Treatment

Abstract

The present invention provides a method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation, which comprises administering an effective amount of an inhibitor of FosB / ΔFosB expression and / or VCAM-1 expression and / or ERK1 / 2 phosphorylation, as well as pharmaceutical compositions and kits comprising the inhibitor of FosB / ΔFosB expression and / or VCAM-1 expression and / or ERK1 / 2 phosphorylation.

Description

[Technical Field]

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

[0002] background Vascular permeability and angiogenesis are key features underlying inflammation, wound healing, tumor growth, and macular edema in both diabetic retinopathy (DR) and neovascular (wet / exudative) age-related macular degeneration (nAMD). DR is the leading cause of vision loss worldwide in patients aged 20 to 74 years. AMD has a global prevalence of 170 million, with approximately 11 million people in the United States affected. Retinal vascular leakage occurs due to disruption of the blood-retinal barrier (BRB), which normally maintains homeostasis. This is driven by endothelial dysfunction, angiogenesis, and inflammatory processes, leading to retinal capillary leakage into the interstitial space and increased osmotic pressure, resulting in edema. Vascular permeability factors include vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), histamine, platelet-activating factor, serotonin, and interleukin-1β (IL-1β).

[0003] Anti-VEGF therapy is widely used clinically to treat DR. However, repeated intravitreal injections are required, and many patients respond suboptimally or do not achieve a sustained improved response. Agents that target not only VEGF but also other important mediators involved in the pathogenesis of nAMD / DR would have particular pharmaceutical appeal in this area of ​​unmet clinical need.

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

[0005] Over the past decade, there have been considerable improvements in the management of RA with biologic 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 disability.

[0006] Given the global aging population, the significant unmet clinical needs for both RA and nAMD / DR, and the global economic burden represented by the impact of these chronic diseases, alternative treatment approaches are needed. Summary of the Invention

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

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

[0009] Thus, a first aspect 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 FosB / ΔFosB expression inhibitor.

[0010] In a first alternative embodiment, there is provided a FosB / ΔFosB expression inhibitor for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or the use of a FosB / ΔFosB expression inhibitor in the manufacture of a medicament for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0011] In a second aspect, there is provided a method for treating or preventing a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, comprising administering to the subject an effective amount of a FosB / ΔFosB expression inhibitor.

[0012] In a second alternative aspect, there is provided a FosB / ΔFosB expression inhibitor for use in the treatment or prevention of a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or the use of a FosB / ΔFosB expression inhibitor in the manufacture of a medicament for the treatment or prevention of a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0013] In a third aspect, there is provided a method for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, comprising administering an effective amount of an inhibitor of FosB / ΔFosB expression, and / or extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) phosphorylation and / or vascular cell adhesion molecule-1 (VCAM-1 or VCAM1) expression.

[0014] A third alternative aspect provides an inhibitor of FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or the use of an inhibitor of FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression in the manufacture of a medicament for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0015] In a fourth aspect, there is provided a method for treating or preventing a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, comprising administering an effective amount of an inhibitor of ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression.

[0016] A fourth alternative aspect provides an inhibitor of FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression for use in the treatment or prevention of a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or the use of an inhibitor of FosB / ΔFosB expression and / or ERK1 / 2 phosphorylation and / or VCAM-1 expression in the manufacture of a medicament for treating or preventing a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0017] In a fifth embodiment, there is provided a compound of Formula I:

[0018] [ka]

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

[0020] [ka]

[0021] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0022] [ka]

[0023] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0024] [ka]

[0025] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but

[0026] [ka]

[0027] wherein q is 1, 2, 3, or 4; and R 5 and n is a straight-chain or branched C1-C6 alkyl group, or a pharmaceutically acceptable salt thereof.

[0028] In a fifth alternative aspect, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or the use of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0029] In a sixth embodiment, there is provided a compound of Formula I:

[0030] [ka]

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

[0032] [ka]

[0033] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0034] [ka]

[0035] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0036] [ka]

[0037] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl), or R 4 but

[0038] [ka]

[0039] The present invention provides a method for treating or preventing an AP-1 and / or ERK1 / 2 mediated disease or condition, comprising administering to a subject an effective amount of a compound of formula (I)), or a pharmaceutically acceptable salt thereof.

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

[0041] In a seventh embodiment, there is provided a compound of Formula I:

[0042] [ka]

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

[0044] [ka]

[0045] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0046] [ka]

[0047] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0048] [ka]

[0049] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but,

[0050] [ka]

[0051] wherein q is 1, 2, 3, or 4; and R 5 is a straight-chain or branched C1-C6 alkyl) or a pharmaceutically acceptable salt thereof, to the subject.

[0052] In a seventh alternative aspect, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in treating or preventing a disease or condition mediated by AP-1, FosB / ΔFosB and / or ERK1 / 2 and / or VCAM-1 and / or VEGF-A and / or IL-1β in a subject; or the use of a compound of 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 FosB / ΔFosB and / or ERK1 / 2 and / or VCAM-1 and / or VEGF-A and / or IL-1β in a subject.

[0053] In an eighth embodiment, there is provided a compound of Formula I:

[0054] [ka]

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

[0056] [ka]

[0057] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0058] [ka]

[0059] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0060] [ka]

[0061] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but,

[0062] [ka]

[0063] wherein q is 1, 2, 3, or 4; and R 5 and a pharmaceutically acceptable salt thereof.

[0064] In an eighth alternative aspect, there is provided a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in treating or preventing a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject; or the use of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject.

[0065] In a ninth aspect, a subject is administered an effective amount of:

[0066] [ka]

[0067] or a pharmaceutically acceptable salt thereof.

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

[0069] [ka]

[0070] or a pharmaceutically acceptable salt thereof; or a compound selected from the following in the manufacture of a medicament for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject:

[0071] [ka]

[0072] The present invention provides the use of a compound selected from the group consisting of:

[0073] In a tenth aspect, a subject is provided with an effective amount of:

[0074] [ka]

[0075] The present invention provides a method for reducing AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression in a subject, comprising administering a compound selected from the group consisting of:

[0076] In a tenth alternative embodiment, there is provided a method for reducing AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression in a subject, comprising administering to a subject the following:

[0077] [ka]

[0078] or a pharmaceutically acceptable salt thereof; or in the manufacture of a medicament for reducing AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression in a subject, a compound selected from:

[0079] [ka]

[0080] The present invention provides the use of a compound selected from the group consisting of:

[0081] In an eleventh aspect, a subject is provided with an effective amount of:

[0082] [ka]

[0083] The present invention provides a method for treating or preventing a disease or condition 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, comprising administering a compound selected from the group consisting of:

[0084] In an eleventh alternative embodiment, the present invention provides a method for treating or preventing an AP-1 and / or FosB / ΔFosB, and / or ERK1 / 2 and / or VCAM-1, and / or VEGF-A, and / or IL-1β mediated disease or condition in a subject, comprising:

[0085] [ka]

[0086] or a pharmaceutically acceptable salt thereof; or a compound selected from the following in the manufacture of a medicament for treating or preventing an AP-1 and / or FosB / ΔFosB, and / or ERK1 / 2 and / or VCAM-1, and / or VEGF-A, and / or IL-1β mediated disease or condition in a subject:

[0087] [ka]

[0088] The present invention provides the use of a compound selected from the group consisting of:

[0089] In a twelfth aspect, a subject is provided with an effective amount of:

[0090] [ka]

[0091] or a pharmaceutically acceptable salt thereof.

[0092] In a twelfth alternative embodiment, there is provided a method for treating or preventing a condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, comprising:

[0093] [ka]

[0094] or a pharmaceutically acceptable salt thereof; or in the manufacture of a medicament for treating or preventing a condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, a compound selected from:

[0095] [ka]

[0096] The present invention provides the use of a compound selected from the group consisting of:

[0097] In a thirteenth embodiment, an effective amount of a compound of Formula I:

[0098] [ka]

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

[0100] [ka]

[0101] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0102] [ka]

[0103] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0104] [ka]

[0105] (In the formula, R 3is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but,

[0106] [ka]

[0107] wherein q is 1, 2, 3, or 4; and R 5 and a pharmaceutically acceptable salt thereof.

[0108] In a fourteenth aspect, an effective amount of:

[0109] [ka]

[0110] 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 a cell, comprising contacting the cell with a compound selected from the group consisting of:

[0111] In a fifteenth embodiment, an effective amount of a compound of Formula I:

[0112] [ka]

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

[0114] [ka]

[0115] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0116] [ka]

[0117] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Compound II:

[0118] [ka]

[0119] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but,

[0120] [ka]

[0121] wherein q is 1, 2, 3, or 4; and R 5 and a linear or branched C1-C6 alkyl) or a pharmaceutically acceptable salt thereof.

[0122] In a sixteenth aspect, an effective amount of:

[0123] [ka]

[0124] and a pharmaceutically acceptable salt thereof.

[0125] In a seventeenth aspect, there is provided a pharmaceutical composition comprising a compound that is an inhibitor of FosB / ΔFosB expression, and optionally an inhibitor of ERK1 / 2 phosphorylation and / or VCAM-1 expression, and a pharmaceutically acceptable carrier.

[0126] In an eighteenth embodiment, a compound represented by the following general formula:

[0127] [ka]

[0128] or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0129] In a nineteenth embodiment, a method for treating rheumatoid arthritis comprising administering an effective amount of an inhibitor of FosB / ΔFosB expression; and optionally an inhibitor of ERK1 / 2 phosphorylation and / or VCAM-1 expression, comprising administering: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, The present invention provides a method for treating or preventing a disease or condition in a subject selected from the group consisting of:

[0130] In a nineteenth alternative aspect, the following: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary 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: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, 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 the group consisting of a FosB / ΔFosB expression inhibitor, a FosB / ΔFosB expression inhibitor, and an ERK1 / 2 phosphorylation and / or VCAM-1 expression inhibitor.

[0131] In a twentieth embodiment, a method for treating a patient with atopic dermatitis comprises administering an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, of the following: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, The present invention provides a method for treating or preventing a condition or disease in a subject selected from the group consisting of:

[0132] In a twentieth alternative aspect, arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of a condition or disease in a subject selected from: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, The present invention provides the use of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a condition or disease in a subject selected from:

[0133] In a twenty-first aspect, arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, The present invention provides a method for treating or preventing a condition or disease in a subject selected from the group consisting of administering an effective amount of:

[0134] [ka]

[0135] or a pharmaceutically acceptable salt thereof.

[0136] For the twentieth aspect, the compound of the general formula:

[0137] [ka]

[0138] or a pharmaceutically acceptable salt thereof.

[0139] In a twenty-first alternative aspect,

[0140] [ka]

[0141] or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, or a pharmaceutically acceptable salt thereof for use in treating or preventing a condition or disease in a subject selected from:

[0142] [ka]

[0143] 1. Use of a compound selected from the group consisting of: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; vascular permeability; retinal vascular permeability; angiogenesis; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; inflammation; cytokine storm; pulmonary inflammation; pulmonary fibrosis, The present invention provides a method for treating or preventing a condition or disease in a subject, comprising administering to said subject a compound selected from the group consisting of:

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

[0145] A twenty-third aspect is a compound represented by the following general formula:

[0146] [ka]

[0147] or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0148] A twenty-fourth aspect provides use of a compound of formula I or II, or a pharmaceutically acceptable salt thereof, for reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression, and / or VEGF-A expression in vitro.

[0149] A twenty-fifth aspect provides a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression, and / or VCAM-1 expression, and / or VEGF-A expression in vitro.

[0150] A twenty-sixth aspect provides a method for reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression in a cell in vitro, comprising contacting the cell with an effective amount of a compound of formula I or II, or a pharmaceutically acceptable salt thereof.

[0151] A twenty-seventh aspect provides a compound of the general formula:

[0152] [ka]

[0153] The present invention provides the use of a compound selected from the group consisting of:

[0154] A twenty-eighth aspect is a compound of the general formula:

[0155] [ka]

[0156] or a pharmaceutically acceptable salt thereof.

[0157] A twenty-ninth embodiment provides an effective amount of a compound of 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 a cell in vitro, comprising contacting the cell with a compound selected from the group consisting of:

[0160] A thirtieth aspect is a compound of Formula I:

[0161] [ka]

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

[0163] [ka]

[0164] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0165] [ka]

[0166] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0167] [ka]

[0168] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but,

[0169] [ka]

[0170] wherein q is 1, 2, 3, or 4; and R 5 is a straight-chain or branched C1-C6 alkyl) or a pharmaceutically acceptable salt thereof.

[0171] A thirty-first aspect is a compound of Formula I:

[0172] [ka]

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

[0174] [ka]

[0175] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0176] [ka]

[0177] (In the formula, R 2 but straight-chain or branched C1-C6 alkyl); or Formula II:

[0178] [ka]

[0179] (In the formula, R 3is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R 4 but,

[0180] [ka]

[0181] wherein q is 1, 2, 3, or 4; and R 5 is a straight-chain or branched C1-C6 alkyl) or a pharmaceutically acceptable salt thereof.

[0182] A thirty-first alternative embodiment provides a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a condition in a subject mediated by expression of a gene referenced in Table 3A, 3B and / or 3C, typically a gene induced by IL-1β referenced in Table 3A, 3B and / or 3C, more typically a gene induced by IL-1β referenced in Table 3B; or the use of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a condition mediated by expression of a gene referenced in Table 3A, 3B and / or 3C, typically a gene induced by IL-1β referenced in Table 3A, 3B and / or 3C, more typically a gene induced by IL-1β referenced in Table 3B.

