Discovery of Anti-MRSA Secondary Metabolites from a Marine-Derived Fungus Aspergillus fumigatus

Methicillin-resistant Staphylococcus aureus (MRSA), a WHO high-priority pathogen that can cause great harm to living beings, is a primary cause of death from antibiotic-resistant infections. In the present study, six new compounds, including fumindoline A–C (1–3), 12β, 13β-hydroxy-asperfumigatin (4), 2-epi-tryptoquivaline F (17) and penibenzophenone E (37), and thirty-nine known ones were isolated from the marine-derived fungus Aspergillus fumigatus H22. The structures and the absolute configurations of the new compounds were unambiguously assigned by spectroscopic data, mass spectrometry (MS), electronic circular dichroism (ECD) spectroscopic analyses, quantum NMR and ECD calculations, and chemical derivatizations. Bioactivity screening indicated that nearly half of the compounds exhibit antibacterial activity, especially compounds 8 and 11, and 33–38 showed excellent antimicrobial activities against MRSA, with minimum inhibitory concentration (MIC) values ranging from 1.25 to 2.5 μM. In addition, compound 8 showed moderate inhibitory activity against Mycobacterium bovis (MIC: 25 μM), compound 10 showed moderate inhibitory activity against Candida albicans (MIC: 50 μM), and compound 13 showed strong inhibitory activity against the hatching of a Caenorhabditis elegans egg (IC50: 2.5 μM).


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as one of the most common bacteria in both community and hospital-acquired infections, causing significant morbidity and mortality [1]. Compared to non-resistant Staphylococcus aureus infections, the mortality rate of MRSA infections increases by 64% [2]. Vancomycin is a last-resort treatment for MRSA infections. However, strains that are less susceptible to vancomycin are emerging in clinics [3,4]. As a result, new antibiotics to treat MRSA infections are desperately needed. In 2017, the development of new antibiotics for the treatment of MRSA infections is listed as a high urgency level by the WHO (World Health Organization) [5].
The marine environment is one of the most complex atmospheres on the earth, due to the huge variations in predation, temperature, pressure, light, and nutrient circumstances, etc. [6]. The organisms that thrive in marine environments could produce extremely diverse and complicated functional secondary metabolites that differ from those observed in terrestrial environments [6][7][8]. In recent decades, an increasing number of bioactive marine natural products (MNPs) have piqued the interest of chemists and pharmacologists for their medicinal values [9,10], such as the earliest marine sponge-derived anticancer drug cytarabine (Cytosar-U ® ), the marine sponge-derived antiviral drug vidarabine (Arasena A ® ), the mollusk-derived ziconotide (Prialt ® ) for the treatment of neuropathic pain, the
Mar. Drugs 2022, 20, 302 5 of 19 H2-16 to C-14, as well as the chemical shifts of C-2 (δ 135.1), C-3 (δ 142.4), C-5 (δ 158.3), C-6 (δ 111.1), and C-14 (δ 164.7), supported a 2-pyridinecarboxylic acid moiety that was connected with a γ-aminobutyric acid moiety through C-14 and linked with a 1,2,4-trisubstituted benzene moiety through C-7, and completed the assignment of the moiety. The HMBC correlations from H-20 to C-2, C-3, and C-21, together with the 1 H-1 H COSY correlations of H-20/H3-22/H3-23, suggested that the isobutenyl group was located at C-3 of the 2-pyridinecarboxylic acid moiety. The key HMBC correlations from H3-24 to C-11 indicated that the methoxy group was located at C-11. Furthermore, these data accounted for 11 of the 12 degrees of unsaturation, implying the presence of an additional cycle, attributed to the NH bridging between C-2 and C-13 to establish the indole-pyridinecarboxylic acid skeleton ( Figure 2). Therefore, the 2D structure of 1 was determined as shown below. Fumindoline B (2) was obtained as a chartreuse powder. Its molecular formula, C22H23N3O4, with 14 degrees of unsaturation, was established on the basis of the HRESIMS data ( Figure S15). The UV spectrum showed absorptions at 282 nm and 343 nm, which