The p53 inhibitor pifithrin-α forms a sparingly soluble derivative via intramolecular cyclization under physiological conditions

The transcription factor p53 coordinates cell cycle arrest and apoptosis in response to DNA damage. Pifithrin-alpha (PFT-alpha) is a small molecule inhibitor of p53 activity that is frequently used in cell culture studies of p53 function. Here we report an investigation of the stability of this compound. PFT-alpha rapidly converts to a planar tricyclic derivative, with a half-life of 4.2 h under physiological conditions. This spontaneous conversion greatly alters the structural and physicochemical properties of the drug. PFT-alpha has a pK(a) of 9.11 and is an ionic species in physiological medium, whereas the tricyclic derivative has a pK(a) of 4.36 and exists as the neutral free base at pH 7. The tricyclic derivative is very hydrophobic, with a log P of 4.26. Although PFT-alpha is generally used at 10-30 mu M concentration, the aqueous solubility of its derivative is only 0.2 mu M, and it can form a visible precipitate under conditions of typical use. The conversion of PFT-alpha proceeds via an intramolecular cyclization reaction involving the imine and carbonyl groups. Modification of the carbonyl function creates a stable analogue of PFT-alpha that remains soluble indefinitely. These results provide a strategy for the rational design of PFT-alpha analogues that exhibit predictable stability, hydrophobicity, and aqueous solubility.