Synthesis of three classes of rhodacyanine dyes and evaluation of their in vitro and in vivo antimalarial activity

Article abstract of DOI:10.1016/j.bmc.2006.08.035

Selected members of three classes of rhodacyanine dyes, [0, 0]-, [1, 0]-, and [0, 0, 0]-rhodacyanines, were synthesized and their in vitro antimalarial activities against Plasmodium falciparum K1 (chloroquine-resistant strain) as well as their in vivo activities against P. berghei in mice were determined. The novel [0, 0, 0]-rhodacynines, 3e and 3h, possessing a benzothiazole moiety, were shown to have highly promising antimalarial activities in vivo. Moreover, the [0, 0, 0]-rhodacyanines were found to be orally bioavailable.

Full text of DOI:10.1016/j.bmc.2006.08.035

Bioorganic & Medicinal Chemistry 14 (2006) 8550–8563
Synthesis of three classes of rhodacyanine dyes and evaluation
of their in vitro and in vivo antimalarial activity
Khanitha Pudhom,a,b,c Kazuki Kasai,a Hiroki Terauchi,a Hiroshi Inoue,a Marcel Kaiser,d
Reto Brun,d Masataka Iharaa,b,e and Kiyosei Takasua,*
aDepartment of Organic Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University,
Aobayama, Sendai 980-8578, Japan
bJapan Science and Technology Agency (JST), 5-3 Yonbanchou, Chiyoda-ku, Tokyo 102-8666, Japan
cDepartment of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand
dSwiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
eHoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
Received 5 July 2006; revised 21 August 2006; accepted 22 August 2006
Available online 12 September 2006
Abstract—Selected members of three classes of rhodacyanine dyes, [0,0]-, [1,0]-, and [0,0,0]-rhodacyanines, were synthesized and
their in vitro antimalarial activities against Plasmodium falciparum K1 (chloroquine-resistant strain) as well as their in vivo activities
against P. berghei in mice were determined. The novel [0,0,0]-rhodacynines, 3e and 3h, possessing a benzothiazole moiety, were
shown to have highly promising antimalarial activities in vivo. Moreover, the [0,0,0]-rhodacyanines were found to be orally
bioavailable.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
malarial activity (EC50 = 12 nM) and significantly selec-
tive toxicity (1000).11 We also demonstrated that the
tricyclic skeleton of the rhodacyanine is required for
activity and a balance between molecular hydrophilicity
and hydrophobicity is important for efficacy.
Malaria is one of the most serious infectious diseases in
tropical and subtropical regions. At least 300 million
people are afflicted and 1–3 million people die from this
disease annually. Another serious issue is that malaria
parasites develop resistance to clinically used chemo-
therapeutic agents such as chloroquine, mefloquine,
and pyrimethamine.1–3 Therefore, a very urgent need ex-
ists to develop new classes of antimalarial drugs that
operate by novel mechanism of actions.4–7
The concept of DLC was originally proposed by Chen in
conjunction with the anticancer research efforts.8 Subse-
quently, it was reported that several DLC compounds
exhibit selective antitumor activity connected with their
selective accumulation in mitochondria of carcinoma
cells.15–18 The structures of members of the rhodacya-
nine family, shown in Figure 1, are comprised of three,
linearly linked heterocycles, in which the two end
Recently, we reported that rhodacyanine dyes 1, de-
signed by using the DLC (p-delocalized lipophilic cat-
ion) hypothesis,8 exhibit potent in vitro antimalarial
activity against Plasmodium falciparum FCR-3 strain
(chloroquine-sensitive).9,10 We also reported that some
DLCs in the rhodacyanine family display antimalari-
al11–13 and antileishmanial activities.13 An in vitro struc-
ture–activity relationship (SAR) investigation showed
that the rhodacyanine MKH-57 (1a) possesses high anti-
R1
Me
N
N
N
N
S
R3
S
Me
S
N
m
n
X
N
R2
TsO
O
O
MKH-57 (1a)
general structure of rhodacyanine
Keywords: Malaria; Rhodacyanines; DLC hypothesis; Drug resistance.
*
Figure 1. General structures of [m,n]-rhodacyanines and MKH-57
(1a).
Corresponding author. Tel.: +81 22 795 6878; fax: +81 22 795
6877; e-mail: kay-t@mail.pharm.tohoku.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.035

K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
R1
8551
dyes. Below, we describe the synthesis of selected mem-
bers of the three rhodacyanine classes [0,0]-, [1,0]-, and
[0,0,0]-rhodacyanines (Fig. 2), and present the results of
an evaluation of their in vitro antimalarial activity
against chloroquine-resistant strains of P. falciparum
K1 and in vivo antimalarial activity against P. berghei
in mice.
R1
N
N
N
N
S
S
R3
R3
N
N
X
X
R2
[1,0]-rhodacyanine (class II, 2)
O
R2
O
[0,0]-rhodacyanine (class I, 1)
O
R3
R1
N
X
N
S
R4
N
Me
S
N
R2
b
N
S
a
N
S
O
SMe
SMe
TsO
S
S
[0,0,0]-rhodacyanine (class III, 3)
N
4
5
6
Et
O
Figure 2. Rhodacyanines probed in this study include [0,0]-rhodacy-
anine (class I, 1), [1,0]-rhodacyanine (class II, 2), and [0,0,0]-
rhodacyanine (class III, 3).
Me
N
Me
N
S
S
N
c
SMe
TsO
S
S
S
N
O
Et
O
Et
heteroaromatic rings (A and C) are fused to a central
rhodanine (4-oxothiazolidine) ring (B). These dyes are
conjugates of two different dye units, a neutral merocy-
anine and a cationic cyanine moiety. Consequently, rho-
dacyanine compounds can be classified as p-delocalized
lipophilic cations (DLCs).
8
7
Me
N
d, e
N
S
(CH2CH2O)3Me
S
I
N
N
Cl
Me (CH2CH2O)3Me
O
Et
1h
9
Prior to our efforts in this area, there was no reports
describing the in vivo antimalarial activity of rhodacya-
nines.19 Thus, the novel and outstanding pharmacologi-
cal profiles displayed by rhodacyanine MKH-57 (1a)
prompted us to conduct a broad screen of the in vivo
activity of a series of structurally related rhodacyanine
Scheme 1. Synthetic route for preparation of [0,0]-rhodacyanines 1
(class I). Reagents and conditions: (a) methyl p-toluenesulfonate,
anisole, 125 ꢁC; (b) NEt3, 6, MeCN, 0 ꢁC to rt; (c) methyl
p-toluenesulfonate, DMF, 130 ꢁC; (d) NEt3, 9, MeCN, 70 ꢁC; (e)
Amberlyteꢂ IRA-400(Clꢀ).
Me
N
Me
N
X
N
S
Me
N
N
S
R3
N
S
Et
R1
Me
S
N
O
N
X
S
Cl
O
R2
N
O
Et
Cl
O
Et
R1
R2
R3
X
1m
1k (X = S), 1l (X = O)
H
H
H
H
H
H
H
H
H
TsO
Cl
1a
1b
1c
1d
1e
1f
CH2CH=CH2
Et
Me
Et
S
Me
Me
N
S
Me
N
N
Et
S
N
Me
Cl
CH2CO2Me
CH2CO2H
Et
Me
Me
S
Cl
O
N
Cl
S
N
Cl
CH2CH2OH
Br
Br
Cl
O
Et
O
Et
1o
1n
CH2CH=CH2 CH2CH2OH
1g
1h
1i
Et
Et
Et
Et
(CH2CH2O)3H
Me
Me
S
(CH2CH2O)3Me
Cl
N
N
N
N
S
S
CH2C6H4-p-CO2H Cl
Cl
Me
Me
N
N
Et
NHCO(CH2)4CO2Me
1j
Me
Cl
Cl
O
Et
O
1q
1p
Et
N
Me
N
X
Me
N
Me
N
S
N
Me
S
S
N
Et
S
N
N
Cl
N
Cl
TsO
O
Et
O
Et
O
Et
1r
1t (X = S), 1u (X = O), 1v (X = NMe)
1s
Chart 1. [0,0]-Rhodacyanines 1a–1v.

