CN102070699B - Trihydroxy-substituted pentacyclic triterpene compounds and preparation method and application thereof - Google Patents

Abstract

The invention discloses a group of trihydroxy-substituted pentacyclic triterpene compounds and a preparation method and application thereof. The general formula of the compounds is shown in the structural formula I, wherein R1 and R2 are selected from hydrogen or methyl, R3 is selected from any one of -COOH, -CH2OH, -CHO, -COOR1, -CONH2, -CONHR1 and -CONR1R2, R1 and R2 are selected from one of alkyl containing 1-8 carbon atoms, substituted or unsubstituted phenyl and substituted or unsubstituted alkylphenyl, R2 and R1 are identical or different, the hydroxide radical substitution configurations of C1, C2 and C3 are alpha-configurations or beta-configurations, and the compound 1 alpha,2 alpha-dyhydroxy oleanolic acid and 1 alpha,2 alpha-dihydroxy ursolic acid are excluded. The invention provides a group of compounds with novel structures and a synthesis method thereof. The compounds and the salts thereof have obvious protection effects of liver injury, and the synthesis method can be used for preparing liver-protecting medicines; and the synthesis method has mild reaction conditions, no special apparatuses are needed, the operation is simple and practical and the synthesis method is easy to enlarge scale and achieve industrial production.

Description

Trihydroxy-replaces pentacyclic triterpenoid and its preparation method and application

Technical field

The present invention relates to 1,2,3-trihydroxy-and replace pentacyclic triterpenoid and its preparation method and application, belong to pharmaceutical chemistry technical field.

Background technology

Hepatocellular injury is the common pathologic basis of different types of liver disease, comprising virus, alcohol and the caused liver injury of chemical substance three large factor.Liver injury disease has become common disease, frequently-occurring disease, and sickness rate is more and more higher.China is the hotspot of hepatopathy, therefore studies hepatic significant.

Current, although China clinically widespread use glycyrrhizin, Biphenylylmethylcarbinol, silybum mariamum and herba swertiae contained liver-benefiting and Radix Astragali injection etc. protect the liver, anti-liver injury Effective Component of Chinese Medicine or compound, but through (the Liu Ping of the autonomous invention of such medicine Jun Bushi of international endorsement China, the connection between theory and practice, basis, in conjunction with clinical, promotes the development of combination of Chinese tradiational and Western medicine hepatopathy subject, and press in conjunction with journal in Chinese and Western, 2003,1 (2): 81-83).Therefore, how from traditional Chinese medicine, excavate there is independent intellectual property right protect the liver class medicine, be the target that we make great efforts always.

The pentacyclic triterpene compound oleanolic acid being prevalent in plant has prolection (Liu J.Pharmacology of oleanolic acid and ursolic acid.J Ethnopharmacol1995 to the liver injury of tetrachloro-methane induction; 49:57-68.); Ursolic acid is to ethanol, thioacetamide, galn and CCL ₄the mouse liver injury of induction has provide protection (Liu J.Pharmacology of oleanolic acid and ursolic acid.JEthnopharmacol 1995; 49:57-68; Jeong HG.Inhibition of cytochrome P450 2E1expression by oleanolic acid:Hepatoprotective effects against carbontetrachloride-induced hepatic injury.Toxicol Lett 1999; 105:215-222; ShuklaB, Visen PKS, PatnaikGK, Tripathi SC, SrimalRC, DayalR, DobhalPC.Hepatoprotective activity in rat of ursolic acid isolated from Eucalyptushybrid.Phytother Res 1992; 6 (2): 74-79.).The inventor is carrying out structural derivative and bioactivity screening research (Bai Yujun, Yang your pupil, the Kang Wenyi etc. of ursolic acid and Oleanolic Acid, structural modification thing and the anti-tumor activity thereof of ursolic acid, West China pharmaceutical journal, 2003,18 (2): 87～90; Chen Lei, Yang your pupil, Yang Juan etc., synthetic and the inhibition activity to alpha-glucosidase of pentacyclic triterpene derivative, China Medicine University's journal, 2010,41 (3): 222-225) time, find, the A ring polyoxy of pentacyclic triterpenoid particularly 1,2,3-trihydroxy-substituent has stronger liver-protecting activity.

Therefore, the present invention carries out structure of modification to ursolic acid and Oleanolic Acid compounds, to finding that the A of high liver-protecting activity encircles the derivative that different trihydroxyies replace configuration.

Summary of the invention

The object of the invention is to: provide the trihydroxy-with liver-protecting activity to replace pentacyclic triterpenoid and its preparation method and application.The present invention has carried out CCl by the series compound that ursolic acid and Oleanolic Acid compounds are carried out after structure of modification ₄due to the prolection screening of hepatocellular injury, find that this compounds has significant protective effect to human liver cell, has the purposes of preparing hepatic.

Technical scheme of the present invention: 1,2,3-trihydroxy-replaces pentacyclic triterpenoid, and its general formula is following structural formula I:

Wherein: R ₁represent hydrogen or methyl; R ₂represent hydrogen or methyl; R ₃be selected from-COOH ,-CH ₂oH, CHO ,-COOR ¹,-CONH ₂,-CONHR ¹,-CONR ¹r ²in any; R ¹be selected from the one in alkyl, replacement or unsubstituted phenyl, replacement or the unsubstituted benzene alkyl that contains 1-8 carbon atom, R ²be selected from the one in alkyl, replacement or unsubstituted phenyl, replacement or the unsubstituted benzene alkyl that contains 1-8 carbon atom, R ²with R ¹can be identical or different; C ₁hydroxyl replace and be configured as α configuration or beta comfiguration, C ₂hydroxyl replace and be configured as α configuration or beta comfiguration, C ₃hydroxyl replace and be configured as α configuration or beta comfiguration; And compound 1 α, 2 alpha-dyhydroxyl oleanolic acids and 1 α, except 2 alpha-dihydroxy-ursolic acid.

Aforesaid compound is oleanane type, i.e. R ₁for hydrogen, R ₂for methyl, R ₃for-COOH; Or be black bearberry alkane type, i.e. R ₁for methyl, R ₂for hydrogen, R ₃for-COOH.

Aforesaid compound is preferably following 14 kinds: (1) 1 α, 2 α, 3 α-trihydroxy-olea-12-alkene-28-acid; (2) 1 α, 2 β, 3 α-trihydroxy-olea-12-alkene-28-acid; (3) 1 β, 2 α, the alkene-28-acid of 3 α-trihydroxy-olea-12; (4) 1 β, 2 beta-dihydroxyl Oleanolic Acids; (5) 1 α, 2 beta-dihydroxyl Oleanolic Acids; (6) 1 β, 2 alpha-dyhydroxyl oleanolic acids; (7) 1 β, 2 β, 3 α-trihydroxy-olea-12-alkene-28-acid; (8) 1 α, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid; (9) 1 α, 2 β, 3 α-trihydroxy-black bearberry-12-alkene-28-acid; (10) 1 β, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid; (11) 1 β, 2 beta-dihydroxyl ursolic acid; (12) 1 α, 2 beta-dihydroxyl ursolic acid; (13) 1 β, 2 alpha-dihydroxy-ursolic acid; (14) 1 β, 2 β, 3 α-trihydroxy-black bearberry-12-alkene-28-acid; Its structural formula is respectively:

Aforesaid compound is more preferably: (1) 1 α, 2 α, 3 α-trihydroxy-olea-12-alkene-28-acid (being called for short A-5); (2) 1 α, 2 β, 3 α-trihydroxy-olea-12-alkene-28-acid (being called for short A-6); (8) 1 α, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid (being called for short U-17); (9) 1 α, 2 β, 3 α-trihydroxy-black bearberry-12-alkene-28-acid (being called for short U-18).

Aforesaid 1,2,3-trihydroxy-replaces one of preparation method of pentacyclic triterpenoid: get ursolic acid U-1, through Jones reagent, (26.72 grams of chromium trioxides are dissolved in 23 milliliters of vitriol oils, then with water, be diluted to the aqueous solution of 100 milliliters of gained) be oxidized to obtain compound U-2, U-2 obtains compound U-3 through benzyl bromine (being cylite) benzyl, and U-3 is through SeO ₂be oxidized to obtain α, beta unsaturated ketone Se-1, Se-1 obtains U-15 under the effect of hydrogen peroxide, and sodium borohydride reduction U-15 obtains R-10-2 intermediate, through perchloric acid hydrolysis, obtain FU-16 again, after palladium carbon catalytic hydrogenation, obtain different isomerization body product (8)～(14); Its reaction scheme is as follows:

Preparation method one detailed process is: 1. get the ursolic acid of 1 molar part, with acetone solution, be placed in ice-water bath and stir 15min, slowly drip the Jones reagent of 1.14 molar part, at N ₂the lower stirring reaction 5h (TLC detection reaction process) of protection, with NaOH aqueous solution adjusting pH, to neutral, pressure reducing and steaming solvent, is extracted with ethyl acetate, and gained resistates obtains U-2 through purification by silica gel column chromatography; 2. the U-2 that gets 1 molar part, adds acetone solution, then adds the K of 10 molar part ₂cO ₃, under room temperature, stir 30min, then add the cylite of 1.17 molar part, at N ₂the lower reaction 18h (TLC detection reaction process) of protection, acetone is removed in decompression, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, removal of solvent under reduced pressure, residue obtains U-3 through silica gel column chromatography; 3. get the U-3 of 1 molar part and the SeO of 1.5 molar part ₂, add diacetyl oxide, at N ₂the lower back flow reaction 5h (TLC detection reaction process) of protection, by 2N aqueous sodium hydroxide solution termination reaction, is extracted with ethyl acetate saturated sodium-chloride water solution washing for organic layer, anhydrous MgSO after adding suitable quantity of water ₄dry, removal of solvent under reduced pressure, resistates obtains Se-1 through silica gel column chromatography; 4. get 1 molar part Se-1, add dissolve with methanol, then add the 10%NaOH aqueous solution (containing NaOH1.5 molar part), stir after 30min, add 30%H ₂o ₂(10 molar part), at N ₂the lower back flow reaction 3h (TLC detection reaction process) of protection, to neutral, reclaims methyl alcohol with aqueous hydrochloric acid adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains U-15 through silica gel column chromatography; 5. get 1 molar part U-15, add dissolve with methanol, at 0～5 ℃, stir after 15min, divide and add 10 molar part NaBH for 2～3 times ₄, at N ₂protection lower reaction 2h (TLC detection reaction process), to neutral, reclaims methyl alcohol with aqueous hydrochloric acid adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains R-10-2 through silica gel column chromatography; 6. get 1 molar part R-10-2, add acetone solution, add 1.5 molar part perchloric acid, then add two of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains FU-16 through silica gel column chromatography; 7. get FU-16 and be dissolved in anhydrous methanol, add 0.1 times of palladium carbon to FU-16 weight as catalyzer, hydro-reduction under room temperature, obtains U-18 and isomer products thereof [compound (8)～(14)].

