WO2006069466A1

WO2006069466A1 - Series of triterpenoid ptp1b inhibitors and their preparing method and use

Info

Publication number: WO2006069466A1
Application number: PCT/CN2004/001525
Authority: WO
Inventors: Lihong Hu; Jia Li; Di Hong; Liqiang Zhang; Qizhuang Ye
Original assignee: Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences; The National Center For Drug Screening
Priority date: 2004-12-27
Filing date: 2004-12-27
Publication date: 2006-07-06
Also published as: WO2006069499A1

Abstract

The invention discloses series of triterpenoid PTP1B inhibitors of following formula (I), and the pharmacologic studies further suggests such compounds or their physiological acceptable salts have the activity of inhibiting PTP1B, thus these compounds can be the euglycemic agents and can be used to prepare the medicament for the treatment of the disorders caused by insulin resistance. The invention also relates to a process for preparing such compounds.

Description

Technical field

The present invention relates to a class of triterpenoids which exhibit high inhibitory activity against protein tyrosine phosphatase ΡΤΡΙ (ΡΤΡΙ Β). These compounds are useful as PTP1B inhibitors and insulin sensitizers for the treatment of diabetes, obesity and its complications caused by insulin resistance. The invention also relates to a process for the preparation of such compounds. · . technical background:

Diabetes mellitus is a group of clinical syndromes caused by the interaction of genetic and environmental factors, due to absolute or relative deficiency of insulin secretion and decreased sensitivity of target tissue cells to insulin, causing sugar, protein, fat, water, and electrolytes. A series of metabolic disorders. Clinically, hyperglycemia is the main common marker. Long-term illness can cause multiple system damage. Acute metabolic disorders such as ketoacidosis can occur when the condition is severe and stress. Serious complications such as coronary heart disease, iron deficiency or hemorrhagic cerebrovascular disease, blindness, and gangrene in the diabetic population were significantly higher than those in the non-diabetic population. Therefore, diabetes and its complications have become a worldwide public health problem that seriously threatens human health.

Currently, diabetes is generally divided into two categories, type I diabetes (insulin-dependent diabetes mellitus, IDDM) and type II diabetes (non-insulin-dependent diabetes mellitus, NIDDM). More than 90% of diabetes is type 2 diabetes. The WHO expects that due to an aging population, obesity, unhealthy diet and a lack of exercise, the number of diabetic patients will increase from 135 million in 1995 to 300 million in 2025.

Type I diabetes patients have genetic susceptibility due to HLA-D gene on the short arm of chromosome 6 and abnormal response to environmental factors, especially viral infection or chemical toxic substances, directly or indirectly through autoimmune reactions, causing islets Beta cells are destroyed, resulting in insufficient insulin. The clinical features are acute onset, more common symptoms such as polyphagia, polyuria, polydipsia, and weight loss. There is a tendency to develop ketoacidosis, and insulin therapy must be relied upon to maintain life.

Type 2 diabetes also has strong hereditary and environmental factors, and it is significantly heterogeneous. The pathogenesis is diverse and complex, and there are large differences among patients. In general, it can be summarized as the relative deficiency of insulin secretion and insulin resistance. A series of studies on people with type 2 diabetes, especially obese diabetics, have confirmed that insulin resistance is a key factor in the development and progression of U-type diabetes. Based on the study of insulin signaling pathways in adipocytes and muscle cells, the development of insulin sensitizers to improve insulin resistance is the focus of new drug research for type II diabetes and one of its main directions. '

Type II diabetes is characterized by resistance to insulin action by insulin-sensitive tissues such as skeletal muscle, liver, and adipose tissue. Although the specific mechanism is still unclear, the attenuation or even blockade of insulin signaling in its conduction pathway must be a direct factor. Insulin binds to its receptor extracellular alpha subunit to activate the intrinsic tyrosine kinase activity of the receptor intracellular beta subunit, resulting in autophosphorylation of key tyrosine residues in the regulatory domain, thereby fully activating insulin receptor tyrosine The kinase activity of the insulin receptor, the insulin receptor tyrosine kinase, then transmits the signal by phosphorylating its substrate. With the deepening of the understanding of reversible tyrosine phosphorylation in the intracellular insulin action pathway, the role of protein tyrosine phosphatase (PTPases) in balancing the levels of related protein tyrosine phosphorylation in this pathway is increasingly Pay attention to it. PTPases may act on multiple pathways in the pathway, such as dephosphorylation of autophosphorylation-activated insulin receptor (1R), thereby reducing receptor kinase activity; Or dephosphorylation of a protein tyrosine residue in a substrate such as insulin receptor substrate 1 (IRS-1), insulin receptor substrate 2 (IRS-2), She, etc., thereby negatively regulating insulin Acting on the receptor pathway. An imbalance in enzyme activity between tyrosine kinases in specific PTPases and insulin pathways may be responsible for insulin resistance in type 2 diabetes. Therefore, it is becoming more and more important to treat type 2 diabetes by finding inhibitors of PTPases that selectively act on this pathway to inhibit its activity, and to enhance and prolong insulin signaling.

PTPases include a large family of transmembrane (receptor) and intracellular (non-receptor) enzymes involved in the regulation of a range of important life processes. Although a variety of PTPases are expressed in insulin-sensitive tissues, such as transmembrane CD45 and LAR-PTPase; intracellular SHPTP-1, SHPTP-2, PTP1B, PTP1C, etc., only a few PTPases may be in the insulin pathway. The receptor or receptor pathway affects normal insulin action. Current research focuses on LAR-PTPase, SHPTP-2 and PTP1B. '

PTP1B is the first PTPase to be purified and characterized biologically, with a total length of approximately 50 KD. Early studies have demonstrated that dephosphorylation of insulin receptors is effective in vitro; microinjection of PTP1 B derived from human placenta into Xenopus oocytes will reduce insulin-induced oocyte maturation and S6 peptide phosphorylation . Subsequently, it was found that PTP1B was highly expressed in all insulin-sensitive tissues; after administration of PTP1B antibody by osmotic shock, DNA synthesis and PI3 kinase activity levels were significantly increased in mouse KRC-7 hepatocytes stimulated by insulin, IR autophosphorylation level, 1R The level of kinase activity and the level of IRS-1 tyrosine phosphorylation were also significantly elevated. Recent studies have shown that PTP1 B interacts directly with the active IR; it also shows the highest selective activity against IRS-1 in vitro; high expression of PTP1B in rat fibroblasts can significantly reduce ligand-induced IR Phosphorylation level: Adenovirus-mediated gene transfection, high expression of PTP1 B in insulin-targeted skeletal and liver model cells L6 muscle cells and Fao cells, significantly inhibiting insulin-induced IR and IRS-1 Tyrosine phosphorylation, and thus significantly inhibits the formation of IRS-1 and PI3 kinase P85 subunit complexes and phosphorylation of Akt, MAPK, and insulin-induced glycogen synthesis is also inhibited [Egawa. et al. J. Biol. Chem. 276(13), 10207-1021 1.]. Using the same method to highly express PTP1 B in another insulin-targeted tissue adipose tissue model cell 3T3-L1, also significantly inhibiting insulin-induced tyrosine phosphorylation of IR, IRS-1 and PI3 kinase, P42 and P44 MAPK Phosphorylation levels were also significantly reduced, while Akt phosphorylation levels and activity were not affected [Venable CL et al. J. Biol. Chem. 275 (24), 18318-18326]. High expression of PTP1B had no effect on basic, moderate, and maximum insulin-induced glucose transport, and had no effect on transported EC50 insulin concentrations. These studies demonstrate that PTP1B is capable of negatively regulating the insulin signaling pathway and acting primarily on the insulin receptor. More important experimental evidence comes from PTP1 B knockout mice. Elchebly et al. reported that PTP1B knockout mice produced by homologous recombination have normal growth, fertility, and significant sensitivity to insulin, and this enhancement is associated with insulin receptors and insulin in the liver and skeletal muscle. Enhancement of phosphorylation levels in human substrate 1 [Elchebly M., et al. Science, 283, 1544-1548.] Surprisingly, PTP1B knockout mice also have food-induced weight gain and insulin resistance. Resistance. The same results were obtained by Klaman and other PTP1B knockout mice produced by the same method, and it was found that the PTP1 B knockout mice were resistant to food-induced weight gain due to the volume of fat cells. Reduced, while the number of fat cells does not change. PTP1B knockout mice have elevated levels of basic metabolism and overall energy expenditure [Klaman LD, et al. Molecular and Cellular Biology, 20(15), 5479-5489]. These experiments have more strongly demonstrated the important role of PTP1 B in insulin sensitivity, energy expenditure and fat storage, thus further clarifying that it is treating type 2 diabetes. A potential drug target for disease and obesity.

