David F. Wiemer

An antifungal terpenoid defends a neotropical tree (Hymenaea) against attack by fungus-growing ants (Atta)

SummaryForagers of the leafcutting ant, Atta cephalotes L. (Formicidae, Attini) seldom or never attack many of the plant species available to them in nature. In the semideciduous forests of lowland Guanacaste Province, Costa Rica, one of the tree species seldom cut is Hymenaea courbaril L. (Leguminosae, Caesalpinioideae). We tested the hypothesis that this species is avoided by the ants because of the presence of ant-repellent secondary compounds in the leaves. A bioassay to test repellency of leaf extracts was developed to guide the chemical isolation of ant repellents, using a laboratory colony of Atta cephalotes.The presence of one or more extractable ant repellents was quickly demonstrated. Subsequent chemical isolation and identification revealed that there was essentially only one terpenoid responsible for the repellency: caryophyllene epoxide. Tests with a concentration series of the pure compound demonstrated that the natural concentration of this terpenoid in Hymenaea could fully account for the observed repellency of intact leaves. Field bioassays of the terpenoid in Costa Rica confirmed this result; leaves of a preferred species, Spondias purpurea L. (Anacardiaceae), became as repellent as Hymenaea leaves when treated with caryophyllene epoxide at natural Hymenaea leaf concentrations. Repellency of the epoxide was 20 times greater than that of caryophyllene, its sesquiterpene hydrocarbon precursor, which is also found in Hymenaea leaves.Attine ants cut leaves to serve as substrate for culturing a specific fungus for food, principally for their larvae. A reasonable hypothesis is that these ants selectively avoid plant species whose leaves contain compounds which are toxic to their fungus. We tested caryophyllene epoxide for antifungal activity and found that it is an extremely potent compound, not only against the attine fungus, but other fungi as well. We speculate that many of the other plant species avoided by these ants in nature may be similarly protected from ant attack by antifungal compounds in their leaves. We further suggest that plant defense against leafcutting ants may be largely an incidental byproduct of selection for fungal resistance in plants.

Chemical Leaf Repellency to an Attine Ant: Seasonal Distribution Among Potential Host Plant Species

The chemical repellency of leaves to the leaf-cutting, fungus-growing ant Atta cephalotes was surveyed for 42 plant species randomly selected from the dry forest of Santa Rosa National Park, Guanacaste, Costa Rica. The sample represents about one-sixth of the potential host plant species available to the ants in the secondary, semideciduous forest at Santa Rosa. Repellencies of leaf extracts were measured by bioassay. A laboratory colony of A. cephalotes collected from Santa Rosa was used. Three-quarters of the species exhibited significantly repellent nonpolar (lipid-soluble) extractables (e.g., terpenoids, steroids, and waxes), and half of the species exhibited repellent polar extractables (e.g., phenols, flavonoids, and glycosides). The occurrence of significant extractable repellents, partic- ularly in the lipid-soluble fraction, correlated closely with species that were avoided in leaf-preference tests and with seasonal patterns of host-plant selection by ant colonies in the Santa Rosa forest. There were no clear correlations of repellency with plant growth form or with evergreen vs. deciduous habit. Further chemical isolation of ant-repellent substances from a number of avoided species revealed that most of the repellent compounds are terpenoids, some of which have already been shown to be highly toxic to the ants fungus and to many other fungi as well. A dramatic decline in the amount of extractable repellent occurred in almost all plant species in the latter half of the wet season, 1-2 mo before the dry season began. Because this decline was prevalent in evergreen as well as deciduous species, we tentatively concluded that the ant-repellent substances in the leaves are mainly the result of selection in plants for fungal resistance, which incidentally confers resistance to attack by leaf-cutting ants. We suggest that the decline in the amount of repellent substance is due to a reduction in the synthesis of antifungal secondary compounds in the dry season, when the risk of fungal attack is low.

Phosphonate and bisphosphonate analogues of farnesyl pyrophosphate as potential inhibitors of farnesyl protein transferase

Several phosphonate and bisphosphonate analogues of farnesyl pyrophosphate have been prepared for an examination of their ability to inhibit farnesyl protein transferase (FPTase). A Horner-Wadsworth-Emmons condensation of farnesal or geranial with tetraethyl methylenediphosphonate gave the desired vinyl phosphonates, while alkylation of the dimethyl methylphosphonate anion with a terpenoid bromide gave the corresponding saturated phosphonates. Alkylation of tetraethyl methylenediphosphonate with farnesyl bromide gave the expected alkyl bisphosphonate, which was converted to its alpha, beta-unsaturated derivative by preparation of the phenyl selenide, oxidation to the selenoxide, and elimination. In a similar fashion, triethyl phosphonoacetate was converted to a farnesyl pyrophosphate analogue by reaction with farnesyl bromide. After preparation of the respective acids, each compound was tested for inhibition of FPTase at concentrations ranging up to 10 microM. The effect of these compounds on FPTase activity varied substantially, ranging from depressed to surprisingly enhanced enzymatic activity.

