In vitro efficiency of 9-(N-cinnamoylbutyl)aminoacridines against blood- and liver-stage malaria parasites

Article abstract of DOI:10.1016/j.bmcl.2012.12.032

Novel 9-aminoacridine derivatives were synthesized by linking the heteroaromatic core to different cinnamic acids through an aminobutyl chain. The test compounds demonstrated mid-nanomolar in vitro activity against erythrocytic stages of the chloroquine-resistant W2 strain of the human malaria parasite Plasmodium falciparum. Two of the most active derivatives also showed in vitro activity against liver-stage Plasmodium berghei, with activity greater than that of the reference liver-stage antimalarial primaquine. The compounds were not toxic to human hepatoma cells at concentrations up to 5 μM. Hence, 9-(N-cinnamoylbutyl)aminoacridines are a new class of leads for prevention and treatment of malaria.

Full text of DOI:10.1016/j.bmcl.2012.12.032

Bioorganic & Medicinal Chemistry Letters 23 (2013) 610–613
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro efficiency of 9-(N-cinnamoylbutyl)aminoacridines against
blood- and liver-stage malaria parasites
Bianca Pérez a, Cátia Teixeira a,b, Ana S. Gomes a, Inês S. Albuquerque c, Jiri Gut d, Philip J. Rosenthal d,
Miguel Prudêncio c, Paula Gomes a,
⇑
a Centro de Investigação em Química da Universidade do Porto, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto,
R. do Campo Alegre, 687, P-4169-007 Porto, Portugal
b CICECO, Departamento de Química, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
c Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
d Department of Medicine, San Francisco General Hospital, University of California, CA 94143-0811, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 9-aminoacridine derivatives were synthesized by linking the heteroaromatic core to different cin-
namic acids through an aminobutyl chain. The test compounds demonstrated mid-nanomolar in vitro
activity against erythrocytic stages of the chloroquine-resistant W2 strain of the human malaria parasite
Plasmodium falciparum. Two of the most active derivatives also showed in vitro activity against liver-stage
Plasmodium berghei, with activity greater than that of the reference liver-stage antimalarial primaquine.
Received 8 November 2012
Revised 10 December 2012
Accepted 11 December 2012
Available online 21 December 2012
The compounds were not toxic to human hepatoma cells at concentrations up to 5 lM. Hence, 9-(N-cin-
namoylbutyl)aminoacridines are a new class of leads for prevention and treatment of malaria.
Keywords:
9-Aminoacridine
Antimalarials
Ó 2012 Elsevier Ltd. All rights reserved.
Blood-stage
Cinnamic acid
Liver-stage
Multi-target drugs
Plasmodium falciparum
Schizontocidal
Malaria, one of the world’s deadliest diseases, primarily affects
developing countries. According to the last World Malaria Report,
there were around 3.3 billion people at risk of infection in 2010.1
Although the malaria mortality rate has probably decreased since
2000, the disease remains widespread in most endemic areas,
and the continued spread of resistance mandates efforts to identify
new antimalarial drugs.1 In particular, the declining efficacy of are-
temisinins, the most active available antimalarials, highlights the
need for the discovery of novel antimalarials (Dondorp, NEJM,
2010).
Quinacrine (QA, 1 in Fig. 1), based on the 9-aminoacridine het-
eroaromatic core, was initially approved in the 1930s as an antima-
larial drug and was one of the first synthetic surrogates for quinine,
whose supply from Indonesia was blocked during World War II.
During that war, quinacrine was the standard suppressive and
therapeutic antimalarial of the Allied forces.2 From 1946, and for
over 50 years, chloroquine (CQ, 2 in Fig. 1), an analogue of QA with
the 6-chloro-2-methoxyacridine core replaced by a 4-amino-7-
antimalarial, given its greater potency and safety, and lower cost.2
However, the use of CQ to treat falciparum malaria has been grad-
ually abandoned worldwide, due to evolution and spread of CQ-
resistant parasites, especially Plasmodium falciparum.1 However,
cross-resistance is not uniformly seen among related compounds,
and new acridine derivatives may arise again as CQ substitutes. If
possible, such derivatives should act as multi-stage antimalarials,
capable of killing liver, blood, and mosquito-stage parasites.3
Recently, our group showed that N-alkylcinnamoylation of the
aminoquinoline core of known antimalarials, such as primaquine
(PQ, Fig. 1) or CQ, led to substantially enhanced antimalarial activ-
ity as compared to those classical drugs.4–6 In view of this, we have
investigated whether a similar effect would be obtained by cou-
pling a butylcinnamoyl moiety to the amino group of 9-aminoacri-
dine, the unsubstituted heterocyclic scaffold of quinacrine. In this
context, we report the synthesis and in vitro evaluation of novel
9-(N-butylcinnamoyl)aminoacridines, 6, which were found to have
greater activity than the acridine analogue of CQ, 3 (Fig. 1), and PQ,
against blood-stage P. falciparum and liver-stage Plasmodium
berghei malaria parasites.
chloroquinoline moiety, replaced quinacrine as
a first-line
The synthesis of the target molecules was straightforward
(Scheme 1) making the chemistry underlying their preparation
⇑
Corresponding author. Tel./fax: +351 220402563.
E-mail address: pgomes@fc.up.pt (P. Gomes).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.032

