beta-Secretase inhibitors: modification at the P4 position and improvement of inhibitory activity in cultured cells

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-570 and KMI-684 in which we replaced carboxylic acid groups at the P(1)(') position of KMI-420 and KMI-429, respectively, with tetrazole derivatives as carboxylic acid bioisosteres. These modifications improved significantly BACE1 inhibitory activity and chemical stability. In this study, the acidic tetrazole ring of the P(4) position of KMI-420 and KMI-570, respectively, was replaced with various hydrogen bond acceptor groups. We found BACE1 inhibitor KMI-574 that exhibited potent inhibitory activity in cultured cells as well as in vitro enzymatic assay.     

Design and synthesis of potent beta-secretase (BACE1) inhibitors with P1' carboxylic acid bioisosteres

Recently, we reported potent and small-sized beta-secretase (BACE1) inhibitors KMI-420 and KMI-429 in which we replaced the Glu residue at the P4 position of KMI-260 and KMI-360, respectively, with a 1H-tetrazole-5-carbonyl DAP (L-alpha,beta-diaminopropionic acid) residue. At the P1' position, these compounds contain one or two carboxylic acid groups, which are unfavorable for crossing the blood-brain barrier. Herein, we report BACE1 inhibitors with P1' carboxylic acid bioisosteres in order to develop practical anti-Alzheimer's disease drugs. Among them, tetrazole ring-containing compounds, KMI-570 (IC50=4.8 nM) and KMI-684 (IC50=1.2 nM), exhibited significantly potent BACE1 inhibitory activities.     

KMI-358 and KMI-370, highly potent and small-sized BACE1 inhibitors containing phenylnorstatine

Recently, we reported a novel substrate-based octapeptide BACE1 inhibitor KMI-008 containing hydroxymethylcarbonyl (HMC) isostere as a transition-state mimic. Using KMI-008 as a lead compound, a small-sized and highly potent BACE1 inhibitor KMI-370 (IC(50)=3.4 nM) was designed and synthesized.     

Novel BACE1 inhibitors with a non-acidic heterocycle at the P1′ position

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P₁′ position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P₁′ position. KMI-1764 (27) exhibited potent inhibitory activity (IC₅₀ = 27 nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure–activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model.     

BACE1 inhibitors: optimization by replacing the P1' residue with non-acidic moiety

Recently, we reported potent BACE1 inhibitors KMI-429, -684, and -574 possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays, especially, KMI-429 was confirmed to significantly inhibit Abeta production in vivo. However, acidic moieties at the P(4) and P(1)' positions of KMI-compounds were thought to be unfavorable for membrane permeability across the blood-brain barrier. Herein, we replaced acidic moieties at the P(4) position with other hydrogen bond acceptor groups, and these inhibitors exhibited improved BACE1 inhibitory activities in cultured cells. In this study, we replaced the acidic moieties at the P(1)' position with non-acidic and low molecular sized moieties.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

Structure-guided design and synthesis of P1' position 1-phenylcycloalkylamine-derived pentapeptidic BACE1 inhibitors

Previously, we reported potent pentapeptidic BACE1 inhibitors with the hydroxymethylcarbonyl isostere as a substrate transition-state mimic. To improve the in vitro potency, we further reported pentapeptidic inhibitors with carboxylic acid bioisosteres at the P(4) and P1' positions. In the current study, we screened new P1' position 1-phenylcycloalkylamine analogs to find non-acidic inhibitors that possess double-digit nanomolar range IC(50) values. An extensive structure-activity relationship study was performed with various amine derivatives at the P1' position. The most potent inhibitor of this pentapeptide series, KMI-1830, possessing 1-phenylcyclopentylamine at the P1' position had an IC(50) value of 11.6 nM against BACE1 in vitro enzymatic assay.     

Structure–activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P2 position

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aβ production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood–brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P₂ position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.     

Novel non-peptidic and small-sized BACE1 inhibitors

Recently, we reported substrate-based beta-secretase (BACE1) inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. These inhibitors showed potent BACE1 inhibitory activities (approximately 1.2 nM IC(50)). In order to improve in vivo enzymatic stability and permeability across the blood-brain barrier, these penta-peptidic inhibitors would need to be further optimized. On the other hand, non-peptidic inhibitors possessing isophthalic residue at the P(2) position were reported from other research groups. We selected isophthalic-type aromatic residues at the P(2) position and an HMC isostere at the P(1) position as lead compounds. On the basis of the design approach focused on the conformer of docked inhibitor in BACE1, we found novel non-peptidic and small-sized BACE1 inhibitors possessing a 2,6-pyridinedicarboxylic, chelidamic or chelidonic residue at the P(2) position.     