[0183] A thirty-second aspect provides a method for reducing expression of ICAM-1, c-Fos, Egr-1, CXCL2, KLF5, and / or VCAM-1 in a cell, comprising contacting the cell with a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0184] A thirty-third aspect provides a method for reducing expression of an IL-1β-induced gene referenced in Table 3A, 3B and / or 3C, more typically an IL-1β-induced gene referenced in Table 3B, comprising contacting a cell with an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0185] Brief description of the figure Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0186] [Figure 1A] Figure 1A shows Western blot images showing 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) and serum-arrested for 20 h. Afterwards, they were treated with 30 μM compounds (T4, T6, T7, BT2, and BT3) in serum-free medium (without EGF or hydrocortisone) at 37°C for 4 h. The medium was then changed to 10% FBS (with EGF and hydrocortisone) with the same concentrations of compounds for 1 h. Lysates were separated by SDS-PAGE and Western blotted for FosB or c-Fos. Experiments were performed biologically independently in duplicate where indicated. The approximate positions of molecular weight markers are indicated. Data represent three biologically independent experiments. [Figure 1B]Figure 1B shows the time-dependent effects of BT2, T4, and T6 on serum-induced endothelial cell proliferation. Serum-starved HMEC-1 cells were treated with compounds in 5% FBS-containing medium (containing EGF and hydrocortisone), and cell proliferation was monitored using the xCELLigence system. (Top panel) Representative real-time profiles from one experiment using the xCELLigence system; concentrations are indicated. Cell index is a quantitative indicator of cell proliferation. (Bottom panel) xCELLigence data represent the mean ± SEM of 5–8 independent experiments after 79 h. Statistical significance was assessed by one-way analysis of variance. [Figure 1C] Figure 1C shows the effects of BT2, T4, and T6 on endothelial migration. BAECs in DMEM containing 10% FBS were seeded into 24-well plates equipped with 0.8 μm Transwell inserts. The medium was changed to DMEM containing 0.01% FBS for 48 h. Compounds were added to the upper chamber at 1 μM in DMEM containing 0.01% FBS, while the medium in the lower chamber was changed to DMEM containing 10% FBS and 50 ng / ml VEGF-A165. Cells were left for 24 h. Nuclei were quantified using NIH ImageJ software. Data represent the mean ± SEM from four to five independent experiments. Statistical significance was assessed using 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 scraped with a sterile toothpick were treated with 0.6 μM of compounds in medium containing 5% FBS. Regeneration of the scraped area was observed 48 h after scraping. 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 analysis of variance. [Figure 1E]Figure 1E shows the effects of BT2, T4, and T6 on endothelial network (tube) formation on Matrigel. HMEC-1 cells in medium containing 1% FBS and 50 ng / ml FGF-2 were mixed with compounds (final concentration of 3 μM) and seeded onto Matrigel-coated wells. Network formation was assessed over 24 h. Networks were quantified using Image-Pro Plus software. Data represent the mean ± SEM from 5–6 independent experiments. Statistical significance was assessed using 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 (at the indicated doses), or vehicle (control) was injected intravenously into both eyes on the day of (day 0) and 7 days after six retinal laser burns. Kenacort was administered intravenously on day 0. Alternatively, aflibercept / Eirea in vehicle (saline) was injected intravenously six times (days 0, 3, 7, 10, 14, and 17). On days 14 and 21, sodium fluorescein was injected subcutaneously, and ocular fluorescence was recorded and scored 10 min later using Heidelberg retinal angiography (HRA). HRA scores are combined for days 14 and 21. Data represent the mean ± SEM. Statistical significance was assessed using one-way analysis of variance (left plot) or a t-test (right plot; BT2 vs. Kenacort in the left plot). n = 5–29 per group. [Figure 2B] Figure 2B shows that BT2 inhibits rhVEGF-A165-induced retinal vascular permeability in rabbits. BT2 or BT3 (600 μg) or vehicle was injected intravenously into the right eyes of rabbits 5 days before inducing vascular leakage with 500 ng of rhVEGF-A165 in 50 μl. Two days after induction, sodium fluorescein was intravenously injected. 1 h later, ocular fluorescence was measured in the right (R) and left (L) eyes using an ocular fluorometer and expressed as a ratio (R / L) for each rabbit. For comparison with the active compound BT2, the ratio data for the vehicle and BT3 groups were pooled (control) because both conditions were inactive and statistically insignificant. Data represent the mean ± SEM. Statistical significance was assessed by t-test. n = 6-8 per group. [Figure 2C-E] Figures 2C–E show immunohistochemical staining for (C) CD31, (D) VEGF-A165, and (E) VEGF-A165 enhancement in rat retinal lesions, with a 100 μm box around the wound. Untreated refers to eyes that were not laser-irradiated or injected with vehicle or drug. The IOD of positive staining (red dye) was assessed using Image-Pro Plus software. Slides were photographed with a 10x or 20x objective and shown at magnification. n = 4–6 per group for CD31, n = 3–6 per group for VEGF-A165, or n = 3–5 per group for VEGF-A165 gradient analysis. Data represent the mean ± SEM per animal. Statistical significance was assessed by one-way ANOVA, Mann-Whitney, or t-test, as appropriate. 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 injected subcutaneously into the left flank of 8-week-old male C57BL / 6 mice. Seven days later, the mice were sacrificed, and the plugs were stained with CD31 antibody. Representative immunohistochemical images of stained CD31 taken with a 10x objective lens are shown, with insets providing magnified views (taken with a 40x objective lens). CD31 staining was quantified using Image-Pro Plus software. Data represent the mean ± SEM per animal. Statistical significance was assessed using the Kruskal-Wallis multiple comparison test. n = 10–11 per group. [Figure 3A]Figure 3A is an image of a Western blot 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 various times up to 4 h. The Western is representative of two to three biologically independent experiments; times are shown in hours (where indicated), and two biologically independent replicates were performed, each in a separate lane. BT2 inhibition of IL-β1-induced VCAM-1 and ERK phosphorylation on the same blot is shown in Figure 3D. [Figure 3B] Figure 3B shows that BT2 inhibits VCAM-1 expression by flow cytometry. Flow cytometry was performed using a BD FACSCanto II with HMEC-1 cells 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 analysis of variance. 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 from HMEC-1 cells pretreated with 30 μM BT2 and incubated with 20 ng / ml IL-1β for 4 h. The PCA plot (top left) shows a close correlation between biological replicates in the UT, IL-1β, and IL-1β + BT2 conditions, as well as a clear separation between the conditions. A heatmap (center, 1579 genes) was generated for all up-regulated genes in the IL-1β vs. UT comparison. Counts per million (cpm) values ​​were used to group genes (rows) by cpm for FosB and VCAM-1 using hierarchical clustering and plotted. The heatmap (right) shows 325 genes with a log fold change (FC) ≥ 2. FosB, c-Fos, and VCAM-1 (the focus of this study) are shown in the figure, along with several other genes inhibited by BT2. Also shown in the figure is a small subset of genes (shown in red) that are additionally induced by BT2.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. Data are representative of three biologically independent experiments. [Figure 3E]Figure 3E shows an image of a Western blot using siRNA demonstrating that VCAM-1 expression is dependent on FosB. HMEC-1 cells treated with 0.6 μM siRNA or control siRNA were stimulated with 20 ng / ml IL-1β for 2 or 4 h. Western blotting was performed using the indicated antibodies. Data are representative of two biologically independent experiments. The approximate positions of molecular weight markers are indicated. [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 in retinal lesions was performed for (A) pERK, (B) FosB, and (C) VCAM-1. The IOD of positive staining (red dye) was evaluated using Image-Pro Plus software. Slides were photographed with a 20x or 40x objective and shown at magnification. n = 3–5 per group for pERK and FosB, and n = 4–6 per group for VCAM-1. Data represent the mean ± SEM per animal. Statistical significance was assessed by one-way ANOVA, Kruskal-Wallis, Mann-Whitney, or t-test, as appropriate. 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 for (D) FosB or (E) VCAM-1. Representative FosB or VCAM-1 staining images were photographed with a 40x or 20x objective, respectively, with insets providing magnification. Staining was quantified using Image-Pro Plus software. Data represent mean ± SEM. Statistical significance was assessed by Kruskal-Wallis multiple comparison test (FosB, n = 9–11 per group) or one-way analysis of variance (VCAM-1, n = 10–11 per group). Arrows indicate examples of positive staining. IOD indicates integrated optical density. [Figure 5A]Figures 5A-D show that the carbamate moiety of BT2 is important for its interaction with MEK1 and its functional effect. Figure 5A shows a proliferation experiment in which serum-starved HMEC-1 cells were treated with compounds (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 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 test. [Figure 5B] Figure 5B shows HMEC-1 network formation after combining compounds (1 μM final concentration) and seeding them onto Matrigel-coated wells in medium containing 1% FBS and 50 ng / ml FGF-2. Networks were quantified using NIH ImageJ software. Data represent the mean ± SEM from three to four independent experiments. Statistical significance was assessed using the Kruskal-Wallis multiple comparison test. [Figure 5C] Figure 5C shows SPR analysis examining the interaction of PD98059, BT2, and BT2 analogs with His-MEK1 (left panel) and His-MEK2 (right panel). Measurements were performed on a Biacore T200 at 15°C in a buffer containing 20 mM HEPES, 150 mM NaCl, 5% DMSO pH 7.4. Data are representative of two independent experiments. [Figure 5D] In Figure 5D, HMEC-1 cells were treated with 1 μM compounds (BT2 and analogs) for 4 h in serum-free medium at 37°C. The medium was changed to 20 ng / ml IL-1β with compounds for 15 min. Lysates were separated by SDS-PAGE and Western blotted for pERK or total ERK. Data are representative of two biologically independent experiments. The approximate positions of molecular weight markers are indicated. [Figure 6A]Figure 6A shows a schematic of the high-throughput compound screening. A luciferase-based high-throughput screen was used to identify hits, including the use of the PAINS frequent hitter filter. Average IC50 data and typical 11-point titration curves for BT2 and Cpd B / X / LK001 are shown. [Figure 6B] FIG. 6B shows reactants in the chemical synthesis of Cpd B / X / LK001 or BT2 analogs. [Figure 7A] Figures 7A-B show that BT2, T4, and T6 inhibit endothelial FosB / ΔFosB and c-Fos expression and block 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 summed. Plotted data represent values ​​from three biologically independent experiments or the mean (when independent biological duplicates were used in one blot) ± SEM. [Figure 7B] Figure 7B shows total cell number and % viable cells as a percentage of total cells, as determined by trypan blue exclusion using a 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 without the primary antibody. Figure 8A shows immunohistochemical staining with the MACH3 AP-Polymer detection system (vehicle group) without the primary antibody in lesion-free or lesion-containing areas (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 using a DAB chromogenic detection system omitting the primary antibody in the Matrigel plugs (vehicle group). [Figure 8C] Figure 8C shows immunohistochemical staining using the MACH3 AP-Polymer detection system (vehicle group) in Matrigel plugs, omitting the primary antibody. No. 10 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 intensity relative to the corresponding control) from Western blot analysis were measured using NIH ImageJ software. FosB / ΔFosB band intensities were summed. Plotted data represent values ​​from two to three biologically independent experiments or the mean (when 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 performing 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 from HMEC-1 cells exposed to BT2 or transfected with plasmids. Figure 11A shows the comparative effects of BT2 and PD98059 on IL-1β-induced VCAM-1 expression and ERK phosphorylation. Band intensities (pixel intensities relative to corresponding controls) from Western blot analysis were 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-induced p-SAPK / JNK or p-p38. Data represent the mean ± SEM of three biologically independent experiments. Approximate positions of molecular weight markers are indicated. [Figure 11C]Figure 11C shows the requirement for ERK phosphorylation for the indication of FosB and VCAM-1 expression by Western blotting. HMEC-1 cells growth-arrested by serum deprivation (without EGF or hydrocortisone) in 6-well plates were transfected with 6 μg of the indicated pcDNA3.1+ / C-(K)DYK-based plasmids containing 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 total protein lysates (harvested 18, 24, 48, and 72 h after plasmid transfection) with the indicated antibodies. L indicates light exposure. The approximate positions of molecular weight markers are indicated. 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 cells in medium containing 1% FBS and 50 ng / ml FGF-2 were combined with various concentrations of BT2 or curcumin compounds and seeded onto Matrigel-coated wells. After 4 h, the networks were quantified using NIH ImageJ software. Data represent the mean ± SEM from three to four independent experiments. Statistical significance was assessed using the Kruskal-Wallis multiple comparison test. [Figure 13A] Figures 13A-B show the biological activity of structural analogs of BT2. In Figure 13A, HMEC-1 cells were treated with 3 μM compounds (BT2 and analogs) for 4 h in serum-free medium at 37°C. The medium was changed to 20 ng / ml IL-1β with compounds for 15 min. Lysates were separated by SDS-PAGE and Western blotted for phosphorylated or total ERK. The approximate positions of molecular weight markers are indicated. [Figure 13B]Figure 13B shows network formation in HMEC-1 cells seeded onto Matrigel-coated wells in a medium containing 1% FBS and 50 ng / ml FGF-2 in combination with compounds (final concentration of 3 μM). Networks were quantified using NIH Image J software. Data represent the mean ± SEM of three to four independent experiments. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 14A-B] Figures 14A-F show that BT2 retains stability and biological activity after boiling or autoclaving. Figures 14A and 14B show RRLC-MS / MS analysis of heat-treated (100°C water bath, 10 min, DL20170921-H) or non-heat-treated (DL20170921) sonicated formulations of BT2 (in saline containing 0.5% Tween 80 and 0.01% DMSO) performed in triplicate 1 week or 6 weeks after preparation of the formulations. Representative chromatograms (deuterated (d3)-BT2 control is shown on the right of each set) are shown. [Figure 14C-D] Figures 14C and 14D show that tubes containing BT2 or BT3 in vehicle (saline containing 0.01% DMSO and 0.5% Tween 80, sonicated) were stored at 22°C (non-heat-treated) or placed in a 100°C water bath for 10 min and then cooled to 22°C (heat-treated, +H) and either used fresh or stored in the dark for 6 weeks or at least 10 months (D, black bars represent 11 months, blue bars represent 10 months, and red bars represent 16 months). Serum-starved HMEC-1 cells were treated with heat-treated or non-heat-treated BT2 or BT3 (0.4, 0.8 μM) in 5% FBS-containing medium, and proliferation was monitored using the xCELLigence system (Roche). Data represent the mean ± SEM from three independent experiments after 79 h. Statistical significance was assessed by one-way analysis of variance. [Figure 14E-F]Figures 14E and 14F show RRLC-MS / MS analysis of heat-treated (100°C, 10 min) or non-heat-treated sonicated formulations of BT2 (in saline containing 0.5% Tween 80 and 0.01% DMSO) performed in triplicate at 10, 11, or 16 months after formulation preparation. Representative chromatograms are shown. [Figure 14G] Figure 14G shows tubes containing BT2 in vehicle (saline containing 0.01% DMSO and 0.5% Tween 80, sonicated) that were either freshly prepared or autoclaved (121°C, 15 psi, 20 min; +A) and stored in the dark for 4 months (orange bars). Serum-starved HMEC-1 cells were treated with autoclaved or freshly prepared BT2 (0.4 or 0.8 μM) in 5% FBS-containing medium, and proliferation was monitored using the xCELLigence system (Roche). Proliferation data represent the mean ± SEM from four independent experiments after 79 h. Statistical significance was assessed by one-way ANOVA. LC / MS analysis of freshly prepared BT2 or BT2 autoclaved and stored in the dark for 4 months is also shown. The figure shows a total ion chromatogram (top, black) integrating the peak intensities of each spectrum, and an extracted ion chromatogram (bottom, brown) integrating the peak intensities of the protonated precursor (m / z 327.1319-327.1361). Table 3 provides genes induced by IL-1β relative to the control (UT) (logFC ≥ 2) (Table 3C) and genes inhibited by BT2 relative to 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 from HMEC-1 cells treated with 30 μM BT2 and incubated with 20 ng / ml IL-1β for 4 h. These data were obtained from the same experiment represented by heatmaps elsewhere. [Figure 15A]Figure 15A is a graph showing the effect of various concentrations of BT2 and BT3 on monocyte cell adhesion to IL-1β-treated endothelium in vitro. THP-1 adhesion to HMECs in vitro was assessed in 96-well plates by first treating HMECs with various concentrations of BT2 or BT3 for 1 h. HMECs were stimulated with 20 ng / ml of IL-1β for 1 h. The fluorescence intensity of calcein-labeled THP-1 cells adhered to the HMEC monolayer was then measured 30 min after cell addition using a fluorescent plate reader. Data are representative of three experiments and are expressed as mean ± SEM. Statistical significance was assessed by one-way analysis of variance. [Figure 15B] Figure 15B is a graph showing the effect of various concentrations of BT2 on monocyte transendothelial cell migration toward MCP-1 in vitro. In vitro migration of THP-1 transendothelial cells was assessed by treating HMECs with various concentrations of BT2 for 1 h in gelatin-coated culture inserts. HMECs were treated with 20 ng / ml IL-1β for 1 h. 24 h later, THP-1 cells that had undergone transendothelial migration toward MCP-1 were measured using a Coulter counter. Data are representative of three experiments and are expressed as mean ± SEM. Statistical significance was assessed by one-way analysis of variance. [Figure 16A] Figure 16A provides a graph showing the effect of vehicle or 3 mg / kg or 30 mg / kg BT2 on hind paw thickness in a collagen antibody-induced arthritis mouse model. Animals were injected i.p. with the antibody cocktail on day 0 and LPS plus BT2 (3 or 30 mg / kg in vehicle) on day 3. Hind paw thickness was measured using digital calipers on day 9. Data are presented as hind paw thickness (mm) for each paw (left and right). n = 8-10 per group. Data are presented as mean ± SEM. Statistical significance was assessed by the Kruskal-Wallis multiple comparison test. [Figure 16B] FIG. 16B provides images (gross specimens) showing the effect of vehicle or BT2 on hind footpad thickness in a collagen antibody-induced arthritis mouse model at 14 days. [Figure 16C]FIG. 16C provides images showing H&E staining of mouse footpads after no treatment or treatment of mice with vehicle or BT2 in a collagen antibody-induced arthritis mouse model on day 14. [Figure 16D] Figure 16D provides images showing the effect of no treatment, or treatment with vehicle or BT2 on bone destruction in a collagen antibody-induced arthritis mouse model. 3D Micro-CT analysis of hind paws on day 14 was quantified with a score of 0 = no bone destruction and a score of 1 = destruction given to each individual paw. Data are presented as the mean bone destruction score per hind paw (left and right) ± SEM (n = 8-10 per group). Statistical significance was assessed using Firth's penalized likelihood test. [Figure 16E] Figure 16E shows Micro-CT images of hind paws in a collagen antibody-induced arthritis mouse model at day 14, either untreated or after treatment with vehicle or BT2. Arrows indicate bone erosion and / or remodeling. [Figure 16F] Figure 16F shows graphs and images depicting tartrate-resistant acid phosphatase (TRAP) staining in hindlimb osteoclasts on day 14 from joints of collagen antibody-induced arthritis model mice untreated or treated with vehicle or BT2. Arrows indicate examples of positive staining. Slides were photographed with a 20x or 40x objective. The IOD of positive staining (red dye) was evaluated using Image-Pro Plus software. Alternatively, osteoclast numbers were counted using NIH Image J. Data are presented as mean ± SEM. Statistical significance was assessed using 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 paws on day 14. IOD / μm under a 20x objective was assessed using Image-Pro Plus software. Data represent the mean ± SEM of the mean. 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, including cytokines, growth factors, stress, and viral and bacterial infections. AP-1 is a heterodimer formed by the dimerization of proteins belonging to the c-Fos, c-Jun, ATF (activating transcription factor), and / or JDP (Jun dimerization protein 2) protein families. The expression and DNA-binding activity of AP-1 family members c-Fos and c-Jun are observed in human rheumatoid synovium and correlate with disease activity. They have 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 factors are expressed in retinal cells after retinal detachment and are elevated in diabetic human retina. Therefore, AP-1 represents an important therapeutic target for various diseases.

