Flavonoid compounds and methods and materials for treating fibrotic conditions using flavonoid compounds

Abstract

This document relates to flavonoid compounds and methods and materials for treating one or more fibrotic conditions (e.g., idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), and / or ocular fibrosis) using flavonoid compounds. For example, one or more flavonoid compounds having a structure of Formula (I) or Formula (II) may 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

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Patent Application Serial No. 63 / 351,485, filed on June 13, 2022. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated by reference therein. Technical Field

[0003] The present invention relates to flavonoid compounds and methods and materials for using flavonoid compounds to treat fibrotic disorders. Background Art

[0004] Fibrotic diseases are a major cause of morbidity and mortality and can affect nearly all tissues and organ systems. The U.S. government estimates that 45% of deaths in the United States are attributable to fibrotic diseases. Summary of the invention

[0005] Provided herein are flavonoid compounds and methods and materials for treating mammals (e.g., humans) with one or more fibrotic conditions (e.g., idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), and / or ocular fibrosis). For example, provided herein are flavonoid compounds having a structure of formula (I) and methods and materials for using one or more flavonoid compounds having a structure of formula (I). In some cases, one or more flavonoid compounds having a structure of formula (I) can be administered to a mammal (e.g., a human) with one or more fibrotic conditions to treat the conditions in the mammal. As demonstrated herein, one or more flavonoid compounds having a structure of formula (I) can induce apoptosis of senescent cells (e.g., senescent fibroblasts) and can be used to treat fibrotic conditions in mammals (e.g., humans).

[0006] Generally speaking, one aspect of the invention features a composition comprising a flavonoid compound having the structure of Formula (I):

[0007]

[0008] or a pharmaceutically acceptable salt thereof, wherein: R 1 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 2 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 3 H, CH2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy. The flavonoid compound of formula (I) may have the structure:

[0009]

[0010] The flavonoid compound of formula (I) may have the structure:

[0011]

[0012] The flavonoid compound of formula (I) may have the structure:

[0013]

[0014] The composition may further include a pharmaceutically acceptable carrier, excipient or diluent.

[0015] In another aspect, the present disclosure features a pharmaceutical composition comprising a flavonoid compound having the structure of Formula (I):

[0016]

[0017] or a pharmaceutically acceptable salt thereof, wherein: R 1 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 2 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 3 H, CH 2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy, and a pharmaceutically acceptable carrier, excipient or diluent.

[0018] In another aspect, the present invention features a method of treating a mammal having a fibrotic disorder. The method may include or consist essentially of administering to a mammal having a fibrotic disorder a composition comprising a flavonoid compound having the structure of formula (I):

[0019]

[0020] or a pharmaceutically acceptable salt thereof, wherein: R 1 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 2 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 3 H, CH 2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4Alkoxy. The mammal may be a human. The method may include identifying a mammal suffering from a fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis. The fibrotic condition may be IPF, and the method may include administering to the mammal an agent for treating IPF. The agent for treating IPF may be pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC 12249, AM966, AM095, talivrin, BI-2545, GLPG1690, BBT 877, SAR100842, BMS-986,020, minaprine, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, ripasudil, netarsudil, belumosudil, lebrikizumab, tralokinumab, dupilumab or pamrevlumab. The fibrotic disorder may be PSC, and the method may include administering to the mammal an agent for treating PSC. The agent used to treat PSC may be ursodeoxycholic acid (UDCA), a corticosteroid, a bile acid sequestrant, an antibiotic, or an antihistamine.

[0021] In another aspect, the present invention features a method of reducing fibrosis in a mammal suffering from a fibrotic disorder. The method may include or consist essentially of administering to a mammal suffering from a fibrotic disorder a composition comprising a flavonoid compound having the structure of formula (I):

[0022]

[0023] or a pharmaceutically acceptable salt thereof, wherein: R 1 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 2H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 3 H, CH 2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 The mammal may be a human. The method may include identifying a mammal suffering from a fibrotic disorder. The fibrotic disorder may be IPF, PSC, NASH, or ocular fibrosis.

[0024] In another aspect, the present invention features a method of reducing the number of senescent cells in a mammal suffering from a fibrotic disorder. The method may include or consist essentially of administering to a mammal suffering from a fibrotic disorder a composition comprising a flavonoid compound having the structure of formula (I):

[0025]

[0026] or a pharmaceutically acceptable salt thereof, wherein: R 1 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 2 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 3 H, CH 2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy. The mammal may be a human. The method may include identifying a mammal having a fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis. The senescent cell may be a fibroblast. The fibrotic condition may be IPF and the senescent cell may be a lung fibroblast. The senescent cell may be an epithelial cell. The fibrotic condition may be PSC and the senescent cell may be a bile duct cell.

[0027] In another aspect, the present invention features a method of inhibiting a serine / threonine kinase 17 (STK17) polypeptide in a mammal. The method may include or consist essentially of administering to the mammal a composition comprising a flavonoid compound having the structure of formula (I):

[0028]

[0029] or a pharmaceutically acceptable salt thereof, wherein: R 1 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 2 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 Alkoxy; R 3 H, CH 2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 H, OH, C 1 -C 4 Alkyl, halogen or C 1 -C 4 The mammal may be a human. The STK17 polypeptide may be a STK17A (DRAK1) polypeptide or a STK17B (DRAK2) polypeptide.

[0030] In another aspect, the present disclosure features a composition comprising a flavonoid compound having the structure of Formula (II):

[0031]

[0032] or a pharmaceutically acceptable salt thereof, wherein X 1 is selected from N and CH; X 2 Selected from N and CR 4 ; R 1 Selected from the following group: H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 2 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 3 Selected from the following group: H, CH 3 , CH 2 CH3 , cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thien-2-yl, thien-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl; and R 4 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy. The flavonoid compound of formula (II) has any one of the following formulae:

[0033]

[0034]

[0035] 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.

[0036] In another aspect, the present invention features a method of treating a mammal having a fibrotic disorder. The method may include or consist essentially of administering to a mammal having a fibrotic disorder a composition comprising a flavonoid compound having the structure of formula (II):

[0037]

[0038] or a pharmaceutically acceptable salt thereof, wherein X 1 is selected from N and CH; X 2 Selected from N and CR 4 ; R 1 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 2 Selected from the following group: H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 3 Selected from the following group: H, CH 3 , CH 2 CH 3 , cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thien-2-yl, thien-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl; and R 4 Selected from the group consisting of H, OH, C 1 -C 4Alkyl, halogen and C 1 -C 4 The mammal may be a human. The method may include identifying a mammal suffering from the fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, or ocular fibrosis. The fibrotic condition may be IPF, and the method may further include administering to the mammal an agent for treating IPF. The agent used to treat the IPF can be pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC 12249, AM966, AM095, taliberin, BI-2545, GLPG1690, BBT 877, SAR100842, BMS-986,020, milnaprim, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, rosudil, nesudil, besudil, nerikizumab, trorocilumab, dupilumab or panrelumab. The fibrotic disorder may be PSC, and the method may further comprise administering to the mammal an agent for treating PSC. The agent for treating PSC may be UDCA, a corticosteroid, a bile acid sequestrant, an antibiotic, or an antihistamine.

[0039] In another aspect, the present invention features a method of reducing fibrosis in a mammal suffering from a fibrotic disorder. The method may include or consist essentially of administering to a mammal suffering from a fibrotic disorder a composition comprising a flavonoid compound having the structure of formula (II):

[0040]

[0041] or a pharmaceutically acceptable salt thereof, wherein X 1 is selected from N and CH; X 2 Selected from N and CR 4 ; R 1 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 2 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 3 Selected from the following group: H, CH 3 , CH 2 CH 3, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thien-2-yl, thien-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl; and R 4 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 The mammal may be a human. The method may include identifying a mammal suffering from the fibrotic disorder. The fibrotic disorder may be IPF, PSC, NASH, or ocular fibrosis.

[0042] In another aspect, the present invention features a method of reducing the number of senescent cells in a mammal suffering from a fibrotic disorder. The method may include or consist essentially of administering to a mammal suffering from a fibrotic disorder a composition comprising a flavonoid compound having the structure of formula (II):

[0043]

[0044] or a pharmaceutically acceptable salt thereof, wherein X 1 is selected from N and CH; X 2 Selected from N and CR 4 ; R 1 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 2 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 3 Selected from the following group: H, CH 3 , CH 2 CH 3 , cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thien-2-yl, thien-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl; and R 4 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4Alkoxy. The mammal may be a human. The method may include identifying a mammal suffering from the fibrotic condition. The fibrotic condition may be IPF, PSC, NASH, and ocular fibrosis. The senescent cell may be a fibroblast. The fibrotic condition may be IPF and the senescent cell may be a lung fibroblast. The senescent cell may be an epithelial cell. The fibrotic condition may be PSC and the senescent cell may be a bile duct cell.

[0045] In another aspect, the present invention features a method of inhibiting STK17 polypeptide in a mammal. The method may include or consist essentially of administering to the mammal a composition comprising a flavonoid compound having the structure of formula (II):

[0046]

[0047] or a pharmaceutically acceptable salt thereof, wherein X 1 is selected from N and CH; X 2 Selected from N and CR 4 ; R 1 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 2 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy; R 3 Selected from the following group: H, CH 3 , CH 2 CH 3 , cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thien-2-yl, thien-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl; and R 4 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 The mammal may be a human. The STK17 polypeptide may be a STK17A (DRAK1) polypeptide or a STK17B (DRAK2) polypeptide.

[0048] Unless otherwise defined, the meaning of all technical and scientific terms used in the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar to or equivalent to those described in the present invention can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references described in the present invention are incorporated herein by reference in their entirety. In the event of a conflict, the present application specification including definitions shall prevail. In addition, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0049] The drawings and the following description further illustrate one or more embodiments of the present invention in detail. Other features, objectives and advantages of the present invention can be clearly seen from the description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A schematic diagram of an exemplary chemical synthesis of a flavonoid compound having the structure of Formula (I) is shown.

[0051] Figure 2 The effect of F-19 on lung cell populations following bleomycin injury and pulmonary fibrosis is shown. Seven days prior to injury, col1a2-mTmG mice were treated with tamoxifen to turn on GFP expression in collagen-producing fibroblasts. F-19 was given daily starting on day 10 post-injury, lungs were isolated, and lung tissue was flow sorted for fibroblasts, epithelial cells, and leukocytes on day 14. RNA was isolated from the sorted cells, followed by qPCR analysis of the indicated genes in each cell population. N = 3 mice / group. No expression of GFP in leukocytes (CD45 + Cells), indicating that they are selective.

[0052] Figures 3A-3C The efficacy of F-4N was shown in a non-regressive model of pulmonary fibrosis (aged mice). Figure 3A is a graph showing the survival rate of mouse groups. C57 / B6 mice aged 10-18 months received intratracheal bleomycin treatment on day 0, and one group received F-4N (10 mg / kg, daily intraperitoneal administration) starting on day 14. Figure 3B The images show lung histology (trichrome staining) of mouse groups (Sham, Bleo+Vehicle and Bleo+F-4N) and the bar graph shows hydroxyproline content analysis. Figure 3C Bar graphs show the expression of collagen I and senescence markers (Cdkn2a, Cdkn1a and Ccl2) in whole lung. N=7-9 male and female mice.

