NOX inhibitors used for cancer treatment

Abstract

The present invention relates to compounds, methods, compositions and uses that can restore responsiveness to immunotherapy, in particular immune checkpoint inhibitors or anti-cancer vaccines, or anti-angiogenic treatments.

Description

Technical Field

[0001] The present invention relates to the use of NADPH oxidase (NOX) inhibitors, particularly NOX4 or NOX4 / 1 dual or NOX1 inhibitors, and related formulations and regimens for the treatment of solid cancers in combination with cancer immunotherapy or anti-VEGF therapy.

Background Art

[0002] Cancer cells are faced with numerous cellular stresses such as hypoxia, increased metabolic demands, genomic instability, immune surveillance, nutritional deficiencies, metastasis, and a changing environment after stress, which lead to treatments such as radiotherapy, chemotherapy, and targeted therapy.

[0003] NADPH oxidase (NOX) is a family of enzymes that have six transmembrane domains and transport electrons across biological membranes. Such enzymes are dedicated, reactive oxygen species-producing enzymes that are involved in the onset and progression of cancer and specifically regulate, in a wide range, redox-sensitive signaling pathways that act on specific cell membranes and microdomains through the activation of oncogenes and the inactivation of tumor suppressor proteins. NOX enzymes are particularly important parts of the adaptive stress response to cancer cells, and thereby these cells are considered to be adaptable and viable (Block et al., 2012, Nature Reviews, pages 627 - 637).

[0004] A marked induction of NOX expression has been reported in cancer cells and host cells within the tumor environment.

[0005] The tumor microenvironment and interactions between cancer cells are recognized to play a major role in tumor growth and metastasis. Cancer-associated fibroblasts (CAFs) are the most abundant cells found in the tumor stroma. Tumor growth is driven by CAFs and their transdifferentiation from fibroblasts to myofibroblasts, and this transdifferentiation generally correlates with poor prognosis in many cancers. While CAFs promote "many malignant features," recent research has focused on their role in promoting tumor immune evasion with CAF-rich cancers, which are characterized by a poor response to cancer immunotherapies such as immune checkpoint inhibitors and cancer vaccines, and a tendency towards metastasis, often referred to as "immune cold."

[0006] Furthermore, high CAF content induces a dense tumor microenvironment, which acts as a barrier to drug delivery due to increased interstitial fluid pressure and compact stroma, leading to poor accumulation of chemotherapy in tumors.

[0007] In particular, melanoma is known as an exceptionally aggressive and treatment-resistant human cancer. While progress has been made over the past decade, including the development of immunotherapies using immune checkpoint inhibitors, limiting response rates, severe side effects, and poor prognosis remain challenges in the treatment of unresectable stage III, stage IV, and recurrent melanoma. Melanoma is driven not only by malignant melanocytes but also by altered signaling between neoplastic and non-malignant cell populations, including fibroblasts, endothelial cells, and immune cells in the tumor stroma. CAFs restructure the extracellular matrix (ECM), as well as disease tissue and secretory chemical factors, which simultaneously promotes a transformative process by accelerating tumor growth, angiogenesis, inflammation, and metastasis, contributing to drug resistance. While it has recently become clear that NOX4 regulates myofibroblastic CAF differentiation in numerous cancers (Hanley et al., 2018, J Natl Cancer Inst., 110), the origin of CAFs and the precise mechanisms by which CAFs contribute to cancer progression and drug resistance remain largely unknown. Furthermore, Hanley et al. (2018) did not include any specific anti-cancer immunotherapy agents as adjunctive treatments in NOX4 inhibition.

[0008] Immunotherapy remains a subject of interest as an effective treatment strategy across several cancer types, including melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, renal cell carcinoma, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer, uterine sarcoma, gastric cancer, esophageal cancer, colon cancer, hepatocellular carcinoma, breast cancer, Merkel cell carcinoma, thyroid cancer, Hodgkin lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mycosis fungoides, and peripheral T-cell lymphoma, encompassing a variety of approaches ranging from stimulating effector mechanisms to counteracting inhibitory and suppressive mechanisms. Strategies to activate effector immune cells include enhancing the patient's own immune system's ability to increase the efficacy of the immune response against neoplasms, vaccination with tumor antigens, or enhancement of antigen presentation (Yaddnapudi et al., 2013, Cancer vaccines, Oncoimmunology, 2(3), e23403). Additional stimulation strategies include adoptive cell therapy (ACT), administration of oncolytic viruses (OV) to initiate systemic antitumor immunity, and the use of antibodies targeting members of the tumor necrosis factor receptor superfamily that enhance T cell activity. Strategies to neutralize immunosuppressive mechanisms include chemotherapy (cyclophosphamide), antibodies that eliminate regulatory T cells (CD25-targeted antibodies), and antibodies against immune checkpoint molecules such as CTLA-4, PD1, and PD-L1.

[0009] The field of cancer immunotherapy has been primarily driven in recent years by the approval of sipuleucel-T, an autologous cell immunotherapy for the treatment of prostate cancer, in 2010 (Topalian et al., 2011, J. Clin. Oncol., 29:48 pp. 28-36), and the approval of anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) antibody, ipilimumab, and anti-programmed cell death protein 1 (PD1) antibody for the treatment of melanoma in 2011 and 2014 (Sharma et al., 2015, Cell, 161:20 pp. 5-14).

[0010] Effective anticancer effects have been demonstrated with the use of immune checkpoint inhibitors targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) / PD-1 ligand (PD-L1), achieving the highest response rates observed in high mutational burden cancers such as melanoma and non-small cell lung cancer (Andrews et al., 2017, Journal for ImmunoTherapy of Cancer, 25:10). However, these therapies have significant limitations, as objective responses to PD-1 inhibitors are observed in only 30-40% of patients, the majority of whom exhibit congenital resistance. Acquired resistance to anti-PD-1 therapy is also a problem in approximately one-quarter of responders who later develop disease progression (Ribas et al., 2016, JAMA, 315:1600-1699).

[0011] Furthermore, resistance to anticancer treatments in solid tumors has also been observed with anti-angiogenic therapy, and despite the promotion of beneficial effects, patients inevitably develop resistance, and significantly better overall survival is frequently not demonstrated, making it a matter of great interest in the use of anti-VEGF therapy (Gardner et al., 2017, Chapter 19, Anti-VEGF Therapy in Cancer: A Double-Edged Sword, http: / / dx.doi.org / 10.5772 / 66763, anti-PDGF agents).

[0012] Therefore, given the recent developments in various strategies in cancer immunotherapy, such as cancer vaccines, adoptive cell therapy, immune checkpoint inhibitors, and oncolytic viruses, as well as anti-angiogenic therapies, and the limitations faced by their efficacy, there is a growing need to develop anti-cancer therapies that are effective against solid cancers, particularly cancers that tend to develop resistance to immunotherapy or anti-angiogenic therapy, that can restore sensitivity to immunotherapy or anti-angiogenic treatment, or that enhance cancer vaccine therapy. [Prior art documents] [Patent Documents]

[0013]

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Non-licensed literature

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Summary of the Invention

Problems to be Solved by the Invention

[0015] This invention addresses the unexpected finding that the recently discovered ability of NOX4 pharmacological inhibition (Hanley et al., 2018, J Natl Cancer Inst., 110) to restore myofibroblastic CAF phenotypes in different cancer cell lines and suppress tumor growth in numerous CAF-rich tumor models (TC1+CAF [HNSCC model], 4T1+CAF [breast cancer], MMTV-PyVT (breast cancer), MMTV-Her2 / neu (breast cancer)) both in vitro and / or in vivo may synergistically enhance cancer immunotherapy or reverse resistance induced by anti-VEGF therapy.

[0016] This invention addresses the unexpected finding that NOX4 / 1 dual inhibitors can restore sensitivity to immunotherapy and / or improve responses to immunotherapy and anti-angiogenic therapy.

[0017] The present invention relates to compositions and methods useful for restoring responsiveness to immunotherapy, particularly to cancer vaccines such as HPV, and to immune checkpoint inhibition with, for example, PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0018] In particular, the present invention addresses the unexpected finding that NOX4 inhibitors can restore sensitivity to antitumor immunotherapy and / or improve the response to immunotherapy.

[0019] In particular, the present invention addresses the unexpected finding that NOX1 inhibitors can improve the response to anti-angiogenic therapy.

[0020] The present invention further relates to compositions and methods useful for restoring responsiveness to anti-angiogenic therapy, particularly to restoring responsiveness to anti-VEGF therapy, and / or for reducing or avoiding the emergence of resistance to anti-VEGF therapy. [Means for solving the problem]

[0021] A first aspect of the present invention provides a NOX4 inhibitor or NOX4 / 1 dual or NOX1 inhibitor for use in the treatment of solid tumor cancers that exhibit or are suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF therapy, which is administered in combination with an anti-cancer immunotherapy agent or an anti-angiogenic agent.

[0022] Another aspect of the present invention provides the use of one or more NOX4 or NOX4 / 1 dual or NOX1 inhibitors for preparing a pharmaceutical composition for the treatment of solid tumor cancers that exhibit or are suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF therapy, wherein the one or more NOX4 or NOX4 / 1 or NOX1 inhibitors are administered in combination with an anti-cancer immunotherapy agent or an anti-angiogenic agent.

[0023] Another aspect of the present invention relates to pharmaceutical compositions comprising at least one NOX4 or NOX4 / 1 or NOX1 inhibitor according to the present invention, in combination with at least one anti-cancer immunotherapy agent or at least one additional hyperangiogenic agent and at least one pharmaceutically acceptable carrier, excipient or additive thereof, as well as tautomers, geometric isomers, optionally active forms, and pharmaceutically acceptable salts thereof.

[0024] Another aspect of the present invention relates to a method for treating subjects with solid tumor cancer that exhibits or is suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF therapy, comprising the step of administering an effective amount of one or more NOX4, NOX4 / 1, or NOX1 inhibitors to a subject in need, in combination with an anti-cancer immunotherapy agent or anti-angiogenic agent.

[0025] Another aspect of the present invention relates to a method for restoring or enhancing responsiveness to anti-cancer immunotherapy in a subject, in particular restoring sensitivity to immunotherapeutic treatment, especially for making a cold tumor hot, and comprising the step of administering an effective amount of one or more NOX4, NOX4 / 1, or NOX1 inhibitors, or a pharmaceutical formulation thereof, in combination with an anti-cancer immunotherapy agent, to a subject in need thereof.