were similar to those of 1, indicating that 2 might have the same conjugation system as 1. The IR spectrum indicated the presence of a secondary amine N-H signal (2980 cm −1 ) and an amide carbonyl signal (1628 cm −1 ). The 1 H NMR and 13 C NMR spectra indicated the presence of two sets of very similar signals, with the same number of carbons (Figures S9 and S10). The spectra of the two sets of signals are well resolved in pairs at 313K and 298K in DMSO-d6, indicating the presence of two relatively stable isomers. From the integrals of the completely resolved signals, a ratio of 1:0.7 was calculated for the two stable isomers. To be better distinguished, we assigned the major isomer as 2a and the minor one as 2b, respectively ( Figure 3). The 1 H NMR and 13 C NMR spectra data of 2 showed close similarity to those of 1, with the biggest difference in the methine . A detailed analysis of the 2D NMR data, including HSQC, HMBC, and COSY spectra, revealed that 2 contained the same indole-pyridinecarboxylic acid skeleton as that of 1 (Figures S11-S13). The HMBC correlations from H2-16 to C-17 and C-18, H2-17 to C-16, C-18, and C-19, H2-18 to C-17, C-19, and C-20, and the 1 H-1 H COSY correlations of H2-16/H2-17/H2-18/H-19, together with the molecular formula, indicated the presence of a proline moiety, and this conclusion was also Fumindoline B (2) was obtained as a chartreuse powder. Its molecular formula, C 22 H 23 N 3 O 4 , with 14 degrees of unsaturation, was established on the basis of the HRESIMS data ( Figure S15). The UV spectrum showed absorptions at 282 nm and 343 nm, which were similar to those of 1, indicating that 2 might have the same conjugation system as 1. The IR spectrum indicated the presence of a secondary amine N-H signal (2980 cm −1 ) and an amide carbonyl signal (1628 cm −1 ). The 1 H NMR and 13 C NMR spectra indicated the presence of two sets of very similar signals, with the same number of carbons (Figures S9 and S10). The spectra of the two sets of signals are well resolved in pairs at 313K and 298K in DMSO-d 6 , indicating the presence of two relatively stable isomers. From the integrals of the completely resolved signals, a ratio of 1:0.7 was calculated for the two stable isomers. To be better distinguished, we assigned the major isomer as 2a and the minor one as 2b, respectively ( Figure 3). The 1 H NMR and 13 C NMR spectra data of 2 showed close similarity to those of 1, with the biggest difference in the methine . A detailed analysis of the 2D NMR data, including HSQC, HMBC, and 1 H-1 H COSY spectra, revealed that 2 contained the same indole-pyridinecarboxylic acid skeleton as that of 1 (Figures 4 and S11-S13). The HMBC correlations from H 2 -16 to C-17 and C-18, H 2 -17 to C-16, C-18, and C-19, H 2 -18 to C-17, C-19, and C-20, and the 1 H-1 H COSY correlations of H 2 -16/H 2 -17/H 2 -18/H-19, together with the molecular formula, indicated the presence of a proline moiety, and this conclusion was also confirmed by the 14 degrees of unsaturation and the chemical shifts of C-16 (δ C 49.6 (2a); δ C 47.5 (2b)) and C-19 (δ C 59.8 (2a); δ C 60.4 (2b)).
The E/Z isomer exists in the tertiary amide. In the solution at room temperature, the slow rotation of the C-N bond in NMR makes it possess the characteristics of a partial double bond [23]. A comparison of the 1 H NMR signals and 13 C NMR signals of 2a and 2b revealed differences in the proline moiety, including variations in the H-19 (δ H 4.48 (2a); δ H 5.3 (2b)), C-16 (δ C 49.6 (2a); δ C 47.5 (2b)), C-17 (δ C 25.2 (2a); δ C 21.9 (2b)), and C-18 (δ C 28.6 (2a); δ C 31.2 (2b)). As shown in Figure 4, strong NOE effects between H-6 and H-16 for 2a and between H-6 and H-19 for 2b were observed in the ROSEY spectrum ( Figure S14). confirmed by the 14 degrees of unsaturation and the chemical shifts of C-16 (δC 49.6 (2a); δC 47.5 (2b)) and C-19 (δC 59.8 (2a); δC 60.4 (2b)). The E/Z isomer exists in the tertiary amide. In the solution at room temperature, the slow rotation of the C-N bond in NMR makes it possess the characteristics of a partial double bond [23]. A comparison of the 1 H NMR signals and 13 C NMR signals of 2a and 2b revealed differences in the proline moiety, including variations in the H-19 (δH 4.48 (2a); δH 5.3 (2b)), C-16 (δC 49.6 (2a); δC 47.5 (2b)), C-17 (δC 25.2 (2a); δC 21.9 (2b)), and C-18 (δC 28.6 (2a); δC 31.2 (2b)). As shown in Figure 4, strong NOE effects between H-6 and H-16 for 2a and between H-6 and H-19 for 2b were observed in the ROSEY spectrum ( Figure  S14).  