A new selective HDAC6 inhibitor that modulates tumor microenvironment, and use in anticancer adjuvant for immunotherapy thereof

Abstract

The present invention relates to a novel HDAC6 selective inhibitor that modulates the tumor microenvironment. The novel HDAC6 selective inhibitor according to the present invention exhibits excellent HDAC6 selective inhibitory activity. Compared to the existing HDAC6 inhibitor Nexturastat A, it significantly increases PD-L1 expression and inhibits the TGF-β signaling pathway in organoids of MSS colorectal cancer patients. Therefore, it can be usefully utilized as an anticancer adjuvant for inducing changes in the cancer cell microenvironment, for targeted cancer therapy, and for immunotherapy.

Description

Technology Field

[0001] The present invention relates to a novel HDAC6 selective inhibitor, a pharmaceutical composition for the prevention or treatment of cancer comprising the same, a composition for increasing PD-L1 expression in cancer cells, a composition for regulating the tumor microenvironment of cancer, an anticancer adjuvant for targeted cancer therapy, and an anticancer adjuvant for cancer immunotherapy. Background Technology

[0002] Histone acetylation is regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC). Since the expression of HDAC is prominent in cancer cells and very little acetylation of histone proteins is found, inhibitors that inhibit HDAC are being studied as a type of anticancer drug.

[0003] HDACs identified to date have four classes (Class I, II, III, IV) and a total of 11 subtypes based on their structure and function. Various research results have been reported regarding the inhibitory effects of HDACs on cancer cells, including killing cancer cells, inducing growth arrest, inhibiting angiogenesis, DNA repair, and regulating the expression of immune-related genes. Based on the various effects of these HDAC inhibitors on cancer cells, the U.S. FDA (Food and Drug Administration) has approved developed pan-HDAC inhibitors as anticancer drugs for the treatment of T-cell lymphoma and multiple myeloma. However, clinical side effects caused by low selectivity (such as liver damage and symptoms of metabolic syndrome with elevated cholesterol and triglycerides) have become a problem. Furthermore, as issues have emerged that the inhibitors worsen atherosclerosis or rapidly accelerate vascular calcification in patients with certain underlying diseases, and particularly in cancer cells, reduce drug delivery by promoting incomplete angiogenesis, thereby accelerating the malignancy of cancer cells, the importance of developing selective inhibitors for HDACs is being emphasized.

[0004] With a series of recent studies showing that histone deacetylase (HDAC) inhibitors activate the immune system, research is actively underway to overcome the limitations of the therapeutic efficacy of immuno-oncology drugs through the combination administration of HDAC inhibitors with immunotherapies.

[0005] Based on PCT International Publication No. WO 2024 / 071935 A1 (Composition for treating colorectal cancer comprising a histone deacetylase inhibitor, and anticancer adjuvant for colorectal cancer immunotherapy) and Korean Patent Application No. 10-2023-0173635 (Pharmaceutical composition for treating cancer comprising a co-inhibitor of histone deacetylase 6 and histone deacetylase 10), the inventors intended to develop a selective HDAC inhibitor involved in inducing the tumor microenvironment (TME) of patients with microsatellite-stable (MSS) colorectal cancer, who do not respond to immunotherapy, into an environment favorable for the action of immunotherapeutic agents. The above two patents are use patents that demonstrate the efficacy of combining an HDAC inhibitor with an immunotherapeutic agent using commercially available HDAC inhibitors.

[0006] Furthermore, while many studies are developing new drugs that may be effective for immunotherapy of colorectal cancer, unfortunately, most patients with colorectal cancer (CRC) are of the microsatellite stable (MSS) type, which differs from the microsatellite instable (MSI) type in that the tumor microenvironment (TME) is unstable, leading to a problem where they do not respond to immunotherapy. MSI-H (high degree of microsatellite instability) colorectal cancer has 10 to 100 times more somatic mutations than MSS colorectal cancer, and immune checkpoint receptors such as PD-1, PD-L1, and CTLA-4, along with their ligands, are overexpressed, making it a good indication for immune checkpoint inhibitor therapy. In fact, the inventors compared the expression of PD-L1, IFN-γ, and CD8 T effector genes in the tumors of 51 MSI-H colorectal cancer patients and 291 MSI-L (low degree of microsatellite instability) / MSS patients, and confirmed that the expression of these genes was significantly lower in the MSI-L / MSS patient group compared to the MSI-H patient group.

[0007] Therefore, the inventors developed a novel HDAC6 selective inhibitor to demonstrate its potential as an anticancer adjuvant for cancer targeted therapy and immunotherapy, and to induce changes in the cancer cell microenvironment. Prior art literature

[0008] WO 2024 / 071935 A1 (Publication date 2024-04-04; composition for treating colorectal cancer comprising a histone deacetylase inhibitor, and anticancer adjuvant for colorectal cancer immunotherapy) The problem to be solved

[0009] The inventors aimed to develop novel HDAC6 selective inhibitors capable of inducing changes in the tumor microenvironment (TME) of MSS colorectal cancer that does not respond to immunotherapy due to low expression of immune checkpoint receptors and their ligands. The inventors developed novel HDAC6 selective inhibitors and confirmed that they exhibit excellent HDAC6 inhibitory activity. Furthermore, compared to the existing HDAC6 inhibitor Nexturastat A, they significantly increased PD-L1 expression and inhibited the TGF-β signaling pathway in organoids of MSS colorectal cancer patients. Consequently, they confirmed that these inhibitors can be usefully employed as anticancer adjuvants for inducing changes in the cancer cell microenvironment, for targeted cancer therapy, and for immunotherapy.

