Stereodivergent Resolution of Oxabicyclic Ketones: Preparation of Key Intermediates for Platensimycin and Other Natural Products

An improved methodology for the preparation of enantiopure oxabicyclo[3.2.1]­octadienes via a stereodivergent resolution is reported. High catalyst control proximal to the oxabridged stereocenter produces readily separable diastereomers in high yield (>92%) and with excellent optical purity (>95% ee). This resolution strategy is amenable to large-scale preparations, and the utility of the resolution was further demonstrated in the asymmetric preparation of a key intermediate used in the synthesis of the antibiotic (−)-platensimycin

Nonracemic Antifolates Stereoselectively Recruit Alternate Cofactors and Overcome Resistance in <i>S</i>. <i>aureus</i>

While antifolates such as Bactrim (trimethoprim-sulfamethoxazole; TMP-SMX) continue to play an important role in treating community-acquired methicillin-resistant <i>Staphylococcus aureus</i> (CA-MRSA), resistance-conferring mutations, specifically F98Y of dihydrofolate reductase (DHFR), have arisen and compromise continued use. In an attempt to extend the lifetime of this important class, we have developed a class of propargyl-linked antifolates (PLAs) that exhibit potent inhibition of the enzyme and bacterial strains. Probing the role of the configuration at the single propargylic stereocenter in these inhibitors required us to develop a new approach to nonracemic 3-aryl-1-butyne building blocks by the pairwise use of asymmetric conjugate addition and aldehyde dehydration protocols. Using this new route, a series of nonracemic PLA inhibitors was prepared and shown to possess potent enzyme inhibition (IC₅₀ values <50 nM), antibacterial effects (several with MIC values <1 μg/mL) and to form stable ternary complexes with both wild-type and resistant mutants. Unexpectedly, crystal structures of a pair of individual enantiomers in the wild-type DHFR revealed that the single change in configuration of the stereocenter drove the selection of an alternative NADPH cofactor, with the minor α-anomer appearing with <b>R-27</b>. Remarkably, this cofactor switching becomes much more prevalent when the F98Y mutation is present. The observation of cofactor site plasticity leads to a postulate for the structural basis of TMP resistance in DHFR and also suggests design strategies that can be used to target these resistant enzymes

Crystal Structures of Trimethoprim-Resistant DfrA1 Rationalize Potent Inhibition by Propargyl-Linked Antifolates

Multidrug-resistant Enterobacteriaceae, notably Escherichia coli and Klebsiella pneumoniae, have become major health concerns worldwide. Resistance to effective therapeutics is often carried by class I and II integrons that can confer insensitivity to carbapenems, extended spectrum β-lactamases, the antifolate trimethoprim, fluoroquinolones, and aminoglycosides. Specifically of interest to the study here, a prevalent gene (<i>dfr</i>A1) coding for an insensitive dihydrofolate reductase (DHFR) confers 190- or 1000-fold resistance to trimethoprim for <i>K. pneumoniae</i> and <i>E. coli</i>, respectively. Attaining inhibition of both the wild-type and resistant forms of the enzyme is critical for new antifolates. For several years, we have been developing the propargyl-linked antifolates (PLAs) as effective inhibitors against trimethoprim-resistant DHFR enzymes. Here, we show that the PLAs are active against both the wild-type and DfrA1 DHFR proteins. We report two high-resolution crystal structures of DfrA1 bound to potent PLAs. The structure–activity relationships and crystal structures will be critical in driving the design of broadly active inhibitors against wild-type and resistant DHFR

Cyclopropene Cycloadditions with Annulated Furans: Total Synthesis of (+)- and (−)-Frondosin B and (+)-Frondosin A

The asymmetric total syntheses of the natural products (+)- and (−)-frondosin B and (+)-frondosin A are reported based on a diastereoselective cycloaddition between tetrabromocyclopropene and an annulated furan to provide a highly functionalized common building block. The bridged bicyclic intermediate could be stereo- and chemoselectively manipulated to produce the two structurally distinct members of the frondosins. Both syntheses feature regioselective palladium-coupling reactions and an unprecedented phosphine-mediated ether bridge cleavage. Surprisingly, the planned enantioselective synthesis of frondosin B led to the opposite epimer of the natural product, suggesting an unusual late stage stereoinversion at C8. Frondosin A, but not frondosin B, was shown to have selective antiproliferative activity against several B-cell lines

Cyclopropene Cycloadditions with Annulated Furans: Total Synthesis of (+)- and (−)-Frondosin B and (+)-Frondosin A

The asymmetric total syntheses of the natural products (+)- and (−)-frondosin B and (+)-frondosin A are reported based on a diastereoselective cycloaddition between tetrabromocyclopropene and an annulated furan to provide a highly functionalized common building block. The bridged bicyclic intermediate could be stereo- and chemoselectively manipulated to produce the two structurally distinct members of the frondosins. Both syntheses feature regioselective palladium-coupling reactions and an unprecedented phosphine-mediated ether bridge cleavage. Surprisingly, the planned enantioselective synthesis of frondosin B led to the opposite epimer of the natural product, suggesting an unusual late stage stereoinversion at C8. Frondosin A, but not frondosin B, was shown to have selective antiproliferative activity against several B-cell lines

Direct Substitution of Arylalkynyl Carbinols Provides Access to Diverse Terminal Acetylene Building Blocks

