WO2015135449A1 - 五环三萜类化合物及其制备方法、药物组合物和用途 - Google Patents

五环三萜类化合物及其制备方法、药物组合物和用途 Download PDF

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WO2015135449A1
WO2015135449A1 PCT/CN2015/073855 CN2015073855W WO2015135449A1 WO 2015135449 A1 WO2015135449 A1 WO 2015135449A1 CN 2015073855 W CN2015073855 W CN 2015073855W WO 2015135449 A1 WO2015135449 A1 WO 2015135449A1
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group
alkyl
hydroxy
compound
hydrogen
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French (fr)
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南发俊
谢欣
王霄音
张书永
李静
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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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to a class of TGR5 (G protein-coupled cholic acid membrane receptor) agonists, in particular, to a class of pentacyclic triterpenoids, a process for the preparation thereof, and a pharmaceutical composition comprising the same, Such compounds are useful as TGR5 agonists.
  • TGR5 G protein-coupled cholic acid membrane receptor
  • Diabetes is caused by different causes (such as genetic factors, immune dysfunction, microbial infections and their toxins, free radicals, mental factors, etc.) leading to islet ⁇ -cell dysfunction and/or body cells to insulin resistance caused by elevated blood glucose Characteristic metabolic disorder syndrome.
  • IDF International Diabetes Federation
  • the drugs for treating diabetes mainly include insulin secretion promoters (sulfonylureas, repaglinide), insulin sensitizers (biguanides, thiazolidinediones) and ⁇ -glucosidase inhibitors (Akapo). Sugar), but they often have varying degrees of side effects such as hypoglycemia, weight gain, cardiovascular side effects, and the like. It is imperative to develop new anti-diabetic drugs that act on new targets and avoid the side effects of traditional anti-diabetic drugs.
  • TGR5 is a G protein-coupled receptor (GPCR) expressed in brown adipose tissue and muscle.
  • GPCR G protein-coupled receptor
  • TGR5 was discovered in 2002 as a specific receptor for endogenous metabolites of bile acids, and prior to this it was long considered to be a detergent capable of dissolving fatty acids, fat-soluble vitamins and cholesterol, thereby promoting their digestion and transport. . Therefore it has only been given limited therapeutic applications.
  • the orphan farnesoid X receptor FXR was the only known receptor activated by the cholic acid analog. By activation of TGR5, cholic acid stimulates the activation of type 2 deiodinase, resulting in increased mitochondrial function and energy expenditure.
  • TGR5 is activated by cholic acid and can cause glucagon-like peptide 1 (GLP1) to be secreted from murine intestinal endocrine cells.
  • GLP1 glucagon-like peptide 1
  • TGR5 agonists mainly include two major categories.
  • One type is chemical synthesis of small molecules. Although these compounds are highly active, some even have EC 50 values below 10 nM, but due to the strong activation of the TGR5 receptor on the gallbladder caused by such compounds, which leads to smooth muscle relaxation and promotes gallbladder filling, it is serious. Increase the side effects of gallbladder volume.
  • Another class of natural product molecules including steroids and other types of natural products, although weaker than synthetic molecules, can overcome the gallbladder toxicity of chemically synthesized molecules due to their structural advantages. The most striking of these is INT-777 (Pellicciari, R. et al. J. Med. Chem.
  • the invention discloses a class of pentacyclic triterpenoids, the main feature of which is the inversion of the hydroxyl group at the 3 position, and the TGR5 agonistic activity is significantly increased after the ⁇ -type hydroxyl group of the natural product betulinic acid is reversed to the ⁇ -type hydroxyl group. And with the modification of other sites in this series of compounds, the degree of activity improvement after hydroxyl inversion is also different.
  • the invention discloses the influence law of the structure on the activity, and obtains a series of compounds with excellent properties. Compared with INT777, the synthesis is not only simple, but also the agonistic activity of TGR5 is significantly better than that of INT777, and it is expected to act on the target. A new drug for the treatment of type 2 diabetes.
  • the object of the present invention is to design and synthesize pentacyclic triterpenoids having the structure of the formula (I).
  • Another object of the invention is to provide a process for the preparation of said compounds.
  • a further object of the invention is to provide the use of a compound of the invention in the manufacture of a medicament for the treatment of type 2 diabetes.
  • a further object of the invention is to provide the use of a compound of the invention in the treatment of diabetes.