[0053] Figure 4A-4BThe mechanism of action of F-4N is shown. Figure 4A Z-score plots for a kinase panel screen of approximately 400 kinases at 1 μM F-4N are shown. Dose response curves for STK17A and STK17B show the half maximal inhibitory concentration (IC 50 ) value is about 200nM. Figure 4B The graph shows the expression of putative targets in senescent lung fibroblasts and demonstrates significant overexpression of STK17A / B.

[0054] Figure 5A-5B The bar graph compares the effects of siRNA targeting STK17A / B in senescent lung fibroblasts (etoposide-induced) and proliferative lung fibroblasts (pro) and shows that STK17A / B selectively regulates senescent lung fibroblasts. Figure 5A The bar graphs show the relative cell number per field of 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 count) and cleaved caspase-3 (apoptosis). Figure 5B The bar graph shows cells counted by automated imaging software. Cells were transfected; 4 days later, total RNA was isolated and subjected to qPCR analysis. N=3.

[0055] Figure 6 Mdr2 treated with vehicle or F-4N - / - Biochemical and histological examination of fibrosis in mice. - / - Mice (18-20 weeks old) were treated with F-4N (10 mg / kg / day ip, n=10) or vehicle (ip, n=10) for 14 days and then sacrificed the next day. Liver tissue and serum were collected for analysis. Left. Hydroxyproline assay of liver tissue showed that F-4N treatment reduced collagen content by approximately 40% compared to vehicle control treated mice. Right. Bitter red staining of mouse livers for selective visualization of collagen fibers showed a reduction in collagen fibers in F-4N treated mice, specifically in the portal vein to portal vein region ("bridging fibrosis").

[0056] Figure 7 .Mdr2 after F-4N treatment - / - Liver function tests were improved in mice treated with vehicle-treated Mdr2 - / - Compared with mice treated with F-4N, Mdr2 - / - The mice had significantly reduced alanine aminotransferase (ALT) (a marker of liver damage) and alkaline phosphatase (ALP), as well as serum bile acid values ​​(a marker of biliary damage and cholestasis).

[0057] Figure 8 Reverse transcription (RT)-PCR of whole liver RNA showed a reduction in markers of fibrosis, inflammation, and senescence. 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 inhibitor 1A (Cdkn1a) and 2A (Cdkn2a). Mdr2 expression was compared between mice treated with F-4N and those treated with vehicle. - / - Mouse, Mdr2 - / - All markers were significantly decreased.

[0058] Figures 9A-9R A 3-point dose response curve of senescent and healthy fibroblasts treated with flavonols is included. Collagen deposition in senescent fibroblasts (circular data points) or low-passage lung fibroblasts stimulated with TGFβ (square data points) was treated with increasing doses of: 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 ) flavonols, ( Fig.9F )4'-methoxy-3-flavonol, ( Figure 9G )4'-Hydroxy-3'-methoxy-flavonoid, ( Figure 9H )3-hydroxy-2-(4-methoxyphenyl)-6-methyl-4h-1-benzopyran-4-one, ( Fig.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-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, ( Fig.9O )2-(3,4-dimethoxyphenyl)-3-hydroxy-7-methyl-4h-1-benzopyran-4-one, ( Figure 9P)3-hydroxy-3',4'-(methylenedioxy)-flavone, ( Figure 9Q )2-(3,4-diethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one, or ( Figure 9R )3-hydroxy-3',4'-dimethoxyflavone. For senescent fibroblast survival experiments (circular data points), human adult lung fibroblasts were repeatedly passaged until senescence (confirmed by RNA and senescence-associated β-galactosidase staining). The cells were then seeded into 96-well plates, treated with the indicated compounds and incubated for 96 hours. The cells were then fixed with 4% PFA, permeabilized with 0.25% triton x-100, and nuclear stained with DAPI. The cells were then imaged using a 4X objective on a Cytation5 microscope, and cell counts were quantified using automated software (Biotek Gen5). The data shown are plotted as survival rate % 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 + 2ng / mL TGFβ to stimulate collagen expression. The cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% triton x-100, and stained with primary and secondary infrared-coupled antibodies that recognize type I collagen. The wells were then imaged at 1X using an Odyssey Lx (LI-CORE) infrared imager, and the collagen intensity was quantified using automated software. The data shown are plotted as collagen intensity % standardized relative to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments.

[0059] Figures 10A-10B Figure 6: Six-point dose-response curves of senescent and healthy fibroblasts treated with flavonols. Collagen deposition in senescent fibroblasts (circular data points) or low-passage lung fibroblasts (square data points) stimulated with TGFβ was treated with increasing doses of ( Fig. 10A )2-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-6-methylchromen-4-one or ( Fig. 10B)2-(3,4-diethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one was treated. For senescent fibroblast survival experiments (circular data points), human adult lung fibroblasts were repeatedly passaged until senescence (confirmed by RNA and senescence-associated β-galactosidase staining). The cells were then seeded into 96-well plates, treated with the indicated compounds and incubated for 96 hours. The cells were then fixed with 4% PFA, permeabilized with 0.25% triton x-100, and nuclear staining with DAPI. The cells were then imaged using a 4X objective on a Cytation5 microscope, and cell counts were quantified using automated software (BiotekGen5). The data shown are plotted as % survival 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 + 2ng / mL TGFβ to stimulate collagen expression. The cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% triton x-100, and stained with primary and secondary infrared-coupled antibodies that recognize type I collagen. The wells were then imaged at 1X using an Odyssey Lx (LI-CORE) infrared imager, and the collagen intensity was quantified using automated software. The data shown are plotted as collagen intensity % standardized relative to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments.

[0060] Figures 11A-11J Figure 6: Six-point dose-response curves of senescent and healthy fibroblasts treated with ethoxylated flavonols. Collagen deposition in senescent fibroblasts (circular data points) or low-passage lung fibroblasts stimulated with TGFβ (square data points) was increased with increasing doses of compound 17 ( Fig.11A )、Compound 18( Fig. 11B )、Compound 19( Fig. 11C )、Compound 20( Fig.11D )、Compound 21( Fig.11E )、Compound 22( Fig.11F )、Compound 23( Fig.11G )、Compound 24( Fig.11H )Compound 25( Fig.11I ) or compound 26 ( Fig.11J) were treated. For the senescent fibroblast survival experiment (circular data points), human adult lung fibroblasts were repeatedly passaged until senescence (confirmed by RNA and senescence-associated β-galactosidase staining (not shown)). The cells were then seeded into 96-well plates, treated with the indicated compounds and incubated for 96 hours. The cells were then fixed with 4% PFA, permeabilized with 0.25% triton x-100, and nuclear staining was performed with DAPI. The cells were then imaged using a 4X objective on a Cytation5 microscope, and cell counts were quantified using automated software (BiotekGen5). The data shown are plotted as % survival normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For the TGFβ-stimulated collagen deposition experiment (square data points), human adult lung fibroblasts (passage 3) were seeded into 96-well plates and treated with the indicated compounds + 2ng / mL TGFβ to stimulate collagen expression. The cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% triton x-100, and stained with primary and secondary infrared-coupled antibodies that recognize type I collagen. The wells were then imaged at 1X using an Odyssey Lx (LI-CORE) infrared imager, and the collagen intensity was quantified using automated software. The data shown are plotted as collagen intensity % standardized relative to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments.

[0061] Figures 12A-12E Figure 6: Six-point dose-response curves of senescent and healthy fibroblasts treated with flavonols having a derivatized flavonol core. Collagen deposition in senescent fibroblasts (circular data points) or low-passage lung fibroblasts stimulated with TGFβ (square data points) was increased with increasing doses of ( Fig. 12A )Compound 27, ( Fig. 12B )Compound 28, ( Fig. 12C )Compound 29, ( Fig.12D ) Compound 30 or ( Fig.12E) Compound 31 was treated. For the senescent fibroblast survival experiment (circular data points), human adult lung fibroblasts were repeatedly passaged until senescence (confirmed by RNA and senescence-associated β-galactosidase staining (not shown)). The cells were then seeded into 96-well plates, treated with the indicated compounds and incubated for 96 hours. The cells were then fixed with 4% PFA, permeabilized with 0.25% triton x-100, and nuclear staining was performed with DAPI. The cells were then imaged using a 4X objective on a Cytation5 microscope, and cell counts were quantified using automated software (BiotekGen5). The data shown are plotted as % survival normalized to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments. For the TGFβ-stimulated collagen deposition experiment (square data points), human adult lung fibroblasts (passage 3) were seeded into 96-well plates and treated with the indicated compounds + 2ng / mL TGFβ to stimulate collagen expression. The cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% triton x-100, and stained with primary and secondary infrared-coupled antibodies that recognize type I collagen. The wells were then imaged at 1X using an Odyssey Lx (LI-CORE) infrared imager, and the collagen intensity was quantified using automated software. The data shown are plotted as collagen intensity % standardized relative to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments.

[0062] Fig.13A –13B shows the efficacy of F-4N in the bleomycin injury model of pulmonary fibrosis. Fig.13A Study Design. Mice received intratracheal bleomycin treatments three times every 2 weeks. 28 days after the last bleomycin insult, mice received daily F-4N or vehicle treatments for 14 days. Fig. 13B . Representative H&E stained histological images of lungs from each group and hydroxyproline analysis of individual lungs from each group. N=3 sham treated mice, N=6 bleo vehicle. N=7 bleo F-4N. 8 week old FVB wild type mice.

[0063] Fig.14 Whole lung RNA expression in repeated bleomycin injury studies. qPCR analysis Figure 1 Mouse lungs in bleo for changes in profibrotic genes, senescence-associated genes, alveolar epithelial type I / II markers, intermediate / transitional alveolar markers specific for fibrosis, and Stk17b. N=3 sham treated mice, N=6 bleo vehicle. N=7 bleo F-4N. 8 week old FVB wild type mice.

[0064] Fig.15 is a schematic diagram of the study design used for Example 8.

[0065] Fig.16 The graph shows that after oral administration of F-4N, Mdr2 - / - The mice had reduced liver mass.

[0066] Figures 17A-17B F-4N showed that after oral treatment with Mdr2 - / - The mice had reduced liver fibrosis. Fig.17A )Microscope image of liver tissue. Fig. 17B ) shows the hydroxyproline (HYP) content in the liver.

[0067] Fig.18 Figure 2 shows that oral delivery of F-4N can reduce Mdr2 - / - Liver fibrosis markers in mice.

[0068] Fig.19 The graph shows that oral delivery of F-4N significantly reduced Mdr2 - / - Markers of liver inflammation and cellular senescence in mice.

[0069] Figures 20A-20D The efficacy of F-4N was shown in a NASH mouse model. Fig. 20A ) Study design and body weight changes in mice treated with daily intraperitoneal administration of 10 mg / kg F-4N for 2 weeks. Fig. 20B ). Representative H&E and Sirius Red liver histology. Fig. 20C )Objective automated quantification of Sirius red staining. Fig.20D ) Changes in liver and colon weights. N=5 chow diet, N=10 choline-deficient high fat diet (CD-HFD) vehicle, N=10 CD-HFD+F-4N.

