Activation of γ-Secretase Trimming Activity by Topological Changes of Transmembrane Domain 1 of Presenilin 1.

γ-Secretase is an intramembrane cleaving protease that is responsible for the generation of amyloid-ß peptides, which are linked to the pathogenesis of Alzheimer disease. Recently, γ-secretase modulators (GSMs) have been shown to specifically decrease production of the aggregation-prone and toxic longer Aß species, and concomitantly increase the levels of shorter Aß. We previously found that phenylimidazole-type GSMs bind to presenilin 1 (PS1), the catalytic subunit of the γ-secretase, and allosterically modulate γ-secretase activity. However, the precise conformational alterations in PS1 remained unclear. Here we mapped the amino acid residues in PS1 that is crucial for the binding and pharmacological actions of E2012, a phenylimidazole-type GSM, using photoaffinity labeling and the substituted cysteine accessibility method. We also demonstrated that a piston-like vertical motion of transmembrane domain (TMD) 1 occurs during modulation of Aß production. Taking these results together, we propose a model for the molecular mechanism of phenylimidazole-type GSMs, in which the trimming activity of γ-secretase is modulated by the position of the TMD1 of PS1 in the lipid bilayer.SIGNIFICANCE STATEMENT Reduction of the toxic longer amyloid-ß peptide is one of the therapeutic approaches for Alzheimer disease. A subset of small compounds called γ-secretase modulators specifically decreases the longer amyloid-ß production, although its mechanistic action remains unclear. Here we found that the modulator compound E2012 targets to the hydrophilic loop 1 of presenilin 1, which is a catalytic subunit of the γ-secretase. Moreover, E2012 triggers the piston movement of the transmembrane domain 1 of presenilin 1, which impacts on the γ-secretase activity. These results illuminate how γ-secretase modulators allosterically affect the proteolytic activity, and highlight the importance of the structural dynamics of presenilin 1 in the complexed process of the intramembrane cleavage.

Enantioselective Total Synthesis of RQN-18690A (18-Deoxyherboxidiene).

The first total synthesis of RQN-18690A (18-deoxyherboxidiene) and the determination of its absolute stereochemical configuration are described. The synthesis features an organocatalytic aldol reaction for the first step, 1,4- and 1,2- dual reductions of α,ß-unsaturated δ-lactone followed by a domino reaction in a one-pot operation, and diastereoselective epoxidation with kinetic resolution.

Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist.

The orexin/hypocretin receptors are a family of G protein-coupled receptors and consist of orexin-1 (OX1) and orexin-2 (OX2) receptor subtypes. Orexin receptors are expressed throughout the central nervous system and are involved in the regulation of the sleep/wake cycle. Because modulation of these receptors constitutes a promising target for novel treatments of disorders associated with the control of sleep and wakefulness, such as insomnia, the development of orexin receptor antagonists has emerged as an important focus in drug discovery research. Here, we report the design, synthesis, characterization, and structure-activity relationships (SARs) of novel orexin receptor antagonists. Various modifications made to the core structure of a previously developed compound (-)-5, the lead molecule, resulted in compounds with improved chemical and pharmacological profiles. The investigation afforded a potential therapeutic agent, (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006), an orally active, potent orexin antagonist. The efficacy was demonstrated in mice in an in vivo study by using sleep parameter measurements.

Design, synthesis, and structure-activity relationships of a series of novel N-aryl-2-phenylcyclopropanecarboxamide that are potent and orally active orexin receptor antagonists.

Herein we describe the design, synthesis, and structure-activity relationships (SARs) of a novel phenylcyclopropane series represented by 7 and 33 b as antagonists of orexin 1 and orexin 2 receptors. With 4 serving as the initial lead for the development of orexin antagonists, exploration of SAR resulted in improved binding affinity for orexin 1 and orexin 2 receptors. Among the synthesized compounds, 33 b ((-)-N-(5-cyanopyridin-2-yl)-2-[(3,4-dimethoxyphenyl)oxymethyl]-2-phenylcyclopropanecarboxamide) exhibited potent in vitro activity and oral efficacy in animal sleep measurement experiments. The results of our study suggest that compound 33 b may serve as a valuable template for the development of new orexin receptor antagonists.

