Process for the preparation of cholic acid derivatives

Abstract

The application discloses a cholic acid derivative as shown in formula (I) and a preparation method thereof. The target product cholic acid derivative is prepared through TBS protection, 4,4-dimethylation, deprotection, oxidation, Witting reaction, reduction, hydrolysis, acylation and deprotection and the like. The application further provides application of the cholic acid derivative in preparation of medicines for preventing and / or treating hypercholesterolemia, hypertriglyceridemia, anti-atherosclerosis and anti-non-alcoholic fatty liver disease. The cholic acid derivative can not only effectively promote degradation of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), but also reduce HMGCR protein increase caused by statins, thereby reducing endogenous cholesterol level, providing a beneficial reference for research and development of new cholesterol-lowering drugs, and having a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and its preparation and application, specifically relating to a bile acid derivative, its preparation method, and its application in anti-atherosclerosis and anti-non-alcoholic steatohepatitis. Background Technology

[0002] Cardiovascular disease is a highly prevalent disease. High cholesterol levels, especially low-density lipoprotein (LDL), are one of the main contributing factors. Currently, the main drugs used to lower high cholesterol are statins, including atorvastatin, simvastatin, lovastatin, pravastatin, fluvastatin, and rosuvastatin. The mechanism of action of statins is primarily due to their structure mimicking HMG-CoA, allowing them to competitively bind to HMG-CoA reductase (HMGCR), thereby inhibiting the conversion of HMG-CoA to mevalonate and limiting cholesterol biosynthesis in the body.

[0003] The safety of statins is receiving increasing attention, with the most serious and common side effects being liver damage and muscle toxicity (myalgia, myositis, and rhabdomyolysis, etc.). Other manifestations include gastrointestinal discomfort, headache, sleep disturbances, and peripheral neuropathy. In particular, long-term use of statins leads to a compensatory increase in HMGCR protein, forcing patients to increase the drug dosage to suppress the increased intracellular HMGCR. This effect severely weakens the efficacy of statins and increases side effects. Although this drawback of statins was recognized from the beginning of their clinical use, there is still no way to prevent statin-induced HMGCR accumulation and its side effects. To target the ubiquitination and degradation of HMGCR protein, our laboratory has designed and synthesized the first generation of lithocholic acid derivatives, from which we screened and obtained several novel and active small molecule compounds. Among them, the representative compound HMG499 has an EC50-60% activity in promoting the ubiquitination and degradation of HMGCR protein. 50=0.41 μM. This compound not only significantly promotes the ubiquitination and degradation of HMGCR protein at the cellular level, reduces statin-induced HMGCR protein accumulation, and effectively inhibits cholesterol production; it also exhibits significant lipid-lowering and cholesterol-lowering effects at the animal level. This research was published in the internationally renowned journal *Nature Communications* (2018, 9, 5138) in December 2018. Subsequent studies revealed that while the first-generation compound mentioned in the article significantly promotes the ubiquitination and degradation of HMGCR protein, reduces statin-induced HMGCR protein accumulation, and effectively inhibits cholesterol synthesis and reduces the formation of atherosclerotic plaques in mice, this first-generation compound also leads to intracellular cholesterol accumulation (e.g., Figure 1 (As shown).

[0004] Cholesterol buildup can impair nerve cell survival, leading to severe neurodegenerative diseases such as Niemann-Pick disease type C. Furthermore, intracellular cholesterol accumulation can cause excessive lipid buildup in the liver, kidneys, spleen, and brain, resulting in organ damage and potentially death. Addressing the cholesterol buildup problem caused by these compounds has become crucial in the development of new drugs containing them.

[0005] Summary of the Invention

[0006] This invention, while retaining the excellent activity of the first-generation lithocholic acid derivatives, further modifies the structure of the compounds to obtain a series of compounds that do not produce cholesterol accumulation at all, which is of great significance for the development of this type of new drug.

[0007] This invention provides a bile acid derivative, the structure of which is shown in formula (I):

[0008]

[0009] Where n is selected from natural numbers.

[0010] R1 is selected from various heterocycles and substituted heterocycles.

[0011] R2 is selected from hydrogen, hydroxyl, carbonyl, halogen, and alkyl.

[0012] R3 is selected from alcohols, carboxyl groups, and amides.

[0013] The C5 and C6 connection keys are either single or double keys.

[0014] Preferably,

[0015] The natural number n is selected from 0, 1, 2, 3, 4.

[0016] R1 includes pyrazine rings, pyrazole rings and substituted pyrazole rings, substituted thiazole rings, 1,2,3-oxadiazole rings, 2-aminopyrimidine rings, isoxazole rings and substituted isoxazole rings;

[0017] The pyrazine ring is

[0018] The pyrazole ring is

[0019] The substituted pyrazole ring includes N-acetylpyrazole. N-propionylpyrazole N-Butyrylpyrazole N-Isobutyrylpyrazole N-Methylacryloylpyrazole 1-Phenylonpyrazole ring 1-(2-Fluoro)phenylpyrazole ring

[0020] Preferably, the substituted pyrazole ring is N-acetylpyrazole. N-Isobutyrylpyrazole

[0021] More preferably, the substituted pyrazole ring is N-acetylpyrazole.

[0022] The substituted thiazole ring includes a 2-methylthiazole ring. 2-Aminothiazole ring

[0023] The 1,2,3-oxadiazole ring is

[0024] The 2-aminopyrimidine ring is

[0025] The isoxazole ring is

[0026] The substituted isoxazole ring is

[0027] The hydroxyl group of R2 is in the α or β configuration, preferably the β configuration. Hydrogen is

[0028] R3 is selected from tertiary alcohols, carboxyl groups, and amides;

[0029] The tertiary alcohol includes diethyl tertiary alcohol. Diallyl tertiary alcohol Dimethyl tertiary alcohol di-n-propyl tertiary alcohol Isopropyl tertiary alcohol di-n-butyl tertiary alcohol Diisopropyltertiary alcohol Diacetylacetyl tert-ol Diphenyltertiary alcohol Dibenzyl tertiary alcohol

[0030] Preferably, the tertiary alcohol comprises diethyl tertiary alcohol. Diallyl tertiary alcohol

[0031] The carboxyl group is

[0032] The amide includes formamide. Acetamide n-Propionamide 2-Methylbenzamide p-Toluamide 5-Nitrothiazolidinamide N-3-pyridineamide Piperidine amide Morpholinamide N-Boc-4-piperidinamide N-acetyl-4-piperidinamide Hexahydropyran-4-amide Morpholine-2-ethylamide

[0033]

[0034] The present invention also provides a bile acid derivative, the structure of which is shown in formula Q27:

[0035]

[0036] The present invention also provides a bile acid derivative, the structure of which is shown in formula Q63:

[0037]

[0038] This invention also proposes a method for preparing a bile acid derivative, using the bile acid derivative shown in formula Q1 as the starting material, and obtaining compound Q7 through TBS protection, 4,4-dimethylation, deTBS protection, oxidation, Witting reaction, and reduction; then, using compound Q5 as the starting material, obtaining compound Q9 through oxidation and esterification; or using compound Q4 as the starting material, obtaining compound Q13 through iodination, cyano substitution, hydrolysis, and esterification respectively; the preparation method is shown in route (1):

[0039]

[0040] Specifically, the method includes the following steps:

[0041] (a) TBS protection response

[0042] Compound Q1 was dissolved in an organic solvent, and the reagent and base used for TBS protection were added at low temperature. The TBS protection reaction yielded compound Q2.

[0043] In step (a), the organic solvent is selected from any one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dichloromethane, chloroform, carbon tetrachloride, etc.; preferably, it is N,N-dimethylformamide.

[0044] In step (a), the base is selected from any one or more of triethylamine, imidazole, diisopropylethylamine, etc.; preferably, it is imidazole.

[0045] In step (a), the molar ratio of compound Q1 to the base is 1:(2-20); preferably, it is 1:8.

[0046] In step (a), the reagent used for TBS protection is selected from any one or more of tert-butyldimethylsilicon chloride (TBSCl), tert-butyldimethylsilyltrifluoromethanesulfonate (TBSOTf), etc.; preferably, it is TBSCl.

[0047] In step (a), the molar ratio of compound Q1 to the reagent used for TBS protection is selected from 1:(1 to 10); preferably, it is 1:4.

[0048] In step (a), the low temperature is -10 to 10°C; preferably, it is 0°C.

[0049] In step (a), the temperature of the TBS protection reaction is 25–70°C; preferably, it is 25°C.

[0050] In step (a), the TBS protection reaction takes 2 to 12 hours; preferably, it takes 5 hours.

[0051] (b) 4,4-Dimethylation reaction

[0052] Compound Q2 was dissolved in an organic solvent, and then a base and a methylating agent were added. The methylation reaction yielded compound Q3.

[0053] In step (b), the organic solvent is selected from one or more of t-BuOH, benzene, carbon tetrachloride, tetrahydrofuran, etc.; preferably, it is t-BuOH.

[0054] In step (b), the alkali is selected from any one or more of t-BuOK, KOC(Et)Me2, etc., preferably t-BuOK.

[0055] In step (b), the methylating agent is any one or more of CH3Cl, CH3Br, CH3I, etc.; preferably, it is CH3I.

[0056] In step (b), the molar ratio of compound Q2 to the base is 1:(2-8); preferably, it is 1:4.

[0057] In step (b), the molar ratio of compound Q2 to the methylating agent is 1:(2-10); preferably, it is 1:8.

[0058] In step (b), the optimal method for adding the methylating agent is to add it slowly dropwise.

[0059] In step (b), the alkali is added in batches.

[0060] In step (b), the reaction temperature during the addition of the methylating agent is 0°C.

[0061] In step (b), the temperature of the methylation reaction is 0–60°C, preferably 25°C.

[0062] In step (b), the methylation reaction takes 2 to 12 hours; preferably, it takes 4 hours.

[0063] (c) Deprotection reaction of TBS

[0064] Compound Q3 was dissolved in an organic solvent, and the acid required for deprotection was added to react and give compound Q4.

[0065] In step (c), the organic solvent is any one or more of diethyl ether, tetrahydrofuran, ethyl acetate, methanol, and ethanol; preferably, it is diethyl ether.

[0066] In step (c), the acid is any one or more of p-toluenesulfonic acid, hydrochloric acid gas, sulfuric acid, and hydrochloric acid solution; preferably, it is hydrochloric acid gas.

[0067] In step (c), the time for the TBS protection removal reaction is 1 to 5 hours; preferably, it is 2 hours.

[0068] In step (c), the temperature of the TBS removal reaction is 25–60°C; preferably, it is 25°C.

[0069] (d) Oxidation reaction

[0070] Compound Q4 was dissolved in an organic solvent, and an oxidizing agent was added to react and give compound Q5.

[0071] In step (d), the organic solvent is selected from one or more of toluene, tetrahydrofuran, dichloromethane, etc.; preferably, it is dichloromethane.

[0072] In step (d), the oxidant is any one or more of PDC, DDQ, PCC, IBX, etc.; preferably, it is PDC.

[0073] In step (d), the molar ratio of compound Q4 to oxidant is 1:(1-3); preferably, it is 1:1.5.

[0074] In step (d), the temperature of the oxidation reaction is 20–35°C; preferably, it is 25°C.

[0075] In step (d), the oxidation reaction takes 7 to 10 hours; preferably, it takes 8 hours.

[0076] (e) Witting reaction

[0077] The base was dissolved in an organic solvent, and Witting reagent and compound Q5 were added respectively to give compound Q6.

[0078] In step (e), the organic solvent is selected from one or more of tetrahydrofuran, toluene, petroleum ether, benzene, etc.; preferably, it is tetrahydrofuran.

[0079] In step (e), the alkali is one or more of sodium methoxide, sodium ethoxide, and sodium hydride (60%); preferably, it is sodium hydride (60%).

[0080] In step (e), the Witting reagent is preferably triethyl phosphoroacetate.

[0081] In step (e), the molar ratio of compound Q5 to base and Witting reagent is 1:(3-8):(3-8); preferably, it is 1:4.5:5.

[0082] In step (e), the Witting reagent is added by dripping for 5 to 30 minutes; preferably, for 10 minutes.

[0083] In step (e), the temperature of the Witting reaction is 25–75°C; preferably, it is 25°C.

[0084] In step (e), the Witting reaction takes 1 to 3 hours; preferably, it takes 1 hour.

[0085] (f) Reduction reaction

[0086] Compound Q6 was dissolved in an organic solvent, a reducing agent was added to obtain a compound intermediate, and then an oxidizing agent was added to oxidize it to obtain compound Q7.

[0087] In step (f), the organic solvent for the reduction reaction is preferably dried anhydrous methanol.

[0088] In step (f), the reducing agent used in the reduction reaction is preferably magnesium shavings.

[0089] In step (f), the molar ratio of compound Q6 to reducing agent is 1:(2-10); preferably, it is 1:10.

[0090] In step (f), the temperature of the reduction reaction is 25–60°C; preferably, it is 25°C.

[0091] In step (f), the reaction time for obtaining the intermediate is 3 to 8 hours; preferably, it is 5 hours.

[0092] In step (f), the organic solvent for the oxidation reaction is selected from one or more of toluene, tetrahydrofuran, DMSO, etc.; preferably, it is a mixed solvent of DMSO and toluene.

[0093] In step (f), the oxidant acts to oxidize the small amount of reduced hydroxyl groups at the C-3 position.

[0094] In step (f), the oxidant is selected from any one or more of IBX, PDC, PCC, DDQ, etc.; preferably, it is IBX.

[0095] In step (f), the molar ratio of compound Q6 to oxidant is 1:(0.5-3); preferably, it is 1:1.

[0096] In step (f), the temperature of the oxidation reaction is 25–60°C; preferably, it is 25°C.

[0097] In step (f), the oxidation reaction takes 2 to 8 hours; preferably, it takes 4 hours.

[0098] (g) Oxidation reaction

[0099] Compound Q5 was dissolved in an organic solvent, and an oxidant was added at low temperature to react and give compound Q8.

[0100] In step (g), the organic solvent for the oxidation reaction is selected from one or more of pyridine, chloroform, dichloroethane, 1,2-dichloropropane, etc.; preferably, it is pyridine.

[0101] In step (g), the temperature of the reaction solution when the oxidant is added is 0 to 10°C; preferably, it is 0°C.

[0102] In step (g), the oxidant is selected from one or more of tetrabutylammonium permanganate, potassium permanganate, potassium dichromate, O3, etc.; preferably, it is tetrabutylammonium permanganate.

[0103] In step (g), the molar ratio of Q5 to oxidant is 1:(1-4); preferably, it is 1:2.

[0104] In step (g), the temperature of the oxidation reaction is 0–60°C; preferably, it is 25°C.

[0105] In step (g), the oxidation reaction takes 1 to 2 hours; preferably, it takes 1 hour.

[0106] (h) Esterification reaction

[0107] Compound Q8 was dissolved in an organic solvent, and an acid was added to carry out an esterification reaction to obtain compound Q9.

[0108] In step (h), the organic solvent is selected from one or more of methanol, tetrahydrofuran, and a mixture of methanol; preferably, it is methanol.

[0109] In step (h), the acid in the esterification reaction is selected from any one or more of concentrated sulfuric acid, p-toluenesulfonic acid, thionyl chloride, etc.; preferably, it is concentrated sulfuric acid.

[0110] In step (h), the molar ratio of compound Q8 to acid is 1:(0.5-2); preferably, it is 1:0.7.

[0111] In step (h), the temperature of the esterification reaction is 25–70°C; preferably, it is 70°C.

[0112] In step (h), the esterification reaction takes 2 to 12 hours; preferably, it takes 2 hours.

[0113] (i) Iodination reaction

[0114] The iodinated reagent, catalyst, and acid-binding agent were dissolved in an organic solvent and stirred for a period of time. Then, compound Q4 was added, and the reaction yielded compound Q10.

[0115] In step (i), the organic solvent is selected from one or more of toluene, dichloromethane, benzene, etc.; preferably, it is toluene.

[0116] In step (i), the iodinated reagent is selected from any one or more of I2, N-iodosuccinimide, etc.; preferably, it is I2.

[0117] In step (i), the catalyst and acid-binding agent in the iodination reaction are PPh3 and imidazole, respectively.

[0118] In step (i), the molar ratio of compound Q4 to the iodinated reagent, catalyst and acid-binding agent is 1:(3-8):(3-8):(6-16); preferably, it is 1:7:7.3:14.

[0119] In step (i), the reaction temperature before adding compound Q4 in the iodination reaction is 25–60°C; preferably, it is 25°C.

[0120] In step (i), the reaction time before adding compound Q4 in the iodination reaction is 0.5 h to 3 h; preferably, it is 1 h.

[0121] In step (i), the reaction temperature after adding compound Q4 in the iodination reaction is 25–60°C; preferably, it is 25°C.

[0122] In step (i), the reaction time after adding compound Q4 in the iodination reaction is 1 to 2 hours; preferably, it is 2 hours.

[0123] (j) Cyano substitution reaction

[0124] Compound Q10 was dissolved in an organic solvent, and a base and a cyanating reagent were added to react and give compound Q11.

[0125] In step (j), the organic solvent is selected from one or more of DMF, tetrahydrofuran, DMSO, toluene, etc.; preferably, it is DMF.

[0126] In step (j), the alkali is selected from one or more of sodium hydroxide, potassium hydroxide, potassium fluoride, sodium fluoride, potassium acetate, and sodium acetate; preferably, it is potassium fluoride.

[0127] In step (j), the cyaniding reagent is selected from one or more of sodium cyanide, trimethylsilyl cyanide, etc.; preferably, it is trimethylsilyl cyanide.

[0128] In step (j), the molar ratio of compound Q10 to the base and cyano reagent is 1:(2-20):(2-10); preferably, it is 1:16:8.

[0129] In step (j), the temperature of the cyanide removal reaction is 40–80°C; preferably, it is 50°C.

[0130] In step (j), the cyanide removal reaction takes 2 to 12 hours; preferably, it takes 4 hours.

[0131] (k) Hydrolysis reaction

[0132] Compound Q11 was dissolved in an organic solvent, and then hydrolyzed with an alkali to obtain compound Q12.

[0133] In step (k), the organic solvent is selected from one or more of methanol, tetrahydrofuran, ethanol, a mixture of water and ethanol, and a mixture of water and methanol; preferably, it is a mixture of water and ethanol.

[0134] In step (k), the volume ratio of the water to ethanol mixed solvent is 1:(1-3); preferably, it is 1:3.

[0135] In step (k), the alkali is selected from one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, it is sodium hydroxide.

[0136] In step (k), the molar ratio of compound Q11 to the base is 1:(1 to 10); preferably, it is 1:10.

[0137] In step (k), the temperature of the hydrolysis reaction is 70–120°C; preferably, it is 100°C.

[0138] In step (k), the hydrolysis reaction takes 24 to 72 hours; preferably, it takes 72 hours.

[0139] (l) Esterification reaction

[0140] The synthesis of compound Q13 is similar to the esterification method of compound Q9 in route (1).

[0141] This invention also proposes a method for preparing a bile acid derivative, using compound Q7 as the starting material, and obtaining Q19 through ethylene glycol protection, reduction, iodination, cyano substitution, hydrolysis, and esterification. The preparation method is shown in route (2):

[0142]

[0143] Specifically, the method includes the following steps:

[0144] (a) Ethylene glycol protection reaction

[0145] Compound Q7 was dissolved in an organic solvent, and a catalyst, a dehydrating agent, and ethylene glycol were added to react and give compound Q14.

[0146] In step (a), the organic solvent is selected from tetrahydrofuran, a mixed solution of tetrahydrofuran and ethylene glycol; preferably, it is a mixed solution of tetrahydrofuran and ethylene glycol.

[0147] In step (a), the volume ratio of tetrahydrofuran to ethylene glycol is 3:(1-3); preferably, it is 3:1.

[0148] In step (a), the catalyst is preferably p-toluenesulfonic acid.

[0149] In step (a), the dehydrating agent is selected from triethyl orthoformate and trimethyl orthoformate; preferably, it is triethyl orthoformate.

[0150] In step (a), the molar ratio of compound Q7 to catalyst and dehydrating agent is 1:(0.2-0.5):(5-10); preferably, it is 1:0.2:5.

[0151] In step (a), the ethylene glycol protection reaction takes 2 to 12 hours; preferably, it takes 3 hours.

[0152] In step (a), the temperature of the ethylene glycol protection reaction is 25–60°C; preferably, it is 25°C.

[0153] (b) Reduction reaction

[0154] Compound Q14 was dissolved in an organic solvent, and a reducing agent was added to react and give compound Q15.

[0155] In step (b), the organic solvent is selected from one or more of tetrahydrofuran, diethyl ether, methanol, ethanol, etc.; preferably, it is tetrahydrofuran.

[0156] In step (b), the reducing agent is any one or more of LiAlH4, NaBH4, red aluminum, diisobutylaluminum hydride, etc.; preferably, it is LiAlH4.

[0157] In step (b), the reducing agent is preferably added in batches.

[0158] In step (b), the molar ratio of compound Q14 to reducing agent is 1:(2-20); preferably, it is 2:10.

[0159] In step (b), the temperature of the reduction reaction is -10 to 10°C; preferably, it is 0°C.

[0160] In step (b), the reduction reaction takes 1 to 5 hours; preferably, it takes 2 hours.

[0161] (c) Iodination reaction

[0162] The synthesis of compound Q16 is similar to the iodination method of compound Q10 in route (1).

[0163] (d) Cyano substitution reaction

[0164] The synthesis of compound Q17 is similar to the substitution method of compound Q11 in route (1).

[0165] (e) Hydrolysis reaction

[0166] The synthesis of compound Q18 is similar to the hydrolysis method of compound Q12 in route (1).

[0167] (f) Esterification reaction

[0168] The synthesis of compound Q19 is similar to the esterification method of compound Q9 in route (1).

[0169] This invention also proposes a method for preparing a bile acid derivative, using compound Q15 as a raw material, through oxidation, Witting reaction, deglycolization protection, and reduction to obtain Q23, as shown in route (3):

[0170]

[0171] Specifically, the method includes the following steps:

[0172] (a) Oxidation reaction

[0173] Compound Q15 was dissolved in an organic solvent, and an oxidizing agent was added to react and give compound Q20.

[0174] In step (a), the organic solvent is selected from one or more of DMSO, toluene, tetrahydrofuran, and a mixed solution of DMSO and toluene; preferably, it is a mixed solution of DMSO and toluene.

