Antiproliferative and antiplasmodial dimeric phloroglucinols from Mallotus oppositifolius from the Madagascar dry forest(1)

Article abstract of DOI:10.1021/np300750q

Bioassay-guided fractionation of an ethanol extract of the leaves and inflorescence of Mallotus oppositifolius collected in Madagascar led to the isolation of the two new bioactive dimeric phloroglucinols mallotojaponins B (1) and C (2), together with the

Full text of DOI:10.1021/np300750q

Article
pubs.acs.org/jnp
Antiproliferative and Antiplasmodial Dimeric Phloroglucinols from
Mallotus oppositifolius from the Madagascar Dry Forest1
Liva Harinantenaina,† Jessica D. Bowman,‡ Peggy J. Brodie,† Carla Slebodnick,† Martin W. Callmander,§
Etienne Rakotobe,⊥ Richard Randrianaivo,§ Vincent E. Rasamison,⊥ Alexander Gorka,∥ Paul D. Roepe,∥
Maria B. Cassera,‡ and David G. I. Kingston*,†
†Department of Chemistry and Virginia Tech Center for Drug Discovery, M/C 0212, Virginia Tech, Blacksburg, Virginia 24061,
United States
‡Department of Biochemistry and Virginia Tech Center for Drug Discovery, M/C 0308, 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
∥Department of Chemistry and Lombardi Cancer Center, Georgetown University, 37th and O Streets NW, Washington, D.C. 20057,
United States
S
* Supporting Information
ABSTRACT: Bioassay-guided fractionation of an ethanol
extract of the leaves and inflorescence of Mallotus oppositifolius
collected in Madagascar led to the isolation of the two new
bioactive dimeric phloroglucinols mallotojaponins B (1) and C
(2), together with the known mallotophenone (3). The
structures of the new compounds were determined on the
basis of spectroscopic evidence, including their 1D- and 2D-
NMR spectra, mass spectrometry, and an X-ray crystal
structure. Compounds 1 and 2 showed potent antimalarial
activity against chloroquine-resistant Plasmodium falciparum,
with IC50 values of 0.75 0.30 and 0.14 0.04 μM, while 3
was inactive in this assay. Compounds 1−3 also displayed strong antiproliferative activity against the A2780 human ovarian
cancer cell line (IC50 1.10 0.05, 1.3 0.1 and 6.3 0.4 μM, respectively).
have been reported to have antifungal activity,11 but no
he tropical genus Mallotus, a member of the family
TEuphorbiaceae, contains about 150 species of trees and
previous work on its constituents has been reported.
shrubs.2 It shares membership in the tribe Acalypheae with the
genus Macaranga,2 a genus that has afforded several promising
RESULTS AND DISCUSSION
■
bioactive compounds.3,4 Mallotus philippinensis is the source of
Isolation of Bioactive Constituents. Initial dereplication
rottlerin, a natural product that first appears to have been
studies using size-exclusion chromatography and HPLC on a
isolated in 18555 and has been the subject of numerous
small amount of an active hexanes-soluble fraction obtained
biological investigations.6−8
from the liquid−liquid partition of the active extract indicated
Our ongoing screening of extracts from plants collected in
the presence of unknown antiproliferative phloroglucinols.
Madagascar as part of the Madagascar International Cooper-
Scale-up of the isolation to 1 g of extract yielded an
ative Biodiversity Group (ICBG) program for antiproliferative
antiproliferative hexanes fraction (IC50 6.7 μg/mL), which
activity toward the A2780 ovarian cancer cell line9 has recently
was subjected to further size-exclusion column chromatography
been supplemented with screening for antiplasmodial activity
(Sephadex LH-20) to furnish two active fractions, with IC50
against the malaria parasite Plasmodium falciparum. An ethanol
values of 1.6 and 2.3 μg/mL, and the known phloroglucinol
extract of Mallotus oppositifolius (Geiseler) Mull. Arg.
̈
mallotophenone (3, IC50 6.3
0.4 μM). The most active
(Euphorbiaceae) was found to display strong activity against
P. falciparum as well as antiproliferative activity against the
A2780 cell line, and this extract was thus selected for further
investigation to isolate the active metabolite(s) responsible for
the observed activities. M. oppositifolius has been used as a
chewing stick in Nigeria,10 and its aqueous and ethanol extracts
fractions were subjected to HPLC and silica gel column
Special Issue: Special Issue in Honor of Lester A. Mitscher
Received: October 30, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 2. 13C NMR Data for Compounds 1−3 (125 MHz,
CDCl3)
chromatography to yield the two new bioactive phloroglucinols
1 and 2.