Some progress has been made in the study of PTP1B selective inhibitors, but most of them are limited to peptides or peptoid compounds, such as the inhibitor EEDE (F2PMP) M based on the substrate sequence of PTP1B dephosphorylation (Ki = 7.2 nM) Glu-F2PMP-F2PMP (IC50 = 40 nM), although these peptide inhibitors have strong inhibitory activity and high selectivity, the fact that they are peptide phosphate compounds makes it difficult to become a drug candidate compound. Recently, a series of non-peptide non-phosphorus compounds, PTP1 B inhibitors, have been reported to have certain selectivity. More importantly, some of these compounds have a significant effect on reducing glucose and insulin levels in plasma of ob/ob mice. . This is the first direct evidence of pharmacology that demonstrates that PTP1B inhibitors have anti-diabetic activity. [Malamas, M. S., et al. J. Med. Chem., 2000, 43, 1293-1310] These undoubtedly provide us with an opportunity to find new small molecule non-peptide organic compounds as efficient, highly selective PTP1B inhibitors. Summary of the invention

The object of the present invention is to provide a class of triterpenoids which are useful as protein tyrosine phosphatase PTP1B inhibitors and insulin sensitizers for the treatment of various diabetes, obesity and complications thereof. Another object of the present invention is to provide a method for synthesizing such compounds by using oleanolic acid or ursolic acid as a raw material. ■ .

The triterpenoid PTP1B inhibitor of the present invention has the following structure:

Wherein is hydrogen, methyl;

R ₂ is hydrogen or methyl;

Is a hydroxyl group, an amino group, a halogen, a fluorenyl group;

X is an alkyl group of Ci-C^, a substituted alkyl group, an unsaturated alkyl group; CONH(CH ₂ )nCH(R4) (n=l-20); CONH(CH ₂ )nCONH(CH ₂ )mCH(R ₅ ) (n=l-20, m-1-20);

R4 is a fluorenyl group, a substituted alkyl group, an unsaturated fluorenyl group, an aryl group, a substituted aryl group;

R ₅ is an indenyl group, a substituted indenyl group, an unsaturated indenyl group, an aromatic group, or a substituted aromatic group.

And physiologically acceptable salts.

A preferred compound of the invention is a triterpenoid PTP1B inhibitor of formula I and a physiologically acceptable salt thereof, wherein Ri is hydrogen;

R ₂ is a methyl group;

R ₃ is a hydroxyl group, an amino group, a halogen, a fluorenyl group;

for. 】-^.垸 group; CONH(CH ₂ )nCH(R4) , CONH(CH ₂ )nCONH(CH ₂ )mCH(R ₅ ), wherein n is 1 -20, m is 1-20; An alkyl group, an aromatic group, a substituted aryl group;

R ₅ is an indenyl group, an aromatic group, or a substituted aromatic group.

Another preferred compound of the invention is a triterpenoid PTP1B inhibitor of structural formula I and is physiologically acceptable

± ιτπ卜.,

Where ^ is methyl;

R ₂ is hydrogen:

Is a hydroxyl group, an amino group, a halogen, a fluorenyl group;

X is a substituted fluorenyl group of d-Czo, an unsaturated fluorenyl group; CONH(CH ₂ )nCH(R4) , CONH(CH ₂ )nCONH(CH ₂ )mCH(R ₅ ), wherein n is 1 -20, m is 20;

R4 is a substituted alkyl group, an unsaturated alkyl group, an aromatic group, or a substituted aromatic group;

It is an alkyl group, an aromatic group, or a substituted aryl group.

The invention is implemented by the following steps:

The screening study found that the chloroform fraction of the ethanol extract of the commonly used hypoglycemic Chinese medicine Cornus officinalis showed better inhibition of PTP1B activity (IC _5Q <2 (^g/mL), separated by activity tracking, from chloroform site It found two active monomer compound ursolic acid (ursolic acid, IC _5Q is 13.45μΜ) and oleanolic acid _(oleanolic acid, IC _{5 ()} of _21.90μΜ) ο

The invention uses oleanolic acid or ursolic acid as raw material to synthesize 28-long-chain fatty acid derivatives by Wittig or Wittig-Homer reaction, and obtains 3 other substituted 28-long-chain fatty acid derivatives by substituting 3-hydroxyl groups; Or by condensing with various amino acids, a 28-peptide chain derivative is synthesized, and 3 other substituted 28-peptide chain derivatives are obtained by substituting the 3-position hydroxyl group.

Synthesis route of 28-long chain fatty acid triterpenoid derivatives

Compound A under the action of NaH/Mel or CH ₂ N ₂ /TBDMSC1 to give 3-methyl ether-28-methyl ester or 3-tert-butyldimethylsilyl ether- 28-methyl ester derivative, and then passed through LiAlH ₄ Reduction of alcohol B, wherein R represents methyl or tert-butyldimethylsilyl. B is oxidized by oxalyl chloride to give aldehyde C. Aldehyde C is reacted by Wittig-Homer to give compound D, where n is 0-20. Compound D is then subjected to hydrogenation reduction and 3-methoxy deprotection to give 28-long chain fatty acid derivatives E and G. Aldehyde C is reacted by Wittig to give compound F, where n is 0-20. Compound F is further deprotected by 3-tert-butyldimethylsilyl sulfonyl group and hydrogenated to give 28-long chain fatty acid derivative G. Compound G gives a 3-keto derivative under the action of IBX, The 3-keto derivative is reacted with benzylamine to give a treatment with sodium borohydride and hydrogenation to give compound M. G is reacted with hydrogen sulfide and trialumina to obtain a 3-mercapto derivative N. Compound G was subjected to HX treatment to give 3 generations of derivative 0. The product obtained was confirmed by NMR.

Or NH-(CH ₂ ) _n CONH(CH ₂ ) _m CH(R ₄ )COOH

Synthesis route of 28-peptide chain triterpene derivatives Compound A is reacted with a mixed solution of acetic anhydride and pyridine (1:1, V/V) to give acetylated product I of compound A. Compound I is treated with a certain amount of oxalyl chloride to give compound J. Compound J is reacted with the methyl ester of the amino acid and the methyl ester of the dipeptide in an anhydrous aprotic solvent (eg, dichloromethane, trichloromethane, 1, 4-dioxane), and some organic base is required to be added. Catalyst (eg triethylamine, pyridine, diisopropylethylamine, N, N-dimethylpiperidine, etc.). The reaction temperature is generally carried out at room temperature, and the reaction time is usually from 1 to 3 h, and TLC is usually used to detect the degree of completion of the reaction. After the completion of the reaction, the product was directly subjected to removal of the solvent under reduced pressure, and the concentrate was subjected to column chromatography to obtain the final products 1 and 1 . The reaction yield is generally from 75% to 90%. The compound L or K gives a 3-keto derivative under the action of IBX, and the 3-keto derivative is reacted with benzylamine to obtain a compound S by sodium borohydride treatment and hydrogenation reduction. Compound G is reacted with hydrogen sulfide and trialuminate to give a 3-mercapto derivative T. Compound G is subjected to hydrazine treatment to give a 3-halo derivative U. The product obtained was confirmed by NMR. DRAWINGS

Figure 1 shows the effect of compound L-5 on the Km value of PTP1B on the substrate pNPP.