[0175] In step (a), the oxidant is selected from any one or more of IBX, DDQ, PCC, PDC, etc.; preferably, it is IBX.

[0176] In step (a), the molar ratio of compound Q15 to oxidant is 1:(1-3); preferably, it is 1:1.5.

[0177] In step (a), the temperature of the oxidation reaction is 20–35°C; preferably, it is 25°C.

[0178] In step (a), the oxidation reaction takes 7 to 10 hours; preferably, it takes 8 hours.

[0179] (b) Witting reaction

[0180] The synthesis of compound Q21 is similar to the synthesis reaction method of compound Q6 in route (1).

[0181] (c) Deprotection reaction of ethylene glycol

[0182] Compound Q21 was dissolved in an organic solvent, and acid was added to react and give compound Q22.

[0183] In step (c), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, a methanol-water mixture, an ethanol-water mixture, and a tetrahydrofuran-water mixture; preferably, it is a methanol-water mixture.

[0184] In step (c), the acid is selected from one or more of dilute sulfuric acid, 2M dilute hydrochloric acid, hydrochloric acid gas, p-toluenesulfonic acid, etc.; preferably, it is a 2M dilute hydrochloric acid aqueous solution.

[0185] In step (c), the volume ratio of the reaction solvent to 2M dilute hydrochloric acid is 5:(1-2); preferably, it is 5:1.

[0186] In step (c), the reaction time is 1 to 5 hours; preferably, it is 2 hours.

[0187] In step (c), the reaction temperature is 0–60°C; preferably, it is 25°C.

[0188] (d) Reduction reaction

[0189] The synthesis of compound Q23 is similar to the reduction reaction method of compound Q7 in route (1).

[0190] This invention also proposes a method for preparing bile acid derivatives, using compound Q7 as the starting material, which undergoes condensation, addition cyclization, hydrolysis, and acetylation to obtain compound Q27; then, using compound Q27 as the starting material, it is condensed with amine compounds to obtain bile acid derivatives as shown in formulas Q28-Q39; and using compound Q25 as the starting material, it undergoes a Grignard reaction with a Grignard reagent to obtain bile acid derivative Q56; the preparation method is shown in route (4):

[0191]

[0192] Specifically, the method includes the following steps:

[0193] (a) Condensation reaction

[0194] Compound Q7 was dissolved in an organic solvent, and a condensing agent was added to react and give compound Q24.

[0195] In step (a), the condensation reaction is preferably carried out under nitrogen protection.

[0196] In step (a), the organic solvent is selected from tetrahydrofuran, ethyl formate, and a mixture of tetrahydrofuran and ethyl formate; preferably, it is ethyl formate.

[0197] In step (a), the condensing agent is selected from one or more of sodium hydride (60%), sodium methoxide, sodium ethoxide, etc.; preferably, it is sodium hydride (60%).

[0198] In step (a), the molar ratio of compound Q7 to sodium hydride is 1:(5-10); preferably, it is 1:10.

[0199] In step (a), the temperature of the condensation reaction is 25–45°C; preferably, it is 25°C.

[0200] In step (a), the condensation reaction takes 20 to 60 minutes; preferably, it takes 20 minutes.

[0201] (b) Addition cyclization reaction

[0202] Compound Q24 was dissolved in an organic solvent, and hydrazine hydrate and a base were added to react and give compound Q25.

[0203] In step (b), the organic solvent is selected from one or more of ethanol, a mixture of ethanol and water, and acetic acid; preferably, it is acetic acid.

[0204] In step (b), the base used in the addition cyclization reaction is selected from one or more of sodium acetate, potassium acetate, sodium ethoxide, and potassium ethoxide; preferably, it is potassium acetate.

[0205] In step (b), the molar ratio of the addition cyclization compound Q24, hydrazine hydrate, and base is 1:(1-3):(1-2); preferably, it is 1:3:2.

[0206] In step (b), the temperature of the addition cyclization reaction is 80–140°C; preferably, it is 130°C.

[0207] In step (b), the addition cyclization reaction takes 3 to 10 hours; preferably, it takes 5 hours.

[0208] (c) Hydrolysis reaction

[0209] Compound Q25 was dissolved in an organic solvent, and then hydrolyzed with an alkali to obtain compound Q26.

[0210] In step (c), the organic solvent is selected from one or more of methanol, tetrahydrofuran, ethanol, a mixture of water and ethanol, and a mixture of water and methanol; preferably, it is a mixture of water and ethanol.

[0211] In step (c), the volume ratio of the water to ethanol mixed solvent is 1:(1-3); preferably, it is 1:3.

[0212] In step (c), the alkali is selected from any one or more of lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, etc.; preferably, it is lithium hydroxide.

[0213] In step (c), the molar ratio of compound Q25 to the base is 1:(1-40); preferably, it is 1:30.

[0214] In step (c), the temperature of the hydrolysis reaction is 10–70°C; preferably, it is 25°C.

[0215] In step (c), the hydrolysis reaction takes 1 to 12 hours; preferably, it takes 8 hours.

[0216] (d) Acetylation reaction

[0217] Compound Q26 was dissolved in an organic solvent, and then an acetylation reagent and a catalyst were added to obtain compound Q27.

[0218] In step (d), the organic solvent is selected from one or more of pyridine, triethylamine, diethylamine, diisopropylethylamine, tetrahydrofuran, DMF, etc.; preferably, it is tetrahydrofuran.

[0219] In step (d), the acetylation reagent is selected from one or more of acetic anhydride, acetyl chloride, etc.; preferably, it is acetic anhydride.

[0220] In step (d), the acetylation reagent is preferably added in batches.

[0221] In step (d), the molar ratio of Q26 to the acetylation reagent is 1:(1-5); preferably, it is 1:3.

[0222] In step (d), the catalyst is selected from DMAP.

[0223] In step (d), the molar ratio of Q26 to DMAP is 1:(0.1~1); preferably, it is 1:0.2.

[0224] In step (d), the temperature of the acetylation reaction is 0–100°C; preferably, it is 60°C.

[0225] In step (d), the acetylation reaction takes 1 to 5 hours; preferably, it takes 2 hours.

[0226] (e) Acylation reaction

[0227] Compound Q27 was dissolved in an organic solvent, and EDCI, HOBt, DMAP and the corresponding amines were added to obtain compounds Q28-Q39.

[0228] In step (e), the organic solvent is selected from one or more of DCM, diethyl ether, acetone, tetrahydrofuran, carbon tetrachloride, toluene, benzene, chloroform, etc.; preferably, it is DCM.

[0229] In step (e), the role of EDCI, HOBt, and DMAP is to promote the amidation reaction.

[0230] In step (e), the molar ratio of compound Q27, EDCI, HOBt, DMAP and the corresponding amine is 1:(1~2):(1~2):(2~4):(1~2); preferably, it is 1:2:2:4:2.

[0231] In step (e), the temperature of the acylation reaction is 25–40°C; preferably, it is 25°C.

[0232] In step (e), the acylation reaction takes 6 to 12 hours; preferably, it takes 12 hours.

[0233] (f) Grignard reaction

[0234] Compound Q25 was dissolved in an organic solvent, and then Grignard reagent was added to obtain compound Q56.

[0235] In step (f), the reaction is preferably carried out under nitrogen protection.

[0236] In step (f), the organic solvent in the reaction is any one or more of diethyl ether, anhydrous tetrahydrofuran, toluene, benzene, etc.; preferably, it is anhydrous tetrahydrofuran.

[0237] In step (f), the reaction reagent is C2H5MgCl or CH2=CHCH2MgCl; preferably, it is C2H5MgCl.

[0238] In step (f), the temperature of the Grignard reaction is 0–60°C; preferably, it is 0°C.

[0239] In step (f), the molar ratio of compound Q25 to Grignard reagent is 1:(10-30); preferably, it is 1:20.

[0240] In step (f), the Grignard reaction takes 0.5 to 5 hours; preferably, it takes 2 hours.

[0241] This invention also proposes a method for preparing a bile acid derivative, using compound Q36 as the starting material, which is hydrolyzed and condensed to obtain bile acid derivative Q43; the preparation method is shown in route (5):

[0242]

[0243] Compound Q36 was dissolved in an organic solvent, and trifluoroacetic acid was added. The Boc group was first removed to obtain an intermediate. Then, the intermediate was acetylated to obtain compound Q43.

[0244] In the reaction of removing the Boc group, the organic solvent is selected from one or more of dichloromethane, tetrahydrofuran, acetone, toluene, etc.; preferably, it is dichloromethane.

[0245] The molar ratio of reactant Q36 to trifluoroacetic acid is 1:(2-3); preferably, it is 1:3.

[0246] The time for the deBoc reaction is 2 to 8 hours; preferably, it is 3 hours.

[0247] The temperature of the deBoc reaction is 0–50°C; preferably, it is 25°C.

[0248] The solvent used in the acetylation reaction is selected from one or more of pyridine, triethylamine, diethylamine, diisopropylethylamine, tetrahydrofuran, DMF, etc.; preferably, it is tetrahydrofuran.

[0249] The catalyst used in the acetylation reaction is selected from DMAP, and the molar ratio of the Q36 compound to DMAP is 1:(0.1-1); preferably, it is 1:0.2.

[0250] The acetylation agent is one or more of acetic anhydride, acetyl chloride, etc.; preferably, it is acetic anhydride.

[0251] The optimal method for adding the acetylation reagent is to add it in batches.

[0252] Wherein, the molar ratio of Q36 to the acetylation reagent is 1:(2-6); preferably, it is 1:4.

[0253] The temperature of the acetylation reaction is 0–100°C; preferably, it is 60°C.

[0254] The acetylation reaction takes 1 to 5 hours; preferably, it takes 2 hours.

[0255] This invention also provides a method for preparing bile acid derivatives, using compound Q24 as the starting material, and obtaining compounds Q45 and Q46 through addition cyclization, hydrolysis, and acylation, or using compound Q44 as the starting material, and obtaining bile acid derivatives as shown in formulas Q49 and Q50 through oxidation, reduction, and Grignard reaction with C2H5MgCl or CH2=CHCH2MgCl, respectively. The preparation method is shown in route (6):

[0256]

[0257]

[0258] Specifically, the method includes the following steps:

[0259] (a) Addition cyclization reaction

[0260] Compound Q24 was dissolved in an organic solvent, and phenylhydrazine hydrochloride was added to react and give compound Q44.

[0261] In step (a), the organic solvent is selected from one or more of ethanol, a mixture of ethanol and water, and acetic acid; preferably, it is a mixture of ethanol and water.

[0262] In step (a), the molar ratio of the reaction compound Q24 to phenylhydrazine hydrochloride is 1:(1-3); preferably, it is 1:3.

[0263] In step (a), the reaction temperature is 80–140°C; preferably, it is 100°C.

[0264] In step (a), the reaction time is 3 to 10 hours; preferably, it is 3 hours.

[0265] (b) Hydrolysis reaction

[0266] The synthesis of compound Q45 is similar to the hydrolysis reaction method of Q26 in route (4).

[0267] (c) Acylation reaction

[0268] The synthesis of compound Q46 is similar to the acylation reaction of Q37 in route (4).

[0269] (d) Oxidation reaction

[0270] Compound Q44 is dissolved in an organic solvent, and when NHPI, acetic acid, and an oxidizing agent are added, the reaction yields compound Q47.

[0271] In step (d), the organic solvent is selected from one or more of acetone, DCM, acetic acid, etc.; preferably, it is acetone.

[0272] In step (d), the oxidant is selected from one or more of sodium dichromate, potassium dichromate, chromium trioxide, etc.; preferably, it is sodium dichromate.

[0273] In step (d), the molar ratio of compound Q44 to NHPI, acetic acid, and oxidant is 1:(1-4):(0.2-2):(1-2); preferably, it is 1:2:1:1.2.

[0274] In step (d), the temperature of the oxidation reaction is 25–50°C; preferably, it is 50°C.

[0275] In step (d), the oxidation reaction takes 1 to 4 hours; preferably, it takes 2 hours.

[0276] (e) Reduction reaction

[0277] Compound Q47 is dissolved in an organic solvent, and a reducing agent is added to react and give compound Q48.

[0278] In step (e), the solvent in the reduction reaction is selected from dichloromethane, methanol, a mixed solution of dichloromethane:methanol = 1:1 or a mixed solution of dichloromethane:methanol = 5:1; preferably, it is methanol.

[0279] In step (e), the temperature of the reduction reaction is 25–50°C; preferably, it is 25°C.

[0280] In step (e), the reducing agent is selected from one or more of hydrogen, sodium borohydride, potassium borohydride, etc.; preferably, it is sodium borohydride.

[0281] In step (e), the molar ratio of compound Q47 to reducing agent is 1:(5-20); preferably, it is 1:10.

[0282] In step (e), the reaction time is 1 to 4 hours; preferably, it is 2 hours.

[0283] (f) Grignard reaction

[0284] The synthesis of compound Q49 is similar to the reaction synthesis of Q56 in route (4).

[0285] The synthesis of compound Q50 is similar to the reaction synthesis of Q56 in route (4).

[0286] This invention also proposes a method for preparing bile acid derivatives, using compound Q26 as the starting material, and through acylation and anhydride protection reactions, obtaining bile acid derivatives as shown in formulas Q51-Q55, the preparation method being shown in route (7):

[0287]

[0288] (a) Acylation reaction

[0289] The synthesis of compound Q51 is similar to the acylation reaction of Q37 in route (4).

[0290] (b) Acid anhydride protection reaction

[0291] The synthesis of compounds Q52-Q55 is similar to the acetylation reaction of Q27 in route (4).

[0292] This invention also provides a method for preparing bile acid derivatives, using compound Q7 as the starting material, and obtaining compounds Q58, Q59, and Q61 through cyclization, hydrolysis, acylation, and carbonyl reduction, as shown in route (8):

[0293]

[0294] (a) Cycling reaction

[0295] Compound Q7 is dissolved in an organic solvent, and sulfur and ethylenediamine are added to react and give compound Q57.

[0296] In step (a), the organic solvent is morpholine.

[0297] In step (a), the sulfur is elemental sulfur powder.

[0298] In step (a), the molar ratio of Q7 to sulfur and ethylenediamine is 1:(5-10):(5-10); preferably, it is 1:10:10.

[0299] In step (a), the temperature of the cyclization reaction is 100–150°C; preferably, it is 120°C.

[0300] In step (a), the cyclization reaction takes 3 to 10 hours; preferably, it takes 5 hours.

[0301] (b) Hydrolysis reaction

[0302] The synthesis of compound Q58 is similar to the hydrolysis reaction method of Q26 in route (4).

[0303] (c) Acylation reaction

[0304] The synthesis of compound Q59 is similar to the acylation reaction of Q37 in route (4).

[0305] (d) Oxidation reaction

[0306] The synthesis of compound Q60 is similar to the oxidation reaction method of Q47 in route (6).

[0307] (e) Reduction reaction

[0308] The synthesis of compound Q61 is similar to the reduction reaction method of Q48 in route (6).

[0309] This invention also provides a method for preparing bile acid derivatives, using compound Q7 as the starting material, and obtaining compounds Q64 and Q66 through bromination, cyclization, and hydrolysis, as shown in route (9):

[0310]

[0311] (a) Bromination reaction

[0312] Compound Q7 was dissolved in an organic solvent, and a brominating agent was added to react and give compound Q62.

[0313] In step (a), the organic solvent is selected from one or more of dichloromethane, methanol, CCl4, DMSO, DMF, benzene, etc.; preferably, it is dichloromethane.

[0314] In step (a), the brominating agent is one or more of pyridinium tribromide, NBS, Br2, etc.; preferably, it is pyridinium tribromide.

[0315] In step (a), the molar ratio of compound Q7 to the brominating agent is 1:(1-2); preferably, it is 1:1.5.

[0316] In step (a), the temperature of the bromination reaction is 0–30°C; preferably, it is 25°C.

[0317] In step (a), the bromination reaction takes 1 to 3 hours; preferably, it takes 2 hours.

[0318] (b) Cycling reaction

[0319] Compound Q62 is dissolved in an organic solvent, and thiourea or thioacetamide is added to react and give compounds Q63 or Q65.

[0320] In step (b), the organic solvent is selected from one or more of ethanol, methanol, and tetrahydrofuran; preferably, it is ethanol.

[0321] In step (b), the molar ratio of compound Q62 to thiourea is 1:(1-3); preferably, it is 1:2.

[0322] The molar ratio of compound Q62 to thioacetamide is 1:(1-3); preferably, it is 1:2.

[0323] In step (b), the temperature of the heterocyclization reaction is 80–120°C; preferably, it is 100°C.

[0324] In step (b), the heterocyclization reaction takes 2 to 5 hours; preferably, it takes 3 hours.

[0325] (c) Hydrolysis reaction

[0326] The synthesis of compounds Q64 and Q66 is similar to the hydrolysis reaction method of Q26 in route (4).

[0327] This invention also provides a method for preparing bile acid derivatives, using compound Q7 as a starting material, which is hydrolyzed and acylated to obtain bile acid derivative Q68; or using Q68 as a starting material, which is condensed, cyclized, and acylated to obtain bile acid derivatives Q71 and Q72; or using compound Q68 as a starting material, which is esterified and cyclized to obtain bile acid derivatives Q74 or Q75 respectively; the preparation method is shown in route (10):

[0328]

[0329] (a) Hydrolysis reaction

[0330] The synthesis of compound Q67 is similar to the hydrolysis reaction method of Q26 in route (4).

[0331] (b) Acylation reaction

[0332] The synthesis of compound Q68 is similar to the acylation reaction of Q37 in route (4).

[0333] (c) Condensation reaction

[0334] Compound Q68 was dissolved in an organic solvent, and alkali was added. After stirring for a period of time, isoamyl nitrite was added dropwise, and the reaction yielded compound Q69.

[0335] In step (c), the alkali is selected from one or more of potassium tert-butoxide, sodium methoxide, sodium formate, sodium ethoxide, potassium hydroxide, etc.; preferably, it is potassium tert-butoxide.

[0336] In step (c), the organic solvent is selected from one or more of tert-butanol, n-butanol, isopropanol, toluene, benzene, carbon tetrachloride, etc.; preferably, it is tert-butanol.

[0337] In step (c), the stirring temperature after adding potassium tert-butoxide is 25–50°C; preferably, it is 30°C.

[0338] In step (c), the stirring time after adding potassium tert-butoxide is 0.5 to 3 hours, preferably 0.5 hours.

[0339] In step (c), the amyl nitrite is added slowly dropwise.

[0340] In step (c), the molar ratio of compound Q68, alkali, and isoamyl nitrite is 1:(2-5):(2-5); preferably, it is 1:5:5.

[0341] In step (c), the reaction temperature is 25–50°C; preferably, it is 25°C.

[0342] In step (c), the reaction time is 3 to 8 hours; preferably, it is 5 hours.

[0343] (d) Condensation reaction

[0344] Compound Q69 was dissolved in an organic solvent, and hydroxylamine hydrochloride and a base were added to react and give compound Q70.

[0345] In step (d), the organic solvent is one or more of methanol, ethanol, pyridine, etc.; preferably, it is pyridine.

[0346] In step (d), the base is one or more of pyridine, triethylamine, Na2CO3, K2CO3, sodium acetate, etc.; preferably, it is pyridine.

[0347] In step (d), the molar ratio of compound Q69 to hydroxylamine hydrochloride is 1:(2-5); preferably, it is 1:3.

[0348] In step (d), the temperature of the condensation reaction is 80–120°C; preferably, it is 100°C.

[0349] In step (d), the condensation reaction takes 2 to 6 hours; preferably, it takes 3 hours.

[0350] (e) Dehydration cyclization reaction

[0351] Compound Q70 dissolves in an organic solvent, and reacts with an alkali to give compound Q71.

[0352] In step (e), the organic solvent is selected from one or more of ethylene glycol, dioxane, and a mixed solvent of ethylene glycol and dioxane; preferably, it is a mixed solvent of ethylene glycol and dioxane.

[0353] In step (e), the optimal ratio of the ethylene glycol to the dioxane mixed solvent is 2:1.

[0354] In step (e), the base for the dehydration cyclization reaction is selected from one or more of potassium hydroxide, sodium hydroxide, potassium acetate, sodium acetate, etc.; preferably, it is potassium hydroxide.

[0355] In step (e), the reaction temperature is 100–220°C; preferably, it is 130°C.

[0356] In step (e), the reaction time is 3 to 8 hours; preferably, it is 5 hours.

[0357] (f) Acylation reaction

[0358] The synthesis of compound Q72 is similar to the acylation reaction of Q37 in route (4).

[0359] (g) Condensation reaction

[0360] Compound Q68 was dissolved in an organic solvent, and a condensing agent and diethyl oxalate were added to react and give compound Q73.

[0361] In step (g), the organic solvent is selected from one or more of anhydrous tetrahydrofuran, dichloromethane, diethyl ether, carbon tetrachloride, benzene, toluene, etc.; preferably, it is anhydrous tetrahydrofuran.

[0362] In step (g), the molar ratio of compound Q68 to diethyl oxalate is 1:(1-5); preferably, it is 1:2.

[0363] In step (g), the condensing agent is selected from one or more of sodium hydride, sodium methoxide, sodium ethoxide, etc.; preferably, it is sodium hydride.

[0364] In step (g), the molar ratio of Q68 to the condensing agent is 1:(5-10); preferably, it is 1:10.

[0365] In step (g), the temperature of the condensation reaction is 25–45°C; preferably, it is 25°C.

[0366] In step (g), the reaction time of the condensation reaction is 20 to 60 minutes; preferably, it is 20 minutes.

[0367] (h) Addition cyclization reaction

[0368] The synthesis of compound Q74 is similar to the cyclization reaction method of Q25 in route (4).

[0369] (i) Addition cyclization reaction

[0370] Compound Q73 was dissolved in an organic solvent, and hydroxylamine hydrochloride was added to react and give compound Q75.

[0371] In step (i), the organic solvent is selected from one or more of ethanol, a mixture of ethanol and water, acetic acid, etc.; preferably, it is a mixture of ethanol and water.

[0372] In step (i), the molar ratio of compound Q73 to hydroxylamine hydrochloride is 1:(1-3); preferably, it is 1:3.

[0373] In step (i), the reaction temperature is 80–140°C; preferably, it is 100°C.

[0374] In step (i), the reaction time is 3 to 10 hours; preferably, it is 3 hours.

