Cytotoxic 4-phenylcoumarins from the leaves of Marila pluricostata.

Abstract

Bioassay-guided fractionation of the CH(2)Cl(2) extract of the leaves of Marila pluricostata led to the isolation of 17 naturally occurring 4-phenylcoumarins, three of them, 5-hydroxy-8,8-dimethyl-4-phenyl-9,10-dihydro-8H-pyrano-[2,3-f]chromen-2-one (1), 5-hydroxy-8,8-dimethyl-4-phenyl-6-propionyl-9,10-dihydro-8H-pyrano-[2,3-f]chromen-2-one (2), and 5,7-dihydroxy-8-(3-methylbut-2-enyl)-4-phenylchromen-2-one (3), are new natural compounds; the remaining (4-17) are known mammea-type coumarins. Their structures were established by spectroscopic means. All compounds were tested in cytotoxicity assays against the MCF-7, H-460, and SF-268 human cancer cell lines.

Full text

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Cytotoxic 4-Phenylcoumarins from the Leaves of Marila pluricostata
Jose ´ L. Lo ´pez-Pe ´rez,*,†Dionisio A. Olmedo,†,‡Esther del Olmo,†Yelkaira Va ´squez,‡Pablo N. Solı ´s,‡
Mahabir P. Gupta,‡and Arturo San Feliciano†
Departamento de Quı ´mica Farmace ´utica, Facultad de Farmacia, Universidad de Salamanca, Campus Unamuno, E-37007,
Salamanca, Spain, and Centro de Investigaciones Farmacogno ´sticas de la Flora Panamen ˜a (CIFLORPAN), Facultad de
Farmacia, Universidad de Panama ´, Apartado 10767, Panama, Repu ´blica de Panama ´
Received November 9, 2004
Bioassay-guided fractionation of the CH2Cl2extract of the leaves of Marila pluricostata led to the isolation
of 17 naturally occurring 4-phenylcoumarins, three of them, 5-hydroxy-8,8-dimethyl-4-phenyl-9,10-dihydro-
8H-pyrano-[2,3-f]chromen-2-one (1), 5-hydroxy-8,8-dimethyl-4-phenyl-6-propionyl-9,10-dihydro-8H-pyrano-
[2,3-f]chromen-2-one (2), and 5,7-dihydroxy-8-(3-methylbut-2-enyl)-4-phenylchromen-2-one (3), are new
natural compounds; the remaining (4-17) are known mammea-type coumarins. Their structures were
established by spectroscopic means. All compounds were tested in cytotoxicity assays against the MCF-
7, H-460, and SF-268 human cancer cell lines.
Clusiaceae or Guttiferae is a plant family generally
confined to the tropics. The genus Marila comprises only
four species growing in Panama. Marila pluricostata is a
tree that has a distribution restricted to regions of
Panama,1-3Costa Rica,1,2and Colombia.4This species has
neither reported common name nor any ethnomedical use.
However, Marila tomentosa is used in Colombia for the
treatment of dysentery.5
Previous studies on species of the genus Marila resulted
in the isolation of betulinic acid,6rhamnetin, 3,4-dihy-
droxybenzoic acid, and several xanthones,7in addition to
a polyisoprenylated compound, laxifloranone.8
As part of a collaborative research program, a CH2Cl2
extract of the leaves of M. pluricostata showed cytotoxic
activitiy against the three human cancer cell lines MCF-
7, H-460, and SF-268. Fractionation of these extracts
afforded three new 4-phenylcoumarins, 5-hydroxy-8,8-
dimethyl-4-phenyl-9,10-dihydro-8H-pyrano-[2,3-f]chromen-
2-one (1), 5-hydroxy-8,8-dimethyl-4-phenyl-6-propionyl-
9,10-dihydro-8H-pyrano-[2,3-f]chromen-2-one (2), and 5,7-
dihydroxy-8-(3-methylbut-2-enyl)-4-phenylchromen-2-one
(3), and 14 known phenylcoumarins (4-17).
Results and Discussion
The dried, powdered leaves of Marila pluricostata Standl.
