Steroidal Adducts and Andrastin-Type Meroterpenoids from the Co-culture of Two Isodon Species-Associated Endophytic Fungi

Abstract

Ergopolyketides A–D, presumably biosynthesized through Diels–Alder additions between ergosterol and polyketides, along with two new andrastin-type meroterpenoids, isopenicins D and E, were isolated from the co-culture of endophytic fungi Penicillium sp. sh18 and Pestalotiopsis sp. HS30, both, inhabiting Isodon species. The structures of these compounds were elucidated using spectroscopic analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Isopenicin D and a known compound, peniandrastin D, exhibited moderate cytotoxic activities against human cancer cell lines HL-60, A549, and SMMC-7721, with IC₅₀ values ranging from 13.37 to 29.17 μM.

Recent research on the secondary metabolites of endophytic fungi associated with Isodon species has confirmed their potential as a valuable source for discovering innovative bioactive compounds. For instance, Penicillium sp. sh18, an endophyte of I. eriocalyx var. laxiflora, has produced penicilfuranone A with antifibrotic activity,[1] and isopenicins A–C, which can inhibit Wnt signaling and suppress cancer cell growth.[2] Additionally, Pestalotiopsis sp. HS30, isolated from I. xerophilus, has yielded novel alkaloids pestaloamides A and B with latent tumor immunotherapy activity.[3] Furthermore, the isolation of ent-kaurane diterpenoids, characteristic metabolites of Isodon species, has been achieved from Geopyxis sp. XY93, which resides within I. parvifolia.[4]

In the present study, the co-culture strategy was employed to reveal more structurally and biologically fascinating natural products from endophytic fungi associated with Isodon species. This method aims to foster a harsh environment that triggers the expression of silent or inactive biosynthetic gene clusters through synergistic and/or competitive interactions among microorganisms.[5] Preliminary screening of various fungal pairs indicated that the co-culture of Penicillium sp. sh18 and Pestalotiopsis sp. HS30 showed an enriched metabolite profile. Subsequent large-scale fermentation under the same conditions yielded six new compounds, comprising four unique steroidal adducts, ergopolyketides A–D (1–4), two new andrastin-type meroterpenoids, isopenicins D (5) and E (6), and a known compound, peniandrastin D (7)[6] (Figure [1]). Notably, 1–4 were deduced to form through Diels–Alder additions between ergosterol and various polyketides. This report details their isolation, structural elucidation, and cytotoxic evaluation.

Ergopolyketide A (1), obtained as a colorless oil, has the molecular formula C₅₄H₇₀O₁₀ as inferred from the (+)-HRESIMS ion at m/z 879.5055 [M + H]⁺ (calcd 879.5042). A careful analysis of the ¹H and ¹³C NMR data (Table S1) of 1 indicated that the chemical structure is composed of two parts: a modified ergosterol part and a furanone part (Figure [2a]). Further analysis of the planar structure of 1 was mainly supported by ¹H–¹H COSY and HMBC correlations. For the structure of part A, the ¹H–¹H COSY spectrum indicated the presence of four spin systems (Figure [2a]). The HMBC spectrum showed correlations of H₃-19 (δ_H 1.19) with C-1 (δ_C 29.2), C-5 (δ_C 77.1), C-9 (δ_C 139.5), and C-10 (δ_C 40.3); H-4b (δ_H 1.98) with C-5 (δ_C 77.1) and C-10; and H-7 with C-9 and C-5 (Figure [2a]). The correlations confirmed that, as compared to ergosterol, part A has oxygenated C-5 and C-6, and includes a C-7/C-8/C-9/C-11 conjugated system. For part B, comparison of the NMR data revealed that its planar structure is identical to that of the known compound penicilfuranone A.[1] The HMBC spectrum revealed a key correlation between H-6 and C-20′, indicating the linkage of C-6 and C-20′ through an oxygen atom. Considering the remaining degree of unsaturation and the chemical shifts of C-5 and C-21′, it is deduced that C-5 and C-21′ are also oxygen-bridged. Collectively, these findings suggest that parts A and B are connected via a 1,4-dioxane ring (Figure [2a]), thereby establishing the planar structure of 1.