[0375] This invention also provides a method for preparing a bile acid derivative, using compound Q24 as a starting material, and obtaining compound Q79 through cyclization, oxidation, reduction, and Grignard reaction, as shown in route (11):

[0376]

[0377] (a) Addition cyclization reaction

[0378] The synthesis of compound Q76 is similar to the cyclization reaction method of Q75 in route (10).

[0379] (b) Oxidation reaction

[0380] The synthesis of compound Q77 is similar to the oxidation reaction method of Q47 in route (6).

[0381] (c) Reduction reaction

[0382] The synthesis of compound Q78 is similar to the reduction reaction method of Q48 in route (6).

[0383] (d) Grignard reaction

[0384] The synthesis of compound Q79 is similar to the reaction method of Q56 in route (4).

[0385] This invention also provides a method for preparing a bile acid derivative, using compound Q68 as a starting material, which is condensed and cyclized to obtain compound Q81. The preparation method is shown in route (12):

[0386]

[0387] (a) Condensation reaction

[0388] The synthesis of compound Q80 is similar to the condensation reaction method of Q24 in route (4).

[0389] (b) Cycling reaction

[0390] Compound Q80 was dissolved in an organic solvent, and o-fluorophenylhydrazine was added to react and give compound Q81.

[0391] In step (b), the organic solvent is selected from one or more of ethanol, a mixture of ethanol and water, acetic acid, etc.; preferably, it is a mixture of ethanol and water.

[0392] In step (b), the molar ratio of Q80 to o-fluorophenylhydrazine hydrochloride is 1:(1-3); preferably, it is 1:3.

[0393] In step (b), the cyclization reaction is carried out at a temperature of 80–140°C; preferably, at 100°C.

[0394] In step (b), the cyclization reaction takes 3 to 10 hours; preferably, it takes 3 hours.

[0395] This invention also provides a method for preparing a bile acid derivative, using compound Q6 as a starting material, reducing it to obtain compound Q82, and then using Q82 as a starting material, undergoing condensation, cyclization, hydrolysis, and acylation to obtain bile acid derivative Q87; the preparation method is shown in route (13):

[0396]

[0397] (a) Reduction reaction

[0398] Compound Q6 was dissolved in an organic solvent, a reducing agent was added, and a reduction reaction was carried out under pressure to obtain compound Q82.

[0399] In step (a), the organic solvent is selected from one or more of methanol, ethanol, tetrahydrofuran, dichloromethane, ethyl acetate, etc.; preferably, it is ethyl acetate.

[0400] In step (a), the reducing agent is selected from one or more of palladium on carbon, Raney nickel, etc.; preferably, it is palladium on carbon.

[0401] In step (a), the mass ratio of compound Q6 to reducing reagent is 1:(0.2-1); preferably, it is 1:0.5.

[0402] In step (a), the reaction is pressurized to 4 MPa.

[0403] In step (a), the reaction temperature is 10–60°C; preferably, it is 25°C.

[0404] In step (a), the reaction time is 24 to 72 hours; preferably, it is 24 hours.

[0405] (b) Condensation reaction

[0406] The synthesis of compound Q83 is similar to the condensation reaction method of Q24 in route (4).

[0407] (c) Addition cyclization reaction

[0408] The synthesis of compound Q84 is similar to the cyclization reaction method of Q25 in route (4).

[0409] (d) Hydrolysis reaction

[0410] The synthesis of compound Q85 is similar to the hydrolysis reaction method of Q26 in route (4).

[0411] (e) Acetylation reaction

[0412] The synthesis of compound Q86 is similar to the acetylation reaction of Q27 in route (4).

[0413] (f) Acylation reaction

[0414] The synthesis of compound Q87 is similar to the acylation reaction of Q37 in route (4).

[0415] This invention also provides a method for preparing bile acid derivatives, using compounds Q9, Q13, Q19 or Q23 as starting materials, and obtaining compounds Q94 to Q111 through condensation, cyclization, hydrolysis and acylation, as shown in route (14):

[0416]

[0417] (a) Aldol condensation reaction

[0418] The synthesis of compounds Q107, Q102, Q88, and Q97 is similar to the condensation reaction method of Q24 in route (4).

[0419] (b) Addition cyclization reaction

[0420] The synthesis of compounds Q108, Q103, Q89, and Q98 is similar to the cyclization reaction method of Q25 in route (4).

[0421] (c) Hydrolysis reaction

[0422] The synthesis of compounds Q109, Q104, Q90, and Q99 is similar to the hydrolysis reaction method of Q26 in route (4).

[0423] (d) Acylation reaction

[0424] The synthesis of compounds Q110 and Q105 is similar to the acylation reaction method of Q54 in route (7).

[0425] The synthesis of compounds Q93 and Q100 is similar to the acetylation reaction of Q27 in route (4).

[0426] (e) Acylation reaction

[0427] The synthesis of compounds Q111, Q106, Q96, and Q101 is similar to the acylation reaction method of Q37 in route (4).

[0428] The synthesis of compound Q94 is similar to the acylation reaction of Q36 in route (4).

[0429] (f) Hydrolysis followed by acylation reaction

[0430] The synthesis of compound Q95 is similar to the reaction method of Q43 in route (5).

[0431] (g) Acylation reaction

[0432] The synthesis of compound Q91 is similar to the acylation reaction of Q37 in route (4).

[0433] (h) Acylation reaction

[0434] The synthesis of compound Q92 is similar to the acylation reaction of Q54 in route (7).

[0435] In the preparation method of this invention, the reaction progress is tracked and measured by thin-plate chromatography. After the reaction is completed, the post-processing methods include concentration, extraction, column chromatography separation, etc. The final product is verified by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry.

[0436] The present invention also proposes the application of the bile acid derivatives in the preparation of drugs that inhibit cellular cholesterol synthesis.

[0437] The present invention also proposes the use of the bile acid derivatives in the preparation of medicaments for the prevention and / or treatment of hypercholesterolemia, hypertriglyceridemia, atherosclerosis, and / or non-alcoholic steatohepatitis.

[0438] The drug can also be used in combination with statins.

[0439] The statins include lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and rosuvastatin.

[0440] The beneficial effects of this invention are as follows: the bile acid derivatives described in this invention can not only effectively promote the degradation of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), but also reduce the increase of HMGCR protein caused by statin drugs, thereby reducing endogenous cholesterol levels. This provides a useful reference for the research and development of new drugs for lowering cholesterol, preventing atherosclerosis, and treating non-alcoholic steatohepatitis, and has good application prospects. Attached Figure Description

[0441] Figure 1 This represents the intracellular cholesterol accumulation of the first-generation compound.

[0442] Figure 2 Western spectroscopy for compounds Q37 and Q54, and their EC50 values; where a is the Western spectroscopy for compound Q37, b is the EC50 for compound Q37, c is the Western spectroscopy for compound Q54, and d is the EC50 for compound Q54.

[0443] Figure 3 This shows the intracellular cholesterol accumulation status of some second-generation compounds (no accumulation at all). Detailed Implementation

[0444] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0445] In the examples below, the structures of the compounds were determined by nuclear magnetic resonance (NMR); the reagents were mainly provided by Shanghai Guoyao Chemical Reagent Co., Ltd.; the product purification was mainly carried out by column chromatography, and the silica gel (200-300 mesh) was produced by Qingdao Ocean Chemical Plant.

[0446] Example 1: Preparation of compounds Q2-Q4

[0447] Compound Q1 (10 g, 0.03 mol) was placed in a 250 mL single-necked flask, and 150 mL of anhydrous DMF was added and stirred until dissolved. Imidazole (16.34 g, 0.24 mol) and TBSCl (18.09 g, 0.12 mol) were added at 0 °C. The mixture was substituted with N2 three times, stirred thoroughly, and then brought to room temperature. After stirring for 5 hours, TLC was performed to confirm the complete reaction of the starting material. The reaction was quenched by adding 50 mL of saturated ammonium chloride solution, followed by extraction with ethyl acetate (100 mL). The aqueous phase was then extracted again with ethyl acetate (30 mL × 3), and the organic phases were combined. The organic phases were washed with saturated ammonium chloride solution (50 mL × 2), water (50 mL × 3), and saturated sodium chloride solution (50 mL × 2), respectively, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain compound Q2 (13.45 g white solid, 99% yield), which was used directly in the next step.

[0448] Compound Q2 (13.45 g, 0.03 mol) was placed in a three-necked flask, and 200 mL of anhydrous tert-butanol was added and stirred until dissolved. The mixture was then substituted three times with N2. Potassium tert-butoxide (13.47 g, 0.12 mol) was added in portions at 0 °C until completely dissolved, and the system turned orange-yellow. Iodomethane (14 mL, 0.24 mol) was slowly added dropwise, and the system gradually turned pale yellow to white. The reaction was exothermic. The mixture was transferred to room temperature and stirred for 4 hours. TLC confirmed complete reaction of the starting material. The reaction was quenched by adding saturated sodium sulfite solution (50 mL). The mixture was then extracted with water (50 mL) and ethyl acetate (100 mL). The aqueous phase was further extracted with ethyl acetate (30 mL × 3), and the organic phases were combined. The organic phase was washed with water (50 mL × 3) and saturated sodium chloride solution (50 mL × 2), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain compound Q3 (white solid), which was used directly in the next step.

[0449] Compound Q3 was directly dissolved in diethyl hydrochloride ether solution and stirred at room temperature for 2 h. The reaction was confirmed to be complete by TLC. 50 mL of water was added, followed by extraction with 100 mL of ethyl acetate. The aqueous phase was then extracted again with ethyl acetate (30 mL × 3), and the organic phases were combined. The organic phases were washed with water (50 mL × 3) and saturated sodium chloride solution (50 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 20:1) to give compound Q4 (6.88 g white solid, two-step yield 64%). 13C NMR (101MHz, CDCl3) δ217.12,149.52,119.47,67.90,56.44,52.38,48.87,48.67,42.46,39.58,3 8.73,37.05,33.71,32.07,31.70,31.22,30.22,27.76,27.23,24.28,21.27,19.33,16.78,11.99.

[0450] Example 2 Preparation of compound Q5

[0451] Compound Q4 (6.88 g, 0.019 mol), PDC (10.84 g, 0.029 mol), and silica gel (10.84 g, 0.029 mol) were placed in a 250 mL single-necked flask, and 100 mL of anhydrous dichloromethane was added. The mixture was stirred at room temperature for 8 hours. The reaction proceeds were monitored by TLC until complete. The silica gel was removed by filtration, and the filter cake was washed with dichloromethane (20 mL × 3). The system was quenched with saturated sodium sulfite for 10 minutes until the system turned light green. The mixture was extracted and separated. The aqueous phase was extracted with dichloromethane (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 2), respectively, dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q5 (6.83 g white solid, 99% yield). 13 C NMR (101MHz, CDCl3) δ216.74,205.03,149.81,119.83,56.02,50.96,49.49,48.88,48.66,42.99,3 9.43,37.05,33.68,32.08,31.65,31.20,30.22,27.23,27.11,24.55,21.22,19.33,13.44,12.28.

[0452] Example 3 Preparation of compound Q6

[0453] In a 250 mL three-necked flask under ice bath conditions, NaH (3.42 g, 0.142 mol) was added, and the mixture was substituted with N2 three times. Then, 50 mL of anhydrous THF was added, and the mixture was stirred until dissolved. Triethyl phosphoroacetate (18.85 mL, 0.095 mol) was slowly added dropwise over approximately 10 minutes, during which bubbles were observed to form in the system. The system was then brought to room temperature and stirred for 10 minutes. Compound Q5 (6.83 g, 0.019 mol) was dissolved in 50 mL of anhydrous THF and added dropwise to the aforementioned system. The mixture was stirred at room temperature for 1 hour, and TLC analysis confirmed complete reaction of the starting material. The reaction was quenched for 10 minutes with 50 mL of saturated ammonium chloride solution. The mixture was then extracted with 60 mL of ethyl acetate, and the aqueous phase was further extracted with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phase was washed with saturated ammonium chloride (30 mL × 2), water (30 mL × 3), and saturated sodium chloride solution (30 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q6 (7.94 g white solid, yield 98%). 13 C NMR (101MHz, CDCl3) δ216.72,167.06,154.60,149.78,119.83,118.99,60.11,56.56,54.88,48.86,48.65,42.70, 39.72,39.57,37.05,33.69,32.07,31.64,31.19,30.22,28.19,27.22,24.18,21.25,19.33,19.22,14.29,12.15.

[0454] Example 4: Preparation of compound Q7

[0455] Compound Q6 (7.94 g, 0.019 mol) and magnesium shavings (4.56 g, 0.19 mol) were placed in a 250 mL single-necked flask, and 100 mL of anhydrous methanol was added. The mixture was stirred at room temperature for 5 hours, during which the system became turbid, bubbles were produced, and the flask became hot. TLC confirmed that the reaction proceeded completely. Water (30 mL), ethyl acetate (50 mL), and 2 M dilute hydrochloric acid were added until the solid was completely dissolved. The mixture was extracted and separated. The aqueous phase was then extracted again with ethyl acetate (30 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and dried to obtain a white solid. A white solid was placed in a 250 mL single-necked flask, and toluene was added until dissolved. IBX (5.32 g, 0.019 mol) was added, followed by DMSO until substantially dissolved. The mixture was stirred at room temperature for 4 hours. TLC analysis confirmed complete reaction of the starting material. 50 mL of water and 50 mL of ethyl acetate were added, resulting in the precipitation of a large amount of insoluble solid. The mixture was filtered, and the filter cake was washed with ethyl acetate (20 mL × 3). The aqueous phase was extracted with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (50 mL × 5) and saturated sodium chloride solution (30 mL × 3), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q7 (7.08 g white solid, 90% yield). 13 C NMR (101MHz, CDCl3) δ216.73,174.36,149.51,119.83,56.72,55.74,51.49,48.90,48.60,42.40,39.70,37.0 4,35.35,33.70,32.06,31.68,31.17,31.03,30.97,30.21,28.13,27.22,24.14,21.26,19.32,18.29,11.91.

[0456] Example 5: Preparation of compound Q8

[0457] Dissolve 2 g (12.66 mmol) of potassium permanganate in 30 mL of water in a 100 mL beaker by stirring. Separately, dissolve 4.3 g (13.34 mmol) of tetrabutylammonium bromide in 8 mL of water in a 50 mL beaker. Add the dissolved tetrabutylammonium bromide to the above system to obtain a purple suspension. Stir at room temperature for 40 minutes, then filter. Wash the filter cake with water (20 mL × 3), and air dry at room temperature to obtain solid tetrabutylammonium permanganate for later use.

[0458] Compound Q5 (2 g, 5.61 mmol) and the tetrabutylammonium permanganate (4.05 g, 11.22 mmol) prepared above were placed in a 100 mL single-necked flask. 40 mL of pyridine was added at 0 °C, and the mixture was stirred for 30 minutes. The mixture was then brought to room temperature and stirred for another hour. TLC was used to confirm that the reaction of the starting material was complete. Water (20 mL) and saturated sodium bisulfite solution (30 mL) were added, and the pH was adjusted to approximately 5 with 2 M dilute hydrochloric acid. 50 mL of ethyl acetate was added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 3), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 10:1) to obtain compound Q8 (1.98 g white solid, 95% yield). 13 C NMR (126MHz, CDCl3) δ216.90,182.52,149.78,119.81,56.38,52.43,48.85,48.69,42.55,42.48,3 9.56,37.06,33.70,32.07,31.64,31.22,30.23,27.34,27.23,24.27,21.23,19.34,17.07,12.09.

[0459] Example 6 Preparation of compound Q9

[0460] Compound Q8 (1.98 g, 5.33 mmol) was added to a 100 mL single-necked flask, followed by 50 mL of methanol. The mixture was stirred until homogeneous, and concentrated sulfuric acid (0.2 mL, 3.73 mmol) was slowly added dropwise. The mixture was heated to reflux at 70 °C and stirred for 2 hours. TLC analysis confirmed that the reaction proceeded completely. Water (20 mL) was added, followed by ethyl acetate (50 mL) for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 3), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to obtain compound Q9 (2.06 g white solid, 99% yield). 13 C NMR (126MHz, CDCl3) δ216.73,177.31,149.79,119.81,56.35,52.84,51.35,48.86,48.67,42.46,42.4 4,39.52,37.05,33.70,32.07,31.64,31.21,30.22,27.23,27.20,24.23,21.23,19.33,17.13,12.08.

[0461] Example 7 Preparation of compounds Q10-Q11

[0462] Iodine (3.47 g, 13.7 mmol) was dissolved in anhydrous toluene (30 mL) in a 100 mL three-necked flask. Triphenylphosphine (3.74 g, 14.3 mmol) and imidazole (1.86 g, 27.4 mmol) were added, and the mixture was protected under nitrogen. The system showed a significant color change, and after stirring at room temperature for 1 hour, a brownish-yellow system was obtained. Compound Q4 (700 mg, 1.95 mmol) was added to the above system, and the mixture was stirred at room temperature for 2 hours. The reaction of the starting material was confirmed by TLC to be complete. The mixture was quenched with saturated sodium sulfite solution (20 mL) for 10 minutes until it was essentially colorless. Ethyl acetate (30 mL) was added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 3), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain compound Q10 (white solid), which was directly added to the next step.

[0463] Compound Q10 (all solid from the previous step) was placed in a 100 mL single-necked flask, and 30 mL of LDM was added and stirred until dissolved. KF (1.81 g, 31.2 mmol) and trimethylsilyl cyanide (1.95 mL, 15.6 mmol) were added, and the mixture was kept at 50 °C for 4 hours. The reaction was monitored by TLC until the starting material was completely reacted. 30 mL of water was added, and the mixture was extracted with 30 mL of ethyl acetate. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 3), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q11 (537 mg white solid, 75% yield in two steps). 13 C NMR (126MHz, CDCl3) δ216.71,149.82,119.76,118.94,56.53,54.69,48.72,48.66,42.49,39.35,37. 04,33.69,33.53,32.05,31.61,31.19,30.22,28.11(2C),27.23,24.82,24.07,21.19,19.33,12.01.

[0464] Example 8 Preparation of compound Q12

[0465] Compound Q11 (537 mg, 1.46 mmol) and NaOH (584 mg, 14.6 mmol) were placed in a 50 mL single-necked flask. A 1:3 mixture of water and ethanol (20 mL) was added, and the mixture was stirred until it was substantially dissolved. The mixture was heated to reflux at 100 °C for 72 hours. The reaction was confirmed by TLC to be complete. The pH was adjusted to approximately 5 by adding 2 M dilute hydrochloric acid solution. Ethyl acetate (20 mL) was added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 3), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 10:1) to obtain compound Q12 (507 mg white solid, 90% yield). 13 C NMR (126MHz, CDCl3) δ216.99,179.43,149.79,119.85,56.76,55.75,48.84,48.69,42.51,41.28,39.5 9,37.06,33.71,33.61,32.08,31.67,31.20,30.21,28.27,27.25,24.12,21.24,19.53,19.34,11.94.

[0466] Example 9 Preparation of compound Q13

[0467] Compound Q12 (507 mg, 1.31 mmol) was placed in a 50 mL single-necked flask, methanol (20 mL) was added, and the mixture was stirred until homogeneous. Concentrated sulfuric acid (0.05 mL, 0.92 mmol) was slowly added dropwise. The mixture was heated to reflux at 70 °C and stirred for 2 hours. The reaction of the starting material was confirmed by TLC to be complete. Water (10 mL) was added, followed by extraction with ethyl acetate (20 mL). The aqueous phase was then extracted again with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phases were washed with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 3), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to obtain compound Q13 (524 mg white solid, 99% yield). 13 C NMR (126MHz, CDCl3) δ216.74,173.97,149.81,119.85,56.75,55.89,51.36,48.85,48.66,42.51,41.42,3 9.60,37.05,33.77,33.71,32.09,31.68,31.20,30.20,28.23,27.25,24.13,21.24,19.55,19.33,11.95.

[0468] Example 10 Preparation of compound Q14

[0469] Compound Q7 (1 g, 2.42 mmol) and ethylene glycol (10 mL) were placed in a 100 mL single-necked flask. Anhydrous THF (30 mL), p-toluenesulfonic acid (96 mg, 0.484 mmol), and triethyl orthoformate (2 mL, 12.1 mmol) were added. The mixture was stirred at room temperature for 3 hours under N2 protection. The reaction proceeded to completion by TLC. Water (30 mL) was added, followed by extraction with ethyl acetate (50 mL). The aqueous phase was then extracted again with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 3), respectively. The mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 20:1) to give compound Q14 (1.11 g white solid, 99% yield). 13 C NMR (126MHz, CDCl3) δ174.86,149.64,119.89,113.22,65.22,64.87,57.17,55.57,51.49,50.54,44.82,42.27,39.7 0,36.26,35.35,35.26,32.30,31.04,31.00,30.88,29.20,28.15,26.83,24.15,22.39,21.70,20.54,18.29,11.86.

[0470] Example 11 Preparation of compound Q15

[0471] Compound Q14 (1.11 g, 2.42 mmol) was dissolved in anhydrous THF (40 mL) by stirring. Lithium aluminum hydride (919 mg, 24.2 mmol) was added in portions at 0 °C under nitrogen protection, and stirring continued for 2 hours. TLC was performed to confirm complete reaction of the starting material. 0.92 mL of water and 1.85 mL of 2 M sodium hydroxide solution were added, followed by quenching with water (0.92 mL). Extraction was performed with water (30 mL) and ethyl acetate (30 mL). The aqueous phase was further extracted with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 3), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 20:1) to obtain compound Q15 (1.04 g white solid, 99% yield). 1H NMR(500MHz, CDCl3)δ5.54–5.49(m,1H),4.03–3.86(m,4H),3.67–3.54(m,2H), 2.09–2.04(m,1H),2.01–1.96(m,2H),1.86–1.80(m,1H),1.74–1.37(m,16H),1. 27(dd,J=9.0,6.0Hz,1H),1.23(s,3H),1.12(s,3H),1.07(dd,J=5.3,3.0Hz,1H ),1.04(s,3H),0.96(dd,J=5.1,3.3Hz,1H),0.93(d,J=6.5Hz,3H),0.67(s,3H).

