WO2015135474A1 - 一类五环三萜类化合物及其在制备治疗阿尔兹海默病的药物中的用途 - Google Patents

一类五环三萜类化合物及其在制备治疗阿尔兹海默病的药物中的用途 Download PDF

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WO2015135474A1
WO2015135474A1 PCT/CN2015/073987 CN2015073987W WO2015135474A1 WO 2015135474 A1 WO2015135474 A1 WO 2015135474A1 CN 2015073987 W CN2015073987 W CN 2015073987W WO 2015135474 A1 WO2015135474 A1 WO 2015135474A1
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substituted
unsubstituted
hydroxy
compound
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南发俊
谢欣
裴钢
王霄音
崔进
赵简
张仰明
张晨露
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中国科学院上海药物研究所
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
    • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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  • the invention belongs to the field of medicinal chemistry, and particularly relates to a class of pentacyclic triterpenoids, a pharmaceutical composition comprising the same, and a compound and a pharmaceutical composition thereof for preparing PS1 (PS1, Presenilin1, Presenilin 1)/BACE1 ( ⁇ site) Amyloid Protein Precursor Cleaving Enzyme 1, the use of amyloid pre-beta-degrading enzyme 1, also known as beta-secretase interaction inhibitor, and its use in the preparation of a medicament for the treatment of Alzheimer's disease.
  • PS1 PS1, Presenilin1, Presenilin 1
  • BACE1 ⁇ site
  • Amyloid Protein Precursor Cleaving Enzyme 1 the use of amyloid pre-beta-degrading enzyme 1, also known as beta-secretase interaction inhibitor, and its use in the preparation of a medicament for the treatment of Alzheimer's disease.
  • AD Alzheimer's Disease
  • AD is an incurable, progressive brain disease that is the leading cause of Alzheimer's disease.
  • AD can cause a large number of nerve cell death, destroy the patient's normal cognitive and behavioral abilities, and ultimately cause the patient to lose basic living self-care and physiological functions until death (Prince et al, World Alzheimer Report 2009. London: Alzheimer's Disease International, 2009, 14) -5; Blennow et al, Alzheimer's disease. Lancet, 2006, 368 (9533): 387-403).
  • AD occurs mainly in the elderly over the age of 60, and the proportion of people with 60-64 years old with AD is below 1%.
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • cholinesterase inhibitors such as Shuangyiping, rivastigmine tartrate, donepezil hydrochloride, galantamine, etc.
  • NMDA N-Methyl-D-Aspartic Acid, N a -methyl-D-aspartate receptor antagonist (such as memantine).
  • these drugs can alleviate the symptoms of AD patients and improve their cognitive and behavioral abilities to a certain extent, they have limited efficacy and cannot fundamentally reverse disease progression (Kastenholz et al., A novel perspective in Dementia therapy. Amyloid-Journal of Protein Folding Disorders, 2009, 16(2): 81-3).
  • a ⁇ Amyloid ⁇ -protein, ⁇ -amyloid
  • a ⁇ is produced by cleavage hydrolysis of amyloid Precursor Protein (APP) on the cell membrane.
  • APP amyloid Precursor Protein
  • the cleavage of APP includes a non-amyloid pathway and an amyloid pathway: the former refers to the hydrolysis of APP by ⁇ -secretase to produce soluble APP (sAPP); the latter refers to the subsequent sequestration of APP by ⁇ - and ⁇ -secretase.
  • a ⁇ deposition is the main cause of senile plaque formation in brain tissue of patients with Alzheimer's disease, and may be a common pathway leading to the pathogenesis of AD. Therefore, drugs developed for A ⁇ production and aggregation can reduce A ⁇ deposition in the brain by reducing A ⁇ production and polymerization, increasing A ⁇ degradation, and promoting A ⁇ clearance, thereby delaying the progression of AD and possibly fundamentally reversing the occurrence of AD. And progress.
  • ⁇ -secretase agonists can increase the production of soluble APP (such as West Vymexin; while ⁇ -secretase inhibitors can indirectly reduce A ⁇ production (such as ⁇ -secretase inhibitors AZD-3293, VTP-37948 and HPP-854 in clinical studies);
  • ⁇ -secretase Inhibitors can directly reduce the production of A ⁇ (such as Avagacestat, Elnd-007, Semagacestat, MK-0752, and PF-3084014); while ⁇ -secretase modulators (such as EVP-0962, CHF-5074, etc.) can regulate ⁇ - Secretion enzyme substrate cleavage mode to reduce A ⁇ 42 production, but no significant inhibition of ⁇ -secretase other substrates (such as Notch), thus reducing other substrate-related signaling pathways for ⁇ -secretase Impact.
  • AD Alzheimer's disease
  • PS1 binds to immature BACE1 and regulates BACE1 maturation and Activity
  • BACE1 beta-site amyloid protein precursor cleaving enzyme
  • Some pentacyclic triterpenoids and some other pentacyclic triterpenoids provided by the application have the activity of inhibiting PS1/BACE1 interaction and can reduce the production of A ⁇ , and thus can be used for the treatment of Alzheimer's disease.
  • the entire disclosure of Chinese Patent Application No. 2014100976413 is incorporated herein by reference.
  • the object of the present invention is to provide a pentacyclic triterpenoid compound represented by the following formula I and its use in the preparation of a PS1/BACE1 interaction inhibitor which inhibits the interaction between PS1/BACE1 and The production of A ⁇ is reduced, and thus it can be used for the preparation of a medicament for treating Alzheimer's disease.
  • the present invention provides a pentacyclic triterpenoid represented by the formula (I),
  • R 1 is hydrogen, halogen, cyano or hydroxy; preferably, R 1 is hydrogen or hydroxy;
  • R 2 is attached to the right A ring in an alpha configuration
  • R 9 is hydrogen or a C 1 -C 4 linear or branched alkyl group
  • R 3 and R 4 are each independently a C 1 -C 4 straight or branched alkyl group which is unsubstituted or substituted by a hydroxy group; preferably, R 3 and R 4 are each independently a C 1 -C 4 straight chain or a branch Alkenylalkyl; more preferably, R 3 and R 4 are both methyl;
  • R 5 is formyl (formaldehyde), carboxyl, unsubstituted or hydroxy-substituted C 1 -C 4 straight or branched alkyl
  • R 5 is a formaldehyde group, a hydroxy-substituted C 1 -C 4 straight or branched alkyl group, a carboxyl group
  • R 5 is a hydroxy-substituted C 1 -C 4 alkyl group, a carboxyl group
  • R 10 is a C 1 -C 8 linear or branched alkyl group, a C 3 -C 8 cycloalkyl group, a C 1 -C 8 linear or branched alkyl group which is unsubstituted or substituted by a carboxyl group, and a carboxyl group C 1 -C 8 alkylene group or C 3 -C 8 cycloalkyl group which is unsubstituted or substituted by a carboxyl group; preferably a C 1 -C 8 linear or branched alkyl group or a carboxy C 1 -C which is unsubstituted or substituted by a carboxyl group 8 alkylene; more preferably C 1 -C 5 straight or branched alkyl or carboxy C 1 -C 7 alkylene unsubstituted or substituted by carboxy;
  • R 8 is hydrogen, substituted or unsubstituted C 1 -C 6 straight or branched alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted C 2 -C 6 straight chain or A branched alkenyl group, a substituted or unsubstituted C 2 -C 6 alkynyl group, a substituted or unsubstituted C 1 -C 6 alkylcarbonyl group, a substituted or unsubstituted C 1 -C 6 alkylamino group,
  • R 8 is hydrogen, substituted or unsubstituted C 1 -C 6 straight or branched alkyl, substituted or unsubstituted C 2 -C 6 straight or branched alkenyl, substituted or unsubstituted C 1- C 6 alkylcarbonyl or The substituent is optionally selected from the group consisting of hydroxyl, C 1 -C 3 alkan
  • R 11 is H, a substituted or unsubstituted C 1 -C 6 alkyl group, preferably a substituted or unsubstituted C 1 -C 4 alkyl group, the substituent optionally having a hydroxyl group, an amino group,
  • R 12 is a substituted or unsubstituted C 1 -C 6 alkyl group, a substituted or unsubstituted C 3 -C 8 cycloalkyl group, a substituted or unsubstituted C 1 -C 4 alkoxy group, a substituted or unsubstituted group a C 6 -C 10 aryl group or a substituted or unsubstituted 5 to 8 membered heteroaryl group containing at least one hetero atom selected from N, S or O, preferably, R 12 is a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 8 cycloalkyl, substituted or unsubstituted C 6 -C 10 aryl or substituted or unsubstituted containing one selected from N, S or O a 5- to 8-membered heteroaryl group of an atom, more preferably, R 12 is a substituted or unsubsti
  • the substituent is optionally selected from the group consisting of a hydroxyl group, a halogen, a cyano group, a C 1 -C 4 alkoxy group, a BocNH-substituted C 1 -C 4 alkoxy group,
  • the substituent is selected from the group consisting of hydroxyl, F, Cl, Br, C 1 -C 4 alkoxy, BocNH-substituted C 1 -C 4 alkoxy,
  • R 13 is C 1 -C 6 alkyl or C 6 -C 10 aryl, preferably C 1 -C 6 alkyl or phenyl;
  • n and n are each independently 1, 2, 3 or 4;
  • Z is methylene or absent
  • the pentacyclic triterpenoid compound of the structure of the formula (I) of the present invention is specifically selected from the following structures:
  • the compound represented by the formula (I) and the specific compounds listed above serve as PS1/BACE1 interaction inhibitors.
  • the present invention also provides a pharmaceutical composition for treating Alzheimer's disease, the composition comprising the one or more selected from the group consisting of pentacyclic triterpenoids represented by the formula (I) as an active ingredient .
  • the composition may further comprise pharmaceutically conventional adjuvants such as dispersing agents, excipients, disintegrating agents, antioxidants, sweeteners, coating agents and the like.
  • the present invention discloses for the first time a class of pentacyclic triterpenoids as inhibitors of PS1/BACE1 interaction, which has the activity of inhibiting the interaction between PS1/BACE1, can reduce the production of A ⁇ , and can be used for the preparation of the treatment of Alzheimer's disease.
  • Drug for the first time a class of pentacyclic triterpenoids as inhibitors of PS1/BACE1 interaction, which has the activity of inhibiting the interaction between PS1/BACE1, can reduce the production of A ⁇ , and can be used for the preparation of the treatment of Alzheimer's disease. Drug.
