CN109223806A - The purposes of dammarane type tetraterpene derivatives - Google Patents

The purposes of dammarane type tetraterpene derivatives Download PDF

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CN109223806A
CN109223806A CN201810930014.1A CN201810930014A CN109223806A CN 109223806 A CN109223806 A CN 109223806A CN 201810930014 A CN201810930014 A CN 201810930014A CN 109223806 A CN109223806 A CN 109223806A
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胡立宏
李佳
刘军华
李静雅
黄勇
陈达锴
许磊
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Shanghai Institute of Materia Medica of CAS
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Shanghai Institute of Materia Medica of CAS
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Abstract

The present invention provides the purposes of a kind of dammarane type tetraterpene derivatives that there is general formula I to indicate or its pharmaceutically acceptable salt, and the pharmaceutical composition comprising the derivative or its pharmaceutically acceptable salt, the purposes of plant extracts.Dammarane type tetraterpene derivatives, pharmaceutical composition and the plant extracts has the function of activating 2 β of AMPK α, 1 γ 1 on a molecular scale, it can be used for the preparation of AMPK small molecule activators, and then be used to prepare the drug of prevention or treatment disease relevant to AMPK.

Description

Application of dammarane type triterpene derivative
The application is a divisional application of an invention patent application with the application date of 2013, 11/1, the application number of 201310534806.4 and the name of 'application of dammarane type triterpene derivatives'.
Technical Field
The invention relates to application of dammarane type triterpene derivatives, in particular to dammarane type triterpene saponins, dammarane type triterpenes, derivatives which are modified by taking the dammarane type triterpenes as parent structures, compositions containing the derivatives and application of plant extracts in preparing AMPK agonists, and application in preparing drugs for treating or preventing AMPK-related diseases.
Background
Currently, type II diabetes has become a major health-threatening disease in humans, the primary pathogenesis of which is insulin resistance. Therefore, promotion of glucose uptake and fatty acid oxidation by non-insulin dependent mechanisms is an important pathway for the prevention and treatment of type II diabetes. Recent studies have found that adenosine monophosphate-activated protein kinase (AMPK) is an important Ser/ThrD protein kinase that senses the cellular and global energy metabolism state and regulates its balance (Zhang B., et al., AMPK: an empirical drug target for diabetes and the metabolic syndrome. cell metabolism.9,407-416 (2009)). When AMPK is activated, catabolic pathways initiate and promote ATP production, such as glucose glycolysis and fatty acid oxidation; metabolic pathways reduce the consumption of ATP, e.g., triglyceride and cholesterol synthesis. Due to its important role in the systemic energy balance (including regulation of plasma glucose, fatty acid oxidation, and glycogen synthesis, among others), AMPK has become a hotspot in the field of type II diabetes. AMPK agonists have also been extensively studied because of their potential class of agents for the treatment of type II diabetes. In addition, due to the important role of AMPK in energy regulation, AMPK agonists are also widely used for the prevention and treatment of various diseases related to AMPK, including metabolic diseases, cancer, cardiovascular diseases, hepatitis, cirrhosis, etc. (yuqing, progress in the study of sinomorin, sinoguan. protein kinase AMPK. life science, 17, 147-. However, very few studies have been reported on AMPK direct agonists. Therefore, the method has important significance for the discovery and research of the AMPK agonist.
Dammarane (dammarane) type triterpenes are tetracyclic triterpenes, which are ring-closed by epoxysqualene in a chair-chair conformation. The dammarane type triterpene exists in the form of saponin, which is an important bioactive component in rare Chinese medicinal materials such as ginseng, American ginseng, gynostemma pentaphylla, pseudo-ginseng and the like. Dammarane triterpenes and saponins thereof, particularly Ginsenoside, have remarkable physiological activities and thus have attracted much attention from the beginning of the century, and various pharmacological activities thereof have been reported in large quantities (Qi, l.w., et al, ginsenosides from American ginseng: chemical and biological chemistry, 72,689-699 2011 (a)). The pharmacological activity mainly comprises: anti-tumor, hypoglycemic, action on the nervous system and cardiovascular system, anti-oxidant, anti-depression, immunity-enhancing etc. (Attale, A.S., et al, Ginseng pharmacology: multiple conjugates and multiple interactions, biochem. Pharmacol.58,1685-1693 (1999)). Nevertheless, only a few documents report that few dammarane triterpenes or their saponins have AMPK agonist activity (numyen, p.h., et al, New dammarane-type glucosides as potent activators of AMP-activated protein kinase (AMPK) from gymnostemma pentaphylum. bioorg.med.chem.19,6254-60 (2011)). Therefore, we have studied the AMPK agonistic activity of this class of compounds in detail.
Disclosure of Invention
The inventor of the application screens a Huizhou gynostemma pentaphylla standard extract and a Shaanxi gynostemma pentaphylla standard extract on a recombinant human-derived AMPK three-subunit protein α 2 β 1 gamma 1 molecular model by establishing a novel screening model based on a homogeneous phase time-resolved fluorescence resonance energy transfer principle (HTRF), finds that the two extracts can activate AMPK α 2 β 1 gamma 1 in a dose-dependent manner, then separates main components in American ginseng, the Huizhou gynostemma pentaphylla and the Shaanxi gynostemma pentaphylla, and carries out structural modification by taking active dammarane type triterpenes as lead compounds, finds that a series of dammarane type triterpenes derivatives can well activate AMPK α 2 β 1 gamma 1 phosphorylation and can be used for preparing AMPK direct agonists.
Therefore, the invention aims to provide the application of the compound represented by the general formula I or the pharmaceutically acceptable salt thereof in preparing the AMPK activator.
Wherein:
R1is OH, Represents a site of attachment in formula I;
R2is H or OH;
R3is C1-C6Straight or branched alkyl, C1-C6Straight or branched alkylcarbonyl or — CHO; preferably C1-C4Straight or branched alkyl, C1-C4Straight or branched alkylcarbonyl or — CHO; more preferably C1-C2Alkyl radical, C1-C2Alkylcarbonyl or — CHO; most preferably methyl or-CHO;
R4is H or OH;
R5is H or OH;
R6is composed of Wherein,represents a site of attachment in formula I;
R7is C1-C6Straight or branched alkyl, - (CH)2) n-OH orn is 1, 2, 3,4, 5 or 6; preferably, R7Is C1-C4Straight or branched alkyl, - (CH)2) n-OH orn is 1, 2, 3 or 4; more preferably, R7Is C1-C2Alkyl, - (CH)2) n-OH orn is 1 or 2; most preferably, R7Is methyl, hydroxymethyl or
R8Is a hydroxyl group,
R9Is composed ofOr
Wherein R is10And R11Each independently is H, C1-C6Straight or branched alkyl, C3-C6Cycloalkyl, hydroxy C1-C6Alkylene radical, C1-C6A straight or branched chain alkyloxy-substituted phenyl or-A-B;
a is C1-C4Alkylene radical, C1-C4Straight or linear alkyl substituted C1-C4Alkylene or carbonyl, B is substituted or unsubstituted C6-C14Aryl or a saturated or unsaturated 5-to 9-membered heterocyclic group containing 1 to 2 heteroatoms;
the substituent of the substituted phenyl is C1-C6Linear or branched alkoxy, -F, -Cl, -Br, -I or nitro, the heteroatom being O, N or S;
preferably, R10And R11Each independently is H, C1-C4Straight or branched alkyl, C3-C4Cycloalkyl, hydroxy C1-C4Alkylene radical, C1-C4Straight or branched chain alkyloxy substituted C6-C10Aryl or-A-B;
a is C1-C3Alkylene radical, C1-C3Straight or linear alkyl substituted C1-C3Alkylene or carbonyl, B is substituted or unsubstituted phenyl or a saturated or unsaturated 5-, 6-or 9-membered heterocyclyl containing 1-2 heteroatoms;
the substituent of the substituted phenyl is C1-C4Linear or branched alkoxy, -F, -Cl, -Br, -I or nitro, the heteroatom is O or N;
more preferably, R10And R11Each independently is H, C1-C4Straight or branched alkyl, cyclopropyl, hydroxy C1-C3Alkylene radical, C1-C3Straight or branched chain alkoxy-substituted phenyl, C1-C3Straight-chain or branched alkoxy-substituted naphthyl, C1-C3A straight or branched chain alkyloxy-substituted biphenyl or-A-B;
a is C1-C2Alkylene radical, C1-C2Alkyl substituted C1-C2Alkylene or carbonyl, B is substituted or unsubstituted phenyl,Furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl or pyrazolyl and pyridyl;
the substituent of the substituted phenyl is C1-C2Alkyloxy, -F, -Cl, -Br, -I or nitro.
Most preferably, the compound of formula I is one of the following compounds:
wherein,represents the site of attachment in formula I.
The term "pharmaceutically acceptable salt" in the present invention refers to a salt of the compound of the general formula I with an inorganic acid such as phosphoric acid, sulfuric acid, hydrochloric acid, or the like, or an organic acid such as acetic acid, tartaric acid, citric acid, malic acid, or the like, or an acidic amino acid such as aspartic acid, glutamic acid, or the like, or a salt of the compound of the general formula I with an inorganic base such as a sodium salt, a potassium salt, a calcium salt, an aluminum salt, and an ammonium salt after forming an ester or an amide with the above.
Another object of the present invention is to provide the use of the compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition or a plant extract comprising the compound of formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing or treating diseases associated with AMPK. Wherein the pharmaceutical composition comprises an optionally pharmaceutically acceptable carrier; the plant extracts are, for example, Gynostemma Pentaphyllum Makino extract and Gynostemma Pentaphyllum Makino extract.
