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• Natural products with novel skeletons not only possess unique chemical structures and remarkable pharmacological activities, but also frequently possess the potential to bind to novel drug target proteins in vivo, offering distinct mechanisms of action compared to existing clinical drugs [1,2]. Consequently, they represent a significant avenue for exploring new mechanism-based drug leads derived from natural sources [3-5]. Among these novel natural products, derivatives with structures derived from steroid skeletons have particularly promising potential [6,7]. Despite the ubiquity of steroid structures, the discovery of novel steroid derivatives with unique skeletons continues to yield compounds with significant pharmacological activities, including anti-microbial, anti-inflammatory, and anti-tumor activities [8,9]. For instance, two ergosteroids featuring a rearranged bicyclo[3.3.1]nonane motif, phomopsterones A and B, isolated from the fungus Phomopsis sp., exhibited anti-inflammatory activity [9]. Nevertheless, the exploration of structurally unique steroids remains in its infancy, and numerous steroids with novel carbon skeletons remain to be discovered. The ongoing exploration of uncommon steroids derived from natural sources will undoubtedly contribute to the development of new drugs.

  The fungal genus Chaetomium is widely dispersed across both marine and terrestrial ecosystems, demonstrating a propensity to biosynthesize a myriad of novel compounds, such as steroids, cytochalasins, and azaphilones. These secondary metabolites exhibited a broad spectrum of pharmacological activities, including potent antifungal, cytotoxic, and antiviral properties [10]. In the course of our continuous exploration for structurally unique natural products from marine-derived fungi [11-13], an unusual octacyclic sterol, chaeglobol A (1), was isolated from the marine-derived fungus Chaetomium globosum HBU-45. The structure of 1, inclusive of its absolute configuration, was meticulously elucidated through a combination of spectroscopic techniques, electronic circular dichroism (ECD) alongside optical rotatory dispersion (ORD) methods, and single-crystal X-ray diffraction analysis. Herein we report the isolation, structural determination, and anti-fungal activity assessment of 1, along with discussing its putative biosynthetic origin.

  Chaeglobol A (1) was obtained as colorless crystals, revealing its molecular formula of C₃₇H₄₈O₆, as confirmed by high resolution electrospray ionization mass spectroscopy (HRESIMS) analysis (m/z 611.3336 [M + Na]⁺, calcd. for C₃₇H₄₈NaO₆⁺ 611.3343), corresponding to 14 sites of unsaturation. The proton nuclear magnetic resonance (¹H NMR) data (Table 1) of 1 showed the presence of seven methyl groups at δ_H 0.84 (d, J = 6.6 Hz, H₃–27), 0.85 (d, J = 7.2 Hz, H₃–26), 0.92 (s, H₃–18), 0.93 (s, H₃–19), 0.94 (d, J = 7.2 Hz, H₃–28), 1.03 (d, J = 6.6 Hz, H₃–21), and 2.00 (s, H₃–9′), two oxymethines at δ_H 4.28 (d, J = 7.8 Hz, H-7′) and 4.40 (t, J = 7.8 Hz, H-3), three olefinic hydrogen at δ_H 5.22 (dd, J = 15.0, 7.2 Hz, H-22), 5.25 (dd, J = 15.0, 7.2 Hz, H-23), and 5.28 (s, H-4), and one hemiacetal oxymethine at δ_H 6.13 (s, H-8′). By combining its heteronuclear single quantum coherence (HSQC) data with the ¹³C NMR of 1, a total of 37 carbon resonances were observed, divided into two saturated ketones (δ_C 199.0 and 199.3), eight olefinic carbon atoms including three methine groups (δ_C 126.7, 132.8, and 135.2) and five quaternary carbons (δ_C 127.6, 132.3, 139.0, 148.6, and 158.1), seven methyl groups, five sp³ methylene groups, eleven sp³ methine groups including two oxygenated carbons(δ_C 68.0 and 84.4) and one hemiacetal carbon (δ_C 97.8), and four quaternary carbons including one oxygenated carbon (δ_C 88.7). The above NMR data accounted for six degrees of unsaturation, suggesting that 1 should be an octacyclic compound.

