All of the AC ring-containing molecules synthesized were subsequently tested for both their ability to bind to tubulin (Kd) and to inhibit microtubule formation (Figure ). MTC (3) had the lowest binding constant of all compounds tested, and was the only compound to inhibit microtubule polymerization at 3 µM. Compounds 4, 5, and (aR)-6 had binding constants 5-30 fold less potent than 3, but were each capable of inhibiting microtubule polymerization at 33 µM. While iso-4 and (aS)-6 each appeared to bind to tubulin near the limits of the assays, Growth inhibition studies were carried out with the library against select cancer cell-lines, and this activity tracked closely with biochemical assays. Specifically, molecules that did not inhibit microtubule polymerization did not have any significant growth inhibition in cells, and the differences in binding constants of active molecules tracked closely with differences in growth inhibition (Figure ). (-)-colchicine was also tested as a control in cell-based assays, and showed comparable activity to MTC (3), consistent with literature precedence. 11a This data supports that the cytotoxicity of (aR)-6 is directly related to microtubule disruption, and the lower cytotoxicity is because of its lower affinity for tubulin. Furthermore, the activity differences between active compounds tracks with prior energetic well profiling of 3, 4, and (aR)-6, which suggested that 4 and 5 prefer an isocolchicine-type pose when unbound, and require uphill energetic climbs to achieve a dihedral angle near that of colchicine (see Scheme 1C). The lower activity of 5 is not as clear based on this data, but one possibility is that its broad energy well could lead to entropic penalties when bound.