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WO2016086824A1 - 一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用 - Google Patents

一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用 Download PDF

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WO2016086824A1
WO2016086824A1 PCT/CN2015/096038 CN2015096038W WO2016086824A1 WO 2016086824 A1 WO2016086824 A1 WO 2016086824A1 CN 2015096038 W CN2015096038 W CN 2015096038W WO 2016086824 A1 WO2016086824 A1 WO 2016086824A1
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bouchardadine
bouchardatine
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French (fr)
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黄志纾
叶冀明
刘宏
饶勇
陈迎春
高琳
余宏
古练权
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the invention belongs to the field of medical technology. More specifically, it relates to a Bouchardadine and Bouchardarine derivative and a preparation method and application thereof.
  • Obesity as a type of metabolic disease, is closely related to the occurrence and development of a variety of other metabolic diseases, such as type 2 diabetes, atherosclerosis, and hypertension. Studies have shown that the formation of obesity is due to the in vitro energy consumption of the body exceeds the body's energy consumption, and the surplus energy is stored in the form of triglycerides, which mediates the metabolic disorder of the body.
  • BMI Body Mass Index
  • the proportion of obese people in the world reached 30% in 2011, among which the number of adolescents (under 18 years old) reached 1.2 billion, accounting for about 10%; in 2012, the number of obese people increased to 1.5 billion; 2013 In the year, the figure rose to 1.7 billion.
  • the growth rate of obese population in China in the past decade has been equal to the growth rate of countries such as Europe and the United States in the past 30 years.
  • the development of effective therapeutic drugs for obesity is relatively lagging behind.
  • Bouchardatine is a kind of ⁇ -quinone quinazolinone alkaloid, and its structure is composed of the 2-position of quinazolinone and the 2-position of hydrazine.
  • Bouchardatine is separated from Bouchardatia neurococca It is found that modern research shows that Bouchardine has many biological activities such as anti-cancer, anti-virus and anti-tuberculosis, but it has not been used in anti-obesity and reducing fatty liver.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the existing obesity and fatty liver treatment drugs, and to provide the application of Bouchardarine and its derivatives in anti-obesity and reduction of fatty liver.
  • the present invention found that the natural product Bouchardatine has a certain activity of inhibiting the differentiation and accumulation of adipocytes, and is a lead structure for anti-obesity drugs with potential development prospects.
  • the inventors have carried out modification and optimization studies based on the Bouchardatine structure, mainly introducing an amine group with a chain structure from the 5 position, in order to improve the interaction ability of the target compound with the target and chemical intervention of the relevant pathway. The effect is to improve the drug properties such as water solubility and bioavailability of the compound.
  • Another object of the present invention is to provide a Bouchardadine derivative and a process for the preparation thereof.
  • a further object of the present invention is to provide the use of Bouchardarine and its derivatives for the preparation of a medicament for the prevention of obesity and/or amelioration of fatty liver.
  • the present invention provides a Bouchardadine derivative which is a derivative of the natural product Bouchardatine.
  • R 1 is independently selected from:
  • R 2 is independently selected from:
  • the R 1 is an aromatic group such as N,N-dimethyl, N,N-diethyl, morphinolinyl, piperidinyl, p-nitrophenyl or p-methoxyphenyl. .
  • the Bouchardadine derivative is the compound SYS-B1 to SYS-B7 prepared in the examples, and the structural formula is as follows:
  • the present invention also provides a preparation method of the above natural product Bouchardadine, and the synthesis steps are as follows:
  • step S3 Substituting compound 2 obtained in step S2 with phenylhydrazine to form an imine after substitution and elimination reaction, and then reacting with one molecule of phenylhydrazine to form compound 3
  • the compound 3 obtained in the step S3 is subjected to a PPA-catalyzed Fischer reaction to obtain a compound 4 having an anthracene ring structure.
  • step S5 The compound 4 obtained in the step S4 is reacted with DMSO-H 2 O and ammonium acetate, and a formyl group is introduced at the 3-position of the oxime to obtain a compound 5, that is, Bouchardadine.
  • the molar ratio of the compound 2 to the phenylhydrazine in the step S3 is 1:3 to 5.
  • reaction in step S5 is carried out under nitrogen.
  • the molar ratio of ammonium acetate to compound 4 in step S5 is 2-6:1, and the DMSO-H 2 O is DMSO-H 2 O solution, and the volume ratio of DMSO to H 2 O in the solution is 10 ⁇ 20:1.
  • Bouchardatine and Bouchardadine derivatives in the preparation of a medicament for the prevention of obesity and/or amelioration of fatty liver production is also within the scope of the present invention.
  • the present inventors have found that the natural product Bouchardatine and the Bouchardadine derivative prepared by the present invention can significantly inhibit the differentiation of pre-adipocyte 3T3-L1, thereby reducing intracellular 3T3-L1 cells. Lipid accumulation; and can reduce the accumulation of lipids in human liver cancer cells induced by sodium oleate, which has the effects of reducing animal cell triglyceride levels and anti-obesity and reducing fatty liver production.
  • the present invention also provides an anti-obesity drug containing the Bouchardatine and Bouchardadine derivatives provided by the present invention, which have the effects of reducing fat accumulation, reducing obesity, relieving fatty liver and the like.
  • Such compounds are useful in the treatment of obesity and related diseases such as diabetes and cardiovascular diseases caused by obesity.
  • the medicament further comprises an adjuvant such as a pharmaceutically acceptable salt or carrier thereof.
  • an adjuvant such as a pharmaceutically acceptable salt or carrier thereof.
  • the pharmaceutical dosage form is an injection, a tablet, a pill, a capsule, a suspension or an emulsion.
  • the route of administration can be oral, transdermal, intravenous or intramuscular.
  • the results of the cell level experiments of the present invention indicate that the Bouchardatine and Bouchardadine derivatives provided by the present invention can not only significantly reduce the triglyceride content in the fat cells of 3T3-L1 mice, but also reduce the sodium oleate-induced human hepatoma cell line HepG-. 2 intracellular triglyceride accumulation.
  • the substance reduces fat formation by inhibiting intracellular lipogenesis, and may promote endogenous fat mobilization by inhibiting exogenous fatty acid uptake to reduce fat accumulation, thereby achieving the effects of treating obesity and reducing fatty liver production.
  • the compound SYS-B3 can effectively inhibit the expression of adipose differentiation-related regulatory factors (such as PPAR ⁇ , C/EBP ⁇ , SREBP-1c) and the related metabolic enzymes that regulate lipogenesis (ACC, FAS, SCD-1).
