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Oct 11, 2016 - (1) The differentiation of the osteoclast is modulated by two major ... (TNFs), regulates the differentiation, survival, and activation...
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Stachybotrysin, an Osteoclast Differentiation Inhibitor from the Marine-Derived Fungus Stachybotrys sp. KCB13F013 Jong Won Kim,† Sung-Kyun Ko,†,‡ Hye-Min Kim,†,‡ Gun-Hee Kim,† Sangkeun Son,†,‡ Gil Soo Kim,†,‡ Gwi Ja Hwang,†,‡ Eun Soo Jeon,† Kee-Sun Shin,§ In-Ja Ryoo,† Young-Soo Hong,†,‡ Hyuncheol Oh,⊥ Kyung Ho Lee,† Nak-Kyun Soung,†,‡ Daisuke Hashizume,∥ Toshihiko Nogawa,# Shunji Takahashi,#,□ Bo Yeon Kim,†,‡ Hiroyuki Osada,# Jae-Hyuk Jang,*,†,‡ and Jong Seog Ahn*,†,‡ †

Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, 28116, South Korea Department of Biomolecular Science, University of Science and Technology, Daejeon 34113, South Korea § Industrial Bio-materials Research Center, Daejeon 34141, South Korea ⊥ College of Pharmacy, Wonkwang University, Iksan 54538, South Korea ∥ Materials Characterization Support Unit, RIKEN Center for Emergent Matter Science (CEMS), Wako, Saitama 351-0198, Japan # Chemical Biology Research Group, RIKEN CSRS, Wako, Saitama 351-0198, Japan □ RIKEN-KRIBB Joint Research Unit, RIKEN Global Research Cluster, Wako, Saitama 351-0198, Japan ‡

S Supporting Information *

ABSTRACT: Two new phenylspirodrimane derivatives, stachybotrysin (1) and stachybotrylactone B (2), were isolated from the cultures of the marinederived fungus Stachybotrys sp. KCB13F013. The structures were determined by analyzing the spectroscopic data (1D and 2D NMR and MS) and chemical transformation, including the modified Mosher’s method and single-crystal X-ray structure analysis. Compound 1 exhibited an inhibitory effect on osteoclast differentiation in bone marrow macrophage cells via suppressing the RANKL-induced activation of p-ERK, p-JNK, pp38, c-Fos, and NFATc1.

flavonoids, ginsenoside Rh2, and agelasine D, have demonstrated the inhibition of osteoclastogenesis.10−15 The Stachybotrys fungi are known to produce various secondary metabolites, such as trichothecenes, diterpenoids, and meroterpenoids.16 The phenyspirodrimanes containing the drimane skeleton attached through a spirobenzofuran belong to the meroterpenoids with a sesquiterpene moiety connected to a phenolic derivative.16,17 They are known as signature secondary metabolites; over 80 compounds of this class have been isolated from the Stachybotrys and Memnoniella fungi. 16 These compounds have exhibited diverse biological activities, such as inhibiting immune complex diseases,18 avian myeloblastosis virus protease (AMV-protease),19 tyrosine kinase receptors,20 and antihyperlipidemic effects on HepG2 hepatocarcinoma cells.21 In our search for new bioactive compounds from marinederived fungi, Stachybotrys sp. KCB13F013 was isolated from the coast sediments of Wi-Island, South Korea. The organic

H

uman bone homeostasis is regulated by activating osteoblasts (bone formation) and osteoclasts (bone resorption), which generates bone remodeling. Many bone diseases, including osteopenia, rheumatoid arthritis, lytic bone metastasis, Paget’s disease, and osteoporosis, are characterized by excessive bone resorption of osteoclasts, which are multinucleate cells formed from mononuclear cells of hematopoietic origin.1 The differentiation of the osteoclast is modulated by two major cytokines: the macrophage colony stimulation factor (M-CSF) and the receptor activator of the NF-κB ligand (RANKL).2−4 RANKL, a family member of the tumor necrosis factors (TNFs), regulates the differentiation, survival, and activation of osteoclasts.4 The interaction of RANKL with RANK (a receptor of RANKL) activates downstream signaling pathways, such as NF-κB, c-jun Nterminal protein kinase (JNK), p38, and extracellular signalrelated kinase (ERK).5−7 This leads to the activation of transcription factors and nuclear factors of activated T cells c1 (NFATc1) and c-Fos.8,9 Therefore, suppressing these signaling pathways is a useful therapeutic target for inhibiting osteoclastogenesis. Diverse natural compounds, such as tannins, © 2016 American Chemical Society and American Society of Pharmacognosy