Aforesaid 1,2,3-trihydroxy-replace pentacyclic triterpenoid preparation method two: get ursolic acid U-1, through Jones reagent oxidation, obtain compound U-2, U-2 is through SeO ₂be oxidized to obtain α, beta unsaturated ketone Se, Se obtains U-15-1 under the effect of hydrogen peroxide, and sodium borohydride reduction U-15-1 obtains R-10-21 intermediate, then obtains different isomerization body product (8)～(14) through perchloric acid hydrolysis; Its reaction scheme is as follows:

Preparation method two detailed process is: 1. get the ursolic acid of 1 molar part, with acetone solution, be placed in ice-water bath and stir 15min, slowly drip the Jones reagent of 1.14 molar part, at N ₂the lower stirring reaction 5h (TLC detection reaction process) of protection, with NaOH aqueous solution adjusting pH, to neutral, pressure reducing and steaming solvent, is extracted with ethyl acetate, and gained resistates obtains U-2 through purification by silica gel column chromatography; 2. get the U-2 of 1 molar part and the SeO of 2 molar part ₂, adding diacetyl oxide, back flow reaction 5h under nitrogen protection (TLC detection reaction process), by 2N aqueous sodium hydroxide solution termination reaction, is extracted with ethyl acetate the saturated NaHCO of organic layer after adding suitable quantity of water ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains Se through silica gel column chromatography; 3. the Se that gets 1 molar part, adds dissolve with methanol, then adds the 10%NaOH aqueous solution (containing NaOH1.5 molar part), stirs after 30min, adds 30%H ₂o ₂(5 molar part), at N ₂the lower back flow reaction 3h (TLC detection reaction process) of protection, to neutral, reclaims methyl alcohol with aqueous hydrochloric acid adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains U-15-1 through silica gel column chromatography; 4. the U-15-1 that gets 1 molar part, adds dissolve with methanol, at 0～5 ℃, stirs after 15min, divides the NaBH that adds 5 molar part for 2～3 times ₄, at N ₂protection lower reaction 2h (TLC detection reaction process), to neutral, reclaims methyl alcohol with aqueous hydrochloric acid adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains R-10-21 through silica gel column chromatography; 5. the R-10-21 that gets 1 molar part, adds acetone solution, adds the perchloric acid of 1.5 molar part, then adds one of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains U-18 and isomer products [compound (8)～(14)] thereof through silica gel column chromatography; The same amount of the obtaining sample U-17 few, that take on a red color under sulfuric acid developer that separates, by EI-MS and ¹h-NMR determines that its structure is 1 α, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid.

Aforesaid 1, 2, the preparation method's of 3-trihydroxy-replacement pentacyclic triterpenoid three: get ursolic acid or its methyl esters or its benzyl ester U-a and obtain U-b under Methanesulfonyl chloride effect, U-b is through Quilonum Retard processing, obtain intermediate U-c, U-c is oxidized to obtain U-d by tin anhydride, after the NaOH aqueous solution is processed, decarboxylation obtains U-e again, intermediate U-e obtains epoxidation intermediate U-f under metachloroperbenzoic acid effect, U-f obtains 1 after perchloric acid hydrolysis, 2, 3-trihydroxy-intermediate U-g, after adopting palladium carbon catalytic hydrogenation or demethylating reaction, obtain 1 of different isomerization body, 2, 3-trihydroxy-black bearberry-12-alkene-28 acid product, be compound (8)～(14), its reaction scheme is as follows:

Preparation method three detailed process is: 1. get the ursolic acid of 1 molar part or its methyl esters or benzyl ester (U-a) and be dissolved in pyridine solvent, under stirring, slowly drip the Methanesulfonyl chloride of 1.2 molar part, at N in ice-water bath ₂the lower stirring reaction 5h (TLC detection reaction process) of protection, is extracted with ethyl acetate, and the aqueous hydrochloric acid of 2N washes away pyridine, and gained resistates obtains U-b through purification by silica gel column chromatography; 2. get the U-b of 1 molar part, with N, N '-dimethyl formamide dissolves, add Quilonum Retard 2 molar part, reflux 1 hour (TLC detection reaction process), is cooled to room temperature, filtering Quilonum Retard, add after suitable quantity of water, be extracted with ethyl acetate, organic phase is washed till neutrality, decompression and solvent recovery, through purification by silica gel column chromatography (sherwood oil: ethyl acetate is eluent), obtain intermediate U-c; 3. get U-c and the 2 molar part tin anhydride (SeO of 1 molar part ₂), add acetic acid to dissolve, reflux 5 hours (TLC detection reaction process), be cooled to room temperature, filtering tin anhydride, saturated sodium bicarbonate is neutralized to neutrality, add after suitable quantity of water, be extracted with ethyl acetate, decompression and solvent recovery, residue, through purification by silica gel column chromatography (sherwood oil: ethyl acetate is eluent), obtains intermediate U-d; 4. the U-d that gets 1 molar part, adds dissolve with methanol, then adds 10%NaOH (2 molar part) aqueous solution, reflux 1 hour, with 2N HCl tune pH value, to neutral, ethyl acetate extracts, after reclaiming solvent, gained residue, through column chromatography for separation, obtains white powder U-e; 5. the U-e of 1 molar part is dissolved in methylene dichloride, adds 1.2 molar part metachloroperbenzoic acids, under room temperature, at N ₂the lower reaction of protection is spent the night (TLC detection reaction process), and with chloroform extraction, after recovery solvent, residue carries out silica gel column chromatography separating purification, obtains white powder U-f; 6. the U-f that gets 1 molar part, adds acetone solution, adds the perchloric acid of 1.5 molar part, then adds one of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains U-g through silica gel column chromatography; 7. get 1 molar part U-g (R is methyl), add dimethyl formamide to dissolve, then add 1.2 molar part lithium iodides, reflux 5 hours, be cooled to after room temperature, add suitable quantity of water, be extracted with ethyl acetate, after recovery solvent, residue is through silica gel column chromatography separating purification, obtain 1 of corresponding different isomerization body, 2,3-trihydroxy-black bearberry-12-alkene-28 acid product, i.e. compound (8)～(14); If the R in U-g is benzyl, adopt palladium carbon catalytic hydrogenation, can obtain equally 1,2 of different isomerization body, 3-trihydroxy-black bearberry-12-alkene-28 acid product, i.e. compound (8)～(14).

Aforesaid 1,2,3-trihydroxy-replace pentacyclic triterpenoid preparation method four: get Oleanolic Acid, through Jones reagent oxidation, become A-1, by SeO ₂be oxidized to obtain α, beta unsaturated ketone A-2, A-2 obtains A-3 under the effect of hydrogen peroxide, and sodium borohydride reduction A-3 obtains A-4 intermediate, through perchloric acid hydrolysis, obtains different isomerization body product (1)～(7); Its reaction scheme is as follows:

Preparation method four detailed process is: the Oleanolic Acid of 1. getting 1 molar part; with acetone solution; be placed in ice-water bath; after stirring 15min, slowly drip the Jones reagent of 1.14 molar part, stirring reaction 5h under nitrogen protection (TLC detection reaction process), with the adjusting of the NaOH aqueous solution, pH is extremely neutral; pressure reducing and steaming acetone; resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains A-1 through silica gel column chromatography (sherwood oil: ethyl acetate); 2. get the A-1 of 1 molar part and the SeO of 2 molar part ₂, adding diacetyl oxide, back flow reaction 5h under nitrogen protection (TLC detection reaction process), by 2N aqueous sodium hydroxide solution termination reaction, is extracted with ethyl acetate the saturated NaHCO of organic layer after adding suitable quantity of water ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, the resistates after concentrating under reduced pressure obtains A-2 through silica gel column chromatography (sherwood oil: ethyl acetate); 3. the A-2 that gets 1 molar part, adds dissolve with methanol, adds the 10%NaOH aqueous solution (containing NaOH1.5 molar part), stirs after 30min, adds 30%H ₂o ₂(5 molar part), at N ₂the lower back flow reaction 3h (TLC detection reaction process) of protection, to neutral, reclaims methyl alcohol with aqueous hydrochloric acid adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃the aqueous solution and saturated common salt water washing, use anhydrous MgSO ₄dry, the resistates after decompression and solvent recovery obtains A-3 through silica gel column chromatography (sherwood oil: ethyl acetate); 4. the A-3 that gets 1 molar part, adds dissolve with methanol, at 0～5 ℃, stirs after 15min, divides the NaBH that adds 5 molar part for 2～3 times ₄, at N ₂protection lower reaction 2h (TLC detection reaction process), to neutral, reclaims methyl alcohol with aqueous hydrochloric acid adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, the resistates after decompression and solvent recovery obtains A-4 through silica gel column chromatography (sherwood oil: ethyl acetate); 5. the A-4 that gets 1 molar part, adds acetone solution, adds the perchloric acid of 1.5 molar part, then adds one of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains A-6 and isomer products [compound (1)～(7)] thereof through silica gel column chromatography (sherwood oil: ethyl acetate); Separate simultaneously and obtain sample A-5 a small amount of, that take on a red color under sulfuric acid developer, by EI-MS and ¹h-NMR can determine that its structure is 1 α, 2 α, 3 α-trihydroxy-olea-12-alkene-28-acid.

Aforesaid 1, 2, the preparation method's of 3-trihydroxy-replacement pentacyclic triterpenoid five: get Oleanolic Acid or its methyl esters or its benzyl ester A-a and obtain A-b under Methanesulfonyl chloride effect, A-b is through Quilonum Retard processing, obtain intermediate A-c, A-c is oxidized to obtain A-d by tin anhydride, after the NaOH aqueous solution is processed, decarboxylation obtains A-e again, intermediate A-e obtains epoxidation intermediate A-f under metachloroperbenzoic acid effect, A-f obtains 1 after perchloric acid hydrolysis, 2, 3-trihydroxy-intermediate A-g, after adopting palladium carbon catalytic hydrogenation or demethylating reaction, obtain 1 of different isomerization body, 2, 3-trihydroxy-olea-12-alkene-28 acid product, be compound (1)～(7), its reaction scheme is as follows:

