Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

Article abstract of DOI:10.1016/j.bmc.2006.07.047

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC₅₀ = 1.4 μM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC₅₀ = 0.28 μM; 15-LOX IC₅₀ = 0.32 μM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC₅₀ = 2.7 μM) and 5-LOX (IC₅₀ = 0.30 μM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.

Full text of DOI:10.1016/j.bmc.2006.07.047

Bioorganic & Medicinal Chemistry 14 (2006) 7898–7909
Synthesis and biological evaluation of a novel class of rofecoxib
analogues as dual inhibitors of cyclooxygenases (COXs)
and lipoxygenases (LOXs)
Qiao-Hong Chen, P. N. Praveen Rao and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2N8
Received 15 June 2006; revised 17 July 2006; accepted 26 July 2006
Available online 10 August 2006
Abstract—A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or
alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substitu-
ents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for
evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory
and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure–activity relationships iden-
tified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of
COX-2 (COX-2 IC50 = 1.4 lM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 lM; 15-LOX IC50 = 0.32 lM),
inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)fura-
none (18a) possesses dual COX-2 (IC50 = 2.7 lM) and 5-LOX (IC50 = 0.30 lM) inhibitor actions. Further in vivo studies
employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent
anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA),
but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of
a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of
the COX and LOX enzymes.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
selective COX-2 inhibitors (see structures in Fig. 1) such
as celecoxib (1) and rofecoxib (2) heralded the start of a
new era for the treatment of inflammatory conditions
such as rheumatoid arthritis (RA) and osteoarthritis
(OA).4 Clinical studies indicated that selective COX-2
inhibitors are effective anti-inflammatory agents with a
reduced risk of undesirable GI effects compared to clas-
sical NSAIDs.5 However, an increased risk of myocardi-
al infarction and cardiovascular thrombotic events
associated with the use of some selective COX-2 inhibi-
tors was subsequently observed. These adverse cardio-
vascular effects, which are attributed to a decreased
level of the vasodilatory prostacyclin (PGI2) and an in-
creased level of the potent platelet aggregator throm-
boxane A2 (TxA2), were primarily responsible for the
recent withdrawal of rofecoxib (Vioxxꢁ) and valdecoxib
(Bextraꢁ) from the market.6
Eicosanoids, the most frequently investigated family of
inflammation mediators, arise from the biotransforma-
tion of arachidonic acid (AA) via the cyclooxygenase
(COX) and lipoxygenase (LOX) pathways.1 In this re-
gard, prostanoids (prostaglandins, prostacyclin, and
thromboxanes) are produced via the COX pathway,
whereas leukotrienes (LTs) are formed by the LOX
pathway. Traditional non-steroidal anti-inflammatory
drugs (NSAIDs) exert their anti-inflammatory effect by
inhibition of the COX-1 and COX-2 isozymes.2 Adverse
side effects, in particular gastrointestinal (GI) irritation,
ulcerogenicity, and renal toxicity, often limit the chronic
use of NSAIDs.3 In the late 1990s, the discovery of
Keywords: Rofecoxib analogues; 3,4-Diaryl-2(5H)furanones; Anti-
inflammatory activity; Oximes; Dual cyclooxygenase and lipoxygenase
inhibitors.
Alternatively, LTs produced via the 5-LOX enzyme
catalyzed pathway are known to play a role in the
pathogenesis of inflammatory and allergic disorders.1
*
Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492
1217; e-mail: eknaus@pharmacy.ualberta.ca
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.047

Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
7899
SO Me
2
SO NH
2
2
N
O
N
F C
3
O
Me
Rofecoxib (2)
Celecoxib (1)
Cl
MeO
HOOC
N
N
N
O
N
Me
HO
Me
Cl
Licofelone (3)
4
Figure 1. Some representative selective COX-2 inhibitors (1–2) and dual COX/LOX inhibitors (3–4).
The related isozyme 15-LOX is linked to cardiovascu-
lar complications since it is known to participate in
2. Chemistry
oxidative modification of low-density lipoproteins
(LDL) leading to the development of atherosclerosis.7
In addition, the 5-LOX pathway is up-regulated during
COX blockade. Accordingly, increased levels of LTs
are potentially responsible for undesirable adverse
effects such as bronchial constriction. Therefore, dual
inhibitors of COXs and LOXs represent an attractive
safer clinical alternative to selective COX-2 inhibitors
in view of their potentially greater anti-inflammatory
efficacy due to a synergistic block of both metabolic
pathways in the AA cascade.8 In this regard, the dual
COX/LOX inhibitor licofelone (3), that has been eval-
uated in a Phase III clinical trial, is effective in the
treatment of OA where it showed reduced GI toxicity
compared to conventional NSAIDs.9 An observation
that arachidonoyl hydroxamate is a potent inhibitor
of 5-LOX,10 presumably because of chelation of the
iron in the enzyme catalytic site, provided a strategy
for the design of hydroxamic acid and N-hydroxy-
urea-based 5-LOX-selective inhibitors.11 This mecha-
nistic information was subsequently used for the
rational design of dual inhibitors such as 4 which is
comprised of a diarylpyrazole moiety present in selec-
tive COX-2 inhibitors such as celecoxib and an iron-
chelating hydroxamic acid moiety present in 5-LOX
inhibitors.12
The synthetic strategies used to prepare the target 3,
4-diaryl-2(5H)furanones are shown in Schemes 1–4. Fri-
edel–Crafts acetylation of ethyl phenylacetate (5) gave a
mixture of the meta- and para-isomers, from which the
para-isomer (6) crystallized from hexanes–acetone on
cooling at 0 ꢂC.15 Subsequent reduction and hydrolysis
of ethyl 4-acetylphenylacetate (6) afforded the 4-(1-hy-
droxyl)ethylphenylacetic acid (8) (Scheme 1). The phen-
ylacetic acid analogue (11) was prepared in high yield
using a dichlorobis(triphenylphosphine)palladium(0)/
CuI catalyzed Sonogashira cross-coupling reaction16 be-
tween 4-iodophenylacetic acid (10) and 3-butyn-2-ol in
the presence of triethylamine (see Scheme 2). The 4-iodo-
phenylacetic acid (10) precursor was prepared by diazo-
CH2COOEt
5
a
O
CH2COOEt
6
b
HO
CH2COOEt
There is currently a renewed interest in the coordination
chemistry of oximes that can bind a metal ion in
different modes.13 In some recent studies, it was shown
that N-hydroxycarbamates exhibit effective 5-LOX-
inhibitory activities.14 It was therefore of interest to de-
sign a new class of hybrid compounds for evaluation as
dual COX/LOX inhibitors, in which the selective COX-2
inhibitor rofecoxib was coupled to an oxime group or an
N-hydroxycarbamate moiety. Accordingly, we now
describe herein the synthesis and biological evaluation
of a group of hybrid 4-(4-methanesulfonylphenyl)-3-
phenyl-2(5H)furanone derivatives.
7
c
HO
CH2COOH
8
Scheme 1. Reagents and conditions: (a) AcCl, AlCl3, CS2, reflux
overnight; (b) NaBH4, MeOH, 25 ꢂC, 1 h; (c) K2CO3, MeOH, H2O,
reflux, 30 min.

