Constituents of the Fruiting Body of Poisonous Mushroom Omphalotus japonicus

Abstract

Six new sesquiterpenes, tsukiyols A–C, neoilludin C, and 4-O-methylneoilludins A and B, were isolated from the fruiting body of Omphalotus japonicus (Kawam.) Kirchm. & O. K. Mill. Additionally, six known compounds, illudin S, neoilludins A–B, 5-hydroxydichomitol, ergosterolperoxide, and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one, were also obtained. Their chemical structures were determined with MS, IR, and NMR spectra and the absolute configurations of neoilludins A–C, 4-O-methylneoilludins A, and B were determined with electronic circular dichroism (ECD). Illudin S and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one showed cytotoxicity against human acute promyelocytic leukemia HL60 cells. Illudin S, 4-O-methylneoilludin A, B, and tsukiyol C showed growth-restoring activity against mutant yeast via Ca²⁺-signal transduction.

Introduction

In Japan, Omphalotus japonicus (Kawam.) Kirchm. & O.K. Mill. (Lampteromyces Japonicus, O. guepiniformis; Tsukiyotake in Japan) is a well-known poisonous mushroom, responsible for the highest number of food poisoning cases with a toadstool.¹⁾ Several cytotoxic compounds were reported, including illudin S,^1–5) neoilludins A, B,^6,7) and dihydroilludin S.⁸⁾ Previously, illudin S has exerted strong antitumor activity, with its semi-synthetic derivatives assessed for augmenting this activity.^9–14) In particular, one semi-synthetic variant of illudin S, hydroxymethylacylfulven (HMAF), was anticipated as a new anticancer drug.^15,16) Omphalotaceae mushrooms are known to produce extensive types of sesquiterpenes.^17–20) However, few studies have investigated compounds obtained from O. japonicus compared to those obtained from other related species. Therefore, in this study, the constituents obtained from the fruiting body of O. japonicus were analyzed. In this article, we reported 12 compounds (Fig. 1), including six new sesquiterpenes isolated from the methanolic extract of the fruiting body of O. japonicus with column chromatography, solid-phase extraction (SPE), and HPLC. The chemical structure was determined with MS, NMR, IR, and electronic circular dichroism (ECD). Tsukiyols A (1) and B (2) were identified as new protoilludane sesquiterpenes, and tsukiyol C (3) as a new fomannosane sesquiterpene. Neoilludin C (4) was a new epimer of neoilludin B (8).^6,7) 4-O-Methylneoilludins A (5) and B (6) were identified as new illudane sesquiterpenes. In addition, the known compounds neoilludins A (7), B (8),^6,7) illudin S (9),^2–5) 5-hydroxydichomitol (10),²¹⁾ ergosterolperoxide (12),^22,23) and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one (11)²⁴⁾ were isolated. Although the cytotoxicity of illudanes has already been established,¹⁴⁾ their growth-restoring activity against a mutant yeast strain has yet to be examined. This assay is a unique phenotypic screening system that detects Ca²⁺-signal transduction inhibitors based on their ability to restore cell growth, demonstrated as a growth zone. A growth defect was observed in the G2 phase of the zds1Δ cells of Saccharomyces cerevisiae in a medium with a high concentration of Ca²⁺. Notably, Ca²⁺-signaling pathways involved in growth regulation are composed of several signaling molecules such as the Ca²⁺ channel (anti-hypertension), calcineurin (immunosuppressant), Mpk1 mitogen-activated protein kinase (MAPK) (anticancer), and Mck1 GSK-3 (anti-diabetes and Alzheimer’s disease).^25,26) Compounds 9 and 11 exhibited cytotoxic activity against HL 60 cells, with compounds 3, 5, 6 and 9 showed weak growth-restoring activity against a mutant yeast.