[0472] Example 12 Preparation of compound Q16

[0473] Iodine (4.31 g, 16.94 mmol) was dissolved in anhydrous toluene (30 mL) in a 100 mL three-necked flask. Triphenylphosphine (4.63 g, 17.67 mmol) and imidazole (2.31 g, 33.88 mmol) were added, and the mixture was protected with nitrogen. The system showed a significant color change, and after stirring at room temperature for 1 hour, a brownish-yellow system was obtained. Compound Q15 (1.04 g, 2.42 mmol) was added to the above system, and the mixture was stirred at room temperature for 2 hours. The reaction of the starting material was confirmed by TLC to be complete. The mixture was quenched with saturated sodium sulfite solution (30 mL) for 10 minutes until it was essentially colorless. Ethyl acetate (30 mL) was added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phase was washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 3), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 30:1) to give compound Q16 (1.18 g white solid, 90% yield). 13 CNMR(126MHz, CDCl3)δ149.68,119.89,113.20,65.24,64.89,57.19,55.68,50.56,44.84,42.28,39.71,36.89, 36.28,35.28,35.08,32.32,30.89,30.35,29.22,28.28,26.85,24.18,22.41,21.72,20.56,18.75,11.88,7.89.

[0474] Example 13 Preparation of compound Q17

[0475] Compound Q16 (1.18 g, 2.18 mmol) was placed in a 100 mL single-necked flask, DMF (30 mL) was added, and the mixture was stirred until dissolved. KF (2.02 g, 34.85 mmol) and trimethylsilyl cyanide (2.18 mL, 17.42 mmol) were added, and the mixture was kept at 50 °C for 4 hours. The reaction of the starting material was detected by TLC to be complete. Water (30 mL) and ethyl acetate (30 mL) were added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 3), respectively, dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q17 (813 mg white solid, 85% yield). 13 CNMR (126MHz, CDCl3) δ149.68,119.87,113.20,65.24,64.89,57.17,55.58,50.54,44.84,42.29,39.72,36.27,35. 27,35.23,35.05,32.29,30.88,29.22,28.26,26.84,24.14,22.40,22.19,21.71,20.55,18.52,17.55(2C),11.86.

[0476] Example 14 Preparation of compound Q18

[0477] Compound Q17 (813 mg, 1.85 mmol) and NaOH (741 mg, 18.5 mmol) were placed in a 50 mL single-necked flask. A 1:3 mixture of water and ethanol (30 mL) was added, and the mixture was stirred until it was substantially dissolved. The mixture was heated under reflux at 100 °C for 72 hours. The reaction was confirmed by TLC to be complete. The pH was adjusted to approximately 5 by adding 2 M dilute hydrochloric acid solution. The mixture was then extracted with 30 mL of ethyl acetate. The aqueous phase was further extracted with ethyl acetate (15 mL × 3), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 3), respectively, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain compound Q18 (690 mg white solid, 90% yield). 1H NMR (500MHz, CDCl3) δ5.54 (s, 1H), 2.56–2.42 (m, 2H), 2.30 (dt, J = 23.4, 7. 9Hz,2H),2.09(dt,J=17.6,4.8Hz,1H),2.00(t,J=11.0Hz,2H),1.86–1.79( m,1H),1.70–1.35(m,10H),1.26(d,J=8.2Hz,1H),1.22(s,6H),1.17–1.04( m,4H),1.04–0.98(m,2H),0.93(d,J=6.2Hz,3H),0.84(s,3H),0.67(s,3H).

[0478] Example 15 Preparation of compound Q19

[0479] Compound Q18 (690 mg, 1.67 mmol) was placed in a single-necked flask, methanol (20 mL) was added, and the mixture was stirred until homogeneous. Concentrated sulfuric acid (0.06 mL, 1.17 mmol) was slowly added dropwise. The mixture was heated to reflux at 70 °C and stirred for 2 hours. The reaction of the starting material was confirmed by TLC to be complete. The mixture was extracted with water (10 mL) and ethyl acetate (20 mL), and the aqueous phase was further extracted with ethyl acetate (15 mL × 3). The organic phases were combined. The organic phases were washed with water (15 mL × 3) and saturated sodium chloride solution (15 mL × 3), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q19 (715 mg white solid, 99% yield). 13 C NMR (126MHz, CDCl3) δ216.84,174.32,149.79,119.91,56.76,55.77,51.43,48.90,48.66,42.38,39.72,37.06,3 5.48,35.39,34.51,33.72,32.09,31.72,31.20,30.21,28.19,27.24,24.16,21.50,21.28,19.34,18.59,11.90.

[0480] Example 15 Preparation of compound Q20

[0481] Compound Q15 (1 g, 2.33 mmol) was dissolved in a 100 mL single-necked flask with toluene. IBX (977 mg, 2.49 mmol) was added, followed by DMSO until substantially dissolved. The mixture was stirred at room temperature for 8 hours. TLC analysis confirmed complete reaction of the starting material. Water (30 mL) and ethyl acetate (30 mL) were added, resulting in the precipitation of a large amount of insoluble solid. The mixture was filtered, and the filter cake was washed with ethyl acetate (20 mL × 3). The mixture was separated, and the aqueous phase was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with water (50 mL × 5) and saturated sodium chloride solution (30 mL × 3), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then purified by silica gel column chromatography (PE:EA = 20:1) to give compound Q20 (998 mg white solid, 99% yield). 13 C NMR (126MHz, CDCl3) δ203.23,149.68,119.86,113.20,65.24,64.88,57.17,55.62,50.53,44.84,42.30,40.90,3 9.70,36.27,35.33,35.27,32.30,30.89,29.22,28.23,27.97,26.84,24.16,22.40,21.70,20.55,18.41,11.89.

[0482] Example 16 Preparation of compound Q21

[0483] Under ice bath conditions, NaH (419 mg, 17.48 mmol) was added to a 100 mL three-necked flask, and the mixture was substituted with N2 three times. 20 mL of anhydrous THF was added, and the mixture was stirred to dissolve. Triethyl phosphoroacetate (2.31 mL, 11.65 mmol) was slowly added dropwise over approximately 10 minutes, during which bubbles were observed to form in the system. The system was then brought to room temperature and stirred for 10 minutes. Compound Q20 (998 mg, 2.33 mmol) was dissolved in anhydrous THF (20 mL) and added dropwise to the aforementioned system. The mixture was stirred at room temperature for 1 hour. TLC analysis confirmed complete reaction of the starting material. The reaction was quenched for 10 minutes with saturated ammonium chloride solution (30 mL), and then extracted with ethyl acetate (30 mL). The aqueous phase was further extracted with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phase was washed with saturated ammonium chloride (20 mL × 2), water (20 mL × 3), and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q21 (1.14 g white solid, yield 98%). 13C NMR (126MHz, CDCl3) δ166.78,149.95,149.66,120.96,119.85,113.17,65.22,64.86,60.08,57.17,55.73,50.53,44.82,42.29, 39.72,36.26,35.46,35.25,34.28,32.30,30.88,29.21,28.97,28.23,26.83,24.15,22.39,21.70,20.55,18.45,14.28,11.88.

[0484] Example 17 Preparation of compound Q22

[0485] Compound Q21 (1.14 g, 2.33 mmol) was placed in a single-necked flask, and a methanol-water mixture was added and stirred until substantially dissolved. 6 mL of 2 M dilute hydrochloric acid aqueous solution was added to the system, and the mixture was stirred at room temperature for 2 hours. TLC was performed to confirm complete reaction of the starting material. Ethyl acetate (30 mL) was added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phase was washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q22 (1.06 g white solid, 99% yield). 13 C NMR (126MHz, CDCl3) δ216.86,166.81,149.91,149.78,121.01,119.91,60.13,56.75,55.91,48.89,48.67,42.44,39.74, 37.06,35.47,34.28,33.72,32.09,31.70,31.20,30.21,29.00,28.22,27.25,24.15,21.28,19.34,18.45,14.29,11.95.

[0486] Example 18 Preparation of compound Q23

[0487] Compound Q22 (1.06 g, 2.33 mmol) and magnesium shavings (560 mg, 23.3 mmol) were placed in a single-necked flask, and anhydrous methanol (40 mL) was added. The mixture was stirred at room temperature for 5 hours, during which the system became turbid, produced bubbles, and the flask became hot. TLC confirmed that the reaction proceeded completely. Water (20 mL), ethyl acetate (30 mL), and 2 M dilute hydrochloric acid were added until the solid was completely dissolved. The mixture was extracted and separated. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phase was washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure, and dried to obtain a white solid. The white solid was placed in a 100 mL single-necked flask, and toluene was added until dissolved. IBX (653 mg, 2.33 mmol) was added, followed by DMSO until substantially dissolved. The mixture was stirred at room temperature for 4 hours. TLC analysis confirmed the reaction was complete. Water (30 mL) and ethyl acetate (30 mL) were added, and a large amount of insoluble solid precipitated. The mixture was filtered, and the filter cake was washed with ethyl acetate (20 mL × 3). The liquid-liquid phase was separated, and the aqueous phase was extracted with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (30 mL × 5) and saturated sodium chloride solution (30 mL × 3), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 15:1) to give compound Q23 (928 mg white solid, 90% yield). 13 C NMR (126MHz, CDCl3) δ216.83,174.30,149.78,119.92,56.77,56.03,51.43,48.91,48.66,42.37,39.74,37.06,35.5 7,35.51,34.16,33.72,32.09,31.73,31.20,30.20,28.25,27.25,25.65,25.39,24.17,21.28,19.33,18.62,11.92.

[0488] Example 19 Preparation of compounds Q24-Q25

[0489] Compound Q7 (1.00 g, 2.41 mmol) was dissolved in a 100 mL single-necked flask with 30 mL of ethyl formate until completely dissolved. NaH (578 mg, 24.1 mmol) was added, and the mixture was stirred at room temperature for 20 minutes under nitrogen protection. The mixture turned orange-yellow. TLC confirmed complete reaction of the reactants. 30 mL of water and 30 mL of ethyl acetate were added. The aqueous phase was extracted once more with 20 mL of ethyl acetate, and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give a pale yellow oil, Q24. This compound was unstable and was directly used in the next step.

[0490] Compound Q24 (the oily substance from the previous step) was dissolved in 30 mL of glacial acetic acid in a 100 mL single-necked flask, along with potassium acetate (473 mg, 4.82 mmol) and 85% hydrazine hydrate (0.42 mL, 8.51 mmol). The mixture was heated to reflux at 130 °C for 5 hours. TLC analysis confirmed the reaction was complete. The mixture was cooled to room temperature, poured into a beaker, and 100 mL of 2 M sodium hydroxide solution was added. The mixture was stirred for 10 minutes, followed by 10 minutes of stirring with saturated sodium bicarbonate solution. Ethyl acetate (30 mL) was added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 5:1) to give compound Q25 (1 g white solid, 91.8% yield). 1 HNMR (500MHz, DMSO) δ12.32(s,1H),7.20(s,1H),5.71(t,J=13.9Hz,1H),4.11–3.96(m,2H),2.63(t,J=24 .9Hz,1H),2.29(td,J=9.5,4.6Hz,1H),2.21–2.14(m,1H),2.10–1.99(m,3H),1.84–1.79(m,1H),1.72–1.6 3(m,2H),1.64–1.55(m,2H),1.52–1.43(m,2H),1.40(d,J=7.5Hz,3H),1.34(t,J=9.0Hz,1H),1.27(s,3H), 1.24–1.20(m,3H),1.19–1.14(m,4H),1.11–1.01(m,3H),0.89(t,J=8.1Hz,3H),0.79(s,3H),0.66(s,3H).

[0491] Example 20 Preparation of compound Q26

[0492] Compound Q25 (1 g, 2.21 mmol) and lithium hydroxide (1.60 g, 66.4 mmol) were added to a 100 mL single-necked flask. 40 mL of a water-ethanol mixture was added, and the mixture was stirred at room temperature for 8 hours. TLC was performed to confirm the reaction was complete. The pH was adjusted to approximately 5 with 2 M dilute hydrochloric acid solution, and 30 mL of ethyl acetate was added. The mixture was extracted and separated. The aqueous phase was further extracted with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (DCM / MeOH = 15:1) to give compound Q26 (1.03 g white solid, 99% yield). 1HNMR(400MHz,DMSO)δ12.05(s,1H),7.19(s,1H),5.68(d,J=25.5Hz,1H),2.6 4(d,J=14.4Hz,1H),2.21(s,1H),2.02(dd,J=30.1,15.3Hz,4H),1.88–1.50( m,6H),1.45(s,1H),1.39(s,3H),1.33(s,1H),1.25(s,3H),1.22–0.98(m,7H ),0.87(d,J=5.2Hz,3H),0.77(s,3H),0.66(d,J=14.9Hz,3H).mp:260-261℃. HRMS(ESI)for C 27 H 41 N₂O₂[M+H] + :calcd 425.3163,found425.3184.

[0493] Example 21 Preparation of compound Q27

[0494] Compound Q26 (1.03 g, 2.21 mmol) and DMAP (73.37 mg, 0.44 mmol) were added to a 100 mL single-necked flask. THF (40 mL) and acetic anhydride (0.63 mL, 6.63 mmol) were added, and the mixture was heated and stirred at 60 °C for 2 hours. The mixture was observed to gradually dissolve completely. TLC showed that the reaction proceeded completely. The mixture was then cooled to room temperature, and the mixture was extracted with water (30 mL) and ethyl acetate (30 mL). The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to give compound Q27 (white solid 927 mg, yield 90%). 1 H NMR (500MHz, CDCl3) δ7.88(d,J=1.2Hz,1H),5.75(dd,J=5.2,2.3Hz,1H),2.77(d,J=14.9Hz,1H),2.65(s,3H),2.44–2.35(m,1H),2.33–

[0495] 2.24(m,1H),2.18–2.09(m,2H),2.08–2.00(m,1H),1.93–1.77(m,2H),1.72–1.54(m,4H),1.52(s,3H) ,1.50–1.39(m,2H),1.35(s,3H),1.31–1.00(m,7H),0.94(t,J=10.0Hz,3H),0.79(s,3H),0.70(s,3H). 13 C NMR (126MHz, CDCl3) δ180.26,169.67,162.93,149.25,124.04,121.19,119.29,56.85,55.73,49.12,42.31,39.71,38.57,36 .35,35.32,33.78,32.24,31.94,31.80,31.59,31.02,30.77,28.14,24.19,21.63,21.14,21.01,18.30,11.92.mp:104-105℃. HRMS(ESI)for C 29 H 42 N₂O₃[M+Na] + :calcd 489.3088,found489.3099.

[0496] Example 22 Preparation of compounds Q28-Q42

[0497] Compound Q27 (145 mg, 0.31 mmol), EDCI (120.8 mg, 0.63 mmol), HOBt (85.2 mg, 0.63 mmol), DMAP (154.0 mg, 1.26 mmol), and the corresponding amine (0.59 mmol) were placed in a 100 mL single-necked flask, and anhydrous DCM (10 mL) was added. The mixture was stirred at room temperature for 12 hours. After the reaction was complete as determined by TLC, 20 mL of water was added, and the aqueous phase was extracted with DCM (20 mL × 3). The organic phases were combined. The organic phases were washed with water (30 mL) and saturated NaCl solution (30 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then purified by silica gel column chromatography to obtain the corresponding compounds.

[0498] Compound Q28 is a white solid with a yield of 95%. 1H NMR (500MHz, CDCl3) δ7.87(d,J=1.3Hz,1H),5.74(dd,J=5.2,2.3Hz,1H),5.53(s,1H),2.80(d,J =4.8Hz,3H),2.76(d,J=14.9Hz,1H),2.64(s,3H),2.28–2.18(m,1H),2.17–2.10(m,2H),2.08–2 .01(m,2H),1.95–1.84(m,1H),1.84–1.77(m,1H),1.71–1.54(m,4H),1.52(s,3H),1.50–1.39(m ,2H),1.34(d,J=6.9Hz,3H),1.32–1.00(m,7H),0.93(d,J=6.5Hz,3H),0.78(s,3H),0.68(s,3H). 13 C NMR (126MHz, CDCl3)δ

[0499] 174.17,169.63,162.91,149.22,123.99,121.21,119.28,56.85,55.82,49.12,42.30,39.73,38.56,36.34,35.55,33 .78,33.56,32.23,31.93,31.83,31.80,31.58,28.21,26.32,24.20,21.63,21.14,21.00,18.43,11.91.mp:178-180℃. HRMS(ESI)forC 30 H 45 N3O2[M+Na] + :calcd 502.3404, found 502.3409.

[0500] Compound Q29 is a white solid with a yield of 95%. 1 H NMR(500MHz, CDCl3)δ7.86(s,1H),5.74(dd,J=5.1,2.1Hz,1H),5.51(s,1H),3.35–3.21(m,2H),2.75(d,J=14.9Hz,1H),2.64(s,3H),2.25–

[0501] 2.18(m,1H),2.17–2.09(m,2H),2.09–2.00(m,2H),1.91–1.85(m,1H), 1.84–1.75(m,1H),1.72–1.54(m,4H),1.51(s,3H),1.49–1.38(m,2H),1 .33(d,J=7.2Hz,3H),1.30–1.15(m,4H),1.12(t,J=7.3Hz,4H),1.08–0.99(m,2H),0.92(t,J=9.3Hz,3H),0.79(d,J=10.8Hz,3H),0.68(s,3H). 13 C NMR (126MHz, CDCl3) δ173.37,169.62,162.91,149.22,123.99,121.21,119.28,56.85,55.83,49.12,42.30,39.73,38.56,36.33,35 .53,34.31,33.78,33.69,32.23,31.93,31.83,31.80,31.58,28.22,24.19,21.63,21.14,21.00,18.45,14.94,11.91.mp:125-126℃. HRMS(ESI)for C 31 H 47 N3O2[M+Na] + :calcd 516.3560, found 516.3578.

[0502] Compound Q30 is a white solid with a yield of 95%. 1 H NMR(400MHz, CDCl3)δ7.86(s,1H),5.73(s,1H),5.60(s,1H),3.22–3.12(m,2H ),2.72(d,J=19.5Hz,1H),2.63(s,3H),2.26–2.17(m,1H),2.11(d,J=15.4Hz,2 H),2.02(d,J=12.5Hz,2H),1.91–1.54(m,6H),1.51(s,3H),1.44–1.37(m,3H) ,1.33(s,3H),1.29–1.05(m,7H),0.93–0.85(m,7H),0.77(s,3H),0.67(s,3H). 13C NMR (101MHz, CDCl3) δ173.58,169.64,162.93,149.21,123.99,121.21,119.29,56.85,55.83,49.12,42.29,41.20,39.72,38.55,36.33 ,35.52,33.77,33.71,32.22,31.92,31.87,31.79,31.57,28.21,24.19,22.90,21.62,21.13,20.99,18.43,11.90,11.37.mp:125-126℃. HRMS(ESI)for C 32 H 49 N3O2[M+Na] + :calcd 530.3717,found530.3707.

[0503] Compound Q31 is a white solid with a yield of 90%. 1 H NMR (500MHz, CDCl3) δ8.56(d,J=2.2Hz,1H),8.32(d,J=4.2Hz,1H),8.20(d,J=8.2Hz,1H),8.03(s,1H),7.87(d,J =1.0Hz,1H),7.26(t,J=5.1Hz,1H),5.75(dd,J=5.1,2.2Hz,1H),2.76(d,J=14.9Hz,1H),2.65(s,3H),2.51–2.41( m,1H),2.35–2.24(m,1H),2.17–2.10(m,2H),2.03(dd,J=9.2,3.3Hz,1H),1.94–1.86(m,2H),1.70–1.55(m,4H), 1.52(s,3H),1.49–1.42(m,2H),1.34(s,3H),1.31–1.00(m,7H),0.96(d,J=6.2Hz,3H),0.78(s,3H),0.67(s,3H). 13C NMR (126MHz, CDCl3) δ172.57,169.75,162.98,149.20,144.91,140.97,1 35.20,127.18,123.99,123.74,121.20,119.33,56.85,55.83,49.11,42. 33,39.75,38.56,36.35,35.53,34.45,33.78,32.24,31.92,31.81,31.5 8,31.50,28.25,24.19,21.66,21.15,21.01,18.49,11.94.mp:174-175℃. HRMS(ESI) for C 34 H 46 N4O2[M+H] + :calcd543.3694,found 543.3697.

[0504] Compound Q32 is a white solid with a yield of 90%. 1 H NMR (400MHz, CDCl3) δ7.87 (s, 1H), 6.00 (s, 1H), 5.72 (d, J = 21.5Hz, 1H), 3.71 (s, 4H) ),3.35(d,J=5.2Hz,2H),2.76(d,J=14.8Hz,1H),2.64(s,2H),2.51–2.42(m,5H),2 .30–2.21(m,1H),2.16–1.56(m,11H),1.52(s,3H),1.44(d,J=8.9Hz,1H),1.34(d, J=6.6Hz,3H),1.29–1.02(m,9H),0.95(d,J=6.1Hz,3H),0.78(s,3H),0.68(s,3H). 13 C NMR(101MHz, CDCl3)δ173.62,169.66,162.93,149.22,124.02,121.21,119.29,66.91(2C),57.10,56.85,55.82,53.33(2C),49.10,42.30,39.7 2,38.56,36.34,35.52,33.78,33.56,32.22,31.93,31.80,31.77,31.58 ,29.70,28.23,24.20,21.65,21.13,21.01,18.43,11.93.mp:108-109℃. HRMS(ESI)for C 35 H 54 N4O3[M+H] +:calcd579.4269,found 579.4215.

[0505] Compound Q33 is a white solid with a yield of 95%. 1 H NMR (500MHz, CDCl3) δ7.87(s,1H),7.79(d,J=7.5Hz,1H),7.19(dd,J=15.4,7.5Hz,2H),7.06(dd,J=15.6,7.8Hz, 1H),7.01(d,J=18.7Hz,1H),5.76(dd,J=5.1,2.2Hz,1H),2.77(d,J=14.9Hz,1H),2.65(s,3H),2.50–2.40(m,1H), 2.31(dd,J=14.9,8.8Hz,1H),2.26(s,3H),2.17–2.10(m,2H),2.09–2.03(m,1H),1.93(s,2H),1.74–1.55(m,4H), 1.53(s,3H),1.51–1.42(m,2H),1.36(s,3H),1.34–1.04(m,6H),1.01(d,J=7.3Hz,3H),0.80(s,3H),0.69(s,3H). 13 C NMR (126MHz, CDCl3) δ171.70,169.64,162.91,149.24,135.76,130.43,126 .78,125.09,124.00,123.23,121.21(2C),119.28,56.87,55.87,49.13,42. 34,39.75,38.58,36.35,35.52,34.52,33.79,32.24,31.94,31.81(2C),31 .60,28.25,24.21,21.64,21.16,21.02,18.48,17.82,11.95.mp:132-133℃. HRMS(ESI) for C 36 H 49 N3O2[M+Na] + :calcd578.3717,found 578.3704.

