The focused set of cyclopentapyrazoles produced six compound
The focused set of cyclopentapyrazoles produced six compounds with sufficient activity to warrant the evaluation of kinetic solubility. Despite the improved activity of the series, these compounds remain stalwartly insoluble (), indicating the need for additional chemical modifications which address aqueous solubility. However, the general tendency of adding of more hydrophobic bulk to the D-ring in order to produce compounds with the desired activity runs counter to the overarching goal of increasing solubility and improving other related physicochemical properties. Therefore, D-ring optimization may not be an ideal avenue to address the underlying property related issues of the cyclopentapyrazole scaffold. Chemical optimization of AGSM through the construction of three related D-ring series of compounds led to the discovery of various novel ligands with good activity for suppressing the formation of Aβ42 through modulating the activity of the γ-secretase enzyme. Unfortunately, many of the ligands identified suffered from poor aqueous solubility. However, a subset of the ligands within the 1-substituted 3--butylpyrazoles series displayed both good activity, as well as improved aqueous solubility. The most potent Losmapimod discovered within the series, -ethylpyrazole , displayed good potency for suppressing Aβ42 with an IC value of 63nM, in addition to moderate solubility (). Consequentially, further in vitro ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicology) analysis of -ethylpyrazole was undertaken in order to identify potential liabilities within this family of compounds which would preclude further development. -Ethylpyrazole demonstrated ideal CyP inhibition potential and hERG ion channel inhibition potential (all IC values >10μM). In addition, compound showed fair microsomal stability. However, poor membrane permeability as measured by MDR1-MDCK assay and high plasma protein binding were also observed for -ethylpyrazole . Despite the limited in vitro membrane permeability, compound was selected for in vivo pharmacokinetic (PK) studies in order to gauge critical PK parameters, determine whether this ligand can reach the intended enzyme target within the brain and establish a baseline for future compound development with respect to SAR/SPR. As shown in , in vivo dosing of -ethylpyrazole in male CD-1 mice revealed that kinetically compound has a relatively high clearance (CL) and a short plasma half-life (), despite the high metabolic stability observed in the in vitro mouse microsomal stability assay. Following IV administration at 1mg/kg in mice, the clearance of compound was 2009mL/hr/kg (or 33.5mL/min/kg) which is about 40% of mouse hepatic blood flow (∼80mL/min/kg), while its half-life () was 2.4h. Although compound has a relatively large volume of distribution (=7.15L/kg), which is about 11-fold higher than body water (∼0.65L/kg), it has a short half-life due mainly to its high rate of clearance. The bioavailability () of -ethylpyrazole is unexpectedly high and is estimated to be 230%. Theoretically, the bioavailability should not exceed 100%, and one would expect based on the clearance of compound being 40% of mouse hepatic blood flow that significant hepatic first-pass metabolism would result in bioavailability much less than 100%, even when the compound is completely absorbed from the intestinal lumen. The high bioavailability is hypothesized to be the result of saturation of hepatic drug metabolism during oral absorption. Following the oral dose of 5mg/kg, the concentration of -ethylpyrazole is very high in the portal vein which saturates the drug metabolizing enzymes in the liver and results in decreased compound clearance leading to a marked increase in the oral AUC and an over estimation of . -Ethylpyrazole also shows good brain penetrance for both routes of administration, in contrast to the in vitro permeability assay results. Kinetically, the mouse PK parameters (CL, and ) of compound are reasonably good and support further development efforts surrounding this class of compounds.