Bioactive Maleic Anhydrides and Related Diacids from the Aquatic

Apr 15, 2015 - Four maleic anhydride derivatives, tricladolides A–D (1–4), and three alkylidene succinic acid derivatives, tricladic acids A–C (...
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Bioactive Maleic Anhydrides and Related Diacids from the Aquatic Hyphomycete Tricladium castaneicola Chunguang Han,† Hiroyuki Furukawa,‡ Tomohiko Tomura,‡ Ryosuke Fudou,§ Kenichi Kaida,⊥ Bong-Keun Choi,∥ Genji Imokawa,# and Makoto Ojika*,‡ †

Research Center for Materials Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan § R&D Planning Department, Ajinomoto Co., Inc., Chuo-ku, Tokyo 104-8315, Japan ⊥ Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Kanagawa 210-8681, Japan ∥ Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Cheoin-gu, Yongin, Gyeonggi-Do 449-728, Korea # Research Institute for Biological Functions, Chubu University, Kasugai 487-8501, Japan ‡

S Supporting Information *

ABSTRACT: Four maleic anhydride derivatives, tricladolides A−D (1−4), and three alkylidene succinic acid derivatives, tricladic acids A−C (5−7), were isolated from the aquatic hyphomycete Tricladium castaneicola. The structures of these compounds were determined by spectroscopic analysis, and all were found to be novel. The compounds exhibited inhibitory activity against fungi, particularly Phytophthora sp., a plant pathogen of oomycetes. The inhibitory activity of these metabolites revealed the importance of the cyclic anhydride structure and the lipophilicity of the alkyl side chain. On the other hand, the cytotoxicity of the compounds against B16 melanoma cells indicated that the cyclic anhydride structure was not essential. quatic hyphomycetes (or “Ingoldian fungi”) are a group of fungi that evolved morphologically and functionally in order to adapt to freshwater environments.1 They are distinguished from other freshwater fungi by their characteristic ecology and morphology: they inhabit deciduous leaves decaying in cool mountain streams and creeks and asexually sporulate underwater to form relatively large multiradiate (often tetraradiate) or sigmoid spores.2,3 Given their specific characteristics and microbiological differentiation from general filamentous fungi, aquatic hyphomycetes are expected to produce novel secondary metabolites. However, because these fungi grow slowly, are difficult to culture, and require the use of special separation techniques to isolate, the discovery of secondary metabolites from these species has been challenging. Only a few such metabolites have been reported, for example, antifungal depsipeptides clavariopsins,4,5 anguillosporal,6 tenellic acids,7 and heliconols.8 Thus, this group of fungi remains a relatively unexplored microbial resource and a promising source for the discovery of new compounds. After collecting specimens of aquatic hyphomycetes in mountain freshwater environments and culturing purified strains, we found an extract of one strain showed potent inhibitory activity against a plant pathogen. As a result, seven novel compounds, the tricladolides A−D (1−4) and tricladic acids A−C (5−7), were isolated from the aquatic hyphomycete Tricladium castaneicola, and the isolation, structural elucidation, and antimicrobial and cytotoxic properties of these metabolites are reported.

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© 2015 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The aquatic hyphomycete T. castaneicola AJ117567 was isolated from leaves recovered from a mountain stream. The fungus cultured on oatmeal medium was extracted with acetone, and the extract partitioned between ethyl acetate and acidic water. The organic fraction was chromatographed on ODS to give fractions that exhibited anti-Phytophthora activity. The active fractions were subjected to HPLC and afforded four maleic anhydride derivatives: tricladolides A−D (1−4). To search for related metabolites, a larger scale culture was performed, and similar chromatographic steps afforded three maleic acid derivatives: tricladic acids A−C (5−7). The structures, except for the cyclic anhydride moieties, of the four maleic anhydrides 1−4 were elucidated using Received: October 3, 2014 Published: April 15, 2015 639

DOI: 10.1021/np500773s J. Nat. Prod. 2015, 78, 639−644

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Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for 1−4 tricladolide A (1) position

δC

1 2 3 4 5 6 7 8 9 10 11 12 13

167.0 136.4 137.2 118.7 147.1 34.4 27.4 32.4 151.7 122.1 171.9 165.3 10.1

δH (J in Hz)

