A diverse series of substituted benzenesulfonamides as aldose reductase inhibitors with antioxidant activity: Design, synthesis, and in vitro activity

Article abstract of DOI:10.1021/jm101008m

We have previously reported the successful replacement of a carboxylic acid functionality with that of a difluorophenolic group on the known aldose reductase inhibitors (ARIs) of 2-(phenylsulfonamido)acetic acid chemotype. In the present work, based on bi

Full text of DOI:10.1021/jm101008m

7756 J. Med. Chem. 2010, 53, 7756–7766
DOI: 10.1021/jm101008m
A Diverse Series of Substituted Benzenesulfonamides as Aldose Reductase Inhibitors
with Antioxidant Activity: Design, Synthesis, and in Vitro Activity
Polyxeni Alexiou and Vassilis J. Demopoulos*
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54 124, Greece
Received July 26, 2010
We have previously reported the successful replacement of a carboxylic acid functionality with that of a
difluorophenolic group on the known aldose reductase inhibitors (ARIs) of 2-(phenylsulfonamido)-
acetic acid chemotype. In the present work, based on bioisosteric principles, additional 2,6-difluor-
ophenol and tetrazole, methylsulfonylamide, and isoxazolidin-3-one phenylsulfonamide derivatives
were synthesized and tested in vitro in protocols primarily related to the long-term diabetic complica-
tions. Most of the compounds were found as ARIs at IC50 <100 μM, while the introduction of the
4-bromo-2-fluorobenzyl group in a phenylsulfonamidodifluorophenol structure resulted in a com-
pound (4c) presenting a submicromolar inhibitory profile. However, the derivatives of tetrazole,
methylsulfonylamine, and the (R)-enantiomer of isoxazolidin-3-one did not exhibit appreciable ARI
activity. The selectivity of the active ARIs is also discussed. Furthermore, the synthesized compounds
exhibited potent antioxidant potential (homogeneous and heterogeneous systems).
Introduction
ARIs with improved pharmacodynamic and pharmacokinetic
properties.5,6
Aldose reductase enzyme (ALR2,a EC 1.1.1.21, AKR1B1)
of the polyol metabolic pathway, and especially its inhibition
by aldose reductase inhibitors (ARIs), has been gaining
attention over the past years from the pharmaceutical com-
munity, as it appears to be a promising pharmacotherapeutic
target. It was first found to be implicated in the etiology of the
long-term diabetic complications, such as retinopathy, ne-
phropathy, and neuropathy.1 However, to date, emerging
reports have suggested that under normal glucose concentra-
tion, ALR2 may be up-regulated by factors other than
hyperglycemia and therefore be involved also in other patho-
logical processes that have become major threats to human
health in the 21st century such as cardiac disorders (including
myocardial ischemia and ischemia-reperfusion injury, conges-
tive heart failure, cardiac hypertrophy, and cardiomyopathy),
inflammation, mood disorders, renal insufficiency, ovarian
abnormalities, and human cancers such as liver, breast, ovarian,
cervical, and rectal cancers.2
Intense efforts have been directed toward the development
of effective ARIs, but still only one (epalrestat) is commer-
cially available and only in Japan.3 Thus, it is becoming
important that novel chemotypes to be developed lacking
the poor pharmacokinetic profile or the side effects of the
various ARIs that have failed in the clinical trials.4 Toward this
direction, many studies have been reported to date, providing
evidence that novel chemotypes, being neither carboxylic acid
nor hydantoin derivatives, could be considered as promising
hits or lead compounds for further development of potent
The present work involves the pharmacochemical study of
novel ARIs. The design of the novel compounds was based on
the concept of bioisosterism,7,8 a strategy used for molecular
modifications on the lead compound. The sulfonamide
(R2SO2NR2) functionality is an acceptable functional group
in medicinal chemistry when incorporated into putative small-
molecule therapeutics, as it has the potential to form several
electrostatic interactions with proteins and other targets (during
the period of 2006-2008, there were nine published patents in
which all or most reported compounds, with a variety of
biological responses, contained the sulfonamide functional
group).9 Furthermore, it has been reported that a series of
compounds, derivatives of 2-(phenylsulfonamido)acetic acid,
show important in vitro ALR2 inhibitory activity.10-12
2-(Phenylsulfonamido)acetic acid (compound 1) was chosen
as the lead compound and a series of nonclassical isosters to the
acetic acid moiety were used for the latter’s replacement in order
for potent (with improved pharmacokinetic and pharmacody-
namic characteristics) ARIs to be developed.
Diverse functional groups are known for their isosteric
relationships with the carboxylate group. The methylsulfony-
lamine and the tetrazole group mimic the carboxylate group
principally in terms of its physicochemical properties related
to acidity, although tetrazole is more stable and lipophilic.13
In order to further balance acidity with lipophilicity, the acetic
acid moiety in the lead compound was replaced with that of
difluorophenol, a reported bioisoster for carboxylate,14,15 and a
limited number of such compounds have already been published
by our research group.5 Furthermore, isoxazolidin-3-one with a
calculated pKa of 8.2616 was studied as a putative isostere for the
carboxylate group, as it has a structure relevant to that of
hydantoin or succinimide derivatives (known classes of ARIs).
*To whom correspondence should be addressed. Phone: þ30 2310
997626. Fax: þ30 2310 997852. E-mail: vdem@pharm.auth.gr.
a Abbreviations: AKR, aldo-keto reductase superfamily; ALR2,
aldose reductase; ALR1, aldehyde reductase; ARIs, aldose reductase
inhibitors; SAR, structure-activity relationship.
r
pubs.acs.org/jmc
Published on Web 10/11/2010
2010 American Chemical Society

Article
The data derived from the in vitro experiments revealed
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7757
Reviews of the literature about ARIs under development
and those already in clinical trials (for example, ranirestat,
multicenter, phase II-III study, http://clinicaltrials.gov/ct2/
show/NCT00927914) highlight the presence of the haloge-
nated benzene ring as a substituent that importantly contrib-
utes to the improvement of the ALR2 inhibitory activity.4,22
This observation, together with the increasing interest in the
employment of fluorine at medical and pharmaceutical
agents,23 led to the choice of 4-bromo-2-fluoromethylbenzyl
moiety as a potent “privileged scaffold”24 that could be
introduced to the phenylsulfonamidedifluorophenol deriva-
tives (compounds 4a-c). Furthermore, N-sulfonylation was
also investigated (compound 4d).
important bioisosterism in the case of the difluorophenol
derivatives (compounds 2a-i), as well as in the case of
(S)-isoxazolidin-3-one derivative 9b but not the (R)-isoxazolidin-
3-one derivative 9a. There is ample evidence that ALR2 may
stereoselectively recognize the inhibitor. Some examples of the
active isomers of known ARIs are sorbinil (S),17 fidarestat
(2S4S),18,19 and ranirestat (R).20,21 The phenylsulfonamides,
derivatives of the tetrazole moiety (compounds 5 and 7), and
the methylsulfonylamine derivative (compound 8) did not
prove to be strong ARIs.
