New eudesmane derivatives from Melampodium camphoratum from the suriname rainforest

Article abstract of DOI:10.1021/np049696q

Bioassay-guided fractionation of an EtOAc extract of the leaves of Melampodium camphoratum using an assay for inhibitors of the degradation of hemin resulted in the isolation of six new eudesmane sesquiterpenes (1-6) and the known 6-epi-β-verbesinol coumarate (7). The structures of compounds 1-6 were established as 6α-(4′-O-methyl-7′E-coumaryloxy)eudesm- 4(14)-ene (1), 6α-({4′-O-stearyl}-7′E-coumaryloxy)eudesm-4(14) -ene (2), 6α-({4′-O-palmityl}-7′E-coumaryloxy)eudesm-4(14)-ene (3), 6α-({4′-O-[9″Z-hexadecenoyl]}-7′E-coumaryloxy) eudesm-4(14)-ene (4), 6α-(7′Z-coumaryloxy)eudesm-4(14)-ene (5), and 6α-({4′-acetoxy}-7′Z-coumaryloxy)eudesm-4(14)-ene (6). Compounds 1-4 showed weak activity in the hemin degradation assay, while compounds 5-7 were inactive.

Full text of DOI:10.1021/np049696q

J. Nat. Prod. 2004, 67, 2053-2057
2053
New Eudesmane Derivatives from Melampodium camphoratum from the
Suriname Rainforest1
V. S. Prakash Chaturvedula,† Afgan Farooq,† Jennifer K. Schilling,† S. Malone,‡ Iwan Derveld,‡
Marga C. M. Werkhoven,§ Jan H. Wisse, Michel Ratsimbason,3 and David G. I. Kingston*,†
Department of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,
Conservation International Suriname, Kromme Elleboogstraat No. 20, Paramaribo, Suriname, The National Herbarium of
Suriname, P.O. Box 9212, Paramaribo, Suriname, Bedrijf Geneesmiddelen Voorziening Suriname,
Commissaris Roblesweg 156, Geyersvlijt, Suriname, and Centre National d’Application et Recherches Pharmaceutiques,
B.P. 702, Antananarivo 101, Madagascar
Received September 14, 2004
Bioassay-guided fractionation of an EtOAc extract of the leaves of Melampodium camphoratum using an
assay for inhibitors of the degradation of hemin resulted in the isolation of six new eudesmane
sesquiterpenes (1-6) and the known 6-epi-â-verbesinol coumarate (7). The structures of compounds 1-6
were established as 6R-(4′-O-methyl-7′E-coumaryloxy)eudesm-4(14)-ene (1), 6R-({4′-O-stearyl}-7′E-cou-
maryloxy)eudesm-4(14)-ene (2), 6R-({4′-O-palmityl}-7′E-coumaryloxy)eudesm-4(14)-ene (3), 6R-({4′-O-[9′′Z-
hexadecenoyl]}-7′E-coumaryloxy)eudesm-4(14)-ene (4), 6R-(7′Z-coumaryloxy)eudesm-4(14)-ene (5), and 6R-
({4′-acetoxy}-7′Z-coumaryloxy)eudesm-4(14)-ene (6). Compounds 1-4 showed weak activity in the hemin
degradation assay, while compounds 5-7 were inactive.
Malaria is a parasitic disease that is a major scourge of
the tropical regions of the world, causing more than 300
million acute illnesses and one million deaths annually.2
Plants have been an important source of antimalarial
compounds, with quinine and artemisinin being clinically
used drugs, but with many other plant natural products
showing activity against the parasite.3 In an extension of
our research on the isolation of bioactive compounds from
plants of the Suriname and Madagascar rainforests, as a
part of the mission of an International Cooperative Biodi-
versity Group (ICBG),4-7 we have recently started working
on the isolation and identification of potential antimalarial
components from these plants. The bioassay used in this
work was a new assay based on the inhibition of the
degradation of hemin by glutathione.