Received: July 16, 2016 Published: October 11, 2016 2703

DOI: 10.1021/acs.jnatprod.6b00641 J. Nat. Prod. 2016, 79, 2703−2708

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extracts of cultures showed weak cytotoxicity (IC50 > 50 μg/ mL) against the Hep-3B and HeLa cell lines. Furthermore, the LC-MS data of the EtOAc extract suggested the presence of unusual metabolites, of which MS and UV data were rare in public and in-house databases. The scale-up culture of the strain and chromatographic separation of the EtOAc extract led to the isolation of two new phenyspirodrimane derivatives. Herein, we report the isolation, structure elucidation, and biological activities of stachybotrysin (1) and stachybotrylactone B (2).

broth and mycelia extracts were partitioned with EtOAc. The EtOAc extracts were purified by silica gel open column chromatography, ODS flash chromatography, and reversedphase HPLC to afford stachybotrysin (1) and stachybotrylactone B (2). Compound 1 was obtained as a yellow, amorphous powder. The molecular formula was deduced as C26H36O5 based on the analysis of HRESIMS in combination with NMR data. The 1H, 13C, and DEPT data in conjunction with the HMQC spectrum of 1 suggested the presence of 26 carbons, including one carbonyl carbon (δC 197.9), six nonprotonated carbons (δC 162.5, 155.6, 142.9, 111.9, 106.0, and 97.8), two quaternary carbons (41.8 and 37.1), five methyl carbons (δC 28.8, 28.4, 22.1, 15.5, and 15.4), six methylene carbons (δC 61.5, 31.4, 30.4, 24.9, 23.8, and 20.6), three olefinic methine carbons (δC 135.8, 125.8, and 108.4), and three sp3 methine carbons (δC 73.2, 39.9, and 36.2). The 1H NMR spectrum of 1 indicated the presence of one doublet methyl proton (δH 0.60), four single methyl protons (δH 2.22, 0.95, 0.92, and 0.80), two coupled olefinic protons (δH 7.62 and 7.16), one single olefinic proton (δH 6.38), one oxymethine proton (δH 3.18), two methine protons (δH 2.14 and 1.80), one oxymethylene proton (δH 4.50), five methylene protons (δH

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RESULTS AND DISCUSSION The fungal strain Stachybotrys sp. KCB13F013 was cultured in potato dextrose broth (PDB, 10 L) for 7 days at 28 °C, and the

Table 1. 1H and 13C NMR Spectroscopic Data for 1 and 2 in DMSO-d6 1 position

δ Ca

type

1

23.8,

CH2

2

24.9,

CH2

3 4 5 6

73.2, 37.1, 39.9, 20.6,

CH C CH CH2

7

31.4,

CH2

8 9 10 11

36.2, 97.8, 41.8, 30.4,

CH C C CH2

15.4, 28.8, 22.1, 15.5, 111.9, 155.6, 108.4, 142.9, 106.0, 162.5, 135.8, 125.8, 197.9, 28.4, 61.5,

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

12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 3-OH 2′-OH 11′-OH a

2 δH (J in Hz)b 1.71, 0.90, 1.80, 1.37, 3.18,

td (13.3, 2.9) m m m brs

2.14, 1.51, 1.42, 1.58, 1.40, 1.80,

d (12.0) m m m m dt (11.9, 6.4)