  • adipose differentiation-related regulatory factors such as PPAR ⁇ , C/EBP ⁇ , SREBP-1c
  • the lipid-lowering activity of the present invention is remarkably improved.
  • the lipid-lowering activity of the compound SYS-B3 is about 23 times higher than that of the Bouchardarine natural product. .
  • IC50 of the acute toxicity of the compound SYS-B3 was greater than 80 mg/kg, which was more than 100 times higher than the effective dose window.
  • Compound SYS-B3 can effectively reduce the weight of white adipose tissue without affecting the feeding condition of mice, and can significantly reduce the content of triglyceride and free fatty acid in plasma.
  • the experimental results are related to the results of cell level research. meets the.
  • the compound SYS-B3 has stable pharmaceutical properties, good membrane permeability and high bioavailability, indicating that SYS-B3 has good drug-forming properties.
  • the Bouchardatine and Bouchardadine derivatives provided by the invention can significantly reduce the lipid accumulation in 3T3-L1 adipocytes, and can reduce the accumulation of lipids in human liver cancer cells induced by sodium oleate, and reduce the triglyceride level and resistance of animal cells. Obesity and the effect of reducing fatty liver production, therefore, have anti-obesity and reduce the ability of fatty liver production, and have high safety and good drug-forming properties.
  • the drug-like properties of the Bouchardadine derivative which has been structurally modified by the preparation method of the present invention, are significantly improved compared to Bouchardadine, and have a prospect of being developed into a new anti-obesity drug.
  • Figure 1 Effect of Bouchardatine and Bouchardadine derivatives provided by the present invention on fat accumulation of 3T3-L1 adipocytes (all compound concentrations were 1 ⁇ M).
  • Figure 2 Comparison of lipid accumulation of 3T3-L1 cells in a concentration-dependent manner by the Bouchardadine derivative SYS-B3 and Bouchardadine natural product provided by the present invention.
  • Fig. 3 is a comparison of cell morphology of Bouchardatine and Bouchardadine derivatives provided by the present invention to 3T3-L1 cell differentiation and BM and DMSO differentiation control groups in the basal control group (cell morphology was observed on day 6 and observed under microscope 40-fold field).
  • Figure 4 Effect of Bouchardatine and Bouchardadine derivatives provided by the present invention on lipid accumulation of human liver cancer cell HepG-2 induced by sodium oleate (all compound concentrations were 1 ⁇ M).
  • Fig. 5 The effect of the Bouchardatine and Bouchardadine derivatives provided by the present invention on the lipid accumulation of human hepatoma cells HepG-2 induced by sodium oleate (the compound concentration is 1 ⁇ M), stained with oil red O, and photographed by a microscope 4 times micrograph.
  • the DMEM complete culture solution containing the differentiation induction solution was used as a diluent, and the drug solution was diluted to a certain concentration, and was added together with Day0 and Day3.
  • the blank control group and the differentiation control group were each added with an equal volume of DMSO solution.
  • oil red O staining photographs and triglyceride content analysis were performed.
  • Differentiation Inducing Solution I containing 500 ⁇ M 3-isobutyl-1-methyl-xanthine, 100 ng/mL dexamethasone, 2 ⁇ g/mL insulin in DMEM complete medium differentiation induction solution II: DMEM complete medium containing 2 ⁇ g/mL insulin
  • the cells were washed twice with pre-cooled PBS, and the PBS was added.
  • the deionized solution containing 0.2% Triton X-100 was added and allowed to stand at room temperature for 1 h.
  • the cell suspension was collected and sonicated for 10 min to fully lyse the cells and centrifuge.
  • the supernatant was collected and the triglyceride content was determined according to the instructions of the triglyceride detection kit.
  • the triglyceride content analysis was expressed as "100% control" in the differentiation control group, that is, the compound triglyceride content/differentiation control content *100%.
  • the experimental results are the average of three independent experiments, and the results were statistically analyzed according to "mean ⁇ standard deviation".
  • the DM group represents a differentiation control group, which represents a normally differentiated cell, and has a large amount of lipid accumulation.
  • Bouchardatine is a natural product, and compounds SYS-B1 and the like are all Bouchardatine derivatives.
  • 1 ⁇ M SYS-B1, SYS-B2, SYS-B3 and SYS-B5 can effectively inhibit lipogenesis (*, p ⁇ 0.05; **, p ⁇ 0.01); among them, compound SYS-B1
  • the inhibition effect with SYS-B3 is most obvious.
  • both Bouchardatine and SYS-B3 were able to reduce lipid content in 3T3-L1 cells in a concentration-dependent manner (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001)
  • the compound SYS-B3 is more active.
  • the triglyceride content was reduced by 18% and 65%, respectively; we calculated that the EC 50 values of Bouchardadine and SYS-B3 were 22.59 ⁇ M, 1.1, respectively.
  • the lipid-lowering activity of the compound SYS-B3 was increased by about 23 times.
  • BM represents a basic control group, representing undifferentiated cells, and no lipid accumulation in the cells;
  • DM represents a differentiation control group, and a large amount of lipid is clearly visible in the cells; and the lipid content in the compound-treated group is accompanied by The concentration of the compound is gradually decreased.
  • compound SYS-B3 is at 0.1, 0.5, 1, 5, 10 ⁇ M
  • the triglyceride content was 73%, 56%, 47%, 28%, and 15% of the control group, respectively.
  • the human liver cancer cell HepG-2 was uniformly inoculated into a 48-well plate and allowed to stand for 24 hours.
  • DMEM complete medium containing 10 mM sodium oleate (DMEM medium containing 10% FBS and 1% double antibody) was replaced with a final concentration of sodium oleate of 1 mM.
  • the compound intervention group was diluted to the desired concentration in a DMEM complete medium containing sodium oleate.
  • the blank control group and the sodium oleate control group were respectively added with the same amount of DMSO.
  • the cells were washed twice with pre-cooled PBS (pH 7.4), and 4% frozen paraformaldehyde fixative was fixed at room temperature for 60 min. 0.3% oil red O staining solution was stained at room temperature for 30 mins. Rinse with deionized water for 2-3 times, and take an inverted microscope (40 ⁇ )
  • the treated cells were washed twice with PBS, PBS was added, and a deionized solution containing 0.2% Triton X-100 was added.
  • the mixture was allowed to stand at room temperature for 1 hour, repeatedly blown to fully lyse the cells, and the cell suspension was collected into a centrifuge tube and sonicated. After 10 min, the supernatant was collected by centrifugation, and the triglyceride content and protein concentration were determined according to the instructions of the triglyceride detection kit and the BCA protein quantitative detection kit. The final triglyceride content is expressed in the form "nmol/mg protein".