Structure Elucidation. Mallotophenone (3) was identified
by single-crystal X-ray analysis and by comparison of its
spectroscopic data with values reported in the literature.12
Compound 1 was obtained as yellowish crystals and gave the
molecular formula C25H30O8, as indicated by high-resolution
ESIMS analysis, which gave a protonated molecular ion peak at
m/z 459.2023 [M+H]+. Its IR spectrum showed an absorption
characteristic of a conjugated hydrogen-bonded carbonyl group
(1620 cm−1). The UV spectrum of 1 was very similar to that of
3, suggesting that the two compounds share the same
carbon
1
2
3
1
108.3
108.5
108.4
1a
2
18.1
157.2
109.2
162.9
110.0
159.8
205.4
33.8
17.9
157.5
114.2
162.8
109.2
159.6
205.4
33.8
18.1
157.1
109.1
163.0
110.2
159.9
205.6
34.1
3
4
5
6
7
1
8
chromophore. The H NMR spectroscopic data of 1 (Table
1) displayed resonances due to an aromatic methyl (δ 2.13, s,
9
8.9
22.9
9.2
1′
2′
3′
4′
5′
6′
7′
8′
9′
OCH3
1″
2″
3″
4″
1″′
2″′
3″′
4″′
108.5
157.7
114.2
162.9
109.2
159.6
205.4
33.8
108.5
157.5
114.2
162.8
109.2
159.6
205.4
33.8
108.4
157.1
109.1
163.0
110.2
159.9
205.6
34.1
1
Table 1. H NMR Data of Compounds 1−3 (500 MHz,
CDCl3)
position
1
2
3
1a
3.68 s
2.71 s
2.13 s
2.71 s
3.68 s
2.70 s
3.66 s
2.70 s
2.11 s
2.70 s
2.11 s
8
9
3.31 d (6.3)
2.70 s
22.9
22.9
9.2
8′
9′
OCH3
62.1 63.0
122.7
132.2
18.0
63.0 63.0
122.7
132.2
17.9
62.2 62.2
3.31 d (6.5)
3.98 s, 3.98 s
5.21 (tq, 6.5, 1.4)
1.68 s
3.31 d (6.3)
3.98 s, 3.98 s
5.21 (brt, 6.0)
1.68 s
3.97 s, 3.97 s
1″
3″
25.8
25.8
4″
1.77 s
1.77 s
122.7
132.2
17.9
1″′
3″′
4″′
OH
5.21 (brt, 6.0)
1.68 s
1.77 s
25.8
8.97 s, 13.64 s
9.05 s, 13.48 s
8.99, 13.66
3H), a 3,3-dimethylallyl group (δ 1.68, s, 3H and 1.77, s, 3H; δ
5.21, tq, J = 6.5, 1.4 Hz, 1H, and δ 3.31, d, J = 6.5 Hz, 2H), two
methoxy groups (δ 3.98, s, 6H), two acyl methyl groups (δ
2.71, s, 6H), and one methylene group at δ 3.68 (s, 2H),
together with signals for two hydroxy groups, one of which was
hydrogen bonded (δ 8.97, s, 1H and 13.64, s, 1H). The 13C
NMR data of 1 (Table 2) exhibited 25 carbon signals that were
identical with those of 3 except for the replacement of the
signal of an aromatic methyl carbon with signals for the carbons
of a 3,3-dimethylallyl unit (δ 18.0, 22.9, 25.8, 122.7, 132.2). On
comparison of the 13C NMR data of 1 with those of 3, the
deshielding of the signal for C-3′ (δ 114.2 instead of 109.1 in 3)
suggested that the 3,3-dimethylallyl group is attached at this
position, which is methylated in 3.13 The locations of the
methyl, methoxy, methylene, carboxyl, hydroxy, and the 3,3-
dimethylallyl groups were confirmed by interpretation of the
1D- and 2D-NMR spectroscopic data of 1, including COSY,
HSQC, HMBC, and nuclear Overhauser effect spectroscopy
experiments. The attachment of the 3,3-dimethylallyl group at
C-3′ was confirmed by the observation of the HMBC long-
range correlation between the two geminal methyls at δ 1.68
and 1.77 to C-1″ (δ 122.7) and from the methine proton at δ
5.21 to C-3′ (δ114.2). The methoxy groups were assigned to C-
2 and C-2′ due to the long-range correlations (Figure 1)
observed between the signals at δ 3.98 and those at δ 157.2 (C-
2) and 157.7 (C-2′), between the C-1a methylene proton signal
(δ 3.68) and C-2 and C-2′, and those observed between the
aromatic methyl protons at δ 2.13 and C-2 and between the
methylene protons signals at δ 3.31 and C-2′. In the same
manner, the acyl group was assigned to C-5 and C-5′ from the
HMBC cross-peaks between the methyl protons at δ 2.71
(CH3-8 and 8′) and C-5 and C-5′. The two hydroxy groups
must be located at C-4, C-4′ and C-6, C-6′, as indicated by the
presence of two hydrogen-bonded hydroxy protons and the
HMBC long-range correlations between the hydroxy group at δ
8.97 and C-3, C-3′, C-5, and C-5′. Moreover, NOESY
correlations were observed between the methoxy protons and
H-1″, H-1a, and CH3-9.
