Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.04.069

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F₂, 4-SO₂Me, 4-OCHMe₂) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC₅₀ = 2.26 μM; COX-2 IC₅₀ = 0.012 μM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC₅₀ >100 μM; COX-2 IC₅₀ = 0.15 μM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC₅₀ = 4.25 μM; COX-2 IC₅₀ = 0.978 μM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl) benzenesulfonamide (17c, COX-1 IC₅₀ = 1.02 μM; COX-2 IC ₅₀ = 1.00 μM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4- methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC₅₀ = 0.109 μM; COX-2 IC₅₀ = 1.14 μM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.

Full text of DOI:10.1016/j.bmc.2005.04.069

Bioorganic & Medicinal Chemistry 13 (2005) 4694–4703
Design, synthesis, and biological evaluation of
N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides
as cyclooxygenase isozyme inhibitors
Qiao-Hong Chen, P. N. Praveen Rao and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
Received 29 March 2005; revised 25 April 2005; accepted 26 April 2005
Available online 23 May 2005
Abstract—A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring sub-
stituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki
cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluo-
robenzenesulfonamide [14, COX-1 IC50 = 2.26 lM; COX-2 IC50 = 0.012 lM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-
carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 lM; COX-2 IC50 = 0.15 lM; COX-2 SI
>667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflam-
matory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-
acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 lM; COX-2 IC50 = 0.978 lM; COX-2 SI = 4.3), N-
acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 lM; COX-2 IC50 = 1.00 lM; COX-2
SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 lM; COX-
2 IC50 = 1.14 lM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflamma-
tion 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral
dose reduced inflammation by 25% at 3 h postdrug administration.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
thrombotic and ulcerogenic effects result from acetyla-
tion of COX-1. Selective cyclooxygenase-2 (COX-2)
inhibitors currently provide effective treatment of
inflammatory disease states such as rheumatoid arthritis
and osteoarthritis circumventing the ulcerogenic effect
associated with the use of nonsteroidal anti-inflamma-
tory drugs (NSAIDs) such as aspirin that inhibit both
COX-1 and COX-2.4 However, a precautionary concern
regarding the use of COX-2 inhibitors in patients at risk
for an adverse cardiovascular event such as myocardial
infarction has been raised and this may be due to a
thromboxane A2/prostacyclin (TxA2/PGI2) imbalance
created by selective COX-2 inhibitors.5 Accordingly,
the ability of aspirin to inhibit blood platelet aggrega-
tion is now viewed as a clinically useful prophylactic ac-
tion that can reduce the incidence of thrombus
formation in individuals with cardiovascular disease.
These observations were exploited in the design of the
aspirin analog o-(acetoxyphenyl)hept-2-ynyl sulfide
(APHS, 2), that is, a selective COX-2 inhibitor.6 In an
earlier study, we reported a novel class of isomeric
acetoxy analogs of rofecoxib (3), which are potent and
Prostaglandins arise from the biotransformation of ara-
chidonic acid by the action of two separate isoforms of
the enzyme cyclooxygenase (COX-1 and COX-2).1 Non-
steroidal anti-inflammatory drugs (NSAIDs) such as
aspirin (1) exert their anti-inflammatory and analgesic
effects via the inhibition of prostaglandin synthesis.2
Aspirin is the only nonsteroidal anti-inflammatory drug
that covalently modifies cyclooxygenases. This unique
property of aspirin is derived from its ability to acety-
late the Ser530 hydroxyl group in the primary COX bind-
ing site of COX-1 and COX-2. In this regard, aspirin is a
10- to 100-fold more potent inhibitor of COX-1 relative
to COX-2.3 Some of aspirinꢀs beneficial therapeutic
effects arise from acetylation of COX-2, whereas its anti-
Keywords: N-Acetyl-2-(or 3-)carboxymethylbenzenesulfonamides; Cyclo-
oxygenase inhibition; Anti-inflammatory activity.
*
Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492
1217; e-mail: eknaus@pharmacy.ualberta.ca
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.069

Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
4695
selective COX-2 inhibitors that, like aspirin, have the
potential to acetylate the COX-2 isozyme.7
2. Chemistry
N-Acetyl-2-(or 3-)carboxymethylbenzenesulfonamides
(12, 14) and N-acetyl-2-(or 3-)methoxycarbonylmethyl-
benzenesulfonamides (13, 15) were synthesized using
the reaction sequences illustrated in Scheme 1. Esterifi-
cation of the 3-halo (6a,b), and 4-halo (7a,b), phenylace-
tic acids using MeOH in the presence of a catalytic
amount of H2SO4 afforded the respective methyl 3-halo-
phenylacetates (8a,b) and methyl 4-halophenylacetates
(9a,b). Chlorosulfonation of the 3-halo compounds 8a,b
using chlorosulfonic acid at ice-salt bath temperature,
followed by ammonolysis in THF under a stream of
ammonia gas, afforded the respective 4-halo-2-methoxy-
carbonylmethylbenzenesulfonamide [10a (36%) or 10b
(64%)]. It is important to carry out this chlorosulfo-
nation reaction at low temperature since chlorosulfo-
nation of methyl 3-bromophenyl acetate (8b) at 25 ꢁC
followed by ammonolysis also produced the 2-bromo-
It was therefore of interest to investigate the N-acetyl-
sulfonamido (SO2NHCOMe) moiety as an pharmaco-
phore that is capable of acetylating the Ser530
hydroxyl moiety in the primary binding site of COX-
2.8 In this regard, incorporation of a para N-acetyl-
sulfonamido substituent on the C-3 phenyl ring of the
rofecoxib regioisomer (4) provided a highly potent and
selective COX-2 inhibitor that has the potential to acet-
ylate the COX-2 isozyme.9 In a recent study, we showed
that the SO2NHCOMe pharmacophore present in N-
acetyl-2-carboxybenzenesulfonamides (5) is a suitable
bioisostere for the acetoxy (OCOMe) group in aspirin.10
As part of our ongoing program to design selective
COX-2 inhibitors, it was of interest to acquire struc-
ture–activity relationships for structurally related
homologs and regioisomers of N-acetyl-2-carboxyben-
zenesulfonamides. We now describe the synthesis and
biological evaluation of a novel group of N-acetyl-2-
(or 3-)carboxymethylbenzenesulfonamides possessing
halogeno (F, Br), or a phenyl ring having a variety of
para (H, F, i-PrO, SO2Me) and ortho (H, F), substitu-
ents in which the interspatial distance between the
COOH and COMe moieties is expected to be larger than
that for the previously investigated10 N-acetyl-2-car-
boxybenzenesulfonamide homologs (5) (benzoic acid
vs phenylacetic acid). Alteration of this interspatial dis-
tance can be used to position the N-acetylsulfonamido
acetylating moiety closer or further from the Ser530 hy-
droxyl group that it, like aspirin, is designed to acetylate
(Fig. 1).