  • control group was "100%” and converted (treatment group triglyceride content (nmol/mg protein) / control triglyceride content ((nmol/mg protein) * 100%).
  • the experimental results are the average of three independent experiments, and the results were statistically analyzed according to "mean ⁇ standard deviation".
  • the intracellular triglyceride content of the sodium oleate-inducing group was significantly higher than that of the blank control group (#, p ⁇ 0.01).
  • Compound intervention can effectively reduce intracellular triglyceride content.
  • the triglyceride content of compounds SYS-B1, SYS-B2, and SYS-B3 is reduced by 43%, respectively. 28%, 54%, with compound SYS-B3 works best.
  • Figure 5 shows that the Blank group represents no lipid accumulation in the cells without sodium oleate treatment; the Control group represents sodium oleate induction treatment, and a large amount of lipid accumulation is clearly visible in the cells.
  • the Control group represents sodium oleate induction treatment
  • a large amount of lipid accumulation is clearly visible in the cells.
  • there was no significant change in intracellular lipid accumulation in Bouchardatin at 10 ⁇ M while intracellular lipid accumulation was significantly reduced in Bouchardatine derivatives at 1 ⁇ M.
  • compound SYS-B1 was the best.

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Abstract

本发明公开了天然产物Bouchardatine和Bouchardatine衍生物在制备抗肥胖症和/或减轻脂肪肝生成的药物中的应用。所述Bouchardatine衍生物的结构式如式(I)所示，其中：R为-(CH2)nR1，n＝0、1、2或3；R1选自：(1)被1个或多个R2取代的取代苯基；(2)吗啡啉基或哌啶基；(3)N,N-二甲基氨基或N,N-二乙基氨基；R2为氟基、甲氧基或硝基。上述天然产物及其衍生物能够显著抑制前脂肪细胞3T3-L1的分化，从而减少3T3-L1脂肪细胞内的脂质累积；并能够减少油酸钠诱导的人肝癌细胞内脂质的积累，具有降低动物细胞甘油三酯水平和抗肥胖症及减轻脂肪肝生成的效果。

Description

一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用 技术领域
本发明属于医药技术领域。更具体地，涉及一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用。
背景技术
肥胖症，作为一类新陈代谢类疾病，与多种其它的代谢类疾病的发生发展存在密切联系，如2型糖尿病、动脉粥样硬化、高血压等。研究表明，肥胖症的形成，是由于机体外源性能量摄入超过机体能量消耗，富余的能量以甘油三酯的形式进行储存，介导机体代谢紊乱的过程。目前，世界卫生组织对肥胖的判定均是基于身体质量指数(Body Mass Index，BMI)，即BMI≥30kg/m2时则定义为肥胖。随着全球经济的发展，肥胖症及其并发症已成为严重影响人类健康的世界性公共卫生问题。据世界卫生健康组织统计，2011年全世界肥胖人口比率达30％，其中青少年(18岁以下)人口数目达12亿，约占10％；2012年，青少年肥胖人口数则增至15亿；2013年，该项数值则升至17亿。而在中国，近十年来我国肥胖人口的增长速率等同于欧美等国家过去30年的增长速率。在世界肥胖人口数目逐渐增多的情况下，针对肥胖症的有效治疗药物研发则相对滞后。
研究表明，肥胖症的形成主要是由于脂肪的过度形成以及脂肪氧化消耗能力减弱所致。脂肪的形成主要是两方面因素共同作用的结果：1)多能/单能干细胞在特定生长因子的诱导下定向分化形成脂肪细胞；2)脂肪细胞内甘油三酯不断累积，形成成熟的脂肪细胞。因此，抑制脂肪细胞分化或促进脂肪氧化消耗，从而减少脂质累积，是发展抗肥胖症药物的基本策略。
目前，世界范围内可供选择的抗肥胖症药物很少。目前临床应用的主要有去甲肾上腺素再摄取抑制剂盐酸芬特明/盐酸芬特明-托吡酯复方胶囊剂，脂酶抑制剂奥利司他，以及中枢神经类药物盐酸劳卡色林等。但是，上述药物在使用过程中，抗肥胖效果有限，副作用明显，限制了抗肥胖药物的使用。因此，研究开发药效更好，安全性更高的抗肥胖药物已成为目前研究热点之一。
天然产物Bouchardatine作为一类β-吲哚喹唑啉酮生物碱，其结构为喹唑啉酮的2-位与吲哚的2-位相连构成。Bouchardatine从Bouchardatia neurococca分离 得到，现代研究表明，Bouchardatine具有抗癌、抗病毒、抗结核等多种生物活性，但未见其在抗肥胖症及减轻脂肪肝中的应用。
发明内容
本发明要解决的技术问题是克服现有肥胖症及脂肪肝治疗药物的不足，提供Bouchardatine及其衍生物在抗肥胖症及减轻脂肪肝中的应用。本发明研究发现，天然产物Bouchardatine具有一定的抑制脂肪细胞分化和蓄积的活性，是一个具有潜在发展前景的抗肥胖症药物先导结构。发明人在Bouchardatine结构基础上进行了修饰和优化研究，主要是从5号位引入具有链状结构特点的胺基，目的是提高目标化合物与作用靶点的相互作用能力和对相关通路的化学干预效果，改善化合物的水溶性和生物利用度等成药性质。实验结果显示，Bouchardatine和Bouchardatine衍生物具有抗肥胖效果好、毒性小、能够减少细胞内脂质累积、降低动物细胞甘油三酯水平等优点，在制备预防和治疗与抗肥胖症及减轻脂肪肝相关疾病的药物上有广阔的应用空间。
本发明的目的是提供一种制备Bouchardatine的方法。
本发明另一目的是提供Bouchardatine衍生物及其制备方法。
本发明的再一目的是提供Bouchardatine及其衍生物在制备抗肥胖症和/或减轻脂肪肝的药物中的应用。
本发明上述目的通过以下技术方案实现：
本发明提供了一种Bouchardatine衍生物，所述Bouchardatine衍生物为天然产物Bouchardatine的衍生物。
天然产物Bouchardatine的结构式为：
Figure PCTCN2015096038-appb-000001
所述Bouchardatine衍生物的结构式如式(I)所示：
Figure PCTCN2015096038-appb-000002
式(I)中：R为-(CH2)n R1，n＝0、1、2或3；
所述R1独立地选自：
(1)被1个或多个R2取代的取代苯基；