[0506] Compound Q34 is a white solid with a yield of 95%. 1H NMR (500MHz, CDCl3) δ7.88(d,J=1.1Hz,1H),7.38(t,J=11.6Hz,2H),7.10(d,J=8.1Hz,2H),5.75(dd, J=5.2,2.2Hz,1H),2.77(d,J=14.9Hz,1H),2.65(s,3H),2.45–2.36(m,1H),2.30(s,3H),2.27–2.20( m,1H),2.18–2.10(m,2H),2.05(t,J=6.2Hz,1H),1.96–1.85(m,2H),1.73–1.55(m,4H),1.53(s,3H), 1.50–1.40(m,3H),1.35(s,3H),1.32–1.00(m,7H),0.97(d,J=6.3Hz,3H),0.79(s,3H),0.70(s,3H). 13 C NMR (126MHz, CDCl3) δ171.71,169.67,162.94,149.22,135.52,133.68,129 .44(2C),124.00,121.22,119.87(2C),119.30,56.86,55.86,49.14,42.32 ,39.75,38.57,36.35,35.53,34.61,33.79,32.25,31.94,31.81,31.66,31 .59,28.24,24.21,21.64,21.16,21.02,20.86,18.50,11.94.mp:159-160℃. HRMS(ESI) for C 36 H 49 N3O2[M+Na] + :calcd578.3717,found 578.3706.

[0507] Compound Q35 is a white solid with a yield of 95%. 1H NMR (500MHz, CDCl3) δ10.56(s,1H),8.32(s,1H),7.88(s,1H),5.76(d,J=2.8Hz,1H),2.75(t, J=19.7Hz,1H),2.64(s,3H),2.63–2.58(m,1H),2.53–2.41(m,1H),2.19–2.10(m,2H),2.06(d ,J=8.9Hz,1H),1.98–1.84(m,2H),1.73–1.56(m,4H),1.53(s,3H),1.51–1.40(m,2H),1.36(s ,3H),1.33–1.04(m,8H),1.00(d,J=6.0Hz,3H),0.95–0.83(m,1H),0.80(s,3H),0.71(s,3H). 13 C NMR(126MHz, CDCl3)δ172.09,162.96,161.67,149.23,143.44,139.93(2C),124.03,121.17,119.30,56.84,55.70,49.08,42.38,39.73 ,38.57,36.36,35.39,33.78,33.17,32.24,31.90,31.81,31.58,30.80,28.26,24.18,21.65,21.14,21.01,18.44,11.94.mp:220-221℃. HRMS(ESI)for C 32 H 43 N5O4S[M+Na] + :calcd 616.2928, found 616.2936.

[0508] Compound Q36 is a white solid with a yield of 95%. 1H NMR(500MHz, CDCl3)δ7.87(s,1H),5.75(d,J=2.9Hz,1H),5.33(d,J=7.7Hz,1H),4.13–3.98(m,2H),3.98–3 .83(m,1H),2.85(d,J=11.3Hz,2H),2.76(d,J=14.9Hz,1H),2.64(s,3H),2.25–2.17(m,1H),2.17–2.09(m, 2H),2.09–2.01(m,2H),1.89(d,J=13.0Hz,3H),1.83–1.75(m,2H),1.71–1.56(m,5H),1.52(s,3H),1.45(s ,9H),1.35(s,3H),1.31–1.23(m,5H),1.21–1.01(m,6H),0.94(d,J=6.5Hz,3H),0.79(s,3H),0.68(s,3H). 13 C NMR (126MHz, CDCl3) δ172.83,169.64,162.91,154.72,149.23,124.00,121 .19,119.28,79.67,56.87,55.81,49.12,46.58,42.31(2C),39.73,38.56, 36.34,35.50,33.78,33.75,32.23,31.93,31.79,31.77,31.58(2C),28.43 (3C),28.24(2C),24.18,21.63,21.14,21.00,18.44,11.91.mp:104-105℃. HRMS(ESI) for C 39 H 60 N4O4[M+Na] + :calcd 671.4507,found671.4548.

[0509] Compound Q37 is a white solid with a yield of 90%. 1H NMR (400MHz, CDCl3) δ7.86 (s, 1H), 5.74 (d, J = 3.0Hz, 1H), 3.64 (t, J = 8.5Hz, 4H), 3.60 (d, J = 4.0Hz, 2H), 3.46(d,J=4.2Hz,2H),2.76(t,J=11.6Hz,1H),2.63(s,3H),2.42–2.32(m,1H),2.25–2.18(m,1H),2.16– 2.08(m,2H),2.03(t,J=6.0Hz,1H),1.95–1.84(m,1H),1.82–1.73(m,1H),1.71–1.54(m,4H),1.50(s,3 H),1.47–1.39(m,1H),1.33(s,3H),1.31–1.00(m,7H),0.95(d,J=6.4Hz,3H),0.77(s,3H),0.68(s,3H). 13 C NMR (101MHz, CDCl3) δ172.30,169.61,162.88,149.22,124.00,121.20,119.26,66.96,66.69,56.83,55.80,49.10,46.08,42.30,41.87,39 .70,38.56,36.33,35.65,33.78,32.23,31.93,31.81,31.57,31.32,3 0.10,28.25,24.22,21.63,21.13,21.00,18.55,11.92.mp:155-156℃. HRMS(ESI)for C 33 H 49 N3O3[M+Na] + :calcd 558.3666, found 558.3675.

[0510] Compound Q38 is a white solid with a yield of 90%. 1H NMR (500MHz, CDCl3) δ7.87 (s, 1H), 5.75 (d, J = 3.0Hz, 1H), 3.59–3.48 (m, 2H), 3.45–3.30 (m, 2H),2.74(t,J=20.9Hz,1H),2.64(s,3H),2.40–2.32(m,1H),2.25–2.16(m,1H),2.16–2.10( m,2H),2.05(d,J=10.7Hz,1H),1.95–1.85(m,1H),1.80–1.53(m,11H),1.52(s,3H),1.50–1. 42(m,2H),1.35(s,3H),1.30–1.03(m,7H),0.96(d,J=6.5Hz,3H),0.79(s,3H),0.69(s,3H). 13 C NMR (126MHz, CDCl3) δ171.91,169.60,162.90,149.23,123.99,121.22,119.28,56.85,55.89,49.13,46.75,42.60,42.31,39.72,38.57,36.3 4,35.76,33.78,32.24,31.94,31.80,31.60,30.50,28.24,26.61,25.5 9,24.61(2C),24.24,21.63,21.15,21.01,18.55,11.92.mp:159-160℃. HRMS(ESI)for C 34 H 51 N3O2[M+Na] + :calcd556.3874,found 556.3873.

[0511] Compound Q39 is a white solid with a yield of 90%. 1H NMR(500MHz, CDCl3) δ7.86(d,J=9.5Hz,1H),5.74(dd,J=5.1,2.2Hz,1H),5.41(s,1H),4.03–3.99(m,1H),3.94( dd,J=8.9,3.0Hz,2H),3.46(td,J=11.6,1.7Hz,2H),2.75(d,J=14.9Hz,1H),2.64(s,3H),2.25–2.19(m,1H),2.1 6–2.10(m,2H),2.07–2.02(m,2H),1.90–1.86(m,2H),1.81-1.75(m,1H),1.72–1.55(m,4H),1.51(s,3H),1.49–1 .40(m,4H),1.33(s,3H),1.28–1.22(m,3H),1.19–1.03(m,5H),0.94(d,J=6.5Hz,3H),0.78(s,3H),0.68(s,3H). 13 C NMR(101MHz, CDCl3)δ172.88,169.67,162.94,149.22,124.01,121.21,119.30,66.82(2C),56.86,55.80,49.10,45.53,42.30,39.72,38.55, 36.33,35.51,33.78,33.74,33.23,32.22,31.92,31.80,31.77,31.57, 29.70,28.25,24.19,21.65,21.13,21.00,18.45,11.90.mp:110-111℃. HRMS(ESI)forC 34 H 51 N3O3[M+Na] + :calcd 572.3823,found572.3815.

[0512] Example 23 Preparation of compound Q43

[0513] Compound Q36 (80 mg, 0.12 mmol) was placed in a 25 mL single-necked flask, and dichloromethane (10 mL) was added. Trifluoroacetic acid (0.026 mL, 0.36 mmol) was added dropwise. The mixture was stirred at room temperature for 3 hours. TLC confirmed that the reaction was complete. The mixture was directly concentrated to obtain a solid. THF (10 mL), DMAP (4 mg, 0.024 mmol), and acetic anhydride (0.045 mL, 0.48 mmol) were then added. The mixture was heated at 60 °C for 2 hours. TLC confirmed that the reaction was complete. The mixture was cooled to room temperature, and water (10 mL) and ethyl acetate (10 mL) were added. The mixture was extracted and separated. The aqueous phase was extracted again with ethyl acetate (10 mL × 3). The organic phases were combined. The organic phases were washed with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 2), respectively. The mixture was dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to obtain compound Q43 (50 mg white solid, 70% yield). 1 H NMR (400MHz, CDCl3) δ7.85(s,1H),6.95(s,1H),5.76(d,J=13.8Hz,1H),5.73(s,1H),4.52(d,J=12.8Hz,1H),3.9 9(s,1H),3.76(d,J=13.3Hz,1H),3.14(t,J=12.7Hz,1H),2.71(dd,J=26.4,12.7Hz,2H),2.63(s,2H),2.24–2.13( m,2H),2.07(s,3H),2.05–1.97(m,3H),1.88(d,J=11.6Hz,2H),1.79–1.58(m,4H),1.54(s,1H),1.50(s,3H),1.40 (s,2H),1.32(d,J=5.6Hz,3H),1.30–1.06(m,10H),0.92(d,J=5.4Hz,3H),0.84(s,1H),0.75(s,2H),0.66(s,3H). 13 C NMR(126MHz, CDCl3)δ173.70,169.66(2C),162.95,149.20,124.01,121.1 9,119.31,56.84,56.73,55.74,49.09,46.52,45.40,42.30,42.25,40.84, 39.71,38.55,36.34,35.52,33.77,33.60,32.61,32.22,31.92,31.80,31. 57,29.70,28.22,24.17,21.65,21.15,21.00,18.40,11.90.mp:140-141℃. HRMS(ESI) for C36 H 54 N4O3[M+Na] + :calcd 613.4088, found 613.4071.

[0514] Example 24 Preparation of compound Q44

[0515] As in Example 19, compound Q7 (500 mg, 1.2 mmol), ethyl formate (30 mL), and NaH (288 mg, 12.0 mmol) reacted to give a pale yellow oily substance Q24.

[0516] Next, compound Q24 (the oily substance from the previous step) was added to a 100 mL single-necked flask with ethanol (30 mL), a few drops of water, and phenylhydrazine hydrochloride (520 mg, 3.6 mmol). The mixture was heated to reflux at 100 °C for 3 hours. TLC was used to confirm the complete reaction of the starting material. The mixture was then extracted with water (20 mL) and ethyl acetate (20 mL), and the aqueous phase was further extracted with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 10:1) to give compound Q44 (570 mg white solid, 90% yield in both steps). 13 C NMR (126MHz, CDCl3) δ174.34,151.33,145.72,142.36,137.66,128.93,128.88(2C),128.55(2C),120.74,114.48,60.19,56.91,55.76,49. 60,42.31,39.76,38.52,36.96,35.38,33.40,32.27,32.21,31.44,3 1.42,31.33,31.02,28.17,24.21,21.30,21.04,18.35,14.28,11.90.

[0517] Example 25 Preparation of compound Q45

[0518] Similar to the synthesis of Q26, compound Q44 (114 mg, 0.22 mmol), lithium hydroxide (160 mg, 6.6 mmol), and anhydrous ethanol (10 mL) were reacted at room temperature to give compound Q45 (110 mg white solid, 99% yield). 1H NMR (500MHz, CDCl3) δ7.47–7.34(m,6H),5.67(d,J=3.2Hz,1H),2.75(d,J=14.6Hz,1H) ,2.42–2.29(m,1H),2.26–2.20(m,1H),2.19(d,J=14.7Hz,1H),2.15–2.06(m,2H),2.0 2(d,J=13.7Hz,1H),1.90–1.75(m,2H),1.71–1.32(m,8H),1.29(d,J=4.2Hz,3H),1.26 –1.16(m,3H),1.13(s,3H),1.11–1.00(m,3H),0.95(s,1H),0.93(s,3H),0.70(s,3H).

[0519] Example 26 Preparation of compound Q46

[0520] Similar to the synthesis of compound Q37, compound Q45 (110 mg, 0.22 mmol) was reacted with EDCI (84.3 mg, 0.44 mmol), HOBt (59.4 mg, 0.44 mmol), DMAP (119.6 mg, 0.66 mmol), and morpholine (0.04 mL, 0.44 mmol) to give compound Q46 (122.7 mg white solid, 98% yield). 1 HNMR (400MHz, CDCl3) δ7.48–7.32(m,6H),5.67(d,J=3.2Hz,1H),3.64(d,J=11.8Hz,4H),3.61(d,J=4. 3Hz,2H),3.46(d,J=4.3Hz,2H),2.75(d,J=14.6Hz,1H),2.41–2.31(m,1H),2.25–2.15(m,2H),2.15–2 .01(m,2H),1.94–1.85(m,1H),1.80(dd,J=11.8,4.3Hz,1H),1.73–1.31(m,9H),1.29(s,3H),1.26–1. 17(m,2H),1.13(s,3H),1.10–1.01(m,2H),0.96(d,J=6.4Hz,3H),0.93(s,3H),0.68(d,J=14.2Hz,3H). 13C NMR (101MHz, CDCl3) δ172.34,151.31,145.73,142.35,137.65,128.95,128.88( 2C),128.57(2C),120.75,114.46,66.97,66.70,56.89,55.80,49.58,46.10,42. 33,41.88,39.76,38.51,36.95,35.66,33.40,32.27,32.20,31.43,31.42,31.3 5,30.10,28.27,24.24,21.30,21.03,18.57,11.92.mp:219-220℃.HRMS(ESI)for C 37 H 51 N3O2[M+H] + :calcd 570.4054,found570.4009.

[0521] Example 27 Preparation of compound Q47

[0522] Compound Q44 (300 mg, 0.57 mmol) and NHPI (186.0 mg, 1.14 mmol) were added to a single-necked flask, dissolved by stirring in 20 mL of acetone. Then, glacial acetic acid (0.033 mL, 0.57 mmol) and sodium dichromate (205.2 mg, 0.69 mmol) were added. The mixture was incubated at 50 °C for 2 hours. After the reaction was complete as determined by TLC, the mixture was extracted with 20 mL of water and 20 mL of ethyl acetate. The aqueous phase was then extracted again with 20 mL of ethyl acetate (20 mL × 2), and the organic phases were combined. The organic phases were washed with 30 mL of water and 20 mL of saturated sodium chloride solution, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 5:1) to give compound Q47 (216.3 mg white solid, 70% yield). 13 C NMR(126MHz, CDCl3)δ202.18,175.90,174.18,143.69,141.78,137.66,129.31,128.82(2C),128.62(2C),125.72,113.53,60.18,54.51,50 .25,49.65,45.04,43.18,40.47,38.70,37.97,35.27,32.24,31.32,3 1.18,31.01,30.69,28.45,26.27,21.22,18.62,18.50,14.28,11.97.

[0523] Example 28 Preparation of compound Q48

[0524] Compound Q47 (100 mg, 0.18 mmol) was added to a 50 mL single-necked flask with 20 mL of methanol. Sodium borohydride (68.4 mg, 1.8 mmol) was slowly added under ice bath conditions. The mixture was stirred at room temperature for 2 hours. After the reaction was complete as determined by TLC, the mixture was extracted with water (20 mL) and ethyl acetate (20 mL). The aqueous phase was then extracted again with ethyl acetate (20 mL × 2), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 3:1) to give compound Q48 (58.8 mg white solid, 60% yield). 13 C NMR (126MHz, CDCl3) δ174.26,153.97,145.10,142.15,137.65,129.06,128.82(2C),128.63(2C),124.96,114.13,73.60,60.21,56.00,55. 13,47.83,42.86,40.52,39.51,38.59,36.84,35.31,33.11,31.97,3 1.41,31.32,31.02,28.47,26.35,21.07,21.04,18.41,14.28,11.84.

[0525] Example 29 Preparation of compounds Q49 and Q50

[0526] Compound Q48 (29 mg, 0.053 mmol) and anhydrous tetrahydrofuran (6 mL) were dissolved in a single-necked flask under nitrogen protection. The mixture was stirred until dissolved, and 2M Grignard reagent (0.5 mL, 1 mmol) was added dropwise under ice bath conditions. The mixture was then transferred to room temperature and stirred for 2 hours. After the reaction was complete as determined by TLC, water (8 mL) and ethyl acetate (10 mL) were added for extraction and separation. The aqueous phase was further extracted with ethyl acetate (5 mL × 2), and the organic phases were combined. The organic phases were washed with water (10 mL × 3) and saturated sodium chloride solution (8 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 1:1) to obtain the compound.

[0527] Compound Q49, white solid, 28 mg, yield 95%. 1HNMR (400MHz, CDCl3) δ7.49–7.33(m,6H),5.60(d,J=23.6Hz,1H),3.95(d,J=5.4Hz,1H),2.76(d,J=14.6Hz, 1H),2.21–2.12(m,1H),2.09–2.02(m,1H),1.95–1.65(m,4H),1.55(d,J=9.1Hz,2H),1.49(s,1H),1.48–1.42 (m,5H),1.41–1.37(m,2H),1.34(s,1H),1.31(s,3H),1.25(s,3H),1.18(s,3H),1.15–1.07(m,2H),1.04(s,1 H),0.98(d,J=6.1Hz,3H),0.94(d,J=12.2Hz,2H),0.87(s,1H),0.85(s,4H),0.82–0.79(m,1H),0.69(s,3H). 13 C NMR (126MHz, CDCl3) δ154.11,145.11,142.15,137.67,129.07,128.82(2C),1 28.64(2C),124.85,114.16,74.72,73.71,56.00,55.10,47.85,42.84,40.58, 39.51,38.61,36.86,36.08,34.26,33.11,31.98,31.41,31.18,30.95,29.27 ,28.60,26.40,21.06,18.92,11.82,7.81,7.72.mp:211-212℃.HRMS(ESI)forC 37 H 54 N₂O₂[M+H] + :calcd 559.4258,found559.4271.

[0528] Compound Q50, white solid, 28 mg, yield 95%. 1HNMR (500MHz, CDCl3) δ7.45–7.33(m,6H),5.91–5.80(m,2H),5.56(d,J=2.5Hz,1H),5.12(t,J=13.3Hz,4H) ,3.94(dd,J=8.1,2.3Hz,1H),2.75(d,J=14.7Hz,1H),2.22–2.17(m,4H),2.16(s,1H),2.06(dd,J=9.5,3.2H z,1H),2.03(s,1H),1.93–1.88(m,1H),1.86–1.80(m,1H),1.68–1.61(m,2H),1.57–1.34(m,7H),1.30(s,3 H),1.24(t,J=7.2Hz,2H),1.18(s,3H),1.14–1.08(m,2H),0.98(s,3H),0.94(d,J=6.5Hz,3H),0.70(s,3H). 13 C NMR (101MHz, CDCl3) δ154.01,145.13,142.13,137.66,133.82(2C),133.79(2C),12 9.08,128.83(2C),128.64(2C),124.94,118.65,118.58,114.16,73.62,56.01,55. 11,47.84,43.83,43.61,42.83,40.53,39.50,38.60,36.85,36.06,35.42,33.11,3 1.97,31.41,29.27,28.62,26.40,21.05,18.90,11.82.mp:188-189℃.HRMS(ESI)for C 39 H 54 N₂O₂[M+H] + :calcd 583.4258, found 583.4285.

[0529] Example 30 Preparation of compound Q51

[0530] Similar to the synthesis of compound Q37, compound Q26 (500 mg, 1.18 mmol), EDCI (450.8 mg, 2.36 mmol), HOBt (318.6 mg, 2.36 mmol), DMAP (783.5 mg, 4.72 mmol), and morpholine (0.21 mL, 2.36 mmol) reacted to give compound Q51 (112.7 mg white solid, 90% yield). 1H NMR (500MHz, CDCl3) δ7.26 (s, 1H), 5.75 (dd, J = 5.2, 2.2Hz, 1H), 3.72–3.65 (m, 4H), 3.65 –3.59(m,2H),3.49–3.43(m,2H),2.75(dd,J=24.6,10.8Hz,1H),2.40–2.34(m,1H),2.25 –2.18(m,1H),2.17–2.10(m,2H),2.06–1.87(m,2H),1.81–1.56(m,5H),1.50(s,3H),1.3 6(d,J=7.6Hz,3H),1.32–1.03(m,9H),0.97(t,J=10.7Hz,3H),0.85(s,3H),0.70(s,3H). 13 C NMR(126MHz, CDCl3)δ172.37,149.89,120.92(2C),112.93(2C),66.97,66.70,56.92,55.80,49.34,46.11,42.31,41.89,39.78,38.86, 35.67,35.51,33.35,32.67,32.41,32.08,31.62,31.36,30.13,28.28,24.25,21.29,21.06,18.56,11.91.mp:200-201℃.HRMS(ESI)for C 31 H 47 N3O2[M+H] + :calcd 494.3741,found 494.3741.

[0531] Example 31 Preparation of compounds Q52-Q55

[0532] Compound Q51 (50 mg, 0.1 mmol) and DMAP (3.5 mg, 0.02 mmol) were placed in a single-necked flask, and THF (10 mL) and different acid anhydrides (0.03 mL, 0.3 mmol) were added. The mixture was heated and stirred at 60 °C for 2 hours. The system gradually dissolved, and TLC showed that the reaction was complete. After returning to room temperature, the mixture was extracted with water (10 mL) and ethyl acetate (10 mL). The aqueous phase was then extracted again with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phases were washed with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 2), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to obtain the compound.

