Isoindolinones, Phthalides, and a Naphthoquinone from the Fruiting

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Isoindolinones, Phthalides, and a Naphthoquinone from the Fruiting Body of Daldinia concentrica Hitoshi Kamauchi, Yuki Shiraishi, Akiyo Kojima, Naoki Kawazoe, Kaoru Kinoshita, and Kiyotaka Koyama* Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan S Supporting Information *

ABSTRACT: A chemical investigation of the ascomycetes of Daldinia concentrica was performed using silica gel column chromatography, ODS column chromatography, and preparative HPLC. Two new isoindolinone compounds, daldinans B (1) and C (2), two new phthalide compounds, daldinolides A (3) and B (4), and a new naphthoquinone, daldiquinone (5), were isolated together with two known compounds (6 and 7). The structures of 1, 2, and 5 were established using NMR, MS, and IR methods, and the structures of 3 and 4 were determined by derivatization from known compounds (6 and 7). 5 exhibited antiangiogenesis activity against HUVECs (IC50 = 7.5 μM). downfield shifted proton at δH 6.00 (brs, H-3), one methyl proton at δH 2.17 (s, H-12-CH3), two methoxy protons at δH 3.85 (s, H-7-OCH3) and 3.86 (s, H-10-OCH3), and a pair of methylene protons at δH 3.26 and 4.39 (each, d, J = 17.5 Hz, H1′). Furthermore, the 13C NMR spectrum showed an amide and carboxyl carbon at δC 166.8 (C-1) and 170.7 (C-2′), 12 aromatic carbons at δC 98.6 (C-6), 101.8 (C-4), 109.5 (C-7a), 112.0 (C-11), 117.5 (C-13), 123.7 (C-8), 128.2 (C-12), 142.5 (C-9), 147.7 (C-10), 151.4 (C-3a), 157.9 (C-7), and 162.6 (C5), one methine carbon at δC 56.4 (C-3), one methyl carbon at δC 20.7 (C-12-CH3), two methoxy carbons at δC 55.3 (C-7OCH3) and 55.7 (C-10-OCH3), and one methylene carbon at δC 41.8 (C-1′) (Table 1). These spectroscopic data for 1 were similar to that of the known isoindolinone compounds daldinan A (8) and entonalactam A (9).14 Therefore, 1 might have the 5hydroxy-7-methoxyisoindolinone skeleton with a 2-hydroxy-3methoxy-5-methylphenyl group at C-3. The NMR assignment of 1 was achieved using the HMBC method and comparison of the NMR data with known isoindolinone derivatives.12,14 The isoindolinone moiety was assigned by HMBC correlations observed in DMSO-d6 from H-4 to C-6 and C-7a and from H-6 to C-5, C-7, and C-7a and the correlations from H-4 to C-3 and C-5 observed in MeOH-d4. Moreover, the HMBC correlations from H-11 to C-9, C-10, and C-13 and from H-12-CH3 to C11, C-12, and C-13 could assign the 5-methylphenyl moiety.

F

ungi from the family Xylariaceae produce a variety of structurally unusual secondary metabolites.1−6 Benzo[j]fluoranthene derivatives isolated from Hypoxylon and Annulohypoxylon spp. showed antiangiogenesis activity.7,8 Azaphilones, benzophenones, binaphthyls, steroids, isoindolinones, and benzo[j]fluoranthene derivatives have been isolated from the ascomycetes of the Xylaria Daldinia concentrica.9−12 Structurally unique polyketides named daeschol A were isolated from D. eschscholzii and showed immunosuppressive activity.13 In this study, we report the isolation and structure determination of two new isoindolinone compounds, daldinans B (1) and C (2), two new phthalide compounds, daldinolides A (3) and B (4), and a new binaphthyl quinone, daldiquinone (5). Their chemical structures were determined by 1D/2D NMR and MS analysis. The fruiting bodies of D. concentrica were extracted with CHCl3 and MeOH. Each extract was subjected to silica gel column chromatography, Sephadex LH-20 column chromatography, octadecyl silyl silica gel column chromatography, and preparative HPLC. Five new compounds, daldinans B (1) and C (2), daldinolides A (3) and B (4), and daldiquinone (5), were isolated. Daldinan B (1) was obtained as a white powder. The molecular formula of 1 was determined to be C19H19NO7 on the basis of HRFABMS. The IR spectrum of 1 suggested the presence of hydroxy group (3307 cm−1) and carbonyl and amide (1662 and 1602 cm−1). The 1H NMR spectrum showed signals attributable to four aromatic protons at δH 6.09 (brs, H13), 6.32 (brs, H-4), 6.42 (brs, H-6), and 6.77 (s, H-11), one © 2018 American Chemical Society and American Society of Pharmacognosy

