Steroid compounds, their preparation methods and applications

Abstract

The present invention discloses a steroid compound, a preparation method thereof and an application. The steroid compound may have a structure as shown in Formula VI etc. The compound of the present invention has SREBP pathway inhibitory activity and can be used for preventing and / or treating diseases such as obesity, hyperlipidemia, fatty liver, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, liver cancer, skin damage, etc.

Description

Technical Field

[0001] The present invention relates to a steroid compound, a preparation method thereof and an application thereof. Background Art

[0002] With the transformation of lifestyle, including the intake of high-calorie foods and high-sugar beverages, lack of exercise and physical activity, etc., globally, metabolic diseases represented by hyperlipidemia, obesity, type 2 diabetes and fatty liver have become increasingly serious health problems. Among them, fatty liver has become an important cause of chronic liver diseases in wealthy regions of Europe, America and China. The prevalence of simple hepatic lipid accumulation in the general adult population is 10% - 30%, and 10% - 20% of them have non-alcoholic steatohepatitis. The incidence of cirrhosis and liver cancer within 10 years of the latter reaches 25%. However, up to now, the pathophysiological mechanism of fatty liver has not been fully elucidated, and there is still a lack of effective and specific therapeutic drugs clinically. It is known that the accumulation of lipids such as cholesterol and triglyceride in the blood and liver is the main cause of hyperlipidemia, and hyperlipidemia is an important pathogenic factor causing atherosclerosis, stroke and fatty liver diseases. Therefore, targeting the lipid metabolism regulation pathway to develop new drugs with lipid reduction as the guide is gradually becoming an important direction for the research and development of new drugs for metabolic diseases.

[0003] The lipid synthesis pathway in mammalian cells is known to be an important factor in regulating lipid metabolic balance. The key factors that regulate cholesterol and fatty acid synthesis are a class of transcription factor proteins called sterol-regulatory element binding proteins (SREBPs). The precursors of these proteins are first synthesized on the endoplasmic reticulum (ER). The precursors are transported to the Golgi apparatus by SREBP cleavage-activating protein (SCAP), and then cleaved by two proteases (Site-1 protease (S1P) and Site-2 protease (S2P)) to release the active domain at their N-terminus, which enters the nucleus to function as a transcription factor and binds to the SREBP response element (SRE) in the promoter region of target genes, initiating the expression of downstream genes. The cleavage and maturation of SREBP proteins are strictly regulated by the intracellular sterol (such as cholesterol, 25-hydroxycholesterol) levels. When cells accumulate sufficient cholesterol in the endoplasmic reticulum, cholesterol binds to SCAP and changes the conformation of SCAP, causing the SCAP-SREBP complex to bind to the protein Insig (Insulin-induced gene), thereby blocking the transport of SREBP to the Golgi apparatus and subsequent activation of SREBP. Conversely, an increase in the nuclear active form of SREBP promotes cellular lipid synthesis. In addition to cholesterol, 25-hydroxycholesterol (25-HC) is another potent endogenous inhibitor of the SREBP pathway. Different from cholesterol binding to SCAP, 25-HC directly binds to Insig and induces the binding of SCAP and Insig.

[0004] Previous studies have found that inhibiting the SREBP pathway is an effective strategy and method for preventing and / or treating metabolic diseases such as obesity, hyperlipidemia, fatty liver, atherosclerosis, diabetes, etc., as well as cardiovascular and cerebrovascular diseases, skin injuries, liver cancer, and other diseases.

[0005] Regarding hyperlipidemia, its pathogenesis is mainly due to factors such as diet or gene mutations that cause increased lipid synthesis or abnormal lipid transport, resulting in excessive accumulation of lipids such as blood cholesterol and fatty acids. Currently, statins and fibrates are the main lipid-lowering drugs in clinical practice. The mechanism of action of statins is to inhibit the cellular cholesterol synthesis pathway and promote the reverse transport of blood cholesterol at the same time. This indicates that targeting the key factors of the cellular lipid synthesis pathway is an important means to effectively reduce lipid levels.

[0006] So far, there are no approved therapeutic drugs for fatty liver disease. Therefore, it is very important to identify therapeutic targets and develop new effective therapies. The pathogenesis of fatty liver disease is known to involve multiple risk factors, such as steatosis that may be triggered by the accumulation of triglycerides in the form of lipid droplets, abnormally increased cholesterol and fatty acids in cells, which can cause endoplasmic reticulum stress and mitochondrial dysfunction, leading to cell death, inflammation, and fibrosis. Among them, the accumulation of free cholesterol has been reported as a key driver for the transition from simple steatosis to aggressive steatohepatitis. Secondly, in establishing a mouse model of fatty liver, a simple cholesterol-free high-fat diet can only induce steatosis even after long-term feeding, while adding 1-2% cholesterol to the diet is a necessary condition for achieving inflammation and fibrosis. Therefore, reducing cholesterol may become a new therapeutic strategy for fatty liver disease. Existing studies have shown that abnormal activation of SREBP is found in patients with fatty liver and in mouse models of fatty liver; deletion or knockout of liver-specific Scap in mice can eliminate the activation of all SREBPs, thereby preventing the occurrence of fatty liver and hyperlipidemia. In addition, recent studies have shown that abnormal activation of SREBP induced by endoplasmic reticulum stress promotes lipogenesis and fatty liver. Therefore, these evidences indicate that reducing the levels of triglycerides and cholesterol in the liver by inhibiting the SREBP pathway is an effective strategy for preventing and / or treating metabolic disorders including fatty liver. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a new compound having inhibitory activity against the SREBP pathway.

[0008] The present invention provides a compound represented by Formula VI or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Wherein, R 3a and R 3b are defined as described in any one of the following groups:

[0011] (i) R 3a is H, R 3b is -NHS(O)2CH3, -NHS(O)2CH2CH3, -NHS(O)2CH2CH2CH3, -NHS(O)2CF3, -NH-C(O)-CH2OH, -CH2OH, -CH2-C(O)-CH2OH, -OR 3c or -CH(OH)-CH2CH2OH; R 3c is -S(O)2OH, -C(O)-CH2COOH, -C(O)-CH2OH, -C(O)-COOH or -CH2CH2OH; and

[0012] (ii) R3a is CH3, R 3b is -OH;

[0013] R 22 is

[0014] In formula VI, when the carbon atom marked with * is a chiral carbon atom, it is in the R configuration, S configuration or a mixture of both.

[0015] In certain preferred embodiments of the present invention, some groups in the compounds of formula VI or XI or their pharmaceutically acceptable salts are defined as follows, and the groups not mentioned are the same as those described in any embodiment of the present invention (abbreviated as "in some embodiments" or "in some preferred embodiments").

[0016] In some embodiments, in the compound of formula VI as described above, R 3a and R 3b are defined as described in any of the following groups:

[0017] (i) R 3a is H, R 3b is -NHS(O)2CH3, -NH-C(O)-CH2OH, -CH2OH, -CH2-C(O)-CH2OH, -OR 3c or -CH(OH)-CH2CH2OH; R 3c is -S(O)2OH, -C(O)-CH2COOH, -C(O)-CH2OH, -C(O)-COOH or -CH2CH2OH; and

[0018] (ii) R 3a is CH3, R 3b is -OH;

[0019] R 22 is

[0020] In formula VI, when the carbon atom marked with * is a chiral carbon atom, it is in the R configuration, S configuration or a mixture of both.

[0021] In some embodiments, in the compound of formula VI as described above, R 3a and R 3b are defined as described in any of the following groups:

[0022] (i) R 3a is H, R 3b is -NHS(O)2CH3, -OR 3c or -CH(OH)-CH2CH2OH; R 3cis -S(O)2OH, -C(O)-CH2COOH, -C(O)-CH2OH, -C(O)-COOH or -CH2CH2OH;

[0023] (ii) R 3a is CH3, R 3b is -OH; and

[0024] R 22 is

[0025] In Formula VI, when the carbon atom marked with * is a chiral carbon atom, it is in the R configuration, S configuration or a mixture of both.

[0026] In some embodiments, in the compounds of Formula VI as described above, R 3a is H, R 3b is -NHS(O)2CH3, -NH-C(O)-CH2OH or -OR 3c ; or, R 3a is CH3, R 3b is -OH.

[0027] In some embodiments, in the compounds of Formula VI as described above, R 3c is -C(O)-CH2COOH, -C(O)-CH2OH or -C(O)-COOH.

[0028] In some embodiments, in the compounds of Formula VI as described above, R 3b is -OR 3c ; R 3c is -C(O)-CH2COOH, -C(O)-CH2OH or -C(O)-COOH.

[0029] In some embodiments, in the compounds of Formula VI as described above, R 3a is H, R 3b is -OR 3c , R 3c is -S(O)2OH.

[0030] In some embodiments, in the compounds of Formula VI as described above, R 3a is H, R 3b is -OR 3c , R 3c is -C(O)-CH2COOH.

[0031] In some embodiments, in the compounds of Formula VI as described above, R 3a is H, R 3b is -OR 3c , R3c is -C(O)-CH2OH.

[0032] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -OR 3c , R 3c is -C(O)-COOH.

[0033] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -OR 3c , R 3c is -CH2CH2OH.

[0034] In some embodiments, in the compound of formula VI as described above, R 3a is CH3, R 3b is -OH.

[0035] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -NHS(O)2CH3.

[0036] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -NH-C(O)-CH2OH.

[0037] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -CH2OH.

[0038] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -CH2-C(O)-CH2OH.

[0039] In some embodiments, in the compound of formula VI as described above, R 3a is H, R 3b is -CH(OH)-CH2CH2OH.

[0040] In some embodiments, in the compound of formula VI as described in any of the previous embodiments, when the carbon atom marked with * in formula VI is a chiral carbon atom, it has the R configuration.

[0041] In some embodiments, in the compound of formula VI as described in any of the previous embodiments, when the carbon atom marked with * in formula VI is a chiral carbon atom, it has the S configuration.

[0042] In some embodiments, in the compound of formula VI as described in any of the previous embodiments, in formula VI, the carbon atom marked with * is a mixture of S configuration and R configuration, for example, S configuration:R configuration = 1:1.

[0043] In some embodiments, the compound of formula VI is any of the following compounds:

[0044]

[0045]

[0046] The present invention also provides a compound of formula XI or a pharmaceutically acceptable salt thereof:

[0047]

[0048] wherein, R 3d is H or R 3c ; the definition of R 3c is as described in any of the embodiments of formula VI;

[0049] R 7a is H, F, CH3 or CF3;

[0050] R 7b is F or OH;

[0051] Alternatively, R 7a and R 7b together with the carbon atom to which they are attached form R 22 is

[0052] In formula XI, the carbon atom marked with * is in R configuration, S configuration or a mixture of both; when the carbon atom marked with # is a chiral carbon atom, it is in R configuration, S configuration or a mixture of both.

[0053] In some embodiments, in the compound of formula XI as described above, R 3d is H.

[0054] In some embodiments, in the compound of formula XI as described above, R 3d is R 3c ; the definition of R 3c is as described in any of the embodiments of formula VI.

[0055] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 22 is

[0056] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 22 is

[0057] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 7a is CH3, R 7b is OH; or, R 7a and R 7b together with the carbon atom to which they are attached form

[0058] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 7a is H, R 7b is F.

[0059] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 7a is CF3, R 7b is OH.

[0060] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 7a is F, R 7b is F.

[0061] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 7a is CH3, R 7b is OH.

[0062] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, R 7a and R 7b together with the carbon atom to which they are attached form

[0063] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, the carbon atom marked with * has the R configuration.

[0064] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, the carbon atom marked with * has the S configuration.

[0065] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, the carbon atom marked with * has a mixture of S configuration and R configuration, for example, S configuration:R configuration = 1:1.

[0066] In some embodiments, in the compound of formula XI as described in any of the previous embodiments, the carbon atom marked with # has the R configuration.

[0067] In some embodiments, in the compounds of formula XI as described in any of the previous embodiments, the #-labeled carbon atom has the S configuration.

[0068] In some embodiments, in the compounds of formula XI as described in any of the previous embodiments, the #-labeled carbon atom is a mixture of the S configuration and the R configuration, for example, S configuration:R configuration = 1:1.

[0069] In some embodiments, the compound of formula XI is any of the following compounds:

[0070]

[0071]

[0072] The compounds of the present invention can be prepared from known starting materials (such as lanosterol) by various conventional reaction methods in the art (such as hydroxyl protection, double bond ozonolysis, wittig reaction, hydrolysis reaction, amide condensation reaction, Grignard reagent addition reaction, reduction reaction, nucleophilic substitution reaction, epoxidation reaction). Exemplary preparation methods are described in the preparation examples of this application.

[0073] For example, the preparation method of compound 37 and its analogs can use lanosterol as the starting material, and through hydroxyl protection, double bond ozonolysis, wittig reaction, hydrolysis, and condensation to obtain compound 37 and its analogs; the reaction route is as follows:

[0074]

[0075] For example, the preparation method of compound 101 and its analogs can use lanosterol as the starting material, and through hydroxyl protection, double bond ozonolysis, wittig reaction, hydrolysis, and Grignard reagent addition reaction to obtain compound 101 and its analogs; the reaction route is as follows:

[0076]

[0077] For example, the preparation method of compound 80 and its analogs can use lanosterol as the raw material, and through hydroxyl protection, double bond ozonation, reduction, two-step substitution, and addition reaction to obtain compound 80 and its analogs; the reaction route is as follows:

[0078]

[0079] For example, the preparation method of compound 125 and its analogs can use lanosterol as the raw material, and through hydroxyl protection, double bond ozonolysis, wittig reaction, hydrolysis, epoxidation, and hydrolysis to finally obtain compound 125 and its analogs; the reaction route is as follows:

[0080]

[0081] For example, the preparation method of compound 193 and its analogs can use lanosterol as the starting material, through hydroxyl protection, double bond ozonation, double bond shift, double bond ozonation, reductive amination, condensation, and hydrolysis to obtain compound 193. The reaction route of the preparation method is shown in the following figure:

[0082]

[0083] For example, the preparation method of compound 199 and its analogs uses (22E,24S)-stigmasta-6(5),22(23)-dien-3β-ol as the starting material, through hydroxyl protection, double bond ozonation, Wittig reaction, hydrolysis, Wittig reaction, double bond reduction, organolithium reagent addition, and epoxidation to obtain product 199. The reaction route of the preparation method is shown in the following figure:

[0084]

[0085] The present invention also provides a pharmaceutical composition, which comprises the compound or its pharmaceutically acceptable salt as described above, and at least one pharmaceutical excipient.

[0086] The present invention also provides the use of the compound or its pharmaceutically acceptable salt as described above or the pharmaceutical composition as described above in the preparation of a drug for preventing and / or treating diseases, wherein the diseases are obesity, hyperlipidemia, fatty liver, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, liver cancer or skin injury.

[0087] The present invention also provides the use of the compound or its pharmaceutically acceptable salt as described above or the pharmaceutical composition as described above in the preparation of a drug for inhibiting the SREBP pathway.

[0088] The present invention also provides a method for inhibiting the SREBP pathway, which comprises administering to a subject an effective amount of the compound or its pharmaceutically acceptable salt as described above.

[0089] The present invention also provides a method for preventing and / or treating diseases, which comprises administering to a subject an effective amount of the compound or its pharmaceutically acceptable salt as described above, wherein the diseases are obesity, hyperlipidemia, fatty liver, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, liver cancer or skin injury.

[0090] Definitions and explanations

[0091] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without a specific definition, but should be understood in its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding product or its active ingredient.

[0092] As used herein, the term "substituted" or "substituent" means that a hydrogen atom in a group is replaced by a specified group. When the substitution position is not specified, the substitution can occur at any position, but only if a stable or chemically feasible compound is formed. Examples are as follows: The structure represents that the hydrogen atoms on the benzene ring are replaced by q R 8 When there are multiple R 8 each R 8 is the same or different.

[0093] When any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted by 0 - 2 R, the group may optionally be substituted by up to two R, and each R has independent options in each case. In addition, combinations of substituents and / or their variants are only permitted if such combinations result in a stable compound.

[0094] When the connecting groups listed herein do not specify their connecting directions, the connecting directions can be arbitrary, including both connecting from left to right and from right to left. Examples are as follows. In -A-L-B, the connecting group L is -C-D-. When the connecting direction of L is not specified, -A-L-B includes -A-C-D-B and -A-D-C-B.

[0095] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

[0096] As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain monovalent hydrocarbon group. C1-C6 alkyl represents an alkyl group having 1 - 6 carbon atoms. In some embodiments, C1-C6 alkyl can be C1-C4 alkyl. C1-C4 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0097] As used herein, the term "alkenyl" refers to a straight-chain or branched-chain monovalent hydrocarbon group containing at least one carbon-carbon double bond. When an alkenyl contains both saturated and unsaturated carbon atoms, it can be linked to other structures either through the saturated carbon atoms or through the unsaturated carbon atoms. C2-C4 alkenyl means an alkenyl having 2, 3 or 4 carbon atoms. Specific examples of alkenyl include, but are not limited to, vinyl and allyl.

[0098] As used herein, the term "alkylene" refers to a saturated straight-chain or branched-chain divalent hydrocarbon group. C1-C4 alkylene refers to an alkylene having 1-4 carbon atoms, specifically methylene, ethylene (e.g., -CH2CH2-, -CH(CH3)-), propylene (e.g., -CH2CH2CH2-, -C(CH3)2-, -CH2CH(CH3)-), butylene (e.g., -CH2CH2CH2CH2-, -CH(CH3)CH(CH3)-, -CH2CH(CH3)CH2-).

[0099] As used herein, the term "fluoroalkyl" refers to a group formed by replacing one or more hydrogen atoms in an alkyl group with fluorine, where the definition of the alkyl group is as described above. Examples of fluoroalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl.

[0100] As used herein, the term "alkoxy" refers to -O-alkyl, where the definition of the alkyl group is as described above. C1-C4 alkoxy refers to -O-(C1-C4 alkyl), where the definition of C1-C4 alkyl is as described above, that is, C1-C4 alkoxy can specifically be methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

[0101] As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic (e.g., fused-ring, spiro-ring or bridged-ring) cyclic hydrocarbon group. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, C 3-10 The cycloalkyl can specifically be C3, C4, C5, C6, C7, C8, C9, C 10 cycloalkyl. C 3-6 The cycloalkyl can specifically be C3, C4, C5, C6 cycloalkyl. In some embodiments, the cycloalkyl is monocyclic. In some embodiments, the cycloalkyl is polycyclic (e.g., fused-ring, spiro-ring or bridged-ring).

