4-Alkyl- and 4-phenylcoumarins from Mesua ferrea as promising multidrug resistant antibacterials

Article abstract of DOI:10.1016/j.phytochem.2004.07.001

Supercritical CO₂ selectively extracted a series of 4-alkyl and 4-phenyl 5,7-dihydroxycoumarins from Mesua ferrea blossoms. Chemical modifications of the isolated compounds allowed us to confirm the structures elucidated by spectroscopic means and to prepare new derivatives amenable to SAR studies and potential pharmaceutical development. Biological investigations towards the screening on a number of bacteria strains and Plasmodium falciparum, identified compounds 1-9 as weak antiprotozoal agents and potent antibacterials on resistant Gram-positive strains.

Full text of DOI:10.1016/j.phytochem.2004.07.001

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2867–2879
www.elsevier.com/locate/phytochem
4-Alkyl- and 4-phenylcoumarins from Mesua ferrea as promising
multidrug resistant antibacterials
a,
a
b
b
Luisella Verotta *, Erminio Lovaglio , Giovanni Vidari , Paola Vita Finzi ,
c
c
c
c
Maria Grazia Neri , Alessandro Raimondi , Silvia Parapini , Donatella Taramelli ,
d
Antonella Riva , Ezio Bombardelli
d
a
`
Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
b
`
Dipartimento di Chimica Organica, Universita degli Studi di Pavia, Via Taramelli 10, 27100 Pavia, Italy
c
`
Istituto di Microbiologia, Universita degli Studi di Milano, Via Pascal 36, 20133 Milano, Italy
d
Indena S.p.A., Viale Ortles 12, 20139 Milano, Italy
Received in revised form 4 July 2004
Available online 31 July 2004
Dedicated to Kurt Hostettmann on the occasion of his 60th birthday
Abstract
Supercritical CO2 selectively extracted a series of 4-alkyl and 4-phenyl 5,7-dihydroxycoumarins from Mesua ferrea blossoms.
Chemical modifications of the isolated compounds allowed us to confirm the structures elucidated by spectroscopic means and
to prepare new derivatives amenable to SAR studies and potential pharmaceutical development. Biological investigations towards
the screening on a number of bacteria strains and Plasmodium falciparum, identified compounds 1–9 as weak antiprotozoal agents
and potent antibacterials on resistant Gram-positive strains.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Mesua ferrea; Guttiferae; 4-Alkyl and 4-phenyl 5,7-dihydroxycoumarins
1. Introduction
activity of isolated phloroglucinols, xanthones, neoflav-
onoids, and coumarins (Verotta, 2003a,b).
In the course of our studies on plants belonging to the
family Guttiferae, we have investigated the lipophilic ex-
tract of the aerial parts of Mesua ferrea L., a well-known
tropical tree, used in folk medicine for the treatment of
fever, dyspepsis and renal diseases (Dennis and Akshaya
Kumar, 1998). Our interest in Guttiferae, which are pro-
lific producers of metabolites, is related to the reported
antibiotic, neuromodulator, antitumor, and antiviral
Different aerial parts of M. ferrea are traditionally
used in the preparation of cosmetics and unguents.
The pounded kernels of the seeds are applied externally
for poulticing wounds and all forms of skin eruptions
(Dennis and Akshaya Kumar, 1998; Perry and Metz-
ger, 1980). Phytochemical investigation of different
parts of the plant showed the occurrence of xanthones,
coumarins, biflavones, cyclohexanedione derivatives,
and an essential oil (Dennis and Akshaya Kumar,
1998).
*
Corresponding author. Tel.: +39-02-5031-4114; fax: +39-02-5031-
A preliminary investigation of a lipophilic extract of
M. ferrea revealed a high antimicrobial activity against
4106.
E-mail address: luisella.verotta@unimi.it (L. Verotta).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.07.001

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L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
Gram-positive and anaerobic bacteria that led to anti-
microbial or preservative formulations for pharmaceuti-
cal or cosmetic uses (Bombardelli and Morazzoni,
1998). Herein, we describe the complete composition
of a lipophilic extract of M. ferrea, obtained under su-
percritical conditions, and the investigation of the bioac-
tivity of the main components and related compounds
on resistant bacteria strains. In addition, given the
known antimalarial or insecticidal properties exhibited
by most plants producing these coumarins (Dennis
and Akshaya Kumar, 1998; Perry and Metzger, 1980;
Neuwinger, 2000; Ito et al., 2000; Nagem and De A. e
Silva, 1988; Bordoloi et al., 1997), the Plasmodium falci-
parum growth inhibitory activity of representative com-
pounds was tested.
2. Results and discussion
Multiple chromatographic separations of an extract
of M. ferrea blossoms, obtained under supercritical
conditions, allowed the identification of eighteen 4-
phenyl-5,7-dihydroxycoumarins, comprising four types
of structures: (a) 6-acyl-8-prenyl derivatives [1 (mesuol)
(Dennis and Akshaya Kumar, 1998; Chakraborty and
Das, 1966; Chakraborty et al., 1985), 1a (mammea A/
AB) (Crombie et al., 1966, 1967), 1b (mammeisin,
mammea A/AA) (Crombie et al., 1966, 1967), 2, 2a];
(b) 8-acyl-6-prenyl derivatives [3 (isomammeisin)
(mammea A/BB) (Crombie et al., 1966, 1967), 3a
(mammea A/BA) (Crombie et al., 1966, 1967), 4, 4a];
(c) 6-acyl-7,8-dihydrofurano derivatives [5 (mammea
A/AD cycloF) (Crombie et al., 1972, 1987), 5a (mam-
mea A/AB cycloF) (Crombie et al., 1972, 1987), 5b
(mammea A/AA cycloF) (Crombie et al., 1972,
1987), 7, 7a]; (d) 6-acyl-7,8-pyrano derivatives [8
(mammea A/AD cycloD, mesuagin) (Chakraborty
and Chatterji, 1969; Crombie et al., 1966, 1967, 1987;
Carpenter et al., 1971), 8a (mammea A/AB cycloD,
mammeigin) (Chakraborty and Chatterji, 1969; Crom-
bie et al., 1966, 1967, 1987; Carpenter et al., 1971), 8b
(mammea A/AA cycloD) (Crombie et al., 1972, 1987),
9, 9a]. In addition, two 4-(20-hydroxypropyl)-5,7-di-
hydroxycoumarins [6 (assamene) (Bordoloi et al.,
1997), 6a (surangin C) (Mahandru and Ravindran,
1986)], were isolated. The compounds were identified
either in a pure state or as a mixture (labeled as
MF) of chromatographically inseparable acyl homo-
logues, through accurate analysis of high field NMR
spectra recorded in two solvents (CDCl3 and ace-
tone-d6). The majority of these data are reported in
Section 3 to complete or update the literature. Com-
pounds 2, 2a, 4, 4a, 7, 7a, 9 and 9a are new metabo-
lites, while the other ones are known constituents of
Mesua species.
The structures of isomeric coumarins 2 and 2a, and 4
and 4a were easily established by comparing their NMR
spectra with those of the corresponding lower homo-
logues 1a and 1b, and 3 and 3a, respectively. Indeed,
the spectra resulted to be superimposable for the most
part, with the notable difference of the characteristic sig-
nals of a geranyl (C10) chain replacing those of a C5
prenyl unit. In analogy, coumarins 9 and 9a resulted
to be the geranyl homologues of 8 and 8a, respectively.
Finally, the NMR signals of dihydrofurans 7 and 7a
were superimposable to 5a and 5b, respectively, except
for a 2-propenyl moiety replacing a 2-hydroxy-2-propyl
group.

