Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine de 
novo biosynthesis pathway. It is located on the mitochondrial inner membrane and 
contributes to the respiratory chain by shuttling electrons to the ubiquinone 
pool. We have discovered ascofuranone (1), a natural compound produced by 
Acremonium sclerotigenum, and its derivatives are a potent class of HsDHODH 
inhibitors. We conducted a structure-activity relationship study and have 
identified functional groups of 1 that are essential for the inhibition of 
HsDHODH enzymatic activity. Furthermore, the binding mode of 1 and its 
derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we 
show that these inhibitors bind at the ubiquinone binding site. In addition, the 
cytotoxicities of 1 and its potent derivatives 7, 8, and 9 were studied using 
human cultured cancer cells. Interestingly, they showed selective and strong 
cytotoxicity to cancer cells cultured under microenvironment (hypoxia and 
nutrient-deprived) conditions. The selectivity ratio of 8 under this 
microenvironment show the most potent inhibition which was over 1000-fold higher 
compared to that under normal culture condition. Our studies suggest that under 
microenvironment conditions, cancer cells heavily depend on the pyrimidine de 
novo biosynthesis pathway. We also provide the first evidence that 1 and its 
derivatives are potential lead candidates for drug development which target the 
HsDHODH of cancer cells living under a tumor microenvironment.