WO2016086825A1 - 一种胺基取代Bouchardatine衍生物及其制备方法和应用 - Google Patents

一种胺基取代Bouchardatine衍生物及其制备方法和应用 Download PDF

Info

Publication number
WO2016086825A1
WO2016086825A1 PCT/CN2015/096044 CN2015096044W WO2016086825A1 WO 2016086825 A1 WO2016086825 A1 WO 2016086825A1 CN 2015096044 W CN2015096044 W CN 2015096044W WO 2016086825 A1 WO2016086825 A1 WO 2016086825A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
group
compound
mmol
amine
Prior art date
Application number
PCT/CN2015/096044
Other languages
English (en)
French (fr)
Inventor
黄志纾
叶冀明
刘宏
饶勇
陈迎春
高琳
余宏
古练权
Original Assignee
中山大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中山大学 filed Critical 中山大学
Publication of WO2016086825A1 publication Critical patent/WO2016086825A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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

Definitions

  • the invention belongs to the field of medical technology. More specifically, it relates to an amine-substituted Bouchardatine derivative and a process for its preparation and use.
  • Obesity a new type of disease that is closely related to a variety of metabolic types of diseases.
  • the occurrence of obesity is due to the imbalance of energy metabolism in the body and mediates the metabolic disorder of the body. When the energy consumed by the body exceeds the energy consumed by the body, the excess energy is stored in the body in the form of fat to promote the occurrence of obesity.
  • BMI Body Mass Index
  • the World Health Organization's determination of obesity is based on the Body Mass Index (BMI), which is defined as obesity when BMI ⁇ 30 kg/m 2 .
  • BMI Body Mass Index
  • the main clinical drugs are central nervous drugs such as laukacillin hydrochloride, norepinephrine reuptake inhibitors such as phentermine hydrochloride/phentermine hydrochloride-topiramate compound capsules.
  • central nervous drugs such as laukacillin hydrochloride, norepinephrine reuptake inhibitors such as phentermine hydrochloride/phentermine hydrochloride-topiramate compound capsules.
  • fat is mainly due to excessive metabolism of fat caused by metabolic disorders in the body, and inhibits the oxidative decomposition ability of fat mobilization.
  • the formation of fat is mainly the result of two factors: 1) balance of glycolipid metabolism, enhanced anabolism, and reduced catabolism; 2) differentiation regulator induces pluripotent/monopotent stem cells to differentiate into adipocytes, such as epidermal Fibroblasts; therefore, inhibiting adipocyte differentiation or promoting glycolipid catabolism, thereby reducing lipid accumulation, is the main basis for the development of anti-obesity drugs This strategy.
  • the natural product Bouchardatine which is a type of ⁇ -quinazolinone alkaloid, is isolated from the plant Bouchardatia neurococca and has a structure in which the 2-position of the quinazolinone is linked to the 2-position of the quinone.
  • Bouchardarine has a wide range of physiological activities, such as anti-cancer, anti-virus, anti-tuberculosis and so on. However, its application in anti-obesity and reduction of fatty liver has not been seen.
  • the technical problem to be solved by the present invention is to overcome the deficiency of the existing obesity and fatty liver treatment drugs, and provide a derivative of the natural product Bouchardatine.
  • the inventors have found that Bouchardarine can inhibit fatty acid synthesis such as 10 ⁇ M concentration at a certain concentration. The lipid content is reduced by about 23%, and it has a certain activity of inhibiting adipocyte differentiation and lipid accumulation. It is a potential anti-obesity drug lead structure. Therefore, the inventors have carried out modification and optimization studies on the basis of Bouchardadine structure, and based on the Bouchardatine base structure, the 3-position modification of the oxime structure is expected to synthesize a lead compound with better anti-obesity effect and less toxicity.
  • the specific scheme is mainly to react the aldehyde group at the 8 position with an amine or an aromatic amine characterized by a chain structure to obtain a Schiff base.
  • the purpose is to improve the interaction ability of the target compound with the target and the chemical intervention effect on the relevant pathway, and improve
  • the compound has the properties of water solubility and bioavailability.
  • Another object of the present invention is to provide a process for the preparation of the amine-substituted Bouchardatine derivative.
  • a further object of the invention is to provide the use of said amine-substituted Bouchardatine derivatives.
  • the present invention provides an amine-substituted Bouchardatine derivative having the structural formula shown in formula (I):
  • R 1 is independently selected from: (1) a substituted phenyl group substituted by one or more R 2 ; (2) a five- or six-membered nitrogen-containing or nitrogen-containing oxyheterocyclic group; (3) one or more a substituted amino group substituted with R 3 ; (4) a C 1-6 fatty alkyl group or a C 3-6 cycloalkyl group; (5) a hydroxyl group; (6) a hydrogen;
  • R 2 is independently selected from: (1) a substituted amino group substituted by one or more R 4 ; (2) a halogen group; (3) a C 1-3 fatty alkyl group; (4) hydrogen;
  • R 3 is a C 1-4 fatty alkyl group
  • R 4 is a C 1-3 linear alkyl group.
  • the R 1 is independently selected from: (1) a substituted phenyl group substituted by one or more R 2 ; (2) a five- or six-membered nitrogen-containing compound Or a nitrogen-containing heterocyclic group; (3) a substituted amino group substituted by one or more R 3 ; (4) a C 1-6 fatty alkyl group or a C 3-6 cycloalkyl group; (5) a hydroxyl group; )hydrogen;
  • R 2 is independently selected from: (1) a substituted amino group substituted by one or more R 4 ; (2) a chloro group; (3) a C 1-3 fatty alkyl group; (4) a hydrogen;
  • R 3 is a C 1-4 fatty alkyl group
  • R 4 is a C 1-3 linear alkyl group.
  • R 1 is independently selected from: (1) a substituted phenyl group substituted by one R 2 ; (2) morphinolyl group, pyrrolyl group, methyl group Piperazinyl or piperidinyl; (3) N,N-dimethylamino or N,N-diethylamino; (4) C 1-4 fatty alkyl or cyclopentyl; (5) hydroxyl; 6) hydrogen;
  • R 2 is independently selected from: (1) N,N-dimethylamino or N,N-diethylamino; (2) chloro; (3) C 1-3 fatty alkyl; (4) hydrogen.
  • the amine-substituted Bouchardadine derivative is the compound SYS-S1 to SYS-S22 prepared in the examples, and the structural formula is as shown in the examples.
  • the present invention provides a synthesis process of the above amine-substituted Bouchardatine derivative as follows:
  • the preparation method of the amine-substituted Bouchardadine derivative includes the following steps:
  • 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, Bouchardarine;
  • 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.
  • An anti-obesity drug comprising the above-mentioned Bouchardadine derivative is also within the scope of the present invention.
  • the medicament has the effects of reducing fat accumulation, reducing obesity, relieving fatty liver, and the like, and can be used for treating obesity and obesity-induced diabetes and heart. Vascular diseases and other related diseases.
  • the medicament further comprises a pharmaceutically acceptable salt or carrier thereof.
  • the drug is an injection, a tablet, a pill, a capsule, a suspension or an emulsion.
  • the drug can be administered orally, transdermally, intravenously or intramuscularly.
