Indolyl esters and amides related to indomethacin are selective COX-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2005.07.073

Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the nonsteroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides displayed time-dependent COX-2 inhibition with IC ₅₀ values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.

Full text of DOI:10.1016/j.bmc.2005.07.073

Bioorganic & Medicinal Chemistry 13 (2005) 6810–6822
Indolyl esters and amides related to indomethacin are
selective COX-2 inhibitors
Amit S. Kalgutkar,ꢀ Brenda C. Crews, Sam Saleh,
Daniel Prudhommeꢁ and Lawrence J. Marnett*
A.B. Hancock, Jr., Memorial Laboratory for Cancer Research, Departments of Biochemistry and Chemistry,
Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, and the Vanderbilt-Ingram Cancer Center,
Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA
Received 5 August 2003; revised 15 June 2005; accepted 25 July 2005
Available online 19 September 2005
Abstract—Previous studies from our laboratory have revealed that esterification/amidation of the carboxylic acid moiety in the non-
steroidal anti-inflammatory drug, indomethacin, generates potent and selective COX-2 inhibitors. In the present study, a series of
reverse ester/amide derivatives were synthesized and evaluated as selective COX-2 inhibitors. Most of the reverse esters/amides dis-
played time-dependent COX-2 inhibition with IC50 values in the low nanomolar range. Replacement of the 4-chlorobenzoyl group
on the indole nitrogen with a 4-bromobenzyl moiety resulted in compounds that retained selective COX-2 inhibitory potency. In
addition to inhibiting COX-2 activity in vitro, the reverse esters/amides also inhibited COX-2 activity in the mouse macrophage-like
cell line, RAW264.7. Overall, this strategy broadens the scope of our previous methodology of neutralizing the carboxylic acid
group in NSAIDs as a means of generating COX-2-selective inhibitors and is potentially applicable to other NSAIDs.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
central nervous system.7–9 In contrast, COX-1 is a con-
stitutive enzyme and appears responsible for the
biosynthesis of cytoprotective prostaglandins in the
gastric mucosa and the kidney.10 Classical NSAIDs
inhibit both isoforms non-selectively.11 The differential
tissue distribution of the COX enzymes was the basis
for the development of COX-2-selective inhibitors as
anti-inflammatory and analgesic agents with reduced
ulcerogenic potency.8
The utility of non-steroidal anti-inflammatory drugs
(NSAIDs) in the treatment of inflammation and pain
is often limited by gastrointestinal liabilities including
ulceration and bleeding. Inhibition of cyclooxygenase
(COX), the enzyme that catalyzes arachidonic acid oxy-
genation, was initially considered to be responsible for
the shared therapeutic benefits and gastrointestinal side
effects of NSAIDs.1,2 However, the discovery of a sec-
ond COX gene, COX-2, provided important insights
into NSAID side effects that translated into more effec-
tive drugs.3,4 COX-2 is inducible, short-lived, and the
product of an immediate early gene.5 Its expression is
stimulated by a host of growth factors, cytokines, and
mitogens.6 Importantly, COX-2 is responsible for pros-
taglandin biosynthesis in inflammatory cells and the
The first COX-2 inhibitors developed clinically were
members of the diarylheterocycle class of compounds
(Fig. 1).12 Extensive structure–activity studies exist in
this series.13 In contrast, relatively few groups have uti-
lized non-selective NSAIDs as leads in the search for
selective COX-2 inhibitors. The Merck–Frosst group
reported that replacement of the 4-chlorobenzoyl group
in the NSAID, indomethacin, with a 4-bromobenzyl
group generates
a
selective COX-2 inhibitor
(Fig. 1).14,15 Our laboratory reported that neutralization
of the carboxylic acid moiety in indomethacin to esters
or amides afforded selective COX-2 inhibitors
(Fig. 1).16 More recently, the Novartis group described
conversion of diclofenac into lumiracoxib, which exhib-
its 500-fold selectivity for COX-2 over COX-1 (Fig. 1).17
Keywords: Indomethacin; COX-2; Indolylester; Indolylamide.
*
Corresponding author. Tel.: +1 615 343 7329; fax: +1 615 343
7354; e-mail: larry.marnett@vanderbilt.edu
Present address: Pfizer Global Research & Development, Groton,
CT 06340, USA.
ꢀ
ꢁ
Present address: Mylan Pharmaceuticals, Morgantown, WV 26504,
USA.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.073

A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
6811
H3C
O
O
O
O
O
O
S
O
O
S
S
CH3
NH2
CH3
S
Cl
NH2
N
N
O
N
N
O
O
CH3
F3C
N
CH3
ETORICOXIB
VALDECOXIB
ROFECOXIB
CELECOXIB
OH
H3CO
O
CH3
N
OH
Br
H3CO
O
CH3
4-BROMOBENZYL DERIVATIVE
R
O
N
O
X
R
Cl
H3CO
H3CO
O
CH3
N
CH3
INDOMETHACIN
N
Cl
O
Cl
O
R = OR' (INDOMETHACIN ESTERS)
R = NHR' (INDOMETHACIN AMIDES)
R' = (CH2)2C6H5 = LM-4108
X = O (REVERSE ESTERS)
X = NH (REVERSE AMIDES)
H3C
CO2H
CO2H
NH
NH
Cl
Cl
F
Cl
DICLOFENAC
LUMIRACOXIB
Figure 1. Structures of selective COX-2 inhibitors from the diarylheterocycle series and those derived from the modification of indomethacin and
diclofenac.
Conversion of non-selective NSAIDs to esters and
amides is a facile strategy for generating COX-2 inhibi-
tors from known drugs but it has the limitation that
indomethacin esters and possibly some amides may be
hydrolyzed to indomethacin in vivo.18,19 Therefore, we
investigated the possibility that indolyl esters and
amides with essentially the ‘‘reverse’’ orientation would
selectively inhibit COX-2. Hypothetically, such com-
pounds eliminate or minimize the generation of
indomethacin in vivo. We report herein that indolyl
esters and amides are potent and selective inhibitors of
COX-2 in vitro and in intact macrophages. Further-
more, reversing the orientation of the carboxyl group
expands the universe of COX-2 inhibitors to 4-bromo-
benzyl analogs of indomethacin.
able 5-methoxy-2-methylindole-3-acetic acid (1)
(Scheme 1). Esterification of 1 in the presence of MeOH
and BOP-Cl20 afforded the corresponding methyl ester
321 that upon reduction with lithium borohydride in
THF–MeOH22 yielded the corresponding alcohol 5.23
Alcohol 5 served as the synthon in the preparation of
all reverse esters. Esterification of appropriate carboxyl-
ic acid derivatives with alcohol 5 employing EDCI as
catalyst resulted in the formation of esters 7–14 in mod-
erate to good yields (60–75%). N-acylation on the indole
nitrogen in esters 7–14 with 4-chlorobenzoyl chloride in
the presence of NaH afforded the final compounds 15–
21 (Table 1). N-alkylation of 11 with 4-bromobenzyl
bromide afforded the N-(4-bromobenzyl)indole deriva-
tive 23. Similar synthetic manipulations on 5-methoxy-
indole-3-acetic acid (2) afforded the corresponding
2-des-methylindolyl alkyl ester 22 (see Scheme 1).
2. Results
Two approaches were employed for the synthesis of
reverse amides. The first utilized 1 as starting material
and proceeded as outlined in Scheme 2. Reaction of 1
with ammonium chloride24 in the presence of EDCI,
HOBt, and DIPEA afforded primary amide 24 in 64%
2.1. Chemistry
The synthesis of the target reverse ester derivatives was
achieved in four steps starting from commercially avail-

6812
A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
R
2
OH
O
COOH
BOP-Cl
COOCH
3
O
H CO
3
H CO
3
H CO
3
H CO
3
LiBH
RCO H
2
4
R
1
R
1
R
1
R
1
N
H
MeOH
Et O-MeOH
2
N
H
N
EDCI/DMAP
N
H
H
1: R = CH
1
3
5: R = CH
3: R = CH
Compounds 7-14
1
3
1
3
2: R = H
1
6: R = H
4: R = H
1
1
NaH
(4-Br)-C H CH Br
6
4
2
R
2
Cl
O
O
H CO
3
R
1
NaH
(4-Cl)-C H COCl
O
N
O
6
4
O
H CO
3
Cl
R
1
N
Compounds 15-22 (Table 1)
Br
23
Scheme 1.
yield. LAH mediated reduction of 24 afforded the pri-
mary amine 25,25 which was characterized as its stable
oxalate salt. EDCI-promoted coupling of 25 with 4-
chlorobenzoic acid, hydrocinnamic acid, or propionic
acid afforded amides 26, 27, and 30, respectively, that
upon N-acylation with 4-chlorobenzoyl chloride or 4-
bromobenzyl bromide furnished target compounds 31–
33, and 37, respectively. N-Acylation/alkylation of
melatonin (28) afforded the N-4-chlorobenzoyl- and
N-4-bromobenzyl des-methylindole analogs 34 and 35,
respectively (see Scheme 2). The synthesis of 36 (see
Scheme 2) was achieved via N-acetylation of 25 with
acetyl chloride in the presence of Et3N resulting in the
formation of the previously described 2-methylmelato-
nin derivative 29.25 N-alkylation on the indole nitrogen
in 29 with 4-bromobenzyl bromide afforded 36.
