Pyridinium cationic-dimer antimalarials, unlike chloroquine, act selectively between the schizont stage and the ring stage of Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmc.2008.04.051

Malaria is a leading cause of death in developing countries, and the emergence of strains resistant to the main therapeutic agent, chloroquine, has become a serious problem. We have developed cationic-dimer type antimalarials, MAP-610 and PMAP-H10, which

Full text of DOI:10.1016/j.bmc.2008.04.051

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6027–6033
Pyridinium cationic-dimer antimalarials, unlike chloroquine,
act selectively between the schizont stage and the
ring stage of Plasmodium falciparum
Mai Yoshikawa, Kazunori Motoshima, Kanji Fujimoto,
Akihiro Tai, Hiroki Kakuta* and Kenji Sasaki
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Division of Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Okayama 700-8530, Japan
Received 24 March 2008; revised 19 April 2008; accepted 22 April 2008
Available online 25 April 2008
Abstract—Malaria is a leading cause of death in developing countries, and the emergence of strains resistant to the main therapeutic
agent, chloroquine, has become a serious problem. We have developed cationic-dimer type antimalarials, MAP-610 and PMAP-
H10, which are structurally different from chloroquine. In this study, we introduced several substituents on the terminal phenyl rings
of PMAP-H10. The electronic and hydrophobic properties of the substituents were correlated with the antimalarial activity and
cytotoxicity of the compounds, respectively. Studies with synchronized cultures of malarial plasmodia showed that our cationic-
dimers act selectively between the schizont stage and the ring stage of the parasitic cycle, unlike chloroquine, which has a stage-inde-
pendent action. Thus, the mechanism of action of our antimalarials appears to be different from that of chloroquine, and our com-
pounds may be effective against chloroquine-resistant strains.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
(2) were developed by Vial et al.7–9 Furamidine (3), a
derivative of the antiparasitic pentamidine, can be
cationized under appropriate conditions, and exerts
antimalarial activity (Fig. 1) through the inhibition of
hemozoin formation.10–12
Malaria kills 1–1.5 million people and affects 300–500
million people every year, and the total number of ma-
laria-affected individuals is estimated to be up to about
800 million.1,2 The problem is most serious in sub-Sah-
aran African countries. Although the number of coun-
tries where malaria is prevalent has been decreasing,2
the movement of people to infected areas has caused
new social problems.3 Pathogenic malaria parasites in-
clude Plasmodium falciparum, Plasmodium vivax, Plas-
modium malariae and Plasmodium ovale.4 Among
them, Plasmodium falciparum (P. falciparum) causes se-
vere infection.1 Chloroquine (1) (Fig. 1) is widely used
for prevention or for initial treatment of malaria, as it
has few side effects. However, the emergence of chloro-
quine-resistant strains remains a problem.5,6
Considering that malaria is mainly prevalent in develop-
ing countries and that chloroquine-resistance is becoming
a problem, we have been seeking new antimalarials that
can be synthesized inexpensively and whose chemical
structures are different from that of chloroquine (1). We
have reported that cationic-dimer type antimalarials of
the MAP series can be synthesized in just two steps from
an inexpensive starting material, isonicotinic acid
(Fig. 1).13 A structure–activity relationship study led to
1,10-(1,10-decanediyl)bis[4-[(hexylamino)carbonyl]
pyridinium bromide] (4: MAP-610) and 1,10-(1,12-
dodecanediyl)bis[4-[(butylamino)-carbonyl]pyridinium
bromide] (5: MAP-412) as potent antimalarials. 1,10-
(1,10-Decanediyl)bis[4-[(phenylamino)carbonyl]pyridini-
um bromide] (6: PMAP-H10), which possesses phenyl
rings at both ends, also showed potent antimalarial activ-
ity, not only in vitro, but also in vivo. It is possible to intro-
duce various substituents onto the phenyl rings of PMAP-
H10 (6), so we examined the effects of introducing several
Among antimalarials active against drug-resistant
strains, bis-cation type antimalarial compounds G25
Keywords: Antimalarials; Cationic-dimer; Hammett’s r; Hansch–Fuj-
ita p; Synchronized culture.
