Amesilide, a New Bicyclic Polyketide from the Marine Fungus Amesia nigricolor MUT6601

Abstract

A new bicyclic polyketide, amesilide (1), along with the previously reported metabolites, chamisides A (2), B (3), and E (4), chaetoconvosins B (5) and C (6), and chaetochromins A (7) and B (8), were isolated from the marine fungus Amesia nigricolor MUT6601. The structures of the compounds were determined by extensive spectrometric (HRMS) and spectroscopic (1D and 2D NMR) analyses, as well as specific rotation. Absolute configurations of the stereogenic centers of amesilide (1) were determined by a comparison of its experimental circular dichroism (CD) spectrum with its time-dependent density functional theory (TD-DFT) electronic circular dichroism (ECD) spectra. Among them, chaetochromins A (7) and B (8) showed strong antibacterial activity against Staphylococcus aureus S25 (MBC values of 12.50 µM and MIC values of 6.25 µM) and a moderate cytotoxicity against monocytes (THP-1) and peripheral blood cells (PBMC) (IC₅₀ values of 33.65–40.01 µM).

1. Introduction

Marine fungi, which usually produce metabolites different from their terrestrial counterparts, represent important sources of bioactive metabolites for drug discovery [1]. Indeed, reports of new natural products from marine-derived fungi have significantly increased over the last few decades [2]. Chaetomium and Chaetomium-like species (Chaetomiaceae) have been proven to be rich sources of specialized metabolites [3,4,5]. The taxonomy of this genus has recently been revised based on phylogenetic analyses and morpho-physiological characteristics [6]. Currently, the family Chaetomiaceae comprises 10 genera which include five newly established genera, including the genus Amesia [6].

Amesia nigricolor (L.M. Ames) X. Wei Wang and Samson (synonym Chaetomium nigricolor) has latterly attracted the attention of several researchers. Several metabolites recently isolated from A. nigricolor have demonstrated anti-inflammatory or cholesterol-lowering activities [4,7,8,9]. Kim et al. identified twelve metabolites from A. nigricolor isolated from soil [7], finding a bis-naphtho-γ-pyrone derivative ((aS)-asperpyrone A) as an important anti-inflammatory component. In another study, Dhayanithy et al. reported that the extract of the endophytic fungus A. nigricolor, which was isolated from Catharanthus roseus, exhibited potent cytotoxicity and antioxidant properties [10]. Chamiside A, a cytochalasin with a tricyclic core skeleton, was identified by Wang et al. and found to display a moderate antibacterial activity against Staphylococcus aureus [8]. Recently, Gu et al. continued the chemical investigation of the same strain of A. nigricolor F5 of the latter study, discovering five novel chytochalasans, Chamisides B-F [10]. Interestingly, Chamiside F showed a promising bioactivity, with inhibitory activity against the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) protein, reducing the cholesterol absorption.

In this context, we investigated the metabolites produced by the marine fungus A. nigricolor MUT6601. Herein, we report the isolation, structural elucidation, and antimicrobial activities of a new bicyclic polyketide, amesilide (1), along with the previously reported metabolites, chamisides A (2), B (3), and E (4), chaetoconvosins B (5) and C (6), and chaetochromins A (7) and B (8) from the marine fungus A. nigricolor MUT6601. All of the isolated compounds were evaluated for their antimicrobial properties and cytotoxicity.

2. Results and Discussion

The EtOAc extract of A. nigricolor MUT6601 was fractionated and purified by a combination of C-18 solid-phase extraction, silica gel column chromatography, and semi-preparative HPLC to yield compounds 1–8 (Figure 1).

