2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

Anchietins A-E: 30-norfriedelane-type triterpenes from Anchietea pyrifolia.

Five undescribed norfriedelane triterpenoids, anchietins A-E, along with three known norfriedelane triterpenoids, 21ß-hydroxycaloncobalactone, and welwitschiilactones B and C, were isolated from the vine of Anchietia pyrifolia. The compounds were characterized by spectroscopic and crystallographic methods, including 2D NMR spectroscopy and single crystal X-ray crystallography. The isolated compounds were evaluated for the cytotoxic activity against HeLa and HL60 cells and for the inhibitory activity against lipopolysaccharide-induced NO production. Further, inhibitory effects on the inducible nitric oxide synthase mRNA expression levels were also assessed.

Chemical and Genetic Diversity of Ligularia kanaitzensis in the Hengduan Mountains Area. Chemical Relationship with L. subspicata.

Root chemicals and the sequences of the internal transcribed spacers (ITSs) were analyzed for 9 Ligularia kanaitzensis and 3 L. subspicata samples collected in northwestern Yunnan and southwestern Sichuan, China. Subspicatins A and C were isolated from two L. kanaitzensis samples. Introgression of genes responsible for these compounds from L. subspicata was suggested by their strong connection with L. subspicata/L. lamarum and the geographical proximity of the samples to L. subspicata. DNA analysis of a set of 27 L. kanaitzensis samples including those analyzed previously showed that they belong to two clades, designated A and B. Together with the presence/absence of furanoeremophilane, the 27 samples were sorted into three groups: clade A/furan, clade B/furan, and clade B/non-furan. The ancestral plant presumably belonged to clade B/non-furan, because furanoeremophilanes are biosynthesized from eremophilan-8-ones. 1ß-Angeloyloxyfukinone, a likely intermediate between fukinone and subspicatin C, was isolated for the first time. This finding allowed us to propose plausible biosynthetic pathways of subspicatins A and C.

Two new alkaloids from Crinum asiaticum var. japonicum.

A new crinine-type alkaloid crijaponine A (1), a new galanthamine-type alkaloid crijaponine B (2), and 11 known alkaloids-ungeremine (3), lycorine (4), 2-O-acetyllycorine (5), 1, 2-O-diacetyllycorine (6), (-)-crinine (7), 11-hydroxyvittatine (8), hamayne (9), (+)-epibuphanisine (10), crinamine (11), yemenine A (12), and epinorgalanthamine (13)-were isolated from the rhizome and fruits of Crinum asiaticum var. japonicum. The structural elucidation of the isolated compounds was performed by spectroscopic methods including 2D NMR. The isolated compounds were evaluated for cytotoxicity against HeLa and HL-60 cells lines and were tested for acetylcholinesterase inhibition activity.

A Polarity-Sensitive Fluorescent Amino Acid and its Incorporation into Peptides for the Ratiometric Detection of Biomolecular Interactions.

A fluorescent amino acid containing our recently developed 1,5-naphthyridin-2(1H)-one based- fluorophore, which is a structural component of the fluorescent natural compounds amarastelline A and nigakinone, has been developed. It has useful functions, that is, solvent-polarity-dependent change of fluorescence ratio at 370/480 nm and strong fluorescence in both aqueous solution and less polar organic solvents. To demonstrate its utility, it was incorporated into a C5 peptide with the aim of detecting the interaction of this peptide with calmodulin. As expected, the fluorescence ratio at 370/480 nm of the peptide was changed by the calmodulin only in the presence of Ca2+ , thus indicating that the fluorescent peptide could sense the conformational change of calmodulin induced by Ca2+ , followed by its interaction. These results also suggest that this fluorescent amino acid as well as its precursor, a succinimidyl ester, could be applicable for detecting various biomolecular interactions.

Solvent-Free Synthesis of 2-Alkylbenzothiazoles and Bile Acid Derivatives Containing Benzothiazole Ring by Using Active Carbon/Silica Gel and Microwave.

