CN117142950A - 澜沧黄杉中萜类化合物及其作为死亡相关凋亡诱导蛋白激酶2抑制剂的用途 - Google Patents
澜沧黄杉中萜类化合物及其作为死亡相关凋亡诱导蛋白激酶2抑制剂的用途 Download PDFInfo
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Abstract
本申请公开一种澜沧黄杉中萜类化合物及其作为死亡相关凋亡诱导蛋白激酶2抑制剂的用途，萜类化合物的结构式如下：
Description
技术领域
本申请涉及医药领域，具体涉及一种分离自澜沧黄杉中的萜类化合物及其作为死亡相关凋亡诱导蛋白激酶2(Drak2)抑制剂的用途。
背景技术
Drak2属于死亡相关蛋白激酶(Death-associated protein kinase,DAPK)(丝氨酸/苏氨酸激酶)家族中的一员，是潜在的抗糖尿原始创新靶点(Cohen,et al.,EMBOJ.1997,16:998-1008；Sanjo,et al.,J.Biol.Chem.1998,273:29066-29071；Zhang,etal.,J.Med.Chem.2023,66:1112-1136)。除Drak2外,迄今为止还确定了其他四个DAPK激酶家族成员,包括DAPK1(Deiss,et al.,Genes Dev.1995,9:15)、DAPK2(Inbal,et al.,Mol.Cell.Biol.2000,20:1044)、DAPK3(Kawai,et al.,Mol.Cell.Biol.1998,18:1642)和DRAK1(Kawai,et al.,Oncogene 1999,18:3471)。
Drak2激酶在免疫系统相关组织，如胸腺、脾脏、淋巴结中表达水平较高，在其他组织和器官，如大脑的嗅叶、脑室区、海马叶、肠上皮细胞和胰腺，也有一定程度的表达(Li,etal.,Cell Metab.2021,33:2004-2020；Mandarano,et al.,Cell Rep.2023,42:112106)。此外，Drak2在中枢神经系统中也广泛存在，表明Drak2不仅与细胞凋亡相关，而且还可能具有其他生物学功能，如：免疫(McGargill,et al.,Immunity,2004,21:781-791)、胰岛细胞凋亡(Delarue,et al.,Curr Opin Clin Nutr Metab Care,2007,10:142-148；Rhodes,etal.,Science,2005,307:380-384)和肿瘤(Yang,et al.,Cell Rep,2012,29,2:1286-1299)。
研究表明,高糖、高脂和某些细胞因子可促进Drak2在细胞水平的高表达(Mao,etal.,Cell.Biochem.2008,105:1073；Mao,et al.,J.Immunol.2009,182:4762)，导致胰岛β细胞的凋亡增加。经反义RNA验证，β细胞凋亡的增加与Drak2基因密切相关，提示Drak2可能在外来刺激诱导糖尿病的发生发展过程中起介导作用。因此靶向Drak2的小分子抑制剂存在治疗糖尿病的潜力(Li,et al.,Cell Metab.2021,33:2004-2020)。
体外实验证明Drak2抑制剂的短期疗效体现为增强葡萄糖刺激的胰岛素释放，长期疗效体现为保护棕榈酸(Palmic acid)诱导的功能损伤及缓解细胞凋亡；体内实验证明Drak2抑制剂能够较好的实现急性降糖，且能改善T2DM模型小鼠(db/db)的高血糖和高血脂病症，减少胰岛细胞凋亡，改善胰岛功能。
发明内容
本申请提供一种澜沧黄杉中萜类化合物及其作为死亡相关凋亡诱导蛋白激酶2(Drak2)抑制剂的用途。
本申请的一个目的是提供具有药用价值的新颖萜类化合物，并提供一种从植物澜沧黄杉中提取本申请化合物的方法。
本申请进一步的目的是提供上述化合物或组合物在制备Drak2所介导疾病药物方面的用途。
