Chaetoxylariones A-G: undescribed chromone-derived polyketides from co-culture of Chaetomium virescens and Xylaria grammica enabled via the molecular networking strategy.

By co-culturing two endophytic fungi (Chaetomium virescens and Xylaria grammica) collected from the medicinal and edible plant Smilax glabra Roxb. and analyzing them with MolNetEnhancer module on GNPS platform, seven undescribed chromone-derived polyketides (chaetoxylariones A-G), including three pairs of enantiomer ones (2a/2b, 4a/4b and 6a/6b) and four optical pure ones (1, 3, 5 and 7), as well as five known structural analogues (8-12), were obtained. The structures of these new compounds were characterized by NMR spectroscopy, single-crystal X-ray diffraction, 13C NMR calculation and DP4+ probability analyses, as well as the comparison of the experimental electronic circular dichroism (ECD) data. Structurally, compound 1 featured an unprecedented chromone-derived sulfonamide tailored by two isoleucine-derived δ-hydroxy-3-methylpentenoic acids via the acylamide and NO bonds, respectively; compound 2 represented the first example of enantiomeric chromone derivative bearing a unique spiro-[3.3]alkane ring system; compound 3 featured a decane alkyl side chain that formed an undescribed five-membered lactone ring between C-7' and C-10'; compound 4 contained an unexpected highly oxidized five-membered carbocyclic system featuring rare adjacent keto groups; compound 7 featured a rare methylsulfonyl moiety. In addition, compound 10 showed a significant inhibition towards SW620/AD300 cells with an IC50 value of PTX significantly decreased from 4.09 µM to 120 nM, and a further study uncovered that compound 10 could obviously reverse the MDR of SW620/AD300 cells.

Dipeniroqueforins A-B and Peniroqueforin D: Eremophilane-Type Sesquiterpenoid Derivatives with Cytotoxic Activity from Penicillium roqueforti.

Guided by the Global Natural Products Social (GNPS) molecular networking strategy, five undescribed eremophilane-type sesquiterpenoid derivatives (1-5) were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of plant Hypericum beanii collected in Shennongjia Forestry District, Hubei Province. Dipeniroqueforins A-B (1-2), representing a lactam-type sesquiterpenoid skeleton with a highly symmetrical and homodimeric 5/6/6-6/6/5 hexacyclic system, are reported within the eremophilane-type family for the first time. Peniroqueforin D (5) represents the first example of a 1,2-seco eremophilane-type sesquiterpenoid derivative featuring an undescribed 7/6-fused ring system. The structures of these compounds were elucidated by various spectroscopic analyses, DP4+ probability analyses, ECD calculations, and single-crystal X-ray diffraction experiments. Furthermore, these isolates were evaluated for cytotoxicity, and the result uncovered that compound 1 displayed broad-spectrum activity. Further mechanistic study revealed that compound 1 could significantly upregulate the mRNA expression of genes related to the oxidative induction, leading to the abnormal ROS levels in tumor cells and ultimately causing tumor cell apoptosis.

New secondary metabolites with cytotoxicity from fungus Penicillium roqueforti.

Two novel compounds including a cyclohelminthol type polyketide (namely oxaleimide K, 1) and a maleimide derivative (namely peniroquefortine A, 2), and a new natural product (namely 2-(acetylamino)-N-[(1E)-2-phenylethenyl]-acetamide, 3), together with four known compounds (4-7), were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of Hypericum beanii N. Robson collected from the Shennongjia Forestry District, Hubei Province. Their structures including absolute configurations were mainly established by the NMR spectroscopy analyses and single-crystal X-ray diffraction experiment. Compound 1 represents the second example of a cyclohelminthol type polyketide, which features a rare 6/6/5/5 tetracyclic system and a branched aliphatic chain containing a terminal olefin (oct-1-en-3-yl) moiety, and compound 2 possesses an unprecedented carbon skeleton that is uniquely defined by a maleimide moiety linked to the respective 4-methylene-2-(3-methylbut-2-en-1-yl)-phenol and para-substituted aromatic moieties via the carbon-carbon bonds. Remarkably, the absolute configuration of a cyclohelminthol type polyketide as exemplified by compound 1 is determined by the single-crystal diffraction analysis for the first time, highlighting an E-configuration for the linkage of a succinimide moiety and a tetrahydrofuran moiety for 1 rather than a Z-configuration as previously reported in the biosynthesis study, which gives a new insight into the structural elucidation of this category of polyketides. Additionally, compound 1 exhibited significant cytotoxic activity against multiple tumor cells, especially against the Farage and SU-DHL-2 cells (IC50 < 20 µM, 48 h). Further mechanism study revealed that compound 1 significantly induced cell cycle arrest in Farage and SU-DHL-2 cells by causing abnormal ROS level and triggering oxidative stress.