Figure 2 shows the effect of compound L-5 on the kcat value of PTP1B versus bottom pNPP.

Figure 3 shows the effect of compound L-5 on the level of tyrosine phosphorylation of Πβ in CHO/IR cells. . 'Benefit effect

The present invention demonstrates that a series of compounds synthesized are a novel class of protein tyrosine gluconate 1B inhibitors which are derived from natural products compared to the now known PTP1B inhibitors; such compounds The parent ursolic acid and oleanolic acid are the drugs used in the bed. They are very toxic and very safe. The raw materials for synthesizing these compounds are ursolic acid and oleanolic acid. detailed description

The invention is further illustrated by the following specific examples, without limiting the invention.

In the following preparations, 1H-NMR was measured with a Varian Mercury AMX300 model; MS was measured with a VG ZAB-HS or VG-7070 type meter, except for the EI source (70 ev) _; all solvents were re-used before use. Distillation, the anhydrous solvent used was obtained by drying according to standard methods; except for the description, all the reactions were carried out under the protection of Ar gas and followed by TLC. After the treatment, the mixture was washed with saturated brine and dried with anhydrous MgS0 ₄ . : Purification of the product except for the use of silica gel (200-300 mesh) column chromatography; the silica gel used, including 200-300 'mesh and GF254 is produced by Qingdao Ocean Chemical Plant or Yantai Yuanbo Silicone Company. In the pharmacological test, the PTPlb high-throughput screening model and the CHO/IR cell model were constructed according to the Guide to Molecular Cloning: PNpp. 2Na (p-nitrophenyl phosphate disodium salt): purchased from Shanghai Shenggong Biological Co., Ltd. Insulin (insulin injection): purchased from Shanghai First Biochemical Pharmaceutical Co., Ltd.; F12 medium and serum were purchased from GibcoTM. Example 1 Preparation of Compound A-1

Sodium hydride (40 mg, 50%, 0.870 mmol) was dissolved in dry DMF (5 mL) and stirred for 10 min. Then, oleanolic acid (60 mg, 0.132 mmol) was added in an ice bath, and after 20 min, a solution of iodothymidine in DMF (2 mL) was added and stirred at room temperature overnight. The unreacted sodium hydride was quenched with EtOAc (EtOAc)EtOAc. /V) Purified white solid A-1 (58 mg, 0.121 mmol).

NMR NMR (CDC1 ₃ , 300 MHz) (δ): 5.28 (m, 1H, -CH in 12), 3.61 (s, 3H, -CH ₃ in -COOCH ₃ ) 3.35 (s, 3H, -CH ₃ in CH3O -), 2.82 (m, 1H, -CH in 18), 2.67 (m, 1H, -CH in 3); ¹³ C NMR (CDC1 ₃ , 75.0 MHz) (<5): 38.51 (Cl), 22.17 (C -2), 88.88 (C-3), 38.86 (C-4), 55.95 (C-5), 18.39 (C-6), 32.84 (C-7), 39.47 (C-8), 47.80 (C- 9), 37.23 (C-10), 23.63 (C-ll), 122.620 (C-12), 143.981 (C-13), 41.804 (C-14), 27.860 (Cl 5), 23.24 (Cl 6), 46.89 (Cl 7), 41.46 (Cl 8), 46.05 (Cl 9), 30.09 (C-20), 34.03 (C-21), 32.56 (C-22), 28.30 (C-23), 16.52 (C- 24), 15.47 (C-25), 17.07 (C-26), 26.13 (C-27), 178.50 (C- 28), 33.32 (C-29), 23.84 (C-30), 57.75 (-CH ₃ In CH ₃ 0-), 51.75 (-CH ₃ in -COOCH ₃ ). Example 2 Preparation of Compound Bl

A-1 (500 mg, 1.093 mmol) was dissolved in dry tetrahydrofuran (25 mL) then EtOAc. Unreacted lithium aluminum hydride was quenched with a small amount of ethyl acetate, then iced water and chloroform (50 mL). The mixture was transferred to a sep. funnel, chloroform layer was dried, dried over anhydrous sodium sulfate, and evaporated to give a white solid B-1 (460 mg, 1.016 mmol).

'HNMR (CDC1 ₃ , 300 MHz) (δ): 5.19 (m, 1H, -CH in 12), 3.53 (d, 15 Hz, 1H, -CH in 28), 3.35 (s, 3H, -CH ₃ in CH ₃ 0-), 3.17 (d, J = 15 Hz, 1H, -CH in 28), 2.67 (m, 1H, -CH in 18); ^l3 C NMR (CDC1 ₃ , 75.0 MHz) (S): 38.73 (Cl), 22.21 (C-2), 88.88 (C-3), 38.93 (C-4), 55.96 (C-5), 18.47 (C-6), 32.19 (C-7), 40.03 (C- 8), 47.81 (C-9), 37.14 (C-10), 22.95 (C-ll), 122.59 (C-12), 144.47 (C-13), 41.90 (C-14), 25.76 (C-15 ), 22.23 (C-16), 34.33 (Cl 7), 42.57 (Cl 8), 46.71 (Cl 9), 30.50 (C-20), 32.80 (C-21), 31.27 (C-22), 28.33 ( C-23), 16.57 (C-24), 15.72 (C-25), 16.95 (C-26), 25.75 (C-27), 69.82 (C-28), 33.45 (C-29), 23.82 (C-30), 57.75 (-CH ₃ in CH ₃ 0-). Example 3 Preparation of Compound C-1

Oxalyl chloride (0.4 mL, 4.4 mmol) was dissolved in dichloromethane (10 mL), cooled to -60 °C, and DMSO (0.68 mL, 8.8 mmol) in dichloromethane (10 mL) , stir 5 niin at -60 °C. A solution of B-l (1.824 g, 4 mmol) in dichloromethane (20 mL) was slowly added dropwise and stirred at -60 °C for 15 min, then triethylamine (2.8 mL, 20 mmol). The temperature was slowly raised to room temperature and stirred for 48 h. The reaction mixture was cooled with EtOAc EtOAc (EtOAc m. , the yield is 90%. .

NMR NMR (CDC1 ₃ , 300 MHz) (δ): 9,39 (s, 1Η, -CH in 28), 5.33 (m, 1 H, -CH in 12), 3.36 (s, 3H, -CH ₃ in CH ₃ 0-), 2.60 (m, 1H, -CH in 18); ^l3 C NMR (CDC1 ₃ , 75.0 MHz) (δ): 38.61 (C-1), 22.21 (C-2), 88.86 (C- 3), 38.90 (C-4), 55.97 (C-5), 18.40 (C-6), 31.82 (C-7), 39.82 (C-8), 47.77 (C-9), 37.02 (C-10 ), 23.66 (Cl l), 123.53 (C-12), 143.21 (C-13), 41.92 (C-14), 26.95 (C-15), 22.33 (C-16), 49.32 (C-17), 40.63 (C-18), 45.82 (C-19), 30.87 (C-20), 33.38 (C-21), 32.97 (C-22), 28.35 (C-23), 16.56 (C-24), 15.55 (C-25), 17.25 (C-26), 25.77 (C-27), 207.76 (C-28), 33.31 (C-29), 22.66 (C-30), 57.77 (-CH ₃ in CH ₃ 0 -). Example 4 Preparation of Compound D1

Add sodium hydride (240 mg, 50%, 1 mmol) to DME (10 mL) under argon, stir for 10 min, cooled to 20 ° C, (diethoxy-phosphoryl)-ethyl acetate (224 mg, 1 mmol) was added and stirred at room temperature for 1 h. Add C-1 (20 mg, 0.043 mmol) in DME (2 mL) at 25 Torr, stir at room temperature for 1 h, then heat to reflux for 3 h. After cooling, a large amount of ice water was added to the reaction solution, which was extracted with chloroform (50 mL), dried over anhydrous sodium sulfate. , V/V) Purified pale yellow powder D-1 (17 mg, 0.032 mmol), yield 75.4%. .