Fig. 1. Chemical Structure of Compounds 1–12

Results and Discussion

Tsukiyol A (1) was obtained as a colorless solid material. The molecular formula was determined as C₁₅H₂₄O₄ from the ion peak at m/z 291.1577 [M + Na]⁺ in high resolution (HR)-FAB-MS. The ¹H-NMR spectrum showed a singlet methyl at δ_H 0.88, 0.90; two hydroxy methylene at δ_H 3.29(2H), 4.02 (1H), and 4.10 (1H); two hydroxy methine at δ_H 3.75, and 3.93; two methine at δ_H 2.12, and 2.22; and six nonequivalent methylene at δ_H 1.20, 1.26, 1.40, 1.59, 2.56, and 2.95. Fifteen carbon resonances were observed in the ¹³C-NMR spectrum, indicated as sesquiterpenes and demonstrating a double bond at δ_c 134.0 and 139.2; and two sp³ quaternary carbons δ_c at 53.1 and 46,52. These spectra were in good agreement with a known protoilludane-type sesquiterpene, 3-epi-illudol, isolated from Clitocybe candicans, except the methyl-15 was replaced with hydroxymethylene at δ_H 3.29.^{27) 1}H–¹H correlation spectroscopy (COSY) observed a correlation between H-8 and H-9; and further between H-9 and H-2, H-10; between H-2 and H-1; and between H-5 and H-4. Heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond connectivity (HMBC) correlations were observed between hydroxy methylene-13 with C-7, C-6, C-8; H-5 with C-6; H-4 with C-12, C-2; Methyl H-12 with C-2, C-3, C-4, C-6; and H-10, H-1, H-14, H-15 with C-11 (Fig. 2). Therefore, 1 was suggested as a protoilludane skeleton and the correlation of two dimensional (2D)-NMR similar to that of 3-epi-illudol in literature.²⁷⁾ Nuclear Overhauser effect (NOE) correlations were observed between methyl-12 to H-8, H-1α; H-9 to H-2, 14-methyl, H-10β; H-2 to H-1β, H-9, 14-methyl, H-4; hydroxyethylene-15 to H-1α, H-10α; and H-4 to H-5β. Furthermore, the vicinal coupling constants of the cyclopentane ring were similar to 3-epi-illudol, i.e., similarities were observed between H-2 and H-1β, H-9 and H-10β (³J = 7.6 Hz and 7.5 Hz), H-2 and H-1α, and H-9 and H-10α (³J = 10.5 Hz and 9.7 Hz).²⁷⁾ The relative configuration of 1 is shown in Fig. 3.

Fig. 2. Key ¹H–¹H COSY and HMBC Correlations between Compounds 1 and 3

Fig. 3. Key NOEs of Compounds 1–6

Tsukiyol B (2) was obtained as a colorless solid material, with the molecular formula C₁₅H₂₄O₄ determined from the ion peak at m/z 291.1569 [M + Na]⁺ in HR-FAB-MS. All NMR data were comparable to those of 1. However, NOE observed a different correlation, i.e., the hydroxy methylene-14 to H-2, H-9, H-10β, H-1β, and the methyl-15 to H-1α, H-10α. Therefore, 2 was indicated as an epimer of 1 at C-11 and chemical structure described in Fig. 3.

Tsukiyol C (3) was obtained as a colorless solid material and the molecular formula was determined as C₁₅H₂₆O₄ from the ion peak at m/z 293.1718 [M + Na]⁺ in HR-FAB-MS. The ¹H-NMR spectrum suggested three methyl groups at δ_H 1.07, 1.66, and 2.18; four nonequivalence hydroxy methylene groups at δ_H 3.63, 3.75, 4.37, and 4.98; two hydroxy methine groups at δ_H 4.23, and 4.98; and a methine group at δ_H 2.61, with six other nonequivalent methylene groups. The ¹³C-NMR spectrum showed 15 carbon resonances suggesting sesquiterpenes; two sp² carbon at δ_C 132.2, and 148.1; and two sp³ quaternary carbon at δ_C 41.8, and 44.7. ¹H–¹H COSY correlation was observed between H-4 and H-5, and between H-8 and H-7, H-11, H-10. HMBC showed correlations of hydroxy methylene-15, methyl-14 to C-9, C-8, C-10; H-11, H-7, H-8 to C-6; methyl-13 to C-5, C-6, C-3; H-5 to C-3; methyl-12 to C-1, C-2; and hydroxymethylene-1 to C-2, C-3 (Fig. 2). Therefore, the planar structure of 3 was suggested as a fomannosane type sesquiterpene similar to illudosin, as mentioned in previous literature.¹⁹⁾ Key NOE correlations were observed between hydroxymethylene-1 and methyl-12, 13, H-7, H-8α; H-8α and hydroxymethylene-1, hydroxymethylene-15, H-7; hydroxymethylene-15 and H-10α; H-7 and H-8α, hydroxymethylene-1, methyl-13; methyl-13 and H-5α; H-5β and H-4, H-11, H-8β; H-8β and H-5β, H-10β, methyl-14; and H-10β and H-8β, H-11, methyl-14 (Fig. 3). These correlations were in good agreement with the relative configuration of one of the fomannosane sesquiterpene illudosin isolated from Omphalotus olearius (O. illudens, Clitocybe illudens).¹⁹⁾ The chemical structure of 3 presented in Fig. 1.