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

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

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

[0191] Another aspect provides a method for treating or preventing a condition associated with vascular permeability, angiogenesis, inflammation, cell migration, and / or cell proliferation, comprising administering an effective amount of an inhibitor of FosB / ΔFosB expression. In one embodiment, the inhibitor is a compound that inhibits FosB / ΔFosB expression.

[0192] As described in the Examples, the inventors further discovered that compound BT2 (a compound of formula II), in addition to inhibiting FosB / ΔFosB expression, inhibits the phosphorylation of ERK1 and ERK2 (ERK1 / 2), as well as VCAM-1 expression and VEGF-A expression.

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

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

[0195] As used herein, a FosB / ΔFosB expression inhibitor is a compound or drug that reduces the amount of FosB / ΔFosB protein produced by cells or tissues after contact with a compound or drug relative to the amount of FosB / ΔFosB protein produced by cells or tissues not contacted with the compound or drug. An ERK1 / 2 phosphorylation inhibitor is a compound or drug that reduces the level of ERK1 / 2 phosphorylation in cells or tissues after contact with a compound or drug relative to the level of ERK1 / 2 phosphorylation in cells or tissues not contacted 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 relative to the amount of VCAM-1 protein produced by cells or tissues not contacted with the compound or drug. A VEGF-A expression inhibitor is a compound or drug that reduces the amount of VEGF-A, typically VEGF-A, produced by cells or tissues after contact with a compound or drug relative to the amount of VEGF-A protein produced by cells or tissues not contacted with the compound or drug. 165 It is a compound or drug that reduces the amount of a protein in the brain.

[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 is an inhibitor of ERK1 / 2 phosphorylation, FosB / ΔFosB expression and VCAM-1 expression.

[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 comprises a carbamate moiety.

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

[0208] In one embodiment, the compound is a compound of Formula I or II, or a pharmaceutically acceptable salt thereof. The compound of Formula I is:

[0209] [ka]

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

[0211] [ka]

[0212] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0213] [ka]

[0214] (In the formula, R 2 is a straight-chain or branched C1-C6 alkyl).

[0215] The compound of formula II is:

[0216] [ka]

[0217] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, Or R 4 but,

[0218] [ka]

[0219] wherein q is 1, 2, 3, or 4; and R 5 is a straight-chain or branched C1-C6 alkyl).

[0220] In some embodiments, the compound that reduces AP-1 dependent gene expression and / or MEK1-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 has formula I:

[0221] [ka]

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

[0223] [ka]

[0224] wherein p is 1, 2, 3, or 4; and R 1 but straight-chain or branched C1-C6 alkyl); Or A:

[0225] [ka]

[0226] (In the formula, R 2 is a straight-chain or branched C1-C6 alkyl)) or a pharmaceutically acceptable salt thereof.

[0227] In some embodiments of Formula (I), X is F. In some embodiments of Formula (I), X is Cl. In some embodiments of Formula (I), X is Br. In some embodiments of Formula (I), X is I. Typically, X is F or Cl.

[0228] In some embodiments of Formula (I), G is C=O. In some embodiments of Formula (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 In some embodiments, p is 2. In some embodiments, R 1 is —CH3. In some embodiments, p is 2 and R 1 is -CH3.

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

[0233] [ka]

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

[0235] In some embodiments, the compound of formula (I) has the 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 is a straight or branched C1-C6 alkyl); or A is:

[0240] [ka]

[0241] (In the formula, R 2 may be a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0242] In some embodiments, the compound of formula (I-1) has the 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 may be a compound in which the alkyl group is a straight or branched C1-C6 alkyl.

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

[0246] [ka]

[0247] may be.

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

[0249] [ka]

[0250] (In the formula, X is F, Cl, Br or I; and R 2 may be a compound in which the alkyl group is a straight or branched C1-C6 alkyl.

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

[0252] [ka]

[0253] (also referred to herein as T6).

[0254] In some embodiments, the compound of formula (I) has the 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 but straight-chain or branched C1-C6 alkyl); Or A:

[0259] [ka]

[0260] (In the formula, R 2may be a straight chain or branched C1-C6 alkyl) compound.

[0261] In some embodiments, the compound of formula (I-2) has the 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 may be a compound in which the alkyl group is a straight or branched C1-C6 alkyl.

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

[0265] [ka]

[0266] (also referred to herein as T4).

[0267] In some embodiments, the compound that reduces 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 has formula (II):

[0268] [ka]

[0269] (In the formula, R 3 is a straight-chain or branched C1-C6 alkyl; and R 4 is a straight-chain or branched C1-C6 alkyl, or R4 but

[0270] [ka]

[0271] wherein q is 1, 2, 3, or 4; and R 5 is straight-chain or branched C1-C6 alkyl) or a pharmaceutically acceptable salt thereof.

[0272] In some embodiments of Formula (II), R 3 is a linear C1-C6 alkyl or a branched C1-C6 alkyl. In some embodiments of Formula (II), R 3 is -CH2CH3 or -CH2CH(CH3)2.

[0273] In some embodiments of Formula (II), R 4 is a linear C1-C6 alkyl or a branched C1-C6 alkyl. In some embodiments of Formula (II), R 4 is -CH2CH3 or -CH2CH(CH3)2.

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

[0275] [ka]

[0276] wherein 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 In some embodiments of Formula (II), q is 2 and R 5 is -CH3.

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

[0278] [ka]

[0279] (In the formula, R 4 is straight-chain or branched C1-C6 alkyl; or R 4 but:

[0280] [ka]

[0281] wherein q is 1, 2, 3, or 4; and R 5 may be a straight chain or branched C1-C6 alkyl) compound.

[0282] For example, the compound of formula (II-1) may be:

[0283] [ka]

[0284] may be selected from:

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

[0286] [ka]

[0287] (In the formula, R 4 is a straight-chain or branched C1-C6 alkyl; or R 4 but:

[0288] [ka]

[0289] wherein q is 1, 2, 3, or 4; and R 5 may be a straight chain or branched C1-C6 alkyl) compound.

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

[0291] [ka]

[0292] may be.

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

[0294] [ka]

[0295] (also referred to herein as BT2).

[0296] In some embodiments, the compound that reduces 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 is one of the following:

[0297] [ka]

[0298] or a pharmaceutically acceptable salt thereof.

[0299] In another aspect, there is provided 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 compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0300] Another aspect provides a method for treating or preventing a condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation, comprising administering an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0301] In one aspect, the following:

[0302] [ka]

[0303] or a pharmaceutically acceptable salt thereof.

[0304] In one embodiment, the compound has the formula:

[0305] [ka]

[0306] or a pharmaceutically acceptable salt thereof.

[0307] In one embodiment, the compound has the formula:

[0308] [ka]

[0309] or a pharmaceutically acceptable salt thereof.

[0310] In one embodiment, the compound has the formula:

[0311] [ka]

[0312] or a pharmaceutically acceptable salt thereof.

[0313] Another embodiment is a compound of the general formula:

[0314] [ka]

[0315] or a pharmaceutically acceptable salt thereof.

[0316] In one aspect, there is provided a method for reducing 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 in a cell, comprising administering an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof. In some embodiments, the cell is a cell of a subject.

[0317] Another aspect provides a method for reducing 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 in a cell, comprising contacting the cell with an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof. In some embodiments, the cell is a subject's cell.

[0318] Another aspect is the following:

[0319] [ka]

[0320] The present invention provides a method for reducing 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 in a cell, comprising contacting the cell with an effective amount of a compound selected from the group consisting of:

[0321] In one embodiment, a compound that reduces 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 is

[0322] [ka]

[0323] or a pharmaceutically 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 VCAM-1 expression and / or VEGF-A expression is reduced in cells of the subject. 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 / or VEGF-A expression is reduced in cells in vitro.

[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; and 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, mucic 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, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0326] In one embodiment, the compound of Formula I or II, or a pharmaceutically acceptable salt thereof, is deuterated.

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

[0328] In one embodiment, the compound of Formula I or II, or a pharmaceutically acceptable salt thereof, is a Z-isomer.

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

[0330] Described herein are pharmaceutical compositions comprising compounds of Formula I or II, or pharmaceutically acceptable salts thereof.

[0331] In one embodiment, a compound of the general formula:

[0332] [ka]

[0333] or a pharmaceutically acceptable salt thereof.

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

[0335] [ka]

[0336] or a pharmaceutically acceptable salt thereof.

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

[0338] [ka]

[0339] or a pharmaceutically acceptable salt thereof.

[0340] The pharmaceutical compositions of the present invention may be used in the inventive methods described herein.

[0341] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier.

[0342] The compounds of Formula 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. The disease or condition is mediated by a protein or protein complex if the activity of that protein or protein complex is necessary for the development and / or maintenance of the disease or condition.

[0343] The compounds of Formula I and II can be used to treat or prevent diseases or conditions associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation.

[0344] In one embodiment, the disease or condition is associated with vascular permeability, which is a key feature in many disease processes, such as cancer, acute and chronic inflammation, wound healing, and pathological angiogenesis. Vascular permeability causes retinal leakage leading to macular edema in diabetic retinopathy and inflammation in rheumatoid arthritis.

[0345] In some embodiments, the disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation is an AP-1, and / or FosB / ΔFosB and / or ERK1 / 2 and / or VCAM-1 and / or VEGF-A and / or IL-1β mediated disease or condition.

[0346] Diseases or conditions associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation 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, pulmonary inflammation, atherosclerosis, myocardial infarction, peripheral vascular disease, stroke, and the like.

[0347] Thus, in some embodiments, the disease or condition associated with vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation is selected from the group consisting of: arthritis; rheumatoid arthritis; Bone destruction; Age-related macular degeneration; diabetic retinopathy; Macular edema; Vascular leakage; retinal vascular permeability; endothelial cell dysfunction; atherosclerosis; stroke; myocardial infarction; peripheral vascular disease; constriction; restenosis; cytokine storm; pulmonary inflammation; pulmonary fibrosis.

[0348] As described in the Examples, the present inventors have demonstrated that administration of compound BT2 inhibits VEGFA 165 We have shown that administration of BT2 inhibits or reduces vascular permeability induced by BT2 and inhibits or reduces laser-induced ocular vascular leakage. Furthermore, we have shown that administration of BT2 reduces inflammation and bone destruction in a collagen antibody-induced arthritis model.

[0349] In one embodiment, there is provided a method for treating or preventing a disease or condition associated with vascular permeability, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0350] In one aspect, there is provided a method for treating or preventing retinal vascular permeability in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0351] In one aspect, there is provided a method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0352] In one aspect, there is provided a method for treating or preventing macular edema in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0353] In one aspect, there is provided a method for treating or preventing age-related macular degeneration in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0354] In one aspect, there is provided a method for treating or preventing bone destruction and / or arthritis in a subject in need thereof, comprising administering an effective amount of a compound of Formula II or a pharmaceutically acceptable salt thereof.

[0355] In one aspect, there is provided a method for treating or preventing rheumatoid arthritis in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0356] In one aspect, there is provided a method for treating or preventing chronic or acute inflammation in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0357] In one embodiment, there is provided a method for reducing angiogenesis in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0358] In one aspect, there is provided a method for treating or reducing endothelial cell dysfunction in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0359] In one aspect, there is provided a method for treating or reducing tissue edema in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0360] In one aspect, there is provided a method for treating or reducing stenosis in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0361] In one aspect, there is provided a method for treating or reducing pulmonary fibrosis in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0362] In one aspect, there is provided a method for treating or reducing pulmonary inflammation in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0363] In one aspect, there is provided a method for treating or reducing atherosclerosis in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0364] In one aspect, there is provided a method for treating or reducing myocardial infarction in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0365] In one aspect, there is provided a method for treating or reducing peripheral vascular disease in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0366] In one aspect, there is provided a method for treating or reducing stroke in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

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

[0368] [ka]

[0369] (In the formula, R 4 is straight-chain or branched C1-C6 alkyl; or R 4 but:

[0370] [ka]

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

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

[0373] [ka]

[0374] may be selected from:

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

[0376] [ka]

[0377] (In the formula, R 4 is straight-chain or branched C1-C6 alkyl; or R 4 but:

[0378] [ka]

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

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

[0381] [ka]

[0382] may be. Typically, the compound of formula (II) is:

[0383] [ka]

[0384] or a pharmaceutically acceptable salt thereof.

[0385] In one embodiment, there is provided a method for treating or preventing an eye disease or condition associated with vascular permeability, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0386] In one embodiment, there is provided a method for treating or preventing retinal vascular permeability in a subject in need thereof, comprising administering an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof.

[0387] In one embodiment, there is provided a method for treating or preventing diabetic retinopathy in a subject in need thereof, comprising administering an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof.

[0388] In one embodiment, there is provided a method for treating or preventing macular edema in a subject in need thereof, comprising administering an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof.

[0389] In one embodiment, there is provided a method for treating or preventing age-related macular degeneration in a subject in need thereof, comprising administering an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof.

[0390] In one embodiment, there is provided a method for treating or preventing bone destruction and / or arthritis in a subject in need thereof, comprising administering an effective amount of a BT2 compound or a pharmaceutically acceptable salt thereof.

[0391] In one embodiment, there is provided a method for treating or preventing rheumatoid arthritis in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0392] In one embodiment, there is provided a method for treating or reducing chronic or acute inflammation in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0393] In one embodiment, there is provided a method for reducing angiogenesis in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0394] In one embodiment, there is provided a method for treating or reducing endothelial cell dysfunction in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0395] In one embodiment, there is provided a method for treating or reducing tissue edema in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0396] In one embodiment, there is provided a method for treating or reducing stenosis in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0397] In one embodiment, there is provided a method for treating or reducing pulmonary fibrosis in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0398] In one embodiment, there is provided a method for treating or reducing pulmonary inflammation in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0399] In one embodiment, there is provided a method for treating or reducing atherosclerosis in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0400] In one embodiment, there is provided a method for treating or reducing myocardial infarction in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0401] In one embodiment, there is provided a method for treating or reducing peripheral vascular disease in a subject in need thereof, comprising administering an effective amount of a compound of BT2 or a pharmaceutically acceptable salt thereof.

[0402] In one embodiment, there is provided a method for treating or reducing stroke in a subject in need thereof, comprising administering an effective amount of a compound of Formula II, or a pharmaceutically acceptable salt thereof.

[0403] The methods described herein can include administering a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0404] Described herein is a pharmaceutical composition comprising a compound of Formula I or II, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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

[0406] In some embodiments, the carrier is a non-naturally occurring carrier.

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

[0408] It will be understood that the administration of the compounds described herein, or pharmaceutically acceptable salts thereof, in combination with one or more other pharmaceutical agents may be simultaneous, sequential or separate administration.

