Rhodacyanine dyes as antimalarials. 1. Preliminary evaluation of their activity and toxicity

Article abstract of DOI:10.1021/jm0155704

The rhodacyanine dye MKT-077 (1), a potent antitumor agent, was found to possess strong in vitro activity against Plasmodium falciparum and a low cytotoxicity. Several new rhodacyanine dyes related to 1, containing a variety of linked heterocyclic moieties, were synthesized, and their antimalarial potencies were evaluated. The synthetic rhodacyanines were found to have EC₅₀ values against P. falciparum in vitro in the range of 4-300 nM. Several compounds in this series have remarkable selective toxicity profiles (> 100).

Full text of DOI:10.1021/jm0155704

© Copyright 2002 by the American Chemical Society
Volume 45, Number 5
February 28, 2002
Letters
Ta ble 1. Antimalarial Activity and Cytotoxicity
Rh od a cya n in e Dyes a s An tim a la r ia ls. 1.
P r elim in a r y Eva lu a tion of Th eir Activity
a n d Toxicity
EC50 (M)
selective
toxicityc
compd
P. falciparuma
FM3Ab
quinine
1.1 × 10-7
1.8 × 10-8
1.7 × 10-8
3.0 × 10-7
7.0 × 10-8
1.0 × 104
910
1800
65
33
210
chloroquine
3.2 × 10-5
1.1 × 10-6
1.0 × 10-5
1.5 × 10-5
Kiyosei Takasu,*,† Hiroshi Inoue,† Hye-Sook Kim,‡
Makoto Suzuki,§ Tadao Shishido,§
methylene blue
rhodamine 123
MKT-077 (1)
Yusuke Wataya,‡ and Masataka Ihara*,†
a
b
Chloroquine sensitive strain (FCR-3). Mouse mammary tu-
mor FM3A cells representing a model of host. c Selective toxicity
) EC50 value for FM3A/EC50 for P. falciparum.
Department of Organic Chemistry, Graduate School of
Pharmaceutical Sciences, Tohoku University, Aobayama,
Sendai 980-8578, J apan, Faculty of Pharmaceutical
Sciences, Okayama University, Tsushima,
Okayama 700-8530, J apan, and Ashigara Research
Laboratories, Fuji Photo Film Company, Ltd.,
210 Nakanuma, Minamiashigara,
larial compounds has become a worldwide issue. New
antimalarials, which have novel mechanisms of action,
are of great importance in this regard since these
substances will likely be effective in the treatment of
patients infected with drug-resistant parasites.4
Kanagawa 250-0193, J apan
Received September 7, 2001
Broad screening of a number of heterocycles and
carbohydrates in our compound library5-7 indicated that
several substances containing delocalized cationic moi-
eties have good antimalarial activity (Table 1). Particu-
larly significant was the finding that the rhodacyanine
dye MKT-077 (1)8 displays strong activity (EC50 ) 7.0
Abstr a ct: The rhodacyanine dye MKT-077 (1), a potent
antitumor agent, was found to possess strong in vitro activity
against Plasmodium falciparum and a low cytotoxicity. Several
new rhodacyanine dyes related to 1, containing a variety of
linked heterocyclic moieties, were synthesized, and their
antimalarial potencies were evaluated. The synthetic rhoda-
cyanines were found to have EC50 values against P. falciparum
in vitro in the range of 4-300 nM. Several compounds in this
series have remarkable selective toxicity profiles (>100).
Malaria is the most serious and widespread parasitic
disease in tropical and subtropical zones. This disease
is estimated to cause millions of deaths each year with
most occurring in infants and young children. Recently,
even inhabitants of temperate zones may be exposed to
the danger of malaria infection owing to global warming.
In addition, the appearance and rapid spread of drug-
resistant malaria parasites has reduced the effective-
ness of commonly used chemotherapeutic agents.1-3
Therefore, the development of new families of antima-
× 10-8 M) in vitro against the erythrocytic stage of
Plasmodium falciparum and a good selective toxicity
(210). These properties make this dye nearly as potent
as traditional antimalarial drugs, such as chloroquine
and quinine (Table 1). Recently, 1 has been developed
as a novel anticancer agent, and it has been subjected
to further clinical investigation for the treatment of solid
tumors.9,10 In this communication, we describe prelimi-
nary results of our studies concentrating on the syn-
thesis of rhodacyanine dyes related to 1 and an evalu-
ation of the antimalarial activity of these substances in
vitro against P. falciparum.
* To whom correspondences should be addressed. (K.T.) Phone:
+81-22-217-6879.
Fax:
+81-22-217-6877.
E-mail:
kay-t@
mail.pharm.tohoku.ac.jp. (M.I.) Phone: +81-22-217-6887. Fax: +81-
22-217-6877. E-mail: mihara@mail.pharm.tohoku.ac.jp.
† Tohoku University.
‡ Okayama University.
§ Fuji Photo Film Company, Ltd.
10.1021/jm0155704 CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/05/2002

