Synthesis and aldose reductase inhibitory activity of a new series of 5- [[2-(ω-carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxothiazolidine derivatives

Article abstract of DOI:10.1016/S0223-5234(99)00128-2

A new series of 5-[[2-(ω-carboxyalkoxy)aryl]methylene]-4-oxo-2- thioxothiazolidine derivatives was synthesized and evaluated for their potency as aldose reductase inhibitors (ARIs). Their activities were examined in terms of their inhibitory effect on rat lens aldose reductase in vitro and in terms of the preventive effect on sorbitol accumulation in the sciatic nerve of streptozotocin (STZ)-induced diabetic rats in vivo. Of these compounds, some of the naphthylmethylene thiazolidine derivatives were comparable to Zenarestat in the inhibitory potency in vitro and in vivo. In particular, compound 30 was 1.5 times more potent than Zenarestat in the in vivo activity, and had an adequate potency for clinical development.

Full text of DOI:10.1016/S0223-5234(99)00128-2

Eur. J. Med. Chem. 34 (1999) 1061−1070
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
1061
Original article
Synthesis and aldose reductase inhibitory activity
of a new series of 5-[[2-(ω-carboxyalkoxy)aryl]methylene]-4-oxo-2-
thioxothiazolidine derivatives
Makoto Murata*, Buichi Fujitani, Hiroyuki Mizuta
Department of Chemistry I. Discovery Research Laboratories, Dainippon Pharmaceutical Company
Ltd., Enoki 33-94, Suita/Osaka 564-0053, Japan
(Received 20 April 1999; accepted 15 July 1999)
Abstract – A new series of 5-[[2-(x-carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxothiazolidine derivatives was synthesized and evaluated
for their potency as aldose reductase inhibitors (ARIs). Their activities were examined in terms of their inhibitory effect on rat lens aldose
reductase in vitro and in terms of the preventive effect on sorbitol accumulation in the sciatic nerve of streptozotocin (STZ)-induced diabetic
rats in vivo. Of these compounds, some of the naphthylmethylene thiazolidine derivatives were comparable to Zenarestat in the inhibitory
potency in vitro and in vivo. In particular, compound 30 was 1.5 times more potent than Zenarestat in the in vivo activity, and had an adequate
potency for clinical development. © 1999 Éditions scientifiques et médicales Elsevier SAS
5-[[2-(x-carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxothiazolidine derivatives / aldose reductase inhibitory activity / sorbitol accu-
mulation inhibition
1. Introduction
ecule [12, 13]. Thus, we gave our attention to the com-
pounds having a 2-(ω-carboxyalkoxy)aryl moiety and
synthesized those compounds. In this paper, we report the
synthesis and AR inhibitory activity of the 5-[[2-(ω-
carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxo-thiazoli-
dine derivatives 1 shown in figure 1.
Aldose reductase (AR), which is the rate limiting
enzyme of the polyol pathway, is implicated in diabetic
complications. Since AR has a low substrate affinity for
glucose, the activity of the polyol pathway is very low at
normal physiological glucose concentrations [1]. How-
ever, when there is elevated glucose concentration in
diabetes, excessive sorbitol production from glucose by
AR is thought to cause cellular damage as a result of
osmotic imbalance [2]. This effect leads to the develop-
ment of diabetic complications such as neuropathy, ret-
inopathy, nephropathy, and cataract formation [3–6].
Recently, numerous compounds have been selected as
potential aldose reductase inhibitors (ARIs) [7–11],
whose representatives are shown in figure 1. These com-
pounds possess an acidic proton which is attached to the
imidic nitrogen or an acetic acid moiety in the mol-
2. Chemistry
The 4-substitued-2-formylphenoxyacetic acid deriva-
tives were prepared according to the method of Emmott
and Livingstone [14] with slight modifications, as shown
in figure 2. The 5-substitued-salicylaldehydes 2–4 were
alkylated with ethyl bromoacetate in the presence of
finely ground potassium carbonate, followed by hydroly-
sis under the alkaline condition to give 4-substitued
2-formylphenoxyacetic acids 5–7.
The ω-(1-formyl-2-naphthyloxy)alkanoic acid deriva-
tives 9–10 were prepared in a similar manner as described
above.
*Correspondence and reprints

1062
Figure 1. Potential aldose reductase inhibitors.
The 3-(1-formyl-2-naphthyloxy)propionic acid 11 was
prepared by the alkylation of the aldehyde 8 with
â-propiolactone in the presence of sodium hydrox-
ide [15].
The configuration of the exocyclic double bond of
1
those thiazolidine derivatives were determined by H-
NMR and 13C-NMR spectroscopy according to the
method of Fresneau et al. and Isida et al. [17, 18].
The 2:3 mixture of compounds 28 and 31 were
obtained by the photoirradation (fluorescent lamp) of 28
in methanol, as shown in figure 4. However, attempted
separation of 28 and 31 failed since smooth reisomeriza-
The 5-(2-carboxymethoxybenzylidene)thiazolidine de-
rivatives 13–24 were prepared according to the method of
Baranov and Komaritsa [16], as shown in figure 3. Con-
densation of the 4-substitued-2-formylphenoxyacetic ac-
ids 12, 5, 6, or 7 with rhodanine under refluxing, in the
presence of 2 eq. anhydrous sodium acetate in acetic acid,
gave compound 13, 16, 19, or 22. Moreover, condensa-
tion of those with N-methylrhodanine gave compound
14, 17, 20, or 23, and with rhodanine-3-acetic acid
likewise gave compound 15, 18, 19, or 24, respectively.