B. Pérez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 610–613
611
Figure 1. Molecular structures of quinacrine (1), chloroquine (2), acridine analogue of chloroquine (3) and primaquine (PQ).
simple and cheap, which must be a constant concern when dealing
with development of potential antimalarials, which should be very
inexpensive. The 9-(N-cinnamoylbutyl)aminoacridines 6 were syn-
thesized by first reacting 9-chloroacridine (4) with a large excess of
butane-1,4-diamine to produce 9-(N-aminobutyl)aminoacridine, 5.
This compound was then condensed to the respective cinnamic
acid by a standard peptide coupling method, namely, activation
of the carboxylic acid with O-(Benzotriazol-1-yl)-N,N,N0,N0-tetram-
ethyluronium tetrafluoroborate (TBTU) in the presence of N-ethyl-
N,N-diisopropylamine (DIEA), in N,N-dimethylformamide (DMF) as
solvent. Crude products thus obtained were further purified by li-
quid chromatography on silica columns, to give the target com-
pounds 6 in high purity, as confirmed by high-performance
liquid chromatography (HPLC). Structural analysis of compounds
6 by electrospray ionization-ion trap mass spectrometry (ESI-IT
MS), as well as by proton (1H) and carbon-13 (13C) nuclear mag-
netic resonance (NMR) provided confirmation of the expected
structures. The synthetic route depicted in Scheme 1 led to low
global synthesis yields (2–22%), but allowed fast production of
compounds 6 for immediate screening as potential dual-stage anti-
malarials. Attempts to increase the yields by optimization of both
SNAr and condensation steps were carried out: (a) pre-activation
of 9-chloroacridine 4 with phenol, followed by addition of an
amine in anhydrous conditions, to avoid extensive formation of
the respective acridones, according to Anderson and co-workers;7
(b) use of condensation reagents of usually higher efficiency than
TBTU, such as (7-azabenzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate (PyAOP).These procedures led to reac-
tion mixtures apparently cleaner by thin layer chromatography
(TLC), especially in the SNAr step, but reaction yields were not sig-
nificantly improved.
The compounds were evaluated for their antiplasmodial activity
against (i) erythrocytic stages of the human parasite P. falciparum
(CQ-resistant W2 strain), Table 1, and (ii) liver stages of the rodent
Scheme 1. Synthesis route towards 9-(N-cinnamoylbutyl)aminoacridines 6. Reagents and conditions: (i) 10 M equiv (equiv) of butane-1,4-diamine, 100 °C, 2 h; (ii) TBTU
(1.1 equiv), respective cinnamic acid (1.1 equiv), DIEA (2 equiv), DMF, 24 h. Substituents R are given on Table 1.

612
B. Pérez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 610–613
Table 1
with IC50s ranging between 126 and 345 nM. These IC50s, deter-
mined against CQ-resistant P. falciparum W2, were significantly
lower than those reported for reference compound 3 against CQ-
sensitive P. falciparum 3D7 (720 nM). Furthermore, compounds
6c, 6d, 6f and 6g displayed activities in the same order of magnitude
as CQ, while the remaining hybrids were slightly less active. The
antiplasmodial activity was significantly improved by introducing
a substituent on the cinnamoyl moiety: the unsubstituted deriva-
tive, 6a, showed an IC50 of 892 nM while the substituted compound,
6e, is approximately 3-fold more active (IC50 345 nM). The most ac-
tive compound of the series, 6c, bears an isopropyl group in the
para-position, similar to what was previously observed by our
group for 4- or 8-(N-cinnamoylalkyl)aminoquinolines,5,6 suggest-
ing that a bulky lipophilic cinnamic substituent is preferred to en-
hance antiparasitic activity. However, in our previous work we
observed the best homogeneity in activity amongst cinnamic deriv-
atives belonging to the same series, meaning that substituent R had
only a slight effect on activity.4,5 In contrast, the influence of
In vitro data on compounds 6 for their blood- and liver-stage antimalarial activity
a
b
Compound
R
Pf W2 IC50 (nM)
Liver stage IC50 (lM)
6a
6b
6c
6d
6e
6f
6g
3
CQ
PQ
H
892 152
—
—
—
3.2
—
—
—
—
>15
8
p-Me
p-iPr
p-OMe
m-F
p-F
m-NO2
225
126
138
3
3
2
345 32
145
142
6
2
cinnamoyl substituent
R on the antiplasmodial activity of
721c
138d
3300e
compounds 6 is apparent, but the SAR is unclear: the best three
compounds, 6c, 6d and 6g (IC50 values of 126, 138 and 142 nM,
respectively), exhibit quite different substituents regarding their
lipophilicity and electron-donating/-withdrawing properties. Yet,
comparison of compounds 6e (R = m-F; IC50 = 345 nM) and 6f
(R = p-F; IC50 = 145 nM) demonstrates a clear preference for substit-
uents in the para- over the meta-position.
a
Blood-stage antiplasmodial activity was determined against the CQ-resistant P.
falciparum strain W2 as previously described.8
b
Inhibition of liver stage infection by compounds 6 was determined by mea-
suring the luminescence intensity in Huh-7 cells infected with a firefly luciferase-
expressing P. berghei line, PbGFP-Luccon, as previously described.9
c
EC50 determined through inhibition of Plasmodium falciparum 3D7 LDH activity,
QA (1), like CQ (2), is thought to act on P. falciparum by disrupt-
ing the storage of toxic heme as polymeric hematin.11 Given that
both QA and compounds 6 share the acridine moiety as a central
scaffold, it was conceivable that they operated via a similar mech-
anism. Thus, compounds 6 were evaluated in vitro as inhibitors of
heme biocrystallization, by methods previously reported by us,4–6
but none of them was found to be capable of inhibiting this process
(data not shown). Still, compounds 6 are active against Plasmo-
dium, and this may be due to one of the other mechanisms earlier
proposed for acridines with antimalarial activity, such as inhibition
of mitochondrial bc1 complex or DNA Topoisomerase II, and inter-
action with DNA.12 Therefore, it would be interesting to explore, in
further studies, if the acridine derivatives 6 act through any of
using an LDH reporter assay; test compounds present at 2 M. Molecule ChEMBL ID:
CHEMBL532286.
d
Value taken from Ref. 10.
Value taken from Ref. 8.
e
parasite P. berghei (Fig. 2, Table 1), as well as for their cytotoxicity
to Huh7 human hepatoma cells (Fig. 2). Methods employed were as
previously reported by us.4,5,8 CQ and its acridine analogue 3 were
chosen as reference drugs for blood-stage assays, whereas PQ was
the reference compound for liver-stage studies.
As shown in Table 1, all the compounds except 6a presented
mid-nanomolar inhibitory activity against erythrocytic parasites,
Figure 2. Activity of compounds 6 against P. berghei liver stages. Anti-infective activity (infection scale, bars) and toxicity to hepatoma cells (cell confluency scale, circles) are
shown. Primaquine (PQ) at 10 M was included for comparison. Infection loads of Huh7 cells, a human hepatoma cell line, were determined by bioluminescence
measurements of cell lysates 48 h after infection with luciferase-expressing P. berghei parasites.
l

B. Pérez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 610–613
613
these mechanisms. Furthermore, work is being carried out on the
Acknowledgments
synthesis and characterization of analogues of compounds 6,
where the 9-aminoacridine moiety has been replaced by the 9-
amino-6-chloro-2-methoxyacridine core of QA (1).
This project was co-funded by FEDER through the POFC-COM-
PETE programme (FCOMP-01-0124-FEDER-020963) and by Portu-
It is now commonly accepted that effective and safe antimalar-
ial drugs active against both liver and erythrocytic stage parasites
will be valuable components of malaria eradication strategies.3
Additionally, our recent findings on the improved liver-stage anti-
malarial activity of N-cinnamoyl derivatives of primaquine4 moti-
vated us to evaluate the activity of compounds 6c, 6d and 6f
against liver-stage P. berghei parasites ( Fig. 2); the compounds
were chosen according to: (i) their antiplasmodial activity against
the erythrocytic stage, 6c being the most active; (ii) their prospec-
tive propensity to present low cytotoxicity, 6d being the most ac-
tive compound complying with lead-likeness properties,13
Lipinski’s Rule of Five,14 and Veber filter,15 and (iii) their likelihood
of good metabolic stability, solubility and bioavailability, the cin-
namic ring of 6f being substituted with a fluorine atom, which is
known for its ability to increase the aforementioned properties
when incorporated into aromatic organic compounds.16 Remark-
ably, the three test compounds were more potent than primaquine
(PQ), the reference drug for the parasite liver stage, and com-
pounds 6d and 6f were non-toxic to Huh7 human hepatoma cells
guese national funds through Fundação para
a Ciência e a
Tecnologia (PTDC/QUI-QUI/116864/2010). Funding through project
PTDC/SAU-MII/099118/2008 (MP), and strategic projects PEst-C/
QUI/UI0081/2011 (PG) and PEst-C/CTM/LA0011/2011 (JRBG) is also
acknowledged to Fundação para a Ciência e Tecnologia (FCT). C.T.
and JRBG thank FCT for the post-doctoral fellowship SFRH/BPD/
62967/2009 and for Programa Ciência 2007, respectively. P.J.R. is
a Doris Duke Charitable Foundation Distinguished Clinical Scientist.
Supplementary data
Supplementary data (details regarding synthetic procedures
and analytical/spectral data for compounds 5 and 6, procedures
for in vitro Plasmodium blood stage infection assays and proce-
dures for in vitro Plasmodium liver stage infection assays) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2012.12.032.
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to 5 lM. Compounds 6 were shown to not exert their antiparasitic
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