L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
2869
9, respectively, in good yields, and devoid of the corre-
sponding dihydrofurano derivatives. Under the same
conditions compound 3 yielded 3b, identical to ponna-
lide isolated from Calophyllum inophyllum (Murti
et al., 1972). As expected, the cyclization of compound
3 occurred between the prenyl chain and the 5-OH sub-
stituent, since the 7-OH group was inhibited by the
strong hydrogen bond with the C-1000 carbonyl group.
Enantioselective total synthesis (Begley et al., 1987)
proved the (S) configuration of the 2-methylbutanone
side-chain of Mammea coumarins, most of which were
isolated also from M. ferrea in the present study. This
stereochemical assignment could not be confirmed by
us, since all our efforts to establish the absolute stereo-
chemistry of isolated compounds failed.
Mixtures of isomeric coumarins (labeled as MF) or
pure compounds, when available, possessing different
prenyl chains (1 and 2, 3 and 4) or devoid of the alkyl
chain (dihydrofurano and pyrano derivatives 5, 7, 8, 9)
were tested as to their growth inhibitory activity on a se-
ries of bacteria strains (Tables 1 and 2) and on P. falci-
parum (Table 3).
The in vitro susceptibility assays performed against
strains of Escherichia coli, Enterobacter cloacae, Serratia
marcescens, Pseudomonas aeruginosa, Acinetobacter
spp., as well as against Candida albicans and Cryptococ-
cus neoformans, did not prove an effective antimicrobial
activity of Mesua coumarins against these Gram-nega-
tive bacteria and fungi. In fact, the minimal inhibitory
concentrations (MICs) against the tested organisms
were above 128 lg/ml. The activity of the Mesua deriv-
atives was also assayed against Gram-positive organ-
isms of the genera Staphylococcus, Enterococcus and a
strain of Streptococcus durans. As shown in Tables 1a
and 1b, the strains expressed different pattern of resist-
ance to several known antibiotics. The choice of this
group of strains aimed to challenge the isolated com-
pounds against the most common and important resist-
ance mechanisms encountered in Gram-positive
bacteria. All the samples tested, except MF5, MF8 and
MF9, showed an activity of potential interest either
against the enterococci (MICs of 8–16 lg/ml) or against
organisms of the genus Staphylococcus. It thus appears
that none of the latter group of assayed strains is en-
dowed with a specific mechanism of resistance to effi-
ciently hinder the antimicrobial activity of Mesua
coumarins. Such observation suggests that these new
compounds may act by an uncommon inhibitory mech-
anism, possibly different from those endowed to other
known antimicrobials.
Differentiation between each pair of regioisomeric 4-
phenyl-5,7-dihydroxycoumarins, i.e. between 2 and 4,
was inferred by the chemical shifts of the corresponding
phenolic hydroxyl groups, which, in case of compounds
like 2 and 2a, resonated in the range of 10.5–10.3 ppm,
both being hydrogen bonded to the ketone carbonyl
group, while, in case of compounds like 4 and 4a, only
one phenolic OH was shifted downfield (d 14.55), due to
chelation with C-1000. These assignments were definitely
confirmed by routine HMBC spectra (Fig. 1).
Structures 8 and 9 were further corroborated by sub-
mitting compounds 1 and 2, separately, to an intramo-
lecular cyclization of the phenolic OH group onto the
ortho-prenyl chain, which, in principle, can afford either
dihydrofurano or pyrano substituted rings. Under suita-
ble experimental conditions, we found that DDQ (Car-
penter et al., 1971), or, more efficiently, Pd2+ catalyzed
intramolecular cyclizations (Larock et al., 1998) of com-
pounds 1 and 2, delivered the pyrano derivatives 8 and
In addition, the anti-Staphylococcus activity of com-
pounds 1–4 was compared with tetracycline, gentamicin,
chloramphenicol, ciprofloxacin and erythromycin in an
assay comprising a group of 42 different Staphylococcus
strains either of the aureus or the non-aureus species. The
results obtained with the individual samples are reported
Fig. 1. Most significant HMBC correlations in compounds 3 and 4.

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L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
in Table 2, in which data are expressed in terms of range
of activity, concentration inhibiting 50% (MIC 50%) or
90% (MIC 90%) of the strains, and geometric mean.
These results show that the reference antibiotics tetracy-
cline, gentamicin and erythromycin were more effective
than the Mesua derivatives against the most susceptible
strains in the group. In fact, 50% of the strains were in-
hibited by the above antibiotics with a MIC of 0.25–0.5
lg/ml, whereas the Mesua derivatives exhibited a higher
MIC 50% (2–4 lg/ml). Conversely, when the concentra-
tions inhibiting 90% of the strains (MIC90%) were com-
pared, some of the Mesua derivatives exhibited the best
activity. Interestingly, this wider selection of strains in-
cluded also organisms resistant to the reference antibiot-
ics (MIC 90% =32 to >128), and all of them could,
however, be inhibited by 4 lg/ml of MF1 and 4.
Infections due to multidrug-resistant Gram-positive
bacteria are a growing worldwide problem, in particular
among seriously ill patients, and antibiotic options for
patients with multidrug-resistant Staphylococcus aureus
infections are severely limited. In this context, our find-
ings confirm the interesting antibiotic properties of a few
coumarins of this group.
Table 3 shows the inhibitory properties of represent-
ative Mesua coumarins on the growth of P. falciparum,
the protozoan responsible for the lethal malaria. The da-
ta point out a weak activity of all tested samples both on
chloroquine-sensitive (D10) and resistant (W2) strains,
at doses comprised between 1.2 lg/ml (W2) for 2 and
15.8 lg/ml (D10) for MF5.
3. Experimental
3.1. General
Melting points were recorded employing a Buchi
¨
SMP-20 apparatus or a Fisher–Johns hot-stage appara-
tus and are uncorrected. UV (in 95% EtOH) spectra
were recorded on a Kontron UVIKON 941. Optical ro-
tation values were recorded on a Perkin–Elmer 241 po-
larimeter. The 1H and 13C NMR spectra were
determined by means of a Bruker CXP 300 spectrometer
(300 and 75.47 MHz, respectively), or, alternatively, on
a Bruker AMX 400 (400 and 100.61 MHz, respectively)
and on a Bruker AC 200 (200 and 50 MHz, respective-
1
ly). The chemical shifts for H and 13C NMR are refer-
enced to CHCl3 at 7.26 ppm and CDCl3
at 77.0 ppm, respectively, or to CH3COCH3 at 2.1
ppm and CD3COCD3 at 28.8 ppm. The spectra were in-
terpreted with the aid of standard COSY, HMBC and
HMQC techniques. EI-MS (probe) 70 eV and CI-MS
(iso-butane, probe), 200 eV spectra were taken on a
VG 7070 EQ spectrometer. Column chromatography
was performed using Silica Gel 60 (70–230 and
40–63 lm, Merck). The reactions were monitored by

Table 1b
In vitro antibacterial activity of 10 Mesua derivatives against twelve strains of the Staphylococcus spp. with defined resistance pattern
Compound
Resistancea
MIC(lg/ml)b
AMPc
E
GM
CIP
T/S
RIF
CL
TET
MF1d
2
3
4
MF5e
MF7f
MF8g
MF9h
Mesua extract
Mesuol (1)
Strains
S. aureus 18268
S. aureus 17380
S. aureus 17592
S. aureus 18110
S. aureus 17547
S. aureus 17728
S. aureus 3012
S. aureus 414
S. epidermidis 3112
S. epidermidis 2515
S. saprophyticus 3010
S. simulans 214
a
R
R
R
R
R
R
S
S
–
R
R
R
R
R
R
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
R
S
S
R
R
S
S
S
S
S
R
S
S
S
S
R
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
4
16
4
4
4
4
2
8
4
32
8
16
32
2
2
4
2
2
4
2
4
64
2
4
2
2
2
16
2
2
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
8
8
8
8
4
4
4
4
4
8
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
–
2
4
4
–
2
–
–
2
–
–
2
–
4
2
2
–
4
–
–
2
–
–
2
R
R
R
S
R
S
R
S
S
S
S
R
S
S
R
64
64
32
16
16
16
4
8
32
4
R
R
R
R
R
R
S
S
S
R
R
S
R
R
S
4
8
4
4
16
2
>128
2
R
S
Breakpoints are those of the National Committee for Clinical Laboratory Standards (1). S, susceptible; R, resistant.
b
c
d
e
f
MIC, minimal inhibitory concentration.
MET, methicillin; E, erythromycin; GM, gentamicin; CIP, ciprofloxacin; T/S, trimethoprim/sulfamethox; RIF, rifampicin; CL, clindamycin; TEICO, teicoplanin.
1a/1b: 4/1.
5a/5b/5: 5/1.5/1.
7/7a: 6/1.
g
h
8a/8b/8: 5/1.5/1.
9a/9: 4/1.