The 5-[(2-ω-carboxyalkoxy)naphthylmethylene]thi-
azolidine derivatives 25–30 were prepared in a similar
manner as described above.
1
tion (31 to 28) occured during separation. H-NMR and
13C-NMR experiments on the mixture of compound 28
and 31 disclosed the following results. (1) Although the
C5 proton of compound 28 showed a signal at δ 8.26 due
to the anisotropic effect by the carbonyl group (C4=O) of
the thiazolidine ring, the same proton signal of 31
1
appeared at δ 7.91 in H-NMR spectroscopy. (2) Al-
though the coupling constant value of the C4 carbon of
compound 28 had J = 6.5 Hz, the same value of 31 had

1063
Reagents: a) K2CO3/toluene, tris(dioxa-3,6-heptyl)amine, BrCH2CO2Et; b) 1 N NaOH/dioxane; c) â-propiolactone, NaOH/H2O.
Figure 2. Synthesis of compounds 5–7, 9, 10 and 11.
J = 12.4 Hz in 13C-NMR spectroscopy. From these
results, we confirmed that the exocyclic double bond of
compound 28 had Z configuration.
4. Results and discussion
Moreover, we postulated that the configuration of other
thiazolidine compounds had Z form on the basis of NMR
data.
The in vitro AR inhibitory activity of the benzylidene
thiazolidine derivatives 13–24 are shown in table I. When
R1 is hydrogen or a carboxymethyl group, introduction of
the substituent R resulted in an increase of AR inhibitory
activity by 1 order of the potency as judged from IC50
values (i.e. compound 13 vs. 16, 19 or 22; or compound
15 vs. 18, 21 or 24). When R1 is a methyl group however,
introduction of the substituent R (i.e. compound 14 vs.
17, 20 or 23) had no influence on the AR inhibitory
activity. Compounds 14 and 19–23, which inhibited the
rat AR in vitro at IC50 values of 10 nM order, were
equipotent compared with the reference compound Ze-
narestat. Furthermore, compounds 16, 17 and 18 were 2
times more potent than Zenarestat. However, these com-
pounds were inactive in inhibiting the sorbitol accumu-
lation in the sciatic nerve of STZ-induced diabetic rats at
100 mg/kg p.o. (table II).
3. Pharmacology
The AR inhibitory activity of the synthesized com-
pounds was assessed by measuring the inhibition of the
enzymatic activity in a partially purified rat lens prepa-
ration [1]. The inhibitory activity was expressed as the
concentration (nM) of the test compound which inhibited
the activity of AR by 50% (IC50). The in vivo AR
inhibitory activity of the test compounds were also
evaluated by measuring their ability to inhibit the sorbitol
accumulation in the sciatic nerve of STZ-induced diabetic
rats [19].

1064
Figure 3. Synthesis of thiazolidine derivatives 13–24 and 25–30.
The in vitro AR inhibitory activity of the naphthyl-
methylene thiazolidine derivatives 25–30 are shown in
table III. When R1 is hydrogen, the increase in the
number of methylene groups gave a remarkable decrease
of the in vitro activity (i.e. compound 25 and 26 vs. 27).
When R1 is a methyl group, the increase in the number of
methylene groups gave a moderate decrease of the in
vitro activity (i.e. compound 28 vs. 29 vs. 30). Of these
compounds, compound 25 was equipotent to Zenarestat,
and compound 26 was 3 times more potent than Zenar-
estat.
Figure 4. Photoirradiation of derivative 28.

1065
Table I. Chemical and biological data, in vitro, of benzylidene thiazolidine derivatives.
AR inhibition in vitro
rat lens ARa
IC50 (nM)b
Compound
M.p. (°C)
Recryst. solvent
Formula
13
14
15
16
17
18
19
20
21
22
23
24
> 250
220–221
> 250
EtOH-H2O
AcOH-H2O
EtOH-H2O
C12H9NO4S 0.2H2O
C13H11NO4S2
560
27
170
17
16
18
C14H10NNaO6S2 H2O
C12H8BrNO4S2
C13H10BrNO4S2
C14H10BrNO6S2
C12H8ClNO4S2
C13H10ClNO4S2
C14H10ClNO6S2 0.5H2O 21
C13H11NO5S2 0.25H2O
C14H13NO5S2
C15H13NO7S2
> 250
(CH3)2CO-H2O
(CH3)2CO-H2O
AcOH-H2O
(CH3)2CO-H2O
(CH3)2CO-H2O
AcOH-H2O
MeOH-H2O
(CH3)2CO-H2O
AcOH-H2O
243–246
204–208
> 250
237–239
209–212
226–228
215–216
213–215
29
18
38
19
86
36
Zenarestat
b
aAR: aldose reductase. The concentration of test compounds required for 50% inhibition of AR.
The in vivo inhibitory activity of the naphthylmethyl-
ene thiazolidine derivatives on the sorbitol accumulation
in the sciatic nerve of STZ-induced diabetic rats is shown
in table II. When R1 is hydrogen, the increase in the
number of methylene groups gave a moderate decrease of
the in vivo activity (i.e. compound 25 vs. 26). When R1
is a methyl group, the increase in the number of methyl-
ene groups unexpectedly gave a moderate increase of the
in vivo activity. Of these, compound 25 had equipotent in
vivo activity to Zenarestat. Of particular interest is that
the in vivo activity of compound 30 was more potent than
that of Zenarestat, although it was 20 times less potent
than Zenarestat in vitro. Therefore, we postulate that
compound 30 possessed good oral absorption and effi-
cient tissue penetration properties because of the in-
creased number of methylene groups.
