Discovery of spirofused piperazine and diazepane amides as selective histamine-3 antagonists with in vivo efficacy in a mouse model of cognition.

A new series of potent and selective histamine-3 receptor (H3R) antagonists was identified on the basis of an azaspiro[2.5]octane carboxamide scaffold. Many scaffold modifications were largely tolerated, resulting in nanomolar-potent compounds in the H3R functional assay. Exemplar compound 6s demonstrated a selective profile against a panel of 144 secondary pharmacological receptors, with activity at only σ2 (62% at 10 µM). Compound 6s demonstrated free-plasma exposures above the IC50 (∼50×) with a brain-to-plasma ratio of ∼3 following intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s demonstrated a statistically significant response compared with the control group. This series represents a new scaffold of H3 receptor antagonists that demonstrates in vivo exposure and efficacy in an animal model of cognition.

Discovery of novel positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5).

Novel in vitro mGlu(5) positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC(50) 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC(50) 156.3 nM). Derivation of a second-generation of mGlu(5) PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC(50) 50.1 nM) as a potent and soluble mGlu(5) PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.

Application of fragment-based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency.

Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.

Synthesis and physical characterization of a P1 arginine combinatorial library, and its application to the determination of the substrate specificity of serine peptidases.

Serine peptidases are a large, well-studied, and medically important class of peptidases. Despite the attention these enzymes have received, details concerning the substrate specificity of even some of the best known enzymes in this class are lacking. One approach to rapidly characterizing substrate specificity for peptidases is the use of positional scanning combinatorial substrate libraries. We recently synthesized such a library for enzymes with a preference for arginine at P1 and demonstrated the use of this library with thrombin (Edwards et al. Bioorg. Med. Chem. Lett. 2000, 10, 2291). In the present work, we extend these studies by demonstrating good agreement between the theroretical and measured content of portions of this library and by showing that the library permits rapid characterization of the substrate specificity of additional SA clan serine peptidases including factor Xa, tryptase, and trypsin. These results were consistent both with cleavage sites in natural substrates and cleavage of commercially available synthetic substrates. We also demonstrate that pH or salt concentration have a quantitative effect on the rate of cleavage of the pooled library substrates but that correct prediction of optimal substrates for the enzymes studied appeared to be independent of these parameters. These studies provide new substrate specificity data on an important class of peptidases and are the first to provide physical characterization of a peptidase substrate library.