CN104910238B

CN104910238B - One class pentacyclic triterpenoid and its purposes in the medicine for preparing treatment alzheimer disease

Info

Publication number: CN104910238B
Application number: CN201510079373.7A
Authority: CN
Inventors: 南发俊; 谢欣; 裴钢; 王宵音; 崔进; 赵简; 张仰明; 张晨露
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2014-03-14
Filing date: 2015-02-13
Publication date: 2017-08-11
Anticipated expiration: 2035-02-13
Also published as: CN104910238A; WO2015135474A1

Abstract

Lead to the pentacyclic triterpenoid shown in formula (I) and the pharmaceutical composition comprising such compound the invention discloses a class, and, the present invention discloses purposes of the compound in PS1/BACE1 interaction inhibitors are prepared, and its purposes in the medicine for preparing treatment alzheimer disease.The pentacyclic triterpenoid led to present invention firstly discloses a class shown in formula (I) can be used as PS1/BACE1 inhibitor, activity with the interphase interaction for suppressing PS1/BACE1, A β generation can be reduced, available for the medicine for preparing treatment alzheimer disease.

Description

One class pentacyclic triterpenoid and its medicine in preparation treatment alzheimer disease In purposes

Technical field

The invention belongs to medicinal chemistry art, and in particular to a class pentacyclic triterpenoid, include such compound Pharmaceutical composition and such compound and pharmaceutical composition are preparing PS1 (PS1, Presenilin1, Presenilin 1)/BACE1 (β catabolic enzymes 1 before β site Amyloid Protein Precursor Cleaving Enzyme 1, amyloid proteins, also known as For beta-secretase) purposes in interaction inhibitor, and its purposes in the medicine for preparing treatment alzheimer disease.

Background technology

Alzheimer disease (Alzheimer ' s Disease, AD) is a kind of can not be cured, progressive cerebral disorders, It is the main reason of alzheimer's disease.AD can cause a large amount of nerve cell deaths, the normal cognitive and behavior energy of destruction patient Power, finally makes the basic self care ability of patient's forfeiture and physiological function until death (Prince etc., World Alzheimer Report 2009.London:Alzheimer's Disease International,2009,14-5； Blennow etc., Alzheimer's disease.Lancet, 2006,368 (9533):387-403).AD is main more than 60 years old Old man in occur, wherein 60-64 Sui interval people suffer from AD ratio below 1%.But with advancing age, often increase by 5 Year, the AD incidences of disease will be doubled, and the AD incidences of disease are up to 23-33% in more than 85 years old old man.Due to lacking effective healing hand Section, the AD illness cycles are many decades, and heavy family burden is brought to family numbers of patients.As China human mortality aging adds Play, Development of Novel AD medicines seem particularly necessary and urgent.

At this stage, AD clinical treatment medicine is mainly anticholinesterase (as double benefits are flat, rivastigmine Spit of fland, Doneppezil Hydrochloride, galanthamine etc.) and NMDA (N-Methyl-D-Aspartic Acid, N-methyl-D-aspartate) Receptor antagonist (such as Memantine hydrochloride).Though these medicines can alleviate AD patient symptoms, improve the cognition of patient to a certain extent And capacity, but offer limited effectiveness, it is impossible to fundamentally reverse disease progression (Kastenholz etc., A novel perspective in dementia therapy.Amyloid-Journal of Protein Folding Disorders, 2009,16(2):81-3)。

It is existing more and more to grind although the pathogenesis currently for Alzheimer's disease does not obtain complete understanding yet Study carefully evidence and show generation and deposition of the AD generation with a large amount of A β of patient's intracerebral (Amyloid β-protein, amyloid-beta) And thus caused neurotoxicity infringement is relevant.A β are the amyloid protein precursor (Amyloid on cell membrane Precursor Protein, APP) shearing hydrolysis generation.APP shearing includes non-starch sample approach and amyloid approach：The former Refer to APP by alpha-secretase enzyme hydrolysis, generation soluble APP (sAPP)；The latter refer to APP successively through β-and gamma-secretase shear production The raw small peptide A β containing 38~43 amino acid, particularly by the insoluble Α β of 42 Amino acid profiles₄₂Polymerization is easiest to be formed Oligomer, fiber and amyloid plaques, wherein A beta oligomers are maximum to neuron and the toxicity of cynapse, cause nerve cell to damage Wound or even death.Because A β depositions are the main causes of Alzheimer's disease patient brain tissue person in middle and old age spot formation, and may It is the common pathway that other factorses cause AD to fall ill.Therefore, the medicine for producing for A β and assembling and researching and developing can be by reducing A β The approach such as generation and polymerization, increase A β degradeds, promotion A β removings reduces intracerebral A β depositions, so as to postpone AD state of an illness processes, and has AD generation and progress may fundamentally be reversed.

Existing multiple targeting A β medicine enters clinical test at present, wherein three secretases related to A β generations are to work as The important target spot of preceding AD medicament research and developments：As alpha-secretase enzyme activator can increase the generation (such as cevimeline) of soluble APP；And β- Secretase inhibitors can then reduce indirectly A β generation (as in clinical stage study beta-secretase inhibitor AZD-3293, VTP-37948 and HPP-854 etc.)；Inhibitors of gamma-secretase can directly reduce A β generation (such as Avagacestat, Elnd- 007, Semagacestat, MK-0752 and PF-3084014 etc.)；And gamma secretase modulators (such as EVP-0962, CHF- 5074 etc.) A β can be reduced by adjusting gamma-secretase substrate cleavage pattern₄₂Generation, but to the other substrates of gamma-secretase The shear action of (such as Notch) does not substantially suppress, therefore reduces the shadow to other substrate associated signal paths of gamma-secretase Ring.

Although the medicament research and development for A β obtains greater advance in recent years, not yet there is related drugs approval listing.By It is a kind of multi-pathogenesis, the nerve retrograde affection for carrying out sexual development in AD, its incidence of occult, the course of disease are long, and pathologic process is complicated, because This is still in the urgent need to developing the AD medicines with novel therapeutic mechanism.As it was previously stated, A β generation is related to two important secretions Enzyme, i.e. BACE1 and the gamma-secretase compound by catalytic subunit of PS1, and they and non-orphaned exist and play hydrolysis function , but there is certain to interact and contact.2003, Sebastien et al. was proved by co-immunoprecipitation experiment, Direct protein-protein interaction is there is between both important albumen of PS1 and BACE1：PS1 can by with prematurity BACE1 combine, regulation and control BACE1 maturation and activity (Hebert etc., Presenilin-1interacts directly with the beta-site amyloid protein precursor cleaving enzyme(BACE1) .Neurobiology of Disease,2003,13(3):238-45).So if PS1/BACE1 interaction suppressions can be developed Preparation, by suppressing protein-protein interaction between PS1/BACE1, reduces A β generation, this class compound can be used for making Into new AD medicines.

The content of the invention

(this is application number to Chinese patent application the 2014100976413rd, and patent application is not yet announced, and the application will Seek the priority of the patent application of above-mentioned Application No. 2014100976413) it is that a Chinese invention before the applicant is special Profit application, it discloses a class pentacyclic triterpenoid, can excitement TGR5 (G-protein coupling cholic acid membrane receptor) biological living Property, and can be used for preparing Remedies for diabetes.In further further investigation, in Chinese invention patent application the On the screening model of PS1/BACE1 inhibitor disclosed in No. 201410673701, the applicant is surprised to find that The there is provided part pentacyclic triterpenoid of No. 201410097641 applications and some other pentacyclic triterpenoid tools There is the activity for suppressing PS1/BACE1 interactions, and A β generation can be reduced, so as to for AD therapy to be made Medicine.No. 2014100976413 Chinese patent application is introduced into the application in full herein.

It is an object of the invention to provide the pentacyclic triterpenoid shown in below formula I and its in preparation PS1/ Purposes in BACE1 interaction inhibitors, the compound can suppress the interaction between PS1/BACE1, and can drop Low A β generation, so as to the medicine for preparing treatment alzheimer disease.

According to the purpose of the present invention, the invention provides the pentacyclic triterpenoid shown in logical formula (I),

Wherein：

R₁For hydrogen, halogen, cyano group or hydroxyl；Preferably, R₁For hydrogen or hydroxyl；

R₂For hydroxyl, halogen, oxo base (=O) ,=N-OH, C₁-C₆Alkyl carbonyl epoxide, C₃-C₈Naphthene base carbonyl epoxide OrPreferably, R₂For hydroxyl, oxo base (=O) ,=N-OH, C₁-C₆Alkyl carbonyl epoxide, C₃-C₈Cycloalkyl carbonyl Base epoxide orPreferably, R₂It is connected with α configurations with right side A rings；

Wherein, R₉For hydrogen or C₁-C₄Straight or branched alkyl；

R₃And R₄It is each independently C that is unsubstituted or being optionally substituted by a hydroxyl group₁-C₄Straight or branched alkyl；Preferably, R₃And R₄ It is each independently C₁-C₄Straight or branched alkyl；It is highly preferred that R₃And R₄It is simultaneously methyl；

R₅The C replaced for formoxyl (carboxaldehyde radicals), carboxyl, unsubstituted or hydroxyl₁-C₄Straight or branched alkyl,Preferably, R₅The C replaced for carboxaldehyde radicals, hydroxyl₁-C₄Straight or branched alkane Base, carboxyl,More preferably R₅The C replaced for hydroxyl₁-C₄Alkyl, carboxyl,

Wherein, R₁₀For C₁-C₈Straight or branched alkyl, C₃-C₈Cycloalkyl, C that is unsubstituted or being replaced by carboxyl₁-C₈Straight chain Or branched alkyl, carboxyl C₁-C₈Alkylidene or C that is unsubstituted or being replaced by carboxyl₃-C₈Cycloalkyl；It is preferably unsubstituted or by The C of carboxyl substitution₁-C₈Straight or branched alkyl or carboxyl C₁-C₈Alkylidene；C that is more preferably unsubstituted or being replaced by carboxyl₁- C₅Straight or branched alkyl or carboxyl C₁-C₇Alkylidene；

R₆And R₇It is each independently hydrogen or C₁-C₄Straight or branched alkyl；It is preferred that hydrogen, methyl, ethyl or propyl group；Or R₆ And R₇Connected carbon atom is collectively forming=CH₂；

R₈For hydrogen, substituted or unsubstituted C₁-C₆Straight or branched alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substitution Or unsubstituted C₂-C₆Straight or branched alkenyl, substituted or unsubstituted C₂-C₆Alkynyl group, substituted or unsubstituted C₁-C₆Alkyl Carbonyl, substituted or unsubstituted C₁-C₆Alkyl amine group,Preferably, R₈For hydrogen, substituted or unsubstituted C₁-C₆Directly Chain or branched alkyl, substituted or unsubstituted C₂-C₆Straight or branched alkenyl, substituted or unsubstituted C₁-C₆Alkyl-carbonyl orThe substituent is optionally from hydroxyl, C₁-C₃Alkanoyl, the C of hydroxyl substitution₁-C₄Alkyl-carbonyl ,=N-OH, amino, The C of hydroxyl substitution₁-C₆Alkyl, the C of unsubstituted or hydroxyl substitution₁-C₆Alkyl amine group orIn one or more, it is excellent Selection of land, the substituent is selected from hydroxyl, formoxyl, acetyl group ,=N-OH, the C of unsubstituted or hydroxyl substitution₁-C₆Alkyl amine group orOne or both of；

