Flavonoid Compounds and Methods and Materials Using Flavonoid Compounds for Treating Fibrotic Conditions - Patent application

Abstract

The present invention relates to flavonoid compounds and methods and materials using the flavonoid compounds for treating one or more fibrotic conditions (e.g., idiopathic pulmonary fibrosis (IPF), nonalcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), and / or ocular fibrosis). For example, one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) to treat the mammal.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application Serial No. 63 / 351,485, filed June 13, 2022. The disclosure of this prior application is considered part of the disclosure of this application and is incorporated by reference into the disclosure of this application.

[0002] The present invention relates to flavonoid compounds and methods and materials that use flavonoid compounds to treat fibrotic conditions. [Background technology]

[0003] Fibrotic diseases are a major cause of morbidity and mortality and can affect nearly every tissue and organ system. The U.S. government estimates that 45% of deaths in the United States can be attributed to fibrotic diseases. Summary of the Invention

[0004] The present invention provides flavonoid compounds and methods and materials using the flavonoid compounds to treat a mammal (e.g., a human) having one or more fibrotic conditions (e.g., idiopathic pulmonary fibrosis (IPF), nonalcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), and / or ocular fibrosis). For example, the present invention provides flavonoid compounds having the structure of Formula (I), as well as methods and materials using one or more flavonoid compounds having the structure of Formula (I). In some cases, one or more flavonoid compounds having the structure of Formula (I) can be administered to a mammal (e.g., a human) having one or more fibrotic conditions to treat the condition in the mammal. As provided herein, one or more flavonoid compounds having the structure of Formula (I) can induce apoptosis in senescent cells (e.g., senescent fibroblasts) and can be used to treat a fibrotic condition in a mammal (e.g., a human).

[0005] Generally, one aspect of the invention is a compound of formula (I):

[0006] [ka] or a pharmaceutically acceptable salt thereof (wherein: R 1 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 2 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 3 is H, CH2CH3, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy. The flavonoid compound of formula (I) has the structure:

[0007] [ka] The flavonoid compounds of formula (I) may have the structure:

[0008] [ka] The flavonoid compounds of formula (I) may have the structure:

[0009] [ka] The composition may also include a pharmaceutically acceptable carrier, excipient, or diluent.

[0010] In another aspect, the present invention provides a compound of formula (I):

[0011] [ka] or a pharmaceutically acceptable salt thereof (wherein: R 1 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R2 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 3 is H, CH2CH3, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy) and a pharmaceutically acceptable carrier, excipient, or diluent.

[0012] In another aspect, the invention features a method of treating a mammal having a fibrotic condition, the method comprising administering to a mammal a compound of formula (I):

[0013] [ka] or a pharmaceutically acceptable salt thereof (wherein: R 1 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 2 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 3 is H, CH2CH3, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4The method may comprise, or consist essentially of, administering to a mammal having a fibrotic condition a composition comprising: (I) a hydroxyl group selected from the group consisting of hydroxyl group (I) and hydroxyl group (I) (wherein I is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy). The mammal may be a human. The method may comprise determining that the mammal has a fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis. The fibrotic condition may be IPF, and the method may comprise administering to the mammal a drug used to treat IPF. The medication used to treat IPF can be pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC12249, AM966, AM095, taribavirin, BI-2545, GLPG1690, BBT877, SAR100842, BMS-986,020, minaprine, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, ripasudil, netarsudil, belmosudil, lebrikizumab, tralokinumab, dupilumab, or pamrevlumab. The fibrotic condition can be PSC, and the method can include administering to the mammal an agent used to treat PSC, which can be ursodeoxycholic acid (UDCA), a corticosteroid, a bile acid sequestrant, an antibiotic, or an antihistamine.

[0014] In another aspect, the invention features a method for reducing fibrosis in a mammal having a fibrotic condition, the method comprising administering to a mammal a compound of formula (I):

[0015] [ka] or a pharmaceutically acceptable salt thereof (wherein: R 1 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 2 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R3 is H, CH2CH3, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4 The method may comprise, or consist essentially of, administering a composition comprising: (I) a fibrotic compound (Is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy) to a mammal having a fibrotic condition. The mammal may be a human. The method may comprise determining that the mammal has a fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis.

[0016] In another aspect, the invention features a method for reducing the number of senescent cells in a mammal having a fibrotic condition. The method comprises administering to a mammal a compound of formula (I):

[0017] [ka] or a pharmaceutically acceptable salt thereof (wherein: R 1 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 2 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 3 is H, CH2CH3, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4 The method may comprise, or consist essentially of, administering a composition comprising a nucleotide sequence comprising: a nucleotide sequence selected from the group consisting of nucleotides (wherein nucleotides are H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy) to a mammal having a fibrotic condition. The mammal may be a human. The method may comprise determining that the mammal has a fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis. The senescent cells may be fibroblasts. The fibrotic condition may be IPF, and the senescent cells may be pulmonary fibroblasts. The senescent cells may be epithelial cells. The fibrotic condition may be PSC, and the senescent cells may be cholangiocytes.

[0018] In another aspect, the invention features a method of inhibiting a serine / threonine kinase 17 (STK17) polypeptide in a mammal. The method comprises inhibiting a serine / threonine kinase 17 (STK17) polypeptide comprising a compound of formula (I):

[0019] [ka] or a pharmaceutically acceptable salt thereof (wherein: R 1 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 2 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy, and R 3 is H, CH2CH3, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4 is H, OH, C1-C4 alkyl, halogen, or C1-C4 alkoxy) to a mammal. The mammal may be a human. The STK17 polypeptide may be an STK17A (DRAK1) polypeptide or an STK17B (DRAK2) polypeptide.

[0020] In another aspect, the present invention provides a compound of formula (II):

[0021] [ka] or a pharmaceutically acceptable salt thereof (wherein X 1 is selected from N and CH, and X 2 N and CR 4 Selected from R 1 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3is selected from the group consisting of H, CH, CHCH, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl, and R 4 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy. The flavonoid compound of formula (II) is represented by the following formula:

[0022] [ka] TIFF2025525323000012.tif83154 or a pharmaceutically acceptable salt thereof. The composition may be a pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically acceptable carrier, excipient, or diluent.

[0023] In another aspect, the invention features a method of treating a mammal having a fibrotic condition, the method comprising administering to a mammal a compound of formula (II):

[0024] [ka] or a pharmaceutically acceptable salt thereof (wherein X 1 is selected from N and CH, and X 2 N and CR 4 Selected from R 1 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3is selected from the group consisting of H, CH, CHCH, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl, and R 4 The method may comprise, or consist essentially of, administering to a mammal having a fibrotic condition a composition comprising: a hydroxyl group selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; the mammal may be a human; and the method may comprise determining that the mammal has the fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis. The fibrotic condition may be IPF, and the method may also comprise administering to the mammal a drug used to treat IPF. The drug used to treat IPF can be pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC12249, AM966, AM095, taribavirin, BI-2545, GLPG1690, BBT877, SAR100842, BMS-986,020, minaprine, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, ripasudil, netarsudil, belmosudil, lebrikizumab, tralokinumab, dupilumab, or pamrevlumab. The fibrotic condition can be PSC, and the method can also include administering to the mammal an agent used to treat PSC, which can be UDCA, a corticosteroid, a bile acid sequestrant, an antibiotic, or an antihistamine.

[0025] In another aspect, the invention features a method for reducing fibrosis in a mammal having a fibrotic condition, the method comprising administering to a mammal a compound of formula (II):

[0026] [ka] or a pharmaceutically acceptable salt thereof (wherein X 1 is selected from N and CH, and X 2 N and CR 4 Selected from R 1 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3 is selected from the group consisting of H, CH, CHCH, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl, and R 4 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy) to a mammal having a fibrotic condition. The mammal may be a human. The method may include determining that the mammal has the fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis.

[0027] In another aspect, the invention features a method for reducing the number of senescent cells in a mammal having a fibrotic condition. The method comprises administering to a mammal a compound of formula (II):

[0028] [ka] or a pharmaceutically acceptable salt thereof (wherein X 1 is selected from N and CH, and X 2 N and CR 4 Selected from R 1 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3 is selected from the group consisting of H, CH, CHCH, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl, and R 4 The method may comprise, or consist essentially of, administering a composition comprising a nucleotide sequence selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy to a mammal having a fibrotic condition. The mammal may be a human. The method may comprise determining that the mammal has the fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, and ocular fibrosis. The senescent cells may be fibroblasts. The fibrotic condition may be IPF, and the senescent cells may be pulmonary fibroblasts. The senescent cells may be epithelial cells. The fibrotic condition may be PSC, and the senescent cells may be cholangiocytes.

[0029] In another aspect, the invention features a method of inhibiting an STK17 polypeptide in a mammal, the method comprising inhibiting an STK17 polypeptide comprising a compound of formula (II):