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L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
Table 2
Comparative in vitro activities of four Mesua derivatives and other
antimicrobial agents against 42 strains of Staphylococcus spp.
ether/CH2Cl2/EtOAc 25:25:1 (1 l), petroleum ether/
CH2Cl2/EtOAc 25:25:2 (2.5 l), CH2Cl2/EtOAc 25:1
(1.5 l), CH2Cl2/EtOAc 25:2 (1.3 l), CH2Cl2/EtOAc 10:1
(1.3 l) and CH2Cl2/MeOH 9:1 (1 l). Eluted material
was collected in 10 · 400 ml and 6 · 1 l fractions. Iden-
tical fractions were combined to give six main fractions:
A (fractions 3–4, 2.3 g), B (fractions 5–7, 3.8 g), C (frac-
tions 8–10, 18.0 g), D (fractions 11–13, 1.8 g), E (frac-
tions 14–15, 9.0 g), F (fraction 16, 3.5 g).
Fraction A was subjected to flash column chroma-
tography over Si gel (254 g, B = 6 cm, h = 18 cm) us-
ing petroleum ether/EtOAc 9:1 (1.5 l) and petroleum
ether/EtOAc 85:15 (0.5 l) as eluent, to yield 3 main
fractions: A1 (fractions 9–10, 160 mg) was further pu-
rified over Si gel (10 g) using petroleum ether/
CH2Cl2/EtOAc 10:10:0.1 (200 ml, 35 · 7 ml fractions)
to afford, in fractions 14–33, 70 mg of MF9 (9a/9: 4/
1); A2 (fractions 14–15, 650 mg) was rechromato-
graphed by flash column chromatography over Si gel
(87 g, B = 3.4 cm, h = 18 cm) using petroleum ether/
CH2Cl2/EtOAc 15:10:0.1 (1.5 l, 120 · 10 ml fractions).
Fractions 48–86 yielded 460 mg of MF4 (4/4a: 4/1). 4
was purified from 4a by repeated crystallizations from
hexane (275 mg); A3 (fractions 18–20, 360 mg) was
crystallized from hexane (5 ml) to obtain 175 mg of
MF3 (3/3a: 25/1).
Compound
MIC (l g/ml)a
Range
50%
90%
Mean
MF1b
2 to 8
4 to 128
2 to 64
2 to >128
0.25 to >128
0.12 to >128
0.06 to >128
4 to 128
4
32
2
4
64
16
4
32
128
>128
64
4.13
23.51
4.00
2.67
0.70
3.16
0.83
6.56
3.22
2
3
4
2
Tetracycline
Gentamicin
Erythromycin
Chloramphenicol
Ciprofloxacin
a
0:25
0:05
0:25
4
0.12 to >128
8
64
50% and 90%: MICs for 50% and 90% of strains tested, respec-
tively. The mean is the geometric mean.
b
Mixture 1a/1b: 4/1.
TLC on Merck 60 F254 (0.25 mm) plates, the spots were
revealed under fluorescence (254 nm) and by spraying
with ethanolic FeCl3 or with 0.5% vanillin in H2SO4/
EtOH (4:1) followed by heating. Commercially available
reagents were used without prior purification. Solvent
extracts of aqueous solutions were dried over anhydrous
Na2SO4.
3.2. Isolation
3.2.1. 5-Hydroxy-6-isobutyryl–8-methyl-8-(4-methylpent-
3-enyl)-4-phenyl-2H-pyrano[2,3-h]chromen-2-one (9)
Yellow gum; Rf = 0.29 (petroleum ether/EtOAc 9:1,
FeCl3: green; vanillin: dark violet); 1H NMR (200
MHz, CDCl3) d 14.60 (1H, s, 5-OH), 7.42–7.20 (5H,
m, Ar), 6.95 (1H, J = 10 Hz, H-1000), 5.98 (1H, s, H-3),
5.58 (1H, d, J = 10 Hz, H-2000), 5.08 (1H, brt, J = 1.0
Hz, H-7000), 3.71 (1H, ept, J = 6.7 Hz, H-200), 2.20–1.75
(4H, m, H-5000 and H-6000), 1.71 and 1.62 (3H and 3H,
two s, H-9000 and H-10000), 1.18 and 1.15 (1.5H and
1.5H, two s, H-4000), 1.19 (6H, d, J = 6.7 Hz, H-300 and
H-400); EI-MS, m/z (rel. int.): 458 [M]+.
A CO2 extract (51.7 g) of M. ferrea dried blossoms
supplied by INDENA SpA (Bombardelli and Morazzo-
ni, 1998) was fractionated by column chromatography
over Si gel (600 g) with a stepwise gradient of petroleum
ether/CH2Cl2/EtOAc, CH2Cl2/EtOAc and CH2Cl2/
MeOH: petroleum ether/CH2Cl2 1:1 (1.4 l), petroleum
Table 3
IC50 values of representative Mesua coumarins tested against D10
(CQ-S) and W2 (CQ-R) strains of P. falciparum
Compounds
IC50 (lg/ml)a
3.2.2. 5-Hydroxy-8-methyl-6-(2-methylbutanoyl)-8-(4-
methylpent-3-enyl)-4-phenyl-2H-pyrano[2,3-h]chromen-
2-one (9a)
D10 (CQ-S)
W2 (CQ-R)
MF1b
8.33 1.06
10.75 0.14
2.74 0.45
11.21 5.40
15.80 3.25
9.19 1.88
12.85 2.69
0.011 0.004
7.50 1.24
8.91 0.27
1.17 0.61
8.38 2.52
13.40 4.25
7.53 2.00
13.75 5.15
0.229 0.090
Mesuol (1)
2
Yellow gum; Rf = 0.29 (petroleum ether/EtOAc 9:1,
FeCl3: green; vanillin: dark violet); 1H NMR (200
MHz, CDCl3) d 14.60 (1H, s, 5-OH), 7.42–7.20 (5H,
m, Ar), 6.95 (1H, J = 10 Hz,00H0 -1000), 5.98 (1H, s, H-3),
5.58 (1H, d, J = 10 Hz, H-2 ), 5.08 (1H, brt, J = 1.0
Hz, H-7000), 3.72 (1H, m, H-200), 2.20–1.75 (5H, m, H-
5000, H-6000 and H-400a), 1.71 and 1.62 (3H and 3H, two
s, H-9000 and H-10000), 1.45 (1H, m, H-400b), 1.18 and
1.15 (1.5H and 1.5H, two s, H-4000), 1.15 (3H, d,
J = 6.7 Hz, H-300), 0.87 (3H, t, J = 7.4 Hz, H-500); EI-
MS, m/z (rel. int.): 472 [M]+ (23), 415 (12), 389 (100),
371 (19).
4
MF5c
MF8d
MF9e
Chloroquine
a
The antimalarial activity was assayed in vitro on P. falciparum
strains using the pLDH assay. The results are expressed as IC50 SD
of three different experiments each performed in triplicate.
b
Mixture 1a/1b: 4/1.
Mixture 5a/5b/5: 5/1.5/1.
c
d
Mixture 8a/8b/8: 5/1.5/1.
Mixture 9a/9: 4/1.
e