Wherein, R₁₁For H, substituted or unsubstituted C₁-C₆Alkyl, preferably substituted or unsubstituted C₁-C₄Alkyl, it is described to take For the optional hydroxyl of base, amino,

Wherein, R₁₂For substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₈Cycloalkyl, substitution do not take The C in generation₁-C₄Alkoxy, substituted or unsubstituted C₆-C₁₀Aryl is substituted or unsubstituted at least containing in N, S or O Heteroatomic 5 to 8 unit's heteroaryl, it is preferable that R₁₂For substituted or unsubstituted C₁-C₆It is alkyl, substituted or unsubstituted C₃-C₈Cycloalkyl, substituted or unsubstituted C₆-C₁₀Aryl is substituted or unsubstituted miscellaneous containing one in N, S or O 5 to 8 unit's heteroaryls of atom, it is highly preferred that R₁₂For substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Ring Alkyl, substituted or unsubstituted phenyl or substituted or unsubstituted thienyl；The substituent is optionally from hydroxyl, halogen, cyanogen Base, C₁-C₄Alkoxy, the C of BocNH- substitutions₁-C₄Alkoxy,Preferably, The substituent is selected from hydroxyl, F, Cl, Br, C₁-C₄Alkoxy, the C of BocNH- substitutions₁-C₄Alkoxy,

Wherein, R₁₃For C₁-C₆Alkyl or C₆-C₁₀Aryl, preferably C₁-C₆Alkyl or phenyl；

Wherein, m and n each stand alone as 1,2,3 or 4；

Z is methylene or is not present；

Represent that this sentences singly-bound or double bond connection.

Preferably, the pentacyclic triterpenoid of the logical formula (I) structure of the present invention is chosen in particular from following structure：

There is provided the compound shown in logical formula (I) and specific chemical combination listed above according to another aspect of the present invention Purposes of the thing in PS1/BACE1 interaction inhibitors are prepared.

There is provided the compound shown in logical formula (I) and specific chemical combination listed above according to another aspect of the present invention Purposes of the thing in the medicine for preparing treatment alzheimer disease；Wherein, mutual-through type (I) compound is as defined above；

In the purposes, compound and particular compound listed above shown in logical formula (I) are used as PS1/BACE1 Interaction inhibitor.

Present invention also offers a kind of pharmaceutical composition for treating alzheimer disease, said composition is comprising described selected from logical One or more in pentacyclic triterpenoid shown in formula (I) are used as active component.The composition simultaneously can be wrapped further Containing pharmaceutically conventional assistant agent, such as dispersant, excipient, disintegrant, antioxidant, sweetener, coating agent.

The present invention has the advantages that：

Can have present invention firstly discloses a class pentacyclic triterpenoid as PS1/BACE1 interaction inhibitors There is the activity for the interphase interaction for suppressing PS1/BACE1, A β generation can be reduced, alzheimer disease is treated available for preparing Medicine.

Brief description of the drawings

Fig. 1 shows the influence that the compounds of this invention C46 is produced for transgenic models mouse A β 42.In figure, control group Mouse n=8, administration group mouse n=6, error line represents standard error (* p<0.05).

Embodiment

First, the source of the application compound and preparation

Compound C1 (3- α-Akebonoic acid, 3- α-akebi terpene acid), C2 (Oleanolic acid, olive Acid), C5 (Betulonic acid, birch bucket acid), C6 (Corosolic acid, Corosolic acid), C7 (Ursolic acid, Ursolic acid), C8 (3-Oxo-Ursolic acid, 3- carbonyl-ursolic acid), C19 (Uvaol, uvaol) and C29 (Betulic Acid, betulic acid) it is the commercially available natural products ((being purchased from Xi'an Hao Xuan bio tech ltd)) bought.Remaining Compound according to following the methods disclosed herein or can refer to Chinese patent application the 2014100976413rd and be prepared into Arrive.

In following preparation embodiments, the Mercury-Vx 300M Instrument measurings that NMR (nuclear magnetic resonance) is produced with Varian, Internal standard：δH 7.26ppm(CDCl₃), 2.50ppm (DMSO-d₆)；The mass spectrum Quadrupole LC/MS liquid of Agilent 1200 Matter combined instrument is determined；Agents useful for same is provided by Solution on Chemical Reagents in Shanghai company；TLC tlc silica gels plate is by Shandong Yantai fellow member of an association or organization's silicon Glue development corporation, Ltd. produces, model HSGF 254；The normal phase column chromatography silica gel that compound purifying is used is Qingdao ocean Chemical plant subsidiary factory produces, model zcx-11,200-300 mesh.

Prepare embodiment 1：Compound C33 synthesis

(1) betulic acid benzyl ester

Raw material betulic acid (4g, 8.76mmol) (being purchased from Xi'an Hao Xuan bio tech ltd) is dissolved at room temperature In DMF (50mL), add Anhydrous potassium carbonate (2.4g, 17.37mmol), be slowly added dropwise under stirring benzyl chloride (1.2mL, Reaction solution 10.52mmol) is moved into 50 DEG C after completion of dropping to be stirred overnight.Next day, mixture is cooled to room temperature, addition go from Sub- water 100mL dilutions, are extracted with ethyl acetate (2 × 100mL), the organic layer of merging are used into deionized water and saturated common salt respectively Water washing, compound as white solid betulic acid benzyl ester (4.62g) needed for rear and vacuum distillation dried over sodium sulfate is obtained, mole Yield：97%.¹H NMR(300MHz,CDCl₃) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J= 11.7Hz),4.75(s,1H),4.62(s,1H),3.21-3.15(m,1H),2.92-2.80(m,1H),2.10-1.90(m, 2H), 1.87-1.69 (m, 2H), 1.64 (s, 3H), 1.64-0.96 (m, other cycloaliphatic ring protons), 1.04 (s, 3H), 1.00 (s, 3H),0.94(s,3H),0.91(s,3H),0.87(s,3H),0.84(s,3H)；ESI-MS(m/z):569.4(M+Na)⁺ (C₃₇H₅₄O₃Theoretical value：546.41).

(2) 3- carbonyls betulic acid benzyl ester

Upper step product (4.62g, 8.64mmol) is dissolved in dichloromethane (100mL) under ice-water bath, Dess- is added portionwise Martin oxidants (4.3g, 10.15mmol), are slowly warmed to room temperature and are stirred overnight.Next day, revolved after reactant mixture is filtered It is dry, it is 20 with petrol ether/ethyl acetate:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- carbonyl betulic acids Benzyl ester (4.39g), is white solid, molar yield：95%.¹H NMR(300MHz,CDCl₃)δ7.34(m,5H),5.09(d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 4.75 (s, 1H), 4.62 (s, 1H), 2.92-2.80 (m, 1H), 2.49-2.39(m,2H),2.10-2.04(m,2H),1.92-1.80(m,2H),1.78-1.68(m,2H),1.65(s,3H), 1.50-1.16 (m, other cycloaliphatic ring protons), 1.04 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s,3H),0.84(s,3H)；ESI-MS(m/z):567.3(M+Na)⁺(C₃₇H₅₂O₃Theoretical value：544.39).

(3) 3- Alpha-hydroxies betulic acid benzyl ester

Step product (1.58g, 2.90mmol) and S- (-) -2- methyl oxazoles on being added in the round-bottomed flask dried to 100mL Borine (80mg, 0.29mmol), and add the treated THF (50mL) of fresh sodium silk.At room temperature slowly be added dropwise 10M borine- Tetrahydrofuran solution (0.32mL), controls rate of addition, was added in ten minutes, stirs ten minutes at room temperature, TLC monitoring displays Reaction has been completed.Reaction bulb is moved into ice-water bath, methanol is slowly added dropwise reaction is quenched, be spin-dried for after there is no bubble formation molten Agent, is 20 with petrol ether/ethyl acetate:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- Alpha-hydroxy white birch fat Acid benzyl ester (790mg), is white solid, molar yield：50%.¹H NMR(300MHz,CDCl₃)δ7.34(m,5H),5.09(d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 4.73 (s, 1H), 4.60 (s, 1H), 3.39 (s, 1H), 3.02-2.96 (m, 1H), 2.28-2.16 (m, 2H), 1.98-1.95 (m, 2H), 1.68 (s, 3H), 1.64-0.96 (m, other cycloaliphatic ring matter Son), 1.04 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H)；ESI-MS (m/z):569.4(M+Na)⁺(C₃₇H₅₄O₃Theoretical value：546.41).

(4) 3- Alpha-hydroxies betulic acid C33

Upper step product (100mg, 0.18mmol) is dissolved in methanol (8mL) and a small amount of ethyl acetate, is changed after nitrogen, rapid to add Enter 10% Pd/C, then change hydrogen is changed after nitrogen, be stirred at room temperature.TLC detections reaction is complete after one hour.Change to filter off after nitrogen and urge Agent, reaction solution uses petrol ether/ethyl acetate to be 10 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound C33 (78mg), is white solid, molar yield：94%.¹H NMR(300MHz,CDCl₃)δ4.73(s,1H),4.60(s,1H), 3.39(s,1H),3.02-2.96(m,1H),2.28-2.16(m,2H),1.98-1.95(m,2H),1.68(s,3H),1.64- 0.96 (m, other cycloaliphatic ring protons), 1.04 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H),0.84(s,3H)；ESI-MS(m/z):479.3(M+Na)⁺(C₃₀H₄₈O₃Theoretical value：456.36).

Prepare embodiment 2：Compound C35 synthesis

Betulic acid (48mg, 0.11mmol) is dissolved in ethanol/methylene (5mL/5mL), is passed through at -78 DEG C smelly Oxygen, TLC display reactions are complete.Stop being passed through the ozone for draining remnants after ozone with oxygen, add dimethyl sulphide (25 μ L) and be quenched Reaction, is slowly warming up to room temperature, and reaction is stayed overnight.Next day, reaction solution is concentrated under reduced pressure, and gained residue is purified through silica gel column chromatography (methylene chloride/methanol=50/1, V/V), obtains compound C35 (21mg, yield 43.6%).¹H NMR(300MHz,CDCl₃), 1.00(s,3H),0.96(s,3H),0.91(s,3H),0.82(s,3H),0.75(s,3H)；ESI-MS(m/z):481.3(M+ Na)⁺(C₂₉H₄₆O₄Theoretical value：458.34).