Corresponding author. Tel.: +81 86 251 7977; fax: +81 86 251
7926; e-mail: kakuta@pharm.okayama-u.ac.jp
*
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.04.051

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M. Yoshikawa et al. / Bioorg. Med. Chem. 16 (2008) 6027–6033
Figure 1. Chemical structures of well-known antimalarials (1–3) and MAPs (4–6).
substituents onto the phenyl rings of the MAP series com-
pounds in order to understand the influence of the elec-
tronic and hydrophobic properties of the substituents
on the antimalarial activity and cytotoxicity. We were
also interested to know whether there is a difference in
the mechanism of action between our cation-dimers and
chloroquine (1), so we examined the site of action of our
compounds in P. falciparum. Here, we describe the results
of a structure–activity relationship study and discuss the
site of action of our cation-dimer type antimalarials.
intermediates were reacted with 1,10-dibromodecane to
afford the target molecules 6–14.
3. Results and discussion
We examined the antimalarial activity against FCR-3
strain (P. falciparum) and the cytotoxicity against
FM3A F28-7 cells (derived from breast cancer of mice)
of new compounds in the MAP series bearing various
substituents on the phenyl rings at both ends (PMAP
series). Chloroquine (1) was used as a positive control.
As shown in Table 1, most of the tested compounds
showed antimalarial activities comparable with that of
chloroquine (1, EC50: 18 nM), indicating that the intro-
duction of substituents into the phenyl rings did not sig-
nificantly decrease the antimalarial activity. In contrast,
IC50 values of cytotoxicity were at the micromolar level.
PMAP-H10 (6) and PMAP-CN10 (13) showed less cyto-
toxicity than chloroquine (1). The nature of the substit-
uents on the phenyl rings at both sides appeared to
influence both antimalarial and cytotoxic activities.
2. Chemistry
Introduction of substituents into the phenyl rings at
both ends of PMAP-H10 (6) was performed with anilino
derivatives possessing substituents with various elec-
tronic or hydrophilic properties. These compounds were
synthesized from isonicotinic acid (15) as a starting
material, according to Scheme 1. Substituted anilines
were reacted with isonicotinic acid (15), HOBt and
EDC in DMF to afford the intermediates 16–24. The
Scheme 1. Reagents and yields: (a) corresponding aniline, HOBt, EDC, DMF, 17–65%; (b) 1,10-dibromodecane, appropriate solvent, 36–82%.

M. Yoshikawa et al. / Bioorg. Med. Chem. 16 (2008) 6027–6033
6029
Table 1. In vitro antimalarial activities and cytotoxicities of chloroquine (1) and MAPs (4–14)a
(CH2)10
N
N
H
H
N
N
O
O
2Br
EC50 (nM)
R
R
Compounds
R
rd
pe
Antimalarial activityb
Cytotoxicityc
Chloroquine (1)
MAP-610 (4)
MAP-412 (5)
—
—
18
5
32,000
10,500
52,900
25,600
42,800
27,800
1420
—
—
—
—
—
—
100
39
29
21
12
8
—
PMAP-M10 (7)
PMAP-H10 (6)
PMAP-F10 (8)
PMAP-I10 (9)
Me
H
ꢀ0.17
0.00
0.06
0.18
0.23
0.23
0.54
0.66
0.78
0.56
0.00
0.14
F
I
Cl
1.12
0.71
PMAP-Cl10 (10)
PMAP-Br10 (11)
PMAP-TF10 (12)
PMAP-CN10 (13)
PMAP-NO10 (14)
1890
1390
Br
CF3
CN
NO2
15
33
35
74
0.86
0.88
5180
42,400
5990
ꢀ0.57
ꢀ0.28
a In vitro antimalarial activities and cytotoxicities were determined as described in Section 5.
b Antimalarial activities were examined against chloroquine-sensitive P. falciparum (FCR-3 strain).
c Cytotoxicity was examined against mouse mammary tumor FM3A cells.
d Electronic parameters are quoted from Ref. 15.
e Hydrophobic parameters are quoted from Ref. 15.
In considering the relationship between substituents on
phenyl rings and bioactivities, Hammett’s r value14,15
and Hansch–Fujita’s p value16–18 are commonly used
as electronic property parameters and hydrophobic
parameters, respectively. Figure 2 shows the relationship
of antimalarial activity with Hammett’s r or Hansch–
Fujita’s p. The results suggest that there is an optimal
electronic character of the substituent on the phenyl
groups for antimalarial activity in the PMAP series com-
pounds. Among them, the non-substituted compound 6
and halogen-substituted derivatives 8–11 exhibited po-
tent antimalarial activities. On the other hand, there ap-
peared to be no correlation between antimalarial activity
and Hansch–Fujita’s p.
with Hammett’s r. Among the potent antimalarial com-
pounds 6 and 8–11, the more hydrophilic compound 7
showed lower cytotoxicity and the more hydrophobic
compounds 9–11 showed higher cytotoxicity. From the
viewpoints of both antimalarial activity and cytotoxic-
ity, compound 6 seems to be the best candidate in the
PMAP series.