Neoilludin C (4) was obtained as a colorless solid material. The molecular formula was C₁₅H₂₂O₆, deduced from the ion peak at m/z 321.1311 [M + Na]⁺ in HR-FAB-MS. The ¹H- and ¹³C-NMR data are shown in Table 1. ¹H-NMR showed three singlet methyl at δ_H 0.88, 1.34, and 1.55; four cyclopropane ring-derived nonequivalent methylene at δ_H 0.38, 0.53, 0.63, and 0.68; a hydroxy methylene at δ_H 3.22; and two singlets hydroxy methine at δ_H 4.49, and 4.50. The ¹³C-NMR spectrum showed one carbon resonance, indicating a sesquiterpene. The important moiety information was α, β-unsaturated carbonyl at δ_C 136.6, 167.4, and 202.2; and four sp³ quaternary carbons at δ_c 38.8, 53.3, 71.1, and 77.0. The data of both 1D NMR are shown in Table 1. ¹H–¹H COSY correlation was observed between H₂-11 and H₂-12. Of the HMBC correlations, the H-11 and H-12 were connected to the sp³ quaternary carbons C-2, C-3, and C-4. Furthermore, the chemical shifts of H-11α, H-11β, H-12α, H-12β, C-11, and C-12 were located on the high magnetic field side, i.e., δ_H: 0.63, 0.68, 0.53, and 0.38; and δ_C: 4.3, and 6.5, respectively (Table 1). Therefore, C-11, C-12, and C-3 formed a spirocyclopropane ring. In addition, the known related substances 7 and 8, demonstrating the spirocyclopropane ring moiety, also presented similar chemical shift values as observed in literature [δ_H: 0.63 (H-11α), 0.72 (H-11β), 0.88 (H-12α), and 0.53 (H-12β); δ_C: 5.1 (C-11), and 8.3 (C-12) in 7; δ_H: 0.65 (H-11α), 0.71 (H-11β), 0.83 (H-12α), and 0.48 (H-12β); and δ_C: 4.98 (C-11), and 7.98 (C-12) in 8].⁶⁾ The methyl H-10 correlated with C-3, C-2, carbonyl carbon C-1, and the methyl H-13 correlated with C-3, C-4, and sp² carbon C-5. The hydroxy methine H-6 exhibited a correlation with the sp² double-bound carbon C-5 and C-9, as well as C-8 and C-7. This moiety suggested the presence of a five-membered ring. In addition, H-8 correlated with C-9, C-1, C-7; and hydroxy methylene H-15 correlated with C-6, C-7, C-8. Therefore, the planar structure of 4 was determined as an illudane type sesquiterpene, comparable to 7 and 8. The NOE correlations of 7 and 8 were observed between the methyl H-13 and H-11β, H-12β, and H-6 in literature. However, 6 correlated with methyl H-13, methyl H-10, and H-11β. Furthermore, other correlations observed good agreement with those of 8, i.e., between H-15 and H-6, H-8, and H-6 and methyl-10, H-11α (Fig. 3). Therefore, neoilludin C was considered a new 4-epimer of 8^6,7) (Fig. 3).