(2)吗啡啉基或哌啶基；
(3)N,N-二甲基氨基或N,N-二乙基氨基；
R2独立地选自：
(1)氟基；
(2)甲氧基；
(3)硝基。
进一步优选地，所述R1为N,N-二甲基、N,N-二乙基、吗啡啉基、哌啶基、对硝基苯基或对甲氧基苯基等芳香类基团。
更优选地，所述Bouchardatine衍生物为实施例所制备的化合物SYS-B1～SYS-B7，结构式如下所示：
Figure PCTCN2015096038-appb-000003
Figure PCTCN2015096038-appb-000004
另外，本发明还提供了一种上述天然产物Bouchardatine的制备方法，合成步骤如下：
S1.将邻氨基苯甲酰胺与原丙酸三乙酯反应得化合物1
Figure PCTCN2015096038-appb-000005
S2.将步骤S1所得化合物1溴化后得到化合物2
Figure PCTCN2015096038-appb-000006
S3.将步骤S2所得化合物2与苯肼经取代、消除反应后先生成亚胺，然后再 与一分子苯肼反应生成化合物3
Figure PCTCN2015096038-appb-000007
S4.将步骤S3所得化合物3经PPA催化的Fischer反应得到含有吲哚环结构特征的化合物4
Figure PCTCN2015096038-appb-000008
S5.将步骤S4所得化合物4与DMSO-H2O及乙酸铵反应，并在吲哚的3-位引入甲酰基得到化合物5，即Bouchardatine。
进一步地，上述Bouchardatine衍生物的制备方法如下：
S6.将上述制备所得Bouchardatine中喹唑啉酮的羰基进行氯代，得到化合物6
Figure PCTCN2015096038-appb-000009
S7.将步骤S6所得化合物6与不同的胺类化合物反应，得到Bouchardatine衍生物。
其中，优选地，步骤S3中所述化合物2与苯肼的摩尔比为1：3～5。
优选地，步骤S5中所述反应在氮气保护下进行。
优选地，步骤S5中所述乙酸铵与化合物4的摩尔比为2～6：1，所述DMSO-H2O为DMSO-H2O溶液，溶液中DMSO与H2O的体积比为10～20：1。
另外，上述制备得到的Bouchardatine以及Bouchardatine衍生物均应在本发明的保护范围之内。
Bouchardatine以及Bouchardatine衍生物在制备抗肥胖症和/或减轻脂肪肝生成的药物中的应用，也均应在本发明的保护范围之内。
本发明研究发现，天然产物Bouchardatine以及本发明制备的Bouchardatine衍生物，能够显著抑制前脂肪细胞3T3-L1的分化，从而减少3T3-L1脂肪细胞内 的脂质累积；并能够减少油酸钠诱导的人肝癌细胞内脂质的积累，具有降低动物细胞甘油三酯水平和抗肥胖症及减轻脂肪肝生成的效果。
本发明还提供了一种抗肥胖药物，该类药物含有本发明提供的Bouchardatine和Bouchardatine衍生物，该类药物具有减少脂肪积累、减轻肥胖、缓解脂肪肝等功效。该类化合物可用于治疗肥胖症以及肥胖引起的糖尿病、心血管疾病等相关疾病。
优选地，所述药物还包括其药学上可接受的盐或载体等辅助剂。
优选地，所述药物的剂型为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂。其给药途径可为口服，经皮，静脉或肌肉注射。
本发明的细胞水平实验结果表明，本发明提供的Bouchardatine和Bouchardatine衍生物不仅能够显著地减少3T3-L1小鼠脂肪细胞内甘油三酯含量，还能够减少油酸钠诱导的人肝癌细胞株HepG-2细胞内甘油三酯累积。如化合物SYS-B3抑制脂肪生成的活性为EC50＝0.78±0.13μM；而抑制人肝癌细胞株HepG-2细胞内甘油三酯累积的活性为EC50＝14.44±0.52μM，表明所述Bouchardatine和Bouchardatine衍生物通过抑制细胞内脂肪生成来减少脂肪形成，并可能通过抑制外源性脂肪酸摄取而促进内源性脂肪动员来减少脂肪累积，达到治疗肥胖症和减轻脂肪肝生成的效果。
通过对化合物SYS-B3降脂机制研究发现，化合物SYS-B3可以有效的抑制脂肪分化相关调控因子的表达(如PPARγ,C/EBPα,SREBP-1c)以及调控脂肪生成的相关代谢酶(ACC,FAS，SCD-1)。其次，我们还发现化合物SYS-B3能够激活AMPK通路以及抑制未折叠蛋白响应通路(unfolded protein response,UPR)，这些结果与其抗肥胖症的治疗作用效果相一致。
通过对本发明提供的Bouchardatine衍生物与天然产物Bouchardatine脂肪抑制效果进行对比发现，本发明降脂活性得到显著提高，特别地，化合物SYS-B3降脂活性比Bouchardatine天然产物降脂活性提高了约23倍。
动物实验结果表明，化合物SYS-B3急毒毒性的IC50大于80mg/kg，比有效剂量窗口高100倍以上。而化合物SYS-B3在不影响小鼠摄食条件下，可有效的减少白色脂肪组织重量，并可显著性的降低血浆中甘油三酯及游离脂肪酸含量，这一实验结果与细胞水平的研究结果相符合。此外，化合物SYS-B3药学性质稳定，膜通透性好，生物利用率较高，均表明SYS-B3具有良好的成药性。
综上所述，药理学和动物水平实验证明，本发明所提供的Bouchardatine和Bouchardatine衍生物，具有抗肥胖症和减轻脂肪肝生成能力，尤其是经过结构改造的Bouchardatine衍生物的成药性相比较Bouchardatine得到显著改善，具有开发成为抗肥胖症新药的前景。
本发明具有以下有益效果：
本发明提供的Bouchardatine和Bouchardatine衍生物能够显著减少3T3-L1脂肪细胞内的脂质累积，并且能够减少油酸钠诱导的人肝癌细胞内脂质的积累，具有降低动物细胞甘油三酯水平和抗肥胖症及减轻脂肪肝生成的效果，因此，具有抗肥胖症和减轻脂肪肝生成能力，并且安全性高，成药性好。
另外，尤其是经过本发明制备方法结构改造的Bouchardatine衍生物的成药性相比较Bouchardatine得到显著改善，具有开发成为抗肥胖症新药的前景。
附图说明
图1：本发明提供的Bouchardatine和Bouchardatine衍生物对3T3-L1脂肪细胞脂肪累积的影响(化合物浓度均为1μM)。
图2：本发明提供的Bouchardatine衍生物SYS-B3与Bouchardatine天然产物以浓度梯度依赖性的方式减少3T3-L1细胞的脂质累积对比图。
图3：本发明提供的Bouchardatine和Bouchardatine衍生物对3T3-L1细胞分化及基础对照组BM和DMSO分化对照组处理的细胞形态对比图(细胞形态为第6天，显微镜40倍视野下观察)。
图4：本发明提供的Bouchardatine和Bouchardatine衍生物对油酸钠诱导人肝癌细胞HepG-2脂质累积的影响(化合物浓度均为1μM)。
图5：本发明提供的Bouchardatine和Bouchardatine衍生物对油酸钠诱导人肝癌细胞HepG-2脂质累积的影响(化合物浓度均为1μM)，经油红O染色，显微镜4倍镜拍照所得图片。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1天然产物Bouchardatine的合成
1、化合物1的合成
50mL单口瓶中投入1.36g 10mmol的邻氨基苯甲酰胺，加入原丙酸三乙酯10mL，将温度调至155℃反应19h，薄层色谱法监测反应完全。将反应液冷却至室温，析出少量白色固体，再放置冰箱析出大量固体，过滤，乙醇洗涤，将母液旋干后，再用乙醇溶解其中杂质，再过滤，合并滤饼，烘干共得到1.51g白色针状固体1。
Figure PCTCN2015096038-appb-000010
产率89.5％；1H NMR(400MHz，CDCl3)δ11.39(s，1H),8.31(d,J＝7.9Hz,1H),7.83-7.67(m,2H),7.48(t,J＝7.4Hz,1H),2.85(q,J＝7.5Hz,2H),1.46(t,J＝7.6Hz,3H)；ESI-MS m/z:175.2[M+H]+.
