Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 3─Development of a Scalable Enzymatic Oxidation Process

Abstract

is an FDA-approved hypoxia inducible factor 2α inhibitor for the treatment of Von Hippel− Lindau disease-associated renal cell carcinoma. One of the key synthetic steps is an enzymatic benzylic hydroxylation of an indanone. Prior to optimization, hydroxylation reactions required dilute conditions (100 volumes of water) and high enzyme loading (50 wt %) to achieve the target conversion, which was unsuitable for a long-term manufacturing process. Reduction of reaction volumes introduced additional complexity for large-scale operations due to the low solubility of the reaction components. In addition to reaction development, it was also necessary to find an optimal method of protein residue removal from the reaction mixture to isolate a high-quality product. We present here our efforts to overcome these challenges and ultimately successfully developed, optimized, and demonstrated at a multikilogram scale a process with improved process mass intensity using only a 7.5 wt % enzyme, 25 volumes of water, and 1-octanol as a unique additive.

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

“…Overall, the combined improvements from the screening hit to the commercial enzyme variant represent a >400,000-fold improvement when considering enzyme loading (267-fold), reaction concentration (40-fold), yield (23-fold), and enantioselectivity (1.7-fold). With this highly engineered enzyme and several process improvements, we could operate under process-intensified conditions that were required for a robust and productive manufacturing process . In total, this single transformation removes 4 steps and reduces the PMI of this transformation by 43% over the former 5-step sequence.…”

Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 4─Applications of Process Analytical Technology in Heterogeneous Biocatalytic Hydroxylation

“…Ferrous iron and α-ketoglutarate (α-KG) dependent hydroxylase has been known to activate the C–H bond and catalyzes reactions using dioxygen as an oxidant. , l -Pipecolic acid 4 hydroxylase from the Fusarium oxysporum family (FoPip4H) has been chosen as the target enzyme to install the hydroxyl group onto the indanone (Figure A). During development, multiple rounds of directed evolution were performed to improve the enzyme activity, stability, and selectivity. , In early process development, the reaction was performed using 100 volumes of water with conditions shown in Figure A (the corresponding effective concentration of indanone is 43.8 mM assuming no volume change during dissolution). Due to the limited solubility of indanone (<1 g/L), the reaction system is a thin slurry with multiple phases (aqueous liquid, organic liquid, solid, and gas).…”

“…During development, multiple rounds of directed evolution were performed to improve the enzyme activity, stability, and selectivity. , In early process development, the reaction was performed using 100 volumes of water with conditions shown in Figure A (the corresponding effective concentration of indanone is 43.8 mM assuming no volume change during dissolution). Due to the limited solubility of indanone (<1 g/L), the reaction system is a thin slurry with multiple phases (aqueous liquid, organic liquid, solid, and gas). The hydroxyindanone has a slightly better solubility of ∼10 g/L; therefore, it is fully dissolved under these conditions (Figure A).…”

“…This enables exact control over the dissolved oxygen concentration and provides a lever for investigating excess oxygen effects on reaction performance. In the early phase of enzyme evolution, some variants of hydroxylase (e.g., mutants from round 6) show overoxidation of hydroxyindanone to form a diketone impurity (see Scheme S1 in the Supporting Information for the structure), which further inhibits the enzyme activity. , Therefore, the reaction was tested with controlled dO at the 10% saturation level to explore possible further improvement (using the round 9 backbone). However, no significant distinctions were observed in kinetics, conversion, and diketone impurity for the reactions performed with and without controlled dO (Figure S1 and Table S1 in the Supporting Information).…”

Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 6─Development of a Nucleophilic Aromatic Substitution in Water

“…Toward this end, a new endgame strategy was developed involving a novel biocatalytic oxidation and an α-fluorination/dynamic kinetic reduction (DKR) sequence using a ketoreductase (Figure ). Furthermore, our previously developed deoxyfluorination conditions were successfully adapted and implemented in the synthesis of the penultimate aryl fluoride 1 . Thus, the final area of focus was the introduction of the biaryl ether via S N Ar of the aryl fluoride with phenol 2 .…”