The disease is selected from one or more of diabetes, chronic complications of diabetes, hyperlipidemia, obesity, hyperinsulinemia, insulin resistance, myocardial hypertrophy, arrhythmia, syndrome X, atherosclerosis, heart failure, ischemic heart disease, upper respiratory infection, chronic obstructive pulmonary disease, chronic bronchitis, pulmonary fibrosis, asthma, hypertension, hepatitis, liver cirrhosis, fatty liver, hepatic fibrosis, alcoholic liver, prostatitis, pancreatitis, nephritis, chronic renal insufficiency, nephrotic syndrome, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, tumor, radiotherapy and chemotherapy insensitivity and radiotherapy and chemotherapy toxic reaction. (Wu Wei Teng, Teng Mei Luo, mangiferin calcium salt as AMPK agonist CN 101919839A; Gengning, Gaoju, protein kinase AMPK research progress Life sciences, 17, 147 + 152 (2005); Xuehe, Jujuanjuan, Jiang Xianjuan, Madong Ming, AMPK research progress with atherosclerosis relation. Tianjin Chinese medicine university report. 31,126 + 128 (2012); Ge, Schemelin, Guzhengxuan, Zhouyuan, Guoex, AMPK research progress as a new target for treating type 2 diabetes mellitus. Chinese pharmacology report. 24,580 + 583 (2008)).
The diabetic complications include diabetic heart disease, type II diabetes complicated with cerebral infarction, type II diabetes complicated with alcoholic fatty liver, diabetic nephropathy, diabetic retinopathy, diabetic peripheral neuropathy, etc.
Drawings
FIG. 1 is a diagram of the main components (HPLC-ELSD (evaporative light scattering)) in the extract of Gynostemma Pentaphyllum;
FIG. 2 is a diagram of the major components (HPLC-ELSD (evaporative light scattering)) in the extract of Gynostemma pentaphyllum in Shanxi.
Detailed Description
The invention is further illustrated with reference to the following specific examples, without limiting the scope of the invention. The experimental procedures of the present invention are versatile and are not limited to the compounds mentioned in the invention.
In the following preparation examples, the following examples were conducted,1H-NMR was measured using a Varian Mercury AMX300 model apparatus. MS with VG ZAB-HS or VG-7070 type and Esquire 3000 plus-01005. All solvents were redistilled before use. All reactions were carried out under nitrogen and followed by TLC, after-treatment with a saturated saline wash and anhydrous sodium sulfate drying, except as indicated. The purification of the product uses silica gel (200-300 mesh) column chromatography except for illustration, the silica gel used comprises 200-300 mesh, and GF254 is produced by Qingdao ocean chemical plant or Tatai Bingbo silica gel company.
Preparation of examples of embodiment
Extract of Gynostemma Pentaphyllum Makino and Gynostemma Pentaphyllum Makino
The extract of Gynostemma Pentaphyllum Makino and Gynostemma pentaphyllum Makino are purchased from Oriental plant health care technology Co., Ltd, and the main components in the extracts of the Gynostemma Pentaphyllum Makino and the Gynostemma pentaphyllum Makino are dammarane type triterpenoid saponin. The two extracts were subjected to compositional analysis by HPLC-ELSD, and the composition charts are shown in FIG. 1 and FIG. 2.
EXAMPLE 1 preparation of Compound 1
100g of American ginseng total saponin (purchased from Nature bioengineering Co., Ltd. in Fusong county) and 1300ml of n-butanol are added into a 2L three-mouth reaction bottle, stirred for 10 minutes, added with 40g of metallic sodium in batches, stirred until no hydrogen is released, added with 10g of benzoyl peroxide, introduced with oxygen, and heated to 120 ℃ for 24 hours. After completion of the TLC detection reaction, the reaction mixture was cooled to room temperature, n-butanol was removed under reduced pressure, and then dissolved in water, followed by extraction with petroleum ether and ethyl acetate in this order, and the ethyl acetate fraction was distilled under reduced pressure to give an extract containing compound 1, which was eluted with silica gel column chromatography (petroleum ether: ethyl acetate: 2:1 (vol.%)) to give compound 1(7g, 7%).1H NMR(300MHz,CDCl3)δ5.19(t,J＝7.3Hz,1H),3.63(td,J＝10.4,5.1Hz,1H),3.22(dd,J＝11.2,5.0Hz,1H),1.72(s,3H),1.66(s,3H),1.22(s,3H),1.01(s,3H),1.00(s,3H),0.91(s,3H),0.90(s,3H),0.80(s,3H),0.74(d,J＝10.6Hz,1H)；ESI-MS461.3[M+H]+。
EXAMPLE 2 preparation of Compound 2
In a 50ml three-necked reaction flask, Compound 1(200mg, 0.435mmol) was dissolved in 15ml CH2Cl2Stirring at-78 deg.C for 10min, and introducing O3The reaction was carried out for 2 min. Then the temperature was raised to-3 ℃ and methylamine (0.1ml), NaBH (OAc) were added3(368.8mg,1.7mmol) and CH3OH (8ml) was stirred overnight, after completion of the reaction 15ml water was added and CH was used2Cl2(30ml) extracted 2 times, dried over anhydrous sodium sulfate and concentrated under reduced pressure CH2Cl2Silica gel column chromatography (dichloromethane/methanol/triethylamine, 40:1:0.5 (vol.%)) afforded compound 2 in 85% yield.1H NMR(300MHz,CDCl3)δ4.12(m,1H),3.52(td,J＝13.0,6.2Hz,1H),3.21(dd,J＝10.9,5.0Hz,1H),2.94(m,1H),2.62(m,1H),2.47(s,3H),1.13(s,3H),0.99(s,3H),0.98(s,3H),0.88(s,6H),0.78(s,3H),0.73(d,J＝11.0Hz,1H)；ESI-MS 450.7[M+H]+。
EXAMPLE 3 preparation of Compound 3
Compound 3 was prepared in the same manner as in example 2, except that ethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 45:1:0.5 (vol.%)) gave a yield of 86%.1H NMR(300MHz,CDCl3)δ3.55(td,J＝12.9,6.3Hz,1H),3.19(dd,J＝10.9,4.9Hz,1H),2.93(m,1H),2.72(m,1H),2.58(m,1H),2.48(m,1H),2.09(m,1H),1.15(s,3H),1.11(t,J＝7.2Hz,3H),0.99(s,3H),0.98(s,3H),0.89(s,3H),0.88(s,3H),0.78(s,3H),0.73(d,J＝10.7Hz,1H)；ESI-MS 464.7[M+H]+。
EXAMPLE 4 preparation of Compound 4
Compound 4 was prepared in the same manner as in example 2, except that propylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave 87% yield.1H NMR(300MHz,CDCl3)δ3.55(td,J＝12.7,6.2Hz,1H),3.20(dd,J＝10.8,5.2Hz,1H),2.94(m,1H),2.67(m,1H),2.52(m,2H),2.11(m,1H),1.14(s,3H),0.99(s,3H),0.97(s,3H),0.93(t,J＝7.3Hz,3H),0.89(s,3H),0.88(s,3H),0.78(s,3H),0.73(d,J＝10.9Hz,1H)；ESI-MS 478.7[M+H]+。
EXAMPLE 5 preparation of Compound 5
Compound 5 was prepared in the same manner as in example 2, except that butylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave 88% yield.1H NMR(300MHz,CDCl3)δ3.54(td,J＝12.6,6.1Hz,1H),3.20(dd,J＝10.7,5.3Hz,1H),2.95(m,1H),2.66(m,4H),1.14(s,3H),0.97(s,3H),0.95(s,3H),0.90(t,J＝7.3Hz,3H),0.87(s,3H),0.86(s,3H),0.76(s,3H),0.73(d,J＝10.5Hz,1H)；ESI-MS 492.6[M+H]+。