Ten undescribed eremophilane and guaiane sesquiterpenes from Penicillium roqueforti.

Nine undescribed eremophilane sesquiterpenes, one undescribed guaiane sesquiterpene, along with ten known analogues were isolated and identified from fungus Penicillium roqueforti, which was separated from the root soil of Hypericum beanii N. Robson collected from the Shennongjia Forestry District, Hubei Province. Their structures were elucidated on the basis of various spectroscopic analyses, mainly including NMR and HRESIMS data, 13C NMR calculations with DP4+ probability analyses, ECD calculations, and single-crystal X-ray diffraction experiments. Furthermore, all twenty compounds were evaluated for the in vitro cytotoxic activities against seven human tumor cell lines, and the result suggested that 14-hydroxymethylene-1(10)-ene-epi-guaidiol A exhibited considerable cytotoxic activity against the Farage (IC50 < 10 µM, 48 h), SU-DHL-2, and HL-60 cells. Further mechanism study demonstrated that 14-hydroxymethylene-1(10)-ene-epi-guaidiol A could significantly promote apoptosis by inhibiting tumor cell respiration and decreasing intracellular ROS levels, thereby inducing S-phase blockade in tumor cells.

A novel Gboxin analog induces OXPHOS inhibition and mitochondrial dysfunction-mediated apoptosis in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive B-cell non-Hodgkin's lymphoma. Currently, moderate efficacy and limitations of approved drugs still exist, and it is necessary to develop newer and more effective drugs. Gboxin is a promising inhibitor of OXPHOS, which specifically inhibits the growth of many kinds of cancer cell lines. In the present study, 21 Gboxin analogs incorporating amide and ester moieties were designed and synthesized. Preliminary screening results show that 5d also has specific selectivity for cancer cells, particularly on the DLBCL cells, which is weaker than that of Gboxin but still good. Thus, the effect and underlying mechanism of 5d on DLBCL cells were further studied. The results showed that 5d exhibits potent proliferation inhibition and cell cycle arrest effects, and its IC50 to DLBCL cells is below 1 µM. In addition, 5d induces apoptosis of DLBCL cells in a time- and dose-dependent manner, and this effect is stronger than that of Gboxin and VP16. Mechanistically, 5d plays its role mainly through the stimulation of metabolic stress in DLBCL cell lines, which induces OXPHOS inhibition, inflammation, DNA damage and mitochondrial dysfunction. These data suggest that 5d has potential as a candidate agent for DLBCL alternative drug development.

Structurally diverse metabolites from a soil-derived fungus Aspergillus calidoustus.

