The synthetic pathway of the target compounds and

The synthetic pathway of the target compounds , , , and is presented in . The intermediate was synthesized according to a reported method. The intermediate was prepared by mixing UA with an equimolar concentration of KCO, a catalytic amount of KI, and more than two equivalents of 1,2-dibromoethane or 1,3-dibromopropane in DMF and stirring at 50 °C for 6 h. The target compounds , , , and were prepared by reacting intermediate with 5-substituted-phenyl-1-tetrazoles () or amines (), respectively, in the presence of KI/KCO in refluxing acetone. The synthetic pathway of the target compounds , , , and is presented in . Jones reagent was used to prepare intermediate with UA, which was subsequently alkylated to yield intermediate . The series and compounds were synthesized using the same method described above to generate series and . The intermediate was reacted with sulfoxide chloride for 4 h in refluxing dichloromethane (DCM) to obtain acyl chloride, which was dissolved in anhydrous DCM and added to a mixture of DMF/DCM (1:1), trimethylamine, and 5-aminotetrazole in an ice bath. Following a 1 h reaction, the mixture was stirred for another 2 days at room temperature to yield compound . Compound was synthesized by reacting with phenyl hydrazine hydrochloride with a few drops of acetic 4sc in refluxing ethanol. Intermediate was prepared by mixing UA with corresponding anhydrides in the presence of DMAP and trimethylamine in an ice bath, followed by stirring overnight at room temperature. Compounds were synthesized by reacting with sulfoxide chloride in refluxing dichloromethane (DCM). Compound was prepared by hydrolyzing compound with 10%NaOH. The structures of all derivatives were characterized by H and C NMR spectroscopy and high-resolution mass spectrometry. The activities of the UA derivatives are summarized in , . To examine the effect of the UA derivatives on hypoxia-induced HIF-1α transcriptional activity, Hep3B cells were transfected with a luciferase reporter construct containing six hypoxia-response elements, and then exposed to hypoxia (1% O) for 24 h. Luciferase activity in the cell culture supernatants was subsequently measured. The cytotoxicity of each of the derivatives against Hep3B cells was evaluated using the MTT assay. UA and compounds A and B were used as positive controls. We found the majority of the synthesized compounds inhibited HIF-1α transcriptional activity. While six of the eight series and compounds exhibited moderate to good inhibitory activities, compound exhibited a potent inhibitory activity (IC 7.5 ± 0.2 µM). The substituents on the benzene ring attached to the tetrazole moiety were found to affect potency in an arbitrary manner, which meant that a meaningful structure–activity relationship could not be determined from the data. Similarly, we did not observe any significant correlation between inhibitory potency and the relative distance between UA and the tetrazole moiety. Most of the series and compounds containing a terminal free tetrazole moiety showed moderate inhibitory activities in the following order: position >  position >  position. Compounds with two carbon linkages between UA and the tetrazole moiety exhibited higher inhibitory activities compared with compounds with three carbon linkages. A comparison of the activities of compounds or with compounds containing a benzene group connected to the tetrazole ring suggested that HIF-1α inhibitory activity was enhanced in the absence of a benzene ring. Next, we compared the inhibitory activities of the compounds in series and to their series and counterparts. The only difference between these compounds is that the hydroxy group at the C-3 position in the series and compounds was oxidized by Jones’ reagent to create the series and compounds. All other substituents were equivalent. We concluded that the series and compounds were slightly less potent than their series and counterparts. Furthermore, the structure–activity relationships of the series and compounds were comparable to the compounds from series and .