The malarial parasite Plasmodium falciparum hydro-
lyzes the hemoglobin content of the erythrocytes of the host
to obtain its essential amino acids, and hemin [Fe(III)-
protoporphyrin IX] is formed as a byproduct; this is toxic
to the parasite. The hemin is detoxified by polymerization
or by reaction with glutathione, and several current anti-
malarial drugs such as chloroquine, quinine, and amodi-
aquine are thought to act by inhibiting the degradation of
hemin by one or both mechanisms, leaving the hemin free
to kill the malarial parasite.8 A simple microtiter plate
based assay has been developed to determine the extent
of hemin degradation in the presence of drug and glu-
tathione.9 Not all antiplasmodial compounds are active in
the hemin degradation assay, and not all compounds that
inhibit the degradation of hemin have antiplasmodial
activity, but the correlation is reasonably good, with the
assay successfully detecting 16 of 19 antiplasmodial com-
pounds from a collection of 42 natural products and giving
only six false positives.9
hemin degradation assay, and it was thus selected for in-
vestigation. M. camphoratum is a small herb that grows
in Brazil, Suriname, and elsewhere; its local name in Suri-
name is “talangpasibita”. The genus Melampodium (Aster-
aceae) has found extensive use in folk medicine for treat-
ment of malaria, stomach pains, and influenza.11 Mono-
terpene glycosides, sesquiterpenes, sesquiterpene lactones,
diterpenes, diterpene lactones, flavonoid glycosides, and
coumarins have been reported previously from this genus.12
The only previous studies on the chemistry of M. camphor-
atum have reported the isolation of flavonoids and a ses-
quiterpene lactone13 and the variation of its essential oils.14
Results and Discussion
Initial liquid-liquid partition of the EtOAc extract of M.
camphoratum indicated that the activity was concentrated
in the hexane- and CHCl3-soluble portions of the hexane/
aqueous MeOH and CHCl3/aqueous MeOH partitions.
Purification of the hexane- and CHCl3-soluble portions by
chromatography over MCI gel followed by reversed-phase
HPLC furnished the six new eudesmane sesquiterpenes
1-6 and the known 6-epi-â-verbesinol coumarate (7).15
A sample of Melampodium camphoratum (L.f.) Baker
(Asteraceae) from Suriname tested positive in an initial
antimalarial assay conducted in Panama10 and also in the
* To whom correspondence should be addressed. Tel: (540) 231-6570.
Fax: (540) 231-3255. E-mail: dkingston@vt.edu.
† Virginia Polytechnic Institute and State University.
‡ Conservation International, Suriname.
§ National Herbarium of Suriname.
Bedrijf Geneesmiddelen Voorziening Suriname.
∇ Centre National d’Application et Recherches Pharmaceutiques.
10.1021/np049696q CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/03/2004

2054 Journal of Natural Products, 2004, Vol. 67, No. 12
Prakash Chaturvedula et al.
and C-10) was assigned as for 1 and 7 on the basis of their
almost identical coupling constants and 13C NMR values.
The structure of 2 was thus assigned as 6R-({4′-O-stearyl}-
7′E-coumaryloxy)eudesm-4(14)-ene.
Compound 3 was isolated as a colorless liquid and was
determined to have the molecular formula C40H62O4 by
1
HRFABMS. The IR and H and 13C NMR spectral data of
3 were almost superimposable on those of 2, suggesting
that it was also a 6-epi-â-verbesinol coumarate moiety with
a fatty acid side chain at the C-4′ position. Alkaline
hydrolysis of 3 furnished 7 and palmitic acid ([M+•], m/z
256), indicating the presence of a palmityl side chain at
the C-4′ position instead of the stearyl side chain in 2. The
structure of compound 3 was thus assigned as 6R-({4′-O-
palmityl}-7′E-coumaryloxy)eudesm-4(14)-ene.
Figure 1. Selected HMBC correlations for 1.