Analysis of the ROESY spectrum, coupling constants, along with NMR data comparison and biosynthetic considerations, indicated that the stereochemistry of part A is mostly consistent with its precursor, ergosterol.[7] [8] Furthermore, the ROESY correlation between H-6 (δ_H 4.76) and H₃-19β (δ_H 1.19) establishes the β-orientation of H-6. Molecular modeling reveals that, due to steric constraints, the C-5–O bond can only adopt the same α-orientation as the C-6–O bond. The H₃-25′/H-14′, H₃-25′/H-15′ correlations in the ROESY spectrum suggest that H-14′, H-15′, and H₃-25′ adopted the same orientation (Figure [2b]). Thus, the relative configuration of 1 has four possibilities, that is: (4′R*,14′S*,15′R*,19′S*)-1 (1a), (4′R*,14′R*,15′S*,19′R*)-1 (1b), (4′S*,14′R*,15′S*,19′R*)-1 (1c), and (4′S*,14′S*,15′R*,19′S*)-1 (1d) (Figure S3, configurations of other chiral centers being equal, that is: 3S*,5R*,6S*,10R*,13R*,14R*,17R*,20R*,24R*). Then, 1a–d were subjected to quantum chemical calculations of chemical shifts at the mPW1PW91-SCRF/6-31+G(d,p)//B3LYP-D3BJ/6-31G(d) level of theory and subsequent DP4+ analysis based on random conformational amplitudes (DP4-BRCA).[9] The calculated NMR chemical shifts of 1a matched well with their experimental counterparts (Figure [2c]), and had an overwhelming DP4+ probability of 95.00%, thus validating the planar structure and establishing the relative configuration of 1. Then, TDDFT ECD calculations of (3S,5R,6S,10R,13R,14R,17R,20R,24R,4′R,14′S,15′R,19′S,22E,5′E, 7′E)-1 (1A) was carried out at the CAM-B3LYP-SCRF/6-31+G(2d,p) level of theory, and the calculated curve matched the experimental spectrum (Figure [2d]), thereby establishing the absolute configuration of 1.

Ergopolyketide B (2), obtained as a colorless oil, had the same molecular formula C₅₄H₇₀O₁₀ as compound 1, as determined by the HRESIMS ion at m/z 879.5048 [M + H]⁺ (calcd 879.5042). A careful analysis of the 1D (Table S1) and 2D (Figure S1) NMR data of 2, combining NMR data comparison with reported structures, indicated that the chemical structure is also composed of a modified ergosterol part and a furanone part structurally similar to penicilfuranone A. The ¹H–¹H COSY spectrum of 2 delineated a H-9/H₂-11/H₂-12 spin system. Furthermore, HMBC correlations were observed between H-6 (δ_H 5.40) and C-4 (δ_C 40.2), C-8 (δ_C 131.2), C-10 (δ_C 37.3), between H-9 (δ_H 2.00) and C-19 (δ_C 15.6), and between H-15 (δ_H 5.00) and C-14 (δ_C 84.6), C-16 (δ_C 34.6). These correlations indicated that part A of 2 is consistent with ergosterol, except for the oxygenation of C-14 and C-15. The HMBC correlation between H-15 and C-21′ (δ_C 134.4), along with its chemical shift, indicated that C-15 (δ_C 76.3) is connected to C-21′ through an oxygen atom. With one remaining degree of unsaturation and one oxygen atom, and considering the chemical shifts of C-14 (δ_C 84.6) and C-20′ (δ_C 137.1), it can be determined that C-14 and C-20 are also linked by an oxygen atom (Figure S2). In the ROESY spectrum, the correlation between H-15 and H₃-18β suggested the β-orientation of H-15. Consequently, both C-14–O and C-15–O bonds were inferred to have α-orientations. The H₃-25′/H-14′, H₃-25/H-15′ correlations in the ROESY spectrum revealed that H-14′, H-15′, and H₃-25′ adopted the same orientation (Figure S2). Subsequently, four candidates of 2 were subjected to quantum chemical calculations of chemical shifts and DP4-BRCA analysis: (4′R*,14′S*,15′R*,19′S*)-2 (2a), (4′R*,14′R*,15′S*,19′R*)-2 (2b), (4′S*,14′R*,15′S*,19′R*)-2 (2c), and (4′S*,14′S*,15′R*,19′S*)-2 (2d) (Figure S4, configurations of other chiral centers being equal, that is: 3S*,9R*,10R*,13R*,14S*,15S*,17R*,20R*,24R*). However, DP4+ failed to distinguish 2b and 2d, which had probabilities of 42.90% and 42.70%, respectively. Considering that the absolute configuration of the ergosterol part should be biosynthetically conserved, (3S,9R,10R,13R,14S,15S,17R,20R,24R,4′R,14′R,15′S,19′R)-2 (2B) and (3S,9R,10R,13R,14S,15S,17R,20R,24R,4′S,14′S,15′R,19′S)-2 (2D) were subjected to TDDFT ECD calculations. The calculated ECD curve for 2B was in better consistence with the experimental spectrum (Figure S5), which implied that the absolute configuration of 2 is as depicted in 2B. In part B of 2, the configurations of C-14′, C-15′, and C-19′ have been inverted relative to penicilfuranone A. These inversions are speculated to have occurred during the formation of the diene prior to the Diels–Alder additions between ergosterol and polyketides.

