Sesteralterin and Tricycloalterfurenes A–D: Terpenes with Rarely

Article pubs.acs.org/jnp

Sesteralterin and Tricycloalterfurenes A−D: Terpenes with Rarely Occurring Frameworks from the Marine-Alga-Epiphytic Fungus Alternaria alternata k21‑1 Zhen-Zhen Shi,†,‡ Feng-Ping Miao,† Sheng-Tao Fang,† Xiang-Hong Liu,† Xiu-Li Yin,† and Nai-Yun Ji*,† †

Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China University of Chinese Academy of Sciences, Beijing 100049, China

‡

S Supporting Information *

ABSTRACT: A new sesterterpene, sesteralterin (1), four new meroterpenes, tricycloalterfurenes A−D (2−5), and a known meroterpene, TCA-F (6), were obtained from the culture extract of an Alternaria alternata strain (k21-1) isolated from the surface of the marine red alga Lomentaria hakodatensis. The structures and relative/absolute configurations of these compounds were identified by spectroscopic analyses, mainly including 1D/2D NMR, ECD, and mass spectra and quantum chemical calculations. Compound 1 represents the first nitidasane sesterterpene naturally produced by fungi, and 2−5 feature a tetrahydrofuran unit rarely occurring in tricycloalternarenes. Compounds 1−6 were assayed for inhibition of the growth of four marine plankton and one marine alga-pathogenic bacterium.

C

resulted in the isolation and identification of one new nitidasane sesterterpene (1) and four new tetrahydrofuranbearing tricycloalternarenes (2−5), and TCA-F (6). Herein, the details of isolation, structure elucidation, and biological evaluation of these compounds are described.

ompared to sesquiterpenes, diterpenes, and triterpenes, a small number of natural sesterterpenes have been found, especially those with a nitidasane framework. Nitidasin as the first nitidasane sesterterpene was obtained from the terrestrial plant Gentianella nitida,1 and the other two members, sesterfisherol and sesterfisheric acid, were discovered from the fungus Neosartorya f ischeri by heterologous expression in Aspergillus oryzae.2 On the other hand, multiple tricycloalternarenes as phytotoxins have been characterized from fungal strains of the genera Alternaria,3−8 Guignardia,9,10 Aspergillus,11 Ulocladium,12 and Septoria,13 with most of them being associated with plants. However, only TCA-F and tricycloalternarene J isolated from marine-derived Alternaria species feature a tetrahydrofuran ring A, and they were identified to be stereoisomers.3,4 As a possible precursor to them, ACTG-toxin C also possesses the same ring A, but it lacks the dihydropyran ring B.5 Thus, both nitidasane and tetrahydrofuran-bearing tricycloalternarene represent rarely occurring natural product frameworks, with three and two members, respectively.1−4 Marine algicolous fungi (MAFs) have contributed more than 400 new secondary metabolites with high diversity in chemical structures and biological activities so far.14,15 It is difficult to elaborate the metabolic specialty of MAFs, but most of them really have different secondary metabolite profiles from the same species of other origin. During our ongoing search for new or bioactive secondary metabolites from MAFs,16−19 a redalga-epiphytic strain (k21-1) of Alternaria alternata was examined. Only one algicolous strain of A. alternata was chemically investigated previously, and two new perylene derivatives were obtained.20 Our effort on A. alternata k21-1 © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The EtOAc extract of the whole cultures of A. alternata k21-1 was fractionated and purified by column chromatography (CC) on silica gel, RP-18, and Sephadex LH-20, preparative TLC, and semipreparative HPLC to yield compounds 1−6. Among them, compound 6 was identified to be TCA-F by their identical NMR, MS, and ECD data. TCA-F was originally obtained from an epiphytic A. alternata strain of the coastal halophyte Scorzonera mongalica and identified by a combination of spectroscopic data, quantum chemical calculations, and Mosher’s reactions (Table S1, Figures S1 and S2).3 Compound 1 was isolated as a white powder and assigned a molecular formula of C25H38O4 by interpretation of HREIMS (m/z 402.2767 [M]+), requiring seven degrees of unsaturation. The 1H NMR spectrum (Table 1) displayed one methyl singlet, four methyl doublets, one broad double doublet, and one doublet of double doublets ascribable to two oxygenated methines, one singlet, and one doublet attributable to two exchangeable protons. According to the [M − H2O]+ peak at m/z 384 and [M − 2H2O]+ peak at m/z 366 in the EIMS spectrum, the exchangeable protons should be derived from Received: June 5, 2017 Published: August 24, 2017 2524

