Niduterpenoids A and B: Two Sesterterpenoids with a Highly

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Niduterpenoids A and B: Two Sesterterpenoids with a Highly Congested Hexacyclic 5/5/5/5/3/5 Ring System from the Fungus Aspergillus nidulans Qin Li,†,⊥ Chunmei Chen,†,⊥ Mengsha Wei,† Chong Dai,† Li Cheng,†,‡ Jiafeng Tao,† Xiao-Nian Li,§ Jianping Wang,† Weiguang Sun,*,† Hucheng Zhu,*,† and Yonghui Zhang*,†

Org. Lett. Downloaded from pubs.acs.org by EAST CAROLINA UNIV on 03/14/19. For personal use only.

†

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ College of Pharmacy, Hubei University of Medicine, Shiyan 442000, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China S Supporting Information *

ABSTRACT: Niduterpenoids A (1) and B (2), two sesterterpenoids with a highly congested hexacyclic 5/5/5/5/3/5 carbon skeleton but no unsaturated functional group, were isolated from Aspergillus nidulans. Their structures were determined by a combination of spectroscopic data and single-crystal X-ray diffraction analyses. Compounds 1 and 2 present the first examples of sesterterpenoids with a hexacyclic carbon ring system. Compound 1 showed no cytotoxicity but abolished 17-estradiol-induced cell proliferation (IC50 = 11.42 ± 0.85 μM).

T

erpenoids are a diverse class of natural products that harbor untapped structural diversity and important biofunctions, mainly from plants and microbes.1−3 Sesterterpenoids are a special subclass of terpenoids that are primarily biosynthesized via cyclization of geranylfarnesyl pyrophosphate (GFPP) with the catalysis of terpene cyclases to construct their skeletons of one to five carbon rings.4,5 Sesterterpenoids are of great interest to scientists because they are architecturally intriguing and exhibit a wide array of biological activities, such as cytotoxic activities, enzymatic inhibitory functions, antimicrobial effects, and defense functions.6−9 Recently, a number of excellent studies on their biosynthetic pathways/mechanisms have made this compound class a hot topic in various scientific communities.10−14 Aspergillus nidulans is known to produce meroterpenoids,15 polyketides,16−18 and alkaloids.19−21 As a continuation of our interest in the discovery of bioactive metabolites from fungi,22,23 the secondary metabolites of A. nidulans were recently investigated, and two sesterterpenoids, niduterpenoids A (1) and B (2) (Figure 1), featuring highly congested hexacyclic 5/5/ 5/5/3/5 carbon skeletons, were isolated and characterized. Notably, niduterpenoids A (1) and B (2) are the first examples of sesterterpenoids with a hexacyclic carbon ring system. Herein, we present their fermentation, isolation, structure elucidation, and bioactivity evaluation as well as a plausible pathway of 1 and 2. The fungus A. nidulans was fermented on a solid-substrate medium (rice 30 kg) for 21 days at 28 °C. Then the mycelia and © XXXX American Chemical Society

Figure 1. Structures of 1 and 2.

rice medium were extracted with ethanol, and the solvent was removed in vacuo to yield 20 g of organic residue. The crude extract was partitioned with EtOAc and fractionated by column chromatography (CC) over silica gel. The crude extract was eluted with a gradient of petroleum ether (60−90 °C)/EtOAc from 10:1 to 1:3 to obtain six fractions (fractions 1−6). Fraction Received: February 15, 2019

A

DOI: 10.1021/acs.orglett.9b00581 Org. Lett. XXXX, XXX, XXX−XXX

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unsaturation, revealed that 1 should be a sesterterpenoid with a hexacyclic ring system without a chromophore group. Further analyses of the 1H−1H COSY and HMBC spectra led to the elucidation of the planar structure of 1. The 1H−1H COSY spectrum (Figure 2) indicated the presence of four

