Molecular properties as well as metabolic stability in

Molecular properties as well as metabolic stability in rat and human liver microsomes for the most active nitro (), nitrile (), methylsulfone (), and sulfonamides () analogues were measured (). All these derivatives possessed excellent membrane permeability as measured in the Pampa assay and furthermore, the sulfone and sulfonamide derivatives (–) exhibited a much higher aqueous solubility (pH 6.5) compared to the nitro and nitrile analogues and . As expected, the two sulfonamides displayed the highest polar surface area (PSA) above 90Å, raising concern regarding the ability of these two compounds to penetrate the central nervous system (CNS). Lower and more favorable PSA values were obtained for the nitrile () and the methylsulfone () derivatives. Finally, encouraging microsomal stability data were obtained for the methylsulfone analogue showing a medium in vitro clearance in both the rat and human species. Altogether, these results demonstrate that among the groups tested at position 5, the methylsulfone function offers the best opportunity to generate compounds having CNS drug-like properties, and as a result constitutes our preferred motif at this position. Pleasingly, in a CEREP selectivity screen performed against a panel of 80 targets including transmembrane and soluble receptors, enzymes, ion channels, and monoamine transporters, the methylsulfone Phleomycin exhibited a highly selective profile (< 20% inhibition at 10μM was measured for all targets). Next, SAR exploration at position 2 of the benzoyl ring in hit and compound was conducted (). Deletion of the morpholine unit () as well as its replacement with smaller alkyl-substituted amino groups (–) resulted in inactive or weakly active analogues. Pleasingly, a dramatic improvement in activity was seen by increasing the size of the alkylamino substituent as seen with the isopropyl (), cyclohexyl (), and piperidinyl () derivatives, which were found to display 1–2nM activity at GlyT1. An abrupt drop of activity was, however, observed upon introduction of more extended substituents like the cyclohexylmethyl group (), suggesting that substituents at position 2 fit in a size limited pocket of the GlyT1 receptor. Further SAR exploration revealed to our delight that activity was in fact not restricted to the presence of an alkylamino residue at this position and that, on the contrary, great latitude for variation was possible. Indeed, low nanomolar GlyT1 activity was also obtained with analogues carrying a wide array of structurally diverse substituents like alkoxy groups (-), alkylsulfanyl groups (), and aromatic groups (–). All these derivatives, in addition, displayed excellent selectivity versus the GlyT2 isoform. Finally, SAR around the left-hand aromatic system was explored (). A rapid scanning of electron-donating (–OMe), electron neutral (–Me), and EWG groups (–Cl and –CF) at the , , and positions of the aromatic ring (–) revealed that best activity was reached with the presence of an EWG group in position, as exemplified with the 4-trifluoromethyl-substituted derivative : 100nM. In fact, further exploration showed that a variety of EWGs were allowed at this position. Indeed, not only lipophilic groups, but also moderately or strongly polar substituents were tolerated such as the cyano () or the methylsulfone group (). Keeping these preferred motifs in place, a significant improvement in GlyT1 potency was measured upon the introduction of an additional EWG like fluorine, or cyano group at either the or position as observed with compounds –. Interestingly, the replacement of the aromatic nucleus with heteroaromatic systems as exemplified with pyridine derivatives and 5 was well tolerated. With all active derivatives, good to excellent selectivity against the GlyT2 isoform was seen (). Having established a preliminary SAR at the three exit vectors around the central benzoylpiperazine core, the next step consisted in combining some of the preferred groups identified at the left-hand side as well as at position 2 on the right-hand benzoyl moiety, while keeping position 5 occupied with the favored methylsulfone group. This approach led to the identification of a number of highly potent GlyT1 inhibitors (), displaying no activity at the GlyT2 isoform (data not shown). Gratifyingly, the great structural diversity seen previously at position 2 was also observed with compounds – incorporating the 4-CF-phenyl group at the left-hand side. Indeed, low nanomolar activity was obtained with the alkylamino (–), the alkoxy (–), the aromatic (–), and the cycloalkyl () derivatives. In view of their favorable pharmacological activities in vitro, these compounds were assessed for solubility and metabolic stability. Interestingly, as seen in , the isopropyloxy and cyclopropylmethylenoxy derivatives and exhibited good aqueous solubility (pH 6.5) and excellent metabolic stability in mouse and human liver microsomes. Much less favorable results were obtained with the corresponding alkylamino, aromatic, and cycloalkyl compounds (–, –), which exhibited, in general, low solubility and medium microsomal clearance, a result likely due to the higher lipophilicity of these analogues compared to the alkoxy derivatives (see log, ). Moreover, the positive influence of alkoxy groups on physico-chemical and metabolic properties was also observed when more polar left-hand aromatic rings than the 4-CF-phenyl group were in place. Indeed, for example, the cyclopropylmethylenoxy derivatives and incorporating, respectively, the cyano and methylsulfone-substituted aromatic rings consistently exhibited higher solubility and metabolic stability than the corresponding piperidinyl and phenyl analogues , and , .