Nine new compounds targeting the transient receptor potential vanilloid-4 (TRPV4) were synthesized and their biological activities toward TRPV4 were determined using freshly isolated mouse skin macrophages through live cell Ca2+ imaging assay. Three compounds 4b, 4c, and 4i exhibited higher percentages of in vitro activation of TRPV4 as 48.1%, 59.3% and 33.5%, which are comparable to 56.4% activation response of the reported TRPV4 agonist GSK1016790A (3). The compound 4i was chosen for 11C-radiosynthesis using its phenol precursor 4g to reacted with [11C]methyl iodide. The radiosynthesis was achieved with good radiochemical yield (16 ± 5%), high chemical and radiochemical purity (>95%), and high molar activity (16–21 GBq/μmol, decay corrected to the end of bombardment, EOB n ≥ 4). Furthermore, the initial ex vivo biodistribution study in rats showed that [11C]4i had higher uptake in kidney, liver and small intestine compared to other tissues with rapid washout.