Solving the supply of resveratrol tetramers from Papua New Guinean rainforest anisoptera species that inhibit bacterial type III secretion systems

Article abstract of DOI:10.1021/np500433z

The supply of (-)-hopeaphenol (1) was achieved via enzymatic biotransformation in order to provide material for preclinical investigation. High-throughput screening of a prefractionated natural product library aimed to identify compounds that inhibit the bacterial virulence type III secretion system (T3SS) identified several fractions derived from two Papua New Guinean Anisoptera species, showing activity against Yersinia pseudotuberculosis outer proteins E and H (YopE and YopH). Bioassay-directed isolation from the leaves of A. thurifera, and similarly A. polyandra, resulted in three known resveratrol tetramers, (-)-hopeaphenol (1), vatalbinoside A (2), and vaticanol B (3). Compounds 1-3 displayed IC₅₀ values of 8.8, 12.5, and 9.9 μM in a luminescent reporter-gene assay (YopE) and IC₅₀ values of 2.9, 4.5, and 3.3 μM in an enzyme-based YopH assay, respectively, which suggested that they could potentially act against the T3SS in Yersinia. The structures of 1-3 were confirmed through a combination of spectrometric, chemical methods, and single-crystal X-ray structure determinations of the natural product 1 and the permethyl ether analogue of 3. The enzymatic hydrolysis of the β-glycoside 2 to the aglycone 1 was achieved through biotransformation using the endogenous leaf enzymes. This significantly enhanced the yield of the target bioactive natural product from 0.08% to 1.3% and facilitates ADMET studies of (-)-hopeaphenol (1).

Full text of DOI:10.1021/np500433z

Article
pubs.acs.org/jnp
Solving the Supply of Resveratrol Tetramers from Papua New
Guinean Rainforest Anisoptera Species That Inhibit Bacterial Type III
Secretion Systems
Rohan A. Davis,†,⊥ Karren D. Beattie,†,⊥ Min Xu,† Xinzhou Yang,† Sheng Yin,† Harish Holla,†
Peter C. Healy,† Melissa Sykes,† Todd Shelper,† Vicky M. Avery,† Mikael Elofsson,‡ Charlotta Sundin,‡,§
and Ronald J. Quinn*,†
†Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia
‡Laboratories for Chemical Biology Umea, Department of Chemistry, Umea University, Umea, SE-90187, Sweden
̊
̊
̊
§Creative Antibiotics Sweden AB, Tvistevagen 48, Umea, SE-90719, Sweden
̈
̊
S
* Supporting Information
ABSTRACT: The supply of (−)-hopeaphenol (1) was
achieved via enzymatic biotransformation in order to provide
material for preclinical investigation. High-throughput screen-
ing of a prefractionated natural product library aimed to
identify compounds that inhibit the bacterial virulence type III
secretion system (T3SS) identified several fractions derived
from two Papua New Guinean Anisoptera species, showing
activity against Yersinia pseudotuberculosis outer proteins E and
H (YopE and YopH). Bioassay-directed isolation from the leaves of A. thurifera, and similarly A. polyandra, resulted in three
known resveratrol tetramers, (−)-hopeaphenol (1), vatalbinoside A (2), and vaticanol B (3). Compounds 1−3 displayed IC50
values of 8.8, 12.5, and 9.9 μM in a luminescent reporter-gene assay (YopE) and IC50 values of 2.9, 4.5, and 3.3 μM in an
enzyme-based YopH assay, respectively, which suggested that they could potentially act against the T3SS in Yersinia. The
structures of 1−3 were confirmed through a combination of spectrometric, chemical methods, and single-crystal X-ray structure
determinations of the natural product 1 and the permethyl ether analogue of 3. The enzymatic hydrolysis of the β-glycoside 2 to
the aglycone 1 was achieved through biotransformation using the endogenous leaf enzymes. This significantly enhanced the yield
of the target bioactive natural product from 0.08% to 1.3% and facilitates ADMET studies of (−)-hopeaphenol (1).
he development pipeline for new antimicrobials is poor,
especially for compounds active against Gram-negative
bacterium adheres to a eukaryotic cell, injects a set of bacterial
effector proteins, Yops (Yersinia outer proteins), into the lumen
of the target cell, and results in the inhibition of the innate
immune response.12,13 Six different effector Yops have been
identified including YopE, which inhibits phagocytosis, pore
formation, and cytokine production, and YopH, a protein
tyrosine phosphatase, which also inhibits phagocytosis and T-
and B-cell activation.14 These Yops are essential for virulence,
and synthetic small molecules that inhibit Yop secretion have
been identified.15,16 It has been shown that the salicylidene
acylhydrazide class of inhibitors can attenuate virulence without
affecting bacterial growth in a number of bacterial species.17−23
Several studies suggest that one of the key advantages of
using virulence systems as targets for novel anti-infectives is a
low probability for development of resistance.4,24 Virulence
blockers target extracellular processes and are therefore less
likely to be impacted by drug efflux mechanisms which have
transmembrane and cytoplasmic domains.25 It has been
demonstrated that the mechanisms of fluoroquinolone
T
pathogens such as Pseudomonas aeruginosa.1 Gram-negative
bacteria are particularly problematic since their defense system
consists of efficient mechanisms to escape antibiotic pressure.2,3
Infections caused by P. aeruginosa and Acinetobacter spp. in
particular have increased in frequency and severity due to their
capacity to generate mutator strains, persister cells, and biofilms
and have therefore become progressively more difficult to
treat.1
In the search for new antibacterial agents, numerous bacteria-
specific cellular machinery or processes have been evaluated,
and of interest, are compounds that disrupt the expression of
bacterial effector proteins, known as exoenzymes, which are
translocated via the type III secretion system (T3SS) from the
bacteria to the infected host cell.4,5 These proteins alter the
function of the host cell, assist the microbe in invading, resist
phagocytosis, grow in deep tissues, and cause disease.6−10 The
T3SS is involved in a range of pathogenic mechanisms, and its
activity correlates closely with disease progression and
outcome.11
The well-studied plasmid-encoded Ysc of Yersinia is
representative of these common virulence systems. The
Received: June 9, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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resistance and T3SS virulence are independently associated in
P. aeruginosa.26 Another advantage of virulence inhibitors is that
cross-resistance is unlikely to evolve within or be transmutable
to the beneficial microflora which do not possess T3SS
machinery.27 Lastly, since virulence blockers have a low impact
on bacterial growth, selective pressure in favor of drug
resistance is reduced, and such mutations might result in
dysfunctional virulence systems and consequently the develop-
ment of less virulent bacteria.4,27,28
To date, multiple approaches toward identifying T3SS
inhibitors have been undertaken. Several studies report on
the utilization of in silico modeling and screening campaigns
with subsequent in vitro evaluations of target compounds.
