CN108341850B

CN108341850B - Saponin derivative, and pharmaceutical composition and application thereof

Info

Publication number: CN108341850B
Application number: CN201710055888.2A
Authority: CN
Inventors: 陈重; 段华庆; 童晓航; 杨世林
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2021-06-01
Anticipated expiration: 2037-01-25
Also published as: CN108341850A

Abstract

The invention discloses a saponin derivative, a pharmaceutical composition and application thereof. The structure of the saponin derivative is shown as a formula (I), wherein X is O, C ═ N-OH and CH-OH, Y is C or CH, or X-Y is CH ═ CH; z is CH₂Or C ═ O; or R₂And Y is connected with the ester through a covalent bond to form the ester. The saponin derivative has high antitumor activity, but its hemolytic toxicity is greatly reduced.

Description

Saponin derivative, and pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of pharmacology, and particularly relates to a saponin derivative, a pharmaceutical composition and application thereof.

Background

Tumors have been receiving wide attention as a major threat to human health, and the search for new antitumor drugs is a key point and a focus of research of medicinal chemists all over the world. The search for new antitumor drugs from natural products (naturalpructs) is one of the major approaches to the discovery of antitumor drugs. Triterpene saponin is a natural product formed by connecting triterpene sapogenin with sugar, uronic acid and the like, and most of glycoside bonds form ether glycoside with C-3 position or hydroxyl at other positions and ester glycoside with 28-position carboxyl. Modern research finds that at least 150 natural triterpenoid saponin components derived from more than 100 plants have potential anti-tumor activity.

Among the numerous triterpene saponins having potential antitumor activity, α -hederin is a typical representative thereof.Alpha-hederagenin is a pentacyclic triterpenoid saponin present in a variety of plants, with a unique disaccharide chain attached to the 3-oxygen atom, namely: 3-O-alpha-L-rhamnopyranosyl- (1 → 2) -alpha-L-arabinopyranose. The literature reports that alpha-hederagenin induces apoptosis of P388 cells at higher than 3 mu M and generates time and dose dependence. Kumara S.S. reports that administration of PSA at a dose of 10mg/kg/d was administered intraperitoneally for 7 consecutive days using LLC-transplanted BDF1 mice as a model, with a tumor suppression rate of 65% on day 8 and 71% on day 15 (planta.Med.,2001,67: 29-32). However, alpha-hederagenin saponin is reported in the literature to have strong hemolytic properties, a concentration that causes 50% red blood cell lysis (HD)₅₀) Is 10. mu.M (bioorg.Med.chem.2009,17, 2002-2008). The results of our earlier studies are consistent with the above literature reports. Alpha-hederagenin, although showing definite anti-tumor activity in tumor animal models, its strong hemolytic property prevents its clinical application. Gauthier c, written in 2009, states that "toxicity of saponin compounds induced by hemolysis in most animals, including humans, is the major stumbling stone that has prevented them as clinical antitumor agents" (bioorg.med.chem.2009,17, 2002-. Therefore, the problem of hemolytic property of the saponin compound is particularly urgent.

The mechanism by which saponins cause hemolysis is not clear up to now. It is thought that saponin contacts with the cell membrane of erythrocyte, and then binds with cholesterol to form an insoluble complex, forming a cavity in the cell membrane, increasing the permeability of the cell membrane, and causing the outflow of hemoglobin in erythrocyte to cause hemolysis. Winter suggests that saponin interacts with aquaporins, increasing water transport in erythrocytes, inducing hemolysis. However, not all saponins have hemolytic effect, such as ginsenosides using protopanaxatriol and oleanolic acid as aglycones, but ginsenosides using protopanaxadiol as aglycones have anti-hemolytic effect, so that the total saponins of ginseng do not show hemolytic effect. The hemolytic activity of saponin is related to sugar chain part, the hemolytic effect of monosaccharidosin is obvious, some biglycan saponin has no hemolytic effect, but has hemolytic effect after partial hydrolysis into monosaccharidosin.

Studies of the haemolytic and cytotoxic activity of 19 hederacosides by chwalekm show that: 1) sugar chains have important effects on cytotoxicity and hemolysis, including configuration of terminal carbon, position of glycosyl substitution, and sugar type; 2) after 28-COOH is formed into methyl ester, the hemolytic property is enhanced, the cytotoxicity is reduced, and the fat solubility of an aglycone part is increased probably due to a methyl esterification product, so that the combination mode of saponin and a cell membrane is changed; 3)28-COOH has a major effect on cytotoxicity; 4) the configuration of the terminal carbon is alpha-L or beta-D type (natural configuration), which is beneficial to improving the hemolytic property and cytotoxicity of the saponin; 5) alpha-L-Rhap- (1 → 2) -alpha-L-Arap is an excellent fragment for cytotoxicity of oleanolic acid saponin or hederagenin saponin (Biochi. Biophy. acta.2006,1760, 1418-1427).

In conclusion, the hemolysis of alpha-hederagenin saponin is caused by the participation of a plurality of factors such as aglycone and sugar chain, and the surface activity of the saponin is determined by the special molecular structure of the saponin; in other words, there is no necessary link between the hemolysis of saponins and the surface activity of saponins. Thus solving the hemolytic problem of saponins is not an insurmountable gap. The alpha-hederagenin saponin is used as a precursor, a plurality of saponin derivatives are designed and synthesized, in-vitro anti-tumor activity research is carried out, the hemolysis of the derivatives is focused, and the patent aims to improve or maintain the anti-tumor activity of the alpha-hederagenin saponin and eliminate or greatly reduce the hemolysis toxicity.

Disclosure of Invention

The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a saponin derivative having an antitumor activity and being capable of eliminating or greatly reducing hemolysis.

In order to achieve the purpose, the invention adopts the technical scheme that:

according to a first aspect of the present invention, there is provided a saponin derivative which is a compound having the general structural formula (I) or a pharmaceutically acceptable salt thereof,

in the formula (I), the compound is shown in the specification,

x is C-O, C-N-OH, CH-OH, Y is C or CH; or X-Y is CH ═ CH;

z is CH₂Or C ═ O;

R₁is hydroxyl, glucosyl, rhamnosyl, arabinosyl, xylosyl, ribosyl, mannosyl, galactosyl or fructosyl;

R₂is COR₃，R₃Is hydroxyl, alkoxy, aromatic oxy, heteroaromatic oxy, oligosaccharide group, aliphatic amine group, cyclic aliphatic amine group, aromatic amine group, heteroaromatic amine group, amino acid, amino oligosaccharide or NO donor; or R₂And Y is connected with the ester through a covalent bond to form the ester.

Further, in formula (I), X is C ═ O or C ═ N-OH, Y is CH, and Z is CH₂，R₂Is COR₃，R₃Is hydroxyl, alkoxy, aromatic oxy, heteroaromatic oxy, oligosaccharide group, aliphatic amine group, cyclic aliphatic amine group, aromatic amine group, heteroaromatic amine group, amino acid, amino oligosaccharide or NO donor.

Specifically, R₂Is COOH.

Further, X-Y is CH ═ CH, Z is C ═ O, R₂Is COR₃，R₃Is hydroxyl, alkoxy, aromatic oxy, heteroaromatic oxy, oligosaccharide group, aliphatic amine group, cyclic aliphatic amine group, aromatic amine group, heteroaromatic amine group, amino acid, amino oligosaccharide or NO donor.

Further, X-Y is CH ═ CH, and Z is CH₂，R₂Is COR₃，R₃Is an amino acid or NO donor.

Further, the NO donor is an alkoxynitrate.

Specifically, the carbon number of the alkoxynitrate is 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12.

Further, the amino acid is-NH (CH)₂)_nCOOH or-NH (CH)₂)_mCONH(CH₂)_pCOOH, wherein n is an integer between 1 and 8, and m and p are independently integers between 1 and 4.

Further, X is C-O, CH-OH or C-N-OH, Y is C, R₂And Y is connected with the ester through a covalent bond to form the ester.

Specifically, R₂is-COO-.

Further, the saponin derivative is one or more of the following compounds:

according to the invention, the compound of the general structural formula (I) includes not only a single compound form, but also a mixture form of a plurality of compounds with structures meeting the requirements of the general formula (I), and different isomer forms of the same compound, such as racemes, enantiomers, diastereoisomers and the like. The pharmaceutically acceptable salts include, but are not limited to, hydrochloride, phosphate, sulfate, acetate, maleate, methanesulfonate, benzenesulfonate, benzoate, methylbenzenesulfonate, succinate, fumarate, tartrate, gallate, citrate, and the like.

According to a second aspect of the present invention, there is provided an intermediate for a saponin derivative as described above, which intermediate is:

according to a third aspect of the present invention, there is provided a pharmaceutical composition for anti-tumor comprising the saponin derivative of the present invention.

Further, saponin derivatives are used as active ingredients in the pharmaceutical composition.

The active ingredient of the above pharmaceutical composition may comprise only the above saponin derivative, in which case the saponin derivative is preferably contained in the pharmaceutical composition in an effective amount; or the active ingredient can also be the combination of the saponin derivative and other anti-tumor active ingredients.

According to a fourth aspect of the present invention, there is provided the use of a saponin derivative according to the above in the preparation of an anti-tumor pharmaceutical composition.

Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:

compared with alpha-hederagenin saponin, the saponin derivative has stronger anti-tumor activity and greatly reduced hemolytic toxicity.

Detailed Description

The compound of the general structural formula (I) can be prepared by utilizing conventional chemical reactions in the field, and a synthetic route can be designed correspondingly according to the specific target compound structure. Examples are as follows:

scheme one, the following synthetic route is shown:

preparing a compound 3 or 4 by using the compound 1 or 2 in a polar solvent and acetic anhydride as an acylating agent, treating the compound with potassium dichromate to obtain a compound 5 or 6, and hydrolyzing the compound to obtain a compound 7 or 8;

taking the compound 3 or 4 as a raw material, obtaining a compound 9 or 10 under the action of m-chloroperoxybenzoic acid, and hydrolyzing to obtain a compound 11 or 12;

taking a compound 3 or 4 as a raw material, reacting with benzyl bromide to prepare a compound 15 or 16, catalyzing with a metal catalyst in a hydrogen environment to prepare a compound 17 or 18, and hydrolyzing to obtain a compound 19 or 20;

taking a compound 17 or 18 as a raw material, reacting with dichlorodicyanobenzoquinone in a nonpolar solvent to obtain a compound 21 or 22, and hydrolyzing to obtain a compound 23 or 24;

the compound 15 is used as a raw material and reacts with hydroxylamine hydrochloride to obtain a compound 25, the compound 26 is prepared by catalysis of a metal catalyst in a hydrogen environment, and the compound 27 is obtained by hydrolysis.

Scheme two, the following synthetic route is shown:

taking a compound 5, 6 or 17 as a raw material, reacting with oxalyl chloride to prepare a compound 28, 29 or 32, directly reacting with glycine ethyl ester hydrochloride to obtain an amide intermediate, and hydrolyzing to obtain a compound 30, 31 or 33.

Scheme three, the following synthetic route shows:

taking a compound 3 as a raw material, reacting with oxalyl chloride to prepare a compound 34, directly reacting with amino acid ethyl ester hydrochloride or glycylglycine ethyl ester hydrochloride to obtain an amide intermediate, and hydrolyzing to obtain a compound 35-38;

and (4) a fourth process: the following synthetic route is shown:

the compound 1 is used as a raw material and reacts with brominated alkanes in a polar solvent and a weak base environment to obtain a compound 39-41, and the treated compound directly reacts with silver nitrate to obtain a compound 42-44.

The invention will be further illustrated by the following specific examples, which are not intended to limit the scope of the invention.

In the examples of the present invention, melting pointMeasuring with an X-4 melting point instrument;¹H-NMR of¹³C-NMR was recorded using a Varian Mercury 400 NMR spectrometer with chemical shifts expressed in delta (ppm); when the silica gel for separation is not particularly described, the silica gel is 200-300 mesh, and the ratio of the eluent is volume ratio.

Example 1: preparation of 11-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranose Oleanolic alkane-28-carboxylic acid (Compound 7)

Step 1-1: preparation of Compound 3

The starting material, alpha-hederagenin (compound 1) (5.00g,6.66mmol), was dissolved in anhydrous pyridine (20mL), acetic anhydride (6.0mL,62.7mmol) was added with stirring, and stirred at room temperature for 12 h. After the reaction is finished, adding 100mL of ethyl acetate, and adjusting the pH value to 4-5 by using 10% HCl; the organic layer was washed with saturated brine (50mL × 3), dried over anhydrous sodium sulfate, filtered, and the solvent was concentrated under reduced pressure, and silica gel column chromatography (petroleum ether: ethyl acetate ═ 2:1) was performed to obtain 6.02g of a white solid, i.e., compound 3, in 90.0% yield.

