Trent D. Ashton

Pharmacological Validation of Trypanosoma brucei Phosphodiesterases B1 and B2 as Druggable Targets for African Sleeping Sickness

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogues, show modest inhibition of TbrPDEB1 and B2 and quickly kill the bloodstream form of the subspecies T. brucei brucei . We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.

Synthesis and characterization of novel 2-amino-3-benzoylthiophene derivatives as biased allosteric agonists and modulators of the adenosine A(1) receptor

A series of novel 2-amino-3-benzoylthiophenes (2A3BTs) were screened using a functional assay of A(1)R mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact CHO cells to identify potential agonistic effects as well as the ability to allosterically modulate the activity of the orthosteric agonist, R-PIA. Two derivatives, 8h and 8i, differing only in terms of the absence or presence of an electron-withdrawing group on the benzoyl moiety of the 2A3BT scaffold, were identified as biased allosteric agonists and positive allosteric modulators of agonist function at the adenosine A(1) receptor (A(1)R) in two different functional assays. Our findings indicate that subtle structural variations can promote functionally distinct receptor conformational states.

Improved synthesis and structural reassignment of MC1568: a class IIa selective HDAC inhibitor.

An improved synthesis and structural reassignment of the class IIa selective histone deacetylase (HDAC) inhibitor MC1568 are described.

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 1. Sildenafil analogs

Parasitic diseases, such as African sleeping sickness, have a significant impact on the health and well-being in the poorest regions of the world. Pragmatic drug discovery efforts are needed to find new therapeutic agents. In this Letter we describe target repurposing efforts focused on trypanosomal phosphodiesterases. We outline the synthesis and biological evaluation of analogs of sildenafil (1), a human PDE5 inhibitor, for activities against trypanosomal PDEB1 (TbrPDEB1). We find that, while low potency analogs can be prepared, this chemical class is a sub-optimal starting point for further development of TbrPDE inhibitors.

In silico identification of an aryl hydrocarbon receptor antagonist with biological activity in vitro and in vivo.

The aryl hydrocarbon receptor (AHR) is critically involved in several physiologic processes, including cancer progression and multiple immune system activities. We, and others, have hypothesized that AHR modulators represent an important new class of targeted therapeutics. Here, ligand shape–based virtual modeling techniques were used to identify novel AHR ligands on the basis of previously identified chemotypes. Four structurally unique compounds were identified. One lead compound, 2-((2-(5-bromofuran-2-yl)-4-oxo-4H-chromen-3-yl)oxy)acetamide (CB7993113), was further tested for its ability to block three AHR-dependent biologic activities: triple-negative breast cancer cell invasion or migration in vitro and AHR ligand–induced bone marrow toxicity in vivo. CB7993113 directly bound both murine and human AHR and inhibited polycyclic aromatic hydrocarbon (PAH)– and TCDD-induced reporter activity by 75% and 90% respectively. A novel homology model, comprehensive agonist and inhibitor titration experiments, and AHR localization studies were consistent with competitive antagonism and blockade of nuclear translocation as the primary mechanism of action. CB7993113 (IC50 3.3 × 10−7 M) effectively reduced invasion of human breast cancer cells in three-dimensional cultures and blocked tumor cell migration in two-dimensional cultures without significantly affecting cell viability or proliferation. Finally, CB7993113 effectively inhibited the bone marrow ablative effects of 7,12-dimethylbenz[a]anthracene in vivo, demonstrating drug absorption and tissue distribution leading to pharmacological efficacy. These experiments suggest that AHR antagonists such as CB7993113 may represent a new class of targeted therapeutics for immunomodulation and/or cancer therapy.

Identification and Characterization of Kava‐derived Compounds Mediating TNF‐α Suppression

There is a substantial unmet need for new classes of drugs that block TNF‐α‐mediated inflammation, and particularly for small molecule agents that can be taken orally. We have screened a library of natural products against an assay measuring TNF‐α secretion in lipopolysaccharide‐stimulated THP‐1 cells, seeking compounds capable of interfering with the TNF‐α‐inducing transcription factor lipopolysaccharide‐induced TNF‐α factor. Among the active compounds were several produced by the kava plant (Piper mysticum), extracts of which have previously been linked to a range of therapeutic effects. When tested in vivo, a representative of these compounds, kavain, was found to render mice immune to lethal doses of lipopolysaccharide. Kavain displays promising pharmaceutical properties, including good solubility and high cell permeability, but pharmacokinetic experiments in mice showed relatively rapid clearance. A small set of kavain analogs was synthesized, resulting in compounds of similar or greater potency in vitro compared with kavain. Interestingly, a ring‐opened analog of kavain inhibited TNF‐α secretion in the cell‐based assay and suppressed lipopolysaccharide‐induced TNF‐α factor expression in the same cells, whereas the other compounds inhibited TNF‐α secretion without affecting lipopolysaccharide‐induced TNF‐α factor levels, indicating a potential divergence in mechanism of action.

Microwave-Assisted Direct Amination: Rapid Access to Multi-Functionalized N6-Substituted Adenosines

Analogues of adenosine have a range of interesting biological activities and potential therapeutic applications. A method for the efficient preparation of highly functionalized N6-substituted adenosines has been developed from the corresponding tert-butyldimethylsilyl-protected inosine. The key step in this procedure is a microwave-assisted amination reaction between an appropriately substituted inosine and an amine in the presence of PyBroP. High yields of desired N6-substituted adenosines were achieved with hindered amines and the reaction was also found to accommodate a range of substituents on the inosine precursor.

Photoredox Catalysis of Intramolecular Cyclizations with a Reusable Silica‐Bound Ruthenium Complex

Photoredox catalysis with the use of a stable, reusable silica‐bound chromophore was applied to the intramolecular cyclization of a series of 2‐benzylidenehydrazinecarbothioamides to give 5‐phenyl‐1,3,4‐thiadiazol‐2‐amines. The catalyst was readily prepared by carbodiimide‐mediated coupling of commercially available amine‐functionalized silica beads to a carboxylic acid functionalized ruthenium complex. The immobilized catalyst was readily removed from the reaction product by filtration and was used eight times without loss of catalytic activity. This simple, safe, and practical method is an attractive alternative to conventional procedures.

NMR studies of dextromethorphan in both isotropic and anisotropic states

Herein, we present the solution-state NMR studies on dextromethorphan (1) under both isotropic and anisotropic conditions. From the measurement of 22 residual dipolar couplings using a stretched polystyrene gel (PS), we show that accurate and detailed structural information is readily determined including the relative stereochemical assignments of chiral centers, validation of diastereomer configuration, and the stereospecific assignment of the seven pairs of prochiral protons. This utility of PS gels is thus showcased to obtain rapid, accurate conformational, and relative configuration information in this important class of compounds without recourse to X-ray analysis.

Synthesis of norbornane bisether antibiotics via silver-mediated alkylation

A small series of norbornane bisether diguanidines have been synthesized and evaluated as antibacterial agents. The key transformation-bisalkylation of norbornane diol 6-was not successful using Williamson methodology but has been accomplished using Ag2O mediated alkylation. Further functionalization to incorporate two guanidinium groups gave rise to a series of structurally rigid cationic amphiphiles; several of which (16d, 16g and 16h) exhibited antibiotic activity. For example, compound 16d was active against a broad range of bacteria including Pseudomonas aeruginosa (MIC = 8 µg/mL), Escherichia coli (MIC = 8 µg/mL) and methicillin-resistant Staphylococcus aureus (MIC = 8 µg/mL).