Synthesis and Evaluation of Habiterpenol Analogs.

Habiterpenol is a G2 checkpoint inhibitor isolated from the culture broth of Phytohabitans sp. 3787_5. Here, we report the synthesis of new habiterpenol analogs through the total synthesis process of habiterpenol and evaluating the analogs for G2 checkpoint inhibitory activity. We investigated two different synthetic approaches for total synthesis, with intramolecular conjugate addition and Ti(III)-mediated radical cyclization as key reactions. Although the former was unsuccessful, the latter reaction facilitated stereoselective total synthesis and determination of the absolute configuration of habiterpenol. The extension of these chemistries to a structure-activity relationship (SAR) study gave new habiterpenol analogs, which could not be derived from natural habiterpenol and only be synthesized by applying the total synthesis. Therefore, this study provides important insights into SAR studies of habiterpenol.

Germacrane sesquiterpenes from leaves of Eupatorium chinense inhibit protein tyrosine phosphatase.

Three new germacrane-type sesquiterpene lactones (1-3) were isolated alongside seven known related congeners (4-10) from the leaves of Eupatorium chinense L. (Compositae). The planar structures of 1-3 were elucidated by their spectroscopic data, including 1D and 2D NMR spectra. The relative and absolute configurations of 1-3 were determined using NOESY experiments and electronic circular dichroism analyses. Compounds 1, 4, 5, and 7 inhibited protein tyrosine phosphatase (PTP) 1B activity with IC50 values of 25, 11, 28, and 24 µM, respectively. Among these, compound 4 exhibited an inhibitory effect on T-cell PTP (TCPTP) with an IC50 value of 25 µM. To our knowledge, this is the first study demonstrating the PTP inhibitory activity of the germacrane sesquiterpenes. The results show that compound 4 acts as an inhibitor of both PTP1B and TCPTP.

Inhibitory effects of sesquiterpene lactones from the Indonesian marine sponge Lamellodysidea cf. herbacea on bone morphogenetic protein-induced osteoblastic differentiation.

A new unique sesquiterpene lactone, bicyclolamellolactone A (1), was isolated together with two known monocyclofarnesol-type sesquiterpenes, lamellolactones A (2) and B (3), from the Indonesian marine sponge Lamellodysidea sp. (cf. L. herbacea). The planar structure of 1 was assigned based on its spectroscopic data (1D and 2D NMR, HRESIMS, UV, and IR spectra). The relative and absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism spectra in combination with NOESY correlations. Compounds 1-3 inhibited bone morphogenic protein (BMP)-induced alkaline phosphatase activity in mutant BMP receptor-carrying C2C12 cells with IC50 values of 51, 4.6, and 20 µM, respectively.

Antifungal trichothecene sesquiterpenes obtained from the culture broth of marine-derived Trichoderma cf. brevicompactum and their structure-activity relationship.

Two new trichothecene sesquiterpenes, trichobreols D (1) and E (2), were isolated from the culture broth of marine-derived Trichoderma cf. brevicompactum together with trichobreol A (3). The structures of 1 and 2 were assigned on the basis of their spectroscopic data. Compound 1 inhibited the growth of two yeast-like fungi, Candida albicans and Cryptococcus neoformans, with equivalent MIC values (6.3 µg/mL), while 2 gave MIC values of 12.5 and 25 µg/mL, respectively. The antifungal activities of five semisynthetic derivatives (4-8) prepared from 3 were evaluated and compared to investigate the preliminary structure-activity relationship.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

Screening of Indonesian Edible Plants for Bioactive Constituents and a New Protein Tyrosine Phosphatase 1B Inhibitory Acylbenzene Derivative from Leaves of Indonesian Syzygium polyanthum.

Bioassay screening using Indonesian plants, such as traditional foods (vegetables, spices, and tea) and folk medicinal herbs, identified eight protein tyrosine phosphatase (PTP) 1B inhibitory and two antibacterial plants. The leaves of Syzygium polyanthum (Wight) Walp. were examined in more detail to define PTP1B inhibitory components, resulting in the isolation of a new active acylbenzene (1) along with four related congeners of 1 (2-5) and four oleanane triterpenes (6-9). The structure of 1 was elucidated as 12-oxo-12-(2,3,5-trihydroxy-4-methylphenyl)dodecanoic acid based on its spectroscopic data. The acylbenzenes 1 and 3-5 inhibited PTP1B activity with IC50 values ranging between 9.5 and 14 µM, whereas the triterpenes 7-9 also suppressed this activity with IC50 values of 3.3-5.7 µM.

Evaluation of Anti-Mycobacterial Compounds in a Silkworm Infection Model with Mycobacteroides abscessus.

