Isolation, structure elucidation and cytotoxic evaluation of endiandrin B from the Australian rainforest plant Endiandra anthropophagorum

Article abstract of DOI:10.1016/j.bmc.2008.12.030

Chemical investigations of the DCM extract from the roots of Endiandra anthropophagorum resulted in the isolation of a new cyclobutane lignan endiandrin B (1), together with the known natural products, endiandrin A (2), and (-)-dihydroguaiaretic acid (3).

Full text of DOI:10.1016/j.bmc.2008.12.030

Bioorganic & Medicinal Chemistry 17 (2009) 1387–1392
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isolation, structure elucidation and cytotoxic evaluation of endiandrin B
from the Australian rainforest plant Endiandra anthropophagorum
*
Rohan A. Davis , Emma C. Barnes, James Longden, Vicky M. Avery, Peter C. Healy
Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, QLD 4111, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical investigations of the DCM extract from the roots of Endiandra anthropophagorum resulted in the
isolation of a new cyclobutane lignan endiandrin B (1), together with the known natural products,
endiandrin A (2), and (ꢀ)-dihydroguaiaretic acid (3). The structure of 1 was determined by extensive
spectroscopic analyses, and confirmed by single crystal X-ray crystallography. Methylation of 1 using
diazomethane afforded the previously reported natural product, cinbalansan (4). All compounds were
evaluated for their cytotoxicity towards human lung carcinoma cells (A549) using high-content screen-
ing. (ꢀ)-Dihydroguaiaretic acid (3) was found to be the most potent cytotoxin against the A549 lung car-
Received 22 October 2008
Revised 3 December 2008
Accepted 5 December 2008
Available online 24 December 2008
Keywords:
Endiandrin B
Endiandrin A
(ꢀ)-Dihydroguaiaretic acid
Cinbalansan
cinoma cell line, with an IC50 value of 7.49 lM.
Ó 2008 Elsevier Ltd. All rights reserved.
Natural products
High content screening
Cytotoxicity
A549
X-ray
NMR
Structure elucidation
1. Introduction
isolation and structure elucidation of a new cyclobutane lignan that
we have named endiandrin B (1). The cytotoxicity of endiandrins A
(2) and B (1), (ꢀ)-dihydroguaiaretic acid (3), and cinbalansan (4)
against human lung carcinoma cells (A549) determined using
high-content screening, is also reported.
The plant family Lauraceae has been the source of over 200 nat-
ural products, however only a small number of these secondary
metabolites have had any bioactivity reported.1 Examples of bioac-
tive Lauraceae compounds include the antibacterial agent
launobine,2,3 the antiviral cryptopleurine,4,5 and the bisbenzyliso-
quinoline antimalarial dehatrine.6 The plant genus Endiandra, which
belongs to the family Lauraceae, has rarely been studied for its
chemical composition and prior to 2007 only five compounds had
been isolated from this genus.1 During 2007 we reported the bioas-
say-guided fractionation of the DCM extract from the roots of Endi-
andra anthropophagorum that resulted in the isolation of a new
cyclobutane lignan, endiandrin A (2), along with previously re-
ported natural products nectandrin B (5) and (ꢀ)-dihydroguaiaretic
acid (3) (Fig. 1).7 These 3 lignans were shown to display glucocorti-
coid receptor (GR) binding activity with IC50 values of 0.9 (2), 27 (5)
2. Results and discussion
The air-dried and ground roots of E. anthropophagorum were ex-
tracted with DCM and the resulting lipophilic material was parti-
tioned between aq MeOH and hexanes. The polar material was
fractionated on a diol bonded silica flash column using a hex-
anes/EtOAc gradient. Further purification of a lignan enriched
fraction using diol bonded silica HPLC (i-PrOH/hexanes) yielded
(ꢀ)-dihydroguaiaretic acid (3, 25.7 mg, 0.010% dry wt), endiandrin
A (2, 75.8 mg, 0.030% dry wt), and endiandrin B (1, 7.1 mg, 0.0028%
dry wt).
