N-Cinnamoylation of Antimalarial Classics: Effects of Using Acyl Groups Other than Cinnamoyl toward Dual-Stage Antimalarials

Article abstract of DOI:10.1002/cmdc.201500164

In a follow-up study to our reports of N-cinnamoylated chloroquine and quinacrine analogues as promising dual-stage antimalarial leads with high in vitro potency against both blood-stage Plasmodium falciparum and liver-stage Plasmodium berghei, we decided

Full text of DOI:10.1002/cmdc.201500164

DOI: 10.1002/cmdc.201500164
Communications
N-Cinnamoylation of Antimalarial Classics: Effects of Using
Acyl Groups Other than Cinnamoyl toward Dual-Stage
Antimalarials
Ana Gomes,[a] Marta Machado,[b] Lis Lobo,[c] Fµtima Nogueira,[c] Miguel PrudÞncio,[b]
Cµtia Teixeira,*[a] and Paula Gomes*[d]
In a follow-up study to our reports of N-cinnamoylated chloro-
quine and quinacrine analogues as promising dual-stage anti-
malarial leads with high in vitro potency against both blood-
stage Plasmodium falciparum and liver-stage Plasmodium ber-
ghei, we decided to investigate the effect of replacing the cin-
namoyl moiety with other acyl groups. Thus, a series of N-acy-
lated analogues were synthesized, and their activities against
blood- and liver-stage Plasmodium spp. were assessed along
with their in vitro cytotoxicities. Although the new N-acylated
analogues were found to be somewhat less active and more
cytotoxic than their N-cinnamoylated counterparts, they equal-
ly displayed nanomolar activities in vitro against blood-stage
drug-sensitive and drug-resistant P. falciparum, and significant
in vitro liver-stage activity against P. berghei. Therefore, it is
demonstrated that simple N-acylated surrogates of classical an-
timalarial drugs are promising dual-stage antimalarial leads.
human body. Another drawback in malaria containment is the
high cost of first-line treatments, such as artemisinin combina-
tion therapies (ACT), which instigates the trafficking of counter-
feit antimalarial drugs.[2,3] One strategy to accelerate the devel-
opment of new drugs is to start from the chemical frameworks
of known antimalarials, i.e., to recycle classical drug scaf-
folds.[4,5] In this regard, our group has been working extensive-
ly on the chemical recycling of classical antimalarials with
rather promising results, including the recent discovery of N-
cinnamoylated chloroquine (3) and N-cinnamoylated quina-
crine (4) analogues as dual-stage antimalarial leads.[6–10] These
conjugates display high in vitro potency against both blood-
stage P. falciparum (CQ-sensitive and -resistant strains), and
liver-stage P. berghei, representing promising dual-stage anti-
malarial leads.[8,10] These were unprecedented findings, as dual-
stage activity for CQ- or QC-based molecules had not been re-
ported previously. Furthermore, some of the compounds de-
veloped in these studies also displayed in vivo antimalarial ac-
tivity.[8]
A child dies nearly every minute from malaria. Despite the fact
that malaria-associated deaths in Africa have decreased by
about 54% since 2000, eradication is far from being attained
in the near future.[1] There are well-identified obstacles to the
eradication of malaria, including the complexity of the malaria
parasite’s life cycle, widespread resistance to less expensive
and most popular antimalarials such as chloroquine (CQ, 1),
the toxicity of some classical antimalarial drugs, e.g., quina-
crine (QC, 2), the lack of effective vaccines, and the scarcity of
multi-stage antimalarials that are able to efficiently deplete
liver- and blood-stage forms of Plasmodium parasites from the
[a] A. Gomes, Dr. C. Teixeira
CICECO, Departamento de Química, Universidade de Aveiro
Campus Universitµrio de Santiago, 3810-193 Aveiro (Portugal)
E-mail: teixeira@ua.pt
Interestingly, compounds 3 and 4 lack a basic aliphatic
amine, classically considered a prerequisite for the potent
blood-stage antimalarial activity of aminoquinolines and relat-
ed structures.[11] Moreover, the potent blood-stage activity of
CQ analogues 3 against CQ-resistant P. falciparum suggests
that insertion of the N-cinnamoyl group produces a resistance-
reversal effect, possibly due to either chemosensitization,[12] or
a mechanism of action (MOA) different from that classically at-
tributed to CQ, i.e., inhibition of b-hematin.[13]
[b] M. Machado, Dr. M. PrudÞncio
Instituto de Medicina Molecular, Faculdade de Medicina
Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)
[c] L. Lobo, Dr. F. Nogueira
Centro de Malµria e Outras DoenÅas Tropicais
Instituto de Higiene e Medicina Tropical
R. da Junqueira 100, 1349-008 Lisboa (Portugal)
[d] Prof. Dr. P. Gomes
UCIBIO-REQUIMTE, Departamento de Química e Bioquímica
Faculdade de CiÞncias da Universidade do Porto
Rua do Campo Alegre 687, 4169-007 Porto (Portugal)
E-mail: pgomes@fc.up.pt
In fact, we found that the antiplasmodial activity of com-
pounds 3 could not be fully explained by b-hematin inhibition.
