Rational Design of Novel 1,3-Oxazine Based β-Secretase (BACE1) Inhibitors: Incorporation of a Double Bond To Reduce P-gp Efflux Leading to Robust Aβ Reduction in the Brain

Abstract

Accumulation of Aβ peptides is a hallmark of Alzheimer's disease (AD) and is considered a causal factor in the pathogenesis of AD. β-Secretase (BACE1) is a key enzyme responsible for producing Aβ peptides, and thus agents that inhibit BACE1 should be beneficial for disease-modifying treatment of AD. Here we describe the discovery and optimization of novel oxazine-based BACE1 inhibitors by lowering amidine basicity with the incorporation of a double bond to improve brain penetration. Starting from a 1,3-dihydrooxazine lead 6 identified by a hit-to-lead SAR following HTS, we adopted a p K lowering strategy to reduce the P-gp efflux and the high hERG potential leading to the discovery of 15 that produced significant Aβ reduction with long duration in pharmacodynamic models and exhibited wide safety margins in cardiovascular safety models. This compound improved the brain-to-plasma ratio relative to 6 by reducing P-gp recognition, which was demonstrated by a P-gp knockout mouse model.

Computer-Aided Drug Design of β-Secretase, γ-Secretase and Anti-Tau Inhibitors for the Discovery of Novel Alzheimer’s Therapeutics

“…Sequence alignment analysis showed that the amino acid sequences of human BACE1 and BACE2 gene coding regions are 45% identical and 75% homologous [22]. Numerus crystal structures of BACE1 have been resolved with and without inhibitor in its active site [23][24][25][26][27][28][29][30][31][32]. BACE1 contains an N-terminal protease domain, a connecting strand, a transmembrane region, and a cytosolic domain [23].…”

Trifluoromethyl Dihydrothiazine‐Based β‐Secretase (BACE1) Inhibitors with Robust Central β‐Amyloid Reduction and Minimal Covalent Binding Burden

“…Considering our experience that lowering the p K a worked well in previous SAR efforts to address these issues, we adopted a similar strategy to optimize the dihydrothiazine series. With detailed knowledge of the optimal p K a range of the amidine moiety (6.5<p K a <7.4) to reduce hERG inhibition and P‐gp efflux as well as retaining cellular potency, our design efforts focused on incorporating electron withdrawing substituents into the dihydrothiazine head group. During this work, an elegant molecular design was disclosed by Hilpert et al.…”

“…Biochemical BACE1 assay . The biochemical BACE1 IC 50 values were determined by HTRF assay using an APP derived peptide as described previously …”

“…Cellular Aβ assay . The cellular Aβ IC 50 values were determined by measuring Aβ 40 using HTRF assay in SH‐SY5Y cells expressing human APP as described previously …”

“…This issue was addressed by a p K a lowering approach culminating in oxazines such as 1 and 2 . During this effort, less basic inhibitors displayed reduced P‐gp efflux and hERG inhibition, although the drop in basicity, in particular compounds with p K a <6.5, was associated with a loss in cellular potency, which led to the proposal of a basicity guideline of 6.5<p K a <7.4 for BACE1 inhibitors . The early efforts also identified dihydrothiazine 4 as a lead, which displayed a higher potency than the corresponding dihydro‐oxazine 3 , though exhibiting high hERG inhibition and P‐gp efflux like 3 .…”