N-cinnamoylation of antimalarial classics: Quinacrine analogues with decreased toxicity and dual-stage activity

Article abstract of DOI:10.1002/cmdc.201300459

Plasmodium falciparum, the causative agent of the most lethal form of malaria, is becoming increasingly resistant to most available drugs. A convenient approach to combat parasite resistance is the development of analogues of classical antimalarial agents, appropriately modified in order to restore their relevance in antimalarial chemotherapy. Following this line of thought, the design, synthesis and in vitro evaluation of N-cinnamoylated quinacrine surrogates, 9-(N-cinnamoylaminobutyl)- amino-6-chloro-2- methoxyacridines, is reported. The compounds were found to be highly potent against both bloodstage P. falciparum, chloroquine-sensitive 3D7 (IC50=17.0- 39.0 nm) and chloroquine-resistant W2 and Dd2 strains (IC50= 3.2-41.2 and 27.1-131.0 nm, respectively), and liver-stage P. berghei (IC50=1.6-4.9 mm) parasites. These findings bring new hope for the possible future rise of a fallen angel in antimalarial chemotherapy, with a potential resurgence of quinacrine- related compounds as dual-stage antimalarial leads.

Full text of DOI:10.1002/cmdc.201300459

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DOI: 10.1002/cmdc.201300459
N-Cinnamoylation of Antimalarial Classics: Quinacrine
Analogues with Decreased Toxicity and Dual-Stage
Activity
Ana Gomes,[a] Bianca Pꢀrez,[a] InÞs Albuquerque,[b] Marta Machado,[c] Miguel PrudÞncio,[b]
Fꢁtima Nogueira,[c] Cꢁtia Teixeira,*[d] and Paula Gomes*[a]
Plasmodium falciparum, the causative agent of the most lethal
form of malaria, is becoming increasingly resistant to most
available drugs. A convenient approach to combat parasite re-
sistance is the development of analogues of classical antimalar-
ial agents, appropriately modified in order to restore their rele-
vance in antimalarial chemotherapy. Following this line of
thought, the design, synthesis and in vitro evaluation of N-cin-
namoylated quinacrine surrogates, 9-(N-cinnamoylaminobutyl)-
amino-6-chloro-2-methoxyacridines, is reported. The com-
pounds were found to be highly potent against both blood-
stage P. falciparum, chloroquine-sensitive 3D7 (IC50 =17.0–
39.0 nm) and chloroquine-resistant W2 and Dd2 strains (IC50 =
3.2–41.2 and 27.1–131.0 nm, respectively), and liver-stage
P. berghei (IC50 =1.6–4.9 mm) parasites. These findings bring
new hope for the possible future “rise of a fallen angel” in anti-
malarial chemotherapy, with a potential resurgence of quina-
crine-related compounds as dual-stage antimalarial leads.
um.[2] Thus, the search for new chemotherapeutic strategies to
fight malaria is urgently needed.
Quinacrine (1), formerly marketed as Atebrin and also
known as mepacrine, is an acridine-based compound that was
first synthesized in 1931 (Bayer, Germany) and initially used for
prophylaxis and treatment of malaria.[3] It was the first synthet-
ic substitute for quinine,[4] a natural product obtained from the
cinchona tree, and the first effective therapy used against ma-
laria. Quinacrine was superseded by chloroquine (2), a more ef-
fective, less toxic, and affordable antimalarial agent, which was
first synthesized in 1934 but only advanced as a therapeutic
agent after World War II.[5] While quinacrine is no longer com-
monly employed as an antimalarial, it has been used “off-label”
to treat giardiasis, systemic lupus erythematosus, and rheuma-
toid arthritis.[6–8]
A convenient approach to antimalarial drug discovery is the
development of analogues of classical antimalarials aiming at
altering their unwanted properties and/or optimizing their
original effects, in order to restore their relevance in antimalari-
al chemotherapy. Following the covalent biotherapy concept
proposed by Meunier and co-workers,[9,10] our group recently
developed hybrid compounds (3–5) through cinnamic acid