[0030] [ka] or a pharmaceutically acceptable salt thereof (wherein X 1 is selected from N and CH, and X 2 N and CR 4 Selected from R 1 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3is selected from the group consisting of H, CH, CHCH, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl, and R 4 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy) to a mammal. The mammal may be a human. The STK17 polypeptide may be an STK17A (DRAK1) polypeptide or an STK17B (DRAK2) polypeptide.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0032] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram showing an exemplary chemical synthesis of a flavonoid compound having the structure of Formula (I). [Figure 2]Figure 1 shows the effect of F-19 on resident lung cell populations after bleomycin injury and pulmonary fibrosis. Seven days before injury, col1a2-mTmG mice were treated with tamoxifen to initiate GFP expression in collagen-producing fibroblasts. F-19 was administered daily starting on day 10 after injury. On day 14, lungs were isolated and flow-sorted into fibroblasts, epithelial cells, and leukocytes. Sorted cells were subjected to RNA isolation, followed by qPCR analysis of the indicated genes in each cell population. N = 3 mice per group. No changes in senescence markers or inflammatory cytokines were measured in leukocytes (CD45+ cells), suggesting selectivity. [Figure 3-1] Figures 3A-3C show the efficacy of F-4N in a treatment-resistant model of pulmonary fibrosis (aged mice). Figure 3A is a graph showing the survival rate of mouse groups. On day 0, 10- to 18-month-old C57 / B6 mice were intratracheally administered bleomycin. On day 14, one group was administered F-4N (10 mg / kg daily, intraperitoneally). Figure 3B includes images showing trichrome staining of lung histology and a bar graph showing hydroxyproline content analysis for mouse groups (sham, Bleo + vehicle, and Bleo + F-4N). Figure 3C includes a bar graph showing the whole-lung expression of type I collagen and senescence markers (Cdkn2a, Cdkn1a, and Ccl2). N = 7-9 male and female mice. [Figure 3-2] Continued from Figure 3-1. [Figure 4] Figures 4A-4B show the mechanism of action of F-4N. Figure 4A shows a Z-score plot for a kinome screen of 1 μM F-4N against approximately 400 kinases. Dose-response curves for STK17A and STK17B show half-maximal inhibitory concentration (IC50) values of approximately 200 nM. Figure 4B includes a graph showing the expression of putative targets in senescent lung fibroblasts, demonstrating that STK17A / B are dramatically overexpressed. [Figure 5-1]Figures 5A-5B include a comparison of the effects of siRNA targeting STK17A / B in senescent (etoposide-induced) and proliferating (pro) lung fibroblasts, showing that STK17A / B selectively regulates senescent lung fibroblasts. Figure 5A includes a bar graph showing the relative cell number per field for senescent (sen) or proliferating (pro) cells transfected with non-targeting (NT) siRNA or STK17A and STK17B (STK17A / B) siRNA. N=2. After 4 days, cells were fixed and stained for DAPI (cell number) and cleaved caspase-3 (apoptosis). Figure 5B includes a bar graph showing cells counted by automated imaging software. Cells were transfected, and after 4 days, total RNA was isolated and qPCR analysis was performed. N=3. [Figure 5-2] Continued from Figure 5-1. [Figure 6] Biochemical and histological examination of fibrosis in Mdr2- / - mice treated with vehicle or F-4N. Mdr2- / - mice (18-20 weeks old) with established liver fibrosis were treated with F-4N (10 mg / kg / day i.p., n=10) or vehicle (i.p., n=10) for 14 days and then sacrificed the following day. Liver tissue and serum were collected for analysis. Left panel. Hydroxyproline assay in liver tissue revealed that F-4N treatment reduced collagen content by approximately 40% compared with vehicle control-treated mice. Right panel. Picrosirius red staining of mouse liver, which selectively visualizes collagen fibers, demonstrates a reduction in collagen fibers in F-4N-treated mice, particularly in the portal-to-portal region ("bridging fibrosis"). [Figure 7] Liver function tests are improved in Mdr2− / − mice after F-4N treatment. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP), markers of liver injury, and serum bile acid levels (a marker of bile duct injury and cholestasis) were significantly reduced in Mdr2− / − mice treated with F-4N compared with vehicle-treated Mdr2− / − mice. [Figure 8]Figure 1 shows that markers of fibrosis, inflammation, and senescence were reduced by reverse transcriptase (RT)-PCR of whole liver RNA. RT-PCR was performed for the fibrosis marker collagen 1A1 (Col1a1), the inflammatory markers interleukin 6 (Il6) and CC motif chemokine ligand 2 (Ccl2), and the senescence markers cyclin-dependent kinase inhibitors 1A (Cdkn1a) and 2A (Cdkn2a). Each marker was significantly reduced in Mdr2- / - mice treated with F-4N compared with vehicle-treated Mdr2- / - mice. [Figure 9-1]9A-9R show three-point dose-response curves of senescent and healthy fibroblasts treated with flavonols: (FIG. 9A) quercetin, (FIG. 9B) fisetin, (FIG. 9C) 2-(3,4-dimethoxyphenyl)-3-hydroxy-6,8-dimethylchromen-4-one, (FIG. 9D) 6-chloro-2-(3,4-dimethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one, (FIG. 9E) flavonol, (FIG. 9F) 4'-methoxy-3-flavonol, (FIG. 9G) 4'-hydroxy-3'-methoxy-flavone, (FIG. 9H) 4'-hydroxy-3'-methoxy-flavone, (FIG. 9I) 4'-hydroxy-3'-methoxy-flavone, (FIG. 9J ... Figure 9H) 3-hydroxy-2-(4-methoxyphenyl)-6-methyl-4h-1-benzopyran-4-one, (Figure 9I) 2-(3,4-dimethoxyphenyl)-3-hydroxy-6-methyl-4h-1-benzopyran-4-one, (Figure 9J) 2-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-6-methylchromen-4-one, (Figure 9K) 3-hydroxy-6-methyl-flavone, (Figure 9L) 3-hydroxy hydroxy-2-(3-methoxyphenyl)-4h-1-benzopyran-4-one, (Figure 9M) 3',4'-dihydroxy-flavone, (Figure 9N) 3-hydroxy-7-(phenylmethoxy)-2-(3,4,5-trimethoxyphenyl)-4h-1-benzopyran-4-one, (Figure 9O) 2-(3,4-dimethoxyphenyl)-3-hydroxy-7-methyl-4h-1-benzopyran-4-one, (Figure 9P) 3-hydroxy-7-(phenylmethoxy)-2-(3,4,5-trimethoxyphenyl)-4h-1-benzopyran-4-one, TGFβ-stimulated collagen deposition in senescent fibroblasts (circle data points) or low-passage lung fibroblasts (square data points) was investigated by treating senescent fibroblasts with increasing doses of either 2-(3,4-diethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one (Figure 9Q), or 3-hydroxy-3',4'-dimethoxyflavone (Figure 9R). For senescent fibroblast viability experiments (circle data points), human adult lung fibroblasts were replicatively passaged until senescence (confirmed by staining for RNA and senescence-associated beta-galactosidase). Cells were then seeded into 96-well plates, treated with the indicated compounds, and incubated for 96 hours. Cells were then fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and nuclear stained using DAPI.Cells were then imaged using a 4x objective on a Cytation5 microscope, and cell number was quantified using automated software (Biotek Gen5). Data shown were plotted as % viability, normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For TGFβ-stimulated collagen deposition experiments (square data points), human adult lung fibroblasts (passage 3) were seeded in 96-well plates and treated with the indicated compound plus 2 ng / mL TGFβ to stimulate collagen expression. Cells were incubated for 96 hours, fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and stained with a primary antibody recognizing type I collagen and an infrared-labeled secondary antibody. Wells were then imaged at 1x using an Odyssey Lx (LI-CORE) infrared imager, and collagen intensity was quantified using automated software. Data shown were plotted as % collagen intensity, normalized to vehicle-treated wells. Mean + / - SEM, N=3 independent experiments. [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 9-4] Continued from Figure 9-3. [Figure 9-5] Continued from Figure 9-4. [Figure 10]Figures 10A-10B show six-point dose-response curves of flavonol-treated senescent and healthy fibroblasts. TGFβ-stimulated collagen deposition (square data points) in senescent fibroblasts (circle data points) or low-passage lung fibroblasts was observed with increasing doses of either (Figure 10A) 2-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-6-methylchromen-4-one or (Figure 10B) 2-(3,4-diethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one. For senescent fibroblast viability experiments (circle data points), human adult lung fibroblasts were replicatively passaged until senescence (confirmed by staining for RNA and senescence-associated beta-galactosidase). Cells were then seeded into 96-well plates and treated with the indicated compounds for 96 hours. Cells were then fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and nuclear stained using DAPI. Cells were then imaged using a 4x objective on a Cytation 5 microscope, and cell numbers were quantified using automated software (Biotek Gen5). Data shown were plotted as percent viability normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For TGFβ-stimulated collagen deposition experiments (square data points), human adult lung fibroblasts (passage 3) were seeded in 96-well plates and treated with the indicated compound plus 2 ng / mL TGFβ to stimulate collagen expression. Cells were incubated for 96 hours, fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and stained with a primary antibody recognizing type I collagen and an infrared-labeled secondary antibody. Wells were then imaged at 1x using an Odyssey Lx (LI-CORE) infrared imager, and collagen intensity was quantified using automated software. Data shown were plotted as % collagen intensity normalized to vehicle-treated wells. Mean + / - SEM, N = 3 independent experiments. [Figure 11-1]Figures 11A-11J show six-point dose-response curves of senescent and healthy fibroblasts treated with paraethoxylated flavonols. TGFβ-stimulated collagen deposition (square data points) in senescent fibroblasts (circle data points) or low-passage lung fibroblasts was measured by treatment with increasing doses of either (Figure 11A) compound 17, (Figure 11B) compound 18, (Figure 11C) compound 19, (Figure 11D) compound 20, (Figure 11E) compound 21, (Figure 11F) compound 22, (Figure 11G) compound 23, (Figure 11H) compound 24, (Figure 11I) compound 25, or (Figure 11J) compound 26. For senescent fibroblast viability experiments (circle data points), human adult lung fibroblasts were replicatively passaged until senescence, as confirmed by staining for RNA and senescence-associated beta-galactosidase (not shown). Cells were then seeded into 96-well plates, treated with the indicated compounds, and incubated for 96 hours. Cells were then fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and nuclear stained using DAPI. Cells were then imaged using a 4x objective on a Cytation 5 microscope, and cell numbers were quantified using automated software (Biotek Gen5). Data shown were plotted as percent viability, normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For TGFβ-stimulated collagen deposition experiments (square data points), human adult lung fibroblasts (passage 3) were seeded into 96-well plates and treated with the indicated compounds plus 2 ng / mL TGFβ to stimulate collagen expression. Cells were incubated for 96 hours, fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and stained with a primary antibody recognizing type I collagen and an infrared-labeled secondary antibody. Wells were then imaged at 1x using an Odyssey Lx (LI-CORE) infrared imager, and collagen intensity was quantified using automated software. Data shown were plotted as % collagen intensity normalized to vehicle-treated wells. Mean + / - SEM, N = 3 independent experiments. [Figure 11-2] Continued from Figure 11-1. [Figure 11-3] Continued from Figure 11-2. [Figure 12-1] Figures 12A-12E show six-point dose-response curves of senescent and healthy fibroblasts treated with flavonols having a derivatized flavonol core. TGFβ-stimulated collagen deposition (square data points) in senescent fibroblasts (circle data points) or low-passage lung fibroblasts was measured with increasing doses of either (Figure 12A) compound 27, (Figure 12B) compound 28, (Figure 12C) compound 29, (Figure 12D) compound 30, or (Figure 12E) compound 31. For senescent fibroblast viability experiments (circle data points), human adult lung fibroblasts were replicatively passaged until senescence (confirmed by staining for RNA and senescence-associated beta-galactosidase (not shown)). Cells were then seeded into 96-well plates, treated with the indicated compounds, and incubated for 96 hours. Cells were then fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and nuclear stained using DAPI. Cells were then imaged using a 4x objective on a Cytation 5 microscope, and cell numbers were quantified using automated software (Biotek Gen5). Data shown were plotted as percent viability normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For TGFβ-stimulated collagen deposition experiments (square data points), human adult lung fibroblasts (passage 3) were seeded in 96-well plates and treated with the indicated compound plus 2 ng / mL TGFβ to stimulate collagen expression. Cells were incubated for 96 hours, fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and stained with a primary antibody recognizing type I collagen and an infrared-labeled secondary antibody. Wells were then imaged at 1x using an Odyssey Lx (LI-CORE) infrared imager, and collagen intensity was quantified using automated software. Data shown were plotted as % collagen intensity normalized to vehicle-treated wells. Mean + / - SEM, N = 3 independent experiments. [Figure 12-2] Continued from Figure 12-1. [Figure 12-3]Continued from Figure 12-2. [Figure 13] Figures 13A-13B show the efficacy of F-4N in the bleomycin injury model of pulmonary fibrosis. Figure 13A. Study design. Mice received three intratracheal bleomycin treatments, 2 weeks apart. 28 days after the final bleomycin injury, mice were treated daily with vehicle or F-4N for 14 days. Figure 13B. Representative H&E-stained histological images of lungs from each group and hydroxyproline analysis of each lung from each group. N = 3 sham-treated mice, N = 6 bleomycin (bleo), vehicle, N = 7 bleomycin (bleo), F-4N. 8-week-old FVB wild-type mice. [Figure 14] Figure 1 shows whole lung RNA expression from a repetitive bleomycin injury study. Mouse lungs from Figure 1 were analyzed by qPCR for changes in fibrosis-specific profibrotic genes, senescence-associated genes, type I / II alveolar epithelial markers, and intermediate / transitional alveolar markers, as well as Stk17b. N=3 sham-treated mice; N=6 bleomycin (bleo), vehicle; N=7 bleomycin (bleo), F-4N. 8-week-old FVB wild-type mice. [Figure 15] FIG. 1 is a schematic diagram of the study design used in Example 8. [Figure 16] FIG. 10 is a graph showing that liver mass was reduced in Mdr2− / − mice after oral treatment with F-4N. [Figure 17] Figures 17A-B show that liver fibrosis was reduced in Mdr2- / - mice after oral treatment with F-4N. Figure 17A) Microscopic image of liver tissue. Figure 17B) Graph showing the amount of hydroxyproline (HYP) in the liver. [Figure 18] 10 is a graph showing that oral delivery of F-4N reduced markers of liver fibrosis in Mdr2− / − mice. [Figure 19] 1 is a graph showing that oral delivery of F-4N significantly reduces markers of hepatic inflammation and cellular senescence in Mdr2− / − mice. [Figure 20-1]Figures 20A-20D show the efficacy of F-4N in a mouse model of NASH. Figure 20A) Study design and weight changes in mice treated intraperitoneally with 10 mg / kg F-4N daily for 2 weeks. Figure 20B) Representative H&E and Sirius Red liver histology. Figure 20C) Objective automated quantification of Sirius Red staining. Figure 20D) Changes in liver and colon weight. N=5 chow, N=10 choline-deficient-high-fat diet (CD-HFD) vehicle, N=10 CD-HFD+F-4N. [Figure 20-2] Continuation of Figure 20-1. [Figure 21] Figure 20 shows analysis of whole liver RNA. Livers from the study in Figure 20 were analyzed by qPCR for expression of profibrotic and inflammatory genes, as well as expression of Stk17b, the molecular target of F-4N. [Figure 22] 22A-22C show additional analyses using samples from the study of FIG. 20, including liver function tests (FIG. 22A), liver triglyceride analysis (FIG. 22B), and hydroxyproline assessment of liver collagen content (FIG. 22C). [Figure 23-1] Figures 23A-C show the in vitro efficacy of F-4N in ocular fibrosis models. Figure 23A) Conjunctival fibroblasts cultured with or without fetal bovine serum (FBS) and with or without F-4N. N=3. Figure 23B) Conjunctival fibroblasts were cultured with + / - 2 ng / mL TGFβ and the indicated concentrations of 4N for 3 days, cells were fixed, stained with DAPI and antibodies against αSMA, and then quantified using automated Cytation 5 software. N=3. Figure 23C) Conjunctival fibroblasts were cultured with + / - 2 ng / mL TGFβ and 2% FBS and the indicated concentrations of 4N for 6 days, cells were fixed, stained with DAPI and antibodies against type I collagen, and then quantified using automated Cytation 5 software. N=3. [Figure 23-2] Continuation of Figure 23-1. [Figure 23-3] Continuation of Figure 23-2. [Figure 24-1]Figures 24A-C show acute bleomycin challenge studies using various doses of F-4N. Figure 24A) Schematic of the study protocol. On day 1, mice underwent intratracheal sham or bleomycin injury. On day 7, mice were assigned to groups: vehicle, 10, 30, and 100 mg / kg F-4N treated daily by oral gavage for 7 days. On day 14, organs and plasma were harvested. Figure 24B) Weight changes over the course of the experiment. Figure 24C) Whole lung RNA expression of pro-fibrotic genes. [Figure 24-2] Continuation of Figure 24-1. [Figure 25] Figure 1 shows F-4N efficacy biomarker discovery. Mice were treated with vehicle or 30 mg / kg F-4N and RNA was analyzed. Eleven genes with decreased expression in mice after 7 days of treatment with F-4N are shown. [Figure 26] FIG. 1 shows levels of F-4N in the plasma (top) and liver (bottom) of mice after F-4N exposure. [Figure 27] Figure 1 shows the stability of F-4N in plasma. Compounds were incubated with human (top) or mouse (bottom) plasma for the indicated times. Warfarin was used as a control for stable compounds, and propantheline was used as a control for unstable compounds. [Figure 28] Figure 1 shows the microsomal stability of F-4N. Compounds were incubated with human (top) or mouse (bottom) liver-derived microsomes for the indicated times. Verapamil was used as a control for a rapidly degraded compound. [Figure 29-1] Figures 29A-B show the results of Stk17b knockdown in a precision-cut lung slice (PCLS) model. Figure 29A) Schematic of the study protocol. PCLS were cultured ex vivo with either non-targeting siRNA or Stk17b-targeting siRNA for 4 days. Figure 29B) RNA was then harvested and analyzed by qPCR. [Figure 29-2] Continuation of Figure 29-1. [Figure 30]FIG. 1 shows the results of a DRAK1 activity assay in the presence of F-4N (top) or the inactive analogue 5-MeOH-F-4N (bottom). [Figure 31-1] Figures 31A-B show the efficacy of F-4N in ex vivo organotypic cultures from IPF patients. Figure 31A) qPCR analysis of lung tissue slices. Figure 31B) ELISA analysis of culture medium IL-6. N=3 patient samples. [Figure 31-2] Continuation of Figure 31-1. [Figure 32-1] Figures 32A-32D are graphs showing the relative efficacy of flavonoids and the absence of toxicity in the aged brain and liver. Five- and 28-month-old wild-type mice were treated orally by gavage with the indicated concentrations of flavonoids (F, Q, compound 19, or compound 20) or vehicle for four consecutive days and then euthanized one week later. Real-time PCR analysis suggests that low doses of compound 19 and / or 20 can reduce the expression of p16ink4a, a key senescence-activated gene, in the brain (Figure 32A) and liver (Figure 32B) more reliably than fisetin (F) or quercetin (Q), which have established senolytic activity at relatively high doses. Analysis of CD68 expression, an indicator of inflammatory activation, reveals no drug-induced toxicity in the brain (Figure 32C) or liver (Figure 32D). [Figure 32-2] Continuation of Figure 32-1. [Figure 33-1] Figures 33A-33C show that quercetin analogs can potently kill senescent fibroblasts. Figures 33A and 33B) Identification of replicatively induced senescent fibroblasts as assessed by senescence marker expression (Figure 33A) and proliferation (Figure 33B). Figure 33C) Proliferation of low-passage proliferating fibroblasts after treatment with quercetin analogs. Five representative derivatives are seen to exhibit nanomolar to low micromolar potency. [Figure 33-2] Continuation of Figure 33-1. [Figure 34-1]Figures 34A-B show that TGFβ and senescent cell-conditioned medium can promote myofibroblast transdifferentiation of fibroblasts. Figure 34A) Representative images observed by staining for αSMA. Figure 34B) Quercetin analogs potently prevent fibroblast activation. NCM: Non-conditioned medium (control). NCM + TGFβ: Non-conditioned medium + 2 ng / mL TGFβ. CCM: Conditioned medium from normal lung fibroblasts. SASP-CM: Conditioned medium from senescent lung fibroblasts. [Figure 34-2] Continuation of Figure 34-1. [Figure 35-1] Figures 35A-B show that quercetin analogs can prevent SASP-CM and TGFβ-induced collagen deposition. Figure 35A) TGFβ and senescent cell-conditioned medium promote type I collagen deposition. Figure 35B) Quercetin analogs potently prevent type I collagen deposition. NCM: non-conditioned medium (control). NCM + TGFβ: non-conditioned medium + 2 ng / mL TGFβ. [Figure 35-2] Continuation of Figure 35-1. [Figure 36-1] Figures 36A-B show that quercetin analogs can prevent TGFβ-induced profibrotic gene expression. Figure 36A) TGFβ promotes profibrotic gene expression. Figure 36B) Quercetin analogs potently block profibrotic gene expression. NCM: Non-conditioned medium (control). NCM + TGFβ: Non-conditioned medium + 2 ng / mL TGFβ. [Figure 36-2] Continuation of Figure 36-1. [Figure 37-1] Figures 37A-C show that quercetin analogs with p-ethoxy have enhanced activity. Figure 37A) Exemplary quercetin analogs with p-ethoxy (highlighted). Figure 37B) Cell proliferation graph showing that quercetin analogs with p-ethoxy induced cellular senescence. Figure 37C) Cell proliferation graph showing that quercetin analogs lacking p-ethoxy did not induce cellular senescence. [Figure 37-2] Continuation of Figure 37-1. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides flavonoid compounds and methods and materials using the flavonoid compounds to treat mammals (e.g., humans) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). For example, the present invention provides a compound represented by formula (I):

