Semisynthesis and cytotoxicity of styryl-lactone derivatives

Article abstract of DOI:10.1021/np990089q

The cytotoxicity and the cell-cycle action of altholactone (1), goniofufurone (2), and eight altholactone derivatives (5-12), were determined in vitro on L-1210 cells. Semisyntheses and structure-activity relationships of these compounds are described. The results of this study suggest that the cytotoxicity of altholactone (1), 11-nitro-altholactone (8), and 7-chloro- 6,7-dihydroaltholactone (10) is due to the accumulation of the cells in the G2 + M phase of the cell cycle.

Full text of DOI:10.1021/np990089q

1106
J . Nat. Prod. 1999, 62, 1106-1109
Sem isyn th esis a n d Cytotoxicity of Styr yl-La cton e Der iva tives
Almudena Bermejo,† Ste´phane Le´once,‡ Nuria Cabedo,† Inmaculada Andreu,† Daniel H. Caignard,‡
Ghanem Atassi,‡ and Diego Cortes*,†
Departamento de Farmacologı´a, Farmacognosia y Farmacodinamia, Facultad de Farmacia, Universidad de Valencia,
46100 Burjassot, Valencia, Spain, and Institut de Recherche SERVIER, 11 rue des Moulineaux, 92150 Suresnes, France
Received March 5, 1999
The cytotoxicity and the cell-cycle action of altholactone (1), goniofufurone (2), and eight altholactone
derivatives (5-12), were determined in vitro on L-1210 cells. Semisyntheses and structure-activity
relationships of these compounds are described. The results of this study suggest that the cytotoxicity of
altholactone (1), 11-nitro-altholactone (8), and 7-chloro-6,7-dihydroaltholactone (10) is due to the
accumulation of the cells in the G2 + M phase of the cell cycle.
The styryl-lactones found abundantly in Goniothalamus
species (Annonaceae) are an interesting group of cytotoxic
and antitumor agents.1 Altholactone (1), a furano-pyrone
first isolated in 19772 and recently identified as the major
compound from the stem bark of Goniothalamus arvensis,3
is known to possess significant cytotoxicity against several
tumor cell lines,4,5 and stereoselective syntheses of 1 were
carried out.6,7 Goniofufurone (2), a known furano-furone
also isolated from G. arvensis8 and other furano or pyrano
styryl-lactone derivatives, showed interesting biological
activities.9,10 The cytotoxic activities previously reported for
eight-membered-ring ú-lactones (heptolides)11,12 were re-
cently explained by a noncompetitive mechanism of action
as inhibitors of the mammalian respiratory chain complex
I for almuheptolide A (3), a 8-phenyl-2-oxocanone isolated
from G. arvensis and also synthesized from 1 via the 6,7-
dihydro-7-ethoxy-altholactone (etharvensin, 4).13
available in large quantity from G. arvensis.3 Treatment
of 1 (previously 3-acetylated) with osmium tetroxide (OsO4)
in water, and N-methylmorpholine N-oxide monohydrate
(NMO) to regenerate the oxidizing agent,14 afforded the
corresponding cis-dihydroxylated derivative 5. The 3-acetyl-
6,7-diacetoxy-6,7-dihydro-altholactone (6) was obtained by
treatment of 1 with NMO and OsO4, followed by acetic
anhydride in dry pyridine. When a solution of 5 was treated
with an excess of triethylamine and a precipitate obtained
from Me3SiCl and Me2SO,15 the 6,7-methylendioxy deriva-
tive was afforded (7) (see Scheme 1).
The methylendioxy moiety that was formed in 7 connects
the diol of 5 but does not change the stereochemistry of
their carbinol centers. A cis-configuration among the 6, 7,
and 7a centers in these three last compounds (5-7) can
be explained as in etharvensin (4)16 by observation of their
1H NMR coupling constants (J 7a,7 and J 7,6 between 3.5 and
4 Hz). On the other hand, when an organic solution of 1
was added to a mixture (1:1) of concentrated nitric acid
and sulfuric acid, two nitro-altholactone derivatives, 8 (11-
nitro-altholactone) and 9 (9,13-dinitro-altholactone), were
obtained in similar yields. The mechanism of aromatic
nitration involves an electrophilic attack of the nitronium
ion at the ortho- or para-position in the monosubstituted
aromatic ring of 1.