[0026] Another aspect of the present invention relates to a method for restoring or enhancing responsiveness to anti-cancer and anti-angiogenic treatment in a subject, particularly restoring sensitivity to anti-VEGF treatment or preventing resistance to anti-VEGF treatment, comprising the step of administering an effective amount of one or more NOX4, NOX4 / 1, or NOX1 inhibitors, or a pharmaceutical formulation thereof, in combination with an anti-angiogenic agent, to a subject in need thereof.

[0027] Other features and advantages of the present invention will become apparent from the following detailed description. [Brief explanation of the drawing]

[0028] [Figure 1A] As described in Example 1, when cancer cells are injected orthotopically into the breast fat pad simultaneously with cancer-associated fibroblasts (CAFs), the relocation of DCD8+ T cells to tumor 4T1 demonstrates the efficacy of NOX4 inhibitor (GKT) treatment. Increase in tumor volume, expressible in mm³, versus the number of days after injection (arrow) of either (1) a combination of tumor cells, CAFs, and a vehicle, or (2) a combination of tumor cells, CAFs, and a NOX4 inhibitor. [Figure 1B] As described in Example 1, when cancer cells are injected orthotopically into the breast fat pad simultaneously with cancer-associated fibroblasts (CAFs), the rearrangement of DCD8+ T cells to tumor 4T1 indicates the efficacy of treatment with a NOX4 inhibitor (GKT). Immunochemical methods and their quantification demonstrate the efficacy of NOX4 inhibitor treatment in reducing SMA-positive CAFs in tumors. [Figure 1C]As described in Example 1, when cancer cells are injected orthotopically into the breast fat pad simultaneously with cancer-associated fibroblasts (CAFs), the rearrangement of CD8+ T cells to tumor 4T1 demonstrates the efficacy of NOX4 inhibitor (GKT) treatment. Immunochemical analysis (and its quantification) shows that CD8+ T cells are rearranged from the tumor periphery to the tumor center as a result of treatment with NOX4 inhibitor. [Figure 2A] As described in Example 1, MC38 cancer cells are injected simultaneously with cancer-associated fibroblasts (CAFs) into mice being treated, demonstrating the efficacy of the combination of αPD1 and NOX4 inhibitor (GKT) in the therapeutic response of CAF-rich tumors. The effects of vehicle alone (Ctl), αPD1, NOX4 inhibitor (GKT) alone, or the combination of αPD1 + NOX4 inhibitor (GKT) are compared in terms of tumor growth after injection. [Figure 2B] As described in Example 1, MC38 cancer cells are injected simultaneously with cancer-associated fibroblasts (CAFs) into mice being treated, demonstrating the efficacy of the combination of αPD1 and NOX4 inhibitors (GKT) in the therapeutic response to CAF-rich tumors. Immunochemical analysis and quantification show that, compared to αPD1 alone, treatment with the combined αD1 / NOX4 inhibitors resulted in the rearrangement of CD8+ T cells from the tumor periphery to the tumor center. [Figure 2C] As described in Example 1, MC38 cancer cells are injected simultaneously with cancer-associated fibroblasts (CAFs) into treated mice, demonstrating the efficacy of the combination of αPD1 and NOX4 inhibitors (GKT) in the therapeutic response in CAF-rich tumors. Kaplan-Meier survival curves for various groups. [Figure 3A] As described in Example 2, the efficacy of the combination of antitumor vaccination and NOX4 inhibitor (GKT) is demonstrated. Tumor growth after injection in mice treated with the combination vaccine / GKT compared to vaccine alone and a control. [Figure 3B]As described in Example 2, the efficacy of the combination of antitumor vaccination and a NOX4 inhibitor (GKT) is demonstrated. Immunochemical analysis and quantification show that, compared to vaccine alone, treatment with the combination vaccine / NOX4 inhibitor resulted in the rearrangement of CD8+ T cells from the tumor periphery to the tumor center. [Figure 3C] As described in Example 2, the effects of the combination of antitumor vaccination and the NOX4 inhibitor (GKT) are shown. Kaplan-Meier survival curves for various groups. [Figure 4] As described in Example 3, the combination of an anti-angiogenic agent and a selective NOX1 inhibitor (GKT2) demonstrated efficacy in inhibiting angiogenesis as measured by CD45- / CD31+ / GP38 cells, compared to the control (*p<0.05; **p<0.01; ***p<0.005; ****p<0.001). [Figure 5] This study demonstrates the tumor size growth in NOX1-KO mice compared to WT mice, and the effect of the anti-VEGFR2 antibody (DC101) on reducing tumor growth in these mice. [Modes for carrying out the invention]

[0029] As used herein, the term “NOX inhibitor” refers to any substance that can inhibit, block, attenuate, or interfere with NOX4 and / or NOX1, either entirely or partially. The term is directly defined as a compound that affects the enzyme activity, cellular localization, protein stability, messenger RNA, or protein expression of an enzyme. Preferably, a NOX4 / NOX1 inhibitor must be able to reduce enzyme activity and ROS production in a cell-free assay using a membrane expressing only the NOX isoform NOX4 / 1 protein, such as recombinant protein NOX4 / 1. Thus, the term “inhibitor” is intended to include, but is not limited to, molecules that completely or partially inhibit the activity of NADPH oxidase 4 and / or NADPH oxidase 1. According to certain embodiments, a NOX4 / 1 inhibitor has a primary NOX inhibitory component against NOX4 and / or NOX1 compared to other NOX proteins, e.g., NOX2 and / or NOX3 / 5. According to certain embodiments, NOX4 / 1 inhibitors have a major NOX inhibitory activity at NOX4 / 1 that is at least about 5 times higher than other NOX proteins.

[0030] For example, NOX4 / 1 inhibitors include small molecules, peptides, peptide mimes, chimeric proteins, native or non-native proteins, nucleic acid-derived polymers (e.g., DNA and RNA aptamers, siRNA, shRNA, PNA, or LNA), fusion proteins with NOX4 / 1 that antagonize its activity, antibody antagonists such as neutralizing anti-NOX4 / 1 antibodies, or gene therapy vectors that induce the expression of such NOX4 / 1 antagonists.

[0031] In particular, NOX4 / 1 inhibitors are drugs that exhibit an inhibition coefficient Ki of less than 5 micromolars in functional ROS production assays, such as those described by Gaggini et al., 2011, Bioorganic and Medicinal Chemistry, Vol. 19(23), pp. 6989-6999. For example, NOX4 / 1 inhibitors are drugs that inhibit ROS production in a range of less than approximately 1 microM, such as approximately 30-300 nanomolars, in cell-free assays using membranes that express only NOX isoforms such as recombinant protein NOX4 or NOX1, such as NOX4 or NOX1 protein.

[0032] The term "siRNA" refers to a small interfering RNA, which is a double-stranded RNA (approximately 19-23 nucleotides) capable of knocking down or silencing target mRNA from a target gene. Artificial siRNA can be chemically synthesized as oligonucleotides or cloned as short hairpin RNA into plasmids or viral vectors (adenovirus, retrovirus, or lentivirus) to produce transient or stable transfection in any type of cell (Martin et al., 2007, Ann. Rev. Genomics Hum. Genet., 8:81-108; Huang et al., 2008, Expert. Opin. Ther. Targets, 12(5), pp. 637-645).

[0033] Examples of cancers described as "solid tumors" include glioblastoma, lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, and brain cancer, particularly glioblastoma.

[0034] As used herein, “treatment” and “to treat” generally mean obtaining a desired pharmacological and physiological effect. As used herein, the term “treatment” encompasses all treatments of diseases in mammals, particularly humans, including inhibiting the disease, i.e., stopping its onset, or alleviating the disease, i.e., causing a reduction in the disease and / or its symptoms or condition, such as cessation or regression of tumor growth.

[0035] As used herein, the term "subject" refers to mammals. For example, mammals considered in this invention include livestock such as humans, primates, cattle, sheep, pigs, horses, laboratory rodents, and dogs.

[0036] As used herein, the term “effective dose” refers to the amount of at least one particle or pharmaceutical formulation thereof according to the present invention that elicits a biological or medical response in a tissue, system, animal, or human being of interest. In one embodiment, the effective dose is a “therapeutic effective dose” for alleviating the symptoms of a disease or condition being treated. Typically, the effective dose may be used to inhibit cancer cell proliferation, i.e., to reduce the rate of cancer cell proliferation compared to the observed or predicted rate of proliferation of untreated control cancer cells, i.e., to delay the rate of cancer cell proliferation and / or migration, to halt cancer cell proliferation and / or migration, or to kill cancer cells. The term “inhibit proliferation” can also refer to a reduction or disappearance of the size of cancer cells or tumors, and a decrease in their metastatic potential. Preferably, such inhibition at the cellular level can reduce the size, delay proliferation, reduce invasiveness, or prevent or inhibit metastasis of a patient’s cancer. Those skilled in the art can easily determine whether cancer cell proliferation is being inhibited from any of a variety of appropriate signs.

[0037] The term "efficacy" of the treatment according to the present invention can be measured based on changes in the course of the disease depending on the use or method according to the present invention. The efficacy of cancer treatment according to the present invention can be measured by a reduction in tumor volume and / or an extension of progression-free survival and / or a higher level of health and well-being of the subject (e.g., suppression of cancer). Inhibition of cancer cell proliferation can be demonstrated, for example, by the arrest of cancer cells at a specific phase of the cell cycle, e.g., arrest at the G2 / M phase of the cell cycle. Inhibition of cancer cell proliferation can also be demonstrated using well-known imaging techniques such as magnetic resonance imaging, computed tomography, PET, SPECT, photoacoustic imaging, X-ray, and fluorescence imaging / excitation. Cancer cell proliferation can also be determined indirectly by determining, for example, the levels of circulating carcinoembryonic antigen, prostate-specific antigen, or other cancer-specific antigens correlated with cancer cell proliferation.

[0038] In particular, the efficacy of the combination therapy according to the present invention can be evaluated by a reduction in tumor size or the disappearance of any biomarker associated with the tumor or carcinoma.

[0039] Unless otherwise limited by the definition of individual substituents, the term "substituted" refers to a group substituted with 1 to 5 substituents selected from the group consisting of "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "C1-C6 alkylaryl", "C1-C6 alkylheteroaryl", "C1-C6 alkylcycloalkyl", "C1-C6 alkylheterocycloalkyl", "amino", "alkylamino", "aminosulfonyl", "ammonium", "alkoxy", "acyl", "acylamino", "aminocarbonyl", "aryl", "heteroaryl", "sulfinyl", "sulfonyl", "sulfonamide", "alkoxy", "alkoxycarbonyl", "carbamate", "sulfanyl", "halogen", trihalomethyl, cyano, hydroxy, mercapto, nitro, etc.