3.02, 2.68, 0.60, 0.92, 0.80, 0.95,

d (16.2) d (16.2) d (6.4) s s s

6.38, s

7.62, d (15.8) 7.16, d (15.8) 2.22, 4.50, 4.07, 9.91, 5.14,

δCc

type

23.8,

CH2

25.0,

CH2

73.3, 37.3, 39.1, 20.5,

CH C CH CH2

30.5,

CH2

36.6, 99.3, 41.8, 30.8,

CH C C CH2

15.4, 28.6, 22.5, 15.9, 113.2, 159.6, 100.8, 149.6, 97.9, 161.1, 68.8, 167.9,

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

s m d (2.6) brs brs

δH (J in Hz)d 1.68, 0.89, 1.79, 1.38, 3.17,

td (13.3, 3.4) m m m brs

2.05, 1.47, 1.40, 1.50, 1.43, 1.78,

dd (12.3, 2.6) m m m m m

3.03, 2.66, 0.62, 0.88, 0.79, 0.93,

d (16.2) d (16.2) d (6.5) s s s

6.31, s

5.13, s

4.08, brs

Recorded at 200 MHz. bRecorded at 800 MHz. cRecorded at 175 MHz. dRecorded at 700 MHz. 2704

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ROESY correlations of H-8/H2-11/H3-14/H3-15 indicated that they were on the same face. However, the lack of correlation between H-5 and H3-15 (δH 0.95) revealed that they were on the opposite face in the molecule (Figure 1) and suggested that the orientations for the A and B ring junctures were trans. Fortunately, single crystals suitable for an X-ray structure analysis of 1 were obtained from a MeOH−H2O (7/3 (v/v)) mixed solution by slow evaporation for 2 weeks. The absolute structure of 1 was proposed by a single-crystal X-ray structure analysis, resulting in 3R, 5S, 8R, 9R, and 10S configurations (Figure 2).

3.02 and 2.68, 1.80 and 1.37, 1.71 and 0.90, 1.58 and 1.40, 1.51 and 1.42), and three exchangeable protons (δH 9.91, 5.14, and 4.07) (Table 1). The interpretation of the 2D NMR data, including COSY, HSQC, and HMBC spectra, led to the construction of the planar structure of 1 (Figure 1). The COSY

Figure 1. Key 2D correlations of compounds 1 and 2.

cross-peaks of H2-1/H2-2/H-3/OH-3 and H-5/H2-6/H2-7/H8/H3-12 and HMBC correlations of H2-2 (δH 1.80) to C-5 (δC 39.9) and C-10 (δC 41.8), H-3 (δH 3.18) to C-1 (δC 23.8) and C-5 (δC 39.9), H-5 (δH 2.14) to C-4 (δC 37.1), C-6 (δC 20.6), C-7 (δC 31.4), C-10 (δC 41.8), C-13 (δC 28.8), C-14 (δC 22.1), and C-15 (δC 15.5), H-8 (δH 1.80) to C-6 (δC 20.6) and C-12 (δC 15.4), H3-12 (δH 0.60) to C-6 (δC 20.6), C-7 (δC 31.4), C-8 (δC 36.2), and C-9 (δC 97.8), H3-13 (δH 0.92) to C-3 (δC 73.2), C-4 (δC 37.1), C-5 (δC 39.9), and C-14 (δC 22.1), H3-14 (δH 0.80) to C-3 (δC 73.2), C-4 (δC 37.1), C-5 (δC 39.9), and C-13 (δC 28.8), and H3-15 (δH 0.95) to C-1 (δC 23.8), C-5 (δC 39.9), C-9 (δC 97.8), and C-10 (δC 41.8) established a drimane moiety that had a hydroxy group at C-3. Furthermore, the HMBC correlations of H-3′ (δH 6.38) to C-1′ (δC 111.9), C-2′ (δC 155.6), C-3′ (δC 108.4), C-4′ (δC 142.9), and C-6′ (δC 162.5) and H2-11 (δH 3.02 and 2.68) to C-1′ (δC 23.8), C-2′ (δC 24.9), C-6′ (δC 162.5), and C-9 (δC 97.8) revealed the presence of a dihydrobenzofuran moiety. The HMBC correlations of the methylene proton H2-11 to C-8, C-9, and C-10 suggested that drimane and dihydrobenzofuran moieties were connected as the phenylspirodrimane skeleton. The COSY correlations of H-7′ and H-8′ and the HMBC correlations of H-7′ to C-4′ (δC 142.9), C-5′ (δC 106.0), C6′ (δC 162.5), C-8′ (δC 125.8), and C-9′ (δC 197.9) and H-8′ to C-5′ (δC 106.0), C-9′ (δC 197.9), and C-10′ (δC 28.4) indicated the presence of a 3-penten-2-one moiety that was connected at C-5′ of the phenylspirodrimane moiety. Another side chain was established by the COSY correlation of H2-11′ to OH-11′ and the HMBC correlations of H2-11′ to C-3′, C-4′, and C-5′. The hydroxylation of C-11′ was supported by the deshielded signals of C-11′ (δC 61.5) and H2-11′ (δH 4.50). Thus, the planar structure of stachybotrysin (1) was assigned as shown in Figure 1. The relative configuration of 1 was determined by a combination of ROESY and 1H−1H coupling constants (Figure 1 and Table 1). The large coupling constants (JH7′−H8′ = 15.8 Hz) revealed that the H-7′ and H-8′ double bond had the trans configuration. Furthermore, the axial orientations of H-5 and H-8 protons were assigned by the large coupling constants (JH5−H6 and JH7−H8 = 12.0 and 11.9 Hz, respectively). The