6.26 7.12 2.31 1.71 2.31 7.06 5.86

d (15.6) dt (15.8, 6.8) m quint (7.6) m dt (15.8, 6.8) dt (16.0, 1.4)

2.12 s

tricladolide B (2) δC 166.4 135.1 137.2 117.5 147.3 33.8 27.9 31.7 132.0 129.9 63.6 164.6 9.3

tricladolide C (3)

δH (J in Hz)

6.24 7.12 2.31 1.61 2.11 5.68 5.68 4.11

d (16.0) dt (16.0, 7.5) q (7.4) quint (7.5) m m m d (4.2)

2.11 s

δC 166.4 135.2 137.2 117.3 147.6 34.4 28.5 25.4 39.0 68.0 23.6 164.6 9.3

δH (J in Hz)

6.24 7.12 2.31 1.52 1.39 1.47 3.81 1.20

d (15.6) dt (15.6, 7.2) q (7.2) m m, 1.47 m m m d (6.6)

2.11 s

tricladolide D (4) δC 166.4 134.9 137.3 117.1 148.0 34.4 28.4 28.9 31.6 22.5 14.0 164.6 9.3

δH (J in Hz)

6.24 7.12 2.28 1.49 1.32 1.32 1.32 0.89

dt (15.6. 1.5) dt (15.6, 7.2) q (7.2) quint (7.2) m m m t (7.2)

2.11 s

the adjacent olefinic protons. A weak rotation value ([α]D −0.4) for tricladolide C (3) suggests that this compound is a racemic mixture because the rotations for 10 methyl carbinols reported in several literature reports ranged from −3.5 to −10.6 (average of −6.6) for the S isomers.12 Preparation of the (R)MTPA ester 3r confirmed this conclusion; a 1:1 diastereomeric mixture was observed in the 1H NMR spectrum of ester 3r. The structures, except for the succinic acid moieties, of the three tricladic acids A−C (5−7) were also elucidated using spectroscopic techniques. Each methylene chain length was automatically assigned based on the MS data. The 1H and 13C chemical shifts for compounds 5−7 are listed in Table 2, and their 2D NMR correlations are summarized in Figure 3. The E geometry at C-3 of 7 was confirmed by the nuclear Overhauser effect spectroscopy (NOESY) correlation between H-5 and H13a (δ 5.62), and similar NMR data suggested the same geometry at C-3 in 5 and 6. However, these data were insufficient to confirm the complete diacid structure. It should be noted that broad absorption bands at 2500−3000 cm−1 and strong bands near 1700 cm−1 in the IR spectra of 5−7 suggest that these compounds are diacids rather than anhydrides. Additional evidence for the diacid nature was obtained from a comparison of the 1H chemical shifts of the olefinic protons for the three compounds 5−7 to those of the known compounds 9−11 (Table 3).13 Thus, the chemical shifts for H-13a of 5−7 and diacid compound 10 resonate at δ 5.60−5.63, whereas that for the anhydride derivative 9 is shifted downfield (δ 6.10). The peak for this proton in dimethyl ester derivative 11 also appears at a similar position to that for the diacid derivatives. Therefore, it can be concluded that the compounds 5−7 are succinic acid derivatives rather than anhydrides. The weak rotation value ([α]D −0.4) of 5 suggests that this compound is a racemic mixture, as is the case for 3, although the modified Mosher’s method was not applied. The absolute stereochemistry of 6 ([α]D +2) was, however, evaluated using the modified Mosher’s method. Although the succinic acid moiety of 6 was transformed to a maleic anhydride structure under the esterification conditions, the method was applicable for determining the stereochemistry at the local position 9. The chemical shift difference (Δδ = δS − δR) of (S)- and (R)-MTPA esters (Figure 4) indicated that 6 is partially racemic with the ratio 9S:9R = 3:1. Tricladolides 1−4 inhibited fungi, particularly oomycetes such as the plant pathogen Phytophthora, selectively but were inactive against yeasts and bacteria.9 The anti-Phytophthora

conventional spectroscopic techniques such as high-resolution MS and two-dimensional (2D) NMR. Their 1H and 13C chemical shifts are listed in Table 1, and their 2D NMR correlations are summarized in Figure 1. The IR absorption at

Figure 1. Two-dimensional NMR correlations in 1−4 (bold bonds: COSY; arrows: HMBC).