[0040] The term "pharmaceutically acceptable salt or complex" refers to a salt or complex of the compounds of the present invention as described below. Examples of such salts include, but are not limited to, base addition salts formed by the reaction of the compounds of the present invention with a metal cation selected from the group consisting of alkali metals (sodium, potassium, or lithium) or alkaline earth metals (e.g., calcium or magnesium) with an organic or inorganic base such as a hydroxide, carbonate, or bicarbonate, or with an organic first, second, or third alkylamine. Other examples of such salts include, but are not limited to, acid addition salts formed by the reaction of the compounds of the present invention with an organic or inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, p-toluenesulfonic acid, 2-naphthalenesulfonic acid, camphosulfonic acid, benzenesulfonic acid, or oxalic acid.

[0041] "Pharmacologically active derivative" means any compound that, upon administration to a recipient, directly or indirectly imparts the activity disclosed herein.

[0042] NOX4 / NOX1 inhibitor according to the present invention In one embodiment, the present invention provides a NOX4 or NOX4 / 1 or NOX1 inhibitor that exhibits an inhibition coefficient (Ki) for NOX4 and / or NOX1 in the range of 60 nM to 300 nM in a functional assay of ROS production, wherein the inhibitory activity against other NOX selected from NOX2, NOX3 and NOX5 is greater than 1 micromolar.

[0043] According to certain embodiments, the NOX4 or NOX4 / NOX1 or NOX1 inhibitor according to the present invention is a pyrazolopyridine compound, a pyrazolindione compound, or an amidethiazole compound, as described, for example, in WO 2008 / 113856, WO 10 / 035217, WO 10 / 035219, WO 10 / 035220, WO 10 / 035221, WO 11 / 036651, WO 2013 / 068972, WO 2015 / 049655 and WO 2016 / 098005.

[0044] According to another specific embodiment, the NOX4 inhibitors according to the present invention are 2,5-disubstituted benzoxazoles and benzothiazole derivatives, as described, for example, in WO 2016 / 207785.

[0045] In one embodiment, the present invention relates to a NOX4 inhibitor, formula (I)

[0046] [ka]

[0047] (wherein G1 is selected from H, optionally substituted alkyl, e.g., aminocarbonylalkyl (e.g., phenylacetamide), optionally substituted C3-C8 cycloalkylalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted arylalkyl, e.g., optionally substituted phenylalkyl, e.g., optionally substituted phenylmethyl (e.g., phenylmethyl or 3-methylphenylmethyl or 4-fluorobenzyl or 2-chlorobenzyl or 4-chlorobenzyl or 4-methylbenzyl or 4-bromobenzyl); and optionally substituted heteroarylalkyl, e.g., optionally substituted pyridinealkyl, e.g., pyridine-2-ylmethyl; G2 is H; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl, e.g., optionally substituted phenyl (e.g., phenyl or 4-fluorophenyl or 4-methoxyphenyl or 4-nitrophenyl or 2-chlorophenyl or 2-methylphenyl or 4-(trifluoromethyl)phenyl or 4-(trifluoromethoxy)phenyl or 2,5-difluorophenyl or 2-methoxyphenyl); optionally substituted alkylaryl; optionally substituted arylalkyl; optionally substituted heteroaryl, e.g., optionally substituted benzothiazolyl (e.g., 1,3-benzothiazole- Selected from: 2-yl) or optionally substituted pyridinyl (e.g., pyridine-2-yl); optionally substituted alkyl heteroaryl; optionally substituted heteroarylalkyl; optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenyl heteroaryl; optionally substituted heteroarylalkenyl; optionally substituted C3-C8 cycloalkyl; optionally substituted heterocycloalkyl; optionally substituted alkylC3-C8 cycloalkyl; optionally substituted C3-C8 cycloalkylalkyl; optionally substituted alkylheterocycloalkyl and optionally substituted heterocycloalkylalkyl; G3 is H; optionally substituted alkyl, e.g., methyl or ethyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl, e.g., optionally substituted phenyl (e.g., phenyl).); optionally substituted alkylaryl; optionally substituted arylalkyl; optionally substituted heteroaryl; optionally substituted alkylhetaryl; optionally substituted heteroarylalkyl; optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenylhetaryl; optionally substituted heteroarylalkenyl; optionally substituted C3-C8 cycloalkyl; optionally substituted heterocycloalkyl; optionally substituted alkylC3-C8 cycloalkyl; optionally substituted C3-C8 cycloalkylalkyl; optionally substituted alkylheterocycloalkyl and optionally substituted heterocycloalkylalkyl; selected from these; G4 is H, optionally substituted alkyl, for example optionally substituted pentyl (e.g., isopentyl) or optionally substituted heteroalkyl, for example optionally substituted methoxy (e.g., 2-methoxyethyl); optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl; optionally substituted alkylaryl; optionally substituted arylalkyl, for example optionally substituted phenylmethyl (e.g., methyl benzoate or benzyl benzoate) or optionally substituted phenylethyl (e.g., 2-phenylethyl, 4-methoxyphenylethyl); optionally substituted heteroaryl; optionally substituted alkylheteroaryl; optionally substituted heteroarylalkyl, for example optionally substituted thiophenylalkyl, for example Optionally substituted thiophenylmethyl (e.g., thiophen-2-ylmethyl) or optionally substituted imidazolylalkyl, optionally substituted imidazolylethyl (e.g., imidazole-4-ylethyl) or optionally substituted indolylalkyl, optionally substituted indolylethyl (e.g., indole-3-ylethyl) or optionally substituted furanylalkyl, optionally substituted furanylmethyl (e.g., furan-2-ylmethyl) or optionally substituted benzodioxolylalkyl, optionally substituted benzodioxolylmethyl (e.g., 1,3-benzodioxol-5-ylmethyl) or optionally substituted pyridinylalkyl, optionally substituted pyridinylmethyl (e.g., pyridine-3-ylmethyl).(I or pyridine-2-ylmethyl); optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenyl heteroaryl; optionally substituted heteroarylalkenyl; optionally substituted C3-C8 cycloalkyl; optionally substituted heterocycloalkyl, e.g., optionally substituted morpholinyl (e.g., 5-morpholin-4-yl) or optionally substituted piperazinyl (e.g., 4-methylpiperazinyl) or optionally substituted piperidinyl (e.g., 4-methylbenzyl)piperidine-4-yl); optionally substituted alkyl C3-C8 cycloalkyl; and optionally substituted C3-C8 cycloalkylalkyl; optionally substituted alkyl heterocycloalkyl and optionally substituted heterocycloalkylalkyl, e.g., optionally A substituted morpholinyl alkyl, for example, optionally substituted morpholinylpropyl (e.g., 3-(morpholin-4-yl)propyl) or optionally substituted morpholinyl ethyl (e.g., 2-morpholin-4-ylethyl); or optionally substituted piperadinyl alkyl, for example, optionally substituted piperadinyl ethyl (e.g., 2-(4-acetylpiperazine-1-yl)ethyl or 2-(4-hexanoylpiperazine-1-yl)ethyl); or optionally substituted pyrrolidinyl alkyl, for example, optionally substituted pyrrolidinylpropyl (e.g., 3-(2-oxopyrrolidine-1-yl)propyl); or optionally substituted tetrahydrofuranyl alkyl, for example, optionally substituted tetrahydrofuranylmethyl (e.g., tetrahydrofuranyl-2-ylmethyl); G5 is H, optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl; optionally substituted alkylaryl; optionally substituted arylalkyl; optionally substituted heteroaryl; optionally substituted alkylhetaryl; optionally substituted heteroarylalkyl; optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenylhetaryl; optionally substituted heteroarylalkenyl; optionally substituted C3-C8 cycloalkyl; optionally substituted heterocycloalkyl; optionally substituted alkylC3-C8 cycloalkyl;Selected from optionally substituted C3-C8 cycloalkylalkyls; optionally substituted alkyl heterocycloalkyls and optionally substituted heterocycloalkylalkyls); Furthermore, the present invention provides pharmaceutically acceptable salts and pharmaceutically active derivatives thereof.

[0048] In another embodiment, the present invention relates to a NOX4 / 1 inhibitor, formula (II)

[0049] [ka]

[0050] (wherein Ar is optionally substituted phenyl, e.g. halogen, e.g. chloro, optionally substituted phenyl (e.g., 2-chlorophenyl), or optionally substituted phenyl (e.g., methoxy); G1 and G4 are H; G2 is selected from optionally substituted C1-C6 alkyl (e.g., methyl) and optionally substituted phenyl (e.g., optionally substituted phenyl with halogen, e.g., 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 4-chloro-2-fluorophenyl, 5-chloro-2-fluorophenyl, optionally substituted phenyl with amino, alkylamino, or alkoxy, e.g., 3-dimethylaminophenyl, 2-trimethylaminophenyl, 3-methylaminophenyl, 3-aminophenyl, 4-methoxyphenyl); G3 is selected from H, optionally substituted C1-C6 alkyl (e.g., methyl, alkoxy-substituted C1-C6 alkyl, e.g., methoxyethyl, e.g., 2-methoxyethyl), optionally substituted heteroaryl C1-C6 alkyl, e.g., optionally substituted pyridinyl C1-C6 alkyl (e.g., optionally substituted pyridinyl methyl, e.g., pyridinyl-2-ylmethyl, pyridinyl-3-ylmethyl, 6-methoxypyridine-3-ylmethyl, 2-methoxypyridine-4-ylmethyl), or optionally substituted pyrazinyl C1-C6 alkyl (e.g., pyrazinyl-2-ylmethyl) and optionally substituted alkoxy C1-C6 alkyl, e.g., methoxyethyl (e.g., 2-methoxyethyl), or G2 and G3 together form an optionally substituted 7-membered heterocycloalkyl ring containing two nitrogen atoms, where the two nitrogen atoms are bonded via an optionally substituted C1-C3 alkyl moiety. The present invention also provides tautomers, geometric isomers, optionally active forms, and pharmaceutically acceptable salts thereof.

