory ability is inactivated by a linker connection in the active camptothecin derivative (a), characterized in that the preparation method comprises: a first step, selecting the active camptothecin derivative (a) from the group consisting of the following compounds of Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8, Formula 9 and isomers thereof; and optionally, a second step, selecting its prodrug (b), which is designed to release the active camptothecin derivative (a) selected in the first step in vivo.

[0050]

[0051]

[0052]

[0053] The preparation method of the third embodiment or the fourth embodiment may further include the following steps: in a tissue biopsy or liquid biopsy, using a complex (c) containing a ligand targeting DDX5 protein to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to be administered with the active camptothecin derivative (a) or its prodrug (b), wherein the complex (c) containing a ligand targeting DDX5 protein is connected in the FL118 compound of Formula 2 or the active camptothecin derivative (a) through a non-cleavable linker to inactivate the type 1 topoisomerase inhibitory ability.

[0054] The fifth embodiment of the present invention provides a compound represented by the following Formula 3, Formula 3-1, Formula 4, Formula 4-1, Formula 5, Formula 5-1, Formula 6, Formula 7, Formula 8 or Formula 9 or a pharmaceutically acceptable salt thereof.

[0055] The sixth embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a compound represented by the following 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 pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof, preferably a carrier-drug conjugate (CDC), and more preferably an antibody-drug conjugate (ADC).

[0056]

[0057]

[0058]

[0059]

[0060] The seventh embodiment of the present invention provides a pharmaceutical composition for killing cells, characterized in that the pharmaceutical composition comprises: an active camptothecin derivative (a) represented by the following formula 1, designed to bind to DDX5 protein and E3 ligase; or a prodrug (b) thereof, designed to release the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), and more preferably an antibody-drug conjugate (ADC).

[0061] The eighth embodiment of the present invention provides an anticancer preparation, characterized in that the active camptothecin derivative (a) represented by the following formula 1 is designed to bind to the DDX5 protein and the E3 ligase; or its prodrug (b) is designed to release the active camptothecin derivative (a) at the target site in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC), which can be administered at a dosage that stimulates antigen presenting cells (APC) through apoptosis of cancer cells to induce an anti-tumor immune response.

[0062] The ninth embodiment of the present invention provides a method for preparing an anticancer drug, wherein the method is a method for preparing an anticancer drug based on an active camptothecin derivative (a), wherein the bystander effect and / or ADME characteristics of the active camptothecin derivative (a) are controlled to prevent T cell exhaustion caused by chronic antigen exposure, or to provide a desired degree of hydrophobicity and / or control aggregation so as to be eliminated quickly or slowly by lymphatic drainage, wherein the anticancer drug based on the active camptothecin derivative (a) is an anticancer preparation comprising the following components: the active camptothecin derivative (a), as a molecular glue degrading agent; degrader), designed in a manner to bind to DDX5 protein and E3 ligase; or a prodrug (b) thereof, designed in a manner to release the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC), the method comprising the following steps: designing the active camptothecin derivative (a) or its prodrug (b) in a manner that it comprises a camptothecin skeleton represented by the following formula 1 as a mother core, or selecting a compound designed in this manner, or synthesizing a compound designed in this manner.

[0063] Hereinafter, the present invention will be described.

[0064] In this article, drugs refer to all substances used to diagnose, cure, alleviate, treat or prevent diseases (except food or equipment) or to affect the structure or function of the body. For example, all chemical or biological substances that affect the body and body metabolism. Preferably, as long as they bind to the DDX5 protein, they belong to the category of drugs.

[0065] For example, the drug of the present invention can be an active camptothecin derivative as a payload of a prodrug and a camptothecin derivative or a prodrug thereof as a diagnostic drug, such as an active camptothecin derivative itself or a prodrug thereof, targeted drug delivery (e.g., carrier-drug conjugate).

[0066] In order for the drug to work effectively in vivo, the concentration of the drug at the target site in vivo must be maintained within the therapeutic range for a predetermined period of time (Example 8, Table 6, Fig.23 If the drug is present in excess in the body, it will be toxic, and if the amount of the drug at the target site is too small, it will have no therapeutic effect.

[0067] Drug efficacy refers to the effect on the target indication without degradation and remaining in the body within the expected time (Example 8, Table 6, Fig.23 ). If the metabolic rate is low, the blood drug concentration will be maintained longer, so the duration of efficacy will be longer.

[0068] On the other hand, the in vivo efficacy and in vivo side effects of anticancer drugs are closely related to the absorption, distribution, metabolism and excretion (ADME) properties of anticancer drugs. The ADME of a drug determines its pharmacokinetics, which describes how a drug moves through the body and interacts with tissues and organs.

[0069] Drug absorption affects efficacy and side effects. Poorly absorbed drugs do not reach therapeutic concentrations in target tissues, which may reduce efficacy. On the other hand, well-absorbed drugs may cause off-target side effects due to increased systemic exposure.

[0070] The distribution of drugs can also affect efficacy and side effects. Drugs that are poorly distributed in target tissues may be ineffective, while drugs that are widely distributed in non-target tissues may induce toxicity in the corresponding tissues. In addition, drugs with high binding capacity to plasma proteins may have reduced distribution to target tissues, which may lead to reduced efficacy.

[0071] The distribution of a drug in the body may be affected by its physicochemical properties, including size, charge, and lipophilicity. The ability of a drug to penetrate into tumor tissue may also be affected by factors such as the tumor microenvironment, such as blood flow and cell density. Some anticancer drugs accumulate in specific tissues, which may cause toxic effects.

[0072] Drug metabolism also affects efficacy and side effects. Rapidly metabolized drugs may have reduced efficacy due to their short half-life, while slowly metabolized drugs may increase toxicity due to accumulation. In addition, some metabolites are more toxic than the parent drug and may therefore cause side effects.

[0073] The rate and route of excretion can also affect the efficacy and side effects of a drug. Rapidly eliminated drugs have a short half-life and may therefore reduce efficacy, while slowly eliminated drugs accumulate and may cause toxicity. In addition, some drugs may be excreted unchanged or as active metabolites, which may result in prolonged or increased exposure and increased risk of side effects.

[0074] Therefore, it is necessary to analyze the ADME properties of anticancer drugs to optimize in vivo efficacy and minimize side effects. That is, evaluating and optimizing ADME characteristics can improve the therapeutic index of these drugs, thereby leading to better outcomes for cancer patients.

[0075] An anti-cancer formulation or anti-cancer composition refers to a medication or therapy designed to treat cancer or to prevent the growth and spread of cancer cells in the body. Anti-cancer formulations may include a combination of drugs, chemical compounds, and / or biological agents that target cancer cells by interfering with their ability to grow and divide.

[0076] The formulation of anti-cancer drugs can represent a complex process involving multiple steps, 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.

[0077] The development of anticancer agents involves designing drugs or drug combinations that can recognize and selectively target cancer cells while leaving healthy cells alone. This can be achieved by targeting specific proteins or enzymes that are overexpressed in cancer cells, or by interfering with the cell cycle, DNA replication, or other cellular processes that are essential for cancer cell growth and survival.

[0078] Once promising drug candidates are identified, preclinical development, which tests the drug in the laboratory and in animal models to evaluate safety and effectiveness, is carried out. If the drug shows promise in preclinical studies, it can enter clinical trials to test it in humans to determine safety, dosage, and effectiveness.

[0079] During clinical trials, drug formulations are optimized to ensure safe administration to patients and achieve the desired therapeutic effect. This may include adjusting the drug dose, route of administration or formulation to improve efficacy and minimize side effects.

[0080] The present invention provides: an active camptothecin derivative (a) represented by the following formula 1, designed in a manner of binding to DDX5 protein and E3 ligase; or a prodrug (b) thereof, designed in a manner of releasing the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC), as a candidate drug for killing target cells.

[0081]

[0082] X 1 and X 3 are independently carbon, oxygen, nitrogen or sulfur, X 1 and X 3 Can be the same or different,

[0083] X 2 is carbon, oxygen, nitrogen, sulfur, single bond or double bond,

[0084] X 1 、(X 2 )n and X 3 It can form a hexagonal ring or a 7-corner ring (n=1 to 2),

[0085] Y 1 , Y 2 and Y 3 They may each independently be hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0086] Among them, non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may include -CHO, -COOH, -NH 2 、-SH、-CONH 2 ,-PO 3 H, -PO 4 H 2 ,-OPO 4 H, -PO 2 (OR 1 )(OR 2 )(R 1 , R 2 =C s H t N u O w S x P y X z , 3 H.-OSO 3 H, -NO 2 、-N 3 、-NR 3 OH(R=C n H 2n+1 ,0≤n≤16),-NR 3 + X - (R=C n H m, 0 ≤ n ≤ 16, 0 ≤ m ≤ 34, X = OH, Cl or Br), NR 4 + 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, -N 3 , -N 2 , -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≤2s), -OH, -O-, >C=O, -SS-, -SO-, -NO 2 , -COX (X = F, Cl, Br or I), -COOCO-, -CONH-, -CN, -SCOCH 3 、-SCN、-NCS、-NCO、-OCN、-CN、-F、-Cl、-I、-Br、epoxy、-hydrazone、-ONO 2 、-PO(OH) 2 、-C=NNH 2 , -HC=CH-, -C=C-, -C≡C- and a functional group in the group consisting of hydrocarbons having two or more carbon atoms.

[0087] According to the present invention, the active camptothecin derivative (a) designed in a manner of binding to the DDX5 protein and the E3 ligase can kill the target cells expressing the DDX5 protein through the molecular glue degrader mechanism (MoA).

[0088] The target cell can be a cancer cell or a senescent cell. Senescent cells also include cells that do not perform long-term intrinsic characteristic functions.

[0089] Preferably, according to the present invention, the active camptothecin derivative (a) designed in such a manner as to bind to DDX5 protein and E3 ligase may have type 1 topoisomerase inhibitory ability and multiple mechanisms of action (MoA) for degrading oncoprotein DDX5.

[0090] For example, the present invention can expose tumor antigens through apoptosis of cancer cells in tumor tissues, thereby inducing anti-tumor immune responses by stimulating antigen presenting cells (APCs) such as dendritic cells and macrophages.

[0091] In addition, the present invention provides in various ways: an active camptothecin derivative (a) represented by Formula 1 designed in a manner to bind to the DDX5 protein and the E3 ligase; or a prodrug (b) thereof designed in a manner to release the active camptothecin derivative (a) at a target site in vivo, as a candidate drug for killing cancer cells, so that anticancer preparations based on the active camptothecin derivative (a), their dose and dosage can be designed in various ways to control the bystander effect and / or ADME characteristics of the active camptothecin derivative (a) to prevent T cell exhaustion caused by chronic antigen exposure, or to provide a desired degree of hydrophobicity and / or control aggregation so as to be quickly or slowly eliminated through lymphatic drainage.

[0092] By applying camptothecin derivatives that exert the effect of type 1 topoisomerase inhibitors, it has been shown that greater tolerability can be achieved when designing camptothecin drugs as shown in general formula 1 regarding changes in R1 and R2 on the A ring (Group A), the active camptothecin derivatives of the present invention are characterized in that they exert an anti-cancer mechanism of degrading oncoprotein DDX5 (Example 2) by being designed in the same manner as the FL118 compound of Formula 2 (Example 1).

[0093]

[0094] The basis for this is that R 1 and R 2 The active camptothecin derivatives of formula 1 designed in the same manner as the FL118 compound, such as the compounds of formula 3, formula 4, or formula 5 below, are transcription cofactors and degrade DDX5 as an oncoprotein, thereby strongly inhibiting the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.) in a concentration-dependent manner ( Figures 11 to 14 ), and through cell viability experiments, it was found that the compound can kill the corresponding cells introduced into the cells ( Fig.15 and Fig.16 ).

[0095]

[0096]

[0097] In Formula 1 or Formula 2, Group C sites bind to type 1 topoisomerase, and Group A sites bind to DNA, stabilizing the covalent bonds of the topoisomerase-DNA complex to prevent the cleaved DNA fragments from reconnecting (Example 1), and / or, in Formula 1 or Formula 2, Group A sites bind to DDX5, and Group C sites bind to E3 ligase, which can induce DDX5 degradation ( Figures 11 to 14 ).

[0098] Inhibitors of type 1 topoisomerase (Topoisomerase I) are an anticancer mechanism whose efficacy and safety have been clinically proven, and camptothecin-based drugs such as Exatecan, Exatecan derivatives such as Dxd, and SN-38 have been developed as payloads for ADCs.

[0099] Exatecan is a drug that has been proven to have cytotoxicity 5 to 10 times stronger than SN-38. Unlike irinotecan, exatecan does not require enzyme activation. In addition, it has stronger type 1 topoisomerase inhibitory activity than irinotecan's main SN-38 or topotecan used in clinical trials, and has stronger cytotoxic activity against various cancer cells in vitro. In particular, it is also effective against cancer cells that show resistance to SN-38 and the like due to the expression of P-glycoprotein. In addition, a strong anti-tumor effect was also shown in a subcutaneous transplantation model of human tumors in mice, so clinical trials were conducted.

[0100] The FL118 drug of Formula 2 is a hydrophobic small molecule that can penetrate the cell membrane. The FL118 drug is a potent type 1 topoisomerase inhibitor and has the same camptothecin nucleus structure as the currently commonly used Top1 inhibitors, namely SN-38, Topotecan, Exatecan, etc. ( Figure 1 and Figure 2 ), however, when administered alone, it has differentiated anticancer efficacy and excellent safety compared to SN-38 in various cancer cells and animal models, ensuring an anticancer drug with a wide therapeutic window.

[0101] The inventors synthesized a camptothecin compound, namely the drug FL118, and continued to conduct various studies on its physicochemical properties. However, due to the limitations of its physicochemical properties, FL118 is restricted in the formulation development.

[0102] The present inventors confirmed that the FL118 drug, when used as a payload of an antibody-drug conjugate (ADC), has stronger cytotoxicity than an ADC with SN-38 as a payload and has the same level of cytotoxicity as the exitecan drug.

[0103] On the other hand, FL118 is a camptothecin-like compound with excellent type 1 topoisomerase inhibition ability and dual MoA of degrading oncoprotein DDX5 (p68).

[0104] Surprisingly, it was found that, like FL118, Exatecan not only strongly inhibited the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), another major cause of anticancer drug resistance, at low concentrations, but also blocked the resistance mechanism ( Fig.10 ).

[0105] Therefore, based on this, Figure 1 As shown, the present invention is characterized in that the following components are designed from the FL118 compound: a novel active camptothecin derivative (a) having excellent type 1 topoisomerase inhibitory ability and a dual mechanism of action (dual MoA) for degrading oncoprotein (oncoprotein) DDX5 (p68); a prodrug (b) thereof, which is designed in a manner to release the active camptothecin derivative (a) in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC); and a complex (c) containing a ligand targeting the DDX5 protein, in which the type 1 topoisomerase inhibitory ability is inactivated by a linker connection in the active camptothecin derivative (a).

[0106] Specifically, the synthetic design concept of active camptothecin derivatives (a) having dual MoA of type 1 topoisomerase inhibition and degradation of oncoprotein DDX5, such as Figure 1As shown, based on the structure activity relationship (SAR), the structure of Group C in the type 1 topoisomerase inhibition region is maintained, and the structure of Group A for the binding site of DDX5 degradation is also maintained the same as that of FL118, and the structure (R3 and R4) of Group B in Formula 1 is adjusted to Formula 1, thereby not only improving the physicochemical properties of the drug, but also being designed to be able to adjust the bystander effect by adjusting the cytotoxicity of the drug and / or the cell membrane permeability of the drug (Example 9, Fig.25 and Fig.26 Embodiment 10, Figure 27b ). In addition, depending on the circumstances, a structure capable of providing a desired degree of hydrophobicity and / or controlled aggregation can be designed so that the active camptothecin derivative (a) released from apoptotic cancer cells is eliminated quickly or slowly through lymphatic drainage.

[0107] When simply comparing cell viability, PBX-7016 (Formula 5) newly designed and synthesized according to the present invention showed an IC level of 1.5 to 2 times that of Dxd. 50 However, surprisingly, when used as an ADC payload, the ADC containing PBX-7016 had about 5 times better cell viability-related cytotoxicity effects than the ADC containing Dxd (Example 7, Fig. 22 ). The compound represented by Formula 5 (PBX-7016) and the compound represented by Formula 5-1 (PBX-7017) as an isomer thereof have a diastereoisomer relationship with each other.

[0108] Therefore, preferably, an active camptothecin derivative is designed in the present invention, and the cell membrane permeability is adjusted as needed by modifying R3 and / or R4 in Formula 1 (for example, PBX-7014, PBX-7016, PBX-7018, PBX-7020, PBX-7022, PBX-7024), thereby exerting an appropriate bystander effect in tumor tissue.

[0109] like Figure 1 As shown, the present invention designs and synthesizes novel compounds such as PBX-7011 similar to Exatecan by modifying R3 and R4 of Group B in Formula 1 (Preparation Example 1). Figure 2As shown, the calculated LogP (or cLogP) and topological polar surface area (tPSA) values ​​confirm that the PBX-7011 of Formula 3 newly designed according to the present invention has a relatively higher hydrophilicity than FL118 of Formula 2.

[0110] Furthermore, by adjusting Group B of Formula 1 in the newly designed PBX-7011 structure, new structure compounds PBX-7014 and PBX-7016 were designed and synthesized (Preparation Examples 2 and 3). It was calculated that PBX-7014 and PBX-7016 showed hydrophilicity values ​​similar to those of Dxd derived from exotecan ( Figure 1 and Figure 2 , Fig.28 ).

[0111] Fig.28 The solubility and topological polar surface area (tPSA) of FL118, exotecan, SN-38, Dxd, exotecan-lactic acid, PBX-7011 / PBX-7012, PBX-7014 / PBX-7015, and PBX-7016 / PBX-7017 are shown for comparison.

[0112] The relative hydrophilicity / hydrophobicity of a drug has a significant impact on its solubility, absorption, distribution, metabolism, excretion (ADME), especially lymphatic drainage, and is very important for the difficulty of a drug to cross the cell membrane and its interaction with drug targets (e.g., receptors).

[0113] The hydrophobicity of a drug is expressed by the partition coefficient (p).

[0114] The p value of hydrophobicity is large, and the p value of hydrophilicity (polarity) is small. In fact, its log P value is used as a measure to evaluate hydrophobicity. Clog P refers to the log P value of a given compound calculated by appropriate software. The smaller the cLogP value, the higher the polarity.

[0115] The polar surface area (PSA) or topological polar surface area (TPSA) of a molecule is defined as the sum of the surface areas of all polar atoms or molecules (primarily oxygen and nitrogen, including attached hydrogen atoms). tPSA is a commonly used medicinal chemistry measurement to optimize the cell penetration ability of a drug. Molecules smaller than 100 μm are often unable to penetrate the cell membrane. Only tPSA can cross the blood-brain barrier and act on receptors in the central nervous system.

[0116] PBX-7016 / PBX-7017 (Formula 5 and Formula 5-1) of the present invention introduces a methyl group (a metabolically unstable functional group) into PBX-7014 / PBX-7015 (Formula 4 and Formula 4-1) to reduce the life span of the drug. Drugs that are extremely stable in metabolism but metabolized very slowly may accumulate and cause toxicity and serious side effects, so it is necessary to ensure an appropriate retention time.

[0117] The newly designed active camptothecin derivative (a) according to the present invention is prepared by modifying R 3 and R 4 The hydrophobic small molecule is designed to be able to penetrate the cell membrane. Therefore, when delivered to the tumor tissue, it can accumulate at a high concentration as it penetrates deeply into the tumor tissue. After the ADC as the active camptothecin derivative (a) or its prodrug (b) penetrates the cell membrane, the active camptothecin derivative (a) exerts cytotoxicity inside the cell to kill the cell, and is released into the extracellular fluid at the same time, and can continuously penetrate the cell membrane in the surrounding cells and move into the cell to exert the cell apoptosis mechanism (Example 9, Fig.25 and Fig.26 Embodiment 10, Figure 27b ). Therefore, the active camptothecin derivatives of the present invention can transform solid tumors that are aggregated due to high cell density into dispersed cancers with low cell density, and / or transform cold tumors with low immune activity into hot tumors with high immune activity. In addition, the bystander effect is high, so it is beneficial for the treatment of heterogeneous tumors.

[0118] In short, Figure 1 , Figure 2 , Fig.28 and Fig.29 As shown, the present invention can not only design various camptothecin derivatives and prodrugs thereof with new structures in various ways Figure 3 ), which exerts a dual mechanism of action (dual MoA) in terms of type 1 topoisomerase inhibition and DDX5 protein degradation, and when designing camptothecin derivatives, hydrophilicity can be controlled in multiple ways to regulate cell membrane permeability, lymphatic drainage and / or bystander effect.

[0119] [FL118 drug]

[0120] FL118 of Formula 2 can act as a molecular glue degrader that degrades DDX5 by directly conjugating DDX5 and ubiquitination regulatory factors.

[0121] FL118 acts as a molecular glue. Without reducing DDX5 mRNA, it directly connects to the oncoprotein DDX5, a multifunctional masterregulator, through the proteasome degradation pathway and has the function of dephosphorylating and degrading it. DDX5 silencing indicates that DDX5 is a master regulator for regulating the expression of multiple oncoproteins including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras.

[0122] FL118 effectively inhibits cancer initiation, development, metastasis, recurrence and treatment resistance by indirectly controlling DDX5 downstream targets, as demonstrated in studies using human colorectal cancer / pancreatic cancer cells and tumor models.

[0123] Genetic manipulation of DDX5 in PDAC cells affects tumor growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Studies in human tumor animal models have shown that FL118 shows high efficiency in eliminating human PDAC and CRC tumors with high DDX5 expression, while FL118 is less effective in PDAC and CRC tumors with low DDX5 expression.

[0124] DDX5 protein is a direct target of FL118 drug and can be used as a biomarker to predict PDAC and CRC tumor sensitivity to FL118.

[0125] On the other hand, FL118 has the same level of Top1 inhibition efficacy as SN-38 in cancer cells and is 5-20 times more potent than SN-38 in various cancer cell lines, i.e., at a low level of IC 50 The numerical values ​​show cytotoxicity, and the evaluation results of 140 cell lines derived from various cancer types also showed very strong anti-cancer efficacy, with IC values ​​of 2.17 for most cancer cells. 50, showing <100nM. FL118 drug ensures excellent safety through GLP-toxicity tests in rats and beagles, and shows better efficacy than SN-38 in various cancer cell line Xenograft models. On the other hand, for Camptothecin anticancer drugs, when used in patients, they initially show excellent anticancer response, but through epigenetic silencing of Top1 gene and Top2 dependence of cancer cells, they show strong resistance to these drugs. On the contrary, FL118 drug shows strong efficacy even in xenograft models of cancer cell lines that do not express Top1 through epigenetic silencing or knockout.

[0126] In addition, FL118 directly targets the established anticancer target type 1 topoisomerase, and is a triple-target anticancer drug that simultaneously inhibits Bcl family members (Bcl family) such as the resistance protein Survivin involved in the drug resistance mechanism, and inhibits the single-acting efflux pump.

[0127] Specifically, SN-38 and other camptothecin anticancer drugs show drug resistance in the way they release drugs outside the cell due to overexpression of ABCG2 Transporter, but FL118 drug is not affected by ABCG2 transporter, so it can overcome drug resistance. FL118 drug can block the expression of drug resistance by strongly inhibiting the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.), which is another major cause of anticancer drug resistance, at low concentrations. Fig.10 ).

[0128] Therefore, FL118 will not be excreted from cells by the efflux pump ABCG2, and can block drug resistance caused by various anti-apoptotic proteins. As a result, FL118 can overcome various drug resistance mechanisms of SN-38 / Exatecan.

[0129] When FL118 was administered in vivo at the same amount as SN-38, it showed a more potent (tumor regression) effect than SN-38 in colon cancer, head and neck cancer, pancreatic cancer, etc.

[0130] After FL118 induced SN-38 resistance in xenograft tumors, it retained strong anticancer efficacy when administered. The potential of FL118 to overcome various resistance mechanisms of SN-38 / exitecan was confirmed in vivo.

[0131] Furthermore, the FL118 drug has an optimal PK / safety profile for targeted drug delivery (e.g., carrier-drug conjugate applications). When the FL118 drug is administered alone systemically, it is rapidly metabolized / excreted from the blood and only shows a low concentration, but it rapidly accumulates in cancer tissues immediately after administration and maintains a high concentration for a long time. For example, when ADC is used, the maximum selectivity between tumor tissues and normal tissues is ensured. This is also true for the active camptothecin derivative (a) represented by Formula 1 (Examples 6 and 8).

[0132] In short, for the FL118 drug,

[0133] (i) In addition to the well-validated anticancer target Top1, other anticancer targets, such as DDX5, UBE2T, and USP2a, can be selectively inhibited / degraded to create a powerful anticancer effect;

[0134] (ii) showed 5-10 times stronger cytotoxicity than SN-38 and equal to or higher than that of exitecan;

[0135] (iii) Unlike SN-38 and ixotecan, it is not a substrate for ABCG2 and P-GP, thus overexpression of these transporters can circumvent drug resistance mechanisms;

[0136] (iv) It exhibits excellent efficacy in cancer animal models that induce epigenetic silencing of Top1 / over-expression of Top2, which is the main mechanism of resistance to Top1 inhibitors;

[0137] (v) Fundamentally blocking the overexpression of anti-apoptotic proteins, which is a common mechanism of drug resistance in most cancers, can avoid drug resistance;

[0138] (vi) Biomarkers (DDX5, K-ras, p53) and companion diagnostic technologies that can predict patients' anti-cancer responses have been established. By ensuring customized biomarkers, personalized treatment through companion diagnostics can be achieved, especially showing strong efficacy in p53 / K-ras mutant cancer cells with poor prognosis.

[0139] KRAS is one of three genes associated with the production of RAS protein, which is involved in cell proliferation signaling. It is known that mutations occur in more than about 20% of cases overall, although they vary by cancer type. However, despite the scientific expectation of anti-cancer efficacy, it is also regarded as an undruggable target. This is because the surface of RAS protein is smooth, so no pockets suitable for binding compounds have been found.

