New antifungal chlorinated orsellinic aldehydes from the deep-sea-derived fungus Acremonium sclerotigenum LW14

ABSTRACT

Two new pairs of chlorinated orsellinic aldehyde enantiomers acresorcinols A and B (1a/1b and 2a/2b), and three orsellinic aldehydes, acresorcinols C−E (3−5) were discovered from the extract of the deep-sea-derived fungus Acremonium sclerotigenum LW14. Their structures, including absolute configurations, were experimentally elucidated by spectroscopic analysis, NMR calculations with DP4+ probability analysis, and ECD calculations. Compounds 1 and 2 exemplify the first reported chlorinated orsellinic aldehydes, characterised by a distinctive 6/5−5 tricyclic core structure with a bridged framework. Acresorcinol C (3) was a rare carbon-bridged resorcinol dimer via a methylene bridge. The bioassay results showed that all compounds exhibited antifungal activities against Cryptococcus gattii 3271G1 at 32 μg/mL. Compounds 1−3 showed antifungal effects against C. gattii 3271G1, displaying MIC values of 8, 16, and 16 µg/mL, respectively.

GRAPHICAL ABSTRACT

1. Introduction

Cryptococcosis, a significant cause of invasive fungal infections, carries a substantial 20% fatality rate, especially among HIV-positive patients (Chayakulkeeree and Perfect 2006; Jarvis et al. 2010). In Sub-Saharan Africa, it is the predominant cause of meningitis among HIV-infected adults (Chayakulkeeree and Perfect 2006; Jarvis et al. 2010; Rajasingham et al. 2017). Cryptococcosis is mainly attributed to C. gattii and C. neoformans (Paccoud et al. 2021; Ke et al. 2022). Due to insufficient safe and effective treatments, as well as limited epidemiological and clinical data, the World Health Organization (WHO) has classified C. gattii as a medium-priority pathogen in its Fungal Priority Pathogen List (Iyer et al. 2021; Beardsley et al. 2022). Current antifungal treatments for C. gattii are limited, primarily relying on drugs such as fluconazole, amphotericin B, and flucytosine, which can be ineffective due to host toxicity and pathogen resistance (Bermas and Geddes-McAlister 2020; Yang et al. 2023). There is an emerging need for new antifungal drugs and treatment strategies to address these challenges.

Throughout the history of drug discovery and development, natural products have played a pivotal role due to diverse chemical frameworks and broad biological activities (Newman and Cragg 2020; Luo et al. 2024; Shi et al. 2024a). In recent years, marine-derived fungi have been recognised as a promising source for the exploration of novel compounds with diverse chemical structures, which possess significant therapeutic potential, making them promising candidates for drug development (Carroll et al. 2024; Li et al. 2024). Among many types of marine-derived fungi, the fungal genus Acremonium has emerged as a tremendous source of new compounds, including alkaloids, terpenoids, isocoumarins, and polyketides, which exhibit diverse biological properties, including antimicrobial effects, cytotoxicity, and anti-inflammatory potential (Tian et al. 2017; Qin et al. 2024). Exploring the metabolic potential of Acremonium species offers promising pathway for the discovery of novel pharmaceutical agents.

In our continuous exploration of bioactive secondary metabolite from marine-derived fungi (Ren et al. 2021; Huo et al. 2022; Lu et al. 2024; Shi et al. 2024b), the strain A. sclerotigenum LW14, obtained from a sediment sample in the Southwest Indian Ridge, was selected for chemical investigation. Fractionation of the ethyl acetate (EtOAc) extract resulted in the identification of 13 new antifungal meroterpenoids, acremorins A−M (Huo et al. 2025). Additionally, purification of the remaining fractions yielded seven new compounds, acresorcinols A−E (1−5) (Figure 1), which were further assessed for their antifungal effects against several species of C. gattii. This study details the isolation, structural characterisation, putative biosynthetic pathway, and biological activities of acresorcinols A−E.

Figure 1.

Chemical structures of compounds 1−5.

2. Materials and methods

2.1. General experimental procedures

The general experimental procedures in this work followed established methodologies (Ren et al. 2021; Huo et al. 2022).

