Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells.

Two toxins, latrunculins A and B, which contain a new class of 16- and 14-membered marine macrolides attached to the rare 2-thiazolidinone moiety, were purified recently from the Red Sea sponge Latrunculia magnifica. The effects of these toxins on cultured mouse neuroblastoma and fibroblast cells have been evaluated. In both types of cells, submicromolar toxin concentrations rapidly induce striking changes in cell morphology that are reversible upon removal of the toxin. Immunofluorescence studies with antibodies specific for cytoskeletal proteins reveal that the toxins cause major alterations in the organization of microfilaments without obvious effects on the organization of the microtubular system.

Crystallization and X-ray Analysis of Stemphyloxin I, a Phytotoxin from Stemphylium botryosum.

Certain isolates of the plant-pathogenic fungus Stemphylium botryosum produce a phytotoxin, stemphyloxin I. This toxin (C(21)H(34)O(6)) was crystallized and its structure was determined by x-ray crystallography to be a beta-ketoaldehyde trans-Decalin. This compound is a highly unusual natural product. Iron (Fe(3+)) controls production of toxin by this fungus. Furthermore, iron reacts with the toxin to yield a colored product which aids in its detection on chromatograms and in its quantitative estimation by colorimetry.;

Effects of natural and novel synthetic jasmonates in experimental metastatic melanoma.

BACKGROUND AND PURPOSE: No current treatment reliably affects the course of metastatic melanoma. Consequently, novel approaches to the control of metastasis are actively sought. The overall goal of the present study was to identify new anti-metastatic agents active against melanoma cells. EXPERIMENTAL APPROACH: Two directions were taken: 1. To determine whether the natural plant hormone methyl jasmonate, which kills cancer cells selectively, can suppress the characteristic metastatic behavior of B16-F10 melanoma cells; 2. To synthesize and identify novel jasmonate derivatives with better cytotoxic and anti-metastatic activities than methyl jasmonate. KEY RESULTS: We found that methyl jasmonate suppressed B16-F10 cell motility and inhibited the development of experimental lung metastases of these cells. Furthermore, methyl jasmonate suppressed the motility of a sub-clone of these cells over-expressing P-glycoprotein and exhibiting multidrug resistance. The synthetic derivative Compound I (5,7,9,10-tetrabromo derivative of methyl jasmonate, the most active derivative) had greater cytotoxic potency (IC(50), 0.04 mM) than methyl jasmonate (IC(50), 2.6mM). Compound I prevented B16-F10 cell adhesion efficiently and inhibited the development of lung metastases at a much lower dose than methyl jasmonate. CONCLUSIONS AND IMPLICATIONS: Natural and synthetic jasmonates have anti-metastatic actions. Further development of these agents for the suppression of metastasis in melanoma and other types of cancer is warranted.

Characterization of non-dialyzable constituents from cranberry juice that inhibit adhesion, co-aggregation and biofilm formation by oral bacteria.

An extract prepared from cranberry juice by dialysis known as nondialyzable material (NDM) has been shown previously to possess anti-adhesion activity toward microbial species including oral bacteria, uropathogenic Escherichia coli and Helicobacter pylori. Bioassay-guided fractionation of cranberry NDM was therefore undertaken to identify the anti-adhesive constituents. An aqueous acetone-soluble fraction (NDMac) obtained from Sephadex LH-20 inhibited adhesion-linked activities by oral bacteria, including co-aggregation of oral bacteria Fusobacterium nucleatum with Streptococcus sanguinis or Porphyromonas gingivalis, and biofilm formation by Streptococcus mutans. Analysis of NDMac and subsequent subfractions by MALDI-TOF MS and 1H NMR revealed the presence of A-type proanthocyanidin oligomers (PACs) of 3-6 degrees of polymerization composed of (epi)catechin units, with some (epi)gallocatechin and anthocyanin units also present, as well as quercetin derivatives. Subfractions containing putative xyloglucans in addition to the mixed polyphenols also inhibit biofilm formation by S. mutans (MIC = 125-250 µg mL-1). These studies suggest that the anti-adhesion activities of cranberry NDM on oral bacteria may arise from a combination of mixed polyphenol and non-polyphenol constituents.

Sensitivity of hematopoietic progenitors of acute myeloblastic leukemia to new compounds derived from marine organisms.