Among four mycobacteria, Mycobacterium avium, M. intracellulare, M. bovis BCG and Mycobacteroides (My.) abscessus, we established a silkworm infection assay with My. abscessus. When silkworms (fifth-instar larvae, n = 5) were infected through the hemolymph with My. abscessus (7.5 × 107 CFU/larva) and bred at 37 °C, they all died around 40 h after injection. Under the conditions, clarithromycin and amikacin, clinically used antimicrobial agents, exhibited therapeutic effects in a dose-dependent manner. Furthermore, five kinds of microbial compounds, lariatin A, nosiheptide, ohmyungsamycins A and B, quinomycin and steffimycin, screened in an in vitro assay to observe anti-My. abscessus activity from 400 microbial products were evaluated in this silkworm infection assay. Lariatin A and nosiheptide exhibited therapeutic efficacy. The silkworm infection model with My. abscessus is useful to screen for therapeutically effective anti-My. abscessus antibiotics.

An Unusual Extender Unit Is Incorporated into the Modular Polyketide Synthase of Scopranones Biosynthesis.

Scopranones, produced by Streptomyces sp. BYK-11038, are the novel bone morphogenetic protein inhibitors characterized by atypical two scoop-like moieties and a 3-furanone moiety. Two scoop-like moieties connected to a 3-furanone have not previously been reported in natural products, and their biosynthesis must occur via a unique pathway. Feeding experiments using 13C-labeled precursors indicated that scopranones were synthesized from three acetates and three butyrates in polyketide-type biosynthesis. Genome mining of Streptomyces sp. BYK-11038 revealed that the candidate biosynthetic gene cluster contains 21 open reading frames (ORFs), including three modular polyketide synthases (PKSs; SprA, SprB, and SprC), which were composed of 4 modules with one loading module and 18 additional ORFs (SprD to SprU) spanning a distance of 55 kbp. The characterization of in-frame deletion mutants and feeding experiments with the predicted extender units indicated that two genes, sprP and sprR, encoding discrete 3-oxoacyl-ACP synthases, and a gene, sprO, encoding crotonyl-CoA reductase, were involved in assembling an unusual C8 branched extender unit, 2-(2-ethylbutyl)malonyl-CoA. Additionally, three ORFs, sprM, sprN, and sprT, encoding cytochrome P450s and a monooxygenase, are important tailoring enzymes in post-PKS modification. SprT is an essential enzyme for decarboxylative ring contraction via oxidation, which converts the 2-pyranone to a 3-furanone.

Inhibition of neutral lipid synthesis by avarols from a marine sponge.

The effects of 14 sesquiterpene hydroquinones, including 8 marine sponge-derived avarols (1-8) and 6 semisynthetic derivatives (9-14), on lipid droplet accumulation and neutral lipid synthesis in Chinese hamster ovary (CHO) K1 cells were investigated. In intact CHO-K1 cell assays, avarol (1) markedly decreased the number and size of lipid droplets in CHO-K1 cells and exhibited the most potent inhibitory activity on the synthesis of cholesteryl ester (CE) and triglyceride (TG) with IC50 values of 5.74 and 6.80 µM, respectively. In enzyme assays, sterol O-acyltransferase (SOAT), the final enzyme involved in CE biosynthesis, and diacylglycerol acyltransferase (DGAT), the final enzyme involved in TG biosynthesis, were inhibited by 1 with IC50 values of 7.31 and 20.0 µM, respectively, which correlated well with those obtained in the intact cell assay. These results strongly suggest that 1 inhibited SOAT and DGAT activities in CHO-K1 cells, leading to a reduction in the accumulation of CE and TG in lipid droplets.

Nectriatide, a Potentiator of Amphotericin B Activity from Nectriaceae sp. BF-0114.

A new compound, designated nectriatide (1), was isolated as a potentiator of amphotericin B (AmB) activity against Candida albicans from the culture broth of Nectriaceae sp. BF-0114. This structure was elucidated based on spectroscopic analyses (1D and 2D NMR data), chemical methods, and total synthesis. Compound 1 was a unique cyclotetrapeptide consisting of l-N-methyltyrosine, anthranilic acid, l-alanine, and l-valine. Compound 1 and several synthetic derivatives, including linear peptides, potentiated AmB activity against C. albicans by up to 16-fold (the MIC value of AmB decreased from 0.5 µg/mL to 0.031 µg/mL in combination with test compound).

Callyspongiamides A and B, sterol O-acyltransferase inhibitors, from the Indonesian marine sponge Callyspongia sp.

Callyspongiamides A (1) and B (2), two new sterol O-acyltransferase (SOAT) inhibitors, were isolated from the Indonesian marine sponge Callyspongia sp. together with a known congener, dysamide A (3). The structures of 1 and 2 were elucidated to be polychlorine-containing modified dipeptides based on their spectroscopic data. Compounds 1-3 inhibited both of the SOAT isozymes, SOAT1 and SOAT2, in cell-based and enzyme-based assays.

Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane.

