New 3- and 4-hydroxyfuranones as anti-oxidants and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2005.04.055

Two series of new furanones substituted by methylsulfonylphenyl or methylsulfamidophenyl moieties were found to protect against oxidation damage by inhibiting or quenching free radicals and reactive oxygen species in in vitro experiments. The effect on lipid peroxidation was also examined. In addition, we investigated the activity of products in two models of inflammation: phorbol ester-induced ear edema in mice and carrageenan-induced paw edema in rat. The most powerful compounds and with reducing activity against DPPH (IC₅₀ = 1779 and 57 μM, respectively), superoxide anion quenching capacity (IC₅₀ = 511 and 49 μM, respectively), lipid peroxidation inhibitory effect and anti-inflammatory properties (about 50-65% inhibition of edema at 200 mg/kg ip in both tests used) were selected for further pharmacological and toxicological tests because of their attractive profile for the treatment of inflammatory diseases.

Full text of DOI:10.1016/j.bmc.2005.04.055

Bioorganic & Medicinal Chemistry 13 (2005) 4552–4564
New 3- and 4-hydroxyfuranones as anti-oxidants and
anti-inflammatory agents
a
b
b
c
´
Valerie Weber, Catherine Rubat, Eliane Duroux, Claire Lartigue,
Michel Madesclairea and Pascal Couderta,*
a
´
´
Laboratoire de Chimie Therapeutique, Faculte de Pharmacie, 28, place Henri Dunant, 63001 Clermont-Ferrand Cedex, France
b
´
Laboratoire de Pharmacologie, Faculte de Pharmacie, 28, place Henri Dunant, 63001 Clermont-Ferrand Cedex, France
´
Laboratoire de Chimie Analytique, Faculte de Pharmacie, 28, place Henri Dunant, 63001 Clermont-Ferrand Cedex, France
c
Received 9 December 2004; accepted 12 April 2005
Available online 23 May 2005
Abstract—Two series of new furanones substituted by methylsulfonylphenyl or methylsulfamidophenyl moieties were found to pro-
tect against oxidation damage by inhibiting or quenching free radicals and reactive oxygen species in in vitro experiments. The effect
on lipid peroxidation was also examined. In addition, we investigated the activity of products in two models of inflammation: phor-
bol ester-induced ear edema in mice and carrageenan-induced paw edema in rat. The most powerful compounds and with reducing
activity against DPPH (IC50 = 1779 and 57 lM, respectively), superoxide anion quenching capacity (IC50 = 511 and 49 lM, respec-
tively), lipid peroxidation inhibitory effect and anti-inflammatory properties (about 50–65% inhibition of edema at 200 mg/kg ip in
both tests used) were selected for further pharmacological and toxicological tests because of their attractive profile for the treatment
of inflammatory diseases.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
plays an important part in the prevention of a large
number of chronic diseases.5,6 However, the low solubil-
ity of ascorbic acid in lipophilic environments, as well as
its susceptibility to thermal and oxidative degeneration,
has led to interest in the synthesis of derivatives with
better lipophilicity and increased stability.7–17 The re-
cent efforts devoted to the development of new ascorbic
acid analogues have resulted in obtaining anti-oxi-
dant,18–22 antitumoural23 and potentially anti-inflamma-
tory19,24 agents.
Free radicals are species of chemical entities, that are
energically unstable and highly reactive. The occurrence
of reactive oxygen species is a characteristic of normal
aerobic organisms. They are generated by-products of
multiple metabolic functions and are thus implicated
in tissue damage seen in various pathological events
such as atherosclerosis, myocardial infarction, asthma,
cancer, liver disease, neurologic trauma. . .1,2 Under nor-
mal conditions, aerobic organisms have developed anti-
oxidant defences to protect themselves against free rad-
icals: enzymes such as superoxide dismutase and gluta-
thione peroxidase, and anti-oxidants and radical
scavengers such as a-tocopherol (vitamin E), b-carotene,
glutathione and ascorbic acid (vitamin C).3 Ascorbate is
one of the most potent naturally occurring anti-oxidants
because it works directly by reaction with aqueous per-
oxyl radicals and indirectly by restoring the anti-oxidant
properties of fat-soluble vitamin E.4 Furthermore, it
Concerning inflammation, free radicals are well known
to play an important part in the inflammatory process.
In the cyclooxygenase pathway, oxygen radicals are lib-
erated during the conversion from PGG2 to PGH2,25,26
and different reports have suggested that an enhance-
ment of free radical generation may contribute to the
development of inflammatory processes27 and particu-
larly to the pathogenesis of airway hyperactivity in asth-
ma.28,29 In these conditions, the use of drugs with both
scavenging activity and anti-inflammatory properties
may be of benefit in the treatment of airway inflamma-
tion and bronchial hyper-responsiveness.
Keywords: Furanones; Anti-oxidants; Anti-inflammatory agents;
Sulfones.
In connection to our previous works19,20 on the synthe-
sis of lactone derivatives 1, 2, 3 (Chart 1) related to
*
Corresponding author. Tel.: +33 4 7317 7986; fax: +33 4 7328
2707; e-mail: pascal.coudert@u-clermont1.fr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.055

V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
4553
OH
OH
OH
OH
HO
HO
HO
O
O
OH
O
OH
Ascorbic acid
(+) Cyanidanol / Catechin
Ar
R
CH3
Ar
Ar2
O
Ar1
O
O
O
O
HO
HO
O
O
O
1 : R = H, Ar
2
3
NH2SO2
CH3SO2
NO2
N
O
N
CF3
O
HN
O
SO2CH3
H3C
Nimesulide
Celecoxib
Rofecoxib
Chart 1.
ascorbic acid, we have found out that some 4,5-diaryl-3-
hydroxy-2(5H)-furanones 3 possess significant anti-in-
flammatory and anti-oxidant activities. These results
prompted us to design and synthesize new furanones
substituted by methylsulfonylphenyl or methylsulfamid-
ophenyl moieties typical of potent non-steroidal anti-
inflammatory drugs (NSAIDs) such as rofecoxib
nimesulide and celecoxib (Chart 1).
condensation of the appropriately substituted benzalde-
hydes with N-acetylglycine and sodium acetate in
refluxing acetic anhydride33 furnished the crystalline
oxazolones 5a,b in high yield. The oxazolones were
converted to the corresponding phenylpyruvic acids
6a,b through a two-step hydrolysis with aqueous so-
dium hydroxide followed by aqueous hydrochloric
acid. Methyl arylpyruvates 7 were prepared by methy-
lating arylpyruvic acids 6 using DBU (1 equiv) and
MeI (5 equiv) in DMF according to the procedure de-
scribed by Namiki et al.30
Thus, the anti-oxidant effects of these new derivatives
have been carried out in vitro by means of three tests:
measurement of the reducing activity on the stable 2,2-
diphenyl-1-picrylhydrazyl radical (DPPH), superoxide
anion scavenging and lipid peroxidation tests. Further-
more, anti-inflammatory activities have been studied
by use of two tests: phorbol 12-myristate 13-acetate
(TPA)-induced ear edema test on the mouse and a
model of carrageenan-induced paw edema on the rat.
In a similar way, the synthesis of derivative 8h (Scheme
2) was achieved by the same addition/cyclization pro-
cess starting from the pyruvate 7a and the sulfonamido
aldehyde 12. This intermediate 12 was obtained from
the commercially available 4-nitrobenzaldehyde 9 in
three steps comprising the formation of amine 10
according to the method of Bellamy,34 the sulfonamide
11 and final benzylation. As mentioned above, the addi-
tion of 12 to the appropriate pyruvate yielded furanone
13. Final debenzylation was achieved through hydrog-
enolysis in the presence of Pd/C to give the target fura-
none 8h.
