Treatment

Abstract

The present invention provides methods, compounds, pharmaceutical compositions, and kits for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation. [Solution] A method is provided comprising administering an effective amount of a FosB / ΔFosB expression and / or VCAM-1 expression and / or ERK1 / 2 phosphorylation inhibitor, as well as pharmaceutical compositions and kits comprising a FosB / ΔFosB expression and / or VCAM-1 expression and / or ERK1 / 2 phosphorylation inhibitor.

Description

[Technical Field]

[0001] This invention relates to methods, compounds, and pharmaceutical compositions for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation, as well as FosB / ΔFosB expression. This also relates to methods for inhibiting ERK1 / 2 phosphorylation and / or VCAM-1 expression. [Background technology]

[0002] background Vascular permeability and neovascularization are associated with diabetic retinopathy (DR) and neovascularization (wet / exudative). In both cases of age-related macular degeneration (nAMD), inflammation, wound healing, tumor growth, and macular edema are key features that confirm the condition. DR is the leading global cause of vision loss in patients aged 20 to 74 years. AMD has a global prevalence of 170 million people, with approximately 11 million people affected in the United States. Leakage from retinal blood vessels usually occurs when the blood-retinal barrier (BRB), which maintains homeostasis, is destroyed. This is caused by the following: This is facilitated by processes of endothelial dysfunction, angiogenesis, and inflammation, leading to edema through leakage of retinal capillaries into the interstitial space and increased osmotic pressure. Factors contributing to vascular permeability include vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), and histamine. Examples include platelet-activating factor, serotonin, and interleukin-1β (IL-1β). ru.

[0003] Anti-VEGF therapy is widely used clinically to treat DR. However, it requires repeated intravitreal injections, and many patients do not respond optimally or their improved response is not sustained. Targeting not only VEGF but also other important mediators involved in the pathogenesis of nAMD / DR is necessary. The drug will have a particular medicinal appeal in this area of ​​unaddressed clinical needs.

[0004] Vascular permeability is also important for the pathogenesis of rheumatoid arthritis (RA), a process mediated by pro-inflammatory cytokines. RA affects approximately 13 million people in the United States alone.

[0005] Over the past decade, there has been considerable improvement in the management of rheumatoid arthritis (RA) with biological agents such as anti-TNF agents and soluble TNF receptors. However, a significant proportion of patients do not achieve clinical remission with current treatment options and remain at risk of progressive joint destruction and functional impairment.

[0006] Globally, the aging population presents significant unaddressed clinical challenges for both RA and nAMD / DR. Given the global economic burden exemplified by the impact of these diseases and their chronic conditions, an alternative treatment approach is necessary. [Overview of the project]

[0007] summary Activator protein-1 (AP-1 or AP1) responds to various pathological stimuli and genes It is a heterodimeric transcription factor involved in regulating gene expression. The inventors reason that compounds that can inhibit AP-1-dependent gene expression may be useful in treating or preventing diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation.

[0008] The inventors identified compounds that inhibit AP-1-dependent gene expression. They investigated the activity of these compounds and found that they inhibit FosB / ΔFosB expression. The author states that such compounds affect vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and We found that it can reduce cell proliferation.

[0009] Therefore, the first embodiment includes administering an effective amount of FosB / ΔFosB expression inhibitor to the subject. The present invention provides a method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation.

[0010] A first alternative embodiment involves the use of FosB / ΔFosB expression inhibitors for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject; or the use of FosB / ΔFosB expression inhibitors in the manufacture of pharmaceuticals for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject. To provide.

[0011] A second embodiment includes administering an effective amount of FosB / ΔFosB expression inhibitor to the subject, The present invention provides methods for treating or preventing diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in the subject.

[0012] A second alternative embodiment provides for use in the treatment or prevention of diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or for use in the manufacture of pharmaceuticals for the treatment or prevention of diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0013] A third aspect involves effective FosB / ΔFosB expression and / or extracellular signal regulation. The treatment involves administering an inhibitor of kinase 1 / 2 (ERK1 / 2) phosphorylation and / or vascular cell adhesion molecule-1 (VCAM-1 or VCAM1) expression to improve vascular permeability, neovascularization, and angiogenesis in subjects. The present invention provides a method for reducing blood, inflammation, cell migration, and / or cell proliferation.

[0014] A third alternative embodiment involves FosB / ΔFosB expression for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in the subject, and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression inhibitors; or vascular permeability in the subject FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or in the manufacture of pharmaceuticals for reducing hyperangiogenesis, neovascularization, inflammation, cell migration and / or cell proliferation. Alternatively, it provides the use of VCAM-1 expression inhibitors.

[0015] A fourth aspect involves effective amounts of ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression, The present invention provides a method for treating or preventing diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering a VCAM-1 expression inhibitor.

[0016] A fourth alternative embodiment involves FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression for use in the treatment or prevention of diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject. Inhibitors; or inhibitors of vascular permeability, neovascularization, angiogenesis, inflammation, and cell migration in the target. FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression inhibition in the manufacture of pharmaceuticals for treating or preventing diseases or conditions related to cell proliferation. Provide the use of harmful agents.

[0017] The fifth aspect is chemical formula I:

[0018] [ka]

[0019] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0020] [ka]

[0021] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0022] [ka]

[0023] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or chemical formula II:

[0024] [ka]

[0025] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but

[0026] [ka]

[0027] (In the formula, q is 1, 2, 3 or 4; and R 5The present invention provides a method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering an effective amount of a linear or branched C1-C6 alkyl compound, or a pharmaceutically acceptable salt thereof.

[0028] A fifth alternative embodiment involves a combination of chemical formulas I or II for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject. A substance, or a pharmaceutically acceptable salt thereof; or vascular permeability, neovascularization, The present invention provides the use of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a pharmaceutical product that reduces angiogenesis, inflammation, cell migration, and / or cell proliferation.

[0029] The sixth aspect is chemical formula I:

[0030] [ka]

[0031] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0032] [ka]

[0033] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0034] [ka]

[0035] (In the formula, R 2is a linear or branched C1-C6 alkyl)); or Chemical Formula II:

[0036] [Chemical Formula]

[0037] (wherein R 3 is a linear or branched C1-C6 alkyl; and R 4 is a linear or branched C1-C6 alkyl), or R 4 is

[0038] [Chemical Formula]

[0039] )) of a compound, or a pharmaceutically acceptable salt thereof, to a subject, comprising the step of administering an effective amount thereof, to provide a method for treating or preventing a disease or condition mediated by AP-1 and / or ERK1 / 2.

[0040] As a sixth alternative aspect, there is provided a compound of Chemical Formula I or II, or a pharmaceutically acceptable salt thereof, for treating or preventing a disease or condition mediated by AP-1 and / or ERK1 / 2 in a subject; or the use of a compound of Chemical Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a disease or condition mediated by AP-1 and / or ERK1 / 2 in a subject. acceptable salt; or the use of a compound of Chemical Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a disease or condition mediated by AP-1 and / or ERK1 / 2 in a subject. in a subject. To provide the use of a pharmaceutically acceptable salt thereof.

[0041] As a seventh aspect, there is provided Chemical Formula I:

[0042] [Chemical Formula]

[0043] (wherein X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0044] [ka]

[0045] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0046] [ka]

[0047] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or chemical formula II:

[0048] [ka]

[0049] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but,

[0050] [ka]

[0051] (In the formula, q is 1, 2, 3 or 4; and R 5 However, linear or branched C1-C6 alkyl groups)) This includes administering an effective amount of the compound or a pharmaceutically acceptable salt thereof to a subject, thereby treating AP-1, and / or FosB / ΔFosB and / or ERK1 / 2 and / or VCAM-1 in the subject. The present invention provides a method for treating or preventing diseases or conditions mediated by VEGF-A and / or IL-1β.

[0052] A seventh alternative embodiment involves AP-1, FosB / ΔFosB and / or ERK1 / 2 in the subject. Compounds of chemical formula I or II for use in the treatment or prevention of diseases or conditions mediated by VCAM-1 and / or VEGF-A and / or IL-1β, or their pharmaceutically permitted A tolerable salt; or a compound of chemical formula I or II in the manufacture of a pharmaceutical for treating or preventing diseases or conditions mediated by AP-1, and / or FosB / ΔFosB, and / or ERK1 / 2, and / or VCAM-1, and / or VEGF-A, and / or IL-1β in a subject, This provides the use of its pharmaceutically acceptable salt.

[0053] The eighth aspect is chemical formula I:

[0054] [ka]

[0055] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0056] [ka]

[0057] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0058] [ka]

[0059] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or chemical formula II:

[0060] [ka]

[0061] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but,

[0062] [ka]

[0063] (In the formula, q is 1, 2, 3 or 4; and R 5 The present invention provides a method for treating or preventing diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering an effective amount of a linear or branched C1-C6 alkyl compound, or a pharmaceutically acceptable salt thereof, to the subject.

[0064] An eighth alternative embodiment is a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or a compound of chemical formula I in the manufacture of a medicament for the treatment or prevention of diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject. The present invention provides the use of compound II or a pharmaceutically acceptable salt thereof.

[0065] The ninth aspect involves providing an effective amount of the following to the target:

[0066] [ka]

[0067] The present invention provides a method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering a compound selected from or a pharmaceutically acceptable salt thereof.

[0068] A ninth alternative embodiment is the following, for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject:

[0069] [ka]

[0070] Compounds selected from or pharmaceutically acceptable salts thereof; or in the manufacture of a pharmaceutical product that reduces vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, the following:

[0071] [ka]

[0072] The present invention provides the use of a compound selected from or a pharmaceutically acceptable salt thereof.

[0073] The tenth aspect is the following effective amount applied to the target:

[0074] [ka]

[0075] The present invention provides a method for reducing AP-1-dependent and / or ERK1 / 2-dependent gene expression in a subject, comprising administering a compound selected from or a pharmaceutically acceptable salt thereof. do.

[0076] A tenth alternative embodiment is the following for use in reducing AP-1-dependent and / or ERK1 / 2-dependent gene expression in the subject:

[0077] [ka]

[0078] Compounds selected from or pharmaceutically acceptable salts thereof; or AP-1 dependence in the subject. In the manufacture of pharmaceuticals that reduce gene expression and / or ERK1 / 2-dependent gene expression Ru, below:

[0079] [ka]

[0080] The present invention provides the use of a compound selected from or a pharmaceutically acceptable salt thereof.

[0081] The eleventh aspect involves providing an effective amount of the following to the target:

[0082] [ka]

[0083] AP of subjects, including administering a compound selected from or a pharmaceutically acceptable salt thereof. -1 and / or FosB / ΔFosB, and / or ERK1 / 2 and / or VCAM-1, and / Alternatively, it provides a method for treating or preventing a disease or condition mediated by VEGF-A and / or IL-1β.

[0084] As an eleventh alternative aspect, AP-1 and / or FosB / ΔFosB in the subject, For use in the treatment or prevention of diseases or conditions mediated by / or ERK1 / 2 and / or VCAM-1, and / or VEGF-A, and / or IL-1β:

[0085] [ka]

[0086] Compounds selected from or pharmaceutically acceptable salts thereof; or AP-1 and in the subject A pharmaceutical product for treating or preventing a disease or condition mediated by / or FosB / ΔFosB, and / or ERK1 / 2 and / or VCAM-1, and / or VEGF-A, and / or IL-1β. In manufacturing, the following:

[0087] [ka]

[0088] The present invention provides the use of a compound selected from or a pharmaceutically acceptable salt thereof.

[0089] The twelfth aspect is the following:

[0090] [ka]

[0091] The present invention provides a method for treating or preventing conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering a compound selected from or a pharmaceutically acceptable salt thereof.

[0092] A twelfth alternative embodiment is the following for use in the treatment or prevention of conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject:

[0093] [ka]

[0094] A compound selected from or a pharmaceutically acceptable salt thereof; or vascular permeability in the subject In the manufacture of pharmaceuticals for the treatment or prevention of conditions related to neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation, the following:

[0095] [ka]

[0096] The present invention provides the use of a compound selected from or a pharmaceutically acceptable salt thereof.

[0097] As the thirteenth aspect, an effective amount of chemical formula I:

[0098] [ka]

[0099] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0100] [ka]

[0101] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0102] [ka]

[0103] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or chemical formula II:

[0104] [ka]

[0105] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but,

[0106] [ka]

[0107] (In the formula, q is 1, 2, 3 or 4; and R 5 However, this method involves contacting cells with a linear or branched C1-C6 alkyl compound, or a pharmaceutically acceptable salt thereof, to achieve ERK1 / 2 phosphorylation in cells, and / or FosB / ΔFosB expression, and / or VCAM-1 expression and / or VEG expression. This invention provides a method for reducing FA expression.

[0108] The fourteenth aspect is the effective amount of the following:

[0109] [ka]

[0110] ERK1 / 2 phosphorylation in cells, and / or FosB / ΔFosB expression and / or VCAM-1 expression, comprising contacting cells with a compound selected from or a pharmaceutically acceptable salt thereof. The present invention provides a method for reducing VEGF-A expression.

[0111] The fifteenth aspect is an effective amount of chemical formula I:

[0112] [ka]

[0113] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0114] [ka]

[0115] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0116] [ka]

[0117] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or compound II:

[0118] [ka]

[0119] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but,

[0120] [ka]

[0121] (In the formula, q is 1, 2, 3 or 4; and R 5 The present invention provides a method for inhibiting ERK1 / 2 phosphorylation, comprising incubating ERK1 / 2 with a linear or branched C1-C6 alkyl compound or a pharmaceutically acceptable salt thereof.

[0122] The sixteenth aspect is the following effective amount:

[0123] [ka]

[0124] The present invention provides a method for inhibiting ERK1 / 2 phosphorylation, comprising incubating ERK1 / 2 with a compound selected from or a pharmaceutically acceptable salt thereof.

[0125] The seventeenth embodiment is a compound that is a FosB / ΔFosB expression inhibitor, and optionally ERK1 / 2 The present invention provides a pharmaceutical composition comprising an oxidation and / or VCAM-1 expression inhibitor, as well as a pharmaceutically acceptable carrier.

[0126] The eighteenth aspect is the following general formula:

[0127] [ka]

[0128] The present invention provides a pharmaceutical composition comprising a compound thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0129] One nineteenth aspect comprises administering an effective amount of a FosB / ΔFosB expression inhibitor; and optionally an ERK1 / 2 phosphorylation and / or VCAM-1 expression inhibitor, as follows: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; Permeability of retinal blood vessels; angiogenesis; endothelial cell dysfunction; Atherosclerosis; stroke; Myocardial infarction; Peripheral vascular disease; constriction; restenosis; inflammation; Cytokine storm; pulmonary inflammation; Pulmonary fibrosis, The present invention provides methods for treating or preventing diseases or conditions in subjects selected from the above.

[0130] In the nineteenth alternative embodiment, the following: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; Permeability of retinal blood vessels; angiogenesis; endothelial cell dysfunction; Atherosclerosis; Stroke; Myocardial infarction; Peripheral vascular disease; Stenosis; Restenosis; Inflammation; Cytokine storm; Lung inflammation; Pulmonary fibrosis, An inhibitor of FosB / ΔFosB expression for use in the treatment or prevention of a disease or condition in a subject selected from: and optionally an inhibitor of ERK1 / 2 phosphorylation and / or VCAM-1 expression; or The following: Arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; Diabetic retinopathy; Macular edema; Vascular leakage; Vascular permeability; Permeability of retinal blood vessels; Angiogenesis; Endothelial cell dysfunction; Atherosclerosis; Stroke; Myocardial infarction; Peripheral vascular disease; Stenosis; Restenosis; Inflammation; Cytokine storm; Lung inflammation; Pulmonary fibrosis, Use of an inhibitor of FosB / ΔFosB expression; and optionally an inhibitor of ERK1 / 2 phosphorylation and / or VCAM-1 expression in the manufacture of a medicament for treating or preventing a disease or condition in a subject selected from.

[0131] As a twentieth aspect, the following, comprising administering an effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof: Arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; Diabetic retinopathy; The following: Macular edema; Vascular leakage; vascular permeability; Permeability of retinal blood vessels; angiogenesis; endothelial cell dysfunction; Atherosclerosis; stroke; Myocardial infarction; Peripheral vascular disease; constriction; restenosis; inflammation; Cytokine storm; pulmonary inflammation; Pulmonary fibrosis, The present invention provides methods for treating or preventing conditions or diseases in subjects selected from the available options.

[0132] A 20th alternative configuration is as follows: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; Permeability of retinal blood vessels; angiogenesis; endothelial cell dysfunction; Atherosclerosis; stroke; Myocardial infarction; Peripheral vascular disease; constriction; restenosis; inflammation; Cytokine storm; pulmonary inflammation; Pulmonary fibrosis, Chemical formula I for use in the treatment or prevention of a condition or disease in a subject selected from the following: Or a compound of type II, or a pharmaceutically acceptable salt thereof; or the following: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; Vascular permeability; Permeability of retinal vessels; Angiogenesis; Endothelial cell dysfunction; Atherosclerotic arteriosclerosis; Stroke; Myocardial infarction; Peripheral vascular disease; Stenosis; Restenosis; Inflammation; Cytokine storm; Pulmonary inflammation; Pulmonary fibrosis, In the manufacture of a medicament for treating or preventing a condition or disease in a subject selected from: The use of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, is provided.

[0133] As a twenty - first aspect, the following: Arthritis; Rheumatoid arthritis; Bone destruction; Age - related macular degeneration; Diabetic retinopathy; Macular edema; Vascular leakage; Vascular permeability; Permeability of retinal vessels; Angiogenesis; Endothelial cell dysfunction; Atherosclerotic arteriosclerosis; Stroke; Myocardial infarction; Peripheral vascular disease; Stenosis; Restenosis; Inflammation; Cytokine storm; Pulmonary inflammation; Pulmonary fibrosis, A method for treating or preventing a condition or disease in a subject selected from: This method comprises an effective amount of the following:

[0134]

Chemical formula

[0135] This includes administering a compound selected from or a pharmaceutically acceptable salt thereof to the target.

[0136] For the 20th aspect, the following general formula applies:

[0137] [ka]

[0138] The present invention provides compounds having the same property or pharmaceutically acceptable salts thereof.

[0139] The 21st alternative form is as follows:

[0140] [ka]

[0141] A compound selected from the following or a pharmaceutically acceptable salt thereof, the following: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; Permeability of retinal blood vessels; angiogenesis; endothelial cell dysfunction; Atherosclerosis; stroke; Myocardial infarction; Peripheral vascular disease; constriction; restenosis; inflammation; Cytokine storm; pulmonary inflammation; Pulmonary fibrosis, Compounds or pharmaceutically acceptable salts thereof for use in the treatment or prevention of conditions or diseases in subjects selected from:

[0142] [ka]

[0143] The use of a compound selected from or a pharmaceutically acceptable salt thereof, the following: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; Permeability of retinal blood vessels; angiogenesis; endothelial cell dysfunction; Atherosclerosis; stroke; Myocardial infarction; Peripheral vascular disease; constriction; restenosis; inflammation; Cytokine storm; pulmonary inflammation; Pulmonary fibrosis, It provides use in the manufacture of pharmaceuticals for treating or preventing conditions or diseases in subjects selected from the above.

[0144] The twenty-second aspect provides a pharmaceutical composition comprising a compound of chemical formula II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0145] The 23rd aspect is given by the following general formula:

[0146] [ka]

[0147] A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. To provide.

[0148] The twenty-fourth aspect is in vitro ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression. , and / or to reduce VCAM-1 expression and / or VEGF-A expression, chemical formula I The present invention provides the use of compound II or a pharmaceutically acceptable salt thereof.

[0149] The twenty-fifth aspect involves in vitro ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression. The present invention provides compounds of chemical formula I or II, or pharmaceutically acceptable salts thereof, for use in reducing VCAM-1 expression and / or VEGF-A expression.

[0150] The twenty-sixth aspect is the presence of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. A method for reducing ERK1 / 2 phosphorylation in cells, and / or FosB / ΔFosB expression, and / or VCAM-1 expression and / or VEGF-A expression in vitro, including contacting the effective dose with cells. provide.

[0151] The 27th aspect is in vitro ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression. The following general formula is used to reduce VCAM-1 expression and / or VEGF-A expression:

[0152] [ka]

[0153] The present invention provides the use of a compound selected from or a pharmaceutically acceptable salt thereof.

[0154] The 28th aspect is in vitro ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression. The following general formula is used for reducing VCAM-1 expression and / or VEGF-A expression:

[0155] [ka]

[0156] The present invention provides compounds selected from or pharmaceutically acceptable salts thereof.

[0157] The 29th aspect is the effective quantity, as shown in the following general formula:

[0158] [ka]

[0159] The present invention provides a method for reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression and / or VEGF-A expression in vitro, comprising contacting cells with a compound selected from or a pharmaceutically acceptable salt thereof.

[0160] The 30th aspect is chemical formula I:

[0161] [ka]

[0162] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0163] [ka]

[0164] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0165] [ka]

[0166] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or chemical formula II:

[0167] [ka]

[0168] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but,

[0169] [ka]

[0170] (In the formula, q is 1, 2, 3 or 4; and R 5 However, linear or branched C1-C6 alkyl groups)) The present invention provides a method for reducing the expression of genes referenced in Tables 3A, 3B, and / or 3C, typically IL-1β-induced genes referenced in Tables 3A, 3B, and / or 3C, more typically IL-1β-induced genes referenced in Table 3B, comprising administering an effective amount of the compound or a pharmaceutically acceptable salt thereof.

[0171] The 31st aspect is chemical formula I:

[0172] [ka]

[0173] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0174] [ka]

[0175] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0176] [ka]

[0177] (In the formula, R 2 (However, linear or branched C1-C6 alkyl groups); Or chemical formula II:

[0178] [ka]

[0179] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but,

[0180] [ka]

[0181] (In the formula, q is 1, 2, 3 or 4; and R 5However, linear or branched C1-C6 alkyl groups)) The present invention provides a method for treating or preventing a subject's condition through the expression of a gene referenced in Tables 3A, 3B, and / or 3C, typically an IL-1β-induced gene referenced in Tables 3A, 3B, and / or 3C, more typically an IL-1β-induced gene referenced in Table 3B, comprising administering an effective amount of the compound or a pharmaceutically acceptable salt thereof.

[0182] The 31st alternative embodiment is a compound of chemical formula I or II, or its pharmaceutically acceptable form, for use in the treatment or prevention of a condition in a subject via the expression of a gene referenced in Tables 3A, 3B and / or 3C, typically an IL-1β-induced gene referenced in Tables 3A, 3B and / or 3C, more typically an IL-1β-induced gene referenced in Table 3B. Possible salts; or genes referenced in Tables 3A, 3B and / or 3C, typically genes induced by IL-1β referenced in Tables 3A, 3B and / or 3C, more typically in Table 3B In the manufacture of a pharmaceutical product for treating or preventing a condition mediated by the expression of a gene referenced by IL-1β, a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. Provides usage.

[0183] The thirty-second aspect involves a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, being used in a finely detailed manner. This includes contacting cells with ICAM-1, c-Fos, Egr-1, CXCL2, KLF5, and / or Alternatively, this provides a method for reducing VCAM-1 expression.

[0184] The thirty-third aspect is the presence of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. The present invention provides a method for reducing the expression of IL-1β-induced genes, more typically the IL-1β-induced gene referenced in Tables 3A, 3B, and / or 3C, by bringing an effective dose into contact with cells.