[0051] In certain embodiments, the present invention provides NOX4 / 1 inhibitors of formula (II), as well as tautomers, geometric isomers, optionally active forms, and pharmaceutically acceptable salts thereof, where G2 and G3 together form an optionally substituted 7-membered heterocycloalkyl ring containing two nitrogen atoms, as shown in formula (I'):

[0052] [ka]

[0053] (In the formulas, Ar, G1 and G5 are defined herein; G6, G8~G 10 is H; G7 is an optionally substituted C1-C6 alkyl, for example an optionally substituted C1-C6 alkyl with optionally substituted phenyl (for example an optionally substituted methyl with optionally substituted phenyl, for example benzyl, a halogen-substituted phenyl-substituted methyl, for example 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, an alkoxy-substituted phenyl-substituted methyl, for example 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl), an optionally substituted aryl C1-C6 alkyl, for example an optionally substituted (Selected from phenyl C1-C6 alkyl groups (e.g., benzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl) and optionally substituted heteroaryl C1-C6 alkyl groups, for example, optionally substituted pyridinyl C1-C6 alkyl groups (e.g., optionally substituted pyridinylmethyl, pyridinyl-2ylmethyl, pyridinyl-3ylmethyl) or optionally substituted furanyl C1-C6 alkyl groups (e.g., optionally substituted furanylmethyl, fran-3ylmethyl)) It forms a compound.

[0054] In certain embodiments, the present invention provides compounds of formula (II) for use according to the present invention, wherein G2 is an optionally substituted C1-C6 alkyl group.

[0055] In another specific embodiment, the present invention provides a compound of formula (II) for use according to the present invention, wherein G2 is an optionally substituted phenyl compound.

[0056] In another specific embodiment, the present invention provides compounds of formula (II) for use according to the present invention, wherein G3 is an optionally substituted C1-C6 alkyl group.

[0057] In another specific embodiment, the present invention provides compounds of formula (II) for use according to the present invention, wherein G3 is an optionally substituted heteroaryl C1-C6 alkyl, such as an optionally substituted pyridinyl C1-C6 alkyl.

[0058] In another specific embodiment, the present invention provides compounds of formula (II) for use according to the present invention, wherein G2 and G3 together form an optionally substituted 7-membered heterocycloalkyl ring containing two nitrogen atoms to form the following compounds of formula (I') (where G7 is an optionally substituted C1-C6 alkyl).

[0059] In addition, the present invention provides a compound of formula (II) for use according to the present invention, wherein G2 and G3 together form an optionally substituted 7-membered heterocycloalkyl ring containing two nitrogen atoms to form the following compound of formula (I') (where G7 is an optionally substituted aryl C1-C6 alkyl).

[0060] In addition, the present invention provides a compound of formula (I) for use according to the present invention, wherein G2 and G3 together form an optionally substituted 7-membered heterocycloalkyl ring containing two nitrogen atoms to form the following compound of formula (I') (where G7 is an optionally substituted heteroaryl C1-C6 alkyl).

[0061] According to another specific embodiment, the NOX1 inhibitor according to the present invention is an amidothiazole derivative, as described, for example, in WO 2016 / 098005.

[0062] In another embodiment, the present invention relates to formula (III):

[0063] [ka]

[0064] (In the formula, X is CR 1 and are selected from N; Y is selected from CH or N; A1 is -OCHR 5 -, -NR 4 -CHR 5 -, -CH2NR 4 - and -CH2-O- are selected; R 1 R is selected from H, halogens, and optionally substituted C1-C6 alkyl groups; 2is selected from H, halogen (for example, chloro, fluoro), optionally substituted alkoxy, for example, optionally substituted methoxy (for example, methoxy, (tetrahydro-2H-pyran-4-yl)methoxy, piperidin-4-ylmethoxy) or optionally substituted ethoxy (for example, 2-(dimethylamino)ethoxy, 2-hydroxyethoxy, 1-phenylethoxy, 2-methoxyethoxy), optionally substituted alkoxy C1-C6 alkyl, optionally substituted C1-C6 alkyl, for example, optionally substituted methyl, optionally substituted amino, for example, optionally substituted C1-C6 alkylamino (for example, methylamino, (tetrahydro-2H-pyran-4-yl)methyl)amino, (1-methylpiperidin-4-yl)methyl)amino, dimethylamino, optionally substituted ethylamino, for example, 2-morpholinoethylamino or 2-(dimethylamino)ethylamino or methoxyethylamino, optionally substituted methylamino, for example, 1-methyl-1H-imidazol-4-ylmethylamino or 2-hydroxyethyl)amino, optionally substituted propylamino, for example, dimethylaminopropylamino), optionally substituted heterocycloalkyl, for example, optionally substituted piperazine (for example, methylpiperazin-1-yl), optionally substituted C1-C6 alkyl heterocycloalkyl, for example, optionally substituted C1-C6 alkyl piperazine (for example, methylpiperazin-1-yl), optionally substituted amino C1-C6 alkyl, optionally substituted alkoxy C1-C6 alkyl, -O-R 8 and -NR 9 R 10 is selected from; R 3 is a group of the formula -(CHR 6 ) n -A2, or R 3 is a part CHR from A1 5and optionally substituted aryls, for example optionally substituted phenyls (e.g., phenyls substituted with phenyl or halogens, e.g., alkoxys, e.g., fluorophenyls substituted with methoxy) and optionally substituted heteroaryls, for example optionally substituted 1,3-dihydro-1H-indenyls (e.g., 1-(dimethylamino)-2,3-dihydro-1H-inden-2-yl, 2,3-dihydro-1H-inden-2-yl, 2,3-dihydro-1H-inden-1-yl) or optionally substituted 6,7-dihydro-5H-cyclopentapyridinyls (e.g., 6,7-dihydro-5H-cyclopentapyridinyl, 2-methylpyridine-3-yl, 5-methylpyridine-2-yl) or optionally It forms an optionally substituted ring selected from optionally substituted 1,2,3,4-tetrahydronaphthalenyl (e.g., 1,2,3,4-tetrahydronaphthalen-1-yl), optionally substituted 2,3-dihydrobenzofuranyl (e.g., 2,3-dihydrobenzofuran-3-yl, 2,3-dihydro-1H-inden-1-yl), optionally substituted thiadiazolyl (e.g., 1,3,4-thiadiazole-2-yl), optionally substituted isoxazolyl (e.g., 5-methylisoxazol-3-yl), optionally substituted pyrazolyl (e.g., 1-methyl-1H-pyrazole-3-yl), or optionally substituted imidazolyl (e.g., 1-methyl-1H-imidazole-2-yl), or R 3 This is a partial NR from A1. 4 This forms an arbitrarily substituted ring selected from arbitrarily substituted aryls and arbitrarily substituted heteroaryls, for example, arbitrarily substituted isoindolinyls (e.g., isoindolin-2-yl, 1H-indole-1-yl); n is an integer from 0 to 4 (e.g., 0, 1, 2, 3, or 4); R 4A2 is selected from H and optionally substituted alkyl, for example, optionally substituted methyl; A2 is optionally substituted aryl, for example, optionally substituted phenyl (e.g., methoxyphenyl, fluorophenyl, chlorophenyl), optionally substituted heteroaryl, for example, optionally substituted pyridine (e.g., pyridine-2-yl, pyridine-3-yl, pyridine-4-yl, 2-methylpyridine-3-yl, 5-methylpyridine-2-yl), or optionally substituted pyrazolyl (e.g., 1,3-dimethyl-1H-pyrazole-5-yl, 1-methyl-1H-pyrazole-3-yl), or optionally substituted thiadiazolyl (e.g., 1,3,4-thiadiazole-2-yl), or optionally substituted An optionally substituted ring selected from imidazolyl (e.g., 1H-imidazole-4-yl, 1-methyl-1H-imidazole-2-yl, 1-methyl-1H-imidazole-5-yl), or optionally substituted 1,2,4-triazolyl (e.g., 1-methyl-1H-1,2,4-triazol-5-yl), or optionally substituted isoxazolyl (e.g., 1-cyclopropylisoxazol-3-yl), or optionally substituted oxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazolyl-3-yl), or optionally substituted pyrimidinyl (e.g., pyrimidinyl-2-yl); R 5This includes H, optionally substituted C1-C6 alkyl groups, for example optionally substituted methyl (e.g., methoxymethyl, 3,3-difluoropyrrolidine-1-ylmethyl, 4-methylpiperazine-1-ylmethyl, hydroxylmethyl) or optionally substituted ethyl or optionally substituted propyl (e.g., methyl, hydroxymethyl, hydroxyethyl, 2-propanolyl, hydroxylisopropyl), optionally substituted amino C1-C6 alkyl groups, for example optionally substituted aminomethyl (e.g., dimethylaminomethyl, methylaminomethyl), optionally substituted alkoxy C1-C6 alkyl groups, optionally substituted heterocycloalkyl C1-C6 alkyl groups, for example optionally substituted heterocycloalkylmethyl, for example optionally substituted pyrrolidine C1-C6 alkyl groups (for example Selected from 3,3-difluoropyrrolidine-1-ylmethyl) or substituted piperazine C1-C6 alkyl (e.g., 4-methylpiperazine-1-ylmethyl) or heterocycloalkylethyl, for example, optionally substituted morpholino C1-C6 alkyl (e.g., morpholinomethyl, morpholinoethyl) or optionally substituted pyrrolidine C1-C6 alkyl (e.g., pyrrolidinemethyl, pyrrolidineethyl), optionally substituted aminocarbonyl (e.g., dimethylaminocarbonyl), optionally substituted C2-C8 cycloalkyl, for example, optionally substituted cyclopropyl and optionally substituted amino C1-C6 alkyl, for example, optionally substituted aminoethyl (e.g., dimethylaminoethyl) or optionally substituted aminomethyl (e.g., dimethylaminomethyl); R 6 R is selected from H, optionally substituted C1-C6 alkyl groups, such as optionally substituted methyl, optionally substituted amino, optionally substituted C1-C6 alkylamino (e.g., dimethylamino), and hydroxy, where R 6 The base is an independent repeating unit (CHR 6 Selected each time; R 7 R is selected from H, halogens (e.g., fluoro) and optionally substituted C1-C6 alkyl groups, such as methyl; R 8This is selected from H, optionally substituted C1-C6 alkyl, e.g., optionally substituted methyl or optionally substituted ethyl (e.g., methoxyethyl, 2-(dimethylamino)ethyl, hydroxyethyl), optionally substituted amino C1-C6 alkyl, optionally substituted heterocycloalkyl, optionally substituted C2-C8 cycloalkyl, optionally substituted heterocycloalkyl C1-C6 alkyl, e.g., optionally substituted heterocycloalkylmethyl, e.g., optionally substituted tetrahydropyran C1-C6 alkyl (e.g., tetrahydro-2H-pyran-4-yl) or optionally substituted piperidine alkyl (e.g., 1-methylpiperidine-4-yl), optionally substituted C2-C8 cycloalkyl C1-C6 alkyl, optionally substituted alkoxy, optionally substituted amino C1-C6 alkyl, e.g., optionally substituted aminoethyl (e.g., 2-(dimethylamino)ethyl); optionally substituted aryl C1-C6 alkyl and optionally substituted heteroaryl C1-C6 alkyl; R 9 and R 10These are independently H, optionally substituted C1-C6 alkyl groups, for example optionally substituted methyl (e.g., 1-methyl-1H-imidazole-4-yl)methyl) or optionally substituted ethyl (e.g., 2-methoxyethyl), optionally substituted amino C1-C6 alkyl groups, for example optionally substituted aminoethyl (e.g., dimethylaminoethyl) or optionally substituted aminopropyl (e.g., dimethylaminopropyl), optionally substituted heterocycloalkyl groups, for example optionally substituted piperidine (e.g., 1-methylpiperidine), optionally substituted C2-C8 cycloalkyl groups, optionally substituted heterocycloalkyl C1-C6 alkyl groups, for example optionally substituted heterocycloalkylethyl groups, for example optionally substituted morpholino C1-C6 alkyl groups (e.g., 2-morpholinoethyl) or optionally substituted heterocycloalkylmethyl groups, for example optionally substituted tetrahydrofuran C1-C6 alkyl groups (e.g., tetrahydro-2H-pyra Selected from: (1-(methyl-1H-imidazole-4-ylmethyl) or piperidine C1-C6 alkyl (e.g., 1-methylpiperidine-4-yl)methyl or optionally substituted imidazolyl C1-C6 alkyl (e.g., 1-methyl-1H-imidazole-4-yl)methyl), optionally substituted C2-C8 cycloalkyl C1-C6 alkyl, optionally substituted alkoxy, optionally substituted alkoxy C1-C6 alkyl, e.g., optionally substituted alkoxyethyl (e.g., 2-methoxyethyl), optionally substituted aryl C1-C6 alkyl and optionally substituted heteroaryl C1-C6 alkyl, e.g., heteroaryl C1-C6 alkylmethyl, e.g., optionally substituted imidazolyl C1-C6 alkyl (e.g., 1-methyl-1H-imidazole-4-ylmethyl), optionally substituted amino C1-C6 alkyl, e.g., optionally substituted aminoethyl or optionally substituted aminopropyl (e.g., 2-(dimethylamino)ethyl, 2-(dimethylamino)propyl)). The present invention provides NOX1 inhibitors, as well as their tautomers, geometric isomers, optionally active forms, pharmaceutically acceptable salts, and pharmaceutically active derivatives.

