Ester and amide derivatives of the nonsteroidal antiinflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1021/jm000004e

Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14- eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with IC₅₀ values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 μM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4- chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.

Full text of DOI:10.1021/jm000004e

2860
J . Med. Chem. 2000, 43, 2860-2870
Ester a n d Am id e Der iva tives of th e Non ster oid a l An tiin fla m m a tor y Dr u g,
In d om eth a cin , a s Selective Cyclooxygen a se-2 In h ibitor s
Amit S. Kalgutkar, Alan B. Marnett, Brenda C. Crews, Rory P. Remmel,† and Lawrence J . Marnett*
A. B. Hancock, J r., Memorial Laboratory for Cancer Research, Departments of Biochemistry and Chemistry,
Center in Molecular Toxicology and the Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-0146
Received J anuary 5, 2000
Recent studies from our laboratory have shown that derivatization of the carboxylate moiety
in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal
antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in
the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the
structure-activity studies involved in the transformation of the arylacetic acid NSAID,
indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin
esters and amides inhibited purified human COX-2 with IC50 values in the low-nanomolar
range but did not inhibit ovine COX-1 activity at concentrations as high as 66 µM. Primary
and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than
the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin
esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds.
Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with
a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin
amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of
the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides
a facile strategy for generating highly selective COX-2 inhibitors and eliminating the
gastrointestinal side effects of the parent compound.
Sch em e 1
In tr od u ction
The committed step in prostaglandin and thrombox-
ane biosynthesis involves the conversion of arachidonic
acid to prostaglandin H2 (PGH2), a reaction catalyzed
by the sequential action of the cyclooxygenase (COX)
and peroxidase activities of prostaglandin endoperoxide
synthase or cyclooxygenase (PGHS or COX, EC 1.14.99.1)
(Scheme 1).1 COX activity originates from two distinct
and independently regulated enzymes, termed COX-1
and COX-2.2-4 COX-1 is the constitutive isoform and is
mainly responsible for the synthesis of cytoprotective
prostaglandins in the gastrointestinal tract (GI) and of
the proaggregatory thromboxane in blood platelets.5
COX-2 is inducible and short-lived; its expression is
stimulated in response to endotoxin, cytokines, and
mitogens.6-8 COX-2 plays a major role in prostaglandin
biosynthesis in inflammatory cells (monocytes/macro-
phages) and in the central nervous system.9-12 Overall,
these observations suggest that COX-1 and COX-2 serve
different physiological and pathophysiological functions.
Classical nonsteroidal antiinflammatory drugs (NSAIDs)
inhibit both COX-1 and COX-2 to varying extents.13 The
differential tissue distribution of COX-1 and COX-2
provides a rationale for the development of selective
COX-2 inhibitors as antiinflammatory and analgesic
agents that lack the GI and hematologic liabilities
exhibited by currently marketed NSAIDs. This hypoth-
esis has been validated in animal models and has led
to the marketing of two diarylheterocycles, celecoxib and
rofecoxib (Figure 1), as COX-2 inhibitors.14-20
Although diarylheterocycles and other compounds
have been extensively studied as selective COX-2 in-
hibitors, there are very few reports on the utilization of
well-established NSAID templates in the design of
selective COX-2 inhibitors.21-24 Of all NSAIDs, in-
domethacin, zomepirac, aspirin, and flurbiprofen are the
only examples of compounds that have been successfully
elaborated into selective COX-2 inhibitors (see Figure
1). However, the methodology utilized in NSAID modi-
fication is not general and consists of extensive modi-
fication of individual compounds. For instance, replace-
ment of the 4-chlorobenzoyl group of indomethacin with
a 4-bromobenzyl moiety generates a COX-2-selective
inhibitor.21 In contrast, exchanging the carboxylate
* To whom correspondence should be addressed. Tel: (615)
343-7329.
Fax:
(615)
343-7534.
E-mail:
marnett@
toxicology.mc.vanderbilt.edu.
† On sabbatical leave from the Department of Medicinal Chemistry,
University of Minnesota.
10.1021/jm000004e CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/08/2000

Ester and Amide Derivatives of Indomethacin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2861
F igu r e 1. Structures of celecoxib and rofecoxib along with examples of conversion of nonselective COX inhibitors to COX-2-
selective inhibitors.
moiety in aspirin with alkyl sulfide functionalities
affords specific COX-2 inhibitors.23
that reveal the molecular basis for selective COX-2
inhibition by the indomethacin derivatives.
We recently described a biochemically based strategy
for the facile conversion of carboxylic acid-containing
NSAIDs. Derivatization of these compounds to esters
or amides produces molecules capable of binding tightly
to COX-2 but not COX-1.25 The facile nature of this
strategy is evident from the observation that a single
chemical derivatization (amidation or esterification) of
the carboxylate moiety of these compounds generates
an impressive array of potent and highly selective
COX-2 inhibitors. Several of the esters or amides tested
exhibit antiinflammatory activity and antiangiogenic
activity.26
In this paper, we disclose the details of the structure-
activity relationship (SAR) analysis on indomethacin
ester and amide derivatives as COX-2-selective inhibi-
tors. These results are discussed with particular refer-
ence to previous site-directed mutagenesis experiments
Resu lts
Ch em istr y. Well-established methodology was uti-
lized in the synthesis of ester and amide derivatives of
indomethacin as indicated in Scheme 2. Indomethacin
esters were prepared by treatment with the appropriate
alcohol or phenol in the presence of dicyclohexylcarbo-
diimide (DCC) and 4-dimethylaminopyridine (DMAP).
Indomethacin amides were prepared in a similar fash-
ion, utilizing bis(2-oxo-3-oxazolidinyl)phosphonic chlo-
ride (BOP-Cl)27 or 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide‚HCl (EDCI) as the carboxylate activator
instead of DCC. The fluorescent indomethacin deriva-
tive 36 was obtained by esterification of the indometha-
cin ethanol amide derivative 35 with N,N-dimethylami-
nocinnamic acid in the presence of EDCI or DCC (see
Scheme 2). No reaction was discernible when BOP-Cl

2862 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Kalgutkar et al.
Sch em e 2
was used as the carboxylate activator instead of EDCI
or DCC. The primary amide derivative 44 was synthe-
sized in near quantitative yields by reacting indometha-
cin with NH4Cl in the presence of (benzotriazol-1-
yloxy)trispyrrolidinophosphonium hexafluorophosphate
(PyBOP), HOBt, and DIPEA.28 The structures of all
compounds were established by NMR and mass spec-
trometry. HPLC analyses in two different solvent
systems of 20 representative target esters and amides
indicated a minimum purity of 96.0%. Most of the
compounds were in excess of 98.5% pure.
The preparation of the 4-bromobenzyl derivatives 76
and 81 is outlined in Scheme 3. Commercially available
5-methoxy-2-methylindole-3-acetic acid (71) was con-
verted to the corresponding phenethyl ester 73 by
reaction with phenethyl alcohol and BOP-Cl. N-Alky-
lation of 73 with 4-bromobenzyl bromide in the presence
of NaH afforded 76. Base-catalyzed hydrolysis of the
ester linkage in 76 afforded the indomethacin analogue
77.21 The corresponding amide 81 was synthesized in a
slightly different fashion. Hydrolysis of the 4-chloroben-
zoyl group in indomethacin phenethyl amide 40 afforded
indole 80 that upon alkylation with 4-bromobenzyl
bromide generated 81. The desmethyl phenethyl ester
and amide analogues 78 and 79 were synthesized in
analogous fashion starting from 5-methoxyindole-3-
acetic acid (72). Esterification or amidation of 72 with
phenethyl alcohol or phenethylamine afforded 74 and
75, respectively, which upon acylation with 4-chloroben-
zoyl chloride gave 78 and 79.