2. Chemistry
For the synthesis of compounds 8 possessing meth-
ylsulfonyl substituents on the phenyl rings, we adopted
the synthetic methods described in a previous commu-
nication.19 Preparation of 4,5-diaryl-3-hydroxyfura-
nones 8a–g was obtained by lactonization after aldol
condensation of methyl arylpyruvates 7 with appropri-
ate benzaldehydes in the presence of 1,5-diazabicy-
clo[5.4.0]undecene (DBU) in dimethylformamide,30–32
as shown in Scheme 1. The requisite a-ketoesters 7a,b
were readily accessible from the commercially available
benzaldehydes 4a,b through the three-step sequence
depicted in Scheme 1 via azlactones 5a,b. Thus, the
The various 4-hydroxy-2(5H)-furanones 18 were syn-
thesized via alkaline cyclization of ethoxycarbonyl-
methyl phenylacetates 17 using sodium hydride in dry
THF (Scheme 3). Intermediates 17 were obtained by
the condensation of a phenylacetic acid derivative 14
with a suitable a-bromo carboxylic ester 16 in the pres-
ence of triethylamine. Bromo compounds 16 were pre-
pared in two steps starting from phenylacetic acids
14a–c after esterification to 15 followed by bromina-

4554
V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
H
O
R1
O
ii
i
R1
OH
N
O
iii
HO
R1
O
4a : R1 = Cl
5a : R1 = Cl
6a : R1 = Cl
4b : R1 = SO2CH3
5b : R1 = SO2CH3
6b : R1 = SO2CH3
R1
R1
iv
OH
OCH3
HO
HO
7a : R1 = Cl
O
O
6a : R1 = Cl
6b : R1 = SO2CH3
6c : R1 = H
6d : R1 = OH
7b : R1 = SO2CH3
7c : R1 = H
7d : R1 = OH
R2
R2
R1
v
7
+
vi
O
O
H
HO
O
8a : R1 = H ; R2 = SO2CH3
8b : R1 = SO2CH3 ; R2 = H
8c : R1 = Cl ; R2 = SO2CH3
8d : R1 = SO2CH3 ; R2 = Cl
8e : R1 = OH ; R2 = SO2CH3
8f : R1 = SO2CH3 ; R2 = OBn
8g : R1 = SO2CH3 ; R2 = OH
vii
Scheme 1. Reagents and conditions: (i) N-acetylglycine, Ac2O, AcONa, 100 ꢁC; (ii) NaOH; (iii) HCl, reflux; (iv) DBU, CH3I, DMF, 0 ꢁC; (v) DBU,
DMF, 0 ꢁC; (vi) HCl; (vii) Pd/C, H2, THF/EtOH.
O
O
O
O
S
H
S
H
NO2
NH2
H3C
NH
H3C
N
ii
iii
i
H
O
H
O
O
O
12
9
10
11
O
S
O
N
CH3
S O
O
H3C
HN
O
O
Cl
Cl
OCH3
OH
7a
Cl
v
O
iv
HO
HO
O
O
13
8h
Scheme 2. Reagents and conditions: (i) SnCl2, EtOH; (ii) CH3SO2Cl, pyridine; (iii) BnCl, NEt3, DMF, 80 ꢁC; (iv) DBU, DMF, 0 ꢁC; (v) Pd/C, H2,
THF/EtOH.
tion. All phenylacetic acids 14 were commercially avail-
able except 14e, which was obtained by benzylation of
14d.35
The structural determination and purity of furanones 8
and 18 were achieved by IR, 1H and 13C NMR. All com-
pounds were obtained as racemates.

V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
4555
Br
i
ii
OEt
OH
OEt
R2
R2
R2
O
O
O
15a : R2 = C6H5
15b : R2 = 4-SO2CH3C6H5
15c : R2 = 4-ClC6H5
16a : R2 = C6H5
16b : R2 = 4-SO2CH3C6H5
16c : R2 = 4-ClC6H5
14b : R2 = 4-SO2CH3C6H5
14c : R2 = 4-ClC6H5
O
R2
EtO
Br
OH
O
OEt
iv
+
R2
O
R1
O
O
R1
14a : R1 = H
17a : R1 = H ; R2 = H
16
14b : R1 = SO2CH3
14c : R1 = Cl
17b : R1 = SO2CH3 ; R2 = H
17c : R1 = H ; R2 = 4-SO2CH3Ph
17d : R1 = SO2CH3 ; R2 = Ph
17e : R1 = Cl ; R2 = 4-SO2CH3Ph
17f : R1 = SO2CH3 ; R2 = 4-ClPh
17g : R1 = OBn ; R2 = 4-SO2CH3Ph
14d : R1 = OH
14e : R1 = OBn
iii
R2
O
HO
v
O
R1
18a : R1 = H ; R2 = H
18b : R1 = SO2CH3 ; R2 = H
18c : R1 = H ; R2 = 4-SO2CH3Ph
18d : R1 = SO2CH3 ; R2 = Ph
18e : R1 = Cl ; R2 = 4-SO2CH3Ph
18f : R1 = SO2CH3 ; R2 = 4-ClPh
18g : R1 = OBn ; R2 = 4-SO2CH3Ph
18h : R1 = OH ; R2 = 4-SO2CH3Ph
vi
Scheme 3. Reagents and conditions: (i) DBU, CH3CH2I, DMF, 0 ꢁC; (ii) NBS, (PhCOO)2, CCl4, reflux; (iii) BnCl, K2CO3, KI, DMF, reflux;
(iv) NEt3, THF, reflux; (v) NaH, THF; (vi) Pd/C, H2, THF/EtOH.
3. Results and discussion
3.1. In vitro anti-oxidant activities
the importance of a phenol group for anti-oxidant activ-
ity. Finally, compared with conventional anti-oxidants,
compounds 8e and 18h showed activity, respectively,
equipotent or two times superior to vitamin
C
As anti-oxidant action is considered to be a complex
process, which may include preventing of both the for-
mation or scavenging of free radicals, it was of interest
to investigate the interaction of the synthesized com-
pounds with the stable free radical 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH). This test is representative of the
capacity of compounds to scavenge free radicals inde-
pendently from any enzymatic activity. With IC50 values
ranging from 9.2 to 1779 lM (Table 1), all derivatives
were found to interact with DPPH but the variant de-
gree of anti-oxidant effect shown by the products cannot
be directly related to their structure. The position of hy-
droxy substituent on furanone ring (8 vs 18) significantly
affect reducing activity on DPPH, the 4-position being
preferred. In series 8, it appeared that the 4-methylsulfo-
nylphenyl group was more favourable to activity when
attached to the phenyl nucleus in the 5-position of the
olide ring (8a,c,e vs 8b,d,g), which was not so evident
in the series 18 (18c,e vs 18d,f). Nevertheless, a 4-
hydroxyphenyl moiety as the Ar1 substituent furnished
in both series the most efficient compounds 8e
(IC50 = 20.1 lM) and 18h (IC50 = 9.2 lM), displaying
(IC50 = 21.0 lM) and vitamin E (IC50 = 22.1 lM), but
less important than (+)-catechin, which is a flavonoid,
a class of molecules well known to have radical-scaveng-
ing properties.36,37
The first product of the univalent reduction of oxygen is
the superoxide anion O2ÅÀ, which is generated in many
biological processes.38,39 As a result, compounds able
ÅÀ
to scavenge O2 could be protecting agents against cel-
lular injury during the reperfusion of ischemic tissues.
Therefore, the superoxide anion scavenging activity of
furanones 8 and 18 was explored using a non-enzymatic
biological generator of superoxide anion radical, for in-
stance, the phenazine methosulfate NADH system.40
This test was limited by the solubility of products in
aqueous solution. In the superoxide anion scavenging
test (Table 1), the presence of a hydroxyl group in the
4-position on the olide ring is favourable to activity
compared with the 3-position (18 vs 8). Compounds
bearing a 4-methylsulfonylphenyl group as Ar1 (8b,d,g)
were more active, or as active as, the analogues 8a,c,e
in contrast to the observations made in the previous test.

4556
V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
Table 1. Anti-oxidant activities of derivatives 8 and 18
Compd
Ar1
Ar2 or R2
Reducing activity on
DPPH IC50 (lM)
Superoxide anion
scavenging activity
IC50 (lM)
Lipid peroxidation
IC50 (lM) or % of
inhibition
8a
8b
C6H5
4-SO2CH3C6H4
4-ClC6H4
4-SO2CH3C6H4
C6H5
530 25
1570 169
383 27
780 127
589 78
22 5a
21 3a
NT
37 8a
91.9 2.3
123 29
NT
Ar2
O
Ar1
HO
8c
8d
4-SO2CH3C6H4
4-ClC6H4
160
122
8
7
4-SO2CH3C6H4
4-OHC6H4
4-SO2CH3C6H4
4-ClC6H4
1014 80
20.1 0.6
1779 161
700 40
8e
8g
4-SO2CH3C6H4
4-OHC6H4
4-NHSO2CH3C6H4
H
488 19
511 76
O
8
8h
18a
NT
C6H5
4-SO2CH3C6H4
C6H5
87
238
2
6
18 15b
1628 199
242 13
21 4a
30 5b
16 4a
R2
O
18b
18c
H
HO
Ar1
4-SO2CH3C6H4
C6H5
119 18
18d
18e
4-SO2CH3C6H4
4-ClC6H4
212
146
57
3
4
1
97
61 31
49
4
3
1a
4-SO2CH3C6H4
4-ClC6H4
4-SO2CH3C6H4
32 4b
22 5b
O
18f
18h
4-SO2CH3C6H4
4-OHC6H4
6
18
9.2 0.4
21.0 0.4
22.1 2.2
7.5 0.3
236 55
24.0 1.5c
NT
169 57
Pro-oxidant
NT
Ascorbic acid
Vitamin E
(+)Catechin
176 10
13.8
3
NT: not tested.
a % inhibition at 0.25 mg/mL.
b % inhibition at 0.5 mg/mL.
c % inhibition at 1 mg/mL.
