Design and synthesis of 1-indol-1-yl-propan-2-ones as inhibitors of human cytosolic phospholipase A2α

Article abstract of DOI:10.1021/jm051243a

The synthesis and structure-activity relationship study of a series of 1-indol-1-yl-3-phenoxypropan-2-one inhibitors of cytosolic phospholipase A ₂α. (cPLA₂α) are described. The compounds were evaluated in a vesicle assay with isolated cPLA₂α and in cellular assays with intact human platelets. Systematic variation led to 3-methylhydrogen 1-[3-(4-decyloxyphenoxy)-2-oxopropyl]indole-3,5-dicarboxylate (57), which revealed the highest activity against the isolated enzyme. With an IC₅₀ value of 4.3 nM in this assay, it is one of the most potent in vitro cPLA₂α inhibitors known today.

Full text of DOI:10.1021/jm051243a

J. Med. Chem. 2006, 49, 2611-2620
2611
Design and Synthesis of 1-Indol-1-yl-propan-2-ones as Inhibitors of Human Cytosolic
Phospholipase A2r
Joachim Ludwig, Stefanie Bovens, Carsten Brauch, Alwine Schulze Elfringhoff, and Matthias Lehr*
Institute of Pharmaceutical and Medicinal Chemistry, UniVersity of Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany
ReceiVed December 13, 2005
The synthesis and structure-activity relationship study of a series of 1-indol-1-yl-3-phenoxypropan-2-one
inhibitors of cytosolic phospholipase A2R (cPLA2R) are described. The compounds were evaluated in a
vesicle assay with isolated cPLA2R and in cellular assays with intact human platelets. Systematic variation
led to 3-methylhydrogen 1-[3-(4-decyloxyphenoxy)-2-oxopropyl]indole-3,5-dicarboxylate (57), which revealed
the highest activity against the isolated enzyme. With an IC50 value of 4.3 nM in this assay, it is one of the
most potent in vitro cPLA2R inhibitors known today.
Introduction
Phospholipase A2 (PLA2) enzymes are a class of esterases
that catalyze the hydrolysis of membrane phospholipids at the
sn-2 position, leading to the production of lysophospholipids
and free fatty acids including arachidonic acid. Arachidonic acid
is further converted by cyclooxygenase and lipoxygenase
enzymes to bioactive eicosanoids, such as prostaglandins and
leukotrienes. In addition, a subset of lysophospholipids re-
leased by PLA2 can be acetylated to the platelet-activating factor
(PAF). Prostaglandins, leukotrienes, lysophospholipids, and
PAFs are potent mediators of inflammation.1-3 Thus, the
inhibition of PLA2 is considered to be an interesting target for
the design of new anti-inflammatory drugs.4-9 The special
attraction of this approach is based on the evidence that un-
Figure 1.
like cyclooxygenase inhibitors the inhibitors of PLA2 not only
reduce the formation of prostaglandins but also suppress the
generation of proinflammatory leukotrienes, lysophospholipids,
and PAFs. Therefore, it can be expected that inhibitors of PLA2
will possess improved therapeutic activities in comparison to
those of the cyclooxygenase inhibitors therapeutically applied
today.
zyme is tightly regulated by different factors, including intra-
cellular Ca2+ levels and phosphorylation. cPLA2R is unique
among the PLA2 enzymes in having a preference for phos-
pholipids with arachidonic acid at the sn-2 position. The cata-
lytic mechanism of cPLA2R is thought to be similar to that of
serine proteases, proceeding through a serine-arachidonoyl
intermediate.
One problem associated with the in vitro search for anti-
inflammatory PLA2 inhibitors is the selection of the appropriate
enzyme because many different PLA2 enzymes are present in
the mammalian organism.10,11 They can be divided into PLA2
enzymes utilizing a catalytic histidine and PLA2 enzymes with
a serine in the active site. The small molecular weight
(approximately 14 kDa) secretory PLA2 enzymes (sPLA2) are
members of the first group. The second group consists of
cytosolic PLA2 enzymes (cPLA2), calcium-independent PLA2
enzymes (iPLA2), and lipoprotein-associated PLA2 enzymes,
which have higher molecular weights than those of the sPLA2
enzymes. From all of these PLA2 enzymes, the R-subtype of
cPLA2 (cPLA2R) seems to play the central role in the arachi-
donic acid cascade and during the inflammatory response as
supported by experiments with cells overexpressing cPLA2R12
and cPLA2R knockout animals.13-18
Although there have been intense efforts made in finding
inhibitors of cPLA2R, no such compound has emerged as an
anti-inflammatory drug to date. The identification of cPLA2R
inhibitors is complicated by the fact that they have to partition
into the phospholipid membrane to interfere with the enzyme
in its active status. For enrichment in the phospholipid layer,
the inhibitors must possess a substantial lipophilicity. Such
compounds, however, often do not have suitable pharmaceutical
properties for in vivo measurements of anti-inflammatory
activity. The only cPLA2R inhibitor reported to undergo clinical
development as an anti-inflammatory drug is the indole deriva-
tive efipladib (1) from Wyeth.19,20 Extreme potent in vitro
cPLA2R inhibitors discovered are pyrrolidine 2 of Shionogi21
and propan-2-one 3 (AR-C70484XX) of AstraZeneca22 (Fig-
ure 1). In vivo data for 2 and 3 have not been published until
now.
cPLA2R, also classified as group IVA PLA2, is an 85 kDa
protein, which is present in the cytosol of resting cells and
translocates to intracellular membranes following stimu-
lation with a variety of agonists.11 The activation of the en-
We have found that several indole-2-carboxylic acid deriva-
tives such as 4 and 5 (Figure 2) are inhibitors of cPLA2R-
mediated arachidonic acid release in intact human platelets in
the low micromolar and submicromolar ranges.23,24 However,
these compounds did not inhibit the isolated enzyme, indicating
that they do not directly interact with the catalytic site of the
enzyme.25
* Corresponding author. Tel: +49-251-83-33331. Fax: +49-251-83-
32144. E-mail: lehrm@uni-muenster.de.
10.1021/jm051243a CCC: $33.50 © 2006 American Chemical Society
Published on Web 03/28/2006

2612 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Ludwig et al.
Scheme 1
Figure 2.
An important pharmacophoric element of the propan-2-one
cPLA2R inhibitor 3 from AstraZeneca is the 4-decyloxyphe-
noxy-2-oxopropyl group. In an effort to gain true inhibitors of
the enzyme, we synthesized and evaluated indole derivatives,
which contain an analogous residue. In this approach, we first
prepared compounds with the related equilipophilic 4-octylphe-
noxy-2-oxopropyl moiety such as indole 13. The reason for the
selection of this residue was the fact that the precursor
4-octylphenol required for its synthesis was commercially
available, in contrast to the 4-decyloxyphenol needed for the
preparation of the 4-decyloxyphenoxy-2-oxopropyl moiety. In
the course of this approach, compounds were obtained, which
proved to be highly active against the isolated enzyme as well
as the cellular production of arachidonic acid.
(a) Epichlorohydrin, KOH, Bu4N+Br-, room temp.; (b) LiBr, silica gel,
CH2Cl2, room temp.; (c) acetyl chloride, pyridine, CH2Cl2, 0 °C; (d) tert-
butyl indole-2-carboxylate, tert-BuOK, DMSO, 110 °C; (e) CH3ONa,
CH3OH; room temp.; (f) acetic anhydride, DMSO, room temp.; (g) TFA,
CH2Cl2, room temp.