[0185] Simple description of the diagram Preferred embodiments of the present invention will be described only illustratively with reference to the accompanying drawings. [Brief explanation of the drawing]

[0186] [Figure 1A] Figure 1A shows Western blot images illustrating the effects of compounds BT2, T4, and T6 on the expression of FosB / ΔFosB and c-Fos. HMEC-1 cells were grown in 6-well plates (in 10% FBS containing EGF and hydrocortisone), serum arrested for 20 hours, and then treated for 4 hours at 37°C with 30 μM of compounds (T4, T6, T7, BT2, and BT3) in serum-free medium (without EGF or hydrocortisone). The medium was changed to 10% FBS (containing EGF and hydrocortisone) with the same concentrations of compounds for 1 hour. Lysates were separated by SDS-PAGE and Western blotting was performed for FosB or c-Fos. Experiments were performed twice biologically independently, as instructed. The approximate location of molecular weight markers is indicated. Data represent three biologically independent experiments. [Figure 1B] Figure 1B shows the time-course effects of BT2, T4, and T6 on serum-induced endothelial cell proliferation. Serum-deficient HMEC-1 cells were treated with the compounds in 5% FBS-containing medium (containing EGF and hydrocortisone), and cell proliferation was monitored using the xCELLigence system. The upper figure shows a representative real-time profile from one experiment using the xCELLigence system, with concentrations indicated. The cell index is a quantitative indicator of cell proliferation. The lower figure shows xCELLigence data representing the mean ± SEM of 5-8 independent experiments after 79 hours. Statistical significance was assessed by one-way ANOVA. [Figure 1C]Figure 1C shows the effects of BT2, T4, and T6 on endothelial migration. BAEC cells in DMEM containing 10% FBS were seeded in 24-well plates fitted with 0.8 μm Transwell inserts. After 48 hours, the medium was changed to DMEM containing 0.01% FBS. 1 μM of the compound was added to the DMEM containing 0.01% FBS in the upper chamber, and the medium in the lower chamber was changed to DMEM containing 10% FBS and 50 ng / ml VEGF-A165. Cells were left for 24 hours. Nuclei were quantified using NIH ImageJ software. Data represent the mean ± SEM from 4-5 independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 1D] Figure 1D shows the effects of BT2, T4, and T6 on endothelial cell regeneration after mechanical injury in vitro using a scratch assay. HMEC-1 monolayers scratched with a sterile toothpick were treated with 0.6 μM of the compound in a medium containing 5% FBS. Regeneration of the scratched area was observed 48 hours after scratching. The regrowth area was determined using Image-Pro Plus software (Cybernetics). Data represent the mean ± SEM of five independent experiments. Statistical significance was assessed by one-way ANOVA. [Figure 1E] Figure 1E shows the effects of BT2, T4, and T6 on endothelial network (tubule) formation on Matrigel. HMEC-1 was mixed with the compound (final 3 μM) in a medium containing 1% FBS and 50 ng / ml FGF-2, and seeded into wells coated with Matrigel. Network formation was evaluated over 24 hours. The network was quantified using Image-Pro Plus software. Data represent the mean ± SEM from 5-6 independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 2A]Figure 2A shows that BT2 inhibits retinal permeability in rats after choroidal laser injury. BT2, T4, T6 (indicated doses), or vehicle (control) were injected bilaterally via IVT on the day of six retinal laser burns (day 0) and 7 days later. Kenacort was administered via IVT on day 0. Alternatively, aflibercept / eirea in a vehicle (saline) was injected via IVT six times (days 0, 3, 7, 10, 14, and 17). On days 14 and 21, fluorescein sodium was subcutaneously injected, and ocular fluorescence was recorded and scored 10 minutes later using Heidelberg retinal angiography (HRA). HRA scores were calculated by combining data from days 14 and 21. Data represent mean ± SEM. Statistical significance was assessed using one-way ANOVA (left plot) or t-test (right plot, left plot comparing BT2 vs. Kenacort). n=5 to 29 per group. [Figure 2B] Figure 2B shows that BT2 inhibits rhVEGF-A165-induced retinal vascular permeability in rabbits. BT2 or BT3 (600 μg) or a vehicle was intravenously injected into the right eye of rabbits 5 days before induction of vascular leakage by intravenous injection of 500 ng rhVEGF-A165 (50 μl). Two days after induction, fluorescein sodium was intravenously injected, and ocular fluorescence of the right (R) and left (L) eyes was measured 1 hour later using an ocular fluorometer, expressed as the ratio (R / L) for each rabbit. For comparison with the active compound BT2, the ratio data for the vehicle group and BT3 group were pooled (control) as both conditions were inactive and showed no statistically significant difference. Data represent mean ± SEM. Statistical significance was assessed by t-tests. n=6-8 per group. [Figure 2C-E]Figures 2C-E show the enlargement of immunohistochemical staining of (C) CD31, (D) VEGF-A165, and (E) VEGF-A165 in rat retinal lesions, enclosed in a 100 μm frame relative to the lesion. Untreated refers to eyes that were not treated with laser irradiation, vehicle, or drug injection. IOD for positive staining (red dye) was evaluated using Image-Pro Plus software. Slides were photographed with 10× or 20× objective lenses, and magnified views are shown. For CD31, n=4-6 per group; for VEGF-A165, n=3-6 per group; or for VEGF-A165 gradient analysis, n=3-5 per group. Data represent the mean ± SEM for each animal. Statistical significance was evaluated as appropriate by one-way ANOVA, Mann-Whitney U test, or t-test. Arrows provide examples of positive staining. [Figure 2F] Figure 2F shows that BT2 inhibits angiogenesis in mouse Matrigel plugs. Matrigel (500 μl) containing VEGF-A165 (100 ng / ml), heparin (10 U), and BT2 or BT3 (2.5 mg / mouse) or vehicle was subcutaneously injected into the left flank of 8-week-old male C57BL / 6 mice. After 7 days, mice were sacrificed and the plugs were stained with CD31 antibody. Representative immunohistochemical images of stained CD31, taken with a 10× objective lens, are shown with insets providing magnified views (taken with a 40× objective lens). CD31 staining was quantified using Image-Pro Plus software. Data represent mean ± SEM for each animal. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. n=10-11 per group. [Figure 3A]Figure 3A is a Western blot image showing that BT2 inhibits ERK phosphorylation, FosB / ΔFosB, and VCAM-1 expression. HMECs treated with 30 μM BT2 or 30 μM PD98059 were stimulated with 20 ng / ml IL-1β for varying durations up to 4 hours. Western blots are representative of 2-3 biologically independent experiments, with durations indicated in hours (where shown), and two biologically independent copies performed in separate lanes. BT2 inhibition of IL-β1-induced VCAM-1 and ERK phosphorylation in the same blot is shown in Figure 3D. [Figure 3B] Figure 3B shows flow cytometry that BT2 inhibits VCAM-1 expression. Flow cytometry was performed using BD FACSCanto II with HMEC-1 treated with 30 μM BT2 or BT3 and 20 ng / ml IL-1β. Data represent the mean ± SEM from three independent experiments. Statistical significance was assessed by one-way ANOVA. n=3 per group. [Figure 3C]Figure 3C shows that BT2 inhibits FosB, c-Fos, VCAM-1, ICAM-1, and other genes involved in cell proliferation, migration, angiogenesis, and / or inflammation. RNA-seq was performed on total RNA prepared by pre-treating HMEC-1 with 30 μM BT2 and incubating it with 20 ng / ml IL-1β for 4 hours. The PCA plot (top left) shows the close association between biological replications under conditions UT, IL-1β, and IL-1β+BT2, and the clear separation between conditions. A heatmap (center, 1579 genes) was created for all upregulated genes in a comparison of IL-1β vs. UT. Using Counts per million (cpm) values, genes (rows) were grouped and plotted using hierarchical clustering by cpm for FosB and VCAM-1. The heatmap (right) shows 325 genes with a log factor change (FC) ≥ 2. FosB, c-Fos, and VCAM-1 (the subject of this study) are shown in the figure along with several other genes inhibited by BT2. A small subset of genes further induced by BT2 (shown in red) is also shown in the figure.BHLHE40, basic helix-loop-helix family member e40; CCL20, CC motif chemokine ligand 20; CXCL2, CXC motif chemokine ligand 2; DUSP1, dual specificity phosphatase 1; EGR1, early growth response 1; ETS1, ETS proto-oncogene 1; FOS, FOS proto-oncogene; FOSB, FosB proto-oncogene; ICAM1, intercellular adhesion molecule 1; IL6, interleukin 6; KLF5, Kruppel-like factor 5; MMP25, matrix metallopeptidase 25; NFKBIA, NFKB inhibitor α; THBS1, thrombospondin 1; TNIP, TNFAIP3 interacting protein 1; PLAT, plasminogen activator, tissue type; VCAM1, vascular cell adhesion molecule 1. [Figure 3D] Figure 3D shows that BT2 inhibits IL-1β-induced VCAM-1 expression and ERK phosphorylation more potently than PD98059. The concentrations of BT2 and PD98059 (1–30 μM) are shown. The data represent three biologically independent experiments. [Figure 3E]Figure 3E is an image of a Western blot using siRNA demonstrating that VCAM-1 expression is FosB-dependent. HMEC-1 cells treated with 0.6 μM siRNA or control siRNA were stimulated with 20 ng / ml IL-1β for 2 or 4 hours. Western blotting was performed using the indicated antibody. The data are representative of two biologically independent experiments. The approximate location of the molecular weight marker is shown. [Figure 4A-E] Figures 4A-E show that BT2 inhibits ERK phosphorylation, FosB / ΔFosB, and VCAM-1 expression in the retina and Matrigel plugs. Immunohistochemical staining of retinal lesions was performed for (A) pERK, (B) FosB, and (C) VCAM-1. IOD of positive staining (red dye) was evaluated using Image-Pro Plus software. Slides are magnified images taken with 20× or 40× objective lenses. n=3-5 per group for pERK and FosB, and n=4-6 per group for VCAM-1. Data represent the mean ± SEM for each animal. Statistical significance was evaluated as appropriate by one-way ANOVA, Kruskal-Wallis, Mann-Whitney, or t-test. Arrows indicate examples of positive staining. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane. Alternatively, Matrigel plugs were stained with (D) FosB or (E) VCAM-1. Representative FosB or VCAM-1 stains were imaged with 40× or 20× objective lenses, respectively, and accompanied by insets providing magnified views. Staining was quantified using Image-Pro Plus software. Data represent mean ± SEM. Statistical significance was evaluated by Kruskal-Wallis multiple comparison tests (FosB, n=9-11 per group) or one-way ANOVA (VCAM-1, n=10-11 per group). Arrows indicate examples of positive staining. IOD represents integrated optical density. [Figure 5A]Figures 5A–D demonstrate the importance of the carbamate site of BT2 for its interaction with MEK1 and its functional effects. Figure 5A shows a proliferation experiment in which serum-deficient HMEC-1 cells were treated with the compound (0.4 or 0.8 μM) in 5% FBS-containing medium, and cell proliferation was monitored using the xCELLigence system (Roche). Left: Representative proliferation profile obtained from one experiment. Right: xCELLigence data representing the mean ± SEM from three independent experiments after 79 h. Statistical significance was assessed by one-way ANOVA or Mann-Whitney U test. [Figure 5B] Figure 5B shows the formation of an HMEC-1 network when a compound (final 1 μM) was seeded in a medium containing 1% FBS and 50 ng / ml FGF-2 in wells coated with Matrigel. The network was quantified using NIH ImageJ software. Data represent the mean ± SEM from 3-4 independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 5C] Figure 5C shows SPR analysis testing the interaction between PD98059, BT2, and BT2 analogs with His-MEK1 (left panel) and His-MEK2 (right panel). Measurements were performed in a Biacore T200 at 15°C in a buffer containing 20 mM HEPES, 150 mM NaCl, and 5% DMSO pH 7.4. Data are representative of two independent experiments. [Figure 5D] In Figure 5D, HMEC-1 was treated with 1 μM of the compound (BT2 and analogues) for 4 hours in serum-free medium at 37°C. The medium was changed to 20 ng / ml IL-1β along with the compound for 15 minutes. Lysates were separated by SDS-PAGE and Western blotting was performed for pERK or total ERK. The data are representative of two biologically independent experiments. The approximate location of molecular weight markers is shown. [Figure 6A]Figure 6A shows a schematic diagram of high-throughput compound screening. Luciferase-based high-throughput screening was used to identify hits, including the use of a PAINS frequent hitter filter. Mean IC50 data and typical 11-point titration curves for BT2 and Cpd B / X / LK001 are shown. [Figure 6B] Figure 6B shows the reactants in the chemical synthesis of Cpd B / X / LK001 or BT2 analogues. [Figure 7A] Figures 7A and 7B show that BT2, T4, and T6 inhibit endothelial FosB / ΔFosB and c-Fos expression, blocking cell proliferation. Figure 7A shows band intensities (pixel intensity relative to the corresponding control) from Western blot analysis measured using NIH ImageJ software. FosB / ΔFosB band intensities were aggregated. Plotted data represent values ​​from three biologically independent experiments or the mean (if independent biological duplicates were used in one blot) ± SEM. [Figure 7B] Figure 7B shows the total cell count and % live cells as determined by trypan blue exclusion using the Countess II Automated Cell Counter. Countess data represent the mean ± SEM from four independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 8A]Figures 8A-C show immunohistochemical staining with the primary antibody omitted. Figure 8A shows immunohistochemical staining using the MACH3 AP-Polymer detection system with the primary antibody omitted (vehicle group) in areas without lesions or areas with lesions (arrows). Vitr, vitreous; ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OLM, outer limiting membrane; IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium; Chor, choroid. [Figure 8B] Figure 8B shows immunohistochemical staining (vehicle group) using a DAB colorimetric detection system with the primary antibody in the Matrigel plug omitted. [Figure 8C] Figure 8C shows immunohistochemical staining (vehicle group) using the MACH3 AP-Polymer detection system with the primary antibody in the Matrigel plug omitted. No. 1o Ab indicates that the primary antibody was omitted. [Figure 9] Figure 9 shows that BT2 inhibits ERK phosphorylation, FosB / ΔFosB, and VCAM-1 expression. Band intensities (pixel intensities relative to the corresponding control) from Western blot analysis were measured using NIH ImageJ software. FosB / ΔFosB band intensities were aggregated. Plotted data represent values ​​from 2-3 biologically independent experiments or the mean (if independent biological duplicates were used in one blot) ± SEM. [Figure 10]Figure 10 shows the gating of VCAM-1+ and VCAM-1- cells by flow cytometry. VCAM-1+ and VCAM-1- cells were gated by flow cytometry (FACSDiva v6.1.3) with or without primary VCAM-1 antibody (non-specific staining), respectively. A representative gating of the latter (i.e., negative control) is shown in the figure. [Figure 11A] Figures 11A–C show Western blotting experiments using extracts of HMEC-1 exposed to BT2 or HMEC-1 transfected with a plasmid. Figure 11A shows the comparative effects of BT2 and PD98059 on IL-1β-inducible VCAM-1 expression and ERK phosphorylation. Band intensity (pixel intensity relative to the corresponding control) from Western blot analysis was measured using NIH ImageJ software. Plotted data represent the mean ± SEM of three biologically independent experiments. [Figure 11B] Figure 11B shows the comparative effects of BT2 and PD98059 (1–30 μM) on IL-1β-inducible p-SAPK / JNK or p-p38. Data represent the mean ± SEM of three biologically independent experiments. Approximate positions of molecular weight markers are shown. [Figure 11C]Figure 11C shows the need for ERK phosphorylation in the signs of FosB and VCAM-1 expression as measured by Western blotting. HMEC-1 cells, quiescent in 6-well plates due to serum deficiency (and without EGF or hydrocortisone), were transfected with 6 μg of the indicated pcDNA3.1+ / C-(K)DYK-based plasmids inserted with ERK1 mutant 1 (NM_002746.2), ERK1 mutant 2 (NM_001040056.3), FosB mutant 1 (NM_006732.2), FosB mutant 2 (NM_001114171.2), or ΔFosB (XM_005258691.1). Western blotting was performed using the indicated antibodies with whole protein lysates (collected 18, 24, 48, and 72 h after plasmid transfection). L indicates mild exposure. The approximate location of the molecular weight marker is shown. The data are representative of two independent experiments. [Figure 12] Figure 12 shows that BT2 is more potent than curcumin in inhibiting endothelial network formation on Matrigel. HMEC-1 in a medium containing 1% FBS and 50 ng / ml FGF-2 was seeded in Matrigel-coated wells in combination with various concentrations of BT2 or curcumin compounds. The network after 4 hours was quantified using NIH ImageJ software. Data represent the mean ± SEM from 3-4 independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 13A] Figures 13A and 13B show the biological activity of structural analogs of BT2. In Figure 13A, HMEC-1 was treated with 3 μM of the compound (BT2 and analogous compounds) for 4 hours in serum-free medium at 37°C. The medium was changed to 20 ng / ml IL-1β along with the compound for 15 minutes. The lysates were separated by SDS-PAGE, and Western blotting was performed for phosphorylated ERK or total ERK. The approximate positions of molecular weight markers are shown. [Figure 13B]Figure 13B shows HMEC-1 network formation seeded in Matrigel-coated wells in a medium combining 1% FBS and 50 ng / ml FGF-2 with the compound (final 3 μM). The network was quantified using NIH Image J software. Data represent the mean ± SEM of 3-4 independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 14A-B] Figures 14A–F show that BT2 retains its stability and biological activity after boiling or autoclaving. Figures 14A and 14B show that RRLC-MS / MS analysis of BT2 (in saline containing 0.5% Tween 80 and 0.01% DMSO) preparations that were heat-treated (100°C water bath, 10 min, DL20170921-H) or non-heat-treated (DL20170921) and sonicated were performed in triplicates one or six weeks after preparation. Representative chromatograms are shown (deuterated (d3)-BT2 control shown to the right of each set). [Figure 14C-D] Figures 14C and 14D show tubes containing BT2 or BT3 in a vehicle (saline solution containing 0.01% DMSO and 0.5% Tween 80, sonicated) stored at 22°C (unheated) or immersed in a 100°C water bath for 10 minutes followed by cooling to 22°C (heated, +H), and then used immediately or stored in the dark for 6 weeks or at least 10 months (D, black bar represents 11 months, blue bar represents 10 months, red bar represents 16 months). Serum-deficient HMEC-1 was treated with heat-treated or unheated BT2 or BT3 (0.4, 0.8 μM) in 5% FBS-containing medium, and growth was monitored using the xCELLigence system (Roche). Data represent the mean ± SEM from three independent experiments at 79 h. Statistical significance was assessed by one-way ANOVA. [Figure 14E-F]Figures 14E and 14F show RRLC-MS / MS analysis of BT2 preparations that were heat-treated (100°C, 10 min) or unheat-treated sonicated (in saline containing 0.5% Tween 80 and 0.01% DMSO) and performed in triplicates 10, 11, or 16 months after preparation. Representative chromatograms are shown. [Figure 14G] Figure 14G shows tubes containing BT2 in a vehicle (saline solution containing 0.01% DMSO and 0.5% Tween 80, sonicated) that was either freshly prepared for use or autoclaved (121°C, 15 psi, 20 min; +A) and stored in the dark for 4 months (orange bars). Serum-deficient HMEC-1 cells were treated with autoclaved or freshly prepared BT2 (0.4, 0.8 μM) in 5% FBS-containing medium, and growth was monitored using an xCELLigence system (Roche). Growth data represent the mean ± SEM from four independent experiments after 79 hours. Statistical significance was assessed by one-way ANOVA. LC / MS analysis results for freshly prepared BT2 and BT2 stored in the dark for 4 months in an autoclave are also shown. The figure shows the total ion chromatogram (top, black) obtained by integrating the peak intensity of each spectrum, and the extracted ion chromatogram (bottom, brown) obtained by integrating the peak intensity of the protonation precursor (m / z 327.1319-327.1361). Table 3 provides genes induced by IL-1β (logFC≧2) compared to the control (UT) (Table 3C), and genes inhibited by BT2 against IL-1β (logFC≧2) (Table 3A). Table 3B shows genes induced by IL-1β and inhibited by BT2. RNA-seq was performed on total RNA prepared by incubating HMEC-1 treated with 30 μM BT2 and 20 ng / ml IL-1β for 4 hours. These data were obtained from the same experiment, which is represented by a heatmap in a different location. [Figure 15A]Figure 15A is a graph showing the effects of various concentrations of BT2 and BT3 on monocyte cell adhesion to IL-1β-treated endothelium in vitro. In vitro THP-1 adhesion to HMEC was evaluated in 96-well plates by first treating HMEC with various concentrations of BT2 or BT3 for 1 hour. HMEC was stimulated with 20 ng / ml IL-1β for 1 hour. Subsequently, 30 minutes after adding cells, the fluorescence intensity of calcein-labeled THP-1 adhered to the HMEC monolayer was measured using a fluorescence plate reader. Data are representative of three experiments and are expressed as mean ± SEM. Statistical significance was assessed by one-way ANOVA. [Figure 15B] Figure 15B is a graph showing the effect of various concentrations of BT2 on monocyte transendothelial cell migration to MCP-1 in vitro. In vitro migration of THP-1 transendothelial cells was evaluated by treating HMEC with various concentrations of BT2 for 1 hour in gelatin-coated culture inserts. HMEC was treated with 20 ng / ml IL-1β for 1 hour. After 24 hours, THP-1 cells that had migrated transendothelially towards MCP-1 were measured using a Coulter counter. The data are representative of three experiments and are expressed as mean ± SEM. Statistical significance was evaluated by one-way ANOVA. [Figure 16A] Figure 16A provides a graph showing the effect of vehicle or 3 mg / kg or 30 mg / kg of BT2 on hind paw thickness in a mouse model of collagen antibody-induced arthritis. Animals were administered an antibody cocktail via ip injection on day 0 and LPS plus BT2 (3 or 30 mg / kg in vehicle) on day 3. Hind paw thickness was measured using a digital caliper on day 9. Data are expressed as hind paw thickness (mm) for each limb (left and right). n=8-10 per group. Data are expressed as mean ± SEM. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 16B] Figure 16B provides images (gross specimens) showing the effect of vehicle or BT2 on hind paw plantar thickness in a collagen antibody-induced arthritis mouse model on day 14. [Figure 16C]Figure 16C provides images showing H&E staining of mouse paw pads on day 14 in a collagen antibody-induced arthritis mouse model, either untreated or treated with vehicle or BT2. [Figure 16D] Figure 16D provides images showing the effects of untreated or vehicle- or BT2-treated mice on bone destruction in a collagen antibody-induced arthritis mouse model. 3D Micro-CT analysis of the hind limbs on day 14 was quantified, with scores of 0 (no bone destruction) and 1 (destruction) assigned to each individual limb. Data were expressed as the mean bone destruction score ± SEM per hind limb (left and right), with n=8-10 per group. Statistical significance was assessed using Firth's penalty-force likelihood test. [Figure 16E] Figure 16E shows micro-CT images of the hind limb at day 14 in a collagen antibody-induced arthritis mouse model, either untreated or after treatment with vehicle or BT2. Arrows indicate bone erosion and / or remodeling. [Figure 16F] Figure 16F shows graphs and images of tartrate-resistant acid phosphatase (TRAP) staining in 14-day hindlimb osteoclasts from the joints of untreated or vehicle- or BT2-treated collagen antibody-induced arthritis model mice. Arrows indicate examples of positive staining. Slides were captured with 20× or 40× objective lenses. IOD of positive staining (red dye) was assessed using Image-Pro Plus software, or the number of osteoclasts was counted using NIH Image J. Data are expressed as mean ± SEM. Statistical significance was assessed by the Wilcoxon signed-rank test. n=6–10 per group. [Figure 16G] Figure 16G shows immunohistochemical staining of VCAM-1 or ICAM-1 in the hind limbs at day 14. IOD / μm2 under a 20× objective lens was evaluated using Image-Pro Plus software. Data represent mean ± SEM. n=3-5 per group. Statistical significance was assessed by one-way ANOVA.