[0065] In certain embodiments, the present invention provides compounds of formula (III) for use according to the present invention, wherein X is CH.

[0066] In certain embodiments, the present invention relates to a compound of formula (III) for use according to the present invention, wherein Y is CR 1 The present invention provides compounds that are, in particular, CH.

[0067] In certain embodiments, the present invention relates to a compound of formula (III) for use according to the present invention, and R 2 The present invention provides a compound which is an arbitrarily substituted alkoxy (e.g., methoxy).

[0068] In certain embodiments, the present invention relates to a compound of formula (III) for use according to the present invention, and R 7 The present invention provides a compound in which H is present.

[0069] In certain embodiments, the present invention relates to a compound of formula (III) for use according to the present invention, wherein A1 is -OCHR 5 And especially R 5 The present invention provides compounds that are optionally substituted morpholino C1-C6 alkyl groups (e.g., morpholinomethyl).

[0070] In another specific embodiment, the present invention relates to a compound of formula (III) for use according to the present invention, wherein A1 is -OCHR 5 And especially R 5 The present invention provides compounds that are optionally substituted amino C1-C6 alkyl groups (e.g., dimethylaminomethyl).

[0071] In another specific embodiment, the present invention relates to a compound of formula (III) for use according to the present invention, wherein A1 is -OCHR 5 And especially R 5 The present invention provides compounds that are optionally substituted with hydroxyl C1-C6 alkyl groups (e.g., hydroxymethyl groups).

[0072] In certain embodiments, the present invention relates to a compound of formula (III) for use according to the present invention, and R 3 However, formula -(CHR 6 ) n The present invention provides a compound that is an A2 group, in particular n is 0, and A2 is an optionally substituted phenyl (e.g., phenyl).

[0073] According to another specific embodiment, the NOX1 inhibitor according to the present invention is 3-methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide, and in particular (R)3-methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide.

[0074] In another embodiment, formula (IV):

[0075] [ka]

[0076] (In the formula, ring (A) represents a non-aromatic 5- to 7-membered heterocycle fused with a phenyl group; the 5- to 7-membered heterocycle contains one oxygen ring atom and one additional ring heteroatom independently selected from oxygen or nitrogen; the 5- to 7-membered heterocycle is independently unsubstituted, monosubstituted, or disubstituted, and the substituents are independently: · A single oxo substituent bonded to the ring carbon atom at the alpha position of a cyclic oxygen and / or cyclic nitrogen atom; and / or • One C bonded to a cyclic nitrogen atom with free valence 1~3 Alkyl; or • Two fluoro substituents bonded to the same ring carbon atom. Selected from; L is -NH-CO- * or -CO-NH- * It represents an asterisk ( *) indicates a bond that binds to the benzoxazole / benzothiazole moiety; X represents O or S, and Y is -NR 1 R 2 (R 1 R 1 C 1~4 Alkyl; di-(C 1~3 Alkyl)amino, hydroxy, or C 1~3 C monosubstituted with alkoxy 2~4 Alkyl; C 3~5 Cycloalkyl-L 1 And, L 1 However, direct bonding or C 1~3 Represents alkylene, C 3~5 The cycloalkyl group optionally contains one oxygen ring atom, and the C 3~5 The cycloalkyl group is either unsubstituted or monosubstituted with methyl or fluoropolymer. 3~5 Cycloalkyl-L 1 ; or, C 3~5 A cycloalkyl piperidine-3-yl, piperidine-4-yl, or pyrrolidine-3-yl group substituted at the cyclic nitrogen atom, wherein the C 3~5 Cycloalkyl represents a piperidine-3-yl, piperidine-4-yl, or pyrrolidine-3-yl group which optionally contains one oxygen ring atom; and R 2 is hydrogen, C 1~3 Alkyl, or C 3~5 (representing cycloalkyl) or, Y is morpholin-4-yl; 2-oxo-pyrrolidine-1-yl; 1,1-dioxidethiomorpholin-4-yl; or optionally oxetane-3-yl or C 1~3 A saturated 4- to 7-membered monocyclic heterocycline selected from alkyl, monosubstituted at the 4-position piperazine-1-yl, or azetidine-1-yl, pyrrolidine-1-yl, or piperidine-1-yl, wherein the azetidine-1-yl, pyrrolidine-1-yl, or piperidine-1-yl is independently unsubstituted or • Two fluoro substituents bonded to the same ring carbon atom; or, • Unsubstituted phenyl, or one substituent selected from unsubstituted or 6-membered heteroaryls; or, ·Hydroxy; C 1~3 Alkoxy;-CO-C 1~4 Alkoxy; di(C) 1~3 Alkyl)amino; and di(C) 1~3 Alkyl)amino, hydroxy, or C 1~3 C monosubstituted with alkoxy 1~3 One substituent selected from alkyl groups; or, • Two substituents, one of which is C 1~4 It is alkyl, and the other is independently hydroxy or di(C) 1~3 Two substituents selected from alkyl)amino; or, Morpholin-4-yl; 1,1-dioxidethiomorpholin-4-yl; or optionally C 1~3 One substituent selected from an alkyl group monosubstituted at the 4-position piperazine-1-yl; • One substituent selected from azetidine-1-yl, pyrrolidine-1-yl, or piperidine-1-yl, which is either unsubstituted, monosubstituted with hydroxyl, or disubstituted with methyl and hydroxyl. It represents a saturated 4-7 member monocyclic heterocycline that is substituted with, Alternatively, Y is a saturated 7-11 member condensed, cross-linked, or spironicyclic heterocycline containing at least one nitrogen atom, wherein the nitrogen atom is bonded to a benzoxazole / benzothiazole moiety, and the heterocycline optionally contains one additional ring heteroatom independently selected from oxygen, nitrogen, and sulfur, and is unsubstituted, or - Two oxo substituents on a cyclic sulfur ring atom; or, - One C bonded to a cyclic nitrogen atom with free valence 1~3 Alkyl substituents (Represents saturated 7-11 member condensed, bridging, or spironicyclic heterocyclines that are substituted with [a specific compound].) NOX4 inhibitors or pharmaceutically acceptable salts thereof are provided.

[0077] In another specific embodiment, a compound of formula (I) for use according to the present invention,

[0078] [ka]

[0079] A compound is provided which is 2-(2-chlorophenyl)-4-methyl-5-(pyridine-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione.

[0080] In another specific embodiment, a compound of formula (I) for use according to the present invention,

[0081] [ka]

[0082] A compound is provided which is 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione.

[0083] In another specific embodiment, a compound of formula (I) for use according to the present invention,

[0084] [ka]

[0085] A compound is provided which is 4-(2-fluoro-4-methoxyphenyl)-2-(2-methoxyphenyl)-5-(pyridine-3-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione.

[0086] In another specific embodiment, a compound of formula (I') for use according to the present invention,

[0087] [ka]

[0088] A compound is provided which is 10-benzyl-2-(2-chlorophenyl)-2,3,8,9,10,11-hexahydro-1H-pyrazolo[4',3':3,4]pyrido[1,2-a][1,4]diazepine-1,5(7H)-dione.

[0089] In another specific embodiment, a compound of formula (IV) for use according to the present invention,

[0090] [ka]

[0091] A compound is provided which is (R)-3-methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide.

[0092] In another specific embodiment, a compound of formula (IV) for use according to the present invention,

[0093] [ka]

[0094] A compound is provided which is (S)-3-methoxy-4-(1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide.

[0095] In another specific embodiment, a compound of formula (IV) for use according to the present invention,

[0096] [ka]

[0097] A compound is provided which is (R)-4-(2-hydroxy-1-phenylethoxy)-3-methoxy-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide.

[0098] In another specific embodiment, a compound of formula (IV) for use according to the present invention,

[0099] [ka]

[0100] A compound is provided which is (R)-4-(2-dimethylamino-1-phenylethoxy)-3-methoxy-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide.