[0010] Accordingly, the object of the present invention is to provide one or more compounds selected from compounds represented by chemical formulas 1 to 10 or pharmaceutically acceptable salts thereof.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the said compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Another object of the present invention is to provide a composition for increasing PD-L1 expression in cancer cells comprising the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another objective of the present invention is to provide a composition for controlling the tumor microenvironment of cancer comprising the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another objective of the present invention is to provide an anticancer adjuvant for targeted cancer therapy comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] Another object of the present invention is to provide an anticancer adjuvant for cancer immunotherapy comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] Another objective of the present invention is to provide a use for the combined administration of the said compound or a pharmaceutically acceptable salt thereof and an anticancer agent.

[0017] Another object of the present invention is to provide a use for the combined administration of the said compound or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor. means of solving the problem

[0018] The present invention relates to a novel HDAC6 selective inhibitor, a pharmaceutical composition for the prevention or treatment of cancer comprising the same, a composition for increasing PD-L1 expression in cancer cells, a composition for regulating the tumor microenvironment of cancer, an anticancer adjuvant for targeted cancer therapy, and an anticancer adjuvant for cancer immunotherapy.

[0019] The inventors anticipated that HDAC6 selective inhibitors would induce increased PD-L1 expression in MSS colorectal cancer, and that the increased PD-L1 expression would alter the tumor microenvironment, thereby enhancing sensitivity to immunotherapy and demonstrating combination effects with immunotherapies. In conclusion, it was expected that HDAC6 selective inhibitors could be presented as an efficient immunotherapy combination therapy for cancer types that do not respond to immunotherapy.

[0020] Accordingly, the inventors confirmed that the developed novel HDAC6 selective inhibitor exhibits excellent HDAC6 inhibitory activity and, compared to the existing HDAC6 inhibitor Nexturastat A, significantly increased PD-L1 expression in MSS colorectal cancer patient organoids and significantly decreased the phosphorylation of SMAD2 and SMAD3, thereby inhibiting the TGF-β signaling pathway and inducing changes in the cancer cell microenvironment.

[0021] It is expected that the novel HDAC6 selective inhibitor of the present invention alters the tumor microenvironment, thereby enhancing sensitivity to immunotherapy and exhibiting combination effects with immunotherapies. Consequently, it is anticipated that the novel HDAC6 selective inhibitor of the present invention can be presented as an efficient immunotherapy combination therapy for cancer types in which immunotherapies are ineffective. Therefore, the novel HDAC6 selective inhibitor of the present invention can be usefully employed as an anticancer adjuvant for inducing changes in the cancer cell microenvironment, for targeted cancer therapy, and for immunotherapy.

[0022] The present invention will be described in more detail below.

[0023] One aspect of the present invention is one or more compounds selected from compounds represented by the following chemical formulas 1 to 10, or pharmaceutically acceptable salts thereof:

[0024] [Chemical Formula 1]

[0025] ,

[0026] [Chemical Formula 2]

[0027] ,

[0028] [Chemical Formula 3]

[0029] ,

[0030] [Chemical Formula 4]

[0031] ,

[0032] [Chemical Formula 5]

[0033] ,

[0034] [Chemical Formula 6]

[0035] ,

[0036] [Chemical Formula 7]

[0037] ,

[0038] [Chemical Formula 8]

[0039] ,

[0040] [Chemical Formula 9]

[0041] , and

[0042] [Chemical Formula 10]

[0043] .

[0044] In the present invention, the term "Nexturastat A" refers to a potent and selective HDAC6 inhibitor. Nexturastat A is known to have inhibitory activity against HDAC6 with an IC50 of 5 nM and is represented by the following chemical formula A.

[0045] [Chemical Formula A]

[0046] .

[0047] The inventors confirmed that novel HDAC6 selective inhibitors represented by compounds 1 to 10 exhibit excellent HDAC6 inhibitory activity and, compared to the existing HDAC6 inhibitor Nexturastat A, significantly increased PD-L1 expression in organoids of MSS colorectal cancer patients and significantly decreased the phosphorylation of SMAD2 and SMAD3, thereby inhibiting the TGF-β signaling pathway and inducing changes in the cancer cell microenvironment. Accordingly, it was confirmed that using the novel HDAC6 selective inhibitor according to the present invention can induce changes in the tumor microenvironment (TME) of MSS colorectal cancer that did not respond to immunotherapy due to low expression of immune checkpoint receptors and their ligands, thereby creating a preventive or therapeutic effect for MSS-type cancer that is difficult to resolve with the existing HDAC6 inhibitor Nexturastat A.

[0048] In the present invention, the compound or a pharmaceutically acceptable salt thereof can inhibit the activity of histone deacetylase (HDAC).

[0049] In the present invention, the histone deacetylase may be HDAC6.

[0050] In this invention, the term "HDAC6" refers to a cytoplasmic deacetylase belonging primarily to class IIb.

[0051] Another aspect of the present invention is a pharmaceutical composition for the prevention or treatment of cancer comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0052] In the present invention, the pharmaceutical composition may further include an anticancer agent.

[0053] In the present invention, the anticancer agent may be an immune checkpoint inhibitor.

[0054] In the present invention, the immune checkpoint inhibitor may be one or more selected from the group consisting of anti-CTLA4 antibody, anti-PD-L1 antibody, and anti-PD-1 antibody.

[0055] The above anti-CTLA4 antibody (Anti-Cytotoxic T-Lymphocyte Antigen 4 antibody) may be ipilimumab, tremelimumab, or BMS-986218, but is not necessarily limited thereto.

[0056] The above anti-PD-L1 antibodies (Anti-Programmed Death-Ligand 1 antibodies) may be atezolizumab, durvalumab, BMS-936559, cemiplimab, MSB0010718C, or TQB2450, but are not necessarily limited thereto.