  • the present inventors have experimentally studied the following results of the above-mentioned amine-substituted Bouchardadine derivatives for inhibiting lipogenesis and accumulation and reducing the ability of fatty liver production:
  • the results of cell experiments showed that the amine-substituted Bouchardadine provided by the present invention can not only significantly reduce the triglyceride content in the fat cells of 3T3-L1 mice, in particular, by comparing the lipid-lowering activity of Bouchardadine natural products with SYS-S2.
  • the lipid-lowering activity of SYS-S2 was increased by about 11 times.
  • Sexual fatty acid intake promotes endogenous fat mobilization to reduce lipid and cholesterol accumulation, and achieves the effects of treating obesity and reducing fatty liver production.
  • the compound SYS-S2 can significantly down-regulate the expression of adipose differentiation-related regulatory factors (such as PPAR ⁇ , C/EBP ⁇ , SREBP-1c) and the key regulator of lipogenesis (ACC, FAS, SCD-1).
  • adipose differentiation-related regulatory factors such as PPAR ⁇ , C/EBP ⁇ , SREBP-1c
  • ACC adipose differentiation-related regulatory factors
  • the IC 50 of the acute toxicity of the compound SYS-S2 was greater than 100 mg/kg, which was much higher than the effective dose window.
  • the compound SYS-S2 can effectively reduce the weight of white adipose tissue without affecting the feeding condition of mice, and can significantly reduce the content of triglyceride, free fatty acid and low density lipoprotein in plasma.
  • the results of the horizontal study are consistent.
  • the compound SYS-S2 has stable pharmaceutical properties, good membrane permeability and high bioavailability, indicating that SYS-S2 has good drug-forming properties.
  • the amine-substituted Bouchardadine derivative provided by the invention can significantly block the differentiation of the pre-adipocyte 3T3-L1 into a lipid process, reduce the lipid content in the 3T3-L1 adipocyte and the cholesterol content in the HepG-2 cell, and can reduce the free fatty acid.
  • the induced lipid accumulation in human hepatoma cells has better anti-obesity and reduced fatty liver production ability than natural Bouchardatine; and it has high safety and good drug-forming properties, and has the prospect of further development into a new anti-obesity drug.
  • Figure 1 Effect of Bouchardadine derivatives provided by the present invention on fat accumulation of 3T3-L1 adipocytes (all compound concentrations are 1 ⁇ M)
  • FIG. 1 Bouchardatine natural product and derivative SYS-S2 reduce the lipid accumulation of 3T3-L1 cells in a concentration-dependent manner.
  • Figure 3 Comparison of cell morphology of Bouchardadine derivatives provided by the present invention to 3T3-L1 cell differentiation and basal control group BM, DMSO differentiation control group (the cell morphology is 3D3-L1 cell differentiation day 6 and microscope 40 times Under vision
  • Figure 4 Effect of the Bouchardadine derivative provided by the present invention on the cholesterol content of human hepatoma cells HepG-2 (the compound concentration was 1 ⁇ M).
  • Figure 5 Effect of compound concentration of 1 ⁇ M on lipid accumulation induced by sodium oleate in human hepatoma cells HepG-2 (all compound concentrations were 10 ⁇ M).
  • the mouse pre-adipocyte 3T3-L1 cell differentiation model was applied and stained with oil red O and photographed in conjunction with a microscope to evaluate the effect of the Bouchardadine derivative provided by the present invention on the triglyceride content in the adipocytes.
  • 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 Day 0 and Day 3.
  • 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.
  • 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-S2 and the like are all Bouchardatine derivatives.
  • 1 ⁇ M SYS-B3, SYS-S22, SYS-S20 and SYS-S7 can effectively inhibit lipogenesis (*, p ⁇ 0.05; **, p ⁇ 0.01); among them, compound SYS-S2 The inhibition effect is most obvious.
  • Figure 2 shows that both Bouchardatine and SYS-S2 can reduce lipid content in 3T3-L1 cells in a concentration-dependent manner (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001), The compound SYS-S2 is more active.
  • the triglyceride content was reduced by 23% and 68%, respectively.
  • the EC 50 values of Bouchardadine and SYS-S2 were 22.59 ⁇ M, 2.21, respectively.
  • the lipid-lowering activity of the compound SYS-S2 was increased by about 11 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.
  • the compound SYS-S2 had a concentration of 0.1, 0.5, 1, 5, and 10 ⁇ M, and the triglyceride content was 73%, 56%, 47%, 62%, and 80% of the control group, respectively.
  • the human hepatoma cell line HpeG-2 was used to evaluate the effect of the Bouchardadine derivative provided by the present invention on the cholesterol content of human hepatoma cell line HpeG-2 cells by using a cholesterol analysis kit and a BCA protein concentration assay kit.
  • the human liver cancer cell HepG-2 was uniformly inoculated into a 48-well plate and allowed to stand for 24 hours.
  • the DMEM complete medium containing the compound (DMEM medium containing 10% FBS and 1% double antibody) was replaced and incubated for 24 h at 37 ° C in 5% CO 2 .
  • the control group was added with an equal amount of DMSO.
  • the treated cells were washed twice with PBS, PBS was added, RIPA lysate was added, and the cells were fully lysed by repeated pipetting.
  • the supernatant was collected by centrifugation, and the cholesterol content was determined according to the cholesterol detection kit instructions and the BCA protein quantitative detection kit. With protein concentration.
  • the final cholesterol content is expressed in the form "nmol/mg protein".
  • the control group was "100%", which was converted (treatment group cholesterol content (nmol/mg protein) / control group cholesterol content ((nmol/mg protein) * 100%).
  • the experimental result was three times.
  • the mean of the independent experiments, the results were statistically analyzed according to "mean ⁇ standard deviation".
  • the human hepatoma cell line HpeG-2 was used, and the Bouchardatine derivative provided by the present invention was used to evaluate the sodium oleate-induced human hepatoma cell line HpeG-2 cell glycerol in vitro using a triglyceride content analysis kit and a BCA protein concentration assay kit. The effect of ester content.
  • the experiment consisted of a blank control group (no sodium oleate), a sodium oleate induction group (sodium oleate), and different concentrations of drug intervention groups.
  • the specific operations are as follows:
  • 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 photograph with 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 compounds in the Bouchardadine and SYS-S2, SYS-S22, and SYS-S1 groups are reduced by 21%, 46%, and 40%, respectively. 35%, with compound SYS-S2, the best lipid-lowering effect.
  • the experimental results again show that after structural modification, the lipid-lowering activity of Bouchardadine derivatives has been significantly improved.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明公开了一种胺基取代Bouchardatine衍生物及其制备和应用。该衍生物如式(I)所示：其中：R为-(CH2)nR1，n＝0、1、2、3或4；R1选自：(1)被1个或多个R2取代的取代苯基；(2)五元或六元含氮或含氮氧杂环基；(3)被1个或多个R3取代的取代氨基；(4)C1-6脂肪烷基或C3-6环烷基；(5)羟基；(6)氢；R2选自：(1)被1个或多个R4取代的取代氨基；(2)卤基；(3)C1-3脂肪烷基；(4)氢；R3为C1-4脂肪烷基；R4为C1-3直链烷基。该衍生物能显著阻断前脂肪细胞3T3-L1的分化成脂，减少HepG-2细胞内胆固醇含量，具有抗肥胖症和减轻脂肪肝生成的作用。