This time the same analysis can be made with the series
18 (18d,f vs 18c,e). Except for compounds 18a and 18b,
all tested 4-hydroxyfuranones 18 showed a noteworthy
superoxide anion scavenging activity with IC50 values
ranging from 49 to 242 lM. The presence of a phenyl
nucleus in the 5-position on the olide appeared to be
essential for the efficiency of furanones 18 (18a,b vs
18c–h). In addition, compounds 18d,e,f displayed a
better activity than previously reported 3-hydroxyfura-
nones 319 and (+)-catechin with IC50 values of, respec-
tively, 97, 61 and 49 lM, and even better than
ascorbic acid with only 24% inhibition at a concentra-
tion of 1 mg/mL. Vitamin E was not tested due to its
insolubility in aqueous medium.
Considering their activity in the three tests used, only
derivatives 8e,g and 18f,h with multiple mechanisms of
protective action appeared useful to minimize tissue
injury in human disease. Indeed, they were able to
scavenge free radicals and/or to trap O2ÅÀ, preventing
its conversion into H2O2. Furthermore, they could con-
tribute to reducing the devastating effects of lipid perox-
idation, taking into account their ability to scavenge
peroxyl and/or hydroxyl radicals (except 18f).
3.2. In vivo anti-inflammatory activities
Reactive oxygen species are well known to be critical
mediators of inflammation and tissue damage.46–48
Therefore, anti-oxidants could be beneficial in the treat-
ment of some acute and chronic inflammatory diseases.
Furthermore, in addition to a well recognized inhibitory
effect on the cyclooxygenase and lipoxygenase activities,
NSAIDs have been reported to affect a variety of other
processes and above all to possess anti-oxidant proper-
ties.49–53 By the structural modifications of previously
described furanones19 consequent to the incorporation
of sulfonyl and methylsulfamido moieties present in
nimesulide and coxibs for instance (Chart 1), we aimed
at enhancing anti-inflammatory properties in com-
pounds 8 and/or 18, which were thus investigated in this
area.
In order to further investigate the radical-trapping
anti-oxidant behaviour of compounds 8 and 18, and
so their ability to prevent continued allylic hydrogen
abstraction from lipids, their effects were evaluated
on the Fe2+/ADP-ascorbate induced lipid peroxidation
of rat liver microsomes. The autoxidation of Fe2+
/
ADP causes a massive lipid peroxidation in liver
microsomes, which can be followed by the formation
of malondialdehyde. Only derivatives 8e,g and 18h
produced a noteworthy activity while other com-
pounds were almost ineffective or could not be tested
due to their poor solubility in the biological reaction
mixture (8c,h). In this test, as previously reported,41–43
vitamin C underwent an autoxidative destruction in
the presence of iron initiating lipid peroxidation. How-
ever, this ÔprooxidantÕ activity in vitro has obviously
no significance in vivo and ascorbic acid works as
an anti-oxidant under most circumstances.42,43 All
compounds are less effective than (+)-catechin, which
is an efficient chain-breaking anti-oxidant with a
good anti-oxidant activity in microsomal lipid
peroxidation.44,45
First, behavioural effects and intraperitoneal acute tox-
icities of compounds 8 and 18 were examined in mice.
Neither of the tested drugs produced significant behav-
ioural effects, even at doses up to 800 mg/kg ip and all
the animals were still alive after a one week observation
period.
With regard to the evaluation of furanones 8 and 18 as
anti-inflammatory compounds, they were first adminis-

V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
Table 2. Anti-inflammatory activity of derivatives 8 and 18
4557
Compound
TPA-induced ear edema test % inhibition of edema
at
Carrageenan-induced paw edema
test % inhibition of edema at
100 mg/kg ip
200 mg/kg ip
200 mg/kg ip
8a
8b
40.4 11.1a
39.2 6.2b
88.0 5.5b
45.1 6.1a
NT
61.7 3.5b
49.7 6.2a
NT
64.1 11.1b
NT
29.1 9.0a
NT
NT
38.3 8.0a
8c
8d
42.4 11.0a
57.0 6.1b
88.1 4.1b
81.0 8.4b
8e
8g
7.8 7.4 (NS)
40.2 9.1a
12.1 8.6 (NS)
65.7 7.3b
8h
18a
1.0 7.8 (NS)
33.9 8.0a
3.5 2.9 (NS)
61.2 9.1b
18b
18c
7.3 5.1 (NS)
11.1 8.1 (NS)
10.1 8.6 (NS)
12.2 7.5 (NS)
47.3 11.8a
20.3 5.2a
9.5 6.7 (NS)
13.4 9.8 (NS)
15.6 6.2 (NS)
16.2 8.4 (NS)
78.4 6.3b
18d
18e
NT
NT
50.8 1.5b
NT
18f
18h
46.4 8.3b
Rofecoxib
Ketorolac
Dexamethasone
12.4 9.5 (NS)
10.6 8.2 (NS)
93.3 2.0b,d
14.8 7.1 (NS)
14.6 6.4 (NS)
NT
52.3 6.1b,c
53.3 9.1b,c
76.5 5.4b,d
NT: not tested. NS: not significant.
a 0.001 < p < 0.05.
b p <0.001.
c Tested at 15 mg/kg ip.
d Tested at 1 mg/kg ip.
tered ip at doses of 100 and 200 mg/kg in the phorbol
12-myristate 13-acetate (TPA)-induced ear edema test
(Table 2). At 100 mg/kg ip, only compounds 8a,c,d,g
and 18f revealed a noteworthy activity and produced
more than 40% inhibitory effects. Anti-inflammatory ef-
fects were dose-dependent since 8a,c,d,g and 18a,f were
particularly effective in reducing TPA inflammation at
200 mg/kg with more than 60% activity. However, the
influence of substituents on anti-inflammatory proper-
ties did not clearly appear: it seemed that a hydroxy
group on aromatic ring Ar1 was not absolutely neces-
sary, as shown by the lower activity of 8e versus 8a
and 8c. Furthermore, the position of the methylsulfonyl-
phenyl moiety on the furan skeleton of 8 did not signif-
icantly affect activity in this test. Contrary to compound
8c showing a good anti-inflammatory efficiency with
88% of inhibition at 200 mg/kg ip in the TPA test, fura-
none 8h, bearing a 4-methylsulfamido group on Ar2, re-
vealed no activity, suggesting that this moiety is not
favourable to anti-inflammatory activity compared to
a methylsulfonyl group. Surprisingly, among furanones
18, the 4-methylsulfonyl group on Ar1 and 4-chloro sub-
stituent on Ar2 provided the most efficient derivative
18f, while the potency of the same substituted furanones
on Ar1 (18b,d) was dramatically reduced in comparison
with that of parent compound 18a. Clearly, 3-hydroxy-
furanones 8 were more active in diminishing TPA
inflammation than 4-hydroxyfuranones 18. Consistent
with the literature data,54 classical NSAIDs rofecoxib
and ketorolac failed to produce any significant effect
after ip administration, since the maximum reduction
of ear thickness was only 13.8% at 100 mg/kg when ste-
roid dexamethasone proved a 93.3% inhibition at 1 mg/
kg ip. Indeed, it has been well established by now that
TPA produces a long-lasting edema that is associated
with a marked influx of neutrophils along with the pre-
dominant formation of leukotriene LTB4.55,56 So it
seems quite natural that, in this model, cycloxygenase
inhibitors show lower anti-inflammatory activity than
dexamethasone, which is well known for inhibiting
phospholipase A2 and so leukotriene release.
The in vivo anti-inflammatory efficiency of furanones
8a,c,d,g and 18a,f was further completed using the func-
tional model of carrageenan-induced paw edema in the
rat, a test particularly efficient in detecting compounds
whose activity is the result of the inhibition of prosta-
glandin amplification.57 Except with the derivative 18a,
all tested drugs were indeed effective at 200 mg/kg ip.
Furanones 8c,g and 18f produced 50–65% of anti-
inflammatory activity and were equipotent to NSAID
administered at the low dose of 15 mg/kg ip.
So, we can see that some furanones (8a,c,d,g and 18f)
were effective in these two models and these results sug-
gest that their anti-inflammatory effects may be due, like
steroids, to the inhibition of both cycloxygenase and
5-lipoxygenase pathways.