Amesilide (1) was isolated as a yellow powder. Its molecular formula, C₂₅H₃₂O₄, was deduced from the HRESI(+)MS analysis which showed a pseudo-molecular ion peak at m/z 397.2369 [M+H]⁺ (calcd for C₂₅H₃₃O₄⁺ 397.2373, Δ = 1.01 ppm, 10 degrees of unsaturation). The ¹H, ¹³C, and ¹H-¹³C HSQC NMR spectra indicated the presence of six methyl groups (δc 32.7, 20.1, 16.8, 14.6, 13.9, and 11.5), one sp³ methylene (δc 41.3), five sp³ methines (δc 50.9, 44.5, 41.2, 37.5, and 33.5), six sp² methines (δc 152.9, 144.7, 139.3, 134.0, 122.9, and 115.6), and seven quaternary sp² carbons (δc 213.8, 198.8, 172.0, 151.1, 135.4, 133.4, and 131.9), including one ketone (δc 213.8), one α,β-unsaturated ketone (δc 198.8), and one α,β-unsaturated carboxylic acid (δc 172.0) (

Table 1. ¹H (400 MHz) and ¹³C NMR (100 MHz) data (in CDCl₃) of amesilide (1) and its calculated chemical shifts.

Position	1		Calculated Shifts
	1H [δ, mult. (J in Hz)]	13C (δ)	1H (δ)	13C (δ)
1	-	172.0	-	172.6
2	5.81 d (15.5)	115.6	5.82	118.5
3	7.37 d (15.5)	152.9	7.68	161.2
4	-	131.9	-	140.4
5	6.27 s	144.7	6.68	154.8
6	-	135.4	-	144.9
7	5.40 d (9.0)	134.0	5.73	144.0
8	5.15 dd (9.0, 2.6)	37.5	6.05	41.9
9	1.81 br d (11.8)	50.9	1.83	56.8
10	2.28 m	44.5	2.27	49.0
11	-	213.8	-	223.4
12	2.42 dq (12.1, 6.1)	41.2	2.40	46.0
13	2.10 ddd (13.5, 5.1, 2.2); 1.60 td (13.5, 5.1)	41.3	2.09 1.59	44.2
14	2.83 br s	33.5	2.91	38.9
15	6.04 s	139.3	6.64	153.1
16	-	133.4	-	142.5
17	-	151.1	-	163.5
18	6.27 s	122.9	6.47	125.3
19	-	198.8	-	206.2
20	2.25 s	32.7	2.27	36.1
21	1.90 br s	13.9	1.83	15.5
22	2.00 br s	16.8	2.07	19.7
23	1.07 d (6.4)	11.5	1.00	13.5
24	0.99 d (6.4)	14.6	0.89	16.5
25	1.96 br s	20.1	2.01	22.4

). The assembly of the ¹H-¹³C HMBC correlations and ¹H-¹H COSY spin systems revealed that 1 has a bicyclic decalin system. The structure of compound 1 was established based on key ¹H-¹³C HMBC correlations, including H-23 to C-9, C-10, and C-11; H-24 to C-11, C-12, and C-13; H-25 to C-15, C-16, and C-17; H-7 to C-8, C-9, and C-17; and H-18 to C-8, C-16, and C-17. The COSY spectrum showed continuous spin systems (H-7 to H-23 and H-24 to H-15), further supporting this framework. The locations of a ketone group at C-11, three methyl groups attached to C-10, C-12, and C-16, and two substituted chains attached to C-8 and C-17 were also determined through the above correlations. One substituent was determined as 4,6-dimethylhepta-2,4,6-trienoic acid by the COSY correlation between H-2 and H-3, and the HMBC correlations from H₃-21 to C-3, C-4, and C-5 and from H₃-22 to C-5, C-6, and C-7. The other substituent was determined by the HMBC correlations from the methyl group H-20 to C-18 and C-19 and from H-18 to C-19 and C-20 (Table 1). The planar structure of 1 was then established as shown in Figure 2.

The relative configuration of 1 was determined by the analyses of vicinal coupling constants and the NOESY correlations. The geometry of the double bond at C-2/C-3 was determined as E, by the large vicinal coupling constant between H-2 and H-3 (J_2,3 = 15.5 Hz), and the ¹H-¹H NOESY correlation of H-2/H-21. Then, the NOESY correlations of H-21/H-22, H-3/H-5, and H-5/H-7 indicated both double bonds C-4/C-5 and C-6/C-7 were E. The geometry of the olefin C-17/C18 was established as E by the key NOESY correlation of H-18/H-25. The NOESY correlations of H-8/H-23 and H-7/H-14 indicated a cis-fused ring junction. A small coupling constant of less than 1 Hz between H-9 and H-14 indicated that the angle between them was near 90°, providing stronger confirmation for the cis-fused decalin system. The NOESY correlation of H-10/H-12 suggested they were both axial and co-facial.