2.2. Fungal material

The strain A. sclerotigenum LW14, obtained from a sediment sample at the depth of 2,398 m of the Southwest Indian Ridge, was identified according to its phylogenetic analysis using ITS (GenBank Accession No. PP033595), LSU (GenBank Accession No. PQ881846), SSU (GenBank Accession No. PQ881845), and TEF 1-α (GenBank Accession No. PO998410) sequences, combined with morphological characterisation.

2.3. Fermentation, extraction, and isolation

Fermentation and extraction were performed following the previously reported methods (Huo et al. 2025). Fraction Fr.4, eluted with 55% EtOAc, was further separated via ODS column chromatography (CC) (20%–100% MeOH/H₂O) into six subfractions (Fr.4.1–Fr.4.6). Fr.4.2, eluted with 40% MeOH/H₂O, was subjected to RP-HPLC (46% MeOH/H₂O for 50 min; 2.0 mL/min), resulting in a mixture of 1 and 2 (3.8 mg; t_R 21.0 min), and compounds 3 (5.7 mg; t_R 22.5 min) and 4 (5.6 mg; t_R 24.0 min). Further purification of Fr.4.3 (eluted with 50% MeOH/H₂O) yielded compound 5 (1.8 mg; t_R 38.0 min) using RP-HPLC (71% MeOH/H₂O for 40 min; 2.0 mL/min). The mixture of 1 and 2 was then subjected to chiral HPLC column (Kromasil 5-CelluCoat column, 250 mm × 4.6 mm, 5 μm) with MeCN/H₂O (46:54, v/v) at a flow rate of 0.4 mL/min, yielding (+)-1a (0.9 mg, t_R 30.0 min), (–)-1b (0.8 mg, t_R 32.0 min), (+)-2a (0.7 mg, t_R 39.0 min), and (–)-2b (0.6 mg, t_R 41.0 min).

(±)-Acresorcinol A (1): amorphous powder; UV (MeOH) λ_max (log ε) 203 (4.02), 231 (3.23), 296 (3.11) nm; IR (neat) ν_max 3,419, 2,977, 2,932, 1,715, 1,633, 1,621, 1,447, 1,381, 1,099 cm⁻¹; HRESIMS at m/z 311.0686 [M + H]⁺ (calcd. for C₁₅H₁₆ClO₅ m/z 311.0681).

(+)-1a: ${\left[\alpha \right]}_{\text{D}}^{25}$ + 30.0° (c 0.10, MeOH); ECD (2.3 × 10⁻⁴ M, MeOH) λ_max (∆ε) 228 (+1.10), 250 (−0.56), 302 (+2.51) nm.

(–)-1b: ${\left[\alpha \right]}_{\text{D}}^{25}$ −30.0° (c 0.10, MeOH); ECD (2.0 × 10⁻⁴ M, MeOH) λ_max (∆ε) 226 (−1.12), 250 (+0.36), 300 (−2.01) nm.

(±)-Acresorcinol B (2): amorphous powder; UV (MeOH) λ_max (log ε) 202 (3.99), 231 (3.10), 295 (3.05) nm; IR (neat) ν_max 3,354, 2,972, 2,936, 1,709, 1,626, 1,453, 1,373, 1,252 cm⁻¹; HRESIMS at m/z 311.0684 [M + H]⁺ (calcd. for C₁₅H₁₆ClO₅ m/z 311.0681).

(+)-2a: ${\left[\alpha \right]}_{\text{D}}^{25}$ +39.9° (c 0.10, MeOH); ECD (2.3 × 10⁻⁴ M, MeOH) λ_max (∆ε) 220 (+3.10), 246 (−1.63), 298 (+4.32) nm.

(–)-2b: ${\left[\alpha \right]}_{\text{D}}^{25}$ −38.9° (c 0.10, MeOH); ECD (2.0 × 10⁻⁴ M, MeOH) λ_max (∆ε) 218 (−4.01), 250 (+0.86), 299 (−2.91) nm.

Acresorcinol C (3): yellow oil; UV (MeOH) λ_max (log ε) 233 (3.98), 295 (3.78), 343 (3.12) nm; IR (neat) ν_max 3,326, 2,972, 2,935, 1,707, 1,626, 1,452, 1,252, 1,106 cm⁻¹; HRESIMS at m/z 323.0684 [M + H]⁺ (calcd. for C₁₆H₁₆ClO₅ m/z 323.0681).