Results of chemotherapy in acute myeloid leukemia (AML) have improved slowly or not at all in the last decade. We evaluated the effect of Eilatin and Norsegoline, two new aromatic alkaloids derived from the Red Sea purple tunicate Eudistoma sp., on in vitro proliferation and differentiation of leukemic cell lines and blast cells of three AML patients. These biological properties were studied in two complementary culture methods. The first is a clonogenic assay that supports colony formation in agar and reflects terminal divisions. The second is a suspension assay where clonogenic cells increase exponentially and reflects self-renewal. Eilatin and Norsegoline, at micromolar concentrations, suppressed, in a dose-dependent manner, both primary colony formation in agar and the recovery of clonogenic cells from suspension culture in the investigated cell lines and in fresh blasts. Furthermore, both alkaloids were more effective in inhibiting clonogenic cells grown in suspension than primary colonies grown in agar. In addition, these agents were able to induce immunophenotypic maturation of leukemic cell lines (upregulation of CD14 and CD11 and down-regulation of CD34 antigens). Our results indicate that Eilatin and Norsegoline significantly inhibit self-renewal capacity of leukemic progenitors and may provide a useful new tool for the treatment of AML patients.

Potent antileukemic activity of the novel agents norsegoline and dibezine.

We examined the effect of norsegoline, a natural marine product, and dibezine, a synthetic product, on the survival of human myeloid progenitor cells [colony-forming unit-cells (CFU-C)] from normal individuals and from 10 patients with Philadelphia-positive chronic myelogenous leukemia (CML) in chronic phase and blastic crisis. We compared their effect to the effect of IFN-alpha. Norsegoline, dibezine, and IFN-alpha inhibited the proliferation of CFU-C in a dose-dependent manner. The number of CFU-C from bone marrow (BM) of five CML patients in chronic phase exposed for 16 h to norsegoline (10(-8)-10(-6)M), dibezine (10(-8)-10(-6)M), and IFN-alpha (500 units/ml) was found to be statistically lower (P < 0.05) than the number of CFU-C derived from normal individuals. A 16-h drug exposure of CD34(+) cells isolated from the peripheral blood of three CML patients in blastic crisis and from BM of two patients in chronic phase resulted in a marked inhibition in the ability of the cells to proliferate in liquid culture and a reduction in CFU-C content. Using the fluorescent in situ hybridization technique, we evaluated detection of the BCR/ABL fusion product in the CD34(+) cells. All five patients were 100% Philadelphia positive at diagnosis. BCR/ABL translocations were detected in 94.6 +/- 0.6% of cells following their growth in liquid culture for 7 days. Following exposure of CD34(+) cells to norsegoline, dibezine, or IFN-alpha, BCR/ABL fusion signals could be detected in 73 +/- 11%, 66.5 +/- 4. 7%, and 66.0 +/- 2.5% of cells from BM and 72.3 +/- 5%, 68.8 +/- 7%, and 60.6 +/- 6.8% of peripheral blood, respectively. Our data indicate that norsegoline and dibezine have in vitro an antileukemic effect against Philadelphia-positive cells and may be used in conjunction with currently available agents for ex vivo purging of BM and/or peripheral blood of CML patients in conjunction with autologous bone marrow transplantation.

Growth modulation and differentiation of acute myeloid leukemia cells by jaspamide.

We have examined the effect of Jaspamide, a peptide isolated from the marine sponge Hemiastrella minor, on in vitro proliferation and differentiation of leukemic cell lines and blast cells of three AML patients and compared it to that of cytosine arabinoside (ARA-C). The biological properties were studied in two complementary culture methods. The first is a clonogenic assay that supports colony formation in agar and reflects terminal divisions. The second is a suspension assay in which clonogenic cells increase exponentially and which reflects self-renewal. Jaspamide, at micromolar concentrations and in a dose-dependent manner, suppressed both primary colony formation in agar and the recovery of clonogenic cells from suspension culture in the investigated cell lines and in fresh blasts. Furthermore, Jaspamide was more effective in inhibiting clonogenic cells grown in suspension than primary colonies grown in agar. In addition, Jaspamide, similarly to ARA-C, was able to induce immunophenotypic maturation of leukemic cell lines (upregulation of CD14 and CD11 and downregulation of CD34 antigens). Our results indicate that Jaspamide significantly inhibits the self-renewal capacity of leukemic progenitors and may provide a new useful tool for the treatment of acute myeloid leukemia (AML) patients.

Eilatin: a novel marine alkaloid inhibits in vitro proliferation of progenitor cells in chronic myeloid leukemia patients.

