Triplinones A-H: Anti-Inflammatory Arylalkenyl α,ß-Unsaturated-δ-Lactones Isolated from the Leaves of Australian Rainforest Plant Cryptocarya triplinervis (Lauraceae).

Our ongoing exploration of Australian rainforest plants for the biodiscovery of anti-inflammatory agents led to the isolation and structural elucidation of eight new arylalkenyl α,ß-unsaturated-δ-lactones, triplinones A-H (1-8), from the leaves of the Australian rainforest plant Cryptocarya triplinervis B. Hyland (Lauraceae). The chemical structures of these compounds were established by NMR spectroscopic data analysis, while their relative and absolute configurations were established using a combination of Mosher ester analysis utilizing both Riguera's and Kishi's methods, ECD experiments, and X-ray crystallography analysis. Compounds 1-8 exhibited good inhibitory activities toward nitric oxide (NO) production in lipopolysaccharide (LPS) and interferon (IFN)-γ induced RAW 264.7 macrophages, in particular compounds 1-3 and 5, with IC50 values of 7.3 ± 0.5, 6.0 ± 0.3, 5.6 ± 0.3, and 5.4 ± 2.5 µM, respectively.

Synthesis and Cheminformatics-Directed Antibacterial Evaluation of Echinosulfonic Acid-Inspired Bis-Indole Alkaloids.

Synthetic efforts toward complex natural product (NP) scaffolds are useful ones, particularly those aimed at expanding their bioactive chemical space. Here, we utilised an orthogonal cheminformatics-based approach to predict the potential biological activities for a series of synthetic bis-indole alkaloids inspired by elusive sponge-derived NPs, echinosulfone A (1) and echinosulfonic acids A-D (2-5). Our work includes the first synthesis of desulfato-echinosulfonic acid C, an α-hydroxy bis(3'-indolyl) alkaloid (17), and its full NMR characterisation. This synthesis provides corroborating evidence for the structure revision of echinosulfonic acids A-C. Additionally, we demonstrate a robust synthetic strategy toward a diverse range of α-methine bis(3'-indolyl) acids and acetates (11-16) without the need for silica-based purification in either one or two steps. By integrating our synthetic library of bis-indoles with bioactivity data for 2048 marine indole alkaloids (reported up to the end of 2021), we analyzed their overlap with marine natural product chemical diversity. Notably, the C-6 dibrominated α-hydroxy bis(3'-indolyl) and α-methine bis(3'-indolyl) analogues (11, 14, and 17) were found to contain significant overlap with antibacterial C-6 dibrominated marine bis-indoles, guiding our biological evaluation. Validating the results of our cheminformatics analyses, the dibrominated α-methine bis(3'-indolyl) alkaloids (11, 12, 14, and 15) were found to exhibit antibacterial activities against methicillin-sensitive and -resistant Staphylococcus aureus. Further, while investigating other synthetic approaches toward bis-indole alkaloids, 16 incorrectly assigned synthetic α-hydroxy bis(3'-indolyl) alkaloids were identified. After careful analysis of their reported NMR data, and comparison with those obtained for the synthetic bis-indoles reported herein, all of the structures have been revised to α-methine bis(3'-indolyl) alkaloids.

Discovery of Latent Cannabichromene Cyclase Activity in Marine Bacterial Flavoenzymes.

Production of phytocannabinoids remains an area of active scientific interest due to the growing use of cannabis by the public and the underexplored therapeutic potential of the over 100 minor cannabinoids. While phytocannabinoids are biosynthesized by Cannabis sativa and other select plants and fungi, structural analogs and stereoisomers can only be accessed synthetically or through heterologous expression. To date, the bioproduction of cannabinoids has required eukaryotic hosts like yeast since key, native oxidative cyclization enzymes do not express well in bacterial hosts. Here, we report that two marine bacterial flavoenzymes, Clz9 and Tcz9, perform oxidative cyclization reactions on phytocannabinoid precursors to efficiently generate cannabichromene scaffolds. Furthermore, Clz9 and Tcz9 express robustly in bacteria and display significant tolerance to organic solvent and high substrate loading, thereby enabling fermentative production of cannabichromenic acid in Escherichia coli and indicating their potential for biocatalyst development.

