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

Abstract

The present invention discloses a new pentacyclic triterpenoid AMPK agonist derivative of oleanolic acid and its medicinal use, specifically a compound represented by formula I or formula II or a pharmaceutically acceptable salt or ester or solvent thereof They can be used to prepare AMPK agonists with the activity of enhancing the phosphorylation level of AMPK and to prepare medicines for preventing or treating AMPK-mediated diseases. The novel pentacyclic triterpenoid compound of the present invention has significant AMPK agonist activity, and its activity is significantly better than the recognized AMPK agonist AICAR, and has better oral bioavailability and other pharmacokinetic properties and very good safety. sex.

Description

Derivatives of oleanolic acid and their medicinal uses

technical field

The present invention relates to the field of biomedicine, to novel pentacyclic triterpenoids with AMPK agonistic activity, in particular to oleanolic acid derivatives and their medicinal uses, and to the preparation of such compounds in the prevention or treatment of AMPK-mediated Use in medicine for diseases and pharmaceutical compositions thereof.

Background technique

AMPK (Adenylate Activated Protein Kinase) is a key kinase that regulates the body's energy metabolism and inflammatory response. Its phosphorylation activation can overcome insulin resistance, lower blood sugar, lower blood lipids (by inhibiting the synthesis of fatty acids and cholesterol), anti-inflammatory and anti-apoptotic. Anti-aging, anti-fibrosis, promoting mitochondrial synthesis, enhancing mitochondrial oxidative metabolism, anti-aging and anti-tumor, etc. (Physiol. Rev. 2009, 89, 1025). In recent years, the anti-inflammatory and anti-fibrotic effects of AMPK have attracted more and more attention (Nature 2013, 493, 346). The possible mechanism is that AMPK exerts anti-inflammatory and anti-fibrotic effects by enhancing the transcriptional function of estrogen-related receptor α (ERRα). chemistry (Immunity 2015, 43, 80).

More and more evidences show that abnormal AMPK function is closely related to the occurrence and development of various diseases. AMPK-mediated diseases include metabolic diseases and cardiovascular and cerebrovascular diseases, 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 and diabetic ulcers, etc.), non-alcoholic fatty liver, non-alcoholic fat Hepatitis, alcoholic fatty liver, cirrhosis, gout, stroke or cerebral infarction, etc.; AMPK-mediated diseases also include inflammatory diseases, autoimmune diseases, organ fibrosis diseases, nerve damage diseases or secondary diseases caused by pathogen infection Diseases such as 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, synovium inflammation, tendonitis, thromboangiitis obliterans, phlebitis, intermittent claudication, keloids, psoriasis, ichthyosis, bullous pemphigoid, dermatitis, contact dermatitis, pancreatitis, chronic nephritis, bladder inflammation, meningitis, gastritis, sepsis, pyoderma gangrenosum, uveitis, Parkinson's disease, Alzheimer's disease, alpha-synucleinopathy, depression, multiple sclerosis, amyotrophic lateral sclerosis , fibromyalgia syndrome, neuralgia, Down syndrome, Hallerwarden-Spaer disease, Huntington's disease or Wilson's disease, etc.

Literature reports that AMPK agonists can 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 DrugDiscov, 2019, DOI: 10.1038/s41573-019-0019-2). For example, metformin, a widely used clinically hypoglycemic drug, is believed to exert various clinical effects mainly by activating AMPK (J. Clin. Invest. 2001, 108, 1167). Although AMPK agonists have broad clinical application prospects, so far, no substantial progress has been made in the development of new safe and effective AMPK agonists. For reasons of safety or efficacy, very few AMPK agonists under investigation have entered the clinical research stage. For example, although the broad-spectrum AMPK-β subunit agonist MK-8722 can reduce blood sugar, it has been found that the animal heart has irreversible cardiac hypertrophy side effects in rat and monkey experiments (Science 2017, 357, 507). In addition, AICAR, as one of the most commonly used AMPK agonists (Eur. J. Biochem. 1995, 229, 558), was also terminated from clinical trials due to its large toxic side effects (J Clin Pharmacol 1991, 31: 342–347) .

In conclusion, there is an urgent need to develop novel AMPK agonists with high activity and less toxic and side effects. On the other hand, oleanolic acid is a pentacyclic triterpenoid commonly found in medicinal plants, which has a wide range of biological activities (Nat Prod Rep 2011, 28, 543). However, the research on pentacyclic triterpenoid derivatives with new structures with stronger activity, such as oleanolic acid derivatives, is of great significance in the field of biomedicine.