The structure of 1 was confirmed by single-crystal X-ray
diffraction (Figure 2). Compound 1 was thus assigned as 3′-
(3,3-dimethylallyl)-1′-(5-acetyl-6-hydroxy-3-methyl-2-methoxy-
benzyl)-2′-methoxyphloracetophenone and has been named
mallotojaponin B based on its relationship to mallotojaponin,
seen here as mallotojaponin A.14
Compound 2, named mallotojaponin C, gave the molecular
formula C29H36O8, as determined by positive ion HRESIMS
(m/z 513.2499, [M + H]+, required for C29H37O8, 513.2488).
Similarly to 1, the IR and UV spectra of 2 were indicative of the
presence of a prenylated phloroglucinol. The 1H and 13C NMR
spectroscopic data of 2 were superposable upon those of 1
(Tables 1 and 2), except for the replacement of the signal due
to the aromatic methyl group with the signals for a second 3,3-
dimethylallyl group. Thus, the signal due to the methine of the
3,3-dimethylallyl group (δ 5.21, brt, J = 6.0 Hz) integrated for
two protons (H-1″ and H-1″′), while the signal at δ 3.68 (s, H2-
1a) also integrated for two protons. In addition, the broad
triplet observed for the 1H NMR signal of the methine protons
at δ 5.21 suggested the presence of two overlapping signals (H-
1″ and H-1″′). These data coupled with the high-resolution
mass spectra allowed the conclusion to be made that
B
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Figure 3. Mass fragmentation observed for 2.
The locations of the hydroxy groups at C-4 (C-4′) and C-6 (C-
6′), the methoxyl groups at C-2 (C-2′), the acetyl group at C-5
(C-5′), and the methylene at C-1 (C-1′) were elucidated in the
same manner as for 1. These data led to the assignment of the
structure of 2 as 1-methylene-bis-4-methoxy-6-hydroxy-3-(3,3-
dimethylallyl)-2-methoxyacetophenone.
Biological Activities. Compounds 1−3 were evaluated for
their activity against P. falciparum Dd2 (a chloroquine/
mefloquine-resistant strain). Compounds 1 and 2 showed
submicromolar activity with half-maximum inhibitory concen-
tration (IC50) values of 0.75
0.30 and 0.14
0.04 μM,
respectively, while compound 3 was not active. In addition to
their cytostatic activity in inhibiting the growth of P. falciparum,
compounds 1 and 2 also showed cytocidal activity vs P.
falciparum. Using a newly developed rapid assay for
determination of cytocidal activity,15 compound 1 was found
to have median lethal dose (LD50) values of 14.6 0.7 and 6.7
0.2 μM vs the drug-sensitive HB3 strain and the drug-
resistant Dd2 strain, respectively, while compound 2 had LD50
values of 0.81 0.05 and 0.80 0.02 μM vs the same two
strains. In these same assays, chloroquine exhibited LD50 values
of 0.10 0.01 and 15.3 0.9 μM (HB3 vs Dd2), so for the
drug-resistant Dd2 strain, compound 2, in particular, is
significantly more cytocidally potent than chloroquine.
