Potent Antimalarial Activity of Two Arenes Linked with Triamine Designed to Have Multiple Interactions with Heme

Article abstract of DOI:10.1021/acsmedchemlett.8b00222

Based on the idea that compounds designed to exhibit high affinity for heme would block hemozoin formation, a critical heme-detoxification process for malarial parasites, we synthesized a series of compounds with two π-conjugated moieties at terminal amino groups of triamine. These compounds exhibited moderate to high antimalarial activities in vitro toward both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. In a P. berghei-infected mouse model, 3a and 12a showed potent antimalarial activities compared to artesunate, as well as a prolonged duration of antimalarial effect. We found a good correlation between protective activity against hemin degradation and antimalarial activity. Compounds 8b and 3a strongly inhibited hemozoin formation catalyzed by heme detoxification protein.

Full text of DOI:10.1021/acsmedchemlett.8b00222

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Letter
Potent Antimalarial Activity of Two Arenes linked with
Triamine, Designed to Have Multiple Interactions with Heme
Yosuke Sakata, Kosuke Yabunaka, Yuko Kobayashi, Hirohisa Omiya, Naoki Umezawa,
Hye-Sook Kim, Yusuke Wataya, Yoshimi Tomita, Yosuke Hisamatsu, Nobuki Kato,
Hirokazu Yagi, Tadashi Satoh, Koichi Kato, Haruto Ishikawa, and Tsunehiko Higuchi
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00222 • Publication Date (Web): 24 Sep 2018
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Potent Antimalarial Activity of Two Arenes linked with Triamine, De-
signed to Have Multiple Interactions with Heme.
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Yosuke Sakata,† Kosuke Yabunaka,† Yuko Kobayashi,† Hirohisa Omiya,† Naoki Umezawa,† Hye-Sook
Kim,‡ Yusuke Wataya,‡ Yoshimi Tomita,† Yosuke Hisamatsu,† Nobuki Kato,† Hirokazu, Yagi,† Tadashi
Satoh,† Koichi Kato,†¶ Haruto Ishikawa,§ Tsunehiko Higuchi*,†
†Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603,
JAPAN. ‡Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-Naka Kita-ku, Okayama 700-8530,
¶
JAPAN. Exploratory Research Center on Life and Living Systems and Institute for Molecular Science, National Institutes of
Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. §Department of Chemistry, Graduate School of
Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, JAPAN
KEYWORDS: Antimalarial, heme, hemozoin, molecular recognition, heme detoxification protein.
ABSTRACT: Based on the idea that compounds designed to exhibit high affinity for heme would block hemozoin formation, a
critical heme-detoxification process for malarial parasites, we synthesized a series of compounds with two -conjugated moieties at
terminal amino groups of triamine. These compounds exhibited moderate to high antimalarial activities towards both chloroquine-
sensitive and chloroquine-resistant Plasmodium falciparum in vitro. In a P. berghei-infected mouse model, 3a and 12a showed
potent antimalarial activities compared to artesunate, as well as a prolonged duration of antimalarial effect. We found a good corre-
lation between protective activity against hemin degradation and antimalarial activity. Compounds 8b and 3a strongly inhibited
hemozoin
formation
catalyzed
by
heme
detoxification
protein.
Malaria is a serious and sometimes fatal disease caused by
several species of parasite of the genus Plasmodium that are
transmitted by mosquitos of the Anopheles genus.1 About 200
million people develop malaria and almost 429,000 people die
every year.1 Relapse after treatment often occurs, and moreo-
ver, resistance to chloroquine, formerly a gold-standard anti-
malarial drug, has become epidemic.1 Global warming is ex-
pected to expand the range of countries at risk. Therefore,
development of new drugs effective against resistant Plasmo-
dium falciparum is important.
stacking forces; in addition, there would be an electrostatic
interaction between the ammonium group of the drug and the
carboxylate group of the heme.2 However, known antimalarial
drugs have not been specifically designed with this mechanism
in mind. Here, we designed new quinolinic compounds in-
tended to interact strongly with heme.
The synthesized compounds indeed showed potent antimalari-
al activity towards both a chloroquine-sensitive and a resistant
P.
falciparum
in
vitro.
The malaria parasite must escape the toxicity of free heme
when it lives within the erythrocyte during its blood stage and
digests hemoglobin, which contain large amounts of heme in
the active site of hemoglobin.2 To achieve this, heme mole-
cules are bound to each other under acidic conditions in the
parasites’ vacuoles to form a heme assembly named hemozoin
(Hz), which is harmless to the parasite, but harmful to humans.