The melting point (mp) of the resulting white solid was 169-170 ℃. The nuclear magnetic data of the obtained white solid are:

¹H NMR(400MHz,CDCl₃,δ):5.21(s,1H,H-1”),4.91–5.03(m,2H,H-1’,H-12),4.42(d,J＝6.4Hz,1H),4.04–4.20(m,3H),3.90(ddd,J＝23.9,14.0,5.0Hz,3H),3.55(dd,J＝16.7,8.7Hz,2H),2.80(d,J＝13.5Hz,1H),2.13(s,3H,CH₃CO),2.10(s,3H,CH₃CO),2.09(s,3H,CH₃CO),2.05(s,3H,CH₃CO),2.03(s,3H,CH₃CO),1.96(s,3H,CH₃CO),1.25(t,J＝7.1Hz,3H),1.20(d,J＝6.2Hz,3H),1.09(s,3H),0.93(s,3H),0.91(s,3H),0.89(s,3H),0.78(s,3H),0.73(s,3H)；

¹³C NMR(100MHz,CDCl₃,δ):183.48(C-28),170.57(CH₃CO),170.53(CH₃CO),170.45(CH₃CO),170.30(CH₃CO),170.19(CH₃CO),169.80(CH₃CO),143.73(C-13),122.59(C-12),103.68(C-1’),98.37(C-1”),82.10(C-3),77.36,74.55,71.98,71.10,69.65,68.71,67.94,67.28,65.18,62.80,60.55,47.94,46.64,45.89,42.03,41.66,41.16,39.37,38.44,36.67,33.89,33.19,32.53,32.47,30.80,27.64,25.88,25.62,23.69,23.51,22.95,21.19,21.17(CH₃CO),21.12(CH₃CO),21.07(CH₃CO),20.95(CH₃CO),20.92(CH₃CO),20.79(CH₃CO),18.08,17.47,17.06,15.95,14.32,12.74。

step 1-2: preparation of Compound 5

Dissolving the compound 3(600mg,0.6mmol) in glacial acetic acid (20mL), adding potassium dichromate (352mg,1.2mmol), adding a condensing device, refluxing in an oil bath kettle at 100 ℃, detecting the reaction progress by thin-layer silica gel chromatography, and stopping the reaction after most of raw materials disappear. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solid was removed by filtration, a large amount of glacial acetic acid was removed under a rotary evaporator, diluted with ethyl acetate (50mL), washed with 5% sodium hydroxide solution (30mL × 2) to remove excess glacial acetic acid, the organic layer was washed with saturated brine (30mL × 3), dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate ═ 2:1) to give 503mg of a yellow solid, i.e., compound 5, in 83% yield.

The melting point (mp) of the resulting solid was 189-190 ℃. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CDCl₃,δ):5.26(s,1H,H-1”),5.22(d,J＝3.2Hz,1H,H-12),5.16–5.20(m,1H,H-1’),5.02(dt,J＝14.4,7.1Hz,3H),4.94(dd,J＝9.0,3.4Hz,1H),4.38(d,J＝6.5Hz,1H),4.04–4.15(m,4H),3.88–3.97(m,3H),3.84(dd,J＝8.9,6.6Hz,2H),3.48–3.60(m,2H),2.11(s,3H),2.08(s,3H),2.08(s,3H),2.02(s,3H),2.00(s,3H),1.94(s,3H),1.19(d,J＝6.2Hz,3H),1.12(s,3H),1.08(s,3H),1.02(s,3H),0.94(s,3H),0.90(s,3H),0.79(s,3H)；

¹³C NMR(75MHz,CDCl₃,δ):200.24(C-11),182.43(C-28),170.53(CH₃CO),170.53(CH₃CO),170.48(CH₃CO),170.28(CH₃CO),170.17(CH₃CO),169.79(CH₃CO),168.52(C-12),128.05(C-13),103.69(C-1’),98.28(C-1”),81.77(C-3),77.36,74.39,72.07,71.11,69.65,68.66,67.94,67.25,65.20,62.92,61.96,60.55,47.83,46.04,45.09,44.22,43.50,42.36,41.51,39.07,36.91,33.74,32.96,32.70,31.64,30.78,27.71,25.64,23.49,22.75,21.19(CH₃CO),21.12(CH₃CO),21.07(CH₃CO),20.93(CH₃CO),20.79(CH₃CO),19.08(CH₃CO),17.46,17.18,16.78,14.32,12.74；

HRMS-ESI(m/z)[M-H]-:calcd.for C₅₃H₇₅O₁₉1015.4903,found 1015.5057。

step 1-3: preparation of Compound 7

Compound 5(503mg,0.495mmol) was dissolved in anhydrous methanol/THF/H₂To a solution of O (2:1:1,20mL) was added potassium hydroxide (332mg,5.93mmol), and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the organic solvent was removed under reduced pressure, the pH was adjusted to 4 to 5 with 10% HCl, n-butanol (50mL) was diluted, the n-butanol layer was washed with saturated brine (30mL × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol: 10:1) was performed to obtain 317mg of a yellow solid, that is, compound 7, in 83.6% yield.

The melting point (mp) of the resulting solid was 212-213 ℃. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,pyridine-d₅,δ):6.28(s,1H,H-1”),5.97(s,1H,H-12),5.10(d,J＝6.3Hz,1H,H-1’),4.75(d,J＝1.8Hz,1H),4.62–4.73(m,2H),4.53–4.61(m,1H),4.30(ddd,J＝16.8,7.9,4.2Hz,2H),4.14–4.25(m,2H),4.10(dd,J＝7.9,3.5Hz,1H),3.76(d,J＝10.7Hz,1H),3.68(d,J＝10.3Hz,1H),3.29(t,J＝13.4Hz,1H),2.63(s,1H),1.63(d,J＝6.2Hz,3H),1.36(s,3H),1.35(s,3H),1.19(s,3H),1.10(s,3H),0.91(s,3H),0.91(s,3H)；

¹³C NMR(100MHz,pyridine-d₅,δ):200.44(C-11),180.05(C-28),170.05(C-12),128.53(C-13),104.87(C-1’),102.03(C-1”),81.36(C-3),76.19,75.25,74.48,72.91,72.71,70.04,69.80,66.19,64.27,62.71,50.03,48.00,46.49,45.71,44.84,44.35,44.29,42.68,40.23,37.77,34.33,33.24,32.59,31.17,30.32,28.73,26.70,23.93,23.77,19.80,18.90,17.58,17.50,14.35；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₄₁H₆₄O₁₃Na 787.4245,found 787.4239。

example 2: preparation of 11-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) [ beta-D-glucopyranose- (1→4)]-alpha-L-arabinopyranoside oleanane-28-oic acid (Compound 8)

Step 2-1: preparation of Compound 4

Starting Compound 2(6.08g,6.66mmol) was dissolved in anhydrous pyridine (20mL), acetic anhydride (8.6mL,89.91mmol) was added with stirring, and the mixture was stirred at room temperature for 12 h. After the reaction is finished, adding 100mL of ethyl acetate, and adjusting the pH value to 4-5 by using 10% HCl; the organic layer was washed with saturated brine (50mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate ═ 2:1) to obtain 7.40g of a white solid, i.e., compound 4, in 86% yield.

The melting point (mp) of the resulting solid was 172-. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CDCl₃,δ):5.26(d,J＝7.1Hz,1H,H-1”),5.12–5.22(m,1H,H-12),5.05(s,1H,H-1’),4.96(d,J＝8.5Hz,1H,H-1”’),4.91(s,2H),4.53(dd,J＝16.1,7.5Hz,1H),4.25(d,J＝13.7Hz,2H),4.13(dd,J＝19.6,10.0Hz,3H),4.02(s,4H),3.68–3.84(m,4H),3.58(s,1H),3.43(d,J＝27.1Hz,1H),2.80(dd,J＝16.5,8.8Hz,1H),2.14(s,3H,CH₃CO),2.08(s,3H,CH₃CO),2.08(s,3H,CH₃CO),2.04(s,9H,CH₃CO),2.01(s,3H,CH₃CO),1.99(s,3H,CH₃CO),1.97(s,3H,CH₃CO),1.25(s,3H),1.18(d,J＝5.3Hz,3H),1.10(s,3H),0.93(s,3H),0.92(s,3H),0.89(s,3H),0.74(s,3H)；

¹³C NMR(100MHz,CDCl₃,δ):183.46(COOH),170.79(CH₃CO),170.68(CH₃CO),170.55(CH₃CO),170.30(CH₃CO),170.22(CH₃CO),170.10(CH₃CO),170.04(CH₃CO),169.57(CH₃CO),169.53(CH₃CO),143.67(C-13),122.63(C-12),101.53(C-1”’),101.31(C-1’),98.24(C-1”),82.03(C-3),77.36,74.72,72.71,71.88,71.06,70.96,69.57,68.99,68.44,67.29,65.39,61.92,60.53,47.96,47.90,46.63,45.97,45.94,42.03,41.79,41.68,41.13,39.38,38.30,36.68,33.18,32.49,30.78,29.81,25.88,23.68,21.17,21.11(CH₃CO),21.06(CH₃CO),21.01(CH₃CO),20.96(CH₃CO),20.93(CH₃CO),20.90(CH₃CO),20.87(CH₃CO),20.82,20.72(CH₃CO),20.69(CH₃CO),17.42,17.04,15.93,14.32,12.74.

step 2-2 preparation of Compound 6

Dissolving compound 4(775mg,0.6mmol) in glacial acetic acid (20mL), adding potassium dichromate (352mg,12mmol), adding a condensing device, refluxing in an oil bath kettle at 100 ℃, detecting the reaction progress by thin-layer silica gel chromatography, and stopping the reaction after most of the raw materials disappear. After the reaction was completed, it was cooled to room temperature, and the solid was removed by filtration, a large amount of glacial acetic acid was removed under a rotary evaporator, diluted with ethyl acetate (50mL), washed with 5% sodium hydroxide solution (30mL × 2) to remove excess glacial acetic acid, the organic layer was washed with saturated brine (30mL × 3), dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate ═ 2:1) to give 634mg of a yellow solid, i.e., compound 6, in 81% yield.

The melting point (mp) of the resulting solid was 168-169 ℃. The nuclear magnetic data of the obtained solid is:

¹H NMR(600MHz,CDCl₃,δ):5.58(s,1H,H-1”),5.18(d,J＝13.2Hz,1H,H-12),5.15–5.09(m,1H,H-1’),4.99(d,J＝10.6Hz,2H),4.89–4.95(m,1H),4.87(s,1H),4.50(d,J＝7.4Hz,1H),4.46(s,1H),4.20(d,J＝9.2Hz,1H),4.11(d,J＝11.6Hz,1H),4.06(d,J＝6.7Hz,1H),3.98(s,2H),3.91(s,1H),3.80(d,J＝11.2Hz,1H),3.71(s,1H),3.67(d,J＝6.8Hz,1H),3.52(d,J＝5.0Hz,1H),3.37(d,J＝7.0Hz,1H),2.91(d,J＝11.2Hz,1H),2.75(d,J＝12.0Hz,1H),2.09(s,3H),2.04(s,6H),2.01(s,3H),1.99(s,3H),1.98(s,3H),1.96(s,3H),1.94(s,3H),1.92(s,3H),1.28(s,3H),1.14(s,3H),1.09(s,3H),0.88(s,3H),0.86(s,3H),0.72(s,3H)；

¹³C NMR(150MHz,CDCl₃,δ):200.07(C-11),182.31(COOH),170.68(CH₃CO),170.57(CH₃CO),17040(CH₃CO)，17020(CH₃CO)，17008(CH₃CO)，16996(CH₃CO)，16993(CH₃CO)，16942(CH₃CO)169.38(CH₃CO),168.41(C-12),127.93(C-13),101.40(C-1”’),101.17(C-1’),98.17(C-1”),81.62,74.67,72.78,72.62,71.77,70.99,70.84,69.48,68.87,68.31,67.17,65.26,61.84,61.74,47.67,45.94,44.97,44.16,43.41,42.25,41.40,38.80,36.83,33.66,32.85,32.62,31.56,30.67,29.68,27.64,25.02,23.41,23.38,22.67,20.97,20.89,20.83,20.78,20.76,20.70,20.60,20.57,19.08,17.32,17.12,16.67,14.21,12.62；

HRMS-ESI(m/z)[M+H]⁺:calcd.for C₆₅H₉₃O₂₇1305.5904,found 1305.5871。

step 2-3: preparation of Compound 8

Compound 6(646mg,0.495mmol) was dissolved in anhydrous methanol/THF/H₂To a solution of O (2:1:1,20mL) was added potassium hydroxide (332mg,5.93mmol), and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the organic solvent was removed under reduced pressure, pH was adjusted to 4 to 5 with 10% HCl, n-butanol (50mL) was diluted, the n-butanol layer was washed with saturated brine (30mL × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol ═ 10:1) was performed to obtain 389mg of a yellow solid, i.e., compound 8, in 81% yield.

