1). Thus, PMC-C was prepared from commercial N-vinylpyrrolidinone towards (1) by formation of the enolate, then acylation with ethyl myristate to give 2. The vinyl protecting group was then removed with aqueous acid, giving PMC-C (3) as a racemic mixture at the readily epimerized stereocentre (Fig. 2). Figure 2 Synthesis of PMC analogs. For analogs PMC-D and �CF, commercial (R)-5-hydroxymethylpyrrolidine-2-one was converted to the protected derivative 4, using a literature procedure [23], then deprotonated and acylated with ethyl myristate, giving 5. Deprotection then afforded PMC-D (6). Alternatively, hydroxylation of 5 with oxygen and cerium chloride as catalyst according to the method of Christoffers [24] gave 7 which was then deprotected to yield PMC-F (8).

Use of the p-fluorophenyl protecting group conveniently resulted in essentially completely stereoselective hydroxylation, whereas other substituents on the benzene ring gave mixtures. The stereochemistry of 8 was established by X-ray crystallography, which revealed that the hydroxy group was trans to the hydroxymethyl substituent, as opposed to the cis relationship in pramanicin. It was therefore of interest to establish whether this hydroxy group and its stereochemistry are important for activity, and thus we included this compound in the test panel. We then performed a 24 hour cytotoxicity assay to search for the most potent analog against HCT116 cells. MTT reduction test which indirectly determines cell viability/proliferation by monitoring metabolic activity, revealed that 100 ��M PMC caused approximately 8% decrease in cell viability (Fig.

3). However, PMC-A which possesses a C=C double bond instead of an epoxy group in its side chain, decreased the cell viability by almost 70% compared to the untreated control. Notably, analogs PMC-C, -E and �CF all retained significant activity, indicating that an acylated pyrrolidinone (as in PMC-C), at most, is the key pharmacophore and that many of the substituents (epoxide, diene, C-5 hydroxymethyl group, and the C-3 and C-4 alcohol groups) are non-essential but enhance activity. Figure 3 PMC-A is the most cytotoxic compound among PMC analogs. PMC-A Induces Cell Death through Induction of Intrinsic Apoptotic Pathway In vitro effective doses of the potent PMC analog PMC-A (25�C100 ��M) caused cell death in a dose-dependent manner among all three HCT116 colon cancer cell lines (wt, p53?/?, and Bax ?/?) as indicated by increased Annexin-V affinities in cell populations (Fig.

GSK-3 4A). In order to analyze cell death and determine the optimum dose to use in a 24 hour time scale, we applied flow cytometry following Annexin-V staining of cells exposed to four physiologically relevant PMC-A concentrations. Cell death induction was evident for all doses in all cell lines and increased dose-dependently.