Miniaturized Cultivation Profiling (MATRIX)-Facilitated Discovery of Noonazines A-C and Noonaphilone A from an Australian Marine-Derived Fungus, Aspergillus noonimiae CMB-M0339.

Subjecting the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339 to cultivation profiling using an innovative miniaturized 24-well plate format (MATRIX) enabled access to new examples of the rare class of 2,6-diketopiperazines, noonazines A-C (1-3), along with the known analogue coelomycin (4), as well as a new azaphilone, noonaphilone A (5). Structures were assigned to 1-5 on the basis of a detailed spectroscopic analysis, and in the case of 1-2, an X-ray crystallographic analysis. Plausible biosynthetic pathways are proposed for 1-4, involving oxidative Schiff base coupling/dimerization of a putative Phe precursor. Of note, 2 incorporates a rare meta-Tyr motif, typically only reported in a limited array of Streptomyces metabolites. Similarly, a plausible biosynthetic pathway is proposed for 5, highlighting a single point for stereo-divergence that allows for the biosynthesis of alternate antipodes, for example, the 7R noonaphilone A (5) versus the 7S deflectin 1a (6).

Bioactive Indole Diketopiperazine Alkaloids from the Marine Endophytic Fungus Aspergillus sp. YJ191021.

Six new prenylated indole diketopiperazine alkaloids, asperthrins A-F (1-6), along with eight known analogues (7-14), were isolated from the marine-derived endophytic fungus Aspergillus sp. YJ191021. Their planar structures and absolute configurations were elucidated by HR-ESI-MS, 1D/2D NMR data, and time-dependent density functional theory (TDDFT)/ECD calculation. The isolated compounds were assayed for their inhibition against three agricultural pathogenic fungi, four fish pathogenic bacteria, and two agricultural pathogenic bacteria. Compound 1 exhibited moderate antifungal and antibacterial activities against Vibrioanguillarum, Xanthomonas oryzae pv. Oryzicola, and Rhizoctoniasolani with minimal inhibitory concentration (MIC) values of 8, 12.5, and 25 µg/mL, respectively. Furthermore, 1 displayed notable anti-inflammatory activity with IC50 value of 1.46 ± 0.21 µM in Propionibacteriumacnes induced human monocyte cell line (THP-1).

Fintiamin: A diketopiperazine from the marine sponge-derived fungus Eurotium sp.

The fungus Eurotium sp., derived from the marine sponge Ircinia variabilis, was found to produce a diketopiperazine-indole alkaloid that we named fintiamin (1). Structural elucidation of 1 was achieved by extensive spectroscopic analysis including nuclear magnetic resonance spectroscopy and mass spectrometry. Compound 1 is a lipophilic terpenoid-dipeptide hybrid molecule that shows affinity for the cannabinoid CB1 receptor at low micromolar concentrations. Docking studies based on previous X-ray structures provide a plausible binding pose for compound 1 in the orthosteric binding site of the CB1 receptor.

Diketopiperazines and other bioactive compounds from bacterial symbionts of marine sponges.

Humanity faces great challenges, such as the rise of bacterial antibiotic resistance and cancer incidence. Thus, the discovery of novel therapeutics from underexplored environments, such as marine habitats, is fundamental. In this study, twelve strains from the phylum Firmicutes and thirty-four strains from the phylum Proteobacteria, isolated from marine sponges of the Erylus genus, collected in Portuguese waters, were tested for bioactivities and the secondary metabolites were characterised. Bioactivity screenings comprised antimicrobial, anti-fungal, anti-parasitic and anti-cancer assays. Selected bioactive extracts were further analysed for already described molecules through high performance liquid chromatography and mass spectrometry. Several bioactivities were observed against the fungus Aspergillusfumigatus, the bacteria (methicillin-resistant Staphylococcus aureus and Escherichia coli), the human liver cancer cell line HepG2 and the parasite Trypanosoma cruzi. Medium scale-up volume extracts confirmed anti-fungal activity by strains Proteus mirabilis #118_13 and Proteus sp. (JX006497) strain #118_20. Anti-parasitic activity was also confirmed in Enterococcus faecalis strain #118_3. Moreover, P. mirabilis #118_13 showed bioactivity in human melanoma cell line A2058 and the human hepatocellular carcinoma cell line HepG2. The dereplication of bioactive extracts showed the existence of a variety of secondary metabolites, with some unidentifiable molecules. This work shows that bacterial communities of sponges are indeed good candidates for drug discovery and, as far as we know, we describe anti-parasitic activity of a strain of E. faecalis and the presence of diketopiperazines in Proteus genus for the first time.

Absolute Configurations and Chitinase Inhibitions of Quinazoline-Containing Diketopiperazines from the Marine-Derived Fungus Penicillium polonicum.

Three new quinazoline-containing diketopiperazines, polonimides A-C (1-3), along with four analogues (4-7), were obtained from the marine-derived fungus Penicillium polonicum. Among them, 2 and 4, 3 and 5 were epimers, respectively, resulting the difficulty in the determination of their configurations. The configurations of 1-3 were determined by 1D nuclear overhauser effect (NOE), Marfey and electron circular dichroism (ECD) methods. Nuclear magnetic resonance (NMR) calculation with the combination of DP4plus probability method was used to distinguish the absolute configurations of C-3 in 3 and 5. All of 1-7 were tested for their chitinase inhibitory activity against OfHex1 and OfChi-h and cytotoxicity against A549, HGC-27 and UMUC-3 cell lines. Compounds 1-7 exhibited weak activity towards OfHex1 and strong activity towards OfChi-h at a concentration of 10.0 µM, with the inhibition rates of 0.7%-10.3% and 79.1%-95.4%, respectively. Interestingly, 1-7 showed low cytotoxicity against A549, HGC-27 and UMUC-3 cell lines, suggesting that good prospect of this cluster of metabolites for drug discovery.

Three New Diketopiperazines from the Previously Uncultivable Marine Bacterium Gallaecimonas mangrovi HK-28 Cultivated by iChip.

The in situ application of iChip cultivation in mangrove sediment from Hainan province, China, led to the isolation of a novel bacterial species Gallaecimonas mangrovi HK-28. The extract of G. mangrovi HK-28 exhibited antibiotic activity against the aquatic pathogen Vibrio harveyi, and its chemical constituents were further investigated by bioactivity-guided isolation. Three new diketopiperazines, gallaecimonamides A-C, were accordingly isolated from the AcOEt extract of the fermentation broth of G. mangrovi HK-28. The planar structures of gallaecimonamides A-C were determined using HR-ESI-MS together with 1D- and 2D-NMR. The absolute configurations of gallaecimonamides A-C were assigned by optical rotation, NOESY experiment and TDDFT ECD calculations. The in vitro antibacterial and antimalarial activities of gallaecimonamides A-C were assessed. Gallaecimonamide A was found to display antibacterial activity against V. harveyi with a MIC value of 50 µm. However, gallaecimonamides B and C showed no antibacterial activity against V. harveyi (MIC >300 µm). In addition, all the isolates did not exhibit any inhibitory activities against V. parahaemolyticus (MIC>300 µm) and Plasmodium falciparum W2 (EC50 >100 µg/mL).

New Indole Diketopiperazine Alkaloids from Soft Coral-Associated Epiphytic Fungus Aspergillus sp. EGF 15-0-3.

Three new indole diketopiperazine alkaloids, 11-methylneoechinulin E and variecolorin M, and (+)-variecolorin G, along with 12 known analogs, were isolated from a soft coral-associated epiphytic fungus Aspergillus sp. EGF 15-0-3. The structures of the new compounds were unambiguously established by extensive spectroscopic analyses including HR-ESI-MS, 1D and 2D NMR spectroscopy and optical rotation measurements. The absolute configurations of (+)- and (-)-variecolorin G were determined by experimental and quantum-chemical ECD investigations and single-crystal X-ray diffraction analysis. Variecolorin G is a pair of enantiomeric mixtures with a ratio of 1 : 2. Moreover, (+)-neoechinulin A is firstly reported as a natural product. The cytotoxic activities of all the isolated compounds against NCI-H1975 gefitinib resistance (NCI-H1975/GR) cell lines were preliminarily evaluated by MTT method.

Diketopiperazine-Type Alkaloids from a Deep-Sea-Derived Aspergillus puniceus Fungus and Their Effects on Liver X Receptor α.

Eight new diketopiperazine-type alkaloids including four oxepin-containing diketopiperazine-type alkaloids, oxepinamides H-K (1-4), and four 4-quinazolinone alkaloids, puniceloids A-D (5-8), together with two known analogues (9 and 10), were isolated from the culture broth extracts of the deep-sea-derived fungus Aspergillus puniceus SCSIO z021. Their structures were elucidated by spectroscopic analyses, and their absolute configurations were determined by Marfey's method along with comparison of their specific rotations and ECD spectra. The absolute configurations of 4 and 5 were further confirmed by a single-crystal X-ray diffraction analysis. Compounds 1-8 showed significant transcriptional activation of liver X receptor α with EC50 values of 1.7-50 µM, and 7 and 8 were the most potent agonists.

Isolation and characterization of three pairs of indolediketopiperazine enantiomers containing infrequent N-methoxy substitution from the marine algal-derived endophytic fungus Acrostalagmus luteoalbus TK-43.

Three pairs of new N-methoxy-containing indolediketopiperazine enantiomers, acrozines A-C (1-3), were isolated from the culture extract of Acrostalagmus luteoalbus TK-43, an endophytic fungus obtained from the marine green alga Codium fragile. The optical resolution of compounds 1-3 by chiral HPLC successfully afforded individual enantiomers (+)-1/(-)-1, (+)-2/(-)-2, and (+)-3/(-)-3, respectively. The structures of all these compounds were established on the basis of detailed interpretation of their NMR and mass spectroscopic data. X-ray crystallographic analysis confirmed the structures of compounds 1-3, while the absolute configurations were determined by TDDFT-ECD calculations. All these compounds containing a N-methoxy group which is uncommon in indolediketopiperazines. The enantiomers, (+)-2/(-)-2, showed different antimicrobial activities against several plant-pathogenic fungi, while (+)-1 displayed better inhibitory activity against acetylcholinesterase than that of (-)-1.

Separation of five diketopiperazines from the marine fungus Alternaria alternate HK-25 by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied to the separation of five diketoperazines from the marine Alternaria alternate HK-25 for the first time using one-step elution method with a pair of two-phase solvent systems composed of petroleum ether/ethyl acetate/methanol/water (5.5:11:5:7, v/v). Where 151.6 mg of crude sample yielded five diketoperazines, 12,13-dihydroxy-fumitremorgin C (1), gliotoxin (2), demethoxyfum itremorgin C (3), bisdethiobis(methylthio)gliotoxin (4), fumitremorgin C (5), and the purities of all compounds were above 94% as determined by high-performance liquid chromatography. The structures of these compounds were identified by 1 H and 13 C NMR spectroscopy. These results showed that high-speed counter-current chromatography can provide a feasible way for highly effective preparation of marine natural products, which ensured the supple of numerous samples for drug development.

Structures and Absolute Configurations of Diketopiperazine Alkaloids Chrysopiperazines A⁻C from the Gorgonian-Derived Penicillium chrysogenum Fungus.

Three new diketopiperazine alkaloids, including two oxepine-containing diketopiperazines, chrysopiperazines A and B (1 and 2), and one quinazoline-containing diketopiperazine, chrysopiperazine C (5), together with three known analogues (3, 4, and 6), were isolated from the gorgonian-derived Penicillium chrysogenum fungus. The relative and absolute configurations of C-3 and C-15 in 1 and 2, C-3 and C-14 in 5 were established by NOE modified Marfey's analysis and electronic circular dichroism (ECD) calculations. Particularly, the absolute configurations of C-19 in 1 and 3, which was very challenging to be identified due to the flexible conformation in a short aliphatic chain, were successfully determined by the vibrational circular dichroism (VCD) method, supplying with a reliable and optional method to define the absolute configurations. Additionally, this is the first report on oxepine-containing diketopiperazines from the genus Penicillium.

New Diketopiperazines from a Marine-Derived Fungus Strain Aspergillus versicolor MF180151.

Six new diketopiperazines, (±)-7,8-epoxy-brevianamide Q ((±)-1), (±)-8-hydroxy-brevianamide R ((±)-2), and (±)-8-epihydroxy-brevianamide R ((±)-3), together with four known compounds, (±)-brevianamide R ((±)-4), versicolorin B (5) and averufin (6), were isolated from a marine-derived fungus strain Aspergillus versicolor MF180151, which was recovered from a sediment sample collected from the Bohai Sea, China. The chemical structures were established by 1D- and 2D-NMR spectra and HR-ESI-MS. 1 is the first sample of brevianamides with an epoxy moiety. Their bioactivities were evaluated against Candida albicans, Bacillus subtilis, Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, and Bacillus Calmette-Guérin. Compounds 1-4 showed no activities against the pathogens, and compounds 5 and 6 showed moderate activities against S. aureus and methicillin-resistant S. aureus.

Thiodiketopiperazines Produced by Penicillium crustosum and Their Activities to Promote Gastrointestinal Motility.

Three new thiodiketopiperazines (1⁻3), along with two known analogues (4 and 5), were isolated from the fermentation broth of Penicillium crustosum. Their structures were elucidated through extensive spectroscopic analysis and the absolute configurations of new compounds were determined by Mosher ester analysis and calculated ECD spectra. Compound 4 and 5 have the activity to promote the gastrointestinal motility of zebrafish via acting on the cholinergic nervous system.

HSQC-TOCSY Fingerprinting-Directed Discovery of Antiplasmodial Polyketides from the Marine Ascidian-Derived Streptomyces sp. (USC-16018).

Chemical investigations on the fermentation extract obtained from an ascidian-derived Streptomyces sp. (USC-16018) yielded a new ansamycin polyketide, herbimycin G (1), as well as a known macrocyclic polyketide, elaiophylin (2), and four known diketopiperazines (3⁻6). The structures of the compounds were elucidated based on 1D/2D NMR and MS data. The absolute configuration of 1 was established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Antiplasmodial activities were tested for the natural products against chloroquine sensitive (3D7) and chloroquine resistant (Dd2) Plasmodium falciparum strains; the two polyketides (1⁻2) demonstrated an inhibition of >75% against both parasite strains and while 2 was highly cytotoxic, herbimycin G (1) showed no cytotoxicity and good predicted water solubility.

Diketopiperazine and Diphenylether Derivatives from Marine Algae-Derived Aspergillus versicolor OUCMDZ-2738 by Epigenetic Activation.

A chemical-epigenetic method was used to enhance the chemodiversity of a marine algicolous fungus. Apart from thirteen known compounds, (+)-brevianamide R ((+)-3), (‒)-brevianamide R ((‒)-3), (+)-brevianamide Q ((+)-4), (‒)-brevianamide Q ((‒)-4), brevianamide V ((+)-5), brevianamide W ((‒)-5), brevianamide K (6), diorcinol B (7), diorcinol C (8), diorcinol E (9), diorcinol J (10), diorcinol (11), 4-methoxycarbonyldiorcinol (12), two new compounds, (+)- and (‒)-brevianamide X ((+)- and (‒)- 2)), as well as a new naturally occurring one, 3-[6-(2-methylpropyl)-2-oxo-1H-pyrazin-3-yl]propanamide (1), were isolated from chemical-epigenetic cultures of Aspergillus versicolor OUCMDZ-2738 with 10 µM vorinostat (SAHA). Compared to cultures in the same medium without SAHA, compounds 1⁻4, 8, 9, 11, and 12 were solely observed under SAHA condition. The structures of these compounds were elucidated based on spectroscopic analysis, specific rotation analysis, ECD, and X-ray crystallographic analysis. (±)-3, (±)-4, and (±)-5 were further resolved into the corresponding optically pure enantiomers and their absolute configurations were determined for the first time. Compounds 11 and 12 showed selective antibacterial against Pseudomonas aeruginosa with a minimum inhibitory concentration (MIC) of 17.4 and 13.9 µM, respectively. Compound 10 exhibited better α-glucosidase inhibitory activity than the assay control acarbose with IC50 values of 117.3 and 255.3 µM, respectively.

Three New Indole Diketopiperazine Alkaloids from Aspergillus ochraceus.