Compound 1 was isolated as a colorless optically active
viscous liquid whose molecular formula was established as
C25H34O3 from HRFABMS, 13C NMR, and APT (attached
proton test) spectral data. The IR absorption bands at 1701
cm-1 suggested the presence of a carbonyl functional group
in 1. The 1H NMR spectrum showed the presence of an
oxymethine group as a triplet at δ 5.17 (J ) 10.3 Hz); one
methyl singlet at δ 0.79; two methyl doublets at δ 0.89 (J
) 6.8 Hz) and 0.90 (J ) 6.8 Hz); an exocyclic methylene
group as singlets at δ 4.56 and 4.75; two olefinic protons
as doublets at δ 6.25 (J ) 15.8 Hz) and 7.59 (2H, J ) 15.8
Hz); four aromatic protons as doublets at δ 6.87 (2H, J )
8.9 Hz) and 7.45 (J ) 8.5 Hz); one methoxy singlet at δ
3.82; and five methylenes and three methines between δ
1.24 and 2.26. The 13C NMR values for all 25 carbons were
assigned on the basis of APT, HMQC, and HMBC spectral
data, which indicated the presence of three sp2 quaternary
carbons, two sp2 methyls, one sp2 methylene, six sp2
methines, one sp3 quaternary carbon, two sp3 methyls, five
sp3 methylenes, four sp3 methines, and a carbonyl group
in its structure. A search in the literature indicated that
the 1H NMR spectral data of 1 were similar to those of the
sesquiterpene 6-epi-â-verbesinol coumarate (7),15 except for
the presence of an additional O-methyl group. In the
absence of any other oxygenated carbons, the methoxy
group in 1 was located at the C-4′ position. Further,
methylation of compound 7 with CH2N2 yielded 1, confirm-
ing the structure and stereochemistry of the latter com-
pound. This structure was fully consistent with the ob-
served COSY (H-2/H-1, H-3; H-6/H-5, H-7; H-8/H-7, H-9;
H-11/H-7, H-12, H-13; H-2′/H-3′; H-4′/H-5′; H-7′/H-8′) and
HMBC correlations (Figure 1). The stereochemistry was
consistent with the ROESY spectrum of 1, in which the
oxymethine proton at C-6 showed a correlation to the
methyl group at C-10 and did not show correlations to the
C-5 or C-7 methine protons. The E stereochemistry of the
double bond between C-7′ and C-8′ was supported by the
J7′-8′ coupling constant of 15.8 Hz. On the basis of the above
spectral data, the structure of 1 was assigned as 6R-(4′-O-
methyl-7′E-coumaryloxy)eudesm-4(14)-ene.
HRFABMS and 13C NMR spectral data deduced the
molecular formula of compound 4 as C40H60O4. The IR
spectrum showed the presence of two carbonyl functional
1
groups similar to those in 2 and 3. The H NMR spectral
data of 4 were similar to those of 3, except for the side chain
at the C-4′ position. The 1H NMR of the side chain showed
the presence of a triplet at δ 0.87 (J ) 7.1 Hz), a broad
singlet for 16 protons, three multiplets centered at δ 1.64
(2H), 2.01 (4H), 5.33 (2H), and a triplet at δ 2.34 (J ) 7.3
Hz), suggesting the presence of a double bond in the ester
side chain of 4. Alkaline hydrolysis of 4 furnished 7 and a
compound identified tentatively as palmitoleic acid from
its mass spectrum ([M+•], m/z 254). The structure of 4 was
confirmed by partial synthesis from 7. Esterification of 7
with palmitoleic acid in the presence of EDCI and DMAP
furnished 4, confirming its structure and stereochemistry.
The 13C NMR values for all the carbons were assigned on
the basis of COSY, HMQC, and HMBC spectral data and
in comparison with 2 and 3. Thus, the structure of 4 was
assigned as 6R-({4′-O-[9′′Z-hexadecenoyl]}-7′E-coumaryl-
oxy)eudesm-4(14)-ene.
Compound 5 was isolated as a colorless oil, with a
molecular formula of C24H32O3 (HRFABMS), identical to
that of 7. The IR spectrum showed the presence of a
1
hydroxy group (3390 cm-1). A comparison of the H NMR
spectral data of 5 with those of 6â-(7′Z-coumaroyloxy)-
eudesm-4(14)-ene (8)16 and 7 indicated that the spectrum
of the sesquiterpene part of compound 5 was superimpos-
able on that of 7 and the spectrum of the coumaryl part of
5 was superimposable on that of 8. This finding indicated
that 5 was the Z isomer of 7. This was supported by the
ROESY spectrum of 5, which showed a strong correlation
between the two protons at C-7′ and C-8′. On the basis of
the above spectral evidence, the structure of 5 was assigned
as 6R-(7′Z-coumaryloxy)eudesm-4(14)-ene.
The molecular formula of 2 was deduced as C42H66O4
from its HRFABMS and 13C NMR spectral data. The H
1
Compound 6 was isolated as a colorless liquid; its
molecular formula was established as C26H34O4 by HR-
FABMS and 13C NMR spectra. Its 1H NMR spectrum was
very similar to that of 5, except for the presence of a signal
for an additional acetate group as a singlet at δ 2.29. The
presence of additional carbon signals at δ 169.2 and 21.1
in the 13C NMR of 6 suggested the replacement of the
hydroxy group at the C-4′ position with an acetoxy group.