The melting point (mp) of the resulting solid was 223-. The nuclear magnetic data of the obtained solid is:

¹H NMR(600MHz,pyridine-d₅,δ):6.26(s,1H,H-1”),5.97(s,1H,H-12),5.11(d,J＝7.9Hz,1H,H-1’),4.96(d,J＝6.7Hz,1H,H-1”’),4.71(s,1H),4.63(d,J＝8.5Hz,1H),4.03(d,J＝7.9Hz,1H),3.88(s,1H),3.74(d,J＝10.4Hz,1H),3.28(dd,J＝33.9,12.8Hz,1H),2.62(s,1H,H-10),1.64(d,J＝5.5Hz,3H,H-6”),1.37(s,3H),1.35(s,3H),1.27(s,3H),1.18(s,3H),1.12(s,3H),0.91(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):200.42(C-11),180.00(COOH),170.03(C-12),128.52(C-13),107.14(C-1”’),104.84(C-1’),102.03(C-1”),81.39(C-3),80.83,79.99,79.16,78.89,76.63,75.81,75.46,74.47,72.80,72.59,71.53,69.98,67.53,65.80,64.21,62.80,62.70,50.02,48.05,46.48,45.71,44.85,44.35,44.27,42.67,40.23,37.77,34.33,34.15,33.35,33.24,32.59,32.46,32.24,31.17,29.95,28.73,26.77,26.01,23.95,23.77,23.28,21.53,19.80,18.98,17.57,17.51；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₄₇H₇₄O₁₈Na 949.4773,found 949.4767。

example 3: preparation of 12, 23-dihydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) [3, 4-O-diacetyl]-α- L-arabinopyranose-oleanane-28-gamma-lactone (Compound 11)

Step 3-1: preparation of Compound 9

Compound 3(482mg, 0.481mmol) was dissolved in anhydrous chloroform (15ml), and m-chloroperoxybenzoic acid (250mg, 1.25mmol) was added in the absence of light, followed by stirring at room temperature in the absence of light for 24 hours. After the reaction was terminated, chloroform was removed in vacuo to obtain a white solid, ethyl acetate (50ml) was added, and the mixture was washed with 10% sodium hydroxide solution (30mlx2), saturated brine (30mlx3), and the organic layer was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (petroleum ether: ethyl acetate: 3:1) to obtain 376mg of a white solid, that is, compound 9, in about 78% yield.

The melting point of the resulting solid was 167-. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CDCl₃,δ):5.24(d,J＝3.3Hz,1H,H-1”),5.18–5.24(m,1H,H-12),5.03–5.07(m,1H,H-1’),4.96(dd,J＝9.0,3.4Hz,1H),4.41(d,J＝6.5Hz,1H),4.12(dd,J＝20.5,9.4Hz,2H),3.90(t,J＝17.8Hz,3H),3.57(d,J＝11.5Hz,1H),2.13(s,3H),2.10(s,3H),2.09(s,3H),2.05(s,3H),2.02(s,3H),1.96(s,3H),1.28(s,3H),1.21(d,J＝6.2Hz,3H),1.13(s,3H),0.97(s,3H),0.91(s,3H),0.89(s,3H),0.79(s,3H)；

¹³C NMR(100MHz,CDCl₃,δ):179.98(C-28),170.51(CH₃CO),170.46(CH₃CO),170.44(CH₃CO),170.29(CH₃CO),170.16(CH₃CO),169.79(CH₃CO),103.78(C-1’),98.22(C-1”),90.59(C-13),81.89(C-3),77.36,76.46,74.26,72.12,71.13,69.69,68.68,68.03,67.24,65.15,63.02,60.55,51.19,47.97,44.89,44.81,42.40,42.15,42.10,39.52,38.79,36.20,34.23,33.76,33.40,31.70,28.97,28.08,27.57,25.71,24.03,21.27,21.20,21.13,21.08,20.95,20.92,20.81,18.74,18.69,17.56,17.46,16.92,14.34,12.60；

HRMS-ESI(m/z)[M-H]-:calcd.for C₅₃H₇₇O₁₉1017.5059,found 1017.5172。

step 3-2: preparation of Compound 11

Compound 9(376mg,0.369mmol) was dissolved in anhydrous methanol/THF/H₂O (2:1:1,20mL) solution was added with potassium hydroxide (248mg,4.42mmol), and stirred at room temperature for 12 h. After the reaction was completed, the organic solvent was removed under reduced pressure, pH was adjusted to 4 to 5 with 10% HCl, n-butanol (50mL) was diluted, the n-butanol layer was washed with saturated brine (30mL × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol: 10:1) was performed to obtain 256mg of a white solid, i.e., compound 11, in 91% yield.

The melting point (mp) of the resulting solid was 206-. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,pyridine-d₅,δ):6.23(s,1H,H-1”),5.06(d,J＝6.1Hz,1H,H-12),4.79(s,1H,H-1’),4.67(dd,J＝9.0,6.2Hz,2H),4.48–4.59(m,1H),4.34(t,J＝9.4Hz,1H),4.16(ddd,J＝24.3,17.0,7.5Hz,6H),3.71(dd,J＝17.7,11.1Hz,2H),1.61(d,J＝5.9Hz,3H),1.54(s,3H),1.32(s,3H),1.04(s,3H),0.92(s,3H),0.86(s,3H),0.79(s,3H)；

¹³C NMR(100MHz,pyridine-d₅,δ):180.05(C-28),104.72(C-1’),101.95(C-1”),91.80(C-13),81.31(C-3),76.04,76.01,75.14,74.40,72.84,72.67,69.98,69.75,66.10,64.19,51.97,49.98,48.06,45.36,45.26,43.93,42.98,42.95,39.76,39.43,36.67,34.78,34.42,33.54,31.92,29.67,28.68,28.57,26.58,24.10,22.02,19.36,19.03,18.86,17.99,17.51,14.02；

HRMS-ESI(m/z)[M⁺Na]⁺:calcd.for C₄₁H₆₆O₁₃Na 789.4401,found 789.4260。

example 4: preparation of 12-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) [ beta-D-glucopyranose- (1→4)]-alpha-L-arabinopyranoside oleanane-28-gamma-lactone (Compound 12)

Step 4-1: preparation of Compound 10

Compound 4(621mg, 0.481mmol) was dissolved in anhydrous chloroform (15ml), and m-chloroperoxybenzoic acid (250mg, 1.25mmol) was added in the absence of light, and the mixture was stirred at room temperature in the absence of light for 24 hours. After the reaction was terminated, chloroform was removed in vacuo to obtain a white solid, ethyl acetate (50ml) was added, and the mixture was washed with 10% sodium hydroxide solution (30mlx2), saturated brine (30mlx3), and the organic layer was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (petroleum ether: ethyl acetate: 3:1) to obtain 503mg of a white solid, i.e., compound 10, in 80% yield.

The melting point of the obtained solid was 155-156 ℃. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CDCl₃,δ):5.23(s,1H,H-1”),5.21(s,1H,H-12),5.16(d,J＝9.5Hz,H-1’),4.99–5.08(m,3H),4.90–4.99(m,3H),4.54(d,J＝7.9Hz,1H),4.50(d,J＝3.4Hz,1H),4.25(dd,J＝12.3,4.6Hz,1H),4.12(dt,J＝14.3,4.3Hz,4H),4.02(t,J＝6.3Hz,1H),3.82–3.91(m,1H),3.68–3.78(m,2H),3.55(q,J＝6.9Hz,1H),3.43(dd,J＝11.7,4.4Hz,1H),2.14(s,3H),2.08(s,3H),2.08(s,3H)2.04(s,3H),2.03(s,3H),2.03(s,3H),2.01(s,3H),1.98(s,3H),1.97(s,3H),1.28(s,3H),1.25(s,3H),1.13(s,3H),0.96(s,3H),0.91(s,3H),0.89(s,3H),0.75(s,3H)；

¹³C NMR(100MHz,CDCl₃,δ):179.97(C-28),170.78(CH₃CO),170.68(CH₃CO),170.51(CH₃CO),170.30(CH₃CO),170.23(CH₃CO),170.10(CH₃CO),170.05(CH₃CO),169.57(CH₃CO),169.56(CH₃CO),101.57(C-1”’),101.44(C-1’),98.20(C-1”),90.59(C-13),81.81(C-3),77.36,76.44,74.63,72.99,72.67,71.88,71.12,70.96,69.59,68.97,68.44,67.27,65.34,61.93,60.53,51.18,47.93,44.91,44.80,42.39,42.14,42.09,39.52,38.62,36.12,34.23,33.77,33.38,31.68,29.81,28.98,28.08,27.57,25.22,24.02,21.27,21.17,21.03,21.02,20.95,20.91,20.88,20.83,20.72,20.70,18.71,17.59,17.41,16.88,14.32,12.53；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₆₅H₉₄O₂7Na 1329.5881,found 1329.5496。

step 4-2: preparation of Compound 12

Compound 10(482mg,0.369mmol) was dissolved in anhydrous methanol/THF/H₂O (2:1:1,20mL) solution was added with potassium hydroxide (248mg,4.42mmol), and stirred at room temperature for 12 h. After the reaction was completed, the organic solvent was removed under reduced pressure, the pH was adjusted to 4 to 5 with 10% HCl, n-butanol (50mL) was diluted, the n-butanol layer was washed with saturated brine (30mL × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol: 10:1) was performed to obtain 308mg of a white solid, i.e., compound 12, with a yield of 90%.

The melting point (mp) of the resulting solid was 198-. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CD₃OD,δ):5.15(s,1H,H-1”),4.46(d,J＝5.9Hz,1H,H-1’),4.44(dd,J＝17.2,6.8Hz,3H),4.41(d,J＝7.6Hz,1H,H-10),4.08(dd,J＝12.1,3.5Hz,1H),1.29(s,3H),1.18(d,J＝6.0Hz,3H,H-6”),1.18(d,J＝6.0Hz,6H),1.05(s,3H),0.91(s,3H),0.86(s,3H),0.86(s,3H),0.63(s,3H)；

¹³C NMR(75MHz,CD₃OD,δ):182.57(C-28),105.86(C-1”’),104.45(C-1’),101.77(C-1”),93.29(C-13),82.17(C-3),79.54(C-12),77.90,77.70,76.78,76.47,75.21,73.81,71.94,71.86,71.20,70.07,64.92,64.46,62.46,52.35,46.12,45.62,44.03,43.32,43.24,40.15,39.70,37.04,35.12,34.47,33.68,32.34,29.53,28.92,28.63,26.56,24.20,22.28,20.92,19.18,19.02,18.20,17.95,17.32,13.56；

HRMS-ESI(m/z)[M+Cl]^-:calcd.for C₄₇H₇₆O₁₈Cl 963.4721,found 963.4853。

example 5: preparation of 12-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranose Oleanorane-28-carboxylic acid (Compound 19)

Step 5-1: preparation of Compound 13

Compound 3(2g, 1.99mmol) and anhydrous potassium carbonate (827mg, 5.99mmol) were placed in a reaction flask, N-dimethylformamide (20ml) was added, benzyl bromide (0.5ml) was added dropwise with stirring, and the mixture was stirred at room temperature for 6 hours. After the reaction, water (100ml) was added to dilute the reaction system to obtain a white emulsion, which was extracted with ethyl acetate (50ml x3), the organic layer was washed with a saturated common salt solution (50ml x3), the organic layer was dried over anhydrous sodium sulfate, and column chromatography on silica gel (petroleum ether: ethyl acetate 4:1) was performed to obtain 1.98g of a white solid, i.e., compound 13, in 92% yield.

The melting point of the obtained solid is 168-169 ℃. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(400MHz,CDCl₃,δ):7.19–7.32(m,5H,Ar-),5.09–5.27(m,5H),4.93–5.06(m,5H),4.90(d,J＝8.7Hz,1H，H-1’),4.35(d,J＝6.2Hz,1H),4.06(t,J＝10.0Hz,3H),3.74–3.94(m,4H),3.50(t,J＝10.4Hz,2H),2.80(t,J＝17.1Hz,1H),2.06(s,3H,CH₃CO),2.03(s,3H,CH₃CO),2.02(s,3H,CH₃CO),1.98(s,3H,CH₃CO),1.96(s,3H,CH₃CO),1.89(s,3H,CH₃CO),1.14(d,J＝6.1Hz,3H),1.02(s,3H),0.84(s,6H),0.82(s,3H),0.72(s,3H),0.52(s,3H)；

¹³CNMR(75MHz,CD Cl₃,δ):177.48(C-28),170.48(CH₃CO),170.46(CH₃CO),170.37(CH₃CO),170.23(CH₃CO),170.12(CH₃CO),169.71(CH₃CO),143.86(C-13),136.49(Ar-C),128.49(Ar-C),128.06(Ar-C),128.00(Ar-C),122.43(C-12),103.70(C-1’),98.20(C-1”),82.02(C-3),74.29,72.07,71.08,69.63,68.65,67.97,67.19,66.00,65.15,62.94,47.89,46.79,45.90,41.98,41.66,41.45,39.34,38.42,36.60,33.91,33.20,32.42,30.78,29.78,27.59,25.80,25.63,23.74,23.48,23.06,21.12,21.08,21.04,20.90,20.87,20.76,18.05,17.41,16.97,15.90,12.78；

HRMS-ESI(m/z)[M+H]⁺:calcd.for C₆₀H₈₅O₁₈1093.5736,found 1093.5579。

step 5-2: preparation of Compound 15

Compound 13(800mg, 0.732mmol) was dissolved in anhydrous chloroform (20ml), and 75% m-chloroperoxybenzoic acid (337mg, 1.25mmol) was added in the absence of light, followed by stirring at room temperature in the absence of light for 24 hours. After the reaction was terminated, chloroform was removed in vacuo to obtain a white solid, ethyl acetate (50ml) was added, the mixture was washed with 10% sodium hydroxide solution (30 ml. times.2), saturated brine (30 ml. times.3), and the organic layer was dried over anhydrous sodium sulfate and subjected to silica gel column chromatography (petroleum ether: ethyl acetate: 3:1) to obtain 707mg of a white solid, that is, compound 15, in about 87% yield.

The melting point of the resulting solid was 154-155 ℃.

The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(400MHz,CDCl₃,δ):7.15–7.31(m,5H,AR-H),5.17(d,J＝2.9Hz,2H),5.14(s,2H),4.97(dd,J＝16.9,7.8Hz,3H),4.88(dd,J＝8.9,3.2Hz,1H),4.31(d,J＝6.6Hz,1H),3.99–4.08(m,2H),3.85(d,J＝11.9Hz,2H),3.78(dd,J＝8.6,7.0Hz,1H),3.39–3.55(m,2H),2.74(d,J＝14.3Hz,1H),2.37(d,J＝3.8Hz,1H),2.06(s,3H,CH₃CO),2.03(s,3H,CH₃CO),2.02(s,3H,CH₃CO),1.98(s,3H,CH₃CO),1.96(s,3H,CH₃CO),1.89(s,3H,CH₃CO),1.14(d,J＝6.1Hz,3H),0.91(s,3H),0.82(s,3H),0.79(s,3H),0.75(s,3H),0.72(s,3H),0.52(s,3H)；

¹³C NMR(75MHz,CDCl₃,δ):211.62(C-12),177.43(C-28),170.42(CH₃CO),170.38(CH₃CO),170.29(CH₃CO),170.16(CH₃CO),170.03(CH₃CO),169.66(CH₃CO),136.31(C-13),128.54(AR-C),128.43(AR-C),128.18(C-12),103.66(C-1’),98.17(C-1”),81.48(C-3),74.29,72.01,70.99,69.53,68.54,67.88,67.14,65.96,64.97,63.01,60.41,51.81,49.86,47.78,47.17,41.87,41.11,38.44,37.79,36.42,36.23,34.43,33.42,32.80,32.02,31.45,30.66,29.69,27.30,25.41,23.21,22.66,21.06(CH₃CO),20.96(CH₃CO),20.83(CH₃CO),20.80(CH₃CO),20.68(CH₃CO),20.56(CH₃CO),17.91,17.34,15.65,14.22,12.46；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₆₀H₈₄O₁₉Na 1131.5505；found:1131.5093。

step 5-3: preparation of Compound 17

Compound 15(707mg, 0.638mmol) was dissolved in anhydrous tetrahydrofuran (15ml), and 10% palladium on carbon (50mg) was added thereto, and the mixture was stirred under hydrogen atmosphere for 4 hours. After the reaction was completed, palladium on carbon was removed by filtration, and column chromatography on silica gel (petroleum ether: ethyl acetate: 5:1) was performed to obtain 620mg of a white solid, i.e., compound 17, in a yield of 97%.

The melting point of the resulting solid was 183-184 ℃. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CDCl₃,δ):5.25(s,1H,H-1”),5.21(s,1H,H-12),5.00–5.08(m,2H),4.96(dd,J＝9.0,3.4Hz,1H,H-1’),4.39(d,J＝6.6Hz,1H),4.07–4.17(m,1H),3.93(d,J＝12.2Hz,2H),3.86(dd,J＝8.9,6.7Hz,1H),3.55(dd,J＝19.6,7.9Hz,2H),2.75(d,J＝10.1Hz,1H),2.67(s,1H),2.13(s,3H),2.10(s,6H),2.05(s,3H),2.03(s,3H),1.96(s,3H),1.21(d,J＝6.2Hz,3H),0.98(s,3H),0.97(s,3H),0.91(s,3H),0.90(s,3H),0.89(s,3H),0.79(s,3H)；

¹³CNMR(100MHz,CD Cl₃,δ):211.40(C-12),182.71(C-28),170.43(CH₃CO),170.41(CH₃CO),170.31(CH₃CO),170.18(CH₃CO),170.05(CH₃CO),169.67(CH₃CO),103.66(C-1’),98.25(C-1”),81.49(C-3),74.48,71.90,70.98,69.55,68.60,67.84,67.18,64.95,62.92,60.45,51.88,49.91,47.80,47.09,46.99,41.90,41.24,38.51,37.80,36.50,36.09,34.41,33.35,33.02,31.84,31.53,30.62,29.71,27.47,25.40,23.10,22.56,21.06,20.99,20.95,20.83,20.79,20.67,20.54,17.95,17.35,16.20,15.70,12.42；

HRMS-ESI(m/z)[M+H]⁺:calcd.for _C53H79O191019.5216；found:1019.4854。

step 5-4: preparation of Compound 19

Compound 17(310mg,0.304mmol) was dissolved in anhydrous methanol/THF/H2O (2:1:1,20mL), potassium hydroxide (205mg,3.65mmol) was added, and the mixture was stirred at room temperature for 12H. After the reaction was completed, the organic solvent was removed under reduced pressure, the pH was adjusted to 4 to 5 with 10% HCl, n-butanol (50ml) was diluted, the n-butanol layer was washed with saturated brine (30 ml. times.3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol: 10:1) was performed to obtain 220mg of a white solid, i.e., compound 19, in 94% yield.

Melting point of the resulting solid was 221-. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(400MHz,pyridine-d₅,δ):6.26(s,1H,H-1”),5.06(d,J＝6.3Hz,1H,H-1’),4.81(s,1H),4.68(d,J＝8.9Hz,1H),4.49–4.62(m,1H),4.35(t,J＝9.4Hz,1H),4.26(dd,J＝11.9,3.7Hz,1H),4.06–4.23(m,2H),3.68(t,J＝10.4Hz,1H),3.33(d,J＝13.4Hz,1H),3.19(d,J＝3.8Hz,1H),1.62(d,J＝6.0Hz,3H),1.11(s,3H),1.02-1.03(d,J＝2.1Hz,9H),0.93(s,3H),0.80(s,3H)；

¹³C NMR(100MHz,DMSO,δ):211.12(C-12),178.63(C-28),103.00(C-1’),99.93(C-1”),79.14(C-3),74.24,72.95,72.06,70.50,70.37,68.13,67.89,64.50,62.43,51.12,49.27,46.16,46.08,42.34,41.42,40.75,38.02,37.59,35.88,33.93,31.54,30.36,25.26,23.12,20.18,17.82,15.81,15.39,12.71；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₄₁H₆₆O₁₃Na 789.4401,found 789.4375.

example 6: preparation of 12-carbonyl-23 hydroxy-3-O-alpha-L-pyranRhamnose- (1 → 2) [ beta-D-glucopyranose- (1→4)]-alpha-L-arabinopyranoside oleanane-28-oic acid (Compound 20)

Step 6-1: preparation of Compound 14

Compound 4(2.57g, 1.99mmol) and anhydrous potassium carbonate (827mg, 5.99mmol) were placed in a reaction flask, N-dimethylformamide (20ml) was added, benzyl bromide (0.5ml) was added dropwise with stirring, and the mixture was stirred at room temperature for 6 hours. After the reaction, water (100ml) was added to dilute the reaction system to obtain a white emulsion, which was extracted with ethyl acetate (50ml × 3), the organic layer was washed with a saturated common salt solution (50ml × 3), the organic layer was dried over anhydrous sodium sulfate, and column chromatography on silica gel (petroleum ether: ethyl acetate 4:1) was performed to obtain 2.47g of a white solid, i.e., compound 14, in 90% yield.

The resulting solid had a melting point of 164-165 ℃. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(600MHz,CDCl₃,δ):7.28–7.37(m,5H,AR-),5.28(t,J＝3.4Hz,1H，H-1”),5.18(t,J＝9.6Hz,1H,H-12),4.92(s,1H,H-1’),4.55(d,J＝7.9Hz,1H,H-1”’),4.52(d,J＝3.4Hz,1H),4.26(dd,J＝12.3,4.7Hz,1H),4.15(dd,J＝12.3,2.2Hz,1H),4.03(dd,J＝9.1,6.2Hz,3H),3.96(dt,J＝16.1,6.2Hz,1H),3.81(d,J＝11.5Hz,1H),3.76(dd,J＝5.6,3.5Hz,1H),3.72(ddd,J＝10.0,4.6,2.4Hz,1H),3.57(q,J＝7.2Hz,1H),3.42(dd,J＝11.8,4.6Hz,1H),2.90(dd,J＝13.6,3.8Hz,1H),2.14(s,3H),2.09(s,3H),2.08(s,1H),2.05(s,3H),2.04(s,3H),2.03(s,3H),2.02(s,3H),1.99(s,3H),1.97(s,3H),1.19(d,J＝6.2Hz,3H),1.09(s,3H),0.91(s,6H),0.89(s,3H),0.75(s,3H),0.59(s,3H)；

¹³C NMR(150MHz,CDCl₃,δ)177.53(C-28),170.77(CH₃CO),170.68(CH₃CO),170.55(CH₃CO),170.29(CH₃CO),170.22(CH₃CO),170.09(CH₃CO),170.04(CH₃CO),169.56(CH₃CO),169.55(CH₃CO),143.85(C-13),136.55(AR-),128.54(AR-),128.12(AR-),128.05(AR-),122.55(C-12),101.58(C-1”’),101.33(C-1)’,98.22(C-1”),82.06(C-3),74.66,72.96,72.73,71.92,71.10,71.00,69.61,69.01,68.47,67.30,66.07,65.44,61.95,47.96,47.92,46.87,45.99,42.05,41.73,41.53,39.42,38.33,36.67,33.99,33.25,32.53,32.49,30.84,29.84,27.68,25.87,25.18,23.79,23.56,23.16,21.09,21.04,20.96,20.93,20.90,20.85,20.75,20.72,18.17,17.44,17.04,15.94,12.83；

HRMS-ESI(m/z)[M+Na]⁺:calcd for C₇₂H₁₀₀O₂₆Na 1403.6401；found:1403.5948。

step 6-2: preparation of Compound 16

Compound 14(1.01g, 0.732mmol) was dissolved in anhydrous chloroform (20ml), and 75% m-chloroperoxybenzoic acid (337mg, 1.25mmol) was added in the absence of light, followed by stirring at room temperature in the absence of light for 24 hours. After the reaction was terminated, chloroform was removed in vacuo to obtain a white solid, and ethyl acetate (50ml) was added, followed by washing with 10% sodium hydroxide solution (30 ml. times.2) and saturated brine (30 ml. times.3), drying the organic layer over anhydrous sodium sulfate, and recovering the solvent to obtain 707mg of a white solid, that is, compound 16, which was directly charged into the next reaction.

Step 6-3: preparation of Compound 18

Compound 16(707mg, 0.51mmol) was dissolved in anhydrous tetrahydrofuran (15ml), and 10% palladium on carbon (50mg) was added thereto, and the mixture was stirred under hydrogen atmosphere for 4 hours. After the reaction, palladium on carbon was removed by filtration, and silica gel column chromatography (petroleum ether: ethyl acetate: 5:1) was performed to obtain 600mg of a white solid, that is, compound 18, in a yield of 90%.

