【作者】 刘军；

【导师】 吉爱国； 娄红祥；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2018， 博士

【摘要】 天然产物是新药发现的重要来源。自然界中存在许多含量及其丰富的大宗天然产物,这类天然产物具有含量大、种类多、结构复杂、廉价易得等特点,同时大宗天然产物价格普遍偏低、生产附加值微薄。萜类化合物在自然界分布广泛、种类繁多,是天然产物中最多的一类,其中又以二萜类化合物结构类型最为丰富,且具有如抗肿瘤、抗微生物、心血管保护等药理活性,成药性强,许多二萜类化合物已成为临床药物上市,如紫杉醇、穿心莲内酯等。借助天然产物复杂的骨架结构、广泛的生物学活性,一方面以大宗天然产物为原料,直接对其进行结构修饰与改造,提高生物活性、降低毒副作用,增强成药性;另一方面可以聚焦结构复杂、制备成本高昂的上市小分子药物,利用与其结构相似的大宗天然产物骨架或结构片断,对天然产物进行有针对性的结构改造,借助其核心骨架或结构片断,同时引入关键药效基团,制备结构新颖的衍生物,研制活性更强、安全性更高的创新药物。同时,能够简化制备工艺、降低生产成本,提高大宗天然产物的利用价值。蛋白酶激活受体-1(Protease-activated receptor 1,PAR1)是近些年新发现的抗血小板药物靶点,抑制PAR1受体能够阻断由凝血酶介导的血小板聚集和病理性血栓扩大的过程,但不影响由TXA2和ADP参与的人体正常保护性止血过程。因此,PAR1抑制剂具有安全高效的特点。美国默沙东公司在天然产物喜巴辛结构的基础上,经结构优化研发出第一个小分子PAR1拮抗剂Vorapaxar,并于2014年在美国上市,主要用于有心脏病发作史的患者和下肢动脉栓塞的患者,临床效果明显。然而其结构复杂(具有7个手性中心)、合成路线长(线性合成步骤为16步)、制备成本高,同时该药生物半衰期较长(126-269 h),当副作用发生时,没有合适的药品能够抵抗和缓解这种副作用,存在安全风险。同时,我们发现二萜类化合物穿心莲内酯结构中稠合二环手性中心的构象与Vorapaxar结构中对应部分的关键手性中心完全一致。因此,我们借助穿心莲内酯的骨架结构,在确保其关键手性中心构象不变的前提下,与Vorapaxar中的重要的药效团杂合,快速高效地制备了一系列结构新颖的Vorapaxar类似物。通过体外活性评价,筛选出活性优良的PAR1小分子拮抗剂,以PAR1抑制活性及药代动力学参数为指导,进行不断的结构优化,提高活性,改善药代动力学性质,最终发现具有全新结构的PAR1小分子拮抗剂1-39。该化合物具有显著的PAR1抑制活性,IC50为1.1 μM;体内外实验均证实其能够显著抑制由凝血酶及TRAP诱导的血小板聚集,IC50分别为0.65 μM和1.6 μM,同时对胶原蛋白和ADP诱导的血小板聚集没有抑制作用,显示出良好的选择性。同时药代动力学研究表明化合物1-39 口服生物利用度高达52.5%,且半衰期为3.1 h,半衰期较Vorapaxar明显缩短,能够大幅降低Vorapaxar由于半衰期过长,药物易体内蓄积导致的出血风险。重要的是,借助于穿心莲内酯的手性骨架,化合物1-39的合成路线(9步)较Vorapaxar明显缩短,收率显著提高(21%)、成本明显降低,是潜在的具有全新结构的新一代PAR1小分子拮抗剂。我们课题组前期在对化合物库中的萜类化合物进行抗真菌活性筛选时,发现许多四环二萜类化合物具有良好的抗真菌活性,其中大多含有α,β-不饱和酮基团,然而由于天然产物量的限制,无法对其进行多样化的修饰以进一步提高活性。大宗天然产物甜菊苷在酸性条件下的水解产物异甜菊醇是具有典型四环二萜结构的化合物。抗真菌活性评价发现其具有较弱的抗真菌活性,MIC为256μg/mL。在异甜菊醇骨架的基础上构建环外α,β-不饱和酮基团,其活性提高了 4倍,MIC=64μg/mL。进一步在其结构的基础上,连接线粒体靶向基团三苯基磷阳离子,靶向线粒体,发现其抗真菌活性又提高了约8倍,MIC = 8μg/mL。我们在此基础上,通过改变连接链的类型及长短等多样化的修饰,衍生出一系列化合物,活性筛选出活性最优化合物11-31,其MIC达到0.5 μg/mL,强于两性霉素B。进一步对其进行多种菌株活性评价,包括外排泵过表达株、临床耐药株等,都显示出强大的抑菌及杀菌作用,能够克服真菌耐药问题。同时继续深入的机制研究发现化合物Ⅱ-31能够升高线粒体膜电位、同时刺激胞内ROS的产生,从而损伤线粒体,另外线粒体外膜蛋白Tom70的分布由正常的管状分布变为聚集状分布,进一步证实化合物Ⅱ-31能损伤线粒体导致细胞死亡。被膜的形成是真菌耐药的主要原因之一,我们研究发现化合物Ⅱ-31不仅能抑制被膜形成,同时具有显著的清除成熟被膜的作用。进一步我们通过构建秀丽隐杆线虫-白色念珠菌感染模型,发现化合物Ⅱ-31能显著提高感染线虫的生存率,且毒性较低。总之,通过借助廉价易得的大宗天然产物异甜菊醇的基本骨架,衍生出一系列具有抗真菌活性的化合物,进一步结构优化得到活性最优化合物Ⅱ-31,其不仅能够抑制真菌生长,也能杀灭真菌,且能杀死耐药菌,同时能够抑制被膜形成,清除成熟被膜,是潜在的能够克服真菌耐药的新型抗真菌药物。另外,我们课题组在前期的研究中还发现,具有不饱和酮基团的四环二萜类化合物具有显著的抗肿瘤活性。我们对以异甜菊醇为骨架构建环外不饱和酮基团的化合物Ⅱ-14进行抗肿瘤活性筛选,发现其具有良好的抗肿瘤活性,IC50值在10μM。我们以其为底物,通过形成二聚体或连接多样化的不饱和酮基团,从而增加药效团。另外,通过连接碱性基团靶向溶酶体,得到一系列衍生物。进一步活性筛选证实部分新得到的衍生物抗肿瘤活性有所提高,然而未达到我们的期望值,我们需要进一步结构优化。由于A环除羧基外,缺少可修饰基团,尚无人做过改造。我们首次在A环构建不饱和酮及五元不饱和内酯活性基团,得到一系列结构新颖的衍生物,对其进行活性筛选,得到具有良好活性的先导化合物Ⅲ-32,IC50值为2μM。进一步以化合物Ⅲ-32为底物,进行结构优化,通过对D环引入曼尼希碱、肟等碱性基团,同时通过Baeyer-Villiger重排、Beckman重排等多样化的构建内酯、内酰胺环等,进一步得到一系列结构新颖的衍生物,对其进行活性筛选,发现D环具有肟甲醚基团的化合物Ⅲ-53活性最优,IC50值为0.29 μM。进一步对其进行机制研究,表明化合物Ⅲ-53主要通过诱导LMP,破坏溶酶体,导致细胞死亡。化合物Ⅲ-53抗肿瘤活性显著,具有发展成为抗肿瘤候选药物的潜力。总之,我们借助大宗天然产物穿心莲内酯及异甜菊醇的骨架结构,简便快捷、有针对性地构建了几个系列结构新颖的衍生物,经过不断的结构优化,发现了几个活性显著,极具成药性的小分子化合物,实现了大宗天然产物的高值化利用。更多还原