Table 1. ¹H- and¹³C-NMR Data of Compounds 4–6

	4		5		6
C	δH	δC	δH	δC	δH	δC
1		202.2		203		203.4
2		77		76.3		77.8
3		38.8		30.3		31.2
4		71.1		77.8		76.7
5		167.4		165.5		162.4
6	4.49 (1H, s)	75	4.47 (1H, s)	78.7	4.52 (1H, d, J = 0.95 Hz)	77.5
7		53.3		52.4		54.9
8	4.50 (1H, s)	77.6	4.94 (1H, s)	79.3	4.56 (1H, d, J = 0.95 Hz)	74.4
9		136.6		136.2		136.8
10	1.34 (3H, s)	26.9	1.58 (3H, s)	27	1.55 (3H, s)	26.5
11α	0.63 (1H, ddd, J = 9.9, 5.9, 4.3 Hz)	4.3	0.25 (1H, ddd, J = 9.9, 5.7, 4.6 Hz)	4.8	0.07 (1H, ddd, J = 9.6, 5.6, 4.6 Hz)	4.5
11β	0.68 (1H, ddd, J = 9.9, 5.9, 4.3 Hz)		0.66 (1H, ddd, J = 9.9, 5.7, 4.6 Hz)		0.49 (1H, ddd, J = 9.6, 5.6, 4.6 Hz)
12α	0.53 (1H, ddd, J = 9.9, 5.9, 3.8 Hz)	6.5	0.94 (1H, m)	6.0	0.85 (1H, m)	6.2
12β	0.38 (1H, ddd, J = 9.9, 5.9, 3.8 Hz)		0.98 (1H, ddd, J = 9.9, 5.7, 5.9 Hz)		0.87 (1H, ddd, J = 9.6, 5.7, 4.9 Hz)
13	1.55 (3H, s)	26.6	1.36 (3H, s)	16.9	1.21 (3H, s)	17.5
14	0.88 (3H, s)	12.9	0.94 (3H, s)	17.2	0.82 (3H, s)	12.6
15	3.22 (2H, s)	67.7	3.63 (1H, d, J = 11.1 Hz)	66.1	3.29 (2H, s)	67.1
			3.79 (1H, d, J = 11.1 Hz)
OMe			3.35 (3H, s)	50.7	3.27 (3H, s)	50.9

4-O-Methylneoilludin A (5) was obtained as a colorless oily material. The molecular formula was determined as C₁₆H₂₄O₆ from the ion peak at m/z 335.1469 [M + Na]⁺ in HR-FAB-MS. The ¹H- and ¹³C-NMR spectra were in good accordance with those of 7,⁶⁾ except for the O-methyl group at δ_H 3.35 and δ_c 50.7. Furthermore, 5 demonstrated a molecular weight larger than that of 7 with one methyl group. The ¹H–¹H COSY, HMQC, and HMBC correlations were in accordance with 7, and HMBC correlation observed between the methoxy group and C-4. The NOE showed correlations between methyl-13 and H-11β, H-12β, H-6, O-methyl; H-6 and hydroxymethylene-15; methyl-14 and H-8, O-methyl; and methyl-10 and H-11α, O-methyl (Fig. 3). Moreover, other correlations indicated good agreement with 7, i.e., hydroxymethylene-15 with H-6 and methyl-14 with H-8 and O-methyl. These correlations demonstrated the same relative configuration of 7.^6,7) Therefore, 5 was considered a new 4-O-methylated variant of 7.

4-O-Methylneoilludin B (6) was obtained as a colorless oil. HR-FAB-MS demonstrated an ion peak at 335.1475 [M + Na]⁺, indicating the molecular formula C₁₆H₂₄O₆. This indicated that the molecular structure of 6 included one methyl group more than that of 8. The ¹H- and ¹³C-NMR spectra of 6 were in good agreement with those of 8,⁶⁾ except for the presence of the O-methyl group at δ_H 3.27 and δ_c 50.9. The ¹H–¹H COSY, HMQC, and HMBC correlations of 6 concurred with those of 8. The methoxy proton correlated with C-4 in the HMBC of 6. The NOE observed correlations between methyl-13 and H-11β, H-12β, H-6, O-methyl; and methyl-10 and H-11α, O-methyl. These moieties suggested the configuration between the spirocyclopropane and cyclohexenone rings as shown in Fig. 3. In addition, H-6 correlated with hydroxymethylene-15; hydroxymethylene-15 correlated with H-8, H-6; and methyl-14 correlated with hydroxymethylene-15, O-methyl. Therefore, 6 suggested the same relative configuration of 8 and was considered a new 4-O-methylated variant of 8 and an epimer of 5.^6,7)

The absolute configurations of 4–8 were determined by comparing the calculated ECD spectrum of 7 and each experimental ECD spectrum (Fig. 4). The calculated ECD model used the time-dependent density-functional theory (TDDFT) method. The experimental ECD curves of 4–8 were in good agreement with the calculated ECD spectrum of (2R, 4S, 6S, 7S, 8S)-7 (Fig. 4). Therefore, the absolute configurations of 4–8 were (2R, 4S, 6S, 7S, 8R)-4, (2R, 4R, 6S, 7S, 8R)-5, (2R, 4S, 6S, 7S, 8S)-6, (2R, 4S, 6S, 7S, 8S)-7 and (2R, 4S, 6S, 7S, 8R)-8.