4 (400 mg) was further refined by a Sephadex LH-20 CC and isostatically eluted with MeOH to obtain fractions 4.1−4.3. Additionally, fraction 4.2 (100 mg) was further separated via semipreparative HPLC (MeOH−H2O, 55:45, v/v) to produce compounds 1 (29.0 mg) and 2 (1.8 mg). Niduterpenoid A (1) was obtained as colorless crystals. The molecular formula of C25H40O4, with six degrees of unsaturation, was deduced by the presence of a [M + Na]+ ion peak at m/z 427.2853 in the HRESIMS spectrum. The IR spectrum of 1 displays an absorption band at 3413 cm−1, suggesting the presence of hydroxy groups. The 1H NMR spectroscopic data (Table 1) of 1 exhibited signals for five methyls at δH 0.91 (d, J = Table 1. 1H (400 MHz) and 13C NMR (100 MHz) Data of 1 and 2 in CD3OD (J, Hz) 1 δH

no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

3.78 d (4.3) 1.66 m 2.12 dd (7.5, 1.9) 2.05 m 1.90 m 2.06 m 1.76 m 1.48 m 1.74 m 1.51 m 1.74 m 1.58−1.53 m 1.55 m 1.21 m 1.32 m 1.85 m 0.82 s 1.16 s 3.54 d (11.0) 3.31a 1.33 s 1.26 s 0.97 s 0.91 d (6.8)

2 δC 42.0 78.7 34.6 50.9 36.2 33.7 57.8 64.9 29.2 40.4 79.5 69.7 28.3 37.0 57.6 50.8 49.5 34.2 10.1 74.7 70.7 25.5 28.7 23.7 13.2

δH 3.76 d (4.9) 1.69 m 1.59 m 1.96 d (7.9) 1.88 m 2.05 m 1.81 m 1.53 m 1.74 m 1.56 m 1.66 m 1.79 m 1.59 m 3.85 dd (9.0, 5.5)

δC

independent spin systems: H-2/H2-3/H-4, H2-6/H-7/H-17/H16/H3-25, H2-9/H2-10, and H-12/H2-13/H2-14. The HMBC correlations (Figure 2) from H3-23 to C-10, C-11, and C-12, from H3-24 to C-8, C-14, C-15, and C-16, and from H2-9 to C-7, C-8, C-12, and C-15, along with the spin systems of H2-9/H2-10, H-12/H2-13/H2-14, and H-16/H-17/H-7 established the structure of rings A−C that are a cyclopenta[c]pentalene skeleton. The structure of the D ring was deduced by HMBC correlations from H2-6 to C-5, C-17, and C-18 and H-18 to C17. Furthermore, the HMBC correlations from H2-6 to C-4 and H3-19 to C-1, C-2, and C-5 were detected, which revealed a spiro[4.4]nonane structure composed by rings D and F. Considering the degrees of unsaturation and the quaternary carbon nature of C-1, the linkage of C-1 and C-18 via a single carbon−carbon bond was proposed, which led to the presence of a cyclopropane ring (ring E). This speculation was supported by the HMBC correlations from H-17 to C-1 and H3-19 to C-18. Finally, the HMBC correlations from H3-22 to C-4, C-20, and C21 indicated C-20 should be linked to C-4. Hence, the planar structure of Niduterpenoid A (1) with a novel hexacyclic 5/5/5/ 5/3/5 carbon skeleton was elucidated as shown in Figure 2. The relative configuration of 1 was determined by analysis of the NOESY spectrum (Figure 3). NOESY correlations of H3-

42.3 78.6 35.2 54.6 36.0 33.7 57.6 64.8 29.7 40.8 79.3 63.5 35.8 76.8

1.35 s

58.1 51.6 49.6 34.6 10.2 72.8 30.3

1.21 s 1.26 s 0.95 s 0.99 d (7.0)

31.2 28.8 18.5 13.1

1.38 m 1.78 m 0.82 s 1.16 s

Figure 2. 1H−1H COSY and key HMBC correlations of 1.

a

Overlapped with the signal of CD3OD.

6.8 Hz, H3-25), 0.97 (s, H3-24), 1.16 (s, H3-19), 1.26 (s, H3-23), 1.33 (s, H3-22); one oxygenated methylene at δH 3.31 (overlapped, H-21) and 3.54 (d, J = 11.0 Hz, H-21); one oxygenated methine at δH 3.78 (d, J = 4.3 Hz, H-2); and other aliphatic protons ranging from δH 1.21 to 2.12. The 13C NMR and DEPT spectra (Table 1) revealed 25 carbon resonances including five methyls, seven methylenes (including an oxygenated carbon at δC 70.7/C-21), seven methines (including an oxygenated carbon at δC 78.7/C-2), and six nonprotonated carbons (including two oxygenated carbons at δC 74.7/C-20 and 79.5/C-11). The above analyses, along with the degrees of

Figure 3. Key NOESY correlations of 1.

23/H-12, H-12/H-7, and H3-23/H-7 revealed they are cofacial and were assigned as α-oriented, which suggested a cis-fusion pattern for rings A and B. Moreover, correlations from H3-24 to H-9 and H-16 indicated that rings B and C were also cis-fused, and H3-24 and H-16 should be β-oriented. In addition, NOESY interactions of H-7/H-17 and H-7/H3-19 suggested their αorientation, while correlations of H-16/H-18, H-18/H-2, and B

DOI: 10.1021/acs.orglett.9b00581 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters H-18/H-4 revealed they were β-oriented. Therefore, the relative configuration of 1, except for C-20, was determined. It is notable that all the rings in compound 1 are cis-fused, including A/B, B/ C, C/D, D/E, and E/F, which is unusual in polycyclic terpenoids. After repeated recrystallization by various solvent systems, 1 furnished a high-quality crystal in methanol at room temperature. Therefore, 1 was successfully subjected to single-crystal Xray diffraction using Cu Kα radiation (Figure 4) with a Flack parameter of 0.06(5) (CCDC 1874514), which confirmed the skeleton and absolute configuration of 1.

Scheme 1. Proposed Biogenetic Pathways of 1 and 2

adjuvant and metastatic settings,25,26 and combined the “probability value”, we assumed ERα was the most likely target of 1 and 2. Therefore, further molecular docking was performed by Sybyl 2.0 to view the potential interactions. As presented in Figure 5, 1 and 2 fit well with the ligand-binding site of Erα and

Figure 4. X-ray crystallographic structure of 1.

Niduterpenoid B (2) was isolated as a colorless powder and assigned a molecular formula of C25H40O4 based on the HRESIMS spectrum with an ion peak at m/z 427.2813 ([M + Na]+, calcd for 427.2824). The 1H and 13C NMR data of 2 closely resembled the data of 1 (Table 1), which revealed that they shared the same carbon skeleton. Nevertheless, detailed comparison of their 1D NMR data disclosed the main differences were the presence of an additional methyl singlet (δH 1.35; δC 30.3) and an oxygenated methine (δH 3.85, dd J = 9.0, 5.5 Hz; δC 76.8), which replaced the hydroxymethy group of C-21 and a methylene, respectively. HMBC correlations from H3-22 to C-4, C-20, and C-21, along with the 1H−1H COSY correlations of H-12/H2-13/H-14 and HMBC interactions from H3-24 to C-14, C-15, and C-16 (Figure S1) confirmed the absence of the hydroxy at C-21 and the presence of an additional hydroxy at C-14. Furthermore, the relative configuration of 2 was determined by NOESY experiments (Figure S1). The α-orientation of H-14 was elucidated by the NOESY interaction between H-14 and H17. On the basis of the closely related optical rotation values of 2 ([α]25D +16 (c 0.1, MeOH)) and 1 ([α]25D +29 (c 0.1, MeOH)), the absolute configuration of 2 was established. To the best of our knowledge, niduterpenoids A and B are the first examples of sesterterpenoids with a hexacyclic carbon ring system containing an unusual cyclopropane ring. A hypothetical biosynthetic pathway for 1 and 2 was proposed, as shown in Scheme 1.11,12 Starting from GFPP, a series of cyclization and Wagner−Meerwein hydride and alkyl shift reactions occurred, leading to the formation of the key intermediate x with a hexacyclic 5/5/5/5/3/5 ring system. Afterward, additional oxidation reactions led to the formation of 1 and 2. To determine potential targets of 1 and 2, we utilized the Swiss Target Prediction tool,24 a web server for the target prediction of bioactive small molecules, to obtain the top 15 potential targets, mainly including transcription factors (estrogen, androgen, oxysterols, and different types of carbonic anhydrase). Given that estrogen receptor α (ERα) was considered a key to the management of breast cancer both in

Figure 5. Low-energy binding conformations the complex between 1 (A), 2 (B), and the LBD of ERα by virtual docking.

partially occupy the whole pocket of lasofoxifene, a well-known potent estrogen receptor modulator. Moreover, several key hydrogen bonds are also observed between 1 and 2 with Met295, Glu353, and Arg346. In addition, many hydrophobic amino acids, including Phe356, Leu298, Met294, Phe377, and Leu354, form a highly hydrophobic envelope, which could form clear hydrophobic interactions with 1 and 2. The above interactions indicated that 1 and 2 are potential ERα inhibitors. To verify the predicted activity in vitro, we chose human breast tumor cell line MCF-7 for further exploration. As shown in Figure 6, 1 showed no obvious cytotoxicity even at the concentration of 80 μM; however, the addition of 1 abolished the 17-estradiol induced cell proliferation in a dose-dependent manner (IC50 = 11.42 ± 0.85 μM), which indicated 1 was a potential ERα antagonist deserved further investegation.27