Scheme 2
Chemistry
Indole-2- and -3-carboxylic acid derivatives 13 and 14 were
synthesized by the route outlined for compound 13 in Scheme
1. 4-Octylphenol (6) was reacted with epichlorohydrin in the
presence of powdered KOH and tetrabutylammonium bromide
to give the intermediate 726 in excellent yield. The epoxide ring
of 7 was opened regioselectively with LiBr, applying silica gel
as a catalyst to afford 8. Acetylation of this compound with
acetyl chloride led to 9, which was coupled with tert-butyl
indole-2-carboxylate27 in DMSO in the presence of potassium
tert-butylate. Treatment of obtained compound 10 with sodium
methoxide in methanol yielded the hydroxy intermediate 11.
The alcohol moiety of 11 was oxidized by the Albright-
Goldman procedure with acetic anhydride-DMSO to provide
keto-ester 12. Deprotection of the tert-butyl ester under standard
conditions afforded target compound 13.
(a) tert-Butyl indole-4-carboxylate, NaH, DMF, 60 °C; (b) acetic
anhydride, DMSO, room temp.; (c) TFA, CH2Cl2, room temp.
Scheme 3
The synthesis of indole-4-, -5-, and -6-carboxylic acids
17-19 was conducted in a manner similar to that described for
the synthesis of cPLA2R inhibitor 3.22 Scheme 2 outlines the
synthesis of compound 17. Thus, tert-butyl indole-4-carboxy-
late27 was reacted with sodium hydride and 7 in DMF to give
the hydroxy intermediate 15. Subsequent oxidation of the
alcohol group of this compound with the acetic anhydride-
DMSO system afforded keto-ester 16, which was treated with
trifluoroacetic acid to yield desired acid 17. During the synthesis
of 18, an indole-5-carboxylic acid derivative with a tert-butyl
group in position 3 of the indole 39 was obtained as a side
product.
The analogues of 18 with varying substituents at the indole-
5-position (20-26) were synthesized in a fashion similar to the
synthesis of 16. For the preparation of indole-5-carboxamide
28, the nitrile moiety of the hydroxy intermediate of 24 was
hydrolyzed to carboxamide 27 by KOH in tert-butyl alcohol.
Oxidation of 27 by acetic anhydride-DMSO yielded keto-amide
product 28.
(a) N-Chlorosuccinimide, CH3OH, room temp.; (b) oxalyl chloride, DMF,
CH2Cl2, 0 °C-room temp; (c) hydroxylammonium chloride, pyridine,
reflux; (d) 2-chloro-1-methylpyridinium iodide, THF, triethylamine, room
temp.; (e) NaCN, activated MnO2, CH3OH, room temp.

Design and Synthesis of 1-Indol-1-yl-propan-2-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2613
Scheme 4
carried out in a fashion similar to that described for the synthesis
of 16. Hydrogenation of intermediate 49 with palladium on
charcoal afforded 50.
Decyloxy substituted target compounds 54 and 57 were
prepared as shown in Scheme 6, starting from 2-(4-decyloxy-
phenoxymethyl)oxirane22 and the appropriate indole-tert-butyl-
ester.
{1-[3-(4-Octylphenoxy)-2-oxopropyl]indol-3-yl}acetic acid
(59) (Figure 3) was synthesized from tert-butyl indol-3-yl-
acetate applying the synthetic route described for the synthesis
of 17.
Evaluation of Inhibitors. All newly synthesized indole
derivatives were evaluated in an assay applying cPLA2R isolated
from human platelets.29 Like other lipases, cPLA2R has evolved
to work optimally at a lipid-water interface. For this reason,
sonicated covesicles consisting of 1-stearoyl-2-arachidonoyl-
sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycerol30 were
used as enzyme substrates. A possible problem of assays using
such an aggregated form of phospholipids is that a test
compound could inhibit the enzyme not by binding to its active
site but by merely altering the substrate assembly and, hence,
causing the enzyme to desorb from the lipid-water interface.
To exclude this path of action, the mole fraction of the inhibitor
in the interface has to be kept low.31 Thus, the highest
concentration of test compounds evaluated was 10 µM, whereas
the concentration of the vesicle-forming lipids was 300 µM.
The enzyme activity was determined by measuring the enzyme
product arachidonic acid formed after an incubation time of 60
min with HPLC and UV detection at 200 nm. Several of the
active compounds were also tested in cellular situations. In this
assay, cPLA2R of intact human platelets was stimulated with
calcium ionophore A23187.24 The cPLA2R-catalyzed liberation
of arachidonic acid from membrane phospholipids was measured
with HPLC and UV detection at 200 nm. To avoid the
metabolism of arachidonic acid via the cyclooxygenase-1 and
12-lipoxygenase pathways, the dual cyclooxygenase/12-lipoxy-
genase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) was
added to the platelets in these experiments.
(a) 1. ZnCl2, BuLi, CH2Cl2, room temp., 2. acetyl chloride, AlCl3, 0
°C-room temp.; (b) KOH, THF, ethylen glycol, reflux; (c) N,N-dimeth-
ylformamide di-tert-butyl acetal, benzene, reflux.
The 3-functionalized indole-5-carboxylic acid derivatives
40-44 were prepared in the same way as 17, starting from the
appropriate 3-substituted tert-butyl indole-5-carboxylates. The
syntheses of the latter compounds are described in Schemes 3
and 4. Chlorination of tert-butyl indole-5-carboxylate (29)27 with
N-chlorosuccinimide28 provided 3-chloro substituted indole 30.
The formylation of 29 in position 3 was achieved with oxalyl
chloride/DMF in CH2Cl2. 3-Formyl intermediate 31 was con-
verted to 3-cyano indole 33 via oxime 32 by reaction with
hydroxylammonium chloride in pyridine followed by dehydra-
tion with 2-chloro-1-methylpyridinium iodide. Treatment of
formylindole derivative 31 with sodium cyanide and activated
MnO2 provided diester intermediate 34. The synthesis of tert-
butyl 3-acetylindole-5-carboxylate (38) started from methyl
indole-5-carboxylate (35) (Scheme 4). This was acetylated in
position 3 to provide 36. Hydrolysis of the methyl ester of 36
by KOH followed by esterfication of obtained carboxylic acid
37 with N,N-dimethylformamide di-tert-butyl acetal led to
intermediate 38.
The indole-3,5-dicarboxylic acid 50 was synthesized as shown
in Scheme 5. Thus, methyl 3-formylindole-5-carboxylate (45)
was converted to dimethylester 46 by reaction with NaCN/
MnO2. Transesterfication with benzyl alcohol/potassium ben-
zylate gave dibenzyl ester 47. The introduction of the 3-aryloxy-
2-oxopropyl residue in position 1 of this indole compound was
Besides, phorbolester 12-O-tetradecanoylphorbol-13-acetate
(TPA) was used as an alternative stimulant. Although A23187
Scheme 5
(a) NaCN, activated MnO2, CH3OH, room temp.; (b) KH (30% dispersion in mineral oil), benzyl alcohol, 100 °C; (c) 2-(4-octylphenoxy)methyloxirane,
NaH, DMF, 100 °C; (d) acetic anhydride, DMSO, room temp.; (e) H2, Pd/C, THF, room temp.

2614 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Ludwig et al.
