Tripterine derivative

Abstract

# imgabs0 # The present disclosure relates to compounds that are derivatives of the compound of formula (I), in which R is an amide group, as well as pharmaceutical compositions comprising these tripterine derivative compounds. The present disclosure also relates to uses of the tripterine derivative compounds described herein, including in medicaments, therapies and methods of treating tripterine responsive diseases or disorders, the tripterine-responsive diseases or disorders such as cancer, inflammatory and / or autoimmune disorders, neurological disorders, brain injury and disorders, obesity-related diseases and disorders, and / or liver-related diseases and disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 389,593, filed on July 15, 2022, the entire contents of which are hereby incorporated by reference into this document.

[0003] field

[0004] The present disclosure relates to compounds that are derivatives of tripterygium wilfordii, pharmaceutical compositions comprising these compounds, and their uses.

[0005] background

[0006] Celastrol, also known as tripterine, is a naturally occurring compound derived from the plant species Tripterygium wilfordii Hook F. Celastrol has been found to exhibit significant anti-tumor activity in preclinical studies, including studies on the treatment of liver cancer, breast cancer, prostate cancer, lung cancer, leukemia, and melanoma (see, e.g., Kashyap et al., 2018; Yadav et al., 2018), and has been shown to have radiosensitizing effects (see, e.g., Dai et al., 2011; Lee et al., 2011). Results from a range of tumor cell lines and animal cancer models have shown that celastrol can kill tumors via a range of mechanisms of action, including: i) induced apoptosis and autophagy, ii) cell cycle arrest, iii) anti-metastatic and anti-angiogenic effects, iv) anti-inflammatory effects, and v) antioxidant activity. (See, e.g., et al., 2017; Kashyap et al., 2018). It is believed that triptolide exerts its effects by targeting multiple signaling pathways, including but not limited to reactive oxygen species (ROS) / JNK and Akt / mTOR (see, e.g., Liu et al., 2019), NF-κb (see, e.g., Chiang et al., 2014), STAT3 / JAK2 (see, e.g., Rajendran et al., 2012), HSP90 (see, e.g., Sreeramulu et al., 2009; Zhang et al., 2009), Cdc37, p23, Iκκb, p-Akt (see, e.g., Kannaiyan et al., 2011), ERα (see, e.g., Jang et al., 2011).

[0007] Celastrol has been shown to act as an inflammasome inhibitor (see, e.g., Lee et al., 2019; Yu et al., 2017) and has been proposed for the treatment of a range of inflammatory indications, autoimmune diseases, and several chronic diseases, including but not limited to rheumatoid arthritis (RA), multiple sclerosis (MS) (see, e.g., Wang et al., 2015; Abdin and Hasby, 2014), ankylosing spondylitis, systemic lupus erythematosus (SLE), inflammatory bowel disease, osteoarthritis (OA), acute respiratory distress syndrome (ARDS) (see, e.g., Wei and Wang, 2017), Guillain-Barré syndrome (GBS) (e.g., Shao et al., 2023), sickle cell disease (SCD) (see, e.g., Kumar et al., 2016), allergies (e.g., asthma), psoriasis and other inflammatory skin conditions (see, e.g., Vankatesha and Moudgil, 2021; Song et al., 2023).

[0008] Celastrol has also been shown to exhibit neuroprotective activity (see e.g., et al., 2017; Cleren et al., 2005; Paris et al., 2010), and have been proposed for the treatment of a range of neurological disorders, including but not limited to Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Gaucher disease (GD) (see, e.g., Vankatesha and Moudgil, 2021).

[0009] Celastrol and celastrol analog compounds have also been proposed as treatments for a variety of metabolic and atherosclerotic diseases, liver diseases, and cardiac disorders, including but not limited to obesity (see, e.g., Liu et al., 2015; Feng et al., 2019), atherosclerosis (see, e.g., Coll et al., 2015; Voutyritsa et al., 2021), type 2 diabetes (T2D) (see, e.g., Liu et al., 2015; Zhou et al., 2021), diabetic nephropathy (see, e.g., Nie et al., 2020), gout (Yan et al., 2021; Wen et al., 2014), cardiac fibrosis (see, e.g., Fan et al., 2023), non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver.

[0010] Due to its anti-inflammatory activity, celastrol has also been shown to be useful for treating and preventing a variety of brain disorders and brain injuries. These include, but are not limited to, brain injury induced by middle cerebral artery occlusion (MCAO), cerebral ischemia / reperfusion (I / R) injury, vascular dementia (VD), and acute ischemic stroke (see, e.g., Jiang et al., 2018).

[0011] In recent years, more and more applications of tripterygium wilfordii have been explored for the treatment of ocular disorders, including but not limited to dry eye disease (see, e.g., Siu Law, 2022), ocular inflammation, age-related macular degeneration (AMD) (see, e.g., Zhang et al., 2019), subconjunctival fibrosis (see, e.g., Li et al., 2023). Tripterygium wilfordii has also been shown to prevent the development of high intraocular pressure-induced retinal ganglion cell degeneration (see, e.g., Gu et al., 2018), bright light-induced degeneration (see, e.g., Bian et al., 2016), and macrophage-induced corneal neovascularization (see, e.g., Li et al., 2016), and support corneal allograft survival (see, e.g., Li et al., 2016) and recovery from damage caused by optic nerve compression (see, e.g., Kyung et al., 2015).

[0012] Although celastrol interacts with many cellular targets and exhibits potent activity for many potential therapeutic indications, the compound exhibits poor ADME characteristics that limit its further clinical development, including aqueous stability, low bioavailability, narrow therapeutic window, and undesirable side effects (see, e.g., et al., 2017; Hou et al., 2020).

[0013] There is a need for tripterygium derivatives or analog compounds that exhibit one or more improved ADME characteristics while retaining some or all of the functional activities of tripterygium related to its therapeutic efficacy.

[0014] Overview

[0015] The present disclosure generally relates to tripterygium wilfordii derivative compounds, compositions, formulations and their uses in medicine and methods of treatment. This overview is intended to introduce the subject matter of the present disclosure, but does not cover every embodiment, combination or variation contemplated and described in the present disclosure. Additional embodiments are contemplated and described by the disclosure of the detailed description, drawings and claims.

[0016] In at least one embodiment, the present disclosure provides a compound of formula (I)

[0017]

[0018] Wherein, R1 is selected from:

[0019]

[0020]

[0021]

[0022]

[0023] or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug thereof.

[0024] In at least one embodiment, the present disclosure provides a compound of formula (I)

[0025]

[0026] in

[0027] R1 is -NH-(CO)2-NR2R3 or -N(CH3)-(CO)2-NR2R3;

[0028] R2 and R3 are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine or heteroaryl, optionally substituted with substituents individually selected from alkyl, alkoxy, cycloalkyl, ether, amine optionally substituted with one or more alkyl, halogen, hydroxy, ether, cyano, nitrile, CF3, ester, amide, cycloalkylamide, sugar, heteroarylamide optionally substituted with alkyl and / or alkoxy, urea, carbamate, thioether, sulfate, sulfonyl, sulfonic acid, carboxylic acid and aryl; or

[0029] R2 and R3 are taken together to form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by substituents individually selected from the group consisting of alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, heteroaryl, amine, halogen, hydroxy, ether, nitrile, cyano, nitro, CF3, ester, amide, urea, carbamate, thioether or carboxylic acid groups;

[0030] or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug thereof.

[0031] In at least one embodiment of any one of the compounds of structural formula (I) of the present disclosure, the compound is selected from the group consisting of: compound 14, compound 15, compound 16, compound 17, compound 22, compound 22-1, compound 42, compound 54, compound 55, compound 56, compound 57, compound 58, compound 59, compound 60, compound 61, compound 62, compound 63, compound 64, compound 65, compound 66, compound 67, compound 68, compound 69, compound 70, compound 71, compound 72, compound 73, compound 74, compound 75, compound 76, compound 77, compound 78, compound 79, compound 80, compound 81, compound 82, compound 83, compound 84, compound 85, compound 86, compound 87, compound 88, compound 89, compound 90, compound 91 and compound 92.

[0032] In at least one embodiment of any one of the compounds of Structural Formula (I) of the present disclosure, the compound is characterized by having an IC in a NF-kB reporter gene assay of about 1200 nM or less, about 1000 nM or less, 750 nM or less, 500 nM or less, or 300 nM or less. 50 .

[0033] In at least one embodiment, the present disclosure provides a pharmaceutical composition comprising a compound of structural formula (I) of the present disclosure and a pharmaceutically acceptable excipient.

[0034] In at least one embodiment, the present disclosure provides a method of treating a tripterine-responsive disease or disorder in a subject suffering from the tripterine-responsive disease or disorder, the method comprising administering to the subject a therapeutically effective amount of a compound of structural formula (I) of the present disclosure or a pharmaceutical composition of the present disclosure.

[0035] In at least one embodiment of the method, the celastrol responsive disease or disorder is cancer; optionally, wherein the cancer is selected from: gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, brain cancer and glioblastoma multiforme (GBM).

[0036] In at least one embodiment of the method, the celastrol responsive disease or disorder is an inflammatory and / or autoimmune disorder; optionally, wherein the inflammatory and / or autoimmune disorder is selected from rheumatoid arthritis (RA), multiple sclerosis (MS), ankylosing spondylitis, systemic lupus erythematosus (SLE), inflammatory bowel disease, osteoarthritis (OA), acute respiratory distress syndrome (ARDS), Guillain-Barré syndrome (GBS), sickle cell disease (SCD), allergies (e.g., asthma), psoriasis and other inflammatory skin conditions.

[0037] In at least one embodiment of the method, the tripterygium responsive disease or disorder is a neurological disorder; optionally, wherein the neurological disorder is selected from Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and Gaucher disease (GD).

[0038] In at least one embodiment of the method, the tripterygium wilfordii responsive disease or disorder is an obesity-related disease or disorder; optionally, wherein the obesity-related disease or disorder is selected from obesity, preobesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi Syndrome, obesity associated with hypothalamic damage, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bardet-Biedl Syndrome, Cohen Syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome and MOMO (Macrosomia Obesity Macrocephaly Ocular abnormalities) syndrome.

[0039] In at least one embodiment of the method, the tripterygium wilfordii responsive disease or disorder is a liver-related disease or disorder; optionally, wherein the liver-related disease or disorder is selected from acute-chronic liver failure (ACLF), alcoholic liver disease, cholestatic liver disease, drug-induced liver disease, hepatocellular carcinoma, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), viral hepatitis and viral liver disease.

[0040] In at least one embodiment of the method, the tripterygium wilfordii responsive disease or disorder is brain injury or brain disorder; optionally, wherein the brain-related injury or disorder is selected from middle cerebral artery occlusion (MCAO)-induced brain injury, cerebral ischemia / reperfusion (I / R) injury, vascular dementia (VD) and acute ischemic stroke-induced brain injury.

[0041] In at least one embodiment of the method, the celastrol-responsive disease or disorder is an ocular disorder or injury; optionally, wherein the ocular-related injury or disorder is selected from dry eye disease, ocular inflammation, age-related macular degeneration (AMD), subconjunctival fibrosis, high intraocular pressure-induced retinal degeneration, bright light-induced degeneration, macrophage-induced corneal neovascularization, corneal allograft survival and optic nerve extrusion.

[0042] In at least one embodiment of the methods of the present disclosure, the compound or pharmaceutical composition is administered in combination with another therapy. In at least one embodiment, administration comprises oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration, or subcutaneous administration.

[0043] In at least one embodiment of the methods of the present disclosure, the pharmaceutical composition is administered in a form selected from the group consisting of pills, capsules, tablets, granules, powders, salts, crystals, liquids, serums, syrups, suspensions, gels, creams, pastes, films, patches, and vapors.

[0044] In at least one embodiment of the methods of the present disclosure, the subject is a mammal. In at least one embodiment, the subject is a human.

