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• Fungi are among the oldest living things on Earth, and their unique evolution has given them the ability to produce a huge number of natural products [1,2]. Unlike plants and marine organisms, the natural products of fungi often have unique structural characteristics, which are determined by fungal genetic levels and enzymes [3]. Fungi are abundant, and a conservative estimate of global fungal diversity exceeds that of land plants by a ratio of 10:1 [4,5], but only the tip of the iceberg has been studied for their chemical composition. Even so, species whose chemical compositions have been studied have produced many star molecules such as penicillins, cephalosporins, statins, mycophenolic acid, pleuromutilins and enfumafungins that have changed the course of our human civilization.

  Sesquiterpenoids are no doubt one of the most abundant classes of natural products from both plants and microbes [6–9], while fungal sesquiterpenes have their unique structural characteristics [10]. Our recent review shows that > 500 new sesquiterpenoids in 20 new carbon skeletons have been discovered from fungi in the last five years [11]. Therefore, fungi are a huge biological treasure house of sesquiterpene sources and have great research potential.

  Our group has been engaged in the study of the chemical composition of fungi for decades, a number of fungal sesquiterpenoids have been reported from basidiomycetes and ascomycetes. For instance, conocilane A, possessing a 6/5/5/5 fused ring system, was isolated from the basidiomycete Conocybe siliginea [12]. Antroalbocin A is an antibacterial sesquiterpenoid having a 5/5/6 bridged carbon skeleton from the basidiomycete Antrodiella albocinnamomea [13]. Trichothecrotocins A‒C are novel antipathogenic sesquiterpenoids from the fungus Trichothecium crotocinigenum [14]. Bipolarithizole A is a novel phenylthiazole-sativene sesquiterpenoid hybrid with antifungal activity from the fungus Bipolaris eleusines [15]. Antroxazole A is an oxazole-containing chamigrane dimer from the fungus Antrodiella albocinnamomea with immunosuppressive activity [16]. These discoveries inspired us to continue the search for more interesting sesquiterpenoids with new scaffolds and potential biological activities.

  Irpex lacteus (I. lacteus) is a wood-decaying fungus belonging to the family Polyporacea. Its crude polysaccharide fraction has long been used as a traditional Chinese medicine for the treatment of chronic glomerulonephritis in clinic [17]. Our previous chemical investigations on this fungus reported tremulane sesquiterpenoids irlactins A‒D that featured a 6/6 backbone [18], as well as a series of tremulane sesquiterpenoids irlactins E–J [19,20]. A recent investigation has characterized the tremulane sesquiterpene synthase of I. lacteus by genome mining, heterologous expression, in vitro assay, and substrate feeding [21].

  In the current study, two novel ring-rearranged sesquiterpenoids, namely irpexlactones A (1) and B (2) (Fig. 1), have been obtained from the fermentation extract of I. lacteus cultured in rice medium. Their structures have been established by extensive spectroscopic methods, as well as single crystal X-ray diffraction. Both compounds possess a novel 5/6/3 fused carbon ring system, containing a γ-lactone moiety. A plausible biosynthetic pathway for them is proposed. In addition, the two compounds have been evaluated for their anti-inflammatory activity. Herein, the details of chemical and biological characterization of 1 and 2 are reported.

  Figure 1

  

  Figure 1.  Structures of irpexlactones A (1) and B (2).

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  Irpexlactone A (1) was isolated as colorless crystals. Its molecular formula C₁₅H₂₀O₄ was determined on the basis of the high resolution electrospray ionization mass spectroscopy (HRESIMS) at m/z 287.12546 [M + Na]⁺ (calcd. for C₁₅H₂₀O₄Na: 287.12538), corresponding to six degrees of unsaturation. The infrared radiation (IR) absorption bands at 3350 and 1668 cm⁻¹, revealed the presence of hydroxy and ester group, respectively. The ¹³C nuclear magnetic resonance (NMR) spectrum displayed 15 carbon resonances, which were classified by distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) technologies (Table 1). Of them, one oxygenated CH₂ (δ_C 65.3), one oxygenated CH (δ_C 80.9), and two keto carbons (δ_C 171.7 and 200.9) were readily identified. Considering the presence of two keto carbons, compound 1 should possess a tetracyclic framework. Three sp³ quaternary carbons at δ_C 46.6, 43.4, and 41.1 suggested that compound 1 might possess a different carbon skeleton with respect to those sesquiterpenoids found from the same resource [18–20].

  Table 1

  

  Table 1.  ¹H (600 MHz) and ¹³C (150 MHz) NMR data for 1 and 2 (methanol-d₄).