We have also ruled out that these compounds could owe their
potent blood-stage activity to falcipain inhibition, through irre-
versible Michael-type addition of the enzyme’s catalytic Cys
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500164.
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Scheme 1. General synthetic route to CQ analogues 9 and QC analogues 10 (note that these compounds differ only in their heteroaromatic cores, where CQ
analogues bear a quinoline core and QC analogues bear an acridine core). Reagents and conditions: a) (starting from 5), butan-1,4-diamine, 1008C, 2 h;
a’) (starting from 6), phenol, Cs2CO3, dry DMSO, 3 Š molecular sieves, 1008C, 2 h, then butan-1,4-diamine, 1008C, 3 h; b) Ac2O (excess), RT, 1 h; c) relevant car-
boxylic acid, TBTU/DIEA (1:2), DMF, RT, 24 h; d) sorbic chloride, Cs2CO3, DMF, RT, 3.5 h.
thiol to the compounds’ a,b-vinylcarbonyl moiety in the cinna-
moyl group.[6,14]
trap quadrupole detection (ESI-IT MS); relevant data are pre-
sented in the Experimental Section.
In the absence of a confirmed MOA for compounds 3 and 4,
we decided to investigate the importance of the cinnamoyl
group for antimalarial activity. To this end, a series of N-acylat-
ed CQ (compounds 9) and QC (compounds 10) analogues
were synthesized (Scheme 1), and their antimalarial activities
against blood- and liver-stage Plasmodium spp., as well as in
vitro toxicity to human hepatocellular carcinoma (HepG2) cells,
were assessed (Table 1). The results are reported and discussed
herein.
Blood-stage activity of compounds 9 and 10 was deter-
mined by previously reported methods[10] against three strains
of P. falciparum: CQ-sensitive 3D7 and CQ-resistant Dd2 and
W2. Notably, compounds 9 and 10 were unable to inhibit b-
hematin formation (i.e., heme polymerization) in vitro (data
not shown). Close inspection of the data listed in Table 1 ena-
bles the following general observations:
1) Remarkably, all compounds display nanomolar activities
against the three P. falciparum strains, with a noteworthy
IC50 value of 4.60 nm for CQ analogue 9b against CQ-re-
sistant strain W2, and with 9 f being the best performer
against the three strains tested (IC50: 5.30, 27.0, and
12.3 nm against 3D7, Dd2, and W2, respectively.
