Insights into the Novel Hydrolytic Mechanism of a Diethyl 2‑Phenyl-2-(2-arylacetoxy)methyl Malonate Ester-Based Microsomal Triglyceride Transfer Protein (MTP) Inhibitor

Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent w...

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Published in:Chemical research in toxicology 2012-10, Vol.25 (10), p.2138-2152
Main Authors: Ryder, Tim, Walker, Gregory S, Goosen, Theunis C, Ruggeri, Roger B, Conn, Edward L, Rocke, Benjamin N, Lapham, Kimberly, Steppan, Claire M, Hepworth, David, Kalgutkar, Amit S
Format: Article
Language:English
Subjects:
Benzamides - metabolism
Benzamides - pharmacology
Carrier Proteins - antagonists & inhibitors
Glutathione - metabolism
Humans
Hydrolysis
Malonates - metabolism
Malonates - pharmacology
Microsomes, Liver - metabolism
Phenylpropionates - metabolism
Tandem Mass Spectrometry
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Summary:Inhibition of intestinal and hepatic microsomal triglyceride transfer protein (MTP) is a potential strategy for the treatment of dyslipidemia and related metabolic disorders. Inhibition of hepatic MTP, however, results in elevated liver transaminases and increased hepatic fat deposition consistent with hepatic steatosis. Diethyl 2-((2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)­phenyl)­acetoxy)­methyl)-2-phenylmalonate (JTT-130) is an intestine-specific inhibitor of MTP and does not cause increases in transaminases in short-term clinical trials in patients with dyslipidemia. Selective inhibition of intestinal MTP is achieved via rapid hydrolysis of its ester linkage by liver-specific carboxylesterase(s), resulting in the formation of an inactive carboxylic acid metabolite 1. In the course of discovery efforts around tissue-specific inhibitors of MTP, the mechanism of JTT-130 hydrolysis was examined in detail. Lack of 18O incorporation in 1 following the incubation of JTT-130 in human liver microsomes in the presence of H2 18O suggested that hydrolysis did not occur via a simple cleavage of the ester linkage. The characterization of atropic acid (2-phenylacrylic acid) as a metabolite was consistent with a hydrolytic pathway involving initial hydrolysis of one of the pendant malonate ethyl ester groups followed by decarboxylative fragmentation to 1 and the concomitant liberation of the potentially electrophilic acrylate species. Glutathione conjugates of atropic acid and its ethyl ester were also observed in microsomal incubations of JTT-130 that were supplemented with the thiol nucleophile. Additional support for the hydrolysis mechanism was obtained from analogous studies on diethyl 2-(2-(2-(3-(dimethylcarbamoyl)-4-(4′-trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)­phenyl)­acetoxy)­ethyl)-2-phenylmalonate (3), which cannot participate in hydrolysis via the fragmentation pathway because of the additional methylene group. Unlike the case with JTT-130, 18O was readily incorporated into 1 during the enzymatic hydrolysis of 3, suggestive of a mechanism involving direct hydrolytic cleavage of the ester group in 3. Finally, 3-(ethylamino)-2-(ethylcarbamoyl)-3-oxo-2-phenylpropyl 2-(3-(dimethylcarbamoyl)-4-(4′-(trifluoromethyl)-[1,1′-biphenyl]-2-ylcarboxamido)­phenyl)­acetate (4), which possessed an N,N-diethyl-2-phenylmalonamide substituent (in lieu of the diethyl-2-phenylmalonate motif in JTT-130) proved to be resistant to t
ISSN:0893-228X
1520-5010
DOI:10.1021/tx300243v