& L.O. Williams (Clusiaceae) were extracted with CH2Cl2.
After elimination of fatty compounds by precipitation with
MeOH followed by treatment with a saturated solution of
urea in MeOH, the soluble part partitioned with a basic
aqueous solution yielded a less cytotoxic acidic fraction and
a neutral part with enhanced cytotoxicity. Repeated chro-
matography of the neutral fraction over silica gel and
Sephadex LH-20, followed by successive recrystallizations
in mixtures of n-hexane/ether and CHCl3/MeOH, afforded
the new 4-phenylcoumarins 1-3, as well as the known
compounds 4-17.
Compound 1 was isolated as a pale yellow amorphous
solid. HRFABMS of 1 showed [M]+at m/z 322.1205,
corresponding to the molecular formula C20H18O4. Com-
pound 1 gave a green coloration with methanolic ferric
chloride, indicating the presence of free phenolic groups;
this was supported by an absorption band at 3503 cm-1in
its IR spectrum. It showed another absorption at 1712 cm-1
corresponding to an R,?-unsaturated lactone. Its1H NMR
spectrum (Table 1) showed one olefinic proton singlet at δ
5.94 characteristic of the H-3 of a 4-substituted coumarin.
The presence of a monosubstituted phenyl group at C-4 was
corroborated by absorptions at 754 and 702 cm-1in the IR
spectrum, whereas five aromatic proton signals were
evident from its1H NMR spectrum. The long-range cor-
relation observed in the HMBC spectrum of 1 between H-3
and the nonprotonated aromatic carbon [δ 138.0 (C-1′)]
supported the localization of this phenyl group at C-4. The
presence in the
reciprocally coupled triplets (δ 1.85, 2H and 2.86 2H, t, J
) 6.8 Hz) together with that of a singlet of a gem-dimethyl
group (δ 1.37) linked to a nonprotonated oxygenated carbon
[δ 75.9 (C-3′′′)] suggested a prenyl group forming a ring
involving C-7 and C-8 resulting in a f-fused pyran ring.
The alternative g-fused pyran ring involving C-5 and C-6
was discarded, taking into account the contour map of
correlations of the HMBC spectrum. In addition, the1H
NMR spectrum showed another aromatic singlet (δ 6.18)
that should be located at C-6. Long-range1H-13C couplings
observed in the HMBC spectrum revealed that the H-3
signal (δ 5.94) and the aromatic singlet at δ 6.18 both
correlated with the same quaternary carbon C-9 (δ 101.0),
thus supporting the presence of a hydrogen atom at C-6.
In addition, H-6 (δ 6.18, s) correlated with two aromatic
oxygenated carbons (δ 153.4 and 158.3). The latter was
assignable to C-7, because it was also connected to the
methylene H-1′′′ (δ 2.86). In consequence, the signal at δ
153.4 was assigned to C-5. Additionally H-1′′′ was con-
nected to another aromatic oxygenated carbon (δ 153.6),
which was assigned to C-10. These data placed the prenyl
substituent at C-8, linked to the oxygen at C-7. The
remaining connectivities observed in the HMBC spectrum
allowed unambiguous assignment of all of the1H and13C
NMR signals (Table 2). Thus, compound 1 was identified
as 5-hydroxy-8,8-dimethyl-4-phenyl-9,10-dihydro-8H-pyrano-
[2,3-f]chromen-2-one. The structure of compound 1 was
confirmed through its preparation by deacylation9of mam-
meisin (4), followed by cyclization of the prenyl chain at
position 8. In this way we obtained a substance whose
spectroscopic and physical properties were identical to
those of the natural compound 1.
1H NMR spectrum of a pair of two
* To whom correspondence should be addressed. Tel: + 34 923 294528.
Fax: + 34 923 294515. E-mail: lopez@usal.es.
†Universidad de Salamanca.
‡Universidad de Panama ´.
369
J. Nat. Prod. 2005, 68, 369-373
10.1021/np049642g CCC: $30.25 © 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/26/2005

Page 2

Compound 2 was obtained as a white amorphous solid.
