Microtubule-Stabilizing 1,2,4-Triazolo[1,5-a]pyrimidines as Candidate Therapeutics for Neurodegenerative Disease: Matched Molecular Pair Analyses and Computational Studies Reveal New Structure-Activity Insights.

Microtubule (MT)-stabilizing 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) hold promise as candidate therapeutics for Alzheimer's disease (AD) and other neurodegenerative conditions. However, depending on the choice of substituents around the TPD core, these compounds can elicit markedly different cellular phenotypes that likely arise from the interaction of TPD congeners with either one or two spatially distinct binding sites within tubulin heterodimers (i.e., the seventh site and the vinca site). In the present study, we report the design, synthesis, and evaluation of a series of new TPD congeners, as well as matched molecular pair analyses and computational studies, that further elucidate the structure-activity relationships of MT-active TPDs. These studies led to the identification of novel MT-normalizing TPD candidates that exhibit favorable ADME-PK, including brain penetration and oral bioavailability, as well as brain pharmacodynamic activity.

Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5-a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies.

Studies in tau and Aß plaque transgenic mouse models demonstrated that brain-penetrant microtubule (MT)-stabilizing compounds, including the 1,2,4-triazolo[1,5-a]pyrimidines, hold promise as candidate treatments for Alzheimer's disease and related neurodegenerative tauopathies. Triazolopyrimidines have already been investigated as anticancer agents; however, the antimitotic activity of these compounds does not always correlate with stabilization of MTs in cells. Indeed, previous studies from our laboratories identified a critical role for the fragment linked at C6 in determining whether triazolopyrimidines promote MT stabilization or, conversely, disrupt MT integrity in cells. To further elucidate the structure-activity relationship (SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68 triazolopyrimidine congeners bearing structural modifications at C6 and/or C7 was designed, synthesized, and evaluated. These studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identification of novel analogues that, relative to the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity, and pharmacokinetics.

Congeners Derived from Microtubule-Active Phenylpyrimidines Produce a Potent and Long-Lasting Paralysis of Schistosoma mansoni In Vitro.

Schistosomiasis is a parasitic disease that affects approximately 200 million people in developing countries. Current treatment relies on just one partially effective drug, and new drugs are needed. Tubulin and microtubules (MTs) are essential constituents of the cytoskeleton in all eukaryotic cells and considered potential drug targets to treat parasitic infections. The α- and ß-tubulin of Schistosoma mansoni have ∼96% and ∼91% sequence identity to their respective human tubulins, suggesting that compounds which bind mammalian tubulin may interfere with MT-mediated functions in the parasite. To explore the potential of different classes of tubulin-binding molecules as antischistosomal leads, we completed a series of in vitro whole-organism screens of a target-based compound library against S. mansoni adults and somules (postinfective larvae), and identified multiple biologically active compounds, among which phenylpyrimidines were the most promising. Further structure-activity relationship studies of these hits identified a series of thiophen-2-yl-pyrimidine congeners, which induce a potent and long-lasting paralysis of the parasite. Moreover, compared to the originating compounds, which showed cytotoxicity values in the low nanomolar range, these new derivatives were 1-4 orders of magnitude less cytotoxic and exhibited weak or undetectable activity against mammalian MTs in a cell-based assay of MT stabilization. Given their selective antischistosomal activity and relatively simple drug-like structures, these molecules hold promise as candidates for the development of new treatments for schistosomiasis.

A brain-penetrant triazolopyrimidine enhances microtubule-stability, reduces axonal dysfunction and decreases tau pathology in a mouse tauopathy model.

