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.

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.

Marine natural products.

Covering: January to the end of December 2022This review covers the literature published in 2022 for marine natural products (MNPs), with 645 citations (633 for the period January to December 2022) referring to compounds isolated from marine microorganisms and phytoplankton, green, brown and red algae, sponges, cnidarians, bryozoans, molluscs, tunicates, echinoderms, the submerged parts of mangroves and other intertidal plants. The emphasis is on new compounds (1417 in 384 papers for 2022), together with the relevant biological activities, source organisms and country of origin. Pertinent reviews, biosynthetic studies, first syntheses, and syntheses that led to the revision of structures or stereochemistries, have been included. An analysis of NP structure class diversity in relation to biota source and biome is discussed.

Letter to the editor on "Antimicrobial compounds isolated from Tropaeolum tuberosum".

A bisphosphonate recently isolated from Tropaeolum tuberosum is almost certainly a contaminant and not a genuine natural product.

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 natural products.

Covering: January to December 2021This review covers the literature published in 2021 for marine natural products (MNPs), with 736 citations (724 for the period January to December 2021) referring to compounds isolated from marine microorganisms and phytoplankton, green, brown and red algae, sponges, cnidarians, bryozoans, molluscs, tunicates, echinoderms, mangroves and other intertidal plants and microorganisms. The emphasis is on new compounds (1425 in 416 papers for 2021), together with the relevant biological activities, source organisms and country of origin. Pertinent reviews, biosynthetic studies, first syntheses, and syntheses that led to the revision of structures or stereochemistries, have been included. An analysis of the number of authors, their affiliations, domestic and international collection locations, focus of MNP studies, citation metrics and journal choices is discussed.

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.

Amphiphilic Polyamine α-Synuclein Aggregation Inhibitors from the Sponge Aaptos lobata.

Bioassay-guided investigation of the sponge Aaptos lobata resulted in the isolation and identification of two new amphiphilic polyamines, aaptolobamines A (1) and B (2). Their structures were determined through analysis of NMR and MS data. MS analysis also indicated that A. lobata contained a complex mixture of aaptolobamine homologues. Both aaptolobamines A (1) and B (2) show broad bioactivity, including cytotoxicity against cancer cell lines, moderate antimicrobial activity against a methicillin-resistant strain of Staphylococcus aureus, and weak activity against a Pseudomonas aeruginosa strain. The mixtures of aaptolobamine homologues were shown to contain compounds that bind to the Parkinson's disease associated amyloid protein α-synuclein and inhibit its aggregation.

Field-scale monitoring of green sea turtles (Chelonia mydas): Influence of site characteristics and capture technique on the blood metabolome.

Given their threatened status, there is considerable interest in establishing monitoring techniques that can be used to evaluate the health of sea turtles in the wild. The present study represents a methodological contribution towards field-scale metabolomic assessment of sea turtles, by exploring differences in blood biochemistry associated with site characteristics and capture technique. We compared the metabolome of blood from animals at three locations (two coastal and one reefal), collected from turtles that were either resting or active, and sampled across multiple seasons at one location. Our results show clear differences in the metabolome of turtles from the three locations, some of which are likely attributable to differences in diet or forage quality and others which may reflect differences in other factors (e.g., occurrence of land-based contaminants or other biotic and/or abiotic stressors) between coastal and reefal sites. Our analysis also revealed the influence of capture technique on metabolite profiles, with numerous markers of physical exertion in animals captured while active that were absent in turtles sampled while resting. We observed a modest potential for temporal differences in the metabolome, but controlling for sampling time did not change the overall conclusions of our study. This suggests that temporal differences in the metabolome warrant consideration when designing studies to evaluate the status of sea turtles in the wild, but that site characteristics and capture technique are bigger drivers. However, sample size for this comparison was relatively small and further investigation of seasonal differences in the metabolome are warranted. Research exploring each of these factors more closely will further contribute towards achieving robust metabolomics analysis of sea turtles across large spatial and temporal scales.

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 binding activity of the plant growth promoter asterubine.

Preventing the aggregation of certain amyloid proteins has the potential to slow down the progression of diseases like Alzheimer's, Parkinson's, and type 2 diabetes mellitus. During a high-throughput screen of 300 Australian marine invertebrate extracts, the extract of the marine sponge Thorectandra sp. 4408 displayed binding activity to the Parkinson's disease-associated protein, α-synuclein. Isolation of the active component led to its identification as the known plant growth promoter asterubine (1). This molecule shares distinct structural similarities with potent amyloid beta aggregation inhibitors tramiprosate (homotaurine) and ALZ-801. Herein we report the isolation, NMR data acquired in DMSO and α-synuclein binding activity of asterubine (1).

Marine natural products.

Covering: 2020This review covers the literature published in 2020 for marine natural products (MNPs), with 757 citations (747 for the period January to December 2020) referring to compounds isolated from marine microorganisms and phytoplankton, green, brown and red algae, sponges, cnidarians, bryozoans, molluscs, tunicates, echinoderms, mangroves and other intertidal plants and microorganisms. The emphasis is on new compounds (1407 in 420 papers for 2020), together with the relevant biological activities, source organisms and country of origin. Pertinent reviews, biosynthetic studies, first syntheses, and syntheses that led to the revision of structures or stereochemistries, have been included. A meta analysis of bioactivity data relating to new MNPs reported over the last five years is also presented.

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.

How different are marine microbial natural products compared to their terrestrial counterparts?

Covering: 1877 to 2020A key challenge in natural products research is the selection of biodiversity to yield novel chemistry. Recently, marine microorganisms have become a preferred source. But how novel are marine microorganism natural products compared to those reported from terrestrial microbes? Cluster analysis of chemical fingerprints and molecular scaffold analysis of 55 817 compounds reported from marine and terrestrial microorganisms, and marine macro-organisms showed that 76.7% of the compounds isolated from marine microorganisms are closely related to compounds isolated from terrestrial microorganisms. Only 14.3% of marine microorganism natural products are unique when marine macro-organism natural products are also considered. Studies targeting marine specific and understudied microbial phyla result in a higher likelihood of finding marine specific compounds, whereas the depth and geographic location of microorganism collection have little influence. We recommend marine targeted strain isolation, incorporating early use of genomic sequencing to guide strain selection, innovation in culture media and cultivation techniques and the application of cheminformatics tools to focus on unique natural product diversity, rather than the dereplication of known compounds.

Sycosterol A, an α-Synuclein Inhibitory Sterol from the Australian Ascidian Sycozoa cerebriformis.

During a recent biodiscovery study to identify new α-synuclein (α-syn) aggregation inhibitors, we screened 29 Australian marine sponge and ascidian extracts in an MS binding assay. This resulted in an extract from the ascidian Sycozoa cerebriformis showing activity toward α-syn. The bioassay and MS guided isolation process led to the identification of one new polyoxygenated sterol sulfate, sycosterol A (1). The structure of this low-yielding steroid was elucidated from HRMS and NMR analysis. Sycosterol A displayed moderate antiaggregation activity with 46.2% (±1.8) inhibition when screened against α-syn at a 5:1 (sycosterol A:α-syn) molar ratio. The α-syn antiaggregation activity displayed by 1 and the recent discovery of similar sterols with α-syn antiaggregation activity and potent antiprion activity suggest this unique class may be useful antineurodegenerative compounds.

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.