Endiandrin B (1) was isolated as clear needles and was assigned
the molecular formula C20H24O4 on the basis of HRESIMS and NMR
data. The small number of resonances in both the 1H and 13C NMR
spectra (Table 1) coupled with the MS data indicated that 1 was a
symmetrical molecule containing nine degrees of unsaturation.
The 1H NMR spectrum of 1 displayed one exchangeable resonance
(dH 5.34), three aromatic resonances indicative of a trisubstituted
and 35 l
M (3), respectively.7 Recollection of this plant sample has
enabled us to further investigate the chemistry of this rainforest
species, as well as obtain several lignan natural products in suffi-
cient quantities for cytotoxicity evaluations. Herein we report the
* Corresponding author. Tel.: +61 7 3735 6043; fax: +61 7 3735 6001.
E-mail address: r.davis@griffith.edu.au (R.A. Davis).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.030

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R. A. Davis et al. / Bioorg. Med. Chem. 17 (2009) 1387–1392
9'
9
7
7'
1'
1
5
5'
3
3'
RO
OR
HO
OH
OMe MeO
OMe MeO
R = H ( Endiandrin B (1)
R = Me Cinbalansan (4)
Endiandrin A (2)
MeO
HO
OMe
OH
OMe
OH
MeO
HO
O
Nectandrin B (5)
(-)-Dihydroguaiaretic acid (3)
Figure 1. Chemical structures for compounds 1–5.
Table 1
NMR data for endiandrin B (1)a
Position
13C
1H (mult., J, int.)
gCOSY
gHMBC
ROESY
1
2
3
133.3
111.4
146.0
55.8
6.23 (d, 1.8, 1H)
3.60 (s, 3H)
3-OMe, 6
2
1, 3, 4, 6, 7
3
3-OMe, 7, 8
2
3-OMe
4
143.5
4-OH
5.34 (s, 1H)
3, 4, 5
5
6
7
8
113.4
120.7
49.9
34.4
15.0
133.3
111.4
146.0
55.8
6.71 (d, 7.8, 1H)
6.58 (dd, 7.8, 1.8, 1H)
3.40 (d, 4.8, 1H)
2.77 (br m, 1H)
1.18 (d, 6.6, 3H)
6
2, 5
8
7, 9
8
1, 3, 4
2, 4, 5, 7
1, 2, 6, 70, 8, 9
1, 7, 80, 90, 9
1, 7, 8, 80, 90
6
5, 7, 8
2, 6, 9
2, 6, 9
7, 8, 80
9
10
20
6.23 (d, 1.8, 1H)
3.60 (s, 3H)
30-OMe, 60
20
10, 30, 40, 60, 70
30
30-OMe, 70, 80
20
30
30-OMe
40
143.5
40-OH
5.34 (s, 1H)
30, 40, 50
10, 30, 40
20, 40, 50, 70
10, 20, 60, 7, 8’, 90
10, 70, 8, 9, 90
10, 70, 80, 8, 9
50
60
70
80
90
113.4
120.7
49.9
34.4
15.0
6.71 (d, 7.8, 1H)
6.58 (dd, 7.8, 1.8, 1H)
3.40 (d, 4.8, 1H)
2.77 (br m, 1H)
1.18 (d, 6.6, 3H)
60
60
20, 50
80
50, 70, 80
20, 60, 90
20, 60, 90
70, 80, 8
70, 90
80
a
Spectra were recorded in CDCl3 at 30 °C.
benzene system [dH 6.23 (d, J = 1.8 Hz), 6.71 (d, J = 7.8 Hz) and 6.58
(dd, J = 7.8, 1.8 Hz)], one methoxy group (dH 3.60), two aliphatic
resonances (dH 3.40 and 2.77) and a methyl resonance (dH 1.18).
