Synthesis and biological assessment of 3,7-dihydroxytropolones.

3,7-Dihydroxytropolones (3,7-dHTs) are highly oxygenated troponoids that have been identified as lead compounds for several human diseases. To date, structure-function studies on these molecules have been limited due to a scarcity of synthetic methods for their preparation. New synthetic strategies towards structurally novel 3,7-dHTs would be valuable in further studying their therapeutic potential. Here we describe the successful adaptation of a [5 + 2] oxidopyrilium cycloaddition/ring-opening for 3,7-dHT synthesis, which we apply in the synthesis of a plausible biosynthetic intermediate to the natural products puberulic and puberulonic acid. We have also tested these new compounds in several biological assays related to human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV) in order to gain insight into structure-functional analysis related to antiviral troponoid development.

Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay.

Cannabinoid (CB) receptors are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed antiproliferative activity against breast cancer and prostate cancer in animal models. To study cannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [(3)H]Win55-212-2 for CB1 and [(3)H]CP55-940 for CB2. The developed plate-based assay was validated by determining IC50 values for known antagonists: AM251, AM281, AM630, and AM6545. The data obtained were consistent with previously reported values, thereby confirming that the assay can be used to determine the functional binding activities (IC50) of antagonists for the CB1 and CB2 receptors. In addition, we demonstrated that the plate-based assay may be used for screening against complex matrices. Specifically, we demonstrated that the plate-based assay was able to identify which extracts of several species of the genus Zanthoxylum had activity at the CB1/CB2 receptors.

Inhibiting Hdm2 and ubiquitin-activating enzyme: targeting the ubiquitin conjugating system in cancer.

The ubiquitin conjugating system represents a rich source of potential molecular targets for cancer and other diseases. One target of great interest is the RING finger ubiquitin ligase (E3) Hdm2/Mdm2, which is frequently overexpressed in cancer and is a critical E3 for the tumor suppressor p53. For those 50% of tumors that express wild-type p53, agents that inhibit Hdm2 have great potential clinical utility. We summarize our ongoing efforts to identify inhibitors of Hdm2 E3 activity by high-throughput screening of both defined small molecules and natural product extracts. Employing a strategy using both enzymatic and cell-based assays, we have identified inhibitors that block the E3 activity of Hdm2, activate a p53 response, preferentially kill p53-expressing cells, and have the capacity to differentially cause death of transformed cells. Therefore, screening for inhibitors of Hdm2 ubiquitin ligase activity through in vitro assays represents a powerful means of identifying molecules that activate p53 in cancer cells to induce apoptosis. We also discuss the potential of inhibitors of ubiquitin-activating enzyme (E1) that were discovered during these screens. E1 inhibitors may similarly serve as the basis for novel therapeutics. Additionally, they represent unique tools for providing new insights into the ubiquitin conjugating system.

Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-atpases.

A series of naturally occurring compounds reported recently by multiple laboratories defines a new small-molecule class sharing a unique benzolactone enamide core structure and diverse biological actions, including inhibition of growth of tumor cells and oncogene-transformed cell lines. Here we show that representative members of this class, including salicylihalamide A, lobatamides A-F, and oximidines I and II inhibit mammalian vacuolar-type (H+)-ATPases (V-ATPases) with unprecedented selectivity. Data derived from the NCI 60-cell antitumor screen critically predicted the V-ATPase molecular target, while specific biochemical assays provided confirmation and further illumination. The compounds potently blocked representative V-ATPases from human kidney, liver, and osteoclastic giant-cell tumor of bone but were essentially inactive against V-ATPases of Neurospora crassa and Saccharomyces cerevisiae and other membrane ATPases. Essential regulation of pH in cytoplasmic, intraorganellar, and local extracellular spaces is provided by V-ATPases, which are ubiquitously distributed among eukaryotic cells and tissues. The most potent and selective V-ATPase inhibitors heretofore known were the bafilomycins and concanamycins, which do not discriminate between mammalian and nonmammalian V-ATPases. Numerous physiological processes are mediated by V-ATPases, and aberrant V-ATPase functions are implicated in many different human diseases. Previous efforts to develop therapeutic pharmacological modulators of V-ATPases have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, availability of the unique benzolactone enamide inhibitor class may enable further elucidation of functional and architectural features of mammalian versus nonmammalian V-ATPase isoforms and provide new opportunities for targeting V-ATPase-mediated processes implicated in diverse pathophysiological phenomena, including cancer.

Cytotoxic cholestane glycosides from the bulbs of Ornithogalum saundersiae.

Further phytochemical analysis of the bulbs of Ornithogalum saundersiae has yielded two new cytotoxic cholestane triglycosides (1 and 2). The structures of these compounds were determined by spectroscopic analysis, including 2D NMR spectroscopic data, and the results of hydrolytic cleavage. Compounds 1 and 2 and several analogues were evaluated for their cytotoxicity against HL-60 cells.

Stolonic acids A and B, new cytotoxic cyclic peroxides from an Indian Ocean ascidian Stolonica species.