To develop next generation antifolates for the treatment of trimethoprim-resistant bacteria, synthetic methods were needed to prepare a diverse array of 3-aryl-propynes with various substitutions at the propargyl position. A direct route was sought whereby nucleophilic addition of acetylene to aryl carboxaldehydes would be followed by reduction or substitution of the resulting propargyl alcohol. The direct reduction, methylation, and dimethylation of these readily available alcohols provide efficient access to this uncommon functional array. In addition, an unusual silane exchange reaction was observed in the reduction of the propargylic alcohols

Measuring Propargyl-Linked Drug Populations Inside Bacterial Cells, and Their Interaction with a Dihydrofolate Reductase Target, by Raman Microscopy

We report the first Raman spectroscopic study of propargyl-linked dihydrofolate reductase (DHFR) inhibitors being taken up by wild type Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus cells. A novel protocol is developed where cells are exposed to the fermentation medium containing a known amount of an inhibitor. At a chosen time point, the cells are centrifuged and washed to remove the extracellular compound, then frozen and freeze–dried. Raman difference spectra of the freeze–dried cells (cells exposed to the drug minus cells alone) provide spectra of the compounds inside the cells, where peak intensities allow us to quantify the number of inhibitors within each cell. A time course for the propargyl-linked DHFR inhibitor UCP 1038 soaking into E. coli cells showed that penetration occurs very quickly and reaches a plateau after 10 min exposure to the inhibitor. After 10 min drug exposure, the populations of two inhibitors, UCP 1038 and UCP 1089, were ∼1.5 × 10⁶ molecules in each E. coli cell, ∼4.7 × 10⁵ molecules in each K. pneumonia cell, and ∼2.7 × 10⁶ in each S. aureus cell. This is the first in situ comparison of inhibitor population in Gram-negative and Gram-positive bacterial cells. The positions of the Raman peaks also reveal the protonation of diaminopyrimidine ring upon binding to DHFR inside cells. The spectroscopic signature of protonation was characterized by binding an inhibitor to a single crystal of DHFR

Tropolones As Lead-Like Natural Products: The Development of Potent and Selective Histone Deacetylase Inhibitors

Natural products have long been recognized as a rich source of potent therapeutics but further development is often limited by high structural complexity and high molecular weight. In contrast, at the core of the thujaplicins is a lead-like tropolone scaffold characterized by relatively low molecular weight, ample sites for diversification, and metal-binding functionality poised for targeting a range of metalloenzyme drug targets. Here, we describe the development of this underutilized scaffold for the discovery of tropolone derivatives that function as isozyme-selective inhibitors of the validated anticancer drug target, histone deacetylase (HDAC). Several monosubstituted tropolones display remarkable levels of selectivity for HDAC2 and potently inhibit the growth of T-cell lymphocyte cell lines. The tropolones represent a new chemotype of isozyme-selective HDAC inhibitors

Charged Propargyl-Linked Antifolates Reveal Mechanisms of Antifolate Resistance and Inhibit Trimethoprim-Resistant MRSA Strains Possessing Clinically Relevant Mutations

Drug-resistant enzymes must balance catalytic function with inhibitor destabilization to provide a fitness advantage. This sensitive balance, often involving very subtle structural changes, must be achieved through a selection process involving a minimal number of eligible point mutations. As part of a program to design propargyl-linked antifolates (PLAs) against trimethoprim-resistant dihydrofolate reductase (DHFR) from <i>Staphylococcus aureus</i>, we have conducted a thorough study of several clinically observed chromosomal mutations in the enzyme at the cellular, biochemical, and structural levels. Through this work, we have identified a promising lead series that displays significantly greater activity against these mutant enzymes and strains than TMP. The best inhibitors have enzyme inhibition and MIC values near or below that of trimethoprim against wild-type <i>S. aureus</i>. Moreover, these studies employ a series of crystal structures of several mutant enzymes bound to the same inhibitor; analysis of the structures reveals a more detailed molecular understanding of drug resistance in this important enzyme

Propargyl-Linked Antifolates Are Potent Inhibitors of Drug-Sensitive and Drug-Resistant <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterium tuberculosis</i> continues to cause widespread, life-threatening disease. In the last decade, this threat has grown dramatically as multi- and extensively-drug resistant (MDR and XDR) bacteria have spread globally and the number of agents that effectively treat these infections is significantly reduced. We have been developing the propargyl-linked antifolates (PLAs) as potent inhibitors of the essential enzyme dihydrofolate reductase (DHFR) from bacteria and recently found that charged PLAs with partial zwitterionic character showed improved mycobacterial cell permeability. Building on a hypothesis that these PLAs may penetrate the outer membrane of <i>M</i>. <i>tuberculosis</i> and inhibit the essential cytoplasmic DHFR, we screened a group of PLAs for antitubercular activity. In this work, we identified several PLAs as potent inhibitors of the growth of <i>M</i>. <i>tuberculosis</i> with several of the compounds exhibiting minimum inhibition concentrations equal to or less than 1 μg/mL. Furthermore, two of the compounds were very potent inhibitors of MDR and XDR strains. A high resolution crystal structure of one PLA bound to DHFR from <i>M</i>. <i>tuberculosis</i> reveals the interactions of the ligands with the target enzyme.</p></div