To evaluate the effect of replacing the N-cinnamoyl moiety
with other N-acyl groups, compounds 9 and 10 were designed
in which the cinnamoyl group of 3 and 4, respectively, was
substituted with either alkanoyl groups of various lengths, in-
cluding linear (9a–d, 10a–d), branched (9e,f, 10e,f), and fluori-
nated (9g, 10g) groups, or by nonaromatic a,b-vinylcarbonyls
(9h–j, 10h–j), as shown in Scheme 1. Accordingly, the CQ and
QC derivatives 9 and 10 reported herein were obtained by
starting from 4,7-dichloroquinoline (5) and 6,9-dichloro-2-
2) CQ analogues
9
(5.30ꢀIC50 ꢀ254 nm) were generally
more active than QC analogues 10 (11.0ꢀIC50 ꢀ493 nm),
with 10h being the best performer amongst the latter
series (IC50: 38.5, 11.0, and 32.4 nm against 3D7, Dd2, and
W2, respectively).
methoxyacridine (6), respectively, through
a couple of
simple steps: a nucleophilic aromatic substitution followed by
a nucleophilic addition–elimination (condensation) reaction
(Scheme 1). Detailed procedures are described under the Ex-
perimental Section below. The small number of synthesis
steps, inexpensive starting materials, and straightforward
workup procedures (cf. Experimental Section) clearly compen-
sate for some of the lower yields obtained in the synthesis of
the target compounds. These were obtained in high purity, as
determined by high-performance liquid chromatography with
diode array detection (HPLC-DAD), and their structures were
3) With only a few exceptions, all compounds are globally
more active than the respective parent drugs, CQ or
QC,[15] against the blood-stage P. falciparum strains used in
these assays.
4) A clear trend regarding the compounds’ differential activi-
ties against the three P. falciparum strains could not be
identified; for instance, for N-alkanoylated CQ analogues
9b–f, compounds 9b,d were increasingly active in the
order Dd2<3D7<W2, 9c in the order Dd2ꢁW2<3D7,
9e in the order Dd2ꢁ3D7<W2, and 9 f in the order
3D7<W2<Dd2.
1
confirmed by H and 13C NMR spectroscopy, as well as by mass
spectrometric analysis with electrospray ionization and ion-
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Table 1. In vitro cytotoxicity (HepG2 cells) and activity against blood-stage P. falciparum strains and liver-stage P. berghei displayed by compounds 9 and
10.[a]
R
Compd
Blood-stage IC50 [nm]
Pf Dd2
HepG2 CC50 [mm]
SI[b]
Liver-stage IC50 [mm]
Pf 3D7
Pf W2
9a
10a
9b
10b
9c
10c
9d
10d
9e
10e
9 f
10 f
9g
10g
134
202
23.6
145
6.60
87.7
30.4
48.2
86.1
48.5
5.30
107
16.5
82.8
254
369
51.0
143
53.4
150
56.9
93.8
87.7
104
27.0
82.9
57.1
126.3
172
493
4.60
213
52.7
185
10.9
70.8
56.0
88.3
12.3
122
13.7
0.76
12.5
1.39
22.3
2.90
13.2
3.65
22.8
3.29
125
53.9
–
–
–
–
7.7
–
2.7
–
4.1
–
5.6
–
Me
Et
1.50
245
6.50
418
15.7
232
38.9
261
31.5
4618
29.8
421
48.8
Pr
Bu
iBu
secBu
3.63
37.6
8.11
89.3
166.1
2.5
0.52
9h
10h
94.7
38.5
202
11.0
125
32.4
9.20
2.07
45.5
53.8
1.2
–
9i
10i
97.3
61.0
114
60.3
153
61.0
4.35
2.86
28.4
46.9
–
–
9j
48.8
34.0
21.0
100[15]
–
170
72.8
108
200[15]
–
64.0
77.9
226
159[15]
–
16.9
3.34
99.4
42.9
22.9
37.0
–
2.9
–
10j
CQ
QC
PQ
–
–
–
97.2[16]
7.40
>15
13
180[18]
7.5[7]
[a] Chloroquine (CQ), quinacrine (QC), and primaquine (PQ) were used as reference classical antimalarial drugs. [b] Selectivity index: ratio of (CC50 HepG2)/
(highest IC50 against Pf strains).
5) N-Acetylated analogues 9a and 10a were substantially
less active than all other compounds tested, with activity
against the 3D7 strain being enhanced by increasing the
length of the R alkyl chain from one to three carbons (9a
to 9c, and 10a to 10c); this enhancement is also ob-
served in terms of the activity against both CQ-resistant
strains, when going from one to two carbons (9a versus
8) Interestingly, introducing a double bond has opposite ef-
fects in CQ and QC analogues: the activity decreases
when going from 9g to its vinylic counterpart 9h, where-
as activity is increased in 10h relative to 10g.