The 13C NMR spectrum of 1 contained only 10 signals, six of which
resonated between dC 110 and 147. The 1H/13C NMR and MS data
readily identified 1 as an isomer of endiandrin A (2) and HMBC
data analysis (Table 1) confirmed that the planar structures of 1
and 2 were identical.7 The major NMR chemical shift differences
between 1 and 2 were noted about the cyclobutane ring moiety,
suggesting that endiandrin B had a different relative configuration
indicated that the methyl and aryl components of 1 had a trans ori-
entation about the cyclobutane ring. On the basis of these data we
assigned the relative configuration of 1 as 8b,80b-dimethyl-7 ,70a
a -
bis(3-methoxy-4-hydroxyphenyl)cyclobutane. Crystals of 1 suit-
able for X-ray diffraction analysis were obtained by slow evapora-
tion of a i-PrOH/hexanes (1:9) mix. An ORTEP8 representation of
the structure of 1 is shown in Figure 2 and confirmed the trans
arrangement of the methyl and phenyl groups on the cyclobutyl
ring. The C9–C8–C18–C19 and C1–C7–C17–C11 torsion angles
were 26.8(4)° and 26.5(4)°, respectively, whilst C6–C1–C7–C17
and C16–C11–C17–C7 were 73.7(3)° and ꢀ78.0(3)°, respectively.
Intramolecular O–HꢂꢂꢂO bonding was observed between the hydro-
xy and methoxy groups. In the crystal lattice, strong inter-molecu-
lar O–HꢂꢂꢂO hydrogen bonds between the O4 and O14 hydroxy
groups link clusters of four molecules to form R44(8) rings9 dis-
posed about crystallographic centres of symmetry.
about the four-membered ring. The optical rotation of endiandrin B
27
(1), ½ ꢁÞD 0 (c 0.12, CHCl3), identified 1 as a meso-isomer. With two
a
meso compounds possible, ROESY data analysis was undertaken in
order to determine the relative configuration of 1. Strong ROESY
correlations between the benzylic protons at dH 3.40 (H-7/H-70)
and the methyl doublets at dH 1.18 (H-9/H-90), as well as ROESY
cross-peaks between the aliphatic multiplets at dH 2.77 (H-8/H-
80) and the aryl signals at dH 6.58 (H-6/H-60) and 6.73 (H-2/H-20),
Methylation of 1 using diazomethane in a MeOH/diethyl ether
mixture afforded a compound that was determined to be cinba-

R. A. Davis et al. / Bioorg. Med. Chem. 17 (2009) 1387–1392
1389
Figure 2. ORTEP plot for endiandrin B (1).
lansan (4), following spectroscopic data comparison with litera-
ture values.10 In
similar manner, endiandrin
and (ꢀ)-
dihydroguaiaretic acid were assigned to compounds 2 and 3,
(Fig. 3). This effect was less pronounced in the mitotracker assay
a
A
with (ꢀ)-dihydroguaiaretic acid found to have an IC50 of
31.2 lM. Endiandrins A (2) and B (1) were found to have a moder-
respectively, after NMR and [
a
]
D
data comparison with literature
ate effect on all three assays giving an inhibition of 76% and 75%
values.7,11–13
at 100 M, respectively. Cinbalansan (4) was found to have a
l
Due to our interest in natural products that exhibit anticancer
activity,14 detailed profiling of the cytotoxic properties of
compounds 1–4 against the lung carcinoma cell line, A549, was
very minor effect in the three assays, giving a maximum inhibi-
tion of 34%, however, as can be seen from the qualitative data,
cinbalasan did have an effect on the uptake of the mitotracker
dye (Fig. 4). This may indicate early stages of apoptosis, however
further work would be required to determine the exact mecha-
nism of action.
TM
determined using a high-content imaging system [Opera (Perk-
TM
inElmer)]. The Opera was utilised due to its ability to simulta-
neously capture and quantify confocal images to measure an array
of cellular changes. Several fluorescent markers were employed to
investigate changes to nuclear morphology, and membrane perme-
ability, which both undergo significant changes during cell death.
