Synthesis and antimalarial efficacy of aza-fused rhodacyanines in vitro and in the P. berghei mouse model

Article abstract of DOI:10.1021/jm0606241

Several aza-fused rhodacyanines were synthesized and assessed for their in vitro and in vivo antimalarial activities against Plasmodium falciparum K1 and P. berghei. All synthetic compounds showed strong selective antimalarial in vitro activity. Class II azarhodacyanines, 3, consisting of four heterocyclic units, were found to display good parasitemia suppression and low acute toxicity in vivo. Among them, 3c appeared to be the most effective at a dose of 20-25 mg kg^-1 day^-1 (ip).

Full text of DOI:10.1021/jm0606241

J. Med. Chem. 2006, 49, 4795-4798
4795
Synthesis and Antimalarial Efficacy of Aza-Fused Rhodacyanines in Vitro and in the P. berghei
Mouse Model
Kiyosei Takasu,*,† Khanitha Pudhom,†,‡,§ Marcel Kaiser,# Reto Brun,# and Masataka Ihara*,†,‡
Department of Organic Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku UniVersity, Aobayama, Sendai 980-8578, Japan,
Japan Science and Technology Agency (JST), 5-3 Yonbanchou, Chiyoda-ku, Tokyo 102-8666, Japan, Department of Chemistry, Faculty of
Science, Chulalongkorn UniVersity, Phayathai Road, Patumwan, Bangkok 10330, Thailand, and Swiss Tropical Institute, Socinstrasse 57,
CH-4002 Basel, Switzerland
ReceiVed May 25, 2006
Several aza-fused rhodacyanines were synthesized and assessed for their in vitro and in vivo antimalarial
activities against Plasmodium falciparum K1 and P. berghei. All synthetic compounds showed strong selective
antimalarial in vitro activity. Class II azarhodacyanines, 3, consisting of four heterocyclic units, were found
to display good parasitemia suppression and low acute toxicity in vivo. Among them, 3c appeared to be the
most effective at a dose of 20-25 mg kg-1 day-1 (ip).
Malaria remains a major health problem in many developing
countries. It affects mainly the population living in tropical and
subtropical areas. Annually, it is estimated that there are 300-
500 million cases of malaria leading to 1-2 million deaths,
most of which are children under 5 years old.1 One of the major
reasons for the morbidity and mortality is the widespread
emergence of drug-resistant strains of the parasite. Particularly,
the efficacy of clinically available antimalarials, such as
Figure 1. General and typical structures of rhodacyanine.
chloroquine (CQa), primaquine, and pyrimethamine, is dramati-
cally decreasing.2,3 Therefore, new antimalarial drugs represent-
ing a new class of molecular framework and displaying novel
mechanism of action compared to clinically used drugs are
urgently needed.4-6 We previously reported that rhodacyanine
dyes (1),7 having a π-delocalized lipophilic cationic (DLC)
structure,8,9 showed strong antimalarial activity in vitro against
Plasmodium falciparum (drug-sensitive FCR-5 strain) with high
selective activity (Figure 1).10-12 However, further investigations
by us revealed that rhodacyanines, such as MKT-077 (1a)7,10
Figure 2. General structures of azarhodacyanines 2 (class I) and 3
and MKH-57 (1b),10 showed poor antimalarial activity in vivo
(class II).
using the rodent malaria parasite P. berghei. In our preliminary
animal experiments it was observed that accumulation of 1 in
tissue levels would prevent drug accumulation into malaria-
affected erythrocytes. To improve the bioavailability by chang-
ing the rhodacyanine skeleton, we have designed aza-fused
analogues 2 and 3 (Figure 2) as a second generation of
antimalarial rhodacyanines. In this communication, we report
the synthesis of two types of azarhodacyanines and the
tether in the original rhodacyanines 1 because introduction of a
heteroatom often leads to improve water solubility and/or
hydrophilicity.10 In this study, we also designed four-rings-linked
analogues 3, which have two rhodanine moieties flanked by
two heterocyclic rings. Synthesis of 2 was accomplished
according to the reported procedures with some modifica-
tions.10,12 Scheme 1 illustrates a typical procedure for the
synthesis of 2a. Condensation of methylthionium salt 4 with
3-ethylrhodanine (5) in the presence of triethylamine afforded
evaluation of their antimalarial activity in vitro and in vivo.
merocyanine 6. S-Methylation of 6 using methyl p-toluene-
sulfonate was further carried out, and the resulting thioiminium
Chemistry
In general, rhodacyanines are composed of three linearly
linked heterocycles involving rhodanine skeleton as a central
core, and the rhodanine ring is tethered with the right cationic
heterocycle by an sp2 carbon atom, whereas the designed
azarhodacyanines 213 possess imine function instead of olefinic
salt 7 was condensed with 2-aminomethylpyridinium salt (8)
in the presence of triethylamine, followed by the treatment with
ion-exchange resin, to provide the desired azarhodacyanine 2a
(class I).