HCl, pH 8.0, containing 500 lM phenol was treated
with several concentrations of inhibitors (0–66 lM) at
25 ꢁC for 20 min. Since the recombinant COX-2 had
specific activity lower than that of ovine COX-1, the
protein concentrations were adjusted such that the per-
centages of total products obtained following arachi-
donic acid oxygenation by the two isozymes were
comparable. The cyclooxygenase reaction was initiated
by the addition of [1-14C]arachidonic acid (50 lM) at
37 ꢁC for 30 s. Control experiments in the absence of
inhibitor indicated ꢀ25–30% conversion of arachidonic
acid to products, which was sufficient for assessing the
inhibitory properties of all compounds described in this
study.
As displayed in Table 1, all of the reverse esters 15–21
displayed potent and selective inhibition of COX-2 with
IC50 values in the low nanomolar range and very high
COX-2 selectivity ratios. No COX-1 inhibition was dis-
cernible with these compounds even at very high concen-
trations (>66 lM). Under these assay conditions,
indomethacin demonstrated modest COX-1-selective
inhibition [(IC50 (COX-1) ꢀ 0.050 lM; IC50 (COX-
2) ꢀ 0.75 lM)]. Removal of the 2-methyl group on the
indole ring of the reverse ester 19 generated 22, an inac-
tive compound. Lack of COX inhibitory potency also
was observed with the 2-des-methyl analogs of the
simple indomethacin esters.26 Replacement of the
4-chlorobenzoyl group on the indole nitrogen in 19 with
Alkylation of the acylindolylamines, 26–30, with 4-bro-
mobenzyl bromide proceeded to some extent on the
amide nitrogen as well as the indole nitrogen. Thus, a
second route was developed for reverse amide synthesis
in which the amine of 25 was BOC-protected then selec-
tively alkylated on the indole nitrogen (Scheme 2).
Removal of the protecting group and acylation of the
amine produced reverse amides 41–43.
The synthesis of N-(4-bromobenzyl)-3-butyl-5-methoxy-
2-methylindole (47) is depicted in Scheme 3. Reaction of
4-methoxyphenylhydrazine (44) and 2-heptanone (45)
following the Fisher-indole protocol afforded indole 46
that upon N-alkylation with 4-bromobenzyl bromide
generated 47.
a
4-bromobenzyl group afforded 23, which also
displayed selective COX-2 inhibitory properties.
Compound 23, however, was 40-fold less potent as a
COX-2 inhibitor than 19. In contrast, replacement of
the 4-chlorobenzoyl group on the indole nitrogen with
a 4-bromobenzyl group in the previously reported indo-
methacin ester series afforded inactive compounds.16
2.2. Enzymology
2.2.1. In vitro inhibition of COX activity. IC50 values for
the inhibition of purified human COX-2 or ovine COX-
1 by test compounds were determined by a thin layer
chromatography (TLC) assay. Hematin-reconstituted
COX-2 (66 nM) or COX-1 (44 nM) in 100 mM Tris–
Examples from the N-(substituted)-5-methoxy-2-methy-
lindole alkyl amide series included the 4-chlorobenzoyl
and the phenethyl amide derivatives 31 and 33, which

A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
Table 1. IC50 values for selective COX-2 inhibition by reverse esters of indomethacin
6813
Cl
R1
O
O
O
O
H3CO
H3CO
CH3
N
R2
N
COC6H4(4-Cl)
22
Compound
R1
R2
IC50 (lM)a
COX-1
Selectivityb
IC50(lM)c
Intact cells
COX-2
O
15
16
17
18
19
20
21
22
23
0.065
0.050
0.040
0.050
0.050
0.040
0.050
>66
>66
>66
>66
>66
>66
>66
>66
66
>1000
>1300
>1500
>1300
>1500
>1300
>1330
—
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
O
O
O
O
O
CH
3
OCH
3
0.32
OCH
3
Cl
Br
I
>0.085
—
—
Cl
2.0
>66
>33
Br
a Ovine COX-1 (44 nM) or human COX-2 (66 nM) was preincubated with inhibitors at 25 ꢁC for 20 min followed by the addition of [1-14C]ara-
chidonic acid (50 lM) at 37 ꢁC for 30 s. All assays were conducted in duplicate.
b Ratio of IC50(COX-1)/IC50 (COX-2).
c RAW264.7 murine macrophages were activated with LPS and IFN-c for 7 h. Vehicle or inhibitor was added for 30 min at 37 ꢁC. Inhibition of
prostaglandin D2 synthesis was determined by adding [1-14C]arachidonic acid (20 lM) for 15 min at 25 ꢁC.
also displayed potent COX-2 inhibition (Table 2). But
these compounds also displayed increased COX-1 inhi-
bition such that the COX-1 over COX-2 selectivity ratio
of 31 and 33 was 80 and 425, respectively, compared to
the esters which displayed selectivity ratios of 1500 or
greater. Interestingly, increasing the length of the incu-
bation time between the COX-1/inhibitor reaction mix-
ture and [1-14C]arachidonic acid from 30 s to 10 min
abolished all COX-1 inhibition by 31 and 33 [compound
31: IC50 (COX-1) ꢀ 4.0 lM (30 s assay); IC50 (COX-
1) > 66 lM (10 min assay); IC50 (COX-2) ꢀ 0.050 lM
(30 s assay); IC50 (COX-2) ꢀ 0.060 lM (10 min assay)].
This result suggests that the off-rates for the dissociation
of 31 and 33 from COX-1 are relatively high so they ex-
change with arachidonic acid substrate on prolonged
incubation. The inhibitory potency of 31 as a competi-
tive inhibitor of COX-1/-2 also was evaluated. Ovine
COX-1 (2.8 nM) or human COX-2 (5 nM) was added
to an incubation mixture containing [1-14C]arachidonic
acid (2 lM) and several concentrations of 31
(0.050 lM, 0.25 lM, 1.0 lM, or 4.0 lM). Although 31
was
a
potent competitive inhibitor of COX-1
(IC50 = 0.25 lM), no competitive inhibition of COX-2
by 31 was discernible under these experimental condi-
tions (IC50 > 4 lM). Competitive inhibition of COX-1
activity has been reported with other time-dependent,
selective COX-2 inhibitors.27,28
Inhibition of COX-1 by 31 and other reverse amides was
abolished by replacement of the 4-chlorobenzoyl group
in these compounds with a 4-bromobenzyl group as
illustrated with 32, 36, and 37, respectively (see Table
2). The 4-bromobenzyl derivative of 2-methylmelatonin
(36) also displayed COX-2-selective inhibitory

6814
A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
NH2
NH2
COOH
H3CO
H3CO
H3CO
O
NH4Cl
LAH
CH3
CH3
CH3
N
N
EDCI, HOBt
DIPEA
N
H
H
H
1
24
25
RCO2H, EDCI
OR
RCOCl, Et3N
O
1. NaH/
HN
NH2
H
R2
O
(4-Br)C6H4CH2Br
OR
(4-Cl)C6H4COCl
OtBu
N
H3CO
H3CO
CH3
H3CO
CH3
N
N
2. HCl
R1
H
R3
N
40
38
H
Compounds 26-30
NaH/
RCO2H, EDCI
OR
RCOCl, Et3N
(4-Br)C6H4CH2Br
OR
(4-Cl)C6H4COCl
H
R2
O
H
R2
O
N
N
H3CO
H3CO
R1
R1
N
N
R3
R3
Compounds 41-43 (Table 2)
Compounds 31-37 (Table 2)
Scheme 2.
H3CO
NHNH2 HCl
H3CO
H3CO
44
+
H
CH3
CH3
NaH
N
N
20 h
O
(4-Br)-C6H4CH2Br
H
Br
46
H3C
47
45
Scheme 3.
properties, albeit with lower COX-2 potency than 32.
Increasing the length of the alkyl substituent on the
amide linkage from methyl (36) to ethyl (37) increased
somewhat COX-2 potency and selectivity. In contrast
to these observations, replacement of the 4-chloro-
benzoyl moiety in simple indomethacin amides with a
4-bromobenzyl group affords inactive compounds.16
As observed with simple indomethacin esters/amides
and with reverse esters, replacement of the 2-methyl
group on the indole ring in the reverse amides with
hydrogen generated inactive compounds (analogs 34
and 35).26 Finally, replacement of the 3-alkylester or
-amide linkage in these compounds with a long chain
alkyl substituent resulted in the 3-butyl derivative, 47,
which was devoid of COX inhibitory activity (Table 2).
assayed in RAW264.7 murine macrophage-like cell
line.29 The cells were treated with lipopolysaccharide
(LPS) (1.0 lg/mL) and c-interferon (10 U/mL) for
7.0 h to induce COX-2 and then treated with several
concentrations of the test compounds. The IC50 values
for inhibition of production of prostaglandin D2
(PGD2) by 17, 19, 31, 32, 36, and 37 were 0.32, 0.085,
0.03, 0.05, 1.5, and 0.14 lM, respectively. Thus, in addi-
tion to inhibition of purified COX-2, these compounds
are potent inhibitors of COX-2 activity in cultured
inflammatory cells.