Scheme 1. Synthesis of N-(3,5-Difluoro-4-hydroxyphenyl)ben-
zenesulfonamides 2a-da
Regarding the desirable pharmacodynamic profile of a
novel ARI, the following points are highlighted:
(a) ARIs that appear to be promising during in vitro studies
often fail to proceed any further into clinical use because of
side effects, which in many cases have been attributed to the
lack of selectivity for other enzymes. ALR2 is a member of the
aldo-keto reductase (AKR) superfamily,25 and another
member of the AKR superfamily is aldehyde reductase
(ALR1, EC 1.1.1.2, AKR1A1). It is believed that the unfavor-
able profile of many ARIs in clinical trials is due to their
concurrent inhibition of these closely related AKRs.26
(b) The limited efficacy of currently known ARIs may also
be related to the post-translational modification of ALR2
activity, which is caused by the hyperglycemia-induced oxida-
tive stress. This modification involves oxidation of a critical
a Reagents and conditions: (i) (CH3)3SiCl/THF; (ii) ArSO2Cl,
DMAP/THF; (iii) Et3N/THF; (iv) H2O.
Scheme 2. Synthesis of N-(3,5-Difluoro-4-hydroxyphenyl)benzenesulfonamides 2e-ia
a Reagents and conditions: (i) cyclohexene, Pd/C /(CH3CH(OH)CH3; (ii) 2,5-dimethoxytetrahydrofuran, 4-chloropyridine hydrochloride/1,4-
dioxane; (iii) RC6H4COCl, Et3N/1,4-dioxane; (iv) 5% aq NaOH; (v) ethyl isocyanate/1,4-dioxane; (vi) 5% aq NaOH.

7758 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Alexiou and Demopoulos
Scheme 3. Synthesis of N-(4-Bromo-2-fluorobenzyl)-N-(3,5-difluoro-4-hydroxyphenyl)arylsulfonamides (4a-c) and N-(3,5-Di-
fluoro-4-hydroxyphenyl)-N-(phenylsulfonyl)benzenesulfonamide (4d)a
a Reagents and conditions: (i) PhCOCl, Et3N/1,4-dioxane; (ii) NaH/DMF; (iii) 4-bromo-1-(bromomethyl)-2-fluorobenzene/DMF; (iv) 5% aq
NaOH; (v) cyclohexene, Pd/C/(CH3CH(OH)CH3; (vi) NaH/THF; (vii) PhSO2Cl/THF; (viii) 2.5% NaOH.
active cysteine thiol (Cys298), which regulates both substrate
and inhibitor binding.27,28 The resulting oxidized form of
ALR2 shows an increase in Km for aldehyde substrates and
a marked reduction in sensitivity to ARIs. Additionally and
taking into consideration the detoxification role of ALR2
against oxidative stress and its potential role as an antioxidant
enzyme (catalysis of the reduction of a wide range of alde-
hydes generated from lipid peroxidation),29 during treatment
with ARIs the concurrent administration of antioxidants
could contribute on the one hand in keeping the enzyme in
its reduced form and on the other hand in counterbalancing its
inhibition. Thus, a bifunctional compound that would com-
bine ALR2 inhibitory activity and antioxidant potential could
be of interest as a pharmacotherapeutic agent.
The general synthetic procedure of the N-(3,5-difluoro-
4-hydroxyphenyl)benzenesulfonamides 2a-d is illustrated in
Scheme 1. The presented compounds 2a-c were prepared in a
one-pot reaction as described previously,5 and 2d was pre-
pared using the same procedure.
The synthesis of compounds 2e-i are presented in Scheme
2. Compound 2e, also reported previously,5 was prepared by a
heterogeneous catalytic hydrogen transfer reaction, and the
formation of the pyrrol ring of compound 2f was attained
by a modified Clauson-Kaas type reaction with the use of
2,5-dimethoxytetrahydrofuran and 4-chloropyridine hydrochlo-
ride as a catalyst.15 The synthetic route for the preparation of
the sulfamoylphenylbenzamide derivatives 2g,h involved a
reaction between 2e and commercially available benzenesul-
fonyl chlorides, followed by a selective hydrolysis in basic
conditions of the intermediate esters. In a similar manner, the
ureido derivative 2i was prepared by using an excess of ethyl
isocyanate for the urea formation,31 followed by selective
hydrolysis in basic conditions. Ultrasound irradiation in the
urea formation step was found to increase the overall yield.
Phenyl benzoates (3a, 3b) were prepared by phenol benzoy-
lation catalyzed with triethylamine in dioxane32 and were
further treated with sodium hydride, followed by alkylation33
with 4-bromo-1-(bromomethyl)-2-fluorobenzene and hydro-
lysis in basic conditions to give the N-(4-bromo-2-fluoro-
benzyl)-N-(3,5-difluoro-4-hydroxyphenyl)arylsulfonamides
(4a, 4b) as shown in Scheme 3. Compound 4c was obtained
from 4b with a heterogeneous catalytic hydrogen transfer
reaction. Furthermore, from 3a and with treatment with
On the basis of the above presented data, the novel com-
pounds developed were tested in vitro for their ALR2 and
ALR1 inhibitory activity and their antioxidant potential in
homogeneous and heterogeneous systems.
Chemistry
Compound 1 was prepared according to the standard
method involving reaction of commercially available benze-
nesulfonyl choride with the appropriate amino acid (glycine)
in aqueous NaOH, which has been described by De Ruiter et
al.10 In a similar manner compound 5 was also prepared using
1H-tetrazol-5-amine as the nucleophilic reactant but with a
lower yieldduetopossibleformation of sulfonylcarbamimidic
azide as reported by Peet et al.30

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7759
Scheme 4. Synthesis of Compounds 6a, 6b, and 7a
a Reagents and conditions: (i) PhSO2Cl, Et3N, DMAP/THF; (ii) PhSO2Cl, DMAP/THF; (iii) (CH3)3SiN3, (CH3CH2CH2CH2)2SndO/toluene.
Scheme 5. Synthesis of N-(Methylsulfonyl)-2-(phenylsulfonamido)acetamide 8a
a Reagents and conditions: (i) PhSO2Cl, aq NaOH; (ii) CDI/THF; (iii) CH3SO2NH2, DBU/THF.
sodium hydride and N-sulfonylation with benzenesulfonyl
chloride followed by hydrolysis in mild basic conditions in
order to avoid the hydrolysis of the N,N-disulfonylated
product,34 4d was obtained.
the acetic acid functionality of the lead compound 1, indicates
effective bioisosteric replacement in the case of the difluorophe-
nol derivative 2a (IC50 = 32.9 μM). Compounds 5, 7, 8, and 9a
are weak ARIs, while 9b exhibits notable activity (IC50
=
For thesynthesisof tetrazole7 a reported one-step synthetic
method for the transformation of nitriles into 5-substituted
tetrazoles35 was adopted. The method involves the use of
trimethylsilyl azide as the azide source and the use of catalytic
dialkyltin oxide. For the preparation of the appropriate nitrile
6a commercial aminoacetonitrile sulfate, benzenesulfonyl
chloride and a catalytic amount of triethylamine and N,N-
dimethylpyridin-4-amine (DMAP) in tetrahydrofurane were
used (method A). By modification of the reaction conditions
(method B), improvement of the overall yield was achieved
but N-(cyanomethyl)-N-(phenylsulfonyl)benzenesulfonamide
(6b) was obtained as a side product. The previous synthetic
routes mentioned are presented in Scheme 4.
The N-(methylsulfonyl)-2-(phenylsulfonamido)acetamide
(8) was prepared according to a reported procedure by Yuan
and Silverman13 that involves treatment of 1 with carbonyl-
diimidazole followed by treatment with methanesulfonamide
in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene as pre-
sented in Scheme 5.
71.2 μM). It is of interest that there is significant difference in
the inhibitory activity of the chiral compounds 9a and 9b. As the
(S)-enantiomer9b was found to be 2 times more potent than the
(R)-enantiomer 9a, the stereocenter in the cycloserine moiety
seems to have an impact on the ALR2 inhibitory activity and
therefore chilarity could be considered as a useful tool in the lead
optimization process, at least in the case of this class of ARIs.
Regarding the difluorophenol derivatives 2a-i the R1
substitution seems to play a significant role in the biological
response. Methoxy (compound 2b) and amino (compound 2e)
substituents increased ALR2 inhibitory activity with amino
(IC50 = 14.1 μM) being slightly superior to methoxy (IC50
=
15.5 μM). In the case of substituents that are electron
acceptors, as in the case of nitro (2c) and trifluoro (2d)
substituent, no improvement in the inhibitory activity was
noticed. A capability for structural modifications is provided
by the amino substituent of the potent ARI 2e. Initially, we
introduced a pyrrole ring (compound 2f) as an attempt to
increase the total aromatic area of the inhibitor, but there
was no improvement in the biological response. On the
contrary, an improvement of efficacy was noticed in the case
of introducing an additional benzene ring in compounds 2g
(IC50 = 7.9 μM) and 2h (IC50 = 12.0 μM). In the case of 2g
and 2h the presence of a carbonyl group between the two
aromatic regions seems to be of significance because it may
be involved in “charge-transfer interactions” in the active site
of ALR2, as this has been previously postulated.40,41
The N-alcylation of the main sulfonamide scaffold with
the “privileged” structure of 4-bromo-2-fluoromethylben-
zene (R2 substitution) resulted in the development of strong
ARIs: 4a (IC50 = 5.7 μM), 4b (IC50 = 9.0 μM), and 4c
(IC50 = 0.397 μM), with 4c to be the most potent one of the
whole series of the synthesized compounds in the present work.
On the other hand, a farther N-sulfonylation (compound 4d)
did not significantly improve the inhibitory activity.
For the synthesis of compounds 9a,b commercial D- or
L-cycloserine was allowed to react with benzenesulfonyl chlo-
ride in an aqueous buffer solution with pKa=10.36
Results and Discussion
Aldose Reductase Inhibitory Activity. The target com-
pounds were tested for their ability to inhibit rat lenses ALR2.
It has been shown that the human and rat sequences of this
enzyme are characterized by 81% identity and 89% homology,
while the proposed active sites of both enzymes are identical.37
The performed assay was based on the spectrophotometric
monitoring of NADPH oxidation, which is proven to be quite
a reliable method.38 Results are shown in Table 1.
The inhibitory activity of compounds 2a, 5, 7, 8, 9a, and
9b, which differ in the R3 substitution and are all isosteres to

7760 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Table 1. ALR2 Inhibitory Activity Data
Alexiou and Demopoulos
a n = 3. b Reported IC50: 134 μM.10 c Reported by Alexiou et al.5 d Reported IC50: 0.25 μM.39

Article
Aldehyde Reductase Inhibitory Activity. Another member
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7761
One of the primary roles of ALR1 in diabetes (as well as of
ALR2) is the detoxification of tissues from the reactive
R-oxoaldehyde glycating agents, like methylglyoxal and
3-deoxyglucosone.45-47 However, other enzymes, such as
glyoxalase-I, betaine aldehyde dehydrogenase, 2-oxoalde-
hyde dehydrogenase, and the dimeric dihydrodiol dehydrog-
enase could also act as the detotoxification enzymes for these
glycating agents.48,49 It is noted that the dimeric dihydrodiol
dehydrogenase is not inhibited by the established ALR1
inhibitors (valproic acid and barbiturates) or the classical
ARIs (sorbinil and tolrestat).50 Furthermore, kidney polyols
may be generated by both aldose and aldehyde reductase,
with ALR1 significantly contributing to the polyol produc-
tion in the kidney cortex, the predominant site of diabetes-
linked kidney lesions.51 It is noted that ALR1, but not
ALR2, is localized in the kidney cortex.52 Although a degree
of selectivity toward ALR2 maybe important for a successful
ARI, on the basis of the above points, this selectivity feature
should not be considered as a negating factor for the
identification of an initial lead ARI.
of the AKR superfamily is aldehyde reductase (ALR1), and
it is believed that the unfavorable profile of many ARIs in
clinical trials is due to their concurrent inhibition of the
closely related ALR1.19 ALR1 and ALR2 share a high
degree of amino acid sequence (∼65%) and structural
homology.42,43 The target compounds were tested for their
ability to inhibit rat kidney ALR1, and the obtained data are
presented in Table 2. Compounds 1, 5, 7, 8, 9a, and 9b, which
were found to be weak ARIs, are also weak inhibitors of
ALR1, with the notable example being 9b, which possesses
the highest selectivity ratio expressed as (% inhibition (C =
100 μM) of ALR2)/(% inhibition (C = 100 μM) of ALR1).
As for the difluorophenol derivatives 2a-i and 4a-d strong
ALR1 inhibitory activity was found in many cases, indicat-
ing lack of selectivity for these compounds.