Received: November 17, 2017 Published: May 1, 2018 1290

DOI: 10.1021/acs.jnatprod.7b00976 J. Nat. Prod. 2018, 81, 1290−1294

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Table 1. NMR Spectroscopic Data of 1−4 daldinan B (1)a position

a

δC

type

1 2 3 3a 4 5 6 7 7a 8 9 10 11 12 13 12-CH3 7-O-CH3 9-O-CH3 10-O-CH3 1′

166.8

C

56.4 151.4 101.8 162.6 98.6 157.9 109.5 123.7 142.5 147.7 112.0 128.2 117.5 20.7 55.3

CH C CH C CH C C C C C CH C CH CH3 CH3

55.7 41.8

CH3 CH2

2′ 3′ 4′

170.7

C

δH (J in Hz)

6.00 (brs) 6.32 (brs) 6.42 (brs)

6.77 (s) 6.09 (brs) 2.17 (s) 3.85 (s) 3.86 (s) 3.26 (d, 17.5) 4.39 (d, 17.5)

daldinan C (2)b,c δC

type

170.3

C

57.6 153.2 102.9 164.8 99.1 159.6 110.9 123.7 144.3 148.9 112.3 130.2 118.9 21.2 55.8

CH C CH C CH C C C C C CH C CH CH3 CH3

56.2 40.3

CH3 CH2

24.7 32.6 177.4

CH2 CH2 C

daldinolide A (3)b

daldinolide B (4)b

δH (J in Hz)

δH (J in Hz)

6.25 (s)

6.24 (d, 1.6)

6.32 (d, 1.8)

6.38 (s)

6.48 (d, 1.6)

6.58 (d, 1.8)

6.69 (s)

6.90 (s)

6.80 (s)

5.91 (s) 2.12 (s) 3.87 (s)

7.22 2.35 3.91 3.38 3.79

6.95 (s) 2.21 (s) 3.88 (s)

δH (J in Hz)

6.06 (s)

3.87 2.83 3.80 1.82 2.25

(s) (dt, 12.9, 7.0) (m)d (t, 7.0) (t, 7.0)

(s) (s) (s) (s) (s)

3.84 (s)

Measured in DMSO-d6. bMeasured in MeOH-d4. cMeasured at −20 °C. dOverlapped signal with MeOH-d4.

Methoxy groups were identified at C-7 and C-10 by the presence of HMBC correlations from H-7-OCH3 to C-7 and H-10-OCH3 to C-10 and by NOESY correlations from 12-CH3 to H-13 and H-11 and from H-11 to 10-OCH3 (Figures 1 and

Figure 2. NOESY correlations of 1 and 2.

show any Cotton effect, indicating that 1 was isolated as a racemic mixture. Daldinan C (2) was isolated as a white powder. The molecular formula of 2 was established to be C21H23NO7 on the basis of HRFABMS. The 1H and 13C NMR data of 2 were similar to those of 1 (Table 1), and thus assignments of the 1H and 13C NMR spectroscopic data of isoindolinone and the 5methylphenyl moiety were made by comparison with those of 1. The HMBC correlations from H-7-OCH3 to C-7 and from H-10-OCH3 to C-10 suggested the presence of two methoxy groups, one each at C-7 and C-10. The difference in the structures of 1 and 2 was the presence of two methylenes (δH 1.82, t, J = 7.0 Hz, δC 24.7 at C-2′ and δH 2.25, t, J = 7.0 Hz, δC 32.6 at C-3′) in 2. The 1H−1H COSY and HMBC correlations

Figure 1. HMBC (blue arrows) and 1H−1H COSY (pink bold lines) correlations of 1 in DMSO-d6 (* was observed in MeOH-d4) and 2 in MeOH-d4.

