CN112142818A

CN112142818A - Oleanolic acid and oleanolic acid derivatives and medical application thereof

Info

Publication number: CN112142818A
Application number: CN201910572404.0A
Authority: CN
Inventors: 孙宏斌; 胡凯文; 刘柳; 程亚龙; 秦鹿柘; 李浩斌; 戴量; 柳军
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2020-12-29
Anticipated expiration: 2039-06-27
Also published as: CN112142818B; WO2020259205A1

Abstract

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

Description

Oleanolic acid and oleanolic acid derivatives 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 oleanolic acid and an oleanolic acid derivative and medical application thereof, and further relates to application of the compound in preparation of 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). While 3 β -hydroxy-oleanane-13 (18) -en-28-oic acid (-oleanolic acid) is a pentacyclic triterpenic acid which is very rare in nature (Phytochemistry 1999,51, 83). To date, very few documents report the biological activity of oleanolic acid.

Disclosure of Invention

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

The present inventors have surprisingly found a series of potent novel AMPK agonists, whose AMPK agonistic activity is significantly superior to the accepted AMPK agonist AICAR, when-oleanolic acid and oleanolic acid are structurally modified.

The technical scheme is as follows: in order to achieve the above objects, an oleanolic acid derivative or an oleanolic acid derivative, a pharmaceutically acceptable salt or ester or solvate thereof, as described in the present invention, represented by the following formula I or formula II:

wherein the content of the first and second substances,

r is H,

Or R^aCO-；

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；

R' is OR²、NR³R⁴Or

R²Is C substituted by a substituent Z₁-C₅Alkyl, the substituent Z being pyrrolidin-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;

R³is H or C₁-C₃An alkyl group;

R⁴is C substituted by a substituent W₁-C₅Alkyl, the substituent W is pyrrolidin-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, diethanolamino or acetylamino;

R⁵is H, OH, F, NH attached to any carbon of the ring₂、C₁-C₃Alkylamino radical, COOH, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, cycloalkyloxy or heterocycloalkyloxy;

q is CH₂、O、NR⁶、S、SO₂、CHR⁷Or a chemical bond;

R⁶is H, C₁-C₃Alkyl radical, C₁-C₃Alkyl acyl radical, C₁-C₃C substituted by alkylsulfonyl or by substituent P₁-C₃Alkyl, the substituent P is OH, COOH, NH₂Or C₁-C₃An alkylamino group;

R⁷is OH, NH₂、COOH、C₁-C₃Alkylamino radical, C₁-C₃Alkoxy, cycloalkyloxy or heterocycloalkyloxy;

n is 0,1 or 2;

m is 0,1 or 2.

In certain preferred embodiments, the compound of formula I or formula II, a pharmaceutically acceptable salt or ester or solvate thereof,

r is H,

Or R^aCO-；

R^ac unsubstituted or substituted by substituents L₁-C₅Alkyl radical ofThe substituent L is 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；

R' is OR²、NR³R⁴Or

R²Is C substituted by a substituent Z₁-C₅Alkyl, the substituent Z being pyrrolidin-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; r³Is H;

R⁵is H, OH, F, NH attached to any carbon of the ring₂、C₁-C₃Alkylamino or COOH;

q is CH₂、O、NR⁶、S、SO₂Or CHR⁷；

R⁷is OH, NH₂COOH or C₁-C₃An alkylamino group;

n is 0 or 1;

m is 0 or 1.

Still further, in said compound of formula II, when substituent Z is N, N-dimethylamino or trimethylammonium, R is not H; when the substituent W is acetylamino, R is not H.

In certain more 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.

Oleanolic acid is commercially available. The synthesis of oleanolic acid can be carried out according to the literature method (org. biomol. chem.,2016,14,11154), i.e. firstly, the benzyl ester of oleanolic acid is prepared according to the literature method, and then, the benzyl ester of oleanolic acid is subjected to the conventional catalytic hydrogenation debenzylation reaction to prepare the oleanolic acid. The synthesis of oleanolic acid and oleanolic acid derivatives can be carried out by referring to the methods of examples or modified methods.

The invention provides application of the compound shown in the formula I or the formula II, and pharmaceutically acceptable salt, ester or solvate thereof in preparing AMPK agonist with the activity of enhancing AMPK phosphorylation level. 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, and 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 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 excipients which can be mixed arbitrarily may vary 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.

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

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

(2) Compared with the lead compounds-oleanolic acid and 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 pentacyclic triterpenoid is used 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 graph of the effect of some compounds on AMPK downstream signaling pathways in 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

(2- (Trimethylammonium) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-1)

Compound I-1(1.27g, 5.0mmol) was placed in a Schlenk tube, and a solution of trimethylamine in tetrahydrofuran (20mL, 40mmol) was added thereto, followed by heating at 55 ℃ for 24 hours under an argon atmosphere. After completion of the reaction, the mixture in the reaction tube was transferred to a bottle in the shape of eggplant with methylene chloride, concentrated under reduced pressure, the residue was suction-filtered, and the filter cake was washed with methylene chloride (5 mL. times.3) to obtain compound I-2 (white solid, 1.166g, yield 75%).

Compound I-2(1.166g, 3.723mmol) was suspended in a mixed solution of chloroform and ethanol (20mL, v: v ═ 7:3), cooled to 0 ℃, and then hydrazine hydrate (340 μ L) was added, and the reaction was heated under reflux at 50 ℃ for 12 hours under an argon atmosphere. After the reaction, the reaction solution was filtered, the filter cake was washed with a mixed solvent of chloroform and ethanol (5mL × 3, v: v ═ 8.5:1.5), the filtrate was concentrated and dried to obtain a crude compound I-3, which was used directly in the next reaction.

The crude compound I-3 from the above step was dissolved in deionized water (2mL), cooled to 0 deg.C, and added aqueous hydrobromic acid (2mL, 48%) and stirred at 0 deg.C for 1 hour. A mixed solvent of toluene and methanol (5mL, v: v ═ 4.5:0.5) was added to the reaction solution, and the mixture was concentrated at 45 ℃ under reduced pressure, and this operation was repeated twice, and then a large amount of white solid was precipitated, which was filtered with suction, and the filter cake was washed with deionized water (1mL × 2). The filtrate was further added with a mixed solvent of toluene and methanol (5mL, v: v ═ 4.5:0.5), concentrated at 45 ℃ under reduced pressure until most of the solvent was removed, acetonitrile (5mL) was added and slurried, filtered with suction, the filter cake was washed with a small amount of methanol (1mL) and dried to give compound I-4 (white solid, 915mg, two-step yield 93%):¹H NMR(300MHz,D₂O)3.82–3.73(m,2H),3.66–3.57(m,2H),3.30(s,9H).

dissolving oleanolic acid (-OA, 100mg, 0.219mmol) in N, N-dimethylformamide (8mL), adding triethylamine (122 mu L, 0.876mmol), adding 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU,108mg, 0.284mmol) at room temperature under stirring, reacting for 1 hour, adding compound I-4(58mg, 0.219mmol), and heating the reaction solution to 50 ℃. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and deionized water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic layers were combined, washed with deionized water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 10: 1) to give compound I-5 (white solid, 41mg, yield 30%).

Compound I-5(175mg, 0.281mmol) was suspended in anhydrous dichloromethane (10mL), and N, N' -dicyclohexylcarbodiimide (DCC,232mg, 1.124mmol), 4-dimethylaminopyridine (DMAP,34mg, 0.281mmol) and monobenzyl phthalate (86mg, 0.337mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, the filtrate was washed with deionized water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to obtain compound I-6 (white solid, 100mg, yield 41%).

Dissolving compound I-6(100mg, 0.116mmol) in tetrahydrofuran10% Palladium on carbon (10mg) was added to pyran (8mL), and the reaction was stirred overnight at room temperature under a hydrogen atmosphere. After completion of the TLC detection reaction, celite was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol 10: 1) to give compound a-1 (white solid, 66mg, 73% yield):¹H NMR(300MHz,DMSO-d₆)13.18(s,1H),7.79–7.69(m,1H),7.69–7.57(m,3H),7.17–7.08(m,1H),4.74–4.60(m,1H),3.59–3.43(m,2H),3.12(s,9H),2.81(d,J＝14.1Hz,1H),2.49–2.37(m,1H),2.17(d,J＝13.2Hz,1H),1.18(s,3H),0.93(s,3H),0.91(s,9H),0.84(s,3H),0.74(s,3H).ESI-MS:m/z 689.6[M-Br]⁺。

example 2

N- (2- (dimethylamino) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-3)

Dissolving oleanolic acid (5g, 0.011mol) in anhydrous pyridine (35mL), slowly adding acetic anhydride (4mL) dropwise under stirring, and heating and refluxing for reaction for 1 hour after dropwise addition. After completion of the TLC detection reaction, the reaction mixture was cooled to room temperature, and dropped into ice water (20 mL. times.2) to precipitate a large amount of white solid, which was filtered, and the filter cake was washed with water (10 mL. times.3) and n-hexane (10 mL. times.3), dried in vacuo, and recrystallized from ethanol to obtain Compound II-1 (white solid, 4.52g, yield 83%).

Compound II-1(150mg, 0.30mmol) was dissolved in anhydrous dichloromethane (5mL), and oxalyl chloride (130. mu.L, 1.50mmol) and N, N-dimethylformamide (1 drop) were slowly added dropwise with stirring and reacted at room temperature for 3 hours. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure to obtain Compound II-2 (yellow solid, 155mg, yield 100%).

N, N-dimethylethylenediamine (66. mu.L, 0.60mmol) was dissolved in anhydrous dichloromethane (5mL), and a solution of Compound II-2(155mg, 0.30mmol) in anhydrous dichloromethane (5mL) and triethylamine (84. mu.L, 0.60mmol) were slowly added dropwise with stirring, and the mixture was reacted at room temperature for 5 hours. After completion of the TLC detection, dichloromethane (10mL) was added to dilute the reaction solution, and the reaction solution was washed with saturated sodium bicarbonate solution (10mL × 3), water (10mL × 3), and saturated brine (10mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, concentrated the filtrate, and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound II-3 (white solid, 143mg, 84% yield).

Compound II-3(141mg, 0.248mmol) was suspended in methanol (10mL), and potassium hydroxide (140mg, 2.48mmol) was added thereto, followed by stirring at room temperature overnight. After completion of the TLC detection reaction, the reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was treated with ethyl acetate (20mL) and water (20mL), the organic phase was washed successively with water (20 mL. times.3) and saturated brine (20 mL. times.3), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound II-4 (white solid, 87mg, yield 67%).

Compound II-4(87mg, 0.165mmol) was dissolved in anhydrous pyridine (10mL), phthalic anhydride (245mg, 1.65mmol) and 4-dimethylaminopyridine (20mg, 0.165mmol) were added, and the reaction mixture was heated to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, dichloromethane (20mL) was added to dilute the reaction solution, which was washed with 1N diluted hydrochloric acid (10mL × 3) and water (10mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound a-3 (white solid, 93mg, yield 84%):¹H NMR(300MHz,CDCl₃)7.84–7.63(m,1H),7.63–7.51(m,1H),7.51–7.30(m,2H),6.72–6.50(m,1H),4.82–4.59(m,1H),3.77–3.43(m,2H),3.07–2.83(m,2H),2.83–2.70(m,1H),2.63(s,6H),2.41(d,J＝13.7Hz,1H),2.36–2.18(m,1H),1.16(s,3H),0.98(s,3H),0.87(s,9H),0.83(s,3H),0.72(s,3H).ESI-MS:m/z 673.3[M-H]^-。

example 3

N- (2- (1-piperidinyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-4)

Referring to the method of example 2, N,replacement of N-dimethylethylenediamine with 1- (2-aminoethyl) piperidine gave compound a-4:¹H NMR(300MHz,CDCl₃)7.74(d,J＝7.1Hz,1H),7.52(d,J＝6.9Hz,1H),7.47–7.29(m,2H),6.77–6.64(m,1H),4.78–4.59(m,1H),3.75–3.46(m,2H),3.08–2.67(m,7H),2.43(d,J＝13.6Hz,1H),2.31(d,J＝10.1Hz,1H),1.16(s,3H),0.98(s,3H),0.88(s,3H),0.86(s,6H),0.79(s,3H),0.72(s,3H).ESI-MS:m/z 715.5[M+H]⁺。

example 4

N- (2- (4-morpholinyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-5)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 4- (2-aminoethyl) morpholine, compound a-5 was prepared:¹H NMR(500MHz,CDCl₃)7.76(d,J＝5.8Hz,1H),7.63(d,J＝5.8Hz,1H),7.52–7.41(m,2H),6.57–6.46(m,1H),4.76–4.67(m,1H),3.76(s,4H),3.57–3.38(m,2H),2.81(d,J＝13.8Hz,1H),2.63(s,6H),2.45(d,J＝13.5Hz,1H),2.42–2.33(m,1H),1.18(s,3H),0.95(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.84(s,3H),0.74(s,3H).ESI-MS:m/z 715.5[M-H]^-。

example 5

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (compound A-6)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 1- (2-aminoethyl) pyrrolidine produced compound a-6:¹H NMR(300MHz,CDCl₃)7.73(d,J＝7.3Hz,1H),7.49(d,J＝7.1Hz,1H),7.37(dt,J＝19.0,7.3Hz,2H),4.78–4.65(m,1H),3.72–3.54(m,2H),3.26–3.08(m,4H),3.08–2.95(m,2H),2.77(d,J＝13.3Hz,1H),2.42(d,J＝13.5Hz,1H),2.31(d,J＝12.0Hz,1H),1.16(s,3H),1.02(s,3H),0.88(s,3H),0.86(s,6H),0.82(s,3H),0.72(s,3H).ESI-MS:m/z 699.3[M-H]^-。

example 6

N- (2- (4-methyl-1-piperazinyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-8)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 1- (2-aminoethyl) -4-methylpiperazine produced compound a-8:¹H NMR(300MHz,CDCl₃)7.75(d,J＝6.9Hz,1H),7.64(d,J＝7.0Hz,1H),7.53–7.35(m,2H),6.46–6.36(m,1H),4.80–4.69(m,1H),3.54–3.30(m,2H),3.04–2.80(m,4H),2.73(s,5H),2.57(s,5H),2.53–2.45(m,1H),2.46–2.38(m,1H),1.24(s,3H),1.00(s,3H),0.97(s,3H),0.93(s,9H),0.78(s,3H).ESI-MS:m/z 730.5[M+H]⁺。

example 7

N- (2- (4-morpholinyl) ethyl) -3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-12)

The oleanolic acid (OA, 20g,0.044mol) is dissolved in N, N-dimethylformamide (200mL), potassium carbonate (6g,0.044mol) is added, benzyl bromide (8mL, 0.044mol) is slowly added dropwise with stirring, and after the dropwise addition is finished, the reaction solution is heated to 85 ℃ to react for 10 hours. After the TLC detection reaction was completed, suction filtration was performed while hot, the filter cake was washed with hot N, N-dimethylformamide (10 mL. times.3), the filtrate was cooled to room temperature and dropped into ice water (100 mL. times.2), and a large amount of white solid was precipitated, suction filtration was performed, the filter cake was washed with water (50 mL. times.3), and N-hexane (50 mL. times.3), and vacuum drying was performed to obtain compound III-1 (white solid, 22.02g, yield 92%).

Compound III-1(22g,0.040mol) was dissolved in dichloromethane (200mL), and protonated montmorillonite (44g) was added to warm the reaction mixture to 40 ℃ for 8 hours. After completion of the TLC reaction, it was cooled to room temperature, filtered with suction, the filter cake was washed with dichloromethane (50mL × 3), the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 10: 1) to obtain compound III-2 (white solid, 16.52g, yield 75%).

Compound III-2(1g, 1.829mmol) was dissolved in anhydrous pyridine (10mL), phthalic anhydride (2.708g, 18.29mmol) and 4-dimethylaminopyridine (223mg, 1.829mmol) were added, and the reaction mixture was warmed to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, dichloromethane (30mL) was added to dilute the reaction solution, which was washed with 1N diluted hydrochloric acid (10 mL. times.3) and water (10 mL. times.3) in this order, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound III-3 (white solid, 925mg, 73% yield).

Compound III-3(150mg, 0.216mmol) was dissolved in anhydrous dichloromethane (6mL), and N, N' -dicyclohexylcarbodiimide (134mg, 0.647mmol), 4-dimethylaminopyridine (27mg, 0.216mmol) and N-acetylethanolamine (30. mu.L, 0.324mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, the filtrate was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 3: 1) to obtain compound III-4 (white solid, 159mg, yield 95%).

Compound III-4(159mg, 0.204mmol) was dissolved in tetrahydrofuran (10mL), 10% palladium on carbon (16mg) was added, and the reaction was stirred at room temperature under a hydrogen atmosphere overnight. After completion of TLC detection, celite was filtered, the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate 1: 1) to give compound III-5 (white solid, 90mg, yield 64%)

Dissolving the compound III-5(100mg, 0.145mmol) in N, N-dimethylformamide (6mL), adding triethylamine (40. mu.L, 0.290mmol), adding 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (72mg, 0.188mmol) under stirring at room temperature, reacting for 1 hour, adding N- (2-aminoethyl) morpholine (23. mu.L, 0.174mmol), and heating the reaction solution to 50 ℃. After completion of the TLC detection, dichloromethane (10) was addedmL) and water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic layers were combined, washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane: methanol 30: 1) purification gave compound a-12 (white solid, 80mg, 69% yield):¹H NMR(300MHz,CDCl₃)7.81(d,J＝7.3Hz,1H),7.66(d,J＝7.0Hz,1H),7.63–7.50(m,2H),6.60–6.49(m,1H),6.49–6.36(m,1H),4.77–4.65(m,1H),4.44(t,J＝4.8Hz,2H),3.76–3.56(m,6H),3.47–3.25(m,2H),2.86(d,J＝15.4Hz,1H),2.53–2.33(m,8H),2.03(s,3H),1.21(s,3H),0.97(s,3H),0.96(s,6H),0.94(s,3H),0.89(s,3H),0.75(s,3H).ESI-MS:m/z 824.6[M+Na]⁺。

example 8

N- (2- (4-methyl-1-piperazinyl) ethyl) -3 beta- (2- (2-acetamidoethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-14)

Referring to the procedure of example 7, substituting 4- (2-aminoethyl) morpholine for 1- (2-aminoethyl) -4-methylpiperazine produced compound a-14:¹H NMR(300MHz,CDCl₃)7.81(d,J＝7.2Hz,1H),7.66(d,J＝7.0Hz,1H),7.62–7.48(m,2H),6.58–6.48(m,1H),6.45–6.36(m,1H),4.76–4.67(m,1H),4.44(t,J＝4.8Hz,2H),3.73–3.55(m,2H),3.43–3.21(m,2H),2.85(d,J＝15.3Hz,1H),2.71–2.51(m,6H),2.51–2.42(m,4H),2.42–2.38(m,1H),2.36(s,4H),2.03(s,3H),1.20(s,3H),0.97(s,3H),0.95(s,9H),0.90(s,3H),0.75(s,3H).ESI-MS:m/z 815.7[M+H]⁺。

example 9

N- (2- (trimethylammonio) ethyl) -3 beta- (2- (2-acetamido-ethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-15)

Compound III-5 was prepared according to the method of example 7, compound III-5(100mg, 0.145mmol) was dissolved in N, N-dimethylformamide (8mL), triethylamine (80. mu.L, 0.580mmol) was added, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (72mg, 0.188mmol) was added under stirring at room temperature, compound I-4(60mg, 0.174mmol) (prepared in example 1) was added after 1 hour of reaction, and the reaction solution was heated to 50 ℃. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and deionized water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic layers were combined, washed with deionized water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to give compound a-15 (white solid, 80mg, yield 65%):¹H NMR(300MHz,DMSO-d₆)8.09–7.98(m,1H),7.83–7.74(m,2H),7.74–7.64(m,2H),7.17–7.06(m,1H),4.74–4.61(m,1H),4.30–4.14(m,2H),3.59–3.45(m,2H),3.44–3.33(m,4H),3.13(s,9H),2.91–2.72(m,1H),2.48–2.39(m,1H),2.18(d,J＝12.1Hz,1H),1.84(s,3H),1.19(s,3H),0.92(s,12H),0.86(s,3H),0.75(s,3H).ESI-MS:m/z 774.6[M-Br]⁺。

example 10

N- (3- (trimethylammonio) propyl) -3 beta- (2- (2-acetamido-ethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-16)

Compound I-4 'was prepared by substituting compound I-1 for compound I-1' according to the procedure of example 1. Referring again to the procedure of example 9, compound I-4 was replaced with I-4' to give compound A-16:¹H NMR(300MHz,DMSO-d₆)8.08–7.96(m,1H),7.79–7.71(m,2H),7.67(d,J＝8.6Hz,2H),7.04–6.88(m,1H),4.71–4.60(m,1H),4.29–4.13(m,2H),3.41–3.29(m,2H),3.29–3.18(m,2H),3.18–3.08(m,2H),3.04(s,9H),2.79(d,J＝12.5Hz,1H),2.43(d,J＝14.3Hz,1H),2.17(d,J＝12.9Hz,1H),1.81(s,3H),1.16(s,3H),0.90(s,12H),0.83(s,3H),0.72(s,3H).ESI-MS:m/z 788.6[M-Br]⁺。

example 11

N- (2- (trimethylammonio) ethyl) -3 beta- (2-methoxylbenzoyl) oxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-17)

Compound III-2 was obtained by the method described in example 7, and Compound III-2(150mg, 0.274mmol) was dissolved in anhydrous dichloromethane (6mL), N' -dicyclohexylcarbodiimide (170mg, 0.823mmol), 4-dimethylaminopyridine (34mg, 0.274mmol) and monomethyl phthalate (54mg, 0.302mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, the filtrate was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 20: 1) to obtain compound IV-1 (white solid, 159mg, yield 82%).

Compound IV-1(159mg, 0.224mmol) was dissolved in tetrahydrofuran (10mL), 10% palladium on carbon (20mg) was added, and the mixture was stirred under hydrogen atmosphere at room temperature overnight. After completion of the TLC reaction, celite was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate 1: 1) to obtain compound IV-2 (white solid, 118mg, yield 85%).

