Ingenol is a diterpenoid with unique architecture and has derivatives possessing important anticancer activity, including the recently Food and Drug Administration-approved Picato, a first-in-class drug for the treatment of the precancerous skin condition actinic keratosis. Currently, that compound is sourced inefficiently from Euphorbia peplus. Here, we detail an efficient, highly stereocontrolled synthesis of (+)-ingenol proceeding in only 14 steps from inexpensive (+)-3-carene and using a two-phase design. This synthesis will allow for the creation of fully synthetic analogs of bioactive ingenanes to address pharmacological limitations and provides a strategic blueprint for chemical production. These results validate two-phase terpene total synthesis as not only an academic curiosity but also a viable alternative to isolation or bioengineering for the efficient preparation of polyoxygenated terpenoids at the limits of chemical complexity.
The complex diterpenoid (+)-ingenol possesses a uniquely challenging scaffold and constitutes the core of a recently approved anti-cancer drug. This full account details the development of a short synthesis of 1 that takes place in two separate phases (cyclase and oxidase) as loosely modeled after terpene biosynthesis. Initial model studies establishing the viability of a Pauson-Khand approach to building up the carbon framework are recounted. Extensive studies that led to the development of a 7-step cyclase phase to transform (+)-3-carene into a suitable tigliane-type core are also presented. A variety of competitive pinacol rearrangements and cyclization reactions were overcome to develop a 7-step oxidase phase producing (+)-ingenol. The pivotal pinacol rearrangement is further examined through DFT calculations, and implications for the biosynthesis of (+)-ingenol are discussed.
The diterpenoid ester
ingenol mebutate (IngMeb) is the active ingredient
in the topical drug Picato, a first-in-class treatment for the precancerous
skin condition actinic keratosis. IngMeb is proposed to exert its
therapeutic effects through a dual mode of action involving (i) induction
of cell death that is associated with mitochondrial dysfunction followed
by (ii) stimulation of a local inflammatory response, at least partially
driven by protein kinase C (PKC) activation. Although this therapeutic
model has been well characterized, the complete set of molecular targets
responsible for mediating IngMeb activity remains ill-defined. Here,
we have synthesized a photoreactive, clickable analogue of IngMeb
and used this probe in quantitative proteomic experiments to map several
protein targets of IngMeb in human cancer cell lines and primary human
keratinocytes. Prominent among these targets was the mitochondrial
carnitine-acylcarnitine translocase SLC25A20, which we show is inhibited
in cells by IngMeb and the more stable analogue ingenol disoxate (IngDsx),
but not by the canonical PKC agonist 12-O-tetradecanoylphorbol-13-acetate
(TPA). SLC25A20 blockade by IngMeb and IngDsx leads to a buildup of
cellular acylcarnitines and blockade of fatty acid oxidation (FAO),
pointing to a possible mechanism for IngMeb-mediated perturbations
in mitochondrial function.
SignificanceSubtype-selective modulation of ion channels is often important, but extremely difficult to achieve for drug development. Using Nav1.7 as an example, we show that this challenge could be attributed to poor design in ion channel assays, which fail to detect most potent and selective compounds and are biased toward nonselective mechanisms. By exploiting different drug binding sites and modes of channel gating, we successfully direct a membrane potential assay toward non–pore-blocking mechanisms and identify Nav1.7-selective compounds. Our mechanistic approach to assay design addresses a significant hurdle in Nav1.7 drug discovery and is applicable to many other ion channels.
Using structure- and ligand-based
design principles, a novel series
of piperidyl chromane arylsulfonamide Nav1.7 inhibitors
was discovered. Early optimization focused on improvement of potency
through refinement of the low energy ligand conformation and mitigation
of high in vivo clearance. An in vitro hepatotoxicity hazard was identified
and resolved through optimization of lipophilicity and lipophilic
ligand efficiency to arrive at GNE-616 (24), a highly
potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response
in a Nav1.7-dependent mouse model, and site-directed mutagenesis
was used to identify residues critical for the isoform selectivity
profile of 24.
Nav1.7 is an extensively investigated target for pain
with a strong genetic link in humans, yet in spite of this effort,
it remains challenging to identify efficacious, selective, and safe
inhibitors. Here, we disclose the discovery and preclinical profile
of GDC-0276 (1) and GDC-0310 (2), selective
Nav1.7 inhibitors that have completed Phase 1 trials. Our
initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable
overall pharmacokinetics profile. To further derisk the predicted
human pharmacokinetics and enable QD dosing, additional optimization
of the scaffold was conducted, resulting in the discovery of a novel
series of N-benzyl piperidine Nav1.7 inhibitors. Improvement
of the metabolic stability by blocking the labile benzylic position
led to the discovery of GDC-0310 (2), which possesses
improved Nav selectivity and pharmacokinetic profile over 1.
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