Anti-inflammatory and antimalarial meroterpenoids from the New Zealand ascidian aplidium scabellum

Article abstract of DOI:10.1021/jo201654h

Bioassay-directed fractionation of an extract of the New Zealand ascidian Aplidium scabellum has afforded the anti-inflammatory secondary metabolite 2-geranyl-6-methoxy-1,4-hydroquinone-4-sulfate (1) and a family of pseudodimeric meroterpenoids scabellone

Full text of DOI:10.1021/jo201654h

Note
pubs.acs.org/joc
Anti-inflammatory and Antimalarial Meroterpenoids from the New
Zealand Ascidian Aplidium scabellum
Susanna T. S. Chan,† A. Norrie Pearce,† Ana H. Januario,‡ Michael J. Page,§ Marcel Kaiser,⊥,∥
Rene J. McLaughlin,∇ Jacquie L. Harper,∇ Victoria L. Webb,# David Barker,† and Brent R. Copp*,†
†School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
‡Nucleo de Pesquisa em Cien
̂
cias Exatas e Tecnológicas da Universidade de Franca, Av. Dr. Armando Salles de Oliveira 201, Parque
́
Universitar
́
io, CEP 14404-600, Franca, SP, Brazil
§National Institute of Water & Atmospheric Research (NIWA) Ltd., P.O. Box 893, Nelson, New Zealand
⊥Swiss Tropical and Public Health Institute, Socinstrasse 57, P.O. Box CH-4002, Basel, Switzerland
∥The University of Basel, CH-4003 Basel, Switzerland
∇Malaghan Institute of Medical Research, P.O. Box 7060, Wellington South, New Zealand
#NIWA, Private Bag 14-901, Kilbirnie, Wellington, New Zealand
S
* Supporting Information
ABSTRACT: Bioassay-directed fractionation of an extract of the
New Zealand ascidian Aplidium scabellum has afforded the anti-
inflammatory secondary metabolite 2-geranyl-6-methoxy-1,4-hydro-
quinone-4-sulfate (1) and a family of pseudodimeric meroterpenoids
scabellones A (2)−D (5). The benzo[c]chromene-7,10-dione scaffold
contained within scabellones A−D is particularly rare among natural
products. The structures were elucidated by interpretation of NMR
data. Scabellone B was also identified as a moderately potent,
nontoxic inhibitor of Plasmodium falciparum.
scidians of the genus Aplidium (Order Enterogona, Family
subjected to a combination of silica, diol, and reversed-phase C2
A
Polyclinidae) are recognized as a valuable source of
flash column chromatography to yield scabellones A−D (2−5).
bioactive marine natural products.1 As part of our ongoing
program aimed at discovering new anti-inflammatory natural
products produced by New Zealand and Antarctic ascidians of
the genus Aplidium,2−4 it was found that a crude organic extract
of the encrusting species Aplidium scabellum (Michaelsen,
1924) collected off Rabbit Island, Hauraki Gulf, New Zealand,
exhibited modest ability to inhibit the respiratory burst of
stimulated human neutrophils.
Bioassay-directed fractionation resulted in the isolation of
five new secondary metabolites, 2-geranyl-6-methoxy-1,4-
hydroquinone-4-sulfate (1) and scabellones A−D (2−5), as
well as the known metabolites verapliquinone A (6)5,6 and 8-
methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzo-pyran-
6-ol (7).7 The identities of 6 and 7 were confirmed by
comparison of spectroscopic data with those found in the
literature.5−7 The isolation, structure elucidation, and biological
evaluation of the five new metabolites are presented.
Specimens of the gray, encrusting ascidian were extracted
with MeOH and fractionated by reversed-phase C18 to give two
fractions. The first fraction was further purified by reversed-
phase cyanopropyl flash column chromatography to give 2-
geranyl-6-methoxy-1,4-hydroquinone-4-sulfate (1), verapliqui-
none A (6), and chromenol 7. The second fraction was
High-resolution negative-ion ESIMS data observed for the
pseudomolecular ion of 1 were consistent with a molecular
formula of C17H24O6S. All 17 carbon resonances required by
this formula were accounted for in the 13C NMR spectrum. In
the H NMR spectrum (CD3OD), resonances attributable to
two aromatic protons (δH 6.75, 6.54), two olefinic protons (δ H
5.29, 5.09), three moderately deshielded methylene signals (δ H
1
Received: August 8, 2011
Published: September 29, 2011
© 2011 American Chemical Society
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3.27, 2.08, 2.00), one methoxyl signal (δ H 3.83), and three
allylic methyl singlets (δH 1.69, 1.65, 1.58) were observed. The
presence of a highly oxygenated benzene ring and geranyl side
chain were deduced from combined analysis of 1H, 13C, COSY,
NOESY, HSQC, and HMBC NMR data. These structural
fragments were reminiscent of those observed for quinol 8, the
semisynthetic reduction product of verapliquinone A (6),
previously reported from a Brittany, France collection of
Aplidium sp.5 While the molecular formula of 1 suggested the
presence of a sulfate group, attempts at desulfation led to
degradation prompting the synthesis of 2-geranyl-6-methoxy-
1,4-hydroquinone (8)5,6 to enable comparative NMR analysis
to establish the position of sulfation in 1. Commercially
available 2,4-dimethoxybenzaldehyde was subjected to Baeyer−
Villiger oxidation to give 2,4-dimethoxyphenol,8 which was
subsequently coupled with geranyl bromide to yield the desired
2-geranyl-4,6-dimethoxyphenol. Oxidation of 2-geranyl-4,6-
dimethoxyphenol with CAN afforded synthetic 2′E-verapliqui-
none A (6),5,6 with subsequent reduction using sodium
dithionite affording the desired 2-geranyl-6-methoxy-1,4-hydro-
quinone (8) (Scheme 1).