Several research groups have undertaken extensive in vitro
screening programs of commercial, predominantly synthetic,
compound libraries (ranging between 9400 and 100 000
compounds).15,16,29−32 This research has resulted in the
identification of several important classes of T3SS inhibitors,
and notable examples include the salicylidene acylhydra-
zides,15,20 salicylanilide esters,33 thiohydrazide hydrazone
derivatives,34 and N-hydroxybenzimidazoles.35 Three smaller
studies have specifically investigated T3SS inhibitors from
natural sources. Vikram et al.36 have evaluated the T3SS
inhibition of 11 citrus flavonoids, while Tsou et al.37 have
evaluated T3SS inhibition of 146 Traditional Chinese Medicine
extracts in a Salmonella typhimurium model, and Moir et al.32
have screened 1873 plant extracts for T3SS inhibition. In
addition, two articles describe activity of T3SS inhibitors in
vivo,38,39 and chemical optimization has resulted in more
potent compounds and quantitative structure−activity relation-
ships.32
In this paper, the bioassay-directed isolation and in vitro
biological activity of three compounds with activity against the
T3SS components Y. psueudotuberculosis YopE and YopH from
two Papua New Guinean Anisoptera species are reported.
Structure elucidation by a combination of NMR, MS, single-
crystal X-ray crystallography, and chemical methods is
discussed. Furthermore, enzymatic hydrolysis of one of the
plant metabolites was achieved through biotransformation
using the endogenous leaf enzymes, which significantly
increased the yield of the target bioactive natural product,
thus enabling and expediting future ADMET and animal
studies.
enzyme activity of the secreted effector protein YopH was
performed in conjunction with a standard antibacterial growth
assay41 and suggested that these fractions selectively inhibited
the T3SS. Bioassay-directed isolation of the CH2Cl2/MeOH
extracts from A. thurifera and similarly A. polyandra resulted in
the purification of three known resveratrol tetramers,
(−)-hopeaphenol (1), vatalbinoside A (2), and vaticanol B (3).
The CH2Cl2/MeOH extracts from the air-dried and ground
leaves of A. thurifera and similarly A. polyandra were initially
chromatographed through polyamide gel. The resulting MeOH
eluent was fractionated by HPLC using C18 bonded silica
(gradient MeOH/H2O/0.1% CF3COOH). Bioassay (YopE
reporter-gene assay and validation of positive hits in the YopH
phosphatase assay) indicated the active fractions, and
subsequent mass-directed isolation of A. thurifera yielded the
previously reported resveratrol tetramers (−)-hopeaphenol (1,
0.08% dry wt),43 vatalbinoside A (2, 1.1% dry wt),44 and
vaticanol B (3, 0.25% dry wt).45 Mass-directed isolation of A.
polyandra yielded 3 (0.26% dry wt). Notably, the chemical
compositions of the leaf samples from A. polyandra and A.
thurifera bear a close similarity by LC-MS.
Stilbene oligomers are biosynthesized via resveratrol or
related phenols (e.g., prenylated stilbenes or aryl benzofuran
derivatives) and can be classified as dimers, trimers, tetramers,
etc., depending on their degree of polymerization.46 Structural
characterization of these compounds is critical for inferring
structure−activity information; however complete assignment
of the tetrameric stilbenoids is particularly challenging since
polymerization can give rise to complex structures with
confusing stereochemistries and the possibility of multiple
epimers, enantiomers, and rotational isomers.46,47 In addition,
further modifications such as glycosylation give rise to
additional molecules with increased complexity.
RESULTS AND DISCUSSION
■
A high-throughput screening (HTS) campaign of the Eskitis
Institute’s Nature Bank prefractionated library40 was under-
taken in order to identify new compounds that potentially
inhibit the T3SS system.41 This library consists of 202 983
unique fractions derived from analytical HPLC (C18 mono-
lithic) fractionation of 18 453 biota samples of plants and
marine invertebrates sourced from tropical and temperate
Australia, China, and Papua New Guinea.42 These samples have
been extracted, prefractionated under optimal conditions for
drug discovery,40 and formatted for HTS.
A primary assay utilizing the bacterial clone Y. pseudotu-
berculosis YPIII(pIB102-Elux),15 with a luminescent reporter
gene under the control of the promoter for the T3SS effector
protein YopE, detected three library fractions derived from
both leaf extracts of two Papua New Guinean Anisoptera
(Dipterocarpaceae) species, A. thurifera and A. polyandra, that
inhibited the luminescent signal. To verify the positive hits, a
colorimetric assay to detect the protein tyrosine phosphatase
In the case of 1, the compact tetrameric structure was
elucidated following 1D and 2D NMR data analysis in
conjunction with comparison to literature values.48 The
absolute configuration was established on the basis of optical
B
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Figure 1. ORTEP view of the compact tetrameric resveratrol rotamer (−)-hopeaphenol (1). The C1−C7−C8−C9 torsion angles for each ring
system in 1 are A −85.6(2)°; B 66.1(2)°; C 64.2 (2)°; and D −88.8(2)° compared to the values of A −83°, −88°; B 60°, 60°; C −60°, 60°; and D
−83°, −88° structures of the dibromodeca-O-methyl derivative (in the Supporting Information S7).50
activity ([α]25 −454 c 0.1, EtOH), which was in agreement
DD
with the reported value ([α]20D −407, EtOH).49 The structure
and absolute configuration of (−)-hopeaphenol had been
determined in 1970 by Coggon et al.50 from X-ray crystallo-
graphic studies of the dibromodeca-O-methyl derivative, which
contained two crystallographic independent molecules, each of
which was located about a 2-fold symmetry axis. A
representative view of one molecule of this structure derived
from the original Coggon data is provided in the supplementary
tables (Supporting Information S7). In the present study, the
high yields of 1 provided sufficient sample to carry out
recrystallization experiments, which resulted in well-formed
crystals of the monohydrate suitable for X-ray diffraction
studies. An ORTEP representation of the structure is shown in
Figure 1. The absolute configuration established for this
structure was consistent with that determined for the structure
of the dibromodeca-O-methyl derivative.
The structure and relative configuration for compound 2 was
assigned from its NMR spectroscopic resemblance to the
aglycone 1 and in comparison to the literature values, with the
Figure 2. ORTEP view of the extended tetrameric resveratrol rotamer
vaticanol B permethyl ether (5). The C1−C7−C8−C9 torsion angles
H-8b and H-8c resonances corrected as two symmetrical and
resolved broad doublets of doublets (J = 3.0, 3.0 Hz) resonating
for each ring system in 5 are A −98.9(5)°; B −59.8(6)°; C 156.8(4)°;
at δH 4.01 and 3.97, respectively, compared to the previous
and D −138.9(5)°.
report in which these protons were assigned as a 2H doublet of
doublets (J = 10.2, 4.2 Hz).44 Crystals of 2 were not suitable for
Vatalbinoside A (2), vaticanol B (3), and (−)-hopeaphenol
X-ray crystallographic studies; the enantiomeric purity of 2 was
(1) exhibited IC50 values of 12.4, 9.9, and 8.8 μM (YopE
confirmed on the basis of its conversion to the aglycone 1 upon
reporter-gene assay) and 4.5, 3.3, and 2.9 μM (YopH
acid hydrolysis (5% H2SO4 in EtOH, 65 °C, 4 h) and
phosphatase assay) without detectable inhibition of bacterial
comparison of the specific rotation of the hydrolysis product
growth (Table 1). These results suggest that this class of
20
([α]D −375, c 0.1, EtOH) to the natural product 1.
molecules targets the T3SS and may be developed as novel
Compound 3 was elucidated as vaticanol B on the basis of
antibacterial agents or as chemical tools for studying the T3SS.