Acresorcinol D (4): pale yellow oil; UV (MeOH) λ_max (log ε) 201 (3.89), 231 (3.53), 296 (3.24) nm; IR (neat) ν_max 3,232, 2,972, 2,936, 1,704, 1,622, 1,454, 1,292, 1,055 cm⁻¹; HRESIMS at m/z 285.0524 [M + H]⁺ (calcd. for C₁₃H₁₄ClO₅ m/z 285.0524).

Acresorcinol E (5): pale yellow oil; ${\left[\alpha \right]}_{\text{D}}^{25}$ −8.3° (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 200 (3.92), 298 (3.46) nm; ECD (1.0 × 10⁻⁴ M, MeOH) λ_max (∆ε) 203 (−3.51), 230 (−1.11) nm; IR (neat) ν_max 3,233, 2,972, 2,937, 1,705, 1,623, 1,454, 1,251, 1,054 cm⁻¹; HRESIMS at m/z 253.1075 [M + H]⁺ (calcd. for C₁₃H₁₇O₅ m/z 253.1071).

2.4. NMR and ECD calculations

The NMR and ECD calculations in this work followed those previously reported (Ren et al. 2021; Huo et al. 2022) with procedural details provided in the Supplementary Material.

2.5. Antifungal assay

The antifungal activity against C. gattii was assessed following established protocols (Duan et al. 2022). Amphotericin B and fluconazole were utilised as positive controls in this study. Specifically, each antimicrobial agent underwent two-fold serial dilution, with 2.5 μL aliquots pipetted into designated wells. Subsequently, fungal suspension (50 μL) was added to achieve the desired inoculum density. Following a 24 h incubation at 37 °C, the MIC values were determined across multiple concentrations, with all measurements conducted in triplicate. The experiment utilised DMSO as the negative control.

3. Results and discussion

3.1. Identification of the strain

The fungus was identified by its phylogenetic analysis based on morphological observation and LSU, SSU, ITS, and TEF 1-α sequences (Figures 2 and 3). To determine the evolutionary position of the fungus LW14, a phylogenetic analysis was performed based on the LSU, SSU, ITS, and TEF 1-α sequence with those from other Acremonium species. The results showed that the strain LW14 was clustered with species in the genus Acremonium (A. sclerotigenum) with high confidence.

Figure 2.

Maximum likelihood tree of Acremonium constructed using ITS, LSU, SSU, and TEF 1-α sequences. Bootstrap support values of maximum likelihood (MLBP) above 50% are shown at the nodes. The tree was rooted to Phialemonium atrogriseum CBS 507.82 and Phialemonium atrogriseum CBS 604.67. The strain LW14 is printed in ped and bold font.

Figure 3.

The phenotypical and morphological characters of Acremonium sclerotigenum LW14. (a) The colony. (b and c) Conidiophores and conidia. Scale bars: 2 µm.