This Hz formation is considered to proceed via hemin dimeri-
zation (Figure 1: hemin dimer) mediated by proteins such as
heme detoxification protein (HDP)3-5 and also via a self-
assembly process (Figure 1),2, 6 although the parasite factors
contributing to Hz formation remain a subject of debate.
Quinoline-containing antimalarial drugs such as chloroquine
are considered to block the fastest growing face of the Hz
crystal by interacting with the surface or with free heme.2, 7-9
The quinoline ring would interact with heme through −
Figure 1. Hemozoin (Hz) formation, and molecular design of
inhibitors of hemin self-assembly as candidate antimalarial
agents.
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takes ammonium form in the vacuoles of malaria parasites (pH
~5) and should interact strongly with carboxylates of hemin
through Coulombic force.
-Electron-conjugated planar molecules interact with heme
via − stacking, so molecules with two planar moieties
should have greater affinity for heme than those with just one.
As shown in Figure 1, we designed compounds with two con-
densed heteroaromatic rings conjugated with terminal amino
groups of Nn+3-methyl[1, n+3, 2n+5]triazaheptane (n=1: 1a) or
triazanonane (n=2: 1b). The compounds bearing two planar
scaffolds are expected to interact with heme by pinching (Fig-
ure 1). Some symmetric bis-quinoline-type compounds have
already been reported to have relatively potent antimalarial
activity, including towards chloroquine-resistant strains.10-21
However, compounds designed to form simultaneous −
interactions with two bicyclic aromatics as well as electrostatic
interaction with heme in a host-guest manner have not been
reported, although several compounds were designed consid-
ering − interaction with heme.16, 22-24 Also, reported mole-
cules bearing two planar scaffolds are mostly symmetric,
probably for reasons of synthetic convenience.25 We synthe-
sized various molecules, including asymmetric ones, based on
the molecular design described above. Here we report that
these compounds bearing two planar scaffolds show potent
antimalarial activity in vitro and in vivo, and also exhibit pro-
tective activity against hemin degradation and HDP-inhibitory
activity.
We prepared 4-(7-chloroquinolyl)-bearing triamines 2a and 2b
as common intermediates (Scheme 1a) by the reaction of tri-
amines 1a or 1b with 4,7-dichloroquinoline. Symmetric com-
pounds 3a, 3b, 4a and 5a with two planar moieties were simp-
ly prepared as shown in Scheme 1b (Ar series). Compound 3a
was originally developed by us as an antimalarial agent,26 and
was recently reported to be a potent inhibitor of autophagy.27
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Next, two series of compounds having asymmetric structure
were prepared (Schemes 1c, 1d and 1e). Only a few asymmet-
ric quinolinic compounds have previously been reported.28-31
We mainly adopted 7-chloroquinoline-attached triamines 2a
and 2b as common structures. The primary amino group on 2
was directly conjugated with chlorinated Ar to afford com-
pounds 6 – 10 (Schemes 1c). Carboxylic acid-bearing -
conjugates planar molecules were condensed with compound
2a or 2b to afford 11 - 13 having an amide bond (Schemes 1d
and 1e).
All the compounds having two -conjugated planes (3-13)
were evaluated in vitro for antimalarial activity against the K1
(chloroquine-resistant) and FCR-3 (chloroquine-sensitive)
strains of P. falciparum according to the procedure reported in
the literature, and the data are presented in Table 1 (triamine-
type compounds and -acyltriamine-type compounds). Com-
pounds not having a 7-chloroquinolin-4-yl group generally
showed almost no antimalarial activity (4a and 5a). These
results indicate the importance of the 7-chloroquinolin-4-yl
group for the activity. Compounds with a monocyclic aromatic
group, 6 and 7, showed poor antimalarial activities towards
both strains. On the other hand, most of the compounds having
a bicyclic aromatic group (3, 8, 9, 12) exhibited much higher
activity than the monocyclic compounds. Especially, 3a, 3b,
Nor-3a, 8b, 12a and 12b showed potent activity against both
strains. It is noteworthy that the 6 compounds were about 10
times more effective against the chloroquine-resistant strain
(K1) than chloroquine itself. There was little difference in
inhibitory activity or toxicity between 3a and Nor-3a. Com-
pound 8b showed potent activity against FCR-3 strain (EC50:
0.92 nM), although its structure resembles that of 3b (EC50: 97
nM). The toxicity of the compounds toward human cells was
also evaluated using MRC-5 cells (human embryonic lung-
derived fibroblast cells). In general, triazaheptane-type com-
pounds (n=1) were less toxic than triazanonane-type ones
(n=2). Compound 12a showed low toxicity to human cells and
its selective toxicity (for K1 versus MRC-5) was the highest
among the compounds tested. We prepared analog of 12a for
structure optimization and evaluated their antimalarial activity
(13) (Table 1). However, the activity and selectivity did not
exceed those of 12a.