[0070] Fig.21 Analysis of whole liver RNA is shown. Liver expression of profibrotic and inflammatory genes from the study in Figure 20 was analyzed by qPCR, as well as expression of the F-4N molecular target Stk17b.

[0071] Figures 22A-22C Further analysis of samples from the study in Figure 20 is shown, including liver function tests ( Fig.22A ), liver triglyceride analysis ( Fig. 22B ) and hydroxyproline assessment of liver collagen content ( Fig. 22C ).

[0072] Fig.23A –23C showed the in vitro efficacy of F-4N in an ocular fibrosis model. Fig.23A ) Conjunctival fibroblasts were cultured with or without fetal bovine serum (FBS) and with or without F-4N. N=3. Fig. 23B ) Conjunctival fibroblasts were cultured with indicated concentrations of + / - 2 ng / mL TGFβ and 4N for 3 days, cells were fixed, stained with DAPI and antibodies against αSMA, and then quantified using automated software on Cytation 5. N=3. Fig.23C ) Conjunctival fibroblasts were cultured with indicated concentrations of + / - 2 ng / mL TGFβ and 4N for 6 days, fixed, stained with DAPI and antibodies against collagen I, and quantified using automated software on Cytation5. N=3.

[0073] Fig.24A –24C shows an acute exposure bleomycin study at multiple doses using F-4N. Fig.24A ) Schematic diagram of the study protocol. On day 1, mice received intratracheal sham treatment or bleomycin injury. On day 7, mice were divided into groups: vehicle, 10, 30, and 100 mg / kg F-4N, treated daily by oral gavage for 7 days. Organs and plasma were collected on day 14. Fig. 24B ) Weight change during the experiment. Fig.24C ) Whole lung RNA expression of profibrotic genes.

[0074] Fig.25 The discovery of biomarkers of F-4N efficacy was shown. Mice were treated with vehicle or 30 mg / kg F-4N and RNA was analyzed. The expression of 11 genes was shown to be reduced after mice were treated with F-4N for 7 days.

[0075] Fig.26 Shown are F-4N levels in plasma (upper panel) and liver (lower panel) of mice after F-4N exposure.

[0076] Fig. 27 The stability of F-4N in plasma is shown. Compounds were incubated with human (upper panel) or mouse (lower panel) plasma for the indicated time. Warfarin was used as a control for stable compounds and propantheline was used as a control for unstable compounds.

[0077] Fig.28 The microsomal stability of F-4N is shown. Compounds were incubated with human (upper panel) or mouse (lower panel) liver-derived microsomes for the indicated times. Verapamil was used as a control for rapidly degrading compounds.

[0078] Fig.29A –29B shows the results of Stk17b knockdown in the precision cut lung slice (PCLS) model. Fig.29A ) Schematic diagram of the study protocol. PCLS were cultured in vitro with non-targeting siRNA or Stk17b-targeting siRNA for 4 days. Fig.29B ) RNA was then collected and analyzed by qPCR.

[0079] Fig.30 Shown are the results of DRAK1 activity assays in the presence of F-4N (upper panel) or the inactive analog 5-MeOH-F-4N (lower panel).

[0080] Fig.31A –31B showed the efficacy of F-4N in IPF patient-derived organotypic ex vivo cultures. Fig.31A ) qPCR analysis of lung tissue sections. Fig.31B ) ELISA analysis of culture medium IL-6. N = 3 patient samples.

[0081] Fig.32A –32D shows the comparative efficacy of flavonoid compounds and the lack of toxicity in the aged brain and liver. Wild-type mice at 5 and 28 months of age were treated with the indicated concentrations of flavonoid compounds (F, Q, compound 19, or compound 20) or vehicle by oral gavage for four consecutive days and were euthanized one week later. Real-time PCR analysis showed that low-dose administration of compounds 19 and / or 20 was more effective than fisetin (F) or quercetin (Q) in reducing brain ( Fig.32A ) and liver( Fig.32B ) expression of the key senescence-activating gene p16ink4a, while fisetin or quercetin have been shown to have senescent cell-clearing effects at relatively high doses. Analysis of the expression of CD68, an indicator of inflammatory activation, showed that brain ( Fig.32C ) or liver ( Fig.32D ) were free of drug-induced toxicity.

[0082] Fig.33A –33C showed that quercetin analogs could effectively kill senescent fibroblasts. Fig.33A and 33B ) through the expression of senescence markers ( Fig.33A ) and proliferation ( Fig.33B ) Validation of measuring replicative induction of senescent fibroblasts. Fig.33C ) Proliferation of low-passage proliferating fibroblasts after treatment with quercetin analogs. Five representative derivatives with nanomolar to low micromolar potency are shown.

[0083] Fig.34A –34B showed that TGFβ and senescent cell conditioned medium can promote the transdifferentiation of fibroblasts into myofibroblasts. Fig.34A ) Representative images observed by αSMA staining. Fig.34B) Quercetin analogs can effectively prevent fibroblast activation. NCM: non-conditioned medium (control). NCM+TGFβ: non-conditioned medium + 2ng / mL TGFβ. CCM: conditioned medium from normal lung fibroblasts. SASP-CM: conditioned medium from senescent lung fibroblasts.

[0084] Fig.35A –35B showed that quercetin analogs could prevent SASP-CM and TGFβ-induced collagen deposition. Fig.35A ) TGFβ and senescent cell-conditioned medium promote collagen I deposition. Fig.35B ) Quercetin analogs can effectively prevent collagen I deposition. NCM: non-conditioned medium (control). NCM+TGFβ: non-conditioned medium + 2ng / mL TGFβ.

[0085] Fig.36A –36B showed that quercetin analogs could prevent TGFβ-induced profibrotic gene expression. Fig.36A )TGFβ promotes the expression of profibrotic genes. Fig.36B ) Quercetin analogs effectively block profibrotic gene expression. NCM: non-conditioned medium (control). NCM+TGFβ: non-conditioned medium + 2ng / mL TGFβ.

[0086] Fig.37A –37C showed that quercetin analogs with p-ethoxy groups had enhanced activity. Fig.37A ) Exemplary quercetin analogs with a p-ethoxy group (highlighted). Fig.37B ) Cell proliferation graph shows that quercetin analogs with p-ethoxy groups induce cell senescence. Fig.37C ) Cell proliferation plots show that quercetin analogs lacking the p-ethoxy group do not induce cell senescence. DETAILED DESCRIPTION

[0087] Provided herein are flavonoid compounds and methods and materials for treating mammals (e.g., humans) with one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) using flavonoid compounds. For example, provided herein are flavonoid compounds having a structure of formula (I):

[0088]

[0089] Where R 1 Can be H, OH, C 1 -C 4 Alkyl (e.g., methyl), halogen or C 1 -C 4 Alkoxy (eg, methoxy and ethoxy); R 2 Can be H, OH, C1 -C 4 Alkyl (e.g., methyl), halogen or C 1 -C 4 Alkoxy (eg, methoxy and ethoxy); R 3 Can be H, CH 2 CH 3 , cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl; and R 4 Can be H, OH, C 1 -C 4 Alkyl (e.g., methyl), halogen or C 1 -C 4 Alkoxy groups (eg, methoxy and ethoxy).

[0090] In some cases, the flavonoid compounds provided herein can have the following structure and can be referred to as F-4N:

[0091]

[0092] In some cases, the flavonoid compounds provided herein can have the following structure and can be referred to as F-5MeO:

[0093]

[0094] In some cases, the flavonoid compounds provided herein can have the following structure and can be referred to as F-4N-5MeO:

[0095]

[0096] Also provided herein are flavonoid compounds and methods and materials for treating mammals (e.g., humans) with one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis) using flavonoid compounds. For example, provided herein are flavonoid compounds having a structure of formula (II):

[0097]

[0098] or a pharmaceutically acceptable salt thereof, wherein:

[0099] X 1 is selected from N and CH;

[0100] X 2 Selected from N and CR 4 ;

[0101] R 1 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C4 Alkoxy;

[0102] R 2 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy;

[0103] R 3 Selected from the following group: H, CH 3 , CH 2 CH 3 , cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl, and tetrahydrofuran-3-yl;

[0104] And R 4 Selected from the group consisting of H, OH, C 1 -C 4 Alkyl, halogen and C 1 -C 4 Alkoxy.

[0105] In some embodiments, X 1 is N. In some embodiments, X 1 In some embodiments, X 2 is N. In some embodiments, X 2 Yes CR 4 In some embodiments, R 3 is H. In some embodiments, R 3 CH 3 In some embodiments, R 3 is phenyl, 4-OH-phenyl, 2-OH-phenyl or 3-OH-phenyl. 3 is 2-pyridyl, 3-pyridyl or 4-pyridyl. 3 It is thiophen-2-yl, thiophen-3-yl, tetrahydrofuran-2-yl or tetrahydrofuran-3-yl.

[0106] In some embodiments, the compound of formula (II) has the formula:

[0107]

[0108] or a pharmaceutically acceptable salt thereof.

[0109] In some embodiments, the compound of formula (II) has the formula:

[0110]

[0111] or a pharmaceutically acceptable salt thereof.

[0112] In some embodiments, the compound of formula (II) is selected from any one of the following compounds:

[0113]

[0114]

[0115]

[0116] or a pharmaceutically acceptable salt thereof.

[0117] In some cases, the flavonoid compounds provided herein (e.g., flavonoid compounds having a structure of formula (I) or formula (II)) can be in the form of a salt (e.g., a pharmaceutically acceptable salt). The salts of the compounds provided herein can be formed by an acid and a basic group (e.g., an amino functional group) of the compound or a base and an acidic group (e.g., a carboxyl functional group) of the compound. When the flavonoid compound having a 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, and organic acids such as p-toluenesulfonic acid, salicylic acid, tartaric acid, ditartaric acid, ascorbic acid, maleic acid, benzenesulfonic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, as well as related inorganic and organic acids. Thus, such pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4- The pharmaceutically acceptable acid addition salts that can be used include, but are 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, alkali metal hydroxides, including sodium, potassium and lithium; alkaline earth metal hydroxides, such as calcium and magnesium; other metal hydroxides, such as aluminum and zinc; ammonia, organic amines, such as unsubstituted or hydroxy-substituted mono-, di- or trialkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, di- or tri-(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, can be substantially isolated.

[0118] At various places herein, substituents of the compounds described herein are described in groups or ranges. Specifically, each individual member or subcombination of all members of such groups and ranges is included and described herein. For example, the term "C 1-6 "Alkyl" specifically means methyl, ethyl, C 3 Alkyl, C 4 Alkyl, C 5 Alkyl and C 6 alkyl.

[0119] Various aryl, heteroaryl, cycloalkyl and heterocycloalkyl rings are described in different places herein. Unless otherwise indicated, these rings can be attached to the rest of the molecule at any ring member where valence permits. For example, the term "pyridine ring" or "pyridyl" can refer to a pyridin-2-yl, pyridin-3-yl or pyridin-4-yl ring.