[0187] Detailed description AP-1 is a transcription factor that regulates gene expression in response to various pathological stimuli such as cytokines, growth factors, stress, and viral and bacterial infections. AP-1 is derived from c-Fos, c-Jun, and ATF(ac It is formed by the dimerization of proteins belonging to the tivating transcription factor and / or the JDP (Jun dimerization protein 2) protein family. It is a heterodimer. It is involved in the expression and DNA binding activity of AP-1 family members c-fos and c-jun. AP-1 is observed in the synovial membrane of human rheumatoid arthritis, is associated with disease activity, and has been shown to regulate gene products involved in angiogenesis, while IL-1β is a mediator of bone and cartilage damage in rheumatoid arthritis. Furthermore, AP-1 factor is expressed in retinal cells after retinal detachment and is elevated in diabetic human retinas. Therefore, AP-1 represents an important therapeutic target for various diseases.

[0188] As described in the examples, the inventors identified and synthesized compounds of formulas I and II that have the ability to inhibit AP-1-dependent gene expression. The inventors further synthesized these compounds We found that it inhibits the phosphorylation of ERK1 / 2, and therefore inhibits ERK1 / 2-dependent gene expression.

[0189] As further described in the examples, the inventors have shown that compounds of formulas I and II inhibit the following The compounds were shown to harm: serum-induced endothelial cell proliferation and migration; endothelial wound repair after in vitro injury; and microtubule formation on the reconstituted basement membrane matrix. The inventors further found that these compounds inhibit the expression of FosB / ΔFosB and c-Fos.

[0190] Accordingly, one embodiment provides a method for reducing vascular permeability, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering an effective amount of a FosB / ΔFosB expression inhibitor. In one embodiment, the inhibitor is a compound that inhibits FosB / ΔFosB expression.

[0191] Another embodiment involves administering an effective amount of FosB / ΔFosB expression inhibitor to improve vascular permeability and blood circulation. The present invention provides a method for treating or preventing conditions related to vascularization, inflammation, cell migration, and / or cell proliferation. In one embodiment, the inhibitor is a compound that inhibits FosB / ΔFosB expression.

[0192] As described in the examples, the inventors further found that compound BT2 (compound of chemical formula II) In addition to inhibiting FosB / ΔFosB expression, it also inhibits the phosphorylation of ERK1 and ERK2 (ERK1 / 2), and We found that it inhibits VCAM-1 expression and VEGF-A expression.

[0193] Therefore, another aspect involves effective amounts of ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression. The present invention provides a method for reducing vascular permeability, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, comprising administering a VCAM-1 expression inhibitor. In one embodiment, the inhibitor is a compound that inhibits ERK1 / 2 phosphorylation and FosB / ΔFosB expression and VCAM-1 expression. FosB dimerizes with proteins of the c-Jun protein family to form AP-1. The Fos protein family, a family of leucine zipper proteins that can perform This is a bar. ΔFosB is a truncated splice variant of FosB. ERK1 ERK2 is a mitogen-activated protein kinase (MAP kinase), and surface tyrosine They are involved in cellular function in response to the activation of surface receptors such as enzymes. ERK1 and ERK2 are related serine / threonine kinases that participate in the Ras-Ras-MEK-ERK signaling cascade. MEK1 / 2 catalyzes phosphorylation at amino acid residues Tyr204 and 187 and Thr202 and 185 of ERK1 / 2. After activation, ERK1 / 2 catalyzes phosphorylation of hundreds of cytoplasmic and nuclear proteins. The Ras-Ras-MEK-ERK signaling cascade is involved in cell proliferation, adhesion, migration, and cell division. It is thought to play a central role in controlling numerous cellular processes, such as cell regeneration and angiogenesis.

[0194] VCAM-1 (also known as CD106) is a cytokine that is expressed in blood vessels in response to cytokine stimulation. It is a cell adhesion molecule. In particular, VCAM-1 expression is upregulated in endothelial cells in response to stimuli such as TNF-alpha and IL-1β.

[0195] When used herein, FosB / ΔFosB expression inhibitors are in contact with compounds or drugs. The amount of FosB / ΔFosB protein produced by cells or tissues that do not contain a compound or drug A compound that reduces the amount of FosB / ΔFosB protein produced by cells or tissues after contact with it. These are substances or drugs. An ERK1 / 2 phosphorylation inhibitor is a compound or drug that reduces the degree of ERK1 / 2 phosphorylation in cells or tissues after contact with a compound or drug, compared to the degree of ERK1 / 2 phosphorylation in cells or tissues that have not been in contact with the compound or drug. A VCAM-1 expression inhibitor is a compound or drug that reduces the amount of VCAM-1 protein produced by cells or tissues after contact with a compound or drug, compared to the amount of VCAM-1 protein produced by cells or tissues that have not been in contact with the compound or drug. A VEGF-A expression inhibitor reduces the amount of VEGF-A protein produced by cells or tissues after contact with a compound or drug, compared to the amount of VEGF-A protein produced by cells or tissues that have not been in contact with the compound or drug, typically VEGF-A 165 The tongue It is a compound or drug that reduces the amount of protein.

[0196] In one embodiment, the compound is a FosB / ΔFosB expression inhibitor.

[0197] In one embodiment, the compound is a VCAM-1 expression inhibitor.

[0198] In one embodiment, the compound is an ERK1 / 2 phosphorylation inhibitor.

[0199] In one embodiment, the compound is an inhibitor of FosB / ΔFosB expression and ERK1 / 2 phosphorylation.

[0200] In one embodiment, the compound is an inhibitor of FosB / ΔFosB and VCAM-1 expression.

[0201] In one embodiment, the compound inhibits ERK1 / 2 phosphorylation, FosB / ΔFosB expression, and VCAM-1 expression. It is a harmful substance.

[0202] In one embodiment, the compound is an inhibitor of ERK1 / 2 phosphorylation, FosB / ΔFosB expression, VCAM-1 expression, and VEGF-A expression.

[0203] In one embodiment, the compound is an inhibitor of ERK1 / 2 phosphorylation, FosB / ΔFosB expression, VCAM-1 expression, and VEGF-A expression.

[0204] In one embodiment, the compound does not inhibit SAPK / JNK or p38 phosphorylation.

[0205] Typically, the compounds are small molecule inhibitors.

[0206] In one embodiment, the compound includes a carbamate moiety.

[0207] In one embodiment, the compound is dibenzoxazepinone or benzophenone.

[0208] In one embodiment, the compound is a compound of chemical formula I or II, or a pharmaceutically acceptable compound thereof. It is a salt. Compounds of chemical formula I are:

[0209] [ka]

[0210] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0211] [ka]

[0212] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0213] [ka]

[0214] (In the formula, R 2 However, they are either linear or branched C1-C6 alkyl groups.

[0215] Compounds of chemical formula II are:

[0216] [ka]

[0217] (In the formula, R 3 However, linear or branched C1-C6 alkyl groups; and R 4 However, linear or branched C1-C6 alkyl groups, Or R 4 but,

[0218] [ka]

[0219] (In the formula, q is 1, 2, 3 or 4; and R 5 However, they are either linear or branched C1-C6 alkyl groups.

[0220] In some embodiments, AP-1-dependent gene expression and / or MEK1-dependent gene expression Reduces the expression of ERK1 / 2-dependent genes and / or ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression. The compound is chemical formula I:

[0221] [ka]

[0222] (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A:

[0223] [ka]

[0224] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0225] [ka]

[0226] (In the formula, R 2 However, it is a linear or branched C1-C6 alkyl compound or a pharmaceutically acceptable salt thereof.

[0227] In some embodiments of equation (I), X is F. is Cl. In some embodiments of equation (I), X is Br. Several implementations of equation (I) Morphologically, X is I. Typically, X is F or Cl.

[0228] In some embodiments of equation (I), G is C=O. In some embodiments of equation (I), G is C=N-OH.

[0229] In some embodiments of formula (I), A is

[0230] [ka]

[0231] (wherein p is 1, 2, 3 or 4; and R 1 However, it is either a linear or branched C1-C6 alkyl group. In some embodiments, p is 2. In some embodiments, R 1 is -CH3. In some embodiments, p is 2 and R 1 It is -CH3.

[0232] In some embodiments of formula (I), A is

[0233] [ka]

[0234] (In the formula, R 2 (is linear or branched C1-C6 alkyl). In some embodiments, R 2 It is -CH3.

[0235] In some embodiments, the compound of formula (I) is formula (I-1):

[0236] [ka]

[0237] (In the formula, X is F, Cl, Br, or I; and A:

[0238] [ka]

[0239] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; also A is:

[0240] [ka]

[0241] (In the formula, R 2However, it may also be a linear or branched C1-C6 alkyl compound, or a pharmaceutically acceptable salt thereof.

[0242] In some embodiments, the compound of formula (I-1) is formula (I-1a):

[0243] [ka]

[0244] (In the formula, X is F, Cl, Br, or I; p is 1, 2, 3 or 4; and R 1 However, it may also be a linear or branched C1-C6 alkyl compound.

[0245] For example, the compound of formula (I-1a):

[0246] [ka]

[0247] That's fine.

[0248] In some embodiments, the compound of formula (I-1) is formula (I-1b):

[0249] [ka]

[0250] (In the formula, X is F, Cl, Br or I; and R 2 However, it may also be a linear or branched C1-C6 alkyl compound.

[0251] In one embodiment, the compound of formula (I-1b) is

[0252] [ka]

[0253] (This is also referred to as T6 in this specification.)

[0254] In some embodiments, the compound of formula (I) is formula (I-2):

[0255] [ka]

[0256] (In the formula, X is F, Cl, Br or I; and A:

[0257] [ka]

[0258] (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C1-C6 alkyl groups; Or A:

[0259] [ka]

[0260] (In the formula, R 2 However, it may also be a linear or branched C1-C6 alkyl compound.

[0261] In some embodiments, the compound of formula (I-2) is formula (I-2a):

[0262] [ka]

[0263] (In the formula, X is F, Cl, Br, or I; p is 1, 2, 3 or 4; and R 1 However, it may also be a linear or branched C1-C6 alkyl compound.

[0264] In one embodiment, the compound of formula (I-2a) is:

[0265] [ka]

[0266] (This is also referred to as T4 in this specification.)

[0267] In some embodiments, compounds that reduce AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression are given by formula (II):

[0268] [ka]

[0269] (In the formula, R 3 However, linear or branched C1-C6 alkyl; and R 4 However, linear or branched C1-C6 alkyl groups, or R 4 but

[0270] [ka]

[0271] (In the formula, q is 1, 2, 3 or 4; and R 5 However, it is a compound that is linear or branched C1-C6 alkyl or a pharmaceutically acceptable salt thereof.

[0272] In some embodiments of equation (II), R 3This refers to linear C1-C6 alkyl or branched C1-C6 alkyl It is R. In some embodiments of equation (II), R 3 This is either -CH2CH3 or -CH2CH(CH3)2.

[0273] In some embodiments of equation (II), R 4 This refers to linear C1-C6 alkyl or branched C1-C6 alkyl It is R. In some embodiments of equation (II), R 4 This is either -CH2CH3 or -CH2CH(CH3)2.

[0274] In some embodiments of equation (II), R 4 teeth,

[0275] [ka]

[0276] (In the formula, q is 1, 2, 3 or 4; and R 5 ( is a linear C1-C6 alkyl or a branched C1-C6 alkyl). In some embodiments of formula (II), q is 2. In some embodiments of formula (II), R 5 is -CH3. In some embodiments of equation (II), q is 2. biR 5 It is -CH3.

[0277] In some embodiments, the compound of formula (II) is formula (II-1):

[0278] [ka]

[0279] (In the formula, R 4 However, linear or branched C1-C6 alkyl groups; or R 4 but:

[0280] [ka]

[0281] (In the formula, q is 1, 2, 3 or 4; and R 5 However, it may also be a linear or branched C1-C6 alkyl compound.

[0282] For example, the compound of formula (II-1) is as follows:

[0283] [ka]

[0284] You may choose from the following.

[0285] In some embodiments, the compound of formula (II) is formula (II-2):

[0286] [ka]

[0287] (In the formula, R 4 However, linear or branched C1-C6 alkyl; or R 4 but:

[0288] [ka]

[0289] (In the formula, q is 1, 2, 3 or 4; and R 5 However, it may also be a linear or branched C1-C6 alkyl compound.

[0290] For example, the compound of formula (II-2) is:

[0291] [ka]

[0292] That's fine.

[0293] In one embodiment, the compound of formula (II) is:

[0294] [ka]

[0295] (This is also referred to as BT2 in this specification.)

[0296] In some embodiments, compounds that reduce AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression are as follows:

[0297] [ka]

[0298] or selected from a pharmaceutically acceptable salt thereof.

[0299] In another form, the effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. The present invention provides a method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in subjects, including administering a substance.

[0300] Another embodiment involves the effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. The present invention provides a method for treating or preventing conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation, including administration of the appropriate medication.

[0301] In one embodiment, the following:

[0302] [ka]

[0303] The present invention provides a method for treating or preventing conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation, comprising administering an effective amount of a compound selected from or a pharmaceutically acceptable salt thereof.

[0304] In one embodiment, the compound is given by formula:

[0305] [ka]

[0306] It is a compound of or a pharmaceutically acceptable salt thereof.

[0307] In one embodiment, the compound is given by formula:

[0308] [ka]

[0309] It is a compound of or a pharmaceutically acceptable salt thereof.

[0310] In one embodiment, the compound is given by formula:

[0311] [ka]

[0312] It is a compound of or a pharmaceutically acceptable salt thereof.

[0313] Another aspect is shown in the following general formula:

[0314] [ka]

[0315] The present invention provides compounds or pharmaceutically acceptable salts thereof.

[0316] In one embodiment, the effective use of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. This includes administering a dose of AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression in cells and / or Alternatively, the present invention provides a method for reducing VCAM-1 expression and / or VEGF-A expression. In some embodiments, the cells are the target cells.

[0317] Another embodiment involves the effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. This includes contacting cells with AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and The present invention provides a method for reducing VCAM-1 expression and / or VEGF-A expression. In some embodiments, the cells are the target cells.

[0318] Another aspect is as follows:

[0319] [ka]

[0320] AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression and / or VCAM-1 expression in cells, comprising contacting cells with an effective amount of a compound selected from or a pharmaceutically acceptable salt thereof, and The present invention provides a method for reducing VEGF-A expression.

[0321] In one embodiment, AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression are performed. Compounds that reduce current and / or VEGF-A expression are

[0322] [ka]

[0323] or a medicinally acceptable salt thereof.

[0324] In one embodiment, AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression and / or in the target cells. Alternatively, VCAM-1 expression and / or VEGF-A expression were reduced. In another embodiment, AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression and / or VCAM-1 expression and / Alternatively, VEGF-A expression was reduced.

[0325] Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, orthophosphoric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid; or acid addition salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, citric acid, lactic acid, mucinic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, trihaloacetic acid (e.g., trifluoroacetic acid), methanesulfonic acid, trihalomethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, EDTA, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0326] In one embodiment, the compound of chemical formula I or II or a pharmaceutically acceptable salt thereof is deuterium It has been transformed.

[0327] In one embodiment, the compound of chemical formula I or II or a pharmaceutically acceptable salt thereof is the E isomer.

[0328] In one embodiment, the compound of chemical formula I or II or its pharmaceutically acceptable salt is Z-isomer.

[0329] In one embodiment, the compound of chemical formula I or II or a pharmaceutically acceptable salt thereof is a mixture of the E isomer and the Z isomer.

[0330] This specification describes compounds of chemical formula I or II, or their pharmaceutically acceptable forms. It is a pharmaceutical composition containing a salt that can be used.

[0331] In one embodiment, the following general formula:

[0332] [ka]

[0333] The present invention provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof.

[0334] In one embodiment, the pharmaceutical composition is a compound:

[0335] [ka]

[0336] or a pharmaceutically acceptable salt thereof.

[0337] In another embodiment, the pharmaceutical composition is a compound:

[0338] [ka]

[0339] or a pharmaceutically acceptable salt thereof.

[0340] The pharmaceutical composition of the present invention may be used in the method of the invention described herein.

[0341] Pharmaceutically, the composition typically contains a pharmaceutically acceptable carrier.

[0342] Compounds of formulas I and II may be used to treat any disease or condition mediated by AP-1 and / or ERK1 / 2 and / or FosB / ΔFosB, and / or VCAM-1, and / or VEGF-A, and / or IL-1β. A disease or condition is mediated by a protein or protein complex if the activity of that protein or protein complex is required for the onset and / or maintenance of the disease or condition.

[0343] The compounds of formulas I and II are involved in vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or it can be used to treat or prevent diseases or conditions related to cell proliferation.

[0344] In one embodiment, the disease or condition is related to vascular permeability. Vascular permeability is a key feature in many disease processes, including acute and chronic inflammation, wound healing, and cancer during pathological angiogenesis. Vascular permeability can lead to retinal leakage, which results in macular edema in diabetic retinopathy, and inflammation in rheumatoid arthritis.

[0345] In some embodiments, diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation are associated with AP-1 and / or FosB / ΔFosB It is a disease or condition mediated by ERK1 / 2 and / or VCAM-1 and / or VEGF-A and / or IL-1β.

[0346] Related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation. Diseases or conditions that may affect this include, for example, retinal vascular permeability, diabetic retinopathy, macular edema, rheumatoid arthritis, tissue edema, inflammation (acute and chronic), stenosis, tissue damage in myocardial infarction, age-related macular degeneration, pulmonary fibrosis, pneumonia, atherosclerosis, myocardial infarction, peripheral vascular disease, and stroke.

[0347] Therefore, in some embodiments, diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation are as follows: arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; Permeability of retinal blood vessels; endothelial cell dysfunction; Atherosclerosis; stroke; Myocardial infarction; Peripheral vascular disease; constriction; restenosis; Cytokine storm; pulmonary inflammation; The group is selected from those with pulmonary fibrosis.

[0348] As described in the examples, the inventors have shown that administration of compound BT2 is effective against VEGFA 165 induced by The inventors demonstrated that BT2 suppresses or reduces vascular permeability and suppresses or reduces laser-induced ocular vascular leakage. Furthermore, the inventors showed that administration of BT2 in a collagen antibody-induced arthritis model It demonstrated a reduction in inflammation and bone destruction.

[0349] In one embodiment, the present invention provides a method for treating or preventing a disease or condition related to vascular permeability, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0350] In one embodiment, a method for treating or preventing retinal vascular permeability in a subject requiring such treatment or prevention is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0351] In one embodiment, a method for treating or preventing diabetic retinopathy in a subject requiring treatment or prevention is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0352] In one embodiment, a method for treating or preventing macular edema in a subject requiring treatment or prevention is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0353] In one embodiment, a method for treating or preventing age-related macular degeneration in a subject requiring such treatment or prevention is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0354] In one embodiment, a method for treating or preventing bone destruction and / or arthritis in a subject requiring such treatment or prevention is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0355] In one embodiment, a method for treating or preventing rheumatoid arthritis in a person requiring treatment or prevention is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0356] In one embodiment, the present invention provides a method for treating or preventing chronic or acute inflammation in a subject requiring such treatment or prevention, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0357] In one embodiment, a method for reducing angiogenesis in subjects requiring reduction is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0358] In one embodiment, the present invention provides a method for treating or reducing endothelial cell dysfunction in a subject requiring treatment or reduction of the condition, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0359] In one embodiment, the present invention provides a method for treating or reducing tissue edema in a subject requiring treatment or reduction, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0360] In one embodiment, a method for treating or reducing stenosis in a subject requiring treatment or reduction is provided, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0361] In one embodiment, the present invention provides a method for treating or reducing pulmonary fibrosis in a subject requiring treatment or reduction, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0362] In one embodiment, the present invention provides a method for treating or reducing pneumonia in a subject requiring treatment or reduction, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0363] In one embodiment, the present invention provides a method for treating or reducing atherosclerosis in a subject requiring treatment or reduction of the disease, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0364] In one embodiment, the present invention provides a method for treating or reducing myocardial infarction in a person requiring treatment or reduction of myocardial infarction, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0365] In one embodiment, the present invention provides a method for treating or reducing peripheral vascular disease in a subject requiring treatment or reduction, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0366] In one embodiment, a method for treating or reducing stroke in a person requiring treatment or reduction of stroke, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof. To provide.

[0367] In some embodiments, the compound of formula (II) is formula (II-1):

[0368] [ka]

[0369] (In the formula, R 4 However, linear or branched C1-C6 alkyl groups; or R 4 but:

[0370] [ka]

[0371] (where q is 1, 2, 3 or 4; and R 5 is a linear or branched C1-C6 alkyl)) may be a compound.

[0372] For example, the compound of formula (II-1) may be the following:

[0373]

Chemical formula

[0374] may be selected from the following.

[0375] In some embodiments, the compound of formula (II) is of formula (II-2):

[0376]

Chemical formula

[0377] (where R 4 is a linear or branched C1-C6 alkyl; or R 4 is:

[0378]

Chemical formula

[0379] (where q is 1, 2, 3 or 4; and R 5 is a linear or branched C1-C6 alkyl)) may be a compound.

[0380] For example, the compound of formula (II-2) may be the following:

[0381]

Chemical formula

[0382] That's fine. Typically, compounds of formula (II) are as follows:

[0383] [ka]

[0384] Alternatively, it may be a pharmaceutically acceptable salt thereof.

[0385] In one embodiment, administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof. The present invention provides a method for treating or preventing eye diseases or conditions related to vascular permeability, including those mentioned above.

[0386] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides a method for treating or preventing retinal vascular permeability in subjects requiring such treatment or prevention.

[0387] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides a method for treating or preventing diabetic retinopathy in subjects requiring treatment or prevention, including those with the condition described above.

[0388] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for the treatment or prevention of macular edema in subjects requiring treatment or prevention of macular edema.

[0389] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or preventing age-related macular degeneration in subjects requiring treatment or prevention.

[0390] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for the treatment or prevention of bone destruction and / or arthritis in subjects requiring such treatment or prevention.

[0391] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for the treatment or prevention of rheumatoid arthritis in subjects requiring treatment or prevention of the disease.

[0392] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or reducing chronic or acute inflammation in subjects requiring treatment or reduction of such inflammation.

[0393] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. This invention provides a method for reducing angiogenesis in subjects requiring reduction of angiogenesis, including [specific example].

[0394] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or reducing endothelial cell dysfunction in subjects requiring treatment or reduction of such dysfunction.

[0395] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or reducing tissue edema in subjects requiring treatment or reduction of such edema.

[0396] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or reducing stenosis in subjects requiring treatment or reduction of stenosis.

[0397] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or reducing pulmonary fibrosis in subjects requiring treatment or reduction of pulmonary fibrosis.

[0398] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides a method for treating or reducing pneumonia in subjects requiring treatment or reduction of pneumonia.

[0399] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides a method for treating or reducing atherosclerosis in subjects requiring treatment or reduction of atherosclerosis.

[0400] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides a method for treating or reducing myocardial infarction in subjects requiring treatment or reduction of myocardial infarction.

[0401] In one embodiment, an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof is administered. The present invention provides methods for treating or reducing peripheral vascular disease in subjects requiring treatment or reduction of such disease.

[0402] One embodiment provides a method for treating or reducing stroke in a person requiring treatment or reduction of stroke, comprising administering an effective amount of a compound of chemical formula II or a pharmaceutically acceptable salt thereof.

[0403] The methods described herein may include the administration of a pharmaceutical composition comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0404] The substances described herein are compounds of chemical formula I or II or their pharmaceutically acceptable salts. The pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0405] In one embodiment, the compound of chemical formula I or II is selected from BT2, T4, and T6.

[0406] In some embodiments, the carrier is a carrier of non-natural origin.

[0407] In some embodiments, the compounds described herein or pharmaceutically acceptable salts thereof may be used in combination with one or more other agents.

[0408] The compound described herein or a pharmaceutically acceptable salt thereof in combination with one or more other agents. It should be understood that the drugs may be administered simultaneously, sequentially, or separately.