µM phenol was treated with several concentrations of
test compounds at 25 °C for 20 min. Since the recom-
binant COX-2 had a lower specific activity than ovine
COX-1, the protein concentrations were adjusted so that
the percentages of total products obtained following
oxygenation of arachidonic acid by the two isoforms
were comparable. The cyclooxygenase reaction was
initiated by the addition of [1-14C]arachidonic acid (50
µM) at 37 °C and continued for 30 s. Control experi-
ments in the absence of inhibitor indicated ∼25-30%
conversion of fatty acid substrate to products which was
sufficient for assessing the inhibitory properties of all
compounds described in this study. Under these condi-
tions, indomethacin displayed selective time- and con-
centration-dependent inhibition of COX-1 [IC50(COX-
1) ∼ 0.050 µM, IC50(COX-2) ∼ 0.75 µM], whereas NS-
39829 and SC-29930 displayed selective COX-2 inhibition
[NS-398: IC50(COX-2) ∼ 0.12 µM, IC50(COX-1) > 66 µM;
SC-299: IC50(COX-2) ∼ 0.060 µM, IC50(COX-1) > 66
µM].
1. Alip h a tic Ester s. Conversion of the free carboxy-
late group in indomethacin to the methyl ester afforded
131 which displayed selective COX-2 inhibition [1: IC50-
(COX-2) ∼ 0.25 µM, IC50(COX-1) ∼ 33 µM] (Table 1).
Thus esterification of the carboxylate moiety in in-
domethacin increased the inhibitory potency against
COX-2 but had a detrimental effect on the potency for
COX-1 inhibition. Chain length extension studies of the
methyl group in 1 to higher alkyl homologues revealed
significant increases in potency and selectivity against
COX-2. For example, the heptyl ester 10 was more
potent and selective as a COX-2 inhibitor than 1
[compound 10: IC50(COX-2) ∼ 0.040 µM, IC50(COX-1)
> 66 µM]. Incorporation of functionalities in the alkyl
chain led to compounds such as the butoxyethyl (11) or
trans-heptenyl (12) ester derivatives, which also dis-
En zym ology. A. Selective COX-2 In h ibition by
Ester Der iva tives of In d om eth a cin . IC50 values for
the inhibition of purified human COX-2 or ovine COX-1
by test compounds were determined by thin-layer chro-
matography (TLC). HoloCOX-2 (66 nM) or holoCOX-1
(44 nM) in 100 mM Tris-HCl, pH 8.0, containing 500

Ester and Amide Derivatives of Indomethacin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2863
Sch em e 3
played potent and selective COX-2 inhibition. Compared
to the olefin 12, the heptynyl ester 13 was less potent
as a COX-2 inhibitor. Likewise, cycloalkyl esters il-
lustrated by the cyclohexyl, cyclohexylethyl, or mor-
pholinoethyl analogues 8, 9, and 16, respectively, were
less potent as COX-2 inhibitors. However, the bulky
BOC-protected 2-aminoethyl ester 17 exhibited potent
and selective COX-2 inhibition, indicating the structural
diversity of functionalities which can be tolerated in the
COX-2 active site.
2. Ar om a tic Ester s. Transformation of the carboxy-
late moiety in indomethacin to a phenyl ester 18 also
led to selective COX-2 inhibition (Table 2). Potency and
selectivity were increased by the introduction of meth-
ylene spacers between the phenyl ring and the ester
oxygen as illustrated with the 2-phenylethyl ester 22.
However, no significant COX inhibition was discernible
with the â- and R-naphthyl esters 19 and 20, respec-
tively, or with the 3,5-dimethylphenyl ester derivative
21, even at very high concentrations. Interestingly,
selective COX-2 inhibition by the aromatic esters was
extremely sensitive to the type and position of substit-
uents on the phenyl ring. For instance, the presence of
a methylmercapto group in the 4-position of the phenyl
group afforded 23 which was only approximately 10-
fold selective as a COX-2 inhibitor. The corresponding
2-methylmercaptophenyl isomer 24 was greater than
1100-fold selective as a COX-2 inhibitor. Furthermore,
replacement of the 4-methylmercapto group with a
4-methoxy group yielded 25, which was one of the most
potent ester derivatives in the series with a COX-1 over
COX-2 selectivity ratio greater than 1650. Like the
4-methylmercaptophenyl ester 23, the 4-fluorophenyl
(27) and 3-pyridyl (28) ester analogues also demon-
strated dramatic losses in selectivity.
B. Selective COX-2 In h ibition by Am id e Der iva -
tives of In d om eth a cin . 1. Alip h a tic Am id es. Like
the methyl ester 1, the N-methyl amide 2931 also
displayed selective COX-2 inhibition [IC50(COX-2) ∼ 0.70
µM, IC50(COX-1) > 66 µM] (Table 3). Increments in
COX-2 potency and selectivity were observed with
higher alkyl homologues such as the octyl derivative 32,
but a further increase in chain length to the nonyl
derivative 33 resulted in a loss of COX-2 selectivity
[octyl amide 32: IC50(COX-2) ∼ 0.040 µM, IC50(COX-1)
∼ 66 µM; nonyl amide 33: IC50(COX-2) ∼ 0.040 µM,
IC50(COX-1) ∼ 17 µM]. Incorporation of terminal func-
tionalities on the alkyl chain afforded compounds that
retained COX-2 potency and selectivity as illustrated
by the 3-chloropropyl and the 2-ethanol amide deriva-
tives 34 and 35. Esterification of the hydroxy group in
35 with N,N-dimethylaminocinnamic acid generated the
corresponding fluorescent analogue 36, which also
exhibited excellent COX-2-selective inhibition. Although
the methyl ester of the indomethacin-glycine amide
conjugate, 37, was a poor inhibitor of COX-2, significant
improvements in COX-2 potency were discernible with
the corresponding methyl esters of indomethacin-ala-

2864 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Kalgutkar et al.
Ta ble 2. In Vitro COX-2 Potencies of Aromatic Esters of
Ta ble 1. In Vitro COX-2 Potencies of Aliphatic Esters of
Indomethacin
Indomethacin
a
IC50 values were determined by incubating several inhibitor
concentrations in DMSO with human COX-2 (66 nM) or ovine
COX-1 (44 nM) for 20 min at rt followed by initiation of the
cyclooxygenase reaction with the addition of 14C-AA (50 µM) at
37 °C for 30 s. Isolation and quantification of prostanoid products
were conducted as described before. Assays were run in duplicate.
b
Ratio of IC50(COX-1):IC50(COX-2). c >90% activity remains at
these inhibitor concentrations.
methyl enantiomer 48. It is noteworthy that all of the
aromatic amides containing the 4-fluoro (compound 50),
4-methylmercapto (compound 52), or 3-pyridyl substitu-
ent (compound 62) displayed potent and selective COX-2
inhibition (Table 4). In contrast, the corresponding
aromatic esters with identical substituents suffered
severe losses in COX-2 selectivity.
a
IC50 values were determined by incubating several inhibitor
SAR studies with the indomethacin amides also
revealed that only the secondary amides displayed
potent and selective COX-2 inhibition, since no signifi-
cant inhibition of either COX isozyme was discernible
with the corresponding tertiary amides at the concen-
tration range studied. This is illustrated by the N,N-
dimethyl (30), N,N-diethyl (31), N,N-methyl-2-pheneth-
yl (41), piperidinyl (42), and morpholide (43) amide
derivatives (see Table 3). The primary amide analogue
44 displayed potency and selectivity similar to many of
the secondary amides.
Although many of the (monosubstituted)phenyl amide
derivatives displayed potent and selective COX-2 inhibi-
tion, the 3,4,5-trimethoxyphenyl amide 56 did not
display COX-2 inhibition over the concentration range
studied. In contrast, COX-2-selective inhibition was
discernible with the bulky 4-benzamido-2-methoxy-5-
methylphenyl and biphenyl amide analogues 60 and 61,
respectively (see Table 4). Several examples of hetero-
cyclic amide derivatives of indomethacin were analyzed
for COX-2-selective inhibition. Although the 3-pyridyl
amide derivative 62 was a potent and selective COX-2
inhibitor, the corresponding pyrazinyl amide 65 was a
poor inhibitor. Likewise, the thiazolyl amide derivative
67 demonstrated modest COX-2-selective inhibitory
properties. Replacement of the methylene group adja-
cent to the amide linkage in the phenethyl amide
analogue 40 generated the previously described hydrox-
concentrations in DMSO with human COX-2 (66 nM) or ovine
COX-1 (44 nM) for 20 min at rt followed by initiation of the
cyclooxygenase reaction with the addition of 14C-AA (50 µM) at
37 °C for 30 s. Isolation and quantification of prostanoid products
were conducted as described before. Assays were run in duplicate.
b
Ratio of IC50(COX-1):IC50(COX-2). c >90% activity remains at
these inhibitor concentrations.
nine amide derivatives 38 and 39, respectively. The
L-enantiomer 39 was 2 times more potent at inhibiting
COX-2 than the corresponding D-enantiomer 38.