Figure 1. Important HMBC correlations observed in 1.
Compounds 1 and 2 were further evaluated for their
gametocytocidal activity against late-stage gametocytes (the
stage responsible for malaria transmission) using the chlor-
oquine-sensitive NF54 strain to generate gametocytes. This
strain was used because it forms gametocytes in culture much
better than the chloroquine-sensitive HB3 strain. Only
compound 2 showed gametocytocidal activity, with an IC50
value of 3.6
0.2 μM. This activity is comparable to the
current antimalarial drug artesunate (IC50 value of 2.3 μM) and
to NPC1161B, an antimalarial drug currently in the develop-
ment pipeline (IC50 value of 3.8 μM).16,17 The IC50 value
determined for compound 2 against asexual stages in the NF54
strain was 0.07 0.01 μM.
In order to address if compounds 1 and 2 were able also to
prevent gametocytogenesis, P. falciparum in vitro cultures were
treated with 0.76 μM (∼IC50) or 44 μM (∼IC100) of compound
1 and 0.14 μM (∼IC50) or 39 μM (∼IC100) of compound 2 for
13 days, as described in the Experimental Section. On day 13,
cultures were recovered and smeared for microscopic
examination. Neither asexual intraerythrocytic stages nor
gametocytes were observed in cultures treated with 0.14 μM
of compound 2 (Figure 4). Compound 1 cleared asexual
intraerythrocytic stages in vitro at 0.76 μM, and the presence of
mature gametocytes was reduced 80% as compared to
untreated parasites (control). This is the first report on
mallotojaponin derivatives showing antimalarial activity.
Figure 2. Anisotropic displacement ellipsoid drawing (50%) of 1.
compound 2 was a symmetrical dimer with two 3,3-
dimethylallyl units, one each at C-3 and C-3′. This was also
confirmed by the observation of the base peak at m/z 263 in its
mass spectrum (Figure 3). Comparison of the 13C NMR
spectroscopic data of 2 with those of 1 demonstrated that the
methyl group at C-3 of 1 is replaced by a 3,3-dimethylallyl unit
in 2. Also, HMBC long-range correlations were observed from
H-1″ and H-1″′ to C-3 and C-3′, respectively, and from H-9
and H-9′ to C-2 and C-4 and to C-2′ and C-4′, respectively.
C
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phloroglucinols in general and of compound 2 in particular.
This conclusion is also supported by the recent report of the
modest antiplasmodial activity of watsonianone A (5), a related
compound lacking phenolic hydroxyl groups isolated from the
Australian tree Corymbia watsoniana.24
Bioactive phloroglucinols have been detected in the
Aspidiaceae, Cannabinaceae, Clusiaceae, Compositae, Crassu-
laceae, Euphorbiaceae, Fagaceae, Guttiferae, Lauraceae, Myrta-
ceae, Rosaceae, and Rutaceae families.18 From the present
study, it may be concluded that Mallotus japonicus and M.
oppositifolius are two Euphorbiaceous species that produce
dimeric phloroglucinols such as mallotophenone (3) and
related compounds.12,25,26 It is also noteworthy that an extract
of M. japonicus inhibited production of pro-inflammatory
cytokines during macrophage activation, and mallotojaponin
was one of the bioactive phloroglucinol derivatives isolated
from this extract that showed this effect.27 Mallotojaponin was
also shown to inhibit HIV-reverse transcriptase activity.28
Figure 4. P. falciparum blood stage distribution after 13 days of
treatment at the indicated concentrations to assess antimalarial activity
against early-stage gametocytes. The control is untreated parasites.
DMSO (drug vehicle at 0.5%) was included as a second control since
it affects the blood-stage distribution but does not affect the total
parasitemia. No parasite growth was observed at doses of 2 of 0.14 and
39 μM and of 1 of 44 μM. Results are presented as means of two
independent experiments SEM, (*) p ≤ 0.02 and (⊥) p ≤ 0.005
compared with the control group.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded on a JASCO P-2000 polarimeter. IR and UV spectra were
measured on MIDAC M-series FTIR and Shimadzu UV-1201
spectrophotometers, respectively. 1H and 13C NMR spectra were
recorded on a JEOL Eclipse 500 spectrometer in CDCl3 with TMS as
internal standard. Mass spectra were obtained on a JEOL JMS-HX-110
and an Agilent 6220 LC-TOF-MS. Preparative HPLC was performed
using Shimadzu LC-10AT pumps coupled with a semipreparative
Varian Dynamax C18 column (5 μm, 250 × 10 mm), a Shimadzu SPD
M10A diode array detector, and a SCL-10A system controller.