Preparation method five detailed process is: 1. get the Oleanolic Acid of 1 molar part or its methyl esters or benzyl ester (A-a) and be dissolved in pyridine solvent, under stirring, slowly drip the Methanesulfonyl chloride of 1.2 molar part, at N in ice-water bath ₂the lower stirring reaction 5h (TLC detection reaction process) of protection, is extracted with ethyl acetate, and the aqueous hydrochloric acid of 2N washes away pyridine, and gained resistates obtains A-b through purification by silica gel column chromatography; 2. get the A-b of 1 molar part, with N, N '-dimethyl formamide dissolves, add Quilonum Retard 2 molar part, reflux 1 hour (TLC detection reaction process), is cooled to room temperature, filtering Quilonum Retard, add after suitable quantity of water, be extracted with ethyl acetate, organic phase is washed till neutrality, decompression and solvent recovery, through purification by silica gel column chromatography (sherwood oil: ethyl acetate is eluent), obtain intermediate A-c; 3. get the A-c of 1 molar part, add acetic acid to dissolve, then add 2 molar part tin anhydride (SeO ₂), 5 hours (TLC detection reaction process) refluxes, be cooled to room temperature, filtering tin anhydride, saturated sodium bicarbonate is neutralized to neutrality, adds after suitable quantity of water, be extracted with ethyl acetate, decompression and solvent recovery, residue, through purification by silica gel column chromatography (sherwood oil: ethyl acetate is eluent), obtains intermediate A-d; 4. the A-d that gets 1 molar part, adds dissolve with methanol, then adds 10%NaOH (2 molar part) aqueous solution, reflux 1 hour, with 2N HCl tune pH value, to neutral, ethyl acetate extracts, after reclaiming solvent, gained residue, through column chromatography for separation, obtains white powder A-e; 5. the A-e of 1 molar part is dissolved in methylene dichloride, adds 1.2 molar part metachloroperbenzoic acids, under room temperature, at N ₂the lower reaction of protection is spent the night (TLC detection reaction process), and with chloroform extraction, after recovery solvent, residue carries out silica gel column chromatography separating purification, obtains white powder A-f; 6. the A-f that gets 1 molar part, adds acetone solution, adds the perchloric acid of 1.5 molar part, then adds one of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains A-g through silica gel column chromatography; 7. get 1 molar part A-g (R is methyl), add dimethyl formamide to dissolve, then add 1.2 molar part lithium iodides, reflux 5 hours, be cooled to after room temperature, add suitable quantity of water, be extracted with ethyl acetate, after recovery solvent, residue is through silica gel column chromatography separating purification, obtain 1 of corresponding different isomerization body, 2,3-trihydroxy-olea-12-alkene-28 acid product, i.e. compound (1)～(7); If R is benzyl in A-g, adopt palladium carbon catalytic hydrogenation, can obtain equally 1,2 of different isomerization body, 3-trihydroxy-olea-12-alkene-28 acid product, i.e. compound (1)～(7).

Aforesaid 1,2,3-trihydroxy-replaces R in pentacyclic triterpenoid (general formula I) ₃for-CH ₂the preparation method of the respective compound of OH is: take compound (1)～(14) as raw material, at LiAlH ₄effect issues raw reduction reaction, obtains; Its reaction scheme is as follows:

The detailed process of the method is: get compound (1)～(14) of 1 molar part, be dissolved in anhydrous tetrahydro furan or anhydrous diethyl ether solvent, divide and add LiAlH 2～3 times ₄(5 molar part), at room temperature reacts 10 hours or reflux 5 hours (TLC detection reaction process); After completion of the reaction, use saturated aqueous ammonium chloride termination reaction, with after the hydrochloric acid neutralization reaction liquid of 2N, be extracted with ethyl acetate, after recovery solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for-CH ₂1,2 of OH, 3-trihydroxy-replaces pentacyclic triterpenoid.

Aforesaid 1,2,3-trihydroxy-replaces R in pentacyclic triterpenoid (general formula I) ₃for the preparation method of the respective compound of-CHO is: take compound (1)～(14) as raw material, with diisobutyl aluminium hydride (i-Bu ₂alH) reaction, obtains; Its reaction scheme is as follows:

The detailed process of the method is: get compound (1)～(14) of 1 molar part, be dissolved in anhydrous tetrahydro furan or anhydrous diethyl ether solvent, divide and add diisobutyl aluminium hydride (i-Bu 2～3 times ₂alH) (5 molar part), under nitrogen protection, reacts 12 hours (TLC detection reaction process) under room temperature; After completion of the reaction, use saturated aqueous ammonium chloride termination reaction, with after the hydrochloric acid neutralization reaction liquid of 2N, be extracted with ethyl acetate, after recovery solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for 1,2 of-CHO, 3-trihydroxy-replaces pentacyclic triterpenoid.

Aforesaid 1,2,3-trihydroxy-replaces R in pentacyclic triterpenoid (general formula I) ₃for-COOR ¹,-CONH ₂,-CONHR ¹or-CONR ¹r ²the preparation method of respective compound be: take compound (1)～(14) as raw material, under dewatering agent DCC (being N, N-dicyclohexylcarbodiimide) or WSC effect, with corresponding alcohol R ¹oH or amine R ¹nH ₂, R ¹r ²nH, BnNH ₂reaction, obtains corresponding ester class or amides, i.e. R in general formula I ₃for-COOR ¹,-CONH ₂,-CONHR ¹or-CONR ¹r ²respective compound; Its reaction scheme is as follows:

The detailed process of aforesaid method is:

Get compound (1)～(14) of 1 molar part and the alcohol R of 1.5 molar part ¹oH or amine R ¹nH ₂, R ¹r ²nH; be dissolved in dry DMF (dimethyl formamide) solvent; add the dewatering agent DCC of 3 molar part or the DMAP (DMAP) of WSC and 0.1 molar part; under nitrogen protection; room temperature reaction 24 hours (TLC detection reaction process), adds after the aqueous hydrochloric acid termination reaction of 2N, is extracted with ethyl acetate; after reclaiming solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for-COOR ¹,-CONHR ¹or-CONR ¹r ²1,2,3-trihydroxy-replace pentacyclic triterpenoid.

Get compound (1)～(14) of 1 molar part and the benzylamine (BnNH of 1.5 molar part ₂); be dissolved in anhydrous 1; in 4-dioxane solvent; add the dewatering agent DCC of 3 molar part or the DMAP (DMAP) of WSC and 0.1 molar part; under nitrogen protection; room temperature reaction 24 hours (TLC detection reaction process); add after the aqueous hydrochloric acid termination reaction of 2N; be extracted with ethyl acetate; after reclaiming solvent, residue is through silica gel column chromatography separating purification; gains are dissolved in methyl alcohol, through 12 hours (under room temperature) of palladium carbon catalytic hydrogenation reduction, obtain R in corresponding general formula I ₃for-CONH ₂1,2,3-trihydroxy-replace pentacyclic triterpenoid.

Aforesaid 1,2,3-trihydroxy-replaces pentacyclic triterpenoid in the application of preparing in hepatic: the pharmacologically acceptable salt that described compound or described compound dissolution are made in sodium hydroxide, potassium hydroxide or ammonia soln is made hepatic preparation with suitable auxiliary material.

Aforementioned pharmaceutical preparation is hard capsule, tablet, oral liquid, soft capsule, granule or pill.

Several preparation method provided by the present invention respectively has superiority, and can select as required.Wherein preparation method the three, the 5th, to supplementing of preparation method one, two, four, can be high yield obtain by the synthetic content of method one, two, four less 1,2,3-trihydroxy-isomer.According to preparation method provided by the present invention, can effectively synthesize the different target products that replace configuration of 1,2,3-trihydroxy-; And method reaction conditions gentleness provided by the invention, without special reagent and strict reaction conditions, operation is simple, is easy to expansion scale, realizes the object of suitability for industrialized production.

Of the present invention 1,2,3-trihydroxy-replaces the application method of pentacyclic triterpenoid, is that described compound is applied as medicine: can add inert auxiliary and/or vehicle that described compound is prepared into suitable form of administration; Also can be using described compound as liver injury protection agent for the control medicine of hepatopathy.Medicine can be tablet, capsule, injection or pill etc.Concrete application method can adopt ordinary method of the prior art, according to preparation method well known in the art, adopts traditional additive, vehicle preparation; One or more active substances and one or more additives, mixed with excipients can be formed to medicinal compositions, then composition is made to medicine.The medicine making thus as required can non-enteron aisle, the administration such as oral.

Through experimental study, find, of the present invention 1,2,3-trihydroxy-replaces pentacyclic triterpenoid tetrachloro-methane induction hepatocellular injury is had to good provide protection, can be used as hepatic application.It is below the biological activity test that the biological activity effect for verifying compound of the present invention is carried out.

1, external to CCl ₄due to the provide protection of hepatocellular injury

Test sample: A-5 (i.e. 1 α, 2 α, 3 α-trihydroxy-olea-12-alkene-28-acid) and sodium salt thereof; A-6 (i.e. 1 α, 2 β, 3 α-trihydroxy-olea-12-alkene-28-acid) and sodium salt thereof; U-17 (i.e. 1 α, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid) and sodium salt thereof; U-18 (i.e. 1 α, 2 β, 3 α-trihydroxy-black bearberry-12-alkene-28-acid) and sodium salt thereof; AM (1,2,3-trihydroxy-olea-12-alkene-28-acid, i.e. the mixture of compound (1)～(7)) and sodium salt thereof; UM (1,2,3-trihydroxy-black bearberry-12-alkene-28-acid, i.e. the mixture of compound (8)～(14)) and sodium salt thereof; FU-16 (embodiment 6 makes); A-6-CH ₂oH (make take A-6 as raw material, see embodiment 31); A-6-CHO (make take A-6 as raw material, see embodiment 32); A-6-CONHBn (make take A-6 as raw material, see embodiment 34); A-6-CONH ₂(take A-6 as raw material, make, see embodiment 35).

Method: conventional vitro culture of human liver HL-7702 cell is inoculated in 96 orifice plates, 17 kinds of samples that give respectively different concns (in Table 1, table 2 and table 3) continue to cultivate after 12h, with three kinds of various dose (8 μ l, 6 μ l, l) CCl of 4 μ ₄cause liver injury, each medicine is all established two parallel control holes.Application MTS method is measured absorbancy at enzyme-linked immunosorbent assay instrument 490nm, observes medicine to CCl ₄due to the provide protection of hepatotoxicity.Relatively normal group is calculated cells survival rate, carries out single dependent variable multifactor analysis of variance with spss software.

Result: CCl ₄model group causes obvious damage to liver cell, and liver cell survival rate and the model group cells survival rate of 17 kinds of example pharmaceuticals of application have significant difference, there is no notable difference (in Table l, table 2 and table 3) with normal group cells survival rate.

Conclusion: all test samples (medicine) all can significantly improve the survival rate of model group cell, has significant protective effect to people liver HL-7702 cell.

Test-results:

L is external to CCl for table ₄due to provide protection (the 8 μ l CCl of hepatocellular injury ₄)

* administration group vs model group, p < 0.01; # model group vs normal group, p < 0.01.

Table 2 is external to CCl ₄due to provide protection (the 6 μ l CCl of hepatocellular injury ₄)

* administration group vs model group, p < 0.01; # model group vs normal group, p < 0.01.

Table 3 is external to CCl ₄due to provide protection (the 4 μ l CCl of hepatocellular injury ₄)

* administration group vs model group, p < 0.01; # model group vs normal group, p < 0.01.

2, body is interior to CCl ₄the provide protection of induced mice hepatocellular injury

A, test materials

(1) tested medicine (sample)

A-6 (i.e. 1 α, 2 β, 3 α-trihydroxy-olea-12-alkene-28-acid) and sodium salt thereof; U-18 (i.e. 1 α, 2 β, 3 α-trihydroxy-black bearberry-12-alkene-28-acid); AM (1,2,3-trihydroxy-olea-12-alkene-28-acid, i.e. the mixture of compound (1)～(7)); UM (1,2,3-trihydroxy-black bearberry-12-alkene-28-acid, i.e. the mixture of compound (8)～(14)) and sodium salt thereof.

