Design and synthesis of N-(3,5-difluoro-4-hydroxyphenyl)benzenesulfonamides as aldose reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.01.042

N-(3,5-Difluoro-4-hydroxyphenyl)benzenesulfonamide (4) and its derivatives 5-7 were prepared as putative bioisosteres of the previously reported aldose reductase inhibitors, which are the N-benzenesulfonylglycine derivatives I-IV. The in vitro aldose reductase inhibitory activity of the prepared compounds is higher than that of the respective glycine derivatives. Furthermore, the parent compound 4 reveals high antioxidant potential. Additionally, the intestine permeability of 4 is determined, and there is initial evidence that there is an operating influx mechanism. Overall, the data indicate that the presented chemotype could serve as a core structure for the design of putative pharmacotherapeutic agents, aiming to the long-term complications of diabetes mellitus.

Full text of DOI:10.1016/j.bmc.2008.01.042

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3926–3932
Design and synthesis of N-(3,5-difluoro-4-hydroxyphenyl)
benzenesulfonamides as aldose reductase inhibitors
Polyxeni Alexiou,a Ioannis Nicolaou,a Milan Stefek,b
Albin Kristlc and Vassilis J. Demopoulosa,*
aDepartment of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
bInstitute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska Cesta 9, Bratislava 84104, Slovakia
cFaculty of Pharmacy, University of Ljubljana, Ljubljana 1000, Slovenia
Received 18 September 2007; revised 17 January 2008; accepted 22 January 2008
Available online 30 January 2008
Abstract—N-(3,5-Difluoro-4-hydroxyphenyl)benzenesulfonamide (4) and its derivatives 5–7 were prepared as putative bioisosteres
of the previously reported aldose reductase inhibitors, which are the N-benzenesulfonylglycine derivatives I–IV. The in vitro aldose
reductase inhibitory activity of the prepared compounds is higher than that of the respective glycine derivatives. Furthermore, the
parent compound 4 reveals high antioxidant potential. Additionally, the intestine permeability of 4 is determined, and there is initial
evidence that there is an operating influx mechanism. Overall, the data indicate that the presented chemotype could serve as a core
structure for the design of putative pharmacotherapeutic agents, aiming to the long-term complications of diabetes mellitus.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Aldose reductase enzyme (AR, ALR2, E.C. 1.1.1.21,
alditol/NADP+ oxidoreductase) of the polyol metabolic
pathway was first found to be implicated in the etiology
of the secondary complications of diabetes.8 Aldose
reductase inhibitors (ARIs) have therefore been noted
as possible pharmacotherapeutic agents. Although sev-
eral ARIs have progressed to the clinical trials’ level,
only one is currently on the market and only in Japan
(Epalrestat, ONO Pharmaceutical, Osaka, Japan).9,10
However, the inhibition of the polyol pathway is consid-
ered to be a promising approach to control diabetic
complications. Thus, attention is currently targeted to
discover ARIs of distinct chemical structures, being
derivatives of neither hydantoin nor carboxylic acid that
are known to cause either toxicity or possess narrow
spectrum of tissue activity.11,12
Diabetes mellitus is a complex metabolic disorder that
affects between 6 and 20% of the population in Western
industrialized societies, with an estimated worldwide
prevalence of 150 million people in 2000. This number
is expected to increase to 220 million people in 2010.1,2
Furthermore, taking into account its present rate of in-
crease, within few decades, it will be one of the world’s
most common diseases and one of the biggest public
health problems, with an estimated minimum of half a
billion cases.3 The diabetic individual is prone to late on-
set complications that are largely responsible for the
morbidity and mortality observed in the patients.4 It
has been demonstrated that the more severe and sus-
tained the degree of hyperglycemia, the more likely it
is that the chronic complications of diabetes will devel-
op.5,6 Pharmaceutical intervention of hyperglycemia
induced diabetic complications is actively pursued since
it is very difficult to maintain normoglycemia by any
means in patients with diabetes mellitus.7,8
In the present work, the synthesis and the AR inhibitory
activity of N-(3,5-difluoro-4-hydroxyphenyl)benzenesul-
fonamide (4) and its derivatives 5–7 are presented. The
design of the compounds 4–7 was based on the previ-
ously reported ARIs I–IV, which are N-benzenesulfo-
nylglycines,13 taking into account the proposal that the
2,6-difluorophenol ring could be a lipophilic isostere
for molecules containing a carboxylic acid group.11,14
The structures of the compounds 4–7 and I–IV are
shown in Figure 1.
