Teratopyrones A–C, Dimeric Naphtho–Pyrones and Other Metabolites

molecules Article Teratopyrones A–C, Dimeric Naphtho-γ-Pyrones and Other Metabolites from Teratosphaeria sp. AK1128, a Fungal Endophyte of Equisetum arvense 1 2 2 3 3 Ya-Ming Xu , A. Elizabeth Arnold , Jana M. U0Ren , Li-Jiang Xuan , Wen-Qiong Wang and A. A. Leslie Gunatilaka 1,* 1 Southwest Center for Natural Products Research, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of Arizona, 250 E, Valencia Road, Tucson, AZ 85706, USA; [email protected] 2 School of Plant Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ 85721, USA; [email protected] (A.E.A.); [email protected] (J.M.U.) 3 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China; [email protected] (L.-J.X.); [email protected] (W.-Q.W.) * Correspondence: [email protected]; Tel.: +520-621-9932 Academic Editors: Muhammad Ilias and Dhammika Nanayakkara Received: 12 October 2020; Accepted: 26 October 2020; Published: 30 October 2020 Abstract: Bioassay-guided fractionation of a cytotoxic extract derived from a solid potato dextrose agar (PDA) culture of Teratosphaeria sp. AK1128, a fungal endophyte of Equisetum arvense, afforded three new naphtho-γ-pyrone dimers, teratopyrones A–C (1–3), together with five known naphtho-γ-pyrones, aurasperone B (4), aurasperone C (5), aurasperone F (6), nigerasperone A (7), and fonsecin B (8), and two known diketopiperazines, asperazine (9) and isorugulosuvine (10). The structures of 1–3 were determined on the basis of their spectroscopic data. Cytotoxicity assay revealed that nigerasperone A (7) was moderately active against the cancer cell lines PC-3M (human metastatic prostate cancer), NCI-H460 (human non-small cell lung cancer), SF-268 (human CNS glioma), and MCF-7 (human breast cancer), with IC50s ranging from 2.37 to 4.12 µM while other metabolites exhibited no cytotoxic activity up to a concentration of 5.0 µM. Keywords: endophytic fungus; Teratosphaeria; teratopyrones; naphtho-γ-pyrones; nigerasperone A 1. Introduction Fungal endophytes constitute an abundant and underexplored group of fungi that inhabit plants in diverse natural and human-managed ecosystems [1,2]. These symbiotic fungi often produce bioactive metabolites, some of which may improve the growth or resiliency of the host plant [3]. Recent studies have demonstrated that fungal endophytes are rich sources of small-molecule natural products with novel structures and biomedical potential [4,5]. In our continuing search for bioactive and/or novel metabolites from endosymbiotic microorganisms [6], we have investigated a cytotoxic extract of the fungal endophyte, Teratosphaeria sp. AK1128, isolated from a photosynthetic stem of Equisetum arvense (field horsetail, Equisetaceae). Herein, we report cytotoxicity assay-guided fractionation of this extract, resulting in isolation and characterization of ten metabolites, including three new naphtho-γ-pyrone dimers, teratopyrones A–C (1–3), and the constituent responsible for cytotoxic activity, nigerasperone A (7). Teratosphaeria is a genus within the newly established fungal family Teratosphaeriaceae (Dothideomycetes, Ascomycota), which has been distinguished recently from Mycosphaerella (Mycosphaerellaceae) [7]. To the best of our knowledge, this constitutes the second report on metabolites of a fungal strain of the family Teratosphaeriaceae [8]. Molecules 2020, 25, 5058; doi:10.3390/molecules25215058 www.mdpi.com/journal/molecules Molecules 2020, 25, x 2 of 10 from Mycosphaerella (Mycosphaerellaceae) [7]. To the best of our knowledge, this constitutes the secondMolecules report2020, 25 ,on 5058 metabolites of a fungal strain of the family Teratosphaeriaceae [8]. 2 of 10 2. Results and Discussion 2. Results and Discussion The EtOAc extract of a PDA (potato dextrose agar) culture of Teratosphaeria sp. AK1128 The EtOAc extract of a PDA (potato dextrose agar) culture of Teratosphaeria sp. AK1128 exhibiting exhibiting cytotoxic activity, on bioassy-guided fractionation involving solvent-solvent partitioning, cytotoxic activity,on bioassy-guided fractionation involving solvent-solvent partitioning, Sephadex LH-20 Sephadex LH-20 size-exclusion and silica gel chromatography followed by HPLC purification, size-exclusion and silica gel chromatography followed by HPLC purification, afforded metabolites afforded metabolites 1–10 (Figure 1). Of these, 4–10 were previously known and were identified as 1–10 (Figure1). Of these, 4–10 were previously known and were identified as naphtho-γ-pyrones, naphtho-γ-pyrones, aurasperone B (4) [9], aurasperone C (5) [10], aurasperone F (6) [11], aurasperone B (4)[9], aurasperone C (5)[10], aurasperone F (6)[11], nigerasperone A (7)[12], nigerasperone A (7) [12], and fonsecin B (8) [9], and diketopiperazines, asperazine (9) [13] and and fonsecin B (8)[9], and diketopiperazines, asperazine (9)[13] and isorugulosuvine (10)[14], isorugulosuvine (10) [14], by comparison of their spectroscopic (1H NMR, 13C NMR, and LR-MS) data by comparison of their spectroscopic (1H NMR, 13C NMR, and LR-MS) data with those reported. with those reported. Figure 1. StructuresStructures of of metabolites metabolites isolated from Teratosphaeria sp. AK1128.AK1128. Spectroscopic ((11HH andand13 13CC NMR, NMR, HRESIMS, HRESIMS, and and UV) UV) data data of teratopyronesof teratopyrones A–C A (1––C3 ),(1 together–3), together with witheirth their common common molecular molecular formula, formula, C31H 26C31OH1126,O suggested11, suggested that that they they are dimericare dimeric naphtho- naphthoγ-pyrones-γ-pyrones [11]. [11].In their In their1H and 1H 13andC NMR13C NMR spectra, spectra, two two sets sets of signals of signals were were observed observed in di infferent different intensity intensity ratios ratios and 7 10′ andthis wasthis suspectedwas suspected to be dueto be to thedue atropisomerism to the atropisomerism around the ar Cound7–C10 the0 axis C [–10C] and axis/or [10] the presenceand/or the of presenceC-2 and C-2 of 0Chemi-ketal-2 and C-2′ stereoisomeric hemi-ketal stereoisomeric mixtures as a mixtures result of non-enzymaticas a result of non formation-enzymatic of this formation moiety ofduring this themoiety biosynthesis during ofthe naphtho- biosynthesisγ-pyrones of [15naphtho]. Although-γ-pyrones several [15] recent. Although publications several on dimericrecent publicationsnaphtho-γ-pyrones on dimeric report naphtho only one-γ-pyrones set of the report NMR only data, one careful set of examinationthe NMR data, of careful the spectra examination of these ofdimeric the spectra naphtho- of theseγ-pyrones dimeric provided naphtho in-γ the-pyrones Supporting provided Information in the Supporting of these papersInformation indicated of these the presencepapers indicated of two sets the of presence signals, corresponding of two sets of to signals, two possible corresponding tautomers to [16 two–18]. possible The atropisomerization tautomers [16– 7 10′ 18around]. The the atropisomerization C7–C100 axis is known around to bethe restricted C –C axis under is known mild conditions to be restricted [10], and under the atropisomer mild conditions ratio [10],for a and given the dimeric atropisomer naphtho- ratioγ-pyrone for a given may dimeric depend naphtho on its producer-γ-pyrone fungus may depend [19]. Although on its prod it isucer not possiblefungus [19]. to obtain Although the atropisomer it is not possible ratio directly, to obtain the use the circular atropisomer dichroism ratio (CD) directly, data for the the use assignment circular dichroismof stereochemistry (CD) data of thefor binaphthylthe assignment moiety of stereochemistry of the major atropisomer of the binaphthyl of dimeric moiety naphtho- of γthe-pyrones major atropisomerby the exciton of chiralitydimeric methodnaphtho has-γ-pyrones been reported by the exciton [20,21]. chirality method has been reported [20,21]. 1 In its H NMR spectrum, teratopyrone A (1) exhibited signals due to two methyls (δH 1.43 (s) and 2.36 (s)), three methoxyls (δH 3.39 (s), 3.60 (s), and 3.90 (s)), five aromatic protons (δH 5.99 (s), 6.12 (br. s), Molecules 2020, 25, 5058 3 of 10 6.26 (br. s), 7.06 (s), and 7.07 (s)), and two hydrogen-bonded phenolic hydroxy groups (δH 14.48 (s) and 14.97 (s)). The 13C NMR spectrum of 1 (Table1) contained 31 carbon signals due to its major tautomer and these were assigned with the help of HSQC and HMBC spectra. The two carbonyl signals at δC 197.9 and 184.6 revealed that 1 was made up of two naphtho-γ-pyrone monomers, of which one was hydrated at C20 –C30 . The HMBC data for 1 (Figure2) revealed that the linkage of two monomers of 1 was the same as that of nigerasperone C (11; Figure1)) in which the hydrated monomer consisted of the upper unit [12]. The 13C NMR data of 1 closely resembled those of 11 [12], except for the signals in the vicinity of C-5–C-8. However, some differences were observed for C-5 (δC 162.5 for 1; 166.1 for 11), C-6 (δC 107.1 for 1; 111.8 for 11), and C-8 (δC 157.3 for 1; 163.1 for 11), suggesting that 1 differed from 11 in the attachment of hydroxy/methoxy groups at C-6/C-8. The attachment of the methoxy group to C-8 in 1 was confirmed by the HMBC correlations of 8-OCH3 (δH 3.38) and H-9 (δH 7.06) to C-8 (δC 157.3). The ECD spectrum of 1 (Figure3) showed Davydov-split Cotton e ffects as negative ([θ] 1.46 105, − × 289.5 nm) first and positive ([θ] +1.78 105, 272 nm) second, suggesting negative chirality [21] and × M-configuration of the 70–100 bond [20]. Thus, the structure of teratopyrone A was determined as (100S)-20,5,500,6-tetrahydroxy-60,8,80-trimethoxy-2,20-dimethyl-20,30-dihydro-4H,40H-[7,100-bibenzo[g] chromene]-4,40-dione (1) (Figure1). Table 1. 13C NMR data (100 MHz, δ) for teratopyrones A–C (1–3).

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