Two new 3,6-epidioxy-7,10-tetrahydrofurano C(26) unsaturated fatty acids, stolonic acids A (1) and B (2), were isolated from a previously undescribed ascidian species, Stolonica sp. collected off the Maldive Islands in the Indian Ocean. The structures and relative stereochemistry of 1 and 2 were determined using conventional spectroscopic methods. Both compounds exhibited antiproliferative activity against selected human melanoma and ovarian tumor cell lines, with IC(50) values of approximately 0.05-0.1 microg/mL.

Characterization of anticancer agents by their growth inhibitory activity and relationships to mechanism of action and structure.

An analysis of the growth inhibitory potency of 122 anticancer agents available from the National Cancer Institute anticancer drug screen is presented. Methods of singular value decomposition (SVD) were applied to determine the matrix of distances between all compounds. These SVD-derived dissimilarity distances were used to cluster compounds that exhibit similar tumor growth inhibitory activity patterns against 60 human cancer cell lines. Cluster analysis divides the 122 standard agents into 25 statistically distinct groups. The first eight groups include structurally diverse compounds with reactive functionalities that act as DNA-damaging agents while the remaining 17 groups include compounds that inhibit nucleic acid biosynthesis and mitosis. Examination of the average activity patterns across the 60 tumor cell lines reveals unique 'fingerprints' associated with each group. A diverse set of structural features are observed for compounds within these groups, with frequent occurrences of strong within-group structural similarities. Clustering of cell types by their response to the 122 anticancer agents divides the 60 cell types into 21 groups. The strongest within-panel groupings were found for the renal, leukemia and ovarian cell panels. These results contribute to the basis for comparisons between log(GI(50)) screening patterns of the 122 anticancer agents and additional tested compounds.

Novel cytotoxic diterpenes from Casearia arborea.

Cytotoxicity-guided fractionation of the dichloromethane-methanol extract of the roots of Casearia arborea yielded five novel clerodane diterpenes, casearborins A-E (1-5), as well as cucurbitacin B. The presence of cucurbitacins glycosides was also detected. The absolute configuration of casearborin E was determined by X-ray crystallography.

Cytotoxic clerodane diterpene esters from Laetia corymbulosa.

Three cytotoxic clerodane diterpene esters, corymbulosins A-C, were isolated from an organic extract of the fruit of Laetia corymbulosa (Flacourtiaceae) from Peru. The structures were determined by spectroscopic methods as clerodane diterpenes unsaturated at C-3, C-13(16) and C-14. Corymbulosin A was esterified at C-2 with a decadienoate moiety, while corymbulosins B and C were C-2 epimers esterified at C-6 with a decanoate moiety.

Cytotoxic geranyl stilbenes from Macaranga schweinfurthii.

Three novel geranyl stilbenes, schweinfurthins A, B, and C (1, 2, and 3), were isolated from the Cameroonian plant Macaranga schweinfurthii (Euphorbiaceae) and their structures determined by NMR and mass spectral methods. The cytotoxicity profile of the schweinfurthins tested in the NCI 60-cell screen was similar to that of the stelletins and cephalostatins, suggesting that these structurally diverse natural products may share similar mechanisms of cytotoxicity.

Structure-activity requirements for flavone cytotoxicity and binding to tubulin.

A series of 79 flavones related to centaureidin (3,6,4'-trimethoxy-5, 7,3'-trihydroxyflavone, 1) was screened for cytotoxicity in the NCI in vitro 60-cell line human tumor screen. The resulting cytotoxicity profiles of these flavones were compared for degree of similarity to the profile of 1. Selected compounds were further evaluated with in vitro assays of tubulin polymerization and [3H]colchicine binding to tubulin. Maximum potencies for tubulin interaction and production of differential cytotoxicity profiles characteristic of 1 were observed only with compounds containing hydroxyl substituents at C-3' and C-5 and methoxyl groups at C-3 and C-4'.

Isolation of a novel Kunitz family protease inhibitor in association with Tethya hemolysin from the sponge Tethya ingalli.

Aqueous extracts from the New Zealand sponge Tethya ingalli (Hadromerida) displayed potent cytotoxicity in the NCI's 60-cell-line human tumor panel. Fractionation of the extract by ammonium sulfate precipitation, gel filtration, ultrafiltration, and both hydrophobic interaction and reversed-phase chromatography resulted in the isolation of two biologically active proteins. The first protein, Tethya protease inhibitor (TPI), which was purified to homogeneity, inhibited trypsin with an EC50 of 65 nM. TPI had a molecular mass of 11,431 Da, and an isoelectric point of 8.2. A partial N-terminal amino acid sequence determined for TPI showed significant homology with protease inhibitors of the Kunitz family. The second isolated protein displayed potent cytotoxicity, with pronounced selectivity for certain tumor cell lines (e.g., ovarian, renal, CNS, and breast). The latter protein, which had an apparent molecular weight of 21 kDa (SDS-PAGE), also lysed human red blood cells (EC50 of 39 nM) and was similar to a hemolysin previously isolated from the sponge Tethya lycinurium.

Isolation and characterization of novel cytotoxic saponins from Archidendron ellipticum.

