Design, Synthesis And Osteoclastogenesis Inhibitory Activity Of Betulinic Acid Derivatives

With the purpose of finding more potent and safe osteoclastogenesis inhibitors which could be used as potential leads for the development of a novel type of anti-osteoporosis agents, a series of betulinic acid (BA) derivatives were designed, synthesized, and evaluated in this dissertation as inhibitors against RANKL-induced osteoclast differentiation on RAW264.7cells.The inhibitory activity of BA on RAW264.7cells was tested and the results showed that BA is a moderate inhibitor of osteoclast differentiation (IC50=20μM). Therefore, we designed and synthesized a series of compounds by modification of A and E ring of BA, and evaluated their inhibitory activities on AW264.7cells.A series of BA derivatives with five-member and six-member heterocyclic rings fused at C-2and C-3positions were synthesized. The inhibitory effects of all the heterocyclic ring-fused betulinic acid derivatives were evaluated against RANKL-induced osteoclast differentiation on RAW.264.7cells. The results showed that the inhibitory effect of these heterocyclic ring-fused derivatives was much stronger than that of their lead compound BA, especially the pyrazole derivative13, its IC50value was0.1μM, which was about200-fold more potent than the lead compound BA. Further, we can see that the inhibition decreased as hydrophilic substituent groups were introduced into the five-membered heterocyclic rings and the inhibition increased as hydrophilic substituent groups were introduced into the six-membered pyrimidine derivatives. To examine whether the impaired osteoclastogenesis in the presence of13is due to the decrease in viability of the precursor cells, we investigated the cytotoxicity of this compound upon osteoclast precursors RAW264.7, and the results showed that13has little cytotoxicity on osteoclasts within the effective concentrations. To examine whether13inhibits osteoclast in vivo, we used the ovariectomy mouse model to mimic menopause-induced bone loss in women. The results showed that13could prevent OVX-induced bone loss and could be used as potential leads for the development of a new type of antiosteoporosis agent. A series of BA derivatives with modification of the C-28and C-19positions were also synthesized. Their structure-activity relationship was studied by evaluation of the inhibitory effects on RAW264.7cells. The results showed that the inhibition decreased as the C-28carboxyl group was modification to C-28methyl ester and C-28hydroxymethyl groups or C-19isopropenyl were modification to hydroxyl and carboxyl groups. The hydrogenation of C-19isopropenyl had only minimal effect on inhibition.In order to make the preparation of BA derivatives easy, we developed a highly efficient reaction system for the preparation of betulonic acid. With commercially natural product betulin as starting material, betulonic acid was prepared efficiently with a total yield of86%by a combination of selective oxidation with IBX, followed with n-Bu4NMnO4. The method is more operable and less toxic due to no heavy-metal-catalyst was used. The IBA, byproduct of IBX, could be recycled and re-oxidized to IBX.In conclusion, we report BA heterocyclic derivatives as a series of new chemical entities for the first time. Especially13, which exhibited potent inhibition of osteoclastogenesis both in vitro and in vivo, could be used as a promising lead for the development of a new class of antiosteoporosis agents. While13had poor oral bioavailability, and how to ameliorate the bioavailability is in progressing.