  • the present invention provides a pentacyclic triterpenoid having a structure represented by the following formula (I):
  • R 1 is hydrogen, hydroxy, halogen or C 1 -C 6 alkyl
  • R 3 and R 8 are each independently hydrogen; hydroxyl; halogen; substituted C 1 -C 6 alkylamino C 1 -C 6 alkyl; an unsubstituted or substituted C 1 -C 6 alkyl, wherein the substituents
  • R 9 is H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, hydroxy, hydroxy C 1 -C 6 alkyl, -CH 2 OC(O)R 10 , -CH 2 OC ( O) OR 11 , -CH 2 OC(O)CH 2 OR 12 ;
  • R 10 , R 11 and R 12 are each independently substituted or unsubstituted 5 to 8 membered aryl; substituted or unsubstituted 3 to 8 membered cycloalkyl; 5 to 8 membered arylamino; containing N, S or a 5- to 8-membered heteroaryl group of at least one hetero atom in O; a C 1 -C 6 alkyl group, a halogenated C 1 -C 6 alkyl group; a hydroxy C 1 -C 6 alkyl group; BocNH(CH 2 ) m O(CH 2 ) n -, wherein each m and n are the same or different and are each independently an integer from 1 to 6; wherein the substituted 5 to 8 membered aryl group or substituted 3 to
  • the substituent in the 8-membered cycloalkyl group is selected from one or more substituents selected from the group consisting of a hydroxyl group, a halogen, a C
  • R 4 is hydrogen, hydroxy, halogen or C 1 -C 6 alkyl
  • R 5 is independently selected from hydrogen; hydroxy; hydroxy C 1 -C 6 alkyl; halogen; C 1 -C 6 alkyl; -C(O)R 13 ;-C(O)O(CH 2 CH 2 O) o CH 2 CH 2 R 14 ;-C(O)NH(CH 2 CH 2 O) p CH 2 CH 2 R 15 ; or -C(O)NH(CH 2 ) q C(O)OH;
  • p are independently 0, 1, 2 or 3; and q is an integer from 3 to 8;
  • R 13 is hydrogen; hydroxycarbonyl C 1 -C 8 alkylamino group; hydroxy group; piperazinyl group unsubstituted or substituted by C 1 -C 6 alkyl group; benzyloxy group; C 1 -C 6 alkoxy group ; tert-butoxycarbonyl C 1 -C 6 alkyloxy; or hydroxycarbonyl C 1 -C 6 alkylamino;
  • R 14 and R 15 are each independently hydrogen; amino; 5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide; C 1 -C 6 amide ; tert-butoxycarboxamide; or C 1 -C 6 alkoxy;
  • R 6 and R 7 are each independently hydrogen, hydroxy, halogen, hydroxycarbonyl or C 1 -C 6 alkoxycarbonyl;
  • R a and R b are each independently hydrogen, hydroxy, halogen, C 1 -C 6 alkyl or hydroxy C 1 -C 6 alkyl;
  • Z is a methylene group or a direct bond
  • R 3 and R 8 are each independently hydrogen, hydroxy, methyl, ethyl,
  • R 5 is methyl, formaldehyde, -COOH, methoxycarbonyl,
  • the general formula (I) according to the present invention has a structure represented by the following formula (III) or (IV):
  • C 1 -C 6 alkyl may be straight-chain or branched C 1 -C 6 alkyl, in particular, may be methyl, ethyl, propyl, isopropyl, butyl, Tert-butyl, isobutyl, pentyl, neopentyl or hexyl; preferably a linear or branched C 1 -C 3 alkyl group.
  • 5 to 8 membered aryl is an aromatic group having a 5- to 8-membered ring, preferably a phenyl group;
  • 5- to 8-membered cycloalkyl is a cycloalkyl group having a 5- to 8-membered ring, and specifically, may be a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group or a cycloheptyl group. Or cyclooctyl;
  • pentacyclic triterpenoids of the structure of the formula (I) of the present invention are specifically:
  • the present invention provides a process for the preparation of a compound of the formula (I).
  • the preparation method includes the following preparation route:
  • Compound 2 undergoes Dess-Martin oxidation in dichloromethane to give compound 16, which is then reacted with borane in a dry tetrahydrofuran under the catalysis of (S)-CBS to give a 3-OH reversal.
  • Compound 17 the last compound 17 is catalytically hydrogenated in methanol to remove the benzyl group to obtain the compound 1', the compound 1' is reacted with an alcohol or an amine to obtain a compound 2', and the compound 2' is reacted with an acid or an acid chloride to obtain a compound 3';
  • R 16 and R 17 are each independently hydrogen, C 1 -C 6 alkyl or hydroxy C 1 -C 6 alkyl;
  • R 18 is C 1 -C 6 alkyl; the remaining substituents are the same as defined in the formula (I).
  • the present invention also provides a pharmaceutical composition for treating type 2 diabetes, which comprises, as an active ingredient, one or more pentacyclic triterpenoids represented by the formula (I) of the present invention.
  • the composition may further comprise pharmaceutically conventional adjuvants such as dispersing agents, excipients, disintegrating agents, antioxidants, sweeteners, coating agents and the like.
  • the invention designs and synthesizes a novel class of pentacyclic triterpenoids, which can effectively stimulate TGR5, and is used for making drugs for treating type 2 diabetes, and overcomes the gallbladder existing in the existing chemical synthesis of small molecule TGR5 agonists. Defects such as toxicity, and a simpler synthesis method and milder reaction conditions than the positive control INT777.
  • the raw materials of the pentacyclic triterpenoids of the present invention are abundant in nature and have the structural advantages of natural products.
  • NMR was measured using a Mercury-Vx 300M instrument manufactured by Varian, NMR calibration: ⁇ H 7.26 ppm (CDCl 3 ), 2.50 ppm (DMSO-d 6 ); mass spectrometry using Agilent 1200 Quadrupole LC/MS LC-MS It is determined by instrument or SHIMADZU GCMS-QP5050A; the reagent is mainly provided by Shanghai Chemical Reagent Co., Ltd.; TLC thin layer chromatography silica gel plate is produced by Shandong Yantai Huiyou Silicone Development Co., Ltd., model HSGF 254; the normal phase column chromatography silica gel used for compound purification is Shandong Qingdao Marine Chemical Plant Branch produces, model zcx-11, 200-300 mesh.
  • 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 (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 C107
  • raw material betulin C29
  • raw material C34
  • 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 (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 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 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.