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Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
tives (8 or 11) proceeded via a 2-step condensation–
H2N
CH2COOH
cyclization reaction that was performed as a one-pot
procedure.17 Thus, treatment of a mixture of the
bromoacetophone (12) and a phenylacetic acid deriva-
tive (8 or 11) in acetonitrile in the presence of triethyl-
amine at 25 ꢂC yielded the ester intermediate.
Subsequent cooling to 0 ꢂC, and addition of DBU,
induced the cyclization reaction to afford the respective
3,4-diaryl-2(5H)furanone (13 or 14). Oxidation of the
alcohols 13 and 14 afforded the target ketones (15
and 16) in 55–70% yield. Subsequent reaction of the
ketones (15 and 16) with hydroxylamine hydrochloride,
or an O-methyl- or O-benzyl derivative thereof, affor-
ded the respective target oxime products (17 and 18)
in 46–100% yield.
9
a
b
I
CH2COOH
10
OH
C
C
HO
11
O
Scheme 2. Reagents and conditions: (a) i—H2SO4, NaNO2, 0 ꢂC,
30 min; ii—KI, 0 ꢂC, 2.5 h; (b) 3-butyn-2-ol, Pd(PPh3)2Cl2, CuI, Et3N,
50 ꢂC, overnight.
The N-hydroxycarbamates 20a–d (R = i-Pr, i-Bu, Ph,
CH2Ph) were prepared employing the reaction sequence
shown in Scheme 4. Thus, reduction of the oxime 17a
using sodium cyanoborohydride gave the intermediate
hydroxylamine 19 which was treated with excess alkyl
chloroformate to afford the respective N,O-bis(alkoxy-
carbonyl)hydroxylamine. Subsequent hydrolysis of the
N,O-bis(alkoxycarbonyl)hydroxylamines in the presence
of ammonia gas furnished the respective target
compounds 20.
tization of commercially available 4-aminophenylacetic
acid (9) and subsequent reaction with potassium iodide.
The target compounds (15–18) were prepared using the
reaction sequence illustrated in Scheme 3. Reaction of
the bromoketone (12) with a phenylacetic acid deriva-
OH
MeO2S
COCH2Br
+
n
HO
8, n = 0
11, n = 1
12
O
a
OH
MeO2S
n
13, n = 0
14, n = 1
O
O
O
b
MeO2S
n
15, n = 0
16, n = 1
O
O
OR
N
c
17a, n = 0, R = H
17b, n = 0, R = Me
MeO2S
n
17c, n = 0, R = CH2Ph
18a, n = 1, R = H
18b, n = 1, R = Me
18c, n = 1, R = CH2Ph
O
O
Scheme 3. Reagents and conditions: (a) i—Et3N, MeCN, 25 ꢂC, 20 min; ii—DBU, 0 ꢂC, 1 h; (b) Jones reagent, 0 ꢂC, 1 h; (c) H2NORÆHCl, Na2CO3,
EtOH, reflux, 1 h.

Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
7901
OH
the structural requirements for 5-LOX inhibition are
more stringent than for 15-LOX inhibition. The C-3
4-(1-hydroxyimino)ethylphenyl compound 17a, an equi-
N
MeO2S
potent inhibitor of 5- and 15-LOX (5-LOX IC50
=
0.28 lM; 15-LOX IC50 = 0.32 lM), was about a 10-fold
more potent inhibitor of both 5- and 15-LOX compared
to the reference drugs caffeic acid (5-LOX IC50 = 3.0 lM)
and luteolin (15-LOX IC50 = 3.2 lM). Replacement of an
ethanone oxime moiety (17a) with an acetyl group (15) led
to a complete loss of 5- and 15-LOX inhibitory activity.
Compounds having a methoxyiminoethyl (17b), or
N-benzyloxymethyl (17c), moiety exhibited selective
15-LOX inhibition (15-LOX IC50 = 1.1–3.0 lM). In con-
trast, the N-hydroxyliminoalkynyl compound 18a exhib-
ited 5-LOX selectivity (5-LOX IC50 = 0.30 lM; 15-LOX
IC50 > 10 lM). Replacement of the hydroxyl substituent
of the oxime moiety present in 18a by a methoxy (18b), or
benzyloxy (18c), substituent abolished both 5- and 15-
LOX inhibitory activity (IC50values > 10 lM). The 4-(3-
oxobut-1-ynyl)phenyl compound (16) also showed potent
dual 5- and 15-LOX inhibition (5-LOX IC50 = 2.0 lM;
15-LOX IC50 = 3.4 lM). Similar in vitro 5- and
15-LOX isozyme inhibition structure–activity relation-
ships for the N-hydroxycarbamate derivatives of rofecox-
ib (20a–d) showed that the alkyl(aryl)oxy moiety present
in the N-alkyl(aryl)oxycarbonyl-N-hydroxylamino moie-
ty was a determinent of LOX inhibitory activity. In this
regard, the N-isopropyloxy compound (20a) did not
inhibit either 5- or 15-LOX at a concentration of
10 lM, whereas the isobutyloxy (20b) and phenoxy
(20c) compounds exhibited potent 15-LOX inhibitory
activity that is comparable to the reference drug luteolin
(15-LOX IC50 = 3.2 lM). In contrast, the benzyloxy
derivative (20d) exhibited selective 5-LOX inhibitory
activity comparable to the reference drug caffeic acid (5-
LOX IC50 = 3.0 lM). The in vitro COX-1/COX-2, and
5-LOX/15-LOX, isozyme inhibition data acquired for
the group of compounds 15–18, 20 showed that 3-[4-(1-
hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphe-
nyl)-2(5H)furanone (17a) exhibited an optimal combina-
tion of COX and LOX inhibition (COX-1 IC50 > 100 lM;
COX-2 IC50 = 1.4 lM; COX-2 SI > 71; 5-LOX
IC50 = 0.28 lM; 15-LOX IC50 = 0.32 lM). Although
17a is a 3-fold less potent inhibitor of COX-2 than the ref-
erence drug rofecoxib, it is a 10-fold more potent inhibitor
of both 5-LOX and 15-LOX compared to the reference
drugs caffeic acid (5-LOX IC50 = 3.0 lM) and luteolin
(15-LOX IC50 = 3.2 lM).
17a
O
O
a
b
NHOH
MeO2S
19
O
O
HO
COOR
N
MeO2S
20a, R = CH(Me)2
20b, R = CH2CH(Me)2
20c, R = Ph
O
O
20d, R = CH2Ph
Scheme 4. Reagents and conditions: (a) NaCNBH3, 12 N HCl,
MeOH, THF, 25 ꢂC, overnight; (b) i—ClCOOR (excess), Et3N,
CH2Cl2, 25 ꢂC, overnight; ii—NH3 (gas), THF, overnight.
