Use of NOX inhibitor for treating cancer

Abstract

To provide a compound, a method, a composition, and use that can recover responsiveness to immunotherapy particularly to immune checkpoint inhibitor or anti-cancer vaccine, or antiangiogenic therapy.SOLUTION: There are provided a composition containing NOX inhibitor selected from NOX4 inhibitor, NOX4 / 1 inhibitor, and NOX1 inhibitor for use in treating solid tumor cancer that presents or is suspected to present resistance to immunotherapy or an antiangiogenic agent, particularly to anti-VEGF therapy, where the NOX4 inhibitor, NOX4 / 1 inhibitor, or NOX1 inhibitor is administered in combination with at least one cancer vaccine, at least one immune checkpoint inhibitor, at least one CD8+T-cell agonist, or an anti-cancer immunotherapeutic agent selected from adoptive T-cell transduction therapy.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Cancer cells face numerous cellular stresses such as hypoxia, increased metabolic demands, genomic instability, immune surveillance, nutrient deprivation, metastasis and an altered environment following stress, which results in treatments such as radiotherapy, chemotherapy and targeted therapy.

[0003] NADPH oxidases (NOXs) are a family of enzymes with six transmembrane domains that transport electrons across biological membranes. These enzymes are specialized reactive oxygen species-producing enzymes that specifically regulate a wide range of redox-sensitive signaling pathways involved in cancer development and progression, acting at specific cell membranes and microdomains through the activation of oncogenes and the inactivation of tumor suppressor proteins. NOX enzymes are considered to be an important part of the adaptive stress response, particularly for cancer cells, enabling these cells to adapt and survive (Block et al., 2012, Nature Reviews, pp. 627-637).

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

[0005] Interactions between the tumor microenvironment and 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. CAFs and their transdifferentiation from fibroblasts to myofibroblasts drives tumor growth, and this transdifferentiation generally correlates with poor prognosis in many carcinomas. While CAFs promote many of the "hallmarks of malignancy," recent studies have focused on their role in promoting tumor immune evasion, with CAF-rich cancers described as "immune cold" due to their poor therapeutic response to cancer immunotherapies such as immune checkpoint inhibitors and cancer vaccines, as well as their propensity to develop metastasis.

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

[0007] Melanoma, in particular, is known as an exceptionally aggressive and treatment-resistant human cancer. Despite advances over the past decade, including the development of immunotherapy using immune checkpoint inhibitors, limited 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 communication between neoplastic and non-malignant cell populations, including fibroblasts, endothelial cells, and immune cells in the tumor stroma. CAFs remodel the structure of the extracellular matrix (ECM) and diseased tissue and secrete chemical factors, which in concert promote tumor growth, angiogenesis, inflammation, and metastasis, contributing to drug resistance and transforming processes. Although NOX4 has recently been shown to regulate myofibroblastic CAF differentiation in multiple cancers (Hanley et al., 2018, J Natl Cancer Inst., 110), the origin of CAFs and the precise mechanisms by which they contribute to cancer progression and drug resistance remain poorly understood. Furthermore, Hanley et al., 2018, did not target any specific anti-cancer immunotherapeutic agents as adjunctive treatment with NOX4 inhibition.

[0008] Immunotherapy continues to be an effective therapeutic strategy of interest 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, and involves a variety of approaches ranging from stimulating effector mechanisms to counteracting inhibitory and suppressive mechanisms. Strategies to activate effector immune cells include vaccination with tumor antigens or enhanced antigen presentation, which enhance the ability of the patient's own immune system to mount an effective immune response against neoplastic cells (Yaddnapudi et al., 2013, Cancer vaccines, Oncoimmunology, 2(3), e23403). Additional stimulatory strategies include adoptive cellular 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 to enhance T cell activity. Strategies to neutralize immunosuppressive mechanisms include chemotherapy (cyclophosphamide), antibodies that eliminate regulatory T cells (CD25-targeting antibodies), and antibodies against immune checkpoint molecules such as CTLA-4, PD1, and PD-L1.

[0009] The field of cancer immunotherapy has been encouraged in recent years primarily by the approval of the autologous cellular immunotherapy, sipuleucel-T, for the treatment of prostate cancer in 2010 (Topalian et al., 2011, J. Clin. Oncol., 29:4828-36), and the approval of the anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody, ipilimumab, and the anti-programmed cell death protein 1 (PD1) antibody for the treatment of melanoma in 2011 and 2014 (Sharma et al., 2015, Cell, 161:205-14).

[0010] Effective anticancer efficacy has been demonstrated through 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), with the highest response rates observed in cancers with high mutational burdens, such as melanoma and non-small cell lung cancer (Andrews et al., 2017, Journal for ImmunoTherapy of Cancer, 25:10). However, a major limitation exists for these therapeutic agents, with objective responses to PD-1 blockade observed in only 30–40% of patients, with the majority of patients exhibiting innate resistance. Acquired resistance to anti-PD-1 therapy is also a problem, with approximately one-quarter of responders subsequently experiencing disease progression (Ribas et al., 2016, JAMA, 315:1600–1609).

[0011] Furthermore, resistance to anticancer treatment in solid cancers has also been observed with antiangiogenic therapies, and despite the promotion of beneficial effects, patients inevitably develop resistance and frequently do not demonstrate significantly better overall survival, making the use of anti-VEGF therapies a high concern (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, in view of the recent development of various strategies in cancer immunotherapy, such as cancer vaccines, adoptive cell therapy, immune checkpoint blockade, and oncolytic viruses, as well as the limitations faced in their efficacy, there is an increasing need to develop anticancer therapies that are effective against solid cancers, particularly those prone to developing resistance to immunotherapy or antiangiogenic therapy, that are capable of restoring sensitivity to immunotherapy or antiangiogenic treatment, or that enhance cancer vaccine therapy. [Prior art documents] [Patent documents]

[0013]

Patent Document 1

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Patent Document 3

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Patent document 8

Patent document 9

Patent document 10

Non-licensed literature

[0014] [Non-licensed document 1] Block et al., 2012, Nature Reviews, pp. 627-637 [Non-licensed document 2] Hanley et al., 2018, J Natl Cancer Inst., 110 [Non-licensed document 3] Yaddnapudi et al., 2013, Cancer vaccines, Oncoimmunology, 2(3), e23403

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Summary of the Invention

Problems to be Solved by the Invention

[0015] The present invention is directed to the unexpected finding that the recently discovered ability of pharmacological inhibition of NOX4 (Hanley et al., 2018, J Natl Cancer Inst., 110) to revert the myofibroblastic CAF phenotype in different cancer cell lines and suppress tumor growth in multiple CAF-rich tumor models (TC1+CAFs [HNSCC model], 4T1+CAFs [breast cancer], MMTV-PyVT (breast cancer), MMTV-Her2 / neu (breast cancer)) both in vitro and / or in vivo may be useful in synergistically enhancing cancer immunotherapy or reversing resistance induced by anti-VEGF treatment.

[0016] The present invention is directed to the unexpected finding that dual NOX4 / 1 inhibitors can restore sensitivity to immunotherapy and / or improve response to immunotherapy and anti-angiogenic therapy.