  • 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 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 intermediate S32 (30 mg, 0.05 mmol), the active ester Biotin-OSu (17 mg, 0.05 mmol) and triethylamine (200 ⁇ L) were dissolved in dry DMF (2 mL) solvent and stirred at 50 ° C overnight. After cooling to room temperature, the reaction system was washed with water (20mL) diluted with ethyl acetate.
  • TGR5 agonism assay Example 1 mediated the accumulation of intracellular cyclic adenosine 3'-5'-cyclic adenosine monophosphate (cAMP) by activating TGR5
  • HEK293 cells transiently transfected with TGR5 were stimulated with compounds and then detected by Homogeneous Time-Resolved Fluorescence (HTRF) to determine whether these compounds mediate intracellular cAMP accumulation via TGR5.
  • HTRF Homogeneous Time-Resolved Fluorescence
  • Gas protein-coupled bile acid receptor TGR5 binds to an agonist and undergoes structural changes, thereby activating adenylate cyclase (AC), further catalyzing ATP to cAMP, and cAMP in phosphodiesterase (PDE) Further degrading into AMP, IBMX can inhibit the activity of PDE, thereby inhibiting the degradation of cAMP into AMP.
  • AC adenylate cyclase
  • PDE phosphodiesterase
  • IBMX can inhibit the activity of PDE, thereby inhibiting the degradation of cAMP into AMP.
  • the cumulative amount of cAMP can be detected by adding IBMX to the experiment, and the amount of cAMP can directly reflect whether the compound is activated or inhibited by GPCR-mediated AC.
  • the cAMP produced by transient transgenic TGR5 HEK293 cells and the cAMP labeled with d2 provided by the kit were used to immunologically compete for the anti-cAMP antibody antigen binding site.
  • a monoclonal antibody labeled with ruthenium or osmium binds to d2-labeled cAMP, there is a relatively large signal.
  • the signal gradually decreases, resulting in a decrease in fluorescence reading. The effect of the compound on the accumulation of intracellular cAMP can therefore be reflected by fluorescence readings.
  • the compound was dissolved in DMSO to prepare a mother liquor.
  • the solution was diluted with the culture solution to the desired concentration, and INT-777 and Lithocholic Acid were used as positive controls for the test. The normality of each test reaction was tested. .
  • the cells are digested with trypsin and then suspended in serum-free medium.
  • EC 50 is the evaluation of TGR5 agonistic activity of sample drugs, half of 50% effective concentration. NR indicates no activity at a concentration of 100 ⁇ M.
  • HEK293 cells transiently transfected with the expression plasmid pBind-FXR and the reporter plasmid pGL4.31 were stimulated with the compound and then used The stable luciferase assay system is tested for the purpose of detecting whether these compounds increase the expression level of luciferase in the cells via FXR.
  • Mammalian one-hybrid is also known as the GAL4 chimera receptor assay, which has been developed in recent years and is mainly used in nuclear receptors (Nuclear).
  • GAL4 chimera receptor assay which has been developed in recent years and is mainly used in nuclear receptors (Nuclear).
  • Nuclear nuclear receptors
  • the mechanism is that the yeast cell transcription factor GAL4 and the nuclear structure of the mammalian nuclear receptor have two similar major domains: a ligand binding domain (LBD) and a DNA binding domain (DBD), which will The ligand binding domain (LBD) is fused to the DNA binding domain (DBD) of the yeast cell transcription factor GAL4 to form a chimeric protein expression plasmid, which is then co-transfected into an animal cell with a reporter plasmid containing a GAL4-specific response element. The expression level of the reporter gene is used to evaluate the agonistic or antagonistic activity of the nuclear receptor ligand.
  • LBD ligand binding domain
  • DBD DNA binding domain
  • the compound was dissolved in DMSO to prepare a mother liquid, which was diluted with the culture solution to the desired concentration, and GW4604 was used as a positive control for the test to test the normality of each test reaction.
  • transient transient transfer of the expression plasmid pBind-FXR and reporter plasmid pGL4.31 to HEK293 cells, then inoculated to 384-well plate at a cell density of 10000 cells / well, in 10% FBS high glucose DMEM, 37 ° C, Incubate for 12 hours under 5% CO2 conditions.
  • EC 50 is the evaluation of TGR5 agonistic activity of sample drugs, half of 50% effective concentration. NR indicates no activity at a concentration of 100 ⁇ M.
  • GW4604 activates FXR, increases luciferase expression in cells by activating FXR, and its activity is dose-dependent. However, these compounds do not increase the expression of luciferase in cells by activating FXR. The results indicate that these compounds do not act through the nuclear receptor FXR, further indicating that these compounds have a certain selection specificity.

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Abstract

本发明涉及一种通式(I)所示的五环三萜类化合物及其制备方法、包含所述化合物的药物组合物，所述化合物在制备治疗二型糖尿病的药物中的应用，以及所述化合物在治疗二型糖尿病中的应用。

Description

五环三萜类化合物及其制备方法、药物组合物和用途 技术领域
本发明涉及一类TGR5(G蛋白偶联胆酸膜受体)激动剂，具体而言，涉及一类五环三萜类化合物，及其制备方法，以及包含所述化合物的药物组合物，和所述类化合物可作为TGR5激动剂。
背景技术
糖尿病是由不同病因(如遗传因素﹑免疫功能紊乱﹑微生物感染及其毒素﹑自由基﹑精神因素等)导致胰岛β细胞功能减退和(或)机体细胞对胰岛素抵抗而引起的以血糖升高为特征的代谢紊乱综合症。根据国际糖尿病联盟(International Diabetes Federation,IDF)统计，2010年中国20～79岁的人群中有4300万以上的糖尿病患者，患病率达到4.5％。糖尿病已经成为危害人类安全健康的疾病。
目前，治疗糖尿病的药物主要有胰岛素分泌促进剂(磺酰脲类﹑瑞格列奈)﹑胰岛素增敏剂(双胍类﹑噻唑烷二酮类)和α-葡萄糖苷酶抑制剂(阿卡波糖)，但它们常具有不同程度的副作用，如低血糖﹑体重增加﹑心血管副作用等。开发作用于新靶点﹑避免传统抗糖尿病药物副作用的新型抗糖尿病药物已经刻不容缓。
TGR5是一种表达于棕色脂肪组织和肌肉的G蛋白偶联受体(GPCR)。TGR5在2002年被发现是胆汁酸内源性代谢产物的特定受体，而在此之前，它长期以来被认为是能够溶解脂肪酸﹑脂溶性维生素以及胆固醇的去垢剂，进而促使它们的消化运输。因此它只被赋予了有限的治疗应用。在TGR5被发现前，孤儿法尼醇X受体(FXR)是唯一已知的被胆酸类似物激活的受体。通过对TGR5的激活，胆酸能够刺激2型脱碘酶的激活，从而导致线粒体功能增加和能量消耗。也有报道证明TGR5被胆酸激活后，能够导致鼠肠内分泌细胞株中分泌胰高血糖素样肽1(GLP1)。这些数据表明TGR5是一个治疗糖尿病和相关代谢紊乱的重要靶点。
目前已知的TGR5激动剂主要包括两大类。一类是化学合成小分子。尽管此类化合物激动活性大都很强，有些甚至有低于10nM的EC50值，但由于此类化合物引起的强烈的激活胆囊上的TGR5受体从而导致平滑肌松弛，促进胆囊充盈，故而有严重的增加胆囊体积的副作用。另一类天然产物类分子，其中包括甾体类和其他类型天然产物，虽然活性相对合成分子来说较弱，但由于其结构优势，这一类化合物能够克服化学合成分子的胆囊毒性。其中最引人注目的当属INT-777(Pellicciari,R.等.J.Med.Chem.2009，52,7958-7961)，这是胆酸的衍生物，对TGR5的激动活性达到EC50为0.82nM,目前已经进入临床阶段。 但化合物INT777具有制备困难的缺点，以胆酸为起点，合成路线多达十二步，且多次用到极为苛刻的反应条件。发明人长期致力于基于化合物INT777的结构改造，以期望发现活性更好，毒性更低的新型糖尿病药物。本发明公开了一类五环三萜类化合物，其主要特点是3位羟基的反转，由天然产物白桦脂酸等的β型羟基反转为α型羟基后，其TGR5激动活性明显增加，且随着该系列化合物其他位点的改造，羟基反转后的活性提高程度也有所不同。本发明公开了这一结构对活性的影响规律，并得到一系列性能优异的化合物，与INT777相比，不仅合成简便，而且其TGR5的激动活性明显优于INT777，有望成为作用于该靶点的治疗二型糖尿病的新型药物。
Figure PCTCN2015073855-appb-000001
发明内容
本发明的目的在于设计与合成具有通式(I)所述结构的五环三萜类化合物。
本发明的另一目的在于提供所述化合物的制备方法。
本发明的还一目的在于提供一种含有所述化合物作为活性成分的药物组合物。
本发明的又一目的是提供本发明所述的化合物在制备治疗二型糖尿病的药物中的应用。
本发明的再一目的在于提供本发明所述的化合物在治疗糖尿病中的应用。
本发明提供了一种五环三萜类化合物，其具有如下通式(I)所示的结构：
Figure PCTCN2015073855-appb-000002
其中：
R1为氢、羟基、卤素或C1-C6烷基；
R2为氢、羟基、卤素、氧代基团(＝O)、＝N-OH、C1-C6烷基羰基氧基团、3至8元环烷基羰基氧基团、C1-C6烷基、3至8元环烷基或
Figure PCTCN2015073855-appb-000003
其中，Rc为C1-C6烷基或氢；
R3和R8各自独立地为氢；羟基；卤素；被C1-C6烷基取代的氨基C1-C6烷基；未取代或取代的C1-C6烷基，其中，取代的C1-C6烷基中的取代基选自羟基、卤素、氧代基团(＝O)、＝N-OH、环氧丙烷基、氨基和羟基C1-C6烷基氨基中的一个以上的取代基；未取代或取代C1-C6链烯基，其中，取代的C1-C6链烯基包括选自羟基、卤素、氧代基团(＝O)、＝N-OH、氨基和羟基C1-C6烷基氨基中的一个以上的取代基；
Figure PCTCN2015073855-appb-000004
其中，R9为H、C1-C6烷基、C1-C6烷氧基、羟基、羟基C1-C6烷基、-CH2OC(O)R10、-CH2OC(O)OR11、-CH2OC(O)CH2OR12；
R10、R11和R12各自独立地为取代或未取代的5至8元芳基；取代或未取代的3至8元环烷基；5至8元芳基氨基；包含N、S或O中的至少一个杂原子的5至8元杂芳基；C1-C6烷基、卤代C1-C6烷基；羟基C1-C6烷基；
Figure PCTCN2015073855-appb-000005
BocNH(CH2)mO(CH2)n-，其中，各m和n相同或不同，并且各自独立地为1至6的整数；其中，取代的5至8元芳基或取代的3至8元环烷基中的取代基选自羟基、卤素、C1-C6烷基、C1-C6烷氧基中的一个以上的取代基；
R4为氢、羟基、卤素或C1-C6烷基；
R5独立地选自氢；羟基；羟基C1-C6烷基；卤素；C1-C6烷基；-C(O)R13；-C(O)O(CH2CH2O)oCH2CH2R14；-C(O)NH(CH2CH2O)pCH2CH2R15；或 -C(O)NH(CH2)qC(O)OH；其中，o和p分别独立地为0、1、2或3；以及q为3至8的整数；