【Abstract】 Natural products are considered to be one of the main sources of medicines.There are many abundant natural products in nature and these kinds of natural products have the characteristics of large content,variety,complex structure,cheap and easy to get.Meanwhile,the added value of these natural products is low.Terpenoids are widely distributed in nature and are the most natural products.Among them,diterpenoids are the most abundant type,and have the pharmacological activity such as anti-tumor,anti-microbial and cardiovascular.Many diterpenoids are listed as clinical drugs,such as paclitaxel,andrographolide and salvia miltiorrhiza.Based on the complex skeleton structure and extensive biological activity of natural products,researchers have always been interested.On the one hand,with the natural products as raw materials,the structural modifications are directly carried out to improve the biological activity,reduce the side effects and enhance the drug-like properties.On the other hand,it can focus on the complex structure and expensive small molecule drugs,looking for natural products with similar structure to targeted structural modification.By this means,it may be possible to improve the probability of finding new lead structures.In addition,this concept could be used in the construction and preparation of complex molecules in a more convenient and efficient way.Protease-activated receptor I(PAR1),a G-protein-coupled thrombin receptor,is the most potent cell surface inducer of platelet activation.The PARI antagonist could be used as potent antiplatelet agent for the prevention of thrombosis and restenosis.Additionally,because PAR1 antagonists target the cellular effector of thrombin,thrombin-mediated fibrin generation is not affected,and therefore normal hemostasis should be maintained when platelet activation is induced.Therefore,a PAR1 antagonist is likely to result in a lower risk of bleeding than conventional antithrombotic agents.So far,only vorapaxar,derived from the natural product himbacine,has been approved by the US Food and Drug Administration as the first-in-class anti-platelet drug targeting PAR1 to reduce the risk of recurrent cardiovascular events.Although vorapaxar exhibits excellent anticoagulant activity,there are still many deficiencies including a complicated structure with seven chiral centers,a long synthetic route with sixteen linear synthetic steps,a low overall yield and high preparation cost.In addition,patients receiving vorapaxar were more likely to experience bleeding events compared with those receiving placebo,which has limited its clinical use.However,this adverse effect is attributed to the long elimination time(126-269 h)of vorapaxar rather than to the PAR1 target itself.Andrographolide,an easily accessible molecule isolated from the leaves of Andrographis paniculata,is an important active natural product.The two fused six-membered rings of andrographolide adopt the chair conformation and,importantly,the key chiral centers on its scaffold are fully consistent with those in vorapaxar.Based on these results,the use of the chiral fragment of andrographolide instead of the tricyclic ring of Vorapaxar while maintaining the key chiral conformation is a convenient approach to quickly develop new PAR1 antagonists.In this study,we prepared two series of novel PAR1 inhibitors by "natural-product