Dissolving a compound IV-2(150mg, 0.242mmol) in N, N-dimethylformamide (8mL), adding triethylamine (135 mu L, 0.970mmol), adding 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (120mg, 0.315mmol) under stirring at room temperature, reacting for 1 hour, adding a compound I-4(128mg, 0.485mmol), and heating the reaction solution to 50 ℃. After completion of the TLC detection reaction, the reaction mixture was treated with dichloromethane (10mL) and deionized water (5mL), the aqueous layer was extracted with dichloromethane (10 mL. times.2), the organic layers were combined, washed with deionized water (5 mL. times.2), dried over anhydrous sodium sulfate,filtration and concentration of the filtrate, purification by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) gave compound a-17 (white solid, 77mg, 40% yield):¹H NMR(300MHz,DMSO-d₆)7.82–7.64(m,4H),7.18–7.06(m,1H),4.74–4.64(m,1H),3.83(s,3H),3.59–3.45(m,2H),3.39–3.34(m,2H),3.13(s,9H),2.83(d,J＝13.4Hz,1H),2.50–2.41(m,1H),2.24–2.11(m,1H),1.19(s,3H),0.93(s,6H),0.92(s,6H),0.86(s,3H),0.75(s,3H).ESI-MS:m/z 703.6[M-Br]⁺。

example 12

N- (3- (trimethylammonio) propyl) -3 beta- (2-methoxylbenzoyl) oxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-18)

By substituting compound I-4 for I-4' according to the procedure of example 11, Compound A-18 was prepared:¹H NMR(300MHz,DMSO-d₆)7.80–7.63(m,4H),7.02–6.89(m,1H),4.76–4.62(m,1H),3.82(s,3H),3.30–3.20(m,2H),3.20–3.10(m,2H),3.06(s,9H),2.81(d,J＝14.7Hz,1H),2.46(d,J＝14.8Hz,1H),2.20(d,J＝13.1Hz,1H),1.19(s,3H),0.93(s,12H),0.86(s,3H),0.74(s,3H).ESI-MS:m/z 717.7[M-Br]⁺。

example 13

N- (3- (dimethylamino) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-19)

Dissolving oleanolic acid (-OA, 100mg, 0.219mmol) in N, N-dimethylformamide (8mL), adding triethylamine (122 mu L, 0.876mmol), adding 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (108mg, 0.285mmol) under stirring at room temperature, reacting for 1 hour, adding 3-dimethylaminopropylamine (55 mu L, 0.438mmol), and heating the reaction solution to 50 ℃. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic layers were combined, washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to obtain compound V-1 (white solid, 113mg, 96% yield).

Compound V-1(113mg, 0.209mmol) was dissolved in anhydrous pyridine (8mL), phthalic anhydride (309mg, 2.09mmol) and 4-dimethylaminopyridine (26mg, 0.209mmol) were added, and the reaction mixture was heated to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, the reaction solution was diluted by adding dichloromethane (20mL), washed with 1N diluted hydrochloric acid (10mL × 3) and water (10mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound a-19 (white solid, 121mg, yield 84%):¹H NMR(300MHz,CDCl₃)7.72(d,J＝7.2Hz,1H),7.55(d,J＝7.1Hz,1H),7.47–7.30(m,2H),6.36–6.27(m,1H),4.76–4.65(m,1H),3.46–3.18(m,2H),2.91–2.72(m,3H),2.62(s,6H),2.43(d,J＝14.2Hz,1H),2.39–2.30(m,1H),1.17(s,3H),0.98(s,3H),0.89(s,3H),0.87(s,6H),0.82(s,3H),0.72(s,3H).ESI-MS:m/z 689.6[M+H]⁺。

example 14

N- (3- (4-morpholinyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-20)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for N- (3-aminopropyl) morpholine, Compound A-20 was prepared:¹H NMR(300MHz,CDCl₃)7.90–7.78(m,1H),7.72–7.59(m,1H),7.59–7.46(m,2H),6.40–6.25(m,1H),4.79–4.66(m,1H),4.13–3.93(m,4H),3.50–3.22(m,2H),3.21–3.04(m,2H),3.04–2.88(m,2H),2.81(d,J＝13.7Hz,1H),2.47(d,J＝13.8Hz,1H),2.35(d,J＝11.9Hz,1H),2.17–2.00(m,2H),1.19(s,3H),0.95(s,3H),0.89(s,6H),0.87(s,6H),0.73(s,3H).

example 15

N- (3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-acyl) -morpholine (compound A-27)

Referring to the procedure of example 2, compound a-27 was prepared by substituting N, N-dimethylethylenediamine for morpholine:¹H NMR(300MHz,DMSO)7.94–7.85(m,1H),7.78–7.68(m,1H),7.63–7.49(m,2H),4.84–4.74(m,1H),3.80(s,4H),3.63(s,4H),2.77(d,J＝14.5Hz,1H),2.46(d,J＝13.2Hz,2H),1.19(s,3H),0.97(s,3H),0.92(s,3H),0.91(s,3H),0.90(s,3H),0.87(s,3H),0.79(s,3H).ESI-MS:m/z 672.5[M-H]^-。

example 16

3 beta- (2- (2-Acylaminoethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2- (4-morpholinyl) ethyl ester (Compound A-44)

Compound III-5 was obtained by the method described in example 7, and compound III-5(120mg, 0.174mmol) was dissolved in N, N-dimethylformamide (10mL), and 4- (2-bromoethyl) morpholine hydrobromide (57mg, 0.208mmol) and potassium carbonate (70mg, 0.522mmol) were added to stir the reaction at room temperature. After completion of the TLC detection reaction, the reaction solution was treated with ethyl acetate (20mL) and (10mL), the aqueous layer was extracted with ethyl acetate (10mL), the organic phases were combined, washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound a-44 (white solid, 87mg, yield 62%):¹H NMR(300MHz,CDCl₃)7.86–7.78(m,1H),7.69–7.63(m,1H),7.63–7.49(m,2H),6.64–6.47(m,1H),4.77–4.67(m,1H),4.44(t,J＝4.8Hz,2H),4.30–4.13(m,2H),3.77–3.55(m,6H),2.75(d,J＝14.0Hz,1H),2.59(t,J＝5.7Hz,2H),2.54–2.46(m,4H),2.42(d,J＝14.1Hz,1H),2.18(d,J＝6.9Hz,1H),2.03(s,3H),1.17(s,3H),0.96(s,6H),0.94(s,3H),0.93(s,3H),0.92(s,3H),0.74(s,3H).ESI-MS:m/z 825.6[M+Na]⁺。

example 17

N- (2- (trimethylammonio) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-66)

Compound a-66 was prepared according to the procedure of example 1:¹H NMR(300MHz,DMSO-d₆)7.16–7.03(m,1H),4.31(d,J＝5.1Hz,1H),3.59–3.41(m,2H),3.08–2.92(m,2H),2.80(d,J＝12.4Hz,1H),2.45(d,J＝14.4Hz,1H),2.17(d,J＝13.4Hz,1H),1.15(s,3H),0.91(s,3H),0.90(s,6H),0.85(s,3H),0.73(s,3H),0.68(s,3H).ESI-MS:m/z541.5[M-Br]⁺。

example 18

N- (3- (trimethylammonio) propyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide bromide (Compound A-67)

By substituting compound I-4 for I-4' according to the procedure of example 1, compound A-67 was prepared:¹H NMR(300MHz,DMSO-d6)7.00–6.89(m,1H),4.31(d,J＝5.0Hz,1H),3.28–3.19(m,2H),3.19–3.09(m,2H),3.05(s,9H),2.79(d,J＝13.4Hz,1H),2.44(d,J＝13.9Hz,1H),2.18(d,J＝13.3Hz,1H),1.15(s,3H),0.90(s,9H),0.85(s,3H),0.72(s,3H),0.68(s,3H).ESI-MS:m/z 555.5[M-Br]⁺。

example 19

N- (2- (dimethylamino) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (compound A-68)

Compound a-68 was prepared according to the procedure of example 2:¹H NMR(300MHz,CDCl₃)6.45(t,J＝4.7Hz,1H),3.45–3.31(m,2H),3.30–3.21(m,1H),2.93–2.80(m,1H),2.54–2.31(m,4H),2.23(s,6H),1.22(s,3H),1.03(s,3H),0.97(s,3H),0.94(s,3H),0.93(s,3H),0.81(s,3H),0.78(s,3H).ESI-MS:m/z 527.5[M+H]⁺。

example 20

N- (2- (1-piperidinyl) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (compound A-69)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 1- (2-aminoethyl) piperidine, compound a-69 was prepared:¹H NMR(300MHz,CDCl₃)6.71–6.26(m,1H),3.47–3.32(m,1H),3.32–3.16(m,2H),2.93–2.78(m,1H),2.55–2.22(m,8H),1.18(s,3H),0.99(s,3H),0.94(s,3H),0.90(s,3H),0.88(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 567.5[M+H]⁺。

example 21

N- (2- (4-morpholinylalkyl) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-70)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 4- (2-aminoethyl) morpholine, compound a-70 was prepared:¹H NMR(300MHz,CDCl₃)6.50–6.40(m,1H),3.76–3.60(m,4H),3.45–3.29(m,2H),3.29–3.18(m,1H),2.90–2.79(m,1H),2.55–2.31(m,8H),1.19(s,3H),0.99(s,3H),0.94(s,3H),0.89(s,3H),0.87(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 569.5[M+H]⁺。

example 22

N- (2- (4-methyl-1-piperazinyl) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-73)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 1- (2-aminoethyl) -4-methylpiperazine produced compound a-73:¹H NMR(300MHz,CDCl₃)6.53–6.44(m,1H),3.47–3.33(m,1H),3.34–3.18(m,2H),2.90–2.80(m,1H),2.64–2.33(m,12H),2.28(s,3H),1.19(s,3H),0.99(s,3H),0.94(s,3H),0.90(s,3H),0.89(s,3H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z 582.5[M+H]⁺。

example 23

N- (3- (dimethylamino) propyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-77)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 3-dimethylaminopropylamine, compound a-77 was prepared:¹H NMR(300MHz,CDCl₃)6.24(t,J＝5.1Hz,1H),3.46–3.32(m,1H),3.29–3.11(m,2H),2.87–2.73(m,1H),2.50–2.42(m,1H),2.41–2.35(m,1H),2.31(t,J＝7.1Hz,2H),2.22(s,6H),1.18(s,3H),0.99(s,3H),0.92(s,3H),0.91(s,3H),0.88(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 541.5[M+H]⁺。

example 24

N- (3- (4-morpholinylalkyl) propyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-78)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for N- (3-aminopropyl) morpholine, compound a-78 was prepared: 1H NMR (300MHz, CDCl)₃)6.03(t,J＝5.7Hz,1H),3.71(d,J＝4.6Hz,4H),3.43–3.28(m,1H),3.28–3.14(m,2H),2.87–2.76(m,1H),2.53–2.30(m,8H),1.19(s,3H),0.99(s,3H),0.91(s,3H),0.90(s,3H),0.88(s,3H),0.78(s,3H),0.74(s,3H).ESI-MS:m/z 583.5[M+H]⁺。

Example 25

N- (2- (acetylamino) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (compound A-83)

Referring to the procedure of example 2, compound a-83 was prepared by substituting N, N-dimethylethylenediamine for N-acetylethylenediamine:¹H NMR(300MHz,CDCl₃)6.49–6.35(m,1H),6.34–6.17(m,1H),3.55–3.28(m,4H),3.27–3.14(m,1H),2.88–2.74(m,1H),2.55–2.42(m,1H),2.42–2.30(m,1H),1.96(s,3H),1.19(s,3H),0.99(s,3H),0.91(s,6H),0.88(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 539.4[M-H]^-。

example 26

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) -morpholine (compound A-84)

Referring to the procedure of example 2, compound a-84 was prepared by substituting N, N-dimethylethylenediamine for morpholine:¹H NMR(300MHz,CDCl₃)4.01–3.69(m,4H),3.68–3.52(m,4H),3.27–3.17(m,1H),2.81–2.70(m,1H),2.54–2.38(m,2H),1.17(s,3H),0.99(s,3H),0.92(s,3H),0.87(s,3H),0.86(s,3H),0.78(s,3H),0.77(s,3H).ESI-MS:m/z 548.4[M+Na]⁺。

example 27

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) -thiomorpholine-1, 1-dioxide (compound A-85)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for morpholine thiomorpholine-1, 1-dioxide, compound a-85 was prepared:¹H NMR(300MHz,CDCl₃)4.38–4.12(m,4H),3.28–3.17(m,1H),3.07–2.93(m,4H),2.81–2.70(m,1H),2.53–2.44(m,1H),2.44–2.33(m,1H),1.19(s,3H),0.99(s,3H),0.92(s,3H),0.87(s,3H),0.86(s,3H),0.78(s,3H),0.77(s,3H).ESI-MS:m/z 572.4[M-H]^-。

example 28

N- (3 beta-hydroxy-oleanane-13 (18) -alkene-28-acyl) -4-methylpiperazine (compound A-87)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for N-methylpiperazine produced compound a-87:¹H NMR(300MHz,CDCl₃)4.01–3.58(m,4H),3.28–3.19(m,1H),2.83–2.71(m,1H),2.54–2.40(m,2H),2.40–2.31(m,4H),2.29(s,3H),1.17(s,3H),0.99(s,3H),0.91(s,3H),0.89(s,3H),0.88(s,3H),0.78(s,3H),0.77(s,3H).ESI-MS:m/z 539.5[M+H]⁺。

example 29

N- (2- (dimethylamino) ethyl) -N-methyl-3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-102)

Referring to the procedure of example 2, compound a-102 was prepared by substituting N, N-dimethylethylenediamine for N, N' -trimethylethylenediamine:¹H NMR(300MHz,CDCl₃)3.66–3.53(m,1H),3.53–3.38(m,1H),3.32–3.24(m,1H),3.22(s,3H),2.83–2.71(m,1H),2.57–2.36(m,4H),2.32(s,6H),1.19(s,3H),1.01(s,3H),0.96(s,3H),0.93(s,3H),0.89(s,3H),0.81(s,3H),0.79(s,3H).ESI-MS:m/z 541.5[M+H]⁺。

example 30

N- (2- (diethylamino) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-103)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for N, N-diethylethylenediamine, compound a-103 was prepared:¹H NMR(300MHz,CDCl₃)6.54–6.29(m,1H),3.50–3.32(m,1H),3.31–3.06(m,2H),2.87–2.78(m,1H),2.60–2.32(m,8H),1.18(s,3H),0.99(s,3H),0.93(s,3H),0.90(s,3H),0.88(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 555.4[M+H]⁺。

example 31

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) -piperidine (compound A-104)

Referring to the procedure of example 2, compound a-104 was prepared by substituting N, N-dimethylethylenediamine for piperidine:¹H NMR(300MHz,CDCl₃)3.88–3.54(m,4H),3.31–3.22(m,1H),2.84–2.74(m,1H),2.54–2.37(m,2H),1.20(s,3H),1.02(s,3H),0.93(s,6H),0.91(s,3H),0.80(s,3H),0.80(s,3H).ESI-MS:m/z 546.4[M+Na]⁺。

example 32

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) -piperazine (compound A-86)

Referring to the procedure of example 2, compound a-86 was prepared by substituting N, N-dimethylethylenediamine for piperazine:¹H NMR(300MHz,CDCl₃)3.87–3.64(m,3H),3.28–3.18(m,1H),2.88–2.78(m,3H),2.78–2.70(m,1H),2.45(d,J＝12.8Hz,2H),2.00–1.87(m,2H),1.17(s,3H),0.99(s,3H),0.91(s,3H),0.88(s,6H),0.77(s,6H).ESI-MS:m/z 525.6[M+H]⁺。

example 33

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) -tetrahydropyrrole (compound A-88)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for tetrahydropyrrole produces compound a-88:¹H NMR(300MHz,CDCl₃)3.66–3.49(m,4H),3.21(dd,J＝10.6,5.6Hz,1H),2.84–2.69(m,1H),2.48–2.38(m,1H),2.38–2.30(m,1H),1.16(s,3H),0.98(s,3H),0.94(s,3H),0.89(s,3H),0.87(s,3H),0.77(s,3H),0.76(s,3H).ESI-MS:m/z 532.5[M+Na]⁺。

example 34

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) - (3S) -hydroxy-tetrahydropyrrole (compound A-91)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for (S) -3-pyrrolidinol, compound a-91 was prepared:¹H NMR(300MHz,CDCl₃)5.29(s,1H),4.49–4.36(m,1H),3.91–3.72(m,1H),3.73–3.54(m,3H),3.22(dd,J＝9.6,4.9Hz,1H),2.76(d,J＝14.9Hz,1H),2.50–2.39(m,1H),2.39–2.29(m,1H),2.22–2.09(m,1H),1.16(s,3H),0.98(s,3H),0.94(s,3H),0.90(s,3H),0.87(s,3H),0.77(s,3H),0.76(s,3H).ESI-MS:m/z 526.4[M+H]⁺。

example 35

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) - (3R) -hydroxy-tetrahydropyrrole (compound A-92)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for (R) -3-pyrrolidinol, compound a-92 was prepared:¹H NMR(300MHz,CDCl₃)4.50–4.34(m,1H),3.80–3.66(m,2H),3.68–3.60(m,2H),3.30–3.12(m,1H),2.75(d,J＝14.8Hz,1H),2.48–2.37(m,1H),2.38–2.26(m,2H),1.15(s,3H),0.97(s,3H),0.95(s,3H),0.89(s,3H),0.86(s,3H),0.77(s,3H),0.75(s,3H).ESI-MS:m/z 526.4[M+H]⁺。

example 36

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) - (2R) -carboxy-tetrahydropyrrole (compound A-93)

Compound VI-1 was prepared by substituting N, N-dimethylethylenediamine for D-proline benzyl ester hydrochloride according to the procedure of example 2.

Compound VI-1(120mg, 0.186mmol) was dissolved in a mixed solution of tetrahydrofuran (1mL) and methanol (1mL), and 2N sodium hydroxide solution (1mL) was added to the solution to react at 80 ℃ for 2 hours. After completion of the TLC detection reaction, the solvent was evaporated under reduced pressure, the pH was adjusted to weak acidity with 1N hydrochloric acid solution, dichloromethane was extracted (5mL × 2), the organic phase was washed with water (5mL × 2), washed with saturated brine (5mL × 2), dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, the residue was treated with a mixed solvent of dichloromethane and acetonitrile (5.5mL, v: v ═ 10: 1) under ultrasound to precipitate a white solid, which was filtered with suction, the cake was washed twice with a mixed solvent of dichloromethane and acetonitrile (5.5mL, v: v ═ 10: 1), and dried under infrared to obtain compound a-93 (white solid, 88mg, yield 85%):¹H NMR(300MHz,CDCl₃)4.70(q,J＝3.8Hz,1H),3.94–3.79(m,1H),3.72–3.59(m,1H),3.25(dd,J＝10.8,5.4Hz,1H),2.79(dd,J＝15.5,4.0Hz,1H),2.53–2.40(m,1H),2.38–2.21(m,2H),1.19(s,3H),1.01(s,3H),0.94(s,6H),0.89(s,3H),0.81(s,3H),0.78(s,3H).ESI-MS:m/z 552.4[M-H]^-。

example 37

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) - (2S) -carboxy-tetrahydropyrrole (compound A-94)

Method according to example 36The method comprises the following steps of replacing D-proline benzyl ester hydrochloride with L-proline benzyl ester hydrochloride to prepare a compound A-94:¹H NMR(300MHz,CDCl₃)4.77–4.62(m,1H),3.90–3.75(m,1H),3.74–3.56(m,1H),3.34–3.18(m,1H),2.78(d,J＝17.1Hz,1H),2.53–2.43(m,1H),2.42–2.33(m,1H),2.28–2.16(m,1H),1.19(s,3H),1.00(s,3H),0.94(s,3H),0.92(s,3H),0.89(s,3H),0.80(s,3H),0.78(s,3H).ESI-MS:m/z 576.53[M+Na]⁺。

example 38

N- (3 β -hydroxy-oleanane-13 (18) -en-28-yl) -azetidine (Compound A-95)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for azetidine produced compound a-95:¹H NMR(300MHz,CDCl₃)4.48–3.89(m,4H),3.30–3.14(m,1H),2.81–2.66(m,1H),2.48–2.34(m,1H),2.26–2.07(m,3H),1.14(s,3H),0.97(s,3H),0.95(s,3H),0.91(s,3H),0.86(s,3H),0.75(s,3H),0.74(s,3H).ESI-MS:m/z518.5[M+Na]⁺。

example 39

N- (3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-acyl) -thiomorpholine-1, 1-dioxide (Compound A-28)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for thiomorpholine-1, 1-dioxide, compound a-28 was prepared:¹H NMR(300MHz,CDCl₃)7.96–7.86(m,1H),7.81–7.70(m,1H),7.65–7.52(m,2H),4.85–4.73(m,1H),4.37–4.12(m,4H),3.08–2.92(m,4H),2.77(d,J＝13.3Hz,1H),2.49(d,J＝13.2Hz,1H),2.44–2.32(m,1H),1.20(s,3H),0.97(s,3H),0.90(s,9H),0.86(s,3H),0.79(s,3H).ESI-MS:m/z 720.5[M-H]^-。

example 40

N- (2- (1, 1-dioxothiomorpholinyl) ethyl) -3 β -hydroxy-oleanane-13 (18) -en-28-amide (Compound A-74)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide gives compound A-74:¹H NMR(300MHz,CDCl₃)6.27–6.11(m,1H),3.48–3.29(m,2H),3.29–3.16(m,1H),3.13–2.91(m,8H),2.82(d,J＝13.0Hz,1H),2.60(t,J＝5.7Hz,2H),2.47(d,J＝13.5Hz,1H),2.43–2.31(m,1H),1.20(s,3H),0.99(s,3H),0.92(s,3H),0.90(s,3H),0.87(s,3H),0.76(s,3H),0.74(s,3H).ESI-MS:m/z 615.5[M-H]^-。