The antivirulence efficacy of 1 in Y. pseudotuberculosis, P.
1D and 2D NMR spectroscopic data and in comparison to the
literature.51 Crystals of 3 could not be obtained from the
nature-derived product; consequently reaction of 3 with MeI/
K2CO3 in acetone at 40 °C for 2 h afforded the decamethyl
ether analogue 5. Purification of the reaction mixture using C18
HPLC afforded 5 (91% yield), which readily formed colorless
prisms upon evaporation of the mobile phase. An ORTEP
representation of the extended tetrameric rotamer vaticanol B
(5) is shown in Figure 2.
aeruginosa, and Chlamydia trachomatis is discussed elsewhere.52
In regard to natural product drug discovery, much concern in
the past and present, particularly from the pharmaceutical
industry, relates to the sufficient supply of a natural product
lead or drug for more detailed pharmacological evaluations, in
the in vitro and/or in vivo setting. The stilbenoid compounds
from these current studies are all structurally complex, and total
syntheses have not been reported. We concluded that
C
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three Gram-negative bacteria are reported by co-workers
elsewhere.52
Table 1. YopE and YopH Anti-infective and Antibacterial
Activities of Compounds 1−3
IC50 (μM)
EXPERIMENTAL SECTION
a
■
compound
YopE
YopH
antibacterial assay
General Experimental Procedures. Optical rotations were
recorded on a Jasco P-1020 polarimeter. NMR spectra were recorded
at 30 °C on either a Varian 500 or 600 MHz Unity INOVA
spectrometer (Varian, Walnut Creek, CA, USA). The latter
spectrometer was equipped with a triple-resonance cold probe. The
1H and 13C NMR chemical shifts were referenced to the solvent peak
for acetone-d6 (δH 2.05 and δC 29.9) or methanol-d4 (δH 3.31 and δC
49.1). LRESIMS were recorded on a Waters ZQ mass spectrometer
(Waters, Milford, MA, USA). HRESIMS were recorded on a Bruker
Daltronics Apex III 4.7e Fourier-transform mass spectrometer (Bruker,
Karlsruhe, Germany). A Bioline orbital shaker (Edwards Instrument
Company, Narellan, NSW, Australia) was used for the large-scale
extraction of plant material. Machery Nagel Polyamide CC6 (0.05−
0.016 mm) was used for tannin/polyphenolic removal (Machery
b
1
2
3
8.8
12.5
9.9
2.9
4.5
not active
b
not active
b
3.3
not active
c
streptomycin
38.0
31.0
36.0
a
b
c
Y. pseudotuberculosis optical density assay. At 364 μM. Commer-
cially available antibiotic used as positive control.
continued investigation of the biological activities of stilbenoids
1−3 must therefore rely on material isolated from natural
sources, at least in the short term. In order to complete
comprehensive structure and pharmacological studies, an
alternative enrichment and efficient large-scale isolation scheme
was developed that has broader application to the purification
of stilbenoids, in general.
It has been shown above that acid hydrolysis could produce 1
in high yield from the more abundant glycosylated compound
2. Compound 1 was slightly more active than 2 and 3, and
consequently, we explored enrichment of 1 by enzymatic
hydrolysis of 2 prior to isolation. In a procedure inspired by the
enzymatic hydrolysis of ruberythric acid to alizarin in madder
root,53 the biotransformation of 2 to 1 was achieved by utilizing
the endogenous enzymes present in the leaf. The dried ground
leaf material of A. polyandra was suspended in tap water for 7
days at 35 °C. Following chromatographic purification, a 16-
fold enrichment of 1 (0.08−1.3% dry wt), but not complete
conversion, was achieved over this time frame.
The genus Anisoptera comprises 11 species [The Plant
List54], which are distributed from Bangladesh to New
Guinea,55 and eight of these species are currently listed as
endangered or critically endangered.56 Anisoptera belongs to the
subfamily Dipterocarpoideae, represented by resinous rainforest
trees, which characteristically produce resveratrol oligostil-
benes.57 The chemistry of only two Anisoptera species has been
investigated: bergenin and six resveratrol oligomers have been
reported from A. marginata,58,59 while the chemical character-
ization of A. scaphula is ongoing.60
Nagel, Duren, Germany). Alltech Davisil 40−60 μm 60 Å C18 bonded
̈
silica was used for preadsorption work (Alltech, Deerfield, IL, USA).
Merck 40−63 μm silica (Kiesselgel 60) was used for flash
chromatography. A Waters 600 pump equipped with a Waters 996
PDA detector and a Waters 717 autosampler (Waters, Milford, MA,
USA) were used for HPLC. ThermoElectron C18 Betasil 5 μm 143 Å
semipreparative (21.2 mm × 150 mm) and preparative (50 mm × 150
mm) columns (Thermo Scientific, Los Angeles, CA, USA) were used
for HPLC separations. All solvents used for chromatography, UV, and
MS were Lab-Scan HPLC grade (RCI Lab-Scan, Bangkok, Thailand),
and the H2O was Millipore Milli-Q PF filtered (Millipore, Billerica,
MA, USA). All synthetic reagents were purchased from Sigma-Aldrich
and used without further purification.
Plant Material. The leaves of Anisoptera thurifera (Blanco) Blume
and A. thurifera subsp. polyandra (Blume) P.S.Ashton (syn. Anisoptera
polyandra) were collected in Papua New Guinea. Voucher samples
(QID016405; A. thurifera and QID018040; A. polyandra) have been
lodged with the Biodiversity Research Pty Ltd., Port Moresby, Papua
New Guinea.
Bioassay-Directed Isolation. In separate extraction processes, the
air-dried and ground leaves of A. thurifera (10 g) and A. polyandra (10
g) were defatted (250 mL of n-hexane, 2 h) and sequentially extracted
with CH2Cl2 (250 mL, 2 h) and MeOH (2 × 250 mL, 2 h then 24 h)
with stirring (200 rpm). The filtered CH2Cl2 and MeOH extractions
were combined and dried under reduced pressure to yield crude
extracts (1.9 g of A. thurifera and 2.0 g of A. polyandra).