Description

一种胺基取代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，涉及一种胺基取代Bouchardatine衍生物及其制备方法和应用。
背景技术
肥胖症，一类与多种代谢类型疾病的密切相关的新型疾病。肥胖症的发生是由于机体能量代谢失衡而介导机体代谢紊乱的过程。当机体摄入的能量多余机体消耗的能量，富余的能量则以脂肪的形式储存于体内，促进肥胖症的发生。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。随着物质生活水平的提高，肥胖症发生率的增多，肥胖症的发病机制研究以及肥胖症的治疗药物研究，受到科学研究者们广泛重视。目前，全球肥胖人口正处于急剧增加的阶段，其中以青少年为主(18岁以下)。据世界卫生健康组织统计，2011年青少年肥胖人口数目达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，近几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，主要以青少年为主(<18岁)。在全球肥胖症广泛存在的情形下，针对肥胖症的有效治疗药物研发则相对滞后。曾获准用于治疗肥胖症的药物西布曲明和奥利司他均存在严重的副作用，前者已遭退市，后者市场前景堪忧。而目前世界范围内可供选择的抗肥胖症药物很少。临床上采用药物的主要有中枢神经类药物如盐酸劳卡色林，去甲肾上腺素再摄取抑制剂如盐酸芬特明/盐酸芬特明-托吡酯复方胶囊剂等。上述药物临床使用表明，抗肥胖效果有限，药物副作用明显，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。
研究表明，肥胖症的形成主要是由于机体内代谢紊乱导致脂肪过度形成，并抑制脂肪动员氧化分解能力。脂肪的形成主要是两方面因素共同作用的结果：1)糖脂代谢平衡破坏，合成代谢增强，分解代谢减弱；2)分化调控因子诱导多潜能/单能干细胞定向分化形成脂肪细胞，如表皮成纤维细胞；因此，抑制脂肪细胞分化或促进糖脂分解代谢，从而减少脂质累积，是发展抗肥胖症药物的主要基 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，是从植物Bouchardatia neurococca分离得到，其结构为喹唑啉酮的2-位与吲哚的2-位相连构成。现代研究表明，Bouchardatine具有广泛的生理活性，如抗癌、抗病毒、抗结核等。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和脂肪肝治疗药物的不足，提供一种天然产物Bouchardatine的一种衍生物，发明人研究发现，Bouchardatine在一定浓度下能抑制脂肪酸合成如10μM浓度下脂质含量约降低23％，具有一定的抑制脂肪细胞分化和脂质蓄积的活性，是一个具有潜在发展前景的抗肥胖症药物先导结构。因此，发明人在Bouchardatine结构基础上进行了修饰和优化研究，基于Bouchardatine碱结构进行吲哚结构的3位修饰，期望合成得到抗肥胖效果更好、毒性更小的先导化合物。具体方案主要是将8号位的醛基与链状结构特点的胺或者芳香胺反应得到希夫碱，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。
本发明的目的是提供一种胺基取代Bouchardatine衍生物。
本发明另一目的是提供所述胺基取代Bouchardatine衍生物的制备方法。
本发明的再一目的是提供所述胺基取代Bouchardatine衍生物的应用。
本发明上述目的通过以下技术方案实现：
本发明提供了一种胺基取代Bouchardatine衍生物，其结构式如式(I)所示：
Figure PCTCN2015096044-appb-000001
式(I)中：R为-(CH2)nR1，n＝0、1、2、3或4；
R1独立地选自：(1)被1个或多个R2取代的取代苯基；(2)五元或六元含氮或含氮氧杂环基；(3)被1个或多个R3取代的取代氨基；(4)C1-6脂肪烷基或C3-6环烷基；(5)羟基；(6)氢；
R2独立地选自：(1)被1个或多个R4取代的取代氨基；(2)卤基；(3)C1-3脂肪烷基；(4)氢；
R3为C1-4脂肪烷基；
R4为C1-3直链烷基。
优选地，上述胺基取代Bouchardatine衍生物的结构式中，所述R1独立地选自：(1)被1个或多个R2取代的取代苯基；(2)五元或六元含氮或含氮氧杂环基；(3)被1个或多个R3取代的取代氨基；(4)C1-6脂肪烷基或C3-6环烷基；(5)羟基；(6)氢；
R2独立地选自：(1)被1个或多个R4取代的取代氨基；(2)氯基；(3)C1-3脂肪烷基；(4)氢；
R3为C1-4脂肪烷基；
R4为C1-3直链烷基。
更优选地，上述胺基取代Bouchardatine衍生物的结构式中，所述R1独立地选自：(1)被1个R2取代的取代苯基；(2)吗啡啉基、吡咯基、甲基哌嗪基或哌啶基；(3)N,N-二甲基氨基或N,N-二乙基氨基；(4)C1-4脂肪烷基或环戊基；(5)羟基；(6)氢；
R2独立地选自：(1)N,N-二甲基氨基或N,N-二乙基氨基；(2)氯基；(3)C1-3脂肪烷基；(4)氢。
另外，更优选地，所述胺基取代Bouchardatine衍生物为实施例中制备的化合物SYS-S1～SYS-S22，结构式如实施例中所示。
另外本发明提供了一种上述胺基取代Bouchardatine衍生物的合成过程如下：
Figure PCTCN2015096044-appb-000002
具体地，所述胺基取代Bouchardatine衍生物的制备方法包括如下步骤：
S1.将邻氨基苯甲酰胺与原丙酸三乙酯反应得化合物1
Figure PCTCN2015096044-appb-000003
S2.将步骤S1所得化合物1溴化后得到化合物2
Figure PCTCN2015096044-appb-000004
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 PCTCN2015096044-appb-000005
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 PCTCN2015096044-appb-000006
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；
S6.将化合物5与不同的胺反应生成希夫碱，得到所述胺基取代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，也都在本发明的保护范围之内。
一种包含上述Bouchardatine衍生物的抗肥胖药物也在本发明的保护范围之内，该类药物具有减少脂肪积累、减轻肥胖、缓解脂肪肝等功效，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。
进一步地，所述药物还包括其药学上可接受的盐或载体。
更进一步地，所述药物为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂。
另外，该药物的给药途径可为口服，经皮，静脉或肌肉注射。
本发明经过实验研究，上述胺基取代Bouchardatine衍生物的抑制脂肪生成和积累以及减轻脂肪肝生成能力的活性的研究结果如下：
细胞实验结果表明，本发明提供的胺基取代Bouchardatine不仅能够显著性地减少3T3-L1小鼠脂肪细胞内甘油三酯含量，特别地，通过对比Bouchardatine天然产物与SYS-S2降脂活性，发现化合物SYS-S2降脂活性提高了约11倍。并且还能够减少HepG-2细胞内胆固醇含量以及油酸钠诱导的人肝癌细胞株HepG-2细胞内甘油三酯累积，例如抑制人肝癌细胞株HepG-2细胞内甘油三酯累积的EC50＝18.7±0.74μM，这显示了本发明所提供的Bouchardatine衍生物的抗肥胖症与减轻脂肪肝生成作用可能是通过两种途径：1)抑制细胞内脂肪生成而减少脂肪积累，2)抑制外源性脂肪酸摄取而促进内源性脂肪动员来减少脂质与胆固 醇累积，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-S2降脂机制研究发现，化合物SYS-S2可显著性的下调脂肪分化相关调控因子的表达(如PPARγ,C/EBPγ,SREBP-1c)以及脂肪生成的关键调节酶(ACC,FAS，SCD-1)。其次，我们还发现化合物SYS-S2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。
动物实验结果表明，化合物SYS-S2急毒毒性的IC50大于100mg/kg，远远高于有效剂量窗口。且化合物SYS-S2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，游离脂肪酸及低密度脂蛋白含量，这一实验结果与细胞水平的研究结果相符合。其次，化合物SYS-S2药学性质稳定，膜通透性好，生物利用率较高，表明SYS-S2具有良好的成药性。
综上所述，药理学和动物实验证明，本发明所涉及的胺基取代Bouchardatine衍生物，比天然的Bouchardatine具有更好的抗肥胖症和减轻脂肪肝生成能力，成药性也得到显著改善，具有进一步开发成为抗肥胖症新药的前景。
本发明具有以下有益效果：
本发明提供的胺基取代Bouchardatine衍生物能够显著阻断前脂肪细胞3T3-L1的分化成脂过程，减少3T3-L1脂肪细胞内的脂质及HepG-2细胞内胆固醇含量，并且能够减少游离脂肪酸诱导的人肝癌细胞内脂质的积累，比天然的Bouchardatine具有更好的抗肥胖症和减轻脂肪肝生成能力；并且安全性高，成药性好，具有进一步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：Bouchardatine天然产物与衍生物SYS-S2以浓度梯度依赖性的方式减少3T3-L1细胞的脂质累积对照图。
图3：本发明提供的Bouchardatine衍生物对3T3-L1细胞分化与基础对照组BM，DMSO分化对照组的细胞形态对比图(所述细胞形态均为3T3-L1细胞分化第6天，显微镜40倍视野下)
图4：本发明提供的Bouchardatine衍生物对人肝癌细胞HepG-2胆固醇含量的影响(化合物浓度均为1μM)。
图5：化合物浓度均为1μM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10μM)。
