In vitro aldose reductase inhibitory activity of 5-benzyl-2,4- thiazolidinediones

Article abstract of DOI:10.1016/j.bmc.2005.08.056

Several 5-benzyl-2,4-thiazolidinediones (5-7) were synthesised and tested as in vitro aldose reductase (ALR2) inhibitors. Most of them, particularly N-unsubstituted 5-benzyl-2,4-thiazolidinediones 5 and (5-benzyl-2,4- dioxothiazolidin-3-yl)acetic acids 7,

Full text of DOI:10.1016/j.bmc.2005.08.056

Bioorganic & Medicinal Chemistry 14 (2006) 567–574
In vitro aldose reductase inhibitory activity of
5-benzyl-2,4-thiazolidinediones
b
b
Dietmar Rakowitz,a Rosanna Maccari,b, Rosaria Ottana and Maria Gabriella Vigorita
*
`
aInstitute of Pharmacy, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
` `
Dipartimento Farmaco-chimico, Facolta di Farmacia, Universita di Messina, Viale SS. Annunziata, 98168 Messina, Italy
b
Received 22 June 2005; revised 6 August 2005; accepted 19 August 2005
Available online 3 October 2005
Abstract—Several 5-benzyl-2,4-thiazolidinediones (5–7) were synthesised and tested as in vitro aldose reductase (ALR2) inhibitors.
Most of them, particularly N-unsubstituted 5-benzyl-2,4-thiazolidinediones 5 and (5-benzyl-2,4-dioxothiazolidin-3-yl)acetic acids 7,
displayed moderate to high inhibitory activity levels. In detail, the insertion of an acetic chain on N-3 significantly enhanced ALR2
inhibitory potency, leading to acids 7 which proved to be the most effective among the tested compounds. In addition, in N-unsub-
stituted derivatives 5 the presence of an additional aromatic ring on the 5-benzyl moiety was generally beneficial. In fact, the ALR2
inhibition results of compounds 5–7, compared to those of the previously assayed corresponding 5-arylidene-2,4-thiazolidinediones,
indicated that N-unsubstituted derivatives 5b, c and d, which bore an additional aromatic group in the para position of the 5-benzyl
residue, were significantly more effective than their 5-arylidene counterparts; in all other cases, the saturation of the exocyclic double
bond C@C in 5 brought about a moderate decrease in activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
NADPH-dependent reduction of glucose to sorbitol.
The deprivation of NADPH and NAD+ and the intra-
cellular accumulation of sorbitol result in biochemical
imbalances which cause damage in target tissues.
ALR2 inhibition thus represents an attractive approach
to prevent or control the progression of chronic diabetic
complications.4–12
Diabetes mellitus is a widespread chronic disease, whose
current worldwide prevalence of 150 million is predicted
to double by 2025.1–3 It is always associated with long-
term complications (retinopathies, nephropathies, neur-
opathies, angiopathies, atherosclerosis and cataracts)
that make it one of the leading causes of blindness, renal
failure and neuronal pathologies. Diabetic complica-
tions also increase the risk of myocardial infarction
and stroke and are thus largely responsible for the mor-
bidity and mortality observed in these patients.1,4
A variety of ALR2 inhibitors (ARIs) have been report-
ed; however, in clinical studies many of them have
exhibited low efficacy or a narrow spectrum of tissue
activity, generally because of unfavourable pharma-
cokinetics, or have proved to produce toxic side-ef-
fects.10,12–15 Currently, epalrestat (Fig. 1) is the only
ARI available on the market.15,16
The increased flux of glucose through the polyol path-
way that occurs in hyperglycaemic conditions in tissues
possessing insulin-independent glucose transport (nerve,
retina, lenses and kidney) is a well-examined factor in-
volved in the onset and progression of such chronic
complications.4–12
The ARIs that have been clinically tested can be divided
into two main classes: (a) cyclic imides, mostly hydanto-
ins (such as sorbinil, Fig. 1) which often have good in vi-
vo activity but can cause hypersensitivity reactions
related to the hydantoin moiety, and (b) carboxylic acids
(such as ponalrestat, epalrestat, Fig. 1), which are gener-
ally less effective in vivo than in vitro.10,12,14
Aldose reductase (EC 1.1.1.21, ALR2) is the first
enzyme of the polyol pathway and catalyses the
In this context, 2,4-thiazolidinedione derivatives can be
considered bioisosteres of hydantoins potentially devoid
of the toxic side-effects linked to the presence of the
hydantoin system.10,15 They have also drawn extensive
Keywords: 2,4-Thiazolidinediones; Aldose reductase; ALR2 inhibitors;
Structure–activity relationships.
*
Corresponding author. Tel.: +39 90 6766406; fax: +39 90 355613;
e-mail: rmaccari@pharma.unime.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.056

568
D. Rakowitz et al. / Bioorg. Med. Chem. 14 (2006) 567–574
O
F
O
H
N
N
N
N
O
F
Br
COOH
O
Ponalrestat
Sorbinil
R
O
O
S
O
N
2 R = H
3 R = CH2COOCH3
COOH
N
4 R = CH2COOH
S
H
S
R'
Epalrestat
O
N
O
N
H
H
S
N
S
O
O
N
O
Pioglitazone
O
N
Rosiglitazone
Figure 1.
interest as antihyperglycaemic compounds.17–22 In fact,
many 2,4-thiazolidinediones have been reported as insu-
lin-sensitizing agents and some of them (pioglitazone,
rosiglitazone, Fig. 1) have been marketed for the treat-
ment of non-insulin-dependent diabetes mellitus
(NIDDM or type 2 diabetes), which accounts for
90–95% of all diabetes.1,23 In addition, some other
2,4-thiazolidinediones with dual activity, having both
antihyperglycaemic and ALR2 inhibitory effects, have
been patented;10,15 they might serve to treat NIDDM
and to control the progression of its long-term
complications.
