CN105017374B - 一种齐墩果酸内酯类衍生物及其制备方法和应用 - Google Patents
一种齐墩果酸内酯类衍生物及其制备方法和应用 Download PDFInfo
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- YBRJHZPWOMJYKQ-UHFFFAOYSA-N Oleanolic acid Natural products CC1(C)CC2C3=CCC4C5(C)CCC(O)C(C)(C)C5CCC4(C)C3(C)CCC2(C1)C(=O)O YBRJHZPWOMJYKQ-UHFFFAOYSA-N 0.000 title claims abstract description 33
- MIJYXULNPSFWEK-UHFFFAOYSA-N Oleanolinsaeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C MIJYXULNPSFWEK-UHFFFAOYSA-N 0.000 title claims abstract description 33
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
本发明提供了一种齐墩果酸内酯类衍生物，如齐墩果烷‑28‑β‑内酯，其制备步骤：先将齐墩果酸和碳酸氢钠依次加入到DMSO中，然后滴加4‑(溴甲基)氧杂环丁烷‑2‑酮，室温下搅拌；反应完毕后，加入水，用乙酸乙酯萃取，有机相用无水硫酸镁干燥，减压浓缩得到粗产品，纯化即可。体外实验证明齐墩果酸内酯类衍生物具有减肥作用，其作用机理是通过抑制胰脂肪酶来达到减肥作用，其作用类型为竞争性抑制类型。齐墩果烷‑28‑β‑内酯抑酶活性(IC50:16.4μmol·L‑1)为齐墩果酸抑酶活性(IC50:87.7μmol·L‑1)的5.35倍。该类衍生物可在制备新型胰脂肪酶抑制剂类减肥药物和减肥保健品中应用。
Description
技术领域
本发明涉及齐墩果酸内酯类衍生物，具体属于一种齐墩果酸内酯类衍生物及其制备方法，以及其在制备减肥药物和减肥保健品中的应用。
背景技术
肥胖已成为全球最大慢性疾病[Haslam DW等,Lancet,2005,366:1197-1209]，可引起II型糖尿病、心脑血管疾病、胆囊疾病、骨关节炎、阻塞性睡眠呼吸暂停、癌症等，每年至少340万成人死于超重或肥胖[Baretic M等，Digest Dis Sci.,2012,30(2):168-172；Derosa G等,Expert Opin Drug Saf.,2012,11(3),459-471]。目前，奥利司他(Orlistat)作为唯一一种长效减肥药物，仍存在较大副作用，尤其会造成肝功能损伤[Derosa G等,Expert Opin Drug Saf.,2012,11(3),459-471；Mary BC等，Case Manager.,2004,15(6):47-49]，而从天然植物中直接分离得到的化合物，减肥效果普遍较低，所以有必要对其进行结构改造以提高它们的活性。
齐墩果酸(Oleanolic acid)是自然界中含量最丰富的五环三萜类化合物，主要存在于油橄榄、夏枯草、怀牛漆、连翘、青叶胆和女贞等植物中[Liu J等，J.Ethnopharmacol.，1995,49(2):57-61；孟艳秋等，药学学报，2015,50(4):469-474]，具有抑制胰脂肪酶(Pancreatic lipase,PL)、降血糖、抗肿瘤、抗HIV、保肝、抗炎等多种药理作用，且副作用小[唐初等，有机化学，2013,33:46-65；李文良等，中国药物化学杂志，2014,24:7-13；马学惠等，药学学报，1982,17:93-97]，是一理想的结构改造目标。
PL由胰腺分泌到十二指肠，可水解50％-70％的食物脂肪成甘油一酯和脂肪酸，抑制PL活性可阻止脂肪分解，进而使其直接通过消化道排出体外，从而达到减肥目的。由于该酶抑制剂直接在肠道内起作用，不需肠吸收，因而副作用较小，所以PL抑制类药物研发成为一种重要研究方向。