Before above sample (medicine) experiment, with 0.5%CMC, be mixed with desired concn suspension.

(2) medicine and reagent

Positive drug: bifendate drop pill Zhejiang Medicine Co lot number: 091206.

Bicyclol Beijing XieHe medicine Factory lot number: 100602.

B, experimental animal

(1) source and kind: KM mouse, male and female dual-purpose, body weight 18～22g.Animal conformity certification number: SCXK (Guizhou Province) 2002-0001 is provided by Guiyang Medical College Experimental Animal Center.

(2) raising condition: KM mouse, male and female sub-cage rearing in clean animal breeding cabinet, 10, the every cage of mouse.Animal housing's illumination abundance, heating ventilation and air-conditioning equipment is good, 18～25 ℃ of room temperatures, relative humidity 50～70%.

(3) data analysis and processing

Data are with mean standard deviation

represent, adopt Excel 2000 to take statistics and learn processing, measurement data adopts Student-t check.

C, route of administration

According to clinical application approach, experimental animal adopts gastric infusion.

D, test method and result

(1) test method:

Get 36 of KM mouse, male and female half and half, body weight 18～22g, be divided at random 6 groups: model group (isometric(al) 0.5%CMC), normal group, Biphenylylmethylcarbinol group (3.75mg/kg), bicyclol group (12.5mg/kg), test sample low dose group (50mg/kg), test sample high dose group (170mg/kg), 6 every group.Positive drug is commercially available bifendate drop pill and Bicyclol.Administration volume 20ml/kg, every day 2 times, successive administration 2 days.Each group last administration 8h pneumoretroperitoneum injection 0.1%CCl ₄peanut oil 10mlkg ^-1.After fasting 16h, put to death mouse, get the centrifugal ALT of being of blood and check.

(2) test-results (in Table 4):

The provide protection of table 4 to the liver injury of tetracol phenixin induced mice

With model group comparison, * P < 0.05, * * P < 0.01.

Result shows, all test samples (medicine) high and low dose group all can obviously reduce CCl ₄the ALT of poisoning mice, with significantly (P < 0.01 of model group comparing difference, P < 0.05), wherein the low dose group of test sample and model group relatively have utmost point significant difference (P < 0.01), also more obvious than positive drug effect.

In sum, provided by the invention 1,2,3-trihydroxy-replaces liver (cell) damage of pentacyclic triterpenoid to tetrachloro-methane induction significant provide protection, and expection can be used as hepatic application.

Compared with prior art, the invention provides 1 of one group of novel structure, 2, pentacyclic triterpene derivatives quasi-compound and synthetic method thereof that 3-trihydroxy-replaces, described synthesising method reacting condition gentleness, without special instruments and equipment, operation is simple, is easy to expansion scale, realizes the object of suitability for industrialized production; And compound provided by the present invention and pharmacologically acceptable salt thereof (sodium salt, sylvite or ammonium salt) have significant liver injury protection effect (body is interior and external), can be used for the preparation of hepatic.

Embodiment

Embodiment 1: compound U-2's is synthetic

Get 1.000g ursolic acid (2.19mmol) (being U-1) in 250mL round-bottomed flask, use 120mL acetone solution, be placed in ice-water bath and stir 15min, slowly drip Jones reagent 0.936mL (2.5mmol), at N ₂the lower stirring of protection 5h has reacted (TLC detection reaction process).Use 2molL ^-1the NaOH aqueous solution regulates pH to neutral, and pressure reducing and steaming solvent, is extracted with ethyl acetate, and gained resistates obtains 946mg U-2 (95.0%) through purification by silica gel column chromatography.

U-2 white powder, 256～258 ℃ of mp, IR (KBr) υ _max1694cm ^-1, 2922cm ^-1, ¹h-NMR (CDCl ₃) δ: 5.25 (1H, s, H-12), 1.08,1.08,1.05,1.02,0.82 (3H × 5, s, 23,24,25,26,27), 0.96～0.95 (3H, d, J=4.0Hz, H-29), 0.87～0.85 (3H, d, J=6.0Hz, H-30), ¹³c-NMR (CDCl ₃) δ: 217.8 (C-3), 183.8 (C-28), 138.0 (C-13), 125.5 (C-12), 55.2 (C-5), 52.5 (C-18), 47.9 (C-17), 47.3 (C-8), 46.7 (C-9), 42.0 (C-14), 39.4 (C-4), 39.2 (C-1), 39.0 (C-20), 38.7 (C-19), 36.6 (C-22), 36.6 (C-10), 34.1 (C-7), 32.4 (C-21), 30.5 (C-15), 27.9 (C-12), 26.5 (C-23), 24.0 (C-16), 23.5 (C-27), 23.3 (C-11), 21.4 (C-30), 31.1 (C-29), 19.5 (C-6), 16.9 (C-25), 16.9 (C-26), 15 (C-24), MS (EI) m/z:454 (M ⁺), 248 (100), 203,133,55,43.

Embodiment 2: compound U-3's is synthetic

Get 414mg (0.912mmol) U-2 in the round-bottomed flask of 250mL, add the acetone solution of 25mL, then add 1.2g (9.12mmol) K ₂cO ₃, under room temperature, stir 30min, then add 128 μ L (1.07mmol) cylites, at N ₂the lower reaction 18h of protection has reacted (TLC detection reaction process), and acetone is removed in decompression, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, removal of solvent under reduced pressure, residue obtains 430mg U-3 (86.7%) through silica gel column chromatography.

U-3 white powder, 96～98 ℃ of mp, IR (KBr) υ _max1715cm ^-1, 2940cm ^-1, 1450cm ^-1, 1379cm ^{- 1}, ¹h-NMR (CDCl ₃) δ: 7.35 (s, 5H), 5.28 (1H, s, H-12), 4.97-5.12 (2H, dd, benzyl), 1.08,1.03,1.02,1.00,0.68 (3H × 5, s, 23,24,25,26,27), 0.94～0.92 (3H, d, J=9.2Hz, H-29), 0.86～0.84 (3H, d, J=6.8Hz, H-29), ¹³c-NMR (CDCl ₃) δ: 217.8 (C-3), 177.1 (C-28), 138.1 (C-13), 136.2 (s), 128.4 (s), 128.2 (s), 128.1 (s), 127.9 (s), 127.9 (s), 125.4 (C-12), 65.9 (benzyl), 55.2 (C-5), 52.9 (C-18), 48.0 (C-17), 47.3 (C-8), 46.7 (C-9), 42.0 (C-14), 39.4 (C-4), 39.2 (C-1), 39.0 (C-20), 38.7 (C-19), 36.5 (C-22), 36.5 (C-10), 34.1 (C-7), 32.4 (C-21), 30.6 (C-15), 27.8 (C-12), 26.5 (C-23), 24.1 (C-16), 23.4 (C-27), 23.3 (C-11), 21.4 (C-30), 21.1 (C-29), 19.5 (C-6), 16.9 (C-25), 16.9 (C-26), 15.1 (C-24), MS (EI) m/z:544 (M ⁺), 91 (100), 248,203,133,55,43.

Embodiment 3: compound S e-1's is synthetic

Get 400mg (0.735mmol) U-3,110mg (1.10mmol) SeO ₂in 50mL round-bottomed flask, add 20mL diacetyl oxide, at N ₂the lower backflow 5h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous sodium hydroxide solution termination reaction, is extracted with ethyl acetate after adding suitable quantity of water, saturated sodium-chloride water solution washing for organic layer, anhydrous MgSO ₄dry, removal of solvent under reduced pressure, resistates obtains 191mg Se-1 (47.9%) through silica gel column chromatography.

Se-1 white powder, 127～130 ℃ of mp, IR (KBr) υ _max1724cm ^-1, 2926cm ^-1, 1667cm ^-1, 1456cm ^-1, 1383cm ^-1, ¹h-NMR (CDCl ₃) δ: 7.35 (s, 5H), 7.06～7.04 (d, J=10.4,1H, H-1), 5.81～5.79 (1H, d, J=10.4Hz, H-2), 5.29 (1H, s, H-12), 4.98-5.13 (2H, dd, benzyl), 2.31～2.28 (1H, d, J=11.2, H-18), 1.14,1.13,1.09,1.03,0.70 (3H × 5, s, 23,24,25,26,27), 0.94～0.93 (3H, d, J=6.8Hz, H-29), 0.87～0.85 (3H, d, J=6.4Hz, H-30) ppm, ¹³c-NMR (CDCl ₃) δ: 205.4 (C-3), 177.2 (C-28), 159.4 (C-1), 138.6 (C-13), 136.2 (s), 128.3 (s), 128.2 (s), 128.0 (s), 127.9 (s), 127.9 (s), 124.9 (C-2), 124.9 (C-12), 65.9 (benzyl), 53.3 (C-5), 53.0 (C-18), 48.1 (C-17), 44.5 (C-14), 42.3 (C-4), 41.6 (C-9), 40.2 (C-8), 39.2 (C-10), 38.9 (C-20), 38.7 (C-19), 36.5 (C-22), 32.7 (C-7), 30.6 (C-21), 29.6 (C-15), 24.1 (C-16), 23.3 (C-23), 23.1 (C-11), 21.6 (C-27), 21.0 (C-30), 18.8 (C-6), 18.7 (C-26), 17.5 (C-25), 16.9 (C-24), MS (EI) m/z 452 (M ⁺), 248 (100), 203,133.

Embodiment 4: compound U-15's is synthetic

Get 200mg (0.442mmo) Se-1 in 50mL round-bottomed flask, add 25mL dissolve with methanol, add the 10%NaOH aqueous solution 265 μ L (0.663mmol), stir after 30min, add 500 μ L (4.42mmol) 30%H ₂o ₂, at N ₂the lower backflow 3h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous hydrochloric acid regulates pH to neutral, reclaims methyl alcohol, and resistates adds water-dispersion, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 152mg U-15 (74%) through silica gel column chromatography.

U-15 white powder, 95～98 ℃ of mp, IR (KBr) υ _max1725cm ^-1, 2926cm ^-1, 1694cm ^-1, 1466cm ^-1, 1385cm ^-1, ¹h-NMR (CDCl ₃) δ: 7.35 (s, 5H), 5.28 (1H, s, H-12), 5.13～4.97 (2H, dd, benzyl), 3.51～3.50 (1H, d, J=4.4Hz, H-2), 3.38～3.37 (1H, d, J=4.4Hz, H-1), 2.31～2.28 (1H, d, J=11.2Hz, H-18), 1.12,1.08,0.99,0.95,0.70 (3H × 5, s, 23,24,25,26,27), 0.94～0.93 (d, J=4.0Hz, 3H, H-29), 0.87～0.86 (d, J=6.4Hz, 3H, H-30) ppm, ¹³c-NMR (CDCl ₃) δ: 212.8 (C-3), 177.2 (C-28), 138.7 (C-13), 136.2 (s), 128.3 (s), 128.1 (s), 128.0 (s), 127.9 (s), 127.9 (s), 124.8 (C-12), 65.9 (C-1), 64.0 (C-2), 56.8 (benzyl), 53.0 (C-18), 48.1 (C-17), 46.7 (C-4), 45.9 (C-5), 42.5 (C-14), 40.4 (C-9), 39.6 (C-8), 39.0 (C-19), 38.7 (C-20), 38.3 (C-10), 36.5 (C-22), 32.4 (C-7), 30.6 (C-21), 27.9 (C-15), 27.9 (C-23), 24.1 (C-16), 23.7 (C-11), 23.5 (C-27), 23.3 (C-30), 21.1 (C-29), 18.7 (C-6), 17.6 (C-26), 17.1 (C-25), 16.9 (C-24), MS (EI) m/z 558 (M ⁺), 91 (100), 133,467,543.

Embodiment 5: compound R-10-2's is synthetic

Get 200mg U-15 (0.358mmol) in 50mL round-bottomed flask, add 25mL dissolve with methanol, at 0～5 ℃, stir after 15min, divide and add 136mg (3.58mmol) NaBH for 3 times ₄, at N ₂the lower reaction 2h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous hydrochloric acid regulates pH to neutral, reclaims methyl alcohol, and resistates adds water-dispersion, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 144mg R-10-2 (72%) through silica gel column chromatography.

R-10-2 white powder, 101～103 ℃ of mp, IR (KBr) υ _max1722cm ^-1, 2925cm ^-1, 1662cm ^-1, 1450cm ^-1, 1384cm ^-1, ¹h-NMR (CDCl ₃) δ: 7.35 (s, 5H), 5.25 (1H, s, H-12), 5.12～4.96 (2H, dd, benzyl), 3.55 (1H, s, H-3), 3.07～3.06 (1H, d, J=4.0Hz, H-2), 2.99～2.98 (1H, d, J=3.6Hz, H-1), 2.28～2.26 (1H, d, J=11.6Hz, H-18), 1.07,0.93,0.93,0.78,0.64 (3H × 5, s, 23,24,25,26,27), 1.06～1.03 (3H, d, J=10.4, H-29), 0.87～0.84 (3H, d, J=6.4, H-30) ppm, ¹³c-NMR (CDCl ₃) δ: 177.4 (C-28), 138.3 (C-13), 136.2 (s), 128.3 (s), 128.0 (s), 127.9 (s), 127.8 (s), 127.8 (s), 124.9 (C-12), 75.7 (C-3), 65.9 (benzyl), 60.3 (C-1), 57.3 (C-2), 52.8 (C-18), 48.0 (C-17), 45.5 (C-5), 41.3 (C-14), 41.0 (C-9), 39.4 (C-8), 39.2 (C-19), 38.9 (C-4), 38.7 (C-20), 36.9 (C-10), 36.5 (C-22), 32.6 (C-7), 30.5 (C-21), 28.5 (C-23), 27.8 (C-15), 24.1 (C-16), 23.4 (C-27), 3.3 (C-11), 21.1 (C-30), 18.1 (C-29), 17.0 (C-26), 16.9 (C-25), 16.7 (C-6), 16.4 (C-24), MS (EI) m/z 560 (M ⁺), 91 (100), 247,433,451,524,542.

Embodiment 6: compound F 17-hydroxy-corticosterone U-16's is synthetic

Get 100mg (0.179mmol) R-10-2 in 50mL round-bottomed flask, add 25mL acetone solution, add 16 μ L (0.268mmol) perchloric acid, then add two of distilled water, at N ₂the lower reaction 18h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous sodium hydroxide solution regulates pH to neutral, reclaims acetone, and resistates adds water-dispersion, is extracted with ethyl acetate saturated common salt water washing for organic layer, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 78mg FU-16 (76.1%) through silica gel column chromatography.

FU-16 white powder, 108～110 ℃ of mp, IR (KBr) υ _max3443cm ^-1, 2925cm ^-1, 1627cm ^-1, 1384cm ^-1, ¹h-NMR (CD ₃oD) δ: 5.24 (1H, s, H-12), 5.12～4.96 (2H, dd, benzyl), 3.93 (1H, d, J=6.8Hz, H-2), 3.61 (1H, d, J=2.4Hz, H-3), 3.45 (1H, J=d, 4.4Hz, H-1), ¹³c-NMR (CD ₃oD) δ: 180.5 (C-28), 139.5 (C-13), 136.2 (s), 128.3 (s), 128.0 (s), 127.9 (s), 127.8 (s), 127.8 (s), 127.1 (C-12), 77.4 (C-3), 75.3 (C-1), 74.9 (C-2), 65.9 (benzyl), 49.6 (C-5), 47.6 (C-17), 47.2 (C-19), 43.4 (C-14), 42.7 (C-18), 41.5 (C-8), 40.3 (C-4), 39.7 (C-9), 39.3 (C-20), 39.2 (C-10), 34.8 (C-21), 33.8 (C-7), 33.6 (C-29), 33.5 (C-22), 30.1 (C-30), 28.8 (C-15), 26.5 (C-27), 24.4 (C-16), 24.0 (C-30), 24.0 (C-11), 19.1 (C-6), 18.1 (C-25), 17.8 (C-24), 14.8 (C-26), MS (EI) m/z:578 (M ⁺), 91 (100), 248,203,469,524,542,556.

Embodiment 7: compound U-18 and isomer thereof synthetic

Getting 100mg (0.173mmol) FU-16 is dissolved in 20mL anhydrous methanol, add 10mg palladium carbon as catalyzer, hydro-reduction under room temperature, obtains 80mgU-18 (95%) and isomer products thereof [compound (8)～(14)].

U-18 white powder, 272～275 ℃ of mp, IR (KBr) υ _max3454cm ^-1, 2924cm ^-1, 1682cm ^-1, ¹h-NMR (CD ₃oD) δ: 5.24 (1H, s, H-12), 3.93 (1H, d, J=6.8Hz), 3.61 (1H, d, J=2.4Hz), 3.45 (1H, d, J=4.4Hz), 1.28,1.23,1.13,1.00,0.87 (3H × 5, s, 23,24,25,26,27), 0.96～0.94 (3H, d, J=6.8Hz, H-29), 0.90～0.89 (3H, d, J=5.2Hz, H-30), ¹³c-NMR (CD ₃oD) δ: 180.5 (C-28), 139.5 (C-13), 127.1 (C-12), 77.4 (C-3), 75.3 (C-1), 74.9 (C-2), 49.6 (C-5), 47.6 (C-17), 47.2 (C-19), 43.4 (C-14), 42.7 (C-18), 41.5 (C-8), 40.3 (C-4), 39.7 (C-9), 39.3 (C-20), 39.2 (C-10), 34.8 (C-21), 33.8 (C-7), 33.6 (C-29), 33.5 (C-22), 30.1 (C-30), 28.8 (C-15), 26.5 (C-27), 24.4 (C-16), 24.0 (C-30), 24.0 (C-11), 19.1 (C-6), 18.1 (C-25), 17.8 (C-24), 14.8 (C-26), MS (EI) m/z:488 (M ⁺), 248 (100), 43,133,203,452,470.

Embodiment 8: compound S e's is synthetic

Get U-2400mg (0.881mmol), SeO ₂176mg (1.76mmol), in 50mL round-bottomed flask, adds 20mL diacetyl oxide, and the 5h that refluxes under nitrogen protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous sodium hydroxide solution termination reaction, is extracted with ethyl acetate the saturated NaHCO of organic layer after adding suitable quantity of water ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 191mg Se (46.7%) through silica gel column chromatography.

Se white powder, 127～130 ℃ of mp, IR (KBr) υ _max1698cm ^-1, 2962cm ^-1, ¹h-NMR (CDCl ₃) δ: 7.07～7.05 (1H, d, J=10.4Hz, H-1), 5.82～5.80 (1H, d, J=10.4Hz, H-2), 5.25 (1H, s, H-12), 2.31～2.28 (1H, d, J=11.2Hz, H-18), 1.17,1.16,1.10,1.08,0.85 (3H × 5, s, 23,24,25,26,27), 0.96～0.94 (3H, d, J=6.4Hz, H-29), 0.87～0.86 (3H, d, J=4.4Hz, H-30) ppm, ¹³c-NMR (CDCl ₃) δ: 205.3 (C-3), 183.8 (C-28), 159.3 (C-1), 138.0 (C-13), 124.9 (C-2), 124.9 (C-12), 53.3 (C-5), 53.0 (C-18), 48.1 (C-17), 44.5 (C-14), 42.3 (C-4), 41.6 (C-9), 40.2 (C-8), 39.2 (C-10), 38.9 (C-20), 38.7 (C-19), 36.5 (C-22), 32.7 (C-7), 30.6 (C-21), 29.6 (C-15), 24.1 (C-16), 23.3 (C-23), 23.1 (C-11), 21.6 (C-27), 21.0 (C-30), 18.8 (C-6), 18.7 (C-26), 17.5 (C-25), 16.9 (C-24), MS (EI) m/z 452 (M ⁺), 248 (100), 203,133,437.

Embodiment 9: compound U-15-1's is synthetic

Get 400mg Se (0.881mmol) in 50mL round-bottomed flask, add 25mL dissolve with methanol, add the 10%NaOH aqueous solution 53 μ L (1.321mmol), stir after 30min, add 30%H ₂o ₂498 μ L (4.40mmol), at N ₂the lower backflow 3h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous hydrochloric acid regulates pH to neutral, reclaims methyl alcohol, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 317mg U-15-1 (76.6%) through silica gel column chromatography.

U-15-1 white powder, 138～140 ℃ of mp, IR (KBr) υ _max1692cm ^-1, 2923cm ^-1, 1468cm ^-1, ¹h-NMR (CDCl ₃) δ: 5.30 (1H, s, H-12), 3.52～3.51 (1H, d, J=4.8, H-2), 3.39～3.37 (1H, d, J=4.8, H-1), 1.25,1.20,1.15,0.96,0.84 (3H × 5, s, 23,24,25,26,27), 0.95～0.93 (3H, d, J=6.4, H-29), 0.86～0.85 (3H, d, J=6.0, H-30), ¹³c-NMR (CDCl ₃) δ: 212.7 (C-3), 183.9 (C-28), 138.5 (C-13), 124.9 (C-12), 63.9 (C-1), 56.9 (C-2), 52.6 (C-18), 48.0 (C-17), 45.9 (C-5), 44.7 (C-14), 42.1 (C-20), 40.4 (C-9), 38.9 (C-19), 38.6 (C-20), 38.4 (C-4), 36.7 (C-10), 36.5 (C-22), 32.3 (C-7), 30.5 (C-21), 28.0 (C-15), 27.9 (C-23), 23.9 (C-16), 23.7 (C-11), 23.4 (C-27), 21.1 (C-30), 20.8 (C-29), 18.7 (C-6), 17.6 (C-26), 16.9 (C-25), 15.0 (C-24), MS (EI) m/z 468 (M ⁺), 202 (100), 43,248,422,450.