1818−1823 cm−1 for these compounds suggested the presence of the cyclic anhydride structures, which was further confirmed as follows. It has been reported that the equilibrium between maleic anhydride (A) and dianion (B) is pH dependent (Figure 2).11 The UV spectrum (λmax 311 nm) of 2 showed no shift

Figure 2. Equilibrium between the maleic anhydride (A) and opened diacid (B) forms.

upon the addition of HCl, whereas a hypsochromic shift of λmax to 264 nm was observed at pH 7.5 (MeOH−HEPES buffer). This result indicates that the opened form is stable only as the corresponding dianion B under mild basic conditions and the diacid form is readily converted to the stable cyclic anhydride A. In fact, compounds 1−4 were extracted from the culture broth at pH 3, and their UV spectra exhibited maxima near 310 nm. The presence of the maleic anhydride moiety was further confirmed by converting 4 to dimethyl ester 8, which like the dianion form, exhibited a maximum at 267 nm. These results support the maleic anhydride form of compounds 1−4. The geometry of 4E,9E for 1 and 2 and of 4E for 3 and 4 was determined by large coupling constants (ca. 16 Hz) between 640

DOI: 10.1021/np500773s J. Nat. Prod. 2015, 78, 639−644

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Table 2. NMR Spectroscopic Data [600 MHz, CDCl3−CD3OD (4:1)] for 5−7 tricladic acid A (5) position

δC

1 2 3 4 5 6 7 8 9 10 11 12 13

168.0 135.9 130.3 146.2 29.1 28.2 28.9 25.1 38.6 67.4 22.6 168.4 129.1

tricladic acid B (6)

δH (J in Hz)

6.99 2.20 1.46 1.32 1.33 1.45 3.73 1.15

δC

δH (J in Hz)

168.5 136.2 130.7 146.7 29.7 28.8 25.4 36.5 73.0 30.0 9.9 168.8 129.7

t (7.5) q (7.5) m m m m, 1.37 m m d (6.0)

5.61 d (1.2), 6.50 d (1.2)

7.00 2.22 1.47 1.34 1.37 3.46 1.41 0.93

t (6.8) q (6.8) m m, 1.46 m m, 1.44 m m m, 1.48 m t (7.2)

5.63 d (1.6), 6.51 d (1.6)

tricladic acid C (7) δC 168.0 135.8 130.2 146.3 29.1 28.2 28.8 28.9 25.3 32.1 62.0 168.4 129.2

δH (J in Hz)

7.00 2.20 1.46 1.32 1.32 1.32 1.53 3.57

t (7.4) q (7.4) quint (7.4) m m m m t (6.6)

5.62 d (1.6), 6.51 d (1.6)

Figure 3. Two-dimensional NMR correlations for 5−7 (bold bonds: COSY; arrows: HMBC).

Table 3. 1H NMR Data for the Olefinic Regions in 5−7 and the Known Related Compounds 9−11a position H-4 H-13a H-13b

5b 6.99 (t, 7.5) 5.61 (d, 1.2) 6.50 (d, 1.2)

6b 7.00 (t, 6.8) 5.63 (d, 1.6) 6.51 (d, 1.6)

7b 7.00 (t, 7.4) 5.62 (d, 1.6) 6.51 (d, 1.6)

9c,d 7.10 (t, 7.0) 6.10 (s) 6.50 (s)

10c,e

11c,e,f

6.92 (t, 8.0) 5.60 (d, 1.5) 6.51 (d, 1.5)

6.92 5.60 6.51

a Chemical shifts δ are in ppm; multiplicities and coupling constants (J in Hz) are in parentheses. bRecorded in CDCl3−CD3OD (4:1). cData reported in ref 13. dRecorded in CCl4. eRecorded in CDCl3. fJ values were not provided in ref 13.

Figure 5. Anti-Phytophthora activity of tricladolides A−D (1−4), tricladic acids A−C (5−7), and dimethyl ester derivative 8 in comparison with control fungicides (Met: metalaxyl; Amp: amphotericin B).