3.2. Structural characterization

Acresorcinols A and B (1 and 2) were initially isolated as a mixture. Subsequent separation using repeated RP-HPLC on a chiral column afforded two sets of enantiomeric pairs (1a/1b, 2a/2b) (Figure S1). Acresorcinol A (1), obtained as an amorphous powder, was determined to have a molecular formula of C₁₅H₁₅ClO₅ (eight degrees of unsaturation) based on HRESIMS data, showing an [M + H]⁺ ion cluster at m/z 311.0686/313.0655 with a 3:1 intensity ratio. Spectroscopic characterisation of compound 1 (Table 1 and Figures S2−S5) revealed diagnostic resonances including a hydroxyl group (δ_H 12.51), an aldehyde group (δ_H/C 10.14/193.6), three methylenes (δ_H/C 3.03, 3.37/28.5; 2.01, 2.25/28.8; 2.67/29.2), one oxygenised sp³ methine (δ_H/C 5.08/89.1), and two singlet methyls (δ_H/C 1.48/22.6; 2.60/14.2), along with eight tertiary carbons, including one carbonyl carbon from an ester group (δ_C 176.1), six aromatic carbons (δ_C 108.5, 111.1, 115.0, 142.2; two oxygenised ones, δ_C 159.5, 163.1), and one oxygenised sp³ carbon (δ_C 86.2). Combined with the eight indices of hydrogen deficiency (IHD), 1 was further established as a tricyclic. The HMBC cross-peaks (Figures 4 and S6) from 2-OH (δ_H 12.51) to C-1 (δ_C 115.0), C-2 (δ_C 159.5), and C-3 (δ_C 111.1), from the aldehyde proton H-8 (δ_H 10.14) to C-1, and C-2, from H₃-7 (δ_H 2.60) to C-1, C-5 (δ_C 108.5), and C-6 (δ_C 142.2), and from H₂-9 (δ_H 3.03, 3.37) to C-2, C-3, and C-4 (δ_C 163.1), as well as from H-10 (δ_H 5.08) to C-3 and C-4, combined with ¹H–¹H COSY cross-peak of H₂-9/H-10 permitted the completion of benzofuran subunit (rings A and B). The aldehyde carbon C-8, methyl carbon C-7, and hydroxyl group were attached to C-1, C-6, and C-2 of benzofuran ring, respectively. Other HMBC correlations from H₂-12 (δ_H 2.01, 2.25) and H₂-13 (δ_H 2.67) to C-14 (δ_C 176.1), and from H₃-15 (δ_H 1.48) to C-11 (δ_C 86.2) and C-12 (δ_C 28.8), along with ¹H–¹H COSY cross-peak of H₂-12/H₂-13 supported the presence of a γ-lactone system (ring C). The connection between C-11 of the γ-lactone moiety and C-10 of the benzofuran core was confirmed by HMBC cross-peaks from H₂-9 and H-10 to C-11, and from the methyl proton H₃-15 to C-10, C-11, and C-12. The chlorine atom was assigned to C-5 based on its characteristic chemical shift (δ_C 108.5). Collectively, 1 was established as a chlorinated orsellinic aldehyde derivative featuring a unique 6/5−5 tricyclic ring system.

Table 1.

¹H (500 MHz) and ¹³C NMR (125 MHz) data of 1 and 2 (in CDCl₃).

Pos.	1		2
	δH (J in Hz)	δC, mult.	δH (J in Hz)	δC, mult.
1		115.0, C		115.0, C
2		159.5, C		159.5, C
3		111.1, C		111.6, C
4		163.1, C		163.1, C
5		108.5, C		108.3, C
6		142.2, C		141.8, C
7	2.60, s	14.2, CH3	2.57, s	14.2, CH3
8	10.14, s	193.6, CH	10.12, s	193.6, CH
9a	3.37, dd (16.0, 10.1)	28.5, CH2	3.31, dd (8.9, 4.0)	28.1, CH2
9b	3.03, dd (16.0, 7.7)
10	5.08, dd (10.0, 7.7)	89.1, CH	4.94, dd (9.6, 8.2)	90.3, CH
11		86.2, C		85.5, C
12a	2.25, m	28.8, CH2	2.12, m	31.6, CH2
12b	2.01, m		2.65, m
13a	2.67, m	29.2, CH2	2.61, m	29.4, CH2
13b			2.93, m
14		176.1, C		176.6, C
15	1.48, s	22.6, CH3	1.49, s	23.5, CH3
2-OH	12.51, s		12.46, s

Figure 4.

Key HMBC and ¹H−¹H COSY correlations of compounds 1−5.

Acresorcinol B (2), isolated as an amorphous powder, shared the same molecular formula C₁₅H₁₅ClO₅ (eight degrees of unsaturation) as 1, confirmed by the HRESIMS ([M + H]⁺ m/z 311.0684). The NMR data of 2 (Table 1 and Figures S8−S11) closely resembled those of 1, with only slight discrepancies in chemical shifts around C-10 and C-11, indicating potential stereochemical distinctions. Further analysis of its 1D and 2D NMR data (Figures 4 and S10−S12) established that compound 2 possessed an identical planar framework as 1.