Its HRFABMS spectra showed a molecular ion m/z 378.1454,
corresponding to the molecular formula C23H22O5. Its1H
NMR spectrum showed a phenolic hydroxyl signal (δ 14.72)
exchangeable with D2O. The strong deshielding of this
signal indicated the existence of an intramolecular hydro-
gen bond of the phenolic hydrogen in proximity to a
carbonyl group. The chemical shift of this signal was
characteristic of the hydroxyl group at C-5 for coumarins
containing an acyl group at C-6.10The IR spectrum, apart
from the absorption at 1716 cm-1of the R,?-unsaturated
lactone, showed an absorption band at 1618 cm-1corre-
sponding to a chelated acyl group.11-13The NMR spectrum
of compound 2 differed from that of 1 in the presence of
additional signals corresponding to a propionyl group,
instead of the aromatic methine of compound 1. The
crowding of signals due to the similarity of several chemical
shifts in the13C NMR spectra (Table 2) of these coumarins
made it necessary to rely on 2D HMBC experiments. On
the basis of the 2D NMR analysis of 2, the propionyl
residue appeared to be linked via the carbonyl carbon to
C-6. These data indicated that it was an almost totally
substituted coumarin, with the exception of position C-3.
This finding was corroborated by further examination of
the HMBC spectrum, which showed a contour map similar
to that of compound 1. Indeed, the singlet at δ 5.96,
characteristic for H-3, correlated with the signal of the
quaternary carbon C-9 (δ 101.8), and with those of the
Chart 1
Table 1.
1H NMR Data of Compounds 1, 2, and 3
1
δH(J in Hz)a
5.94 s
5.11 bs
6.18 s
proton
3
5-OH
6
7-OH
2′,6′
3′, 5′
4′
2′′
3′′
4′′
5′′
1′′′
2′′′
4′′′
5′′′
1
δH(J in Hz)b
6.03 s
2
δH(J in Hz)a
5.96 s
14.72 s
3
δH(J in Hz)a
5.96 s
6.45 bs
6.25 s
6.45 bs
7.37 m
7.54 m
7.54 m
4a
δH(J in Hz)a
6.10 s
3.04 s (OMe)
4b
δH(J in Hz)a
6.24, s
2.29 (OCOMe)
7.50 s
13.14 s
7.40 m
7.44 m
7.44 m
2.82 d (6.8)
2.21 m
0.91 d (6.8)
0.91 d (6.8)
3.47 d (7.3)
5.13 m
1.87 s
1.70 s
2.29 (OCOMe)
7.33 m
7.38 m
7.38 m
2.52 d (6.9)
2.14 m
0.90 d (6.5)
0.90 d (6.5)
3.49 d (8.7)
5.27 m
1.83 s
1.70 s
7.45 m
7.54 m
7.54 m
7.45 m
7.52 m
7.52 m
7.31 m
7.38 m
7.38 m
3.06 q (7.3)
1.15 t (7.3)
2.86 t (6.8)
1.85 t (6.8)
1.37 s
1.37 s
3.00 t (6.8)
2.06 t (6.8)
1.59 s
1.59 s
2.87 t (6.8)
1.89 t (6.8)
1.45 s
1.45 s
3.56 d (6.8)
5.29 t (6.8)
1.85 s
1.75 s
aδ values obtained in CDCl3at 200 MHz.bδ values were obtained in MeOD4at 200 MHz. TMS int. std.
370
Journal of Natural Products, 2005, Vol. 68, No. 3 Lo ´pez-Pe ´rez et al.

Page 3

lactone carbonyl at C-2 (δ 160.8) and the nonprotonated
carbon of the phenyl (δ 139.4) group, which must be
attached at C-4. The HMBC spectrum also showed cor-
relations of the methylene signals (δ 1.89 and 2.87) of the
prenyl group with that of a nonprotonated oxygenated sp3
carbon (δ 77.8), supporting a gem-dimethyl group (δ 26.7).
