Isolation and Pharmacological Evaluation of Minor Cannabinoids from High-Potency Cannabis sativa.

Seven new naturally occurring hydroxylated cannabinoids (1-7), along with the known cannabiripsol (8), have been isolated from the aerial parts of high-potency Cannabis sativa. The structures of the new compounds were determined by 1D and 2D NMR spectroscopic analysis, GC-MS, and HRESIMS as 8α-hydroxy-Δ(9)-tetrahydrocannabinol (1), 8ß-hydroxy-Δ(9)-tetrahydrocannabinol (2), 10α-hydroxy-Δ(8)-tetrahydrocannabinol (3), 10ß-hydroxy-Δ(8)-tetrahydrocannabinol (4), 10α-hydroxy-Δ(9,11)-hexahydrocannabinol (5), 9ß,10ß-epoxyhexahydrocannabinol (6), and 11-acetoxy-Δ(9)-tetrahydrocannabinolic acid A (7). The binding affinity of isolated compounds 1-8, Δ(9)-tetrahydrocannabinol, and Δ(8)-tetrahydrocannabinol toward CB1 and CB2 receptors as well as their behavioral effects in a mouse tetrad assay were studied. The results indicated that compound 3, with the highest affinity to the CB1 receptors, exerted the most potent cannabimimetic-like actions in the tetrad assay, while compound 4 showed partial cannabimimetic actions. Compound 2, on the other hand, displayed a dose-dependent hypolocomotive effect only.

Pharmacophore Modeling, Ensemble Docking, Virtual Screening, and Biological Evaluation on Glycogen Synthase Kinase-3ß.

Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine protein kinase which is engaged in a variety of signaling pathways, regulating a wide range of cellular processes. GSK-3ß, also known as tau protein kinase I (TPK-I), is one of the most important kinases implicated in the hyperphosphorylation of tau that leads to neurodegenerative diseases. Hence, GSK-3ß has emerged as an important therapeutic target. To identify compounds that are structurally novel and diverse compared to previously reported ATP-competitive GSK-3ß inhibitors, we performed virtual screening by implementing a mixed ligand/structure-based approach, which included pharmacophore modeling, diversity analysis, and ensemble docking. The sensitivities of different docking protocols to induced-fit effects were explored. An enrichment study was employed to verify the robustness of ensemble docking, using 13 X-ray structures of GSK-3ß, compared to individual docking in terms of retrieving active compounds from a decoy dataset. A total of 24 structurally diverse compounds obtained from the virtual screening underwent biological validation. The bioassay results showed that 15 out of the 24 hit compounds are indeed GSK-3ß inhibitors, and among them, one compound exhibiting sub-micromolar inhibitory activity is a reasonable starting point for further optimization.

Quantitative Structure-Activity Relationship Analysis and a Combined Ligand-Based/Structure-Based Virtual Screening Study for Glycogen Synthase Kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine protein kinase which regulates a wide range of cellular processes, involving various signalling pathways. GSK-3ß has emerged as an important therapeutic target for diabetes and Alzheimer's disease. To identify structurally novel GSK-3ß inhibitors, we performed virtual screening by implementing a combined ligand-based/structure-based approach, which included quantitative structure-activity relationship (QSAR) analysis and docking prediction. To integrate and analyze complex data sets from multiple experimental sources, we drafted and validated a hierarchical QSAR method, which adopts a two-level structure to take data heterogeneity into account. A collection of 728 GSK-3 inhibitors with diverse structural scaffolds was obtained from published papers that used different experimental assay protocols. Support vector machines and random forests were implemented with wrapper-based feature selection algorithms to construct predictive learning models. The best models for each single group of compounds were then used to build the final hierarchical QSAR model, with an overall R(2) of 0.752 for the 141 compounds in the test set. The compounds obtained from the virtual screening experiment were tested for GSK-3ß inhibition. The bioassay results confirmed that 2 hit compounds are indeed GSK-3ß inhibitors exhibiting sub-micromolar inhibitory activity, and therefore validated our combined ligand-based/structure-based approach as effective for virtual screening experiments.

Neocosmospora sp.-derived resorcylic acid lactones with in vitro binding affinity for human opioid and cannabinoid receptors.

Bioassay-guided fractionation of a fungus Neocosmospora sp. (UM-031509) resulted in the isolation of three new resorcylic acid lactones, neocosmosin A (2), neocosmosin B (3), and neocosmosin C (4). Three known resorcylic acid lactones, monocillin IV (1), monocillin II (5), and radicicol (6), were also isolated and identified. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analysis, mass spectrometric (ESIMS) data, and X-ray crystallography. Compounds 4-6 show good binding affinity for the human opioid receptors. These findings have important implications for evaluating the potential psychoactive effects with this class of compounds.

Benzyl derivatives with in vitro binding affinity for human opioid and cannabinoid receptors from the fungus Eurotium repens.