DOI: 10.1021/acs.jnatprod.7b00478 J. Nat. Prod. 2017, 80, 2524−2529

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time-dependent density function theory (TD-DFT) method at the gas-phase B3LYP/6-31G(d) level using Gaussian 09 software.22 The calculated ECD spectrum was produced by SpecDis software with sigma = 0.2,23 which was in good accordance with the experimental one (Figure 3). Thus, the absolute configuration of 1, trivially named sesteralterin, was assigned to be 2R, 3S, 6S, 7S, 9S, 12R, 14S, 15R, 16R, and 18R. Compound 2 was obtained as a colorless oil with a molecular formula of C21H30O4 established by HREIMS (m/z 346.2140 [M]+), corresponding to seven degrees of unsaturation. In the EIMS, the characteristic [M − H2O]+ fragment ion peak at m/z 328 suggested the presence of a hydroxy group. The 1H NMR spectrum (Table 1) showed four methyl singlets, one double doublet, one broad triplet, and one triple doublet assignable to three oxygenated methines, one broad triplet, and one broad doublet attributable to two olefinic protons, and one broad singlet ascribable to the hydroxy group indicated by EIMS data. The 13C NMR spectrum (Table 2) along with DEPT and HSQC data demonstrated the presence of four methyls, six methylenes, five methines, and six nonprotonated carbons. A detailed comparison of the NMR data with those of TCA-F revealed their similarity.3 Relative to TCA-F, 3 features a deshielded triplet at δH 4.42 (J = 5.8 Hz), which was bonded to a shielded carbon at δC 66.3 by their HSQC correlation. This methine group was hydroxylated and located at C-15 by the COSY correlations of OH-15/H-15/H-16/H-17 and HMBC correlations from H-15 to C-14 and C-16 and from H-17 to C13 and C-18. Other COSY and HMBC correlations (Figure 1) further verified the planar structure of 2. The relative configuration of 2 was established by analysis of NOE correlations (Figure 2) and coupling constants as well as by comparison of NMR data with those of TCA-F. The NOE correlations of H-11 with H-8 and Me-21 positioned them on the same face of the molecule, and the NOE correlation between H-8 and Me-20 allowed an E configuration of the double bond at C-6. The above orientations were supported by the identical NMR data with TCA-F.3 Compared to H-17 of TCA-F and H-4 of guignardone J,3,11 H-15 of 2 was deduced to be equatorial and syn to H-8, H-11, and Me-21 by its splitting pattern and small coupling constant. Furthermore, the weak NOE correlation between H-15 and Me-21 also suggested their cofacial relationship. The ECD spectrum exhibited two positive Cotton effects at 308 and 258 nm and one negative Cotton effect at 202 nm (Figure 4), which was in good agreement with those of TCA-F and guignardone J.3,11 The ECD spectrum was further simulated with the TD-DFT method at the gas-phase B3LYP/6-31G(d) level and then drawn by SpecDis software with sigma = 0.2.22,23 The calculated and experimental ECD spectra also agreed with each other (Figure 4). Thus, the absolute configuration of 2, trivially named tricycloalterfurene A, was assigned to be 8R, 10R, 11R, and 15S. Compound 3 was isolated as a colorless oil, which was determined to possess a molecular formula of C21H30O6 by HREIMS (m/z 378.2035 [M]+), implying seven degrees of unsaturation. The 1H NMR spectrum (Table 1) displayed similarities to the spectrum of compound 2. An analysis of the NMR data revealed that 3 differed from 2 mainly at the side chain moiety (C-1 to C-4, C-19). Replacing a 3-methylbut-2enyl group in 2, a 3-hydroperoxy-3-methylbutenyl group was bonded to C-5 by comparison of the NMR data with those of 25-hydroperoxycholesta-4,23(E)-diene-3,6-dione.24 The connectivity at the side chain moiety was confirmed by the COSY correlations of H-4 with H-3 and H-5 and HMBC

