Synthesis, antiplasmodial activity, and β-hematin inhibition of hydroxypyridone-chloroquine hybrids

Article abstract of DOI:10.1021/ml4001084

A series of noncytotoxic 4-aminoquinoline-3-hydroxypyridin-4-one hybrids were synthesized on the basis of a synergistic in vitro combination of a precursor N-alkyl-3-hydroxypyridin-4-one with chloroquine (CQ) and tested in vitro against CQ resistant (K1 and W2) and sensitive (3D7) strains of Plasmodium falciparum. In vitro antiplasmodial activity of the precursors was negated by blocking the chelator moiety via complexation with gallium(III) or benzyl protection. None of the precursors inhibited β-hematin formation. Most hybrids were more potent inhibitors of β-hematin formation than CQ, and a correlation between antiplasmodial activity and inhibition of β-hematin formation was observed. Potent hybrids against K1, 3D7, and W2, respectively, were 8c (0.13, 0.004, and 0.1 μM); 8d (0.08, 0.01, and 0.02 μM); and 7g (0.07, 0.03, and 0.08 μM).

Full text of DOI:10.1021/ml4001084

Letter
pubs.acs.org/acsmedchemlett
Synthesis, Antiplasmodial Activity, and β‑Hematin Inhibition of
Hydroxypyridone−Chloroquine Hybrids
Warren A. Andayi,† Timothy J. Egan,† Jiri Gut,§ Philip J. Rosenthal,§ and Kelly Chibale*,†,‡
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
‡Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
§Department of Medicine, University of California, San Francisco, California 94143, United States
S
* Supporting Information
ABSTRACT: A series of noncytotoxic 4-aminoquinoline-3-hydroxypyr-
idin-4-one hybrids were synthesized on the basis of a synergistic in vitro
combination of a precursor N-alkyl-3-hydroxypyridin-4-one with chlor-
oquine (CQ) and tested in vitro against CQ resistant (K1 and W2) and
sensitive (3D7) strains of Plasmodium falciparum. In vitro antiplasmodial
activity of the precursors was negated by blocking the chelator moiety via
complexation with gallium(III) or benzyl protection. None of the
precursors inhibited β-hematin formation. Most hybrids were more potent
inhibitors of β-hematin formation than CQ, and a correlation between
antiplasmodial activity and inhibition of β-hematin formation was
observed. Potent hybrids against K1, 3D7, and W2, respectively, were 8c (0.13, 0.004, and 0.1 μM); 8d (0.08, 0.01, and 0.02
μM); and 7g (0.07, 0.03, and 0.08 μM).
KEYWORDS: 4-Aminoquinoline, hydroxypyridinone, antiplasmodial, iron chelators
alaria is estimated to affect 40% of the global population,
with 330−660 million clinical cases reported annually.1
parent aminoalkyl side chain, but many CQ analogues are being
synthesized with either shorter or longer alkyl chains to
maintain full activity against resistant strains.9 Other approaches
have included alterations of side chains via incorporation of
various functional groups10−12 and metalation of the quinoline
nitrogen.13−16 However, changes to the aminoquinoline
nucleus alone have been shown to restore activity marginally.
Drugs with new mechanisms of action have also been
explored. This is exemplified by the iron chelators; a group that
works by arresting parasite growth by iron chelation. Examples
of such drugs include deferiprone (DFP) and desferrioxamine
(DFO), which have been evaluated in vitro, in animal models
and in clinical trials.17,18
Another approach to antimalarial drug discovery is molecular
hybridization. This involves the fusion of two drugs, usually via
a covalent linker. The two drugs may be both active or have
pharmacophoric units derived from known bioactive mole-
cules.19,20 The approach can take advantage of good properties
of one drug; for example, the transport mechanism of one
compound can enhance access of the other drug to the active
site. However, one drug may suffer from liabilities of the other
drug. For instance, incorporation of 7-chloroquinoline could
potentially result in cross-resistance of new hybrids with CQ.
The 3-hydroxylpyridin-4-ones are known to chelate iron;21
thus, these compounds may exert optimal iron abrogation to
M
The disease is caused by mosquito-borne eukaryotic protists of
the genus Plasmodium. Five species, namely, P. ovale, P.
malariae, P. vivax, P. Knowlesi, and P. falciparum, have been
associated with the disease in humans.2 Of these, P. falciparum
is the most problematic, mainly due to its high prevalence,
virulence and drug resistance.2 Currently, there are no vaccines
available for malaria, so control relies on chemotherapy and
insect control measures. However, resistance of malaria
parasites to previously widely used drugs such as chloroquine
(CQ) and of mosquitoes to pesticides remain big challenges.
Artemisinin based therapies (ACTs) are the most important
drugs to treat falciparum malaria and have been recommended
as first line treatment against falciparum malaria globally.3−6
However, early signs of resistance to artemisinins, with delayed
parasite clearance in clinical trials in Southeast Asia are of great
concern.7 There is therefore need for the discovery of new
effective medicines, which are safe and efficacious against
resistant P. falciparum. Many approaches including rescue of old
drugs, synthesis of new pharmacophores, and use of the drugs
in combination have been explored in the fight against malaria.
Toward the rescue of old drugs, efforts have been made to
synthesize analogues of proven pharmacophores such as
endoperoxides and 4-aminoquinoline moieties. Some of the
analogues have proven to be better against resistant parasites
than the parent drugs.8 Knowledge of resistance mechanisms
has also aided the discovery of new antimalarials. Groups that
render drugs unable to act against resistant parasites have been
modified. An example is CQ, where resistance is seen with the
Received: March 15, 2013
Accepted: May 20, 2013
© XXXX American Chemical Society
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a
Scheme 1. Syntheses of N-Alkyl-3-hydroxypyridin-4-ones, Their Gallium(III) Complexes and the Hybrids
a
Reagents and conditions: (i) BnCl, MeOH, 92 °C, 22 h; (ii) RNH2, pH 7 (10 M NaOH), 1:1 EtOH/H2O, 105 °C, 18 h; (iii) H2, Pd/C, 4 atm, 1:9
H2O/EtOH (aq), 4 h; (iv) Ga(NO3)3·9H2O, pH 8 (0.1 M NaOH), 80 °C, 4 h; (v) Me2SO4, (if pyranone R2 = Me) or MeI (if pyranone R2 = Et),
2.5 M KOH, acetone, rt, 12 h; (vi) neat, N2, 80 °C 1 h, then 130 °C, 3 h; (vii) 50% aq EtOH, pH 13 (2 M NaOH), 90−120 °C, 18−24 h; (viii) H2,
Pd/C, 2 M ethanolic HCl, 4 atm or 1:2:3 H2O/EtOH/HCl, 74 °C, 24 h (Note: compd structural details can be found in Table 1).
limit parasite proliferation if it can access the parasitic digestive
vacuole using the CQ transport.
inhibited β-hematin formation up to a concentration of 10 IC50
equivalents of the test compounds. Antiplasmodial activity of
the combinations of 2d or 3d with CQ or dihydroartemisinin
was studied using the isobologram method.26 2d-CQ
combinations were predominantly synergistic in both K1 and
3D7, and this justified the synthesis of the hybrids. In contrast,
the 3d-CQ combinations were predominantly antagonistic.26 In
these tests, up to a maximum concentration of 0.05 μM of 2d
was used.
The hybrids proved to be potent β-hematin inhibitors unlike
the N-alkyl-3-hydroxypyridin-4-ones (Table 1). Overall the β-
hematin inhibition activity of all the hybrids was superior to
that of CQ except for 7e and was in the range of 0.01−1.71
IC50 equiv. For both the benzylated and deprotected analogues,
β-hematin inhibition activity improved with increased lip-
ophilicity or longer alkyl linker. However, no significant
difference was observed between compounds with alkyl linkers
of 4 carbons or 6 carbons, e.g., 8c versus 8d.