EXAMPLE 6 preparation of Compound 6
Compound 6 was prepared in the same manner as in example 2, except that isopropylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave 88% yield.1H NMR(300MHz,CDCl3)δ3.57(td,J＝12.5,6.0Hz,1H),3.19(dd,J＝10.8,5.2Hz,1H),2.95(d,J＝12.6Hz,1H),2.85(m,1H),2.50(m,1H),2.10(m,1H),1.14(s,3H),1.13(d,J＝7.2Hz,6H),0.97(s,3H),0.95(s,3H),0.87(s,3H),0.86(s,3H),0.76(s,3H),0.73(d,J＝10.9Hz,1H)；ESI-MS 478.6[M+H]+。
EXAMPLE 7 preparation of Compound 7
Compound 7 was prepared in the same manner as in example 2, except that tert-butylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave 88% yield.1H NMR(300MHz,CDCl3)δ5.30(s,1H),3.57(td,J＝12.3,6.1Hz,1H),3.19(dd,J＝10.7,5.3Hz,1H),2.93(d,J＝10.8Hz,1H),2.44(t,J＝11.5Hz,1H),2.09(m,1H),1.13(s,12H),0.97(s,6H),0.88(s,6H),0.77(s,3H),0.72(d,J＝10.8Hz,1H)；ESI-MS 492.7[M+H]+。
EXAMPLE 8 preparation of Compound 8
Compound 8 was prepared in the same manner as in example 2, except that n-propanolamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine,40:1:0.5 (vol.)), yield 80%.1H NMR(300MHz,CDCl3)δ3.74(t,J＝5.7Hz,2H),3.54(td,J＝12.6,5.8Hz,1H),3.20(dd,J＝10.8,5.4Hz,1H),2.89(m,2H),2.77(m,1H),2.60(m,1H),1.15(s,3H),0.98(s,3H),0.97(s,3H),0.88(s,3H),0.86(s,3H),0.78(s,3H),0.72(d,J＝10.7Hz,1H)；ESI-MS 494.4[M+H]+。
EXAMPLE 9 preparation of Compound 9
Compound 9 was prepared in the same manner as in example 2, except that p-methoxyaniline was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 90%.1H NMR(CD3OD,300MHz)δ6.76(d,J＝9.0Hz,2H),6.68(d,J＝9.0Hz,2H),3.71(s,3H),3.54(td,J＝12.8,6.3Hz,1H),3.14(dd,J＝11.0,5.2Hz,1H),3.05(t,J＝6.4Hz,2H),2.04(m,1H),1.14(s,3H),0.99(s,3H),0.96(s,3H),0.91(s,6H),0.78(s,3H),0.75(d,J＝10.6Hz,1H)；ESI-MS 542.6[M+H]+。
EXAMPLE 10 preparation of Compound 10
Compound 10 was prepared in the same manner as in example 2, except that benzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 90%.1H NMR(300MHz,CDCl3)δ7.32(m,5H),3.74(s,2H),3.57(td,J＝12.9,6.0Hz,1H),3.20(dd,J＝10.8,5.2Hz,1H),2.97(d,J＝12.3Hz,1H),2.78(dd,J＝12.3,7.2Hz,1H),2.52(t,J＝11.0Hz,1H),2.12(m,1H),1.15(s,3H),0.99(s,3H),0.97(s,3H),0.90(s,3H),0.89(s,3H),0.78(s,3H),0.73(d,J＝10.7Hz,1H)；ESI-MS 526.8[M+H]+。
EXAMPLE 11 preparation of Compound 11
Compound 11 was prepared in the same manner as in example 2, except that (S) -1-phenylethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 86%.1H NMR(300MHz,CDCl3)δ7.29(m,5H),3.82(q,J＝6.7Hz,1H),3.56(td,J＝12.8,5.9Hz,1H),3.19(dd,J＝10.8,5.3Hz,1H),2.69(m,1H),2.40(m,1H),2.11(m,1H),1.40(d,J＝6.7Hz,3H),1.18(s,3H),0.97(s,3H),0.96(s,3H),0.89(s,3H),0.87(s,3H),0.77(s,3H),0.77(d,J＝10.8Hz,1H)；ESI-MS 540.9[M+H]+。
EXAMPLE 12 preparation of Compound 12
Compound 12 was prepared in the same manner as in example 2, except that p-methoxybenzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 91%.1H NMR(CDCl3,300MHz)δ7.21(dd,J＝8.6,4.2Hz,2H),6.87(dd,J＝8.6,4.2Hz,2H),3.80(s,3H),3.79(s,2H),3.69(s,1H),3.57(td,J＝12.8,6.1Hz,1H),3.20(dd,J＝10.8,5.3Hz,1H),2.96(d,J＝12.0Hz,1H),2.52(t,J＝11.0Hz,1H),2.11(m,1H),1.14(s,3H),0.99(s,3H),0.98(s,3H),0.90(s,3H),0.89(s,3H),0.78(s,3H),0.73(d,J＝10.9Hz,1H)；ESI-MS 556.6[M+H]+。
EXAMPLE 13 preparation of Compound 13
Compound 13 was prepared in the same manner as in example 2, except that p-chlorobenzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave 87% yield.1H NMR(300MHz,CDCl3)δ7.29(d,J＝6.1Hz,2H),7.20(d,J＝6.1Hz,2H),3.84(s,1H),3.71(s,2H),3.56(td,J＝12.9,5.9Hz,1H),3.19(dd,J＝10.9,5.3Hz,1H),2.94(m,1H),2.52(t,J＝10.1Hz,1H),2.10(m,1H),1.13(s,3H),0.98(s,3H),0.97(s,3H),0.89(s,3H),0.87(s,3H),0.77(s,3H),0.74(d,J＝10.7Hz,1H)；ESI-MS 560.7[M+H]+。
EXAMPLE 14 preparation of Compound 14
Compound 14 was prepared in the same manner as in example 2, except that p-fluorobenzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave 88% yield.1H NMR(300MHz,CDCl3)δ7.32(dd,J＝8.6,5.4Hz,2H),7.03(dd,J＝8.6,5.4Hz,2H),5.30(s,1H),3.84(s,2H),3.53(td,J＝12.9,6.0Hz,1H),3.20(dd,J＝11.0,5.2Hz,1H),2.98(m,1H),2.65(m,1H),2.09(m,1H),1.12(s,3H),0.98(s,6H),0.88(s,6H),0.78(s,3H),0.73(d,J＝10.8Hz,1H)；ESI-MS 544.6[M+H]+。
EXAMPLE 15 preparation of Compound 15
Compound 15 was prepared in the same manner as in example 2, except that 3, 4-dimethoxybenzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)), yield 92%.1H NMR(300MHz,CDCl3)δ6.93(s,1H),6.86(d,J＝8.4Hz,1H),6.84(d,J＝8.4Hz,1H),5.30(s,1H),3.89(s,3H),3.87(s,3H),3.82(s,2H),3.53(td,J＝12.8,6.0Hz,1H),3.20(dd,J＝10.8,5.3Hz,1H),2.96(m,1H),2.69(m,1H),2.08(m,1H),1.12(s,3H),0.98(s,6H),0.88(s,6H),0.78(s,3H),0.71(d,J＝10.8Hz,1H)；ESI-MS586.6[M+H]+。
EXAMPLE 16 preparation of Compound 16
Compound 16 was prepared in the same manner as in example 2, except that phenethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 90%.1H NMR(300MHz,CDCl3)δ7.23(m,5H),3.56(td,J＝13.1,6.1Hz,1H),3.20(dd,J＝10.9,4.9Hz,1H),2.83(m,5H),2.54(m,1H),2.07(m,1H),1.13(s,3H),0.98(s,3H),0.97(s,3H),0.89(s,3H),0.88(s,3H),0.77(s,3H),0.71(d,J＝10.9Hz,1H)；ESI-MS 540.8[M+H]+。
EXAMPLE 17 preparation of Compound 17
Compound 17 was prepared in the same manner as in example 2, except that p-chlorophenylethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave an 84% yield.1H NMR(300MHz,CDCl3)δ7.29(d,J＝6.2Hz,2H),7.17(d,J＝6.2Hz,2H),3.58(td,J＝12.7,5.9Hz,1H),3.19(dd,J＝10.8,5.2Hz,1H),2.73(m,4H),2.38(m,1H),2.11(m,1H),1.16(s,3H),0.98(s,3H),0.97(s,3H),0.88(s,3H),0.87(s,3H),0.77(s,3H),0.72(d,J＝10.9Hz,1H)；ESI-MS 574.7[M+H]+。
EXAMPLE 18 preparation of Compound 18
Compound 18 was prepared in the same manner as in example 2, except that p-nitroanisole was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)), yield 83%.1H NMR(300MHz,CDCl3)δ8.16(d,J＝8.8Hz,2H),7.40(d,J＝8.8Hz,2H),3.47(m,2H),3.18(m,2H),3.08(m,4H),2.80(m,1H),1.10(s,3H),1.00(s,3H),0.97(s,3H),0.88(s,6H),0.77(s,3H),0.72(d,J＝11.0Hz,1H)；ESI-MS 585.9[M+H]+。
EXAMPLE 19 preparation of Compound 19
Compound 19 was prepared in the same manner as in example 2, except that 3, 4-dimethoxyphenethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)), yield 93%.1H NMR(300MHz,CDCl3)δ6.80(d,J＝8.2Hz,1H),6.73(d,J＝8.2Hz,1H),6.71(s,1H),5.30(s,1H),3.86(s,3H),3.85(s,3H),3.56(td,J＝12.8,6.0Hz,1H),3.20(dd,J＝10.9,5.3Hz,1H),2.80(m,5H),2.52(t,J＝9Hz,1H),1.13(s,3H),0.98(s,3H),0.97(s,3H),0.90(s,3H),0.89(s,3H),0.77(s,3H),0.73(d,J＝11.0Hz,1H)；ESI-MS 600.7[M+H]+。
EXAMPLE 20 preparation of Compound 20
Compound 20 was prepared in the same manner as in example 2, except that homopiperony lamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)), yield 92%.1H NMR(300MHz,CDCl3)δ6.72(d,J＝7.8Hz,1H),6.65(d,J＝1.6Hz,1H),6.61(dd,J＝7.8,1.6Hz,1H),5.91(s,2H),3.56(td,J＝12.8,5.8Hz,1H),3.19(dd,J＝10.9,5.3Hz,1H),2.78(m,6H),2.47(m,1H),2.09(m,1H),1.13(s,3H),0.98(s,3H),0.97(s,3H),0.89(s,3H),0.87(s,3H),0.77(s,3H),0.73(d,J＝10.7Hz,1H)；ESI-MS 584.9[M+H]+。