conjugation with heterocyclic moieties from well-known anti-
malarials, such as 4-amino-7-chloroquinoline from chloroquine,
8-amino-6-methoxyquinoline from primaquine or the acridine
ring from quinacrine. These hybrid agents exhibited improved
antimalarial activity when compared with their parent
drugs.[11–14] Furthermore, hybrids bearing either the chloro-
quine or the acridine core displayed dual-stage activity with
IC50 values of 11–892 nm against P. falciparum W2 strain and
1.1–6.5 mm against liver forms of P. berghei, respectively, which
compares rather favorably with the activities displayed by the
reference drugs against blood (IC50(CQ) =138 nm) and liver
(IC50(PQ) =7.5 mm) stage parasites.[12,13]
Malaria is caused by protozoan parasites of the genus Plasmo-
dium. During the last decade (2000–2010), mortality associated
with malaria has dropped by over 25%. Still, more than half
a million children continue to die from this disease every
year.[1] Moreover, P. falciparum, the organism that causes the
most lethal form of the disease, is becoming increasingly re-
sistant to most drugs available in the antimalarial compendi-
[a] A. Gomes, B. Pꢀrez, Prof. Dr. P. Gomes
Centro de Investigażo em Quꢁmica e Bioquꢁmica
Faculdade de CiÞncias, Universidade do Porto
R. do Campo Alegre, 4169-007 Porto (Portugal)
E-mail: pgomes@fc.up.pt
[b] I. Albuquerque, Dr. M. PrudÞncio
Instituto de Medicina Molecular
Faculdade de Medicina, Universidade de Lisboa
Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)
[c] M. Machado,+ Dr. F. Nogueira
It was previously reported that introduction of a methoxy
group at position 2 and a chlorine at position 6 of the acridine
core increases the inhibitory potency by three- and ninefold
on chloroquine-resistant and chloroquine-sensitive strains, re-
spectively.[15] Based on this, we herein report the synthesis and
in vitro evaluation of N-cinnamoylated quinacrine surrogates,
9-(N-cinnamoylaminobutyl)-amino-6-chloro-2-methoxyacridines
(7a–f), which resulted highly potent against both blood-stage
P. falciparum and liver-stage P. berghei parasites. The choice of
the benzene ring substituents and of the aminobutyl spacer
between the acridine and the cinnamoyl moieties was based
Centro de Malꢂria e Outras DoenÅas Tropicais
Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa
Rua da Junqueira 100, 1349-008 Lisboa (Portugal)
[d] Dr. C. Teixeira
CICECO, Departamento de Quꢁmica, Universidade de Aveiro
Campus Universitꢂrio de Santiago, 3810-193 Aveiro (Portugal)
E-mail: ca.teixeira@ua.pt
[+] Present address: Instituto de Medicina Molecular, Faculdade de Medicina,
Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300459.
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Scheme 1. Synthesis of 9-(N-cinnamoylaminobutyl)amino-6-chloro-2-me-
thoxyacridines 7a–f. Reagents and conditions: a) phenol, Cs2CO3, anhyd
DMSO, 3 ꢃ molecular sieves, 2 h, 1008C; b) butan-1,4-diamine, 1008C, 3 h;
c) cinnamic acid, TBTU, DIEA, DMF, RT, 24 h.
on previously established structure–activity relationships (SAR)
regarding N-cinnamoylated chloroquine analogues.[14]
IC50 values ranging from 17 to 39 nm for chloroquine-sensitive
strain 3D7, and 27.1–131 nm and 3.2–41.2 nm for chloroquine-
resistant strains Dd2 and W2, respectively. Hence, in general,
quinacrine analogues displayed decreasing antiplasmodial ac-
tivity against those three strains in the following order: W2>
3D7>Dd2. Moreover, while all compounds except 7c were
one- to approximately twofold less active than chloroquine
against the chloroquine-sensitive strain, the opposite was ob-
served for both chloroquine-resistant strains: all quinacrine an-
alogues were considerably more active, by two- to fourfold
and five- to 70-fold, than chloroquine against P. falciparum Dd2
and W2, respectively, with the exception of 7d against P. falci-
parum Dd2.