[0120] It should also be understood that, for clarity, some features of this article described in the context of separate embodiments may also be combined in a single embodiment. Conversely, for brevity, various features of this article described in the context of a single embodiment may also be provided individually or in any suitable subcombination.

[0121] The term "n-membered," where n is an integer, generally describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridinyl 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.

[0122] As used herein, the phrase "optionally substituted" refers to unsubstituted or substituted. Substituents are independently selected and may be substituted at any chemically accessible position. As used herein, the term "substituted" refers to hydrogen atoms being removed and replaced by substituents. A single divalent substituent, such as oxo, may replace two hydrogen atoms. It is understood that substitution on a given atom is limited by valence.

[0123] Throughout this definition, the term “C n-m " denotes a range including endpoints, wherein n and m are integers and represent the number of carbons. Examples include C 1-4 , C 1-6 wait.

[0124] As used herein, the term "C n-m"Alkyl" refers to a saturated hydrocarbon group that can be straight or branched and has 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 homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0125] As used herein, the term “C n-m “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 portion contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0126] As used herein, the term “C n-m "Alkoxy", used alone or in combination with other terms, refers to a radical of the formula -O-alkyl, wherein the alkyl radical has n to m carbons. Examples of alkoxy radicals 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 radical has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0127] As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon including a cyclized alkyl and / or alkenyl group. The cycloalkyl group may include a monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) group and a spirocycle. The ring-forming carbon atoms of the cycloalkyl group may be optionally substituted by 1 or 2 independently selected oxo or thioether groups (e.g., C(O) or C(S)). The definition of cycloalkyl also includes a portion having one or more aromatic rings, which is fused to the cycloalkyl ring (i.e., has a common bond), such as benzo or thienyl derivatives of cyclopentane, cyclohexane, etc. The cycloalkyl group containing a fused aromatic ring can be connected by any ring-forming atom including the ring-forming atom of the fused aromatic ring. The cycloalkyl group may have 3, 4, 5, 6, 7, 8, 9 or 10 ring-forming carbons (C 3-10 In some embodiments, the cycloalkyl group is C 3-10 In some embodiments, the cycloalkyl group is C 3-7Monocyclic cycloalkyl. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, etc. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0128] "Heterocycloalkyl" as used herein 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 may also include spirocycles. Examples of heterocycloalkyl groups include, but are not limited to, pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydrofuran (tetrahydropuran), oxetanyl, azetidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene (benzazapene) etc. The ring carbon atoms and heteroatoms of the heterocycloalkyl group may be optionally replaced by 1 or 2 independently selected oxo or thio groups (e.g., C(O), S(O), C(S) or S(O) 2 The heterocycloalkyl group may be connected by ring-forming carbon atoms or ring-forming heteroatoms. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. The definition of heterocycloalkyl also includes a portion having one or more aromatic rings that are fused to the heterocycloalkyl ring (i.e., have a common bond), such as piperidine, morpholine, aza The heterocycloalkyl groups containing fused aromatic rings can be connected by any ring-forming atoms including the ring-forming atoms of the fused aromatic rings. In some embodiments, the heterocycloalkyl group is a monocyclic 4-6 membered heterocycloalkyl group 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 group is a monocyclic or bicyclic 4-10 membered heterocycloalkyl group having 1, 2, 3 or 4 heteroatoms independently selected from nitrogen, oxygen or sulfur and having one or more oxidized ring members.

[0129] The term "compound" as used herein is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. A compound identified herein as one particular tautomeric form by name or structure is intended to include the other tautomeric forms unless otherwise indicated.

[0130] Compounds provided herein also include tautomeric forms.Tautomeric forms are produced by the exchange of a single bond with an adjacent double bond and the accompanying proton migration.Tautomeric forms include proton tautomers, which are isomeric protonation states with the same empirical formula and total charge.Examples of proton tautomers include, but are not limited to, keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and ring forms, in which protons can occupy two or more positions of a heterocyclic system, such as 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindoles and 1H- and 2H-pyrazoles.Tautomeric forms can be in equilibrium or spatially locked into a form by appropriate substitution.

[0131] In some cases, one or more flavonoid compounds provided herein (eg, one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) may lack chirality.

[0132] In some cases, one or more flavonoid compounds provided herein (eg, one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) can be neutral molecules (eg, can lack any charged moieties).

[0133] In some cases, one or more flavonoid compounds provided herein (eg, one or more flavonoid compounds having the structure of Formula (I) or Formula (II)) lack any catechol moieties.

[0134] In some cases, 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 formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a human) suffering from one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). For example, one or more flavonoid compounds having a 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 can be used in the compositions described herein include, but are not limited to, cyclodextrins (e.g., β-cyclodextrins (such as )), dimethyl sulfoxide (DMSO), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified cellulose (such as microcrystalline cellulose) and cellulose ethers (such as hydroxypropyl cellulose (HPC) and the cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), cross-linked polyvinyl pyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers and cross-linked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium dioxide, azo dyes, silica gel gum, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate and selenium), citric acid, sodium citrate, parabens (e.g., methylparaben and propylparaben), petrolatum, 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 hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyacrylates, waxes, lanolin, lecithin and corn oil.

[0135] 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 mammals (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 may be in the form of food supplements. In some cases, compositions suitable for oral administration may be in the form of beverage supplements. Compositions suitable for parenteral administration include but are not limited to aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient.

[0136] Also provided herein are methods for preparing one or more flavonoid compounds described herein (e.g., one or more flavonoid compounds having a structure of formula (I) or formula (II)). Any suitable method can be used to prepare one or more flavonoid compounds provided herein. In some cases, the method can be performed according to Figure 1 In some cases, the flavonoid compound having the structure of formula (I) or formula (II) can be prepared as described in Example 1.

[0137] Also provided herein is a method for using one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds with formula (I) or (II) structure). For example, one or more flavonoid compounds with formula (I) or (II) structure can be administered to a mammal (e.g., human) suffering from one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) to treat the mammal. In some cases, a mammal (e.g., human) suffering from one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) can be administered or guided to self-administer one or more flavonoid compounds with formula (I) or (II) structure.

[0138] 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 a structure of formula (I) or (II)) can be used to reduce or eliminate one or more symptoms of one or more fibrotic disorders. For example, a composition comprising one or more flavonoid compounds having a structure of formula (I) or (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from one or more fibrotic disorders (such as IPF, NASH, PSC, and ocular fibrosis)) to reduce or eliminate one or more symptoms of a fibrotic disorder (e.g., IPF, NASH, and PSC). Examples of symptoms of IPF disease 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 (broadening and rounding of the tips of the fingers or toes). Examples of symptoms of PSC disease include, but are not limited to, feeling tired or weak, itchy skin, abdominal pain, unintentional weight loss, loss of appetite, fever, enlarged liver, enlarged spleen, yellow eyes and skin (jaundice), symptoms of cirrhosis and liver failure (such as bloating, easy bruising and bleeding), confusion, difficulty thinking or memory loss, redness of the palms, 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 a structure of Formula (I) or Formula (II)) can be used to reduce one or more fibrotic disorder symptoms in a mammal suffering from one or more fibrotic disorders, for example, by reducing 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0139] 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 a structure of formula (I) or formula (II)) can be used to reduce or eliminate one or more complications associated with fibrotic disorders. For example, a composition comprising one or more flavonoid compounds having a structure of formula (I) or formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from one or more fibrotic disorders (such as IPF, NASH, PSC, and ocular fibrosis)) to reduce or eliminate one or more complications associated with fibrotic disorders. Examples of complications associated with IPF include, but are not limited to, pulmonary hypertension, acute exacerbation of pulmonary fibrosis, respiratory tract infection, acute coronary syndrome, thromboembolic disease, adverse drug reactions, and lung cancer. Examples of complications associated with primary sclerosing cholangitis 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 disorders in a mammal suffering from one or more fibrotic disorders, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0140] 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 a 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 a structure of formula (I) or formula (II)) can be used to reduce or eliminate fibrotic scars in mammals (e.g., one or more tissues in a mammal). For example, 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 used to slow down the progression of fibrosis in mammals (e.g., one or more tissues in a mammal).

[0141] 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 a structure of formula (I) or formula (II)) can be used to reduce or eliminate fibrotic scars in mammals. For example, a composition comprising one or more (e.g., one, two, three, four or more) flavonoid compounds having a structure of formula (I) or formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a person suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) to reduce or eliminate fibrotic scars in one or more tissues in the mammal. One or more flavonoid compounds provided herein can be used to reduce or eliminate fibrotic scars in any appropriate tissue in the mammal. Examples of tissues that may have fibrotic scars and can be reduced or eliminated with one or more flavonoid compounds provided herein include, but are not limited to, lungs, 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 a structure of Formula (I) or Formula (II)) can be used to reduce fibrotic scarring in one or more tissues in a mammal having fibrotic scarring by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0142] 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 a structure of formula (I) or (II)) can be used to slow down the progression of mammalian fibrosis. For example, a composition comprising one or more flavonoid compounds having a structure of formula (I) or (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) to slow down the progression of fibrosis in the mammal. One or more flavonoid compounds provided herein can be used to slow down the progression of fibrosis in any appropriate tissue in a mammal. Examples of tissues that can be fibrotic and tissues in which the progression of fibrosis can be slowed down by one or more flavonoid compounds provided herein include, but are not limited to, lungs, 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 a structure of Formula (I) or Formula (II)) can be effective in slowing down the progression of fibrosis in a mammal suffering from fibrosis, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. 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 a structure of Formula (I)) can be used to slow down the progression of fibrosis in a mammal suffering from fibrosis, for example, by 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).

[0143] 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 a 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 a structure of Formula (I) or Formula (II)) can be used to induce apoptosis of one or more senescent cells in a mammal. In some cases, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of Formula (I) or Formula (II)) exhibit little or no ability to induce apoptosis of proliferating cells in a mammal (e.g., a human).

[0144] 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 a structure of formula (I) or (II)) can be used to inhibit one or more serine / threonine kinase 17 (STK17) polypeptides. For example, a flavonoid compound having a structure of formula (I) or (II) can bind to an STK17 polypeptide to inhibit the polypeptide function of the STK17 polypeptide. A flavonoid compound having a structure of formula (I) or (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 a structure of formula (I) or (II)) include, but are not limited to, STK17A (DRAK1) polypeptides and STK17B (DRAK2) polypeptides. In some cases, a flavonoid compound having a structure of formula (I) or (II) can inhibit, for example, a flavonoid compound having a structure of formula (I) or (II) can inhibit, for example, a flavonoid compound having a structure of formula (I) or (II) can inhibit, for example, a flavonoid compound having a structure of formula (II) or (I ...I) or (II) can inhibit, for example, a flavonoid compound having a structure of formula (I) or (II) can inhibit, for example, a fla or STK17A (DRAK1) polypeptides shown in any one of Accession Nos. 9263 and Q9UEE5 In some cases, the flavonoid compounds having the structure of formula (I) or formula (II) can inhibit the expression of proteins such as NCBI or The STK17B (DRAK2) polypeptide shown in any one of Accession Nos. 9262 and 094768.