2、化合物2的合成
将1.5g 8.6mmol的化合物1加入到90mL乙酸中，再加入705mg 8.6mmol的乙酸钠，加热到60℃，搅拌一会，直至反应液澄清。取0.5mL的8.6mmol的溴素溶解到10mL乙酸中，用恒压滴液漏斗滴入到反应液中，反应液由棕色变为白色浑浊，反应10个小时左右，将反应液倒入水中，析出大量白色固体，抽滤，大量水洗。红外灯下烘干，得1.93g白色固体2。
Figure PCTCN2015096038-appb-000011
产率88.7％；1H NMR(400MHz,DMSO)δ12.49(s，1H),8.12(d，J＝8.0Hz,1H)，7.86(t，J＝7.8Hz,1H)，7.70(t，J＝8Hz,1H)，7.56(d,J＝8.0Hz,1H)，5.10(m，1H)，2.01(d，J＝8.0Hz,3H)；ESI-MS m/z:255.0[M+H]+，253.0[M-H]-.
3、化合物3的合成
将14.87g 58.7mmol的化合物2加入到450mL乙醇中，再加入20.2mL 0.21mol 的苯肼，加热回流，反应过夜，第二天发现有大量淡黄色固体悬浊。停止反应，冷却静置，过滤，用少量乙醇洗涤，再用乙醇重结晶，烘干得到12.4g淡黄色棉状轻薄的固体3。
Figure PCTCN2015096038-appb-000012
产率76％；1H NMR(400MHz,DMSO)δ11.50(s,1H),9.88(s,1H),8.13(d,J＝7.9Hz,1H),7.81(t,J＝7.6Hz,1H),7.68(d,J＝8.1Hz,1H),7.60(d,J＝7.8Hz,2H),7.49(t,J＝7.5Hz,1H),7.28(t,J＝7.9Hz,2H),6.89(t,J＝7.3Hz,1H),2.35(s,3H)；ESI-MS m/z:279.2[M+H]+，277.2[M-H]-.
4、化合物4的合成
500mL的三颈瓶中加入200mL的PPA，油浴加热到180℃，开启机械搅拌。将12.4g 44.5mmol的化合物3加入到PPA中，反应2h，反应液逐渐变成红褐色。停止反应，冷却静置，倒入大量冰水中，用KOH调pH至中性，析出大量黄绿色固体，过滤，红外灯下烘干，得9.5g黄绿色固体4。
Figure PCTCN2015096038-appb-000013
产率82.1％；1H NMR(400MHz,DMSO)δ11.71(s,1H),8.13(d,J＝7.8Hz,1H),7.80(t,J＝7.5Hz,1H),7.70(d,J＝8.0Hz,1H),7.63(d,J＝7.9Hz,1H),7.58(s,1H),7.53(d,J＝8.2Hz,1H),7.45(t,J＝7.5Hz,1H),7.21(t,J＝7.6Hz,1H),7.05(t,J＝7.5Hz,1H)；ESI-MS m/z:262.1[M+H]+，260.1[M-H]-.
5、化合物5的合成
于三颈瓶中加入1.8g 6.89mmol的化合物4和2.1g 27.56mmol的醋酸铵，用混合溶剂DMSO-H2O(DMSO：H2O＝50mL：2.7mL)溶解，N2保护，于150℃ 反应24小时。将反应液倒入大量冰水中，不断搅拌，析出大量固体，将析出的黄棕色固体过滤，红外灯下烘干，DCM过柱纯化，得1.2g淡黄色固体5，即天然产物Bouchardatine。
Figure PCTCN2015096038-appb-000014
产率61％；1H NMR(400MHz,DMSO)δ13.62(s,1H),13.11(s,1H),10.49(s,1H),8.28(d,J＝8.0Hz,1H),8.22(d,J＝7.1Hz,1H),7.92(dd,J＝11.1,4.1Hz,1H),7.86(d,J＝7.8Hz,1H),7.70(d,J＝8.1Hz,1H),7.61(t,J＝6.9Hz,1H),7.43(t,J＝7.1Hz,1H),7.36(t,J＝7.1Hz,1H)；ESI-MS m/z:290.1[M+H]+，288.0[M-H]-.
实施例2：化合物6的合成
于单口瓶中加入1.1g化合物5、2mL POCl3，0.3mL N，N-二甲基苯胺，回流反应6个小时，将反应液倒入冰水中，不断搅拌，析出大量固体，将析出的淡黄色固体过滤，红外灯下烘干，EA:PE＝1：3过柱纯化，得0.67g淡黄色固体6。
Figure PCTCN2015096038-appb-000015
产率57％；1H NMR(400MHz,DMSO)δ10.68(s,1H),8.28(d,J＝6.6Hz,2H),7.94(t,J＝7.6Hz,1H),7.87(d,J＝7.8Hz,1H),7.74(d,J＝7.8Hz,1H),7.66(t,J＝7.6Hz,1H),7.55(s,1H),7.50(t,J＝7.9Hz,1H),7.43(t,J＝7.8Hz,1H)；ESI-MSm/z:308.1[M+H]+，306.0[M-H]-.