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，本发明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，点板显示反应完全，有两个点。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 PCTCN2015096044-appb-000007
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 PCTCN2015096044-appb-000008
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，加热到86℃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 PCTCN2015096044-appb-000009
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 PCTCN2015096044-appb-000010
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 PCTCN2015096044-appb-000011
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SYS-S1(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及氨乙基吗啡啉(0.08mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中 不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得淡黄色固体。
产率49％；1H NMR(400MHz,DMSO)δ12.62(s,1H),9.12(s,1H),8.18(d,J＝7.8Hz,1H),8.08(d,J＝7.8Hz,1H),7.85(dd,J＝14.0,7.5Hz,2H),7.70(d,J＝8.1Hz,1H),7.53(t,J＝7.1Hz,1H),7.38(t,J＝7.1Hz,1H),7.27(t,J＝7.4Hz,1H),3.98(s,2H),3.57(s,4H),2.88(s,2H),1.23(s,4H).13C NMR(101MHz,CDCl3)δ163.0,155.6,149.2,146.4,135.0,134.2,131.4,129.0,127.2,126.6,126.4,125.6,122.6,122.0,118.8,112.8,112.2,67.0,58.8,53.8,29.7.HRMS(ESI)m/z:calcd for C23H23N5O2,[M+H]+402.1925,found 402.1912.
实施例3  化合物SYS-S2(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及氨乙基吡咯(0.08mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率54％；1H NMR(400MHz,CDCl3)δ8.86(s,1H),8.28(d,J＝7.9Hz,2H),7.79(d,J＝7.9Hz,1H),7.72(s,2H),7.50(d,J＝8.2Hz,1H),7.43(t,J＝4.1Hz,1H),7.36(t,J＝4.2Hz,1H),7.23(t,J＝7.9Hz,1H),4.12(t,J＝6.0Hz,2H),3.31(t,J＝5.7Hz,2H),2.88(s,4H),1.87(s,4H).13C NMR(101MHz,CDCl3)δ163.1,156.0,149.2,146.3,135.0,134.2,131.3,129.0,127.2,126.5,126.3,125.6,122.5,122.0,118.8,112.7,112.1,56.2,54.5,29.7,23.5.HRMS(ESI)m/z:calcd for C23H16N4O,[M+H]+386.1975,found 386.1960.
实施例4  化合物SYS-S3(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及苯胺(0.05mL,0.6mmol)，反应体系颜色逐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝30：1作洗脱剂过中性Al2O3柱纯化得淡黄色固体。
产率53％；1H NMR(400MHz,CDCl3)δ10.39(s,1H),9.20(s,1H),8.35(d,J＝7.9Hz,1H),7.97(d,J＝8.0Hz,1H),7.77(d,J＝4.8Hz,2H),7.72(d,J＝7.7Hz,2H),7.57(d,J＝8.1Hz,1H),7.53(s,1H),7.49(t,J＝87Hz,2H),7.43(dd,J＝13.7,5.8Hz,2H),7.34(dd,J＝13.4,6.4Hz,2H).13C NMR(101MHz,DMSO)δ162.0,153.0,149.0,147.5,146.5,135.9,134.5,131.7,129.4,12 9.0,127.2,126.9,126.5,125.9,125.2,124.0,121.5,119.8,113.1,112.4,104.95.HRMS(ESI)m/z:calcd for C23H16N4O,[M+H]+365.1397,found 365.1386.
实施例5  化合物SYS-S4(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及正丁胺(0.05mL,0.6mmol)，反应体系颜色逐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝25：1作洗脱剂过中性Al2O3柱纯化得淡黄色固体。
产率56％；1H NMR(400MHz,CDCl3)δ8.63(s,1H),8.22(d,J＝7.8Hz,1H),7.71(d,J＝7.9Hz,1H),7.58(s,2H),7.31(d,J＝7.9Hz,3H),7.23(t,J＝7.9Hz,1H),7.14(dd,J＝14.8,7.2Hz,2H),3.72(t,J＝6.8Hz,2H),1.87-1.80(m,2H),1.47-1.38(m,2H),0.92(t,J＝7.3Hz,3H).13C NMR(101MHz,CDCl3)13C NMR(101MHz,CDCl3)δ162.3,153.4,148.3,145.6,134.0,133.0,130.2,127.9,126.0,125.6,125.2,124.4,121.6,120.7,117.9,117.7,111.7,111.0,59.1,31.9,19.5,12.8.HRMS(ESI)m/z:calcd for C21H20N4O,[M+H]+345.1710,found 345.1694.
实施例6  化合物SYS-S5(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及丙胺(0.05mL,0.6mmol)，反应体系颜色逐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝25：1作洗脱剂过中性Al2O3柱纯化得淡棕色固体。
产率62％；1H NMR(400MHz,CDCl3)δ8.82(t,1H),8.33(d,J＝7.9Hz,1H),7.88(d,J＝8.0Hz,1H),7.74(s,2H),7.53(d,J＝7.9Hz,1H),7.44(t,J＝8.2Hz,1H),7.39(t,J＝8.0Hz,1H)7.29(d,J＝7.5Hz,1H),3.83(t,J＝6.7Hz,2H),2.04-1.95(m,2H),1.09(t,J＝7.3Hz,3H).13C NMR(101MHz,CDCl3)δ163.1,154.4,149.2,146.6,135.0,134.1,131.3,129.0,127.1,126.7,126.3,125.5,121.9,118.81,112.8,112.1,100.0,62.2,24.1,11.9.HRMS(ESI)m/z:calcd for C20H18N4O,[M+H]+331.1553,found 331.1540.
实施例7  化合物SYS-S6(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及N,N-二乙基对苯二胺(0.12g,0.6mmol)，反应体系颜色逐渐变深，室温反应6h。后处理：将反应液中不 溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝35：1作洗脱剂过中性Al2O3柱纯化得淡红色固体。
产率64％；1H NMR(400MHz,DMSO)δ12.60(s,1H),9.38(s,1H),8.31(d,J＝8.0Hz,1H),8.20(d,J＝7.8Hz,1H),7.90-7.83(m,4H),7.70(d,J＝8.2Hz,1H),7.55(t,J＝7.3Hz,1H),7.39(t,J＝7.5Hz,1H),7.27(t,J＝7.3Hz,1H),6.80(d,J＝8.9Hz,2H),3.43(dd,J＝13.7,6.7Hz,4H),1.15(t,J＝6.9Hz,6H).13C NMR(101MHz,CDCl3)δ163.1,147.5,146.7,143.9,135.2,133.9,130.5,129.1,127.0,126.5,126.0,125.4,122.7,122.4,121.6,118.9,114.3,112.1,112.0,77.3,77.0,76.7,44.6,12.7.HRMS(ESI)m/z:calcd for C27H25N5O,[M+H]+436.2132,found 436.2127.
