Celastrol derivative

Abstract

The present invention relates to a compound which is a derivative of a compound, cerastrol, and a pharmaceutical composition containing these cerastrol derivative compounds. The present invention also relates to the use of the cerastrol derivative compounds described herein, which includes use in the treatment of cerastrol-responsive diseases or disorders such as cancer, inflammatory and / or autoimmune disorders, neuropathies, brain injuries and disorders, obesity-related diseases and disorders, and / or liver-related diseases and disorders.

Description

Technical Field

[0001] Cross - Reference to Related Applications (Cross - reference to related applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 389,593, filed on July 15, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to compounds that are derivatives of cerastrol, pharmaceutical compositions containing these compounds, and their use.

Background Art

[0003] Celastrol, a natural compound derived from the plant species Tripterygium wilfordii Hook F., also known as triptelin, has been found to exhibit significant antitumor activity in preclinical studies, including research for the treatment of liver cancer, breast cancer, prostate cancer, lung cancer, leukemia, and melanoma (see, for example, Kashyap et al., 2018; Yadav et al., 2018), and has been shown to have a radiation-sensitizing effect (see, for example, Dai et al., 2011; Lee et al., 2011). Results from various tumor cell lines and animal cancer models suggest that celastrol can kill tumors through various 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, for example, Cascao et al., 2017; Kashyap et al., 2018). Celastrol is thought to act by targeting multiple signaling pathways, including but not limited to reactive oxygen species (ROS) / JNK and Akt / mTOR (see, for example, Liu et al., 2019), NF-κb (see, for example, Chiang et al., 2014), STAT3 / JAK2 (see, for example, Rajendran et al., 2012), HSP90 (see, for example, Sreeramulu et al., 2009; Zhang et al., 2009), Cdc37, p23, Iκb, p-Akt (see, for example, Kannaiyan et al., 2011), ERα (see, for example, Jang et al., 2011).

[0004] Celastrol has been shown to act as an inflammasome inhibitor (e.g., Lee et al., 2019; Yu et al., 2017) and has been proposed for the treatment of inflammatory diseases, autoimmune diseases, and many of the following, but not limited to, chronic diseases: 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) (see, 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 (e.g., Vankatesha and Moudgil, 2021; Song et al., 2023).

[0005] Celastrol has also been shown to exhibit neuroprotective activity (see, e.g., Cascao et al., 2017; Cleren et al., 2005; Paris et al., 2010) and has been proposed for the treatment of various neuropathies, including but not limited to Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Gaucher disease (GD) (see, e.g., Vankatesha and Moudgil, 2021).

[0006] Cerastrol and cerastrol analogs have also been proposed for the treatment of multiple metabolic and atherosclerotic diseases, liver diseases, and heart 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, and fatty liver.

[0007] Cerastrol has also been shown to be useful for the treatment and prevention of multiple brain disorders and injuries due to its anti-inflammatory activity. These include, but are not limited to, middle cerebral artery occlusion (MCAO)-induced brain injury, cerebral ischemia / reperfusion (I / R) injury, vascular dementia (VD), and acute ischemic stroke-induced brain injury (see, e.g., Jiang et al., 2018).

[0008] In recent years, an increasing number of uses of cerastrol have been explored for the treatment of eye disorders, including but not limited to dry eye disease (see, e.g., Siu et al., 2022), eye inflammation, age-related macular degeneration (AMD) (see, e.g., Zhang et al., 2019), and subconjunctival fibrosis (see, e.g., Li et al., 2023). Cerastrol has also been shown to protect against the development of high intraocular pressure-induced degeneration of retinal ganglion cells (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 to support corneal allograft survival (see, e.g., Li et al., 2016) and recovery from damage caused by optic nerve crush (see, e.g., Kyung et al., 2015).

[0009] Celastrol interacts with many cellular targets and exhibits potent activity useful for many potential therapeutic indications, but the compound shows insufficient ADME properties, including water instability, low bioavailability, a narrow therapeutic window, and undesirable side effects, which limit its further clinical development (see, for example, Cascao et al., 2017; Hou et al., 2020). SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] There is a need for celastrol derivatives or analogous compounds that exhibit one or more improved ADME properties while retaining some or all of the functional activity of celastrol related to its therapeutic effect. MEANS FOR SOLVING THE PROBLEMS

[0011] The present invention generally relates to celastrol derivative compounds, compositions, formulations, and their use in medicaments and treatment methods. The summary of the present invention is intended to introduce the subject matter of the present invention, but does not cover all embodiments, combinations, or variations contemplated and described within the present invention. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0012] In at least one embodiment, the present invention is a compound of structural formula (I): [Chemical formula] Wherein R1 is selected from the following: [Chemical formula] [Chemical formula] [Chemical formula] [Chemical formula] and / or their pharmaceutically acceptable salts, single stereoisomers, mixtures of stereoisomers, tautomers, or prodrugs.

[0013] In at least one embodiment, the present invention provides a compound of structural formula (I)

Chemical formula

[0014] In at least one embodiment of the compound of structural formula (I) of the present invention, 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, Compound 92.

[0015] In at least one embodiment of any of the compounds of structural formula (I) of the present invention, the compound has an IC in the NF-kB reporter assay that is about 1200 nM or less, about 1000 nM or less, 750 nM or less, 500 nM or less, or 300 nM or less. 50 Characterized by having.

[0016] In at least one embodiment, the present invention provides a pharmaceutical composition comprising a compound of structural formula (I) of the present invention and a pharmaceutically acceptable excipient.

[0017] In at least one embodiment, the present invention provides a method of treating a cholesterol-responsive disease or disorder in a subject suffering from the cholesterol-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 invention, or the pharmaceutical composition of the present invention.

[0018] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is cancer; optionally, 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).

[0019] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is an inflammatory and / or autoimmune disorder; optionally, 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.

[0020] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is a neuropathy; optionally, the neuropathy is selected from Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Gaucher's disease (GD).

[0021] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is an obesity-related disease or disorder; optionally, the obesity-related disease or disorder is selected from obesity, pre-obesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, hypothalamic injury-related obesity, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bardet-Biedl syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macroscopic obesity with ocular anomalies) syndrome.

[0022] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is a liver-related disease or disorder; optionally, 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.

[0023] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is a brain injury or disorder; optionally, 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.

[0024] In at least one embodiment of the present invention, the cerastrol-responsive disease or disorder is an eye disorder or injury; optionally, the eye-related injury or disorder is selected from dry eye disease, eye inflammation, age-related macular degeneration (AMD), subconjunctival fibrosis, retinal high intraocular pressure-induced degeneration, bright light-induced degeneration, macrophage-induced corneal neovascularization, corneal allograft survival, and optic nerve crush.

[0025] In at least one embodiment of the method of the present invention, the compound or pharmaceutical composition is administered in combination with another treatment. In at least one embodiment, the administration includes 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, intra-articular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration, or subcutaneous administration.

[0026] In at least one embodiment of the method of the present invention, the pharmaceutical composition is administered in a form selected from the group including pills, capsules, tablets, granules, powders, salts, crystals, liquid crystals, serums, syrups, suspensions, gels, creams, pastes, films, patches, and vapors.

[0027] In at least one embodiment of the method of the present invention, the subject is a mammal. In at least one embodiment, the subject is a human.

[0028] In at least one embodiment, the present invention provides the use of a compound of structural formula (I) of the present invention, or a pharmaceutical composition of the present invention, as a medicament for treating a cholesterol-responsive disease or disorder in a subject, for treating a cholesterol-responsive disease or disorder in a subject, or for use in the manufacture of a medicament for treating a cholesterol-responsive disease or disorder in a subject.

Brief Description of the Drawings

[0029] A better understanding of the novel features and advantages of the present invention can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and the accompanying drawings (also refer to "Figure: FIG." and "FIG.: FIG." in this specification):

[0030]

Figure 1-1

Figure 1-2

[0031]

Figure 2-1

Figure 2-2

[0032]

Figure 3

[0033]

Figure 4

[0034]

Figure 5-1

Figure 5-2

[0035]

Figure 6-1

Figure 6-2

Figure 6-3

[0036]

Figure 7-1

Figure 7-2

Figure 7-3

Mode for Carrying Out the Invention

[0037] It should be understood that the detailed description provided herein, including the drawings, is merely illustrative and explanatory and does not limit the present invention. The description is not limited to the specific compounds, compositions, methods, techniques, protocols, cell lines, assays, and reagents disclosed herein, which may vary, but is also intended to encompass known variants of these specific embodiments.

[0038] Also, it should be understood that the terms used in this specification are intended to describe particular embodiments and are not intended to limit the scope set forth in the appended claims. Regarding the descriptions in this specification and the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" includes two or more proteins, and a reference to "a compound" refers to two or more compounds. It should further be noted that the claims are written to exclude any optional elements. Thus, this description is intended to serve as a basis for using exclusive terminology such as "solely", "only", etc. or for using "negative" limitations in connection with the recitation of claim elements. The use of "comprise" and "comprises" is interchangeable and not intended to be limiting. The use of "comprise", "comprises", "comprising", "include", "includes" and "including" is interchangeable and is not used in an eliminative or limiting sense. When descriptions of various embodiments use the term "comprising", those skilled in the art will recognize that in some specific instances the embodiments may alternatively be described using the terms "consisting essentially of" or "consisting of".

