Chasing the Elusive Benzofuran Impurity of the THR Antagonist NH-3: Synthesis, Isotope Labeling, and Biological Activity

Abstract

ABSTRACT:We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its formation has enabled isotope (D) labeling. We further developed a highly efficient method for separating EBI from NH-3. Interestingly, EBI was found to be a very potent thyroid hormone receptor (THR) agonist, while NH-3 is an antagonist. In this process, we have also achieved a significantly improved synthesis of NH-3. ■ INTRODUCTIONThyroid hormones regulate growth, metabolism, and homeostasis of vertebrates. They bind to thyroid hormone receptors (THRs) found in almost all cells of the body and exert their effect by altering gene expression. 1 Various developmental and metabolic disorders can result from under-or over-activity of the thyroid. Thus, both THR agonists and antagonists have immense medicinal importance. 2,3 NH-3 (Figure 1) is currently the most potent synthetic small-molecule THR antagonist. 4,5 For a study investigating disrupting effects of environmental toxicants on thyroid hormone regulated neurodevelopmental processes, NH-3 was required as a reference compound. NH-3, which is not commercially available, can only be obtained by following a long synthetic route originally reported by Nguyen et al. 6 In our hands, the synthesis of NH-3 following this procedure resulted in samples of NH-3 that contained high amounts of an inseparable benzofuran impurity (EBI). Through deuterium-labeling experiments, we have demonstrated that the presence of EBI is the result of a base-mediated annulation of an o-alkylnylphenol precursor. The mechanism of formation of benzofurans from o-alkylnylphenols by Pd-, 7a,b Cu-, 7c 7d and Pt-catalyzed 7e reactions has previously been studied. Jacubert et al. 7f have reported an acid-mediated annulation of o-alkylnylphenol methyl ethers and in one case used deuterium labeling to establish the electrophilic nature of the transformation. Our experiment reveals that NH-3 converts into EBI via base-mediated intramolecular annulation without a transition-metal catalyst. There have been speculations about EBI by other research groups, but it could never previously be identified. 8 Therefore, we decided to identify, synthesize, and study the biological activity of EBI and investigate its mechanism of formation along with developing an improved and higher-yielding synthesis of Received: November 20, 2015 Figure 1. Structures of the THR antagonist NH-3 and the benzofuran EBI. ■ RESULTS AND DISCUSSIONOur synthesis of the key intermediates 9 and 10 is depicted in Scheme 1. This sequence is similar to the one reported by Nguyen et al. 6 which includes several steps involving the use of n-BuLi, a pyrophoric substance, posing a safety hazard. The use of BuLi not only necessitates extremely careful handling but also adversely affects reaction scale-up in academic laboratory settings. Therefore, we decided to minimize or eliminate the use of n-BuLi from the sequence and made some cha...

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

“…Using a new, cleaner synthesis of NH-3 (13), we tested whether NH3 antagonized TH-dependent endpoints in our assay suite. At 2 µM, NH-3 significantly inhibits the T3-induced increase in brain width at the optic tectum (Figure 6A).…”

“…Stocks were stored at −20 C. T3 and T4 stock concentrations were verified by spectroscopy (20). NH-3 was synthesized by the Wulff laboratory at UC Davis (13). oLH (ovine luteinizing hormone) was purchased through the National Hormone and Peptide Program (Los Angeles, CA), pregnant mare serum gonadoptropin (PMSG) was purchased from Sigma-Aldrich, and tricaine methansulfonate was purchased from Western Medical Supply (Arcadia, CA).…”

“…We used the TR antagonist NH-3 from a new synthesis that eliminates an agonistic co-purifying compound (13) to validate the assays for antagonism. We also tested three persistent organic pollutants that are ubiquitous in the environment and have been suggested to disrupt TH signaling: the flame retardants tetrabromobisphenol A (TBBPA) (14–16) and tetrabrominated diphenyl ether 47 (BDE-47) (17,18) and the biocide tributyltin (TBT) (19).…”

BDE-99 impairs differentiation of human and mouse NPCs into the oligodendroglial lineage by species-specific modes of action

“…Chemicals were dissolved as follows: 1, 2, 6.66, 10 mM stocks of BDE-99 in dimethyl sulfoxide (DMSO; Carl Roth GmbH), 300 μM T3 or T4 in a 1:1 dilution of 96% ethanol and 1 M HCl (both Carl Roth GmbH) (‘EtOH/HCl’), 100 mM ascorbic acid in H 2 O, 200 mM Trolox in DMSO. TR antagonist NH-375 was diluted in DMSO and a 1 mM stock was prepared. Solvent concentrations used in the experiments were 0.1% (co-treatment experiments) or 0.3% (dose-response experiments) DMSO for BDE-99, 0.1% DMSO for NH-3, 0.01% EtOH/HCl (T3, T4), 0.05% DMSO (Trolox), 0.1% or 0.5% H 2 O (L-ascorbic acid) or the respective combination in the co-treatments.…”

Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos

“…NH-3, a thyroid hormone antagonist18 was synthesized by AGV Discovery (France), absence of contamination by benzofurane was verified56. All chemicals were dissolved at 10 −1 M in DMSO, with the exception of HCB (10 −1 M in acetone) and BDE 209 (10 −2  M in DMSO).…”