[0102] As used herein, the term "heterocycloalkyl" refers to a saturated monocyclic or polycyclic (e.g., fused-ring, spiro-ring or bridged-ring) cyclic group formed by carbon atoms and at least one heteroatom, where the heteroatom is independently selected from N, O and S. The heterocycloalkyl can be linked to other structures through the carbon atoms and heteroatoms on the ring. Examples of heterocycloalkyl include, but are not limited to Tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl. The 3- to 10-membered heteroalkyl group can specifically be a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-membered heteroalkyl group. The 3- to 6-membered heteroalkyl group can specifically be a 3-, 4-, 5- or 6-membered heteroalkyl group. In some embodiments, the heteroalkyl group is monocyclic. In some embodiments, the heteroalkyl group is polycyclic (e.g., fused ring, spiro ring or bridged ring).

[0103] As used herein, the term "C 6-10 aryl" refers to phenyl or naphthyl.

[0104] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or fused ring group formed by carbon atoms and at least one heteroatom, where the heteroatoms are independently selected from N, O, and S. The 5- to 10-membered heteroaryl group can specifically be a 5-, 6-, 7-, 8-, 9- or 10-membered heteroaryl group, such as a 5- to 6-membered heteroaryl group or an 8- to 10-membered fused heteroaryl group. The 5- to 6-membered heteroaryl group is monocyclic, and specific examples include but are not limited to pyrrole, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, pyrazole, imidazole, pyridine, pyrimidine, pyrazine. Examples of the 8- to 10-membered fused heteroaryl group include but are not limited to benzopyrrole, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzopyrazole, benzimidazole, benzopyridine, benzopyrimidine, benzopyrazine, thiazolothiazole, pyridinopyridine, pyridinopyrazine, pyridinopyrimidine.

[0105] As used herein, in a chemical structural formula, represents the connection position. When is included in a cyclic group and the ring atom to which it is attached is not specified, it can be attached to any ring atom, but only a stable or chemically feasible chemical compound is allowed. For example, includes structures such as etc.

[0106] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a suitable non-toxic organic acid, inorganic acid, organic base or inorganic base with a compound, which retains the biological activity of the compound. The organic acids can be various organic acids capable of forming salts that are conventional in the art, preferably one or more of methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, 2-hydroxyethanesulfonic acid, naphthalenesulfonic acid and salicylic acid. The inorganic acids can be various inorganic acids capable of forming salts that are conventional in the art, preferably one or more of hydrochloric acid, sulfuric acid and phosphoric acid. The organic bases can be various organic bases capable of forming salts that are conventional in the art, preferably one or more of pyridines, imidazoles, pyrazines, indoles, purines, tertiary amines and anilines. The tertiary amine organic bases are preferably triethylamine and / or N,N-diisopropylethylamine. The aniline organic base is preferably N,N-dimethylaniline. The pyridine organic bases are preferably one or more of pyridine, methylpyridine, 4-dimethylaminopyridine and 2-methyl-5-ethylpyridine. The inorganic bases can be various inorganic bases capable of forming salts that are conventional in the art, preferably one or more of alkali metal hydrides, hydroxides of alkali metals, alkoxides of alkali metals, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium bicarbonate and sodium bicarbonate. The alkali metal hydrides are preferably sodium hydride and / or potassium hydride. The hydroxides of alkali metals are preferably one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide. The alkoxides of alkali metals are preferably one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide and sodium tert-butoxide.

[0107] In the chemical structure, a solid wedge bond and a dashed wedge bond are used to represent the absolute configuration of a stereocenter, and a solid straight bond and a dashed straight bond are used to represent the relative configuration of a stereocenter. The bond does not specify the configuration, that is, if there is configurational isomerism in the chemical structure, the bond can be or or can contain both and two configurations (for example, the ratio of and is 1:1). When the configuration of a carbon-carbon double bond is not specified, it can be in the E or Z configuration. Stereoisomers can be synthesized using chiral starting materials, prepared by chiral resolution, or can be resolved using conventional techniques such as, but not limited to, high performance liquid chromatography (HPLC) using a chiral column.

[0108] As used herein, the term "subject" includes any animal, preferably a mammal, more preferably a human.

[0109] In this text, the term "effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in a particular case can be determined by those skilled in the art based on routine tests.

[0110] Without departing from the common general knowledge in the art, on the basis of the above preferred conditions, any combination can be made to obtain various preferred examples of the present invention.

[0111] The reagents and raw materials used in the present invention are all commercially available.

[0112] The positive and progressive effects of the present invention are as follows: The present invention provides a class of new compounds that have inhibitory activity against the SREBP pathway and can be used for the prevention and / or treatment of diseases such as obesity, hyperlipidemia, fatty liver, diabetes, atherosclerosis, cardiovascular and cerebrovascular diseases, liver cancer, skin damage, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 Results of 25-hydroxycholesterol inhibiting the weight gain of mice induced by AMLN diet.

[0114] Figure 2 Effect of 25-hydroxycholesterol on the food intake of mice.

[0115] Figure 3 25-Hydroxycholesterol reduces the total cholesterol content in the blood of mice.

[0116] Figure 4 25-Hydroxycholesterol reduces the total triglyceride content in the blood of mice.

[0117] Figure 5 25-Hydroxycholesterol reduces the total cholesterol content in the liver of mice.

[0118] Figure 6 25-Hydroxycholesterol reduces the total triglyceride content in the liver of mice.

[0119] Figure 7 25-Hydroxycholesterol reduces the level of aspartate aminotransferase (AST) in the blood of mice.

[0120] Figure 8 25-Hydroxycholesterol reduces the level of alanine aminotransferase (ALT) in the blood of mice.

[0121] Figure 9 Results of HE staining of mouse liver sections: C57BL / 6J CD group.

[0122] Figure 10 Results of HE staining of mouse liver sections: Ldlr - / -CD group.

[0123] Figure 11 The results of HE staining of mouse liver sections: Ldlr - / - AMLN group.

[0124] Figure 12 The results of HE staining of mouse liver sections: Ldlr - / - AMLN + 25-HL group.

[0125] Figure 13 The results of HE staining of mouse liver sections and the quantitative statistical results of NAFLD activity scores for 4 groups of mice.

[0126] Figure 14 The results of Oil Red O staining of mouse liver sections: C57BL / 6J CD group.

[0127] Figure 15 The results of Oil Red O staining of mouse liver sections: Ldlr - / - CD group.

[0128] Figure 16 The results of Oil Red O staining of mouse liver sections: Ldlr - / - AMLN group.

[0129] Figure 17 The results of Oil Red O staining of mouse liver sections: Ldlr - / - AMLN + 25-HL group.

[0130] Figure 18 The quantitative results of Oil Red O staining of mouse liver sections for 4 groups of mice.

[0131] Figure 19 The results of Sirius red staining of mouse liver sections: C57BL / 6J CD group.

[0132] Figure 20 The results of Sirius red staining of mouse liver sections: Ldlr - / - CD group.

[0133] Figure 21 The results of Sirius red staining of mouse liver sections∶Ldlr - / - AMLN group.

[0134] Figure 22 The results of Sirius red staining of mouse liver sections: Ldlr - / - AMLN + 25-HL group.

[0135] Figure 23 The quantitative results of Sirius red staining of mouse liver sections for 4 groups of mice.

[0136] Figure 24Results of F4 / 80 immunostaining of mouse liver sections and imaging results of polarized light imaging indicating cholesterol crystals: C57BL / 6J CD group.

[0137] Figure 25 Results of F4 / 80 immunostaining of mouse liver sections and imaging results of polarized light imaging indicating cholesterol crystals: Ldlr - / - CD group.

[0138] Figure 26 Results of F4 / 80 immunostaining of mouse liver sections and imaging results of polarized light imaging indicating cholesterol crystals: Ldlr - / - AMLN group.

[0139] Figure 27 Results of F4 / 80 immunostaining of mouse liver sections and imaging results of polarized light imaging indicating cholesterol crystals: Ldlr - / - AMLN + 25-HL group.

[0140] Figure 28 Quantitative results of F4 / 80 immunostaining of mouse liver sections in 4 groups.

[0141] Figure 29 Quantitative imaging results of polarized light imaging indicating cholesterol crystals in mouse liver sections in 4 groups.

[0142] Figure 30 Results of Sudan Red IV staining of mouse aortic tree: C57BL / 6J CD group.

[0143] Figure 31 Results of Sudan Red IV staining of mouse aortic tree: Ldlr - / - CD group.

[0144] Figure 32 Results of Sudan Red IV staining of mouse aortic tree: Ldlr - / - AMLN group.

[0145] Figure 33 Results of Sudan Red IV staining of mouse aortic tree: Ldlr - / - AMLN + 25-HL group.

[0146] Figure 34 Quantitative results of Sudan Red IV staining of mouse aortic tree in 4 groups.

[0147] Figure 35 25-Hydroxycholesterol reduces the expression of lipidogenesis-related genes (Hmgcs, Hmgcr, SCD1, FASN) in liver organoids.

[0148] Figure 3625-Hydroxycholesterol reduces the expression of fibrosis-related genes (Col1a1, aSMA) in liver organoids.

[0149] Figure 37 Bright-field imaging results of mouse liver organoids: vehicle control group.

[0150] Figure 38 Bright-field imaging results of mouse liver organoids: obeticholic acid 1 μM group.

[0151] Figure 39 Bright-field imaging results of mouse liver organoids: obeticholic acid 3 μM group.

[0152] Figure 40 Bright-field imaging results of mouse liver organoids: 25-HL 1 μM group.

[0153] Figure 41 Bright-field imaging results of mouse liver organoids: 25-HL 3 μM group.

[0154] Figure 42 Nile red staining results of mouse liver organoids: vehicle control group.

[0155] Figure 43 Nile red staining results of mouse liver organoids: obeticholic acid 1 μM group.

[0156] Figure 44 Nile red staining results of mouse liver organoids: obeticholic acid 3 μM group.

[0157] Figure 45 Nile red staining results of mouse liver organoids: 25-HL 1 μM group.

[0158] Figure 46 Nile red staining results of mouse liver organoids: 25-HL 3 μM group.

[0159] Figure 47 Quantitative results of Nile red staining of liver organoids from 5 groups of mice.

[0160] Figure 48 Immunofluorescence staining results of the fibrosis marker protein αSMA in mouse liver organoids: vehicle control group.

[0161] Figure 49 Immunofluorescence staining results of the fibrosis marker protein αSMA in mouse liver organoids: obeticholic acid 1 μM group.

[0162] Figure 50 Immunofluorescence staining results of the fibrosis marker protein αSMA in mouse liver organoids: obeticholic acid 3 μM group.

[0163] Figure 51 Immunofluorescence staining results of the fibrosis marker protein αSMA in mouse liver organoids: 25-HL 1 μM group.

[0164] Figure 52 Immunofluorescence staining results of the fibrosis marker protein αSMA in mouse liver organoids: 25-HL 3 μM group.

[0165] Figure 53 Quantitative results of immunofluorescence staining of the fibrosis marker protein αSMA in liver organoids of 5 groups of mice. Detailed implementation manners

[0166] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0167] In the following examples, 25-hydroxycholesterol (25-HL) refers to the compound in Example 68.

[0168] Animals for biological test examples: Adult male C57BL / 6J mice were purchased from Shanghai SLAC Co., Ltd., and low-density lipoprotein receptor gene knockout mice (Ldlr - / - mice, T001464) were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. The mice were all raised under specific pathogen-free conditions, maintained under a 12-hour light / dark cycle, and had free access to water and feed. The gastric lavage vehicle was: 0.5% Tween-80, 0.5% methylcellulose, 0.9% sodium chloride. The AMLN diet (AMLN, Dyets) contained 40% (calorie percentage) fat (80% of which was trans fat), 22% (mass ratio) fructose, and 2% (mass ratio) cholesterol.

[0169] Reagents: Mevalonic acid (41288), paraformaldehyde (P6148), Tween-80 (P8074), methyl cellulose (V900506), Oil Red O (O0625), and the nuclear staining reagent DAPI were purchased from Sigma-Aldrich. Lovastatin (purity ≥98.5%, HPLC) was purchased from Shanghai Pharmaceutical Valley. Dulbecco's modified eagle medium (DMEM) for cell culture was purchased from Thermo Scientific, and fetal bovine serum (S1580) was purchased from Biowest. Deproteinized serum (LPDS) was prepared by our laboratory through ultracentrifugation. Obeticholic acid (OCA, CAS registry number 459789-99-2, purity 98% (HPLC)). Kits for total cholesterol and total triglyceride were purchased from Shanghai Kehua Bio-Engineering Co., Ltd. Kits for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were purchased from Lai Er Bio-tech. Hematoxylin-eosin staining kits (6765001, 6766010) were purchased from Thermo Scientific. Sirius red staining kit (ab150681) was purchased from Abcam. Sudan red IV (A610914) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Nile red (HY-D0718) was purchased from MCE.

[0170] Antibodies: Antibodies used for immunofluorescence staining analysis were as follows: Anti-alpha smooth muscle Actin (αSMA, ab7817, 1:500) antibody was purchased from Abcam; The secondary antibody, fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) (115-095-003), was purchased from Jackson Immunoresearch. Anti-F4 / 80 primary antibody (*14-4801-85, Invitrogen, 1:100), and the fluorescent secondary antibody Alexa Fluor Plus 488-conjugated goat anti-rat IgG (A-11006, 1:500) were purchased from Invitrogen.

[0171] Biological test Example 1: Cell culture

[0172] The human hepatocellular carcinoma cell line Huh-7 / SRE-Luc was grown in a medium containing DMEM, 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin, and 200 μg / ml G418.

[0173] Biological test Example 2: SREBP luciferase reporter gene system

[0174] The Huh-7 / SRE-Luc cell line stably expresses LDLR promotor-luciferase and green fluorescent protein (GFP) in the human hepatocellular carcinoma cell line Huh-7. Among them, the LDLR promotor region contains a sterol-regulatory-element (SRE), which can effectively and sensitively respond to the regulation of the transcription factor SREBP. The GFP signal serves as an internal reference to indicate changes in cell number. Therefore, this cell line can be used to screen for bioactive small molecules that regulate the SREBP signaling pathway. In our work, this cell line was used to screen for inhibitors that can effectively inhibit the SREBP signaling pathway. The cells were incubated in a sterol-deficient medium (5% lipoprotein-depleted serum, 2 μM lovastatin, 10 μM mevalonic acid), and at the same time, treated with the corresponding concentration of the compound for 16 hours. After the compound treatment, the cells were lysed with lysis buffer (E397A, Promega). After adding the luciferase substrate (E1500, Promega), the activity of SRE-driven luciferase was measured by a BioTek SynergyHTX microplate reader (including but not limited to this type of instrument). The fluorescence intensity of green fluorescent protein (EGFP) was also measured by the above-mentioned BioTek microplate reader (including but not limited to this type of instrument) and used as an internal reference. The ratio obtained by dividing the SRE-driven luciferase activity by the fluorescence intensity of green fluorescent protein was used as an index of SREBP pathway activity. The test data of each test compound were analyzed by prism software to obtain the IC 50 parameter.

[0175] Biological Test Example 3: Real-time Fluorescent Quantitative PCR

[0176] Liver or liver organoid samples were homogenized in TRI Reagent (T9424, Sigma), and total RNA was extracted according to the manufacturer's operation manual. Equal amounts of RNA templates were used with oligodT primers and MLV reverse transcriptase (Promega) to synthesize cDNA. The quantitative data of the genes were collected by a Bio-Rad CFX96 real-time PCR System, and the relative amount of gene mRNA was quantified by the relative CT method. The primer sequences used are shown in the following table:

[0177]

[0178]

[0179] Biological Test Example 4 Measurement of Serum and Liver Metabolic Parameters

[0180] After the mice were treated with the drug, the feed was removed and the mice were starved for 4 hours and then sacrificed. Blood and liver were collected. After blood coagulation, it was centrifuged at 1500 g for 10 minutes at 4 °C, and the supernatant was the serum. Lipids in the liver were extracted by the chloroform / methanol method. First, it was homogenized and broken by a homogenizer Precelly24, centrifuged at 16000 g for 10 minutes at 4 °C, the organic phase was transferred to a new tube, dried with nitrogen, and then dissolved in ethanol. The total cholesterol and triglyceride levels in blood and liver were measured by cholesterol and triglyceride assay kits (Shanghai Kehua Bio-Engineering Co., Ltd.) respectively. ALT (LE-M0477, Lai Er Bio-tech) and AST (LE-M0568, Lai Er Bio-tech) in the serum were measured using the analysis system of Sysmex Medical Electronics (Shanghai) Co., Ltd. according to the manuals of the corresponding manufacturers respectively.

[0181] Biological Test Example 5: Histological Section Analysis of Liver

[0182] Hematoxylin-eosin staining: The harvested liver was fixed in 4% paraformaldehyde at 4°C for overnight, embedded in paraffin, sectioned with a paraffin slicer (Leica RM2235) at a thickness of 7 μm, deparaffinized and rehydrated, and then stained with a hematoxylin-eosin staining kit (6765001, 6766010, Thermo Scientific). Images were taken with an Olympus VS120 slide microscope and quantified using ImageJ software. Oil Red O staining: The liver was embedded with OCT compound (Leica), sectioned with a cryostat (Leica CM1950) at a thickness of 7 μm, and stained with Oil Red O (O0625, Sigma). Images were taken with an Olympus VS120 slide microscope and quantified using ImageJ software. Sirius Red collagen staining: After deparaffinization and rehydration of the paraffin sections of the liver, staining was performed with a Sirius Red staining kit (ab150681, Abcam) according to the manufacturer's instructions. Immunofluorescence staining: The liver was embedded with OCT compound (Leica), sectioned with a cryostat (Leica CM1950) at a thickness of 7 μm. The cryosections were stained with anti-F4 / 80 rat monoclonal antibody (14-4801-85, Invitrogen, 1:100), and Alexa Fluor 488-conjugated goat anti-rat IgG secondary antibody (A-11006, Invitrogen, 1:500), and the nuclei were specifically stained with DAPI (Sigma) at 30. After staining and coverslipping, images were taken with a spinning disk confocal microscope (Nikon CSU-W1 SoRa) and quantified using ImageJ software. Polarized light imaging of cholesterol crystals: After F4 / F80 and DAPI staining of the cryosections, images were taken with a spinning disk confocal microscope (Nikon CSU-W1 SoRa) equipped with a polarized light filter and quantified using ImageJ software.

