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  • Tetrazole is one of the

    2021-12-09

    1-Tetrazole is one of the most commonly used bioisosteres for carboxylic acids arising from their similarity in pKa (ca. 4.5–4.9 vs 4.2–4.4, respectively)., Metabolically, tetrazoles may exhibit an advantage over carboxylic acids because they form -glucuronides which are not as chemically reactive as the -glucuronides derived from carboxylic acids. Tetrazole-bearing drugs have not been linked to toxic effects in human due to -glucuronidation. Our earlier efforts on discovery of potent GPR40 agonists led to a novel series of carboxylic apexbio calculator compounds with benzo[]thiophene as the center ring. Based on this scaffold, various carboxylic acid bioisosteres were explored, and tetrazole was identified as the most potent GPR40 agonist among them. Replacement of the carboxylate on different chemical scaffolds of GPR40 agonists with tetrazole frequently resulted in loss of potency. However, the series with a benzo[]thiophene center ring and a 2-propynyl substitution at the β-carbon retained the potency of the acid. In calcium flux assays in cells overexpressing human and rat GPR40 receptors, tetrazole demonstrated an EC of 9 nM and 7 nM for human and rat GPR40, respectively (). To date, there are two major types of GPR40 modulators reported: partial agonists, e.g., TAK-875, and full agonists, e.g., AM-1638. The partial agonists only signal through the Gqα pathway, while the full agonists can signal through not only Gqα but also Gs and cAMP pathways. The different levels of receptor activation afforded by partial vs. full agonists are evident in transiently transfected CHO cells expressing relatively low levels of hGPR40. In the Gqα-mediated inositol monophosphate (IP1) turnover experiments, both partial agonist (TAK-875) and full agonist (AM-1638) stimulated IP1 accumulation, however, the full agonist showed a higher E (). When cAMP levels were measured, only the full agonist (AM-1638) stimulated cAMP production. Since tetrazole behaved similarly to TAK-875 in these assays, we consider it a partial GPR40 agonist. The carboxylic acid and its tetrazole bioisosteric pair exhibit very similar H-bond environments in crystal structures from the CSD, and the attractive energies of these H-bonds are very similar. Recently, the high resolution X-ray structure of GPR40 in complex with the partial agonist, TAK-875 was published. We used this co-crystal structure of hGPR40 (PDB ID: ) to predict the binding mode of tetrazole 1. The binding site was defined as the set of residues in a range of 5 Å around the co-crystallized ligand, TAK-875. The best docking pose of tetrazole 1 in the “pocket #1” binding site, predicted by GOLD (version 5.4.1), is shown in . The -tolyl substituted benzo[]thiophene part of the molecule is exposed to the lipid bilayer of the membrane, in the same area where the (methylsulfonyl)ethoxy biphenyl of TAK-875 projects. The tetrazole group of 1 is buried into the binding site, making ionic H-bonds with key binding site residues Arg183 and Arg258, while the carboxylate in TAK875 involves Tyr91. This tetrazole is also stabilized by pi-pi (π–π) stacking interactions with Phe87. Such interactions are crucial as they serve as anchors for the compound in the binding site. The propyne group is making mainly hydrophobic interactions in a small pocket formed by Tyr91, Leu138, Ala182 and His137. Despite good potency toward GPR40 receptors, had poor microsomal stability with an intrinsic clearance T of 5 min and 16 min in human and rat microsomes, respectively (). To identify the metabolically labile part of the molecule, a metabolic ID study was carried out in rat, dog and human liver S9 fractions. The major metabolite of was the mono-oxidation product M4 in all three species (). The turnover was higher in human and rat than in dog S9 mix. Only 14.1% of unchanged drug (UD) was remaining in human S9 after 1 h of incubation. The phase II metabolite (M17, -glucuronide of the tetrazole moiety) was only 1% in human S9, while the percentage was higher in dog S9 (7.6%). It was suspected that the mono-oxidation metabolite M4 was the benzo[]thiophene 1-oxide shown in . Compound was synthesized by treating with hydrogen peroxide and TFA, and NMR indicated that it was a mixture of - and -enantiomers (at sulfur atom, about 1:1 ratio). and LC–MS confirmed that it was the same compound as M4. The sulfoxide was weakly active in the assay with an EC of 1.4 μM for hGPR40, and it was also metabolically unstable in microsomes (). We initiated an effort to search for benzo[]thiophene tetrazoles with improved metabolic stability.