[0057] The above anti-PD-1 antibodies (Anti-Programmed Death-1 antibodies) may be nivolumab, pembrolizumab, sintilimab, camrelizumab, PDR001, SHR-1210, REGN2810, BMS-936558, ABBV-181, or PF-06801591, but are not necessarily limited thereto.

[0058] In the present invention, the cancer may be one or more selected from the group consisting of colorectal cancer, brain tumor, glioblastoma, glioma, neuroblastoma, head and neck cancer, oral cancer, laryngeal cancer, throat cancer, bronchial cancer, lung cancer, endocrine cancer, thyroid cancer, parathyroid cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, biliary tract cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, kidney cancer, bladder cancer, urethral cancer, testicular cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, adenocarcinoma, skin cancer, melanoma, bone cancer, bone marrow cancer, blood cancer, T-cell lymphoma, and multiple myeloma, but is not necessarily limited thereto, and preferably may be colorectal cancer.

[0059] In the present invention, the arm may be of the MSS type.

[0060] In this invention, the term "MSS (microsatellite stable)" refers to a case where the DNA segments of cancer cells do not change or are free of mutations. When cancer is of the MSS type, it possesses normal levels of mismatch repair gene and protein expression, and MSS cancer cells can skillfully correct DNA mismatch repair errors.

[0061] Microsatellites (MS) consist of repetitive sequences composed of 1 to 6 nucleotides, also known as short tandem repeats (STRs) or simple sequence repeats (SSRs). MS generally occur due to DNA slippage or mismatches during DNA replication and repair processes. While normal tissues can correct errors during DNA replication through DNA repair systems such as mismatch repair (MMR), the likelihood of gene mutations increases in tumor cells due to deficiencies in MMR genes or defects in the replication repair process. Microsatellite instability (MSI) is a significant factor in the development of tumors. Based on the frequency of MSI, it can be classified into three types: high microsatellite instability (MSI-H), low microsatellite instability (MSI-L), and microsatellite stability (MSS). Current clinical studies tend to classify MSS-L and MSS as a single type (Li, K., Luo, H., Huang, L. et al. (2020) Microsatellite instability: a review of what the oncologist should know. Cancer Cell Int 20 , 16).

[0062] ​In the present invention, MSS-type cancers include colorectal carcinoma, malignant solid tumor, colorectal adenocarcinoma, non-small cell lung carcinoma, breast carcinoma, adenocarcinoma of the gastroesophageal junction, gastric adenocarcinoma, endometrial carcinoma, pancreatic carcinoma, head and neck squamous cell carcinoma, melanoma, ovarian carcinoma, rectal adenocarcinoma, urothelial carcinoma, esophageal adenocarcinoma, gastric carcinoma, renal cell carcinoma, and cervical carcinoma. (Cervical Carcinoma), Esophageal Carcinoma, Pancreatic Ductal Adenocarcinoma, Prostate Carcinoma, Small Cell Lung Carcinoma, Bladder Urothelial Carcinoma, Cholangiocarcinoma, Colon Adenocarcinoma, Fallopian Tube Carcinoma, Hepatocellular Carcinoma, High Grade Ovarian Serous Adenocarcinoma, Nasopharyngeal Carcinoma,Ovarian Endometrioid Adenocarcinoma, Pancreatic Adenocarcinoma, Primary Peritoneal Carcinoma, Squamous Cell Lung Carcinoma, Adenocarcinoma, Alveolar Soft Part Sarcoma, Anal Canal Squamous Cell Carcinoma, Appendix Mucinous Adenocarcinoma, Bladder Carcinoma, Breast Adenocarcinoma, Carcinoma, Chordoma, Clear Cell Renal Cell Carcinoma, Colon Carcinoma, Colorectal Mucinous Adenocarcinoma, Cutaneous Melanoma, Diffuse Large B-Cell Lymphoma B-Cell Lymphoma), Digestive System Carcinoma, Endometrial Serous Adenocarcinoma, Esophageal Squamous Cell Carcinoma, Esophagogastric Carcinoma, Fallopian Tube Clear Cell Adenocarcinoma, Fallopian Tube Endometrioid Adenocarcinoma, Glioblastoma, Head and Neck CarcinomaHigh Grade Fallopian Tube Serous Adenocarcinoma, Hypopharyngeal Squamous Cell Carcinoma, Laryngeal Squamous Cell Carcinoma, Lung Adenocarcinoma, Lung Carcinoma, Lymphoma, Malignant Ovarian Epithelial Tumor, Mesothelioma, Multiple Myeloma, Neuroendocrine Tumor, Non-Squamous Non-Small Cell Lung Carcinoma, Oral Cavity Squamous Cell Carcinoma, Oropharyngeal Carcinoma, Oropharyngeal Squamous Cell Carcinoma, Ovary Ovarian Clear Cell Adenocarcinoma, Primary Peritoneal Clear Cell Carcinoma, Primary Peritoneal Endometrioid Adenocarcinoma, Primary Peritoneal High Grade Serous Adenocarcinoma, Prostate Adenocarcinoma, Small Intestinal Carcinoma, Thyroid Gland Undifferentiated (Anaplastic) Carcinoma, Undifferentiated Pleomorphic SarcomaIt may be one or more selected from the group consisting of uterine corpus endometrial stromal sarcoma and uveal melanoma, but is not necessarily limited thereto (see My Cancer Genome, https: / / www.mycancergenome.org / content / alteration / microsatellite-stable-mss / )

[0063] In the present invention, the term “included as an active ingredient” means including a sufficient amount to achieve a specific effect of a compound represented by Formulas 1 to 10, which is a novel HDAC6 inhibitor of the present invention, for example, activity for the prevention or treatment of cancer, activity for increasing PD-L1 expression, activity for inhibiting TGF-β signaling, or activity for regulating the tumor microenvironment of cancer.