9) a,b-Vinylcarbonyl moieties introduced in 9i,j or 10i,j did
not contribute to any significant enhancement in activity,
as compared with the remaining test compounds.
9b, 10a versus 10b), but no other consistent correlations 10) Comparing blood-stage activities reported herein for com-
between activity and alkyl chain length could be identi-
fied.
pounds 9 and 10 with those formerly determined for their
N-cinnamoylated counterparts (compounds 3 and 4) re-
veals that despite the latter having an overall slightly
better performance than the former, differences are not
significant—IC50 values of compounds 3 against Dd2 and
W2 strains were in the ranges of 15.6–78.1 and 11.0–
58.8 nm, respectively,[8] whereas IC50 values for compounds
4 on 3D7, Dd2, and W2 strains were respectively 17.0–
39.0, 27.1–131, and 3.20–41.2 nm.[10]
6) Upon comparing equally sized alkyl groups R, as in 9d,e,f
or 10d,e,f, replacing their linear chain (9d, 10d) by their
b-branched (9e, 10e) or a-branched (9 f, 10 f) isomers
leads to a decrease or an increase, respectively, in activity
against all strains.
7) Replacement of a methyl with a trifluoromethyl group in
CQ analogues, that is, 9e versus 9g, enhances activity
against 3D7 and Dd2, but not against W2; in turn, the
same replacement in QC analogues, i.e., 10e versus 10g,
leads to a decrease in activity against the three strains.
In view of the above, although compounds 3 and 4 display
slightly better activities against intra-erythrocytic parasites[8,10]
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than their analogues 9 and 10 reported herein, overall anti-
plasmodial performance of the latter clearly shows that simple
N-acylation of classical antimalarials like CQ and QC leads to re-
markable nanomolar activities in vitro against CQ-sensitive and
CQ-resistant P. falciparum strains.
group (9d) by a linear alkyldiene (9j) has no apparent effect.
Introduction of a double bond, as in 9g versus 9h, only slight-
ly increases activity, whereas replacing the 4-aminoquinoline
moiety in 9g by an acridine, in 10g, clearly increases activity,
but at the expense of safety. Nevertheless, although 10g is
more cytotoxic, its IC50 against liver-stage parasites (0.52 mm) is
about 16-fold lower than its CC50 against liver HepG2 cells
(8.11 mm). Therefore, in vitro liver-stage activity of compounds
9 and 10 is not markedly lower than those previously reported
The toxicity of compounds 9 and 10 against HepG2 cells
was determined (Table 1) as previously described.[10] All com-
pounds except 9 f (CC50 =125 mm) were more cytotoxic than
CQ (CC50 =97.2 mm[16]), displaying CC50 values below 40 mm. CQ
analogues 9 (9.20 mmꢀCC50 ꢀ125 mm) were less cytotoxic than
QC analogues 10, the latter being more cytotoxic (0.76 mmꢀ
CC50 ꢀ3.65 mm) than the parent drug QC (CC50 =7.40 mm).
Hence, compounds 9 and especially 10 presented poor selec-
tivity indices (SI, taken as the ratio between CC50 and the high-
est IC50 observed amongst the P. falciparum strains used;
Table 1), except in the case of 9 f, the SI for which was 4618.
Comparison of these findings with previous data for N-cinna-
moylated analogues of QC, 4, against the same cell line
(3.4 mmꢀCC50 ꢀ165 mm) shows that replacement of the cinna-
moyl group by other acyl moieties as in 10 is somewhat disad-
vantageous regarding compound cytotoxicity.
for compounds
3
(1.06 mmꢀIC50 ꢀ1.86 mm, average IC50:
1.79 mm)[8] or some of compounds 4, like 4a (R=p-F, IC50 =
1.60 mm), or 4b (R=p-Cl, IC50 =1.70 mm),[10] although none of
them was able to display lower cytotoxicity toward HepG2
cells than the reference drug, PQ (CC50 =180 mm[17]). Altogether,
these results show that replacement of the N-cinnamoyl
moiety with other N-acyl groups is not deleterious concerning
in vitro liver-stage antimalarial activity.