The preparation of compounds 7a–f was inexpensive and
facile; given the areas affected by malaria, a constant concern
when developing potential antimalarial agents is to ensure the
synthesis is as straightforward as possible. Briefly, the synthetic
route to access compounds 7a–f involved two main reaction
steps: a nucleophilic aromatic substitution between butan-1,4-
diamine and 6,9-dichloro-2-methoxyacridine to obtain com-
pound 6, according to an adaptation of the method described
by Anderson and co-workers,[16] followed by a nucleophilic ad-
dition–elimination (condensation) reaction between 6 and the
relevant cinnamic acid (Scheme 1). The global synthesis yields
were around 13–32%, generally higher than those previously
obtained in the synthesis of acridine derivatives 5 (3–22%).[12]
N-Cinnamoylated quinacrine analogues 7a–f were evaluated
in vitro for their 1) activity
Compared with the parent antimalarial quinacrine, com-
pounds 7a–f exhibited more potent activities across all three
strains. Also, the activities of compounds 7a–f against chloro-
against erythrocytic forms of
Table 1. In vitro cytotoxicity and antimalarial activities of test compounds 7a–f and reference drugs against
blood- and liver-stage parasites.
P. falciparum (chloroquine-sensi-
tive 3D7, and chloroquine-resist-
ant W2 and Dd2 strains), 2) activ-
ity against liver-stages of P. ber-
ghei, and 3) cytotoxicity towards
human hepatocellular carcinoma
(HepG2) cells. The corresponding
assays carried out are described
in detail in the Experimental Sec-
tion. Chloroquine and quinacrine
were chosen as reference drugs
for blood-stage activity assays,
whereas primaquine was the ref-
erence drug for liver-stage stud-
ies.
Compd
R
logP[a]
IC50 [nm][b]
Pf Dd2
CC50 [mm][c]
HepG2
SI[f]
IC50 [mm][d]
liver stage
Pf 3D7
Pf W2
7a
7b
7c
7d
7e
7 f
p-Me
p-iPr
p-OMe
p-F
p-Cl
p-Br
5.90
6.63
5.23
5.53
5.99
6.15
33.3
39.0
17.0
29.8
27.6
37.0
21.0
100[e]
–
42.2
27.1
47.9
131.0
28.9
50.6
107.5
200[e]
–
3.2
10.5
41.2
17.8
8.9
11.3
225.8
159[e]
–
8.6
4.1
20.5
164.7
4.3
3.4
–
204
105
428
1257
149
67
–
37
–
–
–
4.9
1.6
1.8
–
Chloroquine
Quinacrine
Primaquine
>15
13
7.4
–
7.5[g]
[a] LogP values were calculated using Marvin 6.1.0 (calculator plugins) from ChemAxon (http://www.chemax-
on.com); [b] Blood-stage antiplasmodial activity was determined against chloroquine-resistant strains W2 and
Dd2 and chloroquine-sensitive strain 3D7 of P. falciparum (Pf); [c] Cytotoxicity was determined against a human
hepatocellular carcinoma (HepG2) cell line; data are expressed as the cytotoxic concentration 50 (CC50), which
is the concentration required to reduce the cell viability by 50%; [d] Liver-stage antiplasmodial activity was de-
termined against P. berghei. [e] Values taken from Ref. [17]. [f] Selectivity index (SI): cytotoxicity/antiplasmodial
ratio, calculated using the least potent IC50 value determined against the P. falciparum strains. [g] Value taken
from Ref. [11].
As shown in Table 1, all com-
pounds
were
exceptionally
active across all three strains of
erythrocytic-stage parasites, with
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quine-resistant strain W2 were significantly better (IC50 =3.2–
41.2 nm) than those of the respective 9-(N-cinnamoylbutyl)ami-
noacridines 5 (IC50 =126–225 nm),[12] as expected due to the
presence of the 2-methoxy and 6-chloro substituents in the ac-
ridine core of 7.[15] For instance, 7a (R=p-Me) displayed an IC50
value of 3.2 nm against P. falciparum W2, almost 70 times more
potent than that displayed by its counterpart in compounds 5
(IC50 =225 nm).