[0409] The term “composition” encompasses formulations comprising an active ingredient and conventional carriers and excipients, as well as formulations comprising an encapsulating material as a carrier to provide a capsule in which the active ingredient (with or without other carriers) is surrounded by an encapsulating carrier. In a pharmaceutical composition, the carrier is “pharmaceutically acceptable” in the sense that it is compatible with other components of the composition and does not dramatically change the substance. The pharmaceutical compositions of the present invention may contain other agents or further activators as described above, and may be formulated by using, for example, conventional solid or liquid vehicles or diluents, and pharmaceutical additives of a type suitable for the desired mode of administration (e.g., excipients, binders, preservatives, stabilizers, fragrances, etc.) in accordance with the art well known in the field of pharmaceutical formulation (see, e.g., Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

[0410] The pharmaceutical composition may be suitable for intravitreal, oral, rectal, nasal, topical (including skin, cheek, and sublingual), vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or it may be in a form suitable for administration by inhalation or gas infusion.

[0411] In this way, the compounds described herein or their pharmaceutically acceptable salts can be put into the form of pharmaceutical compositions and their unit dosage forms together with pharmaceutically acceptable carriers. The pharmaceutical compositions may be solid, such as tablets or filled capsules, for oral administration, or liquid, such as solutions, suspensions, emulsions, elixirs, or capsules filled with them. The pharmaceutical compositions may also be liquid, such as solutions, suspensions, or emulsions, for intravitreal administration. Furthermore, the pharmaceutical compositions may be in the form of suppositories for rectal administration, or in the form of sterile injection solutions for parenteral (subcutaneous, etc.) use.

[0412] Such pharmaceutical compositions and their unit dosage forms may contain conventional components in conventional proportions and may or may not contain additional active compounds or active ingredients, and such unit dosage forms may contain any appropriate effective amount of the active ingredient corresponding to the intended daily dose range adopted.

[0413] For preparing pharmaceutical compositions from the compounds described herein, the pharmaceutically acceptable carrier may be either solid or liquid. Examples of solid formulations include powders, tablets, pills, capsules, cachets, lozanges (solid or chewable), suppositories, and compoundable granules. The solid carrier may be one or more substances that also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrants, or encapsulating materials. .

[0414] Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugars, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting-point wax, and cocoa butter. Suitable solid forms for oral administration include tablets, powders, capsules, pills, cachets, and lozenges.

[0415] Liquid formulations include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions. For example, a liquid formulation for parenteral injection can be formulated as a solution of polyethylene glycol aqueous solution.

[0416] Examples of sterile liquid compositions include sterile solutions, suspensions, emulsions, syrups, and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier such as sterile water, a sterile organic solvent, or a mixture thereof.

[0417] Accordingly, the pharmaceutical compositions according to the present invention may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and may be presented in unit dosage forms in ampoules, pre-filled syringes, small-volume injectors or multi-dose containers with added preservatives. The pharmaceutical compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending agents, stabilizers and / or dispersants. Alternatively, the active ingredient may be in powder form obtained by sterile isolation of a sterile solid or lyophilization from a solution to constitute a suitable vehicle before use, e.g., sterile, pyrogen-free water.

[0418] Suitable pharmaceutical forms for injection include sterile injectable solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions. These should be stored stably under manufacturing and storage conditions and protected from oxidation and contamination by microorganisms such as bacteria and fungi.

[0419] The solvent or dispersion medium of the injectable solution or dispersion may contain any of the conventional solvents or carrier systems of injectable solutions or dispersions, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[0420] Forms of pharmaceuticals suitable for injection can be delivered via any appropriate route, such as intravenous, intramuscular, intracerebral, subarachnoid, or epidural injection or infusion.

[0421] Sterile injectable solutions are prepared by incorporating the required amount of active ingredient, along with various other components as listed above as needed, into a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other components as needed from those listed above. For sterile powders for the preparation of sterile injectable solutions, a preferred preparation method is vacuum drying or freeze-drying of a pre-sterile filtered solution of the active ingredient and any additional desired components.

[0422] The compounds described herein can be formulated into compositions suitable for oral administration, such as with an assimilated food carrier, encapsulated in hard or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into a diet. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0423] The amount of the active compound in a therapeutically useful composition should be sufficient to obtain an appropriate dose.

[0424] Furthermore, the tablets, lozenges, pills, capsules, lozenges, implants, etc. of the present invention may contain the following components: binders such as gum, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, wintergreen, or cherry blossom flavoring. If the dosage unit is in the form of a capsule, a liquid carrier may be included in addition to the above types of materials.

[0425] Various other materials may be present, either as coatings or in other ways, to alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain the active compound, sucrose as a sweetener, methyl and propylparabens as preservatives, colorants, and flavorings such as cherry or orange. Of course, the materials used in the preparation of any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amounts employed. Furthermore, the active ingredient may be incorporated into sustained-release formulations and formulations, such as those that allow for specific delivery of the active ingredient to specific areas of the intestine.

[0426] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding, if desired, appropriate colorants, fragrances, stabilizers, and thickeners. Aqueous suspensions suitable for oral use can be prepared by dispersing finely divided active ingredients in water with a viscous material such as natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose, or other known suspending agents.

[0427] Examples of pharmaceutically acceptable carriers include any pharmaceutically acceptable solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic agent, and absorption retarder.

[0428] Furthermore, solid formulations intended to be converted into an orally administered liquid formulation immediately before use are also included. Such liquid forms include solutions, suspensions, and emulsions. These formulations may contain, in addition to the active ingredient, colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, and the like.

[0429] For topical administration, the compounds described herein may be formulated as aqueous or oily bases by the addition of appropriate thickeners and / or gelling agents. The lotion may be formulated as an aqueous or oily base and generally contains one or more emulsifiers, stabilizers, dispersants, and suspension agents. It will also contain turbidifiers, thickeners, or colorants.

[0430] Formulations suitable for local administration into the oral cavity include lozenges containing the activator in a flavor base, usually sucrose and acacia or tragacanth; pastilles containing the activator in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the activator in a suitable liquid carrier.

[0431] Solutions or suspensions for nasal administration can be applied directly to the nasal cavity by conventional means, such as droppers, pipettes, or sprays. The formulations can be provided in single-dose or multi-dose forms. In the case of droppers or pipettes, this can be achieved by the patient administering an appropriate predetermined amount of the solution or suspension. In the case of sprays, this can be achieved, for example, by a metered spray pump. Delivery and retention in the nasal cavity. To improve retention, the compounds of the present invention may be encapsulated with cyclodextrin or formulated with other agents expected to enhance delivery to and retention of the nasal mucosa.

[0432] For administration to the airways, the active ingredient is supplied in a pressurized pack with a suitable propellant such as chlorofluorocarbons (CFCs), such as dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gases. This can also be achieved with azole formulations.

[0433] The aerosol can conveniently contain surfactants such as lecithin. The dose of the active ingredient may be controlled by providing a metering valve.

[0434] Alternatively, the active ingredient may be a dry powder, such as lactose, starch, hydroxypropyl methylcellulose, and starch derivatives such as polyvinylpyrrolidone (PVP). The compounds may be provided in the form of a powder mixture in a fine powder base. Conveniently, the powder carrier forms a gel in the nasal cavity. The powder composition may also be presented in unit dosage forms, for example, in gelatin capsules or cartridges, or in blister packs, from which the powder can be administered by inhaler.

[0435] In formulations intended for administration to the airway, such as intranasal preparations, the active ingredient will generally have small particle sizes, for example, on the order of 5 to 10 microns or less. It can be obtained by methods well known in the field, such as micronization.

[0436] The compounds described herein can be formulated into compositions for injection into the eyeball, intraocularly, intravitreously, or subconjunctivally. The compounds described herein can be formulated for administration by eye drops, contact lenses, or implants. Implants may be injected intravitreously into the eyeball. Implants may allow for the delivery of a constant therapeutic level of the compound. Such sustained-release implants are typically made of a pellet-shaped compound core surrounded by a non-reactive material such as silicone, ethylene vinyl acetate (EVA), or polyvinyl alcohol (PVA). These implants are not biodegradable and can deliver a sustained dose of the compound for several months to several years. Matrix implants may also be used. Matrix implants are typically used to deliver a loading dose, followed by escalating doses of the compound over a period of one to six months. Polylactic acid (PLA) and / or poly It is most commonly made from glycolic acid lactic acid (PLGA) copolymers, which decompose into water and carbon dioxide.

[0437] The intravitreous administration formulation contains one or more emulsifiers, stabilizers, dispersants, penetrating agents, or suspending agents. It can be formulated as an aqueous base containing [the specified ingredient].

[0438] If desired, a formulation adapted to impart sustained release properties to the active ingredient can also be used.

[0439] Pharmaceutical formulations are preferably in unit dosage forms. In such forms, the formulation is subdivided into unit doses containing an appropriate amount of the active ingredient. A unit dosage form may be a packaged formulation, and the packaging may contain individual quantities of the formulation, such as packaged tablets, capsules, and powders in vials or ampoules. A unit dose may also be a capsule, tablet, cachet, or lozenge itself, or a packaged form of an appropriate number of any of these.

[0440] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dose-unit form. Parenteral compositions may also be in the form of physically individual units suitable as unit doses for the target to be treated, and each unit, in relation to the pharmaceutical carrier, provides the desired therapeutic effect. It contains a predetermined amount of active ingredients calculated to produce the desired effect.

[0441] Furthermore, if the compound is in a single dosage form, it can be administered in the absence of a carrier.

[0442] The term "effective amount" refers to the amount of compound that is effective in achieving the desired reaction.

[0443] The effective amount of the compounds described herein or their pharmaceutically acceptable salts can be determined by those skilled in the art who are interested in a particular compound.

[0444] It will be understood that specific dose levels and administration frequencies for any particular subject may vary and depend on a variety of factors, including the activity of the specific compound employed, its metabolic stability and duration of action, the subject's age, weight, general health, sex and diet, mode and timing of administration, excretion rate, drug combinations, and the severity of the particular condition.

[0445] Appropriate dosages of the compounds described herein or any further activators administered in combination with the compounds described herein can be easily determined by those skilled in the art who are interested in the specific compounds or further activators of the selected invention.

[0446] The compounds described herein are administered in combination with one or more agents or other activators. In such cases, it will be further understood that the dosage form and level may be formulated for simultaneous administration, sequential administration, separate administration, or a combination thereof.

[0447] The method of the present invention is intended for use on any subject that may experience the benefits of the method of the present invention. Therefore, the term "subject" includes not only humans but also non-human mammals. The subject may be, for example, a domesticated animal, a zoo animal, or a livestock.

[0448] The inventors also stated that the compounds of formulas I and II can be used in vitro, for example, in laboratory applications. It is intended to be used for inhibiting AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression and / or ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression.

[0449] One embodiment involves administering an effective amount of the compound of formula I or II, or a pharmaceutically acceptable salt thereof, to cells. The present invention provides a method for reducing AP-1-dependent and / or ERK1 / 2-dependent gene expression in cells in vitro, including contact with the present invention.

[0450] Another aspect is the effective amount, as follows:

[0451] [ka]

[0452] The process involves contacting cells with a compound selected from or a pharmaceutically acceptable salt thereof. AP-1-dependent gene expression and / or ERK1 / 2-dependent inheritance in cells in vitro This provides a method for reducing the expression of offspring.

[0453] Another embodiment involves administering an effective amount of the compound of formula I or II, or a pharmaceutically acceptable salt thereof, to a cell. This invention provides a method for reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression and / or VEGF-A expression in cells in vitro, including contact with the cells. ru.

[0454] Another aspect is the effective amount of the following:

[0455] [ka]

[0456] The present invention provides a method for reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression and / or VEGF-A expression in cells in vitro, comprising contacting cells with a compound selected from or a pharmaceutically acceptable salt thereof.

[0457] Another embodiment provides a method for inhibiting ERK1 / 2 phosphorylation, comprising incubating ERK1 / 2 with an effective amount of a compound of formula I or II or a pharmaceutically acceptable salt thereof.

[0458] Another aspect is the effective amount of the following:

[0459] [ka]

[0460] Provided is a method for inhibiting ERK1 / 2 phosphorylation, which comprises incubating ERK1 / 2 together with a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.

[0461] Also provided is a method for producing a compound of formula I or II or a pharmaceutically acceptable salt thereof.

[0462] Unless otherwise defined herein, the following terms will be understood to have the following general meanings. The terms referred to below will have the following general meanings, unless otherwise indicated, when the term is used alone and when the term is used in combination with other terms. Thus, for example, the definition of "alkyl" applies to "alkyl" as well as "haloalkyl", "heteroalkyl", "arylalkyl", etc.

[0463] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbyl group. Unless otherwise indicated, preference is given to C 1-6 alkyl groups and C 1-4 alkyl groups. The term "C x-y alkyl" refers to an alkyl group having from x to y carbon atoms, where x and y are integers. For example, the term "C 1-6 alkyl" refers to an alkyl group having from 1 to 6 carbon atoms. Examples of C 1-6 alkyl include methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, hexyl, and the like. Unless the context requires otherwise, the term "alkyl" also includes an alkyl group containing one fewer hydrogen atom such that the group is bonded through two positions, i.e., a divalent one.

[0464] As used herein, “to treat” means to affect a subject, tissue, or cell to obtain a desired pharmacological and / or physiological effect, and includes inhibiting a condition, i.e., arresting its onset, or mitigating or improving the effects of a condition, i.e., causing a reversal or regression of its effects. As used herein, “to prevent” means to prevent a condition from developing in a cell or subject at risk of having the condition, but not necessarily that the condition will never develop, or that the subject will never develop the condition. Prevention also includes delaying the onset of a condition in a cell or subject.

[0465] The term "effective dose" refers to the amount of a compound that elicits a biological or medical response in a tissue, system, animal, or human, as sought by researchers, veterinarians, physicians, or other clinicians.

[0466] [Table 1-1]

[0467] [Table 1-2]

[0468] The compounds described herein can be synthesized by methods well known in the art. The compounds referred to herein as BT2 and T6 are commercially available. For example, BT2 can be purchased from Aurora Building Blocks, USA or Life Chemicals HTS Compounds, Canada. T6 is For example, it can be purchased from Sigma-Aldrich, USA.

[0469] The present invention is further described below with reference to the following non-limiting embodiments. [Examples]

[0470] Transcription factors, particularly those encoded by immediate-type genes, integrate signals from the extracellular environment with signal transduction and transcriptional regulation. While it is clear that transcription factors control disease, and despite a strong drug development pipeline (Miyoshi, et al., J Invest Dermatol 131, 108-117 (2011); Cho, EA, et al., The Lancet 381, 1835-1843 (2013)), drugs that directly target such factors are not yet on the market (Mapp et al., Nature Chemical Biology 11, 891-894 (2015)). Basic region leucine zipper (bZIP) factors, including AP-1, respond to various pathological stimuli such as cytokines, growth factors, stress, and viral / bacterial infections, and are involved in genetic processes. It controls the expression of offspring (Hess, et al., Journal of Cell Science 117, 5965-5973 (2004)). AP-1 family members such as FosB / ΔFosB (Chen, G., et al, Front Neurosci 11, 112 (2017)) is mitogen-activated protein kinase (MAPK) (Karin, M. J Biol Chem 270, 16483-16486 (1995)) are under the control of cytokines, growth factors, and various stressors. AP-1 members regulate gene expression in response to various pathological stimuli such as viral and bacterial infections (Hess, et al., Journal of Cell Science 117, 5965-5973 (2004)). Elevated in adult human retinas (Oshitari, T. at el. Current Eye Research 39, 527-531 (2014)) AP-1 is expressed in retinal cells after retinal detachment (Geller, et al., Invest Ophthalmol Vis Sci 42, 1363-1369 (2001)). DNA-binding activity of AP-1 has also been observed in human rheumatoid synovium and is associated with disease activity (Asahara, H., et al., Arthritis Rheum 40, 912-918 (1997)). On the other hand, IL-1β is known as a mediator of bone and cartilage damage in RA (Duff, GW Cytokines and Rheumatoid Arthritis. IN CINICAL APPLICATIONS OF CUTOKINE: Role in Pathogenesis, Diagnosis, and Therapy (eds. Oppenheim, JJ, Rossio, JL & Gearing, AJH) (Oxford University Press, Oxford, 1993). Attempts have been made to bring AP-1 inhibitors to clinical use, but the lack of effective drugs hinders patient access.

[0471] We employed a high-throughput approach to screen approximately 100,000 compounds and identified a novel dibenzoxazepinone, which we named BT2, that had not been previously investigated. They found that BT2 inhibits various proliferation, migratory angiogenesis, and inflammatory processes. BT2 preferentially interacts directly with MEK1 to inhibit ERK activation, and AP-1 protein FosB / ΔFosB, VCAM-1, and VEGF-A 165 It suppresses the inducible expression of BT2. BT2 suppresses the staining of CD31 and tartrate-resistant acid phosphatase (TRAP). Furthermore, BT2 suppresses retinal vascular leakage in rats and rabbits, and inflammation and bone destruction in mice. It suppresses [the following]. BT2 is resistant to boiling and remains biologically stable for up to 16 months. Thus, BT2 is a novel pharmacological inhibitor of angiogenesis, vascular permeability, and inflammation, and is used in patients with nAMD / DR and RA. We will present potential new treatment options.

[0472] material and method High-throughput screening of compound libraries. ~100,000 compounds at the HTS Facility of the Walter & Eliza Hall Institute of Medical Research (WEHI, Bundoora, Vic). From the Lead Discovery Library, AP-1 response e of 384-well microtiter plates. Hits were selected using a commercially available human embryonic kidney (HEK)-293 cell-based assay to drive firefly luciferase in multicopy cells of Fremont (293 / AP-1-luc cell, Panomics, Fremont, CA). In short, the cell-based assay involved 5 x 10⁶ cells in a 384-well plate in pH 7.4 DMEM containing 10% FBS. 3 The procedure involved plating cells. Cells were induced with 10 ng / ml 2-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma, St Louis, MO) in the absence or presence of the test compound, and luciferase activity was measured using a luminometer after ~18 hours. The hit rate for the primary screening was 2.4%. Hit compounds were selected and single-point retesting was performed three times, reconfirming that 931 test compounds were inhibited by more than 50%. Next, interfering compounds from the pan assay were removed. A substructure filter was applied (Baell, JB, et al., J Med Chem 53, 2719-2740 (2010)) and, using the most stringent criteria, 256 hits were selected for further study. This was selected. After dose-response testing, 24 compounds with molecular weight <400 Da were reordered from the supplier. It was written down and tested in a secondary assay.

[0473] Compound synthesis and purification. BT2, Cpd B / X / LK001 and structural analogs are synthesized and purified (>95%) at Advanced Molecular Technologies Pty Ltd (Scoresby, Vic) or as shown below. I obtained it commercially.

[0474] 2-amino-10-methyl-10H-dibenzo[b,f][1,4] in 100ml of dimethylformamide (DMF) Oxazepine-11-one (BT3) (35.0 g, 137 mmol) was mixed with diethyl pyrocarbonate (22.2 ml, 24.43 g, 151 mmol), and the mixture was stirred under a nitrogen atmosphere at 22°C for 1 hour. The solid was filtered and washed with ethyl acetate (SiO4) (100 ml) to obtain a pure first harvest. The combined solvents (DMF and SiO4) were removed, and the mixture was dissolved in dichloromethane (DCM) (200 ml) and then washed twice with water (100 ml). The organic layer was separated, dried over MgSO4, filtered, and the solvent was removed to obtain a yellow solid. This solid was slurryed in SiO4 and filtered to obtain a pure colorless solid. In total, 35.0 g (79% yield) of pure, colorless solid was obtained. 1 7.2 (m, 3H); 7.3 (d, 1H); 7.48 (d, 1H); 7.55 (d, 1H); 7.5 (bs, 1H); 9.7 (s, 1H) ppm.

[0475] Isobutyl (10-ethyl-11-oxo-10,11-dihydrodibenzo[b,f][1,4]oxazepine-2-yl) carbamate (BT2-IC). Under a nitrogen atmosphere, 2-amino-10-methyl-10H-di in 50 ml of DMF. Benzo[b,f][1,4]oxazepine-11-one (1.5g, 5.89mmol, 1.0eq) contains diisobutyldiamine -Boronate (1.55 g, 7.08 mmol, 1.2 eq) was added. The mixture was stirred overnight at 40°C (external). The solvent was removed, the mixture was dissolved in DCM (200 ml), and washed twice with water (150 ml). The organic layer was then dried over MgSO4, filtered over a sintering funnel, and the solvent was removed to obtain 3.0 g of brown as the crude product. A colored solid was obtained. This solid was purified by column chromatography using silica gel and a mixed solvent of hexane:ethyl acetate (starting with 10% ethyl acetate in hexane, then increasing the polarity to 20%) to obtain 1.55 g (74%) of the product as a slightly yellow solid. 1 H-NMR (400 MHz, CDCl3) δ=0.93 (s, 3H); 0.95 (s, 3H); 1.35 (t, 3H); 1.90-2.10 (m, 1H); 3.93 (d, 2H); 4.15 (q, 2H); 6.87 (s, 1H); 7.11-7.21 (m, 3H); 7.23-7.26 (m, 1H); 7.28-7.32 (m, 1H); 7.61 (d, 1H); 7.75 (brs, 1H).

[0476] N-(10-ethyl-11-oxo-10,11-dihydro-dibenzo[b,f][1,4]oxazepine-2-yl)-2-methoxyacetamide (BT2-MeOA). Under a nitrogen atmosphere, carbonyldiimidazole (2.487g, 15.0mmol, 1.3eq) was added to methoxyacetic acid (1.169g, 0.996ml, 12.9mmol, 1.1eq) in 60ml of DMF. The mixture was stirred for 30 minutes. Next, 2-amino-10-methyl-10H-dibenzo[b,f][1,4]oxazepine-11-one (3.0g, 11.8mmol, 1.0eq) was added, and the reaction was stirred overnight at 30°C (external). The solvent was removed, and water (200 ml) and DCM (200 ml) were added to the mixture, and the pH was acidified to 6 with 2 M HCl. The organic phase was washed twice with 50 ml of water. The organic layer was dried over MgSO4, filtered over a sintering funnel, and the solvent was removed to obtain 3.7 g of a sticky yellow solid. The crude product was mixed with 50% acetate in hexane. The product was purified by Chill column chromatography, yielding 3.26 g (85%) as a slightly brown solid. 1 7.8 (d, 1H); 8.03 (s, 1H); 9.9 (s, 1H) ppm.

[0477] (11-Oxo-10-propyl-10,11-dihydro-dibenzo[b,f][1,4]oxazepine-2-yl)- Ethyl carbamate (BT2-Pr). Under a nitrogen atmosphere, 2-amino-10-propyl-10H-dibenzo[b,f][1,4]oxazepine-11-one (2.4g, 9.43 mmol, 1.0eq) in 70 ml of DMF is dissolved in ethyl carbamate. Lupyrocarbonate (2.30 g, 14.16 mmol, 1.5 eq) was added. The mixture was heated at 40°C (externally). The mixture was stirred overnight. The solvent was removed, the mixture was dissolved in DCM (200 ml), and washed twice with water (150 ml). The organic layer was separated, dried over MgSO4, filtered over a sintering funnel, and the solvent was removed to obtain 3.0 g of a brown solid as the crude product. This solid was purified by column chromatography in hexane with 20% ethyl acetate to obtain 2.2 g (72%) of the pure compound as a slightly yellow solid. 1 H-NMR (400 MHz, CDCl3): δ=1.30 (t, 3H); 3.40 (s, 3H); 3.80(t, 2H); 4.20-4.25 (m, 4H); 6.60 (s, 1H); 7.13-7.26 (m, 5H); 7.55-7.60 (m, 2H); 7.68 (s, 1H) ppm.