NMR NMR (CDC1 ₃ , 300 MHz) (δ): 6.90 (d, J = 16.2 Hz, 1H, -CH in -CH=CH-), 5.75 (d, J = 6.2 Hz, 1 H, -CH in 28), 5.29 (m, 1H , -CH in 12), 3.35 (s, 3H, -CH 3 in CH3O-), 2.67 (m, 1 H, -CH in 3), 2.37 (m, 1H, -CH in 18); ¹³ C NMR (CDC1 ₃ , 75.0 MHz) (S): 38.51 (Cl), 22.17 (C-2), 88.88 (C-3) , 38.86 (C-4), 55.48 (C-5), 18.72 (C-6), 32.78 (C-7), 39.92 (C-8), 47.93 (C-9), 37.75 (C-10), 23.80 (C-ll), 123.09 (C-12), 144.20 (C-13), 41.91 (C-14), 27.18 (C-15), 26.41 (C-16), 39.55 (C-17), 44.00 (C-18), 46.65 (C-19), 30.98 (C-20), 34.52 (C-21), 34.23 (C-22), 29.15 (C-23), 16.92 (C-24), 15.92 ( C-25), 17.04 (C-26), 26.15 (C-27), 119.51 (C-28), 32.78 (C-29), 23.80 (C-30), 57.78 (-CH ₃ in CH ₃ 0- ), 159.28 (-CH in -CH=CH-), 167.54 (C in -CO-), 60.30 (-CH ₂ - in - COCH ₂ -), 14.55 (-CH ₃ in -CH ₂ CH ₃ ). Example 5 Preparation of Compound D-2

D-l (50 mg, 0.095 mmol) was dissolved in ethyl acetate (25 mL). Pd-C (10 mg). Filtration over celite, EtOAc (EtOAc)EtOAc.

'HNMR (CDC1 ₃ , 300 MHz) (δ): 5.18 (m, 1Η, -CH in 12), 4.11 (q, J=7.2 Hz, 2H, -CH ₂ - in -COCH2), 3.34 (s, 3H , -CH ₃ in -CH ₂ CH ₃ ), 2.65 (m, 1H, -CH in 3); ^l3 C NMR (CDCI3, 75.0 MHz) (^: 38.74 (Cl), 22.26 (C-2), 88.94 ( C-3), 38.94 (C-4), 55.96 (C-5), 18.47 (C-6), 32.73 (C-7), 40.07 (C-8), 47.86 (C-9), 37.16 (C -10), 23.83 (C-ll), 122.73 (C-12), 144.62 (C-13), 41.79 (C-14), 25.84 (C-15), 22.16 (C-16), 34.56 (C- 17), 47.28 (C-18), 46.87 (C-19), 29.59 (C-20), 34.62 (C-21), 32.77 (C-22), 28.33 (C-23), 16.58 (C-24) ), 15.75 (C-25), 16.87 (C-26), 26.32 (C-27), 32.16 (C-28), 33.48 (C-29), 23.83 (C-30), 57.78 (-CH ₃ in CH ₃ 0-), 34.96 (-CH ₂ in -CH ₂ CO -), 177.94 (C in -CO-), 60.30 (-CH ₂ - in a COCH ₂ -), 14.53 (-CH ₃ in _CH ₂ CH ₃ ). Example 6 Preparation of Compound G1

D-2 (40 mg, 0.080 mmol) was dissolved in acetonitrile (10 mL), sodium iodide (50 mg) was added, and then trimethyl chloride (0.5 mL) was added and refluxed for 6 h. Add a little ice water to the reaction and dilute with chloroform (50 mL) with (3×20 mL) The mixture was washed with saturated brine and dried over anhydrous sodium sulfate. Purification by silica gel column chromatography (EtOAcEtOAcEtOAcEtOAcEtOAc

NMR NMR (CDC1 ₃ , 300 MHz) (S): 5.20 (m, 1H, -CH in 12), 3.21 (m, 1H, -CH in 3); ^l3 C NMR (CDC1 ₃ , 75.0 MHz) (δ) : 39.30 (Cl), 28.47 (C-2), 78.44 (C-3), 39.76 (C-4), 56.18 (C-5), 18.47 (C-6), 32.77 (C-7), 40.07 ( C-8), 47.86 (C-9), 37.75 (C-10), 23.83 (Cl 1), 122.73 (C-12), 144.62 (C-13), 41.79 (C-14), 25.84 (C- 15), 22.16 (C-16), 34.56 (C-17), 47.29 (Cl 8), 46.87 (C-19), 29.59 (C-20), 34.62 (C-21), 32.77 (C-22) , 29.15 (C-23), 16.92 (C-24), 15.75 (C-25), 16.87 (C-26), 26.32 (C-27), 32.16 (C-28), 33.48 (C-29), 23.83 (C-30), 34.96 (-CH ₂ in -CH ₂ CO-), 181.27 (C in-CO -). Example 7 Preparation of Compound E -]

D-l (40 mg, 0.080 mmol) was dissolved in acetonitrile (10 mL), sodium iodide (50 mg) was added, and then trimethylsilyl chlorosilane (0.5 mL) was added and refluxed for 6 h. A little ice water was added to the mixture, which was diluted with chloroform (50 mL). Purification by silica gel column chromatography (EtOAc:EtOAc:EtOAc:EtOAc

NMR NMR (CDC1 ₃ , 300 MHz) (S): 6.98 (d, J- 16.2 Hz, 1 H, -CH in-CH=CH-), 5.78 (d, J = 16.2 Hz, 1H, -CH in 28 ), 5.20 (m, 1H, -CH in 12), 3.21 (m, 1H, -CH in 3); ^l3 C NMR (CDC1 ₃ , 75.0 MHz) (^: 39.30 (Cl), 28.47 (C-2) , 78.44 (C-3), 39.76 (C-4), 56.18 (C-5), 18.47 (C-6), 32.77 (C-7), 40.07 (C-8), 47.86 (C-9), 37.75 (C-10), 23.83 (C-ll), 122.73 (C-12), 144.62 (C-13), 41.79 (C-14), 25.84 (C-15), 22.16 (C-16), 34.56 (C-17), 47.29 (C-18), 46.87 (C-19), 29.59 (C-20), 34.62 (C-21), 32.77 (C-22), 29.15 (C-23), 16.92 ( C-24), 15.75 (C-25), 16.87 (C-26), 26.32 (C-27), 118.96 (C-28), 33.48 (C-29), 23.83 (C-30), 159.28 (- CH in -CH=CH-), 172.67 (C in -CO-). Example 8 Preparation of Compound A-2