In conclusion, we have reported in this article 5-[[2-
(ω-carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxothia-
zolidine derivatives having biological activities which
were comparable to the in vitro and in vivo inhibitory
activities of Zenarestat. Of these, compound 30 was 1.5
times more potent than Zenarestat in the in vivo activity,
and had an adequate potency for clinical development.
Table II. Biological data, in vivo, of benzylidene thiazolidine and naphthylmethylene thiazolidine derivatives.
Aldose reductase inhibition
Compound
Dose (mg/kg)
in vivo % inhibitiona
13
16
17
18
25
26
28
29
100
100
100
100
100
100
30
100
100
100
5.8 ± 3.1
NS
NS
NS
20.1 ± 4.8
8.0 ± 4.8
11.7 ± 5.8
8.6 ± 7.1
34.6 ± 6.4
21.8 ± 7.2
30
Zenarestat
aPercent inhibition of sorbitol accumulation in the sciatic nerve of streptozotocin-induced diabetic rats. Test compounds were orally given at
the single dose indicated. Values are mean ± SEM; mean of 4-6 rats. NS, no significant inhibition.

1066
Table III. Chemical and biological data, in vitro, of naphthylmethylene thiazolidine derivatives.
AR inhibition in vitro
rat lens ARa
IC50 (nM)b
Compound
M.p. (°C)
Recryst. solvent
Formula
25
26
27
28
29
30
231–234
212–216
195–198
206–209
165–168
93–95
AcOH-H2O
AcOH-H2O
AcOEt-hexane
(CH3)2CO-H2O
(CH3)2CO-H2O
AcOEt-hexane
C16H11NO4S2
C17H13NO4S2
C18H15NO4S2
C17H13NO4S2
C18H15NO4S2
33
11
1 100
180
630
C19H17NO4S2 0.25H2O 700
36
Zenarestat
b
aAR: aldose reductase. The concentration of test compounds required for 50% inhibition of AR.
5. Experimental protocols
1 h. After cooling to room temperature, the reaction
mixture was diluted with water (150 mL) and then
acidified with concentrated HCl on an ice bath, and the
precipitated solid was collected by filtration. The solid
was recrystallized from acetone/H2O to give the title
compound as a solid (5.3 g, 62%). M.p. 174–175 °C (lit.
173–174 °C) [20].
5.1. Chemistry
Melting points (m.p.) were determined on
a
Yanagimoto micro-melting apparatus and are uncor-
rected. Proton nuclear magnetic resonance (1H-NMR)
spectra were obtained on a Varian Gemini-200 spectrom-
eter with tetramethylsilane as an internal standard.
Chemical shifts are reported in δ values from internal
tetramethylsilane. Splitting patterns are designated as
follows: s, singlet; d, doublet; dd, double doublet; t,
triplet; q, quartet; br s, broad singlet; m, multiplet.
Coupling constants are reported in hertz (Hz). Infrared
(IR) spectra were recorded on a Shimazu FTIR-8200PC
spectrophotometer. Elemental analyses were carried out
on a Perkin-Elmer 2400 element analyzer and results
obtained for specified elements were within ± 0.4% of the
theoretical values. Visualization was accomplished with
UV light. Unless otherwise noted, all commercially
available materials were used without further purifica-
tion.
The following compounds were prepared in the same
way as described above:
(4-Bromo-2-formylphenoxy)acetic acid 5 (prepared
from 5-bromosalicylaldehyde 2; recrystallized from
acetone/H2O). M.p. 172–174 °C (lit. 174–176 °C) [20].
(2-Formyl-4-methoxyphenoxy)acetic acid 7 (prepared
from 5-methoxysalicylaldehyde 4; recrystallized from
acetone/H2O). M.p. 156–159 °C (lit. 157–159 °C) [20].
5.1.2. (1-Formyl-2-naphthyloxy)acetic acid 9
2-Hydroxy-1-naphthylaldehyde (8; 32 g, 0.2 mol) was
dissolved in dry toluene (200 mL) and finely ground
potassium carbonate (15 g, 0.11 mol) was added. After
the mixture was heated at 100 °C for 4 h, tris(dioxa-3,6-
heptyl)amine (4 mL, 12.5 mmol) and ethyl bromoacetate
(28 mL, 0.25 mmol) were added. The mixture was heated
at 100 °C for 4 h, filtered through Celite and washed with
toluene. The filtrate was washed with water and brine,
dried (anhydrous MgSO4), and evaporated. The residue
was dissolved in dioxane (250 mL) and 1 N NaOH
(250 mL) was added. The mixture was heated at 100 °C
for 1 h. After cooling to room temperature, the reaction
mixture was diluted with water (700 mL) and then
acidified with concentrated HCl on an ice bath, and the
precipitated solid was collected by filtration. The solid
was recrystallized from AcOEt/n-hexane to give the title
compound as a solid (15 g, 65%): M.p. 176–179 °C (lit.
176–177 °C) [14].
5.1.1. Typical procedure for the preparation of 2-
formylphenoxy acetic acids 5–7
5.1.1.1. (4-Chloro-2-formylphenoxy)acetic acid 6
5-Chlorosalicylaldehyde (3; 6.3 g, 40 mmol) was dis-
solved in dry toluene (90 mL) and finely ground potas-
sium carbonate (5.6 g, 40 mmol) were added. After the
mixture was heated at 100 °C for 4 h, tris(dioxa-3,6-
heptyl)amine (1.3 mL, 4.1 mmol) and ethyl bromoacetate
(5.6 mL, 50 mmol) were added. The mixture was heated
at 100 °C for 6 h, filtered through Celite and washed with
toluene. The filtrate was washed with water, and brine,
dried (anhydrous MgSO4), and evaporated. The residue
was dissolved in dioxane (50 mL), and 1 N NaOH
(50 mL) was added. The mixture was heated at 100 °C for

1067
5.1.3. 4-(1-Formyl-2-naphthyloxy)butyric acid 10
5.1.6. 5-(2-Carboxymethoxybenzylidene)-4-oxo-2-thioxo-
thiazolidine 13
Compound 10 was synthesized in the same way as in
5.1.2. Yield 34%. M.p. 179–180 °C (recrystallized from
AcOEt/n-hexane); 1H-NMR (DMSO-d6) d 2.00–2.18
(2H, m), 2.47 (2H, t, J = 7.5 Hz), 4.35 (2H, t, J = 7.5 Hz),
7.40–7.70 (3H, m), 7.90–8.00 (1H, m), 8.27 (1H, d, J = 9
Hz), 9.07–9.17 (1H, m), 10.7 (1H, s), 12.18 (1H, br s); IR
(KBr, cm–1) 1 705 (CO), 1 672 (CO). Anal. calcd. for
C15H14O4: C 69.76; H 5.46; found: C 69.65; H 5.40.
Compound 13 was synthesized in the same way as in
5.1.5. Yield 32%. M.p. > 250 °C (recrystallized from aq.
1
EtOH); H-NMR (DMSO-d6) d 4.88 (2H, S), 7.03–7.18
(2H, m), 7.38–7.52 (2H, m), 7.90 (1H, s), 13.20 (1H, br
s), 13.74 (1H, br s); IR (KBr, cm–1) 1 705 (CO), 1 672
(CO). Anal. calcd. for C12H9NO4S2 0.2H2O: C 48.21; H
3.17; N 3.74; S 21.45; found: C 48.49; H 2.93; N 4.74; S
21.27.
5.1.4. 3-(1-Formyl-2-naphthyloxy)propionic acid 11
5.1.7.
5-(2-Carboxymethoxybenzylidene)-3-methyl-4-
A solution of â-propiolactone (7.6 g, 0.1 mol) in water
(10 mL) was added dropwise to a stirred solution of
2-hydroxy-1-naphthylaldehyde (8; 17.2 g, 0.1 mol) and
NaOH (4 g, 0.1 mol) in water (50 mL) at 100 °C, and the
mixture was stirred for a further 30 min at 100 °C. After
cooling to room temperature, the reaction mixture was
acidified with concentrated HCl on an ice bath, and then
extracted three times with diethyl ether. The combined
ether extracts were shaken three times with saturated
NaHCO3. The solid, which was precipitated on acidifi-
cation of the alkaline solution, was filtered, washed with
water, dried, and recrystallized from AcOEt/n-hexane to
give the title compound as a solid (4.2 g, 17%). M.p.
oxo-2-thioxothiazolidine 14
Compound 14 was synthesized in the same way as in
5.1.5. Yield 77%. M.p. 220–221 °C (recrystallized from
1
AcOH/H2O); H-NMR (DMSO-d6) d 3.43 (3H, s), 4.90
(2H, s), 7.04–7.20 (2H, m), 7.43–7.55 (2H, m), 8.07 (1H,
s), 13.18 (1H, br s); IR (KBr, cm–1) 1 753 (CO), 1 689
(CO). Anal. calcd. for C13H11NO4S2: C 50.47; H 3.58; N
4.53; S 20.73; found: C 50.17; H 3.56; N 4.62; S 21.00.
5.1.8. [5-(2-Carboxymethoxybenzylidene)-4-oxo-2-thio-
xothiazolidin-3-yl]-acetic acid mono sodium salt 15
Compound 15 was synthesized in the same way as in
5.1.5. Yield 11%. M.p. > 250 °C (recrystallized from aq.
EtOH); 1H-NMR (DMSO-d6) d 4.50 (2H, s), 4.69 (2H, s),
6.95–7.15 (2H, m), 7.41–7.51 (2H, m), 8.06 (1H, s); IR
(KBr, cm–1) 1 716 (CO). Anal. calcd. for
C14H10NNaO6S2 H2O: C 42.75; H 3.07; N 3.56; S 16.30;
Na 5.84; found: C 42.82; H 3.07; N 3.59; S 16.01; Na
5.54.
1
157–159 °C (recrystallized from AcOEt/n-hexane); H-
NMR (DMSO-d6) d 2.84 (2H, t, J = 6 Hz), 4.52 (2H, t,
J = 6 Hz), 7.42–7.52 (1H, m), 7.58–7.72 (2H, m),
7.92–8.02 (1H, m), 8.30 (1H, d, J = 9 Hz), 9.07–9.17 (1H,
m), 10.72 (1H, s), 12.48 (1H, br s); IR (KBr, cm–1) 1 705
(CO), 1 672 (CO). Anal. calcd. for C14H12O4: C 68.85; H
4.95; found: C 68.74; H 4.88.
5.1.9.
5-(5-Bromo-2-carboxymethoxybenzylidene)-3-
methyl-4-oxo-2-thioxothiazolidine 17
5.1.5. 5-(5-Bromo-2-carboxymethoxybenzylidene)-4-oxo-
2-thioxothiazolidine 16
Compound 17 was synthesized in the same way as in
5.1.5. Yield 67%. M.p. 243–246 °C (recrystallized from
acetone/H2O); 1H-NMR (DMSO-d6) d 3.41 (3H, s), 4.93
(2H, s), 7.07 (1H, d, J = 9 Hz), 7.53 (1H, d, J = 2.5 Hz),
7.65 (1H, dd, J = 2.5, 9 Hz) 7.91 (1H, s), 13.24 (1H, br
s); IR (KBr, cm–1) 1 705 (CO). Anal. calcd. for
C13H10BrNO4S2: C 40.22; H 2.60; N 3.61; S 16.52; Br
20.58; found: C 40.15; H 2.61; N 3.49; S 16.58; Br 20.55.
A mixture of 4-Bromo-2-formylphenoxyacetic acid (5;
6.0 g, 23.2 mmol), rhodanine (3.7 g, 27.8 mmol) and
anhydrous sodium acetate (3.8 g, 46.3 mmol) in acetic
acid (80 mL) was refluxed for 17 h. Upon cooling, the
reaction mixture was diluted with water and stirred at
room temperature for a further 1 h. The solid was
filtrated, and added to dilute HCl, then the mixture was
stirred at room temperature for 1 h. After filtration, the
solid was recrystallized from acetone/H2O to give the
title compound as a yellow solid (5.9 g, 68%). M.p. >
5.1.10. [5-(5-Bromo-2-carboxymethoxybenzylidene)-4-
oxo-2-thioxo-thiazolidin-3-yl]-acetic acid 18
Compound 18 was synthesized in the same way as in
5.1.5. Yield 55%. M.p. 204–208 °C (recrystallized from
1
250 °C; H-NMR (DMSO-d6) d 4.90 (2H, S), 7.06 (1H,
1
d, J = 9 Hz), 7.49 (1H, d, J = 2.5 Hz), 7.63 (1H, dd, J =
2.5, 9 Hz), 7.76 (1H, s), 13.20 (1H, br s), 13.88 (1H, br
s); IR (KBr, cm–1) 1 714 (CO). Anal. calcd. for
C12H8BrNO4S2: C 38.51; H 2.15; N 3.74; S 17.14; Br
21.35; found: C 38.57; H 1.92; N 3.73; S 17.29; Br;
21.50.
AcOH/H2O); H-NMR (DMSO-d6) d 4.75 (2H, s), 4.95
(2H, s), 7.09 (1H, d, J = 9 Hz), 7.60 (1H, d, J = 2.5 Hz),
7.96 (1H, dd, J = 2.5, 9 Hz) 7.96 (1H, s), 13.36 (2H, br
s); IR (KBr, cm–1) 1 712 (CO). Anal. calcd. for
C14H10BrNO6S2: C 38.90; H 2.33; N 3.24; S 14.84; Br
18.49; found: C 38.73; H 2.39; N 3.15; S 14.59; Br 18.26.

1068
5.1.11.
5-(5-Chloro-2-carboxymethoxybenzylidene)-4-
d, J = 9 Hz), 7.10 (1H, dd, J = 2.5, 9 Hz) 8.03 (1H, s),
13.12 (1H, br s); IR (KBr, cm–1) 1 712 (CO). Anal. calcd.
for C14H13NO5S2: C 49.55; H 3.86; N 4.13; S 18.90;
found: C 49.36; H 3.59; N 4.03; S 18.73.
oxo-2-thioxothiazolidine 19
Compound 19 was synthesized in the same way as in
5.1.5. Yield 62%. M.p. > 250 °C (recrystallized from
acetone/H2O); 1H-NMR (DMSO-d6) d 4.90 (2H, s), 7.14
(1H, d, J = 9 Hz), 7.44 (1H, d, J = 2.5 Hz), 7.52 (1H, dd,
J = 2.5, 9 Hz) 7.84 (1H, s), 13.24 (1H, br s), 13.88 (1H,
br s); IR (KBr, cm–1) 1 733 (CO), 1 683 (CO). Anal.
calcd. for C12H8ClNO4S2: C 43.71; H 2.45; N 4.25; S
19.45; Cl 10.75; found: C 43.79; H 2.42; N 4.19; S 19.12;
Cl 10.75.
5.1.16. [5-(2-Carboxymethoxy-5-methoxybenzylidene)-4-
oxo-2-thioxo-thiazolidin-3-yl]-acetic acid 24
Compound 24 was synthesized in the same way as in
5.1.5. Yield 40%. M.p. 213–215 °C (recrystallized from
acetone/H2O); 1H-NMR (DMSO-d6) d 3.80 (3H, s), 4.75
(2H, s), 4.84 (2H, s), 6.97 (1H, d, J = 2.5 Hz), 7.06 (1H,
d, J = 9 Hz), 7.12 (1H, dd, J = 2.5, 9 Hz), 8.07 (1H, s),
13.30 (2H, br s); IR (KBr, cm–1) 1 716 (CO). Anal. calcd.
for C15H13NO7S2: C 46.99; H 3.42; N 3.65; S 16.73;
found: C 47.09; H 3.26; N 3.75; S 16.67.
5.1.12.
5-(5-Chloro-2-carboxymethoxybenzylidene)-3-
methyl-4-oxo-2-thioxothiazolidine 20
Compound 20 was synthesized in the same way as in
5.1.5. Yield 58%. M.p. 237–239 °C (recrystallized from
acetone/H2O); 1H-NMR (DMSO-d6) d 3.43 (3H, s), 4.93
(2H, s), 7.14 (1H, d, J = 9 Hz), 7.42 (1H, d, J = 2.5 Hz),
7.54 (1H, dd, J = 2.5, 9 Hz) 7.93 (1H, s), 13.24 (1H, br
s); IR (KBr, cm–1) 1 705 (CO). Anal. calcd. for
C13H10ClNO4S2: C 45.42; H 2.93; N 4.07; S 18.65; Cl
10.31; found: C 45.43; H 2.89; N 4.02; S 18.92; Cl 10.11.
5.1.17. 5-(2-Carboxymethoxy-1-naphthylmethylene)-4-
oxo-2-thioxothiazolidine 25
Compound 25 was synthesized in the same way as in
1
5.1.5. Yield 50%. M.p. 231–234 °C; H-NMR (DMSO-
d6) d 5.03 (2H, s), 7.40 (1H, d, J = 9 Hz), 7.43–7.53 (1H,
m), 7.57–7.67 (1H, m), 7.82–7.90 (1H, m), 7.92–8.00
(1H, m), 8.08 (1H, d, J = 9 Hz), 8.09 (1H, s), 13.20 (1H,
br s), 13.70 (1H, br s); IR (KBr, cm–1) 1 712 (CO), 1 662
(CO). Anal. calcd. for C16H11NO4S2: C 55.64; H 3.21; N
4.06; S 18.57; found: C 55.74; H 3.49; N 3.97; S 18.49.
5.1.13. [5-(5-Chloro-2-carboxymethoxybenzylidene)-4-
oxo-2-thioxothiazolidin-3-yl]-acetic acid 21
Compound 21 was synthesized in the same way as in
5.1.5. Yield 64%. M.p. 209–212 °C (recrystallized from
1
AcOH/H2O); H-NMR (DMSO-d6) d 4.75 (2H, s), 4.95
(2H, s), 7.15 (1H, d, J = 9 Hz), 7.49 (1H, d, J = 2.5 Hz),
7.55 (1H, dd, J = 2.5, 9 Hz) 7.97 (1H, s), 13.36 (1H, br
s); IR (KBr, cm–1) 1 716 (CO). Anal. calcd. for
C14H10ClNO6S2 0.5H2O: C 42.38; H 2.79; N 3.53; S
16.16; Cl 8.93; found: C 42.66; H 2.65; N 3.45; S 15.83;
Cl 8.76.
5.1.18. 5-[2-(2-carboxyethoxy)-1-naphthylmethylene]-4-
oxo-2-thioxothiazolidine 26
Compound 26 was synthesized in the same way as in
5.1.5. Yield 31%. M.p. 212–216 °C (recrystallized from
1
AcOH/H2O); H-NMR (DMSO-d6) d 2.78 (2H, t, J = 6
Hz), 4.48 (2H, t, J = 6 Hz), 7.42–7.66 (2H, m), 7.57 (1H,
d, J = 9 Hz), 7.78–7.86 (1H, m), 7.94–8.02 (1H, m), 8.00
(1H, s), 8.11 (1H, d, J = 9 Hz), 12.38 (1H, br s), 13.66
(1H, br s); IR (KBr, cm–1) 1 716 (CO), 1 683 (CO). Anal.
calcd. for C17H13NO4S2: C 56.81; H 3.65; N 3.90; S
17.84; found: C 56.55; H 3.82; N 3.71; S 17.49.
5.1.14. 5-(2-Carboxymethoxy-5-methoxybenzylidene)-4-
oxo-2-thioxothiazolidine 22
Compound 22 was synthesized in the same way as in
5.1.5. Yield 8%. M.p. 226–228 °C (recrystallized from
1
MeOH/H2O); H-NMR (DMSO-d6) d 3.78 (3H, s), 4.83
(2H, s), 6.88 (1H, d, J = 2.5 Hz), 7.03 (1H, d, J = 9 Hz),
7.08 (1H, dd, J = 2.5, 9 Hz), 7.87 (1H, s), 13.10 (1H, br
s), 13.84 (1H, br s); IR (KBr, cm–1) 1 751 (CO), 1 695
(CO). Anal. calcd. for C13H11NO5S2 0.25H2O: C 47.34;
H 3.51; N 4.25; S 19.44; found: C 47.39; H 3.19; N 4.38;
S 19.22.
5.1.19. 5-[2-(3-carboxypropoxy)-1-naphthylmethylene]-
4-oxo-2-thioxothiazolidine 27
Compound 27 was synthesized in the same way as in
5.1.5. Yield 58%. M.p. 195–198 °C (recrystallized from
AcOEt/n-hexane); 1H-NMR (DMSO-d6) d 1.93–2.10
(2H, m), 2.40 (2H, t, J = 7.5 Hz), 4.31 (2H, t, J = 7.5 Hz),
7.41–7.66 (2H, m), 7.55 (1H, d, J = 9 Hz) 7.79–7.87 (1H,
m), 7.93–8.00 (1H, m), 8.05 (1H, s), 8.10 (1H, d, J = 9
Hz), 12.15 (1H, br s), 13.68 (1H, br s); IR (KBr, cm–1)
1 705 (CO). Anal. calcd. for C18H15NO4S2: C 57.89; H
4.05; N 3.75; S 17.17; found: C 57.94; H 4.11; N 3.71; S
16.88.
