Novel limonene phosphonate and farnesyl diphosphate analogues: design, synthesis, and evaluation as potential protein-farnesyl transferase inhibitors.

Limonene and its metabolite perillyl alcohol are naturally-occurring isoprenoids that block the growth of cancer cells both in vitro and in vivo. This cytostatic effect appears to be due, at least in part, to the fact that these compounds are weak yet selective and non-toxic inhibitors of protein prenylation. Protein-farnesyl transferase (FTase), the enzyme responsible for protein farnesylation, has become a key target for the rational design of cancer chemotherapeutic agents. Therefore, several alpha-hydroxyphosphonate derivatives of limonene were designed and synthesized as potentially more potent FTase inhibitors. A noteworthy feature of the synthesis was the use of trimethylsilyl triflate as a mild, neutral deprotection method for the preparation of sensitive phosphonates from the corresponding tert-butyl phosphonate esters. Evaluation of these compounds demonstrates that they are exceptionally poor FTase inhibitors in vitro (IC50 > or = 3 mM) and they have no effect on protein farnesylation in cells. In contrast, farnesyl phosphonyl(methyl)phosphinate, a diphosphate-modified derivative of the natural substrate farnesyl diphosphate, is a very potent FTase inhibitor in vitro (Ki=23 nM).

Stereospecific synthesis and biological evaluation of farnesyl diphosphate isomers.

[formula: see text] A unified, stereospecific synthetic route to the three geometric isomers of (E,E)-farnesyl diphosphate (E,E-FPP) (1, 2, and 3) has been developed. The key feature of this synthesis is the ability to control the stereochemistry of triflation of the beta-ketoester 10 to give either 11 or 14. Preliminary evaluation of these compounds with protein-farnesyl transferase indicates that 1 and 2 are surprisingly effective substrates; however, Z,Z-FPP (3) is a poor substrate and a sub-micromolar inhibitor.

Novel modifications to the farnesyl moiety of the a-factor lipopeptide pheromone from Saccharomyces cerevisiae: a role for isoprene modifications in ligand presentation.

The a-factor of Saccharomyces cerevisiae is a dodecapeptide pheromone [YIIKGVFWDPAC(farnesyl)-OCH3] in which posttranslational modification with a farnesyl isoprenoid and carboxymethyl group is required for full biological activity. Utilizing novel synthetic techniques and a well-characterized array of biological assays, we prepared original modifications to the farnesyl moiety of the pheromone in order to assess the importance of this part of the lipopeptide for biological activity. Specifically, the 3-methyl group was replaced to create analogs containing the ethyl, vinyl, tert-butyl, and phenyl moieties at the 3-position of the farnesyl chain. Subsequent biological analyses demonstrated that all of these modifications render an active pheromone, with the vinyl and ethyl analogs exhibiting higher activity than the native a-factor. However, the level of activity varied with the modification; the bulkier and more hydrophobic groups (tert-butyl and phenyl) exhibited lower biological activity than the smaller moieties (ethyl and vinyl). Furthermore, two analogs with phenyl substitutions that differ only in the presumed isomerization of the allylic double bond show up to an 8-fold difference in bioactivity. It has previously been surmised that the role of isoprenoid additions is solely to target the attached polypeptides to membranes by increasing their hydrophobicity. However, these studies demonstrate that even modest structural changes to the isoprenoid can significantly affect biological activity. These results are clearly inconsistent with a simple hydrophobic role for the isoprenoid and instead illustrate that it plays an active role in mediating optimal a-factor/receptor interaction.