Methyl oleate (470 mg, 1 mmol) was dissolved in 10 mL of anhydrous DMF and added with imidazole (98 mg, 1.2 Methyl), TBDMSCl (180 mg, 1.2 mmol). Diluted with 100 mL of distilled water, suction filtered, washed with EtOAc (EtOAc)EtOAc.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ): 0.03 (s, 6Η, -(CH ₃ ) ₂ Si-), 2.82 (m, -CH in 18), 3.17 ( m, 1H, H in 3) , 3.62 (s, 3H, -CH ₃ in COOCH ₃ ), 5.27 (m, 1H, H in 12); ^l3 C NMR (CDCI3, 75.0 MHz) (^: 39.52 (Cl), 27.84 (C-2), 79.70 (C-3), 39.54 (C-4), 55.54 (C-5), 18.75 (C-6), 32.97 (C-7), 39.47 (C-8), 47.89 (C-9), 37.15 (C-10), 23.66 (C-11), 122.67 (C-12), 143.99 (C-13), 41.85 (Cl 4), 27.92 (Cl 5), 23.30 (Cl 6), 46.95 (Cl 7) , 41.52 (C-18), 46.10 (Cl 9), 30.91 (C-20), 34.03 (C-21)' 32.62 (C-22), 28.76 (C-23), 16.32 (C-24), 15.54 (C-25), 17.05 (C-26), 26.14 (C-27), 178.50 (C-28), 33.34 (C-29), 23.84 (C-30), -3.52 (-CH ₃ in -( CH ₃ ) ₂ Si- ), -4.66 (-CH ₃ in-(CH ₃ ) ₂ Si- ), 26.15 (-C3⁄4 in (CH ₃ ) ₃ C-). Example 9 Preparation of Compound B-2

Compound A-2 (584 mg, 1 mmol) was dissolved in dry THF (20 mL). Add water (0.09 mL) to quench, then add (5 mol/L). NaOH (0.06 mL), water (0.29 mL), stir for 1 h, filter with a sand funnel, and use (20 mL) The mixture was washed with EtOAc (EtOAc m.)

'HNMR (CDC1 ₃ , 300 MHz) (S): 5.19 (m, 1H, -CH in 12), 3.56 (d, 1H, J= 15.2 Hz, -CH in 28), 3.17 (m, 1H, H in 28), 3.17 (m, 1H, H in 3) 0.03 (s, 6H, -(CH ₃ ) ₂ Si-); ¹³ C NMR (CDC1 ₃ , 75.0 MHz) (^: 38.81 (Cl), 28.75 (C -2), 79.66 (C-3), 40.01 (C-4), 55.46 (C-5), 18.77 (C-6), 32.86 (C-7), 40.01 (C-8), 47.82 (C- 9), 37.14 (C-10), 23.77 (C-11), 122.64 (C-12), 144.43 (Cl 3), 41.90 (C-14), 27.86 (C-15), 26.22 (C-16) , 37.02 (Cl 7), 42.55 (C-18), 46.68 (Cl 9), 31.16 (C-20), 34.33 (C-21), 31.26 (C-22), 28.75 (C-23), 16.33 ( C-24), 15.76 (C-25), 16.94 (C-26), 25.92 (C-27), 69.86 (C-28), 33.45 (C-29), 23.82 (C-30), -3.52 ( -CH ₃ in -(CH ₃ ) ₂ Si-), -4.66 (-CH _{3 in -} (CH ₃ ) ₂ Si-), 26.15 (-CH ₃ in (CH ₃ ) ₃ C-). Example 10 Compound Preparation of C-2

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Sial (76 mg, 2 mmol), stirred at room temperature overnight under argon. Add water (0.09 mL) to quench, add (5mol/L) NaOH (0.06 mL), water (0.29 mL), stir for 1 h, filter with a sand core funnel, and wash with (20 mL) dichloromethane. The filtrate was rotary evaporated under reduced pressure to give a white solid. Oxalyl chloride (0.1 mL, 1.1 mmol) was dissolved in dichloromethane (5 mL), cooled to -60 °C, EtOAc (0.17 mL, 2.2 mmol) in dichloromethane (5 mL) Stir at -60 ° C for 5 min. A solution of the above white solid product in dichloromethane (5 mL) was slowly added dropwise and stirred at -60 V for 15 min, then triethylamine (0.7 mL, 5 mmol). The temperature was slowly raised to room temperature and stirred for 48 h. The dichloromethane solution was washed three times with water (10 mL), dried over sodium sulfate and evaporated Take triphenylphosphonium 4-bromobutyrate (1.28 g, 3 mmol) dissolved in (10 mL) dry tetrahydrofuran, add sodium tert-butoxide (168 mg, 1.5 mmol) and stir for 15 min. The product was obtained as a yellow solid (555 mg, 1 mmol). After adding (10 mL) of water, the mixture was extracted with chloroform (3 mL), chloroform layer was dried over anhydrous sodium sulfate and evaporated to dryness, and the obtained oil was subjected to silica gel column chromatography ( petroleum ether: ethyl acetate = 10: 1, V/V) Purified pale yellow powder F-2 (393 mg, 0.58 mmol), yield 58%.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ): 0.03 (s, 3Η, -(CH ₃ ) ₂ Si-), 3.18 (m, IH, H in 3), 5.13 (m, 3H), 5.42 ( m, 2H). Example 15 Preparation of Compound G-4

The compound Fl (678 mg, 1 mmol) was dissolved in (10 mL) tetrahydrofuran, and (10 mL) trifluoroacetic acid: water (9:1) mixture was added, and the mixture was stirred overnight, and extracted with (10 mL) chloroform. The chloroform layer was dried over anhydrous sodium sulfate (MgSO4). Mmmol), yield 38%.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ): 2.37 (t, 2H, J = 5.6 Hz, -CH ₂ - in -CH ₂ -COOH), 3,22 (m, IH, H in 3); 5.21 (m, IH, H in 12), 5.21 (m, 2H, H in -CH=CH-), 5.43 (m, 2H, H in -CH=CH-COO-); ^l3 C NMR (CDC1 ₃ , 75.0 MHz) (δ): 38.67 (Cl), 28.35 (C-2), 79.33 (C-3), 38.97 (C-4), 55.43 (C-5), 18.55 (C-6), 33.64 (C -7), 39.90 (C-8), 48.36 (C-9), 37.24 (C-10), 22.91 (C-11), 127.97 (C-12), 145.40 (C-13), 42.08 (C- 14), 29.22 (C-15), 24.05 (Cl 6), 39.45 (Cl 7), 47.94 (C-18), 46.33 (C-19), 30.87 (C-20), 34.75 (C-21), 32.90 (C-22), 29.92 (C-23), 15.84 (C-24), 15.53 (C-25), 17.34 (C-26), 26.21 (C-27), 122.48 (C-28), 33.68 (C-29), 23.74 (C-30), 34.37 (-CH ₂ - in -CH ₂ -COOH ), 27.67 (-CH ₂ - in =CH-CH ₂ -), 27.60 (,CH ₂ - in - CH ₂ -CH=), 27.39 (-CH ₂ - in =CH-CH ₂ -), 177.86 (C in -COOH). Example 16 Preparation of Compound G-5

Compound G-4 (564 mg, 1 mmol) was dissolved in 5 mL of ethyl acetate. 5% EtOAc (5 mg) was added and stirred overnight under hydrogen atmosphere. White powder G-5 (541 mg, 0.96 mmol) Yield 96

%.