Toxicity of terpenoid deterrents to the leafcutting antAtta cephalotes and its mutualistic fungus.

Four natural products, of varying activity as deterrents of leafcutter attack, were tested for their effects on ant survival and on the growth of the mutualistic attine fungus. The substances were incorporated into an artificial liquid diet for bioassays on the ants or included in an agar culture medium for fungus growth-inhibition studies. Three of the four compounds exhibited deleterious effects on either adult leafcutting ants or their mutualistic fungus, and there appeared to be some correlation between deterrency and activity in these toxicity assays. The implications of these findings for leafcutting ant foraging patterns are discussed.

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.

The intermediate enzymes of isoprenoid metabolism as anticancer targets.

Inhibitors of isoprenoid biosynthesis are widely used to treat human disease including statins and nitrogenous bisphosphonates. Due to the importance of core human isoprenoid biosynthesis for diverse cellular processes related to cancer cell growth and metastasis, inhibition of this pathway may produce beneficial anticancer consequences. For example, ras oncogenes are well known; ras proteins are overexpressed in many human cancers, and these proteins must be isoprenylated to function. The rho proteins are important for regulating cell motility, and also must be isoprenylated. This has drawn significant attention to inhibitors of protein prenyl transferases. In addition to the reactions that are targeted in current clinical applications, there are other enzymes that have not been studied as extensively. Inhibition of these enzymes, from mevalonate kinase to geranylgeranyl diphosphate synthase, could be attractive as a single agent therapy or in combination with current agents for treatment of cancers in which isoprenylated proteins have been implicated. While detailed in vivo data for many of these putative targets is lacking, there have been several breakthroughs in recent years that could facilitate further studies. In particular, compounds that specifically inhibit some of the downstream isoprenoid biosynthesis enzymes have been developed and their effects in cancer models are emerging. This review will discuss current knowledge of these lesser known isoprenoid pathway enzymes, identify trends in the development of their small molecule inhibitors, and describe the applications and effects of these compounds in cancer models.

Enantioselective synthesis of α-hydroxy phosphonates via oxidation with (camphorsulfonyl)oxaziridines

Abstract Reaction of phosphonate anions with enantiomerically pure (camphorsulfonyl)oxaziridines results in formation of nonracemic α-hydroxy phosphonates. This enantioselective hydroxylation methodology provides convenient access to optically active α-hydroxy phosphonates and their corresponding phosphonic acids.

Prodrugs of Phosphonates and Phosphates: Crossing the Membrane Barrier

A substantial portion of metabolism involves transformation of phosphate esters, including pathways leading to nucleotides and oligonucleotides, carbohydrates, isoprenoids and steroids, and phosphorylated proteins. Because the natural substrates bear one or more negative charges, drugs that target these enzymes generally must be charged as well, but small charged molecules can have difficulty traversing the cell membrane by means other than endocytosis. The resulting dichotomy has stimulated a great deal of effort to develop effective prodrugs, compounds that carry little or no charge to enable them to transit biological membranes, but able to release the parent drug once inside the target cell. This chapter presents recent studies on advances in prodrug forms, along with representative examples of their application to marketed and developmental drugs.

Pivaloyloxymethyl-modified isoprenoid bisphosphonates display enhanced inhibition of cellular geranylgeranylation.

Nitrogenous bisphosphonate inhibitors of farnesyl disphosphate synthase have been used clinically for treatment of bone disease. Because many of their effects may be mediated by depletion of geranylgeranyl diphosphate, our group has sought compounds that do this more directly through inhibition of geranylgeranyl diphosphate synthase and we have discovered a number of isoprenoid-containing bisphosphonates that selectively inhibit this enzyme. These compounds have a high negative charge at physiological pH which is necessary for inhibition of the enzyme but may limit their ability to enter cells. Therefore, chemical modifications that mask this charge may enhance their cellular potency. We now have synthesized novel pivaloyloxymethyl-modified isoprenoid bisphosphonates and investigated their ability to inhibit protein geranylgeranylation within cells. We have found that addition of pivaloyloxymethyl moieties to isoprenoid bisphosphonates increases their potency towards cellular geranylgeranylation even though this modification decreases their in vitro inhibition of geranylgeranyl diphosphate synthase. Pivaloyloxymethyl modifications more effectively increase the cellular activity of the more polar isoprenoid bisphosphonates. These results reveal structural relationships between in vitro and cellular activity which may serve as the basis for future development of more potent and/or drug-like inhibitors of geranylgeranyl diphosphate synthase.

Ant-repellent terpenoids from Melampodium divaricatum

Abstract The leaves of Melampodium divaricatum have been systematically fractionated by following biological activity in an assay which measures repellency to the leafcutter ant Atta cephalotes (Formicidae, Attini). Several terpenoids have been isolated which show significant ant-repellency.