Nuclear morphology was visualized using the DNA/RNA binding
dyes DRAQ5, YO-PRO-1, propidium iodide and Hoechst 34580.
DRAQ5 and Hoechst are both cell permeable dyes and therefore
stain all cells, however YO-PRO-1 and propidium iodide are mem-
brane impermeable. Thus, only during the late stages of apoptosis,
or programmed cell death, as the permeability of the cell mem-
brane increases, does nuclear staining with YO-PRO-1 occur. Propi-
dium iodide, however, remains cell impermeable until cell death
has occurred. Cells were also labelled with Mitotracker Red and
phalloidin, which label the mitochondria15 or actin cytoskeleton,16
respectively. The mitotracker dye is sequestered by functioning
mitochondria with the level of staining dependant on mitochon-
drial membrane potential.15 Mitochondria have been shown to un-
dergo major changes in their structure and function early on in the
apoptotic process.17,18 Thus, using these markers in correlation
3. Conclusion
In conclusion this paper describes the isolation and structure
elucidation of the new cyclobutane lignan, endiandrin B (1),
along with the cytotoxicity data for endiandrins A (2) and B
(1), (ꢀ)-dihydroguaiaretic acid (3), and cinbalansan (4) against
the human lung carcinoma cell line, A549. Whilst over 2000
compounds belonging to the lignan structure class have been
isolated, lignans containing a cyclobutane moiety are rare with
less than 30 reported in the literature.1
4. Experimental
4.1. General methods
Optical rotations were recorded on a Jasco P-1020 polarimeter.
UV and IR spectra were recorded on a Camspec M501 spectropho-
tometer and a Bruker Tensor 27 spectrometer, respectively. NMR
spectra were recorded at 30 °C on either a Varian 500 MHz or
600 MHz Unity INOVA spectrometer. The latter spectrometer was
equipped with a triple resonance cold probe. The 1H and 13C chem-
ical shifts were referenced to the proto-deutero solvent peaks for
DMSO-d6 at dH 2.49 and dC 39.5 or for CDCl3 at dH 7.26 and dC
TM
with the Opera high-throughput imaging system we are able to
measure the effect of compounds on multiple stages within the
cytotoxicity pathway. In the HCS assays (ꢀ)-dihydroguaiaretic acid
(3) was found to be the most potent, displaying cytotoxicity to-
wards the A549 cell line with an IC50 value of 7.49
lM after 24 h
incubation in both the propidium iodide and YO-PRO-1 assays

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R. A. Davis et al. / Bioorg. Med. Chem. 17 (2009) 1387–1392
Endiandrin A
Endiandrin B
175
150
125
100
75
175
150
125
100
75
PI
YO-PRO-1
mitotracker
50
50
25
25
0
0
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5
log [compound] (M)
log [compound] (M)
Cinbalansan
(-)-Dihydroguaiaretic acid
175
150
125
100
75
175
150
125
100
75
50
50
25
25
0
0
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5
log [compound] (M)
log [compound] (M)
Figure 3. The effect of the compounds 1–4 on three indicators of cytotoxicity. Data is expressed as a percentage of the negative control i.e. untreated and is mean standard
error of the mean from three separate experiments.
Figure 4. The effect of cinbalansan (4) on the uptake of mitotracker by A549 cells. Untreated cells are shown in (a) whilst cells treated with 30 lM cinbalansan for 24 h are
shown in (b). Mitotracker staining is shown in red, Hoechst 34580 staining in blue and phalloidin staining in green. Images were captured using a ꢃ20 objective on the Evotec
Opera (PerkinElmer).
77.0. LRESIMS were recorded on a Waters ZQ mass spectrometer.
HRESIMS were recorded on a Bruker Daltonics Apex III 4.7e Fou-
rier-transform mass spectrometer. An open glass column
(40 mm ꢃ 110 mm) packed with Alltech Davisil diol-bonded silica
4.2. Biological material
The roots of E. anthropophagorum were collected at State Forest
143, Mt Lewis, Queensland, Australia during 2005. A voucher sam-
ple (AQ603481) has been lodged at the Queensland Herbarium,
Brisbane, Australia.