hybridization",and"borrowed" the two fused six-membered ring moiety from the natural product andrographolid,which led the discovery of the lead compound I-13a.The further structural optimization was guided by the metabolic stability evaluation and finally resulted in the most potent thrombin receptor antagonist Ⅰ-39,which also displayed potent anti-platelet activity both in vitro and ex vivo.Compound Ⅰ-39 inhibited human platelet aggregation induced by thrombin(0.5 U/mL)or TRAP(15μM)in a concentration-dependent manner,with IC50 values of 0.65μM and 1.6 μM,respectively.Meanwhile,the platelet aggregation response following stimulation of human PRP with ADP(10 μM)and collagen(5 μg/mL),was not affected by Ⅰ-39,up to a concentration of 10 μM.These data demonstrated that compound Ⅰ-39 could selectively inhibit PAR1-mediated platelet aggregation.The compound Ⅰ-39 exhibited an excellent oral bioavailability of 52.5%and the oral half-life was 3.1 h.Importantly,with the help of the natural product skeleton,the synthetic route of compound Ⅰ-39 was relatively short and resulted in a high yield.Therefore,compound I-39 could be obtained at low cost,which is an advantage in developing compound Ⅰ-39 as a PAR1 antagonist.These studies suggest that compound 1-39 has a potential to be developed as a new generation of PARI antagonists.In our previous research on the varied natural products with antifungal activities from bryophytes and endolichenic fungi,we found that many tetracyclic diterpenes are active against C.albicans strains,and most of them have α,β-unsaturated ketone motif.However,the activity of these diterpenoids is not good enough and the limited amounts restrict us to directly modify these agents.Isosteviol,easily obtained by acid hydrolysis of stevioside,is a tetracyclic diterpenoid,which has the similar skeleton structure.The MIC assay revealed that the activity of isosteviol was weak.In previous studies,researchers have shown that exo-methylene group is pivotal for the anti-bacterial activity.For this reason,we tried to build up a crucial structure fragment of exo-methylene cyclopentanone in the ring D of isosteviol.And the activity was four-fold more potent than isosteviol with MIC 64 μg/mL.Based on the skeleton,the TPP cation was further connected to target the mitochondria.And the activity was further improved eight-fold with MIC 8 μg/mL.To obtain more active derivatives,several derivatives with different linkers but the same α,β-unsaturated ketone core structure were synthesized,and finally resulted in the most potent antifungal compound Ⅱ-31 with MIC 0.5μg/mL.In addition,we observed that compound Ⅱ-31 was also active against other types of azole-resistant C.albicans strains suggesting that compound Ⅱ-31 could address the drug-resistance problems resulting from efflux pumps and other azole-resistance mechanisms.Flow cytometry analysis revealed that 11-31 remarkably increase the MMP of C.albicans and stimulate the generation of ROS,suggesting that the mitochondrial membrane was impaired.Confocal laser scanning microscopy(CLSM)observation based on Tom70-GFP imaging revealed that non-drug-treated