Discovery of Potent Parthenolide-Based Antileukemic Agents Enabled by Late-Stage P450-Mediated C—H Functionalization

Abstract

The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes for reelaborating this complex natural product have represented a fundamental obstacle toward further optimization of its pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage sp3C—H bond functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for carrying out the highly regioselective hydroxylation of two aliphatic sites (C9and C14) in parthenolide carbocyclic backbone. By chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial structure-activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity against normal mature and progenitor hematopoietic cells. By identifying two ‘hot spots’ for improving the anticancer properties of parthenolide, this study highlights the potential of P450-mediated C—H functionalization as an enabling, new strategy for the late stage manipulation of bioactive natural product scaffolds.

Centrifugal partition chromatography elution gradient for isolation of sesquiterpene lactones and flavonoids from Anvillea radiata

“…The structures of 9␣-hydroxyparthenolide and 9␤-hydroxyparthenolide (Fig. 6) were deduced by comparison of their spectra data with those described in the literature [11][12][13]28] Both compounds 1 and 2 showed similar spectra, thus confirming the assumption of isomeric compounds. The 1 H-NMR spectral data of 9␣-hydroxyparthenolide showed general features similar to those of 9␤-hydroxyparthenolide.…”

“…The chemical shifts are reported in ppm (ı scale) and all coupling constants (J) values are in hertz (Hz). Assignments were based on 1 H NMR and 13 C NMR and two-dimensional NMR spectra as homonuclear and hetero-nuclear correlations, as well as comparison with the data in the literature [11,13,28].…”

Micelle Delivery of Parthenolide to Acute Myeloid Leukemia Cells

“…Unfortunately, PTL has not shown anti-leukemia activity in vivo due to poor bioavailability. 8,25 To increase aqueous solubility and prolong systemic circulation, PTL analogs have been developed 43 and have shown enhanced bioavailability and bioactivity in vivo. 25 Still, systemic delivery of small molecule drugs has many challenges, including nonspecific cell uptake, off-target organ accumulation and toxicity, clearance by extravasation, inability to overcome multidrug resistant mechanisms, and degradation by enzymes and other cell secreted factors.…”

Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis

“…More recently, incorporation of unnatural amino acids into P450 BM3 also allowed for enhancement of activity and alteration of regioselectivity. 15 Effort has also been invested into using computational modelling 16,17 and fingerprinting 17,18,19 of P450s to guide rational engineering of the enzymes to obtain novel regio-and stereoselectivities. Mammalian P450s, on the other hand, have generated great interest mostly because of their important role in drug metabolism.…”