Ergopolyketide C (3), obtained as white powder, has the molecular formula C₄₈H₆₀O₁₀ as inferred from the (+)-HRESIMS ion at m/z 819.4082 [M + Na]⁺ (calcd 819.4079). A careful analysis of the ¹H and ¹³C NMR data (Table S2) of 3, in combination with NMR data with reported structures, indicated that the chemical structure comprises a modified ergosterol part and a furanone part, which is structurally similar to integrastatin B.[10] Based on the evidence from the ¹H–¹H COSY correlation between H-9 (δ_H 2.09) and H₂-11 (δ_H 1.46, 1.53), and the HMBC correlation between H₃-19 and C-9 (δ_C 42.8), the primary difference between part A of 3 and 1 is the reduction of the C-9/C-11 double bond in 1 into a single bond in 3. With one degree of unsaturation and two oxygen atoms remaining, the HMBC correlation between H-6 and C-3′ confirmed that parts A and B are connected through a 1,4-dioxane ring (Figure S1). In the ROESY spectrum of 3, the correlation between H-6 and H₃-19β indicates that both the C-6–O and C-5–O bonds have an α-orientation (Figure S2). In part B, the two chiral centers are situated at the bridgehead, and their chirality should be concerted. Thus, (3S*,5R*,6S*,9S*,10R*,13R*,14R*,17R*,20R*,24R*,1′R*,9′R*)-3 (3a) and (3S*,5R*,6S*,9S*,10R*,13R*,14R*,17R*,20R*,24R*,1′S*,9′S*)-3 (3b) were both subjected to quantum chemical calculations of chemical shifts and subsequent normal DP4+ probability analysis.[11] The calculated NMR chemical shifts of 3a better matched with their experimental counterparts (Table S13), thereby establishing the relative configuration of 3. Then, the absolute configuration of compound 3 was determined as 3S,5R,6S,9S,10R,13R,14R,17R,20R,24R,1′R,9′R through TDDFT ECD calculations (Figure S7).

Ergopolyketide D (4), obtained as colorless oil, has the molecular formula C₃₈H₅₀O₆ as inferred from the (+)-HRESIMS ion at m/z 603.3688 [M + H]⁺ (calcd 603.3680). A careful analysis of the 1D (Table S2) and 2D (Figure S1) NMR data of 4, in combination with NMR data with reported structures, indicated that the chemical structure comprises a modified ergosterol part, which is identical to that of the part A in 2, and a furanone part, which is structurally similar to penicmariae-crucis D.[12] The HMBC correlation between H-15 (δ_H 5.02) and C-5′ (δ_C 134.7), in combination with the remaining one degree of unsaturation and two oxygen atoms, indicated that the two parts are connected via a 1,4-dioxane ring (Figure S1). In the ROESY spectrum of 4, the correlation between H-15 and H₃-18β (δ_H 0.95) indicated that both the C-14–O and C-15–O bonds have an α-orientation (Figure S2). To determine the relative configuration of 4, two C-8′ epimers, (3S*,9R*,10R*,13R*,14S*,15S*,17R*,20R*,24R*,8′S*)-4 (4a) and (3S*,9R*,10R*,13R*,14S*,15S*,17R*,20R*,24R*,8′R*)-4 (4b) were both subjected to quantum chemical calculations of chemical shifts. However, the calculated chemical shifts of both epimers matched well with their experimental counterparts and were not distinguishable. It can be noticed that the calculated H-9/H₃-9′ interproton distances are 2.59 and 4.57 Å in 4a and 4b, respectively. Considering that the correlation between H-9 and H₃-9′ can be observed in the ROESY spectrum (Figure S2), 4a can be determined as the correct configuration. Subsequently, TDDFT ECD calculation successfully determined the absolute configuration of 4 as 3S,9R,10R,13R,14S,15S,17R,20R,24R,8′S (Figure S9).