[0533] Compound Q52, white solid, 49 mg, yield 95%. 1HNMR(400MHz, CDCl3) δ7.88(dd,J=12.7,4.9Hz,1H),5.74(dd,J=5.1,2.1Hz,1H),3.66(dd,J=9.1,4.5Hz,4H) ,3.60(d,J=5.0Hz,2H),3.48–3.42(m,2H),3.10(q,J=7.4Hz,2H),2.75(d,J=14.9Hz,1H),2.42–2.29(m,1H),2 .27–2.07(m,4H),2.07–1.97(m,1H),1.92–1.81(m,1H),1.82–1.54(m,5H),1.50(s,3H),1.47–1.37(m,2H),1 .33(s,3H),1.23(dd,J=14.1,6.6Hz,4H),1.19–1.02(m,5H),0.95(d,J=6.5Hz,3H),0.78(s,3H),0.68(s,3H). 13 C NMR (101MHz, CDCl3) δ173.05,172.39,162.62,149.29,124.08,121.17,118.85,66.95,66.69,56.84,55.79,49.11,46.10,42.31,41.89,39.71 ,38.57,36.32,35.66,33.77,32.25,31.94,31.88,31.58,31.34,30.11 ,28.25,27.31,24.22,21.12,21.02,18.54,11.92,8.58.mp:160-161℃. HRMS(ESI)for C 34 H 51 N3O3[M+Na] + :calcd 572.3823,found572.3842.

[0534] Compound Q53, white solid, 49 mg, yield 95%. 1HNMR(400MHz, CDCl3) δ7.87(d,J=1.2Hz,1H),5.74(dd,J=5.1,2.1Hz,1H),3.66(dd,J=8.7,4.2Hz,4H),3.61 (d,J=4.9Hz,2H),3.50–3.41(m,2H),3.08–3.01(m,2H),2.75(d,J=14.9Hz,1H),2.36–2.26(m,1H),2.14–1. 90(m,5H),1.89–1.79(m,1H),1.78(dd,J=14.8,7.4Hz,3H),1.71–1.54(m,4H),1.51(s,3H),1.43–1.33(m,2 H),1.34(s,3H),1.28–1.05(m,6H),1.01(t,J=7.4Hz,3H),0.95(d,J=6.4Hz,3H),0.78(s,3H),0.68(s,3H). 13 C NMR (101MHz, CDCl3) δ172.33,172.24,162.57,149.32,124.03,121.16,1 18.87,66.96,66.70,56.85,55.81,49.11,46.09,42.31,41.88,39.71,38 .57,36.32,35.66,35.61,33.77,32.26,31.94,31.88,31.59,31.34,30.1 1,28.26,24.22,21.13,21.02,18.55,18.05,13.81,11.92.mp:170-171℃. HRMS(ESI) for C 35 H 53 N3O3[M+Na] + :calcd 586.3979,found586.3991.

[0535] Compound Q54, white solid, 49 mg, yield 95%. 1H NMR(500MHz, CDCl3)δ7.86(s,1H),5.75(d,J=3.0Hz,1H),3.91–3.80(m,1H),3.66(t,J=9.4Hz,4H),3.62(d,J=4.8H z,2H),3.47(d,J=4.4Hz,2H),2.76(d,J=14.9Hz,1H),2.41–2.32(m,1H),2.26–2.18(m,1H),2.18–2.09(m,2H),2.0 8–2.00(m,1H),1.95–1.84(m,1H),1.79–1.69(m,1H),1.70–1.55(m,4H),1.52(s,3H),1.50–1.39(m,2H),1.35(s,3 H),1.34–1.30(m,1H),1.27(d,J=6.9Hz,6H),1.23–1.01(m,6H),0.95(d,J=11.6Hz,3H),0.79(s,3H),0.68(s,3H). 13 C NMR (126MHz, CDCl3) δ176.20,172.34,162.51,149.32,124.21,121.16,1 18.83,66.97,66.70,56.86,55.83,49.14,46.11,42.32,41.90,39.73,38 .60,36.31,35.67,33.74,32.30,31.95,31.91,31.60,31.35,30.12,28.2 6,24.22,21.14,21.04,19.35,18.98,18.89,18.55,11.93.mp:198-199℃. HRMS(ESI) for C 35 H 53 N3O3[M+Na] + :calcd 586.3979,found586.3986.

[0536] Compound Q55, white solid, 49 mg, yield 95%. 1H NMR (500MHz, CDCl3) δ7.89 (s, 1H), 6.13 (s, 1H), 5.80 (s, 1H), 5.75 (d, J = 3.2Hz, 1H), 3.67 (d, J = 3.8Hz, 4H), 3.62 (d,J=4.5Hz,2H),3.46(s,2H),2.77(dd,J=14.7,9.6Hz,1H),2.65(s,1H),2.40–2.31(m,1H),2.26–2.20(m,1H), 2.16(s,3H),2.12(d,J=4.8Hz,1H),2.05(d,J=12.2Hz,1H),1.94–1.87(m,1H),1.79(s,1H),1.68–1.58(m,5H),1 .52(s,3H),1.50–1.43(s,2H),1.36(s,3H),1.29–1.16(m,7H),0.96(d,J=6.3Hz,3H),0.79(s,3H),0.70(s,3H). 13 C NMR (126MHz, CDCl3) δ172.32,167.15,163.20,149.23,137.87,127.18,12 5.97,121.22,118.74,66.97,66.70,56.85,55.83,49.12,46.11,42.33,4 1.90,39.72,38.60,36.35,35.67,33.79,32.21,31.94,31.86,31.60,31. 35,30.13,28.26,24.22,21.15,21.04,20.42,18.55,11.93.mp:140-141℃. HRMS(ESI) for C 35 H 51 N3O3[M+Na] + :calcd584.3823,found 584.3815.

[0537] Example 32 Preparation of compound Q56

[0538] Similar to the synthesis of compound Q49, compound Q25 (50 mg, 0.055 mmol), anhydrous tetrahydrofuran (6 mL), and 2M ethyl magnesium chloride (0.5 mL, 1.1 mmol) reacted to give compound Q56 (50 mg white solid, 98% yield). 1HNMR (500MHz, CDCl3) δ7.26 (s, 1H), 5.75 (dd, J = 5.2, 2.2Hz, 1H), 2.72 (d, J = 14.5Hz, 1 H),2.18–2.02(m,3H),1.91–1.84(m,1H),1.74–1.52(m,5H),1.50(s,3H),1.48–1.41( m,5H),1.39(s,1H),1.37(s,3H),1.35–0.98(m,11H),0.94(t,J=9.3Hz,3H),0.87(d, J=3.5Hz,2H),0.86(s,1H),0.85(d,J=3.5Hz,3H),0.84(d,J=3.7Hz,1H),0.69(s,3H). 13 C NMR (126MHz, CDCl3) δ149.96,120.90,112.90,74.78,73.24,62.97,56.96,5 5.75,49.39,42.26,39.79,38.87,36.13,35.50,34.22,33.92,33.34,32.68, 32.39,32.11,31.64,31.18,30.94,30.35,29.70,29.26,29.15,28.30,24.25 ,21.29,21.07,18.85,11.87,10.02,7.80,7.72.mp:210-211℃.HRMS(ESI)for C 31 H 51 N₂O[M+H] + :calcd 467.3996, found 467.4018.

[0539] Example 33 Preparation of compound Q57

[0540] Compound Q7 (414 mg, 1 mmol), sulfur (320 mg, 10 mmol), and ethylenediamine (0.67 mL, 10 mmol) were added to a 100 mL single-necked flask. Morpholine (40 mL) was added and stirred until homogeneous. The mixture was heated to reflux at 120 °C for 5 hours. After the reaction was confirmed to be complete by TLC, water (30 mL) and ethyl acetate (30 mL) were added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 2), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 20:1) to give compound Q57 (360 mg white solid, yield 80%). 13C NMR (126MHz, CDCl3) δ174.74,159.63,151.19,148.86,142.48,141.15,120.73,56.83,55.76,51.48,48.88,43.86,42 .38,41.25,39.69,37.65,35.38,33.52,31.89,31.72,31.41,31.05,31.01,28.16,24.17,21.07,20.39,18.31,11.91.

[0541] Example 34 Preparation of compound Q58

[0542] Compound Q57 (360 mg, 0.8 mmol) and lithium hydroxide (576 mg, 24 mmol) were added to a 100 mL single-necked flask. A water-ethanol mixture was added, and the mixture was stirred at room temperature for 8 hours. The reaction was confirmed to be complete by TLC. The pH was adjusted to approximately 5 by adding 2 M dilute hydrochloric acid aqueous solution. Ethyl acetate (30 mL) was added, and the mixture was extracted and separated. The aqueous phase was then extracted again with ethyl acetate (30 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 10:1) to give compound Q58 (349 mg white solid, 99% yield). 1 H NMR (400MHz, CDCl3) δ8.45(s,1H),8.28(s,1H),5.79(s,1H),3.09(d,J=15.6Hz,1H),2.63(d,J=15.5H z,1H),2.45–2.35(m,1H),2.33–2.24(m,1H),2.18(d,J=17.9Hz,1H),2.07(d,J=12.5Hz,1H),1.95–1. 78(m,2H),1.71(dd,J=28.3,10.8Hz,3H),1.61(s,3H),1.58–1.40(m,3H),1.33(s,3H),1.29–1.19(m, 3H),1.19–1.05(m,4H),0.96(d,J=6.1Hz,3H),0.78(s,3H),0.70(s,3H).mp:265-266℃.HRMS(ESI)for C 28 H 41 N₂O₂[M+H] + :calcd 437.3163,found437.3190.

[0543] Example 35 Preparation of compound Q59

[0544] Similar to the synthesis of compound Q37, compound Q58 (349 mg, 0.8 mmol), EDCI (306.7 mg, 1.6 mmol), HOBt (243.2 mg, 1.6 mmol), DMAP (531.2 mg, 3.2 mmol), and morpholine (0.14 mL, 1.6 mmol) reacted to give compound Q59 (364 mg white solid, 90% yield). 1 H NMR (500MHz, CDCl3) δ8.40(s,1H),8.31–8.18(m,1H),5.76(d,J=2.2Hz,1H),3.65(s,4H),3.59(s,2H),3.45( d,J=3.1Hz,2H),3.06(d,J=15.6Hz,1H),2.62(d,J=15.4Hz,1H),2.39–2.32(m,1H),2.22–2.14(m,2H),2.03( dd,J=15.7,6.3Hz,1H),1.88(s,1H),1.79–1.63(m,4H),1.59(d,J=2.7Hz,3H),1.45(dd,J=20.7,17.4Hz,2H) ,1.31(s,3H),1.25–1.02(m,7H),1.00–0.89(m,3H),0.86–0.81(m,1H),0.77(s,3H),0.67(d,J=17.8Hz,3H). 13 C NMR (126MHz, CDCl3) δ172.29,159.62,151.17,148.85,142.49,141.16,120.73,66.96,66.69,56.82,55.84,48.87,46.09,43.87,42.40,41.88 ,41.25,39.70,37.65,35.65,33.52,31.89,31.72,31.41,31.33,30.10,28.25,24.19,21.07,20.38,18.54,11.94.mp:151-152℃.HRMS(ESI)for C 32 H 47 N3O2[M+H] + :calcd506.3741,found 506.3762.

[0545] Example 36 Preparation of compound Q60

[0546] Similar to the synthesis of compound Q48, compound Q59 (364 mg, 0.72 mmol), NHPI (234.7 mg, 1.44 mmol), acetone (20 mL), glacial acetic acid (0.042 mL, 0.72 mmol), and sodium dichromate (256.9 mg, 0.864 mmol) reacted to give compound Q60 (262 mg white solid, 70% yield). 13 C NMR (126MHz, CDCl3) δ201.73,173.43,172.25,157.30,149.65,143.15,141.97,125.51,66.97,66.69,54.58,50.27,49.61,46.12 ,45.16,43.30,42.33,42.17,41.91,39.55,38.69,35.56,32.46,31.38,30.38,30.10,28.57,26.31,21.33,18.71,17.66,12.04.

[0547] Example 37 Preparation of compound Q61

[0548] Similar to the synthesis of compound Q48, compound Q60 (100 mg, 0.18 mmol), 20 mL of methanol, and sodium borohydride (68.4 mg, 1.8 mmol) reacted to give compound Q61 (58.8 mg white solid, 60% yield). 1 HNMR (500MHz, CDCl3) δ8.50–8.36(m,1H),8.28(d,J=1.7Hz,1H),5.71(d,J=2.4Hz,1H),4.01(d,J=7.8Hz,1H),3.70–3 .63(m,4H),3.61(d,J=4.8Hz,2H),3.50–3.43(m,2H),3.08(d,J=15.6Hz,1H),2.61(d,J=15.6Hz,1H),2.40–2.32(m,1H ),2.25–2.18(m,1H),2.07(d,J=12.8Hz,1H),1.99–1.92(m,1H),1.89–1.85(m,1H),1.82–1.69(m,3H),1.64(s,3H),1 .54–1.43(m,4H),1.34(s,3H),1.28–1.21(m,3H),1.14–1.07(m,2H),0.97(d,J=6.5Hz,3H),0.83(s,3H),0.72(s,3H). 13C NMR (126MHz, CDCl3) δ172.27,158.94,151.82,150.66,142.72,141.37,124.78,73.32,66.96,66.69,55.96,55.26,47.56,46.09,43.46,42.9 7,41.90,41.11,40.60,39.48,37.74,35.63,33.48,31.35(2C),30.14,28.57,26.33,21.15,20.11,18.60,11.90.mp:185-186℃.HRMS(ESI)for C 32 H 47 N3O3[M+Na] + :calcd 544.3510,found544.3500.

[0549] Example 38 Preparation of compound Q62

[0550] Compound Q7 (300 mg, 0.72 mmol) and tribromopyridinium (278 mg, 0.87 mmol) were added to a 100 mL single-necked flask, and anhydrous DCM (40 mL) was added. The mixture was stirred overnight at room temperature. After the reaction was confirmed to be complete by TLC, water (30 mL) was added for extraction and separation. The aqueous phase was then extracted with dichloromethane (20 mL × 2), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 20:1) to give compound Q62 (white solid 284 mg, yield 80%). 13 C NMR (101MHz, CDCl3) δ209.96,174.74,147.98,120.90,56.62,55.68,51.54,50.42,48.50,44.08,43.74,42.3 8,39.51,38.86,35.35,31.82,31.56,31.03,30.95,30.84,28.37,28.13,24.10,21.24,19.17,18.30,11.93.

[0551] Example 39 Preparation of compound Q63

[0552] Compound Q62 (100 mg, 0.2 mmol), thiourea (30.4 mg, 0.4 mmol), and ethanol (25 mL) were added to a single-necked flask and heated under reflux for 3 hours. After the reaction was complete as determined by TLC, water (30 mL) and ethyl acetate (30 mL) were added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 2), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 10:1) to give compound Q63 (56.4 mg white solid, 60% yield). 13 C NMR (126MHz, CDCl3) δ174.75,164.62,151.43,149.07,120.79,115.28,56.92,55.72,51.49,49.38,42.28,39.69, 38.80,38.77,35.37,35.18,32.19,32.15,31.57,31.17,31.06,31.02,28.16,24.19,21.45,20.93,18.33,11.87.

[0553] Example 40 Preparation of compound Q64

[0554] Similar to the synthesis of Q26, compound Q63 (56 mg, 0.12 mmol) was reacted with lithium hydroxide (87 mg, 3.6 mmol) and hydrolyzed to give compound Q64 (50 mg white solid, 99% yield). 1 HNMR (400MHz, DMSO) δ11.99(s,1H),6.84(s,2H),5.70(s,1H),2.59(d,J=15.5Hz,1H),2.27–2.18( m,1H),2.10–2.02(m,1H),2.07(d,J=21.3Hz,1H),1.97(d,J=9.9Hz,1H),1.81(s,1H),1.73–1.61( m,2H),1.58(d,J=15.5Hz,1H),1.52–1.35(m,4H),1.30(s,3H),1.27(d,J=13.8Hz,1H),1.23(s,3H ),1.18(s,2H),1.12–1.02(m,3H),0.97(s,3H),0.89(d,J=5.5Hz,3H),0.65(s,3H).mp:267-259℃. HRMS(ESI)for C 27 H 40 N₂O₂S[M+H] +:calcd 457.2883, found 457.2889.

[0555] Example 41 Preparation of compounds Q65-Q66

[0556] Compound Q62 (100 mg, 0.2 mmol), thioacetamide (30.1 mg, 0.4 mmol), and ethanol (25 mL) were added to a 50 mL single-necked flask and heated under reflux for 3 hours. After the reaction was complete as detected by TLC, water (30 mL) and ethyl acetate (30 mL) were added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 2), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to obtain a white solid Q65, which was directly used in the next step.

[0557] Similar to the synthesis of Q26, compound Q65, lithium hydroxide (87 mg, 3.6 mmol), and a water-ethanol mixture (20 mL) were reacted and hydrolyzed to give compound Q66 (40 mg white solid, 50% yield in two steps). 1 H NMR (400MHz, CDCl3) δ5.76 (s, 1H), 2.83 (d, J = 15.3Hz, 1H), 2.65 (s, 3H), 2.53 –2.20(m,3H),2.14(d,J=17.6Hz,1H),2.04(d,J=12.1Hz,1H),1.85(d,J=10.0 Hz,2H),1.77–1.52(m,4H),1.49(s,3H),1.40(s,1H),1.36(s,3H),1.32–1.0 2(m,7H),0.98(s,3H),0.95(d,J=5.7Hz,3H),0.90–0.72(m,1H),0.70(s,3H). 13 C NMR (101MHz, CDCl3) δ179.41,162.53,155.82,148.93,125.02,121.08,56.90,55.74,49.34,42.30,39.68,38.93,38.70 ,35.34,35.24,32.52,32.13,31.57(2C),31.09,30.85,28.15,24.20,21.28,20.98,19.02,18.32,11.89.mp:250-251℃. HRMS(ESI)for C 28 H 41 NO2S[M+H] + :calcd456.2931,found 456.2919.

[0558] Example 42 Preparation of compound Q67

[0559] Similar to the synthesis of Q26, compound Q7 (1 g, 2.41 mmol), lithium hydroxide (1.73 g, 72.4 mmol), and a water-ethanol mixture (100 mL) were reacted and hydrolyzed to give compound Q67 (white solid 964 mg, yield 99%). 13 C NMR (101MHz, CDCl3) δ217.14,180.41,149.75,119.91,56.73,55.73,48.85,48.68,42.43,39.71,37.05,3 5.30,33.71,32.06,31.69,31.19,31.05,30.74,30.23,28.13,27.23,24.15,21.27,19.34,18.28,11.94.

[0560] Example 43 Preparation of compound Q68

[0561] Similar to the synthesis of compound Q37, compound Q67 (964 mg, 2.41 mmol), EDCI (924 mg, 4.82 mmol), HOBt (733.4 mg, 4.82 mmol), DMAP (1.6 g, 9.64 mmol), and morpholine (0.42 mL, 4.82 mmol) reacted to give compound Q68 (1.02 g white solid, 90% yield). 13 C NMR (101MHz, CDCl3) δ216.80,172.27,149.74,119.90,66.94,66.68,56.71,55.83,48.85,48.64,46.07,42.42,41.86,3 9.70,37.04,35.64,33.70,32.07,31.68,31.30,31.18,30.21,30.10,28.24,27.23,24.17,21.26,19.32,18.52,11.94.

[0562] Example 44 Preparation of compound Q69

[0563] Compound Q68 (100 mg, 0.21 mmol) and potassium tert-butoxide (100 mg, 1.05 mmol) were dissolved in 10 mL of tert-butanol in a 50 mL single-necked flask. The mixture was heated in a 35 °C water bath for 30 minutes, and then amyl nitrite (0.13 mL, 1.05 mmol) was slowly added dropwise. The mixture was stirred at room temperature for 5 hours. After the reaction was complete as determined by TLC, the mixture was extracted with 30 mL of water and 30 mL of ethyl acetate. The aqueous phase was then extracted again with 20 mL × 3 ethyl acetate solutions, and the organic phases were combined. The organic phases were washed with 20 mL × 3 water solutions and 20 mL × 2 saturated sodium chloride solutions, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to give compound Q69 (90 mg white solid, 86% yield). 13 C NMR (101MHz, CDCl3) δ201.71,172.61,153.22,147.89,121.41,66.95,66.69,56.56,55.77,48.65,48.02,46.14,42.37, 41.95,39.53,35.95,35.71,35.66,31.65,31.41,31.36,30.52,30.10,28.21,27.16,24.17,21.25,20.95,18.54,11.90.

[0564] Example 45 Preparation of compound Q70

[0565] Compound Q69 (90 mg, 0.18 mmol) and hydroxylamine hydrochloride (37.5 mg, 0.54 mmol) were added to a 25 mL single-necked flask. Pyridine (5 mL) was added, and the mixture was heated to reflux at 100 °C for 3 hours. After the reaction was confirmed to be complete by TLC, the mixture was extracted with water (30 mL) and ethyl acetate (30 mL). The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phase was washed with dilute hydrochloric acid (20 mL), water (20 mL × 3), and saturated sodium chloride solution (20 mL × 2), respectively. The mixture was dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to give compound Q70 (83 mg white solid, 90% yield). 1HNMR (400MHz, CDCl3) δ12.88(s,1H),10.76(s,1H),5.65(s,1H),3.67(t,J=7.5Hz,4H),3.63(s,2H),3. 48(d,J=3.7Hz,2H),2.38(dd,J=17.7,7.1Hz,1H),2.28–2.19(m,1H),2.02(d,J=12.9Hz,2H),1.88(d,J= 17.3Hz,1H),1.73(d,J=3.3Hz,1H),1.63–1.56(m,2H),1.43(s,3H),1.34–1.28(m,3H),1.21(s,3H),1. 17–1.07(m,4H),0.96(d,J=6.1Hz,2H),0.88–0.85(m,6H),0.84(s,3H),0.82–0.81(m,1H),0.67(s,3H).

[0566] Example 46 Preparation of compound Q71

[0567] Compound Q70 (83 mg, 0.162 mmol), potassium hydroxide (2 tablets), ethylene glycol (10 mL), and dioxane (5 mL) were added to a 50 mL single-necked flask and heated to reflux at 130 °C for 5 hours. After the reaction was complete as determined by TLC, water (30 mL) and ethyl acetate (30 mL) were added for extraction and separation. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with dilute hydrochloric acid (20 mL), water (20 mL × 3), and saturated sodium chloride solution (20 mL × 2), respectively, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to give compound Q71 (62.11 mg white solid, 90% yield). 1 H NMR(400MHz, CDCl3)δ5.83(d,J=3.2Hz,1H),3.23(d,J=15.5Hz,1H),2.45–2.36(m,1H),2.34–2.25(m,2H),2.24–2.01(m,2H),1.97–1.67(m,4H) ,1.63(s,3H),1.59–1.47(m,3H),1.42(d,J=12.1Hz,3H),1.38–1.19(m, 5H),1.19–1.02(m,3H),0.95(d,J=6.4Hz,3H),0.79(s,3H),0.70(s,3H). 13C NMR (101MHz, CDCl3) δ180.05,159.78,150.69,146.75,122.97,56.65,55.69,48.95,42.26,39.50,38.80,35.30, 35.18,33.45,32.35,31.73,31.50,31.34,30.95,30.72,28.11,24.15,21.12,20.91,18.27,11.89.mp:210-211℃. HRMS(ESI)for C 26 H 38 N₂O₃[M+Na] + :calcd 449.2775, found 449.2778.