[0183] Biological Test Example 6 Isolation of Mouse Arterial Tree and Sudan Red IV Staining of Atherosclerotic Plaques

[0184] After the mouse dosing experiment was completed, the aorta was isolated and fixed in 4% PFA. After removing the perivascular adipose tissue with ophthalmic forceps under a stereomicroscope, it was stained with Sudan IV, and the atherosclerotic plaques were washed with 70% ethanol. After staining, the aortic tree was imaged with a ZEISS Axio Zoom.V16 stereomicroscope. The atherosclerotic lesions were quantified using ImageJ software.

[0185] Biological Test Example 7 Preparation and Culture of Mouse Liver Organoids

[0186] C57BL / 6N mice were fed a fatty liver-inducing diet (Trophic Diet, TP2630052A, containing 10.2% kcal protein, 37.3% kcal carbohydrates, and 52.6% kcal fat) and fructose-containing drinking water (23.1 g fructose and 18.9 g glucose dissolved in 1 L of water, then filter-sterilized) for 16 weeks to generate a fatty liver model. To generate fatty liver liver organoids, the liver tissue of fatty liver mice was minced and digested in digestion buffer at 37 °C for 30 - 60 minutes. The digestion buffer included DMEM / F-12 (Cytiva, SH30023.01), 2.5 mg / mL collagenase D (Roche, COLLD-RO), and 0.1 mg / mL DNase I (Sigma-Aldrich, DN25). The isolated single hepatocytes were filtered through a 70 μm filter membrane and washed once. The cells were collected by centrifugation and resuspended by mixing the medium with basement membrane extract (BME) (R&D Systems, 3533-010-02) in a ratio of 1:3. The medium included AdDMEM / F12, 10 mM Hepes, 1xGlutamax, 1% pen / strep, 1 x B27, 1 x N2, NAC (1 mM), NIC (10 mM), Gastrin (10 nM), EGF (50 ng / mL), FGF10 (100 ng / mL), A83-01 (5 μM), Rki (10 μM), 10% RSPO1 conditioned medium, 30% Wnt3a conditioned medium, and 5% Noggin conditioned medium. Before administration, the organoids were digested with trypsin (Gibco, Cat#25200072) and resuspended. After 24 hours, using DMSO as a blank control, the medium of the organoids was replaced with a medium containing different concentrations of OCA or 25-HL. The fatty liver liver organoids were divided into 5 groups (*1‰ DMSO, 1 μM OCA, 3 μM OCA, 1 μM 25-HL, and 3 μM 25-HL) and treated with the drug for 72 hours respectively.

[0187] Biological Test Example 8 Histochemical Staining of Mouse Liver Organoids

[0188] Immunocytochemistry: The organoids were fixed in immunostaining fixative (Beyotime Biotechnology, P0098) overnight at 4°C. Then they were washed in PBS and then treated with PBS containing 0.5% Triton X-100 for 20 minutes at room temperature. Then the organoids were blocked with a PBS blocking solution containing 10% goat serum for 1 hour at room temperature and incubated with the primary antibody (Anti-alpha smooth muscle Actin, ab7817, Abcam, diluted 1:500) overnight at 4°C. The next day, the organoids were washed and co-incubated with the secondary antibody fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L) (Jackson Immunoresearch, 115-095-003). The cell nuclei were counterstained with Fluoreshield T M and DAPI (Sigma-Aldrich, F6057).

[0189] To observe lipid droplets, the organoids were fixed with 4% paraformaldehyde (PFA) for 1 hour and stained with 250 nmol / L Nile Red (MCE, HY-D0718) for 3 minutes at room temperature. The organoids were rinsed twice with PBS before imaging. The stained organoids were observed under a Dragonfly high-speed confocal microscopy system (Andor, Dragonfly200).

[0190] Biological Test Example 9 Inhibitory Effect of the Compounds of the Present Invention on the SREBP Pathway

[0191] The inhibitory effect of the compounds of the present invention on the SREBP pathway was tested by the method of Biological Test Example 2. The concentration gradient of each compound was designed as 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10 μM, and the control was the solvent DMSO. The IC 50 values of some compounds are shown in Table 1.

[0192] Table 1: Activity Data of Compounds in Some Examples

[0193]

[0194]

[0195]

[0196] Biological Test Example 10 25-Hydroxycholesterol Reduces Blood Lipid Levels, Alleviates Fatty Liver and Liver Injury

[0197] Elevated blood lipid levels and hepatic lipid accumulation are known risk factors for fatty liver. The present invention further analyzed whether 25-hydroxycholesterol can alleviate the typical symptoms of diet-induced fatty liver in mice: lipid accumulation, liver injury, inflammation, and fibrosis.

[0198] Purchase 8-week-old male C57BL / 6J mice and male Ldlr - / - mice (T001464, Jiangsu Genscript Biotech Co., Ltd.), group them and feed them different diets and administer different drug treatments. The mice were randomly divided into 4 groups, with 8-9 mice in each group: 1 group was the C57BL / 6J wild-type mice fed with a basal diet (chow diet, CD) solvent control group. The other 3 groups were all Ldlr - / - gene knockout mice, which were respectively the basal diet (CD) solvent control group, the AMLN diet (AMLNdiet, containing 20% fat, 22% fructose, 2% cholesterol) solvent control group, and the AMLN diet 25-hydroxycholesterol administration group (the administration concentration of 25-hydroxycholesterol was 30 mg / kg / day). The 4 groups of mice were gavaged once a day, and the food intake and body weight changes of the mice in different treatment groups were counted during this period. After 8 weeks, the blood and livers of the mice were collected to analyze the phenotypes of blood lipids, liver lipids, and liver injury.

[0199] The results are as Figures 1-8 shown, among which, Figure 1 is the statistical result of weekly weighing of mice in each group, Figure 2 is the cumulative statistical result of food intake of mice in each group, Figure 3 is that 25-hydroxycholesterol reduces the total cholesterol content in the blood of mice, Figure 4 is that 25-hydroxycholesterol reduces the total triglyceride content in the blood of mice, Figure 5 is that 25-hydroxycholesterol reduces the total cholesterol content in the liver of mice, Figure 6 is that 25-hydroxycholesterol reduces the total triglyceride content in the liver of mice, Figure 7 is that 25-hydroxycholesterol reduces the level of aspartate aminotransferase (AST) in the blood of mice, Figure 8 is that 25-hydroxycholesterol reduces the level of alanine aminotransferase (ALT) in the blood of mice.

[0200] Figure 1 and Figure 2 In, the P value was calculated by two-way ANOVA (Dunnett's multiple comparisons test) statistical analysis; * indicates P < 0.05; ns indicates no statistical difference. Figures 3-8Data are expressed as mean ± standard deviation. P values were determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

[0201] Results showed that after 8 weeks of continuous administration, Ldlr - / - In mice fed with AMLN diet and administered with 25-hydroxycholesterol simultaneously, the weight gain was significantly lower than that in the AMLN diet vehicle control group. This indicates that 25-hydroxycholesterol has a good inhibitory effect on AMLN diet-induced weight gain. The pathological features of fatty liver disease mainly include hepatic steatosis, liver injury, inflammatory infiltration, and fibrosis. After 8 weeks of administration in the 4 groups of mice, we first detected the changes in lipid levels such as total cholesterol and total triglyceride in the blood of mice, as well as the changes in lipid levels of total cholesterol and total triglyceride in the liver. As Figures 3-4 shown: Compared with the control group, 25-hydroxycholesterol significantly decreased the levels of total cholesterol and total triglyceride in the serum. At the same time, as Figures 5-6 shown, 25-hydroxycholesterol significantly decreased the levels of total cholesterol and total triglyceride in the liver of mice. This indicates that 25-hydroxycholesterol has a good effect on reducing blood lipids and liver lipids. As can be seen from Figures 7-8 , the liver injury marker molecules aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were significantly decreased by 25-hydroxycholesterol. This indicates that 25-hydroxycholesterol has a good effect on improving liver injury.

[0202] Biological Test Example 11 25-Hydroxycholesterol Alleviates Fatty Liver and Atherosclerosis

[0203] Furthermore, we performed various staining or immunohistochemical operations on liver tissue sections of each group of mice to analyze the phenotypic changes in liver lipid accumulation and fatty liver. The results are as Figures 9-29 shown, among which, Figures 9-13 are the results of HE staining of mouse liver sections and the quantitative results of NAFLD activity score: 25-hydroxycholesterol reduced hepatic steatosis and significantly decreased the NAFLD activity score; Figures 14-18 are the results of Oil Red O staining and quantification of mouse liver sections: 25-hydroxycholesterol reduced lipid droplets in the liver; Figures 19-23 are the results of Sirius red staining and quantification of mouse liver sections: 25-hydroxycholesterol reduced fibrosis in the liver; Figures 24-29 are the results of F4 / 80 immunohistochemical staining and quantification of mouse liver sections, as well as the imaging and quantification results of polarized light imaging indicating cholesterol crystal formation: 25-hydroxycholesterol reduced the aggregation of Kupffer cells and the formation of cholesterol crystals in the liver.

[0204] Figures 9-29Quantitative analysis was performed using Image J software, and the data were expressed as mean ± standard deviation. P values were determined by one-way ANOVA. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0205] The results were as Figures 9-13 shown by hematoxylin-eosin staining: Male Ldlr - / - mice fed with AMLN diet for 8 weeks had obvious macrovesicular lipid droplets and ballooning degenerated hepatocytes in the liver. Compared with the control group, 25-hydroxycholesterol significantly reduced the above phenotypes. Meanwhile, Figures 14-18 the results of Oil Red O staining of liver tissue sections showed that compared with the control group, 25-hydroxycholesterol significantly reduced the lipid droplet accumulation of neutral lipids including cholesterol and fatty acids in the liver. These results were consistent with Figures 1-8 the results of 25-hydroxycholesterol reducing liver lipid content in Figures 24-29 which indicated that 25-hydroxycholesterol effectively improved the liver lipid accumulation phenotype induced by AMLN diet. In addition, the present invention further analyzed the changes in fatty liver-related inflammation and fibrosis phenotypes in the liver. - / - In Ldlr Figures 24-27 mice fed with AMLN diet, F4 / 80 specific staining showed that Kupffer cells aggregated together and formed a crown-like structure around cholesterol crystals ( Figures 24-29 ) (magnified image). 25-Hydroxycholesterol significantly reduced the crown-like structure formed by Kupffer cells, indicating that 25-hydroxycholesterol could significantly reduce liver inflammatory infiltration. The results of polarized light imaging of cholesterol crystals in liver sections showed that in vehicle control mice, a large number of cholesterol crystals were contained inside the crown-like structure formed by Kupffer cells. Compared with simple steatosis, these cholesterol crystals and the crown-like structure formed by Kupffer cells are the hallmark features of fatty liver, and macrophages are attracted by cholesterol crystals and attempt to clear these residual lipid droplets, similar to the phenomenon described in atherosclerosis. More importantly, 25-hydroxycholesterol significantly reduced the number of crown-like structures and cholesterol crystals ( Figures 19-23 ). Meanwhile, the results of Sirius red staining showed that 25-hydroxycholesterol significantly reduced the fibrotic phenotype of collagen fiber formation in liver tissue sections of mice. The above results suggest that 25-hydroxycholesterol alleviates liver fat accumulation and the symptoms of fatty liver, and can be used for the prevention and / or treatment of diseases such as hyperlipidemia and fatty liver.

[0206] Ldlr - / - mice fed with AMLN are also a commonly used model of atherosclerosis, so we can study fatty liver and atherosclerosis simultaneously. 8-week-old male Ldlr - / -Mice were fed with AMLN feed and administered drugs by gavage once a day for 8 weeks. After the mice were sacrificed, the aorta was isolated and fixed with 4% paraformaldehyde. The perivascular adipose tissue was removed under a stereomicroscope, stained with Sudan Red IV, and rinsed with 70% ethanol. The aortic tree was imaged using a stereomicroscope (Zeiss, Axio Zoom V16, Germany). And the atherosclerotic lesion plaques were quantitatively analyzed using ImageJ software.

[0207] The results are as Figures 30-34 shown, Figures 30-34 for the Sudan Red IV staining and quantitative results of the aortic tree of mice: 25-hydroxycholesterol reduces the formation of atherosclerotic plaques.

[0208] The results showed that after continuous administration for 8 weeks, the aortic trees of mice in each group were isolated, and specific lipid staining of Sudan Red IV for the aorta was performed. As Figures 30-34 shown, compared with the control group of mice, 25-hydroxycholesterol significantly reduced the formation and number of atherosclerotic plaques. 25-Hydroxycholesterol has a slowing effect on the formation of atherosclerosis.

[0209] Figures 1-8 To Figures 9-34 These data indicate that 25-hydroxycholesterol reduces the elevation of blood lipid levels, reduces fat accumulation, cholesterol crystallization, hepatocyte damage, inflammatory infiltration and fibrosis in the liver, and reduces the formation and number of atherosclerotic plaques. It shows that 25-hydroxycholesterol has good curative effects on hyperlipidemia, fatty liver and atherosclerosis.

[0210] Biological Test Example 12 uses in vitro liver organoids to prove that 25-hydroxycholesterol has an inhibitory effect on lipidogenesis and fibrosis in fatty liver

[0211] To distinguish whether the inhibitory effect of 25-hydroxycholesterol is directly based on its impact on the liver or a systemic effect, we used 3D liver organoids as an in vitro research model for fatty liver to analyze the effects of 25-hydroxycholesterol on lipidogenesis and the expression of fibrosis marker molecules in liver organoids. We fed C57BL / 6N mice with a fatty liver-inducing diet (Trophic Diet, TP2630052A, containing 10.2% kcal protein, 37.3% kcal carbohydrates, and 52.6% kcal fat) and fructose-containing drinking water (23.1 g fructose and 18.9 g glucose dissolved in 1 L of water, then filtered and sterilized) for 16 weeks at 12 weeks of age to establish a fatty liver model. Liver organoids were isolated and prepared from the livers of these model mice for in vitro culture. At the same time, the organoids were treated with drugs in vitro. Obeticholic acid (OCA) is an agonist of the farnesoid X receptor (FXR), and this drug has shown good anti-fatty liver effects in phase III clinical trials. In this example, this drug was used as a control drug. The organoids were grouped and treated with the same concentration gradients of obeticholic acid and 25-hydroxycholesterol for 3 days. The organoid RNA was collected and the expression differences of lipid synthesis-related genes (Hmgcs, Hmgcr; SCD1, FASN) and fibrosis marker molecules (αSMA, Col1a1) were analyzed by fluorescence real-time quantitative PCR.

[0212] The results are as Figures 35-53 shown, where Figure 35 25-hydroxycholesterol decreased the expression of lipidogenesis-related genes (Hmgcs, Hmgcr, and SCD1, FASN) in liver organoids, and the effect was better than that of the control drug obeticholic acid at the same concentration; Figure 36 25-hydroxycholesterol decreased the expression of fibrosis-related genes (Col1a1, αSMA) in liver organoids, and the effect was better than that of the control drug obeticholic acid at the same concentration; Figures 37-53 are the bright-field imaging results of liver organoids, the Nile red staining and quantification results, and the immunofluorescence staining and quantification results of the fibrosis marker protein αSMA: 25-hydroxycholesterol decreased lipid accumulation in liver organoids and simultaneously decreased the expression of the fibrosis marker protein αSMA in liver organoids.

[0213] Figure 35 , Figure 36 Data are expressed as mean ± standard deviation. P values were analyzed by one-way ANOVA. ns indicates no statistical difference, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Figure 47 and Figure 53Quantitative analysis was performed using Image J software. Data are expressed as mean ± standard deviation. P values were determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

[0214] The results showed that, compared with obeticholic acid which failed to inhibit lipid synthesis gene expression, 25-hydroxycholesterol significantly inhibited the expression levels of lipid synthesis-related genes ( Figure 35 ) and fibrosis-related genes ( Figure 36 ). Meanwhile, the Nile red staining experiment was used to detect the accumulation of neutral lipids in the organoids. As Figures 37-53 the results showed that 25-hydroxycholesterol and OCA reduced the lipid accumulation in the organoids, and the staining data of the fibrosis marker protein αSMA showed a significant decrease in the expression of the fibrosis protein αSMA. Figures 35-53 The results indicated that 25-hydroxycholesterol directly reduced liver lipid accumulation and fibrosis by inhibiting the expression of lipogenic and fibrotic genes, while OCA might indirectly inhibit liver lipid accumulation by promoting lipid oxidation. These data demonstrated that 25-hydroxycholesterol directly targeted the regulation of the liver lipid synthesis pathway and fibrotic genes.

[0215] Preparation of Key Intermediates

[0216] Preparation of Intermediate II Acetate (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl Ester

[0217]

[0218] Step 1: (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (10.00 g, 23.4 mmol, 1.0 eq) was dissolved in DCM (250 mL). After complete dissolution, acetic anhydride (6.7 mL, 71.0 mmol, 3.0 eq), DMAP (0.57 g, 4.7 mmol, 0.2 eq) and TEA (16.3 mL, 117.2 mmol, 5.0 eq) were successively added to the reaction system. After the addition was completed, the mixture was stirred at room temperature for 2 hours. After monitoring the completion of the reaction by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid baking plate), it was quenched with methanol (10 mL). The reaction solution was washed once with saturated sodium bicarbonate (~100 mL) and water (~100 mL) respectively, dried over anhydrous sodium sulfate, concentrated. When the concentration was almost dry, methanol (~100 mL) was added, and the mixture was stirred in an ice bath for 30 minutes, filtered by suction. The filter cake was rinsed with a small amount of methanol, and the filter cake was dried to obtain white solid acetate (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (I) (9.00 g, 17.3 mmol, purity 90.0%, yield 82.54%). 1 1H NMR (400 MHz, CDCl3): δ 4.50 (dd, J = 11.5, 4.5 Hz, 1H), 2.69 (s, 1H), 2.09 - 1.87 (m, 8H), 1.77 - 1.24 (m, 26H), 1.15 (d, J = 11.4 Hz, 3H), 1.00 (s, 3H), 0.93 - 0.85 (m, 12H), 0.69 (d, J = 2.7 Hz, 3H). 13 13C NMR (400 MHz, CDCl3): δ 203.21, 171.00, 134.37, 134.32, 99.99, 80.90, 77.34, 77.02, 76.71, 50.50, 50.30, 49.82, 44.54, 41.14, 37.81, 36.90, 36.08, 36.03, 35.27, 30.95, 30.78, 28.24, 28.15, 27.91, 26.38, 24.23, 24.17, 21.33, 20.98, 19.19, 18.46, 18.40, 18.11, 16.53, 15.78.