anionic head of the N-3 acetic chain, which favours the
binding with the positively charged recognition region
of the ALR2 active site formed by Tyr48, His110,
Trp111 residues and the nicotinamide ring of
NADP+.10,14,15,24,25,30 Besides, the presence of an addi-
tional aromatic ring or an H-bond donor group in the 5-
benzylidene moiety enhanced ALR2 inhibitory effect; in
fact, except for the polar recognition region, the ALR2 ac-
tive site is highly hydrophobic and thus large lipophilic
moieties can favour the interaction of inhibitors with
the enzyme; otherwise, an H-bond donor group can stabi-
lize the enzyme–inhibitor complex through further H-
bonds.30 Molecular docking simulations into the ALR2
active site allowed the rationalization of the observed
SARs and suggested a pharmacophoric model, consisting
of a lipophilic region and two hydrogen-bond acceptor
features, for this class of ARIs.30
We have recently reported the in vitro ALR2 inhibitory
activity of numerous (Z)-5-arylidene-2,4-thiazolidinedi-
ones (2–4, Fig. 1); they possessed the essential structural
requisites for ALR2 inhibition (an acidic proton, H bond
acceptor groups and a lipophilic aromatic moie-
ty)12,15,24–28 and, in fact, most of them were shown to be
effective as ARIs.29,30 In particular, some N-unsubstitut-
ed derivatives (2) exerted the same activity as sorbinil;
moreover, the introduction of an acetic chain on N-3
brought about a marked increase (from 10 to 100 times)
in inhibitory potency, leading to acids 4, which proved
to be very effective ARIs.29,30 Although devoid of any
acidic functionality, some of the corresponding methyl es-
ters 3 also displayed appreciable ALR2 inhibitory activity
similar to that of the N-unsubstituted derivatives. Howev-
er, acids 4 proved to be more effective than corresponding
esters 3, clearly due to the presence of the carboxylic
We also found that 5-(3-phenoxybenzylidene)- and 5-(4-
phenoxybenzylidene)-2,4-thiazolidinediones, belonging
to series 2, displayed moderate antihyperglycaemic
activity in a preliminary in vivo assay.31
The next step of our ongoing research was to extend
the SARs picture by assessing the significance of the
exocyclic double bond C@C in position 5. Therefore,
we synthesised and assayed as ARIs 5-benzyl-2,4-thiazo-
lidinediones 5–7, which retained the pharmacophoric
elements of the previously explored 5-arylidene
analogues.29,30

D. Rakowitz et al. / Bioorg. Med. Chem. 14 (2006) 567–574
569
At the same time, the reduction of the exocyclic double
bond C@C of 5-arylidene-2,4-thiazolidinediones 2–4
might enhance their potential antihyperglycaemic activ-
ity, similarly to known 5-benzyl-2,4-thiazolidinediones
which are often more effective than their 5-arylidene-
substituted analogues in improving blood glucose levels
in NIDDM.18,19 Recently, the antihyperglycaemic activ-
ity was also reported of several 2,4-thiazolidinediones
having a carboxylic chain inserted on N-3.32
performed in a rapid and safe way.34,35 Upon MW-irra-
diation, the reduction of representative compound 2b by
LiBH4 in pyridine and THF was accomplished in less
than 10 min. However, the yield of the desired derivative
(5b) was similar to that obtained under conventional
conditions.
The treatment of compounds 5 with methyl bromoace-
tate, using potassium carbonate as base, provided esters
6 in high yields; these latter were hydrolysed in acidic
medium to give acids 7 (Scheme 1). [5-(4-Hydroxyb-
enzyl)-2,4-dioxothiazolidin-3-yl]acetic acid (7g) was
prepared by the hydrolysis of the methyl ester of [5-(4-
benzyloxybenzyl)-2,4-dioxothiazolidin-3-yl]acetic acid
(6c) in acidic medium.
It seemed likely, therefore, that compounds 5–7 could
be potentially endowed with both antihyperglycaemic
and ALR2 inhibitory effects. We here report on their
in vitro ALR2 inhibitory activity, which was explored
using partially purified ALR2 from bovine lenses.
The structures of compounds 5–7 were assigned on the
basis of their analytical and spectroscopic data. NMR
spectroscopy proved to be particularly useful for their
2. Chemistry
1
5-Benzyl-2,4-thiazolidinediones 5 were prepared by the
reaction of corresponding 5-benzylidene derivatives 2
with lithium borohydride in pyridine and tetrahydrofu-
ran at reflux for 3–4 h (Scheme 1). Compounds 2 were
in turn obtained, according to a known procedure, by
the Knoevenagel condensation of commercially avail-
able 2,4-thiazolidinedione with suitable benzaldehydes
(Scheme 1).29 The reduction of the exocyclic double
bond C@C of several 5-benzylidene-2,4-thiazolidinedi-
ones (including compounds 2c and 2f) by LiBH4 was
reported by Giles and colleagues; it was shown to be a
rapid and selective hydrogenation method of the double
bond C@C and required simple equipment.33
characterization. In fact, H NMR spectra of all com-
pounds 5–7 showed a diagnostic ABX system, in the
range 3.05–4.62 ppm, consisting of three double dou-
blets due to the resonances of the enantiotopic methy-
lene protons and the methinic proton in 5. In addition,
the absence of any singlet in the range 7.75–7.85 ppm,
attributable to the resonance of the 5-methylidene pro-
ton of parent compounds 2,29,30 irrefutably confirmed
the reduction of the exocyclic double bond C@C.