  • Figure 1 shows the effect of the compound C46 of the present invention on the production of A?42 in a transgenic model mouse.
  • Compound C1 (3- ⁇ -Akebonoic acid, 3- ⁇ - ⁇ ), C2 (Oleanolic acid, oleanolic acid), C5 (Betulonic acid, birch barrel acid), C6 (Corosolic acid, Corosolic acid) ), C7 (Ursolic acid, ursolic acid), C8 (3-Oxo-Ursolic acid, 3-carbonyl-ursolic acid), C19 (Uvaol, ursolic acid) and C29 (Betulic acid, betulinic acid) A natural product commercially available ((purchased from Xi'an Xuanxuan Biotechnology Co., Ltd.)). The remaining compounds are prepared according to the method disclosed in the following application or can be prepared by referring to Chinese Patent Application No. 2014100976413.
  • NMR nuclear magnetic resonance
  • the top step product (1.58 g, 2.90 mmol) and S-(-)-2-methyloxazole borane (80 mg, 0.29 mmol) were added to a 100 mL dried round bottom flask and treated with fresh sodium.
  • THF 50 mL
  • a 10 M borane-tetrahydrofuran solution (0.32 mL) was slowly added dropwise at room temperature to control the dropping rate, and the addition was completed in ten minutes, and stirred at room temperature for ten minutes. TLC monitoring showed that the reaction was completed.
  • the reaction flask was transferred to an ice water bath, and the reaction was quenched by dropwise addition of methanol.
  • Betulinic acid 48 mg, 0.11 mmol was dissolved in methanol/dichloromethane (5 mL/5 mL), and ozone was taken at -78 ° C. After the ozone was stopped, the residual ozone was exhausted with oxygen, and the reaction was quenched by the addition of dimethyl sulfide (25 ⁇ L), gradually warmed to room temperature, and allowed to react overnight. The reaction mixture was concentrated under reduced pressure.
  • the intermediate S20 was dissolved in methanol and a small amount of ethyl acetate. After changing nitrogen, a catalytic amount of Pd/C was added, and after hydrogen substitution, the reaction was stirred at room temperature, and the reaction was completely observed by TLC. Filtration and purification of the obtained crude product were purified by silica gel column chromatography to afford compound C52 (5mg, yield: 84.8%) as white solid.
  • the intermediate S11 (3- ⁇ -hydroxy-20-formyl betulinic acid benzyl ester) is reacted with a n-propyl format reagent, and then hydrogenated to remove the benzyl group.
  • the intermediate S17 was dissolved in methanol and a small amount of ethyl acetate. After changing nitrogen, a catalytic amount of Pd/C was added, and after hydrogen substitution, the reaction was stirred at room temperature under normal pressure, and the reaction was completely confirmed by TLC. Filtration and concentration of the filtrate were carried out, and the obtained crude product was purified by silica gel column chromatography to afford compound C51 (5mg, yield: 71.4%) as white solid.
  • Trimethylsulfoxonium iodide (21 mg, 0.095 mmol) and sodium hydride (2 mg, 0.095 mmol) were poured into dry THF (1 mL) at room temperature, and after half an hour, the intermediate S11 (3- ⁇ -hydroxyl) was added dropwise.
  • a solution of -20-formyl betulinic acid benzyl ester (15 mg, 0.027 mmol) in dry THF (1 mL) The reaction was quenched with EtOAc (EtOAc/EtOAc/EtOAc).
  • EtOAc EtOAc/EtOAc/EtOAc
  • Compound S18 (5 mg, yield: 33.3%), white solid.
  • the intermediate S18 was dissolved in methanol and a small amount of ethyl acetate. After changing nitrogen, a catalytic amount of Pd/C was added, and after hydrogen substitution, the reaction was stirred at room temperature under normal pressure, and the reaction was completely observed by TLC. Filtration and concentration of the obtained crude product were purified by silica gel column chromatography to afford compound C54 (4mg, yield: 94.8%) as white solid.
  • reaction was quenched by the addition of sodium sulphate, and extracted with dichloromethane. The organic phase was washed with water and dried and concentrated. The crude product was dissolved in methanol (20 mL), and sodium borohydride (25 mg, 0.66 mmol) was added portionwise in an ice water bath. After one hour of reaction, the reaction was complete by TLC, and the reaction was quenched by dropwise addition of a saturated aqueous solution of ammonium chloride.
  • the intermediate S15 was dissolved in methanol and a small amount of ethyl acetate. After changing nitrogen, a catalytic amount of Pd/C was added, and after hydrogen substitution, the reaction was stirred at room temperature under normal pressure, and the reaction was completely confirmed by TLC. Filtration and purification of the obtained crude product were purified by silica gel column chromatography to afford compound C55 (6mg, yield: 85.7%) as white solid.
  • the preparation of the intermediate S3 (3- ⁇ -hydroxy betulinic acid benzyl ester) was referred to Preparation Example 1.
  • the intermediate S3 (143 mg, 0.262 mmol) was dissolved in methanol/dichloromethane (5 mL/5 mL), and ozone was passed at -78 ° C.
  • the reaction was completed by TLC, and the residual ozone was blasted with nitrogen after the passage of ozone was stopped.
  • the reaction was then quenched by the addition of dimethyl sulfide (25 ⁇ L) and slowly allowed to warm to room temperature with stirring.
  • the intermediate S21 (19 mg, 0.035 mmol) was reacted with an allyl Grignard reagent to obtain an intermediate S22, which was directly subjected to the next step without purification.
  • the intermediate S14 in the preparation example 9 of the present application the intermediate S22 obtained by the above step is epoxidized with m-chloroperoxybenzoic acid to obtain an intermediate S23, which is obtained by silica gel column chromatography to give a white solid (7 mg, The yield in two steps is 35%).
  • the intermediate S11 was obtained by reacting the intermediate S11 with hydroxylamine hydrochloride, and the intermediate S13 was obtained by hydrogenation to remove the benzyl group to give the compound C58.
  • the obtained intermediate S31 is dissolved in methanol, catalytically hydrogenolysis in the presence of Pd-C under normal pressure, and the benzyloxycarbonyl group and the benzyl group of C-20 are removed to obtain the intermediate S32, and the next step is directly carried out without purification. .
  • the obtained intermediate S32 was dissolved in dichloromethane, and triethylamine and Bo. The reaction mixture was concentrated under reduced pressure.
  • Betulinic acid (1.2g, 2.63mmol) was dissolved in methanol/ethyl acetate (40mL/10mL). After changing nitrogen, a catalytic amount of Pd/C was added. After changing the nitrogen, the hydrogen was exchanged. The mixture was stirred at room temperature for 2 days. The reaction was detected by TLC. complete. After changing the nitrogen gas, the reaction solution was filtered, dried, and then subjected to column chromatography with petroleum ether: ethyl acetate as a 10:1 polarity. The product C34 was obtained as a white solid (1.04 g, 2.27 mmol).
  • raw material oleanolic acid C2
  • raw ursolic acid C7
  • raw glycyrrhetinic acid raw material betulin: C29, raw material C34
  • Betulinic acid (41mg, 0.09mmol) in dry tetrahydrofuran (5 mL), was added dropwise a tetrahydrofuran solution (2M) BH 3 -Me 2 S in an ice-water bath, brought to room temperature After stirring for one hour. The next day, the reaction solution was transferred to an ice water bath, and ethanol (280 ⁇ L), a saturated sodium acetate solution (200 ⁇ L), and a 30% hydrogen peroxide solution (140 ⁇ L) were sequentially added. Stir at room temperature overnight and the reaction was complete by TLC. It was diluted with water, extracted with ethyl acetate and washed with saturated brine.
  • the product of the above step (77 mg, 0.13 mmol) was dissolved in methanol (8 mL) and a small amount of ethyl acetate. After changing nitrogen, 10% of Pd/C was quickly added, and then nitrogen was exchanged, and hydrogen was exchanged and stirred at room temperature. After one hour, the reaction was completely detected by TLC. After changing the nitrogen, the Pd/C was filtered off, and the reaction solution was spun dry, and then subjected to column chromatography with petroleum ether/ethyl acetate as a 10:1 eluent system to obtain a compound 3- ⁇ -acetoxy betulinic acid (60 mg). , 0.12 mmol), as a white solid, m.p.
  • the product of the above step (197 mg, 0.35 mmol) was dissolved in methanol (10 mL) and a small amount of ethyl acetate. After changing nitrogen, 10% of Pd/C was quickly added, and then nitrogen was exchanged, and hydrogen was exchanged and stirred at room temperature. After one hour, the reaction was completely detected by TLC. After changing the nitrogen, the Pd/C was filtered off, and the reaction solution was spun dry, and then subjected to column chromatography with petroleum ether/ethyl acetate as a 2:1 eluent system to obtain a compound 3- ⁇ -hydroxy-22-hydroxy betulinic acid. (140 mg, 0.30 mmol) as a white solid, m.
  • the product of the above step (50 mg, 0.088 mmol) was dissolved in methanol (5 mL) and a small portion of ethyl acetate. After changing nitrogen, 10% of Pd/C was quickly added, and then nitrogen was exchanged and hydrogen was exchanged and stirred at room temperature. After one hour, the reaction was completely detected by TLC. After changing the nitrogen, the Pd/C was filtered off, and the reaction solution was spun dry, and then subjected to column chromatography with petroleum ether/ethyl acetate as a 2:1 eluent system to obtain a compound 3- ⁇ -hydroxy-20-formyl white birch. Acid (36.7 mg, 0.078 mmol) was obtained as a white solid.
  • reaction solution was heated to zero temperature, and a saturated aqueous solution of sodium chloride (50 ⁇ L) was added thereto. After stirring for one hour, the molecular sieve was removed by filtration, and the filtrate was spun dry, and then the petroleum ether/ethyl acetate was used as an eluent of 2:1. The system was subjected to column chromatography to give the title compound 16 mg as a white solid. NMR confirmed that C-17 was the predominant product of the S configuration.
  • the benzyl ester (16 mg, 0.028 mmol) and DMAP (1 mg, 0.1 eq) of the product obtained in Example 26 were obtained from dichloromethane (5 ml), and then, under a water bath, TEA (6 ⁇ L, 0.04 mmol) and acetyl chloride (3 ⁇ L, 0.04 mmol), stirred at room temperature overnight. The next day TLC detected the reaction completely. After direct concentration, column chromatography was carried out using a petroleum ether/ethyl acetate (2:1) eluent system to give the title compound 14 mg as a white solid. The product was directly stripped of the benzyl group by palladium-carbon catalysis to give 6 mg of the desired product as a white solid.