To obtain therapeutically effective new antibiotics, we first searched for bacterial culture supernatants with antimicrobial activity in vitro and then performed a secondary screening using the silkworm infection model. Through further purification of the in vivo activity, we obtained a compound with a previously uncharacterized structure and named it 'lysocin E'. Lysocin E interacted with menaquinone in the bacterial membrane to achieve its potent bactericidal activity, a mode of action distinct from that of any other known antibiotic, indicating that lysocin E comprises a new class of antibiotic. This is to our knowledge the first report of a direct interaction between a small chemical compound and menaquinone that leads to bacterial killing. Furthermore, lysocin E decreased the mortality of infected mice. To our knowledge, lysocin E is the first compound identified and purified by quantitative measurement of therapeutic effects in an invertebrate infection model that exhibits robust in vivo effects in mammals.

Simplifungin and Valsafungins, Antifungal Antibiotics of Fungal Origin.

The targets of antifungal antibiotics in clinical use are more limited than those of antibacterial antibiotics. Therefore, new antifungal antibiotics with different mechanisms of action are desired. In the course of our screening for antifungal antibiotics of microbial origins, new antifungal antibiotics, simplifungin (1) and valsafungins A (2) and B (3), were isolated from cultures of the fungal strains Simplicillium minatense FKI-4981 and Valsaceae sp. FKH-53, respectively. The structures of 1 to 3 including their absolute stereochemistries were elucidated using various spectral analyses including NMR and collision-induced dissociation (CID)-MS/MS as well as chemical approaches including modifications to the Mosher's method. They were structurally related to myriocin. They inhibited the growth of yeast-like and zygomycetous fungi with MICs ranging between 0.125 and 8.0 µg/mL. An examination of their mechanisms of action by the newly established assay using LC-MS revealed that 1 and 2 inhibited serine palmitoyltransferase activity, which is involved in sphingolipid biosynthesis, with IC50 values of 224 and 24 nM, respectively.

Synthesis and Structural Revision of Cyslabdan.

Cyslabdan was isolated from the culture broth of Streptomyces sp. K04-0144 as a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus. We accomplished the synthesis of cyslabdan according to a previously reported structure. However, we subsequently found that this structure was incorrect; our analysis of natural cyslabdan showed that it possessed R stereochemistry at the C8 position, not S, as had previously been reported. Thus, we completed the protecting-group-free synthesis of the correct structure of cyslabdan, which is described herein.

Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil. These effects were partly dependent on nicotinic acetylcholine receptor (nAChR).

Synthesis and biological activity of 5-(4-methoxyphenyl)-oxazole derivatives.

5-(4'-Methoxyphenyl)-oxazole (MPO), originally reported as a synthetic compound, was isolated from fungal culture broth as an inhibitor of hatch and growth of Caenorhabditis elegans. Nineteen MPO derivatives were chemically synthesized, but showed no effect on C. elegans hatch and growth. These findings strongly suggested that the whole structure of MPO is essential for anti-C. elegans activity.

New liposidomycin congeners produced by Streptomyces sp. TMPU-20A065, anti-Mycobacterium avium complex agents with therapeutic efficacy in a silkworm infection model.

Three new liposidomycin congeners (1, 2, and 4), together with 14 known liposidomycins (3 and 5-17), were isolated from the culture broth of Streptomyces sp. TMPU-20A065 as anti-Mycobacterium avium complex agents. The structures of liposidomycins were elucidated by spectroscopic analyses, including NMR and MS. Compounds 1, 2, and 4 belong to type-I liposidomycin-containing sulfate groups and methylglutaric acid, each with a different acyl side chain in the structure. Compounds 1-17 exhibited in vitro anti-M. avium and M. intracellulare activities with MIC values ranging between 2.0 and 64 µg ml-1. Furthermore, 1-17 exerted potent therapeutic effects in an in vivo-mimic silkworm infection model with ED50 values ranging between 0.12 and 3.7 µg larva-1 g-1.

Synthesis and biological evaluation of nectriatide derivatives, potentiators of amphotericin B activity.

Nectriatide 1a, a naturally occurring cyclic tetrapeptide, has been reported to a potentiator of amphotericin B (AmB) activity. In order to elucidate its structure-activity relationships, we synthesized nectriatide derivatives with different amino acids in solution-phase synthesis and evaluated AmB-potentiating activity against Candida albicans. Among them, C-and N-terminal protected linear peptides were found to show the most potent AmB-potentiating activity.

New piericidin rhamnosides as potentiators of amphotericin B activity against Candida albicans produced by actinomycete strain TMPU-A0287.

Four new piericidin rhamnosides (2, 4-6) together with three known piericidins (1, 3, 7) were isolated from the culture broth of the unidentified actinomycete strain TMPU-A0287 as potentiators of antifungal amphotericin B (AmB) activity. The structures of piericidins were elucidated by spectroscopic analyses, including NMR and MS. Compounds 2 and 4-6 possessed a ketone at C-10 and one or two methoxy groups on the rhamnose in their structures. Compounds 1-7 did not exhibit antifungal activity against Candida albicans and all potentiated AmB activity. The MIC values of AmB against C. albicans combined with 1-7 (4.0 µg ml-1) decreased from 0.50 to 0.063 or 0.031 µg ml-1, yielding an 8- or 16-fold increase in AmB activity.