Selective COX-2 inhibitors. Part 1: Synthesis and biological evaluation of phenylazobenzenesulfonamides

Article abstract of DOI:10.1016/j.bmcl.2006.06.036

A series of phenylazobenzenesulfonamide derivatives were designed and synthesized for the evaluation as selective cyclooxygenase-2 (COX-2) inhibitors in a cellular assay using human whole blood (HWB) and an enzymatic assay using purified ovine enzymes. Ex

Full text of DOI:10.1016/j.bmcl.2006.06.036

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4440–4443
Selective COX-2 inhibitors. Part 1: Synthesis and biological
evaluation of phenylazobenzenesulfonamides
Wei-Jern Tsai,a Young-Ji Shiao,a Shwu-Jiuan Lin,b,* Wen-Fei Chiou,a Lie-Chwen Lin,a
Tsang-Hsiung Yang,b Che-Ming Teng,c Tain-Shung Wua and Li-Ming Yanga,b,
*
aDivision of Medicinal Chemistry, National Research Institute of Chinese Medicine, 155-1, Li-Nong Street, Sec. 2, Taipei 112, Taiwan
bDepartment of Medicinal Chemistry, College of Pharmacy, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan
cPharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
Received 10 May 2006; revised 6 June 2006; accepted 12 June 2006
Available online 30 June 2006
Abstract—A series of phenylazobenzenesulfonamide derivatives were designed and synthesized for the evaluation as selective cyclo-
oxygenase-2 (COX-2) inhibitors in a cellular assay using human whole blood (HWB) and an enzymatic assay using purified ovine
enzymes. Extensive structure–activity relationships (SAR) were studied within this series, and several of selective COX-2 inhibitors
have been identified. Among them, compound 8, 4-(4-amino-2-methylsulfanyl-phenylazo)benzenesulfonamide, showed a potent
inhibitory activity to the cyclooxygenase enzymes (IC50’s for COX-1: 23.28 lM; COX-2: 2.04 lM), being active but less COX-2
selective than celecoxib.
Ó 2006 Elsevier Ltd. All rights reserved.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are
widely used for the treatment of pain, fever, and inflam-
mation. Conventional NSAIDs act as nonselective
inhibitors of cyclooxygenase (COX) enzymes, which cat-
alyze the formation of prostaglandins (PGs) from ara-
chidonic acid. There are at least two mammalian COX
isoforms.1,2 COX-2 is induced in response to proinflam-
matory conditions, while COX-1 is constitutive and
responsible for the maintenance of physiological homeo-
stasis, such as gastrointestinal integrity and renal
function. Traditional NSAIDs such as aspirin, indo-
methacin, and diclofenac are nonselective inhibitors
and are associated with gastric ulceration, bleeding,
and renal function suppression.3 Generally, prostacyclin
and thromboxane normally balance each other’s oppos-
ing effects. Introducing the selective COX-2 inhibitor
may bias the balance dangerously toward thromboxane,
raising blood pressure, possibly hardening arteries and
certainly promoting heart-attack and stroke-causing
clots in some patients.4 This couldÒexplain the cardiotox-
icity caused by rofecoxib (Vioxx ),5 and subsequently
valdecoxib (BextraÒ),6 which were recently withdrawn
from the market.7 Thus, there is an urgent need to
search for new selective COX-2 inhibitors with a mild
therapeutic effect on COX-1, which could theoretically
reduce cardiovascular side effects due to their anti-
platelet and anti-thrombotic activities, and improve
the safety profiles.8
The majority of selective COX-2 inhibitors belong to a
class of tricyclic compounds possessing 1,2-diaryl substi-
tution on a central heterocyclic, or carbocyclic ring sys-
tem.9 Extensive structure–activity relationship (SAR)
studies for the diarylheterocycle compounds have shown
that a SO2NH2, or a SO2Me substituent at the para-po-
sition of one of the phenyl rings often provides optimum
selective COX-2 inhibitory potency (Fig. 1).10 Recently,
a number of naturally occurring trans-stilbenoids have
been reported as inhibitors of COX. For example, resve-
ratrol (3,40,5-trihydroxy-trans-stilbene) is a phytoalexin
H2NO2S
MeO2S
H2NO2S
N
N
CF3
O
O
Keywords: Anti-inflammatory; Selective COX-2 inhibitors; Phenylazo-
benzenesulfonamide; Resveratrol; Isosterism; SAR.
N
O
*
Celecoxib (Celebrex®
)
Rofecoxib (Vioxx®
)
Valdecoxib (Bextra®
)
Corresponding authors. Tel.: +886 2 2820 1999x8551; fax: +886 2
2826 4276; e-mail addresses: lmyang@nricm.edu.tw; shwu-lin@
tmu.edu.tw
Figure 1. Selective tricyclic COX-2 inhibitors.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.036