This was supported by the absence of the IR absorption
band corresponding to that of the hydroxy group and the
NMR spectrum of 2 was very similar to that of 1, except
for the replacement of the methoxy signal at δ 3.82 with
signals at δ 0.86 (t, 3H, J ) 7.0 Hz), 1.29 (br s, 28 H), 1.68
(m, 2H), and 2.54 (t, 2H, J ) 7.3 Hz). Alkaline hydrolysis
of 2 furnished 6-epi-â-verbesinol coumarate (7)15 and stearic
acid ([M+•], m/z 284), indicating the presence of a stearyl
side chain at the C-4′ position. The presence of the stearyl
side chain at the C-4′ position was further supported by
the IR spectrum of 2, which showed the presence of two
carbonyl groups. One carbonyl absorption was at 1764
cm-1, and the second, as in 1, was at 1708-1. The 13C NMR
spectrum also showed the presence of two carbonyl carbons
by signals at δ 167.0 and 172.0. The 13C NMR values for
all the carbons were assigned on the basis of HMQC and
HMBC spectral data and by comparison with 1 and 7. The
stereochemistry at the four chiral centers (C-5, C-6, C-7,
presence of an additional carbonyl absorption at 1767 cm-1
.
Further, acetylation of 5 furnished compound 6, confirming
its structure and stereochemistry. Thus, 6 was assigned
as 6R-({4′-acetoxy}-7′Z-coumaryloxy)eudesm-4(14)-ene.
The isolated compounds were tested in a hemin degrada-
tion bioassay, and the four compounds 1-4 were all weakly
active, with DC50 values of 48, 110, 75, and 52 µg/mL,

Eudesmane Derivatives from Melampodium
Journal of Natural Products, 2004, Vol. 67, No. 12 2055
Table 1. 1H NMR Data for Compounds 1-6 (CDCl3)a
position
1
2
3
4
5
6
1
1.96 m
1.65 m
1.50 m, 1.24 m
2.10 d 10.7
5.17 t 10.3
1.42 m
1.54 m, 1.30 m
2.26 m
1.60 m
0.89 d 6.8
0.90 d 6.8
4.75 s, 4.56 s
0.79 s
6.87 d 8.9
7.45 d 8.5
7.59 d 15.8
6.25 d 15.8
1.95 m
1.66 m
1.50 m, 1.26 m
2.10 d 10.7
5.17 t 10.4
1.40 m
1.56 m, 1.23 m
2.27 m
1.62 m
0.89 d 7.0
0.89 d 7.0
4.74 s, 4.54 s
0.78 s
7.07 d 8.5
7.50 d 8.5
7.60 d 15.8
6.33 d 15.8
2.54 t 7.3
1.68 m
1.29 br s
1.29 br s
1.29 br s
1.29 br s
1.29 br s
1.29 br s
1.29 br s
0.86 t 7.0
1.95 m
1.66 m
1.52 m, 1.26 m
2.10 d 10.6
5.17 t 10.1
1.38 m
1.56 m, 1.24 m
2.28 m
1.64 m
0.89 d 6.7
0.89 d 6.7
4.74 s, 4.54 s
0.78 s
1.95 m
1.66 m
1.52 m, 1.30 m
2.10 d 10.6
5.17 t 10.3
1.42 m
1.54 m, 1.24 m
2.28 m
1.60 m
0.89 d 6.9
0.89 d 6.9
4.74 s, 4.55 s
0.78 s
6.81 d 8.5
7.40 d 8.5
7.57 d 15.8
6.24 d 15.8
2.34 t 7.3
1.64 m
1.95 m
1.68 m
1.52 m, 1.32 m
2.04 d 10.8
5.12 t 10.3
1.38 m
1.56 m, 1.26
2.29 m
1.62 m
0.87 d 6.7
0.88 d 6.7
4.77 s, 4.59 s
0.76 s
6.77 d 8.5
7.60 d 8.6
6.78 d 12.8
5.75 d 12.8
1.95 m
1.68 m
1.50 m, 1.30 m
2.09 d 10.6
5.17 t 10.3
1.42 m
1.58 m, 1.26 m
2.26 m
1.60 m
2
3
5
6
7
8
9
11
12
0.89 d 6.7
0.89 d 6.7
4.74 s, 4.54 s
0.78
13
14
15
2′,6′
3′,5′
7′
7.07 d 8.9
7.50 d 8.6
7.60 d 15.8
6.33 d 15.8
2.54 t 7.3
1.68 m
7.05 8.7
7.72 d 8.7
6.85 d 13.3
5.83 d 13.3
8′
2′′
3′′
4′′-7′′
8′′
1.28 br s
1.30 br s
2.01 m
5.33 m
2.01 m
1.30 br s
0.87 t 7.1
1.28 br s
9′′,10′′
11′′
12′′-15′′
16′′
17′′
18′′
OCOCH3
OCH3
1.28 br s
1.28 br s
1.28 br s
0.87 t 7.0
2.29 s
3.82 s
a Assignments made on the basis of COSY, HMQC, and HMBC spectral data and in comparison with the literature data.15,16
Isolation of Bioactive Compounds. The crude EtOAc
extract (0.30 g) was suspended in aqueous MeOH (MeOH-
H2O, 9:1, 50 mL) and extracted with hexane (3 × 50 mL). The
aqueous layer was then diluted to 70% MeOH (v/v) with H2O
and extracted with CHCl3 (3 × 50 mL). The aqueous layer was
concentrated, and the residue obtained was suspended in H2O
(25 mL) and extracted with n-BuOH (2 × 25 mL). The
n-hexane and CHCl3 extracts were selected for further frac-
respectively. Compounds 5-7 were inactive in this assay,
50
with DC values > 200 µg/mL. The compounds were also tested
for antiplasmodial activity, but disappointingly none of them
showed significant activity. It thus appears probable that the
hemin degradation assay did not track well with the presumed
antiplasmodial activity in this case and that further work will be
needed to isolate the compound or compounds giving rise to the
latter activity.
