CN111205346B - Oleanolic acid derivative and medical application thereof - Google Patents

Abstract

The invention discloses a novel pentacyclic triterpenoid AMPK agonist oleanolic acid derivative and a medical application thereof, in particular to a compound shown as a formula I or a formula II or a pharmaceutically acceptable salt, ester or solvate thereof, which can be used for preparing an AMPK agonist with activity of enhancing AMPK phosphorylation level and a medicine for preventing or treating AMPK mediated diseases. The novel pentacyclic triterpenoid has obvious AMPK agonistic activity, and the activity of the novel pentacyclic triterpenoid is obviously superior to that of a recognized AMPK agonistAICAR has better pharmacokinetic properties such as oral bioavailability and the like and very good safety.

Description

Oleanolic acid derivative and medical application thereof

Technical Field

The invention relates to the field of biomedicine, relates to a novel pentacyclic triterpenoid compound with AMPK agonistic activity, and particularly relates to an oleanolic acid derivative and medical application thereof, and also relates to application of the compound in preparing a medicine for preventing or treating AMPK-mediated diseases and a pharmaceutical composition of the compound.

Background

AMPK (adenylate activated protein kinase) is a key kinase for regulating and controlling body energy metabolism and inflammatory reaction, and phosphorylation activation of AMPK can overcome insulin resistance, reduce blood sugar, reduce blood fat (by inhibiting synthesis of fatty acid and cholesterol), resist inflammation, apoptosis, fibrosis, promote mitochondrial synthesis, enhance oxidative metabolism of mitochondria, resist aging, and resist tumors (physiol. rev.2009,89,1025). In recent years, the anti-inflammatory and anti-fibrotic effects of AMPK have been receiving increasing attention (Nature 2013,493,346), and a possible mechanism thereof is that AMPK exerts anti-inflammatory and anti-fibrotic effects by enhancing the transcriptional function of estrogen-related receptor α (ERR α) (Immunity 2015,43, 80).

There is increasing evidence that AMPK dysfunction is closely associated with the development and progression of a variety of diseases. AMPK-mediated diseases include metabolic diseases and cardiovascular and cerebrovascular diseases such as insulin resistance, metabolic syndrome, type 1 or type 2 diabetes mellitus, hyperlipidemia, obesity, atherosclerosis, myocardial ischemia, myocardial infarction, arrhythmia, coronary heart disease, hypertension, heart failure, myocardial hypertrophy, myocarditis, diabetic complications (including diabetic cardiomyopathy, diabetic nephropathy, retinopathy, neuropathy, diabetic ulcer and the like), non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic fatty liver, liver cirrhosis, gout, stroke, cerebral infarction and the like; AMPK-mediated diseases also include inflammatory diseases, autoimmune diseases, organ fibrotic diseases, nerve injury diseases or secondary diseases caused by infection with pathogens, such as pneumonia, asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, bronchiolitis obliterans, idiopathic pulmonary fibrosis, cystic fibrosis lung diseases, allergic rhinitis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), polycystic kidney disease, polycystic ovary syndrome (PCOS), Behcet's disease, systemic lupus erythematosus, rheumatoid arthritis, spondyloarthritis, osteoarthritis, synovitis, tendonitis, thromboangiitis obliterans, phlebitis, intermittent claudication, keloid, psoriasis, ichthyosis, bullous pemphigoid, dermatitis, contact dermatitis, pancreatitis, chronic nephritis, cystitis, meningitis, gastritis, septicemia, pyoderma gangrenosum, psoriasis, chronic nephritis, cystitis, meningitis, and other diseases, Uveitis, Parkinson's disease, Alzheimer's disease, alpha-synucleinopathy, depression, multiple sclerosis, amyotrophic lateral sclerosis, fibromyalgia syndrome, neuralgia, Down's syndrome, Hallervorden-Spatkis disease, Huntington's chorea or Wilson's disease, etc.

AMPK agonists have been reported in the literature to prevent and treat a variety of AMPK-mediated diseases (j.med. chem.,2015,582; Nature 2013,493,346; Experimental Neurology 2017,298, 31; Biochemical Pharmacology 2010,80, 1708; Current Drug Targets,2016,17, 908; Nat Rev Drug Discov,2019, DOI:10.1038/s 41573-019-. For example, metformin, a hypoglycemic agent widely used clinically, is thought to exert various clinical effects mainly by activating AMPK (j.clin.invest.2001,108, 1167). Despite the wide clinical potential of AMPK agonists, however, substantial progress has not been made to develop novel, safe and effective AMPK agonists to date. For reasons of safety or efficacy, AMPK agonists under investigation have entered the clinical study stage for a very limited number of reasons. For example, the broad spectrum AMPK-beta subunit agonist MK-8722, although lowering blood glucose, was found to have irreversible cardiac hypertrophy side effects in the animal heart in rat and monkey experiments (Science 2017,357,507). In addition, AICAR, one of the most commonly used AMPK agonists (Eur. J. biochem.1995,229,558), has also been terminated in clinical trials due to its large toxic side effects (J Clin Pharmacol 1991,31: 342-347).

In conclusion, the development of novel AMPK agonists with high activity and low toxic and side effects is urgently needed in clinic. On the other hand, oleanolic acid is a pentacyclic triterpene commonly found in medicinal plants, which has a wide range of biological activities (Nat Prod Rep 2011,28, 543). The method has great significance in the field of biological medicine for researching pentacyclic triterpenoid derivatives with stronger activity and novel structures, such as oleanolic acid derivatives.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art in the field of AMPK agonist development, the invention provides a novel oleanolic acid derivative; the novel oleanolic acid derivative provided by the invention is a novel AMPK agonist, so that the novel oleanolic acid derivative can be used for preparing a medicine for preventing or treating AMPK-mediated diseases.

The invention unexpectedly discovers a series of potent novel AMPK agonists when the structure of the oleanolic acid is modified, and the AMPK agonistic activity of the novel AMPK agonists is obviously superior to that of the accepted AICAR which is an AMPK agonist.

The technical scheme is as follows: in order to achieve the above objects, the oleanolic acid derivatives represented by the following formula I or formula II or pharmaceutically acceptable salts or esters or solvates thereof according to the present invention:

wherein,

r is H,

R^aCO-or PO₃H₂；

R¹Is H, C₁-C₅C substituted by alkyl or by substituent Y₁-C₅Alkyl, the substituent Y is OH, C (O) NH₂、NH₂、NHC(O)CH₃Pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, 4-methyl-piperazin-1-yl, morpholin-4-yl, thiomorpholin-1, 1-dioxo-4-yl, N-dimethylamino, N-diethylamino, trimethylammonium, or diethanolamino;

R^ac unsubstituted or substituted by substituents L₁-C₅Alkyl, said substituent L being one or two substituents independently selected from the group consisting of: OH, C (O) OH, NH₂Pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, 4-methyl-piperazin-1-yl, morpholin-4-yl, thiomorpholin-1, 1-dioxo-4-yl or NHC (O) CH (CH)₃)NHC(O)CH(CH₃)NH。