D (4), possessing a lipophilic alkyl chain, showed the highest activity. The substitution of a polar functional group on the alkyl chain, particularly via hydroxylation, as in tricladolides B (2) and C (3), strongly reduced the activity, although, despite its polarity, the carboxyl group on the side chain in 1 did not markedly affect the activity. The maleic anhydride moiety may be important, but does not appear to be essential, as the dimethyl ester 8 derived from 4 retains half of the activity of 4, suggesting that the alkyl side chain is more important than the maleic anhydride moiety. In addition, because the succinic acidbased tricladic acids B (6) and C (7) were somewhat active despite the hydroxylation on the alkyl chain, it is thought that the succinic acid moiety may be more effective for antiPhytophthora activity than maleic anhydride. Tricladolide D (4), the most lipophilic compound among the seven natural products, exhibited the highest inhibitory activity against the B16 mouse melanotic melanoma cell line (Figure 6A). The artificial diester derivative 8 was more active than 4. The cytotoxicity of 4 and 8 was further evaluated in a dosedependent manner, providing IC50 values of 80 and 30 μM,

Figure 4. Δδ values (δS − δR) for the two MTPA esters of the major enantiomer of 6.

activity of the compounds in this study was evaluated using a paper disk diffusion assay. Metalaxyl as one of the commercial anti-Phytophthora pesticides and amphotericin B as a typical antifungal antibiotic were used as controls, although the latter was inactive against oomycetes such as Phytophthora. The activity against the vegetable pathogen P. capsici varied with structural differences of the compounds (Figure 5); tricladolide 641

DOI: 10.1021/np500773s J. Nat. Prod. 2015, 78, 639−644

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Figure 6. Cytotoxicity of tricladolides A−D (1−4), tricladic acids A−C (5−7), and dimethyl ester derivative 8 against the B16 melanoma cell line. (A) Relative cell viability of all compounds 1−8 at 100 μg/mL. P: paclitaxel at 100 μg/mL as a positive control. (B) Dose-dependent inhibition curves (sigmoid fitting) for 4 (open circles) and 8 (open squares) with paclitaxel (open triangles, dotted line). 4 times). The combined extracts were concentrated. After adjusting the pH of the aqueous residue (30 mL) to 3 with concentrated hydrochloric acid, it was extracted with ethyl acetate (30 mL, 3 times). The organic layers were then combined, washed with water (90 mL, 3 times), dried over Na2SO4, and concentrated to give a residue (470 mg). This extract was chromatographed on a preparative column {ODS [15 inner diameter (i.d.) × 300 mm], GL Sciences} using 30% to 100% MeCN in water stepwise. The fraction eluted with 30% MeCN, which exhibited anti-Phytophthora activity, was purified by HPLC [CAPCELL PAK C18 UG120 (15 i.d. × 250 mm, Shiseido, Tokyo), 40% MeCN, 8 mL/min, detected at 210 nm] to give 1 (4 mg, tR = 17.7 min), 2 (11 mg, tR = 19.6 min), and 3 (12 mg, tR = 21.5 min). The other active fraction eluted with 80% MeCN was also purified by HPLC [CAPCELL PAK C18 UG120 (15 i.d. × 250 mm), 70% MeCN, 8 mL/min, detected at 210 nm] to give 4 (8 mg, tR = 14.6 min). To obtain other minor metabolites, the mycelia from 72-flask cultures were extracted with 9 L of acetone. After the same protocol described above was followed, the ethyl acetate extract (23 g) was purified by HPLC [CAPCELL PAK C18 UG120 (50 i.d. × 250 mm), 25% MeCN−0.1% trifluoroacetic acid (TFA), 80 mL/min, detected at 210 nm] to give four anti-Phytophthora fractions (fr.1: 413 mg; fr.2: 548 mg; fr.4: 960 mg; and fr.5: 2.16 g). Fraction 1 (56 mg) was purified using a two-step HPLC process [1. CAPCELL PAK C18 UG80 (20 i.d. × 250 mm), 20% MeCN−0.1% TFA, 8 mL/min; 2. Same column, EtOH−MeCN−H2O (2:5:18)−0.1% TFA, 8 mL/min, detected at 210 nm] to give 5 (3.6 mg, tR = 64.6−70.6 min). Fraction 2 (46 mg) was purified by HPLC (same column, 22% MeCN−0.1% TFA, 8 mL/min, detected at 210 nm) to give 6 (7.5 mg, tR = 52.2− 55.1 min) and 7 (7.2 mg, tR = 61.2−66.0 min). Tricladolide A (1): colorless powder; mp 99−102 °C; UV (dioxane) λmax (log ε) 308 (4.05) nm; IR (KBr) νmax 3422 (br), 3400−2400 (br), 1823, 1767, 1751, 1682, 1642, 1286, 1218, 986, 919, 894, 730 cm−1; HRESIMS m/z 273.0771 [M + Na]+ (calcd for C13H14O5Na 273.0734). Tricladolide B (2): pale yellow oil; UV (dioxane) λmax (log ε) 309 (4.14) nm; UV (MeOH−10 mM HEPES, pH 7.5) λmax (log ε) 264 nm (4.14); IR (film) νmax 3374 (br), 1818, 1772, 1653, 1278, 1136, 971, 923, 731, 669 cm−1; HRESIMS m/z 259.0956 [M + Na]+ (calcd for C13H16O4Na 259.0941). Tricladolide C (3): pale yellow oil; [α]27D −0.4 (c 0.78, CHCl3); UV (dioxane) λmax (log ε) 309 (3.71) nm; IR (film) νmax 3567, 3420 (br), 1821, 1771, 1720, 1652, 1277, 1135, 972, 925, 731, 568 cm−1; HRESIMS m/z 261.1119 [M + Na]+ (calcd for C13H18O4Na 261.1097). Tricladolide D (4): pale yellow oil; UV (dioxane) λmax (log ε) 310 (4.01) nm; IR (film) νmax 1820, 1767, 1653, 1276, 1137, 972, 924, 730, 568 cm−1; HRESIMS m/z 223.1304 [M + H]+ (calcd for C13H19O3 223.1329).

