Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial agents with enhanced metabolic stability

Article abstract of DOI:10.1016/j.ejmech.2018.02.048

Hybrid compounds may play a critical role in the context of the malaria eradication agenda, which will benefit from therapeutic tools active against the symptomatic erythrocytic stage of Plasmodium infection, and also capable of eliminating liver stage parasites. To address the need for efficient multistage antiplasmodial compounds, a small library of 1,2,4,5-tetraoxane-8- aminoquinoline hybrids, with the metabolically labile C-5 position of the 8-aminoquinoline moiety blocked with aryl groups, was synthesized and screened for antiplasmodial activity and metabolic stability. The hybrid compounds inhibited development of intra-erythrocytic forms of the multidrug-resistant Plasmodium falciparum W2 strain, with EC₅₀ values in the nM range, and with low cytotoxicity against mammalian cells. The compounds also inhibited the development of P. berghei liver stage parasites, with the most potent compounds displaying EC₅₀ values in the low μM range. SAR analysis revealed that unbranched linkers between the endoperoxide and 8-aminoquinoline pharmacophores are most beneficial for dual antiplasmodial activity. Importantly, hybrids were significantly more potent than a 1:1 mixture of 8-aminoquinoline-tetraoxane, highlighting the superiority of the hybrid approach over the combination therapy. Furthermore, aryl substituents at C-5 of the 8-aminoquinoline moiety improve the compounds' metabolic stability when compared with their primaquine (i.e. C-5 unsubstituted) counterparts. Overall, this study reveals that blocking the quinoline C-5 position does not result in loss of dual-stage antimalarial activity, and that tetraoxane-8- aminoquinoline hybrids are an attractive approach to achieve elimination of exo- and intraerythrocytic parasites, thus with the potential to be used in malaria eradication campaigns.

Full text of DOI:10.1016/j.ejmech.2018.02.048

Accepted Manuscript
Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial agents with
enhanced metabolic stability
Rita Capela, Joana Magalhães, Daniela Miranda, Marta Machado, Margarida
Sanches-Vaz, Inês S. Albuquerque, Moni Sharma, Jiri Gut, Philip J. Rosenthal,
Raquel Frade, Maria J. Perry, Rui Moreira, Miguel Prudêncio, Francisca Lopes
PII:
S0223-5234(18)30184-3
10.1016/j.ejmech.2018.02.048
EJMECH 10225
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 November 2017
Revised Date: 28 January 2018
Accepted Date: 14 February 2018
Please cite this article as: R. Capela, J. Magalhães, D. Miranda, M. Machado, M. Sanches-Vaz, Inê.S.
Albuquerque, M. Sharma, J. Gut, P.J. Rosenthal, R. Frade, M.J. Perry, R. Moreira, M. Prudêncio,
F. Lopes, Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial agents with enhanced
metabolic stability, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.048.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Endoperoxide-8-aminoquinoline hybrids as dual-stage antimalarial
agents with enhanced metabolic stability
Rita Capela,a# Joana Magalhães,a# Daniela Miranda,a# Marta Machado,b Margarida
Sanches-Vaz, b Inês S. Albuquerque,b Moni Sharma,a Jiri Gut,c Philip J. Rosenthal,c
Raquel Frade,a Maria J. Perry,a Rui Moreira,a Miguel Prudênciob* and Francisca Lopesa*
a Instituto de Investigação do Medicamento (iMed.ULisboa), Faculdade de Farmácia,
Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
b Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,
Av. Professor Egas Moniz, 1649-028, Lisboa, Portugal
c Department of Medicine, San Francisco General Hospital, University of California,
San Francisco, Box 0811, CA 94143, USA
# These authors contributed equally to this work
* Corresponding authors: Francisca Lopes, fclopes@ff.ulisboa.pt; Miguel Prudêncio,
mprudencio@medicina.ulisboa.pt
Abstract
Hybrid compounds may play a critical role in the context of the malaria eradication
agenda, which will benefit from therapeutic tools active against the symptomatic
erythrocytic stage of Plasmodium infection, and also capable of eliminating liver stage
parasites. To address the need for efficient multistage antiplasmodial compounds, a
small library of 1,2,4,5-tetraoxane-8- aminoquinoline hybrids, with the metabolically
labile C-5 position of the 8-aminoquinoline moiety blocked with aryl groups, was
synthesized and screened for antiplasmodial activity and metabolic stability. The hybrid
compounds inhibited development of intra-erythrocytic forms of the multidrug-resistant
Plasmodium falciparum W2 strain, with EC50 values in the nM range, and with low
cytotoxicity against mammalian cells. The compounds also inhibited the development
of P. berghei liver stage parasites, with the most potent compounds displaying EC50
values in the low µM range. SAR analysis revealed that unbranched linkers between the
endoperoxide and 8-aminoquinoline pharmacophores are most beneficial for dual

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antiplasmodial activity. Importantly, hybrids were significantly more potent than a 1:1
mixture of 8-aminoquinoline-tetraoxane, highlighting the superiority of the hybrid
approach over the combination therapy. Furthermore, aryl substituents at C-5 of the 8-
aminoquinoline moiety improve the compounds’ metabolic stability when compared
with their primaquine (i.e. C-5 unsubstituted) counterparts. Overall, this study reveals
that blocking the quinoline C-5 position does not result in loss of dual-stage antimalarial
activity, and that tetraoxane-8- aminoquinoline hybrids are an attractive approach to
achieve elimination of exo- and intraerythrocytic parasites, thus with the potential to be
used in malaria eradication campaigns.
Keywords
malaria, hybrid drugs, liver stage, dual-stage antimalarials, metabolism
1. Introduction
Malaria is a major global public health problem, with 214 million new clinical cases and
440,000 deaths estimated in 2015.[1] The disease is caused by Plasmodium parasites,
with P. falciparum and P. vivax causing the heaviest burden. P. falciparum is
responsible for most malaria-associated mortality, while P. vivax can generate cryptic
parasite forms called hypnozoites that persist in the liver for long periods of time,
causing relapsing malaria.[2-4] The life cycles of all Plasmodium spp. also include a
liver stage that precedes erythrocytic infection and a sexual blood form, the gametocyte,
that is responsible for transmission from the mammalian host to the mosquito vector.[5]
There is an urgent need for efficacious and safe tools for malaria prophylaxis and to
effectively prevent the relapse of P. vivax malaria, as well as to treat and block the
transmission of the disease.[6-8] Despite increasing efforts worldwide, available
compounds active against non-erythrocytic stages of malaria parasites remain very
limited.[3]
Primaquine, 1 (Fig. 1), is the only licensed drug active against P. vivax hypnozoites, and
is thus used for radical cure of infections by this parasite.[3, 9] In addition, primaquine
is the only available antimalarial agent that displays marked activity against
gametocytes from all species of parasites causing human malaria, and is therefore
capable of interrupting disease transmission from the host to the mosquito vector.[9]