  Table 1

  

  Table 1.  ¹H (600 MHz) and ¹³C (150 MHz) NMR data for 1 in CDCl₃.

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  The planar structure of 1 was rigorously determined through a comprehensive interpretation of its ¹H–¹H correlation spectroscopy (COSY) and ¹H detected heteronuclear multiple bond correlation (HMBC) spectra (Fig. 1). The ¹H–¹H COSY correlations observed for H₂–1/H₂–2/H-3/H-4, H-6/H-7, H-9/H₂–11/H₂–12, and H-15/H₂–16/H-17, when combined with key HMBC correlations such as those from H₃–19 to C-1/C-5/C-9/C-10, from H₃–18 to C-12/C-13/C-14/C-17, from H-4 to C-2/C-6, from H-9 to C-7, and from H₂–11/H-15 to C-8, unequivocally confirmed the presence of A/B/C/D-rings system. Furthermore, the ¹H–¹H COSY correlations involving H₃–21/H-20/H-22/H-23/H-24/H-25/H₃–26, H-24/H₃–28, and H-25/H₃–27, indicated the presence of 22, 23-dihydro-24-methyl C9 side chain. This side chain was shown to be attached to C-17, based on the ¹H–¹H COSY correlation between H-17 and H-20, and the HMBC correlation from H₃–21 to C-17. The HMBC correlations from OH-5′ to C-4′/C-5′/C-6′, from H-7′ to C-6′, from H-8′ to C-1′/C-3′/C-7′, and from H-9′ to C-2′/C-3′/C-4′ were sufficient to establish the "E + F" 6/5-membered ring system. Finally, the integration of the E/F-ring system with the A/B/C/D-ring system was proposed to occur via bonds between C-5′ and C-6, C-1′ and C-7, and C-7′ and C-15, forming the novel "G + H" 5/6-membered rings. This proposal was supported by the ¹H–¹H COSY correlation between H-7′ and H-15, as well as the HMBC correlations from H-6 to C-4′ and from H-15 to C-1′/C-7′. Thus, the planar structure of 1 was unambiguously established, featuring a unique 6/6/6/5/6/5/6/5 octacyclic ring system. To validate the novel skeleton of 1, we employed the gauge-independent atomic orbital (GIAO) method [14] for ¹³C NMR chemical shift calculation (Fig. 2). The high correlation coefficient (R²) of 0.9986 and all deviation (|Δδ|) between experimental and calculated chemical shifts being less than 5.4 ppm, confirmed the proposed octacyclic structure of 1.

  Figure 1

  

  Figure 1.  Chemical structure, the key HMBC and COSY correlations of chaeglobol A (1).

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  Figure 2

  

  Figure 2.  (A) Regression analysis and (B) individual deviations of experimental versus calculated ¹³C NMR chemical shifts of 1.

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  The relative configuration of the 12 stereogenic centers C-3, C-6, C-7, C-9, C-10, C-13, C-15, C-17, C-1′, C-5′, C-7′, and C-8′ in 1 was assigned through a meticulous analysis of its nuclear overhauser effect spectroscopy (NOESY) spectrum (Fig. 3). The NOESY correlations of H-1α/H-3, H-1α/H-9, H-9/OH-5′, H-1β/H₃–19, H₃–19/H-6, H₃–19/H-7, H₃–19/H₃–18, H₃–18/H-15, H-17/H-7′, and H-7′/H-8′, provided compelling evidence for the proposed relative configuration of the A/B/C/D/E/F/G/H rings: 3S*, 6S*, 7R*, 9R*, 10R*, 13R*, 15S*, 17R*, 1′S*, 5′S*, 7′R*, and 8′R*. Furthermore, the relationship between H-22 and H-23 was ascertained to be E configuration based on their coupling constant (J = 15.0 Hz). Additionally, the configurations of C-20 and C-24 were tentatively determined as R and R since ergosterols of these configurations are commonly found in natural sources [15,16].

  Figure 3

  

  Figure 3.  NOESY correlations of chaeglobol A (1).