[0219] In the second step, acetic acid (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (I) (15.00 g, 32.0 mmol, 1.0 eq) was dissolved in DCM (dichloromethane) (300 mL), purged with nitrogen, the temperature of the system was lowered to 0 °C in an ice bath, ozone was introduced, and the gas was bubbled for 10 min. The reaction was monitored by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid baking plate). The product spot on the TLC plate was significantly thicker than the starting material spot. The ventilation was stopped, and after the system was purged with nitrogen, it was directly concentrated to dryness. The crude product was separated and purified by flash chromatography (PE∶EtOAc = 95∶5 to 90∶10, phosphomolybdic acid baking plate) to obtain white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbut-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (II) (6.50 g, 13.2 mmol, purity 95.5%, yield 41.3%). 1 1H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.9 Hz, 1H), 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.51 - 2.30 (m, 2H), 2.09 - 1.88 (m, 8H), 1.86 - 1.24 (m, 17H), 1.23 - 1.12 (m, 2H), 0.99 (d, J = 11.2 Hz, 3H), 0.93 - 0.84 (m, 12H).

[0220] Preparation of Intermediate VI (5R)-5-[(1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoic acid

[0221]

[0222] In the first step, acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (II) (1.50 g, 3.4 mmol, 1.0 eq) was dissolved in dichloromethane (50 mL). Triethylamine (1.41 mL, 10.2 mmol, 3.0 eq) and tert-butyldimethylsilyl trifluoromethanesulfonate (1.30 g, 5.1 mmol, 1.5 eq) were successively added thereto at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. TLC (PE∶DCM = 3∶1, phosphomolybdic acid plate baking) showed that the starting material spot disappeared. The reaction mixture was concentrated to dryness, n-hexane (50 mL) was added, and the mixture was stirred for 10 min, filtered, and the filtrate was concentrated to dryness by rotary evaporation. DCM (50 mL) was added, and the reaction mixture was cooled to -78 °C. Ozone was introduced, and bubbling was carried out for 10 min. TLC (PE∶DCM = 3∶1) was used to monitor the disappearance of the starting material spot. PPh3 (0.89 g, 3.4 mmol, 1.0 eq) was added and stirred for 30 min. The reaction mixture was directly concentrated, and purified by column chromatography (PE∶EtOAc = 90∶10) to obtain white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-1-formylpropan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (III) (300 mg, 0.560 mmol, 16.52%). 1 H NMR (400 MHz, CDCl3) δ 9.76 (dd, J = 3.3, 1.1 Hz, 1H), 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.47 (dd, J = 16.4, 2.5 Hz, 1H), 2.17 (m, 1H), 2.04 (m, 7H), 1.92 (m, 1H), 1.43 (m, 18H), 0.99 (dd, J = 9.3, 2.8 Hz, 5H), 0.90 (m, 12H), 0.74 (s, 2H).

[0223] In the second step, acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-1-formylpropyl-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (III) (300 mg, 0.700 mmol, 1.0 eq) was dissolved in dichloromethane (10 mL), ethyl (triphenylphosphoranylidene)acetate (243.81 mg, 0.700 mmol, 1.0 eq) was added, and the reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC (PE∶DCM = 3∶1, phosphomolybdic acid plate baking) until completion, and the reaction mixture was directly concentrated. Column chromatography on silica gel (PE∶EtOAc = 95∶5) gave the white solid ethyl (2E,5R)-5-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hex-2-enoate (IV) (250 mg, 0.401 mmol, 57.30%). 1 H NMR (400 MHz, CDCl3) δ 6.96 (m, 1H), 5.81 (d, J = 15.5 Hz, 1H), 4.50 (dd, J = 11.5, 4.5 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H), 1.99 (m, 8H), 1.63 (m, 12H), 1.31 (m, 6H), 1.17 (m, 3H), 1.00 (s, 3H), 0.92 (t, J = 5.3 Hz, 4H), 0.87 (m, 10H), 0.69 (m, 3H). 13 C NMR (101 MHz, CDCl3) δ 171.01, 148.45, 134.35, 122.47, 80.90, 77.33, 77.21, 77.01, 76.69, 60.12, 50.50, 50.24, 49.86, 44.60, 39.33, 37.81, 36.91, 36.38, 35.26, 30.80, 28.22, 27.91, 26.92, 26.38, 24.25, 24.17, 21.33, 20.96, 19.19, 18.96, 18.11, 16.53, 15.78, 14.29, -0.01.

[0224] Step 3: (2E,5R)-Ethyl 5-[(1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hex-2-enoate (IV) (250 mg, 0.55 mmol, 1.0 eq) was dissolved in methanol (10 mL), palladium (palladium on carbon) (30 mg, 0.282 mmol) was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. The reaction was monitored by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid plate). After the reaction was complete, the palladium carbon was filtered off with diatomaceous earth, and the filtrate was concentrated to dryness. Purification by silica gel column chromatography (PE∶EtOAc = 80∶20, phosphomolybdic acid plate) gave the white solid (5R)-ethyl 5-[(1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoate (V) (200 mg, 0.392 mmol, 71.68%). 1 H NMR (400 MHz, CDCl3) δ 4.50 (dd, J = 11.5, 4.5 Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 2.26 (dd, J = 15.5, 8.6 Hz, 2H), 2.04 (m, 8H), 1.68 (m, 7H), 1.50 - 1.37 (m, 9H), 1.26 (t, J = 7.1 Hz, 3H), 1.15 (m, 3H), 1.00 (s, 3H), 0.90 (m, 12H), 0.68 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 173.95, 171.03, 80.95, 77.33, 77.22, 77.02, 76.70, 60.16, 50.52, 50.26, 49.82, 44.49, 37.82, 36.91, 36.21, 35.69, 35.28, 34.82, 30.96, 30.80, 28.16, 27.92, 26.39, 24.25, 24.18, 21.85, 21.34, 21.00, 19.19, 18.61, 18.13, 16.54, 15.75, 14.28,

[0225] Step 4 Ethyl (5R)-5-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoate (V) (200 mg, 0.40 mmol) was dissolved in ethanol (2 mL), sodium hydroxide solution (2 mL, 4 mol / L) was added, the reaction mixture was heated to reflux and stirred overnight. The reaction was monitored by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid plate baking). After the reaction was complete, the reaction mixture was cooled to room temperature, adjusted to pH = 1 with 1N hydrochloric acid, extracted with ethyl acetate three times, the organic phases were combined, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (PE∶EtOAc = 90∶10 to 50∶50) to obtain the white solid (5R)-5-[(1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoic acid (VI) (150 mg, 0.331 mmol, 82.84%). 1 H NMR (400 MHz, CDCl3) δ 3.24 (dd, J = 11.6, 4.5 Hz, 1H), 2.33 (m, 2H), 2.04 (d, J = 8.2 Hz, 4H), 1.91 (dd, J = 13.4, 7.5 Hz, 1H), 1.70 (m, 6H), 1.50 (m, 7H), 1.19 (m, 7H), 0.99 (d, J = 7.8 Hz, 5H), 0.91 (t, J = 5.9 Hz, 3H), 0.87 (s, 3H), 0.81 (s, 3H), 0.69 (s, 3H).

[0226] Preparation of Intermediate VII (4R)-4-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]pentanoic acid

[0227]

[0228] (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl acetate (II) (3.00 g, 6.78 mmol, 1.0 eq.) was dissolved in acetone (50.0 mL) (the solution was turbid and not clear). After replacing nitrogen three times, the temperature was lowered to 0 °C, and Jones reagent (7.8 mL, 2.0 M, 2.0 eq.) was slowly added dropwise. After reacting for 5 minutes, TLC monitoring was carried out (PE∶EA = 5∶1, developed with phosphomolybdic acid, Rf1 = 0.72, Rf2 = 0.34). After the reaction was completed, 15.0 mL of isopropanol was added dropwise at low temperature to quench the reaction, and it was stirred for 30 minutes. After concentration, it was dissolved in 50.0 ml of dichloromethane, washed once with 30.0 mL of 1.5 N NaHSO3 and 30.0 mL of H2O respectively, dried and concentrated, and the crude white solid (4R)-4-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]pentanoic acid (VII) (3.00 g, 4.56 mmol, 67.56%) was obtained. 1 H NMR (400 MHz, Chloroform-d) δ 4.48 (dd, J = 11.6, 4.6 Hz, 1H), 2.40 (ddd, J = 15.4, 9.9, 5.0 Hz, 2H), 2.25 (ddd, J = 15.9, 9.5, 6.4 Hz, 1H), 2.08 - 1.92 (m, 7H), 1.84 - 1.56 (m, 8H), 1.45 (td, J = 13.6, 12.0, 7.2 Hz, 3H), 1.31 (dt, J = 11.8, 4.3 Hz, 2H), 1.23 - 1.07 (m, 3H), 0.98 (s, 3H), 0.88 (q, J = 4.7, 3.8 Hz, 12H), 0.67 (s, 3H). 13 C NMR (100 MHz, Chloroform-d) δ 179.93, 171.11, 134.35, 134.23, 80.94, 50.44, 50.18, 49.78, 44.49, 37.77, 36.86, 35.98, 35.22, 31.14, 30.99, 30.92, 30.74, 28.04, 27.88, 26.33, 24.20, 24.13, 21.32, 20.95, 19.17, 18.24, 18.08, 16.51, 15.75.

[0229] Preparation of Intermediate VIII (4R)-4-[(1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]pentanoic Acid

[0230]

[0231]

[0232] Weigh (4R)-4-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]pentanoic acid (500 mg, 1.09 mmol, 1.0 eq.), add tetrahydrofuran (20.0 mL), methanol (20.0 mL), 1 M lithium hydroxide aqueous solution (20.0 mL), stir overnight at room temperature, monitor by TLC. After the reaction is completed, concentrate to remove tetrahydrofuran and methanol. Then add water (30.0 mL) to the concentrated solution, and extract with ethyl acetate (30.0 mL × 3). Combine the organic phases, dry, and concentrate to obtain (4R)-4-[(1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]pentanoic acid (VIII) (350 mg, 77.1%). 1 HNMR (400 MHz, DMSO-d6) δ 11.99 (s, 1H), 4.35 (d, J = 5.1 Hz, 1H), 3.07 - 2.96 (m, 1H), 2.18 (dddd, J = 22.7, 15.9, 9.3, 6.2 Hz, 2H), 2.04 - 1.84 (m, 5H), 1.73 - 1.06 (m, 16H), 0.92 (ddd, J = 30.4, 18.0, 4.7 Hz, 12H), 0.70 (s, 3H), 0.65 (s, 3H).

[0233] Preparation of Intermediate IX (3R)-3-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]butyric acid

[0234]

[0235] Compound (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl acetate (III) (2.30 g, 5.2 mmol, 1.0 eq) was dissolved in t-BuOH (tert-butanol) (60 mL). 2-Methyl-2-butene (2.19 g, 31.2 mmol, 6.0 eq), aqueous solution of sodium dihydrogen phosphate (2.1 g, 15.6 mmol, 3.0 eq) and sodium chlorite (1.41 g, 15.6 mmol, 3.5 eq) (15 mL) were added under ice bath. The reaction mixture was stirred at room temperature for 2 hrs. TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid developer) showed that the reaction was complete. After concentration of the reaction mixture, it was extracted with EtOAc (50 mL) and washed with saturated sodium bicarbonate (50 mL×2). The organic layer was dried over anhydrous sodium sulfate. The crude product was purified by column chromatography with PE∶EtOAc = 3∶1 to obtain solid (3R)-3-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]butyric acid (IX) (1.6 g, purity 90%, yield 60.42%). 1 H NMR (400 MHz, CDCl3) δ 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.40 (dd, J = 10.2, 5.2 Hz, 1H), 2.29 (dd, J = 9.5, 6.5 Hz, 1H), 2.11 - 1.87 (m, 8H), 1.83 (dd, J = 10.0, 3.5 Hz, 1H), 1.77 - 1.54 (m, 7H), 1.53 - 1.44 (m, 2H), 1.43 - 1.22 (m, 4H), 1.22 - 1.10 (m, 2H), 1.00 (s, 3H), 0.97 - 0.81 (m, 12H), 0.69 (s, 3H).

[0236] Preparation Example

[0237] Example 67

[0238] Preparation of Compound 67 (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-7-hydroxyheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol

[0239]

[0240] In the first step, dissolve acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (II) (500 mg, 1.1 mmol) in tetrahydrofuran (30 mL), change the N2 three times, cool down to 0 °C, add potassium tert-butoxide (253 mg, 2.3 mmol) solid, and stir at 0 °C for 30 min. Dissolve diethyl (2-methoxy-2-oxoethyl)phosphonate (500 mg, 1.1 mmol) in THF (5 ml), add dropwise to the above reaction system, naturally rise to room temperature, and stir for 2 h. TLC (PE∶EtOAc = 10∶1) shows no remaining raw materials. Cool down to 0 °C and add 1 M / HCl (2 mL), separate the layers, extract twice with ethyl acetate, dry over anhydrous sodium sulfate, evaporate to dryness, and purify by column chromatography to obtain the white solid methyl (2E,6R)-6-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hept-2-enoate (67-1) (430 mg, 0.86 mmol, 76.65%). 1HNMR (399 MHz, CDCl3) δ 7.03 - 6.85 (m, 1H), 5.80 (d, J = 15.6 Hz, 1H), 4.48 (dd, J = 11.7, 4.5 Hz, 1H), 3.77 - 3.62 (m, 3H), 2.25 (s, 1H), 2.12 - 1.92 (m, 8H), 1.89 (d, J = 21.3 Hz, 1H), 1.57 - 1.39 (m, 4H) 1.57 - 1.39 (m, 5H), 1.36 - 1.21 (m, 3H), 1.15 (dd, J = 19.7, 10.5 Hz, 3H), 0.98 (s, 3H), 0.87 (dd, J = 14.6, 4.9 Hz, 12H), 0.66 (s, 3H)

[0241] In the second step, methyl (2E,6R)-6-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hept-2-enoate (67-1) (380 mg, 0.762 mmol) was dissolved in ethyl acetate (380 mL), Pd / C (palladium on carbon) (38 mg, 0.762 mmol) was added, the mixture was purged with hydrogen three times, and stirred at room temperature for 30 min. TLC (PE∶EtOAc = 10∶1) showed no remaining starting material, and the reaction was detected to be complete by TLC spotting. It was filtered through diatomaceous earth, rinsed with ethyl acetate, evaporated to dryness, and then purified by column chromatography (PE∶EtOAc = 100% - 20%) to obtain a white solid, methyl (6R)-6-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]heptanoate (67-2) (350 mg, 0.629 mmol, 82.56%). 1 H NMR (400 MHz, CDCl3) δ 4.49 (dd, J = 11.5, 4.6 Hz, 1H), 3.66 (d, J = 1.1 Hz, 3H), 2.30 (t, J = 8.0 Hz, 2H), 2.11 - 1.94 (m, 7H), 1.94 - 1.82 (m, 1H), 1.82 - 1.46 (m, 8H), 1.46 - 1.09 (m, 8H), 0.99 (s, 3H), 0.87 (q, J = 2.7, 2.2 Hz, 12H), 0.67 (s, 3H). 1313C NMR (101 MHz, CDCl3) δ 134.42, 134.20, 51.46, 50.38, 49.77, 44.43, 37.78, 36.86, 36.25, 35.77, 35.24, 34.18, 30.92, 30.78, 28.18, 27.90, 26.35, 25.90, 25.39, 24.23, 24.15, 21.35, 20.97, 19.17, 18.62, 18.10, 16.52, 15.73, 1.02.

[0242] In the third step, methyl (6R)-6-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]heptanoate (67-2) (100 mg, 0.170 mmol) was dissolved in tetrahydrofuran (5 mL), purged with N2, cooled to zero degree, lithium aluminum hydride (35 mg, 0.849 mmol) was added, and the reaction was carried out at zero degree for 2 h. There was no remaining raw material by TLC (PE∶EtOAc = 10∶1). Methanol was added, filtered through diatomaceous earth, concentrated by rotary evaporation, and then purified by column chromatography (PE∶EtOAc = 100%-20%) to obtain a white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-7-hydroxyheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (67) (50 mg, 0.114 mmol, 67.02%). 1 1H NMR (400 MHz, CDCl3) δ 3.64 (t, J = 6.4 Hz, 2H), 3.22 (d, J = 10.7 Hz, 1H), 2.02 (s, 4H), 1.96 - 1.83 (m, 1H), 1.76 - 1.63 (m, 5H), 1.56 (s, 1H), 1.46 (dd, J = 19.2, 10.2 Hz, 2H), 1.41 - 1.11 (m, 13H), 1.04 (d, J = 12.7 Hz, 1H), 0.98 (d, J = 7.8 Hz, 6H), 0.88 (d, J = 8.3 Hz, 6H), 0.80 (s, 3H), 0.68 (s, 3H). 1313C NMR (101 MHz, CDCl3) δ 134.36, 78.98, 77.31, 77.00, 76.68, 63.10, 50.41, 50.37, 49.78, 44.43, 38.87, 36.99, 36.35, 36.15, 35.56, 32.87, 30.95, 30.82, 28.21, 27.94, 27.82, 26.48, 26.19, 26.12, 24.25, 20.98, 19.13, 18.69, 18.23, 15.73, 15.41.

