10
T. Kikuchi et al. / Steroids 115 (2016) 9–17
600 MHz; 13C: 150 MHz) and an Agilent-NMR-vnmrs400 (1H:
400 MHz; 13C: 100 MHz) in CDCl3, C5D5N, and DMSO-d6 with
tetramethylsilane as the internal standard. The conformations for
NOESY experiments correspond to energy-minimized conforma-
tion. Calculation was performed using Chem3D with MM2 force
field (Chem3D Pro version 12.0.2.1076; CambridgeSoft, MA, USA).
EIMS was recorded using a Hitachi 4000H double-focusing mass
spectrometer (70 eV), and FABMS was recorded using a JEOL
JMS-7000 mass spectrometer. Silica gel (70–230 mesh, Merck)
and silica gel 60 (230–400 mesh, Nacalai Tesque, Inc.) were used
for column chromatography. HPLC was carried out using an SiO2
column [Cosmosil 5SL-II column (Nacalai Tesque, Inc.),
25 cm ꢀ 20 mm i.d.] with hexane/AcOEt [1:1 (System I), 3:7 (Sys-
tem II), and 1:4 (System III), and 0:1 (System IV)], and on ODS col-
umn [Cosmosil 5C18-MS-II column (Nacalai Tesque, Inc.)
(25 cm ꢀ 20 mm i.d.) with MeOH/H2O (95:5) (System V), MeOH/
H2O (9:1) (System VI), MeCN/H2O (8:2) (System VII), Cosmosil
5C18-PAQ column (Nacalai Tesque, Inc.) (25 cm ꢀ 20 mm i.d.) with
MeOH/H2O (9:1) (System VIII)] at 35 °C flow rate 4.0 mL/min.
M8 (117.2 mg), eluted with AcOEt, gave 5 fractions; S1-M8-1–S1-
M8-5. Purification of S1-M8-3 (3.9 mg; tR 53.8 min) with HPLC
(System VIII) gave 12 (2.4 mg; tR 36.7 min), Fr. S1-M8-4 (38.9 mg;
56.2 min) (System VIII) gave 14 (1.1 mg; tR 38.6 min), and 13
(1.7 mg; tR 40.2 min).
2.3.2. Sample 2
Sample 2 [the fruiting bodies of P. eryngii (fresh weight 120 kg),
produced in Kagawa, Japan] were subjected to extraction with
MeOH under reflux (3 days, 4 times). The MeOH extract (2625 g)
was then partitioned between AcOEt and H2O (10 L/10 L, 4 times).
The AcOEt-soluble fraction (240 g) was subjected to SiO2 column
chromatography (CC) [SiO2 (2.8 kg); CHCl3/AcOEt (1:0, 5:1, 1:1,
and 0:1), and MeOH in increasing order of polarity] resulting in
37 fractions (Fr. S2-A–S2-Z, S2-a–S2-k). Fr. S2-T (2874.5 mg), eluted
with CHCl3/AcOEt (1:1), was subjected to SiO2 CC to yield 8 frac-
tions, S2-T1–S2-T8. Fr. S2-T6 (961.2 mg), eluted with hexane/AcOEt
(1:1 ? 0:1), was subjected to SiO2 CC to yield 8 fractions, S2-T6-1–
S2-T6-8. Preparative HPLC (System V) of Fr. S2-T6-4 (140.3 mg)
gave 4 (6.28 mg; tR 41.5 min).
2.2. Materials
Fr. S2-U (2004.7 mg), eluted with CHCl3/AcOEt (1:1), was sub-
jected to SiO2 CC to yield 23 fractions, S2-U1–S2-U23. Fr. S2-U11
(168.6 mg), eluted with hexane/AcOEt (1:1), was subjected to
SiO2 CC to yield 10 fractions, S2-U11-1–S2-U11-10. Preparative
HPLC (System V) of Fr. S2-U11-7 (125.3 mg), eluted with hexane/
AcOEt (1:1), gave 16 (2.6 mg; tR 43.0 min).
Fr. S2-W (2626.2 mg), eluted with CHCl3/AcOEt (1:1), was sub-
jected to SiO2 CC to yield 11 fractions, S2-W1–S2-W11. Fr. S2-W7
(369.9 mg), eluted with AcOEt, was subjected to ODS CC to yield
9 fractions, S2-W7-1–S2-W7-9. Preparative HPLC (System VI) of
Fr. S2-W7-3 (154.0 mg), eluted with MeOH, gave 9 (7.0 mg; tR
67.2 min).
Fr. S2-a (911.6 mg), eluted with CHCl3/AcOEt (1:1), was sub-
jected to SiO2 CC to yield 13 fractions, S2-a1–S2-a13. Preparative
HPLC (System VI) of Fr. S2-a7 (69.8 mg), eluted with AcOEt, gave
10 (3.0 mg; tR 82.8 min). Preparative HPLC (System VI) of Fr. S2-
a8 (363.6 mg), eluted with AcOEt, gave 11 (5.9 mg; tR 36.5 min)
and 17 (1.8 mg; tR 63.6 min).
Fr. S2-j (3379.0 mg), eluted with AcOEt, was subjected to SiO2
CC to yield 10 fractions, S2-j1–S2-j10. Fr. S2-j7 (354.3 mg), eluted
with AcOEt, was subjected to ODS CC to yield 4 fractions, S2-j7-
1–S2-j7-4. Preparative HPLC (System VII) of Fr. S2-j4 (161.4 mg),
The fruiting bodies of P. eryngii, produced in Nagano, Japan
(sample 1) and Kagawa, Japan (sample 2), were purchased from
Japan agricultural cooperatives (JA) Zennou Nagano in 2013, and
HOKUTO Corp. in 2014. A voucher specimen was deposited in
the Herbarium of the Laboratory of Medicinal Chemistry, Osaka
University of Pharmaceutical Sciences.