8552
K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
jugated to the rhodanine by double bond and, in class II,
Ac
H
the A-ring is tethered to the rhodanine by two methine
carbons. The class III [0,0,0]-rhodacyanines are unique
in that they possess two linked rhodanine moieties in the
center of the skeleton.
Ph
N
Ph
N
S
S
S
b
a
S
S
S
N
N
N
O
Et
O
Et
O
Et
11
10
6
More than 100 different [0,0]-rhodacyanines, including
1a–1v shown in Chart 1, were prepared by using the pre-
viously reported procedure.11,16 The routes used are
exemplified in Scheme 1 for the synthesis of 1h. Meth-
ylthiobenzothiazolium salt (5), obtained by N-methyla-
tion of methylthiobenzothiazole (4) with methyl
p-toluenesulfonate, was condensed with ethylrhodanine
(6) to afford merocyanine 7. In order to enhance the
electrophilicity of thiocarbonyl group, S-methylation
of 7 was carried out with methyl p-toluenesulfonate to
afford thioiminium salt 8. Treatment of 8 with picolini-
um salt 9 in the presence of triethylamine at 70 ꢁC fol-
lowed by ion exchange using Amberlyteꢂ IRA-400
(Clꢀ) gives the desired rhodacyanine dye 1h.20
Me
N
d
c
S
S
S
-
Me
+
N
TsO
N
Me
O
Et
S
13
12
Me
N
e
S
S
SMe
-
+
+
TsO
N
-
N
TsO
Me Me
O
Et
15
14
The class II [1,0]-rhodacyanines 2a–2g were synthesized
by using a modification of the general procedure.15 An
example of the routes used is shown in Scheme 2 for
the synthesis of 2a. Rhodanine 6 is reacted with N,N0-
diphenylformamidine to give 10, which without purifica-
tion is treated with acetic anhydride to produce acetam-
ide 11. Subsequent reaction of 11 with N-methyl
methylbenzothiazolium salt (12) furnishes merocyanine
13, a vinylog of merocyanine 7. Cation 14, obtained
by S-methylation of 13 with methyl p-toluenesulfonate,
is condensed with picolinium salt 15 to afford the desired
[1,0]-rhodacyanine 2a.
Me
N
+
N
S
S
Me
-
N
TsO
O
Et
2a
Scheme 2. Synthetic route for preparation of [1,0]-rhodacyanines 2
(class II). Reagents and conditions: (a) PhN=CHNHPh, MeCN,
70 ꢁC; (b) Ac2O, NEt3, 110 ꢁC; (c) NEt3, 12, MeCN, 60 ꢁC; (d) methyl
p-toluenesulfonate, DMF, 110 ꢁC; (e) NEt3, 15, MeCN, 75 ꢁC.
2. Results and discussion
A possible method for synthesis of the class III [0,0,0]-
rhodacyanines involves condensation of a cyanine deriv-
ative, as a right half segment consisting of two heterocy-
cles (Fig. 2, class III), with a merocyanine derivative (as
a left half segment). By using this strategy, the synthesis
of cyanine 1815 was accomplished by a route involving
reaction of benzothiazolium salt 16 with ethyl isothiocy-
anate, followed by treatment with bromoacetic acid.
Following the same procedure used for the synthesis
of class I and II rhodacyanines, 18 is condensed with
19 to furnish 3h (Scheme 3). However, the above method
2.1. Synthesis of the rhodacyanines
The rhodacyanine dyes probed in this effort are catego-
rized into three classes in the following manner, [0,0]-
rhodacyanine (class I), [1,0]-rhodacyanine (class II),
and [0,0,0]-rhodacyanines (class III) (Fig. 2). Dyes in
classes I and II are distinguished by the length of the
methine linkage between the heterocyclic A-rings and
rhodanine B-rings. In class I, the A-ring is directly con-
TsO
Et
S
Et
H
N
N
TsO
Me
b
N
N
a
S
Et
Et
S
S
S
N
Br
17
O
Et
16
18
O
Et
Me
Br
Et
c
N
N
S
Me
S
S
N
N
N
S
SMe
S
O
Et
S
N
TsO
O
Et
19
3h
Scheme 3. Synthetic route for preparation of [0,0,0]-rhodacyanines bearing benzothiazolium moiety at the right edge. Reagents and conditions:
(a) ethyl isothiocyanate, NEt3, pyridine, 110 ꢁC; (b) bromoacetic acid, AcOH, 90 ꢁC; (c) NEt3, 19, MeCN, 70 ꢁC.

K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
TsO
8553
Me
TsO
MeS
TsO
a
N
N
NHEt
NHEt
+
N
NHEt
Me Me
S
S
15
20
21
Scheme 4. Formation of bis(thioamide) 21. Reagents and conditions: (a) ethyl isothiocyanate, NEt3, pyridine, 110 ꢁC.
N
S
O
S
c
a, b
N
N
OEt
N
NHEt
24
O
S
Et
22
23
O
Et
Me
N
I
d
e
N
N+
Me
S
Me
N+
Me
S
N
N
S
N
SMe
TsO
I
O
Et
O
Et
N
O
Et
3a
26
25
Scheme 5. Synthetic route for preparation of [0,0,0]-rhodacyanines bearing a pyridinium moiety at the right edge. Reagents and conditions: (a)
EtNH2, MeOH, ꢀ78 ꢁC to rt; (b) Lawesson’s reagent, toluene, 115 ꢁC; (c) bromoacetyl chloride, CH2Cl2, rt; (d) MeI, acetone, 60 ꢁC; (e) NEt3, 26,
MeCN, 70 ꢁC.
cannot be employed to prepare [0,0,0]-rhodacyanines
that possess a pyridinium moiety at the right edge. This
is due to the fact that reaction of picolinium cations,
such as 15, with isothiocyanate affords only bis-thio-
amides (e.g., 21) by secondary reaction of the initially
formed and more highly acidic thioamides 20 with iso-
thiocyanate (Scheme 4). To reduce the acidity of the
methylene protons, the non-ionic intermediate 23 was
synthesized from ethyl pyridylacetate (22) by transami-
dation21 and successive thioamidation using Lawesson’s
reagent.22 Thioamide 23 was then treated with bromo-
acetyl chloride followed by quaternarization of the
resulting product 24 to provide the target cyanine 25.
Finally, condensation of 25 with 26 yields the [0,0,0]-
rhodacyanine 3a (Scheme 5).
summarized in Table 1. All compounds in this group
show promising in vitro antimalarial activities, having
EC50 values <100 nM and low cytotoxicity levels (with
the exception of compounds 1l and 1s). As a result,
the selectivity indices of these substances are estimated
to be quite high. The inhibitory effects of the class I
[0,0]-rhodacyanines against K1 strain of P. falciparum
(chloroquine-resistant) parallel those against the chloro-
quine-sensitive FCR-3 strain, as described in our previ-
ous reports.11,12 For example, the respective EC50 values
for 1a (MKH-57) against FCR-3 strain and K1 strain
are 12 and 19 nM. In contrast, chloroquine shows a
80-fold lower activity against P. falciparum K1
(EC50 = 1500 nM, entry 27) than FCR-311 (EC50
=
18 nM). The results indicate that no chloroquine-resis-
tance appears in the class I [0,0]-rhodacyanines. Similar
effects of hydrophilic groups on in vitro antimalarial
activity, observed in our previous work,11 were noted
with the class I rhodacyanines. Specifically, compounds
1e–1i, bearing hydrophilic substituents such as hydroxyl
groups (1e–1g), polyether chains (1h), and carboxylic
acids (1i) on the central rhodanine ring, have slightly
lower antimalarial activities (entries 6–13).
2.2. Biological results
The in vitro antimalarial activities of the rhodacyanines
against P. falciparum K1 (chloroquine-resistant strain)
were evaluated by using the procedures described by
Desjardins and his co-workers.23 The cytotoxicities of
these compounds to rat skeletal myoblast L-6 cells were
also measured and selective toxicities, defined by the ra-
tio EC50 (L-6)/EC50 (P. falciparum), were determined.
The synthetic rhodacyanines were also subjected to
in vivo antimalarial screening against P. berghei
NK-65 (drug-sensitive strain) in mice. The in vivo assays
were performed by using the Peters’ 4-day suppressive
test protocol.24 The suppression (expressed as a percent-
age) of malaria protozoa is determined by comparing
parasitemia of infected mice that are administered the
compounds by injection for 4 days with those of untreat-
ed control mice (Chart 1–3).
Owing to their high antimalarial activities and low cell
cytotoxicities, several class I [0,0]-rhodacyanines were
subjected to in vivo testing. The results clearly show that
these substances suppress the growth of malaria para-
sites (Table 1). However, despite having good in vitro
activities (EC50 = 19 and 21 nM), the parasite growth
suppression levels of 1a and 1b (10 mg/kg/d, ip) are only
27% and 30%, respectively (entries 1 and 2). In addition,
intravenous administration of 1b (5 mg/kg/d) does not
result in an improvement in parasitemia suppression
(entry 3), and mice death before the end of the drug-
treatment protocol was observed when higher doses of
1b are administered. Among the tested compounds,
2.2.1. Class I [0,0]-rhodacyanines. The antimalarial
potencies of the class
I [0,0]-rhodacyanines are

8554
K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
Me
N
N
S
S
R
N
X
Me
O
Et
S
N
N
S
R
X
S
Et
2a
2b
2c
2d
Me
TsO
Cl
N
CH2CH2OH
O
Et
AcO
(CH2CH2O)3Me
(CH2)4CO2H
Cl
2e
Cl
Et
Et
N
Me
N
N
S
N
Et
S
S
N
N
Cl
O
Et
O
Et
AcO
2f
2g
Chart 2. Prepared [1,0]-rhodacyanines 2a–2g.
O
Et
Me
N
O
Et
O
Et
Me
N
Me
N
Br
X
I
N
N
N
S
S
S
Me
Me
Et
S
S
S
N
N
N
N
N
N
S
O
Et
O
R
O
Et
3e
3f
X
I
R
3a
3b
3c
3d
Et
Ph
Bn
O
Et
Me
O
Et
Me
Br
N
Cl
N
Cl
Cl
I
N
N
N
S
N
S
N
Et
Me
S
S
S
N
S
CH2CHC=H2
S
O
Et
O
Et
3g
3h
O
Et
Me
O
Et
Me
Br
Cl
N
N
S
N
N
S
Et
Me
N
S
S
N
S
N
N
Me
N
S
O
Et
O
Et
3j
3i
Chart 3. [0,0,0]-Rhodacyanines 3a–3j.
[0,0]-rhodacyanines 1d, 1g, 1i, 1p, and 1q (entries 8, 9,
12, 13, 20, and 21) show moderate parasitemia suppres-
sion (50–60%) on intraperitoneal administration (5–
10 mg/kg/d).
potencies (63% and 53% suppression, respectively) at
the same dosages (entries 9 and 13). Second, class I rho-
dacyanines bearing hydrophilic substituents, such as 1d,
1e and 1g–1i, have high in vivo antimalarial activities in
comparison to analogous substances having hydropho-
bic substituents (entries 5, 6, and 8–13). This trend is
opposite to that seen in the in vitro testing results. The
difference could be a consequence of several factors
which can influence in vivo activities including absorp-
tion, metabolism, distribution, and excretion (ADME).
Third, a linear dependence of antimalarial activities on
dosage levels was not observed in the in vivo testing re-
sults (entries 8, 9, and 11–13). This suggests that ADME
factors influence the in vivo antimalarial potencies of the
[0,0]-rhodacyanines. Finally, [0,0]-rhodacyanines that
The results of antimalarial screening of the class I rho-
dacyanines have led to several important conclusions.
First, a good correlation does not exist between the in vi-
tro and in vivo activities of these substances. For exam-
ple, compound 1n shows the highest antimalarial
activity in vitro (EC50 = 1.0 nM) but it exhibits only
29% parasitemia suppression (10 mg/kg/d, ip) in vivo
(entry 18 in Table 1). On the other hand, compounds
1g and 1i, which display much lower in vitro activities
(EC50 = 37 and 83 nM), have markedly higher in vivo

