Synthesis and antimalarial evaluation of a screening library based on a tetrahydroanthraquinone natural product scaffold

Article abstract of DOI:10.1016/j.bmc.2012.09.052

As part of a research program aimed at discovering new antimalarial leads from Australian macrofungi a unique fungi-derived prefractionated library was screened against a chloroquine-sensitive Plasmodium falciparum line (3D7) using a radiometric growth inhibition assay. A library fraction derived from a Cortinarius species displayed promising antimalarial activity. UV-guided fractionation on the CH₂Cl₂/MeOH extract from this fungus resulted in the isolation of four known compounds: (1S,3R)-austrocortirubin (1), (1S,3S)-austrocortirubin (2), 1-deoxyaustrocortirubin (3), and austrocortinin (4). Compound 2 was used as a natural product scaffold in the parallel solution-phase synthesis of a small library of N-substituted tetrahydroanthraquinones (5-15). All compounds (1-15) were tested in vitro against P. falciparum 3D7 parasites and (1S,3S)-austrocortirubin (2), the major fungal constituent, was shown to be the most active compound with an IC ₅₀ of 1.9 μM. This compound displayed moderate cytotoxicity against neonatal foreskin fibroblast (NFF) cells with an IC₅₀ of 15.6 μM.

Full text of DOI:10.1016/j.bmc.2012.09.052

Bioorganic & Medicinal Chemistry 20 (2012) 7167–7174
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimalarial evaluation of a screening library based on a
tetrahydroanthraquinone natural product scaffold
Vanida Choomuenwai a,b, Katherine T. Andrews a,b, Rohan A. Davis a,
⇑
a Eskitis Institute, Griffith University, Brisbane, QLD 4111, Australia
b Queensland Institute for Medical Research, Brisbane, QLD 4006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2012
Revised 18 September 2012
Accepted 25 September 2012
Available online 10 October 2012
As part of a research program aimed at discovering new antimalarial leads from Australian macrofungi a
unique fungi-derived prefractionated library was screened against a chloroquine-sensitive Plasmodium
falciparum line (3D7) using a radiometric growth inhibition assay. A library fraction derived from a
Cortinarius species displayed promising antimalarial activity. UV-guided fractionation on the CH2Cl2/
MeOH extract from this fungus resulted in the isolation of four known compounds: (1S,3R)-austrocortirubin
(1), (1S,3S)-austrocortirubin (2), 1-deoxyaustrocortirubin (3), and austrocortinin (4). Compound 2 was
used as a natural product scaffold in the parallel solution-phase synthesis of a small library of N-substi-
tuted tetrahydroanthraquinones (5–15). All compounds (1–15) were tested in vitro against P. falciparum
3D7 parasites and (1S,3S)-austrocortirubin (2), the major fungal constituent, was shown to be the most
Keywords:
Tetrahydroanthraquinone
Natural product
Austrocortirubin
Scaffold
active compound with an IC50 of 1.9
lM. This compound displayed moderate cytotoxicity against neona-
tal foreskin fibroblast (NFF) cells with an IC50 of 15.6
l
M.
Macrofungi
Malaria
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
1. Introduction
As part of our continuing research into the discovery of new
antimalarial leads from nature8–12 we screened a unique prefrac-
tionated fungi-derived library (2035 fractions) against a chloro-
Malaria is a significant global infectious disease, resulting in the
deaths of 0.8–1.2 million people annually, and predominantly af-
fects children under five years of age.1 Despite recent preliminary
data on a partially effective pre-erythrocytic stage vaccine (RTS,
S),2 malaria prevention and treatment presently relies on vector
control and small molecule drugs. It is forseen that even if the
RTS, S vaccine is approved for clinical use, it will be implemented
as part of a multi-pronged approach that will also include antima-
larial drugs in order to eradicate the parasite. Whilst a number of
drugs and drug combinations are used therapeutically to treat ma-
laria, all have now succumbed to parasite drug resistance and/or
reduced efficacy.3 This, together with the fact that only five of
the ꢀ1700 new drugs registered over the last 30 years were anti-
malarials,4 highlights the need for the discovery and development
of new antimalarial therapeutics.
Historically, natural products have played a significant role in
the development of antimalarial drugs.5–7 Notable examples in-
clude artemisinin from the Chinese medicinal plant Artemisia
annua, and quinine, which was isolated from the bark of the South
American tree Cinchona succirubra.5–7 This research has subse-
quently led to the development of numerous antimalarial drugs
based on these two important plant natural products.7
quine-sensitive Plasmodium falciparum line (3D7), using
a
radiometric growth inhibition assay. A total of 20 fractions derived
from 18 fungal species (10 genera, 8 families) showed promising
antimalarial activity; this equated to a screening hit-rate of
0.98%. UV spectrum analysis of one antimalarial fraction derived
from a Cortinarius species identified kmax values of 230 and
305 nm that were predicted to correspond to the compound(s)
responsible for the observed antiparasitic activity. Large-scale
UV-guided fractionation of the CH2Cl2/MeOH extract from this
fungus resulted in the isolation of four known compounds:
(1S,3R)-austrocortirubin (1),13 (1S,3S)-austrocortirubin (2),14,15
1-deoxyaustrocortirubin (3),14,15 and austrocortinin (4)16 (Fig. 1).
Compound 2 was identified as a natural product scaffold that could
be used in the generation of a tetrahydroanthraquinone-based
compound library. Parallel solution-phase synthesis utilising scaf-
fold 2 with 11 primary amines afforded a small library of N-substi-
tuted tetrahydroanthraquinones (5–15). Herein we report the
design and synthesis of 5–15, along with the in vitro antimalarial
activity and mammalian cell toxicity for the natural products (1–
4) and semi-synthetic analogues (5–15).
2. Results and discussion
The air-dried and ground sample of Cortinarius sp. was
sequentially extracted with CH2Cl2 and MeOH. The combined
⇑
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 Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.052

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V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
O
C
O
OH
B
O
OH
O
O
OH
O
OH
O
OH
O
OH
O
O
OH
A
OH OH
O
OH OH
OH
O
OH
3
1
2
4
Figure 1. Chemical structures for (1S,3R)-austrocortirubin (1), (1S,3S)-austrocortirubin (2), 1-deoxyaustrocortirubin (3) and austrocortinin (4).
CH2Cl2/MeOH extract was subjected to UV-guided fractionation
using semi-preparative C18 HPLC. This yielded the previously iso-
lated tetrahydroanthraquinones, (1S,3R)-austrocortirubin (1),13
(1S,3S)-austrocortirubin (2),14,15 and 1-deoxyaustrocortirubin
(3).14,15 A late eluting fraction from the HPLC fractionation was fur-
ther purified by gel permeation chromatography to afford the
known fungal anthraquinone, austrocortinin (4).16 All compounds
were identified on the basis of spectroscopic and spectrometric
data and in comparison with the literature values.
The polyketide-derived natural products 1–4 were originally
isolated from the Australian toadstool, Dermocybe splendida, by Gill
and co-workers and had their structures tentatively assigned on
chemical and spectroscopic grounds, and were subsequently con-
firmed by single crystal X-ray analyses on several semi-synthetic
derivatives.13–16 The tetrahydroanthraquinone class has been iden-
tified as possessing moderate activity towards a variety of bacteria
and fungi.14,17 For example, (1S,3S)-austrocortirubin (2) displayed
antibacterial activity against the Gram-positive bacteria Staphylo-
4 in the solvent vehicle (DMSO) used for in vitro screening. Cytotox-
icity studies with neonatal foreskin fibroblast (NFF) cells showed
that compounds 1–3 all had approximately 8- to 12-fold better
selectivity for the parasite versus the mammalian cell line (Table 1).
