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

Osteoporosis is the consequence of an imbalance between osteoclastic and osteoblastic activities with reduced bone mass and increased risk of fractures, particularly among postmenopausal women. Treatment of osteoporosis includes either inhibition of the excessive bone-resorbing activity of osteoclasts or stimulation of bone-forming activity of osteoblasts. Present therapeutic strategies concentrate on preventing further bone loss by using antiresorptive drugs, such as bisphosphonates, calcitonins, estrogens, but their effects on increasing or recovering bone mass are relatively small. On the other hand, parathyroid hormone (PTH) as an only clinically available drug with anabolic effect on bone by increasing osteoblast numbers and activity, while PTH increases the incidence of osteosarcoma. Thus, the development of new satisfactory bone anabolic agents that restore lost bone mass and stimulate fracture healing is in considerable demand.Bioassay-guided chemical isolation and structure elucidation revealed that the oleanolic acid (OA) and its glycosides were the bioactive constituents responsible for the antiosteoporosis activity. We previously reported that quinoxaline derivative of oleanolic acid could improve the inhibitory effect on osteoclast formation. Thus, in order to find more potent agent for the treatment of osteoporosis with better water solubility, we designed and synthesized novel quinoxaline derivatives of oleanolic acid modified by different amines at C-28 and different substituents in quinoxaline ring. The identification of isomers was confirmed by NOE (nuclear overhauser effect). The antiosteoporosis activity of the novel compounds was evaluated via MTT assay using osteoblast-like cells isolated from murine calvariae.1. Synthesis of oleanolic acid derivativesNovel oleanolic acid derivatives were prepared from OA. Oxidation of OA using a Na2WO4-H2O2 system gave key intermediate 1, which was sequentially oxidized in t-BuOK/t-BuOH/O2 system to give intermediate 2. Reactions of 2 with benzene-1, 2-diamine,3,4-diaminophenol,4-nitrobenzene-1,2-diamine,4-bromobenzene-1, 2-diamine and 4-iodobenzene-1,2-diamine afforded corresponding isomers 3-7 without further purification, respectively. Compounds 9-29 were prepared by stirring the mixtures 3-7 with oxalyl chloride in anhydrous DCM then treated with different amines. Reduction of 21-29 using Pd/C under H2 yielded compounds 30-38. Compounds 39a and 39b were prepared from 11a and 11b by Williamson reaction, respectively. Their structures were confirmed by 1H-NMR, 13C-NMR and ESI-MS.2. NOE analysisThe identification of the isomers was confirmed by NOE experiment. The nitro group was assigned to C-7’(compound 21a) by the NOE correlations between C23-H [δ1.46 (s,3H)] and C8’-H [δ8.93 (d,J=2.3Hz,1H)]. NOE correlations between signals C8’-H [δ7.33 (d,J= 2.0 Hz,1H)] and C23-H [δ1.42 (s,3H)] in compound 39a implied 3-hydroxypropoxy group should locate at C-7’. The location of the 3-hydroxypropoxy group at C-6’(compound 39b) was confirmed by the NOE correlations between C-23 and C-6’[δ7.87 (d, J= 7.5 Hz,1H)].3. Activity evaluationOsteoblasts are responsible for bone formation and synthesize almost all of the constituents of the bone matrix and direct its subsequent mineralization. The antiosteoporosis activity of the novel compounds was evaluated using osteoblast-like cells isolated from murine calvariae. Most compounds exhibited considerable cell proliferation compared to the control value by MTT assay, compound 31 possessed a higher proliferation rate than other compounds.The current results demonstrate that these new compounds could be used as promising leads for the development of a new class of antiosteoporosis agents. The further pharmacological evaluation of these compounds is underway in our laboratory.