Antiproliferative acetogenins from a Uvaria sp. from the madagascar dry forest(1)

Article abstract of DOI:10.1021/np200697j

Investigation of the endemic Madagascan plant Uvaria sp. for antiproliferative activity against the A2780 ovarian cancer cell line led to the isolation of two new acetogenins. The structures of these two compounds were elucidated on the basis of analysis

Full text of DOI:10.1021/np200697j

Note
pubs.acs.org/jnp
Antiproliferative Acetogenins from a Uvaria sp. from the Madagascar
1
Dry Forest
†
†
†
‡
‡
Yumin Dai, Liva Harinantenaina, Peggy J. Brodie, Martin W. Callmander, Richard Randrianaivo,
§
§
§
⊥
Stephan Rakotonandrasana, Etienne Rakotobe, Vincent E. Rasamison, Yongchun Shen,
⊥
⊥
,†
Karen TenDyke, Edward M. Suh, and David G. I. Kingston*
†
Department of Chemistry, M/C 0212, Virginia Tech, Blacksburg, Virginia 24061, United States
Missouri Botanical Garden, B.P 3391, Antananarivo 101, Madagascar
‡
§
Centre National d’Application des Recherches Pharmaceutiques, B.P 702, Antananarivo 101, Madagascar
⊥Next Generation Systems, Eisai Inc., 4 Corporate Drive, Andover, Massachusetts 01810, United States
*
S Supporting Information
ABSTRACT: Investigation of the endemic Madagascan plant
Uvaria sp. for antiproliferative activity against the A2780
ovarian cancer cell line led to the isolation of two new
acetogenins. The structures of these two compounds were
elucidated on the basis of analysis of their 1D and 2D NMR
spectra, circular dichroism, and mass spectrometric data,
together with chemical modification. The two acetogenins
display weak antiproliferative activity against the A2780
ovarian cancer, the A2058 melanoma, and the H522 lung
cancer cell lines.
s part of our engagement in an International Cooperative
obtain the two new acetogenins uvaricin A (1) and uvaricin B
(2) with modest antiproliferative activity against A2780 ovarian
cancer cells, with IC50 values of 6.4 and 8.8 μM, respectively.
Herein, we report the structural elucidation and antiprolifer-
ative properties of the two isolates.
A
Biodiversity Group (ICBG) program, we are focusing on
searching for antiproliferative natural products from both
tropical dry forests and rain forests in Madagascar. As a part of
this research, an EtOH extract from the aerial parts of a Uvaria
sp. (Annonaceae) from the dry forest of northern Madagascar
exhibited weak antiproliferative activity against the A2780
Uvaricin A (1), [α]21 +23 (c 1.2, MeOH), was isolated as a
D
white, wax-like solid. The positive ion HRESIMS of uvaricin A+
human ovarian cancer cell line, with an IC value of 20 μg/mL.
5
0
revealed a quasi-molecular ion peak at m/z 623.4908 [M + H]
Compounds of the Annonaceae family are well known for their
broad range of bioactivity, including immunosuppression,
antimalarial, insecticidal, antifeedant, and antitumor activ-
+
and adduct ions at m/z 640.5147 [M + NH ] and 645.4726
4
+
[
M + Na] , corresponding to a molecular formula of C H O .
3
7
66
7
−1
A carbonyl absorption at 1747 cm in its IR spectrum, UV
absorption at 210 nm (MeOH), H NMR signals at δ 6.98
H-35), 4.98 (H-36), and 0.88 (H-34), and C NMR
2
−6
ities.
The genus Uvaria has been investigated extensively,
1
H
with over 300 references in the chemical and biological
13
(
literature, and the chemical constituents of Uvaria species have
resonances at δC 174.2 (C-1), 149.1 (C-35), 134.6 (C-2),
7.6 (C-36), and 19.4 (C-37) were indicative of a methylated
7
,8
been summarized. The genus Uvaria is also one of only seven
7
of the 120 genera of the Annonaceae family known to produce
12,13
9
α,β-unsaturated γ-lactone ring (Table 1).
This was
acetogenins, and uvaricin, the first example of these
confirmed by three-bond HMBC correlations observed
between H-35 and C-1 and between H-35 and C-37, as well
as two-bond HMBC correlations between H-35 and C-2 and
between H-36 and C-37 (Figure S1a). Three OH function-
compounds, was isolated from the roots of Uvaria accuminata
10
Oliv. by Jolad et al. in 1982.