We examined the effect of Eilatin, a novel marine product, on the survival of human myeloid progenitor cells (CFU-C) isolated from normal individuals and from 12 patients with Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) in chronic phase and blastic crisis. We compared its effect to the effect of interferon-alpha (IFN-alpha) and cytosine arabinoside (Ara-C). Eilatin, IFN-alpha, and Ara-C inhibited the proliferation of CFU-C from normal individuals and CML patients in a dose-dependent manner. The percent survival of colony-forming units from bone marrow (BM) of seven CML patients in chronic phase exposed for 16 hours to Eilatin (10(-7) and 10(-6) M), IFN-alpha (500 U/mL), or Ara-C (10(-9) M and 10(-8) M) was found to be statistically lower (p < 0.05) than the percent survival of myeloid progenitors from normal individuals. A 16-hour exposure of CD34+ cells isolated from peripheral blood (PB) of three CML patients in blastic crisis and from BM of two patients in chronic phase to Eilatin 10(-7) M, IFN-alpha 500 U/mL, Ara-C 10(-9) M resulted in a marked inhibition in the ability of the cells to proliferate in liquid culture and a reduction in CFU-C content. Using fluorescent in situ hybridization (FISH), we evaluated detection of the BCR/ABL fusion product in the CD34+ cells. All five patients were 100% Ph+ at diagnosis. BCR/ABL translocations were detected in 94.6 +/- 0.6% of CD34+ cells after growth in liquid culture for 7 days. The level of BCR/ABL fusion signals detected after exposure of CD34+ cells for 16 hours to Eilatin 10(-7) M, IFN-alpha 500 U/mL, or Ara-C 10(-9) M were 54.5 +/- 5%, 63.6 +/- 5%, and 70 +/- 4%, respectively (mean +/- SE, n = 5). Our data indicate that Eilatin, a substance isolated from the Red Sea purple tunicate Eudistoma sp., has an antileukemic effect against in vitro Ph+ cells and may be used in conjunction with currently available agents for ex vivo purging of BM and/or PB of CML patients in conjunction with autologous bone marrow transplantation.

Salarin C inhibits the maintenance of chronic myeloid leukemia progenitor cells.

We previously showed that incubation of chronic myeloid leukemia (CML) cells in very low oxygen selects a cell subset where the oncogenetic BCR/Abl protein is suppressed and which is thereby refractory to tyrosine kinase inhibitors used for CML therapy. In this study, salarin C, an anticancer macrolide extracted from the Fascaplysinopsis sponge, was tested as for its activity on CML cells, especially after their incubation in atmosphere at 0.1% oxygen. Salarin C induced mitotic cycle arrest, apoptosis and DNA damage. Salarin C also concentration-dependently inhibited the maintenance of stem cell potential in cultures in low oxygen of either CML cell lines or primary cells. Surprisingly, the drug also concentration-dependently enforced the maintenance of BCR/Abl signaling in low oxygen, an effect which was paralleled by the rescue of sensitivity of stem cell potential to IM. These results suggest a potential use of salarin C for the suppression of CML cells refractory to tyrosine kinase inhibitors.

Structure-activity relationship in a new series of atropine analogues. 1. N,N'-Disubstituted 6,7-diazabicyclo[3.2.2]nonane derivatives.

The synthesis of a new series of N,N'-disubstituted 6,7-diazabicyclo[3.2.2]nonane derivatives is described. The antimuscarinic potency of these drugs was evaluated in the guinea pig ileum and compared to that of atropine sulfate. All the drugs tested competitively inhibited the acetylcholine-induced contractions. Kd values were calculated and, in several cases, compared to those obtained by direct binding to the muscarinic receptor from mouse brain. The order of potencies followed that which is known for various tropine and pseudotropine esters; that is, the 3alpha configuration is more potent than the 3beta configuration, and the quaternary analogues are more potent than the tertiary ones. The antimuscarinic activity of the drugs is dicussed in terms of their acetylcholine-like molecular arrangement that gives rise to a characteristic interaction pharmacophore.

The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum.