2). The assignments of C-3a and C-8 were made by comparison of the NMR data with known isoindolinone derivatives.12,14 The structures of entonalactam A and 1 differed by the presence of a methylene (δH 3.26 and 4.39, each d, J = 17.5 Hz, δC 41.8 at C-1′) and a carboxyl group (δC 170.7 at C2′) in 1. The HMBC correlations from H-1′ to C-1, C-3, and C-2′ indicated that a carboxymethyl group was bonded at N-2. The electronic circular dichroism (ECD) spectrum of 1 did not 1291

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basis of HREIMS. The IR spectrum of 5 showed the presence of hydroxy (3341 cm−1) and two carbonyl (1662 and 1607 cm−1) groups. The 1H NMR spectrum showed signals attributable to nine aromatic protons at δH 6.48 (d, J = 7.8 Hz, H-8), 6.49 (s, H-2), 6.85 (dd, J = 7.3, 1.4 Hz, H-6′), 6.97 (d, J = 7.7 Hz H-3′), 7.07 (d, J = 8.6 Hz, H-6), 7.24 (dd, J = 8.4, 1.4, H-8′), 7.26 (overlapped, H-7′), 7.31 (d, J = 7.7 Hz, H-2′), and 7.34 (dd, J = 8.6, 7.8 Hz, H-7), two methoxy protons at δH 4.03 (s, H-5-OCH3) and 4.12 (s, H-5′-OCH3), and one hydroxy proton at δH 9.64 (s, H-4′-OH). Furthermore, the 13C NMR spectrum showed two carbonyl carbons at δC 178.8 (C3) and 181.1 (C-4), 18 aromatic carbons at δC 104.6 (C-6′), 110.1 (C-3′), 115.1 (C-4a′), 115.2 (C-6), 119.2 (C-4a), 119.8 (C-8′), 123.1 (C-8), 125.5 (C-1′), 126.7 (C-7′), 128.4 (C-2′), 129.3 (C-2), 134.2 (C-8a′), 136.4 (C-7), 138.3 (C-8a), 155.9 (C-4′), 156.4 (C-5′), 157.0 (C-1), and 162.9 (C-5), and two methoxy carbons at δC 56.4 (C-5′-OCH3) and 56.5 (C-5OCH3) (Table 2). These spectroscopic data for 5 were similar

suggested that the side chain of 2 is a carboxypropyl group (Figure 1). Optical rotation and the ECD spectrum suggested that 2 was also a racemic mixture. Daldinolide A (3) was obtained as a white powder. The molecular formula of 3 was determined to be C18H18O7 on the basis of HRFABMS. The IR spectrum of 3 showed absorptions attributable to hydroxy (3348 cm−1) and carbonyl (1719 cm−1) groups. The 1H NMR spectrum of 3 was similar to that of 1 and 2, but 3 had an additional methoxyl group (δH 3.38, s) and lacked a side chain (Table 1). The molecular formula showed that 3 did not contain nitrogen, suggesting that 3 had a phthalide structure, in which the nitrogen of isoindolinone was replaced with oxygen. However, the molecular formula indicated the possibility of two structural isomers: a benzophenone type and a phthalide type. Benzophenone and phthalide compounds have different UV absorbance. The benzophenone compound daldinal A (6) showed absorbance at 321 nm due to the long conjugate system containing a carbonyl group.10 On the other hand, phthalide compounds do not show absorbance over 300 nm because the conjugate system is interrupted by the absence of a carbonyl group. Isoindolinone compounds 1 and 2, in which the phthalide oxygen was replaced with nitrogen, did not show absorbance around 320 nm. Compound 3 was determined to be a phthalide derivative due to its UV absorption at 203, 225, 261, and 294 nm. However, a 13C NMR spectrum of 3 was very broad and did not provide enough signals to determine its chemical structure. Daldinolide B (4) was obtained as a yellow oil. The molecular formula of 4 was determined to be C17H16O7 on the basis of HRFABMS m/z 333.0976 [M + H]+. 1H NMR and the UV spectra of 4 were similar to those of 3 (Table 1). The 1H NMR spectrum showed two methoxy groups, which is one methoxy group less than that of 3. Therefore, the structure of 4 was essentially the same as that of 3 except the number of methoxy groups. Similar to 3, structure determination of 4 was not completed because the 13C NMR spectrum was also broad. We determined the structures of 3 and 4 by semisynthesis. The aldehyde group of known compounds 6 and 7 was oxidized to the carboxyl group using NaClO2. The carboxyl group of the intermediates (10 and 11) spontaneously attacked