Isopenicin D (5) was isolated as a white amorphous powder. The molecular formula of 5 was determined as C₃₂H₄₄O₈ from the HRESIMS ion peak at m/z 579.2938 (m/z 579.2928 calcd. for [M + Na]⁺). A careful analysis of the 1D (Table S3) and 2D NMR data of 5 suggested that its architecture was formed by two parts (A and B) (Figure [3a]). The structure of part A was identical to that of 10-formyl andrastone A.[13] The structure of part B could be determined by the spin systems H₂-1′/H₂-2′ as confirmed by ¹H–¹H COSY spectrum, and the HMBC correlations from H₃-4′ (δ_H 2.09) to C-1′ (δ_C 33.1), C-2′ (δ_C 37.5), and C-3′ (δ_C 206.5). Moreover, the key HMBC correlations from H₂-2′ (δ_H 2.29, 2.57) to C-16, from H₂-1′ (δ_H 1.74, 1.94) to C-16, C-15, C-17, and C-25 (δ_C 20.6) confirmed the connection of parts A and B via the C-16/C-1′ bond (Figure [3a]). The relative configurations of 5 can be determined through analysis of the ROESY spectrum (Figure [3b]) and coupling constants. Moreover, suitable crystals of 5 were obtained by slow solvent evaporation in methanol and then subjected to X-ray diffraction analysis using Cu Kα radiation, which unambiguously determined the absolute configuration of 5 as 3S,5R,8S,9R,10S,13R,14R,16R (Figure [3]) with Flack parameter [0.10(2)] (CCDC 2394745).[14]

Isopenicin E (6) was isolated as a white amorphous powder. The molecular formula of 6 was determined as C₃₂H₄₄O₉ from the HRESIMS ion peak at m/z 571.2904 (m/z 571.2913 calcd. for [M – H]^–). The NMR data of 6 (Table S4) were similar to those of 5, and the obvious difference was that the aldehyde group (C-21, δ_C 204.5) in 5 was oxidized to a carboxyl group (δ_C 178.8) in 6, which could be further verified by HMBC correlations from H₂-1 (δ_H 1.30, 2.36) and H-5 (δ_H 1.54) to C-21 (Figure S1). For configurational determination, most key ROESY correlations observed in 5 could also be found in 6 (Figure S2). However, there was no evidence to assign the orientation of the carboxyl group (C-21). Thus, the theoretical chemical shifts of (3S*,5R*,8S*,9R*,10S*,13R*,14R*,16R*)-6 (6a) and (3S*,5R*,8S*,9R*,10R*,13R*,14R*,16R*)-6 (6b) were calculated and compared to their experimental counterparts. According to the computational results (Table S19), it was concluded that 6a represented the correct configuration of 6. Subsequent TDDFT calculations were carried out and established the absolute configuration of 6 as 3S,5R,8S,9R,10S,13R,14R,16R (Figure [3]).

The cytotoxic effects of isopenicins D (5) and E (6), and peniandrastin D (7) were evaluated against a panel of five human cancer cell lines: HL-60, SMMC-7721, A549, MDA-MB-231, and SW-480, using the MTS assay. Notably, 5 and 7 demonstrated cytotoxic activity against three of these cell lines: HL-60, SMMC-7721, and A549 (Table [1]). Due to insufficient quantities of the compounds after purification, ergopolyketides A–D (1–4) were not subjected to activity screening.