Both methylene signals (δ 1.89 and 2.87) were connected
to C-8 (δ 100.1). Finally, one of these signals (δ 2.87)
correlated with those of the two oxygenated aromatic
carbons (δ 157.6 and 163.3). In consequence the oxygenated
aromatic carbon at δ 158.8 could only be assigned to C-5
and those at δ 157.6 and 163.3 to C-10 and C-7, respec-
tively.
Compound 2 was obtained in good yield by Friedel-Craft
acylation10from compound 1, by refluxing with propionyl
chloride in a carbon disulfide/nitrobenzene mixture and in
the presence of aluminum trichloride. Workup of the crude
reaction product led to the isolation of a substance whose
spectroscopic properties were identical to those of natural
2.
Compound 3 was isolated as a white amorphous solid,
and HRFABMS showed a molecular ion m/z 322.1207,
corresponding to the molecular formula C20H18O4. The IR
spectrum exhibited absorption bands due to -OH (3308
cm-1) and an R,?-unsaturated lactone (1696 cm-1). These
data, together with those of the1H and13C NMR spectra,
indicated that this compound was another coumarin with
a phenyl group at position 4 and phenolic hydroxyl groups
at positions C-5 and C-7. Its1H NMR spectrum showed
the presence of a methine singlet at δ 5.96. It also showed
an aromatic singlet at δ 6.25 (H-6 or H-8). The HMBC
contour map showed correlations between the H-3 singlet
(δ 5.96) and the lactone carbonyl signal (δ 160.9, C-2) and
the quaternary carbons (δ 137.0, C-1′ and 100.8, C-9).
Other long-range correlations were observed between the
proton signal at δ 3.56 and those of C-8, C-7, and C-10,
oxygenated carbon signals of the coumarin nucleus. Cor-
relations between H-2′′′ (δ 5.29, 1H, t, J ) 6.8 Hz), H-4′′′
(δ 1.85, 3H, s), and H-5′′′ (δ 1.75, 3H, s) were also observed.
These data were consistent with a prenyl substituent at
position C-8. In consequence, this compound was identified
as 5,7-dihydroxy-8-(3-methylbut-2-enyl)-4-phenylchromen-
2-one (3).
Coumarins 4-17 were identified as known mammeisin
(mammea A/AA) (4),14,15isomammeisin (mammea A/BA)
(5),14mammeigin (mammea A/AA cyclo D) (6),16,17MAB 5
(mammea A/AB cyclo D) (7),16mesuagin (mammea A/AD
cyclo D) (8),15isomesuol (9)18MAB 1 (mammea A/AB)
(10),19mesuol (mammea A/AD) (11),20(mammea A/BB)
(12),15,16isodispar B (13),21cyclomammeisin (mammea
A/AA cyclo F) (14),13,22disparinol A (15),21MAB 3 (mammea
A/AB cyclo F) (16),19and mesuol cyclo F (mammea A/AD
cyclo F) (17),19through the complete analysis of their NMR
and MS data and comparison with those reported in the
literature for these compounds.13C NMR data for com-
pounds 5, 10, and 17 were not reported before and have,
therefore, been included in this paper (Table 2). NMR data
of the monomethylated derivative 5-O-methylmammeisin
(4a) and the diacetyl derivative 4b are also included here.
Signals corresponding to the labile protons of the phe-
nolic groups at positions C-5 and C-7 were very useful for
the location of the acyl chain, frequently observed in
natural 4-phenylcoumarins and, consequently, for the
structural determination of these compounds. In effect, the
chemical shifts of the signals of these protons in the1H
NMR spectrum are strongly influenced by the presence of
an acyl group at position C-6 or C-8. To clarify these
findings, molecular modeling studies were carried out with
mammeisin (4), which showed singlets at δ 9.84 and 11.03
in its1H NMR spectrum corresponding to both phenolic
protons. After a conformational analysis, two main con-
formers were found for this compound (Figure 1). Relative
stabilities were determined by both molecular mechanics
and semiempirical methods (AM1).23Conformer I, which
is 12 kJ/mol more stable than conformer II, shows an
intramolecular hydrogen bond between the phenolic hy-
drogen 5-OH and the carbonyl of the acyl group of the chain
at position C-6, whereas in conformer II, the hydrogen
bonding appears between the carbonyl and the phenolic
group at position 7 (Figure 1). This information is in
agreement with the model obtained by X-ray diffraction of
this compound (Figure 2). In consequence, the signal at δ
11.03 should be assigned to 5-OH, whereas that at δ 9.84
should correspond to 7-OH.