[0140] [Antitumor mechanism as a selective inhibitor of topoisomerase-1]

[0141] Camptothecin is a selective inhibitor of type 1 topoisomerase (TOPI)-1, an isomerase involved in DNA replication and recombination. Figure 1 Since the discovery of strong cytotoxicity in vitro, the National Cancer Research Center (NCI) of the United States and others have begun to develop through clinical trials, but due to the limitation of extremely poor solubility, various side effects such as bone marrow suppression and hemorrhagic cystitis, the research and development was interrupted. However, after 1990, the unique mechanism of action of camptothecin, that is, different from the inhibition mechanism of DNA type 2 topoisomerase (topoisomerase-2), selectively inhibits DNA type 1 topoisomerase to show anti-tumor effect.

[0142] DNA topoisomerases are enzymes in the cell nucleus that have the function of temporarily cleaving DNA or unwinding the double helix when the cell needs access to genetic material for replication or transcription. They are also involved in various intracellular activities such as chromosome condensation and recombination, and DNA repair. The genetic code of topoisomerases is highly conserved between species.

[0143] It has been observed that the level of type 1 topoisomerase, which is the drug target of camptothecin, is increased in various malignant tumors. Although the drug does not inhibit the free enzyme, it can stabilize the covalent bonds of the topo-DNA complex to prevent the cleaved DNA fragments from reconnecting. Therefore, the sensitivity of cells to this topoisomerase-target drug is related to the level of enzymes present in the cell nucleus. The drug interferes with the recombination of DNA to prevent transcription. The higher the amount of type 1 topoisomerase, the more cleavable complexes are formed, which means that the drug sensitivity is high. This has important clinical relevance, and type 1 topoisomerase inhibitors are used to increase the expression of type 2 topoisomerase, making it more sensitive to type 2 topoisomerase inhibitors. The antagonistic relationship between type 1 topoisomerase and type 2 topoisomerase supports these results. Unlike type 2 topoisomerases, these type 1 topoisomerases are not closely related to proliferation in normal tissues. Compared with the surrounding normal tissues, they are present in large quantities in solid tumors during the "S" phase when cell division is active, such as colon cancer, ovarian cancer, esophageal cancer, etc., including lymphomas. In fact, during the "S" phase of the cell cycle, they may cause cell death by blocking gene replication and transcription in tumor cells.

[0144] [Antitumor mechanism of a molecular glue degrader binding to DDX5 2]

[0145] The ubiquitin-proteasome system (UPS) is an important pathway for degrading intracellular proteins, which are used to regulate a wide range of cellular processes. Ubiquitin is a small protein covalently linked to lysine residues of substrate proteins through a series of enzymatic reactions including ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s), and ubiquitin ligases (E3s). This process is called ubiquitination and is an important mechanism for regulating protein degradation, signal transduction, and transport.

[0146] Ubiquitin ligases are responsible for substrate specificity in the ubiquitination pathway.

[0147] In connection with this, the active camptothecin derivative represented by Formula 1 according to the present invention is designed in such a manner as to bind to the DDX5 protein and the E3 ligase.

[0148] The DDX5 protein (also known as p68) is a multifunctional master regulator that acts on the following mechanisms: (1) the biological process of co-activation of transcription of a large number of oncogenic genes through direct interaction with various transcription factors (e.g., c-Myc) in 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).

[0149] The active camptothecin derivative represented by Formula 1 according to the present invention binds to the DDX5 protein in the absence of DDX5 mRNA reduction and is degraded through functional dephosphorylation and proteasome degradation pathways, which means that the active camptothecin derivative of Formula 1 can attach to both DDX5 and ubiquitin-involved protein stability / degradation regulators and act as a "molecular glue degrader".

[0150] As is well known, DDX5 downstream protein targets are all related to cancer occurrence, development, metastasis, recurrence and treatment resistance. Therefore, when the DDX5 downstream targets are indirectly blocked by the degradation of the DDX5 protein of the active camptothecin derivative represented by Formula 1 according to the present invention, the active camptothecin derivative represented by Formula 1 can exhibit high anti-tumor efficacy.

[0151] [DDX5 as a drug target and / or biomarker]

[0152] Transcription factors are groups of proteins that bind to DNA strands called promoters or enhancers through their DNA binding domains and initiate transcription of genes in cooperation with RNA polymerase and other auxiliary factors. They play an important role in transcriptional regulation during embryogenesis and development. The physiological status of transcription factors in other cell types is also important for maintaining cellular homeostasis. Therefore, the deregulation of transcription factors will lead to the development of cancer cells and the progression of tumors. Depending on the function of transcription factors in cancer cells, transcription factors can be oncogenic or tumor suppressive. In addition, transcription factors have been shown to regulate cancer stem cells, epithelial-mesenchymal transition (EMT), and drug response. Therefore, measuring deregulated transcription factors is considered to predict the treatment outcome of cancer patients, and targeting deregulated transcription factors may be an encouraging strategy for cancer treatment. Major transcription factor deregulation may lead to poor clinical outcomes in cancer patients. Therefore, it is helpful to predict prognosis and drug response and target deregulated transcription factors to design new drugs and therapeutic strategies for cancer treatment.

[0153] As we all know, transcription is the first step of cellular gene expression during embryogenesis, development and homeostasis. During transcription, DNA produces RNA transcripts containing specific coding information. In order to ensure the specificity of gene expression in different cells and tissues, transcription factors (a group of specific proteins) control transcription by selecting genes to be transcribed into RNA transcripts. Transcription factors have DNA binding domains that can bind to specific DNA sequences called gene promoters or enhancers, and can initiate gene transcription with the cooperation of activators, mediators, co-factors and RNA polymerase.

[0154] Transcription factors also control the rate of transcription from DNA to mRNA. Ultimately, the physiological state of transcription factors in different types of cells is very important for maintaining cell homeostasis. Changes in the physiological state of transcription factors in cells cause transcriptional disorders and cause the translation of specific genes, thus causing pathological changes in cells. Therefore, transcription factor dysregulation can lead to many human diseases. A common disease of transcription factor dysregulation is cancer.

[0155] Homeostatic control of human cells normally prevents inappropriate proliferation and inhibits the growth of cells that proliferate abnormally outside their normal niche. As transcriptional control is a key process during homeostasis, dysregulation of the levels or activity of transcription factors can cause cells to escape homeostatic control and contribute to the initiation and progression of human cancers.

[0156] Indeed, many chemotherapy regimens can activate tumor suppressor transcription factors.

[0157] 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. Dysregulation of oncogenic and tumor suppressor transcription factors favors the survival of cancer cells, cancer stem cells, and EMT cells, thereby inducing intrinsic or acquired drug resistance and tumor progression in cancer therapy.

[0158] DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and transcription cofactor. Therefore, when cancer occurs due to transcription factor deregulation caused by the physiological state of DDX5, the transcription cofactor DDX5 (p68) can be selectively degraded to treat cancer diseases. Similarly, cancer diseases can be prevented by selectively degrading the transcription cofactor DDX5 (p68).

[0159] DDX5 is considered a potential biomarker and target for the treatment of various cancer types. Over the years, research has greatly expanded our understanding of the functional diversity of DDX5 in various cancer types and expanded our knowledge of its mechanism of action (MoA). This provides a rationale for the development of new cancer treatments using DDX5 as a biomarker and therapeutic target. However, although most published studies have shown that DDX5 is an oncogenic target and a biomarker of cancer treatment resistance, some studies have reported that DDX5 may act as a tumor suppressor in specific scenarios. The multiple functions of DDX5 make it an excellent independent oncogenic biomarker and target for cancer targeted therapy.

[0160] The transformation of normal cells into cancerous cells occurs through the deregulation of different metabolic pathways involving a complex network of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cellular metabolism, but when deregulated, they can accelerate neoplastic transformation. DDX5 interacts with hundreds of other cellular proteins, and both proteins can act as tumor suppressors or oncogenes, depending on the specific pathways involved.

[0161] The DEAD-box family of RNA helicases participates in a variety of metabolic pathways, from transcription and translation to cell proliferation, innate immunity, and stress responses. Given their diverse roles, their deregulation or mutation has been implicated in a variety of pathological conditions, including cancer. However, in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation, suggesting a tumor suppressor role, whereas in other cases, overexpression of the same enzyme favors cancer progression, acting as a classic oncogene. The role of DDX5 as both an oncogene and a tumor suppressor has been demonstrated in multiple cancer types. Understanding the interplay between different cellular contexts defined by the molecular interaction network of DDX5 in different tumors could help to elucidate the apparently conflicting cancer-specific roles played by these proteins as cancer drivers or suppressors.

[0162] Although DDX5 generally functions as an oncogene, it may also have tumor suppressor functions in certain cancer types (Table 1).

[0163] Source: Secchi, M.; Lodola, C.; Garbelli, A.; Bione, S.; Maga, G. DEAD-Box RNAHelicases DDX3X and DDX5 as Oncogenes or Oncosuppressors: A NetworkPerspective.Cancers 2022, 14, 3820. https: / / doi.org / 10.3390 / cancers14153820

[0164] Table 1

[0165]

[0166] 1 Oncogene(+),oncosuppressor(-).

[0167] Since the DDX5 protein is a drug target, the active camptothecin derivatives represented by Formula 1 according to the present invention can avoid drug resistance, resistance to targeted therapy and / or drug resistance during treatment. In addition, the transcriptional induction of anti-apoptotic genes can be blocked (off) by the active camptothecin derivatives. In addition, the DDX5 protein as a transcription cofactor is degraded by the active camptothecin derivatives, thereby maintaining or improving the sensitivity of cancer cells to chemotherapy or radiotherapy.

[0168] [Camptothecin derivatives represented by Formula 1, Formula 3 to Formula 9 and isomers thereof]

[0169] According to the active camptothecin derivatives represented by Formula 1 of the present invention, wherein R1 and R2 in Formula 1 are designed to be the same as those in the FL118 compound, for example, the following compounds of Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8, Formula 9 and their isomers are also transcription cofactors. They degrade the oncoprotein DDX5, thereby strongly inhibiting the expression of anti-apoptotic proteins (Survivin, cIAP2, XIAP, etc.) in a concentration-dependent manner ( Figures 11 to 14 ), and also kills the corresponding cells into which the compound is introduced through a cell viability experiment ( Fig.15 and Fig.16 ).

[0170] The camptothecin derivatives represented by Formula 1 according to the present invention, for example, the camptothecin derivatives represented by Formulas 3 to 9, are designed as follows: having a five-ring structure having a lactone in the E ring necessary for cytotoxicity, such as Figure 1 The camptothecin shown is designed to maintain the lactone group and α-hydroxyl group located at carbon 20 of the E ring, which is important for the stability of type 1 topoisomerase-DNA byproducts. The structure of the FL118 drug that maintains the advantages of the above-mentioned FL118 drug, R in general formula 1 1 and R 2 (-OCH 2 O-(methylenedioxo) pentagonal ring), the structural characteristics of the drug ixetine (R in general formula 1 3 and R 4 ) is that, compared with SN-38 which induces aggregation through π-π stacking of the aromatic rings formed by the A and B rings, various directions of the continuous carbon-carbon single bonds of the hexagonal rings extending from the A and B rings reach a dynamic equilibrium, thereby weakening or inhibiting the stacking of the aromatic rings formed by the A and B rings.

[0171] In addition, the compound represented by Formula 3 or Formula 3-1 is a compound designed to allow the molecular bond to rotate about the carbon-carbon single bond in the hexagonal ring extending from the A ring and the B ring, so that -NH 2 Exposure to water (H 2 O), so that it carries a (+) charge or forms hydrogen bonds with water to increase water dispersibility.

[0172] In addition, the compound represented by Formula 4 or Formula 4-1 is a compound designed to allow the molecular bond to rotate about the carbon-carbon single bond on the hexagonal ring extending from the A ring and the B ring, so that -NH 2 The glycolic acid form of the functional group CH 2 (OH)CONH- is exposed to water (H 2 O), and forms hydrogen bonds with water while rotating like a propeller to increase water dispersibility.

[0173] In addition, the compound represented by Formula 5 or Formula 5-1 is a compound designed to allow the molecular bond to rotate about the carbon-carbon single bond on the hexagonal ring extending from the A ring and the B ring, so that -NH 2 The functional group CH 3 CH(OH)CONH- is exposed to water (H 2 O), and forms hydrogen bonds with water while rotating like a propeller to increase water dispersibility.

[0174] The compound represented by Formula 5 or Formula 5-1 of the present invention is a compound represented by Formula 4 or Formula 4-1 in which a methyl group is introduced, which is a metabolically unstable functional group, to reduce the life of the drug. Extremely stable and slowly metabolized drugs may cause toxicity and serious side effects due to accumulation, so it is necessary to ensure an appropriate retention time.

[0175] On the other hand, the DXd payload used in Enhertu was originally made of an Exatecan compound that was hardly affected by ABCG2, but due to the presence of the glycolic acid (alpha-hydroxyacetic acid) functional group used to convert Exatecan into DXd, it was strongly affected by ABCG2. However, the glycolic acid functional group plays a very important role in the excellent safety / efficacy characteristics of Enhertu. When it is eliminated, it will bring difficulties to the preparation of ADC, and will also bring problems of deteriorating the characteristics (safety issues or reduced efficacy) of ADC in animal models and clinical trials. Therefore, it is very necessary to be able to easily use it in ADC production and new camptothecin derivatives that are not affected by ABCG2. The compound (PBX-7024) represented by Formula 9 is a compound derived from a compound (PBX-7011) represented by Formula 3, which is a new camptothecin compound that is not affected by ABCG2 and can be easily used in ADC preparation.

[0176] In order to confirm the anticancer efficacy of PBX-7024, efficacy evaluation was performed in FaDu (a cancer cell line that does not express ABCG2) and A549 (a cancer cell line that overexpresses ABCG2). In the cancer cell line A549 that overexpresses ABCG2, it was confirmed that PBX-7024 showed an increased IC 2 with camptothecin compounds including DXd. 50 Compared with the numerical situation, PBX-7016 and PBX-7024 can still maintain powerful functions, such as Fig.15 shown.

[0177] That is, when the novel camptothecin compounds PBX-7016 and PBX-7024 according to the present invention are used, unlike ADCs using existing camptothecin compounds SN-38, DXd, etc., there is an effect of being able to overcome the drug resistance mechanism caused by overexpression of ABCG2.

[0178] Fig.28 and Fig.29 The figure shows a comparison of LogP, cLogP and topological polar surface area (tPSA) of FL118, Exitecan, 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'.

[0179] The compound of formula 3 and the compound of formula 3-1 have a diastereoisomer 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 also have a diastereoisomer relationship with each other. Therefore, compounds that are in a diastereoisomer relationship with the compound of formula 6, the compound of formula 7, the compound of formula 8, or the compound of formula 9 also fall within the scope of the present invention.

[0180] As illustrated in Preparation Example 2, PBX-7014 of Formula 4 can be synthesized from PBX-7011 of Formula 3, and PBX-7015 of Formula 4-1 can be synthesized from PBX-7012 of Formula 3-1.

[0181] As illustrated in Preparation Example 3, the compound of Formula 5 (PBX-7016, Compound E) can be synthesized from the compound of Formula 3 (PBX-7011, Compound A). Similarly, the compound of Formula 5-1 (PBX-7017, Compound F) can be synthesized from the compound of Formula 3-1 (PBX-7012, Compound B).

[0182] In the present specification, the camptothecin drugs 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 include not only the compounds 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 include pharmaceutically acceptable salts thereof, solvates thereof, prodrugs thereof, preferably carrier-drug conjugates (CDCs), and more preferably antibody-drug conjugates (ADCs).

[0183] In this specification, pharmaceutically acceptable salt refers to salts commonly used in the pharmaceutical industry, such as: salts of inorganic ions, sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc.; salts of inorganic acids, hydrochloric acid, phosphoric acid and sulfuric acid, etc.; salts of organic acids, ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, acetylsalicylic acid, etc.; amino acid salts, lysine, arginine, guanidine, etc. And there are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, benzethonium, etc. that may be used in drug reactions, purification and separation processes. However, the types of salts referred to in the present invention are not limited by the types of these salts listed.

[0184] "Solvate" refers to a 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 and a pharmaceutically acceptable salt thereof, which further comprises a stoichiometric or non-stoichiometric amount of a solvent bonded by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0185] [Advantages of the drug model of molecular glue degrader]

[0186] Proteins in cells in the body are naturally degraded within a few hours to a few days after they have fulfilled their functions. In all cells in the body, there is a purification system called the Ubiquitin proteasome system (UPS) that degrades proteins. In this process, ubiquitin plays a marker role to inform proteins that need to be degraded. The proteasome has the function of recognizing ubiquitin tags and destroying the corresponding proteins. That is, next to the protein that has fulfilled its function, multiple substances called ubiquitin are attached like tags. The so-called proteasome only picks up proteins with the tags and then degrades them like a shredder. E3 ligases are enzymes that cause protein degradation systems in the body and are responsible for substrate specificity in the ubiquitination pathway.

[0187] Molecular glue degraders or molecular glues are compounds with adhesive functions that attach target proteins to specific enzymes (E3 ligases) in our body. One of the advantages of molecular glue is the catalytic effect, which degrades the target protein and then detaches again to degrade another target protein.

[0188] When the E3 ligase is attached to the tumor protein through the molecular glue, the tumor protein is degraded, and other tumor proteins are continuously degraded until the targeted tumor protein disappears, thereby preventing the proliferation of cancer cells. Therefore, the molecular glue of tumor protein overcomes the drug resistance that is a problem of targeted anticancer drugs, and has a high therapeutic effect even at a low dosage.

[0189] In addition to the FL118 drug, the active camptothecin derivative designed in Formula 1 of the present invention is a molecular glue degrader that binds to the DDX5 protein and the E3 ligase, that is, a molecular glue degrader that activates the degradation of the cancer protein DDX5. Figures 11 to 14 ).

[0190] Molecular glue degraders are not only ligands (warheads) that bind to target proteins, but can also act as E3 ligase ligands (binders).

[0191] Therefore, the active camptothecin derivative designed in Formula 1 according to the present invention is a molecular glue degrading agent that activates the degradation of the cancer protein DDX5, and can therefore be used as a ligand that targets the DDX5 protein or a ligand that binds to the DDX5 protein.

[0192] Unlike kinase inhibitors, molecular glue degraders are "proximity-driven" and their ability to induce degradation is "event-driven" and depends on the formation of a temporary "target protein-molecular glue-E3 ligase" ternary complex. After degradation occurs, the separated molecular glue forms additional ternary complexes with the target protein, which can undergo multiple degradation processes until the target protein disappears.

[0193] Most proteins that are drug targets develop drug resistance that evolves in response to the drugs. However, molecular glue, a low-molecular compound that functions as an adhesive to bind tumor proteins and E3 ligases, is a suitable model for cancer therapeutic drugs because it is more resistant to drug resistance.

[0194] Every time a gene mutation occurs, the shape of the drug target protein changes slightly. It would be great to block the activity of all variants with one drug, but this is impossible due to selectivity issues. In order to block the activity of all mutations with one drug, the drug target protein is degraded and completely eliminated.

[0195] Therefore, the active camptothecin derivative represented by Formula 1 according to the present invention is a drug that can accurately bind to the DDX5 oncoprotein and can avoid the drug resistance problem of targeted therapeutic drugs through a molecular glue approach method that selectively degrades the protein.

[0196] The active camptothecin derivative represented by Formula 1 according to the present invention determines the binding strength with the target protein DDX5 tumor protein according to its physicochemical properties, and is preferably an irreversible drug, which binds so strongly that it cannot return to its previous state.

[0197] Therefore, the active camptothecin derivative represented by Formula 1 according to the present invention can be obtained by 1 、(X 2 )n、X 3 , Y 1 , Y 2 and / or Y 3 to control the binding strength to the target protein DDX5 oncoprotein.

[0198] Unlike the existing SN38 drug, the active camptothecin derivative designed by Formula 1 according to the present invention has a high affinity for the DDX5 protein, is not easy to fall off, and degrades the DDX5 protein through the molecular glue function, thereby not only irreversibly inhibiting DDX5-related signal transduction in cancer cells, but also adjusting the cell membrane permeability of the drug as needed according to its physicochemical properties to exert a bystander effect or control the extent of its effect, thereby having a high bystander effect, which is beneficial to the treatment of heterogeneous tumors, and can inhibit long-term cancer progression, reduce the risk of drug resistance expression, and improve the treatment response rate.

[0199] [Apoptosis and drug resistance to anticancer drugs]

[0200] The active camptothecin derivative represented by Formula 1 according to the present invention can bind to DDX5, which acts as an oncoprotein in cells, to induce cell death by degradation of DDX5 protein ( Fig.30 ).

[0201] There are two factors that determine tumor growth: cell proliferation and cell death. When the cell cycle of tumor cells is stopped by cytotoxic substances, tumor cells will die by apoptosis.

[0202] Apoptosis plays a very important role in maintaining tissue homeostasis and morphology, regulating cell number, removing damaged or abnormal cells, and serving as a defense mechanism against infection. Organisms maintain normal life by precisely controlling the processes of cell growth, differentiation, and death.

[0203] Apoptosis is described as a complex process that induces cell death through the expression of various genes and the activation of proteins as expression products. This apoptosis is a physiological active suicide mechanism in cells, which plays an important role in the development and differentiation of multicellular organisms and in maintaining homeostasis. Due to a variety of factors, various types of cells will undergo apoptosis. Unlike cell necrosis, there is no free lysosomal enzyme, but apoptotic bodies are found, and cell shrinkage, nuclear condensation and characteristic ladder-shaped DNA fragments are observed.

[0204] Apoptosis is part of the normal developmental program of animals and is important for cancer prevention. Caspases are a family of proteolytic enzymes that participate in apoptosis and cleave many target proteins. When apoptosis is impaired, cells that should be killed can survive and proliferate, forming potentially cancerous clones.

[0205] In cancer treatment, intrinsic drug resistance can be caused by abnormally expressed transcription factors, which are key regulators of cell proliferation and apoptosis. Therefore, the active camptothecin derivatives of the present invention can induce DDX5 protein degradation and the resulting cell death by acting as transcription factor cofactors and molecular glue degraders of oncoproteins that bind to DDX5. In this case, DDX5 protein degradation can downregulate the transcription of anti-apoptotic genes ( Figures 4 to 8 ). Therefore, unlike other targeted therapeutic drugs, drug resistance caused by abnormally expressed transcription factors related to cell proliferation and / or apoptosis does not occur, and / or acquired drug resistance induced by activation of transcription factors that are deregulated during chemotherapy and / or radiotherapy can be suppressed.

[0206] That is, the anti-cancer mechanism of the active camptothecin derivatives according to the present invention can bypass the molecular mechanism of cancer treatment resistance, thus avoiding the problem of drug resistance. When drug resistance occurs, the treatment effect is lower than before, therefore, the active camptothecin derivatives of the present invention can be preferably used as standard treatment drugs or primary treatment drugs after cancer diagnosis.

[0207] In short, the active camptothecin derivative represented by Formula 1 according to the present invention may be a targeted anticancer drug acting on DDX5 protein, which plays an important role in the growth, survival (cell survivor), proliferation, metastasis and / or metabolism of cancer cells.

[0208] [Clinical results and side effects of ADCs with Camptothecin Payloads]

[0209] Trastuzumab Deruxtecan (trade name: Euherde) is a humanized anti-HER2 antibody connected to trastuzumab (camptothecin derivative) through a stable linker. In the first phase 1 dose-escalation study, 22 patients with HER-2-positive advanced or metastatic breast cancer, gastric cancer, or other solid tumors expressing HER-2 received 0.8 mg / kg to 8.0 mg / kg of trastuzumab Deruxtecan every 3 weeks. No dose-limiting toxicity was observed and the MTD was not reached. The target drug exposure dose was 6.4 mg / kg, which was selected as the recommended phase 2 dose.

[0210] The DESTINY-Breast01 clinical trial is a pivotal single-arm Phase 2 clinical trial consisting of two parts. In the first part, patients with advanced / metastatic breast cancer who had previously received more than two anti-HER2 therapies were randomly assigned to receive treatment with 5.4 mg / kg (n=50), 6.4 mg / kg (n=48), or 7.4 mg / kg (n=21), with desutuzumab administered every 3 weeks. In the second part, based on the efficacy and toxicity data obtained in part 1, a dose of 5.4 mg / kg was administered to 134 patients. When 184 patients were treated with trastuzumab at 5.4 mg / kg, the most common adverse reactions of all grades (≥20%) were nausea (77.5%), fatigue (49.8%), alopecia (49.8%), vomiting (44.3%), neutropenia (40.3%), constipation (37.5%), anemia (33.6%), decreased appetite (33.2%), diarrhea (29.2%), leukopenia (26.9%), and thrombocytopenia (24.9%). Adverse reactions of grade 3 or higher occurred in 57.1% of patients, the most common of which were neutropenia (20.7%), anemia (8.7%), nausea (7.6%), leukopenia (6.5%), lymphopenia (6.5%), and fatigue (6.0%). Discontinuation, dose reduction, and treatment cessation due to adverse reactions occurred in 35.3%, 23.4%, and 15.2% of patients, respectively, of which pneumonia (11 cases) and interstitial lung disease (5 cases) were the most common reasons. Black box warnings for patients treated with trastuzumab include interstitial lung disease (ILD) and pneumonia. Treatment-related interstitial lung disease and fatal outcomes occurred in 9% and 2.6% of patients treated with trastuzumab, respectively. Similar to other HER-2-targeted ADCs, patients treated with trastuzumab also have an increased risk of embryo-fetal toxicity and left ventricular dysfunction.