天然产物对于新药开发的指导和借鉴有着独特而重要地位，是新药发现的重要来源(Newman et al.,J.Nat.Prod.2020,83:770-803；Tiago et al.,Nat.Chem.2016,8:531-541)。天然产物是经自然法则长时间选择进化而产生的，往往能与生物大分子有效结合，可以被认为是与人类蛋白高度相关生物系统长年累月筛选淘汰后剩下的精英化合物库。同时，单一靶点的药物往往疗效不佳，而中药/植物药及其组分具有多靶点、多途径的作用特点，在治疗复杂疾病过程中具有独特优势。因此从天然来源(特别是植物来源)化学成分中寻找开发新型、高效、低毒副作用的Drak2抑制剂具有重要的研究价值。具有独特来源和结构特征的天然产物可能可以在目前糖脂代谢紊乱疾病新药研发领域的激烈竞争中获得先机。
澜沧黄杉(Pseudotsuga forrestii Craib)属于松科(Pinaceae)黄杉属(Pseudotsuga)植物，为我国特有种植物。其为常绿乔木，分布于横断山脉中南部中山上部至高山中部。于1992年被《中国植物红皮书—稀有濒危植物》收录，被列为“渐危种”。目前，其化学成分仅有少量报道，如果能保护性地采集少许澜沧黄杉植物样品进行系统的化学成分研究，将会积极促进科学认识、积极保护和利用这一珍稀植物资源为人类服务。
本申请从澜沧黄杉枝叶的甲醇提取物中分离得到两种具有新颖结构的萜类分子，其结构特征为：重排螺环羊毛脂烷型三萜与松香烷二萜分子间通过Diels-Alder[4+2]-环加成而形成的杂二聚五萜化合物(式1)，连接琥珀酸单元的松香烷二萜化合物(式2)，结构式如下：
本申请涉及的化合物为澜沧黄杉分离得到的不同萜类分子化合物，在体外药理活性筛选中发现具有显著的死亡相关凋亡诱导蛋白激酶2(Death Associated ProteinRelated Apoptotic Kinase 2,DRAK2)抑制活性，本申请的两种类型加合物在Drak2抑制活性实验中，显示出较强的抑制作用，且活性与阳性对照REF00107相当。由此可能对高脂血症、非酒精性脂肪肝炎、II型糖尿病、癌症及其它Drak2所介导的疾病具有治疗作用，从而在制药领域有巨大的潜在用途。该化合物可在制备预防、延缓或治疗由Drak2所介导的糖脂代谢紊乱及其他相关疾病药物中应用。本发明亦可为研发糖脂代谢紊乱相关疾病的新型药物提供先导化合物。
基于上述研究结果，本申请提出如下申请：
本申请提供一种结构式如式1或式2所示的萜类化合物或其药学上可接受的盐：
本申请还提供一种死亡相关凋亡诱导蛋白激酶2抑制剂，所述的萜类化合物或其药学上可接受的盐与赋形剂制成片剂、丸剂、胶囊剂或颗粒。该抑制剂中萜类化合物可为式1或式2中的任一一种，也可以是两种的组合。
本申请还提供一种药物组合物，包括治疗有效量的如权利要求1所述萜类化合物或其药学上可接受的盐中的至少一种；其进一步包括药剂学上可接受的药物辅料。该药物组合物中萜类化合物可为式1或式2中的任一一种，也可以是两种的组合。
药物组合物采用上述化合物中的一种或多种作为原料，包含治疗有效量的选自上述化合物中的一种或多种作为活性成分，该组合物可以进一步包括药剂学上可接受的药物辅料，例如载体、赋形剂、佐剂或稀释剂等。所述药物组合可于制备预防、延缓或治疗由Drak2介导的糖脂紊乱相关疾病(特别是高血脂症及其相关的心血管疾病的药物)或是作为该类药物的先导化合物。
本申请还提供一种如所述萜类化合物或其药学上可接受的盐在制备死亡相关凋亡诱导蛋白激酶2抑制剂中的用途。
本申请还提供一种如所述萜类化合物或其药学上可接受的盐在制备预防或治疗由死亡相关凋亡诱导蛋白激酶2所介导疾病药物中的用途。
可选的，由死亡相关凋亡诱导蛋白激酶2所介导疾病包括糖脂代谢紊乱及其并发症；优选地，所述糖脂代谢紊乱及其并发症包括高脂血症、非酒精性脂肪肝炎、II型糖尿病和肥胖症。
本申请所述的化合物可通过从植物中分离纯化得到；也可经本领域技术人员熟知的化学方法合成获得。