Our recent discovery of the exceptional in vitro dual-stage
activity of compounds 3 and 4 unveiled a key role for N-cinna-
moylation of CQ and QC scaffolds as a means to both reverse
resistance of blood-stage parasites to classical antimalarial scaf-
folds and enhance their activity against liver-stage para-
sites.[8,10] Exactly how and why N-cinnamoylation leads to such
remarkable improvement of in vitro antimalarial properties has
yet to be determined. Thus far, it has been possible to rule out
inhibition of either falcipains or b-hematin formation (i.e.,
heme polymerization) as major MOA of those com-
pounds;[6,8,10] additionally, such MOA could only explain blood-
stage activity, not that observed against the parasite’s liver
stages. Preliminary data (not confirmed) have suggested that
inhibition of new permeability pathways (NPPs) formed in
P. falciparum-infected red blood cells might be one possible
MOA underlying blood-stage activity of compounds 3.[6] Al-
though these NPPs have been exclusively associated with
blood-stage infection, recent evidence that similar NPP activity
may be elicited upon invasion of hepatocytes by P. berghei par-
asites might provide a unifying explanation for the dual-stage
activity displayed.[18,19] This hypothesis agrees with earlier evi-
dence that cinnamic acid derivatives are NPP inhibitors.[20] One
other possible and perhaps more likely MOA underlying anti-
malarial performance of compounds 3 and 4 is associated with
the redox properties of cinnamic moieties[21] and other struc-
turally related compounds, e.g., curcumins;[22] hence, N-cinna-
moylated compounds might owe their dual-stage antimalarial
action to the generation of reactive oxygen species in
a manner similar to that described for ferroquine.[23] Still, we
now further demonstrate that using acyl groups other than
cinnamoyl, as in compounds 9 and 10, globally preserves in
vitro nanomolar activities against blood-stage CQ-sensitive and
-resistant P. falciparum strains as well as remarkable activity
against liver forms of P. berghei. Taken together, our findings
show that simple N-acylated surrogates of antimalarial classics
like chloroquine and quinacrine are relevant leads worthy of
further exploration toward the development of dual-stage anti-
malarial candidates.
The activity of compounds 9 and 10 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 (data not shown).[8] Cell con-
fluence measurements confirmed cytotoxicity of most of the
compounds to hepatic cells (data not shown), preventing us
from obtaining reliable quantitative data on their liver-stage
activity. For this reason, we determined IC50 values only for
compounds with SI values >250 (9c,e–g), with the following
exceptions:
*
9d (SI=232), given its interest as a structural isomer of
compounds 9e,f;
*
9h (SI=45.5), to assess the effect of introducing a double
bond, as compared with 9g;
*
9j (SI=99.4), to assess the effect of replacing a linear satu-
rated by a linear unsaturated R group;
*
10g (SI=48.8), to observe the impact on liver-stage activity
upon replacing the 4-aminoquinoline core in 9g with an ac-
ridine moiety.
The results obtained (Table 1) show that, like their N-cinna-
moylated counterparts, these compounds display dual-stage
activity. Hence, the compounds tested are remarkably active in
vitro against liver forms of P. berghei, with IC50 values ranging
from 0.52 to 7.7 mm, all of which are lower than those of their
parent drugs, and lower than or similar to that of the reference
drug for liver-stage malaria, primaquine (PQ): 7.5 mm.[7] The
range of IC50 values is too narrow to build any meaningful
structure–activity relationships, although it can be observed
that increasing the length of the alkyl chain R from three to
four carbons (9c versus 9d) improves activity by a factor of
~3, whereas going from linear (9d) to b-branched (9e) or a-
branched (9 f) isomers only slightly increases IC50 values. Re-
placing a methyl in 9e by a trifluoromethyl in 9g also slightly
improves liver-stage activity, whereas replacing a linear alkane
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dryness on a rotatory evaporator. The impure solid residue was pu-
rified by liquid chromatography on a silica gel column with CH2Cl2/
MeOH 4:1 (v/v) as mobile phase.