4-methoxycarbonylmethylbenzenesulfonamide
regio-
isomer (about 33% yield) that could not be separated
from 10b by silica gel column chromatography. In con-
trast, chlorosulfonation of the methyl 4-halophenylace-
tates (9a,b), and then ammonolysis of the intermediate
sulfonyl chloride product, afforded the respective 6-
halo-3-methoxycarbonylmethylbenzenesulfonamide [11a
(14%) or 11b (36%)]. The subsequent N-acetylation,
and then hydrolysis of the methyl ester, of 10a and
11a yielded the corresponding N-acetyl-2-carboxy-
methyl-4-fluorobenzenesulfonamide (12, 76%) and N-ace-
tyl-3-carboxymethyl-6-fluorobenzenesulfonamide
(14,
93%). During the course of these studies, it was observed
that the methyl ester moiety present in 10a was more dif-
ficult to hydrolyze (NaOH, MeOH) than the methyl
ester group present in the 11a regioisomer (K2CO3,
MeOH). The N-acetyl-4-bromo-2-methoxycarbonyl-
methylbenzenesulfonamide (13, 86%) and N-acetyl-6-bro-
mo-3-methoxycarbonylmethylbenzenesulfonamide (15,
84%) regioisomers were similarly prepared by acetyla-
tion of the sulfonamide substituent present in 10b and
11b. The regiochemistry (relative position of the substit-
uents on the phenyl ring) of compound 11b was deter-
CO2H
SCH2C
C(CH2)3Me
O
C
O
Me
O
C
O
Me
Aspirin (1)
APHS (2)
MeO2S
1
4
3
O
mined by H NMR nuclear Overhauser enhancement
(NOE) studies. The observation that NOE interactions
occurred between CH2 and H-2 (6.7%), and between
CH2 and H-4 (4.3%), in conjunction with the coupling
constants (J values) and chemical shift positions (d val-
ues) of the three phenyl hydrogens in the 1H NMR spec-
trum of 11b, indicates that the sulfonamide moiety is
attached to the 1-position of the phenyl ring (see Fig. 2).
C
O
Me
2
O
O
3
MeCHNO2S
O
SO2Me
The target N-acetyl-2-(or 3-)carboxymethylbenzenesul-
fonamides (17a–d, 19, 20a–e) having a variety (R1 = H,
F, SMe, SO2Me, OCHMe2; R2 = H, F) of substituents
at the ortho and/or para position of a phenyl ring were
prepared using a palladium-catalyzed Suzuki cross-cou-
pling reaction11,12 according to the reaction sequences
shown in Schemes 2 and 3. The cross-coupling reaction
between an aryl bromide (13, 15) and a substituted-
phenylboronic acid (16) in the presence of 2 M aqueous
sodium carbonate in ethylene glycol dimethyl ether,
using tetrakis(triphenylphosphine)palladium(0) as a cata-
lyst, afforded the respective title compounds (17, 18, 20).
O
O
4
R2
CO2H
R1
SO2NHCOMe
1 = H, F, SO2Me, OPr-i; R2 = H, F
R
5
Figure 1. Some representative cyclooxygenase (COX) inhibitors.

4696
Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
CH2CO2H
CH2CO2H
R
R
R
6a, R = F
6b, R = Br
7a, R = F
7b, R = Br
R
a
a
CH2CO2Me
CH2CO2Me
9a, R = F
8a, R = F
R
R
9b, R = Br
8b, R = Br
b,c
b,c
CH2CO2Me
CH2CO2Me
10a, R = F
11a, R = F
SO2NH2
SO2NH2
10b, R = Br
d
11b, R = Br
d,e
d
d,f
CH2CO2H
CH2CO2Me
F
CH2CO2H Br
CH2CO2Me
F
Br
SO2NHCOMe
12
SO2NHCOMe
13
SO2NHCOMe
14
SO2NHCOMe
15
Scheme 1. Reagents and conditions: (a) MeOH, H2SO4, 80 ꢁC, 3 h; (b) ClSO3H, ice-salt bath, 5 h; (c) NH3 (gas), THF, 30 min; (d) Ac2O, pyridine,
DMAP, 25 ꢁC, overnight; (e) NaOH, MeOH, H2O, 80 ꢁC, 3 h; (f) K2CO3, MeOH, H2O, 80 ꢁC, 3 h.
moiety. In order to prepare the corresponding phenyl-
acetic acid compounds 17a–d, it is necessary to change
the solvent from DME to MeOH–H2O (1:1, v/v) to
perform the ester hydrolysis reaction after completion
of the cross-coupling reaction. In contrast, the desired
phenylacetic acid compounds (20a–d) can be obtained
via the conversion of the C-3 methoxycarbonylmethyl
substituent to a C-3 carboxymethyl group during
the one-pot cross-coupling reaction. To circumvent
cyclization13 of ortho-methoxycarbonylmethylbenzene-
sulfonamide compound under acidic oxidation reaction
conditions, the methylsulfonyl compound 18 was syn-
thesized via a two-step process involving a palladium-
4.3%
H
4
CH2CO2Me
6.7%
H
2
Br
SO2NH2
Figure 2. Some NOE studies to determine the relative substituent
positions (regiochemistry) of compound 11b.
Interestingly, the ester hydrolysis of the C-2 methoxy-
carbonylmethyl compounds is more difficult relative to
those compounds having a C-3 methoxycarbonylmethyl
R2
Br
CH2CO2Me
R1
B(OH)2
+
SO2NHCOMe
16, R1 = H, F, OPr-i, SMe
2 = H, F
13
R
a
b,c
R2
R1
MeO2S
CH2CO2H
CH2CO2R
SO2NHCOMe
SO2NHCOMe
d
17a, R1 = R2 = H
18, R = Me
19, R = H
17b, R1 = F, R2 = H
17c, R1 = R2 = F
17d, R1 = OPr-i, R2 = H
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DME, reflux overnight, and then MeOH, H2O, reflux 1.5 h; (b) Pd(PPh3)4, Na2CO3,
DME, reflux 5 h; (c) Oxoneꢂ, MeOH, THF, H2O, 25 ꢁC, 1.5 h; (d) K2CO3, MeOH, H2O, 25 ꢁC, 1.5 h.

Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
4697
R2
p-methanesulfonylphenyl substituent (19) abolished
COX-1 inhibitory activity but exhibited low COX-2
CH2CO2Me
R1
B(OH)2
inhibition (COX-1 IC50 >100 lM; COX-2 IC50
=
+
31.5 lM). The subgroup of N-acetyl-3-carboxymethyl-
benzenesulfonamides (14, 20) showed good to excellent
COX-2 inhibitory activity (IC50 values in the 0.012–
1.76 lM range) with N-acetyl-3-carboxymethyl-6-(4-iso-
propoxyphenyl)benzenesulfonamide (20c), showing the
best combination of COX-2 inhibitory potency and
selectivity (COX-2 IC50 = 0.15 lM; SI >667) as shown
in Table 1. In addition, the N-acetyl-3-carboxymethyl-
6-fluorobenzenesulfonamide (14) is a more potent but
less selective COX-2 inhibitor (COX-1 IC50 = 2.26 lM;
COX-2 IC50 = 0.012 lM; COX-2 SI = 188) relative to
the reference drugs rofecoxib (COX-2 IC50 = 0.50 lM;
SI >200) and celecoxib (COX-2 IC50 = 0.07 lM;
SI = 472). Compounds having a C-6 phenyl substituent
(20a, COX-1 IC50 = 0.92 lM; COX-2 IC50 = 1.76 lM)
or a C-6 p-methanesulfonylphenyl substituent (20e,
COX-1 IC50 = 0.109 lM; COX-2 IC50 = 1.14 lM) exhi-
bit similar COX-1 and COX-2 inhibition profiles to
aspirin (COX-1 IC50 = 0.35 lM; COX-2 IC50 = 2.4 lM).
