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.

Production of an α-pyrone metabolite and microbial transformation of isoflavones by an Indonesian Streptomyces sp.

A benzyl-α-pyrone metabolite, streptpyrone A (1), was obtained together with three known isoflavonoids, daidzein-7-O-α-l-rhamnoside (2), genistein-7-O-α-l-rhamnoside (3), and daidzein (4), from the culture broth of an Indonesian actinomycete Streptomyces sp. TPU1401A. The structure of 1, elucidated based on its spectroscopic data, has been reported as a synthetic compound. However, this is the first report of the isolation of 1 as a metabolite of microbial origin. Strain TPU1401A exhibited the ability to transform the isoflavone aglycones 4 and genistein (5) into the 7-O-glycosides 2 and 3, respectively. Compounds 2 and 3 promoted the growth of strain TPU1401A more effectively than compounds 4 and 5. These results suggest that strain TPU1401A utilizes isoflavone glycosides to promote growth by transforming isoflavones through microbial glycosidation.

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.

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.

Absolute Structures of Wedelolide Derivatives and Structure-Activity Relationships of Protein Tyrosine Phosphatase 1B Inhibitory ent-Kaurene Diterpenes from Aerial Parts of Wedelia spp. Collected in Indonesia and Japan.

Two sesquiterpene lactones with the (9R)-eudesman-9,12-olide framework, wedelolides I and J, have been isolated together with five eudesmanolide sesquiterpenes and twelve ent-kaurene diterpenes from the aerial parts of Indonesian Wedelia prostrata. The absolute configurations of wedelolides I and J, proposed in the previous communication, were proven by comparing their experimental Electronic Circular Dichroism (ECD) spectra with the calculated ECD spectrum of wedelolide I. The phytochemical study on the aerial parts of Okinawan Wedelia chinensis led to the isolation of three other eudesmanolide sesquiterpenes in addition to the three sesquiterpenes and eleven diterpenes isolated from the Indonesian W. prostrata as above. However, the wedelolide derivatives found in the Indonesian plant were not detected. Among these compounds, most of the diterpenes inhibited protein tyrosine phosphatase (PTP) 1B activity, and a structure-activity relationship study revealed that the cinnamoyl group enhanced inhibitory activity. Therefore, two ent-kaurene derivatives with and without a cinnamoyl group were examined for the ability to accumulate phosphorylated-Akt (p-Akt) because PTP1B dephosphorylates signal transduction from the insulin receptor such as phosphorylated Akt, a key downstream effector. However, neither compound enhanced insulin-stimulated p-Akt levels in two human hepatoma cell lines (Huh-7 and HepG2) at non-cytotoxic doses.

A bromopyrrole-containing diterpene alkaloid from the Okinawan marine sponge Agelas nakamurai activates the insulin pathway in Huh-7 human hepatoma cells by inhibiting protein tyrosine phosphatase 1B.

Agelasine G (1), a known bromine-containing diterpene alkaloid, was isolated as a new type of protein tyrosine phosphatase (PTP) 1B inhibitor together with ageline B (2), an inactive debromo-derivative of 1, from the marine sponge Agelas nakamurai collected at Iriomote Island in Okinawa, Japan. Further biological evaluations revealed that compound 1 exhibited selective inhibitory activity against PTP1B over T-cell PTP and CD45 phosphatase. Compound 1 also enhanced the insulin-stimulated phosphorylation levels of Akt in Huh-7 cells more strongly than compound 2. The results obtained in this study suggest that compound 1 activates the insulin signaling pathway by inhibiting PTP1B activity.

Furanoterpenes, new types of protein tyrosine phosphatase 1B inhibitors, from two Indonesian marine sponges, Ircinia and Spongia spp.

