Squaric acid/4-aminoquinoline conjugates: Novel potent antiplasmodial agents

Article abstract of DOI:10.1016/j.ejmech.2013.08.037

We report the synthesis and structure-activity relationship (SAR) analysis of a series of hybrid compounds containing a squaric moiety conjugated with heterocyclic moieties from well-known antimalarials. This novel series of compounds presents improved antiplasmodial activity compared with the squaric derivatives described in our previous work. Three compounds, 8b (IC₅₀ = 99 nM), 8c (IC₅₀ = 95 nM), and 8d (IC₅₀ = 105 nM) had greater in vitro potency than chloroquine 1 (IC₅₀ = 140 nM) against chloroquine resistant Plasmodium falciparum. In addition, they were noncytotoxic against NIH 3T3 and Hek 293T cells.

Full text of DOI:10.1016/j.ejmech.2013.08.037

Accepted Manuscript
Squaric acid/4-aminoquinoline conjugates: Novel potent antiplasmodial agents
Carlos J.A. Ribeiro, S. Praveen Kumar, Jiri Gut, Lídia M. Gonçalves, Philip J.
Rosenthal, Rui Moreira, Maria M.M. Santos
PII:
S0223-5234(13)00554-0
10.1016/j.ejmech.2013.08.037
EJMECH 6384
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 March 2013
Revised Date: 18 August 2013
Accepted Date: 21 August 2013
Please cite this article as: C.J.A. Ribeiro, S.P. Kumar, J. Gut, L.M. Gonçalves, P.J. Rosenthal, R.
Moreira, M.M.M. Santos, Squaric acid/4-aminoquinoline conjugates: Novel potent antiplasmodial agents,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.08.037.
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ACCEPTED MANUSCRIPT
Squaric acid/4-aminoquinoline conjugates: novel potent antiplasmodial agents
Carlos J. A. Ribeiroa, S. Praveen Kumara, Jiri Gutb, Lídia M. Gonçalvesa, Philip J. Rosenthalb,
Rui Moreiraa, Maria M. M. Santosa,*
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Lisbon, Portugal
b San Francisco General Hospital, University of California, San Francisco, USA
Cl
Cl
N
HN
N
H
H
H
N
H
N
N
N
N
N
n
n
Cl
O
O
Chloroquine
Pf W2 IC50 (µM) = 0.095-0.20
Pf W2 IC50 (µM) = 0.14

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•
•
•
•
Squaric acid/4-aminoquinoline conjugates were synthesized.
Compounds were evaluated against papain, falcipain-2 and Pf W2.
Three compounds had higher in vitro potency than chloroquine.
The most active compounds were noncytotoxic.

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Squaric acid/4-aminoquinoline conjugates: novel potent
antiplasmodial agents
Carlos J. A. Ribeiroa, S. Praveen Kumara, Jiri Gutb, Lídia M. Gonçalvesa, Philip
J. Rosenthalb, Rui Moreiraa, Maria M. M. Santosa,*
a
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL),
Faculty of Pharmacy, University of Lisbon. Av. Prof. Gama Pinto, 1649-003
Lisbon, Portugal
b
Department of Medicine, San Francisco General Hospital, University of
California, San Francisco, CA 94143, USA
Corresponding author
Maria M. M. Santos
Medicinal Chemistry Group, iMed.UL
Faculty of Pharmacy, University of Lisbon
Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
Phone: (+) 351 21 794 6451; Fax: (+) 351 21 794 6470
E-mail: mariasantos@ff.ul.pt

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Abstract
We report the synthesis and structure-activity relationship (SAR) analysis of a
series of hybrid compounds containing a squaric moiety conjugated with
heterocyclic moieties from well-known antimalarials. This novel series of
compounds presents improved antiplasmodial activity compared with the
squaric derivatives described in our previous work. Three compounds, 8b (IC50
= 99 nM), 8c (IC50 = 95 nM), and 8d (IC50 = 105 nM) had greater in vitro
potency than chloroquine 1 (IC50 = 140 nM) against chloroquine resistant
Plasmodium falciparum. In addition, they were noncytotoxic against NIH 3T3
and Hek 293T cells.
Keywords: squaramide, 4-aminoquinoline, malaria, hybrid, antiplasmodial
activity

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1. Introduction
Malaria, particularly that caused by Plasmodium falciparum, continues to
represent one of the most deadly parasitic diseases in humans, causing
estimated 655,000 deaths in 2010, mainly in underdeveloped countries [1]. One
of the main difficulties in malaria treatment is the emergence of resistance to
standard antimalarial drugs, including chloroquine (1), primaquine (2) (Figure 1),
antifolates, and artemisinins [2].
To overcome this drawback many efforts are being deployed, such as synthesis
of analogues of known antimalarial drugs and combination therapy.
Furthermore, attention is being given to the development of hybrids, which
consist of a single entity containing two pharmacophores that are connected
directly or by a linker and that act against different targets [3].
Quinoline based analogs of chloroquine remain of interest [4]. In fact, in the last
few years several hybrids containing a quinoline moiety were reported [5-12].
We have studied Michael acceptors as cysteine protease inhibitors [13-17].
Falcipain cysteine proteases play a key role in the metabolism of P. falciparum,
and previous work from our group demonstrated that squaric acid derivatives 3
are active in vitro against this parasite [18].
Based on the hypothesis that linking two active molecules in a hybrid compound
can enhance efficacy and/or reduce propensity to resistance relative to the
parent drugs [19], we synthesised an expanded series of squaric derivatives
conjugated with heterocyclic moieties from known antimalarials. To perform an
SAR study, we synthesized and evaluated compounds containing different

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heterocyclic cores, different aminoaliphatic (alkyl side chain) and
heteroaromatic linkers, and different substituted squaric acid derivatives (Figure
2).
2. Results and discussion
2.1. Chemistry
Thirteen novel compounds (6a-f, 8a-e, 9 and 10) were synthesized.
We started by synthesizing a series of compounds containing the
heteroaromatic core of 4-amino-7-chloroquinoline, linked to the squaric acid
scaffold through a spacer. The rationale to synthesize these compounds was
based on:
•
our previous reports that squaric acid derivatives present good
antiplasmodial activities [15, 18];
•
the role of 4-amino-7-chloroquinoline for inhibition of chloroquine-
resistant P. falciparum strain W2.
Reaction of commercially available squarate 4 with one equivalent of 4-amino-
7-chloroquinolines 5a-b, led to squaramates 6a-b containing a flexible linker
(Scheme 1).
In order to evaluate the effect of the methoxy group on the activity of
compounds 6a-b, we decided to prepare compounds 6c-d (butoxy analogues)
starting from butyl squarate 7. Reaction of squarate 7 with 4-amino-7-
chloroquinolines 5a-b (1 equivalent) at room temperature, led to squaramates
6c-d containing an alkylic linker. Furthermore, the importance of flexibility in the

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linkers to activity was also investigated by synthesizing compounds 6e-f
(analogues of 6c-d, respectively) containing a rigid linker (Scheme 2).
Disubstituted squaramides 8a-d were prepared from reaction of squarate 7 with
two equivalents of 4-amino-7-chloroquinolines 5a-d at reflux conditions
(Scheme 2).
We also evaluated the effect of replacing the 4-amino-7-chloroquinoline moiety
by 8-amino-6-methoxyquinoline (the heteroaromatic ring of the antimalarial
primaquine) (Scheme 3). Starting from squarate 7 and two equivalents of
primaquine, we obtained the monosubstituted compound 9. In the presence of
triethylamine the disubstituted compound 10 was obtained instead.
Finally, we prepared and evaluated compound 8e, containing a Phe residue
between the squaric acid and the aminoquinoline moieties, in order to evaluate
its activity as a falcipain-2 inhibitor and as an antiplasmodial agent (Scheme 2).
All of the proposed structures were established by NMR (1H, 13C, COSY and
HMQC), IR, and MS. For bis-secondary squaramides, especially with side
chains without an H-bond acceptor group, the possibility of establishing
intermolecular hydrogen bonding between the two NH groups of one molecule
with the carbonyls of another, makes the anti/anti conformation the most
preferable one. Conversely for monosubstituted secondary squaramides, the
best intermolecular H bond arrangement seems to favours the syn conformation
[20, 21]. For the monosubstituted compounds synthesized with flexible side
chain two sets of resonance were observed for both the NH, NCH2, and
methoxy or butoxy groups, revealing the presence of a mixture of isomers. The
establishment of the stereochemistry of the major isomer was based on the
evidence that, due to paramagnetic influence of the carbonyl group of the

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squaramide, the N-substituents in a syn conformation are deshielded in relation
to its anti counterpart [22]. Using compound 6a as an example, the proton
chemical shifts observed for the major/minor conformers were 8.86/8.70 ppm,
3.50/3.72 ppm and 3.92/4.25 ppm for the NH, adjacent CH2 and methoxy
groups, respectively in a 1.6:1 relationship.
2.2. Biological activity
The synthesized compounds were examined for their ability to inhibit papain,
falcipain-2 and the chloroquine-resistant W2 strain of P. falciparum. The IC50
values were determined using the cysteine protease inhibitor E-64 (trans-
epoxysucciny-L-leucyl-amido(4-guanidino)butane) and chloroquine as positive
controls. Inspection of the data in Table 1 allows the following observations:
1. As observed before for other squaric derivatives [18], all compounds were
inactive against papain, the prototype for clan CA cysteine proteases.
2. Compounds 6a-c had good antiplasmodial activity, with IC50 values ranging
from 1 to 2 µM, but were not active against falcipain-2 at tested
concentrations.
3. For compounds containing a propyl linker, the substitution of the OMe group
for an OBu group in the squaric moiety resulted in loss of activity against P.
falciparum (i.e. compound 6d, versus the OMe counterpart 6b).
4. Alkylic spacers were prefered to rigid spacers (i.e. compounds 6b and 6c,
versus the counterparts 6e and 6f, respectively).
5. Compounds 8a-d, containing two chloroquinoline moieties, displayed potent
antiplasmodial activity, with an IC50 value of 0.1-0.2 µM against the P.
falciparum W2 strain.

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6. For compounds 8, the effect of carbon chain length between squaric and
quinoline scaffolds had a major impact on activity, with an aminopropyl
spacer preferred over an aminoethyl spacer (3 carbons, 8b vs 2 carbons,
8a). However, the increase of carbon chain length from 3 to 4 carbons had
only modest impact on activity (4 carbons, 8c vs 3 carbons, 8b).
7. Substitution of the chloroquinoline ring by the primaquine ring led to loss of
activity (i.e. compound 8c, versus the counterpart 10).
8. Finally, compounds 8b (IC50 = 99 nM), 8c (IC50 = 95 nM) and 8d (IC50 = 105
nM), had greater in vitro potency than chloroquine 1 (IC50 = 140 nM).
Activity data for hybrid compounds 6 and 8 were also compared with those of P.
falciparum-infected red blood cells incubated with 1:1 and 2:1 mixtures of
chloroquine and squaric acid 7. The results presented in Table 1 show that the
1:1 chloroquine/7 combination was equipotent with chloroquine (IC50 values of
138 and 140 nM, respectively), but significantly more potent than hybrids 6
containing a single 4-aminoquinoline moiety or squaric acid 7. In contrast, the
2:1 chloroquine/7 combination displayed an activity level comparable to that of
hybrids 8b-d, suggesting that the 4-aminoquinoline moiety is contributing
significantly to the overall activity of hybrids 8b-d.
The in vitro cytotoxicity of the compounds active against P. falciparum
(compounds 6a-c, 6e-f, 8a-e, 9 and 10) was evaluated using the NIH 3T3 and
Hek 293T cell lines. Remarkably, most of the squaric derivatives were non-
cytotoxic at concentrations up to 50 µM (Table 1). Only, squaramate 8a led to
cytotoxicity at concentrations < 10 µM, but even in this case, the selectivity
index was ~25, which indicates very low toxicity.

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Although the mechanism by which squaric derivatives reported herein act as
antiplasmodial agents is unknown, there is strong evidence that 4-
aminoquinolines, such as chloroquine, exert their main activity by interfering
with heme detoxification in the acidic digestive vacuole (DV) of erythrocytic
parasites [25-27]. This process requires a proper balance between charged and
uncharged molecular species to allow the appropriate diffusion of the
uncharged form at physiologic pH (7.4) and the retention of the charged
counterpart in the DV (pH= 5.2). To test whether our compounds might in fact
satisfy these requirements, we determined in silico the pH-dependent
distribution of base between water and lipid coefficient (logD) for compounds
6a–f, 8a-b, 8d, 9 and chloroquine, and from these values, the corresponding
vacuolar accumulation ratio (VAR) and lipid accumulation ratio (LAR).
Interestingly, inspection of data presented in Table 2 shows that the VAR values
for disubstituted compounds 8b (IC50= 99nM) and 8d (IC50 = 105 nM) were
respectively 100-fold and 50-fold higher than that of their monosubstituted
counterparts 6c (IC50= 2.04µM) and 6d (IC50> 10µM), suggesting increased
accumulation inside the DV. In addition, high LAR values seem to be important
for activity against chloroquine-resistant strains, since it appears that
compounds more hydrophobic than chloroquine can block its efflux, which is
believed to be mediated by the pfcrt (P. falciparum chloroquine resistance
transporter) gene product [28]. Although the LAR values for the disubstituted
derivatives, 8a and 8b, were similar to those for chloroquine, suggesting good
permeability into the DV, three disubstituted compounds (8b, 8c and 8d) were
more active than chloroquine. Several factors can possibly account for this
observation. First, the squaric acid moiety ameliorates the activity of 4-

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aminoquinolines. Second, 4-aminoquinolines with diamino side chains lengths
of ≥10 carbons and two protonable nitrogens are active against CQR strains
[29]. Compounds 8b-d, which have linkers with 10-12 atoms between the two 4-
aminoquinolines NHs, were more active than chloroquine. In contrast,
compound 8a, which has a chain length of only 8 atoms, was less active than
chloroquine. Third, disubstituted compounds 8 are structurally similar to the
antimalarial piperaquine. Of note, cross-resistance between piperaquine and
chloroquine has generally not been observed [30].
Overall, these results are consistent with the reported observation that
antiplasmodial potency correlates with VAR, suggesting that potency is related
to accumulation inside the DV.
3. Conclusion
Novel antiplasmodial agents have been synthesized by condensation of
commercially available squarates with the heteroaromatic core of the
antimalarial drugs chloroquine and primaquine. The compounds were screened
in vitro for their activity against erythrocytic parasites, and three of them had
about two-fold higher potency than the parent drug, chloroquine against a CQ-
resistant P. falciparum strain and minimal cytotoxicity, thus representing a new
class of antiplasmodial agents.
4. Experimental section
4.1 Chemistry

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All reagents and solvents were obtained from commercial suppliers and were
used without further purification.
Melting points were determined using a Kofler camera Bock monoscope M and
are uncorrected.
The infrared spectra were collected on a Shimadzu FTIR Affinity-1
spectrophotometer.
ESI-MS/MS analysis were performed on Micromass® Quattro Micro triple
quadrupole (Waters®, Ireland) with an electrospray (ESI) ion source (positive
ion mode) at 120oC and capillary voltage of 3.0 kV. High resolution mass
spectra were performed in Unidade de Espectrometria de Masas (HMRS-ESI-
TOF), Santiago de Compostela, Spain and also in a FTICR Bruker Apex Ultra
(7T) at the Faculty of Sciences of Lisbon University, Portugal. Elemental
analysis was performed in a Flash 2000 CHNS-O analyzer (ThermoScientific,
UK), and results were within ± 0.4% of the theoretical values.
Merck Silica Gel 60 F254 plates were used for analytical TLC; flash column
chromatography was performed on Merck Silica Gel (200-400 mesh) and
CombiFlash Rf from Teledyne ISCO (columns RediSep Rf, silica).
1H and 13C NMR spectra were recorded on a Bruker 400 Ultra-Shield (400
1
MHz). H and 13C chemical shifts are expressed in ä (ppm) referenced to the
solvent used and the proton coupling constants (J) in hertz.
4.1.1 General preparation of squaric acid monosubstituted derivatives 6a-b
A reaction mixture of the appropriate 4-aminoquinoline 5 (1 eq.) in dry methanol
(2 ml for 0.22 mmol of amine) and NEt3 (1 eq.) was stirred for 5 min at room
temperature before addition of 3,4-dimetoxy-3-cyclobutene-1,2-dione (1 eq).

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After heating at reflux the solvent was removed under reduced pressure; water
was added, and the product was extracted with EtOAc (3x). The combined
organic extracts were washed with brine, dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure. The obtained residue was
recrystallized from MeOH/EtOAc affording the corresponding product.
4.1.1.1 Compound 6a: Reaction time 5h. Obtained as a white solid.
Yield 69%. Mp: 222-224 °C; IR (KBr): 3356, 2951, 17 05, 1614, 1578 cm-1. 1.6/1.
Major conformer: 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.86 (s, 1H, NH), 8.42
(d, J = 5.4 Hz, 1H, HAr), 8.19 (d, J = 8.8 Hz, 1H, HAr), 7.80 (s, 1H, HAr), 7.51 (s
br, 1H, NH), 7.47 (d, J = 8.8 Hz, 1H, HAr), 6.56 (d, J = 5.4 Hz, 1H, HAr), 3.92 (s,
3H, OCH3), 3.50 (s br, 4H, 2 CH2). 13C NMR (100 MHz, DMSO-d6) δ 189.28
(C=O), 182.32 (C=O), 176.81 (Cq), 172.75 (Cq), 151.69 (CHAr), 150.04 (CqAr),
148.88 (CqAr), 133.61 (CqAr), 127.37 (CHAr), 124.32 (CHAr), 123.99 (CHAr),
117.54 (CqAr), 98.83 (CHAr), 59.77 (OCH3), 42.57 (CH2), 42.35 (CH2). HRMS
(ESI-TOF) (M + 1)+ calcd for C16H15ClN3O3 332.0802 found 332.0796.
4.1.1.2 Compound 6b: Reaction time 8h. Obtained as a light yellow solid.
Yield 66%. Mp: 201-203 °C. IR (KBr): 3231, 2938, 17 05, 1620, 1597 cm-1. 1.5/1.
1
Major conformer: H NMR (400 MHz, DMSO-d6) δ (ppm): 8.82 (s br, 1H, NH),
8.39 (d, J = 5.4 Hz, 1H, HAr), 8.24 (d, J = 9.0 Hz, 1H, HAr), 7.78 (s, 1H, HAr), 7.46
(d, J = 9.0 Hz, 1H, HAr), 7.29 (s br, 1H, NH), 6.47 (d, J = 5.4 Hz, 1H, HAr), 4.12
(s, 3H, OCH3), 3.44–3.27 (m, 4H, 2 CH2), 1.97–1.87 (m, 2H, CH2).13C NMR
(100 MHz, DMSO-d6) δ (ppm): 189.40 (C=0), 182.35 (C=0), 177.01 (Cq),
172.35 (Cq), 151.86 (CHAr), 150.01 (CqAr), 149.00 (CqAr), 133.45 (CqAr), 127.47

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(CHAr), 124.13 (CHAr), 124.06 (CHAr), 117.47 (CqAr), 98.74 (CHAr), 59.97
(OCH3), 41.68 (CH2), 39.25 (CH2), 28.23 (CH2). HRMS (ESI-TOF) (M + 1)+
calcd for C17H17ClN3O3 346.0958 found 346.0953.
4.1.2 General preparation of squaric acid monosubstituted derivatives 6c-d
A reaction mixture of the appropriate 4-aminoquinoline (1 eq.) and 3,4-dibutoxy-
3-cyclobutene-1,2-dione (1 eq.) in dry methanol (2 ml for 0.22 mmol of amine)
was stirred at room temperature for 24 h. The solvent was then removed under
reduced pressure and the obtained residue was recrystallized from
MeOH/EtOAc affording the corresponding product.
4.1.2.1 Compound 6c: Obtained as a white solid.
Yield 65%. Mp: 215-217 °C. IR (KBr): 3360, 1796, 16 97, 1612, 1578 cm-1. 1.6:1.
1
Major conformer: H NMR (400 MHz, DMSO-d6) δ (ppm): 8.86 (s br, 1H, NH),
8.41 (d, J = 5.4 Hz, 1H, HAr), 8.19 (d, J = 9.0 Hz, 1H, HAr), 7.79 (s, 1H, HAr), 7.57
(s br, 1H, NH), 7.47 (dd, J = 9.0, 1.8 Hz, 1H, HAr), 6.56 (d, J = 5.4 Hz, 1H, HAr),
4.26 (t, J = 6.6 Hz, 2H, OCH2), 3.51 (s br, 4H, 2 CH2), 1.41–1.30 (m, 2H,
OCH2CH2), 1.21–1.10 (m, 2H, CH2CH3), 0.77 (t, J = 7.4 Hz, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 189.17 (C=O), 182.17 (C=O), 176.43 (Cq),
172.86 (Cq), 151.51 (CHAr), 150.06 (CqAr), 148.81 (Cq), 133.61 (Cq), 127.26
(CHAr), 124.29 (CHAr), 124.01 (CHAr), 117.55 (CqAr), 98.70 (CHAr), 72.23 (CH2-
butoxi), 42.67 (CH2), 42.44 (CH2), 31.11(CH2-butoxi), 17.95 (CH2-butoxi), 13.41 (CH3-
butoxi). HRMS (ESI-TOF) (M + 1)+ calcd for C19H21ClN3O3 374.1271 found
374.1266.

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4.1.2.2 Compound 6d: Obtained as a white solid.
Yield 63%. Mp: 190-192 °C. IR (KBr): 3331, 2957, 18 02, 1701, 1612, 1582 cm-1.
1
1.6/1. Major conformer: H NMR (400 MHz, DMSO-d6) δ (ppm): 8.83 (s br, 1H,
NH), 8.38 (d, J = 5.4 Hz, 1H, HAr), 8.24 (d, J = 9.0 Hz, 1H, HAr), 7.78 (s, 1H,
HAr), 7.45 (dd, J = 9.0, 1.6 Hz, 1H, HAr), 7.29 (s br, 1H, NH), 6.46 (d, J = 5.4 Hz,
1H, HAr), 4.42 (t, J = 6.6 Hz, 2H, OCH2), 3.45–3.38 (m, 2H, CH2), 3.36–3.28 (m,
2H, CH2), 1.97–1.87 (m, 2H, CH2), 1.51–1.42 (m, 2H, OCH2CH2), 1.26–1.14 (m,
2H, CH2CH3), 0.77 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 189.29 (C=O), 182.20 (C=O), 176.65 (Cq), 172.57 (Cq), 151.91 (CHAr),
150.00 (CqAr), 149.10 (CqAr), 133.42 (CqAr), 127.54 (CHAr), 124.08 (CHAr),
124.05 (CHAr), 117.50 (CqAr), 98.67 (CHAr), 72.30 (CH2-butoxi), 41.69 (CH2),
39.21(CH2), 31.32 (CH2-butoxi), 28.40 (CH2), 18.03 (CH2-butoxi), 13.43 (CH3-butoxi).
HRMS (ESI-TOF) (M + 1)+ calcd for C20H23ClN3O3 388.1428 found 388.1422.
4.1.3 General preparation of squaric acid monosubstituted derivatives 6e-f
A reaction mixture of the appropriate 4-aminoquinoline (2 eq.) and 3,4-dibutoxy-
3-cyclobutene-1,2-dione (1 eq.) in dry methanol (2 ml for 0.22 mmol of amine)
was heated at refluxed for 48 h. The solvent was removed under reduced
pressure and the residue purified by flash chromatography on silica gel using as
eluent n-hexane/EtOAc (7:3) to EtOAc (100%) affording the corresponding
product.
4.1.3.1 Compound 6e: Obtained as a light yellow solid.
Yield 83%. Mp: 162-163 ºC. IR (KBr): 3333, 2949, 1805, 1709, 1611, 1570 cm-1.
1H-RMN (400 MHz, DMSO-d6) δ (ppm) 8.73 (d, J = 5.0 Hz, 1H, HAr), 8.10 (d, J =

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9.0 Hz, 1H, HAr), 8.00 (s, 1H, HAr), 7.57 (d, J = 9.0 Hz, 1H, HAr), 7.06 (d, J = 5.0
Hz, 1H, HAr), 4.65 (t, J = 6.5 Hz, 2H, OCH2), 4.06 (s br, 2H, CH2-piperazine), 3.81 (s
br, 2H, CH2-piperazine), 3.32 (s br, 4H, 2 CH2-piperazine), 1.77–1.67 (m, 2H,
OCH2CH2), 1.45–1.33 (m, 2H, CH2CH3), 0.92 (t, J = 7.4 Hz, 3H, CH3). 13C-RMN
(100 MHz, DMSO-d6) δ (ppm): 188.72 (C=O), 181.78 (C=O), 176.22 (Cq),
169.92 (Cq), 155.81 (CqAr), 152.27 (CHAr), 149.63 (CqAr), 133.78 (CqAr), 128.11
(CHAr), 126.07 (2C, CHAr), 121.31 (CqAr), 110.06 (CHAr), 72.93 (CH2-butoxi), 62.81
(CH2-piperazine), 51.41 (CH2-piperazine), 46.80 (CH2-piperazine), 46.02 (CH2-piperazine),
31.48 (CH2-butoxi), 18.15 (CH2-butoxi), 13.54 (CH3-butoxi). MS (ESI, CP 3.0 kV, SP
30V): m/z calc. = 400 [M]+, m/z found = 400 [M]+. HRMS-ESI-FTICR (M + 1)+
calcd for C21H23ClN3O3 400.14225 found 400.14223.
4.1.3.2 Compound 6f: Obtained as a light yellow solid.
Yield 84%. Mp: 66-68 °C. IR (KBr): 2959, 1707, 1701, 1605 cm-1. 1.1/1 Major
conformer: 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.06 (d, J = 7.6 Hz, 1H, NH),
8.69 (d, J = 5.2 Hz, 1H, HAr), 8.01–7.96 (m, 2H, HAr), 7.59 (d, J = 9.0 Hz, 1H,
HAr), 7.04 (d, J = 5.2 Hz, 1H, HAr), 4.68–4.62 (m, 2H, OCH2), 3.69 (s br, 1H,
CHpiperidine), 3.65–3.55 (m, 2H, CH2-piperidine), 3.04–2.92 (m, 2H, CH2-piperidine),
2.09-1.99 (m, 2H, CH2-piperidine), 1.94–1.80 (m, 2H, CH2-piperidine), 1.77–1.67 (m,
2H, OCH2CH2), 1.44–1.34 (m, 2H, CH2CH3), 0.95–0,86 (m, 3H, CH3).13C NMR
(100 MHz, DMSO-d6) δ (ppm): 189.39 (C=O), 182.44 (C=O), 176.77 (Cq),
171.81 (Cq), 156.41 (CqAr), 152.21 (CHAr), 149.64 (CqAr), 133.60 (CqAr), 128.10
(CHAr), 126.00 (CHAr), 125.88 (CHAr), 121.52 (CqAr), 109.72 (CHAr), 72.50
(OCH2), 51.47 (CHpiperidine), 50.69 (CH2-piperidine), 50.61 (CH2-piperidine), 32.61 (CH2-
piperidine), 32.23 (CH2-piperidine), 31.48 (CH2), 18.24 (CH2), 13.56 (CH3). MS (ESI,

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CP 3.0 kV, SP 30V): m/z calc. = 414 [M]+, m/z found = 414 [M]+. HRMS-ESI-
FTICR (M + 1)+ calcd for C22H25ClN3O3 414.15790 found 414.15864.
4.1.4 General preparation of squaric acid disubstituted derivatives 8a-c
A reaction mixture of the appropriate 4-aminoquinoline (2 eq.) and 3,4-dibutoxy-
3-cyclobutene-1,2-dione (1 eq.) in dry methanol (2 ml for 0.22 mmol of amine)
was heated at reflux for 12 h. The solvent was then removed under reduced
pressure, and the obtained residue recrystallized from MeOH/EtOAc affording
the corresponding product.
4.1.4.1 Compound 8a: Obtained as a light yellow solid.
1
Yield 97%. Mp: 253-255 °C; IR (KBr): 3337, 2957, 15 82, 1533 cm-1. H NMR
(400 MHz, DMSO-d6) δ (ppm): 8.40 (d, J = 5.2 Hz, 2H, HAr), 8.22 (d, J = 9.2 Hz,
2H, HAr), 7.78 (s, 2H, HAr), 7.68 (s br, 2H, NH), 7.47 (s br, 2H, NH), 7.44 (d, J =
9.2 Hz, 2H, HAr), 6.59 (s br, 2H, HAr), 3.76 (s br, 4H, CH2), 3.50–3.37 (m, 4H,
CH2). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 182.64 (C=O), 168.17 (Cq),
151.76 (CHAr), 150.17 (CqAr), 148.81 (CqAr), 133.59 (CqAr), 127.37 (CHAr),
124.29 (CHAr), 124.09 (CHAr), 117.45 (CqAr), 98.95 (CHAr), 43.64 (CH2), 41.77
(CH2). MS (ESI, CP 3.0 kV, SP 30V): m/z calc. = 521 [M]+, m/z found = 521
[M]+. HRMS-ESI-FTICR (M + 1)+ calcd for C26H23Cl2N6O3 521.12541 found
521.12716.
4.1.4.2 Compound 8b: Obtained as a white solid.
1
Yield 77%. Mp: 162-164 °C. IR (KBr): 3246, 2953, 15 82, 1533 cm-1. H NMR
(400 MHz, DMSO-d6) δ (ppm): 8.38 (d, J = 5.4 Hz, 2H, HAr), 8.25 (d, J = 9.2 Hz,
2H, HAr), 7.78 (s, 2H, HAr), 7.45 (d, J = 8.0 Hz, 2H, HAr), 7.30 (s br, 2H, NH),

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6.46 (d, J = 5.4 Hz, 2H, HAr), 3.63 (s br, 4H, CH2), 3.39–3.28 (m, 4H, CH2),
1.98–1.87 (m, 4H, CH2). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 182.47 (C=O),
168.04 (Cq), 151.93 (CHAr), 150.00 (CqAr), 149.07 (CqAr), 133.47 (CqAr), 127.53
(CHAr), 124.17 (CHAr), 124.08 (CHAr), 117.52 (CqAr), 98.71 (CHAr), 41.25 (CH2),
39.51 (CH2), 29.52 (CH2). MS (ESI, CP 3.0 kV, SP 30V): m/z calc. = 549 [M]+,
m/z found = 549 [M]+. HRMS-ESI-FTICR (M + 1)+ calcd for C28H27Cl2N6O3
549.15671 found 549.15740.
4.1.4.3 Compound 8c: Obtained as a white solid.
1
Yield 79%. Mp: 156-158 °C; IR (KBr): 3304, 2940, 16 55, 1605, 1578 cm-1. H
NMR (400 MHz, DMSO-d6) δ (ppm): 8.37 (d, J = 5.0 Hz, 2H, HAr), 8.25 (d, J =
9.2 Hz, 2H, HAr), 7.77 (s, 2H, HAr), 7.43 (d, J = 9.2 Hz, 2H, HAr), 7.33 (s br,
2NH), 6.44 (d, J = 5.0 Hz, 2H, HAr), 3.56 (s br, 4H, CH2), 3.30–3.23 (m, 4H,
CH2), 1.65 (s br, 8H, CH2). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 182.35
(C=O), 167.78 (Cq), 151.92 (CHAr), 150.07 (CqAr), 149.11 (CqAr), 133.42 (CqAr),
127.51 (CHAr), 124.12 (CHAr), 124.06 (CHAr), 117.48 (CqAr), 98.67 (CHAr), 43.06
(CH2), 41.93 (CH2), 28.43 (CH2), 24.74 (CH2). MS (ESI, CP 3.0 kV, SP 30V):
m/z calc. = 577 [M]+, m/z found = 577 [M]+. HRMS-ESI-FTICR (M + 1)+ calcd for
C30H31Cl2N6O3 577.18801 found 577.18913.
4.1.5 Preparation of squaric acid disubstituted derivative 8d
A reaction mixture of the 4-aminoquinoline 5d (115.7 mg, 0.44 mmol, 2 eq.) in
dry methanol (4 ml) and NEt3 (61 µL, 0.44 mmol, 2 eq.) was stirred for 5 min at
room temperature before addition of 3,4-dimetoxy-3-cyclobutene-1,2-dione (48
µL, 0.22 mmol, 1 eq.). After heating at reflux for 48h the solvent was removed

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under reduced pressure. The residue was purified by flash chromatography on
silica gel using as eluent n-hexane/EtOAc (1:1) to EtOAc/MeOH/Et3N (90:9:1).
Compound 8d: Obtained as a white solid.
1
Yield 64%. Mp > 320 °C.IR (KBr): 3441, 2924, 1572 c m-1. H-RMN (400 MHz,
DMSO-d6) ä (ppm): 8.71 (d, J = 4.4 Hz, 2H, HAr), 8.03 (d, J = 8.8 Hz, 2H, HAr),
7.99 (s, 2H, HAr), 7.59 (m, 4H, NH, HAr), 7.06 (d, J = 4.4 Hz, 2H, HAr), 4.08 (s br,
2H, CH), 3.57-3.45 (m, 4H, CH2), 3.07-2.95 (m, 4H, CH2), 2.22-2.09 (m, 4H,
CH2), 1.94-1.80 (m, 4H, CH2). HRMS (ESI) (M + 1)+ calcd for C32H31Cl2N6O2
601.18801 found 601.19037.
4.1.6 Preparation of squaric acid disubstituted derivative 8e
A reaction mixture of BocPheOSu (163 mg, 0.45 mmol) in dry THF (2 mL) and
NEt3 (63 µL, 0.45 mmol) was stirred for 30 min at room temperature under
nitrogen atmosphere. A solution of 4-aminoquinoline 5a (100 mg, 0.45 mmol)
and NEt3 (63 µL, 0.45 mmol) in dry THF (2 mL) was then added and the mixture
was stirred for 40 h at room temperature, under nitrogen atmosphere. The
solvent was removed under reduced pressure; water was added and the
product was extracted with EtOAc (3x). The combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, and the solvent was removed
under reduce pressure. The residue was purified by flash chromatography on
silica gel using as eluent n-hexane/EtOAc (7:3) (180 mg, 85%). A reaction
mixture of the isolated compound (100 mg, 0.21 mmol, 1 eq.) and TFA (64 µL,
0.84 mmol, 4 eq.) in dry DCM (2 ml) was stirred for 2 h at room temperature
under nitrogen atmosphere. The solvent was removed under reduced pressure
and the residue dissolved in dry methanol. NEt3 (59 µL, 0.42 mmol, 2 eq.) was
added and the mixture was stirred for 5 minutes before addition of 3,4-

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dimethoxy-3-cyclobutene-1,2-dione (30 mg, 0.21 mmol). After heating at reflux
for 12h, the solvent was removed under reduced pressure, water was added,
and the product was extracted with EtOAc (3x). The combined organic extracts
were washed with brine and dried over anhydrous Na2SO4. The solvent was
then removed under reduce pressure and the residue purified by flash
chromatography on silica gel using DCM/MeOH (98:2) as eluent.
Compound 8e: Obtained as a light yellow solid.
Yield 32%. Mp: 233- 235 °C. IR (KBr): Cm -1 3357, 3055, 1803, 1672, 1574,
1
1432, 1328. H-RMN (400 MHz, DMSO-d6) δ (ppm): 9.29 (s br, 2H, NH), 8.55–
8.48 (m, 4H, HAr), 8.42 (t br, J = 5.6 Hz, 2H, NH), 7.88 (s, 2H, HAr), 7.72 (d, J =
9.0 Hz, 2H, HAr), 7.25–7.18 (m, 10H, HAr), 7.17–7.10 (m, 2H, NH), 6.88 (d, J =
7.2 Hz, 2H, HAr), 4.52–4.44 (m, 2H, CH), 3.63–3.20 (m, 8H, CH2), 3.09–3.02 (m,
2H, CH2Ph), 2.94–2.85 (m, 2H, CH2Ph). 13C-RMN (100 MHz, DMSO-d6) δ
(ppm): 189.32 (C=O), 181.18 (C=O), 172.55 (Cq), 155.84 (CqAr), 143.98 (CHAr),
139.55 (CqAr), 138.75 (CqAr), 138.10 (CqAr), 129.58 (2 CHAr), 128.52 (2 CHAr),
127.21 (CHAr), 126.60 (CHAr), 126.11 (CHAr), 119.97 (CHAr), 116.11 (CqAr),
98.95 (CHAr), 58.35 (CH), 43.23 (CH2), 38.13 and 37.89 (CH2 and CH2Ph). MS
(ESI, CP 3.0 kV, SP 30V): m/z calc. = 815 [M]+, m/z found = 815 [M]+. HRMS
(ESI) (M+) calcd for C44H41Cl2N8O4 815.26223 found 815.26370.
4.1.7 Preparation of squaric acid monosubstituted derivative 9
A reaction mixture of primaquine diphosphate (402.4 mg, 0.88 mmol, 2eq.) and
NEt3 (245 µL, 1.76 mmol, 4 eq.) in dry methanol (6 mL) was stirred for 30
minutes at room temperature under nitrogen atmosphere before addition of 3,4-
dibutoxy-3-cyclobutene-1,2-dione (96 µL, 0.44 mmol). After heating at reflux for
48h the solvent was removed under reduced pressure; water was added, and

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the product was extracted with ethyl acetate (3x). The combined organic
extracts were washed with brine, dried over anhydrous Na2SO4, and the solvent
was removed under reduced pressure. The residue was purified by flash
chromatography on silica gel using as eluent n-hexane/EtOAc (1:1) to EtOAc
(100%).
Compound 9: Obtained as yellow oil.
Yield 82%. IR (KBr): cm-1 3476, 2961, 1806, 1711, 1693 cm-1. 1.8/1. Major
conformer: 1H-RMN (400 MHz, MeOD) δ (ppm): 8.48–8.45 (m, 1H, HAr), 7.99 (d,
J = 8.2 Hz, 1H, HAr), 7.35–7.30 (m, 1H, HAr), 6.42 (s, 1H, HAr), 6.30–6.24 (m,
1H, HAr), 4.49 (t, J = 6.0 Hz, 2H, OCH2), 3.84 (s, 3H, OCH3), 3.68–3.54 (m, 2H,
CH, CH2), 3.40–3.35 (m, 1H, CH2), 1.78–1.55 (m, 6H, 2 CH2, OCH2CH2), 1.35–
1.23 (m, 5H, CHCH3, CH2CH3), 0.85 (t, J = 7.4 Hz, 3H, CH3). 13C-RMN (100
MHz, MeOD) δ (ppm): 189.74 (C=O), 184.69 (C=O), 177.53 (Cq), 174.60 (Cq),
160.88 (CqAr), 145.99 (CqAr), 145.36 (CHAr), 136.40 (CqAr), 136.35 (CHAr),
131.55 (CqAr), 122.95 (CHAr), 98.49 (CHAr), 93.06 (CHAr), 74.27 (CH2-butoxi),
55.64 (OCH3), 48.64 (CH), 45.32 (CH2), 33.96 (CH2), 32.99 (CH2-butoxi), 28.07
(CH2), 20.80 (CH3), 19.61 (CH2-butoxi), 13.93 (CH3-butoxi). MS (ESI, CP 3.0 kV, SP
30V): m/z calc. = 412 [M]+, m/z found = 412 [M]+. Anal. Calcd for C23H29N3O4
3/4H2O: C, 64.99; H, 7.25; N, 9.89. Found: C, 64.64; H, 7.42; N, 10.21.
4.1.8 Preparation of squaric acid disubstituted derivative 10
A reaction mixture of primaquine diphosphate (402.4 mg, 0.88 mmol, 2eq.) and
NEt3 (368 µL, 2.64 mmol, 6 eq.) in dry methanol (6 mL) was stirred for 30
minutes at room temperature under nitrogen atmosphere before addition of 3,4-
dibutoxy-3-cyclobutene-1,2-dione (96 µL, 0.44 mmol). After heating at reflux for
48h the solvent was removed under reduced pressure; water was added, and

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the product was extracted with EtOAc (3x). The combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, and the solvent was removed
under reduced pressure. The residue was purified by flash chromatography on
silica gel using as eluent n-hexane/EtOAc (1:1) to EtOAc (100%).
Compound 10: Obtained as a yellow solid.
Yield 89% Mp: 81-82 ºC. IR (KBr): cm-1 3385, 2928, 1796, 1578, 1518 cm-1. 1H-
RMN (400 MHz, DMSO-d6) δ (ppm): 8.52 (d, J = 4.0 Hz, 2H, HAr), 8.06 (d, J =
8.2 Hz, 2H, HAr), 7.41 (dd, J = 8.2, 4.0 Hz, 2H, HAr), 7.28 (s br, 2H, NH), 6.46 (s,
2H, HAr), 6.25 (s, 2H, HAr), 6.13 (d, J = 8.8 Hz, 2H, NH), 3.80 (s, 6H, OCH3),
3.63 (s br, 2H, CH), 3.52 (s br, 4H, CH2), 1.72–1.49 (m, 8H, CH2), 1.19 (d, J =
5.9 Hz, 6H, CH3). 13C-RMN (100 MHz, DMSO-d6) δ (ppm): 182.35 (C=O),
167.74 (Cq), 159.02 (CqAr), 144.64 (CqAr), 144.29 (CHAr), 134.85 (CHAr), 134.55
(CqAr), 129.62 (CqAr), 122.16 (CHAr), 96.23 (CHAr), 91.70 (CHAr), 55.03 (OCH3),
46.94 (CH), 43.30 (CH2), 32.89 (CH2), 27.65 (CH2), 20.22 (CH3). MS (ESI, CP
3.0 kV, SP 30V): m/z calc. = 597 [M]+, m/z found = 597 [M]+. HRMS (ESI) (M +
1)+ calcd for C34H41N6O4 597.31838 found 597.32042.
4.2. Biology
4.2.1. Papain assay
Assays were carried out in 200 µL assay buffer (10 mM PBS, pH 7,4, 5 mM
DTT) containing 20 µL of papain activated in assay buffer at 5 µg/mL, and 5
µL of each concentration of tested inhibitors. Reactions were initiated by the
addition of 30 µM fluorogenic substrate (Z-Leu- Leu-Arg-AMC, from Bachem,
Germany) and activity was monitored (excitation 355 nm; emission 460 nm)

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for 30 min, at 37ºC on a Tecan infinite M200 Fluorescence Microplate Reader
(Tecan, Switzerland). The Km of this substrate of papain was previously
determined to be 0.4 µM (data not shown). For all assays, saturated
substrate concentrations were used in order to obtain linear fluorescence
curves. Inhibitor stock solutions and serial dilutions were prepared in DMSO.
Controls were performed using enzyme alone, substrate alone, enzyme with
DMSO and a positive control (E64, Calbiochem, Germany). The IC50 values
were determined by non-linear regression analysis based on the log of
inhibitor concentration versus the percentage of activity using GraphPad
PRISM software. Assays were performed in triplicate [18].
4.2.2.Falcipain-2 assay
The activity of falcipain-2, prepared as previously described [31] was assayed
at 25ºC using 25 µM Z-Leu-Arg-AMC as substrate in 100 mM sodium acetate
pH 6.0, 5 mM DTT, 0.75% DMSO. One microliter of serial dilutions (0.65 nM-
50 µM) of each compound in DMSO were diluted into 100 µL of assay buffer.
Enzyme (1 nM) was added, the mixture was incubated for 10 min, the
reaction was initiated by addition of 50 µL of assay buffer containing the
substrate, and fluorescence was read immediately in a Fluoroskan Ascent
microplate spectrofluorometer. IC50 values were determined by plotting
percentage inhibition relative to controls without inhibitor using GraphPad
Prism 4 (GraphPad Software).

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4.2.3. In vitro antiplasmodial activity
Human red blood cells infected with ~1% parasitemia of ring stage P.
falciparum synchronized with 5% sorbitol were incubated with tested
compounds in 96 well plates at 37°C for 48h in RPMI -1640 medium,
supplemented with 25 mM 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, 91% N2. After 48h the cells were fixed in 2%
HCHO in PBS, transferred into PBS with 100 mM NH4Cl, 0.1% Triton X-100, 1
nM YOYO-1, and then analyzed in a flow cytometer (FACSort, Beckton
Dickinson; EX 488 nm, EM 520 nm) to count infected erythrocytes. IC50s were
calculated using GraphPad PRISM software.
4.2.4. In Vitro Cytotoxicity
Cytotoxicity was assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT), a yellow, water-soluble tetrazolium dye that is
converted by mitochondrial dehydrogenases in viable cells to a water-
insoluble, purple formazan [18]. The day before experiments cells obtained
from the American Type Culture Collection (NIH 3T3 - mouse embryonic
fibroblast cell line (ATCC CRL-1658) or HEK 293T - human embryonic kidney
epithelial cell line, (ATCC CRL-11268) were seeded in 96 well tissue culture
plates, in RPMI 1640 culture medium supplemented with 10% fetal bovine
serum, 100 units of penicillin G (sodium salt), 100 µg of streptomycin sulfate
and 2 mM L-glutamine, at a concentration that allows cells to grow
exponentially during the assay. Compounds to be tested were diluted in

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DMSO, serially diluted in the culture medium and added to the cells. Each
compound concentration was tested in triplicate in a single experiment which
was repeated at least 3 times, .controls contained equivalent concentrations
of DMSO. Cells were incubated at 37°C in humidified 5% CO2 atmosphere.
After 48 hours, cell media was removed and replaced with fresh medium, the
MTT dye solution was added to each well (5mg/mL in 10 mM phosphate
buffer solution at pH 7.4), and after 3h incubations the media was removed
and intracellular formazan crystals were solubilized and extracted with
DMSO. After 15 min at room temperature absorbance was measured at 570
nm in a microplate reader (Infinite M200, Tecan, Austria), and the percentage
of viable cells was determined for each compound concentration as
described previously [32].
IC50s were determined by non-linear regression using GraphPad PRISM
software.
Acknowledgments
This work was supported by Fundação para a Ciência e a Tecnologia (Portugal)
through postdoctoral fellowship to S.P.K. (SFRH/BPD/44481/2008), doctoral
fellowship
to
C.J.A.R.
(SFRH/BD/69258/2010)
and
grant
PEst-
OE/SAU/UI4013/2011. PJR is a Distinguished Clinical Scientist of the Doris
Duke Charitable Foundation.

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