Fused 3-Hydroxy-3-trifluoromethylpyrazoles Inhibit Mutant Huntingtin Toxicity

Abstract

Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragmentbased Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171− 82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.

Recombinant Adeno Associated Viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery

“…In the right striatum (Q138), GFP signal was used to quantify the number and dimensions of GFP positive aggregates in the right striatum. In particular, three slices centered in the injection region spaced at 500µm, were acquired by fluorescence microscopy (Nikon Eclipse 90i, Japan) and subsequently quantified with Matlab (The Mathworks, Natick, USA) using the Otsu's method (Otsu et al, 1979) with minor modifications (La Rosa et al, 2013).…”

The Emerging Landscape of Small-Molecule Therapeutics for the Treatment of Huntington’s Disease

“…3-Hydroxy-3-trifluoromethylpyrazole derivative 111 reduced mHTT toxicity in a phenotypic HD assay and exhibited activity in rat primary striatal neurons (mHtt171-82Q) and PC12 (mHTTex1 expression controlled by tetracycline). Importantly, it was also found to be well-tolerated in the R6/2 mouse model of HD …”

A Michael Equilibration Model To Control Site Selectivity in the Condensation toward Aminopyrazoles

“…The corresponding neutral, i.e., thermodynamic, conditions favored the 5-aminopyrazoles 5a – d with increased selectivity in opposite order to the basic conditions consistent with the dynamic Michael intermediates. Aryl hydrazines are known to strongly favor 5-aminopyrazoles, − , which was observed under the neutral conditions. However, employing the basic conditions with phenyl hydrazine 1e provided an equal mixture of the pyrazoles 3e and 5e , which could be improved to favor the 3-aminopyrazole 3f (78:22) by utilizing the more electronically donating p -methoxyphenyl hydrazine 1f .…”

“…In general, the 5-aminopyrazole isomer was favored through direct condensation . Reversal of this presumed innate reactivity to access the opposite 3-aminopyrazole isomer required indirect approaches such as employing Schmidt and co-workers’ multistep approach, developed in the late 1950s, to direct the initial Michael addition with a suitably protected hydrazine (Scheme ) . Accordingly, specific isomers of aminopyrazoles, resulting from the direct condensation, are readily available while the opposite isomers have limited availability and can only be procured at high cost .…”

“…Accordingly, specific isomers of aminopyrazoles, resulting from the direct condensation, are readily available while the opposite isomers have limited availability and can only be procured at high cost . Rationalization for the site selectivity led to numerous, and at times conflicting, models that differ by the hydrazine and electrophile examined. , The common attribute was based on an initial nucleophilic attack to either the nitrile or activated olefin by the more nucleophilic substituted nitrogen of alkyl hydrazines or the unsubstituted nitrogen of aryl hydrazines. − ,, Although the models provide reasonable rationales, they do not lead to general predictions or provide an avenue toward the uncommon isomer. Only sparse accounts for influence of reaction conditions or unusual site selectivity exist. ,, However, the general consensus relates selectivity to the structure and electronic properties of the reactants.…”