Design, Synthesis And Bioactivity Research Of Steroidal Betulinic Acid Analogues With E Hetero-rings

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-4,C-20 and C-28 pharmacophores of betulinic acid into relative positions in our designed pentacyclic steroids.In this thesis,epiandrosterone and dehydroepiandrosterone were reacted with ethyl formate or dimethyl oxalate to introduce formyl groups or 1'-hydroxy-2'-carboxylic acid methyl ester-ethylidene groups to 16-positions of these two compounds.The products were treated with four different hydrazines to form pyrazol E rings on the steroidal scaffolds.The pentacyclic structures of the forming compounds were similar to the betulinic acid.The further esterification of C-3 hydroxyl group with 2,2-dimethyl-butanedioic anhydride proceeded corresponding esters.During the research,56 compounds have been synthesized in total,of which 41 compounds are unknown compounds,and 16 compounds were target compounds.A part of synthesized compounds 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.In the 10 screened compounds,compound 20,21,30 and 38 exhibited considerable anti-HIV activities under the concentration of 2.5μg/mL.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 of compound 20(EC₅₀=0.024μM,TI=8.40),21(EC₅₀=0.015μM, TI=5.64),31(EC₅₀=0.208μM,TI=4.60) and 37(EC₅₀=0.033μM TI=4.66) were higher than that of positive control compound 2-ME(EC₅₀=0.50μM,TI=3.33)。In synthetic research,several unexpected chemical behaviors of some compounds and reactions were carefully investigated.In the attempt to introduce dimethyls to 4-position of 4-en-3-one steroids or steroidal derivatives,we found that the D ring structure can influence the selectivity between 4-dimethylation and 3-O-methylation through a remote effect.Seven 4-en-3-one steroids or steroidal derivatives with different D ring structures and similar A,B and C ring structures were reacted with the same reagents in the same condition,of which three were converted into 3-O-methylation products and four were converted into 4-dimethylation products.We also found that the structure of A ring can influence on the reactions of D ring too.Such a remote effect on the reactivity in steroidal skeleton was investigated and discussed.