[0478] [10-(2-methoxy-ethyl)-11-oxo-10,11-dihydro-dibenzo[b,f][1,4]oxazepine-2-yl]-carbamate ethyl ester (BT2-EOMe). Under a nitrogen atmosphere, 2-A in 90 ml of DMF Mino-10-(2-methoxy-ethyl)-10H-dibenzo[b,f][1,4]oxazepine-11-one (2.9g, 10. Diethyl pyrocarbonate (1.82 g, 11.22 mmol, 1.1 eq) was added to 2 mmol, 1.0 eq. The mixture was stirred overnight at 40°C (external). The solvent was removed, the mixture was dissolved in DCM (200 ml), and the organic phase was washed twice with water (150 ml). The organic layer was separated, dried over MgSO4, filtered over a sintering funnel, and the solvent was removed. After removing the residue, 3.6 g of a brown solid was obtained as the crude product. This solid was then treated with 30% HCl in hexane. The compound was purified by column chromatography, yielding 3.5 g (96%) of pure compound as a colorless solid. . 1H-NMR (400 MHz, CDCl3) δ=1.30 (t, 3H); 3.40(s, 3H); 3.80(t, 2H); 4.20-4.25 (m, 4H); 6.60 (s, 1H); 7.13-7.26 (m, 5H); 7.55-7.60 (m, 2H); 7.68 (s, 1H) ppm.

[0479] Ethyl (11-(oxetan-3-ylmethyl)dibenzo[b,f][1,4]oxazepine-2-yl)- Rubamate and (BT2-IMO) and ethyl (10-(oxetan-3-ylmethyl)-11-oxo-10,11-dihydro-dibenzo[b,f][1,4]oxazepine-2-yl)-carbamate (BT2-MO). Nitrogen Under atmospheric conditions, 2-nitro-10H-dibenzo[b,f][1,4]oxazepine-11-one (3) (Figure 6B, Scheme 3) (7.5g, 0.029mol, 1.0eq) was added to 100 ml of DMF and stirred for 5 minutes. Then, NaH(1.4g, oil) was added. 2.34 g (60%), 0.058 mol, 2.0 eq) was added in small amounts (exothermic reaction was observed). The mixture was then heated for 40 minutes. The mixture was stirred at °C (external) for 35 minutes. Next, oxetane-3-ylmethylmethanesulfonate (9.73 g, 0.058 mol, 2.0 eq) was added, and the reaction was stirred at 40 °C (external) for 3 hours. After the reaction was complete, TLC was performed with 20% ethyl acetate in hexane. Once the reaction was complete, the solvent was removed using a Kugel (100 °C, complete vacuum). Removed (or using a rotary evaporator with a powerful pump to maintain the bath temperature below 70°C), and 300 ml of water was added. The solid was stirred with a spatula and allowed to settle in the water. Filtration was performed. The solid was then dried overnight in a vacuum oven at 80°C. The crude product mixture was purified by column chromatography over silica gel and a hexane:ethyl acetate mixture (starting with 20% ethyl acetate in hexane, then increasing the polarity to 40%). The first band was the O-alkylated compound (RF=0.65).

[0480] A yield of 1.0 g (13% yield) of the O-alkylated compound was obtained as a slightly yellowish solid. The melting point was 135-137°C (after correction). 1 H-NMR (400 MHz, D6-DMSO): δ=3.42-3.57 (m, 1H); 4.53 (t, 2H); 4.65 (d, 2H); 4.74-4.79 (app. dd, 2H); 7.19-7.27 (m, 3H); 7.32-7.36 (m, 1H); 7.62 (d, 1H); 8.38 (d, 1H); 8.47 (dd, 1H) ppm.

[0481] Next, O-alkyl. 2-nitro-11-(oxetane-3-ylmethyl)dibenzo[b,f][1,4]ox 50 ml of MeOH was added to sazepine-11(10H)-one (1.5 g, 4.6 mmol, 1.0 eq). The mixture was stirred at 40°C (external) for 15 min to dissolve all solids. The reaction mixture was cooled to 22°C and the flask was flushed with nitrogen. 10% Pd / C (200 mg) was added and the mixture was incubated at 40°C (external) under an H2 atmosphere for 1 hour. The chemical reaction was carried out. After removing the solvent, 1.2 g (yield 98%) of a yellow solid was obtained and used in the next step without further purification (purity ≥ 97%). Melting point: 150~152°C (after correction). 1 H-NMR (400 MHz, D6-DMSO): 4.48-4.60 (m, 4H); 4.72-4.78 (app. dd, 2H); 5.2 (s, 2H), 6.71-6.75 (m, 2H); 6.95-6.99 (m, 1H); 7.07-7.20 (m, 4H) ppm.

[0482] Finally, O-alkyl. 2-amino-10-(oxetane-3-ylmethyl)dibenzo[b,f][1,4] Xazepine-11(10H)-one (1.2 g, 4.05 mmol, 1.0 eq) was mixed with diethyl pyrocarbonate (0.98 g, 6.07 mmol, 1.5 eq). The mixture was stirred overnight at 40°C (external). The solvent was removed. The mixture was dissolved in DCM (150 ml) and washed twice with water (150 ml). Next, the organic layer was treated with MgSO4. The mixture was dried, filtered through a sintering funnel to remove the solvent, and 1.31 g of a slightly yellowish solid was obtained as the crude product. The crude product (1.3 g) was purified by column chromatography on silica gel using a hexane:ethyl acetate mixed solvent (starting with 20% ethyl acetate in hexane, then increasing the polarity to 35%).

[0483] BT2-IMO. First, 0.5 g (34% yield) was obtained as a colorless solid. The melting point is 159-162°C. after correction). 1 H-NMR (400 MHz, CDCl3): δ=1.30 (t, 3 H); 3.47-3.59 (m, 1 H), 4.22 (q, 2H); 4.63-4.68 (m, 4H); 4.88-4.93 (m, 2 H); 6.60 (s, 1 H); 7.07-7.260 (m, 5 H); 7.50–7.59 (m, 2 H) ppm.

[0484] 3.5 g of the N-alkylated compound (48% yield) was obtained as a slightly yellowish solid (RF = 0.45). The melting point was 106-109°C (after correction). 1 H-NMR (400 MHz, D6-DMSO) δ=3.17-3.28 (m, 1 H); 4.29 (t, 2 H); 4.47 (br d, 2H); 4.53-4.58 (app. dd, 2H); 7.26-7.37 (m, 2H); 7.46 (dd, 1H); 7.57-7.64 (m, 2H); 8.40 (dd, 1H); 8.46 (d, 1H) ppm.

[0485] Next, 2-nitro-10-(oxetane-3-ylmethyl)dibenzo[b,f][1,4]oxazepine-11(10H)-one (2.5 g, 6.12 mmol, 1.0 eq) and 50 ml of MeOH were added to 250 ml of RBF set up for N-alkyl hydrogenation. The mixture was stirred at 40°C (external) for 15 min to dissolve all solids. The flask was cooled to 22°C and flushed again with nitrogen. 10% Pd / C (200 mg) The mixture was then stirred under a hydrogen atmosphere at 40°C (external) for 1 hour at atmospheric pressure. The mixture was filtered through Celite to remove the solvent and obtain a pure, colorless solid (1.8 g, 99% yield) for use in the next step without further purification. Melting point: 62-72°C (corrected). 1 6.95 (d, 1H); 7.17-7.29 (m, 3H); 7.49 (dd, 1H) ppm.

[0486] Finally, N-alkyl. 2-amino-10-(oxetane-3-ylmethyl)dibenzo[b,f][1,4] Xazepine-11(10OH)-one (1.6 g, 5.49 mmol, 1.0 eq) and diethyl pyrocarbonate (1.44 g, 8.91 mmol, 1.5 eq) were added to 50 ml of DMF. The mixture was stirred at 40°C (external) for 1 hour. The solvent was removed, the mixture was dissolved in DCM (150 ml), and washed twice with water (150 ml). The organic layer was divided into MgSO4. Dry, filter on a sintered funnel to remove the solvent, and use a column with 50% butyl in hexane. Crude product was obtained by chromatography. BT2-MO (1.91g, 87% yield) was a colorless solid. It was obtained as follows: Melting point: 161-162°C (after correction). 16.65 (s, 1H); 7.13-7.26 (m, 5H); 7.57 (d, 1H); 7.70 (s, 1H) ppm.

[0487] 2-Methoxyethyl[[[4-(4-chlorobenzoyl)phenyl]amino]carbonyl]carbamate (Cpd B / X / LK001). A solution of (4-amino-phenyl)-(4-chloro-phenyl)-methanone (49.1g, 210 mmol) in DCM (150 ml) was cooled to ~0°C (internal temperature) in an ice / NaCl bath. DCM (150 ml 2-methoxyethyl carbonisocyanatidate (40 g, 276 mmol) was added dropwise through a funnel while maintaining the internal temperature below 5°C. The ice bath was removed, and the solution was stirred under nitrogen at 22°C for 1 hour. The solution was filtered, and the solid was collected in methanol. Washed with [a solution] to obtain a pure, slightly yellowish yield of the desired product. The filtrate was concentrated (mixture of DCM and MeOH). Further harvests were obtained by filtering and washing with methanol. Combining the fractions, 49 g (62%) of the desired product was obtained. ¹H-NMR (400 MHz, D6-DMSO) δ = 10.52 (s, ¹H, NH), 10.10 (s, ¹H, NH), 7.75-7.68 (m, ⁶H), 7.62 (d, ²H), 4.80 (t, ²H), 3.58 (t, ²H), 3.28 (s, ³H) ppm.

[0488] 2-Methoxyethyl[[[4-(4-chlorophenyl)(hydroxyimino)methyl)phenyl]amino]carbonyl]carbamate (T4). 2-Methoxyethyl[[[4-(4-chlorobenzoyl)phenyl [Amino]carbonyl]carbamate (20.7g, 55mmol), hydroxylamine hydrochloride (11.4g, 165mmol), and sodium acetate (13.5g, 165mmol) were stirred under reflux under a nitrogen atmosphere for 4 hours. The reaction mixture was filtered while hot to remove salts. The filtrate was cooled and the product was filtered. The filtrate was concentrated to two-thirds, cooled to 22°C and filtered to obtain a second harvest. This solid was vacuum-dried at 60°C to obtain the desired product (15.8 g, 73%) as a mixture of E and Z isomers (~1:1). 1 H-NMR (400 MHz, D6-DMSO) δ=11.43 (s, 0.46H, OH), 11.32 (s, 0.62H, OH), 10.42 (bs, 1H, NH), 9.90 (s, 0.49H, NH), 9.88 (s, 0.63H, NH), 7.59 (d, 0.94H), 7.52 (t, 2.44H), 7.42 (q, 1.92H), 7.35-7.25 (m, 3.44H), 4.28 (m, 2H), 3.58 (m, 2H), 3.27 (s, 1.28H), 3.28 (s, 1.72H) ppm.

[0489] Ethyl (10-ethyl(2',2',2'-d3)-11-oxo-10,11-dihydrodibenzo[b,f][1,4]ox Sazepine-2-yl)carbamate (BT2-deut). First, 2-nitro-10H-dibenzo[b,f][1,4]oxazepine-11-one (1 g, 3.9 mmol, 1 eq) was added to 10 ml of DMF and stirred under nitrogen for 5 minutes. Next, NaH (187 mg, 0.32 g in oil, 7.8 mmol, 2 eq) was added in small amounts. The mixture was then removed. The mixture was stirred at 40°C for 35 minutes. Next, ethyliodo-2,2,2-d3 (1.24 g, 0.62 mL, 7.8 mmol, 2 eq) was added, and the reaction mixture was stirred at 40°C for 3 hours. The solvent was evaporated and removed, and the mixture was rinsed three times with water. When rationed, it transforms into a thick paste, which is then eluted using 15% phenylethylamine in hexane. Chromatography was performed to obtain 10-(ethyl-2,2,2-d3)-2-nitrodibenzo[b,f][1,4]oxazepine-11(10H)-one as a yellow solid (0.42 g, 38%). The melting point is 142.3°C to 145.6°C (corrected). 1 ¹H-NMR (400 MHz, D6-DMSO): δ = 4.12 (app s, 2H), 7.25 to 7.38 (m, 2H), 7.45 (dd, 1H), 7.60 (d and dd, 2H), 8.41 (dd, 1H), and 8.45 (d, 1H) ppm.

[0490] Next, 10-(ethyl-2,2,2-d3)-2-nitrodibenzo[b,f][1,4]oxazepine-11(10H)-one (0.4 g, 1.41 mmol, 1 eq) and SnCl2 (0.8 g, 4.2 mmol, 3 eq) were dissolved in 10 ml of EtOH. The mixture was stirred under reflux for 2 hours. The solvent was removed, and the mixture was dissolved in SiO2 (100 ml). The solution was dissolved in 1N NaOH aq. (50 ml). The organic phase was separated, washed with water (2 × 50 ml), dried over MgSO4, filtered, and the solvent was evaporated. The product, 2-amino-10-(ethyl-2,2,2-d3)dibenzo[b,f][1,4]oxazepine-11(10H)-one, was obtained as a pale beige solid (287 mg, 80%) by chromatography using elution with 50% ethyl hexane. The melting point was 165.5°C to 167.0°C (corrected). 1 H-NMR (400 MHz, D6-DMSO): δ = 4.0 (bq, 2H), 5.15 (s, 2H), 6.65 (dd, 1H), 6.84 (d, 1H), 6.95 (d, 1H), 7.15 to 7.30 (m, 3H), and 7.45 (dd, 1H) ppm.

[0491] Finally, 2-amino-10-(ethyl-2,2,2-d3)dibenzo[b,f][1,4]oxazepine-11(10H)-one (0.287 g, 1.2 mmol, 1 eq) in DMF (3 ml) was mixed with diethyl pyrocarbonate (0.183 ml, 0.201 g, 1.24 mmol, 1.1 eq). The mixture was stirred under nitrogen at an external temperature of 25°C for 1 hour. The DMF was removed from the reaction mixture, and the remaining solid was triturated three times with SiO2 to obtain BT2-deut. It was obtained as a colorless solid (260 mg, 66%). The melting point is 184.3°C to 185.7°C (corrected). 1 ¹H-NMR (400 MHz, D6-DMSO): δ = 1.22 (t, 3H), 4.00 to 4.15 (q and br q, 4H), 7.2-7.3 (m, 3H), 7.35 (dd, 1H), 7.50 (dd, 1H), 7.58 (dd, 1H), 7.80 (d, 1H), and 9.75 (s, 1H) ppm.

[0492] Flubendazole (T6), 2-amino-10-ethyldibenzo[b,f][1,4]oxazepine-11(10H)-one (BT3), and (4-aminophenyl)(4-fluorophenyl)methanone (T7) are commercially available from AK Scientific Inc.

[0493] Cell culture. HMEC-1 was obtained from ATCC (Rockville, MD), and treated with 10% FBS, hydrocortisone (1 μg / ml), epidermal growth factor (10 ng / ml), L-glutamine (2 mM), and penicillin / sodium. Cells were grown in MCDB131 medium (Invitrogen, MD) supplemented with leptomycin, pH 7.4. Bovine aortic endothelial cells (BAECs) were obtained as primary cells from Cell Applications (San Diego, CA) and grown in DMEM supplemented with 10% FBS and antibiotics, pH 7.4. BAECs were used in experiments between phases 4 and 6. After detachment with 0.05% trypsin / 5 mM EDTA, the cells were periodically passaged and treated with 5% CO2. The humidified atmosphere was maintained at 37°C.

[0494] Western blot analysis using cell extracts treated with serum. HMEC-1 (80-90% concentration) Fluent cells were arrested for 20 hours in serum-free MCDB131 medium without EGF or hydrocortisone. Cells were treated with 30 μM of the compound in serum-free MCDB131 medium for 4 hours, then the medium was changed to complete medium containing 30 μM of the compound (containing 10% FBS with EGF and hydrocortisone) and treated for 1 hour. As described above, total protein was collected in a radioactive immunoprecipitation (RIPA) lysis buffer containing a protease inhibitor (Li, Y., et al., Int J Cardiol 220, 185-191 (2016)). The proteins were separated on a 4-20% (w / v) sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel (Bio-Rad Mini-PROTEAN TGX) and transferred to an Immobilon-P PVDF membrane (Millipore, USA). The membrane was blocked with 5% skim milk and incubated overnight at 4°C with rabbit monoclonal FosB (cat. 2251, 1:1000, Cell Signaling, USA) or rabbit monoclonal c-Fos antibody (cat. 2250, 1:1000, Cell Signaling, USA), or with mouse monoclonal β-actin antibody. The animal (cat. A5316, 1:30000, Sigma-Aldrich) was incubated at 22°C for 15 minutes, and then the horseradish was incubated. The cells were incubated for 1 hour with peroxidase-conjugated goat anti-rabbit antibody (cat. P0448, 1:1000, DAKO Cytomation, Denmark) or goat anti-mouse antibody (cat. P0447, 1:1000, DAKO Cytomation, Denmark). Chemiluminescence was measured using the Western Lightning Chemiluminescence system (PerkinElmer, USA) and ImageQuant. TM Detection was performed using a LAS 4000 biomolecular imager (GE Healthcare Life Sciences, USA). The images were created using the LAS 4000 with high sensitivity / resolution settings and automatic exposure. The band intensity of the images was quantified using NIH ImageJ.

[0495] Western blot analysis using cell extracts treated with IL-1β. HMEC-1 (80-90% confluent) was treated with serum-free MCDB131 medium (Invitrogen, MD) without growth factors for 48 hours. The cells were treated with a 30 μM compound in serum-free medium for 4 hours, and unless otherwise specified, they were then subjected to a final rinse with 20 ng / ml IL-1β (Sigma, cat. SRE3083) in serum-free medium containing the same concentration of the compound. The proteins were incubated for 4 hours. All proteins were analyzed using RIPA buffer containing a protease inhibitor. As described above, the samples were collected. The protein content was 4-20% (w / v) on an SDS-polyacrylamide gradient. The cells were separated on gel and transferred to an Immobilon-P PVDF membrane. The membrane was blocked with 5% skim milk, and the samples were analyzed using rabbit monoclonal FosB (cat. 2251S, 1:1000, Cell Signaling, USA), rabbit monoclonal VCAM-1 (cat. 13662S, 1:1000, Cell Signaling, USA), and rabbit monoclonal p44 / 42. MAPK (cat. 4695S, 1:1000, Cell Signaling, USA), rabbit polyclonal p38 MAPK (cat. 9212S, 1:1000, Cell Signaling, USA), rabbit polyclonal SAPK / JNK (cat. 9252S, 1:1000, Cell Signaling, USA), rabbit monoclonal phospho-SAPK / JNK (cat. 4671S, 1:1000, Cell Signaling, USA), rabbit monoclonal phospho-p38 MAPK (cat. 4511S, 1:1000, Cell Signaling, USA), or mouse monoclonal phospho-p44 / 42 MAPK antibody (cat. 9106S, 1:2000, Cell Signaling, USA) overnight at 4°C, or mouse monoclonal β-actin antibody (cat. A5316, 1:10000, (Sigma-Aldrich) at 22°C for 1 hour The membranes were incubated. Next, the membranes were incubated for 1 hour with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (cat. P0448, 1:1000, DAKO Cytomation, Denmark) or goat anti-mouse antibody (cat. P0447, 1:1000, DAKO Cytomation, Denmark). Chemiluminescence was measured using the Western Lightning Chemiluminescence system and ImageQuant. TM Detection was performed using a LAS 4000 biomolecular imager. Band intensity was quantified using NIH ImageJ for images created with the same settings on the LAS 4000.

[0496] iRNA experiment. HMEC-1 (70-80% confluent) containing hydrocortisone or EGF. Arrested for 24 hours in serum-free MCDB131 medium, and mixed non-targeting siRNA (cat. D-001810-10-50, Dharmacon, USA), FosB siRNA (cat. L-010086-01-0020, Dharmacon, USA), or VCAM-1 siRNA (cat. L-013351-00-0020, Dharmacon, USA) with Dharma FECT1 transfection reagent (cat. T-2001-03, Dharmacon, USA) for 24 hours. siRNA experiments (using 0.6 μM FosB and 0.6 μM VCAM-1) were performed using the same concentration of non-targeting loading control. The procedure was performed using siRNA in a parallel manner. Cells were further treated with 20 ng / ml of IL-1β in serum-free complete MCDB131 medium. The cells were stimulated for 2 or 4 hours. Total protein was collected using RIPA buffer along with a protease inhibitor, separated on a 4-20% (w / v) SDS-polyacrylamide gradient gel, and then separated onto an Immobilon-P PVDF membrane. The cells were transferred to a separate container. The membrane was blocked with 5% skim milk and incubated overnight at 4°C with rabbit monoclonal FosB (cat. 2251S, 1:1000, Cell Signaling, USA) or rabbit monoclonal VCAM-1 (cat. 13662S, 1:1000, Cell Signaling, USA) antibodies, or mouse monoclonal antibodies. Incubate β-actin (cat. A5316, 1:10000, Sigma-Aldrich) with antibody at 22°C for 1 hour. The membranes were incubated for 1 hour with horseradish peroxidase-conjugated secondary goat anti-rabbit (cat. P0448, 1:1000, DAKO Cytomation, Denmark) or goat anti-mouse (cat. P0447, 1:1000, DAKO Cytomation, Denmark) Ig. Chemiluminescence was measured using the Western Lightning Chemiluminescence system and ImageQuant. TMUsing the LAS 4000 biomolecular imager I took it out.

[0497] Plasmid overexpression. HMEC-1 was seeded in a 6-well plate and expressed at 70-80% confluence. Then, the cells were deprived of serum (or EGF and hydrocortisone) overnight. Following the protocol, Fugene 6 (Promega) was used to transfect the plasmid with 6 μg of the indicated plasmid (in pcDNA3.1+ / C-(K)DYK) (GenScript, USA). Total protein lysates were collected 18, 24, 48, and 72 hours after plasmid transfection in RIPA buffer containing a protease inhibitor. The proteins were 4–20% (w / v) SDS-polyacrylamide. The samples were separated using a gradient gel and transferred to an Immobilon-P PVDF membrane. The membrane was then blocked with 5% skim milk. Rabbit monoclonal p44 / 42 MAPK (cat. 4695S, 1:1000, Cell Signaling), mouse Monoclonal phospho-p44 / 42 MAPK antibody (cat. 9106S, 1:2000, Cell Signaling), rabbit monoclonal FosB (cat. 2251S, 1:1000, Cell Signaling, USA), rabbit monoclonal The cells were incubated overnight at 4°C with either NalVCAM-1 (cat. 13662S, 1:1000, Cell Signaling) or mouse monoclonal α-tubulin (cat. T5168, 1:40000, Sigma). Next, the membrane... The samples were incubated for 1 hour with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (cat. P0448, 1:1000, DAKO Cytomation, Denmark) or goat anti-mouse antibody (cat. P0447, 1:1000, DAKO Cytomation, Denmark). Chemiluminescence was observed. system and Image Quantity TM Detection was performed using the LAS 4000 biomolecular imager.

[0498] RNA-seq. HMEC-1 cells were seeded in nine 100mm petri dishes using complete MCDB131 medium containing 10% FBS. Cells were grown at 70-80% confluence and then arrested for 44 hours in serum-free MCDB131 medium without hydrocortisone or EGF. The cells were pre-treated with 30 μM BT2 for 4 hours in the same medium, followed by further stimulation with 20 ng / mL IL-1β for 4 hours. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Amtsgericht Dusseldorf). In short, the cells were washed twice with pre-cooled 1x PBS, and then lysed with TRIzol (Thermo Fisher Sci, Waltham, MA). Chloroform was then added. The mixture was centrifuged at 13,000 rpm for 15 minutes at 4°C. The upper aqueous layer containing all RNA was then transferred to a new microcentrifuge. Transferred to a tube, isopropanol was added, and loaded onto the RNeasy column. Buffer RW1 The column was washed with RPE. Total RNA was eluted from the column using RNA-free water. The pull was submitted to the Ramaciotti Centre for Genomics (UNSW, Australia) and sequencing was performed using TruSeq Stranded mRNA-seq preparation and One NextSeq 500 1X75bp High Output flowcell (data output up to 400M reads). Sample quality control was set to a value 80% higher than Q30 at 1x75bp.