L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
2873
3.2.3. 5,7-Dihydroxy-8-(2-methylbutanoyl)-6-[(E)-3,7-
dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (4)
Colorless crystals; m.p. 90–92 ꢁC; Rf = 0.21 (pet-
(EtOH) kmax 206, 285, 338, 431 nm; 1H NMR (400
MHz, CDCl3) d 10.52 (1H, s, 5-OH), 10.32 (1H, s, 7-
OH), 7.51–7.38 (5H, m, Ar), 5.96 (1H, s, H-3), 5.27
(1H, t, J = 6.9 Hz, H-2000), 5.06 (1H, brt, J = 6.5 Hz,
H-7000), 3.62 (1H, m, H-200), 3.59 (2H, d, J = 6.9 Hz, H-
1000), 2.12–2.07 (4H, m, H-5000 and H-6000), 1.88 (3H, s,
H-4000), 1.77 (1H, m, H-400a), 1.66 (3H, brs, H-9000), 1.59
(3H, brs, H-10000), 1.36 (1H, m, H-400b), 1.09 (3H, d,
J = 6.7 Hz, H-300), 0.84 (3H, t, J = 7.4 Hz, H-500); 13C
NMR (100 MHz, CDCl3) d 212.0 (s, C-100), 163.0 (s,
C-7), 159.9 (s, C-2 and C-5), 156.8 (s, C-8a), 155.0 (s,
C-4), 139.9 (s, C-10), 137.8 (s, C-3000), 132.1 (s, C-8000),
129.7 (d, C-40), 129.1 (d, C-30 and C-50), 127.6 (d, C-20
and C-60), 123.9 (d, C-7000), 120.6 (d, C-2000), 112.9 (d,
C-3), 107.7 (s, C-8), 107.1 (s, C-6), 101.4 (s, C-4a),
47.0 (d, C-200), 40.0 (t, C-5000), 27.0 (t, C-400), 26.6 (t, C-
6000), 25.9 (q, C-10000), 21.9 (t, C-1000), 17.9 (q, C-9000), 16.8
(q, C-4000), 16.6 (q, C-300), 12.0 (q, C-500); EI-MS, m/z
(rel. int.): 474 [M]+ (23), 417 (27), 405 (19), 389 (11),
351 (100), 333 (22), 293 (66).
roleum ether/EtOAc 9:1, FeCl3: dark purple; vanillin:
25
light violet); – ½aꢀ ¼ 3:83ꢁ (CHCl3; c = 0.73); UV
D
(EtOH) kmax 202, 226, 296, 333 nm; 1H NMR (400
MHz, CDCl3) d 14.55 (1H, s, 7-OH), 7.58–7.38 (5H,
m, Ar), 6.01 (1H, s, H-3), 5.85 (1H, brs, 5-OH), 5.10
(1H, brt, J = 7.4 Hz, H-200), 5.00 (1H, brt, J = 6.5 Hz,
H-700), 3.96 (1H,00sext, J = 6.7 Hz, H-2000), 3.30 (2H, d,
J = 7.4 Hz, H-1 ), 2.1–1.8 (4H, m, H-500 and H-600),
1.93 (1H, m, H-4000b), 1.70 (3H, s, H-400), 1.60 and 1.52
(3H and 3H, two s, H-900 and H-1000), 1.47 (1H, m, H-
4000a), 1.29 (3H, d, J = 6.7 Hz, H-3000), 1.02 (3H, t,
J = 7.4 Hz, H-5000); 13C NMR (100 MHz, CDCl3): d
210.5 (s, C-1000), 166.8 (s, C-7), 158.6 (s, C-2), 157.0 (s,
C-4), 155.4 (s, C-8a), 154.2 (s, C-5), 137.8 (s, C-10),
137.0 (s, C-300), 131.5 (s, C-800), 129.9 (d, C-40), 129.3
(d, C-30 and C-50), 127.4 (d, C-20 and C-60), 123.8 (d,
C-700), 120.5 (d, C-200), 112.4 (s, C-6), 112.0 (d, C-3),
104.0 (s, C-8), 100.4 (s, C-4a), 46.8 (d, C-2000), 39.5 (t,
C-500), 27.1 (t, C-4000), 26.4 (t, C-600), 25.5 (q, C-1000),
21.4 (t, C-100), 17.5 (q, C-900), 16.5 (q, C-3000), 16.1 (q, C-
4000), 11.7 (q, C-5000) EI-MS, m/z (rel. int.): 474 [M]+ (31),
417 (22), 405 (17), 389 (15), 351 (100), 333 (22), 293 (60).
3.2.6. 5,7-Dihydroxy-6-(3-methylbutanoyl)-8-[(E)-3,7-
dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (2a)
1H NMR (400 MHz, CDCl3) d 10.52 (1H, s, 5-OH),
10.32 (1H, s, 7-OH), 7.51–7.38 (5H, m, Ar), 5.96 (1H,
s, H-3), 5.27 (1H, t, J = 6.9 Hz, H-2000), 5.06 (1H, t,
J = 6.5 Hz, H-7000), 3.01 (2H, d, J = 6.6 Hz, H-200), 3.59
(2H, d, J = 6.9 Hz, H-1000), 2.12–2.07 (4H, m, H-5000 and
H-6000), 2.19 (1H, m, H-300), 1.88 (3H, s, H-4000), 1.66
(3H, s, H-9000), 1.59 (3H, s, H-10000), 0.90 (6H, d, J = 6.7
Hz, H-400 and H-500); 13C NMR (100 MHz, CDCl3) d
202.0 (s, C-100), 163.0 (s, C-7), 159.9 (s, C-2 and C-5),
156.8 (s, C-8a), 155.0 (s, C-4), 139.9 (s, C-10), 137.8 (s,
C-3000), 132.1 (s, C-8000), 129.7 (d, C-40), 129.1 (d, C-30
and C-50), 127.6 (d, C-20 and C-60), 123.9 (d, C-7000),
120.6 (d, C-2000), 112.9 (d, C-3), 107.7 (s, C-8), 107.1 (s,
C-6), 101.4 (s, C-4a), 53.5 (t, C-200), 40.0 (t, C-5000), 26.6
(t, C-6000), 25.9 (q, C-10000), 24.8 (d, C-300), 22.6 (q, C-400
and C-500), 21.9 (t, C-1000), 17.9 (q, C-9000), 16.8 (q, C-4000).
Fraction C (18.0 g), was parcelled out in two main
fractions (C1 and C2) by column chromatography over
Si gel (500 g) using toluene/EtOAc 20:1 (2.4 l,
12 · 200 ml fractions) and EtOAc (0.8 l, 4 · 200 ml frac-
tions). C1 (fractions 2–4, 5.3 g) was rechromatographed
by column chromatography over Si gel (200 g), using
petroleum ether/EtOAc 9.5:0.5 (0.3 l) and petroleum
ether/EtOAc 9:1 (1.9 l) to obtain two fractions: C1a
(fractions 41–63, 1.3 g) and C1b (fractions 64–76, 0.8
g). C1a was further purified by flash column chromatog-
raphy over Si gel (220 g, B = 5 cm) using petroleum
ether/t-BuOMe 9.5:0.5 (0.5 l, 20 · 25 ml fractions) and
petroleum ether/t-BuOMe 9:1 (3.5 l, 140 · 25 ml frac-
tions) to afford, in fractions 59–95, a mixture of 3 and
3a (0.63 g). The mixture was added to MF3 of A and
purified by three crystallizations from hexane to give
120 mg of 3.
3.2.4. 5,7-Dihydroxy-8-(3-methylbutanoyl)-6-[(E)-3,7-
dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (4a)
1H NMR (400 MHz, CDCl3) d 14.55 (1H, s, 7-OH),
7.58–7.38 (5H, m, Ar), 6.01 (1H, s, H-3), 5.85 (1H, brs,
5-OH), 5.10 (1H, brt, J = 7.4 Hz, H-200), 5.00 (1H, brt,
J = 1.0 Hz, H-700), 3.30 (2H, d, J = 7.4 Hz, H-100), 3.19
(2H, d, J = 6.7 Hz, H-2000), 2.10 (2H, m, H-3000), 2.1–1.8
(4H, m, H-500 and H-600), 1.70 (3H, s, H-400), 1.60 and
1.52 (3H and 3H, two s, H-900 and H-1000), 1.09 (6H, d,
J = 6.7 Hz, H-4000 and H-5000).
Fraction B (3.86 g) was fractionated by flash column
chromatography over Si gel (250 g, B = 6.0 cm, h = 18
cm) using petroleum ether/EtOAc 85:15 (1 l) and petro-
leum ether/EtOAc 80:20 (1 l) to obtain two fractions. B1
(1.03 g) was purified by column chromatography over Si
gel (flash 140 g, B = 4.0 cm, h = 18 cm) with petroleum
ether/EtOAc 85:15 (1.5 l, 50 · 30 ml fractions); fractions
20–30 gave by crystallization from hexane, 370 mg of
MF2 (2/2a: 9/1); 2 was purified from 2a by repeated crys-
tallizations from hexane (296 mg). B2 (740 mg) was re-
chromatographed by flash column chromatography
over Si gel (113 g, B = 4 cm, h = 18 cm) using petroleum
ether/EtOAc 85:15 (1.6 l, 80 · 20 ml fractions); fractions
19–32 afforded, by crystallization from hexane, 327 mg
of MF1 (1a/1b: 4/1).
3.2.5. 5,7-Dihydroxy-6-(2-methylbutanoyl)-8-[(E)-3,7-
dimethylocta-2,6-dienyl]-4-phenyl-2H-chromen-2-one (2)
Yellow crystals; m.p. 89–90 ꢁC; Rf = 0.16 (petroleum
ether/EtOAc 9:1, FeCl3: green; vanillin: dark violet); UV