Discovery of 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile (perampanel): a novel, noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist.

Dysfunction of glutamatergic neurotransmission has been implicated in the pathogenesis of epilepsy and numerous other neurological diseases. Here we describe the discovery of a series of 1,3,5-triaryl-1H-pyridin-2-one derivatives as noncompetitive antagonists of AMPA-type ionotropic glutamate receptors. The structure-activity relationships for this series of compounds were investigated by manipulating individual aromatic rings located at positions 1, 3, and 5 of the pyridone ring. This culminated in the discovery of 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile (perampanel, 6), a novel, noncompetitive AMPA receptor antagonist that showed potent activity in an in vitro AMPA-induced Ca2+ influx assay (IC50=60 nM) and in an in vivo AMPA-induced seizure model (minimum effective dose of 2 mg/kg po). Perampanel is currently in regulatory submission for partial-onset seizures associated with epilepsy.

Synthesis and structure-activity relationships of 8-substituted-2-aryl-5-alkylaminoquinolines: Potent, orally active corticotropin-releasing factor-1 receptor antagonists.

We previously reported a series of 8-methyl-2-aryl-5-alkylaminoquinolines as a novel class of corticotropin-releasing factor-1 (CRF(1)) receptor antagonists. A critical issue encountered for this series of compounds was low aqueous solubility at physiological pH (pH 7.4). To address this issue, derivatization at key sites (R(2), R(3), R(5), R(5'), and R(8)) was performed and the relationships between structure and solubility were examined. As a result, it was revealed that introduction of a methoxy substituent at the C(8) position had a positive impact on the solubility of the derivatives. Consequently, through in vivo and in vitro biological studies, compound 21d was identified as a potent, orally active CRF(1) receptor antagonist with improved physicochemical properties.

Design, synthesis, and structure-activity relationships of novel pyrazolo[5,1-b]thiazole derivatives as potent and orally active corticotropin-releasing factor 1 receptor antagonists.

This paper describes the design, synthesis, and structure-activity relationships of a novel series of 7-dialkylamino-3-phenyl-6-methoxy pyrazolo[5,1-b]thiazole derivatives for use as selective antagonists of the corticotropin-releasing factor 1 (CRF(1)) receptor. The most promising compound, N-butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]thiazole-7-amine (6t), showed high affinity (IC(50) = 70 nM) and functional antagonism (IC(50) = 7.1 nM) for the human CRF(1) receptor as well as dose-dependent inhibition of the CRF-induced increase in the plasma adrenocorticotropic hormone (ACTH) concentration at a dose of 30 mg/kg (po). Further, in the light/dark test in mice, the compound 6t showed anxiolytic activity at a dose of 30 mg/kg (po).

Design, synthesis, and structure-activity relationships of a series of 2-Ar-8-methyl-5-alkylaminoquinolines as novel CRF1 receptor antagonists.

We designed and synthesized a series of 2-Ar-8-methyl-5-alkylaminolquinolines as potent corticotropin-releasing factor 1 (CRF(1)) receptor antagonists. The structure-activity relationships of substituents at each position (R(3), R(5), R(5'), and R(8)) was investigated. By derivatization, three compounds (6, 14b, and 14c) were identified as orally active CRF(1) receptor antagonists.

Design, synthesis and structure-activity relationships of 5-alkylaminolquinolines as a novel series of CRF1 receptor antagonists.

A series of 5-alkylaminolquinolines was designed and synthesized as potential novel CRF(1) receptor antagonists. The structure-activity relationships (SARs) of the substituents on each position (R(2), R(3), R(5) and R(5')) were investigated.