[0409] The term "composition" also encompasses formulations containing an active ingredient and conventional carriers and excipients, as well as formulations containing encapsulating materials as carriers to provide capsules in which the active ingredient (with or without other carriers) is surrounded by the encapsulating carrier. In pharmaceutical compositions, the carrier is "pharmaceutically acceptable," meaning that it is compatible with the other components of the composition and does not dramatically alter the subject. The pharmaceutical compositions of the present invention may also contain other drugs or additional active agents as described above, and may be formulated, for example, by using conventional solid or liquid vehicles or diluents, and pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavorings, etc.) appropriate for the desired mode of administration, according to techniques well known in the art of pharmaceutical formulation (see, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

[0410] Pharmaceutical compositions may be suitable for intravitreal, oral, rectal, nasal, topical (including cutaneous, buccal, sublingual), vaginal or parenteral (including intramuscular, subcutaneous, intravenous) administration or may be in a form suitable for administration by inhalation or insufflation.

[0411] In this way, the compounds described herein or their pharmaceutically acceptable salts can be combined with a pharmaceutically acceptable carrier to form pharmaceutical compositions and unit dosage forms thereof. Pharmaceutical compositions can be solids, such as tablets or filled capsules for oral administration, or liquids, such as solutions, suspensions, emulsions, elixirs, or filled capsules. Pharmaceutical compositions can also be liquids, such as solutions, suspensions, or emulsions for intravitreal administration. Pharmaceutical compositions can also be in the form of suppositories for rectal administration or in the form of sterile injectable solutions for parenteral (e.g., subcutaneous) use.

[0412] Such pharmaceutical compositions and unit dosage forms thereof contain conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms can contain any suitable effective amount of the active ingredients consistent with the intended daily dosage range employed.

[0413] For preparing pharmaceutical compositions from the compounds described herein, pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powder, tablets, pills, capsules, cachets, lozenges (solid or chewable), suppositories and dispensable granules.Solid carriers can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0414] Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, etc. Solid forms suitable for oral administration can be tablets, powders, capsules, pills, cachets, troches, etc.

[0415] Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions.For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

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

[0417] Therefore, the pharmaceutical composition according to the present invention can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion), and can be presented in unit dosage form in ampoules, pre-filled syringes, small injections, or multi-dose containers containing preservatives.The pharmaceutical composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.Alternatively, the active ingredient can be in powder form, obtained by aseptic isolation of a sterile solid or lyophilization from a solution, which is then reconstituted with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0418] Pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage, and can be protected from oxidation and the contaminating action of microorganisms such as bacteria and fungi.

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

[0420] Pharmaceutical forms suitable for injectable use can be delivered by any suitable route, such as intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

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

[0422] The compounds described herein can be formulated into compositions suitable for oral administration, for example, with assimilable edible carriers, or enclosed in hard or soft shell gelatin capsules, or compressed into tablets, or directly incorporated into the diet.For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0423] The amount of active compound in therapeutically useful compositions should be sufficient so that a suitable dosage will be obtained.

[0424] The tablets, troches, pills, capsules, lozenges, implants, etc. of the present invention may also contain ingredients such as those listed below: binders such as gum, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; flavorings such as peppermint, wintergreen, or cherry flavoring; etc. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to materials of the above type.

[0425] Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavor. Of course, the materials used to prepare any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. Furthermore, the active ingredient can be incorporated into sustained-release preparations and formulations, such as those that allow specific delivery of the active ingredient to a specific region of the intestine.

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

[0427] Pharmaceutically acceptable carriers include any and all pharmaceutically acceptable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0428] Also included are solid form preparations intended to be converted immediately before use into oral liquid form preparations.Such liquid forms include solutions, suspensions and emulsions.These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0429] For topical administration, the compounds described herein may be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. Lotions may be formulated with an aqueous or oily base and will generally also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.

[0430] Formulations suitable for topical administration in the mouth include lozenges containing the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles containing the active agent in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

[0431] The solution or suspension for nasal administration can be directly applied to the nasal cavity by conventional means, such as a dropper, pipette or spray.The formulation can be provided in the form of a single dose or multiple doses.In the case of a dropper or pipette, this can be achieved by the patient administering a predetermined amount of the solution or suspension.In the case of a spray, this can be achieved, for example, by a metered atomizing spray pump.In order to improve delivery and retention to the nasal cavity, the compound of the present invention can be encapsulated with cyclodextrin or formulated with other agents that are expected to enhance delivery and retention to the nasal mucosa.

[0432] Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant, for example a chlorofluorocarbon (CFC), such as dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

[0433] The aerosol may conveniently also contain a surfactant such as lecithin.The dose of active ingredient may be controlled by provision of a metered valve.

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

[0435] In formulations intended for administration to the respiratory tract, such as intranasal formulations, the active ingredient will generally have a small particle size for example of the order of 5 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.

[0436] The compounds described herein can be formulated into compositions for ocular, intraocular, intravitreal, or subconjunctival injection. The compounds described herein can be formulated for administration via eye drops, contact lenses, or implants. Implants may be injected intravitreally into the eye. Implants can deliver a constant therapeutic level of the compound. Such sustained-release implants are typically made with 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 continuous amounts of compound for months to years. Matrix implants can also be used. Matrix implants are typically used to deliver a loading dose followed by incremental administration of the compound over a period of one day to six months. They are most commonly made from polylactic acid (PLA) and / or polylactic-glycolic acid (PLGA) copolymers, which decompose into water and carbon dioxide.

[0437] Formulations for intravitreal administration may be formulated as an aqueous base containing one or more emulsifying agents, stabilizing agents, dispersing agents, osmotic agents, or suspending agents.

[0438] When desired, formulations adapted to give sustained release of the active ingredient may be employed.

[0439] Pharmaceutical preparations are preferably in unit dosage form.In this form, the preparation is subdivided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be a packaged preparation, and the package contains individual amounts of preparations such as packaged tablets, capsules, and powders in vials or ampoules.Also, the unit dosage can be a capsule, tablet, cachet, or lozenge itself, or any appropriate number of these in packaged form.

[0440] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form.The parenteral compositions may be in the form of physically discrete units suitable as a unitary dose to the subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0441] Alternatively, when the compound is in unit dosage form, it can be administered in the absence of a carrier.

[0442] The term "effective amount" means an amount of a compound effective to achieve a desired response.

[0443] An effective amount of a compound described herein or a pharmaceutically acceptable salt thereof can be determined by one of ordinary skill in the art having interest in the particular compound.

[0444] It will be understood that the specific dose level and frequency of administration for any particular subject may vary and will depend on a variety of factors, such as the activity of the particular compound employed, the metabolic stability and duration of action of that compound, the age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0445] Appropriate dosages of the compounds described herein or additional active agents administered in combination with the compounds described herein can be readily determined by one of ordinary skill in the art with an interest in the particular compound of the invention or additional active agent selected.

[0446] It will further be understood that when the compounds described herein are administered in combination with one or more agents or other active agents, the dosage forms and levels may be formulated for either simultaneous, sequential, or separate administration, or any combination thereof.

[0447] The methods of the present invention are intended for use with any subject that may experience the benefits of the methods of the present invention. Thus, the term "subject" includes not only humans but also non-human mammals. The subject may be, for example, a domestic animal, a zoo animal, or a farm animal.

[0448] The inventors also contemplate that the compounds of Formula I and II may be used to inhibit 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 in vitro, for example in laboratory applications.

[0449] One embodiment provides a method for reducing AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression in a cell in vitro, comprising contacting the cell with an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0450] Another embodiment is an effective amount of:

[0451] [ka]

[0452] The present invention provides a method for reducing AP-1-dependent gene expression and / or ERK1 / 2-dependent gene expression in a cell in vitro, comprising contacting the cell with a compound selected from the group consisting of:

[0453] Another aspect provides a method for reducing ERK1 / 2 phosphorylation, and / or FosB / ΔFosB expression and / or VCAM-1 expression and / or VEGF-A expression in a cell in vitro, comprising contacting the cell with an effective amount of a compound of Formula I or II, or a pharmaceutically acceptable salt thereof.

[0454] Another embodiment is an effective amount of:

[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 a cell in vitro, comprising contacting the cell with a compound selected from the group consisting of:

[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 embodiment is an effective amount of:

[0459] [ka]

[0460] and a pharmaceutically acceptable salt thereof.

[0461] Also provided are methods for preparing compounds of formula I or II, or pharmaceutically acceptable salts thereof.

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

[0463] The term "alkyl" refers to a straight or branched chain saturated hydrocarbyl group. Unless otherwise indicated, preferred are C 1-6 Alkyl groups and C 1-4 The term "C" is an alkyl group. 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. 1-6 Examples of 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, etc. Unless the context requires otherwise, the term "alkyl" also includes alkyl groups containing one less hydrogen atom, i.e., divalent, where the group is attached through two positions.

[0464] As used herein, "treating" means affecting a subject, tissue, or cell to achieve a desired pharmacological and / or physiological effect, including inhibiting a condition, i.e., arresting its onset, or alleviating or ameliorating the effects of a condition, i.e., causing a reversal or regression of the effects of a condition. As used herein, "preventing" means preventing a condition from occurring in a cell or subject at risk of having the condition, but does not necessarily mean that the condition will not eventually develop or that the subject will not eventually develop the condition. Prevention also includes delaying the onset of a condition in a cell or subject.

[0465] The term "effective amount" refers to the amount of a compound that elicits the biological or medical response in a tissue, system, animal or human that is desired by a researcher, veterinarian, physician or other clinician.

[0466] [Table 1-1]

[0467] [Table 1-2]

[0468] The compounds described herein can be synthesized by methods 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 can be purchased from, for example, Sigma-Aldrich, USA.

[0469] The invention is further described below by reference to the following non-limiting examples. [Example]

[0470] Transcription factors, particularly those encoded by immediate-early genes, integrate cues from the extracellular environment, signal transduction, and transcriptional regulation. While their role in disease regulation is clear and the drug development pipeline is robust (Miyoshi, et al., J Invest Dermatol 131, 108-117 (2011); Cho, EA, et al., The Lancet 381, 1835-1843 (2013)), no drugs directly targeting such factors are on the market (Mapp et al., Nature Chemical Biology 11, 891-894 (2015)). Basic-domain leucine zipper (bZIP) factors, including AP-1, regulate gene expression in response to various pathological stimuli, including cytokines, growth factors, stress, and viral and bacterial infections (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)) are under the control of mitogen-activated protein kinases (MAPKs) (Karin, M. J Biol Chem 270, 16483-16486 (1995)) and regulate gene expression in response to various pathological stimuli such as cytokines, growth factors, various stresses, and viral and bacterial infections (Hess, et al., Journal of Cell Science 117, 5965-5973 (2004)). AP-1 members are elevated in diabetic human retina (Oshitari, T. at el. Current Eye Research 39, 527-531 (2014)) and are expressed in retinal cells after retinal detachment (Geller, et al., Invest Ophthalmol Vis Sci 42, 1363-1369 (2001)).AP-1 DNA binding activity has also been observed in human rheumatoid synovium and correlates with disease activity (Asahara, H., et al., Arthritis Rheum 40, 912-918 (1997)), while IL-1β is known to mediate bone and cartilage damage in RA (Duff, G.W., Cytokines and Rheumatoid Arthritis. IN CINICAL APPLICATIONS OF CUTOKINE: Role in Pathogenesis, Diagnosis, and Therapy (eds. Oppenheim, J.J., Rossio, J.L. & Gearing, A.J.H.) (Oxford University Press, Oxford, 1993). Attempts to translate AP-1 inhibitors into clinical practice have been made, but the paucity of effective drugs hampers patient access.

[0471] We employed a high-throughput approach to screen ~100,000 compounds and identified a novel, previously unexplored dibenzoxazepinone, named BT2. We found that BT2 inhibits various proliferation, migratory angiogenesis, and inflammatory processes. BT2 preferentially interacts directly with MEK1 to inhibit ERK activation, inhibiting the AP-1 protein FosB / ΔFosB as well as VCAM-1 and VEGF-A. 165 BT2 inhibits the inducible expression of β-glucanase (β-glucanase). BT2 suppresses the staining of CD31 and tartrate-resistant acid phosphatase (TRAP). BT2 also inhibits retinal vascular leakage in rats and rabbits and inflammation and bone destruction in mice. BT2 is resistant to boiling and is biologically stable for up to 16 months. Thus, BT2 is a novel pharmacological inhibitor of angiogenesis, vascular permeability, and inflammation, and represents a potential new therapeutic approach for patients with nAMD / DR and RA.

[0472] material and method High-throughput screening of compound libraries. Hits were selected from the Lead Discovery Library of ~100,000 compounds at the HTS Facility of the Walter & Eliza Hall Institute of Medical Research (WEHI, Bundoora, Vic) using a commercially available human embryonic kidney (HEK)-293 cell-based assay in which firefly luciferase is driven by multiple copies of the AP-1 response element (293 / AP-1-luc cells, Panomics, Fremont, CA) in 384-well microtiter plates. Briefly, the cell-based assay was performed by culturing 5x10 cells in 384-well plates in DMEM, pH 7.4, containing 10% FBS. 3 The primary screening included 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 test compounds. After ~18 h, luciferase activity was measured using a luminometer. The hit rate for the primary screening was 2.4%. Hit compounds were selected and retested three times, and 931 test compounds were reconfirmed to show ≥50% inhibition. Next, a substructure filter was applied to remove pan-assay interfering compounds (Baell, JB, et al., J Med Chem 53, 2719–2740 (2010)). Using the most stringent criteria, 256 hits were selected for further study. After dose-response testing, 24 compounds with molecular weights <400 Da were reordered from the supplier and tested in the secondary assay.

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

[0474] To 2-amino-10-methyl-10H-dibenzo[b,f][1,4]oxazepin-11-one (BT3) (35.0 g, 137 mmol) in 100 mL of dimethylformamide (DMF) was added diethyl pyrocarbonate (22.2 mL, 24.43 g, 151 mmol), and the mixture was stirred at 22 °C under a nitrogen atmosphere for 1 h. The solid was filtered and washed with ethyl acetate (EtOAc) (100 mL) to give a pure initial crop. The combined solvents (DMF and EtOAc) 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 removed to give a yellow solid. This solid was slurried in EtOAc and filtered to give a pure colorless solid. The combined crops gave 35.0 g (79% yield) of a pure colorless solid. 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]oxazepin-2-yl)carbamate (BT2-IC). Under a nitrogen atmosphere, diisobutyl dicarbonate (1.55 g, 7.08 mmol, 1.2 eq) was added to 2-amino-10-methyl-10H-dibenzo[b,f][1,4]oxazepin-11-one (1.5 g, 5.89 mmol, 1.0 eq) in 50 mL of DMF. The mixture was stirred overnight at 40 °C (external). The solvent was removed, and the mixture was dissolved in DCM (200 mL) and washed twice with water (150 mL). The organic layer was then dried over MgSO4, filtered on a sintered funnel, and the solvent was removed to give 3.0 g of a brown solid as the crude product. This solid was purified by column chromatography on silica gel with a mixture of hexane:ethyl acetate (starting with 10% ethyl acetate in hexane, then increasing polarity to 20%) to give 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]oxazepin-2-yl)-2-methoxy-acetamide (BT2-MeOA). Under a nitrogen atmosphere, carbonyldiimidazole (2.487 g, 15.0 mmol, 1.3 eq) was added to methoxyacetic acid (1.169 g, 0.996 ml, 12.9 mmol, 1.1 eq) in 60 ml of DMF. The mixture was stirred for 30 min. Next, 2-amino-10-methyl-10H-dibenzo[b,f][1,4]oxazepin-11-one (3.0 g, 11.8 mmol, 1.0 eq) 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, which was then acidified to pH 6 with 2 M HCl. The organic phase was washed twice with 50 ml of water. The organic layer was dried over MgSO4, filtered on a sintered funnel, and the solvent was removed to give 3.7 g of a sticky yellow solid. The crude product was purified by column chromatography with 50% ethyl acetate in hexane to give 3.26 g (85%) of the product 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]oxazepin-2-yl)-carbamic acid ethyl ester (BT2-Pr). Under a nitrogen atmosphere, 2-amino-10-propyl-10H-dibenzo[b,f][1,4]oxazepin-11-one (2.4 g, 9.43 mmol, 1.0 eq) in 70 ml of DMF was added with diethyl pyrocarbonate (2.30 g, 14.16 mmol, 1.5 eq). The mixture was stirred overnight at 40 °C (external). The solvent was removed, and the mixture was dissolved in DCM (200 ml) and washed twice with water (150 ml). The organic layer was separated, dried over MgSO4, filtered on a sintered funnel, and the solvent was removed to give 3.0 g of a brown solid as the crude product. This solid was purified by column chromatography with 20% ethyl acetate in hexane to give 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]oxazepin-2-yl]-carbamic acid ethyl ester (BT2-EOMe). Under a nitrogen atmosphere, 2-amino-10-(2-methoxy-ethyl)-10H-dibenzo[b,f][1,4]oxazepin-11-one (2.9 g, 10.2 mmol, 1.0 eq) in 90 ml of DMF was added with diethyl pyrocarbonate (1.82 g, 11.22 mmol, 1.1 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 on a sintered funnel, and the solvent was removed to give 3.6 g of a brown solid as the crude product. This solid was purified by column chromatography with 30% EtOAc in hexane to give 3.5 g (96%) of the pure compound as a colorless 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.