EXAMPLE 41

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β -hydroxy-oleanane-13 (18) -en-28-amide (Compound A-81)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide gives compound A-81:¹H NMR(300MHz,CDCl₃)6.05–5.95(m,1H),3.38–3.17(m,3H),3.12–2.90(m,8H),2.81(d,J＝12.5Hz,1H),2.59–2.32(m,4H),1.19(s,3H),0.99(s,3H),0.90(s,6H),0.88(s,3H),0.77(s,3H),0.73(s,3H).ESI-MS:m/z 629.5[M-H]^-。

example 42

N- (2- (1, 1-dioxothiomorpholinyl) ethyl) -3 β - (2-carboxybenzoyl) oxy-oleanane-13 (18) -en-28-amide (Compound A-9)

With reference to the procedure of example 13, 3-dimethylaminopropylamine is replaced by 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide, to give compound a-9:¹H NMR(300MHz,DMSO-d₆)13.20(s,1H),7.79–7.69(m,1H),7.69–7.59(m,3H),6.63–6.52(m,1H),4.73–4.60(m,1H),3.29–3.15(m,2H),3.12–2.98(m,4H),2.98–2.87(m,4H),2.83(d,J＝15.0Hz,1H),2.62–2.53(m,2H),2.47(d,J＝14.2Hz,1H),1.24–1.17(m,3H),0.94(s,6H),0.92(s,6H),0.85(s,3H),0.74(s,3H).ESI-MS:m/z 763.6[M-H]^-。

example 43

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (2-carboxybenzoyl) oxy-oleanane-13 (18) -en-28-amide (Compound A-23)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide gives compound A-23:¹H NMR(300MHz,methanol-d₄)7.82–7.74(m,1H),7.74–7.66(m,1H),7.66–7.57(m,2H),6.86–6.77(m,1H),4.80–4.67(m,1H),3.42–3.23(m,2H),3.18–3.07(m,4H),3.07–2.97(m,4H),2.97–2.86(m,1H),2.64–2.49(m,3H),2.31(d,J＝12.8Hz,1H),1.28(s,3H),1.03(s,6H),1.00(s,3H),0.95(s,3H),0.94(s,3H),0.81(s,3H).ESI-MS:m/z 777.6[M-H]^-。

example 44

N- (3- (1-tetrahydropyrrolyl) propyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (compound A-80)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for 1- (3-aminopropyl) pyrrolidine produced compound A-80:¹H NMR(300MHz,CDCl₃)6.22–6.12(m,1H),3.50–3.34(m,1H),3.29–3.15(m,2H),2.87–2.75(m,1H),2.61–2.37(m,8H),1.21(s,3H),1.18(s,3H),0.99(s,3H),0.91(s,3H),0.88(s,3H),0.77(s,3H),0.73(s,3H).ESI-MS:m/z 567.5[M+H]⁺。

example 45

N- (3- (4-methyl-1-piperazinyl) propyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-79)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for 1- (3-aminopropyl) -4-methylpiperazine gives compound a-79:¹H NMR(300MHz,CDCl₃)6.09–5.96(m,1H),3.41–3.28(m,1H),3.27–3.12(m,2H),2.86–2.72(m,1H),2.65–2.31(m,10H),2.28(s,3H),2.02–1.79(m,2H),1.18(s,6H),0.98(s,3H),0.88(s,6H),0.76(s,3H),0.72(s,3H).ESI-MS:m/z 596.5[M+H]⁺。

example 46

N- (3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide) tetrahydropyrrole (compound A-31)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for tetrahydropyrrole produces compound a-31:¹H NMR(300MHz,methanol-d₄)7.81–7.65(m,2H),7.65–7.51(m,2H),4.80–4.70(m,1H),3.81–3.44(m,4H),2.86(d,J＝15.1Hz,1H),2.52(d,J＝13.4Hz,1H),2.37(d,J＝13.1Hz,1H),1.27(s,3H),1.03(s,3H),1.01(s,6H),0.95(s,6H),0.85(s,3H).ESI-MS:m/z 656.5[M-H]^-。

example 47

N- (2- (acetylamino) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-64)

Referring to the procedure of example 13, 3-Dimethylammonium was addedReplacement of the propylamine with N-acetylethylenediamine produced compound A-64:¹H NMR(300MHz,CDCl₃)7.93–7.77(m,1H),7.77–7.62(m,1H),7.60–7.46(m,2H),6.62–6.45(m,1H),6.38–6.21(m,1H),4.82–4.66(m,1H),3.57–3.20(m,4H),2.82(d,J＝15.1Hz,1H),2.46(d,J＝13.3Hz,1H),2.36(d,J＝11.6Hz,1H),1.97(s,3H),1.19(s,3H),0.95(s,3H),0.90(s,12H),0.74(s,3H).ESI-MS:m/z 687.5[M-H]^-。

example 48

N- (2- (acetylamino) ethyl) -3 beta- (2- (2-acetylamino) ethoxyacylbenzoyl) oxy-oleanane-13 (18) -ene-28-amide (compound A-65)

Referring to the procedure of example 7, substituting N- (2-aminoethyl) morpholine for N-acetylethylenediamine, compound a-65 was prepared:¹H NMR(300MHz,CDCl₃)7.85–7.77(m,1H),7.70–7.63(m,1H),7.62–7.50(m,2H),6.60–6.47(m,1H),6.43–6.34(m,1H),6.33–6.23(m,1H),4.76–4.67(m,1H),4.45(t,J＝4.7Hz,2H),3.71–3.58(m,2H),3.49–3.26(m,4H),2.91–2.76(m,1H),2.48(d,J＝14.3Hz,1H),2.42–2.32(m,1H),2.03(s,3H),1.96(s,3H),1.21(s,3H),0.97(s,3H),0.96(s,6H),0.93(s,3H),0.91(s,3H),0.75(s,3H).ESI-MS:m/z 796.6[M+Na]⁺。

example 49

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (compound A-71)

Referring to the procedure of example 13, substitution of 3-dimethylaminopropylamine for 1- (2-aminoethyl) pyrrolidine affords compound A-71:¹H NMR(300MHz,CDCl₃)6.59–6.45(m,1H),3.46–3.17(m,3H),2.89–2.74(m,1H),2.60–2.30(m,7H),1.18(s,6H),0.99(s,3H),0.93(s,3H),0.89(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 553.5[M+H]⁺。

example 50

1-methyl-4- (3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide) piperazine (compound A-30)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for N-methylpiperazine produced compound a-30:¹H NMR(300MHz,methanol-d₄)7.79–7.72(m,1H),7.72–7.65(m,1H),7.65–7.54(m,2H),4.74(dd,J＝10.7,5.1Hz,1H),4.28–3.84(m,4H),3.28–3.12(m,4H),2.89(s,3H),2.84(d,J＝12.6Hz,1H),2.57(d,J＝13.3Hz,1H),2.38(d,J＝12.6Hz,1H),1.27(s,3H),1.00(s,3H),0.99(s,3H),0.97(s,3H),0.95(s,3H),0.93(s,3H),0.85(s,3H).ESI-MS:m/z 687.5[M+H]⁺。

example 51

N- (3 beta-hydroxy-oleanane-13 (18) -ene-28-acyl) - (3S) -amino-tetrahydropyrrole (compound A-89)

Referring to the procedure of example 2, N-dimethylethylenediamine was replaced with (S) -3-N-benzyloxycarbonylaminopyrrolidine to obtain compound VII-1.

Compound VII-1(200mg, 0.303mmol) was dissolved in tetrahydrofuran (10mL), 10% palladium on carbon (20mg) was added, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. After completion of the TLC detection reaction, celite was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol 10: 1) to give compound a-89 (white solid, 135mg, yield 85%):¹H NMR(300MHz,CDCl₃)3.84–3.75(m,1H),3.74–3.66(m,1H),3.66–3.57(m,1H),3.51(p,J＝6.0Hz,1H),3.24(td,J＝10.6,5.9Hz,2H),2.82–2.69(m,1H),2.48–2.38(m,1H),2.39–2.29(m,1H),2.04(dt,J＝12.4,6.6Hz,1H),1.16(s,3H),0.98(s,3H),0.95(s,3H),0.90(s,3H),0.87(s,3H),0.77(s,3H),0.76(s,3H).ESI-MS:m/z 525.5[M+H]⁺。

example 52

N- (3 β -hydroxy-oleanane-13 (18) -en-28-yl) -3-hydroxy-azetidine (Compound A-96)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for azetidin-3-ol, compound a-96 was prepared:¹H NMR(300MHz,CDCl₃)5.29–5.20(m,1H),3.90–3.72(m,1H),3.72–3.59(m,1H),3.58–3.40(m,2H),3.26–3.04(m,3H),2.13–1.99(m,2H),1.13(s,3H),0.98(s,3H),0.93(s,3H),0.89(s,6H),0.77(s,3H),0.71(s,3H).ESI-MS:m/z 510.5[M-H]^-。

example 53

N- (3 beta-hydroxy-oleanane-13 (18) -en-28-yl) -3-methoxy-azetidine (Compound A-99)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 3-methoxyazetidine produced compound a-99:¹H NMR(300MHz,CDCl₃)4.56–4.17(m,2H),4.11(dt,J＝9.4,4.9Hz,1H),4.07–3.71(m,2H),3.29(s,3H),3.26–3.16(m,1H),2.80–2.68(m,1H),2.46–2.35(m,1H),2.17–2.08(m,1H),1.14(s,3H),0.98(s,3H),0.95(s,3H),0.91(s,3H),0.86(s,3H),0.76(s,3H),0.75(s,3H).ESI-MS:m/z 548.5[M+Na]⁺。

example 55

N- (3 β -hydroxy-oleanane-13 (18) -en-28-oyl) -3-carboxy-azetidine (Compound A-100)

Compound VIII-1(500mg, 2.485mmol) was dissolved in toluene (7.5mL), and 1, 8-diazabicycloundecen-7-ene (576mg, 3.728mmol) and benzyl bromide (467mg, 2.73mmol) were added in that order and the reaction was stirred at room temperature for 4 hours. After completion of the TLC reaction, water (5mL) was added and quenched, extracted with ethyl acetate (10mL × 2), the organic phases were combined, washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 6: 1) to give compound VIII-2 (colorless oily liquid, 296mg, yield 40%).

Compound VIII-2(296mg, 1.016mmol) was dissolved in dichloromethane (5mL), and trifluoroacetic acid (1mL) was added to stir the reaction at room temperature. After TLC detection reaction is completed, the solvent is evaporated under reduced pressure to obtain a compound VIII-3 which is directly used in the next step.

Referring to the procedure of example 36, compound a-100 was prepared by substituting D-proline benzyl ester hydrochloride for compound VIII-3:¹H NMR(300MHz,CDCl₃)4.65–4.01(m,4H),3.42–3.30(m,1H),3.24(dd,J＝10.3,5.5Hz,1H),2.85–2.65(m,1H),2.51–2.32(m,1H),2.26–2.04(m,1H),1.15(s,3H),0.98(s,3H),0.95(s,3H),0.92(s,3H),0.87(s,3H),0.76(s,3H),0.75(s,3H).ESI-MS:m/z 538.4[M-H]^-。

example 55

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (2- (2-acetamidoethoxyacyl) benzoyl) oxy-oleanane-13 (18) -en-28-amide (Compound A-25)

Referring to the procedure of example 7, substituting N- (2-aminoethyl) morpholine for 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide, compound a-25 was prepared:¹H NMR(300MHz,CDCl₃)7.86–7.76(m,1H),7.70–7.63(m,1H),7.63–7.50(m,2H),6.58–6.46(m,1H),6.11–5.97(m,1H),4.77–4.66(m,1H),4.49–4.38(m,2H),3.69–3.59(m,2H),3.38–3.24(m,2H),3.18–2.94(m,6H),2.84(d,J＝15.3Hz,1H),2.68–2.53(m,2H),2.48(d,J＝12.8Hz,1H),2.43–2.34(m,1H),2.03(s,3H),1.21(s,3H),0.96(s,9H),0.93(s,3H),0.92(s,3H),0.75(s,3H).ESI-MS:m/z 862.5[M-H]^-。

example 56

N- (3- (1-tetrahydropyrrolyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (compound A-22)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for 1- (3-aminopropyl) pyrrolidine produced compound A-22:¹H NMR(300MHz,methanol-d₄)7.78(d,J＝7.4Hz,1H),7.69–7.54(m,2H),7.54–7.44(m,1H),4.82–4.72(m,1H),3.46–3.39(m,2H),3.18(t,J＝7.2Hz,2H),2.95(d,J＝14.6Hz,1H),2.62(d,J＝13.8Hz,1H),2.36–2.26(m,1H),2.14(s,3H),1.30(s,3H),1.06(s,6H),1.05(s,3H),1.03(s,3H),0.99(s,3H),0.84(s,2H).ESI-MS:m/z 713.6[M-H]^-。

example 57

N- (3- (4-methyl-1-piperazinyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-amide (Compound A-21)

Referring to the procedure of example 13, 3-dimethylaminopropylamine was substituted for 1- (3-aminopropyl) -4-methylpiperazine to obtain compound a-21:¹H NMR(300MHz,methanol-d₄)7.84–7.77(m,1H),7.74–7.68(m,1H),7.68–7.59(m,2H),4.78(dd,J＝11.0,4.2Hz,1H),3.82–3.43(m,8H),3.42–3.37(m,2H),3.20–3.09(m,2H),3.02(s,3H),2.95(d,J＝13.9Hz,1H),2.62(d,J＝13.7Hz,1H),2.32(d,J＝13.3Hz,1H),1.30(s,3H),1.05(s,3H),1.04(s,3H),1.02(s,3H),0.98(s,3H),0.95(s,3H),0.83(s,3H).ESI-MS:m/z 742.6[M-H]^-。

example 58

N- (2- (1-piperazinyl) ethyl) -3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-13 (18) -ene-28-amide (compound A-13)

Referring to the procedure of example 7, substituting N- (2-aminoethyl) morpholine for N-aminoethyl piperazine produced compound a-13:¹H NMR(300MHz,CDCl₃)7.81(d,J＝7.0Hz,1H),7.66(d,J＝7.0Hz,1H),7.62–7.51(m,2H),6.57–6.47(m,1H),6.19–6.11(m,1H),4.77–4.67(m,1H),4.49–4.41(m,2H),3.69–3.60(m,2H),3.49–3.28(m,2H),3.25–3.09(m,4H),2.90–2.71(m,5H),2.59–2.35(m,4H),2.03(s,3H),1.21(s,3H),0.97(s,3H),0.96(s,3H),0.95(s,3H),0.93(s,3H),0.90(s,3H),0.75(s,3H).ESI-MS:m/z801.6[M+H]⁺。

example 59

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2-dimethylaminoethyl ester (compound A-35)

Oleanolic acid (-OA, 2g, 4.379mmol) was dissolved in N, N-dimethylformamide (20mL), and 1, 2-dibromoethane (3.75mL, 43.79mmol), potassium carbonate (605mg, 4.379mmol) and acetonitrile (2mL) were added in this order, and the reaction mixture was heated to 50 ℃ and stirred for 3 hours. After completion of the TLC detection reaction, the reaction mixture was treated with ethyl acetate (30mL) and water (10mL), the aqueous layer was extracted with ethyl acetate (20mL), the organic phases were combined, washed with water (20mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 5: 1) to obtain compound IX-1 (white solid, 2.25g, 91% yield).

Compound IX-1(150mg, 0.266mmol) was dissolved in anhydrous N, N-dimethylformamide (8mL), dimethylamine hydrochloride (33mg, 0.405mmol) and potassium carbonate (56mg, 0.405mmol) were added in this order, and the reaction mixture was heated to 50 ℃ and stirred. After completion of the TLC detection reaction, the reaction mixture was treated with dichloromethane (10mL) and water (5mL), the aqueous layer was extracted with dichloromethane (10 mL. times.2), the organic phases were combined, washed with water (10 mL. times.2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol. times.30: 1) to obtain compound IX-2 (white solid, 91mg, 65% yield).

Compound IX-2(91mg, 0.172mmol) was dissolved in anhydrous pyridine (8mL), phthalic anhydride (255mg, 1.72mmol) and 4-dimethylaminopyridine (21mg, 0.172mmol) were added, and the reaction mixture was warmed to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and water (10mL), the aqueous layer was extracted with dichloromethane (10mL), the organic phases were combined, washed with 1N dilute hydrochloric acid (10mL × 3) and water (10mL × 3) in order, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to obtain compound a-35 (white solid, 83mg, yield 72%):¹H NMR(300MHz,CDCl₃)7.82(d,J＝7.2Hz,1H),7.58(d,J＝6.6Hz,1H),7.52–7.39(m,2H),4.83–4.74(m,1H),4.41(t,J＝5.5Hz,2H),3.11–2.95(m,2H),2.76(d,J＝14.6Hz,1H),2.61(s,6H),2.45(d,J＝13.7Hz,1H),2.17(d,J＝13.2Hz,1H),1.19(s,3H),1.02(s,3H),0.92(s,9H),0.89(s,3H),0.76(s,3H).ESI-MS:m/z 674.5[M-H]^-。

example 60

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2- (4-morpholinyl) ethyl ester (compound A-39)

Referring to the procedure of example 59, substituting dimethylamine hydrochloride for morpholine, compound a-39 was prepared:¹H NMR(300MHz,CDCl₃)7.92–7.81(m,1H),7.74–7.63(m,1H),7.58–7.46(m,2H),4.83–4.71(m,1H),4.28(t,J＝4.9Hz,2H),3.81–3.68(m,4H),2.81–2.66(m,3H),2.66–2.50(m,4H),2.42(d,J＝13.9Hz,1H),2.22–2.11(m,1H),1.17(s,3H),0.96(s,3H),0.90(s,12H),0.74(s,3H).ESI-MS:m/z 716.5[M-H]^-。

example 61

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2- (1-tetrahydropyrrolyl) ethyl ester (compound A-40)

Referring to the procedure of example 59, substituting dimethylamine hydrochloride for pyrrolidine affords compound a-40:¹H NMR(300MHz,CDCl₃)7.79(d,J＝7.3Hz,1H),7.52(d,J＝6.6Hz,1H),7.47–7.34(m,2H),4.76(dd,J＝10.7,3.2Hz,1H),4.55–4.35(m,2H),3.31–3.05(m,6H),2.73(d,J＝12.0Hz,1H),2.42(d,J＝13.9Hz,1H),2.12(d,J＝13.1Hz,1H),2.08–1.96(m,4H),1.16(s,3H),1.02(s,3H),0.90(s,9H),0.86(s,3H),0.74(s,3H).ESI-MS:m/z 700.5[M-H]^-。

example 62

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2- (1-piperidyl) ethyl ester (compound A-38)

Referring to the procedure of example 59, substituting dimethylamine hydrochloride for piperidine produced compound a-38:¹H NMR(300MHz,methanol-d₄)7.79(d,J＝7.3Hz,1H),7.67(d,J＝7.2Hz,1H),7.59(t,J＝7.0Hz,1H),7.49(t,J＝7.2Hz,1H),4.81–4.73(m,1H),4.45–4.21(m,3H),2.95–2.84(m,1H),2.80(t,J＝5.8Hz,2H),2.74–2.62(m,4H),2.56(d,J＝14.6Hz,1H),2.31–2.22(m,1H),1.31(s,3H),1.07(s,6H),1.06(s,6H),1.02(s,3H),0.86(s,3H).ESI-MS:m/z 714.5[M-H]^-。

example 63

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 3- (1-tetrahydropyrrolyl) propyl ester (Compound A-42)

With reference to the procedure of example 59, 1, 2-dibromoethane was replaced by 1, 3-dibromopropane and dimethylamine hydrochloride by tetrahydropyrroleTo give compound A-42:¹H NMR(300MHz,CDCl₃)7.74(d,J＝7.3Hz,1H),7.55(d,J＝7.0Hz,1H),7.46–7.31(m,2H),4.78–4.69(m,1H),4.24–4.03(m,2H),3.19(s,4H),3.08–2.95(m,2H),2.74(d,J＝13.6Hz,1H),2.43(d,J＝14.1Hz,1H),2.26–2.01(m,7H),1.17(s,3H),0.99(s,3H),0.89(s,12H),0.74(s,3H).ESI-MS:m/z 714.5[M-H]^-。

example 64

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 3-dimethylaminopropyl ester (Compound A-43)

Referring to the procedure of example 59, compound a-43 was prepared by substituting 1, 2-dibromoethane for 1, 3-dibromopropane:¹H NMR(300MHz,methanol-d₄)7.76(d,J＝7.5Hz,1H),7.64–7.53(m,2H),7.50–7.42(m,1H),4.81–4.69(m,1H),4.32–4.14(m,2H),3.18–3.08(m,2H),2.86(s,7H),2.55(d,J＝14.3Hz,1H),2.26–2.20(m,1H),2.20–2.08(m,2H),1.29(s,3H),1.04(s,6H),1.02(s,6H),0.98(s,3H),0.83(s,3H).ESI-MS:m/z 690.6[M+H]⁺。

example 65

3 beta- (2- (2-acetamido ethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-acid 2- (4-methyl-1-piperazinyl) ethyl ester (compound A-48)

Compound III-5 was prepared according to the procedure of example 7. Compound III-5(345mg, 0.500mmol) was dissolved in anhydrous N, N-dimethylformamide (8mL), and 1, 2-dibromoethane (435. mu.L, 5.00mmol) and potassium carbonate (70mg, 0.500mmol) were added in this order, and the reaction mixture was heated to 50 ℃ and stirred for 3 hours. After completion of the TLC detection reaction, the reaction mixture was treated with ethyl acetate (10mL) and water (10mL), the aqueous layer was extracted with ethyl acetate (10mL), the organic phases were combined, washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 1: 1) to obtain compound X-1 (white solid, 348mg, yield 87%).

