Efficient and Stereocontrolled Synthesis of 1,2,4-Trioxolanes Useful for Ferrous Iron-Dependent Drug Delivery

Ferrous iron-promoted reduction of a hindered peroxide bond underlies the antimalarial action of the 1,2,4-trioxane artemisinin and the 1,2,4-trioxolane arterolane. In appropriately designed systems, a 1,2,4-trioxolane ring can serve as a trigger to realize ferrous iron-dependent and parasite-selective drug delivery, both in vitro and in vivo. A stereocontrolled, expeditious (three steps), and efficient (67–71% overall yield) synthesis of 1,2,4-trioxolanes possessing the requisite 3″ substitution pattern that enables ferrous iron-dependent drug delivery is reported. The key synthetic step involves a diastereoselective Griesbaum co-ozonolysis reaction to afford primarily products with a <i>trans</i> relationship between the 3″ substituent and the peroxide bridge, as confirmed by X-ray structural analysis of a 3″-substituted 4-nitrobenzoate analogue

Enantioselective Synthesis and in Vivo Evaluation of Regioisomeric Analogues of the Antimalarial Arterolane

We describe the first systematic study of antimalarial 1,2,4-trioxolanes bearing a substitution pattern regioisomeric to that of arterolane. Conformational analysis suggested that <i>trans</i>-3″-substituted trioxolanes would exhibit Fe­(II) reactivity and antiparasitic activity similar to that achieved with canonical <i>cis</i>-4″ substitution. The chiral 3″ analogues were prepared as single stereoisomers and evaluated alongside their 4″ congeners against cultured malaria parasites and in a murine malaria model. As predicted, the <i>trans</i>-3″ analogues exhibited in vitro antiplasmodial activity remarkably similar to that of their <i>cis</i>-4″ comparators. In contrast, efficacy in the <i>Plasmodium berghei</i> mouse model differed dramatically for some of the congeneric pairs. The best of the novel 3″ analogues (e.g., <b>12i</b>) outperformed arterolane itself, producing cures in mice after a single oral exposure. Overall, this study suggests new avenues for modulating Fe­(II) reactivity and the pharmacokinetic and pharmacodynamic properties of 1,2,4-trioxolane antimalarials

Enantioselective Synthesis and in Vivo Evaluation of Regioisomeric Analogues of the Antimalarial Arterolane

We describe the first systematic study of antimalarial 1,2,4-trioxolanes bearing a substitution pattern regioisomeric to that of arterolane. Conformational analysis suggested that <i>trans</i>-3″-substituted trioxolanes would exhibit Fe­(II) reactivity and antiparasitic activity similar to that achieved with canonical <i>cis</i>-4″ substitution. The chiral 3″ analogues were prepared as single stereoisomers and evaluated alongside their 4″ congeners against cultured malaria parasites and in a murine malaria model. As predicted, the <i>trans</i>-3″ analogues exhibited in vitro antiplasmodial activity remarkably similar to that of their <i>cis</i>-4″ comparators. In contrast, efficacy in the <i>Plasmodium berghei</i> mouse model differed dramatically for some of the congeneric pairs. The best of the novel 3″ analogues (e.g., <b>12i</b>) outperformed arterolane itself, producing cures in mice after a single oral exposure. Overall, this study suggests new avenues for modulating Fe­(II) reactivity and the pharmacokinetic and pharmacodynamic properties of 1,2,4-trioxolane antimalarials

Simple Plate-Based, Parallel Synthesis of Disulfide Fragments using the CuAAC Click Reaction

Disulfide exchange screening is a site-directed approach to fragment-based lead discovery that requires a bespoke library of disulfide-containing fragments. Previously, we described a simple one-pot, two-step synthesis of disulfide fragments from amine- or acid-bearing starting materials. Here, we describe the synthesis of disulfide fragments that bear a 1,4-substituted-1,2,3-triazole linkage between disulfide and molecular diversity element. This work establishes the compatibility of copper­(I)-catalyzed azide–alkyne cycloaddition (CuAAC) chemistry with a one-pot, two-step reaction sequence that can be readily parallelized. We performed 96 reactions in a single deep-well microtiter plate, employing 48 alkynes and two different azide linker reagents. From this effort, a total of 81 triazole-containing disulfide fragments were obtained in useful isolated yields. Thus, CuAAC chemistry offers an experimentally convenient method to rapidly prepare disulfide fragments that are structurally distinct from fragments accessed via amide, sulfonamide, or isocyanate chemistries

Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds <b>2</b> and <b>3</b>) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound <b>2</b> has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) <i>J. Biol. Chem. 266</i>, 15591–15594] show DD2, compound <b>2</b>, and compound <b>3</b> inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions

A High-Throughput Functional Screen Identifies Small Molecule Regulators of Temperature- and Mechano-Sensitive K_2P Channels

K_2P (KCNK) potassium channels generate “leak” potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K_2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K_2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K_2P channel K_2P2.1 (<i>KCNK2</i>, TREK-1). By combining biophysical, structure–activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K_2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K_2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K_2P activity and facilitate the discovery and development of other K_2P small molecule modulators

Toward a Ferrous Iron-Cleavable Linker for Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) are antigen-targeted therapeutics that employ antibodies to deliver potent, cytotoxic effectors to cells with potentially high specificity. While promising clinical results have been achieved, significant pitfalls remain including internalization of ADCs in nontargeted cells expressing target antigen, which can limit therapeutic windows. Novel ADC linkers that are cleaved selectively in cancer cells but not in normal cells could minimize collateral damage caused by ADC uptake in nontargeted tissues. Here, we describe a prototypical ADC linker based on an Fe­(II)-reactive 1,2,4-trioxolane scaffold (TRX) that by itself has demonstrated tumor-selective activity in preclinical cancer models. We prepared TRX-linked ADCs by site-selective conjugation to two sites in trastuzumab and compared their activity in Her2 positive and negative cells to ADC controls based on established linker chemistry. Our results confirm that the TRX moiety efficiently releases its payload following ADC uptake, affording picomolar potencies in antigen-positive cells. We also identified a destabilizing interaction between these initial TRX linkers and nearby antibody residues and suggest an approach to improve upon these prototypical designs

Allosteric Inhibitors, Crystallography, and Comparative Analysis Reveal Network of Coordinated Movement across Human Herpesvirus Proteases

Targeting of cryptic binding sites represents an attractive but underexplored approach to modulating protein function with small molecules. Using the dimeric protease (Pr) from Kaposi’s sarcoma-associated herpes­virus (KSHV) as a model system, we sought to dissect a putative allosteric network linking a cryptic site at the dimerization interface to enzyme function. Five cryogenic X-ray structures were solved of the monomeric protease with allosteric inhibitors bound to the dimer interface site. Distinct coordinated movements captured by the allosteric inhibitors were also revealed as alternative states in room-temperature X-ray data and comparative analyses of other dimeric herpes­virus proteases. A two-step mechanism was elucidated through detailed kinetic analyses and suggests an enzyme isomerization model of inhibition. Finally, a representative allosteric inhibitor from this class was shown to be efficacious in a cellular model of viral infectivity. These studies reveal a coordinated dynamic network of atomic communication linking cryptic binding site occupancy and allosteric inactivation of KHSV Pr that can be exploited to target other members of this clinically relevant family of enzymes

Trioxolane-Mediated Delivery of Mefloquine Limits Brain Exposure in a Mouse Model of Malaria

Peroxidic antimalarial agents including the sequiterpene artemisinins and the synthetic 1,2,4-trioxolanes function via initial intraparasitic reduction of an endoperoxide bond. By chemically coupling this reduction to release of a tethered drug species it is possible to confer two distinct pharmacological effects in a parasite-selective fashion, both in vitro and in vivo. Here we demonstrate the trioxolane-mediated delivery of the antimalarial agent mefloquine in a mouse malaria model. Selective partitioning of the trioxolane–mefloquine conjugate in parasitized erythrocytes, combined with effective exclusion of the conjugate from brain significantly reduced brain exposure as compared to mice directly administered mefloquine. These studies suggest the potential of trioxolane-mediated drug delivery to mitigate off-target effects of existing drugs, including the adverse neuropsychiatric effects of mefloquine use in therapeutic and chemoprophylactic settings

Selected anti-plasmodium lead compounds with associated enzyme and cell-based data.

<p>Selected anti-plasmodium lead compounds with associated enzyme and cell-based data.</p