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Metabolomics can be used to study complex mixtures of natural products, or secondary metabolites, for many different purposes. One productive application of metabolomics that has emerged in recent years is the guiding direction for isolating molecules with structural novelty through analysis of untargeted LC-MS/MS data. The metabolomics-driven investigation and bioassay-guided fractionation of a biomass assemblage from the South China Sea dominated by a marine filamentous cyanobacteria, cf. Neolyngbya sp., has led to the discovery of a natural product in this study, wenchangamide A (1). Wenchangamide A was found to concentration-dependently cause fast-onset apoptosis in HCT116 human colon cancer cells in vitro (24 h IC50 = 38 μM). Untargeted metabolomics, by way of MS/MS molecular networking, was used further to generate a structural proposal for a new natural product analogue of 1, here coined wenchangamide B, which was present in the organic extract and bioactive sub-fractions of the biomass examined. The wenchangamides are of interest for anticancer drug discovery, and the characterization of these molecules will facilitate the future discovery of related natural products and development of synthetic analogues.

Kyanamide (1), a new depsipeptide containing 3-amino-6-hydroxy-2-piperidone moiety, was isolated from the Caldora penicillata marine cyanobacterium collected in Okinawa. Its structure was determined by spectroscopic analysis and Marfey's analysis of acid hydrolysate. Kyanamide exhibited moderate cytotoxicity against HeLa S3 cells. 1 also exhibited potent protease inhibitory activity against elastase and chymotrypsin with IC50 values of 0.13 nM and 1.1 μM.

Minnamide A is a lipopeptide with a unique repeating structure consisting of hydroxy and proposed β-branched methyl groups. The absolute configuration of minnamide A was determined by a combination of chemical degradation, chiral HPLC analyses, and synthetic methods. Minnamide A showed growth-inhibitory activity toward HeLa cells with an IC50 value of 0.17 μM and rapidly induced cell death at a concentration of 2 μM. Minnamide A induced the copper-mediated accumulation of reactive oxygen species.

Croissamide, a proline-rich cyclic peptide that contains an N-prenylated tryptophan, was isolated from a marine cyanobacterium Symploca sp. Its gross structure was determined by spectroscopic analyses, and the absolute configuration was established based on chiral HPLC analyses of acid hydrolysates.

Biselyngbyaside, an 18-membered macrolide glycoside from marine cyanobacteria, and its derivatives are known to be sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Recently, a SERCA orthologue of the malaria parasite, PfATP6, has attracted attention as a malarial drug target. To provide a novel drug lead, we designed new synthetic analogs of biselyngbyolide B, the aglycone of biselyngbyaside, based on the co-crystal structure of SERCA with biselyngbyolide B, and synthesized them using the established synthetic route for biselyngbyolide B. Their biological activities against malarial parasites were evaluated.

Lingaoamide (1), a new cyclic heptapeptide natural product, was isolated from a freshwater Oscillatoria cyanobacterium. The producing organism was collected in Chinese water spinach paddy farm fields in Lingao County, Hainan, China. The structure of 1 was determined by conventional mass spectrometry, NMR spectroscopy, and chemical degradation studies. Lingaoamide concentration-dependently up-regulates the biogenesis of pro-pathogenic melanin pigments (melanogenesis) in the rice blast fungus, Magnaporthe grisea, when tested in vitro. This molecule also exhibits weak in vitro inhibition (MIC 32 μg/mL) of Gram-negative Pseudomonas aeruginosa and Gram-positive Bacillus pumilus, and does not appear to impact plant growth or immune response in vivo using the model organism, Arabidopsis thaliana.

Biseokeaniamides A, B, and C (1-3), structurally novel sterol O-acyltransferase (SOAT) inhibitors, were isolated from an Okeania sp. marine cyanobacterium. Their structures were elucidated by spectroscopic analyses and degradation reactions. Biseokeaniamide B (2) exhibited moderate cytotoxicity against human HeLa cancer cells, and compounds 1-3 inhibited both SOAT1 and SOAT2, not only at an enzyme level but also at a cellular level. Biseokeaniamides (1-3) are the first linear lipopeptides that have been shown to exhibit SOAT-inhibitory activity.

The first total synthesis of janadolide, a new cyclic polyketide-peptide hybrid possessing a tert-butyl group, is described. The synthesis of an unsaturated hydroxycarboxylic acid was effected via the lithiation of vinyl iodide followed by addition to a Weinreb amide with a tert-butyl group and stereoselective 1,2-reduction. The cyclic structure was constructed by macrolactamization at the amide bond between the proline moiety and fatty acid moiety.

A new polyketide with a cis-fused decalin ring scaffold, caldorin, was isolated from the marine cyanobacterium Caldora penicillata. The gross structure and relative configuration were elucidated by spectroscopic analyses. We also clarified that caldorin is a weak SOAT inhibitor and moderate osteoblast differentiation inhibitor. On the other hand, caldorin did not exhibit cytotoxicity against either HeLa or HL60 cells.

The linear lipopeptides komesuamide (1) and odopenicillatamide (2) were isolated from Caldora penicillata a marine cyanobacterium collected in Okinawa. The structures of these compounds were established by spectroscopic analyses, and the absolute configurations were determined by HPLC analyses of the acid hydrolysates. Both compounds showed glucose uptake activity at 40 μM in cultured L6 myotubes.

In 2014, the γ-pyrone-containing polyketide, yoshinone A, was isolated from the marine cyanobacterium Leptolyngbya sp. and its structure was determined. Yoshinone A inhibited differentiation of 3T3-L1 cells into adipocytes, with an EC50 value of 420 nM without any cytotoxicity, and therefore is expected to be a lead compound for obesity drugs. To establish its absolute configuration, and to provide sufficient amounts for further research, the total synthesis of yoshinone A was achieved through synthesis of its two possible diastereomers.

Kanamienamide, an enamide with an enol ether, was isolated from the marine cyanobacterium Moorea bouillonii. The gross structure was established by spectroscopic analyses, and the relative stereochemistry was elucidated on the basis of the analyses of NOESY correlations and 1H–1H coupling constants. The absolute configuration was determined on the basis of the chiral HPLC analysis of the N-Me-Leu derived from kanamienamide. This is the first report of a natural product that possesses an N-Me-enamide adjacent to an enol ether. Kanamienamide showed growth-inhibitory activity toward HeLa cells with an IC50 value of 2.5 μM and induced apoptosis-like cell death.

Urumamide, a novel cyclic depsipeptide that contains a β-amino acid, was isolated from a marine cyanobacterium Okeania sp. Its gross structure was determined by spectroscopic analyses, and the absolute configuration was established based on Marfey’s analyses and chiral HPLC analyses of hydrolysis products. Biologically, urumamide inhibited the growth of human cancer cells. In addition, urumamide inhibited chymotrypsin.

Aglaia odorata Lour. (Meliaceae) was found to have very strong allelopathic activity and a bioherbicide PORGANIC(™) was developed from its leaf extracts. However, the phytotoxic substances causing the strong allelopathic activity of the plants have not yet been determined. Therefore, we investigated allelopathic properties and phytotoxic substances in A. odorata. Aqueous EtOH extracts of A. odorata leaves inhibited root and shoot growth of garden cress (Lepidum sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), timothy (Phleum pratense), ryegrass (Lolium multiflorum), and Echinochloa crus-galli with the extract concentration-dependent manner. The extracts were then purified and a major phytotoxic substance with allelopathic activity was isolated and identified by spectral data as rocaglaol. Rocaglaol inhibited the growth of garden cress and E. crus-galli at concentrations > 0.3 and 0.03 μm, respectively. The concentrations required for 50% inhibition ranged from 0.09 to 2.5 μm. The inhibitory activity of rocaglaol on the weed species, E. crus-galli, was much greater than that of abscisic acid. These results suggest that rocaglaol may be a major contributor to the allelopathic effect of A. odorata and bioherbicide PORGANIC(™) .

Marine cyanobacteria are known as important creators of novel natural products. From this valuable source, various bioactive compounds have been found and characterized in terms of their pharmacological and toxicological activities.1 In the previous work, we have reported on the isolation and structure determination of potent cytotoxic compounds, lyngbyacyclamide A and B;2 an inhibitor of osteoclastogenesis, biselyngbyaside;3 and a protein kinase inhibitor, bisebromoamide.4 In the recent work, we have reported the new marine γ-pyrones yoshinone A, B1 and B2 from Leptolyngbya sp., and determined their planar structures using NMR spectral analysis.5 Yoshinone A, as the major compound among them, showed inhibitory activity against the adipogenic differentiation of 3T3-L1 cells with an half maximal inhibitory concentration (IC50) value of 420 nm without cytotoxicity (IC50>50 μm). On the other hand, the yoshinone B1 and B2 showed only limited activity against 3T3-L1 cells, with higher concentrations compared with yoshinone A. Further studies of the structure–activity relationship lead us to conclude that the position of a pyrone ring and an olefin in the side chain will be important for the inhibition of adipogenic differentiation. These γ-pyrones have olefins in their side chain at positions 7 and 6 in the cases of yoshinones A and B1/B2, respectively. To express the effects on adipocyte, the olefin should not be conjugated with γ-pyrone moiety, such as yoshinone A (Figure 1). In the previous studies, kalkipyrone6 isolated from cyanobacteria, aureothin,7 and actinopyrones A and B8 isolated from streptomyces fell into the same 7-en γ-pyrones. Then, we confirmed that kalkipyrone and aureothin showed this activity, with IC50 values of 67.5 and 54.2 nm, respectively. On the basis of these data, we are focusing on the 7-en γ-pyrone (unconjugated type) compounds. These pyrones are expected to be candidates for novel lead compounds for the treatment of obesity and related diseases.9 Studies on useful tools that regulate adipocytes will contribute to the prevention and treatment of these diseases. At the present stage of our research, we have evaluated the anti-obesity activities of the 7-en γ-pyrones using in vitro and in vivo experiments. In this study, we report on the interesting properties of these pyrones.

Aplog-1 is a simplified analog of debromoaplysiatoxin (DAT) with potent tumor-promoting and proinflammatory activities. Aplog-1 and DAT exhibited anti-proliferative activities against several human cancer cell lines, whereas aplog-1 did not have tumor-promoting nor proinflammatory activities. We have recently found 10-methyl-aplog-1 (1) to have strong anti-proliferative activity compared with aplog-1. To further investigate the structural factors involved in the tumor-promoting, proinflammatory, and anti-proliferative activities, two dimethyl derivatives of aplog-1 (2, 3) were synthesized, where two methyl groups were installed at positions 4 and 10 or 10 and 12. 10,12-Dimethyl-aplog-1 (2) had stronger inhibitory effects on the growth of several human cancer cell lines than 1 and DAT, but exhibited no tumor-promoting and proinflammatory activities. In contrast, 4,10-dimethyl-aplog-1 (3) displayed weak tumor-promoting and proinflammatory activities along with anti-proliferative activity similar to that of 1 and DAT. Compound 2 would be the optimized seed for anticancer drugs among the simplified analogs of DAT.

Wedelia chinensis has been widely used as a traditional medicine and several bioactive compounds have been isolated. We investigated phytotoxic property and phytotoxic substances in the species. An aqueous methanol extract of W. chinensis inhibited the growth of roots and shoots of cress. The extracts were then purified by several chromatographic runs with monitoring the inhibitory activity and a phytotoxic substance was isolated. The substance was determined by spectral data to be a novel kaurene-type compound, wedelienone. Wedelienone inhibited the growth of cress and timothy (Phleum pratense L) at concentrations greater than 10–30 μM. These results suggest that wedelienone may contribute to the phytotoxic effects caused by the extracts.

