High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

Confirmation of the structure of (±)-preisomide by total synthesis.

The structure of preisomide has been confirmed by total synthesis involving chemoselective oxazine formation and vinyl iodide carbonylation in six steps (longest linear sequence) and 23% overall yield.

The Total Synthesis of Raistrickindole A.

The total synthesis of raistrickindole A has been achieved, thereby confirming the proposed structure as an N-hydroxylated DKP. In the first but less selective approach, the DKP was built up by cyclization of a diastereoisomerically mixed N-hydroxylated dipeptide. In the second approach, the same DKP was constructed stereoselectively by the intramolecular Mitsunobu reaction of a hydroxamic acid. The synthesis was completed by a stereoselective oxidative cyclization.

Non-nucleoside Inhibitors of Zika Virus RNA-Dependent RNA Polymerase.

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects, such as microcephaly in newborns and neurological disease, like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here, we report the development of small nonnucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (N pocket) located next to a putative hinge region between the thumb and the palm subdomains that was originally described for dengue virus (DENV) RdRp. We first tested the activity of DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules, and assessed their potency using both enzymatic and cell-based assays. The most potent compound had a 50% inhibitory concentration value of 7.3 µM and inhibited ZIKV replication in a cell-based assay with a 50% effective concentration value of 24.3 µM. Importantly, we report four high-resolution crystal structures detailing how these NNIs insert into the N pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment, and hydration of the N pocket from ZIKV RdRp from those of the N pocket from DENV RdRp that are crucial for the design of improved antiviral inhibitors with activity against ZIKV.IMPORTANCE Zika virus belongs to the Flavivirus genus, which comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The nonstructural protein 5 (NS5) polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthesized inhibitors targeting Zika virus NS5. We show that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrated specific binding to an allosteric site within the polymerase, called the N pocket. This work paves the way for the future structure-based design of potent compounds specifically targeting ZIKV RNA polymerase activity.

Targeting the menaquinol binding loop of mycobacterial cytochrome bd oxidase.

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H2 inhibited both NADH2- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H2 also inhibited NADH2-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H2, reflecting that MQL-H2 targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies. Characterization of novel cytochrome bd oxidase Menaquinol binding domain inhibitor (MQL-H2) using virtual screening and ATP synthesis inhibition assays.

The endo-aza-Michael addition in the synthesis of piperidines and pyrrolidines.

Intramolecular endo-aza-Michael additions are categorised in various ways. Firstly whether they are single or double reactions, secondly whether they are endo- or exo-activated (or both), thirdly whether the Michael acceptor is an alkene or an alkyne, and finally whether the product is a six or a five membered ring. Reactions in the various categories are illustrated by syntheses of piperidines and pyrrolidines, including a range of natural products. The question of the stereochemical outcome and whether it is understood is discussed.

Discovery of a Novel Mycobacterial F-ATP Synthase Inhibitor and its Potency in Combination with Diarylquinolines.

The F1 FO -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors.

Stereocontrol in the synthesis of cyclic amino acids: a new ligand for directed hydrogenation through hydrogen bonding.

A system for the directed hydrogenation of nitrogen heterocycles is described in which hydrogen is delivered cis to a hydroxymethyl group by a rhodium catalyst with a simple phosphine ligand. The chemistry is applied to the synthesis of the hygric acid moiety of lincomycin and the pipecolic acid moiety of Argatroban. A series of control experiments indicate that the stereoselectivity is a result of a combination of both coordination and hydrogen bonding.

How an early or late transition state impacts the stereoselectivity of tetrahydropyran formation by intramolecular oxa-Michael addition.

The intramolecular oxa-Michael addition giving tetrahydropyrans has been examined experimentally using both acidic and basic catalysis. With acidic catalysis, the diequatorial product is exclusively obtained in a kinetically controlled reaction in all cases. Under basic conditions at low temperature, the reaction is again under kinetic control, but formation of the axial-equatorial isomer is generally favoured with an (E)-Michael acceptor, although isomerisation to the diequatorial isomer is observed at higher temperatures. Computationally, it is found that the acid catalysed reaction has a late transition state and the kinetic favouring of the diequatorial isomer has a steric explanation. In contrast, under strongly basic conditions, an early transition state is found. Electrostatic effects are likely to be the main contributor to the stereoselectivity for the (E)-isomer and steric interactions for the (Z)-isomer.

Synthesis of (-)-Cytisine Using a 6- endo aza-Michael Addition.

An asymmetric synthesis of (-)-cytisine has been achieved. The piperidine C-ring was formed using a stereodivergent intramolecular 6- endo aza-Michael addition. The B-ring was established by intramolecular pyridine N-alkylation. The absolute stereochemistry was established by an Evans acyl oxazolidinone enolate alkylation reaction that proceeded with an unexpected stereochemical outcome due to participation of the pyridine nitrogen lone pair.