实施例3：化合物SYS-B1(Bouchardatine衍生物)的合成
将127mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及0.4mLN， N-二乙基乙二胺，于110℃反应12h，反应液呈金黄色，点板显示反应完全。后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，以氯仿：甲醇：氨水＝15：1：0.5％为洗脱剂，过柱纯化，得浅红色固体。
Figure PCTCN2015096038-appb-000016
产率57％；1H NMR(400MHz,CDCl3)δ11.45(s,1H),10.56(s,1H),8.51(d,J＝5.1Hz,1H),7.77(t,J＝9.8Hz,2H),7.71-7.65(m,1H),7.44(d,J＝7.9Hz,1H),7.41-7.37(m,1H),7.27(dd,J＝5.3,1.9Hz,2H),7.14(s,1H),3.66(d,J＝4.1Hz,2H),2.76(t,J＝5.7Hz,2H),2.63(dd,J＝14.4,7.2Hz,4H),1.08(t,J＝7.1Hz,6H).13C NMR(101MHz,CDCl3)δ191.1,159.3,154.3,149.3,142.9,135.1,133.0,128.3,127.0,126.5,124.9,123.2,123.0,121.4,117.5,114.3,111.6,50.9,46.8,38.3,11.4.HRMS(ESI)m/z:calcd for C23H25N5O,[M+H]+388.2132,found 388.2119.
实施例4：化合物SYS-B2(Bouchardatine衍生物)的合成
将127mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及过量的胺链氨丙基吗啡啉0.3mL。于110℃反应12h，反应液呈浅红色，点板显示反应完全。后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，用氯仿：甲醇：氨水＝20：1：0.5％做洗脱剂过柱纯化得淡黄色固体。
Figure PCTCN2015096038-appb-000017
产率51％；1H NMR(400MHz,CDCl3)δ11.48(s,1H),10.09(s,1H),8.53(d,J＝8.2Hz,1H),8.30(s,1H),7.85(d,J＝8.5Hz,2H),7.76(t,J＝7.1Hz,1H),7.48(dd,J＝13.4,7.1Hz,2H),7.35(d,J＝7.1Hz,1H),7.30(t,J＝8.5Hz,1H),.3.87(t,J＝7.8Hz,4H),3.82(dd,J＝10.3,5.7Hz,2H),2.69(t,J＝7.9Hz,2H),2.62(s,4H),1.97(dd,J＝10.8,5.6Hz,2H).13C NMR(101MHz,CDCl3)δ191.0,159.7,154.5,149.3,142.7,134.8,132.9,128.5,127.1,126.1,124.9,123.3,123.0,121.4,117.6,114.4,111.3,67.0,59.3,54.0,43.1,23.1.HRMS(ESI)m/z:calcd for C24H25N5O2,[M+H]+416.2081,found 416.2080.
实施例5：化合物SYS-B3(Bouchardatine衍生物)的合成
将150mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及过量的胺链氨乙基哌啶0.3mL。于110℃反应11h，反应液呈浅红色，点板显示反应完全。后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，用氯仿：甲醇：氨水＝20：1：0.5％做洗脱剂过柱纯化得浅红色固体。
Figure PCTCN2015096038-appb-000018
产率53％；1H NMR(400MHz,CDCl3)δ11.49(s,1H),10.16(s,1H),8.53(d,J＝8.4Hz,1H),7.86(d,J＝9.5Hz,1H),7.80-7.71(m,2H),7.51(d,J＝8.2Hz,1H),7.49-7.44(m,1H),7.35(d,J＝5.7Hz,1H),7.32-7.28(m,1H),7.05(s,1H),3.75(dd,J＝10.5,5.6Hz,2H),2.73(t,J＝5.9Hz,2H),2.52(s,4H),1.72-1.62(m,4H),1.53(d,J＝4.8Hz,2H).13C NMR(101MHz,CDCl3)δ191.0,159.4,154.4,149.4,142.7,134.9,132.9,128.4,127.1,126.4,124.9,123.3,123.0,121.2,117.6,114.3,111.4,56.3,54.1,37.9,26.1,24.3.HRMS(ESI)m/z:calcd for C24H25N5O,[M+H]+400.2132,found 400.2131.
实施例6：化合物SYS-B4(Bouchardatine衍生物)的合成
将150mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及过量的215mg对硝基苯胺。于110℃反应11h，反应液呈浅红色，点板显示反应完全。后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，用氯仿：甲醇：氨水＝35：1：0.5％做洗脱剂过柱纯化得红棕色固体。
Figure PCTCN2015096038-appb-000019
产率50％；1H NMR(400MHz,DMSO)δ10.48(s,1H),8.27(d,J＝7.9Hz,1H),8.21(d, J＝7.8Hz,1H),7.95(d,J＝2.0Hz,1H),7.93(d,J＝1.8Hz,1H),7.92–7.89(m,1H),7.85(d,J＝7.9Hz,1H),7.69(d,J＝8.1Hz,1H),7.61(t,J＝6.9Hz,1H),7.42(t,J＝7.6Hz,1H),7.35(t,J＝7.5Hz,1H),6.70(s,1H),6.64–6.60(m,1H),6.59(d,J＝3.2Hz,1H).
13C NMR(101MHz,DMSO)δ187.49,161.73,156.78,153.88,145.29,135.82,134.85,134.63,128.83,127.58,127.35,126.79,126.34,126.07,125.34,125.03,123.21,122.43,122.23,121.74,120.11,113.23,112.34.ESI-MS m/z:410.2[M+H]+，408.1[M-H]-.
实施例7：化合物SYS-B5(Bouchardatine衍生物)的合成
将117mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及过量的胺链氨丙基哌啶0.3mL。于110℃反应11h，反应液呈浅红色，点板显示反应完全，后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，用氯仿：甲醇：氨水＝15：1：0.5％做洗脱剂过柱纯化得浅红色固体。
Figure PCTCN2015096038-appb-000020
产率58％；1H NMR(400MHz,DMSO)δ9.08(s,1H),8.18(d,J＝7.4Hz,1H),8.07(d,J＝7.7Hz,1H),7.84(dd,J＝14.4,7.5Hz,2H),7.70(d,J＝7.8Hz,1H),7.53(t,J＝8.2Hz,1H),7.37(t,J＝8.0Hz,1H),7.25(t,J＝7.1Hz,1H),3.86(s,2H),2.43(s,4H),2.03(s,2H),1.50(s,4H),1.37(s,2H),1.21(s,2H).13C NMR(101MHz,DMSO)δ188.17,162.2,156.4,135.9,134.3,133.9,128.7,127.2,126.2,126.0,124.9,122.8,121.9,121.5,120.4,119.0,113.2,56.8,56.1,53.8,27.2,25.3,23.9.HRMS(ESI)m/z:calcd for C25H27N5O,[M+H]+414.2288,found 414.2274.