[0064] In this invention, the term "prevention" refers to any act of suppressing cancer or delaying its progression through the administration of the pharmaceutical composition of this invention.

[0065] In the present invention, the term "treatment" means inhibition of cancer development, alleviation of cancer, and elimination of cancer.

[0066] In the present invention, the expression “pharmaceuticalally acceptable salt” refers to a salt of a compound represented by Formulas 1 to 10 of the present invention having a desired pharmacological effect, namely, a preventive or therapeutic effect against cancer such as selective inhibition of HDAC6, anticancer activity, increased PD-L1 expression in cancer cells, and inhibition of TGF-beta signaling protein. These salts can be formed using inorganic acids such as hydrogen chloride, hydrogen bromide, and hydrogen iodide, and organic acids such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, p-toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate, butyrate, citrate, camphorate, camphosulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, 2-hydroxyethanesulfate, lactate, malylate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate, and undecanoate.

[0067] The expression "comprising as an active ingredient" of the present invention means comprising a sufficient amount to achieve pharmacological efficacy or activity of a compound represented by Formulas 1 to 10 of the present invention, or a pharmaceutically acceptable salt thereof, and includes the possibility that various ingredients may be additionally added for the delivery, stabilization, and formulation of the drug.

[0068] In the present invention, any carrier commonly used in the art may be used. Non-limiting examples of the carrier may include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, or combinations thereof.

[0069] The pharmaceutical composition of the present invention may be used with the addition of other pharmaceutically acceptable additives, such as excipients, diluents, antioxidants, buffers, or bacteriostatic agents, if necessary, and may additionally be used with the addition of fillers, extenders, wetting agents, disintegrants, dispersants, surfactants, binders, or lubricants.

[0070] The dosage of the pharmaceutical composition of the present invention can be determined in various ways based on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and can be determined or prescribed as a dosage effective for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001-1000 mg / kg.

[0071] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. The formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or stabilizer, but is not limited thereto.

[0072] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, body weight, gender, form of administration, health condition, and degree of disease, and may be administered in divided doses from once to several times a day at regular intervals at the discretion of a doctor or pharmacist. For example, the daily dosage may be 1 to 1000 ug / mL based on the content of the active ingredient, but this is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0073] Another aspect of the present invention is a composition for increasing PD-L1 expression in cancer cells comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0074] Another aspect of the present invention is a composition for controlling the tumor microenvironment of cancer comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0075] In the present invention, the term "tumor microenvironment" refers to a complex ecosystem surrounding a tumor composed of cancer cells, stromal tissue (including blood vessels, immune cells, fibroblasts, and signaling molecules), and the extracellular matrix.

[0076] In the present invention, the tumor microenvironment regulation may be one or more selected from the group consisting of increased PD-L1 expression in cancer cells and inhibition of TGF-β signaling protein expression.

[0077] The novel HDAC6 selective inhibitor according to the present invention can increase PD-L1 expression in cancer cells and inhibit the expression of TGF-β signaling proteins.

[0078] In the present invention, the TGF-β signaling protein may be one or more selected from the group consisting of p-Smad2 and p-Smad3, but is not necessarily limited thereto.

[0079] In the present invention, the TGF-β signaling protein may be one or more selected from the group consisting of TSP-1 (Thrombospondin-1), TSP-2 (Thrombospondin-2), TGF-β1 (Transforming growth factor beta 1), TGF-β1 receptor 1, TGF-β1 receptor 2, THBS1, Smad2, Smad3, Smad4, p-Smad2, p-Smad3, GSK-3β (Glycogen Synthase kinase-3β), PRKCD (Protein kinase C delta), and Collagen III, but is not necessarily limited thereto.

[0080] In this invention, the term "Smad (SMAD, Suppressor of Mothers against Decapentaplegic)" is a signal transducer and transcriptional modulator that mediates various signaling pathways.

[0081] Smad2 and Smad3 proteins regulate various cellular processes, such as cell proliferation, apoptosis, and differentiation, by mediating TGF-beta (transforming growth factor-beta) signaling. Smad2 and Smad3 proteins are recruited to TGF-beta receptors through interactions with SMAD anchor (SARA) proteins for receptor activation. In response to TGF-beta signaling, Smad2 and Smad3 proteins are phosphorylated by the TGF-beta receptors.

[0082] Another aspect of the present invention is an anticancer adjuvant for targeted cancer therapy comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0083] Another aspect of the present invention is an anticancer adjuvant for cancer immunotherapy comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0084] The above compound or its pharmaceutically acceptable salt can promote histone acetylation by inhibiting the activity of HDAC.

[0085] Another aspect of the present invention is a pharmaceutical composition for the prevention or treatment of diseases mediated by HDAC6, comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0086] In the present invention, the disease mediated by HDAC6 is cancer, Wilson's disease, cerebellar ataxia, prion disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, amyloidosis, Alzheimer's disease, Alexander disease, alcoholic liver disease, cystic fibrosis, Pick's disease, spinal muscular atrophy, Lewy body dementia, rheumatoid arthritis; osteoarthritis; rheumatoid spondylitis; psoriasis; post-ischemic perfusion injury; inflammatory bowel disease; Chronic inflammatory lung disease, eczema, asthma, psoriasis, ischemia / reperfusion injury, ulcerative colitis, acute respiratory distress syndrome, psoriatic arthritis, infectious arthritis, progressive chronic arthritis, degenerative arthritis, osteoarthritis, traumatic arthritis, gouty arthritis, Reiter's syndrome, polychondritis, acute synovitis and spondylitis, glomerulonephritis, hemolytic anemia, aplastic anemia, idiopathic thrombocytopenia, neutropenia, ulcerative colitis, Crohn's disease, graft-versus-host disease, allogeneic rejection, chronic thyroiditis, Graves' disease, scleroderma, diabetes mellitus, active hepatitis, primary binary cirrhosis, myasthenia gravis, multiple sclerosis (MS), systemic lupus erythematosus, atopic dermatitis, contact dermatitis, sunburn, chronic renal failure, Stevens-Johnson syndrome, idiopathic sprue, sarcoidosis, One or more selected from the group consisting of Guillain-Barré syndrome, uveitis, conjunctivitis, keratoconjunctivitis, otitis media, periodontitis, interstitial fibrosis of the lungs, asthma, bronchitis, rhinitis, sinusitis, pneumoconiosis, pulmonary failure syndrome, emphysema, pulmonary fibrosis, and silicosis may be included, but are not necessarily limited thereto, and any disease that occurs or may occur due to increased expression or activity of HDAC6 may be included without limitation.