Table 2. NMR Spectroscopic Data of Daldiquinone (5)a

Scheme 1. Derivatization of 6 and 7 to 3 and 4

position

δC

type

1 2 3c 4c 4a 5 6 7 8 8a 5-OCH3 1′ 2′ 3′ 4′ 4a′ 5′ 6′ 7′ 8′ 8a′ 4′−OH 5′-OCH3

157.0 129.3 178.8 181.1 119.2 162.9 115.2 136.4 123.1 138.3 56.5 125.5 128.4 110.1 155.9 115.1 156.4 104.6 126.7 119.8 134.2

C CH C C C C CH CH CH C CH3 C CH CH C C C CH CH CH C

56.4

CH3

a Measured in CDCl3. interchanged.

b

δH (J in Hz) 6.49 (s)

7.07 (d, 8.6) 7.34 (dd, 8.6, 7.8) 6.48 (d, 7.8) 4.03 (s) 7.31 (d, 7.7) 6.97 (d, 7.7)

6.85 (dd, 7.3, 1.4) 7.26 (m)b 7.24 (dd, 8.4, 1.4) 9.64 (s) 4.12 (s)

Overlapped signal with CDCl3. cMay be

to those of the known binaphthyl compound daldinol.10 The HMBC correlations from H-5-OCH3 to C-5 and H-5′-OCH3 to C-5′ were assigned to two methoxy groups. The 1,2naphthoquinone group at C-3 and C-4 was determined by the 1 H NMR spectrum of the H-2 and HMBC correlations. The 1H NMR chemical shift of H-2 in 1,2-naphthoquinone is 6.38 ppm,15 but 7.07 ppm in 1,4-naphthoquinone.16 The 1H NMR chemical shift of H-2 in 5 was 6.47 ppm, and an HMBC correlation from H-8 to C-1 was consistent with the 1,2naphthoquinone group (Figure 3). Antiangiogenesis activity was evaluated by testing growth inhibition of human umbilical vein endothelial cells (HUVECs). Angiogenesis plays a critical role in the growth of tumor cells, and tumor angiogenesis inhibitors are considered novel targets for anticancer therapy.17 Pathological angiogenesis is

the C-3 carbonyl group to provide the lactone (Scheme 1). The 1 H NMR spectrum of the synthesized 3 and 4 matched that from natural resources. Compounds 3 and 4 were isolated as racemic mixtures, and no ECD spectra could be obtained from either the synthesized or the natural compounds. We conclude that 3 comprised the 5-hydroxy-7-methoxyphthalide group and the 2,3-dimethoxy-5-methylphenyl group, and 4 comprised the 5-hydroxy-7-methoxyphthalide group and the 2-hydroxy-3methoxy-5-methylphenyl group. Daldiquinone (5) was obtained as a brown powder. The molecular formula of 5 was determined to be C22H16O5 on the 1292