Structure Revision of Formyl Phloroglucinol Meroterpenoids: A Unified Approach Using NMR Fingerprinting and DFT NMR and ECD Analyses.

NMR fingerprints are valuable tools for analyzing complex natural product mixtures and identifying incorrectly assigned structures in the literature. Our diagnostic NMR fingerprints for formyl phloroglucinol meroterpenoids revealed discrepancies in the structures reported for eucalyprobusal C (1a) and eucalypcamal K (2a). NMR fingerprinting PCA analyses identified 1a as an oxepine-diformyl phloroglucinol and 2a as an oxepine 3-acyl-1-formyl phloroglucinol, contrary to their initial assignments as pyrano-diformyl and pyrano 3-acyl-1-formyl phloroglucinols, respectively. Extensive reinterpretation of their reported one- and two-dimensional NMR data, coupled with GIAO DFT-calculated 1H and 13C NMR chemical shift and DP4+ analyses, supported the unequivocal reassignment of eucalyprobusal C to 1b and eucalypcamal K to 2b. The absolute configurations of the revised oxepine-containing phloroglucinol meroterpenoids were confirmed via the reinterpretation of their reported ROESY and NOESY NMR data, along with comparative TDDFT-calculated and experimental ECD spectra.

Catecholamine Derivatives: Natural Occurrence, Structural Diversity, and Biological Activity.

Catecholamines (CAs) are aromatic amines containing a 3,4-dihydroxyphenyl nucleus and an amine side chain. Representative CAs included the endogenous neurotransmitters epinephrine, norepinephrine, and dopamine. CAs and their derivatives are good resources for the development of sympathomimetic or central nervous system drugs, while they also provide ligands important for G-protein coupled receptor (GPCR) research. CAs are of broad interest in the fields of chemical, biological, medical, and material sciences due to their high adhesive capacities, chemical reactivities, metal-chelating abilities, redox activities, excellent biocompatibilities, and ease of degradability. Herein, we summarize CAs derivatives isolated and identified from microorganisms, plants, insects, and marine invertebrates in recent decades, alongside their wide range of reported biological activities. The aim of this review is to provide an overview of the structural and biological diversities of CAs, the regularity of their natural occurrences, and insights toward future research and development pertinent to this important class of naturally occurring compounds.

Cyclotheonellazoles D-I, Potent Elastase Inhibitory Thiazole-Containing Cyclic Peptides from Theonella sp. (2131).

Six new thiazole-containing cyclic peptides, the cyclotheonellazoles D-I (1-6), were isolated from the Australian marine sponge Theonella sp. (2131) with their structures assigned by comprehensive 1D and 2D NMR spectroscopic and MS spectrometric analyses, Marfey's derivatization studies, and comparison with time-dependent density functional theory (TDDFT) calculated ECD data. The Type 2 azole-homologated peptides herein comprise up to five nonproteinogenic amino acids, including the protease transition state mimic α-keto-ß-amino acid residue 3-amino-4-methyl-2-oxohexanoic acid (Amoha), while 1-3 also contain a terminal hydantoin residue not previously found in cyclotheonellazoles. The keramamides A (7) and L (8) were reisolated affording expanded exploration of their biological activities. The peptides were examined for protease inhibitory activities against two mammalian serine proteases (elastase and chymotrypsin) and SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), a validated antiviral therapeutic target for COVID-19. Peptides 1-6 and keramamide A (7) displayed potent nanomolar inhibition of elastase (IC50 16.0 to 61.8 nM), while 7 also contained modest inhibition of chymotrypsin and SARS-CoV-2 3CLpro (IC50 0.73 and 1.1 µM, respectively). The cyclotheonellazoles D-E (1-3) do not affect the viability of human breast, ovarian, and colon cancer cells (>100 µM), with the cytotoxicity previously reported for keramamide L (8) not replicated (inactive >20 µM).

α-Synuclein Aggregation Inhibitory and Antiplasmodial Activity of Constituents from the Australian Tree Eucalyptus cloeziana.

Amyloid protein aggregates are linked to the progression of neurodegenerative conditions and may play a role in life stages of Plasmodium falciparum, the parasite responsible for malaria. We hypothesize that amyloid protein aggregation inhibitors may show antiplasmodial activity and vice versa. To test this hypothesis, we screened antiplasmodial active extracts from 25 Australian eucalypt flowers using a binding affinity mass spectrometry assay to identify molecules that bind to the Parkinson's disease-implicated protein α-syn. Myrtucommulone P (1) from a flower extract of Eucalyptus cloeziana was shown to have α-syn affinity and antiplasmodial activity and to inhibit α-syn aggregation. 1 exists as a mixture of four interconverting rotamers. Assignment of the NMR resonances of all four rotamers allowed us to define the relative configuration, conformations, and ratios of rotamers in solution. Four additional new compounds, cloeziones A-C (2-4) and cloeperoxide (5), along with three known compounds were also isolated from E. cloeziana. The structures of all compounds were elucidated using HRMS and NMR analysis, and the absolute configurations for 2-4 were determined by comparison of TDDFT-calculated and experimental ECD data. Compounds 1-3 displayed antiplasmodial activities between IC50 6.6 and 16 µM. The α-syn inhibitory and antiplasmodial activity of myrtucommulone P (1) supports the hypothesized link between antiamyloidogenic and antiplasmodial activity.

Camaldulensals A-C, the First Meroterpenoids Possessing Two Spatially Separated Formyl Phloroglucinols Conjugated to a Terpene Core from the Leaves of Eucalyptus camaldulensis Dehnh.

Three new bis-formyl phloroglucinol-meroterpenoids (1-3), three new euglobal type formyl phloroglucinol-meroterpenoids (4-6), and one new dimeric formyl phloroglucinol (7) were isolated from the leaves of Eucalyptus camaldulensis. Camaldulensal A (1) is the first bis-isovaleryl-formyl-phloroglucinol-sesquiterpenoid. It features a novel 6/6/10/3/6/6 fused ring system and contains six stereogenic centers. Camaldulensals B (2) and C (3) are the first bis-isovaleryl-formyl-phloroglucinols, each conjugated to a monoterpene. Formyl phloroglucinol compounds (FPCs) containing two spatially separated formyl phloroglucinols conjugated to a terpene core such as 1-3 have not been reported previously. The structures of these compounds were elucidated by spectroscopic methods and computational analysis. Camaldulensals B (2) and C (3) exhibited significant antibacterial activity against methicillin-susceptible and methicillin-resistant Staphylococcus aureus. Structure activity relationships are discussed in relation to previously reported antibacterial activities of other molecules from the FPC structure class.

NMR Fingerprints of Formyl Phloroglucinol Meroterpenoids and Their Application to the Investigation of Eucalyptus gittinsii subsp. gittinsii.

NMR fingerprints provide powerful tools to identify natural products in complex mixtures. Principal component analysis and machine learning using 1H and 13C NMR data, alongside structural information from 180 published formyl phloroglucinols, have generated diagnostic NMR fingerprints to categorize subclasses within this group. This resulted in the reassignment of 167 NMR chemical shifts ascribed to 44 compounds. Three pyrano-diformyl phloroglucinols, euglobal In-1 and psiguadiols E and G, contained 1H and 13C NMR data inconsistent with their predicted phloroglucinol subclass. Subsequent reinterpretation of their 2D NMR data combined with DFT 13C NMR chemical shift and ECD calculations led to their structure revisions. Direct covariance processing of HMBC data permitted 1H resonances for individual compounds in mixtures to be associated, and analysis of their 1H/13C HMBC correlations using the fingerprint tool further classified components into phloroglucinol subclasses. NMR fingerprinting HMBC data obtained for six eucalypt flower extracts identified three subclasses of pyrano-acyl-formyl phloroglucinols from Eucalyptus gittinsii subsp. gittinsii. New, eucalteretial F and (+)-eucalteretial B, and known, (-)-euglobal VII and eucalrobusone C, compounds, each belonging to predicted subclasses, were isolated and characterized. Staphylococcus aureus and Plasmodium falciparum screening revealed eucalrobusone C as the most potent antiplasmodial formyl phloroglucinol to date.

α-Synuclein Aggregation Inhibitory Procerolides and Diphenylalkanes from the Ascidian Polycarpa procera.

The aggregation of the neuronal protein α-synuclein (α-syn) is intrinsically linked to the development and progression of Parkinson's disease (PD). Recently we screened the MeOH extracts from 283 marine invertebrates for α-syn binding activity using an affinity mass spectrometry (MS) binding assay and found that the extract of the ascidian Polycarpa procera displayed activity. A subsequent bioassay-guided purification led to the isolation of one new α-syn aggregation inhibitory butenolide procerolide E (3) and one new α-syn aggregation inhibitory diphenylbutyrate methyl procerolate A (5). Herein we report the structure elucidation of procerolide E (3) and methylprocerolate A (5) and α-syn aggregation inhibitory activity of procerolides C-E (1-3), methyl procerolate A (5) and procerone A (4). We also report the α-syn binding activity of 3-bromo-4-methoxyphenylacetamide (6) and a synthetic butenolide library, which has allowed us to determine α-syn aggregation inhibitory structure-activity relationships for this class of compounds.

Marine indole alkaloid diversity and bioactivity. What do we know and what are we missing?

Covering: marine indole alkaloids (n = 2048) and their reported bioactivities up to the end of 2021Despite increasing numbers of marine natural products (MNPs) reported each year, most have only been examined for cytotoxic, antibacterial, and/or antifungal biological activities with the majority found to be inactive in these assays. In this context, why are natural products continuing to be examined in assays they are unlikely to show significant activity in, and what targets might be more useful for expanding knowledge of their biologically relevant chemical space? We have undertaken a meta-analysis of the biological activities for 2048 marine indole alkaloids (MIAs), a diverse sub-class of MNPs reported up to the end of 2021, and this has highlighted that the bioactivity potentials for up to 86% of published MIAs remains underexplored and/or undefined. Although most published MIAs are not cytotoxic or antimicrobial, there is a continued focus on using these assays to evaluate new structurally related analogues. Using cheminformatics analyses, the chemical diversity of the 2048 MIAs were clustered using fragment based fingerprints and their reported bioactivity potency towards specific disease targets was assessed for structure activity trends. These analyses showed that there are groups of MIAs that possess potent and diverse activities and that many analogues, previously tested only in cellular toxicity assays, could be better exploited to generate structure activity relationships associated with leads to treat emerging diseases. A collection of indole drug and drug-lead structures from non-natural sources were also incorporated into the dataset providing complementary bioactivity profiles that were further used to predict underexplored areas of potential new activity and to better direct future testing of MIAs. Our findings clearly suggest the biological evaluation of MIAs continues to be conducted on a narrow range of bioassays and disease targets, and that shifting the focus to non-toxic disease targets should provide expanded knowledge of biologically relevant chemical space aimed at maximising the potential of MIAs for drug discovery.

Echiumin E, an Aryl Dihydronaphthalene Lignan from the Australian Invasive Plant Paterson's Curse (Echium plantagineum).

A new aryl dihydronaphthalene lignan, echiumin E (1), and four known compounds, echiumin A, globoidnan A, (-)-rabdosiin, and rosmarinic acid (2-5), were isolated from the Australian invasive plant Echium plantagineum (Paterson's curse) for the first time. Echiumin E (1) was characterized by 1D/2D NMR spectroscopy and MS spectrometry, with its absolute configuration assigned through comparison of experimental and TDDFT-calculated ECD data. Echiumin E (1) along with compounds 3-5 were screened in vitro against three cancer cell lines (SH-SY5Y, HeLa, and PC-3) and a prostate stromal (normal) cell line (WPMY-1) using a resazurin reduction assay. Echiumin E (1) was found to be active toward HeLa cells (IC50 0.21 µM).