Prepare embodiment 3：Compound C41 synthesis

Compound C42 (12mg, 0.02mmol) is dissolved in ethanol/water (4mL/1mL) in the mixed solvent, adds 2N sodium hydroxides (1mL), is stirred overnight at room temperature.Next day, TLC detection reactions are complete, and the solvent that is concentrated under reduced pressure is removed after ethanol, adjusted with 1N hydrochloric acid solutions To pH=3, then it is extracted with ethyl acetate, is washed after merging organic phase with saturated nacl aqueous solution, organic phase is dried, concentration, Gained residue purifies (methylene chloride/methanol=10/1, V/V) through silica gel column chromatography, obtains compound C41 (6mg, yield： 50%), white solid.¹H NMR(300MHz,CDCl₃) δ 4.78 (s, 1H), 2.28-2.18 (m, 4H), 1.98-1.15 (m, its Remaining aliphatic acyclic hydrocarbon proton), 1.01 (s, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.83 (s, 3H), 0.76 (s,3H),0.74(s,3H)；ESI-MS(m/z):553.3(M+Na)⁺(C₃₂H₅₀O₆Theoretical value：530.36).

Prepare embodiment 4：Compound C45 synthesis

According to the method same with preparing embodiment 23, using compound C38 as raw material, it can synthesize and obtain compound C45 (always Yield 58%, white solid).¹H NMR(300MHz,CDCl3)δ3.78(m,4H),3.39(s,1H),2.78(m,2H),2.67 (m,2H),2.36-2.23(m,8H),1.76-1.09(m,other aliphatic ring protons),0.96(s,3H), 0.93(s,3H),0.92(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.78(s,3H)；ESI-MS(m/ z):649.5(M+Na)⁺, (C₃₉H₆₆N₂O₄Theoretical value：626.50).

Prepare embodiment 5：Compound C52 synthesis

Preparing for intermediate S11 (3- alpha-hydroxy-2 0- formoxyl betulic acids benzyl ester) is specific as follows.

3- Alpha-hydroxy betulic acid benzyl esters are prepared with reference to the method for embodiment 1.

(1) 3- alpha-hydroxy-2s 0,21- epoxy betulic acid benzyl esters

Intermediate 3- Alpha-hydroxy betulic acid benzyl esters (90mg, 0.17mmol) are dissolved in carbon dichloride (10mL), ice-water bath Under be slowly added into after metachloroperbenzoic acid (71mg, 0.34mmol) 3h be stirred at room temperature, TLC detection reactions are complete, add sulfurous Reaction is quenched in sour sodium saturated solution, and organic phase is washed with water, and it is 6 to dry concentration gained residue petrol ether/ethyl acetate:1 Eluant, eluent system carry out column chromatography for separation, obtain compound 3- alpha-hydroxy-2s 0,21- epoxy betulic acid benzyl ester 74mg, are white Color solid, molar yield：80.4%.¹H NMR(300MHz,CDCl₃) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 3.40 (s, 1H), 2.64 (t, 2H), 2.25 (m, 2H), 2.14 (m, 2H), 1.97 (m, 2H), 1.78 (m, 2H), 1.76-1.32 (m, other cycloaliphatic ring protons), 1.28 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.92 (s,3H),0.90(s,3H),0.86(s,3H),0.82(s,3H)；ESI-MS(m/z):562.4(M+Na)⁺(C₃₇H₅₄O₄It is theoretical Value：562.40).

(2) 3- alpha-hydroxy-2s 0- formoxyl betulic acids benzyl ester (S11)

Intermediate 3- alpha-hydroxy-2s 0,21- epoxy betulic acid benzyl esters (68mg, 0.12mmol) are dissolved in carbon trichloride In (10mL), instill two and drip concentrated hydrochloric acid, flow back 1h, TLC detection reactions are complete, convection drying concentration gained residue oil Ether/ethyl acetate is 4:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- alpha-hydroxy-2 0- formoxyl white birch fat Acid benzyl ester 50mg, is white solid, molar yield：73.6%.¹H NMR(300MHz,CDCl₃) δ 9.84 (s, 1H), 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 3.39 (s, 1H), 3.33 (d, 1H, J=4.2Hz), 2.40 (td, 1H, J=11.7,2.7Hz), 2.28 (td, 1H, J=11.7,2.7Hz), 2.30-2.18 (m, 2H), 1.98-1.84 (m, 2H), 1.69-0.96 (m, other cycloaliphatic ring protons), 1.11 (s, 3H), 0.93 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H),0.87(s,3H),0.78(s,3H)；ESI-MS(m/z):585.4(M+Na)⁺(C₃₇H₅₄O₄Theoretical value：562.40).

Intermediate S11 (19mg, 0.034mmol) is dissolved in fresh dry THF (tetrahydrofuran) (2mL), in -78 DEG C The tetrahydrofuran solution (52 μ L, 0.050mmol) of lower dropwise addition 1N methyl-magnesium-bromides, is then slowly increased to ambient temperature overnight.Next day, chlorine Change ammonium saturated solution and reaction is quenched, after ethyl acetate is extracted and dried, be concentrated under reduced pressure to obtain intermediate S19, directly carries out next step Reaction.

Intermediate S19 is dissolved in dichloromethane (2mL), addition Dess-Martin oxidants (DMP) (21mg, 0.050mmol), 3h is stirred at room temperature, TLC detection reactions are complete.Reactant mixture is filtered, filtrate decompression concentration, residue warp Silica gel column chromatography (ethyl acetate/petroleum ether=1/20, V/V), obtains intermediate S20 (7mg, two step yields 35.8%), and white is solid Body.¹H NMR(300MHz,CDCl₃) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 3.39 (s, 1H), 3.24 (td, 1H, J=9.0,3.0Hz), 2.28 (m, 2H), 2.18 (s, 3H), 2.14-2.06 (m, 2H), 2.04-1.88 (m, 4H), 1.64-1.20 (m, remaining aliphatic acyclic hydrocarbon proton), 1.25 (s, 3H), 1.02 (s, 3H), 0.93 (s, 3H),0.91(s,3H),0.83(s,3H),0.81(s,3H)。

Intermediate S20 is dissolved in methanol and a small amount of ethyl acetate, is changed after nitrogen, adds the Pd/C of catalytic amount, hydrogen displacement Afterwards, reaction is stirred at room temperature, the reaction of TLC detecting and trackings is complete.Filtering, gained crude product silica gel column chromatography is purified after filtrate concentration Compound C52 (5mg, yield 84.8%), white solid.¹H NMR(300MHz,CDCl3)δ3.39(s,1H),3.24(td, 1H, J=9.0,3.0Hz), 2.28 (m, 2H), 2.18 (s, 3H), 2.14-2.06 (m, 2H), 2.04-1.88 (m, 4H), 1.64- 1.20 (m, remaining aliphatic acyclic hydrocarbon proton), 1.25 (s, 3H), 1.02 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.83 (s, 3H),0.81(s,3H)；ESI-MS(m/z):509.3(M+Na)⁺(C₃₁H₅₀O₄Theoretical value：486.37).

Prepare embodiment 6：Compound C49 synthesis

The preparation method of compound C52 in embodiment 5, intermediate S11 (3- alpha-hydroxy-2 0- formyls are prepared according to the application Base betulic acid benzyl ester) to be reacted with n-propyl grignard reagent, then hydrogenation removes benzyl, and crude product is purified through silica gel column chromatography again Obtain compound C49, white solid.¹H NMR(300MHz,CDCl₃)δ3.39(m,1H+1H),2.41(m,1H),2.28(m,2H), 1.98-1.80 (m, 2H), 1.69-1.20 (m, remaining aliphatic acyclic hydrocarbon proton), 0.97 (d, 3H, J=6.0Hz), 0.95 (s, 3H), 0.93 (s, 3H), 0.88 (s, 3H), 0.85 (t, 3H, J=3.0Hz), 0.84 (s, 3H), 0.81 (s, 3H)；ESI-MS(m/z): 539.4(M+Na)⁺(C₃₁H₅₀O₄Theoretical value：516.42).

Prepare embodiment 7：Compound C51 synthesis

Intermediate S11 (3- alpha-hydroxy-2 0- formoxyl betulic acids benzyl ester) (8mg, 0.014mmol) and n-amylamine (5 μ L, Absolute ethyl alcohol 0.043mmol) is dissolved in, sodium cyanoborohydride (3mg, 0.043mmol) is then added, reaction system is stirred at room temperature Reaction is stayed overnight.Second day, TLC detection reactions were complete, and reaction is quenched in saturated sodium bicarbonate solution, were diluted with water rear ethyl acetate extraction Take twice, merge organic phase saturated common salt water washing, dry gained residue after concentration purify through silica gel column chromatography (chloroform/ Methanol=20/1, V/V), obtain intermediate S17 (9mg, quantitative yield), white solid.¹H NMR(300MHz,CDCl₃)δ7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 3.39 (s, 1H), 2.90 (t, 2H, J= 4.5Hz), 2.75 (t, 2H, J=4.5Hz), 2.37-2.03 (m, 2H), 1.97-1.67 (m, 4H), 1.65-0.90 (m, remaining fat Fat cyclic hydrocarbon proton), 0.93 (s, 3H), 0.91 (t, 3H, J=3.0Hz), 0.66 (s, 3H), 0.62 (d, 3H, J=6.0Hz), 0.58(s,3H),0.52(s,3H),0.48(s,3H)。

Intermediate S17 is dissolved in methanol and a small amount of ethyl acetate, is changed after nitrogen, puts into the Pd/C of catalytic amount, hydrogen displacement Afterwards, stirring reaction under room temperature normal pressure, TLC detecting and trackings reaction is complete.Filtering, filtrate concentration, gained crude on silica gel post layer Analysis purifies to obtain compound C51 (5mg, yield 71.4%), white solid.¹H NMR(300MHz,CDCl₃+CD₃OD)δ3.39(s, 1H), 2.90 (t, 2H, J=4.5Hz), 2.75 (t, 2H, J=4.5Hz), 2.37-2.03 (m, 2H), 1.97-1.67 (m, 4H), 1.65-0.90 (m, remaining aliphatic acyclic hydrocarbon proton), 0.93 (s, 3H), 0.91 (t, 3H, J=3.0Hz), 0.66 (s, 3H), 0.62 (d, 3H, J=6.0Hz), 0.58 (s, 3H), 0.52 (s, 3H), 0.48 (s, 3H)；ESI-MS(m/z):566.4(M+Na)⁺ (C₃₅H₆₁NO₃Theoretical value：543.46).