The melting point of the resulting solid was 176-177 ℃. The nuclear magnetic data of the obtained solid is:

¹H NMR(400MHz,CDCl₃,δ):5.23(d,J＝1.0Hz,1H,H-1”),5.19–5.22(m,1H,H-12),5.15(d,J＝9.6Hz,1H,H-1’),4.89–5.08(m,6H),4.85(d,J＝9.1Hz,1H),4.53(d,J＝7.9Hz,1H),4.48(d,J＝2.8Hz,1H),3.55(d,J＝8.3Hz,1H),3.42(s,1H),2.75(d,J＝13.7Hz,1H),2.67(d,J＝3.9Hz,1H),2.14(s,3H),2.08(s,6H),2.05(s,3H),2.04(s,6H),2.02(s,3H),2.01(s,3H),1.98(s,3H),1.97(s,3H),1.24(s,6H),0.97(s,3H),0.96(s,3H),0.91(s,3H),0.89(d,J＝2.4Hz,6H),0.75(s,3H)；

¹³C NMR(150MHz,CDCl₃,δ):211.44(C-12),183.16(COOH),170.76(CH₃CO),170.67(CH₃CO),170.54(CH₃CO),170.30(CH₃CO),170.20(CH₃CO),170.07(CH₃CO),170.04(CH₃CO),169.54(CH₃CO),169.52(CH₃CO),101.59(C-1”’),101.44(C-1’),98.32(C-1”),81.60(C-3),74.81,72.98,72.71,71.90,71.09,70.95,69.57,68.96,68.43,67.31,65.28,61.90,60.53,51.97,50.02,47.91,47.23,42.05,42.01,41.39,38.64,37.79,36.65,36.25,34.56,33.46,33.17,31.95,31.70,31.56,30.74,30.31,29.81,27.63,25.05,23.23,22.74,21.17,21.08,21.01,20.94,20.90,20.87,20.82,20.72,20.69,20.67,18.13,17.45,16.31,15.81,14.32,12.55

HRMS-ESI(m/z)[M+Na]⁺:calcd for C₆₅H₉₄O₂₇Na 1329.5881；found:1329.5566。

step 6-4: preparation of Compound 20

Compound 18(397mg,0.304mmol) was dissolved in anhydrous methanol/THF/H2O (2:1:1,20mL), potassium hydroxide (205mg,3.65mmol) was added, and the mixture was stirred at room temperature for 12H. After the reaction was completed, the organic solvent was removed under reduced pressure, the pH was adjusted to 4 to 5 with 10% HCl, n-butanol (50ml) was diluted, the n-butanol layer was washed with saturated brine (30ml × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol 10:1) was performed to obtain 263mg of a white solid, i.e., compound 20, in 93% yield.

Melting point of the resulting solid was 197-. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(600MHz,pyridine-d₅,δ):6.28(s,1H,H-1”),5.13(d,J＝7.4Hz,1H,H-1’),4.95(d,J＝6.4Hz,2H),4.63(d,J＝8.7Hz,2H),4.47–4.55(m,2H),4.24–4.44(m,5H),4.11–4.23(m,4H),4.04(s,2H),3.90(s,1H),3.72(d,J＝10.3Hz,2H),3.33(d,J＝12.8Hz,1H,18βH),3.20(s,1H,13βH),1.66(d,J＝5.2Hz,3H,H-6”),1.12(s,3H),1.07(s,3H),1.04(s,6H),0.94(s,3H),0.82(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):211.78(C-12),180.99(COOH),107.14(C-1”’),104.84(C-1’),102.06(C-1”),81.20(C-3),80.86,79.19,78.94,76.63,75.85,75.54,74.49,72.82,72.63,71.57,69.98,65.88,64.14,62.84,52.60,50.61,50.04,48.07,47.69,43.88,42.57,41.94,39.20,38.74,37.17,37.04,35.21,34.10,33.99,33.19,32.47,32.25,32.20,31.37,30.34,28.52,26.55,23.93,23.76,21.55,21.01,19.00,18.46,16.69,16.20,14.11；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₄₇H₇₆O₁₈Na 951.4930；found:951.4884。

example 7: preparation of 12-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranose Oleanorane-28-gamma-lactone (Compound 23)

Step 7-1: preparation of Compound 21

Compound 17(310mg,0.304mmol) and dichlorodicyanobenzoquinone (135mg, 0.610mmol) were dissolved in anhydrous benzene (20ml) and refluxed at 90 ℃ for 48 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure, methylene chloride (30ml) was added, the mixture was washed with a saturated sodium bicarbonate solution (20ml × 3), the organic layer was dried over anhydrous sodium sulfate, and column chromatography on silica gel (petroleum ether: ethyl acetate ═ 3:1) was performed to obtain 145mg of a white solid, that is, compound 21, in 48% yield.

The melting point of the obtained solid is 208-209 ℃. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(400MHz,CDCl₃,δ):5.24(d,J＝3.2Hz,1H,H-1”),5.17–5.22(m,1H,H-1’),4.99–5.08(m,2H,H-11),4.96(dd,J＝9.0,3.4Hz,1H),4.39(d,J＝6.6Hz,1H),4.05–4.17(m,2H),3.89–4.00(m,2H),3.86(dd,J＝8.9,6.6Hz,1H),3.47–3.61(m,2H),2.70(t,J＝14.2Hz,1H),2.52(t,J＝8.2Hz,1H),2.37(dd,J＝14.5,2.4Hz,3H),2.12(s,3H),2.10(s,3H),2.08(s,3H),2.04(s,3H),2.02(s,3H),1.96(s,3H),1.24(s,3H),1.20(d,J＝6.2Hz,3H),0.96(s,3H),0.95(s,3H),0.94(s,3H),0.93(s,3H),0.80(s,3H)

¹³C NMR(100MHz,CDCl₃,δ):206.14(C-12),178.54(C-28),170.50(CH₃CO),170.42(CH₃CO),170.41(CH₃CO),170.27(CH₃CO),170.12(CH₃CO),169.78(CH₃CO),103.76(C-1’),98.23(C-1”),91.13(C-13),81.43(C-3),77.36,74.30,72.10,71.12,69.67,68.65,67.98,67.25,65.12,63.08,51.45,47.83,44.10,44.05,43.80,42.60,42.11,38.36,37.49,37.42,37.00,34.24,33.29,32.79,31.68,29.81,29.77,29.47,27.33,27.03,25.93,25.51,23.88,22.80,21.12,21.09,21.05,20.92,20.89,20.77,18.66,18.31,17.45,17.35,16.43,14.24,12.47；

HRMS-ESI(m/z)[M+Cl]⁺:calcd.for C₅₃H₇₆O₁₉Cl 1015.4669；found:1015.4671。

step 7-2: preparation of Compound 23

Compound 21(145mg,0.143mmol) was dissolved in anhydrous methanol/THF/H2O (2:1:1,15mL), potassium hydroxide (56mg,1.71mmol) was added, and the mixture was stirred at room temperature for 12H. After the reaction was completed, the organic solvent was removed under reduced pressure, ph was adjusted to 4-5 with 10% HCl, n-butanol (30ml) was diluted, the n-butanol layer was washed with saturated brine (20ml × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol 10:1) was performed to obtain 97mg of a white solid, that is, compound 23, with a yield of 90%.

Melting point of the resulting solid 208-209 ℃. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(400MHz,CDCl₃,δ):6.14(s,1H,H-1”),5.70(s,1H,H-12),4.98(d,J＝5.5Hz,1H,H-1’),4.76(s,1H),4.66(dd,J＝9.3,2.7Hz,1H),4.16(s,1H),3.76–3.92(m,2H),2.32(t,J＝12.1Hz,2H),1.66(d,J＝6.1Hz,3H),1.56(s,3H),1.29(s,3H),1.05(s,3H),0.92(s,3H),0.87(s,3H),0.82(s,3H)；

13C NMR(100MHz,CDCl₃,δ):206.82(C-12),178.56(C-28),104.94(C-1’),102.12(C-1”),91.57(C-13),81.01(C-3),76.22,75.29,74.54,72.96,72.76,70.08,69.85,66.28,64.17,51.81,47.81,44.72,44.60,44.33,43.96,43.11,39.02,38.04,37.72,37.38,34.63,33.38,33.19,32.02,28.25,26.51,26.47,24.15,21.40,19.03,18.95,18.46,17.69,16.80,13.99；

HRMS-ESI(m/z)[M-H]-:calcd.for C₅₃H₇₅O₁₉1015.4903；found:1015.5097。

example 8: preparation of 12-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) [ beta-D-glucopyranose- (1→4)]-alpha-L-arabinopyranoside oleanane-28-lactone (Compound 24)

Step 8-1: preparation of Compound 22

Compound 18(397mg,0.304mmol) and dichlorodicyanobenzoquinone (135mg, 0.610mmol) were dissolved in anhydrous benzene (20ml) and refluxed at 90 ℃ for 48 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure, methylene chloride (30ml) was added, the mixture was washed with a saturated sodium bicarbonate solution (20ml × 3), the organic layer was dried over anhydrous sodium sulfate, and column chromatography on silica gel (petroleum ether: ethyl acetate ═ 3:1) was performed to obtain 195mg of a white solid, that is, compound 22, in 50% yield.

¹H NMR(600MHz,CDCl₃,δ):5.21(s,1H,H-1”),5.19(d,J＝3.1Hz,1H,H-1’),5.15(t,J＝9.5Hz,1H,H-1”’),4.98–5.06(m,2H,H-11),4.94(dd,J＝24.0,14.4Hz,4H),4.50–4.54(m,4H),4.45–4.50(m,1H),4.23(dd,J＝12.3,4.5Hz,1H),4.04–4.15(m,2H),3.91–4.03(m,3H),3.81–3.89(m,2H),3.73(dd,J＝5.4,3.9Hz,1H),3.69(dd,J＝7.7,2.1Hz,1H),3.54(d,J＝8.8Hz,1H),3.41(dd,J＝11.7,4.4Hz,1H),2.12(s,3H),2.06(s,6H),2.02(s,6H),2.01(s,3H),1.99(s,3H),1.97(s,3H),1.95(s,3H),1.26(s,3H),1.16(d,J＝6.1Hz,3H),1.11(s,3H),0.94(s,3H),0.89(s,3H),0.87(s,3H),0.73(s,3H)；

¹³C NMR(150MHz,CDCl₃,δ):206.06(C-12),179.96(C-28),170.71(CH₃CO),170.61(CH₃CO),170.45(CH₃CO),170.23(CH₃CO),170.16(CH₃CO),170.03(CH₃CO),169.98(CH₃CO),169.51(CH₃CO),169.49(CH₃CO),101.49(C-1”’),101.40(C-1’),98.15(C-1”),90.63(C-13),81.77(C-3),76.32,74.58,72.94,72.64,71.84,71.04,70.92,69.55,68.93,68.42,65.30,65.24,61.90,61.85,60.48,51.13,47.85,44.86,44.76,42.34,42.10,42.05,39.45,38.57,36.09,34.19,33.73,33.34,31.63,31.51,30.27,29.75,28.91,28.03,27.53,26.98,25.18,23.97,23.84,21.23,20.98,20.96,20.90,20.86,20.83,20.77,20.67,20.65,18.67,17.55,17.37,16.83,14.27,12.49；

HRMS-ESI(m/z)[M+Cl]⁺:calcd.for C₆₅H₉₂O₂₇Cl 1339.5515；found:1339.5978。

step 8-2: preparation of Compound 24

Compound 22(187mg,0.143mmol) was dissolved in anhydrous methanol/THF/H2O (2:1:1,15mL), potassium hydroxide (56mg,1.71mmol) was added, and the mixture was stirred at room temperature for 12H. After the reaction was completed, the organic solvent was removed under reduced pressure, ph was adjusted to 4-5 with 10% HCl, n-butanol (30ml) was diluted, the n-butanol layer was washed with saturated brine (20ml × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol 10:1) was performed to obtain 117mg of a white solid, i.e., compound 24, with a yield of 90%.