Protein tyrosine phosphatase (PTP) 1B negatively regulates the insulin and leptin signaling pathways, and, thus, the clinical application of PTP1B inhibitors to the prevention and treatment of type 2 diabetes and obesity is expected. During our studies on PTP1B inhibitors, two furanosesterterpenes and a C21 furanoterpene were obtained as new types of PTP1B inhibitors from two Indonesian marine sponges. (7E, 12E, 20Z, 18S)-Variabilin (1) and (12E, 20Z, 18S)-8-hydroxyvariabilin (2) from Ircinia sp. and furospongin-1 (3) from Spongia sp. inhibited PTP1B activity with IC50 values of 1.5, 7.1, and 9.9µM, respectively. The inhibitory activity of compound 1 against T-cell PTP (TCPTP) was approximately 2-fold that against PTP1B, whereas the vaccinia H-1-related phosphatase (VHR) inhibitory effects of 1 were 4-fold weaker than that of its PTP1B inhibitory activity. Compounds 1-3 at 50µM did not show cytotoxicity against two human cancer cell lines, hepatoma Huh-7 and bladder carcinoma EJ-1. Compound 1 did not enhance the phosphorylation level of Akt, a key downstream effector of the cascade, in Huh-7 cells.

Anti-mycobacterial alkaloids, cyclic 3-alkyl pyridinium dimers, from the Indonesian marine sponge Haliclona sp.

Three new dimeric 3-alkyl pyridinium alkaloids, named haliclocyclamines A-C (1-3), were isolated together with five known congeners, cyclostellettamines A (4), B (5), C (6), E (7), and F (8), from the Indonesian marine sponge Haliclona sp. The structures of 1-3 were assigned based on their spectroscopic data (1D and 2D NMR, HRFABMS, ESIMS/MS, UV, and IR). Compounds 1-8 exhibited antimicrobial activities against Mycobacterium smegmatis with inhibition zones of 17, 10, 13, 14, 8, 8, 12, and 12mm, respectively, at 10µg/disc. Compounds 3 and 8 also modestly inhibited the activity of vaccinia H-1-related phosphatase (VHR), a dual-specificity phosphatase, at 17-18µM.

A tetramic acid derivative with protein tyrosine phosphatase 1B inhibitory activity and a new nortriterpene glycoside from the Indonesian marine sponge Petrosia sp.

During the search for protein tyrosine phosphatase 1B (PTP1B) inhibitors from marine organisms, the known tetramic acid derivative, melophlin C (1), was isolated as an active component together with the new nortriterpenoid saponin, sarasinoside S (2), and three homologues: sarasinosides A1 (3), I1 (4), and J (5), from the Indonesian marine sponge Petrosia sp. The structure of 2 was elucidated on the basis of its spectroscopic data. Compound 1 inhibited PTP1B activity with an IC50 value of 14.6µM, while compounds 2-5 were not active at 15.2-16.0µM. This is the first study to report the inhibitory effects of a tetramic acid derivative on PTP1B activity.

Lissoclibadin 1, a Polysulfur Aromatic Alkaloid from the Indonesian Ascidian Lissoclinum cf. badium, Induces Caspase-Dependent Apoptosis in Human Colon Cancer Cells and Suppresses Tumor Growth in Nude Mice.

Lissoclibadins, polysulfur aromatic alkaloids, were isolated from the Indonesian ascidian Lissoclinum cf. badium. Lissoclibadins 1 (1), 3 (2), 4 (3), 7 (4), 8 (5), and 14 (6) inhibited the growth of four human solid cancer cell lines: HCT-15 (colon adenocarcinoma), HeLa-S3 (cervix adenocarcinoma), MCF-7 (breast adenocarcinoma), and NCI-H28 (mesothelioma). Lissoclibadin 1 (1) exerted the most potent cytotoxic effects in vitro and mainly promoted apoptosis through an intrinsic pathway with the activation of a caspase-dependent pathway in HCT-15 cells. In vivo studies demonstrated that 1 suppressed tumor growth in nude mice carrying HCT-15 cells without significant secondary adverse effects. In conclusion, the results obtained in the present study demonstrate that 1 has potential as a chemotherapeutic candidate for preclinical investigations.