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  Position	1			2
  	δH (J in Hz)	δC, type		δH (J in Hz)	δC, type
  1		46.6, C			43.2, C
  2	3.12, dd (5.5, 1.1)	33.6, CH		2.01, d (5.3)	28.9, CH
  3		43.4, C			34.8, C
  4		200.9, C		4.02, dd (9.6, 6.5)	66.4, CH
  5a 5b	2.19, dd (17.8, 4.2) 2.07, dd (17.8, 12.9)	40.2, CH2		1.49, ddd (12.9, 6.5, 2.1) 1.18, m	33.9, CH2
  6	1.88, m	25.0, CH		1.24, m	24.8, CH
  7	2.88, ddd (10.7, 9.0, 4.7)	39.7, CH		2.63, ddd (10.0, 9.3, 4.8)	40.5, CH
  8a 8b	1.69, dd (13.2, 9.0) 1.46, dd (13.2, 10.7)	37.6, CH2		1.54, dd (13.4, 9.3) 1.39, dd (13.4, 10.0)	37.9, CH2
  9		41.1, C			41.4, C
  10	3.40, s	80.9, CH		3.17, s	82.3, CH
  11a 11b	4.56, dd (9.5, 1.1) 4.38, dd (9.5, 5.5)	65.3, CH2		4.37, d (9.3) 4.27, dd (9.3, 5.3)	66.0, CH2
  12		171.7, C			176.6, C
  13	0.96, d (6.8)	17.6, CH3		0.79, d (6.7)	18.2, CH3
  14	1.11, s	22.9, CH3		0.98, s	23.2, CH3
  15	0.97, s	28.7, CH3		0.92, s	28.3, CH3

  The structure of 1 was deduced by comprehensive analysis of its 2D NMR spectra. In the ¹H‒¹H correlation spectroscopy (COSY) spectrum, two fragments were revealed as shown in Fig. 2. In the heteronuclear multiple bond coherence (HMBC) spectrum (Fig. 2), two singlets for Me-14 at δ_H 1.11 (3H, s, H-14) and Me-15 at δ_H 0.97 (3H, s, H-15) showed correlations to δ_C 41.1 (s, C-9), 37.6 (t, C-8), and 80.9 (d, C-10) revealed a gem‑dimethyl moiety, while the HMBC correlations from δ_H 3.40 (1H, s, H-10) to δ_C 46.6 (s, C-1) and 39.7 (d, C-7) suggested the linkage of ‒CH(10)‒C(1)‒CH(7)‒. These HMBC data, in combination with ¹H‒¹H COSY correlations, established a five-membered ring A by C-1, C-7, C-8, C-9, and C-10. Continuous analysis of HMBC spectrum, H-7 showed key correlations to two quaternary carbons at δ_C 46.6 (s, C-1) and 43.4 (s, C-3), while H-5 showed a key correlation to the carbonyl carbon at δ_C 200.9 (s, C-4). These data suggested a six-membered ring B as constructed by C-1, C-3, C-4, C-5, C-6, and C-7. As mentioned above, a fragment of ‒CH(2)‒CH₂(11)‒ was established by the ¹H‒¹H COSY spectrum, while H-2 (δ_H 3.12) showed key HMBC correlations to two sp³ quaternary carbons C-1 and C-3, these data suggested a ternary ring C as built by C-1, C-2, and C-3. So far, only the lactone carbon at δ_C 171.7 (s, C-12) was not assigned. In the HMBC spectrum, both H-11 and H-2 showed key correlations to C-12, constructing a γ-lactone moiety (ring D), which was supported by mass spectroscopy (MS) data analysis. Compound 1 was, therefore, identified as a sesquiterpenoid featured a novel 5/6/3/5 tetracyclic system.

  Figure 2

  

  Figure 2.  Key 2D NMR correlations of 1 and 2.

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  The rotating frame Overhauser effect spectroscopy (ROESY) experiment revealed the relative configuration of 1. As shown in Fig. 2, correlations of H-2 with H-7 and H-6 indicated that H-2, H-6, and H-7 were in the same side (randomly assigned as α orientation). Consequently, correlations of H-7 with H₃–15 and H₃–14 with H-10 suggested that H-10 was β oriented. Due to a novel structure as established as depicted, more solid evidence is necessary. Fortunately, a single crystal of 1 was obtained from methanol, and an X-ray diffraction not only confirmed the structure as elucidated above but also determined the absolute configuration (Fig. 3, Flack parameter = 0.11(7), CCDC: 1893729). Compound 1 was, finally, identified as (1S,2R,3R,6R,7R,10S)-irpexlactone A.

  Figure 3

  

  Figure 3.  Oak Ridge thermal ellipsoid plot (ORTEP) diagram of 1 showing absolute configuration.

