Separation of the four stereoisomers of a potent inhibitor (L-694,458) of human leukocyte elastase and its determination in human plasma using achiral/chiral chromatography with column switching.
A stereoselective method based on high-performance liquid chromatography (HPLC) and ultraviolet detection at 235 nm for the separation of the four possible stereoisomers of compound 1 ((S-(R*,S*))-2-(4-((4-methylpiperazin-1-yl)carbonyl)phenoxy)-3,3-d iethyl-N-(1-(3,4-(methylenedioxy)phenyl)butyl)-4-oxo-1-azetidinecarbo xamide, L-694,458), a potent, selective, and orally active human leukocyte elastase (HLE) inhibitor, in human and monkey plasma has been developed. The molecule of 1 contains two chiral centers and is being developed as a single stereoisomer with the absolute configuration S and R in positions 'a' and 'b', respectively. Although the baseline separation of each of the two pairs of enantiomers (SS/RR and SR/RS) was achieved on a single chiral column (Chiralcel OD-H) using hexane methyl-t-butyl ether (MTBE)-methanol 80/10/10, (v/v/v) as a mobile phase (alpha(RS,SR) = 2.03, alpha(RR,SS) = 4.97), only partial separation of RS from RR was observed under these conditions (k'RS = 3.32, k'RR = 3.08). Baseline separation of all four stereoisomers from each other and from endogenous plasma components required the initial chromatography of the two diastereomeric racemates (SS/RR and SR/RS) on the achiral silica column (50 x 4.6 mm, 5 microm), followed by column switching and further separation of the stereoisomers on a Chiralcel OD-H column (250 x 4.6 mm, 5 microm) using isopropanol (IPA)-hexane diethylamine (DEA), 65/35/0.3, (v/v/v) on both columns as a mobile phase. The drug was extracted from basified (pH 11) plasma (1 ml) using liquid liquid extraction with MTBE. After evaporation of the extract to dryness, the residue was reconstituted in the mobile phase (200 microl) and part of the extract (125 microl) was injected into the HPLC system. Using this method, it was demonstrated that after oral dosing of monkeys at 40 mg kg(-1) with 1 the only stereoisomer detected in the post-dose plasma samples was the starting material 1, and no inversion of the configuration at positions 'a' and 'b' of 1 had occurred in vivo. Based on this observation, a non-chiral assay for 1 in human plasma was also developed. The method was validated in the concentration range 10-500 ng ml(-1) with the assay precision (expressed as the coefficient of variation, CV) better than 9% and assay accuracy in the range of 95-107% of the nominal concentrations at all concentrations within the standard curve range. The total run time in the non-chiral assay was 12 min. The details of both chiral and non-chiral methods are provided.
Zagrobelny
J
J
Merck Research Laboratories, West Point, PA 19486, USA.
Matuszewski
B K
BK
Kline
W F
WF
Vincent
S H
SH
D000818:Animals; D001384:Azetidines / Q000097:blood* / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D008253:Macaca mulatta; D010879:Piperazines / Q000097:blood* / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism
D001384:Azetidines; D010879:Piperazines; C105999:DMP 777; D019272:Leukocyte Elastase