For the most part, deprotection was observed to cause a
decrease in β-hematin inhibition activity as was the replacement
of the 3-benzyl group with a methoxy. This implied the
involvement of the 3-benzyl group or the group at the C-3
position in the inhibition of β-hematin formation.
Strong correlations were observed between β-hematin
inhibition and antiplasmodial activity in 3D7 (R2 = 0.96) and
W2 (R2 = 0.93) for the benzylated analogues.26 At first sight,
the correlation also appears to be strong in 3D7 (R2 = 0.92)
and W2 (R2 = 0.95) for the deprotected analogues. However, if
one data point (2.9, 10.8) is omitted in the determination of
the correlation for deprotected analogues in 3D7, the R2 value
drops to 0.045, strongly suggesting that the correlation is
fortuitous. Despite this, the other significant correlations
observed suggest that β-hematin inhibition is a major mode
of action of these conjugates against both resistant and sensitive
The aims of this study were to synthesize 4-aminoquinoline-
3-hydroxypyridin-4-one hybrid analogues that have activity
against CQ resistant P. falciparum, to study the mechanism of
action of these compounds with respect to inhibition of β-
hematin formation and to characterize these compounds with
respect to cytotoxicity. The hybridization was justified by the
synergistic antiplasmodial effect of combining N-alkyl-3-
hydroxypyridin-4-ones with chloroquine, as observed in the
preliminary studies (Supporting Information).
Documented procedures with some modifications were
followed in the synthesis of 3-hydroxypyridin-4-ones [3,4-
HPOs] (2a−2d; 3a −3d; 5a; and 5b),21N-(7-chloro-4-
quinolinyl)-diaminoalkanes (6a−6d),22 and one of the gallium-
(III) complexes (4)23 (Scheme 1 and Table 1). The Michael
addition of the N-(7-chloro-4-quinolinyl)-diaminoalkanes to
the benzylated pyranones yielded the benzyl-protected hybrids
as free bases (Scheme 1). The benzylated hybrid molecules
were deprotected by catalytic hydrogenolysis at high pressure
or by acid hydrolysis. N-(7-Chloro-4-quinolinyl)-1-(amino-
alkyl)-3-(methoxy)-2-alkyl-4-(1H)-pyridinones [3-methoxy-
lated hybrids] were prepared by reacting 3-methoxylated
pyranones 5a or 5b with N-(7-chloro-4-quinolyl) alkanes
(Scheme 1). Antiplasmodial activity, β-hematin inhibition, and
cytotoxicity of the prepared compounds were evaluated using
standard bioassays,24−26 and the results are shown in Table 1.
The activities of the most potent N-alkyl-3-hydroxypyridin-4-
one 2d were IC50 = 2.38 and 1.8 μM against sensitive strain
3D7 and resistant strain K1 of P. falciparum, respectively
(Supporting Information). In vitro antiplasmodial activity of
these precursors was negated by blocking the chelator moiety
via complexation with gallium(III) or protection by a benzyl
group (Supporting Information). None of these compounds
B
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a
Table 1. Antiplasmodial Activity, Resistance Indices, β-Hematin Inhibition, and Cytotoxicity Data for the Hybrids
a
b
c
SE ≤ 7%, n = 3 IC50 (μM) antiplasmodial activity against P. falciparum. IC50 represent the molar equivalents of test compound relative to β-
hematin required to inhibit β-hematin formation by 50%; A = controls; B = benzylated analogues, C = deprotected analogues, D = methoxy
analogues, and ND = not determined.
strains, at least for the protected analogues. Positive association
between β-hematin inhibition and antiplasmodial activity was
also observed for the W2 strain. Deprotection caused a decline
in β-hematin inhibition but did not translate into decline in
antiplasmodial activity in all compounds. This could be an
indication that other mechanisms may be playing a role in
maintaining antiplasmodial potency.