实施例8  化合物SYS-S7(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及氨丙基甲基哌嗪(0.08mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝15：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率51％；1H NMR(400MHz,CDCl3)δ8.73(s,1H),8.26(d,J＝7.8Hz,1H),7.79(d,J＝8.0Hz,1H),7.79(d,J＝8.0Hz,1H),7.69(d,J＝3.6Hz,2H),7.48(d,J＝8.0Hz,1H),7.39(dd,J＝7.9,4.1Hz,1H),7.35(t,J＝7.8Hz,1H),7.24(t,J＝6.3Hz,1H),3.85(t,J＝6.3Hz,2H),2.71(d,J＝6.8Hz,4H),2.61(s,4H),2.34(s,3H),2.21-2.12(m,2H),0.92-0.78(m,2H).13C NMR(101MHz,CDCl3)δ163.0,155.1,149.1,146.4,135.1,134.1,131.2,129.0,127.1,126.6,126.3,125.5,122.4,121.9,118.7,112.6,112.2,58.1,55.8,54.36,52.4,45.5,29.7.HRMS(ESI)m/z:calcd for C25H28N6O,[M+H]+429.2397,found 429.2380.
实施例9  化合物SYS-S8(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及氨丙基吡咯(0.12mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率54％；1H NMR(400MHz,CDCl3)δ9.07(s,1H),8.14(d,J＝7.7Hz,1H),8.05(d,J＝8.0Hz,1H),7.80(dd,J＝15.6,7.5Hz,2H),7.66(d,J＝8.1Hz,1H),7.49(t,J＝7.1Hz,1H),7.33(t, J＝7.5Hz,1H),7.22(t,J＝7.4Hz,1H),3.85(t,J＝6.3Hz,2H),2.63(t,J＝7.8Hz,2H),2.47(s,4H),2.00(m,2H),1.66(s,4H).13C NMR(101MHz,CDCl3)δ163.0,154.8,149.1,146.5,135.1,134.0,131.3,129.0,127.1,126.1,126.2,125.4,122.5,121.8,118.7,112.7,112.1,58.2,54.1,54.0,29.7,23.5..HRMS(ESI)m/z:calcd for C24H25N5O,[M+H]+400.2132,found 400.2120.
实施例10  化合物SYS-S9(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及N，N-二乙基-1，4-丁二胺(0.09mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得淡红色固体。
产率48％；1H NMR(400MHz,DMSO)δ9.11(s,1H),817(d,J＝8.2Hz,1H),8.09(d,J＝8.2Hz,1H),7.88–7.80(m,2H),7.69(d,J＝8.2Hz,1H),7.52(t,J＝7.6Hz,1H),7.36(t,J＝7.4Hz,1H),7.25(t,J＝7.4Hz,1H),3.85(t,J＝7.4Hz,,2H),2.51(dt,J＝3.5,1.7Hz,2H),2.43(m,4H),1.88–1.80(m,2H),1.59–1.52(m,2H),0.91(t,J＝7.1Hz,6H).13C NMR(101MHz,CDCl3)δ162.1153.6,148.1,145.5,134.1,133.1,130.3,128.7,128.0,126.11,125.6,125.3,124.5,121.6,120.9,117.8,111.1,59.3,51.6,45.8,27.9,23.8,10.4.HRMS(ESI)m/z:calcd for C25H29N5O,[M+H]+416.2445,found 416.2446.
实施例11  化合物SYS-S10(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及N，N-二甲基-1，4-丁二胺(0.09mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得淡红色固体。
产率54％；1H NMR(400MHz,CDCl3)δ8.76(s,1H),8.29(d,J＝7.8Hz,1H),7.82(d,J＝7.6Hz,1H),7.71(s,2H),7.49(d,J＝7.7Hz,1H),7.43–7.33(m,2H),7.25(t,J＝7.6Hz,1H),3.84(t,J＝4.1Hz,2H),2.44(t,J＝7.8Hz,2H),2.28(s,6H),2.01-1.91(m,2H),1.77-1.66(m,2H).13C NMR(101MHz,CDCl3)δ163.1,154.6,149.1,146.5,135.1,134.0,131.2,128.9,127.1,126.5,126.2,125.4,122.5,121.8,118.7,112.7,112.1,60.1,59.3,45.3,28.7,25.3.HRMS(ESI)m/z:calcd for C23H25N5O,[M+H]+388.2132,found 388.2117.
实施例12  化合物SYS-S11(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及氨乙基甲基哌嗪(0.09mL,0.6mmol)，反应体系颜色瞬间变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝15：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率57％；1H NMR(400MHz,CDCl3)δ8.80(s,1H),8.30(d,J＝7.8Hz,1H),7.83(d,J＝8.0Hz,1H),7.72(s,2H),7.51(d,J＝7.8Hz,1H),7.43(t,J＝4.1Hz,1H),7.37(t,J＝7.8Hz,1H),7.26(t,J＝8.0Hz,1H),3.99(t,J＝6.5Hz,2H),3.00(t,J＝6.6Hz,2H),2.66(s,4H),2.46(s,4H),2.26(s,3H).13C NMR(101MHz,CDCl3)δ163.1,155.5,149.1,146.5,135.0,134.1,131.4,129.0,127.1,126.6,126.3,125.5,122.6,121.9,118.8,112.8,112.1,58.3,57.5,55.1,53.3,46.0.HRMS(ESI)m/z:calcd for C24H26N6O,[M+H]+415.2241,found 415.2225.
实施例13  化合物SYS-S12(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及N,N-二甲基对苯二胺盐酸盐(0.10g,0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝30：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率54％；1H NMR(400MHz,CDCl3)δ8.83(s,1H),8.20(d,J＝7.8Hz,1H),7.71(d,J＝7.9Hz,1H),7.57(d,J＝8.5Hz,4H),7.33-7.28(m,2H),7.19(t,J＝7.6Hz,1H),7.10(t,J＝7.4Hz,1H),6.65(d,J＝8.7Hz,2H),2.90(s,6H).13C NMR(101MHz,CDCl3)δ163.1,150.1,146.6,144.7,135.17,133.9,130.7,129.1,127.0,126.5,126.1,125.5,122.4,121.7,118.9,114.2,112.8,112.1,40.4.HRMS(ESI)m/z:calcd for C25H21N5O,[M+H]+408.1819,found 408.1801.
实施例14  化合物SYS-S13(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及异丁胺(0.06mL,0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝25：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率49％；1H NMR(400MHz,MeOD)δ8.87(s,1H),8.18(d,J＝7.8Hz,1H),7.91(d,J＝7.7Hz,1H),7.78(s,2H),7.58(d,J＝8.1Hz,1H),7.46(t,J＝7.4Hz,1H),7.33(t,J＝7.8Hz,1H),7. 23(t,J＝7.4Hz,1H),3.68(d,J＝6.4Hz,2H),2.26-2.19(m,1H),1.08(d,J＝6.6Hz,6H).13C NMR(101MHz,CDCl3)δ162.0,153.6,148.0,145.6,134.1,133.0,130.2,127.9,126.0,125.5,125.2,124.4,121.5,120.7,117.7,111.6,111.1,67.5,28.6,19.7.HRMS(ESI)m/z:calcd for C21H20N4O,[M+H]+345.1710,found 345.1696.
实施例15  化合物SYS-S14(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及叔丁胺(0.06mL,0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝25：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率54％；1H NMR(400MHz,DMSO)δ12.63(s,1H),8.97(s,1H),8.17(dd,J＝7.0,3.6Hz,2H),7.88-7.81(m,3H),7.70(d,J＝8.0Hz,1H),7.52(t,J＝8.0Hz,2H),7.37(t,J＝7.8Hz,1H),7.26(t,J＝8.2Hz,1H),1.52(s,9H).13C NMR(101MHz,DMSO)δ162.6,150.7,135.8,134.3,127.5,127.1,126.1,125.9,124.9,123.1,122.1,121.4,120.3,119.3,113.2,113.0,112.3,29.3,27.1.HRMS(ESI)m/z:calcd for C21H20N4O,[M+H]+345.1710,found 345.1697.