30–40 lm 60 Å was used for flash chromatography. A Waters 600
pump equipped with a Waters 996 PDA detector and a Waters 717
autosampler were used for HPLC. A ThermoElectron C18 Betasil
5
l
m 143 Å column (21.2 mm ꢃ 150 mm) and a YMC diol 5
l
m
4.3. Extraction and isolation
120 Å column (20 mm ꢃ 150 mm) were used for semi-preparative
HPLC separations. All solvents used for chromatography, UV, opti-
cal rotations and MS were Lab-Scan HPLC grade, and the H2O was
Millipore Milli-Q PF filtered. All synthetic reagents were purchased
from Sigma–Aldrich.
The air-dried and ground roots of E. anthropophagorum (250 g)
were exhaustively extracted with DCM (3 ꢃ 1 L). The solvent was
evaporated to yield a dark brown residue (1.23 g) that was dis-
solved in 10% aq MeOH (250 mL) then extracted with hexanes

R. A. Davis et al. / Bioorg. Med. Chem. 17 (2009) 1387–1392
1391
(3 ꢃ 250 mL). The aq MeOH layer was evaporated to dryness (0.7 g)
point half log dose response curves in 100% DMSO before 1 lL of
then pre-adsorbed to diol-bonded silica (ꢄ7 g). The pre-adsorbed
material was loaded onto a diol-bonded silica flash column and a
10% stepwise gradient from 100% hexanes to 100% EtOAc was per-
formed (100 mL washes). The 40% EtOAc/60% hexanes flush con-
tained a complex mixture of lignans (387 mg) that was further
separated by diol semi-preparative HPLC chromatography. A linear
gradient from 100% hexanes to 15% i-PrOH/85% hexanes was run at
a flowrate of 9 mL/min over 60 min, 120 (0.5 min each) fractions
were collected. Fractions 62–65 contained (ꢀ)-dihydroguaiaretic
acid (3, 25.7 mg, 0.010% dry wt), fractions 68–70 yielded endian-
drin A (2, 75.8 mg, 0.030% yield), and fraction 74 afforded endian-
drin B (1, 7.1 mg, 0.0028% dry wt).
each dilution was transferred into the cell plate to give a final con-
centration of 1% DMSO. Cells were incubated with compound for
24 h at 37 °C, 95% humidity, 5% CO2 before being fixed and stained.
Cells were fixed by the addition of 100 lL of 4% paraformaldehyde
into each well and incubated for 20 min at room temperature be-
fore being washed with phosphate buffered saline (PBS) and
stained. Cells were stained with either a mixture of Hoechst
34580, Alexa Fluor 488 phalloidin and mitotracker red, all of which
were diluted in PBS to the required concentration (1/1000, 1/100
and 1/5000, respectively). Alternatively, cells were stained with
DRAQ5, YO-PRO-1 and propidium iodide (1/1000 in PBS). Cells
were incubated with dyes for 20 min in the dark before being
washed with PBS and imaged.
TM
4.3.1. Endiandrin B (1)
Microscopy was performed on an Opera (PerkinElmer), using a
Colourless needles; mp 145–147 °C; ½a D27
ꢁ
0 (c 0.12, CHCl3); UV
e) 233 (3.78), 284 (3.44) nm; IR mmax (KBr)
ꢃ20 objective with a 405 nm excitation line and 420–490 nm band
pass filter for Hoechst 34580 detection, a 488 nm excitation line
and a 510–535 nm band pass emission filter for YO-PRO-1 and
phalloidin detection, a 535 nm excitation line and a 580–620 nm
band pass emission filter for propidium iodide detection and a
635 nm excitation line and a 665–715 nm band pass emission filter
for DRAQ5 and Mitotracker detection.
