Enantioselective synthesis of spirooxindole-pyran derivatives via a remote inverse-electron-demand Diels-Alder reaction of ß,γ-unsaturated amides.

Novel construction methods for obtaining 3,4'-pyran spirooxindole heterocyclic skeletons have always been the focus of attention. Herein, we report a highly enantioselective inverse-electron-demand oxa-Diels-Alder cycloaddition reaction of a ß,γ-unsaturated pyrazole amide and a N-diphenyl isatin-derived oxodiene using a bifunctional catalyst. In addition, large-scale experiments confirmed the reliability of the reaction. The resultant products of this study can be further transformed.

New Pyranone Derivatives and Sesquiterpenoid Isolated from the Endophytic Fungus Xylaria sp. Z184.

The fungus Xylaria sp. Z184, harvested from the leaves of Fallopia convolvulus (L.) Á. Löve, has been isolated for the first time. Chemical investigation on the methanol extract of the culture broth of the titles strain led to the discovery of three new pyranone derivatives, called fallopiaxylaresters A-C (1-3), and a new bisabolane-type sesquiterpenoid, named fallopiaxylarol A (4), along with the first complete set of spectroscopic data for the previously reported pestalotiopyrone M (5). Known pyranone derivatives (6-11), sesquiterpenoids (12-14), isocoumarin derivatives (15-17), and an aromatic allenic ether (18) were also co-isolated in this study. All new structures were elucidated by the interpretation of HRESIMS, 1D, 2D NMR spectroscopy, and quantum chemical computation approach. The in vitro antimicrobial, anti-inflammatory, and α-glucosidase-inhibitory activities of the selected compounds and the crude extract were evaluated. The extract was shown to inhibit nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells, with an inhibition rate of 77.28 ± 0.82% at a concentration of 50 µg/mL. The compounds 5, 7, and 8 displayed weak antibacterial activity against Staphylococcus areus subsp. aureus at a concentration of 100 µM.

seco-Pregnane Glycosides from Australian Caustic Vine (Cynanchum viminale subsp. australe).

Cynanchum viminale subsp. australe, more commonly known as caustic vine, is a leafless succulent that grows in the northern arid zone of Australia. Toxicity toward livestock has been reported for this species, along with use in traditional medicine and its potential anticancer activity. Disclosed herein are novel seco-pregnane aglycones cynavimigenin A (5) and cynaviminoside A (6), together with new pregnane glycosides cynaviminoside B (7) and cynavimigenin B (8). Cynavimigenin B (8) contains an unprecedented 7-oxobicyclo[2.2.1]heptane moiety in the seco-pregnane series, likely arising from a pinacol-type rearrangement. Interestingly, these isolates displayed only limited cytotoxicity in cancer and normal human cell lines, in addition to low activity against acetylcholinesterase and Sarcoptes scabiei bioassays, suggesting that 5-8 are not associated with the reported toxicity of this plant species.

EYES ABSENT and TIMELESS integrate photoperiodic and temperature cues to regulate seasonal physiology in Drosophila.

Organisms possess photoperiodic timing mechanisms to detect variations in day length and temperature as the seasons progress. The nature of the molecular mechanisms interpreting and signaling these environmental changes to elicit downstream neuroendocrine and physiological responses are just starting to emerge. Here, we demonstrate that, in Drosophila melanogaster, EYES ABSENT (EYA) acts as a seasonal sensor by interpreting photoperiodic and temperature changes to trigger appropriate physiological responses. We observed that tissue-specific genetic manipulation of eya expression is sufficient to disrupt the ability of flies to sense seasonal cues, thereby altering the extent of female reproductive dormancy. Specifically, we observed that EYA proteins, which peak at night in short photoperiod and accumulate at higher levels in the cold, promote reproductive dormancy in female D. melanogaster Furthermore, we provide evidence indicating that the role of EYA in photoperiodism and temperature sensing is aided by the stabilizing action of the light-sensitive circadian clock protein TIMELESS (TIM). We postulate that increased stability and level of TIM at night under short photoperiod together with the production of cold-induced and light-insensitive TIM isoforms facilitate EYA accumulation in winter conditions. This is supported by our observations that tim null mutants exhibit reduced incidence of reproductive dormancy in simulated winter conditions, while flies overexpressing tim show an increased incidence of reproductive dormancy even in long photoperiod.

SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila.