3. Discussion
The present study broadens the scope and utility of our
earlier strategy involving the esterification/amidation of
NSAIDs as means of generating selective COX-2 inhib-
itors. As observed with the earlier indomethacin esters/
2.2.2. Inhibition of COX-2 activity in intact cells. The
ability of reverse esters 17 and 19 or reverse amides
31, 32, 36, and 37 to inhibit COX-2 in intact cells was

A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
Table 2. IC50 values for selective COX-2 inhibition by reverse amides of indomethacin
6815
R1
O
NH
OCH3
OCH3
CH3
R2
N
N
R3
44
Br
Compound
R1
R2
R3
IC50 (lM)a
Selectivityb
IC50(lM)c
COX-2
0.05
COX-1
4.0
Intact cells
O
Cl
Cl
31
32
33
34
35
36
37
41
CH3
CH3
CH3
H
80
0.03
Cl
0.04
0.04
>66
>66
2.0
>66
17
>1650
425
—
0.05
Br
Cl
O
O
CH3
>66
>66
>66
>66
>3
Cl
Br
CH3
H
—
CH3
CH3
CH3
CH3
>33
>165
>1.2
1.50
0.14
Br
Br
Br
C2CH3
0.40
2.4
NO2
F
NO2
42
43
47
CH3
CH3
—
0.22
0.05
>66
>3
>1
>14
>20
—
Br
Br
Cl
F
—
>66
a Ovine COX-1 (44 nM) or human COX-2 (66 nM) was preincubated with inhibitors at 25 ꢁC for 20 min followed by the addition of [1-14C]ara-
chidonic acid (50 lM) at 37 ꢁC for 30 s. All assays were conducted in duplicate.
b Ratio of IC50 (COX-1)/IC50 (COX-2).
c RAW264.7 murine macrophages were activated with LPS and IFN-c for 7 h. Vehicle or inhibitor was added for 30 min at 37 ꢁC. Inhibition of
prostaglandin D2 synthesis was determined by adding [1-14C]arachidonic acid (20 lM) for 15 min at 25 ꢁC.
amides, structurally diverse groups can be part of the re-
verse ester/amide linkage in the present series leading to
COX-2 inhibitors. An unexpected outcome from this
study is the potent and selective COX-2 inhibition
exhibited by the reverse ester and amide derivatives, in
which the 4-chlorobenzoyl group on the indole nitrogen
is replaced by the 4-bromobenzyl substituent. Similar
manipulations in the simple indomethacin ester/amide
series afforded inactive compounds.16,26 Thus, in addi-
tion to the array of substituents as part of the reverse es-
ter/amide linkage, we envision broad structural
flexibility on groups linked to the indole nitrogen as
well. The indomethacin template appears to be most
flexible in delivering COX-2-selective inhibitors follow-
ing functional group manipulations. For instance, in
addition to Merck–Frosstꢂs and our own efforts, Woods
et al.27 have reported that replacement of indometha-
cinꢂs carboxylic acid group with 4-substituted thiazolyl
groups results in potent and selective COX-2 inhibitors.
Similarities in the SAR analysis between the reverse es-
ter/amides and the simple indomethacin ester/amide
derivatives include the lack of COX inhibition by the
2-des-methyl analogs in the two series. The 2-methyl
group on the indole ring was recently demonstrated to
insert into a hydrophobic depression in the side of the
active site of both COX-2 and COX-1.30 This appears
to be a major determinant of the time-dependent inhibi-
tion of COX enzymes by indomethacin. It is likely that
the methyl group plays a similar role in COX-2 inhibi-
tion by indomethacin esters and amides.

6816
A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
Preliminary biochemical analysis of the reverse ester/
amide and the simple indomethacin analogs reveals that
they conform to the two-step kinetic mechanism, typical
of slow, tight-binding NSAIDs including selective
COX-2 inhibitors.28,31,32 However, the time course of
COX-2 inhibition is slower for indomethacin derivatives
than diarylheterocycles. These results reflect a slow on-
rate for the initial association of these indomethacin
derivatives with COX-2.30 Although most of the simple
indomethacin amides tested in our previous study were
devoid of COX-1/-2 competitive inhibitory activity,
some reverse amide derivatives (compound 31) in the
present study competitively inhibited COX-1. This fea-
ture seems unique to reverse amide analogs containing
the 4-chlorobenzoyl group on the indole nitrogen, since
its replacement with the 4-bromobenzyl group abolished
all COX-1 competitive inhibition (e.g., 32). These
differences between the two series of indomethacin
analogs suggest subtle differences in their interaction
at the active sites of the two enzymes. Preliminary stud-
ies on the molecular basis for selective COX-2 inhibition
by the reverse esters/amides reveal that these com-
pounds bind at the COX-2 active site in a fashion sim-
ilar to that observed with the simple indomethacin
esters/amides.16,26 For example, the Y355F mutant is
resistant to the inhibitory effects of the simple indo-
methacin esters/amides as well as the reverse esters/
amides but the R120Q mutant is not (data not shown).
These results validate our design strategy and suggest
that hydrogen bonding from the amide nitrogens in
the simple indomethacin amides and the reverse amides
to the hydroxyl group of Tyr-355 is an important deter-
minant of binding.
4.1.1. Methyl ester of 5-methoxy-2-methylindole-3-acetic
acid (3). A reaction mixture containing 5-methoxy-2-
methylindole-3-acetic acid (1, 800 mg, 3.64 mmol) and
BOP-Cl (926 mg, 3.64 mmol) in 10 mL of anhydrous
CH2Cl2 was treated with Et3N (735 mg, 7.28 mmol)
and allowed to stir at rt for 5 min. The mixture was then
treated with anhydrous methanol (0.5 mL) and stirred
overnight at rt. Following dilution with CH2Cl2
(30 mL), the organic solution was washed with water
(2 · 25 mL), dried (MgSO4), filtered, and the solvent
concentrated in vacuo. The crude ester was purified by
chromatography on silica gel (EtOAc:hexanes; 25:75)
to afford a pale yellow oil (648 mg, 76%). 1H NMR
(CDCl3) d 7.75 (br s, 1H, NH), 7.13–7.16 (d, 1H,
J = 8.7 Hz, ArH), 6.98–6.99 (d, 1H, J = 2.2 Hz, ArH),
6.75–6.79 (dd, 1H, J = 8.7, 2.3 Hz, ArH), 3.85 (s, 3H,
OCH3), 3.67 (s, 3H, OCH3), 3.66 (s, 2H, CH2), 2.39 (s,
3H, CH3).
4.1.2. Methyl ester of 5-methoxyindole-3-acetic acid (4).
Compound 4 was obtained upon chromatography on
silica gel (EtOAc:hexanes; 15:85) as a pale yellow oil33
(450 mg, 78%). 1H NMR (CDCl3) d 7.95 (br s, 1H,
NH), 7.14–7.23 (m, 1H, ArH), 7.05 (s, 1H, ArH), 6.96
(s, 1H, ArH), 6.84–6.88 (dd, 1H, J = 8.7, 2.3 Hz,
ArH), 3.86 (s, 3H, CH3), 3.78 (s, 2H, CH2), 3.85 (s,
3H, OCH3), 3.75 (s, 3H, OCH3).
4.1.3. 3-(2-Hydroxyethyl)-5-methoxy-2-methylindole (5).
A reaction mixture containing the methyl ester 2
(648 mg, 2.78 mmol) in anhydrous ether (20 mL) and
dry MeOH (150 lL) was treated with LiBH4 (122 mg,
5.6 mmol) at 0 ꢁC. The reaction mixture was allowed
to attain rt and stirred at that temperature for 5 h.
The mixture was diluted with water and extracted with
ether (2 · 30 mL). The combined organic solution was
washed with water (2 · 25 mL), dried (MgSO4), filtered,
and the solvent was concentrated in vacuo. The crude
alcohol was purified by recrystallization in CH2Cl2/hex-
anes to afford white needles (384 mg, 67%). mp = 102–
103 ꢁC (lit. mp:23 98–101 ꢁC); 1H NMR (CDCl3) d
7.72 (br s, 1H, NH), 7.15–7.18 (d, 1H, J = 8.7 Hz,
ArH), 6.97–6.98 (d, 1H, J = 2.3 Hz, ArH), 6.76–6.80
(dd, 1H, J = 8.7, 2.3 Hz, ArH), 3.83–3.85 (m, 5H, CH2
and OCH3), 2.92–2.97 (t, 2H, J = 6.4 Hz, CH2), 2.39
(s, 3H, CH3).