本发明以PL作为药物筛选靶标，以齐墩果酸为先导化合物，设计合成全新的齐墩果酸内酯类衍生物，并就其减肥作用机理，即抑制胰脂肪酶作用及抑制类型进行了研究。
发明内容
本发明的目的在于提供一种齐墩果酸内酯类衍生物及其制备方法，以及齐墩果酸内酯类衍生物在制备减肥药物和减肥保健品中的应用。
本发明提供的一种齐墩果酸内酯类衍生物，其结构式为：
其中：R1代表氢、烷基、氨基、或酰胺基；R2代表烷基、含亚胺基团、或含硫基团。所述的烷基为甲基、乙基、丙基、丁基或戊基。所述的含亚胺基团为亚胺基、甲亚胺基、乙亚胺基、丙亚胺基、丁亚胺基、或戊亚胺基。所述的含硫基团为甲硫基、乙硫基、丙硫基、丁硫基或戊硫基。
本发明齐墩果酸内酯类衍生物的制备路线：
本发明提供的一种齐墩果酸内酯类衍生物的制备方法，包括如下步骤：
1)先将齐墩果酸和碳酸氢钠加入到DMSO中，然后滴加4-(溴甲基)氧杂环丁烷-2-酮，室温下搅拌,以TLC检测反应终点；其中：齐墩果酸、碳酸氢钠、4-(溴甲基)氧杂环丁烷-2-酮的质量比为5:1-3:2-4；
2)反应完毕后，加入齐墩果酸质量0.1-1倍的蒸馏水，用乙酸乙酯萃取3次，合并有机相，用无水硫酸镁干燥，减压浓缩得到粗产品；
3)粗产品经硅胶柱色谱纯化，得白色粉末，纯化条件：洗脱剂为体积比2-7:1的石油醚和乙酸乙酯混合溶液。
本发明的齐墩果酸内酯类衍生物可在制备减肥药物和减肥保健品中的应用。
本发明的齐墩果酸内酯类衍生物可作为原料药与辅料组合制备成减肥药物。
与现有技术相比本发明的有益效果：
本发明合成的齐墩果酸内酯类衍生物----齐墩果烷-28-β-内酯，其结构经1HNMR、13C NMR及MS的确认。经体外实验证明具有减肥作用，其作用机理是通过抑制胰脂肪酶来达到减肥作用，其作用类型为竞争性抑制类型。齐墩果烷-28-β-内酯抑酶活性(IC50:16.4μmol·L-1)为齐墩果酸抑酶活性(IC50:87.7μmol·L-1)的5.35倍。
附图说明
图1齐墩果烷-28-β-内酯氢谱
图2齐墩果烷-28-β-内酯碳谱
图3齐墩果烷-28-β-内酯质谱图(M＝541.32410)
图4齐墩果烷-28-β-内酯抑制胰脂肪酶活性
图5齐墩果烷-28-β-内酯可逆抑制与不可逆抑制判定
图6齐墩果烷-28-β-内酯抑制胰脂肪酶作用类型
具体实施方式
实施例1齐墩果酸内酯类衍生物的合成和表征
将300mg齐墩果酸和110mg碳酸氢钠加入到4mL DMSO中，滴加163mg 4-(溴甲基)氧杂环丁烷-2-酮，室温下搅拌,以TLC检测反应终点，24h反应完毕后，加入5ml水，用乙酸乙酯萃取3次，合并有机相，无水硫酸镁干燥，减压浓缩得到粗产品，经硅胶柱色谱纯化，洗脱剂为石油醚-乙酸乙酯(5:1)，得白色粉末220.56mg，产率62％。1H-NMR(600MHz,CDCl3),δ0.71(s 0.71(s,3H,CH3),0.78(s,3H,CH3),0.89(s,3H,CH3),0.91(s,3H,CH3),0.93(s,3H,CH3)0.99(s,3H,CH3),1.14(s,3H,CH3),1.98～2.03(m,1H,H-12),2.81～2.87(m,1H,H-2),3.21(dd,1H,J＝3.6,10.8Hz,H-18),3.32～3.58(ddd,J＝4.2,16.2,98.4Hz,2H,CH2O),4.11～4.34(ddd,J＝4.2,12.6,78.0Hz,2H,CH2),4.48(m,1H,CHO),5.31(t,1H,J＝3.6Hz,H-12)；13C NMR(150Hz,CDCl3),δ17.70,17.17,18.47,23.08,23.55,23.68,26.02,27.33,27.79,28.26,30.77,32.52,32.68,32.96,33.14,33.94,34.63,37.19,38.89,39.53,40.16,41.93,45.86,46.85,47.72,55.40,63.00,67.84,69.62,73.19,79.11,123.01,143.31,177.31；HRMS[M+H]calad 540.3815,found 540.3241.
