Bioactive abietane and seco-abietane diterpenoids from Salvia prionitis

Article abstract of DOI:10.1021/np010561j

From the roots of Salvia prionitis a new tricyclic diterpene, saprirearine (1), a new anhydride-type compound, saprionide (2), a new 7,8-seco-abietane diterpene derivative, 7,8-seco-para-ferruginone (3), and two new 4,5-seco-5,10-friedo-abietane diterpeno

Full text of DOI:10.1021/np010561j

1
016
J . Nat. Prod. 2002, 65, 1016-1020
Bioa ctive Abieta n e a n d seco-Abieta n e Diter p en oid s fr om Sa lvia pr ion itis
Xin Chen, J ian Ding, Yong-Mao Ye, and J in-Sheng Zhang*
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
2
94 Tai-Yuan Road, Shanghai 200031, People’s Republic of China
Received November 9, 2001
From the roots of Salvia prionitis a new tricyclic diterpene, saprirearine (1), a new anhydride-type
compound, saprionide (2), a new 7,8-seco-abietane diterpene derivative, 7,8-seco-para-ferruginone (3),
and two new 4,5-seco-5,10-friedo-abietane diterpenoids, 4-hydroxysaprorthoquinone (4) and 3-keto-4-
hydroxysaprorthoquinone (5), were isolated. Their structures were established by spectroscopic methods
and chemical transformation. Compound 3 showed antimicrobial activities against two Gram-positive
organisms, Staphylococcus aureus and Micrococcus luteus, with MIC values of 20.0 and 15.0 µM,
respectively. Compound 4 showed significant inhibition against topoisomerase Ι with an IC50 value of 0.8
µM. Compound 5 exhibited cytotoxic activities against HL-60 human leukemia and the SGC-7901 and
MKN-28 stomach cancer cell lines, with IC50 values of 4.6, 0.2, and 0.3 µM, respectively.
Ta ble 1. 100 Hz 13C NMR Spectral Dataa and HMBC
Correlations for Compounds 1-3 (CDCl3)
As a part of a search for new antitumor agents from
Chinese medicinal plants, the roots of Salvia prionitis
Hance (Labiatae), collected in J iangxi Province, were
investigated. Salvia prionitis is used in Chinese folk
medicine as an antiphlogistic, antibacterial, and antitu-
1
2
3
13C
13C
13C
position
HMBC
HMBC
HMBC
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
25.9
26.0
3
167.0
36.0 3, 20
18.7
40.3 18, 19
34.0 2, 18, 19
42.1 1, 3, 6, 7, 20
42.7 5, 7
202.2 5, 6
188.2 11
154.1 1, 5, 14
43.5 2, 11
1
bercular drug. More than 40 compounds have been
isolated from this plant so far.2 In our continuing study
on its bioactive components, two new abietane (1 and 2)
and three new seco-abietane diterpenoids (3-5) were
obtained. We report herein the isolation, structure elucida-
tion, partial synthesis, and bioactivities of these com-
pounds.
-9
45.8 18, 19
164.9
149.1 2, 3, 18, 19 148.3
128.6 20
130.1
126.5 14
137.3 6,
136.3 1, 7, 14, OH 113.9 5, 17
137.3 2, 6, 20 99.3 OH
74.5
201.7 14, 15
6
129.4 17
134.7 13
124.9
7
5
131.1 6, 13
0
1
2
3
4
5
6
7
8
9
0
2
164.9 13, 14 135.0
138.9 13, 14 187.7 14, 15
141.1 15, 16, 17
135.5
133.7
6
152.8 11, 16, 17
7
27.1 15, 16 133.0 15
26.8 14, 16, 17
21.5 15, 17
21.5 16, 17
111.7 3, 19
24.5 18
22.2 14, 16
22.2 14, 15
23.5
26.2 14, 16, 17
21.1 15
21.4 15
23.0 5, 19
33.5 5, 18
20.6 5
5
19.7
6
a
Assignments based on BB, DEPT, and HMQC spectra; data
are in ppm.