[0035] [ka] (In the formula, R 1 can be H, OH, C1-C4 alkyl (e.g., methyl), halogen, or C1-C4 alkoxy (e.g., methoxy and ethoxy), and R 2 can be H, OH, C1-C4 alkyl (e.g., methyl), halogen, or C1-C4 alkoxy (e.g., methoxy and ethoxy), and R 3 can be H, CHCH, cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl, and R 4 provides flavonoid compounds having the structure: wherein R is H, OH, C1-C4 alkyl (eg, methyl), halogen, or C1-C4 alkoxy (eg, methoxy and ethoxy).

[0036] In some cases, the flavonoid compounds provided herein may have the following structure and may be referred to as F-4N:

[0037] [ka]

[0038] In some cases, the flavonoid compounds provided herein may have the following structure and may be referred to as F-5MeO:

[0039] [ka]

[0040] In some cases, the flavonoid compounds provided herein may have the following structure and may be referred to as F-4N-5MeO:

[0041] [ka]

[0042] The present invention also provides flavonoid compounds and methods and materials using the flavonoid compounds to treat mammals (e.g., humans) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). For example, the present invention provides a compound represented by formula (II):

[0043] [ka] or a pharmaceutically acceptable salt thereof, wherein: X 1 is selected from N and CH; X 2 N and CR 4 is selected from R 1 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3 is selected from the group consisting of H, CH3, CH2CH3, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl; And R 4 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy to provide.

[0044] In some embodiments, X 1 is N. In some embodiments, X 1 is CH. In some embodiments, X 2 is N. In some embodiments, X 2 is CR 4 In some embodiments, R 3 is H. In some embodiments, R 3 is CH3. In some embodiments, R 3 is phenyl, 4-OH-phenyl, 2-OH-phenyl, or 3-OH-phenyl. 3 is 2-pyridinyl, 3-pyridinyl, or 4-pyridinyl. In some embodiments, R 3 is thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, or tetrahydrofuran-3-yl.

[0045] In some embodiments, the compound of formula (II) has the formula:

[0046] [ka] or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments, the compound of formula (II) has the formula:

[0048] [ka] or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, the compound of formula (II) is the following compound:

[0050] [ka] TIFF2025525323000025.tif217147TIFF2025525323000026.tif56141 or a pharmaceutically acceptable salt thereof.

[0051] In some cases, the flavonoid compounds provided herein (e.g., flavonoid compounds having the structure of Formula (I) or Formula (II)) may be in the form of a salt (e.g., a pharmaceutically acceptable salt). The salt of the compounds provided herein may be formed between an acid and a basic group of the compound, such as an amino functional group, or between a base and an acidic group of the compound, such as a carboxyl functional group. When the flavonoid compound having the structure of Formula (I) or Formula (II) is in the form of a salt, the salt may include any suitable acid (e.g., an organic acid or an inorganic acid). Examples of acids that can be used to form pharmaceutically acceptable salts of the compounds described herein include, but are not limited to, inorganic acids such as hydrogen disulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, as well as related inorganic and organic acids.Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-diol ... Examples of suitable salts include hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and other salts. In some embodiments, pharmaceutically acceptable acid addition salts may be used, including, but not limited to, those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid.Examples of bases that can be used to form pharmaceutically acceptable salts of the compounds described herein include, but are not limited to, hydroxides of alkali metals, including sodium, potassium, and lithium; hydroxides of alkaline earth metals, such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia; organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamines), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids, such as arginine, lysine, and the like. In some cases, the compounds described herein or pharmaceutically acceptable salts thereof may be substantially isolated.