Scheme 6
(a) NaH, DMF, tert-butyl indole-5-carboxylate, 60 °C, or 5-tert-butyl-3-methyl indole-3,5-dicarboxylate (34), 100 °C; (b) acetic anhydride, DMSO, room
temp.; (c) TFA, CH2Cl2, room temp.
activates cPLA2R by increasing the cytosolic Ca2+ concentration,
TPA stimulates the enzyme via phosphorylation as a conse-
quence of the activation of protein kinases, probably, in
combination with the elevation of local Ca2+ levels.32 The
kinetics of arachidonic acid formation after stimulation with
A23187 and TPA differ remarkably. Whereas A23187 causes
a fast and short rise of the arachidonic acid liberation, TPA
leads to a slower and long-lasting arachidonic acid release by
the cells. For this reason, different incubation times were used
for both stimuli: 1 min for A23187 and 60 min for TPA.
Because the lysis of the platelets by a test compound may
falsely indicate enzyme inhibition, we also determined the cell
lytic potency of each compound by turbidimetry.33 In these
experiments, it was found that none of the compounds showed
cell lytic properties against human platelets at a concentration
of 10 µM.
the isolated enzyme at 10 µM. Nitrile 24 and carboxylic acid
methylester 25 possessed IC50 values greater than 10 µM in
this assay, whereas the IC50 value of carbaldehyde 26 was 4.3
µM. The most active compound of this series was 5-carboxa-
mide 28. With an IC50 value of 0.12 µM, it was about three
times less active against the isolated enzyme than 5-carboxylic
acid 18. Similar structure-activity relationships could be
observed when the substances were tested in cellular situations
with A23187 as the stimulant.
Next, we investigated the influence of substituents at position
3 of indole 18 on its enzyme inhibitory properties. Although
the introduction of the lipophilic tert-butyl and chloro residues
(39, 40) caused a marked loss of activity against isolated
cPLA2R, the more polar electron-withdrawing groups CN, CHO,
COCH3, and COOCH3 (41-44) significantly increased inhibi-
tory potency (Table 3). The most active compound of this series
was the indole-5-carboxylic acid with a methylester moiety at
position 3 (44). With an IC50 value of 0.0049 µM, this compound
was about 7-fold more active in the assay with the isolated
enzyme than the parent 18. Interestingly, the cleavage of the
ester group of 44 was accompanied by a large decrease of
inhibitory potency. The IC50 value of dicarboxylic acid 50 was
only 0.89 µM.
All derivatives of 18 with substituents at position 3 of the
indole also inhibited the A23187-induced arachidonic acid
release in human platelets. However, the IC50 values obtained
with this assay were much higher than those in the test system
with the isolated enzyme. Furthermore, the inhibition data did
not correlate between the assays. Although 3-acetyl and 3-meth-
oxycarbonyl derivatives 43 and 44 were significantly more
active than the 3-unsubstituted indole 18 in the assay with
isolated cPLA2R, they showed poorer activity than 18 in the
cellular assay.
This decrease of activity in the cellular assay may be because
of an impaired penetration of the compounds into the cells or
the transformation of the compounds into inactive or less active
metabolites in the cells. Besides, in our opinion, a third reason
for the observed results must be considered. The indole-5-
carboxylic acids synthesized are structurally related to the
recently published 1,3-diaryloxypropan-2-ones such as 3.22 This
compound was reported to form covalent bonds with the serine
of the active site of cPLA2R via its activated electrophilic ketone
moiety. With the 3-aryloxy-2-oxopropyl-group in position 1 of
the indole, our cPLA2R inhibitors also possess such a serine
trap. This can be concluded by the fact that the keto group of
Results and Discussion
Our investigations started from the 1-substituted indole-2-
carboxylic acid 13. This compound possessed inhibitory proper-
ties against isolated and cellular cPLA2R similar to those of
structurally related 3-acylindole-2-carboxylic acid 4 (Figure 2).
Although 13 inhibited the cellular arachidonic acid release after
A23187 stimulation with an IC50 value somewhat below 10 µM,
it was inactive against the isolated enzyme at 10 µM (Table 1).
Shifting the carboxylic acid group from position 2 of the indole
ring to the 3- or 4-positions (compounds 14 and 17), slightly
reduced the activity in the cellular assay. However, in contrast
to 13, both substances showed some inhibition of isolated
cPLA2R at 10 µM. A dramatic increase of inhibitory potency
was registered when the carboxy moiety was moved to the
5-position of the indole. With an IC50 value of 0.035 µM against
the isolated enzyme and an IC50 value of 0.41 µM against
A23187-induced cellular arachidonic acid liberation, indole-5-
carboxylic acid 18 was nearly as potent as AstraZeneca
compound 3, which had been tested as a reference (Table 1).
Switching the carboxy group from the 5- to the 6-position led
to a significant decrease in activity. The IC50 value of obtained
compound 19 in the two assays was 1.1 and 4.7 µM, respec-
tively.
The importance of the 5-carboxy moiety for the eminent
activity of 18 was seen when this group was omitted or replaced
by several other substituents (Table 2). Unsubstituted indole
derivative 20 and the compounds with a chloro, methyl, or
methoxy groups in position 5 (21-23) were inactive against

Design and Synthesis of 1-Indol-1-yl-propan-2-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2615
Table 3. Inhibition of cPLA2RActivity
Table 1. Inhibition of cPLA2R Activity
cellular assay
with human
platelets;
cellular assay with
human platelets
vesicle assay
with the
isolated
vesicle assay
position of
the carboxylic
acid moiety at
the indole ring
with the isolated
enzyme
stimulant
A23187
stimulant
TPA
stimulant
enzyme
A23187
IC50 (µM)a
IC50 (µM)a
compd
R
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
compd
18
39
40
41
42
43
44
50
2
H
0.035
0.34
0.11
0.015
0.016
0.016
0.0049
0.89
0.41
3.3
1.5
0.37
0.38
1.0
1.4
6.5
0.019
0.25
0.018
0.23
0.12
0.011
0.0097
0.0064
0.0011
0.56
13
14
17
18
19
3
2
3
4
5
6
n.a.b
>10c
>10c
0.035
1.1
8.0
>10c
>10c
0.41
4.7
C(CH3)3
Cl
CN
CHO
COCH3
COOCH3
COOH
0.011
0.25
a Values are the means of at least two independent determinations; errors
are within (20%. b Not active at 10 µM. c About 40% inhibition of enzyme
activity at 10 µM.
0.031
0.011
0.0045
0.0047
3
a Values are the means of at least two independent determinations; errors
are within (20%.
Table 2. Inhibition of cPLA2R Activity
Table 4. Inhibition of cPLA2R Activity
cellular assay
vesicle assay
with the
with human
platelets; stimulant
A23187
cellular assay with
human platelets
isolated enzyme
vesicle assay
compd
R
IC50 (µM)a
IC50 (µM)a
with the isolated
enzyme
stimulant
A23187
stimulant
TPA
18
20
21
22
23
24
25
26
28
COOH
H
CH3
Cl
OCH3
CN
COOCH3
CHO
CONH2
0.035
n.a.b
n.a.b
n.a.b
n.a.b
>10e
>10g
4.3
0.41
>10c
n.a.b
n.a.b
>10d
>10f
>10h
4.9
compd
R
IC50 (µM)a
IC50 (µM)a
IC50 (µM)a
54
57
H
0.020
0.0043
0.21
0.57
0.0078
0.0009
COOCH3
a Values are the means of at least two independent determinations; errors
are within ( 20%.