实施例8：化合物SYS-B6(Bouchardatine衍生物)的合成
将117mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及101mg 对甲氧基苯胺。反应13h，点板显示反应完全，后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，用氯仿：甲醇：氨水＝35：1：0.5％做洗脱剂过柱纯化得深棕色固体。
Figure PCTCN2015096038-appb-000021
产率54％；1H NMR(400MHz,DMSO)δ12.72(s,1H),10.48(s,1H),9.45(s,1H),8.33(d,J＝8.1Hz,1H),8.20(d,J＝6.8Hz,1H),7.91(d,J＝8.9Hz,2H),7.85(d,J＝7.9Hz,2H),7.70(t,J＝8.2Hz,1H),7.55(t,J＝7.3Hz,1H),7.39(t,J＝7.9Hz,1H),7.29(t,J＝7.4Hz,1H),7.10(d,J＝8.9Hz,2H),3.84(s,3H).13C NMR(101MHz,DMSO)δ187.4,162.1,158.7,150.2,149.1,146.6,140.3,135.9,134.8,134.5,131.2,129.0,127.6,127.2,126.4,125.9,125.1,123.2,122.8,121.8,121.6,120.1,119.8,114.6,113.2,55.4.HRMS(ESI)m/z:calcd for C24H18N4O2,2[M+H]+395.1503,found 395.1491.
实施例9：化合物SYS-B7(Bouchardatine衍生物)的合成
将117mg化合物6加入到15mL的甲苯中，吸取0.1mL三乙胺，以及94.45mg胺链对氟苯胺。反应11h，点板显示反应完全，后处理：将反应液于75℃水浴锅中旋干溶剂和少量三乙胺，再用CHCl3、饱和食盐水萃取产物及分离少量杂质。干燥有机相，旋干溶剂，用氯仿：甲醇：氨水＝35：1：0.5％做洗脱剂过柱纯化得深黄色固体。
Figure PCTCN2015096038-appb-000022
产率57％；1H NMR(400MHz,DMSO):δ12.80(s,1H),9.48(s,1H),8.34(d,J＝7.8H z,1H),8.20(d,J＝7.6Hz,1H),7.98-7.90(m,2H),7.87(dd,J＝13.9,7.4Hz,2H),7.71(d,J＝8.1Hz,1H),7.56(t,J＝7.2Hz,1H),7.39(dd,J＝15.2,7.1Hz,3H),7.30(t,J＝7.3Hz,1H).13C NMR(101MHz,DMSO):δ187.4,162.0,159.9,152.7,149.0,146.3,143.8,135.9,134.5,131.6,129.0,127.3,126.5,125.9,125.2,123.4,123.3,121.8,119.7,116.2,116.0,113.0,112.9；HRMS(ESI)m/z:calcd for C23H15FN4O,[M+H]+383.1303,found 383.1287.
实施例10化合物SYS-B1～SYS-B7(Bouchardatine衍生物)的应用
1、应用小鼠前脂肪细胞3T3-L1细胞分化模型，采用油红O染色并结合显微镜拍照，评价Bouchardatine和Bouchardatine衍生物对脂肪细胞内甘油三酯含量影响。
2、实验步骤
(1)对数生长期的3T3-L1前脂肪细胞，5.0*104细胞/孔，均匀接种至48孔板，细胞培养箱静置培养，每两天更换一次培养液。待细胞生长接近至80％融合，更换培养液，继续培养2天至细胞完全融合(Day0),更换含有分化诱导液Ⅰ的DMEM完全培养液(含10％FBS及1％双抗的DMEM培养液)，37℃5％CO2静置培养3天(Day3)。3天后，更换含有分化诱导液Ⅱ的DMEM完全培养液继续培养3天(Day6)。对于药物干预组，以含分化诱导液的DMEM完全培养液为稀释液，稀释药物溶液至一定浓度，在Day0与Day3时，一同加入。空白对照组与分化对照组，分别加入等体积的DMSO溶液。在Day6时，进行油红O染色拍照以及甘油三酯含量分析。
(2)分化诱导液配制
分化诱导液Ⅰ：含500μM3-异丁基-1-甲基-黄嘌呤，100ng/mL地塞米松， 2μg/mL胰岛素的DMEM完全培养液分化诱导液Ⅱ：含2μg/mL胰岛素的DMEM完全培养液
(3)油红O染色
细胞诱导分化至Day6时，细胞经预冷PBS润洗1次，4％冰冻多聚甲醛固定液室温固定60min。0.3％油红O染色工作液室温染色30min。室温的去离子水漂洗2-3次，倒置显微镜拍照(40×)。
每孔分别加入300uL异丙醇溶液，摇床平缓摇动室温萃取油红O染料30mins，分别移取100μL染液进行510nm吸光度检测。
(4)甘油三酯含量分析
细胞分化结束后，预冷PBS润洗2次，去尽PBS，加入含0.2％Triton X-100的去离子溶液，室温静置1h，收集细胞悬液，超声破碎10min，使细胞充分裂解，离心收集上清液，按照甘油三酯检测试剂盒说明书测定甘油三酯含量。
(5)结果分析
甘油三酯含量分析以分化对照组为“100％对照”表示，即化合物甘油三酯含量/分化对照组含量*100％。实验结果为三次独立实验的平均值，结果按照“平均值±标准差”进行统计学分析。
(6)实验结果
如图1所示，DM组代表分化对照组，代表正常分化的细胞，有大量的脂质累积。Bouchardatine为天然产物，化合物SYS-B1等均为Bouchardatine衍生物。与DM相比，1μM的SYS-B1、SYS-B2、SYS-B3及SYS-B5均能有效的抑制脂肪生成(*，p<0.05；**，p<0.01)；其中以化合物SYS-B1与SYS-B3的抑制效果最明显。
如图2显示，Bouchardatine及SYS-B3均能够以浓度梯度依赖性的方式减少3T3-L1细胞内脂质含量(*，p<0.05；**，p<0.01；***，p<0.001)，而化合物SYS-B3活性更强。由图可知，经过5μM的Bouchardatine及SYS-B3处理过的细胞，甘油三酯含量分别减少了18％、65％；我们通过计算，得到Bouchardatine及SYS-B3的EC50值分别为22.59μM、1.1μM，化合物SYS-B3降脂活性提高了约23倍。