Although the genus Uvaria has been well investigated, no
work has been done on this new species. A total of 17 Uvaria
species are currently known in Madagascar, and 11 remain to
−
1
alities in 1 were evident from the IR absorption at 3376 cm
and the 13C NMR resonances at δ
74.3 (C-21), 72.1 (C-8),
1
1
C
be described. This extract was thus selected for bioassay-
guided fractionation to isolate its active components.
and 71.6 (C-12). The presence of a bis-THF ring system with
13
two flanking OH groups was suggested by the four C NMR
The EtOH extract of the aerial parts of the Uvaria sp. was
subjected to liquid−liquid partitioning to give an active CH Cl2
2
Special Issue: Special Issue in Honor of Gordon M. Cragg
fraction with an IC50 value of 20 μg/mL. Bioassay-guided
separation, including LH-20 size-exclusion, silica gel normal-
phase, and C18 reverse-phase chromatography, was used to
Received: August 24, 2011
Published: December 2, 2011
©
2011 American Chemical Society and
American Society of Pharmacognosy
479
dx.doi.org/10.1021/np200697j | J. Nat. Prod. 2012, 75, 479−483

Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data for 1 and 2 in CDCl (500 MHz)
3
1
2
position
δH (J, Hz)
δC, type
174.2, C
δH (J, Hz)
δC, type
174.0, C
1
2
134.6, C
133.9, C
3
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
a
2.26 tdd (7.2, 1.6, 1.6)
2.26 tdd (7.2, 1.6, 1.6)
1.54 m
25.1, CH2
2.37 dtd (15.2, 7.6, 1.6)
2.51 dtd (15.2, 7.6, 1.6)
1.65 m
21.6, CH2
b
27.6, CH2
29.6−29.9, CH2
25.4, CH2
37.7, CH2
72.1, CH
35.4, CH2
70.9, CH
37.6, CH2
26.1, CH2
1.25 m
3.60 m
1.25 m
1.39 m
1.39−1.42 m
3.60 m
1.48 m
1.26 m
29.6−29.8, CH2
29.6−29.8, CH2
29.6−29.8, CH2
29.6−29.8, CH2
25.6, CH2
1.39−1.42 m
1.25 m
37.4, CH2
22.2, CH2
32.7, CH2
71.6, CH
1.26 m
0
1.26 m
1
1.39−1.42 m
3.87 m
1.26 m
2
1.48 m
3
3.83 m
83.0, CH
1.41 m
32.5, CH2
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
0a
0b
1a
1b
2
1.62 m
28.6, CH2
3.86 m
71.4, CH
1.87 m
1.62 m
29.1, CH2
82.5, CH
82.8, CH
29.1, CH2
28.6, CH2
83.5, CH
74.4, CH
3.83 m
82.9, CH
28.4, CH2
29.0, CH2
82.4, CH
82.6, CH
29.1, CH2
29.4, CH2
1.87 m
3.93 m
1.67 m
1.82 m
1.67 m
1.82 m
3.93 m
3.91 m
1.60 m
1.97 m
1.60 m
1.97 m
3.84 m
3.91 m
1.61 m
1.95 m
3.39 m
1.61 m
1.95 m
1.39−1.42 m
1.25 m
33.5, CH2
25.9, CH2
3.84 m
83.3, CH
74.2, CH
3
3.37 m
4
1.25 m
29.6−29.9, CH2
29.6−29.9, CH2
29.6−29.9, CH2
32.1, CH2
1.41 m
33.5, CH2
25.7, CH2
29.6−29.8, CH2
32.00, CH2
22.8, CH2
14.2, CH3
149.3, CH
77.7, CH
5
1.25 m
1.48 m
6−31
2
1.25 m
1.26 m
1.25 m
1.26 m
3
1.25 m
22.8, CH2
1.26 m
4
0.88 t (6.9)
6.98 td (1.5, 1.5)
4.98 qdd (6.8, 1.5, 1.5)
1.41, d (6.8)
14.3, CH3
0.86 t (6.9)
7.05 td (1.5, 1.5)
5.01 qd (6.8, 1.5)
1.40 d (6.9)
5
149.1, CH
6
77.6, CH
7
19.4, CH3
19.3, CH3
resonances at δC 83.5, 83.0, 82.8, and 82.5, which were
74.4 and 71.6 to the carbons adjacent to the THF rings at C-12
correlated to the proton signals at δ 3.79−3.93 (5H) in the
and C-21, respectively, with the proton signals at δ 3.39 and
H
H
14
HMQC spectrum, as described for similar structures. The
existence of the THF ring was confirmed by the HMBC
3
.87. A third OH group in the hydrocarbon chain was
suggested by the resonances at δ 3.60 and δ 72.1.