[0039] When a range of values is provided, unless the context clearly indicates otherwise, each intervening integer value, and each tenth of an intervening integer value, is within the range between the upper and lower limits of that range, and any other stated or intervening value within the stated range, is understood to be included within the present invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included within the smaller range and are also included within the present invention, subject to any specifically excluded limits within the stated range. When the stated range includes one or both of these limits, ranges excluding (i) either one or (ii) both of those included limits are also included within the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0040] All publications, patents, patent applications, and other documents referenced in the present invention 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 were individually indicated to be incorporated by reference for all purposes.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. For the purpose of interpreting the present invention, the following explanations of terms apply, and where appropriate, terms used in the singular form also include the plural form and vice versa.

[0042] I. Definitions

[0043] "Celastrol" refers to a compound having the chemical structure of compound (1): [Chemical formula]

[0044] "Cereal-responsive disease or disorder" refers to any disease or disorder for which treatment with the compound, cereal, has been proposed or shown to provide a potential therapeutic effect based on clinical trials, preclinical trials, or in vitro tests. Exemplary cereal-responsive diseases or disorders of the present invention include, but are not limited to, the following: 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 diseases (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), neuropathies (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, pre-obesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, hypothalamic injury-related obesity, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bardet-Biedl syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome and MOMO (macrophthalmia with marked obesity) syndrome, brain injury 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 eye diseases (e.g., dry eye disease, eye inflammation, age-related macular degeneration (AMD), subconjunctival fibrosis, retinal hypertension-induced degeneration, photic light-induced degeneration, macrophage-induced corneal neovascularization, corneal allograft survival, and optic nerve contusion), liver-related diseases or disorders (e.g., 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).

[0045] The terms "any" or "optionally" (optionally) mean that the event or situation described thereafter may or may not occur, and that the description includes both the case where the event or situation occurs and the case where it does not occur.

[0046] The term "substituted" includes embodiments in which a monoradical substituent is attached to a single atom of the substituent (e.g., forming a branch), and may also be a diradical bridging group in which the substituent is attached to two adjacent atoms of the substituent, thereby forming a fused ring on the substituent, including such embodiments.

[0047] As described herein, when a given group (or "moiety") is attached to a second group and the attachment site is not explicit, the given group may be attached to any available site of the second group at any available site of the given group. For example, "lower alkyl substituted phenyl" with an unspecified attachment site may have any available site of a lower alkyl group attached to any available site of the phenyl group. In this regard, an "available site" is a site of a group where a hydrogen of the group can be replaced by a substituent.

[0048] It is understood that polymers reached by defining substituents having further substituents on themselves (e.g., a substituted aryl having a substituted aryl group as a substituent where the substituent itself is a substituted aryl group) among all the substituents defined above are not intended to be included herein. Also, numerous substituents are not included, regardless of whether the substituents are the same or different. In such cases, the maximum number of such substituents is 3. Thus, each of the above definitions is restricted, for example, by the limitation that a substituted aryl group is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0049] A compound of a given formula (e.g., "a compound of formula (I)") is intended to include the compounds of the present invention, as well as pharmaceutically acceptable salts, pharmaceutically acceptable esters, hydrates, polymorphs, and prodrugs of such compounds.

[0050] Furthermore, the compounds of the present invention may have one or more asymmetric centers and may be produced as racemic mixtures or as individual enantiomers or diastereoisomers. The number of stereoisomers present in any given compound of a given formula depends on the number of asymmetric centers present (when n is the number of asymmetric centers, 2n stereoisomers are possible). The individual stereoisomers can be obtained by resolution of the racemic or non-racemic mixtures of the intermediates at some suitable stage of the synthesis or by resolution of the compounds by conventional means. The individual stereoisomers (including individual enantiomers and diastereoisomers) as well as racemic and non-racemic mixtures of stereoisomers are included within the scope of the present invention and all of them are intended to be represented by the structures herein unless otherwise specified.

[0051] The term "isomer" means different compounds having the same molecular formula. Isomers include stereoisomers, enantiomers, and diastereomers. The term "stereoisomer" means isomers that differ only in the manner in which the atoms are arranged in space. The term "enantiomer" means a pair of stereoisomers that are mirror images of each other and cannot be superimposed on each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. The term "(±)" is used, where appropriate, to indicate a racemic mixture. The term "diastereoisomer" means stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. The absolute stereochemistry is specified herein according to the Cahn Ingold Prelog R S system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds of unknown absolute configuration are named (+) or (-) according to the direction (dextrorotatory or levorotatory) in which they rotate the plane of polarization at the wavelength of the sodium D line.

[0052] The specific compounds of the present invention contain an asymmetric atom (optical or chiral center) or a double bond. The present invention encompasses compounds described by chemical structures and formulas that do not explicitly show various stereoisomeric or diastereomeric forms, including racemic or optically pure forms of the compounds, and are intended to be included within the scope of the compounds described herein. The optically active (R)- 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, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0053] "Tautomers" or "tautomeric isomers" are isomers that are in equilibrium with each other. For example, amide-containing compounds can exist in equilibrium with their imidic acid tautomers. Specific compounds of the present invention exist as "tautomeric isomers: tautomers" or "tautomers: tautomers". Regardless of which tautomer is shown and regardless of the nature of the equilibrium between tautomers, it is understood by those skilled in the art that the compounds include both amide and imidic acid tautomers. Thus, amide-containing compounds are understood to include their imidic acid tautomers.

[0054] The term "polymorph" refers to different crystal structures of a crystalline compound. Different polymorphs can result from differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism). Specific compounds of the present invention can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0055] The term "solvate" refers to a complex formed by combining a compound with a solvent. The term "hydrate" refers to a complex formed by combining a compound with water. Certain compounds of the present invention can exist in non-solvated forms, solvated forms including hydrate forms. Generally, solvated forms are equivalent to non-solvated forms and are intended to be encompassed within the scope of the compounds described herein even when those compounds are described by chemical structures and formulas that do not explicitly show the solvated forms.

[0056] The term "salt", as used herein, refers to an ionic compound resulting from a neutralization reaction of an acid and a base. Salts are composed of a related 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 ions such as chloride (Cl - ), or organic ions such as acetate (C2H3O2 - ), and can be monoatomic ions such as fluoride (F - ), or polyatomic ions such as sulfate (SO4 2- ).

[0057] 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 is not biologically or otherwise undesirable. Often, the compounds of the present invention can form pharmaceutically acceptable acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto.

[0058] Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Examples of salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Examples of 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, dialkenylamines, trialkenylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, di-substituted cycloalkylamines, tri-substituted cycloalkylamines, cycloalkenylamines, di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, di-substituted cycloalkenylamines, tri-substituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, and mixed di- and triamines in which at least two of the 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 in which two or three substituents together with the amino nitrogen form a heterocyclic or heteroaryl group. Specific examples of suitable amines include, but are not limited to, 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-alkylglucamine, theobromine, purine, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0059] Pharmaceutically acceptable acid addition salts can also be prepared from inorganic and organic acids. Examples of salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Examples of salts derived from organic acids include 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, and the like.

[0060] 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. Their use in therapeutic compositions is contemplated, except where any conventional media or agent is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

[0061] Any formula or structure shown herein that includes formula (I) is also intended to represent compounds in both unlabeled and isotopically labeled forms. Isotopically labeled compounds have the same structure as shown by the 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 invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 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, but are not limited thereto. The various isotopically labeled compounds of the present invention are, for example, 3 H, 13C, and 14 Those incorporating radioisotopes such as C are included. Such isotope-labeled compounds can be useful in metabolic studies, reaction kinetics 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 the radioactive treatment of patients.

[0062] The deuterium-labeled or substituted therapeutic compounds of the present invention can have improved DMPK (drug metabolism and pharmacokinetics) properties related to distribution, metabolism, and excretion (ADME). Substitution with a heavier isotope such as deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or decreased dosage requirements. 18 F-labeled compounds can be useful in PET or SPECT studies. The isotope-labeled compounds and their prodrugs of the present invention can generally be prepared by implementing the schemes or procedures disclosed in the examples and preparations described below by replacing readily available isotope-labeled reagents with non-isotope-labeled reagents. Furthermore, substitution with a heavier isotope, particularly deuterium (i.e., 2 H or D) can provide certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or decreased dosage requirements or improvement of the therapeutic index. It is understood that deuterium in this context is considered a substituent in the compounds of formula (I).

[0063] The concentration of such heavier isotopes, specifically deuterium, can be defined by the isotope enrichment factor. In the compounds of the present invention, any atom not specifically designated as a particular isotope represents any stable isotope of that atom. Unless otherwise specified, when a position is specifically designated as "H" or "hydrogen", that position is understood to have hydrogen with its natural abundance isotope composition. Thus, in the compounds of the present invention, any atom specifically designated as deuterium (D) means deuterium.

[0064] In the description including the examples, all temperatures are in degrees Celsius (°C) unless otherwise specified, and the abbreviations and acronyms have the following meanings listed in Table 1 (below).

