Helenalin Analogues Targeting NF-κB p65: Thiol Reactivity and Cellular Potency Studies of Varied Electrophiles

Abstract

Helenalin is a pseudoguaianolide natural product that targets Cys38 within the DNA binding domain of NF-κB transcription factor p65 (RelA). Helenalin contains two Michael acceptors that covalently modify cysteines: a α-methylene-γ-butyrolactone and a cyclopentenone. We recently reported two simplified helenalin analogues that mimic the biological activity of helenalin and contain both electrophilic moieties. To determine the individual contributions of the Michael acceptors toward NF-κB inhibition, we synthesized a small library of helenalin-based analogues containing various combinations of α-methylene-γ-butyrolactones and cyclopentenones. The kinetics of thiol addition to a subset of the analogues was measured to determine the relative thiol reactivities of the embedded electrophiles. Additionally, the cellular NF-κB inhibitory activities of the analogues were determined to elucidate the contributions of each Michael acceptor to biological potency. Our studies suggest the α-methylene-γ-butyrolactone contributes most significantly to the NF-κB inhibition of our simplified helenalin analogues.

Keywords: Michael acceptors; bis-electrophiles; cysteine reactive; helenalin; p65/RelA transcription factor.

Figure 1

(top) Helenalin is a pseudoguaianolide natural product with two Michael acceptors. 1a and 1b mimic the biological activity of helenalin.^[9b] (bottom) We report a synthesis of helenalin analogues with varied Michael acceptors, the kinetic reactivities of helenalin analogues to thiols, and the NF-κB inhibitory activities of a number of helenalin analogues.

Figure 2

Simplified helenalin derivatives 1a (left spectra) and 1b (right spectra) were reacted with cysteamine in DMSO-d₆.^[12] The reactivities of the cyclopentenone and α-methylene-γ-butyrolactone of 1a and 1b were qualitatively assessed by ¹H NMR analyses of protons H¹–H⁴ as a function of cysteamine concentration and time. Reversibility of cysteamine adducts were determined by dilution studies with CDCl₃. Proton signals were normalized to a TMS internal standard. These data are representative of three independent experiments.

Scheme 1

Reduction of the exocyclic methylene on derivatives 2 and 3.

Scheme 2

Synthesis of simplified helenalin analogues 9 – 20.