α-Synuclein Aggregation Inhibitory Prunolides and a Dibrominated ß-Carboline Sulfamate from the Ascidian Synoicum prunum.

Seven new polyaromatic bis-spiroketal-containing butenolides, the prunolides D-I (4-9) and cis-prunolide C (10), a new dibrominated ß-carboline sulfamate named pityriacitrin C (11), alongside the known prunolides A-C (1-3) were isolated from the Australian colonial ascidian Synoicum prunum. The prunolides D-G (4-7) represent the first asymmetrically brominated prunolides, while cis-prunolide C (10) is the first reported with a cis-configuration about the prunolide's bis-spiroketal core. The prunolides displayed binding activities with the Parkinson's disease-implicated amyloid protein α-synuclein in a mass spectrometry binding assay, while the prunolides (1-5 and 10) were found to significantly inhibit the aggregation (>89.0%) of α-synuclein in a ThT amyloid dye assay. The prunolides A-C (1-3) were also tested for inhibition of pSyn aggregate formation in a primary embryonic mouse midbrain dopamine neuron model with prunolide B (2) displaying statistically significant inhibitory activity at 0.5 µM. The antiplasmodial and antibacterial activities of the isolates were also examined with prunolide C (3) displaying only weak activity against the 3D7 parasite strain of Plasmodium falciparum. Our findings reported herein suggest that the prunolides could provide a novel scaffold for the exploration of future therapeutics aimed at inhibiting amyloid protein aggregation and the treatment of numerous neurodegenerative diseases.

Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Natural products dereplication by diffusion ordered NMR spectroscopy (DOSY).

Diffusion-ordered NMR spectroscopy (DOSY) can be used to analyze mixtures of compounds since resonances deriving from different compounds are distinguished by their diffusion coefficients (D). Previously, DOSY has mostly been used for organometallic and polymer analysis, we have now applied DOSY to investigate diffusion coefficients of structurally diverse organic compounds such as natural products (NP). The experimental Ds derived from 55 diverse NPs has allowed us to establish a power law relationship between D and molecular weight (MW) and therefore predict MW from experimental D. We have shown that D is also affected by factors such as hydrogen bonding, molar density and molecular shape of the compound and we have generated new models that incorporate experimentally derived variables for these factors so that more accurate predictions of MW can be calculated from experimental D. The recognition that multiple physicochemical properties affect D has allowed us to generate a polynomial equation based on multiple linear regression analysis of eight calculated physicochemical properties from 63 compounds to accurately correlate predicted D with experimental D for any known organic compound. This equation has been used to calculate predicted D for 217 043 compounds present in a publicly available natural product database (DEREP-NP) and to dereplicate known NPs in a mixture based on matching of experimental D and structural features derived from NMR analysis with predicted D and calculated structural features in the database. These models have been validated by the dereplication of a mixture of two known sesquiterpenes obtained from Tasmannia xerophila and the identification of new alkaloids from the bryozoan Amathia lamourouxi. These new methodologies allow the MW of compounds in mixtures to be predicted without the need for MS analysis, the dereplication of known compounds and identification of new compounds based solely on parameters derived by DOSY NMR.

Tedaniophorbasins A and B-Novel Fluorescent Pteridine Alkaloids Incorporating a Thiomorpholine from the Sponge Tedaniophorbas ceratosis.