0.12
0.77
our compounds and of reference compound 3 lie in the same
order of magnitude as that of the data obtained in the enzymatic
assay with isolated cPLA2R (Table 3). Furthermore, the cor-
relation of the IC50 values between the cellular and isolated
enzyme assays is much better in this case. Interestingly, potent
pyrrolidine cPLA2R inhibitor 2, which we have also tested as a
reference, does not show such great discrepancies in the IC50
values in the cellular assays than our indoles and 3. In summary,
the high activity of our most active compounds in the platelet
assay applying TPA as the stimulant makes it unlikely that the
substances cannot penetrate into the platelets or that they are
metabolized to inactive compounds in the platelets. The reason
for the lower activity of the inhibitors in the cellular assay using
A23187 for activation of cPLA2R seems to lie in the slow-
binding kinetics of the propane-2-ones.
Because AstraZeneca has found that a 4-decyloxyphenyl-
moiety was the most preferable lipophilic residue in their
cPLA2R inhibitors, we replaced the 4-octylphenyl moiety in
compound 18 and 44 by such a residue. Obtained derivative 54
was about 2-fold more active in the cellular assays as well as
in the assay with the isolated enzyme than parent 18 (Table 4).
For 3-substituted derivative 57, a significant increase of activity
could only be observed in the assay with A23187 compared
with that of 44. However, with an IC50 value of 0.0043 µM in
a Values are the means of at least two independent determinations; errors
are within (20%. b Not active at 10 µM. c Indicates 43% inhibition of
enzyme activity at 10 µM. d Indicates 37% inhibition of enzyme activity at
10 µM. e Indicates 25% inhibition of enzyme activity at 10 µM. f Indicates
46% inhibition of enzyme activity at 10 µM. g Indicates 36% inhibition of
enzyme activity at 10 µM. h Indicates 40% inhibition of enzyme activity
at10 µM.
our active inhibitors easily forms hydrates as indicated by
reversed-phase HPLC/MS investigations performed with aque-
ous eluents.34 In the cellular assay applying A23187 as the
stimulant, an incubation time of only 1 min was applied because
of the fast and short rise of arachidonic acid liberation after the
addition of A23187. This time is probably not sufficient for
the quantitative formation of covalent bonds between serine trap
inhibitors and the enzyme. In the assay with the isolated enzyme,
the incubation time is much longer (60 min), and the inhibitors
can exert their full effect against the enzyme. This assumption
is supported by the results obtained with a cellular inhibitor
assay using the phorbol ester TPA as the stimulant. Here, cellular
liberation of arachidonic acid occurs during a longer period than
that after stimulation with A23187, allowing the enzyme reaction
to be performed for 60 min as in the assay with the isolated
enzyme. Under these conditions, the cellular inhibition data of

2616 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Ludwig et al.
organic phase was washed with water and dried (Na2SO4), and the
solvent was evaporated to give 8 as an oil (1.52 g, 99%).
[1-Bromo-3-(4-octylphenoxy)propan-2-yl]acetate (9). A cooled
(0 °C) mixture of dry pyridine (0.41 g, 5.18 mmol) and dry
CH2Cl2 (10 mL) was treated under a nitrogen atmosphere with
acetyl chloride (0.41 g, 5.22 mmol) and stirred for 30 min. Then,
a solution of 8 (0.50 g, 1.46 mmol) in dry CH2Cl2 (10 mL) was
added dropwise, and the reaction mixture was stirred an additional
3 h at 0 °C. After the addition of CH2Cl2, the organic phase was
washed twice with 5% aqueous NaHCO3 solution. The aqueous
phases were re-extracted with CH2Cl2, and the combined CH2Cl2
phases were washed with water. The organic layer was separated,
dried (Na2SO4), and concentrated. The residue was treated with
toluene, and the solvent was evaporated. This procedure was
repeated twice. Purification by silica gel chromatography (petroleum
ether/ethyl acetate, 19:1) gave 9 as an oil (0.56 g, 99%).
the isolated enzyme assay and 0.0009 µM in the cellular assay
with TPA as the stimulant, compound 57 was found to be one
of the most potent in vitro cPLA2R inhibitors known today.
While this work was in progress, AstraZeneca reported that
1-indol-1-yl-propanone 58 (Figure 3) is an inhibitor of
cPLA2R.35 Closely related compound 59, which we synthesized
independently, inhibited cPLA2R activity in our isolated enzyme
assay with an IC50 value of 1.3 µM.
tert-Butyl 1-[2-acetoxy-3-(4-octylphenoxy)propyl]indole-2-car-
boxylate (10). A solution of tert-butyl indole-2-carboxylate27 (0.33
g, 1.52 mmol) in dry DMSO (15 mL) was treated under a nitrogen
atmosphere with potassium tert-butylate (0.18 g, 1.60 mmol) and
stirred at 110 °C for 15 min. A solution of 9 (0.58 g, 1.51 mmol)
in dry DMSO (15 mL) was added dropwise, and the reaction
mixture was stirred at 110 °C for an additional 30 min. The mixture
was cooled, diluted with brine, and extracted four times with diethyl
ether. The combined organic phases were washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by flash
chromatography on silica gel (petroleum ether/ethyl acetate, 99:1)
to give 10 as an oil (0.25 g, 32%).
tert-Butyl 1-[2-hydroxy-3-(4-octylphenoxy)propyl]indole-2-
carboxylate (11). A solution of 10 (0.21 g, 0.40 mmol) in dry
methanol (10 mL) was treated under a nitrogen atmosphere with
0.5 M sodium methoxide in methanol (1.60 mL). The mixture was
stirred for 15 min at room temperature, concentrated approximately
to one-half of its original volume, and diluted with diethyl ether.
The organic solution was washed with half-saturated brine and dried
(Na2SO4), and the solvent was evaporated. The residue was
separated by silica gel chromatography (petroleum ether/ethyl
acetate, 19:1) to give 11 as an oil (0.15 g, 78%).
tert-Butyl 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-2-car-
boxylate (12). Acetic anhydride (1.02 g, 9.99 mmol) was added to
dry DMSO (10 mL), and the mixture was stirred under a nitrogen
atmosphere at room temperature for 10 min. Then, this solution
was added dropwise to a solution of 11 (120 mg, 0.25 mmol) in
dry DMSO (10 mL). The mixture was stirred under a nitrogen
atmosphere for 8 h and poured into a mixture of 5% aqueous
NaHCO3 and brine (1:1). After 10 min, the mixture was extracted
four times with diethyl ether. The combined organic phases were
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by silica gel chromatography (petroleum ether/ethyl
acetate, 49:1) to give 12 as an oil (113 mg, 96%).
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-2-carboxylic Acid
(13). To the solution of 12 (46 mg, 0.096 mmol) in dry CH2Cl2
(10 mL) was added trifluoroacetic acid (1.49 g, 13 mmol), and the
mixture was stirred at room temperature for 1.5 h. Then, the reaction
mixture was concentrated to dryness. The residue was treated twice
with toluene, and the solvent was evaporated each time. The residue
was recrystallized from petroleum ether/ethyl acetate (2:1) to give
13 as a white solid (31 mg, 77%); mp 173 °C.
Figure 3.
In summary, we have described the synthesis of novel, highly
potent cPLA2R inhibitors with indole-scaffolds. In future articles,
we will report studies on the synthesis of less lipophilic cPLA2R
inhibitors of this series.
Experimental Section
Column chromatography was performed on Merck silica gel 60,
230-400 mesh () flash chromatography) or 70-230 mesh.