In certain preferred embodiments, the compound, or a pharmaceutically acceptable salt or ester or solvate thereof, is selected from the group consisting of:

the compounds of the present invention may also be used as pharmaceutically acceptable salts. The salt may be an acid salt of at least one of the following acids: galactaric acid, D-glucuronic acid, glycerophosphoric acid, hippuric acid, isethionic acid, lactobionic acid, maleic acid, 1, 5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, pivalic acid, terephthalic acid, thiocyanic acid, cholic acid, n-dodecylsulfuric acid, benzenesulfonic acid, citric acid, D-glucose, glycolic acid, lactic acid, malic acid, malonic acid, mandelic acid, phosphoric acid, propionic acid, hydrochloric acid, sulfuric acid, tartaric acid, succinic acid, formic acid, hydroiodic acid, hydrobromic acid, methanesulfonic acid, nicotinic acid, nitric acid, orotic acid, oxalic acid, picric acid, L-pyroglutamic acid, saccharinic acid, salicylic acid, gentisic acid, p-toluenesulfonic acid, valeric acid, palmitic acid, sebacic acid, stearic acid, lauric acid, acetic acid, adipic acid, carbonic acid, benzenesulfonic acid, ethanedisulfonic acid, ethylsuccinic acid, fumaric acid, 3-hydroxynaphthalene-2-carboxylic acid, 1-hydroxynaphthalene-2-carboxylic acid, oleic acid, undecylenic acid, ascorbic acid, camphoric acid, camphorsulfonic acid, dichloroacetic acid, ethanesulfonic acid. Alternatively, the salt may be a salt of a compound of the present invention with a metal ion (including sodium, potassium, calcium, etc.) or a pharmaceutically acceptable amine (including ethylenediamine, tromethamine, etc.), an ammonium ion or choline. The compounds of the present invention may also be comprised in pharmaceutical compositions in the form of esters, prodrugs, N-oxides or solvates thereof.

The invention provides application of the compound shown in the formula I or the formula II or pharmaceutically acceptable salt, ester or solvate thereof in preparing AMPK agonist with AMPK phosphorylation level enhancing activity. The compounds of the present invention have significant agonistic activity against AMPK, and thus may be used to prepare AMPK agonists having activity of enhancing phosphorylation level of AMPK.

The invention also provides application of the compound shown in the formula I or the formula II or pharmaceutically acceptable salt, ester or solvate thereof in preparing a medicament for preventing or treating AMPK mediated diseases.

Wherein the AMPK-mediated disease comprises a metabolic disease, a cardiovascular disease, a cerebrovascular disease, an inflammatory disease, an autoimmune disease, an organ fibrosis disease, a neurodegenerative disease, a secondary disease caused by pathogen infection, a mitochondrial dysfunction or disorder disease or a tumor.

The AMPK mediated diseases, such as metabolic diseases and cardiovascular and cerebrovascular diseases, comprise: such as insulin resistance, metabolic syndrome, type 1 or type 2 diabetes, hyperlipidemia, obesity, atherosclerosis, myocardial ischemia, myocardial infarction, arrhythmia, coronary heart disease, hypertension, heart failure, myocardial hypertrophy, myocarditis, diabetic complications (including diabetic cardiomyopathy, diabetic nephropathy, retinopathy, neuropathy, diabetic ulcer and the like), non-alcoholic fatty liver, non-alcoholic steatohepatitis, alcoholic fatty liver, liver cirrhosis, gout, stroke or cerebral infarction and the like.

The AMPK-mediated diseases, such as inflammatory diseases, autoimmune diseases, organ fibrosis diseases, nerve injury diseases or secondary diseases caused by infection with pathogens, include: pneumonia, asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, bronchiolitis obliterans, idiopathic pulmonary fibrosis, cystic fibrosis lung disease, allergic rhinitis, inflammatory bowel disease (such as crohn's disease and ulcerative colitis), polycystic kidney disease, polycystic ovary syndrome (PCOS), behcet's disease, systemic lupus erythematosus, rheumatoid arthritis, spondyloarthritis, osteoarthritis, synovitis, tendonitis, thromboangiitis obliterans, phlebitis, intermittent claudication, keloid, psoriasis, ichthyosis, bullous pemphigoid, dermatitis, contact dermatitis, pancreatitis, chronic nephritis, cystitis, meningitis, gastritis, septicemia, pyoderma gangrenosum, uveitis, parkinson's disease, alzheimer's disease, alpha-synucleinopathy, depression, multiple sclerosis, amyotrophic lateral sclerosis, fibromyalgia syndrome, Neuralgia, Down's syndrome, Harlervorden-Spatz disease, Huntington's chorea or Wilson's disease, etc.

Such AMPK-mediated diseases, such as mitochondrial dysfunction and disorder diseases, include: myasthenia, myoclonus, exercise intolerance, cahns-seire syndrome, chronic fatigue syndrome, li's syndrome, mitochondrial myopathy-encephalopathy-hyperlactacidemia, stroke syndrome, or stroke-like episodes. Likewise, the compounds of the invention may also be useful in the treatment of muscular dystrophy states, for example, duchenne muscular dystrophy, conchal muscular dystrophy or friedrich's ataxia.

Such AMPK-mediated diseases, such as tumors, include: bone cancer, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, myelodysplastic syndrome, hodgkin's lymphoma, non-hodgkin's lymphoma, hemangioma, granuloma, xanthoma, meningiosarcoma, glioma, astrocytoma, medulloblastoma, ependymoma, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, fibroma, sarcoma, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, colon cancer, rectal cancer, renal cancer, prostate cancer, lymphatic cancer, testicular cancer, interstitial cell cancer, lung cancer, liver cancer, skin cancer, malignant melanoma, basal cell carcinoma, and the like.

The invention relates to a pharmaceutical composition with AMPK phosphorylation level enhancing activity, which comprises a therapeutically effective amount of a compound shown in formula I or formula II or a pharmaceutically acceptable salt, ester or solvate thereof serving as an active ingredient and a pharmaceutically acceptable auxiliary material.

The invention relates to a pharmaceutical composition for preventing or treating AMPK-mediated diseases, which comprises a therapeutically effective amount of a compound shown in formula I or formula II or a pharmaceutically acceptable salt, ester or solvate thereof serving as an active ingredient and a pharmaceutically acceptable auxiliary material.

The above-mentioned adjuvants which can be arbitrarily mixed may be changed depending on the dosage form, administration form and the like. Examples of the auxiliary materials include excipients, binders, disintegrating agents, lubricants, flavoring agents, coloring agents, sweetening agents, and the like. The pharmaceutical composition can be in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches and other pharmaceutically conventional preparations.

If desired, the compounds of formula I or formula II or pharmaceutically acceptable salts or esters or solvates thereof of the present invention may be used in combination with one or more other types of agents for the prophylaxis or treatment of AMPK-mediated diseases, including but not limited to the following combinations.