Melting point 198-. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(600MHz,pyridine-d₅,δ):6.26(s,1H,H-1”),5.13(d,J＝7.7Hz,1H,H-1’),4.96(d,J＝6.9Hz,1H,H-1”’),4.71(s,3H),4.63(dd,J＝9.1,3.3Hz,2H),4.50(dd,J＝15.2,8.5Hz,3H),4.41–4.33(m,3H),4.30(t,J＝9.3Hz,2H),4.25(t,J＝9.1Hz,2H),4.18(dd,J＝17.0,10.0Hz,6H),4.03(t,J＝16.3Hz,3H),3.77(d,J＝10.7Hz,1H),3.67(d,J＝12.4Hz,1H),2.30(t,J＝12.2Hz,2H),1.66(d,J＝6.0Hz,3H),1.56(s,3H),1.34(s,3H),1.11(s,3H),0.94(s,3H),0.88(s,3H),0.81(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):206.80(C-12),180.06(C-28),107.06(C-1”’),104.75(C-1’),102.02(C-1”),91.84(C-13),81.40(C-3),80.76,79.12,78.89,76.62,76.09,75.82,75.39,74.46,72.80,72.61,71.57,69.99,65.77,64.21,62.85,52.04,50.02,48.27,45.43,45.31,43.98,43.05,43.02,39.83,39.50,38.97,37.34,37.28,37.18,36.74,34.83,34.50,33.59,33.35,31.97,29.72,28.75,28.63,26.70,26.50,24.15,22.08,19.42,19.09,18.98,18.44,18.03,17.58,16.78,14.09；

HRMS-ESI(m/z)[M+Cl]⁺:calcd.for C₄₇H₇₄O₁₈Cl 961.4564；found:961.4791。

example 9: preparation of 12-hydroxyimino-23-hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranose Oleanolic alkane-28-carboxylic acid (Compound 27)

Step 9-1: preparation of Compound 25

Compound 15(300mg, 0.270mmol) and hydroxylamine hydrochloride (100mg, 1.36mmol) were dissolved in anhydrous pyridine (15ml), refluxed at 120 ℃ and the progress of the reaction was checked by thin layer silica gel chromatography. After completion of the reaction, it was cooled to room temperature, pyridine was removed under reduced pressure, and thin layer column chromatography (petroleum ether: ethyl acetate 4:1) gave 213mg of compound 25 as a yellow solid in 74% yield.

The melting point of the obtained solid is 161-162 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(400MHz,CDCl₃,δ):7.00-7.50(m,5H,AR-),5.17(s,1H,H-1”),5.13(s,2H),5.11(s,1H),4.99(d,J＝12.0Hz,1H,H-1’),4.96(t,J＝9.5Hz,2H),4.88(dd,J＝9.7,2.1Hz,1H),4.32(d,J＝6.7Hz,1H),4.04(t,J＝8.6Hz,2H),3.81(dt,J＝15.3,9.2Hz,4H),3.46(dd,J＝22.0,8.8Hz,2H),2.93(dd,J＝19.0,2.3Hz,1H),2.84(d,J＝12.0Hz,3H),2.31(s,1H),1.13(d,J＝6.1Hz,3H),0.85(s,3H),0.80(s,3H),0.78(s,3H),0.75(s,3H),0.70(s,3H),0.39(s,3H)；

¹³CNMR(75MHz,CD Cl₃,δ):178.18(C-28),170.81(Ac-),170.74(Ac-),170.58(Ac-),170.47(Ac-),170.06(Ac-),160.16(C-12),136.87(AR-C),128.91(AR-C),128.67(AR-C),128.44(AR-C),104.04(C-1’),98.57(C-1”),82.12(C-3),74.67,72.39,71.43,69.97,68.97,68.30,67.55,66.26,65.43,63.32,48.34,48.22,48.04,43.66,42.34,41.36,40.58,38.38,37.01,36.39,35.06,34.00,33.50,33.09,31.87,31.25,30.13,27.54,27.34,25.91,23.55,23.12,23.05,21.42(CH₃CO),21.38(CH₃CO),21.25(CH₃CO),21.10(CH₃CO),20.23(CH₃CO),18.30,17.76,16.03,15.83,12.90；

HRMS-ESI(m/z)[M+Na]⁺:calcd for C₆₀H₈₅NO₁₉Na 1146.5614；found:1146.5215。

step 9-2: preparation of Compound 26

Compound 25(213mg, 0.189mmol) was dissolved in anhydrous tetrahydrofuran (15ml), and 10% palladium on carbon (20mg) was added thereto, and the mixture was stirred under hydrogen atmosphere for 4 hours. After the reaction was completed, palladium on carbon was removed by filtration, and column chromatography on silica gel (petroleum ether: ethyl acetate: 4:1) gave 186mg of a yellow solid, i.e., compound 26, in 95% yield.

The melting point of the obtained solid was 203-.

¹H NMR(400MHz,CDCl₃,δ):5.24(d,J＝3.5Hz,1H,H-1”),5.18–5.23(m,1H),5.03(t,J＝9.8Hz,2H),4.96(dd,J＝9.0,3.4Hz,1H,H-1’),4.39(d,J＝6.5Hz,1H),4.11(q,J＝7.1Hz,4H),3.93(d,J＝10.9Hz,2H),3.85(dd,J＝8.8,6.7Hz,1H),3.55(dd,J＝16.3,8.8Hz,2H),3.03(dd,J＝18.6,5.5Hz,1H),2.81(d,J＝13.2Hz,1H),2.52(d,J＝2.9Hz,1H),2.13(s,3H,CH₃CO),2.10(s,3H,CH₃CO),2.10(s,3H,CH₃CO),2.04(s,3H,CH₃CO),2.02(s,3H,CH₃CO),1.96(s,3H,CH3CO),1.21(d,J＝6.2Hz,3H),0.87-0.91(m,J＝6.9Hz,12H),0.84(s,3H),0.79(s,3H)；

¹³CNMR(100MHz,CD Cl₃,δ):183.55(C-28),170.53(CH₃CO),170.51(CH₃CO),170.43(CH₃CO),170.28(CH₃CO),170.17(CH₃CO),169.76(CH₃CO),160.61(C-12),103.85(C-1’),98.35(C-1”),81.82(C-3),74.51,72.09,71.15,69.66,68.68,68.03,67.30,65.13,63.12,48.00,47.63,47.30,43.83,42.06,41.03,40.13,37.97,36.79,35.79,34.50,33.76,32.05,30.86,29.82,29.78,23.64,22.82,21.18,21.17,21.11,21.06,20.94,20.90,20.78,20.54,17.47,16.20,15.68,14.32,12.58。

Step 9-3: preparation of Compound 27

Compound 26(186mg, 0.179mmol) was dissolved in anhydrous methanol/THF/H2O (volume ratio 2:1:1,15mL), and potassium hydroxide (120mg,1.71mmol) was added and stirred at room temperature for 12H. After the reaction was completed, the organic solvent was removed under reduced pressure, ph was adjusted to 4-5 with 10% HCl, n-butanol (30ml) was diluted, the n-butanol layer was washed with saturated brine (20ml × 3), the organic layer was dried over anhydrous sodium sulfate, and silica gel column chromatography (dichloromethane: methanol ═ 12:1) was performed to obtain 126mg of a pale yellow solid, that is, compound 27, with a yield of 90%.

The resulting solid had a melting point of 218-219 ℃. The nuclear magnetic data of the obtained solid are as follows:

¹H NMR(400MHz,CD₃OD,δ):5.12(s,1H,H-1”),4.50(d,J＝4.1Hz,1H,H-1’),3.03(d,J＝17.4Hz,1H),2.84(d,J＝12.6Hz,1H),1.20(d,J＝5.7Hz,3H),0.91(s,3H),0.89(s,4H),0.88(s,3H),0.85(s,3H),0.85(s,3H),0.66(s,3H)；

¹³C NMR(100MHz,pyridine-d₅,δ):179.65(C-28),158.25(C-12),104.73(C-1’),101.99(C-1”),81.25(C-3),76.11,75.15,74.44,72.86,72.69,70.00,69.74,66.09,64.18,62.03,49.99,48.69,48.05,43.88,41.85,41.04,39.18,37.27,36.11,31.50,28.41,26.55,23.90,21.45,20.15,19.88,18.97,18.87,18.40,16.43,16.19,14.50,14.06；

HRMS-ESI(m/z)[M+Na]⁺:calcd.for C₄₁H₆₇NO₁₃Na 804.4510；found:804.4604。

example 10: preparation of 11-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranose Sugar oleanane-N-acetic acid-28-formamide (30)

Step 10-1: preparation of Compound 28

Compound 5(300g,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and stirred at room temperature for 4 h. At the end of the reaction, the solvent and excess oxalyl chloride were removed under reduced pressure to give 287g of a yellow solid, compound 28, in 95% yield.

Step 10-2: preparation of Compound 30

Compound 28(287mg,0.277mmol) was dissolved in anhydrous dichloromethane (10mL), and glycine ethyl ester hydrochloride (33mg, 0.554mmol) and triethylamine (84.4mg,0.831mmol) were added under cooling in an ice bath to react at room temperature for 12 hours. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, most of the organic solvent was removed under reduced pressure, adjusted to pH 2 with 10% HCl, and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol ═ 10:1) afforded 184mg of a white solid, compound 30, in about 80% yield.

The melting point of the obtained solid is 246-247 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(400MHz,CD₃OD,δ):5.55(s,1H,H-1”),5.14(s,1H,H-12),4.52(d,J＝2.2Hz,1H,H-1’),3.91(d,J＝17.3Hz,3H),3.78–3.85(m,3H),3.47(d,J＝11.6Hz,3H),3.00(d,J＝10.5Hz,1H),2.69(d,J＝13.6Hz,1H),2.41(s,1H),1.39(s,3H),1.21(d,J＝6.1Hz,3H),1.10(s,3H),0.93(s,3H),0.92(s,3H),0.91(s,3H),0.67(s,3H)；

¹³C NMR(150MHz,CD₃OD,δ):202.99(C-11),179.72(COO),173.31(C-28),172.80(C-12),128.22(C-13),104.27,(C-1’)101.80(C-1”),82.00(C-3),76.60,73.87,73.57,72.07,71.97,70.12,69.07,64.71,64.49,63.16,62.02,49.85,47.88,46.95,46.44,45.83,45.02,44.26,42.85(CH₂COOH),42.08,40.10,37.96,34.95,33.38,33.24,28.40,26.49,24.08,24.06,23.80,19.79,17.96,17.83,17.22,13.65；

HRMS-ESI(m/z)[M+Na]⁺:calcd for C₄₃H₆₇NO₁₄Na 844.4459；found:844.4188。

example 11: preparation of 11-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) [ beta-D-glucopyranose- (1→4)]- α -L-arabinopyranoside oleanane-N-acetic acid-28-carboxamide (compound 31)

Compound 6(385g,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and stirred at room temperature for 4 h. After the reaction was complete, the solvent and excess oxalyl chloride were removed under reduced pressure to give a yellow solid, compound 29. The above compound 29 was dissolved in anhydrous dichloromethane (10mL), glycine ethyl ester hydrochloride (33mg, 0.554mmol) and triethylamine (84.4mg,0.831mmol) were added under cooling in an ice bath, and the mixture was reacted at room temperature for 12 hours. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, most of the organic solvent was removed under reduced pressure, adjusted to pH 2 with 10% HCl, and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol ═ 10:1) afforded 247mg of a white solid, compound 31, in 85% yield.

The melting point of the obtained solid is 253-254 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(600MHz,pyridine-d₅,δ):8.36(s,1H,NHCH₂),6.25(s,1H,H-1”),5.99(s,1H,H-1’),5.70(s,1H,H-1”’),5.11(d,J＝7.8Hz,1H),4.97(d,J＝6.6Hz,1H),4.64(dd,J＝9.2,3.0Hz,1H),4.44–4.55(m,2H),3.88(ddd,J＝8.9,4.3,2.2Hz,1H),3.75(d,J＝10.5Hz,1H),3.29(dd,J＝53.7,12.9Hz,2H),2.79(t,J＝7.4Hz,1H),2.63(d,J＝11.6Hz,1H),1.65(d,J＝6.1Hz,3H),1.37(s,3H),1.36(s,3H),1.21(s,3H),1.12(s,3H),0.88(s,3H),0.85(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):200.58(C-11),177.59(COOH),173.99(C-28),170.13(C-12),128.45(C-11),107.03(C-1”),104.76(C-1’),101.99(C-1”’),81.38(C-3),80.64,79.09,78.81,76.57,75.76,75.24,74.41,72.75,72.54,71.52,70.01,65.67,64.20,62.77,61.74,49.99,48.01,46.22,45.82,45.10,44.32,44.25,43.13,42.40,40.21,37.73,34.53,33.28,33.20,31.10,28.24,26.74,23.99,23.85,19.85,18.94,17.59,17.54,14.33；

HRMS-ESI(m/z)[M-H]-:calcd for C₄₉H₇₆NO₁₉982.5012；found:982.5189。

example 12: preparation of 12-carbonyl-23 hydroxy-3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranose Sugar oleanane-N-acetic acid-28-carboxamide (Compound 33)

Compound 17(290g,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and stirred at room temperature for 4 h. After the reaction, the solvent and excess oxalyl chloride were removed under reduced pressure to obtain compound 32. Compound 32 was dissolved in anhydrous dichloromethane (10mL), glycine ethyl ester hydrochloride (33mg, 0.554mmol) and triethylamine (84.4mg,0.831mmol) were added under cooling in an ice bath, and the mixture was reacted at room temperature for 12 hours. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After the reaction is finished, most of the organic solvent is removed under reduced pressureThe reagent was adjusted to pH 2 with 10% HCl and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol ═ 10:1) afforded 209mg of a white solid, compound 33, in about 86% yield.