2. Ar yla lk yl a n d Ar om a tic Am id es. Incorporation
of a terminal phenyl ring in the alkyl amides also
generated potent and selective COX-2 inhibitors as
illustrated with the 2-phenethyl amide analogue 40 (see
Table 3). As observed with the aromatic esters, COX-2
selectivity was dependent on the type and position of
substituents on the phenyl ring. For instance, the
2-methylbenzyl amide 45 was greater than 440-fold
selective as a COX-2 inhibitor, whereas the 4-methyl-
benzyl isomer 46 was only 130-fold selective as a COX-2
inhibitor (see Table 3). Replacement of the 4-methyl
group in 46 with an acetyl group (analogue 49) led to
significant improvements in COX-2 potency and selec-
tivity. Furthermore, some enantioselective COX-2 in-
hibition also was observed with the R and S enanti-
omers of R-methyl-4-methylbenzyl amide derivatives of
indomethacin. The R-R-methyl enantiomer 47 was a
better inhibitor of COX-2 than the corresponding S-R-

Ester and Amide Derivatives of Indomethacin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2865
Ta ble 3. In Vitro COX-2 Potencies of Aliphatic and Arylalkyl
Ta ble 4. In Vitro COX-2 Potencies of Aromatic and
Amide Derivatives of Indomethacin
Heterocyclic Amide Derivatives of Indomethacin
a
IC50 values were determined by incubating several concentra-
tions of inhibitor in DMSO with human COX-2 (66 nM) or ovine
COX-1 (44 nM) for 20 min followed by treatment with 1-14C-AA
a
IC50 values were determined by incubating several concentra-
b
(50 µM) at 37 °C for 30 s. Assays were run in duplicate. Ratio of
tions of inhibitor in DMSO with human COX-2 (66 nM) or ovine
IC50(COX-1):IC50(COX-2). c >80% remaining COX-1 activity at this
COX-1 (44 nM) for 20 min followed by treatment with 1-14C-AA
concentration.
b
(50 µM) at 37 °C for 30 s. Assays were run in duplicate. Ratio of
IC50(COX-1):IC50(COX-2). c >80% remaining COX-1 activity at this
compounds (ester 78 and ester 79). Likewise, replace-
ment of the 2-methyl group in 77 with hydrogen
resulted in an inactive compound.
concentration.
amate derivative 6832 that was a nonselective COX
inhibitor. Incorporation of a 4-nitro group in the phenyl
ring of 68 or replacement of the hydroxamate linkage
with a hydrazinyl moiety somewhat improved the COX-
2-selective inhibitory properties of the resultant com-
pounds 69 and 70.
Another interesting aspect in the SAR studies with
the indomethacin amides was the observation that the
inhibitory potency of indomethacin esters and amides
was dependent on the N-acyl substituent on the indole
ring (Table 5). Replacement of the 4-chlorobenzoyl
moiety in the potent and COX-2-selective phenethyl
ester 22 and the phenethyl amide 40 with a 4-bro-
mobenzyl group produced inactive compounds (ana-
logues 76 and 81, respectively). The unacylated indole
80 (see Scheme 3) also was devoid of COX-2-selective
inhibitory properties. COX-2 potency also was depend-
ent on the 2-methyl substituent in the indole ring of
indomethacin amides and esters. For instance, its
replacement with hydrogen in the ester and amide
derivatives 22 and 40, respectively, afforded inactive
C. In h ibition of COX-2 Activity in RAW264.7
Mu r in e Ma cr op h a ges. The ability of indomethacin
amide derivatives to inhibit COX-2 in intact cells was
assayed in RAW264.7 macrophages in which COX-2
activity was induced by pathologic stimuli. The mac-
rophages were exposed to lipopolysaccharide and γ-in-
terferon to induce COX-2 and then treated with several
concentrations of 4-methoxyphenyl ester 25 or amide
54. The IC50 values for inhibition of prostaglandin D2
(PGD2) by 25 and 54 were 0.20 and 0.062 µM, respec-
tively. Under comparable conditions, indomethacin
inhibited PGD2 synthesis with an IC50 of 0.010 µM.
Discu ssion
The present report describes our initial SAR studies
of the conversion of indomethacin into COX-2-selective
inhibitors using a biochemically based strategy that we
recently reported.25 Neutral derivatives of NSAIDs such
as indomethacin and meclofenamic acid retain the

2866 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Kalgutkar et al.
Ta ble 5. Replacement of the N-Acyl and 2-Methyl Group in Indomethacin Esters and Amides
a
IC50 values were determined by incubating several inhibitor concentrations in DMSO with human COX-2 (66 nM) or ovine COX-1 (44
nM) for 20 min at rt followed by initiation of the cyclooxygenase reaction with the addition of 14C-AA (50 µM) at 37 °C for 30 s. Isolation
b
and quantification of prostanoid products were conducted as described before. Assays were run in duplicate. Ratio of IC50(COX-1):
IC50(COX-2). c >90% activity remains at these inhibitor concentrations.
ability of the parent compounds to bind tightly to COX-
2, but they do not bind to COX-1. Therefore, the
conversion of these carboxylic acid-containing NSAIDs
into ester or amide derivatives generates derivatives
that are potent and highly selective COX-2 inhibitors.
The potency of some of the derivatives reported in
Tables 1-5 is underscored by the fact that the lowest
IC50 values achieved against COX-2 are comparable to
the enzyme concentration used in the assay (routinely
66 nM). The selectivity for COX-2 exhibited by the some
of the most potent inhibitors is actually underestimated
because many of the compounds assayed exhibited no
inhibition of COX-1 at the highest inhibitor concentra-
tions tested (66 µM). Thus, our relatively straightfor-
ward strategy produces excellent COX-2 inhibitors.
Despite the fact that many of the esters and amides
exhibited high COX-2 selectivity, there were notable
exceptions, particularly in the ester series. For example,
the 4-thiomethoxyphenyl ester of indomethacin was only
10-fold selective for COX-2, whereas the 2-thiomethoxy-
phenyl ester was greater than 1100-fold selective.
Interestingly, the 4-methoxyphenyl ester was greater
than 1700-fold selective, and the 4-thiomethoxyphenyl
amide of indomethacin was greater than 550-fold selec-
tive. Likwise, the 4-fluorophenyl and 3-pyridyl esters
exhibited only 40- and 50-fold selectivity, respectively,
whereas the analogous amides were greater than 1000-
and 1300-fold selective.
Primary and secondary amide derivatives of in-
domethacin were potent inhibitors of COX-2, whereas
tertiary amide derivatives were not. Several dialkyl
amides, cycloalkyl amides, or pyridyl derivatives were
synthesized and did not inhibit COX-2 under our
standard assay conditions. This suggests that hydrogen
bonding from the amide nitrogen to a protein acceptor
is an important determinant of binding. Possible hy-
drogen bond acceptors near the carboxylate-binding
region of COX-2 include Tyr355 and Glu524 (Figure 2).
Mutation of either of these residues to Phe or Ala,
respectively, renders the mutant proteins resistant to
the inhibitory effects of indomethacin amides.25
A striking observation from the present study is the
breadth of substitutions tolerated in the ester or amide
functionality that yield COX-2 inhibitors. Relatively
large alkyl, aryl, aralkyl, and heterocyclic esters or
amides of indomethacin exhibit high potency and se-
lectivity. This is consistent with the model we proposed
for the binding of these esters or amides to COX-2.25
The indomethacin moiety is located in the cyclooxyge-
nase active site, but the ester and amide groups project
through the constriction at the base of the active site
and into a wide “lobby” in the membrane-binding
domain (Figure 2). This model is consistent with the
crystal structure of a complex of COX-2 with a carboxyl
chain-extended analogue of zomepirac.22a A comparable
lobby is present in COX-1, but there are a number of
conserved sequence changes in this region between the
proteins that may contribute to the COX-2 selectivity
exhibited by ester and amide inhibitors.22b
Substitution of the p-chlorobenzoyl group of indometha-
cin with a p-bromobenzyl group generates a molecule,
77, that is a reasonably effective COX-2 inhibitor21a (IC50
) 2.5 µM, selectivity > 30 in our hands). We anticipated
that conversion of this compound to esters or amides
would generate a family of highly selective COX-2
inhibitors, but this was not the case. Neither the

Ester and Amide Derivatives of Indomethacin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2867
F igu r e 2. COX subunit with the key structural elements and important amino acid residues. The model for binding of indomethacin
esters or amides posits that the substituted indole is bound above the constriction comprised of Arg120, Tyr355, and Glu524; the
amide is hydrogen-bonded to residues in the vicinity of the constriction; and the substituted ester or amide group projects down
into the lobby. Other structural elements highlighted are the side pocket into which the sulfonamide or sulfone of celecoxib or
rofecoxib inserts; the heme group ligated by His388; and the top channel into which the ω-end of arachidonic acid inserts.
phenethyl ester 76 nor the phenethyl amide 81 exhib-
ited any COX-2 inhibitory activity. The analogous
derivatives of indomethacin, compounds 22 and 40, were
highly selective inhibitors so it was quite surprising that
the derivatives of 77 were not. This suggests that not
all carboxylate-containing NSAIDs or related com-
pounds will be converted into COX-2 inhibitors by
esterification or amidation.
cally based method for generating COX-2-selective
inhibitors is a promising new strategy for the generation
of antiinflammatory, analgesic, and antiangiogenic
agents.26
Exp er im en ta l Section
Ch em istr y. Melting points were determined using a Gal-
lenkamp melting point apparatus and are uncorrected. Chemi-
cal yields are unoptimized specific examples of one prepara-
tion. Indomethacin was purchased from Sigma (St. Louis, MO).
All other chemicals were purchased from Aldrich (Milwaukee,
WI). Methylene chloride was purchased as “anhydrous” from
Aldrich and was used as received. All other solvents were
HPLC grade. Analytical TLC (Analtech uniplates) was used
to follow the course of reactions. Silica gel (Fisher, 60-100
mesh) was used for column chromatography. 1H and 13C NMR
spectra in CDCl3 were recorded on a Bruker WP-360 or AM-
400 spectrometer; chemical shifts are expressed in parts per
million (ppm, δ) relative to tetramethylsilane as internal
standard. Spin multiplicities are given as s (singlet), d
(doublet), dd (doublet of doublets), t (triplet), q (quartet), and
m (multiplet). Coupling constants (J ) are given in hertz (Hz).
Positive ion electrospray ionization (ESI) and collision-induced
dissociation (CID) mass spectra were obtained on a Finnigan
TSQ 7000 mass spectrometer. CID fragmentations were
consistent with assigned structures.
Gen er a l P r oced u r e for th e Ester ifica tion a n d Am id a -
tion of NSAIDs. Meth od A: 4-(Meth ylm er ca p to)p h en yl
1-p -Ch lor ob e n zoyl-5-m e t h oxy-2-m e t h ylin d ole -3-a ce -
ta te (23). A reaction mixture containing indomethacin (300
mg, 0.84 mmol) in 6 mL of anhydrous CH2Cl2 was treated with
DCC (192 mg, 0.92 mmol), DMAP (10 mg, 84 µmol), and
4-methylmercaptophenol (129 mg, 0.92 mmol). After stirring
at room temperature for 5 h, the reaction mixture was filtered
and the filtrate was concentrated in vacuo. The residue was
diluted with water (∼30 mL) and extracted with EtOAc (2 ×
30 mL). The combined organic solution was washed with 5%
AcOH (2 × 30 mL), 1 N NaOH (2 × 30 mL), and water (∼100
mL), dried (MgSO4), and filtered, and the solvent was removed
in vacuo. The crude product was purified by chromatography
on silica gel (EtOAc:hexanes, 20:80) and obtained as a yellow
oil that solidified upon freezing (307 mg, 76%): mp ) 132-
133 °C; 1H NMR (CDCl3) δ 7.66-7.69 (d, 2 H, J ) 8.4 Hz, ArH),
7.46-7.49 (d, 2 H, J ) 8.5 Hz, ArH), 7.22-7.23 (d, 1 H, J )
Perhaps the most surprising result from our SAR
analysis was the complete absence of inhibitory activity
of indomethacin esters or amides that lacked a methyl
group at the 2-position of the indole ring (Table 5). This
finding uncovers what may be a previously unrecognized
determinant of COX inhibitory activity. Clearly, neither
the 2-desmethylphenethyl ester nor the 2-desmeth-
ylphenethyl amide were active against COX-2. Since
esters or amides of indomethacin are not time-depend-
ent inhibitors of COX-1,38 it is not possible to speculate
whether the 2-methyl group of nonselective inhibitors
is also important for inhibition of COX-1. It will be
interesting to prepare 2-desmethylindomethacin to ex-
plore this possibility.
Some of the molecules described herein (e.g., 40 and
57) have been found to exhibit antiinflammatory activity
in the carageenan-induced foot pad edema assay fol-
lowing oral administration.25 The time dependence of
antiiflammatory activity and in vitro analysis of me-
tabolites generated by liver microsomes suggests that
the antiinflammatory activity is due to the parent
compound and not due to hydrolysis to indomethacin.25
This observation, coupled with the structural flexibility
revealed in the present study, suggests that conversion
of NSAIDs to amide derivatives may be a very useful
approach for the generation of novel and efficacious
COX-2 inhibitors. The ease of amide synthesis by
combinatorial approaches should make it easy to con-
struct highly diverse libraries to maximize not only
COX-2 inhibitory activity but also physical properties,
distribution, metabolic profile, etc. Thus, our biochemi-

2868 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Kalgutkar et al.
2.4 Hz, ArH), 7.04-7.05 (d, 1 H, J ) 2.4 Hz, ArH), 6.97-7.00
(d, 2 H, J ) 8.6 Hz, ArH), 6.87-6.90 (d, 1 H, J ) 9.0 Hz, ArH),
6.67-6.71 (dd, 1 H, J ) 9.0 and 2.5 Hz, ArH), 3.89 (s, 2 H,
CH2), 3.83 (s, 3 H, CH3), 2.46 (s, 3 H, CH3), 2.45 (s, 3 H, CH3);
ESI-CID 480 (MH+), m/z 312, 139.
amides. Chromatography on silica gel (EtOAc:hexanes, 50:50)
gave the title compound as a yellow oil (394 mg, 67%): 1H
NMR (CDCl3) δ 7.72 (bs, 1 H, NH), 7.10-7.25 (m, 6 H, ArH),
6.96-6.97 (d, 1 H, J ) 2.2 Hz, ArH), 6.75-6.79 (dd, 1 H, J )
8.7 Hz & 2.3 Hz, ArH), 4.26-4.30 (t, 2 H, J ) 6.9 Hz, CH2),
3.83 (s, 3 H, OCH3), 3.63 (s, 2 H, CH2), 2.87-2.91 (t, 2 H, J )
6.9 Hz, CH2), 2.34 (s, 3 H, CH3).
Meth od B. N-(4-Aceta m id op h en yl)-1-p-ch lor oben zoyl-
5-m eth oxy-2-m eth ylin d ole-3-a ceta m id e (57). A reaction
mixture containing indomethacin (300 mg, 0.84 mmol) and bis-
(2-oxazolidinyl)phosphinic chloride (218 mg, 0.84 mmol) in 5
mL of anhydrous CH2Cl2 was treated with Et3N (167 mg, 0.84
mmol) and allowed to stir at room temperature for 10 min.
The mixture was then treated with 4′-aminoacetanilide (141
mg, 0.94 mmol) and stirred overnight at room temperature.
The precipitate that formed was filtered and washed with cold
CH2Cl2 and then recrystallized from hot MeOH to afford a pale
yellow solid (221 mg, 54%): mp ) 256-257 °C; 1H NMR
(DMSO-d6) δ 10.14 (s, 1H, NH), 9.86 (s, 1H, NH), 7.62-7.70
(m, 4H, ArH), 7.48 (s, 4H, ArH), 7.18 (d, 1H, J ) 2.3 Hz, ArH),
6.90-6.93 (d, 1H, J ) 9.0 Hz, ArH), 6.68-6.72 (dd, 1H, J )
9.1 and 2.5 Hz, ArH), 3.73 (s, 3H, CH3), 3.71 (s, 2H, CH2), 2.27
(s, 3H, CH3), 1.99 (s, 3H, CH3); ESI-CID 492 (MH+), m/z 340,
312, 174.
P h en eth yl 1-p-Br om oben zyl-5-m eth oxy-3-m eth ylin dole-
3-a ceta te (76). To a solution of 73 (394 mg, 1.2 mmol) in 5
mL of anhydrous DMF was added NaH (53 mg, 1.3 mmol) at
0 °C under argon. The reaction mixture was stirred at 0 °C
for 20 min and then treated with 4-bromobenzyl bromide (330
mg, 1.3 mmol). The reaction mixture was stirred overnight and
then diluted with water. The aqueous solution was extracted
with ether (2 × 20 mL). The combined organic was washed
with water (2 × 25 mL), dried (MgSO4), and filtered, and the
solvent was removed in vacuo. The residue was chromato-
graphed on silica gel (EtOAc:hexanes, 5:95-10:90) to afford
73 as a light brown solid (384 mg, 73%): mp ) 104-106 °C;
1H NMR (CDCl3) δ 7.35-7.37 (d, 2 H, J ) 8.3 Hz, ArH), 7.01-
7.35 (m, 7 H, ArH), 6.96-6.97 (d, 1 H, J ) 2.2 Hz, ArH), 6.75-
6.81 (m, 3 H, ArH), 5.20 (s, 2 H, CH2), 4.27-4.31 (t, 2 H, J )
6.9 Hz, CH2), 3.83 (s, 3 H, OCH3), 3.68 (s, 2 H, CH2), 2.87-
2.92 (t, 2 H, J ) 6.9 Hz, CH2), 2.24 (s, 3 H, CH3); ESI-CID 494
(MH+), m/z 344, 231, 170, 105.
1-p -Br om ob en zyl-5-m et h oxy-3-m et h ylin d ole-3-a cet ic
Acid (77). A reaction mixture containing 76 (150 mg) and 1
N LiOH (∼1.5 mL) in MeOH (3 mL) was stirred at room
temperature for 4 h. The reaction mixture was diluted with
water and extracted with EtOAc (2 × 10 mL) (the organic
washing was discarded) and then acidified with 1 N HCl and
extracted with EtOAc (2 × 20 mL). The combined organic
solution was washed with water (2 × 20 mL), dried (MgSO4),
and filtered, and the solvent was removed in vacuo. The crude
residue was recrystallized from CH2Cl2/hexanes to generate
77 as fluffy white crystals in 90% yield: mp ) 198-199 °C;
1H NMR (CDCl3) δ 7.36-7.38 (d, 2 H, J ) 8.4 Hz & 1.8 Hz,
ArH), 7.02-7.07 (m, 2 H, ArH), 6.75-6.82 (m, 3 H, ArH), 5.21
(s, 2 H, CH2), 3.48 (s, 3 H, OCH3), 3.73 (s, 2 H, CH2), 2.29 (s,
3 H, CH3); NI-ESI 388 (M - H)-.
N-(2-H yd r oxyet h yl)-1-p -ch lor ob en zoyl-5-m et h oxy-2-
m eth ylin d ole-3-a ceta m id e (35). 35 was obtained in a simi-
lar manner as described above. Chromatography of the crude
product on silica gel (EtOAc) afforded 33 as a pale yellow solid
1
(143 mg, 39%): mp ) 162-164 °C; H NMR (CDCl3) δ 7.66-
7.68 (dd, 2 H, J ) 6.7 and 1.7 Hz, ArH), 7.47-7.50 (dd, 2 H,
J ) 6.9 and 1.9 Hz, ArH), 6.85-6.89 (d and s, 2 H, J ) 9.2 Hz,
ArH), 6.68-6.72 (dd, 1 H, J ) 9.0 and 2.5 Hz, ArH), 6.03 (bs,
1 H, NH), 3.82 (s, 3 H, CH3), 3.67 (bs, 4 H, 2CH2), 3.35-3.40
(q, 2 H, J ) 4.8 Hz, CH2), 2.44 (bs, 1 H, OH), 2.39 (s, 3 H,
CH3); ESI-CID 401 (MH+), m/z 375.
N-[2-(N,N-Dim eth ylam in o)cin n am yloxyeth yl]-1-p-ch lo-
r oben zoyl-5-m eth oxy-2-m eth ylin d ole-3-a ceta m id e (36).
To a solution of N,N-dimethylaminocinnamic acid (85 mg, 0.44
mmol) in dry CH2Cl2 (10 mL) were added DCC (99 mg, 0.48
mmol) and DMAP (6 mg, 44 µmol) followed by 33 (200 mg, 0.5
mmol). The reaction was stirred at room temperature over-
night. The mixture was diluted with water (∼30 mL) and
extracted with EtOAc (2 × 30 mL). The combined organic
solution was washed with water (2 × 50 mL), dried (MgSO4),
and filtered, and the solvent was removed in vacuo. The crude
product was purified by chromatography on silica gel (EtOAc:
hexanes, 20:80 then 50:50) and obtained as a bright yellow
5-Meth oxy-2-m eth ylin d ole-3-p h en eth yla ceta m id e (80).
A reaction mixture containing 40 (500 mg, 1.08 mmol) in
MeOH (5 mL) was treated with 1 N NaOH (5 mL) was stirred
at room temperature overnight. The reaction mixture was
diluted with water, extracted with EtOAc (3 × 20 mL). The
organic extracts were washed with water, dried (MgSO4),
filtered, and concentrated in vacuo to essentially yield the pure
compound as a white solid (324 mg, 93%): 1H NMR (CDCl3) δ
7.82 (bs, 1 H, NH), 7.18-7.21 (d, 1 H, J ) 8.2 Hz, ArH), 7.11-
7.13 (m, 3 H, ArH), 6.90-6.93 (m, 2 H, ArH), 6.80-6.84 (m, 2
H, ArH), 5.65 (bt, 1 H, NH), 3.82 (s, 3 H, OCH3), 3.59 (s, 2 H,
CH2), 3.38-3.44 (t, 2 H, J ) 6.6 Hz, CH2), 2.64-2.68 (t, 2 H,
J ) 6.8 Hz, CH2), 2.25 (s, 3 H, CH3).
1
solid (162 mg, 58%): mp ) 138-140 °C; H NMR (DMSO-d6)
δ 8.19 (bt, 1 H, NH), 7.64-7.66 (d, 2 H, J ) 8.5 Hz ArH), 7.59-
7.61 (d, 2 H, J ) 8.6 Hz, ArH), 7.42-7.50 (m, 2 H, ArH), 7.10-
7.11 (d, 1 H, J ) 2.3 Hz, ArH), 6.90-6.92 (d, 1 H, J ) 9.0 Hz,
ArH), 6.68-6.72 (dd, 1 H, J ) 9.0 Hz & 2.4 Hz, ArH), 6.66-
6.69 (m, 3 H, ArH and olefinic H), 6.16-6.20 (d, 1 H, J ) 16
Hz, olefinic H), 4.08-4.11 (t, 2 H, J ) 5.5 Hz, CH2), 3.72 (s, 3
H, CH3), 3.51 (s, 2 H, CH2), 3.32-3.35 (m, 2 H, CH2), 2.96 (s,
6 H, N(CH3)2), 2.20 (s, 3 H, CH3); UV (AcCN) λmax 364 nm;
ESI-CID 574 (MH+), m/z 174.
1-p -Br om ob en zyl-5-m et h oxy-2-m et h ylin d ole-3-p h en -
eth yla ceta m id e (81). This compound was prepared in a
similar manner as described for 76. Compound was obtained
as an off-white solid (recrystallization from CH2Cl2/hexanes)
1-p-Ch lor oben zoyl-5-m et h oxy-2-m et h ylin d ole-3-a cet -
a m id e (44).39 A reaction mixture containing indomethacin
(750 mg, 2.01 mmol), PyBOP (1.57 g, 3.02 mmol), HOBt (409
mg, 3.02 mmol), DIPEA (1.4 mL, 8.04 mmol), and NH4Cl (215
mg, 4.02 mmol) in dry DMF (8 mL) was stirred at room
temperature overnight. The precipitated solid was filtered,
washed with cold CH2Cl2 and then recrystallized from CH2-
Cl2/hexanes to afford a pale yellow solid (600 mg, 84%): mp )
225-227 °C; 1H NMR (DMSO-d6) δ 7.62-7.70 (q, 4 H, J ) 8.6
Hz, ArH), 7.43 (bs, 1 H, NH), 7.09-7.10 (d, 1 H, J ) 2.5 Hz,
ArH), 6.97 (bs, 1 H, NH), 6.90-6.93 (d, 1 H, J ) 8.9 Hz, ArH),
6.68-6.72 (dd, 1 H, J ) 8.9 and 2.5 Hz, ArH), 3.75 (s, 3 H,
CH3), 3.45 (s, 2 H, CH2), 2.21 (s, 3 H, CH3); ESI-MS calcd for
1
(481 mg, 80%): mp ) 141-143 °C; H NMR (CDCl3) δ 7.34-
7.37 (d, 2 H, J ) 8.4 Hz, ArH), 7.07-7.10 (m, 4 H, ArH), 6.88-
6.91 (m, 3 H, ArH), 6.74-7.83 (m, 3 H, ArH), 5.64 (bt, 1 H,
NH), 5.18 (s, 2 H, CH2), 3.83 (s, 3 H, OCH3), 3.64 (s, 2 H, CH2),
3.40-3.46 (t, 2 H, J ) 6.6 Hz, CH2), 2.64-2.69 (t, 2 H, J ) 6.8
Hz, CH2), 2.14 (s, 3 H, CH3); ESI-CID 491 (MH+), m/z 344,
171, 122.
1-p-Ch lor oben zoyl-5-m et h oxyin d ole-3-p h en et h yla ce-
ta m id e (79). To a solution of 5-methoxyindole-3-acetic acid
(72; 450 mg, 2.19 mmol) in dry CH2Cl2 (10 mL) were added
EDCI (497 mg, 2.6 mmol) and DMAP (27 mg, 0.22 mmol),
followed by phenethylamine (315 mg, 2.6 mmol). The reaction
was stirred at room temperature overnight. The mixture was
diluted with water (∼30 mL) and extracted with EtOAc (2 ×
30 mL). The combined organic solution was washed with water
(2 × 50 mL), dried (MgSO4), and filtered, and the solvent was
removed in vacuo. The crude amide 75 upon purification by
C
19H17ClN2O4 (MH+) 357.09, found 356.8; CID m/z 340, 312,
139.
5-Meth oxy-3-m eth ylin d ole 3-P h en eth yla ceta te (73).
This compound was prepared by the reaction of 5-methoxy-2-
methylindole (71) with phenethyl alcohol in the presence of
BOP-Cl in a similar manner as described for indomethacin

Ester and Amide Derivatives of Indomethacin
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2869
chromatography on silica gel (EtOAc:hexanes, 30:70) was
obtained as a yellow oil (600 mg, 88%) and was used in the
next step without any further purification, owing to its
instablity. To this amide (650 mg, 2.11 mmol) in dry DMF (5
mL) was added NaH (60% dispersion in mineral oil, 60 mg,
2.5 mmol) at 0 °C under argon. After stirring at 0 °C for 20
min, 4-chlorobenzoyl chloride (420 mg, 2.4 mmol) was added
to the solution which was then allowed to attain room
temperature and stirred for 15 h. The reaction was quenched
with water and extracted with Et2O (3 × 10 mL). The combined
Et2O extract was washed with water, dried (MgSO4), filtered,
and concentrated in vacuo. The residue was chromatographed
on silica gel (EtOAc:hexanes, 50:50) to afford 79 as a pale
yellow solid (700 mg, 74%): mp ) 125-126 °C; 1H NMR
(CDCl3) δ 8.29-8.32 (d, 1 H, J ) 8.8 Hz, ArH), 7.60-7.62 (d,
2 H, J ) 6.9 Hz, ArH), 7.49-7.52 (d, 2 H, J ) 7.0 Hz, ArH),
7.07-7.13 (m, 5 H, ArH), 6.87-6.95 (m, 3 H, ArH), 5.6 (bs, 1
H, NH), 3.86 (s, 3 H, CH3), 3.57 (s, 2 H, CH2), 3.44-3.46 (m,
2 H, CH2), 2.65-2.69 (t, 2 H, J ) 6.5 Hz, CH2); ESI-CID 447
(MH+), m/z 298, 139, 122, 105.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR data for
all compounds included in the study. This information is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Hawkey, C. J . COX-2 Inhibitors. Lancet 1999, 353, 307-314.
(2) DeWitt, D. L.; Smith, W. L. Primary Structure of Prostaglandin
G/H Synthase From Sheep Vesicular Gland Determined from
the Complementary DNA Sequence. Proc. Natl. Acad. Sci. U.S.A.
1988, 85, 1412-1416.
(3) Yokoyama, C.; Tanabe, T. Cloning of Human Gene Encoding
Prostaglandin Endoperoxide Synthase and Primary Structure
of the Enzyme. Biochem. Biophys. Res. Commun. 1989, 165,
888-894.
(4) Hla, T.; Neilson, K. Human Cyclooxygenase-2 cDNA. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 7384-7388.
(5) Allison, M. C.; Howatson, A. G.; Torrence, C. J .; Lee, F. D.;
Russel, R. I. Gastrointestinal Damage Associated with the Use
of Nonsteroidal Antiinflammatory Drugs. N. Engl. J . Med. 1992,
327, 749-754.
(6) Kujubu, D. A.; Fletcher, B. S.; Varnum, B. C.; Lim, R. W.;
Herschman, H. R. TIS10, A Phorbol Ester Tumor Promoter
Inducible mRNA from Swiss 3T3 Cells, Encodes a Novel Pros-
taglandin Synthase/Cyclooxygenase Homologue. J . Biol. Chem.
1991, 266, 12866-12872.
(7) Lee, S. H.; Soyoola, E.; Chanmugam, P.; Hart, S.; Sun, W.;
Zhong, H.; Liou, S.; Simmons, D.; Hwang, D. Selective Expres-
sion of Mitogen-Inducible Cyclooxygenase in Macrophages Stimu-
lated with Lipopolysaccharide. J . Biol. Chem. 1992, 267, 25934-
25938.
(8) O’Sullivan, M. G.; Huggins, E. M., J r.; Mccall, C. E. Lipopolysac-
charide-Induced Expression of Prostaglandin H Synthase-2 in
Aveolar Macrophages is Inhibited by Dexamethasone but not
by Aspirin. Biochem. Biophys. Res. Commun. 1993, 191, 1294-
1300.
(9) Smith, C. J .; Zhang, Y.; Koboldt, C. M.; Muhammad, J .; Zweifel,
B. S.; Shaffer, A.; Talley, J . J .; Masferrer, J . L.; Seibert, K.;
Isakson, P. C. Pharmacological Analysis of Cyclooxygenase-1 in
Inflammation. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13313-
13318.
(10) Masferrer, J . L.; Zweifel, B. S.; Manning, P. T.; Hauser, S. D.;
Leahy, K. M.; Smith, W. G.; Isakson, P. C.; Seibert, K. Selective
Inhibition of Inducible Cyclooxygenase-2 in vivo is Antiinflam-
matory and Nonulcerogenic. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 3228-3232.
(11) Vane, J . R.; Mitchell, J . A.; Appleton, I.; Tomlinson, A.; Bishop-
Bailey, D.; Croxtall, J .; Willoughby, D. A. Inducible Isoforms of
Cyclooxygenase and Nitric Oxide Synthase in Inflammation.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 2046-2050.
(12) Harada, Y.; Hatanaka, K.; Saito, M.; Majima, M.; Ogino, M.;
Kawamura, M.; Ohno, T.; Yang, Q.; Katori, M.; Yamamoto, S.
Detection of Inducible Prostaglandin H Synthase-2 in Cells in
the Exudate of Rat Carrageenin-Induced Pleurisy. Biomed. Res.
1994, 15, 127-130.
(13) Warner, T. D.; Giuliano, F.; Vojnovic, I.; Bukasa, A.; Mitchell,
J . A.; Vane, J . R. Nonsteroid Drug Selectivities for Cyclo-
oxygenase-1 Rather than Cyclo-oxygenase-2 are Associated with
Human Gastrointestinal Toxicity: A Full In Vitro Analysis. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 7563-7568.
(14) Simon, L. S.; Lanza, F. L.; Lipsky, P. E.; Hubbard, R. C.;
Talwalker, S.; Schwartz, B. D.; Isakson, P. C.; Geis, G. S.
Preliminary Study of the Safety and Eficacy of SC-58635, a Novel
Cyclooxygenase-2 Inhibitor: Efficacy and Safety in Two Placebo-
Controlled Trials in Osteoarthritis and Rheumatoid Arthritis,
and Studies of Gastrointestinal and Platelet Effects. Arthritis
Rheumatism 1998, 41, 1591-1602.
(15) Ehrich, E. W.; Dallob, A.; De Lepeleire, I.; Van Hecken, A.;
Riendeau, D.; Yuan, W.; Porras, A.; Wittreich, J .; Seibold, J . R.;
De Schepper, P.; Mehlisch, D. R.; Gertz, B. J . Characterization
En zym ology. Arachidonic acid was purchased from Nu
Chek Prep (Elysian, MN). [1-14C]Arachidonic acid (∼55-57
mCi/mmol) was purchased from NEN Dupont or American
Radiolabeled Chemicals (ARC, St. Louis, MO). Hematin was
purchased from Sigma Chemical Co. (St. Louis, MO). COX-1
was purified from ram seminal vesicles (Oxford Biomedical
Research, Inc., Oxford, MI) as described earlier.36 The specific
activity of the protein was 20 (µMO2/min)/mg, and the percent-
age of holoprotein was 13.5%. ApoCOX-1 was prepared as
described earlier.37 Apoenzyme was reconstituted by the
addition of hematin to the assay mixtures. Human COX-2 was
expressed in SF-9 insect cells by means of the pVL 1393
expression vector (Pharmingen) and purified by ion-exchange
and gel filtration chromatography. All of the purified proteins
were shown by densitometric scanning of a 7.5% SDS PAGE
gel to be >80% pure. SC-299 was a gift of Dr. J ohn J . Talley,
Monsanto/Searle (St. Louis, MO).
Tim e- a n d Con cen tr a tion -Dep en d en t In h ibition of
Ovin e COX-1 a n d Hu m a n COX-2 Usin g Th in La yer
Ch r om a togr a p h y (TLC) Assa y. Cyclooxygenase activity of
ovine COX-1 (44 nM) or human COX-2 (66 nM) was assayed
by TLC. Reaction mixtures of 200 µL consisted of hematin-
reconstituted protein in 100 mM Tris-HCl, pH 8.0, 500 µM
phenol, and [1-14C]arachidonic acid (50 µM, ∼55-57 mCi/
mmol). For the time-dependent inhibition assay, hematin-
reconstituted COX-1 (44 nM) or COX-2 (66 nM) was preincu-
bated at room temperature for 20 min with varying inhibitor
concentrations in DMSO followed by the addition of [1-14C]-
arachidonic acid (50 µM) for 30 s at 37 °C. Reactions were
terminated by solvent extraction in Et2O/CH3OH/1 M citrate,
pH 4.0 (30:4:1). The phases were separated by centrifugation
at 2000g for 2 min and the organic phase was spotted on a
TLC plate (J . T. Baker, Phillipsburg, NJ ). The plate was
developed in EtOAc/CH2Cl2/glacial AcOH (75:25:1) at 4 °C.
Radiolabeled prostanoid products were quantitated with a
radioactivity scanner (Bioscan, Inc., Washington, D.C.). The
percentage of total products observed at different inhibitor
concentrations was divided by the percentage of products
observed for protein samples preincubated for the same time
with DMSO.
of Rofecoxib as
a Cyclooxygenase-2 Isoform Inhibitor and
In h ibition of COX-2 Activity in Activa ted RAW264.7.
Protocols for COX-2 inhibition in RAW264.7 cells have been
previously described.23 Briefly, cells (6.2 × 106 cells/T25 flask)
were activated with lipopolysaccharide (1 µg/mL) and γ-inter-
feron (10 U/mL) in serum-free DMEM for 7 h and then treated
with inhibitor (0-2 µM) for 30 min at 37 °C. Exogenous
arachidonate metabolism was determined by adding [1-14C]-
arachidonic acid (20 µM) for 15 min at 37 °C. IC50 values are
the average of two independent determinations.
Demonstration of Analgesia in the Dental Pain Model. Clin.
Pharmacol. Ther. 1999, 65, 336-347.
(16) Kalgutkar, A. S. Selective Cyclooxygenase-2 Inhibitors as Non-
Ulcerogenic Antiinflammatory Agents. Exp. Opin. Ther. Patents
1999, 9, 831-849.
(17) Marnett, L. J .; Kalgutkar, A. S. Cyclooxygenase-2 Inhibitors:
Discovery, Selectivity and the Future. Trends Pharmacol. Sci.
1999, 20, 465-469.
(18) Talley, J . J . Selective Inhibitors of Cyclooxygenase-2. Exp. Opin.
Ther. Patents 1997, 7, 55-62.
(19) Carter, J . S. Recently Reported Inhibitors of Cyclooxygenase-2.
Exp. Opin. Ther. Patents 1997, 8, 21-29.
(20) Prasit, P.; Riendeau, D. Selective Cyclooxygenase-2 Inhibitors.
In Annual Reports in Medicinal Chemistry; Hagmann, W. K.,
Ed.; Academic Press Inc.: New York, 1997; Vol 32, pp 211-220.
Ack n ow led gm en t. This research was funded by a
research grant from the National Institutes of Health
(CA47479).

2870 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Kalgutkar et al.
(21) (a) Black, W. C.; Bayly, C.; Belley, M.; Chan, C.-C.; Charleson,
S.; Denis, D.; Gauthier, J . Y.; Gordon, R.; Guay, D.; Kargman,
S.; Lau, C. K.; Leblanc, Y.; Mancini, J .; Quellet, M.; Percival,
D.; Roy, P.; Skorey, K.; Tagari, P.; Vickers, P.; Wong, E.; Xu, L.;
Prasit, P. From Indomethacin to a Selective COX-2 Inhibitor:
Development of Indolalkanoic Acids as Potent and Selective
Cyclooxygenase-2 Inhibitors. Bioorg. Med. Chem. Lett. 1996, 6,
725-730. (b) LeBlanc, Y.; Black, W. C.; Chan, C.-C.; Charleson,
S.; Delorme, D.; Denis, D.; Gauthier, J . Y.; Gordon, R.; Guay,
D.; Hamel, P.; Kargman, S.; Lau, C. K.; Mancini, J .; Quellet,
M.; Percival, D.; Roy, P.; Skorey, K.; Tagari, P.; Vickers, P.;
Wong, E.; Xu, L.; Prasit, P. Synthesis and Biological Evaluation
of Both Enantiomers of L-761,000 as Inhibitors of Cyclooxyge-
nase-1 and 2. Bioorg. Med. Chem. Lett. 1996, 6, 731-736.
