【作者】 李迎红；

【导师】 蒋建东； 宋丹青；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2009， 博士

【摘要】 我组科研人员的发现表明:我国天然药物单体化合物小檗碱（berberine,简写BBR）及其同分异构体—假小檗碱（pseduoberberine,Y53）均为与他汀类药物作用机制不同、安全性高、新结构骨架的降血脂化合物（结构式见图1）。这类化合物在基因转录后水平上,通过作用于3’UTR区域稳定低密度脂蛋白受体（LDLR）的mRNA,显著上调LDLR的表达,在动物体内表现出良好的降血脂活性,显示出明显的应用前景。本项目在此工作基础上,主要开展了以下两方面的研究。一、BBR类似物上调LDLR表达的构效关系为了系统阐明BBR类似物上调LDLR的构效关系,寻找活性必需基团,获得活性更为优异的类似物,本论文在前期工作基础上,以BBR或Y53为先导化合物,通过半合成制备方法,分别对其A、C、D环上的侧链取代进行了较为系统的结构修饰,共设计合成了52个BBR类似物,通过测定其对肝细胞LDLR的表达活性,系统阐明了BBR类似物的构效关系。1、亚甲二氧五元环（E环）结构是活性必需的。将BBR的五元环打开、2-或/和3-羟基烷基化、或将五元环扩至六元环所得产物的活性均减弱或消失。2、C环13位为氢原子时活性最好。在BBR或Y53的13-位引入体积不同的烷基,所得产物活性消失。3、化合物骨架的平面结构为其活性所必需。将C环还原,平面骨架弯曲,化合物活性消失;即使在N7-位引入各种取代基团以保持N7-位的正电荷,化合物上调LDLR活性仍然消失。4、随着9-位侧链取代基体积的增大,活性均有不同程度的降低;9-OH替代BBR的9-OCH₃,所得产物M1（俗称小檗红碱,berberrubine,结构见图1）的活性与BBR相当,为我们提供了一个“-OH”作为连接载体的手臂。因为M1恰为BBR的主要代谢产物之一,使通过M1制备前药提高BBR口服生物利用度的想法成为可能。二、提高小檗碱生物利用度的化学设计与药学实践为了克服BBR口服生物利用度低（仅约5%）的缺陷,并将其转变成自主创新、机制新颖、毒性低的降血脂新药,本论文从以上工作中发现的BBR活性代谢产物—M1着手,通过其结构中裸露出的9-OH“把手”,引入脂溶性增加的载体,制备成M1前药,以改善脂溶性进而提高其口服生物利用度。我们预期这些前药比BBR更易通过肠道进入血液,在机体酯酶或混合氧化酶的催化下,与BBR一样释放出活性代谢产物M1,在机体发挥降血脂作用。本论文参考前药脂水分配系数（ClogP）的预测数据,共设计合成了17个M1前药（8个M1酯型前药,3个M1醚型前药,6个二级前药）。通过在大鼠血浆中的生物转化速率试验的筛选、以及动物体内降血脂活性的评价,从中筛选出高生物利用度、具有显著降血脂活性的酯型前药—M1棕榈酸酯（13）。在大鼠血浆中孵育10 min约有18%的13水解释放M1; 1 h水解百分数为49%。与其它前药相比,水解速度适中。SD大鼠高脂模型实验结果表明,BBR和13（100 mg/kg/天,口服）分别治疗30天后,血浆总胆固醇（CHO）分别下降了27.4%和33.1%,低密度脂蛋白胆固醇（LDL-c）分别下降了28.9%和43.0%,甘油三酯（TG）分别下降32.9%和36.5%,另两个动物模型的数据也证实了SD大鼠实验的结果,表明后者的降血脂效果明显优于BBR。另外,13治疗大鼠30天后,其肝、肾功能的指标均在正常范围之内,未显示肝、肾毒性。本论文共设计合成了52个BBR类似物和17个前药,其结构均经MS及¹H NMR等图谱确证无误。较为系统地阐明了BBR类似物上调LDLR表达活性的构效关系,基本完成了BBR作为LDLR上调剂的化学部分研究。相关研究内容已发表在美国药物化学期刊J.Med.Chem.（2009）杂志上。上述研究还获得活性化合物M1,前药13的结构是由M1和棕榈酸组成,而棕榈酸是临床使用多种前药的载体,具有较高的安全性。化合物13不仅拥有自主知识产权（公开号:WO/2006/029577）,而且还部分克服了BBR生物利用度低的缺陷。与现用的一线降脂药物他汀类相比,13是一个作用机制新颖、安全性高、药效好的新型降血脂候选物,具有良好的应用前景,拟推荐进入临床前研究。更多还原

