Variecolortins A–C, Three Pairs of Spirocyclic Diketopiperazine

Letter Cite This: Org. Lett. 2018, 20, 4593−4596

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Variecolortins A−C, Three Pairs of Spirocyclic Diketopiperazine Enantiomers from the Marine-Derived Fungus Eurotium sp. SCSIO F452 Weimao Zhong,†,‡ Junfeng Wang,† Xiaoyi Wei,§ Yuchan Chen,∥ Tingdan Fu,# Yao Xiang,†,‡ Xinan Huang,# Xinpeng Tian,† Zhihui Xiao,† Weimin Zhang,∥ Si Zhang,† Lijuan Long,*,† and Fazuo Wang*,† Downloaded via DURHAM UNIV on August 3, 2018 at 19:42:41 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China § Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China ∥ State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, 100 Central Xianlie Road, Guangzhou 510070, China # Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510400, China ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China S Supporting Information *

ABSTRACT: Three pairs of spirocyclic diketopiperazine enantiomers, variecolortins A−C (1−3), were isolated from marine-derived fungus Eurotium sp. SCSIO F452. Compound 1 possesses an unprecedented highly functionalized secoanthronopyranoid carbon skeleton featuring a 2-oxa-7azabicyclo[3.2.1]octane core. Compounds 2 and 3 represent rare examples of a 6/6/6/6 tetracyclic cyclohexene−anthrone carbon scaffold. Their structures were determined by spectroscopic analyses, X-ray diffraction, and ECD calculations. Their enantiomers exhibited different antioxidative and cytotoxic activities, and their modes of action were investigated.

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n recent decades, marine-derived fungi have been proven to be a prolific source of structurally diverse and biologically active natural products.1,2 More than 3000 secondary metabolites have been reported from marine fungi, including polyketides, alkaloids, meroterpenoids, and cyclic peptides. These compounds exhibit various biological activities such as antibacterial, antitumor, antiviral, and antiinflammatory activities.1,3−5 In the course of our continuing investigation on marine fungi,6,7 a further chemical investigation was performed on Eurotium sp. SCSIO F452, which was isolated from a South China Sea sediment sample and found to produce diketopiperazine alkaloids and polyketides.8,9 Careful chemical examination led to the isolation and characterization of three pairs of new racemic spirocyclic diketopiperazine enantiomers, variecolortins A−C (1−3) (Figure 1). Structurally, compound 1 possesses an unprecedented highly functionalized seco-anthronopyranoid carbon skeleton featuring a 2oxa-7-azabicyclo[3.2.1]octane moiety. Compounds 2 and 3 contain a rare 6/6/6/6 tetracyclic cyclohexene−anthrone moiety rather than the pyrano−anthrone unit found in variecolortides A−C, which occurred as racemic forms and © 2018 American Chemical Society

Figure 1. Structures of compounds (±)-1, (±)-2, and (±)-3.

exhibited radical-scavenging, cytotoxicity, and caspase-3 inhibitory activities.10,11 Herein, we report the isolation, structure elucidation, plausible biosynthetic pathway, antioxReceived: June 17, 2018 Published: July 16, 2018 4593

DOI: 10.1021/acs.orglett.8b01880 Org. Lett. 2018, 20, 4593−4596

Letter

Organic Letters

HMBC correlations from H2-20 to C-21a (δC 123.8), C-32 (δC 79.0), from H-21 to C-12, C-31, and from H2-27 to C-21 (δC 40.4) implied that 1c was connected to C-20 of 1a and C-32 of 1b by C-21. Hitherto structure moieties 1a, 1b, and 1c had occupied 20 degrees of unsaturation, indicating the other bicyclic units in 1. Through detailed analysis, there are only two possible connecting ways to form the remaining bicyclic units in 1: (i) N-11 of 1a to C-32 of 1b, and C-12−O of 1a to C-22 of 1c; (ii) N-11 of 1a to C-22 of 1c, and C-12−O of 1a to C-32 of 1b. However, the 2D NMR spectra did not provide sufficient information to elucidate the unambiguous connecting pattern. Fortunately, a single crystal of 1 suitable for X- ray diffraction was obtained (CCDC 1849647, Figure 2), which not only confirmed the planar structure of 1 but also determined its relative configurations as (12R*,21S*,32R*) unambiguously. Additionally, the crystals of 1 had a P21/n space group, indicating its racemic nature, which was also supported by the lack of optical activity. Subsequently, 1 was successfully separated via chiral HPLC to two optically pure enantiomers: (+)- and (−)-1, respectively (Figure S2). Furthermore, the calculated ECD spectrum for (12R,21S,32R)-1 agreed well with that of measured for (+)-1 (Figure 3). Thus, the absolute configurations of (±)-1 were unambiguously assigned.