[0101] In another specific embodiment, compounds according to the present invention, selected from the following group, are provided: 2-(2-chlorophenyl)-4-methyl-5-(pyridine-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; 4-(2-fluoro-4-methoxyphenyl)-2-(2-methoxyphenyl)-5-(pyridine-3-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; (R)-3-Methoxy-4-(2-Molfolino-1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide; 10-benzyl-2-(2-chlorophenyl)-2,3,8,9,10,11-hexahydro-1H-pyrazolo[4',3':3,4]pyrido[1,2-a][1,4]diazepine-1,5(7H)-dione; (S)-3-Methoxy-4-(1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide; (R)-4-(2-hydroxy-1-phenylethoxy)-3-methoxy-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide, and (R)-4-(2-dimethylamino-1-phenylethoxy)-3-methoxy-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide.

[0102] In a particular embodiment, a NOX inhibitor selected from NOX4 inhibitors and NOX4 / 1 inhibitors is provided for use in combination with a cancer vaccine or at least one immune checkpoint inhibitor.

[0103] Furthermore, according to a particular embodiment, a NOX inhibitor selected from NOX4 inhibitors and NOX4 / 1 inhibitors is provided for use in combination with a cancer vaccine or at least one immune checkpoint inhibitor.

[0104] According to another, even more specific embodiment, a NOX inhibitor selected from NOX4 inhibitors and NOX4 / 1 inhibitors is provided for use in combination with a cancer vaccine.

[0105] In a further specific embodiment, a NOX inhibitor selected from NOX1 inhibitors and NOX1 / 4 inhibitors is provided for use in combination with at least one anti-angiogenic agent.

[0106] Anti-cancer immunotherapy agent according to the present invention Anticancer immunotherapy agents that can be used in accordance with the present invention include oncolytic or anti-herpes simplex virus vaccines such as those described in Bartlett et al., 2013, Molecular Cancer 2, 12:103 (e.g., talimogene-laharpalepbek (Imritik)) or Fukuhara et al., 2016, Cancer Sci, 107(10), pp. 1373-1379, adoptive cell immunotherapy such as those described in Perica et al., 2015, Rambam Maimonides Med J, 6(1), e0004, Iwai et al., 2017, Journal of Biomedical Science, 24:26 or Mishra, 2017, Future Oncol. doi: 10.2217 / fon-2017-0115 or Soto Chervin et al., 2016, F1000 Research 2016, 5 (F1000 Faculty This includes cancer vaccines such as PD-1 inhibitors like those listed in Rev):803 (e.g., pembrolizumab (Keytruda), nivolumab (Opdivo)), PD-L1 inhibitors like atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), or immune checkpoint inhibitors like CTLA-4 inhibitors like ipilimumab (Yervoy).

[0107] According to another specific embodiment, the immune checkpoint inhibitor according to the present invention may be selected from T cell immunoglobulin and mucin domain 3 (TIM3), lymphocyte activator gene-3 (LAG3), T cell immunoglobulin and ITIM domain (TIGIT), or B and T lymphocyte attenuator (BTLA) inhibitors.

[0108] In a particular embodiment, the immune checkpoint inhibitor according to the present invention is a PD-1 inhibitor.

[0109] According to certain embodiments, the anti-cancer vaccines of the present invention include DNA, RNA, peptides, and oncolytic virus vaccines.

[0110] Furthermore, more generally, since the infiltration of CD8+ T cells into tumors is fundamental to many immunotherapies, the combinations and combined uses according to the present invention are also useful in adoptive T cell induction therapies, including tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR) T cells, and chimeric antigen receptor (CAR) T cells, as described by June et al., 2018, Science, 359: pp. 1361-1365. TILs have been shown to induce a sustained complete response in patients with metastatic melanoma. While CAR T cells offer significant advantages in the treatment of hematological malignancies (Kochenderfer et al., 2010, Blood 116, pp. 4099-4102; Porter et al., 2011, N. Engl. J. Med., 365, pp. 725-733; Brentjens et al., 2013, Sci. Transl. Med., 5, 177ra38; Grupp et al., 2013, N. Engl. J. Med., 368, pp. 1509-1518), the tumor microenvironment remains a major barrier to achieving favorable outcomes in solid tumors.

[0111] Similarly, immunotherapeutic agents that can be used in accordance with the present invention include CD8+ T cell agonists such as α-CD40, α-CD27, α-41BB, α-OX40, and GITR.

[0112] Anti-angiogenic agents for use in combination according to the present invention Anti-angiogenic agents that can be used in accordance with the present invention include anti-VEGF agents such as those described in Gardner et al. in 2017 (above), particularly bevacizumab or sunitinib.

[0113] composition The present invention provides a drug or therapeutic agent as a composition, and a method for treating patients, preferably mammalian patients, most preferably human patients, who have solid tumor cancers that exhibit or are suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF therapy.

[0114] The pharmaceutical compositions of the present invention may contain one or more compounds in any form described herein. The compositions of the present invention may further contain one or more pharmaceutically acceptable additional components, such as alum, solubilizers, stabilizers, antimicrobial agents, buffers, colorants, flavorings, and auxiliary agents.

[0115] The compounds of the present invention can be used in the form of pharmaceutical compositions and their unit dosages, together with conventionally used adjuvants, carriers, excipients, or additives, in which case they can be used as solids such as powders in pouches, tablets, or filled capsules, or as liquids such as solutions, suspensions, emulsions, elixirs, nasal sprays, or capsules filled therein (all for oral use), or as sterile, injectable solutions for parenteral use (including subcutaneous use). Such pharmaceutical compositions and their unit dosage forms may contain conventional ratios of components with or without additional active compounds or components, and such unit dosage forms may contain any appropriate effective amount of active component depending on the range of daily doses intended for use. The compositions according to the present invention are preferably administered orally, sublingually, nasally, and subcutaneously.

[0116] The compositions of the present invention may also be liquid formulations, and are not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, sprays, and elixirs. Suitable liquid forms for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending agents and dispersing agents, colorants, fragrances, etc. The compositions may also be formulated as a dry product to be restored with water or another suitable vehicle before use. Such liquid preparations may contain additives, and are not limited to, suspending agents, emulsifiers, non-aqueous vehicles, and preservatives. Suitable suspending agents include, but are not limited to, sorbitol syrups, methylcellulose, glucose / sugar syrups, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gels, and hydrogenated edible fats. Suitable emulsifiers include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Examples of non-aqueous vehicles include, but are not limited to, edible oils, almond oil, rectified coconut oil, oily esters, propylene glycol, and ethyl alcohol. Examples of preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Further materials and processing techniques are described in The Science and Practice of Pharmacy (Remington: The Science & Practice of Pharmacy), 22nd edition, 2012, edited by Lloyd, Allen, Pharmaceutical Press, which are incorporated herein by reference.

[0117] The solid composition of the present invention may be in the form of a powder in a sachet, tablet, or lozenge formulation prepared by conventional methods. For example, sachets, tablets, and capsules for oral or sublingual administration may contain, but are not limited to, conventional additives, including binders, fillers, lubricants, disintegrants, and wetting agents. Examples of binders, but are not limited to, include syrups, acacia, gelatin, sorbitol, tragacanth, starch viscosities, and polyvinylpyrrolidone. Examples of fillers, but are not limited to, include lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Examples of lubricants, but are not limited to, include magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Examples of disintegrants, but are not limited to, include potato starch and sodium starch glycolate. Examples of wetting agents, but are not limited to, include sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[0118] The injectable compositions are typically based on injectable sterile saline or phosphate-buffered saline, or other injectable carriers known in the art.

[0119] The compositions of the present invention may also be formulated for parenteral administration, including, but not limited to, injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in an oily or aqueous vehicle, and may, but not limited to, contain formulations comprising suspending agents, stabilizers, and dispersants. The compositions may also be produced in powder form for reconstitution with a suitable vehicle, including, but not limited to, sterile water free of pyrogens.

[0120] The compositions of the present invention may also be formulated as depot preparations, which may be administered by injection or intramuscular injection. The compositions may be formulated as suitable polymeric or hydrophobic substances, ion exchange resins (e.g., as emulsions in acceptable oils), or as sparingly soluble derivatives (e.g., as sparingly soluble salts).

[0121] The compounds of the present invention can also be administered via sustained-release or sustained-release drug delivery systems. A description of typical sustained-release substances can also be found in the materials incorporated into Remington's Pharmaceutical Sciences.

[0122] Mode of administration The compositions of the present invention can be administered by any means, but are not limited to, oral, parenteral, sublingual, buccal, nasal, intralesional, or combinations thereof. Parenteral administration includes, but is not limited to, subcutaneous and intramuscular methods. The compositions of the present invention can also be administered in the form of implantation, which allows for sustained release and sustained-release IV infusion of the compositions. In certain embodiments, one or more NOX4, NOX4 / 1, or NOX1 inhibitors are administered orally.

[0123] The dosage administered to an individual, whether as a single or multiple dose, varies depending on various factors, including pharmacokinetic properties, the patient's condition and characteristics (age, weight, health, body dimensions), the severity of symptoms, the frequency of treatment, and the desired effect.

[0124] combination According to one embodiment of the present invention, NOX4, NOX4 / 1, or NOX1 inhibitors and their pharmaceutical formulations are administered in combination with an anti-cancer immunotherapy agent, particularly an anti-cancer vaccine, or at least one immune checkpoint inhibitor such as at least one PD-1, PD-L1, or CTLA4 inhibitor.

[0125] The present invention encompasses the administration of NOX4, NOX4 / 1, or NOX1 inhibitors, or pharmaceutical formulations thereof, wherein the NOX4 / 1 inhibitor or pharmaceutical formulation thereof is administered to the individual before or simultaneously with an anti-cancer immunotherapy agent, for example, simultaneously by the same formulation or by different formulations, particularly separately by different formulation routes.

[0126] According to certain aspects of the present invention, NOX4, NOX4 / 1, or NOX1 inhibitors and their pharmaceutical formulations are administered during the course of treatment and long-term (e.g., daily or weekly) prior to the administration of anti-cancer immunotherapy agents or anti-angiogenic therapies.

[0127] According to another specific aspect of the present invention, NOX4, NOX4 / 1, or NOX1 inhibitors and their pharmaceutical formulations are administered simultaneously with anti-cancer immunotherapy agents.