  • the inhibitory activity of the compound on the PS1/BACE1 interaction was examined by the Split-TEV assay.
  • the two proteins of ⁇ -secretase catalytic center PS1 and BACE1 are respectively fused to the C-terminus and N-terminus of TEV protease of tobacco etch virus; if the two proteins interact, then C and N will be Close, the TEV protease is active, and the cleavage-release transcription factor then initiates expression of the reporter gene, luciferase, Firefly, whose activity reflects upstream protein interactions.
  • the compound was dissolved in DMSO (dimethyl sulfoxide) before the experiment to prepare a mother liquor, which was diluted with the culture solution to the desired concentration.
  • DMSO dimethyl sulfoxide
  • HEK293-MSR cells obtained from Sanofi-Aventis für & Development were cultured in a 37 ° C cell culture chamber containing 5% carbon dioxide in DMEM medium containing 10% fetal calf serum.
  • HEK293-MSR cells suspended after trypsinization were transfected with Fugene HD and plated at 96x10 4 /well cell density in 96-well plates.
  • HEK293APPswe cell line (transfected with 3'HA-APPswe expression plasmid in HEK293 cells and stably stained with APPswe after antibiotic screening) can stably express human APP swedish mutant, APPswedish protein can be endogenous BACE1 and ⁇ -secretase cleavage produces A ⁇ , and the compound can be treated to detect changes in A ⁇ production levels by ELISA.
  • the compound was dissolved in DMSO before the experiment to prepare a mother liquor, which was diluted with the culture solution to the desired concentration.
  • HEK293APPswe cells were cultured in a 37 ° C cell culture chamber containing 5% carbon dioxide in DMEM medium containing 10% fetal calf serum.
  • HEK293APPswe cells suspended after trypsin digestion were plated in 96-well plates at a cell density of 2.5 x 10 4 /well.
  • the cells were exchanged for fluid treatment.
  • the mother liquor of the compound dissolved in DMSO was prepared in PBS to a 10-fold final concentration before the experiment, and 10 ⁇ l/well was added to the 96-well plate to treat the cells.
  • the compound was continuously administered by a micro-sustained-release pump implantation method, and the activity of the compound XERT-472B in the transgenic AD mouse model was examined.
  • the subcutaneous implanted micro-release pump can be used for 4 weeks of continuous administration. After 4 weeks of administration, the A ⁇ level of the brain area of the mouse is measured to evaluate the in vivo activity of the compound XERT-472B.
  • Tg6799 line transgenic model mouse, C3H/C57BL6 inbred line, purchased from Jackson Laboratory
  • aged 3 months was used to identify mice with double positive APP and PS1 genes.
  • a wound of about 1 cm was opened on the back of the patient, and a sustained release pump was placed in the subcutaneous region to suture the mouse wound.
  • Mouse brain tissue samples were weighed, 500 ⁇ l of 2% SDS lysate was added to 20 mg tissue samples, tissue samples were resuspended, and sonicated to clarification. 25,000 g was centrifuged at 20 ° C for 1 hour, and the supernatant was used as an SDS soluble component.
  • Figure 1 shows the level of A ⁇ 42 production in the brain regions of mice after 35 days of administration.
  • the experimental results showed that after 1 month of administration of XYT-472B, the level of A ⁇ 42 production in the brain region was significantly decreased. It is suggested that XYT-472B has good biological activity in mice and can improve the pathogenesis of AD by decreasing the level of A ⁇ production.

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Abstract

本发明公开了一类通式(I)所示的五环三萜类化合物以及包含该类化合物的药物组合物，并且，本发明公开所述化合物在制备PS1/BACE1相互作用抑制剂中的用途，以及其在制备治疗阿尔兹海默病的药物中的用途。本发明首次公开了一类通式(I)所示的五环三萜类化合物可作为PS1/BACE1抑制剂，具有抑制PS1/BACE1之间相互作用的活性，能降低Aβ的生成，可用于制备治疗阿尔兹海默病的药物。

Description

一类五环三萜类化合物及其在制备治疗阿尔兹海默病的药物中的用途 技术领域
本发明属于药物化学领域，具体涉及一类五环三萜类化合物、包含该类化合物的药物组合物和该类化合物和药物组合物在制备PS1(PS1，Presenilin1，早老蛋白1)/BACE1(βsite Amyloid Protein Precursor Cleaving Enzyme 1，淀粉蛋白前β位分解酶1，又称为β分泌酶)相互作用抑制剂中的用途，以及其在制备治疗阿尔兹海默病的药物中的用途。
背景技术
阿尔兹海默病(Alzheimer’s Disease,AD)是一种不可治愈的、进行性大脑疾病，是早老性痴呆的最主要原因。AD可导致大量神经细胞死亡，破坏患者正常的认知和行为能力，最终使病人丧失基本的生活自理能力和生理功能直至死亡(Prince等,World Alzheimer Report 2009.London:Alzheimer's Disease International,2009,14-5；Blennow等，Alzheimer's disease.Lancet,2006,368(9533):387-403)。AD主要在60岁以上的老人中发生，其中60-64岁区间的人患AD的比例在1％以下。但随着年龄的增长，每增加5岁，AD发病率将增加一倍，85岁以上老人中AD发病率可达23-33％。由于缺乏有效的治愈手段，AD患病周期可长达数十年，给患者家属带来沉重的家庭负担。随着我国人口老龄化加剧，发展新型AD治疗药物显得尤为必要和紧迫。
现阶段，AD的临床治疗药物主要为胆碱酯酶抑制剂(如双益平、重酒石酸卡巴拉汀、盐酸多奈哌齐、加兰他敏等)和NMDA(N-Methyl-D-Aspartic Acid，N-甲基-D-天冬氨酸)受体拮抗剂(如美金刚胺)。这些药物虽可缓解AD患者症状，在一定程度上改善患者的认知和行为能力，但疗效有限，不能从根本上逆转疾病进展(Kastenholz等，A novel perspective in dementia therapy.Amyloid-Journal of Protein Folding Disorders,2009,16(2):81-3)。
虽然当前对于阿尔茨海默症的发病机制仍未获得完全了解，但已有越来越多的研究证据表明AD的发生与患者脑内大量Aβ(Amyloidβ-protein，β-淀粉样蛋白)的生成和沉积以及由此所导致的神经毒性损害有关。Aβ是由细胞膜上淀粉样蛋白前体(Amyloid Precursor Protein，APP)剪切水解产生。APP的剪切包括非淀粉样途径和淀粉样途径：前者指APP被α-分泌酶水解，生成可溶性APP(sAPP)；后者是指APP依次经β-和γ-分泌酶剪切产生含有38～43个氨基酸的短肽Aβ，特别是由42个氨基酸构成的不可溶Aβ42最容易聚合形成寡聚体、纤维以及淀粉样斑块，其中Aβ寡聚体对神经元和突触的毒性最 大，致使神经细胞损伤乃至死亡。由于Aβ沉积是阿尔茨海默氏症患者脑组织中老年斑形成的主要原因，并可能是其它因素导致AD发病的共同途径。因此，针对Aβ产生和聚集而研发的药物可通过减少Aβ产生及聚合、增加Aβ降解、促进Aβ清除等途径减少脑内Aβ沉积，从而延迟AD病情进程，并有可能从根本上逆转AD的发生和进展。
目前已有多个靶向Aβ的药物进入临床试验，其中与Aβ生成相关的三个分泌酶是当前AD药物研发的重要靶点：如α-分泌酶激动剂可增加可溶性APP的生成(如西维美林)；而β-分泌酶抑制剂则可间接减少Aβ的产生(如处于临床阶段研究的β-分泌酶抑制剂AZD-3293，VTP-37948和HPP-854等)；γ-分泌酶抑制剂可直接减少Aβ的产生(如Avagacestat，Elnd-007，Semagacestat，MK-0752以及PF-3084014等)；而γ-分泌酶调节剂(如EVP-0962,CHF-5074等)可通过调节γ-分泌酶底物剪切模式从而减少Aβ42的产生，但对γ-分泌酶其它底物(如Notch)的剪切作用没有明显抑制，因此减少了对γ-分泌酶其他底物相关信号通路的影响。