In contrast, incorporation of a C-6 p-fluorophenyl sub-
stituent (20b) did not appreciably change COX-2 inhib-
itory activity but COX-1 inhibitory activity was
Br
16, R1 = H, F, OPr-i, SMe
2 = H
SO2NHCOMe
15
R
a
CH2CO2H
R2
SO2NHCOMe
R1
20a, R1 = R2 = H
20b, R1 = F, R2 = H
20c, R1 = OPr-i, R2 = H
20d, R1 = SMe, R2 = H
b
20e, R1 = SO2Me, R2 = H
Scheme 3. Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DME,
reflux overnight; (b) Oxoneꢂ, MeOH, H2O, 25 ꢁC, 1.5 h.
catalyzed Suzuki cross-coupling reaction followed by
oxidation of the thiomethyl to a methylsulfonyl substi-
tuent in situ. Subsequent ester hydrolysis of the meth-
ylsulfonyl compound 18 in the presence of K2CO3
furnished the target methylsulfonyl compound 19. Oxi-
dation of 20d using aqueous Oxone solution provided
the corresponding methylsulfonyl compound 20e.
abolished (COX-1 IC50 >100 lM; COX-2 IC50
1.52 lM).
=
A molecular modeling experiment was carried out to
determine the binding interactions of N-acetyl-3-car-
boxymethyl-6-(4-isopropoxyphenyl)benzenesulfona-
mide (20c) in the COX-2 binding site (Fig. 3). The
parent aromatic ring possessing the carboxyl and N-
acetyl- sulfonamido substituents is surrounded by non-
polar amino acids such as Leu352, Phe518, Val523, and
Ala527. The N-acetylsulfonamido-substituent is posi-
tioned at the top of the COX-2 binding site in a region
comprised of the amino acids Tyr385, Tyr348, and
Ser530. One of the oxygen atoms of the SO2 (SO2NH-
COMe) participates in a favorable hydrogen bonding
3. Results and discussion
In our recent study,10 a group of N-acetyl-2-carboxy-
benzenesulfonamides (5) possessing an appropriately
substituted-phenyl substituent attached to its C-4 or
C-5 position, was designed for evaluation as selective
cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1
and COX-2 isozyme inhibition structure–activity studies
interaction with the OH of Ser530 (distance = 1.83 A).
˚
identified
benzenesulfonamide as
N-acetyl-2-carboxy-4-(2,4-difluorophenyl)-
highly potent (COX-2
The distance between the OH of Ser530, which is the
a
acetylation site for aspirin, and C@O of the SO2NH-
COMe moiety is about 4.61 A. These observations
˚
IC50 = 0.087 lM), and a highly selective (COX-2 SI
>1149), COX-2 inhibitor that showed superior anti-
inflammatory activity (ED50 = 91 mg/kg) relative to
aspirin (ED50 = 129 mg/kg). This initial study has now
been extended to include the design of N-acetyl-2-(or
3-)carboxymethylbenzenesulfonamide homologs, pos-
sessing either a F, or an appropriately substituted-phen-
yl substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2),
attached to the C-4 or C-6 position of the parent ben-
zenesulfonamide ring system.
suggest that the SO2NHCOMe moiety present in
20c is suitably orientated to potentially acetylate (cova-
lent bond formation) Ser530 present in the COX-2
isozyme.
The carboxylate moiety present in the parent aromatic
ring, is positioned close to Tyr355 and Arg120 near the
mouth of the COX-2 binding site (hydrogen bonding
and electrostatic interactions). This orientation properly
orients the N-acetylsulfonamide substituent closer to
Ser530, the acetylation site for aspirin. In this regard,
In vitro enzyme inhibition studies for the N-acetyl-2-
carboxymethylbenzenesulfonamide subgroups (12, 17,
19) showed a wide range of COX-2 inhibitory activities
(IC50 values in the 0.83 to >100 lM range) with low-to-
moderate COX-2 selectivity indices (see data in Table 1).
In this subgroup, a C-4 p-fluorophenyl substituent (17b)
abolished COX-2 inhibitory activity, but COX-1 inhibi-
tory activity was maintained (COX-1 IC50 = 2.2 lM, see
data in Table 1). In contrast, introduction of a C-4
˚
the OH of the carboxylate is located about 1.99 A from
the NH2 (guanidine moiety) of Arg120, whereas the dis-
tance between the C@O of the COOH and the OH of
Tyr355 is about 4.07 A.
˚
Interestingly, the 4-isopropoxyphenyl ring is oriented in
,
a lipophilic pocket comprised of Leu384, Trp387, Leu507
Met522, Leu525, and Gly526 at the upper apex of the

4698
Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
Table 1. In vitro COX-1/COX-2 enzyme inhibition assay data for 12, 14, 17a–d, 19, and 20a–c,e, in vivo anti-inflammatory assay data for 12, 14, 17c,
20a, 20c, and 20e and analgesic activity assay data for 17c and 20e
R1
R2
CH2COOH
CH2COOH
F
CH2COOH
CH2COOH
F
SO2NHCOMe
R1
SO2NHCOMe
SO2NHCOMe
SO2NHCOMe
12
14
17a-d, 19
20a-c, e
Compds
R1
R2
IC50 (lM)a
COX-2 SIb AI activityc %
inhibition (75 mg/kg)
Analgesic activityd
COX-1 COX-2
% Inhibition (30 min) % Inhibition (60 min)
12
14
—
—
H
F
—
—
H
4.25
2.26
2.83
2.20
1.02
3.74
>100
0.92
>100
>100
0.109
0.35
33.1
>100
0.978
0.012
0.83
>100
1.00
3.16
31.5
1.76
1.52
0.15
1.14
2.4
4.3
188
26.3 18.5e
—
—
—
—
—
—
—
—
Inactive
—
—
17a
17b
3.4
H
<0.02
1.02
1.18
>3.17
0.52
>65.8
>667
0.095
0.14
472
17c
17d
F
OCH(CH3)2
F
14.3 1.9
—
—
69.8 6.8
68.7 8.3
H
—
—
—
—
19
20a
SO2CH3
H
H
—
—
—
—
—
Inactive
—
—
—
—
—
20b
20c
F
OCH(CH3)2
SO2CH3
—
Inactive
20.0 3.0e
25.2 3.3f
—
—
73.77 11.4
56.52 9.8f
—
68.22 7.6
64.30 13.7f
20e
1 (Aspirin)
Celecoxib
Rofecoxib
0.07
0.50
—
—
—
—
>200
—
a Values are means of two determinations acquired using an ovine COX-1/COX-2 assay kit (Catalog No. 560101, 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 Inhibitory activity in a carrageen-induced rat paw edema assay. The results are expressed as mean SEM (n = 4) at 3 h following a 75 mg/kg oral
dose of the test compound.
d Inhibitory activity in the rat 4% NaCl-induced abdominal constriction assay. The results are expressed as mean SEM (n = 6) following a 75 mg/kg
oral dose of the test compound.
e n = 3 animals.
f 50 mg/kg oral dose.
COX-2 binding site. This small lipophilic region has
been exploited in the design of selective COX-2 inhibi-
tors.14 Accordingly, the p-OCH(CH3)2 substituent may
undergo van der Waalꢀs interactions with side chains
of amino acid residues such as Leu384, Leu525, and
N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)ben-
zenesulfonamide (20c), which exhibited high in vitro
COX-2 potency and selectivity, were inactive anti-
inflammatory agents (see data in Table 1). Further
pharmacological studies using systemic routes of admin-
istration may help to explain these observed differences
between in vitro COX-2 inhibition and in vivo anti-
inflammatory activity.
Met522 (distance <5 A).