[0128] According to another specific aspect of the present invention, an anti-cancer immunotherapy agent can be administered in a therapeutically effective dose in combination with other therapeutic regimens or co-reagents (e.g., numerous drug regimens) useful for treating cancer, such as in combination with substances useful for treating, stabilizing, preventing and / or delaying cancer, such as substances used in conventional chemotherapy targeting solid tumors and controlling the establishment of metastasis, or any other molecule that acts by inducing programmed cell death, for example, in combination with angiogenesis inhibitors (e.g., anti-VEGF agents as described above by Gardner et al., 2017), immunotherapy agents (e.g., recombinant cytokines, interferons, interleukins, recombinant antibodies such as Herceptin®), and chemotherapeutic agents (e.g., cisplatin, paclitaxel, methotrexate, 5-fluorouracil, gemcitabine, vincristine, vinblastine, doxorubicin, temozolomide). In particular, according to another specific aspect of the present invention, an anti-cancer immunotherapy agent can be administered in a therapeutically effective dose in combination with other therapeutic regimens or co-agents (e.g., multiple drug regimens) useful for treating cancer, such as in combination with at least one inhibitor of vascular endothelial growth factor (VEGF) (e.g., bevacizumab, sunitinib inhibitor), at least one inhibitor of basic fibroblast growth factor (bFGF), or at least one inhibitor of hypoxia-inducible factor-1 (HIF-1).

[0129] The NOX4 / 1 inhibitor or its pharmaceutical preparation administered simultaneously with the aforementioned anti-cancer immunotherapy agent may be administered in the same or different compositions, or within the same composition, via the same or different routes of administration.

[0130] patient In one embodiment, the subjects of the present invention are those suffering from solid tumor cancers, particularly low-response solid tumor cancers, that exhibit or are suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, especially anti-VEGF therapy.

[0131] In certain embodiments, the subjects of the present invention are individuals suffering from solid tumor cancers selected from lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, and brain cancer, particularly glioblastoma.

[0132] In a particular embodiment, the subject of the present invention is a subject suffering from solid tumor cancer and having high α-smooth muscle actin (α-SMA) expression.

[0133] In another specific embodiment, the subject of the present invention is a subject suffering from hepatocellular carcinoma (HCC).

[0134] In another specific embodiment, the subject of the present invention is a subject suffering from head and neck cancer.

[0135] In another specific embodiment, the subject of the present invention is a subject suffering from melanoma.

[0136] In another specific embodiment, the subject of the present invention is a subject suffering from colon cancer.

[0137] In another specific embodiment, the subject of the present invention is a subject suffering from lung cancer.

[0138] In another specific embodiment, the subject of the present invention is a subject suffering from breast cancer.

[0139] In another specific embodiment, the subject of the present invention is a subject suffering from hepatocellular carcinoma or liver cancer.

[0140] In another specific embodiment, the subject of the present invention is a subject suffering from rectal cancer or colorectal cancer.

[0141] In another specific embodiment, the subject of the present invention is a subject suffering from kidney cancer.

[0142] In another specific embodiment, the subject of the present invention is a subject suffering from pancreatic cancer.

[0143] In another specific embodiment, the subject of the present invention is a subject suffering from brain cancer, particularly glioblastoma.

[0144] In another specific embodiment, the subject of the present invention is a subject having a solid tumor cancer that is at risk of developing resistance or partial resistance to anti-cancer immunotherapy due to another combination therapy or genetic predisposition.

[0145] In another specific embodiment, the subject of the present invention is a hematological malignancy such as lymphoma or leukemia.

[0146] Use according to the present invention In certain embodiments, the present invention provides compounds, methods, uses and compositions useful for the treatment of solid tumor cancer in combination, wherein at least one NOX4 / 1 inhibitor is administered in combination with at least one anti-cancer immunotherapy agent.

[0147] References made herein are incorporated herein by reference in their entirety. The present invention is not limited in scope by the specific embodiments described herein, which are intended as one example of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various modifications of the invention will be apparent to those skilled in the art from the above description and accompanying drawings, in addition to those shown and described herein. Such modifications are intended to be within the scope of the claims.

[0148] The present invention has been described above, and the following examples are provided for illustrative purposes only, but are not limiting. [Examples]

[0149] The efficacy of NOX4 / 1 inhibitors in restoring or enhancing responsiveness to anti-cancer immunotherapy agents can be tested as follows.

[0150] (Example 1) Combination of NOX4 / 1 inhibitors and anti-PD1 inhibitors in cancer treatment To test the efficacy of the combination according to the present invention, the following experiments are performed in a mouse xenograft tumor model as described below.

[0151] Subcutaneous xenograft tumors consisting of C38 cells (colon cancer), CT26 cells (colon cancer), LLC1 cells (lung cancer), B16F10 cells (melanoma), Hepa1-6 cells (liver cancer), or Renca cells (kidney cancer) are subcutaneously injected into the flank of C57Bl / 6 or Balb / c mice (2-3 months old). Alternatively, the MC-38 cell line derived from C57BL6 mouse colon adenocarcinoma cells, or a mouse 4T1 breast cancer model, may be used.

[0152] The tumor is 80-200 mm 3 Combination therapy is initiated once the average tumor volume is reached. Mice are randomized into different groups of 8 to 17 mice based on their individual tumor volume. Each group receives either placebo, a NOX4 / 1 inhibitor alone, a PD-1 antibody alone, or NOX4 / 1 in combination with a PD-1 antibody.

[0153] NOX4 / 1 inhibitors, 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione, or (R)-3-methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide were prepared daily (7 days / week) in 1.2% methylcellulose and 0.8% polysorbate 80 (Sigma), and administered to animals from each group by oral force-feeding via force-feeding tubes at doses of 60-10 mg / kg.

[0154] As a PD-1 inhibitor, an anti-PD-1 antibody (reference: BE0146, BioXcell; clone: ​​RMP1-14, responsiveness: mouse; isotype: rat IgG2a; storage conditions: +4℃) is injected intraperitoneally (intraperitoneal, IP) into mice. The dose volume is adjusted to 10 mL / kg relative to the mouse's current individual body weight.

[0155] Immunochemical methods for tumor sampling and evaluation of T cell infiltration Fourteen days after randomization, if the antitumor activity of NOX4 / 1 compounds alone or in combination is deemed sufficient, tumors from five satellite mice per group are collected, weighed, and cut into two fragments. One fragment is cut into 4 mm thick slices, fixed in 4% neutral buffered formalin for 24–48 hours, and then wrapped in paraffin (Histosec®, Merck, Darmstadt, Germany). The other fragment is wrapped in tissue freezing medium (Microm Microtech, France), rapidly frozen in isopentane cooled with liquid nitrogen, and stored at 80°C until processing. Immunohistochemical staining for CD3, CD4, and CD8 is performed on paraffin-wrapped tissue sections using standard techniques (Biodoxis, France). The number of CD3, CD4, and CD8 immunopositive cells per field is counted.

[0156] Flow cytometry to evaluate tumor biopsy and T cell infiltration. Fourteen days after randomization, tumors from four mice per group will be collected.

[0157] All tumors were collected in RPMI culture medium (reference: BE12-702F, Lonza, Verviers, Belgium). Tumor immunoinfiltrating cells were quantified from each collected sample by flow cytometry. Then, antibodies against the selected markers were added according to the procedures described by the antibody suppliers. All antibodies except FoxP3 were for surface labeling, while FoxP3 was for intracellular labeling. The antibodies used for flow cytometry analysis of effector T lymphocytes (Teff: CD45, CD3, CD8) and regulatory T lymphocytes (Treg: CD45, CD3, CD4, FoxP3) in mouse samples are listed in Table 1 below.

[0158] [Table 1]

[0159] Stained cells are analyzed using a BD® LSR II flow cytometer (BD Biosciences) equipped with three excitation lasers at wavelengths of 405 nm, 488 nm, and 633 nm. Flow cytometry data is acquired for each sample, either until 10,000 mCD45+ events are recorded, or for a maximum duration of 2 minutes.

[0160] Animal monitoring All test data, including animal weight measurements, tumor volume, medical and mortality records, and treatment, will be scheduled and recorded. Survival and behavior will be recorded daily. Weight will be measured twice a week. Tumor length and width will be measured twice a week with calipers, and tumor volume will be estimated using the following formula:

[0161]

number

[0162] Human endpoints. The experiment will end after 5 weeks or in the following cases: In mice, if the weight exceeds 10% of normal body weight or 1,500 mm 3 Tumors exceeding a certain size, • Unstable or nutritionally interfering tumors, ulcerative tumors larger than 8 mm, bleeding infections, • Tissue erosion • Compared to the start of treatment / maximum weight, a 20% weight loss over the two days of monitoring (30% over the first day of monitoring) • Signs of pain, distress, or agony: posture in pain, facial expression in pain, behavior, • Decreased physical condition, emaciation, cachexia, dehydration, • Prolonged absence of voluntary response to external stimuli, • Rapid, labored breathing, anemia, significant bleeding, • Neurological signs: circling, convulsions, paralysis, • Persistent decrease in body temperature, Abdominal distension.

[0163] Efficacy parameters Therapeutic efficacy will be evaluated by comparing the effect of the test substance with that of control animals, based on the tumor volume of the treated animals. The following evaluation criteria for antitumor efficacy will be determined. • resulting in individual and / or average (or median) tumor volume, • Calculate the tumor doubling time (DT). • Calculate the tumor growth inhibition rate (T / C%), defined as the ratio of median tumor volume in the treatment group to the control group:

[0164]

number

[0165] The optimal value is the smallest T / C% ratio that reflects the maximum tumor growth inhibition rate achieved. The effective criterion for the T / C% ratio according to NCI standards is 42%. Calculate the volume V and the time to reach V. Volume V is defined as the target volume selected during the exponential phase of tumor growth, deduced from experimental data. For each tumor, the tumor volume closest to the target volume V is selected in tumor volume measurement. Record this volume V value and the time it takes for the tumor to reach this volume. For each group, calculate the mean tumor volume V and the mean time to reach this volume. Mouse survival rate is also monitored and used as an efficacy parameter. Plot the survival curve.

[0166] MC38 cancer cells (0.5 × 10 5 When using these, they are subcutaneously injected (sc) into the flank of 8-10 week old C57BL / 6 female mice in phosphate-buffered saline (PBS). MC38 cells are injected alone or in combination with C57BL / 6 colon fibroblasts (2.5 × 10⁶) that have been ex vivo pretreated with 2 ng / ml TGFβ1 for 6 days prior to injection to induce the CAF phenotype. 5 It is either mixed with ) or

[0167] 4T1 cancer cells (0.5 × 10) 5 When using these cells, they are sc-injected into the upper mammary fat pads of 8-10 week old female mice in PBS. The cells are injected alone or isolated from 2.5 × 10⁶ cells from spontaneously occurring stroma-rich mammary tumors of transgenic BALBneuT. 5 Either mix it with BALB / C milk CAF.