K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
Table 1. In vitro and in vivo antimalarial activities of [0,0]-rhodacyanines
8555
Entry
Compound
EC50 (M), in vitro
Selectivityc
4 day suppressive test, in vivod
P. falciparuma
L-6b
Dose, method
% suppression
1
2
1a (MKH57)
1b (MKT077)
1.9 · 10ꢀ8
2.1 · 10ꢀ8
1.1 · 10ꢀ4
1.1 · 10ꢀ4
5.4 · 103
5.5 · 103
10 mg/kg/d, ip
10 mg/kg/d, ip
5 mg/kg/d, iv
10 mg/kg/d, ip
5 mg/kg/d, ip
5 mg/kg/d, iv
NTe
27.0
30.0
32.1
32.2
51.9
65.5
—
3
1b
1c
4
NTe
NTe
8.2 · 10ꢀ8
4.0 · 10ꢀ8
3.7 · 10ꢀ8
NTe
NTe
1.8 · 10ꢀ4
1.0 · 10ꢀ4
6.2 · 10ꢀ5
—
5
1d
1e
—
6
2.2 · 103
2.5 · 103
1.7 · 103
7
1f
1g
8
5 mg/kg/d, ip
10 mg/kg/d, ip
5 mg/kg/d, iv
1 mg/kg/d, ip
5 mg/kg/d, ip
10 mg/kg/d, ip
5 mg/kg/d, ip
10 mg/kg/d, ip
10 mg/kg/d, ip
5 mg/kg/d, iv
10 mg/kg/d, ip
NTe
60.9
62.6
58.4
12.4
52.3
53.0
33.4
37.3
38.4
51.9
28.6
—
9
1g
1h
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
5.3 · 10ꢀ8
8.3 · 10ꢀ8
1.3 · 10ꢀ4
5.3 · 10ꢀ5
2.4 · 103
6.4 · 102
1i
1i
1i
1j
1k
1.5 · 10ꢀ8
NTe
NTe
NTe
>2.0 · 10ꢀ4
NTe
NTe
NTe
>1.3 · 104
—
—
1l
1m
—
1n
1o
1.0 · 10ꢀ9
3.8 · 10ꢀ8
NTe
1.4 · 10ꢀ5
1.9 · 10ꢀ4
NTe
1.4 · 104
5.1 · 103
—
1p
1q
10 mg/kg/d, ip
10 mg/kg/d, ip
NTe
NTe
53.7
57.3
—
NTe
NTe
—
1r
1.1 · 10ꢀ8
5.5 · 10ꢀ8
NTe
2.5 · 10ꢀ5
6.0 · 10ꢀ6
NTe
2.2 · 103
1.1 · 102
—
1s
—
1t
1u
5 mg/kg/d, iv
5 mg/kg/d, iv
5 mg/kg/d, iv
10 mg/kg/d, ip
10 mg/kg/d, ip
50.2
51.1
41.4
90.6
18.6
NTe
NTe
NTe
NTe
—
—
1v
Chloroquine
Quinine
1.5 · 10ꢀ6
4.7 · 10ꢀ5
3.2 · 10
—
NTe
NTe
a Chloroquine-resistant strain (K1).
b Rat skeletal myoblast L-6 cells representing a model of host.
c Selective toxicity, EC50 value for L-6/EC50 for P. falciparum.
d Rodent malaria parasites, P. berghei (NK65 strain).
e NT means ‘not tested’.
have pyridinyl A and/or C ring (e.g., 1m, 1p, and 1q)
have contrastingly better in vivo parasitemia suppres-
sion properties.
9.7 nM) and an extremely low cytotoxicity (entry 13 in
Table 3). However, this substance is only weakly active
in vivo, with a suppression level of 29%, even at a high
dosage of 20 mg/kg/d (ip). On the other hand, 3a, which
has pyridine moieties on both edges, is 100-fold less ac-
tive in the in vitro screen but displays high levels of in vi-
vo activity (64% suppression at half dosage, entry 1). It
is noteworthy that compound 3a is not acutely toxic
even when administered at the high dosage of 50 mg/
kg/d (entry 2) but the suppression level of 3a is not
greatly improved at higher dosages.
2.2.2. [1,0]-Rhodacyanines (class II). In vitro and in vivo
antimalarial activities of members of the [1,0]-rhodacy-
anine series (class II) are listed in Table 2. All of the sub-
stances showed strong in vitro antimalarial activity
against the chloroquine-resistant strain with EC50 values
ranging 12–350 nM. However, the cytotoxicities of these
compounds against L-6 cells are almost 10-fold higher
than those of [0,0]-rhodacyanines. In vivo evaluation
of [1,0]-rhodacyanines showed that 2a–2d have low
activities against P. berghei (<25% parasitemia suppres-
sion at a dosage of 10 mg/kg/d (ip), entries 1–4). It was
found that administration of higher dosages (ꢁ50 mg/
kg/d) results in acute toxicity (animal death during the
treatment program).
In vivo screening of the class III rhodacyanines was car-
ried out at the highest dosage levels where no mice tox-
icity was observed. In contrast to the class I and II
rhodacyanines, nearly all of the [0,0,0]-rhodacyanines
could be safely injected at dosages of >50 mg/kg/d (ip).
Several compounds in this series display >50% parasite-
mia suppression at dosages that do not lead to animal
death. Noteworthy are [0,0,0]-rhodacyanines 3e and
3h that possess a benzothiazole ring at the right side.
These substances have high activities, providing 89%
suppression at dosages of 25 mg/kg/d (entries 8 and
12). These activities are comparable to those of chloro-
quine (entry 30 in Table 1). Moreover, in vivo tests show
that 3e and 3h have good dose–activity relationships (en-
tries 7, 8, 11, and 12). Unfortunately, mice treated with
2.2.3. Class III [0,0,0]-rhodacyanines. The results of
in vitro and in vivo antimalarial testing of the class III
[0,0,0]-rhodacyanines are summarized in Table 3. In a
manner similar to results from studies with the [0,0]-rho-
dacyanines, no correlation exists between the in vitro
and in vivo activities of the class III compounds. Com-
pound 3i, containing two side edged benzothiazole rings,
has strong in vitro antimalarial activity (EC50 value of

8556
K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
Table 2. In vitro and in vivo antimalarial activities of [1,0]-rhodacyanines
Entry
Compound
EC50 (M), in vitro
Selectivityc
4 day suppressive test, in vivod
Dose, method % suppression
P. falciparuma
L-6b
1
2
3
4
5
6
7
2a
2b
2c
2d
2e
2f
1.3 · 10ꢀ7
3.5 · 10ꢀ7
1.7 · 10ꢀ7
1.7 · 10ꢀ7
1.2 · 10ꢀ8
1.2 · 10ꢀ8
1.4 · 10ꢀ7
7.6 · 10ꢀ5
4.2 · 10ꢀ5
4.4 · 10ꢀ5
7.7 · 10ꢀ5
3.0 · 10ꢀ5
2.0 · 10ꢀ5
1.0 · 10ꢀ5
5.9 · 102
1.2 · 102
2.6 · 102
1.0 · 102
2.5 · 103
1.7 · 103
7.2 · 10
10 mg/kg/d, ip
10 mg/kg/d, ip
10 mg/kg/d, ip
10 mg/kg/d, ip
NTe
12.6
11.4
16.1
23.3
—
NTe
—
—
2g
NTe
a Chloroquine-resistant strain (K1).
b Rat skeletal myoblast L-6 cells representing a model of host.
c Selective toxicity, EC50 value for L-6/ EC50 for P. falciparum.
d Rodent malaria parasites, P. berghei (NK65 strain).
e NT means ‘not tested’.
Table 3. In vitro and in vivo antimalarial activities of [0,0,0]-rhodacyanines
Entry
Compound
EC50 (M), in vitro
Selectivityc
4 day suppressive test, in vivod
Dose, method % suppression
P. falciparuma
L-6b
1
2
3a
3a
3a
3b
3c
3d
3e
3e
3f
7.1 · 10ꢀ7
7.8 · 10ꢀ5
1.1 · 102
10 mg/kg/d, ip
50 mg/kg/d, ip
100 mg/kg/d, po
50 mg/kg/d, ip
50 mg/kg/d, ip
50 mg/kg/d, ip
10 mg/kg/d, ip
25 mg/kg/d, ip
50 mg/kg/d, ip
50 mg/kg/d, ip
10 mg/kg/d, ip
25 mg/kg/d, ip
20 mg/kg/d, ip
50 mg/kg/d, ip
64.0
68.9
55.0
56.3
23.3
25.9
46.9
88.7
69.1
52.8
56.4
89.0
28.7
53.4
3
4
NTe
NTe
NTe
NTe
NTe
NTe
NTe
NTe
—
—
—
—
5
6
7
8
9
NTe
NTe
NTe
NTe
NTe
NTe
—
—
—
10
11
12
13
14
3g
3h
3h
3i
9.7 · 10ꢀ9
>2.0 · 10ꢀ4
>2.0 · 104
—
3j
NTe
NTe
a Chloroquine-resistant strain (K1).
b Rat skeletal myoblast L-6 cells representing a model of host.
c Selective toxicity, EC50 value for L-6/ EC50 for P. falciparum.
d Rodent malaria parasites, P. berghei (NK65 strain).
e NT means ‘not tested’.
3e and 3h (25 mg/kg/d, ip, 4 days) gradually lose body
weight and survive only 1–2 days longer than untreated
controls. These results point out that 3e and 3h are toxic,
but the origin of the toxicity is not known.
between in vitro and in vivo activities of these sub-
stances. In vivo antimalarial screening revealed that
members of the [0,0,0]-rhodacyanine class, which pos-
sess two central rhodanine moieties, exhibit compara-
bly weak acute toxicities and potent suppressive
effects against malaria parasites. This is especially true
for 3e and 3h that have benzothiazole moieties at the
right edge. These substances are highly active in vivo
with suppressions of parasitemia that approach 89%
at dosages of 25 mg/kg/d (ip). However, further
improvement of toxicological properties of these com-
pounds is still required. Finally, we found that
[0,0,0]-rhodacyanines can be orally administered.
Observations made in this study serve as the rationale
for continuing work on the synthesis and biological
evaluation of novel antimalarial rhodacyanine dyes.
Finally, the oral bioavailability of class III rhodacyanine
3a was determined. Significantly, 3a has a 55% suppres-
sion level at a dosage of 100 mg/kg/d (po) and it does
not have a significant toxicity (entry 3).
3. Conclusions
Members of three classes of rhodacyanines were pre-
pared and evaluated in vitro against P. falciparum
(chloroquine-resistant K1) and cytotoxicity (mammali-
an L-6 cells) screens. Almost all of the substances were
found to possess promisingly high in vitro activities
and several showed both strong activities (EC50 values
ranging from 1 to 20 nM) and excellent selective toxic-
ities (up to 10,000). Finally, the in vivo activities of sev-
eral of rhodacyanines were examined by using P.
berghei in an animal model. The results clearly
demonstrated that no obvious correlation exists
4. Experimental
4.1. General methods
Unless otherwise described, the materials and the solvent
were obtained from commercial suppliers and used with-