EXAMPLE 21 preparation of Compound 21
Compound 21 was prepared in the same manner as in example 2, except that amphetamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 90%.1H NMR(300MHz,CDCl3)δ7.22(m,5H),3.53(td,J＝13.1,6.5Hz,1H),3.19(dd,J＝10.8,5.3Hz,1H),2.90(m,1H),2.66(m,6H),2.07(m,1H),1.10(s,3H),0.98(s,3H),0.97(s,3H),0.88(s,3H),0.87(s,3H),0.72(s,3H),0.72(d,J＝10.7Hz,1H)；ESI-MS 554.4[M+H]+。
EXAMPLE 22 preparation of Compound 22
Compound 22 was prepared in the same manner as in example 2, except that 2-aminomethylpyridine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 82%.1H NMR(300MHz,CDCl3)δ8.53(d,J＝4.9Hz,1H),7.64(t,J＝4.9Hz,1H),7.20(d,J＝4.9Hz,1H),7.16(t,J＝4.9Hz,1H),3.85(s,1H),3.83(s,1H),3.55(d,J＝12.8,5.9Hz,1H),3.19(dd,J＝11.0,5.1Hz,1H),2.92(m,1H),2.47(m,1H),2.06(m,1H),1.15(s,3H),0.98(s,3H),0.97(s,3H),0.88(s,6H),0.77(s,3H),0.73(d,J＝10.9Hz,1H)；ESI-MS 527.8[M+H]+。
EXAMPLE 23 preparation of Compound 23
Compound 23 was prepared in the same manner as in example 2, except that 3-aminomethylpyridine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)) gave a yield of 81%.1H NMR(300MHz,CD3OD)δ8.52(s,1H),8.43(d,J＝4.7Hz,1H),7.85(d,J＝7.4Hz,1H),7.41(dd,J＝7.4,4.7Hz,1H),3.79(s,2H),3.53(d,J＝13.1,5.7Hz,1H),3.14(dd,J＝10.8,5.2Hz,1H),2.63(m,2H),2.02(m,1H),1.14(s,3H),1.00(s,3H),0.96(s,3H),0.91(s,6H),0.77(s,3H),0.74(d,J＝10.7Hz,1H)；ESI-MS 527.8[M+H]+。
EXAMPLE 24 preparation of Compound 24
Compound 24 was prepared in the same manner as in example 2, except that 4-aminomethylpyridine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (volume ratio)), productThe ratio was 81%.1H NMR(300MHz,CDCl3)δ8.56(dd,J＝4.4,1.6Hz,2H),7.23(dd,J＝4.4,1.6Hz,2H),3.77(s,2H),3.58(td,J＝12.9,5.8Hz,1H),3.21(dd,J＝11.0,5.3Hz,1H),2.96(m,1H),2.56(m,1H),2.13(m,1H),1.17(s,3H),0.99(s,3H),0.98(s,3H),0.89(s,3H),0.87(s,3H),0.78(s,3H),0.78(d,J＝10.8Hz,1H)；ESI-MS 527.8[M+H]+。
EXAMPLE 25 preparation of Compound 25
Compound 25 was prepared in the same manner as in example 2, except that cyclopropylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)), yield 80%.1H NMR(300MHz,CDCl3)δ3.54(td,J＝12.6,6.2Hz,1H),3.20(dd,J＝10.7,5.3Hz,1H),3.05(m,1H),2.95(dd,J＝11.0,7.2Hz,1H),2.56(t,J＝11.0Hz,1H),2.11(m,1H),2.07(m,1H),1.11(s,3H),0.97(s,3H),0.96(s,3H),0.87(s,6H),0.76(s,3H),0.72(d,J＝10.9Hz,1H)；ESI-MS 476.6[M+H]+。
EXAMPLE 26 preparation of Compound 26
Compound 26 was prepared in the same manner as in example 2, except that 1- (3-aminopropyl) imidazole was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.%)) gave a yield of 90%.1H NMR(CDCl3,300MHz)δ7.49(s,1H),7.05(s,1H),6.92(s,1H),4.00(m,2H),3.55(td,J＝12.7,6.0Hz,1H),3.19(dd,J＝11.0,5.2Hz,1H),2.88(m,1H),2.68(m,1H),2.52(m,2H),1.13(s,3H),0.98(s,3H),0.97(s,3H),0.89(s,6H),0.78(s,3H),0.72(d,J＝10.9Hz,1H)；ESI-MS 544.7[M+H]+。
EXAMPLE 27 preparation of Compound 27
Compound 27 was prepared in the same manner as in example 2, except that N-aminoethylpiperazine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:1:0.5 (vol.)), yield 80%.1H NMR(CDCl3,300MHz)δ5.31(d,J＝1.4Hz,1H),3.57(td,J＝13.0,5.6Hz,1H),3.20(dd,J＝10.9,5.3Hz,1H),2.80(t,J＝6.0Hz,2H),2.47(m,12H),1.14(s,3H),0.98(s,6H),0.89(s,6H),0.78(s,3H),0.73(d,J＝10.9Hz,1H)；ESI-MS 548.3[M+H]+。
EXAMPLE 28 preparation of Compound 28
Compound 28 was prepared in the same manner as in example 2, except that 4-aminomorpholine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 50:2:0.5 (vol.)), yield 80%.1H NMR(CDCl3,300MHz)δ7.08(s,1H),5.30(s,1H),3.80(t,J＝4.4Hz,4H),3.69(m,1H),3.57(td,J＝13.1,5.7Hz,1H),3.19(dd,J＝10.9,5.3Hz,1H),2.94(d,J＝5.1Hz,4H),2.71(m,1H),2.48(m,2H),1.16(s,3H),0.97(s,6H),0.88(s,6H),0.77(s,3H)；ESI-MS 519.5[M+H]+。
EXAMPLE 29 preparation of Compound 29
In a 50ml round-bottom flask, Compound 1(700mg, 1.52mmol) and 20ml CH were added2Cl2Stirring for dissolving, and introducing O at-78 deg.C3Reacting for 4min, and adding NH after the reaction is completed2OH.HCl (213mg, 3.04mmol) and CH3ONa (300mg,5.5mmol) and stirred at room temperature for 3 h. After the reaction was complete, 20ml of water was added and 60ml of CH was added2Cl2Extracting for 3 times, drying with anhydrous sodium sulfate, and concentrating CH under reduced pressure2Cl2To give Compound 1-1(500 mg).1H NMR(CDCl3,300MHz)δ7.35(t,J＝6.8Hz,1H),3.43(td,J＝12.5,7.1Hz,1H),3.15(dd,J＝10.3,5.8Hz,1H),1.14(s,3H),1.05(s,3H),0.92(s,3H),0.90(s,3H),0.88(s,3H),0.74(s,3H),0.72(d,J＝10.9Hz,1H)；ESI-MS 450.3[M+H]+。
Dissolving compound 1-1500mg in 10ml methanol in 50ml round bottom flask, adding NaBH3CN(1.450g，0.023mol)，NH4OAc (1.345g, 0.017mol), under the protection of nitrogen, slowly dropping TiCl3The solution (2.4ml) was reacted for 6h, after completion of the reaction neutralized with 5N NaOH solution to pH 7, filtered, the residue was rinsed with methanol, the filtrate was concentrated under reduced pressure, extracted with a large amount of ethyl acetate, the ethyl acetate layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure, and then subjected to silica gel column chromatography to give compound 29(120mg, 30%) as a white solid.1H NMR(CDCl3,300MHz)δ3.46(td,J＝13.0,7.0Hz,1H),3.12(dd,J＝10.7,5.5Hz,1H),2.91(t,J＝7.0Hz,2H),1.13(s,3H),1.00(s,3H),0.94(s,3H),0.90(s,3H),0.89(s,3H),0.75(s,3H),0.72(d,J＝10.9Hz,1H)；ESI-MS 436.5[M+H]+。
EXAMPLE 30 preparation of Compound 30
Compound 29(70mg, 0.16mmol) is dissolved in 4ml of methanol solutionTo the mixture was added 0.4ml of a formaldehyde solution, NaBH (OAc)3(67.84mg, 0.32mmol) was stirred overnight, after completion of the reaction 15ml water was added followed by 30ml CH2Cl2Extracting for 2 times, collecting organic phase, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain CH2Cl2And silica gel column chromatography was performed to obtain compound 30 with a yield of 90%.1H NMR(CDCl3,300MHz)δ3.53(td,J＝13.0,6.0Hz,1H),3.19(dd,J＝11.0,5.2Hz,1H),2.71(m,2H),2.50(s,6H),1.11(s,3H),1.01(s,3H),0.97(s,3H),0.88(s,3H),0.77(s,3H),0.71(s,3H),0.70(d,J＝10.9Hz,1H)；ESI-MS 464.6[M+H]+。
EXAMPLE 31 preparation of Compound 31
Compound 31 was prepared in the same manner as in example 30, except that benzaldehyde was used instead of formaldehyde; silica gel column chromatography (dichloromethane/methanol/triethylamine, 70:1:0.5 (vol.)), yield 95%.1H NMR(CDCl3,300MHz)δ7.39(m,10H),4.06(d,J＝7.1Hz,2H),3.89(d,J＝7.1Hz,2H),3.55(td,J＝12.7,6.1Hz,1H),3.19(dd,J＝10.6,5.1Hz,1H),1.16(s,3H),1.00(s,3H),0.98(s,3H),0.89(s,6H),0.79(s,3H),0.71(d,J＝10.8Hz,1H)；ESI-MS 616.5[M+H]+。