R13为氢；羟基羰基C1-C8烷基氨基；羟基；未取代的或被C1-C6烷基取代的哌嗪基；苄基氧基团；C1-C6烷氧基；叔丁氧基羰基C1-C6烷基氧基；或羟基羰基C1-C6烷基氨基；
R14和R15分别独立为氢；氨基；5-(2-氧代六氢-1H-噻吩并[3,4-d]咪唑-4-基)戊酰胺基；C1-C6酰胺基；叔丁氧基甲酰胺基；或C1-C6烷氧基；
R6和R7各自独立地为氢、羟基、卤素、羟基羰基或C1-C6烷氧基羰基；
Ra和Rb各自独立地为氢、羟基、卤素、C1-C6烷基或羟基C1-C6烷基；
Z为亚甲基或直接键；
Figure PCTCN2015073855-appb-000006
表示单键或双键。
进一步优选地，R3和R8各自独立地为氢、羟基、甲基、乙基、
Figure PCTCN2015073855-appb-000007
Figure PCTCN2015073855-appb-000008
在本发明中除非另外指出，
Figure PCTCN2015073855-appb-000009
表示连接位点；
优选地，R5为甲基、甲醛基、-COOH、甲氧基羰基、
Figure PCTCN2015073855-appb-000010
Figure PCTCN2015073855-appb-000011
在本发明的优选实施方案中，进一步优选地，本发明所述的通式(I)具有如下通式(II)所示的结构：
Figure PCTCN2015073855-appb-000012
在通式(II)中的各取代基的定义与通式(I)中的定义相同。
进一步优选地，本发明所述的通式(I)具有如下通式(III)或(IV)所示的结构：
Figure PCTCN2015073855-appb-000013
其中，在通式(III)或(IV)中，各取代基的定义与通式(I)中的定义相同。
在说明书中，术语“C1-C6烷基”可以为直链或支链C1-C6烷基，特别地，可以为甲基、乙基、丙基、异丙基、丁基、叔丁基、异丁基、戊基、新戊基或己基；优选地为直链或支链C1-C3烷基。
在说明书中，术语“5至8元芳基”为5至8元环的具有芳香性的基团，优选为苯基；
在说明书中，术语“5至8元环烷基”为具有5至8元环的环烷基，具体地，可以为，环丙基、环丁基、环戊基、环己基、环庚基或环辛基；
本发明通式(I)结构的五环三萜类化合物具体为：
Figure PCTCN2015073855-appb-000014
Figure PCTCN2015073855-appb-000015
Figure PCTCN2015073855-appb-000016
Figure PCTCN2015073855-appb-000017
Figure PCTCN2015073855-appb-000018
Figure PCTCN2015073855-appb-000019
根据另一方面，本发明提供了通式(I)所示的化合物的制备方法。所述制备方法包括如下制备路线：
路线一：
Figure PCTCN2015073855-appb-000020
路线二：
Figure PCTCN2015073855-appb-000021
路线三：
Figure PCTCN2015073855-appb-000022
具体来说，化合物2在二氯甲烷中发生Dess-Martin氧化反应，得到化合物16，接着在干燥的四氢呋喃中，在(S)-CBS的催化下，与硼烷反应得到3-OH反转的化合物17，最后化合物17在甲醇中催化氢化脱去苄基得到化合物1’，化合物1’与醇或胺反应得到化合物2’，化合物2’与酸或酰氯反应得到化合物3’；
路线四：
Figure PCTCN2015073855-appb-000023
其中，R16和R17分别为氢、C1-C6烷基或羟基C1-C6烷基；
R18为C1-C6烷基；其余取代基与通式(I)中的定义相同。
本发明还提供了一种治疗二型糖尿病的药物组合物，该组合物包含本发明一种或多种通式(I)所示的五环三萜类化合物中作为活性成分。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。
根据本发明的另一方面，提供了通式(I)所示的化合物在制备治疗二型糖尿病的药物中的应用。
根据本发明的又一方面，提供了通式(I)所示的化合物作为TGR5激动剂的应用。
根据本发明的再一方面，提供了通式(I)所示的化合物在治疗糖尿病中的应用。
有益效果
本发明设计与合成了一类新型的五环三萜类化合物，能有效激动TGR5，并有用于制成治疗二型糖尿病的药物，克服了现有化学合成小分子类TGR5激动剂所存在的胆囊毒性等缺陷，并相对于阳性对照INT777有更为简便的合成方法和更为温和的反应条件。本发明五环三萜类化合物的原料在自然界中来源丰富，具有天然产物的结构优势。
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但本发明不局限于这些实施例。
化合物制备实施例
下述制备实施例中，NMR用Varian生产的Mercury-Vx 300M仪器测定，NMR定标：δH 7.26ppm(CDCl3)，2.50ppm(DMSO-d6)；质谱用Agilent 1200Quadrupole LC/MS液质联用仪或SHIMADZU GCMS-QP5050A测定；试剂主要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HSGF 254；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。
制备实施例一(化合物编号：C33)
Figure PCTCN2015073855-appb-000024
(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
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)。
制备实施例二(化合物编号：C34)
Figure PCTCN2015073855-appb-000025
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：C107,原料白桦脂醇：C29,原料：C34)，按照制备实施例一相同的方法，合成以下化合物或中间体：