实施例16  化合物SYS-S15(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及对甲苯胺(0.04g，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝30：1作洗脱剂过中性Al2O3柱纯化得淡黄色固体。
产率49％；1H NMR(500MHz,DMSO)δ12.76(s,1H),9.46(s,1H),8.31(d,J＝8.1Hz,1H),8.19(d,J＝7.9Hz,1H),7.88(t,J＝7.6Hz,1H),7.84(d,J＝7.9Hz,1H),7.79(d,J＝8.2Hz,2H),7.70(d,J＝8.2Hz,1H),7.54(t,J＝7.3Hz,1H),7.38(t,J＝7.6Hz,1H),7.32(d,J＝8.1Hz,2H),7.28(t,J＝7.5Hz,1H),2.37(s,3H).13C NMR(101MHz,DMSO)δ162.0,151.8,149.0,146.6,144.9,136.7,136.0,134.5,131.6,129.9,129.1,127.2,126.5,126.0,125.2,121.8,121.7,121.4,119.8,114.0,113.1,20.7.HRMS(ESI)m/z:calcd for C24H18N4O,[M+H]+379.1553,found379.1537.
实施例17  化合物SYS-S16(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水 MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及间氯苯胺(0.06mL，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝30：1作洗脱剂过中性Al2O3柱纯化得淡黄色固体。
产率57％；1H NMR(400MHz,CDCl3)δ9.08(s,1H),8.31(d,J＝8.4Hz,1H),7.92(d,J＝8.1Hz,1H),7.76-7.71(m,2H),7.61(s,1H),7.59(d,J＝1.9Hz,1H),7.52(d,J＝8.2Hz,1H),7.48-7.43(m,1H),7.42-7.39(m,1H),7.38(d,J＝4.5Hz,1H),7.33-7.28(m,2H).13C NMR(101M Hz,CDCl3)δ162.6,151.6,148.5,145.8,135.2,135.1,134.2,131.6,130.6,129.3,127.4,126.9,126.7,126.6,126.0,122.6,122.3,120.9,120.3,118.7,114.2,112.9,112.4.HRMS(ESI)m/z:calcd for C23H15ClN4O,[M+H]+399.1007,found 399.0990.
实施例18  化合物SYS-S17(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及N，N-二甲基乙二胺(0.06mL，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝15：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率51％；1H NMR(400MHz,CDCl3)δ8.65(s,1H),8.18(d,J＝7.5Hz,1H),7.67-7.54(m,3H),7.33(d,J＝8.3Hz,2H),7.21(d,J＝7.7Hz,1H),7.11(t,J＝8.3Hz,1H),3.86(s,2H),2.90(s,2H),2.32(s,6H).13C NMR(101MHz,CDCl3)δ163.1,155.4,149.1,146.4,135.0,134.0,131.3,128.9,127.1,126.5,126.2,125.4,122.5,121.8,118.7,112.7,112.1,59.7,58.1,45.8.HRMS(ESI)m/z:calcd for C21H21N5O,[M+H]+360.1819,found 360.1803.
实施例19  化合物SYS-S18(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及β-苯乙二胺(0.08mL，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝25：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率50％；1H NMR(400MHz,CDCl3)δ8.61(s,1H),8.35(d,J＝7.8Hz,1H),7.76-7.70(m,3H),7.45(dd,J＝10.0,6.7Hz,2H),7.35(t,,J＝7.8Hz,1H),7.30(t,J＝6.3Hz,3H),7.25(d,J＝5.0Hz,1H),7.21(d,J＝7.5Hz,1H),7.17(t,J＝7.1Hz,1H),4.10(t,J＝7.1Hz,2H),3.32(t,J＝7.2H z,2H).13C NMR(101MHz,CDCl3)δ163.1,155.0,149.2,146.5,144.3,139.5,135.0,134.1,131.3,129.0,128.5,127.1,126.7,126.3,126.3,125.5,122.7,121.9,118.8,112.6,112.0,61.7,37.1.HRMS(ESI)m/z:calcd for C25H20N4O,[M+H]+393.1710,found 393.1692.
实施例20  化合物SYS-S19(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及β-苯乙二胺(0.07mL，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率48％；1H NMR(400MHz,DMSO)δ9.07(s,1H),8.16(d,J＝7.6Hz,1H),8.06(d,J＝7.1Hz,1H),7.82(s,2H),7.68(d,J＝7.2Hz,1H),7.51(t,J＝7.8Hz,1H),7.35(t,J＝4.5Hz,1H),7.23(t,J＝6.5Hz,1H),1.94-1.82(m,4H),1.81–1.65(m,4H),1.46–1.34(m,2H),1.31–1.23(m,1H).13C NMR(101MHz,DMSO)δ162.3,154.1,149.1,147.0,135.8,134.3,131.4,128.6,127.1,126.2,125.9,124.9,122.0,121.4,119.1,113.0,112.2,66.9,33.8,25.1,24.1.HRMS(ESI)m/z:calcd for C23H22N4O,[M+H]+371.1866,found 371.1850.
实施例21  化合物SYS-S20(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及乙醇胺(0.04mL，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝15：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率50％；1H NMR(500MHz,DMSO)δ9.07(s,1H),8.17(d,J＝7.7Hz,1H),8.07(d,J＝7.6Hz,1H),7.89-7.83(m,2H),7.70(d,J＝8.0Hz,1H),7.53(t,J＝7.3Hz,1H),7.37(t,J＝7.3Hz,1H),7.25(t,J＝7.3Hz,1H),3.91(s,4H).13C NMR(126MHz,DMSO)δ162.7,157.4,149.2,147.0,135.8,134.4,131.8,128.6,127.2,126.3,126.0,125.0,121.8,121.6,119.1,113.1,112.1,61.0,60.6.HRMS(ESI)m/z:calcd for C19H16N4O2,[M+H]+333.1346,found 333.1332.
实施例22  化合物SYS-S21(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及乙胺(0.03mL，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再 用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝20：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率63％；1H NMR(400MHz,DMSO)δ9.11(s,1H),8.17(d,J＝7.7Hz,1H),8.08(d,J＝7.8Hz,1H),7.85(m,J＝7.8Hz,2H),7.69(d,J＝8.0Hz,1H),7.52(t,J＝8.0Hz,1H),7.37(t,J＝8.0Hz,1H),7.25(t,,J＝8.0Hz,1H),3.87(q,J＝11.9Hz,2H),1.46(t,J＝7.1Hz,3H).13C NMR(101MHz,CDCl3)δ163.2,153.9,149.2,146.6,135.0,134.1,131.3,129.0.,127.1,126.6,126.2,125.5,122.6,121.8,118.8,112.8,112.1,54.6,16.2.HRMS(ESI)m/z:calcd for C19H16N4O,[M+H]+317.1397,found 317.1385.
实施例23  化合物SYS-S22(胺基取代Bouchardatine衍生物)的合成
将化合物5(0.15g，0.52mmol)加入到10mL的氯仿中，加入无水MgSO4(60mg,0.49mmol)，FeCl3(32mg,0.2mmol)，以及4-甲基戊胺氢溴酸盐(0.11g，0.6mmol)，反应体系颜色渐渐变深，室温反应6h。后处理：将反应液中不溶物过滤掉，再用饱和食盐水洗三次，合并有机相，减压浓缩，再用氯仿：甲醇＝15：1作洗脱剂过中性Al2O3柱纯化得浅棕色固体。