Circadian rhythms are generated by endogenous pacemakers that rely on transcriptional-translational feedback mechanisms conserved among species. In Drosophila, the stability of a key pacemaker protein PERIOD (PER) is tightly controlled by changes in phosphorylation status. A number of molecular players have been implicated in PER destabilization by promoting PER progressive phosphorylation. On the other hand, there have been few reports describing mechanisms that stabilize PER by delaying PER hyperphosphorylation. Here we report that the protein Suppressor of Ras (SUR-8) regulates circadian locomotor rhythms by stabilizing PER. Depletion of SUR-8 from circadian neurons lengthened the circadian period by about 2 hours and decreased PER abundance, whereas its overexpression led to arrhythmia and an increase in PER. Specifically SUR-8 promotes the stability of PER through phosphorylation regulation. Interestingly, downregulation of the protein phosphatase 1 catalytic subunit PP1-87B recapitulated the phenotypes of SUR-8 depletion. We found that SUR-8 facilitates interactions between PP1-87B and PER. Depletion of SUR-8 decreased the interaction of PER and PP1-87B, which supports the role of SUR-8 as a scaffold protein. Interestingly, the interaction between SUR-8 and PER is temporally regulated: SUR-8 has more binding to PER at night than morning. Thus, our results indicate that SUR-8 interacts with PP1-87B to control PER stability to regulate circadian rhythms.

Splice variants of DOMINO control Drosophila circadian behavior and pacemaker neuron maintenance.

Circadian clocks control daily rhythms in behavior and physiology. In Drosophila, the small ventral lateral neurons (sLNvs) expressing PIGMENT DISPERSING FACTOR (PDF) are the master pacemaker neurons generating locomotor rhythms. Despite the importance of sLNvs and PDF in circadian behavior, little is known about factors that control sLNvs maintenance and PDF accumulation. Here, we identify the Drosophila SWI2/SNF2 protein DOMINO (DOM) as a key regulator of circadian behavior. Depletion of DOM in circadian neurons eliminates morning anticipatory activity under light dark cycle and impairs behavioral rhythmicity in constant darkness. Interestingly, the two major splice variants of DOM, DOM-A and DOM-B have distinct circadian functions. DOM-A depletion mainly leads to arrhythmic behavior, while DOM-B knockdown lengthens circadian period without affecting the circadian rhythmicity. Both DOM-A and DOM-B bind to the promoter regions of key pacemaker genes period and timeless, and regulate their protein expression. However, we identify that only DOM-A is required for the maintenance of sLNvs and transcription of pdf. Lastly, constitutive activation of PDF-receptor signaling rescued the arrhythmia and period lengthening of DOM downregulation. Taken together, our findings reveal that two splice variants of DOM play distinct roles in circadian rhythms through regulating abundance of pacemaker proteins and sLNvs maintenance.

Anti-Neuroinflammatory Activity of New Naturally Occurring Benzylated Hydroxyacetophenone Analogs from the Endophytic Fungus Alternaria sp. J030.

Chemical studies on the culture broth of the endophytic fungus Alternaria sp. J030 led to the identification of three benzylated hydroxyacetophenone derivatives, bauvaroalterins A-C (1-3), and 34 structurally diverse metabolites (4-37). The new structures were elucidated by extensive spectroscopic analyses including UV, IR, 1D and 2D NMR, HR-ESI-MS, and further confirmed using single crystal X-ray diffraction. The in vitro anti-neuroinflammatory effects of the co-isolated metabolites were evaluated in lipopolysaccharide (LPS)-stimulated microglial cells. Compounds 1-3 were shown to significantly reduce LPS-induced NO production by inhibiting the expression of iNOS, as well as inhibiting LPS-induced production of the inflammatory factors TNF-α, IL-1ß and IL-6. Further studies revealed that 1-3 were capable of down-regulating the expression of NF-κB subunits p50 and p65, thereby suppressing the activation of NF-κB by inhibiting the LPS-induced phosphorylation of IκB-α. Together these findings demonstrate that bauvaroalterins A-C (1-3) exert anti-neuroinflammatory effects via inhibition of the NF-κB/iNOS signalling pathway in LPS induced BV-2 cells.

Glycosylated coumarins, flavonoids, lignans and phenylpropanoids from Wikstroemia nutans and their biological activities.