Two new jahanyne analogues, jahanene and jahanane, highly N-methylated lipopeptides, were isolated from a marine cyanobacterium Okeania sp., and their structures were determined by NMR and MS. In addition, we achieved total syntheses of the jahanyne family and assessed their activities. The resulting growth-inhibitory activity of jahanyne was nearly one-tenth of the previously reported activity. Furthermore, we found that the degree of unsaturation at the terminus of the fatty acid moiety affected the growth-inhibitory activity against human cancer cells.

Two new pyrrolinone-containing lipopeptides, ypaoamides B (1) and C (2), were isolated from an Okeania sp. marine cyanobacterium collected in Okinawa. Their structures were determined by spectroscopic analysis and Marfey’s analysis of acid hydrolysates. Ypaoamides B (1) and C (2) stimulated glucose uptake in cultured rat L6 myotubes. In particular, ypaoamide B (1) showed potent activity and activated AMP-activated protein kinase.

In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts.

Biselyngbyasides (BLSs), macrolides from a marine cyanobacterium, are cytotoxic natural products whose target molecule is unknown. Here we report that BLSs are high affinity (Ki∼10nM) inhibitors of Ca2+-pumps with a unique binding mode. The crystal structures of the Ca2+-pump in complex with BLSs at 3.2–3.5Å-resolution show that BLSs bind to the pump near the cytoplasmic surface of the transmembrane region. The crystal structures and activity measurement of BLS analogs allow us to identify the structural features that confer high potency to BLSs as inhibitors of the pump.

Mebamamides A and B, new lipopeptides with four d-amino acid residues and a 3,8-dihydroxy-9-methyldecanoic acid residue, were isolated from the green alga Derbesia marina. Their gross structures were elucidated by spectroscopic and ESI-ITMS analyses. The absolute configurations except for the two leucines were revealed based on chiral-phase HPLC analyses of the acid hydrolysate and a modified Mosher's method. A distinction between D-Leu and L-Leu in the sequence was established by the application of a dansyl-Edman method to the partial acid hydrolysate. Mebamamide A did not exhibit any growth inhibitory activity against HeLa and HL60 cells at 10 μM, and mebamamide B did not exhibit any growth inhibitory activity against those cells at 100 μM. Additionally, it was suggested that mebamamide B induced the differentiation of HL60 cells into macrophage-like cells at 100 μM.

An acetylene-containing lipopeptide, jahanyne, was isolated from the marine cyanobacterium Lyngbya sp. Its gross structure was established by spectroscopic analyses, and the absolute configuration was clarified based on a combination of chiral HPLC analyses, spectroscopic analyses, and derivatization reactions. Jahanyne significantly inhibited the growth of human cancer cells and induced apoptosis in HeLa cells.

The first total synthesis of koshikalide, a 14-membered macrolide that contains three olefins, was achieved. The skipped diene in the cyclic system was efficiently constructed by very mild Stille coupling at low temperature. The absolute stereochemistry was established by comparison of the specific optical rotations of natural and synthesized koshikalide.

The first total synthesis of biselyngbyolide B, an 18-membered macrolide, was achieved. The 18-membered ring structure was constructed by esterification using the Shiina reagent and an intramolecular Stille coupling reaction.

Three new compounds of the malyngamide series, 6,8-di-O-acetylmalyngamide 2 (1), 6-O-acetylmalyngamide 2 (2), and N-demethyl-isomalyngamide I (3), were isolated from the marine cyanobacterium Moorea producens. Their structures were determined by spectroscopic analysis and chemical derivatization and degradation. These compounds stimulated glucose uptake in cultured L6 myotubes. In particular, 6,8-di-O-acetylmalyngamide 2 (1) showed potent activity and activated adenosine monophosphate-activated protein kinase (AMPK).

Ninety samples from the extracts of plants from traditional Chinese medicines were screened for antitumor activity. Paeoniflorigenone (PFG) was isolated as an active ingredient from the root of moutan cortex, which showed the strongest activity. In addition, our data indicated that PFG was cytotoxic and induced apoptosis selectively in the cancer cell lines. These effects were cancelled by the addition of caspase inhibitor Z-VAD-FMK, suggesting that it was mediated by caspase-3 activation.

