English

• Meroterpenoids are broadly defined as compounds of mixed terpenoid-nonterpenoid origin, which have attracted broad attention due to their structural complexity and remarkably wide range of bioactivities [1, 2], such as pyripyropene A (cholesterol acyltransferase-2 inhibitor) [3], territrem B (acetylcholinesterase inhibitor) [4], ascofuranone (trypanosome alternative oxidase inhibitor) [5], berkeleyacetal C (anti-inflammatory activity) [6], and so on. Diisoprenyl-cyclohexene/ane-type meroterpenoids, generally possessing two isoprenyl chains (C5 unit) attached to a cyclohexene/ane moiety (C6 unit) at ortho or meta-position, originate from the hybrid terpenoid-shikimate biosynthesis. To date, more than 50 diisoprenyl-cyclohexene/anes have been reported from fungi (such as Pestalotiopsis sp. [7-13], Isariopsis sp. [14-16], and Truncatella sp. [17]) and plants (such as Ophryosporus lorentzii [18] and Cephalozia otaruensis [19]).

  In our previous investigation on metabolites of a fungal strain of Biscogniauxia sp. 71-10-1-1 fermented with rice, a novel diisoprenyl-cyclohexene-type meroterpenoid dimer (dimericbiscognialkyne A) with short-term memory enhancement activity was isolated (Fig. 1), along with three new biscognienynes [20]. Dimericbiscognialkyne A is the first meroterpenoid dimer featuring hexadecahydrobenzo[kl]xanthene ring system, which could derive from two same monomeric diisoprenyl-cyclohexene-type meroterpenoids (biscognienyne B) via a unique intermolecular redox coupling Diels-Alder adduct and a nucleophilic addition reaction. In addition, the dimericbiscognialkyne A and biscognienynes possess aesthetically interesting 'bird'-like topologies, named as huanhuanbirds [20]. In order to obtain more diisoprenyl-cyclohexene/ane-type meroterpenoid dimers, a chemical investigation on the residual fractions of this fungus was carried out, which led to the isolation of two new diisoprenyl-cyclohexene-type meroterpenoid dimers (dimericbiscognienynes B and C, 1 and 2) (Fig. 1). Herein, we describe the isolation and structural elucidation of 1 and 2.

  Figure 1

  

  Figure 1.  Chemical structures of 1, 2, and dimericbiscognialkyne A.

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  The culture was extracted thrice with EtOAc, and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (43.2 g). Then the crude extract was subjected to silica gel CC (4 cm × 15 cm) using cyclohexane-MeOH (100:0 and 0:100, v/v) to afford a cyclohexane extract (C, 12.5 g) and a MeOH extract (w, 21.9 g). The MeOH extract (w, 21.9 g) was separated by ODS CC (4 cm × 30 cm) eluting with MeOH-H₂O (20:80, 50:50, 70:30, and 100:0, v/v) to afford 4 fractions (w1-w4). Fraction w3 (3.1 g) was further subjected to ODS (4 cm × 45 cm) MPLC eluted with a gradient of MeOH-H₂O (20:80 to 100:0, v/v) for 420 min at a flow rate of 20 mL/min to afford fractions w3-1-w3-9. Fraction w3-8 (673.2 mg) was subjected to silica gel CC using cyclohexane-ethyl acetate (100:0 to 0:100, v/v) to afford fractions w3-8-1-w3-8-8. Fraction w3-8-3 (47.3 mg) was subjected to preparative HPLC using MeCN-H₂O (50:50, v/v) at a flow rate of 3 mL/min to yield 1 (t_R: 24.3 min, 2.1 mg) and 2 (t_R: 28.7 min, 5.3 mg).