Table 2. ALR1 Inhibitory Data and Selectivity Ratios
DPPH Scavenging Activity. The radical scavenging po-
tential of the active ARIs 2a-i and 4a-d was assessed in
vitro by using the model reaction with the stable free radical
of 2,2-diphenyl-1-picrylhydrazyl (DPPH). The scavenging
activity data are expressed as IC50 values, EC50 ratios, and an
initial rate of interaction at equimolar concentration of
DPPH and antioxidant (C = 0.2 mM). Results are summarized
in Table 3. In this homogeneous system of the ethanol
solution of DPPH (C = 0.2 mM), antioxidant activity stems
from an intrinsic chemical reactivity toward radicals.53
According to the derived data, the tested compounds possess
lower antioxidant activity, but still significant, compared to
the standard antioxidant Trolox. There is a considerable
difference in the kinetics of the reaction with the DPPH
between the tested molecules and Trolox. The reaction rate is
significantly slower than that with Trolox. The steady state is
achieved after 270 min, while for Trolox it is achieved after
30 min (reported time of 20 min by Silva et al.54) and remains
constant for 270 min. Furthermore, the approximate stoi-
chiometry of the reactions (σ, number of reduced DPPH
molecules per one molecule of antioxidant), defined as
1/[(2)(EC50)],55 was found to be greater than 1 and apparently
not equal to their available hydroxyl groups in all cases. A
putative mechanism of a more complex reaction, justifying
% inhibition (
IC50
(IC50
)
ALR1/ (% inhALR2)/
ALR2
compd
SD a (C = 100 μM) ( SD,a μM (IC50
)
(% inhALR1
)
1
30.52 ( 2.75
1.69
0.89
2a
2b
2c
2d
2e
2f
2g
2h
2i
79.23 ( 1.35
83.02 ( 0.88
55.71 ( 1.21
50.65 ( 1.22
85.40 ( 1.31
41.67 ( 2.35
81.13 ( 2.05
71.68 ( 0.76
68.48 ( 3.07
69.16 ( 0.44
69.78 ( 0.44
95.26 ( 0.26
93.79 ( 0.69
21.71 ( 1.73
19.9 ( 0.72
12.24 ( 0.69
22.43 ( 2.15
2.67 ( 0.57
19.6 ( 1.4
23.8 ( 1.0
0.60
1.54
2.99
1.15
0.73
4.92
0.87
1.03
0.67
2.09
1.71
0.81
0.35
132.9 ( 12.9
105.2 ( 2.61
10.3 ( 0.23
280.7 ( 2.12
6.9 ( 0.9
12.4 ( 0.23
25.5 ( 3.02
11.9 ( 0.58
15.42 ( 0.06
0.323 ( 0.013
9.7 ( 0.68
4a
4b
4c
4d
5
2.06
1.20
7
8
2.69
1.15
9a
9b
18.31
valproic acid
56.1 ( 2.7b
a n = 3. b Reported IC50: 50.1 μM.44
Table 3. Antioxidant Activity Data
homogeneous system of DPPH
heterogeneous system of DOPC liposomes
compd
IC50 ( SD,a μM
EC50
initial ratec ( SDa
IC50 ( SD,a μM
b
2a
2b
2c
49 ( 0.01d
45.2 ( 0.01
54.8 ( 0.08
45.1 ( 0.2
44.7 ( 0.13
48.6 ( 0.23
49.7 ( 0.1
56.9 ( 0.2
51.6 ( 0.1
60.9 ( 0.1
61.5 ( 0.1
61.9 ( 0.06
63.2 ( 0.3
37 ( 0.09
0.25d
0.23
0.27
0.23
0.22
0.24
0.25
0.26
0.28
0.30
0.31
0.31
0.32
0.19e
0.498 ( 0.005d
0.661 ( 0.058
0.465 ( 0.024
0.490 ( 0.037
0.777 ( 0.037
0.489 ( 0.036
0.817 ( 0.032
0.995 ( 0.039
0.859 ( 0.06
1.000 ( 0.033
0.974 ( 0.055
0.977 ( 0.043
0.967 ( 0.031
1.809 ( 0.004
95.9 ( 8.5d
55.1 ( 2.6
45.7 ( 0.7
66.0 ( 1.3
48.1 ( 4.3
97.1 ( 3.4
68.6 ( 1.6
70.7 ( 3.9
81.7 ( 4.3
66.0 ( 6.1
61.82 ( 5.42
73.1 ( 6.7
138.9 ( 13.1
93.5f
2d
2e
2f
2g
2h
2i
4a
4b
4c
4d
Trolox
a n = 3. b IC50/CDPPH
.
c Absorbance decrease at 517 nm after 30 s. d Reported data by Alexiou et al.5 e Reported by Ancerewicz et al.57 f Reported by
Stefek et al.58

7762 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Alexiou and Demopoulos
such a stoichiometry, could involve stabilization of the
generated phenoxyl radicals of the tested compounds to the
more stable sulfonamide/nitrogen centered radical due to the
“capto-dative effect” present56 and further reactions such as
coupling, fragmentation, and intermolecular addition. In the
case of compounds 4a-d there is no possibility of further
stabilization of the generated hydroxyl radical, and this is
depicted on their higher IC50 values and on the higher initial
velocity compared to compounds 2a-i .
Inhibition of Lipid Peroxidation. In membranes, the rela-
tive antioxidant reactivity is probably different from a
homogeneous system, since it is determined by additional
factors, such as location of the antioxidant and radicals, and
ruled to an extent by the partition ratios between water and
lipophilic compartments. As an indicative heterogeneous
assay, the antioxidant inhibitory efficiency of compounds
2a-i and 4a-d was evaluated in the system of unilamellar
DOPC liposomes (C = 0.8 mM) oxidatively stressed by
peroxyl radical generated in the aqueous phase by thermal
decomposition of the hydrophilic azo initiator 2,20-azobis-
(2-amidinopropane) hydrochloride. Results are shown in
Table 3. It is apparent from the inhibition data, which are
expressed as IC50 values, that the tested compounds are
strong antioxidants, comparable to that of the known anti-
oxidant Trolox or even better in most cases.
spectrophotometer. 1H NMR spectra were obtained on a Bruker
AM-300spectrometer with internal TMSstandard. Thepurityof
the compounds was determined by combustion analysis
(elemental analysis) and was confirmed to be g95% for all
compounds. Elemental analyses (C, H, N) were performed at
Galbraith Laboratories, Inc., Knoxville, TN, or with a Perkin-
Elmer 2400 CHN analyzer in the Department of Organic Chem-
istry, School of Chemistry, Aristotle University of Thessaloniki,
Greece. All reactions were routinely checked by TLC on silica gel
Merck 60 F254, and flash chromatography was performed on silica
gel Merck 9385. Compounds 2a-c and 2e have been previously
synthesized by us.5
2-(Phenylsulfonamido)acetic Acid (1). Compound 1 was pre-
pared by the standard method already reported.10 Yield: 2.563 g
(59%). Mp 169-170 °C (lit. 170-171 °C59). IR (Nujol): 3324
(COOH), 1735 (CdO) cm-1 1H NMR (CDCl3/DMSO-d6):
.
3.7 (s, 2H, -CH2COOH), 6.8-8 (m, 7H, -COOH, Ar-H,
-SO2NHCH2-).