[0243] Example 68

[0244] Preparation of Compound 68 (1R, 3aR, 5aR, 7S, 9aS, 11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a, 6, 6, 9a, 11a-pentamethyl-2, 3, 3a, 4, 5, 5a, 6, 7, 8, 9, 9a, 10, 11, 11a-tetradecahydro-1H-cyclopenta[1, 2-a]phenanthren-7-ol

[0245]

[0246]

[0247] In the first step, the starting material lanosterol (3.00 g, 7.03 mmol, 1.0 eq) was dissolved in THF (tetrahydrofuran) (273 mL), water (68 mL) was added, and NBS (0.73 g, 4.08 mmol, 0.58 eq) was added at room temperature. The mixture was stirred at 25 °C for 2 hrs. TLC (n-hexane∶EtOAc = 5∶1, phosphomolybdic acid) showed that the reaction was complete. The mixture was extracted with dichloromethane three times, dried over sodium sulfate, concentrated by evaporation, and then purified by column chromatography (PE∶EtOAc = 50∶1 to 5∶1) to obtain (1R, 3aR, 5aR, 7S, 9aS, 11aR)-1-[(2R)-5-bromo-6-hydroxy-6-methylheptan-2-yl]-3a, 6, 6, 9a, 11a-pentamethyl-2, 3, 3a, 4, 5, 5a, 6, 7, 8, 9, 9a, 10, 11, 11a-tetradecahydro-1H-cyclopenta[1, 2-a]phenanthren-7-ol (68-1) (1.3 g, purity 90%, yield 35.3%). 11H NMR (400 MHz, CDC13) δ 3.98 (dd, J = 21.0, 12.3 Hz, 1H), 3.27 - 3.19 (m, 1H), 2.14 (d, J = 6.0 Hz, 1H), 2.00 (d, J = 11.7 Hz, 5H), 1.84 - 1.61 (m, 6H), 1.58 (s, 9H), 1.33 (t, J = 10.8 Hz, 9H), 1.27 - 1.12 (m, 3H), 1.04 (d, J = 12.2 Hz, 1H), 0.99 (d, J = 7.9 Hz, 7H), 0.94 - 0.84 (m, 7H), 0.80 (s, 3H), 0.69 (d, J = 2.2 Hz, 3H).

[0248] In the second step, (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-5-bromo-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68 - 1) (900 mg, 1.72 mmol, 1.0 eq) was dissolved in THF (tetrahydrofuran) (90 mL), and LAH (433 mg, 12.40 mmol,, 7.2 eq) was added. After the addition was complete, the temperature was raised to 70 °C and refluxed for 2 hrs. After detecting the completion of the reaction by TLC (n-hexane∶EtOAc = 5∶1, phosphomolybdic acid), the reaction system was poured into ice water (10 mL) for quenching, extracted with DCM (dichloromethane) (30 mL x 3), dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68) (0.4 g, purity 90%, yield 48.7%). 1 1H NMR (400 MHz, CDCl3) δ 3.23 (d, J = 8.8 Hz, 1H), 2.02 (s, 4H), 1.92 (d, J = 8.4 Hz, 1H), 1.69 (d, J = 24.3 Hz, 7H), 1.58 (d, J = 11.1 Hz, 2H), 1.52 - 1.33 (m, 8H), 1.30 (s, 2H), 1.18 (d, J = 25.9 Hz, 8H), 1.01 (dd, J = 22.8, 9.8 Hz, 8H), 0.93 - 0.84 (m, 6H), 0.80 (s, 3H), 0.68 (s, 3H). 1313C NMR (101 MHz, CDCl3) δ 134.38, 78.97, 77.33, 77.01, 76.69, 71.13, 50.49, 50.38, 49.79, 44.46, 44.39, 38.87, 37.00, 36.72, 36.45, 35.57, 30.97, 30.82, 29.31, 29.18, 28.23, 27.95, 27.82, 26.48, 24.26, 21.10, 20.99, 19.13, 18.67, 18.24, 15.74, 15.41

[0249] Example 71

[0250] Preparation of Compound 71 (1R,3aR,5aR,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-one

[0251]

[0252] Dissolve the raw material (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68) (1.0 g, 2.0 mmol, 1 eq) in DCM (dichloromethane) (20 mL), under nitrogen protection, add pyridinium chlorochromate (0.87 g, 4.05 mmol, 2 eq) and sodium acetate (0.33 g, 4.05 mmol, 2 eq) at room temperature. React for 2 hours. Monitor the reaction by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid). After the reaction is completed, filter through diatomaceous earth, wash twice with dichloromethane, and concentrate the filtrate. Purify by column chromatography (PE∶EtOAc = 1∶10~1∶3). Obtain a white solid (1R,3aR,5aR,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-one (71) (0.65 g, purity 90%, yield 68.9%). 11H NMR (400 MHz, CDCl3) δ 2.57 (dd, J = 11.2, 7.1 Hz, 1H), 2.43 - 2.41 (m, 1H), 2.08 - 1.96 (m, 6H), 1.76 - 1.59 (m, 7H), 1.56 - 1.34 (m, 9H), 1.22 - 1.20 (m, 8H), 1.10 (dd, J = 12.2, 8.4 Hz, 9H), 0.92 - 0.88 (m, 6H), 0.72 (s, 2H).

[0253] Example 102

[0254] Preparation of Compound 102 (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxyoctan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol

[0255]

[0256]

[0257] Step 1: (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (10.00 g, 23.45 mmol, 1.0 eq) was dissolved in DCM (250 mL). After complete dissolution, acetic anhydride (6.7 mL, 70.36 mmol, 3.0 eq), DMAP (0.57 g, 4.69 mmol, 0.2 eq) and TEA (16 mL, 117.25 mmol, 5.0 eq) were successively added to the reaction system. After the addition was completed, the mixture was stirred at room temperature for 2 h. After monitoring the completion of the reaction by TLC (petroleum ether∶ethyl acetate = 5∶1), the reaction was quenched with methanol (10 mL). The reaction solution was washed once with saturated sodium bicarbonate (~100 mL) and water (~100 mL) respectively, dried over anhydrous sodium sulfate, concentrated. When the concentration was almost dry, methanol (~100 mL) was added, and the mixture was stirred in an ice bath for 30 min, filtered by suction. The filter cake was rinsed with a small amount of methanol, and the filter cake was dried to obtain white solid acetate (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (I) (9 g, 19.2 mmol, purity 90.0%, yield 73.7%).

[0258] 1 H NMR(400MHz,CDCl3): δ5.10(s,1H), 4.50(dd,J = 11.5,4.6Hz,1H), 2.08 - 1.97(m,8H), 1.95 - 1.82(m,2H), 1.76 - 1.58(m,10H), 1.58 - 1.43(m,4H), 1.42 - 1.22(m,6H), 1.20 - 1.14(m,3H), 1.00(s,3H), 0.95 - 0.83(m,15H). 1313C NMR (400 MHz, CDCl3): δ 203.21, 171.00, 134.37, 134.32, 99.99, 80.90, 77.34, 77.02, 76.71, 50.50, 50.30, 49.82, 44.54, 41.14, 37.81, 36.90, 36.08, 36.03, 35.27, 30.95, 30.78, 28.24, 28.15, 27.91, 26.38, 24.23, 24.17, 21.33, 20.98, 19.19, 18.46, 18.40, 18.11, 16.53, 15.78.

[0259] Step 2. Acetic acid (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (I) (15.00 g, 32.00 mmol, 1.0 eq) was dissolved in DCM (dichloromethane) (300 mL), purged with nitrogen, cooled to 0 °C in an ice bath, and ozone was introduced. The gas was bubbled for 10 min. The reaction was monitored by TLC (petroleum ether:ethyl acetate = 5:1). The product spot on the TLC plate was significantly darker than the starting material spot. The gas introduction was stopped, and the system was purged with nitrogen and then directly concentrated to dryness. The crude product was separated and purified by flash chromatography (petroleum ether:ethyl acetate = 95:5 to 90:10) to obtain the white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbut-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (II) (6.50 g, 14.68 mmol, purity 95.5%, yield 43.8%). 1 1H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.9 Hz, 1H), 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.51 - 2.30 (m, 2H), 2.09 - 1.88 (m, 8H), 1.86 - 1.24 (m, 17H), 1.23 - 1.12 (m, 2H), 0.99 (d, J = 11.2 Hz, 3H), 0.93 - 0.84 (m, 12H).

[0260] Step 3. (Methoxymethyl)triphenylphosphonium chloride (3.87 g, 11.32 mmol, 5.0 eq) was dissolved in anhydrous tetrahydrofuran (40 mL). The system was cooled to 0 °C in an ice-water bath, and NaHMDS (11 mL, 1 mol / L, 5.0 eq) was added. After stirring for 20 minutes, (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (II) (1.00 g, 2.26 mmol, 1.0 eq) dissolved in anhydrous tetrahydrofuran (10 mL) was added to the reaction solution. The system was warmed to room temperature and stirred for 2 hours. The reaction was monitored by TLC (petroleum ether∶ethyl acetate = 10∶1). After the reaction was complete, water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 90∶10) to obtain the white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R,4E)-5-methoxypent-4-en-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (37-1) (850 mg, 1.81 mmol, purity 80%, yield 64.1%). 1 H NMR (400 MHz, CDCl3) δ 6.28 (d, J = 12.6 Hz, 1H), 5.85 (d, J = 6.2 Hz, 1H), 4.71 (d, J = 12.6 Hz, 1H), 4.50 (dd, J = 11.5, 4.5 Hz, 1H), 4.31 (d, J = 6.5 Hz, 1H), 3.58 (s, 1H), 3.52 - 3.47 (m, 2H), 2.09 - 1.88 (m, 10H), 1.80 - 1.24 (m, 15H), 1.21 - 1.11 (m, 2H), 1.00 (s, 3H), 0.93 - 0.83 (m, 12H), 0.69 (s, 3H).

[0261] Step 4: (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R,4E)-5-methoxypent-4-en-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (37-1) (700 mg, 1.49 mmol, 1.0 eq) was dissolved in a mixed solvent of dilute hydrochloric acid (5 mol / L, 20 mL) and THF (20 mL). The reaction solution was heated to 50 °C and stirred for 2 h. The reaction was monitored by TLC (petroleum ether∶ethyl acetate = 10∶1). After the reaction was complete, the reaction solution was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sulfuric acid, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 95∶5 to 85∶15) to obtain white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-5-formylpent-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (98-1) (500 mg, 1.09 mmol, purity 80%, yield 58.5%). 1 H NMR (400 MHz, CDCl3) δ 9.77 (t, J = 1.7 Hz, 1H), 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.40 (td, J = 7.9, 1.7 Hz, 2H), 2.12 - 1.97 (m, 7H), 1.96 - 1.85 (m, 1H), 1.78 - 1.63 (m, 7H), 1.59 - 1.24 (m, 9H), 1.21 - 1.06 (m, 3H), 1.00 (s, 3H), 0.90 (dd, J = 17.5, 5.5 Hz, 12H), 0.69 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 202.99, 171.02, 134.43, 134.30, 129.64, 115.29, 80.93, 77.33, 77.02, 76.70, 50.51, 50.24, 49.82, 44.50, 44.35, 37.81, 36.90, 36.31, 35.74, 35.27, 30.96, 30.79, 28.19, 27.92, 26.38, 24.24, 24.18, 21.33, 20.99, 19.19, 18.96, 18.57, 18.12, 16.53, 15.75, -0.00.

[0262] Step 5. Acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-5-formylpentan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (98-1) (80 mg, 0.18 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (5 mL). Ethylmagnesium chloride (0.9 mL, 1.80 mmol, 2.0 mol / L, 10 eq) was added under nitrogen protection at 0 °C. The reaction was monitored by TLC (petroleum ether∶ethyl acetate = 5∶1). After the reaction was complete, the reaction was quenched with ammonium chloride, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 90∶10) to obtain a white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxyoctan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (102) (45 mg, 0.10 mmol, purity 95%, yield 52.8%). 1 H NMR (400 MHz, CDCl3) δ 3.52 - 3.40 (m, 1H), 3.17 (dd, J = 11.5, 4.5 Hz, 1H), 2.00 - 1.91 (m, 4H), 1.88 - 1.81 (m, 1H), 1.68 - 1.58 (m, 5H), 1.54 - 1.42 (m, 6H), 1.35 - 1.22 (m, 7H), 1.20 - 1.05 (m, 4H), 0.98 (dd, J = 12.6, 2.1 Hz, 2H), 0.92 (d, J = 7.8 Hz, 6H), 0.88 (t, J = 7.5 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H), 0.81 (s, 3H), 0.74 (s, 3H), 0.62 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 128.14, 128.11, 56.83, 55.93, 50.50, 47.42, 44.73, 38.67, 38.62, 37.78, 36.88, 30.83, 30.66, 27.82, 27.78, 26.49, 26.42, 24.17, 24.05, 21.23, 21.17, 19.20, 19.16, 16.50, 13.75, 13.56. LC-MS: [M - 17] + = 427.35

[0263] Example 129

[0264] Preparation of Compound 129 (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol

[0265]

[0266] Step 1. Dissolve the white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (I) (4.0 g, 4.2 mmol, 1.0 eq) in DCM (dichloromethane) (100 mL), add sodium bicarbonate (0.39 g, 4.7 mmol, 1.1 eq), and m-chloroperoxybenzoic acid (0.88 g, 5.1 mmol, 1.2 eq). React at room temperature for 1 h. Monitor the reaction completion by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid). Wash the reaction solution twice with saturated sodium bicarbonate solution (100 mL x 2), dry over sodium sulfate, and concentrate. Purify by column chromatography (PE∶EtOAc = 1∶10~1∶3) to obtain the white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (129-1) (1.6 g, purity 85%, yield 65.7%). 1 1H NMR (400 MHz, CDCl3): δ4.50 (dd, J = 11.5, 4.5 Hz, 1H), 2.69 (t, J = 6.2 Hz, 1H), 2.05 - 2.00 (m, 8H), 1.67 - 1.31 (m, 17H), 1.35 - 1.08 (m, 8H), 1.01 - 0.96 (m, 3H), 0.96 - 0.85 (m, 11H), 0.72 - 0.57 (m, 3H).

[0267] Step 3: Dissolve the raw material acetic acid (1R, 3aR, 5aR, 7S, 9aS, 11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (129-1) (1.0 g, 2.1 mmol, 1 eq) in THF (tetrahydrofuran) (40 mL), protect with nitrogen, and add lithium aluminum hydride (0.16 g, 4.2 mmol, 2 eq) under an ice bath. React at room temperature for 1.5 hours. TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid). After the reaction is completed, quench with sodium sulfate decahydrate, filter, and concentrate the filtrate. Purify by column chromatography (DCM∶MeOH = 1∶10~1∶5). Obtain a white solid (1R, 3aR, 5aR, 7S, 9aS, 11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (129) (600 mg, purity 90%, yield 59.13%). 1 1H NMR (400 MHz, CDCl3) δ 3.24 (dd, J = 11.5, 4.5 Hz, 1H), 2.69 (t, J = 6.2 Hz, 1H), 2.11 - 1.88 (m, 6H), 1.76 - 1.51 (m, 12H), 1.32 - 1.22 (m, 8H), 1.07 - 0.97 (m, 8H), 0.93 - 0.86 (m, 7H), 0.81 (s, 3H), 0.70 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 134.45, 134.35, 78.97, 77.34, 77.23, 77.03, 76.71, 64.94, 64.80, 58.43, 58.14, 50.41, 50.38, 50.29, 49.82, 44.51, 38.89, 37.03, 36.34, 36.22, 35.59, 32.81, 32.62, 31.00, 30.98, 30.83, 28.24, 28.20, 27.97, 27.85, 26.50, 25.91, 25.62, 24.95, 24.94, 24.25, 21.00, 19.15, 18.75, 18.68, 18.65, 18.58, 18.26, 15.76, 15.42

[0268] Example 147

[0269] Preparation of Compound 147 (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,5,5a,6,7,8,9,9a,11,11a-dodecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol

[0270]

[0271] In the first step, (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (10.00 g, 23.45 mmol, 1.0 eq) was dissolved in DCM (250 mL). After complete dissolution, acetic anhydride (6.7 mL, 70.36 mmol, 3.0 eq), DMAP (0.57 g, 4.69 mmol, 0.2 eq) and TEA (16 mL, 117.25 mmol, 5.0 eq) were successively added to the reaction system. After the addition was completed, the mixture was stirred at room temperature for 2 hours. After monitoring the completion of the reaction by TLC (petroleum ether∶EtOAc = 5∶1), the reaction was quenched with methanol (10 mL). The reaction solution was washed once with saturated sodium bicarbonate (~100 mL) and water (~100 mL) respectively, dried over anhydrous sodium sulfate, concentrated. When the concentration was almost dry, methanol (~100 mL) was added, and the mixture was stirred in an ice bath for 30 minutes, then filtered by suction. The filter cake was rinsed with a small amount of methanol, and the filter cake was dried to obtain white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl ester (I) (9 g, 19.2 mmol, purity 90.0%, yield 73.7%). 1 H NMR (400 MHz, CDCl3): δ 5.10 (s, 1H), 4.50 (dd, J = 11.5, 4.6 Hz, 1H), 2.08 - 1.97 (m, 8H), 1.95 - 1.82 (m, 2H), 1.76 - 1.58 (m, 10H), 1.58 - 1.43 (m, 4H), 1.42 - 1.22 (n, 6H), 1.20 - 1.14 (m, 3H), 1.00 (s, 3H), 0.95 - 0.83 (m, 15H). 1313C NMR (400 MHz, CDCl3): δ 203.21, 171.00, 134.37, 134.32, 99.99, 80.90, 77.34, 77.02, 76.71, 50.50, 50.30, 49.82, 44.54, 41.14, 37.81, 36.90, 36.08, 36.03, 35.27, 30.95, 30.78, 28.24, 28.15, 27.91, 26.38, 24.23, 24.17, 21.33, 20.98, 19.19, 18.46, 18.40, 18.11, 16.53, 15.78.

[0272] Step 2: (1R,3aR,5aR,7S,9aS,11aR)-3a,6,6,9a,11a-Pentamethyl-1-[(2R)-6-methylhept-5-en-2-yl]-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl acetate (I) (15.00 g, 32.00 mmol, 1.0 eq) was dissolved in DCM (dichloromethane) (300 mL), purged with nitrogen, cooled to 0 °C in an ice bath, and ozone was introduced. The gas was bubbled for 10 min. The reaction was monitored by TLC (petroleum ether:EtOAc = 5:1). After stopping the gas introduction, the system was purged with nitrogen and directly concentrated to dryness. The crude product was separated and purified by flash chromatography (petroleum ether:ethyl acetate = 95:5 to 90:10, phosphomolybdic acid plate baking) to obtain the white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbut-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[2,1-i]phenanthren-7-yl acetate (II) (6.50 g, 14.68 mmol, purity 95.5%, yield 43.8%). 1 1H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.9 Hz, 1H), 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.51 - 2.30 (m, 2H), 2.09 - 1.88 (m, 8H), 1.86 - 1.24 (m, 17H), 1.23 - 1.12 (m, 2H), 0.99 (d, J = 11.2 Hz, 3H), 0.93 - 0.84 (m, 12H).

