Metabolism of Strained Rings: Glutathione S-transferase–Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation

Abstract

AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy) azetidin-1-yl)(5-(4-methoxyphenyl)-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 glutathionerelated metabolites, including the glutathionyl, cysteinyl, cysteinylglycinyl, and mercapturic acid conjugates. The formation of these metabolites was not inhibited by coincubation with the cytochrome P450 (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 GSTA1, 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 GSTA2-2 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 a 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-2oxa-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 ringopening reaction involving a nucleophilic attack by glutathione, which does not involve prior cytochrome P450-catalyzed bioactivation of the substrate and which is catalyzed by glutathione transferases, is reported. We propose a mechanism involving the protonated cyclic aminyl intermediate that undergoes nucleophilic attack by glutathione thiolate anion in this reaction, catalyzed by glutathione transferases.

The 2nd Alpine Winter Conference on Medicinal and Synthetic Chemistry

“…The studies defined oxetanes as the first non‐epoxide class of substrates for human mEH, which was previously known to catalyze the hydrolytic ring opening of electrophilic epoxide‐containing drugs, drug metabolites, and xenobiotics. An analogous metabolic sequence was also revealed with a series of melanin‐concentrating hormone receptor 1 antagonists, which contained a spirocyclic four‐membered azetidine ring [4] . The azetidine ring underwent ring scission in a glutathione transferase/glutathione‐dependent fashion, which did not involve prior cytochrome‐P450‐catalyzed bioactivation of the substrates.…”

Intramolecular Ring-Opening Decomposition of Aryl Azetidines

“…Numerous examples of improved potency and/or pharmacokinetic performance for azetidines vs related heterocycles have been reported in the literature. − However, the small ring size of azetidines introduces strain and the potential for decomposition pathways that may not be observed with larger ring systems . The strain also introduces the potential for metabolic ring-opening via reaction with glutathione . Metabolic ring opening has also been reported for structurally similar oxetanes .…”

Metabolism and Bioactivation: It’s Time to Expect the Unexpected

“…30 The azetidine-containing AZD1979 67 is being developed as a melanin-concentrating hormone receptor 1 antagonist designed for the treatment of obesity, containing a spiromoiety with an azetidine ring and an oxetane ring. 31 When radiolabeled [C 14 ]-AZD-1979 was incubated with human liver S9 fraction in the presence of aminobenztriazole, a pan P450 inhibitor, seven metabolites were identified by liquid chromatography with radiometric detection. One metabolite (59) was characterized by high resolution mass spectrometry to be the m/z of AZD1979 with the addition of 307.0706 Da, consistent with the addition of glutathione.…”