[0045] In at least one embodiment, the present disclosure provides the use of a compound of structural formula (I) of the present disclosure or a pharmaceutical composition of the present disclosure for therapy, as a medicament, for treating a tripterygium-responsive disease or disorder in a subject, or for the manufacture of a medicament for treating a tripterygium-responsive disease or disorder in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] A better understanding of the novel features and advantages of the present disclosure will be obtained by referring to the following detailed description, which sets forth illustrative embodiments in which the principles of the present disclosure are utilized, and the accompanying drawings (also referred to herein as “Figures” and “FIG.”), in which:

[0048] Figure 1A , Figure 1B and Figure 1C Results are shown graphically demonstrating that the tripterygium derivative Compound 22 (also referred to as "CLM-022") robustly inhibits NF-kB inflammatory signaling, as described in Example 14. Figure 1ADepicted is a graph of relative NF-kB luciferase activity (RLU) of Compound 22 and Compound 1 (also referred to as "CLM-001") assayed at 8 point concentrations (10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM, 0.016 μM, and 0 μM). Figure 1B The inhibitory concentration (IC) of compound 22 (CLM-022) and compound 1 (CLM-001) is plotted. 50 )’s picture. Figure 1C Shown are Western blot images of BMDCs pretreated with three different concentrations (10 nM, 25 nM and 50 nM) of compound 1 (CLM-001) or compound 22 (CLM-022), followed by LPS (500 ng / mL) treatment and assayed for known NF-kB target proteins iNOS, NLRP3, Cox-2 and IL-1β.

[0049] Figure 2A , Figure 2B and Figure 2C Results are shown showing that Compound 22 (CLM-022) strongly inhibits IL-1β secretion via inhibition of the NLRP3 inflammasome in bone marrow-derived dendritic cells (BMDCs), as described in Example 15. Figure 2A Depicted are Western blot images showing the relative NLRP3 inflammasome activity of compound 22 (CLM-022) and compound 1 (CLM-001) by detecting the levels of cellular ASC complexes and the mature form of IL-1β (15 kDa) in BMDC conditioned medium treated with LPS (100 ng / mL) for 3 hours and then treated with 50 nM, 100 nM and 500 nM concentrations of compound for 30 min, followed by treatment with ATP or nigericin. ASC monomers and β-actin were used as controls. Figure 2B Depicted are graphs showing the results of secreted IL-1β levels measured by ELISA using conditioned media of BMDCs treated with 63 nM, 125 nM, 250 nM and 500 nM of Compound 22 or Compound 1 upon NLRP3 inflammasome activation induced by LPS+ATP or LPS+Nigericin. Figure 2C Depicted are graphs showing the results of secreted TNF-α levels measured by ELISA using conditioned media of BMDCs treated with 63 nM, 125 nM, 250 nM and 500 nM of Compound 22 or Compound 1 upon NLRP3 inflammasome activation induced by LPS+ATP or LPS+nigericin.

[0050] Figure 3Aand Figure 3B Depicted are results showing that Compound 22 (CLM-022) inhibits NLRP3 inflammasome activity in THP1 and cellular inflammasome complexes, as visualized by the Amnis assay described in Example 16. Figure 3A Images of western blot analysis of THP1 cells treated with LPS and compound 1 (CLM-001) or compound 22 followed by nigericin for NLRP3 inflammasome activation are shown. Inflammasome ASC complexes in control cells (LPS + nigericin) were compared with treatment with compound 1 (CLM-001) (50nM, 100nM), compound 22 (CLM-022) (50nM, 100nM) and MCC950 (100nM, 200nM). FLICA (green) represents active caspase-1, ASC (red) complexes are shown as red spots near the nuclear membrane, and DAPI staining of the nucleus (blue) is captured in the AMNIS analysis. Figure 3B Depicted, the number of cells stained as ASC+ (red) and FLICA+ (green) in AMNIS was counted to compare the level of NLRP3 inflammasome activity in cells treated as indicated (left). ASC+ cells and FLICA+ cells are also plotted as a percentage of the total cell count (right).

[0051] Figure 4A and Figure 4B Depicted are results showing that compound 22 (CLM-022) reduces the production of proinflammatory cytokines in a mouse model of LPS-induced septic shock, as described in Example 17. Compound 1 (CLM-001) and compound 22 (CLM-022) (1 mg / kg) were injected intraperitoneally 1 hour before induction of septic shock by administration of a sublethal dose of LPS (25 mg / kg, ip), and mice were sacrificed 4 hours after LPS injection and subjected to measurement of inflammatory cytokine expression. Figure 4A Depicted are graphs of data showing reduction in TNF-α levels in the serum of Compound 1 and Compound 22 treated mice. Figure 4B Depicted are graphs showing the results of: Gene expression analysis by qPCR has shown that compound 22 pretreatment significantly inhibited IL-β, IL-6, TNF-α and iNOS inflammatory cytokines and molecules in the colon.

[0052] Figure 5A , Figure 5B , Figure 5C and Figure 5DDepicted are results showing that Compound 22 (CLM-022) is superior to the benchmark inflammasome inhibitor compound MCC950 in inhibiting IL-1β secretion (but not TNF-α secretion) in BMDM cells and THP-1 cells whose inflammasome activity and cytokine secretion were activated by LPS and nigericin treatment as described in Example 18.

[0053] Fig. 6A , Figure 6B and Figure 6C Depicted are the results of the DARTS assay described in Example 19, showing significantly improved protection of NLRP3 protein, but not NEK7, by the tripterygium wilfordii derivative Compound 22 (CLM-022) when compared to MCC950 from pronase-induced degradation.

[0054] Fig. 7A , Figure 7B and Figure 7C Depicted are the results of the study described in Example 20, which showed that the tripterygium derivative Compound 22 (CLM-022) had a greater ability to inhibit inflammasome-induced pyroptosis in human THP-1 macrophages when compared to MCC950.

[0055] Detailed Description

[0056] It should be understood that the detailed description provided herein (including the accompanying drawings) is exemplary and explanatory only and is not intended to limit the present disclosure. The description is not limited to the specific compounds, compositions, methods, techniques, protocols, cell lines, assays, and reagents disclosed herein, as these may vary, but is also intended to cover known variations of these specific embodiments.

[0057] It should also be understood that the terms used herein are intended to describe specific embodiments and are not intended to limit the scope as set forth in the appended claims. For the description of this article and the appended claims, unless the context clearly indicates otherwise, the singular forms "a" and "an" include plural indicators. Thus, for example, reference to "protein" includes more than one protein, and reference to "compound" refers to more than one compound. It should also be noted that the claims can be written to exclude any optional elements. Therefore, this statement is intended to be used as a pre-emptive basis for using such exclusive terms such as "solely", "only", etc. or using "negative" restrictions in conjunction with the citation of the claim elements. The use of "comprise", "comprises", "comprising", "include", "includes" and "including" is interchangeable and is not intended to be restrictive. It will also be understood that where the description of various embodiments uses the term “comprising,” those skilled in the art will understand that in some specific instances, the language “consisting essentially of” or “consisting of” may alternatively be used to describe the embodiments.

[0058] In the case of providing a range of values, unless the context clearly indicates otherwise, it should be understood that each intermediate integer of the value between the upper and lower limits of the scope and each tenth of each intermediate integer of the value and any other stated value or intermediate value in the scope of the statement are all included in the present disclosure. The upper and lower limits of these smaller scopes can be included in the smaller scope independently, and are also included in the present disclosure, subject to any specific limiting value excluded in the stated scope. In the case where the scope of statement includes one or two of these limiting values, excluding (i) any one or (ii) two scopes of those included limiting values ​​is also included in the present disclosure. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10" etc.

[0059] All publications, patents, patent applications, and other documents cited in this disclosure are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference herein for all purposes.

[0060] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. It should be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting. For the purpose of explaining the present disclosure, the following description of terms will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0061] I. Definitions

[0062] "Triprolactone" refers to a compound having the chemical structure of compound (1):

[0063]

[0064] "Tripterine-responsive disease or disorder" refers to any disease or disorder for which treatment with the compound tripterine has been suggested or shown, based on clinical studies, preclinical studies, or in vitro studies, to provide a potential therapeutic effect. Exemplary tripterygium wilfordii responsive diseases or disorders of the present disclosure include, but are not limited to, cancer (e.g., gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, brain cancer, and glioblastoma multiforme), inflammatory and / or autoimmune disorders (e.g., rheumatoid arthritis (RA), multiple sclerosis (MS), and experimental autoimmune encephalomyelitis (EAE), ankylosing spondylitis, systemic lupus erythematosus (SLE), ulcerative colitis, inflammatory bowel disease, osteoarthritis (OA), acute respiratory distress syndrome (ARDS), Guillain-Barré syndrome (GBS), sickle cell disease (SCD), asthma, psoriasis, and skin inflammation), neurological disorders (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Gaucher disease (GD)), and obesity-related disorders (e.g., obesity, Premature cataract, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, obesity associated with hypothalamic damage, nonalcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bard-Biedl syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macrosomia) syndrome), brain injuries and disorders (e.g., middle cerebral artery occlusion (MCAO)-induced brain injury, cerebral ischemia / reperfusion (I / R) injury, vascular dementia (VD), and acute ischemic stroke-induced brain injury), and ocular disorders (e.g., dry eye disease, ocular inflammation, age-related macular degeneration (AMD), subconjunctival fibrosis, high intraocular pressure-induced retinal degeneration, bright light-induced degeneration, macrophage-induced corneal neovascularization, corneal allograft survival, and optic nerve compression).

[0065] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0066] The term "substituted" includes embodiments where a monoradical substituent is bonded to a single atom of the substituted group (e.g., forming a branch), and also includes embodiments where the substituent can be a diradical bridging group bonded to two adjacent atoms of the substituted group, thereby forming a fused ring on the substituted group.

[0067] In the case where a given group (or "part") is described as being attached to a second group in this article and the attachment site is unclear, the given group can be attached to any available site of the second group at any available site of the given group. For example, in the case where the attachment site is unclear, a "phenyl substituted with a low alkyl group" can have any available site of a low alkyl group attached to any available site of a phenyl group. In this regard, an "available site" is a group site at which the hydrogen of a group can be replaced by a substituent.

[0068] It should be understood that in all the substituted groups defined above, polymers obtained by defining the substituent as further substituted by itself (e.g., substituted aryl having a substituted aryl group as a substituent substituted by its own substituted aryl group, etc.) are not intended to be included herein. An unlimited number of substituents are also not included, whether the substituents are the same or different. In such a case, the maximum number of such substituents is three. Therefore, each of the above definitions is subject to the restriction that, for example, the substituted aryl group is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0069] A compound of a given formula (eg, "a compound of Formula (I)") is intended to encompass the compounds of the present disclosure, as well as pharmaceutically acceptable salts, pharmaceutically acceptable esters, hydrates, polymorphs, and prodrugs of such compounds.

[0070] In addition, the compounds of the present disclosure may have one or more asymmetric centers and may be produced as racemic mixtures or as individual enantiomers or diastereomers. The number of stereoisomers present in any given compound of a given formula depends on the number of asymmetric centers present (there are 2n possible stereoisomers, where n is the number of asymmetric centers). Individual stereoisomers may be obtained by splitting the racemic or non-racemic mixture of an intermediate at a suitable stage of synthesis or by splitting the compound via conventional means. Individual stereoisomers (including individual enantiomers and diastereomers) and racemic and non-racemic mixtures of stereoisomers are included within the scope of the present disclosure, unless otherwise specifically noted, all of which are intended to be depicted by the structure of this specification.

[0071] The term "isomer" means different compounds with the same molecular formula. Isomers include stereoisomers, enantiomers and diastereomers. The term "stereoisomer" means isomers that differ only in the way atoms are arranged in space. The term "enantiomer" means a pair of stereoisomers of non-superimposable mirror images. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. The term "(±)" is used to specify a racemic mixture where appropriate. The term "diastereomer" means a stereoisomer with at least two asymmetric atoms but not mirror images of each other. Absolute stereochemistry is specified herein according to the CahnIngold Prelog RS system. When a compound is a pure enantiomer, the stereochemistry of each chiral carbon can be specified by R or S. The split compounds with unknown absolute configuration are specified as (+) or (-) according to the direction (right-handed or left-handed) in which they rotate the plane of polarized light at the wavelength of the sodium D line.