[0211] On the other hand, Sacituzumab Govitecan (trade name: Trodelvy) is a humanized anti-TOP-2 IgG connected to the active metabolite of irinotecan (SN-38) through a pH-sensitive linker. In the first-in-human dose-escalation 1 / 2 phase 1 clinical trial, 25 patients with various metastatic solid tumors were treated with Sacituzumab Govitecan at a dose of 8 mg / kg to 18 mg / kg on the first and eighth days of a 21-day cycle. The MTD of the first cycle was determined to be 12 mg / kg, and neutropenia showed dose-limiting toxicity. However, this dose level was too toxic in subsequent cycles, and 8 mg / kg and 10 mg / kg doses were selected in a phase 2 clinical trial. In this clinical phase 2, patients with various metastatic epithelial cancers who had previously received various treatments were given 8 mg / kg (n=81) or 10 mg / kg (n=97) of gosatuzumab. The most common adverse reactions of all grades (≥25%) reported in the 8 mg / kg and 10 mg / kg cohorts were nausea (59% vs. 63%), diarrhea (53% vs. 62%), neutropenia (42% vs. 58%), fatigue (61% vs. 52%), vomiting (36% vs. 43%), anemia (38% vs. 42%), alopecia (46% vs. 37%), and constipation (33% vs. 37%). The most common adverse reactions of grade 3 or above (≥10%) reported in the 8 mg / kg and 10 mg / kg administration groups were neutropenia (30% vs. 36%), anemia (13% vs. 12%), diarrhea (4% vs. 10%), and leukopenia (6% vs. 12%). Dose reductions occurred in 19% and 28% of patients in the 8 mg / kg and 10 mg / kg cohorts, respectively. Neutropenia was the most common adverse reaction leading to dose delays or reductions. Significantly more patients in the 10 mg / kg cohort experienced grade 3 or greater neutropenia after the first dose than in the 8 mg / kg cohort (47% vs. 21%).

[0212] A boxed warning for severe or life-threatening neutropenia and severe diarrhea has been added to the gosartuzumab label. The likelihood that these side effects are mediated by released (“free”) SN-38 is high because it is associated with the same toxicity as iotikon, a prodrug of SN-38. Neutropenia of all grades and grade 3 or greater occurred in 61% and 47% of all patients treated with gosartuzumab, respectively. Febrile neutropenia occurred in 7% of patients. Diarrhea of ​​all grades and grade 3 or greater occurred in 65% and 12% of all patients treated with gosartuzumab, respectively. Neutropenic colitis occurred in 0.5% of patients.

[0213] On the other hand, when patients who have received Enhertu experience ILD / pneumonitis, corticosteroid treatment is indicated (https: / / www.enhertuhcp.com / en / gastric / manage-potential-risks / ild-and-pneumonitis).

[0214] Corticosteroids play an important role in immunosuppression by modulating various aspects of the immune response and reducing inflammation. The mechanism of action of corticosteroids helps to suppress excessive immune activity and alleviate organ damage in conditions where immune regulation is dysregulated.

[0215] Immunosuppressants, especially corticosteroids, are widely used to suppress excessive immune responses and alleviate organ damage in various clinical scenarios, including autoimmune diseases, transplant rejection, and immune-related adverse events (irAEs) associated with checkpoint blockade therapy.

[0216] One of the main mechanisms of corticosteroid action is its ability to inhibit proinflammatory gene transcription. In immune cells, corticosteroids bind to cytoplasmic specific receptors to form corticosteroid-receptor complexes. This complex moves to the nucleus and binds to a specific DNA sequence that becomes a glucocorticoid response element (GRE) in the promoter of the target gene. Thus, corticosteroids interfere with the recruitment of transcription factors and cofactors, ultimately blocking the transcriptional activation of proinflammatory genes. This results in a reduction in the synthesis of inflammatory mediators such as cytokines (such as interleukins and tumor necrosis factor-α) and prostaglandins, thereby causing tissue damage.

[0217] In addition, corticosteroids can directly regulate immune cell function. Corticosteroids inhibit the migration of immune cells to sites of inflammation by reducing the expression of adhesion molecules on endothelial cells and downregulating chemokine receptors on immune cells. Corticosteroids also inhibit the activation and proliferation of T cells, which are important mediators of immune responses. By interfering with the antigen presentation process and the production of co-stimulatory molecules, T cell activation can be weakened and effector function can be reduced. In addition, corticosteroids also inhibit the function of B cells to reduce antibody production.

[0218] Therefore, ADCs with Camptothecin Payloads may also be among the drugs that trigger immune responses (and, depending on the situation, pneumonia / ILD side effects).

[0219] [ADME characteristics of antibody-drug conjugates (ADCs)]

[0220] The in vivo efficacy and in vivo side effects of anticancer drugs can be affected by the absorption, distribution, metabolism, and excretion (ADME) properties of the ADC and the payload released thereby. The ADME characteristics of a drug can affect its ability to reach cancer cells and act on them. In other words, the ADME properties of the ADC and the payload released thereby can have a significant impact on in vivo efficacy and in vivo side effects.

[0221] The ability of ADCs to penetrate tumor tissue, and the payload released thereby, may also be affected by factors such as the tumor microenvironment, including blood flow and cell density.

[0222] Therefore, understanding the ADME properties of ADCs and the payloads released thereby, and optimizing the choice of drug / drug modality and dose / administration, can improve the therapeutic index of these ADCs and the payloads released thereby, leading to better outcomes for cancer patients.

[0223] Due to their different structures and mechanisms of action, antibodies (Ab) and antibody-drug conjugates (ADCs) have different life times and ADME characteristics.

[0224] Antibodies are large proteins that react with foreign substances (antigens) and are naturally produced by the immune system. Due to their size and complex structure, they have a long circulation half-life (weeks to months), which protects them from degradation and elimination. Antibodies are distributed throughout the body, including tissues, and are able to interact with target antigens with high specificity and affinity. Antibodies are mainly eliminated through degradation metabolism by the reticuloendothelial system (RES), including the liver and spleen, and the kidneys and other organs.

[0225] ADC is composed of antibodies (usually monoclonal antibodies) conjugated to cytotoxic drug molecules. The antibody component provides specificity and targeting to tumors or diseased tissues, whereas the drug component (the payload released by ADC) provides cytotoxic activity that kills target cells. The half-life of ADC is shorter than that of antibodies, typically a few days to a week. This is because ADC is internalized into target cells, resulting in lysosomal degradation of ADC and release of drug payload. ADC is mainly eliminated by RES, but the drug payload released by ADC can also be drained by lymphatics, and then metabolized and excreted in the liver and kidneys by the circulatory system (Example 4).

[0226] As expected from the pharmacokinetics and biodistribution of monoclonal antibodies, even in ADCs, high-affinity mAb binding to cell membrane proteins allows the majority of the mAb to be localized to the target cell population, and chemical conjugation of payload and anticancer mAb increases the selectivity of payload delivery to cancer cells to improve the therapeutic index of the payload.

[0227] Antibodies are usually administered by subcutaneous or intravenous injection and absorbed into the bloodstream. They can be distributed throughout the body, including tissues, but due to their large size, they are confined to the extracellular space. ADCs are also administered by injection and absorbed into the bloodstream, but due to the antibody component, after targeting tumors or diseased tissues and, as the case may be, receptor-mediated endocytosis, the drug payload released by the ADC can penetrate into cells and tissues depending on its physicochemical properties (e.g., hydrophobicity, size, aggregation or not) and can produce a bystander effect through non-selective uptake ( Fig.31 ). In addition, the metabolism and excretion of ADCs vary depending on the specific structure and the specific drug or antibody used.

[0228] The drug-to-antibody ratio (DAR) is a very important property in determining the pharmacokinetic properties and in vivo distribution in ADC development.

[0229] The higher the DAR of an ADC, the higher the efficacy in in vitro experiments. However, contrary to expectations, ADCs with higher DARs showed lower in vivo efficacy, which is presumed to be because ADCs with more bound drugs have higher plasma clearance. Therefore, the DAR of ADC preparations has been set at around 2 to 4 for some time. For this reason, the technology of binding drugs to cysteine ​​or lysine residues of antibodies is mainly used.

[0230] Many of the cytotoxic drugs and linkers used are hydrophobic, which can lead to problems such as ADC aggregation, loss of affinity for target antigens, or increased plasma clearance. Hydrophilic linkers containing sulfonates or polyethylene glycol (PEG) solve the problems caused by hydrophobic linkers. PEG linkers have the advantages of water solubility, low toxicity, and low immunogenicity.

[0231] It has long been believed that a DAR of approximately 4 is optimal, but in fact, this applies to second-generation linkers using MMAE or DM1 as the drug, while in third-generation linkers, the higher the DAR, the better. Many recently approved ADCs have DAR values ​​close to 8, and new ADCs currently in clinical trials have DAR values ​​between 1 and 15.

[0232] ADCs (Antibody-Drug Conjugates) using Camptothecin-based payloads have attracted much attention as a new approach to treat various cancers (especially solid tumors). Among these ADCs, new ADCs (e.g., Euhede) containing newly synthesized Camptothecin-based payloads (e.g., DXd) optimized for ADC therapy have recently received special attention due to their successful clinical results.

[0233] The success of these ADCs may be due in part to the following features possessed by the new camptothecins: (1) excellent cell growth inhibition; (2) better safety profiles 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 using camptothecin-based payloads such as Euheride or DS-1062a, as mentioned above, significant unmet needs remain, including improved safety (minimization of interstitial lung disease (ILD) and neutropenia) and the development of new ADCs with multiple MoA payloads to address the heterogeneity of cancer.

[0234] In order to address these unmet needs, the present invention provides active camptothecin derivatives of Formula 1, which are designed as molecular glue degraders to bind to DDX5 protein and E3 ligase and are used as candidate drugs providing various physicochemical properties. Furthermore, new camptothecin-based payloads (PBX series compounds) represented by Formula 1 are synthesized and evaluated (Preparation Examples 1 to 4) (Examples 2 to 10).

[0235] The novel PBX series compound represented by Formula 1 is a derivative with FL118 of Formula 2 as the parent core, and is a strong dual Top1 / anti-apoptotic pathway inhibitor with excellent safety, strong ex-vivo cytotoxicity comparable to Dxd, and shows excellent safety in preliminary toxicity studies in mice, and rapid clearance in PK studies (Example 4, Table 4 and Table 5). When the linker is used as an ADC payload and cleaved early, systemic side effects can be minimized.

[0236] like Fig.17 As shown, it was confirmed that it was the result of the conversion experiment from lactone form to carboxylate at pH 7.4 (Example 5). At pH 7.4 of blood or extracellular fluid, PBX-7014, PBX-7016 and PBX-7024 compounds represented by Formula 1 were converted into inactive carboxylate form (inactive form) faster and at a higher ratio as TOP1 antidote in lactone form (active form) compared with other reference compounds (Ixitect, DXd, SN-38, FL118).

[0237] In particular, at blood pH 7.4, the degree (63-86%) and rate (0.4-0.8% / min) of formation of the carboxylate form, which is inactivated as a TOP1 antidote, differed depending on the type of camptothecin derivative ( Fig.17 PBX-7014 and PBX-7016 formed carboxylate forms at more than 86%, and both were converted from lactone forms to carboxylate forms at a relatively fast rate of 0.8% per minute. DXd, as a competing drug, formed carboxylate forms at a level of 76.5%, and was converted from lactone forms to carboxylate forms at a level of 0.6% per minute.

[0238] The rate of formation of the lactone form (measured as the carboxylate form) active as a TOP1 inhibitor at pH 6.0 in the microtumor environment (TME) was confirmed to be similar for all evaluated drugs (20% carboxylate form was formed after approximately 800 minutes) ( Fig.18 ).

[0239] Thus, compared with the reference compound, the compounds represented by Formula 1, such as PBX-7014, PBX-7016 and PBX-7024 compounds, form an inactive carboxylate form in blood or extracellular fluid (normal tissues other than tumor tissues), rather than an active form, i.e., lactone, which exhibits cytotoxicity rapidly and at a high rate, thereby having relatively low toxicity, i.e., it is expected to exhibit high safety in normal cells. However, in the microtumor environment, the compounds represented by Formula 1, such as PBX-7014, PBX-7016 and PBX-7024, all form lactone forms, whose activity is at the same level as other TOP1 inhibitors, so the efficacy is not reduced.

[0240] Among the PBX series of compounds represented by Formula 1, two lead compounds, PBX-7014 and PBX-7016, were selected for further evaluation as ADC payloads linked to various antibodies, including Trastuzumab (HER2 antibody), Nimotuzumab, and Sacituzumab.

[0241] To compare trastuzumab-7014 and trastuzumab-7016 (DAR 7-8) with euhede (dxd-trastuzumab), PBX-7014 or PBX-7016 were linked to trastuzumab in the same manner using existing enzyme-cleavable linkers that are easily synthesized without aggregation problems (Preparation Examples 5, Fig.19 and Figure 20).

[0242] 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) (Examples 7 and Fig. 22 ).

[0243] In the NCI-N87CDX model with high Her2 expression, trastuzumab-7014 and trastuzumab-7016 not only showed clear dose-dependent anticancer efficacy, but also showed similar or better efficacy than DS-8201 at the same dose (Example 8, Fig.23 and Fig.24 ), and trastuzumab-7016 also showed a similar bystander effect as Uredo (Example 9, Fig.25 and Fig.26 ).

[0244] Trastuzumab-7014 and trastuzumab-7016 not only exert tumor regression effects in tumor tissues for more than 20 days after administration through the bystander effect, but also exert different ADMEs through the difference of only one methyl group, thereby showing dose-dependent anticancer efficacy differences (conc. vs. effect) in target tumor tissues, and also showing anticancer efficacy differences over time (effect vs. time) (Example 8, Fig.23 and Fig.24 ).

[0245] If the hydrophobicity of the linker and the drug increases, the aggregation of the ADC will increase, and the therapeutic coefficient of the drug will decrease accordingly. Surprisingly, although trastuzumab-7016 has increased hydrophobicity due to a difference of one methyl group from trastuzumab-7014, the dose-dependent anticancer efficacy (conc. vs. effect) and the anticancer efficacy over time (effect vs. time) are excellent (Examples 8, Fig.23 and Fig.24 ).

[0246] However, depending on the circumstances, excellent anti-cancer efficacy may also increase side effects over time (due to chronic antigen exposure leading to persistent T cell activation and accumulation of inhibitory signals and the resulting T cell exhaustion (T cell exhaustion), side effects caused by the bystander effect on fibroblasts derived from connective tissue around tumor tissue, ILD, neutropenia). For example, according to the drug model (small molecule vs. ADC) of the active camptothecin derivative of Formula 1, exceeding a specific drug concentration (high) may be important, depending on the circumstances, maintaining a specific drug concentration (low) above the time may be important, and by providing the desired degree of hydrophobicity and / or controlling aggregation, it may also be important to quickly or slowly eliminate through lymphatic drainage.

[0247] Therefore, the main feature of the present invention is that, taking into account not only various side effects but also problems such as reduced therapeutic coefficient, the range of selection of effective loads for ADCs that exert appropriate anti-cancer effects can be expanded to various candidate groups including active camptothecin derivatives of Formula 1, wherein the various candidate groups are designed as molecular glue degraders in a manner that binds to DDX5 protein and E3 ligase.

[0248] Despite the remarkable success of ADCs using camptothecin-based payloads (such as shown in Euhede or DS-1062a), nonetheless, as candidates for multiple MoA payloads for improving safety (minimization of ILD and neutropenia) and addressing cancer heterogeneity, according to the present invention, active camptothecin derivatives represented by Formula 1 designed in a manner to bind to DDX5 protein and E3 ligase are provided in a variety of ways, in order to be able to finely provide the desired efficacy (e.g., anticancer, combination therapy) and side effects (promoting carcinogenic inflammation, drug resistance, ILD, neutropenia), more than one suitable drug in the active camptothecin derivatives of Formula 1 is selected, and then a suitable linker is selected, and a suitable dose-dosage can be used.

[0249] [Toxicity profile of ADCs, non-selective uptake, and bystander effect]

[0250] The therapeutic index of a drug is a measure of the safety and efficacy of a drug in medical treatment. It is the ratio between the dose of a drug that causes a therapeutic effect and the dose that causes toxicity or adverse effects. In other words, it represents the range between the therapeutic dose and the toxic dose of a drug.

[0251] A high therapeutic index indicates a wide margin of safety, where the effective dose is significantly lower than the toxic dose. This means that the drug can be administered at therapeutic levels without inducing severe side effects or toxicity. Drugs with a high therapeutic index are generally considered safer and more suitable for clinical use.

[0252] Conversely, a low therapeutic index refers to a narrow safety margin. In this case, the effective dose and the toxic dose are close, and the risk of side effects or toxicity is higher when the drug is used. Drugs with a low therapeutic index require careful monitoring and accurate dosing to avoid harm to the patient.

[0253] The therapeutic index is an important consideration in drug development because it helps determine the dosage range that provides the desired therapeutic effect while minimizing the risk of side effects. It provides useful information when prescribing a drug and allows for the assessment of the overall benefit-to-risk ratio of a drug.

[0254] Antibody-drug conjugates (ADCs) are a rapidly developing class of anticancer therapeutics, with more than 100 ADCs currently in clinical research. Currently, the U.S. Food and Drug Administration (FDA) has approved 12 ADCs, including gemtuzumab ozogamicin (Mylotarg), brentuximab (Adcetris), oecitabine (Besponsa), trastuzumab enrofloxacin (Kadcyla), vepotuzumab (Polivy), venetoclax (Padcev), detrastuzumab (Uhedre), goxatuzumab (Tontavi), belantamab vedotin (Blenrep), lantuximab (Zynlonta), tisotumab vedotin (Tivdak), and metuximab (Elahere). Furthermore, relatively small numbers of payload molecules (e.g., MMAE, MMAF, DM1, DM4, calicheamicin, SN38, Dxd, PBD) are used in most approved ADCs and those under development.

[0255] Although some ADCs have demonstrated adequate efficacy and safety to obtain FDA approval, clinical use of all ADCs causes significant toxicity to treated patients, and many ADCs fail during clinical development due to unacceptable toxicity profiles. This is because off-site toxicity remains a problem, limiting the tolerated dose of ADCs to doses below those required for significant anticancer efficacy. Even for FDA-approved ADCs, a large proportion of treated patients require adjuvant therapy to reduce the severity of ADC-related toxicity, and many patients require dose reductions, treatment delays, or treatment discontinuation.

[0256] Clinical data analysis shows that dose-limiting toxicities (DLTs) are often shared by multiple ADCs delivering the same cytotoxic payload, regardless of the target antigen and / or the type of cancer being treated. DLTs are often associated with cells and tissues that do not express the target antigen (i.e., off-target toxicity), often limiting ADC doses to below those required for optimal anticancer efficacy.

[0257] ADCs have the potential to safely improve the efficacy of cytotoxic drugs compared to when used as a single agent. ADCs attach drugs to antibodies, allowing the antibodies to specifically target only diseased sites, thereby delivering drugs only to diseased sites and not to normal tissues. In the case of cancer cells, antibodies that specifically bind to specific antigens expressed on the surface of cancer cells are used to specifically deliver highly toxic drugs to cancer cells, killing only cancer cells.

[0258] In order for an ADC to work, it must enter the target cell. The antibody in the ADC specifically binds to a specific antigen expressed on the surface of the target cell (e.g., a cancer cell), and then enters the target cell through a mechanism called clathrin-coated pits in the cell membrane.

[0259] The ADC trapped inside the cell separates from the clathrin, fuses with other vesicles inside the cell, and then follows the endosome-lysosome pathway. After reaching the endosome, the drug is separated from the antibody by specific elements of the internal environment of the specific tumor cell. The independent and free cytotoxic drug separated from the antibody penetrates the lysosomal membrane and enters the cytoplasm. The activated drug exerts its pharmacological effect by binding to its own molecular targets in the surrounding area, inducing cell death and killing cancer cells.

[0260] During these activities, some cytotoxic drugs passively diffuse, actively transport, or escape from the cell through dead cells (the so-called bystander cell-killing phenomenon).

[0261] Compared to Herceptin's DM1, Euheride's DXd has high membrane permeability, so the results show that the intracellularly released payload is delivered to cells adjacent to the target cells that do not express HER2. Due to the characteristics of the Euheride payload, this may also provide clinical advantages for patients who are refractory to Herceptin or Herceptin, and have a potential bystander effect.

[0262] A large number of cancer-specific antigens are expressed only on the surface of cancer cells. In this case, it is not easy to deliver sufficient amounts of cytotoxic drugs into cancer cells using ADCs, so as an alternative, the strength of the toxin is increased.

[0263] In addition, since the drug must be conjugated with minimal impact on the antibody, the amount of payload that can be delivered is limited. This means that cytotoxic drugs applied to ADCs must be able to kill most tumor cells at low concentrations (nM or pM) and must be subject to drug release control to exert therapeutic effects.

[0264] As cytotoxic drugs combined with ADC, drugs that are more potent than general anticancer drugs are used. The potency of cytotoxic drugs (payloads) combined with ADC is generally 100 to 1000 times greater than that of cytotoxic drugs used alone.

[0265] Most potent cytotoxic drugs introduced into ADCs are too toxic, and the drug payload released by ADCs can also affect normal cells due to the bystander effect. Fig.31 ).

[0266] Therefore, it is necessary to develop an ADC that can act very specifically on target cancer cells without causing serious side effects on normal cells.

[0267] Fibroblasts are a member of connective tissue, especially the dominant cells in the stroma in breast cancer, prostate cancer, and pancreatic cancer.

[0268] Stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs), are a specific type of fibroblast found in the tumor microenvironment. Stromal fibroblasts have attracted extensive attention due to their important role in tumor growth, invasion, and metastasis. CAFs are responsible for the production of paracrine growth factors, proteolytic enzymes, and ECM components.

[0269] According to the present invention, the camptothecin drug represented by Formula 1 is a hydrophobic small molecule that can penetrate the cell membrane, so it can penetrate into the tumor tissue and accumulate at a high concentration, penetrate the cell membrane and exert cytotoxicity inside the cell. After the cell apoptosis is released, it can also sustainably penetrate the cell membrane and move into the cell, and act on the surrounding cells. In this case, the bystander effect is high, and the treatment of heterogeneous tumors may have advantages, but according to the circumstances, in order to provide a modality for ADC toxicity based on an ILD such as a camptothecin payload for alleviating or preventing autoimmune diseases, in order to prevent the camptothecin payload released by the ADC from exerting a bystander effect on the fibroblasts around the cancer cells, it can be designed / selected from various candidate groups of active camptothecin derivatives including Formula 1, such as cell membranes that do not penetrate fibroblasts, or IC 50 The effective load that cannot accumulate at high concentrations due to high or rapid elimination through lymphatic drainage. Then, the dose-dose thereof is appropriately applied, thereby precisely controlling the desired efficacy (e.g., anticancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD).

[0270] In addition, the present invention can select the effective load of ADC from the group of camptothecin candidate drugs represented by Formula 1, so as to improve the therapeutic index of the camptothecin drug as the effective load while inhibiting the non-selective uptake of the camptothecin drug and / or ADC released from the apoptotic cells. For example, in a low concentration of drug (effective load), a camptothecin drug with a high killing effect on target cancer cells is selected from the candidate drugs represented by Formula 1, and the ADC dosage is reduced by reducing the total concentration of the effective load of ADC, such as Fig.31 As shown, the by-stander cell-killing phenomenon of free drugs (payload) released from apoptotic cells on normal cells can be inhibited, or the off-target toxicity of free camptothecin drugs (payload) released from apoptotic cells of target / non-target cells can be overcome.

[0271] [Apoptosis of fibroblasts as a member of the connective tissue surrounding cancer cells or tumor tissues and the immune response caused thereby]

[0272] Unlike SN-38, the camptothecin compound represented by Formula 1 is a small molecule drug that binds to oncoprotein DDX5 (p68) for degradation. Oncoprotein DDX5 (p68) inhibits Bcl family such as Survivin, a drug resistance protein involved in the drug resistance mechanism, or controls c-Myc, survivin, and mutant Kras.

[0273] In addition, the camptothecin compound represented by Formula 1 is a camptothecin cytotoxic drug that can degrade DDX5 protein and is designed to bind to DDX5 protein and E3 ligase, has type 1 topoisomerase inhibitory ability, and can kill cells through the mechanism of action (MoA) of degrading oncoprotein DDX5.

[0274] The signaling pathways that regulate apoptosis are a complex network of intracellular signaling pathways that are tightly regulated by various proteins and can be activated by various internal or external stimuli (such as DNA damage, oxidative stress, and growth factor deficiency). Dysregulation of these pathways can lead to various diseases, including cancer and neurodegenerative diseases.

[0275] The major signaling pathways regulating apoptosis all focus on the activation of caspases, a family of caspases that cleave various cellular substrates and ultimately lead to cell death. They are tightly regulated by a variety of proteins, including those of the Bcl-2 family, which can promote or inhibit apoptosis.

[0276] The Bcl-2 protein family includes pro-apoptotic and anti-apoptotic members, which can promote or inhibit apoptosis, respectively. The balance between pro-apoptotic and anti-apoptotic Bcl-2 family members is very important in determining cell fate and sensitivity to apoptosis.

[0277] Other signaling pathways, such as the p53 pathway and the PI3K / Akt pathway, can also regulate apoptosis. The p53 pathway is activated by DNA damage and leads to the transcription of pro-apoptotic genes such as Bax and Puma. On the other hand, the PI3K / Akt pathway can inhibit apoptosis by phosphorylating and inactivating pro-apoptotic proteins such as Bad and Bim.

[0278] Some forms of necroptosis may be a programmed response of cells to stimuli such as infection or DNA damage.

[0279] Immunogenic apoptosis involves changes in the cell surface and the release of cancer antigens and damage-associated molecular patterns during the death of cancer cells due to damage. The released immune-inducing substances can induce anti-tumor immune responses by stimulating dendritic cells and macrophages, which are immune cells.

[0280] Apoptosis is a method by which damaged or aged cells kill themselves, involving the transformation and decomposition of various substances within the cell. During this process, immunogenic substances such as cancer antigens and damage-associated molecular patterns (DAMPs) that induce the body to produce immunity against cancer cells are destroyed by proteolytic enzymes, oxidation, etc., resulting in residual cancer after treatment or limited immunotherapy effects for metastatic cancer.

[0281] The cell death caused by reactive oxygen species produced by chemical anticancer drugs is mainly apoptosis, which involves a proteolytic enzyme caspase.