Wikstroemia nutans Champ. ex Benth., a traditional herbal medicine collected at the Lingnan region of China, was chemically investigated. A new biscoumarin glucoside, wikstronutin (1), along with three known bis- and tricoumarin glucosides (2-4), two flavonoid glycosides (5-6), and eleven lignan glucosides (7-17) were isolated from the stems and roots of W. nutans. The new structure including its absolute configuration was elucidated based on a combination of 1D and 2D NMR, UV, IR, HRESIMS spectroscopic data, as well as chemical transformation. Compounds 1-17 were first isolated from the plant species W. nutans, while compounds 1-3, 8, and 11 were reported from the genus Wikstroemia for the first time. All co-isolates were evaluated for their in vitro inhibitory effects on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in murine RAW264.7 macrophage cells. The antibacterial activity of the selected compounds was also tested. Our work enriches the structure diversity of the secondary metabolites from the genus Wikstroemia.

Regioisomers Salviprolin A and B, Unprecedented Rosmarinic Acid Conjugated Dinorditerpenoids from Salvia przewalskii Maxim.

Salvia przewalskii Maxim is a perennial plant from the genus Salvia (family Lamiaceae). The roots of S. przewalskii were long used as a traditional herb to treat blood circulation related illnesses in China. As part of our continuing interest in polycyclic natural products from medicinal plants, two unprecedented adducts comprised of a dinor-diterpenoid and a 9'-nor-rosmarinic acid derivative, linked by a 1,4-benzodioxane motif (1 and 2), were isolated from the roots of S. przewalskii. Their structures were established by extensive spectroscopic approaches including 1D, 2D NMR, and HRFABMS. Their cytotoxic activities against five human tumor cell lines were evaluated.

Przewalcyrones A-F, epoxychromene-containing polycyclic polyprenylated acylphloroglucinols with immunosuppressive activity from Hypericum przewalskii Maxim.

Chemical investigation of the extracts of the aerial parts of Hypericum przewalskii Maxim. resulted in the isolation and identification of six new epoxychromene-containing polycyclic polyprenylated acylphloroglucinols (PPAPs), named przewalcyrones A-F (1-6), and one known analogue (7). All of the structures were determined based on extensive spectroscopic analyses, X-ray crystallographic analysis, modified Mosher's method, [Rh2(OCOCF3)4]-induced ECD, and electronic circular dichroism (ECD) comparison. Structurally, przewalcyrones A-F represent the first examples of PPAPs containing an unexpected 8,8-dimethyl-3,9-epoxychromene moiety. All these compounds were evaluated for the immunosuppressive activity in anti-CD3/anti-CD28 monoclonal antibody (mAb)-stimulated human T cells. Among them, przewalcyrones C and D exhibited potential in vitro immunosuppressive activity, with IC50 values of (5.01 ± 0.52) µM and (5.26 ± 0.56) µM, respectively, highlighting those compounds as a promising starting point for the development of new immunosuppressive agents.

HPLC-DAD-Directed Isolation of Linearly Fused Prenylated Indole Alkaloids from a Soil-Derived Aspergillus versicolor.

An HPLC-DAD-directed chemical investigation of the soil-derived fungus Aspergillus versicolor QC812 resulted in the isolation and identification of eight new linearly fused prenylated indole alkaloids, asperversiamides I-P (1-8), along with a congener, asperversiamide H (9). Their structures and absolute configurations were determined by spectroscopic analysis including HRESIMS and 1D and 2D NMR, electronic circular dichroism analysis, and single-crystal X-ray diffraction. Asperversiamide I (1), the first diketopiperazine derived from d-proline and l-tryptophan, possesses an unprecedented C-11-spiro-fused 6/6/5/5/6/5 hexacyclic ring system. Asperversiamide J (2) is the first linearly fused 6/6/5 tricyclic prenylated indole alkaloid to be reported. 1 and 2 showed moderate inhibitory activities against HeLa cells with IC50 values of 7.3 and 6.4 µM, respectively.

Asperpyridone A: An Unusual Pyridone Alkaloid Exerts Hypoglycemic Activity through the Insulin Signaling Pathway.

A pyridone alkaloid, asperpyridone A (1), which possesses an unusual pyrano[3,2-c]pyridine scaffold, was isolated from solid cultures of the endophytic fungus Aspergillus sp. TJ23. Its structure, including its absolute configuration, was determined using a combination of nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, quantum chemical calculations (electronic circular dichroism), and X-ray crystallography. In vitro bioassays demonstrated that asperpyridone A (1) could function as a potential hypoglycemic agent, which exhibited pronounced glucose uptake effect in liver HepG2 cells, under both normal and insulin-resistant conditions, with higher efficacy than metformin. The underlying mechanism of asperpyridone A was elucidated by analyzing the genes expressed, the Gene Ontology (GO) function enrichment, the protein interaction network, and real-time quantitative reverse transcription polymerase chain reaction, which suggested that asperpyridone A exhibits hypoglycemic activity by activating the insulin signaling pathway. Moreover, on the basis of the hypoglycemic potency, fibroblast growth factor 21 (FGF21) was determined to be a potential target for asperpyridone A.