Figure 2. X-ray ORTEP diagram of compound 1.

Compound 2 was obtained as a white, amorphous powder. The molecular formula of 2 was established as C23H30O5 by HRESIMS and NMR data. The 1H and 13C NMR spectra of 2 were closely similar to those of 1. The different resonances were a methylene at C-7′ (δC 68.8) and H2-7′ (δH 5.13) and a carbonyl carbon at C-8′ (δC 167.9). The HMBC correlations of H2-7′ to C-4′ (δC 149.6), C-5′ (δC 97.9), and C-8′ (δC 167.9) indicated the presence of a lactone moiety that was connected to the phenylspirodrimane by the HMBC correlations of H2-7′ to C-3′ (δC 100.8), C-4′ (δC 149.6), and C-5′ (δC 97.9). Interestingly, the molecular formula of 2 was the same as that of stachybotrylactone.22,23 The lactone carbonyl of stachybotrylactone was located at C-7′ rather than C-8′ through the HMBC correlations of H-3′ to the carbonyl at C-7′ and lactone methylene protons to C-6′.22 On the other hand, the lactone carbonyl of 2 was located at C-8′. This was supported by the HMBC correlations and the differences in chemical shifts from using the same NMR solvent, acetone-d6 (Figure S26, Supporting Information). Therefore, the planar structure of 2 was assigned as a new member of the phenylspirodrimane family and designated as stachybotrylactone B (2). The relative configuration of 2 was the same as that of 1 (Figure 1). Application of the modified Mosher’s method24,25 determined the absolute configuration of the stereogenic center C-3. First, the hydroxy group in the benzene ring was methylated with TMS−CHN2 to give a methyl ester (2b). (S)- and (R)Methoxy(trifluoromethyl)phenylacetic acid (MTPA) esters were obtained by reacting 2b with (R)- and (S)-MTPA chloride, respectively, in separate experiments (Figure 3). The chemical shift differences (Δδ = δS − δR) calculated from the 2705

DOI: 10.1021/acs.jnatprod.6b00641 J. Nat. Prod. 2016, 79, 2703−2708

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Figure 3. Methylation, modified Mosher’s method, and ΔδS−R values (ppm) obtained for 2c and 2d. 1