An original stereospecific chlorination at C-7 of 1 (â to
the lactonic carbonyl) was carried out under acidic condi-
tions using POCl3 at room temperature. An EIMS fragment
1
ion at m/z 232 [M - HCl]+, as well as a dd at δ 4.54 in H
NMR (H-7) with its characteristic coupling constant values
corresponding to a cis-relationship between the neighbor
protons (J 6a,7 ) 3.7 Hz and J 7a,7 ) 4 Hz) and a signal at δ
50.5 in 13C NMR (C-7), were in agreement with a â-7-
chloro-6,7-dihydro-altholactone (10).
A method for the preparation of both 7-alkoxylated-6,7-
dihydro-δ-lactone (11) and 4,5-dialkoxylated eight-mem-
bered-ring ú-lactones with a heptolide skeleton (12), has
been successfully achieved from 1.13 The enantiospecific
syntheses of compounds 11 and 12 from 1 was carried out
in a one-step reaction, using MeOH and concentrated H2-
SO4. Formation of 7-methoxy-6,7-dihydro-altholactone (11)
is postulated to be a direct Michael-type addition of MeOH
from the less electron-dense position (â to the CdO).16
Subsequent nucleophilic attack with a second MeOH
molecule by a SN2 mechanism would lead to inversion of
the configuration, followed by a concerted opening of the
R-pyrone and the tetrahydrofuran ring and a subsequent
intramolecular eight-ring closure.13
Herein we describe the semisynthesis of eight new styryl-
lactone derivatives in the altholactone series (5-12). The
cytotoxicity of these and the two natural compounds 1 and
2 are also reported.
Resu lts a n d Discu ssion
Altholactone (1), a 2-phenyl-3-hydroxy-6,7-dihydro-furano-
pyrone, represents a good starting material because it is
* To whom correspondence should be addressed. Tel.: (34) 96.386.49.75.
Fax: (34)96.386.49.43. E-mail: dcortes@UV.es.
† Universidad de Valencia.
‡ Institut de Recherche SERVIER.
10.1021/np990089q CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/31/1999

Styryl-Lactone Derivatives
J ournal of Natural Products, 1999, Vol. 62, No. 8 1107
Sch em e 1a
a
Reagents and conditions: (a) Ac2O/Pyr, room temperature; (b) NMO/OsO4 room temperature, followed by 2% NaHSO3, room temperature; (c) Me3SiCl/
Me2SO, followed by (C2H5)3N, room temperature; (d) HNO3/H2SO4, 0 °C; (e) POCl3/CH2Cl2, room temperature, (f) MeOH/H2SO4, reflux.
Ta ble 1. Cytotoxic Activity of Styryl-Lactone Derivativesa
heptolide 12 shows a very important cytotoxicity, explained
by the inhibition of the mammalian respiratory chain
compounds
IC50 (µg/mL)
complex I.13
1
8
9
10
12
2.9
3.2
5.0
3.7
5.2
Perturbation of the cell cycle induced by compounds 1,
8, 9, 10, and 12 was studied using the same cell line.
Altholactone (1), 11-nitro-altholactone (8), and 7-chloro-6,7-
dihydro-altholactone (10) induced a partial accumulation
(35-45%) of the cells in the G2 + M phase on the cell cycle.
However, the 9,13-dinitro-altholactone (9) and the hep-
tolide (12) showed a nonselective accumulation in the cell
cycle.
a
Inhibition of L-1210 cell proliferation measured by the MMT
assay. IC50 of compounds 2, 5-7, and 11 with IC50 > 10 µg/mL.