The organic extracts were resuspended in MeOH (150 mL) and
loaded onto a polyamide gel column (30 g bed volume, pre-
equilibrated with MeOH) to remove pigments. The column was
eluted with MeOH (300 mL), and the eluent evaporated to obtain a
crude extract (1.0 g of A. thurifera and 1.1 g of A. polyandra). A portion
of this crude material (0.8 g) was preadsorbed to C18-bonded silica (1
g) and packed into a stainless steel guard cartridge (10 mm × 30 mm)
that was subsequently attached to a C18 semipreparative HPLC
column. Isocratic HPLC conditions of H2O/MeOH/CF3COOH
(90:10:0.1) were initially employed for the first 10 min; then a linear
gradient to MeOH (0.1% CF3COOH) was run over 40 min, followed
by isocratic conditions of MeOH (0.1% CF3COOH) for a further 10
min, all at a flow rate of 9 mL/min. Sixty fractions (60 × 1 min) were
collected from the start of the run, then submitted to bioassay.
In the case of A. thurifera the bioassay data indicated that fractions
Stilbenoids are biologically active phytoalexins, and their
range of pharmacological activity has been comprehensively
reviewed.46 Of relevance to this study is that the tetrameric
stilbenes have demonstrated antifungal,43 antibacterial,61
antiviral,62 anti-inflammatory,63 cytotoxic,64 and antitumor
activity in vitro.65 The biological activity of hemsleyanol C,
an epimer of 3, is noted as a potent topoisomerase II
inhibitor,66 while an epimer of 1 has demonstrated moderate
antibacterial activity against methicillin-resistant Staphylococcus
aureus (MRSA).61
In this study, three resveratrol tetramers from two Anisoptera
species, which inhibit YopE and YopH from Y. pseudotubercu-
losis in vitro, were identifed. As these compounds did not
inhibit the growth of Y. pseudotuberculosis (Table 1), these
compounds may selectively inhibit the T3SS. T3SS inhibitors
are able to attenuate virulence without effecting bacterial
growth in Y. pseudotuberculosis.20 This is the first report of
chemical constituents of A. thurifera and A. polyandra and the
first report detailing the bioassay-directed isolation of stilbenoid
oligomers against protein components of the T3SS. The
promising in vitro data for stilbenoids 1−3 warranted further
biological evaluation; the anti-infective activity of 1 against
1
25−30 were the active fractions. H NMR analysis of the fractions
identified that fractions 25 and 26 contained vatalbinoside A (2, 44.7
mg, 1.0% dry wt). Fractions 27−30 were a mixture of related
oligostilbenes and were combined (73.4 mg) and further fractionated
using identical C18 semipreparative HPLC conditions to those detailed
above. 1H NMR analyis of all UV-active peaks resulted in the
identification of the previously reported natural products, vatalbinoside
A (2, 4.2 mg, 0.10% dry wt), vaticanol B (3, 10.8 mg, 0.25% dry wt),
and (−)-hopeaphenol (1, 3.4 mg, 0.08% dry wt).
D
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In the case of A. polyandra the bioassay data indicated that fractions
29−31 were active. Fractions 29−31 were combined (32.7 mg), and
the mixtures were fractionated using identical C18 semipreparative
HPLC conditions to those detailed above. 1H NMR analysis of all UV-
active peaks resulted in the identification of the known natural
product, vaticanol B (3, 19.2 mg, 0.26% dry wt). Compounds 1 and 2
were identified in extracts of A. polyandra but were not isolated in
sufficient quantities during the bioassay-directed isolation step.
Alternative Enrichment of 1 and Optimized Large-Scale
Purification of 1−3. Several strategies, with broader application, to
the optimal enrichment or semipurification of the major stilbenoid
tetramers prior to HPLC purification were developed.
(+)-LRESIMS (rel int) m/z 907 (100) [M + H]+. Crystals of the
monohydrate of 1 suitable for X-ray diffraction studies were obtained
by slow evaporation of the MeOH/H2O/0.1% CF3COOH HPLC
eluent of 1.
Vatalbinoside A (2): colorless needles; mp dec ∼280 °C; [α]25
D
−341 (c 0.1, MeOH); lit. [α]25 −320 (c 0.1, MeOH);44 1H and 13C
D
NMR data were consistent with literature values;44 (+)-LRESIMS (rel
int) m/z 1091 (100) [M + Na]+.
Vaticanol B (3): white, amorphous powder; mp dec ∼280 °C;
[α]25 −46 (c 0.1, MeOH); lit. [α]20 −14 (c 0.1, MeOH);51 1H and
D
D
13C NMR data were consistent with literature values;51 (+)-LRESIMS
(rel int) m/z 907 (100) [M + H]+.
Enzymatic Hydrolysis of Stilbenoid Glycosides using
Endogenous Leaf Enzymes. The air-dried and ground leaf material
of A. polyandra (100 g) was suspended in tap H2O (400 mL) in a glass
container. The leaf material/H2O was maintained at 35 °C in a H2O
bath for 7 days. The H2O level was maintained at 400 mL with the
addition of fresh tap H2O. Prior to filtration, MeOH (400 mL) was
added to the plant material to solubilize the aglycone product. The
sample was filtered through a glass frit (pore 1); then the plant
material was sequentially extracted with acetone (2 × 400 mL) at
ambient temperatures over 24 h with shaking. The acetone extracts
were also filtered through a glass frit (pore 1). The resulting H2O/
MeOH and acetone extracts were combined and dried under reduced
pressure to yield a crude extract (14.5 g).
Enrichment of Stilbene Glycosides and Stilbene Aglycones
by Normal-Phase Column Chromatography. The aglycone-
enriched extract of the leaf material was resuspended in acetone and
preadsorbed to silica gel (100 g). The dry preadsorbed sample was
loaded onto a silica flash column (200 g, 12.5 cm diameter × 4 cm
high; pre-equilibrated with 1:1 n-hexane/EtOAc). The extract was
fractionated using a stepwise gradient consisting of 1:1 n-hexane/
EtOAc; 1:9 n-hexane/EtOAc; and 19:1 acetone/MeOH. Fraction 1,
which contained plant pigments, was discarded. Fraction 2 contained a
crude mixture of stilbenoid aglycones, and fraction 3 contained a crude
mixture of stilbenoid glycosides.
Purification of Compounds 1−3 by Reversed-Phase Prepa-
rative HPLC. Fraction 2 (10.3 g) was resuspended in MeOH and
preadsorbed onto C18 bonded silica (40 g). The dried preadsorbed
extract (sufficient for three guard cartridges) was then loaded into a
stainless steel guard cartridge (25 mm × 50 mm). A C18 preparative
HPLC column was pre-equilibrated with the starting mobile phase
H2O/MeOH/CF3COOH (85:15:0.1). Isocratic HPLC conditions of
H2O/MeOH/CF3COOH (85:15:0.1) were employed for the first 2
min; then a linear gradient to H2O/MeOH/CF3COOH (63:37:0.1)
was run over 32 min, followed by isocratic conditions of H2O/MeOH/
CF3COOH (63:37:0.1) for a further 51 min, and last, a linear gradient
to H2O/MeOH/CF3COOH (20:80:0.1) over 68 min, all at a flow rate
of 18 mL/min. A total of 118 fractions (84 s) were collected over 150
min from the start of the run. Pure (−)-hopeaphenol (1) eluted
between fractions 26 and 40 (1.02 g, 99% purity; 0.5 g 60% purity;
1.3% dry wt, cumulative yield). Vaticanol B (3) eluted between
fractions 41−72 (4.15 g, ca. 70−99% purity; ca. 2.9% yield dry wt).
