A Mechanistic and Kinetic Study of the β-Lactone Hydrolysis of Salinosporamide A (NPI-0052), A Novel Proteasome Inhibitor

The aim of the present study was to investigate the mechanism of aqueous degradation of Salinosporamide A (NPI‐0052; 1), a potent proteasome inhibitor that is currently in Phase I clinical trials for the treatment of cancer and is characterized by a unique β‐lactone‐γ‐lactam bicyclic ring structure. The degradation of 1 was monitored by HPLC and by both low‐ and high‐resolution mass spectral analyses. Apparent first‐order rate constants for the degradation at 25°C were determined in aqueous buffer solutions (ionic strength 0.15 M adjusted with NaCl) at various pH values in the range of 1 to 9. Degradation kinetics in water and in deuterium oxide were compared as a mechanistic probe. The studies were performed at pH (pD) 4.5 at 25°C. To further confirm the reaction mechanism, the degradation was also performed in 18O‐enriched water and the degradation products subjected to HPLC separation prior to mass spectral analysis. Solubility and stability in (SBE)7m‐β‐cyclodextrin (Captisol) solutions were also determined. The hydrolytic degradation of 1, followed by both HPLC and LC/MS, showed that the drug in aqueous solutions gives a species with a molecular ion consistent with the β‐lactone hydrolysis product (NPI‐2054; 2). This initial degradant further rearranges to a cyclic ether (NPI‐2055; 3) via an intramolecular nucleophilic displacement reaction. The kinetic results showed that the degradation of 1 was moderately buffer catalyzed (general base) and the rate constants were pH independent in the range of 1-5 and base dependent above pH 6.5. No acid catalysis was observed. The kinetic deuterium solvent isotope effect (KSIE) was 3.1 (kH/kD) and a linear proton inventory plot showed that the rate‐determining step involved only a single proton transfer. This suggested that a neighboring hydroxyl group (as opposed to a second water molecule) facilitated water attack at pD 4.5. Mass spectral analysis from the 18O‐labeling studies proved that the mechanism involves acyl‐oxygen bond cleavage and not a carbonium ion mechanism. 1 is unstable in water (t90% ≤ 33 min at pH <5) and degrades via β‐lactone hydrolysis involving a normal ester hydrolysis mechanism (addition‐elimination) resulting in acyl‐oxygen bond cleavage. Captisol solubilized and stabilized 1 in aqueous solutions. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2037-2047, 2007