Inspired by the literature reports of high-yielding N-alkylations
of quinone motifs, such as those present in 1–3, we decided to ex-
ploit this chemistry for tetrahydroanthraquinone analogue genera-
tion and potential structure activity relationship (SAR) studies.18–21
This additional research had the potential to lead to compounds
with increased potency, better selectivity and/or an increased sta-
bility toward biogenic nucleophiles.18–21 (1S,3S)-Austrocortirubin
(2) was chosen for the semi-synthetic studies since this molecule
displayed the best in vitro antimalarial activity (with a similar
selectivity to 1 and 3), and was available in suitable quantities
(>50 mg) for library generation due to its moderate yield from the
toadstool.8,22,23 Furthermore, the low Mw (320 Da), favourable
cLogP (0.82), and multiple stereogenic centres (n = 2) made 2 a
suitable natural product scaffold for library synthesis (Table 1).24–27
The concept of isolating a natural product scaffold for lead opti-
misation studies or screening library generation has previously
been reported in the literature, although relatively few examples
exist.28 The purification and subsequent use of tambjamine,22 tei-
coplanin,29 yohimbine,30 fredericamycin A21 and muurolane8 scaf-
folds in the synthesis of biodiscovery and lead optimisation
libraries all exemplify this approach. This strategy is dependent
on: (1) access to adequate biota supplies for the large-scale isola-
tion of the desired scaffold; (2) the abundance or high-yielding nat-
ure of the compound of interest from the biota source; and (3) the
presence of chemical handles (e.g. –COOH, –OH, –NH2, Ar–Br) on
the scaffold that allow for simple and high-yielding analogue
generation. Isolation of a desired scaffold, rather than de novo
coccus aureus with an IC50 of 3
lg/mL, and was found to be inactive
(IC50 >50
lg/mL) towards the Gram-negative bacteria Pseudomonas
aeruginosa.17
Compounds 1–4 were tested in vitro against the chloroquine-
sensitive 3D7 and chloroquine-resistant Dd2 P. falciparum lines
and displayed IC50 values of 4.7, 1.9, 4.5, and >50
and 11.1, 2.2, 5.2, and >50 M, respectively. Compound 2 was the
most potent molecule against both P. falciparum lines (IC50 1.9–
2.2 M) while compound 4, the only anthraquinone screened dur-
ing these studies, showed no antimalarial activity (IC50 >50 M).
lM (Table 1)
l
l
l
While the lack of antiparasitic activity of 4 could possibly be due
to the chemical difference in ring A (Fig. 1) of this molecule com-
pared to 1–3, it should be noted that we had difficulty solubilising
Table 1
Physicochemical and biological profiles of compounds 1–15
Compound
Physicochemical parametersa
IC50 SD (lM)
MW
cLogP
HBA
HBD
3D7b
4.7 0.4
1.9 0.4
4.5 1.0
>50
6.7 1.2
4.3 1.9
16.5 3.5
10.3 0.4
3.8 0.5
2.4 0.8
4.2 0.6
15.8 1.9
7.4 0.3
6.9 3.9
6.3 1.8
0.04 0.004
NFFc
SId
9.3
8.2
12.1
>1.5
5.4
18.3
5.4
8.2
0.9
9.7
15.7
3.9
1.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
320
320
304
286
462
347
362
363
409
425
443
418
395
319
402
319
0.82
0.82
2.13
2.70
1.26
1.39
-0.81
0.46
2.52
2.22
3.12
0.31
2.23
0.51
0.93
3.93
7
7
6
5
8
7
8
8
7
8
7
9
7
7
8
4
4
4
3
2
6
5
6
5
5
6
5
5
5
5
5
1
43.7 7.3
15.6 4.1
54.6 9.4
76.0 8.6
36.5 0.8
78.8 15.5
89.8 6.2
84.3 0.3
3.5 0.6
23.3 5.1
66.0 0.6
62.0 1.0
10.7 1.5
59.1 6.9
15.9 5.0
37.3 3.4
8.6
2.5
932.5
Chloroquine
a
In silico calculations performed using Instant JChem software,32 MW, molecular weight (Da); cLogP, calculated partition coefficient; HBA = H-bond acceptors;
HBD = H-bond donors.
b
3D7, P. falciparum chloroquine sensitive line.
NFF, neonatal foreskin fibroblast cells.
SI, selectivity index = NFF cell-line IC50/P. falciparum IC50
c
d
.

V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
7169
O
O
OH
O
OH
H
N
OH
O
OH
R-NH2
R
EtOH : MeCN (1:1)
rt, 16 h
OH OH
O
OH OH
2
5-15
Scheme 1. Reaction conditions for the amino alkylation of (1S,3S)-austrocortirubin (2).
synthesis, can prove to be both a cost- and time-effective mecha-
nism for lead optimisation studies.
In order to obtain larger quantities of (1S,3S)-austrocortirubin
for our semi-synthetic studies additional large-scale extraction
and isolation work was conducted on the air-dried and ground
Cortinarius material, which yielded 91 mg of 2.
drug-like molecules for subsequent synthesis. On the basis of the
in silico data (Table 1), 11 primary amines were initially prioritised
and subsequently reacted with scaffold 2 in EtOH/MeCN at room
temperature for 16 h (Scheme 1). Purification of all reaction mix-
tures by gel permeation chromatography gave the desired products
5–15 in moderate to good yields (Fig. 2).
Prior to semi-synthetic studies commencing on scaffold 2, a vir-
tual library was constructed using a list of commercially available
primary amines (30 in total)31 and Instant JChem software32 and
the data compared to Lipinski’s drug-like ‘‘Rule of Five’’27
(MW < 500; HBA < 10; HBD < 5; LogP < 5) criteria. The physico-
chemical parameter profiles (MW, HBA, HBD, LogP) for all virtual
molecules were analysed in an effort to select the most desirable
The chemical structures and NMR chemical shift assignments of
all new tetrahydroanthraquinone analogues (5–15) were deter-
mined following 1D (1H, 13C) and 2D NMR (COSY, HSQC, HMBC
and ROESY) and (+)-HRESIMS data analysis.
For example, the 1H NMR spectrum for compound 5 in CDCl3
showed signals at dH 1.43 (3H, s), 1.90 (1H, dd, J = 14.2, 4.9 Hz),
2.32 (1H, br d, J = 14.2 Hz), 2.51 (1H, d, J = 18.5 Hz), 3.16 (1H, dd,
O
5
OH
O
OH
H
N
H
N
H
N
10
OH
OH
5a
8a
14
4a
3
O
15
13
11
O
7
9a
9
1
O
OH OH
O
OH OH
6
5
O
O
OH
O
OH
O
OH
H
N
H
H
OH
OH
OH
OH
H2N
OH
N
N
O
OH OH
O
O
OH OH
OH
O
OH OH
9
7
8
O
OH
O
O
OH
H
N
H
H
N
OH
Cl
OH
N
N
O
HO
O
OH OH
OH
O
O
OH OH
10
OH OH
11
12
O
OH
O
OH
H
H
N
H
N
OH
OH
N
N
O
OH OH
O
OH OH
O
OH OH
15
13
14
Figure 2. Chemical structures of the semi-synthetic tetrahydroanthraquinones 5–15.
O
OH
O
OH
H
H
N
H
N
H
N
OH
OH
O
N
O
O
O
O
OH OH
O
OH OH
Figure 3. Key HMBC (?) and ROESY (M) correlations for 5.

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V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
O
O
O
O
O
O
O
OH
HO
O
O
O
O
16
18
17
O
O
O
O
OH
O
OH
Cl
H
H
O
O
20
Cl
19
21
Figure 4. Quinone-based antimalarial compounds.