Eight new coumarin compounds (1-8) were isolated by anti-HIV bioassay-guided fractionation of an extract of Calophyllum lanigerum. The structures of calanolide A (1), 12-acetoxycalanolide A (2), 12-methoxycalanolide A (3), calanolide B (4), 12-methoxycalanolide B (5), calanolide C (6) and related derivatives 7 and 8 were solved by extensive spectroscopic analyses, particularly HMQC, HMBC, and difference NOE NMR experiments. The absolute stereochemistry of calanolide A (1) and calanolide B (4) was established by a modified Mosher's method. Calanolides A (1) and B (4) were completely protective against HIV-1 replication and cytopathicity (EC50 values of 0.1 microM and 0.4 microM, respectively), but were inactive against HIV-2. Some of the related compounds also showed evidence of anti-HIV-1 activity. Studies with purified bacterial recombinant reverse transcriptases (RT) revealed that the calanolides are HIV-1 specific RT inhibitors. Moreover, calanolide A was active not only against the AZT-resistant G-9106 strain of HIV-1 but also against the pyridinone-resistant A17 strain. This was of particular interest since the A17 virus is highly resistant to previously known HIV-1 specific, non-nucleoside RT inhibitors (e.g., TIBO; BI-RG-587; L693,593) which comprise a structurally diverse but apparently common pharmacologic class. The calanolides represent a substantial departure from the known class and therefore provide a novel new anti-HIV chemotype for drug development.

Synergistic effects of interleukin-11 with other growth factors on the expansion of hematopoietic progenitors from normal individuals and chronic myeloid leukemia patients resistant to treatment with cytosine arabinoside or eilatin.

Interleukin-11 (IL-11) is a novel cytokine that has been shown to stimulate human hematopoietic progenitors including the CD34+ CD33- DR- early progenitors. IL-11 has little effect on its own but it synergizes with other hematopoietic growth factors. We investigated the recovery of human myeloid progenitors incubated with IL-11 alone or in combination with other cytokines, including stem cell factor (SCF), interleukin-3 (IL-3) and granulocyte macrophage colony-stimulating factor (GM-CSF) following their in vitro treatment with ARA-C (10(-9) M) or Eilatin (10(-7) M). IL-11 in combination with IL-3 and GM-CSF markedly increased CFU-C colony growth pre- and post-ARA-C or Eilatin incubation from CML and normal individual bone marrow (BM) cells. Similarly, IL-11 alone or in combination with other cytokines increased cell recovery following 7-day suspension culture. A decrease in BCR/ABL fusion product was observed (by FISH analysis) after incubation of BM cells from CML patients in liquid culture for 7 days with 10(-9) M ARA-C or 10(-7) M Eilatin in the presence of IL-11 alone or in combination with other cytokines. These results indicate that following cytoreductive therapy IL-11 may enhance to a greater extent the growth of normal myeloid progenitors than the malignant clone and may, therefore, be of clinical importance for CML patients treated with chemotherapeutic agents.

Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes.

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%

Bilosespens A and B: two novel cytotoxic sesterpenes from the marine sponge Dysidea cinerea.

[formula: see text] Two novel sesterpenes, bilosespens A and B (1 and 2) were isolated from the Red Sea sponge Dysidea cinerea collected in the Dahlak archipelago, Eritrea. The structure of the mixture of the two inseparable compounds was established by spectroscopic analysis, mainly by 1D and 2D NMR measurements. The mixture of bilosespens A and B is cytotoxic to a few human cancer cells.

Neurotoxicity of dipiperidinoethane due to in vivo conversion to a selective cholinesterase inhibitor.

Dipiperidinoethane (DPE) administration produces seizures and CNS lesions. Here we elucidate the cholinergic origin of DPE toxicity. DPE is both an acetylcholinesterase (AChE) inhibitor and a muscarinic antagonist. This dual action negates most of the toxic effects of the compound in vivo. The neurotoxicity is believed to arise from oxidative conversion to DPE-N-oxide, which selectively inhibits AChE. Cytotoxicity does not involve muscarinic neurons, since binding parameters were unchanged following in vivo exposure.

Synthesis of 18,19-dihydroxycorticosterone.

A four-step synthesis of 18,19-dihydroxycorticosterone 5c, starting with 19,21-dihydroxy-3,20-dioxopregn-5-ene-18,11 beta-lactone-di-(ethylene ketal) 2, is presented. Reduction of 2 with sodium aluminum bis-(methoxyethoxy)hydride gave 11 beta,18,19,21-tetrahydroxy-pregn-5-ene-3,20-dione-di-(ethylene ketal) 3a. Acetylation furnished the corresponding 18,19,21-triacetate 3b, which on treatment with a mixture of perchloric and acetic acids gave 18,19-dihydroxycorticosterone 18,19,21-triacetate 4b. Mild saponification yielded the title compound which, on the basis of ir and nmr spectra, exists as one C-20 isomer of the hemiacetal structure 5c. Periodate oxidation of 5c gave the expected 11 beta, 19-dihydroxy-3-oxoandrost-4-ene-17 beta, 18-carbolactone 6b.