[0145] 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 a structure of formula (I) or (II)) can be used to inhibit fibroblast activation. The flavonoid compound having a structure of formula (I) or (II) can inhibit any suitable fibroblast activation. For example, the flavonoid compound having a structure of formula (I) or (II) can inhibit TGFβ-induced fibroblast activation.

[0146] 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 a structure of formula (I) or (II)) can be used to induce apoptosis of cells (e.g., senescent cells) in mammals. For example, a composition comprising one or more flavonoid compounds having a structure of formula (I) or (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) to induce apoptosis of senescent cells in mammals. One or more flavonoid compounds provided herein can be used to induce apoptosis of any appropriate type of senescent cells in mammals. Examples of cell types that can be aged and cell types that can be induced to apoptosis by one or more flavonoid compounds provided herein include, but are not limited to, fibroblasts (e.g., lung fibroblasts) and epithelial cells (e.g., bile duct cells). For example, one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of formula (I) or (II)) can be used to reduce the number of senescent cells in mammals. In some cases, a composition comprising one or more flavonoid compounds having a structure of Formula (I) or Formula (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from 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 a structure of Formula (I) or Formula (II)) can effectively reduce the number of senescent cells in a mammal suffering from one or more fibrotic conditions, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0147] 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 a structure of formula (I) or (II)) can be used to increase the survival rate of a mammal (e.g., a human). For example, a composition containing one or more flavonoid compounds having a structure of formula (I) or (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from 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 a structure of formula (I) or (II)) can be used to increase the survival rate of a mammal suffering from one or more fibrotic conditions, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0148] 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 a structure of formula (I) or (II)) can be used to reduce or eliminate inflammation in one or more tissues in a mammal. For example, a composition comprising one or more flavonoid compounds having a structure of formula (I) or (II) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human suffering from one or more fibrotic conditions such as IPF, NASH, PSC, and ocular fibrosis) to reduce or eliminate inflammation in one or more tissues in a mammal. One or more flavonoid compounds provided herein can be used to reduce inflammation in any appropriate tissue in a mammal. Examples of tissues that are possible and can be used to reduce inflammation with one or more flavonoid compounds provided herein include, but are not limited to, lungs, 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 in a mammal suffering from a fibrotic disorder (e.g., IPF) by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

[0149] Any suitable mammal suffering from one or more fibrotic disorders (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 can suffer from one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis) and 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, a human suffering from one or more fibrotic disorders can be treated by administering one or more flavonoid compounds having the structure of Formula (I) or Formula (II) as described herein.

[0150] When treating a mammal (e.g., a human) suffering from one or more fibrotic disorders, the mammal may suffer from any type of fibrotic disorder. Examples of fibrotic disorders that may be treated as described herein (e.g., by administering one or more flavonoid compounds having a structure of formula (I) or formula (II)) include, but are not limited to, IPF, PSC, NASH, and ocular fibrosis.

[0151] In some cases, the methods described herein may include identifying whether a mammal (e.g., a human) has one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis). Any suitable method may be used to identify whether a mammal has one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis). For example, chest scans (including X-rays and high-resolution computed tomography), breathing tests, pulse oximetry, blood oxygen and CO 2 Tests, exercise capacity and / or lung biopsy (e.g., observation of scarring signs) are used to identify mammals (e.g., humans) with IPF disease. For example, imaging techniques (e.g., magnetic resonance imaging (MRI), magnetic resonance cholangiopancreatography (MRCP) and endoscopic retrograde cholangiopancreatography (ERCP)), cholestatic biochemical signatures and / or liver biopsy can be used to identify mammals (e.g., humans) with PSC disease.

[0152] In some cases, 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 used to treat mammals (e.g., humans) suffering from age-related diseases. For example, 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 mammals (e.g., humans) suffering from age-related diseases (e.g., to treat mammals). 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, asthenia, cardiovascular disease, osteoarthritis, pulmonary fibrosis, kidney disease, neurodegenerative disease, hepatic steatosis, 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 a structure of Formula (I) or Formula (II)) can be administered to a mammal (e.g., a human) suffering from one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis) in any appropriate amount (e.g., any appropriate dose). An effective amount of a composition containing one or more flavonoid compounds having a structure of Formula (I) or Formula (II) can be any amount as described herein that can treat a mammal (e.g., a mammal suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) 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) can be 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, from about 20 μM to about 100 μM). In some cases, an effective amount of one or more flavonoid compounds having the structure of Formula (I) or Formula (II) may be about 200 nM IC. 50To about 800nM IC 50 (e.g., about 200 nM IC 50 To about 700nM IC 50 、About 200nM IC 50 To about 600nM IC 50 、About 200nM IC 50 To about 500nM IC 50 , about 200nMIC 50 To about 400nM IC 50 、About 200nM IC 50 To about 300nM IC 50 、About 300nM IC 50 To about 800nM IC 50 , about 400nMIC 50 To about 800nM IC 50 、About 500nM IC 50 To about 800nM IC 50 、About 600nM IC 50 To about 800nM IC 50 , about 700nMIC 50 To about 800nM IC 50 、About 300nM IC 50 To about 700nM IC 50 、About 400nM IC 50 To about 600nM IC 50 , about 300nMIC 50 To about 400nM IC 50 、About 400nM IC 50 To about 500nM IC 50 、About 500nM IC 50 To about 600nM IC 50 , about 600nMIC 50 To about 700nM IC 50 、About 200nM IC 50 , or about 800nM IC 50) In some cases, an effective amount of one or more flavonoid compounds having the structure of Formula (I) or Formula (II) may include about 0.1 milligrams per kilogram of body weight (mg / kg) to about 200 mg / kg (e.g., about 0.1 mg / kg to about 175 mg / kg, about 0.1 mg / kg to about 150 mg / kg, about 0.1 mg / kg to about 125 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 75 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 200 mg / kg, about 10 mg / kg to about 200 The effective amount can be kept constant or can be adjusted with a sliding amount or a variable dose according to the response of the mammal to the treatment. Various factors can affect the actual effective amount required for use in a particular application. For example, the frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, and / or the severity of the fibrotic disorder (e.g., IPF, NASH, and PSC) in the mammal being treated may require that the actual effective amount administered be increased or decreased.

[0153] 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 suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) at any appropriate frequency. The frequency of administration can be any frequency that is capable of treating a mammal (e.g., a mammal suffering from 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 a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can vary over the duration of treatment. As with the effective amount, a variety of factors can affect the actual frequency of administration for a particular application. For example, the effective amount, duration of treatment, use of multiple therapeutic agents, and / or route of administration may require an increase or decrease in the frequency of administration.

[0154] Compositions containing one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of formula (I) or (II)) can be administered to mammals (e.g., mammals suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) for any appropriate duration. The effective duration of administering or using a composition containing one or more flavonoid compounds having a structure of formula (I) or (II) can be any duration that can treat a mammal (e.g., a mammal suffering from one or more fibrotic conditions (such as IPF, NASH, PSC, and ocular fibrosis)) without causing significant toxicity to the mammal (e.g., human). For example, the effective duration can be from a few weeks to a few months, from a few months to a few years, or from a few years to a lifetime. Various factors can affect the actual effective duration for a particular treatment. For example, the effective duration can vary with the frequency of administration, the effective amount, the use of a variety of therapeutic agents, and / or the route of administration.

[0155] In some cases, a method for treating a mammal (e.g., a mammal such as a human suffering from one or more fibrotic disorders such as IPF, NASH, PSC, and ocular fibrosis) as described herein (e.g., by administering one or more flavonoid compounds having a structure of Formula (I) or (II)) can include administering to the mammal one or more flavonoid compounds having a structure of Formula (I) or (II) as the sole active ingredient to treat the mammal. For example, a composition containing one or more flavonoid compounds having a structure of Formula (I) or (II) can include one or more flavonoid compounds having a structure of Formula (I) or (II) as the sole active ingredient in the composition, which is effective in treating a mammal (e.g., a mammal suffering from one or more fibrotic disorders such as IPF, NASH, PSC, and ocular fibrosis).

[0156] In some cases, the methods for treating a mammal (e.g., a mammal such as a human having one or more fibrotic disorders (such as IPF, NASH, PSC, and ocular fibrosis)) as described herein (e.g., by administering one or more flavonoid compounds having a structure of Formula (I) or Formula (II)) can also include administering to the mammal one or more (e.g., one, two, three, four, five, or more) additional agents / therapies for treating the disorder (e.g., one or more fibrotic disorders (such as IPF, NASH, PSC, and ocular fibrosis). For example, a combination therapy for treating one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis) can include administering to a mammal (e.g., a human) one or more flavonoid compounds having a structure of Formula (I) or Formula (II) as described herein and one or more (e.g., one, two, three, four, five, or more) agents for treating one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis). Examples of agents 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, VPC 12249, AM966, and AM095), autotaxin inhibitors (e.g., talipin, BI-2545, GLPG1690, BBT 877, SAR100842 and BMS-986,020), D1 receptor agonists (e.g., milnaprim, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine and cabergoline), dasatinib, rho kinase inhibitors (e.g., hydroxyfasudil, rosudil, nesudil and besudil), IL-13 neutralizing antibodies (e.g., nerakizumab, trorocirumab and dupilumab), CTGF neutralizing antibodies (e.g., panrelumab) and any combination thereof. Examples of agents 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.

[0157] In the case where one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of formula (I) or formula (II)) are used in combination with an additional agent for treating one or more fibrotic disorders, the one or more additional agents can be administered simultaneously (e.g., in a single composition containing one or more flavonoid compounds having a structure of formula (I) or formula (II) and one or more additional agents) or administered independently. For example, one or more flavonoid compounds having a structure of formula (I) or formula (II) described herein can be administered first, followed by administration of one or more additional agents, or vice versa.

[0158] In some cases, the methods described herein for treating a mammal (e.g., a mammal such as a human suffering from one or more fibrotic conditions such as IPF, NASH, PSC, and ocular fibrosis) (e.g., by administering one or more flavonoid compounds having a structure of Formula (I) or Formula (II)) may also include subjecting the mammal to one or more (e.g., one, two, three, four, five or more) additional therapies for treating one or more fibrotic conditions (e.g., IPF, NASH, PSC, and ocular fibrosis). Examples of therapies for treating IPF disease include, but are not limited to, oxygen therapy, pulmonary rehabilitation, and / or lung transplantation. Therapies for treating PSC disease include, but are not limited to, endoscopic therapy (e.g., balloon dilatation and stenting), percutaneous therapy, non-transplant surgery, liver transplantation, and / or fecal microbiome transplantation.

[0159] In the case where one or more flavonoid compounds provided herein (e.g., one or more flavonoid compounds having a structure of formula (I) or (II)) are used in combination with one or more additional therapies for treating one or more fibrotic disorders (e.g., IPF, NASH, PSC, and ocular fibrosis), the one or more additional therapies may be administered simultaneously with or independently of the one or more flavonoid compounds having a structure of formula (I) or (II) described herein. For example, one or more flavonoid compounds having a structure of formula (I) or (II) may be administered before, during, or after one or more additional therapies.

[0160] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0161] Example

[0162] Example 1: Design and synthesis of flavonoid compounds

[0163] This example describes the design and characterization of flavonoid compounds that may be used to treat one or more fibrotic disorders.