SUMMARY OF THE INVENTION

Purpose of the invention: In view of the problems existing in the prior art in the field of AMPK agonist research and development, the present invention provides a novel oleanolic acid derivative; the novel oleanolic acid derivative provided by the present invention is a new type of AMPK agonist, so It can be used to prepare a medicament for preventing or treating AMPK-mediated diseases.

The present invention unexpectedly discovered a series of potent novel AMPK agonists during the structural modification of oleanolic acid, and its AMPK agonist activity was significantly better than the recognized AMPK agonist AICAR.

Technical solution: In order to achieve the above purpose, as described in the present invention, the oleanolic acid derivative represented by the following formula I or formula II or a pharmaceutically acceptable salt or ester or solvate thereof:

in,

R^aCO-或PO₃H₂；R is H,

R ^a CO- or PO ₃ H ₂ ;

R ¹ is H, C ₁ -C ₅ alkyl or C ₁ -C ₅ alkyl substituted with a substituent Y which is OH, C(O)OH, C(O)NH ₂ , NH ₂ , NHC(O)CH ₃ , pyrrolidin-1-yl, piperidin-1-yl, piperazin-1-yl, 4-methyl-piperazin-1-yl, morpholin-4-yl, thiomo Lino-1,1-dioxo-4-yl, N,N-dimethylamino, N,N-diethylamino, trimethylammonium or diethanolamine;

R ^a is C ₁ -C ₅ alkyl unsubstituted or substituted with a substituent L which is one or two substituents independently selected from the group consisting of OH, C(O)OH, NH ₂ , pyrrole Alk-1-yl, piperidin-1-yl, piperazin-1-yl, 4-methyl-piperazin-1-yl, morpholin-4-yl, thiomorpholin-1,1-dioxo Gen-4-yl or NHC(O)CH( _CH3 )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 can 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-naphthalene Disulfonic acid, naphthalene-2-sulfonic acid, pivalic acid, terephthalic acid, thiocyanic acid, cholic acid, n-dodecyl sulfate, 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, niacin, nitric acid, orotic acid, oxalic acid, bitter 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. On the other hand, the salt can also be a compound of the present invention with metal (including sodium, potassium, calcium, etc.) ions or pharmaceutically acceptable amines (including ethylenediamine, tromethamine, etc.), ammonium ions or choline salt formed. The compounds of the present invention may also be formulated into pharmaceutical compositions in the form of esters, prodrugs, N-oxides or solvates thereof.

The present invention provides the use of the compound of formula I or formula II or a pharmaceutically acceptable salt or ester or solvate thereof in the preparation of an AMPK agonist having the activity of enhancing the phosphorylation level of AMPK. The compounds of the present invention have significant agonistic activity on AMPK, and thus can be used to prepare AMPK agonists with the activity of enhancing the phosphorylation level of AMPK.

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

Wherein, the AMPK-mediated diseases include metabolic diseases, cardiovascular and cerebrovascular diseases, inflammatory diseases, autoimmune diseases, organ fibrosis diseases, neurodegenerative diseases, secondary diseases caused by pathogen infection, mitochondrial dysfunction or disorder or tumor.

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

The AMPK-mediated diseases, such as inflammatory diseases, autoimmune diseases, organ fibrotic diseases, neuro-damaging diseases or secondary diseases caused by pathogen infection, including: pneumonia, asthma, chronic obstructive pulmonary disease, chronic bronchial disease inflammation, emphysema, bronchiolitis obliterans, idiopathic pulmonary fibrosis, cystic fibrosis lung disease, allergic rhinitis, inflammatory bowel diseases (eg, Crohn's disease and ulcerative colitis), polycystic kidney disease, Polycystic ovary syndrome (PCOS), Behçet'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, sepsis, pyoderma gangrenosum, grape Meningitis, Parkinson's disease, Alzheimer's disease, alpha-synucleinopathy, depression, multiple sclerosis, amyotrophic lateral sclerosis, fibromyalgia syndrome, neuralgia, Down's syndrome, Kazakhstan Leverden-Spaer disease, Huntington's disease or Wilson's disease, etc.

The AMPK-mediated diseases, such as mitochondrial dysfunction and disorders, include: myasthenia, myoclonus, exercise intolerance, Kearns-Saier syndrome, chronic fatigue syndrome, Leigh syndrome, mitochondrial Myopathy-encephalopathy-hyperlactatemia, stroke syndrome or stroke-like episode. Likewise, the compounds of the present invention are also useful in the treatment of muscular dystrophic states, eg, Duchenne muscular dystrophy, shell muscular dystrophy, or Friedrich's ataxia.

