Camptothecin derivative combined with DDX5 protein and prodrug thereof

Abstract

The present invention relates to: (a) an active camptothecin derivative represented by Chemical Formula 1 designed to bind to a DDX5 protein and an E3 ligase; (b) a prodrug of an active camptothecin derivative designed to release the active camptothecin derivative (a) at a target site in vivo; or (c) a complex containing a ligand targeting DDX5 protein, wherein the active camptothecin derivative (a) or the FL118 compound of the chemical formula 2 is linked through a linking group, so that the capability of inhibiting I-type topoisomerase is inactivated. The active camptothecin derivative designed according to the invention can be combined with DDX5 protein in cells, and cell death can be induced through the decomposition of the DDX5 protein.

Description

Technical Field

[0001] The present invention relates to: (a) an active camptothecin derivative represented by the following chemical formula 1, which is designed to bind to the DDX5 protein and the E3 ligase; (b) a prodrug of the active camptothecin derivative, which is designed to release the above-mentioned active camptothecin derivative (a) at a target site in vivo; or (c) a complex containing a ligand targeting the DDX5 protein, which is connected to the above-mentioned active camptothecin derivative (a) or the FL118 compound of chemical formula 2 through a connecting group to inactivate the ability to inhibit type I topoisomerase.

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

[0003] Cancer regulatory genes are genes that originally exist to regulate the normal functions of cells, but may cause cancer when they mutate or are regulated abnormally. One of the normal functions of cells is the cell-killing function, and there are cell-killing promoting factors and cell-killing inhibiting factors.

[0004] For example, cancer regulatory 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.29 As shown, cancer may occur if programmed cell death (eg, apoptotic agents) mechanisms are impaired.

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

[0007] Viral oncogenes encode proteins that resemble cellular proteins with important regulatory functions. Such cellular homologs are called proto-oncogenes or normal cellular oncogenes. Mutations in proto-oncogenes can actively promote cell proliferation. Mutated cH-ras proteins have mutations that impair their ability to hydrolyze GTP. This allows the mutant protein to remain in active signaling mode and stimulate cell division. Mutated versions of c-ras are found in many types of tumors.

[0008] A single mutation does not usually cause cancer. Generally, various genes that regulate cell growth mutate before cancer develops.

[0009] HER2-positive breast cancer, in which HER2 is abnormally overexpressed in breast epithelial cells, accounts for about 1 / 4 of all breast cancer patients. Compared with female hormone-related breast cancer, the recurrence rate of chemotherapy anticancer drugs is high and the prognosis is poor. The HER2-targeted therapeutic agent Herceptin (ingredient name: Trastuzumab) treats breast cancer by reducing the amount of HER2 on the cell surface and helping immune cells kill HER2-positive breast cancer cells.

[0010] HER2-positive breast cancer is breast cancer with amplification or overexpression of the ErbB receptor HER2 (ErbB2) cancer gene. Trastuzumab (Herceptin) increases the therapeutic effect of HER2-positive breast cancer, but HER2-positive breast cancer is more aggressive than other breast cancers, and more than half of patients are resistant to existing targeted therapies or develop resistance during treatment.

[0011] The ligands of ErbB receptors attached to the cell membrane include NRG1 and HB-EGF. The activated ErbB ligands bind to their receptors (EGFR, HER3), catalyzing the phosphorylation (phosphorylation, P) caused by the binding between receptors (EGFR-HER2, HER2-HER3), inducing intracellular growth and proliferation signals. This excessive activation of ErbB receptors can lead to resistance to anti-HER2 therapeutic drugs.

[0012] On the other hand, unregulated transcription factors can reduce the sensitivity of cancer cells to chemotherapy or radiotherapy. In cancer treatment, drug resistance can be intrinsic or acquired. In cancer treatment, intrinsic drug resistance may be caused by abnormal expression of transcription factors that are critical regulators of cell proliferation and cell death. Abnormal overexpression of NF-κB, STAT3, HIF-1, AR or ER and inactivation of p53 and FOXO3a in tumor tissues protect cancer cells from killing by anticancer drugs, showing intrinsic drug resistance.

[0013] However, chemotherapy or radiotherapy also induces acquired drug resistance through the activation of unregulated transcription factors. It has been found that ionizing radiation can stimulate the activation and induction of multiple transcription factors including STAT3, NF-κB and HIF-1. Upregulation of NF-κB and STAT3 can induce the transcription of anti-apoptotic genes in cancer cells, promote cancer cells to enter the cell cycle and proliferation, and produce radiation resistance. In addition, radiation-regulated transcription factors NF-κB and HIF-1 can induce EMT cells that are resistant to cancer treatment and genes responsible for maintaining cancer stem cells. In addition, activation of the transcription factor STAT3 also promotes resistance to cancer treatment. Studies have found that feedback activation of STAT3 by chemotherapy or radiotherapy is a molecular mechanism of cancer treatment resistance. Therefore, targeting unregulated transcription factors and their main targets together can be a promising approach to overcome resistance in cancer treatment.

[0014] The STAT (abbreviation for signal transducer and activator of transcription) transcription factor family includes STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6. STAT proteins are growth factors, cytokines, interferons or oncogenes, which can be activated by JAK kinases when stimulated to regulate interferon signal transduction. The DNA binding domain of STAT is an immunoglobulin-like structure that mediates binding to specific DNA target sequences. During activation, STAT3 phosphorylation caused by JAK kinase activates dimerization. Then, the dimer is transferred to the nuclear region, binds to the sequence of the targeted promoter, and induces the expression of the target gene. STAT3 target genes include Survivin, bcl-xl, mcl-1, Cyclin D1, MMP2, MMP9, VEGF, Myc, Sox2, etc. STAT proteins regulate many biological processes, including cell growth, cell death, differentiation and immunity. Within the STAT family, STAT3 and STAT5 are involved in the development of cancer.

[0015] Unregulated STAT3 and activated STAT5 are considered oncogenes that increase angiogenesis and improve cancer cell survival. In studies using many samples from the cBioPortal database, it was found that STAT3 was more often overexpressed in lung cancer, ovarian cancer, gastric cancer, hematopoietic cancer, and brain cancer compared with normal tissues, and overexpression of STAT3 was associated with poor overall survival. JAK kinase-mediated STAT3 phosphorylation is the first step in STAT3 activation. Another study of 90 glioblastoma patients found that high p-STAT3 levels were significantly associated with decreased progression-free survival and overall survival. Multivariate survival analysis showed that high p-STAT3 levels may play an important prognostic role in poor progression-free survival and overall survival.

[0016] Type I topoisomerase (Topoisomerase I) inhibitors (Irinotecan, Topotecan, etc.) are clinically proven anti-cancer mechanisms with proven efficacy and safety. They have been clinically proven to have excellent anti-cancer efficacy in a variety of refractory solid cancers such as colorectal cancer, lung cancer, breast cancer, and ovarian cancer.

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

[0018] Camptothecin (CPT) has low solubility in water, and in preparation for clinical trials, the National Cancer Institute (NCI) prepared a water-soluble sodium salt (NSC100880). Phase I and Phase II clinical trials were not completed due to the high toxicity of the above compounds (hemorrhagic cystitis, gastrointestinal toxicity such as nausea, vomiting, diarrhea, bone marrow suppression, especially leukopenia and low platelet disease).

[0019] Subsequently, many CPT homologs were synthesized to obtain less toxic and more soluble compounds. There are two drugs, irinotecan (CPT-11) and topotecan.

[0020] Irinotecan (CPT-11), developed by Dallchi and Yakult of Japan, was the world's first camptothecin anticancer agent in 1994. It was proven effective in lung cancer (small cell and non-small cell lung cancer) and was marketed in Europe and Japan. In 1995, it was further proven effective in colorectal cancer and breast cancer. In addition, topotecan, developed by GlaxoSmith Kline, was approved by the U.S. FDA for its effectiveness against metastatic ovarian cancer and marketed in April 1995. The new camptothecin derivative CKD-602 (belotecan) recently developed in China, in addition to having a strong I-type topoisomerase inhibitor effect, has also successfully overcome the toxicity caused by the existing poor solubility as a water-soluble drug.

[0021] So far, all confirmed camptothecin derivatives contain a five-ring hair structure that is essential for cytotoxicity ( Figure 1 ). From the perspective of molecular structure, the E-ring and the A- and B-ring sites were identified as important sites. Camptothecin includes an α-hydroxylactone structure or a 6-membered lactone structure in the E-ring necessary for cytotoxicity. The lactone group and α-hydroxyl group located on carbon number 20 of the double E-ring are essential for the stability of the type I topoisomerase-DNA byproduct, and it has been shown that deformation of the A- and B-rings can increase water solubility and activity. Changes on the first ring have been shown, for example, in the case of the above-mentioned drug, to increase solubility in water and allow for greater acceptability.

[0022] CKD-602 also tried substitution of the B ring of carbon 7 to increase its water solubility and anticancer effect. Lee et al. stated that CKD-602 has better anticancer effects than camptothecin and topotecan in a wide range of cancer cell lines. In addition, the maximum tolerated dose (MTD) was confirmed to be 25 mg / kg in the L1210 leukemia nude mouse model, which is a relatively safe drug. The side effects of camptothecin drugs generally known can be roughly divided into hematological side effects and non-hematological side effects. Hematological side effects include fever with neutropenia, sepsis, bleeding, etc. Non-hematological side effects include skin system side effects such as nausea, vomiting, and hair loss, as well as toxicity to the gastrointestinal tract, kidneys, and nervous systems. In animal studies of CKD-602, Kim et al. found that among the side effects of these camptothecin drugs, even if a high dose of the drug was administered that was more than 10 times the clinically applicable dose, no abnormal drug reactions were found except for increased gastric secretion. In addition, recent clinical studies in South Korea have also proven that, compared with severe systemic toxicity, only reversible and adjustable side effects such as neutropenia and leukopenia have been reported, which shows good stability. However, so far, it has been limited to patients who have failed standard chemotherapy or cannot receive standard chemotherapy, i.e., patients who have relapsed or worsened after standard treatment and are difficult to receive anticancer chemotherapy, and patients who believe that surgery is difficult to achieve results, such as refractory or recurrent ovarian cancer and colorectal cancer, refractory or recurrent limited-stage small-cell lung cancer, and extensive-stage small-cell lung cancer, which have failed one chemotherapy.

[0023] SN-38 has an IC in the nanomolar range in multiple cell lines 50 It is a powerful topoisomerase-I inhibitor with high value. It is the active form of the prodrug irinotecan used to treat colorectal cancer, and also shows activity in lung cancer, breast cancer and brain cancer. Taking Trop-2-SN-38ADC as an example, it has been successfully developed in most cancers such as TNBC, bladder cancer, and gastric cancer, but there are still drug resistance problems such as overexpression of drug exux transporter, epigenetic silencing of topoisomerase-I, and increase of anti-apoptotic protein.

[0024] In addition, Daiichi Sankyo The development of Enhertu used the cytotoxic drug DXD (exatecan), which is about 10 times more active than SN-38 in cancer cells. DXD has good solubility, is relatively safe, has a high killing effect on surrounding cells, and is beneficial for the treatment of heterogeneous tumors. However, the half-life, which can reduce non-target effects, is very short. DXD was conjugated to the cysteine ​​residue of the anti-HER2 antibody with a maleimide linker group, and the homogeneous DAR value reached 8. Despite the high DAR value, DXD was only released from plasma by 2.1% within 21 days, and its stability was very high (Ogitani et al., 2016). Enhertu was approved by the US FDA in 2019, and the target patients are adult patients with unresectable metastatic Her2-positive breast cancer who have received more than 2 HER2-targeted therapies in the past.

[0025] Camptothecin derivatives such as SN-38 and Dxd (a derivative of the clinical-stage topoisomerase I inhibitor Exatecan) have recently been successfully developed as new payloads for antibody-drug conjugates (ADCs). As a result, two ADCs using camptothecin payloads, including Trodelvy (Sacituzumab Govitecan) and Enhertu (Trastuzumab Deruxtecan) with SN-38 payloads, have recently been approved by the FDA. Compared with existing ADCs with ultra-toxic payloads such as MMAE and MMAF (DAR = 2 or 4), this camptothecin payload has a moderate degree of cytotoxicity, but due to its excellent safety profile, a higher drug-to-antibody ratio (DAR) such as 8 can be used. In addition, with these camptothecin payloads, linker systems with faster drug release profiles, such as CL-2A or GGFG linker systems, can be used. Through the combination of these components, new ADCs with a wider therapeutic range have been developed.

[0026] In short, since camptothecin compounds acted as topoisomerase I inhibitors and their potential as anticancer drugs was confirmed, many compounds with similar structures have been synthesized and studied. Among them, since topotecan was approved by the FDA in 1996 and irinotecan in 2002, although many compounds have been synthesized, they were in a state of losing development momentum due to poor effects. However, in 2019, Enhertu, an ADC with a new camptothecin derivative Dxd as a payload, and Trodelvy, an ADC with an in vivo active metabolite of irinotecan SN-38 as a payload, were approved by the FDA in 2020, which has revived the vitality of research.

[0027] Antibody-drug conjugates (ADCs) such as Trodelvy and Enhertu that use camptothecin derivatives as payloads can only selectively deliver camptothecin compounds with strong anticancer effects to cancer cells, and can successfully bring excellent anticancer effects without serious systemic side effects, but there are still many shortcomings, the most representative of which is that overexpression of ABCG2 Drug Efflux Pump will produce resistance. Many camptothecin derivatives are rapidly excreted from cells through ABCG2, so compared with cancer cells that do not express ABCG2 or express it at a low level to normal, cancer cells that overexpress ABCG2 have a problem of severely reduced efficacy. This overexpression of ABCG2 is not only the main reason for reduced efficacy in small molecule camptothecin compounds such as irinotecan and topotecan, but also in ADCs such as Trodelvy and Enhertu that use camptothecin compounds as payloads.

[0028] In addition, Enhertu's DXd has a higher membrane permeability than Kadcyla's DM1, and the results also reported that the payload released from the cell interior is delivered to cells that do not express HER2 adjacent to the target cells. This may make Enhertu have a potential bystander effect due to the characteristics of the payload and provide a clinical advantage for patients who are refractory to Herceptin or Kadcyla. Summary of the invention

[0029] Technical issues

[0030] The present invention provides a camptothecin derivative, which is a drug candidate for killing target cells and is designed to bind to DDX5 protein and E3 ligase.

[0031] The inventors synthesized a dual MoA camptothecin drug FL118 compound with excellent ability to inhibit type I topoisomerase and decompose tumor protein (oncoprotein) DDX5 (p68) Figure 2 ) and continue to conduct various studies on its physical and chemical properties. However, due to the limitations of its physical and chemical properties, the formulation development of FL118 compound has limitations.

[0032] Therefore, based on the structure of FL118 which is different from SN38 drug, the inventors aimed to design and synthesize a novel camptothecin derivative with a dual mechanism of action (dual MoA) in terms of type I topoisomerase inhibition and DDX5 degradation, which are the advantages of FL118.

[0033] Therefore, the present invention aims to redesign and provide active camptothecin derivatives having the dual mechanism of action (dual MoA) of inhibiting type I topoisomerase and decomposing oncoprotein DDX5 and / or their prodrug antibody-drug conjugates (ADCs) designed to release the above active camptothecin derivatives in vivo.

[0034] Furthermore, in the present invention, active camptothecin derivatives not only achieve a bystander effect, but also bind to DDX5 protein in cells to induce cell death through DDX5 protein degradation, thereby providing a novel mode of anticancer mechanism that causes cancer cells and / or surrounding cells to die before intrinsic or acquired drug resistance.

[0035] Solutions to the problem

[0036] The first embodiment of the present invention provides (a) an active camptothecin derivative represented by the following chemical formula 1, which is designed to bind to the DDX5 protein and the E3 ligase; or (b) a prodrug of an active camptothecin derivative, which is designed to release the above-mentioned active camptothecin derivative (a) at a target site in vivo; or (c) a complex containing a ligand targeting the DDX5 protein, which is connected to the above-mentioned active camptothecin derivative (a) or the FL118 compound of chemical formula 2 through a linking group to inactivate the ability to inhibit type I topoisomerase.

[0037] [Chemical formula 1]

[0038]

[0039] X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different,

[0040] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0041] X1, (X2)n and X3 form a 6- or 7-corner ring, n = 1 to 2,

[0042] Y1, Y2 and Y3 are each independently hydrogen, or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0043] In the complex containing the ligand targeting the DDX5 protein, the ligand targeting the DDX5 protein may be a ligand that binds to the DDX5 protein.

[0044] Active camptothecin derivatives can be designed as in chemical formula 1, by X1, (X2) n , X3, Y1, Y2 and / or Y3 are deformed to adjust the cell membrane permeability as needed, exert a bystander effect in cancer tissue or control the extent of its effect.

[0045] Preferably, the active camptothecin derivative (a) designed to bind to the DDX5 protein and the E3 ligase plays a ligand (binder) role by binding to the E3 ligase through a molecular glue degrader; and / or causes the death of target cells expressing the DDX5 protein through a molecular glue degrader mechanism (MoA); and / or has the ability to inhibit type I topoisomerase and the mechanism of action (MoA) of decomposing the tumor protein (oncoprotein) DDX5.

[0046] The second aspect of the present invention provides a pharmaceutical composition or cancer diagnostic composition for preventing or treating cancer, comprising the active camptothecin derivative (a) of the first aspect, its prodrug (b) or a complex (c) containing a ligand targeting the DDX5 protein.

[0047] For example, it is administered to treat HER2-positive tumors, breast cancer, lung cancer, and colorectal cancer.

[0048] Preferably, the pharmaceutical composition for preventing or treating cancer is used as a one-time therapeutic agent after cancer diagnosis, or is administered to an individual who (over)expresses DDX5 in cancer tissue.

[0049] The third embodiment of the present invention provides a method for preparing (a) an active camptothecin derivative, (b) a prodrug thereof, or (c) a complex containing a ligand targeting a DDX5 protein, wherein the active camptothecin derivative (a) is designed to bind to a DDX5 protein and an E3 ligase, the prodrug (b) of the active camptothecin derivative is designed to release the active camptothecin derivative (a) at a target site in vivo, and the complex (c) containing a ligand targeting a DDX5 protein is connected to the active camptothecin derivative (a) or the FL118 compound of Chemical Formula 2 through a linking group to inactivate the ability to inhibit type I topoisomerase. The preparation method comprises the steps of designing the active camptothecin derivative (a) to contain a camptothecin skeleton represented by the above Chemical Formula 1 as a parent core, or selecting a compound designed in the above manner, or synthesizing a compound designed in the above manner.

[0050] For example, the step of designing the active camptothecin derivative (a) to include the camptothecin skeleton represented by Chemical Formula 1 as a mother core, or selecting the compound designed in the above manner may be the following step,

[0051] (1) selecting active camptothecin derivatives designed to have a mechanism of action (MoA) for inhibiting type I topoisomerase and / or a mechanism of action (MoA) for decomposing oncoprotein DDX5 from a compound library containing a camptothecin skeleton represented by Chemical Formula 1 as a parent core, and / or

[0052] (2) Confirming whether the compound comprising the camptothecin skeleton represented by Chemical Formula 1 as a core inhibits type I topoisomerase and / or degrades tumor protein DDX5 by in vitro experiments and / or in vivo experiments.

[0053] A fourth aspect of the present invention provides a method for preparing (a) an active camptothecin derivative, (b) a prodrug thereof, or (c) a complex containing a ligand targeting a DDX5 protein, wherein the active camptothecin derivative (a) binds to DDX5, which works as an oncoprotein, in a cell and induces cell death by decomposing the DDX5 protein. death), a prodrug (b) of an active camptothecin derivative is designed to release the above active camptothecin derivative (a) in vivo, the above complex (c) containing a ligand targeting DDX5 protein is connected in the above active camptothecin derivative (a) through a connecting group to inactivate the ability to inhibit type I topoisomerase, and the above preparation method comprises: a first step, selecting the above active camptothecin derivative (a) from a combination consisting of the following chemical formula 3, the compound of chemical formula 4, the compound of chemical formula 5, the compound of chemical formula 6, the compound of chemical formula 7, the compound of chemical formula 8, the compound of chemical formula 9 and their isomers; and a second step, arbitrarily selecting a prodrug (b) of an active camptothecin derivative designed to release the above active camptothecin derivative (a) selected in the first step in vivo.