H
C
1
13
correlations between C-13 and H-16. The one-bond H− C
The locations of the THF rings and the third OH group on
the hydrocarbon chain were determined by analysis of 1D
NMR, HMBC, HMQC, and COSY spectroscopic data and
confirmed by analysis of the EIMS fragmentation shown in
correlations detected in the HMQC spectrum, along with the
1
13
observed mutiple-bond H− C correlations in the HMBC
spectrum, permitted the assignment of the carbon signals at δC
4
80
dx.doi.org/10.1021/np200697j | J. Nat. Prod. 2012, 75, 479−483

Journal of Natural Products
Note
Figure S2a. In the vicinity of the THF moiety, the chemical
HMBC correlations shown in Figure S1b. A third OH group
was also present in the hydrocarbon chain, as indicated by
NMR signals at δ 3.60 and δ 70.9.
1
3
shift at δ 22.2 in the C NMR spectrum was assigned to C-10
by the observation of an HMBC long-range correlation
between H-12 (δ 3.87) and C-10 (δ 22.2). The third OH
H
C
The locations of the THF rings and of the third OH group
on the hydrocarbon chain were determined by analysis of the
1D NMR, HMBC, HMQC, and COSY spectra and confirmed
by analysis of EIMS fragmentations. The two diastereotopic
protons (δ 2.37/2.51) were assigned to H -3 on the basis of
H
C
was assigned to C-8 by the presence of HMBC correlations
between H-9 and C-10, and H-8 and C-9, as well as the COSY
coupling of H-9 and H-8. The presence of an oxygen-bearing
methine at C-8 was confirmed by the HMBC correlations
between H-9 and C-7, H-8 and C-7, H-7 and C-6, H-4 and C-6,
H-4 and C-3, H-3 and C-5, and H-3 and C-35, as well as a
COSY cross-peak between H-4 and H-5. The number of
carbons between the THF rings and the lactone ring was
therefore restricted to 10.
H
2
HMBC correlations between H-3 and C-1, H-3 and C-2, and
H-35 and C-3. The presence of an HMBC long-range coupling
between a methylene carbon at δ 21.5 and H-3, as well as its
C
connected proton (δ 1.65) and C-2, confirmed the assignment
H
of this methylene carbon at C-4. The adjacent oxygen-bearing
carbon was located at C-5 by the presence of HMBC
correlations between H-4 and C-5, and H-3 and C-5. The C-
5 OH was confirmed by comparing the chemical shift of H-35
with those of other acetogenins of the same molecular formula
and with OH groups at C-4, C-5, C-6, C-12, or C-15. As
previously reported, the chemical shift of H-35 in C-5
hydroxylated acetogenins such as narumicin I and calamistrin
The planar structure was further supported by the analysis of
EIMS fragmentation. Intense fragment ions were observed at
m/z 399, 347, 295, and 207. As shown in Figure S2a, these
fragmentations are consistent with those observed for similar
15
acetogenins, and they fully support the assigned structure. In
particular, the unusual loss of H O accompanied by H has
2
2
16
been observed before.
2
4,25
The relative configurations around the bis-THF rings were
F
is always more shielded (δH 7.05) than with C-4
1
determined by analysis of the H NMR spectrum and
hydroxylated acetogenins, such as bullatacin (δ 7.16) and
H‑35
26
comparison with literature data. In Figure S3a, the chemical
squamotacin (δH‑35 7.17). Its chemical shift is however
deshielded compared with the corresponding protons of
acetogenins without an OH group near the lactone ring, such
shifts at δ 3.39 (H-21) and 3.84 (H-20) indicated the S* and
H
S* relative configurations, while the S* and R* relative
configurations were deduced for C-12 and C-13 and for C-16
and C-17, due to the similarity of the chemical shift of H-12
2
7
as the C-12 hydroxylated acetogenin, folianin B (δH‑35 6.98),
and the C-15 hydroxylated acetogenin, guanaconetin-1 (δ
H‑35
28
(
δ 3.87) and H-13 (δ 3.83) and that of H-16 (δ 3.93) and
6.98). Acetogenins with an OH at C-5 could be distinguished
from C-6 hydroxylated acetogenins by their divergent chemical
H
H
H
1
7,18
H-17 (δ 3.91).