(MeOH) kmax (log
3600–3100, 1601, 1515, 1462, 1430, 1376, 1262, 1234, 1155,
1124, 1033, 861, 809 cmꢀ1; 1H and 13C NMR data (CDCl3) see Table
1; (+)-LRESIMS m/z (rel. int.) 351 (100) [M+Na]+; (+)-HRESIMS m/z
351.15,787 (C20H24O4Na [M+Na]+ requires 351.15669).
TM
4.4. X-ray data for endiandrin B (1)
Cytotoxicity was quantified using the Acapella analysis soft-
ware. Firstly, cell nuclei were detected using the Opera Nuclear
Detection Library. This algorithm was used to detect nuclei
(S4a—Supplementary data) stained with DRAQ5 or Hoechst using
the parameters of minimum nuclear area, nuclear intensity and
minimum nuclear distance (the minimum distance between two
nuclear centers). Based on these identified nuclei the fluorescence
was detected within the nuclei itself (S4b—Supplementary data),
for YO-PRO-1 and propidium iodide staining, or in a 2-pixel wide
band around the nucleus (S4c—Supplementary data) for mitochon-
drial staining. Fluorescence intensity readings were captured for
each of these three dyes (Mitotracker, YO-PRO-1 and propidium io-
dide) in each cell, and then averaged for all the cells in each field of
Diffraction data were collected on a crystal of 1 at 295 K on a
Rigaku AFC7R rotating anode four circle diffractometer using
monochromated Mo K
a radiation (k = 0.71069 Å) in the monoclinic
space group P21/n with a = 19.501(5), b = 6.676 (5), c = 14.786(3) Å.
The structure was solved by direct methods using the program SIR
-
9719 with atom positions and displacement parameters refined
using SHELXL9720 within the TEXSAN program package.21 Final refine-
ment to convergence was against F2 with 1835 unique reflections
(2h = 50°) to give a final conventional R-factor (I > 2r(I)) of 0.047
and wR2(all data) = 0.152. Non-hydrogen atoms were refined
anisotropically and the H-atoms placed in idealised geometries.
Full crystallographic data for the structure reported in this paper
have been deposited at the Cambridge Crystallographic Data Cen-
tre (CCDC No. 697474). Copies of the data can be obtained, free
of charge, on application to the Director, CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK (Fax: +44 1223 336033; email:
deposit@ccdc.cam.ac.uk).
view (430 ꢃ 345
l
m2). One field was captured in each experiment
with the experiments repeated three times. The positive controls
were colchicine, wortmannin, staurosporine and taxol, and were
found to be cytotoxic with IC50 values of 10.78 ng/mL, 4.28 ng/
mL, 139.95 ng/mL and 19.64 ng/mL, respectively.22,23
Acknowledgments
4.5. Methylation of endiandrin B (1)
The authors thank Conway Lewis and David Camp from the
Molecular Libraries group at the Eskitis Institute, for their assis-
tance in facilitating the isolation of the natural products described
in this article. We thank Hoan The Vu from Griffith University for
acquiring the HRESIMS measurements. We are also indebted to
Paul Forster and Gordon Guymer of the Queensland Herbarium,
Brisbane, Australia for collection and identification of the plant
material.
Endiandrin B (3.0 mg, 9.1 lmol) was dissolved in dry MeOH
(1.5 mL) and Et2O (1.5 mL) then treated with excess CH2N2-Et2O
at 0 °C for 1 h. The reaction was allowed to warm to rt overnight
and the resulting residue was pre-adsorbed to C18 (ꢄ1 g), packed
into stainless steel cartridge (10 ꢃ 30 mm) then attached to a C18
semi-preparative HPLC column. A linear gradient from 100% H2O
to 100% MeOH over 50 min was run at a flowrate of 9 mL/min, iso-
cratic conditions of 100% MeOH were maintained for a further
10 min at a flowrate of 9 mL/min. Sixty (1 min each) fractions were
collected from time = 0 min. Fraction 49 contained a compound
that was spectroscopically identical to the known cyclobutane, cin-
balansan (4, 2.4 mg, 74% yield).10
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.12.030.
4.6. Biology methods
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