Fig. 4. Experimental ECD Curve of Compound 7 in MeOH (300 µM, Solid Line) and the Calculated ECD (Dashed Line) Curve of (2R, 4S, 6S, 7S, 8S)-7

Compounds 9 and 11 showed cytotoxic activities, each at IC₅₀ = 3.3 nM and 4.1 µM, against the HL60 cells. The remaining compounds showed weak cytotoxicity even at 10 µM. Especially, 9 had potent cytotoxicity owing to the presence of an α, β-unsaturated carbonyl moiety, with reactivity comparable to a Michael acceptor of nucleophilic groups of proteins in the HL60 cells.²⁸⁾ Furthermore, 3, 5, 6, and 9 showed weak growth-restoring activity against mutant yeast YNS17 strain at 1.25, 2.5, 0.625, and 1.25 (µg/spot). These activities were nullified in the absence of 0.3 M CaCl₂. Other compounds observed no growth-restoring activity even at a concentration of 5 µg/spot.

Experimental

General

The NMR spectra were recorded on a JEOL JNM ECX-600 (¹H-NMR: 600 MHz, ¹³C-NMR: 150 MHz). The optical rotation used a HORIBA SEPA-300. IR, UV, and ECD spectra were recorded on HORIBA FT-710, SHIMADZU UV mini 1240, and JASCO J-820 respectively. The HR-FAB-MS was measured with JEOL JMS-700 (Matrix: 3-nitrobenzyl alcohol and glycerol). HPLC was performed with SHIMADZU LC workstation CLASS LC-10 and ODS column (GL science Intersil ODS-3, ϕ = 10 mm, L = 250 mm). Medium pressure liquid chromatography (MPLC) was performed with a pump (KUSANO KAGAKUKIKAI KP-70) and ODS column (NOMURA CHEMICAL Develosil packed column, ϕ = 22 mm, L = 300 mm). Solid-phase column chromatography was performed using silica gel 60 (63-210 mesh, Merck, Germany) and Sephadex LH-20 (GE Healthcare, U.S.A.). The TLC plates were prepared with silica gel F₂₅₄ (Merck) and SPE was performed with Waters Sep-Pak Vac 35cc C₁₈-10g (Waters, U.S.A.).

Screening of Ca²⁺-Signal Transduction Inhibitors

Screening was performed with a mutant yeast utilizing Ca²⁺-signal transduction, namely the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ), and 5 µL of each sample was added to the plate as previously described.²⁶⁾ The inhibitory activity on Ca²⁺-signal transduction was determined by examining the yeast growth zone. The specificity was confirmed using the same plate without the presence of 0.3 M CaCl₂. An immunosuppressive drug, FK506 [tacrolimus; 2.5 ng/spot, calcineurin (protein phosphatase 2B) specific inhibitor], was used as the positive control.

Cytotoxic Activity

Human acute promyelocytic leukemia HL 60 cells (RCB0041, RIKEN BioResource Center, Tsukuba, Japan, 1 × 10⁵ cells/mL) were treated with each compound dissolved in MeOH, using a 96-well microplate at 37°C under a humidified 5% CO₂-containing atmosphere for 2 d, in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich Co., St. Louis, MO, U.S.A.), 50 units/mL of penicillin, and 50 mg/mL of streptomycin (Gibco, Thermo FisherScientific Inc., Waltham, MA, U.S.A.). The cells were subjected to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The positive control camptothecin showed an IC₅₀ value of 54.2 nM.²⁹⁾

Calculated ECD Spectra

The ECD was calculated with TDDFT and discrete Fourier transform (DFT) of the gaseous phase of Gaussian 09.³⁰⁾ The conformational search was performed according to the GMMX program, with the MMFF94 force field and the optimization of the conformer calculated with B3LYP/6-31G(d).³¹⁾ The calculation of the ECD curve was performed with the TDDFT method for 30 excited states with B3LYP/6-331 + G(d) level in the gaseous phase. The spectrum was described with the SpecDis program by the Gaussian band shape 0.3 eV.

Sample

The fruiting body of O. japonicus (Kawam.) Kirchm. & O. K. Mill. (5.4 kg) was collected from the Yamagata University Research Forest in 2018. Identification was performed by one of the authors (D.A.) and a voucher specimen (No. 181010) was deposited in the Laboratory of Bioorganic Chemistry, Faculty of Agriculture, Yamagata University.