5.1.15. 5-(2-Carboxymethoxy-5-methoxybenzylidene)-4-
oxo-2-thioxothiazolidine 23
Compound 23 was synthesized in the same way as in
5.1.5. Yield 35%. M.p. 215–216 °C (recrystallized from
acetone/H2O); 1H-NMR (DMSO-d6) d 3.43 (3H, s), 3.80
(3H, s), 4.83 (2H, s), 6.92 (1H, d, J = 2.5 Hz), 7.05 (1H,

1069
5.1.20. 5-(2-carboxymethoxy-1-naphthylmethylene)-3-
methyl-4-oxo-2-thioxothiazolidine 28
5.2.2. Inhibition of aldose reductase in vitro
The reaction mixture consisted of 100 mM phosphate
buffer (pH 6.5), 0.2 mM NADPH, 1.5 mM D,
L-glyceraldehyde, 0.4 M lithium sulfate, 7.0 mU/mL of
enzyme and test compounds at various concentrations.
The reaction mixture was incubated at 37 °C, and the
absorbance at 340 nm was measured with a spectropho-
tometer (Model 150-20, Hitachi Ltd., Japan). The enzyme
activity was estimated on the basis of its decrease in the
absorbance over a period of 1 min. The concentration of
compounds required for 50% inhibition of enzyme activ-
ity (IC50) was estimated graphically from the log
concentration-inhibition curve.
Compound 28 was synthesized in the same way as in
1
5.1.5. Yield 60%. M.p. 206–209 °C; H-NMR (DMSO-
d6) d 3.43 (3H, s), 5.02 (2H, s), 7.42 (1H, d, J = 9 Hz),
7.43–7.53 (1H, m), 7.57–7.67 (1H, m), 7.82–7.91 (1H,
m), 7.93–8.01 (1H, m), 8.10 (1H, d, J = 9 Hz), 8.26 (1H,
s), 13.22 (1H, br s); IR (KBr, cm–1) 1 757 (CO), 1 668
(CO). Anal. calcd. for C17H13NO4S2: C 56.81; H 3.65; N
3.90; S 17.84; found: C 56.67; H 3.54; N 3.76; S 18.14.
5.1.21. 5-[2-(2-carboxyethoxy)-1-naphthylmethylene]-3-
methyl-4-oxo-2-thioxothiazolidine 29
Compound 29 was synthesized in the same way as in
5.1.5. Yield 28%. M.p. 165–168 °C (recrystallized from
acetone/H2O); H-NMR (DMSO-d6) d 2.78 (2H, t, J = 6
Hz), 3.43 (3H, s), 4.48 (2H, t, J = 6 Hz), 7.43–7.66 (2H,
m), 7.59 (1H, d, J = 9 Hz), 7.79–7.87 (1H, m), 7.94–8.02
(1H, m), 8.13 (1H, d, J = 9 Hz), 8.18 (1H, s), 12.38 (1H,
br s); IR (KBr, cm–1) 1 716 (CO). Anal. calcd. for
C18H15NO4S2: C 57.89; H 4.05; N 3.75; S 17.17; found:
C 57.77; H 4.21; N 3.56; S 16.70.
5.2.3. Inhibition of sorbitol accumulation in vivo
1
Male Wistar rats (200–250 g) were rendered diabetic
by an intravenous injection of streptozotocin (40 mg/kg),
which had been freshly dissolved in physiological saline.
After 7 days, the rats were divided into various groups
with 4–6 animals/group, and orally given test compounds
suspended in 0.5% tragacanth, or an equivalent volume of
0.5% tragacanth. The rats were sacrificed 4 h after the
administration of the test compounds. Tissue sorbitol
levels were determined according to the method of
Clements et al. [17] with slight modifications.
5.1.22. 5-[2-(3-carboxypropoxy)-1-naphthylmethylene]-
3-methyl-4-oxo-2-thioxothiazolidine 30
The sciatic nerve sample (30–60 mg) was quickly
dissected from the hind limb, placed into water (1.0 mL/
40 mg of tissue), heated in a boiling bath for 2 min, and
then homogenized with a Polytron instrument in 6%
perchloric acid (1 mL/10 mg of tissue). The homogenate
was centrifuged at 1 050 g for 15 min at 4 °C. The
supernatant was neutralized with 2 M K2CO3 and used as
tissue extract for the assaying of sorbitol. Sorbitol was
assayed by an enzymatic method in which sorbitol
dehydrogenase catalyses the stoichiometric conversion of
NAD by sorbitol to a fluorogenic product, NADH. The
reaction mixture contained 30 mM glycine buffer (pH
9.4), 1.3 mM NAD, 1.3 U/mL sorbitol dehydrogenase
and 1.0 mL of the tissue extract in a total volume of 3 mL.
After the mixture was allowed to stand for 60 min at
room temperature, the fluorescence intensity was mea-
sured at 365 nm excitation wavelength and 430 nm emis-
sion wavelength using a fluorospectrophotometer (F3000,
Hitachi Ltd., Japan). The sorbitol concentration was
quantitated by comparison with standards of sorbitol. The
sorbitol content in the sciatic nerve of each animal was
expressed as nmole/wet weight.
Compound 30 was synthesized in the same way as in
5.1.5. Yield 43%. M.p. 93–95 °C (recrystallized from
AcOEt/n-hexane); 1H-NMR (DMSO-d6) d 1.80–2.20
(2H, m), 2.40 (2H, t, J = 7.5 Hz), 3.44 (3H, s), 4.30 (2H,
t, J = 7.5 Hz), 7.42–7.66 (2H, m), 7.56 (1H, d, J = 9 Hz),
7.80–7.88 (1H, m), 7.94–8.01 (1H, m), 8.12 (1H, d, J = 9
Hz), 8.23 (1H, s), 12.14 (1H, br s); IR (KBr, cm–1) 1 716
(CO), 1 695 (CO). Anal. calcd. for C19H7NO4S2
0.25H2O: C 58.22; H 4.50; N 3.57; S 16.36; found: C
58.31; H 4.35; N 3.60; S 16.67.
5.2. Pharmacology
5.2.1. Preparation of aldose reductase
Rat lens AR was prepared according to the method of
Hayman and Kinoshita [1] with slight modifications. The
lenses were homogenized in 250 mM phosphate buffer
(pH 7.4) containing 2 mM mercaptoethanol at 0–4 °C,
and the homogenate was centrifuged at 20 000 g for
30 min. The supernatant was subjected to a 40–60%
ammonium sulfate fractionation. The resultant precipitate
was dissolved in 5 mM phosphate buffer (pH 7.4) con-
taining 2 mM mercaptoethanol and used for enzyme
assay. One unit (U) of AR was defined as the enzyme
activity which oxidizes 1 µmol of NADPH in 1 min
under the assay conditions described below.
The activity of test compounds was expressed as the
percent inhibition of sorbitol accumulation at a given
dose, which was calculated according to the following
equation:
% inhibition = (S – T) / (S – N) × 100

1070
[6] Varma S.D., Schocket S.S., Richards R.D., Invest. Ophthalmol. Vis.
Sci. 18 (1979) 237–241.
where S is the sorbitol content in the sciatic nerve of
untreated diabetic control rats, T is the sorbitol content in
the sciatic nerve of diabetic rats given test compounds
and N is the sorbitol content in the sciatic nerve of
age-matched non-diabetic control rats.
[7] Canal N., Comi G., Trends Pharmacol. Sci. 6 (1985) 328–330.
[8] Sestanj K., Bellini F., Fung S., Abraham N., Treasurywala A.,
Humber L., Simard-Duquesne N., Dvornik D., J. Med. Chem. 27
(1984) 255–256.
[9] Kikkawa R., Hatanaka I., Yasuda H., Kobayashi N., Shigeta Y.,
Terashima H., Morimura T., Tsuboshima M., Diabetlogia 24 (1983)
290–292.
Acknowledgements
[10] Mizuno K., Kato N., Matsubara A., Nakano K., Kurono M.,
Metabolism 41 (1992) 1081–1086.
We thank Dr S. Naruto and Dr T. Kadokawa for
encouragement throughout this work, and members of the
analytical section for elemental analyses and spectral
measurements. We also thank Ms. M. Furuichi for mea-
surements of pharmacological studies.
[11] Ao S., Shingu Y., Kikuchi C., Takano Y., Nomura K., Fujiwara T. et
al., Metabolism 40 (1991) 77–87.
[12] Kador P.F., Kinoshita J.H., Sharpless N.E., J. Med. Chem. 28 (1985)
841–849.
[13] Lee Y.S., Pearlstein R., Kador P.F., J. Med. Chem. 37 (1994)
787–792.
[14] Emmott P., Livingstone R., J. Chem. Soc. (1957) 3144–3148.
[15] Gresham T.L., Jansen J.E., Shaver F.W., Bankert R.A., Beears W.L.,
Prendergast M.G., J. Am. Chem. Soc. 71 (1949) 661–663.
References
[16] Baranov S.N., Komaritsa I.D., Khim. Geterotsikl. Soedin. Akad.
Nauk Latv. SSR 1 (1965) 69–73.
[1] Hayman S., Kinoshita J.H., J. Biol. Chem. 240 (1965) 877–882.
[17] Fresneau P., Cussac M., Morand J.M., Szymonski B., Tranqui D.,
Leclerc G., J. Med. Chem. 41 (1998) 4706–4715.
[2] Kador P.F., Akagi Y., Kinoshita J.H., Metabolism 35 (1986) (suppl.
1) 15–19.
[18] Ishida T., In Y., Inoue M., Ueno Y., Tanaka C., Tetrahedron Lett. 30
(1989) 959–962.
[3] Gabbay K.H., New Engl. J. Med. 288 (1973) 831–836.
[4] Akagi Y., Kador P.F., Kuwabara T., Kinoshita J.H., Invest. Ophthal-
mol. Vis. Sci. 24 (1983) 1516–1519.
[19] Clements R.S., Morisson Jr. A.D., Winegrad A.I., Science 166
(1969) 1007–1008.
[5] Cohen M.P., Metabolism 35 (1986) (suppl. 1) 55–59.
[20] Hullar T.L., Failla D.L., J. Med. Chem. 12 (1969) 420–424.