Fraction 3 contained a moderate amount of the unhydrolyzed β-
glycoside 2 (1.7 g, 30% purity; 0.5% yield dry wt). Crystals of 2 were
obtained after further purification by reversed-phase HPLC. A portion
of this crude material (0.1 g) was preadsorbed to C18-bonded silica (1
g), then packed into a stainless steel cartridge (10 × 30 mm) that was
subsequently attached to a C18 semipreparative HPLC column. A
linear gradient from H2O/MeOH/CF3COOH (80:20:0.1) to H2O/
MeOH/CF3COOH (72:28:0.1) was run over 30 min, followed by
isocratic conditions of H2O/MeOH/CF3COOH (72:28:0.1) for a
further 30 min, all at a flow rate of 9 mL/min. Compound 2 eluted
between 36 and 42 min. Crystalline 2 (not suitable for X-ray
crystallographic studies) was obtained by slow evaporation of the
mobile phase.
Acid Hydrolysis of 2. A solution of 2 (10.0 mg in 1 mL of 5%
H2SO4 in EtOH) was refluxed for 4 h at 65 °C. The reaction mixture
was allowed to cool and evaporated to dryness. The crude reaction
mass was partitioned between EtOAc (2 × 10 mL) and saturated
NaHCO3 (10 mL). The organic layer was washed with brine solution
and subsequently dried over Na2SO4. The organic layer was
evaporated, and the reaction mixture was further purified by C18
semipreparative HPLC using the conditions described for fraction 2
(vide supra) to yield (−)-hopeaphenol (1, 8.0 mg, 93% yield, 98%
purity).
Methylation of (−)-Hopeaphenol (1). (−)-Hopeaphenol (1,
20.0 mg, 0.02 mmol) was dissolved in MeOH/CH2Cl2 (1:1, 1.5 mL)
at room temperature before TMS/diazomethane (2.0 M in Et2O, 650
μL, 1.30 mmol) was added dropwise.67 The reaction was stirred
overnight at room temperature and evaporated to dryness. The crude
reaction mixture was adsorbed to C18-bonded silica then purified by
C18 semipreparative HPLC column. A linear gradient from H2O/
MeOH/CF3COOH (50:50:0.1) to H2O/MeOH/CF3COOH
(5:95:0.1) was run over 30 min, followed by a linear gradient to
MeOH (0.1% CF3COOH) over 10 min followed by isocratic
conditions of MeOH (0.1% CF3COOH) for a further 20 min, all at
a flow rate of 9 mL/min. (−)-Hopeaphenol permethyl ether (4, 15.6
mg, 68% yield, ≥95% purity) eluted between 33 and 37 min.
(−)-Hopeaphenol Permethyl Ether (4): colorless needles; mp 169−
172 °C (MeOH/H2O); lit. mp 162−164 °C (benzene/MeOH);49
[α]25 −387 (c 0.1, MeOH); lit. [α]D −378 (c not specified,
D
CHCl3);49 1H NMR (500 MHz, methanol-d4) δH 3.29 (s, H-13a-
OMe/13d-OMe), 3.65 (s, H-13b-OMe/13c-OMe), 3.72 (s, H-4b-
OMe/4c-OMe), 3.77 (s, H-11a-OMe/11d-OMe), 3.80 (s, H-4a-
OMe/4d-OMe), 3.87 (brs, H-8b/8c), 4.15 (d, 11.8, H-8a/8d), 4.92 (d,
2.1, H-14b/14c), 5.73 (brs, H-7b/7c), 5.77 (d, 11.8, H-7a/7d), 5.84
(d, 2.1, H-12b/12c), 6.41 (brd, 2.1, H-14a/14d), 6.66 (d, 2.1, H-12a/
12d), 6.71 (d, 8.5, H-3b,5b/3c,5c), 6.85 (d, 8.5, H-3a,5a/3d,5d), 6.89
(d, 8.5, H-2b,6b/2c,6c), 7.14 (d, 8.5, H-2a,6a/2d,6d); 13C NMR (125
MHz, methanol-d4) δC 41.5 (C-7b/7c), 48.3 (C-8b/8c), 50.8 (C-8a/
8d), 55.9 (C-11a-OMe/11d-OMe), 55.9 (C-4b-OMe/4c-OMe), 56.0
(C-4a-OMe/4d-OMe), 56.1 (C-13a-OMe/13d-OMe), 56.6 (C-13b-
OMe/13c-OMe), 89.0 (C-7a/7d), 95.8 (C-12b/12c), 96.9 (C-12a/
12d), 105.0 (C-14a/14d), 108.8 (C-14b/14c), 114.5 (C-3b,5b/3c,5c),
115.1 (C-3a,5a/3d,5d), 120.7 (C-10b/10c), 124.3 (C-10a/10d), 129.5
(C-2b,6b/2c,6c), 130.3 (C-2a,6a/2d,6d), 132.2 (C-1a/1d), 136.2 (C-
1b/1c), 140.3 (C-9b/9c), 142.6 (C-9a/9d), 159.2 (C-4b/4c), 159.8
(C-11b/11c), 160.9 (C-13a/13d), 161.2 (C-4a/4d), 161.7 (C-11a/
11d), 161.8 (C-13b/13c); (+)-LRESIMS (rel int) m/z 1047 (100) [M
+ H]+; (+)-HRESIMS m/z 1047.4307 (C66H63O12 [M + H]+ requires
1047.4319).
Methylation of Vaticanol B (3). Vaticanol B (3, 22.1 mg, 0.024
mmol) and K2CO3 (320 mg, 2.32 mmol) were dissolved in dry
acetone (5 mL) at room temperature before MeI (200 μL, 3.2 mmol)
was added dropwise. The resulting mixture was heated under reflux for
2 h at 40 °C, then evaporated to dryness. The crude reaction mixture
was adsorbed to C18 bonded silica then purified using the same
conditions described above for compound 4. Evaporation of fractions
35−38 yielded the crystalline vaticanol B permethyl ether (5, 21.0 mg,
90% yield, ≥95% purity) suitable for X-ray diffraction studies.
Vaticanol Permethyl Ether (5): colorless prisms; mp 240−242 °C
(−)-Hopeaphenol (1): colorless plates (MeOH/H2O/0.1%
CF3COOH); mp dec ∼280 °C; lit. mp 351 °C (evac cap);49 [α]25
D
−454 (c 0.1, EtOH); lit. [α]20 −407 (c not specified, EtOH);49 1H
(MeOH/H2O); [α]25 +60 (c 0.01, EtOH) tentative value only; this
D
D
and 13C NMR data were consistent with literature values;48
compound has solubility issues in a range of solvents; H and 13C
1
E
dx.doi.org/10.1021/np500433z | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
NMR data were consistent with literature values;51 (+)-LRESIMS (rel
int) m/z 1048 (100) [M + H]+.
of YopH substrate p-nitrophenyl phosphate (pNPP; Acros Organics;
12.5 mM in 20 mM MES pH 5.0 and 0.8 mM DTT), with a Biomek
FX liquid handler (Beckman Coulter). Plates were incubated for 15
min at 37 °C before addition of 10 μL of NaOH to stop the reaction.