N-(3,5-Difluoro-4-hydroxyphenyl)-4-(trifluoromethyl)benzenesul-
fonamide (2d). To a suspension of 4-amino-2,6-difluorophenol
hydrochloride (275 mg, 1.5 mmol) in THF (78 mL), chlorotri-
methylsilane (0.25 mL, 1.9 mmol) was added. The mixture was
vigorously stirred under a nitrogen atmosphere for 48 h. Then
4-trifluoromethylbenzenesulfonyl chloride (464.5 mg, 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 atmosphere for 24 h. Finally, the
reaction mixture was poured into a stirred water/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 farther 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. The residue
was flash-chromatographed with petroleum ether/EtOAc (2.5:1),
and the title compound was obtained as a beige solid. An
analytical sample was prepared by recrystallization from CH2Cl2/
CH3CN/petroleum ether. Yield: 320 mg (60%). Mp 187-188 °C.
IR (Nujol): 3474-3175 (-NH- and -OH), 1374 (SdO), 1157 (SdO)
cm-1. 1HNMR (CDCl3/DMSO-d6): δ6.7 (d, 2H, difluorophenyl-
2,6H, J = 8.2 Hz), 7.6-8.0 (m, 4H, trifluoromethylphenyl-H),
9.2-9.3 (br s, 1H, phenyl-OH), 9.8-10.0 (br s, 1H, -SO2NH-).
Anal. (C13H8F5NO3S) C, H, N.
Conclusion
The presented pharmacochemical study of a series of
compounds, sulfonamides with diverse functional groups,
shed light into the concept of the bioisterism and its applica-
tion in drug design and indicated a series of ARIs with an IC50
of <100 μM. N-Alcylation of the main difluorophenolic
sulfonamide scaffold with the “privileged” structure of 4-bromo-
2-fluoromethylbenzene resulted in a compound (4c) pre-
senting submicromolar inhibitory profile (IC50 = 0.397 μM),
antioxidant potential, and improved physicochemical profile.
The latter bifunctional compound, although not selective,
could be considered as a lead for further optimization, aimed
for the designand development of potent ARIs with improved
pharmacodynamic and pharmacokinetic profile. At this
point, although a degree of selectivity toward ALR2 maybe
important for a successful ARI, on the basis of the above-
mentioned points, this selectivity feature should not be con-
sidered as a negating factor for the identification of an initial
lead ARI.
Moreover, the data derived from the in vitro experiments
revealed notable bioisosterism in the case of (S)-isoxazolidin-
3-one derivative 9b but not the (R)-isoxazolidin-3-one deriva-
tive 9a. As the (S)-enantiomer 9b was found tobe2 times more
potent than the (R)-enantiomer 9a, the stereocenter in the
cycloserine moiety seems to have an impact on the ALR2
inhibitory activity, and therefore, chilarity could be consid-
eredas a useful toolin the lead optimizationprocess, at least in
the case of this class of ARIs that should be further explored.
N-(3,5-Difluoro-4-hydroxyphenyl)-4-(1H-pyrrol-1-yl)benzenesul-
fonamide (2f). To a solution of 2e (202 mg, 0.67 mmol) in 1,
4-dioxane (10 mL) were added 2,5-dimethoxytetrahydrofuran
(0.15 mL, 1.1 mmol) and 4-chloropyridine hydrochloride (154 mg,
1.03 mmol). The mixture was refluxed under a nitrogen atmo-
sphere for 4 h with vigorous stirring, cooled to room temperature,
and evaporated under reduced pressure. Most of the residue was
dissolved in CH2Cl2 by the gradual addition of several portions of
this solvent, dried over anhydrous Na2SO4, and concentrated
under reduced pressure. The residue was flash-chromatographed
with petroleum ether/EtOAc (3:1) to provide the title compound
as a white solid. An analytical sample was prepared by recrystalli-
zation from CH2Cl2/CH3CN/petroleum ether. Yield: 165 mg
(70%). Mp 202-203 °C. IR (Nujol): 3340-3221 (-NH- and
-OH), 1371 (SdO), 1153 (SdO) cm-1 1H NMR (CDCl3/
.
DMSO-d6): δ 6.2-6.5 (m, 4H, pyrrol-H), 6.7 (d, 2H, difluorophe-
nol-2,6H, J = 8.7 Hz), 7.2-7.8 (m, 4H, 4-pyrrolylbenzenesulfona-
midophenyl-H), 9.5-9.6 (br s, 1H, phenyl-OH), 9.8-10 (br s, 1H,
Experimental Section
Chemistry. Unless otherwise stated, all commercial reagents
were 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
spectrophotometer. Characterization of compounds was estab-
lished by a combination of IR and NMR spectrometry tech-
niques. IR spectra were obtained on a Shimadzu FTIR-8101M
-SO2NH-). Anal. (C16H12F2N2O3S 0.1CH2Cl2) C, H, N.
3
General Procedure for the Preparation of Sulfamoylphenyl-
benzamides (2g,h). To a solution of 2e (295 mg, 0.97 mmol) in
1,4-dioxane (50 mL) were added triethylamine (0.27 mL, 1.94
mmol) and the appropriate aryl chloride (1.94 mmol) (benzoyl
chloride, 0.23 mL; 4-methoxybenzoyl chloride, 0.27 mL). The
mixture was refluxed under a nitrogen atmosphere for 4 h. After

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7763
removal of the solvent under reduced pressure, the residue was
diluted in CH2Cl2 (50 mL), washed with H2O (3 ꢀ 30 mL), brine
(2 ꢀ 20 mL), and concentrated under reduced pressure. The
crude residue was diluted in 1,4-dioxane (15 mL), and 5% cold
aqueous solution of NaOH (15 mL) was added dropwise. The
mixture was stirred vigorously at 0 °C (ice bath) under nitrogen
atmosphere for 2 h. Without removing the ice bath, the reaction
mixture was then acidified with an aqueous 10% HCl solution.
The mixture was stirred with CH2Cl2 (100 mL) for 15 min, and
after the two phases were separated, the aqueous phase was
further extracted with CH2Cl2 (2 ꢀ 20 mL). The combined
organic extracts were washed with H2O (3 ꢀ 20 mL), saturated
aqueous NaHCO3 solution (2 ꢀ 20 mL), brine (2 ꢀ 20 mL),
dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The crude residue was flash-chromatographed with a
mixture of petroleum ether/EtOAc (3:1 and 2.5:1, respectively).
Analytical samples were obtained by recrystallization from
CH2Cl2/CH3CN/petroleum ether as white solids.
0.468 mmol) was then added, and the reaction mixture was
allowed to stir at room temperature and under nitrogen for 72 h.
Then a 5% aqueous NaOH solution (3 mL) was added and the
mixture was stirred under nitrogen overnight and then was
cooled (ice bath) and acidified with concentrated HCl. The
crude product was obtained as a thick precipitate and was
isolated by filtration. Furthermore, the filtrate was treated with
toluene (4 ꢀ 20 mL), washed with H2O (3 ꢀ 30 mL), brine (2 ꢀ
20 mL), dried over Na2SO4, and concentrated under reduced
pressure. The combined crude product was purified by flash
chromatography and with a mixture of petroleum ether/EtOAc
(10:1). Analytical samples were obtained by recrystallization
from CH2Cl2/petroleum ether.