4. Experimental
4.1. Chemistry
Melting points were determined with a Gallenkamp
melting point apparatus and are uncorrected. Chemi-
cal yields are unoptimized specific examples of one
preparation. All chemicals were purchased from Al-
drich (Milwaukee, WI). Methylene chloride was pur-
chased as ‘‘anhydrous’’ from Aldrich and was used
as received. All other solvents were of HPLC grade.
Analytical TLC (Analtech uniplatesTM) was used to
follow the course of reactions. Silica gel (Fisher, 60-
4.1.4. 3-(2-Hydroxyethyl)-5-methoxyindole (6). Com-
pound 6 was obtained in a similar fashion from 4 as a
1
100 mesh) was used for column chromatography. H
1
NMR spectra in CDCl3 or DMSO-d6 were recorded
on Bruker WP-360 or AM-400 spectrometers; chemi-
cal shifts are expressed in parts per million (ppm, d)
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. The purity of products was judged to be
at least 99% on the basis of their chromatographic
homogeneity.
colorless oil34 (306 mg, 74%). H NMR (CDCl3) d 7.92
(br s, 1H, NH), 7.25–7.28 (m, 1H, ArH), 7.05–7.07
(dd, 2H, J = 2.4 Hz, ArH), 6.85–6.89 (dd, 1H, J = 8.8,
2.4 Hz, ArH), 3.88–3.92 (t, 2H, J = 6.3 Hz, CH2), 3.86
(s, 3H, CH3), 2.99–3.03 (t, 2H, J = 6.3 Hz, CH2).
4.1.5. General procedure for the esterification of 5 and 6.
To a solution of the appropriate carboxylic acid
(2.18 mmol) in 5 mL of anhydrous CH2Cl2 were added
EDCI (2.44 mmol), DMAP (0.244 mmol), and 5 or 6
(2.44 mmol) after which time the reaction mixture was
stirred overnight at rt. Upon dilution with water, the
aqueous solution was extracted with CH2Cl2
(2 · 20 mL). The combined organic was washed with

A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
6817
water (2 · 25 mL), dried (MgSO4), filtered, and the sol-
vent concentrated in vacuo. The residue was chromato-
graphed on silica gel to afford the esters 7–14.
4.1.11. 4-Chlorobenzoic acid-2-[5-methoxy-1H-indol-3-
yl]ethyl ester (14). Compound 14 was obtained as a crys-
talline white solid upon chromatography on silica gel
(EtOAc:hexanes; 10:90–20:80). mp = 98–100 ꢁC; 1H
4.1.6. 4-Methylbenzoic acid-2-[5-methoxy-2-methyl-1H-
indol-3-yl]ethyl ester (8). Compound 8 was obtained as a
pale yellow solid upon chromatography on silica gel
(EtOAc:hexanes; 10:90) in 59% yield. 1H NMR
(DMSO-d6) d 10.59 (br s, 1H, NH), 7.73–7.76 (dd, 1H,
J = 6.7, 1.8 Hz, ArH), 7.30–7.44 (m, 1H, ArH), 7.23–
7.30 (m, 2H, ArH), 7.08–7.11 (d, 1H, J = 8.7 Hz,
ArH), 6.96–6.97 (d, 1H, J = 2.0 Hz, ArH), 6.58–6.62
(dd, 1H, J = 8.7, 2.3 Hz, ArH), 4.33–4.37 (t, 2H,
J = 6.8 Hz, CH2), 3.67 (s, 3H, OCH3), 3.01–3.05 (t,
2H, J = 6.8 Hz, CH2), 2.44 (s, 3H, CH3), 2.29 (s, 3H,
CH3).
NMR (CDCl3) d 7.95–7.99 (d and br s, 3H,
J = 8.5 Hz, 2 ArH and NH), 7.37–7.42 (d, 2H,
J = 8.5 Hz, ArH), 7.25–7.29 (m, 1H, ArH), 7.08–7.09
(m, 2H, ArH), 6.84-6.90 (dd, 1H, J = 8.9, 2.4 Hz,
ArH), 4.55–4.62 (t, 2H, J = 7.0 Hz, CH2), 3.85 (s, 3H,
CH3), 3.16–3.24 (t, 2H, J = 7.0 Hz, CH2).
4.1.12. General procedure for the N-acylation and N-
alkylation of esters 7-14. To a solution of the appropri-
ate ester (1.57 mmol) in 5 mL of anhydrous DMF was
added sodium hydride (60% dispersion in mineral oil)
(1.88 mmol) at 0 ꢁC under argon. The reaction mixture
was stirred at 0 ꢁC for 20 min and then treated with
4-chlorobenzoyl chloride or 4-bromobenzyl bromide
(1.88 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
solution was washed with water (2 · 25 mL), dried
(MgSO4), filtered, and the solvent concentrated in vac-
uo. The residue was chromatographed on silica gel
(EtOAc:hexanes; 5:95–10:90) to afford the target
compounds.
4.1.7. 4-Methoxybenzoic acid-2-[5-methoxy-2-methyl-
1H-indol-3-yl]ethyl ester (9). Compound 9 was obtained
as a bright yellow oil upon chromatography on silica gel
(EtOAc:hexanes; 20:80) in 84% yield. 1H NMR (CDCl3)
d 7.60–7.99 (m, 2H, ArH), 7.39 (s, 1H, NH), 7.15–7.17
(d, 2H, J = 8.7 Hz, ArH), 7.02–7.03 (d, 1H, J = 2.1 Hz,
ArH), 6.88–6.91 (d, 1H, J = 9.0 Hz, ArH), 6.75–6.79
(dd, 1H, J = 8.7, 2.1 Hz, ArH), 4.42–4.47 (t, 2H,
J = 7.2 Hz, CH2), 3.83 (s, 3H, OCH3), 3.70 (s, 3H,
OCH3), 3.09–3.14 (t, 2H, J = 7.2 Hz, CH2), 2.40 (s,
3H, CH3).
4.1.13. Valeric acid-2-[1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl]ethyl ester (15). Compound 15 was
obtained as a colorless oil in 34% yield. 1H NMR
(CDCl3) d 7.47–7.50 (d, 2H, J = 8.4 Hz, ArH), 7.29–
7.32 (d, 2H, J = 8.4 Hz, ArH), 6.80–6.81 (d, 1H,
J = 2.4 Hz, ArH), 6.68–6.71 (d, 1H, J = 9.0 Hz, ArH),
6.48–6.51 (d, 1H, J = 9.0 Hz, ArH), 4.07–4.11 (t, 2H,
J = 7.2 Hz, CH2), 3.66–3.71 (s, 3H, CH3), 2.80–2.85 (t,
2H, J = 7.2 Hz, CH2), 2.21 (s, 3H, CH3), 2.10–2.15 (t,
2H, J = 7.2 Hz, CH2), 1.39–1.46 (m, 4 H, CH2), 0.70–
0.76 (t, 3H, CH3).
4.1.8. 4-Chlorobenzoic acid-2-[5-methoxy-2-methyl-1H-
indol-3-yl]ethyl ester (11). Compound 11 was obtained as
a white solid upon chromatography on silica gel
(EtOAc:hexanes; 10:90) in 73% yield. mp = 119–
120 ꢁC; 1H NMR (CDCl3) d 7.92–7.97 (dd, 2H,
J = 6.7, 1.8 Hz, ArH), 7.70 (br s, 1H, NH), 7.37–7.41
(dd, 2H, J = 6.8, 1.9 Hz, ArH), 7.14–7.19 (d, 1H,
J = 8.7 Hz, ArH), 7.01–7.02 (d, 1H, J = 2.0 Hz, ArH),
6.75–6.80 (dd, 1H, J = 8.7, 2.3 Hz, ArH), 4.43–4.51 (t,
2H, J = 7.2 Hz, CH2), 3.83 (s, 3H, OCH3), 3.09–3.16
(t, 2H, J = 7.2 Hz, CH2), 2.39 (s, 3H, CH3).
4.1.14. 4-Methylbenzoic acid-2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethyl ester (16). Com-
pound 16 was obtained as a fluffy white solid upon
recrystallization with CH2Cl2/hexanes in 31% yield.
mp = 127–128 ꢁC; 1H NMR (CDCl3) d 7.83–7.86 (d,
1H, J = 8.7 Hz, ArH), 7.62–7.65 (d, 2H, J = 8.4 Hz,
ArH), 7.37–7.45 (m, 3H, ArH), 7.19–7.26 (m, 2H,
ArH), 7.00–7.01 (d, 1H, J = 2.2 Hz, ArH), 6.90–6.93
(d, 1H, J = Hz, ArH), 6.65–6.69 (dd, 1H, J = 8.9,
2.3 Hz, ArH), 4.47–4.52 (t, 2H,J = 6.9 Hz, CH2), 3.80
(s, 3H, OCH3), 3.11–3.15 (t, 2H, J = 6.9 Hz, CH2),
2.56 (s, 3H, CH3), 2.36 (s, 3H, CH3); ESI-CID 462
(MH+), m/z 326, 139.