idative and cytotoxic bioactivities, and molecular dockings of 1−3. Compound 1 was isolated as a yellow solid. Its molecular formula C36H33N3O9 was determined by HRESIMS at m/z 674.2113 [M + Na]+ (calcd for 674.2109), corresponding to an index of hydrogen deficiency of 22. Its IR spectrum suggested the presence of hydroxyl and amine groups (3503, 3431, 3395 cm−1) and carbonyl functionalities (1705, 1668, 1610 cm−1). The 1H NMR (Table S1) spectrum of 1 recorded in CD3COCD3 showed three tertiary methyls at δH 1.57 (s), 1.59 (s), 2.32 (s), two methoxyls at δH 3.89 (s), 3.95 (s), ten olefinic protons ranging from δH 5.10 to 7.46, and three exchangeable protons at δH 9.07 (br s), 10.39 (br s), 11.10 (br s). The 13C NMR and DEPT (Table S1) revealed the presence of 36 carbon resonances, including three methyls, two methoxyls, three methylenes (one olefinic carbon), nine methines (eight olefinic carbons), 19 nonprotonated carbons (one hemiaminal carbon, one nitrogenated carbon, five carbonyls, 11 olefinic carbons, and one quaternary carbon). By the HSQC spectrum, all proton resonances were unambiguously assigned to their respective carbons, except for the exchangeable protons. Detailed analysis of its 1D and 2D NMR (Figure 2) allowed for the assignment of three partial structures 1a, 1b, and 1c (Figure 2) and their connectivity.

Figure 3. Comparison between calculated (M06-2X/def2-SVP/ IEFPCM) and experimental ECD spectra of 1 in MeCN. Figure 2. Key 2D NMR correlations and X-ray structure of 1.

Compound 2 was isolated as a yellow oil. Its molecular formula was determined as C36H33N3O7 by the positive HRESIMS (m/z 642.2211 [M + Na]+, calcd for 642.2210), indicating 22 degrees of unsaturation. Careful analysis of its 1D (Table S1) and 2D NMR indicated that two partial structures 2a and 2b existed (Figure S1) in 2, which highly resembled that of variecolortide B.10 However, detailed analysis revealed that the double bond (δC 126.1 and 119.7) conjugating to the anthraquinone and the spirocyclic N, O- hemiaminal carbon (δC 84.1) in variecolortide B were transformed into two contiguous methylenes (δC 27.9 and 35.3) and two nonprotonated carbon resonances (δC 61.5 and 66.1) in 2. These differences allowed us to postulate that the pyrano−anthrone unit in variecolortide B was rearranged to a cyclohexene− anthrone moiety in 2. HRESIMS data combined with 1H−1H COSY cross peak of H2-20/H2-21, and HMBC correlations from H-20a (δH 3.12, td, J = 13.5, 2.9 Hz) and H-20b (δH 2.46, dt, J = 13.5, 3.3 Hz) to C-13 (δC 168.9), C-22 (δC 66.1), and C-23 (δC 126.0), from H-21a (δH 2.78, dt, J = 13.9, 3.3 Hz) and H-21b (δH 2.59, td, J = 13.9, 2.9 Hz) to C-12 (δC 61.5), C22a (δC 144.0), and C-31a (δC 150.6), from NH-11 (δH 8.01, s) to C-23, from NH-14 (δH 8.25, s) to C-12, and from OH-22 (δH 5.09, s) to C-21 (δC 27.9), C-22, C-22a, and C-31a could

The 1D NMR data of partial structure 1a showed high similarities with neoechinulin B,12 except that the two olefinic carbons (C-12 and C-20) in neoechinulin B were replaced by an oxygenated nonprotonated carbon (δC 90.9) and one methylene (δC 31.2) in 1a of 1. This assignment was verified by the key HMBC correlations from NH-14 (δH 9.07, br s) to C-12 (δC 90.9), from H-20a (δH 2.53, dd, J = 12.8, 4.5 Hz) and H-20b (δH 2.36, d, J = 12.8 Hz) to C-12, C-13 (δC 160.9). Partial structure 1b was elucidated to be a 2-methoxycyclohex-2-ene-1,4-dione moiety based on the HMBC correlations from H-27a (δH 4.35, d, J = 16.6 Hz) and H-27b (δH 2.49, dd, J = 16.6, 1.1 Hz) to C-28 (δC 193.5), C-29 (δC 114.7), and C31 (δC 188.4), from H-29 (δH 6.34, s) to C-27 (δC 44.4), and C-31, from OMe-30 (δH 3.95, s) to C-30 (δC 162.3). HMBC correlations from H-24 (δH 6.92, s) to C-22 (δC 143.5), C-25a (δC 109.8), and C-26 (δC 171.5), from H-21 (δH 5.04, d, J = 4.5 Hz) to C-22, and C-25a, from H3-33 (δH 2.32, s) to C-22, C-24 (δC 121.1), from OH-25 (δH 11.10, s) to C-24, C-25a, and from OMe-26 (δH 3.89, s) to C-26 indicated that 1c possessed a pentasubstituted benzene ring moiety. Furthermore, 1H−1H COSY cross peak of H2-20/H-21, and key 4594

