Design, synthesis, and evaluation of an anti‐trypanosomal [1,2,4]triazolo[1,5‐ a ]pyrimidine probe for photoaffinity labeling studies

Studies have shown that depending on the substitution pattern, microtubule (MT)‐targeting 1,2,4‐triazolo[1,5‐a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti‐trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin

Structure‐activity relationships, tolerability and efficacy of microtubule‐active 1,2,4‐Triazolo[1,5‐ a ]pyrimidines as potential candidates to treat human African trypanosomiasis

Tubulin and microtubules (MTs) are potential protein targets to treat parasitic infections and our previous studies have shown that the triazolopyrimidine (TPD) class of MT‐active compounds hold promise as antitrypanosomal agents. MT‐targeting TPDs include structurally related but functionally diverse congeners that interact with mammalian tubulin at either one or two distinct interfacial binding sites; namely, the seventh and vinca sites, which are found within or between α,β‐tubulin heterodimers, respectively. Evaluation of the activity of 123 TPD congeners against cultured Trypanosoma brucei enabled a robust quantitative structure‐activity relationship (QSAR) model and the prioritization of two congeners for in vivo pharmacokinetics (PK), tolerability and efficacy studies. Treatment of T. brucei‐infected mice with tolerable doses of TPDs significantly decreased blood parasitemia within 24 h. Further, two once‐weekly doses at 10 mg/kg of a candidate TPD significantly extended the survival of infected mice relative to infected animals treated with vehicle. Further optimization of dosing and/or the dosing schedule of these CNS‐active TPDs may provide alternative treatments for human African trypanosomiasis

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

Development and Target Elucidation of Small Molecules as Candidate Therapeutics

This dissertation is divided into four chapters. Chapter one provides a brief introduction to medicinal chemistry concepts that are relevant to the rest of the dissertation. Chapters two, three, and four are three distinct projects. Chapter two is the target investigation of a class of non-naturally occurring small molecules, the triazolopyrimides (TPDs), against the parasite Trypanosoma brucei via photoaffinity labeling (PAL). TPD-based PAL probes were designed and synthesized, followed by optimization of photoaffinity labeling experiments in mammalian HEK293 cells. Optimized PAL conditions were adapted to whole T. brucei parasites, and these experiments confirmed that TPDs target T. brucei by binding to trypanosome microtubules. Chapter two is the development and target investigation of another class of non-naturally occurring small molecules, the thiophenyl pyrimidines (TPPs) as paralytic agents of the helminth parasite, Schistosoma mansoni. Using an elucidated structure-activity relationship, congeners of the TPP with improved S. mansoni paralysis potency and improved physicochemical properties were developed. Furthermore, using a similar approach used in chapter two, a TPP-based PAL probe was designed and synthesized. PAL experiments in S. mansoni highly suggest that TPPs do not target schistosome microtubules. Chapter three is the design and synthesis of deuterated cystamine derivatives as drug candidates for fatty liver disease. Cystamine is a marketed drug for treatment of cystinosis, and has been studied as a drug candidate for fatty liver disease. Two deuterated cystamine derivatives were designed. While in vitro studies reveal no significant difference between cystamine and its deuterated derivatives, in vivo studies show a deuterium-dependent effect in a fatty liver disease mouse model

Structure Activity Relationships of Microtubule (MT)-Targeting 1,2,4-Triazolo[1,5-a]pyrimidines as Candidates for Neurodegenerative Tauopathies

