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.

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.

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.

Oleanane triterpenes with protein tyrosine phosphatase 1B inhibitory activity from aerial parts of Lantana camara collected in Indonesia and Japan.

During the search for new protein tyrosine phosphatase (PTP) 1B inhibitors, EtOH extracts from the aerial parts of Lantana camara L. (lantana) collected at Manado (Indonesia) and two subtropical islands in Japan (Ishigaki and Iriomote Islands, Okinawa) exhibited potent inhibitory activities against PTP1B in an enzyme assay. Four previously undescribed oleanane triterpenes were isolated together with known triterpenes and flavones from the Indonesian lantana. The EtOH extracts of lantana collected in Ishigaki and Iriomote Islands exhibited different phytochemical profiles from each other and the Indonesian lantana. Triterpenes with a 24-OH group were isolated from the Indonesian lantana only. Five known triterpene compounds were detected in the Ishigaki lantana, and two oleanane triterpenes with an ether linkage between 3ß and 25 were the main components together with five known triterpenes as minor components in the Iriomote lantana. The structures of previously undescribed compounds were assigned on the basis of their spectroscopic data. Among the compounds obtained in this study, oleanolic acid exhibited the most potent activity against PTP1B, and is used as a positive control in studies on PTP1B.

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.

An anti-mycobacterial bisfunctionalized sphingolipid and new bromopyrrole alkaloid from the Indonesian marine sponge Agelas sp.

In the course of our studies on anti-mycobacterial substances from marine organisms, the known dimeric sphingolipid, leucettamol A (1), was isolated as an active component, together with the new bromopyrrole alkaloid, 5-bromophakelline (2), and twelve known congeners from the Indonesian marine sponge Agelas sp. The structure of 2 was elucidated based on its spectroscopic data. Compound 1 and its bis TFA salt showed inhibition zones of 12 and 7 mm against Mycobacterium smegmatis at 50 µg/disk, respectively, while the N,N'-diacetyl derivative (1a) was not active at 50 µg/disk. Therefore, free amino groups are important for anti-mycobacterial activity. This is the first study to show the anti-mycobacterial activity of a bisfunctionalized sphingolipid. Compound 13 exhibited weak PTP1B inhibitory activity (29% inhibition at 35 µM).

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.

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.

Structures and biological activities of triterpenes and sesquiterpenes obtained from Russula lepida.

A seco-cucurbitane triterpene and two aristolane sesquiterpenes, named (24E)-3,4-seco-cucurbita-4,24-diene-3-hydroxy-26,29-dioic acid, (+)-1,2-didehydro-9-hydroxy-aristlone, and (+)-12-hydroxy-aristlone, were isolated from fruiting bodies of the medicinal mushroom Russula lepida, together with (24E)-3,4-seco-cucurbita-4,24-diene-3,26,29-trioic acid and (+)-aristlone. The structures of the first three compounds, including their absolute configurations, were assigned on the basis of their NMR and ECD spectra. Two seco-cucurbitane triterpenes, (24E)-3,4-seco-cucurbita-4,24-diene-3-hydroxy-26,29-dioic acid and (24E)-3,4-seco-cucurbita-4,24-diene-3,26,29-trioic acid, inhibited the activity of protein tyrosine phosphatase 1B (PTP1B), with IC50 values of 20.3 and 0.4µM, respectively. All isolated compounds did not show cytotoxicity against human cancer cell lines, Huh-7 (hepatoma) and EJ-1 (bladder), at 50µM.

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.

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.

Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai.

Three new N-methyladenine-containing diterpenes, 2-oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G (10), and ageline B (11). The structures of 1-3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5-11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 µg/disc, respectively. All compounds were inactive (IC50 > 10 µM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 15 µM.

Two new indole derivatives from a marine sponge Ircinia sp. collected at Iriomote Island.

Two new indole derivatives, 5-hydroxy-1H-indole-3-carboxylic acid ethyl ester (1) and 5-hydroxy-1H-indole-3-glyoxylate ethyl ester (2), and seven known indole alkaloids, dragmacidonamine B (3), gesashidine A (4), hyrtiosulawesine (5), hyrtiomanzamine (6), hyrtimomine D (7), hyrtiosine A (8), and 5-hydroxy-1H-indole-3-carbaldehyde (9), were isolated from a marine sponge Ircinia sp., Irciniidae, collected at Iriomote Island. The structures of 1 and 2 were elucidated on the basis of their spectroscopic data. Compound 1 has previously been reported as a synthetic intermediate and this is the first time that it has been obtained from a natural source.

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.