No obvious influence regarding the stereoelectronic proper-
ties of the cinnamic substituents (R), was observed on the anti-
plasmodial activities of compounds 7a–f. Nevertheless, a similar
trend is observed for the halogenated derivatives against the
chloroquine-resistant strains Dd2
ed us to assess the activity of compounds 7a–f against liver
forms of the rodent malaria parasite P. berghei (Table 1). The ac-
tivity of compounds 7a–f was evaluated at 1, 5 and 10 mm
using a previously described bioluminescence-based method
to quantify overall parasite infection of Huh-7 cells, a human
hepatoma cell line.[19] The toxicity of the compounds was also
assessed against this human cell line through the fluorescence
measurement of cell confluency (Figure 1). Compounds 7a–f
were all active against liver-stage P. berghei parasites, and IC50
values were determined for the three best compounds, accord-
ing to both their activity and cytotoxicity (Table 1 and
Figure 1). Remarkably, as for the blood-stage activities, com-
and W2, as in both cases their
activities followed the order 7e
(p-Cl)>7 f (p-Br)>7d (p-F). Inter-
estingly, para-chloro substitution
of the cinnamoyl moiety has
also been previously found to
lead to most potent activities of
N-cinnamoylated 4- and 8-ami-
noquinolines (3 and 4), and acri-
dine (5) analogues against P. fal-
ciparum W2.[11–13]
In order to assess the selectivi-
ty of compounds 7a–f for para-
site over human cells, their
cytotoxicity was evaluated in
human hepatocellular carcinoma
Figure 1. Activity of compounds 7a–f against P. berghei liver stages. Anti-infective activity (infection scale in rela-
tive luminescence units (RLU), bars) and toxicity to Huh7 cells (cell confluency scale in relative fluorescence units
(RFU), circles) are shown at 1 mm (&), 5 mm (&), and 10 mm (&). Primaquine (PQ) at 10 mm was included in the
assay for comparison. The infection load of human hepatocellular carcinoma (Huh7) cells was determined by bio-
luminescence measurements of cell lysates 48 h after infection with luciferase-expressing P. berghei parasites.[19]
(HepG2) cells. The selectivity
index (SI) is defined as the ratio
of cytotoxicity over antiplasmo-
dial activity—the CC50 value de-
termined against HepG2 cells
over the least potent IC50 value determined against P. falcipa-
rum strains 3D7, W2 or Dd2. Although some compounds pre-
sented moderate cytotoxicity (CC50 =4.1 (7b), 4.3 (7e), and 3.4
(7 f) mm), all compounds except 7 f (SI=67) displayed high se-
lectivity indices (SI>100). Biological efficacy is generally con-
sidered to not be due to in vitro cytotoxicity when an SI value
is ꢀ10.[18] Consequently, these results suggest that the activity
exhibited by compounds 7a–f is unlikely due to general cellu-
lar toxicity but rather to specific antiplasmodial activity. Note-
worthy, compounds with the para-methoxy and para-fluoro
R groups (7c and 7d, respectively) exhibited considerably
higher SI values (428 and 1257, respectively) than the other
quinacrine derivatives, whose SI values ranged from 67 to 204.
This could be related to two key facts: 1) the methoxy- and
fluoro-substituted analogues are the least lipophilic in the
series (Table 1), since high lipophilicity is known to give rise to
increased tendency for toxicity,[30] and 2) aromatic compounds
containing a fluorine atom typically exhibit good stability, solu-
bility, and bioavailability.[27] Thus, compounds 7c and 7d show
a promising profile as potential leads for safe antimalarial ther-
apy.
pounds 7a–f resulted more active against liver-stage P. berghei
(IC50 =1.6–4.9 mm) than the reference drug for this stage, pri-
maquine (IC50 =7.5 mm), or the parent drug, quinacrine (IC50 =
13 mm). The compounds exhibiting the best activity/cytotoxici-
ty ratio, 7d (R=p-F, IC50 =1.6 mm) and 7e (R=p-Cl, IC50 =
1.8 mm) resulted about four to five times more active than pri-
maquine and over ten times more active than quinacrine. Fi-
nally, electron-withdrawing R groups seem to be more effec-
tive than electro-donating groups in terms of the enhance-
ment of liver-stage activity, as suggested by the fact that 7c
(R=p-OMe) was at least twofold less active (IC50 =4.9 mm) than
7d or 7e (R=p-F or p-Cl, respectively).
The mechanism of action (MoA) of quinacrine against malar-
ia parasites remains unclear, but it has been demonstrated that
the drug can interact with b-hematin, consequently inhibiting
hemozoin formation and the development of intraerythrocytic
parasites.[20] In view of this, we further investigated whether
compounds 7a–f could owe their activity against blood-stage
parasites to inhibition of b-hematin polymerization. To this
end, compounds 7a–f were evaluated in vitro as inhibitors of
hemozoin formation by previously described methods.[13,21]
With the exception of compound 7a, which was a moderate
inhibitor of hemozoin formation, as compared to chloroquine,
The recent unprecedented finding of liver-stage antimalarial
activity of N-cinnamoylated chloroquine analogues[14] prompt-
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none of the compounds was able to inhibit hemozoin forma-
tion in vitro (data not shown). This result is in agreement with
our previous observations for acridine analogues 5.[12] Conse-
quently, inhibition of hemozoin formation does not appear to
be the main MoA of compounds 7a–f against blood-stage
Plasmodium spp. Moreover, even if compounds 7a–f were able
to inhibit this intraerythrocytic process, that would not explain
their ability to also inhibit infection by liver-stage parasites.
