Zampanolide and dactylolide are microtubule-stabilizing polyketides possessing potent cytotoxicity towards a variety of cancer cell lines. Using our understanding of the conformational preferences of the macrolide core in both natural products, we hypothesized that analogues lacking the C17-methyl group would maintain the necessary conformation for bioactivity while reducing the number of synthetic manipulations necessary for their synthesis. Analogues <b>3</b>, <b>4</b> and <b>5</b> were prepared <i>via</i> total synthesis, and their conformational preferences were determined through computational and high-field NMR studies. While no observable activities were present in dactylolide analogues <b>3</b> and <b>4</b>, zampanolide analogue <b>5</b> exhibited sub-micromolar cytotoxicity. Herein, we describe these efforts towards understanding the structure- and conformation-activity relationships of dactylolide and zampanolide.