Discovery, total syntheses and potent anti-inflammatory activity of pyrrolinone-fused benzoazepine alkaloids Asperazepanones A and B from *Aspergillus candidus*

The study addresses the need for new anti-inflammatory agents with higher efficacy and lower toxicity, given the cardiovascular side effects associated with current NSAIDs and COX-2 selective inhibitors. Benzoazepines are important pharmacophores present in several marketed drugs and exhibit diverse biological activities. Motivated by the pharmaceutical relevance of benzoazepines and the authors’ ongoing search for bioactive metabolites from marine fungi, the work aimed to discover, elucidate, synthesize, and evaluate new benzoazepine alkaloids from the coral-derived fungus Aspergillus candidus.

The authors outline that although numerous NSAIDs (e.g., aspirin, diclofenac, indomethacin) and COXIBs provide relief in inflammatory diseases, their cardiovascular risks limit use. Benzo[b]azepine motifs occur in marketed tricyclic antidepressants (e.g., anafranil, imipramine, tienopramine, amezepine) and are associated with additional activities (anticonvulsant, anti-HIV-1, antiallergic). Natural products remain a prolific source of drugs, and the authors’ prior work on metabolites from Aspergillus candidus has yielded bioactive compounds. This context supports exploring benzoazepine alkaloids from marine fungi as potential anti-inflammatory leads.

- Source, isolation, and analytical guidance: Chemical investigation of the gorgonian coral-derived fungus Aspergillus candidus (strain CHNSCLM-0393) was guided by LC-MS/MS-based molecular networking and HPLC-UV-DAD, leading to the isolation of two benzoazepine alkaloids with a 6/7/5 tricyclic skeleton, asperazepanones A (1) and B (2). - Structure elucidation: Molecular formulas were determined by positive-mode HRESIMS (1: m/z 231.0763 [M+H]+, C12H10N2O3; 2: m/z 245.0917 [M+H]+, C13H12N2O3). IR indicated carbonyl groups (e.g., 1: 1624 cm−1). 1D/2D NMR (1H, 13C, DEPT, HSQC, COSY, HMBC) established substructures and connectivity, defining a pyrrolinone-fused benzoazepine. Single-crystal X-ray diffraction: 1 crystallized in P21/c indicating racemic nature; 2 afforded absolute configuration 10aS (Cu Kα, Flack −0.1(3)). Time-dependent DFT/ECD calculations corroborated absolute configurations by comparison with experimental ECD spectra. - Chiral analysis and epimerization studies: Chiral HPLC of 1 showed two peaks (3:2). Separation yielded (+)-1 and (−)-1; ECD assigned 10aS to (+)-1 and 10aR to (−)-1. Short-term fermentation followed by rapid isolation showed (+)-1 as the major enantiomer (~95%), indicating (−)-1 as an artifact arising during prolonged fermentation. Stability/epimerization tests: Treating (+)-1 with 0.01% NaOH, 10% AcOH, or 2% TFA in MeOH at room temperature partially converted it to (−)-1 and a dehydrogenation product (3); 2 was stable. A racemization mechanism via enamine–imino tautomerism was proposed. - Proposed biosynthesis: A biogenetic pathway from L-tryptophan via 3-hydroxykynurenine reacting with acetyl-CoA to form intermediates leading to (+)-1 by intramolecular addition and dehydrations; (+)-2 arises by N-methylation of (+)-1. - Total synthesis: Retrosynthesis targeted acid 4 for intramolecular Friedel–Crafts cyclization. Synthesis began from L-aspartic acid diethyl ester hydrochloride (8) and monoethyl malonate (9). Steps: EDCI-mediated amidation to 10 (90%); Dieckmann cyclization (LiHMDS) to racemic tetramic acid 7 (70%), noting C-5 epimerization; decarboxylation of 7 to 3-carbonyl tetramic acid 6 by refluxing in TFA/MeCN; nucleophilic addition of aromatic amine 5 to 6 in AcOH/EtOH at 70 °C to give adduct 11 (30% over two steps); hydrolysis to acid 4 (85%). Key intramolecular Friedel–Crafts acylation was optimized: PPA at 120 °C for 1 h provided tricyclic intermediate 12 in 22% yield (lower yields or failure with TFAA, SOCl2/AlCl3, BF3·Et2O, POCl3, H2SO4). Final steps: BBr3-mediated demethylation to (±)-asperazepanone A (1) (74%); methylation (CH3I/Cs2CO3) followed by BBr3 demethylation to (±)-asperazepanone B (2) (68% over two steps). Chiral HPLC (Chiralpak IC) resolved 2 into (+)-2 and (−)-2. - Biological evaluation: Anti-inflammatory activity was tested in LPS-stimulated RAW 264.7 macrophages. Cell viability was monitored. NO production was measured; TNF-α and IL-6 levels were quantified by ELISA. L-NMMA served as positive control (5 µM). Compounds tested included (±)-1, (+)-2, and (−)-2 at indicated concentrations.

