Design, synthesis and evaluation of wound healing activity for β-sitosterols derivatives as potent Na+/K+-ATPase inhibitors.

β-Sitosterols, is a common steroid that can be identified in a variety of plants and their efficacy in promoting wound healing has been demonstrated. Na⁺/K⁺-ATPase, more than a pump, its signal transduction function for involvement in cell growth regulation attracts widespread concern. The Na⁺/K⁺-ATPase/Src receptor complex can serve as a receptor involved in multiple signaling pathways including promoting wound healing pathways. To finding potent accelerating wound healing small molecular, we choose the high inhibitory activity of Na⁺/K⁺-ATPase and non-cardiotoxic natural compound, β-sitosterol as the substrate. A series of β-sitosterol derivatives were designed, synthesized and evaluated as potential Na⁺/K⁺-ATPase inhibitors. Among them, compounds 31, 47, 49, showed improved inhibitory activity on Na⁺/K⁺-ATPase, with IC₅₀ value of 3.0 μM, 3.4 μM, 2.2 μM, which are more potent than β-sitosterol with IC₅₀ 7.6 μM. Especially, compound 49 can induce cell proliferation, migration and soluble collagen production in L929 fibroblasts. Compared to model, compound 49 can accelerate wound healing in SD rats. Further studies indicated that 49 can activate the sarcoma (Src), uptake the protein kinase B (Akt), extracellular signal-regulated kinase (ERK) proteins expression in a concentration dependent manner. Finally, binding mode of compound 49 with Na⁺/K⁺-ATPase was studied, which provides insights into the determinants of potency and selectivity. These results proved β-stitosterol derivative 49 can serve as an effective inhibitor of Na⁺/K⁺-ATPase and potential candidate for accelerating wound healing agents.