˚
In vivo pharmacological evaluation of a small group of
compounds was carried out to assess their potential
anti-inflammatory and analgesic activities. Initial com-
pound selection for in vivo screening was based on in vi-
tro COX-1/COX-2 enzyme inhibition data. Qualitative
structure–activity relationship data, acquired using the
anti-inflammatory rat paw edema assay, showed that
some of the N-acetyl-2-(or 3-)carboxymethylbenzene-
sulfonamides exhibited moderate anti-inflammatory
activity while others were inactive (inactive to 26% inhi-
bition range for a 75 mg/kg oral dose) (Table 1). N-Acet-
yl-2-carboxymethyl-4-fluorobenzenesulfonamide (12) and
N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)-
benzenesulfonamide (20e) were the most potent anti-
inflammatory agents within this group of compounds,
producing a 26% and 20% reduction in inflammation
at 3 h postdrug administration (75 mg/kg oral dose),
respectively, relative to the reference drug aspirin where
a 50 mg/kg oral dose reduced inflammation by 25% at
3 h postdrug administration. In contrast, N-acetyl-3-
carboxymethyl-6-fluorobenzenesulfonamide (14) and
In a rat model, 4% NaCl-induced abdominal constric-
tion assay, a 75 mg/kg po dose of compounds 17c and
20e exhibited good analgesic activities (68–74% inhibi-
tion range) at 30 or 60 min postdrug administration rela-
tive to the reference drug aspirin (57% and 64%
inhibition) at 30 and 60 min postdrug administration
for a 50 mg/kg oral dose (see data in Table 1).
4. Conclusions
A new class of N-acetyl-2-(or 3-)carboxymethylbenzene-
sulfonamides were designed to develop further struc-
ture–activity relationship data. In vitro enzyme
inhibition studies showed that COX-2 inhibitory po-
tency and selectivity was dependent upon the point of
attachment of the SO2NHCOCH3 moiety. In this
regard, the N-acetyl-3-carboxymethylbenzenesulfon-
amides 14 and 20c are selective COX-2 inhibitors, while

Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
4699
60 ASTM (70–230 mesh). All reagents were purchased
from the Aldrich Chemical Company (Milwaukee, WI)
and used without further purification. Male Sprague–
Dawley rats, used in the anti-inflammatory-analgesic
screens, were purchased from Animal Health Services
at the University of Alberta, and experiments were car-
ried out using protocols approved by the Animal Wel-
fare Committee, University of Alberta.
5.1. General procedure for the synthesis of methyl 3-(or 4)-
halophenylacetates (8a,b and 9a,b)
Concentrated H2SO4 (5 mL) was added dropwise at ice-
bath temperature to a stirred solution of 6a, 6b, 7a, or 7b
(1.0 g) in methanol (50 mL). The reaction mixture was
refluxed for 3 h, cooled to 25 ꢁC, and EtOAc (350 mL)
was added. This solution was washed with water
(3 · 60 mL), the organic fraction was dried (Na2SO4),
and the solvent was removed in vacuo to afford the
respective title compound (8a,b, 9a, or 9b) for which
some physical and spectral data are listed below.
5.1.1. Methyl 3-fluorophenylacetate (8a). Yield, 95%; col-
orless liquid; IR (film): 1727 (C@O), 1622, 1592, 1480
(Ar) cmÀ1; 1H NMR (CDCl3) d 3.63 (s, 2H, CH2COO),
3.71 (s, 3H, OCH3), 6.94–7.04 (m, 3H, H-2, H-4, H-6),
7.27–7.34 (m, 1H, H-5).
Figure 3. Docking of N-acetyl-3-carboxymethyl-6-(4-isopropoxyphen-
yl)benzenesulfonamide (20c) (ball and stick) in the active site of murine
COX-2. Hydrogen atoms of the amino acid residues have been
removed to improve clarity.
5.1.2. Methyl 3-bromophenylacetate (8b). Yield, 89%;
pale yellow liquid; IR (film): 1746 (C@O), 1597, 1498
(Ar) cmÀ1; 1H NMR (CDCl3) d 3.60 (s, 2H, CH2COO),
3.71 (s, 3H, OCH3), 7.17–7.45 (m, 4H, phenyl
hydrogens).
the corresponding regioisomers N-acetyl-2-carboxy-
methylbenzenesulfonamides 12 and 17d, like aspirin, are
nonselective COX-2 inhibitors. N-Acetyl-3-carboxy-
methyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c)
5.1.3. Methyl 4-fluorophenylacetate (9a). Yield, 100%;
colorless liquid; IR (film): 1735 (C@O), 1600, 1570
(Ar) cmÀ1; 1H NMR (CDCl3) d 3.61 (s, 2H, CH2COO),
3.70 (s, 3H, OCH3), 7.02 (t, J = 8.5 Hz, 2H, H-3, H-5),
7.26 (dd, J = 8.5, 5.5 Hz, 2H, H-2, H-6).
exhibited optimal COX-2 inhibitory potency (IC50
=
0.15) and selectivity (COX-2 SI >667). In vivo anti-
inflammatory studies showed that replacement of the
carboxyl substituent in 5 by a homologous acetic acid
substituent that provided N-acetyl-2-(or 3-)carboxy-
methylbenzenesulfonamides resulted in a significant
reduction of in vivo anti-inflammatory activity.
5.1.4. Methyl 4-bromophenylacetate (9b). Yield, 96%;
colorless liquid; IR (film): 1750 (C@O), 1600, 1547,
1465 (Ar) cmÀ1 1H NMR (CDCl3) d 3.59 (s, 2H,
;
CH2COO), 3.70 (s, 3H, OCH3), 7.16 (d, J = 8.2 Hz,
2H, H-2, H-6), 7.46 (d, J = 8.2 Hz, 2H, H-3, H-5).
5. Experimental
Melting points were determined on a Thomas–Hoover
capillary apparatus and are uncorrected. Infrared (IR)
spectra were recorded as films on NaCl plates using
5.2. General procedure for the synthesis of 4-fluoro (or
4-bromo)-2-methoxycarbonylmethylbenzenesulfonamide
(10a,b) and 6-fluoro (or 6-bromo)-3-methoxycarbonyl-
methylbenzenesulfonamide (11a,b)
1
a Nicolet 550 Series II Magna FT-IR spectrometer. H
NMR spectra were measured on a Bruker AM-300 spec-
trometer in CDCl3 or CDCl3 + DMSO-d6 with TMS as
the internal standard, where J (coupling constant) val-
ues are estimated in Hertz. Spin multiples are given as
s (singlet), d (doublet), t (triplet), q (quartet), m (multi-
plet), and br (broad). The NOE studies were performed
under steady-state conditions using the Bruker NOE
DIFF.AU software program (signal:noise ratio of 136
for a single pulse). Microanalyses were performed for
C, H, N (MicroAnalytical Service Laboratory, Depart-
ment of Chemistry, University of Alberta) and were
within 0.4% of theoretical values. Silica gel column
chromatography was performed using Merck silica gel
Chlorosulfonic acid (10 mL) was added slowly at ice-salt
bath temperature to a flask containing a 3-halo (8a,b) or
4-halo (9a,b) methyl phenylacetate (1.0 g). The reaction
was allowed to proceed with stirring for 5 h at the same
low temperature prior to pouring onto crushed ice
(300 mL), and then extracted with CH2Cl2
(3 · 150 mL). The combined CH2Cl2 extracts were
washed with water (3 · 100 mL), and the organic
fraction was dried (Na2SO4). Removal of the solvent
in vacuo gave the respective arylsulfonyl chloride inter-
mediate, which was dissolved in THF (50 mL). This
solution was stirred under a stream of gaseous ammonia

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Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
for 30 min at 25 ꢁC, the insoluble material was removed
by filtration, and the solvent was removed from the fil-
trate in vacuo to yield the respective sulfonamide
(10a,b, 11a, or 11b). Some physical and spectral data
for the title compounds are listed below.