- Discovery and structure: Two novel pyrrolinone-fused benzoazepine alkaloids with a unique 6/7/5 tricyclic scaffold, asperazepanones A (1) and B (2), were isolated from Aspergillus candidus. Compound 1 was isolated as a racemate; X-ray crystallography indicated P21/c (racemic). Compound 2 was optically active; X-ray analysis established absolute configuration 10aS (Flack −0.1(3)). ECD calculations matched experimental spectra, assigning (+)-1 as 10aS and (−)-1 as 10aR. - Racemization and artifact: Short-term fermentation showed (+)-1 predominance (~95%), indicating (−)-1 as an isolation artifact formed via base/acid-promoted racemization through enamine–imino tautomerism; 2 lacked this liability and was stable. - Total synthesis: Efficient total syntheses of (±)-1 (7 steps from 8 and 9) and (±)-2 (8 steps) were achieved. The key step was an intramolecular Friedel–Crafts cyclization promoted by PPA, optimized to 22% yield for intermediate 12 at 120 °C for 1 h. Final transformations furnished (±)-1 (74% from 12 by BBr3 demethylation) and (±)-2 (68% over two steps: methylation then BBr3 demethylation). Spectral data of synthetic compounds matched natural products. - Anti-inflammatory activity: In LPS-stimulated RAW 264.7 cells, only (+)-2 showed activity without affecting viability: NO inhibition 43 ± 4% at 1 µM; TNF-α reduced by 40 ± 2% (p < 0.0001) and IL-6 by 77 ± 7% (p < 0.01) at 0.1 µM. (±)-1 and (−)-2 were inactive. Positive control L-NMMA inhibited NO by 61 ± 11% at 5 µM. Preliminary SAR indicates the absolute configuration (S at 10a) and N-4 methyl group are critical for activity.

The work addresses the need for safer anti-inflammatory agents by identifying a new benzoazepine chemotype from a marine fungus and demonstrating potent, enantioselective anti-inflammatory effects in vitro. Structural elucidation confirmed an unprecedented pyrrolinone-fused benzoazepine 6/7/5 framework. The racemization behavior of 1 explains the occurrence of an artifact enantiomer during prolonged fermentation and underscores process conditions needed to retain stereochemical integrity. The total syntheses, centered on an intramolecular Friedel–Crafts cyclization, provide access to both natural products and enable future analog development. Biologically, only the 10aS enantiomer of asperazepanone B with an N-4 methyl exhibited strong inhibition of NO production and pro-inflammatory cytokines TNF-α and IL-6 at low concentrations, highlighting a strong stereochemical requirement and substituent effect for activity. These findings position (+)-asperazepanone B as a promising lead and the synthetic route as a platform for SAR exploration and mechanism-of-action studies.

In summary, we have isolated and characterized two novel natural products, (+)-asperazepanones A (1) and B (2), from the gorgonian derived fungus Aspergillus candidus. The artifact (−)-asperazepanone A (1) was confirmed and its transformation mechanism was also studied. Moreover, the total syntheses of (±)-1 and (±)-2 featuring intramolecular Friedel-Crafts reaction to construct the tricyclic 6/7/5 architecture were also accomplished. This synthetic process should provide routes for obtaining synthetic analogs. Biological studies revealed that (+)-asperazepanone B (2) showed potent anti-inflammatory activity, which confirmed it as promising new lead for developing anti-inflammatory agent. Further structural optimization, biological evaluation, and mechanism of action are ongoing and will be reported in due course.

- Biological evaluation was limited to in vitro assays in LPS-stimulated RAW 264.7 macrophages; the mechanism of action was not elucidated and is deferred to future work. - The key intramolecular Friedel–Crafts cyclization proceeded in modest yield (up to 22%), which may limit scalability without further optimization. - Enantioselective synthesis was not pursued due to partial racemization under cyclization conditions, necessitating chiral resolution for 2 and complicating asymmetric access. - Racemization of 1 under certain conditions (e.g., prolonged fermentation, acid/base in MeOH) indicates stereochemical lability that may impact isolation and handling.