Ultrasound-assisted synthesis of new bisphosphonate-betulin conjugates and preliminary evaluation of their cytotoxic activity.

Bisphosphonates (BPs) are a well-established group of drugs that have been used for decades in the prevention and treatment of osteoporosis and cancer treatment-induced bone loss. Their unique properties such as high bone affinity, enzymatic stability as well as a multidirectional biological activity prompt the creation of BP conjugates. In this study, we designed and synthesized three new bisphosphonate conjugates with betulin, a natural product with a high safety profile and a broad spectrum of biological activity. The designed conjugates differed in the type of linker used and the number of bisphosphonate moieties attached (mono- or disubstituted derivatives). The proposed method for their synthesis proceeds under mild reaction conditions and gives good yields of products. In addition, as we have shown, the reaction can be assisted by ultrasound, which significantly reduced the reaction time (from 48 hours to 2 hours) and improved the overall product yield (up to 92%). The cytotoxicity of the new conjugates was evaluated against osteosarcoma (U-2 OS), lung adenocarcinoma (A549) and gastric adenocarcinoma (AGS) cell lines. The results of preliminary biological studies showed that the obtained conjugates had improved solubility compared to that of betulin and exhibited a cytotoxic effect on all three tested cell lines at the micromolar level. The betulin analog having two bisphosphonate groups 6 demonstrated the highest cytotoxic activity against tested cell lines (IC50 between 5.16 and 6.21 μM).