Keywords: Antidiabetic aldose reductase inhibitors; Non-classical
bioisosterism; Antioxidant activity; Permeability.
*
Corresponding author. Tel.: +30 2310 997626; fax: +30 2310
997852; e-mail: vdem@pharm.auth.gr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.042

P. Alexiou et al. / Bioorg. Med. Chem. 16 (2008) 3926–3932
3927
formation of 4-amino-2,6-difluorophenyl arylsulfonates
as side products, with a significant lower yield of the
target compounds 4–6.
For the formation of compound 7, we investigated the
heterogeneous catalytic transfer hydrogenation in the
presence of cyclohexene11 in refluxing either ethanol
(57% yield) or isopropanol (77% yield).
2.2. In vitro results
2.2.1. Aldose reductase inhibitory activity. The target
compounds 4–7 were tested for their ability to inhibit
rat lenses AR. It has been shown that there is an approx-
imately 85% sequence similarity between rat lenses and
human AR, while the proposed active sites of both en-
zymes are identical.25 The performed assay was based
on the spectrophotometric monitoring of NADPH oxi-
dation, which is proven to be a reliable method.26 Results
are shown in Table 1 and for comparison, results previ-
ously reported for compounds I–IV in a similar assay
are also included.13 It was found that the putative bio-
isosteres of compounds I–IV, the difluorophenol deriva-
tives 4–7, had improved potency. Although the activities
of 4–7 are not at a submicromolar range, they could be
considered as putative hit inhibitors towards the AR en-
zyme.27 Inspection of the low-energy conformations
(SPARTAN SGI, version 5.1.3)28 reveals that the dis-
tances’ range of the geometric (unweighted) centers of
the aromatic areas of the aryl-sulfonamide substituents
of 4–7 and I–IV (centroids) from the phenolic or carbox-
ylic acidic hydrogens are quite similar (6.91–6.95 and
Figure 1. Structures of N-benzenesulfonylglycines I–IV and N-(3,5-
difluoro-4-hydroxyphenyl)benzenesulfonamides 4–7.
Furthermore, the antioxidant potential of compound 4
was evaluated both in a homogeneous 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay system,15–18 as well as in
a heterogeneous unilamellar L-a-phosphatidylcholine
dioleoyl (DOPC) liposomes’ assay system.18,19 Oxidative
stress is well established as an important biochemical
factor implicated in the long-term complications of dia-
betes mellitus.12,20
Finally, the permeability of compound 4 was determined
in vitro using rat jejunum mounted in side-by-side diffu-
sion cells. We considered that these results could be an
initial indication of the permeability potential of the pre-
sented chemotype.10
˚
7.09–7.11 A, respectively). Furthermore, the analogous
distances of the centroids of the heavy atoms (i.e.,
excluding the hydrogens) of these molecules from the
phenolic or carboxylic acidic hydrogens are also quite
2. Results and discussion
2.1. Chemistry
˚
similar (4.66–4.99 and 4.55–5.15 A, respectively). As a
working hypothesis, we propose that the better inhibi-
tory activities of 4–7 in comparison to I–IV could be
due to their higher molecular surface area [calculated
4-Amino-2,6-difluorophenol has been reported to be
unstable and could be isolated only as its hydrochloride
salt 1.11 We propose that the degradation of the non-
protonated aniline form of 1 is probably due to an auto
oxidative process via an iminoquinone intermediate.
