【作者】 张璐；

【导师】 陈瑛； 夏鹏；

【作者基本信息】 复旦大学 ， 药物化学， 2008， 硕士

【摘要】 桦木酸（betulinic acid）是美国北卡大学药学院于上世纪九十年代末从桃金娘科植物中提取分离出的具有明显的抗HIV-1活性(EC₅₀=1.4μM,TI=13)的五环三萜类化合物,美国北卡大学药学院在其结构基础上对其3-位羟基进行不同酯结构修饰和生物活性观察,发现衍生物PA-457的抗HIV-1活性(EC₅₀＜3.5×10^-4μM,TI＞20000)显著提高。而同时Mayaux等人对桦木酸C-28位侧链进行结构修饰合成了一系列酰胺衍生物,其中IC 9564对HIV-1抑制活性强,治疗指数最佳(EC₅₀=0.053μM,IC⁵⁰＞10μM)。该两类桦木酸衍生物既不是HIV逆转录酶抑制剂也不是蛋白酶抑制剂的作用机制。C-3侧链修饰衍生物可以特异地扰乱CA前体（p25）裂解成成熟的CA（p24）,使得装配完成的病毒颗粒缺损、无感染能力,即在病毒生命周期较晚期阻断HIV-1的复制;而C-28侧链修饰衍生物通过靶向HIV-1囊膜糖蛋白来抑制HIV-1囊膜介导的膜融合,即在病毒生命周期较早阶段发挥抗病毒融合的活性。目前桦木酸的来源主要依赖于植物提取或半合成,规模化生产的原料是目前需要解决的问题之一。本课题组在长期从事甾体及抗病毒（包括HIV-1）药物研究中,首次发现一些简单甾体化合物具有明显的抑制HIV活性。因此本课题旨在以来源方便、易进行化学修饰的甾体骨架为母核,构建与桦木酸类三萜化合物相似的五环结构,并在相应活性位点（对应于桦木酸骨架的C-3,C-20和C-28）进行结构修饰,研究甾体骨架化合物的抗HIV-1活性及其构效关系。本文以孕甾双烯醇酮醋酸酯为原料,利用D环的α,β-不饱和羰基,将20-位羰基转变为羟基后进行消除或直接烯醇化构造二烯结构,然后与带有羧基或酯基的亲二烯体发生Diels-Alder环加成反应,成功构建了甾体的E环并引入游离羧基,并在3-位引入桦木酸结构中的药效团3′,3′-二甲基二酸酯结构,得到目标化合物。全文共合成化合物35个,其中21个未见文献报道的新化合物。在合成的12个目标化合物中,3个为甾体E环侧链具有游离羧基的目标化合物,6个为3-位侧链成3′,3′-二甲基丁二酸酯,3个为既具有E环侧链游离羧基又在3-位侧链成3′,3′-二甲基丁二酸酯的目标化合物。对本论文中合成的部分化合物和目标化合物进行了HIV生物活性检测。但由于HIV生物活性测定在国外进行,周期较长,因此目前手头已获得的活性数据十分有限,且多数为各个设计路线起始步骤的一些结构简单的中间体,参考意义不大,具体构效分析需获得全部目标化合物的活性反馈后再进行讨论和研究。同时对本论文合成的一些化合物进行了血管内皮生长因子的生物活性检测,送检的11个化合物均具有不同程度的抑制活性,尤其是化合物46(EC₅₀=0.0472 mM,TI=18.51)和25(EC₅₀=0.0121 mM,TI=10.03)活性和选择性优于阳性对照2-ME(EC₅₀=0.4964 mM,TI=3.33)。该11个化合物在16、17及20位结构差异较大,但均具一定活性,显示存在较大的进一步修饰空间,同时也暗示与靶点可能存在多个结合点。因此,这对进一步的深入研究构效关系提供了一定的参考。本文在探索三条合成路线的合成研究工作中,对一些化合物和反应的化学行为进行了初步的研究和讨论。a)通过改造甾体D环△¹⁶-α,β-不饱和羰基,完成一条成功构建与桦木酸类似的五环结构合成路线,同时在E环侧链顺利引入游离羧基,为E环C-20进一步进行取代基修饰奠定基础。b)在甾体母核D环△¹⁶-α,β-不饱和羰基的反应活性研究中发现C-18角甲基对20-位基团的反应产生较大的位阻影响。在利用甲基碘化镁对△¹⁶-α,β-不饱和羰基的格氏加成及利用硼氢化钠对其进行还原的反应中,由于C-18角甲基的影响使得相当数量的试剂从空间位阻相对较小的C-16进攻,而生成近半量的1,4-加成副产物:我们通过使用分子体积更小的试剂或活性更强的试剂解决了△¹⁶-α,β-不饱和羰基选择性加成问题。c)本论文还分别摸索了两种适用于甾体△¹⁶-17-叔醇和△¹⁶-17-仲醇的消除反应条件。△¹⁶-17-叔醇在弱碱性条件及酸性条件下均以较高收率得到消除产物;而△¹⁶-17-仲醇,在10%H₂SO₄/Dioxane（wt/wt）条件下可以得到较好收率的消除产物。更多还原

