Cytotoxic and antiplatelet aggregation principles from Aglaia elliptifolia

Article abstract of DOI:10.1021/np970163+

Two related 1H-2,3,3a,8b-tetrahydrocyclopenta[b]benzofurans, aglafolin (1a) and rocaglamide (2), isolated from the stems of Aglaia elliptifolia, showed significant cytotoxicity in six cancer cell lines. Aglafolin (1a) was also found to completely block platelet aggregation caused by arachidonic acid and platelet-activating factor at 100 μM and 2 ng/mL, respectively.

Full text of DOI:10.1021/np970163+

606
J . Nat. Prod. 1997, 60, 606-608
Cytotoxic a n d An tip la telet Aggr ega tion P r in cip les fr om Agla ia elliptifolia
Tian-Shung Wu,*,† Meei-J en Liou,† Chang-Sheng Kuoh,‡ Che-Ming Teng,§ Tsuneatsu Nagao, and
Kuo-Hsiung Lee*,
Department of Chemistry, National Cheng-Kung University, Tainan, Taiwan, Republic of China, Department of Biology,
National Cheng-Kung University, Tainan, Taiwan, Republic of China, Pharmacological Institute, College of Medicine,
National Taiwan University, Taipei, Taiwan, Republic of China, and Natural Products Laboratory, Division of Medicinal
Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599
Received August 18, 1995X
Two related 1H-2,3,3a,8b-tetrahydrocyclopenta[b]benzofurans, aglafolin (1a ) and rocaglamide
(2), isolated from the stems of Aglaia elliptifolia, showed significant cytotoxicity in six cancer
cell lines. Aglafolin (1a ) was also found to completely block platelet aggregation caused by
arachidonic acid and platelet-activating factor at 100 µM and 2 ng/mL, respectively.
Previously, King et al. reported the isolation and
structural characterization of rocaglamide (2), a novel
1H-2,3,3a,8b-tetrahydrocyclopenta[b]benzofuran, from
an alcoholic extract of the dried roots and stems of
Aglaia elliptifolia Merr. (Meliaceae).1 This compound
showed an optimal T/C value of ca. 156% at a dosage of
1.0 mg/kg against the P-388 murine lymphocytic leu-
kemia in vivo test system.1 In 1992, our investigation
of a MeOH extract of the stems of this same plant
resulted in the isolation of a related compound, aglafolin
(1a ), where the dimethylamino group of rocaglamide (2)
is replaced by a methoxy group.2 Aglafolin (1a ) was
described by us as a selective and effective inhibitor of
platelet aggregation induced by platelet-activating fac-
tor (PAF) both in vitro and in vivo.2 The isolation of
this compound from a related species, Aglaia odorata,
and its insecticidal activity was described by Ishibashi
et al.;3 however, the compound was named as methyl
rocaglate in their paper. Herein, we describe the
structural confirmation of aglafolin by NMR spectros-
copy, including 2D methods, and by chemical transfor-
mation to rocaglamide. The antiplatelet aggregation
activity and in vitro cytotoxicity of these two compounds
are also reported.
The stems (2.7 kg) of A. elliptifolia were extracted four
times with MeOH, and the MeOH extract (189.2 g) was
partitioned between CHCl3 and H2O. Repeated column
chromatography of the CHCl3 extract (108.1 g) on Si gel
gave a mixture (2.62 g) of the phytosterols â-sitosterol
and stigmasterol, aglafolin (1a , 580 mg), scopoletin (25
mg), and rocaglamide (2, 135 mg). Like aglafolin (1a ),
scopoletin and rocaglamide (2) were both previously
isolated from A. odorata; however, these latter two
compounds were obtained as a mixture.4
for the absence of the two N-Me singlets and presence
of an OMe singlet in the former. The 13C-NMR spectra
(Table 1) of 1a , 1b, and 2 were also similar to each other,
except for the absence and/or presence of the amide and
ester methyl groups, and are reported here in CDCl3
(the spectra of 1a and 2 were previously reported in
1
acetone-d6).3 The H- and 13C-NMR spectra of 1a , 1b,
and 2 were completely assigned by 2D-NMR techniques
including 1H- and 13C-NMR long-range correlations and
NOESY correlations.
Because aglafolin (1a ) has been previously identified
as a PAF-antagonist, scopoletin and compounds 1a and
2 were tested for inhibition of platelet aggregation
induced by adenosine diphosphate (ADP), arachidonic
acid (AA), PAF, and collagen. As shown in Table 2, at
100 µg/mL aglafolin (1a ) completely blocked the platelet
aggregation caused by PAF and AA but had no effect
on that caused by ADP or collagen. Scopoletin and
rocaglamide (2) were either completely inactive or
showed only slight inhibition at the doses tested.
Also, because rocaglamide (2) has been previously
reported to show antileukemic activity in vitro, com-
pounds 1a and 2 were tested against the KB (nasal
pharyngeal carcinoma), A-549 (human lung carcinoma),
HCT-8 (human colon carcinoma), P-388 (murine leuke-
mia), RPMI-7951 (human melanoma), and TE-671 (hu-
man medulloblastoma) cancer cell lines, and scopoletin
was tested against the KB cell line. As shown in Table
3, compounds 1a and 2 were broadly cytotoxic in all cell
lines, with IC50 values in the ng/mL range. These
compounds deserve further investigation as potentially
useful antitumor agents.
Exp er im en ta l Section
The chemical conversion of 1a to 2 was accomplished
in two steps as shown in Scheme 1. First, saponification
of 1a provided the carboxylic acid 1b; second, amidation
with dimethylamine gave 2.
The 1H-NMR spectra of 1a and 2 were similar to those
reported in the literature3,4 and to each other, except
Gen er al Exper im en tal P r ocedu r es. Melting points
were measured on a Yanagimoto MP-S3 apparatus and
are uncorrected. Optical rotations were measured on
a J ASCO DIP-370 polarimeter in CHCl3. IR spectra
were recorded on a Shimadzu FTIR-8501 spectropho-
tometer as KBr disks. UV spectra were recorded on a
Hitachi UV-3210 spectrophotometer in MeOH. Mass
spectra were determined on a VG 70-250S spectrometer.
1H- and 13C-NMR spectra were recorded on Bruker AC-
300 and AMNX-400 spectrometers with TMS as an
internal standard.
* Authors to whom correspondence should be addressed. Phone:
(919) 962-0066. FAX: (919) 966-3893. E-mail: khlee@unc.edu.
† Department of Chemistry, National Cheng-Kung University.
‡ Department of Biology, National Cheng-Kung University.
§ National Taiwan University.
University of North Carolina.
X Abstract published in Advance ACS Abstracts, J une 1, 1997.
S0163-3864(97)00163-8 CCC: $14.00
© 1997 American Chemical Society and American Society of Pharmacognosy

Notes
J ournal of Natural Products, 1997, Vol. 60, No. 6 607
Sch em e 1. Interconversion of 1a , 1b, and 2
Ta ble 1. 13C-NMR Assignments of Compounds 1a , 1b, and 2
in CDCl3
sterols â-sitosterol and stigmasterol was obtained from
subfraction 6. Subfraction 7 afforded aglafolin (1a , 580
mg, 0.021%). Rocaglamide (2) was isolated from sub-
fraction 12 (135 mg, 0.005%) and also from fraction C
(1.21 g, 0.045%). The physical and spectral data of
compounds 1a and 2 were consistent with those in the
literature; 13C-NMR data in CDCl3 are included in Table
1.
position
1a
1b
2
1
2
79.6
50.5
79.2
51.2
78.5
47.5
3
55.0
55.1
55.8
3a
4a
5
101.9
160.9
89.5
102.1
161.0
89.0
101.5
161.0
89.2
6
7
8
8a
8b
1′
3′,5′
2′,6′
4′
164.1
92.6
157.0
107.7
93.7
126.5
112.7
129.0
158.7
137.0
127.7
127.8
126.5
55.7
163.7
92.2
157.3
107.3
93.6
127.0
112.0
128.8
158.4
137.7
127.6
128.0
126.1
55.4
163.8
92.4
157.1
107.6
93.9
127.1
112.6
128.8
158.5
137.6
127.6
127.7
126.2
55.6
Sa p on ifica tion of 1a a n d Con ver sion to 1b.