4. Conclusion
Although it was difficult, conflicting even with these ser-
ies of furanones, to gather in the same structure anti-oxi-
dant and anti-inflammatory properties, appropriate
modifications of previous derivatives 3 led to active
products in both areas. However, regarding all the data
only furanones 8g and 18f showed significant and potent
biological effects in the different tests used, associated to
a large therapeutic index in view of their LD50 superior
to 800 mg/kg ip. The presence of two aromatic nuclei on
the olide ring confers a high lipophilicity to the mole-

4558
V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
cules, which may contribute to their effective access,
retention and interaction with biological membranes.
If we extrapolate these in vitro results to an in vivo sit-
uation, we can assume that 8g and 18f can interfere at
distinct levels in the radical chain reaction, thus exerting
a synergistic effect in mitigating tissue damage that oc-
curs during inflammatory disease. Thus 8g and 18f were
selected for further pharmacological and toxicological
tests since they have an attractive profile as anti-oxidant
and anti-inflammatory agents.
5.3. General procedure for preparation of phenylpyruvic
acids (6)
The general procedure was adapted from the literature
procedure.33 A suspension of azlactone 5 (7.54 mmol)
in 12 mL of 1.0 N sodium hydroxide solution was
heated at 90 ꢁC until homogeneous. The solution was
acidified to pH 1–2 with a hydrochloric acid solution
3 N and a precipitate was formed. Concentrated hydro-
chloric acid (2 mL) was added, followed by water
(15 mL) and the mixture was heated to reflux for 6 h.
The mixture was cooled in an ice cold bath, and the pre-
cipitate thus formed was isolated by filtration. Washing
with water and drying under reduced pressure produced
phenylpyruvic acids 6 of suitable purity for use in subse-
quent operations.
5. Experimental
5.1. General chemical methods
All starting materials used for synthesis were reagent-
grade from Acros (Noisy-le-Grand, France). Reagents
for biochemical tests were purchased from Sigma (Mon-
tluc¸on, France). Melting points were determined on a
Reichert apparatus (Isi, Paris, France) and were uncor-
rected. Column chromatography was carried out on
SDS silica gel 60 (70–200 mesh). The infrared (IR)
spectra were recorded with a FTIR-Nicolet Impact 410
spectrophotometer (Thermo Optek, Montigny le
Bretonneux, France). The proton and carbon nuclear
magnetic resonance (1H NMR and 13C NMR) spectra
5.3.1. 3-(4-Chlorophenyl)-2-oxo-propionic acid (6a). Yield
43%; mp 188 ꢁC (lit., 182 ꢁC);59 IR (KBr) m 3465, 3200–
1
2500, 1664 cmÀ1; H NMR (DMSO-d6) d 13.34 (br s,
1H, OH), 9.51 (br s, 1H, OH), 7.81 (d, 2H, HAr), 7.41
(d, 2H, HAr), 6.44 (s, 1H, CH); 13C NMR (DMSO-d6)
d 166.3, 142.6, 134.0, 131.5, 130.9, 120.4, 108.3.
5.3.2. 3-(4-Methylsulfonylphenyl)-2-oxo-propionic acid
(6b). Yield 50%; mp 249–250 ꢁC; IR (KBr) m 3445,
1
3200–2400, 1684, 1304, 1149 cmÀ1; H NMR (DMSO-
d6) d 13.60 (br s, 1H, OH), 9.96 (br s, 1H, OH), 8.02
(d, 2H, HAr), 7.91 (d, 2H, HAr), 6.51 (s, 1H, CH), 3.23
(s, 3H, CH3); 13C NMR (DMSO-d6) d 165.9, 144.6,
140.3, 138.4, 129.5, 127.0, 107.4, 43.6.
were recorded on a Bruker AC-400 (400 MHz) spec-
¨
trometer (Bruker, Wissembourg, France). Chemical
¨
shifts are reported in parts per million (d ppm) relative
to tetramethylsilane used as an internal standard. The
abbreviations used for signal patterns are: br s, broad
singlet; s, singlet; d, doublet; m, multiplet. Mass spectral
analyses were performed on a Hewlett-Packard 5985B
or 5989A instrument. The ultra violet (UV) measure-
ments were carried out with a DU-70 spectrometer
(Beckman Instruments, Fullerton, USA).
5.4. General procedure for preparation of 4,5-diaryl-3-
hydroxy-2(5H)-furanones (8)
DBU (0.80 mL, 5.35 mmol) was added under stirring to a
cold (0 ꢁC) solution of a mixture of methyl 2-oxo-3-phe-
nylpropionate 7 (5.27 mmol) and appropriate benzalde-
hyde (5.27 mmol) in dry DMF (24 mL). The mixture
was stirred for 3–5 h at 0 ꢁC and then poured into a mix-
ture of ethyl acetate (10 mL) and HCl 1 M (50 mL). The
organic layer was separated and the aqueous layer was ex-
tracted with AcOEt. The organic layers were combined,
washed with water, dried over Na2SO4 and evaporated
in vacuo. The oily residue was then purified either by col-
umn chromatography on silica gel or by precipitation in
water, filtration and crystallization from ethyl ether.
5.2. General procedure for preparation of 4-benzylidene-
2-methyl-5-oxazolones (5)
The general procedure of the oxazolone preparation was
adapted from the literature procedure.33 A solution of
the appropriate benzaldehyde (8.14 mmol) was dis-
solved in 8 mL of acetic anhydride. N-Acetylglycine
(0.95 g, 8.14 mmol) was added, followed by sodium ace-
tate (0.67 g, 8.14 mmol), and the mixture was heated to
100 ꢁC in an oil bath for 4–5 h. The reaction mixture
was allowed to slowly cool overnight. Meanwhile, the
azlactone precipitated as a yellow/orange solid, which
was isolated by filtration. Washing with cold diethyl
ether afforded quantitative azlactone of suitable purity
for use in subsequent syntheses.
5.4.1. 3-Hydroxy-5-(4-methylsulfonylphenyl)-4-phenyl-
2(5H)-furanone (8a). This was prepared from methyl 2-
oxo-3-phenylpropionate 7c and 4-methylsulfonylbenzal-
dehyde 4b. Precipitation in water and crystallization
from ethyl ether led to compound 8a in 73% yield; mp
1
215 ꢁC; IR (KBr) m 3314, 1762, 1292, 1143 cmÀ1; H
NMR (DMSO-d6) d 11.27 (br s, 1H, OH), 7.94 (d, 2H,
HAr), 7.72 (d, 2H, HAr), 7.65 (d, 2H, HAr), 7.38–7.26
(m, 3H, HAr), 6.75 (s, 1H, H-5), 3.21 (s, 3H, SO2CH3);
13C NMR (DMSO-d6) d 168.9, 142.2, 141.6, 139.1,
130.2, 128.9, 128.7, 127.9, 127.8, 127.4, 78.6, 43.4; EI-
MS m/z 330 [M]+.
5.2.1.
4-(4-Chloro)benzylidene-2-methyl-5-oxazolone
(5a).58 Yield 98%.
5.2.2. 4-(4-Methylsulfonyl)benzylidene-2-methyl-5-oxazo-
lone (5b). Yield 95%; IR (KBr) m 1805, 1302, 1146 cmÀ1
;
1H NMR (DMSO-d6) d 8.27 (d, 2H, HAr), 8.01 (d, 2H,
HAr), 7.14 (s, 1H, CH), 3.09 (s, 3H, SO2CH3), 2.46 (s,
3H, CH3).
5.4.2. 3-Hydroxy-4-(4-methylsulfonylphenyl)-5-phenyl-
2(5H)-furanone (8b). This was prepared from methyl

V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
4559
2-oxo-3-(4-methylsulfonylphenyl)propionate 7b and
benzaldehyde. Purification by column chromatography
on silica gel, eluting with EtOAc/petroleum benzine (5/
5), produced compound 8b in 56% yield; mp 192–
SO2CH3); 13C NMR (DMSO-d6) d 168.7, 159.1, 141.1,
139.6, 136.8, 135.4, 129.4, 128.4, 128.0, 127.9, 127.8,
127.1, 125.3, 115.2, 79.4, 68.3, 43.3.