【Abstract】 Betulinic acid,isolated from the plant Syzygium claviflorum,has a pentacyclic triterpene skeleton with several kinds of biological properties,especially the promising anti-HIV activity.Modifications of its C-3 and C-28 gave two different kinds of betulinic acid derivatives,which demonstrated extremely potent inhibitory activity against HIV-1 in H9 lymphocytic cells.Unlike that of popular NNRTIs and PIs,C-3 side chain derivatives（e.g.PA-457） specifically interfere with the cleavage of p25 to p24,thus resulting in non-infectious virus particles.Whereas C-28 side chain derivatives（e.g.IC9564） was reported to inhibit HIV-1 envelope-mediated membrane fusion by targeting HIV-1 envelope glycoproteins.Therefore,these two derivatives above represented two distinct classes of betulinic acid derivatives with different anti-HIV profiles.Current studies on obtaining betulinic acid mainly rely on the extraction from plants. Although,there are also reports that betulinic acid derivatives can be prepared by semi-synthesis methods from small molecular compounds,the scale production of betulinic acid is still a big problem needing to solve.In our previous research,some steroidal compounds with simple structures showed promising anti-HIV activities.Therefore,the aim of this thesis is to construct pentacyclic steroidal scaffold derivatives simulating betulinic acid in corresponding functional sites.Steroidal intermediates could be gained from a rich resource and they are easier to modify.We hope to explore their anti-HIV activities and SAR by introducing C-3,C-28 pharmacophores of betulinic acid into relative positions in our designed pentacyclic steroids.In this thesis,16-Dehydropregnenolone acetate（16-DPA） was treated with Grignard reagent or reduced directly to offer the 20-hydroxyl compound;then the elimination was followed to produce several diene molecules.These dienes reacted with maleic anhydride or other dienophile with carboxylic or ester group to build up a pentacyclic structure.The further esterification of C-3 hydroxyl group with 2,2-dimethyl-butanedioic anhydride proceeded corresponding esters.During the research,35 compounds have been synthesized in total,of which 21 compounds are new.And 12 target steroidal derivatives containing pentacycles with C-3 and C-28 pharrnacophores of betulinic acid.A few intermediates synthesized have been screened for the HIV-1 replication in H-9 lymphocytes.However,the quantity is not enough for a detailed SAR study at the moment because of the time-consuming bioactivity feedback.More intermediates and target compounds have already been under test and we could have further discussions and studies of structure-activity relationship only when getting the data.In additional,some compounds in this thesis have been sent for bioactivity test of vascular endothelial growth factor inhibition.It has been found that the inhibitory activity and selectivity of compound 46（EC50=0.0472 mM,TI=18.51） and compound 25(EC₅₀=0.0121 mM,TI=10.03) were both higher than that of positive control compound 2-ME(EC₅₀=0.4964 raM,TI=3.33)。These 11 screened compounds,which were quite diversified in their molecular structures,all possessed anti-VEGF activity of a certain extent.This observation reminded us that it needs further modification and implied that it may exist several binding areas to target. Therefore,it provided some reference for further study of SAR.We have finished three synthetic ways,and have taken a careful investigation on several unexpected chemical behaviors of some compounds and reactions encountered in the research.The study results are summarized as followed:a) Completed a general way for synthesize a series of pentacyclic steroids as betulinic acid scaffold via addition or reduction of unsaturated ketones,elimination and coupling with desirable dienophile.Also we have introduced carboxylic group at the side chain of E ring and it benefited to further modifications of C-20 of E ring.b) Studied the special reaction activity of△¹⁶-α,β-unsaturated ketones.Because of the steric effect by angular methyl group at C-18,selective reaction in C-20 of△¹⁶-α,β-unsaturated ketones became more difficult.In Grignard addition with methyl magnesium iodide and the reduction with sodium borohydride,except for getting 20-hydroxyls as target compounds,a substantial amount of 1,4-addition products has been yielded.Angular methyl group at C-18 affected the reagents to choose to attack C-16 from the less hindered side.We have utilized reagents with a stronger reductive activity to solve these problems.c) Found two different methods for the elimination of C-20 tertiary and secondary alcohols.C-20 tertiary alcohol could be eliminated in a high yield under acid or base condition;while C-20 secondary alcohol was eliminated to offer a good yield in the presence of 10%H₂SO₄/dioxane（wt/wt）.更多还原