In conclusion, our research has led to the isolation of four unique steroidal adducts and two new meroterpenoids from the co-culture of Penicillium sp. sh18 and Pestalotiopsis sp. HS30. Notably, ergopolyketides A–D (1–4) were biosynthesized through the fusion of ergosterol with polyketides, likely through a Diels–Alder reaction. This reaction selectively occurs at the C-5/C-6 or C-14/C-15 double bonds of the ergosterol framework, leading to the formation of 1,4-dioxane or morpholine ring structures. Among the previously identified ergosterol-based adducts were sirosterol,[15] dehydroazasirosterol,[15] conio-azasterol,[16] S-dehydroazasirosterol,[16] evanthrasterols A and B,[7] and pestauvicomorpholine A.[8] The present study further highlights the potential of plant endophytic fungi as a sustainable resource for obtaining bioactive compounds with diverse chemical structures.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

We thank the Scientific Data Center, Kunming Institute of Botany, CAS for providing computational resources. We thank the Service Center for Bioactivity Screening, Kunming Institute of Botany, CAS for cytotoxicity evaluation.

• References and Notes

• 1 Wang W.-G, Li A, Yan B.-C, Niu S.-B, Tang J.-W, Li X.-N, Du X, Challis GL, Che Y, Sun H.-D, Pu J.-X. J. Nat. Prod. 2016; 79: 149
  CrossrefPubMedSearch in Google Scholar
• 2 Tang J.-W, Kong L.-M, Zu W.-Y, Hu K, Li X.-N, Yan B.-C, Wang W.-G, Sun H.-D, Li Y, Puno P.-T. Org. Lett. 2019; 21: 771
  CrossrefPubMedSearch in Google Scholar
• 3 Su X.-Z, Zhu Y.-Y, Tang J.-W, Hu K, Li X.-N, Sun H.-D, Li Y, Puno P.-T. Sci. China Chem. 2020; 63: 1208
  CrossrefSearch in Google Scholar
• 4 Xia J.-N, Hu K, Su X.-Z, Tang J.-W, Li X.-N, Sun H.-D, Puno P.-T. Fitoterapia 2022; 158: 105160
  CrossrefPubMedSearch in Google Scholar
• 5a Ochi K, Hosaka T. Appl. Microbiol. Biotechnol. 2013; 97: 87
  CrossrefPubMedSearch in Google Scholar
• 5b Knowles SL, Raja HA, Roberts CD, Oberlies NH. Nat. Prod. Rep. 2022; 39: 1557
  CrossrefPubMedSearch in Google Scholar
• 6 Chang J, Ouyang Q, Peng X, Pei J, Zhang L, Gan Y, Ruan H. Bioorg. Chem. 2023; 139: 106745
  CrossrefPubMedSearch in Google Scholar
• 7 Liangsakul J, Srisurichan S, Pornpakakul S. Steroids 2016; 106: 78
  CrossrefPubMedSearch in Google Scholar
• 8 Hou G.-M, Xu X.-M, Wang Q, Li D.-Y, Li Z.-L. Steroids 2018; 138: 43
  CrossrefPubMedSearch in Google Scholar
• 9 Zanardi MM, Marcarino MO, Sarotti AM. Org. Lett. 2020; 22: 52
  CrossrefPubMedSearch in Google Scholar
• 10 Singh SB, Zink DL, Quamina DS, Pelaez F, Teran A, Felock P, Hazuda DJ. Tetrahedron Lett. 2002; 43: 2351
  CrossrefSearch in Google Scholar
• 11 Grimblat N, Zanardi MM, Sarotti AM. J. Org. Chem. 2015; 80: 12526
  CrossrefPubMedSearch in Google Scholar
• 12 Wang J.-T, Ma Z.-H, Wang G.-K, Xu F.-Q, Yu Y, Wang G.-G, Liu J.-S. Chin. J. Org. Chem. 2019; 39: 1136
  CrossrefSearch in Google Scholar
• 13 Qin Y.-Y, Huang X.-S, Liu X.-B, Mo T.-X, Xu Z.-L, Li B.-C, Qin X.-Y, Li J, Schӓberle TF, Yang R.-Y. Nat. Prod. Res. 2022; 36: 3262
  PubMedSearch in Google Scholar
• 14 CCDC 2394745 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures
• 15 Ayer WA, Ma Y.-T. Can. J. Chem. 1992; 70: 1905
  CrossrefSearch in Google Scholar
• 16 Elsebai MF, Ghabbour HA, Mehiri M. Molecules 2016; 21: 178
  CrossrefPubMedSearch in Google Scholar