two hydroxy groups. The 13C NMR spectrum (Table 2) exhibited 25 resonances, which were classified into five methyls, six methylenes, nine methines, and five nonprotonated carbons by DEPT and HSQC experiments. The COSY correlation between OH-16 and H-16 placed a hydroxy group at C-16, which was extended to C-13 through C-17, C-18, and C-14 by COSY correlations (Figure 1). Additionally, an isopropyl group was bonded to C-18 by the COSY correlations of H-24 and H25 with H-19 and HMBC correlations from H-24 and H-25 to C-18 and C-19. The other large spin system from C-1 to C-9 was also indicated by COSY correlations, which was connected to the above spin system through C-12 and C-15 by the HMBC correlations from OH-12 to C-2, C-12, and C-13 and from H23 to C-1, C-14, C-15, and C-16. The connectivity at C-10 and C-11 was confirmed by the HMBC correlations from OH-12 to C-11 and from H-7 and H-9 to C-10, which along with C-9 and C-22 formed a furanone unit by NMR data comparison with those of plicatilisin G.21 Other HMBC correlations (Figure 1) further corroborated the planar structure of 1. The relative configuration of 1 was determined by NOE correlations (Figure 2) and coupling constants. The NOE correlations of OH-12 with H-1b, H-3, H-13a, and H-14 indicated them to be on the same face of the molecule. H-14 was syn to H-16 and H-18 by the NOE correlations of H-14 with H-16 and H-18, and H-6 was syn to H-3 and H-7 by the NOE correlations of H-6 with H-3 and H-7. The splitting pattern and large coupling constant of H-1b indicated it to be axial and anti to H-2, which was syn to Me-23 by their NOE correlation. Moreover, H-9 and H-21 were oriented on the same face by their NOE correlation. The relative configurations at C-2, C-3, C-6, C-7, C-12, C-14, C-15, and C-18 were identical to those of nitidasin, which were established by analysis of X-ray diffraction data.1 In order to establish the absolute configuration of 1, its electronic circular dischroism (ECD) spectrum was determined and then computed with the 2525

DOI: 10.1021/acs.jnatprod.7b00478 J. Nat. Prod. 2017, 80, 2524−2529

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Table 1. 1H NMR Data for 1−5a pos 1a 1b 2 3 4a 4b 5a 5b 6 7 8a 8b 9a 9b 11 12a 12b 13a 13b 14 15a 15b 16a 16b 17a 17b 18 19 20 21 23 24 25 OH-12 OH-15 OH-16 a

1 1.36, 1.14, 1.72, 2.06, 1.35,

m t (12.8) m m m

1.71, m 2.79, 2.49, 1.99, 1.49, 5.12,

br dd (11.0, 6.5) m dd (10.7, 7.0) m br dd (12.5, 6.9)

2

3

4

5

1.68, s

1.39, s

1.16, s

1.15, s

5.07, 2.07, 1.99, 2.07, 1.99,

5.57, d (15.8) 5.67, dt (15.8, 6.9)

3.36, 1.62, 1.42, 2.28, 2.07,

3.34, 1.60, 1.40, 2.29, 2.05,

br t (6.9) m m m m

2.72, m

dd (10.5, 1.6) m m m m

dd (10.5, 1.9) m m m m

5.17, br d (8.9) 4.76, td (9.1, 4.3)

5.21, br d (9.0) 4.78, td (9.2, 4.0)

5.27, br d (8.5) 4.77, td (8.9, 4.0)

5.24, br d (8.8) 4.76, td (9.1, 4.2)

2.33, 2.08, 3.87, 2.74, 2.26,

2.34, 2.09, 3.89, 2.75, 2.26,

2.34, 2.10, 3.89, 2.75, 2.26,

2.29, 2.07, 3.87, 2.84, 2.23,

dd (13.7, 9.4) dd (13.8, 4.2) dd (5.0, 1.3) br d (18.0) ddd (18.0, 4.9, 2.1)

2.52, 2.44, 2.34, 1.88, 4.04,

br dd (17.8, 12.0) br dd (17.8, 5.0) dtd (12.4, 5.1, 2.3) qd (12.5, 5.6) dd (13.1, 5.5)

dd (13.7, 9.3) dd (13.8, 4.4) dd (4.8, 1.5) br d (18.3) ddd (18.3, 4.8, 1.3)

dd (13.7, 9.5) dd (13.8, 3.9) dd (4.7, 1.5) br d (18.3) ddd (18.3, 4.8, 1.4)

dd (14.0, 9.5) dd (13.9, 4.1) dd (4.8, 1.5) br d (18.2) ddd (18.3, 4.8, 1.4)

2.87, dd (13.1, 2.2) 1.38, t (13.4) 1.83, m

3.40, ddd (9.7, 7.9, 4.8) 1.95, 1.32, 1.50, 1.50, 0.85, 0.97, 0.67, 0.76, 0.83, 4.27,

m m m m d (7.1) d (7.4) s d (5.9) d (5.8) s

4.42, br t (5.8)

4.46, br t (6.4)

4.36, br t (6.3)

2.30, 1.98, 2.60, 2.34,

2.30, 1.98, 2.60, 2.36,

2.30, 1.98, 2.60, 2.36,

m m ddd (16.8, 7.0, 4.8) ddd (16.8, 9.7, 4.9)

1.59, s 1.68, br s 1.35, s

m m ddd (16.8, 6.5, 4.9) ddd (16.8, 10.2, 4.9)

1.39, s 1.68, d (1.1) 1.35, s

m m ddd (16.7, 6.3, 4.7) ddd (16.8, 10.1, 4.9)

1.21, s 1.70,d (1.1) 1.36, s

1.20, s 1.69, d (1.2) 1.31, s

2.56, br s 4.43, d (4.8)

Recorded in DMSO-d6 for 1 and in CDCl3 for 2−5 at 500 MHz.

and 2 suggested the same relative and absolute configurations at C-8, C-10, C-11, and C-15. Unfortunately, the absolute configuration at C-3 remains unknown due to the failure in preparing Mosher’s esters and single crystals. Compound 4 was trivially named tricycloalterfurene C. Compound 5 was isolated as a colorless oil. Its molecular formula was deduced to be C21H32O6, the same as for 4, by HREIMS. The NMR data (Tables 1 and 2) were very similar to those of 4, except for the signals of ring C. A hydroxy group was assigned at C-17 rather than C-15 by the HMBC correlation from the deshielded H-17 to C-18, which was supported by the identical NMR data with TCA-F.3 Other COSY and HMBC (Figure 1) as well as NOE correlations (Figure 2) validated the planar structure and relative configuration of 5, trivially named tricycloalterfurene D. The absolute configurations at C-8, C-10, C-11, and C-17 were deduced to be 8R, 10R, 11R, and 17S by comparison of the ECD spectrum with that of TCA-F (Figure 4), but the configuration at C-3 is unresolved. Compounds 1−6 represent terpene types that have never been reported to be naturally produced by terrestrial-derived fungi. In order to determine the ecological function of 1−6,

correlations from H-1 and H-19 to C-2 and C-3 and from H-20 to C-5, C-6, and C-7. The relative configurations at C-8, C-10, C-11, and C-15 were established by comparison of NMR data with those of 2, which was supported by the NOE correlations of H-11 with H-8 and Me-21. The E double bonds at C-3 and C-6 were indicated by the large coupling constant (15.8) between H-3 and H-4 and by the NOE correlation between H8 and Me-20, respectively, and the absolute configuration was assigned to be 8R, 10R, 11R, and 15S by the identical ECD spectrum with 2 (Figure 4). Compound 3 represents the first hydroperoxy-containing tricycloalternarene, trivially named tricycloalterfurene B. Compound 4 was obtained as a colorless oil. A molecular formula of C21H32O6 with six degrees of unsaturation was given by analysis of HREIMS data. The NMR data (Tables 1 and 2) resembled those of 2, except for the presence of signals for a 1,2-dihydroxyisobutyl group and the lack of signals for an isobutenyl group. The COSY correlation between H-3 and H-4 and HMBC correlations from H-1 and H-19 to C-2 and C-3 confirmed the linkage at the side chain terminus, which was further verified by comparison of the NMR data with those of TCA 6a.6 The identical NMR and ECD data (Figure 4) of 4 2526