For the synthesis of 3 (class II), the promising method was
the condensation of thioiminium salt 7 and cyanine 9, which
correspond to each double-heterocyclic unit on left and right
sides, respectively. A typical procedure for the synthesis of 3a
is summarized in Scheme 2. Synthesis of 9 was accomplished
by a three-step sequence from 2-aminopyridine (10), namely,
the reaction of 10 with isothiocyanate to give thiourea 11, which
was transformed into rhodanine 12 by treatment with chloro-
acetic acid in refluxing EtOH.14 Quaternarization of 12 by
* To whom correspondences should be addressed. For K.T.: phone, +81-
22-795-6878; fax, +81-22-795-6877; e-mail, kay-t@mail.pharm.tohoku.ac.jp.
For M.I.: phone and fax, +81-3-5498-6391; e-mail, m-ihara@hoshi.ac.jp.
† Tohoku University.
‡ JST.
§ Chulalongkorn University.
# Swiss Tropical Institute.
a Abbreviations: CQ, chloroquine; DLC, π-delocalized lipophilic cation;
MSD, mean survival days.
10.1021/jm0606241 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/04/2006

4796 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Scheme 1. Synthesis of Class I Azarhodacyanine 2aa
Brief Articles
Table 1. In Vitro Antimalarial Activity and Cytotoxicity of
Azarhodacyanines
EC50 (nM)
P. falciparuma
entry
compd
L-6
selectivity
1
2
3
4
5
6
7
2a
2b
3c
3d
3j
5.9
4.4
10
22
23
1.2 × 105
1.1 × 104
4.6 × 104
1.4 × 104
1.2 × 104
1.0 × 105
2.0 × 104
2.5 × 103
4.6 × 103
6.4 × 102
5.2 × 102
5.0 × 103
1b
19
150
CQb
a K1 (CQ-resistant) strain. b CQ ) chloroquine.
Table 2. In Vivo Antimalarial Potency of Azarhodacyaninesa
dose
b
entry
compd
(mg kg-1 day-1
)
suppression (%)
MSDc
a Reagents and conditions: (a) NEt3, MeCN, 10 °C; (b) p-TsOMe, DMF,
130 °C; (c) NEt3, MeCN, 70 °C; (d) Amberlyte IRA-400(Cl), MeOH.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
2a
2b
2c
2c
3a
3b
3c
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
10 (ip)
10 (ip)
10 (ip)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
100 (po)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
25 (ip)
10 (ip)
10 (ip)
64.5
54.6
54.1
62.7
78.4
49.7
97.1
41.7
96.4
79.5
98.3
95.5
95.7
95.6
88.4
73.1
95.0
27.0
90.6
NDd
NDd
NDd
5.5
5.6
7.0
25.3
6.5
22.0
10.8
6.3
7.7
18.0
5.3
Scheme 2. Synthesis of Class II Azarhodacyanine 3aa
16.0
12.8
5.0
a Reagents and conditions: (a) EtNCS, benzene, 80 °C; (b) ClCH2CO2H,
NaOAc, EtOH, 100 °C; (c) MeI, MeCN, 70 °C; (d) 7, NEt3, MeCN, 70 °C;
(e) Amberlyte IRA-400(Cl), MeOH.
1b
NDe
22.7
CQe
a In vivo evaluation was carried out according as Peters’ 4-day suppres-
sive protocol using five ICR-mice. b ip ) intraperitoneal administration;
po ) per os administration. c MSD ) mean survival days. MSD for
untreated mice (control) is ∼6 days. d ND ) not determined. e CQ )
chloroquine.
Chart 1. Prepared Azarhodacyanines
mined using a rat skeletal myoblast cell line, L-6. Selectivity
indices, defined as the ratio EC50(L-6)/EC50(P. falciparum), were
determined. The biological results are summarized in Table 1.
All compounds displayed strong antimalarial activity within the
range 4-23 nM and excellent selectivity indices larger than
500. Among the tested compounds, class I compound 2b showed
excellent inhibitory effects against CQ-resistant P. falciparum
with an IC50 of 4.4 nM (entry 2). Its antimalarial activity was
estimated to be 4- and 30-fold stronger than those of rhoda-
cyanine 1b having methyne tether and CQ, respectively (entries
6 and 7). Class II compounds 3 displayed slightly lower
antimalarial activity (EC50 ) 10-23 nM) than 2, but their
cytotoxicity levels were low (entries 3-5). The in vitro results
indicate that azarhodacyanines 2 and 3 would be new lead
structures for antimalarial agents.