Scheme 1. Synthetic scheme of two planar scaffolds linked
with triamines (3-10) and -acyltriamine-type compounds 11-
13
Table 1. In vitro Antimalarial activity and cytotoxicity of tri-
amine-type compounds 3-10 and -acyltriamine-type com-
pounds 11-13
Triamines 1a and 1b (Scheme 1a) having two primary amino
groups and one tertiary amino group were adopted as basic
skeletons and two planar moieties were attached to the prima-
ry amino groups of the triamines. The tertiary amino group
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roquine seems to be better than those of 3a and 12a.32 However,
3a and 12a would be superior to chloroquine against chloroquine-
resistant strains (the P. berghei strain is chloroquine-sensitive).
Table 2. In vivo antimalarial activity of 3a, 12a and ar-
tesunate on P. berghei-infected mice
IC50
(nM)
K1
IC50
(nM)
Cytotoxi-
city MRC-
5 (nM)
Selectivity
Index
Dosage
Comp.
Inhibition (%) Survival (%)
(mg/kg), route
30 x 4, s.c.
30 x 4, p.o.
30 x 4, s.c.
30 x 4, p.o.
15 x 4, p.o.
Comp.
Ar1 Ar2
n
FCR-3
MRC-5/K1
10
11
12
13
14
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19
20
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22
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3a
3a
100
99.5
100
23.5
95.2
>284.6*
115.4
223.1
100
3a
Nor-3a
3b
CQ
CQ
CQ
Q
CQ 1
CQ 1
CQ 2
Q 1
45
20
8800
14000
1400
200
320
66
44
21
37
97
12a
12a
4a
18000
19000
21000
17300
310000
90000
17
artesunate
102.6
5a
BzI BzIm 2
m
4.7
>284.6*:one mice of 3a treatment group was still survive
without malaria parasites on day 35.
6
7
CQ
Py 1
1100
750
510
210
29
470000
30000
9500
430
40
We also examined the duration of the pharmacological effect of
the test compounds by observing prolongation of life of mice after
the end of administration (Table 2, Figure S1). Mean of survival
days in control group were 7.8 days and artesunate-, 3a-, and 12a-
administered group (p.o. treatment) were dead within 9 days. On
the other hand, administration of 3a or 12a (s.c. treatment) pro-
longed the lives of the mice by 15-35 days. Especially, subcutane-
ously inject in to mice of 3a, one mice of them was survive with-
out malaria parasite in the mice. This result indicates that 3a and
12a are superior to artesunate not only in pharmacological activity
but also in the duration of antimalarial effect. Compounds 3a and
12a are thus considered promising lead compounds for drugs to
prevent malarial relapse.
CQ Pym 1
8a
8b
9a
10a
11a
CQ
CQ
CQ
Q 1
Q 2
370
36
37
0.92
24
2200
61
IQ 1
270
9500
36
CQ BzIm 1
3100
18000
230
26000
8.5
> 14
R=4- 1
750 > 250000
imidazolyl
The Clog P values of chloroquine (free base) and primaquine are
reported to be 5.06 and 2.6, respectively.33 Calculated Clog P
values of 3a, 8b, 12a (free base) were 5.09, 5.08 and 5.36,34
which are slightly higher than that of chloroquine. However, at
least each aliphatic amino group of the prepared compounds
should be protonated under physiological conditions. For example,
the Clog P value of mono-protonated 12a was calculated to be 2.2,
which is an appropriate value from the viewpoint of bioavailabil-
ity.