产率58％；1H NMR(400MHz,CDCl3)δ8.79(s,1H),8.33(d,J＝7.8Hz,1H),7.87(d,J＝7.8Hz,1H),7.73(s,1H),7.51(d,J＝7.8Hz,1H),7.43(s,1H),7.40–7.35(m,1H),7.28(d,J＝6.2Hz,1H),3.83(t,J＝6.8Hz,1H),2.00-1.92(m,2H),1.68-1.60(m,1H),1.34(q,J＝11.9Hz,1H),0.93(d,J＝5.6Hz,6H).13C NMR(101MHz,CDCl3)δ163.1,154.3,149.1,146.6,135.0,134.1,131.3,129.0,127.1,126.7,126.3,125.5,122.7,121.8,118.8,112.8,112.1,60.8,36.6,28.7,27.8,22.6.HRMS(ESI)m/z:calcd for C23H24N4O,[M+H]+373.2023,found 373.2008.
上述制备的Bouchardatine衍生物SYS-S1～SYS-S22的结构式如下所示：
Figure PCTCN2015096044-appb-000012
Figure PCTCN2015096044-appb-000013
Figure PCTCN2015096044-appb-000014
Figure PCTCN2015096044-appb-000015
Figure PCTCN2015096044-appb-000016
实施例24  Bouchardatine衍生物SYS-S1～SYS-S22的应用
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，评价本发明提供的Bouchardatine衍生物对脂肪细胞内甘油三酯含量影响。
1、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(Day 0),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(Day 3)。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(Day 6)。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Day 0与Day 3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Day 6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500μM 3-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Day 6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-S2等均为Bouchardatine衍生物。与DM相比，1μM的SYS-B3、SYS-S22、SYS-S20及SYS-S7均能有效的抑制脂肪生成(*，p<0.05；**，p<0.01)；其中以化合物SYS-S2的抑制效果最明显。
图2显示，Bouchardatine及SYS-S2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-S2活性更强。由图可知，经过10μM的Bouchardatine及SYS-S2处理过的细胞，甘油三酯含量分别减少了23％、68％；我们通过计算，得到Bouchardatine及SYS-S2的EC50值分别为22.59μM、2.21μM，化合物SYS-S2降脂活性提高了约11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-S2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％、62％、80％。
实施例25  Bouchardatine衍生物SYS-S1～SYS-S22的应用
应用人肝癌细胞株HpeG-2，采用胆固醇分析试剂盒以及BCA蛋白浓度测定试剂盒，体外评价本发明提供的Bouchardatine衍生物对人肝癌细胞株HpeG-2细胞胆固醇含量的影响。
1、实验步骤：
(1)该实验设置对照组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。更换含有化合物的DMEM完全培养液(含有10％FBS及1％双抗的DMEM培养液)，37℃5％CO2环境下共孵育24h。而对照组加入等量的DMSO。
(2)胆固醇含量与蛋白质含量分析
经过处理的细胞，PBS润洗2次，去尽PBS，加入RIPA裂解液，反复吹打使细胞充分裂解，离心收集上清液，分别按照胆固醇检测试剂盒说明书与BCA蛋白定量检测试剂盒测定胆固醇含量与蛋白浓度。最后胆固醇含量以“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(处理组胆固醇含量(nmol/mg蛋白)/对照组胆固醇含量((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。
(3)实验结果
由图4中可知，与control组相比，化合物干预则可有效的减少细胞内胆固醇含量，如10μM时，化合物Bouchardatine及SYS-S22、SYS-S2、SYS-S1组胆固醇含量分别减少了20％、40％、35％、35％，其中，以化合物SYS-S22效果最好。实验结果表明，Bouchardatine经过结构修饰，降胆固醇活性得到了明显提高。
实施例26  Bouchardatine衍生物SYS-S1～SYS-S22的应用
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，体外评价本发明提供的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。
1、实验步骤：
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5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10μM时，化合物Bouchardatine及SYS-S2、SYS-S22、SYS-S1组甘油三酯含量分别减少了21％、46％、40％、35％，以化合物SYS-S2降脂效果最好。该实验结果再次说明，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. 一种胺基取代Bouchardatine衍生物，其特征在于，其结构式如式(I)所示：
    Figure PCTCN2015096044-appb-100001
    式(I)中：R为-(CH2)nR1，n＝0、1、2、3或4；
    R1独立地选自：(1)被1个或多个R2取代的取代苯基；(2)五元或六元含氮或含氮氧杂环基；(3)被1个或多个R3取代的取代氨基；(4)C1-6脂肪烷基或C3-6环烷基；(5)羟基；(6)氢；
    R2独立地选自：(1)被1个或多个R4取代的取代氨基；(2)卤基；(3)C1-3脂肪烷基；(4)氢；
    R3为C1-4脂肪烷基；
    R4为C1-3直链烷基。
  2. 根据权利要求1所述胺基取代Bouchardatine衍生物，其特征在于，所述R1独立地选自：(1)被1个或多个R2取代的取代苯基；(2)五元或六元含氮或含氮氧杂环基；(3)被1个或多个R3取代的取代氨基；(4)C1-6脂肪烷基或C3-6环烷基；(5)羟基；(6)氢；
    R2独立地选自：(1)被1个或多个R4取代的取代氨基；(2)氯基；(3)C1-3脂肪烷基；(4)氢；
    R3为C1-4脂肪烷基；
    R4为C1-3直链烷基。
  3. 根据权利要求1或2所述胺基取代Bouchardatine衍生物，其特征在于，所述R1独立地选自：(1)被1个R2取代的取代苯基；(2)吗啡啉基、吡咯基、甲基哌嗪基或哌啶基；(3)N,N-二甲基氨基或N,N-二乙基氨基；(4)C1-4脂肪烷基或环戊基；(5)羟基；(6)氢；
    R2独立地选自：(1)N,N-二甲基氨基或N,N-二乙基氨基；(2)氯基；(3) C1-3脂肪烷基；(4)氢。
  4. 据权利要求1～3任一所述胺基取代Bouchardatine衍生物的制备方法，其特征在于，包括如下步骤：
    S1.将邻氨基苯甲酰胺与原丙酸三乙酯反应得化合物1
    Figure PCTCN2015096044-appb-100002
    S2.将步骤S1所得化合物1溴化后得到化合物2
    Figure PCTCN2015096044-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 PCTCN2015096044-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 PCTCN2015096044-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；
    S6.将化合物5与不同的胺反应生成希夫碱，得到所述胺基取代Bouchardatine衍生物。
  5. 根据权利要求4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4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4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所述胺基取代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。
PCT/CN2015/096044 2014-12-04 2015-11-30 一种胺基取代Bouchardatine衍生物及其制备方法和应用 WO2016086825A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410738079.8A CN104447702B (zh) 2014-12-04 2014-12-04 一种胺基取代Bouchardatine衍生物及其制备方法和应用
CN201410738079.8 2014-12-04