The original extract and its successive fractions as well
as all the purified compounds (1-17) were screened against
three human cancer cell lines, MCF-7, H-460, and SF-268,
according to an established protocol. The results are
summarized in Table 3. Cytotoxic activity was concentrated
in the neutral fraction. Cytotoxic potency of the crude CH2-
Table 2.
4a, and 4b
13C NMR Data (δCa) of Compounds 1-3, 5, 10, 17,
carbon
2
3
4
5
6
7
8
9
10
1′
2′,6′
3′,5′
4′
1′′
2′′
3′′
4′′
5′′
1′′′
2′′′
3′′′
4′′′
5′′′
5a
5b
7a
7b
1235 1017 4a4b
161.2 160.8 160.9 157.1 159.1 159.9 159.4 158.7
111.3 112.1 112.0 112.7 112.8 111.9 114.6 118.4
155.0 156.5 154.0 154.1 154.5 155.6 154.5 142.4
153.4 158.8 153.4 158.6 159.6 163.8 159.8 153.1
100.8 107.2 100.8 104.1 107.0 102.4 113.0 122.6
158.3 163.3 159.4 166.8 163.1 164.9 163.3 158.7
102.0 100.1 108.0 112.1 108.1 105.2 114.3 126.5
101.0 101.8 100.8 100.5 101.0 102.4 105.8 111.5
153.6 157.6 153.4 156.1 156.6 156.7 156.6 147.8
138.0 139.4 137.0 134.2 135.4 139.1 138.3 137.3
127.4 127.2 127.5 127.5 127.4 127.2 127.6 128.4
128.7 127.5 129.7 129.6 129.2 127.6 127.4 128.1
129.1 128.1 129.9 130.2 129.7 128.3 128.6 128.9
207.7 21.6 211.9 209.6 207.5 199.8
38.1 120.8
8.9136.8
25.7
17.9
16.4 16.422.1 206.1
31.8 31.2 121.153.6 120.7
75.977.8 135.5 25.6 137.3
26.526.718.122.7
26.526.7 25.9 22.7
46.3
11.8
26.8
18.1
21.8
39.1
18.6
19.2
52.3
25.7
22.6
22.6
21.8
52.8
23.8
22.6
22.6
23.6 26.7
92.9 120.7 119.5
71.5 133.0 133.9
26.2 17.9
24.9 25.7
63.9 168.5
16.6
25.9
18.0
25.7
19.1
168.5
20.6
a 13C spectrum recorded at 400 MHz in CDCl3. Assignments
were made on the basis of1H-1H COSY, HMQC, and HMBC
spectra for compounds 1-3.
Figure 1. Conformers of mammeisin (4).
Cytotoxic 4-Phenylcoumarins from Marila Journal of Natural Products, 2005, Vol. 68, No. 3
371

Page 4

Cl2extract was mainly due to the 4-phenylcoumarins, of
which, mammesin (4), mammea A/AB (10), mesuol (11),
and mammea A/AB (12) were the most cytotoxic.
Some 4-phenylcoumarins described previously have re-
vealed cytotoxic24and anti-HIV25,26activities. Chemopre-
ventive activity against cancer in vitro without cytotoxicity
has also been reported for some of these derivatives.27,28
Experimental Section
General Experimental Procedures. Melting points were
determined on a Bu ¨chi 510-K melting point apparatus and are
uncorrected. IR spectra were recorded (KBr 1%) in a Nicolet
Impact 410 spectrophotometer.1H,13C NMR, COSY, HMQC,
and HMBC were recorded on Bruker AC 200 (200 MHz) and
Bruker DRX 400 (400 MHz) instruments. Sephadex LH-20
(Fluka, 25-100 mm) and silica gel 60 (Merck, 230-400 mesh)
were used for flash chromatography; precoated silica gel plates
(Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC
analysis. For EIMS and HRFABMS analysis, a VG-TS250
mass spectrometer (70 eV) was used.