[0065] Table 1

Table 1-1

Table 1-2

[0066] II. Celastrol Derivative Compounds

[0067] The present invention provides a range of compounds having a chemical structure that is a derivative of the structure of celastrol [compound (1)]. Generally, the celastrol derivative compounds of the present invention retain the 5-cyclic triptylene core structure of compound (1) and substitute the carboxylic acid group with various substituted amine and amide groups as described in more detail below. The celastrol derivative compounds of the present invention retain or improve the functional characteristics of celastrol, including NF-kB cell inhibition and ADME properties, thereby providing alternative molecules for use in the treatment of celastrol-responsive diseases and disorders. Thus, in at least one embodiment, the celastrol derivative compounds of the present invention have the structural formula (I),

Chemical formula

[0068] Table 2: Exemplary Celastrol Derivatives of the R1 Group of Formula (I)

Chemical formula

Chemical formula

[0069] The present invention also contemplates that the cerastrol derivative compound of structural formula (I) may include compounds of a selected subgenus. For example, in at least one embodiment, the present invention provides a cerastrol derivative compound of structural formula (I), wherein the group R1 can be -NH-(CO)2-NR2R3 or -N(CH3)-(CO)2-NR2R3. In at least one embodiment of this subgenus of compounds, the groups R2 and R3 of the structural formula are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine, or heteroaryl group; wherein each of the alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine, or heteroaryl group is optionally substituted with alkyl, alkoxy, cycloalkyl, ether, amine optionally substituted with one or more alkyls, 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, independently selected substituents, and is optionally substituted. In another embodiment of this subgenus, R2 and R3 together form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, Here, each of the cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups is optionally substituted with a substituent independently selected from 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.

[0070] Exemplary cerastrol derivative compounds of the present invention are provided in Table 3 below. The synthesis, characterization, and use of these exemplary compounds are described in the Examples and elsewhere in this specification.

[0071] Table 3: Exemplary Cerastrol Derivative Compounds Compound Number Structure Molecular Weight (m / e)

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0072] As described elsewhere in this specification, those skilled in the art will understand that the celastrol derivative compounds provided herein may exist in various known closely related and / or equivalent forms that are not explicitly described by the chemical structure and formula. The celastrol derivative compounds of structural formula (I) of the present invention include, but are 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, and it is intended to include these closely related forms of the compounds defined by the chemical structure and formula.

[0073] III. Preparation of Celastrol Derivative Compounds

[0074] The celastrol 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 invention provides a general synthetic strategy for preparing the compounds of structural formula (I). The following Schemes A - F provide a series of six reactions starting from celastrol (Compound 1), which can be used, adapted, and / or combined by those skilled in the art with well-known synthetic methods to synthesize the celastrol derivative compounds of the present invention, for example, the compounds in Table 3 (above).

Chemical formula

Chemical formula

[0075] In addition to the reactions of Schemes A - F, Examples 1 - 7 provide specific synthetic protocols demonstrating the preparation of the cerastrol derivative compounds 14, 15, 16, 17, and 22. Those skilled in the art can use and / or adapt these synthetic protocols of Examples 1 - 7 for the preparation of further cerastrol derivative compounds of structural formula (I) described in the present invention. It will be understood by those skilled in the art that, in addition to the typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) described in the examples herein, other process conditions can be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures. Further, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent undesired reactions of specific functional groups. For example, protecting groups can be used to temporarily block functional groups (such as O, S, or N) so that reactions can be selectively carried out at another reaction site in a polyfunctional compound. Protecting groups useful in the synthesis of the present invention are well known in the art and include those described in detail in Protective Groups in Organic Synthesis, Fourth Ed., Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference, and the reference is cited in the present invention.

[0076] As described above, the starting materials and / or reagents used in synthetic reaction schemes A - F are either commercially available, can be prepared by known procedures or obvious modifications thereof, or are generally known compounds 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). Others can be prepared by procedures or obvious modifications thereof 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 Supplementals (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).

[0077] IV. Therapeutic Use and Methods

[0078] As described elsewhere herein, cerastrol has been proposed and shown to provide potential therapeutic effects in several diseases and disorders based on clinical trials, pre - clinical trials, and in vitro tests. These previously identified cerastrol - 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, lung 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)), Allergies (e.g., asthma), psoriasis and skin inflammation, other inflammatory skin conditions), Neurological disorders (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS), and Gaucher's disease (GD)), Obesity-related disorders (e.g., obesity, pre-obesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, hypothalamic injury-related obesity, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bardet-Biedl syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macrosomia, obesity, and ocular anomalies) syndrome), and Liver-related diseases or disorders (e.g., 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).

[0079] Accordingly, the present invention is directed to the use of a cholesterol derivative compound of structural formula (I) of the present invention, or a pharmaceutical composition of such a compound, in the treatment, as a medicament, or in a method of treating a cholesterol-responsive disease or disorder in a subject, or in the manufacture of a medicament for treating a cholesterol-responsive disease or disorder in a subject.

[0080] In at least one embodiment, the present invention contemplates that the cerastrol derivative compounds of structural formula (I) disclosed herein can be used in a method of treating a subject suffering from a cerastrol-responsive disease or a cerastrol-responsive disorder. Generally, the method of treatment comprises administering to a subject in need thereof a therapeutically effective amount of a compound of structural formula (I) disclosed herein. In at least one embodiment, the compound of structural formula (I) to be administered 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 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 one of these compounds.

[0081] In at least one embodiment, the compound of formula (I) to be administered can be in the form of a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients described elsewhere herein. In at least one embodiment, the pharmaceutical composition to be administered 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, single stereoisomer, mixture of stereoisomers, tautomer, or prodrug of any one of these compounds..

[0082] In at least one embodiment of the use of the compound of the formula (I) or the treatment method of the compound of the formula (I), the cerastrol-responsive disease or disorder from which the subject suffers can be cancer. Cancers treatable by this method include, but are 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).

[0083] In at least one embodiment of the use of the cerastrol derivative compound of structural formula (I) or a treatment method, the cerastrol-responsive disease or disorder suffered by the subject may be an inflammatory disorder and / or an autoimmune disorder. The inflammatory and / or autoimmune disorders treated by the method may include 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), allergies (e.g., asthma), psoriasis and skin inflammation, and other inflammatory skin conditions, but are not limited thereto.

[0084] In at least one embodiment of the use of the cerastrol derivative compound of structural formula (I) or a treatment method, the cerastrol-responsive disease or disorder suffered by the subject may be a neuropathy. The neuropathies treated by the method may include Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and Gaucher's disease (GD), but are not limited thereto.

[0085] In at least one embodiment of the use of the cerastrol derivative compound of structural formula (I) or a treatment method, the cerastrol-responsive disease or disorder suffered by the subject may be an obesity-related disease or disorder. The obesity-related diseases or disorders treated by the method may include obesity, pre-obesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, hypothalamic injury-related obesity, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bardet-Biedl syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macroscopic obesity with ocular anomalies) syndrome, but are not limited thereto.

[0086] In at least one embodiment of the use of the cerastrol derivative compound of structural formula (I) or the treatment method, the cerastrol-responsive disease or disorder suffered by the subject can be a liver-related disease or disorder. The liver-related diseases or disorders treated by this method include, but are not limited to, acute-on-chronic liver failure (ACLF), acute hepatic porphyria, alpha-1 antitrypsin deficiency, alcoholic liver disease, alcoholic hepatitis, amebic hepatitis, autoimmune hepatitis, benign liver tumor, biliary atresia, cholestatic liver disease, congestive liver disorder, cirrhosis, Crigler-Najjar syndrome, drug-induced liver disease, Dubin-Johnson, conjugated hyperbilirubinemia, galactosemia, Gilbert syndrome, glycogen storage disease, hemochromatosis, liver abscess, liver cyst, hepatic encephalopathy, hepatitis, hepatorenal syndrome, intrahepatic cholestasis of pregnancy, isoniazid toxicity, jaundice, liver abscess, liver cyst, liver cancer, liver disease during pregnancy, lysosomal acid lipase deficiency (LAL-D), neonatal jaundice, non-alcoholic fatty liver hepatitis (NASH), primary biliary cholangitis, primary sclerosing cholangitis, progressive familial intrahepatic cholestasis, Reye syndrome, type I glycogenosis, unconjugated hyperbilirubinemia, viral hepatitis, viral liver disease, Wilson's disease, etc. In at least one embodiment, the liver-related diseases or disorders treated by this method are selected from acute-on-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.

[0087] V. Pharmaceutical Compositions and Modes of Administration

[0088] The present invention also provides uses and methods in which a compound of the formula (I), such as a cerastrol derivative compound, is administered to a subject in the form of a pharmaceutical composition as described above. For example, in such embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a cerastrol derivative compound of the 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 pharmaceutical compositions can be prepared using methods well known in the pharmaceutical art (e.g., Remington'S Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17th Ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G.S. Banker & C.T. Rhodes)). Methods for preparing pharmaceutical compositions of cerastrol derivative compounds, including the examples disclosed herein, are described in the present invention.

[0089] In general, pharmaceutical compositions can be prepared by diluting the active ingredient with an excipient and / or encapsulating it in a carrier in the form of capsules, sachets, paper, or other containers. When the excipient acts as a diluent, the excipient can be a solid, semi-solid, or liquid material (such as those described above) that acts as a vehicle, carrier, or medium for the active ingredient. Accordingly, pharmaceutical compositions suitable for administration by the methods of the present invention can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid or liquid media), for example, ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.

[0090] The carriers used in the preparation of pharmaceutical compositions can include excipients such as inert solid diluents and fillers, diluents including sterile aqueous solutions and various organic solvents, permeation promoters, solubilizers, and adjuvants. Excipients suitable for use in pharmaceutical compositions containing the cerastrol derivatives of the present invention (for example, the compounds in Table 3) are well known in the art and include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The pharmaceutical composition can further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweetening agents; and flavoring agents.