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

[0018] In particular, the present invention is directed to the unexpected finding that NOX4 inhibitors can restore sensitivity to anti-tumor immunotherapy and / or improve response to immunotherapy.

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

[0020] The present invention is further directed to compositions and methods useful for restoring responsiveness to anti-angiogenic therapy, particularly for 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 a NOX4 / 1 dual or NOX1 inhibitor for use in the treatment of solid tumour cancers that exhibit or are suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF treatment, wherein the NOX4 (or NOX4 / 1 or NOX1) inhibitor is administered in combination with an anti-cancer immunotherapy or 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 the preparation of 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 treatment, wherein the one or more NOX4 or NOX4 / 1 or NOX1 inhibitors are administered in combination with an anti-cancer immunotherapy or anti-angiogenic agent.

[0023] Another aspect of the present invention relates to pharmaceutical compositions containing at least one NOX4 or NOX4 / 1 or NOX1 inhibitor according to the present invention in combination with at least one anti-cancer immunotherapeutic agent or at least one additional anti-angiogenic agent and at least one pharmaceutically acceptable carrier, excipient or additive therefor, 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 a subject suffering from a solid tumor cancer that exhibits or is suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF therapy, comprising administering to a subject in need thereof an effective amount of one or more NOX4 or NOX4 / 1 or NOX1 inhibitors in combination with an anti-cancer immunotherapy 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 particular restoring sensitivity to immunotherapeutic treatment, especially for turning a cold tumor into a hot one, in a subject, comprising administering to a subject in need thereof an effective amount of one or more NOX4 or NOX4 / 1 or NOX1 inhibitors, or pharmaceutical formulations thereof, in combination with an anti-cancer immunotherapeutic agent.

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

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

[0028] [Figure 1A] The effect of treatment with a NOX4 inhibitor (GKT) is shown on the repopulation of DCD8+ T cells into tumor 4T1 when cancer cells are co-injected orthotopically with cancer-associated fibroblasts (CAFs) into the mammary fat pad as described in Example 1. Increase in tumor volume, expressed in mm3, versus days after injection (arrows) of either (1) tumor cells combined with CAFs and vehicle, or (2) tumor cells combined with CAFs and the NOX4 inhibitor. [Figure 1B] The effect of treatment with a NOX4 inhibitor (GKT) on DCD8+ T cell repopulation into tumor 4T1 is demonstrated when cancer cells are co-injected orthotopically with cancer-associated fibroblasts (CAFs) into the mammary fat pad as described in Example 1. Immunochemistry and quantification demonstrate the efficacy of treatment with a NOX4 inhibitor in reducing SMA-positive CAFs in tumors. [Figure 1C]Treatment with a NOX4 inhibitor (GKT) demonstrates the effect of repopulation of DCD8+ T cells into tumor 4T1 when cancer cells are co-injected orthotopically with cancer-associated fibroblasts (CAFs) into the mammary fat pad as described in Example 1. Immunochemistry (and quantification thereof) shows that treatment with the NOX4 inhibitor results in repopulation of CD8+ T cells from the tumor periphery to the tumor center. [Figure 2A] Figure 1 shows the effect of combining αPD1 and a NOX4 inhibitor (GKT) on treatment response in CAF-rich tumors, where MC38 cancer cells are co-injected with cancer-associated fibroblasts (CAFs) into treated mice as described in Example 1. The effects of vehicle alone (Ctl), αPD1, a NOX4 inhibitor (GKT) alone, or the combination αPD1 + a NOX4 inhibitor (GKT) are compared on tumor growth after injection. [Figure 2B] Figure 1 shows the effect of the combination of αPD1 and a NOX4 inhibitor (GKT) on treatment response in CAF-rich tumors, where MC38 cancer cells are co-injected with cancer-associated fibroblasts (CAFs) into treated mice as described in Example 1. Immunochemistry and quantification show that treatment with the combined αD1 / NOX4 inhibitor results in relocation of CD8+ T cells from the tumor periphery to the tumor center compared to αPD1 alone. [Figure 2C] Figure 1 shows the effect of the combination of αPD1 and a NOX4 inhibitor (GKT) on treatment response in cancer-associated fibroblasts (CAFs)-rich tumors, where MC38 cancer cells are co-injected with CAFs into treated mice as described in Example 1. Kaplan Meier survival curves for the various groups. [Figure 3A] Figure 1 shows the effect of combining anti-tumor vaccination with a NOX4 inhibitor (GKT) as described in Example 2. Tumor growth after injection in mice treated with the combined vaccine / GKT compared to vaccine alone and control. [Figure 3B]Figure 1 shows the effect of combining antitumor vaccination with a NOX4 inhibitor (GKT) as described in Example 2. Immunochemistry and quantification show that treatment with the combined vaccine / NOX4 inhibitor results in relocation of CD8+ T cells from the tumor periphery to the tumor center compared to vaccine alone. [Figure 3C] Figure 1 shows the effect of combining antitumor vaccination with a NOX4 inhibitor (GKT), as described in Example 2. Kaplan Meier survival curves of the different groups. [Figure 4] As described in Example 3, the efficacy of a combination of an antiangiogenic agent and a selective NOX1 inhibitor (GKT2) in inhibiting angiogenesis as measured by CD45- / CD31+ / GP38 cells compared to control is shown (*p<0.05; **p<0.01; ***p<0.005; ****p<0.001). [Figure 5] 1 shows the growth of tumor size in NOX1-KO mice compared to WT mice, and the effect of an anti-VEGFR2 antibody (DC101) in reducing tumor growth in these mice. DETAILED DESCRIPTION OF THE INVENTION

[0029] As used herein, the term "NOX inhibitor" refers to any substance capable of fully or partially inhibiting, blocking, attenuating, or interfering with NOX4 and / or NOX1. The term is defined as a compound that directly affects the enzyme's enzymatic activity, cellular localization, protein stability, messenger RNA, or protein expression. Preferably, a NOX4 / NOX1 inhibitor should be able to reduce enzymatic activity and ROS production in a cell-free assay using membranes 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 fully or partially inhibit the activity of NADPH oxidase 4 and / or NADPH oxidase 1. According to certain embodiments, a NOX4 / 1 inhibitor has a predominant NOX inhibitory activity against NOX4 and / or NOX1 compared to other NOX proteins, such as NOX2 and / or NOX3 / 5. According to certain embodiments, the NOX4 / 1 inhibitor has a primary NOX inhibitory activity at NOX4 / 1 that is at least about 5-fold higher than other NOX proteins.

[0030] For example, NOX4 / 1 inhibitors include small molecules, peptides, peptidomimetics, chimeric proteins, natural or non-natural 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, a NOX4 / 1 inhibitor is an agent that exhibits an inhibition coefficient, K, of less than 5 micromolar in a functional ROS production assay, such as that described in Gaggini et al., 2011, Bioorganic and Medicinal Chemistry, vol. 19(23), pp. 6989-6999. For example, a NOX4 / 1 inhibitor is an agent that inhibits ROS production in the range of less than about 1 microM, such as about 30-300 nanomolar, in a cell-free assay using membranes expressing only the NOX isoforms, such as recombinant protein NOX4 or NOX1.