4.1.9. 4-Bromobenzoic acid-2-[5-methoxy-2-methyl-1H-
indol-3-yl]ethyl ester (12). Compound 12 was obtained
as a pale yellow solid upon chromatography on silica
gel (EtOAc:hexanes; 5:95) in 72% yield. mp = 122–
123 ꢁC; 1H NMR (CDCl3) d 7.92–7.97 (dd, 2H,
J = 6.7, 1.8 Hz, ArH), 7.70 (br s, 1H, NH), 7.37–7.41
(dd, 2H, J = 6.8, 1.9 Hz, ArH), 7.14–7.19 (d, 1H,
J = 8.7 Hz, ArH), 7.01–7.02 (d, 1H, J = 2.0 Hz, ArH),
6.75–6.80 (dd, 1H, J = 8.7, 2.3 Hz, ArH), 4.43–4.51 (t,
2H, J = 7.2 Hz, CH2), 3.83 (s, 3H, OCH3), 3.09–3.16
(t, 2H, J = 7.2 Hz, CH2), 2.40 (s, 3H, CH3).
4.1.10. 4-Iodobenzoic acid-2-[5-methoxy-2-methyl-1H-
indol-3-yl]ethyl ester (13). Compound 13 was obtained
as a pale yellow solid upon chromatography on silica
gel (EtOAc:hexanes; 5:95) in 71% yield. mp = 131–
4.1.15. 4-Methoxybenzoic acid-2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethyl ester (17). Com-
pound 17 was obtained as a pale yellow solid upon
recrystallization with CH2Cl2/hexanes in 28% yield.
1
1
132 ꢁC; H NMR (CDCl3) d 7.73–7.76 (m, 4 H, ArH),
mp = 95–97 ꢁC; H NMR (CDCl3) d 7.95–7.97 (d, 2H,
7.14–7.19 (d, 1H, J = 8.7 Hz, ArH), 7.01–7.02 (d, 1H,
J = 2.0 Hz, ArH), 6.75–6.80 (dd, 1H, J = 8.7, 2.3 Hz,
ArH), 4.43–4.51 (t, 2H, J = 7.2 Hz, CH2), 3.83 (s, 3H,
OCH3), 3.09–3.16 (t, 2H, J = 7.2 Hz, CH2), 2.39 (s,
3H, CH3).
J = 8.7 Hz, ArH), 7.62–7.64 (d, 2H, J = 8.4 Hz, ArH),
7.42–7.44 (d, 2H, J = 8.3 Hz, ArH), 7.01–7.02 (d, 1H,
J = 2.2 Hz, ArH), 6.89–6.93 (m, 3H, ArH), 6.66–6.69
(dd, 1H, J = 8.9, 2.3 Hz, ArH), 4.47–4.51 (t, 2H,
J = 6.9 Hz, CH2), 3.86 (s, 3H, OCH3), 3.82 (s, 3H,

6818
A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
OCH3), 3.10–3.14 (t, 2H, J = 6.9 Hz, CH2), 2.36 (s, 3H,
CH3); ESI-CID 478 (MH+), m/z 326, 308, 188, 139.
4.1.21. 4-Chlorobenzoic acid-2-[1-(4-bromobenzyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethyl ester (23). Com-
pound 23 was obtained as a white solid upon recrystal-
lization with CH2Cl2/hexanes in 14% yield. mp = 96–
4.1.16. 2-Methoxybenzoic acid-2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethyl ester (18). Com-
pound 18 was obtained as a white solid upon recrystal-
lization with CH2Cl2/hexanes in 28% yield. mp = 88–
98 ꢁC; 1H NMR (CDCl3)
d 7.89–7.93 (d, 2H,
J = 9.0 Hz, ArH), 7.62–7.66 (d, 2H, J = 9.0 Hz, ArH),
7.33–7.38 (m, 4H, ArH), 7.04-7.07 (d, 1H, J = 9.0 Hz,
ArH), 6.86–6.91 (d, 1H, J = 2.1 Hz, ArH), 6.76–6.79
(dd, 1H, J = 9.0, 2.1 Hz, ArH), 5.20 (s, 2H, CH2),
4.46–4.51 (t, 2H, J = 7.0 Hz, CH2), 3.82 (s, 3H,
OCH3), 3.14–3.19 (t, 2H, J = 7.0 Hz, CH2), 2.24 (s,
3H, CH3); ESI-CID 512 (MH+), m/z 358, 187, 171.
90 ꢁC; 1H NMR (CDCl3)
d 7.95–7.97 (d, 2H,
J = 8.7 Hz, ArH), 7.62–7.66 (m, 2H, ArH), 7.41–7.45
(m, 3H, ArH), 6.89–7.01 (m, 5H, ArH), 6.66–6.69 (dd,
1H, J = 8.9, 2.3 Hz, ArH), 4.47–4.51 (t, 2H,
J = 6.9 Hz, CH2), 3.87 (s, 3H, OCH3), 3.80 (s, 3H,
OCH3), 3.10–3.14 (t, 2H, J = 6.9 Hz, CH2), 2.36 (s,
3H, CH3); ESI-CID 478 (MH+), m/z 326, 139.
4.1.22. 5-Methoxy-2-methylindole-3-acetamide (24). A
reaction mixture containing 1 (880 mg, 4.02 mmol),
EDCI (1.16 g, 6.04 mmol), HOBt (816 mg, 6.04 mmol),
DIPEA (2.8 mL, 16.08 mmol), and NH4Cl (430 mg,
8.04 mmol) in anhydrous (DMF) 16 mL (4 mL DMF/
1 mmol 1) was stirred at rt for 5 h. The reaction was
diluted with water and extracted with EtOAc
(3 · 10 mL). The combined EtOAc extracts were washed
with satd NaHCO3 (2 · 10 mL), water, dried (MgSO4),
filtered, and the solvent concentrated in vacuo until a
minimum volume of EtOAc remained. Upon cooling,
the desired amide crystallized out as a white crystalline
solid. mp = 146–148 ꢁC (lit. mp35 =1 47–150 ꢁC). 1H
NMR (DMSO-d6) d 10.56 (s, 1H, NH), 7.34 (br s, 1H,
CONH), 6.96–7.22 (m, 1H, ArH), 6.81 (s, 1H, ArH),
6.74 (br s, 1H, CONH), 6.58–6.62 (m, 1H, ArH), 3.78
(s, 3H, CH3), 3.34 (s, 2H, CH2), 2.29 (s, 3H, CH3);
ESI-CID 219 (MH+), m/z 187, 174, 148.
4.1.17. 4-Chlorobenzoic acid-2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethyl ester (19). Com-
pound 19 was obtained as a pale yellow solid upon
recrystallization with CH2Cl2/hexanes in 35% yield.
mp = 102–104 ꢁC; 1H NMR (CDCl3) d 7.91–7.96 (d,
2H, J = 8.5 Hz, ArH), 7.62–7.66 (d, 2H, J = 8.5 Hz,
ArH), 7.38-7.46 (m, 4H, ArH), 7.00–7.01 (d, 1H,
J = 2.3 Hz, ArH), 6.86–6.91 (d, 1H, J = 9.0 Hz, ArH),
6.66–6.70 (dd, 1H, J = 9.0, 2.4 Hz, ArH), 4.47–4.54 (t,
2H, J = 7.0 Hz, CH2), 3.82 (s, 3H, OCH3), 3.10–3.17
(t, 2H, J = 7.0 Hz, CH2), 2.38 (s, 3H, CH3); ESI-CID
482 (MH+), 326, 188, 139.
4.1.18. 4-Bromobenzoic acid-2-[1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl]ethyl ester (20). Com-
pound 20 was obtained as a pale yellow solid upon
recrystallization with CH2Cl2/hexanes in 41% yield.
mp = 97–99 ꢁC; 1H NMR (CDCl3) d 7.84–7.88 (m,
3H, ArH), 7.54–7.66 (m, 3H, ArH), 7.42–7.46 (m,
2H, ArH), 6.99–7.00 (d, 1H, J = 2.4 Hz, ArH), 6.86–
6.91 (d, 1H, J = 9.0 Hz, ArH), 6.69–6.70 (dd, 1H,
J = 9.0, 2.4 Hz, ArH), 4.47–4.54 (t, 2H, J = 7.0 Hz,
CH2), 3.82 (s, 3H, OCH3), 3.09–3.16 (t, 2H,
J = 7.0 Hz, CH2), 2.37 (s, 3H, CH3); ESI-CID 527
(MH+), 326, 188, 139.
4.1.23. 3-(2-Aminoethyl)-5-methoxy-2-methylindole (25).
To a suspension of LAH (370 mg, 9.74 mmol) in anhy-
drous tetrahydrofuran (THF) (80 mL) was added 24
(760 mg, 3.38 mmol) under argon at 0 ꢁC. The reaction
mixture was allowed to stir at rt under argon for 60 h.