3. Results and discussion
A group of rofecoxib derivatives possessing an acetyl
(15), 3-oxobut-1-ynyl (16), [hydroxy(or alkoxy)imino]al-
kyl (17a–c), [hydroxy(or alkoxy)imino]alkynyl (18a–c),
or an N-alkoxy(or N-phenoxy)-N-alkylcarbonyl-N-hy-
droxy-N-ethylamino (20a–d) substituent at the para-
position of the rofecoxib C-3 phenyl ring were
evaluated in vitro to determine their COX-1, COX-2,
5-LOX, and 15-LOX inhibitory activities. In vitro
COX-1 and COX-2 isozyme inhibition structure–activi-
ty studies (see data in Table 1) showed that the rofecoxib
analogues possessing an additional acetyl or hydroxyi-
minoethyl substituent with or without an acetylene spac-
er (15, 16, 17a, and 18a), are selective (COX-2 SI > 29–
85), but not particularly potent (IC50 = 1.2–2.7 lM
range), inhibitors of COX-2. Compounds having (meth-
oxyimino)alkyl moieties at the para-position of the C-3
phenyl ring of rofecoxib (17b and 18b) also showed
modest inhibitory potency against COX-2 (17b,
IC50 = 2.9 lM; 18b, IC50 = 11.1 lM) with moderate
COX-2 selective indexes (SI > 34) (17b) and >9 (18b).
Incorporation of a bulky (benzyloxyimino)alkyl moiety
(17c and18c) abolished both COX-1 and COX-2 inhibi-
tory activity (IC50 values > 100 lM). On the other hand,
rofecoxib analogues 20a–d possessing an N-hydroxycar-
bamate moiety at the para-position of the rofecoxib C-3
phenyl ring exhibited equipotent COX-1 inhibitory
activity (IC50’s = 1.0 lM) in conjunction with moderate
(20a, 20c–d) to inactive (20b) COX-2 inhibition (IC50
values from 1.1 to >100 lM range).
A molecular modeling study was performed where the
most selective COX-2 inhibitor 17a was docked in the
binding site of the COX-2 isozyme (Fig. 2). This molec-
ular modeling study reveals that 17a binds in the center
of the primary binding site such that the C-4 para-
SO2Me COX-2 pharmacophore is oriented in the vicin-
ity of amino acid residues lining the COX-2 secondary
(2ꢂ) pocket (His90, Gln192, Ala516, Ile517, Phe518,
and Val523). One of the O-atoms of the SO2Me group
undergoes a dual hydrogen bonding interaction with
the backbone amide hydrogens (NH) of both Ile517
˚
˚
(distance ꢀ 1.96 A) and Phe518 (distance ꢀ 2.42 A).
In vitro 5-LOX and 15-LOX isozyme inhibition struc-
ture–activity studies (see data in Table 1) suggested that
The distance between the second O-atom (of the SO2Me
˚
moiety) and the NH2 of Gln192 is about 4.42 A.The Me

7902
Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
Table 1. In vitro COX-1, COX-2, 5-LOX, and 15-LOX enzyme inhibition assay data for the rofecoxib analogues 15, 16, 17, 18, and 20
HO
O
COOR
OR
N
N
MeO2S
MeO2S
n
MeO2S
n
O
O
O
O
O
O
15, 16
17, 18
20
Selectivity indexb (SI)
IC50 (lM)
a
a
Compound
n
R
IC50 (lM)
COX-1
COX-2
5-LOX
15-LOX
15
16
0
—
—
>100
>100
>100
>100
>100
>100
>100
>100
1.2
3.5
>85
>29
>71
>33.8
—
>10
2.0
>10
3.4
0.32
1.1
3.1
>10
>10
>10
>10
3.2
3.4
>10
3.2
—
1
17a
17b
0
H
CH3
1.4
2.9
0.28
>10
>10
0.30
>10
>10
>10
>10
>10
3.1
0
17c
18a
0
CH2Ph
H
CH3
>100
2.7
1
>37
>9
18b
18c
1
11.1
>100
6.6
1
CH2Ph
—
20a
20b
—
—
—
—
CH(Me)2
CH2CH(Me)2
Ph
1.0
0.15
0.01
0.93
0.08
1.0
1.0
1.0
>100
1.1
12.0
20c
20d
CH2Ph
Luteolin
Caffeic acid
NDGAc
Celecoxib
Rofecoxib
—
3.0
>10
—
3.5
—
—
33.1
0.07
0.5
472
>200
>100
—
a Values are means of two determinations acquired using an ovine COX-1 and COX-2, potato 5-LOX, and soyabean 15-LOX, assay kits (Catalog
Nos. 560101, 60401, and 760700, Cayman Chemicals Inc., Ann Arbor, MI, USA), and the deviation from the mean is <10% of the mean value.
b In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
c NDGA, nordihydroguaiaretic acid.
group of the SO2Me moiety is within van der Waals con-
˚
oriented toward the entrance to the 15-LOX binding
site (Arg403). The Me group of the SO2Me moiety is
located within van der Waals contact range of the
amino acid residues Ile400, Ala404, and Leu408 (dis-
tance < 5 A). The interspatial distance between one of
the O-atoms of the SO2Me group and the NH2 of
˚
Arg403 is about 7.79 A. It is interesting to note that
tact range of Ala516 (distance ꢀ 3.37 A). The C-3
p-C(Me)@N–OH substituted phenyl ring is oriented to-
ward a hydrophobic pocket comprised of Phe381,
Leu384, Tyr385, Trp387, and Met522. It is interesting
to note that the N–OH [of the C(Me)@N–OH moiety]
participates in a hydrogen bonding interaction with
˚
˚
the C@O of Leu384 (distance ꢀ 1.84 A), whereas the
the C-3 phenyl ring possessing the C(Me)@N–OH
LOX pharmacophore is oriented toward the catalytic
center of 15-LOX (His361, His366, His541, and
His545). The OH substituent present in the
–C(Me)@N–OH moiety forms a hydrogen bond with
the peptide backbone (C@O) of His361 (dis-
Me substituent [of the C(Me)@N–OH moiety] is posi-
tioned within van der Waals contact range of Leu384
˚
and Met522 (distance <5 A) at the apex of the COX-2
binding site. It is noteworthy that the C@O of the
central furanone ring forms a hydrogen bond with the
˚
˚
OH of Ser530 (distance ꢀ 1.91 A), the acetylation
tance ꢀ 1.72 A), whereas the N-atom of the
site for aspirin. A recent study has shown the impor-
tance of Ser530 in the COX-2 inhibitory activity of
rofecoxib.18 In addition, the O-atom of the central fura-
C(Me)@N–OH] group forms a weak hydrogen bond
˚
with the imidazole NH of His366 (distance ꢀ 3.57 A).
In addition, the C-3 phenyl ring is involved in a p–p
stacking interaction with the imidazole ring of His361.
The central furanone ring is oriented toward the base
of the 15-LOX binding site where it is positioned in a
hydrophobic pocket comprised of Gln548, Leu549,
˚
none is separated by a distance of 5.77 A from the NH2
˚
of Arg120, and about 5.67 A from the OH of Tyr355,
near the entrance to the COX-2 binding site.
˚
A molecular modeling (docking) simulation was per-
formed to investigate the binding interaction of the dual
5- and 15-LOX selective inhibitor, 3-[4-(1-hydroxyimi-
no)ethylphenyl]-4-(4-methanesulfonyl-phenyl)-2(5H)-
furanone (17a), within the 15-LOX binding site (Fig. 3).
The C-4 p-MeSO2-phenyl COX-2 pharmacophore is
Val594, and Gly598 (distance < 5 A). Interestingly, the
C@O of central furanone ring is hydrogen bonded to
˚
the backbone NH of Leu549 (distance ꢀ 2.85 A).