Synthesis and structure-activity relationships of pyrazolo[1,5-a]pyridine derivatives: potent and orally active antagonists of corticotropin-releasing factor 1 receptor.

Design, synthesis, and structure-activity relationships of a series of 3-dialkylamino-7-phenyl pyrazolo[1,5-a]pyridines (I) as selective antagonists of the corticotropin-releasing factor 1 (CRF(1)) receptor are described. The most prominent compound to emerge from this work, 46 (E2508), exhibits potent in vitro activity, excellent drug-like properties, and robust oral efficacy in animal models of stress-related disorders. It has advanced into clinical trials.

One-pot primary aminomethylation of aryl and heteroaryl halides with sodium phthalimidomethyltrifluoroborate.

A one-pot primary aminomethylation of aryl halides, triflates, mesylates, and tosylates via Suzuki-Miyaura cross-coupling reactions with sodium phthalimidomethyltrifluoroborate followed by deamidation with ethylenediamine is reported.

Palladium-catalyzed direct hydroxymethylation of aryl halides and triflates with potassium acetoxymethyltrifluoroborate.

Suzuki-Miyaura cross-coupling reactions of aryl halides and triflates with potassium acetoxymethyltrifluoroborate afforded the corresponding aryl and heteroaryl methanol products in moderate to excellent yields.

Synthesis and pharmacological profile of serofendic acids A and B.

We present efficient syntheses of serofendic acids A and B (SA-A and SA-B), novel neuroprotective substances isolated from fetal calf serum. Biological and pharmacological evaluation showed that SA-A and SA-B have potent protective action against glutamate-induced neurotoxicity, but do not interact directly with glutamate receptors. A pharmacokinetic study showed that they have good oral bioavailability in rats. The results indicate that SA-A and SA-B are potential lead compounds for candidate drugs to treat various neurological disorders.

Synthesis and neuroprotective effects of serofendic acid analogues.

Analogues of serofendic acid were prepared and their protective effects against L-glutamate (Glu)-induced neurotoxicity were examined using primary cultures of rat cortical neurons. Some analogues exhibited similar neuroprotective activity to that of serofendic acid.

Synthesis of optically active methyl 7 beta-hydroxykaurenoate with potent neuroprotective activity.

(-)-Methyl 7 beta-hydroxykaurenoate (3) and its 4-demethyl acetate (-)-4 were both synthesized via methods that contained radical cyclization and intramolecular Diels-Alder reactions as key steps. Both compounds displayed potent neuroprotective activity against N-methyl-D-aspartate toxicity in cultured cortical neurons.

Serofendic acid prevents acute glutamate neurotoxicity in cultured cortical neurons.

We have previously reported that a novel neuroprotective substance named serofendic acid was purified and isolated from ether extract of fetal calf serum. In the present study, we investigated the effect of serofendic acid on acute neurotoxicity induced by L-glutamate (Glu) using primary cultures of rat cortical neurons. Exposure of cortical cultures to Glu for 1 h caused a marked decrease in cell viability, as determined by trypan blue exclusion. This acute Glu neurotoxicity was prevented by N-methyl-D-aspartate (NMDA) receptor antagonists, extracellular Ca(2+) removal, nitric oxide (NO) synthase inhibitor and NO scavenger. Serofendic acid prevented acute Glu neurotoxicity in a concentration-dependent manner. Acute neurotoxicity was induced by ionomycin, a Ca(2+) ionophore, and S-nitroso-L-cysteine, an NO donor. Serofendic acid also prevented both ionomycin- and S-nitroso-L-cysteine-induced neurotoxicity. Moreover, the protective effect of serofendic acid on acute Glu neurotoxicity was not affected by cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor. These results indicate that serofendic acid protects cultured cortical neurons from acute Glu neurotoxicity by reducing the cytotoxic action of NO and de novo protein synthesis is not required for this neuroprotection.

Discovery of novel neuronal voltage-dependent calcium channel blockers based on emopamil left hand as a bioactive template.