The melting point of the obtained solid is 223-224 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(600MHz,pyridine-d₅,δ):6.23(s,1H,H-1”),5.05(d,J＝5.7Hz,1H,H-12),4.75(s,1H,H-1’),4.60–4.69(m,2H),4.54(d,J＝7.4Hz,2H),4.39–4.48(m,1H),4.30(t,J＝9.1Hz,1H),4.24(d,J＝9.8Hz,1H),4.13(d,J＝10.7Hz,1H),4.08(d,J＝5.2Hz,1H),3.69(dd,J＝20.5,11.0Hz,2H,CH₂COOH),3.38(d,J＝19.9Hz,2H),1.61(d,J＝5.6Hz,3H),1.14(s,3H),1.01(s,9H),0.89(s,3H),0.81(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):211.99(C-12),178.65(COOH),173.93(C-28),104.65(C-1’),101.83(C-1”),81.04(C-3),76.04,74.99,74.30,72.81,72.58,69.92,69.67,66.00,64.10,52.24,50.60,49.95,47.89,47.13,43.77,42.58,42.02,39.17,38.67,37.21,36.95,35.31,34.87,33.94,32.87,32.16,31.18,30.24,28.11,26.36,23.83,23.75,21.01,18.80,18.39,16.84,15.99,13.96；

HRMS-ESI(m/z)[M-H]^-:calcd for C₃₄H₇₀NO₁₄824.4796；found:824.4818。

example 13: 3-O-alpha-L-pyranorhamnose- (1 → 2) -alpha-L-arabinopyranosylivy saponin-N-butyric acid- 28-carboxamide (Compound 35)

Compound 3(291mg,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent and excess oxalyl chloride were removed under reduced pressure to give a yellow solid, compound 34. The resulting compound 34 was dissolved in anhydrous dichloromethane (10mL)Ethyl aminobutyric acid hydrochloride (94mg, 0.554mmol) and triethylamine (84.4mg,0.831mmol) were added under cooling in an ice bath and reacted at room temperature for 12 hours. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, most of the organic solvent was removed under reduced pressure, adjusted to pH 2 with 10% HCl, and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol ═ 10:1) afforded 217mg of white solid, compound 35, in about 88% yield.

The melting point of the obtained solid is 184-185 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(600MHz,pyridine-d₅,δ):7.53(t,J＝5.4Hz,1H,CONH),5.45(s,1H,H-1”),5.12(d,J＝6.2Hz,1H,H-12),4.74(d,J＝2.1Hz,1H,H-1’),4.69(td,J＝12.4,6.2Hz,1H),4.65(dd,J＝9.3,3.4Hz,1H),4.55–4.61(m,1H),4.30(t,J＝9.4Hz,1H),4.24–4.28(m,1H),4.18(d,J＝2.5Hz,1H),4.15(d,J＝10.9Hz,1H),4.09–4.13(m,1H),3.64–3.77(m,3H),3.52(dt,J＝19.4,6.4Hz,1H),3.09(dd,J＝13.1,3.7Hz,1H),2.63(t,J＝7.2Hz,2H),1.64(d,J＝6.2Hz,3H),1.20(s,3H),1.07(s,3H),1.00(s,3H),0.98(s,3H),0.93(s,3H),0.89(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):178.03(COOH),176.31(C-28),145.14(C-13),123.22(C-12),104.69(C-1’),101.95(C-1”),81.34(C-3),76.11,75.01,74.42,72.87,72.69,70.02,69.67,65.98,64.27,48.42,47.97,47.07,46.76,43.83,42.46,42.16,40.12,40.06(CH₂COOH),39.28,37.17,34.69(NHCH₂),34.13,33.53,33.01,31.20,30.30,28.27,26.53,26.47,25.98(CH₂),24.19,24.10,23.96,18.88,18.45,17.88,16.43,14.32；

HRMS-ESI(m/z)[M+Cl]⁺:calcd for C₄₅H₇₃NO₁₃Cl 870.4770；found:870.5009。

examples14: 3-O-alpha-L-pyranorhamnose- (1 → 2) -alpha-L-arabinopyranosylivy saponin-N-valeric acid- 28-carboxamide (Compound 36)

Compound 3(291mg,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent and excess oxalyl chloride were removed under reduced pressure to give a yellow solid, compound 34. The resulting compound 34 was dissolved in anhydrous dichloromethane (10mL), and ethyl aminopentanoate hydrochloride (100mg, 0.554mmol) and triethylamine (84.4mg,0.831mmol) were added under cooling on an ice bath to react at room temperature for 12 hours. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, most of the organic solvent was removed under reduced pressure, adjusted to pH 2 with 10% HCl, and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol 10:1) afforded 216mg of a white solid, compound 36, in about 86% yield.

The melting point of the obtained solid is 185-186 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(600MHz,pyridine-d₅,δ):7.38(t,J＝5.5Hz,1H，CONH),6.27(s,1H,H-1”),5.44(s,1H,H-12),4.75(s,1H,H-1’),4.71(dt,J＝15.4,6.2Hz,1H),4.66(dd,J＝9.3,3.2Hz,1H),4.57–4.61(m,1H),4.24–4.34(m,2H),4.12(dd,J＝7.8,3.3Hz,1H),3.77(d,J＝10.7Hz,1H),3.71(d,J＝11.1Hz,1H),3.48–3.56(m,1H),3.37(dt,J＝19.1,9.4Hz,1H),3.07(dd,J＝13.0,3.5Hz,1H),2.35(t,J＝7.2Hz,2H),2.23(dd,J＝13.0,3.2Hz,1H),1.65(d,J＝6.1Hz,3H),1.21(s,3H),1.10(s,3H),1.02(s,3H),0.99(s,3H),0.94(s,3H),0.90(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):177.89(COOH),174.10(C-28),145.22(C-13),123.21(C-12),104.75(C-1’),102.00(C-1”),81.36(C-3),76.12,75.11,74.47,72.91,72.73,69.98,69.73,66.08,64.30,51.65,48.45,48.02,47.11,46.80,43.87,42.52,42.22,40.15(CH₂),39.75,39.34,37.22,34.73,34.17,34.09(CH₂),33.54,33.06,31.23,29.92(CH₂),28.28,26.57,26.47,24.20,24.09(CH₂),24.02,23.17,18.91,18.48,17.88,16.46,14.37；

HRMS-ESI(m/z)[M+Na]⁺:calcd for C₄₆H₇₅NO₁₃Na 872.5136；found:872.4962。

example 15: 3-O-alpha-L-pyranorhamnose- (1 → 2) -alpha-L-arabinopyranosylivy saponin-N-hexanoic acid- 28-carboxamide (Compound 37)

Compound 3(291mg,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent and excess oxalyl chloride were removed under reduced pressure to give a yellow solid, compound 34. The resulting compound 34 was dissolved in anhydrous dichloromethane (10mL), and ethyl aminocaproate hydrochloride (107mg, 0.554mmol) and triethylamine (84.4mg,0.831mmol) were added under cooling on ice and reacted at room temperature for 12 hours. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, most of the organic solvent was removed under reduced pressure, adjusted to pH 2 with 10% HCl, and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol 10:1) afforded 219mg of a white solid, compound 37, in about 86% yield。

The melting point of the obtained solid was 188-189 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(400MHz,pyridine-d₅,δ):7.32(t,J＝4.0Hz,1H，CONH),6.30(s,1H,H-1″),5.46(s,1H,H-12),5.13(d,J＝6.4Hz,1H,H-1′),4.70-4.74(m,1H),4.67(dd,J＝9.4,3.3Hz,1H),4.61(t,J＝7.1Hz,1H),4.24-4.38(m,2H),4.19(d,J＝3.9Hz,2H),4.12(dd,J＝7.9,3.4Hz,1H),3.74(dd,J＝19.4,10.7Hz,2H),3.49-3.59(m,1H),3.42-3.31(m,1H),3.09(dd,J＝13.7,2.9Hz,1H),2.51(t,J＝7.3Hz,2H),1.66(d,J＝6.0Hz,3H),1.21(s,3H),1.10(s,3H),1.02(d,J＝1.5Hz,6H),0.94(s,3H),0.90(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):177.80(COOH),176.19(C-28),145.31(C-13),123.19(C-12),104.76(C-1′),102.02(C-1″),81.37(C-3),76.12,75.14,74.49,72.91,72.75,70.05,69.74,66.10,64.30,50.02,48.47,48.04,47.14,46.77,43.88,42.54,42.26,40.17,40.13(CH₂),39.34,37.23,35.15(CH₂),34.76,34.14,33.55,33.08,31.23,30.24(CH₂),28.29,27.56(CH₂),26.58,26.47,25.67(CH₂),24.25,24.10,18.92,18.49,17.92,16.48,14.38；

HRMS-ESI(m/z)[M+K]⁺:calcdfor C₄₇H₇₇KNO₁₃902.5032；found:902.4871。

example 16: 3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranosylivy saponin-N-acetyl Glycine-28-carboxamide (Compound 38)

Compound 3(291mg,0.295mmol) was dissolved in anhydrous dichloromethane (15mL), oxalyl chloride (0.1mL,1.17mmol) was added with ice-cooling, and the mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent and excess oxalyl chloride were removed under reduced pressure to give a yellow solid, compound 34. The resulting compound 34 was dissolved in anhydrous dichloromethane (10mL), and amino acid ethyl ester hydrochloride (109 m) was added under ice-coolingg, 0.554mmol), triethylamine (84.4mg,0.831mmol), at room temperature for 12 h. After the reaction, 50mL of dichloromethane was added, and the mixture was washed with saturated brine (50 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Dissolving the white solid in the mixed solvent (CH)₃OH/THF/H₂O2: 1:1(v/v/v), 20mL), potassium hydroxide (187mg, 3.324mmol) was added, and the mixture was stirred at room temperature for 12 h. After completion of the reaction, most of the organic solvent was removed under reduced pressure, adjusted to pH 2 with 10% HCl, and extracted with n-butanol (20 mL. times.3). Dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a white solid. Column chromatography (dichloromethane: methanol ═ 10:1) afforded 211mg of a white solid, compound 38, in about 83% yield.

The melting point of the obtained solid was 290 ℃ and 291 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(600MHz,pyridine-d₅,δ):6.24(s,1H,H-1”),5.42(s,1H,H-12),5.12(d,J＝6.2Hz,1H,H-1’),4.74(d,J＝2.1Hz,1H),4.69(td,J＝12.4,6.2Hz,1H),4.64(dd,J＝9.3,3.4Hz,1H),4.53–4.61(m,1H),4.23–4.32(m,3H),4.17(dd,J＝14.6,6.6Hz,2H),4.10(tdd,J＝10.9,7.0,3.8Hz,2H,NHCH₂CO),3.73(dd,J＝30.6,10.4Hz,2H,CH₂COOH),3.54–3.66(m,1H),3.45(dt,J＝12.7,6.6Hz,1H),3.08(dd,J＝13.0,4.1Hz,1H),2.50(t,J＝7.4Hz,1H),2.25–2.18(m,1H),1.64(s,3H),1.20(s,3H),1.09(s,3H),1.01(s,3H),0.99(s,3H),0.93(s,3H),0.90(s,3H)；

¹³C NMR(150MHz,pyridine-d₅,δ):178.07(COOH),174.03(CONH),173.68(CONHCONH),145.13(C-13),123.17(C-12),104.69(C-1’),101.95(C-1”),81.33(C-3),76.10,75.01,74.41,72.86,72.68,70.02,69.66,65.99,64.55,64.27,51.70,48.41,47.98,47.05,46.76,43.84,42.48,42.12,40.12,39.71,39.30,37.18,34.69,34.17,33.51,33.04,31.24,28.25,26.45,24.17,24.08,23.95,19.68,18.87,17.91,16.45,14.31；

HRMS-ESI(m/z)[M+Cl]⁺:calcd for C₄₅H₇₂NO₁₄Cl 899.4672；found:899.4972。

example 17: 3-O-alpha-L-rhamnopyranose- (1 → 2) -alpha-L-arabinopyranoseHederagenin-2-bromo- 28-Carboxylic acid ethyl ester (Compound 42)

Compound 1(400mg, 0.53mmol) and potassium carbonate (220mg, 1.60mmol) were placed in a reaction flask, N-dimethylformamide (15ml) was added, and dibromoethane (120mg, 0.64mmol) was added dropwise and stirred at room temperature for 6 hours. After completion of the reaction, the reaction mixture was diluted with distilled water (100ml), n-butanol (30 ml. times.3) was extracted, the organic layers were combined, the organic layer was washed with saturated brine (30 ml. times.3), dried over anhydrous sodium sulfate, filtered, and n-butanol was removed under reduced pressure to obtain a white waxy solid, compound 39. The resulting compound 39 was placed in a reaction flask, and silver nitrate (308mg, 1.81mmol) was carefully taken out and added to the reaction flask in the dark, acetonitrile (25ml) was added, stirred at room temperature for ten minutes, and then heated to 60 ℃ and refluxed for 4 hours. After the reaction was terminated, the solid was removed by suction filtration, the solvent was removed under reduced pressure, and silica gel column chromatography (dichloromethane: methanol 15:1) was performed to obtain 364mg of a white waxy solid, that is, compound 42. The yield in two steps was 86%.

