G proteins act as molecular switches in G protein-coupled receptor signaling pathways and are key mediators for numerous important physiological processes. The natural product, cyclic depsipeptide YM-254890, together with the structurally similar FR900359, is the only known selective inhibitor of the G _q/11 subfamily of G proteins. We recently reported the first total synthesis of YM-254890 and FR900359, followed by synthesizing analogues to perform structure–activity relationship studies. However, incomplete information about their structure–activity relationship prevents the further development of potent and structurally simplified analogues. Herein we report the first systematic structure–activity relationship study toward the N-methyldehydroalanine moiety in YM-254890, by designing and synthesizing seven new analogues. Pharmacological characterization of the seven compounds for G _q/11 -, G _i/o - and G _s -mediated signaling showed that the simplified analogue YM-19 is the most potent G _q/11 inhibitor among the new analogues. This study provides information for the future design of potent and simplified YM-254890 analogues.