BACKGROUND: Alzheimer's disease (AD) and related tauopathies are neurodegenerative diseases that are characterized by the presence of insoluble inclusions of the protein tau within brain neurons and often glia. Tau is normally found associated with axonal microtubules (MTs) in the brain, and in tauopathies this MT binding is diminished due to tau hyperphosphorylation. As MTs play a critical role in the movement of cellular constituents within neurons via axonal transport, it is likely that the dissociation of tau from MTs alters MT structure and axonal transport, and there is evidence of this in tauopathy mouse models as well as in AD brain. We previously demonstrated that different natural products which stabilize MTs by interacting with ß-tubulin at the taxane binding site provide significant benefit in transgenic mouse models of tauopathy. More recently, we have reported on a series of MT-stabilizing triazolopyrimidines (TPDs), which interact with ß-tubulin at the vinblastine binding site, that exhibit favorable properties including brain penetration and oral bioavailability. Here, we have examined a prototype TPD example, CNDR-51657, in a secondary prevention study utilizing aged tau transgenic mice. METHODS: 9-Month old female PS19 mice with a low amount of existing tau pathology received twice-weekly administration of vehicle, or 3 or 10 mg/kg of CNDR-51657, for 3 months. Mice were examined in the Barnes maze at the end of the dosing period, and brain tissue and optic nerves were examined immunohistochemically or biochemically for changes in MT density, axonal dystrophy, and tau pathology. Mice were also assessed for changes in organ weights and blood cell numbers. RESULTS: CNDR-51657 caused a significant amelioration of the MT deficit and axonal dystrophy observed in vehicle-treated aged PS19 mice. Moreover, PS19 mice receiving CNDR-51657 had significantly lower tau pathology, with a trend toward improved Barnes maze performance. Importantly, no adverse effects were observed in the compound-treated mice, including no change in white blood cell counts as is often observed in cancer patients receiving high doses of MT-stabilizing drugs. CONCLUSIONS: A brain-penetrant MT-stabilizing TPD can safely correct MT and axonal deficits in an established mouse model of tauopathy, resulting in reduced tau pathology.

Design, synthesis and evaluation of photoactivatable derivatives of microtubule (MT)-active [1,2,4]triazolo[1,5-a]pyrimidines.

The [1,2,4]triazolo[1,5-a]pyrimidines comprise a promising class of non-naturally occurring microtubule (MT)-active compounds. Prior studies revealed that different triazolopyrimidine substitutions can yield molecules that either promote MT stabilization or disrupt MT integrity. These differences can have important ramifications in the therapeutic applications of triazolopyrimidines and suggest that different analogues may exhibit different binding modes within the same site or possibly interact with tubulin/MTs at alternative binding sites. To help discern these possibilities, a series of photoactivatable triazolopyrimidine congeners was designed, synthesized and evaluated in cellular assays with the goal of identifying candidate probes for photoaffinity labeling experiments. These studies led to the identification of different derivatives that incorporate a diazirine ring in the amine substituent at position 7 of the triazolopyrimidine heterocycle, resulting in molecules that either promote stabilization of MTs or disrupt MT integrity. These photoactivatable candidate probes hold promise to investigate the mode of action of MT-active triazolopyrimidines.

Multitargeted Imidazoles: Potential Therapeutic Leads for Alzheimer's and Other Neurodegenerative Diseases.

Alzheimer's disease (AD) is a complex, multifactorial disease in which different neuropathological mechanisms are likely involved, including those associated with pathological tau and Aß species as well as neuroinflammation. In this context, the development of single multitargeted therapeutics directed against two or more disease mechanisms could be advantageous. Starting from a series of 1,5-diarylimidazoles with microtubule (MT)-stabilizing activity and structural similarities with known NSAIDs, we conducted structure-activity relationship studies that led to the identification of multitargeted prototypes with activities as MT-stabilizing agents and/or inhibitors of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways. Several examples are brain-penetrant and exhibit balanced multitargeted in vitro activity in the low µM range. As brain-penetrant MT-stabilizing agents have proven effective against tau-mediated neurodegeneration in animal models, and because COX- and 5-LOX-derived eicosanoids are thought to contribute to Aß plaque deposition, these 1,5-diarylimidazoles provide tools to explore novel multitargeted strategies for AD and other neurodegenerative diseases.

Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer's Disease and Related Tauopathies.