The most unusually distinctive feature of the NMR spectral dataset of 1 is the downfield shift of H-8 at δ_H 5.15, which is directly connected to a carbon at δ_C 37.5. However, this phenomenon has also been reported in polyketides with structures closely related to 1, isolated from a yew-associated Penicillium species by Stierle et al. [11]. This may be due to the anisotropy effect generated by the C-19 ketone group, which deshields H-8. The authors attempted to reduce the C-19 carbonyl group using NaBH₄, yielding two products in which the chemical shifts of H-8 decreased to approximately δ_H 3.82–3.86, confirming the effect of the carbonyl group. However, preussilide C, another polyketide from the endophytic fungus Preussia similis, has an almost identical structure to 1, including a carbonyl group at C-19 but differing in the arrangement of its double bonds [12]. Despite this similarity, it exhibits an H-8 shift of only δ_H 3.26. This suggests that the C-15 to C-19 double bond system also plays a role in causing H-8 to shift toward the downfield region. Hence, the distinctive downfield signal of H-8 is a very coincidental effect of many factors, in which H-8 is both the allylic proton of the two conjugated double bond systems from C-1 to C-7 and C-15 to C-19, and is also influenced by the anisotropic effect from the carbonyl group at C-19. To provide further solid evidence for this phenomenon, the ¹H and ¹³C chemical shifts of 1 were also calculated for comparison.

The experimental CD spectrum of 1 exhibited a negative Cotton effect (CE) at λ_max = 278 nm and one positive CE at λ_max = 310 nm (Figure 3). The Boltzmann-averaged TD-DFT calculated ECD spectrum for the most stable conformers of the enantiomer 1a (8R, 9R, 10R, 12S, and 14S), performed at the CAM-B3LYP/6–31G(d,p) level of theory (IEFPCM, acetonitrile), also showed one negative CE at λ_max = 275 nm and one positive CE at λ_max = 305 nm, which reproduced the signs and differences in amplitude of the experimental CEs. Thus, the absolute configuration of 1 was determined as 8R, 9R, 10R, 12S, and 14S (Figure 3, Tables S1–S25).

Seven already reported compounds were identified as chamisides A (2), B (3), and E (4) [8,9], chaetoconvosins B (5) [13] and C (6) [9], and chaetochromins A (7) and B (8) [14] by comparing their HRESIMS, NMR data, and specific rotations with those reported in the literature. The relative stereochemistry of chaetochromin B (8) remained unclarified.

All isolated compounds were evaluated for their antimicrobial activities against a panel of microorganisms, including Staphylococcus aureus S25, Escherichia coli UTI89, Candida krusei CK01, Candida albicans CA01, and Leishmania infantum (LI01). Only chaetochromins A (7) and B (8) exhibited strong activity against S. aureus S25, with MIC values of 6.25 µM (inhibiting bacterial growth) and MBC values of 12.50 µM (killing 99.9% of bacteria). The strong antibacterial activity of chaetochromin A against S. aureus is also consistent with previous reports [15]. More interestingly, despite showing strong antibacterial activity, these compounds showed no cytotoxicity against human erythrocytes (RBC), and moderate activities against human monocytes (THP-1) and peripheral blood mononuclear cells (PBMC) (IC₅₀ values of 33.65–40.01 µM, Table S1). These results encourage further investigation into the potential clinical applications of compounds 7 and 8.

3. Materials and Methods

3.1. General Experimental Procedure

Optical rotations were determined using an Anton Paar MCP 150 polarimeter (Anton Paar, Graz, Austria). Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet iS50 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA). Semi-preparative chromatography and high-performance liquid chromatography (HPLC) analyses were performed using a Waters Alliance e2695 HPLC system (Waters Corporation, Milford, MA, USA), equipped with a bifunctional Macherey-Nagel NUCLEODUR Sphynx RP column (250 × 4.6 mm or 250 × 10.0 mm, 5 µm) connected to both a Waters 2424 evaporative light scattering (ELS) detector and a Waters 2998 photodiode array (PDA) detector. HPLC-grade solvents were purchased from Sigma-Aldrich (Merck KGaA, Saint-Louis, MO, USA). NMR spectra were recorded on a 400 MHz Bruker Avance NMR spectrometer (Bruker Corporation, Billerica, MA, USA). Circular dichroism (CD) spectra were measured using a JASCO J-810 spectropolarimeter (JASCO International Co., Ltd., Tokyo, Japan). High-resolution mass spectra (HRMS) were acquired using a Thermo Q-Exactive Focus Orbitrap UPLC-HRMS system (Thermo Fisher Scientific, Waltham, MA, USA), and the data were processed with Thermo Xcalibur software version 2.2.44.