respectively, which were comparable to the activity of paclitaxel (IC50 = 56 μM) (Figure 6B).

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific rotations were recorded on a DIP-370 spectrometer (JASCO, Tokyo, Japan). IR spectra were measured using an FT-IR-7000S spectrometer (JASCO). UV spectra were obtained using a V-530 spectrometer (JASCO). High-resolution mass spectra (HRMS) were recorded on a Mariner Biospectrometry workstation (Applied Biosystems, Foster City, CA, USA) in the positive electrospray ionization (ESI) mode using 50% MeCN−0.1% HCOOH as the infusion solvent. NMR spectra were recorded on an AMX2 600 (600 MHz for 1H) or ARX 400 (400 MHz for 1H) spectrometer (Bruker BioSpin, Yokohama, Japan). The chemical shifts (ppm) were referenced to the tetramethylsilane (TMS) peak. Preparative high-performance liquid chromatography (HPLC) was performed using a high-pressure gradient system (JASCO) composed of a PU-1586 pump, DG-1580-53 degasser, and UV 1570 detector. Fungal Strain and Culture. The aquatic hyphomycete (strain AJ117567) was isolated from leaf litter in a mountain stream in Hakone, Kanagawa, Japan. The collected fallen leaves were dipped in water, and a portion of the water surface was placed on low carbon agar (LCA) medium (in 1 L; glucose (1 g), KH2PO4 (0.1 g), MgSO4· 7H2O (0.2 g), KCl (0.2 g), NaNO3 (2 g), yeast extract (0.2 g), agar (15 g), pH 6.5). After detecting the conidia under a microscope, single-spore isolation and culture were performed. Spore germination was detected 2 days after inoculation. On the basis of the gene sequence of the 28S rRNA-D1/D2 region (DDBJ accession number LC033787), this strain was considered to belong to the genus Tricladium, with the closest species being T. castaneicola [100% sequence similarity (582 bp)]. The morphological characteristics of the strain coincide with the taxonomic criteria of this fungal species.9 With the above-mentioned information, the fungus was assigned to Tricladium castaneicola, in Helotiaceae, Helotiales, Leotiomycetes. The strain was deposited as FERM P-19644 in the NITE-International Patent Organism Depositary (NITE-IPOD, Tsukuba, Japan). The strain was cultured on the malt extract (Difco) agar medium at 20 °C for 20 days. For the production of secondary metabolites, pieces of a colony of T. castaneicola AJ117567 were inoculated on an oatmeal medium [oatmeal (20 g, Nippon Food Manufacturer, Sapporo, Japan) and 28 mL of the liquid medium (glucose (2 g), fructose (5 g), sucrose (8 g), NZ amine (2 g, Wako Pure Chemical Industries, Osaka, Japan), MgSO4·4H2O (0.5 g), KCl (0.5 g), ZnSO4·7H2O (0.5 g), and KH2PO4 (1 g)) in 1 L (pH 6)] in a 1 L flask and incubated under static conditions at 20 °C for 14 days. Isolation of 1−7. T. castaneicola AJ117567 grown on the oatmeal production medium (four flasks) was extracted with acetone (125 mL, 642