Fifteen secondary metabolites, including five new sesquiterpenoids (1-5), one new benzofuranoid (10), one new ophiobolin sesterterpenoid (11), and one new 3,5-dimethylorsellinic acid (DMOA)-based meroterpenoid (15), as well as seven known analogues (6-9 and 12-14), were isolated and characterized from fungus Aspergillus calidoustus, which was separated from the wetland soil collected at Dianchi Lake, Yunnan Province. Compound 5 featured an unusual dioxolane moiety, and compound 15 was a rare austin meroterpenoid analogue with the opened A ring, and also featured an undescribed oxygen bridge between C-3 and C-16 to construct an unexpected tetrahydrofuran ring. Their structures were established by widespread spectroscopic methods, single-crystal X-ray diffraction experiments, and ECD calculation. All the isolated drimane sesquiterpenoids were evaluated for the in vitro cytotoxicity against five tumor cell lines, including SW480 (colon cancer), IOMM-Lee (meningioma), HeLa (cervical cancer), FARAGE (diffuse large B-cell lymphoma), and SU-DHL-4 (diffuse large B-cell lymphoma). Compound 9 exhibited significant cytotoxicity against FARAGE and SU-DHL-4 tumor cell lines with IC50 values of 5.54 and 9.78 µM, respectively. Further mechanism study showed that 9 could significantly promote apoptosis in FARAGE and SU-DHL-4 cell lines by interfering with mitochondrial function.

A new pair of cytotoxic enantiomeric isoprenylated chromone derivatives from Pestalotiopsis sp.

A new pair of enantiomeric isoprenylated chromone derivatives, (±)-pestaloficiol X [(±)-1], along with a known compound pestaloficiol J (2), were isolated from the plant endophytic fungus Pestalotiopsis sp. The racemic mixture 1 was separated through chiral HPLC. The structures of new compounds (±)-1 were elucidated on the basis of extensive spectroscopic data and their absolute configurations were further configured through computational analysis of their electronic circular dichroism (ECD) spectra. Compound (+)-1 showed significant inhibitory potency against HL-60 and HEP-3B cell lines, with IC50 values of 1.35 ± 0.15 and 3.70 ± 0.33 µM, respectively, while compound (-)-1 showed significant inhibitory potency against HL-60 and HEP-3B cell lines, with IC50 values of 2.39 ± 0.26 and 2.99 ± 0.35 µM, respectively.

A new abietane-type diterpenoid and a new long-chain alkenone from fungus Daldinia sp. TJ403-LS1.

A new abietane-type diterpenoid, dalterpenoid A (1), a new long-chain alkenone derivative, (3E,5E,10E)-8-hydroxytrideca-3,5,10,12-tetraen-2-one (2), together with six known compounds (3-8), namely epi-guaidiol A (3), xylaranol A (4), daldinone C (5), trans-3,4-dihydroxy-3,4-dihydro-anofinic acid (6), (R)-6-hydroxymellein (7), helicascolide A (8), were obtained from fungus Daldinia sp. TJ403-LS1, which was originally isolated from roots of the medicinally valuable plant Anoectochilus roxburghii. The structures of compounds 1 and 2 were established based on widespread spectroscopic methods, mainly including 1D & 2D NMR and HRESIMS analyses, and the absolute configuration of 1 was further confirmed by electronic circular dichroism (ECD) calculation. All new compounds were tested for the in vitro cytotoxicity against five human cancer cell lines.

Discovery of bioactive polycyclic polyprenylated acylphloroglucinols from Hypericum wilsonii.

Eleven new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperwilsones A-K (1-11), along with five known PPAPs (12-16), were isolated from Hypericum wilsonii. Their structures were established via spectroscopic methods, the careful analysis of calculated and experimental electronic circular dichroism (ECD) spectra, single-crystal X-ray diffraction, the modified Mosher's method, and [Rh2(OCOCF3)4]-induced ECD. Hyperwilsone A (1) and hyperwilsone B (2) possessed the unique acetal functionality. Hyperwilsone C (3) was a rare example of [3.3.1]-type PPAP possessing a 3-isopropylfuran moiety. In bioassay, compounds 9 and 10 showed potent anti-inflammatory activity against LPS-induced NO production by inhibiting the nuclear translocation of NF-κB p65 and thus reducing the production of proinflammatory cytokines. Compounds 5, 8, 11, and 14 exhibited moderate inhibitory activity against SUDHL-4 and HL60 cancer cells with IC50 values in the range of 5.74-19.82 µM.

Asperanstinoids A-E: Undescribed 3,5-dimethylorsellinic acid-based meroterpenoids from Aspergillus calidoustus.