The COX-2, and dual 5-LOX and 15-LOX, inhibitor
3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfo-

Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
7903
Figure 2. Docking of 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) (ball and stick) in the active site of
murine COX-2. Hydrogen atoms of the amino acid residues have been removed to improve clarity.
nylphenyl)-2(5H)furanone (17a), and the dual COX-2/5-
LOX inhibitor 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-
4. Conclusions
4-(4-methanesulfonylphenyl)-2(5H)furanone
(18a),
A new class of hybrid compounds was designed for eval-
uation as dual acting COX/LOX inhibitors where the
selective COX-2 inhibitor rofecoxib was attached to a
5-LOX inhibitory oxime or N-hydroxycarbamate
moiety. In vitro enzyme inhibition, and in vivo anti-in-
flammatory and analgesic, data acquired show that (i)
incorporation of a para-oxime moiety on the C-3 phenyl
ring of rofecoxib provides a suitable template for the de-
sign of dual acting inhibitors of COX and LOX; (ii) the
oxime compound 17a exhibits an optimal combination
of COX-2/5-LOX/15-LOX inhibitory activities; (iii) the
oxime compound 18a is an effective dual inhibitor of
COX-2 and 5-LOX; and (iv) dual inhibitors of COX-2
and 5-/15-LOX such as 17a and 18a possess oral anti-
inflammatory and analgesic activities that may be
clinically relevant.
based on their desirable in vitro COX and LOX isozyme
inhibition effects, were selected for further in vivo phar-
macological evaluation to determine their anti-inflam-
matory (AI) and analgesic activities (see data in Table
2). In a carrageenan-induced rat paw edema assay mod-
el, these two rofecoxib oxime analogues exhibited more
potent anti-inflammatory activities (25–33% inhibition
of inflammation for a 30 mg/kg oral dose), being superi-
or to the respective 15-LOX inhibitor nordihydroguia-
retic acid (NDGA, 15.1% inhibition for a 30 mg/kg
dose), and the 5-LOX inhibitor caffeic acid (8.2% inhibi-
tion of inflammation for a 30 mg/kg oral dose), reference
drugs. However, 17a and 18a were less potent anti-in-
flammatory agents than the selective COX-2 inhibitor
reference drug celecoxib (ED50 = 10.8 mg/kg).
In a rat 4% NaCl-induced abdominal constriction (anal-
gesic) assay, a 30 mg oral dose of the oximes 17a and
18a exhibited moderate analgesic activities (37–58%
range) comparable to the reference drugs caffeic acid,
NDGA, and celecoxib at 30 and 60 min post-drug
administration (see data in Table 2).
5. Experimental
Melting points were determined on a Thomas-Hoover
capillary apparatus and are uncorrected. Infrared (IR)
spectra were recorded on a Nicolet 550 Series II Magna

7904
Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
Figure 3. Docking of 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) (ball and stick) in the active site of
soyabean 15-LOX. Hydrogen atoms of the amino acid residues have been removed to improve clarity.
Table 2. In vivo anti-inflammatory and analgesic activities for 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone
(17a) and 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a)
OH
N
MeO2S
n
O
O
Compound
n
AI activitya
Analgesic activityb
% inhibition (3 h)
% inhibition (30 min)
% inhibition (60 min)
17a
18a
0
33.7 1.7
25.5 4.8
8.2 2.5
37.1 7.6
58.3 10.0
47.2 10.6
45.8 9.2
31.7 9.6c
49.0 13.1
52.2 8.2
58.3 12.8
62.5 4.8
62.0 7.3c
1
Caffeic acid
NDGA
Celecoxib
—
—
—
15.1 1.7
79.9 1.9c,d
a Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as means SEM (n = 4) values at 3 h following a
30 mg/kg oral dose of the test compound.
b Inhibitory activity in the rat 4% NaCl-induced abdominal constriction assay. The results are expressed as means SEM (n = 4–5) values following a
30 mg/kg oral dose of the test compound.
c Percent (%) reduction for a 50 mg/kg oral dose.
d ED50 = 10.8 mg/kg.
1
FT-IR spectrometer. H NMR spectra were measured
on a Bruker AM-300 spectrometer in CDCl3 or
CDCl3 + DMSO-d6with TMS as the internal standard,
where J (coupling constant) values are estimated in
Hertz. Spin multiples are given as s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), and br (broad).
Microanalyses were performed for C, H, N (MicroAna-
lytical Service Laboratory, Department of Chemistry,
University of Alberta) and were within 0.4% of theo-
retical values. Nominal mass, positive polarity, electro-

Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
7905
spray spectra were acquired using a Waters Micromass
5.3. 4-(1-Hydroxy)ethylphenylacetic acid (8)
ZQ mass spectrometer. Silica gel column chromatogra-
phy was performed using Merck silica gel 60 ASTM
(70–230 mesh). Celecoxib and rofecoxib were synthe-
sized according to the literature procedures.4a,19 Luteo-
lin, caffeic acid, and nordihydroguaiaretic acid
(NDGA) were purchased from Cayman Chemicals
Inc. Ann Arbor. Ethyl 4-(1-hydroxyethyl)phenylacetate
(7)20 and 4-(1-hydroxyethyl)phenylacetic acid (8)21 have
been reported previously. Bromoketone 12 was prepared
according to a literature method.22 Other reagents, pur-
chased from the Aldrich Chemical Company (Milwau-
kee, WI), were used without further purification. Male
Sprague–Dawley rats, used in the anti-inflammatory
and analgesic screens, were purchased from Animal
Health Services at the University of Alberta, and exper-
iments were carried out using protocols approved by the
Animal Welfare Committee, University of Alberta.
A solution of K2CO3 (704 mg, 5.1 mmol) in water
(10 mL) was added to a stirred solution of ethyl 4-(1-hy-
droxyl)ethylphenylacetate (7, 353 mg, 1.7 mmol) in
methanol (10 mL). The reaction was allowed to proceed
at reflux for 30 min, the reaction mixture was cooled to
25 ꢂC, and the solvent was removed in vacuo. Ethyl ace-
tate (30 mL) and water (20 mL) were added, and the
mixture was acidified to pH 3 using an aqueous solution
of HCl (5% w/v) prior to extraction with ethyl acetate
(3 · 30 mL). The organic extracts were combined,
washed with brine, and the organic fraction was dried
(Na2SO4). Removal of the solvent under vacuum fur-
nished 8 as a white solid (219 mg, 87%); mp 115–
116 ꢂC. IR (film): 3422 (OH), 2515–3106 (COOH),
1709 (COOH) cmꢁ1 1H NMR (CDCl3 + DMSO-d6;
.
5:1, v/v) d 1.41 (d, J = 6.0 Hz, 3H, CHCH3), 3.53 (s,
2H, CH2), 4.81 (q, J = 6.0 Hz, 1H, CHCH3), 7.21 (d,
J = 8.0 Hz, 2H, phenyl H-3, H-5), 7.29 (d, J = 8.0 Hz,
2H, phenyl H-2, H-6).