A highly efficient and simple method for the synthesis of 2-alkylbenzothiazoles through the condensation of 2-aminothiophenol and aliphatic aldehydes in the presence of active carbon/silica gel is described. The reaction proceeded under solvent-free and microwave irradiation to afford 2-alkylbenzothiazoles in high yields. This method was extended to the synthesis of bile acid derivatives containing a benzothiazole ring and obtained the desired products in high yields. The anti-inflammatory activity and cytotoxicity of the newly synthesized benzothiazole derivatives of bile acid were tested; the results showed that anti-inflammatory activities of all tested compounds tested were higher than that of standard drugs, such as indomethacin.

Two new coumarins and a new xanthone from the leaves of Rhizophora mucronata.

Two new coumarins (1, 2) and a new xanthone (3), together with 14 known compounds-eight coumarins (4, 5, 9, 10, 12-15), three xanthones (11, 16, 17), a benzoic acid (6) and two flavonones (7, 8)-were isolated from the leaves of Rhizophora mucronata. The structures of the compounds were elucidated by spectroscopic (IR, MS, and NMR) analyses. The isolated compounds were tested for cytotoxicity against human cancer cell lines HL-60 and HeLa. Among these compounds, only compound 16 inhibited the growth of both HeLa (IC50 = 4.8 µM) and HL-60 (IC50 = 1.0 µM) cells. Compounds 4, 7, 10, and 12 exhibited moderate activity against HeLa cells (IC50 = 3.8-8.3 µM). Compounds 5, 9, 11, and 17 showed moderate activity against HL-60 cells (IC50 = 2.2-6.3 µM). Higher selectivity against HL-60 cell lines was observed for compounds 5, 9, 11, and 16 with SI values (NIH 3T3/HL-60) of 8.6, 19.2, 9.4, and 10.2, respectively.

Sucupiranins A-L, Furanocassane Diterpenoids from the Seeds of Bowdichia virgilioides.

Twelve new furanocassane diterpenoids, sucupiranins A-L (1-12), and three known compounds (13-15) were isolated from the seeds of Bowdichia virgilioides. The structures of the compounds were elucidated via 1H and 13C NMR analysis, including 2D NMR (1H-1H COSY, HSQC, HMBC, and NOESY); HRMS data; and X-ray crystallographic analysis. The absolute configurations were defined using their electronic circular dichroism (ECD) spectra by applying the exciton chirality method to the bis-p-bromobenzoate of compound 13. Sucupiranin J (10) inhibited lipopolysaccharide-induced nitric oxide production (IC50 30.6 µM), whereas sucupiranins J (10), K (11), and 13 exhibited weak antimalarial activity against Plasmodium falciparum K1 with IC50 values of 32.2, 23.5, and 22.9 µM and selectivity indices of 4.3, 1.9, and >12.0 (MRC-5/K1), respectively.

Two New Diterpenoids from Salvia przewarskii.

One abietane-type and one dinoricetexane-type diterpenoid, salviskin A (1) and salviskin B (2), respectively, together with fourteen known diterpenoids, were isolated from Salvia przewarskii. Structural elucidation of these compounds was performed by spectroscopic methods including 2D NMR. The compounds isolated were evaluated for their cytotoxicity against HeLa and HL-60 cells.

A New Pimarane-type Diterpenoid from the Seeds of Bowdichia virgilioides.

A new pimarane-type diterpenoid, sucupiol (1), and nine known -furanocassane-type diterpenoids, vouacapane (2), 7ß-hydroxyvouacapane (3) 7ß-acetox yvouacapane (4), 6α-hydroxyvouacapane (5), 6α-acetoxyvouacapane (6), sucutinirane F (7), sucutinirane E (8), 6α, 7ß-diacetoxyvouacapane (9), and 6α, 7ß-diacetoxyvouacapane-14ß-al (10), were isolated from the seeds of Bowdichia virgilioides and their structures were elucidated by using 2D NMR data. The isolation of I provides evidence to support the presence of intermediate A in the course of biosynthesis of furanocassane-type diterpenoids.