Figure 6. (A) Treatment with 1 showed no obvious cytotoxicity against the MCF-7. (B) Dose-dependent inhibition of 1 combination with the 100 pM ES is shown. MCF-7 proliferation was stimulated by 100 pM 17-estradiol (ES). C

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(3) Wang, J. P.; Yu, J.; Shu, Y.; Shi, Y. X.; Luo, P.; Cai, L.; Ding, Z. T. Org. Lett. 2018, 20, 5853−5856. (4) Wang, L.; Yang, B.; Lin, X. P.; Zhou, X. F.; Liu, Y. Nat. Prod. Rep. 2013, 30, 455−473. (5) Liu, Z.; Chen, Y.; Chen, S.; Liu, Y.; Lu, Y.; Chen, D.; Lin, Y.; Huang, X.; She, Z. Org. Lett. 2016, 18, 1406−1409. (6) Zhu, T.; Lu, Z.; Fan, J.; Wang, L.; Zhu, G.; Wang, Y.; Li, X.; Hong, K.; Piyachaturawat, P.; Chairoungdua, A.; Zhu, W. J. Nat. Prod. 2018, 81, 2−9. (7) Huang, H.; Huang, H.; Li, H.; Sun, X.; Huang, H.; Lu, Y.; Lin, Y.; Long, Y.; She, Z. Org. Lett. 2013, 15, 721−723. (8) Li, C.-H.; Jing, S.-X.; Luo, S.-H.; Shi, W.; Hua, J.; Liu, Y.; Li, X.-N.; Schneider, B.; Gershenzon, J.; Li, S.-H. Org. Lett. 2013, 15, 1694−1697. (9) Wang, Q. X.; Yang, J. L.; Qi, Q. Y.; Bao, L.; Yang, X. L.; Liu, M. M.; Huang, P.; Zhang, L. X.; Chen, J. L.; Cai, L.; Liu, H. W. Bioorg. Med. Chem. Lett. 2013, 23, 3547−3550. (10) Ye, Y.; Minami, A.; Mandi, A.; Liu, C.; Taniguchi, T.; Kuzuyama, T.; Monde, K.; Gomi, K.; Oikawa, H. J. Am. Chem. Soc. 2015, 137, 11846−11853. (11) Okada, M.; Matsuda, Y.; Mitsuhashi, T.; Hoshino, S.; Mori, T.; Nakagawa, K.; Quan, Z.; Qin, B.; Zhang, H.; Hayashi, F.; Kawaide, H.; Abe, I. J. Am. Chem. Soc. 2016, 138, 10011−10018. (12) Sato, H.; Mitsuhashi, T.; Yamazaki, M.; Abe, I.; Uchiyama, M. Angew. Chem., Int. Ed. 2018, 57, 14752−14757. (13) Qin, B.; Matsuda, Y.; Mori, T.; Okada, M.; Quan, Z.; Mitsuhashi, T.; Wakimoto, T.; Abe, I. Angew. Chem., Int. Ed. 2016, 55, 1658−1661. (14) Huang, A. C.; Kautsar, S. A.; Hong, Y. J.; Medema, M. H.; Bond, A. D.; Tantillo, D. J.; Osbourn, A. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 6005−6014. (15) Lo, H. C.; Entwistle, R.; Guo, C. J.; Ahuja, M.; Szewczyk, E.; Hung, J. H.; Chiang, Y. M.; Oakley, B. R.; Wang, C. C. J. Am. Chem. Soc. 2012, 134, 4709−4720. (16) Ahuja, M.; Chiang, Y.-M.; Chang, S.-L.; Praseuth, M. B.; Entwistle, R.; Sanchez, J. F.; Lo, H.-C.; Yeh, H.-H.; Oakley, B. R.; Wang, C. C. C. J. Am. Chem. Soc. 2012, 134, 8212−8221. (17) Nielsen, M. L.; Nielsen, J. B.; Rank, C.; Klejnstrup, M. L.; Holm, D. K.; Brogaard, K. H.; Hansen, B. G.; Frisvad, J. C.; Larsen, T. O.; Mortensen, U. H. FEMS Microbiol. Lett. 2011, 321, 157−166. (18) Lin, H.; Lyu, H.; Zhou, S.; Yu, J.; Keller, N. P.; Chen, L.; Yin, W. B. Org. Biomol. Chem. 2018, 16, 4973−4976. (19) Scherlach, K.; Schuemann, J.; Dahse, H. M.; Hertweck, C. J. Antibiot. 2010, 63, 375−377. (20) An, C. Y.; Li, X. M.; Luo, H.; Li, C. S.; Wang, M. H.; Xu, G. M.; Wang, B. G. J. Nat. Prod. 2013, 76, 1896−1901. (21) Scherlach, K.; Hertweck, C. Org. Biomol. Chem. 2006, 4, 3517− 3520. (22) Zang, Y.; Genta-Jouve, G.; Zheng, Y.; Zhang, Q.; Chen, C.; Zhou, Q.; Wang, J.; Zhu, H.; Zhang, Y. Org. Lett. 2018, 20, 2046−2050. (23) Wang, W.; Zeng, F.; Bie, Q.; Dai, C.; Chen, C.; Tong, Q.; Liu, J.; Wang, J.; Zhou, Y.; Zhu, H.; Zhang, Y. Org. Lett. 2018, 20, 6817−6821. (24) Gfeller, D.; Grosdidier, A.; Wirth, M.; Daina, A.; Michielin, O.; Zoete, V. Nucleic Acids Res. 2014, 42, 32−38. (25) Early Breast Cancer Trialists’ Collaborative Group.Lancet 2011, 378, 771−784. (26) Jordan, V. C. Nat. Rev. Drug Discovery 2003, 2, 205−213. (27) De Savi, C.; Bradbury, R. H.; Rabow, A. A.; Norman, R. A.; de Almeida, C.; Andrews, D. M.; Ballard, P.; Buttar, D.; Callis, R. J.; Currie, G. S.; Curwen, J. O.; Davies, C. D.; Donald, C. S.; Feron, L. J.; Gingell, H.; Glossop, S. C.; Hayter, B. R.; Hussain, S.; Karoutchi, G.; Lamont, S. G.; MacFaul, P.; Moss, T. A.; Pearson, S. E.; Tonge, M.; Walker, G. E.; Weir, H. M.; Wilson, Z. J. Med. Chem. 2015, 58, 8128−8140.