N-(4-Bromo-2-fluorobenzyl)-N-(3,5-difluoro-4-hydroxyphenyl)-
benzenesulfonamide (4a). Yield: 93 mg (60%). Mp 195-196 °C. IR
1
(Nujol): 3450-3229 (-OH), 1377 (SdO), 1153 (SdO) cm-1. H
NMR (CDCl3/DMSO-d6): δ 4.64 (s, 2H, -CH2-), 6.5 (d, 2H,
difluorophenol-2,6H, J = 11.3 Hz), 6.98-7.90 (m, 9H, phenyl-H
N-(4-(N-(3,5-Difluoro-4-hydroxyphenyl)sulfamoyl)phenyl)-
benzamide (2g). Yield: 133 mg (34%). Mp 244-245.5 °C. IR
(Nujol): 3520-3345 (-NH-), 3160-3070 (-OH), 1670 (CdO), 1370
(SdO), 1160 (SdO) cm-1. 1HNMR(CDCl3/DMSO-d6):δ6.75 (d,
2H, difluorophenol-2,6H, J = 8.7 Hz), 7.4-8.2 (m, 9H, aromatic-
H), 9.4 (br s, 1H, phenyl-OH), 9.8 (s, 1H, -SO2NH-), 10.4 (br s, 1H,
and phenyl-OH). Anal. (C19H13BrF3NO3S 0.4CH2Cl2) C, H, N.
3
N-(4-Bromo-2-fluorobenzyl)-N-(3,5-difluoro-4-hydroxyphenyl)-
4-nitrobenzenesulfonamide (4b). Yield: 135 mg (78.3%). Mp
228-229 °C. IR (Nujol): 3563-3261 (-OH), 1377 (SdO), 1160
(SdO) cm-1. 1HNMR(CDCl3-DMSO-d6):δ4.72 (s, 2H, -CH2-),
6.44-6.66 (d, 2H, difluorophenol-2,6H, J = 17.9 Hz), 7.19-7.51
(m, 4H, Br,F-phenyl-H and phenyl-OH), 7.8 (d, 2H, 4-nitrophenyl-
2,6H, J = 17.4 Hz), 8.28-8.51 (d, 2H, 4-nitrophenyl-3,5H, J =
17.4 Hz). Anal. (C19H12BrF3N2O5S) C, H, N.
-CONH-). Anal. (C19H14F2N2O4S 0.11CH2Cl2) C, H, N.
3
N-(4-(N-(3,5-Difluoro-4-hydroxyphenyl)sulfamoyl)phenyl)-4-
methoxybenzamide (2h). Yield: 108 mg (25%). Mp 229-231 °C.
IR (Nujol): 3400-3196 (-NH- and -OH), 1670 (CdO), 1372
(SdO), 1162 (SdO), 1098 (Ar-O-CH3) cm-1. 1H NMR (CDCl3/
DMSO-d6): δ 3.8 (s, 3H, -OCH3), 6.58-7.14 (m, 4H, difluor-
ophenyl-2,6H and methoxyphenyl-3,5H), 7.54-8.2 (m, 6H,
N-sulfamoylophenyl-H and methoxyphenyl-2,6H), 9.48 (br s,
1H, phenyl-OH), 9.87 (br s, 1H, -SO2NH-), 10.24 (br s, 1H, -
4-Amino-N-(4-bromo-2-fluorobenzyl)-N-(3,5-difluoro-4-hydroxy-
phenyl)benzenesulfonamide (4c). To a solution of 4b (42 mg,
0.08 mmol) in isopropanol (5 mL) were added cyclohexene
(0.06 mL, 0.6 mmol) and Pd/C (21 mg). The mixture was refluxed
under a nitrogen atmosphere for 7.5 h, cooled to room tempera-
ture, filtered through Celite, and evaporated under reduced
pressure. The residue was recrystallized from CH2Cl2/petroleum
ether to provide the 4c as a white solid. Yield: 348 mg (77%). Mp
210-211 °C. IR (Nujol): 3400-3198 (-NH2 and -OH), 1376
(SdO), 1146 (SdO) cm-1. 1H NMR (CDCl3/DMSO-d6): δ 4.34
(s, 2H, -CH2-), 4.87 (s, 1H, -OH), 5.07 (br s, 2H, -NH2-), 6.24 (d,
2H, difluorophenol-2,6H, J = 8.9 Hz), 6.37-7.17 (m, 7H,
phenyl-H). Anal. (C19H14BrF3N2O3S) C, H, N.
N-(3,5-Difluoro-4-hydroxyphenyl)-N-(phenylsulfonyl)benzenesul-
fonamide (4d). Under nitrogen atmosphere, a THF solution (4 mL)
of 2g (232 mg, 0.59 mmol) was added to a suspension of sodium
hydride (135 mg, 2.25 mmol) as a 60% suspension in mineral
oil (225 mg) in dry THF (25 mL), and the mixture was stirred
for 1 h at room temperature. The mixture was cooled to 0 °C
(ice bath), and benzenesulfonyl chloride (0.32 mL, 2.28 mmol) was
added. After being stirred for 72 h at room temperature, the mixture
was diluted with H2O (50 mL) and extracted with AcOEt
(3 ꢀ 60 mL). The AcOEt layer was dried over anhydrous NaSO4,
and the AcOEt was concentrated under reduced pressure. Then the
residue was diluted in 1,4-dioxane (15 mL), a 2.5% aqueous NaOH
solution (15 mL) was added, and the mixture was stirred for 5 h and
then was cooled (ice bath) and acidified with concentrated HCl. The
mixture was stirred with CH2Cl2 (100 mL) for 15 min, the two
phases were separated, and the aqueous phase was further extracted
with CH2Cl2 (2 ꢀ 20 mL). The combined organic phases were
washed with H2O (3 ꢀ 20 mL), brine (2 ꢀ 20 mL), dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The
crude residue was flash-chromatographed with a mixture of petro-
leum ether/EtoAc (5:1), and the title compound was obtained as a
white solid. An analytical sample was obtained by recrystallization
from CH2Cl2/petroleum ether. Yield: 67 mg (26%). Mp 163-164 °C.
IR (Nujol): 3226-3103 (-OH), 1373 (SdO), 1166 (SdO). 1H NMR
(CDCl3/DMSO-d6):δ6.53 (d, 2H, difluorophenol-2,6H, J=8.8Hz),
7.28 (br s, 1H, phenyl-OH), 7.33-7.71 (m, 10H, phenyl-H). Anal.
(C18H13F2NO5S) C, H, N.
CONH-). Anal. (C20H16F2N2O5S 0.2CH2Cl2) C, H, N.