3.2. Microorganism Isolation and Identification

Amesia nigricolor MUT6601 was isolated from the sediments of the Harbor of Livorno, in the Tyrrhenian Sea in Tuscany (IT; 43°33′43.92″ N 10°17′42″ E). The harbor of Livorno is an area affected by several petrochemical and industrial activities, primarily due to contamination from metals and hydrocarbons [16].

As previously described [16], the dilution plate method was used to isolate fungi: the sediment suspensions were plated on Corn Meal Agar (CMA: 2 gL⁻¹ corn meal infusion, 15 g L⁻¹ agar) with antibiotics (Gentamicin 80 mgL⁻¹ and Tazobactam 100 mgL⁻¹) and 2% w/v of sea salts. All of the chemicals were purchased from Sigma-Aldrich. The identification at species level was carried out through morphological and molecular analyses. The internal transcribed spacer region (ITS) gene sequence obtained for A. nigricolor MUT6601 is available at GenBank NCBI under the accession number OP161804.

The strain is deposited at Mycotheca Universitatis Taurinensis (MUT, www.mut.unito.it) of the Department of Life Sciences and Systems Biology, University of Torino, Torino (Italy).

3.3. Fungal Solid State Fermentation

The fermentation was carried out following an adaptation of the method previously described by Yue et al. [17]. Briefly, A. nigricolor MUT6601 was fermented in solid rice media (RM), in 100 mL bottles containing 20 g of rice and 20 mL of mineral medium (MM: 10 mL/L mineral solution—MS and 1 mL/L trace metal solution—TMS. MS (100 mL): KCl 5 g, MgSO₄•7H₂O 5 g, and FeSO₄•7H₂O 0.1 g. TMS (100 mL): ZnSO₄•7H₂O 1 g and CuSO₄ 0.5 g.

Prior to the fermentation A. nigricolor, MUT6601 was cultured on CMA plates for 7–14 days at 24 °C. A mycelium homogenate was prepared as inoculum: the fungal biomass was collected from CMA plates with a sterilized scalpel and blended using distilled sterilized water (1 cm²/1 mL) using an Ultra-turrax^® homogenizer (IKA) (IKA, Staufen, Germany) [18].

The solid-state fermentation was carried out in 80 bottles of RM in which 3 mL of mycelium homogenate was inoculated. The fermentation lasted 2 weeks.

3.4. Extraction and Purification

The colonized rice was extracted with ethyl acetate, and the resulting solution was concentrated in vacuo to yield 8.96 g of crude organic extract. The total extract was then fractionated by C-18 solid-phase extraction, eluting with water/methanol (100:0, 80:20, 60:40, 40:60, 20:80, 10:90, and 0:100, v/v), and then methanol/dichloromethane (50:50, v/v), to give 8 fractions (AN1 → AN8). Fraction AN4 (593.4 mg) was fractionated by silica gel column chromatography, eluting with cyclohexane/ethyl acetate (3:1 → 0:1, v/v) to yield seven sub-fractions (AN4.1 → AN4.7). Sub-fraction AN4.2 (181.2 mg) was purified by analytical HPLC using acetonitrile/water (55:45, 1 mL/min) containing 0.1% formic acid, to yield 3 (4.0 mg, t_R = 8.9 min), 1 (6.4 mg, t_R = 13.1 min), 5 (39.4 mg, t_R = 14.7 min), and 2 (25.5 mg, t_R = 16.2 min). Sub-fraction AN4.3 (43.9 mg) was purified by semi-preparative HPLC using the same mobile phase with a flow rate of 3 mL/min, to yield 6 (2.6 mg, t_R = 12.5 min). Fraction AN5 (707.9 mg) was separated by silica gel column chromatography and eluting with cyclohexane/ethyl acetate (9:1 → 0:1, v/v) to yield 4 (38.2 mg) and eleven other sub-fractions (AN5.1, AN5.3 → AN5.12). Sub-fraction AN5.9 (27.5 mg) was purified by semi-preparative HPLC using acetonitrile/water (65:35, 3 mL/min) containing 0.1% formic acid, to yield 7 (2.0 mg, t_R = 14.5 min) and 8 (3.6 mg, t_R = 12.0 min).