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

[0033] The expression "solid tumor cancer" includes 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, especially glioblastoma.

[0034] As used herein, "treatment" and "treating" and the like generally refer to obtaining a desired pharmacological and physiological effect. As used herein, the term "treatment" encompasses any treatment of a disease in a mammal, particularly a human, including inhibiting the disease, i.e., halting its development, or alleviating the disease, i.e., causing regression of the disease and / or its symptoms or conditions, such as halting or regression of tumor growth.

[0035] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, domestic animals such as cattle, sheep, pigs, horses, laboratory rodents, dogs, etc.

[0036] As used herein, the term "effective amount" refers to an amount of at least one particle according to the present invention or a pharmaceutical formulation thereof that elicits a desired biological or medical response in a tissue, system, animal, or human. In one embodiment, the effective amount is a "therapeutically effective amount" for alleviating the symptoms of the disease or condition being treated. Typically, an effective amount can be used to inhibit cancer cell proliferation, i.e., to somehow slow the rate of cancer cell proliferation and / or migration, stop cancer cell proliferation and / or migration, or kill cancer cells, so as to reduce the rate of cancer cell proliferation compared to the observed or predicted rate of growth of untreated control cancer cells. The term "inhibiting growth" can also refer to a reduction in the size or elimination of cancer cells or tumors, as well as a reduction in their metastatic potential. Preferably, such inhibition at the cellular level can reduce the size, postpone growth, reduce invasiveness, or prevent or inhibit metastasis of the patient's cancer. Those skilled in the art can easily determine whether cancer cell proliferation is inhibited from any of a variety of appropriate indications.

[0037] The term "efficacy" of a 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 a cancer treatment according to the present invention can be measured by a reduction in tumor volume and / or an increase in progression-free survival, and / or an increase in the health and well-being of the subject (e.g., suppression of cancer). Inhibition of cancer cell proliferation can be demonstrated, for example, by arrest of cancer cells in a specific phase of the cell cycle, for example, arrest in the G2 / M phase of the cell cycle. Inhibition of cancer cell proliferation can also be demonstrated using well-known imaging methods 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, for example, by determining the level of circulating carcinoembryonic antigen, prostate-specific antigen, or other cancer-specific antigens correlated with cancer cell proliferation.

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

[0039] Unless otherwise limited by the definition of the 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," "sulfonamido," "alkoxy," "alkoxycarbonyl," "carbamate," "sulfanyl," "halogen," trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like.

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

[0041] "Pharmaceutically active derivative" refers to any compound that, upon administration to a recipient, is capable of directly or indirectly producing an activity disclosed herein.

[0042] NOX4 / NOX1 inhibitors 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) against NOX4 and / or NOX1 in a range of 60 nM or less to 300 nM in a functional assay of ROS production, and has inhibitory activity against other NOXs selected from NOX2, NOX3, and NOX5 of greater than 1 micromolar.

[0043] According to certain embodiments, the NOX4 or NOX4 / NOX1 or NOX1 inhibitor according to the invention is a pyrazolopyridine compound, a pyrazolinedione compound, or an amidothiazole 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 particular embodiment, the NOX4 inhibitors according to the invention are 2,5-disubstituted benzoxazole and benzothiazole derivatives, as described, for example, in WO 2016 / 207785.

[0045] In one embodiment, the present invention provides a NOX4 inhibitor,

[0046] [ka]

[0047] (wherein G1 is selected from H, optionally substituted alkyl, such as aminocarbonylalkyl (e.g., phenylacetamide), optionally substituted C3-C8 cycloalkylalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted arylalkyl, such as optionally substituted phenylalkyl, for example, 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, such as optionally substituted pyridinealkyl, for example, pyridin-2-ylmethyl; G2 is H; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl, such as 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, such as optionally substituted benzothiazolyl (e.g., 1,3-benzothiazolyl). 2-yl) or optionally substituted pyridinyl (e.g., pyridin-2-yl); optionally substituted alkylheteroaryl; optionally substituted heteroarylalkyl; optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenylheteroaryl; 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, such as methyl or ethyl; optionally substituted alkenyl; optionally substituted alkynyl;optionally substituted aryl, such as optionally substituted phenyl (e.g., phenyl); optionally substituted alkylaryl; optionally substituted arylalkyl; optionally substituted heteroaryl; optionally substituted alkylheteroaryl; optionally substituted heteroarylalkyl; optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenylheteroaryl; 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; G4 is H, optionally substituted alkyl, such as optionally substituted pentyl (e.g., isopentyl), or optionally substituted heteroalkyl, such as optionally substituted methoxy (e.g., 2-methoxyethyl); optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl; optionally substituted alkylaryl; optionally substituted arylalkyl, such as 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 (for example, thiophen-2-ylmethyl) or optionally substituted imidazolylalkyl, for example, optionally substituted imidazolylethyl (for example, imidazol-4-ylethyl) or optionally substituted indolylalkyl, for example, optionally substituted indolylethyl (for example, indol-3-ylethyl) or optionally substituted furanylalkyl, for example, optionally substituted furanylmethyl (for example, furan-2-ylmethyl) or optionally substituted benzodioxolylalkyl, for example, optionally substituted benzodioxolylmethyl (for example, 1,3-benzodioxol-5-ylmethyl) or optionally substituted pyridinylalkyl, for example, optionally substituted pyridinylmethyl (for example, pyridin-3-ylmethyl or pyridin-2-ylmethyl); optionally substituted alkenylalkyl. optionally substituted arylalkenyl; optionally substituted alkenylheteroaryl; optionally substituted heteroarylalkenyl; optionally substituted C3-C8 cycloalkyl; optionally substituted heterocycloalkyl, such as 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)piperidin-4-yl); optionally substituted alkylC3-C8 cycloalkyl; and optionally substituted C3-C8 cycloalkylalkyl; optionally substituted alkylheterocycloalkyl and optionally substituted heterocycloalkylalkyl, such as optionally substituted morpholinylalkyl, for example, optionally substituted morpholinylpropyl (e.g., 3-(morpholin-4-yl)propyl)) and optionally substituted morpholinylethyl (e.g., 2-morpholin-4-ylethyl);or optionally substituted piperazinylalkyl, for example optionally substituted piperazinylethyl (for example 2-(4-acetylpiperazin-1-yl)ethyl or 2-(4-hexanoylpiperazin-1-yl)ethyl), or optionally substituted pyrrolidinylalkyl, for example optionally substituted pyrrolidinylpropyl (for example 3-(2-oxopyrrolidin-1-yl)propyl), or optionally substituted tetrahydrofuranylalkyl, for example optionally substituted tetrahydrofuranylmethyl (for example tetrahydrofuran-2-ylmethyl); G5 is selected from H, optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted aryl; optionally substituted alkylaryl; optionally substituted arylalkyl; optionally substituted heteroaryl; optionally substituted alkylheteroaryl; optionally substituted heteroarylalkyl; optionally substituted alkenylaryl; optionally substituted arylalkenyl; optionally substituted alkenylheteroaryl; 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); and pharmaceutically acceptable salts and pharmaceutically active derivatives thereof.

