9
18 J ournal of Natural Products, 1998, Vol. 61, No. 7
Monde et al.
Prefectural Plant Genetic Resources Center, Takikawa,
Hokkaido, J apan.
Bioa ssa y P r oced u r e. For each 2D TLC bioassay,6
NMR (CDCl3, 90 MHz), see Table 1; 13C NMR (CDCl3,
+
22.5 MHz), see Table 2; EIMS m/z 244 [M] (38), 242
+
+
[M] (97), 240 [M] (100), 229 (16), 227 (45), 225 (47),
2
1
01 (17), 199 (46), 197 (48), 164 (19), 162 (28), 135 (23),
33 (34), 127 (12); HREIMS m/z 239.9500 (calcd for
a developed Si gel sheet (i, Et2O; ii, CH2Cl2-MeOH, 49:
1
) was sprayed with a dense conidial suspension of B.
C8H7Cl3O2, 239.9483).
leersiae in a potato-glucose medium, and incubated in
a moist box at 25 °C for 2 days. Fungitoxic spots
appeared white against a dark gray background. The
antifungal activity of each chromatographic fraction was
monitored by 1D TLC using solvent system i or ii. For
semiquantitative bioassays, fresh conidia of B. leersiae
were collected and added to a solution consisting of H2O
2,6-Dich lor o-3,5-d im eth oxytolu en e (3): colorless
crystals; mp 134-135 °C; UV (CH3CN) λmax (log ꢀ) 291
(3.54) nm; IR (CHCl3) νmax 3014, 2962, 2938, 2838, 1578,
-
1 1
1456, 1430, 1339, 1207, 1110, 1081, 940, 804 cm ; H
1
3
NMR (CDCl3, 90 MHz), see Table 1; C NMR (CDCl3,
22.5 MHz), see Table 2; NOE correlation (CDCl3, 400
+
(
100 mL), 1/15 M KH2PO4 (100 mL), and potato dextrose
broth (2 mL). Under stirring with a magnetic stirrer,
mL of the conidial suspension was transferred into
MHz) δ 3.91 (s, 6H)/6.46 (s, 1H); EIMS m/z 224 [M]
+
+
(14), 222 [M] (70), 220 [M] (100), 207 (6), 205 (8), 181
(6), 179 (31), 177 (47), 164 (5), 162 (7), 144 (6), 142 (17),
1
1
2
27 (10); HREIMS m/z 220.0049 (calcd for C9H10Cl2O2,
20.0058).
2,4-Dich lor o-6-h ydr oxy-3,5-dim eth oxytolu en e (4):
each vial (10 mL). Each sample solution in Me2CO (10
µL) was added to the vial, and it was capped and kept
at 20 °C for 24 h. Conidial germination was examined
under a microscope.
colorless crystals; mp 86-87 °C; UV (CH3CN) λmax (log
) 291 (3.51) nm; IR (CHCl3) νmax 3526, 3012, 2936, 2836,
ꢀ
Extr a ction a n d Isola tion . Diseased edible lily bulb
scales (4.9 kg, fresh wt) of L. maximowiczii cv. Hakugin
collected from an infested field in the Kamikawa area
of Hokkaido, J apan, were homogenized in EtOAc using
a Polytron homogenizer, allowed to stand for 3 days,
and centrifuged. The combined supernatant was evapo-
rated under reduced pressure to give a crude extract
1
9
577, 1461, 1407, 1349, 1296, 1191, 1088, 1047, 971,
-
1 1
37, 872 cm ; H NMR (CDCl3, 90 MHz), see Table 1;
C NMR (CDCl3, 22.5 MHz), see Table 2; HMBC
1
3
correlations (CDCl3, 400 MHz) CH3-1/C-1, C-2, C-6:
OCH3-3 or -5/C-3 or C-5; EIMS m/z 240 [M] (11), 238
+
+
+
[M] (87), 236 [M] (87), 225 (12), 223 (67), 221 (100),
1
2
95 (12), 193 (19), 180 (13), 178 (20); HREIMS m/z
36.0009 (calcd for C9H10Cl2O3, 236.0007).
(22 g). To remove tarry materials, the EtOAc-soluble
portion of the extract was passed through a Si gel
column using EtOAc containing 0.005% BHT as eluent.
The eluate from the column was separated, in two
portions, by column chromatography on Sephadex LH-
2
,4-Dich lor o-3,6-d ih yd r oxy-5-m eth oxytolu en e (5)
or 2,4-dich lor o-5,6-dih ydr oxy-3-m eth oxytolu en e (5′):
colorless crystals; mp 146-149 °C; UV (CH3CN) λmax (log
ꢀ) 297 (3.70) nm; IR (CHCl3) νmax 3530, 3010, 2938, 1459,
2
0 (200 g, MeOH containing 0.005% BHT), and the
-
1 1
1
420, 1370, 1312, 1262, 1083, 1038, 941, 891 cm ; H
fractions were combined into three fractions (F-1, F-2,
and F-3) guided by the TLC bioassay. F-1 was inactive,
1
3
NMR (CDCl3, 90 MHz), see Table 1; C NMR (CDCl3,
2
(51), 222 [M] (77), 211 (12), 209 (67), 207 (100), 183
+
+
2.5 MHz), see Table 2; EIMS m/z 226 [M] (9), 224 [M]
and F-3 contained the previously reported yurinelide
+
5
(
1). F-2 (4.3 g) was separated into four fractions (F-
(3), 181 (19), 179 (30).