cooled to 25 ꢁC, water (150 mL) was added, the mixture
was acidified to pH 2–3 using 5% w/v HCl, and the mix-
ture was extracted with EtOAc (3 · 100 mL). The com-
bined EtOAc extracts were washed with water
(2 · 50 mL), the organic fraction was dried (Na2SO4),
and the solvent was removed in vacuo to afford 12
(320 mg, 76%) as a pale yellow solid; mp 195–197 ꢁC;
IR (film): 3670 (NH), 3600–2447 (COOH), 1720
5.2.1. 4-Fluoro-2-methoxycarbonylmethylbenzenesulfon-
amide (10a). Yield, 64%; pale yellow solid; mp 108–
110 ꢁC; IR (film): 3670 (NH2), 1727 (C@O), 1607,
(C@O), 1607, 1592, 1472 (Ar), 1375 (SO2) cmÀ1 1H
;
1
1585, 1457 (Ar), 1352 (SO2) cmÀ1; H NMR (CDCl3)
NMR (CDCl3) d 1.86 (s, 3H, COCH3), 3.96 (s, 2H,
CH2COOH), 6.96–7.02 (m, 2H, H-3, H-5), 8.08 (dd,
J = 5.5, 2.7 Hz, 1H, H-6), 11.03 (br s, 1H, NH). Anal.
Calcd for C10H10FNO5S: C, 43.63; H, 3.66; N, 5.09.
Found: C, 43.72; H, 3.49; N, 4.99.
d 3.68 (s, 3H, OCH3), 4.11 (s, 2H, CH2COO), 6.05 (br
s, 2H, NH2), 6.99 (dd, J = 9.16, 2.4 Hz, 1H, H-3), 7.06
(dt, J = 8.5, 2.4 Hz, 1H, H-5), 8.03 (dd, J = 8.5,
5.5 Hz, 1H, H-6). Anal. Calcd for C9H10FNO4S: C,
43.72; H, 4.08; N, 5.67. Found: C, 43.72; H, 4.10; N,
5.51.
5.2.6. N-Acetyl-4-bromo-2-methoxycarbonylmethylben-
zenesulfonamide (13). To a solution of 4-bromo-2-me-
thoxycarbonylmethylbenzenesulfonamide (10b, 919 mg,
2.98 mmol) in pyridine (3 mL) were added acetic anhy-
dride (2.0 mL, 21 mmol) and 4-dimethylaminopyridine
(109 mg, 0.89 mmol). The reaction solution was stirred
overnight at 25 ꢁC, and EtOAc (350 mL) was added.
This solution was washed successively with saturated
aqueous NH4Cl (2 · 80 mL) and H2O (2 · 80 mL). The
organic fraction was dried (Na2SO4), and the solvent
was removed in vacuo to furnish 13 (898 mg, 86%) as
a pale yellow solid; mp 166–168 ꢁC; IR (film): 3662
(NH), 1742 (C@O), 1622, 1585, 1480 (Ar), 1270 (SO2)
5.2.2. 4-Bromo-2-methoxycarbonylmethylbenzenesulfon-
amide (10b). Yield, 36%; white solid; mp 126–128 ꢁC;
IR (film): 3407 (NH2), 1727 (C@O), 1592, 1555, 1457
1
(Ar), 1352 (SO2) cmÀ1; H NMR (CDCl3) d 3.76 (s,
3H, OCH3), 4.18 (s, 2H, CH2COO), 5.44 (br s, 2H,
NH2), 7.50 (d, J = 1.8 Hz, 1H, H-3), 7.61 (dd, J = 8.2,
1.8 Hz, 1H, H-5), 7.95 (d, J = 8.2 Hz, 1H, H-6). Anal.
Calcd for C9H10BrNO4S: C, 35.08; H, 3.27; N, 4.55.
Found: C, 35.44; H, 2.99; N, 4.34.
5.2.3. 6-Fluoro-3-methoxycarbonylmethylbenzenesulfon-
amide (11a). Yield, 14%; white solid; mp 138–140 ꢁC;
IR (film): 3415 (NH2), 1742 (C@O), 1607, 1487 (Ar),
1
cmÀ1; H NMR (CDCl3) d 1.85 (s, 3H, COCH3), 3.58
(s, 3H, OCH3), 3.98 (s, 2H, CH2COO), 7.39 (d,
J = 1.8 Hz, 1H, H-3), 7.47 (dd, J = 8.5, 1.8 Hz, 1H, H-
5), 7.92 (d, J = 8.5 Hz, 1H, H-6), 11.75 (br s, 1H, NH).
Anal. Calcd for C11H12BrNO5S: C, 37.73; H, 3.45; N,
4.00. Found: C, 38.07; H, 3.17; N, 3.86.
1345 (SO2) cmÀ1 1H NMR (CDCl3) d 3.59 (s, 2H,
;
CH2COO), 3.64 (s, 3H, OCH3), 6.21 (br s, 2H, NH2),
7.10 (dd, J = 9.7, 8.5 Hz, 1H, H-5), 7.38–7.43 (m, 1H,
H-4), 7.74 (dd, J = 6.7, 2.1 Hz, 1H, H-2). Anal. Calcd
for C9H10FNO4S: C, 43.72; H, 4.08; N, 5.67. Found:
C, 43.98; H, 4.42; N, 5.56.
5.2.7. N-Acetyl-3-carboxymethyl-6-fluorobenzenesulfon-
amide (14). Compound 14 was prepared as white crystals
in 93% yield using an acetylation and hydrolysis proce-
dure similar to that described previously for the synthe-
sis of compound 12 where K2CO3 was used for base in
place of NaOH during the hydrolysis; mp 180–182 ꢁC;
IR (film): 3670 (NH), 3587–2455 (COOH), 1720
5.2.4. 6-Bromo-3-methoxycarbonylmethylbenzenesulfon-
amide (11b). Yield, 36%; pale yellow oil; IR (film):
3443, 3347 (NH2), 1739 (C@O), 1607, 1361 (SO2)
cmÀ1 1H NMR (CDCl3) d 3.67 (s, 2H, CH2COO),
;
3.72 (s, 3H, OCH3), 5.22 (br s, 2H, NH2), 7.36 (dd,
J = 8.2, 2.1 Hz, 1H, H-4), 7.71 (d, J = 8.2 Hz, 1H, H-
5), 8.06 (d, J = 2.1 Hz, 1H, H-2); Anal. Calcd for
C9H10BrNO4S: C, 35.08; H, 3.27; N, 4.55. Found: C,
35.41; H, 3.25; N, 4.38.
1
(C@O), 1607, 1502 (Ar), 1375 (SO2) cmÀ1; H NMR
(CDCl3 + DMSO) d 1.95 (s, 3H, COCH3), 3.56 (s, 2H,
CH2COOH), 7.07 (dd, J = 10.0, 8.5 Hz, 1H, H-5), 7.41
(ddd, J = 8.5, 4.6, 2.1 Hz, 1H, H-4), 7.86 (dd, J = 6.7,
2.1 Hz, 1H, H-2), 11.03 (br s, 1H, NH). Anal. Calcd
for C10H10FNO5S: C, 43.63; H, 3.66; N, 5.09. Found:
C, 43.79; H, 3.50; N, 4.97.