[0091] In terms of therapeutic use and methods, pharmaceutical compositions containing cerastrol derivative compounds such as the compounds of structural formula (I) (for example, the compounds in Table 3) can be administered either as a single or multiple administrations and by any of the acceptable modes of administration of active ingredients having similar usefulness. For example, pharmaceutical compositions containing cerastrol derivatives can be administered using various modes including oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal (intramedullary) administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intra-articular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppository administration, inhalation administration, or subcutaneous administration.

[0092] The pharmaceutical composition containing the cerastrol derivative compound of the present invention (for example, the compounds in Table 3) can be used in the area of treatment methods, and a dosage range is included for the administration of a pharmaceutically effective amount. The dosage and frequency (single or multiple dosages) of administration of the pharmaceutical composition to a subject can vary depending on factors such as the route of administration; the size, age, gender, health, weight, and / or diet of the subject; the condition of the disease being treated; whether the subject is suffering from any other diseases, and any concomitant treatments received, etc. Those skilled in the art will understand that adjustment of the dosage (for example, frequency and duration) established to obtain a therapeutically effective amount may be required depending on the subject. Typically, the amount of the pharmaceutical composition containing the cerastrol derivative compound administered to a subject in a treatment method is determined by a physician taking into account the relevant circumstances of the subject being so treated, the selected route of administration, and, of course, age, weight, severity of symptoms, individual response of the subject to the treatment, etc.

[0093] Generally, a therapeutically effective amount is an amount sufficient for the administered composition to achieve the desired therapeutic objective in comparison to the absence of the compound. For example, a therapeutically effective amount can be an amount determined to be sufficient to contribute to the treatment, prevention, or alleviation of the symptoms of a disease or disorder. Methods for determining the dosage to provide a therapeutically effective amount of a compound are known to those skilled in the art and typically are based on analysis of amounts determined in cell assays and / or animal models.

[0094] For example, the dosage for administration to humans can be formulated to achieve the concentration observed to be therapeutically effective in an animal model. The dosage in a pharmaceutical composition for humans can be further adjusted by monitoring effectiveness and adjusting upward or downward. Those skilled in the art can use methods well known in the art to adjust the dosage in the pharmaceutical composition of the present invention (for example, the compounds in Table 3) to achieve maximum therapeutic effectiveness for humans.

[0095] In general, a method for a therapeutic treatment is developed by starting with a pharmaceutical composition containing a cerastrol derivative compound at a dose below the optimal dose. Thereafter, the dose of the compound is increased incrementally until optimal efficacy is achieved. An important factor considered in developing the optimal dose is the ratio between the toxicity and the therapeutic efficacy of the active ingredient. This ratio, called the therapeutic index of the compound, is typically described as the ratio of the LD 50 (the amount of the compound lethal in 50% of the population) to its ED 50 (the amount of the compound effective in 50% of the population). Typically, a higher therapeutic index of the compound is preferred. Therapeutic index data can be obtained from cell culture assays and / or animal model studies and can then be used to determine the safe dose range of the active ingredient in a pharmaceutical composition for administration to humans. Ideally, the determined dose provides the active ingredient at its ED 50 level in the subject with little or no toxicity.

[0096] In some embodiments using the compounds of the present invention (e.g., the compounds in Table 3), the pharmaceutical composition comprises a dose of the cerastrol derivative compound as an active ingredient in an amount of about 0.05 to about 100 mg / kg, about 0.1 to about 0.5 mg / kg, about 0.1 to about 1 mg / kg, about 0.1 to about 5 mg / kg, about 0.1 to about 10 mg / kg, about 0.1 to about 25 mg / kg, about 1 to about 5 mg / kg, about 1 to about 25 mg, about 5 to about 25 mg / kg, about 10 to about 25 mg / kg, about 10 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 75 mg / kg, about 50 to about 100 mg / kg. In some embodiments, the pharmaceutical composition comprises a dose of the cerastrol 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.

[0097] In some embodiments, it is contemplated that pharmaceutical compositions comprising a cerastrol derivative compound of the invention (e.g., the compounds of Table 3) can be formulated to provide 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 polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are disclosed, for example, in U.S. Patent Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345.

[0098] In some embodiments, it is also contemplated that pharmaceutical compositions comprising a cerastrol derivative compound of the invention (e.g., the compounds of Table 3) can be formulated for administration via a transdermal delivery device (e.g., a "patch"). Such transdermal patches can be used to provide continuous or discontinuous infusion of a controlled amount of the pharmaceutical composition. The construction and use of transdermal patches for delivery of pharmaceutical compositions are well known in the art. See, for example, U.S. Patent Nos. 5,023,252; 4,992,445 and 5,001,139. Such patches can be constructed for continuous delivery, pulsatile delivery, or on-demand delivery of the pharmaceutical composition(s).

[0099] In some embodiments, it is contemplated that the pharmaceutical compositions of the present invention (e.g., the compounds of Table 3) can be prepared, for example, as solid formulations for oral administration. Such solid formulations can be prepared by mixing the cerastrol derivative compound - the active ingredient with a pharmaceutical excipient to form a solid preliminary formulation composition containing a homogeneous mixture of the active ingredient and the excipient. When these preliminary formulation compositions are referred to as homogeneous, it means that the active ingredient is evenly dispersed throughout the composition such that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. Tablets or pills can be coated or otherwise formulated to provide a dosage form that provides the advantage of an extended action or to protect from the acidic condition of the stomach. For example, tablets or pills can contain ingredients for internal and external use, and the external use ingredient is in the form of an envelope that covers the former. The two components can be separated by an enteric layer that resists disintegration in the stomach and allows the internal component to pass intact into the duodenum or acts to delay release. Various substances are used for the enteric layer or coating, and the substances include numerous polymeric acids, as well as mixtures of substances such as shellac, cetyl alcohol, and cellulose acetate with polymeric acids.

[0100] In the method of the present invention, another exemplary mode of useful administration is parenteral, particularly by injection. The pharmaceutical compositions of the present invention (e.g., compositions containing the compounds of Table 3) can be incorporated for administration by injection and include aqueous or oily suspensions, or emulsions with sesame oil, corn oil, cottonseed oil, or peanut oil, and elixirs, mannitol, dextrose, or sterile aqueous solutions, and similar pharmaceutical vehicles. Aqueous solutions in physiological saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, etc. (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils can also be used. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of suspensions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0101] Sterile injectable solutions are prepared by incorporating the required amount of the active ingredient of the present invention (e.g., the compounds of Table 3), optionally with the various other ingredients listed above, into a suitable solvent and then filtering to sterilize. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile medium containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, known methods of preparation include vacuum drying and lyophilization techniques that yield any additional desired ingredients from the solution of the active ingredient powder that has been pre-sterilized by filtration.

[0102] Pharmaceutical compositions that can be administered by inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous organic solvents, or mixtures thereof, as well as powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and known in the art. In some embodiments, pharmaceutical compositions of cerastrol derivatives (e.g., the compounds of Table 3) can be administered by the oral or nasal respiratory route for local or systemic effects. In some embodiments, the pharmaceutical compositions are prepared in a pharmaceutically acceptable solvent that can be nebulized by the use of an inert gas. These nebulized solutions can be inhaled directly from the nebulizer device, or the nebulizer device can be attached to a face mask tent or an intermittent positive pressure breathing apparatus. In some embodiments, the pharmaceutical compositions useful in the present method can be solution, suspension, or powder compositions and can be administered orally or nasally from a device that delivers the formulation in a suitable manner.

Example

[0103] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily understand that the specific examples are merely illustrative of the various embodiments of the present invention more fully described in the following claims. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments contained therein.

[0104] 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-octahydropicen-2-one (Compound 4-2)

Chemical formula

Chemical formula

[0105] Step 1 (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-octahydropicen-2-one (Compound 4-1) Synthesis

Chemical formula

[0106] 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-octahydropicene-2-carboxylic acid (4.0 g, 8.88 mmol) in toluene (200 mL), DIPEA (3.44 g, 26.6 mmol) and DPPA (3.66 g, 13.3 mmol) were added under N2 at 25 °C. The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was cooled to 25 °C and poured into water (200 mL). The aqueous phase was extracted with DCM (3 x 100 mL). The combined organic phases were washed with brine (2 x 300 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography (eluent: 0 - 50% ethyl acetate / petroleum ether) to give (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-octahydropicen-2-one (Compound 4-1) (3.0 g, purity 91.4%, yield 76%) and (142 mg, purity 100%) as a yellow solid.

[0107] 1H NMR: In CDCl3 at 400 MHz, δ = 7.05 (d, J = 6.8 Hz, 1H), 6.56 (s, 1H), 6.38 (d, J = 7.2 Hz, 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) LCMS: Rt = 2.949 min in 1.5 min chromatography, 5 - 95 AB, purity 99%, LCMS ESI+ calcd. for C 29 H 37 NO3[M]+ 447.2, found [M + H]+ 448.2, HPLC: Rt = 4.74 min in 8 min chromatography, 50 - 100 AB, purity 96%

[0108] Step 2

[0109] (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 - octahydropicen - 2 - one (Compound 4 - 2) synthesis [Chemical Structure Diagram]

[0110] 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-octahydropicen-2-one (3.0 g, 6.70 mmol) in THF (120 mL) and H2O (12 mL) was added with LiOH-H2O (1.41 g, 33.5 mmol) at 25 °C. The reaction mixture was stirred at 50 °C for 2 h. The reaction mixture was concentrated and then poured into H2O (200 mL), and filtered to obtain 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-octahydropicen-2-one (3.5 g, crude) as a yellow solid. The crude product (50 mg, crude) was purified by silica gel chromatography (eluent: 0 - 20% methanol / dichloromethane) 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-octahydropicen-2-one (Compound 4-2) (11.3 mg, 100% purity) as a yellow solid.