从植物中分离纯化的途径中，所述化合物由澜沧黄杉(Pseudotsuga forrestiiCraib)枝叶经由本领域所涉常规的提取分离方法制备而得，其步骤如下：晾干粉碎的澜沧黄杉枝叶用甲醇/水溶液室温浸泡提取，提取液减压浓缩回收溶剂，合并后得浸膏；浸膏用水分散后依次用石油醚、乙酸乙酯和正丁醇萃取，得石油醚部位、乙酸乙酯部位、正丁醇部位和水溶性部位；乙酸乙酯部位经硅胶、微孔树脂(MCI)、Sephadex LH-20及反相半制备高效液相(semi-preparative RP-HPLC)反复分离纯化，得化合物1和化合物2。
具体的，所述制备方法包括：
(1)澜沧黄杉枝叶的甲醇提取物依次使用等体积的石油醚、乙酸乙酯以及正丁醇各萃取数次，各萃取液经减压浓缩后分别得到石油醚部分、乙酸乙酯部分、正丁醇部分和水4个组分；
(2)对其中的乙酸乙酯部分粗浸膏经硅胶柱色谱分离，以v/v为30:1→0:1的石油醚-乙酸乙酯梯度洗，得到8个组分，Fr.1～Fr.8；
对该步骤中所得洗脱组分中的Fr.3(经石油醚：乙酸乙酯8：1和9：1洗脱得到)经MCI柱色谱进行分离，以v/v为70:30→80:20→90:10→100:0的MeOH-H2O梯度洗脱，得到3个亚组分Fr.3A～Fr.3C，对其中的Fr.3B(MeOH-H2O比例为80:20洗脱得到)经Sephadex LH-2柱层析，得Fr.3B1～Fr.3B2，对其中的Fr.3B1(采用MeOH为流动相洗脱)经半制备HPLC分离纯化得化合物1，制备HPLC的条件为Cosmosil,3mL/min,MeOH-H2O,96:4,v/v，tR＝31.8min；
对该步骤中所得洗脱组分中的Fr.4组分经MCI柱色谱进行分离，以v/v为70:30→80:20→90:10→100:0的MeOH-H2O梯度洗脱，得到3个亚组分Fr.4A～Fr.4C，对其中的Fr.4A(MeOH-H2O比例为80:20洗脱得到)经100-200目硅胶柱层析，以v/v为10:1→0:1石油醚-乙酸乙酯梯度洗，从石油醚-乙酸乙酯v/v为1:1的洗脱组分中富集得到化合物1；
(3)将步骤(2)的乙酸乙酯部分余下组分合并，经硅胶柱色谱分离，以v/v为30:1→0:1的石油醚-乙酸乙酯梯度洗，得到7个组分，Fr.1～Fr.7；
将该步骤所得组分Fr.6(经石油醚：乙酸乙酯2：1洗脱得到)经100-200目硅胶柱层析，以v/v为10:1→0:1的石油醚-丙酮梯度洗，得到3个亚组分Fr.6A～Fr.6C；对其中的Fr.6B(石油醚：丙酮比例为2:1洗脱得到)经Sephadex LH-20柱层析分为三个亚组分Fr.6B1～Fr.6B3；其中的Fr.6B1(采用MeOH为流动相洗脱)经半制备HPLC纯化得到化合物2，半制备HPLC的条件为X-Bridge,3mL/min,MeOH-H2O,65:35,v/v，tR＝27.0min。
可选的，所述甲醇提取物为体积百分浓度70％以上的甲醇提取物。
上述方法中，所述的甲醇/水溶液可以为70％以上的甲醇-水溶液，优选90％的甲醇-水溶液；室温提取的时间没有特殊限制，可以为12小时/次以上；提取次数可以进行一次或多次，优选3次以上。
与现有技术相比，本申请至少具有如下有益效果之一：
(1)基于该类化合物在化学结构新颖性、生物活性显著等方面的优点，使其具有很好的开发前景，有望发展成为结构新颖的针对Drak2所介导疾病的治疗药物或先导化合物。
(2)所述化合物为首次从自然界分离得到的新颖化合物，是具有独特[4+2]-型环化结构的Diels-Alder型加合物(结构式1)或连接琥珀酸单元的松香烷二萜化合物(结构式2)。
(3)同时首次发现该类化合物具有显著的Drak2抑制活性，这对现代人群中高发的糖脂代谢紊乱相关疾病如高脂血症、非酒精性脂肪肝炎、II型糖尿病等疾病将具有重要应用前景。
(4)本申请的两种类型加合物在Drak2抑制活性实验中，显示出较强的抑制作用，且活性与阳性对照REF00107相当。
附图说明
图1为式1所示化合物的1H-NMR图；