【Abstract】 Our previous study has shown that natural compound berberine (BBR) and its isomer pseudoberberine (Y53) were both novel cholesterol-lowering agents with a high safety and a new mechanism distinct from statins. Acting on the post-transcription level, the compounds significantly upregulate expression of the low-density lipoprotein receptor (LDLR) by stabilizing its mRNA and display significant cholesterol-lowering activity in vivo. The present investigation covers two parts.1. Structure-activity relationship (SAR) analysisIn an attempt to systematically elucidate SAR of berberine analogues in LDLR upregulation, 52 derivatives were designed and synthesized. The LDLR up-regulating activity of the compounds was examined and the SAR of this group of chemical entities is summed up.(1) Methylenedioxo cylic group (ring E) was essential for the activity. Enlarging, or opening the five-member ring, or substituting the newly generated hydroxyl with alkyloxy groups made the activity decreased or disappeared.(2) The hydrogen at 13-position was also important. Replacing the 13-hydrogen of BBR or Y53 with different alkyl or benzyl groups decreased the activity consumedly. Retaining 13-hexyl and focusing the structure modification on the skeleton, the resultant derivatives had no up-regulatory effect as well.(3) The planar-like steric configuration was necessary for the activity. Reduction of the double bonds in the ring C resulted in a flexation of the planar skeleton, and caused disappearance of the activity. Introducing substitutes to N7-position to recover its positive charge did not restore the activity.(4) The analogues with increased volume of the side chain at 9-position had lower activity. 9-hydroxyl derivative (M1, berberrubine), one of the main metabolites of BBR (9-methyloxy), owned an activity similar to that of BBR. Furthermore, M1 offers an opportunity of making prodrugs to improve the bioavailability of BBR.2. Improving bioavailability of BBR by making prodrugsIn order to overcome the poor bioavailability of BBR’s (only 5% statistically), BBR’s metabolite berberrubine (M1) was employed to make prodrugs. We introduced lipophilic groups to the naked hydroxyl group in M1 to prepare prodrug compounds, which would deliberate M1 in vivo through enzyme catalyzing. 17 prodrugs (including 8 ester, 3 ether and 6 dual-prodrugs) were designed and synthesized refering to lipid-water distribution coefficient (ClogP) prediction data. A novel compound with improved bioavailability and cholesterol-lowering activity—berberrubine palmitate (13) endured the in vitro hydrolyzation test and in vivo evaluation. 13 displayed a moderate hydrolyze rate in the hydrolyzation test, and its cholesterol-lowering effect was confirmed in three hypercholesterolemic animal models. In the SD rat model, our results showed that 100 mg/kg of BBR or 13 orally administrated twice a day for a month effectively lowered serum CHO levels by 27.4% and 33.1%, LDL-c by 28.9% and 43.0%, TG by 32.9% and 36.5%, respectively. The lipid-lowering activities of 13 were significantly higher than that of BBR in the animals (P＜0.01). Liver and kidney functions were not altered by 13 in the tests. These results indicated that 13 had a better cholesterol-lowering activity than did BBR and did not have toxicity on liver or kidney.69 compounds (including 52 BBR analogues and 17 prodrugs) were designed and synthesized, and their chemical structures were confirmed by MS and ~1H NMR. By systematically elucidating their SAR for LDLR upregulation, we basically fulfilled the chemical study of BBR analogues for their activity on LDLR, and found an active compound M1. Part of the work has been published on Journal of Medicinal Chemistry (2009).Prodrug 13 was composed of M1 and palmitate vector, which had been used in clinic and proved to be safe. 13 had been PCT patent protected (publication No.: WO/2006/029577). Compared with the known cholesterol-lowerering drug statins, 13 is a novel cholesterol-lowering candidate with new mechanism, good safety and high activity, thus promising for preclinical investigation.更多还原