Basimarols A, B, and C, Highly Oxygenated Pimarane Diterpenoids from Basilicum polystachyon.

The highly oxygenated pimarane diterpenoids basimarols A, B, and C (3-5) were isolated from the plant species Basilicum polystachyon, which was collected within the Australian arid zone. Structure elucidation was performed using a suite of spectroscopic techniques, including X-ray crystallography. Anticancer and anti-DENV activity of 3-5 was explored, but only limited activity was observed. More extensive antiviral evaluation of stachyonic acid A (1), which was also isolated from B. polystachyon, revealed broad spectrum antiviral activity against West Nile virus (Kunjin strain, WNVKun) and human influenza viruses H1N1 and H3N2.

Emerging roles for microRNA in the regulation of Drosophila circadian clock.

BACKGROUND: The circadian clock, which operates within an approximately 24-h period, is closely linked to the survival and fitness of almost all living organisms. The circadian clock is generated through a negative transcription-translation feedback loop. microRNAs (miRNAs) are small non-coding RNAs comprised of approximately 22 nucleotides that post-transcriptionally regulate target mRNA by either inducing mRNA degradation or inhibiting translation. RESULTS: In recent years, miRNAs have been found to play important roles in the regulation of the circadian clock, especially in Drosophila. In this review, we will use fruit flies as an example, and summarize the progress achieved in the study of miRNA-mediated clock regulation. Three main aspects of the circadian clock, namely, the free-running period, locomotion phase, and circadian amplitude, are discussed in detail in the context of how miRNAs are involved in these regulations. In addition, approaches regarding the discovery of circadian-related miRNAs and their targets are also discussed. CONCLUSIONS: Research in the last decade suggests that miRNA-mediated post-transcriptional regulation is crucial to the generation and maintenance of a robust circadian clock in animals. In flies, miRNAs are known to modulate circadian rhythmicity and the free-running period, as well as circadian outputs. Further characterization of miRNAs, especially in the circadian input, will be a vital step toward a more comprehensive understanding of the functions underlying miRNA-control of the circadian clock.

Silver-Mediated Cyanomethylation of Cinnamamides by Direct C(sp3)-H Functionalization of Acetonitrile.

An efficient, silver-induced tandem radical addition/cyclization for the synthesis of 3,4-dihydroquinolinones is presented, which exhibits a good functional group tolerance. The reaction is easy to operate and amenable to a multigram-scale synthesis. Additionally, this work illustrates the formation of a key skeleton for the synthesis of biologically interesting 3,4-dihydroquinolinone alkaloids.

Asperversiamides, Linearly Fused Prenylated Indole Alkaloids from the Marine-Derived Fungus Aspergillus versicolor.

Asperversiamides A-H (1-8), eight linearly fused prenylated indole alkaloids featuring an unusual pyrano[3,2- f]indole unit, were isolated from the marine-derived fungus Aspergillus versicolor. The structures and absolute configurations of these compounds were elucidated by extensive spectroscopic analyses, single-crystal X-ray diffraction, electronic circular dichroism (ECD) calculations, and optical rotation (OR) calculations. The relative configuration of C-21 of iso-notoamide B was herein revised, and a new methodology for preliminarily determining if the relative configuration of the bicyclo[2.2.2]diazaoctane moiety of a spiro-bicyclo[2.2.2]diazaoctane-type indole alkaloid is syn or anti was developed. The anti-inflammatory activities of the isolated compounds were all tested, and of these compounds, 7 exhibited a potent inhibitory effect against iNOS with an IC50 value of 5.39 µM.

Bioactive polycyclic polyprenylated acylphloroglucinols from Hypericum perforatum.

Fifteen new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperforatones A-O (1-15), along with 3 structurally related analogues (16-18), were isolated from the stems and leaves of Hypericum perforatum. Their structures and absolute configurations were established by a combination of NMR spectroscopic analyses, experimental and calculated electronic circular dichroism (ECD), modified Mosher's methods, Rh2(OCOCF3)4- and [Mo2(OAc)4]-induced ECD, X-ray crystallography, and the assistance of quantum chemical predictions (QCP) of 13C NMR chemical shifts. Compound 5 was found to be the first PPAP decorated by a rare 2,2,4,4,5-(pentamethyltetrahydrofuran-3-yl)methanol moiety and an oxepane ring. Furthermore, the isolates were screened for their acetylcholinesterase (AChE) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities. Compounds 5, 10, 11, and 15 showed desirable AChE inhibitory activities (IC50 6.9-9.2 µM) and simultaneously inhibited BACE1 (at a concentration of 5 µM) with inhibition rates of 50.3%, 34.3%, 47.2%, and 34.6%, respectively. Interestingly, compound 5 showed the most balanced inhibitory activities against both AChE and BACE1 of all the tested compounds, which means that 5 could serve as the first valuable dual-targeted PPAP for the treatment of Alzheimer's disease. Preliminary molecular docking studies of 5 with BACE1 and AChE were also performed.

