Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity

Article abstract of DOI:10.1021/jm9702692

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC₅₀(CX-1) = 50 nM, IC₅₀(CV-1) = 17.3 μM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor- bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4- oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agent candidates.

Full text of DOI:10.1021/jm9702692

J . Med. Chem. 1997, 40, 3151-3160
3151
Articles
Syn th esis a n d Eva lu a tion of Novel Rh od a cya n in e Dyes Th a t Exh ibit An titu m or
Activity
Masayuki Kawakami,*,† Keizo Koya,† Toshinao Ukai,† Noriaki Tatsuta,† Akihiko Ikegawa,† Keizo Ogawa,†
Tadao Shishido,† and Lan Bo Chen‡
Ashigara Research Laboratories, Fuji Photo Film Company, Ltd., 210 Nakanuma, Minamiashigara, Kanagawa 250-01, J apan,
and Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115
Received April 23, 1997X
Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized
and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic
rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine
(rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure
were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1,
and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) )
50 nM, IC50(CV-1) ) 17.3 µM; selectivity index ) 346 for compound 5). These compounds
were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human
melanoma LOX; T/C (%) ) 168 for compound 5). Structural modifications on rhodacyanine,
including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and
replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-
1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-
activity studies imply that the double-conjugate system with a rhodanine moiety is essential
for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique
antitumor agent candidates.
Ch a r t 1
In tr od u ction
Mitochondria carry out most cellular oxidations and
produce the animal cell’s ATP. In mitochondria the
energy available from combining oxygen with reactive
electrons carried by NADH pumps out protons from the
inner membrane, and the energy is thus stored in the
electrochemical proton gradient which consists of a
membrane potential and a pH gradient. The resultant
transmembrane gradient is in turn used to synthesize
ATP. The membrane potential has been monitored by
organic compounds known as membrane potential sen-
sitive probes.1-3 These probes, most of which are the
positively charged organic compounds, are taken into
cells in response to the high negative charge in the
mitochondria, where they can be toxic preferentially.
Since the membrane potential of the mitochondria
in tumor cells is higher than that of normal cells,
these compounds are accumulated in the tumor mito-
chondria.4-6 Therefore, cytotoxic π electron-delocalized
lipophilic cations (DLCs) are proposed to kill tumor cells
selectively, and DLCs including rhodamine 123,4-7
dequalinium,8 thiacarbocyanines,9,10 and thiopyrylium
AA-1 (Chart 1)11 have been explored as potential
antitumor drugs. In spite of high potential as antitumor
agents, none of them have met the criteria for clinical
development, such as water solubility, stability, toxicity,
and pharmacokinetics.
In an effort to find novel DLCs, we have screened a
wide variety of DLCs from the compound library at Fuji
Photo Film Co., Ltd. which was developed for photo-
graphic systems. Rhodacyanine dyes that were origi-
nally studied as silver halide sensitizers showed high
inhibitory effect in vitro on the growth of tumor cells.
In this paper we focus on synthesis and evaluation of
† Fuji Photo Film Co., Ltd.
‡ Harvard Medical School.
X Abstract published in Advance ACS Abstracts, September 1, 1997.
S0022-2623(97)00269-0 CCC: $14.00 © 1997 American Chemical Society

3152 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kawakami et al.
densed with 3-ethyl-4-oxothiazolidine-2-thione to give
merocyanine dye 3. S-Methylation of 3 was followed
by the reaction of 3-ethyl-2-methylbenzothiazolium
iodide in the presence of triethylamine to give a rhoda-
cyanine dye (5). Other rhodacyanine dyes (n ) 0, n′ )
0) were also synthesized in an analogous manner, and
the results are summarized in Table 1.
3-Ethyl-4-oxothiazolidine-2-thione (6) was reacted
with 1,3-diaza-1,3-diphenylpropene and then with acetic
anhydride to give compound 7, which was condensed
with 3-ethyl-2-methylnaphtho[1,2-d]thiazolinium iodide
to give merocyanine dye 8. A rhodacyanine dye (10) was
synthesized from merocyanine intermediate 8 under
similar conditions as those for the synthesis of 5 from
3. Other rhodacyanine (n ) 1, n′ ) 0) dyes were also
synthesized in an analogous manner, and the results
are summarized in Table 1.
F igu r e 1. General formula of rhodacyanine dye and its
Because the rhodanine moiety is conjugated with two
double bonds, there are several possible geometrical
isomers around this moiety. However, our NMR studies
suggested that these rhodacyanine dyes exist as a single
isomer. As illustrated in Figures 2 and 3, X-ray
crystallographic studies for compounds 32 and 33
revealed that in the merocyanine unit N-substituents
were situated on the opposite side of methine carbons,
while in the cyanine unit N-substituents were sitting
on the same side for both cases. As for the geometrical
isomerism of the merocyanine moiety of 33, it was
determined to be the s-trans configuration (Figure 3).
For structure-activity studies, several DLC com-
pounds analogous to rhodacyanines, including analogs
whose heteroaromatic ring A was replaced with other
moieties (13a ,b, 14, 15, 23) and those whose rhodanine
moiety was substituted with other structurally related
moieties (20, 26, 28, 30), were synthesized. The results
are summarized in Tables 2 and 3.
component parts: merocyanine dye and cyanine dye.
Sch em e 1a
Synthesis of cyanine dye 13b was accomplished by
the reaction of 3-ethyl-2-methylbenzothiazolium iodide
(11) with ethyl thiocyanate followed by the reaction of
bromoacetic acid as illustrated in Scheme 3.13 Treat-
ment of 11 with phenyl thiocyanate gave N-phenyl
derivative 13a , which was converted to 14 and 15 by
the reaction of 1,3-diaza-1,3-diphenylpropene and 1,5-
diaza-1,5-diphenyl-1,3-pentadiene, respectively.
A rhodanine analog (20) was synthesized according
to the reported procedures as illustrated in Scheme
4.14,15 Carbodithioate 1614 was treated with triethyl
orthoformate and then 3-ethyl-2-methylbenzoxazolium
iodide to give merocyanine dye 18, which was further
converted to the target compound 20 under similar
conditions as those for the synthesis of rhodacyanine
dye 10.
Other analogs of the rhodanine moiety (26 and 28)
containing 4-oxoimidazolidine were synthesized by re-
ported procedures similar to those for the synthesis of
the corresponding rhodacyanine dye.16 The other rhoda-
nine analog (30) containing 4-oxo-1,3-dithiolane16 and
cyanine dyes (21 and 22)10 were also synthesized by
reported procedures, respectively.
a
Reagents: (a) methyl p-toluenesulfonate/anisole; (b) 3-ethyl-
4-oxothiazolidine-2-thione/NEt3/MeCN; (c) methyl p-toluene-
sulfonate/DMF; (d) 3-ethyl-2-methylbenzothiazolium iodide/NEt3/
MeCN.
novel rhodacyanine dyes and their analogs to clarify
structure-activity relationships.
Ch em istr y
Rhodacyanine dye consists of three rings, two het-
eroaromatic rings and a central 4-oxothiazolidine (rhoda-
nine), as represented in Figure 1. At the rhodanine
moiety, two dye conjugate systems, neutral merocyanine
with heteroaromatic ring A and cationic cyanine with
heteroaromatic ring B, are integrated. In the present
study, we synthesized two types of rhodacyanine dyes,
(n ) 0, n′ ) 0) and (n ) 1, n′ ) 0) in Figure 1, according
to the reported procedures12 with some modifications.
Typical synthesis examples are illustrated in Schemes
1 and 2, respectively. In both cases, merocyanine dyes
(3 and 8) were synthesized first followed by the conjuga-
tion with cyanine units.
Biologica l Resu lts a n d Discu ssion
In Vitr o Clon ogen ic Assa y. The compounds syn-
thesized here were evaluated for their inhibitory effects
on the growth of human colon carcinoma cell (CX-1) and
toxicity against normal epithelial cell (CV-1). In each
N-Methylation of 2-(methylthio)benzothiazole (1) us-
ing methyl p-toluenesulfonate gave 2, which was con-

Rhodacyanine Dyes That Exhibit Antitumor Activity
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3153
Sch em e 2a
a
Reagents: (a) (i) 1,3-diaza-1,3-diphenylpropene/ligroin, (ii) Ac2O/NEt3; (b) 3-ethyl-2-methylnaphthol[1,2-d]thiazolium p-toluenesulfonate/
Ac2O/NEt3/MeCN; (c) methyl p-toluenesulfonate/DMF/toluene; (d) 3-ethyl-2-methylnaphthol[1,2-d]thiazolium p-toluenesulfonate/NEt3/
MeCN.