H NMR spectra of the two diastereomeric esters 2c and 2d enabled us to determine the absolute configuration of C-3 as R. Therefore, the overall absolute configuration of 2 was determined to be 3R, 5S, 8R, 9R, and 10S. In order to determine the effects of 1 and 2 on osteoclast differentiation, bone marrow macrophage (BMM) cells isolated from mice were pretreated with 1 and 2 followed by both MCSF (30 ng/mL) and RANKL (100 ng/mL) treatment. RANKL induced osteoclast differentiation in the presence of M-CSF, as revealed by the appearance of TRAP-positive multinucleated osteoclasts. Compound 1 significantly reduced osteoclast differentiation in a dose-dependent manner, but compound 2 did not do this (Figure 4). To determine the inhibitory molecular mechanisms of 1 in osteoclastogenesis, the expression of the key transcription factors NFATc1 and c-Fos was tested. Western blot analysis showed that RANKL induced the expression of NFATc1 while c-Fos was significantly suppressed by 1 in a dose- and time-dependent manner (Figure 5A and B). RANK signaling is known to activate downstream signaling pathways, including MAPKs and NF-κB,1 and it was reported that inhibitors of MAPKs suppress osteoclast formation.26,27 To further elucidate the molecular mechanism of 1 on the inhibition of osteoclast differentiation, we investigated the effects of 1 on the RANKL-induced intracellular signaling pathways, MAPKs, and NF-κB. The phosphorylation of ERK, JNK, and p38 was diminished by 1 compared to vehicle in 5 min after RANKL treatment (Figure 5C). However, phosphor-

Figure 5. Effects of compound 1 on RANKL-induced signaling pathway. (A) BMM cells were pretreated with compound 1 (1−5 μg/ mL) or vehicle (DMSO) in the presence of M-CSF for 30 min and then were stimulated with RANKL (25 ng/mL) for 24 h. Cell lysates were prepared for subjection to Western blotting with specific antibodies. (B) Cells were treated with M-CSF, RANKL, and compound 1 (5 μg/mL) as in (A) and were lysed at the indicated times for immune-blot analysis with specific antibodies. (C) Cells were pretreated with compound 1 (5 μg/mL) or vehicle (DMSO) for 6 h in the presence of M-CSF (30 ng/mL) and were then stimulated with RANKL (100 ng/mL) for the indicated times. All of the cell lysates were extracted and subjected to Western blotting analysis with antibodies to p-ERK, ERK, p-p38, p38, p-JNK, JNK, p-IκBα, and IκBα. Actin served as a reference protein. The number of each band is expressed as relative to the control (vehicle, 0 min) after normalization for the levels of actin.

ylation of the inhibitory subunit of NF-κB alpha (IκBα) was not changed in the treatment with 1. This result suggests that the inhibition of ERK, JNK, and p38 phosphorylation might contribute to the inhibitory effect of RANKL-induced osteoclastogenesis. Although many phenylspirodrimane derivatives were shown to exhibit various biological activities, this paper is the first report on the inhibitory effect of osteoclastogenesis on phenylspirodrimane derivatives. Hence, we suggest that compound 1 could be developed as a potent antiosteoporosis therapeutic.

Figure 4. Effects of compounds on osteoclast differentiation. (A) BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) at the indicated doses of new compounds for 4 days. After culturing, the cells were fixed, and TRAP staining was performed. BMMs were cultured for 48 h with M-CSF (30 ng/mL) and RANKL (100 ng/mL) at the indicated doses of compound 1 and compound 2. (B and D) BMM viability was determined by the MTT assay. (C and E) TRAP activity was measured at 405 nm. All the bars represent mean ± SE from three independent experiments. The significance was determined by Dunnett’s multiple comparisons test. *p < 0.05, ***p < 0.001 versus untreated controls. 2706