1
tionation based on their activity and H NMR patterns. The
residue from the n-hexane extract (0.15 g) was chromato-
graphed over MCI gel using MeOH-H2O (1:1 to 100:0) to yield
seven fractions (A-G), of which fractions D and E were found
to be active. Fraction D upon reversed-phase HPLC with the
mobile phase CH3CN-H2O (90:10) yielded the known sesquit-
erpene 7 (4.0 mg) and a new sesquiterpene 6 (2.4 mg). Fraction
E on reversed-phase HPLC with the mobile phase CH3CN-
H2O (98:2) yielded two new sesquiterpenes, 2 (4.3 mg) and 3
(7.6 mg).
The residue from the CHCl3 extract (0.12 g) was chromato-
graphed over MCI gel using MeOH-H2O (1:1 to 100:0) to yield
nine fractions (A-I), of which fractions E-G were found to be
active. Fraction E on reversed-phase HPLC with the mobile
phase CH3CN-H2O (85:15) yielded the known compound 7 (1.8
mg) in addition to the new sesquiterpene 1 (2.1 mg). Fraction
F on reversed-phase HPLC with the mobile phase CH3CN-
H2O (90:10) yielded the known sesquiterpene 7 (14.6 mg) and
the new sesquiterpene 5 (5.2 mg). Fraction G on reversed-
phase HPLC with the mobile phase CH3CN-H2O (70:30)
furnished the new sesquiterpene 4 (2.8 mg) and the known
sesquiterpene 7 (2.1 mg). The known compound 7 was identi-
fied as 6-epi-â-verbesinol coumarate on the basis of the
comparison of its 1H and 13C NMR and HRFABMS spectro-
scopic data with those reported in the literature.15
Experimental Section
General Experimental Procedures. Optical rotations
were recorded on a Perkin-Elmer 241 polarimeter. IR and UV
spectra were measured on MIDAC M-series FTIR and Shi-
madzu UV-1201 spectrophotometers, respectively. NMR spec-
tra were obtained on a JEOL Eclipse 500 spectrometer. The
chemical shifts are given in δ (ppm) with TMS (tetramethyl-
silane) as internal reference, and coupling constants are
reported in Hz. Mass spectra were obtained on a JEOL HX-
110 instrument. MCI gel (CHP20P) was used for column
chromatography. Reversed-phase preparative TLC was per-
formed on Baker Si-C18F plates. HPLC was performed on a
Shimadzu LC-10AT instrument with an ODS A323 column
(250 × 10 mm).
Antimalarial Assays. Activity against P. falciparum was
determined by using the microfluorimetric assay previously
reported;17 results are expressed as IC50 values (µg/mL). A
modified 96-well hemin-glutathione degradation assay was
used for the in vitro antimalarial assay.9 Data are presented
as DC50 values, corresponding to the dose required to reduce
the decomposition of hemin by 50% as compared with a control
experiment without drug. In this determination, the control
drug quinine has a DC50 value of 1.3 µg/mL.
Plant Material and Extract Preparation. The leaves,
stems, and twigs of Melampodium camphoratum (L.f.) Baker
(Asteraceae) were collected in early 2000 on cultivated land
in the district of Sipaliwini, Suriname, and were assigned
collector number 988DeErAK02903. The voucher specimen is
deposited in the National Herbarium of Suriname, Parama-
ribo. The plant samples of M. camphoratum were dried and
extracted with EtOAc to yield crude extract E980306.