K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
8557
out further purification. Melting points were determined
using an AS ONE ATM-01 apparatus and have not been
corrected. IR spectra were recorded with a SHIMADZU
FTIR-8300. The 1H and 13C NMR spectra were recorded
on a Varian Gemini 2000 (300 MHz) and a JEOL JNM-
AL400 (400 MHz) with tetramethylsilane as internal
standard. Chemical shifts are given in ppm, coupling con-
stants are in hertz, and splitting patterns are designated as
follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; q, quartet; m, multiplet. Fast atom bombardment
(FAB) mass spectra were determined with a JEOL JMS
DX-303 mass spectrometer. Electron ionization (EI) mass
spectra were recorded on a JEOL JMS AX-500
instrument. Elemental analyses were performed on
Yanagimoto MT-3, and the results (C, H, N) were within
0.4% of theoretical values. Because of deliquescence and
hygroscopicity, correct elemental analyses for most of the
compounds could only be obtained by factoring in partial
hydration of these organic salts. Compounds 1a, 1b, 1e,
1m–o, 1q–s, 1u, and 2e–g were known compounds.10,11,13,15,16
a solution of the iodide in methanol/dichloromethane
(1/1, v/v) was passed through the basic anion-exchange
resin (Amberliteꢂ IRA-400, chloride form), and the res-
in was washed with methanol. After the concentration
of the eluent chloride, the residue was crystallized from
ethyl acetate to give 1h (389 mg, 52% yield) as orange
solids. Mp 124–127 ꢁC (decomp.); 1H NMR
(400 MHz, DMSO-d6) d 8.61 (1H, d, J = 6.6 Hz), 8.28
(1H, dd, J = 8.0, 7.6 Hz), 8.08 (1H, d, J = 8.6 Hz), 7.86
(1H, d, J = 8.0 Hz), 7.59 (1H, d, J = 8.4 Hz), 7.50–7.45
(2H, m), 7.29 (1H, dd, J = 7.6, 7.6 Hz), 6.11 (1H, s),
4.79 (2H, t, J = 4.8 Hz), 4.08 (2H, q, J = 6.8 Hz), 4.02
(3H, s), 3.90 (2H, t, J = 4.6 Hz), 3.55 (2H, t,
J = 3.9 Hz), 3.46–3.40 (6H, m), 3.20 (3H, s), 1.23 (3H,
t, J = 6.8 Hz); MS (FAB) m/z 514 (M+); Anal.
C26H32ClN3O4S2Æ0.2H2O; C, 56.39; H, 5.90; N, 7.59.
Found: C, 56.64; H, 5.80; N, 7.74.
4.2.4.
2-[3-Methoxycarbonylmethyl-5-(3-methyl-(3H)-
benzothiazol-2-ylidene)-4-oxothiazolidin-2-ylidenemeth-
yl]-1-methylpyridinium chloride (1c). Orange solids; mp
275–277 ꢁC (decomp.); IR (KBr) 2943, 1745, 1656,
4.2. Typical procedure for the synthesis of [0,0]-rhoda-
cyanines (class I)
1548, 682 cmꢀ1 1H NMR (300 MHz, DMSO-d6) d
;
8.69 (d, 1H, J = 6.3 Hz), 8.27 (m, 1H), 8.06 (d, 1H,
J = 8.1 Hz), 7.83 (d, 1H, J = 7.7 Hz), 7.58 (d, 1H,
J = 8.1 Hz), 7.45–7.42 (m, 2H), 7.31–7.26 (m, 1H), 5.90
(s, 1H), 4.96 (s, 2H), 4.07 (s, 3H), 4.04 (s, 3H), 3.73 (s,
3H), 2.27 (s, 3H); MS (FAB) m/z 426 (M+); Anal.
C21H20ClN3O3S2; C, 54.60; H, 4.36; N, 10.39. Found:
C, 54.79; H, 4.59; N, 10.23.
4.2.1. 5-(3-Methyl-(3H)-benzothiazol-2-ylidene)-3-(2-pro-
penyl)-2-thioxo-4-thiazolidinone (7)10. A mixture of 2-
(methylthio)benzothiazole (4; 10.0 g, 55.2 mmol) and
methyl p-toluenesulfonate (15.4 g, 81.7 mmol) in anisole
(14 mL) was stirred at 120 ꢁC for 4 h. After the reaction
mixture was cooled to ambient temperature, to the result-
ing mixture was added a solution of3-ethyl-4-oxothiazoli-
dine-2-thione (6; 8.90 g, 55.2 mmol) in acetonitrile
(200 mL). To this mixture was added triethylamine
(12.6 mL, 90.5 mmol) dropwise under 0 ꢁC with constant
stirring and cooling, and the resulting mixture was stirred
at ambient temperature for 1 h. The yellow precipitate
was collected and washed with acetonitrile to give 7
(13.6 g, 80% yield) as yellow crystals, whose spectral data
were identical with reported ones.10
4.2.5.
2-[3-Carboxymethyl-5-(3-methyl-(3H)-benzothi-
azol-2-ylidene)-4-oxothiazolidin-2-ylidenemethyl]-1-meth-
ylpyridinium chloride (1d). Orange solids; mp
257–258 ꢁC (decomp.); IR (KBr) 3621–3200, 1651,
1
1627, 1506, 956 cmꢀ1; H NMR (300 MHz, DMSO-d6)
d 8.65 (d, 1H, J = 6.3 Hz), 8.31–8.25 (m, 1H), 8.06 (d,
1H, J = 8.5 Hz), 7.86 (d, 1H, J = 7.7 Hz), 7.63 (d, 1H,
J = 8.8 Hz), 7.53–7.42 (m, 2H) 7.31–7.26 (m, 1H), 5.90
(s, 1H), 4.85 (s, 2H), 4.07 (s, 3H), 4.04 (s, 3H); MS
(FAB) m/z 412 (M+); Anal. C20H18ClN3O3S2Æ1.75H2O;
C, 50.10; H, 4.52; N, 8.76. Found: C, 50.32; H, 4.89;
N, 8.53.
4.2.2. 3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-ylidene)-
2-methylsulfanyl-4-oxo-4,5-dihydrothiazol-3-ium p-tolu-
enesulfonate (8)10. A mixture of 7 (14.6 g, 47.4 mmol)
and methyl p-toluenesulfonate (26.5 g, 142 mmol) in
N,N-dimethylformamide (15 mL) was stirred at 130 ꢁC
for 3 h. After the mixture was cooled to 95 ꢁC, acetone
(125 mL) was added. The resulting mixture was further
cooled to ambient temperature with constant stirring.
The precipitate formed was collected and washed with
acetone to yield 8 (21.8 g, 93% yield) as orange crystals,
whose spectral data were identical with reported ones.10
4.2.6.
2-[3-Allyl-5-(3-methyl-(3H)-benzothiazol-2-yli-
dene)-4-oxothiazolidin-2-ylidenemethyl]-1-(2-hydroxyeth-
yl)pyridinium bromide (1f). Orange solids; mp 294–
295 ꢁC (decomp.); 1H NMR (300 MHz, DMSO-d6) d
8.54 (1H, d, J = 6.3 Hz), 8.29 (1H, dd, J = 8.5, 7.6 Hz),
8.05 (1H, d, J = 8.5 Hz), 7.86 (1H, d, J = 7.8 Hz), 7.61
(1H, d, J = 8.5 Hz), 7.52–7.44 (2H, m), 7.29 (1H, dd,
J = 7.6, 6.3 Hz), 5.99 (1H, s), 5.93–5.83 (1H, m), 5.31–
5.19 (2H, m), 4.69 (2H, m), 4.60 (2H, m), 4.05 (3H, s),
3.79 (2H, m); MS (FAB) m/z 424 (M+); Anal.
C22H22BrN3O2S2Æ0.36H2O; C, 51.71; H, 4.48; N, 8.22.
Found: C, 51.63; H, 4.12; N, 8.56.
4.2.3.
2-[3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-yli-
dene)-4-oxothiazolidin-2-ylidenemethyl]-1-{2-[2-(2-meth-
oxyethoxy)ethoxy]ethyl}pyridinium chloride (1h). To a
mixture of 8 (672 mg, 1.36 mmol) and 9 (500 mg,
1.36 mmol) in acetonitrile (6.8 mL) was dropwise added
triethylamine (0.54 mL, 9.08 mmol) at 70 ꢁC, and the
mixture was stirred for 16 h at the same temperature.
After the mixture was cooled to ambient temperature,
EtOAc was poured onto the resulting mixture. The
orange precipitate was collected and washed with ethyl
acetate to give the iodide salt as orange crystals. Then,
4.2.7. 2-[3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-ylid-
ene)-4-oxothiazolidin-2-ylidenemethyl]-1-{2-[2-(2-hydroxy-
ethoxy)ethoxy]ethyl}pyridinium chloride (1g). Orange
solids; mp 216–217 ꢁC (decomp.); IR (KBr) 3423,
1628, 1506, 1445, 1387, 1169 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6) d 8.62 (1H, d, J = 7.8 Hz), 8.27

