Sunday, August 10, 2008
P17

Enzymatic O-demethylation of Apixaban, a potent inhibitor of blood coagulation factor Xa

Thomas P. Tully, Steve Y. Chen, Michael A. Montana, William L. Parker, and Ramesh N. Patel. Process Research & Development, Bristol-Myers Squibb, One Squibb Drive, New Brunswick, NJ 08903

The BMS culture collection was screened for microorganisms capable of the enymatic O-demethylation of BMS-562247, a potent and orally bioavailable coagulation factor Xa inhibitor also known as Apixaban.   Bacterial and fungal strains were initially tested by the addition of 0.5 mg/ml substrate to actively growing cultures followed by whole broth assay.  Strains of both Cunninghamella elegans and Nocardioides albus were found to yield the desired product in low (<5%) yield.  Based upon follow-up studies as well as easier handling of the Nocardioides strain, this organism was selected for pilot plant scale-up.  During the course of this work, yield was found to be significantly dependent upon substrate concentration, medium composition, starting pH, and incubation temperature.  Factorial and central composite DOE analyses to determine critical medium components, interactions, and concentrations were performed and contributed to further improvement in biotransformation efficiency.  For most of the early screening and optimization work described, DMSO was used due to low BMS-562247 solubility, but it was later determined that the substrate could be readily dissolved and heat-sterilized in a water:propylene glycol (1:1) mixture, leading to even higher product yield while minimizing the risk of contamination.  The best medium composition was then used to scale the process into 15-L tanks, and the final biotransformation yield was ca. 80%.  The product was removed from the whole broth by adsorption to XAD-16 resin followed by centrifugation.  This facilitated separation of the cells, leaving product-containing resin for easy processing.  Subsequent purification and isolation of the final demethylated product is also described.

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