996 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Letters
Ta ble 2. Effect of Skeletal Composition on Antimalarial
Activity
F igu r e 1. General formula of the rhodacyanine dye.
Sch em e 1a
P. falciparum
compd
framework
EC50 (M)
1
24
25
26
27
A-B-C
A-B
7.0 × 10-8
>2.8 × 10-5 a
3.7 × 10-6
5.2 × 10-7
2.4 × 10-7
A-B
B-C
A-C
a
EC32 value (68% growth of P. falciparum was observed).
reported procedures (structures see Table 2).11 Each of
these substances lack one of the three ring systems
found in 1. Cyanine 27, which does not contain a
rhodanine (B) ring, was also prepared by the condensa-
tion of 4 and 1,2-dimethylpyridinium p-toluenesulfonate
in the presence of triethylamine (44% yield).
a
Conditions: (a) NEt3, CH3CN, 0 °C; (b) p-TsOMe, DMF, 120
°C; (c) 1,2-dimethylpyridinium p-toluenesulfonate, CH3CN, NEt3
70 °C; (d) 1,4-dimethylpyridinium p-toluenesulfonate, CH3CN,
NEt3 70 °C; (e) Amberlite IRA-400 (Cl).
The antimalarial activities of the synthetic com-
pounds were evaluated in vitro against P. falciparum
(chloroquine sensitive FCR-3 strain), and their cytotox-
icities were determined against mouse mammary tumor
FM3A cells.12 Selective toxicities, defined by the ratio
EC50(FM3A)/EC50(P. falciparum), were determined.
In general, rhodacyanine dyes consist of three, lin-
early linked heterocyclic groups, in which two end
heteroaromatic rings (A and C, Figure 1) flank a
rhodanine (4-oxothiazolidine) B-ring. The dyes are
double conjugates of two different dye units, having left
and right parts comprised of neutral merocyanine and
cationic cyanine structures. MKT-077 (1), a member of
this family, was previously synthesized by means of
sequential condensation of its A and B components
followed by addition of the C-ring moiety. Following this
general strategy (with some modifications),8,11 rhoda-
cyanines 2, 3, 8-23 were prepared. The sequence,
shown in Scheme 1 to prepare 2 and 3, typifies the
procedures used. Accordingly, condensation of thiazo-
lium salt 4, prepared from 2-(methylthio)benzothiazole
and methyl p-toluenesulfonate, with 3-allylrhodanine
(5) in the presence of triethylamine at 0 °C affords the
neutral merocyanine 6 (1 h) in 78% yield (from 5 for
two steps). S-Methylation of 6 is accomplished by
reaction with methyl p-toluenesulfonate at 120 °C for
1.5 h and gives 7 in 99% yield. Treatment of 7 with 1,2-
dimethylpyridinium p-toluenesulfonate in the presence
of triethylamine at 70 °C for 1.5 h then provides the
desired rhodacyanine 2a as bright orange crystals in
41% yield. Treatment of 2a with the chloride ion form
of ion-exchange resins, such as Amberlite IRA-400, gives
the chloride salt 2b quantitatively. Rhodacyanine 3 was
obtained by use of this methodology starting with 7 and
1,4-dimethylpyridinium p-toluenesulfonate. To see if
smaller structural components of these substances are
responsible for their biological activity, we prepared
merocyanine dyes 24 and 25 and cyanine 26 by using
The requirement for each of the heterocyclic compo-
nents of the rhodacyanine dyes (Table 2) was evidenced
by the very low (EC50 values of less than 10-6 M)
antimalarial activities of merocyanines 24 and 25.
Cyanines 26 and 27, which have no merocyanine
conjugation, display moderate activity against P. falci-
parum, but their potency is lower than that of MKT-
077 (1). Thus, tricyclic-rhodacyanine structures, con-
taining both merocyanine and cyanine conjugation, are
required for high antimalarial activity.
The antimalarial potencies of rhodacyanines, which
contain an A-ring 2-benzothiazole moiety, are sum-
marized in Table 3. Compound 8a , which is the N-
methylpyridinium analogue of 1, displays enhanced
antimalarial activity (EC50 value of 2.6 × 10-8 M),
although its cytotoxicity is the same as that of 1. Four
rhodacyanine dyes (8a -d ) with different counteranions
were found to have similar activities. Cytotoxicities
against FM3A cells in vitro are significantly altered by
changing the central oxothiazolidine ring N-substituent.
Replacement of ethyl substituents by methyl or benzyl
leads to 3-fold (8d vs 11) or 14-fold (8d vs 12) increases
in toxicity, respectively, although the antimalarial
activities of these substances are only slightly decreased
by this substitution. In contrast, the N-allylrhodacya-
nine 2a has low toxicity (EC50 value of 1.2 × 10-5 M)
and a high inhibitory effect on malaria parasites (EC50
) 1.2 × 10-8 M). Its selective toxicity is estimated to be
1000.