[0054] [Chemical formula 3]

[0055]

[0056] [Chemical formula 4]

[0057]

[0058] [Chemical formula 5]

[0059]

[0060] [Chemical formula 6]

[0061]

[0062] [Chemical formula 7]

[0063]

[0064] [Chemical formula 8]

[0065]

[0066] [Chemical formula 9]

[0067]

[0068] The preparation method of the third embodiment or the fourth embodiment further comprises the step of quantitatively or qualitatively analyzing intracellular DDX5 protein in a tissue biopsy or a liquid biopsy using a complex (c) containing a ligand targeting DDX5 protein inactivated by a non-cleaving linking group in the FL118 compound of Chemical Formula 2 or the above-mentioned active camptothecin derivative (a) to confirm a patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered, by linking the compound to inactivate the ability to inhibit type I topoisomerase.

[0069] A fifth aspect of the present invention provides a compound represented by the following Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 or a pharmaceutically acceptable salt thereof.

[0070] The sixth embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer containing a compound represented by the following Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof or a prodrug thereof.

[0071] [Chemical formula 3]

[0072]

[0073] [Chemical formula 3-1]

[0074]

[0075] [Chemical formula 4]

[0076]

[0077] [Chemical formula 4-1]

[0078]

[0079] [Chemical formula 5]

[0080]

[0081] [Chemical formula 5-1]

[0082]

[0083] [Chemical formula 6]

[0084]

[0085] [Chemical formula 7]

[0086]

[0087] [Chemical formula 8]

[0088]

[0089] [Chemical formula 9]

[0090]

[0091] The seventh aspect of the present invention provides a pharmaceutical composition for cell death, which contains (a) an active camptothecin derivative represented by the above chemical formula 1, which is designed to bind to the DDX5 protein and the E3 ligase; or (b) a prodrug of the active camptothecin derivative, which is designed to release the above active camptothecin derivative (a) at a target site in vivo.

[0092] The eighth embodiment of the present invention provides an anticancer dosage form, which administers (a) an active camptothecin derivative represented by the above chemical formula 1, or (b) a prodrug thereof, in an amount (dosage) that induces an anti-tumor immune response by stimulating antigen presenting cells (APCs) through cell death of cancer cells, wherein the active camptothecin derivative (a) is designed to bind to the DDX5 protein and the E3 ligase, and the prodrug of the active camptothecin derivative (b) is designed to release the above active camptothecin derivative (a) at a target site in vivo.

[0093] A ninth aspect of the present invention provides a method for preparing an anticancer agent based on an active camptothecin derivative (a), controlling the bystander effect and / or ADME curve of the active camptothecin derivative (a) to prevent the occurrence of T cell exhaustion caused by chronic antigen exposure, characterized in that the anticancer agent based on the active camptothecin derivative (a) is an anticancer dosage form containing (a) an active camptothecin derivative, or (b) a prodrug thereof, wherein the active camptothecin derivative (a) is designed to bind to the DDX5 protein and the E3 ligase through a molecular glue degrader, and the prodrug (b) of the active camptothecin derivative is designed to release the active camptothecin derivative (a) at a target site in vivo, comprising: designing the active camptothecin derivative (a) or its prodrug (b) to contain the camptothecin skeleton represented by the above chemical formula 1 as a mother core, or selecting a compound designed in the above manner, or synthesizing a compound designed in the above manner.

[0094] Hereinafter, the present invention will be described.

[0095] In this specification, a drug is any substance (except food or device) used to diagnose, cure, alleviate, treat or prevent a disease or affect the structure or function of the body. For example, any chemical or biological substance that affects the body and its metabolism. Preferably, as long as it binds to the DDX5 protein, it belongs to the category of drug.

[0096] For example, the drug of the present invention can be an active camptothecin derivative itself or a prodrug thereof, an active camptothecin derivative as an active payload of a prodrug for targeted drug delivery (e.g., carrier-drug conjugate), or a camptothecin derivative or a prodrug thereof as a diagnostic agent.

[0097] In order for the drug to work effectively in vivo, the drug concentration at the target site in the body must be maintained within the therapeutic range for a period of time (Example 8, Table 6, Fig.23 Too much drug in the body will be toxic, and too little in the target area will have no therapeutic effect.

[0098] Drug efficacy is defined as the ability of a drug to remain effective for the target indication in the body for the expected time without being degraded (Example 8, Table 6, Fig.23 ). The lower the metabolic rate, the longer the drug remains in the blood, so the longer the duration of its effectiveness.

[0099] In addition, the in vivo efficacy and in vivo side effects of anticancer agents are closely related to the absorption, distribution, metabolism and excretion (ADME) properties of anticancer agents. The ADME of a drug determines the pharmacokinetics that describe how the drug moves through the body and interacts with tissues and organs.

[0100] Drug absorption affects efficacy and side effects. Poorly absorbed drugs may not reach therapeutic concentrations in target tissues, resulting in decreased efficacy. Conversely, well-absorbed drugs increase systemic exposure and produce non-target side effects.

[0101] 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 cause toxicity in those tissues. In addition, drugs that bind strongly to plasma proteins may reduce distribution to target tissues, thereby reducing efficacy.

[0102] In vivo drug distribution can be influenced by physicochemical properties including size, charge, and lipophilicity. The ability of a drug to penetrate tumor tissue can be affected by factors such as the tumor microenvironment including blood flow and cell density. Some anticancer agents accumulate in specific tissues and induce toxic effects.

[0103] The metabolism of drugs also affects efficacy and side effects. Rapidly metabolized drugs may reduce 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 certain drugs and can cause side effects.

[0104] The rate and route of excretion can also affect the efficacy and side effects of a drug. Rapidly cleared drugs have short half-lives and may have reduced efficacy, while slowly cleared drugs can accumulate and cause toxicity. In addition, some drugs may be excreted unchanged or as active metabolites, resulting in prolonged or increased exposure and an increased risk of side effects.

[0105] Therefore, it is imperative to analyze the ADME properties of anticancer agents to optimize their efficacy in vivo and minimize side effects. That is, evaluating and optimizing ADME profiles can improve the therapeutic index of these drugs and lead to better outcomes for cancer patients.

[0106] An anti-cancer formulation or anti-cancer composition refers to a medicating or therapy designed to treat cancer or prevent the growth and spread of cancer cells in the body. Anti-cancer formulations may include a combination of drugs, chemical compounds, and / or biologics that target cancer cells by interfering with their ability to grow and divide.

[0107] The formulation of an anti-cancer drug may reflect a complex process that includes multiple stages of 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.

[0108] The development of anticancer formulations involves designing drugs or drug combinations that recognize and selectively target cancer cells while activating healthy cells. 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.

[0109] If a promising drug candidate is identified, the drug will be tested in the laboratory and in animal models and undergo preclinical development for safety and efficacy assessment. If the drug shows promise in preclinical studies, it can proceed through clinical trials tested in humans to determine safety, dosage, and efficacy.

[0110] In clinical trials, drug formulations are optimized to be safely administered to patients and to achieve the desired therapeutic effect. This may include adjusting the drug's dose, route of administration, or formulation to improve efficacy and minimize side effects.

[0111] The present invention provides, as a drug cell that causes target cell death, (a) an active camptothecin derivative represented by the following chemical formula 1, which is designed to bind to the DDX5 protein and the E3 ligase; or (b) a prodrug of the active camptothecin derivative, which is designed to release the above-mentioned active camptothecin derivative (a) at the target site in vivo.

[0112] [Chemical formula 1]

[0113]

[0114] X1 and X3 are each independently carbon, oxygen, nitrogen or sulfur, X1 and X3 are the same or different,

[0115] X2 is carbon, oxygen, nitrogen, sulfur, a single bond or a double bond,

[0116] X1, (X2)n and X3 form a 6- or 7-corner ring, n = 1 to 2,

[0117] Y1, Y2 and Y3 are each independently hydrogen, or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

[0118] Non-limiting examples of functional groups containing oxygen, nitrogen, phosphorus or sulfur may include -CHO, -COOH, -NH2, -SH, -CONH2, -PO3H, -PO4H2, -OPO4H, -PO2(OR 1 )(OR 2 )(R 1 , R 2 =C s H t N u O w S x P y X z, X = -F, -Cl, -Br or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -SO3H, -OSO3H, -NO2, -N3, -NR3OH (R = C n H 2n+1 , 0 ≤ n ≤ 16), -NR3 + X - (R = C n H m , 0 ≤ n ≤ 16, 0 ≤ m ≤ 34, X = OH, Cl or Br), NR4 + X - (R = C n H m , 0 ≤ n ≤ 16, 0 ≤ m ≤ 34, X = OH, Cl or Br), -COSH, -COOCO-, -CORCO- (R = C l H m , 0 ≤ l ≤ 3, 0 ≤ m ≤ 2l + 1), -COOR, -CN, -N3, -N2, -NROH (R = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -NR 1 NR 2 R 3 (R 1 , R 2 , R 3 = C s H t N u O w S x P y X z , X = -F, -Cl, -Br or -I, 0 ≤ s ≤ 20, 0 ≤ t ≤ 2(s + u) + 1, 0 ≤ u ≤ 2s, 0 ≤ w ≤ 2s, 0 ≤ x ≤ 2s, 0 ≤ y ≤ 2s, 0 ≤ z ≤ 2s), -CONHNR 1 R 2 (R 1 , R 2 = C s H t N u O w S x P y Xz , 1 R 2 R 3 X'(R 1 , R 2 , R 3 =C s H t N u O w S x P y X z ,X=-F,-Cl,-Br or-I,X'=F - , Cl - ,Br - , or I-, 0≤s≤20, 0≤t≤2(s+u)+1, 0≤u≤2s, 0≤w≤2s, 0≤x≤2s, 0≤y≤2s, 0≤z≤2s), -OH, -O-, >C=O, -SS-, -SO-, -NO2, -COX (X=F, Cl, Br or I), -COOCO-, -CONH-, -CN, -SCOCH3, -SCN, -NCS, -NCO, -OCN, -CN, -F, -Cl, -I, -Br, epoxy, -hydrazone, -ONO2, -PO(OH)2, -C=NNH2, -HC=CH-, -C=C-, -C≡C- and a functional group consisting of a hydrocarbon having 2 or more carbon atoms.

[0119] According to the present invention, the active camptothecin derivative (a) designed to bind to the DDX5 protein and the E3 ligase causes the death of the target cells expressing the DDX5 protein through a molecular glue degrader mechanism (MoA).

[0120] Target cells can be cancer cells or senescent cells. Senescent cells also include cells that do not perform the intrinsic characteristic functions of organs.

[0121] Preferably, the active camptothecin derivative (a) designed to bind to DDX5 protein and E3 ligase according to the present invention may have the ability to inhibit type I topoisomerase and multiple mechanisms of action (MoA) to decompose the oncoprotein DDX5.

[0122] For example, the present invention can expose tumor antigens during cell death of cancer cells in tumor tissues and induce anti-tumor immune responses by stimulating antigen presenting cells (APCs) such as dendritic cells and macrophages.

[0123] In addition, the present invention provides, as drug candidates for killing cancer cells, a variety of (a) active camptothecin derivatives represented by Chemical Formula 1, which are designed to bind to DDX5 protein and E3 ligase; or (b) prodrugs of active camptothecin derivatives, which are designed to release the above-mentioned active camptothecin derivatives (a) at target sites in vivo, control the bystander effect and / or ADME curve of the active camptothecin derivatives (a) to prevent the occurrence of T cell exhaustion caused by chronic antigen exposure, and a variety of anticancer dosage forms based on the active camptothecin derivatives (a), their doses and dosages can be designed.

[0124] Camptothecin derivatives that exert the effect of type I topoisomerase inhibitors, such as general formula 1, are used. When designing camptothecin drugs, regarding the changes in R1 and R2 of the A ring that are verified to allow greater tolerance (Group A), the active camptothecin derivatives of the present invention are designed to be the same as the FL118 compound of chemical formula 2 (Example 1), thereby exerting an anti-cancer mechanism of decomposing the tumor protein (oncoprotein) DDX5 (Example 2).

[0125] [General formula 1]

[0126]

[0127] [Chemical formula 2]

[0128]

[0129] This is based on the active camptothecin derivatives of Formula 1 designed to be the same as R1 and R2 in Formula 1 and the FL118 compound, such as the compounds in the following Formula 3, Formula 4 or Formula 5, which are also transcription cofactors and can decompose the tumor protein (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 through the cell viability experiment (Ce ll viability), it was found that the above compounds killed the corresponding cells introduced into the cells ( Fig.15 and Fig.16 ).

[0130] [Chemical formula 3]

[0131]

[0132] [Chemical formula 4]

[0133]

[0134] [Chemical formula 5]

[0135]

[0136] In Formula 1 or Formula 2, the group C site binds to type I topoisomerase and the group A site binds to DNA to stabilize the covalent bond of the topoisomerase-DNA complex and prevent the reconnection of the cut DNA fragments (Example 1), and / or in Formula 1 or Formula 2, the group A site binds to DDX5 and the group C site binds to E3 ligase to induce DDX5 decomposition ( Figures 11 to 14 ).

[0137] Inhibitors of type I topoisomerase (Topoisomerase I) are anticancer mechanisms with proven efficacy and safety in clinical practice. Exatecan, a camptothecin drug, and exatecan derivatives such as Dxd and SN-38 have been developed as payloads for ADCs.

[0138] Exatecan is a drug that has been confirmed to have 5 to 10 times stronger cytotoxicity than SN-38. Unlike irinotecan, exatecan does not require enzyme activation. In addition, compared with SN-38, the main drug of irinotecan, or topotecan used in clinical practice, it has stronger inhibitory activity against type I topoisomerase and has stronger cytotoxic activity against various cancer cells in vitro. In particular, it has an effect on cancer cells that show resistance to SN-38, etc., through the expression of p-glycoprotein. In addition, it has also shown a strong anti-tumor effect in a subcutaneous transplantation model of human tumors in mice, and clinical trials have been conducted.

[0139] The FL118 drug of chemical formula 2 is a hydrophobic low molecule that can penetrate the cell membrane. The FL118 drug is a powerful type I topoisomerase inhibitor, and has the same camptothecin core structure as the existing commercial Top1 inhibitors SN-38, topotecan, and exotecan ( Figure 1 and Figure 2 ), but when administered alone, it also has differentiated anticancer efficacy and excellent safety compared to SN-38 in a variety of cancer cells and animal models, and is an anticancer agent that ensures a broad therapeutic window.

[0140] The inventors synthesized the camptothecin compound FL118 and continued to conduct various studies on its physicochemical properties. However, due to the limitations of its physicochemical properties, FL118 has limitations in the development of dosage forms.

[0141] The present inventors confirmed that the FL118 drug, when used as a payload of an antibody-drug conjugate (ADC), has stronger cytotoxicity than ADC using SN-38 as a payload, and has the same level of cytotoxicity as the exitecan drug.

[0142] In addition, FL118 is a camptothecin compound with excellent ability to inhibit type I topoisomerase and dual MoA that degrades the oncoprotein DDX5 (p68).

[0143] Surprisingly, it was found that, like FL118, Exitecan can not only strongly inhibit the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.), another major cause of anticancer drug resistance, at lower concentrations, thereby blocking the resistance mechanism ( Fig.10 ).

[0144] Therefore, based on this, the present invention is characterized in that Figure 1 As shown, the FL118 compound is used to design (a) a novel active camptothecin derivative having excellent ability to inhibit type I topoisomerase and a dual MoA for decomposing the tumor protein (oncoprotein) DDX5 (p68), (b) a prodrug of the novel active camptothecin derivative, which is designed to release the above-mentioned active camptothecin derivative (a) in vivo, and (c) a complex containing a ligand targeting the DDX5 protein, which is connected to the above-mentioned active camptothecin derivative (a) through a linker group to inactivate the ability to inhibit type I topoisomerase.

[0145] Specifically, the synthetic design concept of active camptothecin derivatives (a) having the ability to inhibit type I topoisomerase and decompose the oncoprotein DDX5 with a dual mechanism of action (dual MoA) is as follows: Figure 1As shown, the structure can be designed as follows: Based on the structure-activity relationship (SAR), the structure of the type I topoisomerase inhibition region group C is maintained, and the binding site group A for decomposing DDX5 also maintains the same structure as FL118, and the structure (R3 and R4) of group B in general formula 1 is adjusted to be as shown in chemical formula 1, thereby not only improving the physicochemical properties of the drug, but also adjusting the cytotoxicity of the drug and / or the cell membrane permeability of the drug, thereby adjusting the bystander effect (Example 9, Fig.25 and Fig.26 ).

[0146] In a simple comparison of cell viability, the IC of PBX-7016 (Chemical Formula 5) newly designed and synthesized according to the present invention is 50 The values ​​were 1.5 to 2 times that of the Dxd drug, but surprisingly, when used as an ADC payload, the ADC containing PBX-7016 exerted a cell viability-related cytotoxic effect that was approximately 5 times superior to the ADC containing Dxd (Enhertu) (Example 7, Fig. 22 ). The compound represented by Chemical Formula 5 (PBX-7016) and the compound represented by Chemical Formula 5-1 (PBX-7017) as its isomer are partially stereoisomers.

[0147] Therefore, the present invention preferably designs active camptothecin derivatives in the following manner: by modifying R3 and / or R4 in Formula 1 (such as PBX-7014, PBX-7016, PBX-7018, PBX-7020, PBX-7022, PBX-7024), the cell membrane permeability is adjusted as needed to exert an appropriate bystander effect in cancer tissues.

[0148] like Figure 1 As shown, the present invention deforms R3 and R4 of group B in general formula 1 to design and synthesize a compound with a new structure similar to Exa tecan, such as PBX-7011 (Preparation Example 1). Figure 2 As shown, PBX-7011 of Chemical Formula 3 newly designed according to the present invention has relatively high hydrophilicity compared to FL118 of Chemical Formula 2, as confirmed by the calculated LogP (or cLogP) and tPSA values.

[0149] Furthermore, in the newly designed PBX-7011 structure, by adjusting group B of general formula 1, new structure compounds PBX-7014 and PBX-7016 (Preparation Examples 2 and 3) were designed and synthesized. According to calculations, PBX-7014 and PBX-7016 showed similar hydrophilicity values ​​to Dxd derived from exitecan ( Figure 1 and Figure 2 , Fig. 27 ).

[0150] Fig. 27 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 were compared.

[0151] The relative hydrophilicity / hydrophobicity of a drug has a major impact on its solubility, absorption, distribution, metabolism, excretion (ADME). The ease with which a drug crosses cell membranes and is important in receptor interactions.

[0152] The hydrophobicity of a drug is expressed by the partition coefficient (p).

[0153] The p value is large for hydrophobicity and small for hydrophilicity (polarity). In practice, its log P value is used as a measure of hydrophobicity. C log P refers to the log P value of a given compound calculated using appropriate software. The smaller the cLogP value, the higher the polarity.

[0154] The molecular polar surface area (PSA) or phase polar surface area (TPSA) is defined as the surface sum of all polar atoms or molecules including attached hydrogen atoms, primarily oxygen and nitrogen. tPSA is a commonly used medicinal chemistry assay to optimize the cell penetration ability of drugs. Molecules with polar surface areas tend not to penetrate cell membranes. If a molecule is to pass through the blood-brain barrier and act on receptors in the central nervous system, it usually needs to be smaller than of tPSA.

[0155] The PBX-7016 / PBX-7017 (Chemical Formula 5 and Chemical Formula 5-1) of the present invention is to reduce the life span of the drug in PBX-7014 / PBX-7015 (Chemical Formula 4 and Chemical Formula 4-1) by introducing an unstable functional group methyl group in metabolism. For drugs that are extremely stable in metabolism but metabolized very slowly, toxicity and serious side effects may occur due to accumulation, so it is necessary to ensure an appropriate residence time.

[0156] The newly designed active camptothecin derivative (a) according to the present invention is designed to be a hydrophobic low molecule capable of penetrating the cell membrane by deforming R3 and R4 of the general formula 1. If it is delivered to cancer tissue, it can penetrate deep into the cancer tissue and accumulate at a high concentration. After the active camptothecin derivative (a) or its prodrug (b) ADC penetrates the cell membrane, the active camptothecin derivative (a) exerts cytotoxicity in the cell to cause cell death, and is released into the extracellular fluid, continuously penetrating the cell membrane of the surrounding cells to move into the cell, thereby exerting a cell death mechanism (Example 9, Fig.25 and Fig.26 ). Therefore, the active camptothecin derivatives of the present invention can transform solid cancer with high cell density and agglomeration into dispersed cancer with low cell density, and / or transform cold tumor with low immune activity into hot tumor with high immune activity. In addition, it has a high effect on the apoptosis of surrounding cells, which is helpful for the treatment of heterogeneous tumors.

[0157] In summary, in the present invention, Figure 1 , Figure 2 , Fig. 27 and Fig.28 As shown in the examples, it is possible to design a variety of camptothecin derivatives and their prodrugs ( Figure 3 ), when designing camptothecin derivatives, the hydrophilicity can be controlled in a variety of ways to regulate the cell membrane permeability associated with the bystander effect.