2874
L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
C2 (fractions 5–10, 10.5 g) was rechromatographed
(s, C-8a), 154.1 (s, C-5), 136.7 (s, C-10), 134.0 (s, C-300),
130.1 (d, C-40), 129.5 (d, C-30 and C-50), 127.4 (d, C-20
and C-60), 120.7 (d, C-200), 112.6 (s, C-6), 112.1 (d, C-
3), 104.1 (s, C-8), 100.4 (s, C-4a), 53.6 (t, C-2000), 26.4
(d, C-3000), 25.6 (q, C-500), 21.5 (t, C-100), 22.6 (q, C-4000
and C-5000), 17.8 (q, C-400); CI-MS, m/z (rel. int.): 407
[M+H]+ (100), 393 (20), 365 (13), 351 (25).
by column chromatography over Si gel (600 g), using cy-
clohexane/EtOAc 9:1 (3.2 l), cyclohexane/EtOAc 8:2
(0.7 l) and EtOAc (2 l). Eluted material was collected
in 30 · 100 ml and 5 · 400 ml fractions. Similar fractions
were combined to give C2a (fractions 15–20, 1.6 g), C2b
(fractions 21–23, 2.1 g) and C2c (fractions 24–35, 6.4
g). C2a was purified by column chromatography over
Si gel (180 g) using hexane/CHCl3/EtOAc 10:10:0.2
(2.2 l) and hexane/CHCl3/EtOAc 10:10:0.25 (2.0 l).
Fractions 4–8 were purified by crystallization from hex-
ane (6 ml) to yield 504 mg of MF8 (8a/8ba/8: 5/1.5/1).
C2b was purified by crystallization from hexane to give
660 mg of mesuol (1). C2c was purified by column chro-
matography over Si gel (350 g) using petroleum ether/
EtOAc 85:15 (15 · 100 ml fractions). Fractions 4–6
afforded by crystallization from hexane 0.92 g of
MF1 (1a/1b: 4/1). C3 (fractions 11–12, 1.2 g) was added
to D.
3.2.9. 5,7-Dihydroxy-6-(isobutyryl)-8-(3-methylbut-2-
enyl)-4-phenyl-2H-chromen-2-one (mesuol) (1)
Spectral data were identical to those reported in the
literature (Chakraborty et al., 1985).
3.2.10. 5,7-Dihydroxy-6-(2-methylbutanoyl)-8-(3-me-
thylbut-2-enyl)-4-phenyl-2H-chromen-2-one (mammea
A/AB) (1a)
Rf = 0.11 (petroleum ether/EtOAc 9:1, FeCl3: pink;
vanillin: blue); UV kmax (EtOH) 283, 337 nm; 1H
NMR (400 MHz, CDCl3) d 12.15 (1H, brs, 5-OH),
10.70 (1H, brs, 7-OH), 7.48–7.45 (5H, m, Ar), 5.92
(1H, s, H-3), 5.21 (1H, tqq, J = 7.0, 1.2, 0.8 Hz, H-
2000), 3.82 (1H, sext, J = 6.7 Hz, H-200), 3.57 (2H,
dqq, J = 7.0, 1.0 Hz, H-1000), 1.86 (3H, d, J = 0.8 Hz,
H-4000), 1.80 (1H, ddq, J = 14.9, 7.4 Hz, H-400a), 1.70
(3H, d, J = 1.2 Hz, H-5000), 1.38 (1H, ddq, J = 14.9,
7.4 Hz, H-400b), 1.13 (3H, d, J = 6.7 Hz, H-300), 0.86
(3H, t, J = 7.4 Hz, H-500); 13C NMR (100 MHz,
CDCl3) d 212.0 (s, C-100), 161.0 (s, C-7), 160.6 (s, C-
5), 158.5 (s, C-2), 157.4 (s, C-8a), 155.3 (s, C-4),
138.7 (s, C-10), 132.7 (s, C-3000), 128.4 (d, C-40), 127.4
(d, C-30 and C-50), 127.1 (d, C-20 and C-60), 121.0
(d, C-2000), 112.2 (d, C-3), 107.8 (s, C-8), 107.4 (s, C-
6), 101.8 (s, C-4a), 46.4 (d, C-200), 26.7 (t, C-400),
24.9 (q, C-5000), 22.0 (t, C-1000), 17.2 (q, C-4000), 15.8
(q, C-300), 11.1 (q, C-500); EI-MS, m/z (rel. int.): 406
[M]+ (34), 391 (4), 363 (8), 351 (15), 349 (42), 293
(100), 265 (9).
3.2.7. 5,7-Dihydroxy-8-(2-methylbutanoyl)-6-(3-methyl-
but-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/BB)
(isomammeisin) (3)
Colorless crystals; mp 124–125 ꢁC; Rf = 0.16 (petrole-
um ether/EtOAc 9:1, FeCl3: purple; vanillin: pink);
25
D
½aꢀ ¼ ꢂ5:2ꢁ (CHCl3; c = 0.8); UV (EtOH) kmax 293,
1
331 nm; H NMR (400 MHz, CDCl3) d 14.58 (1H, s,
7-OH), 7.59–7.41 (5H, m, Ar), 6.01 (1H, s, H-3), 5.96
(1H, s, 5-OH), 5.10 (1H, tqq, J = 6.9, 1.1 Hz, H-200),
3.97 (1H, sext, J = 6.6 Hz, H-2000), 3.31 (2H, brd,
J = 6.9 Hz, H-100), 1.95 (1H, ddq, J = 13.6, 7.3, 6.5 Hz,
H-4000a), 1.70 (3H, brs, H-400), 1.66 (3H, d, J = 1.1 Hz,
H-500), 1.54 (1H, ddq, J = 13.6, 7.3, 6.5 Hz, H-4000b),
1.30 (3H, d, J = 6.6 Hz, H-3000), 1.03 (3H, t, J = 7.3
Hz, H-5000); 13C NMR (100 MHz, CDCl3) d 210.5 (s,
C-1000), 166.8 (s, C-7), 158.5 (s, C-2), 156.9 (s, C-4),
155.6 (s, C-8a), 154.1 (s, C-5), 136.7 (s, C-10), 134.0 (s,
C-300), 130.1 (d, C-40), 129.5 (d, C-30 and C-50), 127.4
(d, C-20 and C-60), 120.7 (d, C-200), 112.6 (s, C-6),
112.1 (d, C-3), 104.1 (s, C-8), 100.4 (s, C-4a), 46.9 (d,
C-2000), 27.1 (t, C-4000), 25.6 (q, C-500), 21.5 (t, C-100),
17.8 (q, C-400), 16.5 (q, C-3000), 11.7 (q, C-5000); EI-MS,
m/z (rel. int.): 406 [M+] (28), 363 (12), 349 (60), 293
(100), 265 (5).
3.2.11. 5,7-Dihydroxy-6-(3-methylbutanoyl)-8-(3-methyl-
but-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/
AA) (mammeisin) (1b)
1H NMR (400 MHz, CDCl3) d 12.15 (1H, brs, 5-OH),
10.70 (1H, brs, 7-OH), 7.48–7.45 (5H, m, Ar), 5.92 (1H,
s, H-3), 5.21 (1H, tqq, J = 7.0, 1.2, 0.8 Hz, H-2000), 3.57
(2H, dqq, J = 7.0, 1.0 Hz, H-1000), 3.02 (2H, d, J = 6.7
Hz, H-200), 2.25 (1H, m, H-300), 1.86 (3H, d, J = 0.8 Hz,
H-4000), 1.70 (3H, d, J = 1.2 Hz, H-5000), 0.92 (6H, d,
J = 6.6 Hz, H-400 and H-500); 13C NMR (100 MHz,
CDCl3) d 207.5 (s, C-100), 161.0 (s, C-7), 160.6 (s, C-5),
158.5 (s, C-2), 157.4 (s, C-8a), 155.3 (s, C-4), 138.7 (s,
C-10), 132.7 (s, C-3000), 128.4 (s, C-40), 127.4 (d, C-30
and C-50), 127.1 (d, C-20 and C-60), 121.0 (d, C-2000),
112.2 (d, C-3), 107.8 (s, C-8), 107.4 (s, C-6), 101.8 (s,
C-4a), 53.1 (t, C-200), 21.9 (q, C-400 and C-500), 24.9 (q,
C-5000), 24.8 (d, C-300), 22.0 (t, C-1000), 17.2 (q, C-4000).
3.2.8. 5,7-Dihydroxy-8-(3-methylbutanoyl)-6-(3-methyl-
but-2-enyl)-4-phenyl-2H-chromen-2-one (mammea A/BA)
(3a)
1H NMR (400 MHz, CDCl3) d 14.58 (1H, s, 7-OH),
7.59–7.41 (5H, m, Ar), 6.01 (1H, s, H-3), 5.96 (1H, s,
5-OH), 5.10 (1H, tqq, J = 6.9, 1.1 Hz, H-200), 3.31 (2H,
brd, J = 6.9 Hz, H-100), 3.19 (2H, d, J = 6.7 Hz, H-2000),
2.32 (1H, m, H-3000), 1.70 (3H, brs, H-400), 1.66 (3H, d,
J = 1.1 Hz, H-500), 1.06 (6H, d, J = 6.6 Hz, H-4000 and
H-5000); 13C NMR (100 MHz, CDCl3) d 206.5 (s, C-
1000), 166.8 (s, C-7), 158.5 (s, C-2), 156.9 (s, C-4), 155.6