1H NMR、13C NMR及MS确认产物为齐墩果烷-28-β-内酯，见图1、图2、图3所示。
实施例2齐墩果烷-28-β-内酯抑制胰脂肪酶活性实验
采用对硝基苯酚法测定酶活。在反应体系中加入780μL Tris-HCl(pH＝8)，随后分别加入100μL的0.5mg/mL的PL和20μL待测物质溶液，充分混匀，37℃下孵育15min后，立即加入PNPB溶液(12.5mmol/L)，混匀，将溶液点到96孔酶标板内，每个浓度设4个复孔，检测波长为400nm，在酶标仪上进行测定。每3min测定一个点，测定15min，记录全部数据。计算吸光度随时间变化率K，根据如下公式计算抑制率：
PL的抑制率(％)＝(1-K实验/K空白)×100
试验重复3次，结果取平均值。
当抑制剂浓度为2ug/mL时，抑制率为15.75％。
当抑制剂浓度为8ug/mL时，抑制率为46.70％。
当抑制剂浓度为22ug/mL时，抑制率为76.99％。
齐墩果烷-28-β-内酯抑制胰脂肪酶活性IC50值如图4所示。
实施例3可逆抑制与不可逆抑制判定
按照酶活性测试方法，在不同抑制剂浓度条件下，测定酶浓度不同(终浓度为0.1、0.2、0.3、0.4、0.5mg/mL)时残余酶活，求出相应反应速度，以反应速度对加入酶量作图。结果如图5所示。证明为可逆抑制。
实施例4动力学抑制类型判定
在给定抑制剂浓度、不同底物浓度(终浓度为2.5、5.0、7.5、10.0、12.5mmol/L)条件下，按照酶活测试方法测定反应速度，按Lineweaver-Burk双倒数作图法作图，根据对照组和实验组两直线交点位置判断抑制类型。
齐墩果烷-28-β-内酯抑制胰脂肪酶作用类型如图6所示。证明其为竞争性可逆抑制类型。
Claims (5)
1.一种齐墩果酸内酯类衍生物，其特征在于，结构式为：
其中：R1代表氢；R2代表亚甲基。
2.如权利要求1所述的一种齐墩果酸内酯类衍生物的制备方法，包括如下步骤：
1)先将齐墩果酸和碳酸氢钠加入到DMSO中，然后滴加4-(溴甲基)氧杂环丁烷-2-酮，室温下搅拌,以TLC检测反应终点；其中：齐墩果酸、碳酸氢钠、4-(溴甲基)氧杂环丁烷-2-酮的质量比为5:1-3:2-4；
2)反应完毕后，加入齐墩果酸质量0.1-1倍的蒸馏水，用乙酸乙酯萃取3次，合并有机相，用无水硫酸镁干燥，减压浓缩得到粗产品；
3)粗产品经硅胶柱色谱纯化，得白色粉末，纯化条件：洗脱剂为体积比2-7:1的石油醚和乙酸乙酯混合溶液。
3.如权利要求1所述的一种齐墩果酸内酯类衍生物在制备减肥药物中的应用。
4.如权利要求1所述的一种齐墩果酸内酯类衍生物在制备减肥保健品中的应用。
5.一种减肥药物，含有如权利要求1所述的齐墩果酸内酯类衍生物。
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