Thus, in order to overcome the above mentioned insta-
bility, a one-pot reaction (Scheme 1) was implemented
for the formation of the N-(3,5-difluoro-4-hydroxy-
phenyl)benzenesulfonamides (4–6). This involved the
following consecutive steps: (i) protection of the pheno-
lic hydroxyl-group as its corresponding trimethylsilyl
ether, (ii) formation of the sulfonamide bond by the
reaction with the appropriate aryl-sulfonyl chloride, in
the presence of a catalytic amount of N,N-dimethylpyri-
din-4-amine (DMAP), (iii) addition of an equimolar
amount of triethylamine in respect to the staring fluoro-
phenol derivative 1, and (iv) a hydrolytic process in or-
der to obtain the target compounds 4–6. For the above
synthetic route, we suggest an operating cyclic mecha-
nism based on the high affinity of oxygen to silicon as
well as the hypernucleophilic properties of DMAP.21–24
The addition of triethylamine at the latter stage of the
reaction was found to increase the overall yield. It is
worth noting that the addition of triethylamine in higher
amounts or at the start of the reaction resulted in the
electron density surface area range (SPARTAN SGI,
version 5.1.3)28 for compounds 4–7 is 254.73–283.67 A
2
˚
2
while for compounds I–IV is 211.06–239.34 A ]. A num-
˚
ber of works have previously indicated the importance of
the molecular surface area of the compounds for the inhi-
bition of the AR enzyme.29–33
2.2.2. DPPH scavenging activity. The radical scavenging
potential of the title compound 4 was assessed in vitro
by using the model reaction with the stable free radical
of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The scaveng-
ing activity data are expressed as IC50 values, EC50 ra-
tios, as well as an initial rate of interaction. Results
are summarized in Table 2. In this homogeneous system
of the ethanol solution of DPPH, antioxidant activity
stems from an intrinsic chemical reactivity towards rad-
icals. DPPH, as a weak hydrogen radical atom abstrac-
tor, could beÅ considered as a good kinetic model for
peroxyl ROO radicals.34 According to the derived data,
the tested compound 4 possesses high antioxidant activ-
ity, similar to that of trolox,16,35,36 which is a known
reducing agent for DPPH.37 It must be noted that there
is a considerable difference in the kinetics of the reaction

3928
P. Alexiou et al. / Bioorg. Med. Chem. 16 (2008) 3926–3932
Scheme 1. Synthesis of N-(3,5-difluoro-4-hydroxyphenyl)benzenesulfonamides 4–7. Reagents: (a) (CH3)3SiCl/THF; (b) ArSO2Cl, DMAP/THF; (c)
Et3N/THF; (d) H2O; (e) cyclohexene, Pd/C/CH3CH(OH)CH3.
Table 1. Aldose reductase inhibitory activity data
than with trolox, and the steady state is achieved after
b
270 min. In the case of trolox, the steady state is
achieved after 30 min and it is constant after 270 min.
Furthermore, the approximate stoichiometry of the
reactions (r, number of reduced DPPH molecules per
one molecule of antioxidant) were defined as 1/
(2 · EC50),38 that in both tested compounds was found
to be greater than one and apparently not equal to their
available hydroxyl groups. A putative mechanism of a
more complex reaction, justifying such a stoichiometry,
could involve further reactions of the generated phe-
noxyl radicals of compound 4 or trolox, such as cou-
pling, fragmentation and intermolecular addition.
However, it should be pointed out that in the case of
trolox this proposed more complex reaction with DPPH
Compound
IC50 ( SDa) lM
IC50 (lM)
4
32.9 ( 0.4)
15.5 ( 0.1)
44.4 ( 1.9)
14.1 ( 0.5)
0.249 ( 0.006)c
134
31
5
6
79
16
7
Sorbinil
a n = 3.
b Reported IC50 values (lM) for the respective glycine derivatives by
De Ruiter et al.13
c Quoted representative IC50 values: 0.07–0.9 lM by Zaher et al.44
with the DPPH between these two molecules. In the case
of compound 4, the reaction rate is significantly slower
Table 2. Antioxidant and permeability data
Papp (·10ꢀ6 cm/s)
a
Compound
Homogeneous
system of DPPH
Heterogeneous
system of DOPC liposomes
IC50 ( SDb)
·10ꢀ6 M
EC50
Initial
ratec ( SDb)
IC50 ( SDb)
·10ꢀ6 M
M–S ( SDb)
S–M ( SDb)
Rd
4
49 ( 0.01)
37 ( 0.09)
0.25
0.19f
0.498 ( 0.005)
1.809 ( 0.004)
95.9 ( 8.5)
93.5g
6.34 ( 0.17)
4.34h
2.91 ( 0.45)
10.90h
0.46e
2.5h
Trolox
Epalrestat
a Apparent permeability coefficient in mucosal to serosal (M–S) and serosal to mucosal (S–M) direction.
b n = 3.
c Absorbance decrease at 517 nm at 30 s.
d R factor is the ratio between S–M/M–S.
e p = 0.025.
f Reported EC50 = 0.19 by Ancerewicz et al.16
g Reported by Rackova et al.19
h Reported by Sturm et al.10

P. Alexiou et al. / Bioorg. Med. Chem. 16 (2008) 3926–3932
3929
takes place in a shorter period, and probably in parallel
with the initial step of the interaction, while for com-
pound 4 it is significantly more time-dependent.