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5 997
Ta ble 3. Effect of Substituents and Counteranion on Antimalarial Activity and Cytotoxicity
a
b
Chloroquine sensitive strain (FCR-3). Mouse mammary tumor FM3A cells representing a model of host. c Selective toxicity ) EC50
value for FM3A/EC50 for P. falciparum. EC70 value (30% growth of P. falciparum was observed). e EC9 value (91% growth of FM3A cells
d
was observed). f EC17 value (83% growth). NT means not tested. EC19 value (81% growth). EC36 value (64% growth).
g
h
i
Ta ble 4. Effect of Ring A on Antimalarial Activity and Cytotoxicity
a
b
EC14 value (86% growth of FM3A cells was observed). NT means not tested.

998 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 5
Letters
The introduction of hydrophilic groups, such as hy-
droxyl and carboxyl, as R1 or R2 substituent, results in
a 1-2 order of magnitude decrease in activity (e.g., 9,
10, 13). This result points out that hydrophobicity is a
critical factor determining the antimalarial activity of
compounds in this family. The quinolinium ring con-
taining dye 15 displays the most potent inhibitory
activity (EC50 ) 4.6 × 10-9 M), but the cytotoxicity of
this substance is very high. Hydrophobic aromatic
residues on the dye skeleton (e.g., 12, 14, 15) cause
increases in cytotoxicity but not in antimalarial activity.
Thus, it appears that an appropriate balance needs to
be struck between hydrophilicity and hydrophobicity in
order to maximize the efficacy of the rhodacyanines.
Dyes containing a 1,4-pyridinium C-ring moiety (e.g.,
3, 16-18) have nearly the same activities as the
corresponding dyes containing the 1,2-pyridinium group-
ing (e.g., 2a , 8d , 12, and 15).
The effect of the merocyanine component was also
examined (Table 4). Compounds 19 and 20, which
contain substituted benzothiazole A-rings, have strong
antimalarial activities with EC50 values of 10-8 M.
However, other analogues with thiazoline, pyridine, and
quinoline A-rings (e.g., 21-23) are ca.10-fold less active
against P. falciparum than 8d . Thus, a benzothiazole
merocyanine unit is required for high antimalarial
activity.
The preliminary studies described above have uncov-
ered new types of rhodacyanine dyes, which display high
levels of antimalarial activity. Structure-activity stud-
ies indicate that the rhodacyanine skeleton is essential
for strong activity and that a balance between molecular
hydrophilicity and hydrophobicity is important for ef-
ficacy. Finally, compound 2a (named MKH-57) was
found to display high antimalarial activity and a
significantly good selective toxicity.
Currently, we are attempting to optimize the anti-
maliarial properties of the rhodacyanine dyes based on
structure-activity relationships uncovered thus far and
we are carrying out studies to gain information about
the mechanism of antimalarial action of these sub-
stances.
Priority Areas (No. 1147202) and for Exploratory Re-
search (No. 13877378) from the Ministry of Education,
Culture, Sports, Science and Technology, J apan.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and characterization data for compounds 2, 3, 6-9, 11,
12, 14-18, 21-23, and 27. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Ack n ow led gm en t. This work was partially sup-
ported by a Grant-in-Aid for Scientific Research on
J M0155704