Preparative HPLC was performed on a RP18 column (Kromasil
100, 5 µm, 10 mm (i.d.) × 250 mm, protected with an analogously
filled guard column of 10 mm (i.d.) × 50 mm; CS-chromatographie
service, Langerwehe, Germany). Melting points were determined
on a Bu¨chi B-540 apparatus and are uncorrected. 1H NMR spectra
were recorded on a Varian Mercury Plus 400 spectrometer (400
MHz). Mass spectra were obtained on Finnigan GCQ and LCQ
apparatuses applying electron beam ionization (EI) and electrospray
ionization (ESI), respectively. The purity of the target compounds
was determined using two diverse HPLC systems with UV detection
at 254 nm. The first one applied an amino phase (Spherisorb NH2,
5 µm, 4.0 mm (i.d.) × 250 mm; Latek, Heidelberg, Germany),
eluting the compounds with an isohexane/THF gradient at a flow
rate of 0.75 mL/min. In the second system, separation was
performed using a cyano phase (LiChrospher 100 CN, 5 µm, 3.0
mm (i.d.) × 250 mm; Merck, Darmstadt, Germany) with a
isohexane/THF gradient containing 0.1% trifluoroacetic acid at a
flow rate of 0.5 mL/min. With the exception of 41, all target com-
pounds showed purities greater than 97% in both HPLC systems.
The purity evaluated for 41 was 95% (cyano phase) and 99% (amino
phase). Reference inhibitor 2 (N-{(2S,4R)-4-[biphenyl-2-ylmethyl-
(isobutyl)amino]-1-[2-(2,4-difluorobenzoyl)benzoyl]pyrrolidin-2-
ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)phenyl]-
acrylamide)21 was a gift from Merckle (Blaubeuren, Germany).
Reference inhibitor 3 (4-[3-(4-decyloxyphenoxy)-2-oxopropoxy]-
benzoic acid) was synthesized according to published procedures.22
2-(4-Octylphenoxy)methyloxirane26 (7). To a mixture of pow-
dered KOH (88%, 3.10 g, 48.4 mmol), 4-octylphenol (5.00 g, 24.2
mmol) (6), and tetrabutylammonium bromide (0.81 g, 2.44 mmol)
was added epichlorohydrin (13.5 g, 146 mmol). The reaction
mixture was stirred at room temperature for 5 h, treated with water,
and extracted three times with diethyl ether. The combined organic
layers were washed with water and dried (Na2SO4), and the solvent
was evaporated. The residue was purified by silica gel chromatog-
raphy (petroleum ether/ethyl acetate, 95:5) to give 7 as a solid (6.34
g, 99%); mp 25 °C.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-3-carboxylic Acid
(14). Compound 14 was prepared from tert-butyl indole-3-car-
boxylate utilizing the reaction sequence described for the synthesis
of 13; mp 182-183 °C.
tert-Butyl 1-[2-hydroxy-3-(4-octylphenoxy)propyl]indole-4-
carboxylate (15). Under a nitrogen atmosphere, a suspension of
NaH (60% in mineral oil; 48 mg, 1.20 mmol) in dry DMF (10
mL) was stirred at room temperature for 10 min. After the addition
of a solution of tert-butyl indole-4-carboxylate27 (0.25 g, 1.15 mmol)
in dry DMF (10 mL), the mixture was stirred for 1 h. A solution
of 2-(4-octylphenoxymethyl)oxirane (7) (0.30 g, 1.14 mmol) in dry
DMF (10 mL) was added dropwise at room temperature, and the
reaction mixture was heated at 60 °C for 3 h. After it was cooled
1-Bromo-3-(4-octylphenoxy)propan-2-ol (8). To a solution of
7 (1.17 g, 4.46 mmol) in dry CH2Cl2 (10 mL) was added silica gel
(1.34 g) and lithium bromide (1.16 g, 13.4 mmol). The mixture
was evaporated to near dryness and allowed to stand at room
temperature for 3 h. After the addition of CH2Cl2, the reaction
mixture was filtered through a cotton pad. The filtrate was
evaporated, and the residue was treated with diethyl ether. The

Design and Synthesis of 1-Indol-1-yl-propan-2-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2617
to room temperature, the mixture was treated with half-saturated
brine and extracted with diethyl ether four times. The combined
organic phases were washed three times with half-saturated brine
and dried (Na2SO4), and the solvent was evaporated. The residue
was purified by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 9:1) followed by chromatography on an octadecyl
reversed-phase material (acetonitrile/H2O, 4:1) to give 15 as an oil
(0.36 g, 66%).
tert-Butyl 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-4-car-
boxylate (16). Acetic anhydride (1.55 g, 15.2 mmol) was added to
dry DMSO (10 mL), and the mixture was stirred under a nitrogen
atmosphere at room temperature for 10 min. Then, this solution
was added dropwise to a solution of 15 (182 mg, 0.38 mmol) in
dry DMSO (10 mL). The mixture was stirred for 15 h under a
nitrogen atmosphere and poured into a mixture of 5% aqueous
NaHCO3 and half-saturated brine (1:1). After 10 min, the mixture
was extracted four times with diethyl ether. The combined organic
phases were washed three times with brine, dried (Na2SO4), and
concentrated. The residue was purified by flash chromatography
on silica gel (petroleum ether/ethyl acetate, 93:7) to give 16 as a
solid (131 mg, 71%); mp 100 °C.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-4-carboxylic Acid
(17). To the solution of 16 (63 mg, 0.132 mmol) in dry CH2Cl2
(15 mL) was added trifluoroacetic acid (1.1 g, 9.8 mmol), and the
mixture was stirred at room temperature for 2 h. Then, the reaction
mixture was concentrated to dryness. The residue was treated three
times with petroleum ether/ethyl acetate (1:2), and the solvents were
evaporated each time. The residue was recrystallized from petroleum
ether/ethyl acetate (2:1) to give 17 as a solid (48 mg, 86%); mp
160-161 °C.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-5-carbaldehyde (26).
Purification by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 9:1) gave 26 as a solid; mp 96 °C.
1-[2-Hydroxy-3-(4-octylphenoxy)propyl]indole-5-carboxam-
ide (27). 0.18 g (0.44 mmol) of 1-[2-hydroxy-3-(4-octylphenoxy)-
propyl]indole-5-carbonitrile, previously synthesized from indole-
5-carbonitrile and 2-(4-octylphenoxy)methyloxirane (7) using a
method similar to that for 15, was dissolved in tert-butyl alcohol
(15 mL) and treated with 0.23 g (3.6 mmol) powdered KOH (88%).
The mixture was heated under reflux for 11 h. The reaction mixture
was cooled, diluted with water, acidified with 1 M HCl, and
extracted three times with diethyl ether. The combined organic
phases were washed with brine and water, dried (Na2SO4), and
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/ethyl acetate, 1:1) to give 27 as a solid (0.13 g,
70%); mp 118-119 °C.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-5-carboxamide (28).
Compound 27 was oxidized to 28 using the procedure described
for the preparation of 16. The product was recrystallized from
petroleum ether/ethyl acetate (1:1) and further purified by RP-HPLC
applying acetonitrile/H2O (4:1) as the mobile phase. The eluates
were concentrated under reduced pressure until most of the
acetonitrile was distilled off. The remaining solvent was removed
by freeze-drying, yielding 28 as white solid; mp 158-159 °C.
tert-Butyl 3-chloroindole-5-carboxylate (30). A solution of 389
mg (1.79 mmol) of tert-butyl indole-5-carboxylate27 (29) in meth-
anol (12 mL) was treated with 335 mg (2.50 mmol) N-chlorosuc-
cinimide and stirred overnight at room temperature. The solvent
was distilled off and the residue dissolved in 15 mL of ethyl acetate.