18,21-Anhydroaldosterone and derivatives.

18,21-Anhydroaldosterone 8, 18,21-anhydro-19-noraldosterone 9, and 3 alpha, 5 beta-tetrahydro-18,21-anhydro-19-noraldosterone 13, which may be present in acid-processed urine, were prepared by cleaving their 20-ketal derivatives 2, 3, and 12 with hot mineral acid. Compounds 8 and 9 were also made by direct dehydration of aldosterone 5 and 19-noraldosterone 10 in good yield. The reverse ring opening of 8 to 5 could be carried out in moderate yield with an acetic acid-acetic anhydride-perchloric acid mixture, while an analogous ring opening of 9 gave only a poor yield of 10.

Synthesis of 18-hydroxy-19-norcorticosterone and 18-deoxy-19-noraldosterone. Structure determination of related 19-nor steroids by means of 2-D 1H nmr.

The compounds named in the title have been synthesized from the di-(ethylene ketal) of 21-hydroxy-3,20-dioxo-19-norpregn-5-ene-18, 11 beta-lactone and its 5(10)-ene isomer. Reduction of this mixture 1 with sodium aluminum bis-(methoxyethoxy)hydride furnished the 11 beta, 18, 21-triol 2a. Conversion to the 18,21-diacetate 2b, followed by deketalization to the free dione 3 and hydrolysis, afforded 18-hydroxy-19-norcorticosterone 4a which, in the solid state and probably in solution, has the 18,20-hemiacetal structure. Periodate oxidation of 4a gave 11 beta-hydroxy-3-oxo-19-norandrost-4-ene-17 beta, 18-carbolactone 5a, and acid treatment of 4a or its precursor 2a yielded 18-deoxy-19-noraldosterone 6a. The structure of 5a was confirmed by mass spectrometry and 1H nmr, and compared with that of its C-19 methyl homolog 5b and 19-noraldosterone-gamma-etiolactone 8. In particular, 2-D nmr COSY 45 experiments, affording full 1H line assignments, have rigorously established the "natural" beta (axial) configuration of the C-10 hydrogen in the 19-nor lactones 5a and 8, and therefore also in the related 4a, 6a and 19-noraldosterone 7.

Effects of latrunculin A on immunological phagocytosis and macrophage spreading-associated changes in the F-actin/G-actin content of the cells.

Latrunculin A, a toxin from a Red Sea sponge, was shown to be a very potent inhibitor of immunological phagocytosis by normal and activated macrophages (obtained from mice injected i.p. with LPS), as well as by polymorphonuclear leukocytes. This toxin blocks the interiorization of the immune complexes but does not interfere with their binding to the phagocyte (recognition phase); activated macrophages were more susceptible to this inhibition than normal macrophages and polymorphonuclear leukocytes. The effect of the toxin on cellular spreading of macrophages was also studied using two kinds of substrate: glass, and glass covered with IgG immune complexes. Latrunculin A was able to impair the spreading of normal macrophages on glass covered with immune complexes, and could also completely reverse the spreading after it had occurred. Contrarily, in activated macrophages, this toxin could neither impede the spreading nor reverse it, a difference that might be a distinctive property of the activated state. We have also found that latrunculin A can reduce the percentage of F-actin in both normal and activated macrophages, the activated cells being more susceptible to this effect. Since latrunculin A is a blocking agent of actin polymerization in vitro, these results indicate that actin polymerization and assembly must be an essential component of the primary, active event of the engulfment phase of phagocytosis.

Bebryazulene, a new guaiane metabolite from the indian ocean gorgonian coral, bebryce grandicalyx

Bebryazulene (1), a new sesquiterpene having the guaiane skeleton, was isolated from the methanol-chloroform 1:2 extract of the Indian Ocean gorgonian coral Bebryce grandicalyx (Kuekenthal) collected in the lagoon of Mayotte. Structure assignment was based on interpretation of spectroscopic data. This new metabolite was very labile and reacted with 4-phenyl-3H-1,2,4-triazoline-3,5-dione to yield an unexpected adduct.