In view of the biological interest focused on this class of
compounds, a study of the cytotoxic activities of the known
natural (1 and 2) and the new synthetic (5-12) styryl-
lactone derivatives was carried out in vitro using L-1210
leukemia cells. Altholactone (1), some of its furano-pyrone
derivatives (8-10) and the heptolide 12, were more cyto-
toxic than the remaining derivatives (2, 5-7, and 11)
inhibiting L-1210 cell proliferation (see Table 1). Structure-
activity relationships in the styryl-lactone series indicate
that the 6,7-double bond on the pyrone ring or the chlora-
tion at 7 position is the more important structural require-
ment to increase the cytotoxicity. So, altholactone (1), the
two nitro-derivatives on the aromatic ring (8,9), and the
chloro dihydro-altholactone (10) were more cytotoxic than
the furano-furone (2) or the 6,7-dihydro derivatives of
altholactone, whether dihydroxylated (5), triacetylated (6),
methylendioxy (7), or methoxylated (11). Moreover, the
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r e. Optical rotations
were determined on a Perkin-Elmer 241 polarimeter. IR
spectra (film) were run on
a Perkin-Elmer 1750 FTIR
spectrometer. MS were recorded with a VG Auto Spec Fisons
spectrometer, and the liquid secondary ion mass spectrum
(LSIMS) was obtained by fast ion bombardment. 1H NMR (250,
300 and 400 MHz), 13C NMR, and DEPT (62.5, 75 and 100
MHz) spectra were recorded on Bruker AC-250, Varian Unity-
300, and Varian Unity-400 instruments. The signals of 1H
NMR of all the compounds were assigned by COSY 45. Si gel
TLC plates were observed under UV light (254 nm) and after
spraying with anisaldehyde in H2SO4.
P la n t Ma ter ia l. Goniothalamus arvensis Scheff. (Annon-
aceae) was collected in the National Park of Varirata, located

1108 J ournal of Natural Products, 1999, Vol. 62, No. 8
Bermejo et al.
in the Central Province of Papua New Guinea. A voucher
specimen was deposited in the herbarium of the University of
Papua New Guinea.
Cl2 (2 mL), and the mixture was allowed to stand at room
temperature for about 1 h until a white precipitate appeared.
The excess of unreacted reagents was decanted, and the white
precipitate was quickly washed with CH2Cl2 (1 mL). A solution
of 5 (25 mg; 0.08 mmol) in CH2Cl2 (2 mL), and an excess of
(C2H5)3N was added to this precipitate and stirred at room
temperature for 15 h. When the reaction was complete as
estimated by TLC, the mixture was washed using 1% NaHCO3
(5 mL) and H2O (2 × 5 mL), and the CH2Cl2 layer was
evaporated in vacuo. The crude product was subjected to
column chromatography on Si gel 60 H (eluted with CH2Cl2-
EtOAc 60:40) to afford 14 mg of 7 (54%): C16H16O7; IR (dry
film) νmax 2800, 1752 (CdO), 1238, 1155 cm-1; 1H NMR (CDCl3,
300 MHz) δ 7.35-7.30 (m, H-9 to H-13), 5.33 (dd, H-3), 5.11
and 5. 06 (2d, 2 H, OCH2O), 4.99 (dd, H-3a), 4.91 (d, H-2), 4.87
(d, H-6), 4.68 (dd, H-7), 4.54 (t, H-7a), 2.14 (s, 3 H, OCOCH3);
J 2,3 ) 4.2 Hz; J 3,3a ) 1.2 Hz; J 3a,7a ) 3.9 Hz; J 7a,7 ) 4.1 Hz;
J 7,6 ) 2.5 Hz; J OCH2O ) 7.3 Hz;13C NMR (CDCl3, 75 MHz) δ
169.2 (3-COCH3), 167.0 (C-5), 136.9 (C-8), 128.9 (3 CH), 126.2
(2 CH), 94.8 (OCH2O), 85.8, 83.3 and 82.5 (3C, C-2, C-3 and
C-3a), 74.6 and 72.4 (2C, C-6 and C-7), 20.8 (3-OCOCH3); EIMS
m/z (%) [M]+ 320 (3), 261 [M - OCOCH3]+ (20), 233 (12), 145
(64), 133 (85).
