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Application of safety lead optimization screening strategies during the early stage of drug discovery led to the identification of a series of CNS-active small molecule inhibitors with opioid off-target effects, as evidenced by potent agonistic activity in functional cell-based assays for mu (MOP), kappa (KOP) and delta (DOP) opioid receptors. The translation of these effects was confirmed in vivo with the following observations: hypoactivity and decreased fecal production in rats (characteristic of MOP agonism); increased urine production in rats (characteristic of KOP agonism); and decreased intestinal transit time in mice, which was partially blocked by the MOP antagonist naloxone, demonstrating that the in vivo effects were specific for MOP. Based on the confirmation of in vitro-in vivo translatability, an in vitro screening strategy was implemented that resulted in the identification of an optimized backup molecule, devoid of in vivo off-target opioid effects. In addition, in silico modeling by docking of the various molecules to the opioid receptors allowed the identification of the structural drivers of these off-target effects, which can be applied to future chemical-design criteria. Thus, implementation of the safety lead optimization strategy described in this article demonstrates the utility and impact of such approaches on risk mitigation and identification of lead small molecules with improved safety profiles.

A series of 2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromene analogs was developed that demonstrated high selectivity (>2000-fold) for BACE1 vs Cathepsin D (CatD). Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Guided by structure based design, changes to P2' and P3 moieties were explored. A conformationally restricted P2' methyl group provided inhibitors with excellent cell potency (37-137 nM) and selectivity (435 to >2000-fold) for BACE1 vs CatD. These efforts lead to compound 59, which demonstrated a 69% reduction in rat CSF Aβ1-40 at 60 mg/kg (PO).

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50=110 nM) demonstrated a reduction in CSF Aβ in wild type rats after a single dose., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one), a β-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of cytochrome P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation, followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. After hydrolysis of the formamide, a carbon is lost as formic acid, together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.

In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aβ1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aβ1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.

This study was conducted to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of two novel inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1), GNE-629 [(4S,4a'S,10a'S)-2-amino-8'-(2-fluoropyridin-3-yl)-1-methyl-3',4',4a',10a'-tetrahydro-1'H-spiro[imidazole-4,10'-pyrano[4,3-b]chromen]-5(1H)-one] and GNE-892 [(R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one], and to develop a PK-PD model to predict in vivo effects based solely on in vitro activity and PK. GNE-629 and GNE-892 concentrations and PD biomarkers including amyloid β (Aβ) in the plasma and cerebrospinal fluid (CSF), and secreted APPβ (sAPPβ) and secreted APPα (sAPPα) in the CSF were measured after a single oral administration of GNE-629 (100 mg/kg) or GNE-892 (30 or 100 mg/kg) in cynomolgus monkeys. A mechanistic PK-PD model was developed to simultaneously characterize the plasma Aβ and CSF Aβ, sAPPα, and sAPPβ using GNE-629 in vivo data. This model was used to predict the in vivo effects of GNE-892 after adjustments based on differences in in vitro cellular activity and PK. The PK-PD model estimated GNE-629 CSF and free plasma IC₅₀ of 0.0033 μM and 0.065 μM, respectively. These differences in CSF and free plasma IC₅₀ suggest that different mechanisms are involved in Aβ formation in these two compartments. The predicted in vivo effects for GNE-892 using the PK-PD model were consistent with the observed data. In conclusion, a PK-PD model was developed to mechanistically describe the effects of BACE1 inhibition on Aβ, sAPPβ, and sAPPα in the CSF, and Aβ in the plasma. This model can be used to prospectively predict in vivo effects of new BACE1 inhibitors using just their in vitro activity and PK data.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aβ), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aβ is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aβ in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aβ lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aβ in rodents and in monkey.