The absolute configuration of the amino acids from compound 2 was determined by the advanced Marfey's method [24]. The mixture obtained after hydrolyzing compound 2 and further derivatization with L-FDAA was analyzed by HPLC-DAD. The HPLC analyses of the mixture of hydrolysates and appropriate amino acid standards confirmed the L configurations for proline in 2 ( Figure 5). Consequently, the absolute configuration of 2 was elucidated to be 19S. Fumindoline C (3) was obtained as a chartreuse powder. The molecular formula of 3 was established to be C23H25N3O4 from its HREIMS data ( Figure S22). The 1 H and 13 C NMR spectra of 3 were similar to those of 2, possessing two sets of signals (Figures S16 and S17), except for the presence of an additional methoxyl group. The substitution of the methoxyl group was further confirmed by the HMBC correlations from H3-26 to C-20. A further The absolute configuration of the amino acids from compound 2 was determined by the advanced Marfey's method [24]. The mixture obtained after hydrolyzing compound 2 and further derivatization with L-FDAA was analyzed by HPLC-DAD. The HPLC analyses of the mixture of hydrolysates and appropriate amino acid standards confirmed the L configurations for proline in 2 ( Figure 5). Consequently, the absolute configuration of 2 was elucidated to be 19S. confirmed by the 14 degrees of unsaturation and the chemical shifts of C-16 (δC 49.6 (2a); δC 47.5 (2b)) and C-19 (δC 59.8 (2a); δC 60.4 (2b)). The E/Z isomer exists in the tertiary amide. In the solution at room temperature, the slow rotation of the C-N bond in NMR makes it possess the characteristics of a partial double bond [23]. A comparison of the 1 H NMR signals and 13 C NMR signals of 2a and 2b revealed differences in the proline moiety, including variations in the H-19 (δH 4.48 (2a); δH 5.3 (2b)), C-16 (δC 49.6 (2a); δC 47.5 (2b)), C-17 (δC 25.2 (2a); δC 21.9 (2b)), and C-18 (δC 28.6 (2a); δC 31.2 (2b)). As shown in Figure 4, strong NOE effects between H-6 and H-16 for 2a and between H-6 and H-19 for 2b were observed in the ROSEY spectrum ( Figure  S14).  The absolute configuration of the amino acids from compound 2 was determined by the advanced Marfey's method [24]. The mixture obtained after hydrolyzing compound 2 and further derivatization with L-FDAA was analyzed by HPLC-DAD. The HPLC analyses of the mixture of hydrolysates and appropriate amino acid standards confirmed the L configurations for proline in 2 ( Figure 5). Consequently, the absolute configuration of 2 was elucidated to be 19S. Fumindoline C (3) was obtained as a chartreuse powder. The molecular formula of 3 was established to be C23H25N3O4 from its HREIMS data ( Figure S22). The 1 H and 13 C NMR spectra of 3 were similar to those of 2, possessing two sets of signals (Figures S16 and S17), except for the presence of an additional methoxyl group. The substitution of the methoxyl group was further confirmed by the HMBC correlations from H3-26 to C-20. A further Fumindoline C (3) was obtained as a chartreuse powder. The molecular formula of 3 was established to be C 23 H 25 N 3 O 4 from its HREIMS data ( Figure S22). The 1 H and 13 C NMR spectra of 3 were similar to those of 2, possessing two sets of signals (Figures S16 and S17), except for the presence of an additional methoxyl group. The substitution of the methoxyl group was further confirmed by the HMBC correlations from H 3 -26 to C-20. A further comprehensive analysis of its 1 H-1 H COSY, HSQC, and HMBC spectra assigned the planar structure of 3 ( Figures S18-S20). The relative configuration of 3 was determined to be the same as that of 2 by their similar structure and ROESY data ( Figures S14 and S21). Accordingly, 3 was determined to be a methyl ester of 2.