Synthesis of Allahabadolactone A.

A synthesis of allahabadolactone A is described employing diastereoselective Diels-Alder and selenocyclization reactions, starting from (R)-citronellal and propylene oxide. The Diels-Alder substrate is built up in an efficient manner by rhodium-catalyzed alkyne hydroboration and palladium-catalyzed coupling reactions of E-1,2-dichloroethene. It is observed that the Diels-Alder reaction only displays high diastereoselectivity when the diene bears an additional alkene substituent but not an alkyne substituent.

Scandium as a pre-catalyst for the deoxygenative allylation of benzylic alcohols.

Scandium triflate is an effective pre-catalyst for the deoxygenative allylation of benzylic alcohols with a narrow substrate window. The reaction is shown to proceed through a "hidden Brønsted acid" mechanism. The reaction is efficient provided that the aryl group is neither too electron rich nor too electron poor. It is shown that this allows useful selectivity. The reaction also works for benzyhydryl alcohols with broader scope. The reaction may also be catalysed by Nafion.

A total synthesis of (+)-negamycin through isoxazolidine allylation.

The ß-amino acid antibiotic (+)-negamycin has been synthesised in ten steps from epichlorohydrin via Sakurai allylation of an isoxazolidine intermediate. The key allylation reaction proceeded with complete trans-selectivity, which is attributed to electrostatic attraction between the chlorine atom and the iminium ion in the Sakurai intermediate.

Hydroformylation in natural product synthesis.

The application of hydroformylation to the synthesis of natural products and natural product-like molecules is surveyed.

Penicitone: Structural Reassignment of a Proposed Natural Product Acid Chloride.

The proposed structure for the natural product penicitone, which contained a chemically improbable acid chloride functional group, was reassigned to a more probable structure using a combination of chemical knowledge, computer-assisted structure elucidation, and DFT methods.

Synthesis of the stenine ring system from pyrrole.

The skeleton of the stemona alkaloid, stenine, has been synthesized starting from pyrrole, employing an asymmetric organocatalyzed cyclization, Sonogashira coupling, a diastereoselective intramolecular propargylic Barbier reaction, cyclocarbonylation, and diastereoselective alkene reduction. Modulation of the electron-rich nature of the pyrrole nucleus by employing an α-trifluoroacetyl group is essential. The α-trifluoroacetyl group may be rapidly removed under carefully defined, mild conditions.

A synthesis of pseudoconhydrine and its epimer via hydroformylation and dihydroxylation.

A synthesis of the alkaloid pseudoconhydrine and its epimer has been achieved using tandem hydroformylation-condensation to form the six-membered ring and stereoselective dihydroxylation to introduce oxygenation. The stereoselectivity of dihydroxylation can be explained by lipophilic and electrostatic effects, supported by DFT calculations. The alkaloids can be obtained either by regioselective dehydroxylation or by rearrangement, followed by reduction.

A systematic assessment of mycobacterial F1 -ATPase subunit ε's role in latent ATPase hydrolysis.

In contrast to most bacteria, the mycobacterial F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F1 -ATPase component α3 :ß3 :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F1 -ATPase (MsF1 -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF1 αßγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF1 αßγεΔ2-5 , revealed similar ATP hydrolysis as MsF1 αßγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF1 αßγεΔ121 and MsF1 αßγεΔ103-121 with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3 ß3 -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.

Antituberculosis Activity of the Antimalaria Cytochrome bcc Oxidase Inhibitor SCR0911.

The ability to respire and generate adenosine triphosphate (ATP) is essential for the physiology, persistence, and pathogenicity of Mycobacterium tuberculosis, which causes tuberculosis. By employing a lead repurposing strategy, the malarial cytochrome bc1 inhibitor SCR0911 was tested against mycobacteria. Docking studies were carried out to reveal potential binding and to understand the binding interactions with the target, cytochrome bcc. Whole-cell-based and in vitro assays demonstrated the potency of SCR0911 by inhibiting cell growth and ATP synthesis in both the fast- and slow-growing M. smegmatis and M. bovis bacillus Calmette-Guérin, respectively. The variety of biochemical assays and the use of a cytochrome bcc deficient mutant strain validated the cytochrome bcc oxidase as the direct target of the drug. The data demonstrate the broad-spectrum activity of SCR0911 and open the door for structure-activity relationship studies to improve the potency of new mycobacteria specific SCR0911 analogues.

A formal synthesis of porantheridine and an epimer.

A formal synthesis of porantheridine and a synthesis of its C6-epimer have been completed, employing silver-catalyzed allene cyclization to form a common cis-isoxazolidine intermediate and related N-acyl iminium ion intermediates for side-chain introduction. The stereochemistry of this step can be controlled by choice of the N-protection method.