Compound 1a (50 mg) was dissolved in 0.4% KOH/
aqueous MeOH [MeOH-H2O (5:10)] and heated at 50
°C for 4 h. After being cooled to room temperature, the
reaction solution was neutralized with dilute HCl and
then diluted with saturated NaCl solution (30 mL). The
solution was extracted with Et2O four times, and the
ether layers were combined. After evaporation of
solvent, the residue was crystallized from Me2CO to give
1b (32 mg, 66%): colorless needles from Me2CO: mp
144-146 °C; [R]D -70.0° (c 0.62, CHCl3); IR υmax 3450,
1620, 1605, 1515 cm-1; 1H-NMR (CDCl3) δ 3.77 (3H, s,
MeO-4′), 3.83 (6H, overlapping s, MeO-6, MeO-8), 3.85
(1H, dd, J ) 8 Hz, H-2), 4.28 (1H, d, J ) 16 Hz, H-3),
4.86 (1H, d, J ) 8 Hz, H-1), 6.01 (1H, d, J ) 2 Hz, H-7),
6.22 (1H, d, J ) 2 Hz, H-5), 6.59 (2H, dd, J ) 2 and 8
Hz, H-3′, H-5′), 6.93 (2H, dd, J ) 2, 8 Hz, H-2′′, H-6′′),
6.97 (3H, m, H-3′′, H-4′′, H-5′′), 7.04 (2H, dd, J ) 2, 8
Hz, H-2′, H-6′); 13C-NMR data, see Table 1; HRFABMS
m/ z [M + H]+ 497.172 (C27H27O8 requires 479.170).
1′′
3′′, 5′′
2′′, 6′′
4′′
MeO-6
MeO-8
MeO-4′
NMe
55.8
55.1
55.5
54.9
55.6
54.9
35.6
36.9
CO2CH3
CO2CH3
52.0
170.5
175.8
169.5
Ta ble 2. Antiplatelet Aggregation Activity (%) of Compounds
1a and 2 and Scopoletina,b
compd
(100 µg/mL)
ADP
AA
PAF
collagen
(100 µM) (100 µM) (2 ng/mL) (10 µg/mL)
Am id a tion of 1b a n d Con ver sion to 2. Compound
1b (20 mg) was dissolved in CH2Cl2 (2 mL), and
dimethylamine (0.02 mL) was added at 0 °C under a
N2 current. The solution was stirred for 3 min, and then
1,1′-carbonyldiimidazole-CH2Cl2 solution (0.04 mL/1
mL) was added. After being stirred at 0 °C for 6 h, the
solution was warmed to room temperature, and a few
drops of dilute HCl were added. The reaction mixture
was diluted with saturated NaCl solution and extracted
three times with CH2Cl2. The CH2Cl2 layer was com-
bined, dried, and evaporated to afford 2 (5 mg, 24%).
The physical and spectral data of the synthetic com-
pound agreed with those of the known compound.
aglafolin (1a )
85.2 ( 0.7 0.0 ( 0 0.0 ( 0 88.6 ( 8.6
85.5 ( 7.1 83.8 ( 7.9
rocaglamide (2) 77.6 ( 0.8 90.5 ( 0
scopoletin
control
65.3 ( 2.6 79.2 ( 8.0 90.5 ( 0.6 79.2 ( 7.4
88.6 ( 0.6 87.9 ( 7.7 95.2 ( 0.6 90.5 ( 1.3
a
ADP ) adenosine diphosphate, AA ) arachinonic acid, PAF
b
) platelet-activating factor. Platelets were incubated with test
sample or 0.5% DMSO at 37 °C for 1 min, then ADP (100 µm), AA
(100 µm), collagen (10 µg/mL), or PAF (2 ng/mL) was added to
trigger the aggregation. Values are presented as mean ( S.D.
P la n t Ma ter ia l. A. elliptifolia was collected at
Orchid Island, Taiwan, in September 1985. A voucher
specimen is deposited at the Department of Biology,
National Cheng-Kung University, Tainan, Taiwan.
Extr a ction a n d Isola tion of Agla folin (1a ) a n d
Roca gla m id e (2). The stems of A. elliptifolia (2.7 kg)
were extracted four times with MeOH. After concentra-
tion, the MeOH extract (189.2 g) was suspended in H2O
and extracted with CHCl3. After concentration, the
CHCl3 extract (108.1 g) was chromatographed on Si gel
using CHCl3 as eluent to give four fractions, fractions
A-D. Fraction B was chromatographed repeatedly on
Si gel using isopropyl ether-C6H6 (1:3), CHCl3-MeOH
(200:1 to 70:1), and C6H6-Me2CO (4:1) as eluents to
yield 13 subfractions. A mixture (2.62 g) of the phyto-
Cytotoxicity Assa ys. The in vitro KB cytotoxicity
assay was carried out according to procedures described
by Geran et al.5 and Ferguson et al.6 The asssays
against A-549, HCT-8, P-388, RPMI-7951, and TE-671
tumor cells were based on a method reported by Lee et
al.7
P la t elet Aggr ega t ion Assa ys. Collagen (Type 1,
bovine achilles tendon) obtained from Sigma Chemical
Co. was homogenized in 25 mL of HOAc and then stored
at -70 °C. Adachidonic acid, bovine serum albumen
(BSA), EDTA (disodium salt), sodium citrate, dimethyl

608 J ournal of Natural Products, 1997, Vol. 60, No. 6
Ta ble 3. Cytotoxic Activity of 1a and 2 and Scopoletin
Notes
cell line (IC50 µg/mL)b
compda
KB
A-549
HCT-8
P-388
RPMI-7951
TE-671
aglafolin (1a )
rocaglamide (2)
scopoletin
<0.001
0.006
4.0
<0.001
0.006
NTc
0.005
0.007
NTc
0.002
0.005
NTc
<0.001
0.002
NTc
<0.001
0.006
NTc
a
Values (µg/mL) for standard antitumor drugs were:7 etoposide 0.12 (KB) and 2.62 (P-388); vinblastine sulfate 0.002 (A-549), 0.005
b
(HLT-8); doxorubicin HCl 0.15 (A-549) and 0.3 (HCT-8). Values were obtained as described in references 5-7; KB (nasal pharyngeal
carcinoma), A-549 (human lung carcinoma), HCT-8 (human colon carcinoma), P-388 (murine leukemia), RPMI-7951 (human melanoma),
TE-671 (human medullablastoma). c NT ) not tested.
