The cellular activity of 30 and 33 was further validated in a quantitative real-time polymerase chain reaction (qPCR) assay monitoring the relative mRNA level of carbonic anhydrase 9 (CA9), a known HIF target gene and hypoxia-regulated marker (Figure ). Response to the treatment with both compounds as well as 1 (VH298) and VH032, a first-generation VHL inhibitor (Figure ), was assessed in dose-dependent manner in HeLa cells. While VH032 upregulated the target gene CA9 only moderately at high concentrations, significant increase in CA9 mRNA levels was observed with compound 1 (VH298) and inhibitors 30 and 33 already at 50 µM concentration. Despite inducing strong stabilization of HIF-1α and HIF-1α-OH on protein level, compound 33 did not surpass the performance of 1 in inducing CA9 mRNA levels. Compound 30 however greatly outperformed VH298 in the same assay. Together, the cellular data qualifies compound 30 as the most potent VHL inhibitor.