4. Experimental
4.1. General Notes
2.2.3. Inhibition of lipid peroxidation. In membranes, the
relative antioxidant reactivity is probably different from
a homogeneous system since it is determined by addi-
tional factors, such as location of the antioxidant and
radicals and ruled predominantly by the partition ratios
between water and lipophilic compartments. As an
indicative heterogeneous assay, the antioxidant inhibi-
tory efficiency of compound 4 was evaluated in the sys-
tem of unilamellar DOPC liposomes oxidatively stressed
by peroxyl radical generated in the aqueous phase by
thermal decomposition of the hydrophilic azo initiator
2,20-azobis(2-amidinopropane) hydrochloride (AAPH).
Results are shown in Table 2. For comparison, results
previously reported for trolox in an identical assay are
also included.19 It is apparent from the inhibition data,
which are expressed as an IC50 value, that the tested
compound 4 is a potent antioxidant, comparable to that
of the known antioxidant trolox.19
Unless otherwise stated, all commercial reagents were
available from Aldrich or Fluka. Melting points were
determined in open glass capillaries using a Mel-Temp
II apparatus. UV spectra were recorded either on a
Perkin-Elmer 554 or on a Hitachi U-2001 spectropho-
tometer. IR spectra were obtained on a Shimadzu spec-
trophotometer, and 1H NMR spectra on a Bruker AW-
80 spectrometer with internal TMS standard. Elemental
analyses were performed in Galbraith Laboratories,
Inc., Knoxville, TN, USA. Diffusion analyses were per-
formed on EasyMount side-by-side chambers; equipped
with a multi channel voltage–current clamp, model VCC
MC6, Physiologic Instruments, San Diego, USA. HPLC
analyses were performed with a Hewlett-Packard sys-
tem, Series 1100, Waldbrom, Germany. Statistical anal-
yses were performed using SPSS v.12.0.
4.2. Chemistry
2.2.4. Permeability. Apparent permeability coefficients
(Papp) in mucosal-to-serosal (M–S) and serosal-to-muco-
sal (S–M) direction were determined for compound 4.
Results are tabulated in Table 2. For comparison, results
previously reported for epalrestat in an identical assay
are also included.10 The tested compound exhibited
asymmetrical transport properties and its Papp value in
the M–S direction is higher than in the S–M direction.
We propose that there is an operating influx process.
However, at this point, we cannot suggest a molecular
mechanism of this active transport. According to the
Center for Drug Evaluation and Research (CDER) pro-
posed permeability standards (atenolol as low permeable
drug and metoprolol as high permeable drug)39,40 and the
M–S Papp data previously obtained for various drugs,10
the tested compound 4 was classified into biopharmaceu-
tical classification system (BCS) to estimate its in vivo
effective permeability in humans.41 It can be categorized
in intermediate permeability class with the degree of
absorption between 40% and 85% and its bioavailability
would not be limited by the permeability.40
4.2.1. General procedure for the preparation of N-(3,5-
difluoro-4-hydroxyphenyl)benzenesulfonamides (4–6). To
a suspension of 4-amino-2,6-difluorophenol hydrochlo-
ride (1)11 (275 mg, 1.5 mmol) in THF (78 mL), chlorotri-
methylsilane (0.25 mL, 1.9 mmol) was added and the
mixture was vigorously stirred under a nitrogen atmo-
sphere for 48 h. Then, aryl-sulfonyl chlorides (i.e., ben-
zenesulfonyl
chloride,
4-methoxybenzenesulfonyl
chloride and 4-nitrobenzenesulfonyl chloride) (0.26 mL,
393 mg and 421 mg, respectively, 1.9 mmol) and N,N-
dimethylpyridin-4-amine (DMAP, 48 mg, 0.4 mmol)
were added and the mixture was vigorously stirred under
a nitrogen atmosphere for 24 h. Triethylamine (0.21 mL,
1.5 mmol) in THF (11.5 mL) was then added dropwise
and the stirring was continued under a nitrogen atmo-
sphere for 24 h. Finally, the reaction mixture was poured
into a stirred H2O/ice mixture (ꢁ270 mL), and after 2 h
of stirring it was extracted with CH2Cl2 (3· 50 mL).