P. falciparum invades the human body as sporozoites
through the salivary glands of Anopheles mosquitoes.
Sporozoites first invade liver cells, and then transform
into several thousand merozoites after several cycles of
division in hepatocytes over a period of a few days.
After being released from liver cells, merozoites invade
blood cells.19 Infectious plasmodia in blood cells prolif-
erate vigorously through a cycle involving merozoites,
rings, trophozoites, and schizontes.4 Plasmodia can be
cultured at a single stage by means of synchronized cul-
ture methods.20 In order to elucidate the site of action of
Figure 3 shows the relationship of cytotoxicity with
Hammett’s r or Hansch–Fujita’s p. The cytotoxicity ap-
peared to be correlated with Hansch–Fujita’s p, but not
Figure 2. Correlations of antimalarial activities of PMAPs (6–14) with
(a) Hammett substituent constants (r) and (b) Hansch–Fujita p
constants.
Figure 3. Correlations of cytotoxicity of PMAPs (6–14) with (a)
Hammett substituent constants (r) and (b) Hansch–Fujita p constants.

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M. Yoshikawa et al. / Bioorg. Med. Chem. 16 (2008) 6027–6033
our cationic-dimer type antimalarials, we treated plas-
modial cultures synchronized at various stages with
MAP-610 (4) or PMAP-H10 (6), which showed antima-
larial activities in vivo.13 At time zero, plasmodia were
synchronized in the ring, middle trophozoite, late tro-
phozoite, or schizont stage, and then MAP-610 (4) or
PMAP-H10 (6) was added at 100 times the correspond-
ing IC50. The infectious ratio was determined hourly
thereafter. In cultures synchronized at the ring, middle
trophozoite or late trophozoite stage, no significant dif-
ference between the effects of vehicle and drug was
found immediately after drug treatment, indicating that
our compounds did not affect malaria at those stages
(Fig. 4a–c). But, in cultures synchronized at the schizont
stage, the infection ratio was reduced immediately after
drug treatment (Fig. 4d). These results indicate that our
compounds act selectively on malaria parasites between
the schizont stage and the ring stage. In contrast, it was
reported that chloroquine acts at the ring and trophozo-
ite stages, as well as the schizont stage, that is, its activity
is stage-independent.21,22 Therefore, our cationic-dimer
type compounds appear to have a different mechanism
of antimalarial activity from chloroquine, and might
be effective against chloroquine-resistant malaria.
Hammett’s r value for potent antimalarial activity,
while cytotoxicity is correlated with Hansch–Fujita’s p
values. In order to elucidate the site of action of our cat-
ionic-dimer type antimalarials, synchronized culture
methods were employed. The results indicated that
MAP-610 (4) and PMAP-H10 (6) act between the schiz-
ont stage and the ring stage, unlike chloroquine (1),
whose action is stage-independent. Therefore, our cat-
ion-dimers appear to have a different mechanism of ac-
tion from chloroquine, and might be effective against
chloroquine-resistant malaria.
5. Experimental
5.1. Chemistry
Melting points were determined with a Yanagimoto hot-
stage melting point apparatus and are uncorrected. IR
spectra were recorded on a JASCO FT/IR350 (KBr).
NMR spectra were recorded on a VarianVXR-300 (1H
300 MHz) or a VarianVXR-500 (1H 500 MHz) spec-
trometer. Proton chemical shifts were referenced to the
TMS internal standard. Elemental analysis was carried
out with a Yanagimoto MT-5 CHN recorder elemental
analyzer and results were within 0.4% of the theoreti-
cal values. FAB-MS was carried out with a VG70-SE.