The organic solution was washed twice with 1 M aqueous NaHCO3,
dried (Na2SO4), and evaporated. The residue was purified by silica
gel chromatography (hexane/ethyl acetate, 9:1) to give 30 as a solid
(190 mg, 42%); mp 120 °C.
Compounds 18 and 19 were prepared in a manner similar to
that described for the synthesis of 17, utilizing the appropriate tert-
butyl indolecarboxylates and purifying by the method indicated.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-5-carboxylic Acid
(18). Compound 18 was purified by RP-HPLC, applying acetoni-
trile/H2O (4:1) as the mobile phase. The eluates were concentrated
under reduced pressure until most of the acetonitrile was removed.
Freeze-drying the remaining solution gave 18 as a solid; mp 122-
124 °C.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-6-carboxylic Acid
(19). The purification of 19 was performed by RP-HPLC as
described for 18, except that acetonitrile/H2O (9:1) was used as
the eluent; mp 180 °C.
Compounds 20-26 were prepared in a manner similar to that
described for the synthesis of 16 utilizing the appropriate indole
and purified by the method indicated. In case of the synthesis of
23, the coupling of 5-methoxyindole with 7 was performed at room
temperature with protection from light.
1-Indol-1-yl-3-(4-octylphenoxy)propan-2-one (20). Purification
by silica gel chromatography (petroleum ether/ethyl acetate, 19:1)
and recrystallization from petroleum ether gave 20 as a solid; mp
65 °C.
tert-Butyl 3-formylindole-5-carboxylate (31). A solution of
oxalyl chloride (0.4 mL, 4.87 mmol) in dry CH2Cl2 (15 mL) was
treated at 0 °C with a solution of dry DMF (0.4 mL) in dry
CH2Cl2 (15 mL). The mixture was stirred at 0 °C for 20 min. After
the addition of tert-butyl indole-5-carboxylate27 (29) (1.0 g, 4.6
mmol), the mixture was allowed to warm to room temperature and
then stirred for a further 20 min. The solvent was evaporated and
the residue treated with THF (40 mL) and 20% aqueous ammonium
acetate (50 mL). The mixture was heated under reflux for 30 min,
cooled, treated with 5% aqueous NaHCO3, and extracted three times
with ethyl acetate. The combined organic phases were washed with
brine and dried (Na2SO4), and the solvent was evaporated. The
residue was recrystallized from hexane/ethyl acetate to yield 31 as
a solid (0.81 g, 71%); mp 190 °C.
tert-Butyl 3-(hydroxyiminomethyl)indole-5-carboxylate (32).
Hydroxylammonium chloride (0.28 g, 4.08 mmol) was added to a
solution of 31 in pyridine (10 mL) in several portions. The mixture
was heated under reflux for 5 h. Then, the solvent was distilled
off. The residue was dissolved in ethyl acetate and washed with 1
M HCl. The organic phase was separated, and the aqueous layer
was extracted twice with ethyl acetate. The combined organic layers
were washed with water, dried (Na2SO4), and concentrated. The
residue was treated several times with hexane/ethyl acetate (1:1),
and the solvents were evaporated each time to give a mixture of
(E)- and (Z)-tert-butyl 3-(hydroxyiminomethyl)indole-5-carboxylate
as a solid (0.50 g, 94%); mp 188-189 °C (decomp.).
1-(5-Methylindol-1-yl)-3-(4-octylphenoxy)propan-2-one (21).
Purification by silica gel chromatography (petroleum ether/ethyl
acetate, 97:3) gave 21 as a solid; mp 75 °C.
1-(5-Chloroindol-1-yl)-3-(4-octylphenoxy)propan-2-one (22).
Purification by silica gel chromatography (petroleum ether/ethyl
acetate, 19:1) and recrystallization from petroleum ether gave 22
as a solid; mp 77 °C.
tert-Butyl 3-cyanoindole-5-carboxylate (33). 2-Chloro-1-meth-
ylpyridinium iodide (0.29 g, 1.14 mmol) was added under nitrogen
to a solution of 32 (0.27 g, 1.04 mmol) in dry THF (30 mL). The
mixture was stirred at room temperature for 10 min, then treated
dropwise with triethylamine (0.55 mL, 3.94 mmol), and stirred for
a further 20 h at room temperature. Dilute HCl (5%, 20 mL) was
added, and the mixture was extracted three times with ethyl ace-
tate. The combined organic phases were washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by flash
chromatography on silica gel (hexane/ethyl acetate, 3:2) to give
33 as a solid (0.21 g, 87%); mp 188-189 °C.
1-(5-Methoxyindol-1-yl)-3-(4-octylphenoxy)propan-2-one (23).
Purification by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 19:1) gave 23 as a solid; mp 85 °C.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-5-carbonitrile (24).
Purification by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 9:1) and recrystallization from petroleum ether/ethyl
acetate (19:1) gave 24 as a solid; mp 96 °C.
Methyl 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxy-
late (25). Recrystallization from petroleum ether/ethyl acetate (47:
3) gave 25 as a solid; mp 118 °C.

2618 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8
Ludwig et al.
5-tert-Butyl-3-methyl indole-3,5-dicarboxylate (34). A solution
of 31 (0.70 g, 2.83 mmol) in methanol (30 mL) was treated with
NaCN (0.70 g, 14.3 mmol). Then, activated MnO2 (4.92 g, 57.2
mmol) was added in several portions, and the reaction mixture was
stirred at room temperature for 48 h. After the addition of CH2Cl2
(80 mL) and Celite (5 g), the suspension was filtered off by suction
and the residue washed with CH2Cl2. The combined filtrates were
subsequently washed with saturated aqueous FeSO4 solution and
brine, dried (Na2SO4), and concentrated to give 34 as a solid (0.74
g, 94%); mp 196 °C.
Dimethyl indole-3,5-dicarboxylate (46). The treatment of
methyl 3-formylindole-5-carboxylate (45) (2.30 g, 11.3 mmol)
according to the procedure described above for the preparation of
34 provided 46 as a solid (0.75 g, 29%); mp 223-225 °C (decomp.).
Dibenzyl indole-3,5-dicarboxylate (47). A mixture of KH (30%
dispersion in mineral oil, 0.10 g, 0.75 mmol) and dry benzyl alcohol
(10 mL) was stirred at room temperature for 15 min. After the
addition of 46 (0.70 g, 3.0 mmol), the mixture was heated at 100
°C for 5 h. Then, the reaction mixture was cooled, diluted with
brine, and extracted three times with diethyl ether. The com-
bined organic layers were dried (Na2SO4) and concentrated. The
residue was treated twice with methanol, and the solvent was
evaporated each time to give 47 as a solid (0.58 g, 50%); mp
173-174 °C.
Dibenzyl 1-[2-hydroxy-3-(4-octylphenoxy)propyl]indol-3,5-
dicarboxylate (48). Starting from 7 (0.30 g,1.17 mmol) and 47
(0.45 g, 1.17 mmol), 48 was prepared in a manner similar to that
described for the synthesis of 15. The reaction mixture was heated
at 100 °C for 40 h. Purification by silica gel chromatography
(hexane/ethyl acetate, 4:1) gave 48 as a solid (0.20 g 27%); mp
102-103 °C.