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However, primaquine’s clinical value is compromised by its toxic side effects, namely
methaemoglobinaemia and hemolytic anemia in patients with deficiency in glucose-6-
phosphate dehydrogenase (G6PD) activity.[9] The methaemoglobinaemia associated
with primaquine is thought to be a consequence of cytochrome P450-mediated
oxidation of the corresponding 5-hydroxylated metabolites to quinoneimine.[9, 10]
Currently, WHO recommends the use of artemisinin-based combination therapies
(ACTs) as first line treatment for uncomplicated malaria.[11] However, increasing
resistance to ACTs, due to decreased activity of both artemisinins and partner drugs, has
been reported in Southeast Asia, and spread of resistance to other regions may have
devastating consequences.[12-16]
Hybrid compounds capable of hitting more than one molecular target are an attractive
alternative to ACTs.[17-23] Such compounds may be particularly relevant in the
context of the malaria eradication agenda, which benefits from therapeutic tools not
only active against the symptomatic erythrocytic stage of infection, but also capable of
eliminating liver-stages and/or gametocytes.[8, 24] In this context, we recently reported
new peroxide-based hybrid compounds comprising a primaquine moiety combined with
an endoperoxide pharmacophore, e.g. 2 (artemisin-based) and 3 (1,2,4,5-tetraoxane-
based), that showed high potency against both liver and blood stages of malaria
parasites.[25, 26] In addition, hybrid 3 also blocked transmission to the mosquito in an
animal model. However, the most potent compounds displayed high metabolic
susceptibility in rat liver microsomes, dependent on the nature of the linker between the
tetraoxane and primaquine moieties.[26]
Here, we report the design, synthesis and antiplasmodial activity of tetraoxane-8-
aminoquinoline hybrids, 4 (Fig. 1), with an aryl or heteroaryl group installed at the
metabolically labile C-5 position of the 8-aminoquinoline moiety. 5-Hydroxy-8-
aminoquinolines are thought to be the metabolites responsible for the hemolytic toxicity
associated with primaquine and other 8-aminoquinolines, and thus blocking C-5 offers
the potential to resolve the toxicity liability.[9, 27] 5-Aryl-8-aminoquinolines, designed
to probe the possibility of separating antimalarial activity from hemolytic toxicity in 8-
aminoquinolines, have been reported to be metabolically stable, but inactive against the
exoerythrocytic mouse model, with moderate activity against intraerythrocytic
parasites.[28] We now report that tetraoxane-8-aminoquinoline hybrids, 4, are

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metabolically more stable than their primaquine counterparts, 3, while retaining activity
against liver stage parasites.
Figure 1. Chemical structures of primaquine 1, artemisinin-based hybrids 2, 1,2,4,5-
tetraoxane-primaquine hybrids 3, and 1,2,4,5-tetraoxane-8-aminoquinoline hybrids
reported in this study, 4.
2. Results and Discussion
2.1. Chemistry
The library of tetraoxane-8-aminoquinoline hybrids was prepared based on a synthetic
procedure involving the following steps: (i) synthesis of a C-5 substituted 8-
aminoquinoline key intermediate, (ii) incorporation of a diamine spacer, and (iii)
coupling to the appropriately functionalized tetraoxane (4, Fig. 1).
The preparative route for the C-5 substituted 8-aminoquinoline intermediate, 5 (Scheme
1), was an adaptation from that described by Chen[29] and Shiraki et al.[28] First,
bromination of the commercially available 6-methoxy-8-nitroquinoline, 6, was achieved
using bromine and Fe (Scheme 1). The resulting 5-bromo-6-methoxy-8-nitroquinoline 7
was then converted to the appropriate 5-aryl-6-methoxy-8-nitroquinolines, 8, in
satisfactory yields by Suzuki coupling reactions with a series of arylboronic acids using
Pd(OAc)2, PPh3 and Na2CO3 in dimethoxyethane. Finally, reduction of 8 using Sn/SnCl2
[30] gave the intermediates 5 in 76% overall yield.

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Scheme 2 outlines the general route to incorporate the diamine spacer into intermediate
5, which was adapted from Elderfield et al.[31] First, reaction of phthalimide potassium
salt, 10, with the appropriate dibromoalkane afforded the corresponding
bromoalkylphthalimide intermediates 11a-d. Reaction of intermediates 5a-h with 11a-d
gave moderate to excellent yields of the desired 12a-l, which were then converted to the
corresponding amines 13a-l by hydrazinolysis.
In the development of hybrids 4, we explored the nature of the chemical group between
the tetraoxane moiety and the linker. Amide derivatives, 4a-l, were prepared by reacting
tetraoxane 14 [22] with intermediates 13a-l, using TBTU as coupling agent, while their
amine counterparts, 4m-s, were synthesized by reductive amination of tetraoxane 15
[26] with 13a-h and NaBH(AcO)3 (Scheme 3). Finally, hybrid compounds 16a-d,
which do not contain any linker between the tetraoxane and 8-aminoquinoline moieties,
were prepared by reacting the appropriate 8-aminoquinoline 16 with intermediate 14
previously activated with ethyl chloroformate.
Scheme 1. Synthesis of compounds 5 and 7-9a.
a Reagents and conditions: (a) Br2, Fe, CaCO3, DCM, H2O, reflux 15h, then rt 6h; (b)
Pd(OAc)2, PPh3, Na2CO3, TBAB, ArB(OH)2, dimethoxyethane, reflux; (c) Sn, SnCl2,
HCl, 0ºC.

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Scheme 2. Synthesis of intermediates 11-13a.
a Reagents and conditions: (a) 1) KOH, EtOH, reflux, 2) dibromoalkane, acetone, reflux
24h; (b) 5a-h or 9, TEA, reflux, 19h; (c) NH2NH2, EtOH, reflux 7h.

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Scheme 3. Synthesis of hybrids 4 and 16a.
a Reagents and conditions: (a) 1) TBTU, DCM, TEA, 0ºC, 1h, 2) 13a-l for 4a-l or 5a-d
for 16a-d, DCM, TEA, rt.; (b) DCM, 13a-c and 13e-h for 4m-s, AcOH, STAB, rt.

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Table 1. In vitro antimalarial activities (EC50) against intraerythrocytic P. falciparum W2 strain and liver-stage P. berghei, and toxicity (CC50) of hybrid
compounds 4 and 16 against CaCo-2 cells.
Compd
Y
X
R
EC50 / µM
CC50
µM
/
Compd
Y
X
R
EC50 / µM
CC50
µM
/
P.
P.
P.
P.
falciparum
W2 (blood
stage)
berghei
(liver
falciparum
W2 (blood
stage)
berghei
(liver
stage)
stage)
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CO
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
--
0.204+0.013 1.69+0.33
0.057+0.012 2.38+0.56
0.075+0.002 2.81+0.56
0.061+0.020 4.44+0.66
0.060+0.009 4.12+0.49
0.141+0.021 1.65+0.53
0.076+0.010 2.35+0.19
0.045+0.002 2.11+0.13
>50
>50
>50
>50
>50
>50
>50
>50
ND
ND
10.2
ND
ND
ND
ND
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CO
CO
CO
CO
--
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
CH(Me)
--
0.092+0.007
0.099+0.004
ND
Tox.
ND
ND
ND
13.2
10.3
11.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4a
4b
4c
4m
4n
Tox.
62%*
4o
0.019+0.003
0.176+0.009
0.120+0.005
0.081+0.003
0.287+0.015
0.916+0.047
4.19+0.20
5.67+0.43
2.35+0.07
0.021[26]
ND
4d
4e
4p
inhib
Tox.
4q
ND
Tox.
52%*
4f
4r
4g
4h
4i
4s
16a
16b
16c
16d
13f
3a
inhib
ND
Br
0.016+0.004
Tox.
--
37%*
0.015+0.003 2.61+0.08
0.015+0.005 1.11+0.40
0.017+0.003 1.64+0.15
--
4j
inhib
35%*
CH2
--
4k
4l
inhib
CH2CH2
CH(Me)
CH(Me)
5.43+1.17
ART
PQ
0.010+0.001
ND
ND
CO
H
0.538[26]
9.71[26]
7.50 [26]
17.3+3.9
PQ+ART
13f+14
ester
14 ethyl ester
128.2 [26]
ND
3.71+0.32
* Percentage of inhibition at 10 µM; ND: Not determined; Tox: toxic to the host cells;

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2.2. Blood schizontocidal activity and cytotoxicity
Compounds 4 and 16 were first screened for activity against intraerythrocytic parasites,
using cultured chloroquine-resistant W2 strain P. falciparum; the corresponding EC50
values are presented in Table 1. EC50 values for 4a-s ranged from 15 to 200 nM, in line
with the values previously reported for their primaquine counterparts (e.g. 3a, EC50 21
nM, Table 1).[26] This result contrasted sharply with that for derivatives 16a-d, which
lack a linker between the endoperoxide and 8-aminoquinoline moieties. These
compounds were ca. 10 to 50 times less potent than their counterparts 4 (e.g. 4b or 4j
versus 16b or 4d versus 16d), suggesting that the presence of a linker is a major
requirement for activity against intraerythrocytic parasites.
Structure-activity relationship studies indicated that the blood-schizontocidal activity of
hybrids 4a-s was affected by the nature of the linker between the endoperoxide and 8-
aminoquinoline moieties as well as by the substituent on the C-5 position of the
quinoline moiety. For example, compounds containing linear linkers (4j-l) generally
presented better antiplasmodial activity than those with branched linkers (e.g. 4b). In
contrast, the basicity of the linker nitrogen atom close to the endoperoxide moiety had
limited impact on blood-stage activity, as shown by the EC50 values for the amide
series, 4a-h, when compared to those of their amine counterparts, 4m-s. The electronic
effects of the substituent at the C-5 position of the quinoline moiety also influenced
inhibitory activity against erythrocytic parasites. For the amide series, 4a-i, compounds
containing electron withdrawing groups (e.g. 4-Br, 4i, σp = 0.22,[32] or 4-fluorophenyl,
4b, σp = 0.06[32]) showed better activity than those with electron donating substituents
(e.g. 4-methylphenyl, 4f, σp = -0.05[32]). The exception was compound 4h, containing
the electron-rich 3-thienyl substituent at C-5 (σp = -0.02[32]), which also displayed
excellent blood-stage activity.
The in vitro cytotoxicity of compounds 4 was evaluated using mammalian CaCo-2 cells
(Table 1). The selectivity index (SI) as expressed by the ratio CC50CaCo-2/IC50W2, ranged
from ca. 60 to >1000, with compounds 4b-e and 4g,h displaying the highest CC50 and
SI values (>50 and >600, respectively).

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2.3. Liver schizontocidal activity
Hybrid derivatives 4 and 16 were further evaluated for their ability to inhibit the
development of liver-stage malaria parasites. Compounds were initially assayed at two
different concentrations (2 and 10 µM), using an in vitro infection model that employs a
human hepatoma cell line (Huh7) infected with firefly luciferase-expressing P. berghei.
Results were compared to those obtained for primaquine (Fig. 2). Strikingly, hybrids
with an amide linker between the two pharmacophores, 4a-l, were highly active, with
>50% inhibition of infection at 2 µM, and almost complete suppression at 10 µM,
without significantly affecting Huh7 cell proliferation. The exception was the 5-bromo
derivative 4i, which was toxic to Huh7 cells at 10 µM. In contrast, derivatives with an
amine linker, i.e. 4m-s, inhibited infection at both concentrations, but showed toxicity
towards host Huh7 cells at 10 µM. Finally, hybrids lacking a linker between the two
pharmacophores, 16a-d, did not demonstrate significant liver stage activity, indicating
that the presence of a linker is required to convey potency against liver stage parasites.
Based on these observations, we then determined EC50 values for the amide derivatives,
4a-h and 4j-l, against P. berghei infection of Huh7 cells. These compounds presented
EC50 values ranging from 1.1 to 4.3 µM for inhibition of hepatic infection, compared to
0.6 µM for their primaquine hybrid counterpart, 3a (Table 1). In contrast, the parent
tetraoxane (ethyl ester of 14) revealed poor activity (EC50 17 µM), suggesting that this
scaffold is not very effective against the liver stages of the parasite. Furthermore,
compounds 4a-h and 4j-l were significantly more potent than primaquine and the 1:1
combinations of 13f-14 (ethyl ester) and primaquine-artemisinin. This is consistent with
what has been previously reported for hybrids 3.[26]
Results presented in Table 1 indicate that blocking the C-5 position of the quinoline
moiety with an aryl substituent did not result in any significant loss of in vitro
antimalarial potency of the hybrid compound. Furthermore, activity against liver stage
parasites is not significantly affected by the electronic or lipophilicity properties of the
substituent at C-5. Overall, the results contrast sharply with what was observed for 5-
substituted 8-aminoquinolines, which were poorly active even at the highest
concentration tested. For example, the 5-methylphenyl derivative, 4f, is ca. three times
more potent than its 5-methylphenyl-8-aminoquinoline precursor, 13f (Table 1).
Interestingly, these results are in line with those reported for 5-aryl-8-aminoquinolines,

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which have been reported to be inactive in the exoerythrocytic mouse model.[28] Of
note, primaquine is only moderately active in this assay, as the drug requires metabolic
activation to display potent activity.[33] Also, hepatoma cells are metabolically less
competent, compared to hepatocytes, in activating primaquine.[34]

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Figure 2: Hybrid compounds 4 and 16 inhibit the development of Plasmodium hepatic stages in vitro to different extents. Huh7 cells were
infected with luciferase-expressing P. berghei sporozoites and treated with 1 and 10 µM of each hybrid compound or with equivalent amounts of
dimethyl sulfoxide (DMSO; control). Primaquine (PQ) was used as a positive control. Total parasite loads (bioluminescence) and cell viability
(AlamarBlue fluorescence) were assessed at 48 h post infection. Bars represent infection and dots represent Huh7 cell confluency. Error bars
represent standard deviation.

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2.4. Metabolic Stability
Hybrid compounds 4 and 16 underwent NADPH-dependent degradation upon
incubation with rat liver microsomes, with half-lives ranging from 14 min to 2.2 h
(Table 2). Most compounds displayed intermediate (0.3-0.7) or low (<0.3) predicted in
vivo hepatic extraction ratios, EH, representing a significant improvement in metabolic
stability when compared to the primaquine counterpart 3a (EH = 0.81).[26] No obvious
metabolites were detected using the analytical conditions employed for the parent
compounds.
The metabolic susceptibility of compounds 4 and 16 towards microsomes was
significantly affected by the lipophilicity of substituents at C-5, as indicated by the
excellent correlations between the rate of metabolism, as expressed by log t1/2, and the
corresponding Hansch Σπ values for the C-5 substituent (Fig. 3). However, no clear
correlation emerged between liver schizontocidal activities and rates of metabolism. For
example, hybrid compounds with low (e.g. 4c), intermediate (e.g. 4f), and high (e.g. 4i)
susceptibility towards rat liver microsomes were equipotent in inhibiting the growth of
liver stage parasites. These results strongly suggest that liver schizontocidal activity is
not dependent on metabolic activation.
Table 2. In vitro metabolism of selected hybrid compounds 4 and 16 in rat liver
microsomes, and the corresponding predicted in vivo metabolism data.
CLint,invitro
Compd.
t (min)
(µL/min/mg
protein)
33.0
Predicted EH
½
4a
4b
4c
4e
4f
42
40
0.52
0.53
0.26
0.31
0.34
0.55
0.76
0.56
0.75
34.6
129
100
87
10.7
13.9
15.9
4h
4i
37
37.4
14
99.0
4j
35
39.6
15
92.4
4k

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25
42
55.4
33.0
23.1
11.1
11.2
132.8
0.64
0.52
0.43
0.26
0.27
0.81
4l
16a
60
16b
16c
125
124
10
16d
3a[26]
The 5-bromo derivative, 4i, presented the highest susceptibility towards the microsomes
(EH = 0.76). This result is not entirely surprising, as 5-fluoroprimaquine was also shown
to undergo bioactivation with hepatic microsomes from several species, including rat
liver microsomes. According to O’Neill et al., metabolic activation of 5-
fluoroprimaquine may involve epoxidation and dehalogenation,[10] a pathway also
most likely available to the 5-bromo derivative 4i. Further studies are required to
identify the metabolites and to confirm their mechanisms of action.
2.5
y = 0.5498x + 1.6629
R² = 0.9598
2.0
1.5
1.0
y = 0.376x + 0.9549
R² = 0.9367
0.5
0.0
-1.0
0.0
1.0
2.0
3.0
4.0
Σπ
Fig 3. Correlation between log t1/2 values for metabolic activation of compounds 4 (¢)
and 16 (ò), and the corresponding Hansch Σπ values for the C-5 substituents.