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Table 2. 13C NMR Data for 1−5a

a

pos

1

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

36.1, 36.8, 30.1, 34.4, 23.2, 44.8, 36.5, 34.9, 83.4, 171.2, 132.1, 74.6, 34.8, 40.1, 42.1, 79.7, 36.8, 43.5, 30.1, 19.0, 14.6, 174.4, 12.8, 22.1, 23.8,

2 CH2 CH CH CH2 CH2 CH CH CH2 CH C C C CH2 CH C CH CH2 CH CH CH3 CH3 C CH3 CH3 CH3

25.8, 131.9, 124.0, 26.4, 39.7, 139.8, 125.6, 73.1, 46.2, 84.6, 77.2, 19.2, 106.9, 166.4, 66.3, 29.0, 33.6, 197.2, 17.8, 16.6, 20.2,

3 CH3 C CH CH2 CH2 C CH CH CH2 C CH CH2 C C CH CH2 CH2 C CH3 CH3 CH3

24.4, 82.3, 135.7, 129.0, 42.3, 138.1, 126.8, 72.9, 46.0, 84.6, 77.2, 19.3, 106.9, 166.5, 66.4, 29.2, 33.8, 197.2, 24.5, 16.8, 20.1,

4 CH3 C CH CH CH2 C CH CH CH2 C CH CH2 C C CH CH2 CH2 C CH3 CH3 CH3

23.6, 73.2, 78.8, 29.5, 37.1, 139.2, 126.8, 73.2, 46.2, 84.5, 77.2, 19.3, 106.9, 166.6, 66.3, 29.1, 33.8, 197.2, 26.7, 16.6, 20.1,

5 CH3 C CH CH2 CH2 C CH CH CH2 C CH CH2 C C CH CH2 CH2 C CH3 CH3 CH3

23.4, 73.2, 78.5, 29.7, 36.9, 139.2, 126.3, 73.1, 46.1, 84.5, 76.9, 19.2, 104.3, 169.2, 27.8, 29.0, 71.2, 198.1, 26.7, 16.6, 19.9,

CH3 C CH CH2 CH2 C CH CH CH2 C C CH2 C C CH2 CH2 CH C CH3 CH3 CH3

Recorded in DMSO-d6 for 1 and in CDCl3 for 2−5 at 125 MHz.

μM). Compound 1 showed 41−69% inhibition of these three phytoplankton, but it was inactive to the zooplankton A. salina. The similar results of 2 (64, 37, 46% inhibition) and 6 (70, 41, 52% inhibition) against the three phytoplankton, respectively, indicated that the hydroxy group positions on ring C almost had no effect on their activities. Hydroxylation at C-2 and C-3 (4 and 5) slightly reduced the inhibition of the three phytoplankton (range 17−56% inhibition). In addition, compounds 1−6 exhibited no activities against the macroalgapathogenic bacterium P. citrea at 20 μg/disk.