图2为式1所示化合物的13C-NMR图；
图3为式2所示化合物的1H-NMR图；
图4为式2所示化合物的13C-NMR图。
具体实施方式
下面将结合实施例对本申请的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
澜沧黄杉(Pseudotsuga forrestii Craib)枝叶采自云南大理，经阴干后粉碎制成粉末；比旋光测试通过Rudolf Autopol IV旋光仪于25℃下完成；Hitachi U-2900E型紫外光谱仪；Thermo Scientific Nicolet Is5 FT-IR型红外光谱仪；ECD光谱由JASCO-810CD光谱仪测定；HR-ESIMS由AB Sciex TripleTOF 5600型仪测定；所使用的硅胶为青岛海洋化工公司生产；硅胶薄层板为烟台江友硅胶开发有限公司生产，规格为GF254/0.25mm；MCIgel CHP20P为日本三菱公司生产，规格为75-150μm；Sephadex LH-20凝胶为瑞士GEHealthcare Bio-Sciences公司生产；半制备HPLC为Shimadzu LC-20AT，配备SPD-M20APDA检测器以及Waters X-Bridge ODS和Cosmosil半制备柱(250×10mm,5μm)；所有分析纯试剂均为上海国药集团化学试剂有限公司生产；色谱级溶剂由上海星可高纯溶剂有限公司生产；氘代氯仿为Sigma-Aldrich生产。
实施例1：化合物的制备
(1)将采自云南大理的澜沧黄杉枝叶(2019年6月采集，干重15kg)干燥粉碎后，室温下使用90％甲醇(6L)溶液提取7次，每次24小时。提取液合并，减压浓缩除去甲醇后得到浸膏3.9kg(semi-dry)。用水3L分散浸膏，依次使用等体积的石油醚、乙酸乙酯以及正丁醇各萃取三次，各萃取液经减压浓缩后分别得到石油醚部分、乙酸乙酯部分、正丁醇部分和水4个组分。其中，乙酸乙酯部分粗浸膏经硅胶柱色谱(以石油醚-乙酸乙酯梯度洗，30:1→0:1,v/v)分离后得到8个组分，Fr.1～Fr.8。
(2)选择将步骤(1)中的组分Fr.3(44g，步骤1中经石油醚：乙酸乙酯8：1和9：1洗脱得到)经MCI柱色谱进行分离，以MeOH-H2O梯度洗脱(70:30→80:20→90:10→100:0,v/v)得到3个亚组分Fr.3A～Fr.3C。Fr.3B(MeOH-H2O比例为80:20洗脱得到)经Sephadex LH-20柱层析得Fr.3B1～Fr.3B2。Fr.3B1(0.7g，采用MeOH为流动相洗脱)经半制备HPLC(Cosmosil,3mL/min,MeOH-H2O,96:4,v/v)分离纯化得化合物1(70mg,tR＝31.8min)。组分Fr.4(35g，步骤1中经石油醚：乙酸乙酯8：1洗脱得到)经MCI柱色谱进行分离，以MeOH-H2O梯度洗脱(70:30→80:20→90:10→100:0,v/v)得到3个亚组分Fr.4A～Fr.4C。Fr.4A(6.6g，MeOH-H2O比例为80:20洗脱得到)经硅胶柱层析(100-200目，以石油醚-乙酸乙酯梯度洗，10:1→0:1,v/v)，从石油醚-乙酸乙酯v/v为1:1的洗脱组分中富集得到化合物1(1.1g)。
(3)选择将步骤(1)和步骤(2)中乙酸乙酯部分余下组分合并，合并上述剩余各组分浸膏(409g)，经硅胶柱色谱(以石油醚-乙酸乙酯梯度洗，30:1→0:1,v/v)分离后得到7个组分，Fr.1～Fr.7。
(4)选择将步骤(3)组分Fr.6(步骤3中经石油醚：乙酸乙酯2：1洗脱得到)经硅胶(100-200目，以石油醚-丙酮梯度洗，10:1→0:1,v/v)柱层析后得到3个亚组分Fr.6A～Fr.6C。Fr.6B(石油醚：丙酮比例为2:1洗脱得到)则经Sephadex LH-20柱层析后分为三个亚组分Fr.6B1～Fr.6B3。Fr.6B1(采用MeOH为流动相洗脱)经半制备HPLC(X-Bridge,3mL/min,MeOH-H2O,65:35,v/v)纯化得到化合物2(4.6mg,tR＝27.0min)。