L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
2875
3.2.12.
5-Hydroxy-6-isobutyryl-8,8-dimethyl-4-phenyl-
fractions). Fractions 21–45 (846 mg) were crystallized
from MeOH yielding b-sitosterol. The mother liquors
(560 mg) were purified by column chromatography over
Si gel (50 g) with petroleum ether/CH2Cl2/EtOAc
10:10:0.4 (0.3 l), 10:10:0.5 (1 l) and 10:10:1 (1 l).
110 · 30 ml fractions were collected. Fractions 45–75
gave, by crystallization from hexane 254 mg of MF7
(7/7a: 6/1).
2H-pyrano[2,3-h]chromen-2-one (mammea A/AD cy-
cloD) (mesuagin) (8)
1H NMR (300 MHz, CD3COCD3) d 14.70 (1H, s, 5-
OH), 7.42–7.36 (5H, m, Ar), 6.82 (1H, d, J = 10.1 Hz, 0H00 -
1000), 5.92 (1H, s, H-3), 5.85 (1H, d, J = 10.1 Hz, H-2 ),
3.97 (1H, ept, J = 6.7 Hz, H-200), 1.63 (3H, brs, H-4000),
1.62 (3H, brs, H-5000), 1.19 (6H, d, J = 6.7 Hz, H-300
and H-400); 13C NMR (75 MHz, CD3COCD3) d 211.7
(s, C-100), 164.3 (s, C-5), 158.2 (s, C-2), 157.7 (s, C-8a),
155.8 (s, C-4), 154.9 (s, C-7), 139.5 (s, C-10), 127.9 (d,
C-20, C-40 and C-60), 129.4 (d, C-30 and C-50), 127.1 (d,
C-2000), 114.7 (d, C-1000), 112.4 (d, C-3), 106.6 (s, C-6),
102.0 (s, C-4a), 101.4 (s, C-8), 80.1 (s, C-3000), 39.6 (d,
C-200), 28.0 (q, C-4000 and C-5000), 18.6 (q, C-300 and C-400).
3.2.15. 8,9-Dihydro-5-hydroxy-6-(2-methylbutanoyl)-4-
phenyl-8-(prop-1-en-2-yl)furo[2,3-h]chromen-2-one (7)
Rf = 0.10 (petroleum ether/EtOAc 8:2, FeCl3: green,
vanillin: blue); 1H NMR (400 MHz, CDCl3) d 10.18
(1H, s, 5-OH), 7.40–7.26 (5H, m, Ar), 5.95 (1H, s, H-
3), 5.01 and 4.93 (1H and 1H, two brs, H-5000), 4.51
(1H, brt, H-2000), 3.83 (1H, m, H-200), 3.31 (2H, m, H-
1000), 1.93 (3H, s, H-4000), 1.82 (1H, m, H-400a), 1.39 (1H,
m, H-400b), 1.14 (3H, d, J = 6.7 Hz, H-300), 0.89 (3H, t,
J = 7.4 Hz, H-500); 13C NMR (100 MHz CDCl3) d
215.2 (s, C-100), 163.2 (s, C-7), 161.9 (s, C-5), 159.7 (s,
C-2), 157.7 (s, C-8a), 156.5 (s, C-4), 145.9 (s, C-3000),
139.3 (s, C-10), 128.9 (d, C-40), 127.6 (d, C-30 and C-
50), 0010 27.1 (d, C-20 and C-60), 112.1 (d, C-3), 110.9 (t,
C-5 ), 104.6 (s, C-6 and C-8), 102.5 (s, C-4a), 77.6 (d,
C-2000), 46.5 (d, C-200), 28.6 (t, C-1000), 26.8 (t, C-400),
22.6 (q, C-4000), 16.5 (q, C-300), 11.7 (q, C-500); EI-MS,
m/z (rel. int.): 404 [M]+ (31), 353 (100), 347 (75), 333
(60), 295 (50).
3.2.13. 5-Hydroxy-8,8-dimethyl-6-(2-methylbutanoyl)-4-
phenyl-2H-pyrano[2,3-h]chromen-2-one (mammea A/AB
cycloD) (mammeigin) (8a)
Rf = 0.25 (petroleum ether/EtOAc 9:1, FeCl3: purple;
vanillin: blue); 1H NMR (300 MHz, CD3COCD3) d
14.70 (1H, s, 5-OH), 7.42–7.36 (5H, m, Ar), 6.82 (1H,
d, J = 10.1 Hz, H-1000), 5.92 (1H, s, H-3), 5.85 (1H, d,
J = 10.1 Hz, H-2000), 3.86 (1H, sext, J = 6.7 Hz, H-200),
1.86 (1H, ddq, J = 13.4, 7.4 Hz, H-400a), 1.63 (3H, brs,
H-4000), 1.62 (3H, brs, H-5000), 1.43 (1H, ddq, J = 13.4,
7.4 Hz, H-400b), 1.18 (3H, d, J = 6.7 Hz, H-300), 0.92
(3H, t, J = 7.4 Hz, H-500); 13C NMR (75 MHz,
CD3COCD3) d 211.7 (s, C-100), 164.3 (s, C-5), 158.2 (s,
C-2), 157.7 (s, C-8a), 155.8 (s, C-4), 154.9 (s, C-7),
139.5 (s, C-10), 127.9 (d, C-20, C-40 and C-60), 129.4 (d,
C-30 and C-50), 127.1 (d, C-2000), 114.7 (d, C-1000), 112.4
(d, C-3), 106.6 (s, C-6), 102.0 (s, C-4a), 101.4 (s, C-8),
80.1 (s, C-3000), 46.4 (d, C-200), 28.0 (q, C-4000 and C-5000),
27.3 (t, C-400), 16.1 (q, C-300), 11.1 (q, C-500); EI-MS, m/
z (rel. int.): 404 [M]+, (70), 389 (100), 371 (28), 347 (92).
3.2.16. 8,9-Dihydro-5-hydroxy-6-(3-methylbutanoyl)-4-
phenyl-8-(prop-1-en-2-yl)furo[2,3-h]chromen-2-one (7a)
Rf = 0.10 (petroleum ether/EtOAc 8:2, FeCl3: green,
vanillin: blue); 1H NMR (400 MHz, CDCl3) d 10.17
(1H, s, 5-OH), 7.40–7.26 (5H, m, Ar), 5.95 (1H, s, H-
3), 5.01 and 4.93 (1H and 1H, two brs, H-5000), 4.51
(1H, brt, H-2000), 3.19 (2H, d, J = 6.8 Hz, H-200), 3.31
(2H, m, H-1000), 2.22 (1H, m, H-300), 1.93 (3H, s, H-4000),
1.06 (6H, d, J = 6.7 Hz, H-400 and H-500); 13C NMR
(100 MHz, CDCl3) d 212.5 (s, C-100), 163.2 (s, C-7),
161.9 (s, C-5), 159.7 (s, C-2), 157.7 (s, C-8a), 156.5 (s,
C-4), 145.9 (s, C-3000), 139.3 (s, C-10), 128.9 (d, C-40),
127.6 (d, C-30 and C-50), 127.1 (d, C-20 and C-60),
112.1 (d, C-3), 110.9 (t, C-5000), 104.6 (s, C-6 and C-8),
102.5 (s, C-4a), 77.6 (d, C-2000), 53.4 (t, C-200), 28.6 (t,
C-1000), 26.8 (q, C-400 and C-500), 24.9 (d, C-300), 22.6 (q,
C-4000).
Fraction E was fractionated by column chromatogra-
phy over Si gel (400 g) using petroleum ether/CHCl3/Et-
OAc 10:10:2 (0.5 l), CHCl3/EtOAc 10:1 (1.5 l), and
CHCl3/EtOAc 1:1 (1 l) to yield three fractions: E1
(2.44 g), E2 (1.93 g) and E3 (2.76 g). E2 was purified
by column chromatography over Si gel (flash 150 g,
B = 4.0 cm, h = 18 cm) with petroleum ether/EtOAc
9:1 (1.5 l, 100 · 15 ml fractions). Fractions 65–75 yielded,
by crystallization from hexane/EtOAc 9:1, 140 mg of
6; the mother liquors were further purified by silica gel
3.2.14. 5-Hydroxy-8,8-dimethyl-6-(3-methylbutanoyl)-4-
phenyl-2H-pyrano[2,3-h]chromen-2-one (mammea A/
AA cycloD) (8b)
1H NMR (300 MHz, CD3COCD3) d 14.70 (1H, s, 5-
OH), 7.42–7.36 (5H, m, Ar), 6.82 (1H, d, J = 10.1 Hz, H-
1000), 5.92 (1H, s, H-3), 5.85 (1H, d, J = 10.1 Hz, H-2000),
3.06 (2H, d, J = 6.7 Hz, H-200), 2.25 (1H, m, H-300),
1.63 (3H, brs, H-4000), 1.62 (3H, brs, H-5000), 0.98 (6H, d,
J = 6.6 Hz, H-400 and H-500); 13C NMR (75 MHz,
CD3COCD3) d 207.0 (s, C-100), 164.3 (s, C-5), 158.2 (s,
C-2), 157.7 (s, C-8a), 155.8 (s, C-4), 154.9 (s, C-7),
139.5 (s, C-10), 127.9 (d, C-20, C-40 and C-60), 129.4 (d,
C-30 and C-50), 127.1 (d, C-2000), 114.7 (d, C-1000), 112.4
(d, C-3), 106.6 (s, C-6), 102.0 (s, C-4a), 101.4 (s, C-8),
80.1 (s, C-3000), 53.2 (t, C-200), 28.0 (q, C-4000 and C-5000),
24.7 (d, C-300), 21.9 (q, C-400 and C-500).
Fraction D was chromatographed by flash column
chromatography over Si gel (113 g, B = 4 cm, h = 18
cm) using petroleum ether/EtOAc 4:1 (1 l, 55 · 15 ml