5.1. Ethyl 4-acetylphenylacetate (6)
Ethyl phenylacetate (6.5 g, 40 mmol), and then acetyl
chloride (3.8 ml, 53 mmol), was added slowly to a sus-
pension of anhydrous AlCl3 powder (12 g, 90 mmol) in
CS2 (25 mL) at 0 ꢂC with stirring. The temperature
was slowly raised and finally the reaction was allowed
to proceed at reflux overnight. The reaction mixture
was cooled to 25 ꢂC, poured into a mixture of ice and
water, and the subsequent mixture was extracted with
chloroform (3 · 50 mL). The combined extracts were
washed with water (2 · 50 mL) and dried (Na2SO4).
Removal of the solvent in vacuo gave a dark brown
oil which was purified by silica gel column chromatogra-
phy eluting with hexanes–acetone (5:1, v/v) to furnish a
mixture of the para- and meta-isomers. This mixture,
which partially solidified on standing in a refrigerator,
was recrystallized from hexanes–acetone to give the
para-isomer (2.64 g, 32%) as white needles; mp 58–
59 ꢂC (lit.15 64–65 ꢂC). IR (film): 1736 (COO), 1676
5.4. 4-Iodophenylacetic acid (10)
A solution of sodium nitrite (828 mg, 12 mmol) in water
(3 mL) was added slowly with stirring at 0 ꢂC to a solu-
tion of 4-aminophenylacetic acid (1.51 g, 10 mmol) in
water (15 mL) and sulfuric acid (2 mL), and the reaction
mixture was stirred for 30 min prior to the addition of a
cooled solution of KI (3.32 g, 20 mmol) in water
(12 mL). The reaction was allowed to proceed for an
additional 2.5 h at 0 ꢂC, the dark brown mixture was
extracted with ethyl acetate (3 · 50 mL), the combined
organic extracts were washed successively with 5%
HCl (2 · 20 mL), and then saturated sodium thiosulfate
solution (2 · 50 mL), the organic fraction was dried
(Na2SO4), and the solvent was removed in vacuo. The
residue obtained was purified by silica gel column chro-
matography using hexanes–acetone (3:1, v/v) as eluent
to afford 10 (1.81 g, 69%) as a dull white powder; mp
138–140 ꢂC (lit.23 138–140 ꢂC). IR (film): 2589–3227
1
(C@O), 1615, 1474 (aromatic) cmꢁ1. H NMR (CDCl3)
d 1.26 (t, J = 7.0 Hz, 3H, CH2CH3), 2.60 (s, 3H,
COCH3), 3.68 (s, 2H, CH2), 4.17 (q, J = 7.0 Hz, 2H,
CH2CH3), 7.39 (d, J = 8.5 Hz, 2H, phenyl H-2, H-6),
7.93 (d, J = 8.5 Hz, 2H, phenyl H-3, H-5).
(COOH), 1696 (COOH) cmꢁ1 1H NMR (CDCl3) d
.
3.60 (s, 2H, CH2), 7.04 (d, J = 8.2 Hz, 2H, phenyl H-2,
H-6), 7.67 (d, J = 8.2 Hz, 2H, phenyl H-3, H-5).
5.2. Ethyl 4-(1-hydroxy)ethylphenylacetate (7)
5.5. 4-(3-Hydroxybut-1-ynyl)phenylacetic acid (11)
Sodium borohydride (380 mg, 10 mmol) was added to a
solution of ethyl 4-acetylphenylacetate (6, 412 mg,
2 mmol) in methanol (20 mL), the reaction was allowed
to proceed at 25 ꢂC for 1 h with stirring, and the solvent
was removed in vacuo. Ethyl acetate (50 mL) and water
(80 mL) were added, and the mixture was extracted with
ethyl acetate (3 · 50 mL). The organic extracts were
combined, washed with brine (3 · 50 mL), and the
organic fraction was dried (Na2SO4). Removal of the
solvent in vacuo gave 7 as a colorless oil (366 mg,
Cuprous iodide (15 mg, 0.08 mmol) and dichlorobis(tri-
phenylphosphine)palladium (27 mg, 0.04 mmol) were
added to a solution of a 4-iodophenylacetic acid (10,
338 mg, 1.29 mmol) and 3-butyn-2-ol (135 mg,
1.93 mmol) in Et3N (8 mL). The reaction was allowed to
proceed overnight with stirring at 50 ꢂC under an argon
atmosphere, cooled to 25 ꢂC, and then Et3N was removed
under reduced pressure. The residue obtained was puri-
fied by silica gel column chromatography using hex-
anes–acetone (2:1, v/v) as the eluent to furnish 11
(240 mg, 91%) as a pale yellow powder; mp 122–124 ꢂC.
IR (film): 3380 (OH), 2520–3020 (COOH), 1703 (COOH)
cmꢁ1. 1H NMR (CDCl3 + DMSO-d6; 5:1, v/v) d 1.44 (d,
J = 6.7 Hz, 3H, CHCH3), 3.50 (s, 2H, CH2), 4.63 (q,
J = 6.7 Hz, 1H, CHCH3), 7.15 (d, J = 7.0 Hz, 2H, phenyl
H-3, H-5), 7.28 (d, J = 7.0 Hz, 2H, phenyl H-2, H-6).
88%). IR (film): 3422 (OH), 1743 (COO) cmꢁ1 1H
.
NMR (CDCl3) d 1.26 (t, J = 7.0 Hz, 3H, CH2CH3),
1.49 (d, J = 6.4 Hz, 3H, CHCH3), 3.61 (s, 2H, CH2),
4.15 (q, J = 7.0 Hz, 2H, CH2CH3), 4.91 (q, J = 6.4 Hz,
1H, CHCH3), 7.27 (d, J = 8.2 Hz, 2H, phenyl H-3,
H-5), 7.35 (d, J = 8.2 Hz, 2H, phenyl H-2, H-6).

7906
Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
5.6. General procedure for the synthesis of 4-(4-metha-
nesulfonylphenyl)-3-(4-substituted-phenyl)-2(5H)furanon-
es (13 and 14)
15 or 16. Physical, spectral, and microanalytical data
for 15 and 16 are listed below.
5.7.1. 3-(4-Acetylphenyl)-4-(4-methanesulfonylphenyl)-
2(5H)furanone (15). Yield, 70%; pale yellow crystals;
mp 196–197 ꢂC. IR (film): 1756 (furanone CO), 1313,
Et3N (0.38 mL, 2.75 mmol) was added to a solution of
the bromoketone (12, 692 mg, 2.5 mmol) and a 4-substi-
tuted-phenylacetic acid (8 or 11) in acetonitrile (30 mL).
This solution was stirred at 25 ꢂC for 30 min, cooled to
0 ꢂC, and then 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU, 0.56 mL, 3.75 mmol) was added. The reaction
temperature was maintained at 0 ꢂC for an additional
50 min with stirring, and the reaction mixture was acid-
ified with 5% HCl (w/v) until a color change from dark
brown to light yellow was observed. Ice water (30 mL)
was added, stirring was continued for a few minutes,
and the mixture was extracted with EtOAc
(3 · 30 mL). The combined extracts were washed with
brine (30 mL), dried (Na2SO4), and the solvent from
the organic fractions was removed in vacuo. The residue
obtained was subjected to silica gel column chromatog-
raphy eluting with chloroform–acetone (8:1, v/v) to pro-
vide the respective title compound.
1159 (SO2) cmꢁ1 1H NMR (CDCl3) d 2.62 (s, 3H,
.
