Discovery and preliminary structure-activity relationship analysis of 1,14-sperminediphenylacetamides as potent and selective antimalarial lead compounds

Article abstract of DOI:10.1016/j.bmcl.2012.11.072

Screening of synthesized and isolated marine natural products for in vitro activity against four parasitic protozoa has identified the ascidian metabolite 1,14-sperminedihomovanillamide (orthidine F, 1) as being a non-toxic, moderate growth inhibitor of Plasmodium falciparum (IC₅₀ 0.89 μM). Preliminary structure-activity relationship investigation identified essentiality of the spermine polyamine core and the requirement for 1,14-disubstitution for potent activity. One analogue, 1,14-spermine-di-(2- hydroxyphenylacetamide) (3), exhibited two orders of magnitude increased anti-P. f activity (IC₅₀ 8.6 nM) with no detectable in vitro toxicity. The ease of synthesis of phenylacetamido-polyamines, coupled with potent nM levels of activity towards dual drug resistant strains of P. falciparum makes this compound class of interest in the development of new antimalarial therapeutics.

Full text of DOI:10.1016/j.bmcl.2012.11.072

Bioorganic & Medicinal Chemistry Letters 23 (2013) 452–454
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and preliminary structure–activity relationship analysis of
1,14-sperminediphenylacetamides as potent and selective antimalarial
lead compounds
Lydia P. P. Liew a, Marcel Kaiser b,c, Brent R. Copp a,
⇑
a School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b Swiss Tropical and Public Health Institute, Socinstrasse 57, PO Box, CH-4002 Basel, Switzerland
c The University of Basel, CH-4003 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Screening of synthesized and isolated marine natural products for in vitro activity against four parasitic
Received 15 October 2012
Revised 9 November 2012
Accepted 14 November 2012
Available online 29 November 2012
protozoa has identified the ascidian metabolite 1,14-sperminedihomovanillamide (orthidine F, 1) as
being a non-toxic, moderate growth inhibitor of Plasmodium falciparum (IC50 0.89 lM). Preliminary struc-
ture–activity relationship investigation identified essentiality of the spermine polyamine core and the
requirement for 1,14-disubstitution for potent activity. One analogue, 1,14-spermine-di-(2-hydroxyphe-
nylacetamide) (3), exhibited two orders of magnitude increased anti-P. f activity (IC50 8.6 nM) with no
detectable in vitro toxicity. The ease of synthesis of phenylacetamido-polyamines, coupled with potent
nM levels of activity towards dual drug resistant strains of P. falciparum makes this compound class of
interest in the development of new antimalarial therapeutics.
Keywords:
Marine natural product
Ascidian
Plasmodium falciparum
Anti-protozoal
Polyamine
Ó 2012 Elsevier Ltd. All rights reserved.
Parasitic protozoal infection is a cause of suffering for an esti-
mated one billion people worldwide.1 Insect bites that transfer
protozoa to human hosts lead to the diseases malaria (protozoa:
Plasmodium falciparum), Chagas Disease (also known as American
Trypanosomiasis, Trypanosoma cruzi), Leishmaniasis (Leishmania
spp.) and Sleeping Sickness (also known as Human African Try-
panosomiasis, Trypanosoma brucei rhodesiense, Trypanosoma brucei
gambiense). While drugs exist for these diseases, they have unde-
sirable side-effects or are showing reduced efficacy due to the
growing prevalence of drug resistant strains.1
diamide orthidine F (1), previously reported by us as an anti-
inflammatory agent from the New Zealand ascidian Aplidium
orthium.3 Herein we report the in vitro screening data for
orthidine F, and the results of a preliminary structure–activity
relationship study which led to the identification of an analogue
exhibiting two-orders of magnitude increased activity towards
Plasmodium falciparum.
In the context of our continuing search for new leads for the
development of treatments for neglected human diseases,2 we
recently initiated a program of screening a library of synthesized
and isolated marine natural products against a panel of four
parasitic protozoa: Trypanosoma brucei rhodesiense, Trypanosoma
cruzi, Leishmania donovani and Plasmodium falciparum K1 dual
drug-resistant strain, with concurrent counter-screening for
toxicity towards the non-malignant L6 rat myoblast cell line.
One of the first anti-malarial hits identified was the polyamine
The anti-protozoal evaluation of 1 (Table 1, entry 1) established the
natural product to be a moderately potent in vitro growth inhibitor
of P. falciparum K1 strain (IC50 0.89
lM) and a modest inhibitor of
Trypanosoma brucei rhodesiense (IC50 78
lM) while demonstrating
no detectable activity towards T. cruzi and Leishmania donovani
and no cytotoxicity against a mammalian cell-line. Polyamines are
well recognized as a biologically active scaffold,4 exhibiting cyto-
toxicity,5 receptor binding properties (AMPA,6 NMDA7), enzyme
inhibition (trypanothione reductase,8,9 carbonic anhydrase10) and
⇑
Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7422.
E-mail address: b.copp@auckland.ac.nz (B.R. Copp).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.072

L. P. P. Liew et al. / Bioorg. Med. Chem. Lett. 23 (2013) 452–454
453
Table 1
R4
Anti-protozoal and cytotoxic activities of 1–23, 24, 26
R3
5
4
6
O
R1
R4
Entry Compound
IC50
L.
donc
(
l
M)
H2
N
H
N
10
12 13 14
R2
R3
1
9
T. b.
T.
P. falc.
L6e
R2
N
N
H2
3
8
2
R1
11
7
15
rhoda
cruzib
K1d
H
O
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
78
96
51
87
86
3.2
38
120
49
150
120
5.9
110
120
7.5
210
110
3.3
110
63
5.2
150
170
NTf
NT
>120
>140
>130
>130
>130
>120
31
>120
>130
>150
>170
>160
>160
>180
>170
>220
>280
>230
>160
>200
>170
>220
>250
NT
>120
90
110
>130
>130
>120
35
>120
>130
>150
>170
>160
>160
>180
>170
>220
>280
>230
150
>200
>170
>220
>250
NT
0.89
0.14
0.0086
0.17
0.14
4.8
0.019
>6.8
0.61
>8.5
3.6
>120
>140
>130
>130
130
2 R1 = R2 = R3 = R4 = H
3 R1 = OH, R2 = R3 = R4 = H
4 R1 = R3 = R4 = H, R2 = OH
5 R1 = R2 = R4 = H, R3 = OH
6 R1 = R4 = H, R2 = R3 = OH
7 R1 = R4 = OMe, R2 = R3 = H
85
88
>120
>130
>150
>170
97
>160
>180
140
>220
>280
130
>160
>200
96
5
9
R3
R2
4
3
R1
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
O
H2
N
H
N
13
10
12
14
R2
R3
1
9
8
N
H2
N
H
7.9
2
7
11
R1
>8.9
>10
>9.4
>12
>15
>13
>9.1
>11
>9.6
>12
>14
35
O
8 R1 = H, R2 = OMe, R3 = OH
9 R1 = OH, R2 = R3 = H
5
R2
4
3
6
O
H
13
20
22
25
10
12
14
16
1
9
N
19
21
23 R1
R1
N
H
N
H2
8
18
2
7
11
15
17
O
26
R2
24
210
>250
94
10 R1 = OMe, R2 = OH
11 R1 = H, R2 = OH
12 R1 = R2 = OH
26
NT
NT
8.0
110
Melarsoprolg
Benznidazoleg
Miltefosineg
Chloroquineg
Podophyllotoxing
0.005
5
2
R2
4
3
6
1
O
1.8
H2
N
H
10
14
18
20
23
N
R1
21
9
0.53
17
19
R1
N
H
8
16
0.28
7
11
13
15
12
O
24
0.019
R2
22
13
14
R
R
1 = OMe, R2 = OH
IC50 values reported are the average of two independent assays. Assay protocols are
described in Ref. 18.
1 = OH, R2 = H
a
15 R1 = OH, R2 = OH
Trypanosoma brucei rhodesiense, STIB 900 strain, trypomastigotes stage.
Trypanosoma cruzi, Tulahuen C4 strain, amastigotes stage.
Leishmania donovani, MHOM-ET-67/L82 strain, amastigote/axenic stage.
Plasmodium falciparum, K1 strain, IEF stage.
L6 rat skeletal myoblast cell line.
Not tested.
Melarsoprol, benznidazole, miltefosine, chloroquine and podophyllotoxin were
5
b
R2
R1
4
3
6
1
c
O
H
N
10
R1
R2
9
d
8
N
H
e
2
7
11
f
O
g
1
16
17
18
R
= OMe, R2 = OH
= R2 = H
used as positive controls.
1
R
1
R
= R2 = OH
5
2
R2
R1
R2
R1
4
3
6
1
O
O
16
O
10
12
14
anti-protozoal activity towards Trypanosoma sp.11 and P. falcipa-
rum.12,13 Making use of the previously reported facile synthesis of
1,3 we undertook a preliminary structure–activity relationship
study to explore the relevance of the polyamine core and the termi-
nal ‘capping acids’9 towards the observed anti-protozoal activity.
Thus reaction of spermine with six phenylacetic acid derivatives
(phenylacetic acid, 2-, 3- and 4-hydroxyphenyl acetic acid, 3,4-
dihydroxyphenyl acetic acid and 2,5-dimethoxyphenyl acetic acid)
using PyBOP as the coupling reagent (see Supplementary data for
details) afforded, after chromatographic purification, analogues
9
8
N
H
O
O
N
H
11
15
7
13
19
R
1 = OMe, R2 = OH
20 R1 = R2 = H
21 R1 = R2 = OH
5
2
5
2
HO
4
3
HO
4
13
6
1
6
O
O
O
10
10
1
9
9
NH
12
NH
14
8
8
3
N
H
12
11
MeO
MeO
N
H
14
7
11
7
13
15
15
23
22
2
14, 3, 49, 5, 6 and 7 in yields of 45%, 73%, 46%, 65%, 62% and 54%,
H2
N
H
respectively. In a similar fashion, subsets of these capping acids
were reacted with N,N0-bis(3-aminopropyl)ethylenediamine, sper-
midine, N-(2-aminoethyl)-1,3-propanediamine, 1,4-diaminobutane,
or 1,13-diamino-4,7,10-trioxa-tridecane to afford diamide
analogues 8–16, 1715, 18–21. Reaction of homovanillic acid with
2-morpholinoethanamine or N,N-dimethylethane-1,2-diamine
yielded truncated mono-amides 22 and 23 in 73% and 22% yield,
respectively. To explore whether diarylamide substitution of the
polyamine core of 2 was a requirement for activity, spermine
mono-amide 24 was prepared by a two-step route starting with Py-
BOP-activated coupling of spermine-trifluoroacetamide (25)16 with
phenylacetic acid to yield diamide 26 followed by deprotection
using LiOH in THF/H2O to afford 24.
R2
N
N
N
R1
H
H2
1
24
R
= H, R2 = PhCH2CO
25 R1 = COCF3, R2 = H
26 R1 = COCF3, R2 = PhCH2CO
The library of analogues were screened against a set of four proto-
zoa and for cytotoxicity towards the L6 cell line and the results are
summarized in Table 1. A set of spermine analogues that varied in
phenylacetic acid substitution (2–7, entries 2–7) typically exhibited
more potent antimalarial activity than orthidine F, with the

454
L. P. P. Liew et al. / Bioorg. Med. Chem. Lett. 23 (2013) 452–454
2-hydroxyphenylacetamide analogue 3 being the most potent ana-
logue identified in the study. The critical requirement of the hydro-
xyl group at the 2-position is noted, with the 3- and 4-hydroxy
analogues 4 and 5 (entries 4 and 5) being approximately twenty-
fold less active than 3. Also notable in this spermine series was
the pronounced activity of the 3,4-dihydroxy analogue 6 (entry 6)
thank Dr. A. N. Pearce for helpful advice and assistance, M. Cal, S.
Sax and C. Stalder (Swiss TPH) for parasite assay results, the DTP
branch of the National Cancer Institute for cytotoxicity data, Dr.
M. Schmitz for assistance with NMR data acquisition, and Ms. R.
Imatdieva and Dr. N. Lloyd for MS data.
towards T. brucei rhodesiense (IC50 3.2 lM), tempered somewhat
Supplementary data
with the observation of increased cytotoxicity towards the L6 cell
line. From the same series, only the 2,5-dimethoxy analogue 7
exhibited growth inhibitory activity towards Trypanosoma cruzi
and Leishmania donovani, with all other analogues being considered
inactive. In the case of 7 however, pan-panel activity suggests this
particular diamide may be exhibiting activity due to a general cyto-
toxic mechanism. Using a subset of substituted phenylacetic acids, a
variety of diamides were prepared (8–23) that explored the struc-
ture–activity requirement of the spermine fragment of orthidine
F. As summarized in entries 8–23, incorporation of N,N0-bis(3-
aminopropyl)ethylenediamine (8, 9), spermidine (10, 11, 12),
N-(2-aminoethyl)-1,3-propanediamine (13, 14, 15), 1,4-diaminobu-
tane (16, 17, 18), 1,13-diamino-4,7,10-trioxatridecane (19, 20, 21),
2-morpholinoethanamine (22) and N,N-dimethylethane-1,2-
diamine (23) motifs led to substantially reduced anti-malarial
activity. No members of this subset exhibited activity towards T.
cruzi or L. donovani, though the anti-Trypanosoma brucei rhodesiense
activity associated with the 3,4-dihydroxyphenylacetic acid moiety
was again highlighted (12, 15, 18 and 21, entries 12, 15, 18, 21).
Finally, evaluation of the mixed aryl-TFA-diamide 26 and the aryl
mono-amide 24 against P. falciparum found both to be essentially
inactive (entries 24 and 25), indicating the requirement for
diarylamide substitution for optimal anti-malarial activity.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.11.
072. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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Acknowledgments
We acknowledge funding from the University of Auckland
(FDRF 3626196 and Biopharma Thematic Research Initiative). We