化合物1，其核磁及理化数据如下：
片状单晶(MeOH)，[α]D 25+55.4(c 0.27,MeOH)；UV(MeOH)λmax(logε)206(3.57)nm；IR(KBr)vmax 2952,2870,2649,1695,1469,1387,1273,1116,935,882,761cm-1；1H-NMR(CDCl3,600MHz):δ1.62(1H,m,H-1a),1.96(1H,m,H-1b),2.41(1H,m,H-2a),2.65(1H,m,H-2b),1.57(1H,m,H-5),1.63(1H,m,H-6a),1.44(1H,m,H-6b),1.93(2H,m,H-7),2.13(1H,m,H-11a),2.12(1H,m,H-11b),1.40(2H,m,H-12),2.39(1H,m,H-15a),2.27(1H,m,H-15b),1.92(1H,m,H-16a),1.46(1H,m,H-16b),0.77(3H,s,H-18),1.11(3H,s,H-19),2.38(1H,m,H-20),0.74(3H,d,J＝6.5Hz,H-21),2.44(1H,m,H-22a),2.05(1H,m,H-22b),2.79(1H,brs,H-24),1.17(3H,s,H-26),1.09(6H,br s,H-29,30),4.72(1H,br s,H-30a),4.46(1H,brs,H-30b),0.76(1H,m,H-1'a),1.45(1H,m,H-1'b),1.56(1H,m,H-2'a),1.46(1H,m,H-2'b),1.61(1H,m,H-3'a),1.54(1H,m,H-3'b),1.69(1H,m,H-5'),1.58(1H,m,H-6'a),1.33(1H,m,H-6'b),1.59(2H,m,H-7'),1.61(1H,m,H-9'),2.12(1H,m,H-11'a),1.04(1H,m,H-11'b),2.76(1H,br s,H-12'),5.36(1H,s,H-14'),2.34(1H,m,H-15'),1.04(6H,br d,J＝6.5Hz,H-16',17'),1.16(3H,s,H-18'),0.62(3H,s,H-20')；13C-NMR(CDCl3,150MHz):δ35.8(C-1),34.6(C-2),216.7(C-3),47.4(C-4),51.4(C-5),20.9(C-6),26.0(C-7),136.1(C-8),147.9(C-9),35.9(C-10),26.8(C-11),30.9(C-12),67.9(C-13),154.3(C-14),27.0(C-15),38.1(C-16),48.1(C-17),18.3(C-18),18.8(C-19),36.3(C-20),15.4(C-21),44.8(C-22),213.8(C-23),62.2(C-24),49.8(C-25),18.5(C-26),184.2(C-27),21.2(C-28),26.3(C-29),103.7(C-30),38.2(C-1'),17.0(C-2'),37.3(C-3'),46.9(C-4'),48.4(C-5'),22.0(C-6'),30.9(C-7'),45.8(C-8'),49.0(C-9'),37.5(C-10'),20.3(C-11'),34.7(C-12'),149.4(C-13'),125.2(C-14'),32.3(C-15'),20.1(C-16'),20.2(C-17'),16.0(C-18'),186.6(C-19'),17.3(C-20')；ESIMS m/z 769[M+H]+；HRESIMS m/z 769.5392[M+H]+(calcdfor C50H73O6,769.5402,Δ＝+1.02ppm).