Plant Material. Leaves and inflorescences of Mallotus oppositifolius
Compounds 1 and 2 also displayed strong antiproliferative
activity against the A2780 human ovarian cancer cell line with
IC50 values of 1.10 0.05 and 1.3 0.1 μM, respectively. Since
(Geiseler) Mull. Arg. (collection: Richard Randrianaivo et al. 1425)
̈
were collected at an elevation of 137 m in December 2006 near the
village of Befarafara in the dry forest of Solanampilana, 35 km north of
Daraina, Antsiranana, Sava region, 13°05′42″ S 049°34′57″ E,
northern Madagascar. The sample collected was from a shrub 3 m
tall, with white flowers. The plant was determined by Dr. Gordon
McPherson (Missouri Botanical Garden). Duplicate voucher speci-
mens were deposited at the Centre National d’Application des
Recherches Pharmaceutiques, the Herbarium of the Parc Botanique et
Zoologique de Tsimbazaza, Antananarivo, Madagascar, the Missouri
Botanical Garden, St. Louis, Missouri, and the Museum National
d′Histoire Naturelle in Paris, France.
mallotophenone (3) showed weaker activity (IC50 6.3
0.4
μM), it appears that the presence of the 3,3-dimethylallyl group
in the molecule enhances the activity; however, this effect was
more pronounced in the malaria parasite. Phloroglucinols have
been reported to have a wide range of biological activ-
ities.14,18−22 Previous investigation showed that 3′-(3,3-
dimethylallyl)-1′-(5-acetyl-6-hydroxy-3-methyl-2-methoxyben-
zyl)-2′-hydroxyphloracetophenone, a 2′-hydroxylated derivative
of mallotojaponin B (1), displayed cytotoxic activities against
KB and mouse leukemia L-5178Y (ED50 0.58 and 0.74 μg/mL,
respectively).12
The discovery of the potent antimalarial and gametocytocidal
activities of compound 2 raised the question of its possible
mechanism of action. It has been proposed that acylphlor-
oglucinols can function as antimalarial agents by acting as
radical-generating species or by inhibiting hemozoin forma-
tion.23 The latter may occur through binding to precrystalline
forms of heme via π−π interactions (given their electron-rich
structures) and/or coordination with the Fe3+ center of
hematin (such as an Fe−O interaction with the phenolic
moiety of 2).23 As a test of the importance of the phenolic
hydroxy groups of 2, the compound was converted to its
tetramethyl ether 4 by treatment with potassium carbonate and
excess methyl iodide. Compound 4 was active but was 15 times
less potent than its parent compound 2 against P. falciparum,
with IC50 values of 2.2 0.5 and 2.5 0.5 μM to the drug-
sensitive NF54 cell line and the drug-resistant Dd2 cell line,
respectively. It thus appears that the phenolic hydroxy groups
of 2 are important for its antimalarial activity, supporting the
idea that coordination to Fe3+ and/or radical generation plays
an important role in the antimalarial activity of the
Antiproliferative Bioassay. The A2780 ovarian cancer cell line
assay was performed at Virginia Tech as previously reported.29 The
A2780 cell line is a drug-sensitive ovarian cancer cell line.30
Intraerythrocytic Stage Antimalarial Bioassay. The effect of
each fraction and pure compound on parasite growth of the Dd2 strain
was measured in a 72 h growth assay in the presence of drug as
described previously with minor modifications.31,32 Briefly, ring stage
parasite cultures (200 μL per well, with 1% hematocrit and 1%
parasitemia) were then grown for 72 h in the presence of increasing
concentrations of the drug in a 5.05% CO2, 4.93% O2, and 90.2% N2
gas mixture at 37 °C. After 72 h in culture, parasite viability was
determined by DNA quantitation using SYBR Green I (50 μL of SYBR
Green I in lysis buffer at 0.4 μL of SYBR Green I/mL of lysis buffer).32
The half-maximum inhibitory concentration (IC50) calculation was
performed with GraFit software using a nonlinear regression curve
fitting. IC50 values are the average of three independent determinations
with each determination in duplicate and are expressed SEM.