sulfoxide (DMSO), and platelet-activating factor (PAF)
were purchased from Sigma Chemical Co.
Ack n ow led gm en t. This work was supported by
grants from the National Science Council of the Republic
of China [NSC 80-0420-B006-07 (T.-S.W.)] and the
National Cancer Institute, NIH, Bethesda, MD [CA17625
(K.H.L.)].
P la telet Su sp en sion P r ep a r a tion . Blood was col-
lected from the rabbit marginal ear vein and was mixed
with EDTA to a final concentration of 6 mM. It was
centrifuged at 90g for 10 min at room temperature, and
the supernatant was obtained as platelet-rich plasma.
The latter was further centrifuged at 500g for 10 min.
The platelet pellets were washed with Tyrode’s solution
without EDTA. After centrifugation at the same condi-
tions, the platelet pellets were finally suspended in
Tyrode’s solution of the following composition (mM):
NaCl (136.8), KCl (2.8), NaHCO3 (11.9), MgCl2 (1.1),
NaH2PO4 (0.33), CaCl2 (1.0), and glucose (11.2). Platelet
numbers were counted by Coulter Counter (Model ZM)
and adjusted to 4.5 × 108 platelets/mL.
Refer en ces a n d Notes
(1) King, M. L.; Chiang, C. C.; Ling, H. C.; Fujita, E.; Ochiai, M.;
McPhail, A. T. J . Chem. Soc., Chem. Commun. 1982, 1150-1151.
(2) Ko, F. N.; Wu, T. S.; Liou, M. J .; Huang, T. F.; Teng, C. M. Eur.
J . Pharmcol. 1992, 218, 129-135.
(3) Ishibashi, F.; Satasook, C.; Isman, M. B. Phytochemistry 1993,
32, 307-310.
(4) J anprasert, J .; Satasook, C.; Sukumanlanand, P.; Champagne,
D. E.; Isman, M. B.; Wiriyachitra, P. Phytochemistry 1993, 32,
67-69.
(5) Geran, R. I.; Greenberg, N. H.; MacDonald, M. M.; Schumacher,
A. M.; Abbott, B. J . Cancer Chemother. Rep. 1972, 3 (2), 1-103.
(6) Ferguson, P. J .; Fisher, M. H.; Stephenson, J .; Li, D. H.; Zhou,
B. S.; Cheng, Y. C. Cancer Res. 1988, 48, 5956-5964.
(7) Lee, K. H.; Lin, Y. M.; Wu, T. S.; Zhang, D. C.; Yamagishi, T.;
Hayashi, T.; Hall, I. H.; Chang, J . J .; Wu, R. Y.; Yang, T. H.
Planta Med. 1988, 54, 308-311.
P la telet Aggr ega tion . Aggregation was measured
by the turbidimetric method8 with a dual-channel Lumi-
aggregometer (Model 1020, Payton, Canada). All glass-
ware was siliconized. One minute before the addition
of the aggregation inducer, the platelet suspension was
stirred at 900 rpm. The percentage of aggregation was
calculated as described previously.9
(8) O’Brien, J . R. J . Clin. Path. 1962, 15, 446-455.
(9) Teng, C. M.; Chen, W. Y.; Ko, W. C.; Ouyang, C. Biochem.
Biophys. Acta 1987, 924, 375-382.
NP970163+