尽管近年来针对Aβ的药物研发获得了较大进展，但尚未有相关药物批准上市。由于AD是一种多病因、进行性发展的神经退行性病变，其发病隐匿、病程长，病理过程复杂，因此仍迫切需要发展具有新颖治疗机制的AD药物。如前所述，Aβ的生成涉及两个重要的分泌酶，即BACE1和以PS1为催化亚基的γ-分泌酶复合物，且它们并非孤立存在并发挥水解功能的，而是有着某种相互作用和联系。2003年，Sebastien等人在通过免疫共沉淀实验证明，PS1和BACE1这两种重要的蛋白之间存在着直接的蛋白-蛋白相互作用：PS1可通过与未成熟的BACE1结合，调控BACE1的成熟和活性(Hebert等，Presenilin-1interacts directly with the beta-site amyloid protein precursor cleaving enzyme(BACE1).Neurobiology of Disease,2003,13(3):238-45)。因此如果能发展PS1/BACE1相互作用抑制剂，通过抑制PS1/BACE1之间蛋白-蛋白相互作用，减少Aβ的产生，这一类化合物可用于制成新型的AD治疗药物。
发明内容
中国专利申请第2014100976413号(此为申请号，专利申请尚未公布，本申请已要求上述申请号为2014100976413的专利申请的优先权)是本申请人以前的一项中国发明专利申请，其公开了一类五环三萜类化合物，可激动TGR5(G蛋白偶联胆酸膜受体)的生物活性，并可用于制备糖尿病治疗药物。在进一步的深入研究中，在中国发明专利申请第201410673701号所公开的PS1/BACE1抑制剂的筛选模型上，本申请人意外地发现第201410097641号 申请所提供的部分五环三萜类化合物以及一些其它五环三萜类化合物具有抑制PS1/BACE1相互作用的活性，并能减少Aβ的生成，从而可用于制成阿尔兹海默病治疗药物。在此将第2014100976413号中国专利申请全文引入本申请。
本发明的目的在于提供如下通式I所示的五环三萜类化合物及其在制备PS1/BACE1相互作用抑制剂中的用途，所述化合物可以抑制PS1/BACE1之间的相互作用，并可降低Aβ的生成，从而可用于制备治疗阿尔兹海默病的药物。
根据本发明的目的，本发明提供了通式(I)所示的五环三萜类化合物，
Figure PCTCN2015073987-appb-000001
其中：
R1为氢、卤素、氰基或羟基；优选地，R1为氢或羟基；
R2为羟基、卤素、氧代基(＝O)、＝N-OH、C1-C6烷基羰基氧基、C3-C8环烷基羰基氧基或
Figure PCTCN2015073987-appb-000002
优选地，R2为羟基、氧代基(＝O)、＝N-OH、C1-C6烷基羰基氧基、C3-C8环烷基羰基氧基或
Figure PCTCN2015073987-appb-000003
优选地，R2以α构型与右侧A环连接；
其中，R9为氢或C1-C4直链或支链烷基；
R3和R4各自独立地为未取代或被羟基取代的C1-C4直链或支链烷基；优选地，R3和R4各自独立地为C1-C4直链或支链烷基；更优选地，R3和R4同时为甲基；
R5为甲酰基(甲醛基)、羧基、未取代或羟基取代的C1-C4直链或支链烷基、
Figure PCTCN2015073987-appb-000004
优选地，R5为甲醛基、羟基取代的C1-C4 直链或支链烷基、羧基、
Figure PCTCN2015073987-appb-000005
更优选R5为羟基取代的C1-C4烷基、羧基、
Figure PCTCN2015073987-appb-000006
其中，R10为C1-C8直链或支链烷基、C3-C8环烷基、未取代或被羧基取代的C1-C8直链或支链烷基、羧基C1-C8亚烷基或者未取代或被羧基取代的C3-C8环烷基；优选为未取代或被羧基取代的C1-C8直链或支链烷基或羧基C1-C8亚烷基；更优选为未取代或被羧基取代的C1-C5直链或支链烷基或羧基C1-C7亚烷基；
R6和R7各自独立地为氢或C1-C4直链或支链烷基；优选氢、甲基、乙基或丙基；或者R6和R7与其连接的碳原子共同形成＝CH2；
R8为氢、取代或未取代的C1-C6直链或支链烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6直链或支链烯基、取代或未取代的C2-C6链炔基、取代或未取代的C1-C6烷基羰基、取代或未取代的C1-C6烷基胺基、
Figure PCTCN2015073987-appb-000007
优选地，R8为氢、取代或未取代的C1-C6直链或支链烷基、取代或未取代的C2-C6直链或支链烯基、取代或未取代的C1-C6烷基羰基或
Figure PCTCN2015073987-appb-000008
所述取代基任选自羟基、C1-C3烷酰基、羟基取代的C1-C4烷基羰基、＝N-OH、氨基、羟基取代的C1-C6烷基、未取代或羟基取代的C1-C6烷基胺基或
Figure PCTCN2015073987-appb-000009
中的一种或多种，优选地，所述取代基选自羟基、甲酰基、乙酰基、＝N-OH、未取代或羟基取代的C1-C6烷基胺基或
Figure PCTCN2015073987-appb-000010
中的一种或两种；
其中，R11为H、取代或未取代的C1-C6烷基，优选取代或未取代的C1-C4烷基，所述取代基任选羟基、氨基、
Figure PCTCN2015073987-appb-000011
其中，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C6-C10芳基或者取代或未取代的至少含有选自N、S或O中的一个杂原子的5至8元杂芳基，优选地，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C10芳基或者取代或未取代的含有选自N、S或O中的一个杂原子的5至8元杂芳基，更优选地，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的苯基或者取代或未取代的噻吩基；所 述取代基任选自羟基、卤素、氰基、C1-C4烷氧基、BocNH-取代的C1-C4烷氧基、
Figure PCTCN2015073987-appb-000012
优选地，所述取代基选自羟基、F、Cl、Br、C1-C4烷氧基、BocNH-取代的C1-C4烷氧基、
Figure PCTCN2015073987-appb-000013
Figure PCTCN2015073987-appb-000014
其中，R13为C1-C6烷基或C6-C10芳基，优选为C1-C6烷基或苯基；
其中，m和n各自独立为1、2、3或4；
Z为亚甲基或不存在；
Figure PCTCN2015073987-appb-000015
表示此处以单键或双键连接。
优选地，本发明通式(I)结构的五环三萜类化合物具体选自如下结构：
Figure PCTCN2015073987-appb-000016
Figure PCTCN2015073987-appb-000017
Figure PCTCN2015073987-appb-000018
根据本发明的另一方面，提供了通式(I)所示的化合物及以上所列出的具体化合物在制备PS1/BACE1相互作用抑制剂中的用途。
根据本发明的另一方面，提供了通式(I)所示的化合物及以上所列出的具体化合物在制备治疗阿尔兹海默病的药物中的用途；其中，对通式(I)化合物的定义如上所述；
在所述用途中，通式(I)所示的化合物及以上所列出的具体化合物作为PS1/BACE1相互作用抑制剂。
本发明还提供了一种治疗阿尔兹海默病的药物组合物，该组合物包含所述选自通式(I)所示的五环三萜类化合物中的一种或多种作为活性成分。所述组合物并可进一步包含药学上常规的辅剂，例如分散剂、赋形剂、崩解剂、抗氧化剂、甜味剂、包衣剂等。
本发明具有如下有益效果：
本发明首次公开了一类五环三萜类化合物可作为PS1/BACE1相互作用抑制剂，具有抑制PS1/BACE1之间相互作用的活性，能降低Aβ的生成，可用于制备治疗阿尔兹海默病的药物。
附图说明
图1显示了本发明化合物C46对于转基因模型小鼠Aβ42产生的影响。图中，对照组小鼠n＝8，给药组小鼠n＝6,，误差线代表标准误差(*p<0.05)。
具体实施方式
一、本申请化合物的来源和制备
化合物C1(3-α-Akebonoic acid，3-α-木通萜酸)、C2(Oleanolic acid，齐墩果酸)、C5(Betulonic acid，桦木桶酸)、C6(Corosolic acid，科罗索酸)、C7(Ursolic acid，熊果酸)、C8(3-Oxo-Ursolic acid，3-羰基-熊果酸)、C19(Uvaol，熊果醇)和C29(Betulic acid，白桦脂酸)均为可以市售购得的天然产物((购自西安昊轩生物科技有限公司))。其余化合物均按照下面本申请所公开方法或可参照中国专利申请第2014100976413号制备得到。
下述制备实施例中，NMR(核磁共振)用Varian生产的Mercury-Vx 300M仪器测定，内标：δH 7.26ppm(CDCl3)，2.50ppm(DMSO-d6)；质谱用Agilent1200Quadrupole LC/MS液质联用仪测定；所用试剂由上海化学试剂公司提供；TLC薄层层析硅胶板由山东烟台会友硅胶开发有限公司生产，型号HSGF254；化合物纯化使用的正相柱层析硅胶为山东青岛海洋化工厂分厂生产，型号zcx-11，200-300目。
制备实施例1：化合物C33的合成
Figure PCTCN2015073987-appb-000019
(1)白桦脂酸苄酯
室温下将原料白桦脂酸(4g,8.76mmol)(购自西安昊轩生物科技有限公司)溶解在DMF(50mL)中，加入无水碳酸钾(2.4g,17.37mmol)，搅拌下慢慢滴加氯化苄(1.2mL,10.52mmol)滴加完毕后将反应液移至50℃搅拌过夜。次日，将混合物冷却至室温，加入去离子水100mL稀释，用乙酸乙酯(2×100mL)萃取，将合并的有机层分别用去离子水和饱和食盐水洗涤，经硫酸钠干燥后并减压蒸馏得到所需白色固体化合物白桦脂酸苄酯(4.62g)，摩尔收率：97％。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),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,其它脂肪环质子),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)+(C37H54O3理论值：546.41)。
(2)3-羰基白桦脂酸苄酯
在冰水浴下将上步产物(4.62g，8.64mmol)溶于二氯甲烷(100mL),分批加入Dess-Martin氧化剂(4.3g,10.15mmol)，慢慢升至室温并搅拌过夜。次日，将反应混合物过滤后旋干，用石油醚/乙酸乙酯为20:1的洗脱剂体系进行柱层析分离，得到化合物3-羰基白桦脂酸苄酯(4.39g)，为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C37H52O3理论值：544.39)。
(3)3-α-羟基白桦脂酸苄酯
向100mL烘干的圆底烧瓶中加入上步产物(1.58g,2.90mmol)和S-(-)-2-甲基恶唑硼烷(80mg,0.29mmol)，并加入新鲜钠丝处理过的THF(50mL)。室温下慢慢滴加10M的硼烷-四氢呋喃溶液(0.32mL)，控制滴加速度，在十分钟内加完，室温下搅拌十分钟，TLC监测显示反应已经完成。将反应瓶移至冰水浴，慢慢滴加甲醇淬灭反应，待不再有气泡生成后旋干溶剂，用石油醚/乙酸乙酯为20:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基白桦脂酸苄酯(790mg)，为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C37H54O3理论值：546.41)。
(4)3-α-羟基白桦脂酸C33
上步产物(100mg,0.18mmol)溶于甲醇(8mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC 检测反应完全。换氮气后滤去催化剂，反应液旋干后用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到化合物C33(78mg)，为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C30H48O3理论值：456.36)。
制备实施例2：化合物C35的合成
Figure PCTCN2015073987-appb-000020