4. Conclusion
For the structural development of our cationic-dimer
type antimalarials, we synthesized several compounds
bearing various substituents on the phenyl rings of
PMAP-H10 (6) and performed a structure–activity rela-
tionship study using Hammett’s r and Hansch–Fujita’s
p values. The results indicate that there is an optimal
5.2. General procedure for synthesis of substituted N-
phenyl-4-pyridinecarboxamides (GP-A)
To a solution of isonicotinic acid (14) (3.0 mmol) in
DMF (6.0 mL) were added isonicotinic acid (3.0 mmol),
HOBt (3.5 mmol) and EDC (3.6 mmol). The mixture
was stirred overnight at 100 ꢁC, then poured into satd
NaHCO3 and extracted with EtOAc. The organic layer
was washed with H2O and brine, and then dried over
MgSO4. The solvent was removed under reduced pres-
sure. The residue was purified by silica gel column chro-
matography or recrystallization to give the
corresponding amide intermediates 16–24.
5.2.1. N-Phenyl-4-pyridinecarboxamide (16). According
to the general procedure (GP-A), 16 was obtained in
17% yield as colorless plates after recrystallization from
EtOAc/n-hexane; 1H NMR (300 MHz, DMSO-d6) d:
10.49 (br s, 1H), 8.79 (dd, 2H, J = 4.3, 1.7 Hz), 7.86
(dd, 2H, J = 4.3, 1.7 Hz), 7.77 (d, 2H, J = 7.6 Hz), 7.38
(td, 2H, J = 7.6, 1.5 Hz), 7.14 (tt, 1H, J = 7.6, 1.5 Hz).
5.2.2. N-(4-Methylphenyl)-4-pyridinecarboxamide (17).
According to the general procedure (GP-A), 17 was ob-
tained in 41% yield as colorless plates after recrystalliza-
tion from EtOH/n-hexane; 1H NMR (300 MHz,
DMSO-d6) d: 10.41 (br s, 1H), 8.78 (dd, 2H, J = 4.6,
1.6 Hz), 7.85 (dd, 2H, J = 4.6, 1.6 Hz), 7.65 (d, 2H,
J = 8.0 Hz), 7.18 (d, 2H, J = 8.0 Hz), 2.29 (s, 3H).
5.2.3. N-(4-Fluorophenyl)-4-pyridinecarboxamide (18).
According to the general procedure (GP-A), 18 was ob-
tained in 42% yield as a colorless powder after recrystal-
lization from EtOH/n-hexane; 1H NMR (300 MHz,
DMSO-d6) d: 10.55 (br s, 1H), 8.79 (dd, 2H, J = 4.7,
Figure 4. Synchronous growth of P. falciparum in the absence (open
circles) of compounds and in the presence (open triangles) of MAP-610
(4) and in the presence (open squares) of PMAP-H10 (6). Cultures
were initially synchronized at 0 h in (a) ring stage, (b) middle
trophozoite stage, (c) late trophozoite stage and (d) schizont stage.

M. Yoshikawa et al. / Bioorg. Med. Chem. 16 (2008) 6027–6033
6031
1.7 Hz), 7.86 (dd, 2H, J = 4.7, 1.7 Hz), 7.79 (m, 2H),
7.22 (m, 2H).
to the general procedure (GP-B), 6 was obtained in
36% yield as a pale yellow powder after recrystallization
from MeOH/EtOAc. This reaction was carried out in
DMF. Mp 209.0–210.0 ꢁC; IR (KBr) cmꢀ1: 3415
5.2.4. N-(4-Iodophenyl)-4-pyridinecarboxamide (19).
According to the general procedure (GP-A), 19 was ob-
tained in 26% yield as a colorless powder after recrystal-
lization from EtOH/n-hexane; 1H NMR (300 MHz,
DMSO-d6) d: 10.57 (br s, 1H), 8.79 (dd, 2H, J = 4.5,
1.7 Hz), 7.85 (dd, 2H, J = 4.5, 1.7 Hz), 7.72 (dd, 2H,
J = 6.8, 2.1 Hz), 7.62 (dd, 2H, J = 6.8, 2.1 Hz).
1
(NH), 1673 (CO); H NMR (500 MHz, DMSO-d6) d:
10.91 (s, 2H), 9.31 (d, 4H, J = 6.7 Hz), 8.58 (d, 4H,
J = 6.7 Hz), 7.78 (d, 4H, J = 7.7 Hz), 7.42 (t, 4H,
J = 7.7 Hz), 7.20 (t, 2H, J = 7.7 Hz), 4.67 (t, 4H,
J = 7.5 Hz), 1.95 (m, 4H), 1.29 (m, 12H); FAB-MS m/
z: 615, 617 [M+H–HBr]+; Anal. Calcd for
C34H40Br2N4O2Æ1/2H2O: C, 57.88; H, 5.86; N, 7.94.