EXAMPLE 32 preparation of Compound 32
Compound 32 was prepared in the same manner as in example 30, except that 3-methoxybenzaldehyde was used instead of formaldehyde; silica gel column chromatography (dichloromethane/methanol/triethylamine, 80:1:0.5 (vol.%)) gave a yield of 94%.1H NMR(CDCl3,300MHz)δ7.36(t,J＝8.0Hz,2H),7.26(s,2H),6.96(d,J＝8.0Hz,4H),5.30(s,1H),4.12(d,J＝7.1Hz,2H),3.93(dd,J＝7.1Hz,2H),3.84(s,6H),3.51(d,J＝12.9Hz,1H),3.20(dd,J＝10.8,5.3Hz,1H),1.18(s,3H),1.06(s,3H),0.89(s,3H),0.88(s,6H),0.78(s,3H),0.72(d,J＝10.9Hz,1H)；ESI-MS 676.6[M+H]+。
EXAMPLE 33 preparation of Compound 33
Compound 33 was prepared in the same manner as in example 30, except that p-fluorobenzaldehyde was used instead of formaldehyde; silica gel column chromatography (dichloromethane/methanol/triethylamine, 75:1:0.5 (vol.)), yield 93%.1H NMR(CDCl3,300MHz)δ7.26(t,J＝6.8Hz,4H),7.05(t,J＝6.8Hz,4H),5.30(s,1H),4.77(s,1H),3.66(d,J＝7.2Hz,2H),3.60(d,J＝7.2Hz,2H),3.51(m,2H),3.20(dd,J＝10.9,5.1Hz,1H),2.09(m,1H),1.15(s,3H),0.98(s,6H),0.89(s,6H),0.78(s,3H),0.71(d,J＝10.9Hz,1H)；ESI-MS652.3[M+H]+。
EXAMPLE 34 preparation of Compound 34
Compound 29(60mg, 0.138mmol) was charged to a 25mL round bottom flask and 3ml CH was added under nitrogen2Cl2And 0.1ml triethylamine, finally 0.2ml propionyl chloride is added, stirred for 5min, and quenched by adding water after the reaction is completed. With 10ml CH2Cl2Extracting for 2 times, collecting organic phase, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain CH2Cl2Silica gel column chromatography (dichloromethane/methanol, 70:1 (vol.%)) gave product 34 in 90% yield.1H NMR(300MHz,CDCl3)δ5.96(br s,1H),3.60(td,J＝13.2,6.5Hz,1H),3.37(dd,J＝10.9,5.0Hz,1H),3.20(m,2H),2.21(q,J＝7.6Hz,2H),2.05(m,1H),1.15(s,3H),1.14(t,J＝7.6Hz,3H),0.98(s,6H),0.88(s,6H),0.78(s,3H),0.72(d,J＝10.6Hz,1H)；ESI-MS 492.3[M+H]+。
EXAMPLE 35 preparation of Compound 35
Compound 35 was prepared in the same manner as in example 34, except that propionyl chloride was used instead of propionyl chloride; silica gel column chromatography (dichloromethane/methanol, 80:1 (vol.%)) gave 88% yield.1H NMR(300MHz,CDCl3)δ6.18(s,1H),5.30(s,1H),3.58(td,J＝12.8,6.1Hz,1H),3.39(dd,J＝10.9,5.0Hz,1H),3.19(m,2H),2.05(m,1H),1.15(s,3H),0.98(s,6H),0.88(s,6H),0.78(s,3H),0.71(m,4H)；ESI-MS504.3[M+H]+。
EXAMPLE 36 preparation of Compound 36
Compound 36 was prepared in the same manner as in example 34, except that benzoyl chloride was used instead of propionyl chloride; silica gel column chromatography (dichloromethane/methanol, 90:1 (vol.%)) gave 87% yield.1H NMR(300MHz,CDCl3)δ7.80(m,2H),7.43(m,3H),6.92(s,1H),5.30(s,1H),3.60(m,2H),3.36(td,J＝12.8,6.0Hz,1H),3.20(dd,J＝11.0,5.1Hz,1H),2.09(m,1H),1.17(s,3H),0.98(s,3H),0.95(s,3H),0.88(s,3H),0.86(s,3H),0.78(s,3H),0.72(d,J＝10.8Hz,1H)；ESI-MS 540.6[M+H]+。
EXAMPLE 37 preparation of Compound 37
Compound 37 was prepared in the same manner as in example 34, except that methoxybenzoyl chloride was used instead of propionyl chloride; silica gel column chromatography (dichloromethane/methanol, 100:1 (vol.%)) gave 89% yield.1H NMR(300MHz,CDCl3)δ7.77(d,J＝8.7Hz,2H),6.91(d,J＝8.7Hz,2H),6.78(br s,1H),5.30(s,1H),3.84(s,3H),3.62(m,2H),3.33(td,J＝12.6,5.9Hz,1H),3.20(dd,J＝10.6,5.3Hz,1H),2.09(m,1H),1.17(s,3H),0.98(s,3H),0.95(s,3H),0.88(s,3H),0.87(s,3H),0.78(s,3H),0.71(d,J＝10.9Hz,1H)；ESI-MS 570.7[M+H]+。
EXAMPLE 38 preparation of Compound 38
Compound 38 was prepared in the same manner as in example 34, except that benzyl chloroformate was used instead of propionyl chloride; silica gel column chromatography (dichloromethane/methanol, 90:1 (vol.%)) gave a yield of 79%.1H NMR(300MHz,CDCl3)δ7.35(m,5H),5.12(s,2H),3.60(td,J＝13.1,6.2Hz,1H),3.22(m,3H),2.06(m,1H),1.15(s,3H),0.99(s,6H),0.89(s,6H),0.79(s,3H),0.73(d,J＝10.8Hz,1H)；ESI-MS 570.4[M+H]+。
EXAMPLES 39 AND 40 preparation of Compounds 39 and 40
Compound 1(200mg, 0.420mmol) was dissolved in dichloromethane (10ml) in a 50ml round bottom flask, mCPBA (1.5eq) was added and stirred at room temperature for 2 hours, after TLC detection of completion of the reaction, 15ml of water was added, dichloromethane was extracted 2 times, dichloromethane was concentrated under reduced pressure, and white solids 39 and 40 were obtained by silica gel column chromatography.
Compound 39:1H NMR(400MHz,CDCl3)δ5.74(s,1H),3.87(dd,J＝10.8,5.4Hz,1H,H-24),3.52(td,J＝10.3,4.7Hz,1H,H-12),3.19(dt,J＝10.6,5.1Hz,1H,H-3),2.25(td,J＝10.4,4.4Hz,1H,H-17),1.27(s,3H),1.23(s,3H),1.10(s,3H),1.00(s,3H),0.97(s,3H),0.91(s,3H),0.88(s,3H),0.77(s,3H),0.73(dd,J＝10.9,2.6Hz,1H)；ESI-MS 477.4[M+H]+.
compound 40:1H NMR(400MHz,CDCl3)δ3.84(dd,J＝8.9,6.6Hz,1H,H-24),3.50(td,J＝10.5,4.5Hz,1H,H-12),3.18(dd,J＝11.3,4.9Hz,1H,H-3),2.18(td,J＝10.3,9.7,3.6Hz,1H,H-17),1.27(s,3H),1.26(s,3H),1.09(s,3H),0.97(s,3H),0.94(s,3H),0.89(s,3H),0.84(s,3H),0.76(s,3H),0.72(dd,J＝11.2,2.7Hz,1H)；ESI-MS 477.4[M+H]+.
EXAMPLES 41 AND 42 preparation of Compounds 41 and 42
Heizhou Gynostemma total saponin (purchased from Nature bioengineering Co., Ltd., Fusong county) (content: 90%) 10g, and dichloromethane-methanol (15:1-1:1) were subjected to silica gel column chromatography gradient elution to obtain 3 components Fr.1-3. Fr.2(2.3g) gave compound 41(562mg) by silica gel column chromatography and sephadex LH-20 gel, and Fr.3(3.1g) gave compound 42(758mg) by silica gel column chromatography.
Compound 41:13C NMR(400MHz,pyridine-d5)δ15.6,17.1,17.2,17.4,17.5,18.2,22.0,23.0,25.6,26.3,28.0,30.5,30.8,34.8,35.9,37.6,39.8,40.7,47.6,49.2,50.0,51.2,51.4,56.0,62.1,62.6,66.5,62.7,69.9,71.0,71.3,71.6,74.8,76.4,77.9,78.0,78.1,78.2,78.3,79.1,82.0,83.0,95.3,98.0,104.2,105.3,125.7,130.7；ESI-MS985.6[M+Na]+。
compound 42:13C NMR(400MHz,pyridine-d5)δ15.8,17.2,17.4,17.7,18.2,18.9,21.9,22.9,25.6,26.4,28.0,30.4,30.7,34.8,35.9,37.6,39.8,40.7,47.6,49.2,50.0,51.1,51.4,55.9,62.1,62.6,66.5,66.7,69.7,69.8,70.8,71.0,71.1,71.6,74.5,76.4,76.6,77.7,77.8,78.0,78.1,78.2,78.3,79.1,81.8,82.3,95.3,97.8,104.2,105.2,105.5,125.7,130.8；ESI-MS 1117.7[M+Na]+。
EXAMPLE 43 preparation of Compound 43
Compound 43 was prepared in the same manner as in example 1, except that the american ginseng total saponins were replaced with the huizhou gynostemma total saponins; silica gel column chromatography (dichloromethane/methanol, 25:1 (vol.%)) gave a yield of 10%.1H NMR(400MHz,CDCl3)δ5.18(t,J＝7.1Hz,1H),3.68(m,2H),3.02(d,J＝9.4Hz,1H),1.72(s,3H),1.66(s,3H),1.21(s,3H),1.05(s,3H),1.01(s,3H),0.98(s,3H),0.92(s,3H),0.85(s,3H)；ESI-MS 477.4[M+H]+.