The methylene protons H-18 and H-19
H
29
were assigned to signals at δ 1.60 and 1.97 on the basis of
shift at H-3, albeit sharing similar δH‑35 values.
H
COSY correlations between H-20 and H-21, H-19 and H-20,
and H-18 and H-17, combined with HMBC coupling between
H-19 and C-17. The difference between these chemical shifts
indicated a trans configuration for the C-17/C-20 THF ring.
In the same manner, a trans configuration was assigned to the
Analysis of the EIMS fragmentation supported the above
structure. The fragment ions at m/z 155 and 135 are consistent
with the conclusion that the third OH is connected to C-5,
while the fragment ions at m/z 437, 367, 355, and 267 and their
subsequent daughter ions indicated that the bis-THF rings and
the two flanking OH groups are located between C-14 and C-
23 (Figure S2b).
19
C-13/C-16 THF ring based on the divergent chemical shifts at
19
δH 1.62 and 1.87 for H-14 and H-15. Thus, the relative
configuration around the bis-THF rings was concluded to be
S*, R*, R*, S*, S*, S* for C-12, C-13, C-16, C-17, C-20, and C-
The relative configurations around the bis-THF rings were
determined by examination of the data from proton NMR
spectra and comparison to those in the literature. As shown in
2
1. The absolute configuration of C-36 was determined by CD
spectroscopic analysis. The negative n−π* Cotton effect (Δε =
1.13) at 238 nm and a positive π−π* Cotton effect (Δε =
.79) at 208 nm clearly indicated the S configuration at C-36 in
1
Figure S3b, the H NMR chemical shifts around the bis-THF
−
8
rings for compound 2 were similar to those of compound 1,
and the relative configurations were therefore assigned as S*,
R*, R*, S*, S*, S* for C-14, C-15, C-17, C-18, C-22, and C-23.
The CD spectrum of 2 revealed a negative n−π* Cotton effect
(Δε = −1.15) at 238 nm and a positive π−π* Cotton effect
(Δε = 9.64) at 208 nm, indicating an S configuration at the
16
the lactone ring. The absolute configurations of the OH
groups adjacent to the THF rings were determined by Mosher
ester methodology using the (R)-MPA and (S)-MPA
2
0
2
1
22
derivatives and the “in NMR tube” method. As shown in
16
Figure S4a, the Δδ (=δ − δ ) was negative on both chain
stereogenic center (C-36) in the lactone ring. The absolute
configurations of the three secondary alcohols were determined
by Mosher ester methodology in the same way as for 1. As
depicted in Figure S4b, the Δδ (=δ − δ ) is negative on the
H
S
R
sides and positive along the THF rings, indicating S
23
configurations at both C-12 and C-21. The configuration of
C-8 could not be determined due to the negative Δδ values
H
H
S
R
displayed by both H-7 and H-9 on the hydrocarbon chain.
chain side and positive toward the THF rings, indicating the S
2
1
23
Uvaricin B (2), [α] +18 (c 1.2, MeOH), was also isolated
configuration of both C-14 and C-23. The positive values for
D
as a white, wax-like solid. The positive ion HRESIMS of 2
H-3 and H-4 and negative values for H-6 to H-8 indicated an S
configuration for C-5.
+
revealed a quasi-molecular ion peak at m/z 623.4908 [M + H]
+
and adduct ions at m/z 640.5147 [M + NH ] and 645.4726
Uvaricin A (1) and B (2) were evaluated for antiproliferative
activity against the A2780 ovarian cancer, the A2058 melanoma,
and the H522 lung cancer cell lines (Table 2). Both
compounds showed weak inhibition of all three cell lines,
with IC values in the low micromolar range. Several hundred
4
+
[
M + Na] , corresponding to a molecular formula of C H O .