The reaction was carefully quenched by the addition
of ice-cold water (ꢀ100 mL) and then extracted with
Et2O (3 · 25 mL). The combined ether extracts were
washed with 1 N HCl (2 · 25 mL). The combined acidic
extract was washed once with Et2O (50 mL) and then
neutralized with 1 N NaOH. Following neutralization,
the aqueous solution was extracted with Et2O
(3 · 25 mL). The combined organic solution was washed
with water, dried (MgSO4), filtered, and the solvent con-
centrated in vacuo to afford a yellow oil (530 mg, 73%).
A portion of the oil was treated with a solution contain-
ing one equivalent of oxalic acid in Et2O to furnish the
oxalate salt of 25, which was recrystallized from MeOH/
Et2O to afford a light brown crystalline solid. mp = 176–
178 ꢁC; 1H NMR (CD3OD) d 7.12–7.16 (d, 1H,
J = 8.7 Hz, ArH), 6.94–6.95 (d, 1H, J = 2.3 Hz, ArH),
6.67–6.71 (dd, 1H, J = 8.7, 2.3 Hz, ArH), 3.80–3.84 (s,
3H, OCH3), 3.01–3.11 (dd, 4 H, J = 8.3 Hz, CH2), 2.36
(s, 3H, CH3); ESI-CID 205 (MH+), m/z 188, 173, 158,
145, 130.
4.1.19. 4-Iodobenzoic acid-2-[1-(4-chlorobenzoyl)-5-meth-
oxy-2-methyl-1H-indol-3-yl]ethyl ester (21). Compound
21 was obtained as a pale yellow solid upon recrystalli-
zation with CH2Cl2/hexanes in 40% yield. mp = 128–
1
129 ꢁC; H NMR (CDCl3) d 7.84–7.88 (m, 3H, ArH),
7.54–7.66 (m, 3H, ArH), 7.42–7.46 (m, 2H, ArH),
6.99–7.00 (d, 1H, J = 2.4 Hz, ArH), 6.86–6.91 (d, 1H,
J = 9.0 Hz, ArH), 6.69–6.70 (dd, 1H, J = 9.0, 2.4 Hz,
ArH), 4.47–4.54 (t, 2H, J = 7.0 Hz, CH2), 3.82 (s, 3H,
OCH3), 3.09–3.16 (t, 2H, J = 7.0 Hz, CH2), 2.37 (s,
3H, CH3); ESI-CID 574 (MH+), 326, 188, 139.
4.1.20. 4-Chlorobenzoic acid-2-[1-(4-chlorobenzoyl)-5-
methoxy-1H-indol-3-yl]ethyl ester (22). Compound 22
was obtained as a white solid upon recrystallization with
CH2Cl2/hexanes in 67% yield. 1H NMR (CDCl3) d 7.91–
7.96 (d, 2H, J = 8.5 Hz, ArH), 7.62–7.66 (d, 2H,
J = 8.5 Hz, ArH), 7.38–7.46 (m, 4 H, ArH), 7.00–7.01
(d, 1H, J = 2.3 Hz, ArH), 6.86–6.91 (d, 1H, J = 9.0 Hz,
ArH), 6.66–6.70 (dd, 1H, J = 9.0, 2.4 Hz, ArH), 4.55–
4.59 (t, 2H, J = 6.9 Hz, CH2), 3.87 (s, 3H, OCH3),
3.09–3.13 (t, 2H, J = 6.6 Hz, CH2).
4.1.24. 4-Chloro-N-{2-[1-(4-chlorobenzoyl)-5-methoxy-2-
methyl-1H-indol-3-yl]ethyl}-benzamide (31). A reaction
mixture containing 25 (520 mg, 2.55 mmol), EDCI
(489 mg, 2.55 mmol), DMAP (31 mg, 0.255 mmol),
and 4-chlorobenzoic acid (354 mg, 2.26 mmol) in

A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
6819
anhydrous CH2Cl2 (15 mL) was stirred at rt for 3 h. The
reaction mixture was diluted with water and extracted
with CH2Cl2 (2 · 15 mL). The combined CH2Cl2 ex-
tracts were washed with water, dried (MgSO4), filtered,
and the solvent concentrated in vacuo. The crude amide
was chromatographed on silica gel (EtOAc:hexanes;
25:75 then 60:40) to afford 26 as a yellow oil (390 mg,
45%), which was used in the next step without further
characterization owing to its unstable nature. To a reac-
tion mixture comprising 26 (390 mg, 1.14 mmol) in
anhydrous DMF (3 mL) was added sodium hydride
(60% dispersion in mineral oil) (56 mg, 1.4 mmol) at
0 ꢁC under argon. After stirring for 20 min, the reaction
was treated with 4-chlorobenzoyl chloride (180 lL,
1.4 mmol) and the reaction was allowed to stir overnight
at rt. The reaction mixture was quenched with water and
extracted with Et2O (3 · 10 mL). The combined Et2O
extracts were washed with satd NaHCO3 (3 · 10 mL),
water, dried (MgSO4), filtered, and the solvent concen-
trated in vacuo to afford a yellow residue which was
chromatographed (EtOAc:hexanes; 20:80 then 40:60)
to generate 31 as a pale yellow solid (recrystallized from
EtOAc/hexanes) (371 mg, 67%). mp = 165–166 ꢁC; 1H
NMR (CDCl3) d 7.58–7.65 (m, 4 H, ArH), 7.43–7.47
(d, 2H, J = 8.6 Hz, ArH), 7.36–7.39 (d, 2H, J = 8.6 Hz,
ArH), 6.96–6.97 (d, 1H, J = 2.4 Hz, ArH), 6.88–6.90
(d, 1H, J = 9.0 Hz, ArH), 6.65–6.69 (dd, 1H, J = 9.0,
2.5 Hz, ArH), 6.17 (br t, 1H, CONH), 3.76 (s, 3H,
OCH3), 3.67–3.71 (q, 2H, J = 6.7 Hz, CH2), 2.99–3.04
(t, 2H, J = 6.7 Hz, CH2), 2.33 (s, 3H, CH3); ESI-CID
482 (MH+), 312, 173, 139.
matography (EtOAc:hexanes; 20:80 then 40:60) yielded
a yellow oil which upon crystallization from CH2Cl2/
hexanes generated 33 as an off-white solid (recrystallized
from EtOAc/hexanes) (60 mg, 17%). mp = 109–111 ꢁC;
1H NMR (CDCl3) d 7.62–7.66 (d, 2H, J = 8.3 Hz,
ArH), 7.44–7.48 (d, 2H, J = 8.5 Hz, ArH), 7.15-7.23
(m, 5H, ArH), 6.94–6.95 (d, 1H, J = 2.4 Hz, ArH),
6.84-6.88 (d, 1H, J = 9.0 Hz, ArH), 6.65–6.69 (dd, 1H,
J = 9.0, 2.5 Hz, ArH), 5.54 (br t, 1H, CONH), 3.84 (s,
3H, OCH3), 3.45–3.48 (q, 2H, J = 6.4 Hz, CH2), 2.90–
2.98 (t, 2H, J = 7.4 Hz, CH2), 2.80–2.87 (t, 2H,
J = 7 Hz, CH2), 2.38–2.46 (t, 2H, J = 8.1 Hz, CH2),
2.30 (s, 3H, CH3); ESI-CID 475 (MH+), 341, 173, 170.
4.1.27. N-{2-[1-(4-Chlorobenzoyl)-5-methoxy-1H-indol-
3-yl]ethyl}-acetamide (34). Compound 34 was obtained
by the acylation of melatonin (28) with 4-chlorobenzoyl
chloride as described in the preparation of 31. Following
work up, recrystallization from CH2Cl2/hexanes affor-
ded 34 as a white solid in 54% yield. mp = 172–173 ꢁC;
1H NMR (CDCl3) d 8.25–8.28 (d, 1H, J = 8.4 Hz,
ArH), 7.64–7.67 (dd, 2H, J = 6.6, 1.8 Hz, ArH), 7.50–
7.52 (dd, 2H, J = 8.4, 1.8 Hz, ArH), 6.98–7.05 (m, 3H,
ArH), 5.55 (br t, 1H, NH), 3.89 (s, 3H, OCH3), 3.51–
3.58 (q, 2H, J = 6.7 Hz, CH2), 2.85–2.89 (t, 2H,
J = 7.0 Hz, CH2), 1.94 (s, 3H, CH3).
4.1.28. N-{2-[1-(4-Bromobenzyl)-5-methoxy-1H-indol-3-
yl]ethyl}-acetamide (35). Compound 35 was obtained
as a white solid upon recrystallization from EtOAc/hex-
1
anes in 66% yield. mp = 152–153 ꢁC. H NMR (CDCl3)
d 7.39–7.42 (dd, 2H, J = 6.7, 1.76 Hz, ArH), 7.09–7.11
(d, 1H, J = 8.8 Hz, ArH), 7.04–7.05 (d, 1H, J = 2.3 Hz,
ArH), 6.92–6.97 (m, 3H, ArH), 6.82–6.86 (dd, 1H,
J = 8.9, 2.4 Hz, ArH), 5.55 (br t, 1H, NH), 5.19 (s,
2H, CH2), 3.85 (s, 3H, OCH3), 3.54–3.59 (q, 2H,
J = 6.7 Hz, CH2), 2.91–2.96 (t, 2H, J = 6.7 Hz, CH2),
1.92 (s, 3H, CH3).