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  Irpexlactone B (2) was isolated as a colorless oil. Its molecular formula C₁₅H₂₂O₄ was determined on the basis of the HRESIMS at m/z 267.15875 [M + H]⁺ (calcd. for C₁₅H₂₃O₄: 267.15909), corresponding to five degrees of unsaturation. The IR absorption bands at 3342 and 1664 cm⁻¹ inferred the existence of OH and C=O moieties, respectively. The ¹³C NMR spectrum and DEPT data revealed 15 carbon resonances which were closely related to that of 1 (Table 1), except that the signal at δ_C 200.9 (s, C-4) in 1 was replaced by signals of δ_H 4.02 (1H, dd, J = 9.6 and 6.5 Hz, H-4) and δ_C 66.4 (d, C-4) in 2. Detailed analysis of 2D NMR spectra suggested that C-4 in 2 was substituted by a hydroxy group, rather than a carbonyl carbon in 1, as supported by HMBC correlations from δ_H 4.02 (H-4) to δ_C 34.8 (s, C-3) and 33.9 (t, C-5), and by the ¹H‒¹H COSY correlation between H-5 and H-4. One more ROESY correlation between H-4 and H-2 indicated that the hydroxy at C-4 should be β oriented (Fig. 2). Based on this, the results of electronic circular dichroism (ECD) calculations suggested the absolute configuration to be 1S, 2R, 3R, 4S, 6R, 7R, 10S-2a (Fig. 4, details are given in Section S1–1.4 in Supporting information). Therefore, compound 2 was structurally established and named irpexlactone B.

  Figure 4

  

  Figure 4.  Calculated ECD spectra for 2a (red) and 2b (blue) at the mPW1PW91/6–311G(d) level in methanol with PCM model (2a and 2b: σ = 0.3 eV, ultraviolet (UV) shift 10 nm). Experimental CD spectra of 2 (black line) in MeOH.

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  Compounds 1 and 2 should be derived from the common precursor of a tremulane-type sesquiterpenoid which featured with a 5/7 carbon skeleton [19,20]. Recently, our biosynthesis study has characterized an Irpex lacteus iltremulanol synthase Il4946, which produced tremulane sesquterpene iltremulanol A in Aspergillus oryzae [21]. Structurally, compounds 1 and 2 possess a new bond that is suggested to be catalyzed by a cytochrome P450 enzyme via a radical process [22]. Therefore, a plausible hypothesis for the biogenetic pathway for 1 and 2 was afforded. As shown in Scheme 1, iltremulanol A was oxidized and lactonized to give an intermediate Ⅰ, then an enzymatic radical abstracts a hydrogen atom at C-3 to produce Ⅱ. The resulting radical C-3 attacks C-1 to form the C-1‒C-3 bond to give Ⅲ. After further oxidation, the final products of 1 and 2 were constructed.

  Scheme 1

  

  Scheme 1.  Proposed biosynthetic pathway for 1 and 2.

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  Both compounds 1 and 2 were evaluated for their cytotoxicity against MCF-7 cancer cell line and their anti-inflammatory activity by inhibiting nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. As a result, two compounds had no cytotoxicity against MCF-7. However, they exhibited significant inhibitory activity against NO production with half maximal inhibitory concentration (IC₅₀) values of 2.2 ± 0.55 and 1.4 ± 0.04 µmol/L, respectively, better than that of dexamethasone (IC₅₀ = 9.0 ± 1.61 µmol/L). In addition, both compounds exhibited no cytotoxicity to normal RAW264.7 cells at the concentration of 8 µmol/L (Supporting information, Section S1–1.5).

  The effects of 1 and 2 on pro-inflammatory mediators of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were investigated in LPS-stimulated RAW264.7 cells using enzyme linked immunosorbent assay (ELISA) method as we reported previously [23]. As depicted in Fig. 5, the results suggested that compounds 1 and 2 both visibly suppressed the secretion of TNF-α and IL-6 compared to the LPS-only treatment at the concentration of 5 µmol/L, which also exhibited better biological activity than that of dexamethasone (10 µmol/L).

  Figure 5

  

  Figure 5.  Compounds 1 and 2 decreased the production of pro-inflammatory mediators TNF-α and IL-6 were investigated in LPS-induced RAW264.7 cells. Dexamethasone (10 µmol/L) was used as the positive control. Data are expressed as the mean ± SD of triplicate experiments. ^###P < 0.001 vs. the control group; **P < 0.01, ***P < 0.001 vs. the LPS group.

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  Furthermore, we detected the expression of inducible nitric oxide synthase (iNOS) by Western blot [24]. As shown in Fig. 6, the protein expression of iNOS markedly increased upon LPS stimulation. Compounds 1 and 2 dramatically down-regulated the expression of iNOS at the protein level in a dose-dependent manner in LPS-stimulated RAW264.7 cells.