193 ꢁC; IR (KBr) m 3278, 1740, 1311, 1149 cmÀ1
;
1H
5.4.7. 3-Hydroxy-5-(4-hydroxyphenyl)-4-(4-methylsulfo-
nylphenyl)-2(5H)-furanone (8g). Pd/C (10%, 0.30 g) was
added to 5-(4-benzyloxyphenyl)-3-hydroxy-4-(4-meth-
ylsulfonylphenyl)-2(5H)-furanone 8f (1.37 g, 3.14 mmol)
dissolved in THF/EtOH (10/30 mL). The reaction mix-
ture was then submitted to a hydrogen atmosphere for
7 h. After filtration of the catalyst and evaporation of
the solvent, crystallization from ethyl ether produced
0.96 g (88%) of 8g; mp 230 ꢁC; IR (KBr) m 3271, 1734,
NMR (DMSO-d6) d 11.77 (br s, 1H, OH), 7.93 (d, 2H,
HAr), 7.87 (d, 2H, HAr), 7.48–7.39 (m, 5H, HAr), 6.68
(s, 1H, H-5), 3.21 (s, 3H, SO2CH3); 13C NMR
(DMSO-d6) d 168.6, 141.1, 139.7, 136.0, 135.3, 130.2,
129.1, 127.9, 127.8, 127.1, 125.5, 79.6, 43.3; EI-MS m/z
330 [M]+.
5.4.3. 4-(4-Chlorophenyl)-3-hydroxy-5-(4-methylsulfonyl-
phenyl)-2(5H)-furanone (8c). This was prepared from
methyl 2-oxo-3-(4-chlorophenyl)propionate 7a and 4-
methylsulfonylbenzaldehyde 4b. Precipitation in water
and crystallization from ethyl ether led to compound
8c in 66% yield; mp 260 ꢁC; IR (KBr) m 3306, 1743,
1
1312, 1148 cmÀ1; H NMR (DMSO-d6) d 11.64 (br s,
1H, OH), 9.71 (br s, 1H, OH), 7.90 (d, 2H, HAr), 7.82
(d, 2H, HAr), 7.22 (d, 2H, HAr), 6.76 (d, 2H, HAr),
6.52 (s, 1H, H-5), 3.18 (s, 3H, SO2CH3); 13C NMR
(DMSO-d6) d 168.7, 158.4, 141.1, 139.6, 135.5, 129.5,
127.8, 127.1, 126.0, 125.3, 115.8, 79.8, 43.3; EI-MS m/z
346 [M]+.
1
1312, 1150 cmÀ1; H NMR (DMSO-d6) d 11.49 (s, 1H,
OH), 7.97 (d, 2H, HAr), 7.74 (d, 2H, HAr), 7.67 (d,
2H, HAr), 7.47 (d, 2H, HAr), 6.77 (s, 1H, H-5), 3.25 (s,
3H, SO2CH3); 13C NMR (DMSO-d6) d 168.6, 141.8,
141.6, 139.5, 133.0, 129.0, 128.9, 128.7, 127.7, 126.4,
78.3, 43.3; EI-MS m/z 364 [M]+.
5.4.8. N-(4-Formylphenyl)methylsulfonamide (11). Meth-
anesulfonyl chloride (2 mL, 25.8 mmol) was added to a
0 ꢁC solution of 10 (3 g, 24.8 mmol) in pyridine
(30 mL) and the reaction solution was allowed to remain
at this temperature under stirring for 3 h. Most of the
pyridine was evaporated under reduced pressure and
the concentrated solution was diluted in ethyl acetate.
Dilute HCl (1 N) was added, and the organic layer
was separated, washed with water, then brine, dried over
Na2SO4 and evaporated in vacuo. Crystallization from
diethyl ether produced 2.68 g (54%) of the title com-
5.4.4. 5-(4-Chlorophenyl)-3-hydroxy-4-(4-methylsulfonyl-
phenyl)-2(5H)-furanone (8d). This was prepared from
methyl 2-oxo-3-(4-methylsulfonylphenyl)propionate 7b
and 4-chlorobenzaldehyde 4a. Purification by column
chromatography on silica gel, eluting with EtOAc/petro-
leum benzine (5/5), gave compound 8d in 78% yield; mp
199 ꢁC; IR (KBr) m 3466, 1757, 1308, 1151 cmÀ1
;
1H
NMR (DMSO-d6) d 11.83 (br s, 1H, OH), 7.93 (d, 2H,
HAr), 7.86 (d, 2H, HAr), 7.52–7.47 (m, 4H, HAr), 6.71
(s, 1H, H-5), 3.22 (s, 3H, SO2CH3); 13C NMR
(DMSO-d6) d 168.5, 141.3, 139.7, 135.1, 135.0, 134.1,
129.9, 129.1, 127.7, 127.2, 125.2, 78.7, 43.3; EI-MS m/z
364 [M]+.
pound; IR (KBr) m 3242, 1674, 1328, 1145 cmÀ1 1H
;
NMR (DMSO-d6) d 10.53 (s, 1H, NH), 9.92 (s, 1H,
CHO), 7.91 (d, 2H, HAr), 7.39 (d, 2H, HAr), 3.18 (s,
3H, SO2CH3).
5.4.9. N-Benzyl-N-(4-formylphenyl)-methylsulfonamide
(12). N-(4-Formylphenyl)methylsulfonamide 11 (2 g,
10 mmol) was dissolved in DMF (20 mL), triethylamine
(1.2 mL, 12.1 mmol) was added and the mixture was
stirred at room temperature for 30 min. Benzyl chloride
(1.21 mL, 10.5 mmol) was then added dropwise and the
reaction solution heated for 15 h. The mixture was di-
luted with 40 mL of 1 N sodium hydroxide solution,
and extracted with ethyl acetate. The organic layers were
combined, dried over Na2SO4 and concentrated in va-
cuo. The residue was chromatographed on silica gel.
Elution with AcOEt/cyclohexane (5/5) provided com-
pound 12 in 41% yield; mp 81–82 ꢁC; IR (KBr) m 1703,
5.4.5. 3-Hydroxy-4-(4-hydroxyphenyl)-5-(4-methylsulfo-
nylphenyl)-2(5H)-furanone (8e). This was prepared from
methyl 2-oxo-3-(4-hydroxyphenyl)propionate 7d and 4-
methylsulfonylbenzaldehyde 4b. Purification by column
chromatography, eluting with EtOAc/petroleum ben-
zine (5/5), gave compound 8e in 47% yield; mp 241–
242 ꢁC; IR (KBr) m 3390, 3340, 1712, 1314, 1155 cmÀ1
;
1H NMR (DMSO-d6) d 10.81 (s, 1H, OH), 9.87 (s,
1H, OH), 7.98 (d, 2H, HAr), 7.71 (d, 2H, HAr), 7.52
(d, 2H, HAr), 6.78 (d, 2H, HAr), 6.67 (s, 1H, H-5), 3.25
(s, 3H, SO2CH3); 13C NMR (DMSO-d6) d 169.1,
157.9, 142.5, 141.4, 136.7, 129.1, 129.0, 128.8, 127.7,
121.1, 115.5, 78.4, 43.3; EI-MS m/z 346 [M]+.
1
1338, 1149 cmÀ1; H NMR (DMSO-d6) d 9.96 (s, 1H,
CHO), 7.90 (d, 2H, HAr), 7.65 (d, 2H, HAr), 7.35–7.20
(m, 5H, HAr), 5.03 (s, 2H, CH2), 3.22 (s, 3H, SO2CH3);
13C NMR (DMSO-d6) d 192.1, 144.6, 136.3, 134.2,
130.1, 128.4, 127.8, 127.7, 127.4, 52.7, 37.9.
5.4.6. 5-(4-Benzyloxyphenyl)-3-hydroxy-4-(4-methylsul-
fonylphenyl)-2(5H)-furanone (8f). This was prepared
from methyl 2-oxo-3-(4-methylsulfonylphenyl)propio-
nate 7b and 4-benzyloxybenzaldehyde.60 Purification
by column chromatography, eluting with a gradient of
EtOAc/cyclohexane, gave compound 8f in 62% yield;
5.4.10. 5-[4-(N-Benzyl-methylsulfonamido)phenyl]-4-(4-
chlorophenyl)-3-hydroxy-2(5H)-furanone (13). This was
prepared from methyl 3-(4-chlorophenyl)-2-oxo-propio-
nate 7a and N-benzyl-N-(4-formylphenyl)-methylsulf-
onamide 12. Crystallization from ethyl ether gave
compound 13 in 69% yield; mp 187 ꢁC; IR (KBr) m
mp 154 ꢁC; IR (KBr) m 3181, 1760, 1296, 1142 cmÀ1
;
1H NMR (DMSO-d6) d 11.69 (br s, 1H, OH), 7.86 (m,
4H, HAr), 7.40–7.37 (m, 7H, HAr), 7.01 (d, 2H, HAr),
6.59 (s, 1H, H-5), 5.06 (s, 2H, PhCH2O), 3.19 (s, 3H,
1
1757, 1633, 1351, 1156 cmÀ1; H NMR (DMSO-d6) d

4560
V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
11.40 (br s, 1H, OH), 7.60–7.23 (m, 13H, HAr), 6.57 (s,
1H, H-5), 4.88 (s, 2H, CH2), 3.11 (s, 3H, SO2CH3);
13C NMR (DMSO-d6) d 168.7, 140.2, 139.4, 136.5,
135.2, 132.8, 129.2, 128.9, 128.7, 128.6, 128.3, 127.9,
127.3, 126.3, 78.8, 53.4, 37.4.
succinimide. The succinimide was washed with CCl4 and
these extracts were combined with the filtrate before
being evaporated in vacuo. The concentrate was then
purified by means of a silica gel chromatography to give
the desired compound.