12a
12b
13
2-naphthyl 1
2-naphthyl 2
2-quinolinyl 1
28
10
12
4.9
76
34000
1400
1200
140
6.0
870
570
5300
chloro-
quin
46
57000
100
CQ: 7-chloro-4-quinolyl; Q: 2-quinolyl; BzIm: 2-benzimidazolyl;
Py: 2-pyridyl; Pym: 2-pyrimidyl; IQ: 1-isoquinolyl. Nor-3a: N-
demethylated form of 3a.
ITC is an excellent method for evaluation of affinity between
two compounds, because the binding constant, two thermodynam-
ic parameters and binding ratio can be obtained simultaneously.
The interactions of chloroquine with hemin35 or hemin µ-oxo
dimer36-37 have been determined by ITC. Here, we examined the
affinity between the synthesized compounds and hemin by using
ITC after adjusting the pH to 5.2, which is close to that of the
malaria parasites’ vacuoles.38 Table S1 (Supporting Information)
shows the results. Contrary to our expectation, the differences
among 12a, 8b and chloroquine in terms of binding constant, H
and TS were not large. There did not appear to be a clear rela-
tionship between the antimalarial activity and the binding con-
stant.
We examined the in vivo antimalarial activity of compounds 3a
and 12a, which had shown especially good results in vitro. Com-
pounds 3a and 12a were orally (p.o.) or subcutaneously (s.c.)
administered to P. berghei-infected mice. Artesunate, which is one
of principal clinically used antimalarial drugs, was tested as a
positive control. Both 3a (both p.o. and s.c.) and 12a (s.c. only)
(30 mg/kg/day, 4 times) completely eliminated the malaria para-
sites from the mouse body (Table 2). On the other hand, a few
parasites still remained in the case of artesunate. That is to say, the
parasite-inhibitory effects of 3a and 12a are almost equivalent to
that of artesunate, since the molar amounts of 3a (68.3 mol/kg)
and 12a (65.3 mol/kg) used were larger than that of artesunate
(39.0 mol/kg). The reported in vivo therapeutic efficacy of chlo-
Hemin reacts with H2O2 to form hydroxyl radical (•OH) and oxo-
FeIV heme via O-O bond homolysis, and •OH and other oxy radi-
cal species attack the hemin, causing degradation accompanied by
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bleaching.39 Therefore, we used the H2O2-mediated hemin degra-
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dation system to evaluate the protective effect of quinolinic com-
pounds at pH 5.2.3 As shown in Figure 2, chloroquine had moder-
ate protective effect and quinine showed almost no effect. In con-
trast, compounds having two -conjugated planar moieties had
much higher protective activities than chloroquine. These results
strongly suggest that these molecules have high affinity for he-
min. The order of the inhibitory potency is as follows: 12a > 8b >
3a> chloroquine >> quinine.
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DFT calculation was carried out for antimalarial compound–
hematin (HO- coordinated ferric heme) complex in order to clarify
the correlation between complex formation equilibrium and anti-
malarial activity. We chose compounds 8a and 8b for comparison
and a hematin (dianion form) with one 8a or 8b molecule (dica-
tion form) was set for calculation. Geometry optimization (func-
tion set: B97X-D / 6-31G*) successfully converged in both cases
(Table S3). Calculated formed heat in complexation of hematin
with 8b was more than that with 8a with shorter methylene chains
(0.0324 Hartree (85.0 kJ/mol) difference). This result matches
that antimalarial activity of 8b was much higher than that of 8a.
The optimized shape of hematin-8b complex looks like pincer
type one (Figure S3). Next, we examined the complexation with
hemin dimer (-hematin). Calculation in the case of hemin dimer
8b successfully converged (Table S4), but calculation in the case
of 8a did not converge in spite of several trials. As shown in Fig-
ure S3, the optimized structure of hemin dimer 8b complex was
greatly transformed from the original form of hemin dimer. This
complexation would contribute to the prevention of hemozoin
formation.
Figure 2. Protective effect of antimalarial compounds against
degradation of hemin by H2O2.
HDP has a critical role in dimerization of hemin in the food vacu-
oles of malaria parasites to generate hemozoin.3, 40-41 Therefore,
we next examined the inhibitory effect of our compounds on the
production of hemozoin by HDP. HDP was obtained by expres-
sion in Escherichia coli and subsequent purification. The amount
of HDP-produced hemozoin
Figure 3. Inhibitory effect of antimalarial compounds on hemozo-
in formation catalyzed by heme detoxification protein (HDP).