[0087] Another aspect of the present invention relates to a method for preventing or treating cancer, comprising the step of administering to a subject a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0088] In the present invention, the term "object" may be a mammal including humans, and may be, for example, a human, monkey, cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit, or guinea pig, but is not limited thereto.

[0089] In the present invention, "administration" means providing a specific substance to a subject by any appropriate method, and the administration route of a composition comprising a compound represented by Formulas 1 to 10 of the present invention as an active ingredient may be oral or parenteral through any general route as long as it can reach the target tissue. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells. For example, the pharmaceutical composition of the present invention may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, local administration, intranasal administration, intrapulmonary administration, rectal administration, intrathecal administration, ocular administration, skin administration, and transdermal administration, but is not limited thereto.

[0090] The composition for increasing PD-L1 expression in cancer cells, the composition for regulating the tumor microenvironment of cancer, the anticancer adjuvant for targeted cancer therapy, the anticancer adjuvant for cancer immunotherapy, the pharmaceutical composition for the prevention or treatment of diseases mediated by HDAC6, and the method for the prevention or treatment of cancer according to the present invention comprise one or more compounds selected from compounds represented by Formulas 1 to 10, which are novel HDAC6 selective inhibitors identical to the pharmaceutical composition for the prevention or treatment of cancer that is another aspect of the present invention, or pharmaceutically acceptable salts thereof; therefore, to avoid excessive complexity in the description of this specification, redundant content is referenced and the description thereof is omitted. Effects of the invention

[0091] The present invention relates to a novel HDAC6 selective inhibitor that modulates the tumor microenvironment. The novel HDAC6 selective inhibitor according to the present invention exhibits excellent HDAC6 selective inhibitory activity. Compared to the existing HDAC6 inhibitor Nexturastat A, it significantly increases PD-L1 expression and inhibits the TGF-β signaling pathway in organoids of MSS colorectal cancer patients. Therefore, it can be usefully utilized as an anticancer adjuvant for inducing changes in the cancer cell microenvironment, for targeted cancer therapy, and for immunotherapy. Brief explanation of the drawing

[0092] FIGS. 1a to 1c are figures showing the PD-L1 mRNA expression levels confirmed in MSS colorectal cancer cell lines HT29, SW620, and CT26 after treatment with three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13) according to one embodiment of the present invention. FIGS. 1d to 1i show the PD-L1 mRNA expression levels confirmed in MSS colorectal cancer cell lines HT29, SW620, and CT26 after treatment with seven novel HDAC6 inhibitor compounds (A2-21, A1-12, A2-30, C-32, C-31, C-41, C-42) according to one embodiment of the present invention. FIGS. 2a to 2c are figures showing the p-Smad2 protein expression levels confirmed in MSS colorectal cancer cell lines HT29, SW620, and CT26 after treatment with three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13) according to one embodiment of the present invention. Figure 3a shows the PD-L1 mRNA expression levels confirmed in MSS colorectal cancer patient organoids after treatment with novel HDAC6 inhibitor compounds A1-1 and A1-10 according to one embodiment of the present invention at IC50 concentrations. FIG. 3b is a figure showing the expression levels of p-SMAD2 and p-SMAD2 proteins confirmed by Western blot in MSS colorectal cancer patient organoids after treatment with novel HDAC6 inhibitor compounds A1-1 and A1-10 according to one embodiment of the present invention at IC50 concentrations. Specific details for implementing the invention

[0093] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings so that those skilled in the art can easily implement the present invention. Unless otherwise defined, terms used in this specification should be interpreted as generally understood by those skilled in the art.

[0094] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0096] Example 1: 10 Novel HDAC6 Selective Inhibitors

[0097] In the present invention, HDAC6 was selected among 11 HDAC1 to HDAC11, and 10 novel compounds were discovered based on AI (Artificial Intelligence). The inventors confirmed that (i) PD-L1 (Programmed death-ligand 1) expression in MSS-type colorectal cancer increases due to selective inhibition of HDAC6, (ii) changes in the tumor microenvironment are induced by increased PD-L1 expression, which enhances sensitivity to immunotherapy and shows combination effects with immunotherapies, and (iii) consequently, the possibility was confirmed that HDAC6 selective inhibitors could be presented as an efficient immunotherapy combination therapy in cancer types where immunotherapies do not respond.

[0098] As shown in Table 1 below, the inventors discovered the following 10 novel HDAC6 selective inhibitors (A1-1, A1-10, A1-12, A1-13, A2-21, A2-30, C-31, C-32, C-41, and C-42) based on AI.

[0099] Chemical formula 1 Chemical formula 2 Chemical formula 3 Chemical formula 4 Chemical formula 5 Chemical formula 6 Chemical formula 7 Chemical formula 8 Chemical formula 9 Chemical formula 10

[0101] The inventors in order to select a candidate substance for a novel HDAC6 selective inhibitor, in vitro ( in vitro ) Evaluation of drug efficacy and in vitro ( in vitro ) Efficacy evaluation was conducted.