2.3. Extraction and isolation
2.3.1. Sample 1
Sample 1 [the fruiting bodies of P. eryngii (fresh weight 40 kg),
produced in Nagano, Japan] were subjected to extraction with
MeOH under reflux (1 week, 3 times). The MeOH extract (2035 g)
was then partitioned between AcOEt and H2O (5 L/5 L, 4 times).
The AcOEt-soluble fraction (115 g) was subjected to SiO2 column
chromatography (CC) [SiO2 (3.5 kg); CHCl3/AcOEt (1:0, 10:1, 5:1,
1:1, and 0:1), and AcOEt:MeOH (5:1, and 0:1) in increasing order
of polarity] resulting in 18 fractions (Fr. S1-A–S1-R).
Fr. S1-H (490.9 mg), eluted with CHCl3/AcOEt (1:1), was sub-
jected to SiO2 CC to yield 14 fractions, S1-H1–S1-H14. Preparative
HPLC (System III) of S1-H11 (65.0 mg), eluted with hexane/AcOEt
(1:1), gave S1-H11-1–S1-H11-8, and then re-preparative HPLC of
S1-H-11-6 (4.0 mg; tR 47.8 min) gave 18 (1.2 mg; tR 19.2 min) (Sys-
tem V).
Fr. S1-I (397.1 mg), eluted with CHCl3/AcOEt (1:1 and 0:1), was
subjected to SiO2 CC to yield 13 fractions, S1-I1–S1-I13. Preparative
HPLC of S1-I8 (54.8 mg) (System III), eluted with hexane/AcOEt
(1:1), gave 9 fractions, S1-I8-1–S1-I8-9. Fr. S1-I8-8 was 3 (1.1 mg;
tR 54.6 min). Purification of S1-I8-3 (4.2 mg; tR 38.0 min) with HPLC
(System IV) gave 15 (0.9 mg; tR 32.6 min), S1-I8-6 (13.0 mg; tR
43.3 min) gave 5 (2.9 mg; tR 67.8 min). Preparative HPLC of S1-I9
(System IV) gave 2 (4.2 mg; tR 30.9 min).
Fr. S1-J (548.3 mg), eluted with AcOEt, was subjected to SiO2 CC
to yield 16 fractions, S1-J1–S1-J16. Preparative HPLC (System II) of
S1-J8 (83.6 mg), eluted with hexane/AcOEt (1:1), gave 14 fractions;
S1-J8-1–S1-J8-16, and then re-preparative HPLC (System V) of S1-
J8-14 (18.5 mg; tR 84.8 min) gave 8 (11.4 mg; tR 36.5 min).
Fr. S1-K (777.3 mg), eluted with AcOEt, was subjected to SiO2 CC
to yield 22 fractions, S1-K1–S1-K22. Preparative HPLC (System III)
of S1-K15 (93.4 mg), eluted with hexane/AcOEt (1:1), gave 7 frac-
tions; S1-K15-1–S1-K15-8, and then re-preparative HPLC (System
V) of S1-K15-7 (24.9 mg; tR 70.5 min) gave 7 (3.7 mg; tR 35.6 min).
Fr. S1-M (690.8 mg), eluted with AcOEt, was fractionated using
SiO2 CC to give S1-M1–S1-M15. Preparative HPLC (System IV) of S1-
eluted with MeOH, gave
1 (12.0 mg; tR 20.6 min) and 20
(37.6 mg; tR 69.8 min). Fr. S2-j8 (900.8 mg), eluted with AcOEt:
MeOH (1:0 and 10:1), was subjected to ODS CC to yield 10 frac-
tions, S2-j8-1–S2-j8-10. Preparative HPLC (System V) of Fr. S2-j8-
7 (129.0 mg), eluted with MeOH, gave 11 fractions, S2-j8-7-1–S2-
j8-7-11. Fr. S2-j8-7-9 was identified with 19 (10.9 mg; tR
65.9 min). Fr. S2-j8-7-6 was subjected to re-preparative HPLC (Sys-
tem VI), and gave 6 (1.7 mg; tR 85.4 min).
2.3.3. (22E)-3b,5
9-one (1)
a
,6
a
,11-Tetrahydroxy-9(11)-seco-ergosta-7,22-dien-
26
Colorless crystal; mp 145–146 °C; [
a]
35.4 (c = 0.098, EtOH);
D
UV k
nm (log
e
): 205.5 (3.88), 238.0 (3.93); IR
m
max
cmꢁ1
:
EtOH
KBr
max
3340, 2957, 2872, 1670, 1472, 1455, 1372, 1048, 680, 668; FABMS
m/z: 463 [M+H]+, 485 [M+Na]+, 445 [MꢁH2O+H]+; HRFABMS m/z:
463.3430 [M+H]+ (calcd for 463.3416: C28H47O5).
2.3.4. (22E)-8,14-Epoxyergosta-6,22-diene-3b,5
a,9a-triol (2)
Colorless crystal; mp 139–140 °C; [
a
]
20 116.3 (c = 0.091, EtOH);
D
IR
m
max
cmꢁ1: 3336, 2955, 2869, 1559, 1457, 1155, 1101, 1054;
KBr
FABMS m/z: 467 [M+Na]+; HRFABMS m/z: 467.3142 [M+Na]+ (calcd
for 467.3137: C28H44O4Na).