[0273] Step 3 (Methoxymethyl)triphenylphosphonium chloride (3.87 g, 11.32 mmol, 5.0 eq) was dissolved in anhydrous tetrahydrofuran (40 mL). The system was cooled to 0 °C in an ice-water bath, and NaHMDS (11 mL, 1 mol / L, 5.0 eq) was added. After stirring for 20 minutes, acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-formylbutan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (II) (1.00 g, 2.26 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (10 mL) and added to the reaction solution. The system was warmed to room temperature and stirred for 2 hours. The reaction was monitored by TLC (petroleum ether∶ethyl acetate = 10∶1). After the reaction was complete, water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 90∶10) to obtain the white solid acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R,4E)-5-methoxypent-4-en-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (37-1) (850 mg, 1.81 mmol, purity 80%, yield 64.1%). 1 H NMR (400 MHz, CDCl3) δ 6.28 (d, J = 12.6 Hz, 1H), 5.85 (d, J = 6.2 Hz, 1H), 4.71 (d, J = 12.6 Hz, 1H), 4.50 (dd, J = 11.5, 4.5 Hz, 1H), 4.31 (d, J = 6.5 Hz, 1H), 3.58 (s, 1H), 3.52 - 3.47 (m, 2H), 2.09 - 1.88 (m, 10H), 1.80 - 1.24 (m, 15H), 1.21 - 1.11 (m, 2H), 1.00 (s, 3H), 0.93 - 0.83 (m, 12H), 0.69 (s, 3H).

[0274] Step 4 (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R,4E)-5-methoxypent-4-en-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (37-1) (700 mg, 1.49 mmol, 1.0 eq) was dissolved in a mixed solvent of dilute hydrochloric acid (5 mol / L, 20 mL) and THF (20 mL). The reaction solution was heated to 50 °C and stirred for 2 h. The reaction was monitored by TLC (petroleum ether∶ethyl acetate = 10∶1) until completion. The reaction solution was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sulfuric acid, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 95∶5 to 85∶15) to obtain the white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-5-formylpent-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (98-1) (500 mg, 1.09 mmol, purity 80%, yield 58.5%). 1 H NMR (400 MHz, CDCl3) δ 9.77 (t, J = 1.7 Hz, 1H), 4.50 (dd, J = 11.6, 4.5 Hz, 1H), 2.40 (td, J = 7.9, 1.7 Hz, 2H), 2.12 - 1.97 (m, 7H), 1.96 - 1.85 (m, 1H), 1.78 - 1.63 (m, 7H), 1.59 - 1.24 (m, 9H), 1.21 - 1.06 (m, 3H), 1.00 (s, 3H), 0.90 (dd, J = 17.5, 5.5 Hz, 12H), 0.69 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 202.99, 171.02, 134.43, 134.30, 129.64, 115.29, 80.93, 77.33, 77.02, 76.70, 50.51, 50.24, 49.82, 44.50, 44.35, 37.81, 36.90, 36.31, 35.74, 35.27, 30.96, 30.79, 28.19, 27.92, 26.38, 24.24, 24.18, 21.33, 20.99, 19.19, 18.96, 18.57, 18.12, 16.53, 15.75

[0275] Step 5: Weigh (3aR,7S,9aS,11aR)-1-[(2R)-5-formylpentan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl acetate (98-1) (830 mg, 1.82 mmol, 1.0 eq.), add water (14.0 mL), tert-butanol (90 mL), and successively add 2-methyl-2-butene (2.5 g, 36.35 mmol, 20.0 eq.), sodium dihydrogen phosphate (2.4 g, 20.0 mmol, 10.0 eq.), and sodium chlorite (493 mg, 5.4 mmol, 3.0 eq.) at room temperature. Stir at room temperature for 2 hours. After monitoring the reaction by TLC (petroleum ether∶ethyl acetate = 3∶1) until completion, concentrate to remove tert-butanol. Subsequently, add water (10.0 mL) to the concentrated solution, and extract with ethyl acetate (10.0 mL × 3). Combine the organic phases, dry, and concentrate to obtain (5R)-5-[(3aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoic acid (147-1) (1.05 g, 1.77 mmol, 97.78%). 1 H NMR (399 MHz, CDCl3) δ 4.48 (dd, J = 11.6, 4.5 Hz, 1H), 2.42 - 2.22 (m, 2H), 2.03 (s, 4H), 1.98 (s, 2H), 1.94 - 1.85 (m, 1H), 1.76 - 1.63 (m, 8H), 1.51 (dd, J = 20.7, 7.5 Hz, 4H), 1.41 (s, 2H), 1.32 - 1.24 (m, 3H), 1.13 (d, J = 14.2 Hz, 3H), 0.98 (s, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 4.8 Hz, 9H), 0.67 (s, 3H).

[0276] Step 6: Weigh (5R)-5-[(3aR,7S,9aS,11aR)-7-acetyloxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoic acid (147-1) (1.0 g, 2.12 mmol, 1.0 eq.), dissolve it in methanol (100 mL), add concentrated sulfuric acid (3 drops) at room temperature, then heat to 80 °C and stir for 2.5 hours. After monitoring the reaction by TLC (petroleum ether∶ethyl acetate = 10∶1) until completion, concentrate to remove methanol, and then obtain methyl (5R)-5-[(3aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoate (147-2) (0.24 g, 0.54 mmol) by column chromatography (petroleum ether∶ethyl acetate = 50∶1 - 10∶1). 1 H NMR (399 MHz, CDCl3) δ 3.65 (s, 3H), 3.21 (dd, J = 11.6, 4.4 Hz, 1H), 2.25 (dt, J = 16.7, 8.5 Hz, 2H), 2.00 (s, 4H), 1.88 (dd, J = 22.1, 8.8 Hz, 1H), 1.77 - 1.61 (m, 7H), 1.54 (s, 2H), 1.41 (d, J = 16.3 Hz, 3H), 1.32 - 1.11 (m, 5H), 1.03 (d, J = 10.6 Hz, 2H), 0.97 (d, J = 7.9 Hz, 6H), 0.89 (d, J = 6.1 Hz, 3H), 0.85 (s, 3H), 0.79 (s, 3H), 0.67 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 174.34, 134.32, 78.95, 77.30, 77.19, 76.98, 76.67, 51.43, 50.37, 50.18, 49.78, 44.45, 38.86, 36.99, 36.16, 35.67, 35.55, 34.53, 30.93, 30.80, 28.12, 27.94, 27.82, 26.47, 24.23, 21.76, 20.97, 19.12, 18.57, 18.23, 15.71, 15.39.

[0277] Step 7: Weigh methyl (5R)-5-[(3aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoate (147-2) (100 mg, 0.23 mmol, 1.0 eq.), dissolve it in dichloromethane (10 mL), then add m-chloroperoxybenzoic acid (77.61 mg, 0.45 mmol, 2.0 eq.) under an ice bath. After the addition, restore to room temperature and continue stirring. After monitoring the reaction by TLC (petroleum ether∶ethyl acetate = 10∶1), add saturated aqueous sodium bicarbonate solution (5.0 mL), extract with dichloromethane (5.0 mL * 3). The organic phase is dried and concentrated to obtain methyl (5R)-5-[(2S,5S,7S,11R,14R,15R)-5-hydroxy-2,6,6,11,15-pentamethyl-18-oxapentacyclo[8.7.1.01,10.011,15.02,7]octadec-14-yl]hexanoate (147-3) (140 mg, 0.21 mmol, 94.6%). 1 H NMR (399 MHz, CDCl3) δ 3.65 (s, 3H), 3.32 - 3.09 (m, 1H), 2.25 (dd, J = 15.0, 7.7 Hz, 2H), 1.97 - 1.80 (m, 5H), 1.73 - 1.58 (m, 7H), 1.49 (dd, J = 25.3, 12.7 Hz, 6H), 1.36 - 1.24 (m, 6H), 1.10 (s, 3H), 0.92 (s, 3H), 0.87 (d, J = 6.9 Hz, 6H), 0.75 (d, J = 6.8 Hz, 6H).

[0278] Step 8: Weigh methyl (5R)-5-[(2S,5S,7S,11R,14R,15R)-5-hydroxy-2,6,6,11,15-pentamethyl-18-oxapentacyclo[8.7.1.01,10.011,15.02,7]octadec-14-yl]hexanoate (147-3) (140 mg, 0.30 mmol, 1.0 eq.), dissolve it in acetone (10 mL), add concentrated sulfuric acid (3 drops) at room temperature, then stir at room temperature for 5 hours. After monitoring the reaction by TLC (petroleum ether∶ethyl acetate = 10∶1) until it is completed, concentrate to remove acetone, add saturated sodium bicarbonate aqueous solution (10.0 mL), extract with ethyl acetate (8.0 mL * 3), separate the organic phase, dry, and concentrate to obtain methyl (5R)-5-[(3aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,5,5a,6,7,8,9,9a,11,11a-dodecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoate (147-4) (120 mg, 0.22 mmol, 71.36%). 1H NMR (399 MHz, CDCl3) δ 5.44 (s, 1H), 5.29 (d, J = 6.0 Hz, 1H), 3.65 (s, 3H), 3.23 (dd, J = 11.3, 4.5 Hz, 1H), 2.26 (dd, J = 16.1, 8.6 Hz, 2H), 2.15 (s, 1H), 2.05 (dd, J = 17.4, 6.0 Hz, 3H), 2.00 - 1.89 (m, 3H), 1.70 (dd, J = 10.7, 5.9 Hz, 3H), 1.61 - 1.51 (m, 3H), 1.39 (dd, J = 16.0, 7.2 Hz, 3H), 1.31 - 1.17 (m, 2H), 1.11 - 1.04 (m, 2H), 0.98 (t, J = 8.2 Hz, 6H), 0.92 - 0.80 (m, 9H), 0.54 (s, 3H).

[0279] Step 9: Weigh methyl (5R)-5-[(3aR,7S,9aS,11aR)-7-hydroxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,5,5a,6,7,8,9,9a,11,11a-dodecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]hexanoate (147-4) (120 mg, 0.27 mmol, 1.0 eq.), dissolve it in tetrahydrofuran (5.0 mL), cool down to -10 °C to 0 °C, then dropwise add an ethereal solution of methyllithium (59.6 mg, 2.7 mmol, 10.0 eq.) to monitor the reaction. Then restore to room temperature and stir at room temperature for 2 hours. Add saturated aqueous ammonium chloride solution (10.0 mL), extract with ethyl acetate (7.0 mL * 3). Separate the organic phase, dry, concentrate, and purify by preparative plate to obtain (3aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,5,5a,6,7,8,9,9a,11,11a-dodecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (147) (25 mg, 0.056 mmol, 20.53 %). 1 H NMR (399 MHz, CDCl3) δ 5.45 (s, 1H), 5.28 (s, 1H), 3.23 (dd, J = 11.0, 4.1 Hz, 1H), 2.19 (d, J = 18.5 Hz, 1H), 2.07 (t, J = 11.9 Hz, 3H), 1.98 (d, J = 13.1 Hz, 2H), 1.67 (d, J = 11.8 Hz, 2H), 1.52 - 1.48 (m, 2H), 1.47 - 1.34 (m, 6H), 1.25 (s, 2H), 1.24 (s, 3H), 1.20 (s, 6H), 1.07 (dd, J = 10.8, 5.0 Hz, 2H), 0.98 (d, J = 9.6 Hz, 6H), 0.91 - 0.84 (m, 9H), 0.55 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 145.85, 142.65, 120.14, 116.27, 78.93, 77.30, 77.18, 76.98, 76.66, 71.10, 51.00, 50.28, 49.08, 44.36, 43.72, 38.67, 37.79, 37.33, 36.63, 36.21, 35.68, 31.47, 30.91, 29.67, 29.31, 29.17, 28.11, 27.90, 27.77, 25.55, 22.97, 22.71, 21.08, 18.45, 15.76, 15.62, 0.99. LC-MS: [M - 17] += 425.35

[0280] Example 114

[0281] Preparation of Compound 114 (1R,3aR,5aR,7S,9aS,11aR)-7-Hydroxy-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-4-one

[0282]

[0283]

[0284] Dissolve the compound (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,5,5a,6,7,8,9,9a,11,11a-dodecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (147) (240 mg, 0.54 mmol, 1.0 eq.) in glacial acetic acid (5.0 mL). Under nitrogen protection, cool the solution to 0 °C in an ice bath. Then, dropwise add hydrogen peroxide (concentration 31%, 0.7 mL, 7.07 mmol, 13.0 eq.). After the addition, continue to stir in the ice bath for 10 minutes, and then gradually warm to room temperature and stir. Monitor by TLC (PE∶EtOAc = 2∶1, visualized at 254 nm, Rf1 = 0.5, Rf2 = 0.2). After the reaction is completed, the reaction solution is clear. Concentrate and purify by preparative TLC to obtain a white solid (1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-4-one (114) (160 mg, 0.31 mmol, 56.62%). 11H NMR (399 MHz, CDCl3) δ 3.30 - 3.17 (m, 1H), 2.40 (d, J = 13.1 Hz, 1H), 2.28 (dd, J = 11.6, 7.7 Hz, 1H), 2.09 - 1.92 (m, 2H), 1.82 (d, J = 12.4 Hz, 1H), 1.74 (dd, J = 13.8, 6.1 Hz, 2H), 1.66 - 1.58 (m, 4H), 1.46 - 1.30 (m, 8H), 1.23 (s, 3H), 1.19 (s, 6H), 1.14 (s, 3H), 1.07 - 1.02 (m, 1H), 0.97 (s, 3H), 0.87 (d, J = 13.4 Hz, 9H), 0.62 (d, J = 7.9 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 199.11, 164.88, 138.92, 77.90, 77.30, 76.98, 76.67, 71.09, 49.77, 49.03, 47.72, 44.87, 44.33, 39.73, 38.88, 36.61, 36.34, 34.75, 31.96, 30.07, 29.67, 29.31, 29.17, 28.77, 28.06, 27.38, 24.97, 23.64, 21.03, 18.73, 18.32, 15.74, 15.26. LC-MS: [M+1] + = 459.55

[0285] Example 139

[0286] Compound 139

[0287] Preparation of [(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl] oxidosulfonic acid

[0288]

[0289]

[0290] (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68) (100 mg, 0.22 mmol, 1 eq) was dissolved in Pyridine (6 mL) under nitrogen protection, and trimethylammonium trioxide copolymer (93.88 mg, 0.67 mmol, 3 eq) was added at room temperature. The reaction was carried out for 1 hour. TLC (DCM∶MeOH = 10∶1, phosphomolybdic acid). The reaction solution was poured into petroleum ether at 0 °C to precipitate a white solid, which was filtered. Purification by column chromatography (DCM∶MeOH = 1∶10) gave a white solid [(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl] sulfonic acid (139) (19.9 mg, purity 93.2%, yield 21.1%).

[0291] 1 H NMR (400 MHz, DMSO) δ 3.61 (dd, J = 11.8, 4.2 Hz, 1H), 2.06 - 1.85 (m, 6H), 1.82 - 1.50 (m, 6H), 1.48 - 1.38 (m, 3H), 1.31 (dd, J = 23.8, 13.4 Hz, 5H), 1.22 - 1.11 (m, 3H), 1.05 (s, 6H), 1.03 - 0.96 (m, 2H), 0.93 (d, J = 9.1 Hz, 6H), 0.88 (d, J = 6.3 Hz, 3H), 0.84 (d, J = 7.1 Hz, 3H), 0.78 - 0.60 (m, 5H). 13 C NMR (101 MHz, DMSO) δ 134.06, 83.02, 69.25, 51.23, 50.57, 49.85, 44.61, 44.50, 40.60, 40.39, 40.18, 39.97, 39.76, 39.55, 39.34, 38.49, 36.94, 36.90, 36.42, 35.63, 29.87, 29.67, 28.46, 28.25, 24.92, 24.52, 21.04, 20.97, 19.42, 19.03, 18.33, 16.91, 16.06.

[0292] Example 140

[0293] Preparation of Compound 140 3-{[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}-3-oxopropanoic acid

[0294]

[0295]

[0296] Step 1: Dissolve (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68) (200 mg, 0.450 mmol, 1.0 eq) in THF (tetrahydrofuran) (5 mL), displace the reaction system with nitrogen atmosphere, add dropwise methyl 3-chloro-3-oxopropanoate (61.44 mg, 0.450 mmol, 3 eq), and stir the reaction mixture at room temperature for 1 hr. TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid plate baking) shows that the reaction is complete, and stop the reaction. Add water (20 mL) to the reaction system, extract with EA (ethyl acetate) (3×25 mL), combine the organic phases, wash with saturated brine (20 mL), then dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. The crude product is separated and purified by flash chromatography (PE∶EtOAc = 92∶8 to 90∶10) to obtain white solid methyl 3-{[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl]oxy}-3-oxopropanoate (140-1) (160 mg, purity 90%, yield 62.04%). 11H NMR (400 MHz, CDCl3) δ 4.50 (dd, J = 11.7, 4.4 Hz, 1H), 3.68 (s, 3H), 3.32 (s, 2H), 1.96 (dd, J = 13.3, 7.7 Hz, 5H), 1.83 (d, J = 7.5 Hz, 1H), 1.65 (m, 6H), 1.40 (d, J = 15.8 Hz, 8H), 1.36 - 1.17 (m, 8H), 1.15 (s, 5H), 0.95 (d, J = 11.8 Hz, 4H), 0.86 - 0.78 (m, 13H), 0.62 (s, 3H).