白桦脂酸(48mg,0.11mmol)溶于甲醇/二氯甲烷(5mL/5mL)中，在-78℃下通入臭氧，TLC显示反应完全。停止通入臭氧后用氧气排尽残余的臭氧，加入二甲硫醚(25μL)淬灭反应，缓缓升温至室温，并反应过夜。次日，反应液减压浓缩，所得残余物经硅胶柱层析纯化(二氯甲烷/甲醇＝50/1，V/V)，得化合物C35(21mg，收率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)+(C29H46O4理论值：458.34)。
制备实施例3：化合物C41的合成
Figure PCTCN2015073987-appb-000021
化合物C42(12mg,0.02mmol)溶于乙醇/水(4mL/1mL)混合溶剂中，加入2N氢氧化钠(1mL)，室温搅拌过夜。次日，TLC检测反应完全，减压浓缩溶剂除去乙醇后，用1N盐酸溶液调至pH＝3，然后用乙酸乙酯萃取，合并有机相后用饱和氯化钠溶液洗涤，有机相干燥、浓缩，所得残余物经硅胶柱层析纯化(二氯甲烷/甲醇＝10/1，V/V)，得到合物C41(6mg，收率：50％)， 白色固体。1H NMR(300MHz,CDCl3)δ4.78(s,1H),2.28-2.18(m,4H),1.98-1.15(m,其余脂肪环烃质子),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)+(C32H50O6理论值：530.36)。
制备实施例4：化合物C45的合成
Figure PCTCN2015073987-appb-000022
按照与制备实施例23同样的方法，以化合物C38为原料，可合成得到化合物C45(总收率58％，白色固体)。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)+，(C39H66N2O4理论值：626.50)。
制备实施例5：化合物C52的合成
Figure PCTCN2015073987-appb-000023
中间体S11(3-α-羟基-20-甲酰基白桦脂酸苄酯)的制备具体如下。
参照实施例1的方法制备3-α-羟基白桦脂酸苄酯。
(1)3-α-羟基-20，21-环氧白桦脂酸苄酯
中间体3-α-羟基白桦脂酸苄酯(90mg,0.17mmol)溶于二氯化碳(10mL)中，冰水浴下慢慢加入间氯过氧苯甲酸(71mg,0.34mmol)后室温搅拌3h，TLC检测反应完全，加入亚硫酸钠饱和溶液淬灭反应，用水洗涤有机相，干燥浓缩所得残余物用石油醚/乙酸乙酯为6:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-20，21-环氧白桦脂酸苄酯74mg,为白色固体，摩尔收率：80.4％。1H NMR(300MHz,CDCl3)δ7.34(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,其它脂肪环质子),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)+(C37H54O4理论值：562.40)。
(2)3-α-羟基-20-甲酰基白桦脂酸苄酯(S11)
中间体3-α-羟基-20，21-环氧白桦脂酸苄酯(68mg,0.12mmol)溶于三氯化碳(10mL)中，滴入两滴浓盐酸，回流1h，TLC检测反应完全，直接干燥浓缩所得残余物用石油醚/乙酸乙酯为4:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-20-甲酰基白桦脂酸苄酯50mg,为白色固体，摩尔收率：73.6％。1H NMR(300MHz,CDCl3)δ9.84(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,其它脂肪环质子),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)+(C37H54O4理论值：562.40)。
将中间体S11(19mg,0.034mmol)溶于新鲜的干燥THF(四氢呋喃)(2mL)中，于-78℃下滴加1N甲基溴化镁的四氢呋喃溶液(52μL，0.050mmol)，然后缓慢升至室温过夜。次日，氯化铵饱和溶液淬灭反应，乙酸乙酯萃取并干燥后，减压浓缩得中间体S19，直接进行下一步反应。
中间体S19溶于二氯甲烷(2mL)中，加入Dess-Martin氧化剂(DMP)(21mg,0.050mmol)，室温搅拌3h，TLC检测反应完全。将反应混合物过滤，滤液减压浓缩，残余物经硅胶柱层析(乙酸乙酯/石油醚＝1/20，V/V)，得中间体S20(7mg，两步收率35.8％)，白色固体。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),3.24(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,其余脂肪环烃质子),1.25(s,3H),1.02(s,3H),0.93(s,3H),0.91(s,3H),0.83(s,3H),0.81(s,3H)。
中间体S20溶于甲醇和少量乙酸乙酯中，换氮气后，加入催化量的Pd/C,氢气置换后，室温搅拌反应，TLC检测跟踪反应完全。过滤，滤液浓缩后所得粗产物硅胶柱层析纯化得化合物C52(5mg，收率84.8％)，白色固体。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,其余脂肪环烃质子),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)+(C31H50O4理论值：486.37)。
制备实施例6：化合物C49的合成
Figure PCTCN2015073987-appb-000024
按照本申请制备实施例5中化合物C52的制备方法，中间体S11(3-α-羟基-20-甲酰基白桦脂酸苄酯)与正丙基格式试剂反应，然后氢化脱除苄基，粗产物再经硅胶柱层析纯化得化合物C49，白色固体。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,其余脂肪环烃质子),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)+(C31H50O4理论值：516.42)。
Figure PCTCN2015073987-appb-000025
中间体S11(3-α-羟基-20-甲酰基白桦脂酸苄酯)(8mg,0.014mmol)和正戊胺(5μL,0.043mmol)溶于无水乙醇，然后加入氰基硼氢化钠(3mg，0.043mmol)，反应体系室温下搅拌反应过夜。第二天，TLC检测反应完全，饱和碳酸氢钠溶液淬灭反应，加水稀释后乙酸乙酯萃取两次，合并有机相用饱和食盐水洗涤，干燥浓缩后所得残余物经硅胶柱层析纯化(氯仿/甲醇＝20/1，V/V)，得中间体S17(9mg,定量收率)，白色固体。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,其余脂肪环烃质子),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)。
中间体S17溶于甲醇和少量乙酸乙酯中，换氮气后，投入催化量的Pd/C,氢气置换后，室温常压下搅拌反应，TLC检测跟踪反应完全。过滤，滤液浓 缩，所得粗产物经硅胶柱层析纯化得化合物C51(5mg，收率71.4％)，白色固体。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,其余脂肪环烃质子),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)+(C35H61NO3理论值：543.46)。
制备实施例8：化合物C54的合成
Figure PCTCN2015073987-appb-000026
三甲基碘化亚砜(21mg,0.095mmol)和氢化钠(2mg,0.095mmol)于室温下投入干燥的THF(1mL)中，反应半小时后，滴入中间体S11(3-α-羟基-20-甲酰基白桦脂酸苄酯)(15mg,0.027mmol)的干燥THF溶液(1mL)，室温下搅拌反应过夜。次日加水淬灭反应，乙酸乙酯萃取，有机相饱和食盐水洗涤，干燥浓缩后所得残余物经硅胶柱层析纯化(乙酸乙酯/石油醚＝1/6，V/V)，得中间体S18(5mg,收率：33.3％)，白色固体。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,其余脂肪环烃质子),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)。
中间体S18溶于甲醇和少量乙酸乙酯中，换氮气后，加入催化量的Pd/C,氢气置换后，室温常压下搅拌反应，TLC跟踪反应完全。过滤，滤液浓缩后所得粗产物经硅胶柱层析纯化得化合物C54(4mg，收率94.8％)，白色固体。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,其余脂肪环烃质子),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)+(C31H50O4理论值：486.37)。
制备实施例9：化合物C55的合成
Figure PCTCN2015073987-appb-000027
二氧化硒(22mg,0.22mmol)和叔丁基过氧化氢(165μL,5.5M的四氢呋喃溶液)溶于干燥的二氯甲烷(20mL)中，冰水浴下滴加催化量的醋酸(6μL，0.1eq.),在此温度下搅拌十分钟后，慢慢滴加中间体3-α-羟基白桦脂酸苄酯(242mg,0.44mmol)的二氯甲烷溶液(5mL)，缓慢升温至室温并搅拌过夜，TLC检测反应完全，加入亚硫酸钠饱和溶液淬灭反应，二氯甲烷萃取并用水洗涤有机相，干燥浓缩所得残余物直接投下一步。粗品溶于甲醇(20mL),冰水浴下分批加入硼氢化钠(25mg,0.66mmol),反应一小时后，TLC检测反应完全，滴加氯化铵饱和溶液淬灭反应，乙酸乙酯萃取并用饱和食盐水洗涤，有机相干燥浓缩后用石油醚/乙酸乙酯为2:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-22-羟基白桦脂酸苄酯S10197mg,为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C37H54O4理论值：562.40)
S10(96mg,0.17mmol)溶于二氯甲烷(10mL)中，冰水浴下慢慢加入间氯过氧苯甲酸(72mg,0.34mmol)，室温搅拌反应3h，TLC检测反应完全，加入饱和亚硫酸钠溶液淬灭反应，用水洗涤有机相，干燥浓缩后得中间体S14为一白色固体，直接用于下步反应。
S14(120mg,0.207mmol)溶于氯仿(20mL)中，滴入两滴浓盐酸溶液，所得体系加热回流反应，TLC检测S14反应完全后，减压浓缩，残余物硅胶柱层析纯化，得中间体S15(8mg，收率6.7％)，白色固体。1H NMR(300MHz,CDCl3)δ9.60(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,其余脂肪环烃质子),0.94(s,3H),0.92(s,3H),0.81(s,3H),0.73(s,3H),0.65(s,3H)。
中间体S15溶于甲醇和少量乙酸乙酯中，换氮气后，加入催化量的Pd/C,氢气置换后，室温常压搅拌反应，TLC检测跟踪反应完全。过滤，滤液浓缩后所得粗产物硅胶柱层析纯化得化合物C55(6mg，收率85.7％)，白色固体。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,其余脂肪环烃质子),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)+(C30H48O5理论值：488.35)。
制备实施例10：化合物C56的合成
Figure PCTCN2015073987-appb-000028
取中间体S10(100mg)溶于干燥的四氢呋喃(5mL)中，冰水浴下滴入BH3-Me2S的四氢呋喃溶液(2M)，搅拌一小时后移至室温。次日，将反应液移至冰水浴，依次加入乙醇(280μL),饱和醋酸钠溶液(200μL)，30％的过氧化氢溶液(140μL)。室温下搅拌过夜，TLC检测反应完全。加水稀释，乙酸乙酯萃取后饱和食盐水洗涤。有机相干燥浓缩后用氯仿/甲醇为50:1的洗脱剂体系进行柱层析分离，得到中间体S12(88mg，收率85.4％)，白色固体。1H NMR(300MHz,CDCl3)δ7.34(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)+，(C37H56O5理论值：580.41)。
中间体S12经过氢化脱保护后可得到化合物C56，1H 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,其余脂肪环烃质子),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)+，(C30H50O5理论值：490.36)。