Plant Material. Leaves of Marila pluricostata were col-
lected from Llano-Cartı ´ in the province of Panama, Republic
of Panama, in November 2000. Its taxonomic identity was
established by Prof. Mireya Correa, Director of the Herbarium
of the University of Panama (PMA), where voucher specimens
(F-4740 and F-4937) are deposited.
Extraction and Separation. The dried, powdered leaves
(3.4 kg) of M. pluricostata were extracted with CH2Cl2(10 L)
at room temperature for 5 days. Evaporation of the solvent
yielded a crude extract (186.5 g), which was further solubilized
with hot n-hexane (4 L twice) and cooled (-20 °C) overnight,
yielding a soluble fraction (136.1 g), which was defatted
successively with MeOH (3 L twice) and a saturated solution
of urea in MeOH (3 L twice). The finally soluble part (74.5 g)
was partitioned with basic aqueous solution (NaOH 4% × 3
times), yielding an acidic part (3.0 g) and a neutral part (71.1
g). The neutral part was fractionated on a silica gel column
with gradient elution using n-hexane/EtOAc. Fractions (500
mL each) were combined on the basis of their TLC profiles
and after removal of the solvent gave fractions A-J. On the
basis of bioactivity data, fractions E-J were subsequently
chromatographed on silica gel and Sephadex LH 20 columns,
and the compounds were purified through repetitive recrys-
tallization, affording the coumarins 1-17. For chromato-
graphic separations, the appropriate combinations of solvents
(n-hexane, Et2O, CH2Cl2, EtOAc, and MeOH) were used.
Workup of fraction E by repeated column chromatography
using n-hexane/ethyl acetate (85:15) led to the isolation of
coumarins 4 (2.5 g, 1.340%) and 5 (150 mg, 0.080%). Similarly,
fraction F furnished coumarins 6 (242 mg, 0.129%), 7 (56 mg,
0.030%), 8 (164 mg, 0.087%), 9 (118 mg, 0.063%), and 10 (254
mg, 0.136%). Fraction G was chromatographed using CH2Cl2/
EtO2(80:20) and afforded coumarins 11 (220 mg, 0.117%) and
12 (12 mg, 0.0064%). Fraction H, eluting with n-hexane/CH2-
Cl2/MeOH (2:1:1), yielded coumarin 13 (15 mg, 0.0080%).
Workup of fraction I by repeated column chromatography
using n-hexane/CH2Cl2/MeOH (2:1:1) furnished coumarins 14
(53 mg, 0.030%), 15 (160 mg, 0.085%), 16 (46 mg, 0.024%), 17
(146 mg, 0.078%), and 2 (4 mg, 0.0021%). Finally, fraction J,
chromatographed over silica gel using CH2Cl2/MeOH (1:1),
afforded coumarins 1 (27 mg, 0.014%) and 3 (15.3 mg,
0.0082%). Phenylcoumarins 4-17 were identified by compari-
son of their spectroscopic data with those reported in the
literature.14-20
5-Hydroxy-8,8-dimethyl-4-phenyl-9,10-dihydro-8H-pyr-
ano-[2,3-f]chromen-2-one (1): pale yellow amorphous solid;
mp 274-275 °C; IR (CHCl3) νmax3503, 3271, 1712, 1599, 1429,
1362, 1246, 1163, 1111, 1028, 754, 702 cm-1;1H NMR data,
see Table 1;13C NMR data, see Table 2; EIMS m/z 322 [M]+
(28), 281(20), 267(100), 207(43), 153(12), 139(10), 114(15), 105-
(2), 84(9), 77(21), 73(17), 68(18), 65(13), 63(12), 55(14), 50,6);
HRFABMS m/z 322.1205 (calcd for C20H18O4, 322.1205).