[0297] In the second step, the raw material 3 - methoxy - 3 - oxopropanoic acid (1R, 3aR, 5aR, 7S, 9aS, 11aR) - 1 - [(2R) - 6 - hydroxy - 6 - methylheptan - 2 - yl] - 3a, 6, 6, 9a, 11a - pentamethyl - 2, 3, 3a, 4, 5, 5a, 6, 7, 8, 9, 9a, 10, 11, 11a - tetradecahydro - 1H - cyclopenta[1, 2 - i]phenanthren - 7 - yl ester (140 - 1) (100 mg, 0.18 mmol, 1 eq) was dissolved in EtOH (ethanol) (20 mL) and water (5 mL), and 2N aqueous sodium hydroxide solution (1 mL) was added dropwise. The reaction system was replaced with a nitrogen atmosphere and stirred at room temperature for 2 h. The reaction was monitored by TLC (DCM∶MeOH = 10∶1, phosphomolybdic acid plate baking) until completion, and then the reaction was stopped. After the reaction solution was acidified to pH = 3 - 4 with 1 N HCl, ethanol was concentrated off, and the residue was extracted with EA (ethyl acetate) (20 mL × 3). The combined organic phases were washed with saturated brine (50 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was separated and purified by flash chromatography (DCM∶MeOH = 96∶4) to obtain the white solid 3 - {[(1R, 3aR, 5aR, 7S, 9aS, 11aR) - 1 - [(2R) - 6 - hydroxy - 6 - methylheptan - 2 - yl] - 3a, 6, 6, 9a, 11a - pentamethyl - 2, 3, 3a, 4, 5, 5a, 6, 7, 8, 9, 9a, 10, 11, 11a - tetradecahydro - 1H - cyclopenta[1, 2 - a]phenanthren - 7 - yl]oxy} - 3 - oxopropanoic acid (140) (39.29 mg, purity 80.60%, yield 41.06%). 11H NMR (400 MHz, CDCl3) δ 4.63 (dd, J = 11.3, 4.8 Hz, 1H), 3.44 (s, 2H), 2.07 - 1.98 (m, 4H), 1.95 - 1.88 (m, 1H), 1.80 - 1.62 (m, 6H), 1.60 - 1.50 (m, 1H), 1.50 - 1.34 (m, 7H), 1.33 - 1.26 (m, 2H), 1.25 (s, 2H), 1.22 (s, 6H), 1.16 (d, J = 11.9 Hz, 2H), 1.01 (s, 3H), 0.91 (t, J = 3.0 Hz, 6H), 0.88 (d, J = 4.4 Hz, 6H), 0.69 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 168.62, 168.07, 134.64, 134.05, 83.44, 71.34, 50.50, 50.49, 49.81, 44.48, 44.36, 40.16, 37.93, 36.87, 36.71, 36.48, 35.18, 30.94, 30.81, 29.30, 29.18, 28.24, 27.90, 26.34, 24.29, 24.04, 21.12, 21.03, 19.18, 18.69, 18.08, 16.46, 15.77.

[0298] Example 141

[0299] Preparation of (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl glycollate

[0300]

[0301] In the first step, (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68) (200 mg, 0.45 mmol, 1.0 eq) was dissolved in THF (tetrahydrofuran) (5 mL), and the reaction system was replaced with a nitrogen atmosphere. (Benzyloxy)acetyl chloride (124 mg, 0.67 mmol, 1.5 eq) was added dropwise, and the mixture was stirred at room temperature for 18 h. TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid plate baking) showed that the reaction was complete, and the reaction was stopped. Water (20 mL) was added to the reaction system, and the mixture was extracted with EA (ethyl acetate) (3 × 25 mL). The combined organic phases were washed with saturated brine (20 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (PE∶EtOAc = 92∶8 to 90∶10) to obtain the white solid (benzyloxy)acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (141-1) (180 mg, purity 95%, yield 64.13%). 1 H NMR (400 MHz, CDCl3) δ 7.40 - 7.28 (m, 5H), 4.67 - 4.60 (m, 3H), 4.10 (s, 1H), 2.08 - 1.98 (m, 4H), 1.92 (dd, J = 13.4, 7.7 Hz, 1H), 1.79 - 1.62 (m, 6H), 1.61 - 1.55 (m, 1H), 1.52 - 1.44 (m, 6H), 1.41 - 1.29 (m, 5H), 1.26 (s, 1H), 1.22 (s, 6H), 1.16 (d, J = 12.4 Hz, 2H), 1.00 (s, 3H), 0.89 (dd, J = 8.9, 6.3 Hz, 12H), 0.69 (s, 3H).

[0302] Step 2: (Benzyloxy)acetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (141-1) (100 mg, 0.17 mmol, 1 eq) was dissolved in MeOH (methanol) (10 mL), then palladium on carbon (about 55% moistened with water) (10.79 mg, 0.10 mmol, 0.6 eq) was added and the reaction system was replaced with a hydrogen atmosphere, and the reaction was stirred at room temperature for 18 hrs. The reaction was monitored by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid baking plate) until completion, and then the reaction was stopped. The reaction solution was filtered through diatomaceous earth to collect the filtrate, and the filtrate was concentrated to obtain the crude product. The crude product was separated and purified by flash chromatography (PE∶EtOAc = 85∶15 to 80∶20) to obtain the white solid hydroxyacetic acid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-i]phenanthren-7-yl ester (141) (49.86 g, purity 97.54%, yield 57.62%). 1 H NMR (400 MHz, CDCl3) δ 4.65 (dd, J = 11.4, 4.7 Hz, 1H), 4.15 (d, J = 1.3 Hz, 2H), 2.09 - 1.96 (m, 4H), 1.95 - 1.87 (m, 1H), 1.80 - 1.63 (m, 9H), 1.62 - 1.53 (m, 2H), 1.51 - 1.28 (m, 9H), 1.22 (s, 6H), 1.19 - 1.14 (m, 2H), 1.01 (s, 3H), 0.92 - 0.87 (m, 12H), 0.69 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 173.29, 134.62, 134.09, 82.81, 71.14, 60.68, 50.50, 49.81, 44.48, 44.42, 37.95, 36.88, 36.73, 36.48, 35.21, 30.94, 30.81, 29.35, 29.23, 28.24, 27.97, 26.35, 24.29, 24.21, 21.13, 21.02, 19.19, 18.69, 18.09, 16.50, 15.77, 0.01. LC-MS: [M + H]+ = 485.00

[0303] Example 142

[0304] Preparation of Compound 142 2-{[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}-2-oxoacetic acid

[0305]

[0306] Referring to Example 140, methyl 3-chloro-3-oxopropionate in the first step was replaced with methyl 2-chloro-2-oxoacetate, and finally Compound 2-{[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}-2-oxoacetic acid (142) was obtained. 1 H NMR (400 MHz, MeOD) δ 4.63 (s, 1H), 2.20 - 1.93 (m, 5H), 1.91 - 1.73 (m, 5H), 1.72 - 1.86 (m, 15H), 1.26 - 1.20 (m, 6H), 1.14 - 1.08 (m, 3H), 1.08 - 0.88 (m, 12H), 0.85 - 0.72 (m, 3H). 13 C NMR (101 MHz, MeOD) δ 162.25, 162.13, 134.20, 78.28, 70.17, 50.47, 49.65, 44.31, 43.93, 43.73, 37.73, 36.78, 36.66, 36.37, 30.53, 30.52, 29.44, 28.01, 27.94, 27.84, 27.16, 26.50, 26.13, 25.76, 23.47, 20.76, 18.42, 17.98, 17.88, 17.23, 15.73, 15.08. LC-MS: [M+H] + = 499.00

[0307] Example 143

[0308] Preparation of Compound 143 (6R)-6-[(1R,3aR,5aR,7S,9aS,11aR)-7-[(2-hydroxyethyl)oxy]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]-2-methylheptan-2-ol

[0309]

[0310]

[0311] In the first step, (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (129) (360 mg, 0.81 mmol, 1.0 eq) and rhodium(II) acetate (22.77 mg, 0.081 mmol, 0.1 eq) were placed in a sealed tube, added to DCM (dichloromethane) (5 mL), and ethyl diazoacetate (463.89 mg, 4.066 mmol, 5.0 eq) was added dropwise. The reaction mixture was stirred at 40 °C for 18 h under a nitrogen atmosphere. TLC (PE∶EtOAc = 10∶1, phosphomolybdic acid plate baking) showed that the reaction was complete, and the reaction was stopped. The reaction system was evaporated to dryness under vacuum. The crude product was separated and purified by flash chromatography (PE∶EtOAc = 98∶2 to 97∶3) to obtain a white solid ethyl {[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}acetate (143-1) (230 mg, purity 95%, yield 48.15%). 11H NMR (400 MHz, CDCl3) δ 4.26 (s, 2H), 4.09 (dd, J = 20.5, 6.5 Hz, 2H), 2.92 (dd, J = 11.7, 3.9 Hz, 1H), 2.69 (t, J = 6.1 Hz, 1H), 2.02 (d, J = 3.5 Hz, 1H), 1.92 (s, 1H), 1.80 - 1.63 (m, 6H), 1.61 - 1.43 (m, 7H), 1.31 (s, 6H), 1.27 (s, 3H), 1.06 (s, 3H), 0.99 (s, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 4.2 Hz, 6H), 0.69 (s, 3H).

[0312] Step 2. Ethyl {[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}acetate (143 - 1) (180 mg, 0.34 mmol, 1 eq) was dissolved in THF (tetrahydrofuran) (10 mL). The reaction system was cooled to 0 °C, and lithium aluminum hydride (64.59 mg, 1.702 mmol, 5 eq) was slowly added. The reaction mixture was reacted at 80 °C for 18 h under a nitrogen atmosphere. After monitoring the completion of the reaction by TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid plate baking), the reaction was stopped. After the reaction system was cooled to room temperature, the filtrate was collected by filtration, and the filtrate was concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (PE∶EtOAc = 85∶15) to obtain (6R)-6-[(1R,3aR,5aR,7S,9aS,11aR)-7-[(2-hydroxyethyl)oxy]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]-2-methylheptan-2-ol (143) (31.20 mg, purity 98.69%, yield 18.38%). 11H NMR (400 MHz, CDCl3) δ 3.76 - 3.68 (m, 3H), 3.46 - 3.39 (m, 1H), 2.85 (dd, J = 11.7, 4.0 Hz, 1H), 2.07 - 1.98 (m, 4H), 1.96 - 1.86 (m, 1H), 1.83 - 1.66 (m, 5H), 1.56 - 1.34 (m, 9H), 1.33 - 1.24 (m, 2H), 1.22 (s, 6H), 1.16 (d, J = 9.4 Hz, 2H), 1.05 (dd, J = 12.5, 2.1 Hz, 2H), 0.99 (d, J = 3.2 Hz, 6H), 0.93 - 0.86 (m, 6H), 0.82 (s, 3H), 0.69 (s, 3H). 13 13C NMR (101 MHz, CDCl3) δ 134.46, 134.40, 87.28, 71.13, 70.49, 62.30, 50.82, 50.52, 49.81, 44.50, 44.42, 38.98, 36.99, 36.74, 36.47, 35.44, 31.01, 30.83, 29.35, 29.21, 28.25, 28.17, 26.46, 24.27, 23.48, 21.12, 21.04, 19.17, 18.69, 18.15, 16.30, 15.75. LC-MS: [M + H] + = 417.00

[0313] Example 144

[0314] Preparation of Compound 144 {[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}acetic Acid

[0315]

[0316] Referring to Example 140, replace ethyl {[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}acetate (143-1) in the first step with (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (68), and finally obtain the compound {[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]oxy}acetic acid (144). 1 H NMR (400 MHz, CDCl3) δ 4.12 (dd, J = 61.1, 16.2 Hz, 2H), 3.03 (dd, J = 11.8, 3.4 Hz, 1H), 2.09 - 1.97 (m, 4H), 1.97 - 1.87 (m, 1H), 1.83 - 1.65 (m, 5H), 1.63 - 1.52 (m, 3H), 1.49 - 1.29 (m, 8H), 1.25 (s, 2H), 1.22 (s, 6H), 1.19 - 1.12 (m, J = 11.4 Hz, 2H), 1.03 (s, 3H), 1.00 (s, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.87 (s, 6H), 0.69 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 171.09, 134.64, 134.13, 89.01, 71.19, 66.84, 50.68, 50.51, 49.80, 44.48, 44.39, 38.84, 36.92, 36.73, 36.46, 35.21, 30.95, 30.80, 29.70, 29.34, 29.20, 28.22, 26.40, 24.29, 23.30, 21.11, 21.04, 19.14, 18.69, 18.10, 16.31, 15.75. LC-MS: [M+H] + = 485.00

[0317] Example 145

[0318] Preparation of Compound 145 (1R,5aR,9aS,11aR)-1-[(2R)-6-Hydroxy-6-methylheptan-2-yl]-6,6,7,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol

[0319]

[0320]

[0321] (1R,3aR,5aR,9aS,11aR)-1-[(2R)-6-Hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-one (71) (120 mg, 0.280 mmol, 1.0 eq) was dissolved in THF (tetrahydrofuran) (10 mL). The reaction system was cooled to 0 °C, and methylmagnesium chloride (3 M in THF, 0.933 mL, 2.800 mmol, 6 eq) was slowly added. The reaction mixture was reacted at room temperature under a nitrogen atmosphere for 18 h. TLC (PE∶EtOAc = 5∶1, phosphomolybdic acid baking plate) showed that the reaction was complete, and the reaction was stopped. Water (40 mL) was added to the system for dilution, and the mixture was extracted with EtOAc (ethyl acetate) (30 mL×3). The combined organic phases were washed with saturated brine (50 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was separated and purified by flash chromatography (PE∶EtOAc = 92∶8 to 91∶1) to obtain a white solid (1R,5aR,9aS,11aR)-1-[(2R)-6-Hydroxy-6-methylheptan-2-yl]-6,6,7,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (145) (24.63 mg, purity 92.03%, yield 19.39%). 1 H NMR (400 MHz, CDCl3) δ 2.08 - 1.98 (m, 4H), 1.93 - 1.88 (m, 1H), 1.67 - 1.57 (m, 4H), 1.49 - 1.42 (m, 10H), 1.40 - 1.31 (m, 5H), 1.22 (s, 3H), 1.00 (s, 1H), 0.92 - 0.86 (m, 4H), 0.69 (s, 3H). 1313C NMR (101 MHz, CDCl3) δ 134.26, 75.20, 71.13, 50.49, 49.78, 48.53, 44.51, 44.43, 36.75, 36.75, 30.98, 30.84, 29.35, 29.22, 29.22, 28.57, 28.25, 26.60, 25.37, 24.35, 23.32, 21.13, 21.12, 19.40, 18.96, 18.82, 18.69, 18.69, 15.75. LC-MS: [M+H] + = 441.00

[0322] Example 150

[0323] Preparation of Compound 150 (1R,3aR,5aR,7S,9aS,11aR)-7-Hydroxy-1-[(2R)-6-hydroxyoctan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-4-one

[0324]

[0325]

[0326] Step 1: (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxyoctan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (102) (2 g, 4.50 mmol, 1.0 eq) was dissolved in dichloromethane (100 mL). Acetic anhydride (2.1 mL, 22.49 mmol, 5.0 eq), DMAP (0.11 g, 0.90 mmol, 0.2 eq) and triethylamine (3.13 mL, 22.49 mmol, 5.0 eq) were added successively. The mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC (PE∶EtOAc = 10∶1, phosphomolybdic acid plate baking). After the reaction was completed, the reaction solution was quenched with methanol, washed with saturated sodium bicarbonate and saturated brine respectively, dried over anhydrous sodium sulfate, purified by silica gel column chromatography, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain white waxy acetic acid-(7R)-7-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]octan-3-yl ester (150-1) (2.5 g, 4.02 mmol, 89.4%). 1 1H NMR (400 MHz, CDCl3) δ 4.82 (d, J = 5.5 Hz, 1H), 4.50 (dd, J = 11.5, 4.5 Hz, 1H), 2.08 - 1.97 (m, 10H), 1.94 - 1.85 (m, 1H), 1.78 - 1.59 (m, 7H), 1.55 - 1.23 (m, 11H), 1.16 (dd, J = 17.5, 8.2 Hz, 3H), 1.01 (d, J = 7.9 Hz, 4H), 0.88 (t, J = 6.6 Hz, 15H), 0.68 (s, 3H).

[0327] Step 2: Acetic acid-(7R)-7-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]oct-3-yl ester (150-1) (2.5 g, 4.73 mmol, 1.0 eq) was dissolved in dichloromethane (100 mL). Under nitrogen protection and in an ice-water bath, m-CPBA (0.82 g, 4.73 mmol, 1.0 eq) was added portionwise. Subsequently, the reaction was transferred to room temperature. The reaction was monitored by TLC (PE∶EtOAc = 10∶1, phosphomolybdic acid plate baking). After the reaction was completed, it was quenched with saturated sodium bisulfite, washed with saturated sodium bicarbonate, and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (PE∶EtOAc = 95∶5 to 85∶15) to obtain white wax-like acetic acid-2-[(2R,5R,6R,11S)-5-[(2R)-6-acetoxyoct-2-yl]-2,6,10,10-tetramethyl-14-oxatetracyclo[7.4.1.01,9.02,6]tetradecan-11-yl]-2-methylpropyl ester (150-2) (2 g, 2.94 mmol, 62.1%). 1 H NMR (400 MHz, CDCl3) δ 4.81 (s, 1H), 4.46 (s, 1H), 2.04 (d, J = 6.9 Hz, 7H), 1.99 - 1.81 (m, 5H), 1.76 - 1.67 (m, 4H), 1.66 - 1.45 (m, 11H), 1.34 (m, 11H), 0.97 (d, J = 9.5 Hz, 2H), 0.87 (dd, J = 11.7, 7.0 Hz, 13H), 0.82 (s, 3H), 0.76 (s, 3H).

[0328] Step 3: Acetic acid - 2 - [(2R,5R,6R,11S) - 5 - [(2R) - 6 - acetyloxyoctan - 2 - yl] - 2,6,10,10 - tetramethyl - 14 - oxatetracyclo[7.4.1.01,9.02,6]tetradecan - 11 - yl] - 2 - methylpropyl ester (150 - 2) (1.9 g, 3.49 mmol, 1.0 eq) was dissolved in acetone (100 mL), 10 drops of concentrated sulfuric acid were added, and the reaction was monitored by TLC (PE∶EtOAc = 10∶1, phosphorus molybdate baking plate). After the reaction was completed, acetone was removed by rotary evaporation, 100 mL of ethyl acetate was added, washed with saturated sodium bicarbonate, then washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (PE∶EtOAc = 90∶10 to 80∶20) to obtain white solid acetic acid - (7R) - 7 - [(1R,3aR,5aR,7S,9aS,11aR) - 7 - acetyloxy - 3a,6,6,9a,11a - pentamethyl - 2,3,3a,5,5a,6,7,8,9,9a,11,11a - dodecahydro - 1H - cyclopenta[1,2 - a]phenanthren - 1 - yl]octan - 3 - yl ester (150 - 3) (1 g, 1.71 mmol, 49.0%). 1 H NMR(400MHz,CDCl3)δ5.53(s,1H),5.39(d,J = 6.0Hz,1H),4.89(t,J = 6.1Hz,1H),4.59(dd,J = 11.1,4.8Hz,1H),2.20 - 2.10(m,9H),2.10 - 2.03(m,2H),1.81(ddd,J = 11.7,7.5,3.5Hz,2H),1.75 - 1.53(m,9H),1.49 - 1.32(m,8H),1.29 - 1.22(m,2H),1.08(s,3H),1.03(s,3H),0.98 - 0.93(m,15H),0.63(s,3H). 1313C NMR (101 MHz, CDCl3) δ 170.99, 170.96, 145.60, 142.78, 119.87, 116.58, 80.83, 77.33, 77.01, 76.70, 75.58, 75.51, 50.99, 50.32, 49.25, 43.72, 37.82, 37.61, 37.23, 36.20, 36.17, 36.04, 36.00, 35.40, 34.03, 31.88, 31.59, 31.48, 28.09, 27.91, 27.07, 26.91, 26.88, 25.52, 24.26, 22.82, 22.78, 22.66, 22.19, 22.14, 21.33, 21.28, 18.42, 18.40, 16.93, 15.65, 14.12, 9.62, 9.59.