The AMPK-mediated diseases, such as tumors, include: bone cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, multiple myeloma, myeloproliferation Abnormal syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hemangioma, granuloma, xanthoma, meningiosarcoma, glioma, astrocytoma, medulloblastoma, ependyma tumor, germ cell tumor (pineal tumor), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, fibrous neuroma, sarcoma, esophageal cancer, gastric cancer, pancreas cancer, colorectal cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, lymphoma, testicular cancer, stromal cell cancer, lung cancer, liver cancer, skin cancer, malignant melanoma or basal cell cancer, etc.

A pharmaceutical composition with the activity of enhancing the phosphorylation level of AMPK according to the present invention, which contains a therapeutically effective amount of a compound of formula I or formula II or a pharmaceutically acceptable salt or ester or solvate thereof as an active ingredient and a pharmaceutically acceptable amount. acceptable excipients.

A pharmaceutical composition for preventing or treating AMPK-mediated diseases according to the present invention, which contains a therapeutically effective amount of a compound of formula I or formula II or a pharmaceutically acceptable salt or ester or solvate thereof as an active ingredient and a pharmaceutically acceptable acceptable excipients.

The above-mentioned adjuvants that can be arbitrarily mixed can be changed according to dosage forms, administration forms, and the like. Examples of adjuvants include excipients, binders, disintegrating agents, lubricants, flavoring agents, flavoring agents, coloring or 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 conventional formulations in pharmacy.

If desired, a compound of Formula I or Formula II of the present invention, or a pharmaceutically acceptable salt or ester or solvate thereof, may be used in combination with one or more other types of drugs for the prevention or treatment of AMPK-mediated diseases, including but not limited to It is not limited to the following cases of combined medication.

The drugs that can be used in combination with the compounds of the present invention can be one or more antidiabetic drugs, including metformin, sulfonylurea hypoglycemic drugs (such as glyburide and glimepiride, etc.), glucosidase inhibitor Drugs (such as acarbose and miglitol, etc.), PPARγ agonists (such as pioglitazone and rosiglitazone), PPARα/γ dual agonists, dipeptidyl peptidase IV (DPP-IV) inhibitors (such as sitagliptin, saxagliptin, alogliptin and linagliptin, etc.), glinide hypoglycemic agents (such as repaglinide and nateglinide, etc.), SGLT2 inhibitors (such as canger Ligagliflozin, dapagliflozin, empagliflozin, ipagliflozin, lupagliflozin, topagliflozin, etc.), glucokinase agonists (such as HMS5552, etc.), insulin, glucagon-like peptide-1 (GLP-1) drugs (such as exenatide, liraglutide, lixisenatide, dulaglutide, benaglutide and albiglutide, etc.), PTP1B inhibitors, glycogen phosphorylase Inhibitor, glucose-6-phosphatase inhibitor, AMPK agonist, GPR40 agonist, or GPR120 agonist.

Drugs that may optionally be used in combination with the compounds of the present invention may be one or more weight loss drugs, including lorcaserin, orlistat, and glucagon-like peptide-1 (GLP-1) drugs such as senatide, liraglutide, lixisenatide, dulaglutide, benaglutide and albiglutide, etc.).

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

Drugs that may be selected for use in combination with the compounds of the present invention may be one or more hypolipidemic drugs, including niacin, statins (eg, lovastatin, simvastatin, pravastatin, mevastatin, fluvastatin, etc.) statins, atorvastatin, cerivastatin, rovastatin and pitavastatin), cholesterol absorption inhibitors (such as ezetimibe, etc.), fibrates (such as clofibrate, bezafibrate, fenofibrate) Special class), PCSK9 inhibitors (such as Evolocumab and Alirocumab, etc.), CETP inhibitors (such as anacetrapib, etc.), AMPK agonists and ACC inhibitors (such as GS-0976, etc.), etc.

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

Oleanolic acid used in the present invention is commercially available. The synthesis of oleanolic acid derivatives can be carried out with reference to the method of the examples or the improved method.

Beneficial effect: Compared with the prior art, the present invention has the following advantages:

(1) The novel oleanolic acid derivative of the present invention has potent AMPK agonist activity, and its activity is significantly better than the recognized AMPK agonist AICAR.

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

(3) Compared with the existing AMPK agonists AICAR (clinical trials were terminated due to large toxic side effects) and MK-8722 (which can cause irreversible cardiac hypertrophy side effects), the novel pentacyclic triterpenoids of the present invention have very good security.

(4) As an AMPK agonist, the novel oleanolic acid derivative of the present invention has the advantages of low cost, easy preparation and less potential side effects than the existing AMPK agonists, and it can be used alone or in combination with a Or a combination of other types of drugs for preventing or treating AMPK-mediated diseases is expected to become a new type of drugs for preventing or treating AMPK-mediated diseases.