Resu lts a n d Discu ssion
Compound 1, obtained as pale yellow prisms with mp
4
5 °C, was assigned a molecular formula of C20
H
24
O
2
as
+
determined by HREIMS ([M] at m/z 296.1771). The IR
spectrum of 1 showed an R,â-unsaturated carbonyl group
at 1678 cm , which was supported by a C NMR signal δ
-
1
13
-
1
2
01.7, as well as a hydroxyl group at 3442 cm , corre-
1
3
sponding to an oxygenated C NMR signal at δ 74.5 (Table
1
1
). The H NMR spectrum (Table 2) showed the presence
of an aromatic isopropyl group at δ 1.13 (6H, d, J ) 6.6
Hz) and 2.96 (1H, septet, J ) 6.6 Hz), two methyl groups
attached to a benzene ring at δ 2.03 and 2.23, an exo-
methylene singlet at δ 4.90, and a hydroxyl signal at δ 2.88
2
(1H, s, D O exchangeable). Aromatic protons were exhibited
at δ 6.90 (1H, s), and two doublets were observed in an
AB pattern at δ 6.99 (1H, d, J ) 7.7 Hz) and 7.11 (1H, d,
J ) 7.7 Hz). The HMBC correlations for the H-3 methine
proton at δ 2.46 with the carbonyl C-12 at δ 201.7, and
with the exomethylene carbon C-18 at δ 111.7, clearly
indicated a rearranged abietane skeleton.10
*
To whom correspondence should be addressed. Tel: 0086-21-64311833.
Fax: 0086-21-64370269. E-mail: jszhang@mail.shcnc.ac.cn.
1
0.1021/np010561j CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/04/2002

Bioactive Abietane Diterpenoids from Salvia
J ournal of Natural Products, 2002, Vol. 65, No. 7 1017
Ta ble 2. 400 Hz 1H NMR Data for Compounds 1-3 (CDCl3)a
position
1
2
3
1
2
3
5
6
7
1
1
1
1
1
1
1
1
2
2.28 (m), 2.94 (m)
1.82 (m), 2.65 (m)
2.46 (m)
1.31 (m), 2.39 (m)
2.39 (m)
1.51 (m)
2.96 (m)
2.38 (m), 2.10 (m)
9.50 (s)
7.45 (d), J 5,6 ) 8.2 Hz
8.01 (d), J 5,6 ) 8.2 Hz
6.99 (d), J 6,7 ) 7.7 Hz
7.11 (d), J 6,7 ) 7.7 Hz
1
3
4
5
6
7
8
9
0
6.54 (s)
7.98 (s)
3.51 (m)
1.36 (d), J 14,15 ) 6.6 Hz
1.36 (d), J 14,15 ) 6.6 Hz
2.95 (s)
6.90 (s)
2.96 (m)
1.13 (d), J 15,16 ) 6.6 Hz
1.13 (d), J 15,16 ) 6.6 Hz
4.90 (s)
6.42 (s)
2.95 (m)
1.12 (d), J 15,16 ) 7.0 Hz
1.12 (d), J 15,16 ) 7.0 Hz
0.94 (s)
2.23 (s)
2.03 (s)
0.87 (s)
1.17 (s)
OH
2.88 (s)
12.2 (s)
a
Data are in ppm, with multiplicity of signals given in parentheses: s, singlet; d, doublet; m, multiplet.
Sch em e 1
Compound 3 was isolated as yellow needles. Its molec-
ular formula was deduced as C20 by HREIMS. The
IR spectrum showed absorptions for aldehyde (1716 cm ),
28 3
H O
-1
-
1
para-quinone (1643 and 1627 cm ), and aromatic (1591
cm ) functionalities. The 1H NMR spectrum (Table 2)
showed two aromatic proton singlets at δ 6.42 and 6.54,
an isopropyl moiety with a doublet for two methyls at δ
-1
1
.12 (J ) 7.0 Hz) and a septet for a proton at δ 2.95, three
quaternary methyl groups at δ 0.87, 0.94, and 1.17, and
an aldehyde proton at δ 9.50. Its 13C NMR and DEPT
spectra (Table 1) exhibited five methyls, four methylenes,
and four methine groups, and two saturated carbons
together with the signals for a aldehyde carbon and two
quinone carbonyl carbons. The HMBC correlations for the
C-12 carbonyl at δ 187.7 with the H-15 proton in the
isopropyl unit at δ 2.95 and with the H-14 aromatic proton
at δ 6.42, and the C-8 carbonyl at δ 188.2 with the H-11
aromatic proton at δ 6.54, suggested that the isopropyl unit
should be attached to C-13 and that compound 3 must
possess a 7,8-seco-abietane skeleton. Interpretation of the
2D NMR spectra (HMQC, HMBC) supported this assign-
ment unambiguously. The stereochemistry at the C-5
position was determined from the ROESY spectrum. Since
the methyl group at C-10 could be assigned with a
â-configuration for biogenetic reasons and no correlation
between this methyl group and the proton at C-5 was
observed, H-5 was determined to have an R-configuration.