12β,13β-hydroxy-asperfumigatin (4) was obtained as a white amorphous solid. Its molecular formula was determined as C 27 H 33 N 3 O 7 by HRESIMS data ( Figure S31). The 1 H NMR spectrum of 4 ( Figure S25) displayed four singlet methyl groups (δ H 1.17, 1.25, 2.11, and 2.21), one methoxyl group (δ H 3.85) and four olefinic/aromatic protons (δ H 6.40, 6.90, 7.27, and 7.45). The 13 C-NMR spectrum ( Figure S26) exhibited 27 carbon resonances accounted for the functional groups described above and three amide carbonyl carbons (δ C 164.7, 165.5, and 165.9). A comprehensive analysis of its 2D NMR spectra, including 1 H-1 H COSY, HSQC, and HMBC experiments, confirmed the planar structure of 4 ( Table 2, Figures S27-S29), revealing the presence of the indole moiety and the diketopiperazine moiety in 4 ( Figure 6). The planar structure of 4 was determined to be the same as that of asperfumigatin (5), by detailed interpretation of the 2D NMR spectra and NMR data comparison between 4 and 5. Considering the same biosynthesis origin, compound 4 was deduced to share the same absolute configuration at C-3 and C-6 as those of 5-13. comprehensive analysis of its 1 H-1 H COSY, HSQC, and HMBC spectra assigned the planar structure of 3 (Figures S18-S20). The relative configuration of 3 was determined to be the same as that of 2 by their similar structure and ROESY data ( Figures S21 and S24). Accordingly, 3 was determined to be a methyl ester of 2.
12β,13β-hydroxy-asperfumigatin (4) was obtained as a white amorphous solid. Its molecular formula was determined as C27H33N3O7 by HRESIMS data ( Figure S31). The 1 H NMR spectrum of 4 ( Figure S25) displayed four singlet methyl groups (δH 1.17, 1.25, 2.11, and 2.21), one methoxyl group (δH 3.85) and four olefinic/aromatic protons (δH 6.40, 6.90, 7.27, and 7.45). The 13 C-NMR spectrum ( Figure S26) exhibited 27 carbon resonances accounted for the functional groups described above and three amide carbonyl carbons (δC 164.7, 165.5, and 165.9). A comprehensive analysis of its 2D NMR spectra, including 1 H-1 H COSY, HSQC, and HMBC experiments, confirmed the planar structure of 4 ( Table 2, Figures S27-S29), revealing the presence of the indole moiety and the diketopiperazine moiety in 4 ( Figure 6). The planar structure of 4 was determined to be the same as that of asperfumigatin (5), by detailed interpretation of the 2D NMR spectra and NMR data comparison between 4 and 5. Considering the same biosynthesis origin, compound 4 was deduced to share the same absolute configuration at C-3 and C-6 as those of 5-13. Owing to a lack of sufficient NOESY correlations, the relative configurations of C-12 and C-13 were not determined ( Figure S30). The relative configurations of 4 were determined by the DP4+ probability, based on a theoretical NMR calculation that has been proven to be a very powerful tool in natural product structure elucidation [25,26]. The NMR shifts of eight possible relative orientation isomers were calculated with the GIAO method at the MPW1PW91/6-31+G(d,p), and the DP4+ probabilities of each configuration were evaluated based on Boltzmann-averaged theoretical NMR shielding tensors, which provided a 91.55% confidence for the relative configuration 3S*, 6S*, 12S*, 13R* (Table S1 and S2).
To determine the absolute configurations of 4, a ECD calculation method was applied. The two configurations (3S, 6S, 12S, 13R)-4 and (3R, 6R, 12R, 13S)-4 were calculated using time-dependent density functional theory (TDDFT) at PBE1PBE/6-311 G* level, with the PCM model in methanol, and corrected with a 2 nm blue shift according to UV data. A comparison of the experimental ECD spectrum of 4 and the calculated ECD spectra of (3S, 6S, 12S, 13R)-4 and (3R, 6R, 12R, 13S)-4 showed that the experimental ECD spectrum Owing to a lack of sufficient ROESY correlations, the relative configurations of C-12 and C-13 were not determined ( Figure S30). The relative configurations of 4 were determined by the DP4+ probability, based on a theoretical NMR calculation that has been proven to be a very powerful tool in natural product structure elucidation [25,26]. The NMR shifts of eight possible relative orientation isomers were calculated with the GIAO method at the MPW1PW91/6-31+G(d,p), and the DP4+ probabilities of each configuration were evaluated based on Boltzmann-averaged theoretical NMR shielding tensors, which provided a 91.55% confidence for the relative configuration 3S*, 6S*, 12S*, 13R* (Tables S1 and S2).