Compound X-1(116mg, 0.145mmol) was dissolved in anhydrous N, N-dimethylformamide (8mL), N-methylpiperazine (33. mu.L, 0.291mmol) and triethylamine (41. mu.L, 0.291mmol) were added in this order, and the reaction mixture was heated to 50 ℃ and stirred. After completion of the TLC detection reaction, the reaction solution was treated with ethyl acetate (10mL) and water (10mL), the aqueous layer was extracted with ethyl acetate (10mL), the organic phases were combined, washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to obtain compound a-48 (white solid, 63mg, yield 53%):¹H NMR(300MHz,CDCl₃)7.86–7.78(m,1H),7.69–7.63(m,1H),7.63–7.49(m,2H),6.62–6.50(m,1H),4.77–4.67(m,1H),4.45(t,J＝4.8Hz,2H),4.28–4.14(m,2H),3.70–3.59(m,2H),2.75(d,J＝13.0Hz,1H),2.68–2.33(m,11H),2.29(s,3H),2.21–2.15(m,1H),2.03(s,3H),1.17(s,3H),0.96(s,6H),0.95(s,3H),0.93(s,3H),0.92(s,3H),0.74(s,3H).ESI-MS:m/z 816.7[M+H]⁺。

example 66

3 beta- (2- (2-acetamido-ethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-acid 2- (1-piperazinyl) ethyl ester (compound A-47)

Referring to the procedure of example 65, substituting piperazine for N-methylpiperazine produced compound a-47:¹H NMR(300MHz,CDCl₃)7.84–7.79(m,1H),7.69–7.63(m,1H),7.62–7.50(m,2H),6.61–6.50(m,1H),4.77–4.67(m,1H),4.44(t,J＝5.0Hz,2H),4.26–4.12(m,2H),3.71–3.60(m,2H),3.25–3.13(m,4H),2.91–2.70(m,5H),2.66(t,J＝5.5Hz,2H),2.42(d,J＝14.1Hz,1H),2.21–2.11(m,1H),2.03(s,3H),1.18(s,3H),0.97(s,3H),0.96(s,3H),0.95(s,3H),0.91(s,6H),0.74(s,3H).ESI-MS:m/z 802.7[M+H]⁺。

example 67

N- (3 β - (2-carboxybenzoyl) oxy-oleanane-13 (18) -en-28-yl) -azetidine (Compound A-106)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for azetidine produced compound a-106:¹H NMR(300MHz,CDCl₃)8.00–7.36(m,4H),4.88–4.64(m,1H),4.34–3.93(m,4H),2.83–2.66(m,1H),2.48–2.33(m,1H),1.15(s,3H),0.93(s,9H),0.85(s,6H),0.77(s,3H).ESI-MS:m/z 642.5[M-H]^-。

example 68

N- (3 β - (2-carboxybenzoyl) oxy-oleanane-13 (18) -en-28-yl) -3-methoxy-azetidine (Compound A-107)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 3-methoxyazetidine produced compound a-107:¹H NMR(300MHz,DMSO-d₆)13.24(s,1H),7.76–7.68(m,1H),7.66–7.57(m,3H),4.71–4.56(m,1H),4.50–3.58(m,5H),3.19(s,3H),2.79–2.63(m,1H),2.34(d,J＝13.5Hz,1H),2.10–1.97(m,1H),1.14(s,3H),0.91(s,3H),0.88(s,9H),0.82(s,3H),0.73(s,3H).ESI-MS:m/z 672.5[M-H]^-。

example 69

N- (2- (1-piperazinyl) ethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-72)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for N-aminoethylpiperazine gives compound A-72:¹H NMR(300MHz,DMSO-d₆)9.77–9.47(m,2H),7.71–7.58(m,1H),5.30–5.19(m,1H),3.97–3.58(m,4H),3.52–3.32(m,6H),3.14–2.95(m,3H),2.88–2.76(m,1H),1.09(s,3H),0.89(s,6H),0.87(s,3H),0.85(s,3H),0.67(s,3H),0.66(s,3H).ESI-MS:m/z 568.5[M+H]⁺。

example 70

3 beta- (2- (2-Acylaminoethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2-trimethylammonioethyl ester bromide (Compound A-45)

Referring to the procedure of example 65, substituting N-methylpiperazine for trimethylamine yields compound a-45:¹H NMR(300MHz,methanol-d₄)7.78–7.71(m,2H),7.66–7.59(m,2H),4.76–4.67(m,2H),4.61–4.51(m,2H),4.36(t,J＝5.5Hz,2H),3.73(t,J＝5.0Hz,2H),3.53(t,J＝5.4Hz,2H),3.24(s,9H),2.81(d,J＝15.0Hz,1H),2.49(d,J＝13.9Hz,1H),2.19(d,J＝13.5Hz,1H),1.96(s,4H),1.24(s,3H),0.99(s,3H),0.97(s,3H),0.97(s,3H),0.94(s,3H),0.92(s,3H),0.79(s,3H).ESI-MS:m/z 775.6[M-Br]⁺。

example 71

3 β - (2- (2-Acylaminoethoxyacyl) benzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 3-trimethylammoniumpropyl ester bromide (Compound A-46)

Referring to the procedure of example 65, compound a-46 was prepared by substituting 1, 2-dibromoethane for 1, 3-dibromopropane and N-methylpiperazine for trimethylamine:¹H NMR(300MHz,methanol-d₄)7.83–7.76(m,2H),7.71–7.64(m,2H),4.81–4.73(m,1H),4.41(t,J＝5.5Hz,2H),4.23(dd,J＝11.7,5.9Hz,2H),3.58(t,J＝5.5Hz,2H),3.48(dd,J＝10.2,6.5Hz,2H),3.23(s,9H),2.86(d,J＝14.4Hz,1H),2.56(d,J＝14.1Hz,1H),2.30–2.16(m,3H),2.01(s,4H),1.29(s,3H),1.04(s,3H),1.02(s,6H),0.98(s,3H),0.97(s,3H),0.83(s,3H).ESI-MS:m/z 789.6[M-Br]⁺。

example 72

3 beta- (2-carboxybenzoyl) oxy-oleanane-13 (18) -ene-28-oic acid 2- (4-methyl-1-piperazinyl) ethyl ester (compound A-37)

Referring to the procedure of example 59, substituting dimethylamine hydrochloride for N-methylpiperazine gave compound a-37:¹H NMR(300MHz,methanol-d₄)7.84–7.76(m,1H),7.75–7.68(m,1H),7.68–7.60(m,2H),4.77(dd,J＝10.6,5.2Hz,1H),4.52–4.40(m,2H),3.80–3.25(m,10H),3.02(s,3H),2.85(d,J＝14.8Hz,1H),2.53(d,J＝14.2Hz,1H),2.30–2.22(m,1H),1.28(s,3H),1.03(s,3H),1.02(s,3H),1.01(s,3H),0.99(s,3H),0.95(s,3H),0.83(s,3H).ESI-MS:m/z 731.6[M+H]⁺。

example 73

N- (2- (dimethylamino) ethyl) -3 beta-hydroxy-oleanane-12-en-28-amide (compound B-1)

Oleanolic acid (OA, 5g, 0.011mol) was dissolved in anhydrous pyridine (35mL), acetic anhydride (4mL) was slowly added dropwise with stirring, and after completion of the addition, the reaction was heated under reflux for 1 hour. After completion of the TLC detection reaction, the reaction mixture was cooled to room temperature, and dropped into ice water (20 mL. times.2) to precipitate a large amount of white solid, which was filtered, and the filter cake was washed with water (10 mL. times.3) and n-hexane (10 mL. times.3), dried in vacuo, and recrystallized from ethanol to obtain Compound XI-1 (white solid, 4.52g, yield 83%).

Compound XI-1(150mg, 0.301mmol) was dissolved in anhydrous dichloromethane (8mL), and oxalyl chloride (130. mu.L, 1.504mmol) and N, N-dimethylformamide (1 drop) were slowly added dropwise with stirring and reacted at room temperature for 3 hours. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure to give Compound XI-2 (yellow solid, 155mg, yield 100%).

N, N-dimethylethylenediamine (66. mu.L, 0.602mmol) was dissolved in anhydrous dichloromethane (5mL), and a solution of Compound XI-2(155mg, 0.301mmol) in anhydrous dichloromethane (5mL) and triethylamine (84. mu.L, 0.602mmol) were slowly added dropwise with stirring, and reacted at room temperature for 5 hours. After completion of the TLC detection, dichloromethane (10mL) was added to dilute the reaction solution, and the reaction solution was washed with saturated sodium bicarbonate solution (10mL × 3), water (10mL × 3), and saturated brine (10mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 100: 1 to 50: 1) to obtain compound XI-3 (white solid, 156mg, yield 91%).

Compound XI-3(156mg, 0.274mmol) was suspended in methanol (10mL), potassium hydroxide (154mg, 2.74mmol) was added, and the reaction mixture was warmed to 65 ℃ for 5 hours. After completion of the TLC detection reaction, the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was treated with ethyl acetate (20mL) and water (20mL), the organic phase was washed successively with water (20mL × 3) and saturated brine (20mL × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 10: 1) to obtain compound B-1 (white solid, 123mg, yield 85%):¹H NMR(300MHz,CDCl₃)6.59–6.45(m,1H),5.35(t,J＝3.4Hz,1H),3.44–3.29(m,1H),3.27–3.07(m,2H),2.59–2.46(m,1H),2.37(t,J＝6.1Hz,2H),2.23(s,6H),1.16(s,3H),0.99(s,3H),0.91(s,9H),0.79(s,3H),0.78(s,3H).ESI-MS:m/z 527.5[M+H]⁺。

example 74

N- (3 beta-hydroxy-oleanane-12-ene-28-amide) -morpholine (compound B-2)

Referring to the procedure of example 73, compound B-2 was prepared by substituting N, N-dimethylethylenediamine for morpholine:¹H NMR(300MHz,CDCl₃)5.33–5.21(m,1H),3.76–3.54(m,8H),3.28–3.15(m,1H),3.15–3.03(m,1H),1.16(s,3H),1.00(s,3H),0.95(s,3H),0.92(s,6H),0.80(s,3H),0.75(s,3H).ESI-MS:m/z 526.5[M+H]⁺。

example 75

N- (2- (dimethylamino) ethyl) -N-methyl-3 beta-hydroxy-oleanane-12-en-28-amide (compound B-3)

Referring to the procedure of example 73, N, N-dimethylethylenediamine was replaced with N, N, N' -trimethylethylenediamine to obtain a compound B-3:¹H NMR(300MHz,CDCl₃)5.26(t,J＝3.3Hz,1H),3.43(t,J＝7.2Hz,2H),3.26–3.17(m,1H),3.16–3.12(m,1H),3.10(s,3H),2.54–2.31(m,3H),2.25(s,6H),1.14(s,3H),0.99(s,3H),0.93(s,3H),0.91(s,3H),0.89(s,3H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z 541.6[M+H]⁺。

example 76

N- (3- (dimethylamino) propyl) -3 beta-hydroxy-oleanane-12-ene-28-amide (compound B-4)

Referring to the procedure of example 73, substituting N, N-dimethylethylenediamine for 3-dimethylaminopropylamine produced compound B-4:¹H NMR(300MHz,CDCl₃)7.03–6.96(m,1H),5.33–5.28(m,1H),3.59–3.45(m,1H),3.27–3.15(m,1H),3.08–2.94(m,1H),2.60–2.47(m,1H),2.47–2.26(m,2H),2.23(s,6H),1.15(s,3H),0.99(s,3H),0.91(s,9H),0.78(s,3H),0.76(s,3H).ESI-MS:m/z 563.5[M+Na]⁺。

example 77

N- (2- (diethylamino) ethyl) -3 beta-hydroxy-oleanane-12-ene-28-amide (Compound B-5)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with N, N-diethylethylenediamine to obtain a compound B-5:¹H NMR(300MHz,CDCl₃)6.63–6.53(m,1H),5.38–5.33(m,1H),3.53–3.41(m,1H),3.28–3.17(m,1H),3.10–2.97(m,1H),2.65–2.39(m,7H),1.17(s,3H),1.04(t,J＝7.1Hz,6H),1.01(s,3H),0.92(s,9H),0.80(s,3H),0.78(s,3H).ESI-MS:m/z 577.5[M+Na]⁺。

example 78

N- (2- (1-piperidinyl) ethyl) -3 beta-hydroxy-oleanane-12-en-28-amide (compound B-6)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 1- (2-aminoethyl) piperidine to give compound B-6:¹H NMR(300MHz,CDCl₃)6.65–6.55(m,1H),5.43–5.28(m,1H),3.45–3.32(m,1H),3.27–3.08(m,2H),2.57–2.47(m,1H),2.47–2.24(m,6H),1.16(s,3H),0.99(s,3H),0.91(s,9H),0.78(s,3H),0.77(s,3H).ESI-MS:m/z567.5[M+H]⁺。

example 79

N- (2- (4-morpholinylalkyl) ethyl) -3 beta-hydroxy-oleanane-12-en-28-amide (Compound B-7)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with N- (2-aminoethyl) morpholine to give compound B-7:¹H NMR(300MHz,CDCl₃)6.54–6.42(m,1H),5.42–5.30(m,1H),3.78–3.62(m,4H),3.50–3.33(m,1H),3.30–3.08(m,2H),2.58–2.49(m,1H),2.49–2.32(m,6H),1.16(s,3H),0.99(s,3H),0.91(s,6H),0.89(s,3H),0.78(s,3H),0.76(s,3H).ESI-MS:m/z 569.5[M+H]⁺。

example 80

N- (3- (4-morpholinylalkyl) propyl) -3 beta-hydroxy-oleanane-12-ene-28-amide (Compound B-8)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with N- (3-aminopropyl) morpholine to give compound B-8:¹H NMR(300MHz,CDCl₃)6.30–6.21(m,1H),5.40–5.31(m,1H),3.79–3.66(m,4H),3.51–3.36(m,1H),3.27–3.16(m,1H),3.13–2.98(m,1H),2.61–2.50(m,1H),2.50–2.31(m,6H),1.16(s,3H),0.99(s,3H),0.90(s,9H),0.78(s,3H),0.76(s,3H).ESI-MS:m/z 569.5[M+H]⁺。

example 81

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -thiomorpholine-1, 1-dioxide (compound B-9)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with thiomorpholine-1, 1-dioxide to give compound B-9:¹H NMR(300MHz,CDCl₃)5.32–5.23(m,1H),4.09(t,J＝5.2Hz,4H),3.26–3.14(m,1H),3.11–2.90(m,4H),2.29–2.10(m,1H),1.15(s,3H),0.99(s,3H),0.93(s,3H),0.91(s,3H),0.90(s,3H),0.78(s,3H),0.71(s,3H).ESI-MS:m/z 574.4[M+H]⁺。

example 82

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta-hydroxy-oleanane-12-ene-28-amide (compound B-10)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 1- (2-aminoethyl) pyrrolidine to give compound B-10:¹H NMR(300MHz,CDCl₃)6.64–6.52(m,1H),5.37–5.26(m,1H),3.50–3.34(m,1H),3.19(d,J＝4.7Hz,1H),2.63–2.44(m,4H),1.14(s,2H),0.97(s,2H),0.89(s,5H),0.76(s,3H).ESI-MS:m/z 553.5[M+H]⁺。

example 83

N- (2- (1-piperazinyl) ethyl) -3 β -hydroxy-oleanane-12-en-28-amide (compound B-11)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with N-aminoethylpiperazine to obtain compound B-11:¹H NMR(300MHz,DMSO-d₆)9.77–9.47(m,2H),7.71–7.58(m,1H),5.30–5.19(m,1H),3.97–3.58(m,4H),3.52–3.32(m,6H),3.14–2.95(m,3H),2.88–2.76(m,1H),1.09(s,3H),0.89(s,6H),0.87(s,3H),0.85(s,3H),0.67(s,3H),0.66(s,3H).ESI-MS:m/z 568.5[M+H]⁺。

example 84

N- (2- (4-methyl-1-piperazinyl) ethyl) -3 beta-hydroxy-oleanane-12-en-28-amide (compound B-12)

Referring to the procedure of example 73, substituting N, N-dimethylethylenediamine for 1- (2-aminoethyl) -4-methylpiperazine produced compound B-12:¹H NMR(300MHz,CDCl₃)6.55–6.44(m,1H),5.35(s,1H),4.30–4.14(m,1H),3.46–3.31(m,1H),3.28–3.05(m,2H),2.61–2.36(m,10H),2.29(s,3H),1.14(s,3H),0.96(s,3H),0.88(s,9H),0.75(s,3H),0.73(s,3H).ESI-MS:m/z 582.5[M+H]⁺。

example 85

N- (2- (1, 1-dioxothiomorpholinyl) ethyl) -3 β -hydroxy-oleanan-12-en-28-amide (Compound B-13)

Taking olea europaeaAcid (OA, 200mg, 0.438mmol) was dissolved in N, N-dimethylformamide (8mL), triethylamine (243. mu.L, 1.752mmol) was added, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (216mg, 0.569mmol) was added under stirring at room temperature, after 1 hour of reaction, the compound 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide (156mg, 0.875mmol) was added, and the reaction solution was heated to 60 ℃. After completion of the TLC detection reaction, the reaction solution was treated with ethyl acetate (10mL) and water (5mL), the aqueous layer was extracted with ethyl acetate (10mL × 2), the organic layers were combined, washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 50: 1) to obtain compound B-13 (white solid, 175mg, yield 65%):¹H NMR(300MHz,CDCl₃)6.22(s,1H),5.35(s,1H),3.49(dd,J＝13.6,6.2Hz,1H),3.20(d,J＝4.8Hz,2H),3.05(s,8H),2.63(s,2H),2.49(d,J＝11.0Hz,1H),1.17(s,3H),0.99(s,2H),0.90(s,6H),0.78(s,2H),0.76(s,3H).ESI-MS:m/z 617.5[M+H]⁺。

example 86

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) - (2S) -carboxy-tetrahydropyrrole (compound B-16)

Compound XII-1 was prepared by substituting L-proline benzyl ester hydrochloride with N, N-dimethylethylenediamine according to the procedure of example 73.

Compound XII-1(120mg, 0.186mmol) was dissolved in a mixed solution of tetrahydrofuran (1mL) and methanol (1mL), and 2N sodium hydroxide solution (1mL) was added to the solution to react at 80 ℃ for 2 hours. After TLC detection reaction is completed, the solvent is evaporated under reduced pressure, the pH is adjusted to weak acidity by 1N hydrochloric acid solution, dichloromethane is extracted (5mL multiplied by 2), an organic phase is washed (5mL multiplied by 2), saturated brine is washed (5mL multiplied by 2), anhydrous sodium sulfate is dried, the solvent is evaporated under reduced pressure, a residue is treated by a mixed solvent of dichloromethane and acetonitrile (5.5mL, v: v ═ 10: 1) in an ultrasonic mode, a white solid is separated out, suction filtration is carried out, a filter cake is washed twice by the mixed solvent of dichloromethane and acetonitrile (5.5mL, v: v ═ 10: 1), and infrared drying is carried out to obtain a compoundSubstance B-16 (white solid, 88mg, 85% yield):¹H NMR(300MHz,DMSO-d₆)5.32–5.24(m,1H),4.66–4.53(m,1H),3.85–3.67(m,1H),3.53(q,J＝9.3Hz,1H),3.26–3.06(m,2H),2.40(dd,J＝13.0,2.5Hz,1H),1.15(s,3H),0.98(s,3H),0.94(s,3H),0.91(s,3H),0.90(s,3H),0.77(s,3H),0.71(s,3H).ESI-MS:m/z 552.3[M-H]^-。

example 87

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -4-methyl-piperazine (compound B-17)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with N-methylpiperazine to obtain a compound B-17:¹H NMR(300MHz,CDCl₃)5.30–5.24(m,1H),3.76–3.58(m,4H),3.26–3.18(m,1H),3.10(d,J＝12.3Hz,1H),2.45–2.33(m,4H),2.31(s,3H),1.15(s,3H),1.00(s,3H),0.95(s,3H),0.92(s,3H),0.91(s,3H),0.80(s,3H),0.76(s,3H).ESI-MS:m/z 539.5[M+H]⁺。

example 88

N- (2- (4-methyl-1-piperazinyl) propyl) -3 beta-hydroxy-oleanane-12-en-28-amide (compound B-18)

Referring to the procedure of example 85, 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide was replaced with 1- (3-aminopropyl) -4-methylpiperazine to prepare compound B-19:¹H NMR(300MHz,CDCl₃)6.41–6.18(m,1H),5.41–5.27(m,1H),3.54–3.35(m,1H),3.29–3.15(m,1H),3.15–2.99(m,1H),2.70–2.33(m,11H),2.29(s,3H),1.16(s,3H),0.99(s,3H),0.91(s,9H),0.79(s,3H),0.76(s,3H).ESI-MS:m/z 596.6[M+H]⁺。

example 89

N- (2- (1-tetrahydropyrrolyl) propyl) -3 beta-hydroxy-oleanane-12-ene-28-amide (compound B-19)

Referring to the procedure of example 85, 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide was replaced with 1- (3-aminopropyl) pyrrolidine to give compound B-19:¹H NMR(300MHz,CDCl₃)5.63–5.33(m,1H),3.65–3.33(m,4H),3.30–2.98(m,6H),2.75–2.43(m,1H),0.99(s,3H),0.94(s,3H),0.91(s,6H),0.78(s,3H),0.71(s,3H).ESI-MS:m/z 567.5[M+H]⁺。

example 90

N- (2- (1, 1-dioxothiomorpholinyl) propyl) -3 β -hydroxy-oleanan-12-en-28-amide (Compound B-20)

Referring to the procedure of example 85, 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide was replaced with 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide to give compound B-20:¹H NMR(300MHz,CDCl₃)6.10–5.98(m,1H),5.43–5.28(m,1H),3.50–3.33(m,1H),3.30–3.18(m,1H),3.12–2.90(m,9H),2.59–2.42(m,3H),1.17(s,3H),0.99(s,3H),0.91(s,9H),0.79(s,3H),0.76(s,3H).ESI-MS:m/z 631.5[M+H]⁺。

example 91

N- (2- (1-piperidinyl) propyl) -3 beta-hydroxy-oleanane-12-en-28-amide (compound B-22)

Referring to the procedure of example 85, 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide was replaced with 1- (3-aminopropyl) piperidine to give compound B-22:¹H NMR(300MHz,DMSO-d₆)8.82(s,1H),7.39(s,1H),5.22(s,1H),4.22(s,1H),3.43–3.32(m,2H),3.12–3.02(2H),3.01–2.95(s,2H),2.89–2.79(m,2H),1.09(s,3H),0.89(s,6H),0.88(s,3H),0.85(s,3H),0.68(s,3H),0.67(s,3H).ESI-MS:m/z 581.5[M+H]⁺。

example 92

N- (2- (trimethylammonio) ethyl) -3 beta-hydroxy-oleanan-12-en-28-amide bromide (compound B-23)

Oleanolic acid (OA, 100mg, 0.219mmol) was dissolved in N, N-dimethylformamide (8mL), triethylamine (122. mu.L, 0.876mmol) was added, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (HATU,108mg, 0.284mmol) was added under stirring at room temperature, after 1 hour of reaction, compound I-4(116mg, 0.438mmol) was added, and the reaction solution was heated to 50 ℃. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and deionized water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic layers were combined, washed with deionized water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 10: 1) to give compound B-23 (white solid, 43mg, yield 32%):¹H NMR(300MHz,methanol-d₄)7.75–7.66(m,1H),5.46–5.39(m,1H),3.79–3.54(m,2H),3.47(t,J＝6.8Hz,2H),3.30–3.14(m,10H),2.91–2.80(m,1H),1.23(s,3H),1.02(s,3H),1.00(s,6H),0.97(s,3H),0.83(s,3H),0.82(s,3H).ESI-MS:m/z 541.5[M-Br]⁺。

example 93

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -piperazine (compound B-25)

Compound XIII-1 was prepared by substituting 1-benzylpiperazine with N, N-dimethylethylenediamine according to the procedure in example 73.

