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Hypoxia-inducible factor 1 (HIF-1) is the central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. Inhibition of HIF-1 is expected to result in the attenuation of hypoxia-inducible genes, which are vital to many aspects of tumor biology, including adaptative responses for survival under anaerobic conditions. To identify small molecules inhibiting the HIF-1 pathway, we did a biological screen on a 10,000-membered natural product-like combinatorial library. The compounds of the library, which share a 2,2-dimethylbenzopyran structural motif, were tested for their ability to inhibit the hypoxic activation of an alkaline phosphatase reporter gene under the control of hypoxiaresponsive elements in human glioma cells. This effort led to the discovery of 103D5R, a novel small-molecule inhibitor of HIF-1a. 103D5R markedly decreased HIF-1a protein levels induced by hypoxia or cobaltous ions in a dose- and timedependent manner, whereas minimally affecting global cellular protein expression levels, including that of control proteins such as HIF-1h ,I nBaa, and hh-actin. The inhibitory activity of 103D5R against HIF-1a was clearly shown under normoxia and hypoxia in cells derived from different cancer types, including glioma, prostate, and breast cancers. This inhibition prevented the activation of HIF-1 target genes under hypoxia such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (Glut-1). Investigations into the molecular mechanism showed that 103D5R strongly reduced HIF-1a protein synthesis, whereas HIF-1a mRNA levels and HIF-1a degradation were not affected. 103D5R inhibited the phosphorylation of Akt, Erk1/2, and stressactivated protein kinase/c-jun-NH2-kinase, without changing the total levels of these proteins. Further studies on the mechanism of action of 103D5R will likely provide new insights into its validity/applicability for the pharmacologic targeting of HIF-1aa for therapeutic purposes. (Cancer Res 2005; 65(2): 605-12)