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

[0049] [ka]

[0050] (wherein Ar is optionally substituted phenyl, for example phenyl optionally substituted with halogen, for example chloro (for example 2-chlorophenyl) or phenyl optionally substituted with alkoxy (for example methoxy); G1 and G4 are H; G2 is selected from optionally substituted C1-C6 alkyl (for example methyl) and optionally substituted phenyl (for example phenyl optionally substituted with halogen, for example 3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 4-chloro-2-fluorophenyl, 5-chloro-2-fluorophenyl, phenyl optionally substituted with amino or alkylamino or alkoxy, for example 3-dimethylaminophenyl, 2-trimethylaminophenyl, 3-methylaminophenyl, 3-aminophenyl, 4-methoxyphenyl); G3 is H, optionally substituted C1-C6 alkyl (e.g., methyl, C1-C6 alkyl substituted with alkoxy, e.g., methoxyethyl, for example, 2-methoxyethyl), optionally substituted heteroaryl C1-C6 alkyl, e.g., optionally substituted pyridinyl C1-C6 alkyl (e.g., optionally substituted pyridinylmethyl, e.g., pyridinyl-2-ylmethyl, pyridinyl-3-ylmethyl, 6-methoxypyridin-3-ylmethyl, 2-methoxypyridin-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, wherein the two nitrogens are bonded via an optionally substituted C1-C3 alkyl moiety). and their tautomers, geometric isomers, optionally active forms, and pharmaceutically acceptable salts.

[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, wherein G2 and G3 together form an optionally substituted 7-membered heterocycloalkyl ring containing two nitrogen atoms and have the following formula (I'):

[0052] [ka]

[0053] (Wherein, Ar, G1 and G5 are defined herein; G6, G8 to G 10 is H; G7 is optionally substituted C1-C6 alkyl, for example C1-C6 alkyl optionally substituted with optionally substituted phenyl (for example, methyl optionally substituted with optionally substituted phenyl, for example, benzyl, methyl optionally substituted with phenyl substituted with halogen, for example, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, methyl optionally substituted with phenyl substituted with alkoxy, for example, 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl), optionally substituted aryl C1-C6 alkyl, for example, optionally substituted and optionally substituted phenyl C1-C6 alkyl (e.g., benzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-methoxybenzyl, 3-methoxybenzyl, 4-methoxybenzyl) and optionally substituted heteroaryl C1-C6 alkyl, for example, optionally substituted pyridinyl C1-C6 alkyl (e.g., optionally substituted pyridinylmethyl, e.g., pyridinyl-2ylmethyl, pyridinyl-3ylmethyl) or optionally substituted furanyl C1-C6 alkyl (e.g., optionally substituted furanylmethyl, e.g., furan-3ylmethyl)). to form a compound of the formula:

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

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

[0056] In another particular embodiment, the invention provides a compound of formula (II) for use according to the invention, wherein G3 is optionally substituted C1-C6 alkyl.

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

[0058] In another particular embodiment, the present invention provides a compound of formula (II) for use according to the 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') (G7 is optionally substituted C1-C6 alkyl):

[0059] Alternatively, the present invention provides a compound of formula (II) for use according to the 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') (G7 is optionally substituted aryl C1-C6 alkyl):

[0060] Alternatively, the present invention provides a compound of formula (I) for use according to the 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') (G7 is optionally substituted heteroaryl C1-C6 alkyl):

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

[0062] In another embodiment, the present invention provides a compound of formula (III):

[0063] [ka]

[0064] (Wherein, X is CR 1 and N; Y is selected from CH or N; A1 is -OCHR 5 -, -NR 4 -CHR 5 -, -CH2NR 4 - and -CH2-O-; R 1 is selected from H, halogen and optionally substituted C1-C6 alkyl; R 2is H, halogen (e.g., chloro, fluoro), optionally substituted alkoxy, for example, optionally substituted methoxy (e.g., methoxy, (tetrahydro-2H-pyran-4-yl)methoxy, piperidin-4-ylmethoxy) or optionally substituted ethoxy (e.g., 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 (e.g., 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 alkylheterocycloalkyl, for example optionally substituted C1-C6 alkylpiperazine (for example methylpiperazin-1-yl), optionally substituted aminoC1-C6 alkyl, optionally substituted alkoxyC1-C6 alkyl, -OR 8 and -NR 9 R 10 Selected from; R 3 is the formula -(CHR 6 ) n -A2 or R 3 is the partial CHR from A1 5and optionally substituted aryl, such as optionally substituted phenyl (e.g., phenyl or phenyl substituted with halogen, e.g., fluorophenyl substituted with alkoxy, e.g., methoxy), and optionally substituted heteroaryl, such as optionally substituted 1,3-dihydro-1H-indenyl (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-cyclopentapyridinyl (e.g., 6,7-dihydro-5H-cyclopenta[b]pyridin-5-yl, 2-methylpyridin-3-yl, 5-methylpyridin-2-yl) or any 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-thiadiazol-2-yl), optionally substituted isoxazolyl (e.g., 5-methylisoxazol-3-yl), optionally substituted pyrazolyl (e.g., 1-methyl-1H-pyrazol-3-yl), or optionally substituted imidazolyl (e.g., 1-methyl-1H-imidazol-2-yl), or R 3 is the partial NR from A1 4 and R form an optionally substituted ring selected from optionally substituted aryl and optionally substituted heteroaryl, such as optionally substituted isoindolinyl (e.g., isoindolin-2-yl, 1H-indol-1-yl); n is an integer from 0 to 4 (e.g., 0, 1, 2, 3, or 4); 4is 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. pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, 2-methylpyridin-3-yl, 5-methylpyridin-2-yl) or optionally substituted pyrazolyl (e.g. 1,3-dimethyl-1H-pyrazol-5-yl, 1-methyl-1H-pyrazol-3-yl) or optionally substituted thiadiazolyl (e.g. 1,3,4-thiadiazol-2-yl) or optionally substituted an optionally substituted ring selected from optionally substituted imidazolyl (e.g., 1H-imidazol-4-yl, 1-methyl-1H-imidazol-2-yl, 1-methyl-1H-imidazol-5-yl), optionally substituted 1,2,4-triazolyl (e.g., 1-methyl-1H-1,2,4-triazol-5-yl), optionally substituted isoxazolyl (e.g., 1-cyclopropylisoxazol-3-yl), optionally substituted oxadiazolyl (e.g., 5-methyl-1,2,4-oxadiazol-3-yl), or optionally substituted pyrimidinyl (e.g., pyrimidinyl-2-yl); R 5is H, optionally substituted C1-C6 alkyl, such as optionally substituted methyl (e.g., methoxymethyl, 3,3-difluoropyrrolidin-1-ylmethyl, 4-methylpiperazin-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, such as optionally substituted aminomethyl (e.g., dimethylaminomethyl, methylaminomethyl), optionally substituted alkoxy C1-C6 alkyl, optionally substituted heterocycloalkyl C1-C6 alkyl, such as optionally substituted heterocycloalkylmethyl, for example, optionally substituted pyrrolidine C1-C6 alkyl (e.g., 3,3-difluoropyrrolidin-1-ylmethyl) or substituted piperazine C1-C6 alkyl (for example, 4-methylpiperazin-1-ylmethyl) or heterocycloalkylethyl, for example, optionally substituted morpholino C1-C6 alkyl (for example, morpholinomethyl, morpholinoethyl) or optionally substituted pyrrolidine C1-C6 alkyl (for example, pyrrolidinemethyl, pyrrolidineethyl), optionally substituted aminocarbonyl (for example, dimethylaminocarbonyl), optionally substituted C2-C8 cycloalkyl, for example, optionally substituted cyclopropyl and optionally substituted amino C1-C6 alkyl, for example, optionally substituted aminoethyl (for example, dimethylaminoethyl) or optionally substituted aminomethyl (for example, dimethylaminomethyl); R 6 is selected from H, optionally substituted C1-C6 alkyl, such as optionally substituted methyl, optionally substituted amino, optionally substituted C1-C6 alkylamino (e.g., dimethylamino), and hydroxy, where R 6 The groups are independently repeat units (CHR 6 ) selected for each R 7 is selected from H, halogen (e.g., fluoro) and optionally substituted C1-C6 alkyl, e.g., methyl; R 8is selected from H, optionally substituted C1-C6 alkyl, such as 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, such as optionally substituted heterocycloalkylmethyl, for example, optionally substituted tetrahydropyran C1-C6 alkyl (e.g., tetrahydro-2H-pyran-4-yl) or optionally substituted piperidine alkyl (e.g., 1-methylpiperidin-4-yl), optionally substituted C2-C8 cycloalkyl C1-C6 alkyl, optionally substituted alkoxy, optionally substituted amino C1-C6 alkyl, such as 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 10are independently H, optionally substituted C1-C6 alkyl, such as optionally substituted methyl (for example 1-methyl-1H-imidazol-4-yl)methyl) or optionally substituted ethyl (for example 2-methoxyethyl), optionally substituted amino C1-C6 alkyl, such as optionally substituted aminoethyl (for example dimethylaminoethyl) or for example optionally substituted aminopropyl (for example dimethylamino)propyl), optionally substituted heterocycloalkyl, such as optionally substituted piperidine (for example 1-methylpiperidine), optionally substituted C2-C8 cycloalkyl, optionally substituted heterocycloalkyl C1-C6 alkyl, such as optionally substituted heterocycloalkylethyl, for example optionally substituted morpholino C1-C6 alkyl (for example 2-morpholinoethyl) or optionally substituted heterocycloalkylmethyl, such as optionally substituted tetrahydrofuran C1-C6 alkyl (for example tetrahydro-2H-pyra optionally substituted C2-C8 cycloalkylC1-C6 alkyl, optionally substituted alkoxy, optionally substituted alkoxyC1-C6 alkyl, for example optionally substituted alkoxyethyl (for example 2-methoxyethyl), optionally substituted arylC1-C6 alkyl and optionally substituted heteroarylC1-C6 alkyl, for example heteroarylC1-C6 alkylmethyl, for example optionally substituted imidazolylC1-C6 alkyl (for example 1-methyl-1H-imidazol-4-ylmethyl), optionally substituted aminoC1-C6 alkyl, for example optionally substituted aminoethyl or optionally substituted aminopropyl (for example 2-(dimethylamino)ethyl, 2-(dimethylamino)propyl) and tautomers, geometric isomers, optionally active forms, pharmaceutically acceptable salts and pharmaceutically active derivatives thereof.