Prepare embodiment 8：Compound C54 synthesis

Trimethylsulfoxonium Iodide (21mg, 0.095mmol) and sodium hydride (2mg, 0.095mmol) put into drying at room temperature THF (1mL) in, reaction half an hour after, instill intermediate S11 (3- alpha-hydroxy-2 0- formoxyl betulic acids benzyl ester) (15mg, Dry THF solution (1mL) 0.027mmol), stirring reaction is stayed overnight at room temperature.Next day, which adds water, is quenched reaction, ethyl acetate extraction Take, organic phase saturated common salt water washing, dry gained residue after concentration and purify (ethyl acetate/petroleum ether through silica gel column chromatography =1/6, V/V), obtain intermediate S18 (5mg, yield：33.3%), white solid.¹H NMR(300MHz,CDCl₃)δ7.34(m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 3.39 (s, 1H), 3.32 (d, 1H, J=4.5Hz), 2.72 (t, 1H, J=4.5Hz), 2.51 (m, 1H), 2.29-2.23 (m, 2H), 1.96-1.92 (m, 4H), 1.55-1.03 (m, its Remaining aliphatic acyclic hydrocarbon proton), 0.96 (s, 3H), 0.93 (s, 3H), 0.88 (s, 3H), 0.84 (d, 3H, J=6.0Hz), 0.82 (s, 3H),0.80(s,3H)。

Intermediate S18 is dissolved in methanol and a small amount of ethyl acetate, is changed after nitrogen, adds the Pd/C of catalytic amount, hydrogen displacement Afterwards, stirring reaction under room temperature normal pressure, TLC tracking reactions are complete.Filtering, gained crude on silica gel column chromatography is pure after filtrate concentration Change to obtain compound C54 (4mg, yield 94.8%), white solid.¹H NMR(300MHz,CDCl₃+CD₃OD)δ3.39(s,1H), 3.32 (d, 1H, J=4.5Hz), 2.72 (t, 1H, J=4.5Hz), 2.51 (m, 1H), 2.29-2.23 (m, 2H), 1.96-1.92 (m, 4H), 1.55-1.03 (m, remaining aliphatic acyclic hydrocarbon proton), 0.96 (s, 3H), 0.93 (s, 3H), 0.88 (s, 3H), 0.84 (d, 3H, J=6.0Hz), 0.82 (s, 3H), 0.80 (s, 3H)；ESI-MS(m/z):509.3(M+Na)⁺(C₃₁H₅₀O₄Theoretical value： 486.37)。

Prepare embodiment 9：Compound C55 synthesis

Selenium dioxide (22mg, 0.22mmol) and TBHP (165 μ L, 5.5M tetrahydrofuran solutions) are dissolved in In dry dichloromethane (20mL), the acetic acid (6 μ L, 0.1eq.) of catalytic amount is added dropwise under ice-water bath, ten are stirred at this temperature After minute, the dichloromethane solution (5mL) of intermediate 3- Alpha-hydroxy betulic acid benzyl esters (242mg, 0.44mmol) is slowly added dropwise, It is to slowly warm up to room temperature and is stirred overnight, TLC detection reactions are complete, adds sodium sulfite saturated solution and reaction, dichloromethane is quenched Alkane extracts and organic phase is washed with water, and dries concentration gained residue and directly throws next step.Crude product is dissolved in methanol (20mL), frozen water Sodium borohydride (25mg, 0.66mmol) is added portionwise under bath, after reacting one hour, TLC detection reactions are complete, ammonium chloride are added dropwise full Reaction is quenched with solution, ethyl acetate extracts and uses saturated common salt water washing, organic phase, which is dried, uses petroleum ether/acetic acid second after concentration Ester is 2:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- alpha-hydroxy-2 2- hydroxyl betulic acid benzyl esters S10197mg, is white solid, molar yield：79.2%.¹H NMR(300MHz,CDCl₃)δ7.34(m,5H),5.09(d,1H, ), J=11.7Hz 5.17 (d, 1H, J=11.7Hz), 4.97 (s, 1H), 4.92 (s, 1H), 4.12 (m, 2H), 3.39 (s, 1H), 2.88 (td, 1H, J=12.0,3.0Hz), 2.32-2.26 (m, 2H), 2.21-2.10 (m, 2H), 1.98-1.77 (m, 2H), 1.64-0.96 (m, other cycloaliphatic ring protons), 0.99 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H), 0.84 (s,3H)；ESI-MS(m/z):585.4(M+Na)⁺(C₃₇H₅₄O₄Theoretical value：562.40)

S10 (96mg, 0.17mmol) is dissolved in dichloromethane (10mL), and metachloroperbenzoic acid is slowly added under ice-water bath (72mg, 0.34mmol), is stirred at room temperature reaction 3h, TLC detection reaction completely, adds saturated sodium bisulfite solution and reaction is quenched, Organic phase is washed with water, it is a white solid to dry and intermediate S14 is obtained after concentration, is directly used in the next step.

S14 (120mg, 0.207mmol) is dissolved in chloroform (20mL), is instilled two and is dripped concentrated hydrochloric acid solution, the heating of gained system Back flow reaction, TLC detection S14 reaction completely after, be concentrated under reduced pressure, residue silica gel column chromatography purifying, obtain intermediate S15 (8mg, Yield 6.7%), white solid.¹H NMR(300MHz,CDCl₃) δ 9.60 (s, 1H), 7.34 (m, 5H), 5.09 (d, 1H, J= 11.7Hz), 5.17 (d, 1H, J=11.7Hz), 4.00 (m, 1H), 3.77 (m, 1H), 3.39 (s, 1H), 2.65 (m, 1H), 2.28-2.19 (m, 2H), 1.94-1.05 (m, remaining aliphatic acyclic hydrocarbon proton), 0.94 (s, 3H), 0.92 (s, 3H), 0.81 (s, 3H),0.73(s,3H),0.65(s,3H)。

Intermediate S15 is dissolved in methanol and a small amount of ethyl acetate, is changed after nitrogen, adds the Pd/C of catalytic amount, hydrogen displacement Afterwards, room temperature atmospheric agitation reacts, and the reaction of TLC detecting and trackings is complete.Filtering, gained crude product silica gel column chromatography is pure after filtrate concentration Change to obtain compound C55 (6mg, yield 85.7%), white solid.¹H NMR(300MHz,CDCl₃)δ9.60(s,1H),4.00(m, 1H), 3.77 (m, 1H), 3.39 (s, 1H), 2.65 (m, 1H), 2.28-2.19 (m, 2H), 1.94-1.05 (m, remaining aliphatic acyclic hydrocarbon Proton), 0.94 (s, 3H), 0.92 (s, 3H), 0.81 (s, 3H), 0.73 (s, 3H), 0.65 (s, 3H)；ESI-MS(m/z): 511.3(M+Na)⁺(C₃₀H₄₈O₅Theoretical value：488.35).

Prepare embodiment 10：Compound C56 synthesis

Take intermediate S10 (100mg) to be dissolved in dry tetrahydrofuran (5mL), BH is instilled under ice-water bath₃-Me₂S tetrahydrochysene Tetrahydrofuran solution (2M), stirring moves to room temperature after one hour.Next day, reaction solution is moved into ice-water bath, sequentially add ethanol (280 μ L), saturated acetic acid sodium solution (200 μ L), 30% hydrogenperoxide steam generator (140 μ L).It is stirred overnight at room temperature, TLC detection reactions Completely.It is diluted with water, saturated common salt water washing after ethyl acetate extraction.Organic phase, which is dried, uses chloroform/methanol to be 50 after concentration:1 Eluant, eluent system carry out column chromatography for separation, obtain intermediate S12 (88mg, yield 85.4%), white solid.¹H NMR (300MHz, CDCl3) δ 7.34 (m, 5H), 5.08 (dd, 2H, J=15.0,12.0Hz), 3.90 (m, 1H), 3.78 (d, 1H, J= 6.0Hz), 3.72 (d, 4H, J=6.0Hz), 3.38 (s, 1H), 2.21-2.04 (m, 4H), 1.81-1.05 (m, other aliphatic ring protons),0.94(s,3H),0.92(s,3H),0.81(s,6H),0.73(s,3H)；ESI-MS(m/ z):603.4(M+Na)⁺, (C₃₇H₅₆O₅Theoretical value：580.41).

Intermediate S12 can obtain compound C56 after being deprotected through over hydrogenation,¹H NMR(300MHz,CDCl₃)：δ4.30(m, 1H), 4.21 (m, 1H), 4.15 (d, 1H, J=3.0Hz), 3.34 (s, 1H), 3.22-3.13 (m, 2H), 3.56-3.45 (m, 2H), 2.43 (m, 1H), 2.26 (m, 1H), 2.10 (d, 1H, J=9.0Hz), 1.81-1.05 (m, remaining aliphatic acyclic hydrocarbon proton), 0.94(s,3H),0.92(s,3H),0.81(s,3H),0.73(s,3H),0.65(s,3H)；ESI-MS(m/z):513.3(M+ Na)⁺, (C₃₀H₅₀O₅Theoretical value：490.36).

Prepare embodiment 11：Compound C57 synthesis

Intermediate S3 (3- Alpha-hydroxy betulic acids benzyl ester) preparation is with reference to preparation embodiment 1.Intermediate S3 (143mg, 0.262mmol) it is dissolved in ethanol/methylene (5mL/5mL), ozone is passed through at -78 DEG C, TLC tracking reactions is complete, stop It is passed through after ozone and remaining ozone is bloated with nitrogen, then addition dimethyl sulphide (25 μ L), which is quenched under reaction, stirring, is slowly raised to Room temperature.Second day, residue purified to obtain intermediate S21 (94mg, yield 65.8%) through silica gel column chromatography after reaction system concentration, White solid.¹H NMR(300MHz,CDCl₃) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J= 11.7Hz), 3.39 (s, 1H), 2.27 (m, 1H), 2.18 (s, 3H), 2.16-1.92 (m, 4H), 1.64-0.96 (m, remaining fat Cyclic hydrocarbon proton), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H).

With reference to the application prepare embodiment 5 in intermediate S19 preparation method, by intermediate S21 (19mg, Intermediate S22, not purified direct carry out the next step 0.035mmol) is made with allyl grignard reagent reaction.With reference to this Shen The preparation method of intermediate S14 in embodiment 9 please be prepare, intermediate S22 obtained by upper step carries out epoxy with metachloroperbenzoic acid White solid (7mg, two step yields 35%) is obtained after changing the obtained intermediate S23 of reaction, silica gel column chromatography.¹H NMR(300MHz, CDCl₃) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J=11.7Hz), 3.39 (s, 1H), 3.18 (brs, 1H), 2.86-2.79 (m, 1H+1H), 2.49 (m, 2H), 2.26 (m, 4H) 1.97 (m, 4H), 1.84-1.15 (m, remaining Aliphatic acyclic hydrocarbon proton), 1.28 (s, 3H), 1.01 (s, 3H), 0.92 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H), 0.81 (s, 3H)。

Intermediate S23 (7mg) is dissolved in methanol and a small amount of ethyl acetate, is changed after nitrogen and is added the Pd/C of catalytic amount, hydrogen After displacement, stirring reaction under room temperature normal pressure, TLC detecting and trackings reaction is complete.Filtering, gained crude product silicagel column after filtrate concentration Chromatographic purifying obtains compound C57 (3mg, yield 43%), white solid.¹H NMR(300MHz,CDCl₃)δ3.39(s,1H), 3.18(brs,1H),2.86-2.79(m,1H+1H),2.49(m,2H),2.26(m,4H)1.97(m,4H),1.84-1.15(m, Remaining aliphatic acyclic hydrocarbon proton), 1.28 (s, 3H), 1.01 (s, 3H), 0.92 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H), 0.81 (s,3H)；ESI-MS(m/z):539.3(M+Na)⁺(C₃₂H₅₂O₅Theoretical value：516.38).

