The flavone backbone is a well-known pharmacophore present in a number of substrates and inhibitors of various P450 enzymes. In order to find highly potent and novel P450 family I enzyme inhibitors, an acetylene group was incorporated into six different positions of flavone. The introduction of an acetylene group at certain locations of the flavone backbone lead to time-dependent inhibitors of P450 1A1. 3'-Ethynylflavone, 4'-ethynylflavone, 6-ethynylflavone, and 7-ethynylflavone (KI values of 0.035-0.056 μM) show strong time-dependent inhibition of P450 1A1, while 5-ethynylflavone (KI value of 0.51 μM) is a moderate time-dependent inhibitor of this enzyme. Meanwhile, 4'-ethynylflavone and 6-ethynylflavone are highly selective inhibitors toward this enzyme. Especially, 6-ethynylflavone possesses a Ki value of 0.035 μM for P450 1A1 177- and 15-fold lower than those for P450s 1A2 and 1B1, respectively. The docking postures observed in the computational simulations show that the orientation of the acetylene group determines its capability to react with P450s 1A1 and 1A2. Meanwhile, conformational analysis indicates that the shape of an inhibitor determines its inhibitory selectivity toward these enzymes.