5.4.11. 4-(4-Chlorophenyl)-3-hydroxy-5-[4-(methylsulfo-
namido)phenyl]-2(5H)-furanone (8h). Pd/C (10%, 0.20 g)
was added to a solution of 5-[4-(N-benzyl-methylsulfo-
namido)phenyl]-4-(4-chlorophenyl)-3-hydroxy-2(5H)-
furanone 13 (0.90 g, 1.91 mmol) in THF/EtOH (5/
10 mL). The reaction mixture was then submitted to a
hydrogen atmosphere for 12 h. After filtration of the
catalyst and evaporation of the solvent, the concentrate
was then purified by column chromatography on silica
gel with EtOAc/petroleum benzine (4/6) as developing
solvent. Crystallization from diisopropyl ether gave
compound 8h in 55% yield; mp 200 ꢁC; IR (KBr) m
5.6.1. Ethyl a-bromo-(4-methylsulfonylphenyl)acetate
(16b). Elution with EtOAc/cyclohexane (5/5) gave com-
pound 16b as an oil in 63% yield; IR (NaCl) m 1736,
1309, 1150 cmÀ1 1H NMR (CDCl3) d 7.93 (d, 2H,
;
HAr), 7.76 (d, 2H, HAr), 5.37 (s, 1H, CH), 4.24 (q, 2H,
CH2CH3), 3.05 (s, 3H, SO2CH3), 1.28 (t, 3H, CH2CH3);
13C NMR (CDCl3) d 167.6, 141.8, 141.1, 129.8, 127.9,
63.0, 44.9, 44.5, 13.9.
5.7. General procedure of synthesis of compounds (17)
Triethylamine (1 equiv) was added to a solution of the
appropriate phenylacetic acid 14 in dry THF and stir-
ring was maintained for 15 min. The appropriate ethyl
bromoacetate 16 was added and the solution refluxed
for several hours. After cooling, water was added to dis-
solve the precipitate and the mixture was extracted with
ethyl acetate. The organic layers were combined, succes-
sively washed with HCl 3 M twice, water, a saturated
solution of NaHCO3 (two times) and brine. Then the
solution was dried over Na2SO4 and concentrated under
reduced pressure. The residue was then purified to give
the desired compound 17.
1
3303, 1742, 1320, 1151 cmÀ1; H NMR (DMSO-d6) d
11.08 (br s, 1H, OH), 9.94 (br s, 1H, NH), 7.64 (d,
2H, HAr), 7.40–7.27 (m, 4H, HAr), 7.21 (d, 2H, HAr),
6.56 (s, 1H, H-5), 3.03 (s, 3H, CH3); 13C NMR
(DMSO-d6) d 169.1, 139.3, 138.8, 131.5, 130.5, 129.0,
128.5, 127.7, 127.4, 119.4, 79.3, 39.6; EI-MS m/z 379
[M]+.
5.5. General procedure for synthesis of compounds (15b,c)
DBU (3.5 mL; 23.4 mmol) and ethyl iodide (3.8 mL;
47.5 mmol) were added to a solution of the appropriate
phenylacetic acid (23.3 mmol) in dry DMF (80 mL) un-
der stirring at 0 ꢁC, and the mixture was stirred for 3 h
at the same temperature, then poured into dilute HCl
3 N. The product was extracted with ethylacetate and
the organic extract was washed successively with water,
a saturated aqueous Na2CO3 solution, brine and dried
over Na2SO4. Concentration under reduced pressure
gave ester 15 of suitable purity for use in subsequent
synthetic operations.
5.7.1. Ethoxycarbonylmethyl phenylacetate (17a). This
was prepared from phenylacetic acid 14a (3 g,
22.0 mmol)
and
ethylbromoacetate
(2.44 mL,
21.9 mmol) in dry THF (30 mL). The oily residue was
then purified by column chromatography on silica gel,
eluting with EtOAc/cyclohexane (2/8) and compound
17a was obtained as an oil in 83% yield; IR (KBr) m
1747 cmÀ1 1H NMR (CDCl3) d 7.33–7.29 (m, 5H,
;
HAr), 4.60 (s, 2H, OCH2CO), 4.18 (q, 2H, CH3CH2),
3.73 (s, 2H, PhCH2CO), 1.23 (t, 3H, CH3CH2); 13C
NMR (CDCl3) d 171.4, 168.1, 133.9, 129.8, 129.1,
127.7, 61.9, 61.5, 41.2, 14.5.
5.5.1. Ethyl 4-methylsulfonylphenylacetate (15b). Yield
81%; mp 80 ꢁC (lit.,61 79–80 ꢁC); IR (KBr) m 1727,
1297, 1149 cmÀ1 1H NMR (CDCl3) d 7.87 (d, 2H,
;
HAr), 7.48 (d, 2H, HAr), 4.15 (q, 2H, CH2CH3), 3.70
(s, 2H, CH2), 3.03 (s, 3H, SO2CH3), 1.24 (t, 3H,
CH2CH3); 13C NMR (CDCl3) d 170.4, 140.4, 139.3,
130.4, 127.6, 61.3, 44.5, 41.1, 14.1.
5.7.2. Ethoxycarbonylmethyl 4-methylsulfonylphenylace-
tate (17b). This was prepared from 4-methylsulfonyl-
phenylacetic acid 14b (2.5 g, 11.7 mmol) and
ethylbromoacetate (1.29 mL, 11.6 mmol) in dry THF
(25 mL). After evaporation, compound 17b was ob-
tained as a white powder in 75% yield; mp 71 ꢁC; IR
(KBr) m 1757, 1745, 1288, 1149 cmÀ1; 1H NMR (CDCl3)
d 7.93 (d, 2H, HAr), 7.61 (d, 2H, HAr), 4.75 (s, 2H,
OCH2CO), 4.16 (q, 2H, CH2CH3), 4.00 (s, 2H,
PhCH2CO), 3.25 (s, 3H, SO2CH3), 1.21 (t, 3H,
CH2CH3); 13C NMR (CDCl3) d 170.2, 167.6, 140.0,
139.6, 130.5, 127.1, 61.2, 61.0, 43.6, 39.5, 14.0.
5.5.2. Ethyl 4-chlorophenylacetate (15c)62. Yield 99%.
5.6. General procedure for synthesis of a-bromo
4-substituted-phenylacetate (16)
The synthesis of ethyl a-bromophenylacetate 16a and its
purification were performed as described in the litera-
ture63 and the procedure used to obtain compounds
16b and 16c64 was derived from it, therefore we only in-
clude a brief description of the synthesis. The general
procedure is as follows: a solution of the appropriate
ethyl phenylacetate 15, N-bromosuccinimide (1 equiv)
and benzoyl peroxide (catalytic amount) in CCl4 was
heated under reflux for several hours. The reaction solu-
tion was cooled to room temperature and filtered free of
5.7.3. Ethoxycarbonyl-a-(4-methylsulfonylphenyl)methyl-
phenylacetate (17c). This was prepared from phenylace-
tic acid 14a (1.5 g, 11.0 mmol) and ethyl a-bromo-(4-
methylsulfonylphenyl)acetate 16b (3 g, 9.34 mmol) in
dry THF (15 mL). Purification by column chromatog-
raphy, eluting with EtOAc/petroleum benzine (3/7), pro-
duced compound 17c as an oil in 63% yield; IR (KBr) m

V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
4561
1744, 1313, 1151 cmÀ1; 1H NMR (CDCl3) d 7.94 (d, 2H,
HAr), 7.64 (d, 2H, HAr), 7.35–7.26 (m, 5H, HAr), 6.02 (s,
1H, CH), 4.14 (m, 2H, CH2CH3), 3.80 (s, 2H,
PhCH2CO), 3.04 (s, 3H, SO2CH3), 1.18 (t, 3H,
CH2CH3); 13C NMR (CDCl3) d 170.5, 167.6, 141.1,
139.6, 133.0, 129.3, 128.6, 128.2, 127.8, 127.3, 73.8,
62.2, 44.3, 40.8, 13.9.