Extr a ction a n d Isola tion . Dried and powdered stem bark
of G. arvensis (368 g) was macerated with MeOH at room
temperature. The concentrated MeOH extract was partitioned
between hexane and 50% aqueous MeOH. The aqueous MeOH
extract was fractionated successively into CH2Cl2 and EtOAc.
The CH2Cl2 extract (7.0 g) was applied by Si gel 60 H column
chromatography to afford altholactone (1, 2.3 g) and goniofu-
furone (2, 165 mg).
(+)-Alth ola cton e (1): C13H12O4; [R]D +174.2° (c 1.0, EtOH);
EIMS m/z [M]+ 232.3,17,18
(+)-Gon iofu fu r on e (2): C13H14O5; [R]D +12° (c 1.1, EtOH);
mp 154-155 °C (EtOH-hexane); EIMS m/z [M]+ 250.8,17,18
(-)-3-Acetyl-6,7-dih ydr oxy-6,7-dih ydr o-alth olacton e (5).
Treatment of 1 (150 mg, 0.64 mmol) with dry pyridine (1 mL)
and Ac2O (2 mL) afforded 3-acetyl-altholactone (170 mg; 0.62
mmol; 96%) after stirring for 1 h at room temperature. To a
solution of 3-acetyl-altholactone (55 mg; 0.20 mmol) and
N-methylmorpholine N-oxide monohydrate (NMO, 27.1 mg;
0.20 mmol) in 3 mL of Me2CO-H2O (2:1) was added a 4%
solution of OsO4 in H2O (0.3 mL; 0.05 mmol) dropwise. The
reaction mixture was stirred at room temperature for 1.5 h,
and then a 2% solution of NaHSO3 in H2O (2 mL) was added.
After being stirred for 30 min, the reaction mixture was
extracted with EtOAc, which was washed with H2O and brine,
and concentrated to dryness. The reaction residue was sub-
jected to column chromatography on Si gel 60 H (eluted with
CH2Cl2) to afford 46.5 mg of 5 (75% from 3-acetyl-altholac-
tone): C15H16O7; [R]D -12.2° (c 1.8, EtOH); IR (dry film) νmax
3432 (OH), 2920, 1752 (CdO), 1373, 1235, 1117, 1047, 950,
Sem isyn th eses of 8 a n d 9. To 1.5 mL of concentrated
HNO3 was added dropwise at 0 °C concentrated H2SO4 (0.5
mL). To a CH2Cl2 solution (0.5 mL) of altholactone (1, 150 mg,
0.64 mmol) was added dropwise at 0 °C concentrated H2SO4
(2.0 mL). Both solutions were mixed and stirred at room
temperature for 30 min, then H2O was added and the reaction
products extracted into CH2Cl2. The residue was subjected to
column chromatography on Si gel 60 H (eluted with CH2Cl2-
EtOAc 90:10) to afford 39 mg of 8 (22%) and 55 mg of 9 (27%).
914, 864, 763, 735, 700 cm-1 1H NMR (CDCl3, 400 MHz) δ
;
(+)-11-Nit r o-a lt h ola ct on e (8):
C13H11NO6; [R]D +184°
(c 0.7, EtOH); mp 178-180 °C (CHCl3-MeOH); IR (dry film)
νmax 3356 (OH), 1714 (CdO), 1529 (NO2), 1339, 1243, 1089
7.38-7.30 (m, H-9 to H-13), 5.31 (dd, H-3), 5.08 (dd, H-3a),
4.88 (d, H-2), 4.62 and 4.60 (m, 2H, H-6 and H-7), 4.53 (m,
H-7a), 2.13 (s, 3H, 3-OCOCH3); J 2,3 ) 5.1 Hz; J 3,3a ) 1.4 Hz;
J 3a,7a ) 3.9 Hz; J 7a,7 ) 3.2 Hz; 13C NMR (CDCl3, 100 MHz) δ
172.0 (C-5), 169.4 (3-OCOCH3), 136.9 (C-8), 128.8 (3 CH), 126.1
(2 CH), 85.0 (C-3a), 84.8 (C-2), 82.6 (C-3), 77.0 (C-7a), 68.1
(C-6 and C-7), 20.7 (3-OCOCH3); 1H and 13C spectra were
unambiguously assigned by using 2D NMR techniques (COSY
45 and HMQC); EIMS m/z (%) [M]+ 308 (5), 266 [M - COCH2]+
(17), 248 [M - HOCOCH3]+ (74), 232 [M - OCOCH3 - OH]+
(3), 215 [M - OCOCH3 - 2 OH]+ (1), 144 (100), 107 (78), 91
(49), 77 (32).