Next, in vivo evaluation of antimalarial efficacy of 2 and 3
was carried out using the rhodent malaria model P. berghei NK-
65 (drug-sensitive strain) in mice. The experiments were
performed according to Peters’ 4-day suppressive test protocol.17
The reduction of parasitaemia in % was determined by compar-
ing the parasitemia of infected mice that were injected with the
compounds for 4 days with the parasitemia of untreated control
mice. Observation of malaria-affected mice after the end of
treatment was continued in order to record their mean survival
days (MSD). The in vivo results are summarized in Table 2.
Carborhodacyanine 1b showed only weak antimalarial activity
at a dose of 10 mg kg-1 day-1 by intraperitoneal (ip) injection
(entry 18), whereas the newly designed azarhodacyanines 2
methyl iodide furnished cyanine 9. The condensation of 7 and
9 in the presence of triethylamine, followed by an ion exchange
process, gave the desired 3a. By use of the above two
procedures, several azarhodacyanines 2a-c and 3a-l were
synthesized (Chart 1).15
Biological Results and Discussion
The antimalarial potency of several synthetic azarhodacy-
anines was evaluated in vitro against P. falciparum K1 (CQ-
resistant strain) according to the procedures described by
Desjardins and co-workers.16 Their cytotoxicities were deter-

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15 4797
Table 3. In Vivo Antimalarial Potency of 3c and 3d at Various Doses
exhibited good parasitemia suppression (55-65%) at the same
dose (entries 1-3). The results clearly indicate that the
introduction of a nitrogen atom in the cyanine conjugation would
increase antimalarial efficacy in vivo. However, several signs
of acute toxicity, such as diarrhea and body weight loss, were
observed with 2a, 2b, and 1b. Moreover, doses higher than 25
mg kg-1 day-1 (ip) resulted in 100% fatality in mice within 24
h. In contrast, 2c exhibited no apparent acute toxicity at a dose
of 10 mg kg-1 day-1. However, disappointedly, an increase of
the dose (25 mg kg-1 day-1) afforded only slightly improved
activity of 63% suppression and obvious weight loss was
observed (entry 4). Moreover, no survival effect of the treated
mice was observed compared with the control (malaria-affected
mice without drug treatment).
(ip)a
compd
dose (mg kg-1 day-1
)
suppression (%)
MSDb
3c
10
20
40
10
20
40
79.8
97.4
99.1
70.8
92.4
97.8
0
11.8
20.3
7.0
9.8
19.0
7.8
3d
control
5.8
a In vivo evaluation was carried out according as Peters’ 4-day suppres-
sive protocol using five ICR-mice. b MSD ) mean survival days.
almost completely suppressed. The results clearly demonstrate
that ip administration of 3c and 3d at a dose of about 20 mg
kg-1 day-1 resulted in the best suppression of parasitemia and
the best mean survival of the mice.
Further investigation made clear that class II compounds 3
afforded much better results in antimalarial efficacy and toxicity,
although biological results in vitro were inferior to class I. All
compounds could be injected at a dose of 25 mg kg-1 day-1
(ip) and displayed moderate to excellent suppression levels at
the dose. Compounds 3a and 3b, bearing a quinoline moiety at
the left edge, showed 78% and 50% suppression levels,
respectively, but considerable weight loss during the period of
treatment was observed (entries 5 and 6, Table 2). Displacement
of the pyridine ring for quinoline resulted in significant
enhancement of antimalarial potency and survival effect.
Compound 3c resulted in 97% suppression at a dosage of 25
mg kg-1 day-1 by ip administration (entry 7). Although day-
to-day weight loss caused by its acute toxicity was observed
during the medication period, the mice still looked fine and their
body weight was recovered after the treatment. Finally, the mice
treated by 3c survived for 25 days on average. It is noteworthy
In summary, we synthesized two classes of antimalarial
azarhodacyanines 2 and 3 and evaluated the in vitro and in vivo
antimalarial potency against P. falciparum (CQ-resistant K1
strain) and P. berghei (NK-65 strain). Both of them were found
to possess promising in vitro potency with IC50 values ranging
from 4 to 23 nM and good selectivity indices of more than 500.
In vivo results indicated that class I azarhodacyanines 2 showed
higher acute toxicity than 3 (class II). Partial SAR study for 3
suggested that substituents on the heterocyclic moieties remark-
ably influence acute toxicity in vivo. 3c having pyridine and
6-chlorobenzothiazole moieties provided 97% suppression of
parasites at a dose of 20-25 mg/kg without signs of high
toxicity and significantly prolonged the survival of malaria
infected mice. Currently, pharmacokinetic and pharmacody-
namic studies of 3 are under consideration as well as the
synthesis of improved azarhodacyanines with a reduced toxicity
profile.
that the in vivo antimalarial potency of 3c (25 mg kg-1 day-1
)
is comparable to that of CQ at a dose of 10 mg kg-1 day-1
(entry 19). Replacement of chlorine atom at the benzothiazole
ring (a heteroaromatic moiety at the right edge) with a hydrogen
atom retains antimalarial efficacy and survival effect (entry 9),
whereas the fluorinated analogue resulted in lower potency
(entry 10). Interestingly and surprisingly, 3f and 3i, whose
structures are analogous to that of 3c, resulted in excellent
inhibition of parasitic growth comparable to 3c, but its acute
toxicity was significantly high to result in a much earlier death
of the animals (entries 11 and 14). A partial structure-activity
relationship study has revealed that the substituent on the right
rhodanine ring (see Figure 2) also influences the survival,
although the antimalarial activity is retained (entries 7, 12, and
13). Displacement of the pyridine ring of 3c with thiazolidine
(3j) slightly decreased parasites in suppression and survival
(entry 7 vs entry 15). Similar tendencies among structure,
activity, and acute toxicity for thiazolidine analogues 3j-l were
observed (entries 15-17). Thus, the toxicity was considerably
affected by R1 and R2 substituents (see Chart 1). We are now
investigating structure-toxicity relationships uncovered thus far
as well as conducting a pharmacokinetic study. It is noteworthy
that 3c has a 42% suppression level at a dosage of 100 mg
kg-1 day-1 (po) although mice survival was not extended (entry
8). We believe the results indicate that azarhodacyanines have
potential for oral bioavailability.
Experimental Section
Chemistry. Synthesis of 2a. To a mixture of 7 (244 mg, 0.50
mmol) and 2-amino-1-methylpyridinium p-toluenesulfonate (140
mg, 0.50 mmol) in acetonitrile (2.5 mL) was dropwise added
triethylamine (0.21 mL, 1.5 mmol), and the mixture was stirred at
70 °C for 12 h. To the mixture was added ethyl acetate (2.5 mL),
and the mixture was cooled to room temperature. After the mixture
was stirred for 30 min at room temperature, the precipitate formed
was collected and washed with CH3CN/EtOAc (1:1, v/v) to give
the crude product p-toluenesulfonate salt 8. The crude residue was
dissolved in CHCl3/MeOH (1:1, v/v), and the solution was then
passed through an anion-exchange resin (IRA-400(Cl)) by eluting
with CHCl3/MeOH (1:1, v/v). After concentration, recrystallization
from MeOH/EtOAc yielded 2a (147 mg, 71% yield) as orange
solids: mp 239-240 °C; IR (KBr) 1612, 1508, 1481, 1394, 1165
cm-1; 1H NMR (300 MHz, DMSO-d6) δ 8.74 (1H, d, J ) 6.3 Hz),
8.33 (1H, dd, J ) 8.1, 8.1 Hz), 7.98 (1H, d, J ) 9.3 Hz), 7.90 (1H,
d, J ) 8.5 Hz), 7.86-7.72 (4H, m), 7.47 (2H, dd, J ) 6.3, 8.1
Hz), 4.04 (3H, s), 4.01 (2H, q, J ) 7.1 Hz), 3.94 (3H, s), 1.29 (3H,
t, J ) 7.1 Hz); MS (FAB+) m/z 377 (M+). Anal. (C21H21ClN4OS)
C, H, N.
Synthesis of 3a. To a mixture of 7 (122 mg, 0.25 mmol) in
CH3CN (2.5 mL) was dropwise added triethylamine (0.11 mL, 0.75
mmol). The mixture was stirred at 70 °C for 15 h and then cooled
to room temperature. To this mixture was added EtOAc (3.0 mL),
and the mixture was stirred for 1 h. The precipitate was collected
and washed with CH3CN to give the crude residue (mixed salt of
iodide and p-toluenesulfonate). The crude was dissolved in CHCl3/
MeOH (1:1, v/v). The solution was passed through an anion-
exchange resin (IRA-400(Cl)) and eluted with the same mixed
solvent. After concentration, recrystallization from MeOH/EtOAc
yielded 3a (117 mg, 86%) as deep-purple solids: mp >300 °C; IR
Finally, 3c and 3d, showing high antimalarial activity with
low acute toxicity in vivo, were selected to investigate their
dose response by ip administration. As shown in Table 3,
significant improvements on suppression of parasitemia were
observed for 3c and 3d with increased doses. Nevertheless, these
compounds showed remarkable toxicity at a dose of more than
40 mg kg-1 day-1, although growth of malaria parasites was
(KBr) 1653, 1616, 1483, 1435, 1396, 173 cm-1; H NMR (400
1

4798 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 15
Brief Articles
(7) Kawakami, M.; Koya, K.; Ukai, T.; Tatsuta, N.; Ikegawa, A.; Ogawa,
K.; Shishido, T.; Chen, L. B. Synthesis and Evaluation of Novel
Rhodacyanine Dyes That Exhibit Antitumor Activity. J. Med. Chem.
1997, 40, 3151-3160.
MHz, DMSO-d6) δ 8.81 (1H, d, J ) 6.3 Hz), 8.39 (1H, dd, J )
7.2, 8.7 Hz), 7.98-7.85 (2H, m), 7.84-7.70 (4H, m), 7.54 (1H,
dd, J ) 6.3, 7.2 Hz), 7.44 (1H, dd, J ) 7.2, 7.5 Hz), 4.06 (3H, s),
4.04-3.97 (4H, m), 3.95 (3H, s), 1.28 (3H, t, J ) 7.0 Hz), 1.24
(3H, t, J ) 7.0 Hz); MS (FAB) m/z 504 (M+). Anal. (C26H26-
ClN5O2S2‚0.5H2O) C, H, N.
Biological Assays. In vitro antimalarial assay, in vitro cytotoxic
assay, and in vivo antimalarial assay were performed as reported
previously.9
(8) Chen, L. B. Mitochondrial Menmrane Potential in Living Cells. Annu.
ReV. Cell Biol. 1988, 4, 155-181.
(9) Takasu, K.; Shimogama, T.; Saiin, C.; Kim, H.-S.; Wataya, Y.; Brun,
R.; Ihara, M. Synthesis and Evaluation of â-Carbolinium Cations as
New Antimalarial Agents Based on π-Delocalized Lipophilic Cation
(DLC) Hypothesis. Chem. Pharm. Bull. 2005, 53, 653-661.
(10) Takasu, K.; Inoue, H.; Kim, H.-S.; Suzuki, M.; Shishido, T.; Wataya,
Y.; Ihara, M. Rhodacyanine Dyes as Antimalarials. 1. Preliminary
Evaluation of Their Activity and Toxicity. J. Med. Chem. 2002, 45,
995-998.
(11) Takasu, K.; Terauchi, H.; Inoue, H.; Kim, H.-S.; Wataya, Y.; Ihara,
M. Parallel Synthesis of Antimalarial Rhodacyanine Dyes by the
Combination of Three Components in One Pot. J. Comb. Chem. 2003,
5, 211-214.
(12) Takasu, K.; Morisaki, D.; Kaiser, M.; Brun, R.; Ihara, M. Syntheses
and Biological Activities of Structurally Stiff Rhodacyanines as Novel
Antimalarial Candidates. Heterocycles 2005, 66, 161-166.
(13) Ikegawa, A.; Ukai, T.; Kawakami, M. Water-Soluble Thiazolidinone
Merocyanines. JP Patent 06220053, 1994; Chem. Abstr. 123:172637.
(14) Sahu, M.; Garnik, B. K.; Behera, R. Influence of Substituents on the
Synthesis of Thiazolidinones. Indian J. Chem. 1987, 26B, 779-781.
(15) We do not assign all the geometries of synthetic azarhodacyanines
in this study. Although there are several possible geometrical isomers
of azarhodacyanines, the structures shown in the schemes and charts
are depicted as a single geometrical isomer. It is well-known that
the conjugated double bonds, such as the merocyanine and cyanine
moieties, can be easily isomerized in the solution and that the trans
geometrical isomers would be thermodynamic products.
Acknowledgment. We thank Dr. Kozo Sato, Dr. Hiroshi
Kitaguchi, and Dr. Masayuki Kawakami (Fuji Photo Film Co.,
Ltd.) for helpful discussions and for providing several com-
pounds. This work was financially supported by a Grant-in-
Aid from Japan Science and Technology Agency (JST) and a
Grant-in-Aid from the Takeda Science Foundation.
Supporting Information Available: Synthetic procedures and
characterization data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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