Among the natural histone deacetylase inhibitors (HDACi), the bicyclic depsipeptide macrolactone FK228 stands out for its unique chemical structure and mechanism of action. In order to expand the chemical diversity, exploiting the FK228 peculiar structure, we have synthesized a collection of 24 simplified novel analogs. A first series consists of bicyclic macrolactones, where the carboxy terminus of the natural compound was substituted by peptidomimetic aminomethylphenylacetic acid derivatives. These analogs, 7a-i, showed submicromolar cytotoxic activity, even though very low inhibitory activity against HDAC enzymes, suggesting that most probably they behave with a mechanism different from the natural compound. One of the most active members in the group, 7g, was evaluated in vivo and exhibited significant antitumor activity. This evidence supports that the activity is unrelated to HDAC inhibition and these compounds represent a novel series of promising active agents. Another analog series consists of monocyclic macrolactones, 9a-c and 10a-d which lack the disulfide bridge and bear the protected sulfur on the linear external chain; they showed similar cytotoxic activities compared to the natural compound, but proved to be very sensitive to the nature of the sulfur protection. In fact, when the sulfur was protected by an 1-octanoyl residue, like in 9b, the product displayed a one digit nanomolar activity. The results provide evidence that our approach may be followed to develop novel series of FK228 analogs.