  Dimericbiscognienyne B (1): Colorless oil; [α]_D^27.1-123.6 (c 0.25, MeOH); UV (MeOH, nm) λ_max (logε): 205 (4.86), 224 (4.59), 285 (4.30); IR (KBr, cm^-1): ν_max 3421, 2969, 2920, 2363, 1647, 1616, 1540, 1457, 1377, 1089; ECD λ_nm (Δε) (c 9.7 × 10^-4 mol/L, MeOH): 213 (+4.74); ESI-MS (positive): m/z 545 [M+Na]⁺; HR-ESI-MS (positive): m/z 545.2886 [M+Na]⁺ (calcd. for C₃₂H₄₂O₆Na: 545.2879), ¹H and ¹³C NMR data can be found in Table 1.

  Table 1

  

  Table 1.  NMR data of 1 and 2 in CDCl₃.

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  Dimericbiscognienyne C (2): Colorless oil; [α]_D^27.1 +51.6 (c 0.5, MeOH); UV (MeOH, nm) λ_max (logε): 224 (4.43), 315 (3.34); IR (KBr, cm^-1): ν_max 3442, 2965, 2924, 2360, 1718, 1668, 1437, 1376, 1276, 1089; ECD λ_nm (Δε) (c 6.3 × 10^-4 mol/L, MeOH): 213 (+6.70); ESI-MS (positive): m/z 543 [M+Na]⁺; HR-ESI-MS (positive): m/z 521.2899 [M+H]⁺ (calcd. for C₃₂H₄₁O₆: 521.2903); ¹H and ¹³C NMR data can be found in Table 1.

  Dimericbiscognienyne B (1) was obtained as a colorless oil. The molecular formula of 1 was established as C₃₂H₄₂O₆ (12 degrees of unsaturation) from its HR-ESI-MS (m/z 545.2886 [M+Na]⁺, calcd. for C₃₂H₄₂O₆Na: 545.2879), which was 2 Da more than dimericbiscognienyne A. The ¹³C NMR data showed 32 carbon signals (Table 1). Combined with data from the DEPT 135 experiment, these carbons can be categorized as ten aromatic or olefinic carbons (δ_C 135.7, 134.6, 132.9, 132.7, 125.8, 125.6, 122.6, 119.0, 118.2, 116.8), two sp quaternary carbons (δ_C 88.4, 86.1), four sp³ quaternary carbons (δ_C 94.9, 73.5, 64.2, 48.1), six sp³ methine carbons (δ_C 75.2, 66.9, 65.8, 60.1, 41.7, 37.6), four sp³methylene carbons (δ_C 40.5, 38.1, 31.1, 26.3), and six methyl carbons (δ_C 26.0, 25.8, 23.9, 23.5, 18.1, 18.0). The ¹H NMR data (Table 1) of 1 revealed the characteristic signals of six olefinic or aromatic protons [δ_H 5.62 (br s, 1 H), 5.47 (br s, 1 H), 5.25 (br t, 1H, J = 7.5 Hz), 5.19 (1 H), 5.17 (1 H), and 5.03 (br t, 1H, J = 7.5 Hz)] and six methyls [δ_H 1.78 (br s, 3 H), 1.74 (br s, 3 H), 1.73 (br s, 3 H), 1.71 (br s, 3 H), 1.64 (br s, 3 H), 1.64 (br s, 3 H)]. Except for the disappearence of one oxygenated methine signal [δ_C 59.4 (C-3′); δ_H 3.21 (H-3′)], one additional methylene signal [δ_C 40.5 (C-3′); δ_H 2.32 (Ha-3′), 1.32 (Hb-3′)], and the obvious downfield shift of C-2′ (δ_C 73.5), the NMR data of 1 are similar to those of dimericbiscognienyne A [20], which indicated that 1 should have the same skeleton as dimericbiscognienyne A, and 1 is a deoxidized derivative of dimericbiscognienyne A at C-3′. All the proton resonances were associated to the directly attached carbon atoms through the HSQC experiment (Table 1 and Supporting information). The key ¹H-¹H COSY correlations (Table S1 in Supporting information) of H-3′a with H-3′b/H-4′/H-5′, H-3′b with H-3′a/H-4′, and H-4′ with H-3′a/H-3′b/H-5′ and the HMBC cross-peaks (Table S1) from H-3′a/H-3′b to C-1′/C-2′/C-4′/C-5′, and from H-5′ to C-1′/C-3′/C-7′ confirmed the above deduction. The whole planar structure of 1 was established by the analysis of 2D NMR(Table S1), and the assignments of all proton and carbon resonances are shown in Table 1.