[0052] At various places in the present specification, substituents of the compounds described herein are recited in groups or in ranges. It is specifically intended that the invention include and describe each and every individual member of such groups and ranges or each and every subcombination of the members thereof. For example, "C 1~6 The term "alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0053] Various aryl, heteroaryl, cycloalkyl, and heterocycloalkyl rings are described in various places herein. Unless otherwise specified, these rings can be attached to the rest of the molecule at any ring member, if allowed by valence. For example, the term "pyridine ring" or "pyridinyl" can refer to a pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl ring.

[0054] It will be further understood that certain features herein, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features herein, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0055] The term "n-membered" (where n is an integer) generally refers to the number of atoms forming the ring in a moiety where n is the number of atoms forming the ring. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0056] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. The substituents are independently selected, and substitution can be at any chemically available position. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced with a substituent. A single divalent substituent, such as oxo, can replace two hydrogen atoms. It should be understood that substitution at a given atom is limited by the valence of the atom.

[0057] Throughout the definition, "C n~m The term "" indicates a range inclusive of the endpoints, where n and m are integers and indicate the number of carbons. Examples include C 1~4 , C 1~6 These include:

[0058] As used herein, the term "C" used alone or in combination with other terms n~mThe term "alkyl" refers to a saturated hydrocarbon group that may be straight-chained or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, an alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0059] As used herein, the term "C" used alone or in combination with other terms n~m The term "alkylene" refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene groups include, but are not limited to, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0060] As used herein, the term "C" used alone or in combination with other terms n~m "Alkoxy" refers to a group of formula -O-alkyl, where the alkyl group has n to m carbons. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0061] As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon, including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having two, three, or four fused rings) groups and spirocyclic rings. The ring-forming carbon atoms of a cycloalkyl group can be optionally substituted with one or two independently selected oxo or sulfido groups (e.g., C(O) or C(S)). Also included within the definition of cycloalkyl are moieties having one or more aromatic rings fused to (i.e., sharing a bond with) the cycloalkyl ring, e.g., benzo or thienyl derivatives such as cyclopentane, cyclohexane, etc. Cycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms (C 3~10 In some embodiments, cycloalkyl can have C 3~10 In some embodiments, cycloalkyl is C 3~7 Monocyclic cycloalkyl. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0062] As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N, or S. Heterocycloalkyl includes monocyclic 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Examples of heterocycloalkyl groups include, but are not limited to, pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydrofuran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene, and the like. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted with one or two independently selected oxo or sulfido groups (e.g., C(O), S(O), C(S), or S(O)). A heterocycloalkyl group can be bonded through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains zero to three double bonds. In some embodiments, a heterocycloalkyl group contains zero to two double bonds. Also included within the definition of heterocycloalkyl are moieties having one or more aromatic rings fused to (i.e., sharing a bond with) a cycloalkyl ring, e.g., benzo or thienyl derivatives such as piperidine, morpholine, azepine, etc. Heterocycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. In some embodiments, the heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.In some embodiments, the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

[0063] As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. Compounds identified herein by name or structure as a particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

[0064] The compounds provided herein also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond, accompanied by the migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include, but are not limited to, ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position on a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into a certain form by appropriate substitution.

[0065] In some cases, one or more of the flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) may not have chirality.

[0066] In some cases, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be neutral molecules (e.g., can lack any charged moieties).

[0067] In some cases, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) may be completely devoid of a catechol moiety.

[0068] In some cases, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). For example, one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be formulated with one or more pharmaceutically acceptable carriers (additives), excipients, and / or diluents.Examples of pharmaceutically acceptable carriers, excipients, and diluents that may be used in the compositions described herein include, but are not limited to, cyclodextrins (e.g., beta-cyclodextrin, e.g., KLEPTOSE®), dimethyl sulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses, e.g., microcrystalline cellulose, and cellulose ethers such as hydroxypropyl cellulose (HPC) and the cellulose ether hydroxypropylmethylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cross-linked polyvinylpyrrolidone (crospovidone), carboxymethylcellulose, polyethylene-polyoxypropylene-block polymers, and the like). and cross-linked sodium carboxymethylcellulose (croscarmellose sodium)), titanium dioxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinol palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methylparaben and propylparaben), soft paraffin, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, mutton tallow, lecithin, and corn oil.

[0069] In some cases, compositions containing one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of Formula (I) or Formula (II)) can be designed for oral or parenteral administration to a mammal (including, but not limited to, subcutaneous, intramuscular, intravenous, intradermal, intracerebral, intrathecal, or intraperitoneal (ip) injection). Compositions suitable for oral administration include, but are not limited to, liquids, tablets, capsules, pills, powders, gels, and granules. In some cases, compositions suitable for oral administration can be in the form of a dietary supplement. In some cases, compositions suitable for oral administration can be in the form of a dietary supplement drink. Compositions suitable for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.

[0070] The present invention also provides methods of making one or more flavonoid compounds described herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)). Any suitable method can be used to make one or more flavonoid compounds provided herein. In some cases, flavonoid compounds having the structure of Formula (I) or Formula (II) can be made as shown in Figure 1. In some cases, flavonoid compounds having the structure of Formula (I) or Formula (II) can be made as described in Example 1.

[0071] The present invention also provides methods of using one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)). For example, one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) to treat the mammal. In some cases, a mammal (e.g., a human) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) can be administered or instructed to self-administer one or more flavonoid compounds having the structure of Formula (I) or Formula (II).

[0072] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce or eliminate one or more symptoms of one or more fibrotic conditions. For example, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, e.g., IPF, NASH, PSC, and ocular fibrosis) to reduce or eliminate one or more symptoms of the fibrotic condition (e.g., IPF, NASH, and PSC). Examples of IPF disease symptoms include, but are not limited to, shortness of breath (dyspnea), persistent dry cough, fatigue, loss of appetite and weight loss, muscle and joint pain, and clubbing, which is broad, rounded tips of the fingers or toes. Examples of symptoms of PSC disease include, but are not limited to, fatigue or weakness, itchy skin, abdominal pain, weight loss without effort, loss of appetite, fever, enlarged liver, enlarged spleen, yellow eyes and skin (jaundice), symptoms of cirrhosis and liver failure, such as bloating, bruising, and easy bleeding, confusion, difficulty thinking or memory loss, redness of the palms of the hands, and swelling of the legs, ankles, or feet. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce one or more symptoms of a fibrotic condition in a mammal having one or more fibrotic conditions by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0073] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce or eliminate one or more complications associated with a fibrotic condition. For example, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to reduce or eliminate one or more complications associated with the fibrotic condition. Examples of complications associated with IPF include, but are not limited to, pulmonary hypertension, acute exacerbation of pulmonary fibrosis, respiratory infection, acute coronary syndrome, thromboembolic disease, adverse drug reactions, and lung cancer. Examples of complications associated with PSC include, but are not limited to, low levels of fat-soluble vitamins, osteoporosis, bile duct infection, portal hypertension, cirrhosis, liver failure, cholangiocarcinoma, gallbladder cancer, colon cancer, and hepatocellular carcinoma. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I)) can be used to reduce one or more complications associated with one or more fibrotic conditions by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent in a mammal having one or more fibrotic conditions.

[0074] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used as anti-fibrotic agents. For example, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce or eliminate fibrotic scarring in a mammal (e.g., in one or more tissues of a mammal). For example, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to slow the progression of fibrosis in a mammal (e.g., in one or more tissues of a mammal).

[0075] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce or eliminate fibrotic scarring in a mammal. For example, a composition comprising one or more (e.g., one, two, three, four, or more) flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to reduce or eliminate fibrotic scarring in one or more tissues of the mammal. One or more flavonoid compounds provided herein can be used to reduce or eliminate fibrotic scarring in any suitable tissue of a mammal. Examples of tissues that may have fibrous scars and in which one or more flavonoid compounds provided herein can be used to reduce or eliminate fibrous scarring include, but are not limited to, lung, liver, bile duct, kidney, heart, and skin. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce fibrous scarring by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent in one or more tissues of a mammal having fibrous scarring.

[0076] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to slow the progression of fibrosis in a mammal. For example, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to slow the progression of fibrosis in the mammal. One or more flavonoid compounds provided herein can be used to slow the progression of fibrosis in any suitable tissue of a mammal. Examples of tissues that may be fibrotic and in which one or more flavonoid compounds provided herein can be used to slow the progression of fibrosis include, but are not limited to, the lung, liver, bile duct, kidney, heart, and skin. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I)) can be effective to slow the progression of fibrosis in a mammal having fibrosis by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I)) can be used to slow the progression of fibrosis in a mammal having fibrosis by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

[0077] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used as senolytic agents. For example, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to induce apoptosis in one or more senescent cells of a mammal. In some cases, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) exhibit little or no ability to induce apoptosis in proliferating cells of a mammal (e.g., a human).

[0078] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to inhibit one or more serine / threonine kinase 17 (STK17) polypeptides. For example, a flavonoid compound having the structure of Formula (I) or Formula (II) can bind to an STK17 polypeptide to inhibit the polypeptide function of the STK17 polypeptide. A flavonoid compound having the structure of Formula (I) or Formula (II) can inhibit any suitable STK17 polypeptide. Examples of STK17 polypeptides inhibited by one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) include, but are not limited to, STK17A (DRAK1) polypeptide and STK17B (DRAK2) polypeptide. In some cases, flavonoid compounds having the structure of Formula (I) or Formula (II) can inhibit STK17A (DRAK1) polypeptides set forth in any one of the National Center for Biotechnology Information (NCBI) GenBank® or GenPept® accession numbers 9263 and Q9UEE5. In some cases, flavonoid compounds having the structure of Formula (I) or Formula (II) can inhibit STK17B (DRAK2) polypeptides set forth in any one of the NCBI GenBank® or GenPept® accession numbers 9262 and O94768.

[0079] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to inhibit fibroblast activation. Flavonoid compounds having the structure of Formula (I) or Formula (II) can inhibit any suitable fibroblast activation. For example, flavonoid compounds having the structure of Formula (I) or Formula (II) can inhibit TGFβ-induced fibroblast activation.