2
-1 to F-2-4) by column chromatography on Sephadex
LH-20 (200 g, CH2Cl2-MeOH, 4:1, 0.005% BHT). F-2-1
2.5 g) was separated to give two active fractions (F-2-
-1 and F-2-1-2). F-2-1-1 (360 mg) decomposed
4-Ch lor o-2,5-dih ydr oxy-3-m eth oxytolu en e (6): col-
(
1
orless crystals; mp 103-104 °C; UV (CH3CN) λmax (log
ꢀ) 293 (3.36) nm; IR (CHCl3) νmax 3542, 3016, 2940, 2842,
1594, 1475, 1427, 1363, 1280, 1161, 1067, 1001, 926,
865 cm ; H NMR (CDCl3, 90 MHz), see Table 1;
NMR (CDCl3, 22.5 MHz), see Table 2; HMBC correla-
tions (CDCl3, 400 MHz) CH3-1/C-1, C-2, C-6: OCH3-3/
C-3: H-6/C-2, C-4, C-5; EIMS m/z 190 [M] (33), 188
[M] (90), 175 (36), 173 (100), 147 (15), 145 (42).
4-Ch lor o-3,5-dih ydr oxytolu en e (7):11 colorless crys-
tals; mp 134-136 °C; UV (CH3CN) λmax (log ꢀ) 274 (2.99)
nm; IR (CHCl3) νmax 3544, 3254, 3022, 2920, 1591, 1488,
during further separation. F-2-1-2 (460 mg) was
separated by sequential column chromatography on Si
gel (i, 65 g, hexane-CH2Cl2, 2:1; ii, 20 g, hexane-Et2O,
-
1
1
13
C
9
:1) to yield 2 (34 mg) and an active fraction (94 mg),
which gave 3 (7 mg) and 4 (33 mg) on preparative HPLC
Radial-Pak cartridge Resolve C18, MeOH-H2O, 4:1).
+
+
(
F-2-2 (220 mg) was separated by column chromatog-
raphy on Si gel (25 g, hexane-EtOAc, 19:1 to 2:5) to
give two active fractions (F-2-2-1 and F-2-2-2). F-2-
-
1 1
2
-1 (11 mg) gave 5 (8 mg) on preparative HPLC
1460, 1360, 1324, 1262, 1169, 1058, 984, 827 cm ; H
NMR (CDCl3, 90 MHz), see Table 1; C NMR (CDCl3,
22.5 MHz), see Table 2; EIMS m/z 160 [M] (38), 158
[M] (100), 157 (39), 140 (11), 123 (75), 95 (7); HREIMS
1
3
(Radial-Pak cartridge Resolve C18, MeOH-H2O, 11:9).
+
F-2-2-2 (8 mg) on preparative HPLC (Radial-Pak
cartridge Resolve C18, H2O-MeOH, 3:2) gave 6 (4 mg).
F-2-3 (140 mg) gave 7 (23 mg) by column chromatog-
raphy on Si gel (20 g, CH2Cl2). F-2-4 (300 mg) was
separated to give two active fractions (F-2-4-1, 19 mg;
F-2-4-2, 230 mg). F-2-4-1 and F-2-4-2 gave 8 (11
mg) and 9 (16 mg), respectively, on preparative HPLC
on a Radial-Pak cartridge Resolve C18 using H2O-
MeOH (3:2) and H2O-MeOH (7:3).
+
m/z 158.0159 (calcd for C7H7ClO2, 158.0134).
2-Ch lor o-3,5-dih ydr oxytolu en e (8):16 colorless crys-
tals; mp 139-141 °C; UV (CH CN) λ
nm; IR (CHCl ) ν
1259, 1156, 1048, 978, 842 cm ; H NMR (CDCl , 90
MHz), see Table 1; C NMR [(CD ) CO, 22.5 MHz], see
Table 2; EIMS m/z 160 [M] (37), 158 [M] (100), 140
(6), 123 (88), 105 (8), 95 (12).
3
max
(log ꢀ) 282 (2.78)
3
max
3594, 3528, 3314, 1601, 1479, 1350,
-
1 1
3
1
3
3
2
+
+
2
,4,6-Tr ich lor o-3-h yd r oxy-5-m eth oxytolu en e (2):
colorless crystals; mp 119-121 °C; UV (CH3CN) λmax (log
Or cin ol (9): colorless crystals; mp 82-84 °C; UV
(CH3CN) λmax (log ꢀ) 275 (3.20), 281 (3.20) nm; IR
(CHCl3) νmax 3592, 3320, 1600, 1479, 1336, 1299, 1147,
ꢀ
) 284 (3.28), 292 (3.34) nm; IR (CHCl3) νmax 3518, 2938,
-
1 1
1
562, 1458, 1396, 1350, 1284, 1094, 1035, 949 cm ; H