[0479] Ethyl (11-(oxetan-3-ylmethyl)dibenzo[b,f][1,4]oxazepin-2-yl)carbamate (BT2-IMO) and ethyl (10-(oxetan-3-ylmethyl)-11-oxo-10,11-dihydro-dibenzo[b,f][1,4]oxazepin-2-yl)carbamate (BT2-MO). Under a nitrogen atmosphere, 2-nitro-10H-dibenzo[b,f][1,4]oxazepin-11-one (3) (Figure 6B, Scheme 3) (7.5 g, 0.029 mol, 1.0 eq) was added to 100 ml of DMF and stirred for 5 min. Then, NaH (1.4 g, 2.34 g (60%) in oil, 0.058 mol, 2.0 eq) was added in small portions (an exotherm was observed). The mixture was stirred at 40 °C (external) for 35 min. Next, oxetan-3-ylmethyl methanesulfonate (9.73 g, 0.058 mol, 2.0 eq) was added, and the reaction was stirred at 40 °C (external) for 3 h. After completion of the reaction, TLC was performed using 20% ​​ethyl acetate in hexane. Upon completion of the reaction, the solvent was removed using a Kugelrohr (100 °C, full vacuum) (or a rotary evaporator using a powerful pump to keep the bath temperature below 70 °C), and water (300 ml) was added. The solid was stirred with a spatula and precipitated in water. After filtration, the solid was dried in a vacuum oven at 80 °C overnight. The crude product mixture was purified by column chromatography on silica gel and a mixture of hexane:ethyl acetate (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] The O-alkylated compound was obtained in an amount of 1.0 g (13%) as a pale yellow solid, with a melting point of 135-137°C (corrected). 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, 50 mL of MeOH was added to 2-nitro-11-(oxetan-3-ylmethyl)dibenzo[b,f][1,4]oxazepin-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 reaction was hydrogenated under a H atmosphere at 40 °C (external) for 1 h. The solvent was removed to give 1.2 g (98% yield) of a yellow solid, which was used in the next step without further purification (purity ≥ 97%). Melting point: 150-152 °C (corrected). 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-(oxetan-3-ylmethyl)dibenzo[b,f][1,4]oxazepin-11(10H)-one (1.2 g, 4.05 mmol, 1.0 eq) was added with diethyl pyrocarbonate (0.98 g, 6.07 mmol, 1.5 eq). The mixture was stirred at 40 °C (external) overnight. The solvent was removed, and the mixture was dissolved in DCM (150 ml) and washed twice with water (150 ml). The organic layer was then dried over MgSO4, filtered on a sintered funnel, and the solvent was removed to give 1.31 g of a pale yellow solid as the crude product. The crude product (1.3 g) was purified by column chromatography on silica gel using a hexane:ethyl acetate mixture (starting with 20% ethyl acetate in hexane, then increasing the polarity to 35%).

[0483] BT2-IMO. Initially, 0.5 g (34% yield) was obtained as a colorless solid, melting point 159-162°C (corrected). 1H-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 pale yellow solid (RF = 0.45), melting point 106-109°C (corrected). 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, N-alkyl. 2-nitro-10-(oxetan-3-ylmethyl)dibenzo[b,f][1,4]oxazepin-11(10H)-one (2.5 g, 6.12 mmol, 1.0 eq) and 50 mL of MeOH were added to a 250 mL RBF set up for 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 with nitrogen again. 10% Pd / C (200 mg) was added, and the mixture was stirred under a hydrogen atmosphere at 40 °C (external) for 1 h at atmospheric pressure. The mixture was filtered through Celite, and the solvent was removed to give a pure colorless solid (1.8 g, 99% yield), which was used in the next step without further purification. Melting point: 62-72 °C (corrected). 16.95 (d, 1H); 7.17-7.29 (m, 3H); 7.49 (dd, 1H) ppm.

[0486] Finally, N-alkyl 2-amino-10-(oxetan-3-ylmethyl)dibenzo[b,f][1,4]oxazepin-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 h. The solvent was removed, and the mixture was dissolved in DCM (150 mL) and washed twice with water (150 mL). The organic layer was dried over MgSO4, filtered on a sintered funnel, and the solvent was removed to give the crude product, which was purified by column chromatography using 50% EtOAc in hexane. BT2-MO (1.91 g, 87% yield) was obtained as a colorless solid. Melting point: 161-162 °C (corrected). 1 6.65 (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 (49.1 g, 210 mmol) in DCM (150 mL) was cooled to ~0 °C (internal temperature) in an ice / NaCl bath. 2-Methoxyethyl carbonisocyanatidate (40 g, 276 mmol) in DCM (150 mL) was added via a dropping funnel, maintaining the internal temperature below 5 °C. The ice bath was removed, and the solution was stirred at 22 °C under nitrogen for 1 h. The solution was filtered, and the solid was washed with methanol to give a pure, pale yellow crop of the desired product. A further crop was obtained by concentrating the filtrate (a mixture of DCM and MeOH), filtering, and washing with methanol. The combined fractions gave 49 g (62%) of the desired product. 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 (20.7 g, 55 mmol), hydroxylamine hydrochloride (11.4 g, 165 mmol), and sodium acetate (13.5 g, 165 mmol) were stirred at reflux under a nitrogen atmosphere for 4 h. The reaction mixture was filtered 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 give a second crop. The solid was dried under vacuum at 60 °C to give the desired product (15.8 g, 73%) as a mixture of E and Z isomers (~1:1). 1H-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]oxazepin-2-yl)carbamate (BT2-deut). First, 2-nitro-10H-dibenzo[b,f][1,4]oxazepin-11-one (1 g, 3.9 mmol, 1 eq) was added to 10 mL of DMF and stirred for 5 min under nitrogen. Next, NaH (187 mg, 0.32 g in oil, 7.8 mmol, 2 eq) was added in small portions. The mixture was stirred for 35 min at external 40 °C. Next, ethyl iodine-2,2,2-d3 (1.24 g, 0.62 mL, 7.8 mmol, 2 eq) was added, and the reaction was stirred for 3 h at external 40 °C. The solvent was removed by evaporation and triturated three times with water to give a thick paste, which was chromatographed eluting with 15% EtOAc in hexane to give 10-(ethyl-2,2,2-d3)-2-nitrodibenzo[b,f][1,4]oxazepin-11(10H)-one as a yellow solid (0.42 g, 38%), mp 142.3 °C - 145.6 °C (corrected). 1 H-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]oxazepin-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 at reflux for 2 h. The solvent was removed, and the mixture was dissolved in EtOAc (100 ml) and 1 N aqueous NaOH (50 ml). The organic phase was separated, washed with water (2 × 50 ml), dried over MgSO4, filtered, and the solvent was evaporated. Chromatography eluting with 50% EtOAc in hexane gave the product, 2-amino-10-(ethyl-2,2,2-d3)dibenzo[b,f][1,4]oxazepin-11(10H)-one, as a pale beige solid (287 mg, 80%). The melting point is 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, to 2-amino-10-(ethyl-2,2,2-d3)dibenzo[b,f][1,4]oxazepin-11(10H)-one (0.287 g, 1.2 mmol, 1 eq) in DMF (3 ml) was added diethyl pyrocarbonate (0.183 ml, 0.201 g, 1.24 mmol, 1.1 eq). The mixture was stirred under nitrogen at 25 °C for 1 h. The DMF was removed from the reaction mixture, and the remaining solid was triturated three times with EtOAc to give BT2-deut as a colorless solid (260 mg, 66%). The melting point was 184.3 °C to 185.7 °C (corrected). 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]oxazepin-11(10H)-one (BT3) and (4-aminophenyl)(4-fluorophenyl)methanone (T7) are commercially available from AK Scientific Inc.

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

[0494] Western blot analysis was performed using extracts from serum-treated cells. HMEC-1 cells (80-90% confluent) were arrested for 20 h in serum-free MCDB131 medium without EGF or hydrocortisone. Cells were treated with 30 μM compounds in serum-free MCDB131 medium for 4 h, then the medium was changed to complete medium (10% FBS containing EGF and hydrocortisone) containing 30 μM compounds for 1 h. Total protein was harvested in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors as previously described (Li, Y., et al., Int J Cardiol 220, 185-191 (2016)). 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). Membranes were 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 (cat. 2250, 1:1000, Cell Signaling, USA), or mouse monoclonal β-actin (cat. A5316, 1:30000, Sigma-Aldrich) for 15 min at 22°C, followed by horseradish peroxidase-conjugated goat anti-rabbit (cat. P0448, 1:1000, DAKO Cytomation, Denmark) or goat anti-mouse (cat. P0447, 1:1000, DAKO Cytomation, Denmark) antibodies for 1 h. Chemiluminescence was detected 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 sensitivity / resolution was set to high on the LAS 4000, and band intensities in images taken with autoexposure were quantified using NIH ImageJ.

[0495] Western blot analysis was performed using extracts from cells treated with IL-1β. HMEC-1 (80-90% confluent) cells were arrested for 48 h in serum-free MCDB131 medium (Invitrogen, MD) without growth factors. Cells were treated with 30 μM compound for 4 h in serum-free medium and then incubated with 20 ng / ml IL-1β (Sigma, cat. SRE3083) in serum-free medium containing the same compound concentration for up to 4 h, unless otherwise noted. Total protein was harvested as previously described using RIPA buffer containing protease inhibitors. Proteins were separated on a 4-20% (w / v) SDS-polyacrylamide gradient gel and transferred to an Immobilon-P PVDF membrane. The membrane was blocked with 5% skim milk and incubated with antibodies against rabbit monoclonal FosB (cat. 2251S, 1:1000, Cell Signaling, USA), rabbit monoclonal VCAM-1 (cat. 13662S, 1:1000, Cell Signaling, USA), 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), and rabbit monoclonal phospho-p38 MAPK (cat. 4511S, The sections were incubated with mouse monoclonal phospho-p44 / 42 MAPK antibody (cat. 9106S, 1:1000, Cell Signaling, USA) overnight at 4°C, or with mouse monoclonal β-actin antibody (cat. A5316, 1:10000, Sigma-Aldrich) for 1 h at 22°C.The membranes were then incubated 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) antibodies for 1 h. Chemiluminescence was detected using the Western Lightning Chemiluminescence system and ImageQuant. TM Detection was performed using a LAS 4000 biomolecular imager. Band intensities in images generated with the same settings on the LAS 4000 were quantified using NIH ImageJ.

[0496] iRNA experiments. HMEC-1 cells (70–80% confluent) were cultured in serum-free MCDB131 medium without hydrocortisone or EGF for 24 h and then transfected with 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) in Dharma FECT1 transfection reagent (cat. T-2001-03, Dharmacon, USA) for 24 h. siRNA experiments (using 0.6 μM FosB and 0.6 μM VCAM-1) were performed alongside non-targeting control siRNA at the same concentration. Cells were stimulated with 20 ng / ml IL-1β for an additional 2 or 4 h in serum-free complete MCDB131 medium. Total proteins were harvested using RIPA buffer with protease inhibitors, separated on a 4-20% (w / v) SDS-polyacrylamide gradient gel, and transferred to Immobilon-P PVDF membranes. Membranes were blocked with 5% skim milk and incubated overnight at 4°C with rabbit monoclonal FosB (cat. 2251S, 1:1000, Cell Signaling, USA), rabbit monoclonal VCAM-1 (cat. 13662S, 1:1000, Cell Signaling, USA), or mouse monoclonal β-actin (cat. A5316, 1:10000, Sigma-Aldrich) antibodies. The membranes were incubated at 22°C for 1 h. The membranes were incubated 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 for 1 h. Chemiluminescence was detected using the Western Lightning Chemiluminescence system and ImageQuant. TM Detection was performed using a LAS 4000 biomolecular imager.

[0497] Plasmid overexpression. HMEC-1 cells were seeded into 6-well plates at 70-80% confluence and serum (or EGF and hydrocortisone) deprived overnight. Cells were transfected with 6 μg of the indicated plasmid (pcDNA3.1+ / C-(K)DYK) (GenScript, USA) using Fugene 6 (Promega) according to the manufacturer's protocol. Total protein lysates were collected 18, 24, 48, and 72 h after plasmid transfection in RIPA buffer containing protease inhibitors. Proteins were separated on a 4-20% (w / v) SDS-polyacrylamide gradient gel and transferred to an Immobilon-P PVDF membrane. The membranes were blocked with 5% skim milk and incubated overnight at 4°C with rabbit monoclonal p44 / 42 MAPK (cat. 4695S, 1:1000, Cell Signaling), mouse monoclonal phospho-p44 / 42 MAPK (cat. 9106S, 1:2000, Cell Signaling), rabbit monoclonal FosB (cat. 2251S, 1:1000, Cell Signaling, USA), rabbit monoclonal VCAM-1 (cat. 13662S, 1:1000, Cell Signaling), or mouse monoclonal α-tubulin (cat. T5168, 1:40000, Sigma). The membranes were then incubated 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) antibodies for 1 h. Chemiluminescence was detected using the Western Lightning Chemiluminescence system and ImageQuant. TM Detection was performed using a LAS 4000 biomolecular imager.

[0498] RNA-seq. HMEC-1 cells were seeded in nine 100 mm dishes in complete MCDB131 medium containing 10% FBS. At 70–80% confluence, growth arrest was performed for 44 h in serum-free MCDB131 medium without hydrocortisone or EGF. Cells were pretreated with 30 μM BT2 for 4 h in the same medium, followed by stimulation with 20 ng / mL IL-1β for an additional 4 h. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Amtsgericht, Düsseldorf). Briefly, cells were washed twice with pre-chilled 1x PBS and lysed with TRIzol (Thermo Fisher Sci, Waltham, MA). Chloroform was added and the mixture was centrifuged at 13,000 rpm for 15 min at 4°C. The upper aqueous layer containing total RNA was transferred to a new microtube, isopropanol was added, and the mixture was loaded onto an RNeasy column. The column was washed with buffer RW1 and RPE. Total RNA was eluted from the column using RNAse-free water. Samples were submitted to the Ramaciotti Centre for Genomics (UNSW, Australia) for TruSeq Stranded mRNA-seq preparation and sequencing using a One NextSeq 500 1x75bp High Output flowcell (data output up to 400M reads). Sample quality control was set at 80% above Q30 for 1x75bp.

[0499] RNA-seq reads were first assessed for quality using the tool FastQC (v0.11.8) (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Transcript abundance from RNA-seq reads was quantified using the tool Salmon (Patro, R., et al., Nat Methods 14, 417-419 (2017)). Differentially expressed genes between specific comparisons were identified using the R package DESeq2 (Love, M.I., et al., Genome Biol 15, 550 (2014)), which incorporates a method for differential analysis of count data. Heatmaps were generated using the heatmap.2 function in the R package gplots v3.0.1.1 using counts per million (cpm) values ​​for the set of genes of interest. Using the web-based online bioinformatics resource Database for annotation, visualization and integrated discovery (DAVID) (Jiao, X., et al., Bioinformatics 28, 1805-1806 (2012)), we identify gene ontologies, such as bioprocesses (BPs), that were found to be enriched in the differentially expressed gene lists of a particular comparison.