W.-J. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4440–4443
4441
O
S
O
S
found mainly in the skin of grapes and red wine. It has
been demonstrated to have antioxidant, anti-inflamma-
tory, cardioprotective (the so-called ‘French para-
dox’),11 and cancer chemopreventive properties.12 It
has also been shown to exhibit moderate selective
COX-1 inhibitory activity.13
14a-g
N+ NCl−
NaOAc or NaOH
NaNO2
HCl
H2N
NH2
H2N
O
O
13
1. R4= NMe2
2. R4= OH
O
3. R3= OH, R4= OH
4. R3= OMe, R4= OH
8. R2= SMe, R4= NH2
9. R3= CO2H, R4= OH
H2N
S
N
4
N
O
R
2
3
As an alternative to convert a COX-1 selective com-
pound into a COX-2 selective inhibitor, we have de-
signed and modified the basic skeleton of trans-stilbene
by the concept of isosterism,14 which lacks a traditional
central heterocyclic Òor carbocyclic ring template such as
celecoxib (Celebrex ).15 We have previously reported
that the 4-(4-dimethylamino-phenylazo)benzenesulfona-
mide (1) demonstrates a high potency against the amy-
loid-b (Ab) aggregation, which is responsible for
neuro-inflammation in Alzheimer’s disease.16 However,
whether these phenylazobenzenesulfonamide derivatives
possess a selective COX-2 inhibitory activity has not yet
been investigated. In this paper, we describe the synthe-
sis of these compounds and evaluate their inhibitory
potency against COX-1/COX-2 in vitro.
10.R3= Cl,
R4= OH
Scheme 1. Synthesis of phenylazobenzenesulfonamide derivatives 1–4
and 8–10. Reagents: 14a, N,N-dimethylaniline; 14b, phenol; 14c,
catechaol; 14d, guaiacole; 14e, 3-(methylthio)aniline; 14f, salicyclic
acid; 14g, 2-chlorophenol.
O
CH2N2
2
3
H2N
H2N
S
N
N
N
OMe
OMe
O
6
O
S
CH2N2
N
O
5. R= H
7. R= Me
OR
Structural modifications of the phenylazobenzenesulf-
onamide derivatives include (a) replacing the para-hy-
droxyl group of resveratrol by the para-sulfonamide
moiety, and (b) the bioisosteric replacement of the cen-
tral ethylenic linkage with an azo N,N-double bond.
Additionally, we modified the various substituents on
the phenylazo ring B in order to obtain compounds with
the high COX-2 selectivity and potency that we were
seeking. These strategies are illustrated in Figure 2.
Scheme 2. Synthesis of phenylazobenzenesulfonamide derivatives 5–7.
O
15a or 15b
NaH, DMF
H2N
S
N
3
N
OR
OR
O
11. R= -CONHC6H5
12. R= -CONHC6H4-p-iPr
Scheme 3. Synthesis of phenylazobenzenesulfonamide derivatives
11–12. Reagents: 15a, phenyl isocyanate; 15b, 4-isopropylphenyl
isocyante.
The target phenylazobenzenesulfonamides (1–12) were
synthesized in a manner similar to that described in
our previous publication.16 The procedures for prepa-
ration of these compounds are summarized in Schemes
1–3. Although the synthesis of these derivatives has al-
ready been reported,17 neither their selective COXs
inhibitory activity nor the possible chemical conversion
has been described or characterized previously. The
synthesis involved initial diazotization of sulfanilamide
with sodium nitrite in hydrochloride solution to afford
the diazonium salt 13, followed by coupling with a mo-
lar equivalent of the appropriate aromatic compounds
14a–g, in sodium acetate or sodium hydroxide solution
at 0 °C. The reactions were accompanied by a color
change from pale yellow to deep brown or reddish or-
ange and gave the corresponding phenylazobenzene-
sulfonamide derivatives 1–4 and 8–10 (Scheme 1).
The phenol 2 was treated with diazomethane to give
anisole derivative 6 in 94% yield. Similarly, catechol 3
was converted to the monomethylated 5 in 50% yield
and, with a large amount of diazomethane, to veratrole
derivative 7 in almost a quantitative yield (Scheme 2).
The carbamate derivatives were readily prepared from
the catechol 3 with 2.2 molar excess of phenyl isocya-
nate or 4-isopropylphenyl isocyanate in the presence
of sodium hydride in DMF at 0 °C to give the desired
carbamates 11 and 12, respectively, in yields ranging
from 70% to 75% (Scheme 3). The proposed structures
were characterized by detailed 1H, 13C NMR
(HMQC, HMBC, COSY), high resolution electron
impact (HR-EIMS) analyses, and analytical thin-layer
chromatography.
SAR studies of the phenylazobenzenesulfonamides for
the selective COX-2 inhibitors. We have evaluated the
selectivity of COXs inhibitory potency in a cellular assay
using human whole blood (HWB) and in an enzymatic
assay using purified ovine COX enzymes.18,19 The IC50
micromolar values for COXs were determined for select-
ed/promising compounds. Indomethacin, resveratrol,
and celecoxib were evaluated as reference compounds
for the in vitro assay. The COX-1 activity was measured
as the TXB2 production after stimulation of platelet
aggregation by calcium ionophore A-23,187. The
COX-2 activity was measured as the PGE2 levels
produced by leukocytes after stimulation by LPS.20
Classic bioisosterism
O
S
ring B
OH
HO
H2N
N
N
O
R
OH
Resveratrol
Phenylazobenzenesulfonamides
Figure 2. Design concept of phenylazobenzenesulfonamides.

