Troponoid Atropisomerism: Studies on the Configurational Stability of Tropone-Amide Chiral Axes.

Configurationally stable, atropisomeric motifs are an important structural element in a number of molecules, including chiral ligands, catalysts, and molecular devices. Thus, understanding features that stabilize chiral axes is of fundamental interest throughout the chemical sciences. The following details the high rotational barriers about the Ar-C(O) bond of tropone amides, which significantly exceed those of analogous benzamides. These studies are supported by both experimental and computational rotational barrier measurements. We also report the resolution of an axially chiral α-hydroxytropolone amide into its individual atropisomers, and demonstrate its configurational stability at physiological pH and temperatures over 24 h.

Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands.

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to the Herpesviridae family, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.

Catalytic Enantioselective Intermolecular [5 + 2] Dipolar Cycloadditions of a 3-Hydroxy-4-pyrone-Derived Oxidopyrylium Ylide.

The first catalytic enantioselective [5 + 2] dipolar cycloaddition of a 3-hydroxy-4-pyrone-derived oxidopyrylium ylide is described. These studies leveraged the recently recognized ability of oxidopyrylium dimers to serve as the source of ylide, which was found to be key to increasing yields and achieving enantiomeric excesses up to 99%. General reaction conditions were identified for an array of α,ß-unsaturated aldehyde dipolarophiles. Reaction products possess four stereocenters, and subsequent reduction introduced a fifth contiguous stereocenter with total stereocontrol.

Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors.

The natural product α-hydroxytropolones manicol and ß-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an "RNase H-like" fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein-nucleic acid contacts over a region of ∼ 14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme-substrate complex to the enzyme-product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site.

Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses.

Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as ß-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 µM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 µM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC50) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] of 50 to >100 µM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants.

Synthetic α-Hydroxytropolones as Inhibitors of HIV Reverse Transcriptase Ribonuclease H Activity.

HIV Reverse Transcriptase-associated ribonuclease H activity is a promising enzymatic target for drug development that has not been successfully targeted in the clinic. While the α-hydroxytropolone-containing natural products ß-thujaplicinol and manicol have emerged as some of the most potent leads described to date, structure-function studies have been limited to the natural products and semi-synthetic derivatives of manicol. Thus, a library of α-hydroxytropolones synthesized through a convenient oxidopyrylium cycloaddition/ring-opening sequence have been tested in in vitro and cell-based assays, and have been analyzed using computational support. These studies reveal new synthetic α-hydroxytropolones that, unlike the natural product leads they are derived from, demonstrate protective antiviral activity in cellular assays.

Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity.

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 µM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 µM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 µM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

Inhibition of the ANT(2")-Ia resistance enzyme and rescue of aminoglycoside antibiotic activity by synthetic α-hydroxytropolones.

Aminoglycoside-2"-O-nucleotidyltransferase ANT(2")-Ia is an aminoglycoside resistance enzyme prevalent among Gram-negative bacteria, and is one of the most common determinants of enzyme-dependant aminoglycoside-resistance. The following report outlines the use of our recently described oxidopyrylium cycloaddition/ring-opening strategy in the synthesis and profiling of a library of synthetic α-hydroxytropolones against ANT(2")-Ia. In addition, we show that two of these synthetic constructs are capable of rescuing gentamicin activity against ANT-(2")-Ia-expressing bacteria.

The biology and synthesis of α-hydroxytropolones.

α-Hydroxytropolones are a subclass of the troponoid family of natural products that are of high interest due to their broad biological activity and potential as treatment options for several diseases. Despite this promise, there have been scarce synthetic chemistry-driven optimization studies on the molecules. The following review highlights key developments in the biological studies conducted on α-hydroxytropolones to date, including the few synthetic chemistry-driven optimization studies. In addition, we provide an overview of the methods currently available to access these molecules. This review is intended to serve as a resource for those interested in biological activity of α-hydroxytropolones, and inspire the development of new synthetic methods and strategies that could aid in this pursuit.