[0080] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to induce apoptosis in mammalian cells (e.g., senescent cells). For example, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human with one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to induce apoptosis in the mammal's senescent cells. One or more flavonoid compounds provided herein can be used to induce apoptosis in any suitable type of mammalian senescent cells. Examples of cell types that may be senescent and in which one or more flavonoid compounds provided herein can be used to induce apoptosis include, but are not limited to, fibroblasts (e.g., lung fibroblasts) and epithelial cells (e.g., cholangiocytes). For example, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce the number of senescent cells in a mammal. In some cases, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to reduce the number of senescent cells in the mammal. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I)) can be effective to reduce the number of senescent cells in a mammal having one or more fibrotic conditions by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0081] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to increase the survival rate of a mammal (e.g., a human). For example, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to increase the survival rate of the mammal. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I)) can be used to increase the survival rate of a mammal having one or more fibrotic conditions by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0082] In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to reduce or eliminate inflammation in one or more tissues of a mammal. For example, a composition comprising one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) to reduce or eliminate inflammation in one or more tissues of the mammal. One or more flavonoid compounds provided herein can be used to reduce inflammation in any suitable tissue of a mammal. Examples of tissues that may be inflamed and in which one or more flavonoid compounds provided herein can be used to reduce inflammation include, but are not limited to, the lung, liver, bile duct, kidney, heart, and skin. In some cases, one or more (e.g., one, two, three, four, or more) flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I)) can be used to reduce inflammation in one or more tissues of a mammal having a fibrotic condition (e.g., IPF) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0083] Any suitable mammal having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) can be treated as described herein (e.g., by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II)). Examples of mammals that may have one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) and that can be treated as described herein include, but are not limited to, humans, non-human primates, such as monkeys, dogs, cats, horses, cows, pigs, sheep, mice, and rats. In some cases, humans having one or more fibrotic conditions can be treated by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II) as described herein.

[0084] When treating a mammal (e.g., a human) having one or more fibrotic conditions, the mammal may have any type of fibrotic condition(s). Examples of fibrotic conditions that can be treated as described herein (e.g., by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) include, but are not limited to, IPF, PSC, NASH, and ocular fibrosis.

[0085] In some cases, the methods described herein may include determining that a mammal (e.g., a human) has one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). Any suitable method may be used to determine that a mammal has one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). For example, chest scans, including X-rays and high-resolution computed tomography, respiratory function tests, pulse oximetry, blood tests for oxygen and CO2, exercise capacity, and / or lung biopsies (e.g., observing signs of scarring) may be used to determine that a mammal (e.g., a human) has IPF disease. For example, imaging techniques (e.g., magnetic resonance imaging (MRI), magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde cholangiopancreatography (ERCP)), a cholestatic biochemistry profile, and / or a liver biopsy may be used to determine that a mammal (e.g., a human) has PSC disease.

[0086] In some cases, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be used to treat a mammal (e.g., a human) with an age-related disease. For example, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be administered to a mammal (e.g., a human) with an age-related disease (e.g., to treat the mammal). Examples of age-related diseases that can be treated as described herein (e.g., by administering one or more flavonoid compounds provided herein) include, but are not limited to, osteoporosis, frailty, cardiovascular disease, osteoarthritis, pulmonary fibrosis, kidney disease, neurodegenerative disease, fatty liver, and metabolic dysfunction. In some cases, age-related diseases that can be treated as described herein can be as described elsewhere (see, e.g., Kaur et al., Transl. Res., 226:96-104 (2020)). Compositions containing one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be administered in any suitable amount (e.g., any suitable dose) to a mammal (e.g., a human) having one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). An effective amount of a composition containing one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be any amount that can treat a mammal (e.g., a mammal having one or more fibrotic conditions, e.g., IPF, NASH, PSC, and ocular fibrosis) as described herein without causing significant toxicity to the mammal.In some cases, an effective amount of one or more flavonoid compounds having the structure of Formula (I) or Formula (II) is from about 0.1 μM to about 100 μM (e.g., from about 0.1 μM to about 75 μM, from about 0.1 μM to about 60 μM, from about 0.1 μM to about 50 μM, from about 0.1 μM to about 40 μM, from about 0.1 μM to about 30 μM, from about 0.1 μM to about 20 μM, from about 0.1 μM to about 10 μM, from about 0.1 μM to about 1 μM, from about 10 μM to about 100 μM, or from about In some cases, the effective amount of one or more flavonoid compounds having the structure of Formula (I) or Formula (II) may have an IC of about 200 nM. 50 ~800nM IC 50 (e.g., IC of approximately 200 nM) 50 ~700nM IC 50 , IC of approximately 200 nM 50 ~600nM IC 50 , IC of approximately 200 nM 50 ~500nM IC 50 , IC of approximately 200 nM 50 ~400nM IC 50 , IC of approximately 200 nM 50 ~300nM IC 50 , IC of approximately 300 nM 50 ~800nM IC 50 , IC of approximately 400 nM 50 ~800nM IC 50 , IC of approximately 500 nM 50 ~800nM IC 50 , IC of approximately 600 nM 50 ~800nM IC 50 , IC of approximately 700 nM 50 ~800nM IC 50 , IC of approximately 300 nM 50 ~700nM IC 50 , IC of approximately 400 nM 50~600nM IC 50 , IC of approximately 300 nM 50 ~400nM IC 50 , IC of approximately 400 nM 50 ~500nM IC 50 , IC of approximately 500 nM 50 ~600nM IC 50 , IC of approximately 600 nM 50 ~700nM IC 50 , IC of approximately 200 nM 50 , or an IC of approximately 800 nM 50In some cases, the effective amount of one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be from about 0.1 milligrams per kilogram of body weight (mg / kg) to about 200 mg / kg (e.g., from about 0.1 mg / kg to about 175 mg / kg, from about 0.1 mg / kg to about 150 mg / kg, from about 0.1 mg / kg to about 125 mg / kg, from about 0.1 mg / kg to about 100 mg / kg, from about 0.1 mg / kg to about 75 mg / kg, from about 0.1 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 25 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 200 mg / kg, or from about 10 mg / kg). about 200 mg / kg, about 25 mg / kg to about 200 mg / kg, about 50 mg / kg to about 200 mg / kg, about 75 mg / kg to about 200 mg / kg, about 100 mg / kg to about 200 mg / kg, about 150 mg / kg to about 200 mg / kg, about 1 mg / kg to about 150 mg / kg, about 10 mg / kg to about 100 mg / kg, about 25 mg / kg to about 75 mg / kg, about 1 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 25 mg / kg, about 25 mg / kg to about 50 mg / kg, about 50 mg / kg to about 100 mg / kg, about 100 mg / kg to about 150 mg / kg, or about 2 mg / kg) of F-4N. This effective amount may remain constant or may be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors may affect the actual effective amount used for a particular application. For example, the number of doses administered, the duration of treatment, the use of multiple therapeutic agents, the route of administration, and / or the severity of the fibrotic condition (e.g., IPF, NASH, and PSC) in the mammal being treated may require an increase or decrease in the actual effective amount administered.

[0087] Compositions containing one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of Formula (I) or Formula (II)) can be administered to a mammal (e.g., a mammal having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) at any suitable frequency. The frequency of administration can be any frequency that allows treatment of the mammal (e.g., a mammal having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) without causing significant toxicity to the mammal (e.g., a human). For example, the frequency of administration can be from about once daily to about once weekly, from about once weekly to about once monthly, or from about twice monthly to about once monthly. The frequency of administration can remain constant or can vary over the course of treatment. As with the effective amount, various factors can affect the actual frequency of administration used for a particular application. For example, the effective amount, the duration of treatment, the use of multiple therapeutic agents, and / or the route of administration may require an increase or decrease in the frequency of administration.

[0088] Compositions containing one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be administered to a mammal (e.g., a mammal having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) for any suitable period of time. The effective period for administering or using a composition containing one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can be any period that can treat a mammal (e.g., a mammal having one or more fibrotic conditions, such as IPF, NASH, PSC, and ocular fibrosis) without causing significant toxicity to the mammal (e.g., a human). For example, the effective period can vary from several weeks to several months, from several months to several years, or even from several years to a lifetime. Multiple factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary with the number of doses, the effective amount, the use of multiple therapeutic agents, and / or the route of administration.

[0089] In some cases, a method of treating a mammal (e.g., a mammal, e.g., a human, having one or more fibrotic conditions, e.g., IPF, NASH, PSC, and ocular fibrosis) as described herein (e.g., by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can include administering to the mammal one or more flavonoid compounds having the structure of Formula (I) or Formula (II) as the sole active ingredient(s) for treating the mammal. For example, a composition containing one or more flavonoid compounds having the structure of Formula (I) or Formula (II) can include flavonoid compound(s) having the structure of Formula (I) or Formula (II) as the sole active ingredient(s) in a composition that is effective for treating a mammal (e.g., a mammal having one or more fibrotic conditions, e.g., IPF, NASH, PSC, and ocular fibrosis).

[0090] In some cases, a method of treating a mammal (e.g., a mammal, e.g., a human, having one or more fibrotic conditions, e.g., IPF, NASH, PSC, and ocular fibrosis) as described herein (e.g., by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can also include administering to the mammal one or more (e.g., 1, 2, 3, 4, 5, or more) additional agents / therapies used to treat the condition (e.g., one or more fibrotic conditions, e.g., IPF, PSC, and / or ocular fibrosis). For example, a combination therapy used to treat one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) can include administering to a mammal (e.g., a human) one or more flavonoid compounds having the structure of Formula (I) or Formula (II) described herein and one or more (e.g., one, two, three, four, five, or more) agents used to treat one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). Examples of drugs that can be administered to a mammal to treat IPF disease include, but are not limited to, pirfenidone, nintedanib, N-acetylcysteine, phosphodiesterase inhibitors (e.g., sildenafil, vardenafil, tadalafil, and avanafil), lysophosphatidic acid receptor antagonists (e.g., promethazine, FTY720, AM152, BMS-986020, VPC12249, AM966, and AM095), autotaxin inhibitors (e.g., taribavirin, BI-2545, GLPG1690, BBT877, SAR100842, and BMS-986,020), D1 receptor agonists (e.g., minaprine, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, and cabergoline), dasatinib, rho-kinase inhibitors (e.g., hydroxyfasudil, ripasudil, netarsudil, and bermosudil), IL-13 neutralizing antibodies (e.g., lebrikizumab, tralokinumab, and dupilumab), CTGF neutralizing antibodies (e.g., pamrevlumab), and any combination thereof.Examples of drugs that can be administered to a mammal to treat PSC disease include, but are not limited to, ursodeoxycholic acid (UDCA), corticosteroids (e.g., glucocorticoids, such as prednisolone), bile acid sequestrants, antibiotics, antihistamines, and any combination thereof.

[0091] When one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) are used in combination with an additional agent used to treat one or more fibrotic conditions, the one or more additional agents can be administered simultaneously (e.g., in a single composition containing both the one or more flavonoid compounds having the structure of Formula (I) or Formula (II) and the one or more additional agents) or independently. For example, the one or more flavonoid compounds having the structure of Formula (I) or Formula (II) described herein can be administered first and the one or more additional agents can be administered second, or vice versa.

[0092] In some cases, a method of treating a mammal (e.g., a mammal, e.g., a human, having one or more fibrotic conditions, e.g., IPF, NASH, PSC, and ocular fibrosis) as described herein (e.g., by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can also include administering one or more (e.g., one, two, three, four, five, or more) additional therapies used to treat one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) in the mammal. Examples of therapies used to treat IPF disease include, but are not limited to, oxygen therapy, pulmonary rehabilitation, and / or lung transplantation. Examples of therapies used to treat PSC disease include, but are not limited to, endoscopic therapy (e.g., balloon dilation and stent placement), percutaneous therapy, non-transplant surgery, liver transplantation, and / or fecal microbiota transplantation.

[0093] When one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) are used in combination with one or more additional therapies used to treat one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis), the one or more additional therapies can be administered simultaneously with or independently of the administration of one or more flavonoid compounds having the structure of Formula (I) or Formula (II) described herein. For example, one or more flavonoid compounds having the structure of Formula (I) or Formula (II) described herein can be administered before, during, or after the administration of the one or more additional therapies.

[0094] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0095] [Example 1] Design and synthesis of flavonoid compounds This example describes the design and characterization of flavonoid compounds that can be used to treat one or more fibrotic conditions.

[0096] method Compound synthesis Equimolar amounts (2–8 mmol) of hydroxyacetophenone were combined with the aldehyde in 10–50 mL of EtOH. 5–12 mL of 50% NaOH (aq) was added to the reaction solution, and the reaction was allowed to proceed at room temperature for 6–48 hours while monitoring by thin-layer chromatography (TLC). The chalcone obtained after aldehyde consumption was precipitated with 10% HCl (aq). The chalcone was isolated, dried, weighed, and dissolved (1–4 mmol) in 8–30 mL of MeOH containing 1 M KOH. 5–15 mL of H2O2 was added to the reaction solution, and the reaction was allowed to proceed at room temperature for 1–6 hours while monitoring by TLC. The final product was precipitated with 10% HCl (aq) and purified by recrystallization. The structure and purity (>95%) were confirmed by proton nuclear magnetic resonance.

[0097] A schematic diagram of the synthesis of exemplary flavonoid compounds provided herein is shown in FIG.

[0098] apoptosis Human adult lung fibroblasts were replicate-passaged to senescence (confirmed by staining for RNA and senescence-associated beta-galactosidase). In a parallel experiment, low-passage proliferative fibroblasts from the same donor were compared. Cells were seeded in 96-well plates and treated with the indicated compounds (6-point dose-response) for 96 hours. Cells were then fixed using 4% PFA, permeabilized using 0.25% Triton X-100, and nuclear stained using a primary antibody against cleaved caspase-3, a fluorescently labeled secondary antibody, and DAPI. Cells were then imaged using a 4x objective on a Cytation 5 microscope, and cell numbers were quantified using automated software (Biotek Gen5). Data shown are the calculated EC values for the efficacy of each compound to induce apoptosis in senescent versus proliferative lung fibroblasts from dose-response curves calculated from N = 3 independent experiments. 50 value.

[0099] Fibroblast activation Human adult lung fibroblasts (passage 3) were seeded in 96-well plates and treated with the indicated compounds (6-point dose-response) plus 2 ng / mL TGFβ to stimulate fibroblast activation. Cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% Triton X-100, and stained with a primary antibody recognizing alpha-smooth muscle actin, a fluorescently labeled secondary antibody, and DAPI. Cells were then imaged using a 4x objective on a Cytation 5 microscope, and alpha-smooth muscle actin intensity was quantified using automated software (Biotek Gen5). Data shown are IC50 values for the efficacy of each compound in reducing alpha-smooth muscle actin expression, calculated from dose-response curves calculated from N = 3 independent experiments.

[0100] result Flavonoid compounds were evaluated for mechanisms of therapeutic efficacy using high-throughput assays. The properties of exemplary flavonoid compounds are summarized in Table 1.

[0101] [Table 1]

[0102] [Example 2] F-4N and pulmonary fibrosis This example describes the use of one or more flavonoid compounds having the structure of Formula (I) in the treatment of pulmonary fibrosis.

[0103] method Figure 3. On day 0 of the study, pulmonary fibrosis was induced using intratracheal bleomycin (1.1 units / kg) in 10- to 18-month-old C57 / B6 mice (average approximately 15 months). One group received F-4N (10 mg / kg, daily, intraperitoneally) for 14 days before lung removal on day 14 for outcome and clearance of senescent cells and fibrosis. Lung structure and fibrosis were observed by trichrome tissue staining and hydroxyproline content. Hydroxyproline content was measured using a hydroxyproline assay kit (Biovision) according to the manufacturer's instructions with slight modifications. Lung tissue was weighed, homogenized in sterile water (10 mg tissue per 100 μL of HO), and hydrolyzed in 12 M HCl in a pressure-resistant Teflon-capped vial at 120 °C for 3 h, followed by filtration through a 45 μm Spin-X centrifuge tube filter (Corning). Ten microliters of hydrolyzed sample was dried in a Speed-Vac for 2 hours, then incubated with 100 μL of chloramine T reagent at room temperature for 5 minutes and with 100 μL of 4-(dimethylamino)benzaldehyde (DMAB) at 60°C for 90 minutes. The absorbance of oxidized hydroxyproline was measured at 560 nm. Hydroxyproline concentrations were calculated from a standard curve constructed using known concentrations of trans-4-hydroxyl-L-proline. The total amount of protein isolated from the weighed tissue was measured using a protein assay kit (Bio-Rad, absorbance at 595 nm). The amount of collagen was expressed in μg / mg total protein. qPCR: RNA isolation was performed using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. The isolated RNA (250 ng) was then used to synthesize cDNA using SuperScript VILO (Invitrogen). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche) and analyzed using a LightCycler 96 (Roche). Data are expressed as fold change with ΔΔCt compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0104] Figure 4A. F-4N was sent to Reaction Biology and tested against their entire wild-type kinase panel. 1 μM F-4N was tested in an ATP competition assay against 420+ kinases. Z-scores were calculated to identify true hits above noise.

[0105] Figure 4B. Expression of CDKN2A (a senescence marker) in low-passage fibroblasts compared to senescent fibroblasts generated by radiation (10 Gy) or replicative passaging was compared with putative targets identified in a qPCR kinome screen.

[0106] Figure 5A. Cell number and apoptosis were compared in proliferating low-passage fibroblasts (pro) versus senescent high-passage fibroblasts (sen) after transfection with siRNA targeting the putative molecular targets of F-4N-STK-17A and STK17B. Cell number and apoptosis were measured by cleaved caspase-3 and DAPI staining and automated microscopy.

[0107] Figure 5B. qPCR expression of STK17A, STK17B, and senescence markers was compared in proliferating low-passage versus senescent high-passage fibroblasts after transfection with siRNA targeting putative molecular targets of F-4N-STK-17A and STK17B.

[0108] result F-4N and pulmonary fibrosis Mice treated with F-4N showed improved survival after bleomycin-induced fibrosis (Figure 3A). Aged mice exhibit chronic fibrosis after a single dose of bleomycin. The improved survival strongly suggests a beneficial effect of F-4N.

[0109] Compared with mice not treated with F-4N, mice treated with F-4N showed less fibrosis, improved lung structure as assessed by trichrome histological staining, and reduced hydroxyproline content (Figure 3B). These results suggest that F-4N ameliorates pulmonary fibrosis in this chronic model.

[0110] Lung tissue was examined for the expression of type I collagen and senescence markers. Mice treated with F-4N showed decreased expression of type I collagen and decreased expression of senescence markers (Figure 3C). These in vivo results are consistent with our in vitro data and the overarching hypothesis that F-4N has a dual effect on pulmonary fibrosis: clearance of senescent cells and inhibition of fibroblast activation.

[0111] Mechanism of action Using kinome screening, F-4N (1 μM) was tested against approximately 400 kinases. Putative F-4N targets identified by kinome screening include STK17A (also known as DRAK1), STK17B (also known as DRAK2), as well as MYLK4, AURKB, FLT3, and KIT (Figure 4A). The expression of putative F-4N molecular targets was measured, and STK17A and STK17B were found to be highly overexpressed in senescent fibroblasts compared to low-passage fibroblasts.

[0112] F-4N and lung aging To determine whether putative F-4N targets can regulate senescence, apoptosis was assessed in senescent and nonsenescent lung fibroblasts treated with siRNA targeting STK17A and STK17B (Figure 5A). The expression of senescent markers was also assessed in proliferating and senescent cells treated with siRNA targeting STK17A and STK17B polypeptides (Figure 5B). These data further support the idea that F-4N may mediate senolytic activity via inhibition of STK17A / B (DRAK1 / 2).

[0113] [Example 3] F-4N and PSC This example describes the use of one or more flavonoid compounds having the structure of Formula (I) in the treatment of PSC.

[0114] method Hydroxyproline assay Hydroxyproline content was measured using a hydroxyproline assay kit (Biovision) according to the manufacturer's instructions with minor modifications. Liver tissue was weighed, homogenized in sterile water (10 mg tissue per 100 μL of HO), and hydrolyzed in 12 M HCl in a pressure-resistant Teflon-capped vial at 120°C for 3 h, followed by filtration through a 45 μm Spin-X centrifuge filter (Corning). 10 μL of the hydrolyzed sample was dried in a Speed-Vac for 2 h, then incubated with 100 μL of chloramine T reagent at room temperature for 5 min and with 100 μL of 4-(dimethylamino)benzaldehyde (DMAB) at 60°C for 90 min. The absorbance of oxidized hydroxyproline was measured at 560 nm. Hydroxyproline concentrations were calculated from a calibration curve constructed using known concentrations of trans-4-hydroxyl-L-proline. The total amount of protein isolated from the weighed tissue was measured using a protein assay kit (Bio-Rad, absorbance at 595 nm). The amount of collagen was expressed as μg / mg total protein.

[0115] Picrosirius staining Paraffin-embedded liver sections were deparaffinized and rehydrated by heating at 60°C for 10–30 min, and then passaged in the following solutions: - Xylene 1-2 times for 10 minutes - 100% EtOH - 1x 2 min, 95% EtOH - 1 min, 70% EtOH - 1 min, 50% EtOH - 1 min - PBS-3×3 min

[0116] The slides were then stained for 60 minutes at room temperature with a solution containing 0.1% picrosirius red (Direct Red 80) and 0.1% fast green (counterstain) in a saturated aqueous solution of picric acid, followed by one wash with distilled water, dehydration in three changes of 100% EtOH, removal in xylene, and embedding in resin medium.

[0117] Liver function tests Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bile acids were measured using a commercially available veterinary chemistry analyzer (VetScan2, Abaxis).

[0118] Reverse transcription quantitative polymerase chain reaction RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. 250 ng of isolated RNA was then used to synthesize cDNA using SuperScript VILO (Invitrogen). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche) and analyzed using a LightCycler96 (Roche). Data are expressed as fold change with ΔΔCt compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0119] result F-4N and liver fibrosis Compared with mice that did not receive any F-4N, mice that received F-4N showed less collagen fiber deposition, especially in the portal-to-portal region, and a reduced hydroxyproline content (Figure 6). These results suggest that F-4N attenuates established liver fibrosis in a mouse model of PSC.

[0120] F-4N and liver function Livers from mice were examined for the expression of liver function markers. Compared with mice not treated with F-4N, mice treated with F-4N showed lower levels of ALT, ALP, and bile acids (Figure 7). These results suggest that F-4N ameliorates liver injury and cholestasis in a mouse model of PSC.

[0121] F-4N and liver aging Liver tissue was examined for the expression of type I collagen, inflammatory markers, and senescence markers. Mice treated with F-4N showed reduced expression of type I collagen, as well as reduced expression of inflammatory and senescence markers (Figure 8). These results suggest that F-4N effectively targets senescent cells in a mouse model of PSC, resulting in reduced inflammation and liver fibrosis.