Compound XIII-1(100mg, 0.163mmol) was dissolved in tetrahydrofuran (8mL), 10% palladium on carbon (10mg) was added, and the reaction was stirred under hydrogen atmosphere at room temperature overnight. TLC detectionAfter completion of the reaction was detected, celite was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to give compound B-25 (white solid, 61mg, yield 72%):¹H NMR(300MHz,DMSO-d₆)9.28(s,2H),5.15–5.02(m,1H),4.34–4.24(m,1H),3.86–3.60(m,4H),3.09–2.87(m,6H),2.12–1.95(m,1H),1.09(s,3H),0.88(s,9H),0.84(s,3H),0.67(s,3H),0.64(s,3H).ESI-MS:m/z 525.5[M+H]⁺。

example 94

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -tetrahydropyrrole (compound B-26)

Referring to the procedure of example 73, compound B-26 was prepared by substituting N, N-dimethylethylenediamine for tetrahydropyrrole:¹H NMR(300MHz,CDCl₃)7.10–6.85(m,1H),5.44–5.22(m,1H),3.63–3.42(m,1H),3.29–3.13(m,1H),3.12–2.93(m,1H),2.67–2.48(m,2H),2.48–2.34(m,3H),2.27(s,6H),1.15(s,4H),0.99(s,4H),0.91(s,9H),0.78(s,3H),0.76(s,3H).ESI-MS:m/z 541.5[M+H]⁺。

example 95

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) - (3S) -amino-tetrahydropyrrole (compound B-27)

Compound XIV-1 was prepared by substituting (S) -3-N-benzyloxycarbonylaminopyrrolidine for N, N-dimethylethylenediamine in accordance with the procedure of example 73.

Compound XIV-1(216mg, 0.328mmol) was dissolved in tetrahydrofuran (8mL), 10% palladium on carbon (20mg) was added, and the reaction was stirred under hydrogen atmosphere at room temperature overnight. TLC detection reaction is completeAfter that, celite was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound B-27 (white solid, 143mg, yield 83%):¹H NMR(300MHz,CDCl₃)5.29–5.22(m,1H),3.74–3.61(m,2H),3.61–3.49(m,2H),3.37–3.25(m,1H),3.25–3.07(m,2H),1.14(s,3H),0.98(s,3H),0.94(s,3H),0.89(s,6H),0.77(s,3H),0.72(s,3H).ESI-MS:m/z 525.5[M+H]⁺。

example 96

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) - (3R) -amino-tetrahydropyrrole (compound B-28)

By substituting (S) -3-N-benzyloxycarbonylaminopyrrolidine for (R) -3-N-benzyloxycarbonylaminopyrrolidine according to the procedure of example 95, compound B-28 was obtained:¹H NMR(300MHz,CDCl₃)5.29–5.20(m,1H),3.90–3.72(m,1H),3.72–3.59(m,1H),3.58–3.40(m,2H),3.26–3.04(m,3H),2.13–1.99(m,2H),1.13(s,3H),0.98(s,3H),0.93(s,3H),0.89(s,6H),0.77(s,3H),0.71(s,3H).ESI-MS:m/z 525.5[M+H]⁺。

example 97

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) - (3S) -hydroxy-tetrahydropyrrole (compound B-29)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with (S) -3-pyrrolidinol to give compound B-29:¹H NMR(300MHz,CDCl₃)5.26(m,1H),4.50–4.36(m,1H),3.75–3.61(m,2H),3.61–3.49(m,2H),3.24–3.09(m,2H),1.13(s,3H),0.97(s,3H),0.93(s,3H),0.89(s,3H),0.88(s,3H),0.76(s,3H),0.72(s,3H).ESI-MS:m/z 548.4[M+Na]⁺。

example 98

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) - (3R) -hydroxy-tetrahydropyrrole (compound B-30)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with (R) -3-pyrrolidinol to give compound B-30:¹H NMR(300MHz,CDCl₃)5.24(m,1H),4.49–4.31(m,1H),3.81–3.57(m,3H),3.56–3.43(m,1H),3.29–3.13(m,1H),3.14–2.95(m,1H),2.56–2.16(m,1H),1.13(s,3H),0.97(s,3H),0.93(s,3H),0.89(s,3H),0.88(s,3H),0.76(s,3H),0.70(s,3H).ESI-MS:m/z 548.4[M+Na]⁺。

example 99

3 beta-hydroxy-oleanane-12-ene-28-acid 2- (1-piperidyl) ethyl ester (compound B-31)

Oleanolic acid (OA, 5g, 10.9mmol) was dissolved in N, N-dimethylformamide (40mL), and 1, 2-dibromoethane (10.2mL, 109mmol), potassium carbonate (1.506g, 10.9mmol) and acetonitrile (4mL) were added in this order to raise the temperature of the reaction mixture to 50 ℃ and the reaction was stirred for 3 hours. After completion of the TLC detection reaction, the reaction mixture was treated with dichloromethane (100mL) and water (100mL), the aqueous layer was extracted with dichloromethane (50mL), the organic phases were combined, washed with water (100 mL. times.2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate 10: 1) to obtain Compound XV-1 (white solid, 4.44g, yield 72%).

Compound XV-1(200mg, 0.35mmol) was dissolved in anhydrous N, N-dimethylformamide (10mL), piperidine (70. mu.L, 0.70mmol) and potassium carbonate (48mg, 0.35mmol) were added in this order, the reaction mixture was warmed to 50 ℃ and stirred for reaction. After completion of the TLC detection reaction, the reaction mixture was treated with dichloromethane (10mL) and water (5mL), the aqueous layer was extracted with dichloromethane (10 mL. times.2), the organic phases were combined, washed with water (10 mL. times.2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol: 30: 1) to obtain a compoundCompound B-31 (white solid, 47mg, 24% yield):¹H NMR(300MHz,CDCl₃)5.29–5.23(m,1H),4.27–4.09(m,2H),3.26–3.15(m,1H),2.89–2.79(m,1H),2.63(t,J＝5.5Hz,2H),2.54–2.42(m,4H),1.13(s,3H),0.98(s,3H),0.92(s,3H),0.90(s,6H),0.78(s,3H),0.73(s,3H).ESI-MS:m/z 568.6[M+H]⁺。

example 100

3 beta-hydroxy-oleanane-12-en-28-oic acid 2- (4-morpholinyl) ethyl ester (compound B-32)

Referring to the procedure of example 99, substituting piperidine for morpholine, compound B-32 was prepared:¹H NMR(300MHz,CDCl₃)5.30–5.18(m,1H),4.70–4.52(m,2H),4.42–4.11(m,2H),4.11–3.79(m,2H),3.63–3.31(m,2H),3.31–3.08(m,3H),3.04–2.84(m,2H),2.78(d,J＝12.5Hz,1H),1.14(s,3H),0.99(s,3H),0.91(s,9H),0.78(s,3H),0.70(s,3H).ESI-MS:m/z 570.6[M+H]⁺。

example 101

3 beta-hydroxy-oleanane-12-ene-28-oic acid 2- (1-tetrahydropyrrolyl) ethyl ester (compound B-33)

Referring to the procedure of example 99, substituting piperidine for tetrahydropyrrole, compound B-33 is prepared:¹H NMR(300MHz,CDCl₃)5.31–5.21(m,1H),4.67–4.48(m,2H),3.90–3.71(m,2H),3.39–3.12(m,3H),2.92–2.72(m,3H),2.35–2.16(m,2H),2.16–2.02(m,2H),2.02–1.92(m,1H),1.91–1.82(m,2H),1.13(s,3H),0.98(s,3H),0.90(s,9H),0.77(s,3H),0.69(s,3H).ESI-MS:m/z 554.6[M+H]⁺。

example 102

N- (2- (acetylamino) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (Compound B-38)

Compound XVI-1 is obtained by substituting 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide with N-acetylethylenediamine according to the procedure of example 85.

Compound XVI-1(120mg, 0.222mmol) was dissolved in anhydrous pyridine (8mL), phthalic anhydride (329mg, 2.22mmol) and 4-dimethylaminopyridine (28mg, 0.222mmol) were added, and the reaction mixture was heated to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, the reaction solution was diluted by adding dichloromethane (20mL), washed with 1N diluted hydrochloric acid (10mL × 3) and water (10mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound B-38 (white solid, 96mg, yield 63%): to obtain compound B-38:¹H NMR(300MHz,methanol-d₄)7.79–7.72(m,1H),7.68–7.63(m,1H),7.60–7.52(m,2H),7.35–7.24(m,1H),5.52–5.44(m,1H),5.41–5.34(m,1H),4.77–4.61(m,1H),3.20–3.05(m,1H),2.83–2.65(m,1H),1.21–1.19(m,6H),0.99(s,3H),0.97(s,3H),0.95(s,3H),0.91(s,6H),0.78(s,3H).ESI-MS:m/z 687.5[M-H]^-。

example 103

N- (2- (1-piperidinyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (compound B-39)

Referring to the procedure of example 102, N-acetylethylenediamine was replaced with 1- (2-aminoethyl) piperidine to prepare compound B-39:¹H NMR(300MHz,CDCl₃)7.73–7.61(m,2H),7.50–7.40(m,1H),7.39–7.30(m,1H),7.09(d,J＝13.4Hz,1H),5.41–5.25(m,1H),4.79–4.55(m,1H),3.88–3.55(m,3H),3.17–2.76(m,6H),1.17(s,3H),0.99(s,3H),0.95(s,6H),0.89(s,3H),0.87(s,3H),0.77(s,3H).ESI-MS:m/z 715.7[M+H]⁺。

example 104

N- (2- (4-morpholinyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (Compound B-40)

Referring to the procedure of example 102, N-acetylethylenediamine was replaced with N- (2-aminoethyl) morpholine to prepare compound B-40: 1H NMR (300MHz, methane-d)₄)7.74–7.63(m,2H),7.60–7.46(m,2H),5.45–5.32(m,1H),4.74–4.62(m,1H),3.82–3.72(m,4H),3.51–3.38(m,1H),3.38–3.24(m,3H),2.83–2.58(m,7H),1.21(s,3H),1.01(s,3H),0.98(s,3H),0.94(s,6H),0.92(s,3H),0.81(s,3H).ESI-MS:m/z 718.6[M+H]⁺。

Example 105

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-ene-28-amide (compound B-41)

Referring to the procedure of example 102, N-acetylethylenediamine was replaced with 1- (2-aminoethyl) pyrrolidine to obtain compound B-41:¹H NMR(300MHz,methanol-d₄)7.82–7.61(m,1H),7.59–7.45(m,2H),7.44–7.32(m,1H),5.48(s,1H),5.33(s,1H),4.72–4.64(m,1H),3.67–3.45(m,2H),3.23–3.10(m,2H),2.94–2.76(m,1H),1.20(s,3H),1.00(s,9H),0.93(s,3H),0.90(s,3H),0.80(s,3H).ESI-MS:m/z 702.6[M+H]⁺。

example 106

N- (2- (4-morpholinyl) ethyl) -3 beta- (2- (2-acetamido ethoxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide (compound B-47)

Compound III-1(1g, 1.829mmol) was dissolved in anhydrous pyridine (10mL), phthalic anhydride (2.708g, 18.29mmol) and 4-dimethylaminopyridine (223mg, 1.829mmol) were added, and the reaction mixture was warmed to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, dichloromethane (30mL) was added to dilute the reaction solution, which was washed with 1N diluted hydrochloric acid (10 mL. times.3) and water (10 mL. times.3) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain Compound XVII-2 (white solid, 925mg, 73% yield).

Compound XVII-2(150mg, 0.216mmol) was dissolved in anhydrous dichloromethane (6mL), and N, N' -dicyclohexylcarbodiimide (134mg, 0.647mmol), 4-dimethylaminopyridine (27mg, 0.216mmol) and N-acetylethanolamine (30. mu.L, 0.324mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, the filtrate was washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 3: 1) to obtain compound XVII-3 (white solid, 159mg, yield 95%).

Compound XVII-3(159mg, 0.204mmol) was dissolved in tetrahydrofuran (10mL), 10% palladium on carbon (16mg) was added, and the reaction was stirred at room temperature under a hydrogen atmosphere overnight. After completion of the TLC detection reaction, celite was filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate 1: 1) to obtain compound XVII-4 (white solid, 90mg, yield 64%)

Compound XVII-4(100mg, 0.145mmol) is dissolved in N, N-dimethylformamide (6mL), triethylamine (40. mu.L, 0.290mmol) is added, and 2- (7-benzotriazole oxide) -N, N, N 'is added under stirring at room temperature'N' -tetramethyluronium hexafluorophosphate (72mg, 0.188mmol) was added after 1 hour of reaction and N- (2-aminoethyl) morpholine (23. mu.L, 0.174mmol) was added and the reaction mixture was warmed to 50 ℃. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic layers were combined, washed with water (5mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to obtain compound B-47 (white solid, 80mg, yield 69%):¹H NMR(300MHz,CDCl₃)7.78(d,J＝6.1Hz,1H),7.66(d,J＝7.9Hz,1H),7.53–7.42(m,2H),5.34–5.23(m,1H),4.73–4.66(m,1H),4.60–4.49(m,2H),3.52–2.98(m,6H),2.89–2.74(m,1H),1.15(s,3H),0.95(s,3H),0.92(s,9H),0.89(s,3H),0.72(s,3H).ESI-MS:m/z 802.7[M+H]⁺。

example 107

N- (2- (4-morpholinyl) ethyl) -3 beta- (2- (methoxyacyl) benzoyl) oxy-oleanane-12-en-28-amide (compound B-48)

Referring to the procedure of example 106, substituting N-acetylethanolamine for methanol produced compound B-48:¹H NMR(300MHz,CDCl₃)7.77–7.65(m,2H),7.59–7.48(m,2H),6.55–6.45(m,1H),5.43–5.35(m,1H),4.75(dd,J＝11.4,4.5Hz,1H),3.90(s,3H),3.78–3.68(m,4H),3.48–3.37(m,1H),3.28–3.11(m,1H),2.58–2.39(m,7H),1.18(s,3H),0.97(s,3H),0.96(s,3H),0.92(s,9H),0.78(s,3H).ESI-MS:m/z 753.6[M+Na]⁺。

example 108

N- (2- (4-morpholinyl) ethyl) -3 beta- (2- (isopropyloxyacyl) benzoyl) oxy-oleanane-12-en-28-amide (compound B-49)

With reference to the procedure of example 106, N-acetylethanolamineReplacement by isopropanol gave compound B-49:¹H NMR(300MHz,CDCl₃)7.77–7.65(m,2H),7.57–7.48(m,2H),6.59–6.44(m,1H),5.47–5.36(m,1H),5.34–5.20(m,1H),4.77(dd,J＝11.8,4.6Hz,1H),3.83–3.64(m,4H),3.53–3.39(m,1H),3.29–3.14(m,1H),2.62–2.38(m,7H),1.37(d,J＝6.3Hz,6H),1.20(s,3H),1.00(s,3H),0.98(s,3H),0.94(s,9H),0.80(s,3H).ESI-MS:m/z 759.7[M+H]⁺。

example 109

N- (2- (dimethylamino) ethyl) -3 beta- (2- (2-acetamidoethoxyl) benzoyl) oxy-oleanane-12-ene-28-amide (compound B-50)

Referring to the procedure of example 106, substituting N- (2-aminoethyl) morpholine for N, N-dimethylethylenediamine produced compound B-50:¹H NMR(300MHz,CDCl₃)7.87–7.81(m,1H),7.72–7.65(m,1H),7.65–7.53(m,2H),6.93–6.85(m,1H),6.62–6.50(m,1H),5.49–5.41(m,1H),4.79–4.67(m,1H),4.46(t,J＝4.5Hz,2H),3.71–3.63(m,2H),3.60–3.47(m,1H),3.47–3.34(m,1H),3.04–2.92(m,2H),2.75–2.57(m,7H),2.04(s,3H),1.21(s,3H),1.00(s,9H),0.96(s,3H),0.94(s,3H),0.81(s,3H).ESI-MS:m/z 760.6[M+H]⁺。

example 110

N- (2- (dimethylamino) ethyl) -3 beta- (2- (methoxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide (Compound B-51)

Referring to the procedure of example 106, substituting N-acetylethanolamine for methanol and N- (2-aminoethyl) morpholine for N, N-dimethylethylenediamine produced compound B-51:¹H NMR(300MHz,CDCl₃)7.78–7.63(m,2H),7.58–7.48(m,2H),6.60–6.45(m,1H),5.45–5.30(m,1H),4.75(dd,J＝11.1,4.4Hz,1H),3.90(s,3H),3.44–3.30(m,1H),3.23–3.08(m,1H),2.60–2.48(m,1H),2.37(t,J＝5.9Hz,2H),2.23(s,6H),1.18(s,3H),0.97(s,6H),0.91(s,9H),0.81(s,3H).ESI-MS:m/z 689.5[M+H]⁺。

example 111

N- (2- (dimethylamino) ethyl) -3 beta- (2- (isopropyloxyacyl) benzoyl) oxy-oleanane-12-en-28-amide (compound B-52)

Referring to the procedure of example 106, N-acetylethanolamine was replaced with isopropanol and N- (2-aminoethyl) morpholine was replaced with N, N-dimethylethylenediamine to give compound B-52:¹H NMR(300MHz,CDCl₃)7.81–7.65(m,2H),7.61–7.47(m,2H),6.66–6.47(m,1H),5.48–5.36(m,1H),5.35–5.22(m,1H),4.79(dd,J＝11.6,4.6Hz,1H),3.49–3.31(m,1H),3.29–3.09(m,1H),2.67–2.50(m,1H),2.40(t,J＝6.0Hz,2H),2.26(s,6H),1.39(d,6H),1.21(s,3H),1.01(s,6H),0.96(s,3H),0.95(s,6H),0.85(s,3H).ESI-MS:m/z 717.5[M+H]⁺。

example 112

N- (2- (1-piperazinyl) ethyl) -3 beta- (2- (2-acetamidoethoxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide (compound B-53)

Referring to the procedure of example 106, substituting N- (2-aminoethyl) morpholine for N-aminoethyl piperazine produced compound B-53:¹H NMR(300MHz,CDCl₃)7.85–7.77(m,1H),7.71–7.63(m,1H),7.64–7.49(m,2H),6.62–6.40(m,2H),5.47–5.31(m,1H),4.78–4.64(m,1H),4.44(t,J＝4.9Hz,2H),3.74–3.58(m,2H),3.52–3.39(m,1H),3.24–3.11(m,1H),2.99–2.87(m,3H),2.64–2.38(m,6H),2.31(2,3H),2.07–1.99(m,3H),1.18(s,3H),0.97(s,9H),0.92(s,6H),0.79(s,3H).ESI-MS:m/z 801.8[M+H]⁺。

example 113

N- (2- (4-methyl-1-piperazinyl) ethyl) -3 beta- (2- (2-acetamidoethoxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide (compound B-54)

Referring to the procedure of example 106, substituting N- (2-aminoethyl) morpholine for 1- (2-aminoethyl) -4-methylpiperazine produced compound B-54:¹H NMR(300MHz,CDCl₃)7.78(d,J＝6.1Hz,1H),7.66(d,J＝7.9Hz,1H),7.53–7.42(m,2H),5.34–5.23(m,1H),4.73–4.66(m,1H),4.60–4.49(m,2H),3.52–2.98(m,6H),2.89–2.74(m,1H),1.15(s,3H),0.95(s,3H),0.92(s,9H),0.89(s,3H),0.72(s,3H).ESI-MS:m/z 816.7[M+H]⁺。

example 114

N- (2- (trimethylammonio) ethyl) -3 beta- (2- (2-acetamido-ethoxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide bromide (Compound B-55)

Referring to the procedure of example 107, compound B-55 was prepared by substituting N- (2-aminoethyl) morpholine for compound I-4:¹H NMR(300MHz,methanol-d₄)7.84–7.74(m,2H),7.72–7.64(m,2H),5.45–5.39(m,1H),4.80–4.70(m,1H),4.40(t,J＝5.5Hz,2H),3.77–3.61(m,2H),3.58(t,J＝5.4Hz,2H),3.46(t,J＝6.9Hz,2H),3.24(s,9H),2.87(d,J＝9.7Hz,1H),2.25–2.11(m,1H),2.00(s,5H),1.26(s,3H),1.07(s,3H),1.02(s,3H),1.01(s,3H),0.98(s,6H),0.86(s,3H).ESI-MS:m/z 774.6[M-Br]⁺。

example 115

N- (3- (dimethylamino) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (compound B-59)

Referring to the procedure of example 102, substituting N-acetylethylenediamine for 3-dimethylaminopropylamine produced compound B-59:¹H NMR(300MHz,CDCl₃)7.75–7.54(m,2H),7.52–7.30(m,2H),6.97–6.78(m,1H),5.45–5.29(m,1H),4.76–4.62(m,1H),3.92–3.78(m,1H),3.51–3.36(m,1H),3.31–3.14(m,1H),3.07–2.84(m,1H),2.72(s,6H),1.14(s,3H),0.98(s,3H),0.92(s,6H),0.88(s,6H),0.73(s,3H).ESI-MS:m/z 689.7[M+H]⁺。

example 116

N- (3- (4-morpholinyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (Compound B-60)