correlations extending from an oxymethine proton at δH 6.00
(d, J = 9.4 Hz, H-5) to an olefinic proton at δH 5.28 (d, J = 9.4
Hz, H-1′) to a methyl group at δH 1.93 (3H, s, H3-9′). A second
spin-system extended from a methylene resonance at δH 1.94
(H2-3′), to a second methylene group at δH 1.98 (H2-4′), to an
olefinic proton at δH 4.93 (H-5′), which in turn was correlated
to two methyl groups (δH 1.59, 1.50). HMBC correlations
from the methyl group at δH 1.93 to a methylene carbon
resonance at δC 39.8 (C-3′), and from the methylene protons at
δH 1.94 to a carbon resonance at δC 116.9 (C-1′) joined these
two fragments, establishing the presence of a 1,1-disubstituted
oxygeranyl chain fragment. The presence of a second, regular 1-
substituted 2″E-geranyl chain was identified by analysis of the
NMR data and direct comparison with the data observed for 8.
The two remaining highly substituted fragments of the
molecule were deduced to be 2,3,5-trisubstituted 1,4-
benzoquinone and 2,3,4,6-tetrasubstituted phenol rings by
analysis of 13C, NOESY, and HMBC NMR data. HMBC
correlations observed from the proton resonance at δH 5.80
(H-2) to 13C resonances at 182.6 (C-1), 157.8 (C-3), 178.7 (C-
4), and 137.6 (C-10b) and from a methoxyl group (δ H 3.80) to
C-3 defined the presence of the quinonoid ring system, while a
similar set of correlations from the remaining aromtic proton
resonance at 6.40 (H-7) to 13C resonances at δC 150.0 (C-8),
139.2 (C-9), 151.4 (C-6a), and 111.0 (C-10a) and from the
second methoxyl group (δ H 3.89) to C-8 defined the second
ring as a tetrasubstituted phenol.
Scheme 1. Synthesis of 2-Geranyl-6-methoxy-1,4-
hydroquinone (8)
Interfragment HMBC correlations (Figure 1) observed
between quinonoid proton H-2 and phenol ring resonance
1
Direct comparison of the H and 13C chemical shifts of the
aromatic ring signals of 1 and 8 identified an upfield shift of C-4
and downfield shifts of C-3 and C-5 in the 13C NMR spectrum
(of 1 vs 8) and downfield shifts of H-3 and H-5 in the 1H NMR
spectrum which were consistent9 with the placement of the
sulfate group at the C-4 position as shown.
Scabellone A (2) was isolated as an optically inactive brown
oil with a molecular formula of C34H42O6, established using
(+)-HRESI mass spectrometry. Two substructures, a 5-
substituted chromenol (using 7 numbering) and a 3-substituted
2′E-verapliquinone A fragment, were identified by analysis of
1H, 13C, COSY, NOESY, HSQC, and HMBC NMR data and
by direct comparison with the data observed for 7 and 6,
accounting for all the atoms required by the ESIMS molecular
formula. Although no HMBC or NOESY correlations were
observed to provide direct evidence for the linking of the two
substructures, the only logical connection was between C-6 and
C-7 as shown for 2. A lack of optical rotation (at multiple
wavelengths) and no detectable CD spectrum indicated
scabellone A was isolated as a racemate.
Figure 1. Interfragment HMBC correlations observed for scabellone B
(3).
δC 111.0 (C-10a) and between the oxymethine signal at δH
6.00 (H-5) and 13C resonances at δC 178.7 (C-4), 151.4 (C-
6a), and 137.6 (C-10b) established the connectivity between
the 1,1-disubstituted oxygeranyl chain and the hydroquinone−
quinone core. The second geranyl chain (C-1″-C-10″) was
placed at C-10 (δC 126.8) as HMBC correlations from the
diastereotopic methylene protons at δH 3.57 and 3.36 of the
chain (H2-1″) were observed to carbon resonances δC 139.2
(C-9), 126.8 (C-10), and 111.0 (C-10a) of the hydroquinone
ring, completing the planar structure of scabellone B (3) as
shown. A lack of detectable optical rotation and no CD
spectrum led to the conclusion at 3 was isolated as a racemate.