Other mechanisms that might be responsible for the antima-
larial activity of compounds 7a–f include the activation of p53
and the inhibition of NF-kB pathways. It is well known that the
p53 and NF-kB pathways play important roles in diverse cellu-
lar functions, including cell growth, apoptosis, and tumorigen-
esis.[22] Indeed, mutations that inactivate the p53 gene and
trigger NF-kB pathway activation are common occurrences in
human cancers.[22] However, these pathways have also been re-
ported as critical for parasite survival; as the activation of NF-
kB in response to pathogens stimuli is normally associated
with the initiation of protective immunity, it is believed that
several parasites have developed strategies to interfere with
NF-kB activation in order to decrease the host immune re-
sponse to allow parasite survival.[23] In fact, Tato and co-workers
reported the ability of P. falciparum schizonts to activate NF-kB
in host vascular endothelium and, thus, contribute to parasite
survival.[23] Moreover, Kaushansky and co-workers recently dis-
closed their finding that perturbation of the hepatocyte p53
pathway critically impacts parasite survival, as mice that ex-
pressed increased levels of p53 showed decreased liver-stage
parasite burden, whereas p53-knockout mice displayed in-
creased liver-stage parasite burden.[24] In this context, and
based on literature accounts that report quinacrine as a com-
pound that simultaneously activates p53 and inhibits NF-kB
pathways,[22] these two processes arise as putative MoA for
compounds 7a–f, as they would explain both the liver- and
blood-stage activities. Nevertheless, other previously proposed
mechanisms for quinacrine derivatives with antimalarial activi-
ty, such as binding to DNA, either by intercalation or groove
binding, inhibition of mitochondrial bc1 complex or DNA top-
oisomerase II, cannot be ruled out.[25,26] Studies aiming at the
establishment of the MoA of these compounds are currently
underway.
stressed that, altogether, these and previous[11–14] findings from
our group strongly suggest that the N-cinnamoyl moiety is
a relevant pharmacophore to boost antiplasmodial activity of
classic antimalarial agents, based on either the [4-/8-]aminoqui-
noline or acridine cores.
Experimental Section
Chemistry
General: Starting materials 6,9-dichloro-2-methoxy-acridine,
butane-1,4-diamine, and phenol were acquired from Sigma–Al-
drich; O-(Benzotriazol-1-yl)-N,N,N’,N’-tetra-methyluronium tetra-
fluoroborate (TBTU) was bought from Bachem, cinnamic acids
from Acros Organics, and solvents from VWR International. NMR
analyses were carried out on a Brucker Avance III 400 MHz spec-
trometer, and samples were prepared in CDCl3 with tetramethylsi-
lane (TMS) as an internal reference. Mass spectrometry (MS) spectra
were obtained on a Thermo Finnigan LCQ Deca XP Max LC/MSn in-
strument with electrospray ionization and ion-trap mass analysis
(ESI-IT MS). Purity of the compounds was confirmed to be of at
least 98% by HPLC using the following conditions: 10!70% of B
in A (A: 0.05% aq trifluoroacetic acid; B: CH3CN) over 25 min at
a flow rate of 1 mLminꢁ1 on a Purospher STAR, RP-C18 column
(150ꢄ4.0 mm; particle size, 5 mm), using a Merck-Hitachi Lachrom
Elite instrument equipped with a diode-array detector (DAD) and
a thermostated (Peltier effect) autosampler.
General procedure for the synthesis of 9-(N-cinnamoylaminobu-
tyl)amino-6-chloro-2-methoxyacridines (7a–f): 6,9-Dichloro-2-me-
thoxy-acridine (1 equiv), phenol (5 equiv), Cs2CO3 (1 equiv) and
anhyd DMSO (2 mL) were stirred at 1008C for 2 h with 3 ꢃ molecu-
lar sieves. Then, butane-1,4-diamine (10 equiv) was added directly
to the mixture, and the reaction was stirred at 1008C for an addi-
tional 3 h. The mixture was cooled to RT, diluted in CH2Cl2 (25 mL),
and washed with 5% aq Na2CO3 (3ꢄ25 mL), after which the organ-
ic layer was dried over anhyd Na2SO4, filtered and concentrated in
vacuo to yield 9-(N-aminobutyl)amino-6-chloro-2-methoxyacridine
(6) as an orange solid, which was used in the next step without
further purification.