cm-1 1H NMR (CDCl3, 300 MHz) δ 8.16 (d, 2 H, H-10 and
;
H-12), 7.51 (d, 2 H, H-9 and H-13), 7.05 (dd, H-7), 6.25 (d, H-6),
4.94 (dd, H-3a), 4.87 (d, H-2), 4.70 (t, H-7a), 4.38 (dd, H-3);
J 2,3 ) 5.3 Hz; J 3,3a ) 2.0 Hz; J 3a,7a ) 4.9 Hz; J 7,7a ) 5.0 Hz; J 7,6
) 10.0 Hz; J 9,10 ) J 12,13 ) 8.7 Hz; 13C NMR (CDCl3, 75 MHz)
δ 161.2 (C-5), 147.6 (C-8), 145.7 (C-11), 140.1 (C-7), 126.4 (2
CH, C-9 and C-13), 123.9 (C-6), 123.8 (2 CH, C-10 and C-12),
86.3 (C-3a), 84.8 (C-2), 83.5 (C-3), 68.4 (C-7a); LSIMS m/z 300
[M + Na]+, 278 [MH]+; EIMS m/z (%) [M]+ 277 (7), 232
[MH - NO2]+ (10), 213 [M - NO2 - H2O]+ (20), 122 (16), 97
(100).
3-Acetyl-6,7-d ia cetoxy-6,7-d ih yd r o-a lth ola cton e (6). To
a solution of altholactone (1, 50 mg; 0.21 mmol) in 3 mL of
Me2CO-H2O (2:1) was added NMO (27.1 mg; 0.20 mmol) and
catalytic 4% solution of OsO4 in H2O (0.3 mL; 0.05 mmol)
dropwise, and the resulting solution was stirred at room
temperature for 6 h. The reaction mixture was concentrated
to dryness by blowing air over the solution, and the residue
was dissolved in EtOAc and filtered through a plug of Si gel.
The solution was concentrated to dryness, and the residue was
dissolved in dry pyridine (1 mL) and Ac2O (2 mL). The
resulting solution was stirred at room temperature for 4 h.
The residue obtained was extracted with CH2Cl2, washed with
H2O, and dried. The dry extract was subjected to column
chromatography on Si gel 60 H (eluted with CH2Cl2-EtOAc
100:1) to give 39 mg of 6 (46%): C19H20O9; IR (dry film) νmax
(+)-9,13-Din itr o-alth olacton e (9): C13H10N2O8; [R]D +155°
(c 0.8, EtOH); mp 103-106 °C (CHCl3-MeOH); IR (dry film)
νmax 3382 (OH), 1718 (CdO), 1529 (NO2), 1340, 1244, 1102,
1
1052 cm-1; H NMR (CDCl3, 400 MHz) δ 8.23 (d, 2 H, H-10
and H-12), 7.53 (d, H-11), 7.07 (dd, H-7), 6.35 (d, H-6), 5.46
(dd, H-3a), 5.12 (d, H-2), 5.08 (br t, H-3), 4.63 (dd, H-7a);
J 2,3 ) 3.7 Hz; J 3,3a ≈ 1.0 Hz; J 3a,7a ) 3.9 Hz; J 7,7a ) 5.4 Hz;
J 7,6 ) 9.8 Hz; J 10,11 ) J 11,12 ) 9.0 Hz; 13C NMR (CDCl3, 75 MHz)