6r-(4′-O-Methyl-7′E-coumaryloxy)eudesm-4(14)-ene (1):
colorless liquid; [R]25D -54° (c 0.015, CHCl3); UV (MeOH) λmax
(log ꢀ) 218 (3.85), 280 (4.12) nm; IR νmax 2930, 2866, 1701, 1603,
1511, 1252, 1170, 826, 755 cm-1; 1H and 13C NMR, see Tables
1 and 2; HRFABMS m/z 383.2577 [M + H]+ (calcd for C25H35O3,
383.2586).
Methylation of 7. To a solution of compound 7 (2.0 mg) in
diethyl ether (2 mL) was added a freshly prepared solution of
CH2N2 (5 mL), and the mixture was left in the refrigerator

2056 Journal of Natural Products, 2004, Vol. 67, No. 12
Table 2. 13C NMR Data for Compounds 1-6 (CDCl3)a
Prakash Chaturvedula et al.
6r-({4′-O-[9′′Z-Hexadecenoyl]}-7′E-coumaryloxy)-
eudesm-4(14)-ene (4): colorless liquid; [R]25D -18.6° (c 0.015,
CHCl3); UV (MeOH) λmax (log ꢀ) 205 (4.08), 282 (3.85) nm; IR
carbon
1
2
3
4
5
6
1
νmax 2928, 2853, 1708, 1605, 1515, 1190, 1167, 832 cm-1; H
1
2
3
4
5
6
7
8
9
40.3 40.3
24.2 24.4
42.3 42.3
146.4 146.4
56.0 56.0
71.2 71.5
49.7 49.6
18.4 18.4
38.2 38.1
38.3 38.2
26.2 26.2
17.7 17.7
21.5 21.5
106.9 106.8
16.2 16.2
127.4 132.3
129.8 129.2
114.3 122.1
161.3 152.1
144.3 146.4
116.2 118.8
167.5 167.1
172.1
40.3
24.3
42.3
40.3
24.2
42.2
40.3 40.3
24.3 24.3
42.2 42.2
146.5 146.4
55.9 56.0
71.1 71.6
49.5 49.6
18.4 18.4
38.2 38.2
38.3 38.3
26.2 26.2
17.6 17.6
21.4 21.4
106.7 118.8
16.2 16.2
127.6 132.4
132.4 129.3
115.0 122.1
156.4 152.0
143.3 143.5
114.9 115.9
166.6 167.1
and 13C NMR, see Tables 1 and 2; HRFABMS m/z 605.4570
[M + H]+ (calcd for C40H61O4, 605.4570).
146.4
56.0
146.4
56.0
Alkaline Hydrolysis of 4. Hydrolysis of 4 (1.4 mg) as
reported above furnished 7 and palmitoleic acid ([M+•], m/z
254).
71.5
71.4
49.6
49.7
18.4
18.4
Esterification of 7. Compound 7 (5 mg) in CH2Cl2 (1 mL)
and EDCI (6 mg) were added to a solution of palmitoleic acid
(5 mg) and DMAP (2 mg) in CH2Cl2 (1 mL), and the resulting
mixture was stirred at room temperature for 5 h. EtOAc (10
mL) was added to quench the reaction, and the mixture was
concentrated to yield a pale brown viscous liquid. The brown
viscous liquid on purification by reversed-phase preparative
TLC (MeOH-H2O, 80:20) furnished a colorless liquid (1.2 mg),
which was identified as 4 by co-TLC and 1H NMR spectral
data.
38.2
38.1
10
38.3
38.2
11
26.2
26.2
12
17.7
17.7
13
21.5
21.5
14
106.8
16.2
106.8
16.2
15
1′
132.3
129.2
122.1
152.1
146.4
118.8
167.1
172.1
34.5
127.4
130.0
115.8
157.7
144.5
116.0
167.8
179.4
34.0
2′,6′
3′,5′
4′
6r-(7′Z-Coumaryloxy)eudesm-4(14)-ene (5): colorless
7′
liquid; [R]25 -30.8° (c 0.018, CHCl3); UV (MeOH) λmax (log ꢀ)
D
8′
216 (4.12), 279 (4.42) nm; IR νmax 3390, 2930, 2871, 1648, 1604,
1512, 1442, 1156, 855, 747 cm-1; 1H and 13C NMR, see Tables
1 and 2; HRFABMS m/z 369.2428 [M + H]+ (calcd for C24H33O3,
369.2430).