The organic layer was collected and the aqueous layer,
after saturation with NaCl, was further extracted with
CH2Cl2 (2· 20 mL). The combined organic phases were
washed with saturated aqueous NaCl solution, dried
over anhydrous Na2SO4, and evaporated under reduced
pressure and temperature <40 ꢁC. The residues were
flash chromatographed with petroleum ether/EtOAc
2.5:1 (compounds 4 and 6) or 2:1 (compound 5). Analyt-
ical samples were prepared by recrystallization from
CH2Cl2/acetonitrile/petroleum ether.
3. Conclusion
N-(3,5-Difluoro-4-hydroxy-phenyl)benzenesulfonamide
(4) as well as its derivatives 5–7 exhibits a comparatively
improved aldose reductase inhibitory activity. Addition-
ally, the parent compound 4 exhibits a high antioxidant
potential. It should be noted at this point that the anti-
oxidant activity is considered as an additional indication
for the ability of a compound to inhibit the formation of
advanced glycation end products (AGEs),42 that are
implicated in the development of the long-term compli-
cations of diabetes.11 Finally, compound 4 can be char-
acterized as an intermediate membrane permeable
chemical entity. We propose that the presented experi-
mental data could comprise the basis for the design of
novel chemotypes, as pharmacotherapeutic agents for
the treatment of the long-term diabetic complications.
4.2.1.1. N-(3,5-Difluoro-4-hydroxyphenyl)benzenesul-
fonamide (4). 225 mg, yield 52%, mp 178.5–180 ꢁC, IR
(Nujol): 3367–3269 cmꢀ1 (–OH and –NH), 1170 cmꢀ1
(–SO2NHꢀ), 1H NMR (CDCl3/DMSO-d6): d 6.7 (d,
2H, difluorophenyl-2,6H, J = 8.4 Hz), 7.4–7.9 (m, 6H,
phenyl-H and phenyl-OH), 9.6–9.8 (br s, 1H,
–SO2NH–), Anal. Calcd for C12H9F2NO3S: C, 50.52;
H, 3.18; N, 4.91. Found: C, 50.34; H, 3.19; N, 4.84.
4.2.1.2. N-(3,5-Difluoro-4-hydroxyphenyl)-4-methoxy-
benzenesulfonamide (5). 278 mg, yield 58%, mp 165–

3930
P. Alexiou et al. / Bioorg. Med. Chem. 16 (2008) 3926–3932
166 ꢁC, IR (Nujol): 3474–3047 cmꢀ1 (–OH), 1260 cmꢀ1
(C–O), 1162 cmꢀ1 (–SO2NH–), 1H NMR (CDCl3/
DMSO-d6): d 3.8–4.0 (s, 3H, –OCH3), 6.6–7.5 (m, 5H,
methoxyphenyl-3,5H, difluorophenyl-2,6H and phenyl-
OH), 7.7 (d, 2H, methoxyphenyl-2,6H, J = 5.6 Hz),
9.3–9.6 (br s, 1H, –SO2NH–), Anal. Calcd for
C13H11F2NO4S: C, 49.52; H, 3.52; N, 4.44. Found: C,
49.18; H, 3.50; N, 4.37.