Extraction and Isolation

The fruiting body was extracted with MeOH three times and concentrated with a rotary evaporator. Furthermore, the residue was extracted again with EtOAc, and 33.9 g of ethyl acetate extract was obtained. The methanolic extract (143.6 g) was suspended with distilled water and partitioned with hexane, EtOAc, and 1-BuOH. Each organic layer was evaporated, and the hexane (12.0 g), EtOAc (5.5 g), and 1-BuOH (23.4 g) fractions were obtained. The 1-BuOH fraction was removed from 11.1 g of the methanolic insoluble matter and subjected to silica gel column chromatography. The fraction was eluted with chloroform : methanol (85 : 15, 7 : 3, 3 : 2, 2 : 3, and 0 : 1) step-wise, with the 10 separated fractions including B4 (462.5 mg) and B5 (393.9 mg). B5 was subjected to silica gel column chromatography, eluted with chloroform : methanol (9 : 1, 8 : 1, 7 : 1, 6 : 1, 5 : 1, 4 : 1, and 0 : 1) step-wise, and divided into B5-1 and B5-10. Among them, B5-7–9 (94.0 mg) were subjected to Sephadex LH-20 column chromatography (MeOH), and fractions including sesquiterpene mixture (73.0 mg) were identified. The mixture was purified with MPLC (water : methanol = 4 : 1, 7 : 3, 3 : 2, 1 : 1, 2 : 3, and 0 : 1) step-wise, to isolate 10 (5.6 mg), 4 (2.8 mg), 1 (12.8 mg), 2 (3.6 mg), and 3 (4.2 mg). In addition, the residual fraction B5-6 (59.5 mg) was purified with ODS HPLC eluted with water : methanol = 3 : 2 (3.5 mL/min). After separating the peak at 6.4 min, 22.1 mg of 8 was obtained. B4 was subjected to silica gel chromatography, eluted step-wise with chloroform : methanol (95 : 5, 9 : 1, 85 : 15, 4 : 1, 75 : 25, and 0 : 1) to obtain ten fractions, including B4-4 (73.5 mg) and B4-6 (91.9 mg). B4-6 was purified with ODS HPLC eluted with water : methanol [65 : 35 (3.5 mL/min)] and the peak was separated at 9.0 min (24.7 mg of 7). B4-4 was subjected to silica gel column chromatography. After step-wise elution with ethyl acetate : methanol (1 : 0, 15 : 1, 9 : 1, 4 : 1, 0 : 1), 3.7 mg of the fraction including 5 was obtained. The fraction was purified with ODS HPLC, eluted with water : methanol (3 : 2 to 1 : 1 gradient, 4 mL/min). The peak at 14.1 min, which was that of 2.1 mg of 5, was separated. The ethyl acetate fraction was separated with silica gel column chromatography, eluted step-wise with chloroform : methanol (85 : 15, 7 : 3, 1 : 1, and 0 : 1) to obtained six fractions E-1 to E-6. E-2 (924.9 mg) was subjected to ODS Sep-Pak and eluted with water : methanol (35 : 65, 1 : 1, 25 : 75, and 0 : 1). The 0 : 1 fraction was subjected to silica gel column chromatography, eluted in a step-wise manner with hexane : ethyl acetate:methanol (1 : 4 : 0, 0 : 1 : 0, 0 : 9 : 1, 0 : 0 : 1). Further, the identified fractions, including 11, were purified with ODS HPLC and eluted with water : methanol (4 : 1 to 9 : 1 gradient, 4 mL/min). The peak was separated at 21.5 min and was below the peak of 11 (7.5 mg). E-4 (465.2 mg) was separated with silica gel column chromatography, eluted with ethyl acetate : methanol (1 : 0, 15 : 1, 9 : 1, 4 : 1, 0 : 1) step-wise, resulting in 151.3 mg of 9. The residue (131.2 mg) including 6 was subjected to silica gel column chromatography, eluted with chloroform : methanol (15 : 1, 9 : 1, 85 : 15, 4 : 1, 0 : 1) step-wise, and then purified. The ODS HPLC was eluted with water : methanol (65 : 35, 3.5 mL/min) and the peak was separated at 12.1 min to obtain 1.1 mg of 6. The ethyl acetate extract was subjected to silica gel column chromatography, eluted with hexane : ethyl acetate (9 : 1, 4.1, 3 : 2, 2 : 3, 0 : 1) step-wise, and the fractions EE-1 to 10 were obtained. Among these, EE-7 (246 mg) was fractionated with silica gel60 chromatography and eluted with hexane : ethyl acetate (3 : 2, 2 : 3, 1 : 4, and 0 : 1) step-wise to obtain 12 (30.9 mg).