COCH3), 3.09 (s, 3H, SO2CH3), 5.23 (s, 2H, furanone
CH2), 7.52 and 7.53 (two d, J = 8.2 Hz, 2H each, 4-
methanesulfonylphenyl H-2, H-6, 4-acetylphenyl H-2,
H-6), 7.95 and 7.97 (two d, J = 8.2 Hz, 2H each, 4-meth-
anesulfonylphenyl H-3, H-5, 4-acetylphenyl H-3, H-5);
MS (ES+) m/z: Calcd for C19H16O5S (MH+): 357.07.
Found: 356.93; Anal. Calcd for C19H16O5S: C, 64.03;
H, 4.53. Found: C, 64.08; H, 4.47.
5.7.2.
4-(4-Methanesulfonylphenyl)-3-[4-(3-oxobut-1-
ynyl)phenyl]-2(5H)furanone (16). Yield, 55%; pale yellow
foam; mp 88–90 ꢂC. IR (film): 2200 (C ꢂ C), 1745 (fura-
none CO), 1682 (CO), 1327, 1154 (SO2) cmꢁ1. 1H NMR
(CDCl3) d 2.47 (s, 3H, COCH3), 3.09 (s, 3H, SO2CH3),
5.22 (s, 2H, furanone CH2), 7.45 (d, J = 8.5 Hz, 2H, 4-
(3-oxobut-1-ynyl)phenyl H-2, H-6), 7.51 (d, J = 8.5 Hz,
2H, 4-methanesulfonylphenyl H-2, H-6), 7.60 (d,
J = 8.5 Hz, 2H, 4-(3-oxobut-1-ynyl)phenyl H-3, H-5),
7.96 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl
H-3, H-5); MS (ES+) m/z: Calcd for C21H16O5S
(MH+): 381.07. Found: 380.99; Anal. Calcd for
C21H16O5S: C, 66.30; H, 4.24. Found: C, 66.60; H,
4.30.
5.6.1. 3-[4-(1-Hydroxy)ethylphenyl]-4-(4-methanesulfo-
nylphenyl)-2(5H)furanone (13). Yield, 26%; pale yellow
foam; mp 64–66 ꢂC. IR (film): 3489 (OH), 1743 (fura-
1
none CO), 1300, 1152 (SO2) cmꢁ1. H NMR (CDCl3)
d 1.52 (d, J = 6.4 Hz, 3H, CHCH3), 3.08 (s, 3H,
SO2CH3), 4.95 (q, J = 6.4 Hz, 1H, CHCH3), 5.20 (s,
2H, furanone CH2), 7.37–7.44 (AA0BB0 system,
J = 8.5 Hz, 4H, 4-(1-hydroxyl)ethylphenyl H-2, H-3,
H-5, H-6), 7.53 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-
phenyl H-2, H-6), 7.93 (d, J = 8.5 Hz, 2H, 4-metha-
nesulfonylphenyl H-3, H-5); MS (ES+) m/z: Calcd for
C19H18O5S (MH+): 359.09. Found: 358.98.
5.8. General procedure for the synthesis of 3-(4-
alkyloxyiminoalkylphenyl)-4-(4-methanesulfonylphenyl)-
2(5H)furanones (17 and 18)
Sodium carbonate (255 mg, 2.4 mmol) was added to a
stirred solution of the 3-(4-acetyl or 4-acetylethynylphe-
nyl)-4-(4-methanesulfonylphenyl)-2(5H)furanone (15 or
16, 0.3 mmol) and the respective hydroxylamine hydro-
chloride (H2NORÆHCl, R = H, Me, CH2Ph; 2.4 mmol)
in anhydrous ethanol (10 mL). The mixture was refluxed
for 1 h, cooled to 25 ꢂC, and H2O (60 mL) was added
prior to extraction with EtOAc (3 · 50 mL). The com-
bined extracts were washed successively with 5% HCl
(30 mL) and water (30 mL), the organic fraction was
dried (Na2SO4), and the solvent was removed in vacuo.
The crude product was purified by silica gel column
chromatography employing dichloromethane–acetone
(10:1, v/v) as eluent to furnish the respective title com-
pounds. Physical, spectral, and microanalytical data
for 17a–c and 18a–c are listed below.
5.6.2. 3-[4-(3-Hydroxybut-1-ynyl)phenyl]-4-(4-methane-
sulfonylphenyl)-2(5H)furanone (14). Yield, 66%; pale yel-
low foam; mp 96–98 ꢂC. IR (film): 3481 (OH), 2220
(C ꢂ C), 1754 (furanone CO), 1318, 1145 (SO2) cmꢁ1
.
1H NMR (CDCl3) d 1.56 (d, J = 6.7 Hz, 3H, CHCH3),
3.08 (s, 3H, SO2CH3), 4.77 (q, J = 6.7 Hz, 1H, CHCH3),
5.19 (s, 2H, furanone CH2), 7.36 (d, J = 8.2 Hz, 2H,
4-(3-hydroxybut-1-ynyl)phenyl H-2, H-6), 7.45 (d,
J = 8.2 Hz, 2H, 4-(3-hydroxybut-1-ynyl)phenyl H-3,
H-5), 7.50 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl
H-2, H-6), 7.93 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5); MS (ES+) m/z: Calcd for
C21H18O5S (MH+): 383.09. Found: 382.91.
5.7. General procedure for the synthesis of 3-[4-acetyl and
4-(3-oxobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-
2(5H)furanones (15 and 16)
5.8.1. 3-[4-(1-Hydroxyimino)ethylphenyl]-4-(4-methane-
sulfonylphenyl)-2(5H)furanone (17a). Yield, 46.5%; pale
yellow solid; mp 214–216 ꢂC. IR (film): 3361 (OH),
Jones reagent (0.68 mL, 1.8 mmol) was added dropwise
to a solution of a 2(5H)furanone (13 or 14, 0.36 mmol)
at 0 ꢂC, and the mixture was stirred at 0 ꢂC for 1 h.
EtOAc (100 mL) was added, the resulting mixture was
washed with brine (2 · 30 mL), the organic fraction
was dried (Na2SO4), and the solvent was removed in
vacuo. Purification of the residue obtained via silica
gel column chromatography using dichloromethane–
acetone (9:1, v/v) as eluent gave the respective product
1
1750 (furanone CO), 1320, 1145 (SO2) cmꢁ1. H NMR
(CDCl3) d 2.33 (s, 3H, N@C–CH3), 3.09 (s, 3H,
SO2CH3), 5.21 (s, 2H, furanone CH2), 7.45 (d,
J = 8.5 Hz, 2H, (1-hydroxyimino)ethylphenyl H-2,
H-6), 7.52 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl
H-2, H-6), 7.67 (d, J = 8.5 Hz, 2H, (1-hydroxyimi-
no)ethylphenyl H-3, H-5), 7.95 (d, J = 8.5 Hz, 2H,
4-methanesulfonylphenyl H-3, H-5); MS (ES+) m/z:

Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
7907
Calcd for C19H17NO5S (MH+): 372.09. Found: 371.90;
Anal. Calcd for C19H17NO5S: C, 61.44; H, 4.61; N,
3.77. Found: C, 61.62; H, 4.58; N, 4.00.
5.8.6.