[0111] 1H NMR: MeOD 400 MHz δ = 7.22 (d, J = 7.6 Hz, 1H), 6.52 (d, J = 7.2 Hz, 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).

[0112] LCMS: Rt = 0.920 min in 1.5 min chromatography, 5 - 95AB, purity 100%, LCMS ESI+ calcd. for C 28 H 39 NO2[M]+ 421.3, found [M+H]+422.2.

[0113] HPLC: Rt = 2.56 min in 8 min chromatography, 30 - 90AB, purity 100%.

[0114] Example 2: Synthesis 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 - tetradecahydropicen - 2 - yl)amino)-2 - oxoacetate (Compound 14 - 1).

Chemical formula

[0115] Compound 14 - 1 was prepared by the synthesis summarized in Scheme 2 (below). Scheme 2

Chemical formula

[0116] Step 1

[0117] To a solution of (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-dodecahydropicen-2(6bH)-one (1.00 g, 2.37 mmol) in DCM (20 mL) was added Et3N (720 mg, 7.12 mmol) at 0 °C. Then ethyl 2-chloro-2-oxoacetate (291 mg, 2.13 mmol) was added dropwise. The resulting mixture was stirred at 0 °C for 1 h. The reaction mixture was poured into water (50 mL). The aqueous phase was extracted with DCM (3 × 100 mL). The combined organic phases were washed with brine (3 × 30 mL), dried over anhydrous Na2SO4, 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-tetradecahydropicen-2-yl)amino)-2-oxoacetate (1.2 g, 74.7% purity) as a yellow solid. 100 mg of the yellow solid was purified by preparative HPLC (column: Phenomenex Gemini 150*25mm*10μm; 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-tetradecahydropicen-2-yl)amino)-2-oxoacetate (Compound 14-1) (15.25 mg, 96.7% purity) as a yellow solid.

[0118] 11H NMR: In CDCl3 at 400 MHz, δ = 7.08 - 6.78 (m, 2H), 6.63 - 6.26 (m, 2H), 4.42 (dd, J = 4.5, 8.0 Hz, 1H), 4.39 - 3.95 (m, 2H), 2.66 (br d, J = 15.0 Hz, 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 = 7.0 Hz, 2H), 0.72 (br s, 3H).

[0119] LCMS: Rt = 0.595 min in 1.5 min chromatography, 50 - 95 AB, purity 100%, LCMS ESI+ calcd. for C 32 H 43 NO5[M]+ 521.3, found [M + H]+ 522.3.

[0120] HPLC: Rt = 3.101 min in 4 min chromatography, 10 - 80 AB, purity 96.73%.

[0121] 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 - tetradecahydropicen - 2 - yl)-2-(4 - methylpiperazin - 1 - yl)-2 - oxoacetamide (Compound 14).

Chemical Structure

[0122] Compound 14 was prepared by a two - step synthesis summarized in Scheme 3 (below). Scheme 3

Chemical Structure

[0123] Step 1

[0124] 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 - tetradecahydropicen - 2 - yl)amino)-2 - oxoacetic acid (Compound 14 - 2).

Chem.

[0125] 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 - tetradecahydropicen - 2 - yl)amino)-2 - oxoacetate (1.3 g, 2.49 mmol) and LiOH·H₂O (314 mg, 7.48 mmol) in THF (60 mL) and H₂O (12 mL) was stirred at 0 °C for 0.5 h. The reaction mixture was adjusted to pH = 3 by adding aqueous hydrochloric acid (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 - tetradecahydropicen - 2 - yl)amino)-2 - oxoacetic acid (Compound 14 - 2) (1.2 g, yield 80.2%) as a yellow solid.

[0126] LCMS: Rt = 0.507 min in 1.5 min chromatography, 5 - 95 AB, purity 73.919%, LCMS ESI+ calcd. for C 30 H 39NO5[M]+ 493.3, found [M+H]+ 494.3.

[0127] Step 2

[0128] 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-tetradecahydropicen-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14).

[0129] 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-tetradecahydropicen-2-yl)amino)-2-oxoacetic acid (500 mg, 1.01 mmol) in DCM (5 mL) was added DCC (313 mg, 1.52 mmol), and the mixture was 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 h. 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 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-tetradecahydropicen-2-yl)-2-(4-methylpiperazin-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-tetradecahydropicen-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14) (100 mg, yield 15.3%) as a yellow solid.

[0130] 11H NMR: CDCl3 400 MHz δ = 7.09 - 6.90 (m, 3H), 6.51 (d, J = 1.4 Hz, 1H), 6.36 (d, J = 7.1 Hz, 1H), 4.43 - 4.00 (m, 2H), 3.87 - 3.45 (m, 2H), 2.72 (br d, J = 14.0 Hz, 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 = 6.8 Hz, 6H), 1.34 - 1.23 (m, 4H), 1.13 (s, 3H), 1.06 - 0.98 (m, 1H), 0.75 (s, 3H).

[0131] LCMS: Rt = 0.431 min in 1.5 min chromatography, 5-95 AB, purity 98.137%, LCMS ESI+ calcd. for C 35 H 49 N3O4[M]+ 575.4, found [M+H]+ 576.4.

[0132] HPLC: Rt = 2.105 min in 4 min chromatography, 10-80 AB, purity 95.8%.

[0133] 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-tetradecahydropicen-2-yl)-2-oxo-2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)acetamide (Compound 15).

Chem.

[0134] Compound 15 was prepared by the synthesis summarized in Scheme 4 (below). Scheme 4

Chem.

[0135] Step 1

[0136] 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-tetradecahydropicen-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 the mixture was 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 h. 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-tetradecahydropicen-2-yl)-2-oxo-2-(2-oxa-6-azaspiro[3.3]heptan-6-yl)acetamide (Compound 15) (5.5 mg, yield 3.11%), a yellow solid.

[0137] 11H NMR: CDCl3 400 MHz δ = 7.21 (s, 1H), 7.04 (br d, J = 7.0 Hz, 2H), 6.55 (s, 1H), 6.36 (d, J = 7.1 Hz, 1H), 4.90 - 4.68 (m, 7H), 4.19 (s, 2H), 2.63 (br d, J = 14.4 Hz, 1H), 2.27 - 2.10 (m, 4H), 2.04 - 1.83 (m, 4H), 1.71 (br d, J = 8.4 Hz, 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 = 13.6 Hz, 1H), 0.69 (s, 3H).

[0138] LCMS: Rt = 0.590 min in 1.5 min chromatography, 5-95 AB, purity 95.249%, LCMS ESI+ calcd. for C 35 H 46 N2O5[M]+ 574.3, found [M+H]+ 575.3.

[0139] HPLC: Rt = 2.906 min in 4 min chromatography, 10-80 AB, purity 98.90%.

[0140] 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-tetradecahydropicen-2-yl)-N-methyl-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 16).

Chemical formula

[0141] Compound 16 was prepared by the synthesis summarized in Scheme 5 (below). Scheme 5 [Chemical formula]

[0142] Step 1

[0143] 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-tetradecahydropicen-2-yl)-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 14) (180 mg, 313 μmol) in THF (5 mL) was added Mel (44.0 mg, 312 μmol). Subsequently, 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 h. The reaction mixture was poured into a saturated aqueous solution. NH4Cl solution (20 mL) was slowly added at 0 °C and 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 yellow solids 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-tetradecahydropicen-2-yl)-N-methyl-2-(4-methylpiperazin-1-yl)-2-oxoacetamide (Compound 16) (5.75 mg, 2.86% yield).

[0144] 11H NMR: In CDCl3 at 400 MHz, δ = 7.12 - 6.87 (m, 2H), 6.53 (d, J = 1.0 Hz, 1H), 6.36 (d, J = 7.1 Hz, 1H), 5.81 - 5.79 (m, 1H), 3.73 (q, J = 7.0 Hz, 4H), 3.41 (br dd, J = 3.8, 12.9 Hz, 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).

[0145] LCMS: R t = 0.492 min in 1.5 min chromatography, 5-95 AB, purity 96.29%, LCMS ESI+ calcd. for C 36 H 51 N3O4[M]+ 589.4, found [M+H]+ 590.4.

[0146] HPLC: Rt = 2.187 min in 4 min chromatography, 10-80 AB, purity 91.7%.

[0147] 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-tetradecahydropicen-2-yl)-N2-(1-methylazetidin-3-yl)oxalamide (Compound 17).

Chem.

[0148] Compound 17 was prepared by the synthesis summarized in Scheme 6 (below). Scheme 6

Chem.