The drug optionally used in combination with the compound of the present invention may be one or more antidiabetic drugs including metformin, sulfonylurea hypoglycemic agents (e.g., glyburide and glimepiride, etc.), glucosidase inhibitors (e.g., acarbose and miglitol, etc.), PPAR γ agonists (e.g., pioglitazone and rosiglitazone), PPAR α/γ dual agonists, dipeptidyl peptidase IV (DPP-IV) inhibitors (e.g., sitagliptin, saxagliptin, alogliptin and linagliptin, etc.), glinide hypoglycemic agents (e.g., repaglinide and nateglinide, etc.), SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin, engagliflozin, eggliflozin, and troglifloxagliflozin, etc.), glucokinase agonists (e.g., HMS5552, etc.), insulin, glucagon-like peptide-1 (GLP-1) class drugs (e.g., exenatide, gliflozin, and gliflozin, etc.) Liraglutide, lissamide, dolabrupeptide, benalotide, and albiglutide, etc.), a PTP1B inhibitor, a glycogen phosphorylase inhibitor, a glucose-6-phosphatase inhibitor, an AMPK agonist, a GPR40 agonist, or a GPR120 agonist.

The drug optionally used in combination with the compound of the present invention may be one or more of anti-obesity drugs including lorcaserin, orlistat and glucagon-like peptide-1 (GLP-1) drugs (e.g., exenatide, liraglutide, dolabrupeptide, benalolutide, and abiglutide, etc.), and the like.

The drug selected for use in combination with the compounds of the present invention may be one or more anti-non-alcoholic fatty liver disease drugs, including: AMPK agonists (e.g., metformin), Farnesoid X Receptor (FXR) agonists (e.g., obeticholic acid, GS-9674, EDP-305 and LJN452, etc.), acetyl-CoA carboxylase (ACC) inhibitors (e.g., GS-0976, etc.), apoptosis signal-regulating kinase-1 (ASK1) inhibitors (e.g., Selosertib, etc.), PPAR agonists (e.g., Elafibranor, Saroglitazar, IVA337 and MSDC-0602K, etc.), caspase inhibitors (e.g., Emricasan, etc.), stearoyl-CoA desaturase 1(SCD1) inhibitors (e.g., Aramchol, etc.), long-acting glucagon-like peptide-1 (GLP-1) receptor agonists (e.g., Semaglutide, etc.), apical sodium-dependent bile salt transporter (ASBT) inhibitors (e.g., Volixibat, etc.), vascular adhesion protein 1(VAP-1) inhibitors (e.g., BI 1467335, etc.), CCR5R blockers (e.g., Cenicriviroc, etc.), and thyroid hormone receptor beta (THR-beta) agonists (e.g., MGL-3196, etc.), among others.

The drug which can be optionally used in combination with the compound of the present invention may be one or more hypolipidemic drugs, including nicotinic acid, statins (e.g., lovastatin, simvastatin, pravastatin, mevastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin and pitavastatin), cholesterol absorption inhibitors (e.g., ezetimibe, etc.), fibrates (e.g., clofibrate, bezafibrate, fenofibrate, etc.), PCSK9 inhibitors (e.g., evorocumab, Alirocumab, etc.), CETP inhibitors (e.g., anacetrapib, etc.), AMPK agonists and ACC inhibitors (e.g., GS-0976, etc.), and the like.

The amount of the compound of formula I or formula II of the present invention or a pharmaceutically acceptable salt or ester or solvate thereof may be appropriately changed depending on the age, body weight, symptoms and administration route of the patient, etc. When administered to an adult (about 60kg), the compound of formula I or formula II or a pharmaceutically acceptable salt or ester or solvate thereof is administered in an amount of 1mg to 1000mg, preferably 5mg to 500mg, more preferably 10mg to 60mg per time, 1 to 3 times per day. This dosage range may also vary depending on the degree of disease and dosage form.

Oleanolic acid used in the present invention can be commercially available. The synthesis of oleanolic acid derivatives can be carried out by referring to the methods of examples or modified methods.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the novel oleanolic acid derivative has strong AMPK agonistic activity, and the activity of the novel oleanolic acid derivative is obviously superior to that of a recognized AMPK agonist AICAR.

(2) Compared with the lead compound oleanolic acid, the novel pentacyclic triterpenoid compound has better pharmacokinetic properties such as oral bioavailability and the like.

(3) Compared with the existing AMPK agonist AICAR (clinical test is stopped due to large toxic and side effects) and MK-8722 (side effects of irreversible cardiac hypertrophy can be caused), the novel pentacyclic triterpenoid compound has very good safety.

(4) The novel oleanolic acid derivative serving as the AMPK agonist has the advantages of low cost, easiness in preparation, small potential side effect and the like compared with the existing AMPK agonist, can be used independently, can also be used together with one or more other types of drugs for preventing or treating AMPK-mediated diseases, and is expected to become a novel drug for preventing or treating AMPK-mediated diseases.

Drawings

FIG. 1 is a single crystal diffractogram of Compound B-1 in the example;

FIG. 2 is a graph of AMPK agonistic activity of some compounds on Huh-7 cells (Western Blot assay).

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are given for better illustration of the present invention and are not intended to limit the scope of the present invention. Various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

Example 1

12-ene-3 beta-acetoxy-28- (oxazolin-2-yl) -oleanane (Compound A-2)

Oleanolic acid (OA, 10g, 21.9mmol) was dissolved in pyridine (150mL), 4-dimethylaminopyridine (0.26g, 2.19mmol) was added, acetic anhydride (8.3mL, 87.6mmol) was added slowly, and the mixture was stirred at room temperature overnight. After completion of the reaction, 1N hydrochloric acid (300mL) was added, and extraction was performed with ethyl acetate (300mL × 3), and the mixture was washed with saturated brine (300mL × 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and filtered with suction (petroleum ether: dichloromethane 50: 1) to obtain compound I-1 (white solid, 8.2g, yield 75%).

Compound I-1(5g, 10mmol) was dissolved in anhydrous dichloromethane (80mL), and oxalyl chloride (1.7mL, 20mmol) and N, N-dimethylformamide (5 drops) were slowly added dropwise with stirring and reacted at room temperature for 5 hours. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure to obtain Compound I-2 (yellow solid, 5.17g, yield 100%).

Compound I-2(5.17g, 10mmol) was dissolved in anhydrous dichloromethane (80mL), 2-bromoethylamine hydrobromide (4g, 20mmol) was added, triethylamine (5.5mL, 40mmol) was dissolved in anhydrous dichloromethane (20mL), and the triethylamine solution in dichloromethane was slowly added dropwise to the reaction using a constant pressure dropping funnel. Stir at room temperature overnight. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, water (300mL) was added and extracted with ethyl acetate (300 mL. times.3), and the mixture was washed with saturated brine (300 mL. times.3), and the filtrate was concentrated and dried to obtain a crude compound I-3 which was used as it was in the next reaction.