8558
K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
1
(1H, dd, J = 8.5, 8.5 Hz), 8.07 (1H, d, J = 7.8 Hz), 7.84
(1H, d, J = 7.8 Hz), 7.59 (1H, d, J = 8.5 Hz), 7.50–7.43
(2H, m), 7.27 (1H, dd, J = 8.5, 7.8 Hz), 6.10 (1H, s),
4.79 (2H, t, J = 4.9 Hz), 4.59 (1H, s), 4.07 (2H, q,
J = 7.1 Hz), 4.02 (3H, s), 3.90 (2H, t, J = 4.9 Hz),
3.58–3.53 (2H, m), 3.48–3.41 (4H, m), 3.40–3.25 (2H,
m), 1.23 (3H, t, J = 7.1 Hz); MS (FAB) m/z 500 (M+);
Anal. C25H30ClN3O4S2Æ0.4H2O; C, 55.27; H, 5.71; N,
7.73. Found: C, 55.21; H, 5.74; N, 7.69.
IR (KBr) 3430, 1629, 1163 cmꢀ1; H NMR (300 MHz,
DMSO-d6) d 8.56 (1H, d, J = 8.8 Hz), 8.46 (1H, d,
J = 6.3 Hz), 8.08–8.03 (2H, m), 7.66 (1H, dd, J = 8.8,
7.1 Hz), 7.14–7.16 (1H, m), 6.88 (1H, dd, J = 7.1,
6.6 Hz), 5.74 (1H, s), 4.12 (3H, s), 4.07 (2H, q,
J = 7.0 Hz), 4.02 (3H, s), 1.23 (3H, t, J = 7.0 Hz); MS
(FAB) m/z 326 (M+); Anal. C25H27ClN3O4S2Æ0.3H2O; C,
55.76; H, 5.17; N, 7.80. Found: C, 55.38; H, 4.89; N, 8.11.
4.2.13. 2-[3-Ethyl-5-(1-methyl-(1H)-quinolin-2-ylidene)-4-
oxothiazolidin-2-ylidenemethyl]-3-methylbenzothiazolium
chloride (1t). Purple needles; mp 277–280 ꢁC (decomp.);
1H NMR (300 MHz, DMSO-d6) d 8.29–8.25 (1H, m),
8.21 (2H, dd, J = 8.8, 6.8 Hz), 7.99–7.85 (4H, m), 7.72
(1H, dd, J = 9.4, 6.8 Hz), 7.61–7.50 (2H, m), 6.71 (1H,
s), 4.26 (2H, q, J = 6.6 Hz), 4.13 (3H, s), 4.05 (3H, s),
1.29 (3H, t, J = 7.0 Hz); MS (FAB) m/z 432 (M+); Anal.
C24H22ClN3OS2Æ0.6H2O; C, 60.20; H, 4.88; N, 8.78.
Found: C, 60.52; H, 4.91; N, 8.60.
4.2.8. 1-(4-Carboxybenzyl)-2-[3-ethyl-5-(3-methyl-(3H)-
benzothiazol-2-ylidene)-4-oxothiazolidin-2-ylidenemeth-
yl]pyridinium chloride (1i). Orange solids; mp 210–
212 ꢁC (decomp.); IR (KBr) 3412, 1713, 1624, 1497,
1381, 1159 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
;
8.85 (1H, d, J = 6.0 Hz), 8.34 (1H, dd, J = 8.0, 8.0 Hz),
8.06 (1H, d, J = 8.5 Hz), 7.97 (2H, d, J = 8.2 Hz), 7.87
(1H, d, J = 7.5 Hz), 7.63 (1H, d, J = 8.2 Hz), 7.55–7.45
(2H, m), 7.34–7.29 (2H, m), 5.96 (2H, s), 5.74 (1H, s),
4.04 (3H, s), 3.88 (2H, q, J = 7.2 Hz), 0.78 (3H, t,
J = 7.2 Hz); MS (FAB) m/z 502 (M+); Anal.
C27H34ClN3O3S2Æ0.68H2O; C, 57.87; H, 6.36; N, 7.50.
Found: C, 58.09; H, 6.03; N, 7.71.
4.2.14. 2-[3-Ethyl-5-(1-methyl-(1H)-quinolin-2-ylidene)-4-
oxothiazolidin-2-ylidenemethyl]-1,3-dimethyl-(3H)-ben-
zoimidazol-1-ium chloride (1v). Purple amorphous; 1H
NMR (300 MHz, DMSO-d6) d 8.75 (1H, m), 7.99–7.89
(3H, m), 7.63–7.30 (6H, m), 6.02 (1H, s), 3.97 (2H, q,
J = 7.0 Hz), 3.95 (6H, s), 3.78 (3H, s), 1.28 (3H, t,
J = 6.6 Hz); MS (FAB) m/z 429 (M+); Anal.
C25H25ClN4OSÆ2.1H2O; C, 59.71; H, 5.85; N,11.14.
Found: C, 59.60; H, 5.79; N, 11.52.
4.2.9. 2-{5-[6-Ethoxycarbonylpentanoylamino]-3-methyl-
(3H)-benzothiazol-2-ylidene}-3-ethyl-4-oxothiazolidin-2-
ylidenemethyl}-1-ethylpyridinium chloride (1j). Orange
solids; mp 241–243 ꢁC (decomp.); IR (KBr) 1732,
1
1697, 1647, 1541, 866, 761 cmꢀ1; H NMR (300 MHz,
DMSO-d6) d 10.17 (1H, s), 8.65 (1H, d, J = 6.3 Hz),
8.24 (1H, dd, J = 8.1, 8.1 Hz), 8.15 (1H, s), 8.00 (1H,
d, J = 8.3 Hz), 7.60–7.50 (2H, m), 7.40 (1H, dd,
J = 6.6, 6.3 Hz), 5.93 (1H, s), 4.13 (3H, s), 4.09 (2H, q,
J = 7.1 Hz), 4.01 (3H, s), 3.59 (3H, s), 2.34 (4H, m),
1.59 (4H, m), 1.26 (3H, t, J = 7.1 Hz); MS (FAB) m/z
539 (M+); Anal. C28H35ClN4O4S2Æ1.2H2O; C, 54.88; H,
6.15; N, 9.14. Found: C, 54.59; H, 6.01; N, 9.17.
4.3. Typical procedure for the synthesis of [1,0]-rhoda-
cyanines (class II)
4.3.1.
N-(3-Ethyl-4-oxo-2-thioxothiazolidin-5-ylidene-
methyl)-N-phenylacetamide (11). A mixture of 3-ethyl-
rhodanine (6; 9.98 g, 62.0 mmol) and N,N0-
diphenylformamidin (12.1 g, 62.0 mmol) in acetonitrile
(58 mL) was stirred at 70 ꢁC for 1 h and then cooled
to ambient temperature. The precipitate formed was col-
lected and washed with acetone to give 3-ethyl-5-pheny-
laminomethylene-2-thioxothiazolidin-4-one (10) as a
crude product (9.96 g). The crude product was mixed
with acetic anhydride (40.8 mL, 434 mmol) and triethyl-
amine (0.16 mL, 1.28 mmol), and then the resulting mix-
ture was stirred at 100 ꢁC for 30 min. The reaction
mixture was concentrated to about one-half volume un-
der reduced pressure. The residue was added MeOH
(184 mL) and stirred at ambient temperature for 1 h.
The precipitate formed was collected and washed with
MeOH to give 11 (9.46 g, 50% yield) as yellow crystals,
whose spectral data were identical with reported ones.15
4.2.10. 2-[3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-ylid-
ene)-4-oxothiazolidin-2-ylidenemethyl]-3-methylbenzothi-
azol-3-ium chloride (1k). Red amorphous; 1H NMR
(300 MHz, DMSO-d6) d 8.23 (1H, d, J = 8.0 Hz), 7.97–
7.91 (2H, m), 7.76–7.67 (2H, m), 7.54–7.49 (2H, m),
7.35 (1H, dd, J = 8.0, 7.1 Hz), 6.67 (1H, s), 4.26 (2H,
q, J = 7.1 Hz), 4.20 (3H, s), 4.04 (3H, s), 1.28 (3H, t,
J = 7.1 Hz); MS (FAB) m/z 438 (M+); Anal.
C22H20ClN3O2S3Æ0.9H2O; C, 52.19; H, 4.34; N, 8.30.
Found: C, 52.35; H, 4.21; N, 8.68.
4.2.11. 2-[3-Ethyl-5-(3-methyl-(3H)-benzothiazol-2-ylid-
ene)-4-oxothiazolidin-2-ylidenemethyl]-3-methylbenzo-
1
oxazol-3-ium chloride (1l). Red amorphous; H NMR
(300 MHz, DMSO-d6) d 7.97 (1H, d, J = 8.5 Hz), 7.94
(1H, d, J = 8.7 Hz), 7.73 (1H, d, J = 8.0 Hz), 7.71 (1H,
d, J = 8.2 Hz), 7.57–7.45 (3H, m), 7.32 (1H, dd, J = 7.6,
7.5 Hz), 6.28 (1H, s), 4.20 (2H, q, J = 6.9 Hz), 4.15 (3H,
s), 3.86 (3H, s), 1.26 (3H, t, J = 6.9 Hz); MS (FAB) m/z
422 (M+); Anal. C25H27ClN3O4S2Æ0.3H2O; C, 55.93; H,
4.61; N, 8.89. Found: C, 55.76; H, 4.82; N, 8.91.
4.3.2.
dene)-ethylidene]-2-thioxothiazolidin-4-one
3-Ethyl-5-[2-(3-methyl-(3H)-benzothiazol-2-yli-
(13).
A
mixture of 11 (1.00 g, 3.3 mmol), 2,3-dimethyl-
benzothiazol-3-ium p-toluenesulfonate (12; 1.09 g,
3.3 mmol), and acetic anhydride (0.43 mL, 4.6 mmol)
in acetonitrile (33 mL) was stirred at 50 ꢁC for 1 h. After
triethylamine (16.1 mL, 12.1 mmol) was added to the
mixture, the resulting mixture was stirred for an addi-
tional 4 h at 60 ꢁC and, then, cooled to ambient
temperature. The precipitate formed was collected and
washed with acetonitrile to give 13 (0.93 g, 85% yield)
4.2.12. 2-[3-Ethyl-{5-(1-methyl-(1H)-pyridin-2-ylidene)-
4-oxothiazolidin-2-ylidene}methyl]-1-methylpyridinium
chloride (1p). Orange solids; mp 226–229 ꢁC (decomp.);