(d, 2H, HAr), 6.00 (s, 1H, CH), 5.05 (s, 2H, PhCH2O),
4.11 (q, 2H, CH2CH3), 3.74 (s, 2H, PhCH2CO), 3.03
(s, 3H, SO2CH3), 1.18 (t, 3H, CH2CH3); 13C NMR
(CDCl3) d 171.0, 167.9, 158.4, 141.5, 140.0, 137.2,
130.7, 128.8, 128.5, 128.2, 128.1, 127.7, 125.6, 115.3,
74.1, 70.2, 62.4, 44.6, 40.2, 14.2.
5.8. General procedure for synthesis of 5-substituted
3-aryl-4-hydroxy-2(5H)-furanones (18)
5.7.4. Ethoxycarbonyl-a-phenylmethyl 4-methylsulfonyl-
phenylacetate (17d). This was prepared from 4-meth-
ylsulfonylphenylacetic acid 14b (1.5 g, 7.00 mmol) and
ethyl a-bromophenylacetate 16a (1.7 g, 6.99 mmol) in
dry THF (15 mL). Purification by column chromatog-
raphy, eluting with EtOAc/cyclohexane (3/7), gave com-
pound 17d in 28% yield; mp 101–102 ꢁC; IR (KBr) m
A dropwise solution of compound 17 in dry THF was
added to a suspension of NaH (60% dispersion in min-
eral oil, 1.1 equiv) in dry THF in an ice cold bath and
the stirring was maintained at room temperature for sev-
eral hours. Water was added and the solution was ex-
tracted twice with ethyl ether. The aqueous phase was
cooled to 0 ꢁC and then acidified with hydrochloric acid
(3 M) to give a solid precipitate. Filtration and washing
with water or extraction furnished an analytical sample
of the furanones.
1
1750, 1740, 1289, 1149 cmÀ1; H NMR (DMSO-d6) d
7.96 (d, 2H, HAr), 7.66 (d, 2H, HAr), 7.55–7.45 (m,
5H, HAr), 6.03 (s, 1H, CH), 4.15 (m, 2H, CH2CH3),
4.05 (s, 2H, PhCH2CO), 3.26 (s, 3H, SO2CH3), 1.23 (t,
3H, CH2CH3); 13C NMR (DMSO-d6) d 170.2, 168.4,
139.9, 139.6, 133.6, 130.6, 129.4, 128.9, 127.7, 127.1,
74.6, 61.4, 43.6, 39.5, 13.9.
65
5.8.1. 4-Hydroxy-3-phenyl-2(5H)-furanone (18a). This
was prepared from ethoxycarbonylmethyl phenylacetate
17a. Crystallization from diisopropyl ether led to com-
pound 18a in 25% yield.
5.7.5. Ethoxycarbonyl-a-(4-methylsulfonylphenyl)methyl
4-chlorophenylacetate (17e). This was prepared from 4-
chlorophenylacetic acid 14c (0.80 g, 4.69 mmol) and
ethyl a-bromo-(4-methylsulfonylphenyl)acetate 16b
(1.5 g, 4.67 mmol) in dry THF (10 mL). Purification by
column chromatography, eluting with EtOAc/petroleum
benzine (3/7), led to compound 17e in 82% yield; mp 90–
5.8.2. 4-Hydroxy-3-(4-methylsulfonylphenyl)-2(5H)-fura-
none (18b). This was prepared from ethoxycarbonyl-
methyl 4-methylsulfonylphenylacetate 17b. Crystal-
lization from ethyl ether led to compound 18b in 35%
yield; mp 240 ꢁC; IR (KBr) m 1707, 1655, 1308,
93 ꢁC; IR (KBr) m 1758, 1746, 1314, 1154 cmÀ1
;
1H
1
NMR (CDCl3) d 7.96 (d, 2H, HAr), 7.66 (d, 2H, HAr),
7.23–7.33 (m, 4H, HAr), 6.01 (s, 1H, CH), 4.15 (q, 2H,
CH2CH3), 3.78 (s, 2H, PhCH2O), 3.06 (s, 3H, SO2CH3),
1.20 (t, 3H, CH2CH3); 13C NMR (CDCl3) d 170.4,
167.8, 141.5, 139.8, 133.6, 131.8, 131.0, 129.1, 128.5,
128.1, 74.2, 62.6, 44.6, 40.3, 14.2.
1153 cmÀ1; H NMR (DMSO-d6) d 8.24 (d, 2H, HAr),
7.95 (d, 2H, HAr), 4.86 (s, 2H, H-5), 3.23 (s, 3H,
SO2CH3); 13C NMR (DMSO-d6) d 178.1, 172.5, 137.7,
136.1, 126.9, 126.2, 95.8, 66.3, 43.7; EI-MS m/z 254 [M]+.
5.8.3. 4-Hydroxy-5-(4-methylsulfonylphenyl)-3-phenyl-
2(5H)-furanone (18c). This was prepared from ethoxy-
carbonyl-a-(4-methylsulfonylphenyl)methyl phenylace-
tate 17c. The white precipitate thus formed was filtered
and the aqueous phase was extracted with ethyl ether
(three times). The organic layers were combined, dried
over Na2SO4 and evaporated in vacuo. Crystallization
from ethyl ether led to compound 18c in 23% yield;
5.7.6. Ethoxycarbonyl-a-(4-chlorophenyl)methyl 4-meth-
ylsulfonylphenylacetate (17f). This was prepared from 4-
methylsulfonylphenylacetic acid 14b (2.35 g, 11.0 mmol)
and ethyl a-bromo-(4-chlorophenyl)acetate 16c (3.22 g,
11.6 mmol) in dry THF (25 mL). Purification by column
chromatography, eluting with EtOAc/cyclohexane (4/6),
gave compound 17f in 77% yield; mp 94 ꢁC; IR (KBr) m
mp 188 ꢁC; IR (KBr) m 1718, 1647, 1310, 1154 cmÀ1
;
1736, 1303, 1147 cmÀ1; H NMR (DMSO-d6) d 7.92 (d,
1H NMR (DMSO-d6) d 8.05 (d, 2H, HAr), 7.96 (m,
2H, HAr), 7.73 (d, 2H, HAr), 7.45 (t, 2H, HAr), 7.31 (t,
1H, HAr), 6.13 (s, 1H, H-5), 3.28 (s, 3H, SO2CH3); 13C
NMR (DMSO-d6) d 175.0, 171.9, 141.6, 140.8, 130.1,
129.0, 128.1, 127.6, 126.9, 126.8, 98.6, 78.3, 43.5; EI-
MS m/z 330 [M]+
1
2H, HAr), 7.60 (d, 2H, HAr), 7.52 (m, 4H, HAr), 6.05 (s,
1H, CH), 4.11 (q, 2H, CH2CH3), 4.03 (s, 2H,
PhCH2CO), 3.23 (s, 3H, SO2CH3), 1.09 (t, 3H,
CH2CH3); 13C NMR (DMSO-d6) d 170.0, 168.1, 139.8,
139.6, 134.1, 132.5, 130.5, 129.5, 128.9, 127.0, 73.7,
61.5, 43.5, 39.4, 13.8.
5.8.4. 4-Hydroxy-3-(4-methylsulfonylphenyl)-5-phenyl-
2(5H)-furanone (18d). This was prepared from ethoxy-
carbonyl-a-phenylmethyl 4-methylsulfonylphenylace-
tate 17d. Crystallization from ethyl ether led to
compound 18d in 55% yield; mp 226 ꢁC; IR (KBr) m
5.7.7. Ethoxycarbonyl-a-(4-methylsulfonylphenyl)methyl
4-benzyloxyphenylacetate (17g). This was prepared from
4-benzyloxyphenylacetic acid 14e (1.36 g, 5.60 mmol)
and ethyl a-bromo-(4-methylsulfonylphenyl)acetate
16b (1.80 g, 5.60 mmol) in dry THF (15 mL). Purifica-
tion by column chromatography, eluting with EtOAc/
petroleum benzine (3/7), provided compound 17g as an
1
1737, 1660, 1305, 1149 cmÀ1; H NMR (DMSO-d6) d
11.90 (br s, 1H, OH), 8.31 (d, 2H, HAr), 7.99 (d, 2H,
HAr), 7.48–7.44 (m, 5H, HAr), 5.99 (s, 1H, H-5), 3.24
(s, 3H, SO2CH3); 13C NMR (DMSO-d6) d 178.6,
171.8, 137.8, 136.2, 135.0, 129.4, 128.9, 128.1, 126.9,
126.6, 96.5, 78.6, 43.7; EI-MS m/z 330 [M]+.
