Electrochemical generation of electrophilic drug metabolites: characterization of amodiaquine quinoneimine and cysteinyl conjugates by MS, IR, and NMR.
The chemical reactivity of electrophilic metabolites usually prevents their detection in vivo since, by definition, they are relatively short-lived and are likely to undergo one or more structural modifications to form more stable final products. Electrochemical oxidation provides a means to generate reactive metabolites in an environment without the presence of such nucleophiles. This paper describes the results of our MS, MS/MS, NMR, IR, and computational studies on oxidation products (and conjugates) that have been generated electrochemically from the antimalarial agent amodiaquine. The electrophilic quinoneimine metabolite of amodiaquine was the major oxidation product following electrochemical oxidation at +600 mV. The absence of biological nucleophiles in the electrochemical experiment facilitated (i) the acquisition of a clean IR spectrum of the amodiaquine quinoneimine and (ii) the addition of biologically relevant nucleophiles under controlled conditions. The addition of cysteine gave four cysteinyl conjugates, while the addition of glutathione gave four glutathionyl conjugates. The product ion spectra of the conjugates formed in the electrochemical experiment were used to identify suitable fragments for selected reaction monitoring (SRM) to selectively search for these conjugates in human liver microsomal (HLM) incubations. The four cysteinyl conjugates, as well as the four glutathionyl conjugates, were also detected as metabolites in HLM. The experiment with cysteine was repeated on a preparative scale that allowed characterization of the major conjugates by (1)H NMR. Desethylamodiaquine, the major metabolite formed in human liver microsomes, was also generated electrochemically by oxidation of amodiaquine at +1200 mV followed by reduction at -800 mV. In conclusion, the EC-ESI/MS technique provides the unique opportunity to generate reactive metabolites in the absence of biological nucleophiles, which enables studies that can give insight into the nature of these reactive intermediates. Such knowledge is valuable for risk assessment of new compound classes and can be complementary to computer-based structure-activity relationships of carcinogenicity, mutagenicity, and teratogenicity.
Jurva
Ulrik
U
AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden. ulrik.jurva@astrazeneca.com
Holmén
Anders
A
Grönberg
Gunnar
G
Masimirembwa
Collen
C
Weidolf
Lars
L
D000655:Amodiaquine / Q000031:analogs & derivatives* / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004563:Electrochemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013055:Spectrophotometry, Infrared
C094705:amodiaquine quinoneimine; D000655:Amodiaquine; D003545:Cysteine