Arteperoxides A-C, tris-normonoterpene-sesquiterpene conjugates with peroxide-bridges from Artemisia judaica exhibiting antiosteoclastogenic activity.

Antiosteoclastogenic-guided screening was conducted with 120 extracts of the medicinal plants collected in Egypt that led to the selection of Artemisia judaica L. (Asteraceae). Three undescribed davanone-related terpenoids, arteperoxides A-C, were isolated from the extract with two known derivatives, hydroxydavanone and davana acid. Structural analysis revealed that arteperoxides A-C were tris-normonoterpene-sesquiterpene conjugates with peroxide bridges. Although davanone derivatives with peroxides, such as a hydroperoxyl and peroxyhemiketal groups, have been isolated from Artemisia species, arteperoxides A-C are the first variations observed to contain peroxide bridges between two terpene-derived units. The absolute configurations of arteperoxides A and B were studied based on their spectroscopic data compared with those of the semisynthetic analogs that have ether linkages. The natural and synthetic compounds were tested for the antiosteoclastogenic activity, and arteperoxide C and hydroxydavanone were more potent than other compounds at 20 µM.

Structural analysis of previously unknown natural products using computational methods.

Natural products exhibit structural diversity, and biologically active natural products with unprecedented molecular skeletons can potentially be isolated from natural resources in the future. Although it has often been difficult to determine the structures and configurations of new compounds that do not resemble known compounds, the determination of the chemical structures, including the absolute stereo configuration, is very important in drug discovery research. In our efforts to find new bioactive natural products, we have identified novel compounds such as the ubiquitin-proteasome system inhibitors and osteoclast differentiation inhibitors. Various natural products, mixtures of stereoisomers of natural products, and compounds with novel skeletal structures were studied. In cases where it was difficult to determine the structures by NMR spectroscopy, we could successfully determine the chemical structures by computational chemistry. This review presents the results of structural analysis obtained using computational methods for several natural products that we have recently isolated.

Isolation, Synthesis, and Structure-Activity Relationship Study on Daphnane and Tigliane Diterpenes as HIV Latency-Reversing Agents.

Three new diterpenes, stellejasmins A (1) and B (2) and 12-O-benzoylphorbol-13-heptanoate (3), were isolated from the roots of Stellera chamaejasme L. The structures of 1-3 were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 are the first derivatives containing a hydroxy group at C-2 in the family of daphnane and tigliane diterpenes. The presence of a chlorine atom in 1 is unique in the plant metabolite. Compound 3 has an odd-number acyl group, which is biosynthetically notable. Human immunodeficiency virus (HIV) LTR-driven transcription activity was tested with 1-3 and 17 known diterpenes isolated from S. chamaejasme L. and Wikstroemia retusa A.Gray. Among these, gnidimacrin (4), stelleralide A (5), and wikstroelide A (20) were highly potent, with EC50 values of 0.14, 0.33, and 0.39 nM, respectively. The structure-activity relationship (SAR) was investigated using 20 natural and eight synthetic diterpenes. This is the first SAR study on natural daphnane and tigliane diterpenes.

Cell-based screening of extracts of natural sources to search for inhibitors of the ubiquitin-proteasome system and identification of proteasome inhibitors from the fungus Remotididymella sp.

The ubiquitin-proteasome system (UPS) regulates selective protein degradation to maintain protein homeostasis. Small molecules that inhibit the UPS-dependent protein degradation are promising anti-tumor agents. We report a cell-based luminescent assay using HeLa cells expressing luciferase-fused oxygen-dependent destruction domain (ODD) of hypoxia-inducible factor 1 α (HIF-1 α). ODD is degraded by the UPS and this assay system can aid in the identification of natural products that inhibit either process of the UPS, including ubiquitination/deubiquitination and proteasomal degradation. This reporter assay can exclude the influences of coloring or fluorescent compounds in extracts, thereby leading to effective high-throughput processing. The screening of 15,025 extracts of natural sources identified the culture extract of the fungus Remotididymella sp. (18F02908). Bioassay-guided isolation yielded two new polyketides, mellains A (1) and B (2), together with leptosphaerodione (3) and its acetone adduct 4. Compound 1 was revealed to have an unprecedented benzo[g]isoquinoline-8,10-dione skeleton. Evaluation of the biological activities demonstrated that these polyketides inhibit the proteasomal proteolysis. This is the first report of the identification of proteasome inhibitors from natural sources using a cell-based reporter assay targeting UPS inhibitors.

Chamaejasmins, cytotoxic guaiane sesquiterpenes from the root of Stellera chamaejasme L.

Four new guaiane-type sesquiterpenes, chamaejasmins A-D (1-4), were isolated from the root of Stellera camaejasme L. collected in Nepal, together with two known terpenes, stelleraguaianone B (5) and 1α,7α,10αH-guaia-4,11-dien-3-one (6). The structures of 1-4 including their absolute configurations were determined by extensive 2D NMR analyses, mass spectroscopy, and TDDFT calculations of their 13C chemical shifts and ECD spectra. Chamaejasmin A (1) showed cytotoxicity against HeLa cells with an IC50 value of 6.3 µM.

Induction of secondary metabolite production by fungal co-culture of Talaromyces pinophilus and Paraphaeosphaeria sp.

Fungal co-culture is a strategy to induce the production of secondary metabolites by activating cryptic genes. We discovered the production of a new compound, talarodone A (1), along with five known compounds 2-6 in co-culture of Talaromyces pinophilus and Paraphaeosphaeria sp. isolated from soil collected in Miyazaki Prefecture, Japan. Among them, the productions of penicidones C (2) and D (3) were enhanced 27- and sixfold, respectively, by the co-culture. The structure of 3 should be represented as a γ-pyridol form with the reported chemical shifts, but not as a γ-pyridone form, based on DFT calculation.

New inhibitors of RANKL-induced Osteoclastogenesis from the marine sponge Siphonochalina siphonella.

New sipholane type triterpenes, sipholenols N and O (1 and 2) and neviotine D (3), were isolated from the Red Sea marine sponge Siphonochalina siphonella along with four known triterpenes, sipholenone A (4), sipholenol A (5), siphonellinol D (6) and neviotine A (7). Structure elucidation of 1-3 was achieved by extensive 1D and 2D NMR analyses. The isolated compounds were examined for the inhibition of RANKL induced osteoclastogenesis in RAW264 macrophages. Neviotine D (3) and neviotine A (4) showed potent inhibition with IC50 values of 12.8 and 32.8 µM, respectively.

New geranyl flavonoids from the leaves of Artocarpus communis.

Four new geranyl flavonoids 1-4 and four known flavonoids 5-8 were obtained from the leaves of Artocarpus communis collected in Indonesia. The planar structures of flavonoids were elucidated by analyses of MS and NMR spectroscopic data. Absolute configurations of 1 and 2 were determined by ECD spectroscopy. Analyses by HPLC with a chiral-phase column and ECD spectra confirmed that 3 and 4 were stereoisomeric mixtures and 7 and 8 were racemic mixtures. The compounds obtained in this study inhibited the enzymatic activities of ubiquitin-specific protease 7 (USP7) and the chymotrypsin-like activity of the proteasome. Among the geranyl flavonoids tested in this experiment, the USP7 inhibitory activity of 6 (IC50 value, 0.094 µM) was 55 times more potent than the commercially available positive control, P5091 (IC50 value, 5.2 µM).

Ceylonins G-I: spongian diterpenes from the marine sponge Spongia ceylonensis.

Three new spongian diterpenes, ceylonins G-I (1-3), were isolated from the marine sponge Spongia ceylonensis collected in Indonesia, together with five known spongian diterpenes (4-8). Only 4 inhibited USP7 with an IC50 value of 8.2 µM.

Strongylophorines, meroditerpenoids from the marine sponge Petrosia corticata, function as proteasome inhibitors.

Two new strongylophorine derivatives, along with six known strongylophorines, were isolated from the marine sponge Petrosia corticata as proteasome inhibitors. Of these, a hemiacetal mixture of strongylophorines-13/-14 was the strongest inhibitor of the proteasome with an IC50 of 2.1µM.

Manadodioxans A-E: polyketide endoperoxides from the marine sponge Plakortis bergquistae.