[0103] 1-1. In vitro ( in vitro ) Drug efficacy evaluation

[0104] The inventors measured the inhibitory activity of 10 novel HDAC6 selective inhibitors against the HDAC6 enzyme using the HDAC6 Fluorogenic Assay Kit (HDAC6 Fluorogenic Assay Kit, BPS Bioscience). Nexturastat A, a commercially available HDAC6 selective inhibitor, was used as a control. The HDAC6 enzyme activity (IC50, half-maximal inhibitory concentration) is shown in Table 2 below.

[0105] compound HDAC6 enzyme activity (IC50) Nexturastat A 3 nM Chemical formula 1 A1-1 9 nM Chemical formula 2 A1-10 5 nM Chemical formula 3 A1-12 46 nM Chemical formula 4 A1-13 6 nM Chemical formula 5 A2-21 31 nM Chemical formula 6 A2-30 55 nM Chemical formula 7 C-31 13 nM Chemical formula 8 C-32 79 nM Chemical formula 9 C-41 12 nM Chemical formula 10 C-42 11 nM

[0106] As shown in Table 2 above, three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13) with enzymatic activity (IC50) of less than 10 nM against HDAC6, exhibiting IC50 values ​​similar to or superior to the control (Nexturastat A), were selected. in vitro Efficacy evaluation was conducted.

[0107] 1-2. Cell Activity - Confirmation of Inhibitory Activity on Proliferation of MSS Colorectal Cancer Cell Lines

[0108] As shown in Table 3 below, the existing HDAC6 inhibitor Nexturastat A (Next A) and three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13) were treated to MSS colorectal cancer cell lines HT29, SW620, and CT26, and the proliferation rate (%) was confirmed.

[0109] IC50 (μM) Next A A1-1 A1-10 A1-13 HT29 11.3 24.2 37.0 45.8 SW620 6.8 29.6 35.8 48.6 CT26 13.6 > 50 > 50 > 50

[0110] As can be seen in Table 3, although it showed a higher IC50 concentration than the existing HDAC6 inhibitor Nexturastat A, it was confirmed that the three novel HDAC6 inhibitor compounds of the present invention also inhibited the proliferation of MSS colorectal cancer cell lines HT29 and SW620, thus having anticancer activity.

[0111] 1-3. In vivo ( in vitro ) Efficacy evaluation (confirmation of PD-L1, TGF-β changes)

[0112] In the test tube ( in vitroIn the efficacy evaluation, PD-L1 expression and the TGF-β signaling pathway were identified for the following reasons. Through genomic analysis results and a literature review, the inventors anticipated that one of the reasons for the lower responsiveness to immunotherapies in the MSS colorectal cancer patient group compared to the MSI-H colorectal cancer patient group was the significantly lower expression of immune checkpoint receptors (particularly PD-L1). Therefore, the inventors sought to improve responsiveness to immunotherapies by adopting an approach that raises the low expression of PD-L1 to a level where immunotherapies can respond. TGF-β (Transforming growth factor-β) was initially discovered and named for its ability to promote the transformation and attachment-independent growth of normal renal fibroblasts; however, this cytokine is subsequently known to be central to cancer suppression mechanisms due to its ability to inhibit the growth of epithelial cells, endothelial cells, and blood-derived cells. It is known that cancer cells overexpress TGF-β, and this affects not only the cancer cells but also the surrounding microenvironment, thereby promoting cancer growth and metastasis. Therefore, we confirmed whether candidate HDAC6 selective inhibitors increase PD-L1 expression in MSS cell lines such as HT29, SW620, and CT26, and confirmed the expression level of p-Smad2 in the TGF-β signaling pathway. Through the results of numerous animal experiments, the inventors have confirmed that the TGF-β mechanism is critically involved in the inhibition of tumor growth caused by the combination of an existing HDAC6 inhibitor (Nexturastat A) and an anti-PD-L1 inhibitor.

[0113] Based on the results confirmed in Examples 1-2 above, three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13) were treated to MSS colorectal cancer cell lines HT29, SW620, and CT26 at 1 / 2 IC50 and IC50 concentrations, and then in vitro ( in vitro) Efficacy evaluation was conducted. In the CT26 cell line, the IC50 of each compound was set to 50 μM, and the 1 / 2 IC50 was 25 μM.

[0114] MSS colorectal cancer cell lines HT29, SW620, and CT26 were each treated with three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13), and RNA and protein samples were harvested after 48 hours.

[0115] To measure PD-L1 mRNA expression levels, RNA was isolated using Trizol, isopropanol, and chloroform, then synthesized into cDNA using a cDNA synthesis kit (Bioneer, k-2044-B), and real-time polymerase chain reaction (RT-PCR) was performed using the SYBR (Applied Biosystem, 4367659) method.

[0116] To confirm changes in the TGF-β signaling pathway, three types of HDAC6 inhibitor compounds were each treated, and rhTGF-β (Recombinant Human TGF-beta protein) was added at a concentration of 10 ng / ml after 1 hour, and then lysis buffer (Roche, 4719956001) was added to the cells after 24 hours to obtain protein samples. Protein samples were quantified using the Bradford method (Bio-Rad, 50000006), and then Western blot experiments were performed in the order of sample loading-transfer-blocking (3% skim milk; BD232100)-primary antibody (24 hours, 4℃)-secondary antibody (2 hours, room temperature) to measure the expression levels of phosphorylated SMAD2 (p-SMAD2; phosphor-SMAD2; Mothers against decapentaplegic homolog 2, SMAD family member 2) and phosphorylated SMAD3. β-actin was used as an endogenous control.