Ή-NMR (CDC1 ₃ , 300 MHz) (<5): 2.35 (t, 2H, J = 5.8 Hz, -CH ₂ -COO-), 3.22 (m, 1 H, H in 3), 5.16 (t, 1H, J = 3.2 Hz, H in 12). Example 17 Preparation of Compound F-3

Compound F-2 (678 mg, 1 mmol) was dissolved in dry THF (20 mL). Add water (0.09 mL) to quench, add (5 mol / L) NaOH (0.06 mL), water (0.29 mL), stir for 1 h, filter with a sand funnel, and wash with (20 mL) dichloromethane (Additional amount), the filtrate was rotary evaporated under reduced pressure to give a white solid. Oxalyl chloride (0.1 mL, 1.1 mmol) was dissolved in dichloromethane (5 mL), cooled to -60 V, DMSO (0.17 mL, 2.2 mmol) in dichloromethane (5 mL) Add and stir at -60 °C for 5 min. A solution of the above white solid product in dichloromethane (5 mL) was slowly added dropwise and stirred at -60 °C for 15 min, then triethylamine (0.7 mL, 5 mmol). The temperature was slowly raised to room temperature and stirred. The methylene chloride solution was washed three times with water (10 mL), dried over sodium sulfate and evaporated under reduced pressure to give a pale yellow solid. Take triphenylphosphonium 4-bromobutyrate (1.28 g, 3 mmol) dissolved in (10 mL) dry tetrahydrofuran, add sodium tert-butoxide (168 mg, 1.5 mmol) and stir for 15 min. The product was obtained as a yellowish solid, which was reacted for 3 h. After adding (10 mL) of water, the mixture was extracted with EtOAc EtOAc (EtOAc)EtOAc. V/V) Purified pale yellow powder F-3 (440 mg, 0.60 mmol), yield 60%.

Ή-NMR (CDC1 ₃ , 300 MHz) (S): 0.03 (s, 6H, -(CH ₃ ) ₂ Si-), 3.19 (m, 1 H, H in 3), 5.21 (m, 4H), 5.41 (m, 3H Example 18 Preparation of Compound G-6

The compound F-3 (732 mg, 1 mmol) was dissolved in (10 mL) tetrahydrofuran, and (10 mL) trifluoroacetic acid: water (9:1) mixture was added, and the reaction was stirred overnight and extracted with (10 mL) chloroform. After three times, the chloroform layer was dried over anhydrous sodium sulfate and evaporated to dryness, and the obtained oil was purified by silica gel column chromatography ( petroleum ether: ethyl acetate=2:1, V/V) to obtain white powder G-6 ( 229 mg, 0.38 mmol), yield 38%.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ): 3.22 (m, 1 Η, H in 3), 5.21 (m, 4H), 5.42 (m, 3H) ^{: l3} C NMR (CDC1 ₃ , 75.0 MHz) ( S): 38.63 (C-1), 29.36 (C-2), 79.32 (C-3), 38.95 (C-4), 55.38 (C-5), 18.53 (C-6), 33.60 (C-7 ), 39.86 (C-8), 48.33 (C-9), 37.21 (C-10), 22.88 (C-ll), 127.88 (C-12), 145.40 (C-13), 42.05 (C-14) , 29.91 (C-15), 23.72 (C-16), 39.41 (C-17), 47.91 (C-18), 46.30 (C-19), 30.87 (C-20), 34.73 (C-21), 32.86 (C-22), 28.32 (C-23), 15.51 (C-24), 14.35 (C-25), 17.34 (C-26), 26.20 (C-27), 122.40 (C-28), 33.64 (C-29), 24.01 (C-30), 34.40 (-CH ₂ - in -CH ₂ -COOH), 27.36, 27.44, 27.50, 27.58, 27.76, 28.32 (6C, -CH ₂ - in =CH-CH ₂ - or -CH ₂ -CH=), 178.70 (C in_COOH). Example 19 Preparation of Compound G-7

The compound G-6 (618 mg, 1 mmol) was dissolved in 5 mL of ethyl acetate, 5% EtOAc (5 mg) was added, and the mixture was stirred overnight under a hydrogen atmosphere. Yield white powder G-7 (611 mg, 0.98 mmol) Yield 98

%.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ) 2.33 ( t, 2Η, J= 5.5 Hz, -CH ₂ - in -CH ₂ -COOH), 3.24 (m, 1H, H in 3), 5.15 (t , 1H, J = 3.2 Hz, H in 12); ^l3 C NMR (CDC1 ₃ , 75.0. MHz) (S): 38.80 (C-1), 22.53 (C-2), 79.32 (C-3), 38.97 (C-4), 55.93 (C-5), 18.57 (C-6), 32.79 (C-7), 40.21 (C-8), 47.88 (C-9), 37.14 (C-10), 23.77 ( C-11), 122.02 (C-12), 145.44 (C-13), 41.83 (C-14), 26.31(C-15), 23.40 (C-16), 39.45 (C-17), 47.54 (C -18), 46.96 (C-19), 31.20 (C-20), 34.80 (C-21), 40.05 (C-22), 27.38 (C-23), 15.80 (C-24), 15.73 (C- 25), 16.94 (C-26), 26.00 (C-27), 33.56 (C-28), 34.73 (C-29), 23.86 (C-30), 34.23 (-CH ₂ - in -CH ₂ -COOH ), 30.85, 29.92, 29.45, 29.31 (10C, H30i3〇:) (H>ul.

%.

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Compound E-3 (484 mg, 1 mmol) was dissolved in dry tetrahydrofuran (20 niL). Add water (0.09 mL) to quench, add 5 N NaOH (0.06 mL), water (0.29 mL), stir for 1 h, filter with a sifting funnel, wash (20 mL) Rotary evaporation gave a white solid. Oxalyl chloride (0.1 mL, 1.1 mmol) was dissolved in dichloromethane (5 mL), cooled to -60 ° C, and DMSO (0.17 mL, 2.2 mmol) in dichloromethane (5 mL) Stirring 5 min at -60 V. The above white solid product of methylene chloride (5 mL) was slowly added dropwise. After stirring at -60 °C for 15 min, triethylamine (0.7 mL, 5 mmol) was added. . The temperature was slowly raised to room temperature and stirred for 48 h. The solution was washed three times with (10 mL) water, dried over sodium sulfate and evaporated. Take triphenylphosphonium 4-bromobutyrate (1.28 g, 3 mmol) dissolved in (10 mL) dry tetrahydrofuran, add sodium tert-butoxide (168 mg, 1.5 mmol) and stir for 15 min. The product was yellow solid and was reacted for 3 h. After adding (10 mL) of water, the mixture was extracted with EtOAc (EtOAc)EtOAc. Vv) Purified pale yellow powder E-4 (375 mg, 0.65 mmol), yield 65%.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ): 2.66 (m, 1 Η, Η in 3), 3.37 (s, 3Η, -OCH ₃ ), 5.13 (m, 3H), 5.42 (m, 2H) ¹³ C-NMR (CDC1 ₃ , 75.0 MHz) (S): 38.66 (Cl), 22.12 (C-2), 88.95 (C-3), 38.52 (C-4), 57.67 (C-5), 18.37 (C-6), 32.69 (C-7), 40.00 (C-8), 47.78 (C-9), 37.07 (C-10), 23.73 (C-ll), 122.18 (C-12), 145.07 ( C-13), 41.70 (Cl 4), 25.85 (C-15), 23.26 (Cl 6), 33.34 (Cl 7), 47.39 (C-18), 46.79 (Cl 9), 28.25 (C-20), 33.49 (C-21), 39.67 (C-22), 26.20 (C-23), 15.63 (C-24), 14.34 (C-25), 16.88 (C-26), 25.78 (C-27), 31.09 (C-28), 34.66 (C-29), 23.79 (C-30), 34.24 (-CH ₂ - in -CH ₂ -COOH), 179.84 (C in -COOH). Example 23 Preparation of Compound E-5'

The compound E-4 (578 mg, 1 mmol) was dissolved in 5 mL of ethyl acetate. 5% EtOAc (5 mg) was added, and the mixture was stirred overnight under hydrogen atmosphere. White powder E-5 (523 mg, 0.90 mmol) yield 90