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3. Conclusions
We report that tetraoxane-8- aminoquinoline hybrids with an aryl or heteroaryl group
installed at the metabolically labile C-5 position of the 8-aminoquinoline moiety are
efficient dual-stage antiplasmodial agents endowed with good metabolic stability. These
hybrid compounds inhibited the development of intra-erythrocytic forms of P.
falciparum, with EC50 values ranging from the low nanomolar to low micromolar range,
while displaying low cytotoxicity against mammalian cells. Compounds were also
screened for activity against P. berghei liver stages, with the most potent compounds
displaying EC50 values ranging from 1.1 to 4.4 µM. Analysis of SAR revealed that a
linker between the endoperoxide and 8-aminoquinoline pharmacophores is crucial for
dual antiplasmodial activity. In contrast to 5-aryl-8-aminoquinolines, their
endoperoxide-based hybrid derivatives retain liver schizontocidal activity. In general,
hybrids with aryl substituents at C-5 of the 8-aminoquinoline moiety had increased
metabolic stability in microsomes when compared to their primaquine (i.e. C-5
unsubstituted) counterpart, without loss of dual stage antimalarial activity. These results
also suggest that liver schizontocidal activity is not dependent on metabolic activation.
Overall, this study reveals that tetraoxane-8- aminoquinoline hybrids are an attractive
approach to achieve elimination of exo- and intraerythrocytic parasites, thus providing a
new entry in the toolkit of multistage agents with potential utility in malaria eradication
campaigns.
4. Experimental Section
4.1. Chemistry
All chemicals and solvents were of analytical reagent grade and were purchased from
Alfa Aesar or Sigma–Aldrich. Tetrahydrofuran was dried before use. Thin layer
chromatography was performed using Merck silica gel 60F254 aluminum plates with
visualization by UV light, iodine, potassium permanganate dip, and/or p-anisaldehyde
dip. Flash column chromatography was performed using Merck silica gel 60 (230–400
mesh ASTM), eluting with various solvent mixtures and using an air aquarium pump to
apply pressure. NMR spectra were collected using a Bruker 400 Ultra-Shield (400
MHz) or a Bruker 300 Avance (300 MHz) in CDCl3 or MeOD; chemical shifts, δ, are
expressed in ppm, and coupling constants, J, are expressed in Hz. Mass spectra were

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determined using a Micromass Quattro Micro API spectrometer equipped with a Waters
2695 HPLC module and a Waters 2996 photodiode array detector. All target
compounds were determined to be >95% pure by elemental analysis (for C, H, and N),
determined using a FLASH 2000 analyzer. High resolution mass spectrometry (HRMS)
was performed on a Bruker MicrOTOF equipped with ESI ion source from the Mass
Spectrometry and Proteomics Unity of the University of Santiago de Compostela, Spain
and a Bruker Daltonics QqTOF Impact II equipped with ESI ion source from the
Laboratório de Espectrometria de Massa, Instituto Superior Técnico.
Melting points were determined using a Kofler Bock Monoscop M and are uncorrected.
The HPLC system consisted of a LichroCART 125-4 RP-18 (5 mm) analytical column
on a LabChrom L7400 Merck Hitachi instrument. See Supporting Information for
experimental information and data on all intermediates.
4.1.1. General procedure for the synthesis of hybrids 4a–i
To a solution of compound 14 (0.16 mmol) in dry dichloromethane (3 mL) was added
triethylamine (0.24 mmol) and TBTU (0.16 mmol). The reaction mixture was stirred for
60 minutes at 0 °C, under N2 atmosphere. Then a solution of compound 13a-i (0.16
mmol) in dry dichloromethane (2mL) was added and, after 15 minutes, the mixture was
allowed to warm to room temperature and react overnight. After completion of the
reaction, the mixture was diluted with water and extracted with ethyl acetate. The
organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and,
concentrated. Purification by flash column chromatography using ethyl acetate-hexane
(4:6) as eluent gave the pure compound.
4.1.1.1. Compound 4a. Yellow solid (66% yield): mp: 75-76°C; 1H NMR (400 MHz,
CDCl3), δ= 8.53-8.52 (m, 1H), 7.84-7.83 (m, 1H), 7.48-7.44 (m, 2H), 7.38-7.32 (m,
3H), 7.23-7.22 (m, 1H), 6.51 (s, 1H), 6.22 (brs, 1H), 5.65 (brs, 1H), 3.83 (s, 3H), 3.76-
3.74 (m, 1H), 3.39-3.22 (m, 2H), 3.20-3.01 (m, 2H), 2.20-2.09 (m, 1H), 2.03-1.53 (m,
24H), 1.36 (d, J=6.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 176.4, 155.4, 144.8,
143.9, 136.0, 133.0, 131.5, 128.9, 127.7, 126.2, 121.4, 109.9, 106.8, 93.1, 67.7, 55.6,
43.5, 38.7, 36.6, 33.4, 32.7, 27.1, 25.6, 19.5. HRMS [ESI]: m/z [M+H]+ calcd for
C38H48N3O6: 642.3543, found: 642.3523.

ACCEPTED MANUSCRIPT
4.1.1.2. Compound 4b. Yellow solid (79% yield): mp: 81-82°C; 1H NMR (400 MHz,
CDCl3), δ= 8.56 (dd, J=4.1, 1.6 Hz, 1H), 7.79 (dd, J=8.6, 1.5 Hz, 1H), 7.33-7.29 (m,
2H), 7.25 (dd, J=8.6, 4.1 Hz, 1H), 7.19-7.14 (m, 2H), 6.51 (s, 1H), 6.29-6.19 (m, 1H),
5.61-5.53 (m, 1H), 3.85 (s, 3H), 3.82-3.72 (m, 1H), 3.46-3.23 (m, 2H), 2.21-1.65 (m,
27H), 1.38 (d, J=6.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 175.2, 156.0, 145.5,
144.9, 134.4, 133.9, 133.8, 132.3, 129.7, 122.5, 115.9, 115.6, 111.1, 107.7, 93.2, 57.2,
48.3, 44.8, 39.9, 37.5, 34.6, 33.7, 27.6, 26.9, 21.4. MS [ESI]: m/z [M+H]+ calcd for
C38H47FN3O6: 660.34, found: 660.25. Anal. calcd for C38H46FN3O6: C 69.18, H 7.03, N
6.37, found: C 69.26, H 7.18, N 6.56.
4.1.1.3. Compound 4c. Yellow solid (54% yield): mp: 104-105°C; 1H NMR (400 MHz,
MeOD), δ= 8.56-8.46 (m, 1H), 7.80-7.71 (m, 1H), 7.43 (d, J=8.1 Hz, 2H), 7.31-7.20 (m,
3H), 6.62 (s, 1H), 4.66 (brs, 1H), 3.93-3.75 (m, 4H), 3.28-2.99 (m, 4H), 2.27-2.16 (m,
2H), 1.87-1.58 (m, 24H), 1.36 (d, J=6.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 176.4,
155.5, 145.2, 144.0, 134.8, 133.7, 133.1, 132.6, 132.0, 128.7, 127.8, 121.7, 109.9,
109.5, 106.8, 92.6, 60.2, 55.4, 43.9, 41.5, 38.5, 36.6, 33.4, 32.7, 27.1, 25.5, 19.5, 13.1.
Anal. calcd for C38H46ClN3O6: C 67.49, H 6.86, N 6.21, found: C 67.44, H 6.96, N 6.18.
4.1.1.4. Compound 4d. Yellow solid (68% yield): mp: 105-107°C; 1H NMR (400 MHz,
CDCl3), δ= 8.53-8.54 (m, 1H), 7.79 (brs, 1H), 7.58 (d, J= 7.8 Hz, 2H), 7.27-7.25 (m,
1H), 7.20 (d, J= 8.0 Hz, 2H), 6.49 (s, 1H), 6.24 (brs, 1H), 5.56 (brs, 1H), 3.84 (s, 3H),
3.78-3.70 (m, 1H), 3.40-3.22 (m, 2H), 3.20-2.98 (m, 2H), 2.22-2.09 (m, 1H), 2.00-1.52
(m, 24H), 1.35 (d, J=6.2 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 176.4, 155.4, 145.1,
144.0, 135.2, 133.4, 132.7, 130.9, 128.6, 121.7, 120.1, 110.0, 106.8, 92.6, 55.6, 43.6,
38.7, 36.6, 33.4, 32.8, 27.1, 25.6, 19.6. HRMS [ESI]: m/z [M+H]+ calcd for
C38H47BrN3O6: 720.2648, found: 720.2619.
4.1.1.5. Compound 4e. Yellow solid (84% yield): mp: 93-95°C; 1H NMR (400 MHz,
MeOD), δ= 8.69-8.68 (m, 1H), 7.95-7.93 (m, 1H), 7.86 (d, J= 7.9 Hz, 2H), 7.63 (d, J=
7.9 Hz, 2H), 7.44 (dd, J= 8.5, 4.0 Hz, 1H), 6.72 (s, 1H), 4.01 (s, 3H), 3.97-3.96 (m, 1H),
3.42-3.30 (m, 4H), 2.43-2.33 (m, 1H), 2.18-1.69 (m, 24H), 1.54 (d, J= 6.2 Hz, 3H). 13C
NMR (101 MHz, MeOD) δ 176.2, 155.5, 145.3, 144.2, 140.1, 133.6, 132.9, 132.1,
128.6, 124.8, 124.8, 122.0, 110.3, 109.4, 107.0, 92.5, 60.5, 56.1, 43.7, 41.3, 39.0, 36.7,
33.8, 32.9, 27.0, 25.8, 20.1, 13.7. HRMS [ESI]: m/z [M+H]+ calcd for C39H47F3N3O6:
710.3417, found: 710.3394.

ACCEPTED MANUSCRIPT
4.1.1.6. Compound 4f. Yellow solid (78% yield): mp: 93-94°C; 1H NMR (400 MHz,
MeOD), δ= 8.46-8.45 (m, 1H), 7.76-7.74 (m, 1H), 7.23-7.20 (m, 3H), 7.11 (d, J= 8.0
Hz, 2H), 6.58 (s, 1H), 5.46 (s, 1H), 3.82-3.77 (m, 4H), 3.20-3.08 (m, 4H), 2.38 (s, 3H),
2.25-2.11 (m, 1H), 2.00-1.40 (m, 24H), 1.32 (d, J=6.3 Hz, 3H). 13C NMR (101 MHz,
MeOD) δ 176.4, 155.4, 144.7, 143.9, 135.8, 133.8, 133.1, 132.9, 131.3, 129.0, 128.3,
121.3, 111.3, 109.9, 106.8, 93.3, 55.6, 43.5, 38.7, 36.6, 33.4, 32.7, 27.1, 25.5, 19.9,
19.4. HRMS [ESI]: m/z [M+H]+ calcd for C39H50N3O6: 656.3700, found: 656.3685.
4.1.1.7. Compound 4g. Yellow solid (54% yield): mp: 70-71°C; 1H NMR (400 MHz,
CDCl3), δ= 8.54-8.53 (m, 1H), 8.07-7.96 (m, 1H), 7.43-7.41 (m, 1H), 7.31-7.23 (m,
2H), 7.14-7.13 (m, 1H), 6.49 (s, 1H), 6.23 (brs, 1H), 5.62 (brs, 1H), 3.86 (s, 3H), 3.78-
3.74 (m, 1H), 3.34-3.25 (m, 2H), 3.18-3.03 (m, 2H), 2.20-2.08 (m, 1H), 1.95-1.61 (m,
24H), 1.35 (d, J=6.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 174.5, 141.7, 133.2,
121.9, 115.3, 115.1, 113.5, 110.5, 107.1, 60.4, 56.6, 47.9, 44.1, 39.3, 36.9, 34.0, 33.1,
27.0, 26.3, 21.1, 20.7, 14.2. Anal. calcd for C36H45N3O7: C 68.44, H 7.18, N 6.65,
found: C 68.35, H 7.28, N 6.63.
4.1.1.8. Compound 4h. Orange solid (49% yield): mp: 66-68°C; 1H NMR (400 MHz,
CDCl3), δ= 8.54-8.53 (m, 1H), 8.01-7.99 (m, 1H), 7.42 (dd, J= 4.7, 3.1 Hz, 1H), 7.26-
7.23 (m, 2H), 7.14-7.13 (m, 1H), 6.49 (s, 1H), 6.22 (brs, 1H), 5.59 (brs, 1H), 3.86 (s,
3H), 3.75-3.74 (m, 1H), 3.33-3.26 (m, 2H), 3.15-3.04 (m, 2H), 2.17-2.08 (m, 1H), 2.03-
1.51 (m, 24H), 1.35 (d, J= 6.4 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 176.4, 156.0,
144.9, 143.9, 135.4, 133.8, 133.0, 130.6, 129.2, 123.9, 121.5, 109.9, 106.8, 93.1, 55.5,
43.5, 38.7, 37.5, 36.6, 33.3, 32.7, 27.2, 25.5, 19.4. Anal. calcd for C36H45N3O6S: C
66.74, H 7.00, N 6.49, found: C 66.52, H 6.84, N 6.33.
4.1.1.9. Compound 4i. Brown oil (63% yield); 1H NMR (300 MHz, CDCl3), δ= 8.47
(dd, J=4.1, 1.6 Hz, 1H), 8.32 (dd, J=8.6, 1.5Hz, 1H), 7.35 (dd, J= 8.6, 4.2 Hz, 1H), 6.37
(s, 1H), 6.19-6.07 (m, 1H), 5.51-5.34 (m, 1H), 3.94 (s, 3H), 3.85-3.79 (m, 1H), 3.32-
3.12 (m, 2H), 1.93-1.53 (m, 27H), 1.25 (d, J= 6.4 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ 171.4, 147.6, 144.9, 144.7, 134.4, 128.7, 122.9, 110.5, 107.1, 92.9, 56.9, 47.8, 44.1,
36.9, 34.3, 33.9, 33.1, 27.0, 25.5, 21.8, 19.1. HRMS [ESI]: m/z [M+H]+ calcd for
C32H43BrN3O6: 644.2335, found: 644.2313.
4.1.2. General procedure for the synthesis of hybrids 4j–l

ACCEPTED MANUSCRIPT
To a solution of compound 14 (0.31 mmol) in dry dichloromethane (7 mL) was added
triethylamine (0.5 mmol) and TBTU (0.31 mmol). The reaction mixture was stirred for
60 minutes at 0 °C, under N2 atmosphere. Then a solution of compound 13j-l (0.31
mmol) and triethylamine (0.33 mmol) in dry dichloromethane (2 mL) was added and,
after 30 minutes, the mixture was allowed to warm to room temperature and react
overnight. After completion of the reaction, the mixture was diluted with ethyl acetate
and washed with NaHCO3. The combined organic phase was washed with brine, dried
over anhydrous Na2SO4, filtered and, concentrated. Purification by flash column
chromatography using ethyl acetate-hexane (4:6) as eluent gave the pure compound.
4.1.2.1. Compound 4j. Yellow solid (88% yield); mp: 110-112ºC; 1H RMN (300 MHz,
CDCl3) δ 8.47 (dd, J= 4.1, 1.6Hz, 1H), 7.70 (dd, J=8.6, 1.6Hz, 1H), 7.25- 7.19 (m, 2H),
7.18-7.13 (m, 1H), 7.12-7.03 (m, 2H), 6.43 (s, 1H), 6.32 (brs, 1H), 5-86-5.76 (m, 1H),
3.77 (s, 3H), 3.50-3.32 (m, 4H), 2.13-1.48 (m, 25H).13C NMR (75 MHz, CDCl3) δ
174.6, 163.4, 160.2, 155.4, 145.5, 144.5, 133.9, 133.2, 133.1, 131.7, 131.6, 128.9,
122.0, 115.3, 115.0, 110.6, 110.5, 107.1, 92.6, 56.6, 44.1, 41.5, 38.0, 36.9, 33.1, 29.0,
27.0. HRMS [ESI]: m/z [M+H]+ calcd for C36H43FN3O6: 632.3136, found: 632.3107.
4.1.2.2. Compound 4k. Yellow solid (71% yield); mp: 108-110ºC; 1H RMN (300 MHz,
CDCl3) δ 8.54 (dd, J=4.1, 1.5Hz, 1H), 7.77 (dd, J=8.6, 1.5Hz, 1H), 7.32-7.27 (m, 2H),
7.25-7.20 (m, 1H), 7.19-7.10 (m, 2H), 6.48 (s, 1H), 6.31 (brs, 1H), 5.69-5.52 (m, 1H),
3.84 (s, 3H), 3.47-3.30 (m, 4H), 2.23-1.53 (m, 27H). 13C NMR (75 MHz, CDCl3) δ
174.5, 163.4, 160.2, 155.5, 145.8, 144.4, 133.9, 133.3, 133.2, 133.1, 131.8, 131.7,
128.9, 121.9, 115.2, 115.0, 111.0, 110.4, 107.1, 93.0, 56.6, 44.2, 42.9, 39.1, 36.9, 33.1,
27.5, 27.1, 26.6. HRMS [ESI]: m/z [M+H]+ calcd for C37H45FN3O6: 646.3292, found:
646.3263.
4.1.2.3. Compound 4l. Yellow solid (77% yield); mp: 100-102ºC; 1H RMN (300 MHz,
CDCl3) δ 8.46 (dd, J=4.1, 1.6Hz, 1H), 7.69 (dd, J= 8.6, 1.6Hz, 1H), 7.26-7.18 (m, 2H),
7.17-7.12 (m, 1H),7.11-7.01 (m, 2H), 6.40 (s, 1H), 6.23 (brs, 1H), 5.56-5.41 (m, 1H),
3.77 (s, 3H), 3.34-3.15 (m, 4H), 2.22-1.34 (m, 29H). 13C NMR (75 MHz, CDCl3) δ
174.4, 163.4, 160.2, 155.5, 145.9, 144.4, 133.9, 133.3, 133.2, 133.1, 131.8, 131.8,
128.9, 121.9, 115.2, 115.0, 110.5, 110.2, 107.1, 92.4, 56.6, 44.2, 43.2, 39.2, 36.9, 33.1,
29.5, 28.8, 27.1, 24.5. HRMS [ESI]: m/z [M+H]+ calcd for C38H47FN3O6: 660.3449,
found: 660.3417.

ACCEPTED MANUSCRIPT
4.1.3. General procedure for the synthesis of hybrids 4m–s
To a solution of compound 15 (0.052 mmol) in dry dichloromethane (1 mL) was added
the primaquine derivative 13a-c and 13e-h (0.057 mmol). After stirring for 1h at room
temperature under N2 atmosphere, AcOH (0.078 mmol) and STAB (0.078 mmol) were
added. After 16 h, AcOH (0.052 mmol) and STAB (0.052 mmol) were added to allow
the reaction to go to completion. The mixture was diluted and basified to pH 10 with
saturated aqueous NaHCO3 and the aqueous phase was then extracted with
dichloromethane and ethyl acetate. The organic phase was washed with brine, dried
over anhydrous Na2SO4, filtered and concentrated. Purification by flash column
chromatography using ethyl acetate-hexane (4:6) as eluent gave the pure compound.
4.1.3.1. Compound 4m. Yellow solid (42% yield): mp: 52-53°C; 1H NMR (400 MHz,
MeOD), δ= 8.52-8.47 (m, 1H), 7.82 (brs, 1H), 7.80-7.74 (m, 1H), 7.46-7.38 (m, 2H),
7.37-7.29 (m, 2H), 7.28-7.21 (m, 3H), 6.61 (s, 1H), 3.90-3.82 (m, 1H), 3.80 (s, 3H),
3.16-2.93 (m, 2H), 2.79-2.67 (m, 2H), 2.57-2.49 (m, 2H), 2.04-1.49 (m, 25H), 1.36 (d,
J=6.2 Hz, 3H,). 13C NMR (101 MHz, MeOD) δ 144.0, 136.2, 136.0, 133.2, 131.5,
127.8, 126.3, 121.5, 107.4, 93.3, 55.8, 54.0, 49.0, 36.6, 35.6, 33.8, 32.8, 27.1, 19.8.
HRMS [ESI]: m/z [M+H]+ calcd for C38H50N3O5: 628.3750, found: 628.3727.
4.1.3.2. Compound 4n. Yellow solid (62% yield): mp: 75-77°C; 1H NMR (400 MHz,
MeOD), δ= 8.50-8.44 (m, 1H), 7.75-7.69 (m, 1H), 7.29-7.08 (m, 5H), 6.60 (s, 1H),
3.89-3.70 (m, 4H), 3.14-2.88 (m, 2H), 2.75-2.60 (m, 2H), 2.54-2.41 (m, 2H), 2.05-1.39
(m, 25H), 1.33 (d, J=6.2 Hz, 3H).13C NMR (101 MHz, MeOD) δ 155.6, 145.0, 144.0,
133.2, 133.1, 132.8, 132.0, 129.0, 121.6, 114.5, 114.3, 109.8, 107.4, 92.8, 55.5, 53.9,
36.6, 35.6, 33.8, 32.7, 27.1, 24.7, 19.6. HRMS [ESI]: m/z [M+H]+ calcd for
C38H49FN3O6: 646.3656, found: 646.3627.
4.1.3.3. Compound 4o. Yellow solid (78% yield): mp: 79-81°C; 1H NMR (400 MHz,
MeOD), δ= 8.52-8.44 (m, 1H), 7.77-7.68 (m, 1H), 7.38 (d, J= 7.9 Hz, 2H), 7.29-7.16
(m, 3H), 6.60 (s, 1H), 3.88-3.80 (m, 1H), 3.78 (s, 3H), 3.12-2.90 (m, 2H), 2.83-2.72 (m,
2H), 2.60-2.50 (m, 2H), 2.26-2.17 (m, 1H), 2.01-1.48 (m, 24H), 1.32 (d, J=6.2 Hz, 3H).
13C NMR (101 MHz, MeOD) δ 155.5, 145.2, 144.1, 134.7, 133.7, 133.0, 132.6, 132.1,
128.7, 127.8, 121.7, 109.9, 107.2, 92.7, 55.5, 53.4, 36.5, 35.2, 33.6, 32.7, 27.1, 24.1,

ACCEPTED MANUSCRIPT
19.6. Anal. calcd for C38H48ClN3O5: C 68.92, H 7.31, N 6.34, found: C 68.63, H 7.31, N
6.21.
4.1.3.4. Compound 4p. Yellow solid (74% yield): mp: 73-74°C; 1H NMR (400 MHz,
CDCl3), δ= 8.60-8.52 (m, 1H), 7.83-7.76 (m, 1H), 7.75-7.69 (m, 2H), 7.53-7.43 (m,
2H), 7.33-7.24 (m, 1H), 7.22-7.15 (m, 1H), 6.52 (s, 1H), 6.40-6.26 (m, 1H), 3.87 (s,
3H), 3.82-3.71 (m, 1H), 3.27-2.99 (m, 2H), 2.80-2.67 (m, 2H), 2.58-2.47 (m, 2H), 2.13-
1.48 (m, 25H), 1.39 (d, J = 6.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 155.4, 148.9,
145.4, 144.4, 140.1, 133.9, 132.7, 132.1, 129.0, 128.6, 128.2, 125.3, 125.1, 125.0,
122.1, 110.4, 108.0, 92.3, 56.5, 55.0, 49.9, 48.1, 39.1, 36.9, 36.7, 34.6, 33.1, 29.7, 27.0,
26.2, 20.7. Anal. calcd for C39H48F3N3O5: C 67.32, H 6.95, N 6.04, found: C 67.10, H
6.85, N 6.13.
4.1.3.5. Compound 4q. Yellow solid (68% yield): mp: 75-76°C; 1H NMR (400 MHz,
MeOD), δ= 8.49-8.42 (m, 1H), 7.79-7.70 (m, 1H), 7.28-7.17 (m, 3H), 7.14-7.06 (m,
2H), 6.59 (s, 1H), 3.87-3.78 (m, 1H), 3.76 (s, 3H), 3.13-2.91 (m, 2H), 2.74-2.57 (m,
2H), 2.50-2.41 (m, 2H), 2.37 (s, 3H), 2.02-1.47 (m, 25H), 1.32 (d, J=6.3 Hz, 3H). 13C
NMR (101 MHz, MeOD) δ 155.4, 144.7, 143.9, 135.9, 133.2, 132.9, 131.3, 129.1,
128.4, 121.4, 111.4, 109.8, 107.4, 93.3, 55.6, 54.1, 49.1, 36.6, 35.8, 33.9, 32.7, 27.1,
24.8, 19.9, 19.7. Anal. calcd for C39H51N3O5: C 72.98, H 8.01, N 6.55, found: C 73.07,
H 7.98, N 6.62.
4.1.3.6. Compound 4r. Yellow solid (57% yield): mp: 62-64°C; 1H NMR (400 MHz,
MeOD) δ 8.57-8.50 (m, 1H), 8.21-8.11 (m, 1H), 7.68-7.59 (m, 1H), 7.58-7.50 (m, 1H),
7.41-7.29 (m, 1H), 7.27-7.03 (m, 1H), 6.64 (s, 1H), 6.53 (s, 1H), 3.94-3.77 (m, 4H),
3.20-2.95 (m, 2H), 2.82-2.71 (m, 2H), 2.59-2.50 (m, 2H), 2.09-1.48 (m, 25H), 1.38 (d, J
= 6.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 156.2, 144.8, 144.0, 142.1, 141.6, 133.1,
129.1, 121.6, 113.2, 109.8, 107.3, 93.1, 55.6, 53.7, 36.6, 35.5, 33.7, 32.7, 29.4, 27.1,
24.5, 19.6. Anal. calcd for C36H47N3O6: C 69.99, H 7.67, N 6.80, found: C 69.79, H
7.74, N 6.77.
4.1.3.7. Compound 4s. Yellow solid (76% yield): mp: 66-67°C; 1H NMR (400 MHz,
MeOD) δ= 8.55-8.45 (m, 1H), 7.99-7.90 (m, 1H), 7.51-7.40 (m, 1H), 7.34-7.25 (m, 1H),
7.24-7.19 (m, 1H), 7.12-7.03 (m, 1H), 6.59 (s, 1H), 3.82 (s, 3H), 3.17-2.92 (m, 2H),
2.79-2.62 (m, 2H), 2.57-2.42 (m, 2H), 2.34-2.10 (m, 1H), 2.07-1.41 (m, 25H), 1.36 (d, J
= 6.3 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 156.0, 144.8, 144.0, 135.4, 133.8, 133.2,

ACCEPTED MANUSCRIPT
130.6, 129.3, 123.9, 121.6, 109.9, 107.4, 93.2, 55.7, 54.0, 49.0, 36.6, 35.7, 33.8, 32.8,
32.7, 27.1, 24.8, 19.8. HRMS [ESI]: m/z [M+H]+ calcd for C36H48N3O5S: 634.3315,
found: 634.3291.
4.1.4. General procedure for the synthesis of hybrids 16a–d
To a solution of compound 14 (0.25 mmol) in dry dichloromethane (5.62 mL) stirring at
room temperature under N2 atmosphere, was added ethyl chloroformate (0.28 mmol)
and dry triethylamine (0.38 mmol). Product formation was monitored by thin layer
chromatography and after 1 h, compounds 5a-d (0.25 mmol) were added to the reaction
mixture which was then stirred at 60°C. After about 7 h, a saturated solution of sodium
hidrogenocarbonate was added to the reaction and the mixture was then extracted with
dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4,
filtered and, concentrated. Purification by flash chromatography using ethyl acetate-
hexane (4:6) as eluent gave the pure compound.
4.1.4.1. Compound 16a. Yellow solid (58% yield); mp: 184-186⁰C; 1H NMR (400
MHz, CDCl3) δ 10.10 (s, 1H), 8.85 (s, 1H), 8.64 (d, J = 2.8 Hz, 1H), 7.89 (d, J = 8.6 Hz,
1H), 7.49 (t, J = 7.3 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.31-
7.28 (m, 1H), 3.91 (s, 3H), 3.20 (s, 2H), 2.64 (t, J = 10.7 Hz, 1H), 2.25-1.55 (m,
25H).13C NMR (101 MHz, CDCl3) δ 173.9, 154.7, 146.0, 135.5, 131.7, 128.7, 127.7,
122.4, 119.1, 111.0, 107.6, 57.0, 45.8, 37.4, 33.6, 27.5. HRMS [ESI]: m/z [M+H]+ calcd
for C33H37N2O6: 557.2652, found: 557.2653.
4.1.4.2. Compound 16b. Yellow solid (52% yield); mp: 204-206⁰C; 1H NMR (400
MHz, CDCl3) δ 10.09 (s, 1H), 8.84 (s, 1H), 8.64 (d, J = 3.6 Hz, 1H), 7.85 (d, J = 8.4 Hz,
1H), 7.34-7.27 (m, 3H), 7.17 (t, J = 8.6 Hz, 2H), 3.91 (s, 3H), 3.21 (s, 2H), 2.63 (t, J =
7.1 Hz, 1H), 2.20-1.61 (m, 24H); 13C NMR (101 MHz, CDCl3) δ 173.7, 163.6, 161.2,
154.9, 145.8, 135.5, 133.7, 133.0, 130.9, 128.6, 122.5, 117.7, 115.6, 110.7, 107.3, 56.8,
45.7, 37.1, 33.4, 30.2, 27.3. HRMS [ESI]: m/z [M+H]+ calcd for C33H36FN2O6:
575.2557, found: 575.2552.
4.1.4.3. Compound 16c. Yellow solid (51% yield); mp: 208-210⁰C; 1H NMR (400
MHz, CDCl3) δ 10.09 (s, 1H), 8.84 (s, 1H), 8.64 (d, J = 3.0 Hz, 1H), 7.86 (d, J = 8.5 Hz,
1H), 7.46 (d, J = 8.2 Hz, 2H), 7.32 (dd, J = 8.6, 4.1 Hz, 1H), 7.27 (d, J = 7.6 Hz, 3H),
3.91 (s, 3H), 3.20 (s, 1H), 2.63 (t, J = 10.9 Hz, 1H), 2.23-1.54 (m, 26H). 13C NMR (101