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  To determine the absolute configuration of 1, a comprehensive spectral analysis was conducted. The experimental electronic circular dichroism (ECD) and optical rotatory dispersion (ORD) spectra of 1 were compared with the corresponding computed ECD and ORD curves for the structures of (3S, 6S, 7R, 9R, 10R, 13R, 15S, 17R, 20R, 24R, 1′S, 5′S, 7′R, 8′R)−1 and (3R, 6R, 7S, 9S, 10S, 13S, 15R, 17S, 20S, 24S, 1′R, 5′R, 7′S, 8′S)−1 (Fig. 4). Notably, the predicted ECD and ORD curves for the (3S, 6S, 7R, 9R, 10R, 13R, 15S, 17R, 20R, 1′S, 5′S, 7′R, 8′R)−1 matched exquisitely with the measured ECD and ORD spectra of 1, suggesting that the absolute configuration of 1 could be assigned as 3S, 6S, 7R, 9R, 10R, 13R, 15S, 17R, 20R, 24R, 1′S, 5′S, 7′R, 8′R. Finally, the single-crystal X-ray diffraction analysis (Fig. 5) using Cu Kα radiation with a Flack parameter of 0.00(12) (CCDC 2249834) not only validated the novel 6/6/6/5/6/5/6/5 skeleton of 1 but also authenticated its absolute configuration as 3S, 6S, 7R, 9R, 10R, 13R, 15S, 17R, 20R, 24R, 1′S, 5′S, 7′R, 8′R.

  Figure 4

  

  Figure 4.  (A) Experimental versus calculated ECD and (B) ORD spectra of 1.

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  Figure 5

  

  Figure 5.  X-ray structure of 1.

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  Chaeglobol A (1) represents an unparalleled C37 sterol, characterized by a unique 6/6/6/5/6/5/6/5 octacyclic system, prompting the proposal of a plausible biosynthetic pathway (Scheme 1). Initially, mevalonate and CH₃COOH underwent a series of enzymatic catalytic processes to produce ergosterol (a) and 1, 2-benzenedicarboxaldehyde-3, 4, 5-trihydroxy-6-methyl (b), respectively, previously isolated from the fungal genus Chaetomium [17,18]. Then, the oxidative transformation of a would produce intermediate c, while structural resonance of b would produce intermediate d. Subsequently, a pivotal [4 + 2] cycloaddition reaction between the c and d was envisioned to form intermediate e. Ensuing enolization and oxidation of e led to the formation of f. Ultimately, the intermediate f would be transformed into the 1 by an enzymatic cyclization reaction between C-6 and C-5′.

  Figure 1

  

  Figure 1.  Proposed biosynthetic pathway of 1.

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  Chaeglobol A (1) was evaluated for its antifungal activity against Botryosphaeria dothidea (Fig. 6). It was observed that 1 displayed potential inhibitory effect against B. dothidea, with inhibition rates of 37% and 45% at concentrations of 12.5 and 25.0 µg/mL, respectively (Fig. 6A). Carbendazim was used as the positive control with the half maximal inhibitory concentration (IC₅₀) values of 13.6 µg/mL. To explore the antifungal mechanism of 1, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and propidium iodide (PI) and 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining experiments were conducted. In SEM experiment, it was observed that the hyphae of the blank control group were uniform and smooth, while those treated with 1 exhibited shrinkage and protrusion (Figs. 6B and C). The TEM experiment revealed that the cell wall, cell membrane, and organelle structures of the blank control group were clearly visible and orderly. However, after treatment with 1, there were observable changes such as cell deformation, cell membrane damage, and organelle dissolution (Figs. 6D and E). The SEM and TEM results indicated that 1 could affect the normal growth of B. dothidea and potentially inhibited hyphal growth by damaging the cell membrane of B. dothidea. The PI and DCFH-DA staining experiments further confirmed that the cell membrane integrity of B. dothidea was damaged after treatment with 1 and that oxidative damage occurred in the hyphae (Fig. 6F). These findings indicated that 1 can inhibit the growth of B. dothidea by destroying its cell membrane integrity and causing oxidative damage within the cells.