Figure PCTCN2015073855-appb-000026
Figure PCTCN2015073855-appb-000027
制备实施例三3-α-乙酰氧基白桦脂酸(化合物编号：C97)
Figure PCTCN2015073855-appb-000028
(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)。
用实施例三同样的方法和不同的酸酐合成以下化合物：
Figure PCTCN2015073855-appb-000029
Figure PCTCN2015073855-appb-000030
Figure PCTCN2015073855-appb-000031
制备实施例四(化合物编号C46)
Figure PCTCN2015073855-appb-000032
(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):585.4(M+Na)+(C37H54O4理论值：562.40)。
(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)。
用实施例四同样的方法合成C36：
Figure PCTCN2015073855-appb-000033
Figure PCTCN2015073855-appb-000034
制备实施例五(化合物编号：C48)
Figure PCTCN2015073855-appb-000035
(1)3-α-羟基-20-甲酰基白桦脂酸苄酯
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)。
(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)。
制备实施例六(化合物编号：C53)
Figure PCTCN2015073855-appb-000036
(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)。
制备实施例七(化合物编号：C47)
Figure PCTCN2015073855-appb-000037
(1)3-α-羟基-22-羟基白桦脂酸苄酯
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197mg,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)。
(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)。
制备实施例八(化合物编号C111)
Figure PCTCN2015073855-appb-000038
白桦脂醇(250mg,0.56mmol)于二氯甲烷(20mL)中，室温下慢慢加入Dess-Martin氧化剂(718mg,4.5mmol),回流反应4h，TLC检测反应完全。将反应混合物过滤后旋干，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-羰基-白桦脂醛(145mg)，为白色固体，摩尔收率：58.7％。1H NMR(300MHz,CDCl3)δ9.66(s,1H),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):456.3(M+Na)+(C30H46O2理论值：438.35)。
制备实施例九(化合物编号C37)
Figure PCTCN2015073855-appb-000039
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)。
制备实施例十(化合物编号C115)
Figure PCTCN2015073855-appb-000040
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)。
用实施例十同样的方法和不同的胺合成以下化合物：
Figure PCTCN2015073855-appb-000041
Figure PCTCN2015073855-appb-000042
Figure PCTCN2015073855-appb-000043
制备实施例十一(化合物编号C39)
Figure PCTCN2015073855-appb-000044
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)。
制备实施例十二(化合物编号C119)
Figure PCTCN2015073855-appb-000045
Figure PCTCN2015073855-appb-000046
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。再滴入制备实施例七所得产物的苄酯(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)。
用实施例十二同样的方法合成以下化合物：
Figure PCTCN2015073855-appb-000047
制备实施例十三(化合物编号C75)
Figure PCTCN2015073855-appb-000048
制备实施例十二所得产物的苄酯(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)。
用实施例十三同样的方法和不同的酰氯合成以下化合物：
Figure PCTCN2015073855-appb-000049
Figure PCTCN2015073855-appb-000050
Figure PCTCN2015073855-appb-000051
Figure PCTCN2015073855-appb-000052
Figure PCTCN2015073855-appb-000053
Figure PCTCN2015073855-appb-000054
Figure PCTCN2015073855-appb-000055
制备实施例十四：化合物C94的合成
Figure PCTCN2015073855-appb-000056