[0164] method

[0165] Compound synthesis

[0166] Equimolar amounts (2-8 mmol) of hydroxyacetophenone and aldehyde are mixed in 10-50 mL EtOH. 5-12 mL 50% NaOH (aq) is added to the reaction and reacted at room temperature for 6-48 hours while being monitored by thin layer chromatography (TLC). After the aldehyde is exhausted, the resulting chalcone is precipitated with 10% HCl (aq). The chalcone is separated, dried, weighed, and dissolved (1-4 mmol) in 8-30 mL MeOH containing 1 M KOH. 5-15 mL H 2 O 2 Add to the reaction and react 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 NMR.

[0167] Figure 1 Shown are schemes for the synthesis of exemplary flavonoid compounds provided herein.

[0168] Apoptosis

[0169] Human adult lung fibroblasts were repeatedly passaged until senescence (confirmed by RNA and senescence-associated β-galactosidase staining). In parallel experiments, low-passage proliferating fibroblasts from the same donor were compared. Cells were seeded into 96-well plates, treated with the indicated compounds (6-point dose response) and incubated for 96 hours. Cells were then fixed with 4% PFA, permeabilized with 0.25% triton x-100, and nuclei were stained with a primary antibody against cleaved caspase-3, a secondary fluorescent-conjugated antibody, and DAPI. Cells were then imaged using a 4X objective on a Cytation5 microscope, and cell counts were quantified using automated software (Biotek Gen5). The data shown are the EC values ​​for the efficacy of each compound in inducing apoptosis in senescent versus proliferating lung fibroblasts calculated from calculated dose-response curves (N=3 independent experiments) 50 value.

[0170] Fibroblast activation

[0171] Human adult lung fibroblasts (passage 3) were seeded into 96-well plates and treated with the indicated compounds (6-point dose response) + 2ng / 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 primary and secondary fluorescent conjugated antibodies recognizing α-smooth muscle actin and DAPI. Cells were then imaged using a 4X objective on a Cytation5 microscope, and α-smooth muscle actin intensity was quantified using automated software (Biotek Gen5). Data shown are the IC of the efficacy of each compound in reducing α-smooth muscle actin expression. 50 Values, calculated from dose-response curves, N = 3 independent experiments.

[0172] result

[0173] The mechanisms of therapeutic efficacy of flavonoid compounds were evaluated using high throughput analysis. Table 1 summarizes the characteristics of exemplary flavonoid compounds.

[0174] Table 1.

[0175]

[0176]

[0177] Example 2: F-4N and pulmonary fibrosis

[0178] This example describes the use of one or more flavonoid compounds having the structure of formula (I) in the treatment of pulmonary fibrosis.

[0179] method

[0180] FIG3. Lung fibrosis was induced in C57 / B6 mice aged 10-18 months (average age approximately 15 months) using intratracheal bleomycin (1.1 units / kg) on ​​day 0 of the study. On day 14, one group of mice received F-4N (10 mg / kg intraperitoneally daily) for 14 days before harvesting the lungs to observe the outcome and senescent clearance and fibrosis. Lung structure and fibrosis were observed by trichome histological 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 and homogenized in sterile water (10 mg tissue / 100 μL H 2O), then hydrolyzed with 12M HCl at 120°C for 3 hours in a pressure-resistant, Teflon-capped vial, followed by filtration through a 45μm Spin-X centrifuge tube filter (Corning). 10μL of the hydrolyzed sample was dried in a Speed-Vac for 2 hours, then incubated with 100μL of chloramine T reagent at room temperature for 5 minutes and incubated with 100μL of 4-(dimethylamino)benzaldehyde (DMAB) at 60°C for 90 minutes. The absorbance of oxidized hydroxyproline at 560nm was measured. The hydroxyproline concentration was calculated using a standard curve generated with known concentrations of trans-4-hydroxy-L-proline. The total amount of protein isolated from the weighed tissue was determined using a protein assay kit (Bio-Rad, absorbance at 595nm). The amount of collagen is expressed in μg / mg total protein. qPCR: RNA was isolated using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer's instructions. The isolated RNA (250 ng) was then used to synthesize cDNA with SuperScript VILO (Invitrogen). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche) and analyzed using LightCycler 96 (Roche). Data are expressed as ΔΔCt fold change relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0181] Figure 4A F-4N was sent to RBC (Reaction Biology) for testing against their entire wild-type kinase panel. 1 μM F-4N was tested against 420+ kinases in ATP competition assays. Z-scores were calculated to identify true hits from the noise.

[0182] Figure 4B Expression of CDKN2A (a marker of senescence) in low-passage fibroblasts was compared to putative targets identified in a kinome screen by qPCR and to senescent fibroblasts generated by irradiation (10 Gy) or replicating passage.

[0183] Figure 5A Cell counts and apoptosis were compared in proliferating low-passage fibroblasts (pro) and senescent high-passage fibroblasts (sen) after transfection with siRNA targeting putative molecular targets of F-4N-STK-17A and STK17B. Cell counts and apoptosis were determined by cleaved caspase-3 and DAPI staining and automated microscopy.

[0184] Figure 5B qPCR expression of STK17A, STK17B, and senescence markers was compared in proliferating low-passage fibroblasts and senescent high-passage fibroblasts after transfection with siRNAs targeting putative molecular targets of F-4N-STK-17A and STK17B.

[0185] result

[0186] F-4N and pulmonary fibrosis

[0187] Mice that received F-4N showed improved survival after bleomycin-induced fibrosis ( Figure 3A ). Aged mice showed chronic fibrosis after a single bleomycin administration. The improved survival strongly suggests that F-4N has a beneficial effect.

[0188] Mice given F-4N also showed less fibrosis and improved lung structure (assessed by trichome histological staining) as well as reduced hydroxyproline content ( Figure 3B ). These results suggest that F-4N is ameliorating lung fibrosis in this chronic model.

[0189] The expression of collagen I and senescence markers in lung tissue was examined. The expression of collagen I and senescence markers was reduced in mice given F-4N ( Figure 3C ). These in vivo results are consistent with our in vitro data and the overall hypothesis that F-4N has a dual effect on lung fibrosis - clearing senescent cells and blocking fibroblast activation.

[0190] Mechanism of action

[0191] A kinase panel screen was used to test the effects of F-4N (1 μM) on approximately 400 kinases. Putative F-4N targets identified by the kinase panel screen included STK17A (also known as DRAK1), STK17B (also known as DRAK2), as well as MYLK4, AURKB, FLT3, and KIT ( Figure 4A ). The expression of putative molecular targets of F-4N was measured and STK17A and STK17B were found to be highly overexpressed in senescent fibroblasts compared with low-passage fibroblasts.

[0192] F-4N and lung aging

[0193] To determine whether putative F-4N targets could regulate senescence, we assessed apoptosis in lung fibroblasts (senescent and nonsenescent) treated with siRNA targeting STK17A and STK17B. Figure 5AWe also evaluated the expression of senescence markers in proliferating cells and senescent cells treated with siRNA targeting STK17A and STK17B peptides ( Figure 5B These data further support that F-4N can mediate senescent cell clearance activity by inhibiting STK17A / B (DRAK1 / 2).

[0194] Example 3: F-4N and PSC

[0195] This example describes the use of one or more flavonoid compounds having the structure of formula (I) in the treatment of PSC.

[0196] method

[0197] Hydroxyproline test

[0198] Hydroxyproline content was measured using a hydroxyproline assay kit (Biovision) according to the manufacturer's instructions with slight modifications. Liver tissue was weighed and homogenized in sterile water (10 mg tissue / 100 μL HO). 2 O), then hydrolyzed with 12M HCl at 120°C for 3 hours in a pressure-resistant, Teflon-capped vial, followed by filtration through a 45μm Spin-X centrifuge tube filter (Corning). 10μL of the hydrolyzed sample was dried in a Speed-Vac for 2 hours, then incubated with 100μL of chloramine T reagent at room temperature for 5 minutes and incubated with 100μL of 4-(dimethylamino)benzaldehyde (DMAB) at 60°C for 90 minutes. The absorbance of oxidized hydroxyproline at 560nm was determined. The hydroxyproline concentration was calculated using a standard curve generated with known concentrations of trans-4-hydroxy-L-proline. The total amount of protein isolated from the weighed tissue was determined using a protein assay kit (Bio-Rad, absorbance at 595nm). The amount of collagen was expressed in μg / mg total protein.

[0199] Picric acid staining

[0200] Paraffin-embedded liver sections were dewaxed and rehydrated by heating at 60 °C for 10-30 min and then passaged in the following solutions:

[0201] Xylene 1–2 x 10 min

[0202] 100% EtOH – 1x 2 min; 95% EtOH – 1 min; 70% EtOH – 1 min; 50% EtOH – 1 min

[0203] PBS – 3x 3 min

[0204] The slides were then stained in a saturated aqueous solution of picric acid containing 0.1% bitter red (Direct Red 80) and 0.1% fast green (counter-stain) for 60 min at room temperature, followed by washing once with distilled water, dehydrating with 100% EtOH (three times), clearing in xylene, and mounting in resin medium.

[0205] Liver function tests

[0206] Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bile acid were measured using a commercially available veterinary chemistry analyzer (VetScan2, Abaxis).

[0207] Reverse transcription quantitative polymerase chain reaction

[0208] RNA was isolated using the RNeasy Plus Mini kit (Qiagen) according to the manufacturer's instructions. The isolated RNA (250 ng) was then used to synthesize cDNA with SuperScript VILO (Invitrogen). Quantitative PCR was performed using FastStart Essential DNA Green Master (Roche) and analyzed using LightCycler 96 (Roche). Data are expressed as ΔΔCt fold changes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0209] result

[0210] F-4N and liver fibrosis

[0211] Compared with mice that did not receive any F-4N, mice given F-4N showed less collagen fiber deposition, especially in the portal-to-portal area, and decreased hydroxyproline content ( Figure 6 ). These results suggest that F-4N can reduce established liver fibrosis in the PSC mouse model.

[0212] F-4N and liver function

[0213] The livers of the mice were examined for the expression of liver function markers. The mice given F-4N had lower levels of ALT, ALP, and bile acid compared to mice that did not receive any F-4N ( Figure 7 ). These results suggest that F-4N can alleviate liver injury and cholestasis in the PSC mouse model.

[0214] F-4N and liver aging

[0215] The expression of collagen I, inflammatory and senescence markers in liver tissue was examined. Mice given F-4N showed reduced expression of collagen I, as well as reduced expression of inflammatory and senescence markers ( Figure 8 ). These results suggest that F-4N can effectively target senescent cells, thereby reducing inflammation and liver fibrosis in the PSC mouse model.