The crude compound I-3 obtained in the previous step was dissolved in N, N-dimethylformamide (100mL), and potassium carbonate (2.7g, 20mml) was added thereto, followed by reaction with heating at 50 ℃ for 12 hours. After the reaction is finished, pouring the reaction liquid into 500mL of ice water, separating out white solid, carrying out suction filtration, and drying a filter cake. Subjecting to silica gel column chromatography (petroleum ether: acetic acid)Ethyl ester 10:1) purification afforded compound a-2 (white solid, 3.76g, two step yield 72%):¹H NMR(300MHz,CDCl₃)δ5.40-5.16(m,1H),4.62-4.37(m,1H),4.23-4.02(m,2H),3.91-3.66(m,2H),2.94-2.78(m,1H),2.04(s,3H),1.13(s,3H),0.93(s,6H),0.90(s,3H),0.86(s,3H),0.86(s,3H),0.76(s,3H).ESI-MS:m/z 524.5[M+H]⁺。

example 2

Olean-12-en-28- (oxazolin-2-yl) -3 beta-ol

Compound A-2(3.5g, 6.6mmol) was dissolved in 50mL of methanol, potassium hydroxide (3.7g, 66mmol) was added, and the reaction was heated to 50 ℃ and checked for completion by TLC. After completion of the reaction, it was cooled to room temperature, 50mL of water was added, extraction was performed with ethyl acetate (50mL × 3), and the mixture was washed with saturated brine (50mL × 3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain compound a-1 (white solid, 3.0g, yield 95%):¹H NMR(300MHz,DMSO)δ5.36-5.18(m,1H),4.26-4.03(m,2H),3.88-3.63(m,2H),3.27-3.15(m,1H),2.93-2.77(m,1H),1.13(s,3H),0.99(s,3H),0.94(s,3H),0.90(s,6H),0.78(s,3H),0.76(s,3H).ESI-MS:m/z 482.4[M+H]⁺。

example 3

12-ene-3 beta-propionyloxy-28- (oxazoline-2-yl) -oleanane (compound A-3)

Compound a-1(200mg, 0.4mmol) was dissolved in pyridine (3mL), 4-dimethylaminopyridine (5mg, 0.04mmol) and propionic anhydride (133uL, 1mmol) were sequentially added, the reaction was stirred at room temperature, 1N hydrochloric acid (5mL) was added after completion of the reaction by TLC detection, extraction was performed with ethyl acetate (5mL × 3), washing was performed with saturated saline (5mL × 3), drying was performed with anhydrous sodium sulfate, concentration was performed under reduced pressure, and chromatography was performed with a silica gel column (petroleum ether: ethyl acetate ═ 10:1),compound A-3 (white solid, 178mg, 80% yield) was obtained:¹H NMR(300MHz,CDCl₃)δ5.32-5.20(m,1H),4.58-4.42(m,1H),4.22-4.03(m,2H),3.91-3.71(m,2H),2.94-2.79(m,1H),2.32(q,J＝7.6Hz,2H),0.93(s,6H),0.90(s,3H),0.86(s,9H),0.76(s,3H).ESI-MS:m/z 538.5[M+H]⁺。

example 4

12-ene-3 beta-butyryloxy-28- (oxazoline-2-yl) -oleanane (compound A-4)

Referring to the procedure of example 3, substituting propionic anhydride for succinic anhydride produced compound a-4:¹H NMR(300MHz,CDCl₃)δ5.32-5.22(m,1H),4.55-4.42(m,1H),4.21-4.09(m,2H),3.91-3.68(m,2H),2.94-2.78(m,1H),2.28(t,J＝7.4Hz,2H),1.13(s,2H),0.93(s,4H),0.90(s,2H),0.86(s,3H),0.76(s,2H).ESI-MS:m/z 552.5[M+H]⁺。

example 5

12-ene-3 beta-pentanoyloxy-28- (oxazoline-2-yl) -oleanane (compound A-5)

Referring to the procedure of example 3, substituting propionic anhydride for pentanoic anhydride, compound a-5 is prepared:¹H NMR(300MHz,CDCl₃)δ5.31-5.22(m,1H),4.54-4.42(m,1H),4.21-4.06(m,2H),3.85-3.68(m,2H),2.90-2.82(m,1H),2.29(t,J＝7.5Hz,2H),0.93(s,6H),0.91(s,3H),0.90(s,3H),0.86(s,6H),0.76(s,3H).ESI-MS:m/z 566.5[M+H]⁺。

example 6

12-en-3 β - (4-fluorobenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (compound A-6)

Compound A-1(200mg, 0.4mmol) was dissolved in dichloromethane (3mL), N' -dicyclohexylcarbodiimide (247mg, 1.2mmol), 4-dimethylaminopyridine (48mg, 0.4mmol) and p-fluorobenzoic acid (122. mu.L, 1mmol) were added in this order, and the reaction was stirred at room temperature. TLC detection after completion of the reaction was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to give compound a-6 (white solid, 188mg, yield 78%):¹H NMR(300MHz,CDCl₃)δ8.05(dd,J＝8.4,5.7Hz,2H),7.10(t,J＝8.6Hz,2H),5.32-5.22(m,1H),4.82-4.64(m,1H),4.24-4.04(m,2H),3.91-3.64(m,2H),2.95-2.77(m,1H),1.00(s,3H),0.98(s,3H),0.94(s,6H),0.91(s,3H),0.78(s,3H).ESI-MS:m/z 604.5[M+H]⁺。

example 7

12-ene-3 beta- (4-chlorobenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (Compound A-7)

Referring to the procedure of example 6, p-fluorobenzoic acid was substituted for p-chlorobenzoic acid to give compound a-7:¹H NMR(300MHz,CDCl₃)δ7.97(d,J＝8.4Hz,2H),7.41(d,J＝8.6Hz,2H),5.48-5.39(m,1H),4.82-4.63(m,1H),3.93-3.72(m,2H),3.67-3.53(m,2H),2.64-2.48(m,1H),1.01(s,3H),0.99(s,3H),0.92(s,12H),0.81(s,3H).ESI-MS:m/z 620.4[M+H]⁺。

example 8

12-ene-3 beta- (4-methylbenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (Compound A-8)

Referring to the procedure of example 6, p-fluorobenzoic acid was substituted for p-methylbenzoic acid to give compound a-8:¹H NMR(300MHz,CDCl₃)δ8.00(d,J＝8.1Hz,2H),7.27(d,J＝7.2Hz,2H),5.46-5.40(m,1H),4.78-4.66(m,1H),3.65-3.56(m,2H),3.43-3.26(m,2H),2.63-2.50(m,1H),2.43(s,3H),1.19(s,3H),1.01(s,3H),0.99(s,3H),0.94(s,3H),0.92(s,6H),0.81(s,3H).ESI-MS:m/z 600.4[M+H]+。

example 9

12-ene-3 beta- (4-methoxybenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (Compound A-9)

Referring to the procedure of example 6, p-fluorobenzoic acid was substituted for p-methoxybenzoic acid to give compound a-9:¹H NMR(300MHz,CDCl₃)δ8.07(d,J＝8.5Hz,2H),6.95(d,J＝8.5Hz,2H),5.52-5.33(m,1H),4.78-4.60(m,1H),3.88(s,3H),3.68-3.58(m,2H),3.46-3.23(m,2H),2.64-2.48(m,1H),1.19(s,3H),1.01(s,3H),0.99(s,3H),0.92(s,9H),0.81(s,3H).ESI-MS:m/z 616.5[M+H]⁺。

example 10

12-ene-3 beta- (4-trifluoromethoxybenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (Compound A-10)

Referring to the procedure of example 6, p-fluorobenzoic acid was substituted for p-trifluoromethoxybenzoic acid to give compound a-10:¹H NMR(300MHz,CDCl₃)δ8.10(d,J＝8.7Hz,2H),7.35(d,J＝8.5Hz,2H),5.52-5.43(m,1H),4.83-4.74(m,1H),3.69-3.43(m,2H),3.36-3.13(m,2H),2.56-2.43(m,1H),1.23(s,3H),1.03(s,3H),1.01(s,3H),0.95(s,3H),0.93(s,6H),0.81(s,3H).ESI-MS:m/z 692.8[M+Na]⁺。

example 11

12-ene-3 beta- (4-methoxylbenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (Compound A-11)