Embodiment 10: compound R-10-21's is synthetic

Get 200mg U-15-1 (0.427mmol) in 50mL round-bottomed flask, add 25mL dissolve with methanol, at 0～5 ℃, stir after 15min, divide and add 81mg (2.136mmol) NaBH for 3 times ₄, at N ₂the lower reaction 2h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous hydrochloric acid regulates pH to neutral, reclaims methyl alcohol, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 139mg R-10-21 (69%) through silica gel column chromatography.

R-10-21 white powder, 208～210 ℃ of mp, IR (KBr) υ _max1691cm ^-1, 2924cm ^-1, 1457cm ^-1, ¹h-NMR (CDCl ₃) δ: 5.30 (1H, s, H-12), 3.49 (1H, s, H-3), 3.07～3.06 (1H, d, J=3.6Hz, H-2), 3.02～3.01 (1H, d, J=3.6Hz, H-1), 2.82～2.65 (1H, d, J=9.6Hz, H-18), 1.22,1.14,0.93,0.90,0.82,0.80,0.76 (3H × 7, s, 23,24,25,26,27), 0.95～0.93 (3H, d, J=6.4Hz, H-29), 0.86～0.85 (3H, d, J=6.0Hz, H-30) ppm, ¹³c-NMR (CDCl ₃) δ: 183.4 (C-28), 138.3 (C-13), 124.9 (C-12), 75.5 (C-3), 60.3 (C-1), 57.3 (C-2), 52.8 (C-18), 48.0 (C-17), 45.5 (C-5), 41.3 (C-14), 41.0 (C-9), 39.4 (C-8), 39.0 (C-19), 38.9 (C-4), 38.7 (C-20), 36.9 (C-10), 36.5 (C-22), 32.6 (C-7), 30.5 (C-21), 28.5 (C-23), 27.8 (C-15), 24.1 (C-16), 23.4 (C-27), 23.3 (C-11), 21.1 (C-30), 18.1 (C-29), 17.0 (C-26), 16.9 (C-25), 16.7 (C-6), 16.4 (C-24), MS (EI) m/z:470 (M ⁺), 248 (100), 43,133,203,452.

Embodiment 11: compound U-18 and isomer thereof synthetic

Get 100mg R-10-21 (0.213mmol) in 50mL round-bottomed flask, add 25mL acetone solution, add 18 μ L (0.319mmol) perchloric acid, add one of distilled water, at N ₂the lower reaction 18h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous sodium hydroxide solution regulates pH to neutral, reclaims acetone, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains 65mg U-18 (62.7%) and isomer products [compound (8)～(14)] thereof through silica gel column chromatography.

U-18 white powder, 272～275 ℃ of mp, IR (KBr) υ _max3454cm ^-1, 2924cm ^-1, 1682cm ^-1, ¹h-NMR (CD ₃oD) δ: 5.24 (1H, s, H-12), 3.93 (1H, d, J=6.8Hz), 3.61 (1H, d, J=2.4Hz), 3.45 (1H, d, J=4.4Hz), 1.28,1.23,1.13,1.00,0.87 (3H × 5, s, 23,24,25,26,27), 0.96～0.94 (3H, d, J=6.8Hz, H-29), 0.90～0.89 (3H, d, J=5.2Hz, H-30), ¹³c-NMR (CD ₃oD) δ: 180.5 (C-28), 139.5 (C-13), 127.1 (C-12), 77.4 (C-3), 75.3 (C-1), 74.9 (C-2), 49.6 (C-5), 47.6 (C-17), 47.2 (C-19), 43.4 (C-14), 42.7 (C-18), 41.5 (C-8), 40.3 (C-4), 39.7 (C-9), 39.3 (C-20), 39.2 (C-10), 34.8 (C-21), 33.8 (C-7), 33.6 (C-29), 33.5 (C-22), 30.1 (C-30), 28.8 (C-15), 26.5 (C-27), 24.4 (C-16), 24.0 (C-30), 24.0 (C-11), 19.1 (C-6), 18.1 (C-25), 17.8 (C-24), 14.8 (C-26), MS (EI) m/z:488 (M ⁺), 248 (100), 43,133,203,452,470.

Same separate the amount of obtaining few, the sample U-17 that takes on a red color under sulfuric acid developer.By EI-MS and ¹h-NMR determines that its structure is 1 α, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid.

Embodiment 12: compound U-b's is synthetic

Get the ursolic acid of 1mmol or its methyl esters or benzyl ester (U-a) and be dissolved in pyridine solvent, under stirring in ice-water bath, slowly drip the Methanesulfonyl chloride of 1.2mmol, at N ₂the lower stirring reaction 5h (TLC detection reaction process) of protection, is extracted with ethyl acetate, and the aqueous hydrochloric acid of 2N washes away pyridine, and gained resistates obtains U-b (white solid powder) through purification by silica gel column chromatography.

Embodiment 13: compound U-c's is synthetic

Get the U-b of 1mmol, with N, 10 milliliters of dissolvings of N '-dimethyl formamide, add Quilonum Retard 2mmol, reflux 1 hour (TLC detection reaction process), is cooled to room temperature, filtering Quilonum Retard, add after suitable quantity of water, be extracted with ethyl acetate, organic phase is washed till neutrality, decompression and solvent recovery, through purification by silica gel column chromatography (sherwood oil: ethyl acetate mixed solvent is eluent), obtain intermediate U-c (white solid powder).

Embodiment 14: compound U-d's is synthetic

Get the U-c of 1mmol, add 10 milliliters of acetic acid and 2mmol tin anhydride (SeO ₂), 5 hours (TLC detection reaction process) refluxes, be cooled to room temperature, filtering tin anhydride, saturated sodium bicarbonate is neutralized to neutrality, adds after suitable quantity of water, be extracted with ethyl acetate, decompression and solvent recovery, residue, through purification by silica gel column chromatography (sherwood oil: ethyl acetate mixed solvent is eluent), obtains intermediate U-d (white solid powder).

Embodiment 15: compound U-e's is synthetic

Get the U-d of 1mmol, add 10 ml methanol and 5 milliliters of 10%NaOH aqueous solution, reflux 1 hour, use 2N HCl to adjust pH value to neutral, ethyl acetate extraction, after recovery solvent, gained residue, through column chromatography for separation, obtains white powder U-e.

Embodiment 16: compound U-f's is synthetic

The U-e of 1mmol is dissolved in the methylene dichloride of 15 milliliters, adds 1.2mmol metachloroperbenzoic acid, under room temperature, at N ₂the lower reaction of protection is spent the night (TLC detection reaction process), and with chloroform extraction, after recovery solvent, residue carries out silica gel column chromatography separating purification, obtains white powder U-f.

Embodiment 17: compound U-g's is synthetic

The U-f that gets 1mmol, adds acetone solution, adds the perchloric acid of 1.5mmol, then adds one of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains U-g through silica gel column chromatography.

Embodiment 18:1,2,3-trihydroxy-black bearberry-12-alkene-28 acid synthetic

Get 1mmolU-g (R is methyl), add 10 milliliters of dimethyl formamides and 1.2mmol lithium iodide, reflux 5 hours, be cooled to after room temperature, add suitable quantity of water, be extracted with ethyl acetate, after reclaiming solvent, residue, through silica gel column chromatography separating purification, obtains 1,2 of corresponding different isomerization body, 3-trihydroxy-black bearberry-12-alkene-28 acid product, i.e. compound (8)～(14); If U-g (R is benzyl), adopts palladium carbon catalytic hydrogenation, can obtain equally 1,2 of different isomerization body, 3-trihydroxy-black bearberry-12-alkene-28 acid product, i.e. compound (8)～(14).

Embodiment 19: compd A-1 synthetic

Get 1.000g Oleanolic Acid (2.19mmol) in 250mL round-bottomed flask; use 120mL acetone solution; be placed in ice-water bath; after stirring 15min, slowly drip Jones reagent 0.936mL (2.5mmol), under nitrogen protection, stir 5h and reacted (TLC detection reaction process).Use 2molL ^-1the NaOH aqueous solution regulates pH to neutral, pressure reducing and steaming acetone, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, decompression and solvent recovery, resistates obtains 0.946g A-1 (96.3%) through silica gel column chromatography (sherwood oil: ethyl acetate).

A-1 white powder, 128～130 ℃ of mp, IR (KBr) υ _max1697cm ^-1, 2924cm ^-1, ¹h-NMR (CDCl ₃) δ: 5.30 (1H, s, H-12), 1.14,1.07,1.04,1.03,0.98,0.93,0.86 (3H × 7, s, 23,24,25,26,27,29,30) ppm, ¹³c-NMR (CDCl ₃) δ: 217.8 (C-3), 184.4 (C-28), 143.6 (C-13), 122.3 (C-12), 55.2 (C-5), 47.4 (C-17), 46.8 (C-19), 46.5 (C-14), 45.7 (C-8), 41.6 (C-9), 40.9 (C-18), 39.2 (C-4), 39.0 (C-10), 36.7 (C-1), 34.1 (C-22), 33.7 (C-21), 33.0 (C-29), 32.3 (C-7), 32.1 (C-23), 30.6 (C-20), 27.6 (C-15), 26.4 (C-2), 25.8 (C-27), 23.5 (C-30), 23.4 (C-16), 22.8 (C-11), 21.4 (C-26), 19.5 (C-6), 16.9 (C-25), 15.0 (C-24) ppm, MS (EI) m/z 454 (M ⁺), 203 (100), 248,43,55,133,439

Embodiment 20: compd A-2 synthetic

Get A-1400mg (0.881mmol), SeO ₂176mg (1.76mmol), in 50mL round-bottomed flask, adds 20mL diacetyl oxide, and the 5h that refluxes under nitrogen protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous sodium hydroxide solution termination reaction, is extracted with ethyl acetate the saturated NaHCO of organic layer after adding suitable quantity of water ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, the resistates after concentrating under reduced pressure, through silica gel column chromatography (sherwood oil: ethyl acetate), obtains 191mg A-2 (46.7%).

A-2 white powder, 126～129 ℃ of mp, IR (KBr) υ _max1695cm ^-1, 2945cm ^-1, 1459cm ^-1, 1384cm ^-1, ¹h-NMR (CDCl ₃) δ: 7.05～7.02 (1H, d, J=10.4Hz, H-1), 5.82～5.79 (1H, d, J=10.0Hz, H-2), 5.34 (1H, s, H-12), 1.24,1.16,1.14,1.06,0.93,0.90,0.83 (3H × 7, s, 23,24,25,26,27,29,30) ppm, ¹³c-NMR (CDCl ₃) δ: 205.3 (C-3), 184.1 (C-28), 159.0 (C-1), 143.9 (C-13), 125.0 (C-2), 121.8 (C-12), 53.2 (C-5), 46.5 (C-17), 45.5 (C-19), 44.4 (C-14), 41.8 (C-8), 41.6 (C-9), 41.0 (C-18), 39.9 (C-4), 39.4 (C-10), 33.7 (C-22), 33.0 (C-21), 32.3 (C-29), 32.2 (C-7), 30.6 (C-20), 27.7 (C-23), 27.5 (C-15), 25.7 (C-27), 23.4 (C-30), 23.2 (C-16), 22.7 (C-11), 21.5 (C-26), 18.6 (C-6), 18.6 (C-25), 17.4 (C-24), MS (EI) m/z 452 (M ⁺), 203 (100), 55,133,248,437.

Embodiment 21: compd A-3 synthetic

Get 400mg A-2 (0.881mmol) in 50mL round-bottomed flask, add 25mL dissolve with methanol, add the 10%NaOH aqueous solution 53 μ L (1.321mmol), stir after 30min, add 30%H ₂o ₂498 μ L (4.40mmol), at N ₂the lower backflow 3h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous hydrochloric acid regulates pH to neutral, reclaims methyl alcohol, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃the aqueous solution and saturated common salt water washing, then use anhydrous MgSO ₄dry, the resistates after decompression and solvent recovery obtains 317mg A-3 (76.6%) through silica gel column chromatography (sherwood oil: ethyl acetate).

A-3 white powder, 220～224 ℃ of mp, IR (KBr) υ _max1700cm ^-1, 2926cm ^-1, 1468cm ^-1, ¹h-NMR (CDCl ₃) δ: 5.33 (1H, s, H-12), 3.50～3.49 (1H, d, J=4.4Hz, H-2), 3.37～3.36 (1H, d, J=4.4Hz, H-1), 1.20,1.09,0.97,0.95,0.93,0.91,0.80 (3H × 7, s, 23,24,25,26,27,29,30) ppm, ¹³c-NMR (CDCl ₃) δ: 212.8 (C-3), 184.0 (C-28), 143.9 (C-13), 121.6 (C-12), 63.7 (C-1), 56.7 (C-2), 46.3 (C-17), 45.8 (C-5), 46.5 (C-19), 44.6 (C-10), 41.7 (C-14), 41.0 (C-9), 40.4 (C-18), 39.3 (C-8), 38.4 (C-4), 33.7 (C-22), 32.9 (C-29), 32.3 (C-21), 31.9 (C-7), 30.5 (C-20), 29.6 (C-30), 27.8 (C-23), 27.6 (C-15), 25.6 (C-27), 23.6 (C-16), 22.7 (C-11), 20.8 (C-26), 18.6 (C-6), 17.2 (C-25), 14.8 (C-24), MS (EI) m/z 468 (M ⁺), 203 (100), 55,133,248,437,450.

Embodiment 22: compd A-4 synthetic

Get 200mg A-3 (0.427mmol) in 50mL round-bottomed flask, add 25mL dissolve with methanol, at 0～5 ℃, stir after 15min, divide and add NaBH 3 times ₄81mg (2.136mmol), at N ₂the lower reaction 2h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous hydrochloric acid regulates pH to neutral, reclaims methyl alcohol, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, the resistates after decompression and solvent recovery obtains 139mg A-4 (69%) through silica gel column chromatography (sherwood oil: ethyl acetate).

A-4 white powder, 170～172 ℃ of mp, IR (KBr) υ _max1690cm ^-1, 1921cm ^-1, 1434cm ^-1, ¹h-NMR (CDCl ₃) δ: 5.30 (1H, s, H-12), 3.50 (1H, s, H-3), 3.07～3.06 (1H, d, J=3.6Hz, H-2), 3.00～2.99 (1H, d, J=3.6Hz, H-1), 2.82～2.65 (1H, d, J=9.6Hz, H-18), 1.22,1.14,0.93,0.90,0.82,0.80,0.76 (3H × 7, s, 23,24,25,26,27,29,30) ppm, ¹³c-NMR (CDCl ₃) δ: 181.2 (C-28), 143.8 (C-3), 121.5 (C-12), 75.3 (C-3), 60.4 (C-1), 57.6 (C-2), 46.2 (C-17), 45.6 (C-19), 45.5 (C-5), 41.6 (C-14), 41.0 (C-9), 41.0 (C-18), 39.1 (C-4), 36.8 (C-8), 36.2 (C-10), 33.7 (C-22), 32.9 (C-29), 32.3 (C-21), 32.2 (C-7), 30.5 (C-20), 28.2 (C-23), 27.4 (C-15), 25.6 (C-27), 23.3 (C-16), 22.8 (C-11), 17.9 (C-30), 16.8 (C-26), 16.6 (C-25), 16.4 (C-6), 16.2 (C-24), MS (EI) m/z 470 (M ⁺), 248 (100), 55,69,133,203,452.

Embodiment 23: compd A-5, A-6 and isomer thereof synthetic

Get 100mg A-4 (0.213mmol) in 50mL round-bottomed flask, add 25mL acetone solution, add 18 μ L (0.319mmol) perchloric acid, add one of distilled water, at N ₂the lower reaction 18h of protection has reacted (TLC detection reaction process), uses 2molL ^-1aqueous sodium hydroxide solution regulates pH to neutral, reclaims acetone, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains 65mg A-6 (62.7%) and other isomer products [compound (1)～(7)] through silica gel column chromatography (sherwood oil: ethyl acetate).

A-6 white powder, 245～247 ℃ of mp, IR (KBr) υ _max1695cm ^-1, 3443cm ^-1, 2947cm ^-1, 1643cm ^-1, 1386cm ^-1, ¹h-NMR (CD ₃oD) δ: 5.24 (1H, s, H-12), 3.91 (1H, d, J=6.8Hz, H-2), 3.59 (1H, d, J=2.8Hz, H-3), 3.44 (1H, d, J=10.0Hz, H-1), 1.20,1.18,1.04,0.99,0.92,0.88,0.83 (3H × 7, s, 23,24,25,26,27,29,30), ¹³c-NMR (CD ₃oD) δ: 180.5 (C-28), 144.9 (C-13), 123.9 (C-12), 77.4 (C-3), 75.3 (C-1), 74.9 (C-2), 49.6 (C-5), 47.6 (C-17), 47.2 (C-19), 43.4 (C-14), 42.7 (C-18), 41.5 (C-8), 40.3 (C-4), 39.7 (C-9), 39.3 (C-20), 39.2 (C-10), 34.8 (C-21), 33.8 (C-7), 33.6 (C-29), 33.5 (C-22), 30.1 (C-30), 28.8 (C-15), 26.5 (C-27), 24.4 (C-16), 24.0 (C-30), 24.0 (C-11), 19.1 (C-6), 18.1 (C-25), 17.8 (C-24), 14.8 (C-26), MS (EI) m/z:488 (M ⁺), 203 (100), 248,119,133,452,470.

Separate and obtain sample A-5 a small amount of, that take on a red color under sulfuric acid developer simultaneously.By EI-MS and ¹h-NMR can determine that its structure is 1 α, 2 α, 3 α-trihydroxy-olea-12-alkene-28-acid.

Embodiment 24: compd A-b's is synthetic

Get the Oleanolic Acid of 1mmol or its methyl esters or benzyl ester (A-a) and be dissolved in pyridine solvent, under stirring in ice-water bath, slowly drip the Methanesulfonyl chloride of 1.2mmol, at N ₂the lower stirring reaction 5h (TLC detection reaction process) of protection, is extracted with ethyl acetate, and the aqueous hydrochloric acid of 2N washes away pyridine, and gained resistates obtains A-b (white solid powder) through purification by silica gel column chromatography.

Embodiment 25: compd A-c's is synthetic

Get the A-b of 1mmol, with N, 10 milliliters of dissolvings of N '-dimethyl formamide, add Quilonum Retard 2mmol, reflux 1 hour (TLC detection reaction process), is cooled to room temperature, filtering Quilonum Retard, add after suitable quantity of water, be extracted with ethyl acetate, organic phase is washed till neutrality, decompression and solvent recovery, through purification by silica gel column chromatography (sherwood oil: ethyl acetate mixed solvent is eluent), obtain intermediate A-c (white solid powder).

Embodiment 26: compd A-d's is synthetic

Get the A-c of 1mmol, add 10 milliliters of acetic acid and 2mmol tin anhydride (SeO ₂), 5 hours (TLC detection reaction process) refluxes, be cooled to room temperature, filtering tin anhydride, saturated sodium bicarbonate is neutralized to neutrality, adds after suitable quantity of water, be extracted with ethyl acetate, decompression and solvent recovery, residue, through purification by silica gel column chromatography (sherwood oil: ethyl acetate mixed solvent is eluent), obtains intermediate A-d (white solid powder).

Embodiment 27: compd A-e's is synthetic

Get the A-d of 1mmol, add 10 ml methanol and 5 milliliters of 10%NaOH aqueous solution, reflux 1 hour, use 2N HCl to adjust pH value to neutral, ethyl acetate extraction, after recovery solvent, gained residue, through column chromatography for separation, obtains white powder A-e.

Embodiment 28: compd A-f's is synthetic

The A-e of 1mmol is dissolved in the methylene dichloride of 15 milliliters, adds 1.2mmol metachloroperbenzoic acid, under room temperature, at N ₂the lower reaction of protection is spent the night (TLC detection reaction process), and with chloroform extraction, after recovery solvent, residue carries out silica gel column chromatography separating purification, obtains white powder A-f.

Embodiment 29: compd A-g's is synthetic

The A-f that gets 1mmol, adds acetone solution, adds the perchloric acid of 1.5mmol, then adds one of distilled water, at N ₂protection lower reaction 18h (TLC detection reaction process), to neutral, reclaims acetone with aqueous sodium hydroxide solution adjusting pH, and resistates adds water-dispersion, is extracted with ethyl acetate the saturated NaHCO of organic layer ₃solution washing, saturated common salt water washing, anhydrous MgSO ₄dry, after decompression and solvent recovery, resistates obtains A-g through silica gel column chromatography.

Embodiment 30:1,2,3-trihydroxy-olea-12-alkene-28 acid synthetic

Get 1mmolA-g (R is methyl), add 10 milliliters of dimethyl formamides and 1.2mmol lithium iodide, reflux 5 hours, be cooled to after room temperature, add suitable quantity of water, be extracted with ethyl acetate, after reclaiming solvent, residue, through silica gel column chromatography separating purification, obtains 1,2 of corresponding different isomerization body, 3-trihydroxy-olea-12-alkene-28 acid product, i.e. compound (1)～(7); If A-g (R is benzyl), adopts palladium carbon catalytic hydrogenation, can obtain equally 1,2 of different isomerization body, 3-trihydroxy-olea-12-alkene-28 acid product, i.e. compound (1)～(7).

Embodiment 31: R in general formula I ₃for-CH ₂synthesizing of the compound of OH

Get starting compound (1)～(14) of 1mmol, be dissolved in 20mL anhydrous tetrahydro furan solvent, in ice bath, divide and add LiAlH 3 times ₄(5mmol), at room temperature react 10 hours (or refluxing 5 hours) (TLC detection reaction process); After completion of the reaction, use saturated aqueous ammonium chloride termination reaction, with after the hydrochloric acid neutralization reaction liquid of 2N, be extracted with ethyl acetate, after recovery solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for-CH ₂1,2 of OH, 3-trihydroxy-replaces pentacyclic triterpenoid.