In summary, two novel sesterterpenoids, niduterpenoids A (1) and B (2), were isolated from a solid cultivation of the fungus A. nidulans. To the best of our knowledge, compounds 1 and 2 are the first examples of sesterterpenoids with an unprecedented 5/5/5/5/3/5 hexacyclic carbon skeleton, and the involvement of a cyclopropane ring makes them uncommon. Compounds 1 and 2 are ERα inhibitors, and compound 1 abolished 17-estradiol induced human breast tumor cell proliferation in a dose-dependent manner (IC50 = 11.42 ± 0.85 μM). The discovery of compounds 1 and 2 not only expands the sesterterpenoid skeletons with hexacyclic ring systems but also provides promising target molecules for further research.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00581. Experimental section, 1D and 2D NMR, MS, and IR spectra for compounds 1 and 2, and X-ray crystallographic data of 1 (PDF) Accession Codes

CCDC 1874514 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yonghui Zhang: 0000-0002-7222-2142 Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (T2016088); the National Natural Science Foundation for Distinguished Young Scholars (81725021); the Innovative Research Groups of the National Natural Science Foundation of China (81721005); the Natural Science Foundation of Hubei Province of China (2018CFA027); the Academic Frontier Youth Team of HUST; and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST). We thank the Analytical and Testing Center at HUST for IR, UV, and ECD analyses.

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REFERENCES

(1) Huang, A. C.; Hong, Y. J.; Bond, A. D.; Tantillo, D. J.; Osbourn. Angew. Chem., Int. Ed. 2018, 57, 1291−1295. (2) Qiao, Y.; Xu, Q.; Hu, Z.; Li, X. N.; Xiang, M.; Liu, J.; Huang, J.; Zhu, H.; Wang, J.; Luo, Z.; Xue, Y.; Zhang, Y. J. Nat. Prod. 2016, 79, 3134−3142. D

DOI: 10.1021/acs.orglett.9b00581 Org. Lett. XXXX, XXX, XXX−XXX