4.1.25. 4-Chloro-N-{2-[1-(4-bromobenzyl)-5-methoxy-2-
methyl-1H-indol-3-yl]ethyl}-benzamide (32). It was ob-
tained from 26 in a similar manner as 31, using 4-bro-
mobenzyl bromide as the alkylating agent. Upon
workup, silica gel chromatography (EtOAc:hexanes;
20:80 then 25:75) afforded 32 as a pale yellow solid
(recrystallized from CH2Cl2/hexanes) (184 mg, 57%).
mp = 172–173 ꢁC; 1H NMR (CDCl3) d 7.50–7.53 (d,
2H, J = 8.3 Hz, ArH), 7.28–7.43 (m, 4 H, ArH), 7.13-
7.10 (d, 1H, J = 8.9 Hz, ArH), 7.01–7.02 (d, 1H,
J = 2.3 Hz, ArH), 6.77–6.81 (m, 3H, ArH), 6.09 (br t,
1H, CONH), 3.78 (s, 3H, OCH3), 3.66–3.73 (q, 2H,
J = 6.3 Hz, CH2), 3.02–3.07 (t, 2H, J = 6.4 Hz, CH2),
2.24 (s, 3H, CH3); ESI-CID 512 (MH+), 341, 173, 170.
4.1.29.
N-{2-[1-(4-Bromobenzyl)-5-methoxy-2-methyl-
1H-indol-3-yl]ethyl}-acetamide (36). A reaction mixture
containing 25 (520 mg, 2.55 mmol) in 5 mL of anhy-
drous CH2Cl2 was treated with freshly distilled Et3N
(420 lL, 3 mmol) and acetyl chloride (213 lL, 3 mmol)
at 0 ꢁC and the reaction was allowed to proceed at rt
for 5 h. The reaction mixture was quenched with water
and extracted with CH2Cl2 (3 · 10 mL). The combined
organic extracts were washed with satd NaHCO3
(3 · 10 mL), water, dried (MgSO4), filtered, and the sol-
vent concentrated in vacuo. The crude residue was chro-
matographed on silica gel (EtOAc:hexane; 40:60) to
afford 29 as a brown oil (250 mg, 40%), which was alkyl-
ated without further purification. N-alkylation of 29
with 4-bromobenzyl bromide afforded 36 as a white sol-
id recrystallized from CH2Cl2/hexanes in 30% yield.
4.1.26. N-{2-[1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-
1H-indol-3-yl]ethyl}-2-phenethylamide (33). A reaction
mixture containing 25 (277 mg, 1.36 mmol), EDCI
(261 mg, 1.36 mmol), DMAP (17 mg, 0.136 mmol),
and hydrocinnamic acid (179 mg, 1.19 mmol) in anhy-
drous CH2Cl2 (15 mL) was stirred at rt for 3 h. The reac-
tion mixture was diluted with water and extracted with
CH2Cl2 (2 · 15 mL). The combined CH2Cl2 extracts
were washed with water, dried (MgSO4), filtered, and
the solvent concentrated in vacuo. The crude amide
was chromatographed on silica gel (EtOAc:hexanes;
20:80 then 50:50) to afford 27 as a reddish brown oil
(256 mg, 54%), which was used in the next step without
further characterization. Acylation of 27 with 4-chloro-
benzoyl chloride was conducted in a manner similar to
that described in the preparation of 31. Silica gel chro-
1
mp = 168–170 ꢁC; H NMR (CDCl3) d 7.35–7.43 (dd,
2H, J = 6.7, 1.76 Hz, ArH), 7.09–7.11 (d, 1H,
J = 8.8 Hz, ArH), 7.01–7.04 (d, 1H, J = 2.3 Hz, ArH),
6.76–6.81 (m, 3H, ArH), 5.47 (br t, 1H, NH), 5.21 (s,
2H, CH2), 3.85 (s, 3H, OCH3), 3.45–3.52 (q, 2H,
J = 6.7 Hz, CH2), 2.90–2.95 (t, 2H, J = 6.7 Hz, CH2),
2.27 (s, 3H, CH3), 1.90 (s, 3H, CH3); ESI-CID 416
(MH+), 173, 170.

6820
A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
4.1.30.
N-{2-[1-(4-Bromobenzyl)-5-methoxy-2-methyl-
4.1.34. N-{2-[1-(4-Bromo-benzyl)-5-methoxy-2-methyl-
1H-indol-3-yl]-ethyl}-4- fluoro-2-nitrobenzamide (41). 2-
[1-(4-Bromo-benzyl)-5-methoxy-2-methyl-1H-indol-3-
yl]-ethylamine (50 mg, 0.21 mmol), EDCI (53 mg,
0.31 mmol), DIPEA (54 ul, 0.31 mmol), 4-fluoro-2-nitro-
benzoic acid (58 mg, 0.31 mmol), and HOBt (42 mg,
0.31 mmol) were dissolved in DMF (dry, 5 ml) and al-
lowed to stir for 18 h at room temperature. The reaction
was quenched with saturated sodium bicarbonate
(10 mL) and extracted with ethyl acetate (2 · 20 mL).
The combined organic solution was washed with water
(20 mL), dried with sodium sulfate, concentrated, and
purified on silica (ethyl acetate 50% in hexane) to give a
1H-indol-3-yl]ethyl}-propionamide (37). It was obtained
in a similar fashion as 32. Upon workup, the residue
was recrystallized from CH2Cl2/hexanes to afford 37 as
an off-white solid (160 mg, 33%). mp = 168–169 ꢁC; H
NMR (CDCl3) d 7.32–7.39 (dd, 2H, J = 8.7, 2.3 Hz,
ArH), 7.00–7.07 (m, 2H, ArH), 6.76-6.81 (m, 3H,
ArH), 5.45 (br t, 1H, NH), 5.20 (s, 2H, CH2), 3.85 (s,
3H, OCH3), 3.47–3.53 (q, 2H, J = 6.9 Hz, CH2), 2.90–
2.95 (t, 2H, J = 6.9 Hz, CH2), 2.26 (s, 3H, CH3), 2.08–
2.15 (q, 2H, J = 7.6 Hz, CH2), 1.04–1.08 (t, 3H,
J = 7.6 Hz, CH3); ESI-CID 430 (MH+), 343, 173, 170.
1
1
4.1.31. N-{2-[5-Methoxy-2-methyl-1H-indol-3-yl]ethyl}-
carbamic acid tert-butyl ester (38). Compound 25
(50 mg, 0.25 mmol) was stirred in dry methanol (2 mL)
while di-tert-butyldicarbonate (64 mg, 0.29 mmol) in
dry methanol (50 lL) was added dropwise to the stirred
solution at 23 ꢁC. The reaction mixture was stirred at rt
for 18 h. The reaction was concentrated and the residue
was dissolved in ethyl acetate (5 mL), washed with satu-
rated sodium bicarbonate (2 mL), dried over sodium
sulfate, and concentrated. Purification by flash chroma-
tography yielded 75 mg (99%) product. 1H NMR
(CDCl3) d 9.95 (s, 1H, NH), 7.25 (d, J = 8.3 Hz, 1H,
ArH), 6.88 (s, 1H, ArH), 6.70 (d, J = 9.0 Hz, 1H,
ArH), 4.45 (s, 1H, NH), 3.78 (s, 3H, OCH3), 3.60 (d,
J = 2.2 Hz, 2H, CH2), 2.80 (d, J = 2.4 Hz, 2H, CH2),
2.28 (s, 3H), 1.36 (s, 9H, t-Bu); ESI-CID 305 (MÀH+).
yellow solid (52 mg, 72%) H NMR (CDCl3) 8.08 (s,
1H, ArH), 7.63–7.58 (dd, J1 = J2 = 6.24, ArH), 7.37 (d,
J = 8.4 Hz, ArH), 7.30–7.21 (m, 4H, ArH), 7.08 (s, 1H,
ArH), 6.80-6.77 (dd, J1 = J2 = 6.3 Hz, 2H, ArH), 5.20
(s, 2H, CH2), 3.76 (s, 3H, OCH3), 3.69–3.67 (m, 2H,
CH2), 3.08–3.02 (m, 2H, CH2), 2.30 (s, 3H, CH3); ESI-
CID 541 (MÀH+).
4.1.35.