Improved chemical synthesis, X-ray crystallographic analysis, and NMR characterization of (22R)-/(22S)-hydroxy epimers of bile acids.

We report an improved synthesis of the (22R)- and (22S)-epimers of 3α,7α,12α,22-tetrahydroxy-5ß-cholan-24-oic acid and 3α,7α,22-trihydroxy-5ß-cholan-24-oic acid from cholic acid (CA) and chenodeoxycholic acid (CDCA), respectively. The principal reactions involved were as follows: (1) oxidative decarboxylation of the bile acid peracetates with lead tetraacetate, and (2) subsequent Reformatsky reaction of the 23,24-dinor-22-aldehydes with ethyl bromoacetate in the presence of activated Zn as a catalyst with the reaction temperature maintained precisely at 75 °C. The absolute configuration of the chiral center at C-22 of each epimer was established by single-crystal X-ray diffraction data using its ethyl ester-peracetate derivative. The (1)H- and (13)C-NMR spectra that permit the (22R)- and (22S)-epimers to be distinguished are reported as well as the specific (1)H shift effects induced by C(5)D(5)N. Bile acids having hydroxyl groups at C-22 are present in a variety of animal biles, previously have been difficult to identify, and are known to have distinctive physicochemical and biological properties.

A lanostane triterpenoid and three cholestane sterols from Tilia kiusiana.

Kiusianins A-D (1-4) were isolated from the leaves of a Japanese endemic plant, Tilia kiusiana, together with 14 known compounds. The structures of a new lanostane-type triterpenoid 1 and three new cholestane-type sterols 2-4 were elucidated by spectroscopic methods, including two dimensional (2D) NMR. All the compounds isolated were evaluated for their cytotoxicity against two human cancer cell lines, HeLa and HL-60.

N-Methyltaurine N-acyl amidated bile acids and deoxycholic acid in the bile of angelfish (Pomacanthidae): a novel bile acid profile in Perciform fish.

Two novel N-acyl amidated bile acids, N-methyltaurine conjugated cholic acid and N-methyltaurine conjugated deoxycholic acid, were found to be major biliary bile acids in two species of angelfish the regal (Pygoplites diacanthus) and the blue-girdled (Pomacanthus navarchus) angelfish. The identification was based on their having MS and NMR spectra identical to those of synthetic standards. A survey of biliary bile acids of 10 additional species of angelfish found 7 with N-methyltaurine conjugation. In all 12 species, conjugated deoxycholic acid (known to be formed by bacterial 7-dehydroxylation of cholic acid) was a major bile acid. In all previous studies of biliary bile acids in fish, deoxycholic acid has been present in only trace proportions. In addition, bile acid conjugation with N-methyltaurine has not been detected previously in any known vertebrate. N-methyltaurine conjugated bile acids are resistant to bacterial deconjugation and dehydroxylation, and such resistance to bacterial enzymes should aid in the maintenance of high concentrations of bile acids during lipid digestion. Our findings suggest that these species of angelfish have a novel microbiome in their intestine containing anaerobic bacteria, and describe the presence of N-methyltaurine conjugated bile acids that are resistant to bacterial attack.

Inhibitory effect of erythraline on Toll-like receptor signaling pathway in RAW264.7 cells.

Erythraline, isolated from the bark of Erythrina crista-galli which are used as Brazilian medicine plant for the treatment of inflammation diseases, suppressed nitric oxide (NO) production and induction of inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells stimulated by lipopolysaccharide (LPS). Because of Toll-like receptor (TLR) 4 and its signal transduction are indispensable to the production of NO and iNOS expression by LPS, we investigated the effects of erythraline on TLR signaling molecules. Western blot analysis revealed that the degradation of inhibitor of nuclear factor (NF)-κB (IκB) by LPS was suppressed by erythraline. Moreover, erythraline inhibited not only LPS-induced phosphorylation of IκB kinase (Ikk) but also phosphorylation of mitogen-activated protein kinases (MAPKs). However, it showed no effect on LPS -induced phosphorylation of transforming growth factor (TGF)-ß-activated kinase (TAK) 1 that exists upstream of Ikk and MAPKs, and is required for the activation of these signaling molecules on TLR signaling pathway. These results suggested that erythraline might have inhibited the kinase activity of TAK1. Furthermore, these results were supported from the inhibitory pattern of erythraline on TLR signaling molecules when the cells were stimulated by TLR2 ligand, peptidoglycan which activates the same pathway as LPS on TLR signal transduction.

The cytotoxic activity of diterpenoids from Isodon species.

Fifteen Isodon diterpenoids (1-15) were evaluated for their cytotoxic activity against HeLa and HL-60 human cancer cell lines, and against murine vincristine (VCR)-resistant P388 cells. Kamebanin (14) showed efficient cytotoxic activity against HeLa and HL-60 cells. In addition, although dihydroenmein (2) and trichorabdal B (7) were inactive against several tested cell types, they were found to have cytotoxic-enhancing activity of VCR against VCR-resistant P388 cells.

Chemical and genetic study of Ligularia duciformis and related species in Sichuan and Yunnan Provinces of China.

The chemical constituents of the root extracts and the evolutionarily neutral DNA base sequences were studied for 28 samples of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia collected in Sichuan and Yunnan Provinces of China. The samples could be classified into four chemotypes (1-4). Sesquiterpenoids having eremophilane and oplopane skeletons were isolated from two (Chemotype 1) and three (Chemotype 2) samples, respectively. Two new oplopane derivatives were isolated and their structures were determined. In 18 samples, phenylpropenoids were the major components (Chemotype 3). In five samples, neither phenylpropenoids nor sesquiterpenoids were found (Chemotype 4). Despite this large chemical variety, no correlation was found between the chemotype and the morphological criteria of species identification. The analysis of the evolutionarily neutral DNA regions also indicated that the samples were not separated into distinct clades and that introgression was extensive.

Erythrinan alkaloids from seeds of Erythrina velutina.

Four new Erythrinan alkaloids (1-4) were isolated from the seeds of Erythrina velutina. The structures of these new compounds 1-4 were elucidated by spectroscopic methods including 2D-NMR. Three of four were found to be novel sulfated Erythrinan alkaloids.

Structures of new Erythrinan alkaloids and nitric oxide production inhibitors from Erythrina crista-galli.

Two new Erythrinan alkaloids, cristanines A and B (1, 2), were isolated from the bark of Erythrina crista-galli L. together with nine known Erythrinan alkaloids (3-5, 7-12) and an indole alkaloid (13). The structures of the compounds, cristanines A (1) and B (2), were elucidated by spectroscopic methods. Three of the twelve compounds isolated showed significant inhibitory activity on lipopolysaccharide induced nitric oxide (NO) production.

Brartemicin, an inhibitor of tumor cell invasion from the actinomycete Nonomuraea sp.

Brartemicin (1), a new trehalose-derived metabolite, was isolated from the culture broth of the actinomycete of the genus Nonomuraea. Its structure and absolute configuration were determined by spectroscopic analyses. The new compound inhibited the invasion of murine colon carcinoma 26-L5 cells with an IC(50) value of 0.39 microM in a concentration-dependent manner without showing cytotoxic effects.

TRAIL-enhancing activity of Erythrinan alkaloids from Erythrina velutina.

A new Erythrinan alkaloid (1), erythodine N-oxide, was isolated from the seeds of Erythrina velutina together with seven known Erythrinan alkaloids (2-7, 9) and an indole alkaloid (8). The structure of new compound (1) was elucidated by spectroscopic methods. Six of the nine compounds showed enhanced activity when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Erysotrine (4) did not show cytotoxic activity by itself, but exhibited significant cytotoxicity when combined with TRAIL.