This report describes the development of a target-protein-oriented natural-products-isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5'- O-methyl-3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via the TPO-NAPI method developed using GLI1 protein-immobilized beads. 5'- O-Methyl-3-hydroxyflemingin A (3) inhibited Hh signaling (IC 7.3 μM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5'- O-Methyl-3-hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5'- O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 ( K = 7.7 μM). Moreover, 5'- O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cell characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM. These results suggest that Hh inhibitors can efficiently suppress the activity of cancer stem cells.

Pulmonary arterial hypertension (PAH) is a rare and severe progressive disorder characterized by high pulmonary artery pressure. Chronic hypoxia causes a metabolic disorder and the Warburg effect in pulmonary arterial smooth muscle cells (PASMCs). Pyruvate dehydrogenase kinase 1 (PDK1) is a key enzyme in Warburg effect increased by hypoxia‐inducible factor (HIF‐1). We constructed a cell‐based luciferase assay system for HIF‐1 inhibitors. Using this system, six HIF‐1 inhibitors were identified. Among these inhibitors, the effect of tagitinin C (1) on PASMC was investigated. Tagitinin C (1) clearly decreased the amount of HIF‐1β and the HIF‐1 target PDK1. This result indicates that HIF‐1 inhibitors effectively decrease PDK1 activity, which is a cause of the metabolic disorder and Warburg effect observed in PASMCs. Identifying naturally occurring HIF‐1 inhibitors could provide novel insights into the development of PAH medications.

A stereoselective synthetic route to chiral C3-spirooxindole-and C2-spiropseudoindoxyl-pyrrolidines was accomplished by an enantioselective organocatalyzed Pictet-Spengler reaction of tryptamines and isotryptamines followed by a diastereoselective oxidative rearrangement using eco-friendly oxidants (i. e., NaOCl • 5H 2 O and Oxone ® ). This sequential reaction enables rapid access to chiral C3-spirooxindole-and C2-spiropseudoindoxyl-pyrrolidines in a one-pot process. A Wnt signaling inhibitory assay of the prepared enantioenriched spiro compounds demonstrated that they exhibited moderate activities.