Inflammation is a crucial biological response, but inflammatory diseases pose a significant global health problem. While nonsteroidal anti-inflammatory drugs (NSAIDs) and COXIBs exist, they often cause cardiovascular side effects. Natural products, particularly alkaloids, represent a valuable source of new drug candidates. Benzoazepines, a key structural motif in various pharmaceuticals and agrochemicals, are known pharmacophores with diverse biological activities, including antidepressant, anticonvulsant, anti-HIV-1, and antiallergic properties. This research focused on discovering and characterizing new bioactive compounds from marine fungi, specifically *Aspergillus candidus*, a fungus previously shown to produce compounds with antifungal, antimalarial, and antiviral activities. The study aimed to isolate, elucidate the structure of, synthesize, and evaluate the anti-inflammatory activity of novel benzoazepine alkaloids from this fungus.

The literature review extensively covers existing anti-inflammatory drugs, their limitations, and the potential of natural products in drug discovery, especially focusing on benzoazepines and their biological activities. It highlights the successful isolation of bioactive compounds from marine fungi in previous studies, setting the stage for the current investigation of *Aspergillus candidus*. The review emphasizes the importance of benzoazepines as pharmacophores in various therapeutic areas and the need for novel anti-inflammatory agents with improved safety profiles.

The methodology involved the isolation of compounds from *Aspergillus candidus* using various chromatographic techniques (LC-MS/MS-Based Molecular Networking, HPLC-UV-DAD, RP-HPLC, chiral HPLC). Structure elucidation employed extensive spectroscopic methods (HRESIMS, IR, 1H NMR, 13C NMR, DEPT, HSQC, COSY, HMBC), single crystal X-ray diffraction, and ECD calculations. Total synthesis of (±)-1 and (±)-2 started from commercially available L-aspartic acid diethyl ester hydrochloride and monoethyl malonate. The key synthetic step was an intramolecular Friedel-Crafts reaction to construct the tricyclic skeleton. Anti-inflammatory activity was assessed using a macrophage stimulation assay in LPS-stimulated RAW 264.7 cells, measuring NO production and the levels of pro-inflammatory cytokines TNF-α and IL-6 via ELISA. Chiral HPLC was used to separate enantiomers, and the mechanism of racemization of compound 1 was investigated.

Two new benzoazepine alkaloids, (+)-asperazepanone A (1) and (+)-asperazepanone B (2), with a unique 6/7/5 ring system were isolated. The racemic form of asperazepanone A (1) was also obtained. The structures and absolute configurations were fully elucidated using spectroscopic analysis and X-ray crystallography. The total syntheses of (±)-1 and (±)-2 were accomplished in 7 and 8 steps respectively, with the key step being an intramolecular Friedel-Crafts reaction. Importantly, (+)-asperazepanone B (2) exhibited significant anti-inflammatory activity, inhibiting NO production (43 ± 4% at 1 µM) and reducing TNF-α (40 ± 2% at 0.1 µM) and IL-6 (77 ± 7% at 0.1 µM) levels in LPS-stimulated RAW 264.7 cells. The enantiomer (−)-2 showed no activity. A plausible biosynthetic pathway from L-tryptophan was proposed. The racemization of (+)-1 to (−)-1 was attributed to enamine-imino tautomerism under alkaline conditions.

The discovery and synthesis of asperazepanones A and B provide valuable insights into the chemistry and biology of marine-derived fungi. The potent anti-inflammatory activity of (+)-asperazepanone B (2) is particularly noteworthy, highlighting its potential as a lead compound for developing novel anti-inflammatory drugs. The structure-activity relationship study revealed the importance of both the absolute configuration (S) and the presence of a methyl group at N-4 for the observed bioactivity. The proposed biosynthetic pathway suggests a potential route for future metabolic engineering approaches to produce these compounds. The successful total synthesis provides a platform for generating analogues to further optimize the anti-inflammatory activity and reduce potential toxicity.

This study successfully isolated, characterized, and synthesized two novel pyrrolinone-fused benzoazepine alkaloids, asperazepanones A and B, from *Aspergillus candidus*. (+)-Asperazepanone B exhibited potent anti-inflammatory activity, making it a promising lead for drug development. Future work will focus on further structural optimization, in-depth mechanism of action studies, and in vivo evaluation of the lead compound.

The study primarily focused on in vitro anti-inflammatory activity. Further in vivo studies are necessary to confirm the efficacy and safety of (+)-asperazepanone B (2) as a potential therapeutic agent. The yield of the key intramolecular Friedel-Crafts reaction could be further improved for large-scale synthesis. The complete elucidation of the racemization mechanism of asperazepanone A warrants further investigation.