Corresponding Author

Publication History

Received: 04 November 2024

Accepted after revision: 27 January 2025

Accepted Manuscript online:
27 January 2025

Article published online:
14 March 2025

© 2025. Thieme. All rights reserved

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• References and Notes

• 1 Wang W.-G, Li A, Yan B.-C, Niu S.-B, Tang J.-W, Li X.-N, Du X, Challis GL, Che Y, Sun H.-D, Pu J.-X. J. Nat. Prod. 2016; 79: 149
  CrossrefPubMedSearch in Google Scholar
• 2 Tang J.-W, Kong L.-M, Zu W.-Y, Hu K, Li X.-N, Yan B.-C, Wang W.-G, Sun H.-D, Li Y, Puno P.-T. Org. Lett. 2019; 21: 771
  CrossrefPubMedSearch in Google Scholar
• 3 Su X.-Z, Zhu Y.-Y, Tang J.-W, Hu K, Li X.-N, Sun H.-D, Li Y, Puno P.-T. Sci. China Chem. 2020; 63: 1208
  CrossrefSearch in Google Scholar
• 4 Xia J.-N, Hu K, Su X.-Z, Tang J.-W, Li X.-N, Sun H.-D, Puno P.-T. Fitoterapia 2022; 158: 105160
  CrossrefPubMedSearch in Google Scholar
• 5a Ochi K, Hosaka T. Appl. Microbiol. Biotechnol. 2013; 97: 87
  CrossrefPubMedSearch in Google Scholar
• 5b Knowles SL, Raja HA, Roberts CD, Oberlies NH. Nat. Prod. Rep. 2022; 39: 1557
  CrossrefPubMedSearch in Google Scholar
• 6 Chang J, Ouyang Q, Peng X, Pei J, Zhang L, Gan Y, Ruan H. Bioorg. Chem. 2023; 139: 106745
  CrossrefPubMedSearch in Google Scholar
• 7 Liangsakul J, Srisurichan S, Pornpakakul S. Steroids 2016; 106: 78
  CrossrefPubMedSearch in Google Scholar
• 8 Hou G.-M, Xu X.-M, Wang Q, Li D.-Y, Li Z.-L. Steroids 2018; 138: 43
  CrossrefPubMedSearch in Google Scholar
• 9 Zanardi MM, Marcarino MO, Sarotti AM. Org. Lett. 2020; 22: 52
  CrossrefPubMedSearch in Google Scholar
• 10 Singh SB, Zink DL, Quamina DS, Pelaez F, Teran A, Felock P, Hazuda DJ. Tetrahedron Lett. 2002; 43: 2351
  CrossrefSearch in Google Scholar
• 11 Grimblat N, Zanardi MM, Sarotti AM. J. Org. Chem. 2015; 80: 12526
  CrossrefPubMedSearch in Google Scholar
• 12 Wang J.-T, Ma Z.-H, Wang G.-K, Xu F.-Q, Yu Y, Wang G.-G, Liu J.-S. Chin. J. Org. Chem. 2019; 39: 1136
  CrossrefSearch in Google Scholar
• 13 Qin Y.-Y, Huang X.-S, Liu X.-B, Mo T.-X, Xu Z.-L, Li B.-C, Qin X.-Y, Li J, Schӓberle TF, Yang R.-Y. Nat. Prod. Res. 2022; 36: 3262
  PubMedSearch in Google Scholar
• 14 CCDC 2394745 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures
• 15 Ayer WA, Ma Y.-T. Can. J. Chem. 1992; 70: 1905
  CrossrefSearch in Google Scholar
• 16 Elsebai MF, Ghabbour HA, Mehiri M. Molecules 2016; 21: 178
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material