Scabellone C (4) was obtained as an optically inactive purple
oil that gave a pseudomolecular ion at m/z 545.2889 in the
(+)-HRESIMS from which a molecular formula of C34H40O6
Scabellone B (3) was isolated as an optically inactive purple
oil. A molecular formula of C34H42O6 was established by
1
(+)-HRESI mass spectrometry. Analysis of H, 13C, COSY,
NOESY, HSQC, and HMBC NMR data allowed the
identification of four substructures of the molecule: a 1,1-
disubstituted oxygeranyl chain, a regular 1-substituted geranyl
chain, a 2,3,5-trisubstituted 1,4-benzoquinone ring, and a
2,3,4,6-tetrasubstituted phenol. Direct comparison of NMR
data with those of 8 and scabellone A (2) also supported the
presence of these fragments.
The composition of the first substructure, an oxygeranyl
chain, was established by interpretation of COSY, NOESY,
HSQC, and HMBC data. The COSY spectrum of 3 identified
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was derived. Similarities in the NMR data observed for
scabellone C and scabellones A (2) and B (3) were evident,
identifying the presence of a 5-substituted chromenol fragment
(as in 2) and a 1,1-quinonoid-oxy-disubstituted geranyl chain
(as per the fragment defined by C-1−C-5, C-10b, C-1′−C-9′ of
3). HMBC correlations observed from both the quinonoid
proton at δH 5.89 (H-2) and the oxymethine signal at δH 6.04
(H-5) to carbon resonances at δC 179.0 (C-4) and 133.9 (C-
12c) identified the connection between the quinone ring and
the 1,1-disubstituted 2′E-oxygeranyl chain. Additional correla-
tions between both the aromatic proton at δH 6.44 (H-7) and
H-5 to a carbon resonance at δC 151.6 (C-6a) established the
connectivity between the 1,1-substituted oxygeranyl chain and
the 5-substituted chromenol substructures and, therefore,
completed the structure of scabellone C (4) as shown.
viability. Together, these data indicated that 1 and 3 were in
fact inhibiting neutrophil superoxide production whereas the
potent anti-inflammatory activity of 8 was the result of
programmed cell death. Prenylated hydroquinones related to
8 are well-known to exhibit broad ranging biological
activities1,4,16 while their corresponding quinones are noted
for their ability to induce p53-independent apoptosis in
transformed cell lines.6
Scabellone B (3) and quinol 8 were also evaluated against the
neglected disease parasites targets Trypanosoma brucei
rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium
falciparum. While 8 was found to be pan-panel active, likely due
to its general toxicity, 3 exhibited selectivity toward Plasmodium
falciparum (K1 chloroquine-resistant strain) with IC50 4.8 μM
and only poor cytotoxicity (L6 rat myoblast cell line, IC50 65
μM).17
The discovery that quinol sulfate 1 exhibits moderately
potent and selective ability to inhibit neutrophil respiratory
burst suggests that meroterpenoid sulfates could indeed hold
promise for the development of new anti-inflammatory agents.
The core benzo[c]chromene-7,10-dione scaffold of scabellones
A−D is rare among natural products and has previously been
associated with antiproliferative or apoptosis-inducing bio-
logical properties. Our finding of selective antimalarial activity
for 3, combined with no detectable apoptosis toward human
neutrophils, highlights this structural class as a novel lead for
the development of new treatments for malaria. Efforts directed
toward the biomimetic synthesis and structure−antimalarial
activity relationship of these new metabolites are in progress,
the results of which will be reported in due course.
The molecular formula derived from (+)-HRESIMS for the
optically inactive scabellone D (5) was C34H40O6, the same as
1
that observed for scabellone C (4). Analysis of H, 13C, COSY,
NOESY, HSQC, and HMBC NMR data readily identified the
same substructural fragments and interfragment connectivities
as those observed for 4. Close comparison of NMR data
observed for scabellones C (4) and D (5) revealed differences
in chemical shifts centered around C-10, with C-11 in 5 being
modestly deshielded (+0.8 ppm) while shielding was observed
for C-1″ (−2.6 ppm) and C-2″ (−0.8 ppm). These differences
were interpreted as establishing scabellone D as a diastereomer
of scabellone C, depicted with arbitrary relative configuration.
In both cases, scabellones C and D also failed to exhibit optical
rotation at several wavelengths, nor were CD spectra
detectable, indicating both compounds were isolated as
racemates.10
The tricyclic 6H-benzo[c]chromene-7,10-dione core of
scabellone B (3) is rare among natural products, being
contained within the structures of apoptosis-inducing ascidian
metabolites thiaplidiaquinones A (9) and B,11 the sponge-
sourced indoleamine-2,3-dioxygenase inhibitor exiguamine A,12
the microbial metabolite juglochroman B,13 and the cytotoxic
plant metabolite microphyllaquinone,14 while the tetracyclic
core of scabellones C (4) and D (5) has been reported just
once before, contained within the hexacyclic teak wood
metabolite tecomaquinone I.15
EXPERIMENTAL SECTION
■
General Procedures. Optical rotations were recorded using a 0.1
dm cell in CH2Cl2 solvent. Ultraviolet−visible and circular dichroism
spectra were run as methanol solutions. NMR spectra were recorded at
1
either 600, 400, or 300 MHz for H nuclei and 150, 100, or 75 MHz
for 13C nuclei. Residual solvent signals were used as reference
(CD3OD: δH 3.30, δC 49.05; CDCl3: δH 7.25, δC 77.0). 1H NMR data
is reported as position (δ), relative integral, multiplicity (s = singlet, d
= doublet, t = triplet, m = multiplet, br = broad), coupling constant (J,
Hz), and the assignment of the atom. 13C NMR data are reported as
position (δ) and assignment of the atom.