The stereochemistry of 3 was confirmed by chemical
Thus, careful analysis of its HMBC and HMQC spectrum
allowed the structural assignment of 1 as 11R-hydroxy-3-
isopropenyl-13-isopropyl-5-methyl-1,2,3,11-tetrahydrophe-
nalen-12-one, which has been named saprirearine. Com-
pound 1 possibly may be formed from the rearrangement
of sapriorthoquinone11 as shown in Scheme 1. The same
type of rearrangement for aethipinone was proposed by
Ulubelen et al. to explain the existence of candidissiol in
the roots of Salvia candidissima.10
Compound 2 was obtained as a pale yellow amorphous
powder. Its molecular formula was determined as C16
14 4
H O
by HREIMS. The IR spectrum of 2 exhibited bands at-
-
1
tributable to hydroxyl (3435 cm ), conjugated anhydride
1778, 1711 cm 1), and aromatic groups (1599 cm-1). The
H NMR data (Table 2) indicated an AB system with meta-
-
(
1
correlated protons at δ 7.45/8.01 (J ) 8.2 Hz) and an
aromatic proton singlet at δ 7.98. Additional coupled
protons at δ 3.51 (1H) and 1.36 (6H) confirmed an AB
6
system (J ) 6.6 Hz) and were in agreement with an
isopropyl moiety. Finally, signals were observed for a
methyl group at δ 2.95 and a hydroxyl signal at δ 12.20
1
4
transformation. A reference sample, ferruginol, was
treated with Fremy’s salt and KH PO at room tempera-
2
4
(due to chelation between a carbonyl and the hydroxyl) that
ture for 6 h to give compound 3 in a yield of 83%. The
spectral data of the synthetic compound and compound 3
isolated from the plant were identical. Compound 3 is the
first compound possessing a 7,8-seco-abietane skeleton
isolated from a plant in the genus Salvia and has been
assigned as 7,8-seco-para-ferruginone. On the basis of the
fact that it was synthesized from ferruginol and from a
proposal by Gonzalez et al. for the biogenesis of highly
oxidized abietatriene diterpenes,15 this compound may be
produced from the radical oxidation of ferruginol in the
plant with the participation of singlet-state oxygen ap-
pearing to play an important role (Scheme 2). Compound
disappeared with D O. The presence of this chelated proton
2
led to the conclusion that the anhydride moiety was
attached to the ortho position to the hydroxy group in the
aromatic ring. HMBC correlations (Table 1) for the isopro-
pyl methine proton H-14 at δ 3.51 and the aromatic
methine C-13 at δ 133.7, and the aromatic proton H-5 at δ
7
.45 with the methyl group C-17 at δ 23.5, indicated that
the isopropyl and the methyl groups were located at C-12
and C-4, respectively. Compound 2 (11-hydroxy-12-isopro-
pyl-4-methylbenzo[de]isochromene-1,3-dione), which has
been named saprionide, is a rare type of new abietane
derivative with an anhydride moiety. Chang et al. reported
3
is the first 7,8-seco-abietane diterpenoid to have so far
the isolation of similar anhydride-type abietane compounds
from Salvia miltiorrhiza.12 Furthermore, Kusumi et al.
been isolated from a plant in the genus Salvia.
reported the preparation of an anhydride-type abietane
compound with a skeleton similar to 2 by photooxidation
of a diterpenequinone.13 Accordingly, compound 2 might
be produced by the same type of bio-oxidation mechanism
in the plant.
