Two new ring-size varying analogues of ipomoeassin F were synthesized and evaluated.   Improved cytotoxicity (IC₅₀: from 1.8 nM) and in vitro protein translocation inhibition (IC₅₀: 35 nM) derived from ring expansion imply that the binding pocket of Sec61α (isoform 1) can accommodate further structural modifications, likely in the fatty acid portion. Streamlined preparation of a key diol intermediate enabled gram-scale production allowing us to establish ipomoeassin F is biologically active in vivo (MTD: ~3 mg/kg).