Referring to the procedure of example 6, p-fluorobenzoic acid was substituted for p-methoxylbenzoic acid to give compound a-11:¹H NMR(300MHz,CDCl₃)δ8.26-7.96(m,4H),5.46-5.39(m,1H),4.83-4.69(m,1H),3.95(s,3H),3.66-3.56(m,2H),3.42-3.25(m,2H),2.66-2.49(m,1H),1.19(s,3H),1.03(s,3H),1.00(s,3H),0.95(s,3H),0.92(s,6H),0.81(s,3H).ESI-MS:m/z 644.4[M+H]⁺。

example 12

12-ene-3 beta- (3-pyridinoformyl) oxy-28- (oxazolin-2-yl) -oleanane (compound A-12)

Referring to the procedure of example 6, substituting p-fluorobenzoic acid for nicotinic acid gives compound a-12:¹H NMR(300MHz,CDCl₃)δ9.22(s,1H),8.76(d,J＝4.2Hz,1H),8.29(d,J＝7.7Hz,1H),7.44-7.33(m,1H),5.41-5.17(m,1H),4.85-4.67(m,1H),4.27-4.03(m,2H),3.90-3.62(m,2H),2.97-2.76(m,1H),1.15(s,3H),1.01(s,3H),0.98(s,3H),0.94(s,6H),0.91(s,3H),0.78(s,3H).ESI-MS:m/z 587.6[M+H]⁺。

example 13

12-ene-3 beta- (2-carboxybenzoyl) oxy-28- (oxazolin-2-yl) -oleanane (Compound A-13)

Compound A-1(200mg, 0.4mmol) was dissolved in methylene chloride (4mL), and N, N' -dicyclohexylcarbodiimide (247mg, 1.2mmol), 4-dimethylaminopyridine (48mg, 0.4mmol) and monobenzyl phthalate (205mg, 0.8mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, it was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain compound I-4 (white solid, 260mg, yield 78%).

Compound I-4(200mg, 0.277mmol) was dissolved in DCM (4mL), 10% palladium on carbon (10mg) was added, and the reaction was stirred under hydrogen atmosphere at room temperature overnight. After completion of the TLC detection reaction, celite was filtered and the filtrate was concentrated to give Compound A-13 (white solid, 156mg, 90% yield):¹H NMR(300MHz,CDCl₃)δ7.88-7.79(m,1H),7.73-7.64(m,1H),7.59-7.47(m,2H),5.32-5.26(m,1H),4.82-4.67(m,1H),4.28-4.14(m,2H),3.96-3.69(m,2H),2.94-2.79(m,1H),1.15(s,3H),0.97(s,3H),0.94(s,3H),0.94(s,3H),0.90(s,6H),0.76(s,3H).ESI-MS:m/z630.6[M+H]⁺。

example 14

12-ene-3 β -phosphoxy-28- (oxazolin-2-yl) -oleanane (Compound A-14)

Dissolving compound a-1(200mg, 0.4mmol) in dichloromethane (8mL), first adding 1H-tetrazole (109 μ L, 1.2mmol), stirring at room temperature for 10 minutes, then adding dibenzyl N, N-diisopropylphosphoramidite (153 μ L, 0.45mmol), stirring at room temperature for 4 hours under nitrogen atmosphere, after TLC detection reaction is complete, adding m-chloroperoxybenzoic acid (157mg, 0.9mmol), stirring at room temperature for 4 hours, after TLC detection reaction is complete, adding saturated ammonium chloride solution (8mL), quenching, washing with saturated sodium bicarbonate solution (5mL × 2), drying over anhydrous sodium sulfate, filtering, concentrating the filtrate, and purifying by silica gel column chromatography (petroleum ether: ethyl acetate ═ 15: 1) to obtain compound I-4 (white solid, 266mg, 74% yield).

Compound I-4(200mg, 0.23mmol) was dissolved in a mixed solvent of tetrahydrofuran (2mL) and methanol (2mL), 10% palladium on carbon (10mg) was added, and the reaction was stirred under hydrogen atmosphere at room temperature overnight. After TLC detection reaction was complete, celite was filtered, the filtrate was concentrated and slurried with ethyl acetate to give compound a-14 (white solid, 72mg, 56% yield):¹H NMR(300MHz,C₅D₅N)δ5.46-5.38(m,1H),4.90-4.81(m,1H),4.23-4.03(m,2H),3.92-3.73(m,2H),3.28-3.11(m,2H),1.27(s,3H),1.25(s,3H),1.07(s,3H),0.98(s,6H),0.94(s,6H).ESI-MS:m/z560.5[M-H]^-。

example 15

12-ene-3 beta-acetoxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (Compound B-2)

Compound I-1(5g, 10mmol) from example 1 was dissolved in anhydrous dichloromethane (80mL), and oxalyl chloride (1.7mL, 20mmol) and N, N-dimethylformamide (5 drops) were slowly added dropwise with stirring and reacted at room temperature for 5 hours. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure to obtain Compound I-2 (yellow solid, 5.17g, yield 100%).

Compound I-2(5.17g, 10mmol) was dissolved in anhydrous dichloromethane (80mL), 3-bromopropylamine hydrobromide (4.4g, 20mmol) was added, triethylamine (5.5mL, 40mmol) was dissolved in anhydrous dichloromethane (20mL), and a solution of triethylamine in dichloromethane was slowly added dropwise to the reaction solution using a constant pressure dropping funnel. Stir at room temperature overnight. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, water (300mL) was added and extracted with ethyl acetate (300 mL. times.3), and the mixture was washed with saturated brine (300 mL. times.3), and the filtrate was concentrated and dried to obtain a crude compound I-3 which was used as it was in the next reaction.

The crude compound I-3 obtained in the previous step was dissolved in N, N-dimethylformamide (100mL), and potassium carbonate (2.7g, 20mml) was added thereto, followed by reaction with heating at 50 ℃ for 12 hours. After the reaction is finished, pouring the reaction liquid into 500mL of ice water, separating out white solid, carrying out suction filtration, and drying a filter cake. Recrystallization from methanol gave compound B-2 (white solid, 3.57g, 64% over two steps):¹H NMR(300MHz,DMSO)δ5.27-5.14(m,1H),4.56-4.40(m,1H),4.20-3.96(m,2H),3.45-3.24(m,2H),2.89-2.74(m,1H),2.04(s,3H),1.12(s,3H),0.94(s,3H),0.92(s,3H),0.88(s,3H),0.87(s,3H),0.86(s,3H),0.81(s,3H).ESI-MS:m/z 538.5[M+H]⁺。

example 16

Olean-12-ene-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -3 beta-ol (Compound B-1)

Dissolving compound B-2(3.5g, 6.2mmol) in 50mL of methanol, adding potassium hydroxide (3.4g, 62mmol), heating to 50 ℃ and stirring for reaction, detecting completion of the reaction by TLC, after completion of the reaction, cooling to room temperature, adding 50mL of water, extracting with ethyl acetate (50mL × 3), washing with saturated brine (50mL × 3), drying over anhydrous sodium sulfate, concentrating under reduced pressure, and recrystallizing with methanol to obtain compound B-1 (white solid, 2.7g, yield 87%):¹H NMR(300MHz,DMSO)δ5.29-5.12(m,1H),4.17-3.96(m,2H),3.42-3.29(m,2H),3.28-3.14(m,1H),2.90-2.74(m,1H),1.12(s,3H),0.99(s,3H),0.91(s,6H),0.88(s,3H),0.81(s,3H),0.78(s,3H).ESI-MS:m/z 496.4[M+H]⁺. The single crystal diffraction of compound B-1 is shown in FIG. 1.