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured on a JASCO P-1020 polarimeter. The UV and ECD spectra were recorded on a Chirascan CD spectrometer. The IR spectra were obtained on a JASCO FT/IR-4100 spectrometer. The NMR spectra were recorded at 500 and 125 MHz for 1H and 13C, respectively, on a Bruker Avance III 500 NMR spectrometer using tetramethylsilane as an internal standard. The low- and high-resolution EI mass spectra were determined on an Autospec Premier P776 mass spectrometer. HPLC separation was carried out on an Agilent HPLC system (1260 infinity quaternary pump, 1260 infinity diode-array detector) using an Eclipse SB-C18 (5 μm, 9.4 × 250 mm) column. Column chromatography was performed with silica gel (200−300 mesh, Qingdao Haiyang Chemical Co.), RP-18 (AAG12S50, YMC Co., Ltd.), and Sephadex LH-20 (Pharmacia). TLC was carried out with precoated silica gel plates (GF-254, Qingdao Haiyang Chemical Co.). The solvents were of analytical grade except for the spectral-grade MeOH for HPLC. Quantum chemical calculations were performed using Gaussian 09 software (IA32W-G09RevC.01). Fungal Material and Fermentation. The fungal strain Alternaria alternata k21-1 was isolated from the surface of the marine red alga Lomentaria hakodatensis collected from Kongdong Island in July 2015. The fungus was identified by morphological observation and analysis of the ITS regions of its rDNA, whose sequence data have been

Figure 1. Key HMBC (arrows) and COSY (bold lines) correlations of 1−5.

they were evaluated for growth inhibition against three marine phytoplankton (Chattonella marina, Heterosigma akashiwo, and Prorocentrum donghaiense), one marine zooplankton (Artemia salina), and one marine-derived bacterium (Pseudoalteromonas citrea) that can cause Porphyra yezoensis green-spot disease (Table S2).19,25 Among the marine plankton tested, C. marina appeared more sensitive to 1−6, although the inhibition was weak to moderate at the 100 μg/mL concentration used (∼250 2527

DOI: 10.1021/acs.jnatprod.7b00478 J. Nat. Prod. 2017, 80, 2524−2529

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Figure 4. Experimental ECD spectra of 2−6 and calculated ECD spectrum of 2.

directly extracted with EtOAc and then concentrated to give an extract (11.4 g). These two parts were combined due to their similar TLC profiles and then subjected to silica gel CC with step-gradient solvent systems of petroleum ether (PE)/EtOAc (50:1 to 1:1) and CH2Cl2/ MeOH (20:1 to 1:1) to afford 10 fractions (Frs. 1−10). Fr. 6 eluted with PE/EtOAc (1:1) and was further purified by CC on RP-18 (MeOH/H2O, 3:1) and Sephadex LH-20 (MeOH) and preparative TLC (PE/EtOAc, 1:1) to give 1 (11.5 mg) and 6 (7.7 mg). Fr. 7 eluted with CH2Cl2/MeOH (20:1) and was further purified by CC on RP-18 (MeOH/H2O, 7:3) and semipreparative HPLC (MeOH/H2O, 3:2 to 4:1) to produce 2 (7.0 mg) and 3 (3.0 mg). Fr. 8 eluted with CH2Cl2/MeOH (10:1) and was further purified by CC on RP-18 (MeOH/H2O, 2:3 to 1:1) and Sephadex LH-20 (MeOH) and preparative TLC (CH2Cl2/MeOH, 15:1) to yield 4 (2.5 mg) and 5 (2.2 mg). Sesteralterin (1): white powder; [α]24D +130 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 225 (4.20) nm; ECD (0.12 g/L, MeOH), λmax (Δε) 230 (26.8), 262 (−7.8) nm; IR (KBr) νmax 3286, 2947, 2881, 1724, 1643, 1462, 1388, 1041, 953, 733 cm−1; 1H and 13C NMR data, Tables 1 and 2; EIMS m/z (%) 402 [M]+ (12), 384 (65), 366 (49), 323 (33), 247 (85), 231 (100), 149 (62); HREIMS m/z 402.2767 [M]+ (calcd for C25H38O4, 402.2770). Tricycloalterfurene A (2): colorless oil; [α]23D +145 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 260 (4.29) nm; ECD (0.17 g/L, MeOH), λmax (Δε) 202 (−12.33), 258 (13.67), 308 (0.90) nm; IR (KBr) νmax 3421, 2924, 2858, 1724, 1624, 1392, 1165, 1084, 737 cm−1; 1 H and 13C NMR data, Tables 1 and 2; EIMS m/z (%) 346 [M]+ (100), 328 (4), 278 (9), 263 (7), 197 (34), 179 (51), 109 (26), 83 (32), 69 (58); HREIMS m/z 346.2140 [M]+ (calcd for C21H30O4, 346.2144). Tricycloalterfurene B (3): colorless oil; [α]22D +120 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 258 (4.29) nm; ECD (0.14 g/L, MeOH), λmax (Δε) 209 (−8.65), 263 (10.41), 308 (1.71) nm; IR (KBr) νmax 3448, 2924, 2858, 1712, 1628, 1396, 1076 cm−1; 1H and 13 C NMR data, Tables 1 and 2; EIMS m/z (%) 378 [M]+ (0.5%), 344 (14), 221 (17), 179 (100), 139 (52), 83 (51); HREIMS m/z 378.2035 [M]+ (calcd for C21H30O6, 378.2042). Tricycloalterfurene C (4): colorless oil; [α]19D +110 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 261 (4.29) nm; ECD (0.19 g/L, MeOH), λmax (Δε) 201 (−12.02), 257 (10.94), 307 (0.80) nm; IR (KBr) νmax 3359, 2924, 2862, 1720, 1620, 1392, 1073 cm−1; 1H and 13 C NMR data, Tables 1 and 2; EIMS m/z (%) 380 [M]+ (5%), 362 (11), 344 (2), 322 (30), 279 (35), 179 (47), 167 (41), 149 (100), 59 (43); HREIMS m/z 380.2188 [M]+ (calcd for C21H32O6, 380.2199). Tricycloalterfurene D (5): colorless oil; [α]20D +180 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 259 (4.29) nm; ECD (0.20 g/L, MeOH), λmax (Δε) 200 (−12.61), 257 (15.23), 309 (0.73) nm; IR (KBr) νmax 3429, 2931, 2870, 1716, 1620, 1385, 1080 cm−1; 1H and 13 C NMR data, Tables 1 and 2; EIMS m/z (%) 380 [M]+ (2%), 363 (30), 344 (4), 321 (89), 304 (57), 279 (40), 197 (54), 179 (100), 83