制备实施例11：化合物C57的合成
Figure PCTCN2015073987-appb-000029
中间体S3(3-α-羟基白桦脂酸苄酯)的制备参照制备实施例1。中间体S3(143mg,0.262mmol)溶于甲醇/二氯甲烷(5mL/5mL)中，在-78℃下通入臭氧，TLC跟踪反应完全，停止通入臭氧后用氮气鼓出残余的臭氧，然后加入二甲硫醚(25μL)淬灭反应，搅拌下缓缓升至室温。第二天，反应体系浓缩后残余物经硅胶柱层析纯化得中间体S21(94mg，收率65.8％)，白色固体。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),2.27(m,1H),2.18(s,3H),2.16-1.92(m,4H),1.64-0.96(m,其余脂肪环烃质子),1.00(s,3H),0.94(s,3H),0.91(s,3H),0.87(s,3H),0.84(s,3H)。
参照本申请制备实施例5中中间体S19的制备方法，由中间体S21(19mg,0.035mmol)与烯丙基格氏试剂反应制得中间体S22，不经纯化直接进行下步反应。参照本申请制备实施例9中中间体S14的制备方法，上步所得中间体S22与间氯过氧苯甲酸进行环氧化反应制得中间体S23，硅胶柱层析后得白色固体(7mg，两步收率35％)。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),3.18(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,其余脂肪环烃质子),1.28(s,3H),1.01(s,3H),0.92(s,3H),0.88(s,3H),0.84(s,3H),0.81(s,3H)。
中间体S23(7mg)溶于甲醇和少量乙酸乙酯中，换氮气后加入催化量的Pd/C,氢气置换后，室温常压下搅拌反应，TLC检测跟踪反应完全。过滤，滤液浓缩后所得粗产物硅胶柱层析纯化得化合物C57(3mg，收率43％)，白色固体。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,其余脂肪环烃质子),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)+(C32H52O5理论值：516.38)。
制备实施例12：化合物C58的合成
Figure PCTCN2015073987-appb-000030
按照制备实施例21的制备化合物C39类似的方法，以中间体S11为原料，与盐酸羟胺反应可合成得到中间体S13，后者氢化脱去苄基保护后得化合物C58。1H NMR(300MHz,CDCl3)δ6.61(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)+,(C30H49NO4理论值：487.36)。
制备实施例13：化合物C59的合成
Figure PCTCN2015073987-appb-000031
按照制备实施例9的制备方法合成得到中间体S14。中间体S14(10mg，0.017mmol)和催化量的DMAP(1mg)溶于二氯甲烷(5mL)中，加入三乙胺(4mL，0.026mmol)，冰水浴下滴入乙酰氯(2mL，0.030mmol)，室温反应12小时，TLC显示反应完毕。浓缩除去溶剂后，乙酸乙酯稀释后，分别用水和饱和氯化钠水溶液洗涤后，有机相干燥浓缩，硅胶柱层析(乙酸乙酯:石油醚，1:2)，得到化合物S24白色固体(8mg，0.013mmol)，摩尔收率：80％。S24氢化脱去苄基保护即得到化合物C59(收率50％，白色固体)。1HNMR证明为两种构型的混合物(S:R约3:1)，S构型: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 aliphatic ring protons),0.97(s,3H),0.93(s,3H),0.92(s,3H),0.86(s,3H),0.82(s,3H)；R构型:1H NMR(300MHz,CDCl3)δ4.22(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)+，(C32H50O6理论值：530.36)。
制备实施例14：化合物C79的合成
Figure PCTCN2015073987-appb-000032
化合物A2(2mL,19.9mmol)和三乙胺(2.8mL,19.9mmol)于二氯甲烷(40mL)中，冰水浴下慢慢滴加氯甲酸苄醇酯(2.72mL,19.9mmol)，室温下搅拌反应过夜。次日，TLC检测反应完全，反应体系减压浓缩，所得残余物经硅胶柱层析纯化(氯仿/甲醇＝40/1，V/V)，得中间体S29(4.45g，收率93.1％)，无色油状物。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)。
所得中间体S29(1.49g,6.23mmol)溶于乙腈/水(10mL/5mL)混合溶剂中，冰水浴下分别加入二乙酰氧基碘苯(6g,18.68mmol)和四甲基哌啶(TEMPO,292mg,1.87mmol)，室温下搅拌反应过夜。次日，TLC检测反应完全，加入饱和亚硫酸钠溶液搅拌10min，用1N盐酸溶液调至pH＝5,二氯甲烷萃取，合并有机相用饱和食盐水洗涤，有机相干燥，减压浓缩，所得残余物经硅胶柱层析纯化(氯仿/甲醇＝20/1，V/V)，得中间体S30(1.23g，收率77.8％)，黄色油状物。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)。
将中间体S14(31mg,0.054mmol)﹑S30(27mg,0.107mmol)﹑EDCI(1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐)(20mg,0.107mmol)和DMAP(4-二甲氨基吡啶)(7mg,0.054mmol)于DMF(N,N-二甲基甲酰胺)(5mL)中室温反应过夜，第二天反应液加水稀释，乙酸乙酯萃取，有机相用水洗两次，饱和食盐水洗，无水硫酸钠干燥，过滤，滤液浓缩后残余物经硅胶柱层析纯化(乙酸乙酯/石油醚＝1/2)，得中间体S31(33mg，收率76.7％)，白色 固体。1H NMR(300MHz,CDCl3)δ7.36-7.26(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,其余脂肪环烃质子),0.90(s,3H),0.87(s,3H),0.84(s,3H),0.81(s,3H),0.73(s,3H)。
所得中间体S31溶于甲醇中，常压下于Pd-C存在下催化氢解，同时脱去苄氧基羰基和C-20的苄基得到中间体S32，不经纯化，直接进行下步反应。所得中间体S32溶于二氯甲烷中，加入三乙胺和Boc酸酐，室温反应过夜。反应体系减压浓缩，残余物经硅胶柱层析纯化后得到化合物C79(收率76.2％)，白色固体。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,其余脂肪环烃质子),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)+(C39H63NO9理论值：689.45)。
制备实施例15：化合物C80的合成
Figure PCTCN2015073987-appb-000033
参照本申请制备实施例14中化合物C79的制备方法，合成得化合物C80(总收率35.5％)，白色固体。1H NMR(300MHz,CDCl3)δ5.08(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,其余脂肪环烃质子),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)+(C41H67NO10理论值：733.47)。
制备实施例16：化合物C82的合成
Figure PCTCN2015073987-appb-000034
将中间体S32(30mg,0.05mmol)、活性酯Biotin-OSu(17mg,0.05mmol)和三乙胺(200μL)溶解于干燥DMF(2mL)溶剂中，加热到50℃下搅拌反应过夜。冷到室温后，反应体系用水(20mL)稀释，乙酸乙酯萃取，合并有机相后用饱和食盐水洗，无水Na2SO4干燥，过滤浓缩后所得粗产物经硅胶柱层析纯化得化合物C82(收率60％)，白色固体。1H NMR(300MHz,CDCl3)δ6.75(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,其余脂肪环烃质子),0.95(s,3H),0.93(s,3H),0.87(s,3H),0.83(s,3H),0.81(s,3H)。
制备实施例17(化合物编号：C34)
Figure PCTCN2015073987-appb-000035
白桦脂酸(1.2g,2.63mmol)溶于甲醇/乙酸乙酯(40mL/10mL)中，换氮气后加入催化量的Pd/C，再换氮气后换氢气，室温搅拌2天，TLC检测反应完全。换氮气后过滤反应液，旋干后以石油醚：乙酸乙酯为10:1的极性进行柱层析分离。得产物C34为白色固体(1.04g,2.27mmol)，摩尔收率为86％。1H NMR(300MHz,CDCl3)δ3.13(t,1H,J＝9.0,6.9Hz),2.28-2.16(m,2H),1.98-1.78(m,4H),1.64-0.96(m,其它脂肪环质子),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)+(C30H50O3理论值：458.38)。
用同样的方法,分别以不同的天然产物或化合物为原料(原料齐墩果酸：C2，原料熊果酸：C7,原料甘草次酸,原料白桦脂醇：C29,原料C34)，按照制备实施例12相同的方法，合成以下化合物或中间体：
Figure PCTCN2015073987-appb-000036
制备实施例18(化合物编号：C32)
Figure PCTCN2015073987-appb-000037
按照制备实施例1依次制备白桦脂酸苄酯、3-羰基白桦脂酸苄酯、3-α-羟基白桦脂酸苄酯。
3-α-羟基白桦脂酸
上步产物(100mg,0.18mmol)溶于甲醇(8mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC 检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到化合物C32(78mg)，为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C30H48O3理论值：456.36)。
制备实施例19(化合物编号C46)
Figure PCTCN2015073987-appb-000038
(1)3-α-羟基-20，21-环氧白桦脂酸苄酯
按照制备实施例5制备3-α-羟基-20，21-环氧白桦脂酸苄酯74mg。
(2)3-α-羟基-20，21-环氧白桦脂酸
上步产物(74mg,0.14mmol)溶于甲醇(8mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为6:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-20，21-环氧白桦脂酸(38mg,0.08mmol)，为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C30H48O4理论值：472.36)。
用实施例19同样的方法合成C36：
Figure PCTCN2015073987-appb-000039
Figure PCTCN2015073987-appb-000040
制备实施例20(化合物编号C37)
Figure PCTCN2015073987-appb-000041
白桦脂酸(41mg,0.09mmol)于干燥的四氢呋喃(5mL)中，冰水浴下滴入BH3-Me2S的四氢呋喃溶液(2M)，搅拌一小时后移至室温。次日，将反应液移至冰水浴，依次加入乙醇(280μL),饱和醋酸钠溶液(200μL),30％的过氧化氢溶液(140μL)。室温下搅拌过夜，TLC检测反应完全。加水稀释，乙酸乙酯萃取后饱和食盐水洗涤。有机相干燥浓缩后用氯仿/甲醇为50:1的洗脱剂体系进行柱层析分离，得到化合物20(29)-还原-29-羟基白桦脂酸19mg,为白色固体，摩尔收率：45.3％。1H NMR(300MHz,CD3OD)δ3.73(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,其它脂肪环质子),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)+(C30H50O4理论值：474.37)。
