Synthesis of a potent photoreactive acidic γ-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aβ fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.     

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.     

SAR studies of acidic dual γ-secretase/PPARγ modulators

A novel set of dual γ-secretase/PPARγ modulators characterized by a 2-benzyl hexanoic acid scaffold is presented. Synthetic efforts were focused on the variation of the substitution pattern of the central benzene. Finally, we obtained a new class of 2,5-disubstituted 2-benzylidene hexanoic acid derivatives, which act as dual γ-secretase/PPARγ modulators in the low micromolar range. We have explored broad SAR and successfully improved the dual pharmacological activity and the selectivity profile against potential off-targets such as NOTCH and COX. Compound 17 showed an IC(50) Aβ42=2.4 μM and an EC(50) PPARγ=7.2 μM and could be a valuable tool to further evaluate the concept of dual γ-secretase/PPARγ modulators in animal models of Alzheimer's disease.     

Novel γ-secretase enzyme modulators directly target presenilin protein

γ-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid β-peptide (Aβ(42)). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the β-amyloid precursor protein by an intramembrane cleavage of γ-secretase. Because Aβ(42) is widely believed to trigger AD pathogenesis, γ-secretase is a key AD drug target. Unlike inhibitors of the enzyme, γ-secretase modulators (GSMs) selectively lower Aβ(42) without interfering with the physiological function of γ-secretase. The molecular target(s) of GSMs and hence the mechanism of GSM action are not established. Here we demonstrate by using a biotinylated photocross-linkable derivative of highly potent novel second generation GSMs that γ-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of γ-secretase, but not to other γ-secretase subunits. Binding was differentially competed by GSMs of diverse structural classes, indicating the existence of overlapping/multiple GSM binding sites or allosteric alteration of the photoprobe binding site. The β-amyloid precursor protein C-terminal fragment previously implicated as the GSM binding site was not targeted by the compound. The identification of presenilin as the molecular target of GSMs directly establishes allosteric modulation of enzyme activity as a mechanism of GSM action and may contribute to the development of therapeutically active GSMs for the treatment of AD.     

Pyridine-derived γ-secretase modulators

SAR of a novel series of pyridine-derived γ-secretase modulators is described. Compound 5 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aβ42 and Aβ40, and maintain (or increase) the levels of total Aβ. Furthermore, representative compounds 1 and 5 demonstrated in vivo efficacy to lower Aβ42 in the brain without altering Notch processing in the peripheral.     

Pyridazine-derived γ-secretase modulators

SAR of a novel series of pyridazine-derived γ-secretase modulators is described. Compound 25 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aβ42 and Aβ40, and maintain the levels of total Aβ. Furthermore, 25 demonstrated excellent pharmacokinetic parameters as well as good CNS penetration in the rat.     

Triazoles as γ-secretase modulators

Synthesis, SAR, and evaluation of aryl triazoles as novel gamma secretase modulators (GSMs) are presented in this communication. Starting from the literature and in-house leads, we evaluated a range of five-membered heterocycles as replacements for olefins commonly found in non-acid GSMs. 1,2,3-C-aryl-triazoles were identified as suitable replacements which exhibited good modulation of γ-secretase activity, excellent pharmacokinetics and good central lowering of Aβ42 in Sprague-Dawley rats.     

Iminoheterocycles as γ-secretase modulators

The synthesis of a novel series of iminoheterocycles and their structure–activity relationship (SAR) as modulators of γ-secretase activity will be detailed. Encouraging SAR generated from a monocyclic core led to a structurally unique bicyclic core. Selected compounds exhibit good potency as γ-secretase modulators, excellent rat pharmacokinetics, and lowering of Aβ₄₂ levels in various in vivo models.     

Aminothiazoles as γ-secretase modulators

We herein report the discovery of a new γ-secretase modulator class with an aminothiazole core starting from a HTS hit (3). Synthesis and SAR of this series are discussed. These novel compounds demonstrate moderate to good in vitro potency in inhibiting amyloid beta (Aβ) peptide production. Overall γ-secretase is not inhibited but the formation of the aggregating, toxic Aβ42 peptide is shifted to smaller non-aggregating Aβ peptides. Compound 15 reduced brain Aβ42 in vivo in APPSwe transgenic mice at 30mg/kg p.o.     

Quinazolinones as γ-secretase modulators

Synthesis, SAR and evaluation of styrenyl quinazolinones as novel gamma secretase modulators are presented in this communication. Starting from literature and in-house leads we evaluated a range of quinazolinones which showed good modulation of γ-secretase activity.     

