Core Refinement toward Permeable β‑Secretase (BACE-1) Inhibitors with Low hERG Activity

By use of iterative design aided by predictive models for target affinity, brain permeability, and hERG activity, novel and diverse compounds based on cyclic amidine and guanidine cores were synthesized with the goal of finding BACE-1 inhibitors as a treatment for Alzheimer’s disease. Since synthesis feasibility had low priority in the design of the cores, an extensive synthesis effort was needed to make the relevant compounds. Syntheses of these compounds are reported, together with physicochemical properties and structure–activity relationships based on in vitro data. Four crystal structures of diverse amidines binding in the active site are deposited and discussed. Inhibitors of BACE-1 with 3 μM to 32 nM potencies in cells are shown, together with data on in vivo brain exposure levels for four compounds. The results presented show the importance of the core structure for the profile of the final compounds

New Aminoimidazoles as β‑Secretase (BACE-1) Inhibitors Showing Amyloid‑β (Aβ) Lowering in Brain

Amino-2<i>H</i>-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimer’s disease. Synthetic methods, crystal structures, and structure–activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (<i>S</i>)-<b>1m</b> was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (<i>S</i>)-<b>1m</b>, a concentration and time dependent decrease in Aβ40 and Aβ42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aβ was 40–50%, 1.5 h after oral dosing (100 μmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo

Mass spectra of Aβ isoform patterns in all cell models investigated.

<p>SH-SY5Y APP695wt cells treated with DMSO (Panels a and c), 5 µM β-secretase inhibitor IV (Panel b) or 10 µM AZ-20 (Panel d). SH-SY5Y APP695swe cells treated with DMSO (Panel e) or 10 µM AZ-20 (Panel f). 7PA2 APP751 V717F cells treated with DMSO (Panel g) or 10 µM AZ-20 (Panel h). HeLa APPswe cells treated with DMSO (Panel i) or 10 µM β-secretase inhibitor IV (Panel j). HeLa APPswe scrambled siRNA transfected control cells (Panel k) and cells transfected with single oligo siRNAs against BACE1 (Panel l). The mass-to-charge ratio (m/z) of the [M+H]⁺ ion of Aβ5-38 is very close to that of Aβ1-33, causing the peaks to partially overlap and making quantification difficult, wherefore both isoforms were excluded from quantitative analysis. Those peptides are instead presented in these mass spectra as expanded inserts (except for panels g-h where they are clearly visible).</p

CSF Aβ isoform patterns.

<p>Dogs treated with vehicle (N = 15), NB-C8 (N = 3 at 3 hours, N = 3 at 16 hours) and NB-B4 (N = 5 at 6 hours). Mass spectra of the Aβ isoform pattern from dogs treated with placebo (Panel a, upper panel) or NB-C8 16 hours post treatment (Panel a, lower panel). The expanded sections show Aβ1-33, Aβ1-34, Aβ5-38 and Aβ5-40. Aβ1-33 and Aβ5-38 were excluded from quantitative analysis since their peaks partially overlap, making quantification difficult. Absolute (Panels b–c) and normalized (Panels d–e) mass spectral peak intensities of all detected Aβ isoforms. Statistical significances were tested for normalized peak intensities comparing different treatments. For NB-B4, significant differences were seen for Aβ5-40 (P = 0.001), Aβ1-34 (P = 0.001) and Aβ11-40 (P = 0.002). For NB-C8, significant differences were seen between vehicle and treatment at 16 h for Aβ5-40 (P = 0.01) and Aβ1-34 (P = 0.05). Data are means; error bars are SD.</p

Cell medium Aβ isoform patterns.

<p>SH-SY5Y APP695wt cells treated with AZ-20 (Panel a). SHSY-5Y APP695swe cells treated with AZ-20 (Panel b). 7PA2 APP751 V717F cells treated with AZ-20 (Panel c). HeLa-APPswe cells treated with β-secretase inhibitor IV (Panel d). HeLa-APPswe mock and scrambled siRNA-transfected control cells, and cells transfected with single oligo siRNA or pooled siRNA against BACE1 (Panels e). N = 1 for each concentration and treatment.</p

Multivariate analysis and CSF Aβ5-40/Aβ1-34.

<p>Multivariate discriminant analysis of the CSF Aβ pattern for treatment compared with placebo in dogs treated with vehicle (N = 15), NB-C8 (N = 3 at 3 h, N = 3 at 16 h) and NB-B4 (N = 5 at 6 h) (Panels a–b). Score vector results including medians (Panel a). Samples were taken 3 h (open triangles), 6 h (filled squares) or 16 h (filled triangles) after treatment. The groups denoted by “T” were used for constructing the multivariate model while “P” represents the prediction set used for testing the stability of the model. In the construction of the model, all dogs treated with NB-C8 were regarded equal, independent of time after drug administration. Relative contributions of different isoforms to group separations, with increased or decreased relative levels in the treated groups, are shown by white or grey bars, respectively (Panel b). Change of the CSF Aβ5-40/Aβ1-34 ratio in relation to change of CSF Aβ1-40 (Panel c) and CSF Aβ1-42 (Panel d). The Aβ5-40/Aβ1-34 ratio was a more sensitive biomarker than Aβ1-40 and Aβ1-42 in terms of change from baseline (the dotted lines indicate predicted correlations for biomarkers affected equally by treatment). Dogs treated with vehicle (N = 4) and BACE1 inhibitor S obtained from Janssen (N = 4 for each dosage) (Panel e). The CSF Aβ5-40/Aβ1-34 ratio completely separated dogs on active treatment versus placebo (P = 0.005 using the Mann-Whitney U test for comparison of all animals on active treatment versus placebo). Time-dependent dynamics of the ratio in high dose treatment (Panel f).</p

Summary of processing pathways.

<p>The main pathways of Aβ peptide release and how these are affected by BACE1 inhibition (arrows indicate absolute and/or relative changes). The APP box shows major APP cleaving secretases with selected cleavage sites that depend on them.</p

Design and Synthesis of β‑Site Amyloid Precursor Protein Cleaving Enzyme (BACE1) Inhibitors with in Vivo Brain Reduction of β‑Amyloid Peptides

The evaluation of a series of aminoisoindoles as β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer’s disease, (<i>S</i>)-<b>32</b> (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aβ40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (<i>S</i>)-<b>16</b> and (<i>R</i>)-<b>41</b> showing large in vitro margins with BACE1 cell IC₅₀ values of 8.6 and 0.16 nM, respectively, and hERG IC₅₀ values of 16 and 2.8 μM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of β-amyloid peptides in mouse brain following oral dosing