Design,synthesis, and bioactivities of tasiamide B derivatives as cathepsin D inhibitors

Cathepsin D (Cath D) is overexpressed and hypersecreted by malignant tumors and involved in the progress of tumor invasion, proliferation, metastasis, and apoptosis. Cath D has been considered as a potential target to treat cancer. Our previous studies revealed that tasiamide B derivatives TB‐9 and TB‐11 exhibited high potent inhibition against Cath D and other aspartic proteases, but their molecular weights are still high, and the role of each residue is unknown yet. Based on this, two series of tasiamide B derivatives have been designed, synthesized, and evaluated for their inhibitory activity against Cath D/Cath E/BACE1. Enzymatic assays revealed that the target compound 1 with lower molecule weight showed good inhibitory activity against Cath D with IC₅₀ of 3.29 nM and satisfactory selectivity over Cath E (72‐fold) and BACE1 (295‐fold), which could be a valuable template for the design of highly potent and selective Cath D inhibitors.     

Significance of interactions of BACE1–Arg235 with its ligands and design of BACE1 inhibitors with P2 pyridine scaffold

Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across the blood–brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or chelidonic moiety, at the P₂ position were reported. These inhibitors were designed based on the conformer of docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P₂ position, that were optimized for the interactions with Arg235 of BACE1.     

KMI-008, a novel beta-secretase inhibitor containing a hydroxymethylcarbonyl isostere as a transition-state mimic: design and synthesis of substrate-based octapeptides

A novel class of substrate-based β-secretase (BACE1) inhibitors containing a hydroxymethylcarbonyl (HMC) isostere was designed and synthesized. Phenylnorstatine [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid; Pns] was an effective transition-state mimic at the P₁ position. Structure–activity relationships (SARs) of the P₃–P₃′ positions of BACE1 inhibitors were studied.     

Molecular modeling,synthesis, and activity studies of novel biaryl and fused-ring BACE1 inhibitors

A series of transition state analogues of beta-secretases 1 and 2 (BACE1, 2) inhibitors containing fused-ring or biaryl moieties were designed computationally to probe the S2 pocket, synthesized, and tested for BACE1 and BACE2 inhibitory activity. It has been shown that unlike the biaryl analogs, the fused-ring moiety is successfully accommodated in the BACE1 binding site resulting in the ligands with excellent inhibitory activity. Ligand 5b reduced 65% of Aβ40 production in N2a cells stably transfected with Swedish human APP.     

Inhibition of beta-secretase in vivo via antibody binding to unique loops (D and F) of BACE1

β-Secretase (BACE1) is an attractive drug target for Alzheimer disease. However, the design of clinical useful inhibitors targeting its active site has been extremely challenging. To identify alternative drug targeting sites we have generated a panel of BACE1 monoclonal antibodies (mAbs) that interfere with BACE1 activity in various assays and determined their binding epitopes. mAb 1A11 inhibited BACE1 in vitro using a large APP sequence based substrate (IC(50) ～0.76 nm), in primary neurons (EC(50) ～1.8 nm), and in mouse brain after stereotactic injection. Paradoxically, mAb 1A11 increased BACE1 activity in vitro when a short synthetic peptide was used as substrate, indicating that mAb 1A11 does not occupy the active-site. Epitope mapping revealed that mAb 1A11 binds to adjacent loops D and F, which together with nearby helix A, distinguishes BACE1 from other aspartyl proteases. Interestingly, mutagenesis of loop F and helix A decreased or increased BACE1 activity, identifying them as enzymatic regulatory elements and as potential alternative sites for inhibitor design. In contrast, mAb 5G7 was a potent BACE1 inhibitor in cell-free enzymatic assays (IC(50) ～0.47 nm) but displayed no inhibitory effect in primary neurons. Its epitope, a surface helix 299-312, is inaccessible in membrane-anchored BACE1. Remarkably, mutagenesis of helix 299-312 strongly reduced BACE1 ectodomain shedding, suggesting that this helix plays a role in BACE1 cellular biology. In conclusion, this study generated highly selective and potent BACE1 inhibitory mAbs, which recognize unique structural and functional elements in BACE1, and uncovered interesting alternative sites on BACE1 that could become targets for drug development.     