In the search for new antiprotozoal substances, hoshinolactam, an antitrypanosomal lactam, was isolated from a marine cyanobacterium. The gross structure was elucidated by spectroscopic analyses, and the absolute configuration was determined by the first total synthesis. Hoshinolactam showed potent antitrypanosomal activity with an IC50 value of 3.9 nM without cytotoxicity against human fetal lung fibroblast MRC-5 cells (IC50 > 25 μM).

Three novel compounds, yoshinones A, B1, and B2, were isolated from the marine cyanobacterium Leptolyngbya sp., and their structures were elucidated by NMR spectral analysis. Yoshinone A, but not yoshinone B1 or B2, inhibited the differentiation of 3T3-L1 cells into adipocytes. In addition, yoshinone A did not exhibit cytotoxicity, suggesting that yoshinone A may be useful in studies on the treatment of obesity.

The first total synthesis of maedamide, an acyclic peptide isolated from a marine cyanobacterial assemblage of Lyngbya sp., was achieved. This synthesis led to reassignment of the allo - d -Ile of maedamide to be l -Ile, which was supported by 1 H and 13 C NMR data.

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S(3) cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3-6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3-6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.

Bioassay-guided fractionation of the marine cyanobacterium Lyngbya sp. led to the isolation of biselyngbyasides B ( 3 ), C ( 4 ), and D ( 5 ), novel analogs of biselyngbyaside ( 1 ) and biselyngbyolide A ( 2 ). The gross structures of 3 – 5 were determined by NMR spectral analyses, and their stereochemistries were established based on NOESY spectra and CD data. Biselyngbyasides ( 1 – 3 ) showed growth-inhibitory activity and apoptosis-inducing activity against both HeLa S 3 cells and HL60 cells. The fura-2 method revealed that biselyngbyasides ( 1 – 3 ) increased the cytosolic Ca 2+ concentration in HeLa S 3 cells.

The mass and function of bones depend on the maintenance of a complicated balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An inhibitor of osteoclast differentiation and/or function is expected to be useful for treatment of bone lytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis into bone. Biselyngbyaside is a recently isolated macrolide compound from marine cyanobacteria Lyngbya sp. that shows wide-spectrum cytotoxicity toward human tumor cell lines. In this study, we investigated the effects of biselyngbyaside on osteoclast differentiation and function. Biselyngbyaside inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocytic RAW264 cells and primary bone marrow-derived macrophages at a low concentration. Similarly, biselyngbyaside suppressed osteoblastic cell-mediated osteoclast differentiation in cocultures. In the RANKL-induced signaling pathway, biselyngbyaside inhibited the expression of c-Fos and NFATc1, which are important transcription factors in osteoclast differentiation. In mature osteoclasts, biselyngbyaside decreased resorption-pit formation. Biselyngbyaside also induced apoptosis accompanied by the induction of caspase-3 activation and nuclear condensation, and these effects were negated by the pancaspase inhibitor z-VAD-FMK. Taken together, the present findings indicate that biselyngbyaside suppresses bone resorption via inhibition of osteoclastogenesis and induction of apoptosis. Thus, biselyngbyaside may be useful for the prevention of bone lytic diseases.

Five cytotoxic macrolides, aplyronines D–H (4–8), were isolated from the Japanese sea hare Aplysia kurodai. They are new congeners of the antitumor compound aplyronine A (1), which was previously isolated from the same organism. Their structures were determined by spectroscopic analysis (NMR and MS). The cytotoxicity of these new compounds was evaluated in comparison with that of aplyronines A–C (1–3), suggesting the importance of the 7-O-seryl ester group for mediating the potent cytotoxicity of aplyronines.