[0500] Flow cytometry. HMEC-1 (80-90% confluent) cells were arrested for 40 h in serum-free MCDB131 medium without EGF or hydrocortisone and then treated with 30 μM BT2 or BT3 for 4 h. The cells were then incubated in serum-free medium and exposed to 20 ng / ml IL-1β with the same concentrations of BT2 or BT3 for an additional 4 h. Cells were washed with PBS and then detached using Accutase (Stem Cell Technologies, cat. 07920). Cells were centrifuged at 300 g for 5 min and 5 x 10 cells containing BT2 or BT3 were harvested. 6Cells were resuspended at 0.5 ml of 1% paraformaldehyde before flow cytometry using a BD FACSCanto II. Cells were incubated with BV421-conjugated mouse anti-human CD106 (VCAM-1) (BD, cat. 744309) or BV421-conjugated mouse IgG1 (BD, cat. 562438) for 45 min at 22°C. Cells were washed with staining buffer, and the pellet was resuspended in 0.5 ml of 1% paraformaldehyde before flow cytometry using a 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). A representative gating from the latter (i.e., negative control) is shown in Figure 10, demonstrating minimal nonspecific staining. The gating strategy was based on fluorescence excitation from both the 488 nm and 405 nm lasers, with emission filters 670LP for 488 nm and 450 / 50 for 405 nm. Cells with autofluorescence or negative cells (blue population) had the same percentage of fluorescence in both channels, while VCAM-1 positive cells (red) emitted with the 450 / 50 filter.

[0502] SPR. SPR was performed on a Biacore T200. The active and reference flow cells of a Xantec NIHMC Ni sensor chip 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 injected onto separate active flow cells at 10 μl min. -1All immobilizations were 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 running buffer) and BT2 (1.25–15 μM) were injected over the immobilized MEK1 and MEK2 in 30 μl min. -1 The injection was performed at a flow rate of 100 s, and solvent correction was applied to the data using a DMSO standard curve. Data were analyzed using Biacore T200 Evaluation software. Prior to SPR, 1 The solubility limits of the compounds were determined using H 1D NMR.

[0503] Endothelial proliferation assay using the xCELLigence system. HMEC-1 proliferation was assessed using the xCELLigence system (Roche, Castle Hill). Briefly, HMEC-1 (5x10 3 Cells (cells / well) were seeded into 96-well E-plates and inserted into the xCELLigence RTCA station (Roche). After 24 h of serum deprivation, cells were incubated in MCDB131 medium containing 10 ng / ml EGF (Sigma-Aldrich) and 1 μg / ml hydrocortisone (Sigma-Aldrich). Then, cells were treated with compounds (0.2–1 μM) in medium containing 5% FBS, 10 ng / ml EGF (Sigma-Aldrich), and 1 μg / ml hydrocortisone (Sigma-Aldrich). Cell proliferation was automatically monitored every 15 minutes by the xCELLigence system. The Cell Index (CI) provides a quantitative measure of cell proliferation in each well. In this system, CI is a unitless parameter that reports the impedance of electron flow caused by attached cells.

[0504] Endothelial proliferation assay using the Countess system. HMEC-1 proliferation was assessed using the Countess II Automated Cell Counter (ThermoFisher Scientific). Briefly, HMEC-1 (3 x 10 5Cells (cells / well) were seeded in 12-well plates. Cells were serum-deprived for 24 h in MCDB131 medium containing 10 ng / ml EGF and 1 μg / ml hydrocortisone, followed by treatment with compounds (0.1–0.6 μM) in medium containing 5% FBS, 10 ng / ml EGF, and 1 μg / ml hydrocortisone. After 24 h, cells were trypsinized and resuspended in complete medium. A 10 μl aliquot was combined with an equal volume of 4% trypan blue, and the total cell count and the percentage of trypan blue-excluding cells were determined using a Countess™.

[0505] Endothelial dual-chamber migration assay. BAECs (6x10) suspended in DMEM supplemented with 10% FBS were cultured in a 200-well plate. 3 Cells (cells / well) were seeded in the upper chamber of a 24-well plate equipped with Millicell cell culture inserts (cat. PI8P01250, Millipore). After 48 h, the medium was replaced with DMEM containing 0.01% FBS and incubated for 48 h. Compounds prepared in DMEM containing 0.01% FBS were added to the upper chamber. VEGF-A in 10% FBS-containing medium was added to the upper chamber. 165 (50ng / ml, Sigma, cat. V7259) was added to the lower chamber. After 24 h, the medium was removed from the upper chamber, and non-migrating cells and excess liquid were removed with a cotton swab. The inserts were immersed in 70% ethanol for 10 min to fix the cells, and the membranes were allowed to dry for 10–15 min. The filters were then cut and placed on slides. Mounting medium (Fluoroshield with DAPI) was added. TM , Sigma, cat. 6057) was added, and the specimens were visualized under an EVOS FL microscope.

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

[0507] Analysis of BT2 formulations using RRLC-MS / MS. A high-performance liquid chromatography / tandem mass spectrometry (RRLC-MS / MS) method developed by Iris Pharma under GLP conditions was used to measure the BT2 content of heat-treated and non-heat-treated BT2 formulations at room temperature 1 week (T1 week) or 6 weeks (T6 week). Formulations were either heat-treated (tubes placed in a 100°C water bath for 10 min) or non-heat-treated (BT2 formulations sonicated in saline containing 0.5% Tween 80 and 0.01% DMSO). A standard curve was constructed at eight concentrations between the lower limit of quantitation (LLOQ) and upper limit of quantitation (ULOQ). Evaluation was performed using triplicate formulations at identical dilutions. Chromatograms were integrated using MassHunter software. For the BT2 content analysis (T1 week and T6 week), the mean, SD, CV (%) and bias (%) were calculated as follows: For T1, the bias (%) of each formulation, including the test sample, was calculated based on the theoretical concentration (i.e., the weighed amount supplied / formulation):

[0508]

number

[0509] Standard curve in Excel (R) The calibration standard was fitted using version 2011. For each run, the deviation between the standard curve and the back-calculated concentrations of the QCs was determined, and the back-calculated concentrations of the calibration standards were within ±15% of the theoretical values ​​(except for the LLOQ, which was within ±20%). At least 75% of the calibration standards, a minimum of six, had to meet this criterion, and the coefficient of determination (r 2 ) was set to ≥ 0.98.

[0510] Analysis of BT2 formulations was performed 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 HO:CHCN (1:1) 0.1% formic acid (90 μl) for LC / MS analysis. Samples were separated by UPLC using an HPG-3400RS UPLC pump, autosampler, and column compartment system (Thermo Scientific, CA). Samples (0.1 μl) were loaded onto a Hypersil Gold aQ column (2.1 x 50 mm) (Thermo Scientific) containing 1.9 μL of broth. Compounds were eluted using a linear gradient of HO:CHCN, where A contained HO (0.1% formic acid) and B contained HO:CHCN (1:4, 0.1% formic acid). The gradient was: 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, T = 30 min 2% B. 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, applying a high voltage of 3.8 kV. The vaporizer temperature was set to 250°C, with 20 sheath gases and 5 auxiliary gases. The heated capillary was set to T = 290°C. A survey scan was performed from m / z 140 to m / z 800 with the lock mass (m / z 391.28429) enabled (resolution was 70,000 at m / z 200, AGC target value was 3x10). 6 ions, maximum IT is 250 msec). Two microscans combined to capture up to the 10 most abundant ions (minimum AGC target value 5x10 4 The peaks were separated sequentially (width m / z 1.8) with a maximum IT of 110 msec, and 2x10 chromatograms were obtained by HCD (NCE = 20, 30, 50). 5The ion (m / z 200 with a resolution of 17,500) was fragmented to the target value. The m / z ratio selected for MS / MS was dynamically excluded for 12 seconds, and charge state exclusion was not enabled. LC / MS chromatograms were processed with Xcalibur Qual Browser.

[0511] Endothelial network formation assay. HMEC-1 (4x10) cells were cultured in MCDB131 containing 1% FBS. 4 Cells / well) and compounds (1 or 3 μM) or curcumin (1–40 μM) and 50 ng / ml FGF-2 were added to 96-well plates coated with 100 μl of growth factor-reduced reconstituted basement membrane matrix (Matrigel, cat. 354230, Corning, NY) overnight at 4°C. Network formation was observed over the next several hours and photographed using an Olympus CKX41 microscope with a 4x or 10x objective.

[0512] Matrigel plug assay. VEGF-A 165 Matrigel (500 μl) containing BT2 or BT3 (100 ng / ml), heparin (10 U), or their vehicle (saline containing 0.01% DMSO and 0.5% Tween 80) was injected subcutaneously into the left flank of 8-week-old C57BL / 6 male mice. Seven days later, mice were sacrificed by CO2 asphyxiation, and the plugs were carefully removed. Formalin-fixed, paraffin-embedded sections were prepared from the Matrigel plugs for immunohistological evaluation. Heat-induced epitope retrieval was applied to all deparaffinized sections (4 μm Superfrost slides) at 110°C for 5 min in citrate buffer, pH 6. Immunostaining for all groups was performed simultaneously with a given antibody, and development times were identical. Animal experiments were approved by the University of New South Wales Animal Care and Ethics Committee.

[0513] For CD31 staining, sections were blocked with an endogenous enzyme blocking agent (cat. S2003, DAKO) for 10 min, followed by 2% skim milk for 20 min. Slides were incubated with the primary antibody, rabbit polyclonal CD31 antibody, diluted 1:25 (cat. ab28364, Abcam) for 1 h at room temperature. After washing with buffer, slides were incubated with the secondary antibody (goat anti-rabbit (cat. P0448, DAKO)) for 30 min. After washing with buffer, slides were incubated with diaminobenzidine (DAB) chromagen (cat. K3468, DAKO) for 5 min and counterstained with hematoxylin and Scott blue. Slides were dehydrated in 100% ethanol and xylene and coverslipped.

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

[0515] 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 assessed using Image-Pro Plus software (Cybernetics, Bethesda, MD) in 5–12 randomly selected fields of view for each plug, photographed under 10x (CD31), 20x (VCAM-1), and 40x (FosB) objectives. The results were expressed as integrated optical density (IOD) values ​​(mean intensity × area) (Liu, H., et al., Sci Rep 6, 21319 (2016)). Positive immunostaining was also expressed as a percentage of the plug area (Kim, JY, et al., Biomolecules 10, p.i.: E11 (2019)).

[0516] Rabbit retinal vascular hyperpermeability model. Male HY79b pigmented rabbits (8-12 weeks old) were cultured in Rompun. (R) (Xylazine) / Imalgene (R) Animals were anesthetized with an intramuscular injection of ketamine. Five days before rhVEGF-A165 induction, the compounds (600 μg BT2, BT3, or saline vehicle containing 0.5% Tween 80 and 10% DMSO in 100 μl vehicle) were injected into the right eye. Injections were performed under an operating microscope using a 250 μl Hamilton syringe (fitted with a 30G needle) in anesthetized animals. Retinal vascular permeability was measured using 500 ng rhVEGF-A165. 165Animals were induced with a single 50 μl IVT injection of fluorescein (diluted in PBS containing carrier protein) into the right eye. Forty-seven hours (±3 h) after induction, fluorescein sodium (10% in saline, 50 mg / kg) was injected into the marginal ear vein. One hour after fluorescein injection, animals were anesthetized, and pupils were dilated with a single drop of 0.5% tropicamide. Ocular fluorescence in both eyes was measured using an FM-2 Fluorotron Master ocular fluorometer. Animals were euthanized by injection of pentobarbital. This study was approved by the Animal Ethics Committee of Iris Pharma (La Gaude, France) and the Animal Care 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 the Rompun (R) (Xylazine) / Imalgene (R)Eyes were anesthetized with an intramuscular injection of ketamine. One drop of 0.5% tropicamide was instilled into the eye before laser irradiation to dilate the pupils. Six burns were created in both eyes on day 0 using a 170 mW 532 nm laser (Viridis laser, Quantel, France) for 0.1 s at a 75 μm spot around the optic nerve between the main retinal vessel branches, through a slit lamp and contact lens. Rupture of Bruch's membrane was confirmed by the generation of air bubbles during laser irradiation. IVT injections of 2–5 μL of the compound in vehicle (saline containing 0.01% DMSO and 0.5% Tween 80, sonicated) were performed on days 0 and 7 under an operating microscope using a 30-gauge needle attached to a 100 μL Hamilton syringe. Kenacort was administered intravenously to each eye on day 0. Alternatively, aflibercept / Eylea in vehicle (saline) was administered via IVT injection six times (days 0, 3, 7, 10, 14, and 17). Fluorescein angiography was performed using a Heidelberg retinal angiography system. After anesthesia, 10% sodium fluorescein (250 μl / 100 g body weight) was injected subcutaneously, and ocular fluorescence was recorded 10 min after dye injection. On days 14 and 21, angiography was performed by two examiners masked to the study group to assess fluorescence leakage, with the intensity of fluorescence graded as follows: 0 (no leakage), 1 (slight staining), 2 (moderate staining), and 3 (strong staining). This study was approved by the Animal Ethics Committee of Iris Pharma (La Gaude, France) and the Animal Care and Ethics Committee of the University of New South Wales.

[0518] Immunohistochemical staining of rat retina. Rabbit monoclonal anti-CD31 (cat. ab182981), rabbit monoclonal anti-VCAM-1 (cat. ab134047), and rabbit polyclonal anti-VEGF-A (cat. ab46154) were obtained from Abcam. Rabbit monoclonal phospho-p44 / 42 MAPK (pERK1 / 2, Thr 202 / Tyr 204) (cat. 4370) and rabbit monoclonal FosB (cat. 2251) were obtained from Cell Signaling. Rat eyes were excised, formalin-fixed, and paraffin-embedded. Heat-induced epitope retrieval was performed on all deparaffinized sections (4 μm Superfrost slides) at 110°C for 5 min in citrate buffer, pH 6 (VEGF-A, pERK, VCAM-1) or EDTA buffer, pH 9 (CD31). Sections were blocked with a dual endogenous enzyme blocking agent (cat. S2003, DAKO) for 10 min and then with 2% skim milk for 20 min. MACH3 Rabbit AP-Polymer Detection (Biocare Medical, cat. M3R533 G, H, L) was used for 10 min. After washing with buffer, the slides were further incubated for 10 min with the polymer component of MACH3 Rabbit AP-Polymer Detection (Biocare Medical, M3R533 G, H, L). Slides were then incubated for 7 min with a red chromogen (Warp Red™ Chromogen Kit) and counterstained with hematoxylin and Scott Blue. Slides were dried on filter paper, dehydrated in xylene, and coverslipped. Immunostaining with the designated antibodies was performed simultaneously for all groups. Immunostained slides were scanned using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt Waverley, Vic, Australia), and images were captured using ImageScope software (Leica Biosystems). CD31, VEGF-A 165Positive staining (red dye) for pERK, FosB, and VCAM-1 was assessed using Image-Pro Plus software (Cybernetics, Bethesda, MD). The IODs of the IPL and INL were quantified using Image-Pro Plus. The IODs were CD31, VEGF-A165, pERK, INL, ONL, FosB, GCL, OS, and VCAM-1, respectively. The area of ​​positive immunostaining was expressed as a percentage of the retinal tissue area (Kim, JY, et al., Biomolecules 10, pii E11 (2019)). For quantification, 2–4 sections / eye were photographed with a 20x objective lens for the vehicle and BT2 groups, confirming all wounds. For the untreated group, 1–3 sections / eye were photographed with a 20x objective lens. Staining was quantified in n=3–6 groups. When gradient staining for VEGF-A165 was assessed for wounds, immunostaining was assessed in ten consecutive 100 μm boxes starting 150 μm from the center of the wound (double-headed arrow), and the IOD of each box was quantified with Image-Pro Plus.