[0499] RNA-seq reads were first evaluated for quality using the tool FastQC (v0.11.8) (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). The Salmon tool was used to quantify the amount of transcript from the RNA-seq reads (Patro, R., et al., Nat Methods 14, 417-419 (2017)). Then, the R package DESeq2 (Love, MI, et al., Genome Biol 15, 550 (2014)), which incorporates a method for differential count analysis, was used to analyze the difference between specific comparison targets. The expressed genes were identified. Using the heatmap.2 function of the R package gplots v3.0.1.1, related A heatmap using counts per million (cpm) values ​​for sets of genes related to consciousness. The following was generated using the web-based online bioinformatics resource Database for annotation, visualization and integrated discovery (DAVID) (Jiao, X., et al., Bioinformatics 28, 1805-1806 (2012)): differential expression of specific comparison genes. Identify gene ontologies, such as bioprocesses (BPs), that have been found to be concentrated in the list.

[0500] Flow cytometry. HMEC-1 (80-90% confluent) was arrested for 40 hours in serum-free MCDB131 medium without EGF or hydrocortisone, and treated with 30 μM BT2 or BT3 for 4 hours. These cells were incubated in serum-free medium and treated with 20 ng / ml IL-1 along with the same concentration of BT2 or BT3. The cells were further exposed to β for 4 hours. After washing the cells with PBS, they were detached using acutase (Stem Cell Technologies, cat.07920). The cells were centrifuged at 300g for 5 minutes and 5x10⁶ cells containing BT2 or BT3 were extracted. 6The cells were resuspended at a concentration of cells / ml. The cells were incubated with BV421-conjugated mouse anti-human CD106 (VCAM-1) (BD, cat. 744309) or BV421-conjugated mouse IgG1 (BD, cat. 562438) at 22°C for 45 minutes. The cells were washed with staining buffer, and the pellet was resuspended in 0.5 ml of 1% paraformaldehyde before flow cytometry using BD FACSCanto II.

[0501] VCAM-1 + Cells and VCAM-1 - Cells were gated by flow cytometry with or without primary VCAM-1 antibody (nonspecific staining). Representative gating from the latter (i.e., negative control) is shown as Figure 10, showing minimal nonspecific staining. The gating strategy was based on fluorescence excitation from both 488 nm and 405 nm lasers, with the 488 nm off using emission filters 670 LP and the 405 nm off using 450 / 50. Autofluorescence Cells possessing or negative for VCAM-1 (blue population) exhibited the same proportion of fluorescence in both channels, while VCAM-1-positive cells (red) emitted fluorescence through a 450 / 50 filter.

[0502] SPR. SPR was performed on a Biacore T200. Active function of Xantec NIHMC Ni sensor chip. The flow cell and reference flow cell were conditioned with 0.5 M NaEDTA and 5 mM NiCl2 in immobilization buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). Recombinant human His-MEK1 and His-MEK2 (500 nM, ThermoFisher Scientific, cat. PV3303 and PV3615, respectively) were added to separate active flow cells at a rate of 10 μl min. -1The sample was injected for 15 minutes. All immobilization was performed at 25°C. After immobilization, the temperature was lowered to 15°C and the buffer was changed to 20 mM HEPES, 150 mM NaCl, 5% DMSO pH 7.4. Samples of PD98059 (2.5-30 μM in the operating buffer) and BT2 (1.25-15 μM) were used. 30 μl min on immobilized MEK1 and MEK2 -1 Inject at the specified flow rate and solvent supplementation using the DMSO standard curve. The positive result was applied to the data. The data was analyzed with Biacore T200 Evaluation software. Before SPR, 1 The solubility limits of the compounds were measured using 1D NMR.

[0503] Endothelial proliferation assay using the xCELLigence system. HMEC-1 proliferation was evaluated using the xCELLigence system (Roche, Castle Hill). In short, HMEC-1 (5x10) 3 Cells / We The cells were seeded in a 96-well E plate and inserted into an xCELLigence RTCA station (Roche). After removing serum for 24 hours in MCDB131 medium containing 10 ng / ml EGF (Sigma-Aldrich) and 1 μg / ml hydrocortisone (Sigma-Aldrich), 5% FBS, 10 ng / ml EGF (Sigma-Aldrich), and 1 μg / ml hydrocortisone were added. Cells were treated with the compound (0.2-1 μM) in a medium containing locortisone (Sigma-Aldrich). Cell proliferation was observed. The xCELLigence system automatically monitored the cells every 15 minutes. The Cell Index (CI) is a quantitative indicator of cell proliferation in each well. In this system, CI is a unitless parameter that reports the impedance of the electron flow caused by the attached cells.

[0504] Endothelial proliferation assay using the Countess system. HMEC-1 proliferation was evaluated using the Countess II Automated Cell Counter (ThermoFisher Scientific). In short, HMEC-1 (3x10 5 Cells were seeded (per well) into 12-well plates. After serum removal for 24 hours in MCDB131 medium containing 10 ng / ml EGF and 1 μg / ml hydrocortisone, the cells were treated with the compound (0.1–0.6 μM) in medium containing 5% FBS, 10 ng / ml EGF and 1 μg / ml hydrocortisone. After 24 hours, the cells were trypsinized, resuspended in complete medium, and 10 μl aliquots were mixed with an equal volume of 4% trypan blue. In addition, the total cell count and the percentage of trypan blue-excluded cells within the total cell count were measured using Countess.

[0505] Endothelial dual-chamber migration assay. BAEC (6x10) suspended in DMEM with 10% FBS. 3 Cells (per well) were seeded into the upper chamber of a 24-well plate fitted with a Millicell cell culture insert (cat. PI8P01250, Millipore). After 48 hours, the culture medium was replaced with 0.01% FBS-containing DMEM and incubated for another 48 hours. The compound prepared with 0.01% FBS-containing DMEM was added to the upper chamber. VEGF-A in 10% FBS-containing medium 165 (50 ng / ml, Sigma, cat. V7259) was added to the lower chamber. After 24 hours, the culture medium was removed from the upper chamber, and non-migrating cells and excess fluid were removed with a cotton swab. The insert was immersed in 70% ethanol for 10 minutes to fix the cells, and the membrane was dried for 10-15 minutes. The filter was cut and placed on the slide. The mounting medium (Fluoroshield with DAPI) was then added. TM (Sigma, cat. 6057) was added, and the specimen was visualized using an EVOS FL microscope.

[0506] Endothelial repair after in vitro injury. HMEC-1 (90-100% confluent) in 6-well plates was washed with PBS and treated with a 0.6 μM compound in MCDB131 containing 5% FBS. A cell monolayer was scraped with a sterile, sharp toothpick, and the wells were imaged with a 4x objective lens at 0h and 48h. Cell regrowth in the degenerated area was measured using Image-Pro Plus (Cybernetics, USA).

[0507] Analysis of BT2 formulations using RRLC-MS / MS. Using an Agilent 1200 Triple Quad G6410B, Iris Pharma used high-performance liquid chromatography / tandem mass spectrometry (RRLC-MS / MS), developed under GLP (Good Laboratory Practice), to measure the BT2 content of heat-treated or unheat-treated BT2 preparations at room temperature for one week (T1 week) or six weeks (T6 week). Preparations used were either heat-treated (H) (tubes placed in a 100°C water bath for 10 min) or unheat-treated (BT2 sonicated in 0.5% Tween 80 and 0.01% DMSO-containing saline). Standard curves were created for eight concentrations between the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ). Evaluations were performed on three preparations of the same dilution. Chromatograms were integrated using MassHunter software. BT2 content analysis (T1 week) For T1 and week 6, the mean, SD, CV (%), and bias (%) were calculated as follows. For T1, the bias (%) for each formulation, including the test sample, was calculated relative to the theoretical concentration (i.e., supplied weighing / formulation):

[0508]

number

[0509] Standard curves are in Excel (R) The 2011 version was used for calibration. For each run, the bias between the standard curve and the QC inverse concentration was determined, and the calibration standard inverse concentration was set to within ±15% of the theoretical value (however, (LLOQ must be within ±20%). At least 75% of the calibration standards, with a minimum of 6 standards, must meet this standard. It is necessary to have a coefficient of determination (r 2 The value was set to ≥0.98.

[0510] Analysis of BT2 formulations using liquid chromatography-mass spectrometry (LC / MS). MSO (100 μl) and sample (~50 μl) were combined with formic acid (1 μl). These solutions (10 μl) were further diluted with H2O:CH3CN (1:1) 0.1% formic acid (90 μl), and LC / MS analysis was performed. Samples were separated by UPLC using an HPG-3400RS UPLC pump, autosampler, and column compartment system (Thermo Scientific, CA). Sample (0.1 μl) was loaded onto a Hypersil Gold aQ column (2.1 x 50 mm) (Thermo Scientific) containing 1.9 μl of culture medium. The compounds were eluted using a linear gradient of H2O:CH3CN, with A containing H2O (0.1% formic acid) and B containing H2O:CH3CN (1:4, 0.1% formic acid). The gradient was applied over 30 minutes or more with T=0 min 2% B, T=20 min 75% B, T=23 min 95% B, T=25 min 95% B, T=25.2 min 2% B, and T=30 min 2% 200 μl / min. The column oven was heated to 45°C. Positive ions were generated by electrospray, and the QExactive Plus mass spectrometer (Thermo Fisher, Bremen, Germany) was operated in data-dependent acquisition mode (DDA). A heated electrospray source (HESI) was used, with a high voltage of 3.8 kV. The system was set to apply the gas, vaporizer temperature 250°C, sheath gas 20, auxiliary gas 5, and heated capillary temperature T=290°C. Confirmed. Enable Lockmass (m / z 391.28429) and perform a survey scan from m / z 140 to 800. This was done (resolution 70,000 at m / z 200, AGC target value 3x10). 6 Ion, maximum IT is 250 msec). 2 Combining two microscans, the richest ion collection is up to 10 (minimum AGC target value 5x10). 4 The maximum IT (110 msec) is sequentially separated (width m / z 1.8), and 2x10 is processed using HCD (NCE = 20, 30, 50). 5 The ions (with a resolution of 17,500 at m / z 200) were fragmented to the target value. The M / Z ratio selected for MS / MS was dynamically excluded for 12 seconds, and exclusion of charge states was not effective. The LC / MS chromatograms were processed with Xcalibur Qual Browser.

[0511] Endothelial network formation assay. HMEC-1 (4x10) in MCDB131 containing 1% FBS. 4 Cells (per well) and compound (1 or 3 μM) or curcumin (1-40 μM) and 50 ng / ml FGF-2 are used for growth. Factor reduction reconstitution of basement membrane matrix ((Matrigel, cat. 354230, Corning, NY) 100 μl for 4 The sample was added to 96-well plates coated overnight at °C. Network formation was then observed over several hours and imaged using an Olympus CKX41 microscope with 4x or 10x objective lenses.

[0512] Matrigel plug assay. VEGF-A 165 (100 ng / ml), heparin (10 U), BT2 or BT3 (2.5 mg / mouse) or its vehicle (saline solution containing 0.01% DMSO and 0.5% Tween 80) Matrigel (500 μl) containing was subcutaneously injected into the left flank of 8-week-old C57BL / 6 male mice. Seven days later, The mortar was sacrificed by CO2 asphyxiation, and the plug was carefully removed. For immunohistochemical evaluation... Formalin-fixed, paraffin-embedded sections were prepared from Matrigel plugs. Epitope recovery by heat was applied to all deparaffinized sections (4 μm Superfrost slides) at 110°C for 5 minutes in citrate buffer, pH 6. Immunostaining of all groups was performed simultaneously with the given antibodies. The development time was the same for both animals. The animal experiments were approved by the Animal Welfare and Ethics Committee of the University of New South Wales.

[0513] For CD31 staining, sections were blocked for 10 minutes with an endogenous enzyme blocking agent (cat. S2003, DAKO). The sample was then blocked with 2% skim milk for 20 minutes. The slide was then taken with primary antibody rabbit polyp. Ronal CD31 antibody, diluted 1:25 (cat. ab28364, Abcam), was incubated at room temperature for 1 hour. The slides were washed with buffer, incubated with a secondary antibody (goat anti-rabbit (cat. P0448, DAKO)) for 30 minutes, washed with buffer, incubated with diaminobenzidine (DAB) chromagen (cat. K3468, DAKO) for 5 minutes, and counterstained with hematoxylin and Scott Blue. The slides were dehydrated with 100% ethanol and xylene and covered with coverslips.

[0514] For FosB or VCAM-1 staining, sections were blocked with an endogenous enzyme blocking agent (cat. S2003, DAKO) for 10 minutes, followed by blocking with 2% skim milk for 20 minutes. Slides were incubated with primary rabbit monoclonal FosB (cat. 2251, Cell Signaling, USA) or rabbit polyclonal VCAM-1 (cat. sc-8304, Santa Cruz) at room temperature for 1 hour, then incubated with the probe component of MACH3 Rabbit AP-Polymer Detection (Biocare Medical, M3R533 G, H, L) for 10 minutes. After washing with buffer, slides were further incubated with the polymer component of MACH3 Rabbit AP-Polymer Detection (Biocare Medical, M3R533 G, H, L). The samples were incubated with red dye (Warp Red™ Chromogen Kit) for 7 minutes and counterstained with hematoxylin and Scott Blue. The slides were dried on filter paper, dehydrated with xylene, and then covered with coverslips.

[0515] The slides were scanned using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt Waverley, Vic Australia), and images were acquired using ImageScope software (Leica Biosystems). Positive staining within the plugs was performed using 10x (CD31), 20x (VCAM-1), and The images were evaluated and integrated using Image-Pro Plus software (Cybernetics, Bethesda, MD) in 5 to 12 randomly selected fields of view of each plug, captured under a 40x (FosB) objective lens. The optical density (IOD, the product of calibration intensity (optical density) and area, i.e., IOD = intensity (average) × area) was expressed (Media Cybernetics) (Liu, H., et al., Sci Rep 6, 21319 (2016)). Furthermore, positive immunohistochemical staining was expressed as a percentage of the plug area (Kim, JY, et al., Biomolecules 10, pii: E11 (2019)).

[0516] A model of increased retinal vascular permeability in rabbits. Male HY79b pigmented rabbits (8-12 weeks old) were used in Rompun (R) (Xylazine) / Imalgene (R) The animals were anesthetized by intramuscular injection of ketamine. Five days prior to rhVEGF-A165 induction, the compound (a saline vehicle containing 600 μg BT2, BT3, or 0.5% Tween 80 and 10% DMSO in a 100 μl vehicle) was injected into the right eye. The injection was performed under a surgical microscope using a 250 μl Hamilton syringe (equipped with a 30G needle) on anesthetized animals. Retinal blood vessels The permeability is 500ng rhVEGF-A 165 50 μl IVT injection (diluted with PBS containing carrier protein) Induction was achieved by administering the procedure once to the right eye. 47 hours (±3h) after induction, fluorescein sodium (10% in physiological saline, 50 mg / kg) was injected into the auricular vein. One hour after fluorescein injection, the animals were anesthetized, and one drop of 0.5% tropicamide was instilled to dilate the pupils. Ocular fluorescence was observed in both eyes. The ophthalmic fluorescence was measured using an FM-2 Fluorotron Master ophthalmic fluorite meter. The animals were euthanized by injection of pentobarbital. This study was conducted by the Animal Ethics Committee of Iris Pharma (La Gaude, France). This was carried out with the approval of the Animal Welfare and Ethics Committee of the University of New South Wales.

[0517] Rat choroidal laser injury model. Male Brown Norway pigmented rats (8-14 weeks old) were used in Rompun (R) (Xylazine) / Imalgene (R)Anesthesia was administered by intramuscular injection of ketamine. One drop of 0.5% tropicamide was instilled into the eye before laser irradiation to dilate the pupils. A 170mW 532nm laser beam (Viridis laser, Quantel, France) was irradiated for 0.1s for 0 seconds onto a 75μm spot around the optic nerve between the major retinal vessel branches, through a slit lamp and contact lens, six times in both eyes on day 0. A burn was created. The rupture of Bruch's membrane was confirmed by the generation of bubbles during laser irradiation. Compounds in the vehicle (physiological saline containing 0.01% DMSO and 0.5% Tween 80, sonicated) 2-5 μl of the solution was administered under a surgical microscope using a 30G needle attached to a 100 μl Hamilton syringe. IVT injections were administered on day 1 and day 7. Kenacort was administered via IVT to each eye on day 0. Alternatively, aflibercept / Eylea in a vehicle (physiological saline) was administered via IVT six times (days 0, 3, 7, 10, 14, and 17). Fluorescein angiography was performed using the Heidelberg retinal angiography system. The procedure was carried out as follows: After anesthesia, 10% fluorescein sodium (250 μl / 100g body weight) was administered by subcutaneous injection. Fluorescence of the eyeball was recorded 10 minutes after dye injection. Angiography on days 14 and 21 showed that the test group was different. The fluorescence leakage was evaluated by two inspectors, and the fluorescence intensity was assessed as follows: Score: 0: No leakage, 1: Slightly stained, 2: Moderately stained, 3: Strongly stained. This study was conducted by the Animal Ethics Committee of Iris Pharma (La Gaude, France) and the University of New South Wales. This was carried out with the approval of the university's Animal Welfare and Ethics Committee.

[0518] Immunohistochemical staining of rat retina. Rabbit monoclonal anti-CD31 (cat. ab182981), Heron monoclonal anti-VCAM-1 (cat. ab134047) and rabbit polyclonal anti-VEGF-A (cat. ab46154) was obtained from Abcam. Rabbit monoclonal phospho-p44 / 42 MAPK (pERK1 / 2, Thr 202 / Tyr 204 Rat eyeballs (cat. 4370) and rabbit monoclonal FosB (cat. 2251) were obtained from Cell Signaling. Rat eyeballs were excised, fixed in formalin, and paraffin-embedded sections were prepared. Epitope recovery by heat was performed on all deparaffinized sections (4 μm Superfrost slides). ) Add citrate buffer, pH 6 (VEGF-A, pERK, VCAM-1) or EDTA buffer, pH 9 (CD31) and mix until 110°C. The procedure was performed at ℃ for 5 minutes. The sections were blocked with a dual endogenous enzyme blocking agent (cat. S2003, DAKO) for 10 minutes, and then blocked with 2% skim milk for 20 minutes. They were incubated for 10 minutes using MACH3 Rabbit AP-Polymer Detection (Biocare Medical, cat. M3R533 G, H, L). After washing with a buffer solution, the slides were incubated for a further 10 minutes with the polymer component of MACH3 Rabbit AP-Polymer Detection (Biocare Medical, M3R533 G, H, L). The slides were incubated for 7 minutes with a red chromogen (Warp Red™ Chromogen Kit) and counterstained with hematoxylin and Scott Blue. The slides were dried on filter paper, dehydrated with xylene, and then covered with coverslips. Immunostaining with the specified antibodies was performed simultaneously for all groups. The immunostained slides were scanned using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt Waverley, Vic, Scanned in Australia, and the images were captured using ImageScope software (Leica Biosystems). It clicked. CD31, VEGF-A 165The IODs for positive staining (red dye) of pERK, FosB, and VCAM-1 were evaluated using Image-Pro Plus software (Cybernetics, Bethesda, MD). IPL and INL IOD was quantified for CD31, VEGF-A165 for OS from OPL, pERK for ONL from INL, FosB for OS from GCL, and VCAM-1 for OLM using Image-Pro Plus. In addition, positive immunoassay for retinal tissue area was performed. The staining area was expressed as a percentage (Kim, JY, et al., Biomolecules 10, pii E11 (2019)). For image selection for quantification, 2-4 sections per eye were selected for the vehicle group and BT2 group. The wounds were examined and photographed with a 20x objective lens. In the untreated group without wounds, 1-3 sections per eye were photographed with a 20x objective lens. Staining was quantified in each group with n=3-6. When evaluating gradient staining of VEGF-A165 on wounds, immunohistochemistry was evaluated in 10 consecutive 100 μm boxes starting 150 μm from the wound center (double-headed arrow), and the IOD of each box was quantified using Image-Pro Plus.

[0519] Endothelial cell-monocyte cell adhesion assay. HMEC (80-90% confluent) in a 96-well plate. After keeping the sample serum-free for 24 hours, treat it with the compound at the indicated concentration for 1 hour, then treat it with IL-1β (20 ng / ml) and ink for 4 hours. It was devised. On the other hand, THP-1 was 5 μM calcein (5 x 10 6 Cells were labeled with THP-1 (2.5 x 10 ml, BD Bioscience) at 37°C for 30 minutes, then washed three times with PBS. 5 Cells (per well) were added for 30 minutes, and cells that did not bind were washed off three times with PBS. The endothelial layer of calcein-labeled THP-1 was then examined. Adhesion was measured using a fluorescence plate reader at excitation 485 nm and emission 530 nm.

[0520] Monocyte-to-endothelial migration assay. Millicell 8 μm polycarbonate culture plate inserts (Millipore) were coated with 0.1% porcine gelatin type A (Sigma) and placed in a 24-well plate. HMEC (5x10 4 Cells (per well) were seeded into the insert and left to adhere overnight. Cells were made serum-free for 24 hours and treated with various compounds for 1 hour. IL-1β (20 ng / ml) was added to stimulate the cells for 4 hours, and 500 μl of serum-free medium was added to the bottom of a 24-well plate along with the compounds. THP-1 (5 x 10 in 100 μl) 5 Cells were added to the insert, and 100 ng / ml MCP-1 (Sigma) was added to the lower well. After 24 hours, the number of cells that migrated in the endothelial layer was measured using a Coulter cell counter (Beckman Coulter). The evaluation was performed by counting 100 μl of the suspension in the lower chamber using [a specific method / tool].

[0521] Collagen antibody-induced arthritis. Arthritis is caused by five monoclonal antibodies against type II collagen. Using a commercially available antibody cocktail (Chondrex, Inc. Redmond, WA) 2 mg / mouse, female Balb / c mice (6-8 weeks old) were induced as described above, and then BT2(3) in a DMSO vehicle. Alternatively, LPS (50 μg / mouse) with or without (30 mg / kg mouse) is administered via ip on day 3. The thickness of the hind paw pad was measured on day 9 using a digital caliper. The mice were sacrificed on day 14. The animals were sacrificed, and micro-CT scans of their hind limbs were performed. The animal experiment was approved by the Animal Welfare and Ethics Committee of the University of New South Wales.

[0522] Micro-CT scan and analysis. Hind limbs fixed with formalin-ethanol were micro-CT scanned using a Siemens Inveon micro-CT scanner (Victoria, Australia) before histological processing. Data were analyzed using Inveon Acquisition Workplace at 16.84 μm picometric depth. Cell size, 360 projection, 4100ms integration time, 80keV photon energy, and 140μA current were used for acquisition. 3D models were visualized, and limb snapshots were captured using Inveon Research Workplace software. The data was acquired using wearables. For each limb, the data was quantified using a binary value: 0 = no bone destruction, 1 = bone destruction.