Large-scale culture is a complementary and practical method for genome mining and OSMAC approaches to discover novel natural products through accumulation and reprocessing effects. By employing a large-scale culture approach, twelve 3,5-dimethylorsellinic acid (DMOA)-based meroterpenoids, including five undescribed compounds, namely asperanstinoids A-E, were obtained from fungus Aspergillus calidoustus, which was isolated from the wetland soil collected at Dianchi Lake, Yunnan Province. The structures and absolute configurations of asperanstinoids A-E were determined by various spectroscopic analyses, including NMR spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations, and the absolute configurations of three known compounds, dehydroaustinol, austinol, and austin, were confirmed via single-crystal X-ray diffraction for the first time. Notably, asperanstinoid A represents the second example of a DMOA-based meroterpenoid featuring a unique 6/5/6/6/6/5-fused hexacyclic skeleton with a rare "1,13-epoxy" moiety. The cytotoxicity assay of all these isolates revealed that asperanstinoid D, dehydroaustinol, and austin displayed considerable cytotoxicity against the HL-60 and SU-DHL-4 tumor cell lines with IC50 values ranging from 15.7 to 27.8 µM.

Modified Fusicoccane-Type Diterpenoids from Alternaria brassicicola.

Seven new modified fusicoccane-type diterpenoids (1-7), together with two known congeners (8 and 9), were obtained from Alternaria brassicicola. Their structures were elucidated from a combination of NMR and HRESIMS data and 13C NMR calculation as well as DP4+ probability analyses, and the absolute configurations of 1-5 were determined by ECD calculation and single-crystal X-ray diffraction (Cu Kα). Compounds 1-3 belong to a rare class of 16-nor-dicyclopenta[a,d]cyclooctane diterpenoids, and compounds 2 and 4 represent the first examples of fusicoccane-type diterpenoids featuring two previously undescribed tetracyclic 5/6/6/5 ring systems, while compound 5 features a previously undescribed tetracyclic 5/8/5/3 ring system. Compound 7 was moderately anti-inflammatory, and compounds 2, 3, 5, and 7 were weakly cytotoxic.

Fusicoccane-derived diterpenoids with bridgehead double-bond-containing tricyclo[9.2.1.03,7]tetradecane ring systems from Alternaria brassicicola.

Fusicoccane-derived diterpenoids bearing a unique bridgehead double-bond-containing tricyclo[9.2.1.03,7]tetradecane (5-9-5 ring system) core skeleton represent a rarely reported class of rearranged terpenoids, which traced back to fusicoccanes with a classical dicyclopenta[a,d]cyclooctane (5-8-5 ring system) core skeleton via a crucial Wagner-Meerwein rearrangement reaction. In this research, alterbrassicenes B-D (1-3), three new rearranged fusicoccane diterpenoids bearing a rare bridgehead double-bond-containing tricyclo[9.2.1.03,7]tetradecane core skeleton, together with two known congeners, brassicicenes O and K (4 and 5), were isolated from the modified cultures of fungus Alternaria brassicicola. Their structures were elucidated by comprehensive analyses of the NMR and HRESIMS data, and the absolute configurations of 1 and 4 were further confirmed via a combination of 13C NMR and ECD calculations and single-crystal X-ray diffraction analysis (Cu Kα). Interestingly, alterbrassicene B (1) represented the second case of bridgehead C-10-C-11 double-bond-containing natural products with a bicyclo[6.2.1]undecane core skeleton, and also featured an undescribed oxygen bridge between C-6 and C-14 to construct an unprecedentedly caged tetracyclic system. Alterbrassicenes B-D showed moderate cytotoxic activity against certain human tumor cell lines with IC50 values in the range of 15.87-36.85 µM.

Alterbrassinoids A-D: Fusicoccane-Derived Diterpenoid Dimers Featuring Different Carbon Skeletons from Alternaria brassicicola.