A New Pyranonaphtoquinone Derivative, 4-Oxo-rhinacanthin A, from Roots of Indonesian Rhinacanthus nasutus.

A new pyranonaphthoquinone derivative, named 4-oxo-rhinacanthin A (1), was isolated from the roots of the Indonesian Rhinacanthus nasutus together with two known congeners, rhinacanthin A (2) and 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione (3). The structure of 1 was elucidated based on its spectroscopic data. The absolute configuration of 1 was assigned by comparing its experimental Electronic Circular Dichroism (ECD) spectrum with the calculated ECD spectrum. Compounds 2 and 3 inhibited the growth of Staphylococcus aureus with inhibition zones of 16 and 20 mm at 25 µg/disc, respectively. Compound 3 also exhibited inhibitory activity against Mycobacterium smegmatis (20 mm at 25 µg/disc).

A dimeric urea of the bisabolene sesquiterpene from the Okinawan marine sponge Axinyssa sp. inhibits protein tyrosine phosphatase 1B activity in Huh-7 human hepatoma cells.

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator of the insulin and leptin signaling pathways. Therefore, this enzyme is regarded as an attractive therapeutic target for the treatment of type 2 diabetes and obesity. Our screening program for PTP1B inhibitors led to the isolation of four sesquiterpenes and sterol: N,N'-bis[(6R,7S)-7-amino-7,8-dihydro-α-bisabolen-7-yl]urea (1), (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (2), (1R,6S,7S,10S)-10-isothiocyanato-4-amorphene (3), axinisothiocyanate J (4), and axinysterol (5) from the marine sponge Axinyssa sp. collected at Iriomote Island. Of these, compound 1 was the most potent inhibitor of PTP1B activity (IC50=1.9µM) without cytotoxicity at 50µM in two human cancer cell lines, hepatoma Huh-7 and bladder carcinoma EJ-1 cells. Compound 1 also moderately enhanced the insulin-stimulated phosphorylation levels of Akt in Huh-7 cells. Therefore, compound 1 has potential as a new type of anti-diabetic drug candidate possessing PTP1B inhibitory activity.

Sesquiterpene Hydroquinones with Protein Tyrosine Phosphatase 1B Inhibitory Activities from a Dysidea sp. Marine Sponge Collected in Okinawa.

Three new sesquiterpene hydroquinones, avapyran (1), 17-O-acetylavarol (2), and 17-O-acetylneoavarol (3), were isolated from a Dysidea sp. marine sponge collected in Okinawa together with five known congeners: avarol (4), neoavarol (5), 20-O-acetylavarol (6), 20-O-acetylneoavarol (7), and 3'-aminoavarone (8). The structures of 1-3 were assigned on the basis of their spectroscopic data. Compounds 1-3 inhibited the activity of protein tyrosine phosphatase 1B with IC50 values of 11, 9.5, and 6.5 µM, respectively, while known compounds 4-8 gave IC50 values of 12, >32, 10, 8.6, and 18 µM, respectively. In a preliminary investigation on structure-activity relationships, six ester and methoxy derivatives (9-14) were prepared from 4 and 5.

Haliclonadiamine Derivatives and 6-epi-Monanchorin from the Marine Sponge Halichondria panicea Collected at Iriomote Island.

Four new haliclonadiamine analogues, (10Z,12E)-haliclonadiamine (1), (10E,12Z)-haliclonadiamine (2), and halichondriamines A (3) and B (4), were isolated from the Okinawan marine sponge Halichondria panicea together with haliclonadiamine (5) and papuamine (6). The structures of 1-4 were elucidated on the basis of their spectroscopic data by comparisons with those for 5 and 6. Further separation of the remaining fraction led to the isolation of a new bicyclic guanidine alkaloid, 6-epi-monanchorin (7), along with monanchorin (8). Compound 7 is the epimer of 8 at the 6 position. Compounds 1-6 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 12, 7, 8, 7, 16, and 12 mm at 10 µg/disc, respectively. Compounds 2-4 exhibited weak cytotoxicities against the Huh-7 (hepatoma) human cancer cell line and were 2-fold less active than 5 and 6. Compounds 7 and 8 were not active against M. smegmatis at 20 µg/disc or the cancer cell line at 10 µM.