Experimental Section
Chemicals and instrumentation: All solvents and common re-
agents were obtained from Sigma–Aldrich (Spain), whereas the
coupling agent TBTU was from Bachem (Switzerland). NMR spectra
were acquired on a Bruker Avance III400 spectrometer from solu-
tions in either deuterated chloroform (CDCl3), deuterated methanol
(CD3OD) or deuterated dimethyl sulfoxide ([D6]DMSO), containing
tetramethylsilane as internal reference. Multiplicity of proton NMR
signals is given as: s, singlet; d, doublet; t, triplet; q, quartet; qi,
quintuplet; sx, sextet; bs, broad singlet; bt, broad triplet; dd,
double doublet; m, unresolved multiplet.
Complete spectroscopic and analytical data for compounds 9a–j
and 10a–j are provided in the Supporting Information.
Inhibition of b-hematin: The b-hematin inhibition assay was per-
formed as previously described.[24] Various concentrations (0.1–
1 mm) of test compounds dissolved in DMSO were added in tripli-
cate to 50 mL hemin chloride dissolved in DMSO (5.2 mgmLÀ1).
Negative controls were water and DMSO. b-Hematin formation
was initiated by the addition of acetate buffer 0.2m (100 mL,
pH 4.4), plates were incubated at 378C for 48 h, and were then
centrifuged at 3000 rpm for 15 min (SIGMA 3-30K). After discarding
the supernatant, the pellet was washed with DMSO (4”200 mL),
and finally dissolved in 0.2m aq NaOH (200 mL). The solubilized ag-
gregates were further diluted 1:6 with 0.1m aq NaOH, and absorb-
ance values were recorded at l 405 nm (Biotek Powerwave XS with
software Gen5 1.07).
Mass spectrometry (MS) analyses were run on a Thermo Finnigan
LCQ Deca XP Max LC/MSn instrument operating with electrospray
ionization and ion-trap (ESI-IT) quadrupole detection. The target
compounds were confirmed to have at least 95% purity, based on
peak areas obtained through HPLC analyses that were run using
the following gradient elution: 10!70% B in A (A=H2O with
0.05% trifluoroacetic acid; B=acetonitrile) over 25 min, at a flow
rate of 1 mLminÀ1, on a Merck–Hitachi Lachrom Elite instrument
equipped with a diode-array detector (DAD) and thermostated
(Peltier effect) autosampler, using a Purospher STAR RP-18e column
(150”4.0 mm; particle size: 5 mm).
Blood-stage activity assays: Laboratory-adapted P. falciparum Dd2,
W2 (CQ-resistant), and 3D7 (CQ-susceptible) strains were continu-
ously cultured under standard conditions as previously described
by Trager and Jensen.[25] The antimalarial activity was determined
with the SYBR Green I assay as previously described,[26] with modifi-
cations. Briefly, early ring-stage parasites (1% parasitemia and 3%
hematocrit) were tested in triplicate in a 96-well plate and incubat-
ed with 12 concentrations of test compounds serially diluted (1:3),
ranging from 0–10 mm, for 48 h (378C, 5% CO2). After incubation,
a solution of SG (0.001% v/v in PBS) was added to each well, incu-
bated for 60 min, supernatant discarded, and cells re-suspended in
PBS. Fluorescence was detected in a multi-mode microplate reader
(TRIAD Multimode Detector, DYNEX Technologies), with excitation
and emission wavelengths of 485 and 535 nm, respectively, and
analyzed by nonlinear regression using GraphPad Prism 5 demo
version to determine the IC50 values.