Analytical reversed-phase HPLC was run using a C8 column (3 μm,
7 × 33 mm) and eluting with a linear gradient of H2O (0.05% TFA) to
MeCN over 13.5 min at 2 mL/min. Reversed-phase flash column
chromatography was carried out on C18, C8, C2 or CN stationary
support with a particle size of 40−63 μm. Silica gel column
chromatography was carried out on silica media with either 40−63
μm or 15−40 μm particle size. All solvents used were distilled
analytical grade of better. Chemical reagents used were purchased
from standard chemical suppliers.
Extraction and Isolation. Specimens (collection no. MNP9123)
of the gray, encrusting Aplidium scabellum were collected from the reef
at the southern end of Rabbit Island (36 20.9395S, 175 30.308E, 15
m), Great Barrier Island, New Zealand. The ascidian was identified by
M.P., and a voucher specimen is kept at NIWA Wellington under
collection code MNP9123.
Frozen ascidian specimens were freeze-dried (dry weight 200.92 g),
extracted with methanol (5 × 200 mL), and filtered, and solvent was
removed in vacuo. The green crude extract (7.74 g) was subjected to
C18 flash chromatography with a steep gradient from water to
methanol, fractionated at 0%, 25%, 50%, 75%, and 100% methanol in
water. The third fraction (50% MeOH in water) was then subjected to
reversed-phase cyanopropyl flash column chromatography eluting with
water. The first fraction collected yielded 2-geranyl-6-methoxy-1,4-
hydroquinone-4-sulfate (1) (4.0 mg, 0.002%, tR 5.62 min). The fifth
Quinol sulfate 1, scabellone B (3), chromenol 7, and quinol
8 were found to inhibit the superoxide production by PMA-
stimulated human neutrophils in vitro,2 with IC50's of 21, 125,
92, and 0.2 μM, respectively. To determine the effect of the
different treatments on cell survival, drug-treated neutrophils
were stained with the fluorescent markers for necrosis
(propidium iodide) and apoptosis (Annexin V-FITC) and
analyzed by flow cytometry. Treatment with quinol 8 was
found to induce cell death via apoptosis (see the Supporting
Information). In contrast, 1 and 3 had no effect on neutrophil
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fraction (100% MeOH) from the crude C18 column was subjected to
silica flash chromatography, eluting with a gradient of dichloromethane
through to 10% methanol in dichloromethane, and two fractions
(dichloromethane and 1% methanol/dichloromethane) were subjected
to further purification. The 100% dichloromethane fraction was
subjected again to silica flash chromatography where a purple-colored
fraction (hexane/ethyl acetate, 9:1) was collected and by analysis of 1H
NMR data (CDCl3) deduced to be a mixture of two compounds.
Reversed-phase C2 flash column chromatography with a steep gradient
from water to methanol, afforded brown-red 8-methoxy-2-methyl-2-
(4-methyl-3-pentenyl)-2H-1-benzopyran-6-ol (7) (0.8 mg, 0.0004%)
and scabellone B (3) (0.44 mg, 0.0002%), in the fractions eluted with
water/methanol (1:1) and (1:4), respectively. The 1% methanol/
dichloromethane fraction was subjected to silica flash chromatography
with a gradient from hexane to ethyl acetate. A brown-colored fraction
(hexane:ethyl acetate, 4:1) was collected and further subjected to
reversed-phase C2 flash column chromatography with a steep gradient
from water to methanol. Compounds of interest were found in the
70% methanol and 100% methanol fractions. Diol flash column
chromotography with dichloromethane as the eluent was used on the
70% methanol fraction and afforded scabellone A (2) (0.49 mg,
0.0002%). The 100% methanol fraction was subjected to silica flash
chromatography (15−40 μm) where scabellone C (4) (0.85 mg,
0.0004%) and scabellone D (5) (0.53 mg, 0.0003%) were isolated
(hexane/ethyl acetate, 9:1).