Example 17

12-ene-3 beta-propionyloxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-3)

Dissolving compound B-1(200mg, 0.40mmol) in pyridine (3mL), sequentially adding 4-dimethylaminopyridine (5mg, 0.04mmol) and propionic anhydride (133uL, 1mmol), stirring the reaction at room temperature, detecting completion of the reaction by TLC, adding 1N hydrochloric acid (5mL), extracting with ethyl acetate (5mL × 3), washing with saturated saline (5mL × 3), drying over anhydrous sodium sulfate, concentrating under reduced pressure, and subjecting to silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain compound B-3 (white solid, 166mg, yield 75%):¹H NMR(300MHz,CDCl₃)δ5.24-5.16(m,1H),4.55-4.44(m,1H),4.17-4.00(m,2H),3.33(t,J＝9.9Hz,2H),2.87-2.75(m,1H),2.32(q,J＝6.6Hz,2H),0.94(s,3H),0.91(s,3H),0.88(s,3H),0.86(s,9H),0.81(s,3H).ESI-MS:m/z 552.6[M+H]⁺。

example 18

12-ene-3 β -butyryloxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (Compound B-4)

Referring to the procedure of example 17, substituting propionic anhydride for succinic anhydride produced compound B-4:¹H NMR(300MHz,CDCl₃)δ5.24-5.16(m,1H),4.55-4.44(m,1H),4.17-4.00(m,2H),3.33(t,J＝9.9Hz,2H),2.87-2.75(m,1H),2.32(q,J＝6.6Hz,2H),0.94(s,3H),0.91(s,3H),0.88(s,3H),0.86(s,9H),0.81(s,3H).ESI-MS:m/z 566.7[M+H]⁺。

example 19

12-ene-3 beta-valeryloxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (Compound B-5)

Referring to the procedure of example 17, substituting propionic anhydride for pentanoic anhydride, compound B-5 is prepared:¹H NMR(300MHz,CDCl₃)δ5.23-5.13(m,1H),4.55-4.41(m,1H),4.17-4.00(m,2H),3.39-3.27(m,2H),2.89-2.74(m,2H),2.29(t,J＝7.3Hz,2H),1.12(s,3H),0.93(s,3H),0.91(s,3H),0.87(s,3H),0.85(s,6H),0.80(s,3H).ESI-MS:m/z 580.5[M+H]⁺。

example 20

12-ene-3 beta- (4-fluorobenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-6)

Compound B-1(200mg, 0.40mmol) was dissolved in dichloromethane (3mL), and N, N' -dicyclohexylcarbodiimide (247mg, 1.2mmol), 4-dimethylaminopyridine (48mg, 0.4mmol) and p-fluorobenzoic acid (122mg, 1 m) were added in this ordermol), stirring the reaction at room temperature. TLC after completion of the reaction was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to give compound B-6 (white solid, 186mg, yield 80%):¹H NMR(300MHz,CDCl₃)δ8.05(dd,J＝8.3,5.6Hz,2H),7.10(t,J＝8.5Hz,2H),5.42-5.34(m,1H),4.78-4.65(m,1H),3.65-3.44(m,2H),3.27-2.98(m,2H),2.65-2.44(m,1H),1.19(s,3H),1.01(s,3H),0.99(s,3H),0.94(s,3H),0.91(s,6H),0.79(s,3H).ESI-MS:m/z 618.5[M+H]⁺。

example 21

Olean-12-en-3 β - (4-chlorobenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) (Compound B-7)

Referring to the procedure of example 20, p-fluorobenzoic acid was substituted for p-chlorobenzoic acid to give compound B-7:¹H NMR(300MHz,CDCl₃)δ7.97(d,J＝8.4Hz,2H),7.41(d,J＝8.5Hz,2H),5.40(m,1H),4.82-4.61(m,1H),3.66-3.47(m,2H),3.35-3.06(m,2H),2.68-2.39(m,1H),1.19(s,3H),1.01(s,3H),0.99(s,3H),0.94(s,3H),0.92(s,6H),0.79(s,3H).ESI-MS:m/z 635.5[M+H]⁺。

example 22

12-ene-3 beta- (4-methylbenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-8)

Referring to the procedure of example 20, p-fluorobenzoic acid was substituted for p-methylbenzoic acid to obtain compound B-8:¹H NMR(300MHz,CDCl₃)δ7.99(d,J＝8.7Hz,2H),6.92(d,J＝8.7Hz,2H),5.45-5.33(m,1H),4.80-4.57(m,1H),3.86(s,3H),3.64-3.43(m,2H),3.27-3.09(m,2H),2.57-2.38(m,2H),1.18(s,3H),1.01(s,3H),0.99(s,3H),0.94(s,3H),0.91(s,6H),0.79(s,3H).ESI-MS:m/z 614.4[M+H]⁺。

example 23

12-ene-3 beta- (4-methoxybenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-9)

Referring to the procedure of example 20, p-fluorobenzoic acid was substituted for p-methoxybenzoic acid to give compound B-9:¹H NMR(300MHz,CDCl₃)δ8.01(d,J＝8.6Hz,2H),6.93(d,J＝8.7Hz,2H),5.45-5.37(m,1H),4.79-4.63(m,1H),3.87(s,3H),3.67-3.50(m,2H),3.29-3.12(m,2H),2.67-2.49(m,1H),1.20(s,3H),1.03(s,3H),1.01(s,3H),0.95(s,3H),0.93(s,6H),0.81(s,3H).ESI-MS:m/z 630.5[M+H]⁺。

example 24

12-ene-3 beta- (4-trifluoromethoxybenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-10)

Referring to the procedure of example 20, p-fluorobenzoic acid was substituted for p-trifluoromethoxybenzoic acid to give compound B-10:¹H NMR(300MHz,CDCl₃)δ8.10(d,J＝8.7Hz,2H),7.32-7.25(m,2H),5.45-5.34(m,1H),4.83-4.64(m,1H),3.69-3.43(m,2H),3.26-3.03(m,2H),2.66-2.43(m,1H),1.20(s,3H),1.03(s,3H),1.01(s,3H),0.96(s,3H),0.93(s,6H),0.81(s,3H).ESI-MS:m/z 684.4[M+H]⁺。

example 25

12-ene-3 beta- (4-methoxylbenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (Compound B-11)