Alterbrassinoids A-D (1-4), the first examples of fusicoccane-derived diterpenoid dimers furnished by forming an undescribed C-12-C-18' linkage, were isolated from modified cultures of Alternaria brassicicola. Compounds 1 and 2 represent unprecedented heterodimers, whereas 3 and 4 represent unprecedented homodimers, and 4 also features an undescribed anhydride motif. Their structures were assigned via spectroscopic methods, electronic circular dichroism calculations, and single-crystal X-ray diffraction. Putative biosynthetic pathways and a bioactivity evaluation for 1-4 were discussed.

New cytotoxic tricycloalternarenes from fungus Alternaria brassicicola.

Seven previously undescribed metabolites, designated as tricycloalternarenes Q-W (1-7), were isolated and identified from the fermented rice substrate of fungus Alternaria brassicicola. The planar and absolute structures of all new compounds were determined on the basis of extensive NMR spectroscopic data, a modified Mosher's method, X-ray crystallographic analysis, and electronic circular dichroism (ECD) spectral analyses. All the isolates were evaluated for in vitro cytotoxicity against five human tumor MM231, MM468, HeLa, SW1990, and SW480 cell lines, and compounds 1, 2, 5, and 7 showed selective cytotoxicity against certain human tumor cell lines with IC50 values ranging from 12.83 to 32.87 µM, with no obvious cytotoxicity to the normal LO2 cell.

Fusaresters A-E, new γ-pyrone-containing polyketides from fungus Fusarium sp. Hungcl and structure revision of fusariumin D.

Marine-derived fungi have been regarded as an under-explored and promising reservoir of structurally novel and bioactive natural products. In this study, five new γ-pyrone-containing polyketides, fusaresters A-E (1-5), were isolated and identified from the culture extracts of a marine-derived fungus Fusarium sp. Hungcl. The structures of compounds 1-5 were elucidated on the basis of their HRESIMS and NMR spectroscopic data as well as 13C NMR calculation and electronic circular dichroism (ECD) analyses. Remarkably, the structure of fusariumin D was revised to (9S*,11S*)-3. All these isolates were tested for the cytotoxicity against seven human cancer cell lines, including SW480, HL-60, A549, MCF-7, HepG2, HeLa and SMMC-7721, and the inhibitory activity against protein tyrosine phosphatase 1B (PTP1B). The results revealed that only compound 2 showed a weak inhibition rate of 56% at 40 µM.

Alterbrassicicene A, a Highly Transformed Fusicoccane-Derived Diterpenoid with Potent PPAR-γ Agonistic Activity from Alternaria brassicicola.

Alterbrassicicene A (1), a fusicoccane-derived diterpenoid with an unprecedented framework, together with two known biosynthetic intermediates (2 and 3), were characterized from Alternaria brassicicola. The absolute structure of 1 was assigned by extensive spectroscopic analyses and quantum chemical calculations. Compound 1 represents a newly transformed monocyclic carbon skeleton of a highly functionalized diterpenoid bearing unique dihydro-2(3 H)-furanone and 2-cyclopenten-1-one motifs. Compound 1 was the first fusicoccane-derived diterpenoid functioning as a potent PPAR-γ agonist (EC50 = 744.1 nM).

Anti-inflammatory fusicoccane-type diterpenoids from the phytopathogenic fungus Alternaria brassicicola.

Altering the cultivation conditions, such as temperature, media compositions, illumination, aeration, the shape of the culturing flask, etc., is regarded as a useful strategy for the exploitation of structurally novel and bioactive secondary metabolites, which inspired us to explore the chemical and pharmacological diversities from the genetically powerful fungus Alternaria brassicicola. As a result, twelve fusicoccane-type diterpenoids, including eight new ones, termed brassicicenes Q-X (1-8), were isolated from a modified rice medium. Biosynthetically, all these compounds were derived from the mevalonic acid (MVA) pathway, and their structures incorporating absolute configurations were assigned by the interpretation of spectroscopic (HRESIMS and 1D and 2D NMR) analyses, chemical conversion, a modified Mosher's method, 13C NMR calculation, and single-crystal X-ray diffraction (Cu Kα). Structurally, compound 1 was an unusual 16-nor-dicyclopenta[a,d]cyclooctane diterpenoid bearing a fused cyclopent-2-en-1-one moiety. In addition, compound 3 was found to show significant anti-inflammatory activity against the production of NO, TNF-α, and IL-1ß at 10 µM. Further western blot and immunofluorescence experiments found the mechanism of action to be that 3 inhibited the NF-κB-activated pathway, highlighting it as a promising starting point for the development of new anti-inflammatory agents.