Plates were read at 405 nm on a VictorII Wallac plate reader
(PerkinElmer). To the remainder of the assay volume was added
decanal, and luminescence was measured as described above. Controls
were as described for the reporter-gene assay. Fractions were tested at
single-point concentrations of 7.14 μg/μL Active fractions and
controls were retested at 7.14, 1.42, 0.71, 0.14, and 0.071 μg/μL.
Pure compounds were screened at 10, 20, 50, and 100 μM. IC50 values
were calculated from interpolation of the dose−response curves.
Antibacterial Optical Density (OD620) Assay. YPIII(pIB102-
Elux) cultures were grown overnight, then diluted to an OD620 of 0.2
in BHI medium, with 2.5 mM Ca2+. A further 1:4 dilution in BHI with
2.5 mM Ca2+ was prepared before addition to the assay. A 50 μL
amount of the diluted bacteria was added to clear, lidded 384-well
plates (Becton Dickinson), containing 5 μL of fraction/compound or
controls. Active fractions and controls were screened at a single point
concentration of 7.14 μg/μL, then retested at 4.45, 0.91, 0.45, 0.091,
and 0.045 μg/μL to determine a dose response. Plates were incubated
for 3 h at 37 °C and then transferred to room temperature for 15 min
before reading at 620 nm on a VictorII Wallac plate reader.
Streptomycin was used as an in-plate negative growth control, and
an external plate contained a dose response of streptomycin for the
estimation of the antibacterial IC50 values. Pure compounds were
screened at 10, 20, 50, and 100 μM.
X-ray Crystallography. Unique data sets for compounds 1 (Cu
Kα radiation) as the monohydrate and 5 (Mo Kα radiation) were
measured at 200 K on an Oxford-Diffraction Gemini S Ultra CCD
diffractometer utilizing CrysAlis software.68 The structures were solved
by direct methods and refined by full matrix least-squares refinement
on F2. Anisotropic thermal parameters were refined for non-hydrogen
atoms; (x, y, z, Uiso)H were included and constrained at estimated
values. Conventional residuals at convergence are quoted; statistical
weights were employed. Computation used SIR-97,69 SHELX97,70
ORTEP-3,71 and PLATON72 programs and software systems. The
absolute configuration of 1 was determined by anomalous dispersion
effects [3975 Bijvoet pairs, Flack parameter −0.05(12)].73 In the
absence of significant anomalous scatterers in compound 5, Friedel
equivalents were merged with the absolute configuration predicted on
the basis of specific rotation.
Crystal data for (−)-hopeaphenol (1): C56H42O12·H2O, Mr
=
924.9; orthorhombic, space group P212121, a = 11.1051(2) Å, b =
18.7895(4) Å, c = 22.3006(4) Å, V = 4653.2(2)(2) Å3; Dc (Z = 4) =
1.32 g cm−3; μCu = 0.77 mm; specimen 0.36 × 0.23 × 0.08 mm;
Tmin/max = 0.768/0.941; 2θmax = 71.2°; Nt = 32588, N = 8969 (Rint
0.045), N0 = 7876; R1 = 0.037, wR2 = 0.095; S = 1.03, xabs
−0.05(12).
=
=
Crystal Data for Vaticanol B Permethyl Ether (5): C66H62O12, Mr =
1047.2; monoclinic, space group P21, a = 15.5983(6) Å, b =
12.4215(3) Å, c = 16.8136(7) Å, β = 117.230(5), V = 2896.7(2) Å3; Dc
(Z = 2) = 1.20 g cm−3; μMo = 0.08 mm; specimen 0.54 × 0.35 × 0.10
mm; Tmin/max = 0.99/0.96; 2θmax = 55.0°; Nt = 22 650, N = 6728 (Rint
= 0.044), N0 = 3205; R1 = 0.057, wR2 = 0.117; S = 0.84.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full .cif depositions reside with the Cambridge Crystallographic
Data Centre, CCDC numbers 998909 [(−)-hopeaphenol, 1] and
953125 (vaticanol B permethyl ether, 5). Copies can be obtained free
of charge on application at the following address: http://www.ccdc.
cam.ac.uk/cgi-bin/catreqcgi).
1D and 2D NMR spectra for (−)-hopeaphenol permethyl ether
(4) and an ORTEP view of the dibromodeca-O-methyl
derivative of hopephenol. This material is available free of
charge via the Internet at http://pubs.acs.org.
Determination of YopE Expression Using a Reporter-Gene
Assay. YPIII(pIB102-Elux)74 from a culture grown on LB agar
containing 25 μg/mL chloramphenicol (Sigma) for 48 h at 26 °C was
used to inoculate a liquid culture, then grown for 12−16 h in brain
heart infusion broth (BHI; Difco) containing 25 μg/mL chloramphe-
nicol (Sigma) on an orbital shaker at 26 °C. Liquid cultures were
diluted to an OD620 of 0.2, then further diluted 1 in 4 in Ca2+-depleted
media (BHI media with 5 mM EGTA and 20 mM MgCl2) before
addition of 30 μL of bacteria to a 384-well white solid lidded Optiplate
(PerkinElmer). Before bacterial addition, 5 μL of fraction or
compound/controls was added to the plate, by diluting plates of
library fractions or compounds/controls in DMSO with a Minitrak
(PerkinElmer) liquid handler, by addition of 1 μL of fraction to 4 μL
of H2O.
Plates were incubated at 26 °C for 1 h followed by incubation at 37
°C for 3 h and then transferred back to 26 °C for 15 min. A 15 μL
amount of 0.1% decanal (Sigma) emulsified in H2O was added to each
well. Plates were read on a Trilux (PerkinElmer) counter using a
luminescence protocol. A dose response of streptomycin was used as
an antibacterial control. Bacteria in BHI with 2.5 mM Ca2+ was used as
an in-plate negative control for the assay since no Yop protein is
expressed during these conditions. Ca2+ depletion at 37 °C stimulates
Yop production, and the Yop production will eventually suppress
growth.15
Fractions were tested at single-point concentrations of 7.14 μg/μL.
Active fractions and controls were retested at 7.14, 1.42, 0.71, 0.14, and
0.071 μg/μL. Pure compounds were screened at 10, 20, 50, and 100
μM. YopE suppression was calculated using linear regression on the
basis of the reduction in luminescent signal versus sample
concentration (μg/mL). IC50 values were calculated from interpolation
of the dose−response curves.
Combined Reporter-Gene and Phosphatase Assay for the
Determination of YopH Secretion. Before addition of decanal, and
according to the YopH protocol, 5 μL of the final assay volume was
added to a clear 384-well plate (Becton Dickinson) containing 45 μL
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +61-7-3735-6000. Fax: +61-7-3735-6001. E-mail: r.
quinn@griffith.edu.au.