Description of drawings

Fig. 1 is the single crystal diffraction pattern of compound B-1 in the embodiment;

Figure 2 is a graph of the AMPK agonistic activity of some compounds on Huh-7 cells (detected by Western Blot).

Detailed ways

The content of the present invention is specifically described below by means of examples. In the present invention, the embodiments described below are for better illustrating the present invention and are not intended to limit the scope of the present invention. Various changes and modifications of the present invention can be made without departing from the spirit and scope of the inventions.

Example 1

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

Take oleanolic acid (OA, 10 g, 21.9 mmol) and dissolve it in pyridine (150 mL), add 4-dimethylaminopyridine (0.26 g, 2.19 mmol), slowly add acetic anhydride (8.3 mL, 87.6 mmol), stir at room temperature overnight. After the reaction was completed, 1N hydrochloric acid (300 mL) was added, extracted with ethyl acetate (300 mL×3), washed with saturated brine (300 mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, slurried (petroleum ether: dichloromethane) =50:1) Suction filtration to obtain compound I-1 (white solid, 8.2 g, yield 75%).

Compound I-1 (5 g, 10 mmol) was dissolved in anhydrous dichloromethane (80 mL), and oxalyl chloride (1.7 mL, 20 mmol) and N,N-dimethylformamide (5 drops) were slowly added dropwise with stirring, The reaction was carried out at room temperature for 5 hours. After TLC detected that the reaction was complete, the solvent was evaporated under reduced pressure to obtain compound I-2 (yellow solid, 5.17 g, yield 100%).

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

The crude product of compound I-3 obtained in the previous step was dissolved in N,N-dimethylformamide (100 mL), potassium carbonate (2.7 g, 20 mml) was added, and the reaction was heated at 50° C. for 12 hours. After the completion of the reaction, the reaction solution was poured into 500 mL of ice water, a white solid was precipitated, suction filtered, and the filter cake was dried. Purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain compound A-2 (white solid, 3.76 g, 72% yield for two steps): ¹ H NMR (300 MHz, CDCl ₃ ) δ 5. 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

Oleanan-12-ene-28-(oxazolin-2-yl)-3β-ol

Compound A-2 (3.5 g, 6.6 mmol) was dissolved in 50 mL of methanol, potassium hydroxide (3.7 g, 66 mmol) was added, and the reaction was heated to 50°C with stirring. TLC detected the reaction was complete. After the reaction was completed, it was cooled to room temperature, 50 mL of water was added, extracted with ethyl acetate (50 mL×3), washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum Ether:ethyl acetate=10:1) purification to give compound A-1 (white solid, 3.0 g, 95% yield): ¹ H NMR (300 MHz, DMSO) δ 5.36-5.18 (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-3β-propionyloxy-28-(oxazolin-2-yl)-oleanane (Compound A-3)

Compound A-1 (200 mg, 0.4 mmol) was dissolved in pyridine (3 mL), 4-dimethylaminopyridine (5 mg, 0.04 mmol) and propionic anhydride (133 uL, 1 mmol) were added successively, the reaction was stirred at room temperature, and TLC detected that the reaction was complete After that, 1N hydrochloric acid (5 mL) was added, extracted with ethyl acetate (5 mL×3), washed with saturated brine (5 mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether:acetic acid) ethyl ester=10:1) to give compound A-3 (white solid, 178 mg, 80% yield): ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.32-5.20 (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-3β-butyryloxy-28-(oxazolin-2-yl)-oleanane (Compound A-4)

Referring to the method of Example 3, replace propionic anhydride with butyric anhydride to obtain compound A-4: ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.32-5.22 (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-3β-pentanoyloxy-28-(oxazolin-2-yl)-oleanane (Compound A-5)

Referring to the method of Example 3, replace propionic anhydride with valeric anhydride to obtain compound A-5: ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.31-5.22 (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-ene-3β-(4-fluorobenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-6)

Compound A-1 (200 mg, 0.4 mmol) was dissolved in dichloromethane (3 mL), and N,N'-dicyclohexylcarbonimide (247 mg, 1.2 mmol), 4-dimethylaminopyridine (48 mg) were added successively. , 0.4 mmol) and p-fluorobenzoic acid (122 μL, 1 mmol), and the reaction was stirred at room temperature. TLC detected the completion of the reaction, washed with water (5 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated the filtrate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain compound A-6 (white Solid, 188 mg, 78% yield): ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.05 (dd, J=8.4, 5.7 Hz, 2H), 7.10 (t, J=8.6 Hz, 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-3β-(4-chlorobenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-7)

