title: A mechanistic and kinetic study of the beta-lactone hydrolysis of Salinosporamide A (NPI-0052), a novel proteasome inhibitor.
abstract: 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 beta-lactone-gamma-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 degrees 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 degrees C. To further confirm the reaction mechanism, the degradation was also performed in (18)O-enriched water and the degradation products subjected to HPLC separation prior to mass spectral analysis. Solubility and stability in (SBE)(7m)-beta-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 beta-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 (18)O-labeling studies proved that the mechanism involves acyl-oxygen bond cleavage and not a carbonium ion mechanism. 1 is unstable in water (t(90%) <or= 33 min at pH <5) and degrades via beta-lactone hydrolysis involving a normal ester hydrolysis mechanism (addition-elimination) resulting in acyl-oxygen bond cleavage. Captisol solubilized and stabilized 1 in aqueous solutions.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA.
authors.forename: Nunzio
authors.initials: N
authors.lastname: Denora
authors.forename: Barbara C M
authors.initials: BC
authors.lastname: Potts
authors.forename: Valentino J
authors.initials: VJ
authors.lastname: Stella
mesh_terms: D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D017666:Deuterium Oxide / Q000737:chemistry; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007783:Lactones / Q000737:chemistry*; D013058:Mass Spectrometry; D015394:Molecular Structure; D009994:Osmolar Concentration; D061988:Proteasome Inhibitors; D011758:Pyrroles / Q000737:chemistry*; D012995:Solubility; D012996:Solutions / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry; D047392:beta-Cyclodextrins / Q000737:chemistry
chemical_list: D002021:Buffers; D004791:Enzyme Inhibitors; D007783:Lactones; D061988:Proteasome Inhibitors; D011758:Pyrroles; D012996:Solutions; D047392:beta-Cyclodextrins; D014867:Water; C475865:marizomib; D017666:Deuterium Oxide