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with CHCl3−MeOH (50:1, flow rate: 2.0 mL/min) to provide 3 (2.9 mg, tR 14 min). Fraction 2e (557.8 mg) was subjected to three chromatography steps: (1) ODS column chromatography with MeOH−H2O (6:4, 0:10), (2) silica gel column chromatography with CHCl3−MeOH (50:1, 25:1, 10:1), and (3) preparative HPLC using a Pegasil ODS column eluted with MeOH−H2O (6:4, flow rate: 2.0 mL/min) to provide 4 (2.4 mg, tR 15 min). The CHCl3 extract (40.8 g) was subjected to silica gel column chromatography with CHCl3−MeOH (100:0, 100:1, 50:1, 10:1, 2:1) to yield seven fractions (a−g). Fraction b (380.0 mg) was subjected to four chromatography steps: (1) silica gel column chromatography with n-Hex−EtOAc (2:1, 1:1, 0:1) followed by MeOH, (2) silica gel column chromatography with CHCl3−EtOAc (100:0, 100:1, 80:1, 0:100) followed by MeOH, (3) Sephadex LH-20 column chromatography with CHCl3−MeOH−H2O (6:4:1), and (4) ODS column chromatography with MeOH−H2O (75:15, 100:0) followed by CHCl3 to provide 5 (1.2 mg). Fraction e (950.0 mg) was subjected to two chromatography steps: (1) silica gel column chromatography with n-Hex−EtOAc (2:1, 1:1, 0:1) followed by MeOH and (2) ODS column chromatography with MeOH−H2O (7:3, 10:0) to provide 6 and 7 as a mixture (68.8 mg). Daldinan B (1): white powder; [α]24D +3 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 219 (3.57), 223 (sh) (3.55), 259 (3.10), 293 (2.96) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 3307, 1662, 1602 cm−1; 1H and 13C NMR (DMSO-d6) see Table 1; HRFABMS m/z 374.1234 [M + H]+ (calcd for C19H20NO7, 374.1240). Daldinan C (2): white powder; [α]29D ±0 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 206 (4.59), 220 (sh) (4.46), 259 (3.99), 293 (3.84) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 3435, 1651, 1603 cm−1; 1H and 13C NMR (MeOH-d4) see Table 1; HRFABMS m/z 402.1554 [M + H]+ (calcd for C21H24NO7, 402.1553). Daldinolide A (3): white powder; [α]29D −1 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 203 (4.74), 225 (sh) (4.50), 261 (4.09), 294 (sh) (3.84), nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 3348, 1719, 1602 cm−1; 1H NMR (MeOH-d4) see Table 1; HRFABMS m/z 347.1136 [M + H]+ (calcd for C18H19O7, 347.1131). Daldinolide B (4): yellow oil; [α]29D +1 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 207 (4.19) 229 (sh) (3.98), 272 (3.63), 293 (sh) (3.39) nm; ECD (MeOH) λmax (Δε) ±0; IR (ATR) νmax 3365, 1728, 1592 cm−1; 1H NMR (MeOH-d4) see Table 1; HRFABMS m/z 333.0976 [M + H]+ (calcd for C17H17O7, 333.0974). Daldiquinone (5): brown powder; UV (CHCl3) λmax (log ε) 241 (4.39), 309 (3.92), 421 (3.70) nm; IR (ATR) νmax 3341, 1662, 1607 cm−1; 1H and 13C NMR (CDCl3) see Table 2; HREIMS m/z 360.0994 [M]+ (calcd for C22H16O5, 360.0998). Semisynthesis of 3 and 4. Pinnick oxidation was performed as described previously with slight modifications.18 A solution of NaH2PO4 (20 mg, Wako Pure Chemical Industries) in H2O (200 μL) was added to a solution of 6 and 7 (20.0 mg) in DMSO (800 μL), and then the solution was cooled at 0 °C. NaClO2 (20 mg, Wako Pure Chemical Industries) in H2O (200 μL) was added and stirred for 5 min, and then the mixed solution was further stirred at room temperature for 60 min. The reaction mixture was extracted with EtOAc−H2O. The organic layer was purified by silica gel column chromatography (CHCl3−MeOH, 10:1) to give 3 (8.2 mg) and 4 (3.6 mg). Cell Culture. HUVECs were purchased from Lonza Group and cultured using an EGM-2 BulletKit (Lonza Group) at 37 °C in 5% CO2. Cell Proliferation Assay. HUVECs (3× 104 cells/well) were seeded in 96-well plates with an EGM-2 BulletKit for 3 h at 37 °C in 5% CO2. The medium was removed and replaced with EGM-2 BulletKit and incubated for 21 h at 37 °C in 5% CO2. Test samples were added to each well, and the plates were incubated for 18 h. The medium was removed and replaced with EGM-2 BulletKit and incubated for 24 h at 37 °C in 5% CO2. WST-8 reagent (10 μL; Dojindo Molecular Technologies) was added to each well, and cells were incubated at 37 °C in 5% CO2 for 2 h. The inhibition of proliferation was measured by absorbance at 450 nm using an Immuno

Figure 3. HMBC (blue arrows) and 1H−1H COSY (pink bold lines) correlations of 5.