3
N-(3,5-Difluoro-4-hydroxyphenyl)-4-(3-ethylureido)benzenesul-
fonamide (2i). Ethyl isocyanate (0.185 mL, 2.33 mmol) was added
in a solution of 2e (116 mg, 0.38 mmol) in 1,4-dioxane (30 mL),
and the mixture was stirred under a nitrogen atmosphere and
ultrasound irradiation for 8.5 h. More ethyl isocyanate (0.1 mL,
1.26 mmol) was added, and the reaction mixture was heated in
80 °C under nitrogen atmosphere and without ultrasound irradia-
tion for 7 h. After removal of the solvent under reduced pressure the
crude residue was diluted in 1,4-dioxane (10 mL) and a 5% aqueous
solution of NaOH (10 mL) was added dropwise. The mixture was
stirred under nitrogen atmosphere for 15 min in room temperature
and then was cooled (ice bath) and acidified with an aqueous 10%
HCl solution. The mixture was treated with CH2Cl2 (75 mL) for
15 min, and after that the two phases were separated. The aqueous
phase was further extracted with CH2Cl2 (2 ꢀ 20 mL). The
combined organic extracts were washed with H2O, brine, dried
over Na2SO4, and concentrated under reduced pressure. The crude
residue was flash-chromatographed with a mixture of EtOAc/
petroleum ether (1:1) to obtain a white solid. An analytical sample
was obtained by recrystallization from CH2Cl2/CH3CN/petroleum
ether. Yield: 107 mg (74%). Mp 213-214.5 °C. IR (Nujol):
3581-3346 (-NH-), 3100-3050 (-OH), 1687 (CdO), 1370
1
(SdO), 1175 (SdO). H NMR (CDCl3/DMSO-d6): δ 1.11-1.16
(t, 3H, -CH2CH3, J = 7.2 Hz), 3.18-3.27 (m, 2H, -NH-CH2-CH3),
5.94 (br s, 1H, -CO-NH-CH2CH3), 6.70 (d, 2H, difluorophenyl-
2,6H, J = 8.5 Hz), 7.46-7.60 (m, 4H, phenyl-H), 8.54 (br s, 1H,
phenyl-OH), 9.32 (br s, 1H, -SO2NH-), 9.58 (s, 1H, -NH-CON-
HCH2CH3). Anal. (C15H15F2N3O4S 0.4CH2Cl2) C, H, N.
3
General Procedure for the Preparation of N-(4-Bromo-2-
fluorobenzyl)-N-(3,5-difluoro-4-hydroxyphenyl)arylsulfonamides
(4a,b). Sodium hydride (10.05 mg, 0.418 mmol) as a 60%
suspension to mineral oil (16.75 mg) was added portionwise to
a stirred solution of the intermediate benzoate (0.334 mmol) (3a,
130 mg; 3b, 145.8 mg) in DMF (3 mL), and the mixture was
stirred at room temperature for 30 min under nitrogen atmo-
sphere. 4-Bromo-1-(bromomethyl)-2-fluorobenzene (125 mg,
N-(1H-Tetrazol-5-yl)benzenesulfonamide (5). Benzenesulfo-
nyl chloride (2.55 mL, 20 mmol) was added dropwise in a warm
(60-70 °C) solution of 1H-tetrazol-5-amine (1.7 g, 20 mmol)

7764 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Alexiou and Demopoulos
and NaOH (1.6 g, 40 mmol) in water (20 mL). After 3 h of
stirring and heating in the above-mentioned temperature, the
mixture was left to cool down at room temperature. Then it was
acidified with concentrated HCl (ice bath). The crude product
was obtained as a thick precipitate, isolated by filtration, and
recrystallized from EtOAc/petroleum ether to give the title
compound. Yield: 1.8 g (40%). Mp 134-136 °C (lit. 132-
134 °C60). IR (Nujol): 3324 (tetrazole N-H), 1384 (SdO), 1264
(tetrazole C-N), 1170 (SdO) cm-1. 1H NMR (CDCl3/DMSO-d6):
δ 7.34-7.67 (m, 3H, phenyl-3,4,5-H), 7.85-8.12 (m, 2H, phen-
yl-2,6-H), 10.7 (br s, 2H, -SO2NH- and tetrazole-H). Anal.
(C7H7N5O2S) C, H, N.
(S)-N-(3-Oxoisoxazolidin-4-yl)benzenesulfonamide (9b). To
an ice-cold solution of L-cycloserine (250 mg, 2.45 mmol) in
buffer NaHCO3/Na2CO3, pH 10 (12.5 mL), benzenesulfonyl
chloride (0.34 mL, 2.69 mmol) was added portionwise. The
reaction mixture was stirred under nitrogen atmosphere and at
0-4 °C overnight. The crude product was obtained as a thick
precipitate and purified as a white solid by flash chromato-
graphy and with a mixture of petroleum ether/EtOAc (2:1). Yield:
90 mg (17%). Mp 155-156 °C. IR (Nujol): 3324 (NH), 1384
(SdO), 1354-1328 (CN), 1170 (SdO) cm-1. 1H NMR (CDCl3/
DMSO-d6):δ3.961-4.52 (m, 3H, oxoisoxazolidinyl-H), 7.22-7.67
(m, 6H, aromatic-H and -SO2NH-), 8.46 (br s, 1H, oxoisoxazoli-
N-((1H-Tetrazol-5-yl)methyl)benzenesulfonamide (7). To a solu-
tion of N-(cyanomethyl)benzenesulfonamide (70 mg, 0.356 mmol)
and trimethylsilyl azide (0.093 mL, 0.713 mmol) in toluene
(5 mL) was added dibutyltin oxide (8.88 mg, 0.0357 mmol), and
the mixture was refluxed for 47 h under a nitrogen atmosphere. The
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in MeOH and reconcentrated. The residue
was partitioned between EtOAc (25 mL) and aqueous 10%
NaHCO3 solution (26 mL). The organic phase was extracted with
an additional portion of aqueous 10% NaHCO3 solution
(25 mL). The combined aqueous extracts were acidified to pH 2
with 10% HCl and then extracted with EtOAc (2 ꢀ 25 mL). The
combined organic extracts were dried over anhydrous Na2SO4,
filtered, and concentrated to give the title tetrazole as a white solid.
An analytical sample was obtained by recrystallization from
CH2Cl2/petroleum ether. Yield: 45 mg (53%). Mp 181-182 °C.
IR (Nujol): 3324 (tetrazole N-H), 1384 (-NH-), 1350-1324
(tetrazole C-N), 1170 (SdO) cm-1. 1H NMR (CDCl3/DMSO-d6):
δ 4.2-4.4 (m, 2H, -NHCH2-), 7.4-8.2 (m, 7H, phenyl-H,
dinyl-NH). Anal. (C9H10N2O4S 1H2O) C, H, N.
3
Biological Assays. In Vitro Aldose Reductase Enzyme Assay.