[0523] Tartrate-resistant acid phosphatase (TRAP) staining. Osteoclasts were stained using a TRAP kit (Cosmo Bio, Japan, cat. PMC-AK04F-COS). Sections were heated at 65°C for 1 hour before defatting. Tissue sections were deparaffinized with 100% xylene, rehydrated with 100%, 70%, and 30% ethanol, and washed with distilled water for 5 minutes. Sections were covered with TRAP staining solution containing 3 mg of tartaric acid per 50 ml of tartrate buffer. After incubation at 37°C for 1 hour, the sections were washed three times with distilled water to stop the reaction. The sections were stained with hematoxylin for 5S counterstaining, washed with running water until clear, and dried. Sections were dehydrated with xylene, air-dried, and mounted in aqueous permanent mount medium. Six randomly selected regions were selected from the synovial membrane inside the joints of each animal, imaged with a 20x objective lens, in a blinded manner. Osteoclast counts were recorded in NIH Image J, and TRAP staining was quantified using IOD (Image-Pro Plus).

[0524] Immunohistochemical staining and analysis of VCAM-1 and ICAM-1 in hind limbs. Formalin-fixed, paraffin-embedded hind limbs were sectioned (5 μm). The Dako EnVision Rabbit Kit (cat. K4011, Dako) was used for immunohistochemical staining of VCAM-1 and ICAM-1. Briefly, the sections were blocked with peroxidase for 30 minutes, and then... Immunostaining was performed overnight at 4°C with either rabbit monoclonal VCAM-1 (cat. ab134047, 1:100, Abcam) or rabbit polyclonal ICAM-1 (cat. ab124759, 1:100, Abcam). The staining process was as follows: Immunostained slides were visualized using a labeled polymer horseradish peroxidase (HRP) (anti-rabbit) and diaminobenzidine (DAB) system, and counterstained with hematoxylin and Scott Blue. Immunostained slides were scanned with an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt Waverley, Vic, Australia), and images were acquired using ImageScope software (Leica Biosystems). The combined optical density (IOD) of positive staining in articular cartilage of the ankle joint (tibia and talus) was measured using Image-Pro Plus software (Cybernetics, Bethesda, MD, USA). VCAM-1 and ICAM-1 were evaluated using ( ). Area of ​​articular cartilage of the ankle joint (μm 2 The following measurements were taken using Image-Pro Plus software. The total number of cells and the number of positively stained cells in the articular cartilage of the ankle joint were manually counted using Image-Pro Plus software. Data are in IOD / μm 2 This was expressed as the percentage of positively stained cells per 20x objective lens field of view.

[0525] Toxicology. Female Balb / c mice (8-9 weeks old) were administered 3 or 30 mg / kg of BT2 (DMSO vehicle) by intraperitoneal injection (DMSO on days 0 and 5) or by forced oral administration (DMSO / methylcellulose on days 0-4). Alternatively, it was administered by intra-articular injection (DMSO on day 0). Tissue was fixed with 10% formalin and luchi The samples were treated with iodine, sectioned to 4 μm thickness, and stained with hematoxylin and eosin. The sections were American The animals were histologically examined for signs of toxicity by board-certified veterinarians from the College of Veterinary Pathologists. The animal studies were approved by the Animal Welfare and Ethics Committee of the University of New South Wales.

[0526] Statistics. Statistical analysis was performed using PRISM v7.0d as described in the explanation, and the difference was P<0.05. A statistically significant difference was observed. If statistical significance was shown, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0527] result Identification of BT2, T4, and T6. To identify novel small molecule inhibitors of AP-1, the WEHI read discovery library, consisting of approximately 100,000 compounds, was used to identify firefly luciferase as the AP-1 response element. Screening was performed using a 293-cell-based assay driven by multiple copies of AP-1. The inhibitor curcumin (Nelson, KM, et al., J Med Chem 60, 1620-1637 (2017)) is a typical example. A substructure filter was applied during screening to remove PAINS (Pain-Inducing Antimicrobial Agents) that specifically target the PAINS (Bael, JB, et al., J Med Chem 53, 2719-2740 (2010)). This allowed for the removal of PAINS, including dibenzoxazepinone BT2, which were determined using an 11-point titration curve. Chromole or submicromolar range IC 50Twenty-four available hits were obtained. This follows a previous screening of the DIVERSet library (ChemBridge) of 960 compounds that yielded benzophenone Cpd B / X / LK001 (Figure 6A). T4, T6, and T7 are structural analogs of Cpd B / X / LK001, and BT3 is an analog of BT2 (Table 1). BT2 is a commercially available 2-amino-10-e Cpd B / X / LK001 was synthesized after screening by reacting tildibenzo[b,f][1,4]oxazepine-11(10H)-one (BT3) with diethyl pyrocarbonate (Figure 6B, Scheme 1). Cpd B / X / LK001 was synthesized with 2-methoxyethyl carbonisocyanatidate (2) (Krebs, A, et al., European Patent Office EP0230224B1 (1991)) It was produced by reacting commercially available (4-aminophenyl)(4-chlorophenyl)methanone (1). (Figure 6B, Scheme 4). Treatment of Cpd B / X / LK001 with hydroxylamine hydrochloride yielded T4 as a ~1:1 mixture of E and Z isomers (Figure 6B, Scheme 4). Flubendazole (T6) and (4-aminophenyl)(4-fluorophenyl)methanone (T7) were obtained commercially.

[0528] BT2, T4, and T6 inhibit serum-inducible endothelial FosB / ΔFosB and c-Fos expression, and inv It blocks proliferation, migration, and network formation in thoracic cells. We measured the effects of BT2, T4, and T6 on the serum-inducible expression of two AP-1 subunits in cultured human microvascular endothelial cells (HMEC-1). Endothelial cells provide a vital barrier between flowing blood and tissues, but become more permeable when activated or stressed (van Hinsbergh, VW, et al., Arterioscler Thromb Vasc Biol 17, 1018-1023 (1997)). BT2 induces FosB and ΔFosB. Sexual expression was blocked (Figure 1A & 7A). T4 and T6 were not strongly inhibited, and BT3 and T7 showed no inhibition (Figure 1A). BT2 also blocked the inducible expression of c-Fos, a known mediator of angiogenesis (Marconcini, L., et al., Proc Natl Acad Sci USA 96). 9671-9676 (1999) (Figures 1A and 7A).

[0529] Next, we used the xCELLigence system, which monitors cell proliferation in real time. The effects of these compounds on endothelial cell proliferation were investigated. As a result, BT2, T4, and T6 showed Each of the compounds was found to suppress serum-induced proliferation in a dose-dependent manner (Figure 1B). On the other hand, BT3 and T7 did not show any inhibitory effect. This confirms that the inhibition of proliferation is not simply due to cell death. Therefore, one of these compounds (BT2) was tested in a whole-cell proliferation assay combining the Countess system and trypan blue exclusion. BT2 affects trypan blue uptake. Serum-induced proliferation was inhibited without the addition of serum (Figure 7B). In a dual-chamber Transwell system, BT2, T4, and T6 inhibited the migration of bovine aortic endothelial cells (BAECs) to VEGF-A165 in serum-containing medium (Figure 1C). HMEC-1 cells lack VEGFR-2 (Flk / KDR) and only migrate weakly to VEGF, so BAECs were used for this purpose (Shao, R., et al., Biochem Biophys Res Commun 321, 788-794 (2004)). On the other hand, BAECs lack VEGFR-2 It expresses (Lamy, S., et al., Cancer Res 62, 381-385 (2002)) and migrates to VEGF-A (Hussain, S., et al., BMC Cell Biol 9, 7 (2008)). On the other hand, BT3 also expresses PD98059 (allosterone). (Liquid MEK inhibitor), Imatinib (tyrosine kinase inhibitor), Tofacitinib (Janus) Kinase inhibitors also did not show an inhibitory effect at the same concentration (Figure 1C).

[0530] In vitro endothelial cell repair after mechanical injury induces a proliferation and migratory response. BT2, T4, and T6 blocked this repair response within 48 hours, while BT3 or T7 did not show such an effect (Figure 1D). We also evaluated the effects of these compounds in an endothelial network formation assay (also known as tubulation) on a reconstituted basement membrane matrix, which is typically used to characterize angiogenic factors and processes (Arnaoutova, I., et al, Angiogenesis 12, 267-274 (2009)). In this assay, endothelial cells maximally form capillary-like networks within a few hours and then regress. BT2, T4, and T6 inhibited network formation after 2, 4, 6, and 24 hours, respectively (Figure 1E).

[0531] BT2 suppresses retinal vascular permeability and neovascularization. Since retinal vascular permeability is an important pathological feature in nAMD and DME / DR (Campochiaro, PA, et al, J Mol Med (Berl) 91, 311-321 (2013)), we investigated the effects of BT2, T4, and We attempted to measure the effects of T6 (Grossniklaus, HE, et al., Prog Retin Eye Res 29, 500-519 (2010)). BT2 (192 μg) reduced retinal permeability by approximately 50%, and its effect was observed in Afrive. Lucept / Eirea (200 μg administered intravitreal (IVT) in 6 doses over 21 days (days 0, 3, 7, 10, 14, and 17), compared to two injections on BT2 (days 0 and 7)) or triamcinolone acetone The results were similar to those of Nido (Kenacort® 200 μg IVT, 0 days) (Figure 2A). On the other hand, for BT2 No inhibitory effect was observed on T4 and T6 delivered by the drug (Figure 2A). Aflibercept is the first-line therapy for nAMD and DME in the United States, Europe, and the Asia Pacific region (Parikh, R., et al., Ophthalmol Retina 3, 16-26 (2019)), while Kenacort is commonly used to treat DME. It is a corticosteroid (Karacorlu, M., Eye (Lond) 19, 382-386 (2005)). BT2 Also, rhVEGF-A in pigmented rabbits causes fluorescein leakage. 165 Induced by It reduced vascular permeability. A single intravenous administration of BT2 (600 μg) suppressed retinal leakage by ~50% at 2 days post-injury (Figure 2B). Immunohistochemical staining of rat eyes irradiated with laser 21 days after injury showed that BT2 was effective in IPL and INL where CD31 is expressed after laser injury (Figures 2C and 8A). It inhibited inducible CD31 staining (Ju, X, et al., Clin Exp Pharmacol Physiol 46, 75-85 (2019)). Also, BT2 inhibited VEGF-A 165 This suppressed the inducible expression of VEGF (Figure 2D), which is consistent with the finding that VEGF is mainly expressed in the outer retina (Wang, X., et al, Int J Mol Sci 8, 61-69 (2007); Foureaux, G., et al, Braz J Med Biol Res 48, 1109-1114 (2015)). 165 This is due to BT2 The inhibited wounds were stained with a gradient (Figure 2E). The anti-angiogenic effect of BT2 was confirmed by a mouse Matrigel plug assay. VEGF-A 165 Matrigel containing heparin and compounds was subcutaneously implanted into C57BL / 6 mice, and CD31 staining of the plugs was quantified after 7 days. BT2 was Neovascularization was suppressed, but BT3 showed no effect (Figures 2F & 8B).

[0532] BT2 inhibits ERK phosphorylation, FosB / ΔFosB, and VCAM-1 expression. Exposed to IL-1β Endothelial cells undergo rapid phosphorylation of ERK. IL-1β causes endothelial cell permeability. (Puhlmann, M., et al., J Transl Med 3, 37 (2005)), causes leukocytosis in the retina. (Vinores, SA, et al., J Neuroimmunol 182, 73-79 (2007)). Patients with macular edema. Diabetic patients have significantly higher concentrations of IL-1β in aqueous humor compared to other cytokines and VEGFs (Dong, N., et al., PLoS ONE 10, e0125329 (2015)). IL-1β was used as a model agonist with HMEC-1 in Western blotting experiments. BT2 inhibited IL-1β-induced ERK phosphorylation, FosB / ΔFosB, and VCAM-1 expression (Figure 3A and Figure 9). BT2-mediated VCAM-1 The inhibition was further demonstrated by flow cytometry (Figures 3B & 10).

[0533] RNA sequencing analysis confirmed that BT2 suppresses the expression of IL-1β-induced FosB and VCAM-1 (Figure 3C). From a pool of 33,379 gene IDs, 325 genes were more than 2-fold (logFC≧2) induced by IL-1β (Table 3C), of which 89 (27.5%) were inhibited by BT2 (logFC≧2) (Table 3B). Principal component analysis (PCA) (Figure 3C, upper left) showed close associations between biological replicas. BT2 also inhibited a range of other regulatory genes involved in cell proliferation, migration, angiogenesis, and inflammation, including ICAM-1, CXCL2, KLF5, Egr-1, and Fos (Figure 3C).

[0534] Dose escalation and Western blotting experiments showed that BT2 inhibited VCAM-1 and ERK phosphorylation more potently than PD98059 (Figure 3D & 11A). In contrast, BT2, like PD98059, did not affect IL-1β-induced p-SAPK / JNK or p-p38 (Figure 11B). To explore the previously unrecognized dependency of VCAM-1 expression on FosB / ΔFosB, we used siRNA against... Downgrade experiments were performed. FosB siRNA inhibited both FosB / ΔFosB and VCAM-1, while VCAM-1 siRNA inhibited VCAM-1 but not FosB / ΔFosB (Figure 3E). Overexpression of ERK1 did not increase the level of phosphorylated ERK compared to IL-1β stimulation, nor did it increase the levels of FosB, ΔFosB, or VCAM-1 (Figure 11C). Similarly, overexpression of FosB or ΔFosB did not increase VCAM-1 expression compared to IL-1β stimulation (Figure 11C). These findings indicate that in this agonist-free system where ERK is not phosphorylated, FosB and VCAM-1 are not directly activated by ERK1 overexpression, and VCAM-1 is not directly activated by overexpression of FosB or ΔFosB. These data demonstrate our demonstration that BT2, which prevents ERK phosphorylation, neutralizes the induction of FosB / ΔFosB and VCAM-1 in vitro (Figure 3A-D) and in vivo (Figure 4A-E). This complements the agonist stimulation condition. In fact, BT2 physically interacts with MEK1, which phosphorylates ERK (Figure 5C) (Qi, M., et al., Journal of Cell Science 118, 3569-3572 (2005)).

[0535] These findings, along with our demonstration (Figure 3E) that VCAM-1 IL-1β induction is blocked by FosB siRNA, suggest that while FosB is necessary for cytokine-induced VCAM-1 expression, FosB overexpression alone is insufficient to induce VCAM-1 without cytokine stimulation. FosB appears to be cofactor (or post-translational modification) dependent under cytokine-stimulated conditions.

[0536] Immunohistochemical staining of rat retina revealed that BT2 suppressed inducible pERK staining in the INL, OPL, and ONL (Figure 4A), consistent with pERK expression in the vicinity (Takeda, M, et al., Invest Ophthalmol Vis Sci 43, 907-911 (2002); Caicedo, A, et al., Exp Eye Res 81, 38-47 (2005)). Furthermore, BT2 suppressed FosB immunostaining in the retina (Figure 4B). Additionally, BT2... In OLM (Figure 4C), where other people discovered that VCAM-1 was being expressed, the expression of inducible VCAM-1 It suppressed the present (Makhoul, M., et al., Exp Eye Res 101, 27-35 (2012)). BT2 also, Inhibits FosB (Figures 4D & 8C) and VCAM-1 (Figure 4E) immunoactivation in Matrigel plugs. Ta.

[0537] Further experiments have shown that the biological efficacy of BT2 in endothelial network formation assays is BT2 was compared with curcumin (Ye, N., et al., J Med Chem 57, 6930-6948 (2014)). BT2 eliminated network formation after 4 hours at 1 μM, but no inhibition was observed with curcumin at this concentration (Figure 12). Curcumin appeared to inhibit network formation by ~25% at 30 μM and ~50% at 40 μM (Figure 12), indicating that BT2 was more than 40 times potent than curcumin in this assay. It was shown that...

[0538] BT2 structural analogues do not possess the biological efficacy of BT2. Next, we consider the biological efficacy of BT2. We investigated whether the solubility could be improved by structural modification. Dibenzoxazepinone is generally poorly soluble in water. Six BT2 analogues other than BT3 (BT2-MeOA, BT2-EOMe, BT2-Pr, We prepared BT2-IC, BT2-MO, and BT2-IMO (Table 1). BT2-MeOA was synthesized by coupling methoxyacetic acid with 2-amino-10-ethyldibenzo[b,f][1,4]oxazepine-11(10H)-one (BT3). Furthermore, BT2-IC was synthesized using diisobutyl dicarbonate (Figure 6B, Scheme 1). BT2-Pr and BT2-EOMe were synthesized from commercially available (1) and (2) using the same protocol as for the preparation of BT2 (Figure 6B, Scheme 2). Additionally, BT2-Pr and BT2-EOMe were synthesized from commercially available (1) and (2) to prepare BT2. For stability analysis, 2-nitro-10H-dibenzo[b,f][1,4]oxazepine-11-one (3) was alkylated with d3-iodoethane, and the nitro group was reduced to synthesize a trihydrogenated derivative of BT2 (Figure 6B, Scheme 3). This intermediate was reacted with diethyl pyrocarbonate to obtain the desired compound. The product was obtained. When 2-nitro-10H-dibenzo[b,f][1,4]oxazepine-11-one (3) was alkylated with oxetane-3-ylmethylmethanesulfonate, after column chromatography... O- and N-alkylation products were obtained in yields of 13% and 48%, respectively (Figure 6B, Scheme 3). The O- and N-alkylation products were reduced to the corresponding aniline (5) and (6). Obtain (Figure 6B, Scheme 3), then use diethyl pyrocarbonate to produce the desired product by the usual method. The material was converted to BT2-IMO and BT2-MO.

[0539] When these compounds were diluted in a culture medium containing serum, only their analogues (BT2-MeOA) showed higher solubility than BT2, and BT3 was found to be the most soluble of all these dibenzoxazepinones. Adding serum to the diluent increased the solubility of BT2, and serum albumin This is consistent with reports that can increase the solubility of drugs combined with BT2-MeOA (Khoder, M., et al., Pharm Dev Technol 23, 732-738 (2018)). The differential also lacked the ability to inhibit serum-induced proliferation (Figure 5A) or network formation on Matrigel (Figure 5B), similar to or more potently than BT2. BT2-IC showed some inhibition of network formation at higher concentrations (Figure 13B).

[0540] Since BT2 inhibited the phosphorylation of ERK, it appears that BT2 interacts with either MEK1 or MEK2. We hypothesized that this was the case. The binding of BT2 and PD98059 to recombinant His-MEK-1 or His-MEK2 was observed on the surface. Plasmon resonance (SPR) testing was performed. Within the assayable concentration range, BT2 bound significantly better to His-MEK1 than His-MEK2 (Figure 5C). In contrast, and as expected, PD98059 bound to both His-MEK1 and His-MEK2 (Dudley, DT, et al., Proc Natl Acad Sci USA 92, 7686-7689 (1995)) (Figure 5C). Within the comparable concentration range, BT3, BT2-MeOA, and No significant binding of BT2-Pr to MEK1 or MEK2 was observed. BT2-IC showed some interaction with MEK1 (but not MEK2). The decrease in interaction at high concentrations indicates the insolubility of BT2-IC. 1 This is thought to be due to the solubility limit (8±2 μM) determined by H 1D NMR spectroscopy. Western blotting results showed that BT2-IC undergoes ERK phosphorylation (Figure 13A) and network formation (Figure 13B) at 3 μM. It inhibited both (though not as strongly as BT2), but did not inhibit them at 1 μM (Figure 5B & D).

[0541] BT2 maintains its stability and biological activity even after sonication and 100°C treatment or autoclaving. Finally, considering the potential of BT2 as a pharmaceutical, we investigated whether this compound (as a formulation sonicated with saline containing 0.01% DMSO and 0.5% Tween 80) retains its biological efficacy and stability after extreme heat treatment. High-performance liquid chromatography / tandem mass spectrometry (RRLC-MS / MS) showed that BT2 remained stable and active even with and without heat treatment (100°C, 10 min). It remained stable, and even after 6 weeks of storage at 22°C, the BT2 content of the unheated and heat-treated formulations differed by only 0.2% and 1%, respectively (Figure 14A-B). BT2 retained its serum-inducible endothelial proliferation inhibitory ability under these conditions (Figure 14C). Even more surprisingly, it remained stable for up to 16 months. Even after time had passed, there was no loss of biological effect or degradation (Figure 14D-F). Surprisingly, the BT2 formulation remained stable and maintained its biological activity even after 4 months of standard autoclaving and storage at 22°C. (Figure 14G). The antibodies and other proteins that make up all current nAMD / DME therapeutics are, They are typically inactivated by extreme heat (Jones, FS, J Exp Med 46, 291-301 (1927)).

[0542] BT2 inhibits monocyte adhesion to IL-1β-treated endothelium in vitro and monocyte transendothelial migration toward MCP-1 in vitro. VCAM-1 mediates monocyte adhesion in human umbilical vein endothelial cells (Gerszten, RE, et al., Circ Res 82, 871-878 (1998)). Calcein In an in vitro model containing labeled THP-1 monocytes and endothelial cells pretreated with IL-1β Furthermore, the adhesion of THP-1 to endothelial cells is inhibited by BT2 (Figure 15A). BT2 also targets MCP-1. It also inhibits transendothelial migration of THP-1 monocytes from the upper chamber to the lower chamber (Figure 15B).

[0543] Intraperitoneal administration of BT2 resulted in swelling of the paw pads, bone destruction, and expression of VCAM-1 and ICAM-1 in arthritis mice. It inhibits [the process]. Having established the in vitro anti-angiogenic and anti-inflammatory effects of BT2, we hypothesized that BT2 is also useful in complex inflammatory environments such as collagen antibody-induced arthritis (Khachigian, LM Nature Protocols 1, 2512-2516 (2006)). In this model, the induced [effects] were... Hind paw thickness is suppressed by a single dose of 30 mg / kg of BT2 (Figures 16A and B). H&E staining revealed that the significant inflammation in injected CAIA mice was reduced by BT2. (Figure 16C). 3D micro-CT analysis of the hind limb revealed that BT2 suppresses bone destruction (Figures 16D and E). To support these findings, we utilized the fact that tartrate-resistant acid phosphatase (TRAP) activity is an important histochemical marker of osteodegrading osteoclasts (Ballanti, P., et al., Osteoporosis International 7, 39-43 (1997)). BT2 is related to TRAP staining of the nodes was reduced (Figure 16F). BT2 was found to suppress the expression of VCAM-1 and ICAM-1 in bone (Figure 16G). Furthermore, BT2 (30 mg / kg) lowered plasma levels of IL-1β, IL-2, and IL-6 to normal levels, but did not alter IL-4 or IL-10.

[0544] No evidence of toxicity of BT2 with intraperitoneal, intra-articular, or forced oral administration. BT2 (3 or 30 mg / kg) was administered to Balb / c mice via any of the three routes (intraperitoneal injection, intra-articular injection, or forced oral administration). The drug was administered, and tissue samples were evaluated for signs of toxicity. Pathological histology showing toxic damage caused by BT2. There was no concrete evidence (Table 2). The livers of most mice in all groups were minimal to mild. These included rare inflammatory lesions, sometimes accompanied by necrosis of individual hepatocytes or small groups of hepatocytes. This was considered a naturally occurring background lesion commonly seen in laboratory mice (Taylor, I. Mouse. in Background lesions in laboratory animals (ed. McInnes, EF) 45-75 (Saunders Elsevier, Edinburgh, 2012)) and was unrelated to the test item. Most of the ip-administered group Most mice showed minimal to mild inflammation on the capsular surface of their livers, consistent with a nonspecific peritoneal response to injection, and the effect was unrelated to the test item. Rarely, inflammatory foci were observed in the kidneys of 1 out of 5 control mice and 4 out of 30 BT2-treated mice. These are also naturally occurring background lesions common in laboratory mice and are unrelated to the test item. The inflammation of the kidneys, including the pelvis, is thought to be due to ascending bacterial infection of the urinary tract. In the liver and lungs, very rare and minimal changes unrelated to the treatment group were observed. In summary, intraperitoneal administration of BT2, joints There was no evidence of toxicity histopathologically after internal or forced oral administration.