characterized by the persistent proliferation of endothelial cells and blood vessel formation. Isolated compounds were tested for their antiangiogenesis activity, and 5 actively inhibits HUVEC growth (IC50 = 7.5 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Yanaco MP apparatus. Optical rotation was measured with a Horiba SEPA-300 polarimeter. IR spectra were recorded with a Thermo FT-IR Nicolet iS5 spectrophotometer (ATR). UV spectra were recorded with a Thermo Genesys 10S UV−vis spectrophotometer. ECD measurements were carried out on a Jasco 820 spectropolarimeter. 1D and 2D NMR spectra of 1 and 3−5 were measured at room temperature with a JEOL JNM-AL400 MHz spectrometer, using tetramethylsilane as the internal standard. The 1H NMR and 13C NMR spectra of 2 were measured at −20 °C. 2D NMR spectra of 2 were measured at room temperature. Low- and highresolution EI and FABMS spectra were measured with a JEOL JMS700 spectrometer. Column chromatography was performed using silica gel 60N (63-210 μm, Kanto Chemical), ODS silica gel (YMC-GEL ODS-A, YMC), and Sephadex LH-20 (GE Healthcare). Preparative HPLC was performed on a SSC-3461 equipped with a SSC-5410 UV detector at 254 nm (Senshu Scientific) and a Jasco PU-2080 Plus equipped with a Jasco PU-2075 Plus UV detector at 254 nm. The HPLC columns were an Inertsil diol column (10 ϕ × 250 mm, 5 μm, Senshu Scientific), an Inertsustain C18 column (10 ϕ × 250 mm, 5 μm, GL Sciences), and a Pegasil ODS 4251-N column (10 ϕ × 250 mm, 5 μm, Senshu Scientific). Fungal Material. Fruiting bodies of Daldinia concentrica were collected in Yamanashi Prefecture, Japan, in October 2016. A voucher specimen (DC-2016) was deposited at the Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University. Species identification was confirmed by one of the authors (K. Koyama). Extraction and Isolation. Dried and fractured fruiting bodies of D. concentrica (772 g) were extracted twice each with CHCl3, MeOH, and then acetone at room temperature. The MeOH extract (40.8 g) was fractionated by silica gel column chromatography with CHCl3−MeOH (50:1, 30:1, 15:1, 7:1, 3:1, 1:1, 0:10) to yield four fractions (1−4). Fraction 3 (5.7 g) was subjected to two chromatography steps: (1) silica gel column chromatography with CHCl3−MeOH−H2O (30:4:0.5, 15:4:0.5, 10:4:0.5, 0:100:0) and (2) ODS column chromatography with MeOH−H2O (3:7, 4:6, 5:5), to yield six fractions (3a−3f). Fraction 3e (12.7 mg) was subjected to preparative HPLC using an Inertsustain C18 column eluted with MeOH−H2O (7:3, flow rate: 3.0 mL/min) to provide 1 (3.4 mg, tR 5 min). Fraction 3b (33.9 mg) was subjected to preparative HPLC using an Inertsil diol column eluted with CHCl3−MeOH−H2O (90:10:1, flow rate: 2.0 mL/min) to provide 2 (7.0 mg, tR 12 min). Fraction 2 (7.4 g) was subjected to silica gel column chromatography using CHCl3−MeOH (70:1, 50:1, 30:1, 15:1, 0:100) to yield seven fractions (2a−2g). Fraction 2d (2.1 g) was subjected to three chromatography steps: (1) ODS column chromatography with MeOH−H2O (4:6, 6:4, 7:3, 10:0), (2) silica gel column chromatography with CHCl3−MeOH (40:1, 30:1, 20:1, 0:100), and (3) preparative HPLC using an Inertsil diol column eluted 1293

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Mini NJ-2300 microplate reader (Bio-Rad). Cytochalasin B was used as positive control (IC50 = 0.2 μM). The IC50 values were estimated using Prism software (version 5.02; GraphPad).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00976. 1 H and 13C NMR and 2D NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-42-495-8913. Fax: +81-42-495-8912. E-mail: [email protected]. ORCID

Kiyotaka Koyama: 0000-0001-9884-0080 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan. REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00976 J. Nat. Prod. 2018, 81, 1290−1294