Semisynthesis of 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (1), a natural flavone that binds with high affinity to tubulin, was performed from hesperidin, the very abundant Citrus flavanone, by a five-step sequence. The last step of the synthesis also gave rise to 5,3′-dihydroxy-3,6,7,4′-tetramethoxyflavone (= casticin or vitexicarpin) (10), 5,3′-dihydroxy-3,7,8,4′-tetramethoxyflavone (= gossypetin 3,7,8,4′-tetramethyl ether) (11), and, unexpectedly, 5,7,3′-trihydroxy-3,6,8,4′-tetramethoxyflavone (12) and 5,3′-dihydroxy-8-dimethylamino-3,6,7,4′-tetramethoxyflavone (= 8-dimethylaminocasticin) (13). Cytotoxicity and antitubulin activity of these five flavones, as well as 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (= ayanin) (14) and intermediate 6,8-dibromo-ayanin (8), were evaluated. Comparison of the responses confirmed and clarified the influence of the A-ring substitution pattern on the biological activity.