2876
L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
(10 g). Elution with a gradient of hexane/EtOAc yielded
6a (80 mg).
C-10), 128.1 (d, C-40), 127.1 (d, C-30 and C-50), 127.5
(d, C-20 and C-60), 112.0 (d, C-3), 105.1 (s, C-8), 103.0
(s, C-6), 102.5 (s, C-4a), 92.6 (d, C-2000), 71.5 (s, C-3000),
45.6 (d, C-200), 26.7 (t, C-1000), 26.2 (t, C-400), 26.0 (q, C-
4000), 24.7 (q, C-5000), 16.4 (q, C-300), 11.6 (q, C-500); EI-
MS, m/z (rel. int.): 422 [M]+ (45), 408 (19), 365 (97),
347 (40), 307 (23), 293 (100).
3.2.17. 5,7-Dihydroxy-4-(1-hydroxypropyl)-8-(2-methyl-
butanoyl)-6-(3-methylbut-2-enyl)-2H-chromen-2-one
(assamene) (6)
Spectral data were identical to those reported in the
literature (Bordoloi et al., 1997).
3.3. Acylation of MF1
3.2.18. 5,7-Dihydroxy-4-(1-hydroxypropyl)-8-(2-methyl-
butanoyl)-6-[(E)-3,7-dimethylocta-2,6-dienyl]-2H-chr-
omen-2-one (surangin C) (6a)
To a solution of 101 mg of MF1 (80% of compound
1a, 0.199 mmol) in 5 ml of dry CH3CN, p-Br-benzoyl-
chloride (60 mg), and p-dimethylaminopyridine (0.82
mmol, 3.3 mol/eq) were added. After stirring 4 h, the
reaction was worked up by dilution with 0.1 N HCl un-
til pH 6 and extracted with Et2O (2 · 10 ml). The or-
ganic phase was washed with 0.1 N HCl, dried over
Na2SO4 and evaporated to dryness. The residue (200
mg) was purified by flash column chromathography
over Si gel (B = 1.5 cm) using hexane/EtOAc 9:1
(150 ml, 30 · 5 ml fractions) as eluent. Fractions 22–27
by crystallization from MeOH/H2O 9:1, afforded
95.2 mg of 1c (62%).
Yellow crystals; mp 105–106 ꢁC; 1H NMR (200
MHz, CDCl3) d 14.20 (1H, s, 7-OH), 7.25 (1H, s, 5-
OH), 6.09 (1H, s, H-3), 5.20 (1H, brt, J = 7.0 Hz, H-
200), 5.05 (1H, tqq J = 7.0, 1.0 Hz, H-700), 4.72 (1H, t,
J = 7.0 Hz, H-10), 3.72 (1H, m, H-2000), 3.39 (2H, d,
J = 7.0 Hz, H-100), 2.05–1.95 (4H, m, H-500 and H-600),
1.95–1.25 (7H, m, H-20, H-4000 and H-400), 1.63 and
1.56 (6H, two s, H-900 and H-1000), 1.20 (3H, d, J = 7.0
Hz, H-3000), 1.05 (3H, t, J = 7.0 Hz, H-5000), 0.87 (3H, t,
J = 7.0 Hz, H-30); CI-MS, m/z (rel. int.) 456 [M+H]+
(70), 438 (18), 400 (15), 399 (51), 395 (11), 387 (14),
381 (17), 370 (10), 369 (36), 355 (16), 351 (18), 334
(22), 333 (100), 315 (75).
3.3.1. 5,7-Di(4-bromobenzoyloxy)-6-(2-methylbutanoyl)-
8-(3-methylbut-2-enyl)-4-phenyl-2H-chromen-2-one (1c)
Yellow crystals; m.p. 136–137 ꢁC; 1H NMR (300
MHz, CDCl3) d 8.01 (2H, d, J = 10.1 Hz, Br–Ar), 7.69
(2H, d, J = 10.1 Hz, Br–Ar), 7.39 (4H, brs, Br–Ar),
7.25 (2H, d, J = 7.1 Hz, H-20 and H-60), 7.07 (2H, brt,
J = 7.1 Hz, H-30 and H-50), 6.89 (1H, brd, J = 7.4 Hz,
H-40), 6.25 (1H, s, H-3), 5.19 (1H, brt, J = 6.4 Hz, H-
2000), 3.68 (1H, sext, J = 6.9 Hz, H-200), 3.54 (2H, d,
J = 6.5 Hz, H-1000), 1.64 (6H, s, H-4000 and H-5000), 1.57
(1H, m, H-400a), 1.22 (1H, m, H-400b), 0.91 (3H, d,
J = 6.9 Hz, H-300), 0.63 (3H, t, J = 7.2 Hz, H-500); CI-
MS, m/z (rel. int.): 773 [M+H]+ (32), 589 (30), 573
(23), 407 (50), 391 (94), 337 (65), 201 (66), 185 (100).
3.5 g of F were chromatographed by flash column
chromatography over Si gel (255 g, B = 6 cm) using pe-
troleum ether/EtOAc/MeOH 8:2:0.1 (2 l, 10 · 200 ml
fractions) as an eluent. Fractions 7–8 (1.9 g) were re-
chromatographed (flash, 180 g, B = 5 cm) with pet-
roleum ether/EtOAc 7:3 (900 ml, 30 · 30 ml fractions)
to yield, by crystallization from hexane/EtOAc 9:1,
0.55 g of MF5 (5a/5b/5: 5/1.5/1).
3.2.19. 8,9-Dihydro-5-hydroxy-8-(2-hydroxypropan-2-yl)-
6-isobutyryl-4-phenylfuro[2,3-h]chromen-2-one (mammea
a/AD cicloF) (5) and 8,9-dihydro-5-hydroxy-8-(2-hy-
droxypropan-2-yl)-6-(3-methylbutanoyl)-4-phenylfuro-
[2,3-h]chromen-2-one (mammea A/AA cicloF) (5b)
NMR data of these compounds were identical to the
literature (Crombie et al., 1972, 1987).
3.4. Oxidation of MF1, coumarins 2, 3, 4, and mesuol (1)
with PdCl2
3.2.20. 8,9-Dihydro-5-hydroxy-8-(2-hydroxypropan-2-yl)-
6-(2-methylbutanoyl)-4-phenylfuro[2,3-h]chromen-2-one
(mammea A/AB cycloF) (5a)
Reaction of MF1 is described as an example. To a so-
lution of 101 mg of MF1 (80% of compound 1a, 0.2
mmol) in DMSO/H2O 9:1 (15 ml), NaHCO3 (41 mg, 2
mol/eq) and PdCl2 (48 mg, 0.27 mmoli) were added.
The reaction was stirred at room temperature for 8 h,
then diluted with 0.1 N HCl until pH 6 and extracted
with CH2Cl2 (7 · 5 ml). The organic phase was washed
with water, dried with Na2SO4 and evaporated. The res-
idue was purified by silica gel flash column chromatog-
raphy (B = 1.5 cm, h = 15 cm) using petroleum ether/
AcOEt 9:1 as eluent (200 ml, 40 fractions) to afford,
by crystallization from hexane, 68.8 mg of 8a (71%, frac-
tions 30–40).
Rf = 0.12 (petroleum ether/EtOAc 8.5:1.5, FeCl3:
pink-purple, vanillin: blue); 1H NMR (400 MHz,
CDCl3) d 14.59 (1H, s, 5-OH), 7.38–7.28 (5H, m, Ar),
5.92 (1H, s, H-3), 4.91 (1H, t, J = 8.9 Hz, H-2000), 3.61
(1H, sext, J = 6.7 Hz, H-200), 3.31 (2H, d, J = 8.9 Hz,
H-1000), 1.80 (1H, m, H-400a), 1.71 (1H, brs, 3000-OH),
1.42 and 1.31 (3H and 3H, two s, H-4000 and H-5000),
1.41 (1H, m, H-400b), 1.14 (3H, d, J = 6.6 Hz, H-300),
0.91 (3H, t, J = 7.4 Hz, H-500); 13C NMR (100 MHz,
CDCl3) d 209.4 (s, C-100), 164.8 (s, C-7), 163.7 (s, C-5),
159.6 (s, C-2), 156.5 (s, C-4), 155.2 (s, C-8a), 139.1 (s,