Discussion
The marine environmental stress conditions induce many faunae and symbiont microorganisms to synthesize and release secondary metabolites of unique structures and interesting biological activities [61]. These bioactive compounds can serve as an important source for drug discovery. Marine-derived fungi are important sources for the discovery of new antibacterial natural products. Wang et al. isolated the Chaetomium sp. strain NA-S01-R1 from a deep-sea (4050 m) fungus that produced novel chlorinated azaphilone polyketides with antibacterial activity against MRSA [62]. The Emericellopsis minima strain A11, isolated from Talcahuano Bay (Chile), produced an antibiotic called emerimicin IV, with moderate activity against clinical isolates of MDR vancomycin-resistant strains of E. faecalis and MRSA with MIC of 12.5 µg/mL and 100 µg/mL, respectively [63].
A. fumigatus belongs to the filamentous fungi family that is widely distributed in all environments and can cause many diseases and life-threatening conditions in immunocompromised patients [64]. A. fumigatus can produce a wide array of secondary metabolites due to its remarkable adaptability to different environmental conditions, such as fumitremorgins, fumagillins, pseurotins, fumigaclavines, gliotoxins, and helvolic acid derivatives.
Inspired by chemical ecology, we found a marine fungus A. fumigatus H22 with strong antibacterial activities from the marine fungi library. Through in-depth chemical mining, we found 45 compounds, including 6 new compounds, from the culture of this fungus. A evaluation of biological activity showed that nearly half of the compounds exhibit antimicrobial activity. Fumitremorgins derivatives (4-11) have very similar structures, but only a few have strong anti-MRSA activity. Compounds 5, 8 and 11 with strong anti-MRSA activity contain hydroxyl group at C-13, while compounds 6 and 7 without anti-MRSA activity have no hydroxyl group at C-13. In addition, compounds 4 and 5 have the same planar structure, but the 13-OH of compound 4 without anti MRSA activity was α-oriented, while compound 5 and other strongly active compounds were β-oriented. Therefore, it is preliminarily speculated that there is a certain correlation between the substituents and stereoconfiguration in C-12 and C-13 and their anti MRSA activity. Fumitremorgin B (10) was reported with antifungal activity against a variety of phytopathogenic fungi, but it showed weak activity against vancomycin-resistant E. faecalis (VRE), M. bovis, and E. coli in our in vitro assay, which could be involved in fighting against invasion by other pathogens [65].
Pseurotins, with a unique heterospirocyclic furanone-lactam structure, exhibit a broad range of biological activities. In addition to antifungal and antibiotic activities [66,67], pseurotins were also shown to regulate enzymes of cellular metabolism [68], to possess anti-angiogenic activity, to modulate cell differentiation [69], and to inhibit endothelial cell migration [70][71][72]. Fumagillin (33) have been demonstrated to have antitumor, antibacterial and antiparasitic effects [73]. Previous studies revealed that helvolic acid (34) exhibited in vitro antimalarial activity against multidrug resistant Plasmodium falciparum [74], antitrypanosomal activity against Trypanosoma brucei [75], and antimycobacterial activity against M. tuberculosis H37Ra [76]. Our current research showed the strong activities of oxofumitremorgin B (11), helvolic acid (34) From our current findings, it can be found that A. fumigatus from marine sources can produce rich bioactive secondary metabolites, especially in anti-MRSA.

Fungal Material
The fungus H22 was isolated from middle seawater from the Western Pacific. The sample (1 mL) was diluted with sterile H 2 O, 100 µL of which was deposited on a PDA (200 g of potato, 20 g of glucose, 20 g of agar per liter of seawater collected in the Western Pacific) plate containing chloramphenicol (100 µg/mL) and streptomycin (100 µg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and 18S rRNA gene sequences. The phylogenetic tree ( Figure S1), constructed from the ITS gene sequence, indicated that H22 belonged to the genus of Aspergillus, with the highest similarity to A. fumigatus (99.86%, accession number NRRL 163 s). In consideration of the morphological features and phylogeny ( Figure S2), this fungus was identified as A. fumigatus. A reference culture of A. fumigatus H22 maintained at −80 • C was deposited in our laboratory.