3-[4-(3-Benzyloxyiminobut-1-ynyl)phenyl]-4-(4-
methanesulfonylphenyl)-2(5H)furanone (18c). Yield,
85.5%; pale yellow syrup. IR (film): 2226 (C ꢂ C),
1750 (furanone CO), 1313, 1159 (SO2) cmꢁ1. H NMR
1
5.8.2. 4-(4-Methanesulfonylphenyl)-3-[4-(1-methoxyimi-
no)ethylphenyl]-2(5H)furanone (17b). Yield, 61.5%;
yellow oil. IR (film): 1750 (furanone CO), 1326, 1152
(CDCl3) d 2.14 and 2.15 (two s, 3H total, N@C–CH3),
3.08 (s, 3H, SO2CH3), 5.20 (s, 2H, furanone CH2),
5.21 and 5.23 (two s, 2H total, benzyl CH2), 7.30–7.56
(m, 11H, 4-methanesulfonylphenyl H-2, H-6, 4-(3-ben-
zyloxyiminobut-1-ynyl)phenyl hydrogen, benzyl phenyl
hydrogen), 7.95 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5); MS (ES+) m/z: Calcd for
C28H23NO5S (MH+): 486.14. Found: 485.96; Anal.
Calcd for C28H23NO5SÆ1/2H2O: C, 68.00; H, 4.89; N,
2.83. Found: C, 68.25; H, 4.73; N, 3.11.
1
(SO2) cmꢁ1. H NMR (CDCl3) d 2.24 (s, 3H, N@C–
CH3), 3.09 (s, 3H, SO2CH3), 4.01 (s, 3H, OCH3), 5.20
(s, 2H, furanone CH2), 7.43 (d, J = 8.5 Hz, 2H, 4-(1-
methoxyimino)ethylphenyl H-2, H-6), 7.52 (d,
J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-2, H-6),
7.69 (d, J = 8.5 Hz, 2H, 4-(1-methoxyimino)ethylphenyl
H-3, H-5), 7.94 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5); MS (ES+) m/z: Calcd for
C20H19NO5S (MH+): 386.10. Found: 385.98; Anal.
Calcd for C20H19NO5SÆ1/4H2O: C, 61.60; H, 5.04; N,
3.59. Found: C, 61.87; H, 4.94; N, 3.72.
5.9. General procedure for the synthesis of N-alkoxycar-
bonyl-N-hydroxylamines (20)
NaBH3CN (3.0 g, 47.6 mmol) and a trace of methyl
orange were added to a solution of 3-[4-(1-hydroxyimi-
no)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)fura-
none (17a, 2.4 g, 6.5 mmol) in methanol (500 mL) and
THF (100 mL). The reaction mixture was stirred at
25 ꢂC for 5 min prior to the addition of 12 N HCl that
was added dropwise until the color remained pink. The
reaction mixture was maintained at 25 ꢂC for 12 h with
stirring. After removal of the organic solvents, water
was added (200 mL), the mixture was acidified to pH 3
using 5% w/v HCl, and then washed with EtOAc
(3 · 150 mL). The aqueous fraction was basified to pH 9
with sodium carbonate, extracted with EtOAc
(3 · 150 mL), the combined extracts weredried (Na2SO4),
and the organic solvent was removed in vacuo to provide
the hydroxylamine 19 (1.5 g, 72%) as a white foam which
was used immediately without further purification for the
preparation of the N-hydroxycarbamates (20).
5.8.3. 3-[4-(1-Benzyloxyimino)ethylphenyl]-4-(4-metha-
nesulfonylphenyl)-2(5H)furanone (17c). Yield, 65%; yel-
low oil. IR (film): 1756 (furanone CO), 1320, 1152
1
(SO2) cmꢁ1. H NMR (CDCl3) d 2.28 (s, 3H, CNCH3),
3.08 (s, 3H, SO2CH3), 5.20 (s, 2H, furanone CH2), 5.26
(s, 2H, benzyl CH2), 7.29–7.42 (m, 7H, 4-(1-benzyloxyi-
mino)ethylphenyl H-2, H-6, benzyl phenyl hydrogens),
7.52 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-2,
H-6), 7.68 (d, J = 8.5 Hz, 2H, 4-(1-benzyloxyimino)eth-
ylphenyl H-3, H-5), 7.93 (d, J = 8.5 Hz, 2H, 4-metha-
nesulfonylphenyl H-3, H-5); MS (ES+) m/z: Calcd for
C26H23NO5S (MH+): 462.13. Found: 462.01; Anal.
Calcd for C26H23NO5S: C, 67.66; H, 5.02; N, 3.03.
Found: C, 67.52; H, 5.01; N, 3.25.
5.8.4. 3-[4-(3-Hydroxyiminobut-1-ynyl)phenyl]-4-(4- meth-
anesulfonylphenyl)-2(5H)furanone (18a). Yield, 100%;
pale yellow powder; mp 160–162 ꢂC. IR (film): 3325
(OH), 2252 (C ꢂ C), 1750 (furanone CO), 1307, 1150
Under an argon atmosphere, the hydroxylamine (19,
150 mg, 0.4 mmol) obtained above was dissolved in anhy-
drous dichloromethane (8 mL), Et3N (0.09 mL,
0.52 mmol) and an alkyl chloroformate (2.0 mmol) were
added. This reaction was allowed to proceed with stirring
at 25 ꢂC for 12 h, and the organic solvent was removed in
vacuo. The residue was dissolved in THF (20 mL), this
solution was stirred under a stream of gaseous ammonia
for 30 min at 25 ꢂC prior to capping the reaction flask,
and the reaction was allowed to proceed at 25 ꢂC for
12–16 h with stirring. The THF solvent was removed in
vacuo, and the residue was purified by silica gel column
chromatography eluting with chloroform–acetone to fur-
nish the respective N-hydroxycarbamate (20a–d). Physi-
cal and spectral data for 20a–d are listed below.
1
(SO2) cmꢁ1. H NMR (CDCl3) d 2.50 (s, 3H, N@C–
CH3), 3.09 (s, 3H, SO2CH3), 5.22 (s, 2H, furanone
CH2), 6.33 (br s, 1H, OH), 7.50 and 7.57 (two d,
J = 8.5 Hz, 2H each, 4-methanesulfonylphenyl H-2, H-
6, 4-(3-hydroxyiminobut-1-ynyl)phenyl H-2, H-6), 7.81
(d, J = 8.5 Hz, 2H, 4-(3-hydroxyiminobut-1-ynyl)phenyl
H-3, H-5), 7.95 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5); MS (ES+) m/z: Calcd for
C21H17NO5S (MH+): 396.09. Found: 395.90; Anal.
Calcd for C21H17NO5S: C, 63.79; H, 4.33; N, 3.54.
Found: C, 63.40; H, 4.37; N, 3.54.