Author Contributions
⊥R. A. Davis and K. D. Beattie contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to acknowledge Creative Antibiotics
Sweden AB and the Australian Research Council (linkage grant
LP120200339) for financial support. The authors thank D.
Camp, C. Lewis, and K. Watts from the Molecular Libraries
group (Eskitis Institute) for their assistance in the preparation
of the screening library. We thank B. Aldred for technical
assistance with the HTS, T. Rali for the collection and
taxonomic identification of the plant material, and the Central
Analytical Research Facility, Queensland University of Tech-
nology, for access to the single-crystal X-ray diffraction facilities.
REFERENCES
■
(1) Talbot, G. H. Expert. Rev. Anti-Infect. Ther. 2008, 6, 39−49.
(2) Church, D.; Elsayed, S.; Reid, O.; Winston, B.; Lindsay, R. Clin.
Microbiol. Rev. 2006, 19, 403−434.
(3) Erol, S.; Altoparlak, U.; Akcay, M. N.; Celebi, F.; Parlak, M. Burns
2004, 30, 357−361.
(4) Clatworthy, A. E.; Pierson, E.; Hung, D. T. Nat. Chem. Biol. 2007,
3, 541−548.
F
dx.doi.org/10.1021/np500433z | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(5) Dean, S. N.; Bishop, B. M.; van Hoek, M. L. BMC Microbiol.
2011, 11, 114.
III Protein Secretion, 240th ACS National Meeting, August 22−26,
Boston, MA, USA, 2010.
(6) Dacheux, D.; Attree, I.; Schneider, C.; Toussaint, B. Infect.
Immun. 1999, 67, 6164−6167.
(7) Sawa, T.; Yahr, T. L.; Ohara, M.; Kurahashi, K.; Gropper, M. A.;
Wiener-Kronish, J. P.; Frank, D. W. Nat. Med. 1999, 5, 392−398.
(8) Sundin, C. Type III Secretion Mediated Translocation of Effector
Exoenzymes by Pseudomonas aeruginosa. Ph.D. Dissertation, Umea
University, Sweden, 2003.
(9) Hornef, M. W.; Roggenkamp, A.; Geiger, A. M.; Hogardt, M.;
Jacobi, C. A.; Heesemann, J. Microb. Pathog. 2000, 29, 329−343.
(10) Sundin, C.; Hallberg, B.; Forsberg, A. FEMS Microbiol. Lett.
2004, 234, 87−91.
(11) Coburn, B.; Sekirov, I.; Finlay, B. B. Clin. Microbiol. Rev. 2007,
20, 535−549.
(12) Cornelis, G. R. Nat. Rev. Mol. Cell Biol. 2002, 3, 742−752.
(13) Rosqvist, R.; Magnusson, K.-E.; Wolf-Watz, H. EMBO J. 1994,
13, 964−972.
(14) Viboud, G. I.; Bliska, J. B. Annu. Rev. Microbiol. 2005, 59, 69−89.
(15) Kauppi, A. M.; Nordfelth, R.; Uvell, H.; Wolf-Watz, H.;
Elofsson, M. Chem. Biol. 2003, 10, 241−249.
(16) Pan, N. J.; Brady, M. J.; Leong, J. M.; Goguen, J. D. Antimicrob.
Agents Chemother. 2009, 53, 385−392.
(17) Bailey, L.; Gylfe, A.; Sundin, C.; Muschiol, S.; Elofsson, M.;
Nordstroem, P.; Henriques-Normark, B.; Lugert, R.; Waldenstroem,
A.; Wolf-Watz, H.; Bergstroem, S. FEBS Lett. 2007, 581, 587−595.
(18) Muschiol, S.; Bailey, L.; Gylfe, A.; Sundin, C.; Hultenby, K.;
Bergstroem, S.; Elofsson, M.; Wolf-Watz, H.; Normark, S.; Henriques-
Normark, B. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 14566−14571.
(19) Negrea, A.; Bjur, E.; Ygberg, E.; Elofsson, M.; Wolf-Watz, H.;
Rhen, M. Antimicrob. Agents Chemother. 2007, 51, 2867−2876.
(20) Nordfelth, R.; Kauppi, A. M.; Norberg, H. A.; Wolf-Watz, H.;
Elofsson, M. Infect. Immun. 2005, 73, 3104−3114.
(38) Chu, H.; Slepenkin, A.; Elofsson, M.; Keyser, P.; De la Maza, L.
M.; Peterson, E. M. Int. J. Antimicrob. Agents 2010, 36, 145−150.
(39) Slepenkin, A.; Chu, H.; Elofsson, M.; Keyser, P.; Peterson, E. M.
J. Infect. Dis. 2011, 204, 1313−1320.
(40) Camp, D.; Davis, R. A.; Campitelli, M.; Ebdon, J.; Quinn, R. J. J.
Nat. Prod. 2012, 75, 72−81.
̊
(41) Yin, S.; Davis, R. A.; Shelper, T.; Sykes, M. L.; Avery, V. M.;
Elofsson, M.; Sundin, C.; Quinn, R. J. Org. Biomol. Chem. 2011, 9,
6755−6760.
(42) Nature Bank−a Biodiscovery Resource. Available online at
http://www.nature-bank.com.au/ (accessed June 20, 2013).
(43) Liu, J. Y.; Ye, Y. H.; Wang, L.; Shi, D. H.; Tan, R. X. Helv. Chim.
Acta 2005, 88, 8.
(44) Abe, N.; Ito, T.; Ohguchi, K.; Nasu, M.; Masuda, Y.; Oyama, M.;
Nozawa, Y.; Ito, M.; Iinuma, M. J. Nat. Prod. 2010, 73, 1499−1506.
(45) Tanaka, T.; Ito, T.; Nakaya, K.-i.; Iinuma, M.; Takahashi, Y.;
Naganawa, H.; Riswan, S. Heterocycles 2001, 55, 729−740.
(46) Xiao, K.; Zhang, H.-J.; Xuan, L.-J.; Zhang, J.; Xu, Y.-M.; Bai, D.-
L. Stilbenoids: Chemistry and Bioactivities: Studies in Natural Product
Chemistry; Elsevier, 2008; Vol. 34, pp 453−646.
(47) Ito, T.; Oyama, M.; Sajiki, H.; Sawa, R.; Takahashi, Y.; Iinuma,
M. Tetrahedron 2012, 68, 2950−2960.
(48) Kawabata, J.; Fukushi, E.; Hara, M.; Mizutani, J. Magn. Reson.
Chem. 1992, 30, 6−10.
(49) Coggon, P.; Janes, N. F.; King, F. E.; King, T. J.; Molyneux, R. J.;
Morgan, J. W. W.; Sellars, K. J. Chem. Soc. 1965, 406−409.
(50) Coggon, P.; McPhail, A. T.; Wallwork, S. C. J. Chem. Soc. B
1970, 884−897.
(51) Tanaka, T.; Ito, T.; Nakaya, K.; Iinuma, M.; Riswan, S.