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

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

[0067] In certain embodiments, the present invention provides a compound of formula (III) for use according to the invention, wherein R 2 is optionally substituted alkoxy (eg, methoxy).

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

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

[0070] In another particular embodiment, the present invention relates to a compound of formula (III) for use according to the invention, wherein A1 is -OCHR 5 and especially R 5 is an optionally substituted amino C1-C6 alkyl (eg, dimethylaminomethyl).

[0071] In another particular embodiment, the present invention relates to a compound of formula (III) for use according to the invention, wherein A1 is -OCHR 5 and especially R 5 is an optionally substituted hydroxyl C1-C6 alkyl (e.g., hydroxymethyl).

[0072] In certain embodiments, the present invention provides a compound of formula (III) for use according to the invention, wherein R 3 However, the formula -(CHR 6 ) n -A2, particularly compounds wherein n is 0 and A2 is optionally substituted phenyl (eg phenyl).

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

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

[0075] [ka]

[0076] wherein ring (A) represents a non-aromatic 5- to 7-membered heterocyclic ring fused to a phenyl group; said 5- to 7-membered heterocyclic ring contains one oxygen ring atom and optionally one additional ring heteroatom independently selected from oxygen or nitrogen; said 5- to 7-membered heterocyclic ring is independently unsubstituted, monosubstituted, or disubstituted, and the substituents are independently: one oxo substituent attached to a ring carbon atom alpha to the ring oxygen and / or ring nitrogen atom; and / or One C bonded to a cyclic nitrogen atom with a free valence 1~3 alkyl; or Two fluoro substituents attached to the same ring carbon atom Selected from; L is -NH-CO- * or -CO-NH- * and an asterisk ( *) represents the bond connecting the benzoxazole / benzothiazole moiety; X represents O or S; and Y is -NR 1 R 2 (R 1 is R 1 is 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 is a direct bond or C 1~3 represents alkylene, C 3~5 Cycloalkyl optionally contains one oxygen ring atom, and 3~5 cycloalkyl is unsubstituted or monosubstituted with methyl or fluoro; 3~5 Cycloalkyl-L 1 ; or C 3~5 A piperidin-3-yl, piperidin-4-yl or pyrrolidin-3-yl group substituted at the ring nitrogen atom with cycloalkyl, 3~5 cycloalkyl represents a piperidin-3-yl, piperidin-4-yl, or pyrrolidin-3-yl group, optionally containing one oxygen ring atom; and R 2 is hydrogen, C 1~3 Alkyl or C 3~5 represents cycloalkyl), or Y is morpholin-4-yl; 2-oxo-pyrrolidin-1-yl; 1,1-dioxidethiomorpholin-4-yl; or optionally oxetan-3-yl or C 1~3 piperazin-1-yl monosubstituted at the 4-position with alkyl; or saturated 4- to 7-membered monocyclic heterocyclyl selected from azetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl, wherein said azetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl is independently unsubstituted, or two fluoro substituents attached to the same ring carbon atom; or one substituent selected from unsubstituted phenyl or unsubstituted or 6-membered heteroaryl; 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 Alkoxy monosubstituted C 1~3 one substituent selected from alkyl; or two substituents, one of which is C 1~4 alkyl, and the other is independently hydroxy or di(C 1~3 two substituents selected from: morpholin-4-yl; 1,1-dioxidethiomorpholin-4-yl; or optionally C 1~3 one substituent selected from alkyl and piperazin-1-yl monosubstituted in the 4-position; one substituent independently selected from azetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl, which is unsubstituted, monosubstituted with hydroxy, or disubstituted with methyl and hydroxy; represents a saturated 4- to 7-membered monocyclic heterocyclyl substituted with or Y is a saturated 7-11 membered fused, bridged, or spiro bicyclic heterocyclyl containing at least one nitrogen atom, said nitrogen atom being attached to a benzoxazole / benzothiazole moiety, said heterocyclyl optionally containing one additional ring heteroatom independently selected from oxygen, nitrogen, and sulfur, and is unsubstituted; or - two oxo substituents at the cyclic sulfur ring atom; or - One C bonded to a ring nitrogen atom with a free valence 1~3 Alkyl Substituents represents a saturated 7-11 membered fused, bridged, or spiro bicyclic heterocyclyl substituted with or a pharmaceutically acceptable salt thereof.