Method according to example 20Replacing p-fluorobenzoic acid with p-methoxylbenzoic acid to obtain a compound B-11:¹H NMR(300MHz,CDCl₃)δ8.26-7.96(m,4H),5.46-5.39(m,1H),4.83-4.69(m,1H),3.95(s,3H),3.66-3.56(m,2H),3.42-3.25(m,2H),2.66-2.49(m,1H),1.18(s,3H),1.01(s,3H),0.99(s,3H),0.94(s,3H),0.93(s,6H),0.81(s,3H).ESI-MS:m/z 658.4[M+H]⁺。

example 26

12-ene-3 beta- (3-pyridinoformyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-12)

Referring to the procedure of example 20, substituting p-fluorobenzoic acid for nicotinic acid gives compound B-12:¹H NMR(300MHz,CDCl₃)δ9.25(s,1H),8.78(d,J＝3.7Hz,1H),8.31(d,J＝7.9Hz,1H),7.40(dd,J＝7.7,4.9Hz,1H),5.27-5.13(m,1H),4.90-4.68(m,1H),4.26-3.95(m,2H),3.42-3.26(m,2H),2.98-2.77(m,1H),1.17(s,3H),1.04(s,3H),1.01(s,3H),0.97(s,3H),0.94(s,3H),0.91(s,3H),0.85(s,3H).ESI-MS:m/z 601.5[M+H]⁺。

example 27

12-ene-3 beta- (2-carboxybenzoyl) oxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (compound B-13)

Compound B-1(200mg, 0.39mmol) was dissolved in methylene chloride (4mL), and N, N' -dicyclohexylcarbodiimide (247mg, 1.2mmol), 4-dimethylaminopyridine (48mg, 0.4mmol) and monobenzyl phthalate (205mg, 0.8mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, it was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10:1) to obtain compound I-6 (white solid, 208mg, yield 73%).

Compound I-6(200mg, 0.272mmol) was dissolved in DCM (4mL), 10% palladium on carbon (10mg) was added, and the reaction was stirred under hydrogen atmosphere at room temperature overnight. After completion of the TLC detection reaction, celite was filtered and the filtrate was concentrated to give Compound B-15 (white solid, 164mg, 92% yield):¹H NMR(300MHz,CDCl₃)δ8.58(s,1H),7.78(d,J＝7.0Hz,1H),7.61(d,J＝6.6Hz,1H),7.50-7.37(m,2H),5.33-5.18(m,1H),4.80-4.64(m,1H),4.31-4.18(m,2H),3.75-3.38(m,2H),2.95-2.72(m,1H),1.15(s,3H),0.98(s,3H),0.94(s,3H),0.91(s,3H),0.90(s,3H),0.89(s,3H),0.79(s,3H).ESI-MS:m/z 642.4[M-H]^-。

example 28

12-ene-3 beta-phosphoxy-28- (5, 6-dihydro-4H-1, 3-oxazin-2-yl) -oleanane (Compound B-14)

Dissolving compound B-1(200mg, 0.40mmol) in dichloromethane (8mL), first adding 1H-tetrazole (109 μ L, 1.2mmol), stirring at room temperature for 10 minutes, then adding dibenzyl N, N-diisopropylphosphoramidite (153 μ L, 0.45mmol), stirring at room temperature for 4 hours under nitrogen atmosphere, after TLC detection reaction is complete, adding m-chloroperoxybenzoic acid (157mg, 0.9mmol), stirring at room temperature for 4 hours, after TLC detection reaction is complete, adding saturated ammonium chloride solution (8mL), quenching, washing with saturated sodium bicarbonate solution (5mL × 2), drying over anhydrous sodium sulfate, filtering, concentrating the filtrate, and purifying by silica gel column chromatography (petroleum ether: ethyl acetate ═ 15: 1) to obtain compound I-7 (white solid, 275mg, yield 80%).

Compound I-7(200mg, 0.26mmol) was dissolved in a mixed solvent of tetrahydrofuran (2mL) and methanol (2mL), 10% palladium on carbon (10mg) was added, and the reaction was stirred under hydrogen atmosphere at room temperature overnight. After completion of TLC detection, celite was filtered, the filtrate was concentrated and slurried with ethyl acetate to give Compound B-14 (white solid, 61mg, 40% yield)：¹H NMR(300MHz,C₅D₅N)δ5.38-5.31(m,1H),4.89-4.77(m,1H),4.11-3.90(m,2H),3.48-3.30(m,2H),3.23-3.12(m,1H),1.23(s,6H),1.04(s,3H),0.97(s,6H),0.94(s,3H),0.90(s,3H).ESI-MS:m/z 574.5[M-H]^-。

Example 29

Evaluation of Activity of Compounds on Huh-7 cell AMPK agonism

The Western Blot method is adopted to detect the agonistic activity of the compound on Huh-7 cell AMPK.

1. Cell line: huh-7 cells (human hepatoma cells, purchased from the stem cell bank of Chinese academy of sciences) were cultured in DMEM complete medium (containing 10% fetal bovine serum and 1% streptomycin/penicillin) containing 5% CO at 37 deg.C₂Cultured in a cell culture box.

2. Antibody: anti-AMPK (Cell Signaling Technology 2532S); anti-pAMPK (Thr172, Cell Signaling Technology 2535S); gapdh (arigobio), HRP-labeled goat anti-rabbit IgG secondary antibody, HRP-labeled goat anti-mouse IgG secondary antibody (bi yun day).

3. Western Blot experiment: the effect of the compounds on AMPK phosphorylation levels in Huh-7 cells was examined.

The experiment was carried out using cells with a viable cell ratio of 90% or more. In 12-well plates, Huh-7 cells were plated at 20 ten thousand per well. At 37 deg.C, contains 5% CO₂The cells were cultured in the incubator for 24 hours and adhered to the wall. Test compounds were administered under complete medium conditions, with compound final concentrations set at 10 μ M for 12 hours, and AICAR (200 μ M), oleanolic acid (10 μ M) were used as positive control compounds. Proteins were subsequently extracted for Western Blot detection. The method comprises the following specific steps:

protein sample preparation: discarding the original culture solution, washing 3 times with 1 XPBS, discarding PBS, adding 100 μ l RIPA buffer (1 XPBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, PMSF, etc.) per well, incubating on ice for 15min, scraping cells with a cell scraper, sucking into a new 1.5ml EP tube, 4 ℃, 12000g, centrifuging for 15min, transferring the supernatant to a new 1.5ml EP tube, placing on ice, adding 1/5 volume of 6 × loading buffer, heating in a 95 ℃ metal bath for 10min, centrifuging for 1min, and freezing to-20 ℃ for use.

Protein quantification: the protein sample was diluted 20-fold, and 20. mu.l of the diluted protein sample and 200. mu.l of BCA (solution A: solution B ═ 5: 1) reagent were sequentially added to a 96-well plate, incubated at 37 ℃ for 30min, and the OD value was measured at a wavelength of 562nm on a microplate reader, and the protein concentration was calculated from the standard curve.

Electrophoresis: SDS-PAGE gel is poured, the concentration of the separation gel is 10 percent, and the concentration of the concentrated gel is 4 percent. The protein samples were previously heated in a metal bath at 95 ℃ for 4min and then centrifuged for 1min, and 30. mu.g of total protein was applied to each sample, and the samples were individually applied by a microsyringe. The power supply is connected (note that the positive electrode and the negative electrode are connected), constant voltage electrophoresis is carried out at 60V at the beginning, and when the protein sample enters the separation gel, the voltage is adjusted to 100V to continue the constant voltage electrophoresis. When bromophenol blue reaches the bottom of the separation gel, electrophoresis is terminated according to the separation condition of protein marker.