化合物2，其核磁及理化数据如下：
白色粉末,[α]D 25+21.6(c 0.14,MeOH)；UV(MeOH)λmax(logε)206(4.15),252(3.27),294(2.53)nm；IR(KBr)vmax 3444,2972,2927,2855,1741,1439,1379,1367,1160,1073,1028,998cm-1；1H NMR(CDCl3,400MHz):δ2.33(1H,d,J＝12.8Hz,H-1a),1.36(1H,m,H-1b),1.67(2H,m,H-2),2.86(2H,m,H-3),1.63(1H,dd,J＝12.0,2.4Hz,H-5),1.79(1H,m,H-6),2.89(1H,ddd,J＝17.5,13.2,5.1Hz,H-6a),2.84(1H,m,H-6b),7.22(1H,br d,J＝8.4Hz,H-11),7.15(1H,br d,J＝8.3Hz,H-12),7.04(1H,br s,H-14),1.50(6H,s,Me-16,17),3.76(1H,d,J＝11.2Hz,H-18a),4.02(1H,d,J＝11.2Hz,H-18b),0.96(3H,s,Me-19),1.22(3H,s,Me-20),3.07(3H,s,-OCH3),2.58(4H,m,H-2′,H-3′),3.61(3H,s,-COOCH3)；13CNMR(CDCl3,150MHz):δ38.3(C-1),(C-2),35.5(C-3),36.9(C-4),44.3(C-5),19.0(C-6),30.4(C-7),134.5(C-8),148.1(C-9),37.5(C-10),124.1(C-11),123.3(C-12),142.6(C-13),126.2(C-14),76.5(C-15),27.9(C-16),27.8(C-17),72.8(C-18),17.4(C-19),25.3(C-20),50.6(-OCH3),172.6(C-1′),28.9(C-2′),29.3(C-3′),172.2(C-4′),51.7(-COOCH3)；HRESIMS m/z 453.2615[M+Na]+(calcd for C26H38O5Na,453.2611,Δ＝+0.9ppm).
化合物1的1H-NMR图(600MHz，CDCl3)如图1所示；化合物1的13C-NMR(150MHz，CDCl3)图如图2所示；化合物2的1H-NMR图(400MHz，CDCl3)如图3所示；化合物1的13C-NMR(150MHz，CDCl3)图如图4所示。
实施例2：活性检测
实验方法：
本实验中ADP-Glo™；Kinase Assay(ADP-Glo™；激酶检测试剂盒)是一种发光法激酶检测试剂盒，它检测激酶反应中所形成的ADP；在检测中，ADP被转化成ATP，然后ATP再被Ultra-Glo™萤光素酶捕获转化为光信号，发光信号与激酶活性正相关。该试剂盒可用于检测相关化合物对Drak2的抑制活性。
实验前，待测化合物样品溶解于DMSO(二甲基亚砜)配制成母液以便稀释为后续实验所需浓度。
测定过程中，每次向试剂盒孔中添加1μL化合物，除了不含酶的对照孔外，测定板孔中每孔添加2μL激酶溶液(对照改为添加2μL 1x激酶缓冲液)。之后每孔加入2μL ATP溶液，充分混合反应后加入2.5μL ADPGloTM试剂以终止激酶反应并消耗掉未消耗的ATP，仅留下ADP和非常低的ATP背景在室温下孵育60分钟。加入5μL激酶检测试剂以将ADP转化为ATP，并引入荧光素酶和荧光素来检测ATP。通过样品活性对样品浓度进行非线性拟和得到IC50，计算所用软件为GraphPad Prism 4，拟合所使用的模型sigmoidaldose-response(variable slope)。阳性对照为REF00107。
表1.化合物1和2的Drak2抑制活性
a The values(μM)indicate 50％ Drak2 inhibitory effects.These data areexpressed as the mean±SEM of triplicate experiments；
b Positive control for the Drak2 assay.
两种不同类型萜类化合物的Drak2抑制活性数据(IC50值)见表1。测试结果表明本申请中所述两个化合物均对Drak2表现出显著的抑制活性，表明本申请所述化合物可用于制备治疗糖脂代谢紊乱相关疾病的药物或是作为该类药物的先导化合物。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。
Claims (8)
1.结构式如式1或式2所示的萜类化合物或其药学上可接受的盐：
2.一种死亡相关凋亡诱导蛋白激酶2抑制剂，其特征在于，如权利要求1所述的萜类化合物或其药学上可接受的盐与赋形剂制成片剂、丸剂、胶囊剂或颗粒。
3.一种药物组合物，其特征在于，包括治疗有效量的如权利要求1所述萜类化合物或其药学上可接受的盐中的至少一种。
4.如权利要求1所述萜类化合物或其药学上可接受的盐在制备死亡相关凋亡诱导蛋白激酶2抑制剂中的用途。
5.如权利要求1所述萜类化合物或其药学上可接受的盐在制备预防或治疗由死亡相关凋亡诱导蛋白激酶2所介导疾病药物中的用途。
6.根据权利要求5所述的用途，其特征在于，由死亡相关凋亡诱导蛋白激酶2所介导疾病包括糖脂代谢紊乱及其并发症；
优选地，所述糖脂代谢紊乱及其并发症包括高脂血症、非酒精性脂肪肝炎、II型糖尿病和肥胖症。
7.如权利要求1所述萜类化合物的制备方法，其特征在于，包括：
(1)澜沧黄杉枝叶的甲醇提取物依次使用等体积的石油醚、乙酸乙酯以及正丁醇各萃取数次，各萃取液经减压浓缩后分别得到石油醚部分、乙酸乙酯部分、正丁醇部分和水4个组分；
(2)对其中的乙酸乙酯部分粗浸膏经硅胶柱色谱分离，以v/v为30:1→0:1的石油醚-乙酸乙酯梯度洗，得到8个组分，Fr.1～Fr.8；
对该步骤中所得洗脱组分中的Fr.3即石油醚-乙酸乙酯v/v为8：1和9：1洗脱组分经MCI柱色谱进行分离，以v/v为70:30→80:20→90:10→100:0的MeOH-H2O梯度洗脱，得到3个亚组分Fr.3A～Fr.3C，对其中的Fr.3B即MeOH-H2O v/v为80:20洗脱组分经Sephadex LH-2柱层析，得Fr.3B1～Fr.3B2，对其中的Fr.3B1即MeOH为流动相洗脱组分经半制备HPLC分离纯化得化合物1，制备HPLC的条件为Cosmosil,3mL/min,MeOH-H2O,96:4,v/v，tR＝31.8min；
对该步骤中所得洗脱组分中的Fr.4组分即石油醚-乙酸乙酯v/v为8：1洗脱组分经MCI柱色谱进行分离，以v/v为70:30→80:20→90:10→100:0的MeOH-H2O梯度洗脱，得到3个亚组分Fr.4A～Fr.4C，对其中的Fr.4A即MeOH-H2Ov/v为80:20洗脱组分经100-200目硅胶柱层析，以v/v为10:1→0:1石油醚-乙酸乙酯梯度洗，从石油醚-乙酸乙酯v/v为1:1的洗脱组分中富集得到化合物1；
(3)将步骤(2)的余下组分合并，经硅胶柱色谱分离，以v/v为30:1→0:1的石油醚-乙酸乙酯梯度洗，得到7个组分，Fr.1～Fr.7；
将该步骤所得组分Fr.6即石油醚-乙酸乙酯v/v为2：1洗脱组分经100-200目硅胶柱层析，以v/v为10:1→0:1的石油醚-丙酮梯度洗，得到3个亚组分Fr.6A～Fr.6C；对其中的Fr.6B即石油醚-丙酮v/v为2:1洗脱组分经Sephadex LH-20柱层析分为三个亚组分Fr.6B1～Fr.6B3；其中的Fr.6B1即以MeOH为流动相洗脱组分经半制备HPLC纯化得到化合物2，半制备HPLC的条件为X-Bridge,3mL/min,MeOH-H2O,65:35,v/v，tR＝27.0min。
8.根据权利要求7所述的制备方法，其特征在于，所述甲醇提取物为体积百分浓度70％以上的甲醇提取物。
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