[0072] Certain compounds of the present disclosure include asymmetric atoms (optical centers or chiral centers) or double bonds. The present disclosure is intended to be encompassed within the scope of the compounds described herein, even in the case where those compounds described by chemical structures and chemical formulas do not explicitly show multiple stereoisomeric or diastereomeric forms, including racemic or optically pure forms of the compounds. Optically active (R)-isomers and (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers.

[0073] "Tautomeric isomers" or "tautomers" are isomers that are in equilibrium with each other. For example, amide-containing compounds can exist in equilibrium with imidic acid tautomers. Certain compounds of the present disclosure exist as "tautomeric isomers" or "tautomers." Regardless of which tautomer is shown and regardless of the nature of the equilibrium between the tautomers, one of ordinary skill in the art will understand the compounds to include both amide tautomers and imidic acid tautomers. Therefore, amide-containing compounds should be understood to include their imidic acid tautomers.

[0074] The term "polymorph" refers to different crystal structures of a crystalline compound. Different polymorphs can be caused by differences in crystal packing (packing polymorphs) or differences in packing between different conformers of the same molecule (conformational polymorphs). Certain compounds of the present disclosure may exist in polymorphic or amorphous forms. Generally, all physical forms are equivalent for the intended uses of the present disclosure and are intended to be within the scope of the present disclosure.

[0075] The term "solvate" refers to a complex formed by combining a compound and a solvent. The term "hydrate" refers to a complex formed by combining a compound with water. Certain compounds of the present disclosure may exist in unsolvated forms, solvated forms (which include hydrated forms). Generally, the solvated forms are equivalent to the unsolvated forms and are intended to be encompassed within the scope of the compounds described herein, even in those cases where the compounds are described by chemical structures and formulas that do not explicitly show solvated forms.

[0076] As used herein, the term "salt" refers to an ionic compound produced by the neutralization reaction of an acid and a base. A salt contains a relevant number of cations (positively charged ions) and anions (negative ions) such that the product is neutral (has no net charge). These component ions can be inorganic, such as chloride (Cl - ), or organic, such as acetate (C2H3O2 - ); and can be monatomic, such as fluoride (F - ), or polyatomic, such as sulfate (SO4 2- ).

[0077] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological effectiveness and properties of the given compound, and the salt is not biologically or otherwise undesirable. In many cases, the compounds of the present disclosure are capable of forming pharmaceutically acceptable acid salts and / or base salts due to the presence of amino groups and / or carboxyl groups or groups similar thereto.

[0078] Pharmaceutically acceptable base addition salts can be prepared from inorganic bases and organic bases. By way of example only, salts derived from inorganic bases include sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trialnylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines, di(cycloalkenyl)amines Amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines, wherein at least two substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocycle, etc. Also included are amines wherein two or three substituents together with the amino nitrogen form a heterocycle or heteroaryl group. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0079] Pharmaceutically acceptable acid addition salts can also be prepared from inorganic and organic acids. Salts derived from inorganic acids include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Salts derived from organic acids include salts of acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0080] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0081] Any formula or structure given herein, including Formula (I), is also intended to represent unlabeled forms of the compound as well as isotopically labeled forms. An isotopically labeled compound has a structure depicted by a formula given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 36 Cl and 125 I. Various isotopically labeled compounds of the present disclosure, for example, in which radioactive isotopes such as 3 H. 13 C and 14 Such isotopically labeled compounds are useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in radiotherapy of patients.

[0082] Deuterium-labeled or substituted therapeutic compounds of the present disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties related to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. 18 F-labeled compounds can be used for PET or SPECT studies. Isotopically labeled compounds of the present disclosure and their prodrugs can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below, by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents. In addition, heavier isotopes, particularly deuterium (i.e. 2 H or D) may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements or improvement in therapeutic index. It should be understood that deuterium in this context is considered a substituent in the compound of formula (I).

[0083] The concentration of such heavier isotopes (particularly deuterium) can be defined by an isotopic enrichment factor. In the compounds of the present disclosure, any atom that is not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is specifically designated as "H" or "hydrogen", the position is understood to have hydrogen with its natural abundance isotopic composition. Therefore, in the compounds of the present disclosure, any atom that is specifically designated as deuterium (D) is meant to represent deuterium.

[0084] In this specification (including the Examples), unless otherwise indicated, all temperatures are in degrees Celsius (° C.), and abbreviations and acronyms have the following meanings as listed in Table 1 (below).

[0085] Table 1

[0086]

[0087]

[0088]

[0089] II. Tripterygium wilfordii derivative compounds

[0090] The present disclosure provides a series of compounds having chemical structures of derivatives of the structure of tripterygium wilfordii (compound (1)). Generally, the tripterygium wilfordii derivative compounds of the present disclosure retain the 5-ring celastrol core structure of compound (1) and replace the carboxylic acid group with a series of differently substituted amine groups and amide groups, as described in more detail below. The tripterygium wilfordii derivative compounds of the present disclosure retain or improve the functional characteristics of tripterygium wilfordii, including NF-kB cell inhibition and ADME properties, thereby providing alternative molecules for treating tripterygium wilfordii responsive diseases and disorders. Therefore, in at least one embodiment, the tripterygium wilfordii derivative compounds of the present disclosure include those compounds of structural formula (I)

[0091]

[0092] wherein the R1 group (or moiety) of the derivative compound is a group that replaces the carboxylic acid group of compound 1. The present disclosure contemplates a range of possible chemical groups for R1, including those listed in Table 2 (below).

[0093] Table 2: Exemplary tripterygium wilfordii derivatives of the R1 group of formula (I)

[0094]

[0095]

[0096]

[0097]

[0098] The present disclosure also contemplates that the celastrol derivative compounds of formula (I) may include selected subgenus of compounds. For example, in at least one embodiment, the present disclosure provides celastrol derivative compounds of formula (I), wherein the group R1 may be -NH-(CO)2-NR2R3 or -N(CH3)-(CO)2-NR2R3. In at least one embodiment of this subgenus of the compound, the groups R2 and R3 of the formula may be independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine or heteroaryl, optionally substituted by substituents selected individually from the following: alkyl, alkoxy, cycloalkyl, ether, amine optionally substituted with one or more alkyl groups, halogen, hydroxyl, cyano, nitrile, CF3, ester, amide, cycloalkylamide, sugar, heteroarylamide optionally substituted with alkyl and / or alkoxy, urea, carbamate, thioether, sulfate, sulfonyl, sulfonic acid, carboxylic acid and aryl. In another embodiment of this subgenus, the groups R2 and R3 of the structural formula can be combined together to form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by substituents individually selected from the following: alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, heteroaryl, amine, halogen, hydroxyl, ether, nitrile, cyano, nitro, CF3, ester, amide, urea, carbamate, thioether or carboxylic acid group.

[0099] Exemplary tripterygium derivative compounds of the present disclosure are provided below in Table 3. The synthesis, characterization, and use of these exemplary compounds are described in the Examples and elsewhere herein.

[0100] Table 3: Exemplary tripterygium wilfordii derivative compounds

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] As described elsewhere herein, it will be understood by those of ordinary skill that the tripterygium derivative compounds provided herein may exist in a variety of well-known closely related and / or equivalent forms that are not explicitly described by chemical structures and chemical formulas. It is intended that the tripterygium derivative compounds of structural formula (I) of the present disclosure include these closely related forms of compounds defined by chemical structures and chemical formulas, including but not limited to pharmaceutically acceptable salts of the compounds, mixtures of stereoisomers of the compounds, single stereoisomers of the compounds, tautomeric forms of the compounds, and / or prodrug forms of the compounds.

[0112] III. Preparation of tripterygium wilfordii derivatives

[0113] The tripterygium derivative compounds of structural formula (I) can be prepared from readily available starting materials using methods and procedures known in the art. In particular, the present disclosure provides a general synthetic strategy for preparing compounds of structural formula (I). Schemes A-Scheme F presented below provide a series of six reactions starting from tripterygium (compound 1), which can be used, adjusted and / or combined with well-known synthetic methods by ordinary technicians to synthesize the tripterygium derivative compounds of the present disclosure, such as the compounds of Table 3 (above).

[0114] Plan A

[0115]

[0116] Plan B

[0117]

[0118] Plan C

[0119]

[0120] Plan D

[0121]

[0122] Plan E

[0123]

[0124] Plan F

[0125]

[0126] In addition to the reactions of Scheme A-Scheme F, Examples 1-7 provide specific synthetic schemes that demonstrate the preparation of tripterygium wilfordii derivative compounds 14, 15, 16, 17, and 22. Ordinary technicians can use and / or adjust these synthetic schemes of Examples 1-7 to prepare other tripterygium wilfordii derivative compounds of structural formula (I) described in the present disclosure. Ordinary technicians will understand that, except for the typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) described in the embodiments herein, other process conditions may also be used unless otherwise stated. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions may be determined by conventional optimization procedures by those skilled in the art. In addition, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. For example, protecting groups may be used to allow functional groups (such as O, S, or N) to be temporarily blocked so that the reaction can be selectively carried out at another reaction site in the multifunctional compound. Protecting groups useful in the syntheses of the present disclosure are well known in the art and include those described in detail in Protective Groups in Organic Synthesis, 4th edition, Greene, TW and Wuts, PG, eds., John Wiley & Sons, New York: 2007 (the entire contents of which are hereby incorporated by reference) and in the references cited therein.

[0127] As mentioned above, the starting materials and / or reagents used in the synthetic reaction schemes A-Scheme F are commercially available or are generally known compounds that can be prepared by known procedures or obvious modifications thereof or disclosed in the examples herein. For example, many materials / reagents are available from commercial suppliers such as Sigma-Aldrich Chemical Co. (St. Louis, Missouri, USA). Other materials / reagents can be prepared by procedures described in standard reference texts, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and Supplements (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 5th edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or obvious modifications thereof.

[0128] IV. Uses and Methods of Treatment

[0129] As described elsewhere herein, based on clinical studies, preclinical studies or in vitro studies, tripterygium wilfordii has been proposed or shown to provide potential therapeutic effects in a variety of diseases and disorders. These previously determined tripterygium wilfordii responsive diseases and disorders include, but are not limited to, cancer (e.g., gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, brain cancer and glioblastoma multiforme), inflammatory and / or autoimmune disorders (e.g., rheumatoid arthritis (RA), ankylosing spondylitis, systemic lupus erythematosus (SLE), inflammatory bowel disease, osteoarthritis (OA), allergy and skin inflammation), neurological disorders (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS)), obesity-related disorders (e.g., obesity , preobesity, morbid obesity, Prader-Willi syndrome, obesity associated with hypothalamic damage, nonalcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Barder-Biedel syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome and MOMO (macrosomia) syndrome), and liver-related diseases or disorders (e.g., acute-on-chronic liver failure (ACLF), alcoholic liver disease, cholestatic liver disease, drug-induced liver disease, hepatocellular carcinoma, nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease (NASH), viral hepatitis and viral liver disease).

[0130] Therefore, the present disclosure contemplates the use of the tripterygium derivative compounds of structural formula (I) of the present disclosure or pharmaceutical compositions of such compounds in therapy, as medicaments, or in methods of treating a tripterygium responsive disease or disorder in a subject, or in the manufacture of a medicament for treating a tripterygium responsive disease or disorder in a subject.

[0131] In at least one embodiment, the present disclosure contemplates that the tripterygium derivative compounds of structural formula (I) disclosed herein can be used in a method of treating a tripterygium responsive disease or disorder in a subject suffering from a tripterygium responsive disease or disorder. Generally, the method of treatment comprises administering a therapeutically effective amount of a compound of structural formula (I) as disclosed herein to a subject in need thereof. In at least one embodiment, the compound of structural formula (I) administered can be a compound selected from the following: Compound 14, Compound 15, Compound 16, Compound 17, Compound 22, Compound 22-1, Compound 42, Compound 54, Compound 55, Compound 56, Compound 57, Compound 58, Compound 59, Compound 60, Compound 61, Compound 62, Compound 63, Compound 64, Compound 65, Compound 66, Compound 67, Compound 68, Compound 69, Compound 70, Compound 71, Compound 72, Compound 73, Compound 74, Compound 75, Compound 76, Compound 77, Compound 78, Compound 79, Compound 80, Compound 81, Compound 82, Compound 83, Compound 84, Compound 85, Compound 86, Compound 87, Compound 88, Compound 89, Compound 90, Compound 91 and Compound 92, or a pharmaceutically acceptable salt, single stereoisomer, mixture of stereoisomers, tautomer or prodrug of any of these compounds.