Referring to the method in Example 6, p-fluorobenzoic acid was replaced with p-chlorobenzoic acid to obtain compound A-7: ¹ H NMR (300MHz, CDCl ₃ )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-3β-(4-methylbenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-8)

Referring to the method of Example 6, p-fluorobenzoic acid was replaced with p-toluic acid to obtain compound A-8: ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.00 (d, J=8.1 Hz, 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-3β-(4-methoxybenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-9)

Referring to the method of Example 6, p-fluorobenzoic acid was replaced with p-methoxybenzoic acid to obtain compound A-9: ¹ HNMR (300MHz, CDCl ₃ )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-3β-(4-trifluoromethoxybenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-10)

Referring to the method of Example 6, replace p-fluorobenzoic acid with p-trifluoromethoxybenzoic acid to obtain compound A-10: ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.10 (d, J=8.7 Hz, 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-3β-(4-methoxyacylbenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-11)

Referring to the method of Example 6, replace p-fluorobenzoic acid with p-methoxyacylbenzoic acid to obtain compound A-11: ¹ H NMR (300MHz, CDCl ₃ )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-3β-(3-picolinoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-12)

Referring to the method of Example 6, replace p-fluorobenzoic acid with nicotinic acid to prepare compound A-12: ¹ H NMR (300MHz, CDCl ₃ )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-3β-(2-carboxybenzoyl)oxy-28-(oxazolin-2-yl)-oleanane (Compound A-13)

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

Compound I-4 (200 mg, 0.277 mmol) was dissolved in DCM (4 mL), 10% palladium on carbon (10 mg) was added, and the reaction was stirred at room temperature under a hydrogen atmosphere overnight. After the completion of the reaction was detected by TLC, celite was filtered, and the filtrate was concentrated to obtain compound A-13 (white solid, 156 mg, yield 90%): ¹ H NMR (300 MHz, CDCl ₃ ) 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/z 630.6[M+H] ⁺ .

Example 14

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

Dissolve compound A-1 (200 mg, 0.4 mmol) in dichloromethane (8 mL), first add 1H-tetrazolium (109 μL, 1.2 mmol), stir at room temperature for 10 minutes, then add dibenzyl N,N-dibenzyl Isopropyl phosphoramidite (153 μL, 0.45 mmol) was stirred at room temperature for 4 hours under nitrogen atmosphere, after the reaction was detected by TLC, m-chloroperoxybenzoic acid (157 mg, 0.9 mmol) was added, stirred at room temperature for 4 hours, and the reaction was detected by TLC After completion, add saturated ammonium chloride solution (8 mL) to quench, wash with saturated sodium bicarbonate solution (5 mL×2), dry over anhydrous sodium sulfate, filter, concentrate the filtrate, and perform silica gel column chromatography (petroleum ether: ethyl acetate) Ester=15:1) was purified to give compound I-4 (white solid, 266 mg, 74% yield).

Compound I-4 (200 mg, 0.23 mmol) was dissolved in a mixed solvent of tetrahydrofuran (2 mL) and methanol (2 mL), 10% palladium on carbon (10 mg) was added, and the reaction was stirred at room temperature overnight under a hydrogen atmosphere. After the completion of the reaction was detected by TLC, celite was filtered, the filtrate was concentrated, and slurried with ethyl acetate to obtain compound A-14 (white solid, 72 mg, yield 56%): ¹ H NMR (300 MHz, C ₅ D ₅ N)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[MH] ^- .

Example 15

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

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

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

The crude product of compound I-3 obtained in the previous step was dissolved in N,N-dimethylformamide (100 mL), potassium carbonate (2.7 g, 20 mml) was added, and the reaction was heated at 50° C. for 12 hours. After the completion of the reaction, the reaction solution was poured into 500 mL of ice water, a white solid was precipitated, suction filtered, and the filter cake was dried. Recrystallization from methanol gave compound B-2 (white solid, 3.57 g, two-step yield 64%): ¹ HNMR (300 MHz, DMSO) δ 5.27-5.14 (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

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

Compound B-2 (3.5 g, 6.2 mmol) was dissolved in 50 mL of methanol, potassium hydroxide (3.4 g, 62 mmol) was added, heated to 50 ° C and stirred for reaction, TLC detected the reaction was complete, after the reaction was completed, cooled to room temperature, added 50 mL of water, extracted with ethyl acetate (50 mL×3), washed with saturated brine (50 mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and recrystallized from methanol to obtain compound B-1 (white solid, 2.7 g) , 87% yield): ¹ H NMR (300 MHz, DMSO) δ 5.29-5.12 (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-3β-propionyloxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B-3)