DOI: 10.1021/acs.orglett.8b01880 Org. Lett. 2018, 20, 4593−4596

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Organic Letters confirm the existence of a cyclohexene ring rather than a pyranoid ring, which was fused to the anthraquinone unit and connected to the diketopiperazine ring in a spirocyclic manner at C-12 in 2. In addition, a methoxyl group located at C-30 (δC 168.0) was elucidated by the HMBC correlation. The relative configuration of 2 was determined by NOESY (Figure S1) experiments. The diagnostic NOE correlations between NH11 and H-21b, OH-22 and H-21a led to their assignment as αand β-orientation, respectively. Additionally, the geometry of the Δ8 double bond was elucidated to be Z configuration by the downfield shift of H-8 (δH 7.20, s) due to the deshielding effect of the carbonyl group on the β-vinyl proton, which was also coincident with the lack of NOE effect between H-8 and H-14.10 Thus, the structure of 2 (Figure 1) was completed. Compound 3 was obtained as a yellow oil. It had the molecular formula C36H33N3O7 as established by HRESIMS at m/z 642.2185 [M + Na]+ (calcd for 642.2211), which was identical to that of 2. The 1H and 13C NMR spectra (Table S1) of 3 were strikingly similar to those of 2. The main difference between 3 and 2 was that the cyclohexene ring linking the two fragments 3a and 3b (Figure S1) was located at the “up right” part in 3, rather than the “up left” part in 2. This assignment could be confirmed by the key HMBC correlations from NH- 11 (δH 7.71, s) to C-12 (δC 59.5), C-31 (δC 119.1), from H-20a (δH 3.12, td, J = 13.5, 3.0 Hz) and H-20b (δH 2.36, dt, J = 13.5, 3.2 Hz) to C-12, C-22 (δC 65.5), and C-31, from H-21a (δH 2.79, overlap) and H-21b (δH 2.58, td, J = 14.1, 3.0 Hz) to C-12, C-22, and C-31a (δC 150.0), and from OH-22 (4.96, br s) to C-21 and C-31a. The relative configuration of 3 was determined by NOESY (Figure S1) correlations of NH-11 with H-21b as α-orientation and OH-22 with H-21a as βorientation, and by the downfield shift of H-8 (δH 7.07, s) of the Z configuration of the Δ8 double bond. Therefore, the gross structure of 3 was established as shown in Figure 1. Due to the baseline ECD curves and barely measurable specific rotation values of 2 and 3, they were both presumed to be racemic mixtures. Their enantiomers were separated by HPLC using a chiral column (Figures S3 and S4). The absolute configurations of individual (+)-2, (−)-2, (+)-3, and (−)-3 were determined by comparison of their experimental and calculated ECD spectra. As a result, the calculated ECD curves (Figure S6) for 12S,22R-2 and 12S,22R-3 displayed good agreement with the experimental ones of (+)-2 and (−)3, respectively. Thus, the absolute configurations of (±)-2 and (±)-3 were unambiguously assigned. The putative biosynthetic pathway of 1−3 is proposed in Scheme 1. As for (±)-1, nonstereo-selective hetero-Diels− Alder cycloaddition13 and Baeyer−Villiger oxidation14 were key reactions. Products (±)-2 and (±)-3 could be derived from the eurotiumin C9 and physcion,15 which underwent a nucleophilic addition and electrophilic attack on the aromatic carbon by the π-system similar to the Friedel−Crafts alkylation reaction.16 Since enantiomers of substances often have different biological activities, such as R-/S-thalidomide, R-/S-warfarin, R-/S-ehlorpheniramine, and R-/S-ketamine,17 we examined (+)-1, (−)-1, (+)-2, (−)-2, (+)-3, and (−)-3 for their antioxidative activity against DPPH9 and cytotoxic activity against SF-268 and HepG2 cell lines in vitro with the SRB method.18 Interestingly, compound (+)-1 showed significant radical scavenging activity against DPPH with an IC50 value of 58.4 μM, which was comparable to that of the positive control ascorbic acid (Vc) (45.8 μM) and nearly three times stronger

