br Acknowledgements This work was supported by Slovak

Acknowledgements This work was supported by Slovak Academy of Sciences grants VEGA 2/0052/10 and 2/0045/10.
Both clinical observations and preclinical studies suggest that hypofunction of -methyl--aspartate (NMDA) receptor is implicated in the pathophysiology of schizophrenia. Thus, therapeutic intervention aiming at restoring NMDA receptor activity represents a promising novel strategy for the management of schizophrenia. As glycine is an obligatory co-agonist at the NMDA receptor complex, one strategy to enhance NMDA receptor activity is to elevate extracellular levels of glycine in the Psalmotoxin 1 through selective inhibition of glycine uptake mediated by the glycine transporter-1 (GlyT1), which is co-expressed with the NMDA receptor. Strong support for this approach comes from clinical studies where glycine and -serine (co-agonists at the glycine site of NMDA receptor) and sarcosine (a prototypical weak GlyT1 inhibitor) improved positive, negative, and cognitive symptoms in schizophrenic patients, when added to conventional therapy. As a result, considerable efforts have been focused on the development of selective GlyT1 inhibitors. The first examples reported were sarcosine derivatives including ,, and . More recently, non-amino-acid chemotypes like and , have been described (). We have also contributed to this field and reported recently on the optimization of the non-sarcosine based spiropiperidine . During our continued efforts to discover and develop structurally novel and selective GlyT1 inhibitors, the Roche compound collection was screened. This campaign led to the identification of the benzoylpiperazine hit as a potent inhibitor of GlyT1 (15nM), showing more than 300-fold selectivity against the type 2 isoform. is characterized by the presence on the benzoyl moiety of a morpholine and a nitro group occupying positions 2 and 5, respectively. In this letter, we describe the SAR studies obtained in this novel benzoylpiperazine class at positions 2, 5, and at the left-hand aromatic ring attached to the piperazine moiety (). We also report on the strategy we followed that led to the identification of highly potent and selective analogues displaying drug-like properties and in vivo efficacy after oral administration. Our synthetic strategy entailed mainly two straightforward and complementary approaches allowing for rapid modification of the three exit vectors around the central benzoylpiperazine core (). The first route that led to the amino analogues , –, and – involved the reaction under thermal conditions of amines with the 2-halogen-substituted benzoyl piperazine precursors – prepared under standard amide coupling conditions from the 2-halogen-benzoic acids – and piperazine . The second route that led to compounds , , –, , , and consisted in coupling the aryl piperazine with the appropriately functionalized benzoic acids –. Well-established methods were used for the preparation of these acids from their precursors – as indicated in : Mitsunobu alkylation for the 2-alkoxy-substituted acid , thermal conditions for the 2-alkylsulfanyl and 2-alkylamino-substituted acids –, and Suzuki coupling for the 2-aryl- or 2-cycloalkyl-substituted acids . Starting from our first effort was aimed at identifying suitable groups in position 5 of the benzoyl ring to replace the nitro substituent: a moiety often causing safety concerns due to its well-documented mutagenic and carcinogenic potential. summarizes the in vitro potency at GlyT1 and selectivity against GlyT2 of analogues prepared. First, removal of the nitro group to give () resulted in the complete loss of GlyT1 activity. Similar detrimental effect was observed after its replacement with either electron-donating or neutral groups (–) as well as with lipophilic electron-withdrawing (EWG) groups (–). In contrast, in vitro potency recovered with the introduction of polar EWG groups (–). In particular, high activity was obtained with the methylsulfone (), methylsulfonamide (), and sulfonamide () derivatives, respectively, showing 70, 100, and 120nM activity at GlyT1. Gratifyingly, the introduction of such groups simultaneously abolished the low micromolar activity at the GlyT2 isoform seen with the starting hit . Introduction of larger substituents than methyl on the sulfonyl group (–) resulted in a severe drop in GlyT1 activity, which we postulate to be due to limited space available in this region of the receptor.