[0282] Necroptosis is a type of necrosis in which cells kill themselves by collapse of the cell membrane, which differs from apoptosis in that it involves hydrolases such as caspases and does not cause protein degradation or oxidation, thereby minimizing damage to cancer markers or immune-inducing substances.

[0283] Damage-associated molecular patterns (DAMPs) are molecules released during apoptosis that can trigger intracellular immune responses and stimulate immune cells. Representative examples include calreticulin, heat shock protein 70 / 90, high-mobility group box 1, ATP, etc.

[0284] On the other hand, inflammation is the body's natural response to infection or injury and involves the release of inflammatory cytokines and the recruitment of immune cells to the affected area. Furthermore, if inflammation persists for a long time, it can damage tissues and increase the risk of cancer.

[0285] In the case of cancer, T cell exhaustion may occur due to chronic antigen exposure. Tumor cells express antigens that T cells can recognize, but continued exposure to these antigens leads to T cell exhaustion. Therefore, the anti-tumor response is weakened and the tumor grows and spreads.

[0286] Overall, the relationship between T cell exhaustion, chronic antigen exposure, and disease is complex and multifaceted.

[0287] As described above, regarding the clinical results and side effects of ADCs with Camptothecin Payloads, ADCs with Camptothecin Payloads may also correspond to drugs that induce immune responses through cell apoptosis (depending on the situation, pneumonia / ILD side effects).

[0288] One embodiment of the present invention provides an anticancer preparation, characterized in that the following components are used to stimulate antigen presenting cells (APCs) through apoptosis of cancer cells and are administered at a dosage that can induce an anti-tumor immune response, wherein the combination is: the active camptothecin derivative (a) represented by Formula 1, designed to bind to the DDX5 protein and the E3 ligase; or its prodrug (b), designed to release the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), and more preferably an antibody-drug conjugate (ADC).

[0289] Among them, antigen presenting cells (APCs) can be dendritic cells and macrophages.

[0290] In addition, one embodiment of the present invention provides a method for preparing an anticancer drug, wherein in the method for preparing an anticancer drug based on an active camptothecin derivative (a), in order to prevent T cell exhaustion caused by chronic antigen exposure, or, in order to quickly or slowly eliminate through lymphatic drainage by providing a desired degree of hydrophobicity and / or controlling aggregation, the bystander effect and / or ADME characteristics of the active camptothecin derivative (a) are controlled.

[0291] The anticancer drug based on the active camptothecin derivative (a) is an anticancer preparation, which comprises: the active camptothecin derivative (a), as a molecular glue degrader, designed in a manner of binding to the DDX5 protein and the E3 ligase; or a prodrug thereof (b), designed in a manner of releasing the active camptothecin derivative (a) at a target site in vivo, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC);

[0292] The method is characterized in that it comprises the following steps:

[0293] The active camptothecin derivative (a) or its prodrug (b) is designed in such a manner that it contains the camptothecin skeleton represented by the formula 1 as a core, or the compound designed in this manner is selected, or the compound designed in this manner is synthesized.

[0294] As described above, regarding the clinical results and side effects of trastuzumab (Uruheta), the anticancer cell-mediated action caused by ADCs based on camptothecin payloads can provide a therapeutic approach against cancer cells through the immune system. Anticancer cell-mediated action mainly refers to the use of T cells and natural killer cells (NK cells) to attack cancer cells.

[0295] The anticancer cell-mediated effects caused by trastuzumab (Uruheride) are a very useful approach to treating cancer, but side effects often occur. The most common side effect is the development of an autoimmune disease. An autoimmune disease is a condition in which the immune system mistakenly attacks the body's own normal tissues.

[0296] There is a risk of developing autoimmune diseases when receiving anticancer cell-mediated therapy, as the anticancer cell-mediated effects can attack not only cancer cells but also normal cells. Common side effects may include inflammation, fatigue, rash, fever, etc. caused by the drugs that trigger the immune response. However, severe autoimmune diseases (such as ILD) can affect multiple tissues and organs, including the skin, joints, kidneys, liver, and lungs.

[0297] Camptothecin-based payloads released by ADCs not only kill target cells but also have a high killing effect on surrounding cells, which may be beneficial for the treatment of heterogeneous tumors, but depending on the situation, autoimmune diseases such as ILD may occur due to apoptosis of connective tissue-derived fibroblasts surrounding tumor tissue.

[0298] Considering these points, although trastuzumab-7016 has increased hydrophobicity due to the difference of one methyl group from trastuzumab-7014, the dose-dependent anticancer efficacy and the anticancer efficacy over time are excellent (Examples 8, Fig.23 and Fig.24 ), however, side effects such as T-cell exhaustion, ILD, and / or neutropenia may require not only low concentrations / doses of trastuzumab-7016 but also trastuzumab-7014 to control bystander effects.

[0299] Only by understanding the characteristics of drugs can we use them correctly. The pharmacodynamic and pharmacokinetic parameters of drugs help us understand the characteristics of drugs.

[0300] In pharmacodynamics, the magnitude and pattern of changes (effects) (cell viability, clinical effect) (therapeutic action, toxic effect, adverse effect) that occur in cells or in vivo after a drug binds to its receptor are described by their relationship to drug concentration.

[0301] Pharmacokinetics (PK) shows how drug concentration changes as it moves through different compartments of the body through metabolism and excretion (ADME) depending on the drug or drug's mode of action. Example 8, Table 6, Fig.23 and Fig.24 The drug concentration of active camptothecin derivative (a) in tumor tissue over time in various drugs / modes can be predicted.

[0302] From a pharmacokinetic perspective, the reasons for failure in developing new drugs, such as camptothecin-based payloads released by ADCs, are that toxic drugs may accumulate, useful drugs may not have an advantage because doses are too small to establish treatment, and the drugs may be rapidly metabolized.

[0303] Therefore, in order to use the drug correctly, it is necessary not only to select an appropriate camptothecin payload of Formula 1, but also to apply an appropriate dose-dosage, for example, to control the hydrophobicity and / or aggregation of the camptothecin payload of Formula 1 and control the bystander effect through lymphatic drainage.

[0304] Dose refers to the amount of a drug given at one time. The dose of a drug is usually prescribed by a healthcare provider based on factors such as the patient's age, weight, and health status. Conversely, Dosage refers to the frequency and duration of drug administration. It measures the total amount of a drug given over a predetermined period, usually expressed as a daily or weekly amount. Dose and Dosage are important considerations for the safe and effective use of a drug.

[0305] When considering these aspects, the present invention provides various active camptothecin derivatives of Formula 1 that bind to DDX5 protein and E3 ligase as various candidate drugs as molecular glue degraders, thereby selecting more than one appropriate drug and appropriately applying its dosage-dosage regimen, thereby accurately controlling the desired efficacy (e.g., anticancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD).

[0306] For example, depending on the drug model (small molecule, ADC), in terms of apoptosis of heterogeneous tumor cells and / or fibroblasts derived from connective tissue surrounding tumor tissue, it may be important for the active camptothecin derivative of Formula 1 to exceed a specific drug concentration (high) in the tumor tissue, and depending on the situation, the maintenance time above a specific drug concentration (low) in the tumor tissue may be important, and rapid or slow elimination by lymphatic drainage may be important. This can be achieved by selecting an appropriate camptothecin payload of Formula 1 to provide the desired degree of hydrophobicity and / or controlled aggregation.

[0307] [Pro-drug]

[0308] In the present specification, prodrugs include but are not limited to prodrugs that are biologically activated or further activated after being placed in a predetermined physiological environment.

[0309] For example, it refers to a compound designed to chemically modify a physiologically active substance or a therapeutically active organic compound and to free or release the parent compound under enzymatic or other conditions in vivo. Prodrugs are converted into target compounds in vivo after administration. Although they are useful drugs, they have unsuitable characteristics in terms of side effects, stability, solubility, absorbability, duration of action, etc., and are chemically modified to enable them to be used clinically.

[0310] As an example of a prodrug, an antibody-drug conjugate (ADC), in which a cytotoxic drug is conjugated to an antibody, binds to an antigen expressed on the surface of cancer cells and can be internalized into the cells, selectively targeting the cancer cells by delivering the drug to them, where it can be expected that the drug will accumulate and kill the cancer cells.

[0311] Among the elements that make up ADC, the linker is what binds the antibody and the cytotoxic drug together.

[0312] The linker must be stable in the bloodstream, prevent the drug from separating from the antibody, keep it in a prodrug state until it reaches the target, and minimize damage to normal tissues. The most ideal linker is one that remains stable when the ADC circulates throughout the body, but cleaves in the target cell to properly release the cytotoxic drug and safely deliver the drug to the target, ensuring that the ADC is both effective and safe.

[0313] When a drug is linked to an antibody using a linker, the drug should not affect the structural stability, substrate binding properties and pharmacokinetics of the antibody. One of the known reasons for the failure of early ADC drugs is premature release of the drug.

[0314] A large number of ADCs currently in clinical trials use chemical linkers, such as hydrazone, disulfide, peptide or thioether. The process of drug release from the linker takes advantage of the difference in specific pH or enzyme concentration within cancer cells. Typically, hydrazone and disulfide linkers have limited stability in plasma. On the other hand, peptide-based linkers have excellent stability in plasma and are easy to control drug release.

[0315] Some chemical linkers adjust the hydrophobicity balance of antibodies and drugs to prevent ADC aggregation in the bloodstream (hydrophilic environment). Hydrophilic linkers and spacers, such as cyclodextrin, polyethylene glycol (PEG) or other polymers, play a role in bloodstream stability and pharmacokinetic properties.

[0316] The linkers used in ADCs are divided into non-cleavable and cleavable types according to their cleavage ability.

[0317] A representative example of a non-cleavable linker is a thioether linker, which has relatively high plasma stability and resistance to proteolysis compared to a cleavable linker. Unlike a cleavable linker, a non-cleavable linker itself does not decompose the linker, so the drug can only be released after the antibody is introduced into the cell in the form of an ADC and decomposed.

[0318] The drug released in this way is charged and is less likely to diffuse into surrounding cells (bystander effect). There is no bystander effect that is toxic to surrounding cells, but the effect only appears on the target cell after it is internalized by the target cell, which means it is relatively safe. It can be seen that ADCs prepared using non-cleavable linkers are more dependent on biological mechanisms within target cells. In many studies, ADCs with non-cleavable linkers have shown high stability and efficacy and are used as linkers for ADC development. Currently, ADCs using this technology are

[0319] Cleavable linkers cleave in response to specific environmental cues and release the drug into the cytoplasm. There are two types of cleavage: enzymatic and non-enzymatic.

[0320] The enzymatic cleavage type is cleavage by enzymes such as cathepsin B, β-glucuronidase, phosphatase, pyrophosphatase, and sulfatase.

[0321] Peptide linkers are degraded by proteolytic enzymes, and protease inhibitors are present in plasma, resulting in high plasma stability. Cathepsin B is a representative proteolytic enzyme used in ADCs. Cathepsin B is present at high levels in tumor tissues, giving ADCs tumor selectivity. Peptide linkers are mainly developed as dipeptides connected to two amino acids and are used in

[0322] Peptide linkers are composed of dipeptides or tetrapeptides, which are recognized and cleaved by proteolytic enzymes in lysosomes once the ADC is internalized. Tetrapeptides are used in the early stages of development and show limitations such as relatively slow drug release and possible aggregation when combined with hydrophobic drugs. The development of dipeptide linkers such as Val-Cit, Phe-Lys, Val-Lys, and Val-Ala solved this problem and have been successfully applied to And other ADCs.

[0323] The β-glucuronide linker is degraded by β-glucuronidase, a glycolytic enzyme present in lysosomes. β-glucuronidase is overexpressed in some tumor cells, conferring tumor specificity. The enzyme is highly active at low pH, but activity drops to 10% at neutral pH, so ADCs with β-glucuronide linkers have improved stability in plasma and prevent off-target drug release.

[0324] The non-enzymatically cleavable types include acid-labile linkers and oxidation-reduction reaction linkers.

[0325] Acid-labile linkers are chemically unstable linkers that were developed in the early stages of ADC development and have low stability, but are still in use today. A representative linker is the hydrazone linker, which is stable in the neutral environment of blood (pH 7.3-7.5), but is internalized around tumor cells (pH 6.5-7.2) or inside cells, such as endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) and has a mechanism of hydrolyzing and releasing drugs in a weakly acidic environment. However, acidic conditions are not limited to the tumor microenvironment and are often present outside cells, which may lead to nonspecific drug release.

[0326] A representative redox linker is a disulfide linker. A disulfide linker is also a chemically unstable linker based on redox reactions. After internalization, the linker is degraded by disulfide exchange or reducing agents such as glutathione, releasing the cytotoxic drug.

[0327] Glutathione is a low molecular weight thiol that regulates cell proliferation and death and is known as an antioxidant that protects cells from inflammation and oxidative stress. The concentration of intracellular glutathione is 0.5 to 10 mM, but in tumors under hypoxic conditions, its concentration is as high as 1000 times. Disulfide linkers exist in plasma at low concentrations (2-20 μM), have high plasma stability and reduce nonspecific drug release, making them relatively safe linkers that can specifically respond to tumors.

[0328] Depending on the linker used, it can affect the effective release of the cytotoxic drug.

[0329] The development of linkers reflects the long half-life, which is an advantage of monoclonal antibodies (mAbs). Therefore, mAbs must be stable during systemic circulation. It is important that the binding of linkers to cytotoxic drugs does not affect the stability and pharmacokinetics of the antibody.

[0330] In general, the catabolic reactions that may occur during systemic circulation of linker-cytotoxic drugs are as follows, with various other catabolic reactions that have not yet been identified: hydrazone cleavage, protease-mediated dipeptide cleavage, esterase-mediated carbamate cleavage, hydrolysis of acetateester, disulfide cleavage, and succinimide ringopening.

[0331] In some cases, the catabolism occurring at a specific location of the linker-cytotoxic drug can maintain the cytotoxic effect and be active at the target, and may cause toxicity in the systemic blood circulation. Conversely, if the cytotoxic effect is lost, even if the drug reaches the target, it may be difficult to expect the pharmacological effect. For example, thailanstatin, which was developed as a payload for spliceosome inhibitors, can maintain its activity even if the ester group is hydrolyzed, while cyptophycin or tubulysin, which are tubulin inhibitors, lose their activity when the ester group is hydrolyzed.

[0332] Regardless of the type of linker used, such as cleavable or non-cleavable linkers, the stability of the ADC while traveling to the target is very important to achieve the desired therapeutic index.

[0333] An example of the prodrug of the active camptothecin derivative (a) represented by Formula 1 designed in such a manner as to bind to the DDX5 protein and the E3 ligase according to the present invention may be the use of the active camptothecin derivative (a) represented by Formula 1 in DE-310 described later, instead of exitecan.

[0334] As described in WO97 / 46260 (corresponding Korean authorization 10-2087017B1), DE-310 is a complex of isotecan bound to a biodegradable carboxymethyl dextran polyol polymer via a GGFG peptide spacer. By converting isotecan into a polymer prodrug, high blood retention is maintained, and the directionality to the tumor site is passively increased by utilizing the enhanced permeability of tumor neovascularization and the retention of tumor tissue. In DE-310, the active subject isotecan and isotecan bound to glycine and amino groups are continuously released by enzymatic cleavage of the peptide spacer. As a result, pharmacokinetics are improved, and in various tumor evaluation models in non-clinical trials, although the dose of DE-310 is reduced compared to isotecan administered alone, it still shows higher efficacy than when isotecan is administered alone. Regarding DE-310, clinical trials were conducted in humans and its effectiveness was confirmed, and it was reported that the active substance of DE-310 was confirmed to accumulate in tumors rather than normal tissues, the accumulation of DE-310 and the active substance in human tumors was not significantly different from that in normal tissues, and no passive targeting was observed in humans.

[0335] As a related compound of DE-310, -NH(CH 2 ) 4 C(=O)-represented structural part, and -GGFG-NH(CH 2 ) 4 Complexes with C(═O)- as the spacer structure are also known.

[0336] According to the present invention, examples of prodrugs of the active camptothecin derivative (a) represented by Formula 1 designed in a manner of binding to DDX5 protein and E3 ligase may include linkers and / or spacers designed in a manner of releasing the active camptothecin derivative (a) at a target site in vivo.

[0337] Non-limiting examples of self-destructive spacers include Fig.32 shown.

[0338] According to the present invention, the active camptothecin derivatives designed in a manner to bind to the DDX5 protein and the E3 ligase and represented by the following formula 1, for example, camptothecin drugs 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 can be used as various carrier-drug conjugates (Carrier-Drug Conjugate) for payloads (Payload). That is, the various carrier-drug conjugates to which the active camptothecin derivatives are connected as payloads (Payload) are a prodrug of the active camptothecin derivatives, which release the active camptothecin derivatives in vivo. Therefore, the present invention provides a compound, which is an active camptothecin derivative represented by formula 1, preferably, a carrier-drug conjugate containing a 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.

[0339] According to the carrier-drug conjugate of the present invention, the carrier may be an antibody, a peptide, a repebody, and / or a nucleic acid aptamer, but is not limited thereto.

[0340]

[0341] Antibody-drug conjugate (ADC) is a prodrug of the present invention, which includes an immunoconjugate containing an antibody or a fragment thereof containing an antigen binding site, or a pharmaceutically acceptable salt thereof, designed to release the active camptothecin derivative (a) of the present invention in vivo.

[0342] The monoclonal antibodies used in ADC include IgG1, IgG2, and IgG4, of which IgG1 is the most commonly used. In traditional ADCs, full-length antigens are used. As a strategy to improve absorption and penetration, people are trying to use smaller Fabs, scFvs, and diabodies.

[0343] The immunoconjugate of the present invention specifically binds to an antigen of a cancer cell and exhibits cytotoxicity by releasing the drug inside and outside the cancer cell, and thus it can be effectively used for treating or preventing cancer.

[0344] For example, antibody-drug conjugates (ADCs) are new drug platforms that utilize the high tissue selectivity of antibodies to specifically bind to specific antigens expressed on the surface of cancer cells, selectively delivering payloads with strong anti-cancer efficacy only to cancer tissues. ADCs selectively deliver powerful payloads that can kill cancer cells even at concentrations as low as pM, and minimize systemic drug exposure, ensuring anti-cancer efficacy and safety.

[0345] In most cases, the intracellular transduction of ADC is carried out through the function of clathrin-coated pits. The ADC that enters the cell falls off the clathrin, fuses with other intracellular vesicles, and then enters the endosome-lysosome. Then, the proteases in the acidic environment of the endosomes cleave the linker, and the activated "free" drug passes through the lysosomal membrane, moves to the cytoplasm, binds to the molecular target of the drug, blocks the cell cycle of tumor cells and causes apoptosis, which leads to the death of cancer cells. Among them, a certain amount of drugs passively diffuse from the cell, actively transport, or leak out of the cell through dead cells. At this time, if the leaked drug has cell membrane permeability, it may enter the surrounding cells, causing the so-called bystander cell-killing.

[0346] Aptamer-Drug Conjugate (ApDC) is a nucleic acid aptamer introduced in ADC instead of an antibody. Aptamer is a single-stranded nucleic acid with a three-dimensional structure. It was discovered through the 'SELEX' (Systematic Evolution of Ligands by Exponential enrichment) process. Selex is a technology for obtaining functional nucleic acids that bind to target protein molecules in a compound library.

[0347] Aptamers can bind to targets very strongly and selectively, so they are also called chemical antibodies. Aptamers are about 20 kDa in size and are known to have excellent cell penetration and lower immunogenicity compared to antibodies.

[0348] Since aptamers can be chemically synthesized, the conjugation position and the amount of conjugated drugs can be precisely designed when manufacturing aptamer-drug conjugates. The production cost is lower than that of ADC.

[0349] Aptamers are generally made of natural nucleic acids and are therefore degraded by nucleic acid-degrading enzymes in the body, making them less stable in the body. However, by taking advantage of the convenience of chemical modification of aptamers, the stability limitations of modified aptamers can be overcome.

[0350] Peptide-Drug Conjugate (PDC) is a form of ADC in which peptides are introduced instead of antibodies. Peptides are composed of amino acids and range in size from 500 to 5000Da (Daltons). Compared with antibodies above 150kDa (kilodaltons), this size is very small. Therefore, peptide-based PDC has more excellent cell penetration ability than ADC, and the possibility of producing immunogenicity is very low. In addition, peptides can be chemically synthesized. Therefore, PDC not only has very low production costs, but also can accurately control the conjugation position and ratio of peptides to drugs.

[0351] In general, peptides are easily degraded by proteolytic enzymes and thus have a short biological half-life. To overcome these limitations of peptide-based drug conjugates, strategies using modified peptides such as cyclic peptides, unnatural amino acids, etc. have been proposed.

[0352] Repebodies are artificial antibodies that do not have an antibody skeleton but have the ability to recognize antigens like antibodies. Repebodies specific to target proteins can be discovered through phage display technology.

[0353] Phage display is a technology for expressing desired proteins on the surface of phages. The size of repetitive bodies is about 30kDa, which is about 20% of antibodies. Therefore, it is known that compared with antibodies, it has relatively low immunogenicity and improved cell permeability. In addition, it is expected that the structural stability can be improved by controlling the thermal stability and pH stability of repetitive bodies. Compared with antibodies, the production cost is also evaluated as relatively low. Due to these advantages of repetitive bodies, people are increasing their attention to the development of repetitive body-drug conjugates (Repebody-DC) as a strategy for replacing antibodies with repetitive bodies.

[0354] According to the present invention, the carrier-drug conjugate can be a compound in which the carrier is connected to the drug through a linker, that is, the compound is an active camptothecin derivative represented by Formula 1, preferably, a 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 is not limited thereto.

[0355] In the carrier-drug conjugate according to the present invention, the compound is the active camptothecin derivative represented by Formula 1, preferably, 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 can be connected to the linker at an appropriate site, as long as their anticancer activity and other properties are not changed. Therefore, the present invention provides a compound, which is an active camptothecin derivative represented by Formula 1, preferably a drug-linker connected to a linker by a 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.

[0356] Preferably, the drug-linker according to the present invention may have a structure of Formula 3a, Formula 3a-1, Formula 4, Formula 4a-1, Formula 5a, Formula 5a-1, Formula 6a, Formula 7a, Formula 8a or Formula 9a, but is not limited thereto.

[0357]

[0358]

[0359]

[0360] In the structural formula, L represents a linker.

[0361] [Target antigen and antibody]

[0362] In ADCs, the interaction between the antibody and the target antigen is very important to ensure safety and obtain therapeutic efficacy. Two variables in antigen selection are tumor specificity and expression level. Ideally, the antigen is specifically expressed only in tumors and not expressed or expressed minimally in normal cells. Specificity is critical to reduce toxicity and determines the success of an ADC. Cancer-specific antigens are expressed as surface receptors on the surface of tumor cells or within the tumor vasculature and tumor microenvironment.

[0363] As with homogeneous tumors, cancer treatment becomes easier when cancer-specific antigens are uniformly expressed in tumor tissues and all cancer cells respond to the drug. In the case of heterogeneous tumors, non-responsive cancer cells are also mixed together, so there may be cancer cells that survive even after ADC treatment. If ADC has the effect of killing surrounding cells, the problem of tumor heterogeneity can be overcome.

[0364] In solid tumors rich in intracellular matrix, it is challenging to target antigens internalized by tumor cells. In this case, the approach targets the tumor microenvironment rather than cancer cells. Cytotoxic drugs are released to the extracellular space by extracellular proteins, acidic substances in the extracellular matrix, or components such as glutathione.

[0365] Among the ADCs currently undergoing clinical trials, a target antigen that is attractive due to its excellent marketability is called HER2, and three ADCs targeting HER2 are in phase 3 clinical trials (Dean et al., 2021).

[0366] In the case of HER2-positive cancer, the active camptothecin derivative (a) or its prodrug ADC targeting DDX5 protein according to the present invention may be a new therapeutic approach to address anti-HER2 therapy resistance.

[0367] In order to deliver potent cytotoxic drugs only to specific cancer cells, determining the target antigen is the first major step in ADC development. By using antibodies, the high specificity and long half-life of the target can make long-term systemic circulation, so that cytotoxic drugs can be selectively accumulated only to tumor cells and the exposure of normal tissues can be minimized, thereby reducing damage, reducing side effects, and increasing the therapeutic effect. To this end, it is necessary to find a target antigen that can specific tumor cells, which requires the following conditions. First, the target antigen needs to be evenly overexpressed on the surface of tumor cells, and the expression in normal cells should be relatively low or not expressed. A representative example has human epidermal growth factor receptor 2 (HER2), which is known to be expressed more than 100 times as much as normal cells in HER2-positive breast cancer. Therefore, before making antibodies, the tumor expression of the target antigen is analyzed by various analyses, and if the overexpression of a specific antigen is confirmed, a monoclonal antibody that recognizes the antigen is generated. Second, regarding the binding force to the antigen, due to the characteristics of antibodies that cause internalization through receptors, the stronger the binding force to the epitope of the antigen, the more internalization is likely to occur, thereby increasing the therapeutic effect. In addition, it has low immunogenicity. Initially, the first generation of ADCs were produced using antibodies produced in mice. The first generation of ADCs injected mouse antibodies into the human body, but due to side effects and antibody neutralization caused by the human body's immune response to the injected mouse antibodies, it was difficult to see the anti-cancer effect. With the development of genetic engineering technology and the production of chimeric antibodies, humanized antibodies, and fully human antibodies, the immune response problem has been significantly improved.

[0368] Antibodies that recognize cancer cell antigens need to be internalized into cells together with drugs. In order to improve the internalization of cancer cells, bispecific antibodies are being developed.

[0369] MEDI4267 (trastuzumab-META), developed by Medimmune and Astrazeneca, is a biparatopic antibody targeting two non-overlapping epitopes in HER2 that induces HER2 receptor aggregation, thereby inducing cellular internalization, lysosomal trafficking, and degradation.

[0370] Compared with single antibodies, bispecific antibodies that bind to lysosomal markers CD63 or APLP2 and prolactin receptors and tumor-targeting antibodies show improved tumor antigen recognition and cellular internalization. There is a bispecific ADC of trastuzumab and CD63 (HER2xCD63-duostatin-3, Creativebiolabs), targeting HER2, which shows a strong anti-cancer effect by reducing the delivery to normal tissues and delivering it specifically to cancer cells.

[0371] When designing the antibody-drug conjugate (ADC) or immunoconjugate or carrier-drug conjugate of the present invention, the targets may include not only cancer cells but also be expanded to cells associated with infectious organisms and / or autoimmune diseases.

[0372] Thus, the cells targeted by the peptides or fragments thereof containing the antigen binding site may be cancer cells, infectious organisms and / or cells associated with autoimmune diseases.

[0373] Non-limiting examples of target antigens include antigens selectively distributed on the surface of cancer, such as Her2, FolR, PSMA, etc., and antigens overexpressed in cancer cells, such as Trop2, etc., which are distributed in small amounts in normal tissues.

[0374] Cancer cell target antigens can be, for example, 5T4, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALCAM, ALK, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, aromatase, ASPH, ATX, AX1, AXL, AZGP1 (zinc-a-glycoprotein), otein)), B4GALNT1, B7, B7.1, B7.2, B7-H1, B7-H3, B7-H4, B7-H6, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BCMA, BDNF, BLNK, BLR1(MDR15), BIyS, BMP1, B MP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMP10, BMPR1A, BMPR1B, BMPR2, BPAG1 (reticulin), BRCA1, C19orflO (IL27w), C3, C4A, C5, C5R1, CA6, CA9, CANT1, CAPRI N-1, CASP1, CASP4, CAV1, CCBP2 (D6 / JAB61), CCL1 (1-309), CCLI1 (chemokine), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22 (MDC / STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2 / chemokine-2), C CL25 (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIPIb), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM145), CCR2 (mcp-IRB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6),CCR7(CKR7 / EBI1), CCR8 or CDw198(CMKBR8 / TERI / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), CD13, CD164, CD19, CDH6, CDIC, CD2, CD20, CD21, CD200, CD22, CD23, CD24, CD27, CD28, CD29, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD56, C D69, CD70, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD97, CD99, CD117, CD125, CD137, CD147, CD179b, CD223, CD279, CD152, CD274, CDH 1 (E-cadherin), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH3, CDH5, CDH7, CDH8, CDH9, CDH17, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wa p1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEA, CEACAM5, CEACAM6, CEBPB, CERI, CFC1B, CHGA, CHGB, chitinase, CHST1O, CIK, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN6, CLDN7 (Claudin-7), CLDN18, CLEC5A, CLEC6A, CL EC11A, CLEC14A, CLN3, CLU (clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, C-MET, COL18A1, COLIA1, COL4A3, COL6A1, CR2, Cripto, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTAG1B (NY-ESO-1), CTLA4, CTL8, CTNNB1 (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO),CXCLI1(1-TAC / IP-9), CXCL12(SDF1), CXCL13, CXCL14, CXCL16, CXCL2(GRO 2) CXCL3(GRO3), CXCL5(ENA-78 / LIX), CXCL6(GCP-2), CXCL9(MIG), CXCR3 (GPR9 / CKR-L2), CXCR4, CXCR6(TYMSTR / STRL33 / Bonzo), CYB5, CYC1, CYSLT R1, DAB2IP, DES, DKFZp451J0118, DLK1, DNCL1, DPP4, E2F1, Angel, Edge, Fen nel、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG、Enola、ENO2、ENO3、EpCAM、EPHA1 EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, E PHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRINA3, EPHR IN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, E PG, ERBB2(HER-2), ERBB3, ERBB4, EREG, ERK8, Earl, ESR2, F3(TF), F ADD, FAP, smooth muscle cells, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGF1(aFGF), F GF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FG F2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4(HST), FGF5, and FGF 6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR1, FGFR2, FGFR3, FGFR4, FIGF(VEGF). D) FIL1 (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (protein transcription factor), FLT1. FLT-3、FOLR1、FOS、FOSL1(FRA-1)、FR-α、FY(DARC) GABRP(MARKET) GAGEB1 GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GFRA1, GGT1, GM-CSF.GNAS1, GNRH1, GPC1, GPC3, GPNB, GPR2 (CCR10), GPR31, GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, GUCY2C, HAVCR1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HER3, HGF, HIF1A, HIP1, histamine and histamine receptors, HLA-A, HLA-DR, HLA-DRA, HLA-E, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNB1, IFNγ, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1(CD121a), IL1R2(CD121b), IL-IRA, IL-2, IL2RA(CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA, (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCR1(IL8RA), CXCR2, (IL8RB / CD128), IL-9, IL9R(C D129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, IL11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, IL16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHY I, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27,IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, 1L4, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KIT, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (type II hair-specific keratin), L1CAM, LAG3, LAMA5, LAMP1, LEP (leptin), Lewis Y antigen ("LeY"), LILRB1, Lingo-p75, Lingo-Troy, LGALS3BP, LRRC15, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​LY75, LYPD3, MACMARCKS, MAG or OMgp, MAGEA3, MAGEA6, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MLSN, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-UI), mTOR, MTSS1, MUC1 (mucin), MUC16, MYC, MYD88, NCK2, NCR3LG1, neuronal protein Glycan (neurocan), NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOTCH3, NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, NR1H 2. NR1H3, NR1H4, NR112, NR113, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3 C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO1, ODZI, OPRDI,P2RX7, PAP, PART1, PATE, PAWR, P-cadherin, PCA3, PCD1, PD-L1, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, L1-CAM, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 kinase, PIK3CG, PLAU (uPA), PLG, PLXDCI, PKC, PKC-β, PPBP (CXCL 7), PPID, PR1, PRAME, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSMA, PTAFR, PTEN, PTHR2, PTGS2 (COX-2), PTN, PVRIG, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, ROR1, RXR, S100A2, SCGB1D2 (lipophilin B), SCG B2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial monocyte activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (mammaspin), SERPINEI (PAI-I), SERPINFI, SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLAMF7, SLC2A2, SLC33A1, SLC43A1, SLC44A4, SLC34A2, SLIT2, SPP1, SPRR1B (Spr1), ST6GAL1, ST8SIA1, STAB1, STATE, STEAP, STEAP2, TB4R2, TBX21, TCP1O, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor (tissue factor factor), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2(B9 4), TNFAIP3, TNFRSFI1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6(Fas), TNFRSF7, TNFRSF8,TNFRSF9, TNFSF1O(TRAIL), TNFRSF10A, TNFRSF10B, TNFRSF12A, TNFRSF17, TNFSF11(TRANCE), TNFSF12(APO3L), TNFSF13(April), TNFS F13B, TNFSF14(HVEM-L), TNFRSF14(HVEM), TNFSF15(VEGI), TNFSF18, TNFSF4(OX40 ligand), TNFSF5(CD40 ligand), TNFSF6(FasL), TNFSF7(CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TROP2, TRPC6, TSLP, TWEAK, tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, WT1, Wnt-1, XCL1 (lymphatic chemoattractant), XCL2 (SCM-Ib), XCRI (GPR5 / CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303, CDH6, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (dendritic cell-associated lectin 2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (dendritic cell-associated lectin 1), CLEC11A, PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), LILRB1, NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46) , NCR3 (CD337, NKp30), OSCAR, TARM1, CD30, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 (CD314,<h2 style=";text-align:left;direction:ltr">NKG2D)、NCR2(CD336、NKp44)、PILRB、SIGLEC1(CD169、SN)、SIGLEC5、SIGLEC6、SIGLEC7、SIGLEC8、SIGLEC9、SIGLEC10、SIGLEC11、SIGLEC12、SIGLEC14 、SIGLEC15(CD33L3)、SIGLEC16、SIRPA、SIRPB1(CD172B)、TREM1(CD354)、T REM2、KLRF1(NKp80)、17-1A、SLAM7、MSLN、CTAG1B / NY-ESO-1、MAGEA3 / A6、A TP5I(Q06185)、OAT(P29758)、AIFM1(Q9Z0X1)、AOFA(Q64133)、MTDC(P1815) 5)、CMC1(Q8BH59)、PREP(Q8K411)、YMEL1(O88967)、LPPRC(Q6PB66)、LONM( Q8CGK3)、ACON(Q99KI0)、ODO1(Q60597)、IDHP(P54071)、ALDH2(P47738)、ATPB(P56480)、AATM(P05202)、TMM93(Q9CQW0)、ERGI3(Q9CQE7)、RTN4(Q99P 72)、CL041(Q8BQR4)、ERLN2(Q8BFZ9)、TERA(Q01853)、DAD1(P61804)、CALX (P35564)、CALU(O35887)、VAPA(Q9WV55)、MOGS(Q80UM7)、GANAB(Q8BHN3)、 ERO1A(Q8R180)、UGGG1(Q6P5E4)、P4HA1(Q60715)、HYEP(Q9D379)、CALR(P1) 4211)、AT2A2(O55143)、PDIA4(P08003)、PDIA1(P09103)、PDIA3(P27773)、 PDIA6(Q922R8)、CLH(Q68FD5)、PPIB(P24369)、TCPG(P80318)、MOT4(P5778) 7)、NICA(P57716)、BASI(P18572)、VAPA(Q9WV55)、ENV2(P11370)、VAT1(Q6) 2465)、4F2(P10852)、ENOA(P17182)、ILK(O55222)、GPNMB(Q99P91)、ENV1( P10404)、ERO1A(Q8R180)、CLH(Q68FD5)、DSG1A(Q61495)、AT1A1(Q8VDN2)、HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP75 (P38647), ENPL (P08113), CH60 (P63038), or CH10 (Q64433), but not limited thereto.

[0375] The target antigen may be an antigen that is distributed 10 times more in cancer cells than in normal cells.

[0376] Non-limiting examples of antibodies of the present invention that can be used in the antibody-drug conjugates (ADCs) according to the present invention include Urelumab, Utomilumab, Bebtelovimab, Aducanumab, Bapinezumab, Crenezumab, Donanemab, Gantenerumab, Lecanemab, Solanezumab, Nesvacumab, Evizumab, and vinacumab), Enoblituzumab, Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Tilizumab Teplizumab, Ibalizumab, Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab, Veltuzumab uzumab), Epratuzumab, Basiliximab, Daclizumab, Varlilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab, Felzartamab, Isatuximab, Mezagitamab, Bleselumab, Dacetuzumab,Iscalimab, Lucatumumab, Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab, Cusatuzumab, Oleclumab , Milatuzumab, Galiximab, Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab, Eculizumab, Pozelimab, Ravulizumab ), Lacnotuzumab, Axatilimab, Cabiralizumab, Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab, Zaliferimab, Cetuximab, Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Zatuximab (Modotuximab), Necitumumab, Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, Batoclimab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab,Ragifilimab, Gimsilumab, Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Ser Seribantumab, Duligotuzumab, Ficlatuzumab, Rilotumumab, Alomfilimab, Anifrolumab, Emapalumab, Ligelizumab, Omalizumab, Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab, Canakinumab, Gevokizumab, Briakinumab, Ustekinumab, Anrukinzumab, Cendakimab, Lebrikizumab, Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab, Sku Secukinumab, Brazikumab, Guselkumab, Mirikizumab, Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab,Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levilimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Favezelimab, Fulanizumab Fianlimab, Ieramilimab, Relatlimab, Simtuzumab, Abagovomab, Oregovomab, Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonlerolizumab, Alirocumab, Bercovacizumab Bococizumab, Ebronucimab, Evolocumab, Frovocimab, Ongericimab, Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiplimab, Geptanolimab, Nivolumab, Pembrolizumab, Penplimab Penpulimab、Pidilizumab、Prolgolimab、Retifanlimab、Sasanlimab、Serplulimab、Sintilimab、Spartalizumab、Tislelizumab、Toripalimab、Ezabenlimab、Zimberelimab、Atezolizumab、Avelumab, Cosibelimab, Sugemalimab, Durvalumab, Envafolimab, Suvratoxumab, Denosumab, Zilovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociperlimab, Tiragolumab, Vibrio Vibostolimab, Surzebiclimab, Cobolimab, Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Tigatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab, Brolucizumab, Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab, Etrolizumab, Vedolizumab, Intetumumab, Natalizumab )、Obrindatamab、Elranatamab、Linvoseltamab、Teclistamab、Epcoritamab、Glofitamab、Mosunetuzumab、Odronextamab、Flotetuzumab、Vibecotamab、Catumaxomab、Cibisatamab、Talquetamab、Ubamatamab, Emfizatamab, Blinatumomab, Amivantamab, Emicizumab, Zenocutuzumab, Zanidatamab, Tibulizumab, Naptumomab, Belantamab, Pivekimab, Pratumomab luzatamab), coltuximab, denintuzumab, loncastuximab, ibritumomab, inotuzumab, epratuzumab, moxetumomab, brentuximab, gemtuzumab, vadastuximab, lovotuzumab orvotuzumab), Polatuzumab, Tusamitamab, Telisotuzumab, Rovalpituzumab, Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Goxatuzumab Sacituzumab, Vobarilizumab, Cadonilimab, Vudalimab, Tebotelimab, Ivonescimab, Erfonrilimab, Ozoralizumab, Faricimab, Vanucizumab, Navicixizumab, etc.

[0377] [Pharmaceutical composition for preventing or treating cancer]

[0378] The present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned compound represented by Formula 1, such as 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 pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof, preferably a carrier-drug conjugate (CDC), and more preferably an antibody-drug conjugate (ADC) as an active ingredient.

[0379] The active camptothecin derivative (a) or its prodrug (b) represented by Formula 1 according to the present invention, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC) can be contained in a pharmaceutical composition as an effective ingredient, and the pharmaceutical composition is administered to a patient group in which DDX5 acts as an oncoprotein in cells.

[0380] For example, in a tissue biopsy or liquid biopsy, the type 1 topoisomerase inhibitory ability of the FL118 compound of Formula 2 or the active camptothecin derivative (a) is preferably inactivated by non-cleavage linker connection, and a complex (c) containing a DDX5 protein-targeting ligand is used to quantitatively or qualitatively analyze the intracellular DDX5 protein, thereby confirming the patient group to which the active camptothecin derivative (a) or its prodrug (b) is administered.

[0381] According to the active camptothecin derivative (a) represented by Formula 1 of the present invention, for example, compounds 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 are camptothecin derivatives of new structures. Therefore, thus, a candidate drug having more preferred characteristics than Dxd drugs in terms of efficacy, toxicity, selectivity, duration of action, administration, operation, stability and / or production potential of Dxd drugs can be provided. In addition, the interaction between the target and the candidate drug, i.e., the pharmacodynamic properties, can also be optimized, and the proximity of the drug to the target can be optimized, i.e., the pharmacokinetic ability can be improved.

[0382] In addition, one embodiment of the present invention provides a method for treating or preventing cancer, the method comprising the steps of administering a compound represented by Formula 1, such as 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 pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof, preferably a carrier-drug conjugate (CDC), more preferably an antibody-drug conjugate (ADC) in a therapeutically effective amount to a subject in need thereof. The subject may be a mammal including humans.

[0383] In the present invention, the cancer refers to all cancers that can be treated by inhibition of topoisomerase I and / or inhibition of one or more cancer-associated survival genes selected from the group consisting of DDX5, survivin, Mcl-1, XIAP and cIAP2, and may be solid tumors or blood cancers. For example, selected from pseudomyxoma, intrahepatic bile duct 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, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, brain cancer pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampullary cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, Renal 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 cancer, 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 cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer and thymic cancer are composed of one or more of the group, but not limited to this. In addition, the cancer includes not only primary cancer, but also metastatic cancer.

[0384] In this specification, "patient", "subject" and "subject body" refer to animals, such as mammals. In certain embodiments, the patient is a human. In other embodiments, the patient is a non-human animal, such as a dog, a cat, a livestock (e.g., a horse, a pig or a donkey), a chimpanzee or a monkey.

[0385] The term "therapeutically effective amount" used in the present invention means the amount of the active camptothecin derivative (a) represented by Formula 1 that is effective for the treatment or prevention of cancer, such as the amount of a compound represented by Formula 3, Formula 3-1, Formula 4, Formula 4-1, Formula 5 or Formula 5-1, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof. Specifically, "therapeutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio that can be applied to medical treatment, and the effective dose level can be determined according to the type and severity of the object, age, sex, type of disease, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, factors including drugs used simultaneously, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic drug or in combination with other therapeutic drugs, or can be administered sequentially or simultaneously with commercially available therapeutic drugs. In addition, it can be administered once or multiple times. Taking all the above factors into consideration, it is important to administer an amount that can achieve the maximum effect at the minimum amount without side effects. The compounds 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 of the present invention and their pharmaceutically acceptable salts show dose-dependent effects, so the dosage can be simply determined by a person of ordinary skill in the art to which the present invention belongs based on various factors such as the patient's condition, age, gender, and complications. The active ingredients of the pharmaceutical composition of the present invention have excellent safety, and can therefore also be used at a dosage above the determined dosage.

[0386] In addition, according to a specific example of the present invention, the present invention provides the use of the immunoconjugate in the preparation of a drug (medicament) for treating or preventing cancer, for example, a 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, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof, preferably a carrier-drug conjugate (CDC), and more preferably an antibody-drug conjugate (ADC). 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, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof can be mixed with an acceptable adjuvant, diluent, carrier, etc., and can be prepared into a composite preparation together with other active preparations to have a synergistic effect of the active ingredients.

[0387] Matters mentioned in the uses, compositions and methods of treatment of the present invention are equally applicable unless they conflict with each other.

[0388] In the present specification, the anticancer effect or therapeutic effect of an anticancer agent refers to an effect of reducing the severity of cancer, decreasing the size of a tumor, or delaying or slowing down the progression of cancer when suffering from a specific disease.

[0389] For example, the anticancer effect caused by an anticancer drug can be the cell viability (cell viability) (change in the degree of cytotoxicity or the number of cells) of cancer cells after treating cancer cells with anticancer drugs in vitro (in-vitro) and / or in vivo (in-vivo). For example, it can be indirectly confirmed by testing drug response (drug response) using a cell line (Cell line) non-clinical animal model (xenograft). In addition, cancer patients can also directly confirm the anticancer effect of anticancer drugs, derive relevant data, and use it as a database. In addition, when designing anticancer drug dosage guidelines, animal model PK parameters and / or toxicity characteristics (profile) can be considered in parallel.

[0390] The anticancer effect of an anticancer drug can be inferred from in vitro data, such as the maximum effect percentage of the anticancer drug, such as IC 50 ,IC 60 ,IC 70 ,IC 80 and IC 90 and the maximum blood concentration (Cmax) of the drug and / or in vivo data in nonclinical animal models and clinical cancer patients, such as the area under the blood concentration-time curve (AUC).

[0391] The responsiveness of anticancer drugs refers to the clinical sensitivity of the anticancer effect.

[0392] When referring to treatment with anticancer drugs, "sensitivity" and "responsiveness" are relative terms referring to the degree of effectiveness of the compound in alleviating or reducing the progression of the tumor or disease being treated.

[0393] By "effective patient anti-cancer effect / response" is meant an inhibition in the patient's response of, for example, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, as measured by any suitable means, such as gene expression, cell counts, analytical results, etc.

[0394] In this specification, it is the dose expected to have a drug effect. In the present invention, the drug effect can be an anticancer effect. The responsiveness (anticancer effect) of an anticancer drug is the degree of response, which can be a percentage of the maximum effect (Maximum effect) of the anticancer drug, such as IC 50 、IC60 、IC 70 、IC 80 IC 90 , and the toxicity value to normal cells (LC 50 ).

[0395] For example, oral dosage forms can be prepared using a variety of formulation techniques known in the art. For example, it can include a biodegradable (hydrolyzable) polymer carrier for adhering to the oral mucosa. It is manufactured to slowly erode over a predetermined time, providing drug delivery essentially as a whole.

[0396] Drug delivery in oral dosage forms avoids the weaknesses encountered in oral drug administration, such as slow absorption, degradation of activators by liquids present in the gastrointestinal tract and / or first-pass inactivation in the liver. Regarding biodegradable (hydrolyzable) polymer carriers, any such carrier can be used in practice, 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. In general, polymer carriers include hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the oral mucosa. Examples of polymer carriers that can be used herein include acrylic acid polymers (e.g., carbomer). In some instances, non-limiting examples of other ingredients that can be incorporated into oral dosage forms include disintegrants, diluents, adhesives, lubricants, flavoring agents, coloring agents, preservatives, etc. In some instances, it can be in the form of tablets, lozenges, or gels, formulated in a conventional manner for oral or sublingual administration.

[0397] In some instances, if the patient's condition improves, the compound is continued to be administered at the discretion of the physician; alternatively, the dose of the drug to be administered may be temporarily reduced or temporarily suspended for any length of time (i.e., a "drug withdrawal"). The length of a drug withdrawal may vary between 2 days and 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 instances, the dose reduction during the discontinuation period is 10%-100%, and 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% are included.

[0398] Once the patient's condition improves, a maintenance dose will be given if necessary. Thereafter, the dose or frequency of administration, or both, may be reduced to a level that maintains the improved disease, disorder or condition as a function of symptoms. However, if symptoms recur, the patient may require intermittent treatment for a longer period of time.

[0399] The amount of a given pharmaceutical agent corresponding to this amount will vary according to factors such as the specific compound, the severity of the disease and the identity of the subject in need of treatment (e.g., body weight), but will still vary and can be routinely determined in a manner known in the art, depending on, for example, the formulation to be administered, the route of administration, and the specific environment surrounding the subject's body to be treated. However, in general, dosages for treating adults are generally in the range of 0.02-5000 mg / day, or about 1-1500 mg / day.

[0400] In the present specification, 1 administration dose may be administered as a single dose or divided doses administered simultaneously, for example, 2, 3, 4 or more divided doses.

[0401] In some instances, the oral dosage form is a unit dosage form suitable for single administration of precise dosages. In a unit dosage form, the formulation is divided into unit doses containing an appropriate amount of one or more compounds. In some instances, the unit dose is in the form of a packet containing discrete amounts of the dosage form. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose, non-reclosable containers. Alternatively, a multi-dose reclosable container can be used, in which case a preservative is typically included in the composition.

[0402] Formulations for parenteral injection are presented in unit dosage form, including, but not limited to, ampoules, or in multi-dose containers, with an added preservative.

[0403] It is usually prepared for parenteral administration, i.e., in a unit dose injection form for bolus, intravenous and intratumoral injection together with a pharmaceutically acceptable parenteral carrier. It is optionally mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.) in the form of a lyophilized preparation or an aqueous solution.

[0404] [Ligands targeting DDX5 protein]

[0405] In addition to FL118, the active camptothecin derivative represented by Formula 1 according to the present invention is a molecular glue degrading agent that activates the degradation of the cancer protein DDX5, and can therefore be used as a ligand targeting the DDX5 protein or a ligand that binds to the DDX5 protein. Therefore, a complex containing a ligand targeting the DDX5 protein can be provided, wherein the FL118 compound or the active camptothecin derivative (a) is inactivated by connecting the type 1 topoisomerase inhibitory ability to a linker.

[0406] The linker may be a non-cleavable linker, and a non-limiting example of a non-cleavable linker is a thioether linker.

[0407] The FL118 compound or the active camptothecin derivative (a) represented by Formula 1 binds to the DDX5 protein, and the DDX5 protein is a direct target of the active camptothecin derivative (a).

[0408] Therefore, DDX5 can be used as a biomarker for predicting drug sensitivity or tumor sensitivity.

[0409] In tissue biopsy or liquid biopsy, the intracellular DDX5 protein is quantitatively or qualitatively analyzed using the FL118 compound of Formula 2 or the complex (c) containing a ligand targeting the DDX5 protein in which the type 1 topoisomerase inhibitory ability is inactivated by a linker connection in the active camptothecin derivative (a) represented by Formula 1, thereby determining the patient group to be administered with the active camptothecin derivative (a) or its prodrug (b), or predicting the recurrence rate and / or prognosis during chemotherapy anticancer drug treatment.

[0410] [Cancer diagnostic composition]

[0411] The present invention provides a cancer diagnostic composition comprising a complex (c) containing a ligand targeting DDX5 protein, wherein the FL118 compound of Formula 2 or the active camptothecin derivative (a) represented by Formula 1 is inactivated in its type 1 topoisomerase inhibitory ability by connecting with a linker.

[0412] According to the present invention, when the cancer diagnostic composition is used, it can be used to determine the patient group as the administration subject of the active camptothecin derivative (a) or its prodrug (b), or to predict the drug response, recurrence rate and / or prognosis during chemotherapy anticancer drug treatment.

[0413] Effects of the Invention

[0414] According to the present invention, the camptothecin derivative represented by Formula 1, for example, the camptothecin derivative represented by Formula 3 to Formula 9 or its isomers is a compound having a new structure. Thus, it is possible to provide a more preferred candidate drug in terms of the efficacy, toxicity, selectivity, duration of action, administration, operation, stability and / or production potential of the Dxd drug. In addition, the interaction between the target and the candidate drug, i.e., the pharmacodynamic properties, can be optimized, and the proximity of the drug to the target can be optimized, i.e., the pharmacokinetic ability can be improved.

[0415] The present invention provides a compound of Formula 1, such as a compound of Formula 3 to Formula 9 or an isomer thereof, which maintains the structure of the FL118 drug and maintains the advantages of the FL118 drug, which is that the efficacy of the main drug target modulation is maximized by (i) simultaneously inhibiting Bcl family members (Bcl family), such as survivin as a resistance protein, and / or (ii) by an additional mechanism of action of inhibiting the action of an efflux pump.