%. Ή-NMR (CDCI3, 300 MHz) (δ): 2.33 (t, 2H, J = 5.7 Hz, -CH ₂ -COO-) 2.67 (m, 1H, H in 3) 3.36 (s, 3H, -OCH ₃ ), 5.16 (m, 1H, Hin 12). Example 24 Preparation of Compound E-6

Compound E-4 (578 mg, 1 mmol) was dissolved in dry THF (20 mL). Add water (0.09 mL) and quench it, then add 5. M NaOH (0.06 mL), water (0.29 mL), stir for 1 h, filter with a sand funnel, and wash (20 mL) with dichloromethane. The mixture was rotary evaporated to give a white solid. Oxalyl chloride (0.1 mL, 1.1 mmol) was dissolved in dichloromethane (5 mL), cooled to -60 °C, then EtOAc (0.17 mL, 2.2 mmol) Stirring 5 min at -60 °C The solution of the above white solid product in dichloromethane (5 mL) was slowly added dropwise. After stirring at -60 °C for 15 min, triethylamine (0.7 mL, 5 mmol) ) Join. The temperature was slowly raised to room temperature and stirred for 48 h. The solution of methylene chloride was washed three times with water (10 mL), dried over sodium sulfate and evaporated. Take triphenylphosphonium 4-bromobutyrate (1.28 g, 3 mmol) in (10 mL) dry tetrahydrofuran, add sodium tert-butoxide (168 mg, 1.5 mmol), stir for 15 min, add the reaction The product was yellowish solid and was reacted for 3 h. After adding (10 mL of water, and extracting with chloroform (3 mL), chloroform was dried over anhydrous sodium sulfate, and evaporated to dryness, and the obtained oil was subjected to silica gel column chromatography ( petroleum ether: ethyl acetate = 10:1, VV Purified pale yellow powder E-6 (372 mg, 0.59 mmol), yield 59%.

Ή-NMR (CDC1 ₃ , 300 MHz) (S): 2.67 (m , 1H , H in 3) , 3.36 (s , 3H, -OCH ₃ ); 5.17 (m , 4H) , 5.42 (m , 3H); ¹³ C-NMR (CDC1 ₃ , 75.0 MHz) (S) 38.59 (Cl), 22.24 (C-2), 89.07 (C-3), 38.92 (C-4), 57.77 (C-5), 18.44 (C -6), 33.63 (C-7), 39.92 (C-8), 48.38 (C-9), 37.25 (C-10), 22.77 (C-ll), 122.48 (C-12), 145.42 (C- 13), 41.06 (C-14), 28.37 (C-15), 22.77 (C-16), 39.43 (C-17), 47.95 (C-18), 46.34 (Cl 9), 30.88 (C-20) 34.75 (C-21), 31.84 (C-22), 28.37 (C-23), 16.58 (C-24), 14.38 (C-25), 17.37 (C-26), 25.03 (C-27), 127.72 (C-28), 32.90 (C-29), 23.79 (C-30), 31.84 (-CH ₂ - in -CH ₂ -COOH), 55.99 (-CH ₃ in -OCH ₃ ) 140.53, 131.22, 130.01 , 129.57, 128.90, 127.72 (6C in— CH=CH-), 179.57 (C in—COOH). Example 25 Preparation of Compound L1

Acetyl chloride of acetylated oleanolic acid (103.3 mg, 0.2 mmol) was dissolved in (10 mL) anhydrous dichloromethane. (87 mg, 0.4 mmol) of the hydrochloride salt of L-phenylalanine methyl ester was dissolved in (10 mL) anhydrous dichloromethane, and (0.5 mL) triethylamine was added to give L-phenylalanine methyl ester. The hydrochloride salt is completely dissolved. The latter was then added to a solution of the mixture, which was stirred for 1 h, concentrated, and purified using silica gel column ( petroleum ether: ethyl acetate = 10:1 v/v) to afford white crystals 1-1 (111.7 mg, 0.17 Mm), yield 86%.

Ή-NMR (CDC1 ₃ , 300 MHz) (δ): 2.03 (s, 3Η, CH ₃ inAcO), 2.43 (d, J = 11.7 Hz, 1H, - CH- in 18), 3.03 (dd,.J= 5.7 Hz, 13.8 Hz, 1H, CH ₂ in -CH ₂ -ph), 3.17 (dd, J = 5.7 Hz, 13.8 Hz, IH, CH ₂ in -CH ₂ -ph), 3.68 (s, 3H, H in 3'), 4.46 (t, J = 6.6 Hz, IH, -CH-in3), 4.73 (q, J=5.7Hz, IH, H in 1'), 5.27 (s, IH, -CH in 12), 6.63 (d, J = 6.0 Hz, 1 H, -H in -CONH-) , 7.07 (m, 2H, -H in -ph), 7.26 (m, 3H, -H in -ph); ^l3 C-NMR (CDC1 ₃ , 75.0 MHz) (S): 38.03 (Cl), 23.91 (C-2), 80.99 (C-3), 37.83 (C-4), 55.37 (C-5), 18.29 (C-6) , 32.82 (C-7), 39.55 (C-8), 47.64 (C-9), 36.99 (C-10), 23.67 (C-ll), 123.39 (C-12), 143.80 (Cl 3), 42.07 (Cl 4), 27.07 (Cl 5), 23.67 (Cl 6), 46.66 (C-17), 42.07 (C-18), 46.52 (C-19), 30.83 (C-20), 34.31 (C-21 ), 32.60 (C-22), 27.40 (C-23), 16.58 (C-24), 15.57 (C-25), 16.81 (C-26), 25.76 (C-27), 172.16 (C-28) , 53.58 (Cl,), 177.71 (C-2'), 52.26 (C-3'), 38.32 (CH ₂ in -CH ₂ -ph),] 36.29 (C in -ph), 129.49 (mC in -ph ), 127.19 (oC in -ph), 123.39 (pC in -ph), 171.09 (- CO- in CH ₃ -CO-), 21.42 (CH ₃ in CH ₃ -CO -). Example 26 Preparation of Compound L-2

Take Ll (78.9 mg, 0.12 mmol), add THF (12 mL), methanol (8 mL), 4 M aqueous NaOH (5 mL), stir overnight, and adjust pH to about 3 with 1 M diluted hydrochloric acid. </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; /. .

Ή-NMR (pyridine, 300 MHz) (δ): 3.46 (d, J = 6.3 Ηζ, ΙΗ, -CH- in 18), 3.64 (m, 2H, CH ₂ in CH ₂ -ph), 3.65 (m, IH; -CH- in 3), 5.271 (m, IH, - H in -CONH-), 7.34 (m, 2H, -H in -ph), 7.42 (m, 3H, -H in -ph): ^l3 C-NMR (CDC1 ₃ , 75.0 MHz) (S): 39.18 (Cl), 27.95 (C-2), 78.22 (C-3), 38.14 (C-4), 55.93 (C-5), 18.88 (C -6), 33.42 (C-7), 39.54 (C-8), 48.27 (C-9), 37.46

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SZST00/f00ZM3/X3d 99t690/900Z OAV The product of PT/CN2004/001525 has a strong light absorption at 410 _nm , so it is possible to directly observe the change in light absorption at 410 nm to observe changes in enzyme activity and compound-inhibition of enzyme activity. The standard assay system is as follows: 50 mM Mops, ra 7.0, 1 mM EDTA, 2 mM DTT, 2 mM PNPP, 2% DMSO, 40 nM hPTPlB.

pNPP 410 nm II. Observation index: The optical absorption at 410 nm is measured dynamically for 3 min, and the slope of the first-order reaction of the kinetic curve is used as the activity index of the enzyme.