Compound B-1 (200 mg, 0.40 mmol) was dissolved in pyridine (3 mL), 4-dimethylaminopyridine (5 mg, 0.04 mmol) and propionic anhydride (133 uL, 1 mmol) were added successively, the reaction was stirred at room temperature, and TLC detected that the reaction was complete After that, 1N hydrochloric acid (5 mL) was added, extracted with ethyl acetate (5 mL×3), washed with saturated brine (5 mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether:acetic acid) ethyl ester=10:1) to obtain compound B-3 (white solid, 166 mg, 75% yield): ¹ HNMR (300 MHz, CDCl ₃ ) δ 5.24-5.16 (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-3β-butyryloxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B-4)

Referring to the method of Example 17, replace propionic anhydride with butyric anhydride to obtain compound B-4: ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.24-5.16 (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-3β-pentanoyloxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B-5)

Referring to the method of Example 17, replace propionic anhydride with valeric anhydride to obtain compound B-5: ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.23-5.13 (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-3β-(4-fluorobenzoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B-6 )

Compound B-1 (200 mg, 0.40 mmol) was dissolved in dichloromethane (3 mL), and N,N'-dicyclohexylcarbonimide (247 mg, 1.2 mmol), 4-dimethylaminopyridine (48 mg) were added successively. , 0.4 mmol) and p-fluorobenzoic acid (122 mg, 1 mmol), and the reaction was stirred at room temperature. TLC detected the completion of the reaction, washed with water (5 mL×2), dried over anhydrous sodium sulfate, filtered, concentrated the filtrate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain compound B-6 (white Solid, 186 mg, 80% yield): ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.05 (dd, J=8.3, 5.6 Hz, 2H), 7.10 (t, J=8.5 Hz, 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

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

Referring to the method of Example 20, replace p-fluorobenzoic acid with p-chlorobenzoic acid to obtain compound B-7: ¹ H NMR (300MHz, CDCl ₃ )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-3β-(4-methylbenzoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B- 8)

Referring to the method of Example 20, p-fluorobenzoic acid was replaced with p-toluic acid to obtain compound B-8: ¹ HNMR (300 MHz, CDCl ₃ ) δ7.99 (d, J=8.7 Hz, 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-3β-(4-methoxybenzoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B -9)

Referring to the method of Example 20, p-fluorobenzoic acid was replaced with p-methoxybenzoic acid to obtain compound B-9: ¹ HNMR (300MHz, CDCl ₃ )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-3β-(4-trifluoromethoxybenzoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane ( Compound B-10)

Referring to the method of Example 20, replace p-fluorobenzoic acid with p-trifluoromethoxybenzoic acid to obtain compound B-10: ¹ H NMR (300 MHz, CDCl ₃ ) δ 8.10 (d, J=8.7 Hz, 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-3β-(4-methoxyacylbenzoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B -11)

Referring to the method of Example 20, p-fluorobenzoic acid was replaced with p-methoxyacylbenzoic acid to obtain compound B-11: ¹ HNMR (300 MHz, CDCl ₃ ) 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-3β-(3-picolinoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B-12)

Referring to the method of Example 20, replace p-fluorobenzoic acid with nicotinic acid to prepare compound B-12: ¹ H NMR (300MHz, CDCl ₃ )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-3β-(2-carboxybenzoyl)oxy-28-(5,6-dihydro-4H-1,3-oxazin-2-yl)-oleanane (Compound B-13 )

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

Compound I-6 (200 mg, 0.272 mmol) was dissolved in DCM (4 mL), 10% palladium on carbon (10 mg) was added, and the reaction was stirred at room temperature under a hydrogen atmosphere overnight. After the completion of the reaction was detected by TLC, celite was filtered, and the filtrate was concentrated to obtain compound B-15 (white solid, 164 mg, yield 92%): ¹ H NMR (300 MHz, CDCl ₃ ) 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 [MH] ⁻ .

Example 28

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

Dissolve compound B-1 (200 mg, 0.40 mmol) in dichloromethane (8 mL), first add 1H-tetrazolium (109 μL, 1.2 mmol), stir at room temperature for 10 minutes, and then add dibenzyl N,N-dibenzyl Isopropyl phosphoramidite (153 μL, 0.45 mmol) was stirred at room temperature for 4 hours under nitrogen atmosphere, after the reaction was detected by TLC, m-chloroperoxybenzoic acid (157 mg, 0.9 mmol) was added, stirred at room temperature for 4 hours, and the reaction was detected by TLC After completion, add saturated ammonium chloride solution (8 mL) to quench, wash with saturated sodium bicarbonate solution (5 mL×2), dry over anhydrous sodium sulfate, filter, concentrate the filtrate, and perform silica gel column chromatography (petroleum ether: ethyl acetate) Ester=15:1) was purified to give compound I-7 (white solid, 275 mg, 80% yield).