DOI: 10.1021/np500773s J. Nat. Prod. 2015, 78, 639−644

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Tricladic acid A (5): pale yellow oil; [α]25D −0.4 (c 0.35, MeOH); UV (MeOH) λ (log ε) 200 nm (4.08, end abs); IR (film) νmax 1699, 1622, 1279, 1201, 1139, 1047, 962, 757, 721, 688 cm−1; HR ESIMS m/z 221.1188 [M − OH]+ (calcd for C13H17O3 221.1172). Tricladic acid B (6): pale yellow oil; [α]25D +2.0 (c 0.34, MeOH); UV (MeOH) λ (log ε) 200 nm (4.08, end abs); IR (film) νmax 1695, 1622, 1282, 1169, 963, 758 cm−1; HRESIMS m/z 221.1180 [M − OH]+ (calcd for C13H17O3 221.1172). Tricladic acid C (7): pale yellow oil; UV (MeOH) λ (log ε) 200 nm (4.11, end abs); IR (film) νmax 1695, 1622, 1279, 1200, 1051, 963, 758, 721 cm−1; HRESIMS m/z 221.1177 [M − OH]+ (calcd for C13H17O3 221.1172). Preparation of the Mosher Ester of Tricladolide C (3). Compound 3 (0.4 mg, 1.7 μmol) was treated with (S)-methoxy(trifluoromethyl)phenylacetyl (MTPA) chloride (15 μL, 80 μmol) in dry pyridine (0.4 mL) at room temperature for 6 h. The reaction mixture was then purified on an ODS column (2 g, Cosmosil 75C18OPN, Nacalai Tesque Inc., Kyoto, Japan) using MeCN−H2O (5:5, 6:4, 7:3, 8:2, 9:1, 10:0) to give the (R)-MTPA ester 3r (0.1 mg) as a 1:1 mixture of diastereomers: 1H NMR (600 MHz, CDCl3) δ 7.08 (1H, m, H-5), 6.22 and 6.20 (0.5H and 0.5H, d, J = 16.0 Hz, H-4), 5.15 (1H, m, H-9), 2.27 and 2.20 (1H and 1H, q, J = 7.2 Hz, H-6), 2.11 and 2.09 (1.5H and 1.5H, s, H-13), 1.51 and 1.43 (total 2H, m, H-7), 1.60−1.70 (2H, m, H-9), 1.40−1.50 (2H, m, H-8), 1.35 and 1.27 (1.5H and 1.5H, d, J = 6.0 Hz, H-11). Preparation of the Mosher Esters of Tricladic Acid B (6). Compound 6 (0.8 mg, 3.1 μmol) was treated with (R)-MPTA chloride (6 μL, 32 μmol), and then the product was purified under the same conditions as described above for 3r to give the (S)-MTPA ester 6s (0.5 mg, 35%) as a 3:1 mixture of diastereomers in the maleic anhydride form: 1H NMR (600 MHz, CDCl3, major isomer) δ 7.06 (1H, dt, J = 15.6, 6.6 Hz, H-5), 6.23 (1H, d, J = 15.6 Hz, H-4), 5.06 (1H, m, H-9), 2.29 (2H, m, H-6), 2.10 (3H, s, H-13), 1.70 (2H, m, H8), 1.64 (2H, m, H-10), 1.51 (2H, m, H-7), 0.83 (3H, t, J = 7.8 Hz, H11). Using (S)-MPTA chloride, the anhydride form of (R)-MTPA ester 6r (0.4 mg, 29%) was obtained in the same manner as a 3:1 mixture of diastereomers: 1H NMR (600 MHz, CDCl3, major isomer) δ 7.01 (1H, dt, J = 15.6, 6.6 Hz, H-5), 6.19 (1H, d, J = 15.6 Hz, H-4), 5.06 (1H, m, H-9), 2.21 (2H, m, H-6), 2.10 (3H, s, H-13), 1.69 (2H, m, H-10), 1.61 (2H, m, H-8), 1.38 (2H, m, H-7), 0.93 (3H, t, J = 7.8 Hz, H-11). Preparation of Dimethyl Ester 8 from Tricladolide D (4). A mixture of 4 (0.5 mg, 2.2 μmol) and concentrated H2SO4 (15 μL) in 1.5 mL of MeOH was heated at reflux for 5 h. After the removal of MeOH, the residue was dissolved in water (1.0 mL) and extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were evaporated and purified by HPLC [Develosil ODS-UG-5 (10 i.d. × 250 mm), 70% MeCN(aq), 4 mL/min] to