Engineering Hydroxylase and Ketoreductase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of Belzutifan

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Real-Time Monitoring of In Situ Gas-Phase H/D Exchange Reactions of Cations by Atmospheric Pressure Helium Plasma Ionization Mass Spectrometry (HePI-MS)

An enclosed atmospheric-pressure helium-plasma ionization (HePI-MS) source avoids, or minimizes, undesired back-exchange reactions usually encountered during deuterium incorporation experiments under ambient-pressure open-source conditions. A simple adaptation of an ESI source provides an economical way of conducting gas phase hydrogen/deuterium (H/D) exchange reactions (HDX) in real time without the need for complicated hardware modifications. For example, the spectrum of [²H₈]­toluene recorded under exposed ambient conditions showed the base peak at <i>m</i>/<i>z</i> 96 due to fast leaching of ring hydrogens because of interactions with H₂O vapor present in the open source. Such D/H exchanges are rapidly reversed if the deuterium-depleted [²H₈]­toluene is exposed to D₂O vapor. In addition to the enumeration of labile protons, our procedure enables the identification of protonation sites in molecules unambiguously, by the number of H/D exchanges observed in real time. For example, molecules such as tetrahydrofuran and pyridine protonate at the heteroatom and consequently undergo only one H/D exchange, whereas ethylbenzene, which protonates at a ring position of the aromatic ring, undergoes six H/D exchanges. In addition, carbocations generated in situ by in-source fragmentation of precursor protonated species, such as benzyl alcohol, do not undergo any rapid H/D exchanges. Because radical cations, second-generation cations (ions formed by losing a small molecule from a precursor ion), or those formed by hydride abstraction do not undergo rapid H/D exchanges, our technique provides a way to distinguish these ions from protonated molecules

Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 1Process History and Development Strategy

An improved synthesis has been developed for belzutifan, a novel HIF-2α inhibitor for the treatment of Von Hippel–Lindau (VHL) disease-associated renal cell carcinoma (RCC). The efficiency of previous supply and commercial routes was encumbered by a lengthy 5-step sequence, needed to install a chiral benzylic alcohol by traditional methods. Identification and directed evolution of FoPip4H, an iron/α-ketoglutarate dependent hydroxylase, enabled a direct enantioselective C–H hydroxylation of a simple indanone starting material. While this enabling transformation set the stage for a greatly improved synthesis, several other key innovations were made including the development of a base-metal-catalyzed sulfonylation, a KRED-catalyzed dynamic kinetic resolution, and a facile SNAr reaction in water. Together, these improvements resulted in a significantly shorter synthesis (9 steps) versus the supply route (16 steps) and a 75% reduction in process mass intensity (PMI), while also removing the reliance on third-row transition metals and toxic solvents