Rf (100% dichloromethane) 0.38; IR νmax 3484, 1658, 1592, 1481,
1
1441, 1223 cm−1; H NMR (CDCl3, 600 MHz) δ 6.40 (1H, s, H-7),
6.00 (1H, d, J = 9.3 Hz, H-5), 5.80 (1H, s, H-2), 5.48 (1H, br s, OH),
5.28 (1H, d, J = 9.3 Hz, H-1′), 5.06 (1H, m, H-2″), 5.01 (1H, t, J = 6.6
Hz, H-6″), 4.93 (1H, t, J = 6.6 Hz, H-5′), 3.89 (3H, s, 8-OCH3), 3.80
(3H, s, 3-OCH3), 3.57 (1H, m, H-1″a), 3.36 (1H, dd, J = 17.5, 9.1 Hz,
H-1″b), 1.98 (4H, m, H2-4′, H2-5″), 1.94 (2H, m, H2-3′), 1.93 (3H, s,
H3-9′), 1.87 (2H, m, H2-4″), 1.62 (3H, s, H3-9″), 1.59 (3H, s, H3-8′),
1.56 (3H, s, H3-10″), 1.50 (3H, s, H3-7′), 1.49 (3H, s, H3-8″); 13C
NMR (CDCl3, 150 MHz) δ 182.6 (C-1), 178.7 (C-4), 157.8 (C-3),
151.4 (C-6a), 150.0 (C-8), 144.3 (C-2′), 139.2 (C-9), 137.6 (C-10b),
137.1 (C-3″), 131.7* (C-6′), 131.6* (C-7″), 130.8 (C-4a), 126.8 (C-
10), 124.2 (C-2″), 123.8 (C-6″), 123.6 (C-5′), 116.9 (C-1′), 111.0 (C-
10a), 107.3 (C-2), 98.4 (C-7), 67.6 (C-5), 56.1 (3-OCH3), 56.1 (8-
OCH3), 39.8* (C-4″), 39.7* (C-3′), 26.5 (C-1″), 26.3* (C-4′), 26.2*
(C-5″), 25.6* (C-8′), 25.5* (C-9″), 17.6* (C-7′), 17.5* (C-8″), 17.2
(C-9′), 16.5 (C-10″); (+)-ESIMS m/z 547 (100) [M + H]+;
(+)-HRESIMS m/z 547.3063 [M + H]+ (calcd for C34H43O6,
547.3054).
Scabellone C (4): purple oil; Rf (20% ethyl acetate/hexane) 0.33;
[α]20 0 (c 0.005, CH2Cl2); [α]20 0, [α]20 0, [α]20 0 (c 0.007,
D
578
546
436
CH2Cl2); UV (MeOH) λmax (log ε) 205 (3.86), 243 (3.71), 294 sh
1
(3.29), 338 (3.35), 558 (2.82) nm; H NMR (CDCl3, 600 MHz) δ
6.44 (1H, s, H-7), 6.10 (1H, d, J = 9.9 Hz, H-12), 6.04 (1H, d, J = 9.1
Hz, H-5), 5.89 (1H, s, H-2), 5.58 (1H, d, J = 9.9 Hz, H-11), 5.35 (1H,
d, J = 9.1 Hz, H-1′), 5.15 (1H, t, J = 6.7 Hz, H-3″), 4.94 (1H, t, J = 6.7
Hz, H-5′), 3.86 (3H, s, 8-OCH3), 3.84 (3H, s, 3-OCH3), 2.21 (1H, m,
H-2″a), 2.11 (1H, m, H-2″b), 1.99 (2H, m, H2-4′), 1.94 (2H, m, H2-3′),
1.93 (3H, s, H3-9′), 1.93 (1H, m, H-1″a), 1.73 (1H, m, H-1″b), 1.68
(3H, s, H3-6″), 1.60 (3H, s, H3-5″), 1.59 (3H, s, H3-8′), 1.51 (3H, s,
H3-7′), 1.49 (3H, s, H3-13); 13C NMR (CDCl3, 150 MHz) δ 185.4 (C-
1), 179.0 (C-4), 158.3 (C-3), 153.0 (C-8), 151.6 (C-6a), 144.3 (C-2′),
138.3 (C-8a), 133.9 (C-12c), 131.7 (C-6′), 131.7 (C-4″), 131.4 (C-4a),
126.6 (C-11), 124.2 (C-3″), 123.8 (C-12), 123.6 (C-5′), 120.2 (C-
12a), 117.0 (C-1′), 107.6 (C-12b), 107.1 (C-2), 101.2 (C-7), 77.8 (C-
10), 67.7 (C-5), 56.2 (3-OCH3), 56.2 (8-OCH3), 41.1 (C-1″), 39.7
(C-3′), 26.2 (C-4′), 25.7 (C-6″), 25.6 (C-8′), 24.5 (C-13), 23.2 (C-2″),
17.7 (C-7′), 17.7 (C-5″), 17.2 (C-9′); (+)-ESIMS m/z 545 (100) [M +
H]+; (+)-HRESIMS m/z 545.2889 [M + H]+ (calcd for C34H41O6,
545.2898).
Additional biomass (dry weight 40.11 g, crude extract 2.53 g) was
extracted using the same isolation procedure to afford more 8-
methoxy-2-methyl-2-(4-methyl-3-pentenyl)-2H-1-benzopyran-6-ol (7)
(0.73 mg, 0.0018%) and, from the same fraction, verapliquinone A (6)
(1.27 mg, 0.003%).