5.2.5. N-Acetyl-2-carboxymethyl-4-fluorobenzenesulfon-
amide (12). Acetic anhydride (0.3 mL, 3.08 mmol) and
4-dimethylaminopyridine (56 mg, 0.46 mmol) were
added to a solution of 4-fluoro-2-methoxycarbonyl-
methylbenzenesulfonamide (10a, 380 mg, 1.54 mmol)
in pyridine (1 mL), and the reaction was allowed to pro-
ceed overnight at 25 ꢁC with stirring. EtOAc (200 mL)
was added and this solution was washed successively
with saturated aqueous NH4Cl (2 · 50 mL) and H2O
(2 · 50 mL). The organic fraction was dried (Na2SO4)
and the solvent was removed in vacuo to afford the
5.2.8. N-Acetyl-6-bromo-3-methoxycarbonylmethylben-
zenesulfonamide (15). Compound 15 was prepared as a
white solid in 84% yield using an acetylation procedure
similar to that described previously for the synthesis of
compound 13; mp 150–152 ꢁC; IR (film): 3388 (NH),
1739 (C@O), 1348 (SO2) cmÀ1 1H NMR (CDCl3) d
;
2.11 (s, 3H, COCH3), 3.71 (s, 2H, CH2COO), 3.73 (s,
3H, OCH3), 7.44 (dd, J = 8.2, 2.1 Hz, 1H, H-4), 7.72
(d, J = 8.2 Hz, 1H, H-5), 8.20 (d, J = 2.1 Hz, 1H, H-2),
8.88 (br s, 1H, NH). Anal. Calcd for C11H12BrNO5S:
C, 37.73; H, 3.45; N, 4.00. Found: C, 38.00; H, 3.20;
N, 3.86.
intermediate
N-acetyl-4-fluoro-2-methoxycarbonyl-
methylbenzenesulfonamide product, which was dis-
solved in MeOH (15 mL). A solution of NaOH
(123 mg, 3.08 mmol) in H2O (15 mL) was added and
the reaction was allowed to proceed for 3 h at 80 ꢁC,

Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
4701
5.3. General procedure for the synthesis of N-acetyl-2-
carboxymethyl-4-(substituted-phenyl)benzenesulfon-
amides (17a–d)
H-3, H-5), 7.34–7.41 (m, 4H, H-3, H-5, isopropoxyphe-
nyl H-2, H-6), 8.00 (d, J = 8.2 Hz, 1H, H-6). Anal. Calcd
for C19H21NO6S: C, 58.30; H, 5.41; N, 3.58. Found: C,
58.10; H, 5.54; N, 3.32.
N-Acetyl-4-bromo-2-methoxycarbonylmethylbenzene-
sulfonamide (13, 145 mg, 0.41 mmol) and a substituted-
phenylboronic acid (16, 0.61 mmol) were dissolved in
DME (8 mL), and then aqueous Na2CO3 (0.61 mL of
2 M) followed by tetrakis(triphenylphosphine)palladium
(14 mg, 0.012 mmol) were added. The reaction was al-
lowed to proceed overnight at reflux, and the solvent
was removed in vacuo. MeOH (5 mL) and then water
(5 mL) were added, and the reaction was continued at
reflux for 1.5 h, cooled to 25 ꢁC, water (100 mL) was
added, the mixture was acidified to pH 3 using 5% w/v
HCl, and the mixture was extracted with EtOAc
(3 · 60 mL). The combined EtOAc extracts were washed
with water (2 · 50 mL), the organic fraction was dried
(Na2SO4), and the solvent was removed in vacuo to af-
ford the crude product. Purification of the product by
silica gel column chromatography using CHCl3–MeOH
(20:1, v/v) as eluant furnished the respective title com-
pounds (17a–c, or 17d). Some physical and spectral date
for 17a–d are listed below.
5.3.5. N-Acetyl-2-methoxycarbonylmethyl-4-(4-methane-
sulfonylphenyl)benzenesulfonamide (18). To a solution of
the aryl bromide (13, 145 mg, 0.41 mmol) and 4-(meth-
ylthio)phenylboronic acid (16, 102 mg, 0.61 mmol)
in DME (8 mL), aqueous Na2CO3 (0.61 mL of 2 M)
followed by tetrakis(triphenylphosphine)palladium
(14 mg, 0.012 mmol) were added. The reaction was al-
lowed to proceed at reflux for 5 h, and the solvent was
removed in vacuo. The residue obtained was dissolved
in THF (5 mL) and MeOH (5 mL), a solution of Oxone
(636 mg) in water (5 mL) was added, and the reaction
was allowed to proceed at 25 ꢁC for 1.5 h with stirring.
EtOAc (100 mL) was added and this solution was
washed with water (2 · 50 mL). The organic fraction
was dried (Na2SO4), and the solvent was removed in va-
cuo. The residue obtained was purified by silica gel col-
umn chromatography using CHCl3–MeOH (100:3, v/v)
as eluant to afford 18 (136 mg, 77%) as white needles;
mp 197–198 ꢁC; IR (film) 1745 (C@O), 1217 (SO2)
1
cmÀ1; H NMR (CDCl3) d 2.10 (s, 3H, COCH3), 3.12
5.3.1. N-Acetyl-2-carboxymethyl-4-phenylbenzenesulfon-
amide (17a). Yield, 36%, white crystals; mp 194–195 ꢁC;
IR (film): 3670 (NH), 3595–2417 (COOH), 1712 (C@O),
(s, 3H, SO2CH3), 3.75 (s, 3H, OCH3), 4.27 (s, 2H,
CH2COO), 7.59 (d, J = 1.5 Hz, 1H, H-3), 7.72 (dd,
J = 8.6, 1.5 Hz, 1H, H-5), 7.80 (d, J = 8.5 Hz, 2H, meth-
anesulfonylphenyl H-2, H-6), 8.07 (d, J = 8.5 Hz, 2H,
methanesulfonylphenyl H-3, H-5), 8.37 (d, J = 8.6 Hz,
1H, H-6), Anal. Calcd for C18H19NO7S2: C, 50.81; H,
4.50; N, 3.29. Found: C, 50.88; H, 4.13; N, 3.13.
1615, 1457 (Ar), 1247 (SO2) cmÀ1
;
1H NMR
(CDCl3 + DMSO) d 1.78 (s, 3H, COCH3), 3.94 (s, 2H,
CH2COOH), 7.16–8.00 (m, 8H, phenyl hydrogens).