Prepare embodiment 12：Compound C58 synthesis

According to the similar methods of the prepare compound C39 for preparing embodiment 21, using intermediate S11 as raw material, with hydrochloric acid hydroxyl Amine reaction, which can be synthesized, obtains intermediate S13, and the latter's hydrogenation, which is sloughed, obtains compound C58 after benzyl protection.¹H NMR(300MHz, CDCl₃) δ 6.61 (d, 1H, J=8.7Hz), 3.39 (s, 1H), 2.35-2.23 (m, 1H), 2.18-2.08 (m, 1H), 1.98- 1.80 (m, 2H), 1.64-0.96 (m, other aliphatic ring protons), 1.01 (d, 3H, J=6.0Hz), 0.96 (s,6H),0.92(s,3H),0.84(s,3H),0.81(s,3H)；ESI-MS(m/z):510.3(M+Na)⁺,(C₃₀H₄₉NO₄It is theoretical Value：487.36).

Prepare embodiment 13：Compound C59 synthesis

Preparation method synthesis according to embodiment 9 is prepared obtains intermediate S14.Intermediate S14 (10mg, 0.017mmol) It is dissolved in the DMAP (1mg) of catalytic amount in dichloromethane (5mL), adds under triethylamine (4mL, 0.026mmol), ice-water bath and instill Chloroacetic chloride (2mL, 0.030mmol), is reacted at room temperature 12 hours, and TLC shows that reaction is finished.Concentration is removed after solvent, ethyl acetate After dilution, after being washed respectively with water and saturated sodium-chloride water solution, organic phase dries concentration, silica gel column chromatography (ethyl acetate:Stone Oily ether, 1:2) compound S24 white solids (8mg, 0.013mmol), molar yield, are obtained：80%.Benzyl is sloughed in S24 hydrogenations Protection obtains compound C59 (yield 50%, white solid).¹HNMR proves the mixture (S of two kinds of configurations:R about 3:1), S configurations:¹H NMR(300MHz,CDCl₃) δ 4.35 (d, 1H, J=12.0Hz), 4.04 (d, 1H, J=12.0Hz), 3.40 (s, 1H), 2.76 (d, 1H, J=6.0Hz), 2.66 (d, 1H, J=6.0Hz), 2.35-2.24 (m, 2H), 2.09 (s, 3H), 2.01- 1.92(m,4H),1.86-1.75(m,4H),1.57-1.28(m,other aliphatic ring protons),0.97(s, 3H),0.93(s,3H),0.92(s,3H),0.86(s,3H),0.82(s,3H)；R configurations:¹H NMR(300MHz,CDCl₃)δ 4.22 (d, 1H, J=12.0Hz), 4.13 (d, 1H, J=12.0Hz), 3.40 (s, 1H), 2.82 (d, 1H, J=6.0Hz), 2.76 (d, 1H, J=6.0Hz), 2.35-2.24 (m, 2H), 2.09 (s, 3H), 2.01-1.92 (m, 4H), 1.86-1.75 (m, 4H), 1.57-1.28(m,other aliphatic ring protons),0.97(s,3H),0.93(s,3H),0.92(s,3H), 0.86(s,3H),0.82(s,3H)；ESI-MS(m/z):553.3(M+Na)⁺, (C₃₂H₅₀O₆Theoretical value：530.36).

Prepare embodiment 14：Compound C79 synthesis

Compound A2 (2mL, 19.9mmol) and triethylamine (2.8mL, 19.9mmol) are in dichloromethane (40mL), frozen water Chloro-carbonic acid benzyl alcohol ester (2.72mL, 19.9mmol) is slowly added dropwise under bath, stirring reaction is stayed overnight at room temperature.Next day, TLC detection reactions Completely, reaction system is concentrated under reduced pressure, and gained residue purifies (chloroform/methanol=40/1, V/V) through silica gel column chromatography, obtains middle Body S29 (4.45g, yield 93.1%), colorless oil.¹H NMR(300MHz,CDCl₃)δ7.36-7.26(m,5H),5.29 (brs, 1H), 5.10 (s, 2H), 3.71 (t, 2H, J=4.8Hz), 3.54 (m, 4H), 3.40 (t, 2H, J=5.1Hz), 2.31 (brs,1H)。

Gained intermediate S29 (1.49g, 6.23mmol) is dissolved under acetonitrile/water (10mL/5mL) in the mixed solvent, ice-water bath Iodobenzene diacetate (6g, 18.68mmol) and tetramethyl piperidine (TEMPO, 292mg, 1.87mmol) are separately added into, at room temperature Stirring reaction is stayed overnight.Next day, TLC detection reactions are complete, add saturated sodium bisulfite solution stirring 10min, use 1N hydrochloric acid solutions PH=5 is adjusted to, dichloromethane extraction merges organic phase saturated common salt water washing, and organic phase is dried, is concentrated under reduced pressure, gained is residual Excess purifies (chloroform/methanol=20/1, V/V) through silica gel column chromatography, obtains intermediate S30 (1.23g, yield 77.8%), yellow Grease.¹H NMR(300MHz,CDCl₃)δ7.36-7.26(m,5H),5.42(brs,1H),5.10(s,2H),4.12(s, 2H), 3.64 (t, 2H, J=4.8Hz), 3.43 (t, 2H, J=5.1Hz).

By intermediate S14 (31mg, 0.054mmol) ﹑ S30 (27mg, 0.107mmol) ﹑ EDCI (1- ethyls-(3- dimethyl Aminopropyl) carbodiimide hydrochloride) (20mg, 0.107mmol) and DMAP (DMAP) (7mg, 0.054mmol) Room temperature reaction is stayed overnight in DMF (DMF) (5mL), and reaction solution is diluted with water within second day, ethyl acetate extraction, Organic phase is washed with water twice, saturated common salt washing, anhydrous sodium sulfate drying, filtering, and residue is through silica gel column layer after filtrate concentration Analysis purifying (ethyl acetate/petroleum ether=1/2), obtains intermediate S31 (33mg, yield 76.7%), white solid.¹H NMR (300MHz,CDCl₃) δ 7.36-7.26 (m, 5H), 5.45 (brs, 1H), 5.10 (m, 2H), 4.43 (d, 1H, J=12.0Hz), 4.20 (s, 1H), 4.13 (s, 2H), 3.61 (t, 2H, J=5.1Hz), 3.41 (t, 2H, J=5.1Hz), 3.39 (s, 1H), 2.63 (dd, 2H, J=8.7,4.8Hz), 2.30 (m, 2H), 2.10 (m, 2H), 1.90 (m, 4H), 1.82 (m, 4H), 1.76-1.32 (m, Remaining aliphatic acyclic hydrocarbon proton), 0.90 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H), 0.81 (s, 3H), 0.73 (s, 3H).

Gained intermediate S31 is dissolved in methanol, under normal pressure in the presence of Pd-C catalytic hydrogenolysis, while sloughing benzyloxycarbonyl Intermediate S32 is obtained with C-20 benzyl, it is not purified, directly carry out the next step.Gained intermediate S32 is dissolved in dichloromethane In, triethylamine and Boc acid anhydrides are added, room temperature reaction is stayed overnight.Reaction system is concentrated under reduced pressure, residue through silica gel column chromatography after purification Obtain compound C79 (yield 76.2%), white solid.¹H NMR(300MHz,CDCl₃)δ5.12(brs,1H),4.45(d, 1H, J=12.0Hz), 4.25 (s, 1H), 4.10 (m, 4H), 3.47 (m, 4H), 3.61 (t, 2H, J=5.1Hz), 3.39 (s, 1H), 3.34 (m, 2H), 2.64 (dd, 2H, J=8.7,4.8Hz), 2.30 (m, 2H), 2.10 (m, 2H), 1.95 (m, 4H), 1.82-1.32 (m, remaining aliphatic acyclic hydrocarbon proton), 1.44 (s, 9H), 0.95 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.84(s,3H),0.81(s,3H)；ESI-MS(m/z):712.4(M+Na)⁺(C₃₉H₆₃NO₉Theoretical value：689.45).

Prepare embodiment 15：Compound C80 synthesis

The preparation method of compound C79 in embodiment 14 is prepared with reference to the application, compound C80 (total recoverys are synthesized to obtain 35.5%), white solid.¹H NMR(300MHz,CDCl₃) δ 5.08 (brs, 1H), 4.45 (d, 1H, J=12.0Hz), 4.09 (d, 1H, J=12.0Hz), 4.16 (s, 2H), 3.71 (m, 2H), 3.65 (m, 2H), 3.53 (t, 2H, J=5.1Hz), 3.39 (s, 1H), 3.34 (m, 2H), 2.64 (dd, 2H, J=8.7,4.8Hz), 2.30 (m, 2H), 2.10 (m, 2H), 1.95 (m, 4H), 1.82-1.32 (m, remaining aliphatic acyclic hydrocarbon proton), 1.44 (s, 9H), 0.95 (s, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.84(s,3H),0.81(s,3H)；ESI-MS(m/z):756.4(M+Na)⁺(C₄₁H₆₇NO₁₀Theoretical value：733.47).

Prepare embodiment 16：Compound C82 synthesis

By intermediate S32 (30mg, 0.05mmol), active ester Biotin-OSu (17mg, 0.05mmol) and triethylamine (200 μ L) is dissolved in dry DMF (2mL) solvent, is heated to stirring reaction at 50 DEG C and is stayed overnight.It is as cold as after room temperature, reaction system Diluted with water (20mL), ethyl acetate extraction is washed, anhydrous Na after merging organic phase with saturated common salt₂SO₄Dry, filtering and concentrating Gained crude on silica gel column chromatography purifies to obtain compound C82 (yield 60%), white solid afterwards.¹H NMR(300MHz, CDCl₃) δ 6.75 (brs, 1H), 5.33 (brs, 1H), 5.23 (brs, 1H), 4.63 (d, 1H, J=12.0Hz), 4.50 (t, 1H, ), J=6.0Hz 4.31 (t, 1H, J=6.0Hz), 4.10 (m, 2H+2H), 3.94 (d, 1H, J=12.0Hz), 3.65 (m, 2H), 3.48 (m, 2H), 3.39 (s, 1H), 3.19 (m, 1H), 2.90 (m, 1H), 2.72 (d, 1H, J=6.0Hz), 2.67 (d, 1H, J= 6.0Hz), 2.25 (m, 4H), 1.95 (m, 4H), 1.78-1.09 (m, remaining aliphatic acyclic hydrocarbon proton), 0.95 (s, 3H), 0.93 (s, 3H),0.87(s,3H),0.83(s,3H),0.81(s,3H)。

Prepare (the compound number of embodiment 17：C34)

Betulic acid (1.2g, 2.63mmol) is dissolved in methanol/ethyl acetate (40mL/10mL), is changed to add after nitrogen and is urged The Pd/C of change amount, then change hydrogen is changed after nitrogen, it is stirred at room temperature 2 days, TLC detection reactions are complete.Filtering reacting liquid after nitrogen is changed, is revolved With petroleum ether after dry：Ethyl acetate is 10:1 polarity carries out column chromatography for separation.Product C34 be white solid (1.04g, 2.27mmol), molar yield is 86%.¹H NMR(300MHz,CDCl₃) δ 3.13 (t, 1H, J=9.0,6.9Hz), 2.28- 2.16 (m, 2H), 1.98-1.78 (m, 4H), 1.64-0.96 (m, other cycloaliphatic ring protons), 0.96 (s, 3H), 0.93 (s, 3H), 0.92(s,3H),0.90(s,3H),0.89(s,3H),0.87(s,3H),0.78(s,3H)；ESI-MS(m/z):481.3(M+ Na)⁺(C₃₀H₅₀O₃Theoretical value：458.38).