Five new polyketide endoperoxides, manadodioxans A-E, were isolated from the marine sponge Plakortis bergquistae. Manadodioxan E showed antimicrobial activity against Escherichia coli at 10 µg/disk, while its oxo congener, manadodioxan D, was inactive.

Variabines A and B: new ß-carboline alkaloids from the marine sponge Luffariella variabilis.

Two new ß-carboline alkaloids, variabines A (1) and B (2), were isolated from the Indonesian marine sponge Luffariella variabilis. Their structures were elucidated from spectral data, and 1 was found to be a sulfonated derivative of 2. Although numerous ß-carboline alkaloids have been isolated from natural sources to date, 1 is the first ß-carboline derivative containing a sulfate group. Compound 2 inhibited chymotrypsin-like activity of the proteasome and Ubc13 (E2)-Uev1A interaction with IC50 values of 4 and 5 µg/mL, respectively, whereas 1 had little effect on the activity or interaction.

Anti-inflammatory constituents from Tabebuia avellanedae.

Five novel compounds were isolated from the water extract of Tabebuia avellanedae, and their structures were established by analysis of NMR spectroscopy and mass spectrometry. Compounds 1-5 at 25µM showed strong inhibitory activity on the inflammatory cytokine, tumor-necrosis factor-α and interleukin-1ß production in cultured human myeloma THP-1 cells co-stimulated with lipopolysaccharide without any significant cytotoxicity, and their anti-allergic and antioxidant activities were evaluated.

Phenolic lipid ingredients from cashew nuts.

Five new phenolic lipids, 2-(8"Z-eicosenoyl)-6-(8'Z-pentadecenyl) salicylic acid (3), 2-(9"Z-hexadecenoyl)-6-(8'Z, 11'Z-pentadecadienyl) methyl salicylate (5), 2-(10"Z, 13"Z-nonadecadienoyl)-6-(8'Z, 11'Z-pentadecadienyl) salicylic acid (6), 2-(16"Z-pentacosenoyl)-6-(8'Z-pentadecenyl) salicylic acid (7) and 2-(9"Z-octadecenoyl)-6-(8'Z, 11'Z-pentadecadienyl) methyl salicylate (8), and three known compounds, cardols (1), anacardic acid (2) and cardanols (4), were isolated from the nuts of the cashew, Anacardium occidentale L. The structures were established on the basis of detailed MS and NMR spectroscopic analyses. Compound 1 highly enhanced both Th-1 (IL-2, IFN-γ) and Th-2 (IL-4, IL-5) cytokine production, and compounds 7 and 8 highly increased cytokine IL-2 and IFN-γ production in response to concanavalin A in cultured murine Peyer's patch cells ex vivo. The isolated compounds showed moderate inhibitory activities on cytochrome CYP3A4 enzyme.

Stilbenoids from the melinjo (Gnetum gnemon L.) fruit modulate cytokine production in murine Peyer's patch cells ex vivo.

Melinjo fruit ( Gnetum gnemon L.) has been used as a food in Southeast Asia. To investigate if this fruit has regulatory actions on ileal immune responses, we measured T-helper (Th) cytokine production, i.e., interleukin-2 (IL-2), IL-4, IL-5, and interferon-gamma (IFN- γ), in cultured Peyer's patch (PP) cells from mice orally treated with a methanol extract of melinjo fruit. Oral administration of the 50 % ethanol extract at 100 mg/kg/day significantly enhanced the production of the Th1 cytokines IL-2 and IFN- γ irrespective of concanavalin-A stimulation, whereas the production of the Th2 cytokines IL-4 and IL-5 was not affected. We also isolated seven active constituents accompanied with two new stilbenoids from the ethylacetate fraction of the extracts. The structure of the new stilbene glucosides gnemonoside L (5) and gnemonoside M (7) was determined by 1D and 2D NMR and MS analyses. Five known stilbenoids were identified as resveratrol (1), isorhapontigenin (2), gnemonoside D (4), gnetins C (3) and E (6). Among these tested compounds, only new stilbenoid 7 strongly enhanced Th1 cytokine production in cultured PP cells at 10 mg/kg/day. These results indicated that this melinjo extract and its active constituent 7 potentiated T-cell-dependent immune responses in the ileal mucosa.