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MHz, CDCl3) δ 173.6, 154.6, 145.8, 135.6, 133.6, 133.4, 132.8, 128.7, 128.0, 122.2,
117.3, 110.7, 107.2, 104.5, 56.7, 45.5, 37.1, 33.3, 29.9, 27.2. HRMS [ESI]: m/z [M+H]+
calcd for C33H36ClN2O6: 591,2262, found: 591.2260.
4.1.4.4. Compound 16d. Yellow solid (52% yield); mp: 212-214⁰C; 1H NMR (400
MHz, CDCl3) δ 10.09 (s, 1H), 8.83 (s, 1H), 8.64 (d, J = 2.8 Hz, 1H), 7.86 (d, J = 8.5 Hz,
1H), 7.61 (d, J = 8.2 Hz, 2H), 7.32 (dd, J = 8.6, 4.1 Hz, 1H), 7.21 (d, J = 8.3 Hz, 2H),
3.90 (s, 3H), 3.20 (s, 1H), 2.63 (t, J = 10.9 Hz, 1H), 2.17-1.56 (m, 28H). 13C NMR (101
MHz, CDCl3) δ 173.6, 154.4, 145.8, 135.5, 134.1, 133.1, 131.6, 128.0, 122.3, 121.6,
117.2, 110.7, 107.2, 104.4, 56.7, 45.5, 37.1, 33.3, 29.9, 27.2, 19.0. HRMS [ESI]: m/z
[M+H]+ calcd for C33H36BrN2O6: 635,1757, found: 635.1747.
4.2. Biology
4.2.1. Activity against erythrocytic-stage P. falciparum
Human erythrocytes infected with about 1% parasitemia of ring-stage W2 strain P.
falciparum synchronized with 5% sorbitol were incubated with test compounds in 96-
well plates at 37°C for 48 h in RPMI-1640 medium, supplemented with 25 mM 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 10% heat inactivated
human serum(or 0.5% Albumax, 2% human serum), and 100 µM hypoxanthine under
an atmosphere of 3% O2, 5% CO2, and 91% N2. After 48 h, the cells were fixed in 2%
formaldehyde in phosphate-buffered saline (PBS) and transferred into PBS with 100
mM NH4Cl, 0.1% Triton X-100, 1 nM YOYO-1, and infected erythrocytes were
counted in a flow cytometer (FACSort, Beckton Dickinson; EX 488 nm, EM 520 nm).
Values of IC50 based on comparisons with untreated control cultures were calculated by
using GraphPad PRISM software. Two independent experiments were performed, each
with four replicates for each of the experimental conditions.
4.2.2. Activity against P. berghei liver stages
Inhibition of hepatic infection by test compounds was determined by measuring the
luminescence intensity in Huh-7 cells infected with a firefly luciferase-expressing P.
berghei line as previously described.[35] Briefly, Huh-7 cells, a human hepatoma cell
line, were cultured in RPMI-1640 medium supplemented with 10 % (v/v) fetal bovine

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serum, 1 % (v/v) nonessential amino acids, 1 % (v/v) penicillin/streptomycin, 1 % (v/v)
glutamine, and 10 mM HEPES, pH 7, and maintained at 37°C with 5 % CO2. For
infection assays, Huh-7 cells (1.0 × 104 per well) were seeded in 96-well plates the day
before drug treatment and infection. The medium was replaced by medium containing
the appropriate concentration of each compound approximately 1 h prior to infection
with sporozoites freshly obtained through disruption of salivary glands of infected
female Anopheles stephensi mosquitoes. In control wells, the compounds’ vehicle
(DMSO) was added in an amount equivalent to that present in the highest drug
concentration employed. Sporozoite addition was followed by centrifugation at 1700 g
for 5 min. Parasite infection load was measured 48 h after infection by a
bioluminescence assay (Biotium, Hayward, CA). The effect of the compounds on the
viability of Huh-7 cells was assessed by the AlamarBlue assay (Life) using the
manufacturer’s protocol. All compounds were initially screened at 1 and 10 µM and
compounds selected for EC50 determination were additionally assessed at various
concentrations determined on the basis of the initial activity screen. Nonlinear
regression analysis was employed to fit the normalized results of the dose-response
curves, and EC50 values were determined using GraphPad Prism V 5.0.
4.2.3. Metabolism studies
Compounds 4 and 16 were incubated with a reaction mixture consisting of rat liver
microsomal protein (20 µL of a 20 mg mL-1 solution) in 50 mM PBS (160 µL), water
(570 µL), NADPH regenerating system solution A (40 µL, containing 31 mM NADP+,
66 mM glucose 6-phosphatase, and 0.67 mM MgCl2), and NADPH regenerating system
solution B (8 µL, containing 40 U mL-1 glucose-6-phosphate dehydrogenase in 5 mM
sodium citrate). After pre-incubation at 37°C for 5 min, enzyme reactions were initiated
by adding the parent solution of the drug (2 µL, 1 mM) in DMSO. Samples were
aliquoted (80 µL) at various time points and stopped by protein precipitation through
addition of an equal volume of ice-cold acetonitrile. The mixtures were centrifuged at
10,000 g for 10 min at room temperature. The supernatants were immediately analyzed,
and the remaining parent drug versus time was quantified by HPLC. The HPLC system
consisted of a LichroCART 125-4 RP-18 (5 µm) analytical column on a LabChrom
L7400 Merck Hitachi instrument, with a mixture of methanol/water (85:15) as eluent at
a flow rate of 1 mLmin-1. HPLC detection was at the maximum of absorbance of

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compounds 4 and 16 (λ=266 nm). The corresponding half-life values were determined
in triplicate.
4.2.4. Cytotoxicity Assays
Colorectal adenocarcinoma CaCo-2 cells (ATCC) were grown in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS) and antibiotic antimycotic solution
(A5955, Sigma) at 37 ºC in a humidified 5% CO2 atmosphere.
CaCo-2 cells were seeded on 96-well-plates and cultured until reaching confluence.
Stock solutions of the compounds were diluted with the cell culture medium and added
to the confluent cells for 72 h (percentage of organic solvent ≤1%). After this period,
medium was removed from the plates and cells were washed with PBS. Viability was
determined using neutral red as described elsewhere.[36] Each experimental condition
was done in triplicate. CC50 values were determined using GraphPad Prism software.
Acknowledgements
This work was supported by Fundação para a Ciência e Tecnologia (FCT), Portugal,
through grants Pest-OE/SAU/UI4013/2014, REDE/1501/REM/2005,
ROTEIRO/0028/2013, LISBOA-01-0145-FEDER-022125 and PTDC/SAU-
FAR/118459/2010, and fellowships SFRH/BPD/100645/2014 (RC) and Investigator
FCT (MP).
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