Tsukiyol A (1)

Colorless solid material, [α]^D₂₀ = +0.7° (c 0.1, MeOH); IR ν_max (KBr) cm⁻¹: 3363 (hydroxy group); FAB-MS m/z: 291 [M + Na]⁺, HR-FAB-MS m/z: 291.1577 (Calcd for C₁₅H₂₄O₄Na: 291.1572); ¹H-NMR (CD₃OD, 600 MHz) δ_H: 0.88 (3H, s, H-14), 0.90 (3H, s, H-12), 1.20 (1H, dd, J = 12.7 Hz, 9.7 Hz, H-10α), 1.26 (1H, ddd, J = 12.7 Hz, 7.6 Hz, 1.6 Hz, H-1β), 1.40 (1H, dd, J = 12.7 Hz, 10.5 Hz, H-1α), 1.59 (1H, ddd, J = 12.7 Hz, 7.6 Hz, 1.6 Hz, H-10β), 2.12 (1H, m, H-9), 2.22 (1H, ddd, J = 7.6 Hz, 8.8 Hz, 10.5 Hz, H-2), 2.56 (1H, m, H-5α), 2.95 (1H, ddd, J = 14.7 Hz, 7.4 Hz, 1.4 Hz, H-5β), 3.29 (2H, s, H-15), 3.75 (1H, t, J = 7.4 Hz, H-4), 3.93 (1H, br dd, J = 9.2 Hz, 1.6 Hz, H-8), 4.02 (1H, d, J = 12.7 Hz, H-13), 4.10 (1H, d, J = 12.7 Hz, H-13); ¹³C-NMR (CD₃OD, 150 MHz) δ_C: 14.8 (CH₃, C-12), 23.6 (CH₃, C-14), 37.5 (CH₂, C-5), 37.8 (CH₂, C-1), 42.9 (CH₂, C-10), 46.48 (CH, C-2), 46.52 (C, C-11), 51.1 (CH, C-9), 53.1 (C, C-3), 59.7 (CH₂, C-13), 72.7 (CH₂, C-15), 75.1 (CH, C-8), 76.9 (CH, C-4), 134.0 (C, C-7), 139.2 (C, C-6).

Tsukiyol B (2)

Colorless solid material, [α]^D₂₀ = −13.85° (c 0.1, MeOH); IR ν_max (KBr) cm⁻¹: 3363 (hydroxy group), FAB-MS m/z: 291 [M + Na]⁺, HR-FAB-MS m/z: 291.1569 (Calcd for C₁₅H₂₄O₄Na: 291.1572); ¹H-NMR(C₅D₅N, 600 MHz) δ_H: 1.20 (3H, s, H-15), 1.24 (3H, s, H-12), 1.41 (1H, dd, J = 12.8 Hz, 9.8 Hz, H-1α), 1.41 (1H, dd, J = 12.8 Hz, 9.8 Hz, H-10α), 1.81 (1H, dd, J = 12.8 Hz, 7.6 Hz, H-1β), 2.39 (1H, dd, J = 12.8 Hz, 7.6 Hz, H-10β), 2.41 (1H, ddd, J = 7.6 Hz, 8.8 Hz, 9.8 Hz, H-2), 2.57 (1H, m, H-9), 2.88 (1H, m, H-5α), 3.17 (1H, dd, J = 14.4 Hz, 7.2 Hz, H-5β), 3.55 (2H, d, J = 1.6 Hz, H-14), 4.09 (1H, br dd, J = 7.2 Hz, 7.8 Hz, H-4), 4.49 (1H, br, H-8), 4.61 (1H, d, J = 12.1 Hz, H-13), 4.67 (1H, d, J = 12.1 Hz, H-13) ; ¹³C-NMR (C₅D₅N, 150 MHz) δ_C: 14.9 (CH₃, C-12), 25.8 (CH₃, C-15), 37.6 (CH₂, C-1), 37.7 (CH₂, C-5), 42.8 (CH₂, C-10), 45.8 (CH, C-2), 46.3 (C, C-11), 51.2 (CH, C-9), 52.4 (C, C-3), 69.0 (CH₂, C-13), 70.0 (CH₂, C-14), 74.7 (CH, C-8), 76.0 (CH, C-4), 134.4 (C, C-7), 137.0 (C, C-6).