Film transfer: the gel after electrophoresis was gently removed, an unnecessary portion was cut off, and the necessary gel was immersed in a Transfer buffer. PVDF membrane of the same size as the gel was prepared, and the PVDF membrane was soaked with methanol for 1min before use, transferred to a Transfer buffer, and simultaneously the filter paper was soaked in the Transfer buffer. And sequentially paving sponge, filter paper, glue, a PVDF (polyvinylidene fluoride) membrane, filter paper and sponge on an electrode plate of the transfer printing instrument cathode. Air bubbles are avoided between each layer. And switching on a power supply, and transferring for 2.5h by a 200mA constant-current ice bath.

Antibody hybridization: after completion of the membrane transfer, the PVDF membrane was taken out and washed 1 time with l.times.TBST, placed in a blocking solution (5% BSA solution prepared with 1. times.TBS containing 0.1% Tween 20) prepared in advance, and blocked at room temperature for 1 hour. Primary antibody was incubated overnight at 4 ℃. The following day (after 12 h), wash 3 times with 1 × TBST for 10min each. And (3) secondary antibody incubation: incubate with 5% BSA diluted secondary antibody (1: 10000) at room temperature for 1h, wash 3 times with 1 × TBST, 10min each. Excess liquid on PVDF membrane was aspirated, spread on an exposure plate, and equal volume of mixed ECL kit liquid Tanon was added^TMHigh-sig ECL Western Blotting Peroxide Buffer and Tanon^TMHigh-sig ECL Western Blotting lumineol/Enhancer Solution, using Tanon chemiluminescence imager, ECL development, and collecting immunoreaction zone.

4. The experimental results are as follows: after the Western Blot experiment result is subjected to gray scale scanning, the p-AMPK/AMPK ratio of negative control DMSO is defined as 1, the p-AMPK/AMPK ratio of the synthesized test compound is a relative ratio of a negative control group, the larger the value is, the stronger the AMPK agonistic activity of the compound is, and the activity data result is shown in Table 1.

TABLE 1 AMPK agonistic activity of the compounds (positive control AICAR concentration 200. mu.M; oleanolic acid, test compound concentration 10. mu.M)

As shown in table 1 and the experimental results of fig. 2, the novel oleanolic acid derivatives provided by the present invention have significant AMPK agonistic activity at a concentration of 10 μ M. For example, compounds A-1, A-2, A-3, A-4, A-5, A-12, B-5, B-12, B-13, B-14 are potent AMPK agonists with activities significantly better than 200 μ M AICAR and significantly better than oleanolic acid, especially A-1, A3 and B14. The above experimental results show that the compound of the present invention has significant agonistic activity on AMPK, and thus can be used to prepare AMPK agonists having activity of enhancing AMPK phosphorylation level, and further can be used to prepare drugs for preventing or treating AMPK-mediated diseases. Other compounds not listed in the examples of the present invention and methods for synthesizing the same can be found in the above examples, and also have significant agonistic activity against AMPK.

Example 30

Tablet formulation

Compound B-14 obtained in example 28 or the compound of the other examples (50g), hydroxypropylmethylcellulose E (150g), starch (200g), povidone K30, and magnesium stearate (1g) were mixed, granulated, and tabletted.

In addition, the compounds prepared in examples 1 to 28 can be prepared into capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, patches and the like by adding different pharmaceutical excipients according to the conventional preparation method of pharmacopoeia 2015 edition.

Claims (10)

1. Oleanolic acid derivatives represented by the following formula I or formula II:

r is H,

R^aCO-or PO₃H₂；

R¹Is H;

R^ais C₁-C₅An alkyl group.

2. The derivative or the pharmaceutically acceptable salt thereof according to claim 1, wherein the derivative or the pharmaceutically acceptable salt thereof is selected from the following compounds:

3. an oleanolic acid derivative or a pharmaceutically acceptable salt thereof, wherein said derivative or said pharmaceutically acceptable salt thereof is selected from the group consisting of:

4. use of a derivative as claimed in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in the manufacture of an AMPK agonist having activity in enhancing the phosphorylation level of AMPK.

5. Use of a derivative according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention or treatment of AMPK mediated diseases.

6. The use according to claim 5, wherein the AMPK-mediated disease comprises a metabolic disease, a cardiovascular disease, a cerebrovascular disease, an inflammatory disease, an autoimmune disease, an organ fibrosis disease, a neurodegenerative disease, a secondary disease caused by pathogen infection, a mitochondrial dysfunction or disorder disease, or a tumor.

7. The use according to claim 5, wherein the AMPK-mediated disease comprises insulin resistance, metabolic syndrome, type 1 or type 2 diabetes, hyperlipidemia, obesity, atherosclerosis, myocardial ischemia, myocardial infarction, cardiac arrhythmia, coronary heart disease, hypertension, heart failure, myocardial hypertrophy, myocarditis, diabetic complications, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, alcoholic fatty liver disease, liver cirrhosis, gout, stroke, cerebral infarction, pneumonia, asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, bronchiolitis obliterans, idiopathic pulmonary fibrosis, cystic fibrosis lung disease, allergic rhinitis, inflammatory bowel disease, polycystic kidney disease, polycystic ovary syndrome, Behcet's disease, systemic lupus erythematosus, rheumatoid arthritis, spondyloarthritis, osteoarthritis, synovitis, tendonitis, thromboangiitis obliterans, rheumatoid arthritis, polycystic kidney disease, hyperlipidemia, obesity, atherosclerosis, myocardial ischemia, myocardial infarction, chronic bronchitis, emphysema, pulmonary fibrosis, polycystic kidney disease, rheumatoid arthritis, osteoarthritis, synovitis, and rheumatoid arthritis, Phlebitis, intermittent claudication, keloid, psoriasis, ichthyosis, bullous pemphigoid, dermatitis, contact dermatitis, pancreatitis, chronic nephritis, cystitis, meningitis, gastritis, septicemia, pyoderma gangrenosum, uveitis, Parkinson's disease, Alzheimer's disease, alpha-synucleinopathy, depression, multiple sclerosis, amyotrophic lateral sclerosis, fibromyalgia syndrome, neuralgia, Down's syndrome, Hallervorden-Spanish disease, Huntington's chorea, Wilson's disease, myasthenia, myoclonus, exercise intolerance, Kanes-Seir syndrome, chronic fatigue syndrome, Lei's syndrome, mitochondrial myopathy-encephalopathy-lactacidemia, stroke syndrome, stroke-like seizures, Duchenne's muscular dystrophy, conchal muscular dystrophy, Friedrich's ataxia or tumors.

8. A pharmaceutical composition having an activity of enhancing phosphorylation level of AMPK, comprising the derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 as an active ingredient and a pharmaceutically acceptable excipient.

9. A pharmaceutical composition for preventing or treating AMPK-mediated diseases, comprising the derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 2 as an active ingredient and a pharmaceutically acceptable adjuvant.

10. The pharmaceutical composition according to claim 8 or 9, wherein the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

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