Isopetrosynol, a New Protein Tyrosine Phosphatase 1B Inhibitor, from the Marine Sponge Halichondria cf. panicea Collected at Iriomote Island.

A new polyacetylene compound, isopetrosynol (1), was isolated from the Okinawan marine sponge Halichondria cf. panicea together with petrosynol (2), adociacetylene D (3), (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4), and petrosterol (5). The structure of 1 was assigned on the basis of spectroscopic data for 1 and 2. Compound 1 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 8.2±0.3 µM, while compound 2, a diastereomer of 1, showed only 28.9±4.5% inhibition at 21.6 µM. The IC50 values of compounds 3 and 4 were 7.8±0.5 and 12.2±0.5 µM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7±0.1 µM (IC50) in the same experiment. The inhibitory activity of 1 was stronger than that of its diastereomer (2). This is the first study to show the inhibitory effects of polyacetylene compounds on PTP1B.

Strongylophorines, new protein tyrosine phosphatase 1B inhibitors, from the marine sponge Strongylophora strongilata collected at Iriomote Island.

A new meroditerpene, 26-O-ethylstrongylophorine-14 (1), was isolated from the Okinawan marine sponge Strongylophora strongilata together with six known strongylophorines: 26-O-methylstrongylophorine-16 (2) and strongylophorines-2 (3), -3 (4), -8 (5), -15 (6), and -17 (7). The structure of 1 was assigned on the basis of its spectroscopic data. Compound 1 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 8.7 µM, while known compounds 2-8 gave IC50 values of 8.5, >24.4, 9.0, 21.2, 11.9, and 14.8 µM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7 µM (IC50). The inhibitory activities of strongylophorines possessing the acetal moiety at C-26 (1, 2, and 6) were stronger than those of the lactone derivatives (3 and 5). This is the first study to demonstrate that meroditerpenes inhibit PTP1B activity.

Two new protein tyrosine phosphatase 1B inhibitors, hyattellactones A and B, from the Indonesian marine sponge Hyattella sp.

Two unique sesterterpenes, hyattellactones A (1) and B (2), together with two known sesterterpenes, phyllofolactones F (3) and G (4), were isolated from the Indonesian marine sponge Hyattella sp. The structures of the two new compounds, 1 and 2 were assigned based on their spectroscopic data. Hyattellactone A (1) was a scalarane sesterterpene with an α,ß-unsaturated-γ-lactone ring and C-ethyl group, while B (2) was an epimer of 1 at the C-24 position. Compounds 1 and 3 inhibited PTP1B activity with IC50 values of 7.45 and 7.47µM, respectively. On the other hand, compounds 2 and 4 (24S-isomers of 1 and 3, respectively) showed much reduced activity than the 24R-isomers.

Verruculides A and B, two new protein tyrosine phosphatase 1B inhibitors from an Indonesian ascidian-derived Penicillium verruculosum.

Two new merosesquiterpenes, verruculides A (1) and B (2), were isolated from a culture broth of the Indonesian ascidian-derived Penicillium verruculosum TPU1311, together with three known congeners, chrodrimanins A (3), B (4), and H (5). The structures of 1 and 2 were assigned on the basis of their spectroscopic data (1D and 2D NMR, HRMS, UV, CD, and IR). Compound 2 had a linear sesquiterpene moiety and was considered to be the derivative of the biosynthetic precursor for 1 and 3-5. Compounds 1, 3, and 5 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 8.4, 8.5, and 14.9 µM, respectively. Compound 2 showed 40% inhibition at 23.1 µM, while 4 was not active at 20.7 µM.