[0216] Example 4: Flavonol structure optimization

[0217] Materials and methods

[0218] For the senescent fibroblast survival experiment, human adult lung fibroblasts were repeatedly passaged until senescence (confirmed by RNA and senescence-related β-galactosidase staining). The cells were then seeded into 96-well plates, treated with the indicated compounds and incubated for 96 hours. The cells were then fixed with 4% PFA, permeabilized with 0.25% triton x-100, and nuclear staining was performed with DAPI. The cells were then imaged using a 4X objective on a Cytation5 microscope, and cell counts were quantified using automated software (Biotek Gen5). The data shown are plotted as % survival rates 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 + 2ng / mL TGFβ to stimulate collagen expression. The cells were incubated for 96 hours, fixed with 4% PFA, permeabilized with 0.25% triton x-100, and stained with primary and secondary infrared-coupled antibodies that recognize type I collagen. The wells were then imaged at 1X using an Odyssey Lx (LI-CORE) infrared imager, and the collagen intensity was quantified using automated software. The data shown are plotted as collagen intensity % standardized relative to vehicle-treated wells. Mean ± SEM, N = 3 independent experiments.

[0219] result

[0220] The ability of flavonols to induce cell death in senescent fibroblasts and to block collagen deposition in low-passage fibroblasts stimulated by TGFβ was assessed in a 3-point dose response curve (Figure 9). Both 2-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-6-methylchromen-4-one and 2-(3,4-diethoxyphenyl)-3-hydroxy-4h-1-benzopyran-4-one showed potent toxicity against senescent cells and blocked collagen deposition. This was confirmed in a 6-point dose response (Figure 10). Both compounds contain a p-ethoxy modification on the B ring of the flavonol core.

[0221] The second generation of flavonol compounds focused on p-ethoxy modifications on the B ring of the flavonol core and their effects on reducing the survival of senescent fibroblasts and blocking collagen deposition in non-senescent fibroblasts stimulated by TGFβ were evaluated (Figure 11). Based on these results, we identified a structure activity relationship in which p-ethoxy modification of the B ring improved the potency, while the unsubstituted A ring was optimal. The novel synthetic compounds discussed in Table 1, including molecule F-4N, were designed based on this SAR.

[0222] Finally, flavonols containing heteroatom substitutions in the aromatic core were tested for their effects on viability and blockade of collagen deposition in aged fibroblasts. Although these analogs contained a p-ethoxy group in a position consistent with the flavonoid B ring, their potency was significantly reduced.

[0223] Example 5: Treatment of IPF

[0224] A composition comprising one or more flavonoid compounds of formula (I) is administered to a person identified as suffering from IPF or administered by the person himself / herself. In some cases, the one or more flavonoid compounds administered can alleviate the severity of one or more symptoms of IPF. In some cases, the one or more flavonoid compounds administered can reduce the amount of fibrotic scarring in the human lung.

[0225] Example 6: Treatment of PSC

[0226] A composition comprising one or more flavonoid compounds having the structure of formula (I) is administered to or self-administered to a person identified as having PSC. In some cases, the one or more flavonoid compounds administered can reduce the severity of one or more symptoms of PSC. In some cases, the one or more flavonoid compounds administered can reduce the amount of fibrotic scarring in the human liver.

[0227] Example 7: F-4N and pulmonary fibrosis

[0228] The results in this embodiment replicate and extend at least some of the results provided in other embodiments.

[0229] method

[0230] Mice received intratracheal bleomycin treatments three times every 2 weeks. 28 days after the last bleomycin insult, mice received daily F-4N or vehicle treatments for 14 days. Fig.13A Shown is a schematic diagram of the study design.

[0231] result

[0232] The efficacy of F-4N was evaluated in the bleomycin injury model of pulmonary fibrosis. Hydroxyproline analysis was performed on each lung of 8-week-old FVB wild-type mice given bleomycin only (vehicle), bleomycin and F-4N, and sham-treated mice. Fig. 13B Representative H&E-stained histological images of lungs are shown.

[0233] Whole lung RNA expression was also assessed by qPCR analysis Fig. 13B Mouse lungs in order to understand changes in profibrotic genes, aging-related genes, alveolar epithelial type I / II markers, fibrosis-specific intermediate / transitional alveolar markers, and Stk17b ( Fig.14 ).

[0234] Example 8: F-4N and PSC

[0235] The results in this embodiment replicate and extend at least some of the results provided in other embodiments.

[0236] method

[0237] Twenty Mdr2 mice aged about 7 months with liver fibrosis - / - Mice (10 males, 10 females) were assigned to receive F-4N or vehicle treatment (n=5 per group) for 4 weeks. At the end of treatment, mice were sacrificed and livers were harvested to assess liver inflammation and fibrosis. Fig.15 Shown is a schematic diagram of the study design.

[0238] result

[0239] F-4N was orally delivered to a PSC mouse model with liver fibrosis for 4 weeks. The results are as follows:

[0240] ··Improvement of liver fibrosis, especially parenchymal fibrosis ("bridging fibrosis"), was also demonstrated by decreased expression of liver fibrosis genes. See, Figure 16-18 .

[0241] ··Tissue markers of liver inflammation and aging were significantly reduced. See, Fig.19 .

[0242] Example 9: F-4N and Nonalcoholic Steatohepatitis (NASH)

[0243] Mice in the NASH mouse model were given 10 mg / kg F-4N intraperitoneally daily for 2 weeks ( Fig. 20A The livers of treated mice were analyzed by H&E and Sirius red staining ( Fig. 20B Quantification of Sirius Red Staining ( Fig. 20C Changes in liver and colon weights were also assessed ( Fig.20D). These results suggest that F-4N may be effective in treating NASH.

[0244] The expression of profibrotic and inflammatory genes in the liver in Figure 20, as well as the expression of Stk17b (a molecular target of F-4N), were analyzed by qPCR. F-4N effectively reduced the expression of profibrotic genes, inflammatory genes, and Stk17b ( Fig.21 ).

[0245] Further analysis was also performed on the samples in the study of Figure 20. Liver function tests such as Fig.22A As shown, liver triglyceride analysis was Fig. 22B As shown, hydroxyproline assessment of liver collagen content was Fig. 22C These results suggest that F-4N is effective in treating NASH.

[0246] Example 10: F-4N and ocular fibrosis

[0247] Conjunctival fibroblasts were cultured in 2% FBS with or without F-4N for 4 days and their proliferation was assessed. Treated cells were fixed, stained with DAPI, and counted using automated software on a Cytation 5 ( Fig.23A ).

[0248] Conjunctival fibroblasts were cultured in 2% FBS with or without 2 ng / mL TGFβ and with or without F-4N for 3 days and the presence of fibroblasts was assessed. Treated cells were fixed, stained with DAPI, stained with αSMA antibody, and quantified using automated software on Cytation 5 ( Fig. 23B ).

[0249] Conjunctival fibroblasts were cultured in 2% FBS with or without 2 ng / mL TGFβ and with or without F-4N for 6 days and assessed for collagen deposition. Treated cells were fixed, stained with DAPI, stained with collagen I antibody, and quantified using automated software on a Cytation 5 ( Fig.23C ).

[0250] Taken together, these results suggest that F-4N may be useful in the treatment of ocular fibrosis.

[0251] Example 11: F-4N Pharmacology

[0252] Drug administration

[0253] like Fig.24AAs shown in Figure 1, an acute exposure bleomycin study using different doses of F-4N was performed. On day 1, mice received intratracheal sham treatment or bleomycin injury. On day 7, mice were divided into groups and treated with vehicle, 10, 30, or 100 mg / kg F-4N daily by oral gavage for 7 days. Organs and plasma were collected on day 14. Body weight changes ( Fig. 24B Whole lung RNA expression of profibrotic genes was also examined ( Fig.24C In this acute exposure, dose-finding study, oral gavage of 30 and 100 mg / kg / day of F-4N improved body weight changes and reduced the expression of profibrotic genes.

[0254] F-4N efficacy biomarkers were identified. RNA was collected from vehicle-treated mice and 30 mg / kg F-4N-treated mice. RNA was analyzed using RT2 Profiler PCR arrays (Qiagen catalog number-330231), which measures the expression of 89 different cytokines and chemokines. Eleven genes were identified that showed reduced expression in mice after 7 days of treatment with F-4N ( Fig.25 ). Levels of genes whose expression is altered in response to F-4N can be used as biomarkers of F-4N efficacy.

[0255] The F-4N levels in plasma and liver of mice after F-4N exposure are shown ( Fig.26 ). 2 hours and 8 hours after the last dose, Fig.24A Plasma and liver tissue were collected from mice following the protocol described in . The level of unbound (free) F-4N was analyzed by Cyprotex (Framingham, MA) using LC-MS. The effective concentration of F-4N was measured in plasma and liver, with F-4N levels in the liver being higher than in plasma.

[0256] Plasma stability

[0257] The stability of F-4N in plasma was evaluated ( Fig. 27 ). The compounds were incubated with human or mouse plasma. The percentage of F-4N recovered was analyzed by LC-MS. Warfarin was used as a control for stable compounds and propantheline was used as a control for unstable compounds. The analysis was performed by Cyprotex (Framingham, MA). These results indicate that F-4N is stable in both human and mouse plasma.

[0258] The microsomal stability of F-4N was also evaluated ( Fig.28). The compounds were incubated with human or mouse liver derived microsomes. The percentage of F-4N recovered was analyzed by LC-MS. Verapamil was used as a control for rapidly degrading compounds. The analysis was performed by Cyprotex (Framingham, MA). These results indicate that the half-life and clearance of F-4N are consistent with many clinically approved orally administered drugs.

[0259] Plasma protein binding assays were also performed (Table 2). Compounds were incubated with rat plasma for 4 hours. The percentage of F-4N recovered was analyzed by LC-MS. Warfarin was used as a control for highly plasma protein bound compounds. The analysis was performed by Cyprotex (Framingham, MA).

[0260] Table 2. Plasma protein binding assay.

[0261] Test product Test planting Test concentration Unbound fraction in plasma Bound fraction in plasma Recycling after testing F-4N Rat 5μM 0.94％ 99％ 100％ Quercetin Rat 5μM 5.0％ 95％ 0.80％ Warfarin Rat 2μM 0.971％ 99％ 100％

[0262] These results indicate that F-4N exhibits a high plasma protein binding rate.

[0263] Example 12: Mechanism of F-4N

[0264] The effect of Stk17b on fibrotic markers was evaluated. Stk17b was knocked out in the precision cut lung slice (PCLS) model. Fig.29A Shown is a schematic diagram of the study protocol. Six mice received intratracheal bleomycin on day 0. On day 14, at the peak of fibrosis, lungs were harvested and sliced ​​using a vibratome to generate 300 μM PCLS. Tissues were cultured ex vivo for 4 days using non-targeting siRNA or siRNA targeting Stk17b. RNA was then collected and analyzed by qPCR ( Fig.29B The results suggest that Stk17b can be targeted (eg, can be reduced or inhibited) to treat pulmonary fibrosis

[0265] DRAK1 kinase activity was also assessed. Cells were incubated with DRAK1 and radiolabeled ATP and F-4N or the inactive analog 5-MeOH-F-4N. The DRAK1 kinase activity assay was performed by reaction biology. DRAK1 kinase activity ( Fig.30 ). These results suggest that F-4N can inhibit DRAK1 kinase activity. The analogs that were inactive in the phenotypic cell-based assay were inactive against DRAK1.