2-Geranyl-6-methoxy-1,4-hydroquinone-4-sulfate (1):
brown oil; UV (MeOH) λmax (log ε) 210 (4.00), 282 (3.19), 354
(2.00) nm; IR νmax 3401 1669, 1640, 1495, 1433, 1229, 1207 cm−1; 1H
NMR (CD3OD, 600 MHz) δ 6.75 (1H, d, J = 2.1 Hz, H-5), 6.54 (1H,
d, J = 2.1 Hz, H-3), 5.29 (1H, t, J = 6.6 Hz, H-2′), 5.09 (1H, t, J = 6.6
Hz, H-6′), 3.83 (3H, s, OCH3), 3.27 (2H, d, J = 7.3 Hz, H2-1′), 2.08
(2H, m, H2-5′), 2.00 (2H, m, H2-4′), 1.69 (3H, s, H3-10′), 1.65 (3H, s,
H3-9′), 1.58 (3H, s, H3-8′); 13C NMR (CD3OD, 150 MHz) δ 148.6
(C-6), 146.4 (C-4), 141.2 (C-1), 136.7 (C-3′), 132.2 (C-7′), 129.2 (C-
2), 125.5 (C-6′), 123.9 (C-2′), 114.1 (C-3), 103.6 (C-5), 56.6 (OCH3)
41.0 (C-4′), 29.1 (C-1′), 27.9 (C-5′), 25.9 (C-9′), 17.8 (C-8′), 16.3 (C-
10′); (-)-ESIMS m/z 355 [M − H]−; (−)-HRESIMS m/z 355.1219
[M − H]− (calcd for C17H23O6S, 355.1221).
Scabellone D (5): purple oil; Rf (20% ethyl acetate/hexane) 0.44;
[α]20 0, [α]20 0, [α]20 0, [α]20 0 (c 0.007, CH2Cl2); UV
D
578
546
436
(MeOH) λmax (log ε) 203 (3.82), 242 (3.65), 295 sh (3.20), 338
(3.29), 552 (2.74) nm; 1H NMR (CDCl3, 600 MHz) δ 6.43 (1H, s, H-
7), 6.07 (1H, d, J = 10.0 Hz, H-12), 6.02 (1H, d, J = 9.6 Hz, H-5), 5.88
(1H, s, H-2), 5.55 (1H, d, J = 10.0 Hz, H-11), 5.33 (1H, m, H-1′,
obscured by solvent), 5.11 (1H, t, J = 6.8 Hz, H-3″), 4.93 (1H, t, J =
6.8 Hz, H-5′), 3.85 (3H, s, 8-OCH3), 3.82 (3H, s, 3-OCH3), 2.20 (1H,
m, H-2″a), 2.14 (1H, m, H-2″b), 1.99 (2H, m, H2-4′), 1.92 (2H, m, H2-
3′), 1.92 (3H, s, H3-9′), 1.88 (1H, m, H-1″a), 1.80 (1H, m, H-1″b),
1.62 (3H, s, H3-6″), 1.57 (3H, s, H3-5″), 1.58 (3H, s, H3-8′), 1.50 (3H,
s, H3-7′), 1.53 (3H, s, H3-13); 13C NMR (CDCl3, 150 MHz, deduced
from HMBC and HSQC) δ 184.9 (C-1), 178.7 (C-4), 158.0 (C-3),
152.5 (C-8), 151.3 (C-6a), 144.0 (C-2′), 137.9 (C-8a), 133.6 (C-12c),
131.5 (C-6′), 131.5 (C-4″), 131.1 (C-4a), 127.4 (C-11), 124.3 (C-3″),
123.7 (C-5′), 123.6 (C-12), 120.5 (C-12a), 117.0 (C-1′), 107.2 (C-2),
107.1 (C-12b), 101.0 (C-7), 77.4 (C-10), 67.7 (C-5), 56.5 (3-OCH3),
56.5 (8-OCH3), 38.5 (C-1″), 39.4 (C-3′), 26.1 (C-4′), 25.8 (C-6″),
25.9 (C-13), 25.8 (C-8′), 22.4 (C-2″), 17.6 (C-7′), 17.6 (C-5″), 17.2
(C-9′); (+)-ESIMS m/z 545 (100) [M + H]+; (+)-HRESIMS
545.2882 [M + H]+ (calcd for C34H41O6, 545.2898).