Due to the conformational flexibility of the C-10–C-11 bond, NOESY data alone were insufficient to assign the relative configurations of these positions (C-10 and C-11). To resolve this, NMR calculations coupled with DP4+ probability were conducted (Figures 5 and S13). Two calculated conformers, (10S*,11R*)-a and (10S*,11S*)-b, were compared with the experimental NMR data (Table S1). The results revealed that both linear correlation coefficient (R²) and DP4+ probability analysis of (10S*,11R*)-a were higher than thsoe of (10S*,11S*)-b (Figure 5a), suggesting that 1 adopts the 10S*,11R* configuration. Furthermore, the calculated ¹³C NMR data of the isomer (10S*,11S*)-b agreed well with the experimental ¹³C NMR data of 2, supported by a high R² value. DP4+ probability analyses assigned 10S*,11S*-b as the isomer with a 100% probability (Figures 5b and S14). To further confirm these assignments, CP3 analysis was also conducted (Kanehara et al. 2024), which validated the (10S*,11R*)-a for 1 and (10S*,11S*)-b for 2 with probabilities 100% (Figure 5c).

Figure 5.

The ¹³C NMR calculation results of two plausible isomers of 1 and 2. (a) Linear correlation plots of calculated versus experimental ¹³C NMR chemical data of 1 and DP4+ probability. (b) Linear correlation plots of calculated versus experimental ¹³C NMR chemical data of 2, accompanied by DP4+ probability. (c) CP3 probability analysis.

To determine their absolute configurations, experimental ECD spectra of the pure enantiomers were compared with TD-DFT-calculated spectra at the B3LYP/6-311 G (d,p) level. The calculated ECD spectra of the four isomers (10S,11R)-a, (10R,11S)-b, (10S,11S)-c, and (10R,11R)-d closely matched the experimental ECD spectra of 1a/1b and 2a/2b (Figure 6 and Table S2). This allowed the absolute configurations to be assigned as (10S,11R)-a for (+)-acresorcinol A (1a), (10R,11S)-b for (–)-acresorcinol A (1b), (10S,11S)-c for (+)-acresorcinol B (2a), and (10R,11R)-d for (–)-acresorcinol B (2b), respectively.

Figure 6.

Experimental and calculated ECD spectra for 1a/1b, 2a/2b, and 5.

The molecular formula of acresorcinol C (3), obtained as a yellow oil, was deduced as C₁₆H₁₅ClO₅ from negative HRESIMS peaks at m/z 323.0684 [M + H]⁺ (calcd. for C₁₆H₁₆ClO₅ m/z 323.0681) and the NMR data (Table 2, Figures S15 and S16), implying nine IHD. The ¹H NMR spectrum of 3 (Table 2) showed resonances for a hydroxyl proton (δ_H 13.42, br s), an aldehyde proton (δ_H 10.18, s), two meta-coupled aromatic protons (δ_H 6.36, d, J = 2.4 Hz; 6.26, d, J = 2.4 Hz), one methylene proton (δ_H 3.93, s), two methyl protons (δ_H 2.61, s; 2.27, s). The ¹³C NMR spectroscopic data combined with the HSQC spectroscopic data (Figure S17) indicated 16 carbon signals: 2 methyls, 1 methylene, 2 aromatic methines, 1 aldehyde, and 10 nonprotonated carbons. Comprehensive comparison of the 1D NMR spectroscopic data of 3 with those of compounds 1 and 2 suggested the presence of the chlorinated orsellinic aldehyde skeleton in compound 3. Key HMBC cross-peaks (Figures 2 and S18) from methyl proton H₃-16 (δ_H 2.27) to aromatic carbons C-10 (δ_C 115.8), C-11 (δ_C 140.9), and C-12 (δ_C 111.2), from aromatic proton H-12 (δ_H 6.26) to C-11 and C-13 (δ_C 157.4), and from aromatic proton H-14 (δ_H 6.36) to C-10, C-12, C-13, and C-15 (δ_C 155.2) confirmed a tetrasubstituted phenyl group with a methyl carbon C-16 and two hydroxyl groups attached at C-11, C-13, and C-15, respectively. Additionally, HMBC cross-peaks from H₂-9 to C-2 (δ_C 162.6), C-3 (δ_C 113.8), C-4 (δ_C 160.9), C-10, C-11, and C-15 linked the chlorinated orsellinic aldehyde moiety with the tetrasubstituted phenyl subunit via C-9, establishing the planar structure of compound 3.