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。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)。
试验实施例1
TGR5激动剂试验实施例
TGR5激动试验实施例一化合物通过激活TGR5介导细胞内环磷酸腺苷3'-5'-环磷酸腺苷(cAMP)的积累
1、试验目的
利用瞬转TGR5的HEK293细胞用化合物进行刺激，然后用均相时间分辨荧光(Homogeneous Time-Resolved Fluorescence,HTRF)进行检测，目的检测这些化合物是否通过TGR5介导提高细胞内cAMP的累积。
2、试验原理
Gas蛋白偶联的胆汁酸受体TGR5与激动剂结合后其会发生结构改变，从而活化腺苷酸环化酶(AC)，进一步催化ATP生成cAMP,同时cAMP又在磷酸二酯酶(PDE)作用下进一步降解成AMP,而IBMX可以抑制PDE的活性，从而抑制cAMP降解成AMP。因此可以通过在实验中加入IBMX检测累积的cAMP量，而cAMP的量可以直接反映化合物对GPCR介导的AC是活化还是抑制。瞬转TGR5的HEK293细胞所产生的cAMP和试剂盒所提供的标记了d2的cAMP之间进行免疫竞争抗cAMP抗体的抗原结合位点。当标记了铕或铽的单克隆抗体跟d2标记的cAMP结合后会有比较大的信号，随着细胞内产生的cAMP增多，信号逐渐减小,从而导致荧光读数下降。因此可以通过荧光读数来反应化合物对细胞内cAMP的累积的影响。
3、实验样品
试验前将化合物溶于DMSO，配制母液，使用时用培养液稀释至所需浓度，并设INT-777和石胆酸(Lithocholic Acid)作为试验的阳性对照，检测每次试验反应的正常与否。
4、实验方法
4.1、将待测化合物用1xPBS配成终浓度的2倍.其中终浓度为100μM、10μM、1μM、100nM、10nM、1nM、0.1nM、DMSO(每个孔都含有1％的DMSO)。
4.2、细胞处理:
4.2.1、用胰酶消化细胞，然后用无血清培液悬浮。
4.2.2、定细胞密度.并同时在无血清培液中加IBMX(终浓度为500μM)，细胞数为2000/5μl/孔。
4.2.3、加入5μl待测化合物&5μl含IBMX的细胞悬液混合，锡箔纸将384孔板封闭好，室温避光反应不超过30分钟。
4.3.检测底物配置
4.3.1、1μl cAMP-d2用cAMP&cGMP conjugates&lysis buffer稀释到20μl
4.3.2、1μl anti-cAMP-Cryptate用cAMP&cGMP conjugates&lysis buffer稀释至20μl
4.3.3、30分钟后，加入5μl(1.3.1)+5μl(1.3.2)，锡箔纸将384孔板封闭好，室温避光反应分钟。
4.4、60分钟后，Envision2101多功能微孔板酶标仪(PerkinElmer)读数。
5、实验结果：(以C29,C33,C40,C98等十四个化合物为例，但不局限于这些化合物)
表1 化合物TGR5激动活性测试试验
Figure PCTCN2015073855-appb-000057
注：EC50为样品药物对TGR5激动活性的评价，半数50％有效浓度。NR表示在100μM的浓度下没有活性。
6、结果与讨论：
这些化合物在表达TGR5的HEK293细胞中能够提高细胞内的cAMP累积，其活性呈剂量依赖关系，EC50值见表一。而在不表达TGR5的HEK293细胞中，这些化合物则不能导致cAMP的积累。结果进一步说明这些化合物是TGR5受体的激动剂。且在表中表现出3-α构型比3-β构型的TGR5激动活性有明显提高。
试验实施例二：
化合物不能激活核受体FXR介导的报告基因表达
1、试验目的
利用瞬转瞬转表达质粒pBind-FXR和报告基因质粒pGL4.31的HEK293细胞用化合物进行刺激，然后用
Figure PCTCN2015073855-appb-000058
稳定荧光素酶检测系统进行检测，目的检测这些化合物是否通过FXR提高细胞内的荧光素酶的表达水平。
2、试验原理
哺乳动物细胞单杂技术(Mammalian one-hybrid)也称为GAL4嵌合受体基因检测方法(GAL4chimera receptor assay),该技术是近些年发展起来并主要应用于核受体(Nuclear  receptor,NR)功能及其配体生理活性筛选和评价的一种新技术。该技术机理是利用了酵母细胞转录因子GAL4和哺乳细胞核受体的分子结构中都具有2个相似的主要结构域:配体结合结构域(LBD)和DNA结合结构域(DBD),将核受体的配体结合结构域(LBD)与酵母细胞转录因子GAL4的DNA结合结构域(DBD)融合成嵌合蛋白表达质粒,再与含有GAL4特异响应元件的报告质粒共转染动物细胞,通过测定报告基因的表达水平从而评价核受体配体的激动或拮抗活性。
3、实验样品
试验前将化合物溶于DMSO，配制母液，使用时用培养液稀释至所需浓度，并设GW4604作为试验的阳性对照，检测每次试验反应的正常与否。
4、实验方法
4.1、瞬转：瞬转瞬转表达质粒pBind-FXR和报告基因质粒pGL4.31至HEK293细胞，然后按10000个细胞/孔细胞密度接种至384孔板，在10％FBS高糖DMEM，37℃、5％CO2条件下培养12小时。
4.2、加化合物：激动剂测试中将激动剂阳性对照和化合物稀释至10×终浓度直接加入细胞培养板，体积为5μl,继续在37℃、5％CO2条件下培养24小时。
4.3、检测：加药24小时后，用
Figure PCTCN2015073855-appb-000059
稳定荧光素酶检测系统进行检测。每孔吸去25μL培养基，加入25μL荧光素酶活性检测试剂，振荡5min。最后在Envision2101多功能微孔板酶标仪检测化学发光计数值。
5、实验结果：(以C29，C33，C40，C98等十四个化合物为例，但不局限于这些化合物)
表2 化合物FXR激动活性测试试验
Figure PCTCN2015073855-appb-000060
注：EC50为样品药物对TGR5激动活性的评价，半数50％有效浓度。NR表示在100μM的浓度下没有活性。
6、结果与讨论