Scheme 1. Proposed Biosynthetic Pathway of Compounds 1−3

than (−)-1 (159.2 μM) (Table S2). Meanwhile, (+)-2 showed moderate cytotoxicities against SF-268 and HepG2 cell lines with IC50 values of 12.5 ± 0.1, 15.0 ± 0.7 μM, while those of (+)-3 were 30.1 ± 2.8, 37.3 ± 2.2 μM (Table S3). Compounds (−)-2 and (−)-3 were inactive (>100 μM). These results imply that the stereochemistry of the compounds could contribute to their biological activities. To investigate their bioactive mechanism, various types of antioxidative and cytotoxic targets were screened by a molecular docking technique (Tables S5 and S6). The results show that peroxiredoxin was the potential antioxidative target for compounds (+)-1 and (−)-1, and farnesyltransferase was the potential cytotoxic target for compounds (+)-2 and (+)-3 (Table S9). Furthermore, hydrogen bonding and lipophilic contact might play critical roles in stabilizing the complexes of the targets and compounds. In particular, hydrogen bonds (HBs) had a positive correlation with binding affinity for each pair of isomers. Specifically, as for (+)-1, only the secoanthronopyranoid−diketopiperazine part occupied the bioactive pocket of 3HY2 (PDB ID, and so forth) and formed HBs, while (+)-2 and (+)-3 almost embodied their entire structures in the bioactive pocket of 4GTM, which made the indole group contribute to the formation of HBs (Figure 4). It revealed that proper configurations of the compounds were important for bioactivities. In conclusion, (±)-variecolortins A−C (1−3) were isolated from the South China Sea fungus Eurotium sp. SCSIO F452. Compound 1 possesses an unprecedented highly functionalized benzo[f ]pyrazino[2,1-b][1,3]oxazepine new carbon skeleton comprising a 2-oxa-7-azabicyclo[3.2.1]octane core. Compounds 2 and 3 represent rare examples of a 6/6/6/6 tetracyclic cyclohexene-anthrone carbon scaffold. Specifically, (+)-1 exhibited stronger antioxidative activity than (−)-1, while (+)-2 and (+)-3 showed more potent cytotoxicities 4595

DOI: 10.1021/acs.orglett.8b01880 Org. Lett. 2018, 20, 4593−4596

Organic Letters

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 41476136, 41776169, 41230962), the National Key Research and Development Program of China (2017YFC0506300), the Guangdong Province Science and Technology Plan Project (2015B090904003, 2016A020222010), the Pearl River S&T Nova Program of Guangzhou (No. 201710010136), and the Science and Technology Program of Guangzhou (201607020018). We gratefully acknowledge support from the Guangzhou Branch of the Supercomputing Center of the Chinese Academy of Sciences and the analytical facilities in SCSIO.

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Figure 4. Hydrogen bonds and lipophilic properties of the surfaces of (+)-1, (−)-1, (+)-2, and (+)-3 with the bioactive pockets of proteins. (A: (+)-1 and 3HY2; B: (−)-1 and 3HY2; C: (+)-2 and 4GTM; D: (+)-3 and 4GTM; brown is favored regions for lipophilic affinity; red represents the residues to form HBs.).

against SF-268 and HepG2 cell lines than (−)-2 and (−)-3, which indicated that different enantiomers might result in different biological activities. A preliminary molecular docking study provided an inside perspective of the action of their different biological activities, which represented an attractive approach for the better understanding of the bioactivities. The structural complexity and novelty of 1−3 might not only become an attractive target for synthetic organic research in the future but also imply a fascinating biosynthetic mechanism expecting for further biochemical and genetic investigations.

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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.8b01880. Detailed description of the experimental procedures; full spectroscopic data, ECD calculations, and molecular docking for compounds 1−3 (PDF) Accession Codes

CCDC 1849647 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 [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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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

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

Weimao Zhong: 0000-0002-1358-4944 Junfeng Wang: 0000-0001-6702-5366 Xiaoyi Wei: 0000-0002-4053-6999 Notes

The authors declare no competing financial interest. 4596

DOI: 10.1021/acs.orglett.8b01880 Org. Lett. 2018, 20, 4593−4596