Dibenzyl 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-3,5-dicar-
boxylate (49). Compound 49 was prepared from 48 (160 mg, 0.25
mmol) in a manner similar to that described for the synthesis of
16. Purification by flash chromatography on silica gel (hexane/
ethyl acetate, 4:1) gave 49 (63 mg, 39%) as an oil.
1-[3-(4-Octylphenoxy)-2-oxopropyl]indole-3,5-dicarboxylic Acid
(50). A solution of 49 (54.0 mg, 0.08 mmol) in THF (10 mL) was
stirred at room temperature with Pd/C (10%; 15 mg) under H2
supplied by a balloon for 5 h. The mixture was filtered through a
cotton pad and concentrated to give 50 as a solid (21 mg, 56%);
mp 225-225.5 °C (decomp.).
1-[3-(4-Decyloxyphenoxy)-2-oxopropyl]indole-5-carboxylic Acid
(54). Compound 54 was prepared in a manner similar to that
described for the synthesis of 17, starting from tert-butyl indole-
5-carboxylate27 and 2-(4-decyloxyphenoxymethyl)oxirane (51).22 It
was purified by RP-HPLC as described for the purification of 18
using acetonitrile/H2O (9:1) as the eluent; mp 137 °C.
5-tert-Butyl-3-methyl 1-[3-(4-decyloxyphenoxy)-2-hydroxy-
propyl]indole-3,5-dicarboxylate (55). A solution of 5-tert-butyl-
3-methyl indole-3,5-dicarboxylate (34) (0.50 g, 1.82 mmol) in dry
DMF (20 mL) was reacted with NaH (60% in mineral oil; 73 mg,
3.03 mmol) in dry DMF (20 mL) and a solution of 2-(4-
decyloxyphenoxymethyl)oxirane (51)22 (0.56 g, 1.82 mmol) in dry
DMF (20 mL) in a manner similar to that described for the synthesis
of 15, except that the reaction mixture was heated at 100 °C for 26
h. Purification was performed by flash chromatography on silica
gel (petroleum ether/ethyl acetate, 7:3) followed by chromatography
on an octadecyl reversed-phase material (acetonitrile/H2O, 4:1) to
give 55 as an oil (0.26 g, 25%).
5-tert-Butyl-3-methyl 1-[3-(4-decyloxyphenoxy)-2-oxopropyl]-
indole-3,5-dicarboxylate (56). A solution of 55 (240 mg, 0.41
mmol) in dry DMSO (10 mL) was treated with a mixture of acetic
anhydride (1.69 g, 16.5 mmol) and dry DMSO (10 mL) in a manner
similar to that described for the synthesis of 16, except that the
reaction time was 17 h. Purification was achieved by flash
chromatography on silica gel (hexane/ethyl acetate, 4:1) to give
56 as an oil (135 mg, 56%).
Methyl 3-acetylindole-5-carboxylate (36). To a mixture of
ZnCl2 solution in dry diethyl ether (2.2 M, 4.2 mL) and dry
CH2Cl2 (20 mL) was slowly added under nitrogen at 0 °C a solution
of butyllithium in dry hexane (1.6 M, 5.6 mL). The mixture was
allowed to warm to room temperature, stirred for 1 h at room
temperature, treated with a solution of methyl indole-5-carboxylate
(35) (1.5 g, 8.6 mmol) in dry CH2Cl2 (20 mL), and stirred again
for 1 h. The reaction mixture was cooled to 0 °C, acetyl chloride
(1.28 mL, 18 mmol) was added, and stirring was continued for 1
h at room temperature. Then, the mixture was treated with AlCl3
(0.93 g, 7.0 mmol) and further stirred for 1 h. After the addition of
half-saturated brine and THF (20 mL), the mixture was extracted
four times with ethyl acetate. The combined organic layers were
washed with brine, dried (Na2SO4), and concentrated. The residue
was purified by chromatography on silica gel (hexane/ethyl acetate,
3:2) to give 36 as a solid (0.70 g, 38%); mp 235-236 °C.
3-Acetylindole-5-carboxylic Acid (37). Methyl 3-acetylindole-
5-carboxylate (36) (0.69 g, 3.16 mmol) was dissolved in THF (10
mL) and ethylene glycol (10 mL) by heating. After the addition of
KOH (88%, 7.08 g, 0.13 mol), the mixture was heated under reflux
for 30 min. The THF was distilled off under reduced pressure, and
the remaining solution was acidified at 0 °C with dilute HCl. The
precipitate formed was filtered off by suction, washed with water,
and dried to provide 37 as a solid (0.64 g, 99%); mp 364 °C.
tert-Butyl 3-acetylindole-5-carboxylate (38). A solution of N,N-
dimethylformamide di-tert-butyl acetal (90% purity, 6.3 mL, 26.3
mmol) in dry benzene (50 mL) was added dropwise under nitrogen
to the refluxing suspension of 37 (1.34 g, 6.60 mmol) in dry ben-
zene (100 mL) within 30 min. The solution was refluxed for a
further 30 min, cooled, diluted with diethyl ether, and washed with
NaHCO3 solution (5%) and brine. The organic layer was dried
(Na2SO4) and the solvent evaporated. The residue was purified by
silica gel chromatography (hexane/ethyl acetate, 3:2) to give 38
(0.39 g, 23%) as a solid; mp 214 °C.
3-tert-Butyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-car-
boxylic Acid (39). Compound 39 was a side product of the synthesis
of 18. It was separated during the course of the RP-HPLC
purification of 18; mp 146-147 °C.
Compounds 40-44 were prepared in a manner similar to that
described for the synthesis of 17, starting from the appropriate
3-substitued tert-butyl indole-5-carboxylates (30, 33, 31, 38, and
34). The coupling of these compounds with 7 was performed at 60
°C (30, 33) and 120 °C (31, 34, 38), applying a reaction time of
8-26 h. The target compounds were purified by the method
indicated.
3-Chloro-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carbox-
ylic Acid (40). Recrystallization from hexane/THF gave 40 as a
solid; mp 157 °C.
3-Cyano-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carbox-
ylic Acid (41). Recrystallization from hexane/ethyl acetate gave
41 as a solid; mp 197-198 °C (decomp.).
3-Formyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carbox-
ylic Acid (42). The purification of 42 was performed by RP-HPLC
as described for 18 except that acetonitrile/H2O (9:1) was used as
the eluent; mp 193 °C.
3-Acetyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carbox-
ylic Acid (43). Recrystallization from hexane/THF gave 43 as a
solid; mp 197 °C.
3-Methylhydrogen 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-
3,5-dicarboxylate (44). Recrystallization from hexane/THF gave
44 as a solid; mp 208 °C.
3-Methylhydrogen 1-[3-(4-decyloxyphenoxy)-2-oxopropyl]in-
dole-3,5-dicarboxylate (57). A solution of 56 (107 mg, 0.18 mmol)
in dry CH2Cl2 (20 mL) was treated with trifluoroacetic acid (1.68
g, 14.7 mmol) in a manner similar to that described for the synthesis
of 17, except that the reaction time was 3 h. The reaction mixture
was concentrated to dryness. The residue was treated twice with
hexane, and the solvent was evaporated each time. The residue was
recrystallized from hexane/ethyl acetate to give 57 as a solid (79
mg, 82%).