Phytochemistry 2000, 54, 63−69.
(52) Zetterstrom, C. E.; Hasselgren, J.; Salin, O.; Davis, R. A.; Quinn,
̈
(21) Slepenkin, A.; Enquist, P.-A.; Hagglund, U.; de la Maza, L. M.;
Elofsson, M.; Peterson, E. M. Infect. Immun. 2007, 75, 3478−3489.
(22) Duncan, M. C.; Linington, R. G.; Auerbuch, V. Antimicrob.
Agents Chemother. 2012, 56, 5433−5441.
R. J.; Sundin, C.; Elofsson, M. PLoS One 2013, 8, e81969.
(53) Derksen, G. C. H.; Naayer, M.; van Beek, T. A.; Capelle, A.;
Haaksman, I. K.; van Doren, H. A.; de Groot, Æ. Phytochem. Anal.
2003, 14, 137−144.
(23) Tsou, L. K.; Dossa, P. D.; Hang, H. C. MedChemComm 2013, 4,
68−79.
(54) The Plant List Version 1. Published on the internet;. http://
www.theplantlist.org/ (June 12, 2013).
(24) Marra, A. Drugs R&D 2006, 7, 1−16.
(55) Ashton, P. S. In Tree Flora of Sabah and Sarawak; Soepadmo, E.;
Saw, L. G.; Chung, R. C. K., Eds; Forest Research Institute Malaysia:
Kuala Lumpur, 2004; Vol. 5, Chapter 2, pp 71−79.
(56) IUCN Red List of Threatened Species. Version 2012.2. http://
www.iucnredlist.org (June 17, 2013).
(57) Seo, E.-K.; Kinghorn, A. D. Bioactive Constituents of the Family
Dipterocarpaceae: Studies in Natural Product Chemisty; Elsevier, 2000;
Vol. 23, pp 531−561.
(58) Atun, S. Phytochemical Properties of Several Indonesian
Dipterocarpaceae from the Genus Vatica, Anisoptera, Hopea and
Dipterocarpus. Ph.D. Dissertation, Bandung Institute of Technology,
Indonesia, 2004.
(25) Zechini, B.; Versace, I. Recent Pat. Anti-Infect. Drug Discovery
2009, 4, 37−50.
(26) Wong-Beringer, A.; Wiener-Kronish, J.; Lynch, S.; Flanagan, J.
Clin. Microbiol. Infect. 2008, 14, 330−336.
(27) Keyser, P.; Elofsson, M.; Rosell, S.; Wolf-Watz, H. J. Int. Med.
2008, 264, 17−29.
(28) Rasko, D. A.; Moreira, C. G.; Li de, R.; Reading, N. C.; Ritchie,
J. M.; Waldor, M. K.; Williams, N.; Taussig, R.; Wei, S.; Roth, M.;
Hughes, D. T.; Huntley, J. F.; Fina, M. W.; Falck, J. R.; Sperandio, V.
Science 2008, 321, 1078−1080.
(29) Gauthier, A.; R, M.; Lowden, M.; Ibarra, J. A.; Puente, J. L.;
Finlay, B. B. Antimicrob. Agents Chemother. 2005, 49, 4101−4109.
(30) Felise, H. B.; Nguyen, H. V.; Pfuetzner, R. A.; Barry, K. C.;
Jackson, S. R.; Blanc, M. P.; Bronstein, P. A.; Kline, T.; Miller, S. I. Cell
Host Microbe 2008, 4, 325−336.
(31) Mecsas, J. U.S. Patent Application, US20110319398 A1, 2011.
(32) Moir, D. T.; Aiello, D.; Peet, N. P.; Williams, J. D. U.S. Patent
Application, US20120114633 A1, 2010.
(33) Kratky, M.; Vinsova, J. Curr. Pharm. Des. 2011, 17, 3494−3505.
(34) Yarovenko, V. N.; Zayakin, E. S.; Krayushkin, M. M.; Zorina, V.
V.; Kapotina, L. N.; Zigangirova, N. A. J. Chem. Chem. Eng. 2010, 4,
55−59.
(59) Atun, S. Indo. J. Chem. 2009, 6, 151−157.
(60) Parvez, M.; Rahman, A.; Molla, K.; Akter, A.; Int, J. Pharm. Sci.
Rev. Res. 2012, 1, 1−6.
(61) Zgoda-Pols, J. R.; Freyer, A. J.; Killmer, L. B.; Porter, J. R. J. Nat.
Prod. 2002, 65, 1554−1559.
(62) Dai, J.-R.; Hallock, Y. F.; Cardellina, J. H.; Boyd, M. R. J. Nat.
Prod. 1998, 61, 351−353.
(63) Huang, K.-S.; Zhou, S.; Lin, M.; Wang, Y.-H. Planta Med. 2002,
68, 916−920.
(64) Ito, T.; Akao, Y.; Yi, H.; Ohguchi, K.; Matsumoto, K.; Tanaka,
T.; Iinuma, M.; Nozawa, Y. Carcinogenesis 2003, 24, 1489−1497.
(65) Mishima, S.; Matsumoto, K.; Futamura, Y.; Araki, Y.; Ito, T.;
Tanaka, T.; Iinuma, M.; Nozawa, Y.; Akao, Y. J. Exp. Ther. Oncol. 2003,
3, 283−288.
(35) Kim, O. K.; Garrity-Ryan, L. K.; Bartlett, V. J.; Grier, M. C.;
Verma, A. K.; Medjanis, G.; Donatelli, J. E.; Macone, A. B.; Tanaka, S.
K.; Levy, S. B.; Alekshun, M. N. J. Med. Chem. 2009, 52, 5626−5634.
(36) Vikram, A.; Jayaprakasha, G. K.; Jesudhasan, P. R.; Pillai, S. D.;
Patil, B. S. J. Appl. Microbiol. 2010, 109, 515−527.
(66) Yamada, M.; Hayashi, K.-i.; Ikeda, S.; Tsutsui, K.; Tsutsui, K.;
Ito, T.; Iinuma, M.; Nozaki, H. Biol. Pharm. Bull. 2006, 29, 1504−
1507.
(37) Tsou, L. K.; Yount, J.; Dossa, P.; Hang, H. C. In Abstracts of
Papers, Discovery of Antibacterial Virulence Inhibitors Targeted at Type
G
dx.doi.org/10.1021/np500433z | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(67) Garfunkle, J.; Kimball, F. S.; Trzupek, J. D.; Takizawa, S.;
Shimamura, H.; Tomishima, M.; Boger, D. L. J. Am. Chem. Soc. 2009,
131, 16036−16038.
(68) CrysAlis Pro software; Agilent Technologies Inc, S. C. C., USA,
2012.
(69) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.;
Spaagna, R. J. Appl. Crystallogr. 1999, 32, 115−119.
(70) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122.
(71) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
(72) Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7−13.
(73) Flack, H. D. Acta Crystallogr. 1983, A39, 837−838.
(74) Forsberg, A.; Rosqvist, R. Infect. Agents Dis. 1993, 2, 275−278.
H
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