Design,Synthesis And Biological Evaluation Of Pyrazole-Fused Betulinic Acid Derivatives As Anti-osteoarthritis Agents

Osteoarthritis is the most common degenerative bone and joint disease with high morbidity and disability,which brings huge economic costs to society and individuals.In 2018,osteoarthritis was officially identified as a "serious disease with unmet medical need" by the U.S.Food and Drug Administration(FDA).Osteoarthritis affects joints and surrounding tissues such as cartilage,subchondral bone,and synovium.The most common symptom of osteoarthritis is pain.An imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation leads to the bone destruction in osteoarthritis.Inflammatory cytokines such as interleukin-1,interleukin-6 and tumor necrosis factor-alpha are able to stimulate osteoclasts differentiation by promoting the expansion of osteoclast precursors and upregulating the expression of receptor activator for nuclear factor-κB ligand(RANKL),mediating the erosive process in osteoarthritis development.At present,the treatment of osteoarthritis tends to be conservative and the existing drugs mainly focus on relieving pain,with limited efficacy and often accompanied by various side effects.The market prospect of osteoarthritis treatment drugs is broad.The development of new osteoarthritis drugs with fewer systemic adverse reactions and effective efficacy has always been a research focus,but very little progress has been made so far.The strategy of "one drug,multiple targets" is important for developing therapeutic drugs for complex diseases such as osteoarthritis.Natural products are an important source of drug discovery.Their unique skeletons and multi-target advantages have always attracted medicinal chemists to modify and optimize them.Pentacyclic triterpenes,secondary metabolic derivatives of isoprene,are widely distributed in nature as free saponins or aglycones and show a variety of therapeutic effects.Betulinic acid is a natural lupane-type pentacyclic triterpenoid compound.Betulinic acid and its derivatives have a variety of biological activities,including antiviral,antibacterial,antimalarial,anticancer,anti-inflammatory,insect-repellent,anti-influenza,antioxidant and so on.According to existing studies,the pyrazole-fused betulinic acid derivative at C-2 and C-3 positions had a significant inhibitory activity on osteoclastogenesis and a certain anti-inflammatory activity,which gave the clue for structural transformation of this thesis.Starting from the pyrazole-fused betulinic acid derivative,152 novel betulinic acid derivatives were designed and synthesized at the carboxylic acid site,the pyrazole site and the isopropenyl site respectively in this thesis.The osteoclast differentiation inhibitory activity,inflammatory factor production inhibitory activity and cytotoxicity of these newly synthesized derivatives were initially evaluated.The preliminary structure-activity relationship was summarized to guide the further optimization.First,among the derivatives modified at the carboxylic acid site,the results of in vitro activity showed that the osteoclast inhibitory activity of several amine and urea compounds was significantly better than that of betulinic acid;Among the pyrazole site modified compounds,the introduction of long-chain alkanes or nitrogen-containing saturated heterocycles increases the inflammatory factor inhibitory activity of the derivatives;Among the compounds modified at the isopropenyl site,retaining the double bond and introducing flexible side chains or saturated cycloalkanes can help improve the inflammatory factor inhibitory activity of the compounds.And the preferred compound G46 showed potent inhibition of osteoclast differentiation as well as strong inhibitory activity on the production of interleukin-1β secreted by RAW264.7 cells.Compound G46 was confirmed to interfere with the activation of nuclear factor-κB inhibitorα/nuclear factor-κB and p38 signal transduction and prevent the subsequent induced activation of nuclear factor of activated T-cell cytoplasmic 1 and its target genes.In the classic model of rat osteoarthritis induced by sodium iodoacetate,treatment with compound G46 achieved promising therapeutic effects including alleviated pain,improved cartilage damage and subchondral bone change,and the results were dosedependent.This thesis preliminarily verified the therapeutic activity of betulinic acid derivatives on osteoarthritis,and follow-up studies are still in progress.Currently,there are no disease-modifying osteoarthritis drugs(DMOADs)that can improve the pathological process of osteoarthritis.Considering the market gap of novel osteoarthritis drugs,the research of this thesis may provide a new strategy for the development of osteoarthritis treatment candidates.