[0158] [FL118 drug]

[0159] FL118 of Chemical Formula 2 can directly bind to DDX5 and the ubiquitination regulator, and acts as a molecular glue degrader that decomposes DDX5.

[0160] FL118, which acts as a molecular glue, has the function of directly binding to the tumor protein DDX5, a multifunctional masterregulator, at the locus through the proteasome degradation pathway, and dephosphorylating and decomposing it without reducing DDX5 mRNA. The silencing of DDX5 indicates that DDX5 is a regulator of the expression of multiple oncogenic proteins including survivin, Mcl-1, XIAP, cIAP2, c-Myc and Kras.

[0161] As demonstrated in a study of tumor suppression in human colorectal cancer / pancreatic cancer cells by indirectly controlling DDX5 downstream targets, FL118 efficiently inhibits cancer initiation, development, metastasis, recurrence and treatment resistance.

[0162] Genetic manipulation of DDX5 in PDAC cells affects tumor growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. In human tumor animal model studies, FL118 showed high efficacy in eliminating human PDAC and CRC tumors with high DDX5 expression, while FL118 was less effective in PDAC and CRC tumors with low DDX5 expression.

[0163] DDX5 protein is the direct target of FL118 drug and can be used as a biomarker to predict PDAC and CRC tumor sensitivity of FL118.

[0164] In addition, FL118 has a Top1 inhibitory effect in cancer cells at a level equal to or higher than that of SN-38, and is 5 to 20 times stronger than SN-38 in a variety of cancer cell lines, i.e., a low level of IC 50 The numerical value shows cytotoxicity, that is, in the evaluation results of 140 cell lines derived from various cancers, the IC value was <100nM for most cancer cells. 50 Very strong anti-cancer effect. FL118 drug ensured good safety through GLP-toxicity tests on mice and beagles, and showed excellent efficacy compared with SN-38 in various cancer cell line xenograft models. In addition, for camptothecin anticancer agents, patients showed good anti-cancer response in the early stage of use, but showed strong resistance to these drugs through epigenetic silencing of Top1 gene and Top2 dependence of cancer cells. On the contrary, FL118 drug also showed strong efficacy in cancer cell line xenograft models where Top1 could not be expressed through epigenetic silencing or gene knockout.

[0165] In addition, FL118 is a triple-targeted anticancer agent that directly targets type I topoisomerase, an established anticancer target, while inhibiting the Bcl family, such as the drug-resistant protein survivin, involved in the drug resistance mechanism, and inhibiting the action of the extux pump.

[0166] Specifically, camptothecin anticancer drugs such as SN-38 show resistance to drug excretion from cells due to overexpression of the ABCG2 transporter, but FL118 is not affected by the ABCG2 transporter and can overcome the resistance. FL118 can block the expression of resistance by strongly inhibiting the expression of anti-apoptotic proteins (survivin, cIAP2, XIAP, etc.) that are the main cause of resistance to other anticancer drugs at low concentrations. Fig.10 ).

[0167] Therefore, FL118 will not be excreted from the cell by the efflux pump ABCG2, and can block the resistance of multiple anti-apoptotic proteins. As a result, FL118 can overcome the multiple resistance mechanisms of SN-38 / Ixitectonic.

[0168] When FL118 was administered in vivo at the same dose as SN-38, it showed a strong tumor regression effect compared with SN-38 in colorectal cancer, head and neck cancer, pancreatic cancer, etc.

[0169] The drug FL118 has a strong anticancer effect even when administered after inducing SN-38 resistance in tumor xenografts. The possibility of overcoming various resistance mechanisms of the drug FL118 to SN-38 / exitecan was confirmed in vivo.

[0170] Moreover, the FL118 drug has the best PK / safety profile suitable for targeted drug delivery (e.g., carrier-drug conjugate). When the FL118 drug is administered alone, it is rapidly metabolized / excreted in the blood, and only a low concentration is shown, but in cancer tissues, it begins to accumulate rapidly after administration, thereby maintaining a high concentration for a long time. For example, when ADC is applied, the maximum selectivity between tumor tissue and normal tissue is guaranteed. This is also true for the active camptothecin derivative (a) represented by Chemical Formula 1 (Examples 6 and 8).

[0171] In conclusion, among the FL118 drugs,

[0172] (i) In addition to the validated anticancer target Top1, it also selectively inhibits / degrades another anticancer target DDX5, UBE2T, and USP2a to produce a strong anticancer effect;

[0173] (ii) showed 5 to 10 times stronger cytotoxicity than SN-38, and showed cytotoxicity equal to or higher than that of exitecan;

[0174] (iii) Unlike SN-38 and exotecan, they are not substrates for ABCG2 and P-GP, thus avoiding resistance mechanisms caused by overexpression of these transporters;

[0175] (iv) It exhibits excellent efficacy in animal models of cancer caused by epigenetic silencing of Top1 / over-expression of Top2, which is the main mechanism of Top1 inhibitor resistance;

[0176] (v) Overexpression of anti-apoptotic proteins, a common mechanism of resistance in most cancers, can be blocked at the source, thereby avoiding resistance;

[0177] (vi) Biomarkers that can predict patients' anti-cancer response have been established (DDX5, K-ras, p53). By ensuring customized biomarkers, personalized treatment can be carried out through simultaneous diagnosis, especially in p53 / K-ras mutant cancer cells with poor prognosis.

[0178] KRAS is one of the three genes involved in the production of RAS protein that participates in cell proliferation signaling. It is known that although it varies from cancer to cancer, in general, mutations occur in more than 20%. However, although it can be expected to have anti-cancer effects scientifically, it is considered an irreversible target. Because the surface of RAS protein is very smooth, no pocket suitable for binding compounds has been found.

[0179] [Antitumor mechanism as a selective inhibitor of type I topoisomerase (topoisomerase-1) 1]

[0180] Camptothecin is a selective inhibitor of type I topoisomerase, an isomerase involved in DNA replication and recombination. Figure 2). Since it was confirmed to exhibit strong cytotoxicity in vitro, the National Cancer Institute (NCI) of the United States began to develop it through clinical trials, but due to its extremely poor solubility, the development was interrupted due to the emergence of various side effects such as bone marrow suppression and hemorrhagic cystitis. However, after 1990, it was confirmed that it selectively inhibited DNA AI type topoisomerase, which was different from the unique mechanism of action of camptothecin, namely the inhibition mechanism of DNA type 2 topoisomerase (topoisomerase-2), thereby showing anti-tumor effects.

[0181] DNA topoisomerases are enzymes within the nucleus that act to temporarily cut DNA or unwind the double helix when cells need access to genetic material for replication or transcription. They are also involved in a variety of intracellular activities such as chromosome condensation and recombination, DNA repair, etc. The genetic code of topoisomerases is fairly conserved between species.

[0182] It has been observed that the levels of type I topoisomerase, which is the drug target of camptothecin, are increased in a variety of malignancies. Although this drug does not inhibit the free enzyme, it can stabilize the covalent bonds of the topo-DNA complex, thereby preventing the reconnection of the cleaved DNA fragments. Therefore, the sensitivity of cells to this topoisomerase-targeted drug is related to the level of enzyme present in the nucleus. This drug interferes with the recombination of DNA, making transcription impossible. The greater the amount of type I topoisomerase, the more cleavable complexes are formed, which means that the sensitivity of the drug is higher. This has important clinical relevance. Type I topoisomerase inhibitors are used to increase the expression of type 2 topoisomerase, which makes type 2 topoisomerase inhibitors more sensitive. These results are supported by the antagonistic relationship between type 1 topoisomerase and type 2 topoisomerase. It is reported that this type 1 topoisomerase is different from type 2 topoisomerase and is not closely related to proliferation in normal tissues. It exists in large quantities in "S" phase solid cancers with active cell division, including lymphoma, such as colorectal cancer, ovarian cancer, esophageal cancer, etc., compared with the surrounding normal tissues. In fact, it induces cell death by blocking the replication and transcription of genes in tumor cells in the "S" phase of the cell cycle.

[0183] [Antitumor mechanism 2 of DDX5-binding as a molecular glue degrader]

[0184] The ubiquitin-proteasome system (UPS) is an important pathway for intracellular protein degradation that regulates a wide range of cellular processes. Ubiquitin is a small protein that is covalently bonded 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.

[0185] Ubiquitin ligases are responsible for substrate specificity in the ubiquitination pathway.

[0186] In this regard, according to the present invention, the active camptothecin derivative represented by Chemical Formula 1 is designed to bind to the DDX5 protein and the E3 ligase.

[0187] DX5 (also known as p68) is a multifunctional master regulator that acts on the following mechanisms: (1) the biological process of co-activating the transcription of many tumor genes through direct interaction with multiple transcription factors (e.g., c-Myc) in oncogenic gene promoters, (2) the biological process of regulating miRNA and pre-RNA fragmentation (e.g., U1, U2, U3, ... snRNP), and (3) ribosome biogenesis (e.g., 32S rRNA, pre-ribosome).

[0188] According to the present invention, the active camptothecin derivative represented by Chemical Formula 1 binds to DDX5 protein without reducing DDX5 mRNA, and functionally decomposes it through dephosphorylation and proteasome degradation pathways, which indicates that the active camptothecin derivative of Chemical Formula 1 can simultaneously bind to DDX5 and ubiquitin-involved protein stability / degradation regulators, playing the role of "molecular bonding decomposing agent".

[0189] It is known that DDX5 downstream protein targets are involved in the initiation, development, metastasis, recurrence and treatment resistance of cancer. Therefore, according to the present invention, if the DDX5 downstream targets are indirectly blocked by the decomposition of the DDX5 protein by the active camptothecin derivative represented by Chemical Formula 1, the active camptothecin derivative represented by Chemical Formula 1 can produce a high anti-tumor effect.

[0190] [DDX5 as a drug target and / or biomarker]

[0191] Transcription factors are a group of proteins that bind to DNA strands called promoters or enhancers through their DNA binding domains and initiate gene transcription through the cooperation of RNA polymerase and other auxiliary factors. They play an important role in regulating transcription during embryogenesis and development. In other cell types, the physiological state of transcription factors is also important for maintaining cellular homeostasis. Therefore, if transcription factors are deregulated, it will lead to the development of cancer cells and the development of tumors. Depending on their function in cancer cells, transcription factors may be oncogenic or tumor suppressive. In addition, transcription factors also regulate cancer stem cells, epithelial-mesenchymal transition (EMT), and drug response. Therefore, it is believed that the detection of deregulated transcription factors can predict the treatment outcome of cancer patients, and targeting non-deregulated transcription factors may be an encouraging strategy for treating cancer. Deregulated major transcription factors lead to poor clinical outcomes in cancer patients. Therefore, by predicting prognosis and drug response, and targeting deregulated transcription factors, it will help to design new drugs and therapeutic strategies for cancer treatment.

[0192] As is known to all, transcription is the first step of gene expression in cells during embryogenesis, development and homeostasis. During the transcription process, RNA transcripts with specific coding information are generated in DNA. In order to ensure the specificity of gene expression in different cells and tissues, genes that are transcribed into RNA transcripts by transcription factors that are specific protein groups are selected to control transcription. Transcription factors have DNA binding domains and can bind to specific DNA sequences of genes called promoters or enhancers, or they can initiate transcription of genes through the cooperation of activators, mediators, co-factors and RNA polymerase.

[0193] Transcription factors also control the rate of transcription from DNA to mRNA. As a result, in different types of cells, the physiological state of transcription factors is important for maintaining homeostasis within the cell. Changes in the physiological state of transcription factors within cells can cause transcription disorders, leading to the translation of specific genes, thereby causing pathological changes in cells. Therefore, dysregulated transcription factors can cause many human diseases. One of the common diseases with dysregulated transcription factors is cancer.

[0194] Homeostatic control in human cells usually prevents inappropriate proliferation and inhibits the growth of cells that proliferate abnormally in normal, appropriate environments. Since transcriptional control is a core process during homeostasis, if the level or activity of transcription factors is dysregulated, cells will escape homeostatic control, leading to the onset and development of human cancer.

[0195] Indeed, many chemotherapies can activate tumor suppressor transcription factors.

[0196] Dysregulated transcription factors play an important role in tumor formation and tumor progression. They regulate central tumor signaling pathways such as NF-κB, Notch, Wnt / β-catenin, and JAK / STAT. Dysregulated oncogenic and tumor suppressor transcription factors favor the survival of cancer cells, cancer stem cells, and EMT cells, thereby inducing intrinsic or acquired drug resistance and tumor progression in cancer treatment.

[0197] DDX5 (p68) is a well-known multifunctional DEAD-box RNA helicase and a transcription cofactor. Therefore, when cancer occurs due to transcription factor deregulation caused by the physiological state of DDX5, cancer can be treated by selectively degrading the transcription cofactor DDX5 (p68). Similarly, cancer can be prevented by selectively degrading the transcription cofactor DDX5 (p68).

[0198] DDX5 is considered a potential biomarker and target for the treatment of multiple cancer types. Years of research have greatly expanded the understanding of the functional diversity of DDX5 in various cancer types and expanded the knowledge of the mechanism of action (MoA). This provides a rationale for the development of new cancer therapeutics using DDX5 as a biomarker and therapeutic target. However, while DDX5 was found to be an oncogenic target and a biomarker of cancer treatment resistance in most published studies, some studies report that DDX5 may function as a tumor suppressor in specific circumstances. The multifunctionality of DDX5 makes DDX5 an excellent independent oncogenic biomarker and target for targeted cancer therapy.

[0199] The transformation of normal cells into cancerous cells is caused by the deregulation of different metabolic pathways that comprise a complex network of protein-protein interactions. Cellular enzymes and DDX5 play important roles in maintaining normal cellular metabolism, but if they are deregulated, they may accelerate tumor metamorphosis. Both DDX5 interact with hundreds of different cellular proteins, and both proteins can function as tumor suppressors or oncogenes, depending on the specific pathway involved.

[0200] The DEAD-box family of RNA helicases participates not only in transcription and translation but also in a variety of metabolic pathways including cell proliferation, innate immunity and stress responses. Given their various roles, their deregulation or their mutations are associated with various pathological conditions including cancer. But in some cases, loss of function of a given DEAD-box helicase promotes tumor transformation, thereby exhibiting a tumor suppressor role, while in other cases, overexpression of the same enzyme favors cancer progression, becoming a classic oncogene. The role of DDX5 as both an oncogene and a tumor suppressor has been documented in multiple cancer types. Understanding the interplay of different cellular conditions defined by the molecular interaction network of DDX5 in different tumors could help explain the apparently conflicting roles played by these proteins as oncogenes or cancer suppressors, which are cancer-specific.

[0201] These results suggest that DDX5 more frequently functions as an oncogene, but may have tumor suppressor functions in specific cancer types (Table 1).

[0202] Source: Secchi, M.; Lodola, C.; Garbelli, A.; Bione, S.; Maga, G.

[0203] Table 1

[0204]

[0205] 1 Oncogene (+), tumor suppressor (-).

[0206] According to the present invention, in the active camptothecin derivative represented by Chemical Formula 1, the DDX5 protein is a drug target, and therefore, drug resistance, resistance to targeted therapy, and / or resistance during treatment can be avoided. In addition, the transcriptional induction of anti-apoptotic genes can be blocked by the active camptothecin derivative. Furthermore, by the active camptothecin derivative, the DDX5 protein as a transcription cofactor is degraded to maintain or improve sensitivity to chemotherapy or radiotherapy.

[0207] [Camptothecin derivatives represented by Chemical Formula 1, Chemical Formula 3 to Chemical Formula 9 and isomers thereof]

[0208] According to the present invention, R1 and R2 in Formula 1 are designed to be the same as the FL118 compound, and the active camptothecin derivative represented by Chemical Formula 1, such as the following compounds of Chemical Formula 3, Chemical Formula 4, Chemical Formula 5, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, Chemical Formula 9 and their isomers are also transcription cofactors, and degrade tumor protein (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 through cell viability experiments, it was found that the above compounds killed the corresponding cells introduced into the cells ( Fig.15 and Fig.16 ).

[0209] According to the present invention, the camptothecin derivatives represented by Chemical Formula 1, for example, by Chemical Formula 3 to Chemical Formula 9 are as follows Figure 1 The camptothecin shown has a pentyl ring structure with a lactone on the E-ring which is essential for cytotoxicity. In order to stabilize the type 1 topoisomerase-DNA byproduct, it is designed to maintain the lactone group and α-hydroxyl group located at the 20th carbon of the important E-ring, maintain the structure of the FL118 drug (maintain the advantages of the above-mentioned FL118 drug), R1 and R2 in general formula 1 (-OCH2O-(methylenedioxo) 5-sided ring), and the structural characteristics of the ixitecan drug (R3 and R4 in general formula 1) are compared with SN-38, which is induced by the π-π enrichment (stacking) of the aromatic ring formed by the A- and B-rings. The various orientations of the continuous carbon-carbon single bonds of the hexagonal ring extending from the A- and B-rings form a dynamic equilibrium, thereby weakening or inhibiting the enrichment (stacking) of the aromatic ring formed by the A- and B-rings.

[0210] Furthermore, the compound represented by Chemical Formula 3 or Chemical Formula 3-1 is a compound designed in the following manner: for the carbon-carbon single bonds on the hexagonal rings extending from the A- and B-rings, molecular bond rotation can be achieved, thereby allowing -NH2 with a larger degree of freedom to be exposed to water (H2O), carrying a (+) charge or hydrogen bonding with water, and increasing water dispersibility.

[0211] In addition, the compound represented by Chemical Formula 4 or Chemical Formula 4-1 is a compound designed in the following manner: for the carbon-carbon single bonds on the hexagonal rings extending from the A- and B-rings, molecular bond rotation can be achieved, so that the functional group CH2(OH)CONH- of -NH2 with a larger degree of freedom is exposed to water (H2O) and rotates like a propeller to hydrogen bond with water and increase water dispersibility.

[0212] Furthermore, the compound represented by Chemical Formula 5 or Chemical Formula 5-1 is a compound designed in the following manner: for the carbon-carbon single bonds on the hexagonal rings extending from the A- and B-rings, molecular bond rotation can be achieved, so that the functional group CH3CH(OH)CONH- in the form of lactic acid (Lactic acid) with a larger degree of freedom -NH2 is exposed to water (H2O) and rotates like a propeller to hydrogen bond with water, thereby increasing water dispersibility.

[0213] The compound represented by Chemical Formula 5 or Chemical Formula 5-1 of the present invention is a compound represented by Chemical Formula 4 or Chemical Formula 4-1 in which a metabolically unstable functional group methyl is introduced to reduce the life span of the drug. Drugs that are extremely metabolically stable but metabolized very slowly may cause toxicity and serious side effects due to accumulation, so it is necessary to ensure an appropriate residence time.

[0214] In addition, the DXd payload used for Enhertu was originally created from the isotecan compound that was hardly affected by ABCG2, but due to the presence of the glycolic acid (alpha-hydroxyacetic acid) functional group for converting isotecan to DXd, it was strongly affected by ABCG2. However, the glycolic acid functional group plays a very important role in the excellent safety / efficacy curve of Enhertu. If it is removed, it will lead to difficulties in ADC preparation, as well as problems of ADC performance degradation (safety issues or reduced efficacy) in animal models and clinical trials. Therefore, the demand for new camptothecin derivatives that can easily prepare ADC but are not affected by ABCG2 is very high. The compound (PBX-7024) represented by Chemical Formula 9 is a compound derived from the compound (PBX-7011) represented by Chemical Formula 3, and is a new camptothecin compound that is not affected by ABCG2 and can easily prepare ADC.

[0215] To confirm the anticancer efficacy of PBX-7024, the efficacy was evaluated in FaDu cancer cells that do not express ABCG2 and A549 cancer cells that overexpress ABCG2. Fig.15 As shown, it can be confirmed that the IC 50 Numerically similar ratios, PBX-7016 and PBX-7024 still maintain strong effectiveness.

[0216] That is, when the novel camptothecin compounds PBX-7016 and PBX-7024 of the present invention are used, unlike ADCs using existing camptothecin compounds SN-38, DXd, etc., they have the effect of overcoming the resistance mechanism caused by overexpression of ABCG2.

[0217] Fig. 27 and Fig.28 The 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' were compared.

[0218] The compound of Chemical Formula 3 and the compound of Chemical Formula 3-1 are partial stereoisomers with each other. Similarly, the compound of Chemical Formula 4 and the compound of Chemical Formula 4-1; and the compound of Chemical Formula 5 and the compound of Chemical Formula 5-1 are also partial stereoisomers with each other. Therefore, the compound in a partial stereoisomer relationship with the compound of Chemical Formula 6, the compound of Chemical Formula 7, the compound of Chemical Formula 8 or the compound of Chemical Formula 9 also belongs to the scope of the present invention.