4-Hydroxysaprorthoquinone (4) was isolated as red
syrup. Its molecular formula was deduced as C20
26 3
H O by
+
HREIMS ([M] at m/z 314.1825). Its IR spectrum showed
-
1
peaks at 3390 cm (hydroxyl) and 1662, 1640, and 1630
-
1
cm (ortho-quinoid). This compound exhibited NMR data

1
018 J ournal of Natural Products, 2002, Vol. 65, No. 7
Chen et al.
Ta ble 3. NMR Data for Compounds 4, 4a , and 5a
4
4a
5
position
1H (J )
13C
1H (J )
1H (J )
13C
HMBC
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2.99 (m)
1.62 (m)
1.62 (m)
30.6
23.8
44.2
2.98 (m)
1.50 (m)
1.95 (m)
3.25 (m)
2.78 (m)
24.9
23.2
213.6
76.4
2, 3, 5, 10
1, 3,
71.1
148.6
140.3
136.7
128.2
134.9
140.0
182.3
181.4
144.6
128.1
26.9
146.2
134.0
137.0
128.6
135.0
140.0
182.4
181.2
144.9
128.6
26.9
7.03 (d, 7.5)
7.34 (d, 7.5)
7.03 (d, 7.6)
7.34 (d, 7.6)
7.07 (d, 7.6)
7.37 (d, 7.6)
8, 10, 20
5, 9, 14
0
1
2
3
4
5
6
7
8
9
0
7.07 (s)
7.06 (s)
7.07 (s)
7, 9, 12, 13, 15
12, 14, 16(17)
13, 15
13, 15
3, 19
2.99 (m)
1.14 (d, 7.0)
1.14 (d, 7.0)
1.21 (s)
1.21 (s)
2.36 (s)
2.98 (m)
1.14 (d, 7.0)
1.14 (d, 7.0)
1.42 (s)
1.42 (s)
2.34 (s)
2.99 (m)
1.14 (d, 7.0)
1.14 (d, 7.0)
1.40 (s)
1.40 (s)
2.35 (s)
21.5
21.5
29.2
29.2
21.4
21.4
26.5
26.5
3, 18
6, 10
19.8
19.7
OAc
1.93 (s)
a
1H NMR, 400 MHz; 13C NMR, 100 MHz; run in CDCl3; data in ppm (J in Hz). Multiplicity of signals is given in parentheses: s,
singlet; d, doublet; m, multiplet.
Sch em e 2
derivative 4a (Table 3) in 44% yield, along with the
dehydration product saprorthoquinone (Scheme 3). This
result further proved the structure of compound 4, which
is a 4,5-seco-5,10-friedo-abietane diterpenoid.
3
-Keto-4-hydroxysaprorthoquinone (5), a red syrup, was
assigned a molecular formula of C20 , as determined
by HREIMS. The IR spectrum exhibited frequencies for a
24 4
H O
-
1
-1
hydroxyl at 3487 cm , a carbonyl group at 1708 cm , and
-1
an ortho-qunonoid group at 1688, 1658, and 1633 cm . The
1
H NMR data (Table 3) were also very similar to those of
sapriorthoquinone,11 except for the loss of an olefinic
proton, which suggested that both compounds possess the
same skeleton but with different side chains. The chemical
1
3
shifts of the two methyl groups at δ 1.40 as well as the
C
NMR signal at δ 76.4 indicated the presence of a hydroxyl
group at C-4. The HMBC spectrum showed correlations for
CH -4 at δ 1.40 with the carbonyl group at δ 213.6,
3
suggesting that the carbonyl group was placed at C-3.