L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
2877
The same reaction applied to compound 3 afforded
3b (22%) as major product, together with two more po-
lar by-products 3c (17 mg, 16%) and 3d (10 mg, 9.5%).
Compounds 4b, 8 and 9a were obtained, in a similar
way, from 4 (11%), mesuol (1) (69%), and 2 (33%), re-
spectively.
3.5.3. 3,4-Dihydro-5-hydroxy-2,2-dimethyl–6-(2-methyl-
butanoyl)-4-oxo-10-phenyl-2H-pyrano[2,3-f]chromen-8-
one (3d)
1
Colorless oil; H NMR (200 MHz, CDCl3) d 12.70
(1H, s, 7-OH), 7.44–7.22 (5H, m, Ar), 6.01 (1H, s, H-
3), 3.12 (1H, ddq, J = 6.6, 6.8 Hz, H-2000), 2.62 (2H, s,
H-200), 1.92 (1H, m, H-4000a), 1.53 (1H, m, H-4000b), 1.26
(3H, d, J = 6.9 Hz, H-3000), 1.02 (6H, s, H-400 and H-
500), 1.02 (3H, t, J = 7.0 Hz,H-5000); 13C NMR (50
MHz, CDCl3) d 204.5 (s, C-1000), 197.4 (s, C-100), 162.3
(s, C-7), 159.6 (s, C-4), 158.6 (s, C-2), 155.3 (s, C-5
and C-8a), 139.7 (s, C-10), 128.5 (d, C-40), 128.2 (d, C-
30 and C-50), 127.2 (d, C-20 and C-60), 113.6 (d, C-3),
112.0 (s, C-6), 104.0 (s, C-8), 102.4 (s, C-4a), 81.3 (s,
C-300), 49.4 (d, C-2000), 47.7 (t, C-200), 26.1 (q, C-400 and
C-500), 25.7 (t, C-4000), 15.3 (q, C-3000), 12.0 (q, C-5000);
CI-MS, m/z (rel. int.): [M+H]+ (100), 375 (20), 363 (45).
3.5. Oxidation of MF1 and coumarin 3 with DDQ
To a solution of MF1 (25 mg, 0.05 mmol) in MeOH
(2 ml), an excess of DDQ (43 mg, 0.19 mmol, 4 molar
equivalents) was added. After stirring 6 h at room tem-
perature, the reaction was worked up by dilution with
water and extraction with CH2Cl2 (3 · 3 ml). The
organic phase was washed with H2O, dried with Na2SO4
and concentrated. The residue was purified by silica gel
(flash, B = 0.8 cm, h = 15 cm) using petroleum ether/
EtOAc 9:1 as eluent (100 ml, 20 fractions) to afford 5
mg of 8a (22%, fractions 9–15).
3.5.4. 5-Hydroxy-2-methyl-6-(2-methylbutanoyl)-2-(4-
methylpent-3-enyl)-10-phenyl-2H-pyrano[2,3-f]chromen-
8-one (4b)
The same reaction applied to compound 3 gave cou-
marin 3b (20%).
Colorless crystals; m.p. 95–96 ꢁC; 1H NMR (200
MHz, CDCl3) d 14.65 (1H, s, 7-OH), 7.40–7.24 (5H,
m, Ar), 6.69 (1H, d, J = 10.3 Hz, H-100), 6.01 (1H, s,
H-3), 5.35 (1H, d, J = 10.3 Hz, H-200), 4.89 (1H, brt,
H-700), 3.97 (1H, sext, J = 6.5 Hz, H-2000), 1.93 (1H, m,
H-4000a), 1.65 (6H, brs, H-900 and H-1000), 1.59–1.44
(4H, m, H-500 and H-600), 1.47 (1H, m, H-4000b), 1.30
(3H, d, J = 6.8 Hz, H-3000), 1.03 (3H, t, J = 7.4 Hz, H-
5000), 0.98 (3H, s, H-400); CI-MS, m/z (rel. int.): 473
[M+H]+ (100), 405 (12), 389 (46).
3.5.1. 5-Hydroxy-2,2-dimethyl-6-(2-methylbutanoyl)-10-
phenyl-2H-pyrano[2,3-f]chromen-8-one (3b)
25
Yellow crystals; m.p. 125–126 ꢁC; ½aꢀ ¼ ꢂ1:2ꢁ
D
(CHCl3; c = 0.01); 1H NMR (300 MHz, CDCl3) d
14.59 (1H, s, 7-OH), 7.38–7.21 (5H, m, Ar), 6.62 (1H,
d, J = 9.8 Hz, H-100), 5.99 (1H, s, H-3), 5.37 (1H, d,
J = 9.8 Hz, H-200), 3.95 (1H, ddq, J = 6.4, 6.7 Hz, H-
2000), 1.95 (1H, ddq, J = 13.6, 7.4, 6.2 Hz, H-4000a), 1.50
(1H, ddq, J = 13.6, 7.4, 6.2 Hz, H-4000b), 1.28 (3H, d,
J = 6.7 Hz, H-3000), 1.01 (3H, t, J = 7.4 Hz, H-5000), 0.95
(3H, s, H-400), 0.94 (3H, s, H-500); 13C NMR (75 MHz,
CDCl3) d 210.4 (s, C-1000), 163.8 (s, C-7), 158.8 (s, C-2),
156.7 (s, C-4), 156.0 (s, C-5 and C-8a), 140.0 (s, C-10),
127.7 (d, C-40), 127.5 (d, C-30 and C-50), 127.0 (d, C-20
and C-60), 126.7 (d, C-200), 115.2 (d, C-100), 111.8 (d, C-
3), 105.8 (s, C-6), 103.6 (s, C-8), 102.1 (s, C-4a), 79.0
(s, C-300), 46.8 (d, C-2000), 27.3 (q, C-400 and C-500), 27.1
(t, C-4000), 16.5 (q, C-3000), 11.7 (q, C-5000); CI-MS, m/z
(rel. int.): 405 [M+H]+ (100), 389 (34), 347 (20).
3.6. Biological evaluation
3.6.1. Bacterial strains
The bacterial strains employed were recent clinical
isolates from different hospital laboratories and were
maintained in the culture collection of the Microbiology
Institute of the University of Milano. Bacteria were
stored frozen at ꢂ80 ꢁC in brain heart infusion (BHI)
broth supplemented with 20% (v/v) glycerol (Sigma
Chemical Co.).
3.5.2. 3,4-Dihydro-4,5-dihydroxy-2,2-dimethyl-6-(2-methyl-
butanoyl)-10-phenyl-2H-pyrano[2,3-f]chromen-8-one
(3c)
3.6.2. Susceptibility assay
The minimal inhibitory concentration (MIC) was de-
termined by an agar dilution method in Mueller–Hinton
(M–H) agar according with the protocol of the National
Committee for Clinical Laboratory Standards NCCLS,
2000. Stock solutions of the compounds were prepared
by dissolving them in sterile 0.1 M potassium phosphate
buffer (PB), pH 7.4 or in dimethyl sulfoxide (DMSO)
(Sigma Chemical Co.) at 2560 lg/ml. Serial doubling
dilutions of the stock solution were prepared in PB to
obtain concentrations twenty times higher than those
to be tested. One ml of these were mixed with 19 ml of
melted M–H agar in a dish plate.
25
Yellow crystals; m.p. 138–139 ꢁC; ½aꢀ ¼ 0; 1H NMR
D
(300 MHz, CDCl3) d 15.39 (1H, s, 7-OH), 7.21–7.17
(5H, m, Ar), 6.02 (1H, s, H-3), 4.97 (1H, td, J = 6.5,
1.7 Hz, H-100), 3.95 (1H, ddq, J = 6.4, 6.7 Hz, H-2000),
3.43 (1H, s, 100-OH), 1.93 (1H, ddq, J = 13.6, 7.4, 6.2
Hz, H-4000a), 1.91 (2H, d, J = 6.5 Hz, H-200), 1.50 (1H,
ddq, J = 13.6, 7.4, 6.2 Hz, H-4000b), 1.29 (3H, d, J = 6.7
Hz, H-3000), 1.01 (3H, t, J = 7.4 Hz, H-5000), 0.81 (6H, s,
H-400 and H-5000);CI-MS, m/z (rel. int.): 423 [M+H]+
(15), 405 (100), 389 (37), 363 (17), 347 (14).

2878
L. Verotta et al. / Phytochemistry 65 (2004) 2867–2879
The inocula were prepared by a direct saline suspen-
otide (APAD) as co-factor. Briefly, at the end of the in-
cubation, the cultures are carefully resuspended,
aliquots of 20 ll are removed and added to 0.1 ml of
the Malstat reagent in a 96-well microtiter plate. The
Malstat reagent is made with 0.125% Triton X-100,
sion of isolated colonies from a 24 h M–H agar plate.
The suspension was adjusted to obtain a turbidity com-
parable to that of the 0.5 McFarland standard and fur-
ther on diluted 1:10 to obtain a bacterial concentration
of approximately 107 CFU/ml. Few microliters of such
dilution were deposited on the agar surface of the plates
130 mM
L-lactic acid, 30 mM Tris buffer and 0.62
lM APAD. The spectrophotometric assessment of
pLDH activity is facilitated by adding 25 ll of a solu-
tion of 1.9 lM nitro blue tetrazolium (NBT) and 0.24
lM phenazine ethosulfate (PES) to the Malstat rea-
gent. As APADH is formed, the NBT is reduced and
forms a blue formazan product that can be measured
at 650 nm. The antimalarial activity of test compound
was expressed as the 50% inhibitory concentration
(IC50); each IC50 value is the mean and standard devi-
ation of at least three separate experiments performed
in triplicate. Statistical analysis was performed using
the paired t test with Statview software.
by
a multipoint inoculating device (Denley-Tech
Ltd., Billingshurst, UK) producing an inoculum of ap-
proximately 104–105 CFU per spot. The plates were
incubated in air at 35 ꢁC for 24 h and the MIC was de-
fined as the minimum concentration at which no visible
grown was observed.
3.6.3. Parasite cultures
RPMI 1640 medium was purchased from Gibco-
BRL, human A-positive red blood cells and plasma
were kindly provided by the Blood Bank of the
National Cancer Institute, Milano, Italy. Chloroquine
diphosphate (CQ) (C-6628), and DMSO were all ob-
tained from Sigma–Aldrich, Milan, Italy. P. falciparum
cultures were carried out according to the method de-
scribed by Trager and Jensen (1976). Briefly, the CQ-
sensitive (CQ-S), moderately mefloquine-resistant clone
D10 and the CQ-resistant (CQ-R), mefloquine-suscepti-
ble clone W2 were maintained at 5% haematocrit (hu-
man type A-positive red blood cells) in complete
culture medium at 37 ꢁC. Complete medium contained
RPMI 1640 medium (Gibco-BRL, NaHCO3 24 mM)
with the addition of 10% heat-inactivated A-positive
human plasma, 20 mM Hepes (Biological Industries,
Kibbutz, Israel), 2 mM Glutammine (Biological Indus-
tries). All the cultures were maintained in a standard
gas mixture consisting of 1% O2, 5% CO2, 94% N2.
When parasitemia exceeded 5%, subcultures were
taken; the culture medium was changed every second
day.
Acknowledgement
Work was in part supported by MIUR (funds PRIN)
of Italy.
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