4442
W.-J. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4440–4443
Table 1. Inhibitory activity of phenylazobenzenesulfonamides using human whole blood assays
a
Compound
R
HWB IC50 (lM)
Selectivity index
COX-1/COX-2
COX-1
COX-2
1
4-N(CH3)2
4-OH
3,4-(OH)2
>100
>100
na
na
ns
ns
2
3
59.96
>100
12.42
16.85
4.83
7.91
ns
4
3-OCH3, 4-OH
3-OH, 4-OCH3
4-OCH3
5
66.41
>100
na
14.63
6
9.48
14.42
11.40
3.55
2.94
3.17
2.12
0.13
0.09
60.25
7
3,4-(OCH3)2
61.73
23.28
4.28
2.04
8
2-SCH3, 4-NH2
3-CO2H, 4-OH
3-Cl, 4-OH
9
10
31.57
33.32
8.89
11.34
11
12
3,4-(OCONHC6H5)2
3,4-(OCONHC6H4–p-i-C3H7)2
35.07
26.26
11.07
12.37
Indomethacin
Resveratrol
Celecoxib
0.005 0.001
2.84 1.90
20.03 1.58
0.036 0.004
33.15 3.50
0.33 0.10
na, no activity; ns, nonselective.
a Values are means SEM from three independent experiments using COXs assay kits (Catalog Nos. 519031 and 514010 Cayman Chemicals Inc.,
Ann Arbor, MI, USA). Since SEM values never exceeded 15% of the media, they have been omitted.
inhibitory activity. Interestingly, introduction of the
3,4-dimethoxy moiety, 7 (COX-2 IC50 = 4.28 lM), pro-
vided excellent activity and selectivity than the parent
compound 3 (COX-2 IC50 = 12.42 lM).
Table 2. In vitro COXs enzyme inhibition data for compounds 7–10
a
Compound
IC50 (lM)
Selectivity index
COX-1/COX-2
COX-1
COX-2
7
>100
34.09
84.5
18.0
ns
8
1.89
1.24
1.02
0.01
0.11
60.48
Replacement of the 3,4-dihydroxy moiety of 3 by a 3,4-
di-N-phenylcarbamate, 11, and a 3,4-di-N-(4-isopropyl-
phenyl)carbamate, 12, group was investigated for selec-
tivity and potency. As shown in Table 1, the selective
COX-2 inhibitory activities were decreased gradually
in proportion to the size of the substituents. But interest-
ingly, the COX-1 activities were enhanced as the substit-
uents became bulkier. Consequently, compound 7 was
found to be a preferential COX-2 inhibitor (SI value
of 14.42). Indeed, compound 8 displayed a greater
COX-2 inhibitory profile when compared to phenylazo-
9
10
29.25
40.53
23.6
39.7
Indomethacin
Resveratrol
Celecoxib
0.004 0.001
0.38 0.17
26.61 8.49
0.34 0.07
3.49 1.44
0.44 0.06
ns, nonselective.
a Values are means SEM from three independent experiments using
an ovine COXs assay kit (Catalog No. 560101, Cayman Chemicals
Inc., Ann Arbor, MI, USA). Since SEM values never exceeded 15%
of the media, they have been omitted.
benzenesulfonamide in this series (COX-2 IC50
=
The selectivity index (SI, COX-1 IC50/ COX-2 IC50) was
calculated. The results of inhibition are summarized in
Tables 1 and 2.