37
66
7
The NMR, IR, and UV data of compound 2 were very similar
to those of compound 1. As with compound 1, absorptions
typical of a methylated α,β-unsaturated γ-lactone ring in its IR
and UV spectra and of a bis-THF ring system with two flanking
50
acetogenins have been identified from these plants, but not all
of them have antiproliferative activity. According to previous
studies of the structure−activity relationships of acetogenins,
1
13
OH groups in the H and C NMR spectra were observed
Table 1). These structures were supported by the long-range
(
4
81
dx.doi.org/10.1021/np200697j | J. Nat. Prod. 2012, 75, 479−483

Journal of Natural Products
Note
had an IC50 >20 μg/mL. LH-20 size exclusion open column
Table 2. Antiproliferative Activities of Compounds 1 and 2
chromatography of the CH Cl2 fraction was used to obtain four
2
IC50 (μM)
fractions, of which the most active fraction (106 mg) had an IC50 of
6
.9 μg/mL. This fraction was applied to a silica gel column eluted with
compound
A2780
A2058
H522
a gradient of hexane−EtOAc, 9:1, 4:1, and 0:1, to give four fractions,
of which the 100% EtOAc fraction (71.0 mg) was the most active
1
6.4
6.6
7.2
<10
<10
ND
2
8.8
(
IC50 5.5 μg/mL). A C-18 open column was employed for further
paclitaxel
0.02
ND
separation, eluted by 90% MeOH to give eight fractions, and the most
active one (IC50 4.3 μg/mL) was separated by C-18 HPLC with the
the relative configuration of the THF rings and the positions of
the OH groups on the hydrocarbon chains have a significant
influence on their activities. The threo-cis-threo-cis-erythro
configuration was described as the most potent subgroup of
solvent system MeOH−H O, 85:15, and a flow rate of 2 mL/min.
2
This yielded two active compounds, with IC50 values of 4.0 and 5.5
μg/mL and with elution times of 28.3 and 35 min, respectively.
Uvaricin A (1): white, wax-like solid; [α]21 +23 (c 1.2, MeOH);
D
−1
30
UV (MeOH) λ (ε) 210 (2.60); IR νmax cm 3376, 2920, 1748,
max
acetogenins with bis-THF rings; as an example, rolliniastatin-
−
1
1
13
1
466, 1027 cm ; H NMR (500 MHz, CDCl ) and C NMR (125+
−
7
3
1
has an IC50 value of 0.7 × 10 μM against SW480 human
31
MHz, CDCl ), see Table 1; HRESIMS m/z 645.4726 [M + Na]
3
colon cancer cells. In addition, the presence of two OH
groups adjacent to the THF rings is necessary for potent
inhibitory effects. The moderate cytotoxicity of uvaricins A
and B can thus be explained by the stereochemistry of the bis-
THF rings flanked by the two OH groups, whose configuration
is different from that of rolliniastatin-1. The weak antiprolifer-
ative activity may also be due to the number and the location of
(
calcd for C H NaO , 645.4726).
Uvaricin B (2): white, wax-like solid; [α] +18 (c 1.2, MeOH);
37 66 7
21
D
32
−1
UV (MeOH) λ (ε) 210 (2.20); IR νmax cm 3449, 2923, 1753,
max
−
1
1
13
1460, 1024 cm ; H NMR (500 MHz, CDCl ) and C NMR (125+
3
MHz, CDCl
calcd for C H NaO , 645.4726).
), see Table 1; HRESIMS m/z 645.4726 [M + Na]
3
(
37 66
7
Preparation of the (R)- and (S)-MPA Ester Derivatives of 1
3
2,33
and 2. In a 25 mL round-bottom flask, 5.00 mg of (R)-(−)-α-
the OH groups.
methoxyphenylacetic acid ((R)-MPA) was dissolved in 5 mL of
CH Cl2 and 15 mg of oxalyl chloride. Several drops of
2
EXPERIMENTAL SECTION
dimethylformamide were added to the reaction system as a catalyst.
The reactants were mixed with a magnetic stir bar in an ice bath for 1 h
to obtain 4.98 mg of (R)-(−)-α-methoxyphenylacetyl chloride (91%
yield). The in-NMR-tube reaction was carried out to prepare the MPA
■
General Experimental Procedures. Optical rotations were
recorded on a JASCO P-2000 polarimeter. UV and IR spectroscopic
data were measured on a Shimadzu UV-1201 spectrophotometer and a
MIDAC M-series FTIR spectrophotometer, respectively. CD spectra
were obtained on a JASCO J-815 circular dichroism spectrometer.
NMR spectra were recorded in CD OD or CDCl on either JEOL
2
2
ester derivatives. The selected acetogenin (0.2 mg) dissolved in
CDCl was transferred to a clean NMR tube, and the solvent was
3
completely evaporated. (R)-(−)-α-Methoxyphenylacetyl chloride (0.5
3
3
mg) was dissolved in CDCl (0.5 mL) and added to the NMR tube
3
Eclipse 500 or Bruker Avance 600 spectrometers. The chemical shifts
are given in δ (ppm), and coupling constants (J) are reported in Hz.
Mass spectra were obtained on an Agilent 6220 LC-TOF-MS in the
immediately under an N gas flow, and the NMR tube was shaken
2
carefully to mix the acetogenin and MPA chloride. The reaction NMR
+
tube was kept at room temperature under N for 6 h to form the (R)-
2
positive ion mode; NH4 adducts are commonly observed with this
1
MPA ester and was then analyzed by H NMR spectroscopy. The (S)-
MPA ester of the acetogenin was prepared from (S)-MPA by the same
method.
instrument. HPLC was performed on a Shimadzu LC-10AT
instrument with a semipreparative C18 Varian Dynamax column (5
μm, 250 × 10 mm).
Plant Material. The above-ground parts of Uvaria sp. were
collected in July 2005. The plant was a liana with immature yellowish-
green fruit and probably represents a new species still to be
described. The collection was made in dry degraded evergreen
broadleaf forest near Betsimiranjana, in the Ambilobe region of
Madagascar, at coordinates 13°02′35″ S, 049°09′15″ E. Voucher
specimens have been deposited at the Parc Botanique and Zoologique
de Tsimbazaza (TAN), the Centre National d’Application des
Recherches Pharmaceutiques in Antananarivo, Madagascar
ASSOCIATED CONTENT
■
*
S
Supporting Information
34
Figures S1−S4 and H and 13C NMR spectra of 1 and 2 are
1
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Tel: +1-540-231-6570. Fax: +1-540-231-3255. E-mail:
dkingston@vt.edu.
■
*
(
(
(
CNARP), the Missouri Botanical Garden in St. Louis, Missouri
MO), and the Museum National d’Histoire Naturelle in Paris, France
P), voucher Stephan Rakotonandrasana et al. 923.
Antiproliferative Bioassays. Antiproliferative activities were
́
́
ACKNOWLEDGMENTS
■
obtained at Virginia Polytechnic Institute and State University against
the drug-sensitive A2780 human ovarian cancer cell line as previously
This project was supported by the Fogarty International
Center, the National Cancer Institute, the National Science
Foundation, the National Heart, Lung and Blood Institute, the
National Institute of Mental Health, the Office of Dietary
Supplements, and the Office of the Director of NIH, under
Cooperative Agreement U01 TW000313 with the International
Cooperative Biodiversity Groups. This project was also
supported by the National Research Initiative of the
Cooperative State Research, Education and Extension Service,
USDA, Grant #2008-35621-04732. These supports are grate-
fully acknowledged. This work was also supported by the
National Science Foundation under Grant CHE-0619382 for
purchase of the Bruker Avance 600 NMR spectrometer and
Grant CHE-0722638 for the purchase of the Agilent 6220 mass
spectrometer. We thank Mr. B. Bebout for obtaining the mass
35
described. Antiproliferative activities against the A2058 melanoma
and the H522 lung cancer cell lines were determined at Eisai Inc. by
36
similar procedures to those used for the H460 cell line.
Extraction and Isolation. Dried aerial parts of Uvaria sp. (250 g)
were ground in a hammer mill, then extracted with EtOH by
percolation for 24 h at room temperature to give the crude extract MG
3
352 (24.9 g), of which 6.9 g was shipped to Virginia Tech for
bioassay-guided isolation against the A2780 cell line. A 1.0 g sample of
MG 3352 (IC50 20.0 μg/mL) was suspended in aqueous MeOH
(
MeOH−H O, 9:1, 100 mL) and extracted with hexane (3 × 100 mL
2
portions). The aqueous layer was then diluted to 60% MeOH (v/v)
with H O and extracted with CH Cl (3 × 150 mL portions). The
2
2
2
hexane fraction was evaporated in vacuo to leave 567 mg of material
with IC50 >20 μg/mL. The residue from the CH Cl fraction (200 mg)
2
2
had an IC50 of 20 μg/mL, and the remaining aqueous MeOH fraction
4
82
dx.doi.org/10.1021/np200697j | J. Nat. Prod. 2012, 75, 479−483

Journal of Natural Products
Note
spectra and Dr. H. Azurmendi for assistance with the NMR
spectra. We also thank Dr. T. Deroin, a specialist on the
Annonaceae family, for confirming the determination of the
collection. Field work essential for this project was conducted
under a collaborative agreement between the Missouri
Botanical Garden and the Parc Botanique et Zoologique de
Tsimbazaza and a multilateral agreement between the ICBG
partners, including the Centre National d’Applications des
Recherches Pharmaceutiques. We gratefully acknowledge
courtesies extended by the Government of Madagascar
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DEDICATION
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Tormo, J. R.; De Pedro, N.; Estornell, E.; Zafra-Polo, M. C.; Pelae
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