Referring to the procedure of example 102, N-acetylethylenediamine was replaced with N- (3-aminopropyl) morpholine to prepare compound B-60:¹H NMR(300MHz,CDCl₃)7.82–7.67(m,1H),7.69–7.56(m,1H),7.55–7.36(m,2H),6.54–6.35(m,2H),5.51–5.24(m,1H),4.95–4.60(m,1H),4.04–3.77(m,4H),3.54–3.34(m,1H),3.24–3.03(m,1H),2.91–2.55(m,6H),1.15(s,3H),0.98(s,3H),0.91(s,3H),0.89(s,3H),0.87(s,3H),0.74(s,3H).ESI-MS:m/z 731.7[M+H]⁺。

example 117

N- (3- (4-methyl-1-piperazinyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (compound B-61)

Referring to the procedure of example 102, substituting N-acetylethylenediamine for 1- (3-aminopropyl) -4-methylpiperazine produced compound B-61:¹H NMR(300MHz,CDCl₃)7.77–7.60(m,2H),7.60–7.37(m,2H),6.60–6.19(m,3H),5.44–5.34(m,1H),4.79–4.57(m,1H),3.57–3.33(m,1H),3.27–3.04(m,8H),2.84–2.72(m,2H),1.19–1.15(m,3H),0.96(s,3H),0.92(s,3H),0.90(s,6H),0.74(s,3H).ESI-MS:m/z 744.7[M+H]⁺。

example 118

N- (3- (1-tetrahydropyrrolyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (compound B-62)

Referring to the procedure of example 102, N-acetylethylenediamine was substituted with 1- (3-aminopropyl) pyrrolidine to give compound B-62:¹H NMR(300MHz,CDCl₃)7.70–7.54(m,2H),7.46–7.38(m,1H),7.36–7.30(m,1H),6.99–6.88(m,1H),5.39–5.27(m,1H),4.76–4.60(m,1H),3.52–3.37(m,1H),3.32–2.91(m,7H),2.81–2.65(m,1H),1.13(s,3H),0.99(s,3H),0.93(s,6H),0.88(s,6H),0.73(s,3H).ESI-MS:m/z 715.7[M+H]⁺。

example 119

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (Compound B-63)

Referring to the procedure of example 102, N-acetylethylenediamine was replaced with 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide to give compound B-63:¹H NMR(300MHz,CDCl₃)7.90–7.78(m,1H),7.75–7.65(m,1H),7.59–7.50(m,2H),6.17–6.02(m,1H),5.40–5.34(m,1H),5.32–5.24(m,1H),4.83–4.62(m,1H),3.53–3.33(m,1H),3.16–2.96(m,8H),2.62–2.42(m,3H),1.17(s,3H),0.96(s,3H),0.91(s,12H),0.76(s,3H).ESI-MS:m/z 779.7[M+H]⁺。

example 120

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (2- (2-acetylamino) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-carboxamide (Compound B-65)

Referring to the procedure for example 106, substituting N- (2-aminoethyl) morpholine for 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide gave compound B-65:¹H NMR(300MHz,CDCl₃)7.84–7.77(m,1H),7.69–7.63(m,1H),7.62–7.50(m,2H),6.56–6.46(m,1H),6.05–5.96(m,1H),5.40–5.33(m,1H),4.74–4.65(m,1H),4.44(t,J＝4.9Hz,2H),3.69–3.58(m,2H),3.42(dd,J＝13.3,6.5Hz,1H),3.10–2.93(m,8H),2.58–2.45(m,3H),2.02(s,4H),1.19(s,3H),0.97(s,9H),0.91(s,6H),0.78(s,3H).ESI-MS:m/z 866.6[M+Na]⁺。

example 121

N- (2- (1-piperidinyl) ethyl) -3 beta- (2- (methoxyacyl) benzoyl) oxy-oleanane-12-en-28-amide (compound B-67)

Referring to the procedure of example 106, substituting N-acetylethanolamine for methanol and N- (2-aminoethyl) morpholine for 1- (2-aminoethyl) piperidine provided compound B-67:¹H NMR(300MHz,CDCl₃)7.77–7.64(m,2H),7.58–7.48(m,2H),6.68–6.58(m,1H),5.42–5.34(m,1H),4.75(dd,J＝11.3,4.4Hz,1H),3.90(s,3H),3.46–3.32(m,1H),3.27–3.09(m,1H),2.59–2.48(m,1H),2.48–2.28(m,6H),1.18(s,3H),0.96(s,6H),0.92(s,9H),0.79(s,3H).ESI-MS:m/z 729.6[M+H]⁺。

example 122

N- (2- (1-piperidinyl) ethyl) -3 beta- (2- (isopropyloxyacyl) benzoyl) oxy-oleanane-12-en-28-amide (compound B-68)

Referring to the method of example 106, N-Replacement of acetylethanolamine with isopropanol and N- (2-aminoethyl) morpholine with 1- (2-aminoethyl) piperidine gave compound B-68:¹H NMR(300MHz,CDCl₃)7.76–7.62(m,2H),7.56–7.44(m,2H),6.67–6.57(m,1H),5.43–5.34(m,1H),5.32–5.16(m,1H),4.75(dd,J＝11.7,4.5Hz,1H),3.47–3.31(m,1H),3.25–3.10(m,1H),2.59–2.47(m,1H),2.47–2.27(m,6H),1.35(d,J＝6.2Hz,6H),1.17(s,3H),0.98(s,3H),0.96(s,3H),0.93(s,3H),0.92(s,6H),0.79(s,3H).ESI-MS:m/z757.6[M+H]⁺。

example 123

N- (2- (1-piperidinyl) ethyl) -3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-amide (compound B-69)

By substituting N- (2-aminoethyl) morpholine for 1- (2-aminoethyl) piperidine according to the procedure of example 106, compound B-69 was prepared:¹H NMR(300MHz,CDCl₃)7.81(d,J＝6.7Hz,1H),7.66(d,J＝6.6Hz,1H),7.56(p,J＝7.0Hz,2H),7.08–6.93(m,1H),6.59–6.46(m,1H),5.48(s,1H),4.71(t,J＝7.8Hz,1H),4.52–4.35(m,2H),3.72–3.35(m,5H),3.25–2.90(m,5H),2.69–2.56(m,1H),2.01(s,3H),1.18(s,3H),0.97(s,9H),0.94(s,3H),0.92(s,3H),0.77(s,3H).ESI-MS:m/z 800.7[M+H]⁺。

example 124

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta- (2- (methoxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide (compound B-70)

Referring to the procedure of example 106, substituting N-acetylethanolamine for methanol and N- (2-aminoethyl) morpholine for 1- (2-aminoethyl) pyrrolidine provided compound B-70:¹H NMR(300MHz,CDCl₃)7.77–7.64(m,2H),7.58–7.47(m,2H),6.62–6.52(m,1H),5.40–5.28(m,1H),4.75(dd,J＝11.4,4.4Hz,1H),3.90(s,3H),3.50–3.38(m,1H),3.25–3.12(m,1H),2.68–2.41(m,7H),1.81–1.76(m,4H),1.18(s,3H),0.97(s,6H),0.91(s,9H),0.81(s,3H).ESI-MS:m/z 715.5[M+H]⁺。

example 125

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta- (2- (isopropyloxyacyl) benzoyl) oxy-oleanane-12-ene-28-amide (compound B-71)

Referring to the procedure of example 106, N-acetylethanolamine was replaced with isopropanol and N- (2-aminoethyl) morpholine was replaced with 1- (2-aminoethyl) pyrrolidine to provide compound B-71:¹H NMR(300MHz,CDCl₃)7.81–7.60(m,2H),7.59–7.44(m,2H),6.64–6.46(m,1H),5.40–5.31(m,1H),5.30–5.19(m,1H),4.75(dd,J＝11.3,4.1Hz,1H),3.53–3.32(m,1H),3.26–3.09(m,1H),2.69–2.35(m,7H),1.81–1.75(m,4H),1.18(s,3H),0.98(s,6H),0.93(s,3H),0.91(s,6H),0.81(s,3H).ESI-MS:m/z 743.6[M+H]⁺。

example 126

N- (2- (1-tetrahydropyrrolyl) ethyl) -3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-12-ene-28-amide (compound B-72)

Referring to the procedure of example 106, substituting N- (2-aminoethyl) morpholine for 1- (2-aminoethyl) pyrrolidine produced compound B-72:¹H NMR(300MHz,CDCl₃)7.85–7.78(m,1H),7.70–7.63(m,1H),7.56(qd,J＝7.3,3.6Hz,2H),7.08–6.96(m,1H),6.64–6.48(m,1H),5.52–5.42(m,1H),4.78–4.65(m,1H),4.45(tq,J＝12.3,6.5,5.6Hz,2H),3.69–3.59(m,2H),3.58–3.34(m,6H),2.71–2.56(m,1H),2.01(s,3H),1.18(s,3H),0.99(s,3H),0.97(s,6H),0.94(s,3H),0.91(s,3H),0.76(s,3H).ESI-MS:m/z 786.7[M+H]⁺。

example 127

N- (3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-oyl) -morpholine (compound B-73)

Referring to the procedure of example 102, Compound XVI-1 was substituted for Compound B-2 to give Compound B-73:¹H NMR(300MHz,DMSO-d₆)13.18(s,1H),7.69(s,1H),7.60(s,3H),5.08(s,1H),4.66–4.55(m,1H),3.50(s,8H),2.97–2.88(m,1H),1.10(s,3H),0.89(s,6H),0.85(s,6H),0.80(s,3H),0.65(s,3H).ESI-MS:m/z 672.5[M-H]^-。

example 128

N- (3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-oyl) -thiomorpholine-1, 1-dioxide (Compound B-74)

Referring to the procedure of example 102, Compound B-74 was prepared by substituting Compound XVI-1 for Compound B-9:¹H NMR(300MHz,DMSO-d₆)7.71(s,1H),7.58(s,3H),5.12(s,1H),4.62(s,1H),3.94(s,4H),3.15–2.96(m,4H),2.92(s,1H),1.13(s,3H),0.92(s,3H),0.91(s,3H),0.88(s,3H),0.87(s,3H),0.82(s,3H),0.67(s,3H).m/z 720.4[M-H]^-。

example 129

N- (3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-oyl) -tetrahydropyrrole (compound B-77)

Referring to the procedure of example 102, Compound B-77 was prepared by substituting Compound XVI-1 for Compound B-26:¹H NMR(300MHz,DMSO-d₆)13.20(s,1H),7.71(s,1H),7.63(s,3H),5.11(s,1H),4.62(t,J＝7.8Hz,1H),3.70–3.33(m,4H),3.08–2.98(m,1H),1.12(s,3H),0.90(s,6H),0.89(s,6H),0.82(s,3H),0.65(s,3H).ESI-MS:m/z 656.5[M-H]^-。

example 130

N- (3 beta- (2-methoxylbenzoyl) oxy-oleanane-12-ene-28-acyl) -morpholine (compound B-78)

Referring to the procedure of example 106, substituting N-acetylethanolamine for methanol and N- (2-aminoethyl) morpholine for morpholine yields compound B-78:¹H NMR(300MHz,CDCl₃)7.77–7.65(m,2H),7.57–7.48(m,2H),5.34–5.22(m,1H),4.75(dd,J＝11.3,4.7Hz,1H),3.89(s,3H),3.76–3.54(m,8H),3.17–3.01(m,1H),1.16(s,3H),0.96(s,6H),0.94(s,3H),0.91(s,6H),0.75(s,3H).ESI-MS:m/z 688.5[M+H]⁺。

example 131

N- (3 beta- (2-isopropyloxyacyl benzoyl) oxy-oleanane-12-alkene-28-acyl) -morpholine (compound B-79)

Referring to the procedure of example 106, substituting N-acetylethanolamine for isopropanol and N- (2-aminoethyl) morpholine for morpholine produced compound B-79:¹H NMR(300MHz,CDCl₃)7.77–7.60(m,2H),7.58–7.42(m,2H),5.37–5.15(m,2H),4.75(dd,J＝11.6,4.7Hz,1H),3.84–3.42(m,8H),3.18–3.01(m,1H),1.35(d,J＝6.1Hz,6H),1.16(s,3H),0.97(s,3H),0.96(s,3H),0.94(s,3H),0.93(s,3H),0.91(s,3H),0.76(s,3H).ESI-MS:m/z 738.5[M+Na]⁺。

example 132

N- (3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-oyl) -morpholine (compound B-80)

Referring to the procedure of example 106, substituting N- (2-aminoethyl) morpholine for morpholine, compound B-80 was prepared:¹H NMR(300MHz,CDCl₃)7.87–7.75(m,1H),7.72–7.61(m,1H),7.61–7.48(m,2H),6.64–6.50(m,1H),5.34–5.24(m,1H),4.80–4.63(m,1H),4.53–4.33(m,2H),3.80–3.41(m,9H),3.20–2.99(m,1H),2.32–2.08(m,1H),2.05(s,3H),1.16(s,3H),0.97(s,9H),0.94(s,3H),0.91(s,3H),0.76(s,3H).ESI-MS:m/z781.7[M+Na]⁺。

example 133

3 beta- (2-carboxybenzoyl) oxy-oleanane-12-ene-28-oic acid 2- (1-piperidyl) ethyl ester (compound B-87)

Referring to the procedure of example 102, Compound B-87 was prepared by substituting Compound XVI-1 for Compound B-31:¹H NMR(300MHz,CDCl₃)7.78(d,J＝6.1Hz,1H),7.66(d,J＝7.9Hz,1H),7.53–7.42(m,2H),5.34–5.23(m,1H),4.73–4.66(m,1H),4.60–4.49(m,2H),3.52–2.98(m,6H),2.89–2.74(m,1H),1.15(s,3H),0.95(s,3H),0.92(s,9H),0.89(s,3H),0.72(s,3H).ESI-MS:m/z 717.6[M+H]⁺。

example 134

3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-oic acid 2- (4-morpholinyl) ethyl ester (compound B-88)

Referring to the procedure of example 102, Compound B-88 was prepared by substituting Compound XVI-1 for Compound B-32:¹H NMR(300MHz,CDCl₃)7.88–7.79(m,1H),7.71–7.63(m,1H),7.56–7.46(m,2H),5.31–5.24(m,1H),4.76(dd,J＝11.5,4.3Hz,1H),4.35–4.22(m,2H),3.88–3.75(m,4H),2.91–2.78(m,3H),2.77–2.62(m,4H),1.15(s,3H),0.97(s,3H),0.93(s,3H),0.92(s,3H),0.91(s,3H),0.90(s,3H),0.73(s,3H).ESI-MS:m/z 716.5[M+H]⁺。

example 135

3 beta- (2-carboxybenzoyl) oxy-oleanane-12-ene-28-oic acid 2- (1-tetrahydropyrrolyl) ethyl ester (compound B-89)

Referring to the procedure of example 102, Compound B-89 was prepared by substituting Compound XVI-1 for Compound B-33:¹H NMR(300MHz,CDCl₃)7.76(d,J＝7.3Hz,1H),7.59(d,J＝7.5Hz,1H),7.50–7.35(m,2H),5.33–5.25(m,1H),4.79–4.67(m,1H),4.52–4.42(m,2H),3.34–3.11(m,6H),2.91–2.76(m,1H),1.15(s,4H),0.98(s,2H),0.96–0.90(m,9H),0.89(s,3H),0.72(s,3H).ESI-MS:m/z 702.5[M+H]⁺。

example 136

3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-oic acid 2- (4-morpholinyl) ethyl ester (compound B-93)

Referring to the preparation of compound XVII-4 in example 106, compound XVII-4(300mg, 0.435mmol) was dissolved in N, N-dimethylformamide (8mL), and 1, 2-dibromoethane (375. mu.L, 4.348mmol) and potassium carbonate (60mg, 0.435mmol) were added in this order to stir the reaction mixture at 50 ℃ for 3 hours. After completion of the TLC detection reaction, the reaction solution was diluted with dichloromethane (8mL), washed with water (8mL), and the aqueous layer was extracted with dichloromethane (8mL), the organic phases were combined, washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 2: 1) to give compound XVIII-1 (white solid, 331mg, 96% yield).

The compound XVIII-1(200mg, 0.251mmol) was dissolved in anhydrous N, N-dimethylformamide (10)mL), morpholine (45. mu.L, 0.52mmol) and potassium carbonate (48mg, 0.35mmol) were added in this order, and the reaction mixture was heated to 50 ℃ and stirred. After completion of the TLC detection reaction, the reaction solution was treated with dichloromethane (10mL) and water (5mL), the aqueous layer was extracted with dichloromethane (10mL × 2), the organic phases were combined, washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30: 1) to obtain compound B-93 (white solid, 97mg, yield 48%):¹H NMR(300MHz,CDCl₃)7.88–7.76(m,1H),7.71–7.63(m,1H),7.62–7.49(m,2H),6.62–6.51(m,1H),5.37–5.18(m,1H),4.86–4.59(m,1H),4.52–4.36(m,2H),4.31–4.08(m,2H),3.82–3.58(m,6H),2.99–2.81(m,1H),2.71–2.58(m,2H),2.58–2.44(m,3H),2.02(s,3H),1.16(s,3H),0.97(s,9H),0.93(s,3H),0.91(s,3H),0.76(s,3H).ESI-MS:m/z 803.8[M+H]⁺。

example 137

3 beta- (2- (2-acetylamino) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-oic acid 2-trimethylammonioethyl ester bromide (compound B-94)

Referring to the procedure of example 136, compound B-94 was prepared by substituting morpholine into a solution of trimethylamine in tetrahydrofuran:¹H NMR(300MHz,methanol-d₆)7.84–7.74(m,2H),7.71–7.63(m,2H),5.37–5.31(m,1H),4.79–4.70(m,1H),4.61–4.49(m,2H),4.41(t,J＝5.4Hz,2H),3.86–3.71(m,2H),3.58(t,J＝5.4Hz,2H),3.29(s,9H),2.92(d,J＝10.1Hz,1H),2.23–2.10(m,1H),2.00(s,5H),1.26(s,3H),1.07(s,3H),1.03(s,3H),1.00(s,3H),0.99(s,6H),0.85(s,3H).ESI-MS:m/z 775.7[M-Br]⁺。

example 138

3 beta- (2- (2-acetylamino) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-oic acid 3-trimethylammoniumpropyl ester bromide (Compound B-95)

Referring to the procedure of example 136, compound B-95 was prepared by substituting 1, 2-dibromoethane for 1, 3-dibromopropane and morpholine for trimethylamine:¹H NMR(300MHz,methanol-d₆)7.84–7.75(m,2H),7.72–7.63(m,2H),5.39–5.33(m,1H),4.76(dd,J＝10.2,5.8Hz,1H),4.41(t,J＝5.5Hz,2H),4.25–4.12(m,2H),3.58(t,J＝5.5Hz,2H),3.48(dd,J＝11.0,5.7Hz,2H),3.23(s,9H),2.99–2.89(m,1H),2.28–2.16(m,2H),2.16–2.06(m,1H),2.00(s,5H),1.26(s,3H),1.07(s,3H),1.02(s,3H),1.01(s,3H),0.99(s,6H),0.85(s,3H).ESI-MS:m/z 789.6[M-Br]⁺。

example 139

3 beta- (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-12-ene-28-oic acid 2- (4-methyl-1-piperazinyl) ethyl ester (compound B-97)

Referring to the procedure for example 136, morpholine was substituted with N-methylpiperazine to give compound B-97:¹H NMR(300MHz,CDCl₃)7.85–7.78(m,1H),7.69–7.63(m,1H),7.62–7.50(m,2H),6.59–6.48(m,1H),5.30–5.23(m,1H),4.77–4.63(m,1H),4.51–4.37(m,2H),4.28–4.04(m,2H),3.73–3.57(m,2H),2.93–2.80(m,1H),2.73–2.38(m,8H),2.34–2.26(m,2H),2.02(s,3H),1.15(s,3H),0.97(s,9H),0.92(s,3H),0.91(s,3H),0.75(s,3H).ESI-MS:m/z 816.8[M+H]⁺。

example 140

N- (2- (acetylamino) ethyl) -3 beta- (2- (2-acetylamino) ethoxyacylbenzoyl) oxy-oleanane-12-ene-28-amide (compound B-105)

Referring to the procedure of example 106, substituting N- (2-aminoethyl) morpholine for N-acetylethylenediamine, compound B-105 was prepared:¹H NMR(300MHz,CDCl₃)7.86–7.78(m,1H),7.70–7.63(m,1H),7.57(qt,J＝7.3,3.6Hz,2H),6.68–6.52(m,2H),6.42(t,J＝4.8Hz,1H),5.42(s,1H),4.77–4.64(m,1H),4.44(t,J＝4.8Hz,2H),3.71–3.57(m,2H),3.54–3.16(m,4H),2.62–2.51(m,1H),2.02(s,3H),1.97(s,3H),1.18(s,3H),0.97(s,9H),0.92(s,6H),0.77(s,3H).ESI-MS:m/z 796.7[M+Na]⁺。

example 141

N- (3 β -hydroxy-oleanane-12-en-28-oyl) -azetidine (Compound B-106)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with azetidine to give compound B-106:¹H NMR(300MHz,CDCl₃)5.24(t,J＝3.2Hz,1H),4.50–3.83(m,4H),3.29–3.12(m,1H),2.85(dd,J＝13.4,3.5Hz,1H),2.21(t d,J＝7.5Hz,2H),1.12(s,3H),0.98(s,3H),0.92(s,3H),0.91(s,3H),0.89(s,3H),0.78(s,3H),0.77(s,3H).ESI-MS:m/z 518.4[M+Na]⁺。

example 142

N- (3 β -hydroxy-oleanane-12-en-28-oyl) -3-hydroxy-azetidine (Compound B-107)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with azetidin-3-ol to give compound B-107:¹H NMR(300MHz,CDCl₃)5.25(m,1H),4.68–4.53(m,1H),4.49–4.20(m,2H),4.14–3.77(m,2H),3.28–3.12(m,1H),2.87–2.77(m,1H),1.13(s,3H),0.98(s,3H),0.92(s,3H),0.90(s,6H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z510.4[M-H]^-。

example 143

N- (3 β -hydroxy-oleanane-12-en-28-oyl) -3-methoxy-azetidine (Compound B-110)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 3-methoxyazetidine to give compound B-110:¹H NMR(300MHz,CDCl₃)5.28–5.21(m,1H),4.55–3.75(m,5H),3.28(s,3H),3.25–3.13(m,1H),2.90–2.78(m,1H),1.12(s,3H),0.98(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z 548.5[M+Na]⁺。

example 144

N- (3 β -hydroxy-oleanane-12-en-28-oyl) -3-carboxy-azetidine (Compound B-111)