[0122] [Example 4] Flavonol structure optimization Materials and Methods For senescent fibroblast viability experiments, human adult lung fibroblasts were replicate-passaged to senescence (confirmed by staining for RNA and senescence-associated beta-galactosidase). Cells were then seeded into 96-well plates, treated with the indicated compounds, and incubated for 96 hours. Cells were then fixed using 4% PFA, permeabilized with 0.25% Triton X-100, and nuclear stained with DAPI. Cells were then imaged using a 4x objective on a Cytation 5 microscope, and cell numbers were quantified using automated software (Biotek Gen5). Data shown were plotted as % viability, normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For TGFβ-stimulated collagen deposition experiments (square data points), human adult lung fibroblasts (passage 3) were seeded into 96-well plates and treated with the indicated compounds plus 2 ng / mL TGFβ to stimulate collagen expression. Cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% Triton X-100, and stained with a primary antibody recognizing type I collagen and an infrared-labeled secondary antibody. Wells were then imaged at 1x using an Odyssey Lx (LI-CORE) infrared imager, and collagen intensity was quantified using automated software. Data shown were plotted as % collagen intensity, normalized to vehicle-treated wells. Mean + / - SEM, N = 3 independent experiments.

[0123] result Flavonols were evaluated in three-point dose-response curves for their ability to specifically induce cell death in senescent fibroblasts, as well as to inhibit collagen deposition in low-passage fibroblasts stimulated with TGFβ (Figure 9). 2-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-6-methylchromen-4-one and 2-(3,4-diethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one both exhibited potent toxicity toward senescent cells and inhibited collagen deposition. This was confirmed by six-point dose-response curves (Figure 10). Both compounds contain a para-ethoxy modification on the B ring of their flavonol cores.

[0124] A second generation of flavonol compounds featuring this paraethoxy modification on the B ring of the flavonol core was evaluated for their effect on reducing the viability of senescent fibroblasts and on blocking collagen deposition in TGFβ-stimulated nonsenescent fibroblasts (Figure 11). Based on these results, we identified a structure-activity relationship in which the paraethoxy modification on the B ring was potent and the unsubstituted A ring was optimal. The novel synthetic compounds surveyed in Table 1, including molecule F-4N, were designed based on this SAR.

[0125] Finally, flavonols containing heteroatom substitutions within the aromatic core were tested for their effect on senescent fibroblast viability and inhibition of collagen deposition. Although these analogs contain a paraethoxy at a position consistent with the flavonoid B ring, they showed significantly reduced potency.

[0126] [Example 5] Treatment of IPF A person diagnosed with IPF is administered or self-administered a composition comprising one or more flavonoid compounds having the structure of Formula (I). In some cases, the administered flavonoid compounds can reduce the severity of one or more symptoms of IPF. In some cases, the administered flavonoid compounds can reduce the amount of fibrous scarring in the person's lungs.

[0127] [Example 6] Treatment of PSC A human diagnosed with PSC is administered or self-administered a composition comprising one or more flavonoid compounds having the structure of Formula (I). In some cases, the administered flavonoid compounds can reduce the severity of one or more symptoms of PSC. In some cases, the administered flavonoid compounds can reduce the amount of fibrous scarring in the human's liver.

[0128] [Example 7] F-4N and pulmonary fibrosis The results of this example correspond to at least some of the results achieved in other examples, at least some of which will be described in more detail below.

[0129] method Mice received three intratracheal bleomycin treatments, 2 weeks apart. 28 days after the final bleomycin insult, mice were treated daily with vehicle or F-4N for 14 days. A schematic diagram of the study design is shown in Figure 13A.

[0130] result The efficacy of F-4N in a bleomycin injury model of pulmonary fibrosis was evaluated.

[0131] Hydroxyproline analysis was performed on individual lungs from 8-week-old FVB wild-type mice treated with bleomycin alone (vehicle), bleomycin and F-4N, and sham treatment. Representative histological images of H&E-stained lungs are shown in Figure 13B.

[0132] Whole lung RNA expression was also assessed. Mouse lungs from Figure 13B were analyzed by qPCR for changes in fibrosis-specific profibrotic genes, senescence-associated genes, type I / type II alveolar epithelial markers, and intermediate / transitional alveolar markers, as well as Stk17b (Figure 14).

[0133] [Example 8] F-4N and PSC The results of this example correspond to at least some of the results achieved in other examples, at least some of which will be described in more detail below.

[0134] method Approximately 7-month-old Mdr2 with established liver fibrosis - / - Twenty mice (10 males, 10 females) were assigned to receive either F-4N or vehicle (n=5 per group) for 4 weeks. At the end of treatment, mice were sacrificed and livers were harvested to evaluate liver inflammation and fibrosis. A schematic diagram of the study design is shown in Figure 15.

[0135] result Oral delivery of F-4N for 4 weeks to a mouse model of PSC with established liver fibrosis resulted in: - Improvement in liver fibrosis, especially parenchymal fibrosis ("bridging fibrosis"), which is also confirmed by a reduction in the expression of fibrogenic genes in the liver. See Figures 16-18. - Significant reduction in tissue markers of inflammation and aging in the liver. See Figure 19.

[0136] [Example 9] F-4N and Nonalcoholic Steatohepatitis (NASH) Mice in a mouse model of NASH were intraperitoneally treated with 10 mg / kg of F-4N daily for 2 weeks (Figure 20A). Livers from treated mice were analyzed by H&E and Sirius Red staining (Figure 20B). Sirius Red staining was quantified (Figure 20C). Changes in liver weight and colon weight were also assessed (Figure 20D). These results suggested that F-4N is effective for treating NASH.

[0137] Livers from the study in Figure 20 were analyzed by qPCR for the expression of profibrotic and inflammatory genes and Stk17b (the molecular target of F-4N). F-4N efficiently reduced the expression of profibrotic and inflammatory genes and Stk17b (Figure 21).

[0138] Additional analyses of samples from the study in Figure 20 were also performed. Figure 22A shows liver function tests, Figure 22B shows liver triglyceride analysis, and Figure 22C shows hydroxyproline assessment of liver collagen content. These results suggested that F-4N is effective for treating NASH.

[0139] [Example 10] F-4N and ocular fibrosis Conjunctival fibroblasts were cultured in 2% FBS with or without F-4N for 4 days and assessed for proliferation. Treated cells were fixed, stained with DAPI, and counted using automated Cytation 5 software (Figure 23A).

[0140] Conjunctival fibroblasts were cultured for 3 days in 2% FBS with or without 2 ng / mL TGFβ and F-4N, and assessed for the presence of fibroblasts. Treated cells were fixed, stained with DAPI, stained with αSMA antibody, and counted using automated Cytation 5 software (Figure 23B).

[0141] Conjunctival fibroblasts were cultured for 6 days in 2% FBS with or without 2 ng / mL TGFβ and F-4N, and assessed for collagen deposition. Treated cells were fixed, stained with DAPI, and stained with an antibody against type I collagen, and quantified using automated Cytation 5 software (Figure 23C).

[0142] Together, these results demonstrate that F-4N can be used to treat ocular fibrosis.

[0143] [Example 11] Pharmacology of F-4N Administration An acute bleomycin challenge study using various doses of F-4N was performed as shown in Figure 24A. On day 1, mice were subjected to intratracheal sham or bleomycin injury. On day 7, mice were assigned to groups and treated daily for 7 days with vehicle, 10, 30, or 100 mg / kg F-4N administered orally by gavage. On day 14, organs and plasma were harvested. Body weight changes were measured throughout the experiment (Figure 24B). Whole lung RNA expression of profibrotic genes was also examined (Figure 24C). In this acute challenge, dose-finding study, daily oral administration of 30 and 100 mg / kg F-4N administered orally by gavage improved body weight changes and reduced profibrotic gene expression.

[0144] Biomarkers of F-4N efficacy were identified. RNA was collected from vehicle-treated mice and mice treated with 30 mg / kg F-4N. The RNA was analyzed using an RT2 Profiler PCR array (Qiagen catalog number 330231), which measures the expression of 89 different cytokines and chemokines. After 7 days of treatment with F-4N, eleven (11) genes were identified that showed decreased expression in mice (Figure 25). The levels of genes whose expression changes in response to F-4N can be used as biomarkers for the efficacy of F-4N.

[0145] The levels of F-4N in the plasma and liver of mice after F-4N exposure were examined (Figure 26). Plasma and liver tissue from mice subjected to the protocol described in Figure 24A were collected 2 and 8 hours after the final dose. The levels of unbound (free) F-4N were analyzed by Cyprotex (Framingham, MA) using LC-MS. The effective concentration of F-4N was measured in both plasma and liver, with F-4N levels being higher in the liver than in plasma.

[0146] plasma stability The stability of F-4N in plasma was evaluated (Figure 27). Compounds were incubated with human or mouse plasma. The percentage of recovered F-4N was analyzed by LC-MS. Warfarin was used as a control for stable compounds, and propantheline was used as a control for unstable compounds. Analysis was performed by Cyprotex (Framingham, MA). These results demonstrate that F-4N is stable in both human and mouse plasma.

[0147] The microsomal stability of F-4N was also evaluated (Figure 28). Compounds were incubated with human or mouse liver-derived microsomes. The percentage of recovered F-4N was analyzed by LC-MS. Verapamil was used as a control for a rapidly degraded compound. Analysis was performed by Cyprotex (Framingham, MA). These results demonstrate that the half-life and clearance rate of F-4N are consistent with many clinically approved orally administered drugs.

[0148] Plasma protein binding assays were also performed (Table 2). Compounds were incubated with rat plasma for 4 hours. The percentage of recovered F-4N was analyzed by LC-MS. Warfarin was used as a control for compounds with high plasma protein binding. Analysis was performed by Cyprotex (Framingham, MA).

[0149] [Table 2]

[0150] These results demonstrate that F-4N exhibits high plasma protein binding.

[0151] [Example 12] Mechanism of the F-4N The effect of Stk17b on fibrosis markers was evaluated. Stk17b was knocked down in a precision-cut lung slice (PCLS) model. A schematic diagram of this study protocol is shown in Figure 29A. On day 0, six mice were intratracheally administered bleomycin. On day 14, lungs at the peak of fibrosis were excised and sliced using a vibratome to generate 300 μM PCLS. The tissue was cultured ex vivo with either non-targeting siRNA or siRNA targeting Stk17b for 4 days. RNA was then harvested and analyzed by qPCR (Figure 29B). These results demonstrated that Stk17b could be a target (e.g., reduced or inhibited) for treating pulmonary fibrosis.

[0152] DRAK1 kinase activity was also assessed. Cells were incubated with DRAK1, radiolabeled ATP, and F-4N or the inactive analog 5-MeOH-F-4N. DRAK1 kinase activity was measured by Reaction Biology. DRAK1 kinase activity was measured and plotted (Figure 30). These results demonstrated that F-4N can inhibit DRAK1 kinase activity. Analogs that were inactive in the phenotypic cell-based assay were inactive against DRAK1.

[0153] The efficacy of F-4N was assessed using ex vivo organ cultures derived from IPF patients. Lung tissue slices (approximately 500 μM) were dissected from transplanted lungs of IPF patients and cultured ex vivo with DMSO (0.1%) or F-4N (3 μM) for 4 days. Lung tissue slices were harvested, RNA was isolated, and qPCR analysis was performed (Figure 31). Culture medium was also collected for ELISA analysis of IL-6 (Figure 31B). N = 3 patient samples. These results demonstrated that F-4N reduced the expression of fibrotic markers and enhanced the expression of mature alveolar epithelial cell markers.