[0117] As shown in Figures 1a to 1c, it was confirmed that PD-L1 mRNA expression increased compared to the control group that was not treated with the compounds in all cases where MSS colorectal cancer cell lines HT29, SW620, and CT26 were treated with three novel HDAC6 inhibitor compounds (A1-1, A1-10, A1-13) at 1 / 2 IC50 and IC50 concentrations. Among the three novel HDAC6 inhibitor compounds, compound A1-10 increased the expression of PD-L1 in MSS colorectal cancer cell lines HT29 and SW620 by 2 to 3 times compared to the control group when treated at 1 / 2 IC50 and IC50 concentrations, and compound A1-1 increased the expression of PD-L1 in MSS colorectal cancer cell line CT26 by 2 to 3 times compared to the control group when treated at 1 / 2 IC50 and IC50 concentrations.

[0118] However, as shown in Figures 1d to 1i, when checking for changes in PD-L1 expression for the remaining 7 compounds (A2-21, A1-12, A2-30, C-32, C-31, C-41, C-42) excluding 3 compounds (A1-1, A1-10, A1-13) with enzymatic activity for HDAC6 of less than 10 nM in MSS colorectal cancer cell lines (HT29, SW620, CT26), there was no significant change in the increase of PD-L1 expression, so the next experiment was not conducted.

[0119] The seven compounds (A2-21, A1-12, A2-30, C-32, C-31, C-41, C-42) have the same core structure as the three compounds (A1-1, A1-10, A1-13) but differ in their substituents. It was confirmed that different effects were exerted by changing the substituents despite containing the same core structure, as the three compounds increased the PD-L1 expression of MSS colorectal cancer cell lines by 2 to 3 times, whereas the seven compounds did not have this effect, and there was also a significant difference in HDAC6 inhibitory activity.

[0120] As shown in Figure 2a, when MSS colorectal cancer cell line HT29 was treated with rhTGF-β, the phosphorylation of SMAD2 protein was significantly increased, but when treated with the novel HDAC6 inhibitor compound A1-10 at an IC50 concentration, the phosphorylation of SMAD2 protein was decreased.

[0121] As shown in Figure 2b, when MSS colorectal cancer cell line SW620 was treated with rhTGF-β, the phosphorylation of SMAD2 protein was significantly increased, but when the novel HDAC6 inhibitor compound A1-10 was treated at 1 / 2 IC50 and IC50 concentrations, the phosphorylation of SMAD2 protein was decreased.

[0122] In addition, as shown in Figure 2c, when MSS colorectal cancer cell line CT26 was treated with rhTGF-β, the phosphorylation of SMAD2 protein increased, but when treated with the novel HDAC6 inhibitor compound A1-1 at an IC50 concentration, the phosphorylation of SMAD2 protein decreased.

[0123] 1-4. Results of Selection of Novel HDAC6 Selective Inhibitor Compounds

[0124] From the above results, the inventors have presented in Tables 4 to 6 below compounds that satisfy all of the following criteria in each MSS colorectal cancer cell line (HT29, SW620, CT26): (i) excellent HDAC6 enzyme inhibitory activity, (ii) an increase in PD-L1 expression within 2 to 3 times, and (iii) a decrease in TGF-β signaling.

[0125] HT29 (human) A1-1 A1-10 A1-13 HDAC6 enzyme activity 8 nM 5 nM 6 nM Increased PD-L1 expression 7.7 times 1.9 times 3.5 times Reduction in TGF-β signaling X O X

[0126] SW620 (human) A1-1 A1-10 A1-13 HDAC6 enzyme activity 8 nM 5 nM 6 nM Increased PD-L1 expression 4.6 times 3.0 times 2.0 times Reduction in TGF-β signaling X O X

[0127] CT26 (mouse) A1-1 A1-10 A1-13 HDAC6 enzyme activity 8 nM 5 nM 6 nM Increased PD-L1 expression 2.7 times 0.4 times 1.3 times Reduction in TGF-β signaling O X X

[0129] Synthesizing the above experimental results, novel HDAC6 selective inhibitor compound A1-10 was selected for the MSS colorectal cancer human cell line, and novel HDAC6 selective inhibitor compound A1-1 was selected for the MSS colorectal cancer mouse cell line.

[0131] Example 2: Verification of HDAC6 selective inhibitor compounds A1-1 and A1-10 in human organoids

[0132] The inventors intended to confirm whether the compounds could be practically applied to patients by examining changes in PD-L1 expression and TGF-β signaling caused by HDAC6 selective inhibitor compounds A1-1 and A1-10 in the organoid PM-PS-326T of MSS colorectal cancer patients with liver metastasis.

[0133] The organoid PM-PS-326T from an MSS colorectal cancer patient with liver metastasis was obtained by the following method. After cutting the sample into small pieces on a blade, an enzyme reaction (Collagenase type IV; Gibco, 17104-019, Dispase II; Gibco, 17105-041) was carried out by adding digestion buffer (DMEM; Gibco, 11965-092, P / S; Gibco, 15140-122, FBS; Gibco, 26140079), and dead cells were removed using a strainer (Corning, CLS431752). Then, Matrigel (Corning, 354234) was added to obtain organoids in a 24-well plate (Thermo Fisher Scientific, 142475).

[0134] After treating the organoid PM-PS-326T of MSS colorectal cancer patients with HDAC6 selective inhibitor compounds A1-1 and A1-10 according to the present invention, the cell viability (%) was measured, and the IC50 concentrations capable of reducing the viability of cancer cells to 50% compared to the untreated group were 19 μM and 42 μM, respectively, confirming that there is an anticancer effect on the organoid of MSS colorectal cancer patients.