Tsukiyol C (3)

Colorless solid material, [α]^D₂₀ = +89.45° (c 0.1, MeOH); IR ν_max (KBr) cm⁻¹: 3381 (hydroxy group), FAB-MS m/z: 293 [M + Na]⁺, HR-FAB-MS m/z: 293.1718 (Calcd for C₁₅H₂₆O₄Na: 293.1729); ¹H-NMR (C₅D₅N +2.5% D₂O, 600 MHz) δ_H: 1.07 (3H, s, H-14), 1.15 (1H, dd, J = 13.0 Hz, 11.1 Hz, H8-β), 1.66 (3H, s, H-13), 1.85 (1H, dd, J = 11.7 Hz, 7.7 Hz, H-10β), 1.90 (1H, dd, J = 12.2 Hz, 5.5 Hz, H-5α), 2.04 (1H, dd, J = 11.7 Hz, 6.2 Hz, H-10α), 2.16 (3H, s, H-12), 2.18 (1H, dd, J = 13.0 Hz, 8.3 Hz, H-8α), 2.55 (1H, dd, J = 12.2 Hz 8.8 Hz, H-5β), 2.61 (1H, ddd, J = 7.7 Hz, 8.3 Hz, 11.1 Hz, H-7), 3.63 (1H, d, J = 16.7 Hz, H-15), 3.75 (1H, d, J = 16.7 Hz, H-15), 4.23 (1H, br dd, J = 6.2 Hz, 7.7 Hz, H-11), 4.37 (1H, d, J = 11.6 Hz, H-1), 4.41 (1H, d, J = 11.6 Hz, H-1), 4.98 (1H, brt, H-4); ¹³C-NMR (C₅D₅N +2.5% D₂O, 150 MHz) δ_C: 16.1 (CH₃, C-12), 26.9 (CH₃, C-14), 28.1 (CH₃, C-13), 39.5 (CH₂, C-8), 40.9 (CH₂, C-5), 41.8 (C, C-9), 44.7 (C, C-6), 48.2 (CH₂, C-10), 55.8 (CH, C-7), 62.7 (CH₂, C-1), 67.6 (CH, C-4), 71.1 (CH₂, C-15), 75.2 (CH, C-11), 132.2 (C, C-2), 148.1 (C, C-3).

Neoilludin C (4)

Colorless solid material, [α]^D₂₀ −16.4° (c 0.1, MeOH); IR ν_max (KBr) cm⁻¹: 3390 (hydroxy group), 1683 (α,β-unsaturated carbonyl); UV λ_max (MeOH): 230 (log ε = 2.99); ECD nm (Δɛ): 253 (−8.39), 217(+5.59); FAB-MS m/z: 321 [M + Na]⁺, HR-FAB-MS: m/z: 321.1311 (Calcd for C₁₅H₂₂O₆Na: 321.1314); ¹H-NMR and ¹³C-NMR data are shown in Table 1.

4-O-Methylneoilludin A (5)

Colorless oily material, [α]^D₂₀ = −3.2° (c 0.1, MeOH); IR ν_max (NaCl) cm⁻¹: 3435 (hydroxy group), 1683 (α,β-unsaturated carbonyl); UV λ_max (MeOH): 242 (log ε = 2.95); ECD nm (Δɛ): 249 (−8.68), 212 (+11.63); FAB-MS m/z: 335 [M + Na]⁺, HR-FAB-MS m/z: 335.1469 (Calcd for C₁₆H₂₄O₆Na: 335.1470); ¹H-NMR and ¹³C-NMR data are shown in Table 1.

4-O-Methylneoilludin B (6)

Colorless oily material, [α]^D₂₀ = −5.15° (c 0.1, MeOH); IR ν_max (NaCl) cm⁻¹: 3398 (hydroxy group), 1682 (α,β-unsaturated carbonyl); UV λ_max (MeOH): 242.5(log ε = 2.95); ECD nm (Δɛ): 246 (−6.04), 211(+5.82); FAB-MS m/z: 335 [M + Na]⁺, HR-FAB-MS m/z: 335.1475(Calcd for C₁₆H₂₄O₆Na: 335.1470); ¹H-NMR and ¹³C-NMR data are shown in Table 1.

Acknowledgment

I thank Prof. Hiroyuki Konno for providing the ECD equipment.

Conflict of Interest

The authors declare no conflict of interest

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