Studies toward the Unique Pederin Family Member Psymberin: Full Structure Elucidation, Two Alternative Total Syntheses, and Analogs

Two synthetic approaches to psymberin have been accomplished. A highly convergent first generation synthesis led to the complete stereochemical assignment and demonstrated that psymberin and irciniastatin A are identical compounds. This synthesis featured a diastereoselective aldol coupling between the aryl fragment and a central tetrahydropyran core and a novel one-pot procedure to convert an amide, via intermediacy of a sensitive methyl imidate, to the <i>N</i>-acyl aminal reminiscent of psymberin. The highlights of the second generation synthesis include an efficient iridium-catalyzed enantioselective bisallylation of neopentyl glycol and a stepwise Sonogashira coupling/cycloisomerization/reduction sequence to construct the dihydroisocoumarin unit. The two synthetic avenues were achieved in 17–18 steps (longest linear sequence, ∼14–15 isolations) from 3 fragments prepared in 7–8 (first generation) and 3–8 (second generation) steps each. This convergent approach allowed for the preparation of sufficient amounts of psymberin (∼ 0.5 g) for follow-up biological studies. Meanwhile, our highly flexible strategy enabled the design and synthesis of multiple analogs, including a psymberin–pederin hybrid, termed psympederin, that proved crucial to a comprehensive understanding of the chemical biology of psymberin and related compounds that will be described in a subsequent manuscript

Rifamycin Biosynthetic Congeners: Isolation and Total Synthesis of Rifsaliniketal and Total Synthesis of Salinisporamycin and Saliniketals A and B

We describe the isolation, structure elucidation, and total synthesis of the novel marine natural product rifsaliniketal and the total synthesis of the structurally related variants salinisporamycin and saliniketals A and B. Rifsaliniketal was previously proposed, but not observed, as a diverted metabolite from a biosynthetic precursor to rifamycin S. Decarboxylation of rifamycin provides salinisporamycin, which upon truncation with loss of the naphthoquinone ring leads to saliniketals. Our synthetic strategy hinged upon a Pt­(II)-catalyzed cycloisomerization of an alkynediol to set the dioxabicyclo[3.2.1]­octane ring system and a fragmentation of an intermediate dihydropyranone to forge a stereochemically defined (<i>E</i>,<i>Z</i>)-dienamide unit. Multiple routes were explored to assemble fragments with high stereocontrol, an exercise that provided additional insights into acyclic stereocontrol during stereochemically complex fragment-assembly processes. The resulting 11–14 step synthesis of saliniketals then enabled us to explore strategies for the synthesis and coupling of highly substituted naphthoquinones or the corresponding naphthalene fragments. Whereas direct coupling with naphthoquinone fragments proved unsuccessful, both amidation and C–N bond formation tactics with the more electron-rich naphthalene congeners provided an efficient means to complete the first total synthesis of rifsaliniketal and salinisporamycin

Studies toward the Unique Pederin Family Member Psymberin: Structure–Activity Relationships, Biochemical Studies, and Genetics Identify the Mode-of-Action of Psymberin

Psymberin is the only member of the pederin natural product family that contains a dihydroisocoumarin side chain. Structural modifications of psymberin uncoupled inhibition of protein translation from cytotoxicity, suggesting that psymberin has more than one bioactivity. A forward genetic screen in Caenorhabditis elegans was conducted to identify the molecular target(s) of psymberin. Multiple independent psymberin-resistant mutants were isolated, each containing the same point mutation in a gene encoding a ribosomal protein. However, a psymberin-resistant mutant strain bearing this mutation was not cross-resistant to the pederin family member mycalamide A, which binds to the archaeal form of the same protein. Thus, two pederin family members likely differ in how they bind the same molecular target. The accumulation of psymberin in cells was sensitive to the stereochemistry of the amide side chain at C4 or C8 and the presence of the dihydroisocoumarin side chain. The observation that psymberin diastereomers or dihydroisocoumarin-truncated analogs lose all cytotoxic activity while retaining the ability to inhibit protein translation in a cell-free in vitro assay can be explained in the context of these differential cell uptake issues. Finally, we also demonstrate that the blistering activity associated with pederin and other members of the family is not due to their protein synthesis inhibiting activity. Unlike pederin and mycalamide, psymberin does not display irritant or blistering activity

SAR-Based Optimization of a 4‑Quinoline Carboxylic Acid Analogue with Potent Antiviral Activity

It is established that drugs targeting viral proteins are at risk of generating resistant strains. However, drugs targeting host factors can potentially avoid this problem. Herein, we report structure–activity relationship studies leading to the discovery of a very potent lead compound 6-fluoro-2-(5-isopropyl-2-methyl-4-phenoxyphenyl)­quinoline-4-carboxylic acid (<b>C44</b>) that inhibits human dihydroorotate dehydrogenase (DHODH) with an IC₅₀ of 1 nM and viral replication of VSV and WSN-Influenza with an EC₅₀ of 2 nM and 41 nM. We also solved the X-ray structure of human DHODH bound to <b>C44</b>, providing structural insight into the potent inhibition of biaryl ether analogues of brequinar

Species-Selective Pyrimidineamine Inhibitors of <i>Trypanosoma brucei S</i>-Adenosylmethionine Decarboxylase