Scabellone A (2): brown oil; [α]20 0, [α]20 0, [α]20 0,
D
578
546
[α]20 0 (c 0.005, CH2Cl2); UV (MeOH) λmax (log ε) 207 (4.58),
436
230 sh (4.48), 269 (4.27), 315 (3.81), 450 sh (3.11) nm; Rf (50% ethyl
acetate/hexane) 0.44; IR νmax (ATR) 3447, 1677, 1634, 1436, 1226
1
cm−1; H NMR (CDCl3, 600 MHz) δ 6.43 (1H, s, H-9), 6.01 (1H, s,
H-2), 5.92 (1H, d, J = 10.0 Hz, H-14), 5.59 (1H, d, J = 10.0 Hz, H-13),
5.07 (1H, t, J = 6.6 Hz, H-3″), 5.00 (1H, t, J = 6.6 Hz, H-6′), 4.90 (1H,
t, J = 6.6 Hz, H-2′), 3.85 (3H, s, 3-OCH3), 3.85 (3H, s, 10-OCH3),
3.10 (1H, m, H-1′a), 2.97 (1H, m, H-1′b), 2.10 (2H, m, H2-2″), 1.95
(2H, m, H2-5′), 1.86 (2H, m, H2-4′), 1.74 (1H, m, H-1″a), 1.65 (1H,
m, H-1″b), 1.64 (3H, s, H3-6″, obscured by water), 1.64 (3H, s, H3-9′,
obscured by water), 1.55 (3H, s, H3-8′), 1.55 (3H, s, H3-5″), 1.44 (3H,
s, H3-15), 1.36 (3H, s, H3-10′); 13C NMR (CDCl3, 150 MHz, carbon
1
resonances deduced from H−13C HMBC and HSQC NMR data) δ
186.4 (C-1), 181.5 (C-4), 158.3 (C-3), 149.4 (C-10), 145.7 (C-8),
145.7 (C-5), 138.4 (C-6), 138.1 (C-3′), 136.2 (C-10a), 131.3 (C-13),
131.5 (C-4″), 131.2 (C-7′), 124.1 (C-6′), 124.0 (C-3″), 120.4 (C-14a),
120.1 (C-14), 118.0 (C-2′), 109.1 (C-7), 107.6 (C-2), 101.4 (C-9),
77.5 (C-12),56.2 (3-OCH3), 56.2 (10-OCH3), 39.6 (C-1″), 39.2 (C-
4′), 26.6 (C-1′), 26.3 (C-5′), 25.4 (C-15), 25.4 (C-9′), 25.4 (C-6″),
22.2 (C-2″), 17.3 (C-8′), 17.3 (C-5″), 15.6 (C-10′); (+)-ESIMS m/z
547 (100) [M + H]+; (+)-HRESIMS 547.3020 [M + H]+ (calcd for
C34H43O6, 547.3054).
2-Geranyl-4,6-dimethoxyphenol. 2,4-Dimethoxyphenol (10)8
(0.52 g, 3.38 mmol) was dissolved in dry toluene (25 mL), and
sodium hydride (0.15 g, 3.73 mmol) was added. The reaction mixture
was stirred for 1 h at room temperature under nitrogen, and then
geranyl bromide (0.73 g, 3.38 mmol) was added dropwise at 0 °C. The
mixture was stirred for an additional 3 h before it was poured into ice−
water and acidified with 2 M acetic acid. The acidic solution was
extracted with diethyl ether (100 mL). The organic layer was washed
with saturated aqueous sodium bicarbonate, followed by saturated
Scabellone B (3): purple oil; [α]20 0, [α]20 0, [α]20 0,
D
578
546
[α]20436 0 (c 0.004, CH2Cl2); (MeOH) λmax (log ε) 209 (4.57), 238 sh
(4.25), 303 (3.94), 340 sh (3.78), 358 sh (3.59), 546 (3.32) nm; UV−
vis (MeOH/KOH) λmax 212 nm (log ε 4.55), 303 (4.01), 546 (3.44);
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The Journal of Organic Chemistry
Note
aqueous sodium chloride, and dried over anhyd MgSO4 and solvent
removed in vacuo. Purification by silica gel column chromatography
eluting with ethyl acetate (0−20%) in hexane and subsequently 100%
dichloromethane gave the product as a yellow oil (0.47 g, 48%): Rf
(100% dichloromethane) 0.82; IR νmax (ATR) 3546, 2914, 1611,
1224, 1053 cm−1; 1H NMR (CDCl3, 400 MHz) δ 6.35 (1H, d, J = 2.6
Hz, H-5), 6.30 (1H, d, J = 2.6 Hz, H-3), 5.33 (1H, m, H-2′), 5.29 (1H,
m, OH), 5.10 (1H, m, H-6′), 3.84 (3H, s, 4-OCH3), 3.74 (3H, s, 6-
OCH3), 3.35 (2H, d, J = 7.2 Hz, H2-1′), 2.10 (2H, m, H2-5′), 2.04 (2H,
m, H2-4′), 1.72 (3H, s, H3-10′), 1.67 (3H, s, H3-9′), 1.59 (3H, s, H3-8′);
13C NMR (CDCl3, 100 MHz) δ 152.8 (C-4), 146.7 (C-6), 137.4 (C-
1), 136.5 (C-3′), 131.4 (C-7′), 127.5 (C-2), 124.3 (C-6′), 122.0 (C-2′),
105.4 (C-3), 96.7 (C-5), 56.0 (6-OCH3), 55.7 (4-OCH3), 39.7 (C-4′),
28.1 (C-1′), 26.7 (C-5′), 25.6 (C-9′), 17.6 (C-8′), 16.1 (C-10′);
(+)-ESIMS m/z 291 [M + H]+; (+)-HRESIMS m/z 291.1952 [M +
H]+ (calcd for C18H27O3, 291.1955).