A series of novel neuronal voltage-dependent calcium channel (VDCC) blockers, with inhibitory activity at low micromolar and moderate solubility in water, was discovered by constructing and screening a focused library based on emopamil (1) left hand (ELH) as a bioactive template.

Sulfonamide derivative, E7820, is a unique angiogenesis inhibitor suppressing an expression of integrin alpha2 subunit on endothelium.

In the process of angiogenesis, endothelial adhesion molecules play a significant role in vascular morphogenesis, in coordination with angiogenic factor signaling. Here we report that a novel angiogenesis inhibitor, E7820 (an aromatic sulfonamide derivative), inhibited in vitro proliferation and tube formation of human umbilical vascular endothelial cell (HUVEC). E7820 decreased integrin alpha2, 3, 5, and beta1 in confluent culture of HUVEC, and integrin alpha2 was initially suppressed in mRNA level, followed by decrement of integrins alpha3, 5, and beta1. The inhibition of integrin alpha2 expression in HUVEC showed dose dependence but did not alter the level of CD31. Up-regulation of integrin alpha2 by phorbol 12-myristate 13-acetate abrogated the inhibitory effect of E7820 on tube formation within type I collagen gel, whereas addition of antibody against integrin alpha2 canceled the phorbol 12-myristate 13-acetate effect. These results suggest that E7820 inhibited tube formation through the suppression of integrin alpha2. Oral administration of E7820 remarkably resulted in inhibition of tumor-induced angiogenesis in mouse dorsal air sac model, and tumor growth of human colorectal tumor cell lines (WiDr and LoVo) was inhibited in xenotransplanted model in mice. This is the first time that a small molecule has been shown to modulate integrins, and this finding may provide the basis for a new approach to antiangiogenic therapy through the suppression of integrin alpha2 on endothelium.

Practical synthesis of chiral emopamil left hand as a bioactive motif.

An asymmetric synthesis of (2S)-2-(2-isopropyl)-5-hydroxy-2-phenylpentanenitrile (emopamil left hand, 2) has been completed by use of the MAD (methyl aluminum bis(4-methyl-2,6-di-tert-butylphenoxide)-induced rearrangement of a chiral epoxyalcohol as the key reaction. The stereochemistry of the chiral quaternary center was confirmed by transformation of 2 to (S)-noremopamil. This method requires minimal purification procedures and affords high chemical and optical yields. Acid-catalyzed isomerization of an allylaldehyde and retro-aldol type racemization at the quaternary carbon of a nitrile-alcohol were encountered.

Isolation of a diterpenoid substance with potent neuroprotective activity from fetal calf serum.

Excess activation of glutamate receptors and production of free radicals including nitric oxide may result in severe and irreversible damage to the mammalian central nervous system (CNS), but endogenous defense systems that protect neurons from these insults are poorly understood. Here, we purified and isolated a neuroprotective substance, which has been named "serofendic acid," from a lipophilic fraction of FCS based on the ability to protect rat primary cortical neurons against nitric oxide cytotoxicity. Mass spectrometry and NMR spectroscopy revealed the chemical structure of serofendic acid (15-hydroxy-17-methylsulfinylatisan-19-oic acid) as a sulfur-containing atisane-type diterpenoid, which is unique among known endogenous substances. Synthetic serofendic acid exhibited potent protective actions on cortical neurons against cytotoxicity of a nitric oxide donor as well as of glutamate, although it did not show appreciable influences on glutamate receptor-mediated responses in these neurons. Electron spin resonance analysis demonstrated that serofendic acid had no direct scavenging activity on nitric oxide radicals but was capable of inhibiting the generation of hydroxyl radical, a presumed "executor" radical in the nitric oxide-mediated neurotoxic cascade. These findings suggest that serofendic acid is a low-molecular-weight bioactive factor that promotes survival of CNS neurons, probably through the attenuation of free radical-mediated insults.