Discovery and characterization of novel indole and 7-azaindole derivatives as inhibitors of ß-amyloid-42 aggregation for the treatment of Alzheimer's disease.

The aggregation of amyloid-ß peptides into cytotoxic oligomeric and fibrillary aggregates is believed to be one of the major pathological events in Alzheimer disease. Here we report the design and synthesis of a novel series of indole and 7-azaindole derivatives containing, nitrile, piperidine and N-methyl-piperidine substituents at the 3-position to prevent the pathological self-assembly of amyloid-ß. We have further demonstrated that substitution of the azaindole and indole derivatives at the 3 positions is required to obtain compounds with improved physicochemical properties to allow brain penetration.

Synthesis and structure-activity relationship of 2,6-disubstituted pyridine derivatives as inhibitors of ß-amyloid-42 aggregation.

It is assumed that amyloid-ß aggregation is a crucial event in the pathogenesis of Alzheimer's disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the ß-sheet conformation of Aß via donor-acceptor-donor hydrogen bond formation. A series of pyridine derivatives were synthesized and tested regarding their potential to inhibit the aggregation of Aß. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aß aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at least one C2- or C3-linker displayed the most potent inhibition of Aß aggregation.

Discovery and structure activity relationship of small molecule inhibitors of toxic ß-amyloid-42 fibril formation.

Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.

Release of bioactive volatiles from supramolecular hydrogels: influence of reversible acylhydrazone formation on gel stability and volatile compound evaporation.

In the presence of alkali metal cations, guanosine-5'-hydrazide (1) forms stable supramolecular hydrogels by selective self-assembly into a G-quartet structure. Besides being physically trapped inside the gel structure, biologically active aldehydes or ketones can also reversibly react with the free hydrazide functions at the periphery of the G-quartet to form acylhydrazones. This particularity makes the hydrogels interesting as delivery systems for the slow release of bioactive carbonyl derivatives. Hydrogels formed from 1 were found to be significantly more stable than those obtained from guanosine. Both physical inclusion of bioactive volatiles and reversible hydrazone formation could be demonstrated by indirect methods. Gel stabilities were measured by oscillating disk rheology measurements, which showed that thermodynamic equilibration of the gel is slow and requires several cooling and heating cycles. Furthermore, combining the rheology data with dynamic headspace analysis of fragrance evaporation suggested that reversible hydrazone formation of some carbonyl compounds influences the release of volatiles, whereas the absolute stability of the gel seemed to have no influence on the evaporation rates.

PI-2620 Lead Optimization Highlights the Importance of Off-Target Assays to Develop a PET Tracer for the Detection of Pathological Aggregated Tau in Alzheimer's Disease and Other Tauopathies.

The first candidate PI-2014 was tested in healthy controls and subjects with Alzheimer's disease (AD). As PI-2014 displayed off-target binding to monoamine oxidase A (MAO-A), a new lead with improved binding to Tau and decreased MAO-A binding was required. For compound optimization, Tau binding assays based on both human AD brain homogenate and Tau-paired helical filaments were employed. Furthermore, two MAO-A screening assays based on (1) human-recombinant MAO-A and (2) displacement of 2-fluoro-ethyl-harmine from mouse brain homogenate were employed. Removing the N-methyl group from the tricyclic core resulted in compounds displaying improved Tau binding. For the final round of optimization, the cyclic amine substituents were replaced by pyridine derivatives. PI-2620 (2-(2-fluoropyridin-4-yl)-9H-pyrrolo[2,3-b:4,5-c']dipyridine) emerged as a best candidate displaying high Tau binding, low MAO-A binding, high brain uptake, and fast and complete brain washout. Furthermore, PI-2620 showed Tau binding on brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease.

Discovery of 2-(4-(2-fluoroethoxy)piperidin-1-yl)-9-methyl-9H-pyrrolo[2,3-b:4,5-c']dipyridine ([18F]PI-2014) as PET tracer for the detection of pathological aggregated tau in Alzheimer's disease and other tauopathies.

The compound screening was initiated with a direct staining assay to identify compounds binding to Tau aggregates and not Abeta plaques using human brain sections derived from late stage Alzheimer's disease donors. The binding of Tau aggregate selective compounds was then quantitatively assessed with human brain derived paired helical filaments utilizing the label-free Back Scattering Interferometry assay. In vivo biodistribution experiments of selected fluorine-18 labeled compounds were performed in mice to assess brain uptake, brain washout, and defluorination. Compound 11 emerged as the most promising candidate, displaying high in vitro binding affinity and selectivity to neurofibrillary tangles. Fluorine-18 labeled compound 11 showed high brain uptake and rapid washout from the mouse brain with no observed bone uptake. Furthermore, compound 11 was able to detect Tau aggregates in tauopathy brain sections from corticobasal degeneration, progressive supranuclear palsy, and Pick's disease donors. Thus, 2-(4-(2-fluoroethoxy)piperidin-1-yl)-9-methyl-9H-pyrrolo[2,3-b:4,5-c']dipyridine (PI-2014, compound 11) was selected for characterization in a first-in-human study.

