To test this concept we took advantage of

To test this concept, we took advantage of our large supply of various 2-pyridyl containing [3.1.0] cores (inactive with alkyl or aryl sulfonamides) and prepared the -methylimidazole sulfonamide analogs and (), as work form Merck demonstrated that the 2-pyridyl moiety was superior to the original 4-phenyl moiety in ., These analogs all displayed sub-micromolar potency at GlyT1 (ICs of 247nM for and 185nM for ), clogPs of 2.3, large fraction unbound in plasma ( 7–14%), clean CYP profiles (IC >30μM) and in oral c-src tissue distribution studies, ([brain]/[plasma]) ratios of 1.1. Interestingly, replacement of the -methylimidazole with either imidazole or an -methyl pyrazole led to a complete loss of GlyT1 activity. As incorporation of the -methyl imidazole sulfonamide increased potency in the 2-pyridyl [3.1.0] core from ICs >30μM to ICs <250nM, we were excited to see the impact of this modification to the already very potent cyclopropyl methyl [3.1.0] core of . With a slight modification of the chemistry in , we were able to readily prepare a library of analogs , where the benzamide moiety was varied in the context of the cyclopropyl methyl [3.1.0] core containing an -methyl imidazole sulfonamide. As shown in , this endeavor was very productive, affording potent (ICs from 4 to 119nM) and selective GlyT1 inhibitors with excellent DMPK profiles and CNS penetration in rat with oral dosing ( of 0.3–0.8). While many analogs displayed excellent potency at GlyT1, such as , and , analog stood out as having the best balance of potency (GlyT1 IC=5nM), fraction unbound in plasma ( 7%), and in an oral brain tissue distribution study, a ([brain]/[plasma]) ratio of 0.8. After assessing ancillary pharmacology in a Eurofin Lead Profiling panel of radioligand binding assays against 68 GPCRs, ion channels and transporters, our focus narrowed on , as it displayed no inhibition >50%@10μM against any target in the panel, yet was a potent and selective GlyT1 inhibitor (GlyT1 IC=5±0.5nM (=3), GlyT2 IC >30μM) (). Eadie–Hoffstee plots, where the reaction rate is plotted versus the ratio of the reaction rate and substrate concentration, provide useful insight into the mechanism of enzymatic inhibition, with competitive and noncompetitive enzymatic inhibition demonstrating distinct patterns. An Eadie–Hoffstee plot () of the effect of this series, represented by , on the enzyme kinetics of [C]-glycine transport showed that this series is competitive with respect to glycine, in accordance with the known mechanism of action for and , , , and distinct from the non-competitive mechanism of action of the sarcosine-derived GlyT1 inhibitors, such as NFPS., , , In addition, possessed a clean CYP profile (IC >30μM against 1A2, 2C9 and 2D6; 14.9μM against 3A4) and good unbound fraction in both human (=6.8%) and rat (=44.8%). We assessed stability in rat plasma for 4h at 37°C, and was stable, indicating the free fraction in rat plasma is truly high. In vitro intrinsic clearance experiments suggest possesses moderate to high predicted clearance for both human (Cl=17.7mL/min/kg) and rat (Cl=43.0mL/min/kg). A rat IV PK study was conducted with and it displayed moderate in vivo clearance (0.5mg/kg) (Cl=31mL/min/kg) with a short half-life (=14.5min). This is in-line with data reported for Merck’s , which was moderate to high clearance in rat, but low clearance in dog. We evaluated in two separate rat brain tissue distribution studies: one with subcutaneous (10mg/kg sc in 10% tween 80) dosing and one with oral (10mg/kg po in 0.5% methocellulose) dosing. Both dosing routes exhibited good exposure (sc plasma AUC: 976nMh; sc brain, AUC: 431nMh (or =0.44); po plasma AUC: 1156nMh, po brain, AUC: 956nMh (or =0.83)) with unbound concentrations above the GlyT1 IC at 6h. Due to the higher exposure, for an in vivo proof of concept study in a preclinical model of schizophrenia, we proceeded with oral dosing.