Identification of a novel in vitro metabonate from liver microsomal incubations.
In vitro drug metabolism studies during the early drug discovery stage are becoming increasingly important. With the increasing demand for high throughput and quick turnaround time for in vitro metabolism studies, however, careful examination of the results and proper design of the experiments are still crucial. In this communication, we report the identification and mechanism of formation of a novel metabonate from incubations of a diamine-containing compound with liver microsomes. The metabonate appeared to be the major product, and its formation was NADPH- and microsomal protein-dependent. Liquid chromatography/mass spectrometry and NMR analysis of the metabonate indicated an extra carbon and unusual formation of an imidazolidine ring. Further studies revealed that this metabonate was not a true biotransformation product from the diamine compound itself in the microsomal incubation, but rather a product resulting from a condensation reaction between the compound and a metabolite of the solvent (alcohol) used in the incubation. When the microsomal incubations contained a small amount of methanol or ethanol as solvent, the alcohols were metabolized to formaldehyde or acetaldehyde, which then condensed with the diamine compound through an imine intermediate to form the metabonate. The compound itself was metabolically stable in vitro when acetonitrile or dimethyl sulfoxide was used as solvent. During the study of in vitro microsomal stability and metabolite identification of amine-containing compounds, the use of alcohol as solvent should be avoided.
Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Dr., Thousand Oaks, CA 91320-1799, USA. chunl@amgen.com
Chun
C
Li
Sekhar
S
Surapaneni
QingPing
Q
Zeng
Brian
B
Marquez
David
D
Chow
Gondi
G
Kumar
D000818:Animals; D016477:Artifacts; D004353:Drug Evaluation, Preclinical; D000431:Ethanol / Q000737:chemistry; D005029:Ethylenediamines / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D000432:Methanol / Q000737:chemistry; D008862:Microsomes, Liver / Q000378:metabolism*; D015394:Molecular Structure; D051381:Rats; D012997:Solvents / Q000737:chemistry
D005029:Ethylenediamines; D012997:Solvents; D000431:Ethanol; D000432:Methanol