Thus, compound 5 was characterized as 3-keto-4-hydroxy-
4,5-seco-5,10-friedo-abieta-3,5(10),6,8,13-pentaene-11,12-di-
one and has been given the trivial name 3-keto-4-hydrox-
ysaprorthoquinone. Earlier, Ulubelen et al. reported the
compound sclareapinone, isolated from Salvia sclarea, with
the same structure as 5 based on interpretation of its 1D
(
Table 3) very similar to that of saprorthoquinone.11 The
main differences observed were the missing signals due to
an olefinic methine at δ 5.28/123.8 and additional signals
due to a methylene at δ 1.62/44.2, which indicated that
there was an oxygenated carbon instead of two olefinic
carbons in the side chain of this new compound. Its 13
C
1
13
16
NMR spectrum showed two carbonyl signals, eight aro-
matic signals, and an oxygenated quaternary carbon.
Careful analysis of its NMR spectrum led to the deduction
of its structure as 4-hydroxysaprothoquinone (4). To sup-
port this assignment, compound 4 was synthesized from
saprorthoquinone11 (Scheme 3). All the spectral data of the
synthetic compound were identical with those of the
isolated compound. Compound 4 was treated with acetic
anhydride with p-TsOH used as a catalyst to give its acetyl
H and C NMR spectra. However, on comparing the
spectral data of both compounds, we found some differ-
ences. The isolate obtained in the present study was
assigned with the structure 5 not only from the HMBC
spectrum but also by chemical transformation. Compound
5 was synthesized from the known compound sapriortho-
quinone11 in three steps (Scheme 4) to confirm its structure.
According to the structure of the starting material, two
hydroxyl groups in the intermediate 6 could be formed only
Sch em e 3

Bioactive Abietane Diterpenoids from Salvia
J ournal of Natural Products, 2002, Vol. 65, No. 7 1019
Sch em e 4
EtOAc (4:1) to afford compound 4 (R
compound 5 (R ) 0.3, 15 mg).
f
) 0.7, 60 mg) and
f
Com p ou n d 1: pale yellow prisms, mp 45 °C; UV (MeOH)
max (log ꢀ) 243 (4.22), 335 (3.85) nm; IR (KBr) νmax 3442, 2958,
λ
-
1
1
13
1
678, 1633, 1458, 1377, 868 cm ; H and C NMR data, see
+
Tables 1 and 2; EIMS m/z 296 [M] (40), 268 (56), 253 (32),
28 (38), 225 (100), 185 (18); HREIMS m/z 296.1771 (calcd for
, 296.1776).
Com p ou n d 2: pale yellow powder; UV (CHCl
2
20 24 2
C H O
3
) λmax (log ꢀ)
3
1
33 (3.17), 358 (3.14), 374 (3.43) nm; IR (KBr) νmax 3435, 2953,
-
1 1
778, 1711, 1648, 1599, 1506, 1159, 1095, 959 cm ; H and
1
3
+
C NMR data, see Tables 1 and 2; EIMS m/z 270 [M] (84),
2
55 (100), 242 (31), 237 (20), 224 (6), 209 (10), 185 (17), 153
14 4
(16), 149 (30); HREIMS m/z 270.0905 (calcd for C16H O ,
2
70.0892).
Com p ou n d 3: yellow needles; mp 151-152 °C; UV (MeOH)
λ
max (log ꢀ) 260 (4.07) nm; IR (KBr) νmax 2964, 1716, 1643, 1627,
-
1
1
13
1
591, 1236, cm ; H and C NMR data, see Tables 1 and 2;
+
EIMS m/z 316 [M] (23), 301 (6), 298 (8), 283 (7), 272 (100),
57 (24), 229 (12), 216 (20), 203 (40); HREIMS m/z 316.2029
(calcd for C20 , 316.2038 ).
Oxid a tion of F er r u gin ol to Com p ou n d 3. Fremy’s salt
0.7 g) and KH PO (2.12 g, 15.6 mmol) were dissolved in water
60 mL). To this solution was added ferruginol (0.26 g, 0.91
2
at C-3 and C-4, respectively. Furthermore, only the hy-
droxyl group at C-3 could be oxidized to a carbonyl group.
Like compound 4, 5 is also a 4,5-seco-5,10-friedo-abietane
diterpenoid.
The bioactivities of compounds 1-5 have been tested.
Compound 3 displayed antimicrobial activity against the
Gram-positive organisms, Staphylococcus aureus and
Micrococcus luteus, when tested according to established
protocols17 (MIC values of 20.0 and 15.0 µM, respectively).
