Synthesis And Proliferation Activity On Osteoblast-like Cells Of Oleanolic Acid Derivatives

Osteoporosis is a systematic bone disease, characterized by low bone mass and structural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk. It is a global public health problem currently affecting hundreds of millions of people worldwide, particularly postmenopausal women and older men. In the case of osteoporosis, efforts have been primarily made on the development of drugs that block bone resorption by inhibiting osteoclasts differentiation or activity, or promote bone formation by increasing osteoblasts functions.Bisphosphonates are widely used agents in the management of osteoporosis by prevention of excessive osteoclast-mediated bone resorption, however, they associate with unpleasant gastric side effects. Recombinant parathyroid hormone (PTH) is the only anabolic agent currently used for treatment of osteoporosis by stimulating bone formation. But PTH is an injectant and its clinical use is limited to a 2-year period due to its increased incidence of osteosarcoma. Therefore, development of new alternative agents for the treatment of osteoporosis is still highly desirable.Oleanolic acid (OA) and its glycosides have a wide spectrum of important biological activities including anti-osteoporosis effect. In our previous studies, we have already synthesized and evaluated anti-osteoporosis activity of several heterocyclic derivatives in A-ring of oleanolic acid and C28-amides using natural amino acids, and most of the compounds inhibited osteoclast formation. In this paper, as a continuing research of our project to further improve the antiosteoporosis activity, series of novel oleanolic acid derivatives via modifications in A-ring and C-28 position with heterocycles were designed and synthesized, and their bioactivity were evaluated via MTT assay using osteoblast-like cells isolated from murine calvariae.1. Synthesis of oleanolic acid derivativesOleanolic acid derivatives were prepared from OA. Oxidation of OA using a Na2WO4-H2O2 system gave key intermediate 1. The key intermediate 2 was obtained in a high yield by Claisen condensation of 1 with ethyl formate in the presence of sodium methoxide in toluene. Then, enamines were obtained by a condensation reaction of 2 with various amines; pyrazole and ioxazole OA were prepared via a reaction of 2 with hydrazine hydrochloride and hydroxylamine hydrochloride in EtOH, respectively. Final amides were obtained as follows:17-Acyl chlorides of OA derivatives were prepared by stirring amines or pyrazole or ioxazole OA with thionyl chloride, then removed the solvent and treated with corresponding amines in the presence of triethylamine afforded amides。2. Activity evaluationOsteoblasts are responsible for bone forming, producing most of the proteins present in the bone extracellular matrix (ECM) and controls the mineralization of this ECM. The antiosteoporosis activity of the novel compounds were evaluated using osteoblast-like cells isolated from murine calvariae. Most compounds exhibited considerable cell proliferation compared to the control value by MTT assay, and compound C5 possessed a higher proliferation rate than others. The further pharmacological evaluation of these compounds is underway in our laboratory.