oil in 88% yield; IR (KBr) m 1742, 1304, 1145 cmÀ1
;
1H NMR (CDCl3) d 7.94 (d, 2H, HAr), 7.65 (d, 2H,
HAr), 7.40–7.32 (m, 5H, HAr), 7.22 (d, 2H, HAr), 6.94

4562
V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
5.8.5. 3-(4-Chlorophenyl)-4-hydroxy-5-(4-methylsulfonyl-
phenyl)-2(5H)-furanone (18e). This was prepared from
a 5 · 10À3 M sample solution. The absorbance at
517 nm was measured every 5 min for 3 h. The absor-
bance of the control sample without test compound
was measured simultaneously. The difference in absor-
bance between the control and the test compound was
taken as the reducing activity.
ethoxycarbonyl-a-(4-methylsulfonylphenyl)methyl
chlorophenylacetate 17e. Crystallization from diisopro-
4-
pyl ether led to compound 18e in 51% yield; mp 122–
125 ꢁC; IR (KBr) m 1728, 1667, 1298, 1152 cmÀ1
;
1H
NMR (DMSO-d6) d 8.20–7.98 (m, 4H, HAr), 7.67 (d,
2H, HAr), 7.46 (d, 2H, HAr), 6.25 (s, 1H, H-5), 3.25 (s,
3H, SO2CH3); 13C NMR (DMSO-d6) d 176.0, 171.9,
141.6, 140.7, 131.0, 130.3, 129.3, 129.1, 128.3, 127.6,
97.2, 77.5, 43.5; EI-MS m/z 364 [M]+.
5.9.2. Superoxide anion scavenging assay. The technique
of Slater and Eakins40 based on the interactions of
NADH, phenazine methosulfate (PMS), molecular oxy-
gen and nitro blue tetrazolium (NBT) was used to eval-
uate the superoxide anion scavenging. The NADH/
PMS/O2/NBT system involves the intermediate forma-
tion of the superoxide anion radical (O2ÅÀ) from the
interaction of reduced PMS with O2; the superoxide an-
ion radical then reduces NBT to the highly coloured for-
mazan. The reaction can be followed by measuring the
absorbance of the formazan at 578 nm. The incubation
mixture contained disodium hydrogen phosphate buffer
(200 lL, 19 mM, pH = 7.4), PMS (200 lL, 10.8 lM),
NBT (200 lL, 172 lM) and NADH + H+ (200 lL,
360 lM). In order to assess their activity, the com-
pounds were dissolved in the buffer at different concen-
trations and 200 lL of buffer in the reaction mixture
were replaced by 200 lL of these solutions of com-
pounds. Meanwhile, blanks were realized by replacing
200 lL of NADH + H+ by 200 lL of buffer. Each test
was performed after a 5 min incubation.
5.8.6. 5-(4-Chlorophenyl)-4-hydroxy-3-(4-methylsulfonyl-
phenyl)-2(5H)-furanone (18f). This was prepared from
ethoxycarbonyl-a-(4-chlorophenyl)methyl 4-methylsul-
fonylphenylacetate 17f. Crystallization from diisopropyl
ether led to compound 18f in 51% yield; mp 262 ꢁC; IR
1
(KBr) m 3019, 1707, 1651, 1309, 1151 cmÀ1; H NMR
(DMSO-d6) d 11.86 (br s, 1H, OH), 8.28 (d, 2H, HAr),
7.96 (d, 2H, HAr), 7.52 (d, 2H, HAr), 7.44 (d, 2H,
HAr), 5.99 (s, 1H, H-5), 3.22 (s, 3H, SO2CH3); 13C
NMR (DMSO-d6) d 178.6, 171.8, 137.8, 136.1, 134.1,
130.0, 128.9, 126.9, 126.6, 96.4, 77.8, 43.7; EI-MS m/z
364 [M]+.
5.8.7. 3-(4-Benzyloxyphenyl)-4-hydroxy-5-(4-methylsul-
fonylphenyl)-2(5H)-furanone (18g). This was prepared
from ethoxycarbonyl-a-(4-methylsulfonylphenyl)methyl
4-benzyloxyphenylacetate 17g. Crystallization from
diisopropyl ether led to compound 18g in 66% yield; mp
5.9.3. Lipid peroxidation assay. Iron-dependent peroxi-
dation of male rat liver microsomes was tested as previ-
ously described.66 Liver microsomes were prepared
according to the technique of May and McCay.67 Lipid
peroxidation was initiated by the ADP-Fe2Å+/ascorbate
system,68 which produced hydroxyl radical ( OH). Incu-
bations of 200 lL microsomal fractions (1 mg protein/
mL) were carried out at 37 ꢁC with 200 lL potassium
phosphate buffer (0.1 M, pH = 7.4), and 200 lL of
ADP solution in buffer (2 mM), 200 lL of aqueous
FeSO4 solution (16 lM). Lipid peroxidation was initi-
ated by adding a 200 lL aqueous ascorbate solution
(0.5 mM) to the above incubation mixture. The extent
of polyunsaturated fatty acid peroxidation was mea-
sured spectrophotometrically by the rate of malondial-
dehyde formed after a 90 min microsomial incubation.
The reaction was quenched by the addition of 0.5 mL
of 20% trichloroacetic acid; after centrifugation, the
supernatant fraction was collected and then boiled with
0.5 mL of 0.67% thiobarbituric acid solution for 20 min.
After cooling, samples were diluted with 1 mL of dis-
tilled water and extinction was read at 535 nm. In order
to assess their activity, the compounds were dissolved
into disodium hydrogen phosphate buffer (19 mM,
pH = 7.4) at different concentrations and 200 lL of buf-
fer in the reaction mixture was replaced by 200 lL of the
solution of compounds. In parallel, blanks were realized
by replacing 200 lL of ascorbate by 200 lL of buffer.
169 ꢁC; IR (KBr) m 3032, 1714, 1637, 1302, 1148 cmÀ1
;
1H NMR (DMSO-d6) d 8.02 (d, 2H, HAr), 7.89 (d, 2H,
HAr), 7.68 (d, 2H, HAr), 7.46–7.34 (m, 5H, HAr), 7.09 (d,
2H, HAr), 6.08 (s, 1H, H-5), 5.15 (s, 2H, PhCH2O), 3.26
(s, 3H, SO2CH3); 13C NMR (DMSO-d6) d 173.5, 172.1,
157.1, 141.6, 140.9, 137.1, 129.0, 128.5, 128.2, 127.8,
127.7, 127.6, 122.6, 114.6, 98.5, 77.3, 69.2, 43.5.
5.8.8. 3-(4-Hydroxyphenyl)-4-hydroxy-5-(4-methylsulfo-
nylphenyl)-2(5H)-furanone (18h). Pd/C (10%, 80 mg)
was added to 3-(4-benzyloxyphenyl)-4-hydroxy-5-(4-
methylsulfonylphenyl)-2(5H)-furanone 18g (313 mg,
0.72 mmol) dissolved in THF/EtOH (5/10 mL) and sub-
mitted to a hydrogen atmosphere for 4 h. After filtration
of the catalyst and evaporation of the solvent, the resi-
due was purified by column chromatography on silica
gel, eluting with EtOAc/cyclohexane (8/2) and com-
pound 18h was obtained in 42% yield; IR (KBr) m
1
3326, 1719, 1669, 1302, 1144 cmÀ1; H NMR (DMSO-
d6) d 9.51 (br s, 1H, OH), 8.00 (d, 2H, HAr), 7.75 (d,
2H, HAr), 7.65 (d, 2H, HAr), 6.80 (d, 2H, HAr), 6.04 (s,
1H, H-5), 3.25 (s, 3H, SO2CH3); 13C NMR (DMSO-
d6) d 173.2, 172.6, 156.7, 141.9, 141.4, 129.3, 128.7,
128.0, 121.1, 115.4, 99.4, 77.6, 43.9; EI-MS m/z 346
[M]+.
5.9. Anti-oxidant assays
5.10. Pharmacological assays
5.9.1. Measurement of the reducing activity against the
stable radical DPPH. A solution of 4 mL of DPPH
(10À4 M, 2 mg) in EtOH (50 mL) was added to 1 mL
of a solution of the test compound in DMF, to prepare
All tests were performed in accordance with the recom-
mendations and policies of the International Associa-
tion for the Study of Pain (IASP).69

V. Weber et al. / Bioorg. Med. Chem. 13 (2005) 4552–4564
4563
5.10.1. Acute toxicity in mice. The compounds were
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