Preparation of Compound 1. Compound 4 (100 mg) was
stirred with 1 mL of 75% sulfuric acid at room temperature
for 35 h. The mixture was poured onto ice, washed with H2O,
and filtered. The air-dried solid, 0.70 mg, was recrystallized
twice, affording a product whose spectroscopic properties were
identical to those of the natural compound 1.
5-Hydroxy-8,8-dimethyl-4-phenyl-6-propionyl-9,10-di-
hydro-8H-pyrano-[2,3-f]chromen- 2-one (2): yellow amor-
phous solid; mp 274-275 °C; IR (CHCl3) νmax3474, 2975, 2933,
2872, 1746, 1716, 1618, 1599, 1439, 1364, 1246, 1165, 1110,
1030 cm-1;1H NMR data, see Table 1;13C NMR data, see Table
2; EIMS m/z 378 [M]+(64), 349(46), 323(100), 293(86), 265(4),
171(10), 141(5), 115(15), 77(7), 55(5); HRFABMS m/z 378.1454
(calcd for C23H22O5, 378.1467).
Preparation of Compound 2. Anhydrous aluminum
trichloride (150 mg) was added to a stirred suspension of
compound 1 (50 mg) in carbon disulfide (5 mL). Nitrobenzene
(2 mL) was then added over 40 min, forming a homogeneous
solution with evolution of HCl. The solution was heated under
reflux for 30 min, and then propionyl chloride (15 mg) in
nitrobenzene (1 mL) was added over 40 min before allowing it
to cool with stirring. The mixture was poured onto ice/water
and aqueous HCl and was extracted with ethyl acetate (25
mL, twice). Workup of the crude product by chromatography
on silica gel led to the isolation of 2 (30 mg), which was
identical to the isolated natural product.
5,7-Dihydroxy-8-(3-methylbut-2-enyl)-4-phenylchromen-
2-one (3): white amorphous solid; mp 130-132 °C; IR (CHCl3)
νmax3308, 3271, 2959, 2931, 1696, 1559, 1449,1439 1367, 1080
cm-1;1H NMR data, see Table 1;13C NMR data, see Table 2;
EIMS m/z 322 [M]+(34), 307(20), 305(4), 279(17), 268(17), 267-
(100), 251(7), 238(5), 226(4), 210(2), 197(2), 181(3), 171(4), 165-
Figure 2. ORTEP drawing of mammeisin (4).
Table 3. Cytotoxic Activity of Compounds 1-17a
GI50(µg/mL)
H-460
6.7
5.7
4.4
0.2
0.5
0.6
0.8
>10.0
>10.0
>10.0
0.6
0.1
0.3
0.3
1.9
4.6
0.6
4.4
6.1
7.2 × 10-7
compound
1
2
3
4
4a
4b
5
6
7
8
9
10
11
12
13
14
15
16
17
adriamycin
MCF-7
5.7
4.7
3.4
0.2
0.4
0.6
0.7
>10.0
9.6
>10.0
0.7
0.2
0.2
0.2
1.5
4.2
0.9
3.4
5.8
6.5 × 10-7
SF-268
7.8
6.8
4.8
0.1
0.4
0.5
0.6
>10.0
>10.0
>10.0
0.6
0.1
0.1
0.1
1.8
5.2
0.5
4.8
6.4
8.6 × 10-7
aFor cell lines used, see the Experimental Section.
372
Journal of Natural Products, 2005, Vol. 68, No. 3 Lo ´pez-Pe ´rez et al.

Page 5

(9), 152(7), 139(7), 128(5), 115(14), 105(7), 91(5), 77(10), 69(15),
55(5); HRFABMS found m/z 322.1205 (calcd for C20H18O4,
322.1203).