δ 159.5 (C-5), 148.1 (C-8), 143.6 (2 C, C-9 and C-13), 138.1
(C-7), 126.9 (2 CH, C-10 and C-12), 125.2 (C-11), 124.1 (C-6),
90.8 (C-3a), 83.0 (C-2), 82.5 (C-3), 69.5 (C-7a); LSIMS m/z 345
[M + Na]+, 323 [MH]+; EIMS m/z (%) [MH]+ 323 (10), 277
[MH - NO2]+ (40), 231 [MH - 2 NO2]+ (19), 213 [M - OH -
2 NO2]+ (10), 167 (87), 165 (100).
2927, 1752 (CdO), 1372, 1218, 1047, 702 cm-1 1H NMR
;
(+)-6,7-Dih yd r o-7-ch lor o-a lth ola cton e (10). To a dry
CH2Cl2 solution (10 mL) of altholactone (1, 50 mg, 0.21 mmol)
was added POCl3 (1.0 mL; 0.5 mmol) dropwise. After stirring
for 4 h at room temperature, the reagents were removed under
reduced pressure, then H2O was added and the reaction
mixture extracted into CH2Cl2. The organic solution after usual
workup was purified by column chromatography on Si gel 60
H (eluted with CH2Cl2-EtOAc 90:10), to afford 30 mg of 10
(52%). C13H13O4Cl; [R]D +22.3° (c 2.8, EtOH); IR (dry film) νmax
(CDCl3, 250 MHz) δ 7.37-7.28 (m, H-9 to H-13), 5.90 (d, H-6),
5.78 (t, H-7), 5.35 (dd, H-3), 5.02 (dd, H-3a), 4.94 (d, H-2), 4.49
(t, H-7a), 2.17, 2.15 and 2.13 (3s, 9H, 3-, 6-, and 7-OCOCH3);
J 2,3 ) 4.7 Hz; J 3,3a ) 1.3 Hz; J 3a,7a ) 4.0 Hz; J 7a,7 ) 3.8 Hz;
J 7,6 ) 3.4 Hz; 13C NMR (CDCl3, 62.5 MHz) δ 169.2 (3 C, 3-, 6-,
and 7-OCOCH3), 164.3 (C-5), 136.4 (C-8), 128.9 (3 CH), 126.0
(2 CH), 85.4 (C-3a), 84.4 (C-2), 82.5 (C-3), 75.1 (C-7a), 68.2
and 65.8 (C-6 and C-7), 20.7, and 20.4 (3 C, 3-, 6-, and
7-OCOCH3); EIMS m/z (%) [MH]+ 393 (5), 334 [M - OCOCH3]+
(2), 274 [M - 2 OCOCH3]+ (3), 215 [M - 3 OCOCH3]+ (50),
185 (100), 155 (75), 105 (94), 91 (70), 77 (24).
3421 (OH), 3035, 2920, 1733 (CdO), 1386, 1055, 762, 700 cm-1
;
1H NMR (CDCl3, 400 MHz) δ 7.37-7.28 (m, H-9 to H-13), 5.04
(dd, H-3a), 4.78 (d, H-2), 4.54 (dd, H-7), 4.51 (t, H-7a), 4.34
(dd, H-3), 3.21 (dd, H-6b), 2.93 (dd, H-6a); J 2,3 ) 5.6 Hz;
J 3,3a ) 1.4 Hz; J 3a,7a ) 4.0 Hz; J 7a,7 ) 4.1 Hz; J 7,6b ) J 7,6a ) 3.7
3-Acetyl-6,7-d ih yd r o-6,7-m eth ylen d ioxy-a lth ola cton e
(7). To Me3SiCl (2 mL) was added Me2SO (2 mL) and dry CH2-

Styryl-Lactone Derivatives
J ournal of Natural Products, 1999, Vol. 62, No. 8 1109
Refer en ces a n d Notes
Hz; J 6a,6b ) 17.5 Hz; 13C NMR (CDCl3, 100 MHz) δ 167.1 (C-
5), 137.7 (C-8), 128.8 (2 CH), 128.5 (CH), 125.9 (2 CH), 86.9
(C-3a), 86.8 (C-2), 83.4 (C-3), 75.4 (C-7a), 50.6 (C-7), 34.9 (6-
CH2); 1H and 13C spectra were unambiguously assigned by
using 2D NMR techniques (COSY 45 and HMQC); EIMS m/z
(%) [M]+ 268.5 (14), 251 [M - OH]+ (15), 232 [M - HCl]+ (64),
215 [M - OH - HCl]+ (5), 107 (83), 97 (100), 91 (62), 77 (60).