Structural selection in G-quartet-based hydrogels and controlled release of bioactive molecules.

A guanosine-5'-hydrazide can entrap biologically interesting molecules such as acyclovir, vitamin C, and vancomycin into its hydrogel network. Controlled release of these molecules was monitored by 1H NMR spectroscopy. The hydrazide may potentially form mixed G-G quartets with analogous compounds containing a guanine group. 1H NMR spectroscopy was used to study the inclusion of various guanine derivatives into the hydrogel. The structural selectivity was found to depend strongly on both the shape and the charge of the additive and may arise from the strong cohesion of the supramolecular architecture of the gel and the resulting resistance to perturbation by foreign bodies. Hydrogels thus offer a promising medium for highly selective, controlled release of bioactive substances.

Molecular structure of guanine-quartet supramolecular assemblies in a gel-state based on a DFT calculation of infrared and vibrational circular dichroism spectra.

The infrared (IR) and vibrational circular dichroism (VCD) spectra of guanosine-5'-hydrazide ( G-1), a powerful hydrogelator, have been measured and analyzed on the basis of ab initio modeling. B3LYP/6-31G** DFT calculations predict that G-1, forming a clear solution in deuterated DMSO, is present in monomeric form in this solvent, whereas strong gelation in a phosphate buffer is due to the formation of a guanine-quartet structure, ( G-1)4, in which the four G-1 are linked by hydrogen-bonded guanine moieties and stabilized by an alkali metal cation. The B3LYP/6-31G** IR and VCD spectra of the nearly planar G-quartet, whose structure is slightly distorted from the C4h symmetry, in which the G-bases interact via four Hoogsteen-type hydrogen bonds and a sodium cation is positioned in the middle of the G-quartet, are in very good agreement with the experimental spectra, indicating that this structure is the predominant structure in the gel state. The geometric parameters are discussed. This study is the first to use IR and VCD spectroscopies coupled with DFT calculations to elucidate the structure of a supramolecular species in a gel state and shows the VCD spectroscopy as a powerful method for investigating the structure of complex supramolecular self-assemblies where the use of other structural methods is limited.

DyNAs: constitutional dynamic nucleic acid analogues.

Dynamic cationic polymers were generated in aqueous media from functionally complementary monomers bearing nucleobase groups. (1)H NMR spectroscopy was used to follow the polycondensation reaction of the nucleobase-appended dihydrazides 1 and 2 with the dialdehydes B and C. The reversibility of these polymers was established by proton NMR spectroscopy through exchange of the dihydrazide 2 with polymer 1 B. The polymers 1 B, 2 B, 1 C, and 2 C represent dynamic biopolymers of nucleic acid type, DyNAs. Electrostatic interaction of these polymers with polyanionic entities, such as polyphosphates, polynucleotides, and polyaspartic acid, was shown to take place. It induces a change in size of the dynamic polymer, as it responds by an increase in degree of polymerization to an increase of the overall anionic charge introduced, that is, to the total electrostatic interaction.

Gelation-driven component selection in the generation of constitutional dynamic hydrogels based on guanine-quartet formation.

The guanosine hydrazide 1 yields a stable supramolecular hydrogel based on the formation of a guanine quartet (G-quartet) in presence of metal cations. The effect of various parameters (concentration, nature of metal ion, and temperature) on the properties of this gel has been studied. Proton NMR spectroscopy is shown to allow a molecular characterization of the gelation process. Hydrazide 1 and its assemblies can be reversibly decorated by acylhydrazone formation with various aldehydes, resulting in formation of highly viscous dynamic hydrogels. When a mixture of aldehydes is used, the dynamic system selects the aldehyde that leads to the most stable gel. Mixing hydrazides 1, 9 and aldehydes 6, 8 in 1:1:1:1 ratio generated a constitutional dynamic library containing the four acylhydrazone derivatives A, B, C, and D. The library constitution displayed preferential formation of the acylhydrazone B that yields the strongest gel. Thus, gelation redirects the acylhydrazone distribution in the dynamic library as guanosine hydrazide 1 scavenges preferentially aldehyde 8, under the pressure of gelation because of the collective interactions in the assemblies of G-quartets B, despite the strong preference of the competing hydrazide 9 for 8. Gel formation and component selection are thermoreversible. The process amounts to gelation-driven self-organization with component selection and amplification in constitutional dynamic hydrogels based on G-quartet formation and reversible covalent connections. The observed self-organization and component selection occur by means of a multilevel self-assembly involving three dynamic processes, two of supramolecular and one of reversible covalent nature. They extend constitutional dynamic chemistry to phase-organization and phase-transition events.