[0266] The efficacy of F-4N was determined using organotypic ex vivo cultures derived from IPF patients. Lung tissue sections (approximately 500 μμM) were cut from IPF patient transplanted lungs and cultured in vitro for 4 days with DMSO (0.1%) or F-4N (3 μμM). Lung tissue sections were collected, RNA was isolated, and qPCR analysis was performed (Figure 31). Culture medium was also collected for ELISA analysis of IL-6 ( Fig.31B ). N = 3 patient samples. These results indicate that F-4N reduces the expression of fibrosis markers and enhances the expression of mature alveolar epithelial cell markers.

[0267] Example 13: Structural optimization of quercetin to enhance pharmacological properties

[0268] This example describes the identification of quercetin structures that can enhance senescent cell clearance and can enhance transdifferentiation blockade in lung fibroblasts.

[0269] Comparison of efficacy and nontoxicity of flavonoid compounds on the aged brain and liver.

[0270] Wild-type mice aged 5 and 28 months were treated with the indicated concentrations of flavonoid compounds (F, Q, compound 19, or compound 20) or vehicle by oral gavage for four consecutive days and were euthanized one week later. Real-time PCR analysis showed that low-dose administration of compound 19 and / or 20 could reduce brain ( Fig.32A ) and liver( Fig.32B ) expression of the key senescence-activating gene p16ink4a, while fisetin or quercetin have been shown to have anti-aging effects at relatively high doses. Analysis of the expression of CD68, an indicator of inflammatory activation, showed that brain ( Fig.32C ) or liver( Fig.32D ) were free of drug-induced toxicity.

[0271] Quercetin analogues effectively kill senescent fibroblasts

[0272] The expression of senescence markers was measured in senescent fibroblasts treated with quercetin analogs ( Fig.33A ) and its proliferation ( Fig.33B ). More than 30 different quercetin analogs were screened for their ability to kill senescent cells more effectively than they killed low-passage proliferating fibroblasts. Fig.33C The most potent derivatives are shown, with some exhibiting nanomolar to low micromolar potency.

[0273] TGFβ and senescent cell culture medium promote transdifferentiation of fibroblasts into myofibroblasts

[0274] Fibroblasts were detected by αSMA staining and the staining intensity was quantified ( Fig.34A Fibroblast activation markers ( Fig.34B ). These results suggest that quercetin analogs can effectively prevent fibroblast activation.

[0275] Quercetin analogs prevent SASP-CM- and TGFβ-induced collagen deposition.

[0276] TGFβ and senescent cell conditioned medium promote collagen I deposition ( Fig.35A ). Quercetin analogs are effective in preventing collagen I deposition ( Fig.35B ).

[0277] Quercetin analogs can block TGFβ-induced profibrotic gene expression

[0278] TGFβ promotes the expression of profibrotic genes ( Fig.36A ). Quercetin analogs effectively block profibrotic gene expression ( Fig.36B ).

[0279] Quercetin analogs with p-ethoxylation have enhanced activity

[0280] Fig.37A Exemplary quercetin analogs with p-ethoxy groups are shown. Cell proliferation graphs show that quercetin analogs with p-ethoxy groups induce cell senescence ( Fig.37B ). Cell proliferation plots show that quercetin analogs lacking the p-ethoxy group do not induce cell senescence ( Fig.37C ).

[0281] Example 14: Treatment of IPF

[0282] A composition comprising one or more flavonoid compounds with formula (II) structure is administered or administered voluntarily to a person identified as suffering from IPF. In some cases, the one or more flavonoid compounds administered can alleviate the severity of one or more symptoms of IPF. In some cases, the one or more flavonoid compounds administered can reduce the amount of fibrotic scarring in human lungs.

[0283] Example 15: Treatment of PSC

[0284] A composition comprising one or more flavonoid compounds having the structure of formula (II) is administered to a person identified as having PSC or self-administered to a person identified as having PSC. In some cases, the one or more flavonoid compounds administered can reduce the severity of one or more symptoms of PSC. In some cases, the one or more flavonoid compounds administered can reduce the amount of fibrotic scarring in the human liver.

[0285] Other Implementations

[0286] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and improvements are within the scope of the claims.

Claims

1. A composition comprising a flavonoid compound having a structure of formula (I): or a pharmaceutically acceptable salt thereof, wherein: R 1 Selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 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, CH2CH3, cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl and 4-pyridyl; And R 4 Selected from the group consisting of H, OH, C1-C4 alkyl, halogen and C1-C4 alkoxy.

2. The composition according to claim 1, wherein the flavonoid compound of formula (I) has the structure:

3. The composition according to claim 1, wherein the flavonoid compound of formula (I) has the structure:

4. The composition according to claim 1, wherein the flavonoid compound of formula (I) has the structure:

5. The composition according to any one of claims 1-4, further comprising a pharmaceutically acceptable carrier, excipient or diluent.

6. A pharmaceutical composition comprising a flavonoid compound having the structure of formula (I): or a pharmaceutically acceptable salt thereof, wherein: R 1 Selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 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, CH2CH3, cyclopropyl, phenyl, 2-pyridyl, 3-pyridyl and 4-pyridyl; And R 4 is selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; and A pharmaceutically acceptable carrier, excipient or diluent.

7. A method of treating a mammal suffering from a fibrotic disorder, wherein the method comprises administering to the mammal a composition as claimed in any one of claims 1 to 6.

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

9. The method of claim 7 or claim 8, wherein the mammal is identified as suffering from the fibrotic disorder.

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

11. The method of claim 10, wherein the fibrotic disorder is IPF, and wherein the method further comprises administering to the mammal an agent for treating IPF.

12. The method of claim 11, wherein the agent used to treat IPF is selected from the group consisting of pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC 12249, AM966, AM095, taliberine, BI-2545, GLPG1690, BBT 877, SAR100842, BMS-986,020, milnaprim, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, rosudil, nesudil, besudil, nerakizumab, trorocirumab, dupilumab, and panrelumab.

13. The method of claim 10, wherein the fibrotic disorder is PSC, and wherein the method further comprises administering to the mammal an agent for treating PSC.

14. The method of claim 13, wherein the agent for treating PSC is selected from the group consisting of ursodeoxycholic acid (UDCA), corticosteroids, bile acid sequestrants, antibiotics, and antihistamines.

15. A method of reducing fibrosis in a mammal suffering from a fibrotic disorder, wherein the method comprises administering to the mammal a composition as claimed in any one of claims 1 to 6.

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

17. The method of claim 15 or claim 16, wherein the mammal is identified as suffering from the fibrotic disorder.

18. The method according to any one of claims 15-17, wherein the fibrotic disorder is selected from the group consisting of IPF, PSC and NASH.

19. A method of reducing the number of senescent cells in a mammal suffering from a fibrotic disorder, wherein the method comprises administering to the mammal a composition as claimed in any one of claims 1 to 6.

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

21. The method of claim 19 or claim 20, wherein the mammal is identified as suffering from the fibrotic disorder.

22. The method according to any one of claims 19-21, wherein the fibrotic disorder is selected from the group consisting of IPF, PSC and NASH.

23. The method of any one of claims 19-22, wherein the cells are fibroblasts.

24. The method of claim 23, wherein the fibrotic disorder is IPF, and wherein the senescent cells are lung fibroblasts.

25. The method of any one of claims 19-22, wherein the senescent cells are epithelial cells.

26. The method of claim 25, wherein the fibrotic disorder is PSC, and wherein the senescent cell is a cholangiocyte.

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

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

29. The method according to claim 27 or 28, wherein the STK17 polypeptide is selected from the group consisting of a STK17A (DRAK1) polypeptide and a STK17B (DRAK2) polypeptide.

30. The method of claim 9, wherein the fibrotic disorder is ocular fibrosis.

31. The method of any one of claims 15-17, wherein the fibrotic disorder is ocular fibrosis.

32. A composition comprising a flavonoid compound having the structure of formula (II): or a pharmaceutically acceptable salt thereof, wherein: X 1 is selected from N and CH; X 2 Selected from N and CR 4 ; R 1 Selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 2 Selected from the group consisting of H, OH, C1-C4 alkyl, halogen, and C1-C4 alkoxy; R 3 Selected from the group consisting of H, CH3, CH2CH3, cyclopropyl, phenyl, 4-OH-phenyl, 2-OH-phenyl, 3-OH-phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, thien-2-yl, thien-3-yl, tetrahydrofuran-2-yl and tetrahydrofuran-3-yl; And R 4 Selected from the group consisting of H, OH, C1-C4 alkyl, halogen and C1-C4 alkoxy.

33. The composition of claim 32, wherein the flavonoid compound of formula (II) has any one of the following formulae: 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 of treating a mammal suffering from a fibrotic disorder, wherein the method comprises administering to the mammal a composition as described in any one of claims 32-33.

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

37. The method of claim 35 or claim 36, wherein the mammal is identified as suffering from the fibrotic disorder.

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

39. The method of claim 38, wherein the fibrotic disorder is IPF, and wherein the method further comprises administering to the mammal an agent for treating IPF.

40. The method of claim 39, wherein the agent used to treat IPF is selected from the group consisting of pirfenidone, nintedanib, N-acetylcysteine, sildenafil, vardenafil, tadalafil, avanafil, promethazine, FTY720, AM152, BMS-986020, VPC 12249, AM966, AM095, taliberine, BI-2545, GLPG1690, BBT 877, SAR100842, BMS-986,020, milnaprim, dopamine, levodopa, apomorphine, fenoldopam, pergolide, bromocriptine, cabergoline, dasatinib, hydroxyfasudil, rosudil, nesudil, besudil, nerakizumab, trorocirumab, dupilumab, and panrelumab.

41. The method of claim 38, wherein the fibrotic disorder is PSC, and wherein the method further comprises administering to the mammal an agent for treating PSC.

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

43. A method of reducing fibrosis in a mammal suffering from a fibrotic disorder, wherein the method comprises administering to the mammal a composition as described in any one of claims 32-33.

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

45. The method of claim 43 or claim 44, wherein the mammal is identified as suffering from the fibrotic disorder.

46. ​​The method of any one of claims 43-45, wherein the fibrotic disorder is selected from the group consisting of IPF, PSC, NASH, and ocular fibrosis.

47. A method of reducing the number of senescent cells in a mammal suffering from a fibrotic disorder, wherein the method comprises administering to the mammal a composition as described in any one of claims 32-33.

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

49. The method of claim 47 or claim 48, wherein the mammal is identified as suffering from the fibrotic disorder.

50. The method of any one of claims 47-49, wherein the fibrotic disorder is selected from the group consisting of IPF, PSC, NASH, and ocular fibrosis.

51. The method of any one of claims 47-50, wherein the senescent cells are fibroblasts.

52. The method of claim 51, wherein the fibrotic disorder is IPF, and wherein the senescent cells are lung fibroblasts.

53. The method of any one of claims 47-50, wherein the senescent cells are epithelial cells.

54. The method of claim 53, wherein the fibrotic disorder is PSC, and wherein the senescent cell is a cholangiocyte.

55. A method of inhibiting a STK17 polypeptide in a mammal, wherein the method comprises administering to the mammal a composition according to any one of claims 32-33.

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

57. The method of claim 55 or 56, wherein the STK17 polypeptide is selected from the group consisting of a STK17A (DRAK1) polypeptide and a STK17B (DRAK2) polypeptide.

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