9′
1′′
2′′
34.5
3′′
25.0
25.0
24.7
4′′-6′′
7′′
29.3-29.8 29.3-29.8 29.2-29.8
29.3-29.8 29.3-29.8 31.9
29.3-29.8 29.3-29.8 27.2
29.3-29.8 29.3-29.8 130.1
29.3-29.8 29.3-29.8 130.1
29.3-29.8 29.3-29.8 27.2
29.3-29.8 29.3-29.8 31.9
6r-({4′-Acetoxy}-7′Z-coumaryloxy)eudesm-4(14)-ene (6):
colorless liquid; [R]25 -56.3° (c 0.019, CHCl3); UV (MeOH)
D
8′′
λmax (log ꢀ) 218 (3.92), 282 (4.14) nm; IR νmax 2930, 2866, 1767,
9′′
1704, 1636, 1506, 1201, 1176, 1009, 912, 833, 756 cm-1 1H
;
10′′
11′′,12′′
13′′
14′′
15′′
16′′
17′′
18′′
OCOCH3
OCOCH3
OCH3
and 13C NMR, see Tables 1 and 2; HRFABMS m/z 411. 2519
[M + H]+ (calcd for C26H35O4, 411.2535).
29.3-29.8 32.0
29.3-29.8 22.8
29.2-29.8
22.7
14.2
Acetylation of 5. Compound 5 (1.2 mg) was dissolved in
pyridine (0.4 mL) and acetic anhydride (0.4 mL), and the
mixture was stirred for 20 h at room temperature. The product
was dried under vacuum, and the residue obtained was
purified by reversed-phase HPLC with the mobile phase
MeOH-H2O (80:20) to furnish 6 (0.7 mg), identified by co-
32.0
22.8
14.2
14.2
169.2
21.2
1
TLC and H NMR spectral data.
55.4
a Assignments made on the basis of COSY, HMQC, and HMBC
spectral data and comparison with the literature values.15,16
Acknowledgment. We gratefully acknowledge financial
support from International Cooperative Biodiversity Grant
U01 TW/CA-00313 from the Fogarty Center, National Insti-
tutes of Health. We are also grateful to Drs. E. Ortega-Barria
and T. Capson of the Panama ICBG for the initial antimalarial
bioassay of M. camphoratum and for training two scientists
from CNARP in their bioassay methodology. We thank Mr.
W. Bebout and Mr. T. Glass (Virginia Polytechnic and State
University, VPISU) for obtaining the mass and NMR spectra,
respectively.
for 16 h. The product was dried under vacuum and the residue
was purified over reversed-phase preparative HPLC with the
mobile phase MeOH-H2O (85:15) to furnish a product (1.2 mg)
that was identified as 1 by co-TLC and its 1H NMR spectrum.
6r-({4′-O-Stearyl}-7′E-coumaryloxy)eudesm-4(14)-
ene (2): colorless liquid; [R]25 -55.4° (c 0.035, CHCl3); UV
D
(MeOH) λmax (log ꢀ) 217.8 (4.21), 280 (4.08) nm; IR νmax 2918,
1
2849, 1764, 1708, 1637, 1506, 1209, 1176, 758 cm-1; H and
13C NMR, see Tables 1 and 2; HRFABMS m/z 635.5034 [M +
Supporting Information Available: 1H and 13C NMR spectra
of compounds 1-6. This material is available free of charge via the
Internet at http://pubs.acs.org.
H]+ (calcd for C42H67O4, 635.5039).
Alkaline Hydrolysis of 2. To a solution of compound 2 (2
mg) in MeOH (1 mL) was added 5% methanolic KOH (2 mL),
and the reaction mixture was refluxed for 1 h. The mixture
was concentrated, and water (5 mL) was added. The aqueous
layer was extracted with CH2Cl2 (3 × 10 mL), and the
combined organic layer was concentrated to yield a brown
solid, which on purification by preparative TLC (hexane-
EtOAc, 75:25) furnished a colorless solid (0.5 mg) identified
as 6-epi-â-verbesinol coumarate (7) by spectral (1H NMR and
HRFABMS) data.15 The aqueous layer was acidified with 1 N
HCl and extracted with EtOAc (3 × 10 mL) to yield a brown
viscous liquid, which on preparative TLC (hexane-EtOAc, 50:
50) furnished stearic acid (0.4 mg, [M+•], m/z 284).
References and Notes
(1) Biodiversity Conservation and Drug Discovery in Suriname, Part 15.
For part 14, see: Williams, R. B.; Hoch, J.; Glass, T. E.; Evans, R.;
Miller, J. S.; Wisse, J. H.; Kingston, D. G. I. Planta Med. 2003, 69,
861-864.