[0545] Finally, in a GLP-compliant pharmacokinetic and intraocular tolerance study conducted by Iris Pharma (France), a single intravitreal injection (10 μg / 50 μl BT2) in rabbits showed an intraocular half-life of 28 days (t 1 / 2 The tolerance level was 3.3 days, indicating good tolerance both macroeconomically and organizationally.

[0546] Table 2. Severity assessment of histopathological findings after intra-articular, intraperitoneal, or forced oral administration of BT2. The severity of the abnormalities was histologically graded as follows: 0 = no abnormality, 1 = minimal change, 2 = mild change, 3 = moderate change, 4 = severe change, NA = not evaluated. n=5 mice per group. IA indicates intra-articular, and IP indicates intraperitoneal. The vehicle was DMSO.

[0547] [Table 2-1]

[0548] [Table 2-2]

[0549] [Table 2-3]

[0550] [Table 2-4]

[0551] Consideration A new therapeutic approach is needed to complement existing VEGF-based strategies for nAMD / DR. (Apte, RS, et al., Cell 176, 1248-1264 (2019)). IVT anti-VEGF remains Although it is the first-line treatment for retinal leak, many patients do not show an optimal response or the response is not sustained, necessitating alternative therapies. The Comparison of AMD Treatments Trials (CATT) trial, involving 647 nAMD patients treated with ranibizumab or bevacizumab, showed that the visual improvement achieved in the first two years was not maintained at five years (Maguire, MG, et al., Ophthalmology 123, 1751-1761 (2016); Pedrosa, AC, et al., Clin Ophthalmol 10, 541-546 (2016)). Furthermore, the AURAiv trial, which involved 2,227 nAMD patients in eight European countries, revealed that while anti-VEGF therapy initially improved visual acuity, this improvement was not sustained long-term and declined mainly due to insufficient treatment (Holz, FG, et al., Br J Ophthalmol 99, 220-226 (2015)).

[0552] This specification reports on the discovery of a novel dibenzoxazepinone obtained from a high-throughput screening of approximately 100,000 compounds and its biological properties. BT2 is an in vitro dibenzoxazepinone. It blocks cell proliferation, migration, wound repair, and network formation. This compound has shown efficacy in animal models of vascular leakage and angiogenesis (Carneiro, A., et al., Acta Ophthalmol 87, 517-523 (2009); Ameri, H., et al., Invest Ophthalmol Vis Sci 48, 5708-5715 (2007); Pan, CK, et al., J Ocul Pharmacol Ther 27, 219-224 (2011)), and these have become a major platform in the development of nAMD / DR therapies, which are used by millions of people today. It has played a role in this. BT2 showed efficacy in reducing retinal vascular permeability after choroidal laser injury in rats, comparable to first-line therapy for nAMD and DME, and suppressed retinal vascular permeability in a comparison of six doses of aflibercept with two doses of BT2 at the same dose. BT2 reduced CD31 staining in IPL and INL, and was used in the cross between Ptf1a-Cre mice and floxed Vhl (Vhlf / f) mice. In studies inducing pseudo-hypoxia, large-scale neovascularization in IPL and INL was revealed, which was consistent with VEGF-A gain-of-function studies in amacrine and horizontal cells (Usui, Y, et al., J Clin Invest 125, 2335-2346 (2015)). In rabbits, BT2 is associated with VEGF-A 165 We found that it suppresses retinal vascular leakage induced by [the substance].

[0553] BT2 is VEGF-A 165 It suppressed the inducible expression of [substance], but its effect in the retina was limited to VEGF. This was not the case. BT2 suppressed both ERK activation and VCAM-1 expression, which are involved in the pathogenesis of nAMD and DR (Kyosseva, SV, et al., Ophthalmol Eye Dis 8, 23-30 (2016); Ye, X, et al., Invest Ophthalmol Vis Sci 53, 3481-3489 (2012); Jonas, JB, et al., Arch Ophthalmol 128, 1281-6 (2010); Barile, GR, et al., Curr Eye Res 19, 219-227 (1999). Our findings suggest the existence of a pERK-FosB / ΔFosB-VCAM-1 cascade under cytokine-stimulated conditions. Furthermore, BT2 is involved in cell proliferation, migration, angiogenesis, and inflammation. It suppressed various genes. BT2 is more potent than PD98059 and more than 40 times more potent than curcumin, the main active ingredient in turmeric, the golden spice that inhibits AP-1 (Ye, N., et al., J Med Chem 57, 6930-6948 (2014), in a double-blind, placebo-controlled clinical trial of curcumin). Despite unsuccessful trials, it is widely used for medicinal purposes (Nelson, KM, et al., J Med Chem 60, 1620-1637 (2017))).

[0554] We synthesized BT2 analogs with various substituents at the 2 and 10 positions of the 2-amino-dibenzo[b,f][1,4]oxazepine-11(10H)-one ring system. Minor changes to the carbamic acid moiety (BT2-MeOA and BT2-IC) had a significant effect on activity, as did modifications at the 10 position (BT2-Pr, BT2-EOMe, BT2-MO, and BT2-IMO). BT2-EOMe, BT2-MO, and BT2-IMO all exhibited low calculated log Ps. We expected that water solubility would improve. BT2-MeOA (and BT3) had higher solubility than BT2, but the results of two assays showed that BT2 remained the most biologically active of these compounds, and that larger substituents at positions 2 and 10 are not necessarily better. This was shown. Comparing BT2 with its isomer BT2-MeOA (amide bond), the 2- The carbamate site at position A is found to be important for the function of BT2. BT2 may be compatible with lipid-based drug delivery systems such as self-emulsification delivery methods, which have improved the oral absorption of poorly water-soluble drugs and facilitated high-dose toxicity studies (Chen, XQ, et al., J Pharm Sci 107, 1352-1360 (2018).

[0555] While rodent and rabbit models are useful for replicating certain features of human retinal diseases, nAMD and DR are complex, multifactorial chronic diseases that cannot be accurately reproduced in acute experiments using a single stimulus, and therefore cannot fully replicate the human condition (Robinson, R., et al, Dis Model Mech 5, 444-456 (2012)). Rats are relatively inexpensive and exhibit rapid disease progression. This has the advantage of being able to see the macula, but rats (like mice) do not have a macula (Pennesi, ME, et al., Mol Aspects Med 33, 487-509 (2012)). The size of a rabbit's eye is close to that of a human's. Furthermore, the blood flow to the posterior segment of the eye differs from that of primates and rodents, and rabbits also lack a macula (Chen, S., et al., Expert Rev Opthalmol 9, 285-295 (2014)). BT2 is a humanization test in animal models. Overcoming the limitations of translatability that have hindered the widespread use of drugs and species-specific reagents. It can be taken (Lu, F., et al., Graefes Arch Clin Exp Ophthalmol 247, 171-177 (2009)).

[0556] Extraretinal effects of BT2. New and effective anti-inflammatory and anti-arthritis agents are also needed. Approximately one-third of patients treated with TNF inhibitors do not achieve 20% improvement according to the American College of Rheumatology criteria (Klak, A., et al., Rheumatologia 54, 177-186 (2016); Rubbert-Roth, A. & Finckh, A. Arthritis Research & Therapy 11 Suppl 1, S1 (2009)), which is thought to be related to the serum IFN-β / α ratio (Wampler Muskardin, T, et al., Annals of the Rheumatic Diseases 75, 1757-1762 (2016)). Synovial tissue of RA patients has elevated p-ERK levels compared to healthy individuals (Thiel, MJ, et al., Arthritis Rheum 56, 3347-3357 (2007)). difference Furthermore, serum sVCAM1 levels reflect the clinical state in RA (Navarro-Hernandez, RE et al., Disease Markers 26, 119-126 (2009)), and decrease with symptom relief in RA patients (Wang, L., et al., Experimental and Therapeutic Medicine 10, 1229-1233 (2015)). We found that systemic administration of BT2 to CAIA mice suppresses joint inflammation and bone erosion. Furthermore, BT2 inhibited monocyte adhesion to endothelial cells and transendothelial migration of monocytes to MCP-1 in vitro. In addition, systemic administration of BT2 to mice resulted in swelling of the soles of the feet, TRAP staining, and bone destruction. Inflammation is suppressed. Furthermore, inflammation is thought to promote all aspects of atherosclerosis, from the onset and progression of arteriosclerosis to ultimately causing plaque rupture and infarction, which in turn triggers further inflammation. Recent clinical trials of CANTOS (Hansson, GK Circulation 136, 1875-7 (2017); Ridker, PM, et al. N Engl J Med 377, 1119-31 (2017)), COLCOT (Tardif, JC, et al. N Engl J Med 381:2497-2505 (2019)), and tocilizumab (Kleveland, O, et al. Eur Heart J 37, 2406-13 (2016)) have revealed that inflammation is a therapeutic mechanism in cardiovascular diseases. However, patients treated with existing anti-inflammatory approaches (such as canakinumab and colchicine) remain at considerable risk of major cardiac adverse events, even with the widespread use of statins and antiplatelet therapies (Ridker, PM, et al. N Engl J Med 377, 1119-31 (2017); Tardif, JC, et al. N Engl J Med 381:2497-2505 (2019); Thompson, PL Clin Ther. 41, 41:8-10 (2019). Also, cardiovascular blood other than statins. There is a shortage of clinically effective anti-inflammatory small molecule drugs for vascular diseases (Collins, R. et al.). Lancet 388, 2532-61 (2016). This indicates the therapeutic potential of BT2 in inflammatory diseases, including rheumatoid arthritis (RA).

[0557] In conclusion, BT2 is a medical device that targets signs of vascular permeability, angiogenesis, and inflammation. This provides a new means in which BT2 establishes the ERK-FosB-VCAM1 axis that mediates vascular permeability. It functioned as a molecular tool for this purpose. BT2 improves vascular permeability via the ERK-FosB-VCAM1 axis. This molecular tool establishes a promising clinical utility for retinal diseases and rheumatoid arthritis, along with a favorable toxicity profile. BT2 is used in antibodies and proteins that primarily target VEGF. Unlike current clinical treatments using cereals, it suppresses the inductive expression of multiple genes that underlie angiogenesis and inflammatory responses, not just VEGF. Furthermore, its biological activity is maintained even after boiling, autoclaving, and storage at room temperature for several months, adding to its medicinal appeal. Similar to triamcinolone acetonide, BT2 is poorly soluble in water, and therefore, bolus injection is required. This may offer the further advantage of forming a depot agent at the site that promotes sustained release (Yang, Y., et al, Retina 35, 2440-2449 (2015)). Furthermore, BT2 promotes sustained release. It can be used in in-vivo reservoir or implant strategies and ocular delivery systems (Kang-Mieler, JJ, et al. Eye (Lond) 34, 1371-1379 (2021)).

[0558] In the following claims and prior specifications of the present invention, unless the context requires otherwise by explicit language or necessary implication, variations of the word “comprise,” “comprises,” or “comprising,” etc., are used in a comprehensive sense, i.e., as stated. This term is used to identify the presence of a feature, but without precluding the presence or addition of further features in various embodiments of the present invention.

[0559] [Table 3A-1]

[0560] [Table 3A-2]

[0561] [Table 3A-3]

[0562] Table 3A-4

[0563] Table 3A-5

[0564] Table 3A-6

[0565] Table 3A-7

[0566] Table 3A-8

[0567] Table 3A-9

[0568] Table 3A-10

[0569] Table 3A-11

[0570] Table 3A-12

[0571] Table 3A-13

[0572] Table 3A-14

[0573] Table 3A-15

[0574] Table 3A-16

[0575] Table 3A-17

[0576] Table 3A-18

[0577] Table 3A-19

[0578]

Table 3A-20

[0579]

Table 3A-21

[0580]

Table 3A-22

[0581] Table 3A-23

[0582]

Table 3A-24

[0583] Table 3A-25

[0584] Table 3A-26

[0585] Table 3A-27

[0586] Table 3A-28

[0587] Table 3A-29

[0588] Table 3A-30

[0589]

Table 3A-31

[0590] Table 3A-32

[0591] Table 3A-33

[0592] Table 3A-34

[0593] Table 3A-35

[0594] Table 3A-36

[0595] Table 3A-37

[0596] Table 3A-38

[0597] Table 3A-39

[0598]

Table 3A-40

[0599] Table 3A-41

[0600]

Table 3A-42

[0601]

Table 3A-43

[0602] Table 3A-44

[0603]

Table 3A-45

[0604] Table 3A-46

[0605] Table 3A-47

[0606] Table 3A-48

[0607] Table 3A-49

[0608]

Table 3A-50

[0609] Table 3A-51

[0610]

Table 3A-52

[0611]

Table 3A-53

[0612] Table 3A-54

[0613] Table 3A-55

[0614]

Table 3A-56

[0615] Table 3A-57

[0616] Table 3A-58

[0617] Table 3A-59

[0618]

Table 3A-60

[0619]

Table 3A-61

[0620]

Table 3A-62

[0621] Table 3A-63

[0622] Table 3A-64

[0623] Table 3A-65

[0624] Table 3A-66

[0625] Table 3A-67

[0626] Table 3B-1

[0627] Table 3B-2

[0628] Table 3B-3

[0629] Table 3C-1

[0630]

Table 3C-2

[0631] Table 3C-3

[0632]

Table 3C-4

[0633] Table 3C-5

[0634] Table 3C-6

[0635]

Table 3C-7

[0636]

Table 3C-8

[0637]

Table 3C-9

[0638]

Table 3C-10

[0639]

Table 3C-11

Claims

1. FosB / ΔFosB expression and / or VCAM-1 expression and / or ERK1 / 2 phosphorylation inhibitors A method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation, including administering an effective dose.

2. The method according to claim 1, wherein the inhibitor is a compound that inhibits FosB / ΔFosB expression.

3. The method of claim 2, wherein the compound is also an ERK1 / 2 phosphorylation inhibitor.

4. The method according to claim 2 or 3, wherein the compound is also a VCAM-1 expression inhibitor.

5. The compound is an inhibitor of ERK1 / 2 phosphorylation, FosB / ΔFosB expression, and VCAM-1 expression, claim Method 2.

6. The compounds are not limited to, but include those that promote cell proliferation such as ICAM-1, CXCL2, KLF5, Egr-1, and c-Fos. The method according to any one of claims 1 to 5, wherein the inhibitor is a regulatory gene involved in migration, angiogenesis and / or inflammation.

7. The method according to any one of claims 1 to 6, wherein the compound does not inhibit SAPK / JNK or p38 phosphorylation.

8. The method according to any one of claims 1 to 7, wherein the compound comprises a carbamate moiety.

9. The method according to any one of claims 1 to 8, wherein the compound is dibenzoxazepinone or benzophenone.

10. The compound has chemical formula I: 【Chemistry 1】 (In the formula, X is F, Cl, Br, or I; G is either C=O or C=N-OH; and A: 【Chemistry 2】 (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C 1 -C 6 Alkyl) Or A: 【Transformation 3】 (In the formula, R 2 However, linear or branched C 1 -C 6 Alkyl); or formula II: 【Chemistry 4】 (In the formula, R 3 is a linear or branched C 1 -C 6 alkyl; and R 4 However, linear or branched C 1 -C 6 Alkyl, or R 4 but 【Transformation 5】 (In the formula, q is 1, 2, 3 or 4; and R 5 However, linear or branched C 1 -C 6 (It is alkyl.) The method according to any one of claims 1 to 9, wherein the compound is or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein X is F or Cl.

12. The compound of formula (I) is formula (I-1): 【Transformation 6】 (In the formula, X is F, Cl, Br, or I; and A: 【Transformation 7】 (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C 1 -C 6 Alkyl) Or A: 【Transformation 8】 (In the formula, R 2 However, linear or branched C 1 -C 6 The method of claim 10 or 11, wherein the compound is alkyl.

13. The compound of formula (I-1) is, formula (I-1a): 【Chemistry 9】 (In the formula, X is F, Cl, Br, or I; p is 1, 2, 3 or 4; and R 1 However, linear or branched C 1 -C 6 The method of claim 12, wherein the compound is alkyl.

14. The compound of formula (I-1a): 【Chemistry 10】 The method of claim 13.

15. The compound of formula (I) is given by formula (I-2): 【Chemistry 11】 (In the formula, X is F, Cl, Br or I; and A: 【Chemistry 12】 (wherein p is 1, 2, 3 or 4; and R 1 However, linear or branched C 1 -C 6 Alkyl) Or A: 【Chemistry 13】 (In the formula, R 2 However, linear or branched C 1 -C 6 The method of claim 10, wherein the compound is alkyl.

16. The compound of formula (I-2) is, formula (I-2a): 【Chemistry 14】 (In the formula, X is F, Cl, Br, or I; p is 1, 2, 3 or 4; and R 1 However, linear or branched C 1 -C 6 Alkyl) The method of claim 15, wherein the compound is [the compound].

17. The compound of formula (I-2a): 【Chemistry 15】 The method according to claim 16.

18. The compound is given by formula (II): 【Chemistry 16】 (In the formula, R 3 However, linear or branched C 1 -C 6 alkyl; and R 4 However, linear or branched C 1 -C 6 Alkyl, or R 4 but 【Chemistry 17】 (In the formula, q is 1, 2, 3 or 4; and R 5 However, linear or branched C 1 -C 6 The method of claim 10, wherein the compound is alkyl.

19. R 3 However, linear C 1 -C 6 Alkyl or branched C 1 -C 6 The method of claim 18, wherein the alkyl group is used.

20. R 3 However, -CH 2 CH 3 or -CH 2 CH(CH 3 ) 2 The method according to claim 18.

21. R 4 However, linear C 1 -C 6 Alkyl or branched C 1 -C 6 The method of claim 18, wherein the alkyl group is used.

22. R 4 However, -CH 2 CH 3 or -CH 2 CH(CH 3 ) 2 The method according to claim 18.

23. R 4 but, [Chemistry 18] (In the formula, q is 1, 2, 3 or 4; and R 5 However, linear C 1 -C 6 Alkyl or branched C 1 -C 6 The method of claim 18, wherein the alkyl is

24. The method of claim 23, wherein q is 2.

25. R 5 ga-CH 3 The method of claim 23.

26. q is 2 and R 5 ga-CH 3 The method of claim 23.

27. The compound of formula (II) is, formula (II-1): 【Chemistry 19】 (In the formula, R 4 However, linear or branched C 1 -C 6 alkyl; or R 4 but: 【Chemistry 20】 (In the formula, q is 1, 2, 3 or 4; and R 5 However, linear or branched C 1 -C 6 The method of claim 18, wherein the compound is alkyl.

28. The compound of formula (II-1): 【Chemistry 21】 The method of claim 18, selected from among.

29. The compound of formula (II): 【Chemistry 22】 The method of claim 28.

30. Administer an effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. A method for treating a disease or condition related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration, and / or cell proliferation in a subject, including,

31. An effective dose of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, is administered to the target. This includes AP-1 and / or FosB / ΔFosB and / or ERK1 / 2 in the target. Methods for the treatment or prevention of diseases or conditions mediated by VCAM-1 and / or VEGF-A and / or IL1-β.

32. An effective dose of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, is administered to the target. This includes reducing AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression. How to reduce.

33. Compounds of chemical formula I or II: 【Chemistry 23】 A compound selected from the group consisting of the above, or a pharmaceutically acceptable salt thereof, The method according to any one of claims 30 to 32.

34. Diseases or conditions related to vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation are associated with AP-1 and / or FosB / ΔFosB and / or ERK1 / 2 and The method according to claim 30 or 33, wherein the disease or condition is mediated by VCAM-1 and / or VEGF-A and / or IL1-β.

35. The method of any one of claims 30, 31, 33, or 34, wherein the disease or condition is selected from the group consisting of the following: ·arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; ・Diabetic retinopathy; Macular edema; -Vascular leakage; ・Vascular permeability; • Permeability of retinal blood vessels; ・Angiogenesis; -Endothelial cell dysfunction; • Atherosclerosis; ·stroke; Myocardial infarction; Peripheral vascular disease; ·constriction; ・Restenosis; ·inflammation; • Cytokine storm; ・Pulmonary inflammation; ・Pulmonary fibrosis.

36. below: 【Chemistry 24】 AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression in cells, comprising contacting cells with an effective amount of a compound selected from or a pharmaceutically acceptable salt thereof. Reduction of FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression method.

37. Effective amount: 【Chemistry 25】 A method for inhibiting ERK1 / 2 phosphorylation, comprising incubating an ERK1 / 2 molecule with a pharmaceutically acceptable salt thereof or the same.

38. The method according to any one of claims 36 to 37, wherein the method is performed in vitro.

39. AP-1-dependent compounds, including compounds of chemical formula I or II, or pharmaceutically acceptable salts thereof. A kit for reducing gene expression and / or ERK1 / 2-dependent gene expression and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression.

40. The compounds are as follows: 【Chemistry 26】 The kit according to claim 39, wherein the compound selected from there is a pharmaceutically acceptable salt thereof.

41. The following general formula: 【Chemistry 27】 A compound having or a pharmaceutically acceptable salt thereof.

42. Administer an effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof. A method of treatment or prevention of a condition or disease selected from the following groups in the subject: ·arthritis; Rheumatoid arthritis; Bone destruction; Age-related macular degeneration; ・Diabetic retinopathy; Macular edema; -Vascular leakage; ・Vascular permeability; • Permeability of retinal blood vessels; ・Angiogenesis; -Endothelial cell dysfunction; • Atherosclerosis; ·stroke; Myocardial infarction; Peripheral vascular disease; ·constriction; ・Restenosis; ·inflammation; • Cytokine storm; ・Pulmonary inflammation; ・Pulmonary fibrosis.

43. The compounds are as follows: 【Chemistry 28】 The method of claim 42, wherein the compound selected from therefor or a pharmaceutically acceptable salt thereof.

44. The compound, 【Chemistry 29】 The method of claim 42.

45. The compound, 【Transformation 30】 The method of claim 42.

46. A pharmaceutical composition comprising a compound of chemical formula II or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

47. The following general formula: 【Chemistry 31】 A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

48. Use of a compound of chemical formula I or II or a pharmaceutically acceptable salt thereof for in vitro reduction of ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression.

49. A compound of chemical formula I or II for use in reducing ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression in vitro. A substance, or a medicinally acceptable salt thereof.

50. An effective amount of a compound of chemical formula I or II, or a pharmaceutically acceptable salt thereof, is brought into contact with cells. This includes ERK1 / 2 phosphorylation in cells in vitro, and / or FosB / ΔFosB A method for reducing expression and / or VCAM-1 expression and / or VEGF-A expression.

51. The following general formula is used to reduce ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression, and / or VEGF-A expression in vitro. 【Chemistry 32】 The use of a compound selected from or a pharmaceutically acceptable salt thereof.

52. The following general formula can be used to reduce ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression, and / or VEGF-A expression in vitro. 【Transformation 33】 A compound selected from or a pharmaceutically acceptable salt thereof.

53. The following general formula 【Transformation 34】 In vitro ERK1 / 2 phosphorylation and / or FosB / ΔFosB expression in cells, comprising the step of contacting cells with an effective amount of a compound selected from or a pharmaceutically acceptable salt thereof. / or a method for reducing VCAM-1 expression and / or VEGF-A expression.

54. A compound of chemical formula I or II is either an E isomer or a Z isomer, or both an E isomer and a Z isomer. The method according to any one of claims 10 to 38, 42 to 45, 50, or 53, wherein the mixture is a mixture.

55. A compound of chemical formula I or II is either an E isomer or a Z isomer, or both an E isomer and a Z isomer. A kit according to claim 39 or 40, which is a mixture.

56. The compound according to claim 41 or 52, wherein the compound is an E isomer, a Z isomer, or a mixture of an E isomer and a Z isomer.

57. Claims 10 to 38, 42 to 45, wherein a compound of chemical formula I or II is deuterated. or any one of the methods in item 50.

58. A kit according to claim 39 or 40, wherein a compound of chemical formula I or II is deuterated.

59. The compound of claim 41 or 52, wherein the compound is deuterated.

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