  The relative configuration of 1 was deduced by comparison with dimericbiscognienyne A, which was previously established by X-ray crystallography analysis [20]. Their planar structures can be divided into two parts (A and B), and the planar structures of part A in 1 and dimericbiscognienyne A were identical. Because the ¹³C chemical shifts of part A in 1 were substantially identical with those of dimericbiscognienyne A [20], the relative configuration of part A in 1 was considered as the same as that of dimericbiscognienyne A, which was assigned as 1R^*, 2S^*, 3S^*, 4R^*, 5S^*, 6R^*, 7′R^*. In part B of 1, the large value of ³J_{Hb-3′, H-4′} (9.5 Hz) indicated that H-4′ was on the pseudoaxial orientation in the cyclohexene ring (C1′-C6′), and Hb-3′ was on the opposite axial orientation in the cyclohexene ring (C1′-C6′) of 1 (Fig. 2). In the NOESY experiment of 1, the observed correlations between H-1′ and Hb-3′/Ha-12′/Hb-12′ demonstrated that H-1′, Hb-3′, and C-12′ were on the same face in the cyclohexene ring (C1′-C6′) of 1 (Fig. 2 and Table S1). On the basis of the analyses of ³J_{Hb-3′, H-4′} and the NOESY correlations, the relative configuration of the part B in 1 was assigned as 1′R^*, 2′S^*, 4′S^* (Fig. 2). Combined with the above deductions and the key NOESY correlation between H-1′ and H-7′ (Fig. 2 and Table S1), the whole relative configuration of 1 was assigned as 1R^*, 2S^*, 3S^*, 4R^*, 5S^*, 6R^*, 1′R^*, 2′S^*, 4′S^*, 7′R^* (Fig. 2).

  Figure 2

  

  Figure 2.  Key NOESY or ROESY correlations of 1 and 2.

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  Dimericbiscognienyne C (2) was obtained as a colorless oil. The molecular formula of 2 was established as C₃₂H₄₀O₆ (13 degrees of unsaturation) from its HR-ESI-MS (m/z 521.2899 [M+H]⁺, calcd. for C₃₂H₄₁O₆: 521.2903), which was 2 Da less than dimericbiscognienyne B (1). The ¹³C NMR data showed 32 carbon signals (Table 1). Combined with data from the DEPT 135 experiment, these carbons can be categorized as eleven sp² carbons [including one carbonyl (δ_C197.0)], two sp quaternary carbons, four sp³ quaternary carbons [including three oxygenated ones (δ_C 94.9, 75.4, 64.2)], five sp³ methine carbons [including three oxygenated ones (δ_C 75.0, 66.7, 60.4)], four sp³ methylene carbons, and six methyl carbons. The ¹H NMR data (Table 1) of 2 revealed that the characteristic signals of six olefinic or aromatic protons [δ_H 5.95 (br s, 1 H), 5.44 (br s, 1 H), 5.20 (1 H), 5.18 (2 H), 5.05 (br t, 1H, J = 6.9 Hz)], and six methyls [δ_H 1.76 (br s, 3 H), 1.76 (br s, 3 H), 1.73 (br s, 3 H), 1.73 (br s, 3 H), 1.65 (br s, 3 H), 1.64 (br s, 3 H)]. Except for the lacking of an oxygenated methine signal [δ_C 65.8 (C-4′); δ_H 4.56 (H-4′)] and the appearance of a carbonyl [δ_C 197.0 (C-4′)], the NMR data of 2 highly resemble those of 1 (Table 1), which indicated that 2 is an oxidation product of 1 at C-4′. The detailed analyses of 2D NMR (Table S2 in Supporting information) established the whole planar structure of 2, and the assignments of all proton and carbon resonances are shown in Table 1.