Next, a mixture of the appropriate cinnamic acid (1.1 equiv), TBTU
(1.1 equiv) and N,N-diisopropylethylamine (2 equiv) in DMF (2 mL)
was stirred at 08C for 10 min. Then, a solution of 6 (1 equiv) in
DMF (2 mL) at RT was added, and the reaction was stirred for 24 h
at RT. The reaction mixture was diluted with CH2Cl2 (25 mL),
washed with 5% aq Na2CO3 (3ꢄ25 mL), dried over anhyd Na2SO4,
filtered and concentrated in vacuo to yield the crude product as
an oil. Purification by flash chromatography using a silica gel
column (H2Cl2/MeOH, 4:1 v/v) gave target compounds 7a–f as
orange solids (13–32%). Complete spectroscopic and analytical
data are provided in the Supporting Information.
In summary, we have presented a novel family of N-cinna-
moylated quinacrine analogues as dual-stage antimalarial com-
pounds. The compound with the best profile of the series was
7d (R=p-F), which exhibits 1) activities comparable or higher
than those of reference chloroquine against blood-stage 3D7,
Dd2 and W2 P. falciparum parasites, 2) the most potent activity
against liver-stage P. berghei, and 3) the weakest cytotoxicity
against human cells. The latter adds to the fact that fluorinated
compounds are known to have higher bioavailability, solubility,
and metabolic stability compared with their nonfluorinated an-
alogues.[27] The MoA of the compounds remains to be elucidat-
ed, but results suggest that inhibition of hemozoin formation
can be ruled out. Ongoing studies will hopefully elucidate the
MoA, bringing new insights into the development of new anti-
malarial agents and enabling resurgence for quinacrine-related
compounds as dual-stage antimalarial leads. Finally, it must be
Biology
In vitro inhibition of b-hematin polymerization: Stock solutions of
compounds 7a–f were prepared at 0.1–1 mm in DMSO. The assays
were run in triplicate, and DMSO and water were used as negative
controls, and chloroquine was used as a positive control. Each well
of a 96-well plate contained varying concentrations of test com-
pound, hemin chloride in DMSO (5.2 mgmLꢁ1; 50 mL) and acetate
buffer (0.2m, pH 4.4, 100 mL), and the plate was incubated at 378C
for 48 h. Samples were centrifuged at 3000 rpm for 15 min. After
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discarding the supernatant, the pellet was washed with DMSO (4ꢄ
200 mL) and then dissolved in 0.2m aq NaOH (200 mL). The solubi-
lized aggregates were further diluted with 0.1m aq NaOH (1:6),
and absorbance was recorded at 405 mm.
each well, the plates were incubated for 3 h under standard culture
conditions, and the supernatant was removed and substituted by
200 mL of acidified isopropanol. Results were obtained by ELISA
reading at 570 nm, to produce a log dose-dependent curve. The
CC50 value was estimated for each compound by nonlinear interpo-
lation of the dose-dependent curve using GraphPad Software (trial
version).
Drugs and antibodies: The two commercial monoclonal antibodies
(Immunology Consultants Laboratory Inc., Newberg, OR, USA) di-
rected against P. falciparum-specific HRP2 were MPFM- 45A (MPFM-
55A), an immunoglobulin M (IgM) antibody used as the capture an-
tibody, and MPFG-45P (MPFG-55P), a horseradish peroxidase-conju-
gated IgG antibody used as the indicator antibody. Chloroquine
(C6628), bovine serum albumin (BSA; A2153), Tween 20 (P1379),
3,3’,5,5’-tetramethylbenzidine (T0440), thiazolyl blue tetrazolium
bromide (M2128) were purchased from Sigma–Aldrich and
RPMI 1640 medium (51800019), AlbuMAX II (11021037) from Life
Technologies.