[0219] As illustrated in Preparation Example 2, PBX-7014 of Chemical Formula 4 can be synthesized using PBX-7011 of Chemical Formula 3, and PBX-7015 of Chemical Formula 4-1 can be synthesized using PBX-7012 of Chemical Formula 3-1.

[0220] As illustrated in Preparation Example 3, the compound of Chemical Formula 5 (PBX-7016, Compound E) can be synthesized using the compound of Chemical Formula 3 (PBX-7011, Compound A), and by the same method, the compound of Chemical Formula 5-1 (PBX-7017, Compound F) can be synthesized using the compound of Chemical Formula 3-1 (PBX-7012, Compound B).

[0221] In the present specification, the camptothecin drugs of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 of the present invention include not only the compounds of Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, but also pharmaceutically acceptable salts thereof, solvates thereof, and prodrugs thereof.

[0222] In this specification, the salts allowed in pharmacy refer to salts commonly used in the pharmaceutical industry, for example, including: salts of inorganic ions, such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc.; inorganic acid salts, such as hydrochloric acid, phosphoric acid and sulfuric acid, in addition, including: organic acid salts, such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid and acetylsalicylic acid; and amino acid salts, such as lysine, arginine, guanidine, etc. In addition, organic ion salts that can be used for drug reactions, purification and separation processes, such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium and benzethonium chloride, etc., are also included. However, the type of salt in the present invention is not limited to these salts exemplified.

[0223] "Solvate" refers to a compound represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 and a pharmaceutically acceptable salt thereof, which further contains a chemical amount or a non-chemical amount of a solvent bound by a non-covalent molecular force. When the volume is water, the solvate is a hydrate.

[0224] [Advantages of a drug model called molecular glue degrader]

[0225] Proteins in cells in the body are naturally degraded within hours to days after performing their functions. All cells in the body have a purification function called the ubiquitin proteasome system (UPS) that degrades proteins. In this process, ubiquitin acts as a marker to inform proteins that need to be degraded, and the proteasome acts as a shredder that recognizes ubiquitin and destroys the corresponding proteins. That is, next to the protein that performs its function, there are multiple substances called ubiquitin attached like a marker, and a substance called proteasome only selects proteins with this marker and degrades them like a shredder. E3 ligase is an enzyme that triggers the protein degradation system in the body and is responsible for substrate specificity in the ubiquitination pathway.

[0226] Molecular glue degrader or molecular glue is a compound with adhesive function that binds the target protein and the specific enzyme (E3 ligase) of our body to each other. One of the advantages of molecular glue is catalysis, which can be re-detached after degrading the target to degrade another target protein.

[0227] If the E3 ligase is attached to the tumor protein through a molecular glue, the tumor protein will be degraded until the targeted tumor protein disappears, and other tumor proteins will be continuously degraded, thereby preventing the proliferation of cancer cells. Therefore, the molecular glue targeting tumor proteins overcomes the drug resistance problem of targeted anticancer drugs, and the efficacy is high even at a small dose.

[0228] In addition to the FL118 drug, the active camptothecin derivative designed in Chemical Formula 1 according to the present invention is a molecular glue degrader that binds to the DDX5 protein and the E3 ligase, that is, a molecular glue that activates the degradation of the tumor protein DDX5 ( Figures 11 to 14 ).

[0229] Molecular glue degrader is not only a ligand (Warhead) that binds to the target protein, but can also act as a ligand (binder) for the E3 ligase.

[0230] Therefore, not only FL118, the active camptothecin derivative designed in Chemical Formula 1 according to the present invention is a molecular adhesive that activates the degradation of tumor protein DDX5, and therefore, can be used as a ligand that targets DDX5 protein or binds to DDX5 protein.

[0231] Molecular glue degraders are different from kinase inhibitors in that they are "proximity-driven" and their degradation-inducing ability is "event-driven" through the temporary formation of a triple complex of "target protein-molecular glue-E3 ligase". After degradation occurs, the separated molecular glue forms an additional triple complex with the target protein, which can undergo multiple degradation processes until the target protein disappears.

[0232] Most proteins that become drug targets develop resistance to drugs through evolution. However, low-molecular-weight compounds that function as adhesives to bind tumor proteins and E3 ligases to each other have strong resistance and are therefore suitable models for cancer therapeutics.

[0233] Whenever a gene mutation occurs, the shape of the drug target protein gradually changes. It would be best to use a drug to block the activity of all variants, but this is impossible due to selectivity issues, so a drug is used to degrade the drug target protein in order to block the activity of all variants and simply eliminate it.

[0234] Therefore, according to the present invention, the active camptothecin derivative represented by Chemical Formula 1 is a drug that can accurately bind to the DDX5 tumor protein and avoid the resistance problem of targeted therapeutic agents through a molecular glue method that directly and selectively degrades the protein.

[0235] According to the present invention, the active camptothecin derivative represented by Chemical Formula 1 determines the binding strength to the target protein DDX5 tumor protein according to its physicochemical properties, and is preferably an irreversible drug that binds too strongly to restore the previous state.

[0236] The active camptothecin derivatives designed from Chemical Formula 1 according to the present invention, including FL118, are different from the existing SN38 drug. They have a high affinity with DDX5 and are not easy to fall off. They degrade DDX5 through the molecular glue function, not only irreversibly inhibiting the signal transduction of cancer cells related to DDX5, 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 killing effect on surrounding cells, which is beneficial to the treatment of heterogeneous tumors, and can also inhibit the development of cancer for a long time, reduce the risk of resistance, and improve the treatment response rate.

[0237] [Cell Death and Anticancer Drug Resistance]

[0238] According to the present invention, the active camptothecin derivative represented by Chemical Formula 1 can bind to DDX5, which works as an oncoprotein in cells, and can induce cell death by degrading DDX5 protein ( Fig.29 ).

[0239] There are two factors that determine tumor growth: first, cell proliferation, and second, cell death. If the cell cycle of tumor cells is stopped by cytotoxic substances, tumor cells die by apoptosis.

[0240] Cell death plays a very important role in maintaining tissue homeostasis and morphology, regulating cell number, removing damaged or abnormal cells, and defending against infection. Living organisms maintain normal life by accurately regulating the growth, differentiation, and death of cells.

[0241] Apoptosis is described as a complex process that induces cell death accompanied by 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 indispensable role in maintaining the development, differentiation and homeostasis of multicellular organisms. Apoptosis occurs in different types of cells due to many factors. Unlike cell necrosis, there is no free lysosomal enzyme, apoptotic bodies are found, and cell shrinkage, nuclear condensation and characteristic ladder-shaped DNA segmentation are found.

[0242] Apoptosis is part of the normal developmental program of animals and is important for preventing cancer. Caspases, a family of protein-degrading enzymes, are involved in cell death and cleave many target proteins. If cell death is impaired, cells that need to be killed can survive and proliferate, forming potentially cancerous clones.

[0243] In cancer treatment, drug resistance in nature may be caused by abnormally expressed transcription factors that are decisive regulators of cell proliferation and cell death. Therefore, the active camptothecin derivatives of the present invention can induce DDX5 protein degradation and the resulting cell death by the mechanism of action of molecular glue degraders that bind to the oncoprotein DDX5 as transcription cofactors. At this time, DDX5 protein degradation can downregulate the transcription of anti-apoptotic genes. Therefore, unlike other targeted therapeutic agents, drug resistance caused by abnormally expressed transcription factors related to cell proliferation and / or cell death is not easy to occur, and / or acquired drug resistance induced by activating transcription factors that are dysregulated during chemotherapy and / or radiotherapy can be inhibited.

[0244] That is, the anti-cancer mechanism of the active camptothecin derivatives according to the present invention may bypass the molecular mechanism of cancer treatment resistance, thus getting rid of the problem of drug resistance. Since the efficacy after resistance is not as good as before, the active camptothecin derivatives of the present invention can be used as the standard therapeutic agent or the first therapeutic agent after cancer diagnosis.

[0245] In conclusion, the active camptothecin derivative represented by Chemical Formula 1 according to the present invention can become a targeted anticancer agent acting on the DDX5 protein which plays an important role in the growth, survival (cell survivor), proliferation, metastasis and / or metabolism of cancer cells.

[0246] [ADME curve of antibody-drug conjugate (ADC)]

[0247] The in vivo efficacy of anticancer agents may be affected by the absorption, distribution, metabolism, and excretion (ADME) properties. The ADME profile of a drug may affect its ability to reach cancer cells and exert its effects. That is, the ADME properties of anticancer drugs can have a significant impact on in vivo efficacy.

[0248] The ability of drugs to penetrate tumor tissue may be affected by factors such as the tumor microenvironment, including blood flow and cell density.

[0249] Therefore, understanding the ADME properties of anticancer agents and optimizing the selection and dosage / dosage of drugs / drug modalities can improve the therapeutic index of these anticancer agents and lead to better outcomes for cancer patients.

[0250] Antibodies (Ab) and antibody-drug conjugates (ADCs) have different life times and ADME curves due to their different structures and mechanisms of action.

[0251] Antibodies are large proteins naturally produced by the immune system in response to foreign substances (antigens). Due to their size and complex structure, they have a long circulation half-life (weeks to months), which prevents degradation and removal. Antibodies are distributed throughout the body, including tissues, and interact with target antigens with high specificity and affinity. Antibodies are primarily cleared by the reticuloendothelial system (RES), including the liver and spleen, and by catabolism in the kidneys and other organs.

[0252] ADC is composed of antibodies (usually monoclonal antibodies) conjugated to cytotoxic drug molecules. The antibody component provides specificity and targeting for tumors or diseased tissues, while the drug component provides cytotoxic activity that kills target cells. ADC has a shorter half-life than antibodies, generally 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 cleared by RES, but the drug payload may be metabolized and excreted by the liver and kidneys (Metabolism and Excretion) (Example 4).

[0253] Antibodies are typically administered subcutaneously or intravenously and absorbed into the bloodstream. They can be distributed throughout the body, including tissues, but are usually confined to the extracellular space due to their size. ADCs are also administered by injection and absorbed into the bloodstream, but because the antibody component is targeted to tumor or diseased tissues, and in some cases, after receptor-mediated endocytosis, the drug payload released by the ADC can penetrate cells and tissues. In turn, the metabolism and excretion of ADCs vary depending on the specific structure and the specific drug or antibody used.

[0254] Antibody-Drug Conjugates (ADCs) based on camptothecin payloads have attracted great attention as a new way to treat a variety of solid tumors. Among these ADCs, in particular, new ADCs (e.g., Enhertu) including newly synthesized camptothecin payloads (e.g., DXd) optimized for ADC mode have recently received special attention due to their successful clinical results.

[0255] The success of these ADCs is due in part to the novel camptothecins' (1) superior cell growth inhibition, (2) better safety profiles than existing ADC payloads, (3) optimized bystander effects, and (4) superior physicochemical properties suitable for generating high-DAR ADCs. However, despite the stunning success of ADCs using camptothecin-based payloads such as Enhertu or DS-1062a, there are clear demands for the development of new ADCs with multiple MoA payloads that have improved safety profiles (minimizing interstitial lung disease (ILD) and neutropenia) and address cancer heterogeneity.

[0256] In order to address these unmet needs, the present invention provides a variety of active camptothecin derivatives of Chemical Formula 1 as candidate drugs, which are designed to bind to DDX5 protein and E3 ligase through molecular adhesives, and further, novel camptothecin payloads (PBX series compounds) based on Chemical Formula 1 are synthesized (Preparation Examples 1 to 4) and evaluated (Examples 2 to 9).

[0257] The new PBX series compound represented by Chemical Formula 1 is a derivative with a good safety profile, based on FL118 of Chemical Formula 2 as a dual Top1 / anti-apoptotic pathway inhibitor, exhibiting strong ex-vivo cytotoxicity comparable to Dxd, an excellent safety profile in a pre-toxicity study on mice, and rapid clearance in a PK study (Example 4, Table 4 and Table 5). When used as a payload for ADC, systemic side effects are minimized if the linker is cut off early.

[0258] Of the PBX series of compounds, two lead compounds, PBX-7014 and PBX-7016, were selected for further evaluation as ADC payloads attached to a variety of HER2 antibodies, including trastuzumab.

[0259] Trastuzumab-7014 and Trastuzumab-7016 (DAR7-8) were linked to trastuzumab in the same manner by PBX-7014 or PBX-7016 through an existing enzyme-cleavable linker for comparative evaluation with Enhertu (dxd-trastuzumab), and were easily synthesized without agglutination issues (Preparation Example 5, Fig.19 and Fig. 20 ).

[0260] The two new ADCs showed better cytotoxicity than DS-8201a (anti-HER2-DXd) in Her2-high cell lines (MDA-MB-453) and Her2-low / mid cell lines (FaDu), and similar levels of cytotoxicity as DS-8201a in Her2-negative cell lines (MDA-MB-468).

[0261] In the NCI-N87CDX model with high Her2 expression, trastuzumab-7014 and trastuzumab-7016 showed the same or more obvious dose-dependent anticancer efficacy as DS-8201, and trastuzumab-7016 also had a bystander effect similar to Enhertu (Example 9, Fig.25 and Fig.26 ).

[0262] Trastuzumab-7014 and trastuzumab-7016 not only exerted tumor regression effects in tumor tissues through the bystander effect for more than 20 days after administration, but also exerted different ADMEs through only one methyl difference, and showed not only dose-dependent anticancer efficacy differences (conc. vs. efficacy) in targeted tumor tissues, but also anticancer efficacy differences over time (effect vs. time) (Example 8, Fig.23 and Fig.24 ).

[0263] If the hydrophobicity of the linker and the drug increases, problems such as increased cohesion of the ADC and a resulting decrease in the drug therapeutic coefficient will arise. Surprisingly, although the hydrophobicity of trastuzumab-7016 increased by one methyl group compared with trastuzumab-7014, its dose-dependent anticancer efficacy (conc. vs. effect) and anticancer efficacy over time (effect vs. time) were very excellent (Examples 8, Fig.23 and Fig.24 ).

[0264] However, in some cases, the side effects of excellent anticancer efficacy (persistent T cell activation caused by chronic antigen exposure, accumulation of inhibitory signals and resulting T cell exhaustion (T cell exclusion), ILD, neutropenia) will also increase over time. For example, according to the drug model of the active camptothecin derivative of Chemical Formula 1 (small molecule vs. ADC), exceeding a specific drug concentration (high) may be important, and in some cases, the time maintained above a specific drug concentration (low) may be important.

[0265] Therefore, the main feature of the present invention is that the selection range of ADC payloads that exert appropriate anti-cancer effects is expanded to include a variety of candidate groups of active camptothecin derivatives of Chemical Formula 1, which are designed to bind to DDX5 protein and E3 ligase through a molecular gluedegrader, taking into account not only various side effects but also problems such as reduced therapeutic coefficient.

[0266] Despite the astonishing success of ADCs using payloads based on camptothecin, such as Enhertu or DS-1062a, it remains a candidate for multi-MoA payloads with improved safety profiles (minimization of ILD and neutropenia) and for addressing cancer heterogeneity. According to the present invention, a variety of active camptothecin derivatives represented by Chemical Formula 1 designed to bind to DDX5 protein and E3 ligase are provided, whereby, in order to be able to precisely control the desired efficacy (e.g., anticancer, combination therapy) and side effects (oncogenic inflammation, drug resistance, ILD, neutropenia), more than one appropriate drug can be selected from the active camptothecin derivatives of Chemical Formula 1, and then a linker suitable therefor can be selected, and an appropriate dose-dosage can be applied.

[0267] [Cell death of cancer cells and the immune response it induces]

[0268] One embodiment of the present invention provides an anticancer dosage form, which administers (a) an active camptothecin derivative represented by the above chemical formula 1, or (b) a prodrug thereof, in an amount (dosage) that induces an anti-tumor immune response by stimulating antigen presenting cells (APCs) through cell death of cancer cells, wherein the active camptothecin derivative (a) is designed to bind to the DDX5 protein and the E3 ligase, and the prodrug of the active camptothecin derivative (b) is designed to release the above active camptothecin derivative (a) at a target site in vivo.

[0269] Among them, antigen presenting cells (APC) can be dendritic cells and macrophages.

[0270] In addition, one embodiment of the present invention provides a method for preparing an anticancer agent based on an active camptothecin derivative (a), wherein the bystander effect and / or ADME curve of the active camptothecin derivative (a) is controlled to prevent the occurrence of T cell exhaustion caused by chronic antigen exposure, wherein:

[0271] The anticancer agent based on the active camptothecin derivative (a) is an anticancer dosage form containing (a) an active camptothecin derivative, or (b) a prodrug thereof, wherein the active camptothecin derivative (a) is designed to bind to the DDX5 protein and the E3 ligase via a molecular glue degrader, and the prodrug of the active camptothecin derivative (b) is designed to release the active camptothecin derivative (a) at a target site in vivo.

[0272] The method comprises: designing an active camptothecin derivative (a) or a prodrug thereof (b) to include a camptothecin skeleton represented by the above chemical formula 1 as a parent core, or selecting a compound designed in the above manner, or synthesizing a compound designed in the above manner,

[0273] The signal transduction pathways that regulate cell death are a complex network of intracellular signal transduction pathways that are tightly regulated by multiple proteins and can be activated by a variety of internal or external stimuli such as DNA damage, oxidative stress, and growth factor deficiency. Dysregulation of these pathways may induce a variety of diseases, including cancer and neurodegenerative disorders.

[0274] The major signaling pathways regulating cell death all converge on the activation of caspases, a family of cysteine ​​proteases that cleave a variety of cellular substrates and ultimately lead to cell death. Caspase activation is tightly regulated by a variety of proteins, including the Bcl-2 family of proteins, which can promote or inhibit apoptosis.

[0275] The Bcl-2 protein family includes pro-apoptotic and anti-apoptotic members that can promote or inhibit cell suicide. The balance between pro-apoptotic and anti-apoptotic Bcl-2 family members is crucial in determining the fate of cells and their sensitivity to cell death.

[0276] Other signaling pathways, such as the p53 pathway and the PI3K / Akt pathway, can also regulate cell death. The p53 pathway is activated in response to DNA damage and leads to the transcription of pro-apoptotic genes such as Bax and Puma. However, the PI3K / Akt pathway can inhibit apoptosis by phosphorylating and inactivating pro-apoptotic proteins such as Bad and Bim.

[0277] Some forms of necroptosis may be programmed cellular responses to stimuli such as infection or DNA damage.

[0278] Immunogenic cell death is accompanied by changes in the cell surface, release of cancer antigens and damage-associated molecular patterns, etc. during the death of cancer cells due to damage. The released immune-inducing substances can stimulate dendritic cells and macrophages as immune cells to induce anti-tumor immune response.

[0279] Apoptosis is a method of cell death by damaged or aged cells, which is accompanied by the deformation and degradation of various intracellular substances. In this process, immune-inducing substances such as cancer antigens and damage-associated molecular patterns (DAMPs) that induce in vivo immunity against cancer cells are damaged by protease and oxidation, which limits the effect of immunotherapy on residual cancer or metastatic cancer after treatment.

[0280] The cell death caused by reactive oxygen species generated by chemical anticancer agents is mainly apoptosis involving the protein degrading enzyme caspase.

[0281] Necroptosis, a type of necrosis in which cells destroy their membranes and die spontaneously, is different from apoptosis, which involves the involvement of degradative enzymes such as caspases. It does not cause protein degradation or oxidation, and can minimize the damage to cancer markers or immune-inducing substances.

[0282] Damage-associated molecular patterns (DAMPs) are molecules that can trigger intracellular immune responses released during cell death and stimulate immune cells. Representative examples include calreticulin, heat shock protein 70 / 90, high-mobility group box 1, ATP, etc.

[0283] On the other hand, inflammation is the body's natural response to infection or injury, involving the release of inflammatory cytokines and the recruitment of immune cells to the affected area. However, if inflammation persists for a long time, it can damage tissues and increase the risk of cancer.

[0284] T cell exhaustion can also occur due to chronic antigen exposure associated with cancer. Tumor cells express antigens that T cells can recognize, but if they are continuously exposed to these antigens, T cells may become exhausted. As a result, the anti-tumor response is weakened and the tumor may grow and spread.

[0285] Overall, the relationship between T cell exhaustion, chronic antigen exposure, and disease is complex and multifaceted.

[0286] Although the hydrophobicity of trastuzumab-7016 and trastuzumab-7014 increased to one methyl group, their dose-dependent anticancer efficacy and anticancer efficacy over time were excellent (Examples 8, Fig.23 and Fig.24 ), but side effects such as T-cell depletion, ILD, and / or neutropenia may require treatment with lower concentrations of trastuzumab 7014.