[0077] In another particular embodiment, the compound of formula (I) for use according to the invention is

[0078] [ka]

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

[0080] In another particular embodiment, the compound of formula (I) for use according to the invention is

[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 particular embodiment, the compound of formula (I) for use according to the invention is

[0084] [ka]

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

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

[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 particular embodiment, a compound of formula (IV) for use according to the invention,

[0090] [ka]

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

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

[0093] [ka]

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

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

[0096] [ka]

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

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

[0099] [ka]

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

[0101] In another particular embodiment, there is provided a compound according to the invention selected from the group consisting of: 2-(2-chlorophenyl)-4-methyl-5-(pyridin-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-(pyridin-3-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; (R)-3-Methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-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-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)benzamide; (R)-4-(2-hydroxy-1-phenylethoxy)-3-methoxy-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)benzamide, and (R)-4-(2-dimethylamino-1-phenylethoxy)-3-methoxy-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)benzamide.

[0102] According to certain aspects, there is provided a NOX inhibitor selected from a NOX4 inhibitor and a NOX4 / 1 inhibitor for use in combination with a cancer vaccine or at least one immune checkpoint inhibitor.

[0103] According to a further particular aspect, there is provided a NOX inhibitor selected from a NOX4 inhibitor and a NOX4 / 1 inhibitor for use in combination with a cancer vaccine or at least one immune checkpoint inhibitor.

[0104] According to another more particular aspect, there is provided a NOX inhibitor selected from a NOX4 inhibitor and a NOX4 / 1 inhibitor for use in combination with a cancer vaccine.

[0105] According to another more particular embodiment, there is provided a NOX inhibitor selected from a NOX1 inhibitor and a NOX1 / 4 inhibitor for use in combination with at least one anti-angiogenic agent.

[0106] Anti-cancer immunotherapeutic agent according to the present invention Anti-cancer immunotherapeutic agents that can be used in accordance with the present invention include oncolytic or anti-herpes simplex virus vaccines as described in Bartlett et al., 2013, Molecular Cancer 2, 12:103 (e.g., talimogene laherparepvec (Imlizic)) or Fukuhara et al., 2016, Cancer Sci, 107(10), pp. 1373-1379, adoptive cellular immunotherapy as 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, F1000Research 2016, 5 (F1000 Faculty These include cancer vaccines such as PD-1 inhibitors such as those described in Rev:803 (e.g., pembrolizumab (Keytruda), nivolumab (Opdivo)), PD-L1 inhibitors such as atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), or immune checkpoint inhibitors such as CTLA-4 inhibitors such as ipilimumab (Yervoy).

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

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

[0109] According to particular embodiments, anti-cancer vaccines according to the present invention include DNA, RNA, peptide, and oncolytic viral vaccines.

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

[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, GITR.

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

[0113] composition The present invention provides drugs or therapeutic agents as compositions and methods for treating patients, preferably mammalian patients, most preferably human patients, suffering from 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 can contain one or more compounds in any form described herein. The compositions of the present invention may further comprise one or more additional pharmaceutically acceptable ingredients, such as alum, solubilizers, stabilizers, antibacterial agents, buffers, colorants, flavoring agents, adjuvants, etc.

[0115] The compounds of the present invention, together with conventionally used adjuvants, carriers, excipients, or additives, can be made into the form of pharmaceutical compositions and their unit dosages, and can be used in such forms as solids such as powders in sachets, tablets, or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, nasal sprays, or filled capsules (all for oral use), or in the form of sterile injectable solutions for parenteral use (including subcutaneous). Such pharmaceutical compositions and their unit dosage forms can contain conventional ratios of ingredients, with or without additional active compounds or ingredients, and such unit dosage forms can contain any suitable effective amount of active ingredient, depending on the intended daily dosage range for use. The compositions according to the present invention are preferably oral, sublingual, nasal, and subcutaneous.

[0116] The compositions of the present invention may also be liquid preparations, including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, sprays, and elixirs. Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispersing agents, colorants, flavors, and the like. The compositions may also be formulated as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain additional substances, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Non-aqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Additional 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 is 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 formulated in a conventional manner. For example, sachets, tablets, and capsules for oral or sublingual administration may contain conventional additives, including, but not limited to, binders, fillers, lubricants, disintegrants, and wetting agents. Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage starch, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets can be coated according to methods well known in the art.

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

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

[0120] The compositions of the present invention can also be formulated as a depot preparation, which can be administered by infusion or intramuscular injection. The compositions can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0121] The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in the incorporated materials of Remington's pharmaceutical sciences.

[0122] Mode of administration The compositions of the present invention can be administered by any method, including but not limited to, oral, parenteral, sublingual, buccal, nasal, intralesional, or a combination thereof. Parenteral administration includes but is not limited to subcutaneous and intramuscular. The compositions of the present invention can also be administered in the form of an implant, which allows for sustained release of the composition and sustained intravenous injection. In certain embodiments, one or more NOX4, NOX4 / 1, or NOX1 inhibitors are administered orally.

[0123] The dosage administered to an individual as a single or multiple doses will vary depending on a variety of factors, including pharmacokinetic properties, the patient's condition and characteristics (age, weight, health, size), the severity of symptoms, frequency of treatment, and the desired effect.

[0124] combination According to one embodiment of the present invention, the NOX4, NOX4 / 1 or NOX1 inhibitors according to the present invention and pharmaceutical formulations thereof are administered in combination with an anti-cancer immunotherapeutic agent, in particular 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 a NOX4, NOX4 / 1 or NOX1 inhibitor, or a pharmaceutical formulation thereof, wherein the NOX4 / 1 inhibitor or pharmaceutical formulation thereof is administered to an individual before or simultaneously with an anti-cancer immunotherapeutic agent, e.g., simultaneously in the same formulation or separately in different formulations, particularly by different formulation routes.

[0126] According to certain aspects of the present invention, the NOX4, NOX4 / 1 or NOX1 inhibitors according to the present invention and pharmaceutical formulations thereof are administered chronically (e.g., daily or weekly) for the duration of treatment and prior to administration of anti-cancer immunotherapeutic agents or anti-angiogenic treatments.

[0127] According to another particular embodiment of the present invention, the NOX4, NOX4 / 1 or NOX1 inhibitors according to the present invention and pharmaceutical formulations thereof are administered simultaneously with anti-cancer immunotherapeutic agents.