K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
8559
as yellow amorphous. 1H NMR (400 MHz, DMSO-d6) d
7.81 (1H, d, J = 7.8 Hz), 7.52–7.41 (3H, m), 7.26 (1H,
dd, J = 7.8, 7.8 Hz), 5.74 (1H, d, J = 13.2 Hz), 4.01
(2H, q, J = 7.1 Hz), 3.66 (3H, s), 1.15 (3H, t, J = 7.1 Hz);
MS (EI) m/z 334; Anal. C15H14N2OS3; C, 53.86; H,
4.22; N, 8.38. Found: C, 53.92; H, 4.36; N, 8.16.
(1H, dd, J = 7.5, 7.5 Hz), 8.19 (1H, d, J = 8.2 Hz), 7.73
(1H, d, J = 7.8 Hz), 7.65 (1H, m), 7.42–7.39 (3H, m),
7.18 (1H, m), 6.34 (1H, s), 5.58 (1H, d, J = 12.6 Hz),
4.83 (2H, m), 3.99 (2H, d, J = 6.8 Hz), 3.90 (2H, m),
3.57 (3H, s), 3.53 (2H, m), 3.42 (2H, q, J = 7.0 Hz),
3.36 (2H, m), 3.20 (3H, s), 1.21 (3H, t, J = 7.0 Hz);
MS (FAB) m/z 540 (M+); Anal. C28H34ClN3O4S2; C,
58.37; H, 5.95; N, 7.29. Found: C, 58.24; H, 6.95; N,
7.28.
4.3.3. 3-Ethyl-5-[2-(3-methyl-(3H)-benzothiazol-2-ylid-
ene)-ethylidene]-2-methylsulfanyl-4-oxo-4,5-dihydrothia-
zol-3-ium p-toluenesulfonate (14). A mixture of 13
(1.50 g, 4.47 mmol) and methyl p-toluenesulfonate
(2.50 g, 13.4 mmol) in DMF (6 mL) was stirred at
135 ꢁC for 3 h. After being cooled to ambient tempera-
ture to the mixture was added acetone. The resulting
mixture was stirred for 30 min. The precipitate formed
was collected and washed with acetone to yield 14
(2.12 g, 91% yield) as green solids. This compound was
used for next reaction without further purification.
4.3.7.
1-(4-Carboxy-butyl)-2-{3-ethyl-5-[2-(3-methyl-
(3H)-benzothiazol-2-ylidene)-ethylidene]-4-oxothiazoli-
din-2-ylidenemethyl}pyridinium chloride (2d). Green
amorphous; mp 189–201 ꢁC (decomp.); IR (KBr) 1652,
1521, 1369 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
;
8.82 (1H, m), 8.43–8.17 (2H, m), 7.73 (1H, d,
J = 8.1 Hz), 7.53–7.10 (6H, m), 5.46 (1H, d,
J = 12.5 Hz), 3.60 (2H, q, J = 6.8 Hz), 3.56 (3H, s),
3.13–3.07 (4H, m), 1.24–1.16 (4H, m), 1.11 (3H, t,
J = 6.8 Hz); MS (FAB) m/z 494 (M+); Anal.
C26H28ClN3O3S2Æ0.8H2O; C, 57.35; H, 5.48; N, 7.72.
Found: C, 57.43; H, 5.72; N, 7.61.
4.3.4. 2-{3-Ethyl-5-[2-(3-methyl-(3H)-benzothiazol-2-yli-
dene)-ethylidene]-4-oxothiazolidin-2-ylidenemethyl}-1-meth-
ylpyridinium p-toluenesulfonate (2a). To a mixture of 14
(100 mg, 0.19 mmol) and 1,2-dimethylpyridinium p-tol-
uenesulfonate (15; 53.5 mg, 0.19 mmol) in acetonitrile
(5.8 mL) was added triethylamine (80 lL, 0.58 mmol)
dropwise. The reaction mixture was stirred at 75 ꢁC
for 2.5 h and, then, cooled to ambient temperature.
The precipitate formed was collected and washed with
acetonitrile to give 2a (33.0 mg, 29% yield) as green sol-
ids. Mp 245–248 ꢁC (decomp.); IR (KBr) 1674, 1539,
4.4. Typical procedure for the synthesis of [0,0,0]-
rhodacyanines (class III) outlined in Scheme 3
(for 3c, 3h, and 3i)
4.4.1.
2-(3-Ethyl-4-oxothiazolidin-2-ylideneamino)-3-
methyl-3-benzothiazolium bromide (18). To a mixture
of 2-amino-3-methyl-3-benzothiazolium p-toluenesulfo-
nate (16; 1.74 g, 4.70 mmol) and ethyl isothiocyanate
(0.42 mL, 4.70 mmol) in pyridine (2.5 mL) was added
dropwise triethylamine (0.66 mL, 4.70 mmol). The
reaction mixture was stirred at 110 ꢁC for 45 min
and then poured into a mixture of crushed ice and
water. The precipitate formed was collected and
washed with water and then dried up. To the residue
obtained were added bromoacetic acid (1.29 g,
9.26 mmol) and acetic acid (4 mL), and then the mix-
ture was stirred at 90 ꢁC for 30 min. After being
cooled to ambient temperature, to the mixture was
added diethyl ether (10 mL). The resulting mixture
was stirred for 30 min at the same temperature. The
precipitate formed was collected and washed with
diethyl ether. The crude product was recrystallized
from methanol to give 18 (1.72 g, 90%) as yellow sol-
ids. Mp 233–235 ꢁC (decomp.); 1H NMR (400 MHz,
DMSO-d6) d 8.51 (1H, d, J = 2.0 Hz), 8.14 (1H, d,
J = 9.0 Hz), 7.88 (1H, dd, J = 9.0, 2.0 Hz), 4.57 (2H,
s), 4.06 (3H, s), 3.93 (2H, q, J = 7.1 Hz), 1.26 (3H,
t, J = 7.1 Hz); MS (FAB) m/z 326 (M+); Anal.
C15H17BrN2OS2Æ0.7H2O; C, 45.27; H, 4.66; N, 7.04.
Found: C, 45.33; H, 4.69; N, 7.12.
1371, 1199 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d
;
8.77 (1H, d, J = 6.3 Hz), 8.35 (1H, dd, J = 8.4, 8.4 Hz),
8.13 (1H, d, J = 8.4 Hz), 7.74 (1H, d, J = 7.7 Hz), 7.58
(1H, dd, J = 7.5, 7.5 Hz), 7.47 (2H, d, J = 8.0 Hz), 7.44
(1H, s), 7.41–7.39 (2H, m), 7.21–7.09 (1H, m), 7.10
(2H, d, J = 8.0 Hz), 6.16 (1H, s), 5.63 (1H, d, J =
12.5 Hz), 4.19 (3H, s), 4.02 (2H, q, J = 7.0 Hz), 3.58
(3H, s), 2.28 (3H, s), 1.23 (3H, t, J = 7.0 Hz); MS
(FAB) m/z 408 (M+); Anal. C29H29N3O4S3Æ1H2O; C,
58.27; H, 5.23; N, 7.03. Found: C, 58.19; H, 5.08; N, 6.85.
4.3.5. 2-{3-Ethyl-5-[2-(3-methyl-(3H)-benzothizol-2-yli-
dene)ethylidene]-4-oxothiazolidin-2-ylidenemethyl}-1-(2-
hydroxyethyl)pyridinium chloride (2b). Green solids; mp
276–279 ꢁC (decomp.); IR (KBr) 3566, 1652, 1521,
1369 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 8.72
;
(1H, d, J = 5.8 Hz), 8.38 (1H, dd, J = 8.5, 8.5 Hz), 8.19
(1H, d, J = 8.0 Hz), 7.73 (1H, d, J = 7.8 Hz), 7.66 (1H,
dd, J = 6.3, 6.3 Hz), 7.42–7.39 (2H, m), 7.20–7.16 (1H,
m), 6.35 (1H, s), 5.59 (1H, d, J = 12.6 Hz), 5.32 (1H, t,
J = 4.8 Hz), 4.70 (2H, t, J = 4.8 Hz), 4.00 (2H, q,
J = 7.0 Hz), 3.85 (2H, t, J = 4.8 Hz), 3.57 (3H, s), 1.20
(3H, t, J = 7.0 Hz); MS (FAB) m/z 438 (M+); Anal.
C23H24ClN3O2S2Æ1H2O; C, 56.14; H, 5.33; N, 8.54.
Found: C, 55.92; H, 5.27; N, 8.77.
4.4.2. Compound 3h. To a mixture of 18 (106 mg,
0.24 mmol) and compound 19 (91.3 mg, 0.24 mmol) in
acetonitrile (2.4 mL) was dropwise added triethylamine
(0.71 mmol, 0.10 mL) at ambient temperature. The reac-
tion mixture was stirred at 70 ꢁC for 15 h. After being
cooled to ambient temperature, the mixture was stirred
for an additional 1 h. The precipitate formed was col-
lected and washed with acetonitrile to give 3h (108 mg,
73% yield) as green solids. Mp 258–259 ꢁC (decomp.);
4.3.6. 2-{3-Ethyl-5-[2-(3-methyl-(3H)-benzothizol-2-yli-
dene)-ethylidene]-4-oxothiazolidin-2-ylidenemethyl}-1-{2-
[2-(2-methoxyethoxy)ethoxy]ethyl}pyridinium
(2c). Green amorphous; mp 80–86 ꢁC (decomp.); IR
(KBr) 1541, 1386, 1199, 1147 cmꢀ1 1H NMR
(400 MHz, DMSO-d6) d 8.75 (1H, d, J = 5.8 Hz), 8.38
chloride
;