[0154] [Example 13] Structural optimization of quercetin to enhance its pharmacological properties This example describes the identification of quercetin structures that can enhance senescent cell clearance capacity and potentiate transdifferentiation inhibition in lung fibroblasts.

[0155] Relative flavonoid efficacy and loss of toxicity in the aging brain and liver. Five- and 28-month-old wild-type mice were treated orally by gavage with the indicated concentrations of flavonoids (F, Q, compound 19, or compound 20) or vehicle for 4 consecutive days and then euthanized 1 week later. Real-time PCR analysis suggests that low doses of compound 19 and / or 20 can reduce the expression of p16ink4a, a key senescence-activated gene, in the brain (Figure 32A) and liver (Figure 32B) more reliably than fisetin (F) or quercetin (Q), which have established senolytic activity at relatively high doses. Analysis of CD68 expression, an indicator of inflammatory activation, demonstrated no drug-induced toxicity in the brain (Figure 32C) or liver (Figure 32D).

[0156] Quercetin analogues can potently kill senescent fibroblasts Senescence marker expression (Figure 33A) and proliferation (Figure 33B) were measured in induced senescent fibroblasts treated with quercetin analogs. More than 30 diverse quercetin analogs were screened for their ability to more potently kill senescent cells than low-passage proliferating fibroblasts. Shown in Figure 33C are the most potent derivatives, several of which exhibit nanomolar to low micromolar potencies.

[0157] TGF-β and senescent cell-conditioned medium can promote transdifferentiation of fibroblasts into myofibroblasts Fibroblasts were detected by αSMA staining, and staining intensity was quantified (Figure 34A). Markers of fibroblast activation were also assessed (Figure 34B). These results demonstrated that quercetin analogs potently prevented fibroblast activation.

[0158] Quercetin analogs can prevent SASP-CM and TGFβ-induced collagen deposition. TGFβ and senescent cell-conditioned medium promote type I collagen deposition (Figure 35A). Quercetin analogs potently prevent type I collagen deposition (Figure 35B).

[0159] Quercetin analogs can prevent TGFβ-induced profibrotic gene expression. TGFβ promotes the expression of profibrotic genes (Figure 36A), and quercetin analogs potently inhibit the expression of profibrotic genes (Figure 36B).

[0160] Quercetin analogs with p-ethoxy had enhanced activity Exemplary quercetin analogs with p-ethoxy are shown in Figure 37A. A cell proliferation graph showing that quercetin analogs with p-ethoxy induced cellular senescence (Figure 37B). A cell proliferation graph showing that quercetin analogs without p-ethoxy did not induce cellular senescence (Figure 37C).

[0161] [Example 14] Treatment of IPF A person diagnosed with IPF is administered or self-administered a composition comprising one or more flavonoid compounds having the structure of formula (II). In some cases, the administered flavonoid compounds can reduce the severity of one or more symptoms of IPF. In some cases, the administered flavonoid compounds can reduce the amount of fibrous scarring in the person's lungs.

[0162] [Example 15] Treatment of PSC A human diagnosed with PSC is administered or self-administered a composition comprising one or more flavonoid compounds having the structure of Formula (II). In some cases, the administered flavonoid compounds can reduce the severity of one or more symptoms of PSC. In some cases, the administered flavonoid compounds can reduce the amount of fibrous scarring in the human liver.

[0163] Other embodiments While the present invention has been described in conjunction with a detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Equation (I): 【Chemistry 1】 Flavonoid compounds having the structure or pharmaceutically acceptable salts thereof (In the formula: R 1 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 Selected from the group consisting of alkoxys, R 2 is selected from the group consisting of H, OH, C 1 to C 4 alkyl, halogen, and C 1 to C 4 alkoxy, and is selected from the group consisting of R 3 H, CH 2 CH 3 Selected from the group consisting of cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, and 4-pyridinyl, And R 4 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 (Selected from the group consisting of alkoxys) A composition containing the following:

2. The flavonoid compound of formula (I) above has the structure: 【Chemistry 2】 The composition according to claim 1, having the following characteristics.

3. The flavonoid compound of formula (I) above has the structure: 【Transformation 3】 The composition according to claim 1, having the following characteristics.

4. The flavonoid compound of formula (I) above has the structure: 【Chemistry 4】 The composition according to claim 1, having the following characteristics.

5. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

6. Equation (I): 【Transformation 5】 Flavonoid compounds having the structure or pharmaceutically acceptable salts thereof (In the formula: R 1 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 Selected from the group consisting of alkoxys, R 2 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 Selected from the group consisting of alkoxys, R 3 H, CH 2 CH 3 Selected from the group consisting of cyclopropyl, phenyl, 2-pyridinyl, 3-pyridinyl, and 4-pyridinyl, And R 4 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 (Selected from the group consisting of alkoxys), and Pharmacologically acceptable carriers, excipients, or diluents A pharmaceutical composition containing the following:

7. A method for treating a mammal having a fibrotic state, comprising administering to the mammal a composition according to any one of claims 1 to 6.

8. The method according to claim 7, wherein the mammal is a human.

9. The method according to claim 7, wherein a mammal is determined to have the fibrotic state.

10. The method according to claim 9, wherein the fibrotic state is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), primary sclerosing cholangitis (PSC), and non-alcoholic steatohepatitis (NASH).

11. The method according to claim 10, wherein the fibrotic state is IPF, and the method further comprises administering to the mammal an agent used to treat IPF.

12. The method according to claim 11, wherein the agent used to treat the IPF is selected from the group consisting of pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC12249, AM966, AM095, taribavirin, BI-2545, GLPG1690, BBT877, SAR100842, BMS-986020, minaprin, dopamine, levodopa, apomorphine, phenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, ripasudil, netalusdil, bermosudil, lebrikizumab, tralokinumab, dupilumab, and pamlevlumab.

13. The method according to claim 10, wherein the fibrotic state is PSC, and the method further comprises administering to the mammal an agent used to treat PSC.

14. The method according to claim 13, wherein the agent used to treat the PSC is selected from the group consisting of ursodeoxycholic acid (UDCA), corticosteroids, bile acid blockers, antibiotics, and antihistamines.

15. A method for alleviating fibrosis in a mammal having a fibrotic state, comprising administering a composition according to any one of claims 1 to 6 to the mammal.

16. The method according to claim 15, wherein the mammal is a human.

17. The method according to claim 15, wherein a mammal is determined to have the fibrotic state.

18. The method according to claim 15, wherein the fibrotic state is selected from the group consisting of IPF, PSC, and NASH.

19. A method for reducing the number of senescent cells in a mammal having a fibrotic state, comprising administering to the mammal a composition according to any one of claims 1 to 6.

20. The method according to claim 19, wherein the mammal is a human.

21. The method according to claim 19, wherein a mammal is determined to have the fibrotic state.

22. The method according to claim 19, wherein the fibrotic state is selected from the group consisting of IPF, PSC, and NASH.

23. The method according to claim 19, wherein the senescent cells are fibroblasts.

24. The method according to claim 23, wherein the fibrotic state is IPF and the senescent cells are lung fibroblasts.

25. The method according to claim 19, wherein the senescent cells are epithelial cells.

26. The method according to claim 25, wherein the fibrotic state is PSC and the senescent cells are bile duct cells.

27. A method for inhibiting serine / threonine kinase 17 (STK17) polypeptide in a mammal, comprising administering a composition according to any one of claims 1 to 6 to the mammal.

28. The method according to claim 27, wherein the mammal is a human.

29. The method according to claim 27, wherein the STK17 polypeptide is selected from the group consisting of STK17A(DRAK1) polypeptide and STK17B(DRAK2) polypeptide.

30. The method according to claim 9, wherein the fibrotic state is ocular fibrosis.

31. The method according to claim 15, wherein the fibrotic state is ocular fibrosis.

32. Formula (II): 【Transformation 6】 Flavonoid compounds having the structure or pharmaceutically acceptable salts thereof (In the formula: X 1 It is selected from N and CH, X 2 N and CR 4 Selected from, R 1 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 Selected from the group consisting of alkoxys, R 2 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 Selected from the group consisting of alkoxys, R 3 H, CH 3 CH 2 CH 3 Selected from the group consisting of cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, thiophene-2-yl, thiophene-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl, And R 4 H, OH, C 1 ~C 4 Alkyl, halogen, and C 1 ~C 4 (Selected from the group consisting of alkoxys) A composition containing the following:

33. The flavonoid compound of formula (II) above is given by the following formula: 【Transformation 7】 【change】 【change】 The composition according to claim 32, comprising any one of the or a pharmaceutically acceptable salt thereof.

34. A pharmaceutical composition comprising the composition of claim 32 or 33 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or diluent.

35. A method for treating a mammal having a fibrotic state, comprising administering to the mammal a composition according to any one of claims 32 and 33.

36. The method according to claim 35, wherein the mammal is a human.

37. The method according to claim 35, wherein a mammal is determined to have the fibrotic state.

38. The method according to claim 37, wherein the fibrotic state is selected from the group consisting of IPF, PSC, NASH, and ocular fibrosis.

39. The method according to claim 38, wherein the fibrotic state is IPF, and the method further comprises administering to the mammal an agent used to treat IPF.

40. The method according to claim 39, wherein the agent used to treat the IPF is selected from the group consisting of pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC12249, AM966, AM095, taribavirin, BI-2545, GLPG1690, BBT877, SAR100842, BMS-986020, minaprin, dopamine, levodopa, apomorphine, phenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, ripasudil, netalusdil, bermosudil, lebrikizumab, tralokinumab, dupilumab, and pamlevlumab.

41. The method according to claim 38, wherein the fibrotic state is PSC, and the method further comprises administering to the mammal an agent used to treat PSC.

42. The method according to claim 41, wherein the agent used to treat the PSC is selected from the group consisting of UDCA, corticosteroids, bile acid blockers, antibiotics, and antihistamines.

43. A method for alleviating fibrosis in a mammal having a fibrotic state, comprising administering the composition according to any one of claims 32 and 33 to the mammal.

44. The method according to claim 43, wherein the mammal is a human.

45. The method according to claim 43, wherein a mammal is determined to have the fibrotic state.

46. The method according to claim 43, wherein the fibrotic state is selected from the group consisting of IPF, PSC, NASH, and ocular fibrosis.

47. A method for reducing the number of senescent cells in a mammal having a fibrotic state, comprising administering the composition according to any one of claims 32 and 33 to the mammal.

48. The method according to claim 47, wherein the mammal is a human.

49. The method according to claim 47, wherein a mammal is determined to have the fibrotic state.

50. The method according to claim 47, wherein the fibrotic state is selected from the group consisting of IPF, PSC, NASH, and ocular fibrosis.

51. The method according to claim 47, wherein the senescent cells are fibroblasts.

52. The method according to claim 51, wherein the fibrotic state is IPF and the senescent cells are lung fibroblasts.

53. The method according to claim 47, wherein the senescent cells are epithelial cells.

54. The method according to claim 53, wherein the fibrotic state is PSC and the senescent cells are bile duct cells.

55. A method for inhibiting the STK17 polypeptide in a mammal, comprising administering the composition according to any one of claims 32 and 33 to the mammal.

56. The method according to claim 55, wherein the mammal is a human.

57. The method according to claim 55, wherein the STK17 polypeptide is selected from the group consisting of STK17A(DRAK1) polypeptide and STK17B(DRAK2) polypeptide.

Images