[0287] Only by understanding the characteristics of a drug can we use it correctly. The pharmacodynamics and pharmacokinetic parameters of a drug help us understand the characteristics of a drug.

[0288] Pharmacodynamics uses the relationship with drug concentration to explain the magnitude and form of changes (effect) (cell viability, clinical effect) (therapeutic action, toxic effect, adverse effect) in cells or the body caused by drug binding to receptors.

[0289] Pharmacokinetics (PK) shows how drug concentration changes as it moves through different areas of the body (Compartment) via ADME, depending on the drug or drug mode. Example 8, Table 6, Fig.23 and Fig.24 The drug concentration of active camptothecin derivative (a) in tumor tissue at different times in multiple drugs / modes can be predicted.

[0290] From a pharmacokinetic perspective, the development of new drugs may fail because toxic drugs may accumulate, useful drugs may be taken in doses too small to establish treatment and therefore may have no benefit, and the drugs may be rapidly metabolized.

[0291] Therefore, in order to use the medicine correctly, it is necessary to select the appropriate medicine and apply the appropriate dose-dosage.

[0292] Dose indicates the amount of medication given at one time. The dosage of a medication is usually prescribed by a healthcare provider based on factors such as the patient's age, weight, and health status. In contrast, Dosage indicates how often and when a medication is taken. The total amount of medication given over a period of time is measured and is usually shown as a daily or weekly dose. Dose and Dosage are important considerations when using medications safely and effectively.

[0293] In this regard, the present invention provides a variety of active camptothecin derivatives of Chemical Formula 1 that are bound to DDX5 protein and E3 ligase via a molecular glue degrader as multiple drug candidates, appropriately applying more than one appropriate drug and its dosage-usage, and can precisely control the desired efficacy (e.g., anticancer, combination therapy) and side effects (chronic cancer antigen exposure, drug resistance, ILD).

[0294] For example, for the active camptothecin derivative of Formula 1, depending on the drug mode (small molecule, ADC), exceeding a specific drug concentration (high) in tumor tissue may be important, and in some cases, the time to maintain above a specific drug concentration (low) in tumor tissue may be important.

[0295] [Pro-drug]

[0296] 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.

[0297] For example, the above-mentioned prodrug refers to a compound designed by chemically modifying a physiologically active substance or a therapeutically active organic compound and freeing or releasing the parent compound under enzyme or other conditions in vivo. The prodrug becomes a target compound in vivo after administration. Although it is a useful drug, it has unsuitable properties in terms of side effects, stability, solubility, absorbability, duration of action, etc., and is chemically modified so that it can be used clinically.

[0298] According to the present invention, the active camptothecin derivatives preferably represented by the following chemical formula 1, for example, the camptothecin drugs of chemical formula 3, chemical formula 3-1, chemical formula 4, chemical formula 4-1, chemical formula 5, chemical formula 5-1, chemical formula 6, chemical formula 7, chemical formula 8 or chemical formula 9 designed to bind to the DDX5 protein and the E3 ligase can be used as payloads for various carrier-drug conjugates. That is, the various carrier-drug conjugates to which the active camptothecin derivatives are connected by the payload are a prodrug of the active camptothecin derivatives that release the active camptothecin derivatives in vivo. At this time, the carrier can be an antibody, a peptide, a repetitive body and / or an aptamer.

[0299]

[0300] For example, antibody-drug conjugates (ADCs) are a new drug platform that utilizes the high tissue selectivity of antibodies that specifically bind to specific antigens expressed on the surface of cancer cells to selectively deliver payloads with strong anti-cancer efficacy only to cancer tissues. ADCs selectively deliver powerful payloads that kill cancer cells only to cancer tissues at lower concentrations at the pM level and minimize systemic drug exposure, thereby ensuring both anti-cancer efficacy and safety.

[0301] Most ADCs are introduced into cells through the function of clathrin-coated pits. ADCs that move into cells fall off clathrin, fuse with other vesicles in the cell, and then proceed through the endosome-lysosome pathway. Next, proteases in the acidic environment of the endosome cut the linker group, and the activated "free" drug is transferred to the cytoplasm through the lysosomal membrane, where it binds to the molecular target of the drug, causing the cell cycle of tumor cells to stop and cancer cells to die due to apoptosis. Among them, a certain amount of drugs passively diffuse in the cell, actively transport, or flow out of the cell through dead cells. At this time, if the outflowing drug has cell membrane permeability, it may also enter the surrounding cells, resulting in the so-called bystander cell-killing phenomenon.

[0302] For HER2-positive cancer, according to the present invention, active camptothecin derivatives (a) or prodrugs thereof ADCs that use DDX5 protein as a drug target can become new therapeutic approaches to address resistance to anti-HER2 therapy.

[0303] The prodrug of the present invention may be an immunoconjugate or a pharmaceutically acceptable salt thereof, wherein the immunoconjugate comprises an antibody or a fragment containing the antigen binding site thereof designed to release the active camptothecin derivative (a) of the present invention in vivo.

[0304] The immunoconjugate of the present invention can bind to cancer cell antigen-specific antigens and release drugs inside and outside cancer cells, showing cytotoxicity, and therefore can be used for the treatment or prevention of cancer.

[0305] Aptamer-Drug Conjugate (ApDC) is an antibody that introduces an aptamer instead of an ADC. Aptamers are single-stranded nucleic acids with a three-dimensional structure. They are discovered through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. SELEX is a technology that adds targeted protein molecules to a compound library to obtain functional nucleic acids that bind to them.

[0306] Aptamers can bind to targets very strongly and selectively, so they are also called chemical antibodies. It is reported that aptamers are about 20 kDa in size and have excellent cell permeability and lower immunogenicity compared to antibodies.

[0307] Aptamers can be chemically synthesized, so when preparing aptamer-drug conjugates, the conjugation position and amount of the drug can be precisely designed. Compared with ADC, the production cost is low.

[0308] The conjugated aptamer is composed of natural nucleic acids, which will be decomposed by in vivo nucleases, resulting in decreased in vivo stability. However, the stability limitations of deformed aptamers can be overcome by utilizing the fact that aptamers are easily chemically deformed.

[0309] Peptide-Drug Conjugate (PDC) is a form of ADC that introduces peptides instead of antibodies. Peptides are composed of amino acids and range in size from 500 to 5000 Da (Daltons). This is very small compared to antibodies above 150 kDa (kilodaltons). Therefore, peptide-based PDC has better cell penetration ability than ADC, and the possibility of immunogenicity is very low. In addition, peptides can be chemically synthesized. Therefore, PDC not only has a very low production cost, but also can accurately control the conjugation position and ratio of peptides and drugs.

[0310] In general, peptides are easily degraded by proteases and thus have a short biological half-life. To overcome the limitations of peptide-based drug conjugates, strategies such as the use of cyclic peptides and the introduction of unnatural amino acids have been proposed to modify peptides.

[0311] 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.

[0312] Phage display is a technology that expresses desired proteins on the surface of bacterial phages. The size of a repetitive body is about 30 kDa, which is about 20% of an antibody. Therefore, compared with antibodies, it has relatively low immunogenicity and enhanced cell permeability. In addition, the thermal stability and pH stability of the repetitive body can also be adjusted, which is expected to improve structural stability. Compared with antibodies, the production cost is also evaluated as low. Due to the advantages of repetitive bodies, as a strategy to replace antibodies with repetitive bodies, the development of repetitive body-drug conjugates (Repebody-DC) has attracted increasing attention.

[0313] [Pharmaceutical composition for preventing or treating cancer]

[0314] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the compound represented by the above-mentioned Chemical Formula 1, for example, by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof or a prodrug thereof as an active ingredient.

[0315] The active camptothecin derivative (a) or its prodrug (b) represented by Chemical Formula 1 according to the present invention may be contained as an effective ingredient in a pharmaceutical composition for administration to a patient group in which DDX5 functions as an oncoprotein in cells.

[0316] For example, in a tissue biopsy or liquid biopsy, a complex (c) containing a ligand targeting DDX5 protein inactivated by linking the FL118 compound of Chemical Formula 2 or the above-mentioned active camptothecin derivative (a) preferably via a non-cleaving linking group to inactivate the ability to inhibit type I topoisomerase is used to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered.

[0317] According to the present invention, the active camptothecin derivative (a) represented by Chemical Formula 1, for example, the compound represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 is a camptothecin derivative of a new structure. Therefore, a drug candidate substance having more preferred properties in terms of efficacy, toxicity, selectivity, duration of action, administration, handling, stability and / or production possibility of a Dxd drug can be provided. In addition, the interaction between the target and the drug candidate substance, that is, the pharmacodynamic properties, can also be optimized, and the target of the drug can also be improved to be close to the optimization, that is, the pharmacokinetic ability.

[0318] In addition, one embodiment of the present invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of a compound represented by Chemical Formula 1, for example, Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8, or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof to a subject in need thereof. The subject may be a mammal including a human.

[0319] In the present invention, the above-mentioned cancers include 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 cancers or blood cancers. For example, the cancer can be one or more selected from the group consisting of pseudomyxoma, intrahepatic biliary 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, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, sinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningeal cancer. tumors, 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 cancer, gastrointestinal stromal cancer, Wilms' cancer, 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 cancer, 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 thymus cancer, but are not limited to these. Furthermore, the above-mentioned cancers include not only primary cancers but also metastatic cancers.

[0320] In this specification, "patient", "subject" and "object" refer to animals such as mammals. In a specific embodiment, the patient is a human. In another embodiment, the patient can be a non-human animal such as a dog, cat, livestock (e.g., a horse, pig or donkey), chimpanzee or monkey.

[0321] The term "therapeutically effective amount" used in the present invention refers to the amount of the compound represented by the above-mentioned Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5 or Chemical Formula 5-1, its pharmaceutically allowed salt, its solvate or its prodrug (prodrug) that is effective for treating or preventing cancer. Specifically, "therapeutically effective amount" refers to an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including individual type, severity, age, sex, disease type, drug activity, drug sensitivity, duration of administration, route of administration, excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent, or can be administered in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. In addition, it can be administered once or multiple times. Taking all the above factors into consideration, it is important to obtain the maximum effect with the minimum amount without causing side effects. The compounds represented by Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9 of the present invention and their pharmaceutically acceptable salts show dose-dependent effects. Therefore, a person of ordinary skill in the art can easily determine the dosage according to various factors such as the patient's condition, age, gender and complications. Since the active ingredients of the pharmaceutical composition of the present invention have excellent safety, it can be used even in excess of the determined dosage.

[0322] In addition, according to a specific example of the present invention, the present invention provides a use of a compound represented by the above Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof or a prodrug thereof for preparing a medicament for treating or preventing cancer. The compound represented by the above Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof or a prodrug thereof for preparing a medicament can be mixed with an acceptable auxiliary agent, diluent, carrier, etc., and prepared as a composite preparation together with other active agents, so as to have an ascending effect of the active ingredient.

[0323] Matters mentioned in the uses, compositions and methods of treatment of the present invention apply in the same manner unless they contradict each other.

[0324] In the present specification, the anticancer effect or therapeutic effect of an anticancer agent may refer to the effect of reducing the severity of cancer, shrinking the size of a tumor, or delaying or slowing down the progression of cancer, which occurs when a patient suffers from a specific cancer.

[0325] For example, the anticancer effect caused by the anticancer agent can be a change in the cell viability of the cancer cell (change in the degree of cytotoxicity or the number of cells) after the cancer cell is treated with the anticancer agent in vitro (in-vitro) and / or in vivo (in-vivo). For example, it can be indirectly determined by testing the drug response in a cell line (Cell line) or a non-clinical animal model (xenograft). In addition, the anticancer effect of the anticancer agent can be directly confirmed in cancer patients, and relevant data can be derived and used as a database. In addition, when designing the dosage guide of the anticancer agent, animal model PK parameters and / or toxicity characteristics (profile) can be considered in parallel.

[0326] The anticancer effect of an anticancer agent can be inferred from in vitro data, such as the maximum effect percentage of the drug, such as IC 50 、IC 60 、IC 70 、IC 80 and IC 90 , or inferred from in vivo data, such as peak blood concentration (Cmax) and / or area under the blood concentration-time curve (AUC) of the drug in nonclinical animal models and clinical cancer patients.

[0327] The responsiveness of an anticancer agent refers to the clinical sensitivity of its anticancer effect.

[0328] When referring to anticancer treatment, "sensitivity" and "responsive" are relative terms referring to how effective a compound is in reducing or decreasing the progression of the tumor or disease being treated.

[0329] An "effective patient anti-cancer effect / response" can be an inhibition of a patient's response, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, as measured by any suitable method, e.g., gene expression, cell counts, assay results, etc.

[0330] In this specification, the dosage refers to the dosage expected to produce the drug effect. In the present invention, the drug effect can be an anticancer effect. The responsiveness (anticancer effect) of the anticancer agent can be the degree of responsiveness, such as the maximum effect percentage (Maximum effect) of the anticancer agent, such as IC 50 、IC 60 、IC 70、IC 80 and IC 90 , or its toxic value to normal cells (LC 50 ).

[0331] For example, oral formulations can be formulated using various formulation techniques known in the art. For example, it can include a biodegradable (hydrolyzable) polymer carrier for adhering to the oral mucosa. They are formulated to be gradually eroded over a predetermined period of time, wherein the drug delivery is substantially intact.

[0332] In oral formulations, drug delivery avoids the attenuation encountered in oral drug administration, such as slow absorption, degradation of the active agent by the liquid present in the gastrointestinal tract and / or first-pass inactivation in the liver. For biodegradable (hydrolyzable) polymer carriers, virtually any such carrier can be used, as long as the desired drug release profile is not compromised, and the carrier is compatible with any other ingredients present in the oral dosage unit. Typically, polymer carriers include hydrophilic (water-soluble and water-swellable) polymers that adhere to the wetted surface of the oral mucosa. Examples of polymer carriers that can be used in this specification include acrylic polymers (e.g., carbomer). In some embodiments, non-limiting examples of other ingredients that can be incorporated into oral formulations include disintegrants, diluents, binders, lubricants, flavorings, colorants, preservatives, etc. In some embodiments, the preparation can be in the form of conventionally formulated tablets, lozenges, or gels for oral or sublingual administration.

[0333] In some embodiments, if the patient's condition improves, the compound may continue to be administered at the doctor's discretion; alternatively, the dose of the drug to be administered may be temporarily reduced or the drug may be temporarily discontinued for a period of time (i.e., a "drug break"). The length of the drug break may vary from 2 days to 1 year, and includes, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dosage reduction during the drug holiday is 10%-100%, including by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0334] If the patient's condition improves, a maintenance dose may be administered as necessary. Thereafter, the dose or frequency of administration, or both, may be reduced, depending on the symptoms, to maintain the improved level of the disease, disorder or condition. However, the patient may require intermittent treatment for a longer period of time upon any recurrence of symptoms.

[0335] The amount of a given preparation corresponding to this amount will vary depending on different factors, such as the specific compound, the severity of the disease, and the identity of the subject to be treated (e.g., body weight), but can still be routinely determined in a manner known in the art, for example, based on the preparation to be administered, the route of administration, and the specific circumstances of the subject to be treated. However, in general, the dosage for treating an adult is usually in the range of 0.02-5000 mg / day, or about 1-1500 mg / day.

[0336] In the present specification, a single dose may be provided as a single dose or may be provided as divided doses administered simultaneously, for example, in two, three, four or more divided doses.

[0337] In some embodiments, the oral formulation 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 appropriate amounts of one or more compounds. In some embodiments, the unit dose is in the form of a sachet containing different amounts of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-resealable containers. Alternatively, a multi-dose resealable container can be used, in which case the composition typically contains a preservative.

[0338] In some embodiments, parenteral injection formulations are provided in unit dosage form or in multi-dose containers, including but not limited to ampoules, together with an added preservative.

[0339] The unit dose injection form is usually formulated with a pharmaceutically acceptable parenteral carrier for parenteral administration, i.e., bolus, intravenous, and intratumoral injection. It can be freely mixed with a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1980), 16th edition, Osol, A. ed.) to form a lyophilized preparation or aqueous solution.

[0340] [Ligands that target DDX5 protein]

[0341] Therefore, not only FL118, but also the active camptothecin derivative represented by Chemical Formula 1 according to the present invention is a molecular adhesive that activates the degradation of tumor protein DDX5, and therefore, can be used as a ligand that targets DDX5 protein or a ligand that binds to DDX5 protein. Therefore, a complex containing a ligand that targets DDX5 protein can be provided, which is connected to the FL118 compound or the above-mentioned active camptothecin derivative (a) through a linking group to inactivate the ability to inhibit type I topoisomerase.

[0342] The linking group may be a non-cleaving linking group, and a non-limiting example of the non-cleaving linking group is a thioether linking group.

[0343] The FL118 compound or the active camptothecin derivative (a) represented by Chemical Formula 1 binds to the DDX5 protein, which is a direct target of the active camptothecin derivative (a).

[0344] Therefore, DDX5 can be used as a biomarker to predict drug sensitivity or tumor sensitivity.

[0345] In tissue biopsy or liquid biopsy, a complex (c) containing a ligand targeting DDX5 protein in which the ability to inhibit type I topoisomerase is inactivated by a linker in the FL118 compound of Chemical Formula 2 or the active camptothecin derivative (a) represented by Chemical Formula 1 is used to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to which the active camptothecin derivative (a) or its prodrug (b) is administered, or when a chemical anticancer agent is treated, the recurrence rate and / or prognosis can be predicted.

[0346] [Cancer diagnostic composition]

[0347] The present invention provides a cancer diagnostic composition, which comprises a complex (c) containing a ligand targeting DDX5 protein, wherein the complex (c) containing a ligand targeting DDX5 protein is connected to the FL118 compound of Chemical Formula 2 or the active camptothecin derivative (a) represented by Chemical Formula 1 through a linking group to inactivate the ability to inhibit type I topoisomerase.

[0348] The cancer diagnostic composition according to the present invention can be used to determine the patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered, or to predict the recurrence rate and / or prognosis when undergoing chemotherapy.

[0349] Effects of the Invention

[0350] According to the present invention, the camptothecin derivative represented by Chemical Formula 1, such as the camptothecin derivative represented by Chemical Formula 3 to Chemical Formula 9 or its isomers, is a compound of a new structure. Drug candidate substances with more preferred properties in terms of efficacy, toxicity, selectivity, duration of action, administration, handling, stability and / or production feasibility of Dxd drugs can be provided. In addition, the interaction between the target and the drug candidate substance, i.e., the pharmacodynamic properties, can also be optimized, and the target of the drug can be close to optimization, i.e., the pharmacokinetic ability can also be improved.

[0351] The present invention provides a compound of Chemical Formula 1 that maintains the drug structure of FL118, for example, compounds of Chemical Formulas 3 to 9 or isomers thereof, which maintain the advantage of FL118 of maximizing the modulation efficacy of the main drug target through an additional mechanism of action of (i) simultaneously inhibiting the Bcl family such as the resistance protein survivin, and / or (ii) inhibiting the action of an efflux pump.

[0352] Therefore, the compound of Chemical Formula 1, such as the compounds or isomers of Chemical Formulas 3 to 9, such as the FL118 drug, is designed based on a well-designed polypharmacology approach, which achieves excellent therapeutic effects by targeting multiple drug targets simultaneously with one drug, thereby treating refractory diseases for which existing therapeutic agents have shown significant therapeutic effects. In addition, recurrent / resistant cancers cannot obtain sufficient efficacy by regulating the activity of only one drug target, and when targeting multiple unspecific drug targets at the same time, there is a problem that safety cannot be guaranteed. However, the FL118 drug, which utilizes two or more selected drug targets, can reflect a synergistic effect in terms of efficacy while ensuring sufficient safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0353] Figure 1 The synthetic design concept of a novel active camptothecin derivative (a) having a dual mechanism of action (dual MoA) of inhibiting type I topoisomerase and degrading oncoprotein DDX5 by improving the structure of FL118 is shown.

[0354] Figure 2 The structural formula of camptothecin (CPT) and its binding to type I topoisomerase (topoisomerase-1) are shown, and the molecular design concepts of FL118 and PBX-7011 derived from camptothecin are expanded, as well as the new structural compounds PBX-7014 and PBX-7016 expanded from PBX-7011 and their hydrophilicity calculation results.

[0355] Figure 3 The following is an example of a method for synthesizing 25-4 and 25-6 using PBX-7014 and PBX-7016, similar to the process for synthesizing Deruxtecan using Dxd.

[0356] Figure 4 Molecular dynamics simulation and docking score were used to analyze the interaction between existing camptothecin drugs and TOP1.