New therapeutic options are needed for treatment of human African trypanosomiasis (HAT) caused by protozoan parasite <i>Trypanosoma brucei</i>. <i>S</i>-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the polyamine pathway of <i>T. brucei</i>. Previous attempts to target this enzyme were thwarted by the lack of brain penetration of the most advanced series. Herein, we describe a <i>T. brucei</i> AdoMetDC inhibitor series based on a pyrimidineamine pharmacophore that we identified by target-based high-throughput screening. The pyrimidineamines showed selectivity for <i>T. brucei</i> AdoMetDC over the human enzyme, inhibited parasite growth in whole-cell assay, and had good predicted blood–brain barrier penetration. The medicinal chemistry program elucidated structure–activity relationships within the series. Features of the series that were required for binding were revealed by determining the X-ray crystal structure of <i>Tb</i>AdoMetDC bound to one analog. The pyrimidineamine series provides a novel starting point for an anti-HAT lead optimization

Species-Selective Pyrimidineamine Inhibitors of <i>Trypanosoma brucei S</i>-Adenosylmethionine Decarboxylase

New therapeutic options are needed for treatment of human African trypanosomiasis (HAT) caused by protozoan parasite <i>Trypanosoma brucei</i>. <i>S</i>-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the polyamine pathway of <i>T. brucei</i>. Previous attempts to target this enzyme were thwarted by the lack of brain penetration of the most advanced series. Herein, we describe a <i>T. brucei</i> AdoMetDC inhibitor series based on a pyrimidineamine pharmacophore that we identified by target-based high-throughput screening. The pyrimidineamines showed selectivity for <i>T. brucei</i> AdoMetDC over the human enzyme, inhibited parasite growth in whole-cell assay, and had good predicted blood–brain barrier penetration. The medicinal chemistry program elucidated structure–activity relationships within the series. Features of the series that were required for binding were revealed by determining the X-ray crystal structure of <i>Tb</i>AdoMetDC bound to one analog. The pyrimidineamine series provides a novel starting point for an anti-HAT lead optimization

Species-Selective Pyrimidineamine Inhibitors of <i>Trypanosoma brucei S</i>-Adenosylmethionine Decarboxylase

New therapeutic options are needed for treatment of human African trypanosomiasis (HAT) caused by protozoan parasite <i>Trypanosoma brucei</i>. <i>S</i>-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the polyamine pathway of <i>T. brucei</i>. Previous attempts to target this enzyme were thwarted by the lack of brain penetration of the most advanced series. Herein, we describe a <i>T. brucei</i> AdoMetDC inhibitor series based on a pyrimidineamine pharmacophore that we identified by target-based high-throughput screening. The pyrimidineamines showed selectivity for <i>T. brucei</i> AdoMetDC over the human enzyme, inhibited parasite growth in whole-cell assay, and had good predicted blood–brain barrier penetration. The medicinal chemistry program elucidated structure–activity relationships within the series. Features of the series that were required for binding were revealed by determining the X-ray crystal structure of <i>Tb</i>AdoMetDC bound to one analog. The pyrimidineamine series provides a novel starting point for an anti-HAT lead optimization

Inhibition of Influenza A Virus Infection <i>in Vitro</i> by Saliphenylhalamide-Loaded Porous Silicon Nanoparticles

Influenza A viruses (IAVs) cause recurrent epidemics in humans, with serious threat of lethal worldwide pandemics. The occurrence of antiviral-resistant virus strains and the emergence of highly pathogenic influenza viruses have triggered an urgent need to develop new anti-IAV treatments. One compound found to inhibit IAV, and other virus infections, is saliphenylhalamide (SaliPhe). SaliPhe targets host vacuolar-ATPase and inhibits acidification of endosomes, a process needed for productive virus infection. The major obstacle for the further development of SaliPhe as antiviral drug has been its poor solubility. Here, we investigated the possibility to increase SaliPhe solubility by loading the compound in thermally hydrocarbonized porous silicon (THCPSi) nanoparticles. SaliPhe-loaded nanoparticles were further investigated for the ability to inhibit influenza A infection in human retinal pigment epithelium and Madin-Darby canine kidney cells, and we show that upon release from THCPSi, SaliPhe inhibited IAV infection <i>in vitro</i> and reduced the amount of progeny virus in IAV-infected cells. Overall, the PSi-based nanosystem exhibited increased dissolution of the investigated anti-IAV drug SaliPhe and displayed excellent <i>in vitro</i> stability, low cytotoxicity, and remarkable reduction of viral load in the absence of organic solvents. This proof-of-principle study indicates that PSi nanoparticles could be used for efficient delivery of antivirals to infected cells

Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei (<i>Tb</i>). Available treatments are difficult to administer and have significant safety issues. <i>S</i>-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop <i>Tb</i>AdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible <i>Tb</i>AdoMetDC inhibitors with low-micromolar potency (IC₅₀) against both <i>Tb</i>AdoMetDC and <i>T. brucei</i> parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood–brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular <i>Tb</i>AdoMetDC in the parasite, although evidence for a secondary off-target component was also present. The discovery of several new <i>Tb</i>AdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT