Structure-activity of novel rhodacyanine dyes as antitumor agents

Article abstract of DOI:10.1021/jm970590k

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3- ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothiazolidin-2- ylidene]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.

Full text of DOI:10.1021/jm970590k

1
30
J . Med. Chem. 1998, 41, 130-142
Str u ctu r e-Activity of Novel Rh od a cya n in e Dyes a s An titu m or Agen ts
,
†
†
†
†
†
†
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
X
Received September 3, 1997
We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent
inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine)
was an essential moiety for antitumor activity. On the basis of our foregoing work, two types
of rhodacyanine dyes, which categorized into class I and II depending on the methine length,
were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused
on the structure-activity study of two heteroaromatic rings. In class I, where the A rings
were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found
to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical
properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A
rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a
benzothiazole moiety for the A ring, exhibited the favarable biological and chemical properties.
Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various
heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal
moiety for the B ring (compound 13). Further, the variation of counteranion had a profound
effect on solubility in water without influence on antitumor activity. Chloride anion was selected
as the favorable anion with respect to synthetic method as well as solubilty in water. Our
study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-
(
methylbenzothiazolin-2-ylidene)-4-oxothiazolidin-2-ylidene]methyl]pyridinium chloride) as the
candidate for clinical trials and is currently subjected to further investigation as a potent
antitumor agent in phase I clinical trial for the treatment of solid tumors.
In tr od u ction
that some “π electron-delocalized lipophilic cations
denoted as DLCs)” exhibited marked and selective
(
During the past 20 years, a variety of approaches have
been taken for cancer chemotherapy, and many anti-
tumor drugs have been developed for clinical use. In
the treatment of solid tumors, however, the conventional
approaches have met with only limited success, and
cancer still remains as one of the leading causes of
human mortality. Current chemotherapeutic antitumor
drugs suffer two major drawbacks: adverse effects and
drug resistance. Adverse effects associated with con-
ventional antitumor drugs are usually caused by their
indiscriminate cytotoxic effect on normal cells. In drug
resistance, the use of combination chemotherapy, which
is the administration of several drugs with different and
complementary mechanisms of action, is regarded as the
effective approach. Therefore, to overcome the short-
comings of the present cancer chemotherapy, an anti-
tumor drug with a new mechanism of action, which is
capable of discriminating tumor cells from normal
proliferative cells and exhibits selective toxicity against
cancer, is the subject of our current research.
antitumor activity. They rationalized that this effect
of DLCs was caused by their selective accumulation in
the negatively charged mitochondria in carcinoma cells
due to the electrochemical proton gradient.1
the compounds described in the studies were still rather
toxic to normal cells and have never been developed for
clinical use.
-7
However,
In our search for a DLC compound possessing satis-
factory chemical and biological properties, we conducted
an extensive screening of numerous cationic dye mol-
ecules, which were originally developed for silver halide
photographic systems in our research laboratories. As
a result, we found that rhodacyanine dyes exhibited
selective antitumor activity against the human colon
carcinoma cell line, CX-1, in comparison to its activity
against the indicator cell for normal epithelial cell line,
8
CV-1. In our previous paper, we reported that two
classes of rhodacyanine dyes, such as 1 (class I) and 2
(class II) (Chart 1), exhibited a potent inhibitory effect
in vitro on the growth of several tumor cell lines and
were also efficacious in tumor-bearing nude mice mod-
els. Furthermore, our structure-activity study revealed
that rhodanine and π electron delocalization were the
essential structural requirements and that any slight
modification on the moiety resulted in a loss of the
antitumor activity.
As a new mechanism of action, we focused our
attention on the studies by Chen et al. which reported
†
Fuji Photo Film Co., Ltd.
Harvard Medical School.
Abstract published in Advance ACS Abstracts, December 15, 1997.
‡
X
S0022-2623(97)00590-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/01/1998

Rhodacyanine Dyes as Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 131
Ch a r t 1
rhodacyanine 12 was accomplished via reactions similar
to those in the synthesis of 1 from 6 in Scheme 1. The
chloride compound 3 was prepared from p-toluene-
sulfonate salt 12 by passing it through the commercially
available strongly basic anion-exchange resin (chloride
form), and the acetate compound 13 was obtained from
1
2 by using weakly basic anion-exchange resin which
was converted to acetate form by acetic acid before use.
As for the synthesis of a structurally restricted rhoda-
cyanine 17, the condensation of 2-aminothiophenol and
5
-bromovaleryl chloride was followed by an intramo-
lecular N-alkylation to give intermediate 16, which was
converted into the desired compound 17 by the usual
methods (Scheme 3). Other class II rhodacyanine dyes,
whose structures are summarized in Tables 2 and 3,
were also synthesized in an analogous manner.
Class II rhodacyanine dye 3 was subjected to X-ray
crystallography analysis. As shown in Figure 1, the
study revealed that all three rings (A, B, rhodanine)
were almost coplanar, and both of the N-ethyl groups
on the B ring and rhodanine were situated on the same
side of the molecule while the N-methyl group on the A
ring was sitting on the opposite side. NOE signals
between the methine proton and the two N-ethyl groups
were observed in an NMR study (Figure 2). This
indicated that the molecular geometry of Figure 2, at
least in the cationic dye unit (rhodanine-B ring), was
conserved in a 10:1 solution of methanol-d4 and chlo-
roform-d.
On the basis of these findings, we designed and
synthesized a wide variety of rhodacyanine dyes and
evaluated them for biological (in vitro activity, in vivo
efficacy, and toxicity) and chemical (stability, water
solubility) properties. We placed attention on the
structure-activity relationship of two heteroaromatic
groups which are conjugated to the rhodanine moiety.
This led to the identification of compound 3 (MKT 077),
which is currently undergoing evaluation in phase I
clinical trial.
Ch em istr y
Resu lts a n d Discu ssion
Rhodacyanine dye consists of three rings, two het-
eroaromatic rings (A and B) and a central 4-oxothiazol-
idine (rhodanine), in which two-dye-conjugate systems,
a neutral and a cationic dye unit, are integrated at the
rhodanine moiety. The dye molecules discussed in this
study are structurally categorized into two classes, class
I/class II, depending on the methine length between A
ring and the rhodanine moiety. Class I has two methine
groups (for general formula, see Table 1) and class II
has the A ring conjugated directly to the rhodanine
moiety (for general formula, see Table 2). Syntheses of
these compounds were accomplished according to re-
ported procedures,9 but with some modifications.
Scheme 1 illustrates a typical synthesis scheme of a
class I rhodacyanine dye. 3-Ethyl-4-oxothiazolidine-2-
thione (4) was reacted with 1,3-diaza-1,3-diphenylpro-
pene and then with acetic anhydride to give com-
pound 5, which was condensed with 3-ethyl-2-methyl-
naphtho[1,2-d]thiazolinium p-toluenesulfonate to give
neutral dye 6. S-Methylation of 6 was followed by the
reaction of 3-ethyl-2-methylnaphtho[1,2-d]thiazolinium
p-toluenesulfonate in the presence of triethylamine to
give the desired rhodacyanine dye 1. Other class I
rhodacyanine dyes, whose structures are summarized
in Table 1, were also synthesized in an analogous
manner.
We previously reported that both class I and II
rhodacyanine dyes displayed marked and selective
antitumor activity and that rhodanine was the essential
8
moiety. In our present study, rhodacyanine dyes
having a variety of heteroaromatic groups were evalu-
ated for their in vitro and in vivo antitumor activity.
The compounds, which exhibited antitumor activity,
were also tested on thier toxicity and chemical proper-
ties as well to select an optimized compound for clinical
trials.
We first devoted our efforts to the study of class I,
the results of which are summarized in Table 1. Most
of the compounds in this class, except for 18 and 21e,
exhibited high inhibitory in vitro effect on the growth
of human carcinoma, the colon (CX-1) and epidermoid
(KB), with IC50 values of below 0.5 and 1.0 µM,
respectively. These active compounds were further
evaluated for their prolongation activities against hu-
man melanoma LOX cell lines in nude mice model.
,10
6
After the intraperitoneal (ip) implantation of 2 × 10
human melanoma LOX cells, appropriately determined
doses of the compounds were intraperitoneally admin-
istered according to schedule. The evaluation of efficacy
was determined by the ratio of the survival periods of
the treated group against the control group (T/C).
Compounds 1, 20, 23, and 24 exhibited modest in vivo
efficacy with T/C values of 126, 122, 138, and 143,
respectively, among the class I rhodacyanine dyes
evaluated.
In Scheme 2, an example of a typical synthesis of a
class II rhodacyanine dye is described. N-Methylation
of 2-methylthiobenzothiazole (8) using methyl p-tolu-
enesulfonate was followed by the condensation with
We also examined the in vitro inhibitory effect of
N-substituents of the B ring, and the results are
summarized in Table 1. The increase in size from the
3
1
-ethyl-4-oxothiazolidine-2-thione (4) to give neutral dye
0. Conversion of the intermediate 10 to the target

1
32 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Kawakami et al.
Ta ble 1. Effect of Heterocycles on Antitumor Activity
methyl to the butyl group had little effect (21a -c), while
introduction of the hydrophilic or anionic substituents,
such as the hydroxyl (21d ) or the carboxyl group (21e),
led to a decrease or even a loss of the in vitro activities.
This indicated the importance of the hydrophobic char-
acter of the rhodacyanine dyes for antitumor activities,
which was, in turn, consistent with the hypothesis of
the DLCs.

Rhodacyanine Dyes as Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 133
Ta ble 2. Effect of Heterocycles on Antitumor Activity
Compounds 1, 20, 23, and 24 which exhibited efficacy
in the LOX nude mice model, were further evaluated
for chemical stability and water solubility, which are
important factors for clinical development. These com-
pounds did not satisfy the criteria. Compound 20
decomposed rapidly in aqueous solution, and compound
24 decomposed in its solid state by 5% at 50 °C in a
month. Although compounds 1 and 23 were chemically
stable, they were poorly soluble in plasma because of
their high hydrophobicity. They were observed to
precipitate in several organs, especially in the lung, even
when administered at effective doses to mice, and such

1
34 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Kawakami et al.
Sch em e 1a
a
Reagents: (a) (i) 1,3-diaza-1,3-diphenylpropene/ligroin, (ii) Ac2O/NEt3; (b) 3-ethyl-2-methylnaphtho[1,2-d]thiazolium p-toluenesulfonate/
Ac2O/NEt3/MeCN; (c) methyl p-toluenesulfonate/DMF/toluene; (d) 3-ethyl-2-methylnaphtho[2,1-d]thiazolium p-toluenesulfonate/NEt3/
MeCN.
Sch em e 2a
a
Reagents: (a) methyl p-toluenesulfonate/anisole; (b) 3-ethyl-4-oxothiazolidine-2-thione (4)/NEt3/MeCN; (c) methyl p-toluenesulfonate/
DMF; (d) (i) 1-ethyl-2-methylpyridinium p-toluenesulfonate/NEt3/MeCN, (ii) PA-318/MeOH/CH2Cl2.
Sch em e 3a
a
Reagents: (a) 5-bromovaleryl chloride/xylene; (c) (i) 11/NEt3/MeCN, (ii) WA-21/MeOH/CH2Cl2.
precipitations might be conducive to the development
of serious adverse effects.
II rhodacyanine dyes, which have the thiazole (including
benzo- and naphtho-fused) or thiazoline as their A ring,
exhibited significant in vitro antitumor activity, in
general. Replacing sulfur with oxygen (13 vs 27) led to
a decrease in the activity. Reversing the A and the B
ring of compound 13 also resulted in a decrease in
activity for compound 26.
Being unable to find a compound for further develop-
ment from class I series, we next worked on the class
II series. In this series, on the basis of our previously
obtained data in Table 1 (21a -e: note that a longer
alkyl chain had an unfavorable effect on the solubility),
the N-substituent of the B ring was retained as the
constant ethyl group. As summarized in Table 2, class
On the basis of these findings, class II rhodacyanine
dyes having the thiazole or thiazoline as the A ring

Rhodacyanine Dyes as Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 135
Ta ble 3. Effect of Heterocycles on Antitumor Activity and Toxicity
a
T/C represents the ratio of the median survival time of drug-treated to control, untreated tumor-bearing mice, expressed as 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
c
group consisted of five nude mice. The statistical significance of difference between the control and the drug-treated group was determined
by applying the Bartlett’s, Dunnett’s, and Scheffer’s test. *P < 0.01, **P < 0.001.
shown in Table 2 were further evaluated for their in
vivo efficacy. Compound 13 was the most efficacious
in prolongation of survival time with T/C ) >310%,
which implied a cure, followed by compound 25 with T/C
them, compound 13, in particular again, was found to
have exceptionally low toxic activity against the normal
kidney cell line, CV-1 (IC50 ) 63.2 µM), and conse-
quently it also exhibited selective toxicity against tumor
cells. An in vivo study (the LOX ip xenograft model) of
the compounds demonstrated that compounds 13, 17,
25, 33, and 35 displayed marked efficacy, with T/C
>140% at doses of 4-5 mg/kg (ip administration).
Among the compounds, compound 17 showed a rela-
tively high acute toxicity (single intravenous (iv) ad-
ministration into ICR mice) with LD50 ) 3 mg/kg, and
compound 35 was chemically unstable because the
oxazolium moiety was easily hydrolyzed. These two
compounds were, therefore, eliminated from the list of
candidates for further study. The remaining com-
pounds 13, 25, and 33 were chemically stable. They
were further evaluated for their inhibitory effect using
)
163%. These two compounds were also found to have
satisfactory chemical properties, i.e., good solubility in
water (>10 mg/mL) and chemical stability (little de-
composition either in solid at 95 °C for 2 weeks or in
aqueous solution at 50 °C for 2 weeks).
Compounds 13 and 25, which exhibited the most
favarable biological and chemical properties, both pos-
sessed a benzothiazole moiety for the A ring. Therefore,
we decided to have a benzothiazole moiety as ring A and
introduce various heterocyclic groups for the B ring in
the class II series. Most of the compounds thus designed
displayed significant in vitro activities against CX-1 and
KB, with IC50 values of below 1.1 µM (Table 3). Among

1
36 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Kawakami et al.
After finding that the best ring combination through
our experiments with the class II series was that of A
(benzothiazole) and B (pyridine), we focused our final
attention on the counteranion, which might affect the
chemical (stability, solubility) and the biological (anti-
tumor effect, toxicity) properties. As shown in Table 4,
while the iodide 36 was poorly soluble in water and
saline, conversion to the chloride and acetate (3 and 13)
apparently enhanced the solubility by more than 25-
fold. However, despite its significant effect on the
chemical properties, the variation of the counteranion
had little effect on its biological properties. Compounds
3
, 13 and 36 displayed quite similar in vitro activities
against KB cells. The former two compounds, whose
water solubility were high enough for in vivo studies,
exhibited virtually the same in vivo efficacy (against
LOX and CA755) and toxicity. While there were no
significant differences found in the chemical and the
biological properties of the compounds 3 and 13, the
former could be more readily prepared from p-toluene-
sulfonate salt (12 in Scheme 2) by using a commercially
available anion-exchange resin (quaternary ammonium
chloride form). Thus, we selected chloride as the
preferred counteranion.
F igu r e 1. X-ray-determined structure of rhodacyanine dye
3
.
On the basis of the data obtained from its in vitro
activity, in vivo efficacy and toxicity, chemical stability,
water solubility, and synthetic availability, we finally
selected compound 3 (named MKT 077) as the candidate
for clinical trials. Extensive studies on pharmacology,
pharmacokinetics, toxicity, and mechanism of action
1
1-13
were carried out.
Mechanistic studies have dem-
onstrated that 3 inhibited respiratory activity in mito-
chondrial membrane fragments in a dose-dependent
1
1
manner. Further, our study showed that under the
same conditions and without a loss of nuclear DNA, 3
caused a selective loss of mitochondrial DNA in CX-1
cells (carcinoma), but not CV-1 cells (normal epithelial).
3
is the first antitumor agent which selectively inhibits
mitochondrial function, and the phase I clinical trials
are in progress.
Con clu sion
Various class I and II rhodacyanine dyes were syn-
thesized and evaluated for their chemical (water solu-
bility, chemical stability) and biological (in vitro anti-
tumor activity, in vivo efficacy and toxicity) properties.
Our attention and effort was particularly focused on the
structure-activity study of heteroaromatic rings, which
were conjugated to rhodanine moiety. This led to the
finding of benzothiazole (A ring) plus pyridinium ring
(B ring) as the best combination in class II series. The
reversal of these positions resulted in a decrease in
activity. The counteranion had a marked effect on
solubility in water, while it had virtually no effect on
the biological properties. The selected compound 3
(MKT 077) displayed high-level efficacy in both LOX
xenograft and CA755 allograft models with low acute
toxicity. From this work, compound 3 emerged as a
promising antitumor agent and has entered into phase
I clinical trials.
F igu r e 2. ROESY spectrum of rhodacyanine dye 3 (600 MHz,
DC OD/CDCl (10/1)).
3
3
the adenocarcinoma CA755 subcutaneous (sc) allograft
model and iv treatment in BDF1 mice. Compound 13
displayed the highest efficacy with tumor inhibi-
tion ratio (TI) ) 76.5%, while compounds 25 and 33
were also moderately effective with TI ) 59.4 and
4
5.6%, respectively. An acute toxicity study demon-
strated that compound 13 showed the lowest toxicity
LD50 ) 20 mg/kg (iv), 50 mg/kg (ip)). The pyridinium
(
ring was, therefore, selected as the optimal moiety for
the B ring.

Rhodacyanine Dyes as Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 137
Ta ble 4. Effect of Anions on Solubility and in Vitro Activity
solubility (mg/mL)
IC50 (µM)
in vivo
toxicity LD50 mg/kg
X-
H2O
0.9% saline
KB cell
CV-1
T/C (%) (LOX)
a
TI (%) (CA755)
b
iv
ip
compd
3
Cl-
>5.0
>5.0
0.81
0.86
0.80
>69.0
>344**c
75.5**
(7 36 .m5 g/kg/day × 4)
(3 mg/kg/day × 4)
20
50
(
5 mg/kg/day × 4)
1
3
3
6
AcO-
I-
>5.0
>5.0
<0.1
63.2
46.8
>310*
20
50
(3 mg/kg/day × 4)
0.2
a
T/C represents the ratio of the median survival time of drug-treated to control, untreated tumor-bearing mice, expressed as 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
c
group consisted of five nude mice. The statistical significance of difference between the control and the drug-treated group was determined
by applying the Bartlett’s, Dunnett’s, and Scheffer’s test. *P < 0.01, **P < 0.001.
Exp er im en ta l Section
the precipitate was collected to give 6 (27.4 g, 70.7%) as purple-
red crystals: 1H NMR (DMSO-d
) δ 1.16 (t, J ) 7.2 Hz, 3H),
6
1
1.59 (t, J ) 7.2 Hz, 3H), 4.04 (q, J ) 7.2 Hz, 2H), 4.66 (q, J )
The H NMR spectra were recorded on a Bruker AMX-600,
7
7
.2 Hz, 2H), 5.58 (d, J ) 13.2 Hz, 1H), 7.54-7.69 (m, 3H),
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; f, fifthtet; m, multiplet; dd,
doublet of doublets. Ultraviolet-visible (UV-vis) absorption
spectra were recorded on a Shimadzu UV-260 spectrophotom-
eter. 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.
Because of hygroscopicity, correct elemental analyses for most
of the compounds could only be obtained by factoring in partial
hydration of these organic salts.
.85-7.93 (m, 2H), 8.08 (d, J ) 9.0 Hz, 1H), 8.46 (d, J ) 9.0
Hz, 1H); MS (HRMS) for C20
H
18
N
2
OS
3
calcd 398.0581, found
3
2
98.0572. Anal. (C20
4.58.
18 2 3
H N OS ) C, H; N: calcd, 24.13; found,
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 (1). A
mixture of 6 (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-
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 the
mixture was cooled to 25 °C, the precipitate was collected and
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 (5). To a solution of 3-ethyl-4-
oxothiazolidine-2-thione (4) (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
the mixture was cooled to room temperature, the precipitate
was collected and washed with acetone (100 mL) to give
washed with acetonitrile (300 mL). Compound 1 (20.4 g,
1
74.1%) was obtained as green crystals: H NMR (DMSO-d
6
)
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,
3
-et hyl-4-oxo-5-[(phenyla m ino)m et hyliden e]t hia zolin e-2-
thione (45.0 g). This product was mixed with acetic anhydride
110 g, 1080 mmol) and triethylamine (0.18 g, 2 mmol), and
+
J ) 8.4 Hz, 1H), 8.52 (d, J ) 8.4 Hz, 1H); MS (FAB ,
-
(
nitrobenzyl alcohol) for C34
zyl alcohol) for C
OS calcd 592.1551, found 592.1561. Anal. (C41
2.7H O) C, N, S; H: calcd, 5.26; found, 4.84.
H
30
N
3
OS
3
m/z 592, (FAB , nitroben-
+
the mixture was stirred at 110 °C for 30 min. The reaction
mixture was concentrated to about 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 5 (37.0 g, 77.9%) as yellow crystals: H NMR (DMSO-d
7
H
7
O
3
S m/z 171; HRMS (FAB ) for C34
H
30
N
3
-
3
H
37
N
3
O
4
S
4
‚
2
3-E t h yl-5-(3-m et h ylb en zot h ia zolin -2-ylid en e)-4-oxo-
th ia zolid in e-2-th ion e (10). A mixture of 2-(methylthio)-
benzothiazole (8) (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 (4) (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
the mixture was cooled to 25 °C, the precipitate was collected
and washed with methanol (140 mL) to give 10 (59.0 g, 87.0%)
1
6
)
δ 1.08 (t, J ) 7.2 Hz, 3H), 2.02 (s, 3H), 3.95 (q, J ) 7.2 Hz,
H), 7.47-7.57 (m, 2H), 7.59-7.72 (m, 3H), 8.47 (s, 1H); MS
EI) m/z 306. Anal. (C14 ) C, H, N, S.
-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-
2
(
14 2 2 2
H N O S
3
id en e)eth ylid en e]-4-oxoth ia zolid in e-2-th ion e (6). A mix-
ture of 5 (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 the mixture was cooled 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 the mixture was cooled to 25 °C,
as yellow crystals: 1H NMR (DMSO-d
3H), 3.98 (s, 3H), 4.10 (q, J ) 7.2 Hz, 2H), 7.35 (t, J ) 8.0 Hz,
) δ 1.20 (t, J ) 7.2 Hz,
6

1
38 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
H), 7.52 (t, J ) 8.0 Hz, 1H), 7.78 (d, J ) 8.0 Hz, 1H), 7.93 (d,
Kawakami et al.
1
1-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
4-oxot h ia zolid in -2-ylid en e]m et h yl]p yr id in iu m Acet a t e
(13). This compound was prepared from p-toluenesulfonate
salt (12) using the anion-exchange resin (Diaion WA-21,
J ) 8.0 Hz, 1H); MS (EI) m/z 308. Anal. (C13
N, S.
12 2 3
H N OS ) C, H,
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 (11).
mixture of 10 (58.0 g, 188 mmol), methyl p-toluenesulfonate
105.0 g, 564 mmol), and N,N-dimethylformamide (58 mL) was
acetate form): 1H NMR (DMSO-d
1
7
)
1
) δ 1.22 (t, J ) 7.2 Hz, 3H),
.45 (t, J ) 7.2 Hz, 3H), 1.74 (s, 3H), 4.03 (s, 3H), 4.10 (q, J )
.2 Hz, 2H), 4.61 (q, J ) 7.2 Hz, 2H), 5.98 (s, 1H), 7.28 (dd, J
8.0, 8.0 Hz, 1H), 7.43-7.51 (m, 2H), 7.59 (d, J ) 8.0 Hz,
H), 7.86 (d, J ) 8.0 Hz, 1H), 8.06 (d, J ) 8.0 Hz, 1H), 8.28
A
6
(
stirred at 130-145 °C for 2.5 h. After the mixture was 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 being cooled to 25 °C, the precipitate was
collected and washed with acetone (150 mL) to give 11 (86.5
g, 93.0%) as orange crystals: H NMR (DMSO-d
)
2
2
+
(
dd, J ) 8.0, 8.0 Hz, 1H), 8.71 (d, J ) 8.0 Hz, 1H); MS (FAB ,
-
glycerine) for C21
for Cl m/z 35; HRMS (FAB ) for C21
22 3 2
H N OS m/z 396, (FAB , triethanolamine)
+
22 3 2
H N OS calcd 396.1204,
found 396.1217.
Tetr a h yd r oben zo[b]ben zoth ia zoliu m Br om id e (16).
To a solution of 2-mercaptoaniline (14.9 g, 0.12 mol) in xylene
(150 mL) was added 5-bromovaleryl chloride (25.0 g, 0.13 mol)
dropwise at room temperature. The mixture was stirred under
reflux for 1.5 h. After the mixture was cooled to room
temperature, the supernatant was removed, and to the re-
sidual oil were added methanol (20 mL) and then ethyl acetate
(100 mL). The precipitate formed was collected and washed
with ethyl acetate (30 mL) to give 16 (9.7 g, 30.7%) as pale
1
6
) δ 1.33 (t, J
7.2 Hz, 3H), 2.27 (s, 3H), 3.05 (s, 3H), 4.17 (q, J ) 7.2 Hz,
H), 4.24 (s, 3H), 7.10 (d, J ) 8.9 Hz, 2H), 7.46 (d, J ) 8.9 Hz,
H), 7.52 (t, J ) 8.0 Hz, 1H), 7.70 (t, J ) 8.0 Hz, 1H), 8.00 (d,
+
J ) 8.0 Hz, 1H), 8.18 (t, J ) 8.0 Hz, 1H); MS (FAB , glycerine)
-
for C14
m/z 171. Anal. (C21
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
-oxot h ia zolid in -2-ylid en e]m et h yl]p yr id in iu m p -Tolu -
en esu lfon a te (12). To a mixture of 11 (24.7 g, 50 mmol) and
-ethyl-2-methylpyridinium p-toluenesulfonate (14.7 g, 50
H
15
N
2
OS
3
m/z 323, (FAB , triethanolamine) for C
7 7 3
H O S
H
22
N
2
O
4
S
4
‚0.3H O) C, H, N, S.
2
brown crystals: 1H NMR (DMSO-d
.18-2.23 (m, 2H), 3.58 (t, J ) 7.0 Hz, 2H), 4.58 (t, J ) 7.0
Hz, 2H), 7.79-7.92 (m, 2H), 8.24 (d, J ) 8.4 Hz, 1H), 8.45 (t,
) δ 2.02-2.08 (m, 2H),
6
1
2
4
+
J ) 8.4 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C11H12NS
m/z 190, (FAB , triethanolamine) for Br m/z 79, 81; HRMS
3
-
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 temperature. 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
+
(
FAB ) for C11
12BrNS‚0.5H
1-[3-E t h yl-5-(3-m e t h ylb e n zot h ia zolin -2-ylid e n e )-4-
H
12NS calcd 190.0690, found 190.0699. Anal.
(C
11
H
2
O) C, H, Br, N, S.
oxot h ia zolid in e -2-ylid e n e ]t e t r a h yd r ob e n z[b]b e n zo-
th ia zoliu m Aceta te (17). This compound was synthesized
from intermediates 11 and 16, in a manner analogous to the
1
preparation of rhodacyanine dye 13: H NMR (DMSO-d
6
) δ
°
C with constant stirring. The precipitate was collected and
1.34 (t, J ) 7.2 Hz, 3H), 1.56 (s, 3H), 2.20-2.22 (m, 2H), 3.05-
3.08 (m, 2H), 4.20 (s, 3H), 4.23 (t, J ) 7.2 Hz, 2H), 4.32-4.35
(m, 2H), 7.41 (t, J ) 8.4 Hz, 1H), 7.52 (t, J ) 8.4 Hz, 1H), 7.57
(t, J ) 8.4 Hz, 1H), 7.68 (t, J ) 8.4 Hz, 1H), 7.77-7.83 (m,
2H), 8.01 (t, J ) 8.4 Hz, 1H), 8.15 (t, J ) 8.4 Hz, 1H); MS
washed with ethyl acetate (110 mL) to give 12 (14.2 g, 50.0%)
as orange crystals: H NMR (DMSO-d ) δ 1.24 (t, J ) 7.2 Hz,
3
J ) 7.2 Hz, 2H), 4.60 (q, J ) 7.2 Hz, 2H), 5.98 (s, 3H), 7.11 (d,
J ) 8.9 Hz, 2H), 7.28 (dd, J ) 8.0, 8.0 Hz, 1H), 7.47 (dd, J )
8
8
1
1
6
H), 1.43 (t, J ) 7.2 Hz, 3H), 2.28 (s, 3H), 4.03 (s, 3H), 4.11 (q,
+
-
(FAB , nitrobenzyl alcohol) for C24
triethanolamine) for C
OS
BrN
H
22
N
3
OS
3
m/z 464, (FAB ,
+
.0, 8.0 Hz, 1H), 7.49 (d, J ) 8.9 Hz, 2H), 7.61 (dd, J ) 8.0,
.0 Hz, 1H), 7.88 (dd, J ) 8.0, 8.0 Hz, 1H), 8.06 (d, J ) 8.0 Hz,
H), 8.26 (t, J ) 8.0 Hz, 1H), 8.70 (d, J ) 8.0 Hz, 1H); MS
2
H
3
O
2
m/z 59; HRMS (FAB ) for C24
calcd 464.0925, found 464.0921. Anal. (C26
‚3.5H O) C, N; S: calcd, 5.50; found, 4.91.
H
22
N
H
25
3
-
-
3
3
O
3
S
3
2
+
-
(FAB , glycerine) for C21
H
22
N
3
OS
2
m/z 396, (FAB , triethanol-
‚0.7H O)
3-Eth yl-2-[[3-eth yl-5-[2-(3-m eth yln a p h th o[1,2-d ]th ia zo-
lin -2-ylid en e)et h ylid en e]-4-oxot h ia zolid in -2-ylid en e]-
m eth yl]-4-m eth ylth ia zoliu m Aceta te (18). The p-toluene-
sulfonate salt of the target compound was synthesized from
2,3-dimethylnaphtho[1,2-d]thiazolinium p-toluenesulfonate,
3-ethyl-4-oxothiazolidine-2-thione (4), and 3-ethyl-2,4-dimeth-
ylthiazolium p-toluenesulfonate in a manner analogous to the
preparation of rhodacyanine dye 1, and it was converted to
acetate salt (18) using anion-exchange resin (Diaion WA-21,
amine) for C
C, H, N, S.
7
H
7
SO
3
m/z 171. Anal. (C28
H
29
N
3
O
4
S
3
2
1
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
-oxoth ia zolid in -2-ylid en e]m eth yl]p yr id in iu m Ch lor id e
3, MKT 077). A solution of 12 in the solution (375 mL) of
4
(
chloroform/methanol (1/2, v/v) was passed through the basic
anion-exchange resin (about 350 mL, Diaion PA-318, chloride
form), and the resin was washed with a solution (375 mL) of
chloroform/methanol (1/2, v/v) and then methanol (250 mL).
The eluent was evaporated, and the residue was dissolved in
ethanol (275 mL) at 60 °C and then cooled to 45 °C. To this
solution was added ethyl acetate (725 mL) at 40-45 °C with
constant stirring. The precipitate was collected and washed
with ethyl acetate (175 mL) to give 3 (9.0 g, 42.0%) as orange
crystals: mp 253-257 °C dec; IR (KBr) 760, 1050, 1160, 1385,
acetate form): 1H NMR (DMSO-d
) δ 1.18 (t, J ) 7.2 Hz, 3H),
6
1
7
1
2
.29 (t, J ) 7.2 Hz, 3H), 1.69 (s, 3H), 2.38 (s, 3H), 4.04 (t, J )
.2 Hz, 2H), 4.19 (s, 3H), 4.35 (t, J ) 7.2 Hz, 2H), 5.86 (d, J )
3.0 Hz, 1H), 6.45 (s, 1H), 7.50-7.65 (m, 4H), 7.78-7.89 (m,
H), 8.01 (d, J ) 8.3 Hz, 1H), 8.58 (d, J ) 8.3 Hz, 1H); MS
+
-
(
FAB , nitrobenzyl alcohol) for C26
nitrobenzyl alcohol) for C
C H N OS calcd 492.1238, found 492.1234.
H
26
N
3
OS
3
m/z 492, (FAB ,
+
2
H
3
O
2
m/z 59; HRMS (FAB ) for
-1
1
515, 1550, 1635, 1650, 2900-3000 cm ; UV-vis λmax ) 494.0
26 26
3
3
4
4
1
(
MeOH, ꢀ ) 4.94 × 10 ), 486 nm (H
NMR (CD OD:CDCl ) 10:1) δ 1.34 (t, J ) 7.2 Hz, 3H), 1.57
t, J ) 7.2 Hz, 3H), 4.05 (s, 3H), 4.09 (q, J ) 7.2 Hz, 2H), 4.54
2
O, ꢀ ) 4.56 × 10 ); H
3-Eth yl-2-[[3-eth yl-5-[2-(3-eth yln aph th o[2,1-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[1,2-d]oxazoliu m p-Tolu en esu lfon ate (19). This
compound was synthesized from 2,3-dimethylnaphtho[2,1-d]-
thiazolinium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-
thione (4), and 3-ethyl-2-methylnaphtho[1,2-d]oxazolium p-
3
3
(
(
q, J ) 7.2 Hz, 2H), 5.86 (s, 1H), 7.25 (ddd, J ) 8.0, 7.0, 1.4
Hz, 1H), 7.34 (ddd, 7.5, 6.6, 1.4 Hz, 1H), 7.40 (dd, J ) 8.3, 1.4
Hz, 1H), 7.44 (ddd, J ) 8.3, 7.0, 1.4 Hz, 1H), 7.65 (dd, J ) 8.0,
1
.4 Hz, 1H), 8.04 (dd, J ) 8.5, 1.4 Hz), 8.19 (ddd, 8.5, 7.5, 1.9
toluenesulfonate in a manner analogous to the preparation of
1
3
1
Hz, 1H), 8.49 (dd, 6.6, 1.9 Hz, 1H); C NMR (CD
δ 12.5, 14.7, 35.2, 39.8, 53.8, 80.2, 84.4, 112.4, 120.5, 122.8,
1
1
3
OD + CDCl
3
)
rhodacyanine dye 1: H NMR (DMSO-d
6
) δ 1.25 (t, J ) 7.2
Hz, 3H), 1.41 (t, J ) 7.2 Hz, 3H), 1.58 (t, J ) 7.2 Hz, 3H), 2.29
(s, 3H), 4.15 (q, J ) 7.2 Hz, 2H), 4.47 (q, J ) 7.2 Hz, 2H), 4.92
(q, J ) 7.2 Hz, 2H), 6.09 (d, J ) 13.1 Hz, 1H), 6.50 (s, 1H),
7.10 (d, J ) 7.9 Hz, 2H), 7.43-7.52 (m, 3H), 7.59 (t, J ) 8.6
Hz, 1H), 7.70-7.90 (m, 5H), 8.02-8.11 (m, 3H), 8.19 (d, J )
8.6 Hz, 1H), 8.26 (d, J ) 8.6 Hz, 1H), 8.48 (d, J ) 8.6 Hz, 1H);
25.0, 125.1, 127.5, 128.3, 141.4, 143.6, 144.8, 152.0, 153.5,
+
57.0, 165.7; MS (FAB , glycerine) for C21
22 3 2
H N OS m/z 396,
-
+
(
C
FAB , triethanolamine) for Cl m/z 35; HRMS (FAB ) for
OS calcd 396.1204, found 396.1186. Anal. (C21
) C, H, Cl, N, S.
21
H
22
N
3
2
22
H -
3 2
ClN OS

Rhodacyanine Dyes as Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 139
+
MS (FAB , nitrobenzyl alcohol) for C34
H
30
N
3
O
2
S
2
m/z 576,
a manner analogous to the preparation of rhodacyanine dye
-
1
(
(
FAB , triethanolamine) for
‚0.5H O) C, H, N, S.
-E t h yl-2-[[3-et h yl-5-[2-(3-et h yl-4-m et h ylt h ia zolin -2-
C
7
H
7
O
3
S
m/z 171. Anal.
1: H NMR (DMSO-d
2H), 3.72 (q, J ) 7.2 Hz, 2H), 3.93 (t, J ) 7.2 Hz, 2H), 4.07-
6
) δ 1.20-1.31 (m, 6H), 3.40-3.45 (m,
C
41
H
37
N
3
O
5
S
3
2
4
1
8
1
C
1
5
.17 (m, 4H), 4.92 (t, J ) 7.2 Hz, 2H), 5.78 (d, J ) 13.2 Hz,
H), 6.94 (s, 1H), 7.61 (d, J ) 13.2 Hz, 1H), 7.74 (dd, J ) 8.0,
.0 Hz, 1H), 7.86 (dd, J ) 8.0, 8.0 Hz, 1H), 8.14 (d, J ) 8.0 Hz,
3
ylid en e)eth ylid en e]-4-oxoth ia zolid in -2-ylid en e]m eth yl]-
ben zoth ia zoliu m Aceta te (20). The p-toluenesulfonate salt
of the target compound was synthesized from 3-ethyl-2,4-
dimethylthiazolinium p-toluenesulfonate, 3-ethyl-4-oxothia-
zolidine-2-thione (4), and 3-ethyl-2-methylbenzothiazolium
p-toluenesulfonate in a manner analogous to the preparation
of rhodacyanine dye 1, and it was converted to acetate salt 20
+
H), 8.19-8.31 (m, 3H). MS (FAB , nitrobenzyl alcohol) for
-
26
H
28
N
3
O
2
S
3
m/z 510, (FAB , nitrobenzyl alcohol) for I m/z
+
27; HRMS (FAB ) for C26
10.1333. Anal. (C26
H
28
N
3
O
2
S
3
calcd 510.1343, found
2
O) C, H, I, N, S.
H
28IN
3
O
2
S
3
‚2.0H
3-(2-Ca r boxyeth yl)-2-[[3-eth yl-5-[2-(3-eth ylth ia zolid in -
using anion-exchange resin (Diaion WA-21, acetate form): 1
NMR (DMSO-d ) δ 1.23 (t, J ) 7.2 Hz, 3H), 1.28-1.39 (m, 6H),
.76 (s, 3H), 2.33 (s, 3H), 4.11-4.24 (m, 4H), 4.69 (q, J ) 7.2
H
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[2,1-d ]th ia zoliu m Iod id e (21e). This compound
was synthesized from 3-ethyl-2-methylthiazolinium p-toluene-
sulfonate, 3-ethyl-4-oxothiazolidine-2-thione (4), and 3-(2-
carboxyethyl)-2-methylnaphtho[2,1-d]thiazolium iodide in a
6
1
Hz, 2H), 6.00 (d, J ) 13.5 Hz, 1H), 6.69 (s, 1H), 6.95 (s, 1H),
7
7
)
.53 (dd, J ) 8.4, 8.4 Hz, 3H), 7.71 (dd, J ) 8.4, 8.4 Hz, 1H),
.74 (d, J ) 13.5 Hz, 1H), 7.94 (d, J ) 8.4 Hz, 1H), 8.20 (d, J
manner analogous to the preparation of rhodacyanine dye 1:
+
1
8.4 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C23
H
26
N
3
-
6
H NMR (DMSO-d ) δ 1.22-1.30 (m, 6H), 2.90 (t, J ) 7.2 Hz,
-
OS
HRMS (FAB ) for C23
-[[3-E t h yl-5-[2-(3-e t h ylt h ia zolid in -2-ylid e n e )e t h yl-
3
m/z 456, (FAB , nitrobenzyl alcohol) for C
2
H
3
O
2
m/z 59;
2H), 3.40-3.46 (m, 2H), 3.72 (q, J ) 7.2 Hz, 2H), 4.05-4.13
(m, 4H), 4.97 (t, J ) 7.2 Hz, 2H), 5.80 (d, J ) 13.2 Hz, 1H),
6.95 (s, 1H), 7.61 (d, J ) 13.2 Hz, 1H), 7.73 (dd, J ) 8.0, 8.0
Hz, 1H), 7.84 (dd, J ) 8.0, 8.0 Hz, 1H), 8.11 (d, J ) 8.0 Hz,
+
26 3 3
H N OS calcd 456.1238, found 456.1231.
2
iden e]-4-oxoth iazolidin -2-yliden e]m eth yl]-3-m eth yln aph -
th o[2,1-d ]th ia zoliu m Iod id e (21a ). This compound was
synthesized from 3-ethyl-2-methylthiazolinium p-toluene-
sulfonate, 3-ethyl-4-oxothiazolidine-2-thione (4), and 2,3-di-
methylnaphtho[2,1-d]thiazolium iodide in a manner analogous
+
1H), 8.16-8.28 (m, 3H); MS (FAB , nitrobenzyl alcohol) for
-
C
27
H
28
N
3
O
3
S
3
m/z 538, (FAB , nitrobenzyl alcohol) for I m/z
+
127; HRMS (FAB ) for C27
538.1290.
28 3 3 3
H N O S calcd 538.1293, found
1
to the preparation of rhodacyanine dye 1: H NMR (DMSO-
3-E t h yl-2-[[3-e t h yl-5-[2-(1,3,3-t r im e t h ylin d olin -2-yl-
id en e)eth ylid en e]-4-oxoth ia zolid in e-2-ylid en e]m eth yl]-
n a p h th o[2,1-d ]oxa zoliu m p-Tolu en esu lfon a te (22). This
compound was synthesized from 1,2,3,3-tetramethylindolinium
p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-thione (4), and
3-ethyl-2-methylnaphtho[1,2-d]oxazolium p-toluenesulfonate
in a manner analogous to the preparation of rhodacyanine dye
d
6
) δ 1.26 (t, J ) 7.2 Hz, 6H), 3.40-3.47 (m, 2H), 3.72 (q, J )
7
(
.2 Hz, 2H), 4.09 (q, J ) 7.2 Hz, 2H), 4.16-4.28 (m, 5H), 5.80
d, J ) 13.2 Hz, 1H), 6.83 (s, 1H), 7.62 (d, J ) 13.2 Hz, 1H),
7
8
.74 (dd, J ) 7.9, 7.9 Hz, 1H), 7.87 (dd, J ) 7.9, 7.9 Hz, 1H),
.18 (d, J ) 7.9 Hz, 1H), 8.21-8.28 (m, 2H), 8.32 (d, J ) 7.9
+
Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C25
26 3 3
H N OS m/z
-
1: 1H NMR (DMSO-d ) δ 1.27 (t, J ) 7.2 Hz, 3H), 1.48 (t, J )
4
26
80, (FAB , nitrobenzyl alcohol) for I m/z 127. Anal. (C25H -
6
IN
3
OS
3
‚0.5H
2
O) C, H, I, N, S.
7.2 Hz, 3H), 1.65 (s, 6H), 2.28 (s, 3H), 3.58 (s, 3H), 4.17 (q, J
) 7.2 Hz, 2H), 4.65 (q, J ) 7.2 Hz, 2H), 5.84 (d, J ) 13.5 Hz,
1H), 6.64 (s, 1H), 7.10 (d, J ) 7.6 Hz, 2H), 7.11-7.14 (m, 1H),
7.22 (d, J ) 7.8 Hz, 1H), 7.30-7.39 (m, 1H), 7.47 (d, J ) 7.6
Hz, 2H), 7.49-7.52 (m, 1H), 7.76 (dd, J ) 7.9, 7.9 Hz, 1H),
7.92 (dd, J ) 7.9, 7.9 Hz, 1H), 8.05 (d, J ) 7.8 Hz, 1H), 8.07
3
-Eth yl-2-[[3-eth yl-5-[2-(3-eth ylth ia zolid in -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-
2,1-d ]th ia zoliu m Iod id e (21b). This compound was syn-
thesized from 3-ethyl-2-methylthiazolinium p-toluenesulfonate,
-ethyl-4-oxothiazolidine-2-thione (4) and 3-ethyl-2-methyl-
naphtho[2,1-d]thiazolium iodide in a manner analogous to the
preparation of rhodacyanine dye 1: H NMR (DMSO-d
.20-1.29 (m, 6H), 1.44 (t, J ) 7.2 Hz, 3H), 3.40-3.50 (m, 2H),
[
3
(d, J ) 13.5 Hz, 1H), 8.21-8.28 (m, 2H), 8.58 (d, J ) 7.8 Hz,
1
+
) δ
1H); MS (FAB , nitrobenzyl alcohol) for C32
H
32
N
3
O
2
S m/z 522,
6
-
1
3
7
1
7
8
(FAB , nitrobenzyl alcohol) for C
‚1.0H O) C, H, N, S.
7 7 3
H O S m/z 171. Anal.
.72 (q, J ) 7.2 Hz, 2H), 4.07 (t, J ) 7.2 Hz, 2H), 4.19 (q, J )
.2 Hz, 2H), 4.86 (q, J ) 7.2 Hz, 2H), 5.80 (d, J ) 13.2 Hz,
H), 6.82 (s, 1H), 7.62 (d, J ) 13.2 Hz, 1H), 7.75 (dd, J ) 7.9,
.9 Hz, 1H), 7.86 (d, J ) 7.9, 7.9 Hz, 1H), 8.14-8.36 (m, 3H),
(C39
H
39
N
3
O
5
S
2
2
3-E t h yl-2-[[3-e t h yl-5-[2-(1-e t h ylp yr id in -2-ylid e n 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 Ch lor id e (23). The p-toluenesulfonate
salt of the target compound was synthesized from 1-ethyl-2-
methylpyridinium p-toluenesulfonate, 3-ethyl-4-oxothia-
zolidine-2-thione (4), and 3-ethyl-2-methylnaphtho[1,2-d]-
thiazolium p-toluenesulfonate in a manner analogous to the
preparation of rhodacyanine dye 1 to convert to chloride salt
22 using anion-exchange resin (Diaion PA-318, chloride
+
.33 (d, J ) 7.9 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for
-
C
26
H
28
N
3
OS
3
+
m/z 494, (FAB , nitrobenzyl alcohol) for I m/z 127;
HRMS (FAB ) for C26
Anal. (C26 OS
-Bu t yl-2-[3-et h yl-5-[2-(3-et h ylt h ia zolid in -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-
2,1-d ]th ia zoliu m Iod id e (21c). This compound was syn-
thesized from 3-ethyl-2-methylthiazolinium p-toluenesulfonate,
-ethyl-4-oxothiazolidine-2-thione (4), and 3-buthyl-2-meth-
ylnaphtho[2,1-d]thiazolium iodide in a manner analogous to
the preparation of rhodacyanine dye 1: 1H NMR (DMSO-d
δ 0.94 (t, J ) 7.2 Hz, 3H), 1.22-1.29 (m, 6H), 1.40-1.50 (m,
H), 1.83 (t, J ) 7.2 Hz, 2H), 3.42 (t, J ) 7.2 Hz, 2H), 3.73 (q,
J ) 7.2 Hz, 2H), 4.08 (t, J ) 7.2 Hz, 2H), 4.20 (q, J ) 7.2 Hz,
H), 4.87 (t, J ) 7.2 Hz, 2H), 5.80 (d, J ) 13.2 Hz, 1H), 6.80
s, 1H), 7.63 (d, J ) 13.2 Hz, 1H), 7.74 (dd, J ) 7.9, 7.9 Hz,
H
28
N
3
OS
3
calcd 494.1394, found 494.1396.
H
28IN
3
3
‚1.3H O) C, H, I, N, S.
2
3
form): 1H NMR (DMSO-d
) δ 1.27 (t, J ) 7.2 Hz, 3H), 1.42 (t,
[
6
J ) 7.2 Hz, 3H), 1.71 (t, J ) 7.2 Hz, 3H), 4.23 (q, J ) 7.2 Hz,
2H), 4.35 (q, J ) 7.2 Hz, 2H), 5.01 (q, J ) 7.2 Hz, 2H), 5.64 (d,
J ) 13.5 Hz, 1H), 6.70 (s, 1H), 6.91-6.98 (m, 1H), 7.62-7.84
(m, 4H), 7.83 (d, J ) 7.8 Hz, 1H), 7.96-8.03 (m, 1H), 8.05 (d,
3
6
)
J ) 13.5 Hz, 1H), 8.12-8.23 (m, 3H), 8.62 (d, J ) 7.8 Hz, 1H);
+
2
MS (FAB , nitrobenzyl alcohol) for C28
H
28
N
3
OS
2
m/z 486,
-
+
(FAB , nitrobenzyl alcohol) for Cl m/z 35; HRMS (FAB ) for
OS calcd 486.1674; found 486.1671.
-E t h yl-2-[[3-e t h yl-5-[2-(1-e t h ylq u in olin -2-ylid e n e )-
2
(
C
28
H
28
N
3
2
3
1
(
H), 7.87 (dd, J ) 7.9, 7.9 Hz, 1H), 8.14-8.36 (m, 4H); MS
FAB , nitrobenzyl alcohol) for C28
nitrobenzyl alcohol) for I m/z 127; HRMS (FAB ) for C28
et h ylid en e]-4-oxot h ia zolid in -2-ylid en e]m et h yl]t h ia zo-
lin iu m Aceta te (24). The p-toluenesulfonate salt of the
target compound was synthesized from 1-ethyl-2-methylquino-
linium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-thione,
and 3-ethyl-2-methylthiazolinium p-toluenesulfonate in a
manner analogous to the preparation of rhodacyanine dye 1,
+
-
H
32
N
3
OS
3
m/z 522, (FAB ,
+
H
H32IN
32
N
3
-
-
OS
OS
3
calcd 522.1707, found 522.1697. Anal. (C28
‚0.5H O) C, H, I, N, S.
-[[3-E t h yl-5-[2-(3-e t h ylt h ia zolid in -2-ylid e n e )e t h yl-
3
3
2
2
id en e]-4-oxoth ia zolid in -2-ylid en e]m eth yl]-3-(2-h yd r oxy-
eth yl)n a p h th o[2,1-d ]th ia zoliu m Iod id e (21d ). This com-
pound was synthesized from 3-ethyl-2-methylthiazolinium
p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-thione (4), and
and it was converted to acetate salt 24 using anion-exchange
1
resin (Diaion WA-21, acetate form): H NMR (DMSO-d
6
) δ
1.15 (t, J ) 7.2 Hz, 3H), 1.22 (t, J ) 7.2 Hz, 3H), 1.37 (t, J )
7.2 Hz, 3H), 1.50 (s, 3H), 3.67 (t, J ) 7.2 Hz, 2H), 3.83 (q, J )
7.2 Hz, 2H), 4.09 (q, J ) 7.2 Hz, 2H), 4.18 (t, J ) 7.2 Hz, 3H),
3
-(2-hydroxyethyl)-2-methylnaphtho[2,1-d]thiazolium iodide in

1
40 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Kawakami et al.
4
1
1
1
.39 (q, J ) 7.2 Hz, 2H), 5.62 (d, J ) 12.9 Hz, 1H), 6.13 (s,
H), 7.38 (dd, J ) 7.8, 7.8 Hz, 1H), 7.70 (dd, J ) 7.8, 7.8 Hz,
thesized from 2,3-dimethylnaphtho[1,2-d]thiazolium p-
toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-thione, and 3-ethyl-
H), 7.73-7.84 (m, 1H), 7.88 (d, J ) 7.8 Hz, 1H), 8.26 (d, J )
2-methylnaphtho[2,1-d]oxazolium p-toluenesulfonate in a man-
+
1
2.9 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C24
H
28
N
3
-
ner analogous to the preparation of rhodacyanine dye 13:
H
-
OS
HRMS (FAB ) for C24
Anal. (C26
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
-oxot h ia zolid in -2-ylid e n e ]m e t h yl]b e n zot h ia zoliu m
Aceta te (25). This compound was synthesized from 3-ethyl-
-methylbenzothiazolium p-toluenesulfonate and 3-ethyl-4-
oxothiazolidine-2-thione in a manner analogous to the prepa-
2
m/z 438, (FAB , nitrobenzyl alcohol) for C
2
H
3
O
2
m/z 59;
calcd 438.1674, found 438.1655.
2
O) C, H, N, S.
NMR (DMSO-d ) δ 1.32 (t, J ) 7.2 Hz, 3H), 1.48 (t, J ) 7.2
6
+
H
28
N
‚4.3H
3
OS
2
Hz, 3H), 1.81 (s, 3H), 4.00 (q, J ) 7.2 Hz, 2H), 4.60-4.70 (m,
5H), 6.49 (s, 1H), 7.53 (dd, J ) 7.8, 7.8 Hz, 2H), 7.63-7.74 (m,
2H), 7.88-8.05 (m, 4H), 8.21 (d, J ) 7.8 Hz, 1H), 8.32 (d, J )
7.8 Hz, 1H), 8.21 (d, J ) 7.8 Hz, 2H), 8.32 (d, J ) 7.8 Hz, 1H),
H
31
N
3
O
3
S
2
3
4
+
8.63 (d, J ) 7.8 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for
-
2
C
31
H
26
N
3
O
2
S
2
m/z 536, (FAB , nitrobenzyl alcohol) for C
2
H
3
O
2
+
m/z 59; HRMS (FAB ) for C31
536.1464. Anal. (C33
H
26
N
3
O
2
S
2
calcd 536.1466, found
1
ration of rhodacyanine dye 13: H NMR (DMSO-d
J ) 7.1 Hz, 3H), 1.37 (t, J ) 7.1 Hz, 3H), 1.68 (s, 4.5 H), 4.21
s, 4.5H), 4.28 (q, J ) 7.2 Hz, 2H), 4.70 (q, J ) 7.2 Hz, 2H),
.67 (s, 1H), 7.33 (dd, J ) 7.8, 7.8 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),
6
) δ 1.26 (t,
H
29
N
3
O
4
S
2
‚3.0H
2
O) C, H, N, S.
3
-Eth yl-2-[[3-eth yl-5-(3-m eth ylth ia zolid in -2-ylid en e)-4-
(
6
oxot h ia zolid in -2-ylid e n e ]m e t h yl]n a p h t h o[1,2-d ]t h ia -
zoliu m Aceta te (30). This compound was synthesized from
3
-methyl-2-(methylthio)thiazolidinium p-toluenesulfonate, 3-eth-
+
8
.24 (d, J ) 8.1 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for
yl-4-oxothiazolidine-2-thione (4), and 3-ethyl-2-methylnaph-
tho[1,2-d]thiazolium p-toluenesulfonate in a manner analogous
-
C
23
H
22
N
3
OS
m/z 59; HRMS (FAB ) for C23
52.0891.
-E t h yl-2-[[3-e t h yl-5-(1-m e t h ylp yr id in -2-ylid e n e )-4-
3 2 3 2
m/z 452, (FAB , nitrobenzyl alcohol) for C H O
+
H
22
N
3
OS
3
calcd 452.0925, found
1
to the preparation of rhodacyanine dye 13: H NMR (DMSO-
4
d
3
4
7
8
7
6
) δ 1.25 (t, J ) 7.2 Hz, 3H), 1.66 (s, 3H), 1.71 (t, J ) 7.2 Hz,
3
H), 3.30-3.41 (m, 2H), 3.57 (s, 3H), 4.01 (q, J ) 7.2 Hz, 2H),
.22 (q, J ) 7.2 Hz, 2H), 5.06 (q, J ) 7.2 Hz, 2H), 6.70 (s, 1H),
.78 (dd, J ) 7.8, 7.8 Hz, 1H), 7.84 (dd, J ) 7.8, 7.8 Hz, 1H),
.08 (d, J ) 7.8 Hz, 1H), 8.22 (d, J ) 7.8 Hz, 1H), 8.26 (d, J )
oxoth ia zolid in -2-ylid en e]m eth yl]ben zoth ia zoliu m p-Tol-
u en esu lfon a te (26). This compound was synthesized from
1
zolidine-2-thione, and 3-ethyl-2-methylbenzothiazolium p-tolu-
enesulfonate in a manner analogous to the preparation of
rhodacyanine dye 12: H NMR (DMSO-d
Hz, 3H), 1.35 (t, J ) 7.2 Hz, 3H), 2.28 (s, 3H), 4.20 (s, 3H),
4
7
7
,2-dimethylpyridinium p-toluenesulfonate, 3-ethyl-4-oxothia-
+
.8 Hz, 1H), 8.64 (d, J ) 7.8 Hz, 1H); MS (FAB , nitrobenzyl
-
alcohol) for C23
for C
54.1081, found 454.1085. Anal. (C25
H, N, S.
-Eth yl-2-[[3-eth yl-5-(3-m eth yl-4,5,6,7-tetr a h yd r oben -
H
24
N
3
OS
3
m/z 454, (FAB , nitrobenzyl alcohol)
1
6
) δ 1.26 (t, J ) 7.2
H
O
m/z 59; HRMS (FAB ) for C23
+
H
N
OS
calcd
2
O) C,
2
3
2
24
3
3
4
H
27
N
3
O
3
S
3
‚3.0H
.22 (q, J ) 7.2 Hz, 2H), 4.60 (q, J ) 7.2 Hz, 2H), 6.60 (s, 1H),
.10 (d, J ) 7.8 Hz, 2H), 7.21 (dd, J ) 7.8, 7.8 Hz, 1H), 7.41-
.44 (m, 1H), 7.47 (d, J ) 7.8 Hz, 2H), 7.65 (dd, J ) 7.8, 7.8
3
zot h ia zolid in -2-ylid en e]-4-oxot h ia zolid in e-2-ylid en e}-
m eth yl]th ia zolin iu m Aceta te (31). This compound was
synthesized from 3-methyl-2-(methylthio)-4,5,6,7-tetrabenzo-
thiazolidinium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-
Hz, 1H), 7.82 (d, J ) 7.8 Hz, 1H), 7.93 (dd, J ) 7.8, 7.8 Hz,
H), 8.13 (d, J ) 7.8 Hz, 1H), 8.37 (d, J ) 7.8 Hz, 1H), 8.50 (d,
1
+
J ) 7.8 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C21
22 3
H N -
-
OS
Anal. (C28
-E t h yl-2-[[3-et h yl-5-(3-m et h ylben zoxa zol-2-ylid en e)-
-oxoth ia zolid in -2-ylid en e]m eth yl]p yr id in iu m Ch lor id e
27). This compound was synthesized from 2,3-dimethylbenz-
2
m/z 396, (FAB , nitrobenzyl alcohol) for C
7 7 3
H SO m/z 171.
2
-thione (4), and 3-ethyl-2-methylnaphtho[1,2-d]thiazolium
29 3 4 3
H N O S
) C, H, N, S.
p-toluenesulfonate in a manner analogous to the preparation
1
1
of rhodacyanine dye 13: H NMR (DMSO-d
7
(
6
) δ 1.19 (t, J )
4
(
.2 Hz, 3H), 1.22 (t, J ) 7.2 Hz, 3H), 1.57 (s, 3H), 1.70-1.87
m, 4H), 2.58-2.64 (m, 4H), 3.60 (t, J ) 7.2 Hz, 2H), 3.77 (q,
oxazolium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-
thione and 1-ethyl-2-methylpyridinium p-toluenesulfonate in
a manner analogous to the preparation of rhodacyanine dye
3
7
7
J ) 7.2 Hz, 2H), 3.84 (s, 3H), 4.08 (t, J ) 7.2 Hz, 2H), 4.15 (q,
+
J ) 7.2 Hz, 2H), 6.00 (s, 1H); MS (FAB , nitrobenzyl alcohol)
-
for C19
H
26
N
3
OS
m/z 59. Anal. (C21
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iaolin -2-yliden e)-
-oxoth ia zolid in -1-ylid en e]m eth yl]qu in olin iu m Aceta te
32). This compound was synthesized from 3-ethyl-2-methyl-
3
m/z 408, (FAB , nitrobenzyl alcohol) for
1
:
H NMR (DMSO-d
6
) δ 1.15 (t, J ) 7.2 Hz, 3H), 1.41 (t, J )
C
2
H
3
O
2
H
29
N
3
O
3
S
3
2
‚2.0H O) C, H, N, S.
.2 Hz, 3H), 3.22 (s, 3H), 3.89 (q, J ) 7.2 Hz, 2H), 4.70 (q, J )
.2 Hz, 2H), 6.40 (s, 1H), 6.95 (dd, J ) 7.8, 7.8 Hz, 1H), 7.08
1
4
(
(
d, J ) 7.8 Hz, 1H), 7.23 (dd, J ) 7.8, 7.8 Hz, 1H), 7.34 (d, J
)
7.8 Hz, 1H), 7.86 (dd, J ) 7.8, 7.8 Hz, 1H), 8.13 (d, J ) 7.8
benzothiazolium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-
2-thione, and 1-ethyl-2-methylquinolinium p-toluenesulfonate
in a manner analogous to the preparation of rhodacyanine dye
Hz, 1H), 8.49 (dd, J ) 7.8, 7.8 Hz, 1H), 9.04 (d, J ) 7.8 Hz,
+
1
22 3 2
H); MS (FAB , nitrobenzyl alcohol) for C21H N O S m/z 380,
-
+
(FAB , nitrobenzyl alcohol) for Cl m/z 35; HRMS (FAB ) for
13: 1H NMR (DMSO-d
) 7.2 Hz, 3H), 1.50 (s, 3H), 4.12 (s, 3H), 4.22 (q, J ) 7.2 Hz,
) δ 1.29 (t, J ) 7.2 Hz, 3H), 1.48 (t, J
C
ClN
21
H
22
N
3
O
2
S calcd 380.1432, found 380.1429. Anal. (C21
H
22
-
6
3
O
2
S
2
‚2.0H O) C, H, Cl, N, S.
2
2
7
1
7
7
H), 4.78 (q, J ) 7.2 Hz, 2H), 6.11 (s, 1H), 7.30 (dd, J ) 7.8,
.8 Hz, 1H), 7.48 (dd, J ) 7.8, 7.8 Hz, 1H), 7.68 (d, J ) 7.8 Hz,
H), 7.71 (dd, J ) 7.8, 7.8 Hz, 1H), 7.91 (d, J ) 7.8 Hz, 1H),
.98 (dd, J ) 7.8, 7.8 Hz, 1H), 8.11-8.18 (m, 2H), 8.23 (d, J )
3
-Eth yl-2-[[3-eth yl-5-(3-m eth yln a p h th o[1,2-d ]th ia zolin -
2
-ylid en e)-4-oxot h ia zolid in -2-ylid en e]m et h yl]n a p h t h o-
[
2,1-d ]th ia zoliu m Ch lor id e (28). This compound was syn-
thesized from 2,3-dimethylnaphtho[1,2-d]thiazolium p-
toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-thione, and 3-ethyl-
2
ner analogous to the preparation of rhodacyanine dye 3:
NMR (DMSO-d ) δ 1.23 (t, J ) 7.2 Hz, 3H), 1.45 (t, J ) 7.2
+
.8 Hz, 1H), 8.58 (d, J ) 7.8 Hz, 1H); MS (FAB , nitrobenzyl
-
alcohol) for C25
H
24
N
3
OS
2
m/z 446, (FAB , nitrobenzyl alcohol)
-methylnaphtho[2,1-d]thiazolium p-toluenesulfonate in a man-
+
1
2 3 2 25 24 3 2
for C H O m/z 59; HRMS (FAB ) for C H N OS calcd
H
4
46.1361, found 446.1366. Anal. (C27
H
27
N
3
O
3
S
2
2
‚3.0H O) C,
6
H, N, S.
Hz, 3H), 4.32 (q, J ) 7.2 Hz, 2H), 4.63 (s, 3H), 4.83 (q, J ) 7.2
Hz, 2H), 6.76 (s, 1H), 7.43 (dd, J ) 7.8, 7.8 Hz, 1H), 7.61 (dd,
J ) 7.8, 7.8 Hz, 1H), 7.71 (dd, J ) 7.8, 7.8 Hz, 1H), 7.76-7.87
3-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
4-oxot h ia zolid in -2-ylid e n e ]m e t h yl]-4-m e t h ylt h ia zoli-
u m Aceta te (33). This compound was synthesized from
3-ethyl-2-methylbenzothiazolium p-toluenesulfonate, 3-ethyl-
4-oxothiazolidine-2-thione, and 1-ethyl-2-methylquinolinium
p-toluenesulfonate in a manner analogous to the preparation
(
8
m, 2H), 7.94 (d, J ) 7.8 Hz, 1H), 8.00 (d, J ) 7.8 Hz, 1H),
.13 (d, J ) 7.8 Hz, 1H), 8.16-8.24 (m, 2H), 8.31 (d, J ) 7.8
+
Hz, 1H), 8.53 (d, J ) 7.8 Hz, 1H); MS (FAB , nitrobenzyl
alcohol) for C31
for Cl m/z 35; HRMS (FAB ) for C31
found 552.1242. Anal. (C31
S.
-
26 3 3
H N OS m/z 552, (FAB , nitrobenzyl alcohol)
+
1
H
26
N
3
OS
3
calcd 552.1238,
O) C, H, Cl, N,
of rhodacyanine dye 13: H NMR (DMSO-d ) δ 1.25 (t, J )
6
H
26ClN
3
OS
3
‚3.0H
2
7.2 Hz, 3H), 1.32 (t, J ) 7.2 Hz, 3H), 1.67 (s, 3H), 2.47 (s, 3H),
4.06 (s, 3H), 4.18 (q, J ) 7.2 Hz, 2H), 4.43 (q, J ) 7.2 Hz, 2H),
6.55 (s, 1H), 7.34 (dd, J ) 7.5, 7.5 Hz, 1H), 7.52 (dd, J ) 7.5,
3
-Eth yl-2-[[3-eth yl-5-(3-m eth yln a p h th o[1,2-d ]th ia zolin -
-ylid en e)-4-oxot h ia zolid in -2-ylid en e]m et h yl]n a p h t h o-
2,1-d ]oxa zoliu m Aceta te (29). This compound was syn-
2
[
7.5 Hz, 1H), 7.54 (s, 1H), 7.68 (d, J ) 7.5 Hz, 1H), 7.92 (d, J
+
) 7.5 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C20
H
22
N
3
-

Rhodacyanine Dyes as Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 141
In Vitr o Cytotoxic Assa y. For the cytotoxic assay, cells
-
OS
3
m/z 416, (FAB , nitrobenzyl alcohol) for C
2
H
3
O
2
m/z 59;
calcd 416.0925, found 416.0923.
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
-oxoth ia zolid in -2-ylid en e]m eth yl]th ia zolin iu m Aceta te
34). This compound was synthesized from 3-ethyl-2-methyl-
benzothiazolium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-
-thione, and 3-ethyl-2-methylbenzoxazolium p-toluenesulfonate
in a manner analogous to the preparation of rhodacyanine dye
+
4
HRMS (FAB ) for C20
H
22
N
3
OS
3
were seeded at 4 × 10 cells/20 mm plate and incubated at 37
3
2
°C in 5% CO . On the following day, cells were treated with
4
(
each compound at varying concentrations and then incubated
for 48 h (KB-cell) or 1 week (CV-1). Cells were stained with
crystal violet, and the number of viable cells were determined
by monocellater.
2
In Vivo Hu m a n Tu m or Xen ogr a fts in Nu d e Mice a n d
Tu m or Allogr a fts in BDF 1 Mice. Male Swiss nu/nu mice
about 5-weeks old) were obtained from Taconic Farm, Inc.
Germantown, NY). Group housing (five/cage) was provided
1
1
6
3: H NMR (DMSO-d ) δ 1.20 (t, J ) 7.2 Hz, 3H), 1.25 (t, J
)
)
)
(
)
7.2 Hz, 3H), 1.57 (s, 3H), 3.66 (t, J ) 7.2 Hz, 2H), 3.82 (q, J
(
(
7.2 Hz, 2H), 4.09 (s, 3H), 4.12 (t, J ) 7.2 Hz, 2H), 4.19 (q, J
7.2 Hz, 2H), 6.06 (s, 1H), 7.37 (dd, J ) 7.5, 7.5 Hz, 1H), 7.55
in polycarbonate cages with 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
dd, J ) 7.5, 7.5 Hz, 1H), 7.72 (d, J ) 7.5 Hz, 1H), 7.96 (d, J
+
22 3
7.5 Hz, 1H); MS (FAB , nitrobenzyl alcohol) for C19H N -
-
OS
HRMS (FAB ) for C19
Anal. (C21
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
-oxoth ia zolid in -2-ylid en e]m eth yl]ben zoxa zoliu m Ace-
3
m/z 404, (FAB , nitrobenzyl alcohol) for C
2
H
3
O
2
m/z 59;
calcd 404.0925, found 404.0923.
2
O) C, H, N, S.
+
H
22
N
3
OS
3
H
25
N
3
O
3
S
3
‚3.1H
3
6
chloride. Each mouse received 2 × 10 LOX cells (trypan blue
4
negative) in 0.2 mL of PBS by ip injection. On the day after
ip implantation, test compounds (0.2 mL/20 g mouse body
weight, 45% Encapsin HPB (hydroxypropyl â-cyclodextrin
t a t e (35). This compound was synthesized from 3-ethyl-2-
methylbenzothiazolium p-toluenesulfonate, 3-ethyl-4-oxothia-
zolidine-2-thione, and 3-methyl-2-(methylthio)benzoxazolium
(
American Maise-Products Co.) in water) were administered
ip for LOX tumor, bearing with appropriately determined doses
and schedules.
p-toluenesulfonate in a manner analogous to the preparation
1
of rhodacyanine dye 13: H NMR (DMSO-d
6
) δ 1.27 (t, J )
7
4
7
7
.2 Hz, 3H), 1.39 (t, J ) 7.2 Hz, 3H), 1.77 (s, 3H), 4.18 (s, 3H),
.23 (q, J ) 7.2 Hz, 2H), 4.48 (q, J ) 7.2 Hz, 2H), 6.37 (s, 1H),
.38 (d, J ) 7.5 Hz, 2H), 7.48-7.58 (m, 2H), 7.80 (dd, J ) 7.5,
Inbred 5-week-old male BDF1 mice were obtained from
Charles River J apan Inc. and were allowed to acclimate for
.5 Hz, 2H), 7.96 (d, J ) 7.5 Hz, 1H), 8.01 (d, J ) 7.5 Hz, 1H);
approximately 1 week prior to use. Each mouse received 5 ×
+
MS (FAB , nitrobenzyl alcohol) for C23
H
22
N
3
O
2
S
2
m/z 436,
5
1
0 CA755 cells by sc injection. Test compounds (0.2 mL/20 g
-
+
(FAB , nitrobenzyl alcohol) for C
2
H
3
O
2
m/z 59; HRMS (FAB )
calcd 436.1153, found 436.1159. Anal.
‚2.4H O) C, H, N, S.
mouse body weight, 0.9% saline) were administered iv for
CA755 tumor, bearing with appropriately determined doses
and schedules. For both in vivo evaluations, the statistical
significance of difference between the control and the drug-
treated groups was determined by applying the Bartlett’s,
for C23
H
N
22
N
3 2 2
O S
2
(C
25
H
25
3
O
4
S
2
1
-Eth yl-2-[[3-eth yl-5-(3-m eth ylben zoth iazolin -2-yliden e)-
4
-oxoth iazolidin -2-yliden e]m eth yl]pyr idin iu m Iodide (36).
This compound was synthesized from 3-ethyl-2-methylben-
zothiazolium p-toluenesulfonate, 3-ethyl-4-oxothiazolidine-2-
thione, and 1-ethyl-2-methylpyridinium iodide in a manner
analogous to the preparation of rhodacyanine dye 12: 1H NMR
14-16
Dunnett’s, and Scheffet’s test.
Ack n ow led gm en t. We are indebted to Dr. K. Aoki,
Y. Matsumoto, Mr. E. Fujioka, K. Aikawa, M. Hiraya-
ma, M. Yoshikane, Ms. A. Nakazawa, M. Watanabe, N.
Suzuki, and T. Mochizuki for their contributions to the
chracterization of rhodacyanines. We thank Mr. D.
Nishio for helpful discussion and Dr. H. Kitaguchi for
his advice about preparing the manuscript. We also
thank Professor G. Sheldrick of Institut f u¨ r Anor-
ganische Chemie der Universitat for providing us with
SHELXS86 for X-ray structure analysis.
(
6
DMSO-d ) δ 1.25 (t, J ) 7.2 Hz, 3H), 1.45 (t, J ) 7.2 Hz, 3H),
4
5
7
8
1
.03 (s, 3H), 4.11 (q, J ) 7.2 Hz, 2H), 4.60 (q, J ) 7.2 Hz, 2H),
.98 (s, 1H), 7.29 (dd, J ) 8.0, 8.0 Hz, 1H), 7.42-7.50 (m, 2H),
.59 (d, J ) 8.0 Hz, 1H), 7.86 (d, J ) 8.0 Hz, 1H), 8.06 (d, J )
.0 Hz, 1H), 8.27 (dd, 8.0, 7.0 Hz, 1H), 8.79 (d, J ) 8.0 Hz,
+
H); MS (FAB , nitrobenzyl alcohol) for C21
22 3 2
H N OS m/z 396,
-
(
FAB , triethanolamine) for I m/z 127. Anal. (C21
3
H22IN -
OS ‚1.5H O) C, H, I, N, S.
2
2
X-r a y Str u ctu r e An a lysis of 3. Compounds 3 was crys-
tallized from a 1:3 solution of methanol and ethyl acetate.
X-ray diffraction data was measured by a Rigaku AFC-5R
using a Cu KR radiation and graphite monochromator. Struc-
tures were determined by a direct method SHELXS86 and
succesive Fourier syntheses and refined by a full-matrix least-
squares method. Full crystallographic details are available
as Supporting Information.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data for compound 3 (7 pages). Ordering information
is given on any current masthead page.
In Vitr o Clon ogen ic Assa y. CX-1 cell line was grown in
a 50:50 mix of Dulbecco’s modified Eagle’s (DMEM) and RPMI
1
640 medium (GIBCO Laboratories, Grand Island, NY) supple-
mented with 10% calf serum (Hyclone Laboratories Inc.,
Logan, UT) and antibiotics at 37 °C under 5% CO , 95% air,
Refer en ces
2
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wells for CX-1 in 96-well plates (Becton Dickinson Labware,
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% crystal violet in 70% ehtanol and counted by an automated
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J M970590K