A series of new ester saponins, elliptosides A-J, has been isolated from the tropical plant Archidendron ellipticum (Leguminosae). These saponins were particularly cytotoxic to certain renal and melanoma cancer cell lines in the NCI's 60-cell line human tumor screen. The structures of elliptosides A, E, and F were elucidated by spectroscopic and chemical means. Elliptoside A showed in vivo antitumor activity against the LOX melanoma cell line.

Majapolene A, a cytotoxic peroxide, and related sesquiterpenes from the red alga Laurencia majuscula.

Seven new sesquiterpenes, majapolenes A [1] and B [2], majapolone [3], and majapols A [4], B [5], C [6], and D [7], were isolated from a Philippine collection of Laurencia majuscula. With the exception of majapolene B [2], all compounds were isolated as inseparable diastereomeric mixtures. Structure elucidation was achieved by spectroscopic methods. Majapolene A [1], a dioxabicyclo[2.2.2]-alkene, displayed modest activity in the NCI 60-cell line cytotoxicity screen. Majapolene A was also found as a major component of a Philippine collection of Laurencia caraibica.

A reinvestigation of Maprounea triterpenes.

Anti-HIV activity and the inhibition of phorbol ester receptor binding activity in two species of Maprounea were traced to small amounts of highly potent phorbol esters of the daphnane type. The triterpenes previously isolated from this genus were found to be devoid of biological activity when scrupulously purified. Four new triterpene esters were elucidated; two [3,4] were found in M. africana, while three [4,6,7] were found in M. membranacea. Nmr assignments have also been made for two previously known compounds [2,5] in this group.

Esperamicin P, the tetrasulfide analog of esperamicin A1.

A minor congener of esperamicin A1 [1], designated esperamicin P (BMY-41339, 2), was isolated from a fermentation broth of Actinomadura verrucosospora and determined to differ from esperamicin A1 by having a methyl tetrasulfide moiety instead of a methyl trisulfide. It was active against xenografted tumors in mice and exhibited antimicrobial activity. Interconversions of 2 and 1 have been observed for DMSO solutions of both congeners at room temperature.

Two new cytotoxic chalcones from Calythropsis aurea.

The crude extract of Calythropsis aurea (Myrtaceae) produced a pattern of differential cytotoxicity in the NCI 60 cell line assay which was similar to those of known tubulin-interactive compounds. Cytotoxicity-guided fractionation led to the isolation of two new chalcones, calythropsin [1] and dihydrocalythropsin [2], which were responsible for the activity. Calythropsin was demonstrated to have a weak effect on mitosis, and presumably also on tubulin polymerization.

A cytotoxic beta-carboline from the bryozoan Catenicella cribraria.

1-Vinyl-8-hydroxy-beta-carboline was identified as the cytotoxic constituent of the bryozoans Catenicella cribraria and Cribricellina cribraria. Literature nmr data for this previously known compound, now reported from a new source, were corrected.

A chemical screening strategy for the dereplication and prioritization of HIV-inhibitory aqueous natural products extracts.

A relatively high percentage (ca. 15%) of aqueous extracts from terrestrial plants, cyanobacteria, and marine invertebrates and algae has exhibited activity in the National Cancer Institute's primary AIDS-antiviral screen. By removal of anionic polysaccharides in a first stage of dereplication, we have eliminated from further consideration a considerable number of these extracts. However, a still substantial proportion of the active extracts remained, from which we wished to select and prioritize a small percentage for our detailed bioassay-directed fractionation studies. Therefore, a chemical screening protocol, utilizing various solid-phase extraction cartridges, has been developed for a second-stage dereplication and to assist in prioritization of these extracts for our further investigations.

A nonpromoting phorbol from the samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1.

Extracts of Homalanthus nutans, a plant used in Samoan herbal medicine, exhibited potent activity in an in vitro, tetrazolium-based assay which detects the inhibition of the cytopathic effects of human immunodeficiency virus (HIV-1). The active constituent was identified as prostratin, a relatively polar 12-deoxyphorbol ester. Noncytotoxic concentrations of prostratin from greater than or equal to 0.1 to greater than 25 microM protected T-lymphoblastoid CEM-SS and C-8166 cells from the killing effects of HIV-1. Cytoprotective concentrations of prostratin greater than or equal to 1 microM essentially stopped virus reproduction in these cell lines, as well as in the human monocytic cell line U937 and in freshly isolated human monocyte/macrophage cultures. Prostratin bound to and activated protein kinase C in vitro in CEM-SS cells and elicited other biochemical effects typical of phorbol esters in C3H10T1/2 cells; however, the compound does not appear to be a tumor promoter. In skin of CD-1 mice, high doses of prostratin induced ornithine decarboxylase only to 25-30% of the levels induced by typical phorbol esters at doses 1/30 or less than that used for prostratin, produced kinetics of edema formation characteristic of the nonpromoting 12-deoxyphorbol 13-phenylacetate, and failed to induce the acute or chronic hyperplasias typically caused by tumor-promoting phorbols at doses of 1/100 or less than that used for prostratin.