Synthesis of 4-(N-aminobutyl)amino-7-chloroquinoline (7): Com-
pound 7 was prepared as previously described, and its structural
analyses were in agreement with formerly reported data.[6]
Synthesis of 9-(N-aminobutyl)amino-6-chloro-2-methoxyacridine
(8): Compound 8 was prepared as previously described, and used
without further purification.[10]
Synthesis of compounds 9a and 10a: Synthesis of N-acetylated
surrogates of CQ (9a) and QC (10a) was carried out by addition of
acetic anhydride (20 equiv) to 7 and 8, respectively (1 equiv), allow-
ing the reaction to run for 1 h at room temperature (RT). The reac-
tion mixture was then taken to dryness on a rotatory evaporator,
and the solid residue was re-dissolved in CH2Cl2 (20 mL). The result-
ing organic layer was washed three times with 5% aqueous
Na2CO3 (25 mL), dried with anhydrous Na2SO4, filtered, and evapo-
rated to dryness on a rotatory evaporator. The impure solids ob-
tained were purified by liquid chromatography on a silica gel
column using CH2Cl2/MeOH 4:1 (v/v) as mobile phase to obtain the
final products in high purity.
In vitro liver-stage infection assays: Inhibition of liver-stage infection
by test compounds was determined as previously described,[8,10] by
measuring the luminescence intensity in Huh-7 cells infected with
a firefly luciferase-expressing P. berghei line, PbGFP-Luccon. Huh-7
cells, a human hepatoma cell line, were cultured in 1640 RPMI
medium supplemented with 10% v/v fetal calf serum, 1% v/v non-
essential amino acids, 1% v/v penicillin/streptomycin, 1% v/v glu-
tamine, and 10 mm 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic
acid (HEPES), pH 7, and maintained at 378C with 5% CO2. For infec-
tion assays (performed in triplicate for each compound), Huh-7
cells (1.2”104 per well) were seeded in 96-well plates the day
before drug treatment and infection. Medium in the cells was re-
placed by medium containing the appropriate concentration of
each compound approximately 1 h prior to infection with sporo-
zoites freshly obtained through disruption of salivary glands of in-
fected female Anopheles stephensi mosquitoes. Sporozoite addition
was followed by centrifugation at 1700 g for 5 min. At 24 h post-in-
fection, the medium was replaced by fresh medium containing the
appropriate concentration of each compound. Inhibition of para-
site development was measured 48 h after infection. The effect of
the compounds on the viability of Huh-7 cells was assessed by the
AlamarBlue assay (Invitrogen, UK), using the manufacturer’s proto-
col.
Synthesis of compounds 9b–i and 10b–i: The relevant carboxylic
acids (1.1 equiv) were activated with O-(benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium tetrafluoroborate (TBTU; 1.1 equiv) in the pres-
ence of N-ethyl-N,N-diisopropylamine (DIEA; 2.0 equiv) in N,N-di-
methylformamide (DMF; 2 mL) for 20 min at 08C. Then, a solution
of 7 (for compounds 9b–i) or 8 (for compounds 10b–i) (1 equiv) in
DMF (2 mL) was added, and the reaction was allowed to proceed
at RT for 24 h. CH2Cl2 (25 mL) was added to the reaction mixture,
and the resulting solution was washed three times with 5% aque-
ous Na2CO3 (30 mL), dried with anhydrous Na2SO4 and filtered. The
solvent was evaporated under reduced pressure (rotatory evapora-
tor), and the crude residue was purified by liquid chromatography
on a silica gel column with CH2Cl2/MeOH 4:1 (v/v) as eluent.
Synthesis of compounds 9j and 10j: Either 7 (for the synthesis of
9j) or 8 (for the synthesis of 10j) (1.0 equiv) was added to a solu-
tion of sorbic chloride (1.1 equiv) and Cs2CO3 (2.5 equiv) in DMF
(2 mL), and the reaction was allowed to proceed for 3.5 h at RT.
CH2Cl2 (10 mL) was then added to the reaction mixture, and the re-
sulting solution was washed three times with 5% aqueous Na2CO3
(15 mL), dried with anhydrous Na2SO4, filtered and evaporated to
ChemMedChem 2015, 10, 1344 – 1349
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Received: April 13, 2015
Published online on June 2, 2015
ChemMedChem 2015, 10, 1344 – 1349
www.chemmedchem.org
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim