Isomeric Acetoxy Analogues of Rofecoxib: A Novel Class of
Highly Potent and Selective Cyclooxygenase-2 Inhibitors
M. Abdur Rahim, P. N. Praveen Rao and Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
Received 26 March 2002; accepted 17 June 2002
Abstract—A group of isomers possessing a 2-, 3-, or 4-acetoxy moiety on the 3-phenyl substituent of rofecoxib were synthesized
that exhibit highly potent, and selective, COX-2 inhibitory activity that have the potential to acetylate the COX-2 isozyme.
# 2002 Elsevier Science Ltd. All rights reserved.
The historical belief that a single cyclooxygenase (COX)
enzyme catalyzed the bioconversion of arachidonic acid
to prostaglandinsand thromboxanes,
responsible for both the therapeutic anti-inflammatory
and associated gastrointestinal and renal toxicity exhib-
(NSAIDs), required modification following the dis-
covery that there are two isozymes, COX-1 and COX-
2.1The constitutive COX-1 isozyme is produced in a
variety of tissues and appears to be important to the
maintenance of physiological functions such as gastro-
protection and vascular homeostasis.2Alternatively, the
COX-2 isozyme is induced by mitogenic and proin-
flammatory stimuli3linking its involvement to inflam-
matory processes.4The initial concept that a selective
COX-2 inhibitor would illicit effective antiinflammatory
activity without the adverse ulcerogenic effect associated
with the use of NSAIDs that inhibit both COX-1 and
COX-2 has been validated by postmarket clinical stud-
ies which attest to the efficacy of the selective COX-2
inhibitors rofecoxib (1)5and celecoxib (2).6
Aspirin (3) is a unique non-selective COX inhibitor due
to its ability to acetylate the serine hydroxyl group in
the COX binding 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-2.7Some of aspirin’s bene-
ficial therapeutic effects can be attributed to acetylation
of COX-2, while its antithrombotic and ulcergenic effects
are due to acetylation of COX-1. These observations
were elegantly exploited in the design of the aspirin
analogue o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS,
4) that selectively acetylated, and irreversibly inacti-
vated, COX-2.8More recently biological data was
acquired that suggests the diastereomeric acyl-CoA-
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
Bioorganic & Medicinal Chemistry Letters 12 (2002) 2753–2756
*Corresponding author. Tel.: +1-780-492-5993; fax: +1-780-492-
1217; e-mail: email@example.com
ketoprofen conjugates (5) are reversible inhibitors of
COX-1 and irreversible inhibitors of COX-2.9
A special communication has raised a cautionary flag
regarding the use of COX-2 inhibitors in patients at risk
for cardiovascular morbidity such as myocardial infarc-
tion that has been explained using the following facts.10
The COX-1 isozyme is expressed in platelets and it
mediates production of the potent platelet activator and
aggregator thromboxane A2(TxA2). On the other hand,
COX-2 produces prostaglandins at the sites of inflam-
mation as well as PGI2, which is a vasodilator and
inhibitor of platelet aggregation. Although selective
COX-2 inhibitors have no effect on TxA2production,
by decreasing PGI2production, selective COX-2 inhibi-
tors may tip the natural balance between prothrombotic
TxA2and antithrombotic PGI2that could potentially
increase the possibility of a thrombotic cardiovascular
event.10As part of our ongoing program to design
COX-2 inhibitors, we now describe a novel class of
highly selective and potent inhibitors of COX-2 that
also have the potential to selectively acetylate COX-2 at
The target 3-(2-, 3-, or 4-acetoxyphenyl)-4-(4-methane-
sulfonylphenyl)-2(5H)furanone isomers (13a–c) were
synthesized using the reaction sequence illustrated in
Scheme 1. Accordingly, bromination of 4-methylthio-
acetophenone (7), prepared in 95% yield by Friedel–
Crafts acetylation of thioanisole (6), afforded the bro-
moacetyl derivative (8, 90%). Condensation of 8 with
either 2-, 3-, or 4-methoxyphenylacetic acid in the pre-
sence of Et3N yielded the respective isomeric 4-methyl-
thiophenacyl 2-, 3-, or 4-methoxyphenyl acetate (9a–c,
41–58%). Cyclization of the isomers (9a–c) using NaH
in DMSO gave the respective 3-(2-, 3-, or 4-methoxy-
(10a–c, 58–75%) which on O-demethylation using neat
pyridinium chloride11at 190–210?C yielded the corre-
sponding phenol derivative (11a–c, 57–77%). Acetyla-
tion of 11a–c gave the respective 3-(2-, 3-, or 4-
acetoxyphenyl) isomer (12a–c, 78–100%). Subsequent
oxidation of 12a–c using Oxone1(-SMe!-SO2Me)
afforded the corresponding isomeric product (13a–c,
The 3-(2-, 3-, or 4-hydroxyphenyl)-4-(4-methanesulfo-
nylphenyl)-2(5H)furanone isomers (15a–c) were synthe-
sized using the reaction sequence illustrated in Scheme 2.
Thus, oxidation of the methylthio isomers (10a–c) to the
corresponding methanesulfonyl derivative (14a–c, 77–
82%) using Oxone1, and subsequent O-demethylation
using pyridinium hydrochloride gave the respective 3-(2-,
3-, or 4-hydroxyphenyl) product (15a–c, 32–46%).
A group of 3-(2-, 3-, and 4-acetoxyphenyl) analogues
(13a–c) of rofecoxib were prepared to investigate the
effect of isomeric 2-, 3-, and 4-acetoxy substituents on
COX-2 selectivity and potency. In vitro COX-1/COX-2
inhibition studies showed that 13a–c, which do not
inhibit COX-1 (IC50values >100 mM), are potent inhib-
itors of COX-2 (IC50values in the 0.00126–0.00350 mM
range) with high COX-2 selectivity indexes (SIs in the
28,482 to >79,365 range) relative to the reference drug
rofecoxib (COX-2 IC50=0.4279 mM; SI >1168) as
summarized in Table 1. These data suggest that the
acetoxy isomers 13a–c should inhibit the synthesis of
inflammatory prostaglandins via the cyclooxygenase
pathway at sites of inflammation and be devoid of
ulcerogenicty due to the absence of COX-1 inhibition.
Aspirin treatment of human prostaglandin endoper-
oxide H synthase (hPGHS-1, hCOX-1) expressed in cos-
1 cells causes a time dependent inactivation of oxygenase
Scheme 1. Reagents and conditions: (a) AlCl3, AcCl, CHCl3, 0–10?C, 1 h; (b) Br2, CHCl3, 25?C, 30 min; (c) 2-, 3-, or 4-MeO–C6H4–CH2CO2H,
Et3N, MeCN, 25?C, 1 h; (d) NaH, DMSO, 25?C, 1 h; (e) pyridinium hydrochloride, 190–210?C, 1 h; (f) AcCl, Et3N, 0!25?C, 1 h; (g) Oxone1,
MeOH, THF, H2O, 25?C, 18 h.
Scheme 2. Reagents and conditions: (a) Oxone1, MeOH, THF, H2O,
25?C, 18 h; (b) pyridinium hydrochloride, 190–210?C, 1 h.
2754 M. Abdur Rahim et al./Bioorg. Med. Chem. Lett. 12 (2002) 2753–2756
activity. In contrast, treatment of PGHS-2 (COX-2)
produced an enzyme that retained oxygenase activity,
butwhich formed the
5,8,11,13-eicosatetraenoic acid [(15R)-HETE] exclu-
sively that is a precursor to leukotrienes via the lipoxy-
genase (5-LO) pathway rather than prostaglandin H2
(PGH2) produced via the cyclooxygenase pathway. The
Kmvalues for arachidonate of native and aspirin-treated
hPGHS-2 were similar suggesting that arachidonate
binds to both aspirin-treated and native hPGHS-2 in a
similar manner.12A recent study has shown that (15R)-
HETE inhibits the release of the potent inflammatory
mediator LTB4from blood polymorphonuclear cells via
the 5-LO pathway.13Based on these reports, it is possible
that the acetoxy compounds 13a–c, in addition to inhibit-
ing COX-2, could alsoacetylate COX-2 to produce (15R)-
HETE that would prevent the formation of inflammatory
leukotrienes such as LTB4via the 5-LO pathway.
In view of the fact that the acetoxy compounds 13a–c
may undergo in vivo bioconversion by esterases to the
phenolic compounds 15a–c, the ability of 15a–c to inhi-
bit COX-1/COX-2 was also investigated. All three phe-
nolic isomers 15a–c were inactive inhibitors of COX-1
(IC50values >250 mM). The relative COX-2 inhibition
potency order for 15a–c, which were much less potent
inhibitors of COX-2 than the corresponding acetoxy
analogues, was 2-OH (15a)>3-OH (15b)>inactive 4-
OH (15c). Compounds 15a and 15b exhibited COX-2
selectivity indexes of >136 and >63, respectively
A molecular modeling study was performed where 3-(3-
anone (13b) was docked in the active site of human
COX-2 (1CX2 PDB file) using a procedure previously
reported.14The objective of this docking experiment
was to determine the orientation of 13b within the
COX-2 binding site and the spatial orientation of the
acetoxy group relative to the serine hydroxyl group
which it could potentially acetylate to produce acety-
lated COX-2. This docking study showed (see Fig. 1)
13b binds in the center of human COX-2 primary active
site such that the phenolic OH of Ser530is about 6.05 A˚
from the O-atom of the C¼O (3-OAc phenyl), and that
the S-atom of the MeSO2moiety is inserted deep inside
(4.53 A˚) the entrance to the secondary COX-2 pocket
(Val523). In addition, the carbonyl O-atom of the central
lactone ring is about 4.31 A˚from one hydrogen atom of
The results of this investigation show (i) incorporation
of a 2-, 3-, or 4-OAc substituent on the 3-phenyl ring of
rofecoxib provides highly potent, and selective, COX-2
inhibitors, (ii) molecular modeling studies indicate the
3-OAc substituent of 13b is suitably positioned to acet-
ylate the serine hydroxyl group in the COX-2 primary
binding site, and (iii) the acetoxy compounds 13a–c
could serve as useful probes to study the function and
catalytic activity of the COX-2 isozyme.
We are grateful to the Canadian Institutes of Health
Research (MOP-14712) for financial support of this
research and to Rx&D-HRF/CIHR for a postdoctoral
fellowship (to A.R.) and a graduate scholarship (to P.R.).
References and Notes
1. (a) Fu, J. Y.; Masferrer, J. L.; Seibert, K.; Raz, A.; Nee-
dleman, P. J. Biol. Chem. 1990, 265, 16737. (b) Xie, W. L.;
Chipman, J. G.; Robertson, D. L.; Erikson, R. L.; Simmons,
D. L. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2692.
2. Smith, W. L.; DeWitt, D. L. Adv. Immunol. 1996, 62, 167.
3. Herschman, H. R. Biochem. Biophys. Acta 1996, 1299, 125.
4. Dubois, R. N.; Abramson, S. B.; Crofford, L.; Gupta,
R. A.; Simon, L. S.; Van de Putta, L. B. A.; Lipsky, P. E.
FASEB J. 1998, 12, 1063.
Figure 1. Docking the 3-OAc analogue of rofecoxib (13b) (ball-and-
stick) in the active site of human COX-2 (line and stick) (Eintermolecular
= ?60.74 kcal/mol).
acetoxyphenyl) (13a–c), and 3-(2-, 3-, and 4-hydroxyphenyl) (15a–c)
analogues of rofecoxib
In vitro inhibition of COX-1 and COX-2 by 3-(2-, 3-, and 4-
aValues 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.
bIn vitro COX-2 selectivity index (IC50COX-1/IC50COX-2).
M. Abdur Rahim et al./Bioorg. Med. Chem. Lett. 12 (2002) 2753–2756 2755
5. Hawkey, Download full-text
Maldonado-Cocco, J.; Acevedo, E.; Shahane, A.; Quan, H.;
Bolognese, J.; Mortensen, E. Arthritis Rheum. 2000, 43, 370.
6. Goldstein, J. L.; Silverstein, F. E.; Agrawal, N. M.;
Hubbard, R. C.; Kaiser, J.; Maurath, C. I.; Verburg, K. M.;
Geis, G. S. Am. J. Gastroenterol. 2000, 95, 1681.
7. Meade, E. A.; Smith, W. L.; DeWitt, D. L. J. Biol. Chem.
1993, 268, 6610.
8. (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, J. R. J. Med. Chem. 1998, 41,
C.; Laine, L.;Simon, T.;Beaulieu, A.;
9. Levoin, N.; Chretien, F.; Lapicque, F.; Chapleur, Y.
Bioorg. Med. Chem. 2002, 10, 753.
10. Mukherjee, D.; Nissen, S. E.; Topol, E. J. J. Am. Med.
Assoc. 2001, 286, 954, and references cited therein.
11. Dikshit, D. K.; Singh, S.; Singh, M. M.; Kamboj, V. P.
Ind. J. Chem. 1990, 29B, 954.
12. Lecomte, M.; Laneuville, O.; Ji, C.; DeWitt, D. L.; Smith,
W. L. J. Biol. Chem. 1994, 269, 13207.
13. Vachier, I.; Chanez, P.; Bonnans, C.; Godard, P.; Bous-
quet, J.; Chavis, C. Biochem. Biophys. Res. Commun. 2002,
14. Habeeb, A. G.; Praveen Rao, P. N.; Knaus, E. E. J. Med.
Chem. 2001, 44, 2921.
2756 M. Abdur Rahim et al./Bioorg. Med. Chem. Lett. 12 (2002) 2753–2756