Anal. Calcd for C16H15NO5S: C, 57.65; H, 4.54; N,
4.20. Found: C, 58.03; H, 4.62; N, 3.99.
5.3.6. N-Acetyl-2-carboxymethyl-4-(4-methanesulfonylphen-
yl)benzenesulfonamide (19). An aqueous solution of
K2CO3 (65 mg) in water (5 mL) was added to a stirred
solution of N-acetyl-2-methoxycarbonylmethyl-4-(4-
methanesulfonylphenyl)benzenesulfonamide (18, 100
mg, 0.23 mmol) in MeOH (5 mL). The reaction was al-
lowed to proceed with stirring at 80 ꢁC for 1.5 h, cooled
to 25 ꢁC, water (100 mL) was added, the mixture was
acidified to pH 3 using 5% w/v HCl, and the mixture
was extracted with EtOAc (3 · 60 mL). The combined
EtOAc extracts were washed with water (2 · 50 mL),
the organic fraction was dried (Na2SO4), and the solvent
was removed in vacuo to give 19 (78 mg, 81%) as white
crystals; mp 215–217 ꢁC; IR (film): 3677 (NH), 3602–
2440 (COOH), 1712 (C@O), 1630, 1465 (Ar), 1240
5.3.2. N-Acetyl-2-carboxymethyl-4-(4-fluorophenyl)ben-
zenesulfonamide (17b). Yield, 83%; pale yellow crystals;
mp 202–203 ꢁC; IR (film): 3670 (NH), 3580–2725
(COOH), 1712 (C@O), 1622, 1472 (Ar), 1247 (SO2)
1
cmÀ1; H NMR (CDCl3) d 1.79 (s, 3H, COCH3), 3.95
(s, 2H, CH2COOH), 6.96 (t, J = 8.6 Hz, 2H, fluorophen-
yl H-3, H-5), 7.33 (s, 1H, H-3), 7.40–7.50 (m, 3H, H-5,
fluorophenyl H-2, H-6), 8.02 (d, J = 8.2 Hz, 1H, H-6),
11.6 (br s, 1H, NH). Anal. Calcd for C16H14FNO5S: C,
54.70; H, 4.02; N, 3.99. Found: C, 55.05; H, 4.04; N, 3.69.
5.3.3. N-Acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)-
benzenesulfonamide (17c). Yield, 59%; white crystals;
mp 199–200 ꢁC; IR (film): 3670 (NH), 3587–2432
(COOH), 1720 (C@O), 1622, 1472 (Ar), 1240 (SO2)
(SO2) cmÀ1 1H NMR (CDCl3 + DMSO) d 1.75 (s,
;
cmÀ1 1H NMR (CDCl3 + DMSO) d 1.70 (s, 3H,
;
3H, COCH3), 2.91 (s, 3H, SO2CH3), 3.96 (s, 2H,
CH2COO), 7.41 (br s, 1H, H-3), 7.45 (br d, J = 8.2 Hz,
1H, H-5), 7.61 (d, J = 8.2 Hz, 2H, methanesulfonylphe-
nyl H-2, H-6), 7.81 (d, J = 8.2 Hz, 2H, methanesulfonyl-
phenyl H-3, H-5), 8.06 (d, J = 8.5 Hz, 1H, H-6). Anal.
Calcd for C17H17NO7S2Æ1/2H2O: C, 48.56; H, 4.32; N,
3.33. Found: C, 48.44; H, 4.26; N, 3.24.
COCH3), 3.86 (s, 2H, CH2COOH), 6.64–6.76 (m, 2H,
difluorophenyl H-3, H-5), 7.13–7.33 (m, 3H, difluo-
rophenyl H-6, H-3, H-5), 7.92 (d, J = 8.2 Hz, 1H, H-
6). Anal. Calcd for C16H13F2NO5S: C, 52.03; H, 3.55;
N, 3.79. Found: C, 51.94; H, 3.53; N, 3.69.
5.3.4. N-Acetyl-2-carboxymethyl-4-(4-isopropoxyphenyl)-
benzenesulfonamide (17d). Yield, 52%; white crystals;
mp 193–194 ꢁC; IR (film): 3677 (NH), 3602–2717
(COOH), 1727 (C@O), 1630, 1525, 1472 (Ar), 1240
5.4. General procedure for the synthesis of N-acetyl-3-
carboxymethyl-6-(substituted-phenyl)benzenesulfon-
amides (20a–d)
(SO2) cmÀ1 1H NMR (CDCl3 + DMSO) d 1.18 [d,
;
J = 5.6 Hz, 6H, CH(CH3)2], 1.80 (s, 3H, COCH3), 3.95
(s, 2H, CH2COOH), 4.43 [hept, J = 5.6 Hz, 1H,
CH(CH3)2], 6.77 (d, J = 8.5 Hz, 2H, isopropoxyphenyl
N-Acetyl-6-bromo-3-methoxycarbonylmethylbenzene-
sulfonamide (15, 100 mg, 0.29 mmol) and a substituted-

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Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
phenylboronic acid (16, 0.43 mmol) were dissolved in
DME (5 mL), and then aqueous Na2CO3 (0.42 mL of
2 M) followed by tetrakis(triphenylphosphine)palladium
(10 mg, 0.0086 mmol) were added. The reaction was re-
fluxed overnight, and the solvent was removed in vacuo.
Water (80 mL) was added, the mixture was acidified to
pH 3 using 5% w/v HCl, and the mixture was extracted
with EtOAc (3 · 50 mL). The combined EtOAc extracts
were washed with water (2 · 30 mL), the organic frac-
tion was dried (Na2SO4), and the solvent was removed
in vacuo to afford the crude product. Purification of
the product by silica gel column chromatography using
CHCl3–MeOH (20:1, v/v) as eluant gave the respective
title compound (20a–d). Some physical and spectral data
for 20a–d are listed below.
5.4.5. N-Acetyl-3-carboxymethyl-6-(4-methanesulfonyl-
phenyl)benzenesulfonamide (20e). An aqueous solution
of Oxone (270 mg, 0.42 mmol, 5 mL) was added to a
stirred solution of the methylthiophenyl compound
(20d, 80 mg, 0.21 mmol) in methanol (5 mL), and the
reaction was allowed to proceed with stirring at 25 ꢁC
for 1.5 h. Addition of H2O (60 mL), extraction with
EtOAc (3 · 50 mL), drying the combined EtOAc ex-
tracts (Na2SO4), and removal of the solvent in vacuo
afforded 20e (50 mg, 58%) as a pale yellow foam; mp
117–118 ꢁC; IR (film): 3685 (NH), 3602–2492 (COOH),
1727 (C@O), 1630, 1465 (Ar), 1375 (SO2) cmÀ1 1H
;
NMR (CDCl3 + DMSO) d 1.81 (s, 3H, COCH3), 3.10
(s, 3H, SO2CH3), 3.74 (s, 2H, CH2COOH), 7.20 (d,
J = 8.0 Hz, 1H, H-5), 7.55 (d, J = 8.2 Hz, 2H, meth-
anesulfonylphenyl H-2, H-6), 7.59 (br d, J = 8.0 Hz,
1H, H-4), 7.95 (d, J = 8.2 Hz, 2H, methanesulfonylphe-
nyl H-3, H-5), 8.19 (br s, 1H, H-2). 11.1 (br s, 1H, NH),
Anal. Calcd for C17H17NO7S2: C, 49.62; H, 4.16; N,
3.40. Found: C, 49.83; H, 4.16; N, 3.31.
5.4.1. N-Acetyl-3-carboxymethyl-6-phenylbenzenesulfon-
amide (20a). Yield, 50%, white foam; mp 189–190 ꢁC; IR
(film): 3663 (NH), 3601–2467 (COOH), 1719 (C@O),
1623, 1478 (Ar), 1238 (SO2) cmÀ1
;