[0357] Figure 5 Molecular dynamics simulation and docking score (Docking Score) are used to analyze the interaction between the novel camptothecin drugs of the present invention and TOP1.

[0358] Figure 6 Molecular dynamics simulation to analyze the interaction between camptothecin and mutant TOP1 (E418K).

[0359] Figure 7 The SN38-TOP1 interaction in Normal TOP1 is shown.

[0360] Figure 8 SN38-TOP1 interaction in E418K mutant TOP1 is shown.

[0361] Fig. 9 The compound of Chemical Formula 1-1 (Compound A)-TOP1 interaction in E418K mutant TOP1 is shown.

[0362] Figures 10 to 14 In order to verify the dual MoA (topoisomerase I inhibitor & DDX5 degradation) according to Example 2-1, the protein blotting results of the inhibition degree of anti-regression protein by various camptothecin drugs (FL118 drug, SN-38 drug, exitecan drug, PBX-7011, PBX-7014, PBX-7016) in FaDu cell line and A549 cell line are shown. 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 through a curve graph.

[0363] Fig.15 The results are the results of comparative evaluation of in vitro cell viability of various camptothecin drugs in FaDu cell line or A549 cell line.

[0364] Fig.16 The results are the results of a comparative evaluation of in vitro cell viability in MDA-MB-453 (HER2++) cell lines and FaDu (HER2+) cell lines in response to various camptothecin drugs.

[0365] Fig.17This is the result of carboxylate to lactone form conversion in pH 6.0.

[0366] Fig.18 This is the result of the conversion of lactone to carboxylate form in pH 7.4.

[0367] Fig.19 and Fig. 20 Results were confirmed for ADC preparation using SEC and SDS PAGE.

[0368] Fig.21 The results are to compare the binding ability of trastuzumab and ADCs conjugated with trastuzumab and PBX-7014 and PBX-7016 to Her2.

[0369] Fig. 22 The results of in vitro cell viability comparison were evaluated for various camptothecin drugs and ADCs with them as payloads in MDA-MB-453 (HER2++), FaDu (HER2+) and MDA-MB-468 (HER2-) cell lines.

[0370] Fig.23 The results are shown in an in vivo xenograft model of ADCs containing various camptothecin drugs as payloads in mice using the JIMT-1 cell line.

[0371] Fig.24 The results are as follows: body weight changes of mice using JIMT-1 cell line in an in vivo xenograft model for ADCs with various camptothecin drugs as payloads.

[0372] Fig.25 The results are as follows: MDA-MB-453 (HER2++) cell line and GFP-overexpressing MDA-MB-468 (HER2-) cell line were co-cultured, and the number of cells was counted by FACS based on the expression of GFP for ADCs using camptothecin drugs as the payload.

[0373] Fig.26The results are as follows: MDA-MB-453 (HER2++) cell lines and MDA-MB-468 (HER2-) cell lines were co-cultured, and the number of cells was counted by FACS using a HER2-FITC antibody based on the expression of HER2 for ADCs containing camptothecin drugs as the payload.

[0374] Fig. 27 A graph comparing the 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).

[0375] Fig.28 This is a graph comparing LogP, cLogP and tPSA of various camptothecin drugs derived from Chemical Formula 1, Compound G, Compound H, Compound I, Compound J, Compound G', Compound H', Compound I' and Compound J'.

[0376] Fig.29 A detailed classification diagram of cell death associated with programmed cell death. DETAILED DESCRIPTION

[0377] The present invention will be described in more detail below by way of examples. However, the following examples are only intended to illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[0378] Preparation Example 1: Synthesis of active camptothecin derivatives (PBX-7011 and PBX-7012) of Chemical Formula 3 or Chemical Formula 3-1

[0379]

[0380] 1-1. Synthesis of Compound 2

[0381]

[0382] Silver triflate (57.7 g, 224 mmol) and iodine (57.0 g, 224 mmol) were added to a solution of 5-nitrobenz[d][1,3]dioxole (25 g, 150 mmol) in a dichloromethane (748 ml) flask. The solution was stirred in a dark room at room temperature under N2 atmosphere.

[0383] 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 distributed between EtOAc (250 mL) and 5% (v / v) NH4OH / H2O solution (200 mL). The organic layer was separated and washed with 1M Na2SO3 (5×200 mL) and brine (200 mL), dried with Na2SO4, treated with activated carbon, and filtered through diatomaceous earth. The solvent was evaporated under reduced pressure to provide an unpurified product as a brown solid. It was crushed and filtered from ice-cold EtOH (400 mL), and the solid was washed with ice-cold EtOH (100 mL) to provide the resultant as a light brown-grey solid (14.3 g). The solvent was removed from the filtrate and the unpurified product was crushed a second time from ice-cold EtOH (300 mL). The solids were collected by filtration and washed with EtOH (50 mL) to provide an additional amount of product as a dark brown-grey solid (10.2 g). Both batches were used directly without further purification.

[0384] SC_ACID:m / z 294.2[M+H] +

[0385] 1-2. Synthesis of Compound 3

[0386]

[0387] 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]dioxole (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 (3×50 mL). The mixture was transferred to a separatory funnel and water was added and the aqueous layer was removed. The organic layer was washed with saturated aqueous NaHCO3 solution (150 mL) and brine (150 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure to obtain a brown solid (4.75 g, 60% yield). The iron residue was washed thoroughly with ethyl acetate to recover additional product. The organic fraction was then washed with saturated aqueous NaHCO3 solution (150 mL), brine (150 mL), and dried over Na2SO4. The solvent was removed under reduced pressure to obtain a brown solid (1.74 g, 22% yield).

[0388] SC_ACID:m / z 264.0[M+H] +

[0389] 1-3. Synthesis of Compound 4

[0390]

[0391] To a solution of 7-iodobenzoic acid [d][1,3]dioxol-5-amine (6.39 g, 24.29 mmol) in dichloromethane (49 ml) was added acetic anhydride (2.75 mL, 29.2 mmol) and triethylamine (4.06 mL, 29.2 mmol). The reaction mixture was stirred overnight at room temperature. 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 resultant was further dried under reduced pressure to obtain a light grey solid (4.46 g). The remaining solution was concentrated under reduced pressure and the residue was dissolved in EtOAc, washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure to obtain a brown solid (~3 g). The brown solid was purified by flash column chromatography (80 g Si, 0-100% acetoacetate in heptane). All batches of the resultant were crushed from ice-cold Et OAc (5-10 ml). The two batches were added together and further dried to give an off-white solid.

[0392] Total yield: 5.0 g, 66%

[0393] SC_ACID:m / z 306.0[M+H] +

[0394] 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).

[0395] 1-4. Synthesis of Compound 5

[0396]

[0397] In a three-necked flask equipped with a condenser, a slurry of N-(7-iodobenzoic acid [d][1,3] dioxol-5-yl) acetamide (5.06 g, 16.59 mmol), but-3-enoic acid (1.69 mL, 19.90 mmol) and potassium carbonate (2.98 g, 21.56 mmol) in acetonitrile (40 mL) was cooled to 0-5°C. Water (13.33 mL) was slowly added to generate gas. If the generation of gas was interrupted, the mixture was degassed with Ar for 30 minutes. Tri-O-tolylphosphine (0.505 g, 1.659 mmol) and palladium acetate (0.186 g, 0.829 mmol) were added, and the mixture was degassed for another 30 minutes and then heated under reflux under Ar. The reaction was cooled to room temperature and filtered through celite. The filter cake was washed with H2O and EtOAc. The organic solvent was removed from the filtrate under vacuum and the aqueous material was acidified 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, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude material was crushed from ice-cold EtOAc (30 mL), the solids were collected by filtration, and the resultant was provided as a brown solid. The mother liquor was concentrated under reduced pressure and purified by flash column chromatography (80 g Si, 0-100% EtOAc in heptane). The resultant was fractionated and concentrated under reduced pressure to obtain a light brown foam. Total yield: 3.46 g, 75% of a mixture of E / Z isomers was obtained.

[0398] SC_ACID:m / z 264.4[M+H] +

[0399] 1-5. Synthesis of Compound 6

[0400]

[0401] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)but-3-enoic acid (3.47 g, 13.18 mmol) in tetrahydrofuran (50 mL) / water (50 mL) was degassed with N2 for 15 min. Pd / C (2.97 g, 1.397 mmol) was added and the suspension was degassed with H2 for 5 min before heating to 40 °C under H2 (balloon). After 24 h, nitrogen and additional Pd / C (2.97 g, 1.397 mmol) were added to the reaction mixture. The reaction mixture was bubbled with hydrogen for 10 min and stirred at 45 °C under hydrogen overnight. The reaction was filtered through celite. The filter cake was washed with H2O (50 mL) and the filtrate was used to remove EtOAc (50 mL) and EtOAc (50 mL) and organic solvents under vacuum. The aqueous solution was acidified to pH=1 with concentrated HCl and a dark brown / green precipitate was collected by filtration through a sinter funnel. The aqueous filtrate was extracted. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent was removed under vacuum to obtain a brown oil. Due to the low recovery of the product, the filter cake was added to a flask containing solids from MeOH rinses, such as EtOAc (50ml), water (200ml) and NaOH (400ml), water / EtOAc rinses. The aqueous layer was acidified to a concentrated concentration. HCl and an off-white precipitate were formed until pH=1 and collected by filtration through a sinter funnel. After the aqueous filtrate was extracted with EtOAc (3×200mL), LCMS showed that all products had been removed from the aqueous. The combined organic phases were washed with brine, dried over Na2SO4, filtered, and the solvent was removed under vacuum to obtain a bright brown solid. All product batches were combined and purified by flash column chromatography (40 g Si, 0-10% MeOH in DCM). The product was fractionally concentrated to give a light brown solid. Yield: 2.52 g, 72%

[0402] SC_ACID:266.2[M+H] +

[0403] 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).

[0404] 1-6. Synthesis of Compound 7

[0405]

[0406] A suspension of 4-(6-acetamidobenzo[d][1,3]dioxol-4-yl)butanoic acid (150 mg, 0.565 mmol) in TFA (433 μL, 5.65 mmol) was cooled on ice. TFAA (157 μL, 1.131 mmol) was added. The mixture was stirred at 0-5°C for 1 hour, turning into a dark solution over time. The reaction mixture was added dropwise to an ice-cold saturated aqueous NaHCO3 solution (10 mL), and the aqueous solution was extracted with ethyl acetate (3×25 mL). The combined organic layers sat, the NaHCO3 and brine were dried over Na2SO4 and filtered, and the solvent was removed under vacuum to obtain a salmon pink solid. Yield: 140 mg, 100%

[0407] SC_ACID:m / z 248.2[M+H] +

[0408] 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).

[0409] 1-7. Synthesis of Compound 8

[0410]

[0411] A suspension of N-(6-oxo-6,7,8,9-tetrahydronaphthalene[1,2-d][1,3]dioxol-5-yl)acetamide (50 mg, 0.202 mmol) in tetrahydrofuran (1.2 mL) was cooled to 0°C, and potassium tert-butoxide (27.2 mg, 0.243 mmol) and isoamyl nitrosate (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, dust (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 obtain a black milky substance. The crude product was purified by flash column chromatography (4 g Si, 0-4% MeOH in DCM). The product was fractionated and concentrated to obtain a gray solid (32 mg, 52%) with a purity of 80-90%. A higher purity sample was obtained by purification using MPLC.

[0412] SC_ACID:m / z 305.4[M+H] +

[0413] 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).

[0414] 1-8. Synthesis of Compound 9

[0415]

[0416] N,N'-(6-oxo-6,7,8,9-tetrahydronaphthalene[1,2-d][1,3]dioxole-5,7-diyl)diethylamide (244 mg, 0.802 mmol) was suspended in 2M hydrochloric acid (4.69 mL, 9.38 mmol) in ethanol / water (5 / 1). The mixture was heated to 55°C 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 partitioned between water and DCM. The layers were separated and the aqueous layer was extracted once with DCM. The combined organic layers were dried over Na2SO4 and concentrated to obtain a brown solid product (178 mg, 73%) with 86% purity.

[0417] SC_ACID:m / z 263.0[M+H] +

[0418] 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).

[0419] 1-9. Synthesis of Compound 10

[0420]

[0421] (4S)-4-ethyl-7,8-dihydroxy-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-tetrahydronaphthalen[1,2-d][1,3]dioxol-7-yl)acetamide (112 mg, 0.427 mmol) were dried, toluene (4.5 ml) and PPTS (21 mg, 0.085 mmol) were added, and the reaction mixture was stirred at 115° C. for 40 hours.

[0422] 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.

[0423] 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%).

[0424] LCMS analysis showed 2 partial stereoisomers.

[0425] SC_ACID:m / z 490.2[M+H] +

[0426] 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).

[0427] 1-10. Synthesis of PBX-7011 and PBX-7012

[0428]

[0429] N-((10S)-10-ethyl-10-hydroxy-11,14-dioxo-2,3,10,11,14,16-hexahydroxy-1H,13H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (73.5 mg, 0.150 mmol) was dissolved in 1.0 mL of 6N HCl and stirred at 90°C for 8 hours and then at room temperature overnight. The reaction mixture was concentrated under reduced pressure and purified by MPLC twice (Luna2-30) by acidic fractionation. The fraction containing the separated stereoisomers was acidified with 5 drops of 3N HCl and freeze-dried to obtain a yellow solid product.

[0430] The first eluting isomer (1 st eluting isomer): PBX-7011, 23 mg, 34% yield

[0431] U_AN_ACID:m / z 448.4[M+H] +

[0432] 1 H NMR (400MHz, DMSO-d6) δ8.54(s,3H),7.50(s,1H),7.27(s,1H),6.52(s,1H),6.34(d,J=13.3Hz,2H),5.77(d,J=19.3Hz,1H),5.44(s,2 H),5.37(d,J=19.3Hz,1H),5.05(s,1H),3.19-3.02(m,2H),2.46(s,1H),2.20-2.03(m,1H),1.94-1.81(m,2H),0.88(t,J=7.3Hz,3H).

[0433] The second eluting isomer (2 nd Eluting isomer): PBX-7012, 28 mg, 41% yield

[0434] U_AN_ACID:m / z 448.2[M+H] +

[0435] 1H 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).

[0436] Preparation Example 2: Synthesis of active camptothecin derivatives (PBX-7014 and PBX-7015) of Chemical Formula 4 or Chemical Formula 4-1

[0437] In this example, the synthesis of two separated partial stereoisomers of exitecan-mixed compounds (PBX-7011 and PBX-7012) and compounds PBX-7014 and PBX-7015 are described.

[0438]

[0439] 2-1: Synthetic PBX-7014

[0440]

[0441] Prepare a stock solution of activated glycolic acid as follows.

[0442] 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.

[0443] Next, to a suspension of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydroxy-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (40 mg, 0.089 mmol) and triethylamine (0.025 ml, 0.179 mmol) in N,N-dimethylformamide (2.5 mL) was added 0.4 mL of the active acid solution. The mixture was stirred at room temperature for 3 hours. 0.05 mL of the freshly prepared active acid solution was added. Next, the reaction mixture was stirred at room temperature for an additional 2 hours. The reaction mixture was evaporated to dryness. The crude product was purified by column chromatography (0-8% methanol in chloroform). It provides a yellow solid containing PBX-7014 product and residual succinimide. The product is further purified by acid fractionation using MPLC (Luna5-40) and freeze-dried to obtain a bright yellow solid. Yield: 20 mg, 50%

[0444] U_AN_ACID:m / z 506.2[M+H] +

[0445] 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).

[0446] 2-2: Synthetic PBX-7015

[0447]

[0448] Prepare stock solutions of activated acids as follows.

[0449] 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.

[0450] Next, to a suspension of (1R,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydroxy-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyran[3',4':6,7]indolizino[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) was added 0.25 mL of the active acid solution. The mixture was stirred overnight at room temperature. 0.03 mL of the freshly prepared active acid solution was added. Next, the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was combined with the previous smaller batch and evaporated to dryness. The crude product was purified by column chromatography. It provides a yellow solid containing PBX-7015 product and residual succinimide. The product is purified by acid extraction with MPL C (Luna5-40). The product is fractionated and freeze-dried to obtain a bright yellow solid. Yield: 15 mg, 38%

[0451] U_AN_ACID:506.2[M+H] +

[0452] 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).

[0453] Preparation Example 3: Synthesis of the active camptothecin derivative (PBX-7016) of Chemical Formula 5

[0454] In this example, the synthesis of exitecan-mix (PBX-7011) and compound PBX-7016 are described.

[0455]

[0456] Prepare a stock solution of activated D-lactic acid as follows.

[0457] D-lactic acid (22 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (27 mg, 0.235 mmol) and EDC (38.6 mg, 0.201 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours.

[0458] Next, 0.3 mL of the active acid solution was added to a solution of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydroxy-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (36 mg, 0.080 mmol) and triethylamine (0.022 ml, 0.161 mmol) in N,N-dimethylformamide (2.5 ml). The mixture was stirred at room temperature for 6 hours. 0.05 mL of the prepared active acid solution was added. Next, the reaction mixture was stirred overnight at room temperature. The reaction mixture was directly purified by MPLC (Luna 10-50) for acid fractionation, and the product was freeze-dried and fractionated to obtain a bright yellow solid.

[0459] Yield: 22 mg, 52%

[0460] U_AN_ACID:m / z 520.2[M+H] +

[0461] 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).

[0462] As described above, PBX-7016 of Chemical Formula 5 can be synthesized using PBX-7011 of Chemical Formula 3. Therefore, PBX-7017 of Chemical Formula 5-1 can be synthesized using PBX-7012 of Chemical Formula 3-1 by the same method.

[0463] Preparation Example 4: Synthesis of the active camptothecin derivative (PBX-7016) of Chemical Formula 9

[0464]

[0465] The PBX-7011 compound was combined with (2S)-2-cyclopropyl-2-hydroxyacetic acid to prepare PBX-7024 in high yield.

[0466] (2S)-2-Cyclopropyl-2-hydroxyacetic acid (26 mg, 0.244 mmol) was dissolved in 1 mL of N,N-dimethylformamide. HOSu (26 mg, 0.226 mmol) and EDC (42 mg, 0.219 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. Next, 0.6 mL of the active acid solution was added to a solution of (1S,10S)-1-amino-10-ethyl-10-hydroxy-1,2,3,10,13,16-hexahydroxy-11H,14H-benzo[de][1,3]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11,14-dione (40 mg, 0.089 mmol) and DIPEA (0.047 ml, 0.268 mmol) in N,N-dimethylformamide (2.5 ml). The mixture was stirred overnight at room temperature. The reaction mixture was directly purified by acidic fractionation MPLC (Luna 10-50), and the product was freeze-dried and fractionated to obtain a bright white solid.

[0467] Yield: 32 mg, 65%

[0468] U_AN_ACID:m / z 520.2[M+H] +

[0469] 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).

[0470] Example 1: Analysis of TOP1 / DNA Binding Mode of Camptothecin Derivatives

[0471] Table 2

[0472]

[0473] According to the binding mode prediction results (Docking Score) simulated by molecular dynamics, all camptothecin derivatives in Table 2 are basically bound by strong π-π stacking between DNA bases ( Figure 4 and Figure 5 ), the lactone ester hydrogen bonds with Arg488 of TOP1, and the ethyl and OH groups hydrogen bonds with Asp533 of TOP1 (H-bonding)( Figure 2 ) to support the good binding of camptothecin derivatives between TOP1 protein and DNA.

[0474] SN-38 drug hydrogen bonds (H-bonding) with Glu356 and Arg364 of TOP1 and stabilizes ( Figure 4 ).

[0475] Exitecan forms hydrogen bonds (H-bonding) with the surrounding DNA bases and further stabilizes ( Figure 4 ).

[0476] The -OCH2O- (methylenedioxy) pentagonal ring of the FL118 drug is weaker than hydrogen bonding (unlike actual cytotoxicity) and shows a worse docking score than the SN-38 drug or the exotecan drug, but this binding is perfect between DNA bases and also helps solubilization ( Figure 4 ).

[0477] like Figure 5 As shown, according to the present invention, the compound of Chemical Formula 3 (PBX-7011) and the compound of Chemical Formula 3-1 (PBX-7012) designed by mixing the functional groups of the FL118 drug and the Ixitect drug are partial stereoisomers and are expected to share the same binding posture and stably bind to TOP1. In particular, the compound of Chemical Formula 3 (PBX-7011) is calculated to have a higher docking score than the SN-38 drug.

[0478] The compound of Chemical Formula 3 (PBX-7011) having the same chirality as the exitecan drug showed slightly better binding than the compound of Chemical Formula 3-1 (PBX-7012).