Novel potent cytotoxic peptides bisebromoamide (1) and norbisebromoamide (2) have been isolated from the marine cyanobacterium Lyngbya sp. The planar structure of these peptides was elucidated through the extensive application of 1D and 2D NMR techniques. The absolute stereostructure of 1 was determined by chemical degradation followed by chiral HPLC analysis. Recently, Tao and co-workers achieved synthesis of bisebromoamide, and the configuration of thiazoline moiety was revised. We re-investigated the stereochemistry of thiazoline moiety of 1. The structure–activity relationships of bisebromoamide (1) were investigated with the use of natural and synthetic analogs. Furthermore, bisebromoamide (1) potently inhibited protein kinase: the phosphorylation of ERK in NRK cells by PDGF-stimulation was selectively inhibited by treatment with 10–0.1 μM of 1.

N,N′ -diphenethylurea isolated from Okinawan ascidian Didemnum molle enhances adipocyte differentiation in 3T3-L1 cells

Maedamide, a novel chymotrypsin-inhibiting depsipeptide, was isolated from a cyanobacterial assemblage that mostly consisted of Lyngbya sp. Its structure was elucidated by spectroscopic analyses and chiral HPLC analyses of hydrolysis products. Maedamide selectively inhibited chymotrypsin but not elastase and trypsin. In addition, Maedamide strongly inhibited the growth of HeLa cells and HL60 cells.

A novel cytotoxic peptide, termed bisebromoamide (1), has been isolated from the marine cyanobacterium Lyngbya sp. Its planar structure was determined by 1D and 2D NMR spectroscopy. The absolute stereostructure of 1 was determined by chemical degradation followed by chiral HPLC analysis. Bisebromoamide (1) exhibited potent protein kinase inhibition: the phosphorylation of ERK in NRK cells by PDGF-stimulation was selectively inhibited by treatment with 10-0.1 microM of 1.

Covering: up to 2007 Aplyronines A–H are cytotoxic macrolides isolated from the sea hare Aplysia kurodai. Aplyronine A is the major constituent among the aplyronines, and this review concentrates on the results of chemical and biological research into this natural product. The isolation, determination of stereostructure and enantioselective total synthesis of the aplyronines are covered, together with discussion of their antitumour activity and structure–activity relationships, and the three-dimensional X-ray structure of the actin–aplyronine A complex.

Halichonine B is a sesquiterpene alkaloid isolated from the marine sponge Halichondria okadai Kadota. Halichonine B has exhibited cytotoxicity against mammalian cancer cells and induced apoptosis in the human leukemia cell line HL60. Here we established a practical route for the synthesis of halichonine B and its analogues, and we evaluated their biological activities. It was revealed that the secondary amino groups in the side chain portion are important for the strong cytotoxicity of halichonine B and that the N(11)-prenyl group is unimportant. Halichonine B and its analogues were also observed to induce apoptosis in HL60 cells.

Mycalolide analog 4, consisting only of the side chain of mycalolide B (2), a trisoxazole macrolide of marine origin, was stereoselectively synthesized using Roush crotylboration, an Evans aldol reaction, and a Paterson aldol reaction as key steps. The analog 4 was found to have strong actin-depolymerizing activity.

Three saponins, isotheasaponins B1-B3, were isolated from the leaves of the tea plant Camellia sinensis var. sinensis, and their structures were determined to be theasapogenol B [beta-D-galactopyranosyl(1-->2)][beta-D-xylopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->3)]-beta-D-gulcopyranosiduronic acid with two acyl groups by spectroscopic analysis.

The interaction between actin and aplyronine A, a potent antitumor and actin-depolymerizing substance of marine origin, was investigated by photoaffinity labeling experiments. Photoaffinity probes consisting of a side-chain portion of aplyronine A as a ligand, a diazirine moiety as a photoaffinity group, and a fluorophore as a detecting group were synthesized. Photolabeling experiments between actin and the probe were carried out. Actin was successfully photolabeled by the fluorescent probe and visualized clearly. The present results provide the first chemical evidence for the direct interaction between actin and the side-chain portion of aplyronine A.