Purine derivatives as potent γ-secretase modulators

The development of a novel series of purines as γ-secretase modulators for potential use in the treatment of Alzheimer’s disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based γ-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Αβ42 in an APP-YAC transgenic mouse model.     

Design and synthesis of an aminopiperidine series of γ-secretase modulators

The design, synthesis, and SAR of cyclic diamines as novel γ secretase modulators (GSMs) are presented in this Letter. Starting from information in the literature and in-house cyclic diamines library, we have found a 3(S)-aminopiperidine as a potent structure for lowering Aβ42 production both in vitro and in vivo.     

Discovery of fused 5,6-bicyclic heterocycles as γ-secretase modulators

We herein report the discovery of four series of fused 5,6-bicyclic heterocycles as γ-secretase modulators. Synthesis and SAR of these series are discussed. These compounds represent a new class of γ-secretase modulators that demonstrate moderate to good in vitro potency in inhibiting Aβ₄₂ production.     

Membrane anchoring γ-secretase modulators with terpene-derived moieties

Modulation of γ-secretase activity is a promising therapeutic strategy for the treatment of Alzheimer’s disease. Herein we report on the synthesis of carprofen- and tocopherol-derived small-molecule modulators carrying terpene moieties as lipophilic membrane anchors. Additionally, these modulators are equipped with an acidic moiety, which contributes to the desired modulatory effect on the γ-secretase with decreased formation of Aβ₄₂ and increased Aβ₃₈ production.     

Fluorinated piperidine acetic acids as γ-secretase modulators

We report herein a novel series of difluoropiperidine acetic acids as modulators of γ-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective β-difluorination with Selectfluor®. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Aβ42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250 mg/kg per day, AUC_0–24 = 2100 μM h) did not exhibit Notch-related effects.     

Design and synthesis of bicyclic heterocycles as potent γ-secretase modulators

The evolution of amide 3 into conformationally restricted bicyclic triazolo-piperidine 14-S as a γ-secretase modulator is described. This is a potential disease modifying anti-Alzheimer’s drug which demonstrated high in vitro and in vivo potency against Aβ42 peptide, reduced lipophilicity and enhanced brain free fraction compared to the previous series.     

Lipid bilayer position and orientation of novel carprofens,modulators of γ-secretase in Alzheimer's disease

γ-Secretase is an integral membrane protein complex and is involved in the cleavage of the amyloid precursor protein APP to produce amyloid-β peptides. Amyloid-β peptides are considered causative agents for Alzheimer's disease and drugs targeted at γ-secretase are investigated as therapeutic treatments. We synthesized new carprofen derivatives, which showed γ-secretase modulating activity and determined their precise position, orientation, and dynamics in lipid membranes by combining neutron diffraction, solid-state NMR spectroscopy, and molecular dynamics simulations. Our data indicate that the carprofen derivatives are inserted into the membrane interface, where the exact position and orientation depends on the lipid phase. This knowledge will help to understand the docking of carprofen derivatives to γ-secretase and in the design of new potent drugs. The approach presented here promises to serve as a general guideline how drug/target interactions in membranes can be analyzed in a comprehensive manner.     

NSAID-based γ-secretase modulators do not bind to the amyloid-β polypeptide

γ-Secretase modulators (GSMs) have received much attention as potential therapeutic agents for Alzheimer's disease (AD). GSMs increase the ratio between short and long forms of the amyloid-β (Aβ) polypeptides produced by γ-secretase and thereby decrease the amount of the toxic amyloid species. However, the mechanism of action of these agents is still poorly understood. One recent paper [Richter et al. (2010) Proc. Natl. Acad. Sci. U. S. A.107, 14597-14602] presented data that were interpreted to support direct binding of the GSM sulindac sulfide to Aβ(42), supporting the notion that GSM action is linked to direct binding of these compounds to the Aβ domain of its immediate precursor, the 99-residue C-terminal domain of the amyloid precursor protein (C99, also known as the β-CTF). Here, contrasting results are presented that indicate there is no interaction between monomeric sulindac sulfide and monomeric forms of Aβ42. Instead, it was observed that sulindac sulfide is itself prone to form aggregates that can bind nonspecifically to Aβ42 and trigger its aggregation. This observation, combined with data from previous work [Beel et al. (2009) Biochemistry48, 11837-11839], suggests both that the poor behavior of some NSAID-based GSMs in solution may obscure results of binding assays and that NSAID-based GSMs do not function by directly targeting C99. It was also observed that another GSM, flurbiprofen, fails to bind to monomeric Aβ42 or to C99 reconstituted into bilayered lipid vesicles. These results disfavor the hypothesis that these NSAID-based GSMs exert their modulatory effect by directly targeting a site located in the Aβ42 domain of free C99.