(0.4–0.025 mM) and added to 1 mL solution of DPPH
in absolute ethanol (0.4 mM) to give final concentra-
tions of 0.2–0.0125 mM and 0.2 mM for the tested com-
pounds and DPPH, respectively. The continual
absorbance decrease of the ethanol solution of the stable
free radical at 517 nm, in the presence of the tested com-
pounds, was measured. The IC50 values were determined
by least-square analysis of the linear portion of the logC
versus
%
scavenging
of
DPPH
curves
4.2.1.3. N-(3,5-Difluoro-4-hydroxyphenyl)-4-nitroben-
zenesulfonamide (6). 300 mg, yield 60%, mp 182–183 ꢁC,
IR (Nujol): 3517–3132 cmꢀ1 (–OH), 1163 cmꢀ1 (–SO2-
NH–), 1H NMR (CDCl3/DMSO-d6): d 6.7 (d, 2H,
difluorophenyl-2,6H, J = 8.1 Hz), 7.3–8.4 (m, 5H, phe-
nyl-OH and phenyl-H), 9.8–10.1 (br s, 1H, –SO2NH–),
Anal. Calcd for C12H8F2N2O5S: C, 43.64; H, 2.44; N,
8.48. Found: C, 43.69; H, 2.44; N 8.56.
(0.904 < r2 < 0.977) of nine concentrations of the tested
compounds after 270 min (steady state). The effective
concentration value (EC50, that is, the IC50 value di-
vided by the concentration of DPPH) was also calcu-
lated.16 Finally, the initial rate of the reaction was
estimated from the approximately linear absorbance de-
crease during the initial 30 s, of equimolar concentra-
tions (0.2 mM) of the tested compounds and DPPH.18
The experiments were performed in triplicate.
4.2.2. 4-Amino-N-(3,5-difluoro-4-hydroxyphenyl)benzene-
sulfonamide (7). To a solution of N-(3,5-difluoro-4-
hydroxyphenyl)-4-nitrobenzenesulfonamide (6) (500 mg,
1.51 mmol) in isopropanol (12.5 mL), cyclohexene
(1.15 mL, 11.36 mmol) and Pd/C (375 mg) were added.
The mixture was refluxed under a nitrogen atmosphere
for 4 h, cooled to room temperature, filtered through
Celite, and evaporated under reduced pressure. The res-
idue was recrystallized from CH2Cl2/acetonitrile/petro-
leum ether to provide compound 7 (348 mg, yield
77%): mp 158–159 ꢁC, IR (Nujol): 3410–3196 cmꢀ1
(–OH and –NH2), 1151 cmꢀ1 (–SO2NH–), 1H NMR
(CDCl3/DMSO-d6): d 3.4–4.4 (br s, 2H, phenyl-NH2),
6.4–6.8 (m, 5H, phenyl-OH, aminophenyl-3,5H, difluo-
rophenyl-2,6H), 7.6 (d, 2H, aminophenyl-2,6H,
J = 8.0 Hz), 9.2–9.4 (br s, 1H, –SO2NH–), Anal. Calcd
for C12H10F2N2O3S(0.4CH2Cl2): C, 44.56; H, 3.26; N,
8.38. Found: C, 44.39; H, 3.22; N, 8.89.
4.3.3. Liposome preparation, incubation and LOOH
determination. The experimental protocol is based on a
previously described methodology.18,19 A suspension
of unilamellar L-a-phosphatidylcholine dioleoyl (C18:1,
[cis]-9; DOPC; 99% grade) liposomes (1 mM) in phos-
phate buffer (20 mL, 20 mM, pH 7.4) was prepared.
The liposomes (final concentration 0.8 mM DOPC) were
incubated in the presence of different concentrations of
compound 4 (10–300 lM) with the water-soluble initia-
tor AAPH (final concentration 10 mM) at 50 ꢁC for
80 min. Aliquots (1 mL) of the incubation mixtures were
extracted with 2 mL portions of an ice-cold mixture of
CHCl3/MeOH (2:1, v/v) containing 2,6-di-tert-butyl-p-
cresol (BHT) (0.05%). The lipid hydroperoxide content
was determined by the thiocyanate method by sequen-
tially adding the CHCl3/MeOH (2:1, v/v) mixture
(1.4 mL) and the thiocyanate reagent (0.1 mL).43 The
thiocyanate reagent was prepared by mixing equivalent
volumes of a methanolic solution of KSCN (3%) and
a ferrous ammonium sulfate solution (45 mM in
0.2 mM HCl). After the mixture had been left at ambi-
ent temperature for at least 5 min, the absorbance at
500 nm was recorded. The lipid peroxide value was
determined using a calibration curve prepared with stan-
dard cumene hydroperoxide. The value of IC50 was ob-
tained by least-square analysis of the linear part of the
semi logarithmic plot of I (%, percentage of inhibition)
versus antioxidant concentration (0.965 < r2 < 0.971).
The experiment was performed in triplicate.