J = 18.5, 1.7 Hz), 5.21 (1H, br d, J = 4.9 Hz), and 12.24 (1H, s), 14.10
(1H, s), which were essentially identical, in respect to magnitude
and multiplicities, to the proton resonances corresponding to the
2.2- and 1.9-fold better selectivity. The lack of significant SAR for
analogues 5–15 indicates that ring A plays a more important role
for bioactivity than ring C (Fig. 1) and thus any future medicinal
chemistry on this unique tetrahydroanthraquinone core should
focus on generating analogues that would probe ring A for SAR.
A variety of quinone-based antimalarial natural products have
been isolated from fungi, lichens, plants and marine inverte-
brates.35 Plant-derived examples include the napthoquinoids,
2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (16) and
isopinnatal (17) from Kigelia pinnata, which both inhibited the P.
3-methyl-1,2,3,4-tetrahydronaphthalene-1,3,5,8-tetraol
system
(rings A and B) present in the known natural product, (1S,3S)-
austrocortirubin (2).14,15 Additional proton signals for 5 were iden-
tified at dH 1.46 (9H, s), 1.84 (2H, q, J = 6.7 Hz), 3.25 (2H, m), 3.29
(2H, m), 4.77 (1H, br s) and 6.61 (1H, br s), and were assigned to a
mono-Boc protected 1,3-diaminepropyl moiety following interpre-
tation of the gCOSY, gHSQC and gHMBC data. The N-alkylation of
the tetrahydroanthraquinone core was deemed to have taken place
at C-6, on the basis of a literature report on the related mono-meth-
oxylated quinone, fredericamycin A.21 Semi-synthetic studies on
this Streptomyces-derived natural product had shown that the
methoxy group was readily replaced by nucleophiles such as pri-
mary amines.21 Confirmation of the N-alkylation position for 5
was also supported by the absence of methoxy NMR signals in con-
junction with gHMBC and ROESY correlations (Fig. 3). These data
enabled the chemical structure of 5 to be unequivocally assigned.
The relative configuration and absolute stereochemistry of 5–15
were all shown to be identical to that of the starting material,
(1S,3S)-austrocortirubin (2), based on 1H–1H coupling constants,
the magnitude of the 1H NMR chemical shifts, and specific rotation
comparisons.14,15 It is noteworthy that the magnitude of the spe-
cific rotation values for the semi-synthetic analogues 5–15 all
showed larger than expected values (+725 to +1520) in MeOH
compared to the natural product scaffold and starting material,
falciparum K1 line with IC50 values of 0.63 and 0.76 lM, respec-
tively (Fig. 4).36 The benzoquinone metabolites, xylariaquinone A
(18) and 2-chloro-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-
dione (19), were isolated from an endophytic fungus Xylaria sp.
and were reported to display IC50 values towards P. falciparum K1
line of 1.84 and 6.68 l
M, respectively.37 It is noteworthy that lapa-
chol (20), a hydroxynapthoquinone isolated from several South
American species belonging to the plant family Bignoniaceae,38
served as a drug lead for the synthetic antimalarial drug atovaqu-
one (21), which is typically co-administered with proguanil in the
treatment of P. falciparum infections. The mechanism of action for
atovaquone has been determined and involves the inhibition of the
cytochrome bc1 complex in Plasmodium parasites.39–41 On the
basis of this data it has been hypothesized that many of the previ-
ously reported quinone-based antimalarial natural products may
also interfere with the mitochondrial electron transport system
in the malaria parasite.42–44
20
25
(1S,3S)-austrocortirubin {[
a]
+34 (c 0.543, CHCl3),14,15
[a]
D
D
3. Conclusion
+172 (c 0.2, MeOH)}. Subsequent literature searching for related
optically active N-alkylated quinones identified examples such as
+1010 (c, 0.1, CHCl3)},33 and rubradirin {[
a]
20
25
In summary, UV-guided fractionation on the bioactive CH2Cl2/
MeOH extract from the Australian toadstool Cortinarius sp. resulted
in the isolation of four known compounds: (1S,3R)-austrocortiru-
bin (1), (1S,3S)-austrocortirubin (2), 1-deoxyaustrocortirubin (3),
and austrocortinin (4). Compound 2 was isolated in quantities that
allowed its use as a natural product scaffold in the generation of a
small tetrahydroanthraquinone-based compound library (5–15).
All compounds were tested in vitro against a chloroquine-sensitive
P. falciparum (3D7) malaria parasite line and (1S,3S)-austrocortiru-
bin (2), the major fungal constituent, was shown to be the most ac-
awamycin {[
a
]
D
D
+777 (c 0.028, (CH3)2CO)},34 which also displayed large specific
rotations. Furthermore, compounds 5–15, awamycin and rubradi-
rin are all red, and have UV chromophores that typically extend
past 500 nm.33,34 We postulate that the UV absorption characteris-
tics of these molecules may be the cause for the large specific
rotations.
Compounds 5–15 were all tested for in vitro growth inhibitory
activity against P. falciparum 3D7 parasites and mammalian cell
toxicity using the NFF cell line (Table 1). Eight of the 11 semi-
synthetic analogues (5, 6, 9–11, 13–15) displayed IC50 values
<7.4 lM against the malaria parasite that were comparable to
the natural products 1–3. While none of the synthons displayed
tive compound with an IC50 of 1.9
moderate cytotoxicity against neonatal foreskin fibroblast (NFF)
cells with an IC50 of 15.6 M. This is the first report of antimalarial
activity for the tetrahydroanthraquinone structure class.
lM. Compound 2 displayed
l
improved potency compared to 2, compounds 6 and 11 showed

V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
7171
(2, 6.0 mg, 0.24% dry wt) and 1-deoxyaustrocortirubin (3, 4.5 mg,
0.18% dry wt). In addition, fraction 55 obtained from the first iso-
lation step, was further purified by gel permeation chromatogra-
phy [Sephadex LH20, MeOH/CH2Cl2 (1:1)] at a flow rate of
1.0 mL/min to yield austrocortinin (4, 12.0 mg, 0.48% dry wt). Iden-
tical extraction and isolation conditions were employed on 50 g of
Cortinarius sp. in order to obtain larger quantities of scaffold 2
(91.0 mg, 1.68% dry wt.) for the semi-synthetic studies.
4. Experimental section
4.1. Materials and methods
Optical rotations were recorded on a JASCO P-1020 polarimeter.