Found: C, 57.79; H, 5.72; N, 7.82.
5.2.5. N-(4-Chlorophenyl)-4-pyridinecarboxamide (20).
According to the general procedure (GP-A), 20 was ob-
tained in 52% yield as a colorless powder after recrystal-
lization from EtOAc/n-hexane; 1H NMR (300 MHz,
DMSO-d6) d: 10.61 (br s, 1H), 8.79 (dd, 2H, J = 4.7,
1.4 Hz), 7.85 (dd, 2H, J = 4.7, 1.4 Hz), 7.81 (dd, 2H,
J = 7.0, 1.9 Hz), 7.44 (dd, 2H, J = 7.0, 1.9 Hz).
5.3.2. 1,10-(1,10-Decanediyl)bis[4-[[4-(methyl)phenylami-
no]carbonyl]pyridinium bromide] (7): PMAP-M10.
According to the general procedure (GP-B), 7 was ob-
tained in 79% yield as yellow needles after recrystalliza-
tion from MeOH. This reaction was carried out in
DMF. Mp 238.0–239.0 ꢁC; IR (KBr) cmꢀ1: 3283
1
5.2.6. N-(4-Bromophenyl)-4-pyridinecarboxamide (21).
According to the general procedure (GP-A), 21 was ob-
tained in 65% yield as colorless plates after recrystalliza-
tion from EtOAc/n-hexane; 1H NMR (300 MHz,
DMSO-d6) d: 10.62 (br s, 1H), 8.79 (dd, 2H, J = 4.5,
1.7 Hz), 7.85 (dd, 2H, J = 4.5, 1.7 Hz), 7.76 (d, 2H,
J = 9.0 Hz), 7.57 (d, 2H, J = 9.0 Hz).
(NH), 1678 (CO); H NMR (500 MHz, DMSO-d6) d:
10.85 (s, 2H), 9.30 (d, 4H, J = 6.7 Hz), 8.56 (d, 4H,
J = 6.7 Hz), 7.67 (d, 4H, J = 8.2 Hz), 7.22 (d, 4H,
J = 8.2 Hz), 4.67 (t, 4H, J = 7.5 Hz), 2.31 (s, 6H), 1.96
(m, 4H), 1.29 (m, 12H); FAB-MS m/z: 643, 645
[M+H–HBr]+; Anal. Calcd for C36H44Br2N4O2Æ1/
2H2O: C, 58.94; H, 6.18; N, 7.64. Found: C, 59.08; H,
6.07; N, 7.82.
5.2.7. N-[4-(Trifluoromethyl)phenyl]-4-pyridinecarboxa-
mide (22). According to the general procedure (GP-A),
22 was obtained in 35% yield as colorless needles after
recrystallization from EtOAc/n-hexane; 1H NMR
(300 MHz, DMSO-d6) d: 10.82 (br s, 1H), 8.81 (dd,
2H, J = 4.4, 1.7 Hz), 8.01 (d, 2H, J = 8.8 Hz), 7.88 (dd,
2H, J = 4.4, 1.7 Hz), 7.76 (dd, 2H, J = 8.8 Hz).
5.3.3. 1,10-(1,10-Decanediyl)bis[4-[[4-(fluoro)phenylami-
no]carbonyl]pyridinium bromide] (8): PMAP-F10.
According to the general procedure (GP-B), 8 was ob-
tained in 72% yield as a yellow powder after recrystalli-
zation from MeOH/EtOAc. This reaction was carried
out in dioxane. Mp 239.0–240.0 ꢁC; IR (KBr) cmꢀ1
:
1
3410 (NH), 1662 (CO); H NMR (300 MHz, DMSO-
d6) d: 10.98 (s, 2H), 9.32 (d, 4H, J = 6.7 Hz), 8.58 (d,
4H, J = 6.7 Hz), 7.82 (m, 4H), 7.24 (m, 4H), 4.67 (t,
4H, J = 7.4 Hz), 1.94 (m, 4H), 1.29 (m, 12H); FAB-
MS m/z: 651, 653 [M+H–HBr]+; Anal. Calcd for
C34H38Br2F2N4O2Æ1/2H2O: C, 55.07; H, 5.30; N, 7.56.