[0132] In at least one embodiment, the compound of structural formula (I) administered can be in the form of a pharmaceutical composition comprising a compound of structural formula (I) or a pharmaceutically acceptable salt or ester thereof and one or more pharmaceutically acceptable excipients as described elsewhere herein. In at least one embodiment, the administered pharmaceutical composition may contain one or more pharmaceutically acceptable excipients, and the compound is selected from Compound 14, Compound 15, Compound 16, Compound 17, Compound 22, Compound 22-1, Compound 42, Compound 54, Compound 55, Compound 56, Compound 57, Compound 58, Compound 59, Compound 60, Compound 61, Compound 62, Compound 63, Compound 64, Compound 65, Compound 66, Compound 67, Compound 68, Compound 69, Compound 70, Compound 71, Compound 72, Compound 73, Compound 74, Compound 75, Compound 76, Compound 77, Compound 78, Compound 79, Compound 80, Compound 81, Compound 82, Compound 83, Compound 84, Compound 85, Compound 86, Compound 87, Compound 88, Compound 89, Compound 90, Compound 91 and Compound 92, or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug of any of these compounds.

[0133] In at least one embodiment of the therapeutic use or method of treating a tripterygium derivative compound of structural formula (I), the tripterygium responsive disease or disorder suffered by the subject may be cancer. The cancer treated by the method may include, but is not limited to, gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, brain cancer, and glioblastoma multiforme (GBM).

[0134] In at least one embodiment of the therapeutic use or method of treating a tripterygium derivative compound of structural formula (I), the tripterygium responsive disease or disorder suffered by the subject may be an inflammatory and / or autoimmune disorder. The inflammatory and / or autoimmune disorders treated by the method may include, but are not limited to, rheumatoid arthritis (RA), ankylosing spondylitis, systemic lupus erythematosus (SLE), inflammatory bowel disease, osteoarthritis (OA), allergy, and skin inflammation.

[0135] In at least one embodiment of the therapeutic use or method of treating a tripterygium derivative compound of structural formula (I), the tripterygium responsive disease or disorder suffered by the subject may be a neurological disorder. The neurological disorder treated by the method may include, but is not limited to, Parkinson's disease, Huntington's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS).

[0136] In at least one embodiment of the therapeutic use or method of treating the tripterygium derivative compound of structural formula (I), the tripterygium responsive disease or disorder suffered by the subject may be an obesity-related disease or disorder. Obesity-related diseases or disorders treated by the method may include, but are not limited to, obesity, pre-obesity, morbid obesity, Prader-Willi syndrome, obesity associated with hypothalamic damage, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bard-Biedel syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macrosomic obesity macrophthalmia) syndrome.

[0137] In at least one embodiment of the therapeutic use of the tripterygium derivative compound of structural formula (I) or the method of treatment, the tripterygium responsive disease or disorder suffered by the subject may be a liver-related disease or disorder. Liver-related diseases or disorders treated by the method may include, but are not limited to, acute-on-chronic liver failure (ACLF), acute hepatic porphyria, Alagille syndrome, alpha-1 antitrypsin deficiency, alcoholic liver disease, alcoholic hepatitis, amoebic liver, autoimmune hepatitis, benign liver tumors, biliary atresia, cardiac cirrhosis, cholestatic liver disease, congestive liver disease, cirrhosis, Crigler-Najjar syndrome, drug-induced liver disease, Dubin-Johnson syndrome, conjugated hyperbilirubinemia, galactosemia, Gilbert syndrome, glycogen storage disease, hemochromatosis, hepatic abscesses, hepatic cysts, hepatic encephalopathy, hepatitis, hepatorenal syndrome, intrahepatic cholestasis of pregnancy, isoniazid poisoning, jaundice, liver abscesses, The method is directed to treating a liver-related disease or disorder selected from the group consisting of acute-chronic liver failure (ACLF), alcoholic liver disease, cholestatic liver disease, drug-induced liver disease, hepatocellular carcinoma, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), primary biliary cholangitis, primary sclerosing cholangitis, progressive familial intrahepatic cholestasis, Reye syndrome, type I glycogen storage disease, unconjugated hyperbilirubinemia, viral hepatitis, viral liver disease, and Wilson's disease. In at least one embodiment, the liver-related disease or disorder treated by the method is selected from the group consisting of acute-chronic liver failure (ACLF), alcoholic liver disease, cholestatic liver disease, drug-induced liver disease, hepatocellular carcinoma, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), viral hepatitis, and viral liver disease.

[0138] V. Pharmaceutical Compositions and Modes of Administration

[0139] The present disclosure also provides uses and methods in which a tripterygium derivative compound, such as a compound of structural formula (I), is administered to a subject in the form of a pharmaceutical composition, as described above. For example, in such an embodiment, the pharmaceutical composition includes a therapeutically effective amount of a tripterygium derivative compound of structural formula (I) (e.g., a compound of Table 3), or a pharmaceutically acceptable salt or ester of such a compound and one or more pharmaceutically acceptable carriers. Such a pharmaceutical composition can be prepared using methods well known in the pharmaceutical field (see, for example, Remington's Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th edition (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd edition (GS Banker & C.T. Rhodes, ed.). Methods for preparing pharmaceutical compositions of tripterygium derivative compounds are described in the present disclosure, including embodiments disclosed herein.

[0140] Typically, pharmaceutical compositions can be prepared by diluting the active ingredient with an excipient and / or enclosing it in a carrier in the form of a capsule, pouch, paper or other container. When an excipient is used as a diluent, it can be a solid material, a semisolid material, or a liquid material (as above) that acts as a vehicle, carrier or medium for the active ingredient. Thus, pharmaceutical compositions suitable for administration in the methods of the present disclosure can be in the form of tablets, pills, powders, lozenges, pouches, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

[0141] The carrier for preparing the pharmaceutical composition may include excipients such as inert solid diluents and fillers, diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers and excipients. Suitable excipients for pharmaceutical compositions containing the tripterygium wilfordii derivatives (e.g., compounds of Table 3) of the present disclosure are well known in the art and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, sterile water, syrups and methylcellulose. Pharmaceutical compositions may additionally include lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl benzoate and propylhydroxy-benzoate; sweeteners; and flavoring agents.

[0142] In the use and method of treatment, it is expected that the pharmaceutical composition containing the tripterygium derivative compound, such as the compound of structural formula (I) (e.g., the compound of Table 3) can be administered as a single dose or multiple doses, and by any acceptable mode of administration of active ingredients with similar efficacy. For example, the pharmaceutical composition containing the tripterygium derivative can be administered using a variety of different modes, including oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration or subcutaneous administration.

[0143] Pharmaceutical compositions comprising the tripterygium derivative compounds of the present disclosure (e.g., compounds of Table 3) can be used in a range of therapeutic treatment methods, and a range of doses are expected for the administration of pharmaceutically effective amounts. The dose and frequency (single or multiple doses) of administering the pharmaceutical composition to the subject may vary according to a range of factors, such as the route of administration; the size, age, sex, health, weight and / or diet of the subject; the state of the disease being treated; whether the subject suffers from any other disease, and any concurrent treatment being received. Those of ordinary skill will understand that it may be necessary to adjust the determined dose (e.g., frequency and duration) according to the subject to obtain a therapeutically effective amount. Typically, the amount of the pharmaceutical composition comprising the tripterygium derivative compound to be administered to the subject in the therapeutic treatment method will be determined by the physician according to the relevant circumstances of the subject being treated, the route of administration selected, and of course the age, weight, severity of symptoms, response of the individual subject to treatment, etc.

[0144] Typically, a therapeutically effective amount is an amount sufficient to achieve the desired therapeutic purpose of the administered composition relative to the absence of the compound. For example, a therapeutically effective amount can be an amount determined to be sufficient to help treat, prevent, or alleviate one or more symptoms of a disease. Methods for determining a dosage of a compound that provides a therapeutically effective amount are well known to those of ordinary skill in the art and are generally based on analysis of amounts determined in cell assays and / or animal models.

[0145] For example, the dosage for administration to humans can be formulated to reach a concentration that has been observed to be therapeutically effective in animal models. The dosage in the pharmaceutical composition for humans can be further adjusted by monitoring effectiveness and adjusting upward or downward. Ordinary technicians can use methods well known in the art to adjust the dosage in the pharmaceutical composition of the present disclosure (e.g., the compound of Table 3) to achieve maximum therapeutic efficacy to humans.

[0146] Typically, methods for therapeutic treatment are developed by starting with a pharmaceutical composition containing less than the optimal dose of a tripterygium wilfordii derivative compound. Thereafter, the dose of the compound is incrementally increased until the optimal efficacy is reached. The key factor to consider when developing the optimal dose is the ratio between the toxicity and therapeutic efficacy of the active ingredient. This ratio (known as the therapeutic index of the compound) is usually described as the LD50 of the active ingredient. 50 (the amount of compound that is lethal in 50% of the population) and its ED 50 The ratio of the amount of compound effective in 50% of the population. Generally, a higher therapeutic index for a compound is preferred. Therapeutic index data can be obtained from cell culture assays and / or animal model studies and then used to determine a safe dosage range for the active ingredient in a pharmaceutical composition for administration to humans. Ideally, the determined dose provides a 50% increase in the ED in a subject. 50 Levels of active ingredients with little or no toxicity.

[0147] In some embodiments using a compound of the present disclosure (e.g., a compound of Table 3), a pharmaceutical composition comprises a dose of a celastrol derivative compound as an active ingredient in an amount of about 0.05 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 25 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 25 mg / kg, about 5 mg / kg to about 25 mg / kg, about 10 mg / kg to about 25 mg / kg, about 10 mg / kg to about 50 mg / kg, about 25 mg / kg to about 50 mg / kg, about 25 mg / kg to about 75 mg / kg, or about 50 mg / kg to about 100 mg / kg. In some embodiments, the pharmaceutical composition comprises a dose of a celastrol derivative compound of formula (I) in an amount of about 0.05 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg or about 100 mg / kg.

[0148] In some embodiments, it is expected that the pharmaceutical composition containing the tripterygium derivative compound of the present disclosure (e.g., the compound of Table 3) can be formulated so as to provide a rapid, sustained or delayed release of the active ingredient after administration. For example, controlled release drug delivery systems for oral administration are known in the art and include osmotic pump systems and dissolution systems containing reservoirs or drug-polymer matrix formulations of polymer coatings. Examples of controlled release systems are given in, for example, U.S. Patent Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345.

[0149] In some embodiments, it is contemplated that the pharmaceutical composition comprising the tripterine derivative compound of the present disclosure (e.g., the compound of Table 3) can also be formulated for administration via a transdermal delivery device (e.g., a "patch"). Such a transdermal patch can be used to provide a continuous or discontinuous infusion of a controlled amount of a pharmaceutical composition. The construction and use of transdermal patches for delivering pharmaceutical compositions are well known in the art. See, for example, U.S. Patents Nos. 5,023,252, 4,992,445, and 5,001,139. ​​Such a patch can be constructed for continuous, pulsatile, or on-demand delivery of a pharmaceutical composition.