[0329] Step 4. Acetic acid-(7R)-7-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-2,3,3a,5,5a,6,7,8,9,9a,11,11a-dodecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]oct-3-yl ester (150-3) (0.9 g, 1.71 mmol, 1.0 eq) was dissolved in glacial acetic acid (30 mL). Under nitrogen protection and in an ice-water bath, 30% hydrogen peroxide (1.7 mL, 17.1 mmol, 10.0 eq) was added dropwise. After stirring at a constant temperature for 10 minutes, the reaction was warmed to room temperature and stirred. The reaction was monitored by TLC (PE∶EtOAc = 10∶1, phosphomolybdic acid baking plate). After the reaction was completed, it was diluted with ethyl acetate and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness to obtain the crude product acetic acid-(7R)-7-[(1R,3aS,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-4-oxo-2,3,3a,3b,4,5,5a,6,7,8,9,9a,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]oct-3-yl ester (150-4) (350 mg, 0.52 mmol, 30.2%). The crude product was directly used in the next step.

[0330] Step 5 Acetic acid-(7R)-7-[(1R,3aS,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-4-oxo-2,3,3a,3b,4,5,5a,6,7,8,9,9a,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]oct-3-yl ester (150-4) (200 mg, 0.37 mmol) was dissolved in a mixed solvent of chloroform (3 mL) and methanol (1.5 mL). Under nitrogen protection and in an ice-water bath, concentrated hydrochloric acid (36%-38%) (0.18 mL) was added dropwise. After stirring at the same temperature for 10 minutes, the reaction was transferred to room temperature. The reaction was monitored by TLC (PE∶EtOAc = 10∶1, phosphomolybdic acid plate baking). After the reaction was completed, the reaction was diluted with water and extracted with ethyl acetate three times. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (PE∶EtOAc = 80∶20) to obtain acetic acid-(7R)-7-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-4-oxo-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]oct-3-yl ester (150-5) (100 mg, 0.12 mmol, 32.5%). 1 H NMR (400 MHz, CDCl3) δ 4.85-4.76 (m, 1H), 4.51 (dd, J = 11.6, 4.3 Hz, 1H), 2.42 (t, J = 8.6 Hz, 1H), 2.29 (dt, J = 17.0, 6.9 Hz, 1H), 2.06 (d, J = 5.6 Hz, 6H), 2.00-1.90 (m, 1H), 1.84-1.69 (m, 5H), 1.60-1.46 (m, 5H), 1.41-1.20 (m, 8H), 1.19-1.13 (m, 3H), 1.02 (m, 2H), 0.96 (d, J = 8.2 Hz, 3H), 0.86 (m, 14H), 0.63 (d, J = 8.5 Hz, 2H). 1313C NMR (101 MHz, CDCl3) δ 198.73, 171.01, 170.83, 79.62, 77.34, 77.02, 76.70, 75.53, 49.86, 49.03, 47.79, 44.90, 39.62, 37.76, 36.45, 36.27, 34.50, 34.05, 31.99, 30.09, 27.38, 27.10, 26.91, 25.02, 23.85, 23.70, 22.12, 21.29, 21.24, 18.72, 18.44, 16.37, 15.79, 9.62, 9.59.

[0331] Step 6. Acetic acid-(7R)-7-[(1R,3aR,5aR,7S,9aS,11aR)-7-acetoxy-3a,6,6,9a,11a-pentamethyl-4-oxo-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]oct-3-yl ester (150-5) (100 mg, 0.18 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (1 mL) solution, protected by nitrogen, and methylmagnesium chloride solution (0.307 mL) was added dropwise at 0 °C. Then it was transferred to room temperature for reaction. The reaction was monitored by TLC (PE∶EtOAc = 10∶1, phosphomolybdic acid baking plate). After the reaction was completed, it was quenched with saturated ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (PE∶EtOAc = 80∶20) to obtain a white solid (1R,3aR,5aR,7S,9aS,11aR)-7-hydroxy-1-[(2R)-6-hydroxyoctan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-4-one (150) (14 mg, 0.03 mmol, 16.6%). 1 1H NMR (400 MHz, CDCl3) δ 3.58 - 3.48 (s, 1H), 3.28 (dd, J = 11.5, 4.5 Hz, 1H), 2.50 - 2.34 (m, 2H), 2.34 - 2.20 (m, 2H), 2.10 - 1.90 (m, 2H), 1.85 (d, J = 13.0 Hz, 1H), 1.80 - 1.69 (m, 4H), 1.63 (dd, J = 12.3, 5.4 Hz, 1H), 1.46 - 1.23 (m, 12H), 1.17 (s, 3H), 1.00 (s, 3H), 1.10 - 1.04 (m, 1H), 0.94 (dd, J = 13.6, 5.2 Hz, 9H), 0.88 (s, 3H), 0.65 (s, 3H).13 13C NMR (101 MHz, CDCl3) δ 199.03, 164.75, 139.01, 77.95, 77.32, 77.01, 76.69, 73.43, 73.35, 49.85, 49.01, 47.77, 44.92, 39.77, 38.92, 37.50, 37.37, 36.65, 36.39, 36.28, 36.17, 34.83, 32.00, 30.28, 30.14, 28.77, 27.44, 25.00, 23.66, 22.44, 22.30, 18.77, 18.34, 15.79, 15.27, 9.84. LC-MS: [M+1] + = 459.3

[0332] Example 192

[0333] Preparation of Compound 192 (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxyoctan-2-yl]-3a,4,6,6,9a,11a-hexamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthrene-4,7-diol

[0334]

[0335] (7R)-7-[(1R,3aR,5aR,7S,9aS,11aR)-7-Acetoxy-3a,6,6,9a,11a-pentamethyl-4-oxo-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]octan-3-yl acetate (150-5) (100 mg, 0.18 mmol, 1.0 eq) was dissolved in anhydrous tetrahydrofuran (1 mL) solution. Under nitrogen protection, methylmagnesium bromide solution (0.5 mL, 3.0 mol / L, 10.0 eq) was added dropwise at 0 °C, and then the reaction was transferred to room temperature. The reaction was monitored by TLC (petroleum ether∶ethyl acetate = 1∶1). After the reaction was completed, it was quenched with saturated sodium chloride solution under an ice-water bath, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 90∶10) to obtain the crude product. The crude product was further prepared by reverse phase to obtain a white solid (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-Hydroxyoctan-2-yl]-3a,4,6,6,9a,11a-hexamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthrene-4,7-diol (192) (3.5 mg, 0.006 mmol, purity 81%, yield 7%). 1 H NMR (400 MHz, CDCl3) δ 3.56 - 3.49 (m, 1H), 3.28 - 3.17 (m, 1H), 2.15 - 1.88 (m, 6H), 1.81 - 1.66 (m, 6H), 1.60 - 1.43 (m, 11H), 1.36 - 1.24 (m, 10H), 1.23 - 1.15 (m, 2H), 1.07 (d, J = 4.4 Hz, 3H), 1.03 - 0.99 (m, 3H), 0.98 - 0.87 (m, 11H), 0.86 - 0.60 (m, 6H). 13 C NMR (101 MHz, CDCl3) δ 140.54, 139.00, 78.59, 77.32, 77.00, 76.68, 75.06, 73.47, 55.26, 50.63, 50.51, 50.32, 48.60, 46.11, 39.65, 39.09, 38.33, 37.56, 35.92, 31.66, 31.40, 31.23, 30.28, 30.06, 28.82, 27.86, 27.55, 25.91, 23.34, 22.40, 21.57, 18.79, 18.47, 17.03, 15.51, 9.85 LCMS (ESI) [M - OH] + = 457.4

[0336] Example 189

[0337] Preparation of Compound 189 2-Hydroxy-N-[(1R,3aR,5aR,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]acetamide

[0338]

[0339]

[0340] In the first step, the raw material (1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ol (129) (3 g, 6.776 mmol, 1.0 eq), sodium acetate (1.84 g, 13.552 mmol, 2.0 eq) were dissolved in DCM (dichloromethane) (20 mL), and pyridinium chlorochromate (2.92 g, 13.552 mmol, 2.0 eq) was added. The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 18 hr. TLC (petroleum ether∶ethyl acetate = 5∶1) was used for monitoring. After the reaction was completed, the reaction solution was filtered through diatomaceous earth, and the filtrate was rotary evaporated under vacuum to obtain a crude product. The crude product was separated and purified by flash chromatography (petroleum ether∶ethyl acetate = 90∶10) to obtain a white solid (1R,3aR,5aR,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-one (189-1) (1.8 g, purity 90%, yield 54.2%). 1 H NMR (400 MHz, CDCl3) δ 2.69 (t, J = 6.1 Hz, 1H), 2.65 - 2.36 (m, 2H), 2.13 - 1.90 (m, 2H), 1.81 - 1.37 (m, 5H), 1.31 (s, 1H), 1.27 (s, 1H), 1.11 (d, J = 9.3 Hz, 2H), 1.07 (s, 1H), 0.92 (d, J = 6.1 Hz, 3H), 0.89 (s, 3H), 0.72 (s, 3H).

[0341] Step 2: Dissolve the raw material (1R,3aR,5aR,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxiran-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-one (189-1) (1.8 g, 4.08 mmol, 1.0 eq) and sodium acetate (1.67 g, 12.253 mmol, 3.0 eq) in EtOH (ethanol) (20 mL), add hydroxylamine hydrochloride (0.85 g, 12.253 mmol, 3.0 eq), and stir the reaction mixture at 60 °C for 3 h under a nitrogen atmosphere. Monitor by TLC (petroleum ether∶ethyl acetate = 5∶1). After the reaction is completed, dilute the system with water (40 mL), extract with ethyl acetate (30 mL×3), wash the combined organic phases with saturated brine (50 mL), then dry over anhydrous sodium sulfate, filter, and concentrate to obtain a crude product. The crude product is separated and purified by flash chromatography (petroleum ether∶ethyl acetate = 95∶5 to 92∶8) to obtain the white solid [(1R,3aR,5aR,7E,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxiran-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ylidene]hydroxylamine (189-2) (1.7 g, purity 90%, yield 82.2%). 1 1H NMR (400 MHz, CDCl3) δ 3.16 - 3.08 (m, 1H), 2.69 (t, J = 6.1 Hz, 1H), 2.25 (ddd, J = 15.5, 12.8, 5.8 Hz, 1H), 2.06 (dt, J = 15.9, 9.1 Hz, 4H), 1.96 - 1.85 (m, 2H), 1.74 - 1.33 (m, 14H), 1.31 (s, 4H), 1.27 (s, 3H), 1.20 (s, 4H), 1.11 (s, 3H), 1.10 (s, 3H), 0.92 (d, J = 6.0 Hz, 3H), 0.87 (s, 3H), 0.71 (s, 3H).

[0342] Step 3: Dissolve [(1R,3aR,5aR,7E,9aS,11aR)-1-[(2R)-4-(3,3-dimethyloxetan-2-yl)butan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-ylidene]hydroxylamine (189-2) (50 mg, 0.22 mmol, 1.0 eq) in THF (tetrahydrofuran) (5 mL). Cool the reaction system to 0 °C and slowly add lithium aluminum hydride (41.56 mg, 1.095 mmol, 5.0 eq). React the reaction mixture at 80 °C for 18 h under a nitrogen atmosphere. Monitor by TLC (DCM∶MeOH = 10∶1). After the reaction is complete, cool the reaction system to room temperature, filter to collect the filtrate, and concentrate the filtrate to obtain the crude product. Purify the crude product by flash chromatography (DCM∶MeOH = 95∶5 to 94∶6) to obtain the white solid (6R)-6-[(1R,3aR,5aR,9aS,11aR)-7-amino-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]-2-methylheptan-2-ol (189-3) (20 mg, purity 90%, yield 12.3%).

[0343] 1 H NMR (400 MHz, CDCl3) δ 2.65 (dd, J = 12.0, 3.3 Hz, 1H), 2.09 - 1.88 (m, 11H), 1.82 - 1.57 (m, 8H), 1.52 - 1.35 (m, 9H), 1.22 (s, 6H), 1.06 (s, 3H), 0.98 (s, 3H), 0.92 - 0.87 (m, 9H), 0.68 (s, 3H).

[0344] Step 4: Dissolve glyoxylic acid (18.85 mg, 0.25 mmol, 1.1 eq) in DMF (N,N-dimethylformamide) (10 mL). After adding HATU (102.82 mg, 0.27 mmol, 1.2 eq) and N,N-diisopropylethylamine (0.074 mL, 0.451 mmol, 2 eq), replace the reaction system with a nitrogen atmosphere. After stirring the reaction mixture at room temperature for 30 min, add (6R)-6-[(1R,3aR,5aR,9aS,11aR)-7-amino-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]-2-methylheptan-2-ol (189-3) (100 mg, 0.225 mmol, 1 eq), and continue stirring at room temperature for 3 h. Monitor by TLC (petroleum ether∶ethyl acetate = 1∶2). After the reaction is complete, dilute the system with water (40 mL), extract with ethyl acetate (40 mL×3), wash the combined organic phases with saturated brine (50 mL), then dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. The crude product is separated and purified by flash chromatography (petroleum ether∶ethyl acetate = 45∶55 to 34∶66) to obtain the white solid 2-hydroxy-N-[(1R,3aR,5aR,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]acetamide (189) (19.64 mg, purity 84.66%, yield 16.44%). 19.64 mg, 0.037 mmol, 16.44%. 1 H NMR (400 MHz, DMSO) δ 7.03 (d, J = 9.9 Hz, 1H), 5.49 (t, J = 5.8 Hz, 1H), 4.03 (s, 1H), 3.87 - 3.75 (m, 2H), 3.55 - 3.44 (m, 1H), 2.05 - 1.83 (m, 6H), 1.74 - 1.52 (m, 7H), 1.48 - 1.28 (m, 8H), 1.24 - 1.13 (m, 4H), 1.05 (s, 6H), 0.94 (d, J = 4.6 Hz, 3H), 0.90 - 0.86 (m, 7H), 0.79 (s, 3H), 0.77 (s, 6H), 0.67 (s, 3H). 1313C NMR (101 MHz, DMSO) δ 171.40, 134.55, 134.21, 69.24, 61.82, 55.72, 51.43, 50.56, 49.89, 44.62, 44.48, 38.18, 36.99, 36.94, 36.51, 36.42, 31.04, 30.86, 29.89, 29.70, 28.86, 28.25, 26.48, 25.94, 24.53, 21.03, 20.91, 19.30, 19.03, 18.59, 16.84, 16.10. LCMS [M+H] + = 502.5

[0345] Example 190

[0346] Preparation of Compound 190 N-[(1R,3aR,5aR,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]methanesulfonamide

[0347]

[0348] Dissolve the raw material (6R)-6-[(1R,3aR,5aR,9aS,11aR)-7-amino-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-1-yl]-2-methylheptan-2-ol (189-3) (50 mg, 0.11 mmol, 1.0 eq) and triethylamine (0.047 mL, 0.34 mmol, 3.0 eq) in DCM (dichloromethane) (10 mL). After cooling the reaction system to 0 °C, add methanesulfonyl chloride (15.53 mg, 0.14 mmol, 1.2 eq), and stir the reaction mixture at room temperature under a nitrogen atmosphere for 2 h. Monitor by TLC (petroleum ether∶ethyl acetate = 1∶2). After completion of the reaction, dilute the system with water (40 mL), extract with DCM (dichloromethane) (20 mL × 3), wash the combined organic phases with saturated brine (50 mL), then dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purify the crude product by flash chromatography (petroleum ether∶ethyl acetate = 20∶80 to 10∶90) to obtain the white solid N-[(1R,3aR,5aR,7S,9aS,11aR)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-3a,6,6,9a,11a-pentamethyl-2,3,3a,4,5,5a,6,7,8,9,9a,10,11,11a-tetradecahydro-1H-cyclopenta[1,2-a]phenanthren-7-yl]methanesulfonamide (190) (1.7 g, purity 88.38%, yield 24.08%). 1 HNMR (400 MHz, DMSO) δ 7.41 (d, J = 9.5 Hz, 1H), 4.03 (s, 1H), 3.46 (d, J = 9.2 Hz, 1H), 2.10 - 1.85 (m, 6H), 1.82 (s, 3H), 1.77 - 1.52 (m, 6H), 1.50 - 1.40 (m, 3H), 1.39 - 1.28 (m, 6H), 1.21 - 1.11 (m, 4H), 1.05 (s, 6H), 0.94 (s, 3H), 0.89 - 0.85 (m, 6H), 0.77 (s, 3H), 0.75 (s, 3H), 0.67 (s, 3H). 1313C NMR (101 MHz, DMSO) δ 137.00, 136.98, 69.24, 56.33, 56.11, 51.45, 50.56, 49.88, 44.61, 44.48, 38.27, 36.98, 36.94, 36.66, 36.42, 31.04, 30.85, 29.89, 29.70, 28.88, 28.24, 25.99, 24.52, 23.29, 21.03, 20.89, 19.39, 19.03, 18.60, 17.17, 16.09 LCMS: [M+H]+ = 527.5.

Claims

1. A compound as shown below or a pharmaceutically acceptable salt thereof:

2. A pharmaceutical composition comprising: (1) The compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof; and (2) at least one pharmaceutical excipient.

3. Use of a compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as claimed in claim 2 in the preparation of a medicament for inhibiting the SREBP pathway.

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