[0416] Therefore, the compounds of Formula 1, such as the compounds of Formulas 3 to 9 or their isomers, are designed based on a well-designed polypharmacology approach to obtain excellent therapeutic efficacy by simultaneously targeting multiple drug targets that need to be controlled together, as in the case of the FL118 drug, and are different from the existing drug development methods that have limited therapeutic efficacy of targeting one drug target with one drug. They can treat difficult and complicated diseases for which existing therapeutic drugs do not show obvious therapeutic effects. In addition, in recurrent / resistant cancers, sufficient efficacy cannot be obtained by regulating the activity of only one drug target, and at the same time, when targeting multiple unspecified drug targets, there is a problem that safety cannot be guaranteed. However, by using two or more carefully selected drug targets, as in the case of the FL118 drug, synergistic effects can be shown in terms of efficacy, while sufficient safety can be guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0417] Figure 1 The synthetic design concept of a novel active camptothecin derivative (a) having dual MoA of inhibiting type 1 topoisomerase and degrading oncoprotein DDX5 by improving the structure of FL118 is shown.

[0418] Figure 2 The structural formula of camptothecin (CPT) and its binding to type 1 topoisomerase (topoisomerase-1) are shown, the molecular design concepts of FL118 and PBX-7011 are expanded from camptothecin, and the expanded new structural compounds PBX-7014 and PBX-7016 and the calculated structures of their hydrophilicity are derived from PBX-7011.

[0419] Figure 3 To be similar to the procedure for synthesizing Deruxtecan from Dxd, the syntheses of 25-4 and 25-6 are exemplified from PBX-7014 and PBX-7016.

[0420] Figure 4 It is a molecular dynamics simulation and docking score (Docking Score) that analyzes the interaction between existing camptothecin drugs and TOP1.

[0421] Figure 5 The molecular dynamics simulation and docking score (Docking Score) are used to analyze the interaction between the novel camptothecin drug of the present invention and TOP1.

[0422] Figure 6 This is a molecular dynamics simulation analyzing the interaction between camptothecin and mutant TOP1 (E418K).

[0423] Figure 7 The SN38-TOP1 interaction in Normal TOP1 is shown.

[0424] Figure 8 Diagram showing SN38-TOP1 interaction in E418K mutated TOP1.

[0425] Fig. 9 The interaction between the compound of formula 1-1 (Compound A) and TOP1 in E418K mutated TOP1 is shown.

[0426] Figures 10 to 14 The purpose is to verify the protein blotting results of the inhibition degree of anti-apoptotic proteins of various camptothecin drugs (FL118 drug, SN-38 drug, exitecan drug, PBX-7011, PBX-7014, PBX-7016) in Dual MoA (Topoisomerase I inhibition & DDX5 degradation), FaDu cell line and A549 cell line according to Example 2-1. Fig.12 and Fig.14 The results of western blot experiments performed in FaDu cell lines and A549 cell lines are shown in Figure 2. Fig.11 and Fig.13 ) is the result of quantifying the concentration using a graph.

[0427] Fig.15The results are the results of the in vitro cell viability comparison of various camptothecin drugs in FaDu cell line or A549 cell line.

[0428] Fig.16 The results are a comparison of the in vitro cell viability of various camptothecin drugs in MDA-MB-453 (HER2++) and FaDu (HER2+) cell lines.

[0429] Fig.17 It is the result of carboxylate to lactone form conversion in pH 7.4.

[0430] Fig.18 It is the result of carboxylate to lactone form conversion in pH 6.0.

[0431] Fig.19 20 is the ADC production confirmation result using SEC and SDS PAGE.

[0432] Fig.21 The results are a comparison of the binding ability of trastuzumab and ADCs conjugated with trastuzumab and PBX-7014 and PBX-7016 against Her2.

[0433] Fig. 22 The results are a comparative study of the in vitro cell viability of various camptothecins and ADCs using them as payloads in MDA-MB-453 (HER2++), FaDu (HER2+) and MDA-MB-468 (HER2-) cell lines.

[0434] Fig.23 The results are of efficacy tests on ADCs using various camptothecin drugs as payloads in an in vivo xenograft model in mice using the JIMT-1 cell line.

[0435] Fig.24 The results are as follows: body weight changes of mice using JIMT-1 cell line were evaluated by in vivo xenograft model using various camptothecin drugs as payloads for ADC.

[0436] Fig.25 The results are obtained by co-culturing MDA-MB-453 (HER2++) cell line and GFP-expressing MDA-MB-468 (HER2-) cell line, and then using FACS to count the number of cells using GFP expression as a criterion for ADC with camptothecin as the payload.

[0437] Fig.26 The results are the result of co-culturing MDA-MB-453 (HER2++) cell line and MDA-MB-468 (HER2-) cell line, then using camptothecin drugs as the payload of ADC, and using HER2-FITC antibody to count the number of cells using FACS based on the expression of HER2.

[0438] FIG. 27 is a comparative evaluation result of in vitro cell viability (ADC treatment for 6 days) of cetuximab-25-6 in MDA-MB-468 (EGFR positive) cell line and SW480 (EGFR negative) cell line ( Fig.27a ); and the results of the in vitro cell viability (ADC treatment for 6 days) comparison evaluation of Sacituzumab-25-6 using FaDu (Trop2+++) cell line and Calu-6 (Trop2--) cell line as the subjects ( Figure 27b ).

[0439] Fig.28 It is a comparison of LogP, cLogP and topological polar surface area (tPSA) of FL118, Exitecan, 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).

[0440] Fig.29 It is a comparison of LogP, cLogP and topological polar surface area (tPSA) of various camptothecin drugs, compound G, compound H, compound I, compound J, compound G', compound H', compound I' and compound J' which can be derived from formula 1.

[0441] Fig.30It is a detailed classification diagram of apoptosis related to programmed cell death.

[0442] Fig.31 Showing the mechanism of ADC toxicity (Source: Cancers 2023, 15(3), 713; https: / / doi.org / 10.3390 / cancers15030713)

[0443] Fig.32 The working mechanisms of various self-immolative spacers are shown as examples. DETAILED DESCRIPTION

[0444] The following is further described in detail by the present invention. However, the following examples are only used to illustrate the technical features of the present invention and are not intended to limit the scope of protection of the present invention.

[0445] Preparation Example 1: Synthesis of active camptothecin derivatives (PBX-7011 and PBX-7012) of Formula 3 or Formula 3-1

[0446]

[0447] 1-1. Synthesis of Compound 2

[0448]

[0449] In a dichloromethane (748 ml) flask, silver trifluoromethanesulfonate (57.7 g, 224 mmol) and iodine (57.0 g, 224 mmol) were added to a solution of 5-nitrobenz[d][1,3]dioxolane (25 g, 150 mmol). 2 Stir under atmosphere and avoid light.

[0450] 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 dissolved in EtOAc (250 mL) and 5% (v / v) NH 4 OH / H 2 O solution (200 mL). The organic layer was separated and washed with 1 M Na 2 SO 3 (5x200 mL) and brine (200 mL), washed with Na 2 SO 4The product of 4-nitropropene (2-nitropropene) (2-nitropropene) (4-nitropropene) (2-nitropropene) (3-nitropropene) (4-nitropropene) (5 ...

[0451] SC_ACID:m / z 294.2[M+H] +

[0452] 1-2. Synthesis of Compound 3

[0453]

[0454] Iron powder (6.67 g, 119 mmol) and ammonium chloride (6.39 g, 119 mmol) were added to a suspension of 4-iodo-6-nitrobenzo[d][1,3]dioxolane (8.75 g, 29.9 mmol) in a mixture of water (80 ml), methanol (40.0 ml) and tetrahydrofuran (40.0 ml). The suspension was heated to 75 °C and stirred for 16.5 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting black solid was suspended in ethyl acetate (100 ml) and the solution was decanted. The residue was washed with EtOAc (3x50 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 NaHCO 3 The organic layer was washed with aqueous solution (150 mL) and brine (150 mL). 2 SO 4 The aqueous solution was dried and the solvent was removed under reduced pressure to obtain a brown solid (4.75 g, 60% yield). The iron residue was washed thoroughly with ethyl acetate to recover additional product. The organic portion was then washed with saturated NaHCO 3 The mixture was washed with aqueous solution (150 mL), brine (150 mL) and then with Na 2 SO 4 The solvent was removed under reduced pressure to obtain a brown solid (1.74 g, 22% yield).

[0455] SC_ACID:m / z 264.0[M+H] +

[0456] 1-3. Synthesis of Compound 4

[0457]

[0458] To a solution of 7-iodobenzo[d][1,3]dioxolane-5-amine (6.39 g, 24.29 mmol) 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 a few hours, additional DCM (10 ml) was added. The suspension was filtered through a sinter 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, and purified by Na 2 SO 4 Drying and concentration under reduced pressure gave 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 ground in ice-cold EtOAc (5-10 ml). Two batches were combined and further dried to give an off-white solid.

[0459] Total yield: 5.0 g, 66%

[0460] SC_ACID:m / z 306.0[M+H] +

[0461] 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).

[0462] 1-4. Synthesis of Compound 5

[0463]

[0464] In a three-necked flask equipped with a condenser, a slurry of N-(7-iodobenzo[d][1,3]dioxolan-5-yl)acetamide (5.06 g, 16.59 mmol), but-3-enoic acid (1.69 mL, 19.90 mmol) and potassium carbonate (2.98 g, 21.56 mmol) in acetonitrile (40 mL) was cooled to 0-5 ° C. Water (13.33 mL) was slowly added to generate gas. Once gas evolution stopped, the mixture was degassed with Ar for 30 minutes. Tri-o-tolylphosphine (0.505 g, 1.659 mmol) and palladium acetate (0.186 g, 0.829 mmol) were added, and the mixture was degassed again for 30 minutes and then added to reflux under Ar. The reaction was cooled to room temperature and filtered through celite. The filter cake was washed with H 2O and EtOAc. The organic solvent was removed from the filtrate under vacuum and the aqueous material was acidified to a concentrated solution. The HCl aqueous layer was extracted with EtOAc until pH = 1-2, and the combined organic phases were washed with brine and purified by Na 2 SO 4 The crude product was ground in ice-cold EtOAc (30 mL) and the solid was collected by filtration to obtain a brown solid product. The mother liquor was concentrated under reduced pressure and purified by flash chromatography (80 g Si, 0-100% EtOAc in heptane). The product fraction was concentrated under reduced pressure to obtain a light brown foam. Total yield: 3.46 g, 75%. A mixture of E / Z isomers was obtained.

[0465] SC_ACID:m / z 264.4[M+H] +

[0466] 1-5. Synthesis of Compound 6

[0467]

[0468] A suspension of 4-(6-acetylaminobenzo[d][1,3]dioxolan-4-yl)but-3-enoic acid (3.47 g, 13.18 mmol) was suspended in tetrahydrofuran (50 mL) / water (50 mL) and stirred with N 2 Degas for 15 min. 2 Before heating to 40 °C under low temperature (balloon), Pd / C (2.97 g, 1.397 mmol) was added and the suspension was heated with H 2 Degas for 5 minutes. After 24 hours, the reaction mixture was purged with nitrogen and additional Pd / C (2.97 g, 1.397 mmol). The reaction mixture was bubbled with hydrogen for 10 minutes and stirred at 45 °C under hydrogen overnight. The reaction was filtered through celite. The filter cake was washed with H 2 O (50 mL) and EtOAc (50 mL), and the organic solvent was removed from the filtrate under vacuum. The aqueous solution was acidified with concentrated HCl until pH = 1 and the dark brown / green precipitate was collected by filtration on a sintered funnel. The aqueous filtrate was extracted. The combined organic phase was washed with brine and purified by Na 2 SO 4Dry, filter and remove the solvent in vacuo to give a brown oil. Due to low product recovery, the filter cake was mixed with EtOAc (50 ml), water (200 ml) and MeOH (400 ml). The water / EtOAc rinse was added to the flask containing the solid from the MeOH rinse. The aqueous layer was acidified to a thick consistency. HCl and an off-white precipitate formed until pH = 1, and the precipitate was collected by filtration through a sintered funnel. After extracting the aqueous filtrate with EtOAc (3x200 mL), LCMS showed that all products were removed from the aqueous. The combined organic phases were washed with brine and purified by Na 2 SO 4 Dry, filter and remove the solvent in vacuo to give a light brown solid. All batches of product are combined and purified by flash column chromatography (40 g Si in DCM, 0-10% MeOH). The product fractions are concentrated to give a light brown solid. Yield: 2.52 g, 72%.

[0469] SC_ACID:266.2[M+H] +

[0470] 1 H 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).

[0471] 1-6. Synthesis of Compound 7

[0472]

[0473] A suspension of 4-(6-acetylaminobenzo[d][1,3]dioxolan-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 hour and became a dark solution over time. The reaction mixture was added dropwise to an ice-cold saturated NaHCO 3 The combined organic layers were added to saturated NaHCO 3 (sat.NaHCO 3 ) and saline solution 2 SO 4 Dry, filter and remove the solvent in vacuo to give a light pink solid. Yield: 140 mg, 100%

[0474] SC_ACID:m / z 248.2[M+H] +

[0475] 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).

[0476] 1-7. Synthesis of Compound 8

[0477]

[0478] A suspension of N-(6-oxo-6,7,8,9-tetrahydronaphthyl[1,2-d][1,3]dioxolan-5-yl)acetamide (50 mg, 0.202 mmol) in tetrahydrofuran (1.2 mL) was cooled to 0 ° C, and potassium tert-butoxide (27.2 mg, 0.243 mmol) and isoamyl nitrite (35.0 μL, 0.263 mmol) were added. The dark green mixture was stirred on ice (<5 ° C) for 1.5 hours. Acetic acid (170 μL, 2.94 mmol), acetic anhydride (170 μL, 1.810 mmol) and zinc powder (66.1 mg, 1.011 mmol) were added to the reaction mixture. The suspension was stirred at 0 ° C for 2 hours. The reaction mixture was filtered on diatomaceous earth and rinsed with DCM. The filtrate was concentrated under reduced pressure to give a black oily substance. The crude product was purified by flash column chromatography (4 g Si, 0-4% MeOH in DCM). The product fractions were concentrated to give a grey solid with a purity of 80-90% (32 mg, 52%). A higher purity sample was obtained when purified by MPLC for purification.

[0479] SC_ACID:m / z 305.4[M+H] +

[0480] 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).

[0481] 1-8. Synthesis of Compound 9

[0482]

[0483] N,N'-(6-oxo-6,7,8,9-tetrahydronaphthyl[1,2-d][1,3]dioxolane-5,7-diyl)diethylamide (244 mg, 0.802 mmol) was suspended in ethanol / water (5 / 1) with 2M hydrochloric acid (4.69 mL, 9.38 mmol). The mixture was heated to 55 °C and maintained for 4 hours. The black mixture was cooled to 0-5 °C. Triethylamine (1.4 mL, 10.04 mmol) was added dropwise while stirring. The mixture was then diluted with EtOH and evaporated to dryness. The residue was distributed between water and DCM. The layers were separated and the aqueous layer was extracted once with DCM. The combined organic layers were purified by Na 2 SO 4 Drying and concentration gave the product (178 mg, 73%) as a brown solid with a purity of 86%.

[0484] SC_ACID:m / z 263.0[M+H] +

[0485] 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).

[0486] 1-9. Synthesis of Compound 10

[0487]

[0488] (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-tetrahydronaphthyl[1,2-d][1,3]dioxolan-7-yl)acetamide (112 mg, 0.427 mmol) were added to anhydrous toluene (4.5 ml), PPTS (21 mg, 0.085 mmol), and the reaction mixture was stirred at 115° C. for 40 hours.

[0489] The reaction mixture was cooled to room temperature. The suspension was diluted with 2 mL of DCM and filtered. A black residue (210 mg) was obtained.

[0490] The crude product was purified by column chromatography (12 g Si, 0-7% methanol in DCM) to give the product as a brown solid (45 mg, 21%).

[0491] LCMS analysis showed two diastereomers.

[0492] SC_ACID:m / z 490.2[M+H] +

[0493] 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).

[0494] 1-10. Synthesis of PBX-7011 and PBX-7012

[0495]

[0496] N-((10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydro-1H,13H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizinyl[1,2-b]quinolin-1-yl)acetamide (73.5mg, 0.150mmol) was dissolved in 1.0mL 6N HCl and stirred overnight at room temperature for 8 hours at 90°C. The reaction mixture was concentrated under reduced pressure and purified by acidic preparative MPLC twice (Luna2-30). The fraction containing the separated diastereomers was acidified with 5 drops of 3N HCl and lyophilized to give a yellow solid product.

[0497] The first eluting isomer (1 st eluting isomer):PBX-7011,23mg,34%yield

[0498] U_AN_ACID:m / z 448.4[M+H] +

[0499] 1H 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).

[0500] The second eluting isomer (2 nd eluting isomer):PBX-7012,28mg,41%yield

[0501] U_AN_ACID:m / z 448.2[M+H] +

[0502] 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).

[0503] Preparation Example 2: Synthesis of active camptothecin derivatives (PBX-7014 and PBX-7015) of Formula 4 or Formula 4-1

[0504] This example describes the synthesis of compounds PBX-7014 and PBX-7015 starting from two separated diastereomers of exotecan-hybrid compounds (PBX-7011 and PBX-7012).

[0505]

[0506] 2-1: Synthesis of PBX-7014

[0507]

[0508] A stock solution of activated glycolic acid was prepared as follows:

[0509] Glycolic acid (17 mg, 0.224 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour.

[0510] Next, 0.4 mL of the activated acid solution was added to a suspension of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizinyl[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 reaction mixture was stirred at room temperature for 3 hours. 0.05 mL of the freshly prepared activated acid solution was added. Next, the reaction mixture was stirred for an additional 2 hours at room temperature. The reaction mixture was evaporated and dried. 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 light yellow solid after lyophilization of the product fractions. Yield: 20 mg, 50%

[0511] U_AN_ACID:m / z 506.2[M+H] +

[0512] 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).

[0513] 2-2: Synthesis of PBX-7015

[0514]

[0515] Stock solutions of activated acids were prepared as follows:

[0516] Glycolic acid (17.00 mg, 0.223 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (25.7 mg, 0.223 mmol) and EDC (42.8 mg, 0.223 mmol) were added. The reaction mixture was stirred at room temperature for 1 hour.

[0517] Next, 0.25 mL of activated acid solution was added to a suspension of (1R, 10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizinyl[1,2-b]quinoline-11,14-dione (25 mg, 0.056 mmol) and triethylamine (0.016 ml, 0.112 mmol) in N,N-dimethylformamide (2.5 mL). The mixture was stirred at room temperature overnight. 0.03 mL of freshly prepared activated acid solution was added. Next, the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then combined with a smaller batch from before and evaporated to dryness. The crude product was purified by column chromatography. This gave 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 light yellow solid. Yield: 15 mg, 38%

[0518] U_AN_ACID:506.2[M+H] +

[0519] 1 H 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).

[0520] Preparation Example 3: Synthesis of the active camptothecin derivative (PBX-7016) of Formula 5

[0521] This example illustrates the synthesis of compound PBX-7016 starting from the exotecan-hybrid compound (PBX-7011).

[0522]

[0523] Follow the steps below to prepare a stock solution of activated D-lactic acid.

[0524] 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, and the reaction mixture was stirred at room temperature for 2 hours.

[0525] Then, in N, N-dimethylformamide (2.5ml), (1S, 10S) -1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H, 14H-benzo [de] [1,3] dioxol [4,5-g] pyrans [3', 4': 6,7] indolizinyl [1,2-b] quinoline -11,14- dione (36mg, 0.080mmol) and triethylamine (0.022ml, 0.161mmol) solution was added 0.3mL of activated acid solution. The mixture was stirred at room temperature for 6 hours. 0.05mL of the prepared activated acid solution was added. Then, the reaction mixture was stirred at room temperature overnight. The reaction mixture was purified by acidic preparative MPLC (Luna10-50) to obtain a light yellow solid after the product fraction was lyophilized.

[0526] Yield: 22 mg, 52%

[0527] U_AN_ACID:m / z 520.2[M+H] +

[0528] 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).

[0529] As described above, PBX-7016 of Formula 5 can be synthesized from PBX-7011 of Formula 3, and thus, in the same manner, PBX-7017 of Formula 5-1 can be synthesized from PBX-7012 of Formula 3-1.

[0530] Preparation Example 4: Synthesis of the active camptothecin derivative (PBX-7024) of Formula 9

[0531]

[0532] (2S)-2-cyclopropyl-2-hydroxyacetic acid was conjugated to the PBX-7011 compound and PBX-7024 was prepared in high yield.

[0533] (2S)-2-cyclopropyl-2-hydroxyacetic acid (26 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (26 mg, 0.226 mmol) and EDC (42 mg, 0.219 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Then, in N, N-dimethylformamide (2.5ml), (1S, 10S) -1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydro-11H, 14H-benzo [de] [1,3] dioxol [4,5-g] pyrano [3', 4': 6,7] indolizinyl [1,2-b] quinoline -11,14- dione (40mg, 0.089mmol) and DIPEA (0.047ml, 0.268mmol) solution was added 0.6mL of activated acid solution. The mixture was stirred at room temperature overnight. The reaction mixture was directly purified by acidic preparative MPLC (Luna10-50), and a bright white solid was obtained after the product fraction was lyophilized.

[0534] Yield: 32 mg, 65%

[0535] U_AN_ACID:m / z 520.2[M+H] +

[0536] 1 H NMR(400MHz,DMSO-d6)δ8.33(d,J=8.7Hz,1H),7.39(s,1H),7.23(s,1H),6.4 8(s,1H),6.28(d,J=4.9Hz,2H),5.50(q,J=6.7Hz,1H),5.40(s,3H),5.23-5.0 6(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=7.3Hz,3H),0.47-0.30(m,4H).

[0537] Example 1: Analysis of the binding mode of camptothecin derivatives to TOP1 / DNA

[0538] Table 2

[0539]

[0540] According to the binding mode prediction results (DockingScore) of molecular dynamics simulation, all camptothecin derivatives in Table 2 are basically bound by strong π-π stacking between DNA bases ( Figure 4 and Figure 5 ), the ester of lactone forms a hydrogen bond (H-bonding) with Arg488 of TOP1, and the ethyl and OH groups form a hydrogen bond (H-bonding) with Asp533 of TOP1 ( Figure 1 ), camptothecin derivatives play a role in supporting the good binding between TOP1 protein (Protein) and DNA.

[0541] SN-38 forms hydrogen bonds (H-bonding) with Glu356 and Arg364 of TOP1 and stabilizes ( Figure 4 ).

[0542] Exatecan forms hydrogen bonds with surrounding DNA bases to further stabilize Figure 4 ).

[0543] FL118 Drug-OCH 2 The O-(methylenedioxo) pentagonal rings are weaker than hydrogen bonds (which are not the same as actual cytotoxicity) and have a poorer docking score than the SN-38 drug or the ixotecan drug, but these bonds are perfect between DNA bases and also help solvation ( Figure 4 ).

[0544] like Figure 5As shown, 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 ixitecan drug according to the present invention are diastereoisomers, and therefore share the same binding posture and are expected to be stably bound to TOP1. In particular, it is calculated that the compound of formula 3 (PBX-7011) has a higher docking score than the SN-38 drug.

[0545] The compound of Formula 3 (PBX-7011), which has the same chirality as the exitecan drug, showed slightly better binding than the compound of Formula 3-1 (PBX-7012).

[0546] On the other hand, for the TOP1 mutation (E418K) showing resistance to Trodelvy (trade name) (payload: SN-38), such as Figure 6 As shown, the E418K mutation is far away from the drug binding site.

[0547] like Figure 7 and Figure 8 As shown in the figure, energy minimization was performed after the E418K mutation. As a result, the newly introduced Lys418 changed part of the drug binding site to a relatively polar environment and continued to ionically bond with the surrounding Glu356, which was confirmed by performing MD simulations. When part of the binding site becomes polar due to mutation, it was observed in the MD simulation that substances with polar functional groups, such as SN-38 drugs, move slightly away from the existing binding site to interact ionically with Lys418 and Glu356. This may weaken the interaction between lactone and Asp533 and Lys532, which is a common inhibitory activity principle shared by the same series of TOP1 inhibitors.

[0548] When the SN-38 drug was docked on the mutated TOP1 structure, although the SN-38 drug left the main binding site and moved to one side, a new hydrogen bond (H-bonding) was obtained, showing a good docking score for binding. The docking score has limitations in explaining the phenomenon that the mutated TOP1 shows resistance to SN-38.

[0549] like Figure 7 and Figure 8 As shown, when analyzing the interaction between the SN-38 drug and TOP1 during MD simulation, it was confirmed that due to mutation, the interaction between SN-38 and Asp533 and Arg488 (89%) was reduced to less than 10%, but the interaction with Asn352 and Glu356 increased to a level of 40% to 80%.

[0550] When docking was performed after TOP1 mutation, the SN-38 drug moved to the newly modified binding site and bound in a new form, showing a score similar to the existing score (Table 2).

[0551] On the contrary, as shown in Table 2, it was confirmed that the relative docking scores between the ixitecan drug, FL118 drug, compound of Formula 3 (PBX-7011) and compound of Formula 3-1 (PBX-7012) without a polar functional group (-OH) at carbon 10 of ring A deteriorated due to the environmental changes of some new binding sites and the TOP1 of mutation. Nevertheless, it was confirmed by MD simulation that the ixitecan drug, FL118 drug, compound of Formula 3 (PBX-7011) and compound of Formula 3-1 (PBX-7012) maintained the existing interaction between lactone and Asp533 and Lys532.

[0552] like Fig. 9 As shown, even after TOP1 mutation, the compound of Formula 3 (PBX-7011) still maintains its existing binding mode (especially lactone and -OH groups).

[0553] In summary, the E418K mutation does not directly interact with the TOP1 inhibitor, but to explain the situation that the FL118 drug inhibits the TOP1 mutation that exhibits resistance to the SN-38 drug at a high level through E418K, the mutation is different from the docking results, showing that the new binding of SN-38 to the modified binding site is carried out in a way that weakens the action of lactone with Asp533 and Lys532. This can consider the hypothesis that it is a binding that cannot inhibit the action of TOP1.

[0554] Under this assumption, the drug Exitecan, the drug FL118, the compound of Formula 3 (PBX-7011), and the compound of Formula 3-1 (PBX-7012) continue to act on Asp533 and Lys532 of mutated TOP1, showing less drug resistance.