[0150] In some embodiments, it is expected that the pharmaceutical composition of the present disclosure (e.g., the compound of Table 3) can be prepared as a solid preparation, for example for oral administration. Such a solid preparation can be prepared by mixing the active ingredient of the tripterygium wilfordii derivative compound with a pharmaceutical excipient to form a solid preformulation composition containing a uniform mixture of the active ingredient and the excipient. When referring to these preformulation compositions as uniform, it means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily redivided into equivalent unit dosage forms such as tablets, pills and capsules. Tablets or pills can be coated or otherwise compounded to provide a dosage form that gives the advantage of prolonged action, or to protect from the acidic conditions of the stomach. For example, a tablet or pill can include an internal dosage component and an external dosage component, the latter being in the form of being encapsulated in the former. The two components can be separated by an enteric layer, which is used to resist decomposition in the stomach and allows the internal component to pass intact into the duodenum or delay release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0151] Another exemplary mode of administration that can be used for the method of the present disclosure is parenteral, particularly by injection. In order to be administered by injection, the pharmaceutical composition of the present disclosure (for example, the composition comprising the compound of Table 3) can be incorporated into an aqueous or oily suspension or emulsion with sesame oil, corn oil, cottonseed oil or peanut oil, and in elixir, mannitol, dextrose or sterile aqueous solution and similar pharmaceutical vehicles. The aqueous solution in saline is also generally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol etc. (and suitable mixtures thereof), cyclodextrin derivatives and vegetable oils can also be adopted. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant, suitable fluidity can be maintained. The effect of preventing microorganisms can be achieved by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal etc.

[0152] Sterile injectable solutions are prepared by incorporating a desired amount of an active ingredient of the present disclosure (e.g., a compound of Table 3) into an appropriate solvent, followed by filtration sterilization, wherein the solvent has a variety of other ingredients as listed above as required. Typically, dispersions are prepared by incorporating a variety of sterilized active ingredients into a sterile vehicle comprising a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, known preparation methods include vacuum drying techniques and freeze drying techniques, which produce powders of the active ingredient plus any additional desired ingredients from their previously sterile filtered solutions.

[0153] The pharmaceutical composition that can be administered by inhalation or insufflation includes solutions and suspensions in pharmaceutically acceptable aqueous organic solvents or mixtures thereof, and powders. Liquid compositions or solid compositions may include suitable pharmaceutically acceptable excipients as described herein and as known in the art. In some embodiments, the pharmaceutical composition of the tripterygium wilfordii derivative (e.g., the compound of Table 3) may be administered by oral routes or nasal respiratory routes for local or systemic effects. In some embodiments, the pharmaceutical composition is prepared in a pharmaceutically acceptable solvent that can be atomized using an inert gas. These atomized solutions can be inhaled directly from the atomizing device, or the atomizing device may be attached to a face mask tent (face masks tent) or an intermittent positive pressure breathing machine. In some embodiments, the pharmaceutical composition that can be used for the method may be a solution, a suspension, or a powder composition, and may be administered orally or nasally from a device that delivers the preparation in an appropriate manner. Example

[0154] Various features and embodiments of the present disclosure are shown in the following representative examples, which are intended to be illustrative and non-restrictive. It will be readily appreciated by those skilled in the art that the specific examples are merely illustrations of various embodiments of the present disclosure as more fully described in the appended claims. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained therein.

[0155] Example 1: Synthesis of (6aS, 6bS, 8aS, 11R, 12aR, 14aR)-11-amino-3-hydroxy-4,6a, 6b, 8a, 11, 14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydroquinone-2-one (Compound 4-2)

[0156]

[0157] Compound 4-2 was prepared via a 2-step synthesis summarized in Scheme 1 (below).

[0158] Solution 1

[0159]

[0160] Step 1

[0161] Synthesis of (6aS,6bS,8aS,11R,12aR,14aR)-3-hydroxy-11-isocyanato-4,6a,6b,8a,11,14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydroquinone-2-one (Compound 4-1)

[0162]

[0163] To a solution of (2R, 4aS, 6aR, 6aS, 14aS, 14bR) -10-hydroxy-2,4a, 6a, 6a, 9, 14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-carboxylic acid (4.0 g, 8.88 mmol) in toluene (200 mL) was added DIPEA (3.44 g, 26.6 mmol) and DPPA (3.66 g, 13.3 mmol) at 25 ° C under N2. The reaction mixture was stirred at 100 ° C for 16 hours. The reaction mixture was cooled to 25 ° C and poured into water (200 mL). The aqueous phase was extracted with DCM (3 × 100 mL). The combined organic phase was washed with brine (2 × 300 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica gel chromatography (0-50% ethyl acetate / petroleum ether eluent) to provide both (6aS,6bS,8aS,11R,12aR,14aR)-3-hydroxy-11-isocyanato-4,6a,6b,8a,11,14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydroquinone-2-one (Compound 4-1) (3.0 g, 91.4% purity, 76% yield) and (142 mg, 100% purity) as yellow solids.

[0164] 1 H NMR: CDCl3 400MHzδ=7.05(d,J=6.8Hz,1H),6.56(s,1H),6.38(d,J=7.2Hz,1H),2.24-1.90(m,8H),1. 85-1.52(m,10H),1.46(s,3H),1.42(s,3H),1.28(s,3H),1.10-0.99(m,4H),0.95(s,3H).

[0165] LCMS: Rt = 2.949 min in 1.5 min chromatography, 5-95 AB, purity 99%, LCMS ESI + for C 29 H 37 NO3[M] + Calculated value 447.2, found value [M+H] + 448.2.

[0166] HPLC: Rt = 4.74 min, 50-100 AB, purity 96% in 8 min chromatography.

[0167] Step 2

[0168] Synthesis of (6aS,6bS,8aS,11R,12aR,14aR)-11-amino-3-hydroxy-4,6a,6b,8a,11,14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydroquinone-2-one (Compound 4-2)

[0169]

[0170] To a solution of (6aS,6bS,8aS,11R,12aR,14aR)-3-hydroxy-11-isocyanato-4,6a,6b,8a,11,14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydroquinone-2-one (3.0 g, 6.70 mmol) in THF (120 mL) and H2O (12 mL) was added LiOH-H2O (1.41 g, 33.5 mmol) at 25°C. The reaction mixture was stirred at 50°C for 2 hours. The reaction mixture was concentrated and then poured into H2O (200 mL) and filtered to give a solid. The solid was dissolved in MeOH and concentrated in vacuo to give (6aS,6bS,8aS,11R,12aR,14aR)-11-amino-3-hydroxy-4,6a,6b,8a,11,14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydronaphthol-2-one (3.5 g, crude) as a yellow solid. The crude product (50 mg, crude) was purified by silica gel chromatography (0-20% methanol / dichloromethane eluent) to give (6aS,6bS,8aS,11R,12aR,14aR)-11-amino-3-hydroxy-4,6a,6b,8a,11,14a-hexamethyl-7,8,9,10,12,12a,13,14-octahydroquinone-2-one (Compound 4-2) (11.3 mg, 100% purity) as a yellow solid.

[0171] 1 H NMR:MeOD 400MHzδ=7.22(d,J=7.6Hz,1H),6.52(d,J=7.2Hz,1H),6.47(s,1H),2.34-2.15(m,4H),2.05-1.61(m,14H),1.61-1.13(m,15H),0.85(s,3H).

[0172] LCMS: Rt = 0.920 min in 1.5 min chromatography, 5-95 AB, purity 100%, LCMS ESI + for C 28 H 39 NO2[M] + Calculated value 421.3, found value [M+H]+ 422.2.

[0173] HPLC: Rt = 2.56 min, 30-90 AB in 8 min chromatography, purity 100%.

[0174] Example 2: Synthesis of (2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydrophthalic acid-2-yl)amino)-2-oxoacetic acid ethyl ester (Compound 14-1).

[0175]

[0176] Compound 14-1 was prepared via the synthesis summarized in Scheme 2 (below).

[0177] Solution 2

[0178]

[0179] Step 1

[0180] At 0 ° C, to (6bS, 8aS, 11R, 12aR, 12bS, 14aR) -11-amino-3-hydroxy-4, 6b, 8a, 11, 12b, 14a-hexamethyl-7, 8, 8a, 9, 10, 11, 12, 12a, 12b, 13, 14, 14a- dodecahydro-2 (6bH) -one (1.00g, 2.37mmol) in DCM (20mL) solution was added Et3N (720mg, 7.12mmol). Then, 2-chloro-2-oxo-ethyl acetate (291mg, 2.13mmol) was added dropwise. The resulting mixture was stirred at 0 ° C for 1 hour. The reaction mixture was poured into water (50mL). The aqueous phase was extracted with DCM (3 × 100mL). The combined organic phases were washed with brine (3×30 mL), dried over anhydrous Na 2 SO 4 , filtered and concentrated to give ethyl 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydrophthalim-2-yl)amino)-2-oxoacetate as a yellow solid (1.2 g, 74.7% purity). 100 mg of yellow solid was purified by preparative HPLC (column: Phenomenex Gemini 150*25 mm*10 μm; mobile phase: [water (0.05% NH3H2O)-ACN]; B%: 62%-92%, 10 min) to give ethyl 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydrophthalimide-2-yl)amino)-2-oxoacetate (Compound 14-1) (15.25 mg, 96.7% purity) as a yellow solid.

[0181] 1 H NMR: CDCl3 400MHzδ=7.08-6.78(m,2H),6.63-6.26(m,2H),4.42(dd,J=4.5,8.0Hz,1H),4.39-3.95(m,2H),2.66(br d,J=15.0Hz,1H),2.32-2.11(m,4H),2.02-1.52(m,15H),1.39-1.28(m,6H),1.22-1.00(m,5H),0.87(br d,J=7.0Hz,2H),0.72(br s,3H).

[0182] LCMS: Rt = 0.595 min in 1.5 min chromatography, 50-95 AB, purity 100%, LCMS ESI + for C 32 H 43 NO5[M] + Calculated value 521.3, found value [M+H] + 522.3.

[0183] HPLC: Rt = 3.101 min, 10-80 AB, purity 96.73% in 4 min chromatography.

[0184] Example 3: Synthesis of N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14).

[0185]

[0186] Compound 14 was prepared via a 2-step synthesis summarized in Scheme 3 (below).

[0187] Solution 3

[0188]

[0189] Step 1

[0190] Synthesis of 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydro-2-yl)amino)-2-oxoacetic acid (Compound 14-2)

[0191]

[0192] A mixture of ethyl 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydrophthalim-2-yl)amino)-2-oxoacetate (1.3 g, 2.49 mmol) and LiOH.HO (314 mg, 7.48 mmol) in THF (60 mL) and HO (12 mL) was stirred at 0° C. for 0.5 h. The reaction mixture was adjusted to pH=3 by addition of aqueous HCl (1 M). The aqueous phase was extracted with DCM (30 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydrophthalimide-2-yl)amino)-2-oxoacetic acid (Compound 14-2) (1.2 g, 80.2% yield) as a yellow solid.

[0193] LCMS: Rt = 0.507 min in 1.5 min chromatography, 5-95AB, purity 73.919%, LCMS ESI+ for C 30 H 39 NO5[M] + Calculated value 493.3, found value [M+H] + 494.3.

[0194] Step 2

[0195] Synthesis of N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14)

[0196] To a solution of 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydro-2-yl)amino)-2-oxoacetic acid (500 mg, 1.01 mmol) in DCM (5 mL) was added DCC (313 mg, 1.52 mmol) and stirred at 0°C. 1-Methylpiperazine (152 mg, 1.52 mmol) and DMAP (61.9 mg, 506 μmol) were added at 0°C. The resulting mixture was stirred at 20°C for 0.5 hours. The reaction mixture was poured into water (5 mL). The aqueous phase was extracted with DCM (3×10 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol=1 / 0 to 20 / 1, Rf=0.32) to afford N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydro-2-yl)-2-(4-methylpiperazine-1-yl)-2-oxoacetamide (200 mg) as a brown solid. The brown solid was purified by preparative-TLC (SiO2, dichloromethane:methanol=10 / 1, Rf=0.32) to give N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14) (100 mg, 15.3% yield) as a yellow solid.

[0197] 1H NMR: CDCl3 400MHzδ=7.09-6.90(m,3H),6.51(d,J=1.4Hz,1H),6.36(d,J=7.1Hz,1H),4.43-4.00(m,2H),3.87-3.45(m,2H),2.72(br d,J=14.0Hz,1H),2.64-2.39(m,4H),2.31(s,3H),2.24-2.11(m,4H),2.06-1.67(m,11H),1.51-1. 50(m,1H),1.45(d,J=6.8Hz,6H),1.34-1.23(m,4H),1.13(s,3H),1.06-0.98(m,1H),0.75(s,3H).