[0135] As can be seen in Figure 3a, in the organoid PM-PS-326T of MSS colorectal cancer patients with liver metastasis, the existing HDAC6 selective inhibitor Nexturastat A (Next A) did not increase the mRNA expression of PD-L1 at all at an IC50 concentration (10 μM), whereas the novel HDAC6 selective inhibitors A1-1 and A1-10 according to the present invention statistically significantly increased the mRNA expression of PD-L1 by 2 to 3 times at IC50 concentrations (19 μM and 42 μM, respectively).

[0136] Next, changes in TGF-β signaling were examined, and as can be seen in Figure 3b, when rhTGF-β 10 ng / ml was applied to PM-PS-326T organoids from MSS colorectal cancer patients with liver metastasis, phosphorylation of SMAD2 and SMAD3 increased. When the existing HDAC6 selective inhibitor Nexturastat A (Next A) was additionally applied at an IC50 concentration (10 μM), there was no change in the phosphorylation levels of SMAD2 and SMAD3 that had increased due to rhTGF-β treatment. On the other hand, when the novel HDAC6 selective inhibitors A1-1 and A1-10 were applied at IC50 concentrations (19 μM and 42 μM, respectively), the phosphorylation of SMAD2 and SMAD3 that had increased due to rhTGF-β was significantly reduced.

[0137] Therefore, it was confirmed that the novel HDAC6 selective inhibitors A1-1 and A1-10 of the present invention significantly increased PD-L1 mRNA expression and significantly inhibited TGF-β signaling in organoids of MSS colorectal cancer patients with liver metastasis, compared with the existing HDAC6 selective inhibitor Nexturastat A.

[0139] Sintering

[0140] From the above results, the inventors confirmed that the 10 novel HDAC6 selective inhibitors according to the invention have excellent HDAC6 inhibitory activity, and that the novel HDAC6 selective inhibitors A1-1, A1-10, and A1-13 have anticancer activity. In addition, since the novel HDAC6 selective inhibitors A1-1 and A1-10 increase PD-L1 expression in MSS colorectal cancer organoids by 2 to 3 times, it is predicted that effective anticancer activity can be exerted by improving responsiveness to immunotherapies through the PD-L1 expression increased by the novel HDAC6 selective inhibitors A1-1 and A1-10 in the MSS colorectal cancer patient group, which has significantly lower PD-L1 expression compared to the MSI-H colorectal cancer patient group and thus low responsiveness to immunotherapies. Furthermore, the novel HDAC6 selective inhibitors A1-1 and A1-10 according to the invention are expected to inhibit the development and progression of cancer and suppress cancer growth and metastasis by regulating the tumor microenvironment, as they reduce the phosphorylation of Smad2 and Smad3 in MSS colorectal cancer organoids and inhibit the activation of the TGF-β signaling pathway. Moreover, it is expected that the novel HDAC6 selective inhibitors A1-1 and A1-10 according to the present invention can effectively inhibit tumor growth when administered in combination with immunotherapies such as anti-PD-L1.

Claims

Claim 1 One or more compounds selected from compounds represented by the following chemical formulas 1 to 10, or pharmaceutically acceptable salts thereof: [Chemical Formula 1] ,[Chemical Formula 2] ,[Chemical Formula 3] ,[Chemical Formula 4] ,[Chemical Formula 5] ,[Chemical Formula 6] ,[Chemical Formula 7] ,[Chemical Formula 8] ,[Chemical Formula 9] , and [Chemical Formula 10] . Claim 2 In claim 1, the compound or the pharmaceutically acceptable salt thereof is a compound or the pharmaceutically acceptable salt thereof that inhibits the activity of histone deacetylase (HDAC). Claim 3 In paragraph 2, the compound or pharmaceutically acceptable salt thereof in which the histone deacetylase is HDAC6. Claim 4 A pharmaceutical composition for the prevention or treatment of cancer comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 5 A pharmaceutical composition according to claim 4, wherein the pharmaceutical composition further comprises an anticancer agent. Claim 6 A pharmaceutical composition according to claim 5, wherein the anticancer agent is an immune checkpoint inhibitor. Claim 7 A pharmaceutical composition according to claim 6, wherein the immune checkpoint inhibitor is one or more selected from the group consisting of anti-CTLA4 antibody, anti-PD-L1 antibody and anti-PD-1 antibody. Claim 8 A pharmaceutical composition according to claim 4, wherein the cancer is one or more selected from the group consisting of colorectal cancer, brain tumor, glioblastoma, glioma, neuroblastoma, head and neck cancer, oral cancer, laryngeal cancer, throat cancer, bronchial cancer, lung cancer, endocrine cancer, thyroid cancer, parathyroid cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, biliary tract cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, kidney cancer, bladder cancer, urethral cancer, testicular cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, endometrial cancer, cervical cancer, adenocarcinoma, skin cancer, melanoma, bone cancer, bone marrow cancer, blood cancer, T-cell lymphoma, and multiple myeloma. Claim 9 A pharmaceutical composition according to claim 4, wherein the above-mentioned cancer is of the MSS (microsatellite stable) type. Claim 10 A composition for increasing PD-L1 expression in cancer cells comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 11 A composition for controlling the tumor microenvironment of cancer comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 12 A composition for regulating the tumor microenvironment of cancer, wherein, in claim 11, the tumor microenvironment regulation is one or more selected from the group consisting of increased PD-L1 expression in cancer cells and inhibition of TGF-β signaling protein expression. Claim 13 A composition for regulating the tumor microenvironment of cancer, wherein, in claim 12, the TGF-β signaling protein is one or more selected from the group consisting of p-Smad2 and p-Smad3. Claim 14 An anticancer adjuvant for targeted cancer therapy comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 15 An anticancer adjuvant for cancer immunotherapy comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

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