N-{2-[1-(4-bromo-benzyl)-5-methoxy-2-methyl-
1H-indol-3-yl]-ethyl}-4-chloro-2-nitrobenzamide (42). 2-
[1-(4-bromo-benzyl)-5-methoxy-2-methyl-1H-indol-3-yl]-
ethylamine (50 mg, 0.21 mmol), EDCI (53 mg,
0.31 mmol), DIPEA (54 lL, 0.31 mmol), 4-chloro-2-
nitrobenzoic acid (63 mg, 0.31 mmol), and HOBt
(42 mg, 0.31 mmol) were dissolved in DMF (dry,
5 mL) and allowed to stir for 18 h at room temperature.
The reaction was quenched with saturated sodium bicar-
bonate (10 mL) and extracted with ethyl acetate
(2 · 20 mL). The combined organic solution was washed
with water (20 mL), dried with sodium sulfate, concen-
trated, and purified on silica ( ethyl acetate 50% in hex-
ane) to give a yellow solid (56 mg, 74%) 1H NMR
(CDCl3) 8.0 (s, 1H, ArH), 7.62–7.57 (dd,
J1 = J2 = 6.24, ArH), 7.38 (d, J = 8.4 Hz, ArH), 7.29–
7.22 (m, 4H, ArH), 7.06 (s, 1H, ArH), 6.80–6.77 (dd,
J1 = J2 = 6.3 Hz, 2H, ArH), 5.20 (s, 2H, CH2), 3.76 (s,
3H, OCH3), 3.75–3.73 (m, 2H, CH2), 3.16–3.10 (m,
2H, CH2),2.30 (s, 3H, CH3); ESI-CID 557 (MÀH+).
4.1.32. N-{2-[1-(4-Bromo-benzyl)-5-methoxy-2-methyl-
1H-indol-3-yl]-ethyl}-carbamic acid tert-butyl ester (39).
NaH (7 mg, 0.29 mmol) was stirred in DMF (2 mL) at
0 ꢁC. Compound 38 (74 mg, 0.25 mmol) in DMF
(1 mL) was added dropwise to the stirred solution.
The reaction mixture was stirred for 20 min at 0 ꢁC at
which time p-bromobenzyl bromide (72 mg, 0.29 mmol)
was added. The reaction mixture stirred overnight at
room temperature. The reaction mixture was diluted
slowly with water, extracted with ethyl acetate
(2 · 10 mL) and washed with water (2 · 5 mL), dried
over sodium sulfate, concentrated, and purified on silica
10:90 ethyl acetate/hexane to give 52 mg of a yellow sol-
4.1.36.
N-{2-[1-(4-bromo-benzyl)-5-methoxy-2-methyl-
1
id, 40%. H NMR (CDCl3) d 7.39 (d, J = 8 Hz, 2H,
1H-indol-3-yl]-ethyl}-4-fluorobenzamide (43). 2-[1-(4-
Bromobenzyl)-5-methoxy-2-methyl-1H-indol-3-yl]-eth-
ylamine (42 mg, 0.11 mmol), EDCI (25 mg, 0.13 mmol),
DIPEA (23 lL, 0.13 mmol), p-fluorobenzoic acid
(18 mg, 0.13 mmol), and HOBt (18 mg, 0.13 mmol) were
dissolved in DMF (dry, 5 mL) and allowed to stir for
18 h at room temperature. The reaction was quenched
with saturated sodium bicarbonate (10 mL) and extracted
with ethyl acetate (2 · 20 mL). The combined organic
solution was washed with water (20 mL), dried with sodi-
um sulfate, concentrated, and purified on silica (ethyl ace-
tate 50% in hexane) to give a yellow solid (30 mg, 56%) 1H
NMR (CDCl3) 8.11–8.08 (m, 1H, ArH), 7.61–7.56 (m,
2H, ArH), 7.34 (d, J = 8.4 Hz, ArH), 7.16–6.98 (m, 5H,
ArH), 6.77 (d, J = 8.5 Hz, 2H, ArH), 5.19 (s, 2H, CH2),
3.76 (s, 3H, OCH3), 3.69-3.67 (m, 2H, CH2), 3.06–3.01
(m, 2H, CH2), 2.23 (s, 3H, CH3); ESI-CID 496 (MÀH+).
ArH), 7.10 (s, 1H, ArH), 7.06 (d, J = 8.6 Hz, 1H,
ArH), 6.8 (d, J = 8.2 Hz, 2H, ArH), 6.77 (d,
J = 8.7 Hz, 1H, ArH), 5.2 (s, 2H, CH2), 3.86 (s, 3H,
OCH3), 3.4 (d, J = 2.2 Hz, 2H, CH2), 2.85 (d,
J = 2.6 Hz, 2H, CH2), 2.28 (s, 3H, CH3), 1.40 (s, 9H,
t-Bu); ESI-CID 474 (MÀH+).
4.1.33. 2-[1-(4-Bromo-benzyl)-5-methoxy-2-methyl-1H-
indol-3-yl]-ethylamineÆHCl (40). HCl(g) was slowly bub-
bled through a 1 mL solution of compound 39 in dichlo-
romethane. After 1 h, the product precipitated. The
solvent was removed in vacuo to give 13 mg (84%) of
brown solid, which was used as is without further purifica-
tion. 1H NMR (300 MHz, DMSO) 7.3 (s, 1H, ArH), 7.20
(d, J = 8.2 Hz, 1H, ArH), 7.05 (d, J = 2.4 Hz, 2H, ArH),
6.85 (d, J = 8.9 Hz, 2H, ArH), 6.65 (d, J = 8.9 Hz, 1H,
ArH), 5.2 (s, 2H, CH2), 3.72 (s, 3H, OCH3), 3.2 (d,
J = 2.4 Hz, 2H, CH2), 2.9 (d, J = 2.3 Hz, 2H, CH2), 2.20
(s, 3H, CH3); ESI-CID 373 (MÀH+).
4.1.37. N-2-[1-(4-Bromobenzyl)-3-butyl-5-methoxy-2-
methyl]-1H-indole (47). A suspension containing 4-meth-

A. S. Kalgutkar et al. / Bioorg. Med. Chem. 13 (2005) 6810–6822
6821
oxyphenylhydrazineÆHCl salt (44, 5 g, 28.63 mmol) and
2-heptanone (45, 3.4 g, 30 mmol) in 50 mL of glacial
acetic acid was stirred at rt for 30 min, heated at 60 ꢁC
for 30 min and then at 120 ꢁC for 20 h. The dark solu-
tion was cooled and diluted with 20 mL heptane and
concentrated in vacuo. The residue was partioned be-
tween EtOAc and water. The organic layer was washed
with brine, water, dried (MgSO4), filtered, and the sol-
vent concentrated in vacuo. Silica gel chromatography
(EtOAc:hexanes; 10:90) gave the desired indole as a pale
yellow oil 46, which eventually solidified upon cooling
(2.1 g, 33%). The crude indole 46 was alkylated with 4-
bromobenzyl bromide as described in the preparation
of 32. Following chromatography on silica gel
(EtOAc:hexanes; 5:95), 47 was obtained as a gray solid
in 26% yield. mp = 89–90 ꢁC; 1H NMR (CDCl3) d
7.34–7.38 (m, 2H, ArH), 7.00–7.03 (m, 2H, ArH),
6.72–6.82 (m, 3H, ArH), 5.19 (s, 2H, CH2), 3.83 (s,
3H, CH3), 2.67-2.72 (t, 2H, J = 7.5 Hz, CH2), 2.24 (s,
3H, CH3), 1.54–1.64 (m, 2H, CH2), 1.33-1.43 (m, 2H,
CH2), 0.91–0.95 (t, 3H, J = 7.2 Hz, CH3).
centage 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.
4.2.2. Inhibition of COX-2 activity in activated
RAW264.7 cells. Low passage number murine
RAW264.7 cells were grown in DMEM containing
10% heat-inactivated FBS. Cells (6.2 · 106 cells/T25
flask) were activated with 500 ng/mL LPS and 10 U/
mL IFN-c in serum-free DMEM for 7 h. Vehicle
(DMSO) or inhibitor in DMSO (0–1 lM) was added
for 30 min at 37 ꢁC. Inhibition of exogenous arachidonic
acid metabolism or inhibition of PGD2 synthesis was
determined by incubating the cells with 20 lM [1-14C]ar-
achidonic acid, respectively, for 15 min at 25 ꢁC. Ali-
quots (200 lL) were removed into termination solution
and total products were quantitated by the TLC assay
as described earlier.29
Acknowledgment
4.2. Enzymology
This study was supported by a grant from the National
Institutes of Health (CA89450).
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). Hema-
tin was purchased from Sigma Chemical Co. (St. Louis,
MO). COX-1 was purified from ram seminal vesicles
(Oxford Biomedical Research, Inc., Oxford, MI) as de-
scribed earlier.36 The specific activity of the protein
was 20 lM O2/min/mg, and the percentage of holopro-
tein 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 from the pVL1393 expres-
sion vector (Pharmingen), and purified by ion-exchange
and gel filtration chromatography.38 All of the purified
proteins were shown by densitometric scanning of a
7.5% SDS–PAGE gel to be >80% pure.
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