{1-[3-(4-Octylphenoxy)-2-oxopropyl]indol-3-yl}acetic Acid
(59). Indol-3-ylacetic acid was converted to its tert-butylester with
N,N-dimethylformamide di-tert-butyl acetal applying the method
described for the synthesis of 38. tert-Butyl indol-3-yl acetate was

Design and Synthesis of 1-Indol-1-yl-propan-2-ones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 8 2619
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reacted with 2-(4-octylphenoxymethyl)oxirane (7) at room tem-
perature for 14 h using a procedure analogous to the procedure
described for the synthesis of 15. The intermediate obtained was
converted to 59 using the procedures for the synthesis of 16 and
17. Compound 59 was purified by RP-HPLC applying acetonitrile/
H2O/formic acid (85:15:0.05) as the mobile phase. The eluates were
concentrated under reduced pressure until most of the acetonitrile
was removed. Freeze-drying the remaining solution gave 59 as a
solid; mp 121-122 °C.
Assay of cPLA2R Inhibition Using the Isolated Enzyme. The
inhibition of cPLA2R isolated from human platelets was performed
as previously described.29 Briefly, sonicated co-vesicles consisting
of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (0.2 mM)
and 1,2-dioleoyl-sn-glycerol (0.1 mM) were used as enzyme
substrates. The cPLA2R activity was determined by measuring the
arachidonic acid released by the enzyme with reversed-phase HPLC
and UV detection at 200 nm after cleaning up the samples using
solid-phase extraction.
Ionophore- and Phorbolester-Induced Arachidonic Acid
Release from Human Platelets. The ability of the compounds to
inhibit cPLA2R activity in intact cells was determined by monitoring
calcium ionophore A23187-induced and phorbolester-induced
arachidonic acid release from human platelets by HPLC with UV
detection.
(16) Hegen, M.; Sun, L.; Uozumi, N.; Kume, K.; Goad, M. E.; Nickerson-
Nutter, C. L.; Shimizu, T.; Clark, J. D. Cytosolic phospholipase A2R-
deficient mice are resistant to collagen-induced arthritis. J. Exp. Med.
2003, 197, 1297-1302.
(17) Miyaura, C.; Inada, M.; Matsumoto, C.; Ohshiba, T.; Uozumi, N.;
Shimizu, T.; Ito, A. An essential role of cytosolic phospholipase A2R
in prostaglandin E2-mediated bone resorption associated with inflam-
mation. J. Exp. Med. 2003, 197, 1303-1310.
(18) Bonventre, J. V. Cytosolic phospholipase A2R reigns supreme in
arthritis and bone resorption. Trends Immunol. 2004, 25, 116-
119.
(19) McKew, J. C.; Tam, S. Y.-K.; Clark, J. D.; Lee, K. L.; Chen, L.;
Thakker, P.; Sum, F. W.; Behnke, M. L.; Hu, B. Preparation of indolyl
containing sulfonamides as inhibitors of cytosolic phospholipase A2.
PCT Int. Appl. WO2003048122, 2003.
The inhibition of the A23187-induced arachidonic acid release
was measured according to a procedure previously described,
applying a final A23187 concentration of 1 µM and an incubation
time of 1 min.24 Deviating, the HPLC separation of arachidonic
acid was achieved on a RP18 multospher 100 column, 3 µm, 3.0
mm (i.d.) × 125 mm, with a RP18 multospher 100 guard column,
5 µm, 3.0 mm (i.d.) × 20 mm (CS-chromatographie service,
Langerwehe, Germany). The mobile phase consisted of an aceto-
nitrile/(NH4)2HPO4 buffer (10 mM) adjusted to pH 7.4 with ortho-
phosphoric acid (50:50, v/v). The flow rate was 0.33 mL/min, and
the injected sample volume was 300 µL. The detection wavelength
was 200 nm, applying a Waters 2487 UV-detector. After each run,
the column was washed with 0.6 mL of methanol. 3-(4-Decyl-
oxyphenyl)propanoic acid was applied as internal standard.
The inhibition of the phorbolester (TPA)-induced arachidonic
acid release was measured similarly, applying a final TPA
concentration of 2 µM and an incubation time of 60 min. The
sample volume injected to HPLC was 600 µL. Control incubations
in the absence of the stimulant TPA were carried out in parallel
and used to calculate specific hydrolysis. In intact human platelets,
iPLA2 is not involved in TPA-induced arachidonic acid release
because the iPLA2 inhibitor bromoenol lactone (BEL) did not reduce
arachidonic acid liberation at a test concentration of 10 µM.
(20) Khurdayan, V.; Cullell-Young, M. Cronicles in drug discovery. Drug
News & Perspect. 2005, 18, 227-280.
(21) Seno, K.; Okuno, T.; Nishi, K.; Murakami, Y.; Watanabe, F.;
Matsuura, T.; Wada, M.; Fujii, Y.; Yamada, M.; Ogawa, T.; Okada,
T.; Hashizume, H.; Kii, M.; Hara, S.; Hagishita, S.; Nakamoto, S.;
Yamada, K.; Chikazawa, Y.; Ueno, M.; Teshirogi, I.; Ono, T.; Ohtani,
M. Pyrrolidine inhibitors of human cytosolic phospholipase A2. J.
Med. Chem. 2000, 43, 1041.
(22) Connolly, S.; Bennion, C.; Botterell, S.; Croshaw, P. J.; Hallam, C.;
Hardy, K.; Hartopp, P.; Jackson, C. G.; King, S. J.; Lawrence, L.;
Mete, A.; Murray, D.; Robinson, D. H.; Smith, G. M.; Stein, L.;
Walters, I.; Wells, E.; Withnall, W. J. Design and synthesis of a novel
and potent series of inhibitors of cytosolic phospholipase A2 based
on a 1,3-disubstituted propan-2-one skeleton. J. Med. Chem. 2002,
45, 1348-1362.
(23) Lehr, M. Synthesis, biological evaluation, and structure-activity
relationships of 3-acylindole-2-carboxylic acids as inhibitors of
the cytosolic phospholipase A2. J. Med. Chem. 1997, 40, 2694-
2705.
Supporting Information Available: 1H NMR and MS data of
all compounds, purity of all target compounds evaluated with two
diverse HPLC systems, and HRMS data of target compounds
42-44, 54, and 57. This material is available free of charge via
the Internet at http://pubs.acs.org.
(24) Lehr, M.; Schulze Elfringhoff, A. Comparison of the inhibition of
the cytosolic phospholipase A2-mediated arachidonic acid release by
several indole-2-carboxylic acids and 3-(pyrrol-2-yl)propionic acids
in bovine and in human platelets. Arch. Pharm. (Weinheim, Ger.)
2000, 333, 312-314.
(25) Ghasemi, A.; Schulze Elfringhoff, A.; Lehr, M. Structure-activity
relationship studies of 3-dodecanoylindole-2-carboxylic acid inhibitors
of cytosolic phospholipase A2R-mediated arachidonic acid release
in intact platelets: variation of the keto moiety. J. Enzyme Inhib.
Med. Chem. 2005, 20, 429-437.
(26) Wiesner, I.; Kolinsky, J.; Bruthans, V.; Sieber, J. Glycidyl ethers of
monovalent phenols. CS 107473, 1963 CA: 60: 30832.
(27) Ludwig, J.; Lehr, M. Convenient synthesis of pyrrole- and indole-
carboxylic acid tert-butylesters. Synth. Commun. 2004, 34, 3691-
3695.
(28) Gwaltney, S. L.; Jae, H.-S.; Kalvin, D. M.; Liu, G.; Sham, H. L.; Li,
Q.; Claiborne, A. K.; Wang, L.; Barr, K. J.; Woods, K. W. Substituted
oxazolines as antiproliferative agents. PCT Int. Appl. WO0006556,
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