[0479] In addition, regarding the TOP1 mutation (E418K) that exhibits resistance to Trodelvy (payload: SN-38), Figure 6 As shown, the E418K mutation is far from the drug binding site.

[0480] like Figure 7 and Figure 8 As shown in the figure, the results of energy minimization after the E418K mutation were confirmed by molecular dynamics simulation (MD simulation) to confirm that the newly introduced Lys418 converts part of the drug binding site into a relatively polar environment and continues to bind ionically with the surrounding Glu356. Molecular dynamics simulation observed that if part of the binding site is polarized by mutation, the substance with polar functional groups, such as the SN-38 drug, will move slightly away from the existing binding site in order to interact ionically with Lys418 and Glu356. This will weaken the interaction between the lactone and Asp533 and Lys532, which is the principle of inhibitory activity shared by the same series of TOP1 inhibitors.

[0481] When the SN-38 drug was docked in the mutant TOP1 structure, although the SN-38 drug moved sideways from the original binding site, a new hydrogen bond (H-bonding) was obtained, showing a good binding docking score. Therefore, the docking score is limited to explain the phenomenon that the mutant TOP1 shows resistance to SN-38.

[0482] like Figure 7 and Figure 8 As shown, the interaction of SN-38 drug with TOP1 during molecular dynamics simulation was analyzed and it was found that the mutation reduced the interaction of Asp533 and Arg488 with SN-38 (89%) to less than 10%, but increased the interaction with Asn352 and Glu356 from 40% to 80%.

[0483] If docking is performed after TOP1 mutation, the SN-38 drug shifts to the newly deformed binding site, binds in a new form, and shows a score similar to the existing score (Table 2).

[0484] In contrast, as shown in Table 2, the relative docking scores of the ixotecan drug, FL118 drug, the compound of Chemical Formula 3 (PBX-7011), and the compound of Chemical Formula 3-1 (PBX-7012) without a polar functional group (-OH) at carbon No. 10 of the A-ring with the mutated TOP1 deteriorated due to partial environmental changes in the new binding site. Nevertheless, it was confirmed by molecular dynamics simulation that the ixotecan drug, FL118 drug, the compound of Chemical Formula 3 (PBX-7011), and the compound of Chemical Formula 3-1 (PBX-7012) maintained interactions with the existing lactone and Asp533 and Lys532.

[0485] like Fig. 9 As shown, even if TOP1 is mutated, the compound of Chemical Formula 3 (PBX-7011) still retains the existing binding mode (especially, lactone and -OH groups).

[0486] In summary, although the E418K mutation has no direct interaction with the TOP1 inhibitor, in order to illustrate that the FL118 drug has a high level of inhibition on the TOP1 mutation, the above-mentioned TOP1 mutation shows resistance to the SN-38 drug through E418K. It can be assumed that the new binding of SN-38 at the binding site deformed by the mutation is the following binding: Unlike the docking results, it is carried out in the form of a weakened action of lactone and Asp533 and Lys532, thereby failing to inhibit the action of TOP1.

[0487] Under the above assumption, the drug Exitecan, the drug FL118, the compound of Chemical Formula 3 (PBX-7011), and the compound of Chemical Formula 3-1 (PBX-7012) will also continuously act with Asp533 and Lys532 of mutated TOP1, thereby reducing resistance.

[0488] Example 2

[0489] For FL118, exotecan, SN-38, Dxd, exotecan-lactic acid, PBX-7011, PBX-7014, PBX-7016, and PBX-7024, an anti-apoptotic protein expression inhibition assay and an in vitro cell viability assay were performed as described below.

[0490] 2-1: Confirmation of the expression inhibitory activity of cancer-associated survival genes DDX5, survivin, Mcl-1, XIAP, and cIAP2 by Western blot

[0491] (1) Protein extraction

[0492] 200,000 FaDu cells were seeded per well of a 6-well plate and cultured at a constant temperature (37°C, 5% CO2). 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 0nM, 10nM, and 100nM, respectively. Cultured at a constant temperature (37°C, 5% CO2) for 24 hours. 100 μl of RIPA buffer containing a protease inhibitor cocktail was treated in the wells. The plate was embedded in ice and cultured at a constant temperature in an orbital shaker for 2 hours. The RIPA buffer containing the lysed cells was added to an ep tube and centrifuged (16000rcf, 20 minutes, 4°C). Afterwards, only the supernatant was moved to a new ep tube. The protein concentration was confirmed by protein assay.

[0493] (2) Protein separation by electrophoresis

[0494] Mix protein sample and 4x SDS-PAGE loading buffer in a ratio of 3:1, boil at 95°C for 10 minutes and then cool. Load the sample into the wells of the gel to make the protein amount equal. Perform electrophoresis in the gel at 60V.

[0495] (3) Transferring proteins from gel to membrane

[0496] exist Turbo TM On the cassettes of the Transfer System, place 7 activated filter papers, PVDF membrane, gel and 7 filter papers in sequence and close the lid, insert into the machine and start the protocol.

[0497] (4) Antibody incubation

[0498] The transferred membrane was immersed in blocking buffer and incubated at a constant temperature (RT, 1 hour). Then, incubated at a constant temperature (4°C, overnight) in the primary antibody solution. Rinse with TBST buffer for 3 minutes (repeat 3 times). Incubate at a constant temperature in the secondary antibody solution conjugated with HRP (RT, 1 hour). Rinse with TBST buffer for 3 minutes (repeat 3 times).

[0499] (5) Analysis of results with images

[0500] After the membrane is immersed in the ECL substrate for 3 to 5 minutes, use ChemiDoc TM The MP Imaging System confirms the signal. The HRP bound to the secondary antibody detects the light released by the luminol oxidized by the ECL and displays it 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.

[0501] like Fig.10 and Fig.12 As shown, the changes in protein expression levels were confirmed by drug treatment (FL118 drug, SN-38 drug, Exitecan drug, PBX-7011, PBX-7014, PBX-7016). GAPDH is an enzyme involved in glycolysis, an essential metabolic process in cells. It is a gene that is constantly expressed in cells and its expression level remains constant. It is an indicator that can tell whether the same amount of protein in the sample is loaded on the gel.

[0502] Instead of using the same method to seed A549 cells that overexpress ABCG2, 4 μg of protein was loaded and Western blotting was performed ( Fig.13 and Fig.14 ).

[0503] Discussion: Review of PBX-7011, PBX-7014 and PBX-7016

[0504] The results of Western blotting to confirm the degree of inhibition of the expression of anti-apoptotic proteins in the FaDu cell line showed that PBX-7011, PBX-7014 and PBX-7016 showed that the expression of survivin, cIAP2, Mcl-1, and XIAP decreased with increasing concentrations, and compared with the existing FL118, SN-38, and isotecan drugs, they showed levels similar to FL118 and isotecan drugs, and better than SN-38.

[0505] In Example 2-1, it was confirmed that the dual inhibitory action of inhibiting topoisomerase 1 and simultaneously degrading DDX5 was shown, and that the so-called heterogeneous characteristics of DDX5 degradation and its anticancer effect were possessed.

[0506] Specifically, two cell lines (FaDu, A549) were treated with drugs at certain concentrations (0nM, 10nM, 100nM), and the expression levels of each protein (DDX, survivin, Mcl-1, XIAP) were confirmed by western blotting. The results showed that FL118 had better DDX5 degradation than other substances, followed by 7011, 7016, and 7014.

[0507] Except for DDX5, other downstream anti-apoptotic proteins have similar tendencies.

[0508] In conclusion, PBX-7014 and PBX-7016 not only inhibit type I topoisomerases, but also display dual MoA as degradation of the tumor protein DDX5 (p68) that regulates survivin, Mcl-1, XIAP, etc.

[0509] The evaluation results showed that, among PBX-7014 and PBX-7016, PBX-7016 inhibited DDX5 in a concentration-dependent manner, and the results showed that it inhibited survivin, Mcl-1, and XIAP. Thus, PBX-7016 showed its role as a topoisomerase I inhibitor and DDX5 degradation of FL118, and it was also confirmed that PBX-7016 worked better than PBX-7014 ( Figures 11 to 14 ).

[0510] 2-2: Cell viability assay for FaDu cell line / A549 cell line

[0511] In a 96-well plate, 3000 FaDu cell lines (cancer cells that do not express ABCG2) or A549 cell lines (cancer cells that overexpress ABCG2) were inoculated per well and cultured at a constant temperature (37°C, 5% CO2). After 24 hours, 100 μl of drugs at 9 concentrations (serial dilutions from 1000 nM to 1 / 5) were treated in the cells. 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. Culture was carried out at a constant temperature for 3 or 6 days (37°C, 5% CO2). 100 μl of CellTiter-Glo reagent (using Luminescent Cell Viability Assay kit (Promega, G7571)) was used for pipetting. After 10 minutes of constant temperature incubation (RT), luminescence was measured. When the luminescence value at drug concentration 0 was 100%, the concentration representing 50% luminescence value was IC 50 value.

[0512] Two cell lines (FaDu, A549) were treated with two compounds (PBX-7014, PBX-7016, PBX-7024) and reference compounds (Dxd, SN-38, Exitecan, FL118), and cell viability was observed by incubation for 3 days.

[0513] like Fig.15 As shown in the results, PBX-7024 showed strong cell killing effect not only in the FaDu cell line that does not express ABCG2, but also in the cancer cell line A549 that overexpresses ABCG2. In other words, it was confirmed that the IC 50 .

[0514] In the cancer cell line A549 overexpressing ABCG2, it was confirmed that Fig.15 The camptothecin compounds such as Dxd shown in FIG. 50 Compared with the numerical values, PBX-7016 and PBX-7024 still maintain strong effectiveness.

[0515] That is, when the novel camptothecin compounds PBX-7016 and PBX-7024 of the present invention are used, unlike ADCs using existing camptothecin compounds SN-38, DXd, etc., they have the effect of overcoming the resistance mechanism caused by overexpression of ABCG2.

[0516] Example 3. New PBX-series compounds as candidate for ADC payload: in vitro cell viability test

[0517] The cell viability analysis was performed on MDA-MB-453 (HER2++) cell line and FaDu (HER2+) cell line in the same manner as in Example 2-2.

[0518] 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 the cell viability was observed by incubation for 3 days. The results are shown in Fig.16 middle.

[0519] like Fig.16 As shown in the evaluation results, PBX-7016, PBX-7018, PBX-7020, and PBX-7022 have ICs that are at or above the same level as Dxd. 50 In addition, it was confirmed that the FaDu cell line that does not express ABCG2 has an IC level equivalent to or higher than that of Dxd. 50 value.

[0520] Example 4: PBX-7016 IV and IP Pharmacokinetic Profile in NOD SCID Mice

[0521] The experimental methods are shown in Table 3 below. The IV plasma concentration-time data of PBX-7016 are shown in Table 4, and the IP plasma concentration-time data of PBX-7016 are shown in Table 5.

[0522] Table 3

[0523]

[0524] Table 4

[0525]

[0526] Table 5

[0527]

[0528] From the results in Tables 4 and 5, when the PBX-7016 compound is administered intravascularly or intraperitoneally to mice, clearance in the blood can be confirmed in a very short time, and it is expected that the PBX-7016 compound is highly safe when administered to the body alone, rather than in the form of a prodrug or ADC. When the PBX-7016 compound is administered IV, it can be released from the body in a very short time and enter the body, and when the drug is released prematurely, it can also be used as a safety device to reduce systemic exposure of the drug.

[0529] In addition, this experiment is a PK study of the PBX-7016 compound used as a payload alone, suggesting that it is a safe payload that produces clearance of the PBX-7016 drug in the blood in a short period of time. In other words, after the PBX-7016 compound exerts a bystander effect at the tumor site, it can be safely released to the body in a short period of time while circulating in the blood.

[0530] Example 5. Carboxylate to Lactone conversion rate at pH 6.0 in PBX-7016

[0531] Example 5 Fig.17 and Fig.18) is an experiment related to the activation of the lactose portion of camptothecin, which is known as a Top1 inhibitor activity factor.

[0532] 5-1. Carboxylate to lactone form conversion in pH 6.0

[0533] PBX-7016 powder was dissolved in DMSO at 5 mM and prepared. PBX-7016 dissolved in DMSO was present in lactone form and was converted to carboxylate form by diluting 1 / 10 with 0.1 N NaOH and incubating in a 25°C water bath for 30 minutes. The carboxylate form of PBX-7016 was diluted to 3 μM with 4°C PBS (pH 6.0) containing 10% DMSO. At this time, HCl was added in an amount equal to the amount of 0.1 N NaOH used to minimize pH changes. After dilution, the mixture was centrifuged at 4°C, 2 minutes, 13,000 RCF, and the supernatant was used as a sample.

[0534] When the lactone form of PBX-7016 was generated, two solvents were used to separate it by RP-HPLC, namely, solvent A: 100 mm acetic acid buffer, pH 5.5, and solvent B: ACN. The column used was a Cortecs C18 2.7 μm, 4.6×50 mm column, and the analysis was performed under the following conditions: 1) 20 minutes: 5% B, 2) sample injection, 3) 2 minutes: 5% B, 4) 10 minutes: 5% B→50% B, 5) 1 minute: 50% B.

[0535] In order to measure the change over time, the sample chamber was maintained at 37°C, and the sample was injected at each measurement time. The measurement time was 0 min, 20 min, 40 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min, 480 min, 600 min, 720 min, 840 min, 960 min, 1200 min, 1440 min.

[0536] The contents were calculated using the peak areas of the lactone and carboxylic acid ester. Since the extinction coefficients of lactone and carboxylic acid ester are different, the lactone area was calculated by multiplying the carboxylic acid ester area by the following formula.

[0537] Lactone EC 390 / Carboxylate EC 390 =0.918

[0538] At pH 6.0, the results of the conversion experiment from carboxylate to lactone form showed that Fig.17 In. Fig.17 As shown, it was confirmed that various camptothecin compounds were converted from carboxylic acid esters to active lactones that inhibit topoisomerase I at similar rates and in equivalent amounts at pH 6.0, which can be predicted to have equivalent activities in a micro-tumor environment (Tumor Micro-environment) at a pH of 6.0.

[0539] 5-2. Lactone to carboxylate form conversion in pH 7.4

[0540] PBX-7016 powder was prepared by dissolving it in DMSO at 5 mM, and then diluted with 4°C PBS (pH 7.4) containing 10% DMSO to prepare 3 μM PBX-7016. After that, it was centrifuged at 4°C, 2 minutes, 13000 RCF, and the supernatant was used as a sample. PBX-7016 dissolved in DMSO exists in the form of lactone.

[0541] When the lactone form of PBX-7016 was generated, two solvents were used to separate it by RP-HPLC, namely, solvent A: 100 mm acetic acid buffer, pH 5.5, and solvent B: ACN. The column used was a Cortecs C18 2.7 μm, 4.6×50 mm column, and the analysis was performed under the following conditions: 1) 20 minutes: 5% B, 2) sample injection, 3) 2 minutes: 5% B, 4) 10 minutes: 5% B→50% B, 5) 1 minute: 50% B.

[0542] In order to measure the change over time, the sample chamber was kept at 37°C and the sample was injected at each measurement time. The measurement time was 0 min, 20 min, 40 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min, 480 min, 600 min, and 720 min.

[0543] The contents were calculated using the peak areas of the lactone and carboxylic acid ester. Since the extinction coefficients of lactone and carboxylic acid ester are different, the lactone area was calculated by multiplying the carboxylic acid ester area by the following formula.

[0544] Lactone EC 390 / Carboxylate EC 390 =0.918

[0545] At pH 7.4, the results of the conversion experiment from lactone to carboxylate form showed that Fig.18 In. By Fig.18The results confirmed that at pH 7.4 in the blood, the PBX-7016 compound not only converts from the lactone form to the carboxylate form faster than other reference compounds (exitecan, DXd, SN-38, FL118), but also converts at a higher ratio from the lactone form to the carboxylate form. It is predicted that the PBX-7016 compound is not an active lactone that exhibits cytotoxicity relatively quickly and at a high ratio in the blood, but exists as an inactive carboxylate, compared with the reference compounds, and thus exhibits high safety in normal cells.

[0546] Preparation Example 5: Synthesis of 25-4 and 25-6 using PBX-7014 and PBX-7016 and preparation of their ADC (DAR7-8) (trastuzumab-25-4 and trastuzumab-25-6)

[0547] Linker-payloads 25-4 (Chemical Formula 10) and 25-6 (Chemical Formula 11) were synthesized, which included two compounds (PBX-7014, PBX-7016) and introduced GGFG as an enzymatically cleavable linker system.

[0548] [Chemical formula 10]

[0549]

[0550] [Chemical formula 11]

[0551]

[0552] The molecular structure of 25-4 is GGFG-PBX-7014, and the molecular structure of 25-6 is GGFG-PBX-7016. That is, each uses the same Same GGFG linker.

[0553] Furthermore, using The same GGFG linker and trastuzumab were used to prepare their ADCs with PBX-7014 and PBX-7016 as payloads.

[0554] ADCs were synthesized by conjugating the linker-payload compounds of Chemical Formula 10 and Chemical Formula 11 to trastuzumab, a Her2-targeting antibody (Tra-25-4 and Tra-25-6, both of which are DAR8).

[0555] Trastuzumab-25-4 (trastuzumab-7014) was prepared by the following method. The prepared trastuzumab was subjected to buffer exchange with a reaction buffer (150 mM NaCl, 50 mM histidine, pH 6.0) using a PD-10 desalting column, and then 825 μM TC EP was treated on 27.5 μM antibody at 25°C for 2 hours to obtain the thiol site required for the reaction from the disulfide of the antibody.

[0556] After that, excess TCEP was removed using a PD-10 desalting column, and 61.9 μM 25-4 drug linker (Chemical 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.

[0557] Trastuzumab-25-6 (Trastuzumab-7016) was prepared by the same process, using the same concentration of 25-6 drug linker (Chemical Formula 11) instead of 61.9 μM of 25-4 drug linker (Chemical Formula 10).

[0558] Afterwards, each ADC was purified using SEC and confirmed to be free of aggregation and purified as a monomer ( Fig.19 The drug was conjugated by SDS-PAGE, and the band shift of the light chain and the heavy chain confirmed that the drug was conjugated. Fig. 20 ).

[0559] Example 6. Anti HER2 ADCs with PBX-payloads: Binding affinity analysis

[0560] The coating buffer is prepared by diluting the target antigen to 0.66μg / ml using ELISA plate coating buffer (R&D system, #DY006). Use 100μL of the prepared coating buffer to coat each well. Cover the plate lid, maintain at 2-8°C, and perform overnight (12 hours to 18 hours). The plate used is Corning, #3590. Wash three times with more than 200μL of TBS Tween-20 buffer (thermo, #28360). When washing, first remove the liquid with a pipette, and gently tap it on a paper towel to remove excess buffer. Block with 200μL of detection buffer at room temperature for 1 hour. Remove the liquid with a pipette, and gently tap it on a paper towel to remove excess buffer. Prepare the control antibody (Control antibody) and ADC to be measured at 1μg / ml in the detection buffer, and prepare the required amount or more at the following concentrations by 1 / 5 serial dilution. For example, the samples can be prepared by diluting the duplicate standards [240 μl detection buffer + 60 μl sample of the previous concentration] to the following concentrations: 1 μg / ml, 0.4 μg / ml, 0.08 μg / ml, 0.016 μg / ml, 0.0032 μg / ml, 0.00064 μg / ml, 0.000128 μg / ml. Repeat the binding of the diluted control antibody and ADC in 100 μL in each coated well. Perform 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, and gently tap on a paper towel to remove excess buffer. Prepare the detection antibody by diluting it in the detection buffer according to the manufacturer's instructions. For human antibodies, use loading anti-human Fc-HRP (Novex, #A18817) as the detection antibody and prepare it at a dilution of 1 / 2000. After adding 100 μL of the diluted detection antibody to each well, 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 and gently tap on a paper towel to remove excess buffer. React at room temperature for 30 minutes with 100 μL of TMB substrate solution (Thermo, #N301). Stop the reaction with 100 μL of stop solution (Thermo, N600). Stop the reaction and measure the absorbance at 450 nm. Complete within 30 minutes after adding the stop solution. When foam is present, move 100-150 μL to a new plate and measure. Thus, the ADC conjugated with trastuzumab, trastuzumab and PBX-7014, and trastuzumab and PBX-7016 was measured. Fig.21 The result is Fig.21As shown, when the binding strength of trastuzumab to Her2 antigen and the binding strength of trastuzumab-conjugated ADC to Her2 antigen were compared at various concentrations, no difference in binding strength was confirmed.