(22) (a) Luong, C.; Miller, A.; Barnett, J .; Chow, J .; Ramesha, C.;
Browner, M. F. Flexibility of the NSAID Binding Site in the
Structure of Human Cyclooxygenase-2. Nat. Struct. Biol. 1996,
3, 927-933. (b) Picot, D.; Loll, P. J .; Garavito, R. M. The X-ray
crystal structure of the membrane protein prostaglandin H2
synthase-1. Nature 1994, 367, 243-249.
(23) (a) Kalgutkar, A. S.; Crews, B. C.; Rowlinson, S. W.; Garner, C.;
Seibert, K.; Marnett, L. J . Aspirin-Like Molecules that Co-
valently Inactivate Cyclooxygenase-2. Science 1998, 280, 1268-
1270. (b) Kalgutkar, A. S.; Kozak, K. R.; Crews, B. C.; Hochge-
sang, J r., G. P.; Marnett, L. J . Covalent Modification of
Cyclooxygenase-2 (COX-2) by 2-(Acetoxyphenyl)alkyl Sulfides,
a New Class of Selective COX-2 Inactivators. J . Med. Chem.
1998, 41, 4800-4818.
Inhibitors of Cyclooxygenase-2 (COX-2). Med. Res. Rev. 1999,
19, 199-208.
(31) Shen, T. Y. Antiinflammatory Amides. U.S. Patent 3,285,908,
1966.
(32) Flynn, D. L.; Capiris, T.; Cetenko, W. J .; Connor, D. T.; Dyer,
R. D.; Kostlan, C. R.; Nies, D. E.; Schrier, D. J .; Sircar, J . C.
Nonsteroidal Antiinflammatory Drug Hydroxamic Acids. Dual
Inhibitors of Both Cyclooxygenase and 5-Lipoxygenase. J . Med.
Chem. 1990, 33, 2070-2072.
(33) (a) Kurumbail, R. G.; Stevens, A. M.; Gierse, J . K.; McDonald,
J . J .; Stegeman, R. A.; Pak, J . Y.; Gildehaus, D.; Miyashiro, J .
M.; Penning, T. D.; Seibert, K.; Isakson, P. C.; Stallings, W. C.
Structural Basis for Selective Inhibition of Cyclooxygenase-2 by
Antiinflammatory Agents. Nature 1996, 384, 644-648. (b)
Gierse, J . K.; McDonald, J . J .; Hauser, S. D.; Rangwala, S. H.;
Koboldt, C. M.; Seibert, K. A Single Amino Acid Difference
Between Cyclooxygenase-1 (COX-1) and -2 (COX-2) Reverses
the Selectivity of COX-2-Specific Inhibitors. J . Biol. Chem. 1996,
271, 15810-15814. (c) Wong, E.; Bayly, C.; Waterman, H. L.;
Riendeau, D.; Mancini, J . A. Conversion of Prostaglandin G/H
Synthase-1 Into an Enzyme Sensitive to PGHS-2-Selective
Inhibitors by a Double His513Arg and Ile523Val Mutation. J .
Biol. Chem. 1997, 272, 9280-9286. (d) Guo, Q.; Wang, L.-H.;
Ruan, K.-H.; Kulmacz, R. J . Role of Val509 in Time-Dependent
Inhibition of Prostaglandin H Synthase-2 Cyclooxygenase Activ-
ity by Isoform-Selective Agents. J . Biol. Chem. 1996, 271,
19134-19139.
(24) Bayly, C. I.; Black, W. C.; Leger, S.; Quimet, N.; Quellet, M.;
Percival, M. D. Structure-Based Design of COX-2 Selectivity Into
Flurbiprofen. Bioorg. Med. Chem. Lett. 1999, 9, 307-312.
(25) Kalgutkar, A. S.; Crews, B. C.; Rowlinson, S. W.; Marnett, A.
B.; Kozak, K. R.; Remmel, R. P.; Marnett, L. J . Biochemically
Based Design of Cyclooxygenase (COX-2) Inhibitors. Facile
Conversion of Nonsteroidal Antiinflammatory Drugs to Potent
and Highly Selective COX-2 Inhibitors. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 925-930.
(26) Daniel, T. O.; Liu, H.; Morrow, J . D.; Crews, B. C.; Marnett, L.
J . Thromboxane A2 is a mediator of Cyclooxygenase-2-Dependent
Endothelial Migration and Angiogenesis. Cancer Res. 1999, 59,
9, 4574-4577.
(27) Diago-Meseguer, J .; Palomo-Coll, A. L.; Fernandez-Lizarbe, J .
R.; Zugaza-Bilbao, A. A New Reagent for Activating Carboxyl
Groups; Preparation and Reactions of N,N-Bis[2-oxo-3-oxazo-
lidinyl]phosphorodiamidic Chloride. Synthesis 1980, 547-551.
(28) Wang, W.; McMurray, J . S. A Selective Method for the Prepara-
tion of Primary Amides: Synthesis of Fmoc-L-4-Carboxami-
dophenylalanine and Other Compounds. Tetrahedron Lett. 1999,
40, 2501-2504.
(34) Penning, T. D.; Talley, J . J .; Bertenshaw, S. R.; Carter, J . S.;
Collins, P. W.; Docter, S.; Graneto, M. J .; Lee, l. F.; Malecha, J .
W.; Miyashiro, J . M.; Rogers, R. S.; Rogier, D. J .; Yu, S. S.;
Anderson, G. D.; Burton, E. G.; Nita Cogburn, J .; Gregory, S.
A.; Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A.
W.; Zhang, Y. Y.; Isakson, P. C. Synthesis and Biological
Evaluation of the 1,5-Diarylpyrazole Class of Cyclooxygenase-2
Inhibitors: Identification of 4-[5-(4-Methylphenyl)-3-(trifluorom-
ethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, Celecox-
ib). J . Med. Chem. 1997, 40, 1347-1365.
(35) American Home Products Corp. WO9821195, 1997.
(36) Marnett, L. J .; Siedlik, P. H.; Ochs, R. C.; Pagels, W. D.; Das,
M.; Honn, K. V.; Warnock, R. H.; Tainer, B. E.; Eling, T. E.
Mechanism of the Stimulation ofProstaglandin H Synthase by
the Antithrombotic and Antimetastatic Agent, Nafazatrom. Mol.
Pharmacol. 1990, 26, 328-335.
(37) Odenwaller, R.; Chen, Y.-N. P.; Marnett, L. J . Preparation and
Proteolytic Cleavage of Apoprostaglandin Endoperoxide Syn-
thase. Methods Enzymol. 1990, 187, 479-485.
(38) Rome, L. H.; Lands, W. E. M. Structural requirements for time-
dependent inhibition of prostaglandin biosynthesis by antiin-
flammatory drugs. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 4863-
4865.
(29) Futaki, N.; Yoshikawa, K.; Hamasaka, Y.; Arai, I.; Higuchi, S.;
Iizuka, H.; Otomo, S. NS-398, A Novel Nonsteroidal Antiinflam-
matory Drug With Potent Analgesic and Antipyretic Effects,
Which Causes Minimal Stomach Lesions. Gen. Pharmacol. 1993,
24, 105-110.
(30) Talley, J . J .; Bertenshaw, S. R.; Brown, D. L.; Carter, J . S.;
Graneto, M. J .; Koboldt, C. M.; Masferrer, J . L.; Norman, B. H.;
Rogier, J r., D. J .; Zweifel, B. S.; Seibert, K. 4,5-Diaryloxazole
(39) De Martiis, F.; Franzone, J . S.; Tamietto, T. Synthesis and
antiphlogistic properties of some indolylacetohydroxamic acids.
Boll. Chim. Farm. 1976, 114, 309-318.
J M000004E