Design,synthesis and evaluation of 2-amino-imidazol-4-one derivatives as potent β-site amyloid precursor protein cleaving enzyme 1 (BACE-1) inhibitors

Inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) to prevent brain β-amyloid (Aβ) peptide’s formation is a potential effective approach to treat Alzheimer’s disease. In this report we described a structure-based optimization of a series of BACE1 inhibitors derived from an iminopyrimidinone scaffold W-41 (IC₅₀ = 7.1 μM) by Wyeth, which had good selectivity and brain permeability but low activity. The results showed that occupying the S₃ cavity of BACE1 enzyme could be an effective strategy to increase the biological activity, and five compounds exhibited stronger inhibitory activity and higher liposolubility than W-41, with L-5 was the most potent inhibitor against BACE1 (IC₅₀ = 0.12 μM, logP = 2.49).     

Novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P2 position

Recently, we reported substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays. We also designed and synthesized non-peptidic and small-sized inhibitors possessing a heterocyclic scaffold at the P(2) position. By studying the structure-activity relationship of these inhibitors, we found that the σ-π interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the inhibition mechanism. Hence, we optimized the inhibitors with a focus on their P(2) regions. In this Letter, a series of novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P(2) position are described along with the results of the related structure-activity relationship study. These small-sized inhibitors are expected improved membrane permeability and bioavailability.     

Phenylimino-2H-chromen-3-carboxamide derivatives as novel small molecule inhibitors of β-secretase (BACE1)

The inhibition of β secretase (BACE1) is potentially important approach to treatment of Alzheimer disease (AD). A novel series of 4-bromophenyl piperazine derivatives coupled to the phenylimino-2H-chromen-3-carboxamide scaffold were investigated as BACE1 inhibitors in this study. Docking study suggested that the phenyl-imino group of the scaffold establishes favorable π–π stacking interaction with side chain of Phe108 of flap pocket. Some of the docking proposed derivatives were synthesized and evaluated for BACE1 inhibitory activity using a FRET-based assay. High BACE1 inhibitory activities were observed from derivatives containing fused heteroaromtic groups attached through the aliphatic linkage to the N4-piperazine moiety, which may be attributed to the engagement of effective interactions with S1–S′1 sub-pocket residues. Of the most potent compounds, 9e displayed an IC₅₀ value for BACE1 of 98 nM. Some of these derivatives demonstrated good inhibitory activity on Aβ production in N2a-APPswe cells at 5 and 10 μM. These compounds might be considered as promising BACE1 inhibitory agents that could lower Aβ production in AD.     

基于作用机制的氨基恶唑啉呫吨类BACE1抑制剂的分子设计

天冬氨酰蛋白酶(β-site amyloid precursor protein cleaving enzyme 1, BACE1)作为治疗阿尔兹海默症的潜在靶点,其抑制剂的开发已成为医学领域的重要研究方向。本文以59个氨基恶唑啉呫吨类BACE1抑制剂为研究对象,运用比较分子相似性指数(comparative molecular similarity index, CoMSIA)和分子对接方法,深入挖掘影响抑制剂活性的特征结构,以及抑制剂与BACE1间的结合模式和作用力类型,并以此为基础设计新型抑制剂并预测其活性。CoMSIA模拟结果表明,由立体场、静电场、疏水场和氢键供体场4个场组合建立的构效关系模型具有较强的预测能力,交叉验证相关系数Q2=0.48, 非交叉验证相关系数Rncv2=0.94, 外部预测相关系数Rpre2=0.85;通过分子对接,发现抑制剂占据了靶标的S3、S1和S2'位点,与BACE1之间的结合主要是通过氢键作用力和π-π堆积作用实现的;占据S2'位点的R取代基是立体场、静电场和疏水场影响的敏感区域,氨基恶唑啉核心官能团是氢键供体场的敏感区域。基于以上分析获得的抑制剂特征结构信息及其与蛋白质受体的作用机制,成功设计出了新的分子并预测了抑制活性。实验所得模型和信息,为后续新型BACE1抑制剂的结构优化和改造提供了重要理论依据     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.     

Preparation and biological evaluation of BACE1 inhibitors: Leveraging trans-cyclopropyl moieties as ligand efficient conformational constraints

Inhibition of BACE1 has become an important strategy in the quest for disease modifying agents to slow the progression of Alzheimer’s disease. We previously reported the fragment-based discovery of LY2811376, the first BACE1 inhibitor reported to demonstrate robust reduction of human CSF Aβ in a Phase I clinical trial. We also reported on the discovery of LY2886721, a potent BACE1 inhibitor that reached phase 2 clinical trials. Herein we describe the preparation and structure activity relationships (SAR) of a series of BACE1 inhibitors utilizing trans-cyclopropyl moieties as conformational constraints. The design, details of the stereochemically complex organic synthesis, and biological activity of these BACE1 inhibitors is described.