[0198] LCMS: Rt = 0.431 min in 1.5 min chromatography, 5-95AB, purity 98.137%, LCMS ESI+ for C 35 H 49 N3O4[M] + Calculated value 575.4, found value [M+H] + 576.4.

[0199] HPLC: Rt = 2.105 min, 10-80 AB, purity 95.8% in 4 min chromatography.

[0200] Example 4: Synthesis of N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)-2-oxo-2-(2-oxa-6-azaspiro[3.3]hept-6-yl)acetamide (Compound 15).

[0201]

[0202] Compound 15 was prepared via the synthesis summarized in Scheme 4 (below).

[0203] Solution 4

[0204]

[0205] Step 1

[0206] To a solution of 2-(((2R, 4aS, 6aS, 12bR, 14aS)-10-hydroxy-2,4a, 6a, 9, 12b, 14a-hexamethyl-11-oxo-1,2,3,4,4a, 5,6,6a, 11, 12b, 13, 14, 14a, 14b-tetrahydro-2-yl)amino)-2-oxoacetic acid (compound 14-2) (100 mg, 203 μmol) in DCM (1.5 mL) was added DCC (105 mg, 506 μmol) and stirred at 0 °C. Then, 2-oxa-6-azaspiro[3.3]heptane (22.1 mg, 223 μmol) and DMAP (37.1 mg, 304 μmol) were added. The resulting mixture was stirred at 20 °C for 12 hours. The reaction mixture was poured into water (5 mL). The aqueous phase was extracted with DCM (3×10 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether:ethyl acetate=1 / 0 to 0 / 1, Rf=0.26) to give a crude brown solid (60 mg). The brown solid was purified by preparative TLC to give N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthoyl)-2-oxo-2-(2-oxa-6-azaspiro[3.3]hept-6-yl)acetamide (Compound 15) (5.5 mg, 3.11% yield) as a yellow solid.

[0207] 1 H NMR: CDCl3 400MHzδ=7.21(s,1H),7.04(br d,J=7.0Hz,2H),6.55(s,1H),6.36(d,J=7.1Hz,1H),4.90-4.68(m,7H),4.19(s,2H),2.63(br d,J=14.4Hz,1H),2.27-2.10(m,4H),2.04-1.83(m,4H),1.71(br d,J=8.4Hz,4H),1.64-1.58(m,3H),1.48-1.40(m,6H),1.28(s,3H),1.12(s,3H),1.02(br d,J=13.6Hz,1H),0.69(s,3H).

[0208] LCMS: Rt = 0.590 min in 1.5 min chromatography, 5-95AB, purity 95.249%, LCMS ESI+ for C 35 H 46N2O5[M] + Calculated value 574.3, found value [M+H] + 575.3.

[0209] HPLC: Rt = 2.906 min, 10-80 AB, purity 98.90% in 4 min chromatography.

[0210] Example 5: Synthesis of N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)-N-methyl-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 16).

[0211]

[0212] Compound 16 was prepared via the synthesis summarized in Scheme 5 (below).

[0213] Solution 5

[0214]

[0215] Step 1

[0216] To a solution of N-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14) (180 mg, 313 μmol) in THF (5 mL) was added MeI (44.0 mg, 312 μmol). Then, NaH (75.0 mg, 1.88 mmol, 60% in mineral oil) was added at 0°C under N2. The resulting mixture was stirred at 20°C for 0.5 hours. The reaction mixture was slowly poured into saturated NH4Cl aqueous solution (20 mL) at 0 ° C, and then extracted with DCM (3 × 20 mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane: methanol = 1: 0 to 50 / 1; Rf = 0.54) to obtain N-((2R, 4aS, 6aS, 12bR, 14aS)-10-hydroxy-2,4a, 6a, 9, 12b, 14a-hexamethyl-11-oxo-1,2,3,4,4a, 5, 6, 6a, 11, 12b, 13, 14, 14a, 14b-tetrahydro-2-yl)-N-methyl-2-(4-methylpiperazine-1-yl)-2-oxoacetamide (Compound 16) (5.75 mg, 2.86% yield) as a yellow solid.

[0217] 1 H NMR: CDCl3 400MHzδ=7.12-6.87(m,2H),6.53(d,J=1.0Hz,1H),6.36(d,J=7.1Hz,1H),5.81-5.79(m,1H),3.73(q,J=7.0Hz,4H),3.41(br dd,J=3.8,12.9Hz,4H),2.87(s,3H),2.50-2.37(m,4H),2.31(s,3H),2.21(s,3H),2.17-1.97(m,3H ),1.96-1.81(m,4H),1.46(s,3H),1.37-1.31(m,4H),1.23(s,3H),1.20-1.09(m,5H),0.87(s,3H).

[0218] LCMS: R in 1.5 min chromatography t = 0.492 min, 5-95AB, purity 96.29%, LCMS ESI+ for C 36 H 51 N3O4[M] +Calculated value 589.4, found value [M+H] + 590.4.

[0219] HPLC: Rt = 2.187 min in 4 min chromatography, 10-80 AB, purity 91.7%.

[0220] Example 6: Synthesis of N1-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydro-2-yl)-N2-(1-methylazetidin-3-yl)oxalamide (Compound 17)

[0221]

[0222] Compound 17 was prepared via the synthesis summarized in Scheme 6 (below).

[0223] Solution 6

[0224]

[0225] Step 1

[0226] To a solution of 2-(((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydronaphthalene-2-yl)amino)-2-oxoacetic acid (compound 14-2) (200 mg, 405 μmol) in DCM (5 mL) at 0°C was added DCC (125 mg, 608 μmol). Then, 1-methylazetidin-3-amine (38.4 mg, 446 μmol) and DMAP (24.8 mg, 203 μmol) were added. μmol). The resulting mixture was stirred at 20 ° C for 0.5 hours. The reaction mixture was poured into water (5mL). The aqueous phase was extracted with DCM (3×3mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane: methanol = 1 / 0 to 50 / 1, Rf = 0.40) to obtain a crude product (100 mg). The crude product was diluted with EtOAc (10mL) and washed with HCl aqueous solution (0.02M, 10mL×2). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give N1-((2R,4aS,6aS,12bR,14aS)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydrophthalimide-2-yl)-N2-(1-methylazetidin-3-yl)oxalamide (Compound 17) (60.0 mg, 22.2% yield) as a yellow solid.

[0227] 1 H NMR: CDCl3 400MHzδ=7.74(br d,J=8.5Hz,1H),7.23(s,1H),7.01(dd,J=0.9,7.1Hz,1H),6.52(d,J=1.0Hz,1H),6.35(d,J=7 .1Hz,1H),4.39(dd,J=6.6,14.9Hz,1H),3.63(t,J=7.5Hz,2H),2.95(q,J=6.7Hz,2H),2.63(br d,J=15.4Hz,1H),2.32(s,3H),2.22-2.12(m,4H),1.90-1.69(m,10H),1.44(d,J=11.8Hz,5H),1.29-1.25(m,6H),1.13(s,3H),1.04(br d,J=14.0Hz,1H),0.91-0.86(m,1H),0.68(s,3H).

[0228] LCMS ： Rt = 0.437 min in 1.5 min chromatography, 5-95AB, purity 92.577%, LCMS ESI+ for C 34 H 47 N3O4[M] + Calculated value 561.4, found value [M+H] + 562.5.

[0229] HPLC: Rt = 2.206 min in 4 min chromatography, 10-80 AB, purity 91.886%.

[0230] Example 7: Synthesis of N'-[(2R,4aS,6aR,6aS,14aS,14bR)-10-methoxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-yl]-N,N,N'-trimethyl-oxalamide (Compound 22):

[0231]

[0232] Compound 22 was prepared via a 2-step synthesis summarized in Scheme 7 (below).

[0233] Solution 7

[0234]

[0235] Step 1

[0236] Synthesis of N-[(2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-yl]-N',N'-dimethyl-oxalamide (Compound 22-1):

[0237]

[0238] At 25 ° C, to (6aS, 6bS, 8aS, 11R, 12aR, 14aR) -11-amino-3-hydroxy-4, 6a, 6b, 8a, 11, 14a-hexamethyl-7, 8, 9, 10, 12, 12a, 13, 14-octahydro-2-one (50mg, 119μmol) and 2- (dimethylamino) -2-oxo-acetic acid (18mg, 154μmol) in DCM (1mL) solution, DCC (36.7mg, 178μmol) and DMAP (7.24mg, 59.3μmol) were added. The reaction was stirred at 25 ° C for 16 hours. The residue was poured into ice water (30mL) and the aqueous phase was extracted with DCM (3×10mL). The combined organic phase was washed with brine (30mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with petroleum ether / ethyl acetate=1:1 to give a crude product (90 mg). The crude product was further purified by preparative HPLC (column: Phenomenex Gemini-NX C1875*30 mm*3 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 60%-90%, 7 min) to give N-[(2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-yl]-N',N'-dimethyl-oxamide (compound 22-1) (2.0 mg, purity: 100%) as a yellow solid.

[0239] 1 H NMR: CDCl3 400MHzδ=7.13(s,1H),7.04-6.93(m,2H),6.51(s,1H),6.35(d,J=7.6Hz,1H),3.37(s,3H),2.93(s,3H),2. 77(d,J=14.0Hz,1H),2.21(s,3H),2.01-1.65(m,10H),1.47-1.40(m,7H),1.30-0.99(m,9H),0.73(s,3H).

[0240] LCMS: Rt = 1.120 min in 1.5 min chromatography, 5-95AB, purity 100%, LCMS ESI+ for C 32 H 44 N2O4[M] + Calculated value 520.3, found value [M+H] + 521.1.

[0241] HPLC: Rt = 6.12 min in 8 min chromatography, 10-80 AB, purity 100%.

[0242] Step 2

[0243] Synthesis of N'-[(2R,4aS,6aR,6aS,14aS,14bR)-10-methoxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-yl]-N,N,N'-trimethyl-oxalamide (Compound 22):

[0244] To a solution of N-[(2R,4aS,6aR,6aS,14aS,14bR)-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-yl]-N',N'-dimethyl-oxamide (300 mg, 576 μmol) in THF (3 mL) was added NaH (69.13 mg, 1.73 mmol, 60% in mineral oil) at 20 ° C and stirred for 30 minutes. At 20 ° C, MeI (81.8 mg, 576 μmol) was added and the reaction mixture was stirred for 2 hours. The reaction mixture was poured into H2O (50 mL). The mixture was extracted with DCM (4×10 mL). The combined organic phases were washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (eluted with ethyl acetate / petroleum ether = 0% to 100%) to afford N'-[(2R, 4aS, 6aR, 6aS, 14aS, 14bR)-10-hydroxy-2,4a, 6a, 6a, 9, 14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydroquinone-2-yl]-N,N,N'-trimethyl-oxamide (Compound 22) (20 mg, 6% yield) as a yellow solid.

[0245] 1 H NMR: CDCl3 400MHzδ=7.02-6.95(m,2H),6.51(d,J=1.2Hz,1H),6.34(d,J=7.2Hz,1H),3.48-3.33(m,1H),2.93-2.89(m,6H),2.84(s,3H),2. 20(s,3H),2.15-1.65(m,11H),1.59-1.52(m,2H),1.46-1.41(m,6H),1.30(s,3H),1.16(s,3H),1.09-1.02(m,1H),0.86(s,3H).

[0246] LCMS: Rt = 1.119 min in 1.5 min chromatography, 5-95AB, purity 96.0%, LCMS ESI+ for C 33 H 46 N2O4[M] + Calculated value 534.3, found value [M+H] + +535.4.

[0247] HPLC: Rt = 2.103 min, 5-95 AB, purity 92.0% in 4 min chromatography.

[0248] Example 8: Synthesis of Compound 14-4

[0249]

[0250] Compound 14-4 can be prepared via a 6-step synthesis as summarized in Scheme 8 (below).

