The discovery, synthesis and antimalarial evaluation of natural product-based polyamine alkaloids

Article abstract of DOI:10.1016/j.tetlet.2013.07.058

Bioassay-guided fractionation of an antimalarial extract derived from the fungus Ramaria subaurantiaca afforded the known polyamine alkaloid, pistillarin. Nine pistillarin analogues were synthesised via EDC-mediated chemistry and these compounds along with the previously reported natural product polyamines, ianthelliformisamines A-C and spermatinamine, were evaluated against Plasmodium falciparum (3D7) parasites and a normal human cell line to determine parasite-specific activity. Spermatinamine (IC₅₀ 0.23 μM) and pistillarin (IC₅₀ 1.9 μM) were the two most potent antimalarials identified during these studies.

Full text of DOI:10.1016/j.tetlet.2013.07.058

Tetrahedron Letters 54 (2013) 5188–5191
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
The discovery, synthesis and antimalarial evaluation of natural
product-based polyamine alkaloids
Vanida Choomuenwai a,b, Brett D. Schwartz a, Karren D. Beattie a, Katherine T. Andrews a,b
,
Shahan Khokhar a, 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:
Bioassay-guided fractionation of an antimalarial extract derived from the fungus Ramaria subaurantiaca
afforded the known polyamine alkaloid, pistillarin. Nine pistillarin analogues were synthesised via
EDC-mediated chemistry and these compounds along with the previously reported natural product poly-
amines, ianthelliformisamines A–C and spermatinamine, were evaluated against Plasmodium falciparum
(3D7) parasites and a normal human cell line to determine parasite-specific activity. Spermatinamine
Received 15 May 2013
Revised 1 July 2013
Accepted 9 July 2013
Available online 16 July 2013
(IC50 0.23
these studies.
l
M) and pistillarin (IC50 1.9
l
M) were the two most potent antimalarials identified during
Keywords:
Macrofungus
Ramaria subaurantiaca
Natural product
Pistillarin
Ó 2013 Elsevier Ltd. All rights reserved.
Polyamine
Antimalarial
Polyamines are aliphatic alkaloids that have been reported from
various natural sources including terrestrial and marine animals,
plants, fungi and bacteria.1 Two of the simplest naturally occurring
polyamines include spermidine and spermine. Substructure
searching of the Dictionary of Natural Products database1 against
these two molecules identified >400 natural products that contain
these aliphatic alkaloid moieties. Knowledge associated with poly-
amine chemistry and metabolism in human cells (both normal and
tumour lines), bacteria and parasites is expanding rapidly.2,3 The
polyamine chemotype has been shown to effect multiple biological
processes including: the regulation of gene expression, cell prolif-
eration and translation, the modulation of cell signalling, mem-
brane stability, inhibition of carbonic anhydrase function,
inhibition of certain ion channels and free radical-scavenging.2,4–7
It has been reported that, at physiological pH, polyamines can bind
with various anionic macromolecules in the cell including DNA,
RNA, ATP, proteins and phospholipids.8 Consequently, polyamine-
based drug discovery is an evolving field with various compounds
currently under development as potential therapeutics.2,3 Further-
more, clinical success has been achieved for the polyamine-deriv-
ative eflornithine [( )-2,5-diamino-2-(difluoromethyl)pentanoic
acid; syn. difluoromethylornithine (DFMO)], which was approved
by the FDA for both the systemic treatment of African trypanoso-
miasis9,10 [OrnidylÒ, Sanofi-Aventis] and the topical treatment of
hirsuitism [VaniqaÒ, Gillette]. Of significance, this compound has
also progressed to phase III clinical trials as an anticancer agent.3,11
Mechanism of action studies for DFMO has shown that this com-
pound is a potent suicide inhibitor of the polyamine biosynthetic
enzyme, ornithine decarboxylase.12,13
As part of our continuing research into the discovery of new
antimalarial leads from nature14–18 we screened a unique fungi-de-
rived fraction library (2035 fractions) against a chloroquine-sensi-
tive Plasmodium falciparum line (3D7), using a radiometric growth
inhibition assay.19,20 A total of 20 fractions (0.98% hit rate) derived
from 18 fungal species (10 genera, 8 families) showed promising
antimalarial activity. One fraction derived from Ramaria subauran-
tiaca Corner was prioritised for further investigation since it dem-
onstrated potent malarial growth inhibition along with no
cytotoxicity towards neonatal foreskin fibroblast (NFF) cells at
the highest concentration tested.21 Bioassay-guided fractionation
of the large-scale CH2Cl2/MeOH extract from R. subaurantiaca using
reversed phase semi-preparative C18 HPLC [MeOH/H2O/0.1%
CF3CO2H] yielded the trifluoroacetate salt of the known fungal
metabolite pistillarin (1).22–24 This molecule was identified on
the basis of spectroscopic and spectrometric data, and by compar-
ison with literature values.22–24 Pharmacological evaluation of 1
(Table 1) showed that it inhibited the growth of a drug-sensitive
⇑
Corresponding author. Tel.: +61 7 3735 6043; fax: +61 7 3735 6001.
E-mail address: r.davis@griffith.edu.au (R.A. Davis).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.07.058