Referring to the procedure of example 86, compound B-111 was prepared by substituting L-proline benzyl ester hydrochloride for compound VIII-3:¹H NMR(300MHz,CDCl₃)5.34–5.16(m,1H),4.71–4.02(m,4H),3.40–3.28(m,1H),3.28–3.17(m,1H),2.94–2.71(m,1H),1.13(s,3H),0.99(s,3H),0.92(s,3H),0.90(s,6H),0.79(s,3H),0.75(s,3H).ESI-MS:m/z 538.4[M-H]^-。

example 145

N- (2- (1, 1-dioxothiomorpholinyl) ethyl) -3 β - (2- (2-acetamido) ethoxyacylbenzoyl) oxy-oleanane-12-en-28-amide (Compound B-118)

Referring to the procedure for example 106, substituting N- (2-aminoethyl) morpholine for 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide affords compound B-118:¹H NMR(300MHz,CDCl₃)7.81(d,J＝6.9Hz,1H),7.66(d,J＝6.7Hz,1H),7.62–7.51(m,2H),6.58–6.47(m,1H),6.27–6.12(m,1H),5.40–5.33(m,1H),4.77–4.66(m,1H),4.48–4.38(m,2H),3.69–3.60(m,2H),3.56–3.43(m,1H),3.23–3.01(m,9H),2.69–2.58(m,2H),2.57–2.45(m,1H),1.19(s,3H),0.98(s,9H),0.92(s,3H),0.91(s,3H),0.79(s,3H).ESI-MS:m/z 872.7[M+Na]⁺。

example 146

N- (3- (1-piperidinyl) propyl) -3 beta- (2-carboxybenzoyl) oxy-oleanane-12-en-28-amide (compound B-119)

Referring to the procedure of example 102, N-acetylethylenediamine was replaced with 1- (3-aminopropyl) piperidine to prepare compound B-119:¹H NMR(300MHz,DMSO-d₆)7.70(s,1H),7.55(s,2H),7.31(s,1H),5.22(s,1H),4.61(s,1H),3.30(s,3H),3.04(s,3H),2.80(s,2H),1.12(s,3H),1.05(s,3H),0.92(s,3H),0.90(s,3H),0.88(s,3H),0.82(s,3H),0.68(s,3H).ESI-MS:m/z 727.6[M-H]^-。

example 147

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) - (2R) -carboxy-tetrahydropyrrole (compound B-120)

Referring to the procedure of example 86, substituting L-proline benzyl ester hydrochloride for D-proline benzyl ester hydrochloride yielded compound B-120:¹H NMR(300MHz,DMSO-d₆)5.26(m,1H),4.64–4.56(m,1H),3.84–3.73(m,1H),3.73–3.61(m,1H),3.26–3.15(m,1H),3.12–2.98(m,1H),2.41–2.26(m,1H),1.15(s,3H),0.98(s,3H),0.93(s,3H),0.91(s,3H),0.89(s,3H),0.77(s,3H),0.72(s,3H).ESI-MS:m/z 552.4[M-H]^-。

example 148

N- (2- (N, N-dihydroxyethyl) aminoethyl) -3 beta-hydroxy-oleanane-13 (18) -ene-28-amide (Compound A-76)

Referring to the procedure of example 13, substituting 3-dimethylaminopropylamine for N, N-bis (2-hydroxyethyl) ethylenediamine produced compound A-76:¹H NMR(300MHz,DMSO-d₆)6.61–6.53(m,1H),4.42–4.32(m,2H),4.30(d,J＝5.0Hz,1H),3.49–3.38(m,4H),3.27–3.16(m,1H),3.15–2.96(m,2H),2.85–2.72(m,1H),2.61–2.54(m,4H),2.43(d,J＝14.1Hz,1H),2.25–2.15(m,1H),1.15(s,3H),0.91(s,3H),0.90(s,6H),0.85(s,3H),0.72(s,3H),0.69(s,3H).ESI-MS:m/z 587.5[M+H]⁺。

example 149

N- (2- (N, N-dihydroxyethyl) aminoethyl) -3 β -hydroxy-oleanane-12-ene-28-amide (Compound B-15)

Referring to the procedure of example 85, 4- (2-aminoethyl) thiomorpholine-1, 1-dioxide was replaced with N, N-bis (2-hydroxyethyl) ethylenediamine to obtain compound B-15:¹H NMR(300MHz,DMSO-d₆)7.14–7.05(m,1H),5.27–5.22(m,1H),4.45–4.33(m,2H),4.29(d,J＝5.1Hz,1H),3.49–3.39(m,4H),3.23–3.11(m,1H),3.07–2.92(m,2H),2.80–2.69(m,1H),2.63–2.54(m,4H),1.11(s,3H),0.91(s,3H),0.90(s,6H),0.87(s,3H),0.69(s,6H).ESI-MS:m/z 587.5[M+H]⁺。

example 150

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β -acetoxy-oleanane-13 (18) -en-28-amide (Compound A-108)

Compound II-1 was obtained by the method of example 2. Dissolving the compound II-1(150mg, 0.300mmol) in tetrahydrofuran (8mL), sequentially adding N, N-diisopropylethylamine (50 μ L, 0.300mmol) and O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (116mg, 0.361mmol), and stirring at room temperature for reaction. After completion of the TLC detection reaction, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (8mL), washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 20: 1) to give compound XIX-1, which was used in the next step as it was.

Compound XIX-1 was dissolved in N, N-dimethylformamide (8mL), and 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide (58mg, 0.300mmol) and sodium carbonate (32mg, 0.300mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, the reaction solution was diluted with ethyl acetate (8mL), washed with water (8mL), and the aqueous layer was extracted with ethyl acetate (8mL), the organic phases were combined, washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to give compound a-108 (white solid, 179mg, 89% yield):¹H NMR(300MHz,CDCl₃)5.99(t,J＝5.8Hz,1H),4.51(dd,J＝10.6,5.7Hz,1H),3.34–3.22(m,2H),3.09–2.93(m,8H),2.87–2.76(m,1H),2.57–2.34(m,4H),2.05(s,3H),1.19(s,3H),0.90(s,9H),0.87(s,3H),0.85(s,3H),0.74(s,3H).ESI-MS:m/z 671.5[M-H]^-。

example 151

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (3-carboxypropionyl) oxy-oleanane-13 (18) -en-28-amide (Compound A-109)

Referring to the procedure of example 13, compound a-109 was prepared by substituting 3-dimethylaminopropylamine for 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide and phthalic anhydride for succinic anhydride:¹H NMR(300MHz,CDCl₃)6.06–5.95(m,1H),4.59–4.48(m,1H),3.39–3.18(m,2H),3.13–2.91(m,8H),2.87–2.75(m,1H),2.73–2.58(m,4H),2.59–2.33(m,4H),1.19(s,3H),0.90(s,9H),0.87(s,3H),0.85(s,3H),0.74(s,3H).ESI-MS:m/z 729.5[M-H]^-。

example 152

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (3-picolinoyl) oxy-oleanane-13 (18) -en-28-amide (Compound A-110)

Compound A-81(100mg, 0.158mmol) was dissolved in anhydrous dichloromethane (8mL), N' -dicyclohexylcarbodiimide (66mg, 0.317mmol), 4-dimethylaminopyridine (20mg, 0.158mmol) and nicotinic acid (30mg, 0.238mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, the filtrate was washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound a-110 (white solid, 83mg, yield 72%):¹H NMR(300MHz,methanol-d₄)5.99(t,J＝5.8Hz,1H),4.51(dd,J＝10.6,5.7Hz,1H),3.34–3.22(m,2H),3.09–2.93(m,8H),2.87–2.76(m,1H),2.57–2.34(m,4H),2.05(s,3H),1.19(s,3H),0.90(s,9H),0.87(s,3H),0.85(s,3H),0.74(s,3H).ESI-MS:m/z 736.6[M+H]⁺。

example 153

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (2S-amino-3-methylbutyryl) oxy-oleanane-13 (18) -ene-28-amide dihydrochloride (Compound A-111)

Compound A-81(100mg, 0.158mmol) was dissolved in anhydrous dichloromethane (8mL), and N, N' -dicyclohexylcarbodiimide (66mg, 0.317mmol), 4-dimethylaminopyridine (20mg, 0.158mmol) and N-Boc-L-valine (52mg, 0.238mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, suction filtration was performed, the filtrate was washed with water (8 mL. times.2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound XX-1 (white solid, 127mg, yield 97%).

Compound XX-1(127mg, 0.158mmol) was dissolved in absolute ethanol (10mL), and the reaction was stirred at room temperature with hydrogen chloride gas being introduced for 30 minutes. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate and methanol to give Compound A-111 (white solid, 95mg, yield 77%):¹H NMR(300MHz,methanol-d₄)4.77–4.69(m,1H),4.00(d,J＝3.8Hz,1H),3.34–3.13(m,8H),2.95(d,J＝13.9Hz,1H),2.87–2.71(m,2H),2.61(d,J＝14.3Hz,1H),2.46–2.37(m,1H),2.37–2.27(m,1H),1.29(s,3H),1.06(s,3H),1.04(s,3H),1.01(s,3H),0.99(s,6H),0.83(s,3H).ESI-MS:m/z 730.6[M+H]⁺。

example 154

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β -hydroxy-oleanane-13 (18) -en-28-amide hydrochloride (Compound A-112)

Compound A-81(100mg, 0.158mmol) was dissolved in absolute ethanol (10mL), and the reaction was stirred at room temperature with hydrogen chloride gas introduced for 30 minutes. After the reaction, the solvent was evaporated under reduced pressure until 2mL remained, and the residue was filtered, and the filter cake was washed with ethyl acetate (1 mL. times.2), and dried to obtain Compound A-111 (white solid, 52mg, yield 49%)¹H NMR(300MHz,DMSO-d₆)6.99–6.91(m,1H),3.90–3.51(m,8H),3.21–3.08(m,4H),3.06–2.97(m,1H),2.77(d,J＝13.7Hz,1H),2.42(d,J＝13.7Hz,1H),2.21–2.11(m,1H),1.13(s,3H),0.89(s,9H),0.83(s,3H),0.71(s,3H),0.67(s,3H).ESI-MS:m/z 631.5[M+H]⁺。

Example 155

3 β -hydroxy-oleanane-13 (18) -en-28-oic acid 3- (1, 1-dioxothiomorpholinyl) propyl ester (Compound A-113)

Compound II-1(200mg, 0.401mmol) was dissolved in N, N-dimethylformamide (8mL), and 3-bromo-1-propanol (73. mu.L, 0.802mmol) and potassium carbonate (111mg, 0.802mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, the reaction mixture was diluted with ethyl acetate (8mL), washed with water (8mL), and the aqueous layer was extracted with ethyl acetate (8mL), the organic phases were combined, washed with water (8 mL. times.2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate 5: 1) to obtain compound XXI-1 (white solid, 197mg, 88% yield).

Compound XXI-1(197mg, 0.354mmol) was dissolved in methylene chloride (8mL), pyridinium chlorochromate (153mg, 0.707mmol) was added, and the reaction was stirred at room temperature. After completion of the TLC detection, celite was filtered, and the filtrate was spun dry and purified by silica gel column chromatography (petroleum ether: ethyl acetate 10: 1) to give compound XXI-2 (white solid, 157mg, yield 80%).

Compound XXI-2(157mg, 0.283mmol) and 1, 1-thiomorpholine dioxide (39mg, 0.283mmol) were dissolved in dichloromethane (8mL), stirred at room temperature for 1 hour, then added with sodium triacetoxyborohydride (90mg, 0.424mmol), and the reaction was stirred at room temperature. After completion of the TLC detection reaction, saturated sodium bicarbonate solution (5mL) was added, the mixture was separated, the organic phase was washed with water (5 mL. times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 3: 1) to obtain compound XXI-3 (white solid, 69mg, yield 36%).

Compound XXI-3(69mg, 0.102mmol) was dissolved in a mixed solution of methanol (5mL) and tetrahydrofuran (5mL), and potassium hydroxide (57mg, 1.024mmol) was added to stir the reaction at room temperature. After completion of the TLC detection reaction, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (10mL), washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 3: 1) to obtain compound a-113 (white solid, 45mg, yield 69%):¹H NMR(300MHz,CDCl₃)4.21–4.02(m,2H),3.28–3.17(m,1H),3.14–2.89(m,8H),2.74(d,J＝11.3Hz,1H),2.57(t,J＝7.0Hz,2H),2.43(d,J＝14.0Hz,1H),2.22–2.11(m,1H),1.16(s,3H),0.99(s,3H),0.90(s,3H),0.90(s,3H),0.87(s,3H),0.77(s,3H),0.74(s,3H).ESI-MS:m/z 632.5[M+H]⁺。

example 156

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β -acetoxy-oleanane-12-en-28-amide (Compound B-121)

By a method similar to that in example 73, Compound XI-1 was obtained. Dissolving a compound XI-1(150mg, 0.300mmol) in tetrahydrofuran (8mL), sequentially adding N, N-diisopropylethylamine (50 mu L, 0.300mmol) and O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (116mg, 0.361mmol), and stirring at room temperature for reaction. After completion of the TLC detection reaction, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (8mL), washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 20: 1) to give compound XXII-1, which was used directly in the next step.

Compound XXII-1 was dissolved in N, N-dimethylformamide (8mL), 4- (3-aminopropyl) thiomorpholine-1, 1-dioxide (58mg, 0.300mmol) and sodium carbonate (32mg, 0.300mmol) were successively added, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, the reaction solution was diluted with ethyl acetate (8mL), washed with water (8mL), and the aqueous layer was extracted with ethyl acetate (8mL), the organic phases were combined, washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to give compound B-121 (white solid, 172mg, yield 85%):¹H NMR(300MHz,CDCl₃)6.10–5.97(m,1H),5.41–5.33(m,1H),4.54–4.43(m,1H),3.50–3.34(m,1H),3.17–2.92(m,8H),2.63–2.43(m,3H),2.05(s,3H),1.16(s,3H),0.93(s,3H),0.91(s,3H),0.90(s,3H),0.87(s,3H),0.86(s,3H),0.76(s,3H).ESI-MS:m/z673.6[M+H]⁺。

example 157

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (3-carboxypropionyl) oxy-oleanane-12-en-28-amide (Compound B-122)

Compound B-20(100mg, 0.158mmol) was dissolved in anhydrous pyridine (8mL), succinic anhydride (159mg, 1.585mmol) and 4-dimethylaminopyridine (20mg, 0.158mmol) were added, and the reaction mixture was heated to 115 ℃ and stirred overnight. After completion of the TLC detection reaction, the reaction solution was diluted by adding dichloromethane (20mL), washed with 1N diluted hydrochloric acid (10mL × 3) and water (10mL × 3) in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound B-122 (white solid, 64mg, yield 55%):¹H NMR(300MHz,CDCl₃)6.07–5.97(m,1H),5.39–5.33(m,1H),4.59–4.45(m,1H),3.50–3.34(m,1H),3.12–2.90(m,8H),2.74–2.57(m,4H),2.57–2.42(m,4H),1.16(s,3H),0.93(s,3H),0.91(s,3H),0.90(s,3H),0.86(s,6H),0.76(s,3H).ESI-MS:m/z 731.6[M+H]⁺。

example 158

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (3-picolinoyl) oxy-oleanan-12-en-28-amide (Compound B-123)

Compound B-20(141mg, 0.223mmol) was dissolved in anhydrous dichloromethane (8mL), and N, N' -dicyclohexylcarbodiimide (184mg, 0.894mmol), 4-dimethylaminopyridine (28mg, 0.223mmol) and nicotinic acid (55mg, 0.447mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, the filtrate was washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound B-123 (white solid, 118mg, yield 72%):¹H NMR(300MHz,CDCl₃)9.23(s,1H),8.77(d,J＝4.7Hz,1H),8.29(d,J＝8.0Hz,1H),7.39(dd,J＝8.0,4.9Hz,1H),6.07–5.96(m,1H),5.41–5.33(m,1H),4.83–4.70(m,1H),3.51–3.34(m,1H),3.11–2.92(m,8H),2.59–2.45(m,3H),1.19(s,3H),1.02(s,3H),0.99(s,3H),0.95(s,3H),0.92(s,3H),0.91(s,3H),0.78(s,3H).ESI-MS:m/z 736.6[M+H]⁺。

example 159

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β - (2S-amino-3-methylbutyryl) oxy-oleanane-12-en-28-amide dihydrochloride (Compound B-124)

Compound B-20(100mg, 0.158mmol) was dissolved in anhydrous dichloromethane (8mL), and N, N' -dicyclohexylcarbodiimide (66mg, 0.317mmol), 4-dimethylaminopyridine (20mg, 0.158mmol) and N-Boc-L-valine (52mg, 0.238mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC reaction, suction filtration was performed, and the filtrate was washed with water (8mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 20: 1) to obtain compound XXIII-1 (white solid, 125mg, yield 95%).

Compound XXIII-1(125mg, 0.158mmol) was dissolved in absolute ethanol (10mL), and the reaction mixture was stirred at room temperature with hydrogen chloride gas introduced thereinto for 30 minutes. After completion of the TLC detection reaction, the solvent was distilled off under reduced pressure, and the residue was slurried with ethyl acetate, suction-filtered, and the cake was washed with ethyl acetate (2 ml. times.2) and dried to obtain Compound B-124 (white solid, 110mg, yield 91%):¹H NMR(300MHz,methanol-d₄)5.45–5.39(m,1H),4.76–4.65(m,1H),4.00(d,J＝3.7Hz,1H),3.64–3.40(m,8H),3.32–3.18(m,1H),3.17–2.94(m,2H),2.93–2.82(m,1H),2.49–2.33(m,1H),1.25(s,3H),1.05(s,3H),1.01(s,6H),1.00(s,3H),0.97(s,3H),0.86(s,3H).ESI-MS:m/z 730.6[M+H]⁺。

example 160

N- (3- (1, 1-dioxothiomorpholinyl) propyl) -3 β -hydroxy-oleanane-12-en-28-amide hydrochloride (Compound B-125)

Compound B-20(70mg, 0.111mmol) was dissolved in absolute ethanol (10mL), and the reaction mixture was stirred at room temperature with hydrogen chloride gas introduced for 30 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was slurried with ethyl acetate, suction-filtered, and the filter cake was washed with ethyl acetate (2ml × 2) and dried to obtain compound B-125 (white solid, 69mg, yield 93%):¹H NMR(300MHz,methanol-d₄)5.47–5.41(m,1H),3.96–3.78(m,4H),3.73–3.60(m,4H),3.46–3.26(m,4H),3.26–3.17(m,1H),2.92–2.82(m,1H),1.25(s,3H),1.04(s,3H),1.03(s,3H),1.01(s,3H),0.99(s,3H),0.85(s,3H),0.84(s,3H).ESI-MS:m/z 631.6[M+H]⁺。

example 161

3 beta-hydroxy-oleanane-12-en-28-oic acid 3- (1, 1-dioxothiomorpholinyl) propyl ester hydrochloride (Compound B-126)

Compound XI-1(200mg, 0.401mmol) was dissolved in N, N-dimethylformamide (8mL), and 3-bromo-1-propanol (73. mu.L, 0.802mmol) and potassium carbonate (111mg, 0.802mmol) were added in this order, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, the reaction mixture was diluted with ethyl acetate (8mL), washed with water (8mL), and the aqueous layer was extracted with ethyl acetate (8mL), the organic phases were combined, washed with water (8 mL. times.2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and purified by silica gel column chromatography (petroleum ether: ethyl acetate 5: 1) to obtain compound XXIV-1 (white solid, 220mg, yield 98%).

Compound XXIV-1(220mg, 0.395mmol) was dissolved in dichloromethane (10mL), pyridinium chlorochromate (170mg, 0.790mmol) was added, and the reaction was stirred at room temperature. After completion of the TLC detection, celite was filtered, and the filtrate was spun dry and purified by silica gel column chromatography (petroleum ether: ethyl acetate 4: 1) to give compound XXIV-2 (white solid, 164mg, 75% yield).

Compound XXIV-2(220mg, 0.396mmol) and 1, 1-thiomorpholine dioxide (54mg, 0.396mmol) were dissolved in methylene chloride (8mL), and after stirring at room temperature for 1 hour, sodium triacetoxyborohydride (126mg, 0.595mmol) was added, and the reaction was stirred at room temperature. After completion of the TLC detection reaction, saturated sodium bicarbonate solution (5mL) was added, the mixture was separated, the organic phase was washed with water (5 mL. times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 3: 1) to obtain compound XXIV-3 (white solid, 131mg, yield 49%).

Compound XXIV-3(131mg, 0.194mmol) was dissolved in a mixed solution of methanol (5mL) and tetrahydrofuran (5mL), and potassium hydroxide (109mg, 1.943mmol) was added to stir the reaction at room temperature. After completion of the TLC detection reaction, the solvent was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (10mL), washed with water (10mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate ═ 2: 1) to obtain compound XXIV-4 (white solid, 75mg, yield 61%).