Asperochramides A - D (1 - 4), a new natural product and three new indole diketopiperazine alkaloids, along with seven known analogs (5 - 11), were isolated from the ethyl acetate extract of Aspergillus ochraceus. Their structures were elucidated by extensive spectroscopic analyses, ECD calculation, and single-crystal X-ray diffraction analysis. Compounds 3 and 4 represent a rare group of indole diketopiperazine alkaloid with a 3-hydroxyl-2-indolone moiety. The in vitro anti-inflammatory effects of compounds 1 and 3 - 11 were investigated by using LPS-stimulated murine macrophage RAW 264.7 cells. Compounds 1, 8, 10, and 11 showed potential anti-inflammatory activities.

Cytotoxic and Antimicrobial Compounds from the Marine-Derived Fungus, Penicillium Species.

The organic extract of liquid cultures of the marine-derived Penicillium sp. was investigated. Fractionation of the extracts of the fungus led to the purification and identification of two new compounds, penicillatides A (1) and B (2), together with the previously reported cyclo(R-Pro-S-Phe) (3) and cyclo(R-Pro-R-Phe) (4). The structures of compounds 1-4 were assigned by extensive interpretation of their NMR and high-resolution mass spectrometry (HRMS). The antiproliferative and cytotoxic activities of the compounds against three human cancer cell lines as well as their antimicrobial activity against several pathogens were evaluated. Compounds 2-4 displayed variable cytotoxic and antimicrobial activities.

Waspergillamide A, a Nitro depsi-Tetrapeptide Diketopiperazine from an Australian Mud Dauber Wasp-Associated Aspergillus sp. (CMB-W031).

Chemical profiling of extracts from a mud dauber wasp-associated fungus, Aspergillus sp. (CMB-W031), revealed a remarkably diverse array of secondary metabolites, with many biosynthetic gene clusters being transcriptionally responsive to specific culture conditions. Chemical fractionation of a jasmine rice cultivation yielded many known fungal metabolites, including the highly cytotoxic (-)-stephacidin B and an unprecedented nonribosomal peptide synthase derived nitro depsi-tetrapeptide diketopiperazine, waspergillamide A (1). All structures were assigned by detailed spectroscopic analysis and, where appropriate, chemical degradation and Marfey's analysis.

Butyrolactones and Diketopiperazines from Marine Microbes: Inhibition Effects on Dengue Virus Type 2 Replication.

Two new compounds, 4S,10R-dihydroxy-11-methyl-dodec-2-en-1,4-olide (1) (butyrolactone-type) and cyclo-(4-trans-6-dihydroxy-proline-D-leucine) (2) (diketopiperazine-type), as well as one known 4S,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (3) and three known diketopiperazines, cyclo-(L-proline-L-leucine) (4), cyclo-(4-trans-hydroxy-L-proline-L-leucine) (5), and cyclo-(4-trans-hydroxy-L-proline-L-phenylalanine) (6), were isolated from the ethyl acetate extracts of Streptomyces gougerotii GT and Microbulbifer variabilis C-03. Compounds 3, 4, 5, and 6 exhibited a significant reduction effect on dengue virus type 2 replication with EC50 values of 21.2, 16.5, 12.3, and 11.2 µM, respectively.

Secondary Metabolites from the Marine-Derived Fungus Dichotomomyces sp. L-8 and Their Cytotoxic Activity.

Bioassay-guided isolation of the secondary metabolites from the fungus Dichotomomyces sp. L-8 associated with the soft coral Lobophytum crassum led to the discovery of two new compounds, dichotones A and B (1 and 2), together with four known compounds including dichotocejpin C (3), bis-N-norgliovictin (4), bassiatin (5) and (3R,6R)-bassiatin (6). The structures of these compounds were determined by 1D, 2D NMR and mass spectrometry. (3R,6R)-bassiatin (6) displayed significant cytotoxic activities against the human breast cancer cell line MDA-MB-435 and the human lung cancer cell line Calu3 with IC50 values of 7.34 ± 0.20 and 14.54 ± 0.01 µM, respectively, while bassiatin (5), the diastereomer of compound 6, was not cytotoxic.