Compound 4 showed a significant inhibitory effect (IC50 0.8
28 3
H O
(
(
2
4
1
4
mmol) in 40 mL of acetone. The mixture was stirred in the
dark under N overnight. On concentration in vacuo, the
mixture was extracted with ether three times. The organic
2
phase was washed with water and brine, dried (MgSO ), and
4
evaporated. The residue was purified by flash chromatography
on a silica gel column (20 g) eluted with cyclohexane-EtOAc
(20:1) to give compound 3 (0.64 g, 83% yield).
Com p ou n d 4: red syrup, UV (MeOH) λmax (log ꢀ) 211 (4.48),
µM) on topoisomerase Ι according to a method developed
by Isabella et al.18 Compound 5 was evaluated for its
249 (4.47), 342 (3.57), 436 (3.25) nm; IR (film) ν
max 3390, 2930,
-
1
1
13
1
662, 1640, 1508, 1457, 1251, 1035 cm ; H and C NMR
cytotoxic activities against HL-60 human leukemia cells
and the SGC-7901 and MKN-28 stomach cancer cell lines
by a microculture tetrazolium colorimetric assay (MTT).19
This substance exhibited potent activities against the three
cancer cell lines with IC50 values of 4.6, 0.2, and 0.3 µM,
respectively. Compounds 1 and 2 were not found to be
active in any of these three assays.
+
data, see Table 3; EIMS m/z 314 [M] (3), 312 (28), 286 (5),
68 (7), 253 (3), 243 (100), 227 (10); HREIMS m/z 314.1825
calcd for C20 , 314.1882).
Acetyla tion of 4. A portion (20 mg, 0.064 mmol) of
compound 4 was dissolved in 2 mL of Ac O, and a trace amount
2
(
26 3
H O
2
of p-TsOH was added. The mixture was stirred at room
temperature with protection from light for 2 h. After the usual
workup, the reaction mixture was purified by preparative TLC
(
cyclohexane-EtOAc, 10:1; R ) 0.4) to afford 10 mg of 4a .
f
Exp er im en ta l Section
Compound 4a : red syrup, UV (MeOH) λmax (log ꢀ) 213 (3.81),
Gen er a l Exp er im en ta l P r oced u r es. The melting points
were determined on a Kofler hot-stage apparatus and are
uncorrected. UV spectra were measured on a Beckman DU-
220 (3.83), 247 (3.87), 407 (2.65), 443 (2.60) nm; IR (film) νmax
2860, 1732, 1662, 1433, 1367, 1256, 1175, 1020, 756 cm ; 1H
-1
+
NMR data, see Table 3; EIMS m/z 356 [M] (4), 298 (12), 296
(
36), 268 (36), 227 (100), 141 (14); HREIMS m/z 356.1993 (calcd
for C22 , 356.1988).
2
Syn th esis of 4 fr om Sa p r ior th oqu in on e. Hg(OAc) (190
6
00 spectrophotometer. IR spectra were measured on a Nicolet
1
13
28 4
H O
Magna 750 spectrophotometer. H, C, HMBC, and ROESY
1
NMR spectra were recorded on a Bruker AM-400 ( H) or a
1
3
mg, 0.50 mmol) was placed in a 50 mL round-bottomed flask,
and 5 mL of water together with 10 mL of THF were added,
followed by 150 mg of sapriorthoquinone11 (0.50 mmol). The
mixture was stirred at room temperature for 30 min. Then,
Bruker AC-100 ( C) spectrometer with TMS as internal
standard and CDCl as solvent. Mass spectra were obtained
3
on a MAT 711 mass spectrometer. Silica gel (200-300 mesh)
was used for column chromatography and silica gel GF254 for
preparative TLC.
0
.5 mL of a 3 M NaOH solution and 0.5 mL of 0.5 M NaBH
4
in a 3 M NaOH solution were added, and the reaction was
stopped immediately. After the usual workup, the crude
product was chromatographed by silica gel column chroma-
tography (cyclohexane-EtOAc, 7:1) to furnish 40 mg of
compound 4 (yield 20%).