  Figure 6

  

  Figure 6.  Western blot experiments analysis (A) and relative protein expression of iNOS in LPS-stimulated RAW264.7 cells (B). Dexamethasone (20 µmol/L) was used as the positive control. Data are expressed as the mean ± SD of triplicate experiments. ^###P < 0.001 vs. the control group; **P < 0.01, ***P < 0.001 vs. the LPS group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  In conclusion, two ring-rearranged sesquiterpenoids, irpexlactones A and B bearing an unprecedented 5/6/3/5 tetracyclic system, were obtained from cultures of the fungus I. lacteus in rice medium. It is noteworthy that a centered cyclopropane ring containing two quaternary carbons fused with other three rings is very rare in natural products. The whole molecule occurs in a unique way with very special ring angles, which looks like structural stiffness under large ring tension. A plausible enzyme catalytic radical biosynthesis pathway for them infers new biosynthetic enzymes in I. lacteus. Moreover, the anti-inflammatory assay revealed that compounds 1 and 2 had strong inhibitory activity against NO production in LPS-stimulated RAW264.7 cells with IC₅₀ values better than that of dexamethasone, which highlights irpexlactones as a new research hotspot for potential anti-inflammatory lead compounds.

  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

  Juan He: Writing – original draft, Funding acquisition, Data curation. Jiao-Xian Du: Methodology, Investigation, Data curation. Meng Wang: Writing – original draft, Investigation, Data curation. Xiao-Dong Luo: Writing – review & editing, Methodology, Conceptualization. Tao Feng: Writing – review & editing, Supervision, Project administration.

  Acknowledgments

  This work was financially supported by the National Natural Science Foundation of China (Nos. 22277147, 22177139) and the State Key Laboratory of Applied Microbiology South China (No. SKLAM003–2022).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110769.

  
  
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• 

  Figure 1  Structures of irpexlactones A (1) and B (2).

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  Figure 2  Key 2D NMR correlations of 1 and 2.

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  Figure 3  Oak Ridge thermal ellipsoid plot (ORTEP) diagram of 1 showing absolute configuration.

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  Figure 4  Calculated ECD spectra for 2a (red) and 2b (blue) at the mPW1PW91/6–311G(d) level in methanol with PCM model (2a and 2b: σ = 0.3 eV, ultraviolet (UV) shift 10 nm). Experimental CD spectra of 2 (black line) in MeOH.

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  Scheme 1  Proposed biosynthetic pathway for 1 and 2.

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  Figure 5  Compounds 1 and 2 decreased the production of pro-inflammatory mediators TNF-α and IL-6 were investigated in LPS-induced RAW264.7 cells. Dexamethasone (10 µmol/L) was used as the positive control. Data are expressed as the mean ± SD of triplicate experiments. ^###P < 0.001 vs. the control group; **P < 0.01, ***P < 0.001 vs. the LPS group.

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  Figure 6  Western blot experiments analysis (A) and relative protein expression of iNOS in LPS-stimulated RAW264.7 cells (B). Dexamethasone (20 µmol/L) was used as the positive control. Data are expressed as the mean ± SD of triplicate experiments. ^###P < 0.001 vs. the control group; **P < 0.01, ***P < 0.001 vs. the LPS group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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  Table 1.  ¹H (600 MHz) and ¹³C (150 MHz) NMR data for 1 and 2 (methanol-d₄).

  Position	1			2
  	δH (J in Hz)	δC, type		δH (J in Hz)	δC, type
  1		46.6, C			43.2, C
  2	3.12, dd (5.5, 1.1)	33.6, CH		2.01, d (5.3)	28.9, CH
  3		43.4, C			34.8, C
  4		200.9, C		4.02, dd (9.6, 6.5)	66.4, CH
  5a 5b	2.19, dd (17.8, 4.2) 2.07, dd (17.8, 12.9)	40.2, CH2		1.49, ddd (12.9, 6.5, 2.1) 1.18, m	33.9, CH2
  6	1.88, m	25.0, CH		1.24, m	24.8, CH
  7	2.88, ddd (10.7, 9.0, 4.7)	39.7, CH		2.63, ddd (10.0, 9.3, 4.8)	40.5, CH
  8a 8b	1.69, dd (13.2, 9.0) 1.46, dd (13.2, 10.7)	37.6, CH2		1.54, dd (13.4, 9.3) 1.39, dd (13.4, 10.0)	37.9, CH2
  9		41.1, C			41.4, C
  10	3.40, s	80.9, CH		3.17, s	82.3, CH
  11a 11b	4.56, dd (9.5, 1.1) 4.38, dd (9.5, 5.5)	65.3, CH2		4.37, d (9.3) 4.27, dd (9.3, 5.3)	66.0, CH2
  12		171.7, C			176.6, C
  13	0.96, d (6.8)	17.6, CH3		0.79, d (6.7)	18.2, CH3
  14	1.11, s	22.9, CH3		0.98, s	23.2, CH3
  15	0.97, s	28.7, CH3		0.92, s	28.3, CH3

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•