DOI: 10.1021/acs.jnatprod.6b00641 J. Nat. Prod. 2016, 79, 2703−2708

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1457 cm−1; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS m/z 409.1985 [M + Na]+ (calcd for C23H30O5Na, 409.1991). Crystallization and Single-Crystal X-ray Structure Analysis of Stachybotrysin (1). Pillar-shaped, pale yellow crystals were grown from a MeOH−H2O (7:3 (v/v)) mixed solution. A single crystal with dimensions of 0.35 × 0.14 × 0.10 mm was mounted on a glass capillary and set on a Rigaku RAXIS-RAPID diffractometer. X-rays generated with a Cu sealed tube (40 kV, 40 mA) were monochromated to 1.541 84 Å by a graphite monochromator. The measurements were carried out by an oscillation method at 90 K. The unit cell dimensions were determined using 27 813 reflections with 4.460° ≤ 2θ ≤ 68.285°. The diffraction data of 30 809 up to (sin θ/ λ)max = 0.60 Å−1 were collected. Lorentz, polarization, and numerical absorption corrections were applied during the scaling processes, affording 4312 unique reflections with an Rint of 0.0328. The initial structure of 1 was solved by a direct method using the SIR2004 program28 and refined by a full matrix least-squares method that uses the SHELXL2014 program.29 The absolute structure of 1 was determined by using the anomalous dispersion effects of X-rays, which afforded a 3R, 5S, 8R, 9R, and 10S configuration with χ = 0.04(6)30 (Figure 2). Crystallographic data of compound 1 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1487073. Crystal Data of Stachybotrysin (1). C26H36O5·2H2O, fw = 464.58, monoclinic, space group P21, a = 10.09710(18) Å, b = 6.95501(13) Å, c = 18.0231(3) Å, β = 100.8400(7)°, V = 1243.10(4) Å3; DX = 1.241 Mg m−3; Z = 2; μ(Cu Kα) = 0.723 mm−1, R(F) = 0.0343, wR(F2) = 0.0744, and S = 1.146 for 3874 reflections of (sin θ/λ)max = 0.60 Å−1 with I > 2σ(I). Δρmin,max = −0.155, 0.218 e Å−3. Methylation of 2. The solution of compound 2 (1.5 mg) in MeOH (0.5 mL) was treated with 100 μL of trimethylsilyldiazomethane (2 M in diethyl ether) at room temperature for 24 h. The product formation was confirmed by LC/MS (ESIMS m/z 401 [M + H]+), and the reaction mixture was purified by semipreparative HPLC (Optimapak, C18, 10 × 250 mm, 10 μm, 3 mL/min) eluting with a gradient solvent system of 40−100% MeCN−H2O over 35 min to afford methylated product 2b (1.3 mg). 1H NMR (DMSO-d6, 700 MHz) of 2b: δH 6.66 (1H, s, H-3′), 5.24 (2H, s, H-7′), 4.09 (1H, d, J = 3.2 Hz, OH-3), 3.86 (3H, s, OMe), 3.17 (1H, d, J = 2.4 Hz, H-3), 3.07 (1H, d, J = 16.4 Hz, H-11), 2.72 (1H, d, J = 16.5 Hz, H-11), 2.05 (1H, dd, J = 12.3, 2.6 Hz, H-5), 1.83−1.77 (2H, m, H-2, 8), 1.68 (1H, td, J = 13.3, 3.4 Hz, H-1), 1.52 (1H, d, J = 10.1 Hz, H-7), 1.47 (1H, dd, J = 12.8, 6.7 Hz, H-6), 1.36−1.44 (3H, m, H-2, 6, 7), 0.94 (3H, s, H-14), 0.92 (1H, d, J = 12.5 Hz, H-1), 0.89 (3H, s, H-13), 0.79 (3H, s, H-14), 0.61 (3H, d, J = 6.5 Hz, H-12). MTPA Esterification of 2b. A slight excess of dimethylaminopyridine (DMAP) was added to a solution of 2b (0.65 mg) in 1.0 mL of anhydrous pyridine. The reaction mixture was stirred for 5 min and treated with 40 μL of (R)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl). The reaction continued for 24 h at room temperature. After confirming the successful product formation by LC/MS (ESIMS m/z 616 [M + H]+), the reaction was quenched by the addition of 50 μL of H2O. The product mixture was purified by semipreparative HPLC (Optimapak, C18, 10 × 250 mm, 10 μm, 3 mL/ min) that used a gradient solvent system of 60−100% MeCN−H2O to afford the (S)-MTPA ester of 2b (2c). The corresponding (R)-MTPA ester of 2b (2d) was obtained from the same esterification reaction with (S)-MTPA-Cl. 1H NMR (DMSO-d6, 900 MHz) of 2c: δH 7.41 (5H, m, aromatic), 6.67 (1H, s, H-3′), 5.24 (2H, q, J = 15.4 Hz, H-7′), 4.77 (1H, s, H-3), 3.87 (3H, s, OMe), 3.41 (3H, s, OMe), 3.10 (1H, d, J = 16.3 Hz, H-11), 2.75 (1H, d, J = 16.4 Hz, H-11), 2.04 (1H, t, J = 14.2 Hz, H-2), 1.91 (1H, dd, J = 10.8, 4.2 Hz, H-5), 1.83 (1H, dt, J = 11.0, 5.5 Hz, H-8), 1.64 (1H, m, H-2), 1.51 (2H, m, H-1, 7), 1.43 (2H, m, H-6), 1.41 (1H, m, H-7) 1.22 (1H, dd, J = 9.8, 3.3 Hz, H-1), 0.99 (3H, s, H-15), 0.92 (3H, s, H-13), 0.63 (3H, s, H-14), 0.59 (3H, d, J = 6.5 Hz, H-12). 1H NMR (DMSO-d6, 900 MHz) of 2d: δH 7.37 (2H, m, aromatic), 7.33 (3H, m, aromatic), 6.683 (1H, s, H-3′), 5.25 (1H, d, J = 15.6 Hz, H-7′), 5.20 (1H, d, J = 15.7 Hz, H-7′), 4.78 (1H, s, H3), 3.85 (3H, s, OMe), 3.42 (3H, s, OMe), 3.03 (1H, d, J = 16.3 Hz, H-11), 2.69 (1H, d, J = 16.3 Hz, H-11), 2.00 (1H, t, J = 14.1 Hz, H-2), 1.95 (1H, dd, J = 12.1, 2.8 Hz, H-5), 1.83 (1H, m, H-8), 1.59 (1H, d, J

EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured on a JASCO P-1020 polarimeter that uses a 100 mm glass microcell. UV spectra were obtained on a Shimadzu UV-1601 spectrophotometer. IR spectra were recorded on a Bruker VERTEX 80 V FT-IR spectrometer. The melting point was recorded on a Yanaco melting point apparatus. The NMR spectra were recorded on a Bruker AVANCE HD 900, 800, and 700 NMR spectrometer at the Korea Basic Science Institute (KBSI) in Ochang, South Korea. The proton and carbon NMR spectra were measured in DMSO-d6 solution at 800 and 200 MHz (1) and in DMSO-d6 and acetone-d6 solution at 700 and 175 MHz (2), respectively. For MTPA esters of 2, proton NMR spectra were recorded in DMSO-d6 solution at 900 MHz. Chemical shifts were referenced to a residual solvent signal (DMSO-d6 1 H δH 2.50, 13C δC 39.51; acetone-d6 1H δH 2.05, 13C δC 206.68). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired via a Q-TOF mass spectrometer on a SYNAPT G2 (Waters) at KBSI in Ochang, South Korea. Open column chromatography was performed with silica gel 60 (Merck, 0.063−0.200 mm) and ODS (Cosmosil, 75 μm). Analytical C18 (YMC, 5 μm, 4.6 × 150 mm) and semipreparative C18 (Optimapak, 10 μm, 10 × 250 mm) columns were used for HPLC on a Waters 515 HPLC system equipped with a photodiode array detector (Waters 2996) that uses HPLC grade solvents (Burdick & Jackson). α-MEM, fetal bovine serum, and penicillin were purchased from Invitrogen. The TRAP staining solution was from Sigma-Aldrich. Soluble human recombinant M-CSF and mouse RANKL were purchased from PeproTech EC. Specific antibodies against IκBα, c-Fos, and NFATc1 were obtained from Santa Cruz Biotechnology, Inc., and phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, and phospho-IκBα were from Cell Signaling Technology. Fungal Material. The fungal strain KCB13F013 was isolated from a marine sediment collected from the coast of Wi-island, South Korea. A GenBank search with the 26S and ITS rRNA genes of KCB13F013 indicated Stachybotrys eucylindrospora, Stachybotrys chartarum, and Stachybotrys subreniformis as the closest matches, which show sequence identities of 100%, 99%, and 99%, respectively. Therefore, the fungal strain was identified and named Stachybotrys sp. KCB13F013. Extraction and Isolation. For a scale-up culture, one agar plug taken from solid cultures was maintained on potato dextrose agar (PDA: 4 g of potato starch, 20 g of dextrose, and 18 g of agar per 1 L). This plug was then transferred into four 500 mL baffled Erlenmeyer flasks, respectively, each containing 100 mL of PDB (4 g of potato starch and 20 g of dextrose per 1 L). After incubating for 3 days at 28 °C in a rotary shaker (125 rpm), 1000 mL baffled Erlenmeyer flasks containing 250 mL of PDB, respectively, were inoculated with 2.5 mL of culture broth. These broth cultures (10 L) were incubated at 28 °C for 7 days in a rotary shaker (125 rpm). The broth and mycelia extracts were partitioned with EtOAc−H2O, and the EtOAc layer was dried in vacuo. The EtOAc extract was subjected to silica gel open column chromatography that used a stepwise gradient elution with CHCl3−MeOH (50:1 to 1:1) to yield eight fractions (Fr. 1−Fr. 8). Fr. 2 (CHCl3−MeOH, 20:1) was subjected to reversed-phase (ODS, 75 μm) flash column chromatography that used a stepwise gradient elution with MeOH−H2O (2:8 to 10:0) and further purified by semipreparative HPLC with an ODS column (Optimapak, C18, 10 × 250 mm, 10 μm) that used an isocratic elution with MeCN−H2O (40:60, 3 mL/min) to yield compounds 1 (5.8 mg) and 2 (2.6 mg), respectively. Stachybotrysin (1): pale yellow crystals; mp 125−127 °C; [α]23D −210 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 232 (3.97), 258 (3.88), and 353 (4.13) nm; IR (ATR) νmax 3244(br), 2948, 2876, 1572, 1443 cm−1; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS m/z 451.2456 [M + Na]+ (calcd for C26H36O5Na, 451.2460). Stachybotrylactone B (2): white, amorphous powder; [α]23D −54 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 226 (4.01), 259 (3.58), and 301 (4.26) nm; IR (ATR) νmax 3248(br), 2947, 2877, 1732, 1617, 2707

