Design and synthesis of bicyclic acetals as Beta Secretase (BACE1) inhibitors

Abstract

Taking advantage of the structural similarity between aspartic proteases, small-molecule peptidomimetic inhibitors that already showed activity towards Secreted Aspartic Protease 2 as anti-Candida agents and HIV protease inhibitors were exploited as potential BACE1 inhibitors. A focused library of 6,8-dioxa-3-azabicyclo[3.2.1]-octane peptidomimetic scaffolds was synthesized and assayed towards BACE1 enzyme, resulting in the identification of a thiolactam-containing hit compound possessing IC₅₀ in the low micromolar range, and confirming the bicyclic acetal portion as a potential transition state analogue in the interaction with catalytic aspartic acid residues.

Introduction

According to the World Health Organization (WHO), about 35.6 millions people have been diagnosed for dementia in 2010, and the number is projected to double every twenty years.¹ The overall cost for dementia worldwide has been estimated approximately 604 billion dollars.² Alzheimer’s Disease (AD) is the most common cause of dementia in older people, consisting of a chronic and progressive neurodegenerative disorder.³ The classic clinical symptoms of the disease are memory loss and difficulties with thinking, problem-solving or language. Actually, the accessible therapies for such disease succeeded only in slowing the cognitive decline due to AD. The pathological hallmarks of AD include two types of lesions in the brain, the extracellular accumulation of amyloid plaques composed of the β-amyloid (Aβ) peptide, and intracellular neurofibrillary tangles (NFT) resulting from the aggregation of hyperphosphorylated microtubule-associated protein tau.⁴

The formation of Aβ is a sequential proteolytic process consisting of the cleavage of the amyloid precursor protein (APP) by the β- and γ-secretase enzymes. The β secretase, referred to as Beta-site Amyloid precursor protein (APP) Cleaving Enzyme 1 (BACE1), is the enzyme that initiates Aβ production by cleaving the extracellular domain of APP, generating a N-terminal Aβ fragment and the membrane bound C-terminal fragment C99.⁵ Successively, intramembrane processing of the latter fragment by γ-secretase yields Aβ.⁶ Most Aβ peptides terminate at residue 40 but about 5–10% ends at residue 42, which is a major player in AD pathogenesis. Thus, both β and γ secretases are required for the production of Aβ, and the inhibition or modulation of these enzymes has been considered a key therapeutic approach for reducing cerebral Aβ concentrations in patients with AD. The important role of BACE1 in APP processing was evinced in vivo from studies with knockout mice showing that both wild type and mutant forms of APP failed to produce Aβ in the absence of BACE1,⁷ further corroborating the importance of BACE1 as a target for the therapeutic intervention of AD.

The first-peptide based BACE1 inhibitors used a transition-state mimetic approach using a non-hydrolyzable peptide isostere at the scissile site. Successively, other non-peptide inhibitors based on diverse heterocyclic scaffolds were developed, all addressing the catalytic aspartic acid diad through acid-base and hydrogen-bond interactions.⁸ Some of these molecules possessing high efficacy in vitro proved to reduce the secretion of Aβ in cell cultures.⁹ Indeed, an important issue in the development of BACE1 inhibitor drugs encompasses the difficulty in crossing the blood-brain barrier (BBB), thus resulting in low potency in vivo, and recent papers reported interesting improvements towards brain penetration.10, 11

Several years ago our research group introduced a novel class of bicyclic peptidomimetic scaffolds showing dipeptide isostere potential.¹² Such compounds are easily achieved from the synthetic combination of suitable amino acids and tartaric acid or carbohydrates derivatives, and show remarkable stability of the acetal portion to acidic conditions.¹³ More recently, we identified this class of peptidomimetic compounds as potential aspartic protease inhibitors taking advantage of their role as dipeptide isosteres, and of the bicyclic acetal portion as a potential transition-state analogue in the interaction with key residues of enzyme’s catalytic site. Among this library of peptidomimetics, two hit candidates proved to be effective against drug-resistant C. albicans strains in in vivo experiments.14, 15, 16 The structural analogy of aspartic proteases expressed in different pathological systems suggested the screening of this library of bicyclic compounds towards other important therapeutic targets, such as the HIV protease. Indeed, sub-micromolar hit compounds were identified as HIV protease inhibitors,¹⁷ thus corroborating the capability of these compounds as aspartic protease inhibitors by means of a transition-state isosterism.

Accordingly, in this work we envisaged the application of these bicyclic compounds against BACE1 as an additional aspartic protease target. The analysis of the similarity of BACE1 with SAP2 from C. albicans and HIV-1 protease was carried out through molecular modeling, and the key structural features of the active site were investigated by a three-dimensional structural superimposition of BACE1 with SAP2 and the HIV protease. Also, we expanded the array of this class of bicyclic compounds by the synthesis of novel bicyclic thiolactams, with aim to explore the potential of the sulfur atom as a hydrogen-bond acceptor in the key functional group at position 2 of these scaffolds, and we applied them together with representative library members in enzyme inhibition assays for the identification of novel BACE1 inhibitors.