Butenolides from a marine-derived fungus Aspergillus terreus with antitumor activities against pancreatic ductal adenocarcinoma cells.

Chemical study on the extract of a marine-derived fungus Aspergillus terreus yielded twelve butenolide derivatives, including three new compounds, namely asperlides A-C (1-3) and nine known butenolides (4-12). The structures of 1-3 were confirmed by comprehensive spectroscopic analysis, including HRESIMS, NMR spectroscopy, and calculated electronic circular dichroism (ECD). The cytotoxicity of the compounds was evaluated using PANC-1, HCC1806, HepG2, BEAS-2B and HT-29 cancer cells. The results showed that (+)-3',3'-di-(dimethylallyl)-butyrolactone II (4) and versicolactone B (6) exhibited the most potent cytotoxin of PANC-1 cell line, with the IC50 values of 5.3 and 9.4 µM, respectively. Morphological features of apoptosis were observed in 4 and 6-treated PANC-1 cells, including apoptotic body formation, membrane blebbing, cell shrinkage and nuclear condensation. Cell cycle analysis with propidium iodide staining exhibited that 4 inhibits proliferation of PANC-1 cells via the induction of G2/M and S phase arrest, while 6 could retard the PANC-1 cells via the induction of S phase arrest. Flow cytometric analysis suggested that treatment with 4 and 6 significantly induced PANC-1 cells apoptosis. These findings indicated that 4 and 6 might serve as a starting point for the development of an anticancer drug for the treatment of pancreatic ductal adenocarcinoma.

Terrusnolides A-D, new butenolides with anti-inflammatory activities from an endophytic Aspergillus from Tripterygium wilfordii.

Terrusnolides A-D (1-4), four butenolides were isolated from an endophytic Aspergillus from Tripterygium wilfordii. The structures of 1-4 were established by comprehensive spectroscopic analyses and electronic circular dichroism (ECD) calculation. It is interesting that 1 was a butenolide derived by a triple decarboxylation, while 2-4 were the metabolites with 4-benzyl-3-phenyl-5H-furan-2-one motif possessing an isopentene group fused to the benzene ring. In vitro anti-inflammatory effects of these isolates were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. 1-4 exhibited excellent inhibitory effects on the production of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in LPS-induced macrophages, comparable with the positive control (indomethacin). Those results indicated that, terrusnolides A-D might serve as new potential natural remedies for the treatment of inflammation.

A New Breviane Spiroditerpenoid from the Marine-Derived Fungus Penicillium sp. TJ403-1.

Marine-derived fungi are a promising and untapped reservoir for discovering structurally interesting and pharmacologically active natural products. In our efforts to identify novel bioactive compounds from marine-derived fungi, four breviane spiroditerpenoids, including a new compound, brevione O (1), and three known compounds breviones I (2), J (3), and H (4), together with a known diketopiperazine alkaloid brevicompanine G (5), were isolated and identified from an ethyl acetate extract of the fermented rice substrate of the coral-derived fungus Penicillium sp. TJ403-1. The absolute structure of 1 was elucidated by HRESIMS, one- and two-dimensional NMR spectroscopic data, and a comparison of its electronic circular dichroism (ECD) spectrum with the literature. Moreover, we confirmed the absolute configuration of 5 by single-crystal X-ray crystallography. All the isolated compounds were evaluated for isocitrate dehydrogenase 1 (IDH1) inhibitory activity and cytotoxicity, and compound 2 showed significant inhibitory activities against HL-60, A-549, and HEP3B tumor cell lines with IC50 values of 4.92 ± 0.65, 8.60 ± 1.36, and 5.50 ± 0.67 µM, respectively.