Found: C, 55.15; H, 5.21; N, 7.78.
5.2.8. N-(4-Cyanophenyl)-4-pyridinecarboxamide (23).
According to the general procedure (GP-A), 23 was ob-
tained in 22% yield as colorless plates after recrystalliza-
tion from EtOAc/n-hexane; 1H NMR (300 MHz,
DMSO-d6) d: 10.90 (br s, 1H), 8.81 (dd, 2H, J = 4.3,
1.7 Hz), 7.98 (d, 2H, J = 9.0 Hz), 7.87 (dd, 2H, J = 4.3,
1.7 Hz), 7.84 (d, 2H, J = 9.0 Hz).
5.3.4.
1,10-(1,10-Decanediyl)bis[4-[[4-(iodo)phenylami-
5.2.9. N-(4-Nitrophenyl)-4-pyridinecarboxamide (24).
According to the general procedure (GP-A), 24 was ob-
tained in 30% yield as yellow needles after recrystalliza-
tion from EtOH/n-hexane; 1H NMR (500 MHz,
DMSO-d6) d: 11.02 (br s, 1H), 8.81 (dd, 2H, J = 4.9,
1.5 Hz), 8.29 (d, 2H, J = 9.2 Hz), 8.06 (d, 2H,
J = 9.2 Hz), 7.88 (dd, 2H, J = 4.9, 1.5 Hz).
no]carbonyl]pyridinium bromide] (9): PMAP-I10.
According to the general procedure (GP-B), 9 was ob-
tained in 82% yield as a yellow powder after recrystalli-
zation from MeOH. This reaction was carried out in
DMF. Mp > 300.0 ꢁC; IR (KBr) cmꢀ1: 3347 (NH),
1
1681 (CO); H NMR (500 MHz, DMSO-d6) d: 10.86
(br s, 2H), 9.30 (d, 4H, J = 6.9 Hz), 8.56 (d, 4H,
J = 6.9 Hz), 7.66 (d, 4 H, J = 8.4 Hz), 7.22 (d, 4H,
J = 8.4 Hz), 4.67 (t, 4H, J = 7.3 Hz), 1.94 (m, 4H),
1.29 (m, 12H); FAB-MS m/z: 867, 869 [M+H–HBr]+;
Anal. Calcd for C34H38Br2I2N4O2Æ1/2H2O: C, 42.66;
H, 4.11; N, 5.85. Found: C, 42.47; H, 4.10; N, 5.81.
5.3. General procedure for synthesis of PMAPs (GP-B)
To a solution of the appropriate N-phenyl-4-pyridine-
carboxamide (0.5 mmol) in an appropriate solvent was
added 1,10-dibromodecane (0.2 mmol). The mixture
was stirred overnight at 100 ꢁC and concentrated under
reduced pressure. The residue was recrystallized or trit-
urated using an appropriate solvent to give the target
compounds 6–14.
5.3.5. 1,10-(1,10-Decanediyl)bis[4-[[4-(chloro)phenylami-
no]carbonyl]pyridinium bromide] (10): PMAP-Cl10.
According to the general procedure (GP-B), 10 was ob-
tained in 48% yield as yellow plates after recrystalliza-
tion from MeOH/EtOAc. This reaction was carried
5.3.1.
bonyl]pyridinium bromide] (6): PMAP-H10. According
1,10-(1,10-Decanediyl)bis[4-[(phenylamino)car-
out in DMF. Mp 261.5–263.0 ꢁC; IR (KBr) cmꢀ1
:
3432 (NH), 1665 (CO); H NMR (300 MHz, DMSO-
1

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M. Yoshikawa et al. / Bioorg. Med. Chem. 16 (2008) 6027–6033
d6) d: 11.05 (s, 2H), 9.32 (d, 4H, J = 6.7 Hz), 8.57 (d, 4H,
J = 6.7 Hz), 7.83 (dd, 4H, J = 6.8, 2.1 Hz), 7.49 (dd, 4H,
J = 6.8, 2.1 Hz), 4.67 (t, 4H, J = 7.3 Hz), 1.94 (m, 4H),
1.28 (m, 12H); FAB-MS m/z: 683, 685 [M+H–HBr]+;
Anal. Calcd for C34H38Br2Cl2N4O2ÆH2O: C, 52.13; H,
5.15; N, 7.15. Found: C, 52.10; H, 5.13; N, 7.09.