[0555] Example 2

[0556] For FL118, exotecan, SN-38, Dxd, exotecan-lactic acid, PBX-7011, PBX-7014, PBX-7016, and PBX-7024, expression inhibition analysis of anti-apoptotic proteins and in vitro cell viability assay were performed.

[0557] 2-1: Confirmation of the expression inhibitory activity of cancer-associated survival genes DDX5, survivin, Mcl-1, XIAP, and cIAP2 by Western blot

[0558] (1) Protein extraction

[0559] FaDu cells were seeded at 200,000 cells / well in a 6-well plate and incubated at a constant temperature (37°C, 5% CO 2 ). After 24 hours, drugs (FL118 drug, SN-38 drug, Exitecan drug, PBX-7011, PBX-7014, PBX-7016) were treated in the wells at concentrations of 0 nM, 10 nM, and 100 nM, respectively. Constant temperature incubation (37°C, 5% CO 2 ) for 24 hours. 100ul of RIPA buffer containing protease inhibitor cocktail was treated in the well. The plate was placed on ice and incubated on an orbital shaker for 2 hours. The RIPA buffer containing lysis cells was transferred to an ep tube and centrifuged (16000rcf, 20min, 4°C). Then, only the supernatant was transferred to a new ep tube. The protein concentration was confirmed by protein assay.

[0560] (2) Protein separation by electrophoresis

[0561] Protein sample and 4x SDS-PAGE loading buffer were mixed at a ratio of 3:1, boiled at 95°C for 10 minutes, and then cooled. The samples were loaded into the gel wells so that the amount of protein was the same. Electrophoresis was performed at 60V in the gel.

[0562] (3) Transfer of proteins from gel to membrane

[0563] Turbo TM Transfer system( Turbo TM Into the cassettes of the Transwell Transfer System, 7 activated filter papers, PVDF membrane, gel, and 7 filter papers were placed in sequence, then the lid was closed, inserted into the instrument, and the experimental protocol was started.

[0564] (4) Antibody incubation

[0565] Immerse the transferred membrane in blocking buffer and incubate at a constant temperature (RT, 1h). Then, incubate at a constant temperature (4°C, overnight) in the primary antibody solution. Rinse with hybridization membrane cleaning solution (TBST buffer) for 3 minutes (min) (repeat 3 times). Incubate at a constant temperature (RT, 1h) in the secondary antibody solution conjugated with HRP (horseradish peroxidase). Rinse with hybridization membrane cleaning solution (TBST buffer) for 3 minutes (min) (repeat 3 times).

[0566] (5) Imaging and result analysis

[0567] After the membrane is immersed in ECL (enhanced chemiluminescence) substrate for about 3 to 5 minutes, use ChemiDoc TM MP Imaging System (ChemiDocTM MP Imaging System) to confirm the signal. HRP (horseradish peroxidase) bound to the secondary antibody oxidizes the luminol of ECL, and the released light is detected to display as an image. The thickness of the band is proportional to the amount of protein, so the amount of protein can be compared by the thickness of the band.

[0568] like Figures 10 to 12 As shown, it was confirmed how the protein expression level changes due to drug (FL118 drug, SN-38 drug, Exitecan drug, PBX-7011, PBX-7014, PBX-7016) treatment. Glyceraldehyde phosphate dehydrogenase (GAPDH) is an enzyme involved in glycolysis, which is an essential metabolic process in cells. GAPDH is a gene that is always expressed in cells and its expression level is not easily changed, which can indicate whether the sample is loaded with the same amount of protein on the gel.

[0569] In the same manner, A549 cells overexpressing ABCG2 were seeded instead of FaDu cells not expressing ABCG2, 4 μg protein was loaded, and western blot was performed ( Fig.13 and Fig.14 ).

[0570] Review: PBX-7011, PBX-7014 and PBX-7016 evaluation

[0571] The degree of inhibition of anti-apoptotic protein expression in the FaDu cell line was confirmed by western blot. The results showed that as the concentration of PBX-7011, PBX-7014 and PBX-7016 increased, the expression of Survivin, cIAP2, Mcl-1 and XIAP decreased. When compared with the existing FL118, SN-38 and Ixitecan drugs, the levels were similar to those of FL118 and Ixitecan drugs, and were superior to SN-38.

[0572] In Example 2-1, it was confirmed that the compound exhibited dual inhibition of topoisomerase 1 and degradation of DDX5, and had heterogeneous properties of DDX5 degradation and corresponding anticancer effects.

[0573] Specifically, two cell lines (FaDu, A549) were treated with drugs at predetermined concentrations (0, 10, 100 nM), and the expression levels of each protein (DDX, Survivin, Mcl-1, XIAP) were confirmed by western blot. As a result, FL118 was superior to other substances in DDX5 degradation, followed by 7011, 7016, and 7014.

[0574] Not only DDX5, but other anti-apoptotic proteins in its downstream also showed similar changing trends.

[0575] In short, PBX-7014 and PBX-7016 not only inhibit type 1 topoisomerase, but also display dualMoA, acting as degraders of DDX5 (p68), an oncoprotein that regulates Survivin, Mcl-1, XIAP, etc.

[0576] The evaluation results showed that, among PBX-7014 and PBX-7016, PBX-7016 inhibited DDX5 in a concentration-dependent manner. As a result, inhibition of Survivin, Mcl-1, and XIAP was observed. In addition, FL118 was shown to act as a DDX5 degrader not only in Topoisomerase I inhibitor, but also in PBX-7016. Moreover, PBX-7016 had superior performance compared to PBX-7014 ( Figures 11 to 14 ).

[0577] 2-2: Cell viability assay for FaDu cell line / A549 cell line

[0578] FaDu cell line (cancer cells that do not express ABCG2) or A549 cell line (cancer cells that overexpress ABCG2) were seeded at 3000 cells / well in a 96-well plate and incubated at a constant temperature (37°C, 5% CO 2 ). After 24 hours, the cells were treated with 100 μl of drugs at 9 concentrations (serial dilution starting from 1000 nM in 1 / 5 steps). At this time, Dxd drugs, PBX-7014, PBX-7016, and PBX-7024 were treated. At this time, a control group without drug treatment (drug concentration 0) was also prepared. Constant temperature incubation (37°C, 5% CO2 ) for 3 or 6 days. 100 μl of CellTiter-Glo reagent (using Luminescent Cell Viability Assay Kit ( Luminescent cell viability Assay kit (Promega, G7571))) was used, and then pipetted. After incubation at constant temperature (RT) for 10 minutes, the luminescence was measured. When the luminescence value when the drug concentration was 0 was regarded as 100%, the concentration representing the luminescence value of 50% was the IC 50 value.

[0579] Two cell lines (FaDu, A549) were treated with two compounds (PBX-7016, PBX-7024) and reference compounds (Dxd, SN-38, Exitecan, FL118), and cell viability was observed after 3 days of incubation.

[0580] like Fig.15 As shown in the results, PBX-7024 showed strong apoptotic effect 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, the loading agent used for the existing Euheride, i.e., Dxd, was confirmed to have an IC value equal to or higher than that of Dxd. 50 .

[0581] like Fig.15 As shown in Figure 2, it was confirmed that the cancer cell line A549 overexpressing ABCG2 showed increased IC with camptothecin compounds including Dxd. 50 Compared with the previous models, PBX-7016 and PBX-7024 still maintain strong performance.

[0582] That is, when the new camptothecin compounds PBX-7016 and PBX-7024 according to the present invention are used, unlike ADCs using existing camptothecin compounds SN-38, DXd, etc., they have the ability to overcome the drug resistance mechanism caused by overexpression of ABCG2.

[0583] Example 3. New PBX-series compounds as candidate for ADC payload: in vitro cell viability test

[0584] Cell viability assays were performed on MDA-MB-453 (HER2++) cell lines and FaDu (HER2+) cell lines in the same manner as in Example 2-2.

[0585] Two cell lines (MDA-MB-453, FaDu) were treated with two compounds (PBX-7014, PBX-7016, PBX-7018, PBX-7020, PBX-7022) and one reference compound (Dxd), and cell viability was observed after 3 days of incubation. The results are shown in Fig.16 .

[0586] like Fig.16 As shown in the evaluation results, PBX-7016, PBX-7018, PBX-7020, and PBX-7022 have ICs equal to or higher than Dxd. 50 In addition, it was confirmed that the FaDu cell line that does not express ABCG2 has an IC value equal to or higher than Dxd. 50 value.

[0587] Example 4: Pharmacokinetic Profile of PBX-7016 IV and IP in NOD SCID Mice

[0588] The experimental method is shown in Table 3. The IV plasma concentration-time data of PBX-7016 is shown in Table 4, and the IP plasma concentration-time data of PBX-7016 is shown in Table 5.

[0589] Table 3

[0590]

[0591] Table 4

[0592] IV dose 1 mg / kg

[0593]

[0594] Table 5

[0595] IP dose 1 mg / kg

[0596]

[0597] The results in Tables 4 and 5 confirm that when intravascularly or intraperitoneally administered to mice, the PBX-7016 compound is cleared from the blood in a very short time, and when administered in vivo in the form of a PBX-7016 compound alone rather than a prodrug or ADC, it is expected to exhibit high safety. When the PBX-7016 compound is injected intravenously (IV), it can also serve as a safety device to reduce systemic exposure of the drug by releasing it from the body in a very short time and prematurely releasing the drug very early after entering the body.

[0598] In addition, this experiment is a separate PK of the PBX-7016 compound used as a payload, indicating that it is a safe payload that can clear the clearance of the PBX-7016 drug from the blood in a short time. In other words, this shows that after the PBX-7016 compound exerts a bystander effect at the tumor site, it will be safely released outside the body in a very short time even if it circulates in the blood.

[0599] Example 5. Carboxylate to Lactone conversion rate of PBX-7016 at pH 6.0

[0600] Example 5 Fig.17 and Fig.18 ) is an experiment related to the activation of the lactyl portion of camptothecin, a known Top1 inhibitor activator.

[0601] 5-1. Carboxylate to lactone form conversion at pH 6.0

[0602] PBX-7016 powder was dissolved to 5 mM in DMSO for preparation. PBX-7016 dissolved in DMSO exists in lactone form, so it was converted into carboxylate form by diluting it 1 / 10 with 0.1 N NaOH and incubating it in a 25° C. water bath for 30 minutes. PBX-7016 in carboxylate form was diluted to 3 μM using 4° C. PBS (pH 6.0) containing 10% DMSO. At this time, HCl in the same amount as the 0.1 N NaOH used was added to minimize pH changes. After dilution, centrifugation was performed at 4° C., 2 mins, 13000 RCF, and the supernatant was used as a sample.

[0603] When the lactone form of PBX-7016 was generated, two solvents were used, solvent A: 100 mM acetate buffer, pH 5.5; solvent B: ACN, and it was separated by RP-HPLC. The chromatographic column was Cortecs C18 2.7 μm, 4.6 x 50 mm Column, and the analysis conditions were as follows: 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.

[0604] To measure changes over time, the sample chamber was kept at 37°C and set to inject the sample at each measurement. The measurement times were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, 720, 840, 960, 1200, 1440 mins.

[0605] The content was calculated from the peak area of ​​each lactone and carboxylate. Lactone and carboxylate have different absorption coefficients, so the lactone area was calculated by multiplying the carboxylate area by the following formula.

[0606] Lactone EC 390 / Carboxylate EC 390＝ 0.918

[0607] The same experiment as Example 5-1 was performed except that FL118, SN38, DxD, exotecan, PBX-7014, and PBX-7024 were used instead of PBX-7016.

[0608] At pH 6.0, the results of the conversion experiment from carboxylate to lactone are as follows Fig.18 shown. Fig.18 As shown, it has been demonstrated that various camptothecin compounds are converted from carboxylate to lactone (active form that inhibits topoisomerase I) at similar rates and levels at pH 6.0, and thus, in this way, they are expected to have the same level of activity in the micro-tumor environment (Tumor Micro-environment) where the pH is usually 6.0.

[0609] 5-2. Lactone to carboxylate form conversion at pH 7.4

[0610] Dissolve PBX-7016 powder in DMSO to 5mM for preparation. Prepare 3μM PBX-7016 by sacrificing 10% DMSO in 4℃ PBS (pH7.4). Then, centrifuge at 4℃, 2mins, 13000RCF, and remove the supernatant as a sample. PBX-7016 dissolved in DMSO exists in lactone form.

[0611] When the PBX-7016 carboxylate salt form was generated, two solvents were used, solvent A: 100 mM acetate buffer, pH 5.5; solvent B: ACN, and it was separated by RP-HPLC. The chromatographic column used was Cortecs C18 2.7 μm, 4.6 x 50 mm Column, and the analysis was performed 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.

[0612] To measure changes over time, the sample chamber was kept at 37°C and set to inject the sample at each measurement. The measurement times were 0, 20, 40, 60, 90, 120, 180, 240, 300, 360, 480, 600, 720 mins.

[0613] The content was calculated from the peak area of ​​each lactone and carboxylate. Lactone and carboxylate have different absorption coefficients, so the lactone area was calculated by multiplying the carboxylate area by the following formula.

[0614] Lactone EC 390 / Carboxylate EC 390 =0.918

[0615] The same experiment as Example 5-2 was performed except that FL118, SN38, DxD, exitecan, PBX-7014, and PBX-7024 were used instead of PBX-7016.

[0616] At pH 7.4, the results of the conversion experiment from lactone to carboxylate are as follows Fig.17 As shown, it has been confirmed that at pH 7.4 in liquid or extracellular fluid, PBX-7014, PBX-7016 and PBX-7024 compounds represented by Formula 1 are converted from the lactone form (active form) to the carboxylate form (inactive form) which is inactive as a TOP1 antidote not only faster but also at a higher rate compared to other reference compounds (exitecan, DXd, SN-38, FL118).

[0617] In particular, the degree of formation (63-86%) and rate (0.4-0.8% / min) of the carboxylate form, which loses its activity as a TOP1 antidote at blood pH 7.4, vary greatly depending on the type of camptothecin derivative. PBX-7014 and PBX-7016 formed more than 86% of the carboxylate form, and both were converted from the lactone form to the carboxylate form relatively quickly at a level of 0.8% per minute. The competing drug DXd formed the carboxylate form at a level of 76.5%, and was converted from the lactone form to the carboxylate form at a level of 0.6% per minute.

[0618] Thus, compared with the reference compound, compounds PBX-7014 and PBX-7016 are expected to exhibit high safety in normal cells because they exist in the inactive carboxylate form rather than in the active lactone form which exhibits cytotoxicity relatively quickly and at a high rate in the blood.

[0619] Preparation Example 5: Synthesis of 25-4 and 25-6 from PBX-7014 and PBX-7016 and preparation of their ADC (DAR7-8) (Trastuzumab-25-4 and Trastuzumab-25-6)

[0620] Linker-payloads 25-4 (Formula 10) and 25-6 (Formula 11) containing two compounds (PBX-7014, PBX-7016) and introducing GGFG (enzymatically cleavable linker system) were synthesized.

[0621]

[0622]

[0623] The molecular structure of 25-4 is GGFG-PBX-7014, and the molecular structure of 25-6 is GGFG-PBX-7016. In other words, they used the same Same GGFG connector.

[0624] In addition, use with UHD The same GGFG linker and trastuzumab antibody were used to prepare ADCs with PBX-7014 and PBX-7016 as payloads.

[0625] 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, both with DAR 8).

[0626] Trastuzumab-25-4 (trastuzumab-7014) was prepared as follows. The prepared trastuzumab buffer was exchanged into reaction buffer (150 mM NaCl, 50 mM Histidine pH 6.0) using a PD-10 desalting column, and then 825 μM TCEP was treated at 25°C for 2 hours in 27.5 μM antibody to remove the thiol sites required for the reaction prepared by the disulfide bonds of the antibody.

[0627] Then, excess TCEP was removed using a PD-10 desalting column, and 61.9 μM 25-4 drug linker (Formula 10) and 13.8 μM reduced trastuzumab were reacted in a reaction buffer containing 10% DMSO at 25° C. for 1 hour to perform a conjugation reaction.

[0628] Trastuzumab-25-6 (trastuzumab-7016) was prepared by the same process, and the same concentration of 25-6 drug linker (Formula 11) was used instead of 61.9 μM 25-4 drug linker (Formula 10).

[0629] Each ADC was then purified using SEC to confirm that it was purified as a monomer and without aggregation ( Fig.19 ). SDS-PAGE confirmed that the drug was conjugated through the band shift of the light chain and the heavy chain ( Fig.20a ).

[0630] Preparation Example 6: Synthesis of 25-6 from PBX-7016 and preparation of its ADC (DAR7-8) (Nimotuzumab-25-6 and Gosartuzumab-25-6)

[0631] Nimotuzumab-25-6, gosartuzumab-25-6, and cetuximab-25-6 were synthesized in the same manner as in Preparation Example 5, except that Nimotuzumab, gosartuzumab, or cetuximab was used instead of trastuzumab.

[0632] Each ADC was then purified using SEC to confirm that it was purified as a monomer and without aggregation. SDS-PAGE confirmed that the drug was conjugated by the band shift of the light and heavy chains ( Fig.20b , Fig.20c as well as Fig.20d ).

[0633] Example 6. Anti-HER2 ADCs with PBX-payloads: binding affinity analysis

[0634] Prepare the coating buffer by diluting the target antigen to 0.66 μg / ml using ELISA plate coating buffer (R&D Systems, #DY006). Use 100 μL per well to coat the prepared coating buffer. Cover the plate lid, maintain at 2-8°C, and leave overnight (12-18 hours). The plate used is Corning, #3590. Wash three times with more than 200 μL of TBS Tween-20 buffer (Thermo, #28360). When washing, first aspirate the liquid with a pipette, then gently tap on a paper towel to remove excess buffer. Use 200 μL of assay buffer for blocking (Assay buffer) at room temperature for 1 hour. Remove the liquid with a pipette and remove excess buffer by gently tapping on a paper towel. The control antibody and the ADC to be tested are prepared at 1 μg / ml in the assay buffer, and then prepared to the following concentrations by 1 / 5 serial dilution. For example, the sample can be prepared by diluting to the following concentrations based on duplication [240ul assay buffer + sample of previous concentration] 60ul]: 1μg / ml, 0.4μg / ml, 0.08μg / ml, 0.016μg / ml, 0.0032μg / ml, 0.00064μg / ml, 0.000128μg / ml. The diluted control antibody and ADC are repeated (duplicate) at a concentration of 100μL per coated well. Perform for 1 hour at room temperature. Wash 3 times with 100ul of TBS Tween-20 buffer (Thermo, #28360). When washing, first remove the liquid with a pipette, and then gently tap on a paper towel to remove excess buffer. Prepare the detection antibody by diluting it in the assay buffer with reference to the manufacturer's manual. In the case of human antibody, use goat anti-human Fc-HRP (Novex, #A18817) as the detection antibody and prepare it by diluting it 1 / 2000. Add 100 μL of the diluted detection antibody to each well and incubate at room temperature for 1 hour. Wash 3 times with 100 μL of TBS Tween-20 buffer (Thermo, #28360). When washing, first remove the liquid with a pipette, then gently tap on a paper towel to remove excess buffer. Use 100 μL of TMB substrate solution (Thermo, #N301) to react at room temperature for 30 minutes.The reaction was terminated using 100 μL of Stopsolution (Thermo, N600). The reaction was stopped and the absorbance was measured at 450 nm. It was completed within 30 minutes after the addition of the stop solution. When bubbles appeared, 100 to 150 μl were transferred to a new plate and measured. Thus, ADCs conjugated with trastuzumab, trastuzumab and PBX-7014, and trastuzumab and PBX-7016 were measured to obtain. Fig.21 The results are shown. Fig.21 As shown, when the binding intensity of ADC conjugated with PBX-7014 or PBX-7016 to the Her2 antigen of trastuzumab was compared at different concentrations, it was confirmed that there was no difference in the binding intensity.

[0635] Example 7. Anti-HER2 ADCs with PBX-payloads: in vitro potency and target selectivity

[0636] In the same manner as in Example 2-2, for ADCs with Dxd, PBX-7014 and PBX-7016 as effective loads, respectively, in vitro cell viability assays were performed on MDA-MB-453 (HER2++) cell lines, FaDu (HER2+) cell lines and MDA-MB-468 (HER2-) cell lines as follows.

[0637] At this time, the HER2 targeted therapeutic agent Herceptin (ingredient name: trastuzumab), Dxd drug, PBX-7014 (Preparation Example 2), PBX-7016 (Preparation Example 3), Tra-25-4, Tra-25-6 (Preparation Example 5) and Tra-Dxd (Uherde) as its ADC ) was processed.

[0638] As a reference, three cell lines, MDA-MB-453 (Her2++), FaDu (Her2+), and MDA-MB-468 (Her2-), were treated with Tra-Dxd (DAR 8) according to the Her2 expression level, and the cell viability was observed after 3 days of incubation. Fig. 22 ).

[0639] Fig. 22As shown, anti-Her2 ADCs with PBX payloads have been demonstrated to have excellent target selectivity.

[0640] Review: Evaluation of PBX-7014 and PBX-7016 as ADC Payloads

[0641] like Fig. 22 As shown, Her2++ has been confirmed, which is the IC of ADC (Tra-25-6) in MDA-MB-453 with PBX-7016 as the effective load 50 The value is about 5 times better than Tra-Dxd, and the negative cell line MDA-MB-468 has been confirmed to have the same IC level. 50 The values ​​indicate that when the linker-payload system of Formula 10 and Formula 11 was developed as ADC, it was confirmed that it could not only work by distinguishing positive / negative cell lines, but also showed excellent efficacy compared with the reference, i.e., Euheride.

[0642] Review: The originality and excellence of PBX-7016

[0643] According to the present invention, the camptothecin compound newly designed and synthesized according to formula 1 of the present invention, i.e., PBX-7016, is a new compound derived from the MD-CPT skeleton FL118, and like FL118, it not only acts as a topoisomerase I inhibitor, but also acts as a DDX5 degrader, thereby proposing a new topoisomerase 1 inhibitor for degrading DDX5. Figures 11 to 14 ).

[0644] In addition, when simply comparing cell viability, PBX-7016 showed an IC 50 The value was 1.5 to 2 times that of Dxd, but surprisingly, it was confirmed that when used as an ADC payload, the ADC containing PBX-7016 had about 5 times better cell viability than the ADC containing Dxd (Uhude) ( Fig. 22 ).

[0645] Example 8. Anti HER2 ADCs with PBX-payloads: in vivo efficacy

[0646] 8-1. Cell culture

[0647] JIMT-1 breast cancer cells were cultured in 5% CO 2 The cells were cultured in DMEM containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C and routinely passaged twice a week by trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0648] 8-2.BALB / c nude mice

[0649] Both SCID mice and BALB / c nude mice are used as animal models to study the immune system and test treatments for various diseases.

[0650] Although both SCID mice and BALB / c nude mice have impaired immune systems, the mechanisms of immunodeficiency are different.

[0651] SCID mice are genetically engineered mice that lack functional T cells and B cells, resulting in a severely impaired immune system. They are used to study the development and function of the immune system and to test potential treatments for diseases that affect the immune system, such as autoimmune diseases and certain types of cancer.

[0652] BALB / c nude mice, on the other hand, are mice that cannot produce T cells because they do not have a thymus gland. They are also born hairless due to a hairless gene defect. These mice are used to study the role of T cells in immune responses and to test potential treatments for diseases that affect T cell function, such as certain types of cancer.

[0653] 8-3. Tumor inoculation and drug treatment

[0654] JIMT-1 tumor cells (5x10 6 ) were subcutaneously inoculated into the right anterior flank of each BALB / c nude mouse. When the average tumor volume reached approximately 215 mm 3 When, animals were randomized and treated for efficacy studies.

[0655] Treatment: IV, Q1W

[0656] Vehicle group: Vehicle, 0 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0657] Isotype-Dxd (3) group: CTP63-Dxd, 3 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0658] Isotype-Dxd (10) group: CTP63-Dxd, 10 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0659] Isotype-PBX7016 (3) group: CTP63-PBX7016, 3 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0660] Isotype-PBX7016 (10) group: CTP63-PBX7016, 10 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0661] Isotype-PBX7014 (3) group: CTP63-PBX7014, 3 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0662] Isotype-PBX7014 (10) group: CTP63-PBX7014, 10 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0663] Trastuzumab-PBX7014 (3) group: CTP6-PBX7014, 3 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0664] Trastuzumab-PBX7014 (10) group: CTP6-PBX7014, 10 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0665] Trastuzumab-PBX7016 (3) group: CTP6-PBX7016, 3 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0666] Trastuzumab-PBX7016 (10) group: CTP6-PBX7016, 10 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0667] Euheride (3) group: Euheride, 3 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0668] Euheride (10) group: Euheride, 10 mg / kg, 5 μL / g, iv, twice (day 0, day 6)

[0669] 8-4. Tumor Measurements and Endpoints

[0670] The primary endpoint was whether tumor growth could be delayed or whether the mice could be cured. The two-dimensional dimensions of the tumor were measured twice a week using a caliper and the formula (V = 0.5 axb 2 , where a and b are the long and short diameters of the tumor, respectively) in mm 3 Indicates volume.

[0671] The tumor growth inhibition (TGI) of each group was calculated.

[0672] TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )]×100

[0673] (T i is the mean tumor volume of the treatment group on a given day, T 0 is the mean tumor volume of the treatment group on the first day of treatment, V i is with T i The mean tumor volume of the vehicle control group on the same day, V 0 is the average tumor volume of the first-line drug group).

[0674] As humane endpoints, weight loss equal to or greater than 15% or tumor volume equal to or greater than 3000 mm 3 The animals were euthanized.

[0675] 8-5. Statistical analysis

[0676] The results are expressed as mean ± SEM. The data were analyzed using Graphpad Prism 6.0 software with two-way RM ANOVA Dunnett's multiple comparison test, and p < 0.05 was considered statistically significant.

[0677] Table 6 below shows the tumor volume (Tumor Volume) (mm 3 )Trends over time (effectvs.time).

[0678] Table 6

[0679]

[0680] Fig.23Table 6 is presented in graphical form, showing the results of efficacy testing of ADCs containing various camptothecin drugs as payloads in mice transplanted with the JIMT-1 cell line (ie, in vivo xenograft model).

[0681] 8-6. Body weight of JIMT-1 tumor model

[0682] The body weight of JIMT-1 tumor model mice was monitored regularly ( Fig.24 ).

[0683] Example 9: Anti-HER2 ADCs with PBX-payload: Bystander effect study

[0684] The bystander effect is one of the factors that significantly affect the efficacy of ADC drugs. To confirm the bystander effect, it was analyzed by FACS under 40nM ADC treatment conditions.

[0685] In order to verify the efficacy and practicality of using PBX-7016 (the lead material of the present invention) as an ADC drug, as an effective load in an antibody-drug conjugate (ADC), wherein the effective load-linker is targeted to the HER2 protein, the bystander effect was confirmed in vitro by flow cytometry analysis experiments. ADC treatment of the HER2-positive cell line MDA-MB-453 induces apoptosis, and the drug (effective load) released during apoptosis is toxic to the surrounding HER2-negative cell lines, thereby causing apoptosis, which is defined as the bystander effect in this embodiment.

[0686] In Preparation Example 5, in order to evaluate the in vitro bystander effect of the manufactured ADC, HER2 expression-negative cell lines and HER2 expression-positive cell lines were mixed in a 6-well cell culture plate and co-cultured in proportion to determine whether there was a bystander effect for evaluating ADC treatment.

[0687] Specifically, GFP-MDA-MB-468 cells (3x10^5 cells) (prepared to express GFP (Green fluorescent protein) in MDA-MB-468, a HER2 expression-negative cell line) and MDA-MB-453 cells (1x10^5 cells) (as a HER2 expression-positive cell line) were mixed in a ratio of 3:1. After 24 hours of culture, trastuzumab antibody and a total of 5 ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, and Euheride) were treated to 40 nM concentrations, respectively. After 6 days of cell culture, the GFP fluorescence and cell number of GFP-MDA-MB-468 cells, a HER2 expression-negative cell line, were analyzed in living cells by flow cytometry.

[0688] In addition, in a 6-well cell culture plate, MDA-MB-468 cells (3x10^5 cells) as a HER2 expression-negative cell line and MDA-MB-453 cells (1x10^5 cells) as a HER2 expression-positive cell line were mixed in a ratio of 3:1. After 24 hours of culture, trastuzumab antibody and a total of 5 ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, and Euheride) were treated to 40 nM concentrations, respectively. After 6 days of cell culture, the cells were stained with anti-HER2-FITC antibody (an antibody that can recognize HER2 protein and labeled with fluorescein isothiocyanate (FITC) fluorescent dye to show fluorescence), and the FITC fluorescence measurement and cell number of MDA-MB-453 cells as a HER2 expression-positive cell line in living cells were identified by flow cytometry.

[0689] The flow cytometry method was to collect cells from each well of a 6-well cell culture plate, dilute with buffer (400 μl), and then analyze GFP or FITC simultaneously (1 min each) using the FL1 laser and measure the fluorescence of each sample with the same flow rate.

[0690] like Fig.25 and Fig.26As shown, flow cytometry confirmed that the ADC based on PBX-7016, i.e., trastuzumab-25-6, not only killed HER2-positive cells, but also killed HER2-negative cells when compared with the ADCs based on DXd, i.e., trastuzumab-Dxd and euheride, and was judged to have a bystander effect comparable to that of the existing DXd-based euheride. In contrast, it was confirmed that the negative control ADCs, i.e., isotype IgG-DXd and isotype IgG-25-6, did not induce apoptosis of HER2-negative cells compared with the untreated conditions without ADC treatment.

[0691] In short, the anti-HER2 ADC using PBX-7016 showed a similar bystander effect as Ulcetrimonium.

[0692] Example 10: In vitro cell cytotoxicity test of cetuximab-25-6 and gosartuzumab-25-6

[0693] In the same manner as in Example 2-2, MDA-MB-468 (EGFR positive) and SW480 (EGFR negative) cell lines were treated with cetuximab-25-6 ( Fig.27a ); and FaDu (Trop2+++) cell line and Calu-6 (Trop2--) cell line were treated with gosartuzumab-25-6 ( Figure 27b ), after 6 days of incubation, a cell viability assay was performed. The results are shown in FIG27 .

[0694] Figure 27 shows that in ADCs such as cetuximab-25-6 and gosartumumab-25-6, most mAbs can also be localized to the target cell population by binding to high-affinity mAbs of cell membrane proteins. Chemical conjugation of the PBX-7016 payload and anticancer drug mAbs increases the selectivity of payload delivery to cancer cells, indicating that it improves the therapeutic index of the payload.

[0695] In particular, the in vitro cell viability (ADC 6daysTreatment) of gosartuzumab-25-6 and PBX-7016 was compared and evaluated using FaDu (Trop2+++) cell line and Calu-6 (Trop2--) cell line. Figure 27b) Taking the active camptothecin derivative (a) represented by Formula 1 as an example, PBX-7016 designed to be released at the target site in vivo can move into the cell by penetrating the cell membrane of non-target cells to exert a cell apoptosis mechanism, which means that the ADC based on PBX-7016 can exert a bystander effect.

Claims

1. An active camptothecin derivative (a) represented by the following formula 1, designed to bind to DDX5 protein and E3 ligase; or a prodrug (b) thereof, designed to release the active camptothecin derivative (a) at a target site in vivo, preferably an antibody-drug conjugate (ADC); or a complex (c) containing a ligand targeting DDX5 protein, wherein the type 1 topoisomerase inhibitory ability of the active camptothecin derivative (a) or the FL118 compound of formula 2 is inactivated by a linker connection, X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 form a hexagonal ring or a heptagonal ring, and n is a value of 1 to 2, Y1, Y2 and Y3 are each independently 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 in such a manner as to bind to the DDX5 protein and the E3 ligase functions as a molecular glue degrading agent and as a ligand binding to the E3 ligase.

3. The camptothecin derivative (a) or its prodrug (b) according to claim 1, characterized in that: Active camptothecin derivatives (a) designed in a way to bind to DDX5 protein and E3 ligase induce apoptosis of target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).

4. The camptothecin derivative (a) or its prodrug (b) according to claim 1, characterized in that: The active camptothecin derivative (a) designed in such a way as to bind to the DDX5 protein and the E3 ligase has the ability to inhibit type 1 topoisomerase and also has a mechanism of action (MoA) of degrading the oncoprotein DDX5.

5. The camptothecin derivative (a) or its prodrug (b) according to claim 4, characterized in that: (1) In Formula 1 or Formula 2, optionally, the C group site binds to type 1 topoisomerase and the A group site binds to DNA to stabilize the covalent bonds of the topoisomerase-DNA complex to prevent the cleaved DNA fragments from rejoining, and / or, (2) In Formula 1 or Formula 2, the A group site binds to DDX5 and optionally, the C group site binds to E3 ligase to induce DDX5 degradation, 6. A pharmaceutical composition or cancer diagnostic composition for preventing or treating cancer, wherein: The invention comprises the active camptothecin derivative (a) according to any one of claims 1 to 5, the prodrug thereof (b), preferably the antibody-drug conjugate (ADC) thereof, or the complex (c) containing a ligand targeting DDX5 protein.

7. The pharmaceutical composition according to claim 6, characterized in that Used as a first-line treatment drug after cancer diagnosis or administered to individuals whose cancer tissues (over)express DDX5.

8. The pharmaceutical composition according to claim 6, characterized in that It is administered for the treatment of HER2-positive cancers, breast cancer, lung cancer, and colon cancer.

9. The pharmaceutical composition or cancer diagnostic composition according to claim 6, characterized in that: DDX5 protein was used as a biomarker to predict drug sensitivity or tumor sensitivity.

10. The pharmaceutical composition according to claim 6, characterized in that The DDX5 protein is a drug target for active camptothecin derivatives to avoid drug resistance, resistance to targeted therapy and / or resistance during treatment.

11. The pharmaceutical composition according to claim 6, characterized in that Transcriptional induction of anti-apoptotic genes is blocked (off) by active camptothecin derivatives.

12. The pharmaceutical composition according to claim 6, characterized in that Through active camptothecin derivatives, DDX5 protein, which acts as a transcriptional cofactor, is degraded to maintain or enhance the sensitivity of cancer cells to chemotherapy or radiotherapy.

13. The pharmaceutical composition according to claim 6, characterized in that Converting a solid tumor that is agglomerated due to high cell density into a dispersed cancer with low cell density and / or converting a cold tumor with low immune activity into a hot tumor with high immune activity.

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

15. A method for preparing: an active camptothecin derivative (a) designed in a manner to bind to a DDX5 protein and an E3 ligase; That The prodrug (b) is designed to release the active camptothecin derivative (a) at the target site in vivo, preferably an antibody-drug conjugate (ADC) thereof; or a complex (c) containing a ligand targeting DDX5 protein, which is connected by a linker to inactivate the type 1 topoisomerase inhibitory ability in the active camptothecin derivative (a) or the FL118 compound of formula 2, wherein the method is characterized in that it comprises the following steps: The active camptothecin derivative (a) is designed in such a way that it contains a camptothecin skeleton represented by the following formula 1 as a mother core, or a compound designed in this way is selected, or a compound designed in this way is synthesized, X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 form a hexagonal ring or a heptagonal ring, and n is a value of 1 to 2, Y1, Y2 and Y3 are each independently hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

16. The method for preparing the active camptothecin derivative, the prodrug thereof or the complex containing the DDX5 protein targeting ligand according to claim 15, characterized in that: The active camptothecin derivative (a) designed in such a way as to bind to the DDX5 protein and the E3 ligase has the ability to inhibit type 1 topoisomerase and also has a mechanism of action for degrading the oncoprotein DDX5.

17. The method for preparing the active camptothecin derivative, the prodrug thereof or the complex containing the DDX5 protein targeting ligand according to claim 15, characterized in that: In the step of designing the active camptothecin derivative (a) to include the camptothecin skeleton represented by Formula 1 as a core, or selecting the compound designed in this way, (1) selecting an active camptothecin derivative designed to have a mechanism of action (MoA) for inhibiting type 1 topoisomerase and / or a mechanism of action (MoA) for degrading oncoprotein DDX5 from a library of compounds comprising a camptothecin skeleton represented by Formula 1 as a core, and / or (2) Whether the compound comprising the camptothecin skeleton represented by Formula 1 as a core inhibits type 1 topoisomerase and / or degrades oncoprotein DDX5 is confirmed by in vitro experiments and / or in vivo experiments.

18. The method for preparing an active camptothecin derivative or a prodrug thereof according to claim 15, characterized in that: Active camptothecin derivatives are designed to be n , X3, Y1, Y2 and / or Y3 can be modified to adjust their hydrophobicity or cell membrane permeability as needed to exert a bystander effect in tumor tissue or control the extent of its effect.

19. The method for preparing an active camptothecin derivative or a prodrug thereof according to claim 15, characterized in that: The active camptothecin derivative (a) or its prodrug (b) is used for administration to a patient group in which DDX5 acts as an oncoprotein in cells.

20. The method for preparing the camptothecin derivative, the prodrug thereof or the complex containing the DDX5 protein targeting ligand according to claim 15, characterized in that: The method further comprises the following steps: in a tissue biopsy or liquid biopsy, using a complex (c) containing a ligand targeting DDX5 protein to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to be administered with the active camptothecin derivative (a) or its prodrug (b), wherein the complex (c) containing a ligand targeting DDX5 protein is connected to the FL118 compound of Formula 2 or the active camptothecin derivative (a) via a linker to inactivate the type 1 topoisomerase inhibitory ability.

21. A preparation method for preparing an active camptothecin derivative (a), which binds to DDX5, which acts as an oncoprotein in cells, and induces cell apoptosis through degradation of DDX5 protein; The prodrug (b) thereof is designed in a manner to release the active camptothecin derivative (a) in vivo, preferably an antibody-drug conjugate (ADC) thereof; or a complex (c) containing a ligand targeting DDX5 protein, wherein the type 1 topoisomerase inhibitory ability is inactivated in the active camptothecin derivative (a) by connecting with a linker, and the preparation method is characterized in that it comprises the following steps: The first step is to select the active camptothecin derivative (a) from the group consisting of the following compounds of Formula 3, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8, Formula 9 and isomers thereof; as well as Optionally, in a second step, a prodrug (b) thereof is selected, wherein the prodrug (b) is designed to release the active camptothecin derivative (a) selected in the first step in vivo, 22. The preparation method according to claim 21, characterized in that: The active camptothecin derivative (a) or its prodrug (b) is used for administration to a patient group in which DDX5 acts as an oncoprotein in cells.

23. The preparation method according to claim 21, characterized in that: The method further comprises the following steps: in a tissue biopsy or liquid biopsy, a complex (c) containing a ligand targeting DDX5 protein is used to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to be administered with the active camptothecin derivative (a) or its prodrug (b), wherein the complex (c) containing a ligand targeting DDX5 protein is inactivated in the FL118 compound of Formula 2 or the active camptothecin derivative (a) by a linker connection to inactivate the type 1 topoisomerase inhibitory ability.

24. A compound represented by the following Formula 3, Formula 3-1, Formula 4, Formula 4-1, Formula 5, Formula 5-1, Formula 6, Formula 7, Formula 8 or Formula 9, or a pharmaceutically acceptable salt thereof, 25. A pharmaceutical composition for preventing or treating cancer, wherein: A compound represented by the following 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 pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof, preferably an antibody-drug conjugate (ADC) thereof, 26. A pharmaceutical composition for killing cells, characterized in that: The invention comprises: an active camptothecin derivative (a) represented by the following formula 1, designed to bind to DDX5 protein and E3 ligase; or a prodrug (b) thereof, designed to release the active camptothecin derivative (a) at a target site in vivo, preferably an antibody-drug conjugate (ADC) thereof, X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 form a hexagonal ring or a heptagonal ring, and n is a value of 1 to 2, Y1, Y2 and Y3 are each independently hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

27. The pharmaceutical composition for killing cells according to claim 26, characterized in that: Active camptothecin derivatives (a) designed in a way to bind to DDX5 protein and E3 ligase induce apoptosis of target cells expressing DDX5 protein through a molecular glue degrader mechanism (MoA).

28. The pharmaceutical composition for killing cells according to claim 26, characterized in that: The active camptothecin derivative (a) designed in such a way as to bind to the DDX5 protein and the E3 ligase has the ability to inhibit type 1 topoisomerase and also has a mechanism of action (MoA) of degrading the oncoprotein DDX5.

29. The pharmaceutical composition for killing cells according to claim 26, characterized in that: Active camptothecin derivatives are designed to adjust their hydrophobicity or cell membrane permeability as needed by modifying X1, (X2)n, X3, Y1, Y2 and / or Y3, so as to exert a bystander effect or control the extent of its effect in tumor tissues.

30. The pharmaceutical composition for killing cells according to claim 27, characterized in that: The target cells are cancer cells or senescent cells.

31. An anticancer preparation, characterized in that The active camptothecin derivative (a) represented by the following formula 1 is designed to bind to the DDX5 protein and the E3 ligase; or its prodrug (b) is designed to release the active camptothecin derivative (a) at the target site in vivo, preferably its antibody-drug conjugate (ADC), which can be administered at a dose that stimulates antigen presenting cells (APCs) through apoptosis of cancer cells to induce anti-tumor immune response, X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 form a hexagonal ring or a heptagonal ring, and n is a value of 1 to 2, Y1, Y2 and Y3 are each independently hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

32. The anticancer preparation according to claim 31, characterized in that Anticancer agents are designed in such a way that they target tumor tissue.

33. A method for preparing an anticancer drug based on an active camptothecin derivative (a), wherein the bystander effect and / or ADME characteristics of the active camptothecin derivative (a) are controlled to prevent T cell exhaustion due to chronic antigen exposure, or to provide a desired degree of hydrophobicity and / or controlled aggregation for rapid or slow elimination by lymphatic drainage, characterized in that The anticancer drug based on the active camptothecin derivative (a) is an anticancer preparation comprising the following components: the active camptothecin derivative (a), as a molecular glue degrader, designed in a manner to bind to the DDX5 protein and the E3 ligase; or its prodrug (b), designed in a manner to release the active camptothecin derivative (a) at the target site in vivo, preferably its antibody-drug conjugate (ADC), The method comprises the following steps: designing an active camptothecin derivative (a) or a prodrug thereof (b) in a manner that it contains a camptothecin skeleton represented by the following formula 1 as a parent core, or selecting a compound designed in this manner, or synthesizing a compound designed in this manner, X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different, X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond, X1, (X2)n and X3 form a hexagonal ring or a heptagonal ring, and n is a value of 1 to 2, Y1, Y2 and Y3 are each independently hydrogen or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

34. The preparation method according to claim 33, characterized in that: The anticancer drug based on the active camptothecin derivative (a) is a preparation comprising the following components: the active camptothecin derivative (a), which is designed in a manner that it has the ability to inhibit type 1 topoisomerase and also has a mechanism of action (MoA) of decomposing the cancer protein DDX5; or its prodrug (b), which is designed in a manner that the active camptothecin derivative (a) is released in vivo.

35. A carrier-drug conjugate, wherein: The invention comprises the active camptothecin derivative represented by formula 1 as claimed in claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

36. The carrier-drug conjugate according to claim 35, characterized in that The active camptothecin derivative of Formula 1 is selected from the group consisting of Formula 3 to Formula 9, 37. The carrier-drug conjugate according to claim 35, characterized in that The carrier is an antibody, a peptide, a repebody, or a nucleic acid aptamer.

38. The carrier-drug conjugate according to claim 37, characterized in that The carrier-drug conjugate is in the form of a carrier conjugated to a camptothecin derivative represented by Formula 1 via a linker.

39. The carrier-drug conjugate according to claim 38, characterized in that The linker comprises GGFG.

40. The carrier-drug conjugate according to claim 37, characterized in that The vector is selected from 4-1BB, 5T4, integrin, activin, β-amyloid protein, angiopoietin, i.e., angiopoietin 1 or 2, angiopoietin-like protein 3, B cell maturation antigen, B cell activating factor, B7-H3, complement component 5, CCR4, CCR5, CCL11, CD2, CD3, CD4, CD6, CD11a, CD16A, CD19, CD20, CD22, CD25, CD27, CD 28. CD30, CD32B, CD33, CD38, CD40, CD45, CD46, CD47, CD52, CD56, CD62, CD70, CD73, CD74, CD79b, CD80, CD105, CD123, CD154, CD166, CD262, CD278, CD319, CD326, carcinoembryonic antigen, CGRP, Claudin-18, c-Met, CSF-1, CSF-1 receptor, CTLA4, DLL3, EGF receptor, hemophilin factor, Fc receptor, FGF23, folate receptor, GD2, glucocorticoid-induced tumor necrosis factor receptor, glypican 3, GM-CSF, HER2, HER3, TROP2, hepatocyte growth factor, interferon receptor, interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 8, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 23, interleukin 31 receptor, interleukin 36 receptor, lymphocyte-activation gene 3,LAG3), lysyl oxidase 2, mesothelin, Mucin-1, Mucin-16, Netin-4, nerve growth factor, OX40, proprotein convertase subtilisin 9, PD-1, PD-L1, phospholipase C, receptor activator of nuclear factors kappa B ligand, tyrosine-protein kinase transmembrane receptor, sialic acid-binding immunoglobulin-like lectin 15, transforming growth factor-β, T-cell innunoreceptor with immunoglobulin and ITIM domain, T-cell immunoglobulin and mucin domain-containing protein 3, tissue factor, tissue factor pathway inhibitor, TORP-2, tumor necrosis factor, thymic stromal lymphopoietin, colony stimulating factor 1 receptor, vascular endothelial growth factor, VEGF receptor and vWF (von Willebrand Factor). Factor), namely, an antibody, peptide, repebody, or nucleic acid aptamer that specifically binds to the antigen.

41. The carrier-drug conjugate according to claim 37, characterized in that The carrier is an antibody.

42. The carrier-drug conjugate according to claim 41, characterized in that The antibody is selected from the group consisting of Urelumab, Utomilumab, Bebtelovimab, Aducanumab, Bapinezumab, Crenezumab, Donanemab, Gantenerumab, Lecanemab, Solanezumab, Nesvacumab, Evinacumab, Enoblituzumab , Omburtamab, Belimumab, Ianalumab, Tabalumab, Bertilimumab, Mogamulizumab, Leronlimab, Siplizumab, Foralumab, Muromonab-CD3, Otelixizumab, Teplizumab, Ibalizumab, Qulizumab Tregalizumab, Zanolimumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Tositumomab, Ocrelizumab, Ofatumumab, Rituximab, Ublituximab, Veltuzumab, Epratuzumab, Basiliximab siliximab), Daclizumab, Varlilumab, Lulizumab, Iratumumab, Lintuzumab, Daratumumab, Felzartamab, Isatuximab, Mezagitamab, Bleselumab, Dacetuzumab, Iscalimab, Lucatumumab,Mitazalimab, Sotigalimab, Dapirolizumab, Apamistamab, Ligufalimab, Magrolimab, Alemtuzumab, Crizanlizumab, Inclacumab, Cusatuzumab, Oleclumab, Milatuzumab, Galiximab , Carotuximab, Adecatumumab, Eptinezumab, Erenumab, Fremanezumab, Galcanezumab, Zolbetuximab, Onartuzumab, Eculizumab, Pozelimab, Ravulizumab, Lacnotuzumab, Axatilimab b), Cabiralizumab, Emactuzumab, Ipilimumab, Quavonlimab, Tremelimumab, Zaliferlimab, Cetuximab, Depatuxizumab, Futuximab, Imgatuzumab, Matuzumab, Modotuximab, Necitumumab, Nimotuzumab Nimotuzumab, Panitumumab, Tomuzotuximab, Zalutumumab, Batoclimab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Dinutuximab, Naxitamab, Ragifilimab, Gimsilumab,Lenzilumab, Mavrilimumab, Namilumab, Otilimab, Plonmarlimab, Codrituzumab, Margetuximab, Pertuzumab, Trastuzumab, Datopotamab, Patritumab, Seribantumab, Duligotuzumab ), Ficlatuzumab, Rilotumumab, Alomfilimab, Anifrolumab, Emapalumab, Ligelizumab, Omalizumab, Cixutumumab, Dalotuzumab, Figitumumab, Ganitumab, Teprotumumab, Bermekimab, Card Canakinumab, Gevokizumab, Briakinumab, Ustekinumab, Anrukinzumab, Cendakimab, Lebrikizumab, Tralokinumab, Brodalumab, Bimekizumab, Ixekizumab, Secukinumab, Brazikumab, Gusekizumab Guselkumab, Mirikizumab, Risankizumab, Tildrakizumab, Nemolizumab, Imsidolimab, Spesolimab, Pascolizumab, Dupilumab, Depemokimab, Mepolizumab, Reslizumab, Benralizumab,Clazakizumab, Olokizumab, Siltuximab, Sirukumab, Ziltivekimab, Levilimab, Sarilumab, Satralizumab, Tocilizumab, Abituzumab, Favezelimab, Fianlimab, Ieramilimab, Relalimab Relatlimab, Simtuzumab, Abagovomab, Oregovomab, Tanezumab, Ivuxolimab, Rocatinlimab, Tavolimab, Telazorlimab, Vonlerolizumab, Alirocumab, Bococizumab, Ebronucimab, Elocumab Evolocumab, Frovocimab, Ongericimab, Tafolecimab, Dostarlimab, Balstilimab, Camrelizumab, Cemiplimab, Geptanolimab, Nivolumab, Pembrolizumab, Penpulimab, Pidilizumab, Palolimab Prolgolimab, Retifanlimab, Sasanlimab, Serplulimab, Sintilimab, Spartalizumab, Tislelizumab, Toripalimab, Ezabenlimab, Zimberelimab, Atezolizumab, Avelumab, Cosibelimab,Sugemalimab, Durvalumab, Envafolimab, Suvratoxumab, Denosumab, Zilovertamab, Elotuzumab, Domvanalimab, Etigilimab, Ociperlimab, Tiragolumab, Vibostolimab, Surzebiclimab, Coberli Cobolimab, Sabatolimab, Concizumab, Marstacimab, Adalimumab, Golimumab, Infliximab, Certolizumab, Conatumumab, Tigatuzumab, Tezepelumab, Gatipotuzumab, Cabiralizumab, Bevacizumab cizumab), Brolucizumab, Ranibizumab, Olinvacimab, Icrucumab, Ramucirumab, Caplacizumab, Abrilumab, Etrolizumab, Vedolizumab, Intetumumab, Natalizumab, Obrindatamab, Elranatama b), Linvoseltamab, Teclistamab, Epcoritamab, Glofitamab, Mosunetuzumab, Odronextamab, Flotetuzumab, Vibecotamab, Catumaxomab, Cibisatamab, Talquetamab, Ubamatamab, Emfizatamab,Blinatumomab, Amivantamab, Emicizumab, Zenocutuzumab, Zanidatamab, Tibulizumab, Naptumomab, Belantamab, Pivekimab, Praluzatamab, Coltuximab mab), Denintuzumab, Loncastuximab, Ibritumomab, Inotuzumab, Epratuzumab, Moxetumomab, Brentuximab, Gemtuzumab, Vadastuximab, Lorvotuzumab, Vepotuzumab Polatuzumab, Tusamitamab, Telisotuzumab, Rovalpituzumab, Depatuxizumab, Farletuzumab, Mirvetuximab, Disitamab, Anetumab, Enfortumab, Sacituzumab ), Vobarilizumab, Cadonilimab, Vudalimab, Tebotelimab, Ivonescimab, Erfonrilimab, Ozoralizumab, Faricimab, Vanucizumab and Navicixizumab.

43. A pharmaceutical composition for preventing or treating cancer, wherein: Comprising the carrier-drug conjugate according to any one of claims 35 to 42.

44. The pharmaceutical composition according to claim 43, characterized in that The cancer is selected from pseudomyxoma, intrahepatic bile duct 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, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, brain cancer pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampullary cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer , heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid, gastrointestinal stromal cancer, Wilms tumor, breast cancer, triple negative breast cancer (TNBC), sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, blood cancer, and thymic cancer.

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