DOI: 10.1021/acs.jnatprod.6b00641 J. Nat. Prod. 2016, 79, 2703−2708

Journal of Natural Products

Article

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= 12.0 Hz, H-2), 1.53 (1H, m, H-6), 1.52 (1H, m, H-7), 1.44 (1H, m, H-6), 1.43 (1H, m, H-7), 1.27 (1H, td, J = 13.6, 3.6 Hz, H-1), 1.08 (1H, m, H-1), 0.98 (3H, s, H-15), 0.95 (3H, s, H-13), 0.88 (3H, s, H14), 0.57 (3H, d, J = 6.5 Hz, H-12). Biological Assays. The inhibitory effects on osteoclast differentiation in BMMs were evaluated by the procedure described previously.14

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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.6b00641. Crystallographic data (CIF) NMR and HRESIMS spectroscopic data of compounds of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (J.-H. Jang): +82-43-240-6164. Fax: +82-43-240-6169. Email: [email protected]. *Tel (J. S. Ahn): +82-43-240-6160. Fax: +82-43-240-6169. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support from a grant by the Global R&D Center (NRF-2010-00719) programs of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning of Korea (MSIFP). This research was also supported by grants from the Chungcheongbuk-do, KRIBB Research Initiative Program, and the Medicinal Crops Division, Ginseng and Medicinal Plants Research Institute, Rural Development Administration (PJ010221). We acknowledge the Korea Basic Science Institute, Ochang, South Korea, for providing NMR (700, 800, and 900 MHz) and HRESIMS data.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.6b00641 J. Nat. Prod. 2016, 79, 2703−2708