(-)-6,7-Dih yd r o-7-m eth oxy-a lth ola cton e (11): C14H16O5;
[R]D -69.2° (c 1.3, EtOH); EIMS m/z [M]+ 264; proposed
according to literature.13
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proposed according to literature.13
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(13) Bermejo, A.; Tormo, J . R.; Cabedo, N.; Estornell, E.; Figade`re, B.;
Cortes, D. J . Med. Chem. 1998, 41, 5158-5166.
Cytotoxicity a n d Cell Cu ltu r e. L-1210 cells were culti-
vated in RPMI 1640 medium supplemented with 10% fetal calf
serum, 2 mM L-glutamine, 50 units/mL of penicillin, 50 µg/
mL of streptomycin, and 10 mM of Hepes buffer (pH ) 7.4).
Cells were exposed to graded concentrations of drug for 48 h.
Cytotoxicity was measured by the microculture tetrazolium
assay.19 Results are expressed as IC50, the concentration
needed to reduce by 50% the optical density of treated cells
with respect to the optical density of untreated controls.
For the cell-cycle analysis, L-1210 cells were incubated for
21 h at 37 °C with various concentrations of drugs. Cells were
then fixed by 70% EtOH (v/v), washed, and incubated with
PBS containing 100 µg/mL RNAse and 25 µg/mL propidium
iodide for 30 min at 20 °C. Results are expressed as percentage
of accumulated cells in the G2 + M phase after 21 h compared
with the control.
(14) Elomri, A.; Mitaku, S.; Michel, S.; Skaltsounis, A. L.; Tillequin, F.;
Koch, M.; Pierre´, A.; Guilbaud, N.; Le´once, S.; Kraus-Berthier, L.;
Rolland, Y.; Atassi, G. J . Med. Chem. 1996, 39, 4762-4766.
(15) Queiroz, E. F.; Roblot, F.; Figade`re, B.; Laurens, A.; Duret, P.;
Hocquemiller, R.; Cave´, A.; Serani, L.; Lapre´vote, O.; Cotte-Laffitte,
J .; Que´ro, A. M. J . Nat. Prod. 1998, 61, 34-39.
(16) Bermejo, A.; Bla´zquez, M. A.; Serrano, A.; Zafra-Polo, M. C.; Cortes,
D. J . Nat. Prod. 1997, 60, 1338-1340.
(17) Bermejo, A.; Bla´zquez, M. A.; Rao, K. S.; Cortes, D. Phytochem. Anal.
1999, 10, 127-131.
(18) Bla´zquez, M. A.; Bermejo, A.; Zafra-Polo, M. C.; Cortes, D. Phytochem.
Anal., in press.
Ack n ow led gm en t. This research was supported by the
Spanish Direccio´n General de Investigacio´n Cient´ıfica y Te´c-
nica (DGICYT) under grant SAF 97-0013. We are grateful to
Prof. Kundar S. Rao of the University of Papua New Guinea,
for help in obtaining the plant material.
(19) Pierre´, A.; Dunn, T. A.; Kraus-Berthier, L.; Le´once, S.; Saint-Dizier,
D.; Regnier, G.; Dhainaut, A.; Bizzari, J . P.; Atassi, G. Invest. New
Drugs 1992, 10, 137-141.
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