5.8.5. 4-(4-Methanesulfonylphenyl)-3-[4-(3-methoxyimi-
nobut-1-ynyl)phenyl]-2(5H)furanone (18b). Yield, 88%;
yellow oil. IR (film): 2220 (C ꢂ C), 1756 (furanone
1
CO), 1313, 1145 (SO2) cmꢁ1. H NMR (CDCl3) d 2.10
5.9.1. N-Isopropyloxycarbonyl-N-{1-[4-(4-methanesulfo-
nylphenyl)-2(5H)furanon-3-yl]phenyl}ethylhydroxylamine
(20a). Yield, 18%; pale yellow foam; mp 83–85 ꢂC. IR
(film): 3368 (OH), 1750 (furanone CO), 1689 (carbamate
CO), 1306, 1145 (SO2) cmꢁ1. 1H NMR (CDCl3) d 1.27 and
1.31 [two d, J = 6.4 Hz, 6H total, CH(CH3)2], 1.63 (d,
J = 7.0 Hz, 3H, CHCH3), 3.09 (s, 3H, SO2CH3), 5.00 [sep-
tet, J = 6.4 Hz, 1H, CH(CH3)2], 5.20 (s, 2H, furanone
CH2), 5.30 (q, J = 7.0 Hz, 1H, CHCH3), 7.38 and 7.43
(AA0BB0 system, J = 8.0 Hz, 4H total, disubstituted-
and 2.14 (two s, 3H total, N@C–CH3), 3.09 (s, 3H,
SO2CH3), 3.98, 4.00 (s, 3H, OCH3), 5.20 (s, 2H, fura-
none CH2), 7.38–7.57 (m, 6H, 4-methanesulfonylphenyl
H-2, H-6, 4-(3-methoxyiminobut-1-ynyl)phenyl H-2,
H-3, H-5, H-6), 7.95 (d, J = 8.2 Hz, 2H, 4-methanesulfo-
nylphenyl H-3, H-5); MS (ES+) m/z: Calcd for
C22H19NO5S (MH+): 410.11. Found: 409.98; Anal.
Calcd for C22H19NO5S: C, 64.53; H, 4.68; N, 3.42.
Found: C, 64.38; H, 4.71; N, 3.53.

7908
Q.-H. Chen et al. / Bioorg. Med. Chem. 14 (2006) 7898–7909
phenyl hydrogens), 7.53 (d, J = 7.9 Hz, 2H, 4-metha-
nesulfonylphenyl H-2, H-6), 7.94 (d, J = 7.9 Hz, 2H,
4-methanesulfonylphenyl H-3, H-5); MS (ES+) m/z:
Calcd for C23H25NO7S (MH+): 460.14. Found: 460.01.
Chemical, Ann Arbor, MI, USA) and soybean
15-LOX (Catalog No. 760700, Cayman Chemical, Ann
Arbor, MI, USA) (IC50 values, lM) was determined
using an enzyme immunoassay (EIA) kit according to
our previously reported method.25
5.9.2. N-Isobutyloxycarbonyl-N-{1-[4-(4-methanesulfo-
nylphenyl)-2(5H)furanon-3-yl]phenyl}ethylhydroxylamine
(20b). Yield, 13%; pale yellow powder; mp 97–99 ꢂC. IR
(film): 3355 (OH), 1757 (furanone CO), 1703 (carbamate
CO), 1315, 1142 (SO2) cmꢁ1. 1H NMR (CDCl3) d 0.92 (d,
J = 6.7 Hz, 6H, CH(Me)2), 1.64 (d, J = 7.0 Hz, 3H,
CHMe), 1.87–2.01 [m, 1H, CH2CH(Me)2], 3.09 (s, 3H,
SO2CH3), 3.96 (d, J = 6.7 Hz, 2H, CH2CH), 5.19 (s, 2H,
furanone CH2), 5.31 (q, J = 7.0 Hz, 1H, CHCH3), 7.37
and 7.43 (AA0BB0 system, J = 8.5 Hz, 4H total, disubsti-
tuted-phenyl hydrogens), 7.53 (d, J = 8.5 Hz, 2H, 4-meth-
anesulfonylphenyl H-2, H-6), 7.93 (d, J = 8.5 Hz, 2H,
4-methanesulfonylphenyl H-3, H-5); MS (ES+) m/z:
Calcd for C24H27NO7S (MH+): 474.15. Found: 474.07;
Anal. Calcd for C24H27NO7S: C, 60.87; H, 5.75; N,
2.96. Found: C, 60.99; H, 5.78; N, 2.99.
8. In vitro cyclooxygenase (COX) inhibition assay
The ability of the test compounds listed in Table 1 to
inhibit ovine COX-1 and COX-2 (IC50 value, lM) was
determined using an enzyme immunoassay (EIA) kit
(Catalog No. 560101, Cayman Chemical, Ann Arbor,
MI, USA) according to our previously reported
method.26
9. Anti-inflammatory assay
Anti-inflammatory activity was performed using a meth-
od described by Winter et al.27
5.9.3. N-Phenoxycarbonyl-N-{1-[4-(4-methanesulfonyl-
phenyl)-2(5H)furanon-3-yl]phenyl}ethylhydroxylamine
(20c). Yield, 21%; white foam; mp 128–130 ꢂC. IR (film):
3348 (OH), 1750 (furanone CO), 1703 (carbamate CO),
10. Analgesic assay
Analgesic activity was determined using a 4% sodium
chloride-induced abdominal constriction assay previous-
ly reported.28
1
1313, 1152 (SO2) cmꢁ1. H NMR (CDCl3) d 1.70 (d,
J = 7.0 Hz, 3H, CHCH3), 3.07 (s, 3H, SO2CH3), 5.19
(s, 2H, furanone CH2), 5.45 (q, J = 6.7 Hz, 1H,
CHCH3), 7.22 (t, J = 7.7 Hz, 1H, phenyl H-4), 7.33–
7.39 (m, 4H, disubstituted-phenyl hydrogens), 7.47–
7.50 (m, 4H, 4-methanesulfonylphenyl H-2, H-6, phenyl
H-3, H-5), 7.92 (d, J = 8.5 Hz, 2H, 4-methanesulfonyl-
phenyl H-3, H-5); MS (ES+) m/z: Calcd for
C26H23NO7S (MH+): 494.13. Found: 493.88.
Acknowledgments
We are grateful to (i) the Canadian Institutes of
Health Research (CIHR) (MOP-14712) for financial
support of this research, (ii) Rx&D-HRF/CIHR for
a postdoctoral fellowship (to Q.H.C.), and to the
Alberta Heritage Foundation for Medical Research
(AHFMR) for a postdoctoral fellowship award (to
Q.H.C.).
5.9.4. N-Benzyloxycarbonyl-N-{1-[4-(4-methanesulfon-
ylphenyl)-2(5H)furanon-3-yl]phenyl}ethylhydroxylamine
(20d). Yield, 23%; white foam; mp 95–96 ꢂC. IR (film):
3388 (OH), 1750 (furanone CO), 1320, 1152 (SO2)
1
cmꢁ1. H NMR (CDCl3) d 1.63 (d, J = 6.7 Hz, 3H,
CHCH3), 3.08 (s, 3H, SO2CH3), 5.19 and 5.21 (two s,
2H each, furanone CH2, benzyl CH2), 5.35 (q,
J = 6.7 Hz, 1H, CHCH3), 7.30–7.43 (m, 9H, disubstitut-
ed-phenyl hydrogens, benzyl phenyl hydrogens), 7.51 (d,
J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-2, H-6),
7.93 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-3,
H-5); MS (ES+) m/z: Calcd for C27H25NO7S (MH+):
508.14. Found: 507.95.
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