The tested compounds and the reference compound sorbinil
(C11H9FN2O3, Pfizer, Inc., Central Research Division, Groton,
CT) were dissolved in 10% aqueous solution of DMSO. Lenses
were quickly removed 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 Administration of the Republic
of Greece. The enzyme preparation and assay were performed as
previously described.58 Compounds were tested at five concen-
trations, the log(dose)-response curves were then constructed
from the inhibitory data, and the IC50 values were calculated by
least-squares analysis of the linear portion of the log(dose) vs
response curves (0.889 < r2 < 0.994). The experiments were
performed in triplicate.
In Vitro Aldehyde Reductase Enzyme Assay. The tested com-
pounds and the reference compound valproic acid were dis-
solved in 10% aqueous solution of DMSO. Kidneys were
quickly removed from Fischer-344 rats of both sexes following
euthanasia. The experiments conform to the law for the protec-
tion of experimental animals (Republic of Greece) and are
registered at the Veterinary Administration of the Republic of
Greece. The enzyme preparation and assay were performed as
previously described.44 Compounds were tested at five concen-
trations, the log(dose)-response curves were then constructed
from the inhibitory data, and the IC50 values were calculated by
least-squares analysis of the linear portion of the log(dose) vs
response curves (0.863 < r2 < 0.987). The experiments were
performed in triplicate.
DPPH Assay. To investigate the antiradical activity of the
tested compounds in a homogeneous system, a method based on
the scavenging of the stable free radical 2,2-diphenyl-1-picryl-
hydrazyl (DPPH) was used.53 The experimental protocol was
applied as described previously.5 The tested compounds and the
reference compound 6-hydroxy-2,5,7,8-chroman-2-carboxylic
acid (Trolox) were dissolved in ethanol, and the IC50 values
were determined by least-squares analysis of the linear portion
of the log(dose)-percentage of scavenging of DPPH curves
(0.895<r2 <0.988) of nine concentrations of the tested com-
pounds after 270 min (steady state). The effective concentration
value (EC50, i.e., the IC50 value divided by the concentration of
DPPH) was also calculated. Finally, the initial rate of the
reaction was estimated from the approximately linear absor-
bance decrease during the initial 30 s, of equimolar concentra-
tions (0.2 mM) of the tested compounds and DPPH. The
experiments were performed in triplicate.
Liposome Preparation, Incubation, and LOOH Determina-
tion. Unilamellar L-R-phosphatidylcholine dioleoyl (C18:1,
[cis]9, DOPC, 99% grade) liposomes were used as a heteroge-
neous system61 for the evaluation of the antioxidant activity of
the tested compounds in comparison with that of the standard
Trolox. Peroxidation of the liposomal membrane was triggered
by thermal decomposition of the hydrophilic azo compound
2,2-azobis(2-amidinopropane)hydrochloride (AAPH), and the
procedure for the liposome preparation, incubation, and
LOOH determination was applied as previously described.5 The
lipid peroxide value was determined using a calibration curve
-SO2NH- and tetrazole-H). Anal. (C8H9N5O2S 0.1CH2Cl2) C,
3
H, N.
N-(Methylsulfonyl)-2-(phenylsulfonamido)acetamide (8). A solu-
tion of 1 (640 mg, 2.98 mmol) in THF (6 mL) was added dropwise
to a stirred solution of carbonyldiimidazole (CDI) (726 mg, 4.46
mmol) in THF (7.5 mL) under a nitrogen atmosphere. The
mixture was stirred for 30 min, refluxed for 1 h, and allowed
to cool to room temperature. Methylsulfonamide (425 mg,
4.45 mmol) was added in one portion, and the mixture was stirred
for 10 min before a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) (0.69 mL, 4.45 mmol) in THF (4.5 mL) was added
dropwise. The resulting mixture was stirred for 72 h under a
nitrogen atmosphere, poured into an ice-cold aqueous 1 N HCl
solution (40 mL), and then was extracted with EtOAc (3 ꢀ 50 mL).
After the two phases were separated, the aqueous phase was
further washed with Et2O (30 mL), acidified to pKa = 2, and
extracted with EtOAc (2 ꢀ 20 mL). The combined EtOAc extracts
were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. An analytical sample was obtained by recrys-
tallization from CH2Cl2/petroleum ether as a light yellow solid.
Yield: 252 mg (29%). Mp 118-119 °C. IR (Nujol): 3282 (-NH-),
1
1340 (SdO), 1324 (SdO), 1166-1149 (SdO) cm-1. H NMR
(CDCl3/DMSO-d6): δ 3.08 (s, 3H, -SO2CH3), 3.57-4.06 (br s, 3H,
-SO2NHCH2-), 7.29-7.62 (m, 3H, phenyl 3,4,5-H), 7.65-8.08
(m, 2H, phenyl 2,6-H), 11,7 (br s, 1H, -CONHSO2-). Anal.
(C9H12N2O5S2) C, H, N.
(R)-N-(3-Oxoisoxazolidin-4-yl)benzenesulfonamide (9a). To
an ice-cold solution of D-cycloserine (204 mg, 2 mmol) in buffer
NaHCO3/Na2CO3, pH 10 (10 mL), benzenesulfonyl chloride
(0.28 mL, 2.2 mmol) was added portionwise. The reaction
mixture was stirred under nitrogen atmosphere and at 0-4 °C
overnight. The crude product was obtained as a thick precipitate
and purified as a white solid by flash chromatography and with a
mixture of petroleum ether/EtOAc (2:1). Yield: 151 mg (39%).
Mp 155-156 °C. IR (Nujol): 3324 (NH), 1380 (SdO), 1354-1328
(CN), 1162 (SdO) cm-1 1H NMR (CDCl3/DMSO-d6): δ
.
3.961-4.52 (m, 3H, oxoisoxazolidinyl-H), 7.22-7.67 (m, 6H,
phenyl-H and -SO2NH-), 8.46 (br s, 1H, oxoisoxazolidinyl-NH).
Anal. (C9H10N2O4S 1H2O) C, H, N.
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7765
prepared with standard cumene hydroperoxide. The values of
IC50 were obtained by least-squares analysis of the linear part of
the semilogarithmic plot of I (%, percentage of inhibition) vs
antioxidant concentration (0.860 < r2 < 0.988). The experi-
ment was performed in triplicate.
(17) Kador, P. F.; Goosey, J. D.; Sharpless, N. E.; Kolish, J.; Miller,
D. D. Stereospecific inhibition of aldose reductase EC-1.1.1.21.
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Acknowledgment. This work was supported by grants
from the State Scholarships Foundation of Greece and the
Research Committee of Aristotle University of Thessaloniki,
Greece. Acknowledgments are made to Dr. Ioannis Nicolaou
and Dr. Dionysia Papagiannopoulou (School of Pharmacy,
Aristotle Universityof Thessaloniki) for their contributions in
obtaining the 1H HMR spectral data and to Professor Anna
Tsantili-Kakoulidou (School of Pharmacy, University of
Athens) for permission to use the Pallas program.
Supporting Information Available: Elemental analysis data of
all synthesized compounds and experimental and spectroscopic
details for nonkey compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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