[0128] According to another particular aspect of the invention, the anti-cancer immunotherapeutic agent may be administered in a therapeutically effective amount in combination with other therapeutic regimens or co-agents (e.g., multi-drug regimens) useful in the treatment of cancer, such as in combination with substances useful for treating, stabilizing, preventing, and / or delaying cancer, e.g., substances used in conventional chemotherapy directed at solid tumors to control the establishment of metastases, or any other molecule that acts by inducing programmed cell death, e.g., in combination with a co-agent selected from angiogenesis inhibitors (e.g., anti-VEGF agents as described in Gardner et al., 2017, supra), immunotherapeutic 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 particular aspect of the present invention, the anti-cancer immunotherapeutic agent can be administered in a therapeutically effective amount in combination with other therapeutic regimens or co-agents (e.g., multi-drug regimens) useful in the treatment of cancer, such as in combination with at least one inhibitor of vascular endothelial growth factor (VEGF) (e.g., bevacizumab, sunitinib inhibitors), 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 pharmaceutical formulation thereof administered simultaneously with the anti-cancer immunotherapeutic agent can be administered in or within the same or different composition and by the same or different administration routes.

[0130] patient In one embodiment, the subject according to the invention is a subject suffering from a solid tumor cancer, particularly a poorly responsive solid tumor cancer, which exhibits or is suspected of exhibiting resistance to immunotherapy or anti-angiogenic agents, particularly anti-VEGF treatment.

[0131] In certain embodiments, the subject according to the invention is a subject suffering from a solid tumor cancer 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, renal cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, and brain cancer, particularly glioblastoma.

[0132] In certain embodiments, a subject according to the present invention is a subject suffering from solid tumor cancer and has high alpha-smooth muscle actin (α-SMA) expression.

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

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

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

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

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

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

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

[0140] In another particular embodiment, the subject according to the invention is a subject suffering from rectal cancer or colorectal cancer.

[0141] In another specific embodiment, the subject according to the present invention is a subject suffering from renal cancer.

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

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

[0144] In another specific embodiment, the subject according to the invention is a subject with a solid tumor cancer who is at risk of developing resistance or partial resistance to anti-cancer immunotherapy due to another concomitant treatment or genetic predisposition.

[0145] In another specific embodiment, a subject according to the present invention is a subject with 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 a combination form, wherein at least one NOX4 / 1 inhibitor is administered in combination with at least one anti-cancer immunotherapeutic agent.

[0147] The entire contents of all documents cited herein are incorporated herein by reference. The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

[0148] Having described the invention, the following examples are offered by way of illustration and not limitation. [Example]

[0149] The efficacy of NOX4 / 1 inhibitors to restore or enhance responsiveness to anti-cancer immunotherapeutic agents can be tested as follows.

[0150] Example 1 Combination of NOX4 / 1 inhibitors and anti-PD1 inhibitors in the treatment of cancer To test the efficacy of the combination according to the invention, the following experiments are carried out 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 (renal cancer) were injected subcutaneously into the flanks 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 the murine 4T1 breast cancer model were used.

[0152] Tumors between 80 and 200 mm 3 Once tumors reach a mean tumor volume of 1000 mcg, combination treatment begins. Mice are randomized into different groups of 8-17 mice based on individual tumor volume. Each group receives either a placebo, a NOX4 / 1 inhibitor alone, a PD-1 antibody alone, or a NOX4 / 1 inhibitor in combination with a PD-1 antibody.

[0153] The 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-(pyridin-4-yl)-1,3,4-thiadiazol-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 gavage via gavage tube at doses of 60-10 mg / kg, respectively.

[0154] As a PD-1 inhibitor, an anti-PD-1 antibody (reference: BE0146, BioXcell; clone: ​​RMP1-14, reactivity: mouse; isotype: rat IgG2a; storage condition: +4°C) is injected into the abdominal cavity of the mice (intraperitoneally, IP). The administration volume is adjusted to 10 mL / kg based on the latest individual body weight of the mouse.

[0155] Tumor harvesting and immunohistochemistry to assess T cell infiltration Fourteen days after randomization, when the antitumor activity of the NOX4 / 1 compounds alone or in combination was deemed sufficient, tumors from five satellite mice per group were harvested, weighed, and cut into two pieces. One piece was cut into 4 mm thick slices, fixed in 4% neutral buffered formalin for 24-48 hours, and then embedded in paraffin (Histosec®, Merck, Darmstadt, Germany). One piece was embedded in tissue freezing medium (Microm Microtech, France), flash-frozen in liquid nitrogen-cooled isopentane, and stored at 80°C until processing. Immunohistochemical staining for CD3, CD4, and CD8 was performed on paraffin-embedded tissue sections using standard techniques (Biodoxis, France). The number of CD3, CD4, and CD8 immunopositive cells per field was counted.

[0156] Tumor harvesting and flow cytometry to assess T cell infiltration Fourteen days after randomization, tumors from four mice per group are harvested.

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

[0158] [Table 1]

[0159] Stained cells are analyzed on 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 are acquired either until 10,000 mCD45+ events are recorded for each sample or for a maximum duration of 2 minutes.

[0160] Animal Monitoring All study data, including animal weight measurements, tumor volume, medical and mortality records, and treatments, are scheduled and recorded. Survival and behavior are recorded daily. Body weights are measured twice weekly. Tumor length and width are measured twice weekly with calipers, and tumor volume is estimated by the following formula:

[0161]

number

[0162] Human endpoints. The study will be terminated after 5 weeks or if: In mice, more than 10% of normal body weight or 1,500 mm 3 tumors exceeding Unstable or nutritionally interfering tumors, ulcerated tumors larger than 8 mm, bleeding infections, Tissue erosion A 20% weight loss (30% on one monitoring day) lasting for 2 monitoring days compared to the treatment start date / maximum weight. Signs of pain, distress, or agony: pain postures, pain facial expressions, behavior, Decreased physical condition, emaciation, cachexia, dehydration, - prolonged lack of voluntary response to external stimuli, Rapid labored breathing, anemia, severe bleeding, Neurological signs: circling, convulsions, paralysis, A persistent decrease in body temperature, Abdominal bloating.

[0163] Efficacy parameters Therapeutic efficacy is assessed in terms of the effect of the test substance on tumor volume in treated animals compared to control animals. The following evaluation criteria for antitumor efficacy are determined: resulting in individual and / or mean (or median) tumor volumes, Calculate tumor doubling time (DT), Calculate the tumor growth inhibition rate (T / C%), defined as the ratio of median tumor volumes 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 standard for 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 deduced from experimental data and selected during the exponential phase of tumor growth. 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 the tumor reaches this volume. For each group, calculate the average tumor volume V and the average time to reach this volume. Mouse survival is also monitored and used as an efficacy parameter. Depict a survival curve.

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

[0167] 4T1 cancer cells (0.5 × 10 5 ), they are injected sc in PBS into the upper mammary fat pad of 8-10 week old female mice. Cells are injected alone or in a concentration of 2.5 x 10 cells isolated from spontaneous stroma-rich breast tumors of transgenic BALBneuT mice. 5 BALB / C milk or mixed with CAF.