In vitro liver-stage activity assays: In vitro inhibition of liver-stage in-
fection by test compounds was determined by measuring the lu-
minescence intensity in Huh-7 cells infected with a firefly luciferase
expressing P. berghei line, PbGFP-Luccon, as previously described.[19]
Huh-7 cells, from a human hepatoma cell line, were cultured in
RPMI 1640 medium supplemented with 10% v/v fetal calf serum,
1% v/v nonessential amino acids, 1% v/v penicillin/streptomycin,
1% v/v glutamine, and 10 mm 4-(2-hydroxyethyl)-1-piperazineetha-
nesulfonic acid (HEPES), pH 7, and maintained at 378C with 5%
CO2. The Huh-7 cells (1.2ꢄ104 per well) were seeded in 96- well
plates the day before compound treatment and infection. Medium
in the cells was replaced by medium containing the appropriate
concentration of each compound approximately 1 h prior to infec-
tion with sporozoites freshly obtained through disruption of saliva-
ry glands of infected female Anopheles stephensi mosquitoes. Spor-
ozoite addition was followed by centrifugation at 1700 g for 5 min.
At 24 h after infection, medium was replaced by fresh medium
containing the appropriate concentration of test compound. Inhib-
ition of parasite development was measured 48 h after infection.
The effect of the compounds on the viability of Huh-7 cells was as-
sessed by the AlamarBlue assay (Invitrogen, UK) using the manu-
facturer’s protocol.
Parasite cultivation: Laboratory-adapted P. falciparum Dd2 (chloro-
quine-resistant, mefloquine-resistant), W2 (chloroquine-resistant
and mefloquine-sensitive) and 3D7 (chloroquine and mefloquine-
sensitive) strains were continuously cultured and synchronized as
previously described.[28] Staging and parasitaemia were determined
by light microscopy of Giemsa-stained thin blood smears.
HRP2 drug assay: Synchronized 3D7, W2 and Dd2 parasites with
>90% ring forms were diluted to 0.5% parasitaemia with 1.5%
haematocrit. Cultures were then incubated with serial dilutions of
each compound at 378C with 5% CO2, 5% O2, and 90% N2. After
72 hours, cultures were diluted by adding 100 mL/well of distilled
water and stored at ꢁ208C until reading.
HRP2 double-site antigen capture ELISA: High-binding 96-well ELISA
plates (Costar 3590; Corning Inc., NY, USA) were coated with
100 mL/well of a 1.0 mgmLꢁ1 solution of anti-HRP2 IgM antibody so-
lution (MPFM-45 A) in phosphate-buffered saline (PBS). Then, the
plates were sealed and incubated overnight at 48C. The superna-
tant was discarded, and plates were saturated for 2 h with 200 mL/
well of a 2% BSA solution in PBS. The supernatant was discarded,
and plates were washed three times with 200 mL/well of washing
solution (0.05% Tween 20 in PBS). 100 mL of each hemolyzed (by
freeze-thawing at least twice) cultured sample were added to the
precoated ELISA plates. The ELISA plates were incubated for 1 h at
room temperature and washed three times with washing solution.
Then 100 mL of the antibody conjugate (0.05 mgmLꢁ1 of MPFG-45P
in 2% BSA and 1% Tween 20 in PBS) were added to each well.
After incubation for 1 h at room temperature, the plate was
washed three times with washing solution and 100 mL of 3,3’,5,5’-
tetramethylbenzidine was added to each well, and the plates were
incubated in the dark for 5–10 min. The reaction was stopped with
50 mL of 1m H2SO4. Plates were read at 450 nm using an ELISA
plate reader (Dynex Triad, Alfagene). IC50 values were estimated for
each compound by nonlinear interpolation of the dose-dependent
curve using GraphPad Software (trial version).
Acknowledgements
This project was co-funded by the European Regional Develop-
ment Fund (FEDER), Portugal through the POFC-COMPETE pro-
gram (FCOMP-01–0124-FEDER-020963) and by the Portuguese
Foundation for Science and Technology (FCT) (PTDC/QUI-QUI/
116864/2010). Additional funding from the FCT through project
PTDC/SAU-MII/099118/2008 (M.P.) and strategic project PEst-C/
QUI/UI0081/2011 (P.G.) is also acknowledged. C.T. and B.P. also
thank the FCT for the post-doctoral grant SFRH/BPD/62967/2009
and the doctoral grant SFRH/BD/86166/2012, respectively.
Keywords: N-cinnamoylation · drug resistance · dual-stage
antimalarial agents · Plasmodium falciparum · quinacrine
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Received: November 11, 2013
Published online on December 27, 2013
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