Ize the 3-hydroxyl group on the pyrazole ring from dehydration by

Ize the 3-hydroxyl group on the pyrazole ring from dehydration by an intramolecular hydrogen bond interaction (see compound 4a in Figure 1). In the optimization program, we opted for maintaining the main structural features of the molecules in order to keep the general pharmacophore shape and focused on the exploration of three main points: (a) the carbocyclic ring, (b) the linker, and (c) the R1 ring (see Figure 1). Initial hit 4a showed acceptable solubility and permeability, but a far too high metabolism rate in human and mouse. In an effort to improve the overall profile of 4a, mitigating its metabolic stability and moving to a IP-free chemical space, we decided to explore the insertion of different heterocycle rings in R1 position, and few analogues were synthesized (see Scheme 1). The presence of an heterocycle in R1 not only produced a Scheme 1. General Synthetic Route for the Fused 3Hydroxy-3-trifluoromethylpyrazole DerivativesaFigure 2. General workflow.An HTS method was developed in-house creating a stable recombinant 293/T-Rex cell line generated with both a CREluciferase (CRE-LUC) reporter gene and with the full-length mutant Htt gene under control of an inducible CMV promoter; it has been shown that mutated Htt sequesters the cAMP response element-binding protein (CREB) coactivator, CREBbinding protein (CBP) through direct protein interactions, which leads to decreased CREB-mediated transcription.Momelotinib 14 In addition to this, we planned to use another in vitro model of HD based on Htt expression via LV infection on primary striatal rat neurons as a secondary screening assay.Palmitoylethanolamide This assay relies on the incorporation of a Htt-derived sequence expressing an N-terminal 171 aa fragment of mutant or wildtype Htt (Htt171-82Q or Htt171-18Q, respectively; see Supporting Information).PMID:25027343 15 For the HTS screening campaign we selected 24,000 small organic molecules from the diverse Siena Biotech compound collection. Among the most promising hit compounds, a small set of molecules containing a fused 3-hydroxy-3-trifluoromethylpyrazole moiety, initially consisting of 4 compounds and exemplified by compound 4a, displayed an activity range between 5.9 and 18 M with fold increase (FI) values between 30 and 50 as a measure of efficiency of the compound to restore the CREB-mediated transcriptional activity in cells expressing mutant Htt. A set of nonfused analogues represented by compound 5 proved inactive in the screening when tested up to 50 M, showing the selectivity of this specific chemotype only when fused to a cyclic ring. A major concern of this series was the presence of the geminal 3-hydroxy-3-trifluoromethyl functionality and its stability to dehydration. Indeed, it is reported in the literature that 2-aryl or 2-alkyl substituted 3-hydroxy-3-trifluoromethyl hexahydroindazoles undergo dehydration in acidic conditions to afford the corresponding 3-trifluoromethyl tetrahydroindazoles derivatives.16 After retest from a new batch and a preliminary stability test conducted at pH = 7.4 and pH = 3, the 2-acyl and 2-sulphonyl hexahydroindazoles confirmed activityReagents and conditions: (a) ethyl trifluoroacetate, NaOMe, Et2O, -10 to RT; (b) glacial AcOH; (c) acylhydrazide, pyrrolidine, mol sieves, 0 to RT, THF; (d) sulphonylhydrazide, pyrrolidine, mol sieves, 0 to RT, THF.ageneral improvement in the metabolic stability of the compounds but also favored solubility and permeability across the series (see Table 1). As shown in Scheme 1, the fused.