With same method, respectively using different natural products or compound as raw material (raw material oleanolic acid：C2, raw material Ursolic acid：C7, raw material enoxolone, raw material betulin：C29, raw material C34), according to the preparation identical method of embodiment 12, Synthesize following compound or intermediate：

Prepare (the compound number of embodiment 18：C32)

Betulic acid benzyl ester, 3- carbonyl betulic acids benzyl ester, 3- Alpha-hydroxy white birch fat are sequentially prepared according to embodiment 1 is prepared Acid benzyl ester.

3- Alpha-hydroxy betulic acids

Upper step product (100mg, 0.18mmol) is dissolved in methanol (8mL) and a small amount of ethyl acetate, is changed after nitrogen, rapid to add Enter 10% Pd/C, then change hydrogen is changed after nitrogen, be stirred at room temperature.TLC detections reaction is complete after one hour.Change after nitrogen and filter off Pd/C, reaction solution uses petrol ether/ethyl acetate to be 10 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound C32 (78mg), is white solid, molar yield：94%.¹H NMR(300MHz,CDCl₃)δ4.73(s,1H),4.60(s,1H), 3.39(s,1H),3.02-2.96(m,1H),2.28-2.16(m,2H),1.98-1.95(m,2H),1.68(s,3H),1.64- 0.96 (m, other cycloaliphatic ring protons), 1.04 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H),0.84(s,3H)；ESI-MS(m/z):479.3(M+Na)⁺(C₃₀H₄₈O₃Theoretical value：456.36).

Prepare embodiment 19 (compound number C46)

(1) 3- alpha-hydroxy-2s 0,21- epoxy betulic acid benzyl esters

3- alpha-hydroxy-2s 0,21- epoxy betulic acid benzyl esters 74mg are prepared according to embodiment 5 is prepared.

(2) 3- alpha-hydroxy-2s 0,21- epoxy betulic acids

Upper step product (74mg, 0.14mmol) is dissolved in methanol (8mL) and a small amount of ethyl acetate, is changed after nitrogen, rapid to add Enter 10% Pd/C, then change hydrogen is changed after nitrogen, be stirred at room temperature.TLC detections reaction is complete after one hour.Change after nitrogen and filter off Pd/C, reaction solution uses petrol ether/ethyl acetate to be 6 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- alpha-hydroxy-2s 0,21- epoxies betulic acid (38mg, 0.08mmol) is white solid, molar yield：58%.¹H NMR (300MHz,CDCl₃)δ3.40(s,1H),2.64(t,2H),2.25(m,2H),2.14(m,2H),1.97(m,2H),1.78(m, 2H), 1.76-1.32 (m, other cycloaliphatic ring protons), 1.28 (s, 3H), 0.98 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.90(s,3H),0.86(s,3H),0.82(s,3H)；ESI-MS(m/z):495.3(M+Na)⁺(C₃₀H₄₈O₄Theoretical value： 472.36)。

C36 is synthesized with the same method of embodiment 19：

Prepare embodiment 20 (compound number C37)

Betulic acid (41mg, 0.09mmol) instills BH in dry tetrahydrofuran (5mL) under ice-water bath₃-Me₂S's Tetrahydrofuran solution (2M), stirring moves to room temperature after one hour.Next day, reaction solution is moved into ice-water bath, sequentially add ethanol (280 μ L), saturated acetic acid sodium solution (200 μ L), 30% hydrogenperoxide steam generator (140 μ L).It is stirred overnight at room temperature, TLC inspections Survey reaction complete.It is diluted with water, saturated common salt water washing after ethyl acetate extraction.Organic phase uses chloroform/methanol after drying concentration For 50:1 eluant, eluent system carries out column chromatography for separation, obtains compound 20 (29)-reduction -29- hydroxyl betulic acid 19mg, is White solid, molar yield：45.3%.¹H NMR(300MHz,CD₃OD) δ 3.73 (dd, 1H, J=9.0,3.0Hz), 3.34 (s, 1H), 3.13 (dd, 1H, J=9.0,6.0Hz), 2.36-2.12 (m, 4H), 1.83-1.08 (m, other cycloaliphatic ring protons), 0.99 (s,3H),0.96(s,3H),0.95(s,3H),0.94(s,3H),0.87(s,3H),0.75(s,3H)；ESI-MS(m/z): 497.3(M+Na)⁺(C₃₀H₅₀O₄Theoretical value：474.37).

Prepare embodiment 21 (compound number C39)

Intermediate 3- carbonyls -20 (29)-reduction betulic acid (82mg, 0.18mmol), hydroxylamine hydrochloride (25mg, 0.37mmol) it is stirred overnight at room temperature with pyridine (37 μ L, 0.46mmol) in absolute ethyl alcohol (5mL), next day TLC detection reactions Completely.It is 10 that petrol ether/ethyl acetate is used after directly concentrating:1 eluant, eluent system carries out column chromatography for separation, obtains target chemical combination Thing 77mg, is white solid, molar yield：91.2%.¹H NMR(300MHz,CDCl₃)ClR(300MHz,：Slough benzyl simultaneously Double bond is reduced, 0.92 (s, 3H), 0.90 (s, 3 other cycloaliphatic ring protons), 0.94 (s, 3H), 0.91 (s, 3H), 0.90 is obtained (s,3H),0.88(s,3H),0.85(s,3H),0.84(s,3H),0.76(s,3H)；ESI-MS(m/z):494.3(M+Na)⁺ (C₃₀H₄₉NO₃Theoretical value：471.37).

Prepare the 3- α of embodiment 22-acetoxyl group betulic acid (compound number：T2)

(1) 3- α-acetoxyl group betulic acid benzyl ester

The DMAP (10mg, 0.08mmol) of 3- Alpha-hydroxy betulic acid benzyl esters (107mg, 0.20mmol) and catalytic amount is dissolved in In dichloromethane (10mL), add and acetic anhydride (42mL, 0.60mmol) instilled under triethylamine (82mL, 0.60mmol), ice-water bath, Room temperature reaction 12 hours, TLC shows that reaction is finished.Concentration is removed after solvent, after ethyl acetate dilution, respectively with water and saturation chlorine Change after sodium water solution washing, organic phase dries concentration, and gained residue is using petrol ether/ethyl acetate as 20:1 eluant, eluent post layer Analysis obtains compound 3- α-acetoxyl group betulic acid benzyl ester white solid (77mg, 0.13mmol), molar yield after purification： 65%.¹H NMR(300MHz,CDCl₃) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J= 11.7Hz), 4.62 (s, 1H), 2.84-2.20 (m, 4H), 2.08 (s, 3H), 1.98-1.15 (m, other cycloaliphatic ring protons), 1.01(s,3H),0.94(s,3H),0.92(s,3H),0.85(s,3H),0.83(s,3H),0.76(s,3H),0.74(s,3H)； ESI-MS(m/z):611.4(M+Na)⁺(C₃₉H₅₆O₄Theoretical value：588.42).

(2) 3- α-acetoxyl group betulic acid

Upper step product (77mg, 0.13mmol) is dissolved in methanol (8mL) and a small amount of ethyl acetate, is changed after nitrogen, rapid to add Enter 10% Pd/C, then change hydrogen is changed after nitrogen, be stirred at room temperature.TLC detections reaction is complete after one hour.Change after nitrogen and filter off Pd/C, reaction solution uses petrol ether/ethyl acetate to be 10 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- α-acetoxyl group betulic acid (60mg, 0.12mmol), are white solid, molar yield：92%.¹H NMR(300MHz, CDCl₃) δ 4.62 (s, 1H), 2.84-2.20 (m, 4H), 2.08 (s, 3H), 1.98-1.15 (m, other cycloaliphatic ring protons), 1.01 (s,3H),0.94(s,3H),0.92(s,3H),0.85(s,3H),0.83(s,3H),0.76(s,3H),0.74(s,3H)；ESI- MS(m/z):521.3(M+Na)⁺(C₃₂H₅₀O₄Theoretical value：498.37).

Following compound is synthesized with the same method of embodiment 22 and different acid anhydrides：

Prepare embodiment 23 (compound number C115)

20 (29)-reduction betulic acid (37mg, 0.08mmol) and EDCI (21mg, 0.12mmol) in DMF (2mL), Addition triethylamine (15 μ L), HOBt (15mg, 0.12mmol) and N-Boc-2- (2- aminoethoxies) ethylamine (15mg, 0.08mmol), 50 DEG C are stirred overnight.Next day TLC detection reactions are complete, and reaction solution is diluted with water, and satisfy after being extracted with ethyl acetate And brine It.Organic phase, which is dried, uses chloroform/methanol to be 80 after concentration:1 eluant, eluent system carries out column chromatography for separation, obtains Target compound 45mg, is white solid, molar yield：86.5%.¹H NMR(300MHz,CDCl₃) δ 5.93 (t, 1H, J= 5.1Hz), 4.84 (brs, 1H), 3.48 (m, 4H), 3.13 (dd, 1H, J=9.0,6.0Hz), 3.30 (m, 4H), 2.40 (td, 1H, J=12.0,3.0Hz), 2.23 (td, 1H, J=12.0,3.0Hz), 1.44 (s, 9H), 1.96-1.09 (m, other cycloaliphatic rings Proton), 0.96 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.78 (s,3H)；ESI-MS(m/z):667.5(M+Na)⁺(C₃₉H₆₈N₂O₅Theoretical value：644.51).

Following compound is synthesized with the same method of embodiment 23 and different amine：

Prepare (the compound number of embodiment 24：C47)

(1) 3- alpha-hydroxy-2s 2- hydroxyls betulic acid benzyl ester

3- alpha-hydroxy-2 2- hydroxyl betulic acid benzyl esters are prepared with reference to embodiment 9 is prepared.

