Jeffrey D. Neighbors

Total Synthesis of (+)-Schweinfurthins B and E

The first total synthesis of (+)-schweinfurthin B, a potent and differentially active cytotoxic agent, has been accomplished. Completion of the synthesis required just 16 steps in the longest linear sequence from commercially available vanillin. Key synthetic transformations included a Shi epoxidation and an efficient cascade cyclization initiated by treatment of the resulting epoxide with BF(3).OEt(2). Furthermore, use of a methyl ether as a stable protecting group for benzylic alcohols dramatically increased the efficiency of the overall sequence. The benzylic ether can be removed from this electron-rich aromatic system through oxidation with DDQ that provided the desired aldehyde intermediate in quantitative yield and shortened the synthetic sequence. Introduction of the A-ring diol in the required cis stereochemistry then became viable through a short sequence highlighted by an aldol condensation with benzaldehyde to introduce an olefin as a latent carbonyl group at the C-3 position. This synthesis established for the first time the absolute stereochemistry of the natural product, and provides access to material on a scale that will advance biological studies. The total synthesis of the closely related compound (+)-schweinfurthin E also is reported.

Hypericum in infection: Identification of anti-viral and anti-inflammatory constituents.

The Iowa Center for Research on Botanical Dietary Supplements seeks to optimize Echinacea, Hypericum, and Prunella botanical supplements for human-health benefit, emphasizing anti-viral, anti-inflammatory, and anti-pain activities. This mini-review reports on ongoing studies on Hypericum. The Center uses the genetically diverse, well-documented Hypericum populations collected and maintained at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), and the strength of research in synthetic chemistry at Iowa State University to tap natural diversity, to help discover key constituents and interactions among constituents that impact bioactivity and toxicity. The NCRPIS has acquired more than 180 distinct populations of Hypericum, with a focus on Hypericum perforatum L. (Hypericaceae), representing about 13% of currently recognized taxa. Center chemists have developed novel synthetic pathways for key flavones, acyl phloroglucinols, hyperolactones, and a tetralin that have been found in Hypericum, and these compounds are used as standards and for bioactivity studies. Both light-dependent and light-independent anti-viral activities have been identified by using bioactivity-guided fractionation of H. perforatum and a HIV-1 infection test system. Our Center has focused on light-independent activity, potentially due to novel chemicals, and polar fractions are undergoing further fractionation. Anti-inflammatory activity has been found to be light-independent, and fractionation of a flavonoid-rich extract revealed four compounds (amentoflavone, chlorogenic acid, pseudohypericin, and quercetin) that interacted in the light to inhibit lipopolysaccharide-induced prostaglandin E2 activity. The Center continues to explore novel populations of H. perforatum and related species to identify constituents and interactions of constituents that contribute to potential health benefits related to infection.

A Tandem Cascade Cyclization-Electrophilic Aromatic Substitution: Application in the Total Synthesis of (+)-Angelichalcone

When cascade cyclizations initiated by Lewis acid-mediated opening of an epoxide are terminated through reaction with a MOM-protected phenol, a tandem electrophilic aromatic substitution can be obtained. This highly regioselective tandem process has been employed in the first synthesis of (+)-angelichalcone.

Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at -80 degrees C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355+/-0.030 and 0.827+/-0.082 units of nmol/g wet tissues in brain, 0.320+/-0.019 and 0.293+/-0.035 units of nmol/g wet tissues in kidney, 0.326+/-0.064 and 0.213+/-0.029 units of nmol/g wet tissues in liver, and 0.364+/-0.015 and 0.349+/-0.023 units of nmol/g wet tissues in heart (means+/-SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.

Synthesis and biological activity of a fluorescent schweinfurthin analogue

Most of the natural schweinfurthins are potent and selective inhibitors of cell growth as measured by the National Cancer Institutes 60-cell line screen. Due to the limited supply of these natural products, we have initiated a program aimed at their synthesis. To date, this effort has led to the preparation of three natural schweinfurthins and more than 40 analogues, and assays on these compounds have afforded some understanding of structure-activity relationships in this family. Further development of schweinfurthins as chemotherapeutic agents would benefit from characterization of their mechanism(s) of action. This perspective led to development of a fluorescent schweinfurthin analogue that retains the differential activity of the natural products, and yet has properties that facilitate its visualization within cells.

Pseudohypericin is necessary for the Light-Activated Inhibition of Prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E2 production (PGE2)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1microM chlorogenic acid (compound 1), 0.08microM amentoflavone (compound 2), 0.07microM quercetin (compound 3), and 0.03microM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A2, two enzymes in the PGE2-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.

Identification of light-independent inhibition of human immunodeficiency virus-1 infection through bioguided fractionation of Hypericum perforatum

BackgroundLight-dependent activities against enveloped viruses in St. Johns Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated.ResultsHere, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material.ConclusionThrough bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.

Targeting the isoprenoid pathway to abrogate progression of pulmonary fibrosis

Fibrotic remodeling in lung injury is a major cause of morbidity. The mechanism that mediates the ongoing fibrosis is unclear, and there is no available treatment to abate the aberrant repair. Reactive oxygen species (ROS) have a critical role in inducing fibrosis by modulating extracellular matrix deposition. Specifically, mitochondrial hydrogen peroxide (H2O2) production by alveolar macrophages is directly linked to pulmonary fibrosis as inhibition of mitochondrial H2O2 attenuates the fibrotic response in mice. Prior studies indicate that the small GTP-binding protein, Rac1, directly mediates H2O2 generation in the mitochondrial intermembrane space. Geranylgeranylation of the C-terminal cysteine residue (Cys(189)) is required for Rac1 activation and mitochondrial import. We hypothesized that impairment of geranylgeranylation would limit mitochondrial oxidative stress and, thus, abrogate progression of pulmonary fibrosis. By targeting the isoprenoid pathway with a novel agent, digeranyl bisphosphonate (DGBP), which impairs geranylgeranylation, we demonstrate that Rac1 mitochondrial import, mitochondrial oxidative stress, and progression of the fibrotic response to lung injury are significantly attenuated. These observations reveal that targeting the isoprenoid pathway to alter Rac1 geranylgeranylation halts the progression of pulmonary fibrosis after lung injury.

A concise synthesis of pawhuskin A.

Pawhuskin A is an isoprenylated stilbene that was isolated from Dalea purpurea and reported to have affinity for the opioid receptor in vitro. It has been synthesized through a convergent sequence that joins a prenylated aldehyde with a geranylated phosphonate in a stereoselective Horner-Wadsworth-Emmons condensation to afford the target E olefin isomer. This synthesis confirms the structure assigned to the natural product and establishes a route that may be used to explore its biological activity and to prepare more active analogues.

Stilbenes as κ-Selective, Non-nitrogenous Opioid Receptor Antagonists

The natural stilbene pawhuskin A has been shown to function as an opioid receptor antagonist, with preferential binding to the κ receptor. This finding encouraged assembly of a set of analogues to probe the importance of key structural features. Assays on these compounds determined that one (compound 29) shows potent opioid receptor binding activity and significantly improved selectivity for the κ receptor. These studies begin to illuminate the structural features of these non-nitrogenous opioid receptor antagonists that are required for activity.