Generally, deprotection was found to lower the resistance
indices of most of the compounds.26 Thus, the chelation or the
free chelator moiety in these hybrid molecules could be
responsible for enhancing the potency of the hybrids against
resistant strains. However, data from the N-alkyl-3-hydroxypyr-
idin-4-ones indicate that iron chelation by itself is not enough
to cause significant antiplasmodial activity, as demonstrated by
the poor activity of the precursor compounds.26 It may be
hypothesized that the antiplasmodial activity of the precursor
was enhanced in hybrid molecules due to accumulation in the
digestive vacuole mediated by the CQ moiety.
Cytotoxicity studies in the mammalian cell line KB revealed
that most of the hybrids (11 out of 16) tested were not
cytotoxic as evidenced by IC50 > 10 μg/mL (Table 1). These
hybrid compounds had a toxicity profile comparable to or
better than that of CQ, and they were far less toxic than the
standard podophyllotoxin (POD). However, there is a
possibility that solubility may have precluded detecting
cytotoxicity of a few of the compounds whose solubility was
C
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<10 μg/mL. For the majority compounds (12 out of 16), the
cytotoxicity IC50 values were ≥10 μg/mL implying that they
were soluble throughout the assay conditions. Greater selective
indices (Supporting Information) were observed in the 3D7
strain compared to the KB line, with the exception of 8c. None
of the compounds had better selective indices than CQ,
although compounds 7c, 7g, 8c, 8g, and 8d·2HCl were
comparable to CQ.
This study confirms that the antiplasmodial activity of the
synthesized hybrid molecules depends largely on β-hematin
inhibition. However, the iron chelator group has potential to
enhance activity against resistant strains of P. falciparum for
these types of compounds. Conjugation with 3,4-HPOs seems
to lower the cytotoxicity of CQ at least in vitro. Currently, we
are investigating the in vitro and in vivo ADME/PK properties
of these hybrids alongside in vivo efficacy.
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and analytical data for some
compounds including 2d, 3d, 8c, 8d, and 7g and a table
showing antiplasmodial activity of N-alkyl-3-hydroxypyridin-4-
ones and a gallium(III) complex. This material is available free
of charge via the Internet at http://pubs.acs.org.
■
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in anti-malarial drug discovery and development. Curr. Top. Med.
Chem. 2011, 11, 1226−1254.
(12) Wells, T. N. C.; Alonso, P. L.; Gutteridge, W. E. New medicines
to improve control and contribute to the eradication of malaria. Nat.
Rev. Drug. Discovery 2009, 8, 879−891.
AUTHOR INFORMATION
Corresponding Author
*(K.C.) Tel: +27 21 650 2553. Fax: +27 21 650 5195. E-mail:
kelly.chibale@uct.ac.za.
(13) Navarro, M. Gold complexes as potential anti-parasitic agents.
Coord. Chem. Rev. 2009, 253, 1619−1626.
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Transition Met. Chem. 2008, 33, 493−497.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. All the authors contributed equally to this
manuscript.
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Hider, R. The effect of N-alkyl modification on the antimalarial activity
of 3-hydroxypyridin-4-one oral iron chelators. Blood 1991, 77, 637−
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Funding
T.J.E. is grateful for the funding from National Research
Foundation (NRF) and the Medical Research Council (MRC)
of South Africa. K.C. gratefully acknowledges funding from the
MRC of South Africa, University of Cape Town, and South
African Research Chairs Initiative of the Department of Science
and Technology administered through the NRF.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Susan Little of the London School of Hygiene and Tropical
Medicine is acknowledged for performing the antiplasmodial
tests against the K1 and 3D7 strains of P. falciparum as well as
cytotoxicity assays.
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(26) See the Supporting Information for full details.
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