give dimethyl ester 8 (0.6 mg, tR = 19.6 min): UV (MeCN) λmax (log ε) 267 nm (4.23); IR νmax (film) 1738, 1718, 1265, 1198, 1170, 961, 769 cm−1; 1H NMR (400 MHz, CDCl3) δ 6.33 (1H, d, J = 16.0 Hz), 5.95 (1H, dt, J = 16.0, 7.3 Hz), 3.86 (3H, s), 3.74 (3H, s), 2.20 (2H, quart, J = 7.3 Hz), 2.00 (3H, s), 1.42 (2H, m), 1.27 (6H, m), 0.88 (3H, t, J = 7.3 Hz); 13C NMR (100 MHz, CDCl3) δ 169.6, 167.6, 141.8, 141.7, 123.8, 123.6, 52.2, 52.3, 33.6, 31.6, 28.8, 28.6, 22.5, 14.1, 13.5; HRESIMS m/z 291.1583 [M + Na]+ (calcd for C15H24O4Na 291.1567). Anti-Phytophthora Activity. The phytopathogenic oomycete Phytophthora capsici NBRC 30696, purchased from NITE-Biological Resource Center (NBRC, Chiba, Japan), was cultured on a potato-agar medium [in 1 L, potato broth from 200 g of fresh potato, glucose (20 g), and agar (20 g)] in a 9 cm dish at 25 °C for 7 days in the dark. A piece of the colony was then inoculated on the center of a 5% V8-agar medium [V8 vegetable juice (5 mL) and water (95 mL)] in a 9 cm dish and incubated at 25 °C for 48 h in the dark until the colony grew to approximately 3−4 cm in diameter. A paper disc (8 mm in diameter) impregnated with a sample was placed 1 cm away from the front of the colony. After incubating for 22−24 h, the distance between the edge of the colony and the paper disc (control: 0 mm) was measured.

Cytotoxicity Assay. B-16 mouse melanotic melanoma cells10 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (MP Biomedicals, CA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., MA, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific Inc.). A total of 5000 cultured cells were seeded into each well of a 96-well plate containing 99 μL of the same medium. After preincubation for 24 h at 37 °C in an atmosphere of 5% CO2, a compound in 1 μL of dimethyl sulfoxide (DMSO) was added to each well, and the cells were incubated an additional 48 h. A solution (10 μL) of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer saline (PBS) (5 mg/mL) was then added to each well, and the plate was incubated for an additional 3 h. Subsequently, the medium was removed by aspiration, any generated formazan was dissolved in 100 μL of DMSO, and the absorbance was measured at 595 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific).

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of all new compounds (1−7); a photomicrograph of conidia of T. castaneicola. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-52-789-4116. Fax: +81-52-789-4118. E-mail: ojika@ agr.nagoya-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Mr. M. Ishihara for culturing the fungus and chromatographic analysis and to Ms. M. Komizu for helping us to purify the metabolites.

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REFERENCES

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DOI: 10.1021/np500773s J. Nat. Prod. 2015, 78, 639−644

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DOI: 10.1021/np500773s J. Nat. Prod. 2015, 78, 639−644