[0149] Step 1

[0150] 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-tetradecahydropicen-2-yl)amino)-2-oxoacetic acid (Compound 14-2) (200 mg, 405 μmol) in DCM (5 mL) was added DCC (125 mg, 608 μmol) at 0 °C. Then, 1-methylazetidin-3-amine (38.4 mg, 446 μmol) and DMAP (24.8 mg, 203 μmol) were added. The resulting mixture was stirred at 20 °C for 0.5 h. The reaction mixture was poured into water (5 mL). The aqueous phase was extracted with DCM (3 × 3 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 50 / 1, Rf = 0.40) to give the crude product (100 mg). The crude product was diluted with EtOAc (10 mL) and washed with aqueous HCl solution (0.02 M, 10 mL × 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-tetradecahydropicen-2-yl)-N2-(1-methylazetidin-3-yl)oxalamide (Compound 17) (60.0 mg, yield 22.2%) as a yellow solid.

[0151] 11H NMR: In CDCl3 at 400 MHz, δ = 7.74 (broad doublet, J = 8.5 Hz, 1H), 7.23 (singlet, 1H), 7.01 (doublet of doublets, J = 0.9, 7.1 Hz, 1H), 6.52 (doublet, J = 1.0 Hz, 1H), 6.35 (doublet, J = 7.1 Hz, 1H), 4.39 (doublet of doublets, J = 6.6, 14.9 Hz, 1H), 3.63 (triplet, J = 7.5 Hz, 2H), 2.95 (quartet, J = 6.7 Hz, 2H), 2.63 (broad doublet, J = 15.4 Hz, 1H), 2.32 (singlet, 3H), 2.22 - 2.12 (multiplet, 4H), 1.90 - 1.69 (multiplet, 10H), 1.44 (doublet, J = 11.8 Hz, 5H), 1.29 - 1.25 (multiplet, 6H), 1.13 (singlet, 3H), 1.04 (broad doublet, J = 14.0 Hz, 1H), 0.91 - 0.86 (multiplet, 1H), 0.68 (singlet, 3H).

[0152] LCMS: Retention time (Rt) = 0.437 min in a 1.5 - minute chromatography, with a 5 - 95 AB gradient, purity 92.577%, LCMS ESI+ calculated for C 34 H 47 N3O4[M]+ 561.4. Found [M + H]+ 562.5.

[0153] HPLC: Rt = 2.206 min in a 4 - minute chromatography, with a 10 - 80 AB gradient, purity 91.886%.

[0154] 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 - octahydropicen - 2 - yl]-N,N,N'-trimethyloxamide (Compound 22):

Chemical Structure

[0155] Compound 22 was prepared by a two-step synthesis summarized in Scheme 7 (below). Scheme 7

Chem.

[0156] Step 1

[0157] 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-octahydropicen-2-yl]-N',N'-dimethyl-oxamide (Compound 22-1):

Chem.

[0158] To a solution 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-octahydropicen-2-one (50 mg, 119 μmol) and 2-(dimethylamino)-2-oxoacetic acid (18 mg, 154 μmol) in DCM (1 mL), DCC (36.7 mg, 178 μmol) and DMAP (7.24 mg, 59.3 μmol) were added at 25 °C. The reaction mixture was stirred at 25 °C for 16 h. The residue was poured into ice water (30 mL), and the aqueous phase was extracted with DCM (3 × 10 mL). The combined organic phases were washed with brine (30 mL), 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 the crude product (90 mg). The crude product was further purified by preparative HPLC (column: Phenomenex Gemini-NX C18 75*30mm*3um; mobile phase: [water (0.225% FA)-ACN]; B%: 60% - 90%, 7 min) 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-octahydropicen-2-yl]-N',N'-dimethyloxamide (Compound 22-1) (2.0 mg, purity: 100%) as a yellow solid.

[0159] 1H NMR: CDCl3 400 MHz δ = 7.13 (s, 1H), 7.04 - 6.93 (m, 2H), 6.51 (s, 1H), 6.35 (d, J = 7.6 Hz, 1H), 3.37 (s, 3H), 2.93 (s, 3H), 2.77 (d, J = 14.0 Hz, 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).

[0160] LCMS: Rt = 1.120 min in 1.5 min chromatography, 5 - 95 AB, purity 100%, LCMS ESI+ calcd. for C32H44N2O4 [M]+ 520.3, found [M+H]+521.1.

[0161] HPLC: Rt = 6.12 min in 8 min chromatography, 10 - 80 AB, purity 100%.

[0162] Step 2

[0163] 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 - octahydropicen - 2 - yl]-N,N,N'-trimethyl - oxamide (Compound 22):

[0164] 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-octahydropicen-2-yl]-N',N'-dimethyl-oxamide (300 mg, 576 μmol) in THF (3 mL) was added with NaH (69.13 mg, 1.73 mmol, 60% in mineral oil) at 20 °C and stirred for 30 minutes. Mel (81.8 mg, 576 μmol) was added at 20 °C 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 (eluting with ethyl acetate / petroleum ether = 0 - 100%) 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-octahydropicen-2-yl]-N,N,N'-trimethyl-oxamide (Compound 22) (20 mg, yield 6%) as a yellow solid.

[0165] 1H NMR: CDCl3 400 MHz δ = 7.02 - 6.95 (m, 2H), 6.51 (d, J = 1.2 Hz, 1H), 6.34 (d, J = 7.2 Hz, 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).

[0166] LCMS: Rt = 1.119 min in 1.5 min chromatography, 5 - 95AB, purity 96.0%, LCMS ESI+ calcd. for C33 H 46 N2O4[M]+ 534.3, found [M+H]+535.4.

[0167] HPLC: Rt = 2.103 min in 4 min chromatography, 5-95AB, purity 92.0%.

[0168] Example 8: Synthesis of Compound 14-4

Chem.

[0169] Compound 14-4 can be prepared by a six-step synthesis as outlined in Scheme 8 (below). Scheme 8

Chem.

Chem.

[0170] Example 9: Synthesis of Compound 54:

Chem.

[0171] Compound 54 can be prepared from Compound 14-4 via the synthesis outlined in Scheme 9: Scheme 9

Chem.

[0172] Example 10: Synthesis of Compound 55:

Chem.

[0173] Compound 55 can be prepared from Compound 14-4 via the synthesis summarized in Scheme 10: Scheme 10

Chem.

[0174] Example 11: Synthesis of Compound 56:

Chem.

[0175] Compound 56 can be prepared from Compound 14-4 via the synthesis summarized in Scheme 11: Scheme 11

Chem.

[0176] Example 12: Synthesis of Compound 57:

Chem.

[0177] Compound 57 can be prepared from Compound 14-4 via the synthesis summarized in Scheme 12: Scheme 12

Chem.

[0178] Example 13: Inhibition of NF-κB Signaling by a Cerastrol Derivative Compound

[0179] This example describes the examination of the inhibition of NF-κB signaling by the following exemplary cerastrol derivative compounds of the present invention: Compound 14, Compound 15, Compound 16, Compound 17, and Compound 22.

[0180] Materials and Methods

[0181] The HepG2 NF-kB-Luc reporter cells stimulated with IL-1 (20 ng / ml) and the relative NF-kB luciferase activity (RLU) were determined for each of celastrol (Compound 1) and the representative derivative compounds at the following 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. The assay values were plotted (not shown), and the IC 50 inhibitory concentration was calculated after normalization using Prizm 9.0 software.

[0182] Results

[0183] The data from the NF-kB inhibition assay were analyzed, and the obtained IC 50 values and their % IC 50 values relative to celastrol for the celastrol derivative compounds (Compound 1; IC 50 = 667 nM) are summarized in Table 4 (below).

[0184] Table 4

Table A

[0185] Example 14: Anti-inflammatory properties of celastrol derivative compounds

[0186] This example describes the examination of the anti-inflammatory ability of an exemplary celastrol derivative (Compound 22) of the present invention using an NF-kB luciferase promoter reporter gene assay in the human hepatoma cell line HepG2.

[0187] Materials and methods

[0188] NF-κB luciferase reporter assay: Celastrol derivative, compound 22 (prepared as described in Example 13), and the parent molecule, compound 1 (celastrol), were freshly dissolved in DMSO. HepG2 NF-κB reporter cells were seeded in 96-well plates (2.5 x 10 4 / well), then pretreated with either compound 22 or compound 1 at eight concentrations of 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.016, or 0 μM, and the NF-κB promoter was activated by adding IL-1β (20 ng / ml) to the cells. After 18 hours, 1 x passive cell lysis buffer (Promega) was added, and the cells were harvested by measuring the relative NF-κB luciferase activity (RLU) using a GloMax Discover microplate reader (Promega). The inhibitory concentration (IC 50 ) was obtained after normalization using Prizm 9.0 software.

[0189] LPS-induced NF-κB target protein expression: Bone marrow-derived dendritic cells (BMDC) were pretreated with three concentrations (10 nM, 25 nM, 50 nM) of compound 22 or compound 1 for 1 hour and then treated with LPS (500 ng / mL) overnight. The next day, the cells were subjected to Western blotting for iNOS, COX2, NLRP3, and IL-1β.

[0190] Results

[0191] As shown in Figure 1(a), the celastrol derivative compound 22 has a stronger inhibitory ability on the IL-1β-induced NF-κB inflammatory signaling pathway at a lower concentration than celastrol (compound 1). As shown by the analysis of the normalized percentage response curve in Figure 1B, compound 22 has an IC of 268 nM for NF-κB promoter activity. 50It was shown to have one, and its value was 50% lower than that of Compound 1 (IC50 = 528 nM). Furthermore, as shown by the Western blot image shown in FIG. 1C, Compound 22, a cerastrol derivative compound, significantly inhibited the expression of LPS-induced NF-kB target proteins iNOS, COX2, NLRP3, and IL-1β in a dose-dependent manner.