5-O-Methylmammeisin (4a). A cooled ethereal diazo-
methane solution (3 mL) was added dropwise to a cooled
solution of mammeisin (10 mg) in ether (2 mL), and the
reaction mixture was maintained at 0 °C for 3 h, then
concentrated in vacuo to give 4a (11 mg): white amorphous
solid (CHCl3); mp 130-132 °C;1H NMR data, see Table 1;13C
NMR data, see Table 2; EIMS m/z 420 [M]+(50), 406(24), 405-
(91), 377(17), 366(13), 365(55), 363(38), 349(21), 347(12), 308-
(22), 307(100), 293(9), 253(16), 205(7), 178(6), 165(13), 152(11),
139(13), 115(13), 105(22), 91(14), 77(16), 69(15), 57(17).
Mammeisin diacetate (4b). Acetic anhydride (0.2 mL) was
added to a solution of 4 (15 mg) in dry pyridine (0.5 mL), and
the reaction mixture was allowed to stand for 3 h at room
temperature, diluted with H2O (15 mL), extracted with CH2-
Cl2, and washed with diluted HCl and H2O four times. Workup
led to the isolation of 4b (17 mg): colorless oil; IR (CHCl3) νmax
2959, 2931, 2871, 1738, 1623, 1581, 1446,1407 1374, 1162,
1123, 1112 cm-1;1H NMR data, see Table 1;13C NMR data,
see Table 2.
Cytotoxicity Bioassay. The cytotoxicity bioassay was
performed against breast (MCF-7), lung (H-460), and CNS (SF-
268) human cancer cell lines according to the method of Monks
et al.29During the isolation process, the activity of all fractions
and compounds was monitored using all three cell lines.
Molecular Modeling. Calculations were performed on a
Silicon Graphics Indigo computer. Compounds were built using
Macromodel v.4.30Conformational analysis was performed by
a Monte Carlo random search. All freely rotating bonds were
searched with MM231minimization to a gradient of less than
0.001 kcal/mol. Full geometry optimization of the two main
conformers of each compound was performed using Stewart’s
AM1 and PM3 Hamiltonian in MOPAC 6.0.
X-ray Analysis of Compound 4. Compound 4, C25H26O5,
crystallizes in orthorhombic space group P212121, with Z ) 4,
and unit cell parameters a ) 13.675(3) Å, b ) 14.243(2) Å, c
) 15.810(3) Å, R ) 116.46(1)°, ? ) 69.46(2)°, γ ) 85.22(1)°.
X-ray diffraction data were collected on a four-circle Seifert
XRD 3003 SC diffractometer (Cu FR, λ ) 1.5418 Å), graphite
monochromator, room temperature, ω-2? scan. The unit cell
parameters were determined by least-squares refinement on
the 2? values of 25 strong well-centered reflections in the
range 16° < 2? < 40°. Scattering factors for neutral atoms
and anomalous dispersion corrections for C and O were taken
from ‘International Tables for X-Ray Crystallography (1995,
Vol. C, Kluwer Academic Publishers: Dordrecht). The struc-
ture of C25H26O5was resolved by direct methods and refined
in the space group P212121. Full matrix least-squares refine-
ment with anisotropic thermal parameters for non-H atoms
was carried out by minimizing w(Fo2- Fc2)2. Refinement on
F2for all reflections, weighted R factors (Rw), and all goodness
of fit S are based on F2, while conventional R factors (R) are
based on F; R factors based on F2are statistically about twice
as large as those based on F, and R factors based on all data
will be even larger. Resulting absolute structure parameter:
0.36 (155).
All calculations were performed using CRYSOM,32software
for data collection, XRAY80,33and for data reduction, SHELX-
TLTM (Siemens SHELXTLTM version 5.0, Siemens Analytical
X-ray instruments Inc., Madison, WI, 1995) to resolve and
refine the structure and to prepare material for publication.
Full crystallographic details have been deposited at the
Cambridge Crystallographic Data Centre (CCDC No. 2506931).
Acknowledgment. Thanks are due to European Commis-
sion ALFA grant EC-1233, for a fellowship to D.A.O., Orga-
nization of American States for financial support to the Project
AE-106/3, Spanish grants MCyT SAF2001-0037 and FIS RIS-
G03-173, and the University of Panama for granting a study
leave to D.A.O.
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