  The planar structures of part A in 2 and 1 were also identical. Because the ¹³C chemical shifts of part A in 2 were substantially identical with those of 1 [20], the relative configuration of part A in 2 was considered as the same as that of 1, which was assigned as 1R^*, 2S^*, 3S^*, 4R^*, 5S^*, 6R^*, 7′R^*. The ROESYcorrelations of between H-5 and 1-OH, and between H-4 and H-7′ in 2 were consistent with the above deduction (Fig. 2 and Table S2). In addition, the observed ROESY correlations between H-1′ and Hb-3′/Ha-12′/Hb-12′ demonstrated that H-1′, Hb-3′, and C-12′ were on the same face in the cyclohexene ring (C1′-C6′) of 2 (Fig. 2 and Table S2), and the relative configuration of part B in 2 was assigned as 1′R^*, 2′S^*. Combined with the above deductions and the key ROESY correlation between H-1′ and H-7′ (Fig. 2 and Table S2), the whole relative configuration of 2 was assigned as 1R^*, 2S^*, 3S^*, 4R^*, 5S^*, 6R^*, 1′R^*, 2′S^*, 7′R^* (Fig. 2).

  The ECD curves (Fig. 3) of 1 and 2 were similar to that of dimericbiscognienyne A, which suggested that 1 and 2 shared the same obsolute configurations with dimericbiscognienyne A. Therefore, the obsolute configurations of 1 and 2 were assigned as 1R, 2S, 3S, 4R, 5S, 6R, 1′R, 2′S, 4′S, 7′R, and 1R, 2S, 3S, 4R, 5S, 6R, 1′R, 2′S, 7′R, respectively.

  Figure 3

  

  Figure 3.  ECD spectra of 1, 2 and dimericbiscognialkyne A.

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  Since the first report on the diisoprenyl-cyclohexene/ane-type meroterpenoidsfrom Beauveria felina SANK 13682 in 1983 [21], more than 50 diisoprenyl-cyclohexene/ane-type meroterpenoids have been reported, most of which are monomeric (C16). Only three dimers have been reported, including pestalofones B and C with unique 2-cyclohexylspiro[5.5] undecane ring system (Pestalotiopsis sp.) [12] and dimericbiscognialkyne A with unusual hexadecahydrobenzo[kl]xanthene ring system (Biscogniauxia sp.) [20]. In our chemical investigation, two new diisoprenyl-cyclohexene-type meroterpenoid dimers (dimericbiscognialkynes B and C) were obtained, which derive from two different monomeric diisoprenyl-cyclohexene-type meroterpenoids (biscognienynes A and B [20]) via a unique intermolecular redox coupling Diels-Alder adduct and a nucleophilic addition reaction (Scheme 1, Figs. S1 and S2). The result of this study added the members of the rare class of diisoprenyl-cyclohexene/ane-type meroterpenoid dimers.

  Scheme 1

  

  Scheme 1.  Plausible biosynthetic pathways of 1 and 2.

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  Acknowledgments

  This work was financially supported by grants from the National Natural Science Foundation of China (No. 3171101305), Chang Jiang Scholars Program (Hao Gao, 2017) from the Ministry of Education of China, the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2017A03036027), Guangdong Special Support Program (No. 2016TX03R28), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (Hao Gao, 2014), Pearl River Nova Program of Guangzhou (No. 201610010021), and K. C. Wong Education Foundation (Hao Gao, 2016).

  Appendix A. Supplementary data

  Supplementary material related to this article can befound, in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.05.019.

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• 

  Figure 1  Chemical structures of 1, 2, and dimericbiscognialkyne A.

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  Figure 2  Key NOESY or ROESY correlations of 1 and 2.

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  Figure 3  ECD spectra of 1, 2 and dimericbiscognialkyne A.

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  Scheme 1  Plausible biosynthetic pathways of 1 and 2.

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  Table 1.  NMR data of 1 and 2 in CDCl₃.

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•