[0568] Example 47 Preparation of compound Q72

[0569] Similar to the synthesis of compound Q27, compound Q71 (42 mg, 0.1 mmol), DMAP (3.5 mg, 0.02 mmol), and acetic anhydride (0.03 mL, 0.3 mmol) were reacted by heating to give compound Q72 (45 mg, 95%). 1 H NMR (500MHz, CDCl3) δ5.83(dd,J=5.3,2.3Hz,1H),3.67(d,J=4.3Hz,4H),3.62(d,J=5.0Hz,2H),3.50–3.43(m,2H ),3.23(d,J=15.5Hz,1H),2.40–2.83(m,1H),2.29(d,J=15.5Hz,1H),2.26–2.14(m,2H),2.08(dd,J=9.4,3.3Hz, 1H),1.94–1.87(m,1H),1.85–1.75(m,1H),1.74–1.64(m,2H),1.62(s,3H),1.60–1.46(m,3H),1.43(s,3H),1.34 (dt,J=13.5,9.1Hz,2H),1.29–1.20(m,3H),1.20–1.02(m,3H),0.96(d,J=6.5Hz,3H),0.78(s,3H),0.70(s,3H). 13C NMR (126MHz, CDCl3) δ172.25,159.78,150.69,146.71,122.98,66.97,66.70,56.65,55.82,48.95,46.08,42.27,41.89,39.52, 38.80,35.65,35.18,33.45,32.35,31.73,31.50,31.34,31.29,30.11,28.22,24.18,21.12,20.91,18.53,11.91.mp:250-251℃. HRMS(ESI)forC 30 H 45 N3O3[M+Na] + :calcd518.3352, found 518.3359.

[0570] Example 48 Preparation of compounds Q73-Q74

[0571] Compound Q68 (50 mg, 0.11 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL) in a 50 mL single-necked flask. NaH (28.8 mg, 1.2 mmol) and diethyl oxalate (0.03 mL, 0.22 mmol) were added under nitrogen protection. The mixture was stirred at room temperature for 20 minutes, and the system turned orange-yellow. TLC confirmed complete reaction of the reactants. The mixture was extracted with water (10 mL) and ethyl acetate (10 mL), and the aqueous phase was extracted again with ethyl acetate (10 mL). The organic phases were combined. The organic phases were washed with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 2), dried over anhydrous Na₂SO₄, and concentrated under reduced pressure to give a pale yellow solid, Q73. This compound was unstable and was directly used in the next step.

[0572] Compound Q73 (the solid from the previous step) was added to a 50 mL single-necked flask with acetic acid (20 mL), potassium acetate (22 mg, 0.22 mmol), and hydrazine hydrate (0.18 mL, 3.6 mmol). The mixture was heated to reflux at 130 °C for 5 hours. TLC was used to confirm the complete reaction of the starting material. 2 M sodium hydroxide solution (60 mL) was added and the mixture was stirred for 10 minutes. 30 mL of saturated sodium bicarbonate solution and 20 mL of ethyl acetate were added, and the mixture was extracted and separated. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to give compound Q74 (56 mg white solid, 90% yield in both steps). 1HNMR(500MHz, CDCl3)δ5.75(dd,J=5.2,2.2Hz,1H),4.44–4.27(m,2H),3.70–3.63(m,4H),3.61(d,J=5.2Hz,2H), 3.51–3.42(m,2H),3.19(d,J=15.7Hz,1H),2.43–2.29(m,1H),2.26–2.16(m,2H),2.13(dt,J=17.7,5.4Hz,1H),2 .04(dd,J=9.8,2.8Hz,1H),1.92–1.82(m,1H),1.81–1.74(m,1H),1.71–1.54(m,4H),1.51(s,3H),1.50–1.40(m, 2H),1.39(s,1H),1.37(s,2H),1.36(s,3H),1.33–1.01(m,7H),0.95(d,J=6.5Hz,3H),0.85(s,3H),0.69(s,3H). 13 C NMR (126MHz, CDCl3) δ172.42,161.30,149.21(2C),121.07(2C),117.66,66.97,66.70,60.69,56.90,55.79,49.37,46.12,42.31,41.91,39 .75,38.48,35.67,35.66,33.20,32.91,32.35,32.05,31.59,31.37,3 0.12,28.26,24.24,21.42,21.07,18.56,14.38,11.92.mp:144-145℃. HRMS(ESI)for C 34 H 51 N3O4[M+Na] + :calcd588.3772,found 588.3798.

[0573] Example 49 Preparation of compound Q75

[0574] Equivalent to the synthesis of compound Q73 in Example 48, compound Q68 (50 mg, 0.11 mmol), NaH (28.8 mg, 1.2 mmol), and diethyl oxalate (0.03 mL, 0.22 mmol) reacted to give a pale yellow solid Q73.

[0575] Compound Q73 (the solid from the previous step) and hydroxylamine hydrochloride (23 mg, 0.33 mmol) were placed in a 50 mL single-necked flask, and an ethanol-water mixture (20 mL) was added. The mixture was heated to reflux at 100 °C for 3 hours. The reaction proceeded to completion as determined by TLC. After returning to room temperature, the mixture was extracted with water (20 mL) and ethyl acetate (20 mL). The aqueous phase was then extracted again with ethyl acetate (20 mL × 3), and the organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to obtain compound Q75 (42 mg, 90%). 1 H NMR (500MHz, CDCl3) δ5.83–5.67(m,1H),4.48–4.33(m,2H),3.71–3.62(m,4H),3.59(d,J=4.7Hz,2H),3.45(d,J=4.4 Hz,2H),2.97(t,J=17.1Hz,1H),2.38–2.31(m,1H),2.24–2.19(m,1H),2.16(dd,J=13.9,5.0Hz,1H),2.10(dd,J=14.7 ,9.3Hz,1H),2.02(d,J=12.5Hz,1H),1.91–1.83(m,1H),1.81–1.74(m,1H),1.73–1.49(m,6H),1.47(d,J=9.4Hz,3H) ,1.39(dd,J=8.2,6.0Hz,5H),1.34–1.26(m,3H),1.24–0.98(m,7H),0.94(d,J=3.5Hz,1H),0.93(s,3H),0.68(s,3H). 13 C NMR (101MHz, CDCl3) δ175.71,172.27,160.90,153.61,147.52,122.23,110.80,66.93,66.67,61.69,56.76,55.74,49.36,46.06,42.25,41.86 ,39.57,38.60,36.98,35.60,32.33,31.96,31.46,31.29,30.50,30.04 ,29.82,28.21,24.20,21.72,20.93,18.54,14.18,11.87.mp:140-141℃. HRMS(ESI)for C 34 H 50 N₂O₅[M+Na] + :calcd 589.3612, found 589.3638.

[0576] Example 50 Preparation of compound Q76

[0577] As in Example 19, compound Q7 (1.00 g, 2.41 mmol), ethyl formate (30 mL), and NaH (578 mg, 24.1 mmol) reacted to give a pale yellow oily substance Q24.

[0578] Compound Q24 (the oily substance from the previous step) and hydroxylamine hydrochloride (502 mg, 7.23 mmol) were added to a 100 mL single-necked flask. An ethanol-water mixture (50 mL) was added, and the mixture was heated to reflux at 100 °C for 3 hours. TLC analysis confirmed the reaction was complete. After returning to room temperature, the mixture was extracted with water (30 mL) and ethyl acetate (30 mL). The aqueous phase was then extracted again with ethyl acetate (30 mL × 3), and the organic phases were combined. The organic phases were washed with water (30 mL × 3) and saturated sodium chloride solution (30 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 2:1) to obtain compound Q76 (952 mg, 90%). 1 H NMR (500MHz, CDCl3) δ8.02(s,1H),5.77(d,J=3.4Hz,1H),3.84(s,1H),3.66(s,3H),2.57(d,J=14.9Hz,1H ),2.39–2.31(m,1H),2.27–2.19(m,1H),2.14–2.10(m,1H),2.05(d,J=12.6Hz,1H),1.92–1.85(m,1H),1.8 3–1.75(m,1H),1.67–1.61(m,3H),1.54(dd,J=10.7,6.7Hz,2H),1.48(s,3H),1.39(s,3H),1.34–1.30(m, 2H),1.26(d,J=8.4Hz,3H),1.18(s,1H),0.94(s,3H),0.93(s,3H),0.79(s,1H),0.69(s,3H),0.68(s,1H).

[0579] Example 51 Preparation of compounds Q77-Q79

[0580] Similar to the synthesis of compound Q48, compound Q76 (250 mg, 0.57 mmol), NHPI (186.0 mg, 1.14 mmol), 20 mL of acetone, glacial acetic acid (0.033 mL, 0.57 mmol), and sodium dichromate (205.2 mg, 0.69 mmol) reacted to give compound Q77 (182 mg white solid, 70% yield).

[0581] Similar to the synthesis of compound Q49, compound Q77 (82 mg, 0.18 mmol), 20 mL of methanol, and sodium borohydride (68.4 mg, 1.8 mmol) were reacted and reduced to give compound Q78 (50 mg white solid, 60% yield).

[0582] Similar to the synthesis of compound Q49, compound Q79 was obtained by reacting compound Q78 (50 mg, 0.11 mmol), anhydrous tetrahydrofuran (6 mL), and 2M ethyl magnesium chloride reagent (1 mL, 2.2 mmol). 1 HNMR (400MHz, CDCl3) δ8.06(d,J=24.1Hz,1H),5.70(t,J=10.3Hz,1H),3.97(t,J=10.7Hz,1H),2.59(d,J=15.0Hz,1H),2.12(d ,J=15.1Hz,1H),2.04(d,J=5.3Hz,1H),1.93(dd,J=12.1,7.8Hz,1H),1.87–1.80(m,1H),1.68–1.62(m,2H),1.60–1.55(m,2H), 1.53(s,3H),1.50(s,1H),1.48–1.43(m,5H),1.42(s,3H),1.39–1.34(m,2H),1.34–1.28(m,2H),1.26(d,J=7.2Hz,3H),1.20( d,J=4.6Hz,1H),1.19–1.13(m,2H),1.11–1.03(m,2H),1.01(s,3H),0.96(d,J=6.4Hz,3H),0.86(d,J=2.4Hz,3H),0.72(s,3H). 13 C NMR (101MHz, CDCl3) δ172.01,151.16,149.63,125.94,108.56,74.73,73.38,55.95,55.09,47.79,42.80,40.71,39.41,38.97,36.68,36 .06,34.24,32.04,31.18,30.95,30.31,29.91,29.26,28.55,26.38,21.47,21.04,18.89,11.78,7.80,7.71.mp:191-192℃.HRMS(ESI)for C 31 H 49 NO3[M+H] + :calcd 484.3785,found484.3811.

[0583] Example 52 Preparation of compounds Q80-Q81

[0584] Similar to the synthesis of Q24, compound Q68 (100 mg, 0.21 mmol), 20 mL of ethyl formate, and NaH (51 mg, 2.1 mmol) react to yield a pale yellow oily substance Q80.

[0585] Compound Q80 (the oily substance from the previous step) was added to a 50 mL single-necked flask along with 20 mL of ethanol, a few drops of water, and 80 mg (0.63 mmol) of o-fluorohydrazine hydrochloride. The mixture was heated to reflux at 100 °C for 3 hours. TLC analysis confirmed the reaction was complete. 10 mL of water and 20 mL of ethyl acetate were added, and the mixture was extracted and separated. The aqueous phase was then extracted again with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then subjected to silica gel column chromatography (PE:EA = 10:1) to give compound Q81 (111 mg of white solid, 90% yield in both steps). 1 H NMR (500MHz, CDCl3) δ7.52–7.37(m,3H),7.21(dd,J=16.1,8.4Hz,2H),5.68(d,J=3.4Hz,1H) ,3.71–3.63(m,4H),3.61(d,J=5.0Hz,2H),3.50–3.41(m,2H),2.75(d,J=14.6Hz,1H),2.41–2 .31(m,1H),2.26–2.14(m,2H),2.14–2.02(m,2H),1.94–1.83(m,1H),1.83–1.75(m,1H),1.71 –1.41(m,6H),1.41–0.99(m,14H),0.97(d,J=3.8Hz,2H),0.95(d,J=6.1Hz,3H),0.70(s,3H). 13 C NMR(126MHz, CDCl3)δ172.34(2C),138.68(2C),131.19,131.12,123.99,120.80(3C),114.67(2C),66.96,66.69,56.88,55.80,49.58(2C),46 .10,42.32,41.89,39.75,38.53,36.73,35.66,33.37,32.19,31.44(2C ),31.35(2C),30.11,28.27,24.23,21.03,18.56,11.92.mp:250-251℃. HRMS(ESI)for C 37 H 50 FN3O2[M+H]+ :calcd 588.3960, found 588.3952.

[0586] Example 53 Preparation of compound Q82

[0587] Compound Q6 (1 g, 2.35 mmol), palladium on carbon (500 mg), and 30 mL of ethyl acetate were placed in an autoclave and stirred at room temperature for 24 hours at 4 MPa. The reaction was confirmed by TLC to be complete. The mixture was filtered, and the filter cake was washed with ethyl acetate (10 mL × 3). After direct concentration, the mixture was subjected to silica gel column chromatography (PE:EA = 10:1) to obtain compound Q82 (1 g of white solid, 99% yield in two steps). 13 C NMR (126MHz, CDCl3) δ174.36,79.09,60.17,56.58,56.34,55.84,54.72,42.50,39.88,38.94,37.36,36.48,3 5.35,35.07,32.89,31.32,31.00,28.14(2C),27.62,24.19,21.29,20.63,18.27,15.50,14.37,14.26,12.02.

[0588] Example 54 Preparation of compounds Q83-Q84

[0589] Similar to the synthesis of Q24-Q25, compound Q82 (1.00 g, 2.41 mmol), 30 mL of ethyl formate, and NaH (578 mg, 24.1 mmol) react to give a pale yellow oily substance Q83.

[0590] Then, compound Q83 (the oily substance from the previous step), 30 mL of glacial acetic acid, potassium acetate (473 mg, 4.82 mmol), and 85% hydrazine hydrate (0.42 mL, 8.51 mmol) were heated under reflux to give compound Q84 (1 g of white solid, yield 91.8%). 13 C NMR (126MHz, CDCl3) δ174.37,149.38,134.09,112.57,60.19,56.47,55.88,54.97,52.85,42.41,39.95,37.90,35.6 1,35.36,34.76,33.40,32.56,31.44,31.33,31.00,28.14,24.39,24.27,22.40,20.85,18.30,14.27,13.72,11.94.

[0591] Example 55 Preparation of compounds Q85-Q86

[0592] Similar to the synthesis of Q26–Q27, compound Q84 (1 g, 2.21 mmol) was hydrolyzed with lithium hydroxide (1.60 g, 66.4 mmol) to give compound Q85 (1.03 g, white solid, 99% yield). Then, compound Q85 (1.03 g, 2.21 mmol), DMAP (73.37 mg, 0.44 mmol), and acetic anhydride (0.63 mL, 6.63 mmol) reacted to give compound Q86 (927 mg, white solid, 90% yield). 1 H NMR (500MHz, CDCl3) δ7.84 (d, J=5.6Hz, 1H), 2.71–2.66 (m, 1H), 2.64 (s, 3H), 2.4 5–2.35(m,1H),2.30–2.21(m,1H),2.08–1.99(m,2H),1.87–1.75(m,3H),1.72–1. 66(m,1H),1.66–1.57(m,2H),1.51–1.33(m,8H),1.29(s,3H),1.24(s,3H),1.19– 1.05(m,4H),0.94(d,J=6.5Hz,3H),0.89–0.83(m,2H),0.79(s,3H),0.67(s,3H). 13 C NMR (126MHz, CDCl3) δ180.06,169.65,162.87,124.41,119.37,56.43,55.84,54.74,52.77,42.40,39.89,37.49,35.60,35. 32,34.67,34.55,32.48,31.63,30.97,30.75,28.12,24.95,24.24,22.69,21.61,20.88,18.26,13.70,11.95.mp:135-136℃. HRMS(ESI)for C 29 H 44 N₂O₃[M+Na] + :calcd491.3244,found 491.3243.

[0593] Example 56 Preparation of compound Q87

[0594] Similar to the synthesis of compound Q37, compound Q86 (145 mg, 0.31 mmol), EDCI (120.8 mg, 0.63 mmol), HOBt (85.2 mg, 0.63 mmol), DMAP (154.0 mg, 1.26 mmol), and morpholine (0.59 mmol) reacted to give compound Q87 (140 mg white solid, 95% yield).1 H NMR (500MHz, CDCl3) δ7.81 (s, 1H), 3.64 (s, 4H), 3.59 (s, 2H), 3.45 (s, 2H), 2.65 (d, J = 10.8Hz ,1H),2.62(s,3H),2.39–2.30(m,1H),2.19–2.08(m,1H),2.05–1.94(m,2H),1.88–1.74(m,3 H),1.68(d,J=14.9Hz,1H),1.63–1.54(m,2H),1.52–1.32(m,7H),1.28(s,3H),1.22(s,3H), 1.16–1.02(m,4H),0.93(d,J=6.7Hz,3H),0.88–0.81(m,2H),0.77(s,3H),0.67–0.62(s,3H). 13 C NMR (126MHz, CDCl3) δ172.32,169.59,162.83,124.38,119.34,66.95,66.69,56.42,55.93,54.74,52.75,46.09,42.40,41.88,39.89,37. 48,35.65,35.58,34.66,34.53,32.48,31.63,31.32,30.10,28.22,2 4.95,24.26,22.67,21.60,20.87,18.51,13.69,11.96.mp:135-136℃. HRMS(ESI)for C 33 H 51 N3O3[M+Na] + :calcd 560.3823, found 560.3817.

[0595] Example 57 Preparation of compounds Q88-Q89

[0596] Similar to the synthesis of Q24-Q25, compound Q19 (1.00 g, 2.41 mmol), 30 mL of ethyl formate, and NaH (578 mg, 24.1 mmol) react to give a pale yellow oily substance Q88.

[0597] Then, compound Q88 (the oily substance from the previous step), 30 mL of glacial acetic acid, potassium acetate (473 mg, 4.82 mmol), and 85% hydrazine hydrate (0.42 mL, 8.51 mmol) were heated to give compound Q89 (1 g of white solid, yield 91.8%). 13CNMR(101MHz, CDCl3)δ174.00,150.02(2C),120.87(2C),112.81,60.20,56.95,55.78,49.38,42.25,39.78,38.87 ,35.52,35.40,34.81,33.33,32.71,32.45,32.11,31.63,28.23,24.23,21.56,21.31,21.06,18.63,14.29,11.86.

[0598] Example 58 Preparation of compounds Q90-Q92

[0599] Similar to the synthesis of Q26, compound Q89 (104 mg, 0.221 mmol) and lithium hydroxide (160 mg, 6.64 mmol) were hydrolyzed to give compound Q90 (103 mg white solid, 99% yield).

[0600] Similar to the synthesis of compound Q37, compound Q90 (100 mg, 0.23 mmol), EDCI (90 mg, 0.46 mmol), HOBt (62 mg, 0.46 mmol), DMAP (152.7 mg, 0.92 mmol), and morpholine (0.04 mL, 0.46 mmol) were reacted and condensed to give compound Q91 (111 mg white solid, 95% yield).

[0601] Compound Q91 (111 mg, 0.22 mmol) and DMAP (7.337 mg, 0.044 mmol) were added to a 50 mL single-necked flask. 20 mL of THF and isobutyric anhydride (0.063 mL, 0.663 mmol) were added, and the mixture was heated and stirred at 60 °C for 2 hours. The mixture gradually dissolved, and TLC confirmed complete reaction of the starting material. The mixture was then cooled to room temperature, and 10 mL of water and 20 mL of ethyl acetate were added. The aqueous phase was extracted again with ethyl acetate (20 mL × 3). The organic phases were combined. The organic phases were washed with water (20 mL × 3) and saturated sodium chloride solution (20 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then purified by silica gel column chromatography (PE:EA = 2:1) to give compound Q92 (114 mg white solid, 90% yield). 1HNMR(500MHz, CDCl3)δ7.88–7.85(m,1H),5.75(dd,J=5.2,2.2Hz,1H),3.92–3.81(m,1H),3.66(d,J=4.4Hz,4 H),3.62(d,J=5.2Hz,2H),3.49–3.44(m,2H),2.77(d,J=14.9Hz,1H),2.33–2.22(m,2H),2.17–2.10(m,2H),2 .04(dd,J=9.4,3.2Hz,1H),1.88–1.81(m,1H),1.75–1.56(m,5H),1.52(s,3H),1.50–1.39(m,4H),1.35(s,3H ),1.29–1.25(m,6H),1.22–1.01(m,7H),0.95(d,J=6.5Hz,3H),0.90–0.82(m,1H),0.79(s,3H),0.69(s,3H). 13 C NMR (126MHz, CDCl3) δ176.22,172.03,162.55,149.35,124.23,121.17,11 8.89,66.97,66.70,56.88,55.75,49.15,46.09,42.26,41.92,39.71,38.6 0,36.31,35.74,35.66,33.75,33.59,32.28,31.97,31.94,31.91,31.60,2 8.28,24.21,21.78,21.14,21.04,19.35,18.98,18.68,11.89.mp:96-97℃. HRMS(ESI) for C 36 H 55 N3O3[M+Na] + :calcd 600.4136, found 600.4138.

[0602] Example 59 Preparation of compound Q93

[0603] Similar to the synthesis of compound Q27, compound Q90 (200 mg, 0.46 mmol), DMAP (15.16 mg, 0.091 mmol), 20 mL THF, and acetic anhydride (0.14 mL, 1.37 mmol) were heated to give compound Q93 (199 mg white solid, 90% yield). 13C NMR (126MHz, CDCl3) δ179.87,169.68,162.90,149.30,124.05,121.18,119.25,56.90,55.87,49.18,42.28,39.75,38.57 ,36.34,35.59,35.47,35.02,33.77,32.26,31.98,31.83,31.60,28.25,24.24,21.63,21.57,21.16,21.02,18.63,11.90.