Ta ble 1. Structure of Rhodacyanine Dyes
F igu r e 2. X-ray-determined structure of rhodacyanine dye
32.
rhodanine moiety. The rhodacyanine dyes thus syn-
thesized were found to be equipotent (5) or 1.6 times
more potent (10) to their cyanine counterparts as
depicted in Table 4. Another interesting finding about
rhodacyanines in Table 4 is their low toxicity against
CV-1 and the resulting high selectivity defined by
case, the cells were incubated with a compound for 24
h and then in compound-free medium for 2 weeks. The
number of colonies was measured by a crystal violet-
staining method, and the results are expressed as the
IC50 value.
IC50(CV-1)/IC50(CX-1): they were more selective than
the corresponding cyanines by 17-fold (5 vs 21) and 109-
fold (10 vs 22).
To understand why rhodacyanine dyes have such a
high activity specifically against tumor cells, our first
structure-activity study focused on deletion or replace-
ment of the heteroaromatic ring of the merocyanine
conjugate system (ring A in Figure 1). Simple deletion
of the N-ethylbenzothiazolidene group of rhodacyanine
5 led to a decrease of the activity by 26-fold (5 vs 13b
in Table 5). Replacement with a cyclohexylidene group
(23) resulted in further loss of activity. While com-
pounds 13b and 23 still retain some moderate activities,
it may be due to the delocalized cationic cyanine
Cyanine dyes such as thiacarbocyanine (S23)9 and
other DLCs4-8,11 were reported to exhibit selective
activity against carcinoma cells. In this study two
cyanine dyes with different methine lengths, 21 (S23)
and 22, showed high inhibitory effect on CX-1 with IC50
values of 60 nM (Table 4). As a part of structure
modifications of cyanine dyes in an attempt to find a
novel class of DLCs with higher activity and selectivity,
a methine unit of the cyanine dyes was replaced with a

3154 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kawakami et al.
Ta ble 3. Structure of Rhodacyanine Dye Analogs
F igu r e 3. X-ray-determined structure of rhodacyanine dye
33.
Ta ble 2. Structure of Rhodacyanine Dye Analogs
Sch em e 3a
p
a
Reagents: (a) 12a sphenyl thiocyanate/NaH/THF, 12bsethyl
thiocyanate/pyridine/NEt3; (b) 13a sbromoacetic acid/1-BuOH,
13bsbromoacetic acid/acetic acid; (c) 1,3-diaza-1,3-diphenylpro-
pene/ethylene glycol; (d) 1,5-diaza-1,5-diphenyl-1,3-pentadiene‚HCl
salt/NEt3/NaI/MeOH.
conjugate system (between rhodanine and ring B) in
these compounds. The fact that relatively low selectiv-
ity of these compounds (30 for 13b, 9 for 23) compared
to a rhodacyanine (346 for 5) are within the same range
for cyanine dyes 21 and 22 in Table 4 supports this
hypothesis. The results of N-phenyl derivatives were
quite similar to those of N-ethyl derivatives (Table 5).
Deletion (13a ) or replacement (14, 15) of the benzothia-
zolidene moiety of rhodacyanine 24 made the activity
and selectivity as low as those for 13b and 23. In
contrast, elongation of the methine unit between the A
ring and rhodanine moiety in rhodacyanine dye (from
n ) 0 to n ) 1 in Figure 1) affected the quite selective
activity of 24 only in a moderate way, and the resultant
compound 25 showed high activity and selectivity (IC50
against CX-1 ) 60 nM, selectivity ) 50).
An attempt to measure the IC50 values for merocya-
nine dyes such as 3 failed due to their poor solubility.
In our preliminary experiments, however, they did not
exhibit any appreciable activity at 3 µM. A comparison
of 3 (merocyanine), 5 (rhodacyanine), and 13b (cyanine),
together with other results in Table 5, led us to the
hypothesis that the merocyanine conjugate system in
rhodacyanine dyes enhanced moderate activity which

Rhodacyanine Dyes That Exhibit Antitumor Activity
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3155
Sch em e 4a
a
Reagents: (a) CH(OEt)3/Ac2O; (b) 3-ethyl-2-methylbenzoxazolium iodide/NEt3/EtOH; (c) methyl p-toluenesulfonate; (d) 3-ethyl-2-
methylbenzothiazolium iodide‚HCl salt/NEt3/EtOH.
Ta ble 4. Clonogenic Assay of Rhodacyanine Dyes and Cyanine
Dyes on Human Colon Carcinoma (CX-1) and Monkey Normal
Kidney Epithelial Cells (CV-1)
Ta ble 6. Clonogenic Assay of Rhodacyanine Dyes and Their
Analogs of the 4-Oxothiazolidine (Rhodanine) Moiety
IC50 (µM)
compd
CX-1
CV-1
ratio CV-1/CX-1
5
27
29
0.05
0.05
0.03
17.3
26.2
1.7
346
524
57
20
26
28
30
0.07
1.3
0.1
3.4
25.4
0.8
49
20
8
>1.7
3.4
<2
Ta ble 7. Clonogenic Assay on Four Human Cancer Cell Linesa
IC50 (µM)
compd
anion
EJ
LOX
MFC-7
CRL1420
5
31
I-
0.07
0.06
0.02
0.01
0.09
0.4
0.05
0.06
AcO-
24
25
p-TsO-
0.04
0.4
0.04
0.04
0.06
0.04
0.04
0.07
I-
a
EJ , bladder carcinoma; LOX, melanoma; MCF-7, breast
carcinoma; CRL1420, pancreatic carcinoma.
Ta ble 5. Clonogenic Assay of Rhodacyanine Dyes and Their
Cyanine Dye Analogs
Our physical chemistry studies including X-ray crys-
tallography, absorption and NMR spectra, and molec-
ular orbital calculations revealed that all three rings
(A, B, rhodanine) were almost coplanar and that π
electrons delocalized throughout the molecule. This
electron delocalization across the two dye conjugate
systems was partly indicated from a marked bathochro-
mic shift of rhodacyanine 5 (λmax: 500.0 nm in methanol)
compared to the partial structured merocyanine 3
IC50 (µM)
compd
CX-1
CV-1
ratio CV-1/CX-1
5
24
25
0.05
0.01
0.06
17.3
2.2
346
220
50
20
30
1
3.0
13a
13b
14
2.3
1.3
1.5
0.8
1.8
46.1
38.9
1.5
15
9.8
12
9
23
16.2
(λmax: 428.0 nm in methanol) and cyanine 13b (λmax
:
arised from the cationic cyanine conjugate system in a
selective manner.
390.4 nm in methanol). A molecular orbital calculation
suggested that the p orbital of rhodanine’s sulfur atom
was involved in such an electron delocalization, which
was thought to be a key factor for the selective activity
of rhodacyanine dyes against tumor cells. Further
studies concerning the relationship between physical
property and activity are in progress.
Further in vitro studies of rhodacyanines 5, 31, 24,
and 25 were conducted using four human tumor cell
lines: EJ (human bladder carcinoma), LOX (human
melanoma), MCF-7 (human breast carcinoma), and
CRL-1420 (human pancreatic carcinoma). As sum-
marized in Table 7, all the compounds showed high
inhibitory activities against the cell lines with IC50
values of 10-90 nM, suggesting that rhodacyanine dyes
are potential medications for a wide spectrum of tumors.
The highly water-soluble compound 31 (solubility > 10
To obtain further insights into the role of the mero-
cyanine conjugate system, we modified the structure of
the rhodanine moiety. Replacement with 4-oxoimida-
zolidine resulted in a decrease of the activity against
CX-1 cells (5 vs 26, 27 vs 28 in Table 6). Compound 28
was more toxic against CV-1 than the corresponding
rhodacyanine 27 by 33-fold, resulting in the marked
decrease of the selectivity (66-fold). Analysis of Table
6 reveals that an analog with the 4-oxo-1,3-dithiolan
moiety (30) exhibited further decreased activity (>50
times), while substitution with 5-oxothiazoline (20)
made the activity only one-half that of the parent com-
pound (29). Thus, our structure-activity studies so far
concluded that the rhodanine moiety is indispensable
for the quite high and selective activity for tumor cells.

3156 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kawakami et al.