[0192] Example 15: Anti-inflammatory properties of cerastrol derivative compounds

[0193] This example discloses an examination of the anti-inflammatory ability of an exemplary cerastrol derivative (Compound 22) of the present invention using a specific NLRP3 inflammasome assay using bone marrow-derived dendritic cells (BMDCs) activated with LPS+ATP or LPS+ nigericin.

[0194] Materials and methods

[0195] NLRP3 inflammasome assay

[0196] 1. BMDCs were treated with LPS (100 ng / mL) for 3 hours (Signal 1) to prime the NLRP3 inflammasome, and then treated with cerastrol (Compound 1) or Compound 22, a cerastrol derivative compound, 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 the treated cells with the ASC complex (>48 kDa). Also, the level of secreted IL-1β in the conditioned medium represents active caspase-1, a downstream molecule of the NLRP3 inflammasome that cleaves pro-IL1β, resulting in the secreted form of mature IL-1β.

[0197] 2. The secreted form of IL-1β, an NLRP3 inflammasome-dependent cytokine, was measured by an ELISA assay using the conditioned medium of BMDCs treated with compound 1 (CLM-1) or compound 22 (CLM-022) at concentrations of 63, 125, 250, 500 nM under NLRP3 inflammasome activation induced by either LPS + ATP or LPS + nigericin.

[0198] 3. The secreted form of TNF-α, an NLRP3 inflammasome-independent cytokine induced by LPS + ATP or LPS + nigericin, was measured by an ELISA assay using the conditioned medium of BMDCs treated with compound 1 (CLM-1) or compound 22 (CLM-022) at concentrations of 63, 125, 250, 500 nM under NLRP3 inflammasome activation induced by either LPS + ATP or LPS + nigericin.

[0199] Results

[0200] Compound 22 showed a better and more potent inhibitory function against NLRP3 inflammasome activity than that observed for the parent molecule, cerastrol (compound 1). As shown by the results presented in Figure 2A, compound 22 more strongly suppressed ASC complex formation than compound 1. Furthermore, as shown by the plots presented in Figure 2B, compound 22 more strongly inhibited IL-1β secretion induced by both LPS + ATP and LPS + nigericin. As shown in Figure 2C, compound 22 (and compound 1) did not show a significant inhibitory effect on TNF-α inflammatory cytokine secretion under the same NLRP3 inflammasome activation. The lack of inhibitory effect on TNF-α secretion, which is independent of NLRP3 inflammasome activity, indicates the specificity of compound 22 for the NLRP3 inflammasome. Thus, compound 22 has the ability to inhibit the NF-kB inflammatory response and specifically suppresses IL-1β secretion induced by the NLRP3 inflammasome in BMDCs.

[0201] Example 16: Inhibition of the NLRP3 inflammasome in human THP-1 cells by a cerastrol derivative compound.

[0202] In this example, the anti-inflammatory potential of an exemplary cerastrol derivative (Compound 22) disclosed in the present invention was examined using an NLRP3 inflammasome assay in human monocytic cell line THP-1 activated with LPS + nigericin in the presence of Compound 22 or the known NLRP3 inhibitor compound MCC950 (CAS 256373-96-3) as a benchmark molecule.

[0203] Materials and methods

[0204] ASC complex formation in THP-1 cells.

[0205] Human THP-1 cells were treated with LPS + nigericin as shown previously (Figure 2), where increasing doses of Compound 1, Compound 22, or MCC950 were added as shown. To demonstrate relative NLRP3 inflammasome activity, the levels of ASC complex formation were compared by Western blotting.

[0206] Detection of ASC complex and caspase-1 activity in THP-1 cells by Amnis.

[0207] Amnis Imaging flow cytometry (Luminex Co.) is a high-throughput single-cell fluorescence-based image analysis method that can detect inflammasomes by acquiring images of the cellular distribution of ASC complexes and active caspase-1. THP-1 cells were treated with LPS + nigericin in the presence of inhibitors. The treated cells were stained for ASC and active caspase-1 was visualized using FLICA, a caspase-1 substrate that fluoresces upon cleavage. Finally, ASC+ / FLICA+ cells were counted as THP-1 cells having the active component NLRP3 inflammasome complex.

[0208] Results

[0209] THP-1 human monocytes form strong ASC complexes in response to LPS + nigericin treatment. As shown by the Western blot in Figure 3A, the presence of compound 22 at 50 nM and 100 nM concentrations strongly inhibited this ASC complex formation, and the inhibitory ability was stronger than that observed for celastrol (compound 1) and the benchmark molecule MCC950. As shown by the Amnis imaging results in Figure 3B, the strong inhibitory effect observed for compound 22 was further verified by counting THP-1 cells carrying cellular ASC+ FLICA+ speckles. As shown by the plot in Figure 3C, compound 22 was observed to exert a strong inhibitory function at a low concentration of about 25 nM, while the benchmark molecule MCC950 showed an inhibitory efficacy 5 to 10 times lower at 100 nM concentration.

[0210] Example 17: Inhibition of inflammation by celastrol derivative compounds in a LPS-induced septic shock mouse model.

[0211] This example describes the examination of the inhibitory function of an exemplary celastrol derivative (compound 22) of the present invention against LPS-induced acute inflammation in vivo. The inventors established a LPS-induced septic shock mouse model (Yu et al., 2017) and detected the expression levels of inflammatory cytokines in the colon.

[0212] Materials and Methods

[0213] Mice (n = 3 per group) were intraperitoneally (i.p.) administered a control vehicle, compound 1 (1 mg / kg), or compound 22 (1 mg / kg). One hour later, the mice were administered a sub-lethal dose of LPS (25 mg / kg) by intraperitoneal injection. Four hours later, the mice were bled and colonic mucosa was collected by scraping the colon. ELISA (Catalog number 900-T54, PeproTech) was performed for TNF-α, cytokine expression was measured in serum samples, and qPCR was performed on colonic mucosa samples for cytokines IL-1 (TaqMan, Mm00434228), IL-6 (TaqMan, Mm00446190), TNF-α (TaqMan, 00443258), and iNOS (TaqMan, 00440502).

[0214] Results

[0215] As shown by the results presented in the plots of FIGS. 4A and 4B, the cerastrol derivative compound, compound 22, successfully inhibited the LPS-induced acute inflammatory response in a mouse septic shock model, which was equivalent to or lower than the levels observed in the group administered cerastrol (compound 1), efficiently suppressing the expression of inflammatory mediators and inflammatory cytokines including IL-1, IL-6, TNF-α, and iNOS in blood serum and colonic mucosa.

[0216] Example 18: Comparison of CLM-022 (Compound 22) and MCC950 (Known Compound) NLRP3 Inflammasome Inhibitory Activity in Mice and Human Macrophages.

[0217] This example illustrates a comparative study of CLM-022 and MCC950 in the inhibition of NLRP3 inflammasome activity in mice and human macrophages.

[0218] Materials and Methods

[0219] A. BMDM Cell Study

[0220] Bone marrow-derived macrophages (BMDMs) 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, BMDMs were treated with CLM-022 or MCC950 in the dose range of 0 - 10 μM for 30 minutes. Then, nigericin (10 μM) was further added to BMDMs for another 30 minutes to fully activate NLRP3-dependent inflammasome activity and cytokine secretion.

[0221] B. THP-1 cell study.

[0222] To confirm the inhibitory effect of CLM-022 in human macrophages, the human leukemia monocyte cell line THP-1 cells were differentiated into macrophages using phorbol 12-myristate-13-acetate (PMA, 100 ng / ml) treatment for 1 day. Subsequently, THP-1 macrophages were 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 in the dose range of 0 - 10 μM for 30 minutes. Then, nigericin (10 μM) was further added to THP-1 cells for another 60 minutes to fully activate NLRP3-dependent inflammasome activity.

[0223] Results

[0224] As shown by the results presented in Figure 5A, dose-dependently, BMDMs treated with CLM-022 showed significant inhibition of IL-1β secretion. The normalized IC 50 calculations demonstrated that CLM-022 showed greater potential in the inhibition of IL-1β compared to MCC950 (IC 50: CLM-022 = 9 nM vs MCC950 = 70 nM). However, as shown in Figure 5B, since TNF-α is not regulated by the NLRP3 inflammasome, neither CLM-022 nor MCC950 inhibited TNF-α secretion. As shown by the results in Figures 5C and 5D, similar to the results obtained in BMDMs, in human THP-1 macrophages, IL-1β secretion was strongly inhibited by CLM-022, but TNF-α was not inhibited. CLM-022 showed a more potent inhibitory ability than MCC950 (IC 50 : CLM-022 = 158.9 nM vs MCC950 = 1248 nM). These results clearly show that CLM-022 has the potential to be an excellent inflammasome inhibitor compared to MCC950, the most advanced NLRP3 inflammasome inhibitor.

[0225] Example 19: DARTS assay for the protection of NLRP3 by CLM-022 (Compound 22).