Bioassay-guided fractionation of the fungus Eurotium repens resulted in the isolation of two new benzyl derivatives, (E)-2-(hept-1-enyl)-3-(hydroxymethyl)-5-(3-methylbut-2-enyl)benzene-1,4-diol (1) and (E)-4-(hept-1-enyl)-7-(3-methylbut-2-enyl)-2,3-dihydrobenzofuran-2,5-diol (2), along with seven known compounds (3-9) including five benzaldehyde compounds, flavoglaucin (3), tetrahydroauroglaucin (4), dihydroauroglaucin (5), auroglaucin (6), and 2-(2',3-epoxy-1',3'- heptadienyl)-6-hydroxy-5-(3-methyl-2-butenyl)benzaldehyde (7), one diketopiperazine alkaloid, echinulin (8), and 5,7-dihydroxy-4-methylphthalide (9). The chemical structures of these compounds were established on the basis of extensive 1D and 2D NMR and HRMS data. Compounds 1-4 and 6 showed good binding affinity for human opioid or cannabinoid receptors. These findings have important implications for psychoactive studies with this class of compounds.

Biological activity of peanut (Arachis hypogaea) phytoalexins and selected natural and synthetic Stilbenoids.

The peanut plant (Arachis hypogaea L.), when infected by a microbial pathogen, is capable of producing stilbene-derived compounds that are considered antifungal phytoalexins. In addition, the potential health benefits of other stilbenoids from peanuts, including resveratrol and pterostilbene, have been acknowledged by several investigators. Despite considerable progress in peanut research, relatively little is known about the biological activity of the stilbenoid phytoalexins. This study investigated the activities of some of these compounds in a broad spectrum of biological assays. Since peanut stilbenoids appear to play roles in plant defense mechanisms, they were evaluated for their effects on economically important plant pathogenic fungi of the genera Colletotrichum, Botrytis, Fusarium, and Phomopsis. We further investigated these peanut phytoalexins, together with some related natural and synthetic stilbenoids (a total of 24 compounds) in a panel of bioassays to determine their anti-inflammatory, cytotoxic, and antioxidant activities in mammalian cells. Several of these compounds were also evaluated as mammalian opioid receptor competitive antagonists. Assays for adult mosquito and larvae toxicity were also performed. The results of these studies reveal that peanut stilbenoids, as well as related natural and synthetic stilbene derivatives, display a diverse range of biological activities.

Indolizidine, antiinfective and antiparasitic compounds from Prosopis glandulosa var. glandulosa.

A new potent antiinfective and antiparasitic 2,3-dihydro-1H-indolizinium chloride (1) was isolated from Prosopis glandulosa var. glandulosa. Three additional new (2-4) and one known (5) indolizidines were also isolated, and the dihydrochloride salts of 1-3 (compounds 6, 7, and 8) were prepared. Structures were determined by 1D and 2D NMR and mass spectra. Compound 1 showed potent in vitro antifungal activity against Cryptococcus neoformans and Aspergillus fumigatus (IC(50) values = 0.4 and 3.0 microg/mL, respectively) and antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare (IC(50) values of 0.35 and 0.9 microg/mL, respectively). The remarkable in vitro fungicidal activity of 1-4 against C. neoformans (MFCs = 0.63-1.25 microg/mL) and 2, 3, and 5 against A. fumigatus (MFCs = 0.63-2.5 microg/mL) were similar to amphotericin B, but >2-4-fold more potent than 6-8. Prosopilosidine (1) showed potent in vivo activity at 0.0625 mg/kg/day/ip for 5 days in a murine model of cryptococcosis by eliminating approximately 76% of C. neoformans infection from brain tissue compared to approximately 83% with amphotericin B at 1.5 mg/kg/day. Compounds 1 and 4 exhibited potent activity and high selectivity index (SI) values against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum, with IC(50) values of 39 and 95 ng/mL and 42 and 120 ng/mL, respectively (chloroquine, IC(50) = 17 and 140 ng/mL). Prosopilosine (1) also showed in vivo antimalarial activity, with an ED(50) value of approximately 2 mg/kg/day/ip against Plasmodium berghei-infected mice after 3 days of treatment.

Sepicanin A- A new geranyl flavanone from Artocarpus sepicanus with activity against methicillin-resistant Staphylococcus aureus (MRSA).

Bioassay-guided fractionation of the EtOH extract of Artocarpus sepicanus Diels leaves has led to the isolation of a new geranyl flavanone (1), along with the known compounds, afzelechin-3-O-α-L-rhamnopyranoside and ß-sitosterol glucoside. The structure of the new compound was established by UV, IR, HRESIMS, 1D and 2D NMR experiments. Antimicrobial testing of the three compounds indicated that 1 displayed a significant selective antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with IC50 and MIC values of 1.4 and 2.9 µM, respectively.

Sorocenols G and H, anti-MRSA oxygen heterocyclic Diels-Alder-type adducts from Sorocea muriculata roots.