IC50 against FCR-3 strain: Compound 3a (20 nM), 8b (0.92 nM),
12a (12 nM), chloroquine (46 nM).
was determined by absorption spectrometry of an alkaline solu-
tion of isolated hemozoin. Concentration-dependent inhibition by
the test compounds is shown in Figure 3. Compounds 8b and 3a
were much more potent inhibitors than quinine and chloroquine,
and 8b showed very high inhibitory activity at low concentration.
Hence, the extremely high antimalarial activity (FCR-3) of 8b
may be explained mainly in terms of this potent HDP inhibition.
Compound 12a was the most potent protector of hemin decompo-
sition by H2O2, whereas it was only an intermediate-strength in-
hibitor of HDP-catalyzed hemozoin formation. These results
strongly suggest that the antimalarial effect of the compounds we
developed is due not only to interference with the self-assembly
of heme dimer by direct interaction with hemin, but also to inhibi-
tion of heme dimerization by HDP. Hence, the antimalarial activi-
ty of the developed compounds involves at least these two mech-
anisms.
In summary, we designed and synthesized a new series of antima-
larial compounds based on the idea that high affinity for heme
would block hemozoin polymer formation in malaria parasites.
Compounds with a 7-chloroquinolin-4-yl group and another -
conjugated moiety at the two terminal amino groups of triamine
1a or 1b generally exhibited moderate to high antimalarial activi-
ties in vitro. Especially, 3a, 3b, Nor-3a, 8b, 12a and 12b had
potent activities, with EC50 toward a chloroquine-resistant strain
(K1) reaching the 10-8 M level. It is well established that 4-amino-
7-chloroquinolines bearing a basic side-chain are strongly active
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against malaria parasites. However, there has been no explicit
REFERENCES
attempt to design molecules with two -stacking groups that can
pincer heme. On the other hand, there is recent evidence that
quinoline-type drugs may inhibit Hz formation by binding the
surfaces of the crystal.9 An alternative mechanism has also been
suggested, i.e., that these compounds could bind to more than one
surface site on the Hz crystal. Compounds 3a and 12a showed
more potent in vivo antimalarial activity than artesunate in s.c.
treatment, and provided longer-lasting protection against P.
berghei-infected mice. The plot of protective activity against he-
min degradation by H2O2 versus antimalarial activity showed a
good correlation, supporting the validity of our molecular design
concept. However, the order of inhibitory activity on HDP-
catalyzed hemozoin formation was somewhat different from that
of protective activity against hemin degradation. This result indi-
cates that the effect on HDP catalysis is also significant. These
findings results should contribute to the development of antima-
larial drugs with excellent activity against drug-resistant malaria.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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Buller, R.; Peterson, M. L.; Almarsson, Ö.; Leiserowitz, L.,
Quinoline Binding Site on Malaria Pigment Crystal:ꢀ A Rational
Pathway for Antimalaria Drug Design. Cryst. Growth Des. 2002, 2
(6), 553-562.
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Ketchum, M. A.; Lee, A. M.; Vekilov, P. G.; Rimer, J. D.,
Biomimetic Assay for Hematin Crystallization Inhibitors: A New
Platform To Screen Antimalarial Drugs. Cryst. Growth Des. 2017, 17
(1), 197-206.
Experimental data of synthesis, experimental methods of assays,
ITC, Suppressing effect of 3a or 12a, curve fitting data for Figure
2(a), DFT calculations (PDF).
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Olafson, K. N.; Nguyen, T. Q.; Rimer, J. D.; Vekilov, P.
G., Antimalarials inhibit hematin crystallization by unique drug–
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†If an author’s address is different than the one given in the affili-
ation line, this information may be included here.
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The manuscript was written through contributions of all authors.
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Funding Sources
This work was supported in part by Grants-in-Aid for Scientific
Research (A) from the Japan Society for the Promotion of Science
(JSPS). This work was also supported by the Platform Project for
Supporting in Drug Discovery and Life Science Research from
MEXT, and Japan Agency for Medical Research and Develop-
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ACKNOWLEDGMENT
We are grateful to Dr. Ken-ichi Sato and Dr. Tadashi Hashimoto
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Figure 1. Hemozoin (Hz) formation, and molecular design of inhibitors of hemin self-assembly as candidate
antimalarial agents.
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Scheme 1. Synthetic scheme of bisꢀplanar triamines (3ꢀ10) and αꢀacyltriamineꢀtype compounds 11ꢀ13.
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