FACS buffer (0.1% BSA, PBS) containing 250 ng/mL propidium
iodide (PI, Sigma-Aldrich, Auckland, NZ). Cells were then analyzed by
flow cytometry (FACSCalibur; Becton Dickinson) to identify live cells
(AnV−/PI−), apoptosing cells (AnV+/PI−), necrotic cells (AnV−/PI
+), and dead cells (AnV+/PI+).
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, NMR spectra for 1−8, and cytometry
plots. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: b.copp@auckland.ac.nz.
2-Geranyl-6-methoxy-1,4-benzoquinone (synthetic verapli-
quinone A) (6).5,6 A solution of cerium ammonium nitrate (0.96 g,
1.76 mmol) solution in a 1:2 mixture of CH3CN:H2O (15 mL) was
added dropwise at 0 °C to a solution of 2-geranyl-4,6-dimethox-
yphenol (11) (0.26 g, 0.88 mmol) in CH3CN (5 mL) with stirring.
The mixture was stirred overnight at 0 °C, poured into a 10% sodium
chloride solution, and then extracted with diethyl ether. The organic
extract was dried over anhyd MgSO4 and solvent evaporated in vacuo.
Purification by silica gel column chromatography eluting with ethyl
acetate (0−10%) in hexane gave the product as a bright yellow oil
(0.16 g, 66%): Rf (10% ethyl acetate/hexane) 0.22; IR νmax (ATR)
ACKNOWLEDGMENTS
■
We acknowledge funding from the Foundation for Research
Science and Technology, contract CO1X0205 and the
University of Auckland. We thank M. Cal, S. Sax, and C.
Stalder (Swiss TPH) for parasite assay results and Professor T.
Brittain (UoA) for access to the circular dichroism spectropho-
tometer.
1
2914, 1678, 1230 cm−1; H NMR data agreed with that previously
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123.9 (C-6′), 117.7 (C-2′), 107.1 (C-5), 56.3 (OCH3), 39.6 (C-4′),
27.1 (C-1′), 26.4 (C-5′), 25.7 (C-9′), 17.7 (C-8′), 16.1 (C-10′);
(+)-ESIMS m/z 297 [M+Na]+; (+)-HRESIMS m/z 297.1459 [M +
Na]+ (calcd for C17H22NaO3, 297.1461).
■
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(1H, d, J = 2.8 Hz, H-5), 6.15 (1H, d, J = 2.8 Hz, H-3), 5.28 (1H, m,
H-2′), 5.09 (1H, m, H-6′), 3.78 (3H, s, OCH3), 3.23 (2H, d, J = 7.6
Hz, H2-1′), 2.09 (2H, m, H2-5′), 2.01 (2H, m, H2-4′), 1.69 (3H, s, H3-
10′), 1.64 (3H, s, H3-9′), 1.58 (3H, s, H3-8′); 13C NMR (CD3OD, 100
MHz) δ 151.0 (C-4), 149.3 (C-6), 138.0 (C-1), 136.6 (C-3′), 132.3
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(+)-HRESIMS m/z 299.1611 [M + Na]+ (calcd for C17H24NaO3,
299.1618).
Biological Assays. The neutrophil respiratory burst assay, details
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organism parasite assay protocols have been reported elsewhere.18
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Trypanosoma brucei rhodesiense, STIB 900 strain, trypomastigotes stage
(positive control melarsoprol, IC50 0.01 μM), Trypanosoma cruzi,
Tulahuen C4 strain, amastigotes stage (positive control benznidazole,
IC50 1.35 μM), Leishmania donovani, MHOM-ET-67/L82 strain,
amastigote/axenic stage (positive control miltefosine, IC50 0.52 μM),
Plasmodium falciparum, K1 strain, IEF stage (positive control
chloroquine, IC50 0.20 μM), L6 rat skeletal myoblast cell line for
cytotoxicity (positive control podophyllotoxin, IC50 0.01 μM).
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washed in chilled annexin binding buffer (10 mM HEPES, 140 mM
NaCl, 2.5 mM CaCl2). Neutrophils were incubated 15 min in the dark
with FITC labeled annexin V (AnV-FITC, BD Pharmingen, NJ),
washed with annexin binding buffer and resuspended in 200 μL of
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hydroquinone 8 in MeOH open to air overnight in the presence of
either H2SO4 (0.1%) or triethylamine (0.1%), mimicking extremes of
pH that may have occurred during extraction, led to either recovered
starting material (acidic) or extensive decomposition (basic). Dimers
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The possibility that quinone 6 and hydroquinone 1 could have reacted
in the extraction process was investigated by stirring a mixture of 6 and
1 in CH2Cl2 (+ triethylamine, 1%) overnight open to air: the reaction
returned starting materials. We consider 2−5 to be natural products
and not artefacts of isolation as they could be detected, by ESIMS and
NMR, in the fractions resulting from the first C18 chromatography step
and none were observed to interconvert during purification.
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9155
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The Journal of Organic Chemistry
Note
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