The microtubule (MT)-stabilizing protein tau disengages from MTs and forms intracellular inclusions known as neurofibrillary tangles in Alzheimer's disease and related tauopathies. Reduced tau binding to MTs in tauopathies may contribute to neuronal dysfunction through decreased MT stabilization and disrupted axonal transport. Thus, the introduction of brain-penetrant MT-stabilizing compounds might normalize MT dynamics and axonal deficits in these disorders. We previously described a number of phenylpyrimidines and triazolopyrimidines (TPDs) that induce tubulin post-translational modifications indicative of MT stabilization. We now further characterize the biologic properties of these small molecules, and our results reveal that these compounds can be divided into two general classes based on the cellular response they evoke. One group composed of the phenylpyrimidines and several TPD examples showed a bell-shaped concentration-response effect on markers of MT stabilization in cellular assays. Moreover, these compounds induced proteasome-dependent degradation of α- and ß-tubulin and caused altered MT morphology in both dividing cells and neuron cultures. In contrast, a second group comprising a subset of TPD molecules (TPD+) increased markers of stable MTs in a concentration-dependent manner in dividing cells and in neurons without affecting total tubulin levels or disrupting MT architecture. Moreover, an example TPD+ compound was shown to increase MTs in a neuron culture model with induced tau hyperphosphorylation and associated MT deficits. Several TPD+ compounds were shown to be both brain penetrant and orally bioavailable, and a TPD+ example increased MT stabilization in the mouse brain, making these compounds potential candidate therapeutics for neurodegenerative tauopathies such as Alzheimer's disease.

Pharmacokinetic, pharmacodynamic and metabolic characterization of a brain retentive microtubule (MT)-stabilizing triazolopyrimidine.

Previous studies revealed that examples of the non-naturally occurring microtubule (MT)-stabilizing triazolopyrimidines are both brain penetrant and orally bioavailable, indicating that this class of compounds may be potentially attractive in the development of MT-stabilizing therapies for the central nervous system (CNS). We now report on the pharmacokinetics (PK), pharmacodynamics (PD), and metabolism of a selected triazolopyrimidine congener, (S)-3-(4-(5-chloro-7-((1,1,1-trifluoropropan-2-yl)amino)-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl)-3,5-difluorophenoxy)-propan-1-ol (4). These studies revealed that 4 exhibits longer brain than plasma half-life that may be exploited to achieve a selective accumulation of the compound within the CNS. Furthermore, compound metabolism studies suggest that in plasma 4 is rapidly oxidized at the terminal hydroxyl group to form a comparatively inactive carboxylic acid metabolite. Peripheral administration of relatively low doses of 4 to normal mice was found to produce a significant elevation in acetylated α-tubulin, a marker of stable MTs, in the brain. Collectively, these results indicate that 4 may effectively target brain MTs at doses that produce minimal peripheral exposure.

Potent, long-acting cyclopentane-1,3-Dione thromboxane (A2)-receptor antagonists.

A series of derivatives of the known thromboxane A2 prostanoid (TP) receptor antagonists, 3-(6-((4-chlorophenyl)sulfonamido)-5,6,7,8-tetrahydronaphthalen-1-yl)propanoic acid and 3-(3-(2-((4-chlorophenyl)sulfonamido)ethyl)phenyl) propanoic acid, were synthesized in which the carboxylic acid functional group was replaced with substituted cyclopentane-1,3-dione (CPD) bioisosteres. Characterization of these molecules led to the discovery of remarkably potent new analogues, some of which were considerably more active than the corresponding parent carboxylic acid compounds. Depending on the choice of the C2 substituent of the CPD unit, these new derivatives can produce either a reversible or an apparent irreversible inhibition of the human TP receptor. Given the potency and the long-lasting inhibition of TP receptor signaling, these novel antagonists may comprise promising leads for the development of antithromboxane therapies.

Brain-penetrant, orally bioavailable microtubule-stabilizing small molecules are potential candidate therapeutics for Alzheimer's disease and related tauopathies.

Microtubule (MT) stabilizing drugs hold promise as potential treatments for Alzheimer's disease (AD) and related tauopathies. However, thus far epothilone D has been the only brain-penetrant MT-stabilizer to be evaluated in tau transgenic mice and in AD patients. Furthermore, this natural product exhibits potential deficiencies as a drug candidate, including an intravenous route of administration and the inhibition of the P-glycoprotein (Pgp) transporter. Thus, the identification of alternative CNS-active MT-stabilizing agents that lack these potential limitations is of interest. Toward this objective, we have evaluated representative compounds from known classes of non-naturally occurring MT-stabilizing small molecules. This led to the identification of selected triazolopyrimidines and phenylpyrimidines that are orally bioavailable and brain-penetrant without disruption of Pgp function. Pharmacodynamic studies confirmed that representative compounds from these series enhance MT-stabilization in the brains of wild-type mice. Thus, these classes of MT-stabilizers hold promise for the development of orally active, CNS-directed MT-stabilizing therapies.