在瞬转核受体FXR中，GW4604能激活FXR，通过激活FXR提高细胞内的荧光素酶的表达，并且其活性呈剂量依赖关系。但是这些化合物不能通过激活FXR提高细胞内的荧光素酶的表达。结果说明这些化合物并不能通过核受体FXR发挥作用，进一步说明这些化合物具有一定的选择特异性。

Claims (10)

  1. 一种具有通式(I)所示结构的化合物，
    Figure PCTCN2015073855-appb-100001
    其中，R1为氢、羟基、卤素或C1-C6烷基；
    R2为氢；羟基；卤素；氧代基团(＝O)；＝N-OH；C1-C6烷基羰基氧基团；3至8元环烷基羰基氧基团；C1-C6烷基；3至8元环烷基；或
    Figure PCTCN2015073855-appb-100002
    其中，Rc为C1-C6烷基或氢；
    R3和R8各自独立地为：氢；羟基；卤素；被C1-C6烷基取代的氨基C1-C6烷基；未取代或取代的C1-C6烷基，其中，取代的C1-C6烷基中的取代基选自羟基、卤素、氧代基团(＝O)、＝N-OH、环氧丙烷基、氨基或羟基C1-C6烷基氨基；未取代或取代C1-C6链烯基，其中，取代的C1-C6链烯基中的取代基选自羟基、卤素、氧代基团(＝O)、＝N-OH、氨基或羟基C1-C6烷基氨基；
    Figure PCTCN2015073855-appb-100003
    其中，R9为H、C1-C6烷基、C1-C6烷氧基、羟基、羟基C1-C6烷基、-CH2OC(O)R10、-CH2OC(O)OR11、-CH2OC(O)CH2OR12；
    其中，R10、R11和R12各自独立地为取代或未取代的5至8元芳基；取代或未取代的3至8元环烷基；5至8元芳基氨基；包含N、S或O中的至少一个杂原子的5至8元杂芳基；C1-C6烷基；卤代C1-C6烷基；羟基C1-C6烷基；
    Figure PCTCN2015073855-appb-100004
    Figure PCTCN2015073855-appb-100005
    BocNH(CH2)mO(CH2)n-，其中，各m和n相同或不同，并且各自独立地为1至6的整数；其中，取代的5至8元芳基或取代的3至8元环烷基中的取代基选自羟基、卤素、C1-C6烷基或C1-C6烷氧基；
    R4为氢、羟基、卤素或C1-C6烷基；
    R5独立地选自氢；羟基；羟基C1-C6烷基；卤素；C1-C6烷基；-C(O)R13；-C(O)O(CH2CH2O)oCH2CH2R14；-C(O)NH(CH2CH2O)pCH2CH2R15；其中，o和p分别独立地为0、1、2或3；或-C(O)NH(CH2)qC(O)OH，q为3至8的整数；
    R13为氢；羟基羰基C1-C8烷基氨基；羟基；未取代的或被C1-C6烷基取代的哌嗪基；苄基氧基团；C1-C6烷氧基；叔丁氧基羰基C1-C6烷基氧基；或羟基羰基C1-C6烷基氨基；
    R14和R15分别独立为氢；氨基；5-(2-氧代六氢-1H-噻吩并[3,4-d]咪唑-4-基)戊酰胺基；C1-C6酰胺基；叔丁氧基甲酰胺基；或C1-C6烷氧基；
    R6和R7各自独立地为氢、羟基、卤素、羟基羰基或C1-C6烷氧基羰基；
    Ra和Rb各自独立地为氢、羟基、卤素、C1-C6烷基或羟基C1-C6烷基；
    Z为亚甲基或直接键；
    Figure PCTCN2015073855-appb-100006
    表示单键或双键。
  2. 根据权利要求1所述的化合物，其中，R3和R8各自独立地为氢、羟基、甲基、乙基、
    Figure PCTCN2015073855-appb-100007
    Figure PCTCN2015073855-appb-100008
    Figure PCTCN2015073855-appb-100009
  3. 根据权利要求1所述的化合物，其中，R5为甲基、甲醛基、-COOH、甲氧基羰基、
    Figure PCTCN2015073855-appb-100010
  4. 根据权利要求1所述的化合物，其中，所述化合物具有如下通式(II)所示的结构：
    Figure PCTCN2015073855-appb-100011
    其中，各取代基的定义如在权利要求1中的定义相同。
  5. 根据权利要求1所述的化合物，其中，所述化合物具有如下通式(III)或(IV)的结构：
    Figure PCTCN2015073855-appb-100012
    其中，各取代基的定义如在权利要求1中的定义相同。
  6. 根据权利要求1所述的化合物，其中，所述化合物具有如下结构：
    Figure PCTCN2015073855-appb-100013
    Figure PCTCN2015073855-appb-100014
    Figure PCTCN2015073855-appb-100015
    Figure PCTCN2015073855-appb-100016
    Figure PCTCN2015073855-appb-100017
    Figure PCTCN2015073855-appb-100018
  7. 一种权利要求1所述的化合物的制备方法，其包括如下制备路线：
    路线一：
    Figure PCTCN2015073855-appb-100019
    路线二：
    Figure PCTCN2015073855-appb-100020
    路线三：
    Figure PCTCN2015073855-appb-100021
    路线四：
    Figure PCTCN2015073855-appb-100022
    其中，R16和R17分别为氢、C1-C6烷基或羟基C1-C6烷基；
    R18为C1-C6烷基；其余取代基与权利要求1中的定义相同。
  8. 一种药物组合物，其包含根据权利要求1至6中任一项所述的化合物作为活性成分。
  9. 根据权利要求1至6中任一项所述的化合物在制备治疗二型糖尿病的药物中的应用。
  10. 根据权利要求1至6中任一项所述的化合物作为TGR5激动剂的应用。
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