Ta ble 8. In Vivo Antitumor Activities of Rhodacyanine Dyes in the Tumor-Bearing Nude Mice Model
in vivo activity
T/Ca (%)
compd
treatment
LOX
168
OVCARIII
TIb (%) CX-1
Pc
5
at 5 mg/kg ip on days 1, 5, 8, 12, and 15
at 5 mg/kg ip on days 1, 3, 7, 10, and 16
at 2 mg/kg ip on days 1, 2, 4, 5, 7, 8, and 10
at 4 mg/kg ip on days 1, 2, 4, 5, 7, 8, and 10
<0.05
<0.01
<0.05
<0.01
<0.01
<0.05
180
31
24
156
163
at 5 mg/kg ip on days 1, 5, 8, and 12
at 5 mg/kg ip on days 1, 3, 7, 10, and 16
at 5 mg/kg ip on days 1, 5, and 7
183
>300
42
25
at 1.5 mg/kg ip on days 1, 3, 4, 7, and 9
256
<0.05
a
T/C represents the ratio of the median survival time of drug-treated to control, untreated tumor-bearing mice, expressed as a percentage.
b
Each group consisted of five nude mice. TI represents tumor inhibition ratio, which was evaluated at day 11 and calculated as follows:
inhibition ratio (%) ) (A - B)/A × 100, where A is the average tumor weight in the control group and B is that in the treated group. Each
group consisted of five nude mice. c The statistical significance of difference between the control and the drug-treated group was determined
by applying the Bartlett’s, Dunnett’s, and Scheffet’s test.
mg/mL), which has an acetate anion and was prepared
from the corresponding less soluble iodide 5 (solubility
< 0.1 mg/mL) using an anion-exchange method, has
similar IC50 values to those for 5, which implies that
the cation dye is the active component while the
counteranion has little influence upon activities.
subjected to further extensive structure-activity stud-
ies, results of which will be presented elsewhere.
Exp er im en ta l Section
1
The H-NMR spectra were recorded on a Bruker AMX-600,
ARX-300, or AC-200 spectrometer with tetramethylsilane as
internal standard. Chemical shifts are given in ppm, coupling
constants are in hertz, and splitting patterns are designated
as follows: s, singlet; brs, broad singlet; d, doublet; dd, doublet
of doublets; t, triplet; q, quartet; m, multiplet; dd, doublet of
doublets. Ultraviolet-visible (UV-vis) absorption spectra
were recorded on a Shimadzu UV-260 spectrophotometer. Fast
atom bombardment (FAB) mass spectra were determined with
a J EOL DX303 mass spectrometer, and high-resolution mass
spectrometry (HRMS) was recorded on a J EOL SX-102A mass
spectrometer; electron ionization (EI) mass spectra were
determined with a J EOL J MS-D300 instrument. Elemental
analyses were performed on Yanagimoto MT-3 and Dionex
2000i/SP instruments, and the results (C, H, N) were within
(0.4% of theoretical values unless indicated otherwise. Cor-
rect elemental analyses for most of the compounds could only
be obtained by factoring in partial hydration of these organic
salts.
3-Eth yl-5-(3-m eth ylben zoth ia zolin -2-ylid en e)-4-oxoth i-
a zolid in e-2-th ion e (3). A mixture of 2-(methylthio)ben-
zothiazole (1) (40.0 g, 220 mmol), methyl p-toluenesulfonate
(61.5 g, 330 mmol), and anisole (56 mL) was stirred at 120-
136 °C for 4 h. After the mixture was cooled to room
temperature, 3-ethyl-4-oxothiazolidine-2-thione (6) (35.0 g, 220
mmol) and acetonitrile (800 mL) were added. To this mixture
was added triethylamine (36.4 g, 360 mmol) dropwise under
15 °C with constant stirring and cooling, and the resulting
mixture was stirred at 10 °C for 4 h. The yellow precipitate
was collected and washed with acetonitrile (40 mL) and then
with methanol (140 mL). The crude product thus obtained
was suspended in acetone (210 mL) and methanol (420 mL),
and the mixture was stirred under reflux for 15 min. After
cooling to 25 °C, the precipitate was collected and washed with
methanol (140 mL) to give 3 (59.0 g, 87.0%) as yellow
crystals: UV-vis (MeOH) λmax 428.0 nm (ꢀ 6.62 × 104); 1H-
NMR (DMSO-d6) δ 1.20 (t, J ) 7.2 Hz, 3H), 3.98 (s, 3H), 4.10
(q, J ) 7.2 Hz, 2H), 7.35 (t, J ) 8.0 Hz, 1H), 7.52 (t, J ) 8.0
Hz, 1H), 7.78 (d, J ) 8.0 Hz, 1H), 7.93 (d, J ) 8.0 Hz, 1H); MS
(EI) m/ z 308. Anal. (C13H12N2OS3) C, H, N, S.
In Vivo An titu m or Activities. Rhodacyanine dyes
5, 31, 24, and 25 were evaluated for their inhibitory
activities against the growth of human cell lines in the
nude mice model. After intraperitoneal (ip) implanta-
tion of 2 × 106 human melanoma LOX cells (to which
the compounds exhibited the highest activity in Table
7), male Swiss nu/nu mice had a median survival time
of 24 days. In contrast, mice that received ip adminis-
tration of compound 5 at 5 mg/kg on days 1, 5, 8, 12,
and 15 showed a median survival time of 40 days with
tumor/control (T/C) ) 168% (Table 8). Administration
of other compounds with appropriately determined
doses and schedules made the survival period similar
or even longer: T/C ) 156% (2 mg/kg) or 163% (4 mg/
kg) for 31, 183% for 24, 256% for 25. Compounds 5 and
24 were also tested against human ovarian carcinoma
OVCARIII, and both were found to be quite efficacious
(T/C ) 180% for 5, >300% for 24), and compound 24 in
the human colon carcinoma CX-1 nude mice model
displayed marked inhibition of tumor growth (tumor
inhibition ratio (TI) ) 42%). Thus simple anion ex-
change had little influence upon in vivo antitumor
efficacy and was a useful method to increase the water
solubility which was an important criterion for clinical
use. Any significant body weight loss was not obsesrved
in these nude mice models, and also the acute toxicity
(LD50, ip administration into ICR mice) of compound 31
observed was over 30 mg/kg.
Con clu sion
The results described in this study demonstrated that
rhodacyanine dyes exhibited selective inhibitory activi-
ties in vitro against the growth of several human cell
lines over CV-1, an indicator cell for normal epithelial
cells. They also displayed high-level efficacy against
LOX and OVCARIII in vivo, with low toxicity. Structure-
activity studies indicated that rhodanine was an indis-
pensable moiety for the antitumor activity of rhodacy-
anines, which was markedly improved from those of
cyanines or other DLCs. This class of compounds is
expected to be a novel antitumor agent and is being
3-Eth yl-5-(3-m eth ylben zoth ia zolin -2-ylid en e)-2-(m eth -
ylth io)-4-oxoth ia zolin iu m p-Tolu en esu lfon a te (4). A mix-
ture of 3 (58.0 g, 188 mmol), methyl p-toluenesulfonate (105.0
g, 564 mmol), and N,N-dimethylformamide (58 mL) was stirred
at 130-145 °C for 2.5 h. After the mixture cooled to 95 °C,
acetone (500 mL) was added. The mixture was further cooled
to 25 °C with constant stirring, and the precipitate formed was
collected and washed with acetone (150 mL). The crude
product thus obtained was suspended in acetone (400 mL), and
the mixture was stirred under reflux for 15 min. After cooling
to 25 °C, the precipitate was collected and washed with acetone

Rhodacyanine Dyes That Exhibit Antitumor Activity
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3157
(150 mL) to give 4 (86.5 g, 93.0%) as orange crystals: UV-vis
(MeOH) λmax 420.7 nm (ꢀ 3.68 × 104); 1H-NMR (DMSO-d6)
δ 1.33 (t, J ) 7.2 Hz, 3H), 2.27 (s, 3H), 3.05 (s, 3H), 4.17 (q,
J ) 7.2 Hz, 2H), 4.24 (s, 3H), 7.10 (d, J ) 8.9 Hz, 2H), 7.46
(d, J ) 8.9 Hz, 2H), 7.52 (dd, J ) 8.0, 8.0 Hz, 1H), 7.70 (dd, J
) 8.0, 8.0 Hz, 1H), 8.00 (d, J ) 8.0 Hz, 1H), 8.18 (d, J )
8.0 Hz, 1H); MS (FAB+, glycerine) for C14H15N2OS3 m/ z
323, (FAB-, triethanolamine) for C7H7O3S m/ z 171. Anal.
(C21H22N2O4S4‚0.3H2O) C, H, N, S.