[0168] Once tumors were 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 by oral gavage at 40 mg / kg, 5 times per week. Control mice received vehicle by oral gavage. During chronic administration, an initial dose of 15 mg / kg was administered as described, but was reduced to 50 mg / kg, 3 times per week for 3 weeks, and then to 60 mg / kg, 2 times per week for 3 weeks. An anti-PD-1 antibody (Bioxcell; RMP1-14) was administered via intraparietal (ip) injection. Every other day, when tumors were palpable, 300 μg of antibody or IgG2a isotype control (Bioxcell) was given for a total of three doses.

[0169] For the data presented in Figure 1, tumors were measured every 2–3 days using electronic skin calipers for their longest width and length. Tumor volume was calculated using the formula 4 / 3π × r3, where the radius (r) was calculated from the tumor width and length measurements to represent the mean diameter. Mice were randomized into groups based on tumor volume, resulting in no statistically significant differences in mean tumor volume between groups before treatment. Figure 1A shows that on day 15, i.e., 8 days after treatment, tumors were significantly smaller in mice treated with the NOX4 inhibitor compared to vehicle alone. Furthermore, immunohistochemistry (performed as described above) revealed that treatment with the NOX4 inhibitor significantly reduced intratumoral SMA-positive CAFs, resulting in the relocation of CD8+ T cells from the tumor periphery to the tumor center, as shown in Figures 1B and 1C, respectively. Using a 4T1 breast cancer model, these results demonstrate that GKT treatment inhibits CAF formation, as indicated by a reduced myofibroblast (SMA-positive cell) population, and CD8 + T cells are clearly shown to be able to access tumors and kill cancer cells, reducing tumor size. This is due to the CD8 +This supports the beneficial effect of combining a NOX4 inhibitor and an anti-cancer immunotherapy agent that further activates T cells.

[0170] The beneficial effect of this combination is further supported by the results presented in Figure 2, in which the combination of the PD-1 inhibitor (αPD1) and the NOX4 inhibitor GKT137831 significantly improved 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). Following administration of the αPD1 / GKT831 combination, CD8+ T cells significantly relocated from the tumor periphery to the tumor center (Figure 2B). Survival outcomes were also significantly increased compared to αPD1 alone (Figure 2C). Using the MC38 colon cancer model, the beneficial effect of GKT / αPD1 combination therapy was confirmed by demonstrating a highly significant reduction in tumor volume. This reduction was accompanied by increased survival in mice. Furthermore, this effect was shown to be mediated by CD8+ T cell infiltration into the NOX inhibitor tumors. These results strongly suggest that the NOX4 inhibitors of the present invention, particularly GKT137831, are promising candidates for PD1 combination therapy of all CAF-rich cancers.

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

[0172] TC1 cancer cells (0.5 × 10 5 ) (prostate cancer) were injected subcutaneously (sc) in phosphate-buffered saline (PBS) into the flanks of 8-10 week-old C57BL / 6 female mice. TC1 cells were injected alone or with C57BL / 6 lung fibroblasts (2.5 × 10 cells) that had been pretreated ex vivo with 2 ng / ml TGFβ1 for 6 days prior to injection to induce the CAF phenotype. 5 ) or mixed with

[0173] Tumors were measured every 2-3 days for maximum width and length with electronic skin calipers. Tumor volume measurements, mouse randomization, and oral gavage dosing 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) fused to the immunodominant CD8 epitope of tetanus β-glucanase (TGF-β) (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) once tumors were palpable. A single injection containing 50 μg of DNA in PBS was administered, and optional repeat doses were given 3 weeks after the initial immunization. Treatment with the NOX4 inhibitor (GKT137831), reconstituted as described in Example 1, was administered to mice once tumors were palpable.

[0175] Figure 3 supports the finding that the combination of antitumor vaccination with a NOX4 inhibitor significantly improved the therapeutic response in CAF-rich tumors, as tumors were significantly smaller in mice treated with the vaccine / NOX4 inhibitor combination compared with those treated with the vaccine alone at day 24. Following administration of the vaccine / NOX4 inhibitor combination, CD8+ T cells significantly relocated from the tumor periphery to the tumor center (Figure 3B), and survival outcomes were also significantly increased compared with those treated with the vaccine alone (Figure 3C). Effective immunotherapy, whether based on checkpoint inhibitors, T cell agonists, vaccination, or adoptive T cell transfer, requires the presence of CD8+ effector T cells in the tumor. Cancer-associated fibroblasts are found in many solid cancers and play a key role in tumor immune evasion, rendering immunotherapy ineffective by eliminating CD8+ T cells from the tumor. 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 the infiltration of CD8+ T cells into tumors and restoring responses to vaccine- and PD1-based immunotherapy. These data suggest that combination immunotherapy with the NOX4 inhibitors of the present invention, particularly GKT137831, significantly improves the response rate to this type of treatment.

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

[0177] MC38 xenograft mouse models of tumors were grown in wild-type C57 / BL6 mice or NOX1-deficient (NOX1-KO) mice with PBS (5.10 for MC38). 5 MC38 tumor cells diluted in HCl were injected subcutaneously. Tumors were 50 mm 3Once tumor size reached 100 mg / kg, purified anti-VEGF antibody, either DC101 or irrelevant rat IgG (as a control), was administered intraperitoneally twice weekly. DC101 was administered at a dose of 600 μg per mouse per injection. Vehicle (VL) (i.e., methylcellulose and Tween 80) or the NOX1-selective inhibitor, (R)-3-methoxy-4-(2-morpholino-1-phenylethoxy)-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)benzamide (GKT2) (10 mg / kg twice daily) was administered by oral gavage until sacrifice. Tumor size was measured with a vernier caliper, and tumor volume was determined according to the formula: (length * width * thickness). Tumor size was measured in vivo with a vernier caliper every 5 days (D-0 to D-15). After sacrifice, the tumors were removed without fixation with paraformaldehyde (PFA), 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) can inhibit angiogenesis. Furthermore, GKT2 and DC101 act synergistically to enhance the inhibition of angiogenesis.

[0179] Figure 5 shows that tumors in NOX1-KO mice showed reduced growth kinetics compared with tumors in WT mice, clearly indicating the involvement of NOX1. Furthermore, treatment with an anti-VEGFR2 antibody (DC101) reduced tumor growth in NOX1-deficient mice, and this effect was even more pronounced compared with WT mice. This clearly suggests a distinct mechanism of action between VEGFR2 and NOX1 signaling.

[0180] Thus, taken together, these data support that the combination of a NOX1 inhibitor and an anti-angiogenic agent, such as an anti-VEGF inhibitor, can achieve synergistic effects on tumor treatment.

Claims

1. A composition 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, 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.

2. The composition according to claim 1, 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 at least one immune checkpoint inhibitor.

3. The composition according to claim 1 or 2, 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, and brain cancer.

4. The composition according to claim 3, wherein the brain cancer is glioblastoma.

5. The composition according to claim 1 or 2, wherein the solid tumor cancer is selected from lung cancer, breast cancer, head and neck cancer, colorectal cancer, prostate cancer, and pancreatic cancer.

6. The composition according to claim 1 or 2, wherein the solid tumor cancer is esophageal cancer.

7. A pharmaceutical formulation 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.

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