(2) Gelb, M. H.; Hol, W. G. J. Science 2002, 297, 343-344.
(3) Schwikkard, S.; van Heerden, F. R. Nat. Prod. Rep. 2002, 19, 675-
692.
(4) Zhou, B.-N.; Baj, N. J.; Glass, T. E.; Malone, S.; Werkhoven, M. C.
M.; van Troon, F.; David, M.; Wisse, J. H.; Kingston, D. G. I. J. Nat.
Prod. 1997, 60, 1287-1293.
(5) Kingston, D. G. I.; Abdel-Kader, M.; Zhou, B.-N.; Yang, S.-W.; Berger,
J. M.; van der Werff, H.; Miller, J. S.; Evans, R.; Mittermeier, R.;
Famolare, L.; Guerin-McManus, M.; Malone, S.; Moniz, E.; Wisse, J.
H.; Vyas, D.; Wright, J. J. K. Pharm. Biol. 1999, S37, 22-34.
(6) Chaturvedula, V. S. P.; Schilling, J. K.; Miller, J. S.; Andriantsiferana,
R.; Rasamison, V. E.; Kingston, D. G. I. J. Nat. Prod. 2004, 67, 895-
898.
6r-({4′-O-Palmityl}-7′E-coumaryloxy)eudesm-4(14)-
ene (3): colorless liquid; [R]25 -15.5° (c 0.065, CHCl3); UV
D
(MeOH) λmax (log ꢀ) 218 (4.32), 273 (3.93) nm; IR νmax 2918,
2849, 1762, 1705, 1637, 1506, 1463, 1210, 1165, 756 cm-1; 1H
and 13C NMR, see Tables 1 and 2; HRFABMS m/z 607.4730
[M + H]+ (calcd for C40H63O4, 607. 4726).
(7) Cao, S.; Schilling, J. K.; Miller, J. S.; Andriantsiferana, R.; Rasamison,
V. E.; Kingston, D. G. I. J. Nat. Prod. 2004, 67, 454-456.
(8) Ginsburg, H.; Ward, S. A.; Bray, P. G. Parasitol. Today 1999, 15,
357-360.
(9) Steele, J. C.; Phelps, R. J.; Simmonds, M. S. J.; Warhurst, D. C.;
Meyer, D. J. J. Antimicrob. Chemother. 2002, 50, 25-31.
Alkaline Hydrolysis of 3. Hydrolysis of 3 (2.5 mg) as
reported above furnished 7 and palmitic acid ([M+•], m/z 256).

Eudesmane Derivatives from Melampodium
Journal of Natural Products, 2004, Vol. 67, No. 12 2057
(10) Initial antimalarial testing was carried out in Panama by Dr. E.
Ortega-Barria at the Smithsonian Tropical Research Institute (STRI)
and a member of the Panama ICBG program. This technology was
then transferred to CNARP in Madagascar through a visit of two
researchers from CNARP to STRI.
(15) Cardona, L.; Garcia, B.; Gimenez, J. E.; Pedro, J. R. Tetrahedron 1992,
48, 851-860.
(16) Jakupovic, J.; Ellmauerer, E.; Jia, Y.; Bohlmann, F.; Dominguez, X.
A.; Schmeda- Hirschmann, G. Planta Med. 1987, 53, 39-42. Com-
pound 8 was reported as 6â-(7′Z-coumaroyloxy)eudesm-4(15)-ene in
this reference, which used the older numbering scheme for eudesmane
sesquiterpenes. The recommended numbering scheme is given by:
Giles, P. M. Pure Appl. Chem. 1999, 71, 587-643.
(11) Giron, L, M.; Freire, V.; Alonzo, A.; Caceres, A. J. Ethnopharmacol.
1991, 34, 173-176.
(12) Huther, J.; Passreiter, C. M.; Wray, V.; Willuhn, G. Phytochemistry
1999, 51, 979-986.
(17) Corbett, Y.; Herrara, L.; Gonzalez, J.; Cubilla, L.; Capson, T. L.; Coley,
P. D.; Kursar, T. A.; Ortega-Barria, E. Am. J. Trop. Med. Hyg. 2004,
70, 119-124.
(13) Jacobs, H.; Bunbury, M.; Hylands, P. J.; McLean, S. J. Nat. Prod.
1986, 49, 1163-1164.
(14) Maia, J. G. S.; Zoghbi, M. das G. B.; da Silva, M. H. L.; Luz, A. I. R.
J. Essent. Oil Res. 1998, 10, 109-110.
NP049696Q