P la n t Ma ter ia l. Salvia prionitis was collected at Yujiang,
J iangxi Province, People’s Republic of China, in J uly 1995, and
authenticated by Prof. X. L. Huang of our institute. A voucher
specimen is maintained in the Herbarium of Shanghai Insti-
tute of Materia Medica (SIMMP 95068).
Extr a ction a n d Isola tion . Dried roots of S. prionitis (5
kg) were extracted with EtOAc for 6 days at room temperature.
The solvent was removed in vacuo to yield 350 g of a gummy
residue. The extract was subjected to repeated chromatography
over silica gel eluted with mixture of cyclohexane-EtOAc of
increasing polarity (100:1 to 5:1). The fraction that eluted with
cyclohexance-EtOAc (30:1) was further chromatographed
using the same adsorbent and solvent system as presented
above to give 90 mg of compound 1 together with a mixture
containing compounds 2 and 3. This mixture was purified by
preparative TLC using cyclohexane-EtOAc (10:1) to afford
Com p ou n d 5: red syrup; UV (MeOH) λmax (log ꢀ) 353 (3.21),
435 (3.41) nm; IR (KBr) νmax 3487, 2966, 2936, 2872, 1709,
1689, 1659, 1633, 1568, 1464, 1254 cm ; H and C NMR
data, see Table 3; EIMS m/z 328 [M] (2), 316 (8), 286 (2), 270
(100), 242 (50), 228 (90), 224 (64), 200 (47); HREIMS m/z
-
1
1
13
+
328.1654 (calcd for C20
Syn th esis of Com p ou n d 5. To a stirred solution of
m-chloroperbenzoic acid (43 mg, 0.025 mmol) in CHCl (10 mL)
held at 0 °C was added a solution of sapriorthoquinone (60
mg, 0.02 mmol) dissolved in 6 mL of CHCl . The mixture was
stirred overnight at room temperature, washed with 10%
NaHCO solution, and dried over anhydrous MgSO . The
solvent was concentrated in vacuo, and the residue was
purified by silica gel column chromatography by elution with
cyclohexane-EtOAc (9:1) to afford epoxide 6 (50 mg, 79%
24 4
H O 328.1675).
3
1
1
3
3
4
compound 2 (R
f
) 0.4, 10 mg) and compound 3 (R
f
) 0.3, 15
mg). The fraction that eluted with cyclohexane-EtOAc (5:1)
was further purified by preparative TLC using cyclohexane-

1
020 J ournal of Natural Products, 2002, Vol. 65, No. 7
Chen et al.
yield). Then, 50 mg (0.16 mmol) of 6 were dissolved in 12 mL
of tetrahydrofuran. After the addition of 2 mL of water,
followed by 0.4 mL of 8% perchloric acid, the mixture was
stirred under N at room temperature for 6 h. Then, 30 mL of
2
brine were added and the solution was extracted several times
with ether. The organic phase was washed with dilute NaH-
(Project No. 39830440). The authors thank the National Center
for Drug Screening of China for the antimicrobial assays, and
the Division of Antitumor Pharmacology, Shanghai Institute
of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, for the topoisomerase Ι inhibi-
tion and cytotoxicity assays.
CO
was purified by preparative TLC (cyclohexane-EtOAc 1:2) to
give red crystals of compound 7 (R ) 0.6, 34 mg, 65% yield).
A portion (34 mg, 0.1 mmol) of 7 was further treated with PCC
110 mg, 0.51 mmol) in 10 mL of CH Cl . The solution was
stirred at room temperature for 30 min. Then, the mixture
was filtered and the filtrate dried over MgSO and evaporated.
3 4
and brine and dried (MgSO ). On evaporation, the residue
f
Refer en ces a n d Notes
(1) Luo, X. R., Ed. Colored Icons of Chinese Medicine; Guangdong Science
and Technology Press: Guangdong, 1994; Vol. 3.
(
2
2
(2) Zhang, J . S.; Huang, Y. Nat. Prod. Res. Dev. 1995, 7, 1-4.
4
(3) Huang, X. L.; Wang, X. M.; Huang, Y.; Zhang, J . S.; Lin L. Z. Acta
Bot. Sin. 1990, 32, 490-491.