Intraerythrocytic Stage Cytocidal Antimalarial Bioassay. The
effectiveness of compounds 1 and 2 at killing intraerythrocytic stages
of the chloroquine-sensitive HB3 and chloroquine-resistant Dd2
strains of P. falciparum was assessed as previously reported.15 LD50
values are the average of three replicate determinations and are
expressed SEM.
D
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Late Gametocyte Stage Antimalarial Bioassay. To test
compounds for their effectiveness in killing late-stage (stage V)
gametocytes, late-stage gametocytes were generated using a combina-
tion of established methods.16,33 Initial gametocyte cultures were
developed from the P. falciparum NF54 strain (chloroquine-sensitive
strain). All cultures were maintained in 75 cm2 culture flasks in a
reduced oxygen environment (5% O2, 5% CO2, 95% N2) at 37 °C.
Parasitaemia was calculated by counting the percentage of infected
RBCs by Giemsa staining of thin smears and light microscopy. Asexual
stages were synchronized by sorbitol treatment at least two days before
setting gametocyte cultures.34 Thin blood smears were made and
stained with Giemsa to check parasite development on days 4, 8, 12,
and 13 after the initial subculture. On day nine of the gametocyte
cultures, parasites were treated with 5% sorbitol for 10 min at 37 °C to
start removing asexual stages. Sorbitol treatment was performed for
four consecutive days, which effectively removes >99% of asexual
parasites. Gametocyte recovery and concentration was achieved on day
13 using a NycoPrep 1.077 cushion,16 and the number of gametocytes
was calculated using a Neubauer chamber. About 30 000 to 50 000
gametocytes per well were added to the black flat-bottom half-area 96-
well plates containing drug candidates in a 100 μL final volume. The
plate was incubated in a humidified chamber at 37 °C and low oxygen
conditions (5% O2, 5% CO2, 95% N2) for 72 h. Alamar Blue was
added on day 16 postinduction at 10% of the well volume.33 The plate
was returned to the chamber for an additional 24 h and then was read
in a microplate reader at 585 nm after excitation at 540 nm. IC50 values
were calculated using a dose−response curve fitting with GraFit. IC50
values are the average of two independent determinations, each
determination in duplicate and are expressed SEM.
Early Gametocyte Stage Antimalarial Bioassay. To test
efficacy in preventing gametocytogenesis, 24-well plates were set at
0.75% parasitemia (NF54 strain) and 1% hematocrit and cultured for
13 days with or without the presence of compounds 1 and 2. The plate
was incubated in a humidified chamber at 37 °C and low oxygen
conditions for the duration of the experiment. Medium or medium
supplemented with drug was replaced on days 4, 6, 8, and 9−12. On
day 13, each well was recovered and the parasitaemia (both asexual
and sexual) was calculated from Giemsa-stained smears.
Extraction and Isolation. A ground sample of M. oppositifolius
leaves and inflorescences (137 g) was extracted with ethanol at room
temperature to yield 6.0 g of crude ethanol extract, designated MG
4129. A total of 1.8 g of this extract was made available to Virginia
Polytechnic Institute and State University. In order to locate the
biological activity and to have an idea about the types of metabolites
responsible for the activity of the active fraction, 100 mg of the crude
ethanol extract of M. oppositifolius was subjected to a liquid−liquid
partition using hexanes, EtOAc, and H2O to afford 42.5 mg of an
active hexanes fraction (IC50 6.7 μg/mL). Size-exclusion chromatog-
raphy on Sephadex LH-20 of the hexanes fraction eluted with
Mallotojaponin B (1): colorless prisms (EtOAc−hexanes); mp 175
1 °C; UV (MeOH) λmax (log ε) 283 (4.06) nm; IR (film) νmax 3450,
1
3223, 1620, 1596, 1405, 1283, 1128 cm−1; H NMR and 13C NMR
data, see Tables 1 and 2; positive HRESIMS m/z 459.2023 [M + H]+
(calcd for C25H31O8, 459.2019).