Amesilide (1): yellow powder. [α]_D²⁰ -12.0 (c 0.6, CHCl₃). Molecular formula: C₂₅H₃₂O₄. HRESIMS m/z 397.2369 [M+H]⁺ (calcd for C₂₅H₃₃O₄⁺ 397.2373). IR υ_max: 2928.3, 1706.5, 1618.2, 1582.3, 1452.9, 1376.7, 1183.4, and 1024.1. CD (acetonitrile) λ (Δε): 214 (+6.11), 277 (−18.76), and 310 (+12.21). ¹H and ¹³C NMR data see Table 1.

3.5. Computational Details

The conformational searches of each possible isomer were performed by employing MAESTRO software (Schrodinger LLC, New York, NY, USA), using an energy window of 5 kcal/mol, applying 10,000 steps of the Monte Carlo multiple minimum method with PRCG energy minimization using the Merck Molecular Force Field (MMFF) in gas phase, yielding 42 conformers of the isomer 1a (8S, 9S, 10S, 12R, and 14R) and 43 conformers of the enantiomer 1b (8R, 9R,10R, 12S, and 14S). Those occurring conformers were then subjected to geometrical optimization and vibrational frequencies calculation using the DFT/B3LYP/6-31G(d,p) (IEFPCM, acetonitrile) level with the Gaussian 16 A.03 package (Gaussian Inc., Wallingford, CT, USA) [19]. The low-energy conformers over 0.5% Boltzmann population were chosen for ECD calculation at TD-DFT/CAM-B3LYP/6-31G(d,p) (IEFPCM, acetonitrile) level. ECD curves were Boltzmann averaged and extracted by SpecDis v.1.7 software with a half-band of 0.3 eV.

For NMR calculation, all 43 conformers of 1b (8R, 9R,10R, 12S, and 14S) were further geometrically optimized using the DFT/B3LYP/6-31G(d,p) (IEFPCM, chloroform) level. They were then calculated for NMR shielding tensors at the IEFPCM/mpw1pw91/6-311G(d,p) level with chloroform as a solvent. The calculated NMR shielding tensors were averaged based on the Boltzmann populations, and the chemical shift values were calculated by the equation below (Equation (1)) where δ^x is the calculated NMR shift for nucleus x, and σ⁰ is the shielding tensor for the proton or carbon nuclei in tetramethylsilane calculated at the same condition.

δ^x = (σ⁰ − σ^x)/(1 − σ⁰/10⁶)

(1)

3.6. Antimicrobial Assay

Stock solutions of all compounds were prepared in DMSO. Two bacterial (E. coli UTI89 and S. aureus S25) [20,21] and two yeast strains (C. krusei CK1 and C. albicans CA01) have been used for this study. For antibacterial assays, the two strains E. coli UTI89 and S. aureus S25 were inoculated in LB liquid medium and incubated at 37 °C with agitation until the culture had an optical density (OD_600nm) of 1.2. Each culture was diluted 12 times to have a final OD value of 0.1. Then, 100 µL from the diluted (OD of 0.1) bacterial culture and 0.5 μL of each stock solution was added to the corresponding wells (in triplicate) in a 96-well plate to a final concentration of 50 μM. For the antifungal assay, the two strains C. krusei CK1 and C. albicans CA01 were spread on Petri plates with YPD (yeast-extract peptone dextrose) agar. Later, an isolated colony was collected in a tube containing 5mL of RGM (RPMI 1640^® (21875, Invitrogen GIBCO^®) + 34.56g/L MOPS) to have an OD of 0.05. The fungal culture and stock solutions of compounds were diluted and added to the wells as indicated for the antibacterial activity assay. To determine the potential inhibitory effect of extracts on fungal growth, absorbance at t = 0 h, t = 20 h (for antibacterial assay), and t = 48 h (for antifungal assay) were measured with the Biotek Synergy 2 Plate Reader at a wavelength of 600 nm and analyzed with the software Gen5 v3.08. Then, the differential OD was calculated (ODT48/20-ODT0) and compared to solvent control. A two-fold dilution concentration series of each compound that showed no growth (at 50 μM) was prepared to repeat the experiment to determine MIC/MBC. Ampicillin was used as positive control (MIC of 0.5 µg/mL for all bacteria and 1.0 µg/mL for all fungi).