3. Sample test: 1. Dissolve the l mg sample in 200 L DMSO. 20 L was added to the A2-H11 sample well of a %-well polypropylene plate, and then 80 L of DMSO was added as a mother plate. 2. Use the Biomek 2000 Automated Loading System to take 2 samples into the corresponding sample wells of the %-well polystyrene plate for use as a daughter plate for screening. 3. Sample wells 2 DMSO was added to Al-DK E12-H12 as a 100% enzyme activity control. 4. Add 2 L of different concentrations of positive control (four concentrations diluted by 10 g/mL) to sample wells A12-D12 and E1-H1. 5. Sample wells A1-H12 are added to the 88 μί Assay mix. 6. Sample wells A1-H12 are added to 10 μί1ιΡΤΡ1Β. 7. Measure the light absorption at 410 nm on a SpectraMAX 340 for 3 minutes. 8. Output the data as a text file and open it in Excel. The Vmax values of sample wells A1-D1 and E12-H12 are averaged as 100% enzyme activity. The inhibition rate of the compound to PTP1B was obtained by the following formula:

% inhibition rate = (1 - Vmax value for each screening well I blank control Vmax average) χ 100%

4. Test results: The screening result is the percent inhibition of enzyme activity when the concentration of the compound is 20 g/mL. When the inhibitory activity is higher than 50%, the IC ₅ o is obtained by routine screening, and the positive control 4-[4 - (4-oxalyl - phenoxymethyl) - phenylmethoxy] - phenylethyl acid (4- [4- (4-oxalyl -phenoxymethyl) -benzyloxy] -phenyl-oxo-acetic acid) of the IC ₅₀ It is 5.4 μΜ [Christopher T. Seto, et al. J. Med. Chem., 2002, 45, 3946-3952]. The IC50 values of each test compound for inhibiting hPTP-ΙΒ are shown in Table 1.

4-[4-(4-oxalyl-phenoxymethyl)-benzyloxy]-phenyl-oxo-acetic acid Test compound inhibits PTP1B activity data

Example 32 Effect of Compound L-5 on the Binding Capacity and Catalytic Rate of PTP1 B to Substrate pNPP

First, the test method: 1, in the 96-well polystyrene plate sample hole A1 - HI; A3-H3; A5-H5; A7-H7; A9-H9; A1 1 - H11 respectively added 625nM start with DMSO two Double dilutions of 6 L of L-5 in DMSO solution. 2. In the sample wells A1-A12; B1-B12; C1-C12; D1-D12; E1-E12; F1-F12; G1-G12; H1-H12, add eight dilutions of 40 mM starting with DMSO Concentration of substrate pNPP in DMSO solution 10 μΙ. 3. The sample wells A1-H12 were added to the 88 L Assay mix. 6. Sample well A H12 is added with 10 μί ΡΤΡ1 分别. 7. Measure the light absorption at 410 nm on a SpectraMAX 340 for 3 minutes.

2. Test results: Compound L-5 inhibits PTP1B with an IC50 of 250 nM and has high inhibitory activity. Moreover, as the concentration of the inhibitor increased, the binding capacity of PTP1B to the substrate pNPP, that is, Km became larger, and the catalytic rate (kcat) of the substrate did not change significantly (see Fig. 1 and Fig. 2). Thus, the compound is a competitive inhibitor of a class of PTP1 B. Example 33 Effect of Compound L-5 on IR Phosphorylation Level in CHO/IR Cells

I. Test principle: The phosphorylation level of insulin receptor IR in insulin-sensitive cells is tightly regulated by protein tyrosine kinases and protein tyrosine phosphatases, PTP. 1B acts as a negative regulator in this process. By detecting changes in IR phosphorylation levels in CHO/IR cells (National New Drug Screening Center), it is possible to determine whether PTP1 B inhibitors enter cells and function intracellularly.

2. Test methods: 1. Cell seeding: Cells with good growth state were connected to a 6-well plate at a density of 3-4 X 105 cells/well, and 2 mL of medium F12 + 10% NCS per well. 2. Hunger: After 24 hours, serum-free F12 medium, 2 mL, overnight for 12 h. 3. Administration: Remove the old medium, add fresh F12, lmL, and incubate for 20 minutes. The compound 1 μ υ 1 mL F12 serum-free medium was diluted and added to each well for 2 to 4 hours. Positive and negative controls were made with 1 mM Na ₃ V0 ₄ and 0.1% DMSO, respectively. 4. Insulin stimulation: Insdin with a final concentration of 10 nM was added to each well for 10 minutes. Or no insulin stimulation. 5, take the sample: Discard 'to the medium, add 100 μL X 1 per well to the buffer to lyse the cells.

III. Test results: Studies on compound L-5 showed that the phosphorylation level of IR increased with the increase of inhibitor concentration. There is now a clear upward trend. Thus, L-5 inhibits intracellular IR dephosphorylation, indicating that this compound may have an enhanced effect in maintaining insulin-stimulated signals' ability to alleviate insulin resistance to some extent.

Claims

Rights request

1. A class of triterpenoid PTP1B inhibitors having the following structure and physiologically acceptable salts:

Wherein is hydrogen, methyl;

R ₂ is hydrogen or methyl;

For hydroxyl, amino, halogen, fluorenyl:

X is an alkyl group of a Cr o , a substituted fluorenyl group, an unsaturated fluorenyl group; CONH(CH ₂ )nCH(R 4 ) . , CONH(CH ₂ )nCONH(CH ₂ )mCH(R ₅ ), wherein η is 1-20 , m is 1-20;

a fluorenyl group, a substituted fluorenyl group, an unsaturated fluorenyl group, an aromatic group, a substituted aryl group;

R ₅ is an alkyl group, a substituted alkyl group, an unsaturated fluorenyl group, an aromatic group, or a substituted aryl group.

2. The triterpenoid PTP1 B inhibitor according to claim 1 and a physiologically acceptable salt thereof, wherein is hydrogen;

!3⁄4 is a methyl group;

R ₃ is a hydroxyl group, an amino group, a halogen, a fluorenyl group;

Is ^-^. Alkyl; CONH(CH ₂ )nCH(R4) , CONH(CH ₂ )nCONH(CH ₂ )mCH(R ₅ ), wherein n is 1-20 and m is 1-20;

a thiol group, an aromatic group, a substituted aryl group;

It is an alkyl group, an aromatic group, or a substituted aryl group.

3. The triterpenoid PTP1B inhibitor according to claim 1 and a physiologically acceptable salt thereof, wherein ^ is a methyl group;

R ₂ is hydrogen;

Is a hydroxyl group, an amino group, a halogen, a fluorenyl group;

X is a substituted thiol group of C !-CM, an unsaturated alkyl group; CONH(C3⁄4)nCH(R4) , CONH(CH ₂ )nCONH(CH ₂ )mCH(R ₅ ), wherein η is 1-20, m is 20;

a substituted fluorenyl group, an unsaturated alkyl group, an aromatic group, a substituted aryl group;

It is an alkyl group, an aromatic group, or a substituted aryl group.

The method for preparing a triterpenoid PTP1 B inhibitor according to claim 1, wherein the 28-long-chain fatty acid derivative is synthesized by Wittig or Wittig-Horner reaction using oleanolic acid or ursolic acid as a raw material. By replacing 3 The hydroxyl group obtains 3 other substituted 28-long chain fatty acid derivatives; or by condensing with various amino acids, synthesizing the 28-peptide chain derivative, and obtaining 3 other substituted 28-peptide chain derivatives by substituting the 3-position hydroxyl group .

5. Use of a triterpenoid PTP1B inhibitor according to claim 1 as an insulin sensitizer.

The triterpenoid PTP1B inhibitor according to claim 1 which has activity for inhibiting PTP1B activity as a medicament for treating diabetes, obesity and complications thereof caused by insulin resistance.