X-ray Crystallography of 1. A colorless prism of 1 was centered
on the goniometer of an Oxford Diffraction SuperNova A
diffractometer operating with Cu Kα radiation. The data collection
routine, unit cell refinement, and data processing were carried out with
the program CrysAlisPro.35 The Laue symmetry and systematic
absences were consistent with the monoclinic space group P21/c. The
structure was solved using SHELXS-9736 and refined using SHELXL-
9736 via OLEX2.37 The final refinement model involved anisotropic
displacement parameters for non-hydrogen atoms. A riding model was
used for the aromatic and alkyl hydrogens. The −OH hydrogen
positions were located from the residual electron density map and
refined independently.
Crystal data: colorless prism; C25H30O8, Mr = 458.49, monoclinic,
P21/c, a = 12.88614(12) Å, b = 12.34409(7) Å, c = 14.84644(13) Å, β
= 110.4761(10)°, V = 2212.38(3) Å3, 37 837 reflections, 317
parameters; crystal size 0.4149 × 0.1458 × 0.1287 mm3. The final
indices were R1 = 0.0367, wR2 = 0.0973 [I > 2σ(I)]. Crystallographic
data for compound 1 have been deposited as Supporting Information
at the Cambridge Crystallographic Data Centre (deposition no.
CCDC 874720).38
Mallotojaponin C (2): amorphous powder; UV (MeOH) λmax (log
ε) 283 (4.12) nm; IR (film) νmax 3440, 3220, 1620, 1595, 1434, 1405,
1
1280, 1121 cm−1; H NMR and 13C NMR data, see Tables 1 and 2;
positive HRESIMS m/z 513.2499 [M + H]+ (calcd for C29H37O8,
513.2488).
Mallotophenone (3). The structure of 3 was identified by single-
crystal X-ray analysis and by comparison of its spectroscopic data with
values reported in the literature.12 Crystallographic data for compound
3 has been deposited as Supporting Information at the Cambridge
Crystallographic Data Centre (deposition no. CCDC 874719).38
Methylation of Mallotojaponin B (2). Compound 2 (0.9 mg) was
dissolved in acetone (1.5 mL) and treated with K2CO3 (120 mg) and
methyl iodide (100 μL). The mixture was stirred at room temperature
for 17 h. The reaction mixture was evaporated, dissolved in water, and
extracted with EtOAc to give compound 4 (1 mg). The structure of 4
was confirmed by interpretation of its 1H NMR spectrum and by
HRESIMS (see Supporting Information).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 1 and 2, H NMR
1
spectrum of 4, and an ORTEP drawing of 3. This information
is available free of charge via the Internet at http://pubs.acs.org.
MeOH−CH2Cl2 gave mallotophenone (3, IC50 6.3
0.4 μM) and
two active fractions (Fr. 3, 13 mg, IC50 2.3 μg/mL, and Fr. 4, 8.4 mg,
IC50 1.6 μg/mL). High-performance liquid chromatography (HPLC)
on a C18 column with a solvent gradient from water−MeOH (system
I) 40:60 to 30:70 for 10 min, to 20:80 from 10 to 15 min, to 15:85
from 15 to 20 min, maintained at 15:85 for 5 min, to 10:90 from 25 to
30 min, and to 0:100 from 30 to 35 min, ending with 100% MeOH for
50 min of fractions 3 and 4 showed the presence of two major and
active phloroglucinols (tR 39.79 min; IC50 0.5 μg/mL and tR 44.15
min; IC50 0.61 μg/mL). To isolate more material for structure
elucidation and for bioctivity evaluations, the isolation was scaled up
by starting with 1 g of ethanol extract. Liquid−liquid partion (hexanes,
3 × 200 mL) followed by Sephadex LH-20 of the hexanes fraction
(407 mg) afforded two active fractions (Fr. 3, 125.2 mg, IC50 2.4 μg/
mL and Fr. 4, 137.1 mg, IC50 2.2 μg/mL). Mallotophenone (3) was
obtained from fractions 3 and 4 by precipitation. The mother liquid of
Fr. 3 was subjected to silica gel column chromatography to give
compounds 1 (8.3 mg) and 2 (6 mg). Also, HPLC of the mother
liquid of Fr. 4 on a C18 column using isocratic 100% MeOH gave two
active peaks (tR 39.79 and 44.15 min), which were purified by silica gel
CC to yield compounds 1 (3.1 mg) and 2 (5 mg).
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1-540-231-6570. Fax: +1-540-231-3255. E-mail:
dkingston@vt.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
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,
E
dx.doi.org/10.1021/np300750q | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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No. CHE-0619382 for purchase of the Bruker Avance 600
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