3.7. Hemolytic and Cytotoxicity Assay

Each compound which showed antimicrobial activity at a concentration of 50 μM was selected for the cytotoxicity assay. Human erythrocytes (RBC), human monocytes (THP-1), and peripheral blood mononuclear cells (PBMC) were used for this study. THP-1 cells (TIB-202) were obtained from ATCC (https://www.atcc.org (accessed on 26 January 2025)) and activated with PMA (phorbol myristate acetate) (100 ng/mL) for 48 h. Cells were grown in RPMI 1640 medium supplemented with fetal bovine serum (FBS) 10% (10270-098, GIBCO^®) and penicillin/streptomycin (1%) (15140-122 GIBCO^®) at 37 °C under 5% CO₂ atmosphere.

For the hemolytic assay, freshly obtained human erythrocytes (RBCs) from patients were washed twice with PBS (pH 7.4). This assay was performed at 10% hematocrit with two-fold serial dilutions from 200 μM of test compounds in a final volume of 100 μL, followed by incubation at 37 °C under 5% CO₂ atmosphere for 30 min. Later, the plates were centrifugated at 3000g for 5 min and supernatant was transferred onto a new 96-well plate. Finally, RBC lysis was determined by the absorbance of the supernatant at 540 nm. Results were calculated with the following formula (Equation (2)):

$$\% \mathrm{hemolysis} = 100 \times {\displaystyle \frac{\mathrm{ODt} - \mathrm{OD}100}{\mathrm{OD}0 - \mathrm{OD}100 }}$$

(2)

where ODt = the absorbance value of test compounds; OD100 = the absorbance value of the medium; and OD0 = the absorbance value of the wells treated with 1% Triton-X.

For the cytotoxicity assay, THP-1 and PBMC medium cultures were added into 96-well plates with 5 × 10³ cells per well. A two-fold dilution concentration series of each compound was added to the plates to final concentrations of 200 − 6.25 μM. AlamarBlue (DAL1025, ThermoFisher, Waltham, MA, USA), a ready-to-use resazurin-based solution, was used to measure cell viability after treatment with the extracts. A total of 10 µL was added to each well and the plate was incubated for 6–8 h at 37 °C. Fluorescence was then measured with the Biotek Synergy 2 Plate Reader using excitation at 530 nm and emission at 590 nm. The percentage of cell death was then calculated using the following formula (Equation (3)):

$$\% \mathrm{Cellular} \mathrm{Death} = 100 \times {\displaystyle \frac{\mathrm{ODt} - \mathrm{OD}100}{\mathrm{OD}0 - \mathrm{OD}100}}$$

(3)

where ODt = the fluorescence value of the test compound, OD100 = the fluorescence value for the culture medium, and OD0 = the fluorescence value for Triton 0.12%.

4. Conclusions

The marine fungus Amesia nigricolor MUT6601 has proven to be a promising source of bioactive compounds. In our study, a new bicyclic polyketide, amesilide (1), along with the previously reported metabolites, chamisides A (2), B (3), and E (4), chaetoconvosins B (5) and C (6), and chaetochromins A (7) and B (8), were isolated from the marine fungus Amesia nigricolor MUT6601. Notably, chaetochromins A (7) and B (8) exhibited significant antibacterial activity against Staphylococcus aureus, with minimal cytotoxicity, suggesting their potential for further development as therapeutic agents. These results highlight the potential of marine fungi as prolific sources of novel bioactive compounds, and lay the groundwork for future pharmacological development and optimization.