NMR spectra were recorded at 30 °C on either a Varian 500 MHz or
a 600 MHz Unity INOVA spectrometer. 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 CD3OD at
dH 3.31 and dC 49.0, and for CDCl3 at dH 7.26 and dC 77.0, respec-
tively. LRESIMS spectra were recorded on a Waters ZQ mass spec-
trometer. HRESIMS data were recorded on a Bruker Daltronics
Apex III 4.7e Fourier-transform mass spectrometer. A BIOLINE orbi-
tal shaker was used for the large-scale extractions of the fungal
4.3.1. (1S,3R)-Austrocortirubin (1)
Red amorphous solid; NMR, MS, and [
a
a
a
]
D
]
D
]
D
data were consistent
data were consistent
data were consistent
with the literature values.13
4.3.2. (1S,3S)-Austrocortirubin (2)
Red amorphous solid; NMR, MS, and [
with the literature values.14,15
material. Alltech Davisil 40–60 lm 60 Å C18 bonded silica was used
for pre-adsorption of extracts or fractions and was packed into an
open glass column (100 mm  200 mm) for C18 flash chromatogra-
phy. A Waters 600 pump equipped with a Waters 996 PDA detec-
tor, and either a Waters 717 autosampler or Gilson 215 liquid
handler were used for HPLC. An Alltech stainless steel guard car-
tridge (10 mm  30 mm) was used for loading pre-adsorbed ex-
tracts or fractions onto the semi-preparative HPLC columns. A
Phenomenex C18 ONYX column (4.6 mm  100 mm) was used for
analytical HPLC associated with the library generation. A Thermo
4.3.2.1. 1-Deoxyaustrocortirubin (3)
Red amorphous solid; NMR, MS, and [
with the literature values.14,15
4.3.3. Austrocortinin (4)
Orange amorphous solid; NMR and MS were consistent with the
literature values.16
Electron C18 Betasil column (5
and a Phenomenex Luna C18 column (5
l
m, 143 Å, 21.2 mm  150 mm)
4.4. General procedure for the amino alkylation reaction18
l
m, 21.2 mm  250 mm)
were used for semi-preparative HPLC separation. Gel permeation
chromatography was used for natural product purification (Sepha-
dex LH20, 50 mm  120 mm, open column) and synthetic reaction
products purification (Sephadex LH20, 25 mm  290 mm, open
Scaffold 2 (5.0 mg, 0.016 mmol), EtOH (0.5 mL), MeCN (0.5 mL)
and the relevant primary amine (0.08 mmol, 5 mol excess) were
stirred for 16 h at room temperature under Ar. The solution was
evaporated to dryness and redissolved in CH2Cl2 (20 mL), before
being extracted with H2O (20 mL). The CH2Cl2 extract was purified
by gel permeation chromatography [Sephadex LH20, CH2Cl2:MeOH
(1:1)] at a flow rate of 1.0 mL/min. Nine fractions (1 min each)
were collected after 20 min, evaporated to dryness and analysed
by analytical HPLC to determine purity.
column). All solvents used for chromatography, [a]D, UV, and MS
were Lab-Scan HPLC grade, and the H2O was Millipore Milli-Q PF
filtered. All amine reagents were purchased from Sigma-Aldrich.
4.2. Fungal material
The toadstool Cortinarius sp. (Family: Cortinariaceae) was col-
lected in South-East Queensland, Australia, on the 1st May 2003.
A voucher sample (0801050310001075) has been lodged at the
Queensland Herbarium, Brisbane, Australia.
4.4.1. Compound 5
Dark red powder, (4.4 mg, 77%); [
UV (MeOH) kmax (log
29
a
]
D
+725 (c 0.004, MeOH);
e
) 213 (3.82), 266 (3.83), 325 (3.33), 511
(3.45) nm; 1H NMR (500 MHz, CDCl3) dH 1.45 (3H, s, 3-Me), 1.46
(9H, s, H-16), 1.84 (2H, tt, J = 6.7, 6.7 Hz, H-12), 1.90 (1H, dd,
J = 14.2, 4.9 Hz, H-2ax), 2.32 (1H, ddd, J = 14.2, 1.7, 1.7 Hz, H-2eq),
2.51 (1H, d, J = 18.5 Hz, H-4ax), 3.16 (1H, dd, J = 18.5, 1.7 Hz,
H-4eq), 3.25 (2H, m, H-13), 3.29 (2H, m, H-11), 4.77 (1H, br s,
13-NH), 5.21 (1H, dd, J = 4.9, 1.7 Hz, H-1), 5.62 (1H, s, H-7), 6.61
(1H, br s, 6-NH), 12.24 (1H, s, 10-OH), 14.10 (1H, s, 9-OH); 13C
NMR (125 MHz, CDCl3) dC 28.4 (C-16), 28.7 (3-Me), 28.7 (C-12),
37.3 (C-4), 37.6 (C-13), 39.8 (C-11), 40.0 (C-2), 63.1 (C-1), 68.1
(C-3), 80.0 (C-15), 108.9 (C-8a), 100.1 (C-7), 110.0 (C-5a), 133.1
(C-4a), 138.6 (C-9a), 149.2 (C-6), 155.2 (C-9), 156.7 (C-14), 157.3
(C-10), 183.3 (C-5), 186.9 (C-8); (À)-LRESIMS m/z (rel. int.) 461
(100) [MÀH]À; (+)-LRESIMS m/z (rel. int.) 445 (20) [MÀOH]+, 463
(100) [M+H]+; (+)-HRESIMS m/z 485.1899 (C23H30N2O8Na
[M+Na]+ requires 485.1894).
4.3. Extraction and isolation
The air-dried and ground fruit-bodies of Cortinarius sp. (10 g)
were transferred to a conical flask (1 L) and extracted with CH2Cl2
(250 mL), the mixture was shaken at 200 rpm for 2 h. The resulting
extract was filtered under gravity, and set aside. MeOH (250 mL)
was added to the fungal sample, and the flask shaken at 200 rpm
for 2 h before filtration. Another volume of MeOH (250 mL) was
then added and the MeOH/fungal mixture shaken for a further
16 h at 200 rpm, followed by gravity filtration. Finally, all CH2Cl2
and MeOH extracts were combined and dried under reduced pres-
sure to yield a red gum (3.57 g). A portion of the CH2Cl2/MeOH ex-
tract (0.5 g) was re-suspended in MeOH/CH2Cl2, pre-adsorbed to
C18 silica and packed into a stainless steel guard cartridge that
was subsequently attached to a semi-preparative C18 Betasil HPLC
column. Isocratic conditions of 10% MeOH/90% H2O were initially
performed for the first 10 min, followed by a linear gradient to
MeOH in 40 min, then isocratic conditions of MeOH were run for
10 min all at a flow rate of 9 mL/min. Sixty fractions (60 Â 1 min)
were collected from the start of the HPLC run and fractions con-
taining UV absorbing material at k 230 and 305 nm were analysed
by ( )-LRESIMS and 1H NMR spectroscopy. Three pure compounds
were obtained after the first C18 HPLC fractionation; (1S,3R)-
austrocortirubin (1, 3.0 mg, 0.12% dry wt), (1S,3S)-austrocortirubin
4.4.2. Compound 6
29
Red powder, (4.0 mg, 59%); [
a]
+1450 (c 0.001, MeOH); UV
D
(MeOH) kmax (log
e
) 213 (4.08), 265 (3.99), 325 (3.49), 510 (3.61)
nm; 1H NMR (500 MHz, CDCl3) dH 1.06 (3H, t, J = 7.5 Hz, H-13),
1.48 (3H, s, 3-Me), 1.78 (2H, qt, J = 7.5, 7.0 Hz, H-12), 1.93 (1H,
dd, J = 14.8, 5.0 Hz, H-2ax), 2.35 (1H, ddd, J = 14.8, 1.7, 1.7 Hz,
H-2eq), 2.54 (1H, d, J = 18.4 Hz, H-4ax), 3.17 (1H, d, J = 18.4 Hz,
H-4eq), 3.20 (2H, dt, J = 7.0, 5.8 Hz, H-11), 5.25 (1H, dd, J = 5.0,
1.7 Hz, H-1), 5.68 (1H, br s, H-7), 6.13 (1H, t, J = 5.8 Hz, 6-NH),
12.28 (1H, s, 10-OH), 14.18 (1H, s, 9-OH); 13C NMR (125 MHz,

7172
V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
CDCl3) dC 11.7 (C-13), 21.7 (C-12), 30.2 (3-Me), 37.6 (C-4), 40.3
(C-2), 44.6 (C-11), 63.4 (C-1), 68.0 (C-3), 100.6 (C-7), 109.0
(C-8a), 110.4 (C-5a), 133.2 (C-4a), 138.7 (C-9a), 149.1 (C-6), 155.2
(C-9), 157.3 (C-10), 183.5 (C-5), 187.1 (C-8); (À)-LRESIMS m/z
(rel. int.) 346 (100) [MÀH]À; (+)-LRESIMS m/z (rel. int.) 330 (100)
[MÀOH]+, 348 (80) [M+H]+, 717 (80) [2M+Na]+; (+)-HRESIMS m/z
370.1266 (C18H21NO6Na [M+Na]+ requires 370.1261).
5.08 (1H, br s, H-1), 5.61 (1H, s, H-7), 6.74 (2H, br d, J = 8.0 Hz,
H-15, H-17), 7.10 (2H, br d, J = 8.0 Hz, H-14, H-18); 13C NMR
(125 MHz, CD3OD) dC 30.1 (3-Me), 34.3 (C-12), 38.2 (C-4), 41.6
(C-2), 45.4 (C-11), 63.8 (C-1), 69.5 (C-3), 100.4 (C-7), 109.9
(C-8a), 111.4 (C-5a), 116.6 (2C, C-15, C-17), 130.5 (C-13), 130.8
(C-14, C-18), 134.0 (C-4a), 139.9 (C-9a), 150.9 (C-6), 155.9 (C-16),
156.0 (C-9), 157.2 (C-10), 184.8 (C-5), 188.3 (C-8); (À)-LRESIMS
m/z (rel. int.) 424 (100) [MÀH]À; (+)-HRESIMS m/z 448.1388
(C23H23NO7Na [M+Na]+ requires 448.1367).
4.4.3. Compound 7
29
Red powder, (5.0 mg, 76%); [
(MeOH) kmax (log
a]
+1050 (c 0.003, MeOH); UV
D
e) 213 (4.09), 265 (3.92), 325 (3.50), 507 (3.46)
4.4.7. Compound 11
nm; 1H NMR (500 MHz, CD3OD) dH 1.39 (3H, s, 3-Me), 1.86 (2H,
tt, J = 7.1, 7.1 Hz, H-12), 1.92 (1H, br dd, J = 14.4, 4.3 Hz, H-2ax),
2.16 (1H, br d, J = 14.4 Hz, H-2eq), 2.55 (1H, br d, J = 18.0 Hz,
H-4ax), 2.78 (2H, br t, J = 7.1 Hz, H-13), 3.29 (2H, br t, J = 7.1 Hz,
H-11), 3.02 (1H, br d, J = 18.0 Hz, H-4eq), 5.07 (1H, br s, H-1), 5.59
(1H, br s, H-7); 13C NMR (150 MHz, CD3OD) dC 30.1 (3-Me), 31.3
(C-12), 38.3 (C-4), 39.9 (C-13), 41.3 (C-11), 41.6 (C-2), 63.9 (C-1),
69.5 (C-3), 100.2 (C-7), 110.1 (C-8a), 111.6 (C-5a), 134.6 (C-4a),
139.7 (C-9a), 151.4 (C-6), 156.2 (C-9), 157.8 (C-10), 185.8 (C-5),
188.0 (C-8); (À)-LRESIMS m/z (rel. int.) 361 (100) [MÀH]À; (+)-LRE-
SIMS m/z (rel. int.) 363 (100) [M+H]+; (+)-HRESIMS m/z 363.1561
(C18H23N2O6 [M+H]+ requires 363.1551).
Red powder, (8.0 mg, 90%); [
a]
+1520 (c 0.001, MeOH); UV
29
D
(MeOH) kmax (log
e
) 214 (3.95), 266 (3.74), 325 (3.23), 512
(3.33) nm; 1H NMR (500 MHz, CD3OD) dH 1.39 (3H, s, 3-Me), 1.91
(1H, dd, J = 14.5, 4.0 Hz, H-2ax), 2.17 (1H, br d, J =14.5 Hz, H-2eq),
2.56 (1H, br d, J = 18.5 Hz, H-4ax), 2.77 (2H, br t, J = 7.0 Hz, H-12),
3.04 (1H, br d, J = 18.5 Hz, H-4eq), 3.48 (2H, br t, J = 7.0 Hz, H-11),
5.08 (1H, br s, H-1), 5.61 (1H, s, H-7), 7.28 (2H, br dd, J = 7.6,
2.5 Hz, H-14, H-18), 7.32 (2H, br d, J = 7.6 Hz, H-15, H-17); 13C
NMR (125 MHz, CD3OD) dC 30.1 (3-Me), 34.3 (C-12), 38.2 (C-4),
41.6 (C-2), 44.8 (C-11), 63.9 (C-1), 69.5 (C-3), 100.4 (C-7), 109.8
(C-8a), 111.4 (C-5a), 129.6 (2C, C-15, C-17), 131.4 (2C, C-14,
C-18), 133.5 (C-16), 134.4 (C-4a), 138.7 (C-13), 139.9 (C-9a),
151.0 (C-6), 156.1 (C-9), 158.5 (C-10), 184.5 (C-5), 188.1 (C-8);
(À)-LRESIMS m/z (rel. int.) 442 (100) [MÀH]À; (+)-HRESIMS m/z
466.1047 (C23H22ClNO6Na [M+Na]+ requires 466.1027).
4.4.4. Compound 8
29
Red powder, (5.1 mg, 75%); [
a]
+1500 (c 0.002, MeOH); UV
D
(MeOH) kmax (log e) 213 (4.13), 264 (4.03), 325 (3.54), 508 (3.63)
nm; 1H NMR (500 MHz, CD3OD) dH 1.40 (3H, s, 3-Me), 1.93 (1H,
dd, J = 15.0, 4.8 Hz, H-2ax), 2.18 (1H, ddd, J = 15.0, 2.2, 2.2 Hz, H-
4.4.8. Compound 12
29
Red powder, (6.9 mg, 97%); [
a]
+1450 (c 0.002, MeOH); UV
D
2
eq), 2.58 (1H, bd, J = 19.9 Hz, H-4ax), 3.05 (1H, br d, J = 19.9 Hz,
(MeOH) kmax (log e) 214 (3.53), 264 (3.38), 323 (2.93), 510
H-4eq), 3.41 (3H, s, H-13), 3.43 (2H, t, J = 6.3 Hz, H-11), 3.64 (2H,
t, J = 6.3 Hz, H-12), 5.09 (1H, dd J = 4.8, 2.2 Hz, H-1), 5.68 (1H, br
s, H-7); 13C NMR (125 MHz, CD3OD) dC 30.0 (3-Me), 38.2 (C-4),
41.6 (C-2), 43.3 (C-11), 59.1 (C-13), 63.9 (C-1), 69.5 (C-3), 70.8
(C-12), 100.7 (C-7), 109.9 (C-8a), 111.4 (C-5a), 134.2 (C-4a), 139.9
(C-9a), 151.2 (C-6), 156.2 (C-9), 157.9 (C-10), 184.6 (C-5), 188.3
(C-8); (À)-LRESIMS m/z (rel. int.) 362 (100) [MÀH]À; (+)-LRESIMS
m/z (rel. int.) 364 (100) [M+H]+; (+)-HRESIMS m/z 386.1203
(C18H21NO7Na [M+Na]+ requires 386.1210).
(2.95) nm; 1H NMR (500 MHz, CD3OD) dH 1.40 (3H, s, 3-Me), 1.92
(1H, br dd, J = 15.5, 5.0 Hz, H-2ax), 2.17 (1H, br d, J =15.5 Hz,
H-2eq), 2.52 (4H, m, H-13, H-16), 2.58 (1H, br d, J = 18.5 Hz,
H-4ax), 2.69 (1H, br t, J = 6.5 Hz, H-12), 3.04 (1H, br d, J = 18.5 Hz,
H-4eq), 3.34 (2H, br t, J = 6.5 Hz, H-11), 3.70 (4H, m, H-14, H-15),
5.09 (1H, br s, H-1), 5.64 (1H, s, H-7); 13C NMR (125 MHz, CD3OD)
dC 30.1 (3-Me), 38.3 (C-4), 40.0 (C-2), 41.6 (C-11), 54.6 (C-13, C-16),
54.8 (C-12), 63.9 (C-1), 67.8 (C-14, C-15), 69.5 (C-3), 100.6 (C-7),
109.9 (C-8a), 111.5 (C-5a), 134.3 (C-4a), 140.0 (C-9a), 151.2 (C-6),
156.3 (C-9), 158.2 (C-10), 185.9 (C-5), 188.3 (C-8); (À)-LRESIMS
m/z (rel. int.) 417 (100) [MÀH]À; (+)-LRESIMS m/z (rel. int.) 401
(20) [MÀOH]+; 419 (100) [M+H]+; (+)-HRESIMS m/z 441.1639
(C21H26N2O7Na [M+Na]+ requires 441.1632).
4.4.5. Compound 9
29
Red powder, (5.9 mg, 70%); [
a
]
D
+1450 (c 0.001, MeOH); UV
(MeOH) kmax (log e) 213 (3.92), 264 (3.74), 325 (3.23), 506 (3.34)
nm; 1H NMR (500 MHz, CDCl3) dH 1.45 (3H, s, 3-Me), 1.89 (1H,
dd, J = 14.8, 5.0 Hz, H-2ax), 2.32 (1H, ddd, J = 14.8, 1.7, 1.7 Hz, H-
4.4.9. Compound 13
29
2eq), 2.51 (1H, d, J = 18.5 Hz, H-4ax), 3.00 (2H, t, J = 7.0 Hz, H-12),
Red powder, (3.3 mg, 80%); [
a]
+1440 (c 0.002, MeOH); UV
D
3.14 (1H, dd, J = 18.5, 1.5 Hz, H-4eq), 3.48 (2H, dt, J = 7.0, 6.5 Hz,
H-11), 5.22 (1H, dd, J = 5.0, 1.7 Hz, H-1), 5.69 (1H, br s, H-7), 6.12
(1H, br t, J = 6.5 Hz, 6-NH), 7.23 (2H, br d, J = 7.5 Hz, H-14, H-18),
7.29 (1H, br d, J = 7.5 Hz, H-16), 7.35 (2H, br d, J = 7.5 Hz, H-15,
H-17); 13C NMR (125 MHz, CDCl3) dC 30.1 (3-Me), 34.5 (C-12),
37.6 (C-4), 40.3 (C-2), 44.0 (C-11), 63.3 (C-1), 68.0 (C-3), 100.8
(C-7), 108.9 (C-8a), 110.4 (C-5a), 127.3 (C-16), 128.8 (C-14, C-18),
129.1 (C-15, C-17), 133.4 (C-4a), 137.7 (C-13), 138.8 (C-9a), 148.9
(C-6), 155.2 (C-9), 157.3 (C-10), 183.3 (C-5), 187.0 (C-8); (À)-LRE-
SIMS m/z (rel. int.) 408 (100) [M-H]À; (+)-LRESIMS m/z (rel. int.)
410 (80) [M+H]+, 432 (70) [M+Na]+, 841 (100) [2 M+Na]+; (+)-HRE-
SIMS m/z 432.1430 (C23H23NO6Na [M+Na]+ requires 432.1418).
(MeOH) kmax (log e) 213 (3.64), 264 (3.50), 329 (2.95), 509 (3.10)
nm; 1H NMR (500 MHz, CD3OD) dH 1.38 (3H, s, 3-Me), 1.92 (1H,
br dd, J = 15.5, 5.0 Hz, H-2ax), 2.17 (1H, d, J = 15.5 Hz, H-2eq), 2.59
(1H, d, J = 18.5 Hz, H-4ax), 3.06 (1H, d, J = 18.5 Hz, H-4eq), 4.49
(2H, s, H-11), 5.09 (1H, dd, J = 5.0, 3.0 Hz, H-1), 5.59 (1H, s, H-7),
7.28 (1H, dd, J = 8.3, 4.4 Hz, H-15), 7.36 (4H, br d, J = 4.4 Hz, H-13,
H-14, H-16, H-17); 13C NMR (125 MHz, CD3OD) dC 30.1 (3-Me),
38.2 (C-4), 41.6 (C-2), 47.2 (C-11), 63.9 (C-1), 69.5 (C-3), 101.6
(C-7), 109.9 (C-8a), 111.5 (C-5a), 128.4 (2C, C-13, C-17), 128.7
(C-15), 129.9 (C-14, C-16), 134.2 (C-4a), 139.9 (C-9a), 151.2 (C-6),
156.2 (C-9), 157.8 (C-10), 185.0 (C-5), 188.3 (C-8); (À)-LRESIMS
m/z (rel. int.) 394 (100) [MÀH]À; (+)-LRESIMS m/z (rel. int.) 396
(70) [M+H]+, 418 (40) [M+Na]+, 813 (80) [2M+Na]+; (+)-HRESIMS
m/z 418.1268 (C22H21NO6Na [M+Na]+ requires 418.1261).
4.4.6. Compound 10
29
Red powder, (7.2 mg, 85%); [
a]
+1120 (c 0.0025, MeOH); UV
D
(MeOH) kmax (log
e
) 215 (3.53), 277 (3.53), 264 (3.32), 311 (2.95),
4.4.10. Compound 14
504 (2.99) nm; 1H NMR (500 MHz, CD3OD) dH 1.39 (3H, s, 3-Me),
1.92 (1H, dd, J = 14.0, 4.8 Hz, H-2ax), 2.17 (1H, d, J = 14.0 Hz, H-
Red powder, (3.3 mg, 82%); [
a]
+1510 (c 0.001, MeOH); UV
29
D
(MeOH) kmax (log e) 213 (3.54), 264 (2.82), 322 (2.96), 510 (3.05)
2eq), 2.57 (1H, d, J = 18.5 Hz, H-4ax), 2.87 (2H, t, J = 7.3 Hz, H-12),
nm; 1H NMR (500 MHz, CD3OD) dH 1.40 (3H, s, 3-Me), 1.94 (1H,
br dd, J = 14.4, 4.2 Hz, H-2ax), 2.18 (1H, br d, J =14.4 Hz, H-2eq),
3.04 (1H, br d, J = 18.5 Hz, H-4eq), 3.43 (2H, t, J = 7.3 Hz, H-11),

V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
7173
2.59 (1H, br d, J = 18.6 Hz, H-4ax), 2.91 (3H, s, H-11), 3.05 (1H, br d,
J = 18.6 Hz, H-4eq), 5.10 (1H, br s, H-1), 5.56 (1H, s, H-7); 13C NMR
(150 MHz, CD3OD) dC 29.4 (C-11), 30.1 (3-Me), 38.2 (C-4), 41.6 (C-
2), 64.0 (C-1), 69.5 (C-3), 99.9 (C-7), 110.0 (C-8a), 111.4 (C-5a),
134.1 (C-4a), 139.8 (C-9a), 152.2 (C-6), 156.0 (C-9), 157.7 (C-10),
184.9 (C-5), 188.2 (C-8); (À)-LRESIMS m/z (rel. int.) 318 (100)
[MÀH]À, 637 (30) [2MÀH]À; (+)-LRESIMS m/z (rel. int.) 302 (100)
[MÀOH]+, 320 (90) [M+H]+; (+)-HRESIMS m/z 342.0946
(C16H17NO6Na [M+Na]+ requires 342.0948).
interpolation of inhibition curves.45 The mean IC50 ( SD) is shown
for three independent experiments, each carried out in triplicate.
4.7. Cytotoxicity assay46
Neonatal foreskin fibroblast (NFF) cells were cultured in RPMI
1640 (Life Technologies, Inc., Rockville, MD) supplemented with
10% FCS (CSL Biosciences, Parkville, Victoria, Australia), 1% strepto-
mycin (Life Technologies, Inc., Rockville, MD; complete medium) at
37 °C and 5% CO2. Cells were maintained in log phase growth and
seeded (3000/well) into 96-well tissue culture plates (Corning,
USA) then cultivated for 24 h before treatment. Compounds were
dissolved in DMSO and diluted in complete medium; the DMSO
concentration in the medium did not exceed 1%. Control cells were
treated with the equivalent dose of DMSO. Three days after treat-
ment initiation, the cells were washed with PBS and fixed in meth-
ylated spirits, total protein was determined using sulforhodamine
B as described previously.46 Compounds were tested in triplicate
in three independent experiments.
4.4.11. Compound 15
29
Red powder, (5.5 mg, 98%); [
a
]
D
+1450 (c 0.001, MeOH); UV
(MeOH) kmax (log
e
) 213 (3.74), 264 (3.62), 323 (3.18), 509 (3.85)
nm; 1H NMR (500 MHz, CD3OD) dH 1.40 (3H, s, 3-Me), 1.86 (4H,
br s, 1.94, H-14, H-15), 1.92 (1H, br dd, J = 14.4, 3.9 Hz, H-2ax),
2.17 (1H, d, J = 14.4 Hz, H-2eq), 2.56 (1H, d, J = 18.0 Hz, H-4ax),
2.67 (4H, br s, H-13, H-16), 2.82 (2H, br s, H-12), 3.03 (1H, br d,
J = 18.0 Hz, H-4eq), 3.39 (2H, br s, H-11), 5.07 (1H, br s, H-1), 5.61
(1H, s, H-7); 13C NMR (150 MHz, CD3OD) dC 24.6 (2C, C-14, C-15),
30.1 (3-Me), 38.1 (C-4), 41.7 (C-2), 42.3 (C-11), 54.4 (C-12), 55.2
(C-13, C-16), 63.9 (C-1), 69.6 (C-3), 100.4 (C-7), 109.8 (C-8a),
111.5 (C-5a), 134.2 (C-4a), 139.8 (C-9a), 151.3 (C-6), 156.0 (C-9),
157.9 (C-10), 184.4 (C-5), 188.1 (C-8); (À)-LRESIMS m/z (rel. int.)
401 (100) [MÀH]À; (+)-LRESIMS m/z (rel. int.) 403 (100) [M+H]+;
(+)-HRESIMS m/z 425.1673 (C21H26N2O6Na [M+Na]+ requires
425.1683).
Acknowledgments
We are indebted to N. Fechner, P. Forster and G. Guymer of the
Queensland Herbarium, Brisbane, Australia for collection and iden-
tification of the fungal material. V.C. would like to acknowledge
Nakhonratchasima Ratjabhat University (Thailand) for financial
support along with Griffith University for a Ph.D. scholarship (GUI-
PRS). We thank R. Quinn for access to the fungal sample, which is
part of the Eskitis Institute’s Nature Bank biota library. We thank
H. Vu from Griffith University for acquiring the HRESIMS measure-
ments. R.A.D. and K.T.A thank the National Health and Medical
Research Council (NHMRC) for financial support towards this re-
search through a project Grant (APP1024314). K.T.A. acknowledges
the Australian Research Council (ARC) for a Future Fellowship. We
thank Mark Brundrett from the Western Australian Department of
Environment and Conservation for the graphical abstract image.
We also acknowledge the Australian Red Cross Blood Service for
the provision of Type O+ erythrocytes.
4.5. Compound purity analysis
Compounds 1–15 were all prepared at a concentration of
0.2 mg/200 lL in DMSO and injected (100 lL) onto an analytical
Phenomenex C18 ONYX monolithic column. HPLC fractionation
conditions consisted of a linear gradient (curve #6) from 90%
H2O (0.1% TFA)/10% MeOH (0.1% TFA) to 50% H2O (0.1% TFA)/50%
MeOH (0.1% TFA) in 3 min at a flow rate of 4 mL/min, a convex gra-
dient (curve #5) to MeOH (0.1% TFA) in 3.50 min at a flow rate of
3 mL/min, held at MeOH (0.1% TFA) for 0.50 min at a flow rate of
3 mL/min, held at MeOH (0.1% TFA) for a further 1.0 min at a flow
rate of 4 mL/min, then a linear gradient (curve #6) back to 90% H2O
(0.1% TFA)/10% MeOH (0.1% TFA) in 1 min at a flow rate of 4 mL/min,
then held at 90% H2O (0.1% TFA)/10% MeOH (0.1% TFA) for 2 min at
a flow rate of 4 mL/min. Total run time for each analytical injection
was 11 min. Compound purity was determined by extracting each
chromatogram at 210 nm and integrating all UV peaks. The % pur-
ity and retention time for 1–15 were as follows: 1 (89%, 4.36 min),
2 (98%, 5.43 min), 3 (96%, 5.93 min), 4 (99%, 7.20 min), 5 (99%,
6.22 min), 6 (92%, 6.05 min), 7 (96%, 5.60 min), 8 (96%, 5.68 min),
9 (97%, 6.46 min), 10 (97%, 6.0 min), 11 (98%, 6.65 min), 12 (96%,
4.07 min), 13 (99%, 6.27 min), 14 (99%, 5.52 min), 15 (87%,
4.23 min).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.052.
References and notes
1. Murray, C. J.; Rosenfeld, L. C.; Lim, S. S.; Andrews, K. G.; Foreman, K. J.; Haring,
D.; Fullman, N.; Naghavi, M.; Lozano, R.; Lopez, A. D. Lancet 2012, 379, 413.
2. Agnandji, S. T.; Lell, B.; Soulanoudjingar, S. S.; Fernandes, J. F.; Abossolo, B. P.;
Conzelmann, C.; Methogo, B. G.; Doucka, Y.; Flamen, A.; Mordmuller, B.; Issifou,
S.; Kremsner, P. G.; Sacarlal, J.; Aide, P.; Lanaspa, M.; Aponte, J. J.; Nhamuave, A.;
Quelhas, D.; Bassat, Q.; Mandjate, S.; Macete, E.; Alonso, P.; Abdulla, S.; Salim,
N.; Juma, O.; Shomari, M.; Shubis, K.; Machera, F.; Hamad, A. S.; Minja, R.;
Mtoro, A.; Sykes, A.; Ahmed, S.; Urassa, A. M.; Ali, A. M.; Mwangoka, G.; Tanner,
M.; Tinto, H.; D’Alessandro, U.; Sorgho, H.; Valea, I.; Tahita, M. C.; Kabore, W.;
Ouedraogo, S.; Sandrine, Y.; Guiguemde, R. T.; Ouedraogo, J. B.; Hamel, M. J.;
Kariuki, S.; Odero, C.; Oneko, M.; Otieno, K.; Awino, N.; Omoto, J.; Williamson,
J.; Muturi-Kioi, V.; Laserson, K. F.; Slutsker, L.; Otieno, W.; Otieno, L.; Nekoye,
O.; Gondi, S.; Otieno, A.; Ogutu, B.; Wasuna, R.; Owira, V.; Jones, D.; Onyango, A.
A.; Njuguna, P.; Chilengi, R.; Akoo, P.; Kerubo, C.; Gitaka, J.; Maingi, C.; Lang, T.;
Olotu, A.; Tsofa, B.; Bejon, P.; Peshu, N.; Marsh, K.; Owusu-Agyei, S.; Asante, K.
P.; Osei-Kwakye, K.; Boahen, O.; Ayamba, S.; Kayan, K.; Owusu-Ofori, R.; Dosoo,
D.; Asante, I.; Adjei, G.; Chandramohan, D.; Greenwood, B.; Lusingu, J.; Gesase,
S.; Malabeja, A.; Abdul, O.; Kilavo, H.; Mahende, C.; Liheluka, E.; Lemnge, M. N.
Engl. J. Med. 1863, 2011, 365.
4.6. Antimalarial assays
P. falciparum growth inhibition assays were carried out using an
isotopic microtest, as previously described.45 Briefly, ring-stage P.
falciparum infected erythrocytes (0.5% parasitemia and 2.5%
hematocrit) were seeded into 96 well tissue culture plates contain-
ing serial dilutions of control (chloroquine) or test compounds. Fol-
lowing 48 h incubation under standard P. falciparum culture
conditions, 0.5 l
Ci 3H-hypoxanthine was added to each well after
3. Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong, M.;
Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.;
Day, N. P.; Lindegardh, N.; Socheat, D.; White, N. J. N. Engl. J. Med. 2009, 361,
455.
which the plates were cultured for a further 24 h. Cells were har-
vested onto 1450 MicroBeta filter mats (Wallac) and 3H incorpora-
tion was determined using a 1450 MicroBeta liquid scintillation
counter. P. falciparum growth inhibition was compared to DMSO
controls (0.5%), and IC50 values were calculated using linear
4. Fattorusso, E.; Taglialatela-Scafati, O. Mar. Drugs 2009, 7, 130.
5. Kajimoto, T. Kagaku Kyoiku 2007, 55, 418.

7174
V. Choomuenwai et al. / Bioorg. Med. Chem. 20 (2012) 7167–7174
6. Foye’s Principles of Medicinal Chemistry; Williams, D. A., Lemke, T. L., Eds., 5th
ed.; Lippincott Williams & Wilkins, 2002.
7. Kayser, O.; Kiderlen, A. F.; Croft, S. L. Stud. Nat. Prod. Chem. 2002, 26, 779.
8. Barnes, E. C.; Choomuenwai, V.; Andrews, K. T.; Quinn, R. J.; Davis, R. A. Org.
Biomol. Chem. 2012, 10, 4015.
25. Grabowski, K.; Baringhaus, K.-L.; Schneider, G. Nat. Prod. Rep. 2008, 25, 892.
26. Gierasch, T. M.; Shi, Z.; Verdine, G. L. Org. Lett. 2003, 5, 621.
27. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3.
28. Kingston, D. G. I. J. Nat. Prod. 2011, 74, 496.
9. Mueller, D.; Davis, R. A.; Duffy, S.; Avery, V. M.; Camp, D.; Quinn, R. J. J. Nat.
Prod. 2009, 72, 1538.
10. Davis, R. A.; Buchanan, M. S.; Duffy, S.; Avery, V. M.; Charman, S. A.; Charman,
W. N.; White, K. L.; Shackleford, D. M.; Edstein, M. D.; Andrews, K. T.; Camp, D.;
Quinn, R. J. J. Med. Chem. 2012, 55, 5851.
29. Seneci, P.; Sizemore, C.; Islam, K.; Kocis, P. Tetrahedron Lett. 1996, 37, 6319.
30. Atuegbu, A.; MacLean, D.; Nguyen, C.; Gordon, E. M.; Jacobs, J. W. Bioorg. Med.
Chem. 1996, 4, 1097.
31. Sigma-Aldrich Castle Hill, NSW; www.sigmaaldrich.com/chemistry/chemistry.
32. Instant JChem; ChemAxon: version 5.5, 2011; www.chemaxon.com/products/
instant-jchem.
33. Umezawa, I.; Oka, H.; Komiyama, K.; Hagiwara, K.; Tomisaka, S.; Miyano, T. J.
Antibiot. 1983, 36, 1144.
34. Bannister, B.; Zapotocky, B. A. J. Antibiot. 1992, 45, 1313.
35. Dictionary of Natural Products (DVD); Version 18.2; Taylor & Francis Group/
CRC Press: London, UK, 2009.
36. Weiss, C. R.; Moideen, S. V. K.; Croft, S. L.; Houghton, P. J. J. Nat. Prod. 2000, 63,
1306.
37. Tansuwan, S.; Pornpakakul, S.; Roengsumran, S.; Petsom, A.; Muangsin, N.;
Sihanonta, P.; Chaichit, N. J. Nat. Prod. 2007, 70, 1620.
38. Carvalho, L. H.; Rocha, E. M. M.; Raslan, D. S.; Oliveira, A. B.; Krettli, A. U. Braz. J.
Med. Biol. Res. 1988, 21, 485.
39. Mather, M. W.; Henry, K. W.; Vaidya, A. B. Curr. Drug Targets 2007, 8, 49.
40. Hudson, A. T. Parasitol. Today 1993, 9, 66.
41. Olliaro, P. Pharmacol. Ther. 2001, 89, 207.
11. Davis, R. A.; Duffy, S.; Avery, V. M.; Camp, D.; Hooper, J. N. A.; Quinn, R. J.
Tetrahedron Lett. 2010, 51, 583.
12. Davis, R. A.; Carroll, A. R.; Andrews, K. T.; Boyle, G. M.; Tran, T. L.; Healy, P. C.;
Kalaitzis, J. A.; Shivas, R. G. Org. Biomol. Chem. 2010, 8, 1785.
13. Elsworth, C.; Gill, M.; Ten, A. Aust. J. Chem. 1999, 52, 1115.
14. Gill, M.; Smrdel, A. F.; Strauch, R. J.; Begley, M. J. J. Chem. Soc., Perkin Trans. 1990,
1, 1583.
15. Gill, M.; Steglich, W. Prog. Chem. Org. Nat. Prod. 1987, 51, 1.
16. Archard, M. A.; Gill, M.; Strauch, R. J. Phytochemistry 1985, 24, 2755.
17. Beattie, K. D.; Rouf, R.; Gander, L.; May, T. W.; Ratkowsky, D.; Donner, C. D.; Gill,
M.; Grice, I. D.; Tiralongo, E. Phytochemistry 2010, 71, 948.
18. Bolognesi, M. L.; Calonghi, N.; Mangano, C.; Masotti, L.; Melchiorre, C. J. Med.
Chem. 2008, 51, 5463.
19. Mecklenburg, S.; Shaaban, S.; Ba, L. A.; Burkholz, T.; Schneider, T.; Diesel, B.;
Kiemer, A. K.; Röseler, A.; Becker, K.; Reichrath, J. Org. Biomol. Chem. 2009, 7,
4753.
20. Wang, J.; Bourguet-Kondracki, M. L.; Longeon, A.; Dubois, J.; Valentin, A.; Copp,
B. R. Bioorg. Med. Chem. Lett. 2011, 21, 1261.
42. Schwikkard, S.; Heerden, F. R. Nat. Prod. Rep. 2002, 19, 675.
43. Kaur, K.; Jain, M.; Kaur, T.; Jain, R. Bioorg. Med. Chem. 2009, 17, 3229.
44. Ioset, J.-R. Curr. Org. Chem. 2008, 12, 643.
21. Abel, U.; Simon, W.; Eckard, P.; Hansske, F. G. Bioorg. Med. Chem. Lett. 2006, 16,
3292.
45. Andrews, K. T.; Walduck, A.; Kelso, M. J.; Fairlie, D. P.; Saul, A.; Parsons, P. G. Int.
J. Parasitol. 2000, 30, 761.
22. Davis, R. A.; Carroll, A. R.; Quinn, R. J. Aust. J. Chem. 2001, 54, 355.
23. Davis, R. A.; Pierens, G. K.; Parsons, P. G. Magn. Reson. Chem. 2007, 45, 442.
24. Feher, M.; Schmidt, J. M. J. Chem. Info. Comp. Sci. 2003, 43, 218.
46. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.