Compound XXIV-4(75mg, 0.119mmol) was dissolved in absolute ethanol (10mL), and the reaction mixture was stirred at room temperature with hydrogen chloride gas introduced thereinto for 30 minutes. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was slurried with ethyl acetate, suction-filtered, and the filter cake was washed with ethyl acetate (2ml × 2) and dried to obtain compound B-126 (white solid, 78mg, yield 60%):¹H NMR(300MHz,methanol-d₄)5.36–5.27(m,1H),4.16(t,J＝6.3Hz,2H),3.28–3.09(m,8H),2.99–2.89(m,1H),2.82–2.71(m,2H),1.23(s,3H),1.03(s,3H),1.01(s,3H),1.00(s,3H),0.97(s,3H),0.84(s,3H),0.83(s,3H).ESI-MS:m/z 654.5[M+Na]⁺。

example 162

N- (3 beta-acetoxy-oleanane-13 (18) -ene-28-acyl) -thiomorpholine-1, 1-dioxide (compound A-114)

Referring to the procedure of example 2, substituting N, N-dimethylethylenediamine for 1, 1-thiomorpholine dioxide, compound a-114 was prepared:¹H NMR(300MHz,CDCl3)4.55–4.46(m,1H),4.36–4.13(m,4H),3.05–2.93(m,4H),2.76(d,J＝13.0Hz,1H),2.48(d,J＝13.5Hz,1H),2.39(d,J＝12.5Hz,1H),2.05(s,3H),1.18(s,3H),0.92(s,3H),0.90(s,3H),0.87(s,3H),0.86(s,3H),0.85(s,3H),0.79(s,3H).ESI-MS:m/z 616.5[M+H]⁺。

example 163

N- (3 beta- (3-picolinoyl) oxy-oleanane-13 (18) -en-28-yl) -thiomorpholine-1, 1-dioxide (Compound A-115)

Referring to the procedure of example 152, compound A-115 was prepared by substituting compound A-81 for compound A-85:¹H NMR(300MHz,CDCl₃)9.27–9.19(m,1H),8.77(d,J＝3.8Hz,1H),8.29(d,J＝7.8Hz,1H),7.47–7.34(m,1H),4.85–4.72(m,1H),4.39–4.11(m,4H),3.13–2.90(m,4H),2.78(d,J＝13.3Hz,1H),2.49(d,J＝13.1Hz,1H),2.43–2.32(m,1H),1.21(s,3H),1.01(s,3H),0.95(s,6H),0.92(s,3H),0.89(s,3H),0.80(s,3H).ESI-MS:m/z 679.5[M+H]⁺。

example 164

N- (3 beta- (3-carboxypropionyl) oxy-oleanane-13 (18) -ene-28-acyl) -thiomorpholine-1, 1-dioxide (Compound A-116)

Referring to the procedure of example 2, compound a-116 was prepared by substituting N, N-dimethylethylenediamine for 1, 1-thiomorpholine and phthalic anhydride for succinic anhydride:¹H NMR(300MHz,CDCl₃)4.59–4.48(m,1H),4.38–4.12(m,4H),3.06–2.92(m,4H),2.83–2.73(m,1H),2.71–2.57(m,4H),2.48(d,J＝13.2Hz,1H),2.38(d,J＝12.4Hz,1H),1.18(s,3H),0.91(s,3H),0.90(s,3H),0.86(s,6H),0.85(s,3H),0.79(s,3H).ESI-MS:m/z 674.5[M+H]⁺。

example 165

N- (3 beta- (2S-amino-3-methylbutyryl) oxy-oleanane-13 (18) -en-28-yl) -thiomorpholine-1, 1-dioxide hydrochloride (A-117)

By substituting compound a-81 for compound a-85 according to the procedure of example 153, compound a-117 was prepared:¹H NMR(300MHz,DMSO-d₆)8.55–8.28(m,3H),5.61(d,J＝8.1Hz,1H),4.63–4.52(m,1H),4.15–3.93(m,4H),3.88(d,J＝3.8Hz,1H),3.22–3.01(m,4H),2.71(d,J＝11.9Hz,1H),2.38(d,J＝12.9Hz,1H),2.31–2.14(m,2H),1.14(s,3H),0.89(s,3H),0.88(s,6H),0.85(s,3H),0.83(s,3H),0.75(s,3H).ESI-MS:m/z 673.6[M+H]⁺。

example 166

N- (3 beta-acetoxy-oleanane-12-en-28-oyl) -thiomorpholine-1, 1-dioxide (compound B-127)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 1, 1-thiomorpholine dioxide to give compound B-127:¹H NMR(300MHz,CDCl₃)5.32–5.25(m,1H),4.53–4.44(m,1H),4.16–4.05(m,4H),3.12–2.90(m,5H),2.21(t,J＝13.6Hz,1H),2.04(s,3H),1.14(s,3H),0.93(s,6H),0.91(s,3H),0.86(s,3H),0.85(s,3H),0.71(s,3H).ESI-MS:m/z 616.5[M+H]⁺。

example 167

N- (3 beta- (3-picolinoyl) oxy-oleanane-12-en-28-oyl) -thiomorpholine-1, 1-dioxide (compound B-128)

Referring to the procedure of example 152, compound B-128 was prepared by substituting compound A-81 for compound B-9:¹H NMR(300MHz,CDCl₃)9.23(s,1H),8.77(d,J＝4.0Hz,1H),8.29(d,J＝8.0Hz,1H),7.42–7.35(m,1H),5.32–5.27(m,1H),4.82–4.72(m,1H),4.18–4.02(m,4H),3.14–2.91(m,5H),2.30–2.13(m,1H),1.17(s,3H),1.02(s,3H),0.98(s,3H),0.94(s,6H),0.92(s,3H),0.74(s,3H).ESI-MS:m/z 679.5[M+H]⁺。

example 168

N- (3 beta- (3-carboxypropionyl) oxy-oleanane-12-en-28-oyl) -thiomorpholine-1, 1-dioxide (Compound B-129)

Compound B-129 was prepared by substituting compound B-2 for compound B-9 with reference to the procedure of example 127:¹H NMR(300MHz,DMSO-d₆)12.19(s,1H),5.14–5.06(m,1H),4.45–4.35(m,1H),4.01–3.84(m,4H),3.15–2.88(m,5H),1.10(s,3H),0.88(s,9H),0.81(s,6H),0.66(s,3H).ESI-MS:m/z 674.5[M+H]⁺。

example 169

N- (3 β - (2S-amino-3-methylbutyryl) oxy-oleanan-12-en-28-oyl) -thiomorpholine-1, 1-dioxide hydrochloride (Compound B-130)

Referring to the procedure of example 159, Compound B-20 was substituted for Compound B-9 to give Compound B-130:¹H NMR(300MHz,DMSO-d₆)8.53–8.37(m,3H),5.15–5.06(m,1H),4.60–4.50(m,1H),4.02–3.82(m,5H),3.18–2.87(m,5H),1.11(s,3H),1.01(s,3H),0.99(s,3H),0.90(s,3H),0.87(s,6H),0.66(s,3H).ESI-MS:m/z 671.5[M+H]⁺。

example 170

3 beta- (2- (2-Acylaminoethoxyacyl) benzoyl) oxy-oleanane-12-en-28-oic acid 3- (4-morpholinyl) propyl ester (Compound B-131)

Referring to the procedure of example 136, 1, 2-dibromoethane was replaced with 1, 3-dibromopropane to produce compound B-131:¹H NMR(300MHz,CDCl₃)7.85–7.79(m,1H),7.69–7.63(m,1H),7.62–7.51(m,2H),6.60–6.50(m,1H),5.32–5.26(m,1H),4.75–4.66(m,1H),4.48–4.39(m,2H),4.13–4.01(m,2H),3.75–3.68(m,4H),3.68–3.59(m,2H),2.87(d,J＝10.3Hz,1H),2.52–2.31(m,6H),2.02(s,3H),1.15(s,3H),0.97(s,9H),0.93(s,3H),0.91(s,3H),0.75(s,3H).ESI-MS:m/z 817.7[M+H]⁺。

example 171

3 β - (2- (2-Acylaminoethoxyacyl) benzoyl) oxy-oleanane-12-en-28-oic acid 3- (1, 1-dioxothiomorpholinyl) propyl ester (Compound B-132)

Referring to the procedure of example 161, Compound XI-1 was substituted for Compound XVII-4 to give Compound B-132:¹H NMR(300MHz,CDCl₃)7.89–7.77(m,1H),7.73–7.63(m,1H),7.62–7.50(m,2H),6.60–6.45(m,1H),5.34–5.24(m,1H),4.75–4.64(m,1H),4.52–4.36(m,2H),4.14–4.01(m,2H),3.73–3.54(m,2H),3.02(d,J＝13.2Hz,8H),2.86(d,J＝14.1Hz,1H),2.65–2.51(m,2H),1.16(s,3H),0.97(s,9H),0.92(s,6H),0.75(s,3H).ESI-MS:m/z 865.6[M+H]⁺。

example 172

N- (3 beta-hydroxy-oleanane-12-ene-28-acyl) -4-benzyl-piperazine (compound B-133)

Method according to reference example 73Replacing N, N-dimethylethylenediamine with 1-benzylpiperazine to produce compound B-133:¹H NMR(300MHz,CDCl₃)7.39–7.27(m,5H),5.28–5.21(m,1H),3.78–3.60(m,4H),3.57(s,2H),3.27–3.16(m,1H),3.14–3.01(m,1H),2.58–2.31(m,4H),2.16–2.02(m,1H),1.13(s,3H),0.99(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.78(s,3H),0.71(s,3H).ESI-MS:m/z 615.6[M+H]⁺。

example 173

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -4-cyclopropyl-piperazine (compound B-134)

Referring to the procedure of example 73, substituting N, N-dimethylethylenediamine for 1-cyclopropylpiperazine produced compound B-134:¹H NMR(300MHz,CDCl₃)5.32–5.21(m,1H),3.72–3.44(m,4H),3.27–3.15(m,1H),3.15–3.03(m,1H),2.70–2.45(m,4H),2.19–2.03(m,1H),1.14(s,3H),0.99(s,3H),0.93(s,3H),0.90(s,3H),0.90(s,3H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z 565.6[M+H]⁺。

example 174

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -4-ethyl-piperazine (compound B-135)

Referring to the procedure of example 73, substituting N, N-dimethylethylenediamine for N-ethylpiperazine gave compound B-135:¹H NMR(300MHz,CDCl₃)5.32–5.23(m,1H),3.77–3.58(m,4H),3.29–3.16(m,1H),3.16–3.02(m,1H),2.54–2.30(m,6H),2.20–2.03(m,1H),1.14(s,3H),0.99(s,3H),0.93(s,3H),0.90(s,6H),0.78(s,3H),0.74(s,3H).ESI-MS:m/z 553.6[M+H]⁺。

example 175

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -4-oxo-piperidine (compound B-136)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 4-piperidone to obtain compound B-136:¹H NMR(300MHz,CDCl₃)5.33–5.26(m,1H),3.98–3.79(m,4H),3.26–3.16(m,1H),3.15–3.04(m,1H),2.56–2.33(m,4H),2.27–2.11(m,1H),1.16(s,3H),0.99(s,3H),0.95(s,3H),0.91(s,3H),0.91(s,3H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z 560.5[M+Na]⁺。

example 176

N- (3 beta-hydroxy-oleanane-12-en-28-oyl) -4-hydroxy-piperidine (compound B-137)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 4-hydroxypiperidine to obtain compound B-137:¹H NMR(300MHz,CDCl₃)5.30–5.23(m,1H),4.18–3.99(m,2H),3.97–3.84(m,1H),3.28–3.01(m,4H),1.14(s,3H),0.99(s,3H),0.93(s,3H),0.90(s,6H),0.78(s,3H),0.75(s,3H).ESI-MS:m/z 540.5[M+H]⁺。

example 177

N- (3 β -hydroxy-oleanane-12-en-28-oyl) -3-hydroxymethyl-azetidine (Compound B-138)

Referring to the procedure of example 73, N-dimethylethylenediamine was replaced with 3-azetidinemethanol to obtain compound B-138:¹H NMR(300MHz,CDCl₃)5.26(t,J＝3.0Hz,1H),4.54–3.82(m,4H),3.81–3.70(m,2H),3.27–3.14(m,1H),2.86(dd,J＝13.6,3.1Hz,1H),2.79–2.63(m,1H),1.13(s,3H),0.99(s,3H),0.93(s,3H),0.91(s,3H),0.89(s,3H),0.78(s,3H),0.77(s,3H).ESI-MS:m/z 524.5[M-H]^-。

example 178

N- (3 β -hydroxy-oleanane-12-en-28-oyl) -3-methoxycarbonyl-azetidine (Compound B-139)

Referring to the procedure of example 73, substituting N, N-dimethylethylenediamine for 3-methyl formate azetidine hydrochloride produced compound B-139:¹H NMR(300MHz,CDCl₃)5.38–5.19(m,1H),4.69–4.00(m,4H),3.75(s,3H),3.42–3.29(m,1H),3.21(dd,J＝9.1,4.7Hz,1H),2.82(dd,J＝13.8,3.5Hz,1H),1.13(s,3H),0.99(s,3H),0.93(s,3H),0.91(s,3H),0.90(s,3H),0.79(s,3H),0.76(s,3H).ESI-MS:m/z 552.5[M-H]^-。

example 179

N- (3 β - (pyridine-3-formyl) oxy-oleanane-12-en-28-oyl) -3-methoxy-azetidine (compound B-140)

Referring to the procedure of example 158, compound B-20 was substituted with compound B-110 to give compound B-140:¹H NMR(300MHz,CDCl₃)9.23(s,1H),8.77(d,J＝3.8Hz,1H),8.37–8.23(m,1H),7.39(dd,J＝7.9,4.9Hz,1H),5.27(t,J＝2.9Hz,1H),4.86–4.70(m,2H),4.44–3.86(m,5H),3.30(s,3H),2.87(dd,J＝13.5,3.6Hz,1H),1.15(s,3H),1.02(s,3H),0.99(s,3H),0.95(s,3H),0.93(s,3H),0.90(s,3H),0.78(s,3H).ESI-MS:m/z 631.5[M+H]⁺。

example 180

N- (3 β - (pyridine-2-formyl) oxy-oleanane-12-en-28-oyl) -3-methoxy-azetidine (compound B-141)

Referring to the procedure of example 158, compound B-20 was replaced with compound B-110 and nicotinic acid was replaced with 2-picolinic acid to give compound B-141:¹H NMR(300MHz,CDCl₃)8.83–8.74(m,1H),8.06(d,J＝7.8Hz,1H),7.82(td,J＝7.7,1.6Hz,1H),7.49–7.40(m,1H),5.27(d,J＝2.9Hz,1H),4.93–4.80(m,1H),4.45–3.81(m,5H),3.30(s,3H),2.93–2.79(m,1H),1.15(s,3H),1.04(s,3H),0.99(s,3H),0.96(s,3H),0.93(s,3H),0.90(s,3H),0.78(s,3H).ESI-MS:m/z 631.5[M+H]⁺。

example 181

N- (3 β - (furan-2-formyl) oxy-oleanane-12-en-28-oyl) -3-methoxy-azetidine (compound B-142)

Referring to the procedure of example 158, compound B-142 was prepared by substituting compound B-20 for compound B-110 and nicotinic acid for furoic acid:¹H NMR(300MHz,CDCl₃)8.06–7.98(m,1H),7.45(t,J＝1.7Hz,1H),6.81–6.73(m,1H),5.30(t,J＝3.2Hz,1H),4.70(dd,J＝9.3,6.8Hz,1H),4.55–3.86(m,5H),3.33(s,3H),2.89(dd,J＝13.3,3.2Hz,1H),1.18(s,3H),1.00(s,3H),0.98(s,3H),0.96(s,3H),0.95(s,3H),0.93(s,3H),0.81(s,3H).ESI-MS:m/z 631.5[M+H]⁺。

example 182

N- (3 β - (6-fluoropyridine-2-formyl) oxy-oleanane-12-en-28-yl) -3-methoxy-azetidine (compound B-143)

Referring to the procedure of example 158, compound B-143 was prepared by substituting compound B-20 for compound B-110 and nicotinic acid for 2-fluoropyridine-6-carboxylic acid:¹H NMR(300MHz,CDCl₃)8.00–7.87(m,2H),7.17–7.09(m,1H),5.30–5.24(m,1H),4.38–3.87(m,5H),3.30(s,3H),2.92–2.81(m,1H),1.15(s,3H),1.03(s,3H),0.98(s,3H),0.95(s,3H),0.93(s,3H),0.90(s,3H),0.78(s,3H).ESI-MS:m/z 649.5[M+H]⁺。

example 183

N- (3 β - (6-chloropyridine-2-formyl) oxy-oleanan-12-en-28-yl) -3-methoxy-azetidine (compound B-144)

Referring to the procedure of example 158, compound B-144 was prepared by substituting compound B-20 for compound B-110 and nicotinic acid for 2-chloropyridine-6-carboxylic acid:¹H NMR(300MHz,CDCl₃)7.97(d,J＝7.5Hz,1H),7.78(t,J＝7.7Hz,1H),7.49(d,J＝7.9Hz,1H),5.30–5.24(m,1H),4.88–4.76(m,1H),4.43–3.85(m,5H),3.30(s,3H),2.92–2.81(m,1H),1.15(s,3H),1.02(s,3H),0.98(s,3H),0.94(s,3H),0.93(s,3H),0.90(s,3H),0.78(s,3H).ESI-MS:m/z 665.5[M+H]⁺。

example 184

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. Administration of test Compounds under complete Medium conditionsBoth final concentrations of compound were set at 10 μ M for 12 hours, and AICAR (100 μ M) was used as a positive control compound. 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 with l.times.TBST 1 time, and placed in a previously prepared blocking solution (containing 0.1 part of% Tween20 in 5% BSA in 1 × TBS), blocked at room temperature for 1 h. 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. And the experimental result is 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 compounds

(Positive control AICAR concentration 100. mu.M; test compound concentration 10. mu.M)

As shown in the experimental results of table 1, the novel oleanolic acid and the-oleanolic acid derivatives provided by the present invention have significant AMPK agonistic activity at a concentration of 10 μ M. For example, compounds A-9, A-12, A-23, A-25, A-27, A-28, A-68, A-81, A-85, A-108, A-109, A-110, A-112, B-9, B-20, B-63, B-65, B-80, B-94, B-110, B-121, B-122, B-123, B-125, and the like are potent AMPK agonists with AICAR and oleanolic acid activities significantly better than 100 μ M. Wherein the AMPK agonistic activity of compound a-68 (pAMPK/AMPK ═ 5.48) is more than 3 times higher than that of-oleanolic acid (pAMPK/AMPK ═ 1.45). 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 185

Effect of Compounds on AMPK downstream Signaling pathways in Huh-7 cells

And detecting the influence of the compound on an AMPK downstream signal channel of the Huh-7 cell by adopting a Western Blot method.

Cell culture conditions: huh-7 cells: DMEM complete medium (containing 10% fetal bovine serum and 1% streptomycin/penicillin) in 5% CO₂Cultured in an incubator at 37 ℃.

Antibody: anti-ACC (CST, 3676S); anti-pACC (CST, 11818S); anti-mTOR (CST, 2983S); anti-pmTOR (CST, Ser 2448).

Western Blot experiment: detecting the effect of the compound on AMPK downstream signaling pathway of Huh-7 cells:

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 25 ten thousand per well and placed in a 5% CO₂After 12 hours, the original medium was discarded, and a complete medium containing the compound was added to the medium, the final concentration of each test compound was set to 10. mu.M, and the administration time was 12 hours. AICAR (100. mu.M) was used as the positive control compound. Then extracting the protein to carry out Western Blot detection, and detecting the protein change of pACC/ACC and pmTOR/mTOR. The results of the experiment are shown in FIG. 1.

The experimental results (figure 1) show that the compounds of the invention can effectively cause the change of AMPK downstream protein, and the change trend is related to AMPK excitement. For example, compounds B-125, A-112, B-122, A-109, B-121, A-108, B-123, A-110, B-20, A-81, etc., all increase the phosphorylation level of AMPK, decrease the phosphorylation level of mTOR, and simultaneously increase the phosphorylation level of ACC. In addition, other compounds of the invention increase phosphorylation of AMPK, decrease phosphorylation of mTOR, and simultaneously increase phosphorylation of ACC.

Example 186

Tablet formulation

The compound a-81 obtained in example 41 or the compound of other examples (50g), hydroxypropylmethylcellulose E (150g), starch (200g), appropriate amount of povidone K30 and magnesium stearate (1g) were mixed, granulated and tabletted.

In addition, the compounds prepared in examples 1 to 183 can be added with different pharmaceutical excipients according to the conventional preparation method of pharmacopoeia 2015 edition to prepare capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, patches and the like.

Claims

1. An oleanolic acid derivative or an oleanolic acid derivative, a pharmaceutically acceptable salt or ester or solvate thereof, represented by the following formula I or formula II:

r is H,

Or R^aCO-；

R' is OR²、NR³R⁴Or

R²Is C substituted by a substituent Z₁-C₅Alkyl, said substituent Z being pyrrolidin-1-ylPiperidin-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;

R³is H or C₁-C₃An alkyl group;

q is CH₂、O、NR⁶、S、SO₂、CHR⁷Or a chemical bond;

n is 0,1 or 2;

m is 0,1 or 2.

2. The compound, pharmaceutically acceptable salt or ester or solvate thereof, according to claim 1,

r is H,

Or R^aCO-；

R' is OR²、NR³R⁴Or

R³is H;

q is CH₂、O、NR⁶、S、SO₂Or CHR⁷；

R⁷is OH, NH₂COOH or C₁-C₃An alkylamino group;

n is 0 or 1;

m is 0 or 1.

3. The compound, pharmaceutically acceptable salt or ester or solvate thereof, according to claim 1, wherein in said compound of formula II, when substituent Z is N, N-dimethylamino or trimethylammonium, R is not H; when the substituent W is acetylamino, R is not H.

4. The compound, pharmaceutically acceptable salt or ester or solvate thereof, according to claim 1, wherein said compound, pharmaceutically acceptable salt or ester or solvate thereof is selected from the group consisting of:

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

6. Use of a compound, pharmaceutically acceptable salt or ester or solvate thereof according to any one of claims 1 to 4 in the manufacture of a medicament for the prevention or treatment of AMPK mediated diseases.

7. The use according to claim 6, 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.

8. The use of claim 6, 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, chronic bronchitis, 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.

9. A pharmaceutical composition for preventing or treating AMPK-mediated diseases, comprising the compound, pharmaceutically acceptable salt or ester thereof, or solvate thereof according to any one of claims 1 to 4 as an active ingredient and pharmaceutically acceptable excipients.

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