1H NMR
(CDCl3 + DMSO) d 1.72 (s, 3H, COCH3), 3.67 (s, 2H,
CH2COOH), 7.18 (d, J = 8.0 Hz, 1H, H-5), 7.38–7.24
(m, 5H, C-6 phenyl hydrogens); 7.51 (dd, J = 8.0,
1.8 Hz, 1H, H-4), 8.11 (d, J = 1.8 Hz, 1H, H-2). Anal.
Calcd for C16H15NO5SÆ1/4H2O: C, 56.87; H, 4.62; N,
4.15. Found: C, 56.79; H, 4.52; N, 4.00.
5.5. Molecular modeling (docking) study
Docking experiments were performed using Insight II
software Version 2000.1 (Accelrys Inc.) running on a Sili-
con Graphics Octane 2 R14000A workstation according
to a previously reported method.15
5.4.2. N-Acetyl-3-carboxymethyl-6-(4-fluorophenyl)ben-
zenesulfonamide (20b). Yield, 26%; white foam; mp
114–115 ꢁC; IR (film): 3482 (NH), 3395–2468 (COOH),
5.6. In vitro cyclooxygenase (COX) inhibition assay
1716 (C@O), 1635, 1461 (Ar), 1380 (SO2) cmÀ1
;
1H
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 number 560101, Cayman Chemical, Ann Ar-
bor, MI, USA) according to our previously reported
method.15
NMR (CDCl3 + DMSO) d 1.68 (s, 3H, COCH3), 3.59
(s, 2H, CH2COOH), 6.98 (t, J = 8.6 Hz, 2H, fluorophe-
nyl H-3, H-5), 7.08 (d, J = 8.0 Hz, 1H, H-5), 7.18 (m,
2H, fluorophenyl H-2, H-6), 7.43 (br d, J = 8.0 Hz,
1H, H-4), 8.01 (br s, 1H, H-2). Anal. Calcd for
C16H14FNO5SÆ1/2H2O: C, 53.33; H, 4.20; N, 3.89.
Found: C, 53.37; H, 4.34; N, 3.63.
5.7. Anti-inflammatory assay
5.4.3. N-Acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)-
benzenesulfonamide (20c). Yield, 54%; white powder; mp
100–102 ꢁC; IR (film): 3388–2492 (COOH), 1716
Anti-inflammatory activity was performed using a meth-
od described by Winter et al.16
1
(C@O), 1630, 1474 (Ar), 1380 (SO2) cmÀ1; H NMR
5.8. Analgesic assay
(CDCl3 + DMSO) d 1.39 [d, J = 5.8 Hz, 6H, CH(CH3)2],
1.80 (s, 3H, COCH3), 3.81 (s, 2H, CH2COOH), 4.62
[hept, J = 5.8 Hz, 1H, CH(CH3)2], 6.97 (d, J = 8.6 Hz,
2H, isopropoxyphenyl H-3, H-5), 7.29 (d, J = 7.0 Hz,
1H, H-5), 7.32 (d, J = 8.6 Hz, 2H, isopropoxyphenyl
H-2, H-6), 7.59 (br d, J = 7.0 Hz, 1H, H-4), 8.19 (br s,
1H, H-2). Anal. Calcd for C19H21NO6SÆ1/4 H2O: C,
57.63; H, 5.47; N, 3.54. Found: C, 57.57; H, 5.26; N,
3.29.
Analgesic activity was determined using a 4% sodium
chloride-induced writhing (abdominal constriction) as-
say previously reported.17
Acknowledgments
We are grateful to the Canadian Institutes of Health Re-
search (CIHR) (MOP-14712) for financial support of
this research, Rx&D-HRF/CIHR for a postdoctoral fel-
lowship (to Q.H.C.), and to the Alberta Heritage Foun-
5.4.4. N-Acetyl-3-carboxymethyl-6-(4-methylthiophenyl)-
benzenesulfonamide (20d). Yield, 70%; white powder; mp
172–174 ꢁC; IR (film): 3664–2569 (COOH), 1727
dation for Medical Research (AHFMR) for
postdoctoral fellowship award (to Q.H.C.).
a
1
(C@O), 1630, 1465 (Ar), 1375 (SO2) cmÀ1; H NMR
(CDCl3) d 1.83 (s, 3H, COCH3), 2.54 (s, 3H, SCH3),
3.82 (s, 2H, CH2COOH), 7.21–7.34 (m, 5H, H-5, meth-
ylthiophenyl H-2, H-3, H-5, H-6), 7.63 (d, J = 7.8 Hz,
1H, H-4), 8.18 (br s, 1H, H-2). Anal. Calcd for
C17H17NO5S2Æ1/2H2O: C, 52.56; H, 4.67; N, 3.60.
Found: C, 52.45; H, 4.32; N, 3.50.
References and notes
1. (a) Hamberg, M.; Samuelsson, B. J. Biol. Chem. 1967, 242,
5336; (b) Miyamoto, T.; Ogino, N.; Yamamoto, S.;

Q.-H. Chen et al. / Bioorg. Med. Chem. 13 (2005) 4694–4703
4703
Hayaishi, O. J. Biol. Chem. 1976, 251, 2629; (c) Fletcher,
B. S.; Kujubu, D. A.; Perrin, D. M.; Herschman, H. R. J.
Biol. Chem. 1992, 267, 4338.
F.; Rowlinson, S. W.; Caprioli, R. M.; Marnett, L. Arch.
Biochem. Biophys. 2003, 409, 127.
7. Rahim, M. A.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med.
Chem. Lett. 2002, 12, 2753.
8. Sykes, N. O.; Macdonald, S. J. F.; Page, M. I. J. Med.
Chem. 2002, 45, 2850.
9. Zarghi, A.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med.
Chem. Lett. 2004, 14, 1975.
10. Chen, Q. H.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med.
Chem. 2005, 13, 2459.
11. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
12. Stanforth, S. P. Tetrahedron 1998, 54, 263.
13. Sianesi, E.; Redaelli, R.; Bertani, M.; Da Re, P. Chem.
Ber. 1970, 103, 1992.
14. Bayly, C. I.; Black, W. C.; Leger, S.; Ouimet, N.; Ouellet,
M.; Percival, M. D. Bioorg. Med. Chem. Lett. 1996, 9, 307.
15. Uddin, M. J.; Rao, P. N. P.; Knaus, E. E. Bioorg. Med.
Chem. 2004, 12, 5929.
16. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp.
Biol. Med. 1962, 111, 544.
2. Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu. Rev.
Pharmacol. Toxicol. 1998, 38, 97.
3. Meade, E. A.; Smith, W. L.; DeWitt, D. L. J. Biol. Chem.
1993, 268, 6610.
4. (a) Silverstein, F. E.; Faich, G.; Goldstein, J. L.; Simon, L.
S.; Pincus, T.; Whelton, A.; Makuch, R.; Eisen, G.;
Agrawal, N. M.; Stenson, W. F.; Burr, A. M.; Zhao, W.
W.; Kent, J. D.; Lefkowith, J. B.; Verburg, K. M.; Geis,
G. S. J. Am. Med. Assoc. 2000, 284, 1247; (b) Hawkey, C.;
Laine, L.; Simon, T.; Beaulieu, A.; Maldonado, C. J.;
Acevedo, E.; Shahane, A.; Quan, H.; Bolognese, J.;
Mortensen, E. Arthritis Rheum. 2000, 43, 370.
5. Mukherjee, D.; Nissen, S. E.; Topol, E. J. J. Am. Med.
Assoc. 2001, 286, 954, and references cited therein.
6. (a) Kalgutkar, A. S.; Crews, B. C.; Rowlinson, S. W.;
Garner, C.; Seibert, K.; Marnett, L. J. Science 1998, 280,
1268; (b) Kalgutkar, A. S.; Kozak, K. R.; Crews, B. C.;
Hochgesang, G. P., Jr.; Marnett, L. J. J. Med. Chem. 1998,
41, 4800; (c) Hochgesang, G. P., Jr.; Nemeth-Cawley, J.
17. Fukawa, K.; Kawano, O.; Hibi, M.; Misaka, N.; Ohba, S.;
Hatanaka, Y. J. Pharmacol. Methods 1980, 4, 251.