Synthetic Studies Directed toward Dideoxy Lomaiviticinone Lead to Unexpected 1,2-Oxazepine and Isoxazole Formation

In the course of studies directed toward the synthesis of dideoxy lomaiviticinone, 3-(nitromethyl)cyclohexenones <b>2a</b> (X = H) and <b>2b</b> (X = I) were prepared. The corresponding enolates were reacted with naphthazarin (<b>1</b>) and unexpectedly afforded 1,2-oxazepine <b>3</b> and isoxazole <b>4</b>, respectively. Rationale for their formation is proposed

Integrated Platform for Expedited Synthesis–Purification–Testing of Small Molecule Libraries

The productivity of medicinal chemistry programs can be significantly increased through the introduction of automation, leading to shortened discovery cycle times. Herein, we describe a platform that consolidates synthesis, purification, quantitation, dissolution, and testing of small molecule libraries. The system was validated through the synthesis and testing of two libraries of binders of polycomb protein EED, and excellent correlation of obtained data with results generated through conventional approaches was observed. The fully automated and integrated platform enables batch-supported compound synthesis based on a broad array of chemical transformations with testing in a variety of biochemical assay formats. A library turnaround time of between 24 and 36 h was achieved, and notably, each library synthesis produces sufficient amounts of compounds for further evaluation in secondary assays thereby contributing significantly to the shortening of medicinal chemistry discovery cycles

Personalized medicine approach for optimizing the dose of tafamidis to potentially ameliorate wild-type transthyretin amyloidosis (cardiomyopathy)

<div><p></p><p>Placebo-controlled clinical trials are useful for identifying the dose of a drug candidate that produces a meaningful clinical response in a patient population. Currently, Pfizer, Inc. is enrolling a 400-person clinical trial to test the efficacy of 20 or 80 mg of tafamidis to ameliorate transthyretin (TTR)-associated cardiomyopathy using clinical endpoints. Herein, we provide guidance for how to optimize the dose of tafamidis for each WT TTR cardiomyopathy patient using its mechanism of action as the key readout, i.e. we identify the dose of tafamidis that maximally kinetically stabilizes TTR in the blood. Tetramer dissociation is rate limiting for TTR aggregation, which appears to drive the pathology of the TTR amyloidoses. Hence, we measure the TTR tetramer dissociation rate (kinetic stability) in the patient's plasma as a function of tafamidis dose to optimize the dose employed to maximize kinetic stability. Historical data tell us that a subset of patients exhibiting higher tafamidis plasma concentrations are maximally kinetically stabilized at the 20-mg tafamidis dose, whereas the patient studied herein required a 60 mg once daily dose to achieve maximum kinetic stabilization. We anticipate that establishing the dose of tafamidis that achieves maximal TTR kinetic stabilization will translate into a maximal clinical effect, but that remains to be demonstrated.</p></div

Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange

The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration

A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in <i>Caenorhabditis elegans</i>

Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe <b>4</b>, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe <b>4</b> comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission maximum of probe <b>4</b> in comparison to its nonfluorosulfated analogue. We demonstrate that probe <b>4</b> is dark in aqueous buffers, whereas the TTR·<b>4</b> complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe <b>4</b> to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live <i>Caenorhabditis elegans</i> expressing human TTR transgenically and treated with probe <b>4</b> display TTR·<b>4</b> fluorescence in macrophage-like coelomocytes. An analogue of fluorosulfate probe <b>4</b> does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analogue suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes