Antiplasmodial activities of 4-aminoquinoline-statine compounds

Article abstract of DOI:10.1016/j.bmcl.2012.07.069

We report the discovery of new potent inhibitors of the growth of Plasmodium falciparum chloroquine (CQ)-resistant W2 strain. These compounds were designed using the double drug approach by introducing a residue able to enhance the accumulation of plasmep

Full text of DOI:10.1016/j.bmcl.2012.07.069

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5915–5918
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiplasmodial activities of 4-aminoquinoline–statine compounds
Nadia Vaiana a, Melissa Marzahn b, Silvia Parapini c, Peng Liu b, Mario Dell’Agli c, Andrea Pancotti a,
Enrico Sangiovanni c, Nicoletta Basilico d, Enrica Bosisio c, Ben M. Dunn b, Donatella Taramelli c,
Sergio Romeo a,
⇑
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy
b Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA
c Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy
d Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Via Pascal 36, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery of new potent inhibitors of the growth of Plasmodium falciparum chloroquine
(CQ)-resistant W2 strain. These compounds were designed using the double drug approach by introduc-
ing a residue able to enhance the accumulation of plasmepsins inhibitors into the food vacuole. Some of
the molecules were more active than CQ against CQ-resistant strain and showed good selectivity against
cathepsin D.
Received 7 June 2012
Revised 16 July 2012
Accepted 18 July 2012
Available online 27 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Double drugs
Plasmodium falciparum
Aminoquinolines
Plasmepsin inhibitors
Antimalarials
Malaria is one of the major health problems worldwide, com-
mon in the tropics, especially south of Sahara. In 2010 malaria
caused about 216 million infections and it was responsible for
approximately 655,000 deaths and about 86% of these occurred
in children under 5 years of age.1 Five parasite species are respon-
sible for human malaria: Plasmodium vivax (Pv), Plasmodium ovale
(Po), Plasmodium malariae (Pm), Plasmodium falciparum (Pf) and
more recently Plasmodium knowlesi (Pk).2 Pf infections, if not trea-
ted in a timely fashion and with effective drugs, progress to severe
forms of disease that are frequently fatal. From the WHO World
Malaria Report 2011, Pf infection is one of the leading causes of
deaths worldwide from a single infectious agent.1 Due to the rapid
spread of parasite resistance to available antimalarial drugs there
is an urgent need for new drugs with novel mechanisms of action.1
Vacuolar plasmepsins are a family of plasmodial aspartic prote-
ases comprising plasmepsin 1 (PLM 1), plasmepsin 2 (PLM 2),
plasmepsins 4 (PLM 4) and a histo-aspartic protease (HAP). These
enzymes are localized in the food vacuole of Pf and are involved
in the degradation of hemoglobin during the erythrocytic stage
of the parasite.3 Vacuolar PLMs are considered promising targets
for the development of new antimalarial compounds.3 However,
PLMs inhibitors, including molecules with a statine-based core,
have shown potent enzyme inhibition, but limited efficacy in kill-
ing the parasite (IC50 range 2–20 l
M).3 The current view is that the
drug discovery efforts focused on vacuolar plasmepsins should
consider the development of compounds that can inhibit two or
more of this enzyme family.4,5
During the hemoglobin degradation process, the heme group is
detoxified by crystallization into hemozoin. 4-Aminoquinolines
(Chloroquine (CQ), Amodiaquine, Tebuquine) have been histori-
cally among the most popular and effective antimalarials acting
by inhibition of hemozoin formation. In vitro antagonism has been
demonstrated by the combination of a protease inhibitor with a
4-aminoquinoline, since both molecules interfere with the hemo-
globin catabolism process.6 It is also known that the antiplasmo-
dial activity of the 4-aminoquinolines is increased by their
selective accumulation into the acidic parasite food vacuole thanks
to their peculiar basic character.7
In our previous studies,8–11 we synthesized a series of PLM(s)
inhibitors based on the double drug approach12 that combines
two different moieties, acting against different targets, into a single
chemical entity. The synthesized compounds showed a remarkable
improvement of inhibition of Pf growth with good selectivity
towards cathepsin and low toxicity against human dermal fibro-
blasts. In particular, compound 1, (Fig. 1) presented relevant activ-
ities against CQ-sensitive (D10) and CQ-resistant (W2) Pf strains
⇑
Corresponding author. Tel.: +39 02 503 19363; fax: +39 02 503 19364.
E-mail address: sergio.romeo@unimi.it (S. Romeo).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.069

5916
N. Vaiana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5915–5918
O
O
O
OH O
+
H
N
NHC4H9
BnOOC
COOH
N
H
X
N
H
NHC4H9
HCl Ile
O
8
a
9
OH
O
O
O
O
X
H
N
NHC4H9
OMe
BnOOC
N
H
O
OH O
6
1
N
NH
N
b
LeuLysHN
HN
N
Cl
N
Cl
a
c
2
H2N
11
NH
Cl
10
2
3
4
N
H
6
d
Cl
b
N
Cl
N
N
e
b
a
3
Ala N
H
N
H
BocAlaHN
NH
e, f
Cl
12
6
b
LeuAla N
N
H
Cl
N
H
g
4
Figure 1. Structures of reference compound
compounds 2–4.
1 and 4-aminoquinoline–statine
CbzLeuAlaHN
NH
7
Scheme 1. Reagents and conditions: (a) HBTU,HOBt, NMM; (b) H2, Pd/C; (c)
Diaminopropane, 140 °C; (d) BocAlaOH, HBTU, HOBt; (e) HCl 4N, dioxane; (f)
CbzLeuOH, HBTU, HOBt; (g) HATU.
(IC50 400–700 nM) and displayed low cytoxicity against human
dermal fibroblasts (50% @ 100
M).8
l
To further improve the antiplasmodial activities of our inhibi-
tors, we reasoned that the introduction of the 7-chloro-4-amino-
quinoline ring system derived from CQ in the statine double drugs
could improve the accumulation of plasmepsins inhibitors into
the food vacuole. Compounds 2–4 (Fig. 1) were designed and syn-
thesized to define the effects of CQ on the double drugs. Compound
2 shows the aminoquinoline ring system bound to the oxybisbenzo-
ic acid through an aminoalkyl spacer analogue to CQ, while com-
pound 3 presents an additional alanine spacer between the two
molecules. This substitution was inspired by our previous work on
primaquine-statine double drugs where a 10-fold improvement in
antiplasmodial activity was observed by introducing a spacer com-
posed of one or two amino acids between primaquine and the aro-
matic acid.9 Compound 4 was designed considering that the
chlorine atom in position 7 is essential for inhibition of b-hematin
formation, but its removal improves vacuolar accumulation.13 In or-
der to further explore the contribution of PLMs inhibition to the
antiplasmodial activity of the double drugs, compounds 5, 6 and 7
were synthesized (Scheme 1 and Scheme 2). In these compounds
the quinolinic ring system has been replaced with a benzylic amide
(5) or a benzylic ester (6) and the statine portion of the double drug
has been removed (7).
O
O
a
BnOOC
COOH BnOOC
CONHBn
13
8
b, c
O
O
H
N
O
NHC4H9
BnHNOC
N
H
OH
O
5
Scheme 2. Reagents and conditions: (a) Benzylamine, HBTU; (b) H2, Pd/C; (c) 9,
NMM, HOBt, HBTU.
followed by coupling with CbzLeuOH (Scheme 1). Finally com-
pound 5 (Scheme 2) was prepared by coupling monobenzylester
8 with benzylamine followed by removal of the benzyl ester and
reaction with IleStatine hydrochloride 9. All synthesized com-
pounds gave satisfactory analytical (purity P95% by HPLC) and
spectroscopic data. 1H NMR and HRMS spectra were consistent
with the assigned structures.
Compounds 2–7 were prepared according to Schemes 1 and 2.
Compound 2 was obtained by coupling IleStatine hydrochloride
99 with the monobenzylester of 4,40-oxybisbenzoic acic 8,9 fol-
lowed by coupling the deprotected benzyl ester 6 with amine 11,
obtained by the nucleophilic attack of 1,3-diaminopropane to
4,7-dichloroquinoline 10 (Scheme 1). Compound 3 was obtained
by coupling deprotected ester 6 with deprotected compound 12.
Compound 12 was obtained by coupling compound 11 with Boc–
Ala. Compound 4 was obtained by coupling deprotected dipeptide
7 with deprotected ester 6 using HATU as coupling reagent. As
expected during catalytic hydrogenation of compound 7 the chlo-
rine atom in position 7 was removed (Scheme 1).14 Compound 7
was obtained by removal of the Boc group from compound 12
Compounds 2–7, have been tested in vitro against recombinant
Pf PLM 2 and Pf, Po, Pv and Pm PLMs 4; and against D10 (CQ sensi-
tive) and W2 (CQ resistant) Pf strains (see Supplementary data).
9
The selectivity versus human cathepsin D, which shares a high
homology with PLMs, was also investigated for the most promising
compounds (Table 1).
As expected, compounds 2–6 containing the b-hydroxyamino
acid statine inhibited all PLMs at nanomolar concentrations, while
CQ and the peptidic derivative of CQ 7 were completely inactive.
Considering PLM 2, the Ki values of compounds 2–6 are comprised
in a small range (1.3 nM–3.8 nM), while for Pf PLM 4 the inhibition
constants are distributed in a greater range (2.6 nM–20 nM).
Among orthologous PLM 4, Pm appears to be less susceptible to