(2) 3- alpha-hydroxy-2s 2- hydroxyl betulic acids

Upper step product (197mg, 0.35mmol) is dissolved in methanol (10mL) and a small amount of ethyl acetate, is changed after nitrogen, rapidly 10% Pd/C is added, then changes hydrogen is changed after nitrogen, is stirred at room temperature.TLC detections reaction is complete after one hour.Change after nitrogen and filter off Pd/C, reaction solution uses petrol ether/ethyl acetate to be 2 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- alpha-hydroxy-2 2- hydroxyls betulic acids (140mg, 0.30mmol), are white solid, molar yield：85%.¹H NMR (300MHz,CDCl₃) δ 4.97 (s, 1H), 4.92 (s, 1H), 4.12 (m, 2H), 3.39 (s, 1H), 2.88 (td, 1H, J= 12.0,3.0Hz), 2.32-2.26 (m, 2H), 2.21-2.10 (m, 2H), 1.98-1.77 (m, 2H), 1.64-0.96 (m, it is other Cycloaliphatic ring proton), 0.99 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.87 (s, 3H), 0.84 (s, 3H)；ESI-MS(m/ z):495.3(M+Na)⁺(C₃₀H₄₈O₄Theoretical value：472.36).

Prepare (the compound number of embodiment 25：C48)

(1) 3- alpha-hydroxy-2s 0- formoxyls betulic acid benzyl ester

3- alpha-hydroxy-2 0- formoxyl betulic acid benzyl esters are prepared according to embodiment 5 is prepared.

(2) 3- alpha-hydroxy-2s 0- formoxyl betulic acids

Upper step product (50mg, 0.088mmol) is dissolved in methanol (5mL) and a small amount of ethyl acetate, is changed after nitrogen, rapid to add Enter 10% Pd/C, then change hydrogen is changed after nitrogen, be stirred at room temperature.TLC detections reaction is complete after one hour.Change after nitrogen and filter off Pd/C, reaction solution uses petrol ether/ethyl acetate to be 2 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- alpha-hydroxy-2 0- formoxyls betulic acids (36.7mg, 0.078mmol), are white solid, molar yield：87.3%.¹H NMR(300MHz,CDCl₃) δ 9.84 (s, 1H), 3.39 (s, 1H), 3.33 (d, 1H, J=4.2Hz), 2.58 (m, 1H), 2.40 (m, 1H), 2.23 (m, 2H), 1.98-1.84 (m, 2H), 1.69-0.96 (m, other cycloaliphatic ring protons), 1.12 (d, 3H, J= 9.0Hz),0.96(s,3H),0.93(s,3H),0.87(s,3H),0.84(s,3H),0.81(s,3H)；ESI-MS(m/z): 495.2(M+Na)⁺(C₃₀H₄₈O₄Theoretical value：472.36).

Prepare embodiment 26 (compound number C119)

Molecular sieve and the dichloromethane (10mL) dried are placed in dry 50mL round-bottomed flasks, at -20 DEG C respectively The tetra isopropyl oxygen titanium (1 μ L, 0.0037mmol) and D- (-)-DIPT (1 μ L, 0.0056eq) that instillation is steamed are stirred 15 minutes again after 5.5M TBHP THF solution (10 μ L, 0.056mmol) is added, is stirred 30 minutes.The preparation products therefrom of embodiment 24 is instilled again Benzyl ester (21mg, 0.037mmol) dichloromethane solution (2mL), be placed in -20 DEG C of refrigerator overnights.Next day, by reaction solution liter Temperature adds the saturated common salt aqueous solution (50 μ L) of 5N sodium hydroxides to zero degree, and stirring is filtered to remove molecular sieve, filtrate after one hour It is 2 that petrol ether/ethyl acetate is used after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains target compound 16mg, is White solid, molar yield：76.2%.It is the product based on S configurations that NMR, which proves C-17,.¹H NMR(300MHz,CDCl₃)δ 7.34 (m, 5H), 5.09 (s, 2H), 3.80-3.64 (m, 2H), 3.40 (s, 1H), 2.92 (d, 1H, J=4.5Hz), 2.63 (d, 1H, J=4.5Hz), 2.35-2.24 (m, 2H), 2.01-1.92 (m, 4H), 1.86-1.75 (m, 4H), 1.57-1.28 (m, it is other Cycloaliphatic ring proton), 0.97 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.86 (s, 3H), 0.82 (s, 3H).Product is directly pressed Above the step of, sloughs benzyl under palladium carbon catalysis, obtains 12mg target products, is white solid, molar yield：89.2%.¹H NMR(300MHz,CDCl₃) δ 3.76 (d, 1H, J=12.0Hz), 3.67 (d, 1H, J=12.0Hz), 3.40 (s, 1H), 2.91 (d, 1H, J=6.0Hz), 2.62 (d, 1H, J=6.0Hz), 2.35-2.24 (m, 2H), 2.01-1.92 (m, 4H), 1.86-1.75 (m, 4H), 1.57-1.28 (m, other cycloaliphatic ring protons), 0.97 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.86 (s, 3H),0.82(s,3H)；ESI-MS(m/z):511.3(M+Na)⁺(C₃₀H₄₈O₅Theoretical value：488.35).

Following compound is synthesized with the same method of embodiment 26：

Prepare (the compound number of embodiment 27：C53)

(1) 3- alpha-hydroxy-2s 0- (2 '-aminoethyle alcohol base) betulic acid benzyl ester

Intermediate 3- alpha-hydroxy-2 0- formoxyl betulic acid benzyl esters (20mg, 0.036mmol) and sodium cyanoborohydride (11mg, 0.018mmol) stirs 2h at room temperature, and monoethanolamine (11 μ L, 0.18mmol) is instilled afterwards and is stirred overnight at room temperature, TLC Detection reaction is complete, and reaction is quenched in saturated sodium bicarbonate solution, is diluted with water rear ethyl acetate and is extracted twice, saturated common salt washing After washing, organic phase dries concentration, and gained residue chloroform/methanol is 20:1 eluant, eluent system carries out column chromatography for separation, obtains It is white solid, molar yield to compound 3- alpha-hydroxy-2s 0- (2 '-aminoethyle alcohol base) betulic acid benzyl ester 12mg：60% 。¹H NMR(300MHz,CDCl₃+CD₃OD) δ 7.34 (m, 5H), 5.09 (d, 1H, J=11.7Hz), 5.17 (d, 1H, J= 11.7Hz), 3.49 (t, 2H, J=4.5Hz), 3.00 (s, 1H), 2.75 (t, 2H, J=4.5Hz), 2.57-2.46 (m, 2H), 2.10-1.85 (m, 4H), 1.55-0.90 (m, other cycloaliphatic ring protons), 0.93 (s, 3H), 0.66 (s, 3H), 0.62 (d, 3H, J =6.0Hz), 0.58 (s, 3H), 0.52 (s, 3H), 0.48 (s, 3H)；ESI-MS(m/z):608.5(M+H)⁺(C₃₈H₅₉NO₄It is theoretical Value：607.91).

(2) 3- alpha-hydroxy-2s 0- (2 '-aminoethyle alcohol base) betulic acid

Upper step product (12mg, 0.020mmol) is dissolved in methanol (2mL) and a small amount of ethyl acetate, is changed after nitrogen, rapid to add Enter 10% Pd/C, then change hydrogen is changed after nitrogen, be stirred at room temperature.TLC detections reaction is complete after one hour.Change after nitrogen and filter off Pd/C, reaction solution uses petrol ether/ethyl acetate to be 4 after being spin-dried for:1 eluant, eluent system carries out column chromatography for separation, obtains compound 3- alpha-hydroxy-2s 0- (2 '-aminoethyle alcohol base) betulic acid (9.3mg, 0.018mmol), is white solid, molar yield： 91%.¹H NMR(300MHz,CDCl₃+CD₃OD) δ 3.49 (t, 2H, J=4.5Hz), 3.00 (s, 1H), 2.75 (t, 2H, J= 4.5Hz), 2.57-2.46 (m, 2H), 2.10-1.85 (m, 4H), 1.55-0.90 (m, other cycloaliphatic ring protons), 0.93 (s, 3H), 0.66 (s, 3H), 0.62 (d, 3H, J=6.0Hz), 0.58 (s, 3H), 0.52 (s, 3H), 0.48 (s, 3H)；ESI-MS(m/ z):518.4(M+H)⁺(C₃₂H₅₅NO₄Theoretical value：517.41).

Prepare embodiment 28 (compound number C74)

The benzyl ester (16mg, 0.028mmol) and DMAP (1mg, 0.1eq) of the products therefrom of embodiment 26 are prepared in dichloromethane TEA (6 μ L, 0.04mmol) and chloroacetic chloride (3 μ L, 0.04mmol) is added dropwise under (5ml), ice-water bath successively, is stirred overnight at room temperature.It is secondary The detection reaction of day TLC is complete.It is 2 that petrol ether/ethyl acetate is used after directly concentrating:1 eluant, eluent system carries out column chromatography for separation, Target compound 14mg is obtained, is white solid, molar yield：82.3%.The step of product is directly pressed above is in palladium carbon catalysis Under slough benzyl, obtain 6mg target products, be white solid, molar yield：50%.¹H NMR(300MHz,CDCl₃)δ4.35 (d, 1H, J=12.0Hz), 4.04 (d, 1H, J=12.0Hz), 3.40 (s, 1H), 2.76 (d, 1H, J=6.0Hz), 2.66 (d, 1H, J=6.0Hz), 2.35-2.24 (m, 2H), 2.09 (s, 3H), 2.01-1.92 (m, 4H), 1.86-1.75 (m, 4H), 1.57- 1.28 (m, other cycloaliphatic ring protons), 0.97 (s, 3H), 0.93 (s, 3H), 0.92 (s, 3H), 0.86 (s, 3H), 0.82 (s, 3H)；ESI-MS(m/z):553.3(M+Na)⁺(C₃₂H₅₀O₆Theoretical value：530.36).

Following compound is synthesized with the same method of embodiment 28 and different acyl chlorides：

2nd, test example

(1) inhibitory activity of the Split-TEV experiments detection the compounds of this invention to PS1/BACE1 interphase interaction

1. experiment purpose

The inhibitory activity that detection compound interacts to PS1/BACE1 is tested with Split-TEV.

2. experimental principle

The two albumen with the catalytic center PS1 and BACE1 of gamma-secretase are merged in marmor erodens respectively In the C-terminal and N-terminal of TEV protease；If the two albumen have interaction, then C, N two ends can be approached, TEV protease hair Wave activity, shearing release transcription factor and then the expression for starting reporter gene luciferase Firefly, its active reaction upstream egg White interaction.

3. laboratory sample

Compound is dissolved in DMSO (dimethyl sulfoxide) before experiment, mother liquor is prepared, it is dense needed for being diluted to when using with nutrient solution Degree.

4. experimental method

4.1 HEK293-MSR cells (be obtained from Sanofi-Aventis Recherche＆Development companies) are with containing There are the DMEM medium cultures of 10% hyclone in 37 DEG C of cell culture incubators containing 5% carbon dioxide.

The HEK293-MSR cells suspended after the digestion of 4.2 pancreatin are transfected using Fugene HD, with 2.5x10⁴/well Cell density is plated on 96 orifice plates.

Compound processing is given after 4.3 cultures 4 hours.The compound stock solutions for being dissolved in DMSO are prepared with PBS before experiment Into 10 times of ultimate density, 10 μ l/ holes are taken to add 96 orifice plates processing cell.

4.4 compounds handled cell after 16-18 hours, and luciferase is detected using SteadyGlo kits (Promega) Activity.

Obtained experimental data is shown in Table 1.

(2) enzyme linked immunosorbent assay detection the compounds of this invention produces the effect of level to A β

1. experiment purpose

Cell produces the change of A β levels after being handled with enzyme linked immunosorbent assay detection compound.

2. experimental principle

(HEK293 3 ' HA-APPswe expression plasmids of intracellular transfection are simultaneously sieved HEK293APPswe cell lines using antibiotic Stable expression APPswe cell line is obtained after choosing) expression people source APP swedish mutant, APPswedish eggs can be stablized A β can be produced by BACE1 the and γ-secretase shearings of cellular endogenous in vain, can be detected with ELISA method after compound processing A β produce the change of level.

3. laboratory sample

Compound is dissolved in DMSO before experiment, mother liquor is prepared, is diluted to required concentration when using with nutrient solution.

4. experimental method

4.1 HEK293APPswe cells are with the DMEM medium cultures containing 10% hyclone in containing 5% titanium dioxide 37 DEG C of cell culture incubators of carbon.

The HEK293APPswe cells suspended after the digestion of 4.2 pancreatin are with 2.5x10⁴/ hole cell density is plated on 96 orifice plates.

Cell changes liquid while giving compound processing after 4.3 cultures 16-18 hours.DMSO chemical combination will be dissolved in before experiment Thing mother liquor is configured to 10 times of ultimate density with PBS, takes 10 μ l/ holes to add 96 orifice plates processing cell.

4.4 compounds collect training liquid after handling 8 hours, the experiment provided according to the biological Co., Ltd's kits of Yi Kesai Method carries out Total A β detections.

Obtained experimental data is shown in Table 1.

Table 1

Above-mentioned active testing result is shown, under 10 μM of concentration, and the compound shown in the logical formula (I) of the present invention is to PS1/ BACE1 interacts and Α β secretion has good inhibiting effect, and 3- α configurations activity is generally significantly higher than 3- beta comfigurations Compound.

(3) vivo detection compound XYT-472B (the compound C46 for corresponding to the present invention) activity

1. experiment purpose

Compound continued administration is carried out by the method for miniature slow-releasing pump heeling-in, detection compound XYT-472B is in transgenosis Activity in AD mouse models.

2. experimental principle

The miniature slow-releasing pump of subdermal implantation can complete to be administered continuously for 4 weeks, and administration refers to after 4 weeks to brain area A β levels of mouse etc. Mark is measured, and assesses compound XYT-472B activity in vivo.

3. laboratory sample

3.1 ages, (transgenic models mouse, C3H/C57BL6 outbreeding systems, was purchased from the Tg6799 strains of 3 months Jackson Laboratory) the positive mouse of PCR identification APP and PS1 Gene Doubles.

Compound is dissolved in DMSO (dimethyl sulfoxide) before 3.2 experiments：Propane diols (v/v=1:1), it is poured into miniature sustained release Pump.

4. experimental method

After 4.1 anesthetized mices, about 1cm wound is scratched at its back, slow-releasing pump is inserted into subcutaneous area, suture mouse wound Mouthful.

4.2 miniature slow-releasing pump dosage XYT-472B 10mg/kg/ days, start to be denoted as administration the 1st day to perform the operation, to By mouse anesthesia during medicine 35 days, PBS cardiac perfusions are carried out, its brain is dissected, prefrontal cortex region and hippocampus is obtained, point EP pipes are not placed in, and liquid nitrogen flash freezer is after -80 DEG C of storages.

4.3 are weighed Mice brain tissues sample, and 500 μ l 2%SDS lysates, weight are added according to 20mg tissue samples Outstanding tissue sample, ultrasonic degradation to clarification.25,000g centrifuge 1 hour in 20 DEG C, and supernatant is used as SDS soluble constituents.

4.4 experimental methods provided according to the biological Co., Ltd's kits of Yi Kesai carry out A β 42 and detected.

Fig. 1 show administration 35 days after the generation levels of mouse brain area A β 42.Test result indicates that, XYT-472B gives mouse After 1 month, the generation levels of A β 42 are significantly reduced in its brain area.XYT-472B is pointed out in Mice Body to there is preferable biology to live Property, AD morbidities can be improved by reducing A β generations level.

Claims

1. purposes of the pentacyclic triterpenoid in the medicine for preparing treatment alzheimer disease shown in below formula I,

Wherein：

R₁For hydrogen, halogen, cyano group or hydroxyl；

R₂For hydroxyl, halogen, C₁-C₆Alkyl carbonyl epoxide, C₃-C₈Naphthene base carbonyl epoxide orWherein, R₉For hydrogen Or C₁-C₄Straight or branched alkyl；And R₂It is connected with α configurations with right side A rings；

R₃And R₄It is each independently C that is unsubstituted or being optionally substituted by a hydroxyl group₁-C₄Straight or branched alkyl；

R₅The C replaced for formoxyl, carboxyl, unsubstituted or hydroxyl₁-C₄Straight or branched alkyl, Wherein, R₁₀For C₁-C₈Straight or branched alkyl, C₃-C₈It is cycloalkyl, unsubstituted or by carboxyl Substituted C₁-C₈Straight or branched alkyl, carboxyl C₁-C₇Alkylidene or C that is unsubstituted or being replaced by carboxyl₃-C₈Cycloalkyl；

R₆And R₇It is each independently hydrogen or C₁-C₄Straight or branched alkyl；Or R₆And R₇The connected common shape of carbon atom Into=CH₂；

R₈For hydrogen, substituted or unsubstituted C₁-C₆Straight or branched alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substitution or not Substituted C₂-C₆Straight or branched alkenyl, substituted or unsubstituted C₂-C₆Alkynyl group, substituted or unsubstituted C₁-C₆Alkyl oxycarbonyl Base, substituted or unsubstituted C₁-C₆Alkyl amine group orWherein, the substituent is optionally from hydroxyl, C₁-C₃Alkane acyl Base, the C of hydroxyl substitution₁-C₄Alkyl-carbonyl ,=N-OH, amino, the C of hydroxyl substitution₁-C₆Alkyl, unsubstituted or hydroxyl replace C₁-C₆Alkyl amine group orIn one or more；

Wherein, R₁₁For H, substituted or unsubstituted C₁-C₆Alkyl, the optional hydroxyl of substituent, amino,

Wherein, R₁₂For substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₈It is cycloalkyl, substituted or unsubstituted C₁-C₄Alkoxy, substituted or unsubstituted C₆-C₁₀Aryl is substituted or unsubstituted at least containing one in N, S or O Individual heteroatomic 5 to 8 unit's heteroaryl, wherein, the substituent is optionally from hydroxyl, halogen, cyano group, C₁-C₄Alkoxy, BocNH- Substituted C₁-C₄Alkoxy,

Wherein, R₁₃For C₁-C₆Alkyl or C₆-C₁₀Aryl；

Wherein, m and n each stand alone as 1,2,3 or 4；

Z is methylene or is not present；

Represent to connect with singly-bound or double bond.

2. purposes according to claim 1, wherein,

R₁For hydrogen or hydroxyl；

R₂For hydroxyl, C₁-C₆Alkyl carbonyl epoxide, C₃-C₈Naphthene base carbonyl epoxide or

R₃And R₄It is each independently C₁-C₄Straight or branched alkyl；

R₅The C replaced for formoxyl, carboxyl, hydroxyl₁-C₄Alkyl,

R₈For hydrogen, substituted or unsubstituted C₁-C₆Straight or branched alkyl, substituted or unsubstituted C₂-C₆Straight or branched alkenyl, Substituted or unsubstituted C₁-C₆Alkyl-carbonyl orWherein, the substituent be selected from hydroxyl, formoxyl, acetyl group ,= N-OH, the C1-C6 alkyl amine groups of unsubstituted or hydroxyl substitution orOne or both of；

Wherein, R₁₁For substituted or unsubstituted C₁-C₄Alkyl, the optional hydroxyl of substituent, amino,

Wherein, R₁₂For substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₈It is cycloalkyl, substituted or unsubstituted C₆-C₁₀Aryl or substituted or unsubstituted heteroatomic 5 to 8 unit's heteroaryl containing in N, S or O, wherein, institute State substituent and be selected from hydroxyl, F, Cl, Br, C₁-C₄Alkoxy, the C of BocNH- substitutions₁-C₄Alkoxy,

Wherein, R₁₃For C₁-C₆Alkyl or phenyl.

3. purposes according to claim 1, wherein,

R₁For hydrogen or hydroxyl；

R₃And R₄It is simultaneously methyl；

R₅The C replaced for formoxyl, hydroxyl₁-C₄Alkyl, carboxyl,

Wherein, R₁₂For substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆It is cycloalkyl, substituted or unsubstituted Phenyl or substituted or unsubstituted thienyl, wherein, the substituent is selected from hydroxyl, F, Cl, Br, C₁-C₄Alkoxy, The C of BocNH- substitutions₁-C₄Alkoxy,

R₁₃For C₁-C₆Alkyl or phenyl.

4. purposes according to claim 1, wherein,

R₁For hydrogen or hydroxyl；

R₃And R₄It is simultaneously methyl；

R₅The C replaced for formoxyl, carboxyl, hydroxyl₁-C₄Alkyl,Wherein, R₁₀To be unsubstituted or The C replaced by carboxyl₁-C₅Straight or branched alkyl or carboxyl C₁-C₇Alkylidene；

R₆And R₇It is each independently hydrogen, methyl, ethyl or propyl group, or R₆And R₇Connected carbon atom is collectively forming= CH₂；

R₈For hydrogen, substituted or unsubstituted C₁-C₆Straight or branched alkyl, substituted or unsubstituted C₂-C₆Straight or branched alkenyl, Substituted or unsubstituted C₁-C₆Alkyl-carbonyl orWherein, the substituent be selected from hydroxyl, formoxyl, acetyl group ,= N-OH, the C of unsubstituted or hydroxyl substitution₁-C₆Alkyl amine group orOne or both of；

R₁₃For C₁-C₆Alkyl or phenyl.

5. the purposes according to any one of claim 1-4, wherein, the pentacyclic triterpenoid conduct shown in formula I PS1/BACE1 interaction inhibitors.

6. purposes of the pentacyclic triterpenoid selected from following structure in the medicine for preparing treatment alzheimer disease：

7. purposes according to claim 6, wherein, the pentacyclic triterpenoid interacts as PS1/BACE1 Inhibitor.