制备实施例21(化合物编号C39)
Figure PCTCN2015073987-appb-000042
中间体3-羰基-20(29)-还原白桦脂酸(82mg,0.18mmol)，盐酸羟胺(25mg,0.37mmol)和吡啶(37μL，0.46mmol)于无水乙醇(5mL)中，室温搅拌过夜，次日TLC检测反应完全。直接浓缩后用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到目标化合物77mg，为白色固体，摩尔收率：91.2％。1H NMR(300MHz,CDCl3)ClR(300MHz,：脱去苄基同时将双键还原，得到0.92(s,3H),0.90(s,3其它脂肪环质子),0.94(s,3H),0.91(s,3H),0.90(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)+(C30H49NO3理论值：471.37)。
制备实施例22 3-α-乙酰氧基白桦脂酸(化合物编号：T2)
Figure PCTCN2015073987-appb-000043
(1)3-α-乙酰氧基白桦脂酸苄酯
3-α-羟基白桦脂酸苄酯(107mg，0.20mmol)和催化量的DMAP(10mg,0.08mmol)溶于二氯甲烷(10mL)中，加入三乙胺(82mL，0.60mmol)，冰水浴下滴入乙酸酐(42mL，0.60mmol)，室温反应12小时，TLC显示反应完毕。浓缩除去溶剂后，乙酸乙酯稀释后，分别用水和饱和氯化钠水溶液洗涤后，有机相干燥浓缩，所得残余物以石油醚/乙酸乙酯为20:1的洗脱剂柱层析纯化后得到化合物3-α-乙酰氧基白桦脂酸苄酯白色固体(77mg，0.13mmol)，摩尔收率：65％。1H NMR(300MHz,CDCl3)δ7.34(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,其它脂肪环质子),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)+(C39H56O4理论值：588.42)。
(2)3-α-乙酰氧基白桦脂酸
上步产物(77mg,0.13mmol)溶于甲醇(8mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为10:1的洗脱剂体系进行柱层析分离，得到化合物3-α-乙酰氧基白桦脂酸(60mg,0.12mmol)，为白色固体，摩尔收率：92％。1H NMR(300MHz,CDCl3)δ4.62 (s,1H),2.84-2.20(m,4H),2.08(s,3H),1.98-1.15(m,其它脂肪环质子),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)+(C32H50O4理论值：498.37)。
用实施例22同样的方法和不同的酸酐合成以下化合物：
Figure PCTCN2015073987-appb-000044
Figure PCTCN2015073987-appb-000045
Figure PCTCN2015073987-appb-000046
制备实施例23(化合物编号C115)
Figure PCTCN2015073987-appb-000047
20(29)-还原白桦脂酸(37mg,0.08mmol)和EDCI(21mg,0.12mmol)于DMF(2mL)中，加入三乙胺(15μL)，HOBt(15mg,0.12mmol)和N-Boc-2-(2-胺基乙氧基)乙基胺(15mg,0.08mmol),50℃搅拌过夜。次日TLC检测反应完全，反应液加水稀释，用乙酸乙酯萃取后饱和食盐水洗涤。有机相干燥浓缩后用氯仿/甲醇为80:1的洗脱剂体系进行柱层析分离，得到目标化合物45mg,为白色固体，摩尔收率：86.5％。1H NMR(300MHz,CDCl3)δ5.93(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,其它脂肪环质子),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)+(C39H68N2O5理论值：644.51)。
用实施例23同样的方法和不同的胺合成以下化合物：
Figure PCTCN2015073987-appb-000048
Figure PCTCN2015073987-appb-000049
制备实施例24(化合物编号：C47)
Figure PCTCN2015073987-appb-000050
(1)3-α-羟基-22-羟基白桦脂酸苄酯
参照制备实施例9制备3-α-羟基-22-羟基白桦脂酸苄酯。
(2)3-α-羟基-22-羟基白桦脂酸
上步产物(197mg,0.35mmol)溶于甲醇(10mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为2:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-22-羟基白桦脂酸(140mg,0.30mmol)，为白色固体，摩尔收率：85％。1H NMR(300MHz,CDCl3)δ4.97(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,其它脂肪环质子),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)+(C30H48O4理论值：472.36)。
制备实施例25(化合物编号：C48)
Figure PCTCN2015073987-appb-000051
(1)3-α-羟基-20-甲酰基白桦脂酸苄酯
按照制备实施例5制备3-α-羟基-20-甲酰基白桦脂酸苄酯。
(2)3-α-羟基-20-甲酰基白桦脂酸
上步产物(50mg,0.088mmol)溶于甲醇(5mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为2:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-20-甲酰基白桦脂酸(36.7mg,0.078mmol)，为白色固体，摩尔收率：87.3％。1H NMR(300MHz,CDCl3)δ9.84(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,其它脂肪环质子),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)+(C30H48O4理论值：472.36)。
制备实施例26(化合物编号C119)
Figure PCTCN2015073987-appb-000052
Figure PCTCN2015073987-appb-000053
分子筛和干燥的二氯甲烷(10mL)置于干燥的50mL圆底烧瓶中，在-20℃下分别滴入重蒸的四异丙基氧钛(1μL，0.0037mmol)和D-(-)-DIPT(1μL,0.0056eq).搅拌15分钟后加入5.5M的TBHP的THF溶液(10μL,0.056mmol)，搅拌30分钟。再滴入制备实施例24所得产物的苄酯(21mg,0.037mmol)的二氯甲烷溶液(2mL)，置于-20℃冰箱中过夜。次日，将反应液升温至零度，加入5N氢氧化钠的饱和食盐水溶液(50μL)，搅拌一小时后过滤除去分子筛，滤液旋干后用石油醚/乙酸乙酯为2:1的洗脱剂体系进行柱层析分离，得到目标化 合物16mg,为白色固体，摩尔收率：76.2％。NMR证明为C-17为S构型为主的产物。1H NMR(300MHz,CDCl3)δ7.34(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,其它脂肪环质子),0.97(s,3H),0.93(s,3H),0.92(s,3H),0.86(s,3H),0.82(s,3H)。产物直接按前面的步骤在钯碳催化下脱去苄基，得到12mg目标产物，为白色固体，摩尔收率：89.2％。1H NMR(300MHz,CDCl3)δ3.76(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,其它脂肪环质子),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)+(C30H48O5理论值：488.35)。
用实施例26同样的方法合成以下化合物：
Figure PCTCN2015073987-appb-000054
制备实施例27(化合物编号：C53)
Figure PCTCN2015073987-appb-000055
(1)3-α-羟基-20-(2’-羟基乙胺基)白桦脂酸苄酯
中间体3-α-羟基-20-甲酰基白桦脂酸苄酯(20mg,0.036mmol)和氰基硼氢化钠(11mg，0.018mmol)于室温下搅拌2h,之后滴入乙醇胺(11μL,0.18mmol)室温搅拌过夜，TLC检测反应完全，饱和碳酸氢钠溶液淬灭反应， 加水稀释后乙酸乙酯萃取两次，饱和食盐水洗涤后，有机相干燥浓缩，所得残余物用氯仿/甲醇为20:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-20-(2’-羟基乙胺基)白桦脂酸苄酯12mg,为白色固体，摩尔收率：60％。1H NMR(300MHz,CDCl3+CD3OD)δ7.34(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,其它脂肪环质子),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)+(C38H59NO4理论值：607.91)。
(2)3-α-羟基-20-(2’-羟基乙胺基)白桦脂酸
上步产物(12mg,0.020mmol)溶于甲醇(2mL)和少量乙酸乙酯中，换氮气后，迅速加入10％的Pd/C,再换氮气后换氢气，室温搅拌。一小时后TLC检测反应完全。换氮气后滤去Pd/C，反应液旋干后用石油醚/乙酸乙酯为4:1的洗脱剂体系进行柱层析分离，得到化合物3-α-羟基-20-(2’-羟基乙胺基)白桦脂酸(9.3mg,0.018mmol)，为白色固体，摩尔收率：91％。1H NMR(300MHz,CDCl3+CD3OD)δ3.49(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,其它脂肪环质子),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)+(C32H55NO4理论值：517.41)。
制备实施例28(化合物编号C74)
Figure PCTCN2015073987-appb-000056
制备实施例26所得产物的苄酯(16mg,0.028mmol)和DMAP(1mg,0.1eq)于二氯甲烷(5ml)，冰水浴下依次滴加TEA(6μL,0.04mmol)和乙酰氯(3μL，0.04mmol),室温搅拌过夜。次日TLC检测反应完全。直接浓缩后用石油醚/乙酸乙酯为2:1的洗脱剂体系进行柱层析分离，得到目标化合物14mg,为白色固体，摩尔收率：82.3％。产物直接按前面的步骤在钯碳催化下脱去苄基，得到6mg目标产物，为白色固体，摩尔收率：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,其它脂肪环质子),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)+(C32H50O6理论值：530.36)。
用实施例28同样的方法和不同的酰氯合成以下化合物：
Figure PCTCN2015073987-appb-000057
Figure PCTCN2015073987-appb-000058
Figure PCTCN2015073987-appb-000059
Figure PCTCN2015073987-appb-000060
Figure PCTCN2015073987-appb-000061
Figure PCTCN2015073987-appb-000062
Figure PCTCN2015073987-appb-000063
二、试验实施例
(一)Split-TEV实验检测本发明化合物对PS1/BACE1之间相互作用的抑制活性
1.实验目的
用Split-TEV实验检测化合物对PS1/BACE1相互作用的抑制活性。
2.实验原理
将以γ-分泌酶的催化中心PS1和BACE1这两个蛋白分别融合在烟草蚀纹病毒的TEV蛋白酶的C端和N端上；如果这两个蛋白有相互作用，那么C、N两端会接近，TEV蛋白酶发挥活性，剪切释放转录因子然后启动报告基因荧光素酶Firefly的表达，其活性反映上游蛋白相互作用。
3.实验样品
实验前将化合物溶于DMSO(二甲亚砜)，配制母液，使用时用培养液稀释至所需浓度。
4.实验方法
4.1HEK293-MSR细胞(获自Sanofi-Aventis Recherche&Development公司)用含有10％胎牛血清的DMEM培养基培养于含有5％二氧化碳的37℃细胞培养箱。
4.2胰酶消化后悬浮的HEK293-MSR细胞采用Fugene HD进行转染，以2.5x104/well细胞密度铺板于96孔板。
4.3培养4小时后给予化合物处理。实验前将溶解于DMSO的化合物母液用PBS配制成最终浓度的10倍，取10μl/孔加入96孔板处理细胞。
4.4化合物处理细胞16-18小时后，使用SteadyGlo试剂盒(Promega)检测荧光素酶活性。
所得实验数据见表1所示。
(二)酶联免疫吸附法检测本发明化合物对Aβ产生水平的作用
1.实验目的
用酶联免疫吸附法检测化合物处理后细胞产生Aβ水平的变化。
2.实验原理
HEK293APPswe细胞株(HEK293细胞内转染3’HA-APPswe表达质粒并使用抗生素筛选后得到稳定表达APPswe的细胞株)能够稳定表达人源APP swedish突变体，APPswedish蛋白能够被细胞内源的BACE1和γ-secretase剪切产生Aβ，化合物处理后用ELISA法能够检测Aβ产生水平的变化。
3.实验样品
实验前将化合物溶于DMSO，配制母液，使用时用培养液稀释至所需浓度。
4.实验方法
4.1HEK293APPswe细胞用含有10％胎牛血清的DMEM培养基培养于含有5％二氧化碳的37℃细胞培养箱。
4.2胰酶消化后悬浮的HEK293APPswe细胞以2.5x104/孔细胞密度铺板于96孔板。
4.3培养16-18小时后细胞换液同时给予化合物处理。实验前将溶解于DMSO的化合物母液用PBS配制成最终浓度的10倍，取10μl/孔加入96孔板处理细胞。
4.4化合物处理8小时后收集培液，按照依科赛生物有限公司试剂盒所提供的实验方法进行Total Aβ检测。
所得实验数据见表1所示。
表1
Figure PCTCN2015073987-appb-000064
Figure PCTCN2015073987-appb-000065
Figure PCTCN2015073987-appb-000066
上述活性测试结果显示，在10μM的浓度下，本发明通式(I)所示的化合物对PS1/BACE1相互作用以及Aβ的分泌具有较好的抑制作用，且3-α构型活性一般明显高于3-β构型化合物。
(三)体内检测化合物XYT-472B(对应于本发明的化合物C46)活性
1.实验目的
通过微型缓释泵埋植的方法进行化合物持续给药，检测化合物XYT-472B在转基因AD小鼠模型中的活性。
2.实验原理
皮下埋植微型缓释泵可以完成4周持续给药，给药4周后对小鼠的脑区Aβ水平等指标进行测定，评估化合物XYT-472B的体内活性。
3.实验样品
3.1年龄在3个月的Tg6799品系(转基因模型小鼠，C3H/C57BL6远交系，购自Jackson Laboratory)PCR鉴定APP和PS1基因双阳性的小鼠。
3.2实验前将化合物溶于DMSO(二甲亚砜)：丙二醇(v/v＝1:1)，灌注于微型缓释泵。
4.实验方法
4.1麻醉小鼠后，在其背部划开约1cm的创口，将缓释泵置入皮下区域，缝合小鼠创口。
4.2微型缓释泵给药剂量XYT-472B 10mg/kg/天，以手术开始记作给药第1天，在给药35天时将小鼠麻醉，进行PBS心脏灌流，解剖其大脑，获得前额叶皮层区域和海马区域，分别置于EP管，液氮速冻后于-80℃储存。
4.3将小鼠脑组织样品进行称重，按照20mg组织样品加入500μl 2％SDS裂解液，重悬组织样品，超声裂解至澄清。25,000g于20℃离心1小时，上清作为SDS可溶组分。
4.4按照依科赛生物有限公司试剂盒所提供的实验方法进行Aβ42检测。
图1显示了给药35天后小鼠脑区Aβ42产生水平。实验结果表明，XYT-472B给予小鼠1个月后，其脑区内Aβ42产生水平显著降低。提示XYT-472B在小鼠体内具有较好的生物学活性，可通过降低Aβ产生水平改善AD发病。

Claims (10)

  1. 如下通式I所示的五环三萜类化合物，
    Figure PCTCN2015073987-appb-100001
    其中：
    R1为氢、卤素、氰基或羟基；
    R2为羟基、卤素、氧代基、＝N-OH、C1-C6烷基羰基氧基、C3-C8环烷基羰基氧基或
    Figure PCTCN2015073987-appb-100002
    其中，R9为氢或C1-C4直链或支链烷基；
    R3和R4各自独立地为未取代或被羟基取代的C1-C4直链或支链烷基；
    R5为甲酰基、羧基、未取代或羟基取代的C1-C4直链或支链烷基、
    Figure PCTCN2015073987-appb-100003
    Figure PCTCN2015073987-appb-100004
    其中，R10为C1-C8直链或支链烷基、C3-C8环烷基、未取代或被羧基取代的C1-C8直链或支链烷基、羧基C1-C7亚烷基或者未取代或被羧基取代的C3-C8环烷基；
    R6和R7各自独立地为氢或C1-C4直链或支链烷基；或者R6和R7与其连接的碳原子共同形成＝CH2；
    R8为氢、取代或未取代的C1-C6直链或支链烷基、取代或未取代的C3-C6环烷基、取代或未取代的C2-C6直链或支链烯基、取代或未取代的C2-C6链炔基、取代或未取代的C1-C6烷基羰基、取代或未取代的C1-C6烷基胺基、或者
    Figure PCTCN2015073987-appb-100005
    其中，所述取代基任选自羟基、C1-C3烷酰基、羟基取代的C1-C4烷基羰基、＝N-OH、氨基、羟基取代的C1-C6烷基、未取代或羟基取代的C1-C6烷基胺基或
    Figure PCTCN2015073987-appb-100006
    中的一种或多种；
    其中，R11为H、取代或未取代的C1-C6烷基，所述取代基任选羟基、氨基、
    Figure PCTCN2015073987-appb-100007
    其中，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C1-C4烷氧基、取代或未取代的C6-C10芳基或者取代或未取代的至少含有选自N、S或O中的一个杂原子的5至8元杂芳基，其中，所述取代基任选自羟基、卤素、氰基、C1-C4烷氧基、BocNH-取代的C1-C4烷氧基、
    Figure PCTCN2015073987-appb-100008
    其中，R13为C1-C6烷基或C6-C10芳基；
    其中，m和n各自独立为1、2、3或4；
    Z为亚甲基或不存在；
    Figure PCTCN2015073987-appb-100009
    表示以单键或双键连接。
  2. 根据权利要求1所述的化合物，其中，
    R1为氢或羟基；
    R2为羟基、氧代基、＝N-OH、C1-C6烷基羰基氧基、C3-C8环烷基羰基氧基或
    Figure PCTCN2015073987-appb-100010
    R3和R4各自独立地为C1-C4直链或支链烷基；
    R5为甲酰基、羧基、羟基取代的C1-C4烷基、
    Figure PCTCN2015073987-appb-100011
    R8为氢、取代或未取代的C1-C6直链或支链烷基、取代或未取代的C2-C6直链或支链烯基、取代或未取代的C1-C6烷基羰基或
    Figure PCTCN2015073987-appb-100012
    其中，所述取代基选自羟基、甲酰基、乙酰基、＝N-OH、未取代或羟基取代的C1-C6烷基胺基或
    Figure PCTCN2015073987-appb-100013
    中的一种或两种；
    其中，R11为取代或未取代的C1-C4烷基，所述取代基任选羟基、氨基、
    Figure PCTCN2015073987-appb-100014
    其中，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的C6-C10芳基或者取代或未取代的含有选自N、S或O中的一个杂原子的5至8元杂芳基，其中，所述取代基选自羟基、F、Cl、Br、C1-C4烷氧基、BocNH-取代的C1-C4烷氧基、
    Figure PCTCN2015073987-appb-100015
    其中，R13为C1-C6烷基或苯基。
  3. 根据权利要求1所述的化合物，其中，
    R1为氢或羟基；
    R2以α构型与右侧A环连接；
    R3和R4同时为甲基；
    R5为甲酰基、羟基取代的C1-C4烷基、羧基、
    Figure PCTCN2015073987-appb-100016
    R8为氢、取代或未取代的C1-C6直链或支链烷基、取代或未取代的C2-C6直链或支链烯基、取代或未取代的C1-C6烷基羰基或
    Figure PCTCN2015073987-appb-100017
    其中，所述取代基选自羟基、甲酰基、乙酰基、＝N-OH、未取代或羟基取代的C1-C6烷基胺基或
    Figure PCTCN2015073987-appb-100018
    中的一种或两种；
    其中，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的苯基或者取代或未取代的噻吩基，其中，所述取代基选自羟基、F、Cl、Br、C1-C4烷氧基、BocNH-取代的C1-C4烷氧基、
    Figure PCTCN2015073987-appb-100019
    Figure PCTCN2015073987-appb-100020
    R13为C1-C6烷基或苯基。
  4. 根据权利要求1所述的化合物，其中，
    R1为氢或羟基；
    R2以α构型与右侧A环连接；
    R3和R4同时为甲基；
    R5为甲酰基、羧基、羟基取代的C1-C4烷基、
    Figure PCTCN2015073987-appb-100021
    其中，R10为未取代或被羧基取代的C1-C5直链或支链烷基或羧基C1-C7亚烷基；
    R6和R7各自独立地为氢、甲基、乙基或丙基，或者R6和R7与其连接的碳原子共同形成＝CH2；
    R8为氢、取代或未取代的C1-C6直链或支链烷基、取代或未取代的C2-C6直链或支链烯基、取代或未取代的C1-C6烷基羰基或
    Figure PCTCN2015073987-appb-100022
    其中，所述取代基选自羟基、甲酰基、乙酰基、＝N-OH、未取代或羟基取代的C1-C6烷基胺基或
    Figure PCTCN2015073987-appb-100023
    中的一种或两种；
    其中，R11为H、取代或未取代的C1-C6烷基，所述取代基任选羟基、氨基、
    Figure PCTCN2015073987-appb-100024
    其中，R12为取代或未取代的C1-C6烷基、取代或未取代的C3-C6环烷基、取代或未取代的苯基或者取代或未取代的噻吩基，其中，所述取代基选自羟基、F、Cl、Br、C1-C4烷氧基、BocNH-取代的C1-C4烷氧基、
    Figure PCTCN2015073987-appb-100025
    Figure PCTCN2015073987-appb-100026
    R13为C1-C6烷基或苯基。
  5. 根据权利要求1所述的化合物，其中通式(I)所示结构的五环三萜类化合物具体选自如下结构：
    Figure PCTCN2015073987-appb-100027
    Figure PCTCN2015073987-appb-100028
    Figure PCTCN2015073987-appb-100029
  6. 根据权利要求1-5中任一项所述的化合物在制备PS1/BACE1相互作用抑制剂中的用途。
  7. 根据权利要求1-5中任一项所述的化合物在制备治疗阿尔兹海默病的药物中的用途。
  8. 根据权利要求7所述的用途，其中，通式I所示的五环三萜类化合物作为PS1/BACE1相互作用抑制剂。
  9. 一种治疗阿尔兹海默病的药物组合物，其中，所述药物组合物包含选自根据权利要求1-5中任一项所述的化合物中一种或多种作为活性成分。
  10. 根据权利要求9所述的药物组合物，其中，所述药物组合物进一步包含药学上常规的分散剂、赋形剂、崩解剂、抗氧化剂、甜味剂、包衣剂。
PCT/CN2015/073987 2014-03-14 2015-03-11 一类五环三萜类化合物及其在制备治疗阿尔兹海默病的药物中的用途 WO2015135474A1 (zh)

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