N. Vaiana et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5915–5918
5917
Table 1
Effect of compounds 1–7 and CQ on PLMs activities and on growth of CQ-sensitive (D10) and CQ-resistant (W2) P. falciparum strains
b
c
Compd
Kib (nM) PfPLM2
Kib (nM) PfPLM4
Kib (nM) PmPLM4
Kib (nM) PoPLM4
Kib (nM) PvPLM4
IC50 (nM) hCatD
IC50 (nM) D10
IC50(nM)c W2
1a
2
3
4
5
6
7
CQ
0.4 0.1
2.3 0.2
3.8 0.3
2.6 0.2
1.3 0.2
1.9 0.3
Inactive
Inactive
0.5 0.1
5.4 0.5
1.2 0.1
0.93 0.05
2.4 0.2
0.87 0.04
5.8 0.6
2.1 0.1
n.t.
10 1.4
1.2 0.2
n.t.
400
177
114
161
>7000
3850
197
23
700
78
64
474
>7000
4680
1675
412
11
1
0.70 0.06
0.48 0.05
0.43 0.04
220 10
540 60
300 30
n.t.
n.t.
n.t.
8.7
6.7 0.8
20
1
8.5 0.6
8.1 0.9
1.1 0.06
0.34 0.03
n.t.
2
19
2
2.6 0.1
n.t.
n.t.
4.7 0.4
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.: not tested.
a
Reference compound.9
Results are the mean S.D. of three experiments performed in triplicate.
Results are the mean of two experiments performed in duplicate.
b
c
the structural modifications, and the benzylamide 5 was the less
active compound. In the case of Po PLM 4, the quinolinic deriva-
tives 2–4 are less active than the benzylic derivatives 5–6, while
the opposite effect was observed against Pv PLM 4. In addition,
compound 6 containing the ester function was more potent than
the amidic analogue 5 in inhibiting PLMs 4 from all the strains
tested.
The introduction of one amino acid (compound 3) or a dipeptide
(compound 4) in compound 2 did not alter significantly the activity
against all the PLMs.
Inhibition of human cathepsin D, a high human homologue of
PLMs, has been determined for compounds 2–4 and no inhibition
was observed at the concentrations required to inhibit PLMs (Table
1), indicating a good selectivity for the plasmodial enzymes.
Compounds 2–4 showed different parasite inhibition profiles
against D10, CQ sensitive and W2, CQ resistant strains. The IC50
values against the D10 strain are significantly lower than that of
the reference compound 1 (400 nM), thus possibly reflecting the
increased accumulation of the PLMs inhibitors in the food vacuole
due to the presence of the 4-aminoquinoline portion. The effect of
compounds 2–4 on W2 strain was improved with respect to com-
inhibition of b-hematin formation using the BHIA method.16 CQ
was used as control, and as expected, compound 4 yielded consid-
erably less activity than CQ or compound 3 (Table 2).
We next planned to verify the effect of the quinolinic system on
the antiplasmodial activity of PLMs inhibitors. When tested on Pf
D10, CQ sensitive strain, compounds 5 and 6 were found to be inac-
tive although they were highly potent against PLMs. As expected,
the introduction of a quinolinic ring system led to the recovery
of antiplasmodial activity (compound 4). However, compound 4
not having the chlorine atom in position 7 is not able to inhibit
b-hematin formation, thus the improvement in activity can be as-
cribed to an enhanced accumulation into the food vacuole. The
introduction of the chlorine atom in position 7 (2 and 3) did not
lead to an increase in the antiplasmodial activities, as the amidic
analogue of CQ (7).
In this work, we obtained new potent Pf inhibitors using the
double drug approach by introducing a residue able to enhance
the accumulation of PLMs inhibitors into the food vacuole. Some
of the molecules were more active than CQ against CQ resistant
strain and showed good selectivity against cathepsin D. These
derivatives represent therefore new leads that will be further
explored.
pound 1, however
a significative difference in activity was
observed between 2–3 and 4, thus implying a direct effect of the
7-chloro-4-aminoquinoline system on the antiplasmodial activity
of these compounds. The low efficacy of 4 in inhibiting the W2
growth could be explained by the lack of chlorine in position 7,
which is essential for the inhibition of b-hematin formation as con-
firmed by the BHIA test (Table 2).
Interestingly, compounds 2 and 3 were more active than CQ
against W2 (CQ-R) strain; these data are consistent with Moura
et al. which report the resistance to CQ is attenuated in parasites
lacking vacuolar plasmepsins.15
Compounds 5 and 6 deprived of the quinolinic residue showed
negligible activities against all strains. Compound 7, deprived of
statine and therefore inactive against plasmepsins, showed a sharp
decrease in activity against the W2 strain, while retaining inhibi-
tion of the D10 strain.
The inhibitory activities of compound 3, the most potent Pf
growth inhibitor of this series, and compound 4, the analogue
lacking the chlorine substituent in position 7, were tested for
Acknowledgments
This work was supported by MIUR (PRIN2008 20084MMXN:
Leads ad Attività Antimalarica di Origine Naturale: Isolamento,
Ottimizzazione e Valutazione Biologica), and by the AntiMal pro-
ject, funded under the sixth Framework Programme of the Euro-
pean Community (Contract No. IP-018834). The authors are
solely responsible for its content, which does not represent the
opinion of the European Community and the Community is not
responsible for any use that might be made of the information con-
tained herein.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.07.
069. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Table 2
b-Hematin inhibitory assay for derivatives 3, 4 and CQ.a
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Compd
BHIA
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