[0519] Endothelial cell-monocyte cell adhesion assay. HMECs (80-90% confluent) in 96-well plates were serum-deprived for 24 h, treated with the indicated concentrations of compounds for 1 h, and then incubated with IL-1β (20 ng / ml) for 4 h. THP-1 cells were incubated with 5 μM calcein (5x10 6 The cells were labeled with THP-1 (2.5 × 10 cells / ml, BD Bioscience) at 37°C for 30 min and then washed three times with PBS. 5 After 30 min, unbound cells were washed three times with PBS. Adhesion of calcein-labeled THP-1 to the endothelial layer was measured using a fluorescence plate reader at excitation 485 nm and emission 530 nm.

[0520] Monocyte transendothelial migration assay. Millicell 8 μm polycarbonate culture plate inserts (Millipore) were coated with 0.1% porcine gelatin type A (Sigma) and placed in 24-well plates. HMECs (5 × 104 THP-1 (5x10 cells / well) were seeded onto the inserts and allowed to attach overnight. Cells were then serum-deprived for 24 h and treated with various compounds for 1 h. IL-1β (20 ng / ml) was added to stimulate the cells for 4 h, and 500 μl of serum-free medium was added to the bottom of the 24-well plate along with the compounds. THP-1 (5x10 cells / well in 100 μl) was added to the bottom of the 24-well plate. 5 Cells were added to the insert, and 100 ng / ml MCP-1 (Sigma) was added to the lower well. After 24 h, the number of cells that had migrated through the endothelial layer was assessed by counting 100 μl of the suspension in the lower chamber using a Coulter cell counter (Beckman Coulter).

[0521] Collagen antibody-induced arthritis. Arthritis was induced in female Balb / c mice (6–8 weeks old) as previously described using a commercially available cocktail of five monoclonal antibodies against type II collagen (Chondrex, Inc. Redmond, WA) at 2 mg / mouse. LPS (50 μg / mouse) with or without BT2 (3 or 30 mg / kg mouse) in DMSO vehicle was then administered i.p. on day 3. Hind footpad thickness was measured on day 9 using digital calipers. Mice were sacrificed on day 14, and micro-CT scans of the hind limbs were performed. Animal experiments were approved by the University of New South Wales Animal Care and Ethics Committee.

[0522] Micro-CT scanning and analysis. Formalin-ethanol-fixed hind limbs were subjected to micro-CT scanning using a Siemens Inveon micro-CT scanner (Victoria, Australia) before histological processing. Data were acquired using Inveon Acquisition Workplace with a 16.84 μm pixel size, 360 projections, a 4100 ms integration time, 80 keV photon energy, and 140 μA current. 3D models were visualized, and snapshots of the limbs were taken using Inveon Research Workplace software. Data were quantified for each limb using a binary scale: 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 h before dewaxing. Tissue sections were deparaffinized in 100% xylene, rehydrated in 100, 70, and 30% ethanol, and then washed in distilled water for 5 min. Sections were covered with TRAP staining solution containing 3 mg of tartaric acid per 50 ml of tartrate buffer. Sections were incubated at 37°C for 1 h and then washed three times with distilled water to stop the reaction. Sections were counterstained with hematoxylin for 5 s, washed under running water until clear, and then dried. Sections were dehydrated in xylene, air-dried, and mounted with aqueous permanent mounting medium. Six random fields were selected from the synovial membrane inside the joints of each animal in a blinded study, photographed with a 20x objective. The number of osteoclasts was counted using NIH Image J, and TRAP staining was quantified using IOD (Image-Pro Plus).

[0524] Immunohistochemical staining and analysis of hind paws for VCAM-1 and ICAM-1. Formalin-fixed, paraffin-embedded hind paws were sectioned (5 μm). Immunohistochemical staining for VCAM-1 and ICAM-1 was performed using the Dako EnVision Rabbit Kit (cat. K4011, Dako). Briefly, sections were blocked with peroxidase for 30 min and then immunostained overnight at 4°C with rabbit monoclonal VCAM-1 (cat. ab134047, 1:100, Abcam) or rabbit polyclonal ICAM-1 (cat. ab124759, 1:100, Abcam). Staining was visualized using a polymer-conjugated 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 captured with ImageScope software (Leica Biosystems). The integrated optical density (IOD) of positive staining in the articular cartilage of the ankle joint (tibia and talus) was assessed for VCAM-1 and ICAM-1 using Image-Pro Plus software (Cybernetics, Bethesda, MD, USA). The area (μm) of the articular cartilage of the ankle joint was calculated. 2 ) was measured 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 counted manually using Image-Pro Plus software. Data are expressed as IOD / μm 2 and expressed as the percentage of positively stained cells per 20x objective field.

[0525] Toxicology. Female Balb / c mice (8–9 weeks old) were administered 3 or 30 mg / kg of BT2 (DMSO vehicle) via intraperitoneal injection (DMSO on days 0 and 5), oral gavage (DMSO / methylcellulose on days 0–4), or intra-articular injection (DMSO on day 0). Tissues were fixed in 10% formalin, routinely processed, sectioned at 4 μm, and stained with hematoxylin and eosin. Sections were examined histologically for signs of toxicity by a board-certified physician from the American College of Veterinary Pathologists. Animal studies were approved by the University of New South Wales Animal Care and Ethics Committee.

[0526] Statistics. Statistical analyses were performed using PRISM v7.0d as described in the legend. Differences were considered significant when P<0.05. Where indicated, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0527] result Identification of BT2, T4, and T6. To identify novel small molecule inhibitors of AP-1, the WEHI lead discovery library of ~100,000 compounds was screened using a 293 cell-based assay in which firefly luciferase is driven by multiple copies of the AP-1 response element. Substructure filters were applied during screening to remove pan-assay interfering compounds (PAINS) (Bael, JB, et al., J Med Chem 53, 2719-2740 (2010)), which typically capture the AP-1 inhibitor curcumin (Nelson, KM, et al., J Med Chem 60, 1620-1637 (2017)). This resulted in the discovery of compounds with IC values ​​in the micromolar or submicromolar range, as determined using an 11-point titration curve, including the dibenzoxazepinone BT2. 50Twenty-four usable hits with the benzophenone Cpd B / X / LK001 were obtained. This follows a previous screening of the DIVERSet library (ChemBridge) of 960 compounds, which yielded the 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 was synthesized after screening by reacting commercially available 2-amino-10-ethyldibenzo[b,f][1,4]oxazepin-11(10H)-one (BT3) with diethyl pyrocarbonate (Figure 6B, Scheme 1). Cpd B / X / LK001 was prepared by reacting 2-methoxyethyl carbonisocyanatidate (2) (Krebs, A, et al., European Patent Office EP0230224B1 (1991)) with commercially available (4-aminophenyl)(4-chlorophenyl)methanone (1) (Figure 6B, Scheme 4). Treatment of Cpd B / X / LK001 with hydroxylamine hydrochloride afforded 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-induced endothelial FosB / ΔFosB and c-Fos expression, blocking proliferation, migration, and network formation in vitro. We measured the effects of BT2, T4, and T6 on serum-induced expression of two AP-1 subunits in cultured human microvascular endothelial cells (HMEC-1). Endothelial cells provide an important barrier between the flowing blood and tissues, but they become highly permeable upon activation or stress (van Hinsbergh, VW, et al., Arterioscler Thromb Vasc Biol 17, 1018-1023 (1997)). BT2 blocked the inducible expression of FosB and ΔFosB (Figures 1A & 7A). T4 and T6 were less potent inhibitors, 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)) (FIGS. 1A and 7A).

[0529] Next, we examined the effects of these compounds on endothelial cell proliferation using the xCELLigence system, which monitors cell proliferation in real time. We found that BT2, T4, and T6 each dose-dependently inhibited serum-induced proliferation (Figure 1B). In contrast, BT3 and T7 showed no inhibitory effect. To confirm that the growth inhibition was not simply due to cell death, we tested one of these compounds (BT2) in a whole-cell proliferation assay using the Countess system and trypan blue exclusion. BT2 inhibited serum-induced proliferation without affecting trypan blue uptake (Figure 7B). In a dual-chamber transwell system, BT2, T4, and T6 inhibited bovine aortic endothelial cell (BAEC) migration toward VEGF-A165 in serum-containing medium (Figure 1C). For this purpose, we used BAECs because HMEC-1 cells lack VEGFR-2 (Flk / KDR) and exhibit only weak migration in response to VEGF (Shao, R., et al., Biochem Biophys Res Commun 321, 788-794 (2004)). On the other hand, BAECs express VEGFR-2 (Lamy, S., et al., Cancer Res 62, 381-385 (2002)) and migrate in response to VEGF-A (Hussain, S., et al., BMC Cell Biol 9, 7 (2008)). Neither BT3, PD98059 (an allosteric MEK inhibitor), imatinib (a tyrosine kinase inhibitor), nor tofacitinib (a Janus kinase inhibitor) had any inhibitory effect at the same concentrations (Figure 1C).

[0530] Endothelial cell repair after mechanical injury in vitro induces a proliferative and migratory response. BT2, T4, and T6 blocked this repair response within 48 h, whereas BT3 or T7 had no such effect (Figure 1D). We also evaluated the effects of these compounds in an endothelial network formation assay (also known as tube formation) on reconstituted basement membrane matrix, which is typically used to characterize angiogenic factors and processes (Arnaoutova, I., et al., Angiogenesis 12, 267-274 (2009)). Endothelial cells in this assay maximally form capillary-like networks within a few hours, followed by regression. BT2, T4, and T6 inhibited network formation after 2, 4, 6, and 24 h (Figure 1E).

[0531] BT2 inhibits retinal vascular permeability and neovascularization. Because retinal vascular permeability is a key pathological feature in nAMD and DME / DR (Campochiaro, PA, et al, J Mol Med (Berl) 91, 311-321 (2013)), we sought to measure the effects of BT2, T4, and T6 on fluorescein leakage induced in Brown Norway pigmented rat eyes after multiple laser ablation of Bruch's membrane around the optic nerve (Grossniklaus, HE, et al., Prog Retin Eye Res 29, 500-519 (2010)). BT2 (192 μg) reduced retinal permeability by ~50%, an effect similar to that of aflibercept / Eilea (200 μg given as six intravitreal (IVT) injections over 21 days (days 0, 3, 7, 10, 14, and 17) compared with two injections of BT2 (days 0 and 7)) or triamcinolone acetonide (Kenacort® 200 μg IVT, day 0) (Figure 2A). In contrast, T4 and T6 delivered on BT2 had no inhibitory effect (Figure 2A). Aflibercept is the first-line treatment for nAMD and DME in the United States, Europe, and the Asia-Pacific region (Parikh, R., et al., Ophthalmol Retina 3, 16-26 (2019)), and Kenacort is a corticosteroid commonly used to treat DME (Karacorlu, M., Eye (Lond) 19, 382-386 (2005)). BT2 also inhibits rhVEGF-A in pigmented rabbits, causing fluorescein leakage. 165 BT2 reduced vascular permeability induced by VEGF-A. A single intravenous administration of BT2 (600 μg) suppressed retinal leakage by ~50% after 2 days (Figure 2B). Immunohistochemical staining of laser-irradiated rat eyes 21 days after injury revealed that BT2 inhibited induced CD31 staining in the IPL and INL (Figures 2C and 8A), where CD31 is expressed, after laser injury (Ju, X, et al., Clin Exp Pharmacol Physiol 46, 75-85 (2019)). BT2 also inhibited VEGF-A. 165VEGF-A suppressed the inducible expression of VEGF (Figure 2D), consistent with the finding that VEGF is primarily 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 The staining of the BT2-inhibited wound was performed in a gradient manner (Figure 2E). The anti-angiogenic effect of BT2 was confirmed by a mouse Matrigel plug assay. VEGF-A 165 Matrigel containing BT2, heparin, and the compounds was implanted subcutaneously into C57BL / 6 mice, and CD31 staining in the plugs was quantified 7 days later. BT2 inhibited neovascularization, whereas BT3 had no effect (Figures 2F & 8B).

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

[0533] RNA-seq analysis confirmed that BT2 suppressed IL-1β-induced FosB and VCAM-1 expression (Figure 3C). From a pool of 33,379 gene IDs, there were 325 genes induced by IL-1β by more than two-fold (logFC ≥ 2) (Table 3C), of which 89 (27.5%) were inhibited by BT2 (logFC ≥ 2) (Table 3B). Principal component analysis (PCA) (Figure 3C, top left) demonstrated close correlation between biological replicates. 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 FosB (Figure 3C).

[0534] Dose-escalation and Western blotting experiments demonstrated that BT2 inhibited VCAM-1 and ERK phosphorylation more potently than PD98059 (Figures 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 dependence of VCAM-1 expression on FosB / ΔFosB, we performed siRNA knockdown experiments. FosB siRNA inhibited both FosB / ΔFosB and VCAM-1, whereas VCAM-1 siRNA inhibited VCAM-1 but not FosB / ΔFosB (Figure 3E). ERK1 overexpression did not increase the levels of phosphorylated ERK, nor did it increase the levels of FosB, ΔFosB, or VCAM-1 compared with IL-1β stimulation (Figure 11C). Similarly, overexpression of FosB or ΔFosB did not increase VCAM-1 expression compared with 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 FosB or ΔFosB overexpression. These data complement our demonstration (under agonist-stimulated conditions) that BT2, which prevents ERK phosphorylation, abolishes the induction of FosB / ΔFosB and VCAM-1 in vitro (Figure 3A-D) and in vivo (Figure 4A-E). Indeed, 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, together with our demonstration that IL-1β induction of VCAM-1 was blocked by FosB siRNA (Fig. 3E), suggested that while FosB is required for cytokine-induced VCAM-1 expression, overexpression of FosB alone is insufficient to induce VCAM-1 without cytokine stimulation. FosB appears to be dependent on cofactors (or post-translational modifications) under cytokine-stimulated conditions.

[0536] Immunohistochemical staining of rat retinas revealed that BT2 suppressed inducible pERK staining in the INL, OPL, and ONL (Figure 4A), consistent with pERK expression in these areas (Takeda, M, et al., Invest Ophthalmol Vis Sci 43, 907-911 (2002); Caicedo, A, et al., Exp Eye Res 81, 38-47 (2005)). BT2 also suppressed FosB immunostaining in the retina (Figure 4B). Furthermore, BT2 suppressed inducible VCAM-1 expression in the OLM (Figure 4C), where others have found VCAM-1 expression (Makhoul, M., et al., Exp Eye Res 101, 27-35 (2012)). BT2 also inhibited FosB (Figures 4D & 8C) and VCAM-1 (Figure 4E) immunoreactivity in Matrigel plugs.

[0537] In further experiments, the biological potency of BT2 was compared to that of curcumin (Ye, N., et al., J Med Chem 57, 6930-6948 (2014)) in an endothelial network formation assay. BT2 abolished network formation after 4 h at 1 μM, whereas 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 is over 40-fold more potent than curcumin in this assay.

[0538] BT2 structural analogs lack the biological potency of BT2. Next, we investigated whether structural modifications could improve the biological potency and solubility of BT2. Dibenzoxazepinones are generally poorly soluble in water. Six BT2 analogs (BT2-MeOA, BT2-EOMe, BT2-Pr, BT2-IC, BT2-MO, and BT2-IMO) were prepared (Table 1), excluding BT3. BT2-MeOA was synthesized by coupling 2-amino-10-ethyldibenzo[b,f][1,4]oxazepin-11(10H)-one (BT3) with methoxyacetic acid, and 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). BT2-Pr and BT2-EOMe were also prepared from commercially available compounds (1) and (2). For stability analysis, 2-nitro-10H-dibenzo[b,f][1,4]oxazepin-11-one (3) was alkylated with d3-iodoethane and the nitro group was reduced to synthesize the tritiated derivative of BT2 (Figure 6B, Scheme 3). This intermediate was reacted with diethyl pyrocarbonate to obtain the desired product. Alkylation of 2-nitro-10H-dibenzo[b,f][1,4]oxazepin-11-one (3) with oxetan-3-ylmethyl methanesulfonate afforded the O- and N-alkylated products in 13% and 48% yields, respectively, after column chromatography (Figure 6B, Scheme 3). The O- and N-alkylated products were reduced to the corresponding anilines (5) and (6) (Figure 6B, Scheme 3), which were then converted to the desired products BT2-IMO and BT2-MO using diethylpyrocarbonate in the usual manner.