EXAMPLE 44 preparation of Compound 44
In a 50ml three-necked reaction flask, compound 43(200mg, 0.420mmol) was dissolved in 15ml CH2Cl2Stirring at-78 deg.C for 10min, and introducing O3The reaction was carried out for 2 min. Then the temperature was raised to-3 ℃ and methylamine (0.1ml), NaBH (OAc) were added3(368.8mg,1.7mmol) and CH3OH (8ml) was stirred overnight, after completion of the reaction 15ml water was added and CH was used2Cl2(30ml) extracted 2 times, dried over anhydrous sodium sulfate and concentrated under reduced pressure CH2Cl2Silica gel column chromatography (dichloromethane/methanol/triethylamine, 20:1:0.5) afforded product 44 in 70% yield.1H NMR(400MHz,CD3OD)δ3.61(m,1H),2.80(m,2H),2.57(m,1H),2.04(m,1H),1.889(m,3H),1.15(s,3H),1.01(s,3H),1.00(s,3H),0.97(s,3H),0.93(s,3H),0.80(s,3H)；ESI-MS 466.7[M+H]+。
EXAMPLE 45 preparation of Compound 45
Compound 45 was prepared in the same manner as in example 44, except that ethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 20:1:0.5 (vol.)), yield 69%.1H NMR(400MHz,MeOD)δ3.63(m,1H),2.93(d,J＝9.6Hz,1H),2.84(m,2H),2.06(m,2H),1.91(m,2H),1.77(s,3H),1.04(m,3H),1.03(s,3H),1.00(s,3H),0.95(s,3H),0.83(s,3H)；ESI-MS 480.3[M+H]+。
EXAMPLE 46 preparation of Compound 46
Compound 46 was prepared in the same manner as in example 44, except that ethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 20:1:0.5 (vol.)), yield 71%.1H NMR(400MHz,CD3OD)δ3.61(m,1H),3.18(m,2H),2.94(m,3H),2.05(m,1H),1.29(t,J＝7.2Hz,3H),1.01(s,6H),1.00(s,3H),0.97(s,3H),0.93(s,3H),0.80(s,3H)；ESI-MS 493.6[M+H]+。
EXAMPLE 47 preparation of Compound 47
Compound 47 was prepared in the same manner as in example 44, except that isopropylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 20:1:0.5 (vol.%)) gave a yield of 68%.1H NMR(400MHz,CDCl3)δ5.32(s,1H),3.69(m,1H),3.59(td,J＝10.3,5.1Hz,1H),2.99(d,J＝9.6Hz,1H),2.95(m,1H),2.82(m,1H),2.47(td,J＝11.5,2.5Hz,1H),2.11(m,2H),1.16(s,3H),1.12(d,J＝6.3Hz,3H),1.10(d,J＝6.3Hz,3H),1.03(s,3H),0.99(s,3H),0.97(s,3H),0.91(s,3H),0.83(s,3H)；ESI-MS 494.5[M+H]+。
EXAMPLE 48 preparation of Compound 48
Compound 48 was prepared in the same manner as in example 44, except that tert-butylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 20:1:0.5 (vol.%)) gave a yield of 70%.1H NMR(400MHz,CD3OD)δ3.58(m,3H),2.90(d,J＝9.6Hz,1H),2.04(m,2H),1.35(s,9H),1.14(s,3H),1.01(m,3H),1.00(m,3H),0.97(s,3H),0.92(s,3H),0.80(s,3H)；ESI-MS 508.4[M+H]+。
EXAMPLE 49 preparation of Compound 49
Same as example 44 except that benzylamine was used instead of methylamineManner of preparing compound 49; silica gel column chromatography (dichloromethane/methanol/triethylamine, 25:1:0.5 (vol.%)) gave a yield of 68%.1H NMR(400MHz,CDCl3)δ7.31(m,5H),3.72(m,4H),2.96(t,J＝9.9Hz,2H),2.55(t,J＝11.1Hz,1H),2.11(s,2H),1.14(s,3H),1.04(s,3H),0.98(s,3H),0.95(s,3H),0.90(s,3H),0.81(s,3H)；ESI-MS 542.6[M+H]+。
EXAMPLE 50 preparation of Compound 50
Compound 50 was prepared in the same manner as in example 44, except that p-methoxybenzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 25:1:0.5 (vol.%)) gave a yield of 67%.1H NMR(400MHz,CDCl3)δ7.23(d,J＝6.3Hz,2H),6.89(d,J＝6.3Hz,2H),3.842(s,3H),3.70(m,2H),3.61(td,J＝10.3,5.2Hz,1H),2.98(m,2H),2.53(t,J＝11.3Hz,1H),2.14(dt,J＝12.8,4.6Hz,2H),1.16(s,3H),1.03(s,3H),1.00(s,3H),0.97(s,3H),0.92(s,3H),0.83(s,3H)；ESI-MS 572.4[M+H]+。
EXAMPLE 51 preparation of Compound 51
Compound 51 was prepared in the same manner as in example 44, except that p-fluorobenzylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 25:1:0.5 (vol.%)) gave a yield of 70%.1H NMR(400MHz,CDCl3)δ7.26(m,2H),7.08(m,2H),3.71(m,3H),3.61(td,J＝10.4,5.0Hz,1H),2.98(m,2H),2.68(m,1H),2.54(m,1H),2.13(m,2H),1.89(m,2H),1.16(s,3H),1.02(s,3H),1.00(s,3H),0.97(s,3H),0.92(m,3H),0.83(s,3H)；ESI-MS560.4[M+H]+。
EXAMPLE 52 preparation of Compound 52
Compound 52 was prepared in the same manner as in example 44, except that phenethylamine was used instead of methylamine; silica gel column chromatography (dichloromethane/methanol/triethylamine, 25:1:0.5 (vol.)), yield 71%.1H NMR(400MHz,MeOD)δ7.29(m,5H),3.62(m,1H),2.90(m,3H),2.74(m,2H),2.06(m,2H),1.91(m,2H),1.16(s,3H),1.03(s,6H),1.00(s,3H),0.95(s,3H),0.83(s,3H)；ESI-MS 556.3[M+H]+.
EXAMPLE 53 preparation of Compound 53
Compound 43(200mg, 0.420mmol) was dissolved in dichloromethane (10ml) in a 50ml round bottom flask as in equation 6, Pd/C (32mg) was added, and the solution was taken up in H2Stirring overnight at room temperature, detecting by TLC, filtering, concentrating under reduced pressure dichloromethane, and performing silica gel column Chromatography (CH)2Cl2MeOH,60:1 (vol.)) gave compound 53 as a white solid in 85% yield.1HNMR(300MHz,CDCl3)δ3.66(m,2H),2.99(d,J＝9.4Hz,1H),1.15(s,3H),1.02(s,3H),0.98(s,3H),0.94(s,3H),0.89(d,J＝3.6Hz,6H),0.86(s,3H),0.81(s,3H)；ESI-MS 479.4[M+H]+。
Examples 54 and 55 preparation of Compounds 54 and 55
Dissolving compound 43(200mg, 0.420mmol) in dichloromethane (10ml) in a 50ml round bottom flask, adding mCPBA (1.5eq), stirring at room temperature for 2 hours, detecting completion of the reaction by TLC, adding 15ml water, extracting with dichloromethane 2 times, concentrating dichloromethane under reduced pressure, and performing silica gel column Chromatography (CH)2Cl2MeOH,30:1 (vol.)) gave compound 54 and compound 55 as white solids in 43% and 40% yields, respectively.
Compound 54:1H NMR(300MHz,CDCl3)δ3.88(m,1H),3.61(m,1H),3.52(m,1H),2.98(d,J＝9.6Hz,1H),2.70(s,1H),1.26(s,3H),1.21(s,3H),1.10(s,3H),1.01(s,3H),0.99(s,3H),0.95(s,3H),0.91(s,3H),0.81(s,3H)；13C NMR(75MHz,CDCl3)δ87.8,87.3,83.6,70.7,70.6,69.1,56.2,52.4,50.4,49.1,48.9,47.6,40.0,39.4,38.8,34.9,32.4,31.9,31.8,29.8,29.1,28.7,27.9,25.5,24.1,18.4,18.0,17.8,16.7,15.7；ESI-MS 493.4[M+H]+。
compound 55:1H NMR(300MHz,CDCl3)δ3.83(t,J＝8.4Hz,1H),3.71(m,1H),3.68(m,1H),3.50(s,1H),2.96(d,J＝4.2Hz,1H),1.28(s,3H),1.26(s,3H),1.09(s,3H),1.01(s,3H),0.97(s,3H),0.92(s,3H),0.90(s,3H),0.81(s,3H)；13C NMR(75MHz,CDCl3)δ86.6,85.3,83.4,71.1,70.5,68.8,56.2,52.2,50.5,49.3,48.0,47.6,39.9,39.4,38.6,34.9,32.6,31.6,31.2,28.7,28.1,27.5,26.1,25.1,18.4,18.3,17.7,16.7,15.6；ESI-MS 493.4[M+H]+。
EXAMPLE 56 preparation of Compound 56
In a 50ml three-necked reaction flask, compound 43(200mg, 0.420mmol) was dissolved in methylene chloride (15ml), and after stirring at-78 ℃ for 10min, ozone was introduced to the mixture to react for 3-5 min. Then heating to 0 ℃, stirring1h, after TLC detection reaction is completed, 15ml of water is added, and CH is used2Cl2(30ml) extracted 2 times, dried over anhydrous sodium sulfate, concentrated dichloromethane under reduced pressure, and chromatographed on silica gel Column (CH)2Cl2MeOH,20:1 (vol.)) gave compound 56 in 75% yield.1H NMR(300MHz,CDCl3)δ5.54(d,J＝6.3Hz,1H),5.43(s,1H),5.29(s,1H),5.02(s,1H),3.66(m,1H),3.47(m,1H),3.45(s,3H),2.98(dd,J＝9.6,3.0Hz,1H),1.01(s,6H),0.96(s,3H),0.93(s,3H),0.90(s,3H),0.82(s,3H)；ESI-MS 465.3[M+H]+。
EXAMPLE 57 preparation of Compound 57
30g of total gypenosides (content: 90%) in Heizhou were added to 600mL of 10% aqueous hydrochloric acid, and the mixture was refluxed under stirring at 70 ℃ for 6 hours. The reaction solution was cooled to room temperature, extracted 3 times with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate, and 6.7g of the crude product was distilled under reduced pressure through filtration. The crude product (6.7g) was chromatographed on a silica gel column with a gradient elution using dichloromethane-methanol (160:1-40:1) to give three fractions Fr.1-3. Fr.2(1.6g) was eluted with dichloromethane-methanol 90:1 to give a fraction, which was recrystallized to give compound 57(102 mg).1H NMR(400MHz,pyridine-d5)δ6.35(1H,s),3.70(1H,m),3.55(1H,td,J＝10.4,5.2Hz),3.01(1H,dd,J＝9.6,3.6Hz),1.28(3H,s),1.24(3H,s),1.20(3H,s),1.03(3H,s),0.99(3H,s),0.97(3H,s),0.90(3H,s),0.84(3H,s)；13C NMR(400MHz,CDCl3)δ88.4,73.3,69.9,68.9,59.9,54.6,51.3,49.8,49.0,47.3,39.8,39.1,38.5,36.4,35.7,34.7,33.0,31.2,30.6,28.6,27.1,25.2,19.4,18.2,17.4,17.1,16.6,16.3,15.7；ESI-MS 477.6[M+H]+。
EXAMPLES 58-61 preparation of Compound 58-
40g of total saponins of gynostemma pentaphylla (40% of total saponins) from Shanxi province, purchased from Nature bioengineering Co., Ltd, Fusong county, and the content of the total saponins is subjected to silica gel column chromatography gradient elution to obtain 3 components Fr.1-3. Fr.2(2.7g) was subjected to multiple reverse phase C-18 column (methanol-water) to give compounds 58(72mg), 59(35mg) and 60(43 mg); fr.3(2.2g) was subjected to reverse phase C-18 column (methanol-water) multiple times to give compound 61(53 mg).