Figure 2. Key NOE correlations of 1, 2, and 5.

Figure 3. Experimental and calculated ECD spectra of 1. deposited at GenBank with the accession number MF179020. The initial cultures were maintained on potato dextrose agar plates. Pieces of mycelia were cut into small segments and aseptically inoculated into 100 Erlenmeyer flasks (1 L), each containing 300 mL of potato dextrose broth media. The media were prepared by adding 500 mL of potato (100 g) broth, 20 g of glucose, 5 g of peptone, and 5 g of yeast extract powder into 500 mL of natural seawater from the coast of Yantai. Static fermentations were performed at room temperature for 30 days. Extraction and Isolation. The mycelia of cultures (300 mL × 100 flasks) were obtained by filtration, and they were dried, smashed, and then exhaustively extracted with a mixture of CH2Cl2 and MeOH (1:1, v/v). The organic solvents were evaporated to dryness under reduced pressure, and the residue was partitioned between EtOAc and H2O to give an EtOAc-soluble extract (13.6 g). Additionally, the filtrate was 2528

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(92), 59 (77); HREIMS m/z 380.2184 [M]+ (calcd for C21H32O6, 380.2199).

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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.jnatprod.7b00478. 1D/2D NMR and mass spectra of 1−6 as well as Cartesian coordinates of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-535-2109176. Fax: +86535-2109000. ORCID

Nai-Yun Ji: 0000-0002-6526-4731 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Open Fund of Key Laboratory of Experimental Marine Biology, Chinese Academy of Sciences (KF2017NO4), Key Cutting-Edge Research Program of the Chinese Academy of Sciences (QYZDB-SSW-DQC013), National Natural Science Foundation of China (31670355), Natural Science Foundation for Distinguished Young Scholars of Shandong Province (JQ201712), and Yantai Municipal SciTech Development Program (2015ZH076) is gratefully acknowledged.

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DOI: 10.1021/acs.jnatprod.7b00478 J. Nat. Prod. 2017, 80, 2524−2529