[0168] When a tumor was palpable, mice were administered the NOX4 inhibitor, 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione (GKT137831). GKT137831 was reconstituted with 0.1% polysorbate (Sigma) in 1.2% methylcellulose (Sigma) and administered orally at a dose of 40 mg / kg five times / week via force-feeding. Control mice received vehicle via force-feeding. During long-term administration, the initial dose was given as described in 15, but was reduced to 50 mg / kg for 3 weeks, three times / week, and then to 60 mg / kg for 3 weeks, twice / week. Anti-PD-1 antibody (Bioxcell; RMP1-14) was administered via intraparietal (ip) injection. Every other day, when a tumor was palpable, 300 μg of antibody or IgG2a isotype control (Bioxcell) was administered, for a total of three doses.

[0169] For the data presented in Figure 1, the longest width and length of the tumor were measured every 2-3 days using an electronic skin caliper. Tumor volume was calculated using the formula 4 / 3π×r3, with the radius (r) calculated from the measured width and length of the tumor to show the average diameter. When mice were randomized into multiple groups based on tumor volume, no statistically significant difference was observed between the groups in average tumor volume before the start of treatment. Figure 1A shows that on day 15, i.e., 8 days after treatment, the tumors were significantly smaller when mice were treated with a NOX4 inhibitor compared to those treated with vehicle alone. Furthermore, immunochemistry (performed as described above) revealed, as shown in Figures 1B and 1C, that treatment with a NOX4 inhibitor significantly reduced SMA-positive CAFs within the tumor, resulting in the rearrangement of CD8+ T cells from the tumor periphery to the tumor center. Using a 4T1 breast cancer model, these results indicate that GKT therapy inhibits CAF formation, as demonstrated by a reduced myofibroblast (SMA-positive cell) population, and CD8 + T cells are clearly shown to be able to approach tumors, kill cancer cells, and reduce tumor size. This is due to CD8 +This supports the beneficial effects of a combination of NOX4 inhibitors and anti-cancer immunotherapy agents that further activate T cells.

[0170] The beneficial effects of such combinations are further supported by the results presented in Figure 2 for the combination of the PD-1 inhibitor (αPD1) and the NOX4 inhibitor GKT137831, which significantly improves the therapeutic response in CAF-rich tumors. Tumors were significantly smaller when mice were treated with the αPD1 / GKT831 combination compared to αPD1 alone (Figure 2A), and following administration of the αPD1 / GKT831 combination, CD8+ T cells significantly relocated from the tumor periphery to the tumor center (Figure 2B), and survival outcomes were also significantly improved compared to αPD1 alone (Figure 2C). Using the MC38 colon cancer model, the beneficial effects of GKT / αPD1 combination therapy were confirmed by showing a very significant reduction in tumor volume. This reduction was accompanied by increased survival in mice. Furthermore, it was shown that this effect arises from the infiltration of CD8+ T cells into the tumor by the NOX inhibitor. These results strongly suggest that the NOX4 inhibitor of the present invention, particularly GKT137831, is a promising candidate for PD1 combination therapy in all CAF-rich cancers.

[0171] (Example 2) Combination of NOX4 / 1 inhibitors and cancer vaccines in cancer treatment To test the efficacy of the combination according to the present invention, a NOX4 / 1 inhibitor is combined with a vaccine, such as an anti-HPV vaccine.

[0172] TC1 cancer cells (0.5 × 10 5 TC1 cells (prostate cancer) were subcutaneously injected into the flanks of 8-10 week old C57BL / 6 female mice in phosphate-buffered saline (PBS). TC1 cells were either injected alone or ex vivo pre-treated with 2 ng / ml TGFβ1 for 6 days before injection to induce the CAF phenotype in C57BL / 6 lung fibroblasts (2.5 × 10⁻¹⁴). 5 It was either mixed with ) or something else.

[0173] The longest width and length of the tumors were measured every 2-3 days using an electronic skin caliper. Tumor volume measurement, mouse randomization, and oral force-feeding were performed as described above.

[0174] E7 HPV RAHYNIVTF (RAH, E7 49-57 Vaccination with a DNA vaccine encoding tetanus C fragment domain 1 (Dom), which condenses to the immunodominant CD8 epitope of (Rice et al., 2002, J Immunol., 169:3908-13; Rice et al., 2008, Nat Rev Cancer, 8:108-20), was administered via intramuscular injection (im) when a tumor was palpable. A single injection containing 50 μg of DNA in PBS was given, and any repeat doses were administered for 3 weeks after the initial immunization. Treatment with a reconstituted NOX4 inhibitor (GKT137831) as described in Example 1 was administered to mice when a tumor was palpable.

[0175] Figure 3 shows that on day 24, tumors were significantly smaller when mice were treated with the combination vaccine / NOX4 inhibitor compared to vaccine alone, supporting the idea that the combination of antitumor vaccination with a NOX4 inhibitor significantly improves the therapeutic response in CAF-rich tumors. Following administration of the combination vaccine / NOX4 inhibitor, CD8+ T cells were significantly relocated from the tumor periphery to the tumor center (Figure 3B), and survival outcomes were also significantly increased compared to vaccine alone (Figure 3C). Effective immunotherapy requires the presence of CD8+ effector T cells in the tumor, whether based on checkpoint inhibitors, T cell agonists, vaccination, or adoptive T cell introduction. Cancer-associated fibroblasts are found in many solid cancers and play a major role in tumor immune evasion, as they render immunotherapy ineffective by eliminating CD8+ T cells from the cancer. Therefore, the NOX inhibitors of the present invention, particularly GKT831, effectively target CAFs, as demonstrated by the reduction of SMA-positive cells in the 4T1 model, thereby promoting CD8+ T cell infiltration into tumors and restoring responses to vaccine-based and PD1-based immunotherapies. These data suggest that combination immunotherapy with the NOX4 inhibitors of the present invention, particularly GKT137831, significantly improves response rates to this type of therapy.

[0176] (Example 3) Combination of NOX4 / 1 inhibitors and anti-VEGF agents in cancer treatment To test the efficacy of the combination according to the present invention, a NOX4 / 1 inhibitor is combined with anti-VEGF therapy.

[0177] In a mouse model of tumor MC38 xenograft, PBS (5.10 compared to MC38) was used in wild-type C57 / BL6 mice or NOX1-deficient (NOX1-KO) mice. 5 The tumor was generated by subcutaneous injection of diluted MC38 tumor cells into a solution. 3Once the target was reached, purified antibodies against either anti-VEGF:DC101 or unrelated rat IgG (as a control) were administered intraperitoneally twice a week. DC101 was administered at a dose of 600 μg per mouse in a single injection. The mice were given by oral force-feeding of a vehicle (VL) (i.e., methylcellulose and Tween 80) or a NOX1 selective inhibitor, (R)-3-methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridine-4-yl)-1,3,4-thiadiazole-2-yl)benzamide (GKT2) (10 mg / kg twice daily) until euthanasia. Tumor size was measured with calipers, and tumor volume was determined according to the formula: (length * width * thickness). Tumor size was measured in vivo with calipers every 5 days (D-0 to D-15). After euthanasia, the tumor was removed without fixation with PFA (paraformaldehyde), isolated, and vascular endothelial cells (CD45- / CD31+ / GP38-) were analyzed by flow cytometry.

[0178] Figure 4 shows that the combination of a highly selective NOX1 inhibitor (GKT2) and an anti-VEGF-R2 blocking antibody (DC101) enables the inhibition of angiogenesis. Furthermore, GKT2 and DC101 act synergistically to enhance the inhibition of angiogenesis.

[0179] As shown in Figure 5, tumors in NOX1-KO mice exhibit reduced proliferation dynamics compared to tumors in WT mice, clearly indicating the involvement of NOX1. Furthermore, treatment with the anti-VEGFR2 antibody (DC101) reduced tumor growth in NOX1-deficient mice, and this effect was even more pronounced compared to WT mice. This clearly suggests distinct mechanisms of interaction between VEGFR2 and NOX1 signaling.

[0180] Therefore, in summary, these data support the idea that a combination of NOX1 inhibitors and anti-angiogenic agents such as anti-VEGF inhibitors can achieve synergistic effects in tumor treatment.

Claims

1. A composition for use in the treatment of solid tumor cancer exhibiting or suspected of exhibiting resistance to immunotherapy, comprising 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione, wherein the 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione is administered in combination with an anti-cancer immunotherapy agent selected from at least one cancer vaccine and at least one immune checkpoint inhibitor. The aforementioned immune checkpoint inhibitor is a PD-1 inhibitor or a CTLA-4 inhibitor. The aforementioned cancer vaccine is selected from DNA, RNA, peptides, oncolytic viruses, and anti-herpes simplex virus vaccines. composition.

2. The composition according to claim 1, for use in combination with a cancer vaccine selected from oncolytic and anti-herpes simplex virus vaccines.

3. The composition according to claim 1, wherein the anti-cancer immunotherapy agent is an anti-herpes simplex virus vaccine, T-Vec (Imritsic, Tarimodine / Laharpalebeck).

4. The composition according to claim 1, wherein the PD-1 inhibitor is selected from pembrolizumab (Keytruda) and nivolumab (Opdivo).

5. The composition according to claim 1, wherein the CTLA-4 inhibitor is ipilimumab (Yervoy).

6. The composition according to any one of claims 1 to 5, wherein the solid tumor cancer is selected from lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, melanoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and glioblastoma.

7. The composition according to any one of claims 1 to 6, wherein the solid tumor cancer is selected from lung cancer, breast cancer, head and neck cancer, colorectal cancer, prostate cancer, and pancreatic cancer.

8. The composition according to any one of claims 1 to 5, wherein the solid tumor cancer is esophageal cancer.

9. A pharmaceutical formulation for use in the treatment of solid tumor cancers that are resistant to immunotherapy or suspected to be resistant to immunotherapy, comprising 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione in combination with at least one anti-cancer immunotherapy agent and at least one pharmaceutically acceptable carrier, wherein the at least one anti-cancer immunotherapy agent is selected from at least one cancer vaccine and at least one immune checkpoint inhibitor. The aforementioned immune checkpoint inhibitor is a PD-1 inhibitor or a CTLA-4 inhibitor. A pharmaceutical formulation wherein the cancer vaccine is selected from DNA, RNA, peptides, oncolytic viruses, and anti-herpes simplex virus vaccines.

10. The pharmaceutical preparation according to claim 9, wherein the anti-cancer immunotherapy agent is an anti-herpes simplex virus vaccine.

11. The pharmaceutical formulation according to claim 9, wherein the PD-1 inhibitor is selected from pembrolizumab (Keytruda) and nivolumab (Opdivo).

12. The pharmaceutical formulation according to claim 9, wherein the CTLA-4 inhibitor is ipilimumab (Yervoy).

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