Embodiment 32: R in general formula I ₃synthetic for the compound of-CHO

Get 1mmol starting compound (1)～(14), be dissolved in 25mL anhydrous tetrahydro furan solvent, divide and add diisobutyl aluminium hydride (i-Bu 2 times ₂alH) (5mmol), under nitrogen protection, under room temperature, react 12 hours (TLC detection reaction process); After completion of the reaction, use saturated aqueous ammonium chloride termination reaction, with after the hydrochloric acid neutralization reaction liquid of 2N, be extracted with ethyl acetate, after recovery solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for 1,2 of-CHO, 3-trihydroxy-replaces pentacyclic triterpenoid.

Embodiment 33: R in general formula I ₃for-COOR ¹respective compound synthetic

Get the correspondent alcohol R of 1mmol starting compound (1)～(14) and 1.5mmol ¹oH; be dissolved in 30mL dry DMF (dimethyl formamide) solvent; adding the dewatering agent DCC of 3mmol (is N; N-dicyclohexylcarbodiimide) and the DMAP (DMAP) of 0.1mmol, under nitrogen protection, room temperature reaction 24 hours (TLC detection reaction process); add after the aqueous hydrochloric acid termination reaction of 2N; be extracted with ethyl acetate, after recovery solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for-COOR ¹1,2,3-trihydroxy-replace pentacyclic triterpenoid.

Embodiment 34: R in general formula I ₃for-CONHR ¹or-CONR ¹r ²respective compound synthetic

Get the corresponding amine R of 1mmol starting compound (1)～(14) and 1.5mmol ¹nH ₂or R ¹r ²nH; be dissolved in 30mL dry DMF solvent; add the dewatering agent DCC of 3mmol and the DMAP of 0.1mmol; under nitrogen protection; room temperature reaction 24 hours (TLC detection reaction process), adds after the aqueous hydrochloric acid termination reaction of 2N, is extracted with ethyl acetate; after reclaiming solvent, residue, through silica gel column chromatography separating purification, obtains R in corresponding general formula I ₃for-CONHR ¹or-CONR ¹r ²1,2,3-trihydroxy-replace pentacyclic triterpenoid.

Embodiment 35: R in general formula I ₃for-CONH ₂compound synthetic

Get the benzylamine (BnNH of 1mmol starting compound (1)～(14) and 1.5mmol ₂); be dissolved in 30mL anhydrous 1; in 4-dioxane solvent, add the dewatering agent DCC of 3mmol and the DMAP of 0.1mmol, under nitrogen protection; room temperature reaction 24 hours (TLC detection reaction process); add after the aqueous hydrochloric acid termination reaction of 2N, be extracted with ethyl acetate, after recovery solvent, residue is through silica gel column chromatography separating purification; gains are dissolved in 20 ml methanol, obtain R in corresponding general formula I after palladium carbon catalytic hydrogenation reduction (lower 12 hours of room temperature) ₃for-CONH ₂1,2,3-trihydroxy-replace pentacyclic triterpenoid.

Embodiment 36: get the pentacyclic triterpene derivatives quasi-compound that 1,2,3-trihydroxy-replaces, cross 80 mesh sieves after pulverizing, add appropriate amount of starch, dextrin, lactose to mix by equivalent multiplication method, granulate, dry, whole grain, is sub-packed in capsule shell, obtains hard capsule; Be sub-packed in aluminum foil bag, obtain granule; Particle compressing tablet obtains tablet.Each preparation is oral, one day twice, each serving dose, with bulk drug, counts 5-30 milligram.

Embodiment 37: get the pentacyclic triterpene derivatives quasi-compound that 1,2,3-trihydroxy-replaces, cross 80 mesh sieves after pulverizing, add the appropriate vegetables oil containing 10% beeswax, mix; With gelatin: glycerine: distilled water: sanitas=1: prepare capsule material at 0.4: 0.8: 0.003, compacting, obtains soft capsule.Said preparation is oral, one day twice, each serving dose, with bulk drug, counts 5-30 milligram.

Embodiment 38: get the pentacyclic triterpene derivatives quasi-compound that 1,2,3-trihydroxy-replaces, cross 80 mesh sieves after pulverizing, add in the polyoxyethylene glycol of melting, mix, dripping becomes ball in vegetables oil under heat-retaining condition, obtains pill.Said preparation is oral, one day twice, each serving dose, with bulk drug, counts 5-30 milligram.

Claims (13)

1.1,2,3-trihydroxy-replaces pentacyclic triterpenoid, it is characterized in that: the general formula of this compound is following structural formula I:

Ⅰ

Wherein: R ₁represent hydrogen or methyl; R ₂represent hydrogen or methyl; R ₃be selected from-COOH ,-CH ₂oH, CHO ,-COOR ¹,-CONH ₂,-CONHR ¹in any; R ¹be selected from the alkyl that contains 1-8 carbon atom; C ₁hydroxyl replace and be configured as α configuration, C ₂hydroxyl replace and be configured as α configuration or beta comfiguration, C ₃hydroxyl replace and be configured as α configuration.

2. according to claim 11,2,3-trihydroxy-replaces pentacyclic triterpenoid, it is characterized in that: R in formula I ₁for hydrogen, R ₂for methyl, R ₃for-COOH; Or R ₁for methyl, R ₂for hydrogen, R ₃for-COOH.

3. according to claim 21,2,3-trihydroxy-replaces pentacyclic triterpenoid, it is characterized in that: described compound is (1) 1 α 2 α, 3 α-trihydroxy-olea-12-alkene-28-acid; (2) 1 α, 2 β, 3 α-trihydroxy-olea-12-alkene-28-acid; (8) 1 α, 2 α, 3 α-trihydroxy-black bearberry-12-alkene-28-acid; (9) 1 α, 2 β, 3 α-trihydroxy-black bearberry-12-alkene-28-acid.

4. as claimed in claim 21,2,3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: get ursolic acid U-1, through Jones reagent oxidation, obtain compound U-2, U-2 obtains compound U-3 through benzyl bromobenzyl, and U-3 is through SeO ₂be oxidized to obtain α, beta unsaturated ketone Se-1, Se-1 obtains U-15 under the effect of hydrogen peroxide, sodium borohydride reduction U-15 obtains R-10-2 intermediate, then obtains FU-16 through perchloric acid hydrolysis, obtains 1 of different isomerization body after palladium carbon catalytic hydrogenation, 2,3-trihydroxy-black bearberry-12-alkene-28 acid; Its reaction scheme is as follows:

.

5. as claimed in claim 21,2,3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: get ursolic acid U-1, through Jones reagent oxidation, obtain compound U-2, U-2 is through SeO ₂be oxidized to obtain α, beta unsaturated ketone Se, Se obtains U-15-1 under the effect of hydrogen peroxide, and sodium borohydride reduction U-15-1 obtains R-10-21 intermediate, then obtains 1,2 of different isomerization body through perchloric acid hydrolysis, 3-trihydroxy-black bearberry-12-alkene-28 acid; Its reaction scheme is as follows:

.

6. as claimed in claim 21, 2, 3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: get ursolic acid or its methyl esters or its benzyl ester U-a and under Methanesulfonyl chloride effect, obtain U-b, U-b is through Quilonum Retard processing, obtain intermediate U-c, U-c is oxidized to obtain U-d by tin anhydride, after the NaOH aqueous solution is processed, decarboxylation obtains U-e again, intermediate U-e obtains epoxidation intermediate U-f under metachloroperbenzoic acid effect, U-f obtains 1 after perchloric acid hydrolysis, 2, 3-trihydroxy-intermediate U-g, after adopting palladium carbon catalytic hydrogenation or demethylating reaction, obtain 1 of different isomerization body, 2, 3-trihydroxy-black bearberry-12-alkene-28 acid product, its reaction scheme is as follows:

。

7. as claimed in claim 21,2,3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: get Oleanolic Acid, become A-1, by SeO through Jones reagent oxidation ₂be oxidized to obtain α, beta unsaturated ketone A-2, A-2 obtains A-3 under the effect of hydrogen peroxide, and sodium borohydride reduction A-3 obtains A-4 intermediate, through perchloric acid hydrolysis, obtains 1,2 of different isomerization body, 3-trihydroxy-olea-12-alkene-28 acid; Its reaction scheme is as follows:

.

8. as claimed in claim 21, 2, 3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: get Oleanolic Acid or its methyl esters or its benzyl ester A-a and under Methanesulfonyl chloride effect, obtain A-b, A-b is through Quilonum Retard processing, obtain intermediate A-c, A-c is oxidized to obtain A-d by tin anhydride, after the NaOH aqueous solution is processed, decarboxylation obtains A-e again, intermediate A-e obtains epoxidation intermediate A-f under metachloroperbenzoic acid effect, A-f obtains 1 after perchloric acid hydrolysis, 2, 3-trihydroxy-intermediate A-g, after adopting palladium carbon catalytic hydrogenation or demethylating reaction, obtain 1 of different isomerization body, 2, 3-trihydroxy-olea-12-alkene-28 acid product, its reaction scheme is as follows:

。

9. as claimed in claim 11,2,3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: with the acid of 1,2,3-trihydroxy-olea-12-alkene-28 or the acid of 1,2,3-trihydroxy-black bearberry-12-alkene-28 for raw material, at LiAlH ₄effect issues raw reduction reaction, obtains R in formula I ₃for-CH ₂the respective compound of OH; Its reaction scheme is as follows:

。

10. as claimed in claim 11,2,3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: with the acid of 1,2,3-trihydroxy-olea-12-alkene-28 or the acid of 1,2,3-trihydroxy-black bearberry-12-alkene-28 for raw material, with diisobutyl aluminium hydride i-Bu ₂alH reaction, obtains R in formula I ₃for the respective compound of-CHO; Its reaction scheme is as follows:

。

11. is as claimed in claim 11,2, and 3-trihydroxy-replaces the preparation method of pentacyclic triterpenoid, it is characterized in that: with 1,2,3-trihydroxy-olea-12-alkene-28 acid or 1,2, the acid of 3-trihydroxy-black bearberry-12-alkene-28 is raw material, under dewatering agent DCC or WSC effect, with corresponding alcohol R ¹oH or amine R ¹nH ₂, BnNH ₂reaction, obtains corresponding ester class or amides, i.e. R in formula I ₃for-COOR ¹,-CONH ₂or-CONHR ¹respective compound; Its reaction scheme is as follows:

。

12. as described in any one in claim 1-3 1,2,3-trihydroxy-replaces pentacyclic triterpenoid in the application of preparing in hepatic, it is characterized in that: the pharmacologically acceptable salt that described compound or described compound dissolution are made in sodium hydroxide, potassium hydroxide or ammonia soln is made hepatic preparation with suitable auxiliary material.

13. is according to claim 12 1,2, and 3-trihydroxy-replaces pentacyclic triterpenoid in the application of preparing in hepatic, it is characterized in that: described pharmaceutical preparation is hard capsule, tablet, oral liquid, soft capsule, granule or pill.