[0251] Solution 8

[0252]

[0253]

[0254] Example 9: Synthesis of Compound 54:

[0255]

[0256] Compound 54 can be prepared from compound 14-4 via the synthesis summarized in Scheme 9:

[0257] Solution 9

[0258]

[0259] Example 10: Synthesis of Compound 55:

[0260]

[0261] Compound 55 can be prepared from compound 14-4 via the synthesis summarized in Scheme 10:

[0262] Solution 10

[0263]

[0264] Example 11: Synthesis of Compound 56:

[0265]

[0266] Compound 56 can be prepared from compound 14-4 via the synthesis summarized in Scheme 11:

[0267] Solution 11

[0268]

[0269] Example 12: Synthesis of Compound 57:

[0270]

[0271] Compound 57 can be prepared from compound 14-4 via the synthesis summarized in Scheme 12:

[0272] Solution 12

[0273]

[0274] Example 13: Inhibitory effect of tripterygium wilfordii derivative compounds on NF-kB signal transduction

[0275] This example illustrates the study of the inhibitory effects of the following exemplary tripterygium wilfordii derivative compounds of the present disclosure on NF-kB signaling: Compound 14, Compound 15, Compound 16, Compound 17, and Compound 22.

[0276] Materials and methods

[0277] HepG2 NF-kB-Luc reporter cells were stimulated with IL-1β (20 ng / ml), and the relative NF-kB luciferase activity (RLU) of each tripterygium wilfordii (Compound 1) and each exemplary derivative compound was measured at the following eight point concentrations: 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM, 0.016 μM, and 0 μM. The measured values ​​were plotted (not shown), and the IC was calculated after normalization using Prizm 9.0 software 50 Inhibitory concentration.

[0278] result

[0279] Data from the NF-kB inhibition assay were analyzed and the IC values ​​of the resulting tripterygium wilfordii derivative compounds were calculated. 50 Values ​​and their %IC relative to triptolide 50 Values ​​(Compound 1; IC 50 =667 nM) are summarized in Table 4 (below).

[0280] Table 4

[0281]

[0282] Example 14: Anti-inflammatory Potential of Tripterygium Wilfordii Derivative Compounds

[0283] This example illustrates the study of the anti-inflammatory potential of an exemplary tripterygium wilfordii derivative of the present disclosure (Compound 22) using a NF-kB luciferase promoter reporter assay in the human liver cancer cell line HepG2.

[0284] Materials and methods

[0285] NF-kB Luciferase Reporter Assay: The tripterygium derivative compound 22 (prepared as described in Example 13) and the parent molecule compound 1 (tripterygium wilfordii) were freshly dissolved in DMSO. HepG2 NF-kB reporter cells (2.5×10 4 / well) in a 96-well plate, and then pretreated with compound 22 or compound 1 at 8 points of 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM, 0.016 μM or 0 μM, respectively, and NF-kB promoter was activated by adding IL-1β (20 ng / ml) to the cells. After 18 hours, cells were harvested by adding 1× passive cell lysis buffer (Promega), and then relative NF-kB luciferase activity (RLU) was measured using a GloMax discover microplate reader (Promega). The inhibitory concentration (IC) was calculated after normalization using Prizm 9.0 software. 50 ).

[0286] LPS-induced NF-kB target protein expression: Bone marrow derived dendritic cells (BMDCs) were pretreated with three concentrations (10 nM, 25 nM, 50 nM) of compound 22 or compound 1 for one hour and then treated with LPS (500 ng / mL) overnight. The next day, cells were subjected to Western blotting for iNOS, COX2, NLRP3 and IL-1β.

[0287] result

[0288] If through Figure 1A As shown in the results graph depicted in FIG, the tripterygium wilfordii derivative compound 22 has a more robust inhibitory potential than tripterygium wilfordii (compound 1) on the IL-1β-induced NF-kB inflammatory signaling pathway at low concentrations. Figure 1B As shown, compound 22 had an IC of 268 nM for NF-kB promoter activity as indicated by analysis of the normalized percentage response curve. 50 , which is higher than the IC 50 (IC 50=528nM) is 50% lower. In addition, Figure 1C As shown in the Western blot images shown in , the tripterygium wilfordii derivative compound 22 significantly inhibited the LPS-induced expression of NF-kB target proteins iNOS, COX2, NLRP3 and IL-1β in a dose-dependent manner.

[0289] Example 15: Anti-inflammatory Potential of Tripterygium Wilfordii Derivative Compounds

[0290] This example illustrates the investigation of the anti-inflammatory potential of an exemplary tripterygium wilfordii derivative of the present disclosure (Compound 22) using a specific NLRP3 inflammasome assay employing bone marrow-derived dendritic cells (BMDCs) activated with LPS+ATP or LPS+nigericin.

[0291] Materials and methods

[0292] NLRP3 inflammasome assay

[0293] 1. BMDCs were treated with LPS (100 ng / mL) for 3 hours (signal 1) to induce NLRP3 inflammasomes, and then treated with tripterine (compound 1) or tripterine derivative compound compound 22 at concentrations of 50 nM, 100 nM, and 500 nM. After 30 minutes, nigericin (10 μM) or ATP (5 mM) was added (signal 2), and the level of NLRP3-dependent inflammasome activity was determined by Western blotting of treated cells with ASC complex (>48 kDa). In addition, the level of secreted IL-1β in the conditioned medium represents active caspase-1, which is a downstream molecule of the NLRP3 inflammasome that cleaves pre-IL1β, resulting in the secretion of mature IL-1β.

[0294] 2. Under NLRP3 inflammasome activation induced by LPS+ATP or LPS+nigericin, the secreted form of IL-1β, a NLRP3 inflammasome-dependent cytokine, was measured by ELISA assay using conditioned medium from BMDCs treated with compound 1 (CLM-1) or compound 22 (CLM-022) at concentrations of 63nM, 125nM, 250nM, 500nM.

[0295] 3. Under NLRP3 inflammasome activation induced by LPS+ATP or LPS+nigericin, the secreted form of TNF-α (an NLRP3 inflammasome-independent cytokine) induced by LPS+ATP or LPS+nigericin was measured by ELISA assay using conditioned medium of BMDCs treated with compound 1 (CLM-1) or compound 22 (CLM-022) at concentrations of 63nM, 125nM, 250nM, and 500nM.

[0296] result

[0297] Compound 22 exhibited robust inhibitory activity against NLRP3 inflammasome activity, superior to that observed for the parent molecule, tripterygium wilfordii (compound 1). Figure 2A As shown in the results depicted in FIG, compound 22 inhibits the formation of the ASC complex more strongly than compound 1. Figure 2B As shown in the graph depicted in FIG, compound 22 more strongly inhibited IL-1β secretion induced by LPS+ATP and LPS+nigericin. Figure 2C As shown in , compound 22 (and compound 1) did not show significant inhibitory effects on TNF-α inflammatory cytokine secretion under the same NLRP3 inflammasome activation. The lack of inhibitory effects on TNF-α secretion (which is independent of NLRP3 inflammasome activity) indicates the specificity of compound 22 for NLRP3 inflammasomes. Therefore, compound 22 has the ability to inhibit NF-kB inflammatory response and also specifically inhibits IL-1β secretion induced by NLRP3 inflammasomes in BMDCs.

[0298] Example 16: Inhibitory effect of tripterygium wilfordii derivative compounds on NLRP3 inflammasome in human THP-1 cells

[0299] This example illustrates the investigation of the anti-inflammatory potential of an exemplary celastrol derivative of the present disclosure (Compound 22) using the NLRP3 inflammasome assay in THP-1, a human monocytic cell line activated with LPS + nigericin in the presence of Compound 22 or a known NLRP3 inhibitor compound, MCC950 (CAS 256373-96-3), as a benchmark molecule.

[0300] Materials and methods

[0301] Formation of the ASC complex in THP-1 cells

[0302] As previously shown (Figure 2), human THP-1 cells were treated with LPS + nigericin, with increasing doses of compound 1, compound 22 or MCC950 added as indicated. To demonstrate relative NLRP3 inflammasome activity, the levels of ASC complex formation were compared by Western blotting.

[0303] Detection of ASC complex and caspase-1 activity in THP-1 cells by Amnis

[0304] Amnis imaging flow cytometry (Luminex Co.) is a high-throughput, single-cell, fluorescence-based image analysis method that can detect inflammasomes by acquiring cellular distribution images of ASC complexes and active caspase-1. THP-1 cells were treated with LPS+nigericin in the presence of inhibitors. The treated cells were subjected to ASC staining and active caspase-1 was visualized using FLICA, a caspase-1 substrate that is fluorescent when cleaved. Finally, ASC+ / FLICA+ cells were counted as THP-1 cells with active NLRP3 inflammasome complexes.

[0305] result

[0306] THP-1 human monocytes form strong ASC complexes in response to LPS+nigericin treatment. Figure 3A As shown by Western blot, the presence of 50 nM and 100 nM concentrations of compound 22 strongly inhibited the formation of this ASC complex, with an inhibitory ability stronger than that of tripterygium wilfordii (compound 1) and the benchmark molecule MCC950. Figure 3B The strong inhibitory effect observed with compound 22 was further verified by counting THP-1 cells carrying ASC+FLICA+ spots, as shown by the Amnis imaging results of Figure 3C As shown in the figure, compound 22 was observed to exert strong inhibitory function at a concentration as low as 25 nM, while the benchmark molecule MCC950 showed a 5- to 10-fold reduction in inhibitory potency at a concentration of 100 nM.

[0307] Example 17: Inflammation inhibition effect of tripterygium wilfordii derivative compounds in LPS-induced septic shock mouse model

[0308] This example illustrates the study of the inhibitory function of the exemplary tripterygium wilfordii derivative (Compound 22) of the present disclosure on LPS-induced acute inflammation in vivo. We established an LPS-induced septic shock mouse model (Yu et al., 2017) and detected the expression levels of inflammatory cytokines in the colon.

[0309] Materials and methods

[0310] Mice (n=3 per group) were administered control vehicle, compound 1 (1 mg / kg) or compound 22 (1 mg / kg) by intraperitoneal (ip). After 1 hour, mice were administered sublethal doses of LPS (25 mg / kg) by ip injection. Four hours later, mice were subjected to blood collection and colon mucosa was harvested by scraping colon. Cytokine expression was measured by ELISA (Catalog No. 900-T54, PeproTech) of TNF-α to serum samples and qPCR of cytokines IL-1 (TaqMan, Mm00434228), IL-6 (TaqMan, Mm00446190), TNF-α (TaqMan, 00443258), iNOS (TaqMan, 00440502) to colon mucosal samples.

[0311] result

[0312] If through Figure 4A and Figure 4B As shown by the results depicted in the figure, the tripterygium wilfordii derivative compound Compound 22 successfully inhibited the LPS-induced acute inflammatory response in the mouse septic shock model, which effectively suppressed the expression of inflammatory mediators and inflammatory cytokines (including IL-1, IL-6, TNF-α and iNOS) in serum and colonic mucosa to levels comparable to or lower than those observed in the group administered tripterygium wilfordii (Compound 1).

[0313] Example 18： Effect of CLM-022 and MCC950 on the proliferation of mouse and human macrophages Compare NLRP3 inflammasome inhibitory activity

[0314] This example illustrates Effects of CLM-022 and MCC950 on inhibiting NLRP3 inflammasome activity in mouse and human macrophages Comparative Study .

[0315] Materials and methods

[0316] A. BMDM cell research

[0317] Bone marrow-derived macrophages (BMDM) were treated with LPS (100 ng / mL) for 3 hours to induce the expression of inflammatory genes, including TNF-α, NLRP3, and Pro-IL-1β. Subsequently, BMDM were treated with CLM-022 or MCC950 at a dose range of 0 μM-10 μM for 30 minutes. Then, nigericin (10 μM) was added to BMDM for another 30 minutes to fully activate NLRP3-dependent inflammasome activity and cytokine secretion.