The residue was purified by preparative TLC (cyclohexane-
(
4) Lin, L. Z.; Wang, X. M.; Huang, X. L.; Huang, Y. Acta Pharm. Sin.
990, 25, 154-156.
5) Lin, L. Z.; Wang, X. M.; Huang, X. L.; Yang, B. J . Planta Med. 1988,
EtOAc 4:1) to furnish compound 5 (R ) 0.3, 20 mg, 30% yield
f
1
from sapriorthoquinone).
(
Bioa ssa ys. An tim icr obia l Assa y. The microbial strains
were from the American Type Culture Collection, and the
antimicrobial susceptibility tests were carried out by a mi-
5
4, 443-444.
(6) Lin, L. Z.; Blasko, G.; Cordell, G. A. Phytochemistry 1989, 28, 177-
181.
1
7
(7) Lin, L. Z.; Cordell, G. A.; Lin, P. Phytochemistry 1995, 40, 1469-
crodilution assay. The microbial cells were suspended in
-
5
-6
1471.
Mueller-Hinton broth to form a final density of 5 × 10 -10
(
8) Li, M.; Zhang, J . S.; Ye, Y. M.; Fang, J . N. J . Nat. Prod. 2000, 63,
39-40.
CFU/mL and incubated at 37 °C for 18 h under aerobic
conditions with the respective compounds, which were dis-
solved in DMSO. The blank controls of microbial culture were
incubated with a limited amount of DMSO under the same
conditions. DMSO was determined not to be toxic under these
experimental conditions.
1
(9) Li, M.; Zhang, J . S.; Chen, M. Q. J . Nat. Prod. 2001, 64, 971-972.
(10) Ulubelen, A.; Topcu, G.; Tan, N. Tetrahedron Lett. 1992, 33, 7241-
7244.
(
11) Yang, B. J .; Huang, X. L.; Huang, Y.; Wang, X. M.; Lin, L. Z.; But, P.
H.; Zhuang, G. F. Acta Bot. Sin. 1988, 30, 524-527.
12) Chang, H. M.; Choang, T. F.; Chui, K. Y.; Hon, P. M.; Lee, C. M.;
Mak, T. C. W.; Wong, H. N. C. J . Chem. Res. (S) 1990, 114-115.
(
Top oisom er a se Ι In h ibition Assa y. The topoisomerase Ι
inhibition assay was performed using the topoisomerase Ι
extracted from Ehrlich carcinoma cells according to a method
(13) Kusumi, T.; Kishi, T.; Kakisawa, H.; Kinoshita, T. J . Chem. Soc.,
Perkin Trans. 1 1976, 1716-1718.
(14) Mayer, W. L.; Clemans, G. B.; Manning, R. A. J . Org. Chem. 1975,
1
8
developed by Isabella et al. The activity was measured by
topoisomerase Ι mediated supercoiled pBR322 relaxation.
Cytotoxicity Assa y. Cytotoxic activities were carried out
against HL-60 human leukemia cells and two stomach cancer
cell lines (SGC-7901 and MKN-28). The three cell lines were
exposed to the compounds for 48 h. The cytotoxic activities
were measured by a microculture tetrazolium colorimetric
4
6, 3686-3694.
(
(
15) Gonzalez, A. G.; Andes, L. S.; Aguiar, Z. E.; Luis, J . G. Phytochemistry
1
992, 31, 1297-1305.
16) Ulubelen, A.; Sonmez, U.; Topcu, G. Phytochemistry 1997, 44, 1297-
1299.
(17) Demetzos, C.; Stahl, B.; Anastassaki, M.; Tzouveleki, L.; Rallis, M.
Planta Med. 1999, 65, 76-80.
1
9
(18) Isabella, D. P.; Capranico, G.; Binashi, M.; Tinelli, S.; Zunino, F. Mol.
Pharmacol. 1990, 37, 11-16.
assay (MTT).
(19) Mossman, T. J . Immunol. Methods 1983, 65, 55-63.
Ack n ow led gm en t. This work was supported by a grant
from the National Natural Science Foundation of China
NP010561J