Publications (1)

Publication Number Publication Date
WO2016086825A1 true WO2016086825A1 (zh) 2016-06-09

Family

ID=52894526

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/096044 WO2016086825A1 (zh) 2014-12-04 2015-11-30 一种胺基取代Bouchardatine衍生物及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN104447702B (zh)
WO (1) WO2016086825A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104447702B (zh) * 2014-12-04 2016-12-07 中山大学 一种胺基取代Bouchardatine衍生物及其制备方法和应用
CN114044769B (zh) * 2021-11-25 2023-12-12 中山大学 一种β-吲哚喹唑啉酮衍生物及其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104367575A (zh) * 2014-12-04 2015-02-25 中山大学 一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用
CN104447702A (zh) * 2014-12-04 2015-03-25 中山大学 一种胺基取代Bouchardatine衍生物及其制备方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104367575A (zh) * 2014-12-04 2015-02-25 中山大学 一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用
CN104447702A (zh) * 2014-12-04 2015-03-25 中山大学 一种胺基取代Bouchardatine衍生物及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RAO, YONG ET AL.: "synthesis and", JOURNAL OF MEDICINAL CHEMISTRY, vol. 58, no. 23, 17 November 2015 (2015-11-17), pages 9395 - 9413 *
YANG DAOSHAN;: "Study of Synthesis of Nitrogen Heterocycle Antiviral Drugs", BEIJING UNIVERSITY OF TECHNOLOGY MASTER'S DISSERTATION, 31 December 2009 (2009-12-31), pages 61 *

Also Published As

Publication number Publication date
CN104447702B (zh) 2016-12-07
CN104447702A (zh) 2015-03-25

Similar Documents

Publication Publication Date Title
WO2016086824A1 (zh) 一种Bouchardatine和Bouchardatine衍生物及其制备方法和应用
JP7296318B2 (ja) Ｓｉｋインヒビターとしてのピリミドピリミジノンの使用
US10059712B2 (en) N-benzyl tryptanthrin derivative, and preparation method and application thereof
RU2608306C2 (ru) Обогащенный дейтерием 4-гидрокси-5-метокси-n,1-диметил-2-оксо-n-[(4-трифтор-метил)фенил]-1,2-дигидрохинолин-3-карбоксамид
US20130281396A1 (en) Treatment of diseases by epigenetic regulation
AU2017247282B2 (en) Nitrogen heterocyclic tryptamine ketone derivative and application as IDO1 and/or TDO inhibitor
TWI576339B (zh) 治療β-地中海型貧血及鐮刀型貧血症之方法及組合物
JP2001501197A (ja) 抗―腫瘍剤としての置換された複素環式化合物類
CN103502219A (zh) 作为治疗剂的新型小分子
WO2018223969A1 (zh) N-苄基色胺酮类衍生物作为色氨酸双加氧酶(tdo)抑制剂的用途
WO2016086825A1 (zh) 一种胺基取代Bouchardatine衍生物及其制备方法和应用
WO2020142748A1 (en) Methods and materials for increasing transcription factor eb polypeptide levels
WO2018086242A1 (zh) pH敏感的轴向取代硅酞菁配合物及其制备方法和在医药上的应用
KR100229049B1 (ko) 치료용 벤즈아제핀 화합물
JP2000034230A (ja) 含硫黄抗真菌剤
WO2020238917A1 (zh) 作为钾通道调节剂的四氢-1h-苯氮杂卓类化合物及其制备和应用
WO2022021981A1 (zh) 二-(苯并咪唑)-1,2,3-三唑衍生物及其制备和应用
CN107827837A (zh) 鞘氨醇‑1‑磷酸受体调节剂化合物及其制备方法与应用
CN110041335B (zh) 一种提高色胺酮的水溶性的方法、色胺酮衍生物及其制备方法和应用
CN108689958B (zh) 一种含有肼基的吲哚胺2,3-双加氧化酶抑制剂
CN112703190B (zh) Hdac6选择性抑制剂的晶型及其应用
WO2022022678A1 (zh) 一种吡嗪类化合物及其制备方法和应用
CN109748914A (zh) 吡啶并嘧啶类化合物及其应用
CN112062732B (zh) 一种ido抑制剂及其组合物、制备方法和用途
WO2021121012A1 (zh) 一种氨基二硫代过酸硫酯类化合物及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15864690

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15864690

Country of ref document: EP

Kind code of ref document: A1