[0561] Example 7. Anti HER2 ADCs with PBX-payloads: in vitro potency and target selectivity

[0562] For ADCs with Dxd, PBX-7014 and PBX-7016 as payloads, respectively, the following in vitro cell viability analysis was performed on MDA-MB-453 (HER2++) cell line, FaDu (HER2+) cell line and MDA-MB-468 (HER2-) cell line using the same method as in Example 2-2.

[0563] 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 as its ADC were treated.

[0564] As a reference, Enhertu (Tra-Dxd, DAR8) was treated in three cell lines MDA-MB-453 (Her2++), FaDu (Her2+), and MDA-MB-468 (Her2-) according to the expression level of Her2, and the cell viability was observed by culturing for 3 days ( Fig. 22 ).

[0565] like Fig. 22 As shown, it was confirmed that the anti-Her2 ADC with PBX-payload had good target selectivity.

[0566] Discussion: Evaluation of PBX-7014 and PBX-7016 as ADC payloads

[0567] like Fig. 22 As shown in the figure, it was confirmed that in the MDA-MB-453 as Her2++, the ADC (Tra-25-6) with PBX-7016 as the payload has an excellent IC of about 5 times higher than that of Enhertu (Tra-Dxd). 50The IC values ​​were also confirmed to be equivalent in the negative cell line MDA-MB-468. 50 The results also confirmed that when the linker-payload systems of Chemical Formula 10 and Chemical Formula 11 were developed as ADCs, they not only distinguished between positive / negative cell lines to work, but also showed excellent efficacy compared to Enhertu as a reference.

[0568] Discussion: The originality and excellence of PBX-7016

[0569] According to the present invention, the camptothecin compound PBX-7016 redesigned and synthesized in Chemical Formula 1 is a compound newly derived from the MD-CPT skeleton FL118, which not only acts as a topoisomerase I inhibitor, but also acts as a DDX5 degrader, and a new topoisomerase 1 inhibitor is newly proposed, which can degrade DDX5 ( Figures 11 to 14 ).

[0570] In addition, when simply comparing cell viability, PBX-7016 showed 1.5- to 2-fold higher IC compared to Dxd. 50 values, but surprisingly, when used as ADC payload, ADCs containing PBX-7016 had approximately 5-fold increased cell viability compared to ADCs containing Dxd (Enhertu) ( Fig. 22 ).

[0571] Example 8. Anti HER2 ADCs with PBX-payloads: in vivo efficacy

[0572] 8-1. Cultivation of cells

[0573] JIMT-1 breast cancer cells were cultured in DMEM containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin at 5% CO2 and 37°C, treated with trypsin-EDTA, and subcultured twice a week by conventional methods. Cells growing in the exponential growth phase were harvested and counted for tumor inoculation.

[0574] 8-2.BALB / c nude mice

[0575] Both SCID mice and BALB / c nude mice are used as animal models to study the immune system and test treatments for various diseases.

[0576] The immune systems of both SCID mice and BALB / c nude mice are impaired, but the underlying mechanisms of the immunodeficiency are different.

[0577] SCID mice are genetically engineered mice that lack functional T cells and B cells and have a severely compromised immune system. They are used to study immune system development and function and to test potential treatments for conditions such as autoimmune disorders and specific types of diseases that affect the immune system.

[0578] In contrast, BALB / c nude mice are mice that cannot produce T cells because they do not have a thymus gland. They are also born without hair due to a genetic defect that causes hairlessness. This mouse is 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.

[0579] 8-3. Tumor inoculation and drug treatment

[0580] JIMT-1 tumor cells (5×10 6 ) were subcutaneously inoculated into the right anterior rib of BALB / c nude mice. When the average tumor volume reached about 215 mm 3 At 14:00, the animals were randomly divided into groups and the treatment for the drug efficacy study was started.

[0581] Treatment: IV, Q1W

[0582] Blank control group: vehicle, 0 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0583] Isotype Dxd (3) group: CTP63-Dxd, 3 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0584] Isotype Dxd (10) group: CTP63-Dxd, 10 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0585] Isotype PBX7016 (3) group: CTP63-PBX7016, 3 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0586] Isotype PBX7016 (10) group: CTP63-PBX7016, 10 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0587] Isotype PBX7014 (3) group: CTP63-PBX7014, 3 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0588] Isotype PBX7014 (10) group: CTP63-PBX7014, 10 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0589] Trastuzumab PBX7014 (3) group: CTP6-PBX7014, 3 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0590] Trastuzumab PBX7014 (10) group: CTP6-PBX7014, 10 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0591] Trastuzumab PBX7016 (3) group: CTP6-PBX7016, 3 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0592] Trastuzumab PBX7016 (10) group: CTP6-PBX7016, 10 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0593] Enhertu (3) group: Enhertu, 3 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0594] Enhertu (10) group: Enhertu, 10 mg / kg, 5 μL / g, intravenous injection, twice (day 0, day 6)

[0595] 8-4. Tumor Measurements and Endpoints

[0596] The primary endpoint was a time point that would determine whether tumor growth was delayed or whether mice could be treated. The two-dimensional size of the tumor was measured twice a week using a caliper, and the volume was calculated using the formula (V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively) and are expressed in mm 3 express.

[0597] Tumor growth inhibition (TGI) was calculated for each group.

[0598] TGI (%) = [1-(T i -T0) / (V i -V0)]×100

[0599] (T i is the mean tumor volume of the treatment group on a given day, T0 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 blank control group on the same day, V0 is the mean tumor volume of the blank control group on the first day of treatment).

[0600] Weight loss of more than 15% or tumor volume 3000mm 3 The above animals were euthanized as a humane endpoint.

[0601] 8-5. Statistical analysis

[0602] Results are presented as mean ± SEM. Data were analyzed using Graphpad Prism 6.0 software using two-way RM ANOVA Dunnett's multiple comparison test, and p < 0.05 was considered statistically significant.

[0603] Table 6 below shows the tumor volume (Tumor Volume) (mm 3 ) over time (effect vs. time).

[0604] Table 6

[0605]

[0606] The curve diagram shows that Fig.23 The figures show the results of efficacy tests on ADCs with various camptothecin drugs as payloads in an in vivo xenograft model in mice transplanted with JIMT-1 cells.

[0607] 8-6. Body weight of JIMT-1 tumor model

[0608] The body weight of JIMT-1 tumor model mice was monitored regularly ( Fig.24 ).

[0609] Example 9: Anti HER2 ADCs with PBX-payload: Bystander effect study

[0610] The bystander effect is one of the important factors affecting the efficacy of ADC drugs. In order to confirm the bystander effect, FACS analysis was performed under 40 nM ADC treatment conditions.

[0611] In the antibody-drug conjugate (ADC) with a payload-linker group bound to trastuzumab targeting the HER2 protein, in order to verify the efficacy and utility of the lead substance PBX-7016 of the present invention as a payload as an ADC drug, a flow cytometric analysis experiment was performed in vitro to confirm the bystander effect. The ADC was treated on the HER2-positive cell line MDA-MB-453 to induce cell death. The drug (payload) released during the cell death process was toxic to the surrounding HER2-negative cell lines, resulting in cell death, which is defined as the bystander effect in this embodiment.

[0612] In Preparation Example 5, in order to evaluate the in vitro bystander effect of the prepared ADC, HER2-negative cell lines and HER2-positive cell lines were incorporated into a 6-well cell plate to evaluate whether ADC treatment produced a bystander effect under proportional co-culture conditions.

[0613] Specifically, the HER2 expression negative cell line MDA-MB-468 was mixed with GFP-MDA-MB-468 cells (3×10 5 cells) and HER2-positive cell line MD A-MB-453 cells (1×10 5 Cells) were mixed at a ratio of 3:1 and co-cultured for 24 hours. Trastuzumab and a total of five ADC samples (trastuzumab-DXd, isotype IgG-DXd, trastuzumab-25-6, isotype IgG-25-6, Enhertu) were treated at a concentration of 40 nM, respectively. After 6 days of cell culture, the GFP fluorescence measurement and cell number of the HER2 expression-negative cell line GFP-MDA-MB-468 cells in living cells were confirmed by flow cytometry.

[0614] At the same time, HER2 negative cell line MDA-MB-468 cells (3×10 5 cells) and HER2-positive cell line MDA-MB-453 cells (1×10 5The cells were mixed at a ratio of 3:1 and co-cultured for 24 hours. Trastuzumab and a total of 5 ADC samples (trastuzumab DXd, isotype IgG-DXd, trastuzumab 25-6, isotype IgG-25-6, Enhertu) were treated at a concentration of 40 nM, respectively. After 6 days of cell culture, for antibodies that can recognize HER2 protein, cells were stained with anti-HER2-FITC antibodies labeled with fluorescein isothiocyanate (FITC) fluorescent dye to show fluorescence. FITC fluorescence measurement and cell number of HER2-positive cell line MDA-MB-453 cells in living cells were confirmed by flow cytometry analysis.

[0615] For flow cytometry analysis, cells present in each well of a 6-well cell culture plate were collected and diluted with a buffer (400 μl). Then, GFP or FITC fluorescence was measured for the same time (1 min each) and at the same flow rate for each sample using an FL1 laser.

[0616] like Fig.25 and Fig.26 As shown, the results confirmed by flow cytometry analysis showed that the PBX-7016-based ADC trastuzumab-25-6 killed not only HER2-positive cells, but also HER2-negative cells compared with the DXd-based ADCs trastuzumab-Dxd and Enhertu, thereby showing a bystander effect at the same level as the DXd-based Enhertu, while in contrast, the isotype IgG-DXd and isotype IgG-25-6, which served as negative control ADCs, did not induce significant HER2-negative cell death compared with the condition of untreated ADC.

[0617] In conclusion, the anti-HER2 ADC using PBX-7016 demonstrated a similar bystander effect as Enhertu.

Claims

1. (a) an active camptothecin derivative represented by the following chemical formula 1, which is designed to bind to the DDX5 protein and the E3 ligase; or (b) a prodrug of an active camptothecin derivative, which is designed to release the active camptothecin derivative (a) at a target site in vivo; or (c) a complex containing a ligand targeting the DDX5 protein, which is connected to the active camptothecin derivative (a) or the FL118 compound of chemical formula 2 through a linking group to inactivate the ability to inhibit type I topoisomerase, [Chemical formula 1] 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 6- or 7-corner ring, n = 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 to bind to the DDX5 protein and the E3 ligase functions as a ligand that binds to the E3 ligase via a molecular adhesive.

3. The camptothecin derivative (a) or its prodrug (b) according to claim 1, characterized in that: Active camptothecin derivatives (a) designed to bind to DDX5 protein and E3 ligase cause the death of target cells expressing DDX5 protein through a molecular adhesive mechanism.

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

5. The camptothecin derivative (a) or its prodrug (b) according to claim 4, characterized in that: (1) In Formula 1 or Formula 2, the group C site selectively binds to type I topoisomerase and the group A site binds to DNA to stabilize the covalent bonds of the topoisomerase-DNA complex and prevent the reconnection of the cut DNA fragments and / or, (2) In Formula 1 or Formula 2, the group A site binds to DDX5 and selectively, the group C site binds to E3 ligase to induce the decomposition of DDX5, [General formula 1] [Chemical formula 2] 6. A pharmaceutical composition or cancer diagnostic composition for preventing or treating cancer, The invention comprises the active camptothecin derivative (a), the prodrug thereof (b), or the complex (c) containing a ligand targeting DDX5 protein according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, characterized in that After cancer diagnosis, it is used as a one-time therapeutic agent or administered to individuals who express or overexpress DDX5 in cancer tissue.

8. The pharmaceutical composition according to claim 6, characterized in that It is used to treat HER2-positive tumors, breast cancer, lung cancer, and colorectal cancer.

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

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

11. The pharmaceutical composition according to claim 6, characterized in that Blocking the transcriptional induction of anti-apoptotic genes through active camptothecin derivatives.

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

13. The pharmaceutical composition according to claim 6, characterized in that Convert a solid cancer that is clumpy due to high cell density into a dispersed cancer with low cell density, and / or convert 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 is used to identify a group of patients to be administered with an active camptothecin derivative (a) or a prodrug thereof (b), or to predict drug response, recurrence rate and / or prognosis when undergoing chemotherapy for anticancer agents.

15. A method for preparing (a) an active camptothecin derivative, (b) a prodrug thereof, or (c) a complex containing a ligand targeting DDX5 protein, wherein the active camptothecin derivative is designed to bind to the DDX5 protein and the E3 ligase, the prodrug of the active camptothecin derivative is designed to release the active camptothecin derivative (a) at a target site in vivo, and the complex containing a ligand targeting DDX5 protein is connected to the active camptothecin derivative (a) or the FL118 compound of Chemical Formula 2 through a linking group to inactivate the ability to inhibit type I topoisomerase, characterized in that: include: The active camptothecin derivative (a) is designed to contain a camptothecin skeleton represented by the following chemical formula 1 as a mother core, or a compound designed in the above manner is selected, or a step of synthesizing a compound designed in the above manner, [Chemical formula 1] 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 6- or 7-corner ring, n = 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 ligand targeting DDX5 protein according to claim 15, characterized in that: The active camptothecin derivative (a) designed to bind to the DDX5 protein and E3 ligase has the ability to inhibit type I topoisomerase and the mechanism of action of degrading the tumor protein DDX5.

17. The method for preparing the active camptothecin derivative, the prodrug thereof or the complex containing the ligand targeting DDX5 protein according to claim 15, characterized in that: In the step of designing the active camptothecin derivative (a) to include the camptothecin skeleton represented by Chemical Formula 1 as a mother core, or selecting a compound designed in the above manner, (1) selecting active camptothecin derivatives designed to have a mechanism of action for inhibiting type I topoisomerase and / or degrading tumor protein DDX5 from a compound library containing a camptothecin skeleton represented by Chemical Formula 1 as a parent core, and / or (2) Confirming whether the compound comprising the camptothecin skeleton represented by Chemical Formula 1 as a core inhibits type I topoisomerase and / or degrades tumor protein DDX5 by in vitro experiments and / or in vivo experiments.

18. The method for preparing the active camptothecin derivative or its prodrug according to claim 15, characterized in that: Active camptothecin derivatives are designed through X1, (X2) n , X3, Y1, Y2 and / or Y3 are deformed to adjust the cell membrane permeability as needed, exert a bystander effect in cancer tissue or control the extent of its effect.

19. The method for preparing the active camptothecin derivative or its prodrug according to claim 15, characterized in that: The active camptothecin derivative (a) or its prodrug (b) is used for administration to a group of patients in which DDX5 functions as a tumor protein in cells.

20. The method for preparing the camptothecin derivative, the prodrug thereof or the complex containing the ligand targeting DDX5 protein according to claim 15, characterized in that: The method further comprises: in a tissue biopsy or liquid biopsy, using a complex (c) containing a ligand targeting DDX5 protein inactivated by a linker group in the FL118 compound of Chemical Formula 2 or the above-mentioned active camptothecin derivative (a) to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered.

21. A method for preparing (a) an active camptothecin derivative, (b) a prodrug thereof, or (c) a complex containing a ligand targeting DDX5 protein, wherein the active camptothecin derivative binds to DDX5, which works as a tumor protein, in cells and induces cell death by decomposing the DDX5 protein, the prodrug of the active camptothecin derivative is designed to release the active camptothecin derivative (a) in vivo, and the complex containing a ligand targeting DDX5 protein is connected to the active camptothecin derivative (a) through a linking group to inactivate the ability to inhibit type I topoisomerase, characterized in that: include: The first step is to select the above active camptothecin derivative (a) from the group consisting of the compound of the following Chemical Formula 3, the compound of the following Chemical Formula 4, the compound of the following Chemical Formula 5, the compound of the following Chemical Formula 6, the compound of the following Chemical Formula 7, the compound of the following Chemical Formula 8, the compound of the following Chemical Formula 9 and their isomers; and In the second step, a prodrug (b) of an active camptothecin derivative designed to release the above active camptothecin derivative (a) selected in the first step in vivo is arbitrarily selected, [Chemical formula 3] [Chemical formula 4] [Chemical formula 5] [Chemical formula 6] [Chemical formula 7] [Chemical formula 8] [Chemical formula 9] 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 group of patients in which DDX5 functions as a tumor protein in cells.

23. The preparation method according to claim 21, characterized in that: The method further comprises: in a tissue biopsy or liquid biopsy, using a complex (c) containing a ligand targeting DDX5 protein inactivated by a linker group in the FL118 compound of Chemical Formula 2 or the above-mentioned active camptothecin derivative (a) to quantitatively or qualitatively analyze the intracellular DDX5 protein to confirm the patient group to which the active camptothecin derivative (a) or its prodrug (b) is to be administered.

24. A compound represented by the following Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, or a pharmaceutically acceptable salt thereof, [Chemical formula 3] [Chemical formula 3-1] [Chemical formula 4] [Chemical formula 4-1] [Chemical formula 5] [Chemical formula 5-1] [Chemical formula 6] [Chemical formula 7] [Chemical formula 8] [Chemical formula 9] 25. A pharmaceutical composition for preventing or treating cancer comprising a compound represented by the following Chemical Formula 3, Chemical Formula 3-1, Chemical Formula 4, Chemical Formula 4-1, Chemical Formula 5, Chemical Formula 5-1, Chemical Formula 6, Chemical Formula 7, Chemical Formula 8 or Chemical Formula 9, a pharmaceutically acceptable salt thereof, a solvate thereof, or a prodrug thereof, [Chemical formula 3] [Chemical formula 3-1] [Chemical formula 4] [Chemical formula 4-1] [Chemical formula 5] [Chemical formula 5-1] [Chemical formula 6] [Chemical formula 7] [Chemical formula 8] [Chemical formula 9] 26. A pharmaceutical composition for cell death, characterized in that: Containing (a) an active camptothecin derivative represented by the following chemical formula 1, which is designed to bind to the DDX5 protein and the E3 ligase; or (b) a prodrug of the active camptothecin derivative, which is designed to release the active camptothecin derivative (a) at a target site in vivo, [Chemical formula 1] 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 6- or 7-corner ring, n = 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 cell death according to claim 26, characterized in that: Active camptothecin derivatives (a) designed to bind to DDX5 protein and E3 ligase cause the death of target cells expressing DDX5 protein through a molecular adhesive mechanism.

28. The pharmaceutical composition for cell death according to claim 26, characterized in that: The active camptothecin derivative (a) designed to bind to the DDX5 protein and E3 ligase has the ability to inhibit type I topoisomerase and the mechanism of action of degrading the tumor protein DDX5.

29. The pharmaceutical composition for cell death according to claim 26, characterized in that: The active camptothecin derivatives are designed to modulate cell membrane permeability as required through deformation of X1, (X2)n, X3, Y1, Y2 and / or Y3, thereby exerting a bystander effect or controlling the extent of its effect in cancer tissues.

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

31. An anticancer dosage form, characterized in that: administering (a) an active camptothecin derivative represented by the following chemical formula 1, or (b) a prodrug thereof, in an amount that induces an anti-tumor immune response by stimulating antigen-presenting cells through cell death of cancer cells, wherein the active camptothecin derivative is designed to bind to the DDX5 protein and the E3 ligase, and the prodrug of the active camptothecin derivative is designed to release the active camptothecin derivative (a) at a target site in vivo, [Chemical formula 1] 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 6- or 7-corner ring, n = 1 to 2, Y1, Y2 and Y3 are each independently hydrogen, or a functional group containing oxygen, nitrogen, phosphorus or sulfur.

32. The anticancer dosage form according to claim 31, characterized in that: Anticancer dosage forms are designed to target tumor tissue.

33. A method for preparing an anticancer agent based on an active camptothecin derivative (a), controlling the bystander effect and / or ADME curve of the active camptothecin derivative (a) to prevent the occurrence of T cell exhaustion caused by chronic antigen exposure, characterized in that: The anticancer agent based on the active camptothecin derivative (a) is an anticancer dosage form containing (a) an active camptothecin derivative, or (b) a prodrug thereof, wherein the active camptothecin derivative is designed to bind to the DDX5 protein and the E3 ligase via a molecular adhesive, and the prodrug of the active camptothecin derivative is designed to release the active camptothecin derivative (a) at a target site in vivo, The method comprises: designing an active camptothecin derivative (a) or a prodrug thereof (b) to include a camptothecin skeleton represented by the following chemical formula 1 as a parent core, or selecting a compound designed in the above manner, or synthesizing a compound designed in the above manner, [Chemical formula 1] 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 6- or 7-corner ring, n = 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 agent based on the active camptothecin derivative (a) is a dosage form comprising (a) an active camptothecin derivative or (b) a prodrug thereof, wherein the active camptothecin derivative is designed to have the ability to inhibit type I topoisomerase and a mechanism of action for decomposing the tumor protein DDX5, and the prodrug of the active camptothecin derivative is designed to release the active camptothecin derivative (a) in vivo.

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