Compound 58:13C NMR(100MHz,pyridine-d5)δ17.0,17.8,17.9,18.7,19.1,19.7,19.9,23.1,24.6,26.0,27.1,28.2,28.3,29.1,32.8,36.9,37.8,38.2,39.9,41.0,42.0,43.0,47.4,51.7,52.3,57.9,64.1,68.5,71.0,71.1,71.9,73.0,73.7,73.8,73.9,75.1,76.1,76.7,77.6,77.7,78.2,79.3,79.5,79.8,79.9,89.5,90.2,103.0,106.2,106.3,107.5,127.3,132.1；ESI-MS 1085.6[M+Na]+。
compound 59:13C NMR(100MHz,pyridine-d5)δ15.6,16.5,16.6,16.8,17.7,18.4,18.5,21.6,23.3,24.6,25.8,26.7,26.8,27.6,31.4,36.0,36.5,36.8,39.5,39.7,40.6,41.7,46.1,50.3,50.9,56.5,62.4,62.7,62.8,69.7,69.8,71.4,71.5,72.3,72.4,73.7,75.0,75.4,76.1,76.1,76.8,77.6,78.0,78.1,78.2,78.3,88.6,89.3,101.1,103.9,104.8,106.0,126.0,130.7；ESI-MS 1093.2[M+H]+。
compound 60:13C NMR(100MHz,pyridine-d5)δ17.3,17.7,18.4,18.9,19.0,19.8,23.5,24.6,25.9,27.1,27.7,28.9,29.0,33.2,34.8,35.9,37.9,41.3,41.7,42.8,47.2,51.6,54.0,54.1,56.1,64.0,66.4,68.3,69.7,71.3,72.2,73.0,73.7,73.8,75.1,75.7,75.8,76.7,77.5,77.6,79.0,79.8,79.9,83.0,88.4,103.3,106.0,106.5,107.4,127.2,132.2,206.8；ESI-MS 1069.7[M+Na]+。
compound 61:13C NMR(100MHz,pyridine-d5)δ15.8,15.9,16.4,17.0,17.5,17.6,18.3,22.2,23.0,24.5,25.6,26.2,27.3,27.7,31.9,33.4,34.4,36.4,40.2,41.4,45.9,50.1,52.6,52.7,54.6,64.9,66.4,66.8,68.2,69.8,70.7,72.2,72.3,73.7,74.2,74.4,76.3,77.5,81.5,86.9,101.8,104.6,105.0,125.6,130.6,205.4；ESI-MS 907.5[M+Na]+。
EXAMPLES 62 AND 63 preparation of Compounds 62 and 63
Compounds 62 and 63 were prepared in the same manner as in example 2, except that total saponins of gynostemma pentaphyllum from shanxi was used instead of total saponins of panax quinquefolium; silica gel column chromatography (dichloromethane/methanol, 25:1 (vol.%)) gave 1% and 0.5% yields, respectively.
Compound 62:1H NMR(400MHz,CDCl3)δ5.14(t,J＝6.4Hz,1H),3.53(d,J＝3.2Hz,2H),3.22(dd,J＝11.2,5.0Hz,1H),1.70(s,3H),1.65(s,3H),0.99(s,3H),0.98(s,3H),0.90(s,3H),0.87(s,3H),0.79(s,3H),0.75(dd,J＝11.4,2.0Hz,1H)；13C NMR(100MHz,CDCl3)δ132.2,124.7,79.2,77.2,67.2,56.1,50.8,50.3,45.9,41.6,40.6,39.2,39.1,37.3,35.4,35.0,31.4,28.2,27.6,27.5,25.9,24.4,22.7,21.7,18.5,17.9,16.5,16.4,15.7,15.6；ESI-MS 483.2[M+Na]+。
compound 63:1H NMR(400MHz,CDCl3)δ10.15(s,1H),5.10(t,J＝6.1Hz,1H),3.56(d,J＝3.2Hz,2H),3.12(dd,J＝11.4,5.0Hz,1H),1.69(s,3H),1.63(s,3H),0.98(s,3H),0.97(s,3H),0.91(s,3H),0.79(s,3H),0.72(dd,J＝11.4,2.0Hz,1H)；13C NMR(100MHz,CDCl3)δ206.1,132.3,124.6,78.2,77.0,67.2,54.4,53.1,50.3,45.7,41.3,40.3,39.5,34.9,34.3,33.4,32.4,31.7,28.3,27.4,26.5,26.2,25.4,24.7,22.7,21.9,17.9,17.7,17.4,16.7,14.6；ESI-MS 497.4[M+Na]+。
EXAMPLE 64 preparation of Compound 64
Compound 64 was prepared in the same manner as in example 4, except that compound 62 was used instead of compound 1; silica gel column chromatography (dichloromethane/methanol/triethylamine, 30:1:0.5 (vol.)), yield 70%.1H NMR(400MHz,MeOD)δ3.43(s,1H),3.30(s,1H),3.09(dd,J＝11.3,4.9Hz,1H),2.88(m,2H),1.85(s,1H),0.97(t,J＝6.3Hz,3H),0.96(s,3H),0.91(s,3H),0.86(s,3H),0.84(s,3H),0.72(s,3H)；ESI-MS478.6[M+H]+。
EXAMPLE 65 preparation of Compound 65
Compound 65 was prepared in the same manner as in example 7, except that compound 62 was used instead of compound 1; silica gel column chromatography (dichloromethane/methanol/triethylamine, 30:1:0.5 (vol.)), yield 71%.1H NMR(400MHz,CDCl3):δ3.41(s,2H),3.19(dd,J＝11.2,4.9Hz,1H),2.65(m,1H),2.51(m,1H),1.86(m,2H),1.11(s,9H),0.96(s,3H),0.95(s,3H),0.86(s,3H),0.83(s,3H),0.76(s,3H),0.72(m,1H)；ESI-MS492.7[M+H]+。
EXAMPLES 66-69 preparation of Compounds 66-69
40g of total saponins (content 40%) of gynostemma pentaphylla in Shaanxi, and gradient elution of dichloromethane-methanol (15:1-1:1) through silica gel column chromatography to obtain 3 components Fr.1-3. Fr.1(841mg) was subjected to reverse phase C-18 column (methanol-water) to give compounds 66(36mg), 67(53mg), 68(46mg) and 69(62 mg).
Compound 66:13C NMR(100MHz,pyridine-d5)δ15.4,16.1,16.4,16.7,17.9,18.2,18.4,21.4,25.3,25.6,26.7,27.1,27.7,31.5,35.5,36.7,39.5,39.6,40.4,40.5,43.0,45.5,50.4,50.9,56.4,62.4,67.0,69.6,69.7,70.3,72.2,72.3,73.7,74.0,74.5,76.6,77.8,78.0,78.8,88.0,88.7,101.5,104.7,104.8,125.3,138.2,179.3；ESI-MS 935.6[M+Na]+。
compound 67:13C NMR(100MHz,pyridine-d5)δ15.4,16.1,16.4,16.7,18.0,18.2,18.4,21.5,25.4,26.0,26.7,27.6,27.8,31.5,35.5,36.8,38.8,39.5,39.6,40.4,44.7,45.1,50.0,50.9,56.4,62.4,67.0,69.6,69.7,70.4,72.2,72.3,73.7,74.6,75.0,76.7,77.8,78.0,80.9,88.0,88.7,101.6,104.7,104.8,123.7,139.2,178.1；ESI-MS 935.6[M+Na]+。
compound 68:13C NMR(100MHz,pyridine-d5)δ15.4,16.1,16.2,16.5,17.7,18.1,18.3,20.5,21.5,25.3,25.4,26.5,27.1,27.4,31.4,35.3,36.9,39.4,39.5,40.2,40.4,43.0,45.6,50.3,50.9,56.4,63.6,66.7,69.5,69.6,70.1,71.7,72.2,73.4,74.0,74.1,74.4,76.3,77.8,79.8,87.4,89.0,101.4,104.5,104.6,125.0,138.2,170.4,179.0；ESI-MS 957.2[M+H]+。
compound 69:13C NMR(100MHz,pyridine-d5)δ15.4,16.0,16.4,16.6,18.0,18.2,18.4,20.6,21.6,25.4,26.0,26.6,27.6,27.8,31.5,35.5,36.9,38.8,39.5,39.6,40.5,44.7,45.1,50.0,50.9,56.5,63.8,67.0,69.6,69.7,70.3,72.1,72.2,73.6,74.1,74.5,75.0,76.4,78.0,80.9,87.5,89.2,101.5,104.7,104.8,123.7,139.3,170.5,178.1；ESI-MS 979.7[M+Na]+。
examples 70 and 71 preparation of Compounds 70 and 71
Compounds 70 and 71 were prepared in the same manner as in example 1, except that total saponins of gynostemma pentaphyllum from shanxi was used instead of total saponins of panax quinquefolium; silica gel column chromatography (dichloromethane/methanol, 30:1 (vol.%)) gave 2% and 3% yields, respectively.