8560
K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
1
IR (KBr) 1657, 1504, 1411, 1367, 1271, 1063 cmꢀ1; H
with 1 N HCl. The resulting aqueous phase separated
was brought to pH 10 with K2CO3 and then extracted
again with CHCl3. The combined organic layers were
dried over Na2SO4 and concentrated to yield 23 as
yellow oil (1.45 g, 86% yield): 1H NMR (300 MHz,
CDCl3) d 9.82 (1H, s), 8.55 (1H, d, J = 5.8 Hz), 7.69
(1H, dd, J = 7.7, 7.6 Hz), 7.30 (1H, d, J = 7.7 Hz), 7.22
(1H, dd, J = 7.6, 5.8 Hz), 4.21 (2H, s), 3.67 (2H, q,
J = 7.2 Hz), 1.26 (3H, t, J = 7.2 Hz); MS (EI) m/z 180
(M+); Anal. C9H12N2S; C, 59.96; H, 6.71; N, 15.54.
Found: C, 60.12; H, 6.91; N, 15.63.
NMR (400 MHz, DMSO-d6)
d
8.29 (1H, d,
J = 7.7 Hz), 8.01 (1H, d, J = 8.5 Hz), 7.76 (1H, dd,
J = 8.5, 7.2 Hz), 7.58 (1H, dd, J = 7.7, 7.2 Hz), 6.75
(1H, s), 4.75 (2H, q, J = 7.2 Hz), 4.30–4.17 (4H, m),
3.93 (2H, t, J = 7.6 Hz), 3.47 (3H, s), 3.25 (2H, t,
J = 7.6 Hz), 1.41–1.32 (6H, m), 1.26 (3H, t,
J = 7.2 Hz); MS (FAB) m/z 531 (M+); Anal.
C24H27BrN4O2S2; C, 52.65; H, 4.97; N, 10.23. Found:
C, 52.73; H, 5.05; N, 10.26.
4.4.3. Compound 3c. Deep purple solids; mp 199–
201 ꢁC (decomp.); IR (KBr) 1636, 1545, 1491, 1377,
4.5.2. 3-Ethyl-2-pyridin-2-ylmethylenethiazolidin-4-one
(24). To a solution of 23 (2.70 g, 15 mmol) in dichloro-
methane (20 mL) was added bromoacetyl chloride
(3.75 mL, 45 mmol) at 0 ꢁC. The reaction mixture was
stirred at ambient temperature for 2 days. After addition
of saturated NaHCO3, the mixture was extracted with
EtOAc and washed with brine. The combined organic
layers were dried over Na2SO4 and concentrated under
reduced pressure to give 24 as colorless oil: IR (KBr)
1283, 1165, 1067 cmꢀ1
;
1H NMR (400 MHz,
DMSO-d6) d 8.60 (1H, d, J = 6.0 Hz), 8.47 (1H, d,
J = 8.7 Hz), 8.23 (1H, dd, J = 8.7, 7.3 Hz), 8.13 (1H,
d, J = 6.0 Hz), 7.70 (1H, dd, J = 7.3, 6.0 Hz), 7.61
(1H, d, J = 8.7 Hz), 7.44–7.33 (3H, m), 7.31–7.20
(3H, m), 6.94 (1H, dd, J = 7.8, 6.8 Hz), 5.82 (1H,
s), 5.42 (2H, s), 4.14 (3H, s), 4.06 (3H, s), 3.99
(2H, q, J = 7.2 Hz), 1.18 (3H, t, J = 7.2 Hz); MS
(FAB) m/z 515 (M+); Anal. C28H27ClN4O2S2Æ1.1H2O;
C, 58.90; H, 5.15; N, 9.81. Found: C, 58.97; H, 5.11;
N, 9.89.
1701, 1603, 1545, 1391 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 8.57 (1H, d, J = 5.7 Hz), 7.55 (1H, dd,
J = 7.8, 7.5 Hz), 7.11 (1H, d, J = 7.8 Hz), 6.97 (1H, dd,
J = 7.5, 5.7 Hz), 6.08 (1H, s), 3.83 (2H, q, J = 7.2 Hz),
3.68 (2H, s), 1.28 (3H, t, J = 7.2 Hz); MS (EI) m/z 220
(M+); Anal. C11H12N2OS; C, 59.97; H, 5.49; N, 12.72.
Found: C, 59.78; H, 5.46; N, 12.62.
4.4.4. Compound 3i. Green solids; mp >300 ꢁC; IR (KBr)
1664, 1641, 1539, 1478, 1318, 1275, 1065 cmꢀ1 1H
;
NMR (300 MHz, DMSO-d6)
d
8.29 (1H, d,
J = 8.0 Hz), 7.93 (1H, d, J = 8.4 Hz), 7.87 (1H, d,
J = 7.7 Hz), 7.72 (1H, dd, J = 8.4, 7.6 Hz), 7.57 (1H,
dd, J = 8.0, 7.6 Hz), 7.49 (1H, d, J = 8.2 Hz), 7.35 (1H,
dd, J = 8.2, 7.4 Hz), 7.26 (1H, dd, J = 7.7, 7.4 Hz),
6.66 (1H, s), 4.65 (2H, m), 4.27 (4H, m), 4.10 (3H, s),
1.42 (3H, t, J = 6.9 Hz), 1.34 (3H, t, J = 6.9 Hz), 1.28
(3H, t, J = 7.1 Hz); MS (FAB) m/z 579 (M+); Anal.
C28H27BrN4O2S4Æ0.5H2O; C, 50.29; H, 4.22; N, 8.38.
Found: C, 50.27; H, 4.22; N, 8.41.
4.5.3.
2-(3-Ethyl-4-oxothiazolidin-2-ylidenemethyl)-1-
methylpyridinium iodide (25). A mixture of 24 (363 mg,
1.65 mmol) and methyl iodide (0.64 mL, 9.89 mmol) in
acetone (4 mL) was stirred at 60 ꢁC for 3 h. The mixture
was concentrated under reduced pressure, and then
EtOAc (5 mL) was added to the residue. The precipitate
was collected and washed with EtOAc to give 25 (0.56 g,
93% yield) as light brown amorphous: IR (KBr) 1711,
1545, 1383, 1119 cmꢀ1; H NMR (300 MHz, DMSO-
1
4.5. Typical procedure for the synthesis of [0,0,0]-
rhodacyanines (class III) outlined in Scheme 5
(for 3a–3d, 3f, 3g, 3i, and 3j)
d6) d 8.87 (1H, d, J = 6.3 Hz), 8.43 (1H, dd, J = 8.0,
7.4 Hz), 8.13 (1H, d, J = 8.0 Hz), 7.73 (1H, dd, J = 7.4,
6.3 Hz), 6.23 (1H, s), 4.20 (3H, s), 4.16 (2H, s), 3.87
(2H, q, J = 7.2 Hz), 1.18 (3H, t, J = 7.2 Hz); MS
(FAB) m/z 235 (M+); Anal. C12H15IN2OSÆ0.5H2O; C,
38.82; H, 4.27; N, 7.43. Found: C, 39.01; H, 4.46; N,
7.51.
4.5.1. N-Ethyl-2-pyridylthioacetamide (23). To anhy-
drous ethylamine, which was collected from the heating
of 70% aq EtNH2 (14 mL) on KOH (0.50 g) at 70 ꢁC for
1 h, was added ethyl pyridylacetate (20, 1.52 mL,
10 mmol) at ꢀ78 ꢁC. After being stirred at the same tem-
perature for 5 h, the mixture was allowed to warm to
ambient temperature and was stirred for an additional
4 days. The mixture was evaporated to give N-ethyl-2-
pyridylacetamide as a pure form (1.7 g, quant.). 1H
NMR (300 MHz, CDCl3) d 8.56 (1H, d, J = 6.3 Hz),
7.67 (1H, dd, J = 7.8, 7.5 Hz), 7.28 (1H, d, J = 7.8 Hz),
7.21 (1H, dd, J = 7.5, 6.3 Hz), 3.72 (2H, s), 3.29 (2H,
q, J = 7.2 Hz), 1.12 (3H, t, J = 7.2 Hz); MS (EI) m/z
164 (M+).
4.5.4. Compound 3a. To a mixture of 25 (109 mg,
0.30 mmol) and compound 26 (132 mg, 0.30 mmol) in
acetonitrile (3 mL) was dropwise added triethylamine
(0.13 mL, 0.90 mmol). The mixture was stirred at
70 ꢁC for 15 h. After being cooled to ambient tempera-
ture, EtOAc (3 mL) was added to the mixture, and the
resulting was stirred for 1 h. The precipitate formed
was collected and washed with EtOAc. The crude prod-
uct was recrystallized from MeOH/EtOAc to give 3a
(118 mg, 68% yield) as green solids. Mp 251–252 ꢁC (de-
comp.); IR (KBr) 1634, 1543, 1489, 1375, 1167,
A solution of the amide (1.55 g, 9.3 mmol) and Lawes-
son’s reagent (1.88 g, 4.7 mmol) in toluene (10 mL)
was refluxed for 1 h. After the solvent was removed
under reduced pressure, 20% aq K2CO3 was added to
the residue. Then, the mixture was extracted with CHCl3
three times. The combined organic layer was washed
1003 cmꢀ1 1H NMR (300 MHz, DMSO-d6) d 8.62
;
(1H, d, J = 6.3 Hz), 8.43 (1H, d, J = 8.8 Hz), 8.23 (1H,
dd, J = 8.8, 7.5 Hz), 8.05 (1H, d, J = 6.6 Hz), 7.95 (1H,
d, J = 8.8 Hz), 7.64 (1H, dd, J = 8.8, 7.8 Hz), 7.37 (1H,
dd, J = 7.5, 6.6 Hz), 6.87 (1H, dd, J = 7.8, 6.3 Hz),
5.91 (1H, s), 4.20–3.95 (10H, m), 1.34–1.18 (6H, m);