The melting point of the obtained solid is 153-154 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(400MHz,pyridine-d₅,δ):6.28(s,1H,H-1”),5.37(t,J＝3.2Hz,1H,H-12),5.11(d,J＝6.3Hz,1H,H-1’),4.89(dd,J＝6.2,2.6Hz,2H,CH₂ONO₂),4.75(d,J＝4.3Hz,1H),4.68–4.74(m,1H),4.66(dd,J＝9.3,3.4Hz,1H),4.56(ddd,J＝9.2,6.3,5.2Hz,4H),4.22–4.38(m,3H),4.04–4.21(m,4H),3.73(dd,J＝24.2,11.3Hz,2H,COOCH₂),3.05(dd,J＝15.1,2.0Hz,1H),2.21(dd,J＝12.6,3.0Hz,1H),1.64(d,J＝6.2Hz,3H),1.15(s,3H),1.08(s,3H),0.97(s,3H),0.91(s,3H),0.88(s,3H),0.86(s,3H)；

¹³C NMR(100MHz,pyridine-d₅,δ):177.61(C-28),143.98(C-13),123.32(C-12),104.75(C-1’),101.98(C-1”),81.23(C-3),76.08,75.09,74.43(CH₂ONO₂),72.83,72.66,72.05,69.94,69.72,66.10,64.19,61.04(COOCH₂),48.28,47.92,47.30,46.13,43.78,42.20,41.98,39.95,39.23,37.10,34.09,33.61,33.32,32.96,31.02,28.25,26.50,26.30,24.06,23.87,23.52,18.86,18.38,17.49,16.35,14.30；

HRMS-ESI(m/z)[M+H]⁺:calcdfor C₄₃H₇₀NO₁₅840.4745；found:840.4564。

example 18: 3-O-alpha-L-pyranorhamnose- (1 → 2) -alpha-L-arabinopyranosylivy saponin-4-bromine- 28-Carboxylic acid butyl ester (Compound 43)

Compound 1(400mg, 0.53mmol) and potassium carbonate (220mg, 1.60mmol) were placed in a reaction flask, N-dimethylformamide (15ml) was added, and dibromobutane (138mg, 0.64mmol) was added dropwise and stirred at room temperature for 6 hours. After the reaction, the mixture was diluted with distilled water (100ml), n-butanol (30 ml. times.3) was extracted, the organic layers were combined, the organic layer was washed with saturated brine (30 ml. times.3), dried over anhydrous sodium sulfate, filtered, and n-butanol was removed under reduced pressure to obtain a white waxy solid, i.e., compound 40. The resulting compound 40 was placed in a reaction flask, and silver nitrate (308mg, 1.81mmol) was carefully taken and added to the reaction flask in the dark, acetonitrile (25ml) was added, stirred at room temperature for ten minutes, and then heated to 60 ℃ and refluxed for 4 hours. After the reaction was terminated, the solid was removed by suction filtration, the solvent was removed under reduced pressure, and silica gel column chromatography (dichloromethane: methanol 15:1) was performed to obtain 372mg of a white waxy solid, that is, compound 43. The yield of the two steps is 81%.

The melting point of the obtained solid is 141-142 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(400MHz,pyridine-d₅,δ):6.24(s,1H,H-1”),5.35(s,1H,H-12),5.07(d,J＝6.3Hz,1H,H-1’),4.72(d,J＝1.9Hz,1H),4.67(dd,J＝9.4,6.2Hz,1H),4.62(dd,J＝9.3,3.3Hz,1H),4.54(dt,J＝10.3,7.1Hz,2H,CH₂ONO₂),4.26(dd,J＝20.9,11.3Hz,3H),4.17(t,J＝5.9Hz,2H,COOCH₂),4.07–4.14(m,2H),3.70(dd,J＝23.6,10.5Hz,2H),3.04(dd,J＝12.9,3.0Hz,1H),2.17(dd,J＝12.8,3.2Hz,1H),1.60(d,J＝6.2Hz,3H),1.15(s,3H),1.05(s,3H),0.94(s,3H),0.91(s,3H),0.87(s,3H),0.85(s,3H)；

¹³C NMR(100MHz,pyridine-d₅,δ):177.51(C-28),144.25(C-13),123.02(C-12),104.55(C-1’),101.71(C-1”),81.00(C-3),75.82,74.88,74.21,73.71(CH₂ONO₂),72.67,72.45,69.71,69.53,65.91,63.98,63.78(COOCH₂),49.83,48.09,47.72,47.02,46.07,43.59,42.09,41.88,39.81,39.06,36.92,34.01,33.20,32.98,32.86,30.88,30.04(CH₂),28.10,26.29,26.14,25.40(CH₂),23.97,23.74,23.42,18.68,18.19,17.50,16.16,14.15；

HRMS-ESI(m/z)[M+H]⁺:calcdfor C₄₅H₇₄NO₁₅868.5058；found:868.4859。

example 19: 3-O-alpha-L-pyranorhamnose- (1 → 2) -alpha-L-arabinopyranosylivy saponin-6-bromo- 28-hexyl formate (Compound 44)

Compound 1(400mg, 0.53mmol) and potassium carbonate (220mg, 1.60mmol) were placed in a reaction flask, N-dimethylformamide (15ml) was added, and dibromohexane (156mg, 0.64mmol) was added dropwise and stirred at room temperature for 6 hours. After the reaction, the mixture was diluted with distilled water (100ml), n-butanol (30 ml. times.3) was extracted, the organic layers were combined, the organic layer was washed with saturated brine (30 ml. times.3), dried over anhydrous sodium sulfate, filtered, and n-butanol was removed under reduced pressure to obtain a white waxy solid, Compound 41. The resulting compound 41 was placed in a reaction flask, and silver nitrate (308mg, 1.81mmol) was carefully taken out and added to the reaction flask in the dark, acetonitrile (25ml) was added, stirred at room temperature for ten minutes, and then heated to 60 ℃ and refluxed for 4 hours. After the reaction was terminated, the solid was removed by suction filtration, the solvent was removed under reduced pressure, and silica gel column chromatography (dichloromethane: methanol 15:1) was performed to obtain 370mg of a white waxy solid, that is, compound 44. The yield in two steps is 78%.

The melting point of the obtained solid was 126-127 ℃. The solid nuclear magnetic data obtained were as follows:

¹H NMR(400MHz,pyridine-d₅,δ):6.27(s,1H,H-1”),5.39(s,1H,H-12),5.10(d,J＝6.3Hz,1H,H-1’),4.74(dd,J＝3.2,1.4Hz,1H),4.70(dd,J＝9.4,6.2Hz,1H),4.65(dd,J＝9.3,3.4Hz,1H),4.54–4.61(m,1H),4.43(t,J＝6.6Hz,2H,CH₂ONO₂),4.28(dt,J＝12.0,6.7Hz,3H),4.15–4.21(m,4H,COOCH₂),4.12(dd,J＝7.8,3.3Hz,1H),3.73(dd,J＝26.2,10.4Hz,2H),3.10(dd,J＝14.1,1.3Hz,1H),2.21(dd,J＝13.0,3.5Hz,1H),1.63(d,J＝6.2Hz,3H),1.18(s,3H),1.08(s,3H),0.98(s,3H),0.95(s,3H),0.90(d,J＝1.8Hz,6H)；

¹³C NMR(100MHz,pyridine-d₅,δ):177.74(C-28),144.45(C-13),123.14(C-12),81.20(C-3),76.02,75.06,74.40,74.18(CH2ONO2),72.83,72.63,69.91,69.69,66.07,64.47,64.18(COOCH₂),48.28,47.92,47.16,46.26,43.76,42.26,42.04,40.00,39.12,37.10,34.21,33.36,33.21,33.04,31.07,28.99(CH₂),28.30,27.03(CH₂),26.49(CH₂),26.31,26.14,25.72(CH₂),24.06,23.90,23.61,18.84,18.35,17.70,16.33,14.29；

HRMS-ESI(m/z)[M+H]⁺:calcdfor C₄₇H₇₈NO₁₅896.5371；found:896.5157。

example 20: cytotoxicity assay

Purpose of the experiment:

the in vitro anti-tumor activity of the tested compound is screened by culturing human lung cancer cell A549, human liver cancer cell SMMC-7721 and human gastric cancer cell BGC-823 in vitro, and detecting the cell proliferation activity by adopting an MTT method.

Experimental materials:

1. test cell lines: human lung cancer cell A549, human liver cancer cell SMMC-7721 and human gastric cancer cell BGC-823 cell strain, which are purchased from cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences.

2. Thiazole blue MTT, purchased from shanghai nationality medicinal group.

3. The tested drugs are: the invention relates to a saponin derivative.

4. Positive control: alpha-hederin (PSA) in the number, pulsatilla saponin D (PSD in the number) and cisplatin (cis-DDP).

The experimental method comprises the following steps:

1. test drug formulation each test drug concentration setting: 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M; dissolved in a small amount of DMSO (within 0.5%), prepared in the corresponding medium, and filtered at 0.22. mu.M for use.

2. The cell proliferation inhibition experiment takes cells in logarithmic growth phase, adjusts the density to be 8 multiplied by 104, adds 100 mu L of culture medium containing medicine with different concentrations, adds 100 mu L of culture medium into each hole of a control group, arranges 3 multiple holes for the control group and the medicine group, arranges a blank control group, only adds culture medium without adding cells as the control group, and adjusts zero. Placing 96-well plate in 5% CO₂Culturing in an incubator (37 ℃) for 48 hours, adding 20 mu L of MTT with the concentration of 5mg/ml, continuously culturing for 4 hours, removing supernatant, adding 150 mu L of DMSO, shaking and dissolving for 10min, and measuring the OD value at 490nm by using an enzyme-linked immunosorbent assay.

3. Observation index

Calculating the inhibition rate according to a formula, and calculating the IC of three parallel tests₅₀See table 1 for average values and results.

The inhibition ratio (%) × 100% (1-mean OD value in drug administration group/mean OD value in control group).

Table 1: in vitro anti-tumor activity and hemolytic toxicity data of PSA/PSD derivatives

Example 21: hemolysis test

21.1 blood cell suspension preparation

1 healthy rabbit was taken, about 10mL of blood was taken from the marginal ear vein, placed in a triangular flask containing glass beads in advance, shaken for 10min, and stirred to remove fibrin, so that defibrinated blood was obtained. Then moving the blood into a graduated centrifuge tube, adding about 10 times of 0.9% sodium chloride solution, uniformly mixing, centrifuging for 15min (1000-.

21.2 test methods

The number of the clean test tube 7 is taken for numbering, the tubes No. 1 to No. 6 are test drug tubes, the final concentration is 100 mu g/m, 50 mu g/m, 25 mu g/m, 12.5 mu g/m, 6.25 mu g/m and 3.125 mu g/mL in sequence, the tube No. 7 is a negative control tube, and the tube No. 8 is a positive control tube. Adding 2% erythrocyte suspension, 0.9% sodium chloride solution or distilled water, and tested medicine in sequence, mixing, immediately placing in 37 + -0.5 deg.C water bath for incubation, observing for 1 time every 15 minutes, and observing for 1 time every 1 hour after 1 hour for 3 hours. According to the principle that heme released by red blood cells has maximum absorption in a visible wavelength range, the hemolytic degree of each tested medicine is determined by a spectrophotometric method. After incubation for 3h, the test drug solution in each tube was centrifuged, the supernatant was collected, and the OD value of each tube was read in a microplate reader at 545nm in the absence of distilled water.

21.3 determination of results: the hemolysis ratio (%) of each test tube was calculated by the following formula.

Hemolysis ratio (%) (ODt-ODnc)/(ODpc-ODnc)

Wherein: ODt is the test drug tube absorbance; ODnc is the negative control tube absorbance; ODpc is the positive control tube absorbance.

The inhibition rate was calculated according to the equation and HD50 was calculated using software, the results of which are shown in table 1.

It can be seen from the above examples 20 and 21 that the saponin derivative of the present invention has stronger anti-tumor activity but greatly reduced hemolytic toxicity compared to α -hederin, and is suitable for treating or adjunctively treating tumors.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A saponin derivative, which is any one of the following compounds:

2. a pharmaceutical composition for antitumor comprising the saponin derivative according to claim 1.

3. Use of a saponin derivative according to claim 1 in the preparation of an anti-tumor pharmaceutical composition.