Bioassay-guided fractionation of a root extract of Sorocea muriculata led to the isolation and identification of two new oxygen heterocyclic Diels-Alder-type adducts, sorocenols G (1) and H (2), along with lupeol-3-(3' R-hydroxytetradecanoate) and oxyresveratrol. The structures of 1 and 2 were elucidated using 1D and 2D NMR spectroscopic and HRMS data and by comparison with reported values. The absolute configurations of 1 and 2 were established by analysis of their experimental and theoretically calculated CD spectra. Compounds 1 and 2 showed significant and selective activity against methicillin-resistant Staphylococcus aureus with IC50 values of 1.5 and 0.5 microM, respectively. Compound 2 also displayed antifungal activity against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, with IC 50 values of 5.4, 5.4, and 10.0 microM, respectively.

Sinulodurins A and B, antiproliferative and anti-invasive diterpenes from the soft coral Sinularia dura.

Two new diterpenes, sinulodurin A (1) and sinulodurin B (2), along with two known sterols, 24 S-methyl cholesterol and 24-methylene cholesterol, were isolated from the Palau soft coral Sinularia dura. The structures of the new metabolites were determined on the basis of spectroscopic methods and by comparison of NMR data with those of related metabolites. Sinulodurin A (1) and sinulodurin B (2) showed antiproliferative activity against highly malignant +SA mammary epithelial cells with an IC 50 range of 20-30 microM. They also displayed anti-invasive activity against human highly metastatic prostate cancer PC-3M-CT+ cells in the spheroid disaggregation assay. Furthermore, the antimicrobial activities of the isolates were tested.

Potent in vitro antifungal activities of naturally occurring acetylenic acids.

Our continuing effort in antifungal natural product discovery has led to the identification of five 6-acetylenic acids with chain lengths from C(16) to C(20): 6-hexadecynoic acid (compound 1), 6-heptadecynoic acid (compound 2), 6-octadecynoic acid (compound 3), 6-nonadecynoic acid (compound 4), and 6-icosynoic acid (compound 5) from the plant Sommera sabiceoides. Compounds 2 and 5 represent newly isolated fatty acids. The five acetylenic acids were evaluated for their in vitro antifungal activities against Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Trichophyton mentagrophytes, and Trichophyton rubrum by comparison with the positive control drugs amphotericin B, fluconazole, ketoconazole, caspofungin, terbinafine, and undecylenic acid. The compounds showed various degrees of antifungal activity against the 21 tested strains. Compound 4 was the most active, in particular against the dermatophytes T. mentagrophytes and T. rubrum and the opportunistic pathogens C. albicans and A. fumigatus, with MICs comparable to several control drugs. Inclusion of two commercially available acetylenic acids, 9-octadecynoic acid (compound 6) and 5,8,11,14-eicosatetraynoic acid (compound 7), in the in vitro antifungal testing further demonstrated that the antifungal activities of the acetylenic acids were associated with their chain lengths and positional triple bonds. In vitro toxicity testing against mammalian cell lines indicated that compounds 1 to 5 were not toxic at concentrations up to 32 muM. Furthermore, compounds 3 and 4 did not produce obvious toxic effects in mice at a dose of 34 mumol/kg of body weight when administered intraperitoneally. Taking into account the low in vitro and in vivo toxicities and significant antifungal potencies, these 6-acetylenic acids may be excellent leads for further preclinical studies.

DNA synthesis and neuronal apoptosis caused by familial Alzheimer disease mutants of the amyloid precursor protein are mediated by the p21 activated kinase PAK3.

Apoptotic pathways and DNA synthesis are activated in neurons in the brains of individuals with Alzheimer disease (AD). However, the signaling mechanisms that mediate these events have not been defined. We show that expression of familial AD (FAD) mutants of the amyloid precursor protein (APP) in primary neurons in culture causes apoptosis and DNA synthesis. Both the apoptosis and the DNA synthesis are mediated by the p21 activated kinase PAK3, a serine-threonine kinase that interacts with APP. A dominant-negative kinase mutant of PAK3 inhibits the neuronal apoptosis and DNA synthesis; this effect is abolished by deletion of the PAK3 APP-binding domain or by coexpression of a peptide representing this binding domain. The involvement of PAK3 specifically in FAD APP-mediated apoptosis rather than in general apoptotic pathways is suggested by the facts that a dominant-positive mutant of PAK3 does not alone cause neuronal apoptosis and that the dominant-negative mutant of PAK3 does not inhibit chemically induced apoptosis. Pertussis toxin, which inactivates the heterotrimeric G-proteins Go and Gi, inhibits the apoptosis and DNA synthesis caused by FAD APP mutants; the apoptosis and DNA synthesis are rescued by coexpression of a pertussis toxin-insensitive Go. FAD APP-mediated DNA synthesis precedes FAD APP-mediated apoptosis in neurons, and inhibition of neuronal entry into the cell cycle inhibits the apoptosis. These data suggest that a normal signaling pathway mediated by the interaction of APP, PAK3, and Go is constitutively activated in neurons by FAD mutations in APP and that this activation causes cell cycle entry and consequent apoptosis.