In 13C NMR spectra, the methylene carbon and the thia-
zolidinedione C-5 of compounds 5–7 resonated at 36.7–
38.8 and 51.6–54.0 ppm, respectively.
1
In an attempt to increase the yields of compounds 5, we
also tried carrying out the same reduction by irradiation
with microwaves (MW). In fact, although reduction
reactions were the last to appear on the scene of MW-
assisted chemistry, it has been demonstrated that MW-
assisted hydrogenation reactions can be successfully
The H and 13C NMR spectra of esters 6 and acids 7
showed an additional signal due to the resonance of
N-CH2 (the proton in the range 4.30–4.38 ppm and
the carbon at 41.5–41.9 ppm); in their 13C NMR spec-
tra, besides two signals attributable to the resonances
of 2- and 4-carbonylic groups at 170.0–175.0 ppm,
CHO
O
H
O
H
N
a
N
S
+
S
O
O
R
H
R
R
2
a
b
c
3-OC6H5
b
4-OC6H5
4-OCH2C6H5
O
S
O
S
O
S
CH2COOH
O
CH2COOCH3
c
d
e
4-C6H5
3-OCH3
4-OCH3
4-OH
H
O
N
N
d
N
O
f
g
R
R
R
7
6
5
Scheme 1. Reagents and conditions: (a) C5H11N, C2H5OH, D; (b) LiBH4, Py, THF, D; (c) K2CO3, acetone, BrCH2COOCH3, D; (d) AcOH, HCl, D.

570
D. Rakowitz et al. / Bioorg. Med. Chem. 14 (2006) 567–574
another signal due to the resonance of the carboxylic
carbon appeared in the same range.
moderate ALR2 inhibition at 50 lM dose (Table 1).
Compounds 5b, 5c and 5d, which bore an aromatic sub-
stituent in the para position of the 5-benzyl group, were
significantly more potent than the corresponding 5-ary-
lidene analogues (2b, 2c and 2d), which in turn had pro-
duced 41%, 10%, and 20% ALR2 inhibition,
respectively, at 50 lM dose.30 This appreciable increase
in potency was not observed either when the phenoxy
group was displaced in meta position (5a) or when it
was replaced by a methoxy group (5e and 5f); in fact,
compounds 5a, 5e and 5f proved to be less effective than
corresponding compounds 2.29,30
3. Results and discussion
Compounds 5–7 were tested in vitro for their ability to
inhibit partially purified ALR2 from bovine lenses,
using sorbinil and ponalrestat as reference drugs (Table
1). The enzyme activity was assayed by spectrophoto-
metrically monitoring NADPH oxidation, which
accompanies the reduction of D,L-glyceraldehyde used
as substrate.
The insertion of an acetic chain on N-3 led to (5-benzyl-
2,4-dioxothiazolidin-3-yl)acetic acids 7, which, analo-
gously to what had been observed in the series of 5-ary-
lidene-2,4-thiazolidinediones,29,30 were shown to be
from 15- to 80-fold more effective than N-unsubstituted
analogues 5; they displayed IC50 values ranging between
1.01 and 2.96 lM (Table 1).
Taking into account the SARs defined in our previous
studies relevant to 2,4-thiazolidinediones 2–4,29,30 we
prepared analogues 5–7 a–d which had an additional
aromatic moiety, that is, a phenoxy, benzyloxy or phen-
yl group, on the 5-benzyl residue (Scheme 1). In addi-
tion, we synthesised derivatives 5–7 e,f in which these
additional aromatic groups were replaced by a methoxy
group. Finally, we also prepared [5-(4-hydroxybenzyl)-
2,4-dioxothiazolidin-3-yl]acetic acid (7g) as the 5-benzyl
analogue of 4g, which in turn had proved to be one of
the most active among the previously explored 5-arylid-
ene-2,4-thiazolidinediones, with IC50 = 0.15 lM.30
The ALR2 inhibitory potency of acids 7 appeared to be
independent of the pattern substitution on the 5-benzyl
moiety; indeed, the most active derivatives were [5-(3-
phenoxybenzyl)-2,4-dioxothiazolidin-3-yl]acetic
acid
(7a, IC50 = 1.01 lM) and [5-(4-methoxybenzyl)-2,4-
dioxothiazolidin-3-yl]acetic acid (7f, IC50 = 1.07 lM).
Among N-unsubstituted 5-benzyl-2,4-thiazolidinedi-
ones, derivatives 5 a–d displayed IC50 values ranging
from 31.4 to 78.9 lM, whereas 5e and 5f produced only
In comparison with previously studied acids 4, ana-
logues 7a, b and d–f were shown to be from 3- to almost
8-fold less effective, whereas 5-(4-hydroxybenzyl) substi-
tuted acid 7g was 14 times less active than its 5-arylidene
counterpart.30
Table 1. In vitro bovine lensesÕ ALR2 inhibitory activity of 2,4-
thiazolidinediones 5–7
Among methyl esters 6, only compound 6e produced
appreciable ALR2 inhibition (IC50 = 21.1 lM), proving
to be at least 2.5-fold more active than the correspond-
O
S
R
N
5 R = H
6 R = CH2COOCH3
ing
N-unsubstituted
2,4-thiazolidinedione
5e
O
(IC50 > 50 lM) but 7-fold less effective than acid 7e
(IC50 = 2.96 lM) (Table 1). Ester 6a, which was the 5-
benzyl analogue of compound 3a (IC50 = 1.32 lM),29,30
was also shown to be inactive (Table 1).