图3中，BM代表基础对照组，代表未分化的细胞，细胞内无脂质累积；DM代表分化对照组，细胞内清晰可见大量的脂质；而化合物处理组细胞内的脂质含量随着化合物浓度的升高而逐渐减少。如化合物SYS-B3在0.1、0.5、1、5、10μM 浓度下，甘油三酯含量分别为对照组的73％、56％、47％、28％、15％。
实施例11化合物SYS-B1～SYS-B7(Bouchardatine衍生物)的应用
1、应用人肝癌细胞株HpeG-2，采用甘油三酯含量分析试剂盒以及BCA蛋白浓度测定试剂盒，体外评价Bouchardatine和Bouchardatine衍生物对油酸钠诱导的人肝癌细胞株HpeG-2细胞甘油三酯含量的影响。
2、实验步骤：
(1)该实验设置空白对照组(不加油酸钠)、油酸钠诱导组(含油酸钠)，以及不同浓度的药物干预组。具体操作如下：
将人肝癌细胞HepG-2均匀接种至48孔板，静置培养24h。更换含有0.5mM油酸钠的DMEM完全培养液(含有10％FBS及1％双抗的DMEM培养液)，油酸钠终浓度为1mM。37℃5％CO2环境下共孵育24h。此实验中，化合物干预组是化合物经含有油酸钠的DMEM完全培养液稀释至所需浓度。而空白对照组与油酸钠对照组分别加入等量的DMSO。
(2)油红O染色及吸光度分析
细胞经预冷PBS(PH7.4)润洗2次，4％冰冻多聚甲醛固定液室温固定60min。0.3％油红O染色液室温染色30mins。去离子水漂洗2-3次，倒置显微镜拍照(40×)
(3)甘油三酯含量与蛋白含量分析
经过处理的细胞，PBS润洗2次，去尽PBS，加入含0.2％Triton X-100的去离子溶液，室温静置1h，反复吹打使细胞充分裂解，收集细胞悬液至离心管，超声破碎10min，离心收集上清液，分别按照甘油三酯检测试剂盒说明书与BCA蛋白定量检测试剂盒测定甘油三酯含量与蛋白浓度。最后甘油三酯含量以“nmol/mg蛋白”形式表示。为了便于与对照组比较，以对照组为“100％”，分别换算(处理组甘油三酯含量(nmol/mg蛋白)/对照组甘油三酯含量((nmol/mg蛋白)*100％)。实验结果为三次独立实验的平均值，结果按照“平均值±标准差”进行统计学分析。
(4)实验结果
由图4中可知，与空白对照Blank组相比，油酸钠诱导组细胞内甘油三酯含量显著升高(#，p<0.01)。而化合物干预则可有效的减少细胞内甘油三酯含量，如1μM时，化合物SYS-B1、SYS-B2、SYS-B3组甘油三酯含量分别减少了43％、 28％、54％，以化合物SYS-B3效果最好。
图5显示，Blank组代表未经油酸钠处理的，细胞内无明显的脂质积累；Control组代表油酸钠诱导处理的，细胞内清晰可见大量的脂质积累。而Bouchardatine在10μM浓度时细胞内脂质积累无明显变化，而Bouchardatine衍生物1μM时细胞内脂质积累均明显减少，其中以化合物SYS-B1效果最好，该实验再次说明，经过结构改造，Bouchardatine衍生物的降脂活性得到了明显提高。

Claims (10)

  1. 一种Bouchardatine衍生物，其特征在于，所述Bouchardatine衍生物为天然产物Bouchardatine的衍生物，其结构式如式(I)所示：
    Figure PCTCN2015096038-appb-100001
    其中：R为-(CH2)nR1，n＝0、1、2或3；
    所述R1独立地选自：
    (1)被1个或多个R2取代的取代苯基；
    (2)吗啡啉基或哌啶基；
    (3)N,N-二甲基氨基或N,N-二乙基氨基；
    R2独立地选自：
    (1)氟基；
    (2)甲氧基；
    (3)硝基。
  2. 根据权利要求1所述Bouchardatine衍生物，其特征在于，所述R1为N,N-二甲基、N,N-二乙基、吗啡啉基、哌啶基、对硝基苯基或对甲氧基苯基。
  3. 一种权利要求1所述天然产物Bouchardatine的制备方法，其特征在于，合成步骤如下：
    S1.将邻氨基苯甲酰胺与原丙酸三乙酯反应得化合物1
    Figure PCTCN2015096038-appb-100002
    S2.将步骤S1所得化合物1溴化后得到化合物2
    Figure PCTCN2015096038-appb-100003
    S3.将步骤S2所得化合物2与苯肼经取代、消除反应后先生成亚胺，然后再与一分子苯肼反应生成化合物3
    Figure PCTCN2015096038-appb-100004
    S4.将步骤S3所得化合物3经PPA催化的Fischer反应得到含有吲哚环结构特征的化合物4
    Figure PCTCN2015096038-appb-100005
    S5.将步骤S4所得化合物4与DMSO-H2O及乙酸铵反应，并在吲哚的3-位引入甲酰基得到化合物5，即Bouchardatine。
  4. 一种权利要求1所述Bouchardatine衍生物的制备方法，其特征在于，合成步骤如下：
    S6.将权利要求3制备所得Bouchardatine中喹唑啉酮的羰基进行氯代，得到化合物6
    Figure PCTCN2015096038-appb-100006
    S7.将步骤S6所得化合物6与不同的胺类化合物反应，得到Bouchardatine衍生物。
  5. 根据权利要求3所述的制备方法，其特征在于，步骤S3中所述化合物2与苯肼的摩尔比为1：3～5。
  6. 根据权利要求3所述的制备方法，其特征在于，步骤S5中所述反应在氮气保护下进行。
  7. 根据权利要求3所述的制备方法，其特征在于，步骤S5中所述乙酸铵与化合物4的摩尔比为2～6：1，所述DMSO-H2O为DMSO-H2O溶液，溶液中DMSO与H2O的体积比为10～20：1。
  8. 权利要求1所述天然产物Bouchardatine或Bouchardatine衍生物在制备抗肥胖症和/或减轻脂肪肝生成的药物中的应用。
  9. 根据权利要求8所述应用，所述药物还包括其药学上可接受的盐或载体。
  10. 根据权利要求8所述应用，其特征在于，所述药物的剂型为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂。
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