New 3,5-dimethylorsellinic acid-based meroterpenoids with BACE1 and AchE inhibitory activities from Aspergillus terreus.

Chemical investigation of the extracts of Aspergillus terreus resulted in the identification of terreusterpenes A-D (1-4), four new 3,5-dimethylorsellinic acid-based meroterpenoids. The structures and absolute configurations of 1-4 were elucidated by spectroscopic analyses including HRESIMS and 1D- and 2D-NMR, chemical conversion, and single crystal X-ray diffraction. Terreusterpenes A (1) and B (2) featured 2,3,5-trimethyl-4-oxo-5-carboxy tetrahydrofuran moieties. Terreusterpene D (4) was characterized by a 4-hydroxy-3-methyl gamma lactone fragment that was generated by accident from the rearrangement of 3 in a mixed tetrahydrofuran-H2O-MeOH solvent. All these compounds were evaluated for the ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and acetylcholinesterase (AchE) inhibitory activities. Among them, compounds 1 and 2 showed potentially significant BACE1 inhibitory activity, with IC50 values of 5.98 and 11.42 µM, respectively. Interestingly, compound 4 exhibited promising BACE1 and AchE inhibitory activities, with IC50 values of 1.91 and 8.86 µM, respectively, while 3 showed no such activity. Taken together, terreusterpenes A and B could be of great importance for the development of new BACE1 inhibitors, while terreusterpene D could serve as the first dual-targeted 3,5-dimethylorsellinic acid-based meroterpenoid for the treatment of Alzheimer's disease.

Cytochalasans Produced by the Coculture of Aspergillus flavipes and Chaetomium globosum.

The cocultivation of Aspergillus flavipes and Chaetomium globosum, rich sources of cytochalasans, on solid rice medium, resulted in the production of 13 new, highly oxygenated cytochalasans, aspochalasinols A-D (1-4) and oxichaetoglobosins A-I (5-13), as well as seven known compounds (14-20). Of these compounds, 13 is a novel cytochalasan with an unexpected 2-norindole group. The isolated compounds were characterized by NMR spectroscopy, single-crystal X-ray crystallography, and ECD experiments. Compounds 1-4 represent the first examples of Asp-type cytochalasans with C-12 hydroxy groups, which may be a result of the coculture, as hydroxylated Me-12 groups are frequently found in Chae-type cytochalasans from C. globosum. In addition, 5-10 are unusual cytochalasans with an oxygenated C-10. Interestingly, 13 is the first example of a naturally occurring cytochalasan possessing a uniquely degraded indole ring that is derived from chaetoglobosin W, with 11 and 12 both serving as its biosynthetic intermediates. In the coculture of A. flavipes and C. globosum, most of these cytochalasans are more functionalized than normal cytochalasans, and the underlying causes may attract substantial attention from synthetic biologists. The cytotoxicities against five human cancer cell lines (SW480, HL-60, A549, MCF-7, and SMMC-7721) and the immunomodulatory activities of these new compounds were evaluated in vitro.

BACE1 Inhibitory Meroterpenoids from Aspergillus terreus.

Sixteen 3,5-dimethylorsellinic acid-based (DMOA-based) meroterpenoids, including 10 new compounds, asperterpenes D-M (1-10), were obtained from Aspergillus terreus. The structures and absolute configurations of the new compounds were confirmed by extensive spectroscopy, single-crystal X-ray diffraction analysis, and experimental electronic circular dichroism (ECD) measurements. Compounds 2, 3, and 7 are the first 3,5-dimethylorsellinic acid-based meroterpenoids possessing a unique cis-fused A/B ring system. These new compounds were evaluated for their inhibitory activity against ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1). Compounds 2, 3, and 7, the first 3,5-dimethylorsellinic acid-based meroterpenoids possessing cis-fused A/B rings, exhibited significant inhibitory activities against BACE1 with IC50 values of 3.3, 5.9, and 31.7 µM, respectively.