The Construction Of Similar D/E Ring Moiety Of Betulinic Acid On Steroidal Scaffold Via Diels-Alder Reaction And Preliminary Bioactivity Test

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 ofâ–³¹⁶-α,β-unsaturated ketones.Because of the steric effect by angular methyl group at C-18,selective reaction in C-20 ofâ–³¹⁶-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).