[0226] The NLRP3 inflammasome is composed of a multi-protein complex, and the exact molecular mode of NLRP3 activation remains unknown. Currently, there are no clinically available NLRP3 inflammasome inhibitors, and it is important to identify the mechanism of action and molecular targets of small molecules that may interact with NLRP3. This example discloses an examination of the ability of CLM-022 to protect NLRP3 molecules, determined using a drug affinity responsive target stability (DARTS) assay. The DARTS assay is a powerful method for identifying small molecule target proteins. In the case of drug-target interactions, small molecules protect the target protein from degradation by a protease (pronase), while free protein can be degraded very efficiently under given conditions.

[0227] Materials and Methods

[0228] Using THP-1 lysates pretreated with LPS and nigericin, a DARTS assay was performed to induce the activation of the NLRP3 inflammasome. THP-1 cells (1x105 Cells were treated with LPS (100 ng / mL) for 3 hours and then further treated with 10 μM nigericin for 30 minutes. Proteinase (50 - 500 ng / μg of protein) was added to the lysates containing CLM-022 (10 μM) or MCC950 (10 μM) at room temperature for the indicated time (10 - 15 minutes). The protein components of the NLRP3 inflammasome were visualized using antibodies that recognize the NLRP3-Natch domain (Figure 6A), the NLRP-PYD domain (Figure 6B), or the NEK7 protein (Figure 6C). After densitometric scanning, the relative band intensity was normalized by GAPDH.

[0229] Results

[0230] As shown by the results presented in Figures 6A, 6B, and 6C, CLM-022 significantly protected NLRP3 protein from degradation that was clearly more efficient than the degradation of MCC950. CLM-022, however, did not protect NEK7 from protease-induced degradation. This result demonstrates that CLM-022 directly interacts with the NLRP3 protein in a target-specific manner.

[0231] Example 20: Protection of THP-1 cells from inflammasome-induced pyroptosis.

[0232] Pyroptosis, an inflammation-induced form of cell death, is induced by the activation of NLRP3 inflammasome components (GSDMD and caspase-1), leading to the release of inflammatory cytokines IL-1β and IL-18, as well as damage-associated molecular patterns (DAMPs). This process triggers a cascade of inflammatory responses, which can persist if dysregulated in affected tissues. Considering the significant inhibitory ability of CLM-022 against NLRP3 inflammasome and IL-1β secretion, it was speculated that CLM-022 could effectively enhance cell survival rate 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.

[0233] Materials and Methods

[0234] After treatment with LPS and nigericin, THP-1 cells were treated with either CLM-022 or MCC950 over a dose range of 0 μM to 10 μM, and cell viability was determined by an impermeable "live / dead" staining dye and flow cytometry analysis. Furthermore, NLRP3 inflammasome-induced cytotoxicity was examined by measuring the levels of LDH in the conditioned medium after treatment with CLM-022 and MCC950. In addition, Western blotting of samples from the THP-1 cell viability assay was performed to determine the levels of pyroptosis-mediated molecules, GSDMD and caspase-1.

[0235] Results

[0236] As shown by the results presented in Figure 7A, when THP-1 cells were treated with CLM-022 within a dose range of 0 μM to 10 μM, the cell viability increased up to 60%. A similar effect was also observed in cells treated with MCC950 (Figure 7A).

[0237] As shown by the results presented in Figure 7B, consistent with the results of the cell viability assay, both CLM-022 and MCC950 significantly inhibited the release of LDH. In particular, CLM-022 demonstrated superior inhibitory ability compared to MCC950 even at lower concentrations (Figure 7B, left). The normalized IC 50 values calculated from these LDH release results revealed that CLM-022 was 10-fold more potent in preventing pyroptosis (IC 50 : CLM-022 = 12.4 nM vs MCC950 = 123.5 nM) compared to MCC950.

[0238] As shown by the Western blot images presented in Figure 7C, CLM-022 inhibited GSDMD and caspase-1 cleavage at low concentrations of around 100 nM, while MCC950 required 1000 nM for inhibition.

[0239] Overall, these comparative analyses clearly demonstrated that CLM-022 has a higher ability to inhibit inflammasome-induced pyroptosis in human THP-1 macrophages than the benchmark molecule MCC950.

[0240] The foregoing disclosure of the present invention has been described in some detail for purposes of clarity and understanding by way of example and illustration. However, the present invention, including the examples, descriptions, and embodiments set forth herein, is for illustrative purposes only, is intended to be exemplary, and should not be construed as limiting the present invention. It will be apparent to those skilled in the art that various modifications or changes can be made to the examples, descriptions, and embodiments described herein and should be within the spirit and scope of the present invention and the appended claims. Furthermore, those skilled in the art will recognize numerous methods and procedures equivalent to those described herein. All such equivalents are to be understood as being within the scope of the present invention and are encompassed by the appended claims.

[0241] Further embodiments of the present invention are described in the following claims.

[0242] The disclosure of all publications, patent applications, patents, or other documents referred to in this specification is specifically shown to be incorporated by reference in its entirety herein for all purposes as if each such individual publication, patent, patent application, or other document were set forth in full herein. To the extent that the entire document is described herein, it is incorporated by reference in its entirety herein for all purposes as if it were set forth in full herein. In case of conflict, the specification, including specific terms, shall control.

Claims

1. Compound (I) of Structural Formula (I), 【Chemical 1】 Here, R 1 is selected from the following 【Chemical Formula 2-1】 [[Chemical Formula 2-2]] 【Chemical 2-3】 [Chemical 2-4] or a pharmaceutically acceptable salt, single stereoisomer, mixture of stereoisomers, tautomer, or prodrug thereof.

2. R 1 The compound according to claim 1, wherein R is selected from the following: 【Chemical Formula 3】

3. Compound (I) of Structural Formula (I) 【Chemical 4】 wherein, R 1 is -NH-(CO) 2 -NR 2 R 3 , or -N(CH 3 )-(CO) 2 -NR 2 R 3 ; R 2 and R 3 are each independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine, or heteroaryl groups; wherein each of said alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine, or heteroaryl group is optionally substituted with a substituent independently selected from alkyl, alkoxy, cycloalkyl, ether, amine optionally substituted with one or more alkyls, 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; or R 2 and R 3 together form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group, Here, each of the cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with a substituent independently selected from alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, heteroaryl, amine, halogen, hydroxyl, ether, nitrile, cyano, nitro, CF 3 , ester, amide, urea, carbamate, thioether, or carboxylic acid group; or a pharmaceutically acceptable salt, single stereoisomer, mixture of stereoisomers, tautomer, or prodrug thereof.

4. R 1 is the compound according to claim 3 selected from the following: 【Chemical Formula 5-1】 【Chemical Formula 5-2】 【Chemical Formula 5-3】 【Chemical Formula 5-4】

5. The compound according to any one of claims 1 to 4, wherein the compound is selected from the following Compound 14, Compound 15, Compound 16, Compound 17, Compound 22, and Compound 22-1: [Chemical Formula 6] or a pharmaceutically acceptable salt, single stereoisomer, mixture of stereoisomers, tautomer, or prodrug thereof.

6. The compound according to any one of claims 1 to 4, wherein the compound is selected from the following 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: 【Chemical Formula 7-1】 【Chemical Formula 7-2】 【Chemical Formula 7-3】 【Chemical Formula 7-4】 or a pharmaceutically acceptable salt, single stereoisomer, mixture of stereoisomers, tautomer, or prodrug thereof.

7. An IC in an NF-kB reporter 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 The compound according to any one of claims 1 to 6, characterized by having

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

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

10. The method according to claim 9, wherein the cerastrol-responsive disease or disorder is cancer; optionally, 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, glioblastoma multiforme (GBM).

11. The method according to claim 9, wherein the cerastrol-responsive disease or disorder is an inflammatory and / or autoimmune disorder; optionally, the inflammatory and / or autoimmune disorder is selected from 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), allergies (e.g., asthma), psoriasis and skin inflammation, other inflammatory skin conditions.

12. The method according to claim 9, wherein the cerastrol-responsive disease or disorder is a neuropathy; optionally, the neuropathy is selected from Parkinson's disease, Huntington's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (ALS), and Gaucher's disease (GD).

13. The method according to claim 9, wherein the cerastrol-responsive disease or disorder is an obesity-related disease or disorder; optionally, the obesity-related disease or disorder is obesity, pre-obesity, morbid obesity, type 2 diabetes (T2D), atherosclerosis, diabetic nephropathy, gout, cardiac fibrosis, Prader-Willi syndrome, hypothalamic injury-related obesity, non-alcoholic steatohepatitis, hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver, Bardet-Biedl syndrome, Cohen syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO (macroscopic obesity with ocular anomalies) syndrome.

14. The cerastrol-responsive disease or disorder is a liver-related disease or disorder; optionally, 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, the method according to claim 9.

15. The cerastrol-responsive disease or disorder is a brain injury or disorder; optionally, 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, the method according to claim 9.

16. The cerastrol-responsive disease or disorder is an eye disorder or injury; optionally, the eye-related injury or disorder is selected from dry eye disease, eye inflammation, age-related macular degeneration (AMD), subconjunctival fibrosis, retinal hypertension-induced degeneration, bright light-induced degeneration, macrophage-induced corneal neovascularization, corneal allograft survival, and optic nerve crush, the method according to claim 9.

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

18. The administration is 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, intra-articular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation (spray) administration, suppository administration, inhalation administration, or subcutaneous administration, the method according to any one of claims 9 to 15.

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

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

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

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 cerastrol-responsive disease or disorder in a subject.

23. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 8 in treatment.

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 cerastrol-responsive disease or disorder in a subject.

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