7 R = CH2COOH
R'
a
R0
Compound
IC50
In conclusion, although the saturation of the exocyclic
double bond C@C in position 5 of 5-arylidene-2,4-thia-
zolidinediones generally appeared to be not very benefi-
cial for ALR2 inhibitory activity, 5-benzyl-2,4-
thiazolidinediones 5 and (5-benzyl-2,4-dioxothiazoli-
din-3-yl)acetic acids 7 proved to be moderately to highly
effective in vitro ARIs. Ester 6e also displayed ALR2
inhibitory properties.
3-OC6H5
4-OC6H5
4-OCH2C6H5
4-C6H5
5a
5b
78.9 (62.3–99.9)
40.4 (34.8–47.1)
31.4 (29.4–33.5)
65.8 (47.8–90.7)
16% (50 lM)
21% (50 lM)
26% (50 lM)
1% (50 lM)
24% (50 lM)
5c
5d
3-OCH3
4-OCH3
3-OC6H5
4-OC6H5
4-OCH2C6H5
4-C6H5
5e
5f
6a
6b
6c
6d
6e
15% (50 lM)
21.1 (16.4–27.0)
On the basis of the biological results so far acquired, we
could identify some structural features which influenced
their effectiveness as ARIs. In N-unsubstituted deriva-
tives 5, the presence of an additional aromatic ring in
the 5-benzyl moiety, especially in the para position, ap-
peared to have a beneficial influence, whereas the meth-
oxy group was a less favourable substituent. As
expected, the insertion of an acetic chain on N-3 in-
creased activity up to almost 100 times and led to acids
7, which were the most effective among the tested
compounds.
3-OCH3
4-OCH3
3-OC6H5
4-OC6H5
4-C6H5
6f
7a
24% (50 lM)
1.01 (0.92–1.11)
2.83 (2.68–3.00)
1.68 (1.09–2.59)
2.96 (2.37–3.70)
1.07 (0.96–1.20)
2.10 (1.81–2.43)
3.42 (3.39–3.46)
0.08 (0.073–0.087)
7b
7d
3-OCH3
4-OCH3
4-OH
7e
7f
7g
Sorbinil
Ponalrestat
a IC50 (lM) (95% CL) or % inhibition at the given concentration.

D. Rakowitz et al. / Bioorg. Med. Chem. 14 (2006) 567–574
571
Taking into account that compounds 5b and 5f are al-
ready present in the literature as antihyperglycaemic
agents,17,36 the logical continuation of this research
may be the evaluation, in a suitable animal model of
NIDDM, of the antihyperglycaemic activity of the most
promising ARIs among the 5-benzyl-2,4-thiazolidinedi-
ones here reported, in order to assess their ability to
act as potential antidiabetic agents with dual effect.
(3 Cq arom); 170.1 and 173.9 (2 CO). Anal.
(C16H13NO3S) C, H, N.
4.2.2. 5-(4-Phenoxybenzyl)-2,4-thiazolidinedione (5b).
1
Yield 41%; mp 120–123 ꢁC. H NMR (CDCl3): d 3.18
(dd, J = 14.1 and 9.9 Hz, 1H, part A of ABX system,
CH2); 3.55 (dd, J = 14 and 3.6 Hz, 1H, part B of ABX
system, CH2); 4.57 (dd, J = 9.9 and 3.6 Hz, 1H, part X
of ABX system, 5-CH); 7.01–7.53 (m, 9H, CH arom);
8.57 (br s, 1H, NH). 13C NMR (CDCl3): d 37.9 (CH2);
53.5 (5-CH); 118.9, 119.2, 123.6, 129.8 and 130.6 (9
CH arom); 130.3, 156.8 and 157.0 (3 Cq arom); 170.5
and 174.3 (2 CO). Anal. (C16H13NO3S) C, H, N.
4. Experimental section
4.1. Chemistry
Melting points were recorded on a Kofler hot-stage
apparatus and are uncorrected. TLC controls were car-
ried out on precoated silica gel plates (F 254 Merck).
Elemental analyses (C, H, N), determined by means of
a C. Erba mod. 1106 elem. Analyzer, were within
0.4% of theory. 1H and 13C NMR spectra were record-
ed on a Varian 300 magnetic resonance spectrometer
Compound 5b was also synthesised according to the fol-
lowing procedure: a mixture of 2b (0.25 g, 0.84 mmol),
LiBH4 (0.04 g, 1.85 mmol), pyridine (1.6 ml) and anhy-
drous THF (0.58 ml), in a hermetically sealed vial, was
irradiated with microwaves (300 W, 120 ꢁC, max pres-
sure 300 psi) for 5–6 min. The vial was cooled on an
ice bath and a solution of HCl 12 N (0.42 ml) and
H2O (2.6 ml) was slowly introduced by means of a syr-
inge. The mixture was again irradiated with microwaves
(300 W, 110 ꢁC, max pressure 300 psi) for 2 min. After
evaporation of the solvent in vacuo, the crude solid
was treated as above described in the general method
for the synthesis of compounds 5, to give pure 5b (yield
42%).
1
(300 MHz for H and 75 MHz for 13C). Chemical shifts
are given in d units (ppm) relative to internal standard
Me4Si and refer to CDCl3 solutions. Coupling constants
(J) are given in hertz (Hz). 13C NMR spectra were deter-
mined by Attached Proton Test (APT) experiments and
the resonances were always attributed by proton–carbon
heteronuclear chemical shift correlation. A microwave
oven Discover (CEM) was used to carry out micro-
wave-assisted reactions. Unless stated otherwise, all
materials were obtained from commercial suppliers
and used without further purification. All synthesised
compounds were assayed as racemic mixtures.
4.2.3. 5-(4-Benzyloxybenzyl)-2,4-thiazolidinedione (5c).
1
Yield 42%; mp 115–118 ꢁC. H NMR (CDCl3): d 3.11
(dd, J = 14.1 and 9.3 Hz, 1H, part A of ABX system,
CH2); 3.47 (dd, J = 14.1 and 3.9 Hz, 1H, part B of
ABX system, CH2); 4.51 (dd, J = 9.3 and 3.9 Hz, 1H,
part X of ABX system, 5-CH); 5.06 (s, 2H, OCH2);
6.96 (m, 2H, CH arom); 7.16 (m, 2H, CH arom);
7.35–7.47 (m, 5H, CH arom); 8.72 (br s, 1H, NH). 13C
NMR (CDCl3): d 36.7 (CH2); 53.6 (5-CH); 69.5
(OCH2); 115.1, 127.5, 128.0, 128.6 and 130.3 (9 CH
arom); 130.7, 138.9 and 158.0 (3 Cq arom); 171.3 and
175.0 (2 CO). Anal. (C17H15NO3S) C, H, N.
4.2. General method for the synthesis of 5-benzyl-2,4-
thiazolidinediones 5
Lithium borohydride (1.54 g, 70.6 mmol) was slowly
added to a stirred solution of 229,30 (32 mmol) in pyri-
dine (60 ml) and anhydrous THF (22 ml) at room tem-
perature; effervescence was controlled by the addition
rate. When the addition of LiBH4 was completed and
effervescence finished, the stirred mixture was refluxed
for 3–4 h. A solution of hydrochloric acid 12 N (16 ml)
and deionised water (100 ml) was carefully added to
the stirred mixture, which was cooled on an ice bath,
and then the mixture was heated to reflux for 1 h. After
evaporation of the solvent under reduced pressure, the
residue was dissolved in chloroform, washed with water
(3 · 100 ml) and dried with anhydrous Na2SO4. After
evaporation of the solvent in vacuo, the solid was puri-
fied by column chromatography on silica gel, using
diethyl ether/light petroleum 40–60 ꢁC (4:6) as eluant.
4.2.4. 5-Biphenyl-4yilmethyl-2,4-thiazolidinedione (5d).
1
Yield 46%; mp 138–140 ꢁC. H NMR (CDCl3): d 3.24
(dd, J = 14.1 and 9.9 Hz, 1H, part A of ABX system,
CH2); 3.65 (dd, J = 14.1 and 3.6 Hz, 1H, part B of
ABX system, CH2); 4.62 (dd, J = 9.9 and 3.6 Hz, 1H,
part X of ABX system, 5-CH); 7.34–7.64 (m, 9H, CH
arom); 8.36 (br s, 1H, NH). 13C NMR (CDCl3): d
38.3 (CH2); 53.3 (5-CH); 127.0, 127.4, 127.5, 128.8
and 129.6 (9 CH arom); 134.7, 140.4 and 140.6 (3 Cq
arom); 170.1 and 173.9 (2 CO). Anal. (C16H13NO2S)
C, H, N.
4.2.1. 5-(3-Phenoxybenzyl)-2,4-thiazolidinedione (5a).
Yield 43%; mp 98 ꢁC. H NMR (CDCl3): d 3.11 (dd,
4.2.5. 5-(3-Methoxybenzyl)-2,4-thiazolidinedione (5e).
Yield 40%; mp 115 ꢁC. H NMR (CDCl3): d 3.08 (dd,
1
1
J = 13.8 and 9.9 Hz, 1H, part A of ABX system,
CH2); 3.52 (dd, J = 13.8 and 3.8 Hz, 1H, part B of
ABX system, CH2); 4.52 (dd, J = 9.9 and 3.8 Hz, 1H,
part X of ABX system, 5-CH); 6.88–7.39 (m, 9H, CH
arom); 8.68 (br s, 1H, NH). 13C NMR (CDCl3): d 38.4
(CH2); 53.1 (5-CH); 117.9, 119.1, 119.3, 123.6, 123.8,
129.8 and 130.2 (9 CH arom); 137.7, 156.8 and 157.8
J = 14.1 and 10.2 Hz, 1H, part A of ABX system,
CH2); 3.55 (dd, J = 14.1 and 3.9 Hz, 1H, part B of
ABX system, CH2); 3.81 (s, 3H, OCH3); 4.53 (dd,
J = 10.2 and 3.9 Hz, 1H, part X of ABX system, 5-
CH); 6.78–6.85 (m, 3H, CH arom); 7.26 (dd, J = 7.8
and 7.8 Hz, 1H, CH arom); 9.36 (br s, 1H, NH). 13C
NMR (CDCl3): d 38.7 (CH2); 53.4 (5-CH); 55.2

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D. Rakowitz et al. / Bioorg. Med. Chem. 14 (2006) 567–574
(OCH3); 112.9, 114.8, 121.3 and 129.9 (4 CH arom);
137.5 and 159.8 (2 Cq arom); 170.9 and 174.6 (2 CO).
Anal. (C11H11NO3S) C, H, N.
4.3.4.
(5-Biphenyl-4-ylmethyl-2,4-dioxothiazolidin-3-
yl)acetic acid methyl ester (6d). Yield 80%; mp 95 ꢁC.
1H NMR (CDCl3): d 3.14 (dd, J = 14.4 and 10.5 Hz,
1H, part A of ABX system, CH2); 3.66 (dd, J = 14.4
and 3.9 Hz, 1H, part B of ABX system, CH2); 3.76 (s,
3H, OCH3); 4.34 (s, 2H, N-CH2); 4.57 (dd, J = 10.5
and 3.9 Hz, 1H, part X of ABX system, 5-CH); 7.30–
7.37, 7.42–7.46, 7.55–7.59 (3m, 9H, CH arom). 13C
NMR (CDCl3): d 38.6 (CH2); 41.9 (N-CH2); 51.9 (5-
CH); 52.8 (OCH3); 127.1, 127.5, 127.6, 128.8 and
129.6 (9 CH arom); 135.0, 140.5 and 140.6 (3 Cq arom);
171.6, 172.9 and 173.8 (3 CO). Anal. (C19H17NO4S) C,
H, N.
4.2.6. 5-(4-Methoxybenzyl)-2,4-thiazolidinedione (5f).
1
Yield 47%; mp 110 ꢁC. H and 13C NMR data were
reported in Ref. 33. Anal. (C11H11NO3S) C, H, N.
4.3. General method for the synthesis of (5-benzyl-2,4-
dioxothiazolidin-3-yl)acetic acids methyl esters 6
A
mixture of 5-benzyl-2,4-thiazolidinedione
5
(17 mmol), methyl bromoacetate (5.2 g, 34 mmol) and
potassium carbonate (4.7 g, 34 mmol) in acetone
(150 ml) was refluxed for 24 h. After evaporation of
the solvent under reduced pressure, the residue was dis-
solved in chloroform, washed with water (3 · 100 ml)
and dried with anhydrous Na2SO4. After evaporation
of the solvent, the solid was recrystallized from ethanol
providing pure ester 6.
4.3.5.
[5-(3-Methoxybenzyl)-2,4-dioxothiazolidin-3-
yl]acetic acid methyl ester (6e). Yield 60%; mp 92 ꢁC.
1H NMR (CDCl3): d 3.06 (dd, J = 14.1 and 10.5 Hz,
1H, part A of ABX system, CH2); 3.65 (dd, J = 14.1
and 3.6 Hz, 1H, part B of ABX system, CH2); 3.78
and 3.82 (2s, 6H, 2 OCH3); 4.35 (s, 2H, N-CH2); 4.57
(dd, J = 10.5 and 3.6 Hz, 1H, part X of ABX system,
5-CH); 6.80–6.86 (m, 3H, CH arom); 7.26 (dd J = 7.5
and 7.5 Hz, 1H, CH arom). 13C NMR (CDCl3): d 38.8
(CH2); 41.8 (N-CH2); 51.8 (5-CH); 52.8 and 55.2 (2
OCH3); 113.0, 115.1, 123.3 and 129.9 (4 CH arom);
132.1 and 161.5 (2 Cq arom); 170.8, 171.3 and 173.6
(3 CO). Anal. (C14H15NO5S) C, H, N.
4.3.1.
[2,4-Dioxo-5-(3-phenoxybenzyl)thiazolidin-3-
yl]acetic acid methyl ester (6a). Yield 60%; mp 80 ꢁC.
1H NMR (CDCl3): d 3.06 (dd, J = 14.1 and 10.2 Hz,
1H, part A of ABX system, CH2); 3.59 (dd, J = 14.1
and 3.9 Hz, 1H, part B of ABX system, CH2); 3.77
(s, 3H, OCH3); 4.33 (s, 2H, N-CH2); 4.52 (dd,
J = 10.2 and 3.9 Hz, 1H, part X of ABX system, 5-
CH); 6.88–7.03 (m, 5H, CH arom); 7.15 (m, 1H, CH
arom); 7.30–7.39 (m, 3H, CH arom). 13C NMR
(CDCl3): d 38.8 (CH2); 41.9 (N-CH2); 51.7 (5-CH);
52.9 (OCH3); 117.9, 119.1, 119.2, 123.6, 123.7, 129.9
and 130.3 (9 CH arom); 138.0, 157.1 and 158.6 (3 Cq
arom); 170.1, 170.4 and 173.2 (3 CO). Anal.
(C19H17NO5S) C, H, N.
4.3.6.
[5-(4-Methoxybenzyl)-2,4-dioxothiazolidin-3-
yl]acetic acid methyl ester (6f). Yield 75%; mp 88 ꢁC.
1H NMR (CDCl3): d 3.05 (dd, J = 13.8 and 9.9 Hz,
1H, part A of ABX system, CH2); 3.54 (dd, J = 13.8
and 3.9 Hz, 1H, part B of ABX system, CH2); 3.76
and 3.79 (2s, 6H, 2 OCH3); 4.31 (s, 2H, N-CH2); 4.50
(dd, J = 9.9 and 3.9 Hz, 1H, part X of ABX system, 5-
CH); 6.85 (m, 2H, CH arom); 7.15 (m, 2H, CH arom).
13C NMR (CDCl3): d 38.2 (CH2); 41.5 (N-CH2); 51.9
(5-CH); 52.8 and 55.2 (2 OCH3); 113.9 and 129.9 (4
CH arom); 128.0 and 159.2 (2 Cq arom); 170.4, 171.0
and 173.4 (3 CO). Anal. (C14H15NO5S) C, H, N.
4.3.2.
[2,4-Dioxo-5-(4-phenoxybenzyl)thiazolidin-3-
yl]acetic acid methyl ester (6b). Yield 94%; mp 70 ꢁC.
1H NMR (CDCl3): d 3.10 (dd, J = 13.8 and 9.3 Hz,
1H, part A of ABX system, CH2); 3.58 (dd, J = 13.8
and 3.0 Hz, 1H, part B of ABX system, CH2); 3.76 (s,
3H, OCH3); 4.33 (s, 2H, N-CH2); 4.52 (dd, J = 9.3 and
3.0 Hz, 1H, part X of ABX system, 5-CH); 6.94–7.34
(m, 9H, CH arom). 13C NMR (CDCl3): d 38.2 (CH2);
41.9 (N-CH2); 52.0 (5-CH); 52.8 (OCH3); 118.9, 119.1,
123.5, 129.8 and 130.5 (9 CH arom); 130.4, 155.2 and
157.0 (3 Cq arom); 170.3, 173.0 and 173.8 (3 CO). Anal.
(C19H17NO5S) C, H, N.
4.4. General method for the synthesis of (5-benzyl-2,4-
dioxothiazolidin-3-yl)acetic acids 7
A mixture of ester 6 (10 mmol), glacial AcOH (120 ml)
and HCl 12N (30 ml) was refluxed for 1.5 h. After evap-
oration in vacuo, the residue was refluxed again with
AcOH (120 ml) and HCl (30 ml) for 1.5 h. After evapo-
ration to dryness in vacuo, the crude solid was washed
with H2O and recrystallized from ethanol providing
pure carboxylic acid 7.
4.3.3.
[5-(4-Benzyloxybenzyl)-2,4-dioxothiazolidin-3-
yl]acetic acid methyl ester (6c). Yield 80%; mp 86 ꢁC.
1H NMR (CDCl3): d 3.07 (dd, J = 14.4 and 10.2 Hz,
1H, part A of ABX system, CH2); 3.56 (dd, J = 14.4
and 3.9 Hz, 1H, part B of ABX system, CH2); 3.78 (s,
3H, OCH3); 4.33 (s, 2H, N-CH2); 4.51 (dd, J = 10.2
and 3.9 Hz, 1H, part X of ABX system, 5-CH); 5.07
(s, 2H, OCH2); 6.95 (m, 2H, CH arom); 7.17 (m, 2H,
CH arom); 7.34–7.46 (m, 5H, CH arom). 13C NMR
(CDCl3): d 38.1 (CH2); 41.8 (N-CH2); 52.2 (5-CH);
52.8 (OCH3); 70.0 (OCH2); 115.2, 127.5, 128.0, 128.6
and 130.3 (9 CH arom); 130.2, 138.4 and 160.7 (3 Cq
arom); 170.0, 172.3 and 172.5 (3 CO). Anal.
(C20H19NO5S) C, H, N.
4.4.1.
[2,4-Dioxo-5-(3-phenoxybenzyl)thiazolidin-3-
yl]acetic acid (7a). Yield 60%; mp 150 ꢁC. 1H NMR
(CDCl3): d 3.07 (dd, J = 14.1 and 10.2 Hz, 1H, part A
of ABX system, CH2); 3.57 (dd, J = 14.1 and 3.9 Hz,
1H, part B of ABX system, CH2); 4.36 (s, 2H, N-
CH2); 4.53 (dd, J = 10.2 and 3.9 Hz, 1H, part X of
ABX system, 5-CH); 6.88–7.03 (m, 5H, CH arom);
7.15 (m, 1H, CH arom); 7.29–7.39 (m, 3H, CH arom).
13C NMR (CDCl3): d 38.6 (CH2); 41.6 (N-CH2); 51.6
(5-CH); 117.9, 119.1, 119.3, 123.6, 123.8, 129.8 and
130.2 (9 CH arom); 137.7, 156.8 and 157.7 (3 Cq arom);

D. Rakowitz et al. / Bioorg. Med. Chem. 14 (2006) 567–574
573
170.3, 170.7 and 173.0 (3 CO). Anal. (C18H15NO5S) C,
H, N.
(CH2); 41.7 (N-CH2); 51.9 (5-CH); 115.8 and 131.9 (4
CH arom); 129.9 and 159.8 (2 Cq arom); 170.2, 170.5
and 172.8 (3 CO). Anal. (C12H11NO5S) C, H, N.
4.4.2.
[2,4-Dioxo-5-(4-phenoxybenzyl)thiazolidin-3-
yl]acetic acid (7b). Yield 50%; mp 160 ꢁC. 1H NMR
(CDCl3): d 3.11 (dd, J = 14.1 and 9.9 Hz, 1H, part A
of ABX system, CH2); 3.56 (dd, J = 14.1 and 3.9 Hz,
1H, part B of ABX system, CH2); 4.38 (s, 2H, N-
CH2); 4.53 (dd, J = 9.9 and 3.9 Hz, 1H, part X of
ABX system, 5-CH); 6.94–7.03, 7.10–7.21, 7.32–7.38
(3m, 9H, CH arom). 13C NMR (CDCl3): d 38.0 (CH2);
41.6 (N-CH2); 51.9 (5-CH); 118.9, 119.2, 123.6, 129.8
and 130.5 (9 CH arom); 130.3, 156.8 and 157.0 (3 Cq
arom); 170.3, 170.9 and 173.1 (3 CO). Anal.
(C18H15NO5S) C, H, N.
4.5. Enzyme section
NADPH, DL-glyceraldehyde and dithiothreitol (DTT)
were purchased from Sigma Chemical Co. DEAE-cellu-
lose (DE-52) was obtained from Whatman. Sorbinil
was a gift from Professor L. Costantino, University
of Modena (Italy). All other chemicals were commer-
cial samples of good grade. Calf lenses for the purifica-
tion of ALR2 were obtained locally from freshly
slaughtered animals. The enzyme was purified by a
chromatographic procedure as previously described.37
Briefly, ALR2 was released by carving the capsule
and the frozen lenses were suspended in potassium
phosphate buffer, pH 7, containing 5 mM DTT and
stirred in an ice-cold bath for 2 h. The suspension
was centrifuged at 4000 rpm at 4 ꢁC for 30 min and
the supernatant was subjected to ion exchange chroma-
tography on DE52. Enzyme activity was assayed spec-
trophotometrically on a Cecil Super Aurius CE 3041
spectrophotometer by measuring the decrease in
absorption of NADPH at 340 nm which accompanies
the oxidation of b-NADPH catalysed by ALR2. The
assay was performed at 37 ꢁC in a reaction mixture
containing 0.25 M sodium phosphate buffer, pH 6.8,
0.38 M ammonium sulfate, 0.11 mM NADPH and
4.7 mM DL-glyceraldehyde as substrate in a final vol-
ume of 1.5 ml. All inhibitors were dissolved in DMSO.
The final concentration of DMSO in the reaction mix-
ture was 1%. To correct for the non-enzymatic oxida-
tion of NADPH, the rate of NADPH oxidation in
the presence of all of the reaction mixture components,
except the substrate, was subtracted from each experi-
mental rate. Each dose–effect curve was generated using
at least three concentrations of inhibitor causing an
inhibition between 20% and 80%. Each concentration
was tested in duplicate, and IC50 values as well as the
95% confidence limits (95% CL) were obtained by using
CalcuSyn software for dose effect analysis.38
4.4.3.
(5-Biphenyl-4-ylmethyl-2,4-dioxothiazolidin-3-
yl)acetic acid (7d). Yield 56%; mp 145 ꢁC. 1H NMR
(CDCl3): d 3.18 (dd, J = 13.8 and 9.6 Hz, 1H, part A
of ABX system, CH2); 3.65 (dd, J = 13.8 and 3.0 Hz,
1H, part B of ABX system, CH2); 4.38 (s, 2H, N-
CH2); 4.57 (dd, J = 9.6 and 3.0 Hz, 1H, part X of
ABX system, 5-CH); 7.30–7.60 (m, 9H, CH arom). 13C
NMR (CDCl3): d 38.6 (CH2); 41.6 (N-CH2); 51.9 (5-
CH); 127.0, 127.5, 127.6, 128.8 and 129.5 (9 CH arom);
134.9, 140.7 and 140.8 (3 Cq arom); 170.4, 171.4 and
172.3 (3 CO). Anal. (C18H15NO4S) C, H, N.
4.4.4.
[5-(3-Methoxybenzyl)-2,4-dioxothiazolidin-3-
1
yl]acetic acid (7e). Yield 57%; mp 88–90 ꢁC. H NMR
(CDCl3): d 3.05 (dd, J = 13.8 and 10.5 Hz, 1H, part A
of ABX system, CH2); 3.60 (dd, J = 13.8 and 3.6 Hz,
1H, part B of ABX system, CH2); 3.80 (s, 3H, OCH3);
4.37 (s, 2H, N-CH2); 4.55 (dd, J = 10.5 and 3.6 Hz,
1H, part X of ABX system, 5-CH); 6.81 (m, 3H, CH
arom); 7.25 (m, 1H, CH arom). 13C NMR (CDCl3): d
38.8 (CH2); 41.7 (N-CH2); 51.8 (5-CH); 55.2 (OCH3);
113.0, 114.7, 121.3 and 129.9 (4 CH arom); 137.4 and
159.8 (2 Cq arom); 170.5, 170.8 and 173.2 (3 CO). Anal.
(C13H13NO5S) C, H, N.
4.4.5.
[5-(4-Methoxybenzyl)-2,4-dioxothiazolidin-3-
yl]acetic acid (7f). Yield 55%; mp 84 ꢁC. 1H NMR
(CDCl3): d 3.08 (dd, J = 13.8 and 9.6 Hz, 1H, part A
of ABX system, CH2); 3.53 (dd, J = 13.8 and 3.9 Hz,
1H, part B of ABX system, CH2); 3.81 (s, 3H, OCH3);
4.37 (s, 2H, N-CH2); 4.53 (dd, J = 9.6 and 3.9 Hz, 1H,
part X of ABX system, 5-CH); 6.87 (m, 2H, CH arom);
7.16 (m, 2H, CH arom). 13C NMR (CDCl3): d 38.0
(CH2); 41.6 (N-CH2); 52.2 (5-CH); 55.3 (OCH3); 114.3
and 130.3 (4 CH arom); 127.7 and 159.1 (2 Cq arom);
170.5, 170.9 and 173.2 (3 CO). Anal. (C13H13NO5S) C,
H, N.
Acknowledgments
The authors acknowledge Schlachthof Salzburg-Berg-
heim (Austria) (KR Ing. Sebastian Grießner and Mag.
Verena Klob) for providing calf lenses.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.08.056.
4.4.6.
[5-(4-Hydroxybenzyl)-2,4-dioxothiazolidin-3-
yl]acetic acid (7g). was synthesised as above described,
strarting from compound 6c: yield 60%; mp 130–
132 ꢁC. 1H NMR (CDCl3): d 3.08 (dd, J = 13.8 and
9.3 Hz, 1H, part A of ABX system, CH2); 3.46 (dd,
J = 13.8 and 3.9 Hz, 1H, part B of ABX system, CH2);
4.30 (s, 2H, N-CH2); 4.51 (dd, J = 9.3 and 3.9 Hz, 1H,
part X of ABX system, 5-CH); 6.76 (m, 2H, CH arom);
7.29 (m, 2H, CH arom). 13C NMR (CDCl3): d 38.1
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