Compound 70:1H NMR(400MHz,CDCl3)δ5.30(d,J＝8.8Hz,1H),5.15(q,J＝8Hz,1H),3.22(q,J＝5.2Hz,1H),1.80(s,3H),1.78(s,3H),1.00(s,3H),0.99(s,3H),0.92(s,3H),0.87(s,3H),0.80(s,3H)；13C NMR(100MHz,CDCl3)δ179.0,140.0,123.2,78.9,78.3,74.2,55.9,50.7,50.5,45.5,42.3,40.5,40.5,39.0,39.0,37.1,35.2,31.4,28.0,27.3,26.4,25.8,25.1,21.3,18.4,18.2,16.4,16.3,15.4,15.4；ESI-MS 473.6[M+H]+。
compound 71:1H NMR(400MHz,CDCl3)δ5.33-5.41(m,1H),5.22(d,J＝8.8Hz,1H),3.23(q,J＝5.2Hz,1H),1.82(s,3H),1.79(s,3H),1.00(s,3H),0.98(s,3H),0.93(s,3H),0.87(s,3H),0.80(s,3H)；13C NMR(100MHz,CDCl3)δ177.7,140.4,122.5,81.2,78.9,75.3,55.8,50.7,49.9,45.3,44.4,40.4,39.1,38.9,37.8,35.3,31.4,28.0,27.3,27.3,25.8,25.6,21.4,18.5,18.2,16.2,16.1,15.4,15.4；ESI-MS(M/Z):473.5([M+H]+)。
EXAMPLE 72 preparation of Compound 72
In the same manner as in example 1, silica gel column chromatography (petroleum ether: ethyl acetate: 1:2 (vol.%)) was performed to give compound 72(1g, 1%).1H NMR(400MHz,CDCl3)δ5.14(t,J＝7.1Hz,1H),3.99(td,J＝9.7,5.5Hz,1H),3.58(td,J＝10.4,5.0Hz,1H),2.72(ddd,J＝15.3,12.0,6.5Hz,1H),2.32–2.22(m,1H),1.68(s,3H),1.62(s,3H),1.33(s,3H),1.31(s,3H),1.20(s,3H),1.03(s,3H),0.92(s,3H),0.78(s,3H)；ESI-MS(M/Z):477.4([M+H]+)。
EXAMPLES 73 AND 74 preparation of Compounds 73 and 74
Compound 72(200mg, 0.420mmol) was dissolved in dichloromethane (10ml) in a 50ml round bottom flask, mCPBA (1.5eq) was added and stirred at room temperature for 2 hours, after TLC detection of completion of the reaction, 15ml of water was added, dichloromethane was extracted 2 times, dichloromethane was concentrated under reduced pressure, and white solids 73 and 74 were obtained by silica gel column chromatography.
Compound 73:1H NMR(400MHz,CDCl3)δ5.77(s,1H),4.17–4.05(m,1H,H-6),3.86(dd,J＝10.8,5.2Hz,1H,H-24),3.51(td,J＝10.3,4.8Hz,1H,H-6),3.17(brd,J＝10.6Hz,1H,H-3),2.24(td,J＝10.5,4.5Hz,1H,H-17),1.30(s,3H),1.26(s,3H),1.22(s,3H),1.09(s,3H),1.08(s,3H),0.97(s,3H),0.92(s,6H)；ESI-MS(M/Z):493.4([M+H]+)。
compound 74:1H NMR(400MHz,CDCl3)δ4.10(td,J＝10.6,3.8Hz,1H,H-6),3.84(dd,J＝8.7,6.6Hz,1H,H-24),3.50(td,J＝10.5,4.5Hz,1H,H-12),3.16(dd,J＝11.5,4.8Hz,1H,H-3),2.18(ddd,J＝10.9,9.2,3.7Hz,1H,H-17),1.30(s,3H),1.263(s,3H),1.257(s,3H),1.08(s,3H),1.05(s,3H),0.96(s,3H),0.92(s,3H),0.88(s,3H)；ESI-MS(M/Z):493.4([M+H]+)。
EXAMPLE 75 preparation of Compound 10. HCl
Dissolving the compound 10(1mmol) in 30mL of 1% diluted hydrochloric acid, stirring at room temperature for 2h, filtering after the reaction is finished to obtain a crude product, and recrystallizing the crude product with ethanol to obtain a white solid compound 10 & HCl with the yield of 80%.
All other hydrochloride salts of the compounds can be prepared by reacting the corresponding compound with dilute hydrochloric acid as described in example 75.
The organic acid salts or inorganic acid salts of the compounds mentioned in the present invention can be prepared by reacting the compounds with the corresponding organic acids or inorganic acids in a similar manner to example 75.
Test examples
Test example Activity test on AMPK α 2 β 1. gamma.1 molecular model
1. Material
Human AMPK α 2 β 1 gamma 1 protein is obtained by expression and purification of an escherichia coli expression system (please describe the method for obtaining the human AMPK α 2 β 1 gamma 1 protein)
HTRF KinEASE-STK S1kit purchased from Cisbio was used to detect activation of AMPK (α 2 β 1. gamma.1) by small compounds at the molecular level.
2. Process for producing a metal oxide
The HTRF KinEASE-STK S1kit has the following principle: the substrate peptide fragment contains a site which can be phosphorylated by AMPK, after 45 minutes of phosphorylation reaction in a reaction system (containing 2mM MgCl2, 400. mu.M DTT, 160nM substrate peptide, 4. mu.M ATP and 1.6nM AMPK), the generated phosphorylated substrate (i.e., product) is combined with an antibody (labeled Donor) which recognizes phosphorylation of the site, biotin on the other end is connected with streptavidin-XL665 (receptor), and FRET signals generated are detected by using Evison of PE company. The final AMPK activity is expressed as the ratio of the reading at 665nm wavelength to the reading at 615nm wavelength (665nm/615 nm).
3. Sample processing
Samples to be tested were dissolved in DMSO and stored cryogenically. Usually, the concentration of DMSO in the final system is controlled within a range that allows the compound to dissolve without precipitation and without affecting the kinase activity, and is optimized to 2%.
4. Data processing
Single concentration conditions (e.g., 20. mu.g/ml) were selected for preliminary testing of sample activity. For compounds that exhibit some activity in a preliminary activity assay, such as a Fold activation (Fold) greater than 2, it is desirable to further test the dose-dependent relationship of their activity, i.e., EC, by a multiple concentration activation assay50The value is obtained. EC was obtained by non-linear fitting of AMPK activity to compound concentration50Values, the computational software used was Graphpad Prism 4, the fitting model used was sigmoidal-response (variable slope), and the bottom of the fitting curve for most agonist screening models was set at 100. In general, each compound requires multiple wells (n.gtoreq.2) for activity testing, and the results are expressed as Standard Deviation (SD) or Standard Error (SE). Generally, each test has a reported compound as a reference.
5. Results of the experiment
The results are shown in Table 1, where the positive control compounds A-769662 and AMP were able to dose-dependently activate AMPK α 2 β 1. gamma.1 phosphorylation, with a fold activation of A-769662 of 6.7 and EC50The value was 21.7nM, the fold activation of AMP was 5.1, EC50The Heizhou gynostemma pentaphylla standard extract and the Shanxi gynostemma pentaphylla standard extract with the value of 320 nM. can activate the phosphorylation and EC of AMPK α 2 β 1 gamma 1 in a dose-dependent manner50Values of 1.32. + -. 0.25. mu.g/ml and 1.62. + -. 0.62. mu.g/ml, respectively, most of the tested compounds showed better activity in activating phosphorylation of AMPK α 2 β 1. gamma.1, with the most active compounds being 6, 7, 10, 11, 15, 20, 24 and 49.
TABLE 1 evaluation of AMPK direct agonistic Activity of dammarane-type triterpene derivatives (AMPK α 2 β 1. gamma.1)

Claims (6)

1. Use of a compound having the formula:
wherein R is1Is OH; r2Is OH; r3Is CH3；R4Is H; r5Is OH; r6Is composed of
2. Use of a compound according to claim 1 for the preparation of a medicament for the prevention or treatment of diseases associated with AMPK.
3. Use of a pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 in the manufacture of a medicament for the prevention or treatment of a disease associated with AMPK.
4. The use of claim 3, wherein the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.
5. The use according to any one of claims 2-4, wherein the disease is selected from one or more of diabetes, chronic complications of diabetes, hyperlipidemia, obesity, hyperinsulinemia, insulin resistance, fatty liver.
6. The use according to claim 5, wherein the diabetic chronic complication is diabetic heart disease, type II diabetes complicated with cerebral infarction, type II diabetes complicated with alcoholic fatty liver, diabetic nephropathy, diabetic retinopathy and/or diabetic peripheral neuropathy.
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