Fermentation and Extraction
The isolate was grown for 7 days at 28 • C, on slants of a PDA medium. The spores of the strain on the plate were collected using 0.01% sterile Tween 80 (BTL, Warsaw, Poland) and adjusted to 1 × 10 6 CFU/mL to make inoculum. A large-scale fermentation was carried out in 50 × 500 mL Fernbach culture flasks, holding 100 g of rice in 110 mL of distilled water (each with 0.5 mL of spore suspension) and incubated for 4 weeks at 28 • C. With the help of ultrasonication, the fermented rice substrates were extracted with ethyl acetate (3 × 5 L), and the organic solvent was filtered and evaporated to dryness under a vacuum to obtain the crude extract (78.0 g).

Marfey's Analysis of Compound 2
Compound 2 (2.0 mg) was dissolved in 6 N HCl (2.0 mL) and heated at 100 • C for 24 h. The solutions were then evaporated to dryness and placed in a 4 mL reaction vial and treated with a 10 mg/mL solution of FDAA (200 µL) in acetone, followed by 1 M NaHCO 3 (40 µL). The reaction mixtures were heated at 45 • C for 90 min, and the reactions were quenched by the addition of HCl (1 N, 40 µL). In a similar fashion, the standard L-proline and D-proline were derivatized separately. The derivatives of the acid hydrolysate and the standard amino acids were subjected to RP HPLC analysis (Kromasil C18 column; 5 µM, 4.6 × 250 mm; 1.0 mL/min; UV detection at 340 nm), with a linear gradient of acetonitrile (30-40%) in water (TFA, 0.01%) over 30 min. The retention times for the authentic standards were as follows: L-proline derivative (8.91 min) and D-proline derivative (9.88 min). The absolute configuration of the chiral amino acid in 2 was determined by comparing the retention times.

Computational Details for NMR and ECD
The GMMX software tool was used to undertake the systematic conformational evaluations for 4 and 17, utilizing the MMFF94 molecular mechanics force field. Gaussian 16 software was used to further improve the MMFF94 conformers, utilizing the M062X/6-31G(d) basis set level in gas for NMR calculations and B3LYP/6-31+G(d,p) basis set level in methanol, with a PCM model for ECD calculations. The shielding constants were calculated using the GIAO technique in chloroform, using the SMD solvent model and Gaussian function at mPW1PW91/6-31+G(d,p). A previously documented approach was used to calculate the 1 H and 13 C chemical shifts for the DP4+ probability analysis [77]. ECD spectra were stimulated in methanol with a Gaussian function at the B3LYP/6-311+G(2d,p) level using the PCM model, and 60 NStates were calculated. Boltzmann statistics were used to compute the equilibrium populations of the conformers at 298.15 K, based on their respective free energies (∆G). The Boltzmann weighting of the key conformers was then used to construct the overall ECD spectra. UV correlation was used to correct the systematic mistakes in predicting the wavelength and excited-state energy [78].

Antimicrobial Assay
An antimicrobial assay was performed according to the Antimicrobial Susceptibility Testing Standards, outlined by the Clinical and Laboratory Standards Institute against MRSA (clinical strain from Chaoyang Hospital, Beijing, China), Pseudomonas aeruginosa (ATCC 15692), Escherichia coli (O57:H7), Mycobacterium bovis (ATCC35743), vancomycinresistant Enterococci faecalis (VRE) (clinical strain from 309 Hospital, Beijing, China), and pathogen fungi Candida albicans SC5314. The protocol was performed as previously reported [58,59]. The positive controls were vancomycin against MRSA, E. faecalis, ciprofloxacin against P. aeruginosa and E. coli, amphotericin B for C. albicans, and rifampicin for M. bovis. All the experiments were performed in triplicate.

Conclusions
In summary, we isolated forty-five compounds from A. fumigatus H22, including six new compounds 1-4, 17, and 37. The stereochemistry of the new compounds was determined by quantum calculations of NMR, ECD calculations and chemical derivatizations. Bioactivity screening indicated that compounds 5, 8, 10, 11, 16, 21, 23, 29-38, and 41 exhibited antimicrobial activities against MRSA, with MIC values ranging from 1.25 to 25 µM. Compound 8 also exhibited strong activity against M. bovis, with a MIC of 25 µM. To the best of our knowledge, this is the first report for the antimicrobial activities of compounds 5, 10, 11, 16, 30, 31, and 37. The strains of A. fumigatus from ocean environments are a good source of antibacterial natural products, deserving further exploitation.