V. Choomuenwai et al. / Tetrahedron Letters 54 (2013) 5188–5191
5189
Table 1
iformisamines A–C (2–4) were isolated initially from the marine
sponge Suberea ianthelliformis7 and displayed varying levels of
inhibitory activity against the Gram-negative bacteria Pseudomo-
nas aeruginosa.7 Specifically, ianthelliformisamine A was the most
Biological activities for compounds 1–12, 16 and 17
Compound
IC50 SD (
l
M)
NFFb
SIc
3D7a
potent antibacterial agent with an IC50 value of 6.8
lM (MIC
1
2
3
4
5
6
7
8
9
10
11
12
16
17
1.9 0.6
14.5 0.4
12.1 1.0
4.4 0.5
0.23 0.04
>25
>100
>100
>100
17.5 0.4
2.1 0.4
78.8 15.5
89.8 6.2
>100
>100
>100
>100
>100
>100
>100
40.2 3.4
>53
>7
>12
4
35
l
M).7 Spermatinamine (5) was originally isolated from the
sponge Pseudoceratina sp. and was the first natural product inhib-
itor of isoprenylcysteine carboxyl methyltransferase (ICMT), which
catalyses the carboxyl methylation of oncogenic proteins in the fi-
nal step of a series of post-translational modifications.4 ICMT has
been proposed as an attractive and novel anticancer target.4 More
recently spermatinamine (5) and a series of related natural prod-
ucts have been shown to inhibit Gram-negative bacteria.32
In order to evaluate the pharmacological activity of similar
polyamines, we embarked on a synthetic programme that aimed
to generate several modified pistillarin analogues. It was hoped
that these compounds, in conjunction with the previously isolated
natural products 2–5, would enable us to elucidate preliminary
structure activity relationships.
9
<3
<4
>5
>5
>4
>4
>4
>6
>6
1005
>25
20.3 0.4
20.5 0.5
>25
>25
>25
16.7 2.7
17.2 13.8
0.04 0.004
Chloroquine
a
b
c
3D7 = Plasmodium falciparum chloroquine-sensitive line.
NFF = neonatal foreskin fibroblast cells.
SI = selectivity index = NFF cell line IC50 / P. falciparum IC50
.
The approach undertaken for the target analogues 6–12, 16
and 17 (Fig. 2) relied on an EDC-mediated amide formation be-
tween benzoic acid or 3,4-dimethoxybenzoic acid with either
spermidine or N1,N5-bis-Boc-spermidine; yields ranged from 2–
30%.33,34 Treatment of analogue 8 with boron tribromide-dimeth-
ylsulfide complex conveniently effected global de-protection and
afforded the desired catechol 10.35,36 Analogues 11 and 12 were
synthesised in a similar manner through a two-step coupling/
deprotection protocol from 3,4-dimethoxybenzoic acid with
either N-Boc-1,4-diaminobutane or N-Boc-1,3-diaminopropane.
During the preparation of this manuscript, we identified the liter-
ature that reported the antimalarial activity of the marine natural
product orthidine F (13), and several synthetic analogues, two of
which (14 and 15) displayed potent antimalarial activity (with
IC50 values of 19.0 and 8.6 nM respectively, against the P. falcipa-
rum K1 line).37 Due to these data, we elected to synthesise and
screen the related compounds 16 and 17. These molecules were
generated by coupling 2,5-dimethoxybenzoic acid or acetylsali-
cylic acid with spermidine by using EDC as the coupling reagent,
which afforded 16 and 17 in yields of 6% and 2%, respectively.33,34
The acetylated derivative of 17 was not identified during these
studies and we propose that ester hydrolysis occurred during
the purification step. In order to obtain larger quantities of 17,
a different coupling methodology was employed. Accordingly,
spermidine was exposed to salicyloyl chloride at À20 °C in
CH2Cl2, and after work-up and purification, 17 was afforded in
an improved yield of 8%.
P. falciparum line (3D7) with an IC50 value of 1.9 lM, and displayed
no cytotoxicity against NFF cells at 100 lM.
Pistillarin (1) has been isolated from a variety of fungal species
including Penicillium bilaii,22 Gomphus floccosus,23 Clavariadelphus
pistillaris and several Ramaria species24 and is a known siderophore
(i.e., a low molecular weight ferric-ion specific ligand produced by
microorganisms to sequester iron under aerobic conditions and at
low iron concentrations).25–27 Note that many reported sidero-
phores possess a hydroxamic acid or a catechol moiety, such as
that present in 1.26 Since iron is essential for parasitic growth
and multiplication, it was reasoned that chelation within infected
erythrocytes may be a selective mechanism to control P. falcipa-
rum.25–29 Importantly, the antimalarial activity of iron-chelating
agents has been established both in vitro and in vivo.28–31 In addi-
tion, compound 1 has been shown to exhibit significant protective
effects against DNA damage by hydroxyl radicals generated from
the Fenton reaction via iron chelation, as well as free radical-scav-
enging activity.23
Encouraged by the antimalarial data of 1, we subsequently per-
formed substructure searching on an in-house natural product li-
brary using the spermidine fragment in an attempt to identify
additional polyamine alkaloids for antiplasmodial evaluation. This
process identified four previously reported polyamines, ianthelli-
formisamines A–C (2–4), and spermatinamine (5; Fig. 1). Ianthell-
Br
Br
OH
O
O
H
HO
HO
N
H
N
H
N
N
H
N
H
OH
Br
N
H
NH2
O
1
3
O
O
2
4
Br
O
O
O
H
N
H
N
H
N
H
N
Br
O
Br
N
H
N
H
Br
NH2
O
Br
Br
O
HO
N
O
H
N
N
Br
O
Br
N
N
H
O
N
OH
5
Br
Figure 1. Chemical structures of the natural product polyamines pistillarin (1), ianthelliformisamines A–C (2–4) and spermatinamine (5).

5190
V. Choomuenwai et al. / Tetrahedron Letters 54 (2013) 5188–5191
O
19
21
O
O
H
H
1
11
13
N
O
O
N
15
7
N
N
N
H
N
O
9
17
H
H
H
O
5
O
3
6
7
O
O
H
H
N
N
O
O
N
N
Boc
N
N
Boc
H
H
Boc
Boc
8
9
O
O
O
HO
HO
NH2
HO
HO
HO
HO
NH2
N
H
N
NH2
N
H
N
H
H
10
11
12
HO
O
O
H
N
H
N
O
N
N
H
H
O
OH
13
14
O
O
O
O
H
H
N
N
N
N
H
H
O
O
O
OH
H
H
N
N
N
N
H
H
OH
O
15
O
OH
O
O
O
H
H
N
N
N
N
N
N
H
H
H
H
O
OH
O
O
16
17
O
Figure 2. Chemical structures of synthetic polyamine analogues (6–12, 14–17) and the ascidian natural product orthidine F (13).
All synthetic analogues generated during these studies were
NFF cells, and modifying the length of the amide side chain did not
affect the activity in this study. Whilst the monoaryl brominated
natural products 2 and 3 showed slight antimalarial activity, they
characterised using 1D (1H, 13C) and 2D (gCOSY, gHSQC, gHMBC)
NMR, UV and MS data. The majority of these compounds have been
previously reported as either natural products (6,38 7,39 and 940), or
they have been synthesised (1041,42 and 1143), but only partially
characterised. We report the full spectroscopic characterisation of
6, 8, 10–12, 16 and 17 (see Supplementary Data for details). Prior
to pharmacological evaluation all compounds were assessed for
purity by 1H NMR spectroscopic analysis and confirmed to be
P95%.
displayed no cytotoxicity against NFF cells at 100 lM. In contrast,
the diaryl brominated natural products 4 and 5 were significantly
more active against 3D7 parasites and more cytotoxic than their
monoaryl congeners 2 and 3. Lastly, Boc protection of the two
amine groups (monoaryl synthetic compounds 8 and 9) main-
tained some antimalarial activity; a similar pattern of activity for
Boc-substituted compounds has been reported previously.44 The
improved activity associated with Boc protection was proposed
by others44 to be due to enhancement of the membrane permeabil-
ity of the cationic amine analogues, which subsequently increased
accumulation of the compound(s) within the digestive vacuole of
the malaria parasite.44
Compounds 1–12, 16 and 17 were all tested for in vitro growth
inhibitory activity against P. falciparum 3D7 parasites and for
mammalian cell toxicity using the NFF cell line (Table 1). Pistillarin
(1) showed the best selectivity index (SI >53), whilst spermatin-
amine (5) displayed the most potent antimalarial and cytotoxic
activities. The poor selectivity of 5 (SI = 9) prevented further eval-
uation of this compound as a potential antimalarial lead. Com-
pounds 6 and 7 were both significantly less active than pistillarin
indicating that the catechol group(s) present in 1 are important
for antimalarial activity. However, the moderate antimalarial data
for 16 and 17 suggest substitution variability about the aryl units is
tolerated, and this is supported by other literature reports.37
When comparing the significance of the number of aryl substit-
uents it was observed that the series of monoaryl amides 2, 3 and
8–12 were the least active against P. falciparum 3D7 parasites and
The micromolar antimalarial activity for compounds 16 and 17
(IC50 16.7–17.2 lM, 3D7) compared to the nanomolar activity for
the previously reported analogues 14 and 15 (IC50 <20 nM, K1),37
indicated that the extended polyamine chain length [spermine
(14 and 15) versus spermidine (16 and 17)] and/or the amide func-
tionality [phenylacetamide (14 and 15) versus benzamide (16 and
17)] is critical for parasite growth inhibition in the 2,5-dimethoxy-
and 2-hydroxy-aryl series. A caveat to this observation is that the
screening for the previously reported 14 and 15 (IC50 <20 nM),
was conducted against P. falciparum K1 parasites,37 which is a

V. Choomuenwai et al. / Tetrahedron Letters 54 (2013) 5188–5191
5191
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support along with Griffith University for a PhD scholarship
(GUIPRS). S.K. acknowledges the Australian Government for an
Australian Postgraduate Award. R.A.D. and K.T.A. thank the
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Supplementary data (general experimental procedures, fungus
collection and identification details, isolation and extraction proto-
cols, synthetic reaction details, spectroscopic/spectrometric data
for compounds 6, 8, 10–12, 16 and 17, malaria and cytotoxicity
assay protocols and 1H and 13C NMR spectra for compounds 6, 8,
10–12, 16 and 17) associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.tet-
let.2013.07.058. MOL files and InChiKeys of the most important
compounds described in this article are also included.
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