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Drug Metab Dispos. 2019 Sep 6. pii: dmd.119.088658. doi: 10.1124/dmd.119.088658. [Epub ahead of print]

Metabolism of strained rings: glutathione S-transferase catalyzed formation of a glutathione conjugated spiro-azetidine without prior bioactivation.

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AstraZeneca R&D Molndal.
AstraZeneca R&D.
Virginia Tech.
AstraZeneca R&D;


AZD1979, 3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone, is a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity. In this study, metabolite profiles of AZD1979 in human hepatocytes revealed a series of glutathione (GSH) related metabolites including the glutathionyl, cysteinyl-, cysteinylglycinyl- and mercapturic acid conjugates. The formation of these metabolites was not inhibited by co-incubation with the P450 inhibitor 1-aminobenzotriazole. In efforts to identify the mechanistic features of this pathway, investigations were performed to characterize the structure of the glutathionyl conjugate M12 of AZD1979 and to identify the enzyme system catalyzing its formation. Studies with various human liver subcellular fractions established that the formation of M12 was NAD(P)H-independent and proceeded in cytosol and S9 fractions but not in microsomal or mitochondrial fractions. The formation of M12 was inhibited by ethacrynic acid, an inhibitor of glutathione S-transferases (GSTs). Several human recombinant GSTs including GST A1, A2-2, M1a, M2-2, T1-1, and GST from human placenta were incubated with AZD1979. All GSTs tested catalyzed the formation of M12 with GST A2-2 being the most efficient. Metabolite M12 was purified from rat liver S9 incubations and its structure elucidated by NMR. These results establish that M12 is the product of the GST-catalyzed glutathione attack on the carbon atom α to the nitrogen atom of the strained spiro-azetidinyl moiety to give, after ring opening, the corresponding amino-thioether conjugate product, a direct conjugation pathway that occurs without the prior substrate bioactivation by P450. SIGNIFICANCE STATEMENT: The investigated compound, AZD1979, contains a 6-substituted-2-oxa-6-azaspiro[3.3]heptanyl derivative that is an example of strained heterocycles, including spiro-fused ring systems, that are widely used in synthetic organic chemistry. An unusual azetidinyl ring-opening reaction involving nucleophilic attack by glutathione, that does not involve prior P450-catalyzed bioactivation of the substrate and that is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by GS- in this reaction, catalyzed by glutathione transferases.


NMR; UPLC; drug design; glutathione conjugates/glutathione transferases/GST; hepatocytes; mass spectrometry/MS; metabolite identification

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