[0604] Example 60 Preparation of compounds Q94-Q95

[0605] Compound Q93 (110 mg, 0.23 mmol), EDCI (90 mg, 0.46 mmol), HOBt (62 mg, 0.46 mmol), DMAP (152.7 mg, 0.92 mmol), and N-Boc-4-aminopiperidine (92 mg, 0.46 mmol) were placed in a 25 mL single-necked flask. Anhydrous DCM (10 mL) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete as determined by TLC, 20 mL of water was added, and the aqueous phase was extracted with DCM (10 mL × 3). The organic phases were combined. The organic phases were washed with water (10 mL) and saturated NaCl solution (10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and then purified by silica gel column chromatography to give compound Q94 (145 mg white solid, 95% yield).

[0606] Compound Q94 (145 mg, 0.22 mmol) was added to a 50 mL single-necked flask along with 15 mL of dichloromethane. Trifluoroacetic acid (0.048 mL, 0.66 mmol) was added dropwise, and the mixture was stirred at room temperature for 3 hours. TLC confirmed the reaction was complete. The mixture was directly concentrated to obtain a solid, and then 15 mL of THF, DMAP (7.3 mg, 0.044 mmol), and acetic anhydride (0.083 mL, 0.88 mmol) were added. The mixture was heated at 60 °C for 2 hours, and TLC confirmed the reaction was complete. The mixture was cooled to room temperature, and 10 mL of water and 10 mL of ethyl acetate were added. The mixture was extracted and separated. The aqueous phase was then extracted again with ethyl acetate (10 mL × 3), and the organic phases were combined. The organic phases were washed with water (10 mL × 3) and saturated sodium chloride solution (10 mL × 2), dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and then purified by silica gel column chromatography (PE:EA = 2:1) to obtain compound Q95 (93 mg white solid, 70% yield). 1H NMR (500MHz, CDCl3) δ7.87(s,1H),5.75(dd,J=5.2,2.2Hz,1H),5.38(d,J=18.1Hz,1H),4.53(d,J=19.7Hz,1H),4.0 3–3.94(m,1H),3.76(d,J=18.1Hz,1H),3.20–3.07(m,1H),2.79–2.74(m,1H),2.74–2.67(m,2H),2.64(s,3H),2.17 –2.10(m,4H),2.09(s,3H),2.06–2.02(m,2H),1.91(dd,J=16.7,9.4Hz,1H),1.85–1.77(m,1H),1.74–1.55(m,6H), 1.52(s,3H),1.48–1.37(m,4H),1.35(s,3H),1.31–1.00(m,15H),0.94(d,J=6.5Hz,3H),0.78(s,3H),0.68(s,3H). 13 C NMR(126MHz, CDCl3)δ172.56,168.92,162.92,149.25(2C),124.01,121.20, 119.30,56.86,55.73,49.12,46.50,45.31,42.25,40.56,39.70,38.57,37. 28,36.34,35.58,35.48,33.78,32.84,32.22,31.95,31.80,31.58,29.71,2 8.28,24.20,22.27,21.65,21.46,21.13,21.01,18.67,11.88.mp:110-111℃. HRMS(ESI)for C 37 H 56 N4O3[M+Na] + :calcd 627.4245,found 627.4245.

[0607] Example 61 Preparation of compound Q96

[0608] Similar to the synthesis of compound Q37, compound Q93 (110 mg, 0.23 mmol), EDCI (90 mg, 0.46 mmol), HOBt (62 mg, 0.46 mmol), DMAP (152.7 mg, 0.92 mmol), and morpholine (0.04 mL, 0.46 mmol) reacted to give compound Q96 (120 mg white solid, 95% yield). 1HNMR(500MHz, CDCl3) δ7.86(d,J=1.1Hz,1H),5.73(dd,J=5.2,2.3Hz,1H),3.64(d,J=4.1Hz,4H) ,3.60(d,J=5.2Hz,2H),3.48–3.39(m,2H),2.75(d,J=14.9Hz,1H),2.63(s,3H),2.32–2.19(m,2H ),2.16–2.07(m,2H),2.05–1.99(m,1H),1.83–1.75(m,1H),1.73–1.54(m,5H),1.50(s,3H),1.4 7–1.36(m,3H),1.33(s,3H),1.27–1.01(m,8H),0.94(d,J=6.5Hz,3H),0.77(s,3H),0.67(s,3H). 13 C NMR (126MHz, CDCl3) δ171.90,169.59,162.90,149.26,124.01,121.20,119.29,66.96,66.69,56.86,55.74,49.13,46.05,42.25,41.88,39.7 0,38.56,36.32,35.73,35.64,33.78,33.57,32.21,31.94,31.79,31.5 8,28.27,24.20,21.74,21.62,21.13,21.00,18.68,11.88.mp:90-91℃. HRMS(ESI)for C 34 H 51 N3O3[M+Na] + :calcd 572.3823, found 572.3822.

[0609] Example 62 Preparation of compounds Q97-Q98

[0610] Similar to the synthesis of Q24-Q25, compound Q23 (1.07 g, 2.41 mmol), 30 mL of ethyl formate, and NaH (578 mg, 24.1 mmol) react to give a pale yellow oily substance Q97.

[0611] Then, compound Q97 (the oily substance from the previous step), 30 mL of glacial acetic acid, potassium acetate (473 mg, 4.82 mmol), and 85% hydrazine hydrate (0.42 mL, 8.51 mmol) were heated under reflux to give compound Q98 (1.03 g of white solid, yield 91.8%). 13CNMR(126MHz, CDCl3)δ173.96,150.01(2C),120.89(2C),112.87,60.18,56.96,56.01,49.40,42.25,39.81,38.88,35.61 ,35.55,35.50,34.47,33.35,32.70,32.42,32.12,31.64,28.28,25.64,25.45,24.24,21.30,21.07,18.66,14.27,11.89.

[0612] Example 62 Preparation of compound Q99

[0613] Similar to the synthesis of Q26, compound Q98 (103 mg, 0.221 mmol) and lithium hydroxide (160 mg, 6.64 mmol) were hydrolyzed to give compound Q99 (103 mg white solid, 99% yield). 1 H NMR(500MHz,MeOD)δ7.90(s,1H),5.97–5.85(m,1H),2.91(d,J=15.0Hz,1H),2.31–2.10(m,5H),1.93– 1.86(m,1H),1.78–1.59(m,5H),1.57(s,3H),1.55–1.50(m,1H),1.44(s,4H),1.42–1.39(m,1H),1.38–

[0614] 1.04(m,11H),0.97(d,J=6.5Hz,3H),0.90(s,3H),0.79–0.71(m,3H).

[0615] Example 63 Preparation of compound Q100

[0616] Similar to the synthesis of compound Q27, compound Q99 (100 mg, 0.23 mmol), DMAP (7.8 mg, 0.046 mmol), 20 mL THF, and acetic anhydride (0.07 mL, 0.69 mmol) were heated to give compound Q100 (102 mg white solid, 90% yield). 13C NMR (101MHz, CDCl3) δ179.55,169.68,162.98,149.24,124.05,121.24,119.35,56.87,56.01,49.15,42.26,39.73,38.58,36 .34,35.61,35.52,34.08,33.78,32.23,31.97,31.81,31.59,28.28,25.61,25.15,24.22,21.64,21.15,21.01,18.65,11.90.

[0617] Example 64 Preparation of compound Q101

[0618] Similar to the synthesis of compound Q37, compound Q100 (102 mg, 0.21 mmol), EDCI (80 mg, 0.42 mmol), HOBt (57 mg, 0.42 mmol), DMAP (139.4 mg, 0.84 mmol), and morpholine (0.04 mL, 0.42 mmol) underwent a condensation reaction to yield compound Q101 (112 mg white solid, 95% yield). 1 HNMR (500MHz, CDCl3) δ7.86 (d, J=4.8Hz, 1H), 5.74 (dd, J=5.2, 2.3Hz, 1H), 3.68–3.64 (m, 4H), 3.61 (d, J=5. 1Hz,2H),3.51–3.41(m,2H),2.76(d,J=14.9Hz,1H),2.64(s,3H),2.30(td,J=15.2,8.8,5.5Hz,2H),2.18–

[0619] 2.09(m,2H),2.08–2.01(m,1H),1.88–1.81(m,1H),1.75–1.55(m,6H),1.51(s,3H),1.48–

[0620] 1.38(m,3H),1.34(s,3H),1.28–1.00(m,9H),0.92(d,J=6.5Hz,3H),0.78(s,3H),0.67(s,3H). 13C NMR (126MHz, CDCl3) δ171.92,169.64,162.93,149.26,124.01,121.23,1 19.31,66.98,66.71,56.89,56.04,49.16,46.09,42.26,41.88,39.74,38 .58,36.34,35.71,35.63,33.78,33.22,32.23,31.97,31.80,31.59,28.3 0,26.00,25.73,24.21,21.63,21.15,21.01,18.68,11.90.mp:105-106℃. HRMS(ESI) for C 35 H 53 N3O3[M+Na] + :calcd586.3979,found 586.3981.

[0621] Example 65 Preparation of compounds Q102-Q106

[0622] Similar to the synthesis of Q24 to Q26, compound Q13 (400 mg, 1.0 mmol), 30 mL of ethyl formate, and NaH (240 mg, 10.0 mmol) react to give a pale yellow oily substance Q102.

[0623] Then, compound Q102 (the oily substance from the previous step), 20 mL of glacial acetic acid, potassium acetate (196.2 mg, 2.0 mmol), and 85% hydrazine hydrate (0.15 mL, 3.0 mmol) were heated under reflux to give compound Q103 (386 mg of white solid, 91% yield).

[0624] Compound Q103 (386 mg, 0.91 mmol) and lithium hydroxide (655 mg, 27.3 mmol) were hydrolyzed to give compound Q104 (380 mg white solid, 99% yield).

[0625] Similar to the synthesis of compound Q92, compound Q104 (380 mg, 0.91 mmol), DMAP (30 mg, 0.182 mmol), 30 mL THF, and isobutyric anhydride (0.37 mL, 3.64 mmol) were heated to give compound Q105 (393 mg white solid, 90% yield).

[0626] Similar to the synthesis of compound Q37, compound Q105 (393 mg, 0.82 mmol), EDCI (312 mg, 1.64 mmol), HOBt (222 mg, 1.64 mmol), DMAP (543.7 mg, 3.28 mmol), and morpholine (0.16 mL, 1.64 mmol) reacted to give compound Q106 (white solid, 427.7 mg, yield 95%). 1 H NMR(500MHz, CDCl3)δ7.86(d,J=4.4Hz,1H),5.74(dd,J=5.2,2.2Hz,1H),3.91–3.80(m,1H),3.71–3.57(m ,6H),3.48(s,2H),2.76(d,J=15Hz,1H),2.39(d,J=11.9Hz,1H),2.18–2.10(m,2H),2.09–1.97(m,3H),1.8 9–1.80(m,1H),1.73–1.55(m,4H),1.51(s,3H),1.49–1.38(m,1H),1.35(s,3H),1.34–1.30(m,1H),1.27(d ,J=1.7Hz,3H),1.26(d,J=1.7Hz,3H),1.22–1.03(m,5H),1.00(d,J=6.0Hz,3H),0.79(s,3H),0.74(s,3H). 13 C NMR (126MHz, CDCl3) δ176.17,171.53,162.45,149.39,124.22,121.06,118.78,67.04,66.72,56.92,56.46,49.11(2C),46.34,42.48,41.96, 39.66,39.62,38.59,36.31,33.86,33.74,32.29,31.94,31.91,31.59, 28.39,24.19,21.11,21.03,19.62,19.34,18.98,11.96.mp:230-231℃. HRMS(ESI)for C 34 H 51 N3O3[M+Na] + :calcd 572.3823, found 572.3825.

[0627] Example 66 Preparation of compounds Q107-Q111

[0628] The synthesis of compounds Q107 to Q111 uses Q9 as the starting material and is similar to the synthesis of compounds Q102 to Q106.1 H NMR (500MHz, CDCl3) δ7.86 (s, 1H), 5.74 (dd, J = 5.1, 2.1Hz, 1H), 3.91–3.80 (m, 1H), 3. 72–3.50(m,8H),2.76(d,J=14.9Hz,1H),2.72–2.68(m,1H),2.17–2.08(m,2H),2.04–

[0629] 1.95(m,1H),1.88–1.80(m,2H),1.76–1.54(m,5H),1.51(s,3H),1.49–1.36(m,2H),1.35(s,3H),1.27(s,3H), 1.26(s,3H),1.20(dd,J=11.0,3.9Hz,1H),1.16(d,J=6.7Hz,3H),1.13–1.08(m,2H),0.79(s,3H),0.73(s,3H). 13 C NMR (126MHz, CDCl3) δ176.19,175.36,162.47,149.32,124.23,121.10,118.79,67.12,66.89,56.30,52.78,49.05,46.32,42.18,42.07, 39.54(2C),38.58,36.31,33.76,32.26,31.91(2C),31.87,31.59,27.87,24.29,21.08,21.06,19.35,18.96,17.42,12.30.mp:248-249℃. HRMS(ESI)forC 33 H 49 N3O3[M+Na] + :calcd 558.3666,found 558.3670. Example 67: Activity test of bile acid derivatives in degrading HMGCR-GFP

[0630] The following method was used to determine the HMGCR degradation activity of the compounds prepared in the embodiments of the present invention using the CHO-7 / HMGCR-GFP cell line. The CHO-7 / HMGCR-GFP cell line was formed by stably transfecting Chinese hamster ovarian epithelial cells CHO-7 with the expression of the HMGCR-GFP fusion protein.

[0631] The experimental method is briefly described as follows: CHO-7 / HMGCR-GFP cells were cultured in DMEM / F12 (1:1) medium (containing 5% FBS, 100 units / ml Penicillin-Streptomycin). When the cell confluence reached 80%, the cells were digested with 0.25% trypsin (containing EDTA) and then diffused into 3 × 10⁻⁶ cells per well. 4 Cells were seeded into 24-well plates. After culturing at 37°C and 5% CO2 for 24 hours, the culture medium was aspirated, and the cells were washed once with PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4). Then, delipoprotein DMEM / F12 medium (containing 5% delipoprotein serum, 1μM lovastatin, 10μM mevalonic acid, 100 units / ml penicillin-streptomycin) containing different concentrations of the compounds prepared in the embodiments of this invention was added. The statin helps to maximize the induction of HMGCR accumulation. After culturing for 16 hours, the culture medium was aspirated, and 150μl of Reporter Lysis Buffer (Promega, E397A) was added to each well. The 24-well plates were frozen at -80°C, thawed once at room temperature, and then vortexed at 800 rpm for 10 minutes to achieve complete cell lysis.

[0632] 100 μl of lysis buffer was added to a 96-well black microplate (PerkinElmer, 6005329). The fluorescence intensity of green fluorescent protein (EGFP) was measured using an EnVsion microplate reader (PerkinElmer) with excitation at 485 nm and emission at 535 nm. The compound prepared according to the embodiments of the present invention was used with concentration gradients of 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM, normalized to 100% with the untreated DMSO group. The half-maximal inhibitory concentration (EC50) was calculated using GraphPad Prism software. The activity of the compound in degrading HMGCR was determined using the above method, and the degradation effect is shown in Table 1.

[0633] Table 1. Activity data of the compounds prepared in the embodiments of the present invention for degrading HMGCR-GFP.

[0634]

[0635]

[0636]

[0637]

[0638]

[0639] As can be seen from the data in Table 1, the compounds prepared in the embodiments of the present invention all have significant effects on the degradation of HMGCR, among which the EC50 of the most active compounds Q37 and Q54 are 0.49 μM and 0.45 μM, respectively.

[0640] Example 68: Experiment on the degradation of HMGCR by the compound prepared according to the embodiment of the present invention.

[0641] The following method was used to determine the degradation of HMGCR by the compound prepared according to embodiments of the present invention. The experimental method is briefly described below: CHO-7 cells were incubated at 6 × 10⁻⁶ cells / day. 5 Transfer the cells to 60mm culture dishes and incubate for 1 day. Remove the culture medium, wash once with PBS, remove the PBS, and add DMEM / F12 medium containing different concentrations of the compounds prepared in the embodiments of this invention (containing 5% delipoprotein and fatty acid-free serum, 1μM lovastatin, 10μM mevalonic acid, 100 units / ml penicillin-streptomycin). Treat for 16 hours. Scrape the cells from the culture dish with a cell scraper, transfer to a 15ml centrifuge tube, and place on ice. Centrifuge at 1000g for 5 minutes at 4°C, discard the culture medium, add 1ml PBS buffer, transfer to a 1.5ml centrifuge tube, and centrifuge at 1000g for 5 minutes at 4°C. Remove the PBS, add 300μl SDS Lysis Buffer (10mM Tris-HCl (pH 7.6), 100mM sodium chloride, 1% sodium dodecyl sulfate, and protease inhibitor), and pass through a No. 7 needle 15 times. Centrifuge at 13200 rpm at room temperature for 10 minutes, transfer 270 μl of supernatant to a new 1.5 ml centrifuge tube, add 90 μl of 4x Loading Buffer (1.2% sodium dodecyl sulfate, 30% glycerol, 150 mM Tris-HCl (pH 6.8), 0.6% β-mercaptoethanol and appropriate amount of bromophenol blue), mix well, boil at 95°C for 10 minutes, and store in a -20°C freezer.

[0642] Western blot detection of HMGCR degradation

[0643] 1) Sample loading: Load 30 μl of each protein sample into the wells of an 8% SDS-PAGE gel.

[0644] 2) Electrophoresis: First, use 85 volts for 30 minutes to allow the protein to enter the stacking gel, and then use 120 volts for 1 hour to allow the protein to enter the separating gel.

[0645] 3) Transfer: After electrophoresis, remove the gel and place it into the transfer apparatus in the order of filter paper, nitrocellulose membrane, gel, and filter paper, taking care to remove air bubbles. Transfer at a constant voltage of 100 volts for 1.5 hours.

[0646] 4) Blocking: After the transfer, the nitrocellulose membrane was immersed in TBST buffer (20mM Tris-HCl (pH 7.4), 150mM sodium chloride and 0.05% Tween-20) containing 5% skim milk powder and incubated on a shaker at room temperature for 1 hour.

[0647] 5) Primary antibody incubation: After blocking, wash the membrane three times with TBST buffer. Add 4 μg / ml of HMGCR antibody (IgG-A9, a mouse monoclonal antibody that recognizes hamster HMGCR) and incubate at room temperature for 2 hours or at 4°C overnight. Recover the antibody and wash the membrane four times with TBST for 8 minutes each time.

[0648] 6) Secondary antibody incubation: Discard TBST, add goat anti-mouse HRP-conjugated secondary antibody diluted 1:5000, and incubate at room temperature for 1 hour. Wash 4 times with TBST, 8 minutes each time.

[0649] 7) Color development and photography: Cover the ECL luminescent substrate onto the nitrocellulose membrane, seal it with plastic wrap, and take pictures using the LAS4000 cold light / bioluminescence imaging analyzer (Fujifilm).

[0650] 8) Quantitative analysis of HMGCR protein: The obtained grayscale images were quantitatively analyzed using ImageJ (NIH) software, with the untreated group as 100%.

[0651] The measurement results are as follows Figure 2 As shown in a and c: The compounds prepared in the embodiments of the present invention can significantly degrade HMGCR. Figure 2 The quantitative analysis results of b and d show that the compounds prepared in the embodiments of the present invention have very strong activity in degrading HMGCR, with EC50 values ​​of 0.22 μM and 0.26 μM for Q37 and Q54, respectively.

[0652] Example 69: Detection of intracellular cholesterol accumulation

[0653] The following method was used to determine the HMGCR degradation activity of the compounds prepared in the embodiments of the present invention using the CHO-7 / HMGCR-GFP cell line. The CHO-7 / HMGCR-GFP cell line was formed by stably transfecting Chinese hamster ovarian epithelial cells CHO-7 with the expression of the HMGCR-GFP fusion protein.

[0654] The experimental method is briefly described as follows: CHO-7 / HMGCR-GFP cells were cultured in DMEM / F12 (1:1) medium (containing 5% FBS, 100 units / ml Penicillin-Streptomycin). When the cell confluence reached 80%, the cells were digested with 0.25% trypsin (containing EDTA) and then diffused into 3 × 10⁻⁶ cells per well. 4 Cells were seeded into 12-well plates. After incubation at 37°C and 5% CO2 for 24 hours, the culture medium was aspirated and the cells were washed once with PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4).

[0655] Add 1 ml of delipoprotein DMEM / F12 medium (containing 10% delipoprotein serum, 1 μM lovastatin, 10 μM mevalonic acid, and 100 units / ml penicillin-streptomycin) containing 0 μM, 1 μM, and 10 μM concentrations of the compounds prepared in the embodiments of this invention. Incubate at 37°C in a 5% CO2 incubator for 16 hours. Remove the medium, wash once with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), fix with 4% paraformaldehyde (PFA) for 30 minutes, and then rinse with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Wash three times with KH2PO4, then dilute the ethanol-dissolved Filipin stock solution (5 mg / ml) with PBS containing 10% FBS to a final concentration of 50 μg / ml, and incubate at room temperature in the dark for 30 minutes. Wash three times with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), and twice with double-distilled water. Mount the slide, air dry overnight, and then observe intracellular cholesterol accumulation under a microscope (Zeiss Z2 wide-field microscope (non-confocal) DAPI channel). Figure 3 As shown, no cholesterol accumulation was detected in cells after treatment with compounds Q37 and Q46.

[0656] The bile acid derivatives proposed in this invention, as compounds that lower cholesterol and triglycerides in the body, have potential drug research value in the prevention and / or treatment of hypercholesterolemia, hypertriglyceridemia, atherosclerosis, and non-alcoholic steatohepatitis. They provide new ideas for finding novel drugs for the prevention and / or treatment of hypercholesterolemia, hypertriglyceridemia, atherosclerosis, and non-alcoholic steatohepatitis.

[0657] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing a bile acid derivative, characterized in that, Starting with compound Q68, cholic acid derivative Q74 was obtained by ester condensation and cyclization; the preparation method is shown in route (7): ； Route (7).

2. The preparation method according to claim 1, characterized in that, Starting with compound Q7, the bile acid derivative Q68 was obtained by hydrolysis and acylation; the preparation method is shown in route (5): ； Route (5).

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