5.4. Culture of P. falciparum
Antimalarial activities of synthesized compounds
against P. falciparum (FCR-3 strain) were determined.
P. falciparum was cultivated by a modification of the
method of Trager and Jensen using a 5% hematocrit sus-
pension of type A human red blood cells (RBCs) in
RPMI 1640 medium supplemented with 44 mM
HEPES, 24 mM NaHCO3, 25 lg/mL gentamicin, and
heat-inactivated 10% type A human serum. The plates
were held under an atmosphere of 5% CO2, 5% O2,
and 90% N2 at 37 ꢁC. The medium was changed daily
until 5% parasitemia (five parasite-infected RBCs per
100 RBCs).
5.3.6. 1,10-(1,10-Decanediyl)bis[4-[[4-(bromo)phenylami-
no]carbonyl]pyridinium bromide] (11): PMAP-Br10.
According to the general procedure (GP-B), 11 was ob-
tained in 47% yield as a yellow powder after recrystalliza-
tion from MeOH/EtOAc. This reaction was carried out in
DMF. Mp 280.5–282.0 ꢁC; IR (KBr) cmꢀ1: 3402 (NH),
1
1664 (CO); H NMR (300 MHz, DMSO-d6) d: 11.06 (s,
2H), 9.32 (d, 4H, J = 6.8 Hz), 8.57 (d, 4H, J = 6.8 Hz),
7.78 (dd, 4H, J = 6.9, 2.0 Hz), 7.63 (dd, 4H, J = 6.9,
2.0 Hz), 4.68 (t, 4H, J = 7.3 Hz), 1.94 (m, 4H), 1.28 (m,
12H); FAB-MS m/z: 771, 773, 775, 777 [M+H–HBr]+;
Anal. Calcd for C34H38Br4N4O2: C, 47.80; H, 4.48; N,
6.56. Found: C, 47.66; H, 4.53; N, 6.48.
5.5. Culture of mammalian cells
Cytotoxicity of the test compounds toward mouse
mammary tumor FM3A cells (wild-type, subclone
F28-7) was determined. FM3A cells were maintained
in suspension culture at 37 ꢁC in a 5% CO2 atmo-
sphere in plastic bottles containing ES medium sup-
plemented with 2% heat-inactivated fetal bovine
serum. FM3A cells grew with a doubling time of
about 12 h.
5.3.7. 1,10-(1,10-Decanediyl)bis[4-[[4-(trifluoromethyl)phe-
nylamino]carbonyl]pyridinium bromide] (12): PMAP-
TF10. According to the general procedure (GP-B), 12
was obtained in 74% yield as a yellow powder after
recrystallization from MeOH/EtOAc. This reaction
was carried out in dioxane. Mp 215.0–217.0 ꢁC; IR
5.6. In vitro antimalarial activity assay23
(KBr) cmꢀ1
:
3400 (NH), 1681 (CO); 1H NMR
(300 MHz, DMSO-d6) d: 11.23 (s, 2H), 9.33 (d, 4H,
J = 6.7 Hz), 8.59 (d, 4H, J = 6.7 Hz), 8.02 (d, 4H,
J = 8.5 Hz), 7.81 (d, 4H, J = 8.5 Hz), 4.68 (t, 4H,
J = 7.3 Hz), 1.95 (m, 4H), 1.27 (m, 12H); FAB-MS
m/z: 751, 753 [M+H–HBr]+; Anal. Calcd for
C36H38Br2F6N4O2Æ2H2O: C, 49.78; H, 4.87; N, 6.45.
Found: C, 49.92; H, 4.76; N, 6.43.
Five microliters of solution of a test compound in
DMSO at several concentrations was added to indi-
vidual wells of a 24-well plate. RBCs with 0.3% para-
sitemia were added to each well, containing 995 lL of
culture medium, to give a final hematocrit level of 3%.
Plates were incubated at 37 ꢁC for 72 h under an
atmosphere of 5% CO2, 5% O2, and 90% N2. To eval-
uate the antimalarial activities of the test compounds,
thin blood films from each culture were prepared and
stained with Diff-Quik stain. Test compounds were
tested in duplicate at each concentration. Com-
pound-free control cultures were run simultaneously.
All data points represent the mean of three experi-
ments (parasitemia in controls reached between 4%
and 5% at 72 h). The 50% inhibitory concentration
(IC50), which is the concentration giving 50% inhibi-
tion of parasite proliferation compared with the con-
trol, was calculated from the growth inhibition curve
for each compound.
5.3.8. 1,10-(1,10-Decanediyl)bis[4-[[4-(cyano)phenylami-
no]carbonyl]pyridinium bromide] (13): PMAP-CN10.
According to the general procedure (GP-B), 13 was ob-
tained in 66% yield as a colorless powder after recrystal-
lization from MeOH. This reaction was carried out in
DMF. Mp 284.5–285.5 ꢁC; IR (KBr) cmꢀ1: 3414
1
(NH), 1679 (CO); H NMR (300 MHz, DMSO-d6) d:
11.34 (s, 2H), 9.37 (d, 4H, J = 6.3 Hz), 8.61 (d, 4H,
J = 6.3 Hz), 8.02 (d, 4H, J = 8.7 Hz), 7.91 (d, 4H,
J = 8.7 Hz), 4.70 (t, 4H, J = 7.1 Hz), 1.95 (m, 4H),
1.28 (m, 12H); FAB-MS m/z: 665, 667 [M+H–HBr]+;
Anal. Calcd for C36H38Br2N6O2ÆH2O: C, 56.55; H,
5.27; N, 10.99. Found: C, 56.38; H, 5.11; N, 10.94.
5.7. Cytotoxicity against mammalian cell line23
5.3.9. 1,10-(1,10-Decanediyl)bis[4-[[4-(nitro)phenylami-
no]carbonyl]pyridinium bromide] (14): PMAP-NO10.
According to the general procedure (GP-B), 14 was ob-
tained in 82% yield as a pale brown powder after recrys-
tallization from MeOH. This reaction was carried out in
DMF. Mp 240.0–241.0 ꢁC; IR (KBr) cmꢀ1: 3382 (NH),
Prior to exposure to the test compounds, cell density
was adjusted to 5 · 104 cells/mL. Cell suspension
(995 lL/well) was dispensed into the 24-well test plate,
and each test compound at various concentrations sus-
pended in DMSO (5 lL) was added to individual wells.
The plate was incubated at 37 ꢁC for 48 h under an
atmosphere of 5% CO2 in air. All the test compounds
were assayed in duplicate at each concentration. Cell
numbers were counted microscopically. All data points
represent the mean of three experiments. The IC50 value
refers to the concentration of the compound necessary
to inhibit the increase in cell density at 48 h by 50% of
the control.
1
1687 (CO); H NMR (500 MHz, DMSO-d6) d: 11.44 (s,
2H), 9.35 (d, 4H, J = 6.9 Hz), 8.60 (d, 4H, J = 6.9 Hz),
8.33 (d, 4H, J = 9.2 Hz), 8.07 (d, 4H, J = 9.2 Hz), 4.69
(t, 4H, J = 7.3 Hz), 1.95 (m, 4H), 1.28 (m, 12H); FAB-
MS m/z: 705, 707 [M+H–HBr]+; Anal. Calcd for
C34H38Br2N6O6ÆH2O: C, 50.76; H, 5.01; N, 10.45.
Found: C, 50.67; H, 4.97; N, 10.57.

M. Yoshikawa et al. / Bioorg. Med. Chem. 16 (2008) 6027–6033
6033
5.8. Synchronization of parasites in culture21,23,24
pound-added groups and the control groups were
judged as the stage at which the test compound inhibits
the growth of the parasites.
The culture was synchronized by D-sorbitol treatment. It
is known that P. falciparum at the trophozoite and schiz-
ont stages is sensitive to D-sorbitol, and ring-stage para-
sites were obtained in synchronous culture. The culture
was centrifuged at 2000 rpm for 5 min and the medium
was removed. The pellet was resuspended in 5% D-sorbi-
tol in distilled water for 20 min at room temperature and
washed twice with culture medium and RPMI. Then, the
residue was added to freshly erythrocytes. The synchro-
nous culture was maintained in wells containing 2 mL
5% erythrocytes (v/v) in RPMI under an atmosphere
of 5% CO2, 5% O2, and 90% N2. At 40 h after the first
synchronization, the culture was treated again with D-
sorbitol in the same manner, and culture in which the ra-
tio of the ring stage was more than 0.8 (>80%) was used
in this experiment. At the start of every assay, the ratio
of each stage was checked under a microscope in the
same manner as described for the ring stage.
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