4.3. In vitro assays
4.3.1. In vitro aldose reductase enzyme assay. The target
compounds 4–7 as well as the reference compound
sorbinil (C11H9FN2O3, Phizer, Inc., Central Research
Division, Groton, CT, USA) were dissolved in 10%
aqueous solution of DMSO. Lenses were quickly re-
moved from Fischer-344 rats of both sexes following
euthanasia. The experiments conform to the law for
the protection of experimental animals (Republic of
Greece) and are registered at the Veterinary Administra-
tion of the Republic of Greece. The enzyme preparation
and assay were performed as previously described.11,33
Compounds 4–7 were tested at five concentrations, the
log (dose)–response curves were then constructed from
the inhibitory data, and the IC50 values were calculated
by least-square analysis of the linear portion of the log
(dose) versus response curves (0.952 < r2 < 0.994). The
experiments were performed in triplicate.
4.3.4. In vitro transport study across rat jejunum. The
study was performed in a similar manner as described
previously.10 The experiments conform to the law for
the protection of experimental animals (Republic of
Slovenia) and are registered at the Veterinary Adminis-
tration of the Republic of Slovenia. Rat jejunum was
obtained from starved male Wistar rats (250–320 g)
after euthanasia and laparotomy. The tissue was opened
along the mesenteric border, stretched onto special in-
serts with exposed tissue area of 1 cm2 and then placed
between two compartments of side-by-side diffusion
chambers. Each compartment was filled with 2.5 mL
of incubation saline containing 10 mM glucose and
10 mM mannitol on serosal and mucosal side of the tis-
4.3.2. DPPH assay. To investigate the antiradical activ-
ity of the title compound 4 in a homogeneous system, a
method based on the scavenging of the stable free radi-
cal DPPH was used.15–18 Compound 4 or, as a reference
compound, 6-hydroxy-2,5,7,8-chroman-2-carboxylic
acid (trolox) was dissolved in 1 mL of absolute ethanol

P. Alexiou et al. / Bioorg. Med. Chem. 16 (2008) 3926–3932
3931
10. Sturm, K.; Levstik, L.; Demopoulos, V. J.; Kristl, A. Eur.
J. Pharm. Sci. 2006, 28, 128.
11. Nicolaou, I.; Zika, C.; Demopoulos, V. J. J. Med. Chem.
2004, 47, 2706.
sue, respectively. After preincubation for 25 min, the
incubation salines on mucosal side, if studying muco-
sal-to-serosal (M–S) transport, or serosal side, if study-
ing serosal-to-mucosal (S–M) transport, were replaced
by 2.5 mL donor solution containing 0.479 mM of the
title compound 4, 10 lM fluorescein as a paracellular
transport marker and 10 mM glucose (S–M) or 10 mM
mannitol (M–S) in Ringer buffer. Samples of 250 lL
were withdrawn from acceptor side every 25 min up to
175 min and replaced by fresh Ringer buffer containing
all necessary ingredients at appropriate concentrations.
The diffusion chambers were equipped with two pairs
of Ag/AgCl electrodes, connected to the chambers via
3 M KCl/3.5% agar bridges for measuring transepitheli-
al potential difference (PD) and short circuit current (Isc)
with multi channel voltage–current clamp in order to
check/control the tissue viability and integrity by moni-
toring PD, Isc and transepithelial electrical resistance
(TEER) every 25 min. The viability was additionally
checked by recording the rise of Isc and PD after the
addition of concentrated glucose solution to the mucosal
compartment at the end of the experiment. Tissue integ-
rity was assessed also by determining fluorescein perme-
ability coefficient. The samples were analyzed by HPLC.
A Chromolith C8 100 · 4.6 mm column was used. The
mobile phase was composed of 0.05 M KH2PO4 (ad-
justed to pH 3.5 by orthophosphoric acid) and acetoni-
trile with 80:20 portions, respectively (flow = 2.2 mL/
min). For detection, UV diode array detector was used
at 220 nm. Fluorescence detector Tecan GENious was
used for fluorescein detection, and the apparent perme-
ability coefficient (Papp) was calculated.
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This work was supported by the General Secretariat of
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P.D.E., E.P.A.N. 4.3.6.1/2000SE 01330005 2003–2005
and 2005–2007, the State Scholarships Foundation of
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