[0318] B.THP-1 cell studies

[0319] To confirm the inhibitory effect of CLM-022 in human macrophages, human leukemia monocytic cell line THP-1 cells were differentiated into macrophages using phorbol 12-myristate-13-acetate (PMA, 100 ng / ml) for one day. THP-1 macrophages were then treated with LPS (100 ng / mL) for 3 hours to induce the expression of inflammatory genes. Subsequently, THP-1 cells were treated with CLM-022 or MCC950 at a dose range of 0 μM-10 μM for 30 minutes. Then, in order to fully activate NLRP3-dependent inflammasome activity, nigericin (10 μM) was added to THP-1 cells for another 60 min.

[0320] result

[0321] If through Figure 5A The results depicted in , CLM-022-treated BMDMs showed significant inhibition of IL-1β secretion in a dose-dependent manner. Normalized IC 50 Calculations showed that CLM-022 exhibited greater potential in inhibiting IL-1β compared with MCC950 (IC 50 : CLM-022 = 9 nM vs. MCC950 = 70 nM). However, Figure 5B As shown in , neither CLM-022 nor MCC950 inhibits TNF-α secretion because TNF-α is not regulated by the NLRP3 inflammasome. Figure 5C and Figure 5D As shown by the results depicted in Figure 2, similar to those obtained with BMDM, CLM-022 robustly inhibited IL-1β secretion, but not TNF-α, in human THP-1 macrophages. CLM-022 exhibited a more potent inhibitory potential than MCC950: (IC 50: CLM-022 = 158.9 nM vs. MCC950 = 1248 nM). These results clearly indicate that CLM-022 has superior inflammasome inhibitor potential compared to the state-of-the-art NLRP3 inflammasome inhibitor MCC950.

[0322] Example 19： DARTS assay for protection of NLRP3 by CLM-022

[0323] The NLRP3 inflammasome contains a multiprotein complex, and the exact molecular mode of NLRP3 activation remains unclear. Currently, there are no NLRP3 inflammasome inhibitors in the clinic, and it is important to identify the mechanism of action and molecular targets of potential small molecules that interact with NLRP3. This example illustrates a study of the ability of CLM-022 to protect NLRP3 molecules, as determined using the drug affinity responsive target stability (DARTS) assay. The DARTS assay is a robust method for identifying small molecule target proteins. In the case of drug-target interactions, small molecules protect target proteins from being degraded by proteases (pronase), while free proteins can be degraded very effectively under given conditions.

[0324] Materials and methods

[0325] DARTS assay was performed using THP-1 lysates pretreated with LPS and nigericin to induce activation of NLRP3 inflammasome. THP-1 cells (1 × 10 5 / mL) were treated with LPS (100 ng / mL) for 3 hours, followed by 10 μM nigericin for another 30 minutes. Pronase (50-500 ng / μg protein) was added to the lysate together with CLM-022 (10 μM) or MCC950 (10 μM) for the indicated time (10 min-15 min) at room temperature. The protein components of the NLRP3 inflammasome were identified using the NLRP3-Natch domain ( Fig. 6A ), NLRP-PYD domain ( Figure 6B ) or NEK7 protein ( Figure 6C After densitometry scanning, relative band intensities were normalized by GAPDH.

[0326] result

[0327] If through Fig. 6A , Figure 6B and Figure 6C As shown in the results depicted inCLM-022 significantly protected NLRP3 protein from degradation, which was significantly more effective than MCC950. However, CLM-022 failed to protect NEK7 from pronase-induced degradation. This result emphasizes that CLM-022 will directly interact with NLRP3 protein in a target-specific manner.

[0328] Embodiment 20: Protection of THP-1 cells against inflammasome-induced pyroptosis

[0329] Pyroptosis, a proinflammatory form of cell death, is triggered by the activation of NLRP3 inflammasome components (GSDMD and caspase-1), leading to the release of proinflammatory cytokines IL-1β and IL-18, as well as damage-associated molecular patterns (DAMPs). This process triggers a cascade of inflammatory responses that may persist if dysregulated in affected tissues. Given the significant inhibitory potential of CLM-022 on NLRP3 inflammasome and IL-1β secretion, it is proposed that CLM-022 can effectively enhance cell viability by inhibiting pyroptosis in the context of inflammation and inflammasome activation. This example illustrates a comparative study of the ability of CLM-022 and MCC950 to protect THP-1 cells from inflammasome-induced pyroptosis.

[0330] Materials and methods

[0331] After LPS and nigericin treatment, THP-1 cells were treated with CLM-022 or MCC950 in a dose range of 0 μM to 10 μM, and cell viability was determined by non-permeable live / dead staining dye and flow cytometric analysis. In addition, NLRP3 inflammasome-induced cytotoxicity was studied by measuring the level of LDH in the conditioned medium after CLM-022 and MCC950 treatment. In addition, Western blotting of samples from THP-1 cell viability assays was performed to determine the levels of pyroptosis-mediating molecules GSDMD and caspase-1.

[0332] result

[0333] If through Fig. 7A As shown in the results depicted in Figure 2, when THP-1 cells were treated with CLM-022 in a dose range of 0 μM to 10 μM, cell viability increased by up to 60%. Similar effects were observed in cells treated with MCC950 ( Fig. 7A ).

[0334] If through Figure 7BAs shown by the results depicted in , consistent with the cell viability assay results, both CLM-022 and MCC950 significantly inhibited the release of LDH. Notably, CLM-022 showed superior inhibitory potential to MCC950 even at lower concentrations ( Figure 7B , left). The normalized IC calculated from these LDH release results 50 The values ​​clearly show that CLM-022 exhibits 10-fold higher potency (IC 50 : CLM-022 = 12.4 nM relative to MCC950 = 123.5 nM).

[0335] If through Figure 7C As shown in the Western blot images depicted in , CLM-022 inhibited GSDMD and caspase-1 cleavage at concentrations as low as 100 nM, while MCC950 required 1000 nM for inhibition.

[0336] Overall, these comparative analyses clearly demonstrate that CLM-022 has a greater ability than the benchmark molecule MCC950 to inhibit inflammasome-induced pyroptosis in human THP-1 macrophages.

[0337] Although for the purpose of clarity and understanding, the foregoing disclosure of the present disclosure has been described in some detail by way of example and illustration, the present disclosure including the examples, descriptions and embodiments described herein is for illustrative purposes, is intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to those skilled in the art that various modifications or changes may be made to the examples, descriptions and embodiments described herein, and these modifications or changes will be included in the spirit and authority of the present disclosure and the appended claims. In addition, those skilled in the art will recognize many methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of the present disclosure and covered by the appended claims.

[0338] Further embodiments of the present disclosure are set out in the dependent claims.

[0339] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes, to the same extent as if each such individual publication, patent, patent application, or other document was individually specifically indicated to be incorporated by reference in its entirety for all purposes and was set forth in its entirety herein. In the event of a conflict, the present specification, including the designated term, will control.

Claims

1. A compound of structural formula (I) in, R1 is selected from: or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug thereof.

2. The compound according to claim 1, wherein R1 is selected from:

3. A compound of structural formula (I) in, R1 is -NH-(CO)2-NR2R3 or -N(CH3)-(CO)2-NR2R3; R2 and R3 are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine or heteroaryl, optionally substituted with substituents individually selected from alkyl, alkoxy, cycloalkyl, ether, amine optionally substituted with one or more alkyl, halogen, hydroxy, ether, cyano, nitrile, CF3, ester, amide, cycloalkylamide, sugar, heteroarylamide optionally substituted with alkyl and / or alkoxy, urea, carbamate, thioether, sulfate, sulfonyl, sulfonic acid, carboxylic acid and aryl; or R2 and R3 are taken together to form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, optionally substituted by substituents individually selected from the group consisting of alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, heteroaryl, amine, halogen, hydroxy, ether, nitrile, cyano, nitro, CF3, ester, amide, urea, carbamate, thioether or carboxylic acid groups; or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug thereof.

4. The compound according to claim 3, wherein R1 is selected from:

5. The compound according to any one of claims 1 to 4, wherein the compound is selected from compound 14, compound 15, compound 16, compound 17, compound 22 and compound 22-1 or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug thereof.

6. The compound according to any one of claims 1 to 4, wherein the compound is selected from compound 42, compound 54, compound 55, compound 56, compound 57, compound 58, compound 59, compound 60, compound 61, compound 62, compound 63, compound 64, compound 65, compound 66, compound 67, compound 68, compound 69, compound 70, compound 71, compound 72, compound 73, compound 74, compound 75, compound 76, compound 77, compound 78, compound 79, compound 80, compound 81, compound 82, compound 83, compound 84, compound 85, compound 86, compound 87, compound 88, compound 89, compound 90, compound 91 and compound 92 or a pharmaceutically acceptable salt, a single stereoisomer, a mixture of stereoisomers, a tautomer or a prodrug thereof.

7. The compound of any one of claims 1 to 6, wherein the compound is characterized by having an IC in a NF-kB reporter gene assay of about 1200 nM or less, about 1000 nM or less, 750 nM or less, 500 nM or less, or 300 nM or less. 50 .

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. A method of treating a tripterine-responsive disease or disorder in a subject suffering from the same, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8.

10. The method of claim 9, wherein the celastrol responsive disease or disorder is cancer; optionally, wherein the cancer is selected from the group consisting of gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, hepatocellular carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, brain cancer, and glioblastoma multiforme (GBM).

11. The method of claim 9, wherein the celastrol responsive disease or disorder is an inflammatory and / or autoimmune disorder; optionally, wherein the inflammatory and / or autoimmune disorder is selected from rheumatoid arthritis (RA), multiple sclerosis (MS), ankylosing spondylitis, systemic lupus erythematosus (SLE), inflammatory bowel disease, osteoarthritis (OA), acute respiratory distress syndrome (ARDS), Guillain-Barré syndrome (GBS), sickle cell disease (SCD), allergies (e.g., asthma), psoriasis and other inflammatory skin conditions.

12. The method of claim 9, wherein the tripterygium wilfordii responsive disease or disorder is a neurological disorder; optionally, wherein the neurological disorder is selected from Parkinson's disease, Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis (ALS) and Gaucher disease (GD).

13. The method of claim 9, wherein the tripterygium wilfordii responsive disease or disorder is an obesity-related disease or disorder; optionally, wherein the obesity-related disease or disorder is selected from obesity, preobesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, obesity associated with hypothalamic damage, nonalcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bard-Biedel syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macrosomia) syndrome.

14. The method of claim 9, wherein the celastrol responsive disease or disorder is a liver-related disease or disorder; optionally, wherein the liver-related disease or disorder is selected from acute-chronic liver failure (ACLF), alcoholic liver disease, cholestatic liver disease, drug-induced liver disease, hepatocellular carcinoma, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), viral hepatitis, and viral liver disease.

15. The method of claim 9, wherein the tripterygium wilfordii responsive disease or disorder is brain injury or brain disorder; optionally, wherein the brain-related injury or disorder is selected from middle cerebral artery occlusion (MCAO)-induced brain injury, cerebral ischemia / reperfusion (I / R) injury, vascular dementia (VD), and acute ischemic stroke-induced brain injury.

16. The method of claim 9, wherein the celastrol-responsive disease or disorder is an ocular disorder or injury; optionally, wherein the ocular-related injury or disorder is selected from dry eye disease, ocular inflammation, age-related macular degeneration (AMD), subconjunctival fibrosis, high intraocular pressure-induced retinal degeneration, bright light-induced degeneration, macrophage-induced corneal neovascularization, corneal allograft survival, and optic nerve extrusion.

17. The method of any one of claims 9 to 14, wherein the compound or pharmaceutical composition is administered in combination with another therapy.

18. The method of any one of claims 9 to 15, wherein administration comprises oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration, or subcutaneous administration.

19. The method according to any one of claims 9 to 16, wherein the compound or pharmaceutical composition is administered in a form selected from the group consisting of pills, capsules, tablets, granules, powders, salts, crystals, liquids, serums, syrups, suspensions, gels, creams, pastes, films, patches and vapors.

20. The method of any one of claims 9 to 17, wherein the subject is a mammal.

21. The method of any one of claims 9 to 18, wherein the subject is a human.

22. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for treating a tripterine-responsive disease or disorder in a subject.

23. A compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 for use in therapy.

24. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 as a medicament.

25. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 in the manufacture of a medicament for treating a tripterine-responsive disease or disorder in a subject.

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