A subset of neurodegenerative diseases known as tauopathies, of which Alzheimer’s disease (AD) is the most prominent example, are defined by the presence of proteinaceous inclusions comprised of hyperphosphorylated tau protein within neurons. Tau has been shown to provide stability to microtubules (MTs), key constituents of the cytoskeleton that are involved in a variety of cellular functions, including cell communication and transport. Tau hyperphosphorylation and mutation causes it to detach from MTs and causes them to destabilize leading to axonal transport deficits and, ultimately, neuronal loss. A possible strategy of therapeutic intervention calls for the development of brain penetrant MT-stabilizing agents that could compensate for the loss of tau function in neurons. Through a screen of non-naturally occurring small molecules, a few selected classes of molecules were found to have advantages over MT stabilizing natural products, in terms of synthetic accessibility and drug like physicochemical properties. Preliminary evaluation and SAR of those selected classes led to the identification of a preferred subset of triazolopyrimidines which were shown to be brain penetrant, orally bioavailable, and possess other favorable pharmacokinetic properties. The initial cellular assessment of triazolopyrimidines (TPDs) found that depending on the substitution pattern TPDs can either preserve or disrupt MT integrity in cells. Presented here are the design, synthesis and in vitro evaluation of a set of triazolopyrimidine congeners bearing a range of structural modifications at position 6 and/or 7. These studies complement and expand prior SAR studies and characterize the role that substituents of the triazolopyrimidine scaffold can have in determining MT-stabilizing activity

Structure Activity Relationships of Microtubule (MT)-Targeting 1,2,4-Triazolo[1,5-a]pyrimidines as Candidates for Neurodegenerative Tauopathies

A subset of neurodegenerative diseases known as tauopathies, of which Alzheimer’s disease (AD) is the most prominent example, are defined by the presence of proteinaceous inclusions comprised of hyperphosphorylated tau protein within neurons. Tau has been shown to provide stability to microtubules (MTs), key constituents of the cytoskeleton that are involved in a variety of cellular functions, including cell communication and transport. Tau hyperphosphorylation and mutation causes it to detach from MTs and causes them to destabilize leading to axonal transport deficits and, ultimately, neuronal loss. A possible strategy of therapeutic intervention calls for the development of brain penetrant MT-stabilizing agents that could compensate for the loss of tau function in neurons. Through a screen of non-naturally occurring small molecules, a few selected classes of molecules were found to have advantages over MT stabilizing natural products, in terms of synthetic accessibility and drug like physicochemical properties. Preliminary evaluation and SAR of those selected classes led to the identification of a preferred subset of triazolopyrimidines which were shown to be brain penetrant, orally bioavailable, and possess other favorable pharmacokinetic properties. The initial cellular assessment of triazolopyrimidines (TPDs) found that depending on the substitution pattern TPDs can either preserve or disrupt MT integrity in cells. Presented here are the design, synthesis and in vitro evaluation of a set of triazolopyrimidine congeners bearing a range of structural modifications at position 6 and/or 7. These studies complement and expand prior SAR studies and characterize the role that substituents of the triazolopyrimidine scaffold can have in determining MT-stabilizing activity

1,2,4-Triazolo[1,5-a]pyrimidines in drug design

The 1,2,4-triazolo[1,5-a]pyrimidine (TP) heterocycle, in spite of its relatively simple structure, has proved to be remarkably versatile as evidenced by its use in many different applications reported over the years in different areas of drug design. For example, as the ring system of TPs is isoelectronic with that of purines, this heterocycle has been proposed as a possible surrogate of the purine ring. However, depending on the choice of substituents, the TP ring has also been described as a potentially viable bio-isostere of the carboxylic acid functional group and of the N-acetyl fragment of ε-N-acetylated lysine. In addition, the metal-chelating properties of the TP ring have also been exploited to generate candidate treatments for cancer and parasitic diseases. In the present review article, we discuss recent applications of the TP scaffold in medicinal chemistry, and provide an overview of its properties and methods of synthesis

Protein–protein interactions: developing small-molecule inhibitors/stabilizers through covalent strategies

The development of small-molecule inhibitors or stabilizers of selected protein-protein interactions (PPIs) of interest holds considerable promise for the development of research tools as well as candidate therapeutics. In this context, the covalent modification of selected residues within the target protein has emerged as a promising mechanism of action to obtain small-molecule modulators of PPIs with appropriate selectivity and duration of action. Different covalent labeling strategies are now available that can potentially allow for a rational, ground-up discovery and optimization of ligands as PPI inhibitors or stabilizers. This review article provides a synopsis of recent developments and applications of such tactics, with a particular focus on site-directed fragment tethering and proximity-enabled approaches

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 Aβ 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