3-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
4-oxoth ia zolid in -2-ylid en e]m eth yl]ben zoth ia zoliu m Io-
d id e (5). To a mixture of 4 (24.7 g, 50 mmol) and 3-ethyl-2-
methylbenzothiazolium iodide (15.3 g, 50 mmol) in acetonitrile
(250 mL) was added triethylamine (15.2 g, 150 mmol) dropwise
at 70 °C, and the mixture was stirred for 1.5 h at the same
tempetature. To the reaction mixture was added ethyl acetate
(250 mL), and the mixture was cooled to 30 °C with constant
stirring. The orange precipitate was collected and washed
with ethyl acetate (125 mL). The crude product thus obtained
was dissolved in methanol (80 mL), and then to this solution
was added ethyl acetate (250 mL) at 50°C with constant
stirring. The precipitate was collected and washed with ethyl
acetate (110 mL) to give 5 (14.5 g, 50.0%) as orange crystals:
UV-vis (MeOH) λmax 500.0 nm (ꢀ 7.49 × 104); 1H-NMR
(DMSO-d6) δ 1.28 (t, J ) 7.1 Hz, 3H), 1.38 (t, J ) 7.1 Hz, 3H),
4.22 (s, 3H), 4.30 (q, J ) 7.2 Hz, 2H), 4.71 (q, J ) 7.2 Hz, 2H),
6.69 (s, 1H), 7.36 (t, J ) 7.8 Hz, 1H), 7.52-7.58 (m, 2H), 7.70-
7.79 (m, 2H), 7.94-7.98 (m, 2H), 8.26 (d, J ) 7.2 Hz, 1H); MS
(FAB+, nitrobenzyl alcohol) for C23H22N3OS3 m/ z 452, (FAB-,
glycerine) for I m/ z 127. Anal. (C23H22IN3OS3‚1.5H2O) C, H,
I, N, S.
5-[(N-Acet yl-N-p h en yla m in o)m et h ylid en e]-3-et h yl-4-
oxoth ia zolid in e-2-th ion e (7). To a solution of 3-ethyl-4-
oxothiazolidine-2-thione (6) (25.0 g, 155 mmol) in ligroin (145
mL) was added 1,3-diaza-1,3-diphenylpropene (32.5 g, 166
mmol), and the mixture was stirred at 70 °C for 1 h. After
cooling to room temperature, the precipitate was collected and
washed with acetone (100 mL) to give 3-ethyl-4-oxo-5-[(phe-
nylamino)methylidene]thiazoline-2-thione (45.0 g). This prod-
uct was mixed with acetic anhydride (110 g, 1080 mmol) and
triethylamine (0.18 g, 2 mmol), and the mixture was stirred
at 110 °C for 30 min. The reaction mixture was concentrated
to about one-half volume under reduced pressure. To this
residue was added methanol (225 mL), and the mixture was
stirred at 10 °C for 1 h. The precipitate formed was collected
and washed with methanol (100 mL) to give 7 (37.0 g, 77.9%)
as yellow crystals: 1H-NMR (DMSO-d6) δ 1.08 (t, J ) 7.2 Hz,
3H), 2.02 (s, 3H), 3.95 (q, J ) 7.2 Hz, 2H), 7.47-7.57 (m, 2H),
7.59-7.72 (m, 3H), 8.47 (s, 1H); MS (EI) m/ z 306. Anal.
(C14H14N2O2S2) C, H, N, S.
3-E t h y l-5-[2-(3-e t h y ln a p h t h o [1,2-d ]t h ia zo lin -2-y l-
id en e)eth ylid en e]-4-oxoth ia zolid in e-2-th ion e (8). A mix-
ture of 7 (29.8 g, 97 mmol), 3-ethyl-2-methylnaphtho[1,2-d]-
thiazolinium p-toluenesulfonate (38.8 g, 97 mmol), and acetic
anhydride (14.2 g, 140 mmol) in acetonitrile (1000 mL) was
stirred at 50 °C for 1 h. To this was added triethylamine (36.3
g, 359 mmol) at 50 °C, and the mixture was stirred for an
additional 4 h at 60 °C. After cooling to 25 °C, the precipitate
formed was collected and washed with acetonitrile (250 mL).
The crude product thus obtained was suspended in methanol
(750 mL), and the mixture was stirred under reflux for 1 h.
After cooling to 25 °C, the precipitate was collected to give 8
(27.4 g, 70.7%) as purple-red crystals: 1H-NMR (DMSO-d6) δ
1.16 (t, J ) 7.2 Hz, 3H), 1.59 (t, J ) 7.2 Hz, 3H), 4.04 (q, J )
7.2 Hz, 2H), 4.66 (q, J ) 7.2 Hz, 2H), 5.58 (d, J ) 13.2 Hz,
1H), 7.54-7.69 (m, 3H), 7.85-7.93 (m, 2H), 8.08 (d, J ) 9.0
Hz, 1H), 8.46 (d, J ) 9.0 Hz, 1H); MS (HRMS) for C20H18N2-
OS3 calcd 398.0581, found 398.0572. Anal. (C20H18N2OS3) C,
H; N: calcd, 24.13; found, 24.58.
enesulfonate (14.7 g, 36 mmol) in acetonitrile (1080 mL). To
this was added triethylamine (11.0 g, 109 mmol) at 75 °C, and
the resulting mixture was stirred at 75 °C for an additional 1
h and then cooled to 30 °C. The precipitate formed was
collected and washed with acetonitrile (300 mL). The crude
product thus obtained was suspended in methanol (720 mL),
and the mixture was stirred under reflux for 1 h. After cooling
to 25 °C, the precipitate was collected and washed with
acetonitrile (300 mL). Compound 10 (20.4 g, 74.1%) was
obtained as green crystals: UV-vis (MeOH) λmax 620.0 nm (ꢀ
1
1.00 × 105); H-NMR (DMSO-d6) δ 1.28 (t, J ) 7.2 Hz, 3H),
1.65-1.72 (m, 6H), 2.29 (s, 3H), 4.17 (q, J ) 7.2 Hz, 2H), 4.72
(q, J ) 7.2 Hz, 2H), 4.98 (q, J ) 7.2 Hz, 2H), 6.02 (d, J ) 12.9
Hz, 1H), 6.62 (s, 1H), 7.05-7.82 (m, 12H), 7.97 (d, J ) 8.4 Hz,
1H), 8.03-8.12 (m, 2H), 8.30 (d, J ) 8.4 Hz, 1H), 8.52 (d, J )
8.4 Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for C34H30N3OS3
m/ z 592, (FAB-, nitrobenzyl alcohol) for C7H7O3S m/ z 171;
HRMS (FAB+) for C34H30N3OS3 calcd 592.1551, found 592.1561.
Anal. (C41H37N3O4S4‚2.7H2O) C, N, S; H: calcd, 5.26; found,
4.84.
3-E t h yl-2-[[(N -p h e n yla m in o)t h ioca r b on yl]m e t h yl]-
ben zoth ia zoliu m Iod id e (12a ). To a suspension of sodium
hydride (36.0 g, 1.5 mol) in tetrahydrofuran (1.1 L) was added
3-ethyl-2-methylbenzothiazolium iodide (11) (154.1 g, 0.5 mol)
in a small portion at room temperature. To this was added
dropwise a solution of phenyl thiocyanate (68.9 g, 0.5 mol) in
tetrahydrofuran over a period of 1 h at 20-30 °C, and the
resulting mixture was stirred under reflux for 30 min. The
solvent was evaporated, and to the residue was added water
(500 mL) dropwise under 10 °C. The precipitate formed was
collected and washed with water (500 mL) to give 12a (131.3
g, 84.0%) as yellow crystals. Compound 12a was used for the
next step without further purification.
3-Eth yl-2-[(3-p h en yl-4-oxoth ia zolid in -2-ylid en e)m eth -
yl]ben zoth ia zoliu m Br om id e (13a ). A mixture of com-
pound 12a (11.0 g, 35 mmol) and bromoacetic acid (11.0 g, 79
mmol) in 1-butanol (22 mL) was stirred at 100 °C for 10 min.
After cooling to room temperature, to the mixture was added
diethyl ether (100 mL). The precipitate formed was collected
and washed with diethyl ether (50 mL) to give the crude
product, which was recrystallized from methanol (75 mL) to
give 13a (10.9 g, 73.0%) as brown crystals: UV-vis (MeOH)
1
λmax 377.8 nm (ꢀ 3.70 × 104); H-NMR (DMSO-d6) δ 1.13 (t, J
) 7.2 Hz, 3H), 4.29 (q, J ) 7.2 Hz, 2H), 4.58 (s, 2H), 5.97 (s,
1H), 7.52 (dd, J ) 7.5, 1.5 Hz, 2H), 7.63-7.75 (m, 4H), 7.79 (t,
J ) 7.2 Hz, 1H), 8.11 (d, J ) 7.9 Hz, 1H), 8.41 (d, J ) 7.9 Hz,
1H); MS (FAB+, glycerine) for C19H17N2OS2 m/ z 353, (FAB-,
glycerine) for Br m/ z 79, 81. Anal. (C19H17BrN2OS2‚1.1H2O)
C, H, Br, N, S.
3-E t h y l-2-[[(N -e t h y la m in o )t h io c a r b o n y l]m e t h y l]-
ben zoth ia zoliu m Iod id e (12b). A solution of 3-ethyl-2-
methylbenzothiazolium iodide (11) (36.0 g, 118 mmol) and
triethylamine (4.4 g, 43 mmol) in pyridine (60 mL) was stirred
at 110 °C for 20 min, and then the reaction mixture was poured
into water (2000 mL). The precipitate formed was collected
to give 12b (19.0 g, 60.9%) as purple crystals. Compound 12a
was used for the next step without further purification.
3-Eth yl-2-[(3-eth yl-4-oxoth ia zolid in -2-ylid en e)m eth yl]-
ben zoth ia zoliu m Br om id e (13b). A solution of compound
12b (6.0 g, 23 mmol) and bromoacetic acid (6.0 g, 43 mmol) in
acetic acid (20 mL) was stirred at 90 °C for 5 min. After the
mixture cooled to room temperature, diethyl ether (30 mL) was
added. The precipitate formed was collected and washed with
diethyl ether (20 mL) to give the crude product, which was
recrystallized from methanol (350 mL) to give 13b (5.6 g,
77.1%) as yellow crystals: UV-vis (MeOH) λmax 390.4 nm (ꢀ
1
4.20 × 104); H-NMR (DMSO-d6) δ 1.29 (t, J ) 7.2 Hz, 3H),
1.39 (t, J ) 7.2 Hz, 3H), 4.09 (q, J ) 7.2 Hz, 2H), 4.45 (s, 2H),
4.86 (q, J ) 7.2 Hz, 2H), 6.82 (s, 1H), 7.67 (t, J ) 7.5 Hz, 1H),
7.82 (t, J ) 7.5 Hz, 1H), 8.15 (d, J ) 7.9 Hz, 1H), 8.39 (d, J )
7.9 Hz, 1H); MS (FAB+, glycerine) for C15H17N2OS2 m/ z 305,
(FAB-, glycerine) for Br m/ z 79, 81. Anal. (C15H17BrN2-
OS2‚0.7H2O) C, H, Br, N, S.
3-Eth yl-2-[[3-eth yl-5-[2-(3-eth yln aph th o[1,2-d]th iazolin -
2-ylid en e)eth ylid en e]-4-oxoth ia zolid in -2-ylid en e]m eth -
yl]n a p h th o[1,2-d ]th ia zoliu m p-Tolu en esu lfon a te (10). A
mixture of 8 (14.5 g, 36 mmol) and methyl p-toluenesulfonate
(20.2 g, 109 mmol) in N,N-dimethylformamide (35 mL) and
toluene (13 mL) was stirred at 115 °C for 6 h. To the mixture
was added 3-ethyl-2-methylnaphtho[1,2-d]thiazolinium p-tolu-
3-Eth yl-2-[[3-p h en yl-5-[(N-p h en yla m in o)m eth ylid en e]-
4-oxoth ia zolid in -2-ylid en e]m eth yl]ben zoth ia zoliu m Br o-
m id e (14). A mixture of 13a (13.0 g, 30 mmol) and 1,3-diaza-

3158 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kawakami et al.
1,3-diphenylpropene (29.4 g, 150 mmol) in ethylene glycol (65
mL) was stirred at 80 °C for 30 min. After cooling to room
temperature, to this mixture was added diethyl ether (30 mL),
and the precipitate formed was collected. The crude product
thus obtained was suspended in 2-propanol (60 mL) and stirred
at room temperature for 10 min. The precipitate was collected
and washed with isopropyl alcohol (30 mL) to give 14 (18.0 g,
quantitative) as yellow crystals: UV-vis (MeOH) λmax 480.3
3-Eth yl-2-[3-(3-eth ylben zoth ia zolin -2-ylid en e)-1-p r op e-
n yl]ben zoth ia zoliu m Iod id e (21). This compound was
synthesized from 3-ethylbenzothiazolinium iodide and triethyl
orthoformate in pyridine according to the reported method:10
UV-vis (MeOH) λmax 555.8 nm (ꢀ 1.56 × 104); 1H-NMR
(DMSO-d6) δ 1.35 (t, J ) 7.1 Hz, 6H), 4.38 (q, J ) 7.1 Hz, 4H),
6.67 (d, J ) 12.6 Hz, 2H), 7.41 (t, J ) 7.8 Hz, 2H), 7.58 (t, J
) 7.8 Hz, 2H), 7.72-7.83 (m, 3H), 8.01 (d, J ) 7.8 Hz, 2H);
MS (FAB+, nitrobenzyl alcohol) for C21H21N2S2 m/ z 365,
1
nm (ꢀ 6.26 × 104); H-NMR (DMSO-d6) δ 1.14 (t, J ) 7.2 Hz,
3H), 4.26 (q, J ) 7.2 Hz, 2H), 6.00 (s, 1H), 7.12-7.21 (m, 1H),
7.40-7.51 (m, 3H), 7.53-7.82 (m, 7H), 8.06 (d, J ) 7.5 Hz,
1H), 8.36 (d, J ) 7.5 Hz, 1H), 8.46 (t, J ) 7.5 Hz, 1H), 11.01
(d, J ) 13.2 Hz, 1H); MS (FAB+, glycerine) for C26H22N3OS2
m/ z 456, (FAB-, nitrobenzyl alcohol) for Br m/ z 79, 81. Anal.
(C26H22BrN3OS2‚0.5H2O) C, H, Br, N, S.
(FAB-, nitrobenzyl alcohol) for I m/ z 127. Anal. (C21H21
IN2S2‚1.1H2O) C, H, I, N, S.
-
3-Eth yl-2-[3-ch lor o-5-(3-eth yln a p h th o[1,2-d ]th ia zolin -
2-ylid en e)-1,3-p en ta d ien yl]n a p h th o[1,2-d ]th ia zoliu m p-
Tolu en esu lfon a te (22). This compound was synthesized
from 3-ethylbenzothiazolinium iodide and 3-chloro-1,5-diaza-
1,5-diphenyl-1,3-pentadiene in pyridine in a manner analogous
to the preparation of 21: UV-vis (MeOH) λmax 681.0 nm (ꢀ
3-E t h yl-2-[[3-p h en yl-5-[(N-p h en yla m in o)-2-p r op en yl-
id en e]-4-oxot h ia zolid in -2-ylid en e]m et h yl]b en zot h ia zo-
liu m Iod id e (15). A mixture of 13a (8.7 g, 20 mmol), 1,5-
diaza-1,5-diphenyl-1,3-pentadiene hydrochloride salt (25.9 g,
100 mmol), and triethylamine (13.0 g, 128 mmol) in methanol
(170 mL) was stirred at 50 °C for 20 min. After the mixture
cooled to room temperature, a solution of sodium iodide (9.0
g, 60 mmol) in methanol (45 mL) was added. The precipitate
formed was collected to give the crude product, which was
suspended in ethanol (160 mL) and stirred at 50 °C for 20 min.
The precipitate was collected and washed with ethanol (30 mL)
to give 14 (9.8 g, 80.3%) as dark-green crystals: UV-vis
1
2.42 × 105); H-NMR (DMSO-d6) δ 1.66 (t, J ) 7.2 Hz, 6H),
2.29 (s, 3H), 4.73 (q, J ) 7.2 Hz, 4H), 6.32 (d, J ) 12.9 Hz,
2H), 7.11 (d, J ) 8.1 Hz, 2H), 7.48 (d, J ) 8.1 Hz, 2H), 7.55 (t,
J ) 7.5 Hz, 2H), 7.70 (t, J ) 7.5 Hz, 2H), 7.90 (d, J ) 12.9 Hz,
2H), 7.97 (d, J ) 8.7 Hz, 2H), 8.03-8.11 (m, 4H), 8.42 (d, J )
8.7 Hz, 2H); MS (FAB+, nitrobenzyl alcohol) for C31H26ClN2S2
m/ z 525, (FAB-, nitrobenzyl alcohol) for C7H7O3S m/ z 171.
Anal. (C38H33ClN2O3S3‚1H2O) C, H, Cl, N, S.
2-[(5-Cycloh exyliden e-3-eth yl-4-oxoth iazolidin -2-yliden -
e)m eth yl]-3-eth ylben zoth ia zoliu m p-Tolu en esu lfon a te
(23). This compound was synthesized from 5-cyclohexylidene-
3-ethyl-4-oxothiazolidine-2-thione and 3-ethyl-2-methylben-
zothiazolium p-toluenesulfonate in a manner analogous to
the preparation of rhodacyanine dye 5: UV-vis (MeOH) λmax
1
(MeOH) λmax 547.4 nm (ꢀ 7.46 × 104); H-NMR (DMSO-d6) δ
1.13 (t, J ) 7.2 Hz, 3H), 4.22 (q, J ) 7.2 Hz, 2H), 5.89 (t, J )
12.0 Hz, 1H), 6.04 (s, 1H), 7.07 (t, J ) 7.5 Hz, 1H), 7.19 (d, J
) 8.1 Hz, 2H), 7.36 (t, J ) 7.5 Hz, 2H), 7.56-7.78 (m, 7H),
7.95-8.06 (m, 2H), 8.30-8.45 (m, 2H), 10.67 (d, J ) 12.6 Hz,
1H); MS (FAB+, glycerine) for C28H24N3OS2 m/ z 482, (FAB-,
nitrobenzyl alcohol) for I m/ z 127. Anal. (C28H24IN3OS2‚
1.0H2O) C, H, I, N, S.
1
425.8 nm (ꢀ 5.97 × 104); H-NMR (CDCl3) δ 1.29 (t, J ) 7.1
Hz, 3H), 1.52 (t, J ) 7.1 Hz, 3H), 1.68-1.80 (m, 2H), 1.92-
2.10 (m, 4H), 2.31 (s, 3H), 2.47 (t, J ) 6.6 Hz, 2H), 3.22 (t, J
) 6.6 Hz, 2H), 4.33 (q, J ) 7.1 Hz, 2H), 5.06 (q, J ) 7.1 Hz,
2H), 7.10 (d, J ) 7.8 Hz, 2H), 7.19 (s, 1H), 7.52-7.58 (m, 1H),
7.68 (d, J ) 7.8 Hz, 1H), 7.77 (d, J ) 8.1 Hz, 1H), 8.02 (d, J )
7.8 Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for C21H25N2OS2
m/ z 385, (FAB-, nitrobenzyl alcohol) for C7H7O3S m/ z 171.
Anal. (C28H32N2O4S3‚1H2O) C, H, N, S.
4-[2-(3-Eth ylben zoxazolin -2-yliden e)eth yliden e]-2-(eth -
ylth io)-5-oxoth ia zolin e (18). To a solution of carbodithioate
16 (17.9 g, 0.10 mol) in acetic anhydride (80 mL) was added
triethyl orthoformate (21.3 g, 0.14 mol), and the mixture was
refluxed 1.5 h. After the acetic anhydride was evaporated, to
the residue were added 3-ethyl-2-methylbenzoxazolinium io-
dide (28.9 g, 0.10 mol) in ethanol (80 mL) and then triethy-
lamine (18.6 g, 0.18 mol). The resulting mixture was refluxed
for 15 min and then cooled to 10 °C. The precipitate formed
was collected to give merocyanine dye intermediate 18 (25.9
g, 78.0%). Compound 18 was used for the next step without
further purification.
3-E t h yl-2-[[3-p h en yl-5-(3-m et h ylb en zot h ia zolin -2-yl-
id en e)-4-oxot h ia zolid in -2-ylid en e]m et h yl]ben zot h ia zo-
liu m p-Tolu en esu lfon a te (24). This compound was synthe-
sized from 2-(methylthio)benzothiazole and 4-oxo-3-phenylth-
iazolidine-2-thione in a manner analogous to the preparation
of rhodacyanine dye 5: UV-vis (MeOH) λmax 501.0 nm (ꢀ 6.83
× 104); 1H-NMR (DMSO-d6) δ 1.14 (t, J ) 7.1 Hz, 3H), 2.28 (s,
3H), 4.20 (q, J ) 7.1 Hz, 2H), 4.30 (s, 3H), 5.93 (s, 1H), 7.10
(d, J ) 7.8 Hz, 2H), 7.39 (t, J ) 8.1 Hz, 1H), 7.48 (d, J ) 7.8
Hz, 2H), 7.51-7.62 (m, 4H), 7.66-7.76 (m, 4H), 7.82 (d, J )
7.8 Hz, 1H), 7.94 (d, J ) 7.8 Hz, 1H), 7.98 (d, J ) 7.8 Hz, 1H),
8.28 (d, J ) 7.6 Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for
C27H22N3OS3 m/ z 500, (FAB-, nitrobenzyl alcohol) for C7H7O3S
m/ z 171; HRMS (FAB+); for C27H22N3OS3 calcd 500.0925,
found 500.0908.
3-Eth yl-2-[[3-ph en yl-5-[2-(3-eth ylben zoth iazolin -2-ylide-
n e)et h ylid en e]-4-oxot h ia zolid in -2-ylid en e]m et h yl]b en -
zoth ia zoliu m Iod id e (25). This compound was synthesized
from 3-ethyl-2-methylbenzothiazolium p-toluenesulfonate and
4-oxo-3-phenylthiazolidine-2-thione in a manner analogous to
the preparation of rhodacyanine dye 10: UV-vis (MeOH) λmax
595.4 nm (ꢀ 1.07 × 105); 1H-NMR (DMSO-d6) δ 1.13 (t, J ) 7.1
Hz, 3H), 1.33 (t, J ) 7.1 Hz, 3H), 4.19 (q, J ) 7.1 Hz, 2H),
4.38 (q, J ) 7.1 Hz, 2H), 5.99 (s, 1H), 6.18 (d, J ) 13.2 Hz,
1H), 7.32 (t, J ) 7.2 Hz, 1H), 7.45-7.95 (m, 12H), 8.29 (d, J )
7.2 Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for C30H26N3O1S3
m/ z 540, (FAB-, nitrobenzyl alcohol) for I m/ z 127. Anal.
(C30H26IN3O1S3‚0.3H2O) C, H, I, N, S.
4-[2-(3-Eth ylben zoxazolin -2-yliden e)eth yliden e]-2-(eth -
ylth io)-3-m eth yl-5-oxoth ia zolin iu m p-Tolu en esu lfon a te
(19). A mixture of compound 18 (24.9 g, 75 mmol) and methyl
p-toluenesulfonate (44.7 g, 240 mmol) was stirred at 130 °C
for 1 h. After cooling to 40 °C, to the reaction mixture were
added ethanol (30 mL) and then diethyl ether (100 mL). The
precipitate was collected to give 19 (30.1 g, 77.4%): 1H-NMR
(DMSO-d6) δ 1.41 (t, J ) 7.2 Hz, 6H), 2.29 (s, 3H), 3.92 (s,
3H), 4.35 (q, J ) 7.2 Hz, 2H), 7.03-7.13 (m, 3H), 7.47 (d, J
) 8.9 Hz, 2H), 7.51-7.61 (m, 2H), 7.81-7.92 (m, 2H), 7.98 (d,
J
) 13.8 Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for
C17H19N2O2S2 m/ z 347, (FAB-, nitrobenzyl alcohol) for C7H7O3S
m/ z 171; HRMS (FAB+) for C17H19N2O2S2 calcd 347.0888,
found 347.0886.
3-Eth yl-2-[[3-m eth yl-4-[2-(3-eth ylben zoxa zolin -2-ylid e-
n e)et h ylid en e]-5-oxot h ia zolid in -2-ylid en em et h yl]b en -
zoth ia zoliu m Iod id e (20). To a mixture of 19 (10.4 g, 20
mmol) and 3-ethyl-2-methylbenzothiazolium iodide (6.1 g, 20
mmol) in ethanol (65 mL) was added triethylamine (2.6 g, 26
mmol) at room temperature, and the reaction mixture was
refluxed for 30 min. After cooling to room temperature, the
precipitate was collected and washed with ethanol (30 mL) to
yield 20 (7.46 g, 63.3%): UV-vis (MeOH) λmax 574.1 nm (ꢀ 2.09
× 105); 1H-NMR (DMSO-d6) δ 1.31 (t, J ) 7.2 Hz, 3H), 1.34 (t,
J ) 7.2 Hz, 3H), 3.83 (s, 3H), 4.15 (q, J ) 7.2 Hz, 2H), 4.52 (q,
J ) 7.2 Hz, 2H), 6.46 (s, 1H), 6.78 (d, J ) 13.2 Hz, 1H), 7.29-
7.55 (m, 4H), 7.61-7.69 (m, 4H), 7.97 (d, J ) 7.0 Hz, 1H); MS
(FAB+, glycerine) for C25H24N3O2S2 m/ z 462, (FAB-, glycerine)
for I m/ z 127. Anal. (C25H24IN3O2S2‚1H2O) C, H, I, N, S.
2-[[1,3-Dieth yl-5-(3-m eth ylben zoth ia zolin -2-ylid en e)-4-
oxoim id a zolid in -2-ylid en e]m eth yl]-3-eth ylben zoth ia zo-
liu m Iod id e (26). This compound was synthesized from
2-(methylthio)benzothiazole and 1,3-diethyl-4-oxoimidazoli-
dine-2-thione in a manner analogous to the preparation of
rhodacyanine dye 5: UV-vis (MeOH) λmax 481.0 nm (ꢀ 4.83 ×
104); 1H-NMR (DMSO-d6) δ 1.01 (t, J ) 6.9 Hz, 3H), 1.23 (t, J
) 7.1 Hz, 3H), 1.35 (t, J ) 7.1 Hz, 3H), 3.95 (q, J ) 7.2 Hz,

Rhodacyanine Dyes That Exhibit Antitumor Activity
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3159
2H), 4.05 (s, 3H), 4.08 (q, J ) 7.2 Hz, 2H), 4.42 (q, J ) 7.2 Hz,
2H), 5.88 (s, 1H), 7.36 (t, J ) 8.0 Hz, 1H), 7.44 (t, J ) 8.0 Hz,
1H), 7.54 (t, J ) 8.0 Hz, 1H), 7.49-7.75 (m, 3H), 7.94 (d, J )
7.5 Hz, 1H), 8.04 (d, J ) 7.5 Hz, 1H); MS (FAB+, nitrobenzyl
alcohol) for C25H27N4OS2 m/ z 463, (FAB-, nitrobenzyl alcohol)
for I m/ z 127. Anal. (C25H27IN4OS2) C, H, I, N, S.
4-oxoth ia zolid in -2-ylid en e]m eth yl]ben zoth ia zoliu m Ac-
eta te (31). A solution of 5 (4.2 g, 7 mmol) in a 0.1 M acetic
acid solution (300 mL) of chloroform/methanol (1/2, v/v) was
passed through the basic anion-exchange resin (about 120 mL,
Diaion WA-21, acetate form), and the resin was washed with
a 0.1 M acetic acid solution (200 mL) of methanol. The eluent
was concentrated to about 50 mL, to which was added ethyl
acetate (300 mL). The precipitate formed was collected and
washed with ethyl acetate (50 mL) to give 31 (2.8 g, 67.4%) as
orange crystals: UV-vis (MeOH) λmax 501.2 nm (ꢀ 6.88 × 104);
1H-NMR (DMSO-d6) δ 1.26 (t, J ) 7.1 Hz, 3H), 1.37 (t, J ) 7.1
Hz, 3H), 1.68 (s, 3H), 4.21 (s, 4.5H), 4.28 (q, J ) 7.2 Hz, 2H),
4.70 (q, J ) 7.2 Hz, 2H), 6.67 (s, 1H), 7.33 (t, J ) 7.7 Hz, 1H),
7.50 (q, J ) 7.8 Hz, 2H), 7.71 (q, J ) 8.5 Hz, 2H), 7.94 (d, J )
8.1 Hz, 2H), 8.24 (d, J ) 8.1 Hz, 1H); MS (FAB+, nitrobenzyl
alcohol) for C23H22N3OS3 m/ z 452, (FAB-, nitrobenzyl alcohol)
for C2H3O2 m/ z 59; HRMS (FAB+) for C23H22N3OS3 calcd
452.0925, found 452.0891. Anal. (C25H25N3O3S3‚2.85H2O‚
0.5C2H4O2) C, S; H: calcd, 5.56; found, 4.98. N: calcd, 7.09;
found, 6.43.
3-Eth yl-2-[[3-eth yl-5-[2-(3-eth yln aph th o[1,2-d]th iazolin -
2-ylid en e)eth ylid en e]-4-oxoth ia zolid in -2-ylid en e]m eth -
yl]n aph th o[2,1-d]th iazoliu m p-Tolu en esu lfon ate (27). This
compound was synthesized from 3-ethyl-2-methyl[1,2-d]naph-
thothiazolium p-toluenesulfonate and 3-ethyl-4-oxothiazoli-
dine-2-thione in a manner analogous to the preparation of
rhodacyanine dye 10: UV-vis (MeOH) λmax 622.9 nm (ꢀ 1.07
× 105); 1H-NMR (DMSO-d6) δ 1.27 (t, J ) 6.9 Hz, 3H), 1.38 (t,
J ) 7.0 Hz, 3H), 1.72 (t, J ) 6.9 Hz, 3H), 2.30 (s, 3H), 4.08 (q,
J ) 6.9 Hz, 2H), 4.52-4.70 (m, 4H), 5.94 (d, J ) 13.2 Hz, 1H),
6.39 (s, 1H), 6.77-6.88 (m, 2H), 7.14 (d, J ) 8.1 Hz, 2H), 7.35-
7.7.48 (m, 2H), 7.50-7.67 (m, 6H), 7.78-7.95 (m, 5H); MS
(FAB+, nitrobenzyl alcohol) for C34H30N3OS3 m/ z 592, (FAB-,
nitrobenzyl alcohol) for C7H7O3S m/ z 171; HRMS (FAB+) for
C34H30N3OS3 calcd 592.1551, found, 592.1558.
2-[[1,3-Dieth yl-5-[2-(3-eth yln a p h th o[1,2-d ]th ia zolin -2-
yliden e)eth yliden e]-4-oxoim id a zolid in -2-yliden em eth yl]-
3-eth yln a p h th o[2,1-d ]th ia zoliu m Iod id e (28). This com-
pound was synthesized from 3-ethyl-2-methyl[1,2-d]naphtho-
thiazolium p-toluenesulfonate and 1,3-diethyl-4-oxoimidazo-
lidine-2-thione in a manner analogous to the preparation of
rhodacyanine dye 10: UV-vis (MeOH) λmax 606.3 nm (ꢀ 1.04
× 105); 1H-NMR (DMSO-d6) δ 1.23-1.30 (m, 6H), 1.38 (t, J )
7.2 Hz, 3H), 1.74 (t, J ) 7.2 Hz, 3H), 3.95 (q, J ) 7.2 Hz, 2H),
4.30 (q, J ) 7.2 Hz, 2H), 4.41 (q, J ) 7.2 Hz, 2H), 4.69 (t, J )
7.2 Hz, 2H), 5.77 (s, 1H), 7.50-7.58 (m, 3H), 7.62-7.84 (m,
5H), 7.95-8.18 (m, 5H), 8.47 (1H, J ) 8.5 Hz, 1H); MS (FAB+,
nitrobenzyl alcohol) for C36H35IN4OS2 m/ z 603, (FAB-, ni-
trobenzyl alcohol) for I m/ z 127. Anal. (C36H35IN4OS2‚1.5H2O)
C, H, I, N, S.
2-[[3-Cycloh exyl-5-(3-m eth ylben zoth ia zolin -2-ylid en e)-
4-oxoth ia zolid in -2-ylid en e]m eth yl]-3-eth ylben zoxa zoli-
u m Ch lor id e (32). This compound was synthesized from
2-(methylthio)benzothiazole and 4-oxo-3-cyclohexylthiazoli-
dine-2-thione in a manner analogous to the preparation of
rhodacyanine dye 5: UV-vis (MeOH) λmax 488.1 nm (ꢀ 6.73 ×
1
104); H-NMR (DMSO-d6) δ 1.24-1.90 (m, 10H), 4.18 (s, 3H),
4.50 (q, J ) 7.0 Hz, 2H), 4.60 (brs, 1H), 6.36 (s, 1H), 7.48 (dd,
J ) 7.8, 7.8 Hz, 1H), 7.48-7.61 (m, 3H), 7.79 (dd, J ) 8.0, 8.0
Hz, 2H), 7.98 (dd, J ) 7.8, 7.8 Hz, 2H); MS (FAB+, nitrobenzyl
alcohol) for C27H28N3O2S2 m/ z 490, (FAB-, nitrobenzyl alcohol)
for Cl m/ z 35. Anal. (C27H28ClN3O2S2‚2.5H2O) C, H, Cl, N,
O, S.
3-Eth yl-2-[[3-eth yl-5-[2-[3-eth yl-5-(tr iflu or om eth yl)ben -
zot h ia zolin -2-ylid e n e ]e t h ylid e n e ]-4-oxot h ia zolid in -2-
ylid en e]m eth yl]-4-m eth ylth ia zoliu m Iod id e (33). This
compound was synthesized from 3-ethyl-2-methyl-5-trifluo-
robenzothiazolium p-toluenesulfonate and 3-methyl-4-oxothia-
zolidine-2-thione in a manner analogous to the preparation of
rhodacyanine dye 10: UV-vis (MeOH) λmax 559.2 nm (ꢀ 8.39
× 104); 1H-NMR (CD3OD) δ 1.32 (t, J ) 7.1 Hz, 3H), 1.40 (t, J
) 7.1 Hz, 3H), 1.45 (t, J ) 7.1 Hz, 3H), 2.53 (s, 3H), 4.14 (q, J
) 7.1 Hz, 2H), 4.31 (q, J ) 7.1 Hz, 2H), 4.46 (q, J ) 7.2 Hz,
2H), 5.87 (d, J ) 12.5 Hz, 1H), 6.59 (s, 1H), 7.49 (s, 1H), 7.51
(d, J ) 8.9 Hz, 1H), 7.65 (s, 1H), 7.84 (d, J ) 12.5 Hz, 1H),
7.85 (d, J ) 8.9 Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for
C24H25F3N3OS3 m/ z 524, (FAB-, nitrobenzyl alcohol) for I m/ z
127; HRMS (FAB+) for C24H25F3N3OS3 calcd 524.1112, found
524.1108. Anal. (C24H25F3IN3OS3‚2H2O) H, F, N, S; C: calcd,
4.25; found, 3.69. I: calcd, 18.46; found, 17.85.
X-r a y Str u ctu r e An a lysis of 32 a n d 33. Compounds 32
and 33 were crystallized from ethanol and acetonitrile solu-
tions, respectively. X-ray diffraction data were measured by
a Rigaku AFC-5R instrument using Cu KR radiation and a
graphite monochromometer. Structures were determined by
direct-method SHELXS86 and successive Fourier syntheses
and refined by a full-matrix least-squares method. Full
crystallographic details are available as Supporting Informa-
tion.
In Vitr o Clon ogen ic Assa y. CX-1 and CV-1 cell lines were
grown in a 50:50 mix of Dulbecco’s modified Eagle’s medium
(DMEM) and RPMI 1640 medium (GIBCO Laboratories,
Grand Island, NY) supplemented with 10% calf serum (Hy-
clone Laboratories Inc., Logan, UT) and antibiotics at 37 °C
under 5% CO2, 95% air, and 100% humidity. CV-1 (normal
African green monkey kidney) was obtained from the American
Type Culture Collection (Rockville, MD), and CX-1 (human
colon carcinoma) from Dr. M. Wolpert (National Cancer
Institute).
For the clonogenic assay, cells were seeded at 1500 cells/
wells for CX-1 and 1000 cells/wells for CV-1 in 96-well plates
(Becton Dickinson Labware, Lincoln Park, NJ ). The assay was
performed in duplicate. The drugs were first dissolved in
dimethyl sulfoxide, to prepare 10 mg/mL stock solutions. The
final drug solution was made by mixing 100 µL of this stock
solution with 10 mL of 5% CS DME media solution. On the
3-Eth yl-2-[[3-m eth yl-5-[2-(3-eth ylben zoxa zolin -2-ylid e-
n e)et h ylid en e]-4-oxot h ia zolid in -2-ylid en e]m et h yl]b en -
zoth ia zoliu m Iod id e (29). This compound was synthesized
from 3-ethyl-2-methylbenzoxazolium p-toluenesulfonate and
3-methyl-4-oxothiazolidine-2-thione in a manner analogous to
the preparation of rhodacyanine dye 10: UV-vis (MeOH) λmax
564.3 nm (ꢀ 8.76 × 104); 1H-NMR (DMSO-d6) δ 1.33-1.43 (m,
6H), 3.60 (s, 3H), 4.23 (q, J ) 7.1 Hz, 2H), 4.72 (q, J ) 7.1 Hz,
2H), 5.62 (d, J ) 13.2 Hz, 1H), 6.70 (s, 1H), 7.28-7.42 (m,
2H), 7.53-7.75 (m, 4H), 7.96-8.08 (m, 2H), 8.24 (d, J ) 7.2
Hz, 1H); MS (FAB+, nitrobenzyl alcohol) for C25H24N3O2S2 m/ z
462, (FAB-, nitrobenzyl alcohol) for I m/ z 127; HRMS (FAB+)
for C25H24N3O2S2 calcd 462.1310, found 462.1309. Anal.
(C25H24IN3O2S2‚1.75H2O) C, I, N, S; H: calcd, 4.46; found, 3.92.
3-E t h yl-2-[[5-[2-(3-et h ylb en zoxa zolin -2-ylid en e)et h -
ylid en e]-4-oxo-1,3-d ith iola n -2-ylid en e]m eth yl]ben zoth ia -
zoliu m iod id e (30). This compound was synthesized accord-
ing to the reported method16 from 3-ethyl-2-methyl-
benzothiazolinium iodide and carbon disulfide: UV-vis (MeOH)
λ
max 586.8 nm (ꢀ 6.54 × 104); NMR studies (COSY and ROESY
spectra (data are not shown)) revealed that compound 30 was
a mixture (1:1) of geometrical isomers at the merocyanine
moiety, 1H-NMR (CDCl3:DMSO-d6 ) 3.5:1.5) (one isomer) δ
1.47-1.55 (m, 6H), 4.33 (q, J ) 7.2 Hz, 2H), 4.71-4.77 (m,
2H), 5.54 (d, J ) 13.5 Hz, 1H), 7.38 (dd, J ) 7.6, 7.6 Hz, 1H),
7.43 (dd, J ) 7.6, 7.6 Hz, 1H), 7.49 (d, J ) 7.6 Hz, 1H), 7.58
(d, J ) 7.6 Hz, 1H), 7.68 (dd, J ) 7.6, 7.6 Hz, 1H), 7.83 (dd, J
) 7.6, 7.6 Hz, 1H), 7.84 (s, 1H), 8.00 (d, J ) 7.6 Hz, 1H), 8.17
(d, J ) 13.5 Hz, 1H), 8.25 (d, J ) 7.6 Hz, 1H), (another isomer)
δ 1.47-1.55 (m, 6H), 4.25 (q, J ) 7.2 Hz, 2H), 4.71-4.77 (m,
2H), 5.39 (d, J ) 13.5 Hz, 1H), 7.38 (dd, J ) 7.6, 7.6 Hz, 1H),
7.43 (dd, J ) 7.6, 7.6 Hz, 1H), 7.49 (d, J ) 7.6 Hz, 1H), 7.58
(d, J ) 7.6 Hz, 1H), 7.64 (dd, J ) 7.6, 7.6 Hz, 1H), 7.67 (s,
1H), 7.78 (dd, J ) 7.6, 7.6 Hz, 1H), 7.95 (d, J ) 7.6 Hz, 1H),
8.10 (d, J ) 13.5 Hz, 1H), 8.20 (d, J ) 7.6 Hz, 1H); MS (FAB+,
nitrobenzyl alcohol) for C24H21N2O2S3 m/ z 465, (FAB-,
nitrobenzyl alcohol) for
I
m/ z 127; HRMS (FAB+) for
C24H21N2O2S3 calcd 465.0765, found 465.0765. Anal. (C24H21
-
IN2O2S3‚1.5H2O) C, H, N, S; I: calcd, 20.48; found, 19.85.
3-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-

3160 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kawakami et al.
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Selective Toxicity of Rhodamine-123 in Carcinoma Cells in Vitro.
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following day, cells were treated with test compounds at varied
concentrations and cultured precisely for 24 h in the media.
After rinsing, cells were incubated in drug-free medium for 2
weeks. Colonies were stained with 2% crystal violet in 70%
ethanol and counted by an automated colony counter (Artek
counter model 880, Dynatech Laboratories Inc., Chantilly, VA).
In Vivo Hu m a n Tu m or Xen ogr a fts in Nu d e Mice. Male
Swiss nu/nu mice (about 5 weeks old) were obtained from
Taconic Farm, Inc. (Germantown, NY). Group housing (5/cage)
was provided in polycarbonate cages with a wire top and
filters. Mice were allowed to acclimate for 1 week prior to
experiments. Only normal, healthy mice were used. Human
melanoma LOX cells used in this model were first grown sc
in nude mice. On the day of ip implantation, tumors were
excised and a single cell suspension was prepared. RBCs were
lysed by ammonium chloride. Each mouse received 2 × 106
LOX cells (trypan blue-negative) in 0.2 mL of PBS by ip
injection. Test compounds (0.2 mL/20 g of mouse body weight,
45% encapsin HPB (hydroxypropyl â-cyclodextrin; American
Maise-Products Co.) in water) were administered ip for LOX
tumor-bearing mice with appropriately determined doses and
schedules. Evaluation against human ovarian carcinoma
OVCARIII cells and human colon carcinoma CX-1 cells was
performed in a manner analogous to the LOX ip tumor model.
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(11) Sun, X.; Wong, J . R.; Song, K.; Hu, J .; Garlid, K. D.; Chen, L. B.
Ack n ow led gm en t. We thank Dr. Hiroshi Kitaguchi
for helpful discussions and Mr. Hiroo Fukunaga for
advice about a molecular orbital calculation. We also
thank Professor George Sheldrick of Institut fu¨r Anor-
ganische Chemie der Universitat for providing us with
SHELXS86 for the X-ray structure analysis.
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