Table 2.

¹H (500 MHz) and ¹³C NMR (125 MHz) data of 3−5 (in acetone-d₆).

Pos.	3		4		5
	δH (J in Hz)	δC, mult.	δH (J in Hz)	δC, mult.	δH (J in Hz)	δC, mult.
1		113.5, C		114.3, C		113.6, C
2		162.6, C		162.7, C		164.6, C
3		113.8, C		112.9, C		113.8, C
4		160.9, C		158.5, C		163.8, C
5		115.5, C		114.3, C	6.35, s	111.0, CH
6		139.9, C		140.2, C		143.1, C
7	2.61, s	14.5, CH3	2.65, s	14.7, CH3	2.50, s	18.0, CH3
8	10.18, s	195.0, CH	10.19, s	195.6, CH	10.09, s	194.5, CH
9	3.93, s	19.1, CH2	3.57, d (7.3)	23.2, CH2	2.68, t (7.8)	20.3, CH2
10a		115.8, C	6.80, t (7.3)	139.6, CH	1.91, m	33.3, CH2
10b					1.60, m
11		140.9, C		128.9, C	2.43, q (7.0)	39.6, CH
12	6.26, d (2.4)	111.2, CH		169.2, C		177.9, C
13		157.4, C	1.97, s	12.5, CH3	1.19, d (7.0)	17.4, CH3
14	6.36, d (2.4)	101.0, CH
15		155.2, C
16	2.27, s	20.6, CH3
2-OH	13.42, br s		12.83, br s		12.83, br s

The molecular formula of acresorcinol D (4), a pale yellow oil, was deduced through analysis of its HRESIMS and 1D NMR data, showing a value of m/z 285.0524 [M + H]⁺ and suggesting seven IHD. From the NMR data (Table 2 and Figures S19−S22), it was evident that compound 4 has the same chlorinated orsellinic aldehyde skeleton as compounds 1–3, except that notable differences exist in the terminal part of C-10 in 4. HMBC correlations (Figures 4 and S23) from methyl proton H₃-13 (δ_H 1.97) to C-10 (δ_C 139.6), C-11 (δ_C 128.9), and ester carbonyl carbon C-12 (δ_C 169.2), from H₂-9 (δ_H 3.57) to C-2 (δ_C 162.7), C-3 (δ_C 112.9), C-4 (δ_C 158.5), and C-11, and from olefinic hydrogen H-10 (δ_H 6.80) to C-12 and C-13 (δ_C 12.5), together with ¹H–¹H COSY cross-peak of H₂-9/H-10 permitted the completion of 2-methylbut-2-enoic acid subunit with C-9 located at C-3 position of the chlorinated orsellinic aldehyde core. The NOESY correlation (Figures 7 and S24) between H₃-13 and H₂-9 suggested the E geometry of Δ¹⁰ double bond.

Figure 7.

Key NOESY correlation of compound 4.

The molecular formula of acresorcinol E (5) was established as C₁₃H₁₆O₅ (six IHD) by HRESIMS (m/z 253.1075 [M + H]⁺), indicating a 32 mass unit reduction compared to 4. NMR spectroscopic (Figures S25−S28) analysis revealed structural similarities between 5 and 4, with key differences including the replacement of a trisubstituted olefin C-10/C-11 (δ_H/C 6.80/139.6; 128.9) in 4 by a methylene (δ_H/C 1.91, 1.60/33.3, C-10) and a methine (δ_H/C 2.43/39.6, C-11) in 5, along with the presence of an aromatic methine (δ_H/C 6.35/111.0, C-5). This observations were supported by HMBC correlations (Figure S29) from aromatic proton H-5 (δ_H 6.35) to C-1 (δ_C 113.6) and C-3 (δ_C 113.8), from methyl proton H₃-7 (δ_H 2.50) to C-1, C-5, and C-6 (δ_C 143.1), and from H₂-10 (δ_H 1.91, 1.60) to C-3 and C-12 (δ_C 177.9), as well as the ¹H–¹H COSY cross-peaks of H₂-9/H₂-10/H-11/H₃-13. The absolute configuration of 5 was determined by comparing experimental ECD data with TD-DFT-calculated ECD spectra for the isomers (11S)-5a and (11R)-5b, optimised at the B3LYP/6-31 G(d) level (Table S3). The agreement between the calculated ECD spectrum of (11R)-5b and the experimental data confirmed the absolute configuration of 5 as 11R (Figure 6).