  Figure 6

  

  Figure 6.  Inhibitory effects of 1 on B. dothidea. (A) Inhibition rate of mycelial growth on potato dextrose agar (PDA) medium. Data are presented as mean ± standard deviation (SD) (n = 3). Values with different letters (a–d, at 7 days) are statistically significantly different (P  <  0.05). (B, C) SEM microscopic observations of the B. dothidea mycelia. B1 and B2, control; C1 and C2, 1 of 25.0 µg/mL. Scale bars: 10 µm (B1, C1) and 5 µm (B2, C2), respectively. (D, E) TEM microscopic observations of the B. dothidea mycelia. Scale bars: 2 µm (D1, E1) and 1 µm (D2, E2), respectively. D1 and D2, control; E1 and E2, 1 of 25.0 µg/mL. (F) Effect of 1 treatment on the cell membrane and oxidative stress of B. dothidea. Scale bars: 100 µm.

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  In summary, the fungal genus Chaetomium is renowned for its production of diverse natural products with novel skeletons [19]. To the best of our knowledge, previous research has primarily focused on the discovery of novel cytochalasins and azaphilones [20-22]. In the present study, we isolated an unprecedented C37 sterol (1) from the marine-derived fungus C. globosum HBU-45, characterized by a unique 6/6/6/5/6/5/6/5 octacyclic system that is highly fascinating to the organic chemistry community. Notably, compound 1 exhibited potent antifungal activity against B. dothidea, offering remarkable potential for agricultural fungicide development.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Yun-Feng Liu: Writing – original draft. Hui-Fang Du: Visualization, Project administration, Investigation. Ya-Hui Zhang: Formal analysis. Zhi-Qin Liu: Supervision. Xiao-Qian Qi: Methodology. Du-Qiang Luo: Writing – review & editing. Fei Cao: Writing – review & editing, Supervision, Funding acquisition.

  Acknowledgments

  This research was funded by the S&T Program of Hebei (No. 21323202D), the Natural Science Interdisciplinary Research Program of Hebei University (No. DXK201913), the Natural Science Foundation of Hebei Province of China (Nos. H2024201028, H2020201029), the Excellent Youth Research Innovation Team of Hebei University (No. QNTD202406), the Hebei University Research and Innovation Team (No. IT2023C1), the Innovation Capacity Improvement Plan of Hebei Province (No. 20567605H), the National Training Program of Innovation and Entrepreneurship for Undergraduates (No. DC2024177), and the High Performance Computer Center of Hebei University.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.109858.

  
  
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• 

  Figure 1  Chemical structure, the key HMBC and COSY correlations of chaeglobol A (1).

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  Figure 2  (A) Regression analysis and (B) individual deviations of experimental versus calculated ¹³C NMR chemical shifts of 1.

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  Figure 3  NOESY correlations of chaeglobol A (1).

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  Figure 4  (A) Experimental versus calculated ECD and (B) ORD spectra of 1.

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  Figure 5  X-ray structure of 1.

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  Figure 1  Proposed biosynthetic pathway of 1.

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  Figure 6  Inhibitory effects of 1 on B. dothidea. (A) Inhibition rate of mycelial growth on potato dextrose agar (PDA) medium. Data are presented as mean ± standard deviation (SD) (n = 3). Values with different letters (a–d, at 7 days) are statistically significantly different (P  <  0.05). (B, C) SEM microscopic observations of the B. dothidea mycelia. B1 and B2, control; C1 and C2, 1 of 25.0 µg/mL. Scale bars: 10 µm (B1, C1) and 5 µm (B2, C2), respectively. (D, E) TEM microscopic observations of the B. dothidea mycelia. Scale bars: 2 µm (D1, E1) and 1 µm (D2, E2), respectively. D1 and D2, control; E1 and E2, 1 of 25.0 µg/mL. (F) Effect of 1 treatment on the cell membrane and oxidative stress of B. dothidea. Scale bars: 100 µm.

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  Table 1.  ¹H (600 MHz) and ¹³C (150 MHz) NMR data for 1 in CDCl₃.

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