Compound I-7 (200 mg, 0.26 mmol) was dissolved in a mixed solvent of tetrahydrofuran (2 mL) and methanol (2 mL), 10% palladium on carbon (10 mg) was added, and the reaction was stirred at room temperature under a hydrogen atmosphere overnight. After the completion of the reaction was detected by TLC, celite was filtered, the filtrate was concentrated, and the filtrate was slurried with ethyl acetate to obtain compound B-14 (white solid, 61 mg, yield 40%): ¹ H NMR (300 MHz, C ₅ D ₅ N)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 [MH] ⁻ .

Example 29

Activity evaluation of compounds on AMPK agonism in Huh-7 cells

The agonistic activity of compounds on AMPK in Huh-7 cells was detected by Western Blot.

1. Cell line: Huh-7 cells (human hepatoma cells, purchased from the Stem Cell Bank of the Chinese Academy of Sciences), in DMEM complete medium (containing 10% fetal bovine serum and 1% streptomycin/penicillin), containing 5% CO at 37°C ₂ in a cell incubator.

2. Antibodies: 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 Anti (Biyuntian).

3. Western Blot experiment: To detect the effect of compounds on the phosphorylation level of AMPK in Huh-7 cells.

Cells with a viable cell ratio of more than 90% were used for experiments. In 12-well plates, Huh-7 cells were plated at 200,000 per well. Incubate in an incubator at 37 °C with 5% _CO for 24 h, adherent. The test compounds were administered under the condition of complete medium. The final concentration of the compounds was set to 10 μM, and the administration time was 12 hours. AICAR (200 μM) and oleanolic acid (10 μM) were used as positive control compounds. The protein was then extracted for Western Blot detection. Specific steps are as follows:

Protein sample preparation: discard the original culture medium, wash three times with 1×PBS, discard the PBS, and add 100 μl RIPAbuffer (1×PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, PMSF, etc.) to each well , incubate on ice for 15min, scrape the cells with a cell scraper, suck into a new 1.5ml EP tube, centrifuge at 12000g at 4°C for 15min, transfer the supernatant to a new 1.5ml EP tube, place on ice, add 1/5 volume of 6× loading buffer, heated in a metal bath at 95°C for 10 min, centrifuged for 1 min, and frozen at -20°C for later use.

Protein quantification: Dilute the protein sample by 20 times, in a 96-well plate, add 20 μl of the diluted protein sample and 200 μl of BCA (solution A: solution B = 5:1) reagent, incubate at 37°C for 30 min, and place on a microplate reader. The OD value was measured at a wavelength of 562 nm, and the protein concentration was calculated according to the standard curve.

Electrophoresis: cast SDS-PAGE gel, the concentration of separating gel is 10%, and the concentration of stacking gel is 4%. The protein samples were pre-heated in a 95°C metal bath for 4 min and then centrifuged for 1 min. Each sample was loaded with 30 μg of total protein, which was added one by one with a micro-injector. Connect the power supply (pay attention to the connection of the positive and negative electrodes), and start electrophoresis at a constant voltage of 60V. When the protein sample enters the separation gel, adjust the voltage to 100V to continue the constant voltage electrophoresis. When bromophenol blue reaches the bottom of the separation gel, the electrophoresis is terminated according to the separation of protein markers.

Transfer membrane: Gently remove the gel after electrophoresis, cut off the unnecessary part, and immerse the required gel in Transferbuffer. Prepare a PVDF membrane with the same size as the gel, soak the PVDF membrane with methanol for 1 min before use, transfer it to the Transferbuffer, and at the same time put the filter paper into the Transfer buffer to soak. Sponge, filter paper, glue, PVDF membrane, filter paper and sponge are laid on the electrode plate of the cathode of the transfer instrument in sequence. Avoid air bubbles between each layer. The power was turned on, and the 200mA constant current ice bath was transferred for 2.5h.

Antibody hybridization: After membrane transfer, take out the PVDF membrane and wash it once with 1×TBST, put it into the pre-prepared blocking solution (5% BSA solution in 1×TBS containing 0.1% Tween20), and block at room temperature for 1 h. The primary antibody was incubated at 4°C overnight. The next day (after 12h), wash three times with 1×TBST for 10min each time. Secondary antibody incubation: Incubate with 5% BSA-diluted secondary antibody (1:10000) at room temperature for 1 h, and wash 3 times with 1×TBST for 10 min each time. Aspirate the excess liquid on the PVDF membrane, spread it on the exposure plate, and add an equal volume of mixed ECL kit liquid Tanon ^TM High-sig ECL Western Blotting Peroxide Buffer and Tanon ^TM High-sig ECL Western Blotting Luminol/Enhancer Solution, using Tanon Chemiluminescence imager, ECL imaging, collection of immunoreactive bands.