cells had an extensive network of mitochondria displaying a tubular structure,whereas those in Ⅱ-31-treated cells presented an aggregated distribution and a disrupted tubular structure.Most Candida infections are associated with the formation of biofilms,which are highly recalcitrant to antifungal agents,on inert or biological surfaces.Our findings here showed that the compound 11-31 could not only prevent C.albicans biofilm formation but also kill preformed mature biofilms.Using Caenorhabditis elegans as an infectious model,compound Ⅱ-31 was observed to prolong the survival of C.albicans-infected C.elegans.In short,based on the skeleton of isosteviol,series of derivatives were synthesized with potent antifungal activity.The further structural optimization was guided by activity and finally resulted in the most potent antifungal compound 11-31.Further investigations revealed that compound 11-31 not only elicited mitochondria dysfunction and dramatically increased intracellular reactive oxygen species,but also it exhibited comparable ability to amphotericin B to eradicate preformed C.albicans biofilms.These studies suggest that compound 11-31 has a potential to be developed as a new generation of antifungal drug.The previous research of our group found that tetracyclic diterpenes withα,β-unsaturated ketone motif have potent antitumor activity.The antitumor activity of compound 11-14 was evaluated and we found that it has a moderate activity with IC5010 μM.Take it as a substrate,some derivatives were synthesized by forming a dimmer or by connecting a variety of unsaturated ketone groups.Meanwhile,we further access diverse nitrogen-containing electrophiles for targeting lysosome to improve anticancer activity.Although the activity of some derivatives were improved,it still haven’t met our expectation,and the structure needs to be further optimized.To date,little chemical effort has been directed toward modifications of the A-ring of isosteviol.Herein,we,for the first time,introduce α,β-unsaturated ketone or a five-membered unsaturated lactone in isosteviol A-ring and resulted in the lead compound III-32 with potent anticancer activity.In addition,modifications of the D-ring with Baeyer-Villiger rearrangement,Beckmann rearrangement and Mannich reaction monoconfigurational generated a series of diverse heterocyclic compounds.Not surprisingly,these new molecules have demonstrated potent antiproliferative effects against various human cancer cell lines,especially,compound Ⅲ-53 with the IC50 value of 0.29 μM against A549 cell lines.Further mechanisms research on compound III-53 proved that it damaged lysosome and induced LMP to cause cell death and had a potential to be developed as a noval anticancer agent.In conclusion,we synthesized several series of novel derivatives in a more convenient and efficient way by borrowing the skeleton of andrographolide and isosteviol,two easily accessible natural molecules.After structural optimization,several compounds with potent activity and powerful druggability were found,and higher value application of bulk natural products was realized.更多还原