Two new fluorescent pteridine alkaloids, tedaniophorbasins A (1) and B (2), together with the known alkaloid N-methyltryptamine, were isolated, through application of mass directed purification, from the sponge Tedaniophorbas ceratosis collected from northern New South Wales, Australia. The structures of tedaniophorbasins A and B were deduced from the analysis of 1D/2D NMR and MS data and through application of 13C NMR DFT calculations. Tedaniophorbasin A possesses a novel 2-imino-1,3-dimethyl-2,3,7,8-tetrahydro-1H-[1,4]thiazino[3,2-g]pteridin-4(6H)-one skeleton, while tedaniophorbasin B is its 2-oxo derivative. The compounds show significant Stokes shifts (~14,000 cm-1) between excitation and emission wavelengths in their fluorescence spectra. The new compounds were tested for bioactivity against chloroquine-sensitive and chloroquine-resistant strains of the malaria parasite Plasmodium falciparum, breast and pancreatic cancer cell lines, and the protozoan parasite Trypanosoma brucei brucei but were inactive against all targets at 40 µM.

Citronamine A, an Antiplasmodial Isoquinoline Alkaloid from the Australian Marine Sponge Citronia astra.

Citronamine A (1), an isoquinoline alkaloid containing an unprecedented pentacyclic ring system, was isolated from the Australian marine sponge Citronia astra. Based on the combination of MS and NMR analyses and comparison of experimental and TDDFT calculated ECD spectra, the absolute structure of 1 was determined. Compound 1 displayed moderate activity against drug sensitive (3D7) and drug resistant (Dd2) strains of the parasite, Plasmodium falciparum, responsible for malaria.

Antiplasmodial Alkaloids from the Australian Bryozoan Amathia lamourouxi.

An extract from the bryozoan Amathia lamourouxi with antiplasmodial activity was identified through high-throughput screening of an Australian marine invertebrate extract library against Plasmodium falciparum. Chemical investigation of A. lamourouxi resulted in the isolation of six new brominated alkaloids, convolutamines K and L (1 and 2), volutamides F-H (3-5), and 2,5-dibromo-1-methyl-1H-indole-3-carbaldehyde (6). Three of the compounds (2-4) displayed moderate to potent antiplasmodial activity against both the chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) parasite strains of Plasmodium falciparum with an IC50 range of 0.57-1.7 µM and a high selectivity index against a human cell line (HEK293).

Sulfadiazine Masquerading as a Natural Product from Scilla madeirensis (Scilloideae).

The structure of 2,4-(4'-aminobenzenamine)pyrimidine (1), a pyrimidine alkaloid previously isolated from the bulbs of Scilla madeirensis (Asparagaceae, synonym Autonoë madeirensis), has been revised. These conclusions were met via comparison of reported NMR and EIMS data with those obtained from synthetic standards. The corrected structure is the antibiotic sulfadiazine (2), which has likely been isolated as a contaminant from the site of collection. The reported bioactivity of 1 as an α1-adrenoceptor antagonist should instead be ascribed to sulfadiazine. Our findings appear to show another example of an anthropogenic contaminant being identified as a natural product and emphasize the importance of considering the biosynthetic origins of isolated compounds within a phylogenetic context.

Structure Revisions of the Sponge-Derived Dibrominated Bis-indole Alkaloids, Echinosulfone A and the Echinosulfonic Acids A to D.

The structures of the sponge-derived dibrominated bis-indole alkaloids, namely, echinosulfone A (2) and the echinosulfonic acids A to D (9-12), have been revised based upon reanalysis of their NMR spectroscopic and MS spectrometric data, comparison of this data with those reported for structurally related compounds, and based on their common biogenesis. The reinterpreted spectroscopic evidence has been corroborated by the total synthesis of the revised structure of echinosulfone A (2). This was achieved by bis-carbonylation at C-3 of the magnesium salt of 6-bromoindole with triphosgene to afford the new dibrominated bis-indole ketone, bis(6-bromo-1H-indol-3-yl)methanone (3), followed by N-sulfonation of one indole moiety to furnish 6-bromo-3-(6-bromo-1H-indole-3-carbonyl)-1H-indole-1-sulfonate (2). The five marine alkaloids corrected herein each contain an indole sulfamate and are all carbon-bridged dibrominated bis-indoles: echinosulfone A (2) is a di(1H-indol-3-yl)methanone, while the echinosulfonic acids A to D (9-12) are methyl 2,2-bis(1H-indol-3-yl) acetates.