[0539] When these compounds were diluted in serum-containing medium, only one of these analogs (BT2-MeOA) was found to be more soluble than BT2, while BT3 was found to be the most soluble of all these dibenzoxazepinones. The addition of serum to the diluent increased the solubility of BT2, consistent with reports that serum albumin can enhance the solubility of drug combinations (Khoder, M., et al., Pharm Dev Technol 23, 732-738 (2018)). Neither BT2-MeOA nor other BT2 analogs had the ability to inhibit serum-induced proliferation (Figure 5A) or network formation on Matrigel (Figure 5B) as well as or more potently than BT2. BT2-IC showed some inhibition of network formation at higher concentrations (Figure 13B).

[0540] Because BT2 inhibited ERK phosphorylation, we hypothesized that BT2 might interact with MEK1 or MEK2. We tested the binding of BT2 and PD98059 to recombinant His-MEK-1 or His-MEK-2 by surface plasmon resonance (SPR). Over the assayable concentration range, BT2 bound significantly better to His-MEK1 than to 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). Over comparable concentration ranges, no significant binding of BT3, BT2-MeOA, or BT2-Pr to MEK1 or MEK2 was observed. BT2-IC showed some interaction with MEK1 (but not MEK2). The decreased interaction at high concentrations is due to the insolubility of BT2-IC ( 1 This is likely due to its solubility limit of 8 ± 2 μM as determined by H 1D NMR spectroscopy. Western blotting revealed that BT2-IC inhibited both ERK phosphorylation (Figure 13A) and network formation (Figure 13B) at 3 μM (although less potently than BT2) but not at 1 μM (Figure 5B & D).

[0541] BT2 maintains stability and biological activity after sonication and autoclaving at 100°C. Finally, considering the potential of BT2 as a pharmaceutical, we investigated whether this compound (as a sonicated formulation in saline containing 0.01% DMSO and 0.5% Tween 80) retains its biological potency and stability after extreme heat treatment. High-performance liquid chromatography / tandem mass spectrometry (RRLC-MS / MS) demonstrated that BT2 was stable with or without heat treatment (100°C, 10 min), with only a 0.2% and 1% difference in BT2 content between unheated and heat-treated formulations, respectively, after 6 weeks of storage at 22°C (Figure 14A-B). BT2 retained its ability to inhibit serum-induced endothelial proliferation under these conditions (Figure 14C). Even more striking, there was no loss of biological potency or degradation after up to 16 months (Figure 14D-F). Remarkably, the BT2 formulation remained stable and biologically active after 4 months of standard autoclaving and storage at 22°C (Figure 14G). Antibodies and other proteins that comprise all current nAMD / DME treatments 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 to human umbilical vein endothelial cells (Gerszten, RE, et al., Circ Res 82, 871-878 (1998)). In an in vitro model involving calcein-labeled THP-1 monocytes and endothelial cells pretreated with IL-1β, THP-1 adhesion to endothelial cells is inhibited by BT2 (Figure 15A). BT2 also inhibits transendothelial migration of THP-1 monocytes from the upper to the lower chamber toward MCP-1 (Figure 15B).

[0543] Intraperitoneal administration of BT2 inhibited footpad swelling, bone destruction, and VCAM-1 and ICAM-1 expression in arthritic mice. Having established the antiangiogenic and anti-inflammatory effects of BT2 in vitro, we hypothesized that BT2 might also be useful in complex inflammatory environments such as collagen antibody-induced arthritis (Khachigian, LM Nature Protocols 1, 2512-2516 (2006)). Hind footpad thickness induced in this model was suppressed by a single dose of 30 mg / kg BT2 (Figures 16A and 16B). H&E staining revealed that BT2 attenuated the significant inflammation in injected CAIA mice (Figure 16C). 3D microCT analysis of the hind paws revealed that BT2 suppressed bone destruction (Figures 16D and 16E). To support these findings, we utilized tartrate-resistant acid phosphatase (TRAP) activity, an important histochemical marker of bone-degrading osteoclasts (Ballanti, P., et al., Osteoporosis International 7, 39-43 (1997)). BT2 reduced TRAP staining in the joints (Figure 16F). BT2 was found to suppress VCAM-1 and ICAM-1 expression in bone (Figure 16G). Furthermore, BT2 (30 mg / kg) reduced 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 BT2 toxicity following intraperitoneal, intraarticular, or oral gavage administration. BT2 (3 or 30 mg / kg) was administered to Balb / c mice by one of three routes (intraperitoneal injection, intraarticular injection, or oral gavage), and tissues were evaluated for signs of toxicity. There was no histopathological evidence of BT2-induced toxic injury (Table 2). The livers of most mice in all groups contained minimal to mild, infrequent inflammatory foci, occasionally accompanied by necrosis of individual hepatocytes or small clusters of hepatocytes. This was considered a spontaneous background lesion common 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. The livers of most mice in the ip-administered group showed minimal to mild inflammation on the capsule, consistent with a nonspecific peritoneal reaction to the injection, an effect unrelated to the test item. Rare inflammatory foci were observed in the kidneys of 1 of 5 control mice and 4 of 30 BT2-treated mice. These were also spontaneous background lesions common in experimental mice and unrelated to the test item. The inflammation involving the renal pelvis was likely due to ascending bacterial infection of the urinary tract. Other very rare and minimal changes unrelated to treatment group were observed in the liver and lungs. In summary, there was no histopathological evidence of toxicity following intraperitoneal, intra-articular, or oral gavage administration of BT2.

[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 demonstrated an intraocular half-life (t 1 / 2 ) was found to be well tolerated both macroscopically and organizationally, with a survival time of 3.3 days.

[0546] Table 2. Severity assessment of histopathological findings after intra-articular, intraperitoneal, or oral gavage administration of BT2. The severity of the lesions was histologically graded as follows: 0 = no abnormalities, 1 = minimal changes, 2 = mild changes, 3 = moderate changes, 4 = severe changes, NA = not assessed. n = 5 mice per group. IA indicates intra-articular, 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 Novel therapeutic approaches to complement existing VEGF-based strategies for nAMD / DR are needed (Apte, RS, et al., Cell 176, 1248-1264 (2019)). While IVT anti-VEGF remains the first-line treatment for retinal leakage, many patients experience suboptimal or unsustained responses, necessitating alternative therapies. The Comparison of AMD Treatments Trials (CATT) study, which included 647 nAMD patients treated with ranibizumab or bevacizumab, showed that visual gains over the first 2 years were not maintained at 5 years (Maguire, MG, et al., Ophthalmology 123, 1751-1761 (2016); Pedrosa, AC, et al., Clin Ophthalmol 10, 541-546 (2016)). In addition, the AURAiv trial, which involved 2,227 nAMD patients in eight European countries, found that although anti-VEGF therapy resulted in initial improvements in vision, the gains were not sustained over the long term and declined primarily due to undertreatment (Holz, FG, et al., Br J Ophthalmol 99, 220-226 (2015)).

[0552] Herein, we report the discovery and biological properties of a novel dibenzoxazepinone obtained from a high-throughput screen of ~100,000 compounds. BT2 blocks cell proliferation, migration, wound repair, and network formation in vitro. This compound has demonstrated 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, C.K., et al., J Ocul Pharmacol Ther 27, 219-224 (2011)), and has served as a primary platform for the development of nAMD / DR therapies that are used by millions of people today. BT2 demonstrated comparable efficacy to first-line therapy for nAMD and DME in reducing retinal vascular permeability after choroidal laser injury in rats, with six doses of aflibercept compared with two doses of BT2. BT2 reduced CD31 staining in the IPL and INL, consistent with VEGF-A gain-of-function studies in amacrine and horizontal cells after crossing Ptf1a-Cre mice with floxed Vhl (Vhlf / f) mice to induce simulated hypoxia, revealing extensive neovascularization in the IPL and INL (Usui, Y, et al., J Clin Invest 125, 2335-2346 (2015)). In rabbits, BT2 inhibited VEGF-A. 165 We found that this compound inhibited retinal vascular leakage induced by α-glucan.

[0553] BT2 is VEGF-A 165Although BT2 inhibited the inducible expression of FosB, its effects in the retina were not limited to VEGF. BT2 inhibited 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. BT2 also inhibited various other genes involved in cell proliferation, migration, angiogenesis, and inflammation. BT2 is more potent than PD98059 and over 40 times more potent than curcumin, the main active ingredient in the golden spice turmeric, which inhibits AP-1 (Ye, N., et al., J Med Chem 57, 6930-6948 (2014)). Curcumin is widely used medicinally despite the failure of double-blind, placebo-controlled clinical trials (Nelson, KM, et al., J Med Chem 60, 1620-1637 (2017)).

[0554] We synthesized BT2 analogs bearing various substituents at the 2- and 10-positions of the 2-amino-dibenz[b,f][1,4]oxazepin-11(10H)-one ring system. Minor modifications of the carbamate moiety (BT2-MeOA and BT2-IC) significantly affected 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 had lower calculated log Ps, which we predicted would improve water solubility. Although BT2-MeOA (and BT3) were more soluble than BT2, two assays demonstrated that BT2 remained the most biologically active of these compounds, suggesting that larger substituents at the 2- and 10-positions were not necessarily more effective. Comparison of BT2 with its isomer, BT2-MeOA (amide bond), reveals that the 2-carbamate moiety of BT2 is important for its function. BT2 may be compatible with lipid-based drug delivery systems, such as self-emulsifying delivery systems, which have improved the oral absorption of poorly water-soluble drugs and facilitated high-dose toxicity testing (Chen, XQ, et al., J Pharm Sci 107, 1352-1360 (2018)).

[0555] Although rodent and rabbit models are useful for reproducing certain features of human retinal disease, they cannot fully replicate the human condition because nAMD and DR are complex, multifactorial, and chronic diseases that cannot be accurately reproduced through acute experiments with a single stimulus (Robinson, R., et al., Dis Model Mech 5, 444-456 (2012)). Rats have the advantage of rapid disease progression and being relatively inexpensive, but rats (like mice) do not have a macula (Pennesi, ME, et al., Mol Aspects Med 33, 487-509 (2012)). Although rabbit eyes are similar in size to humans, the posterior segment of the eye is vascularized differently from primates and rodents, and rabbits do not have a macula (Chen, S., et al., Expert Rev Opthalmol 9, 285-295 (2014)). BT2 can overcome the translatability limitations that have hindered the widespread use of humanized and species-specific reagents in animal models (Lu, F., et al., Graefes Arch Clin Exp Ophthalmol 247, 171-177 (2009)).

[0556] Extraretinal effects of BT2. New, effective anti-inflammatory and anti-arthritic drugs are also needed. Approximately one-third of patients treated with TNF inhibitors do not achieve a 20% improvement based on 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 appears to be related to the serum IFN-β / α ratio (Wampler Muskardin, T, et al., Annals of the Rheumatic Diseases 75, 1757-1762 (2016)). p-ERK levels are elevated in the synovial tissue of RA patients compared with normal controls (Thiel, MJ, et al., Arthritis Rheum 56, 3347-3357 (2007)). Furthermore, serum sVCAM1 levels reflect the clinical status of RA (Navarro-Hernandez, RE et al., Disease Markers 26, 119-126 (2009)) and decrease with symptomatic improvement in RA patients (Wang, L., et al., Experimental and Therapeutic Medicine 10, 1229-1233 (2015)). We found that systemic administration of BT2 in CAIA mice suppressed joint inflammation and bone erosion. BT2 also inhibited monocyte adhesion to endothelial cells and transendothelial migration toward MCP-1 in vitro. Furthermore, systemic administration of BT2 in mice suppressed footpad swelling, TRAP staining, and bone destruction. Inflammation is thought to promote all aspects of atherosclerosis, from the initiation to progression of atherosclerosis, ultimately leading to plaque rupture and infarction, which then trigger further inflammation.Recent clinical trials, including 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 demonstrated that inflammation is a therapeutic mechanism in cardiovascular disease. However, patients treated with existing anti-inflammatory approaches (e.g., canakinumab and colchicine) remain at substantial risk for major adverse cardiac events, even with the widespread use of statins and antiplatelet therapy (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)). Furthermore, clinically effective anti-inflammatory small molecule drugs for cardiovascular disease other than statins are lacking (Collins, R. et al. Lancet 388, 2532-61 (2016)). This highlights the therapeutic potential of BT2 in inflammatory diseases, including RA.

[0557] In conclusion, BT2 offers a new tool in the medical armamentarium for targeting vascular permeability, angiogenesis, and inflammatory conditions. BT2 served as a molecular tool for establishing the ERK-FosB-VCAM1 axis, which mediates vascular permeability. This, along with its favorable toxicity profile, suggests the compound's clinical utility in retinal diseases and rheumatoid arthritis. Unlike current clinical treatments using antibodies and proteins that primarily target VEGF, BT2 suppresses the inducible expression of multiple genes underlying 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 pharmaceutical appeal. Like triamcinolone acetonide, BT2 is poorly soluble in water, which may offer the added advantage that bolus injection can form a depot at the injection site that promotes sustained release (Yang, Y., et al., Retina 35, 2440-2449 (2015)). Furthermore, BT2 can be used in internal reservoir or implant strategies and ocular delivery systems that promote sustained release (Kang-Mieler, JJ, et al. Eye (Lond) 34, 1371-1379 (2021)).

[0558] In the claims that follow and in the preceding specification of the invention, unless the context requires otherwise by express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., a sense that specifies the presence of stated features but does not exclude the presence or addition of further features in various embodiments of the 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. A pharmaceutical composition for reducing vascular permeability, neovascularization, angiogenesis, inflammation, cell migration and / or cell proliferation in a subject, comprising: A compound of the formula: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for reducing AP-1 subunit expression, and / or reducing ERK1 / 2 phosphorylation, and / or reducing FosB / ΔFosB expression, and / or reducing VCAM-1 expression, and / or reducing VEGF-A expression in a subject, comprising: A compound of the formula: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

3. 1. A pharmaceutical composition for reducing AP-1 subunit expression, and / or reducing ERK1 / 2 phosphorylation, and / or reducing FosB / ΔFosB expression, and / or reducing VCAM-1 expression, and / or reducing VEGF-A expression in a cell, comprising: A compound of the formula 【Transformation 3】 or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition for inhibiting ERK1 / 2 phosphorylation in a subject, comprising: an effective amount of: 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

5. An in vitro method for inhibiting ERK1 / 2 phosphorylation in a subject, comprising: Incubating the ERK1 / 2 molecule with an effective amount of a compound of the formula: 【Transformation 5】 A method comprising: 【Request Item 6】 【Transformation 6】 or a pharmaceutically acceptable salt thereof. 【Request Item 7】 【Chemistry 7】 or a pharmaceutically acceptable salt thereof for treating or preventing a condition or disease in a subject selected from the group consisting of: ·arthritis; - rheumatoid arthritis; - bone destruction; Age-related macular degeneration; ・Diabetic retinopathy; -Macular edema; ・Vascular leakage; ・Vascular permeability; - retinal vascular permeability; ・Angiogenesis; ・Endothelial cell dysfunction; - atherosclerosis; ·stroke; Myocardial infarction; peripheral vascular disease; ·constriction; ・Restenosis; ·inflammation; - Cytokine storm; ・Pulmonary inflammation; -Pulmonary fibrosis. 【Request Item 8】 【Transformation 8】 or a pharmaceutically acceptable salt thereof.

9. The pharmaceutical composition of claim 1, wherein the compound is an E isomer or a Z isomer or a mixture of E and Z isomers.

10. The kit of claim 6, wherein the compound is an E isomer or a Z isomer or a mixture of E and Z isomers.

11. The pharmaceutical composition of any one of claims 1 to 4, or 7, wherein the compound is deuterated.

12. The kit of claim 6, wherein the compound is deuterated.

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