This is the first report on the discovery and successful resolution of the 2,3-dihydrobenzofuran orsellinic aldehyde mixture consisting of all possible stereoisomers, that is, two pairs of enantiomers and their configurational assignment. The biosynthetic pathway of 1−5 was proposed (Figure 8). Biogenetically, the formation of the key precursor ilicicolinic acid B (a) was catalysed by prenyltransferase, utilising orsellinic acid and farnesyl pyrophosphate (FPP) as substrate (Araki et al. 2019; Quan et al. 2019). Then, the process of reduction and halogenation resulted in the formation of ilicicolin A (c). The decarboxylation and halogenation of a led to the formation of d. The oxidative cleavage of the double bond in b and c afford e and f, respectively. The β-oxidation and reduction of e would yield 5. Similarly, the β-oxidation of f provided j and 4. Notably, the epoxidation and cyclisation of j resulted in the formation of 1 and 2 bearing a rare benzofuran core, whose biosynthesis mechanism has not been reported before. Furthermore, the existence of a methylene bridge is highly unusual within natural compounds. It was hypothesised that 3 was formed by orsellinic acid via a nonenzymatic dimerisation process involving a highly reactive C1 electrophilic intermediate (Fan et al. 2022).

Figure 8.

Putative biosynthetic pathways for the formation of compounds 1−5.

3.3. Antifungal activity

The in vitro antifungal effects of 1−5 were assessed against three strains of C. gattii (3271G1, 3284G14, and R265) (Table 3). At a concentration of 32 µg/mL, all tested compounds exhibited antifungal effects against these strains. Notably, compound 1 demonstrated potent effect against C. gattii 3271G1 with an MIC value of 8 µg/mL, comparable to that of fluconazole (positive control, MIC 8 µg/mL). Compounds 2 and 3 showed moderate inhibitory effects against C. gattii 3271G1 both with MIC values of 16 µg/mL. Furthermore, compounds 1 and 2 also displayed inhibitory activities against C. gattii R265, both displaying MIC values of 16 µg/mL, while the MIC value for fluconazole was 8 µg/mL.

Table 3.

The inhibitory effects of 1−5 against Cryptococcus gattii strains, MIC (μg/mL).

Strains	Compounds
	1	2	3	4	5	Fluconazole	Amphotericin B
C. gattii 3271G1	8	16	16	32	32	8	1
C. gattii 3284G14	32	32	32	32	32	8	1
C. gattii R265	16	16	32	32	32	8	1

4. Conclusions

To summarise, we have identified two new pairs of enantiomers, acresorcinols A and B (1 and 2), and three orsellinic aldehydes, acresorcinols C−E (3−5) from the fungus A. sclerotigenum LW14. Compounds 1 and 2 were the first reported chlorinated orsellinic aldehydes featuring a unique bridged 6/5−5 tricyclic core skeleton. Acresorcinol C (3) was a rare carbon-bridged resorcinol dimer connected via a methylene bridge. Compounds 1−3 showed inhibitory effects against C. gattii. These findings not only broaden the chemical scope of the chlorinated orsellinic aldehyde family, but also offer promising avenues for the development of more potent antifungal agents.

Funding Statement

This work was supported by the National Key R&D Program of China [2024YFC2309600] and the National Natural Science Foundation of China [32472320, 32170067, and 32022002].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, Ling Liu; methodology, Ruiyun Huo; writing original draft, Ruiyun Huo; investigation, Ruiyun Huo, Yu Tu, Chang Liu, and Gangrong Zi; formal analysis, Ruiyun Huo, Yu Tu, Ying Shi, and Jinwei Ren; editing, Ruiyun Huo and Ling Liu; resources, Lei Cai; project administration, Ling Liu; supervision, Ling Liu; funding acquisition, Ling Liu.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21501203.2025.2485477