4. Experimental results: After grayscale scanning of the Western Blot experimental results, the p-AMPK/AMPK ratio of the negative control DMSO was defined as 1, and the p-AMPK/AMPK ratio of the synthesized test compound of the present invention was the relative ratio of the negative control group. , the larger the value, the stronger the AMPK agonistic activity of the compound. The activity data results are shown in Table 1.

Table 1. AMPK agonistic activity of compounds (positive control AICAR concentration is 200 μM; oleanolic acid, test compound concentration is 10 μM)

As shown in the experimental results in Table 1 and Figure 2, at a concentration of 10 μM, the novel oleanolic acid derivatives provided by the present invention have significant AMPK agonistic activity. For example, Compounds A-1, A-2, A-3, A-4, A-5, A-12, B-5, B-12, B-13, B-14 compounds are all potent AMPK agonists and the activity was significantly better than 200 μM AICAR and oleanolic acid, especially A-1, A3 and B14. The above experimental results show that the compounds of the present invention have significant agonistic activity on AMPK, so they can be used to prepare AMPK agonists with the activity of enhancing the phosphorylation level of AMPK, and further can be used to prepare medicines for preventing or treating AMPK-mediated diseases. Other compounds not listed in the examples of the present invention can refer to the above-mentioned examples for their synthesis methods, and also have significant agonistic activity on AMPK.

Example 30

tablet

Compound B-14 prepared in Example 28 or compounds of other embodiments (50g), hydroxypropyl methylcellulose E (150g), starch (200g), an appropriate amount of povidone K30 and magnesium stearate (1g) ) mixing, granulation, and tableting.

In addition, according to the conventional preparation method of the 2015 edition of the Pharmacopoeia, the compounds prepared in Examples 1 to 28 can be given different pharmaceutical excipients to make capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments tablets, suppositories, or patches.

Claims (10)

1. The oleanolic acid derivative represented by the following formula I or formula II or a pharmaceutically acceptable salt thereof:

R is H,

R ^a CO- or PO ₃ H ₂ ; R ¹ is H; R ^a is C ₁ -C ₅ alkyl. 2. The derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the derivative or a pharmaceutically acceptable salt thereof is selected from the following compounds:

3. An oleanolic acid derivative or a pharmaceutically acceptable salt thereof, characterized in that the derivative or a pharmaceutically acceptable salt thereof is selected from the following compounds:

4. A use of the derivative according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of an AMPK agonist having the activity of enhancing the phosphorylation level of AMPK. 5. Use of a derivative according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating AMPK-mediated diseases. 6. purposes according to claim 5, described AMPK-mediated diseases include metabolic diseases, cardiovascular and cerebrovascular diseases, inflammatory diseases, autoimmune diseases, organ fibrosis diseases, neurodegenerative diseases, caused by pathogen infection secondary disease, mitochondrial dysfunction or disorder, or tumor. 7. The use according to claim 5, wherein the AMPK-mediated diseases include 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, non-alcoholic fatty liver, non-alcoholic steatohepatitis, alcoholic fatty liver, cirrhosis, gout, stroke, brain 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, phlebitis, intermittent claudication , keloid, psoriasis, ichthyosis, bullous pemphigoid, dermatitis, contact dermatitis, pancreatitis, chronic nephritis, cystitis, meningitis, gastritis, sepsis, pyoderma gangrenosum, uveitis , Parkinson's disease, Alzheimer's disease, alpha-synucleinopathy, depression, multiple sclerosis, amyotrophic lateral sclerosis, fibromyalgia syndrome, neuralgia, Down's syndrome, Halover Den-Spar disease, Huntington's disease, Wilson disease, myasthenia, myoclonus, exercise intolerance, Kearns-Saier syndrome, chronic fatigue syndrome, Leigh syndrome, mitochondrial myopathy-encephalopathy- Hyperlactatemia, stroke syndrome, stroke-like episodes, Duchenne muscular dystrophy, Shell muscular dystrophy, Friedrich's ataxia, or tumor. 8. A pharmaceutical composition having the activity of enhancing the phosphorylation level of AMPK, comprising the derivative according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable Accessories. 9. A pharmaceutical composition for preventing or treating AMPK-mediated diseases, comprising the derivative according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable Accessories. 10. The pharmaceutical composition according to claim 8 or 9, wherein the pharmaceutical composition is capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment tablets, suppositories or patches.

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