K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
8561
MS (FAB) m/z 453 (M+); Anal. C23H25IN4O2S2Æ1H2O;
C, 46.15; H, 4.55; N, 9.36. Found: C, 46.09; H, 4.60;
N, 9.40.
C25H25IN4O2S3Æ1H2O; C, 45.87; H, 4.16; N, 8.56.
Found: C, 45.68; H, 4.32; N, 8.61.
4.5.10. Compound 3j. Deep purple solids; mp 242–243 ꢁC
(decomp.); IR (KBr) 1626, 1541, 1485, 1369, 1285, 1155,
1003 cmꢀ1; 1H NMR (400 MHz, DMSO-d6) d 8.61 (1H,
d, J = 6.0 Hz), 8.24 (1H, dd, J = 8.2, 7.5 Hz), 7.95 (1H,
d, J = 8.2 Hz), 7.78–7.69 (2H, m), 7.50–7.41 (2H, m),
7.36 (1H, dd, J = 6.0, 7.5 Hz), 5.91 (1H, s), 4.24–4.05
(7H, m), 3.84 (6H, s), 1.31 (3H, t, J = 7.0 Hz), 1.25
(3H, t, J = 7.0 Hz); MS (FAB) m/z 506 (M+); Anal.
C26H28IN5O2S2Æ0.5H2O; C, 48.60; H, 4.55; N, 10.90.
Found: C, 48.49; H, 4.57; N, 10.81.
4.5.5. Compound 3b. Deep purple solids; mp 246–247 ꢁC
(decomp.); IR (KBr) 1649, 1545, 1491, 1375, 1283, 1167,
1076 cmꢀ1; 1H NMR (400 MHz, DMSO-d6) d 8.57 (1H,
d, J = 6.3 Hz), 8.38 (1H, d, J = 8.5 Hz), 8.12–8.03 (2H,
m), 7.72–7.58 (4H, m), 7.52–7.46 (2H, m), 7.32 (1H,
dd, J = 6.3, 6.3 Hz), 7.21 (1H, d, J = 8.5 Hz), 6.89 (1H,
dd, J = 8.0, 6.8 Hz), 5.75 (1H, s), 4.11 (3H, s), 4.01
(3H, s), 3.98 (2H, q, J = 7.0 Hz), 1.16 (3H, t,
J = 7.0 Hz); MS (FAB) m/z 501 (M+); Anal.
C27H25ClN4O2S2Æ2.5H2O; C, 55.71; H, 5.19; N, 9.62.
Found: C, 55.67; H, 5.28; N, 9.74.
4.6. Biological experiment (malaria parasites)
4.6.1. In vitro antimalarial assay. Plasmodium falciparum
K1 strain (a clone originating from Thailand) was used
in this study. The strain was maintained in RPMI 1640
medium with 0.36 mM hypoxanthine, supplemented
with 25 mM N-2-hydroxyethylpiperazine-N0-2-ethane-
sulfonic acid (HEPES), 25 mM NaHCO3, neomycin
(100 U/mL), and 5 g/L of Albumaxꢂ II (lipid-rich bo-
vine serum albumin, GIBCO, Grand Island, NY,
USA), together with 5% washed human A+ erythro-
cytes. All cultures and assays were conducted at 37 ꢁC
under an atmosphere of 4% CO2, 3% O2, and 93% N2.
Cultures were kept in incubation chambers filled with
the gas mixture. Subcultures were diluted to a parasite-
mia of between 0.1% and 0.5% and the medium was
changed daily. Stock drug solutions were prepared in
100% DMSO at 10 mg/mL and heated or sonicated if
necessary to dissolve the sample. For the assay, the com-
pound was further diluted in serum-free culture medium
and finally to the appropriate concentration in complete
medium without hypoxanthine. The DMSO concentra-
tion in the wells with the highest drug concentration
did not exceed 1%.
4.5.6. Compound 3d. Green solids; mp 211–212 ꢁC (de-
comp.); IR (KBr) 1639, 1541, 1489, 1373, 1165 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6) d 8.61 (1H, d,
J = 6.1 Hz), 8.44 (1H, d, J = 8.5 Hz), 8.23 (1H, dd,
J = 8.5, 7.6 Hz), 8.08 (1H, d, J = 6.1 Hz), 7.90 (1H, d,
J = 8.5 Hz), 7.65 (1H, dd, J = 8.5, 8.5 Hz), 7.37 (1H,
dd, J = 6.1, 7.6 Hz), 6.89 (1H, dd, J = 8.5, 8.5 Hz),
6.10–5.99 (1H, m), 5.89 (1 H, s), 5.18 (1H, d,
J = 11.2 Hz), 4.93 (1H, d, J = 17.1 Hz), 4.77 (2H, m),
4.10 (3H, s), 4.09 (3H, s), 4.04 (2H, q, J = 7.1 Hz),
1.23 (3H, t, J = 7.1 Hz); MS (FAB) m/z 465 (M+); Anal.
C24H25IN4O2S2; C, 48.65; H, 4.25; N, 9.46. Found: C,
48.67; H, 4.18; N, 9.55.
4.5.7. Compound 3e. Green solids; mp 284–285 ꢁC (de-
comp.); IR (KBr) 1639, 1541, 1489, 1435, 1367,
1271 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 8.46
;
(1H, d, J = 8.5 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.16 (1H,
d, J = 6.5 Hz), 7.93 (1H, d, J = 8.5 Hz), 7.75 (1H, dd,
J = 8.5, 7.3 Hz), 7.69 (1H, dd, J = 8.5, 7.3 Hz), 7.51
(1H, dd, J = 8.0, 7.3 Hz), 6.98 (1H, dd, J = 7.3,
6.5 Hz), 6.66 (1H, s), 4.69 (2H, q, J = 7.2 Hz), 4.33–
4.20 (4H, m), 4.41 (3H, s), 1.43–1.32 (m, 6H), 1.27
(3H, t, J = 7.2 Hz); MS (FAB) m/z 523 (M+); Anal.
C26H27BrN4O2S3; C, 51.73; H, 4.51; N, 9.28. Found:
C, 52.04; H, 4.49; N, 9.48.
Assays were performed in sterile 96-well microtiter
plates, each well containing 200 lL of parasite culture
(0.15% parasitemia, 2.5% hematocrit) with or without
serial drug solutions. Seven 2-fold dilutions were used,
covering a range from 5 to 0.078 lg/mL. Each drug
was tested in duplicate and the assay was repeated
for active compounds showing an EC50 below
1.0 lg/mL. After 48 h of incubation at 37 ꢁC,
0.5 mCi 3H-hypoxanthine was added to each well.
Cultures were incubated for a further 24 h before
being harvested onto glass-fiber filters and washed
with distilled water. The radioactivity was counted
using a BetaplateTM liquid scintillation counter (Wallac,
Zurich, Switzerland). The results are recorded as
counts per minute per well at each drug concentration
and expressed as percentage of the untreated controls.
EC50 values are calculated from the sigmoidal inhibi-
tion curves.
4.5.8. Compound 3f. Green solids; mp 259–261 ꢁC (de-
comp.); IR (KBr) 1647, 1504, 1375, 1168 cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6)
d
8.68 (1H, d,
J = 6.5 Hz), 8.29 (1H, dd, J = 8.5, 7.2 Hz), 8.00 (1H, d,
J = 8.5 Hz), 7.77–7.66 (3H, m), 7.50–7.43 (2H, m), 7.40
(1H, dd, J = 7.2, 6.5 Hz), 7.10 (1H, d, J = 8.0 Hz), 6.01
(1H, s), 4.15 (3H, s), 4.13–4.02 (4H, m), 1.32–1.21
(6H, m); MS (FAB) m/z503 (M+); Anal. C27H27IN4O2-
S2Æ1.1H2O; C, 49.86; H, 4.53; N, 8.61. Found: C,
49.79; H, 4.62; N, 8.91.
4.5.9. Compound 3g. Red solids; mp >300 ꢁC; IR (KBr)
1655, 1497, 1379, 1285, 1173, 1065 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6) d 8.67 (1H, d, J = 6.5 Hz), 8.30
(1H, dd, J = 8.2, 7.7 Hz), 7.99 (1H, d, J = 8.2 Hz), 7.83
(1H, d, J = 7.7 Hz), 7.57–7.40 (4H, m), 7.25 (1H, dd,
J = 7.5, 7.5 Hz), 5.97 (1H, s), 4.20–4.09 (5H, m), 4.08–
3.98 (5H, m), 1.29 (3H, t, J = 7.0 Hz), 1.24
(3H, t, J = 7.0 Hz); MS (FAB) m/z 509 (M+); Anal.
4.6.2. In vitro cytotoxicity assay. The rat skeletal myo-
blast cell line (L-6 cells) was used to assess cytotoxicity
in host cells. The cells were grown in RPMI 1640 medi-
um supplemented with 1% L-glutamine (200 nM) and
10% fetal bovine serum in T-25 tissue culture flasks at

8562
K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
37 ꢁC in 5% CO2 in air. The cultures were subpassaged
three times a week using trypsin to detach the cells
and split in a 1:2 or 1:3 ratio depending on the density
of the parent culture. Stock drug solutions were pre-
pared in 100% DMSO at 10 mg/mL. For the assays,
the compound is further diluted to the appropriate con-
centration using complete medium. The DMSO concen-
tration in the wells with the highest drug concentration
did not exceed 1%.
difference between the mean of the control group and
those of the experimental groups was calculated and
expressed as a percent relative to the control group (=
%suppression). The care and treatment of mice were in
accordance with the guidelines (No. 141, 1987) issued
by the Science and International Affairs Bureau of the
Japanese Ministry of Education, Culture, Science and
Technology.
Assays were performed in 96-well microtiter plates, each
well receiving 100 lL of culture medium with
4 · 104 cells. After 24 h, the medium was removed from
all wells and replaced by 100 lL of fresh medium in all
wells except for those in row H of the plate. Fresh medi-
um (150 lL) containing the highest drug concentration
was added to wells of row H. Serial drug dilutions were
prepared by transferring 50 lL from wells of row H to
wells of row G. After gentle mixing 50 lL from row G
was transferred to row F, and so on. The highest con-
centration for the test compounds was 200 lg/mL. Seven
3-fold dilutions were used, covering a range from 200 to
0.274 lg/mL. Each drug was tested in duplicate. After
72 h of incubation the plates were inspected under an
inverted microscope to assure growth of the controls
and sterile conditions. Then, 10 lL of Alamar blue
(12.5 mg resazurin dissolved in 100 mL distilled water)
was added to each well and the plates were incubated
for another 2 h. Then the plates were read with a Spec-
tramax Gemini XS microplate fluorometer (Molecular
Devices Cooperation, Sunnyvale, CA, USA) using an
excitation wavelength of 536 nm and an emission wave-
length of 588 nm. EC50 values are determined using the
microplate reader software Softmax Pro (Molecular
Devices Cooperation, Sunnyvale, CA, USA).
Acknowledgments
We thank Dr. Kozo Sato, Dr. Hiroshi Kitaguchi, and
Dr. Masayuki Kawakami (Fuji Film Co., Ltd.) for help-
ful discussions and providing several compounds. 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.
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K. Pudhom et al. / Bioorg. Med. Chem. 14 (2006) 8550–8563
8563
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