2.04 1.57 lM), although it appeared to be less selective
against COX-2 (SI of 11.40 and 60.25 for 8 and celecox-
ib, respectively).
Compound 1 showed a remarkable inhibitory activity
against amyloid-b (Ab) aggregation.16 Surprisingly, this
compound completely lost the COX-2 inhibitory activity
in the HWB assay.19,20 Compounds with various substit-
uents on the phenylazo ring B were examined to deter-
mine whether the substituents affect the COX-2
activity and selectivity, as outlined in Table 1. The intro-
duction of a 4-hydroxy, 2, or a 3-hydroxy-4-methoxy, 5,
group showed to be devoid of COX-2 inhibitory activi-
ty. The presence of a 4-hydroxy-3-methoxy, 4, or a 4-
methoxy, 6, group led to a complete loss of both
COX-2 and COX-1 inhibitory activities.
From these results, we further evaluated more potent
derivatives, 7–10, as selective COX-2 inhibitors in puri-
fied ovine enzymes assays.21 As described in Table 2, all
of the substituents mentioned above lost or showed
diminished COX-2 selectivity. However, compound 8
showed a remarkable increase in COX-2 selectivity
(IC50 = 18.0 lM; SI = 1.89). The reasons for these dis-
crepancies are at present unknown.
In conclusion, we have identified a series of novel phenyl-
azobenzenesulfonamides that are potent but moderately
selective COX-2 inhibitors with an appropriate balance
between COX-1 and COX-2 inhibition. These results con-
firm that the dihydroxy compound 3 was a less potent
COX-2 selective inhibitor than its corresponding dime-
thoxy compound 7. However, replacement of the dime-
thoxy moiety in 7 with carbamate analogues yielded 11
and 12, which showed diminished COX-2 inhibitory
activity. Among the SAR examined, compound 8 was
The SAR studies demonstrated that the parent com-
pound, 3,4-dihydroxyl (3), exhibited moderate COX-2
inhibitory potency. However, replacement of the 3-hy-
droxy moiety in 3 with a 3-chloro, 10, retained some
COX-2 potency, but less active than that of a 3-carboxy,
9, group. Also, these compounds enhanced COX-1

W.-J. Tsai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4440–4443
4443
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Acknowledgments
We gratefully thank the National Science Council of
Republic of China Grants NSC-94-2320-B-077-005
(L.-M.Y.) and NSC-94-2320-B-038-030 (S.-J.L.), and
National Research Institute of Chinese Medicine for
research funding (Grant NRICM-95-DMC-007).
13. Szewczuk, L. M.; Forti, L.; Stivala, L. A.; Penning, T. M.
J. Biol. Chem. 2004, 279, 22727.
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