1-(5-carboxyindol-1-yl)propan-2-one inhibitors of human cytosolic phospholipase A2α: Effect of substituents in position 3 of the indole scaffold on inhibitory potency, metabolic stability, solubility, and bioavailability

Article abstract of DOI:10.1021/jm101094p

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be potent inhibitors of human cytosolic phospholipase A ₂α (cPLA₂α). In the course of structure-activity relationship studies, we investig

Full text of DOI:10.1021/jm101094p

8298 J. Med. Chem. 2010, 53, 8298–8308
DOI: 10.1021/jm101094p
1-(5-Carboxyindol-1-yl)propan-2-one Inhibitors of Human Cytosolic Phospholipase A2r: Effect of
Substituents in Position 3 of the Indole Scaffold on Inhibitory Potency, Metabolic Stability,
Solubility, and Bioavailability
†
Stefanie Bovens, Alwine Schulze Elfringhoff, Martina Kaptur, Dirk Reinhardt, Michael Schafers, and Matthias Lehr*
†
†
‡
‡
,†
€
†Institute of Pharmaceutical and Medicinal Chemistry, University of Mu€nster, Hittorfstrasse 58-62, 48149 Mu€nster, Germany, and
‡European Institute for Molecular Imaging (EIMI), University of Mu€nster, Mendelstrasse 11, 48149 Mu€nster, Germany
Received August 24, 2010
Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 have been found to be
potent inhibitors of human cytosolic phospholipase A2R (cPLA2R). In the course of structure-activity
relationship studies, we investigated the effect of a substitution of indole 3 position with acyl, alkyl, and
oxadiazole residues. The highest increase of inhibitory potency could be achieved by a 3-methyl-1,2,
4-oxadiazol-5-yl-moiety. Appropriate compound 40 revealed an IC50 of 0.0021 μM against isolated
cPLA2R. In a cellular assay applying human platelets 40 blocked cPLA2R activity even with an IC50 of
0.0006 μM. Metabolic stability and aqueous solubility of the target compounds were also determined.
Furthermore, one selected compound was tested for peroral bioavailability in mice.
Introduction
and benzhydrylquinazolinediones21 as well as 1,3-diaryloxy-
propan-2-ones like 1 (AR-C70484XX) (Figure 1).22,23
Cytosolic phospholipase A2R (cPLA2Ra) specifically cata-
lyzes the hydrolysis of the sn-2 ester of arachidonate-containing
membrane phospholipids.1,2 The released arachidonic acid is
rapidly oxidized via cyclooxygenase (COX) and lipoxygenase
(LO) pathways to eicosanoids such as prostaglandins and
leukotrienes. Remaining lysophospholipids with ether re-
sidues in position 1 and choline phosphate head groups in
position 3 of the glycerol backbone can be acetylated to platelet
activating factor (PAF). Prostaglandins, leukotrienes, and
PAF are known to be important mediators of inflammatory
processes. Although several other phospholipases A2 are
present in the mammalian organism, the pre-eminence
of cPLA2R for lipid mediator generation was demonstrated
especially by studies with cPLA2R deficient mice. These
animals, which display a reduced eicosanoid production, are
resistant to disease in a variety of models of inflammation,
including collagen-induced arthritis.3-8 Therefore, cPLA2R is
considered as a target for inflammatory diseases.9
We have published effective cPLA2R inhibitors like com-
pound 2, structurally related to 1.24-28 In these derivatives,
heteroaryl substituents are tethered to aryloxypropan-2-one
scaffolds. Structure-activity relationship studies have revealed
that introduction of a methoxycarbonyl or an acetyl moiety in
position 3 of the indole nucleus of compound 2 significantly
increases inhibitory potency. IC50 of 3 and 4 against cPLA2R
was about 3- and 5-fold lower than that of the lead compound 2.
In this study we investigated the effect of a substitution of the
acetyl group of 4 by other acyl and by alkyl residues. Further-
more, we replaced the metabolically less stable ester moiety of
3 by a bioisosteric oxadiazole. Besides inhibition of cPLA2R,
metabolic stability and aqueous solubility of the target
compounds were determined. Finally, one selected compound
was tested for peroral bioavailability in mice.
Chemistry
Although there have been intense efforts for developing
inhibitors of cPLA2R,10-13 only few substances with high
in vitro potency have been found until now, such as
certain thiazolidinediones,14,15 benzhydrylindoles,16-20
The preparationof 3-butanoyl-substituted indole10 started
from indole-5-carboxylic acid benzyl ester (5) (Scheme 1).
After acylation with butanoyl chloride, the obtained inter-
mediate 6 was reacted with epichlorohydrin to afford 7.
Regioselective opening of the epoxy ring of 7 with 4-octyl-
phenol was achieved without solvent in the presence of catalytic
amounts of 4-dimethylaminopyridine. Oxidation of the re-
sulting alcohol intermediate 8 to the ketone 9 was carried out
withacetic anhydride/DMSO. Hydrogenolytic cleavageof the
benzyl ester group of 9 yielded the desired target compound
10. The same reaction sequence was applied for the synthesis
of the 3-hexanoyl-, 3-hexyl-, and 3-(3-carboxypropanoyl)indole-
5-carboxylic acid derivatives 11, 21, and 22 (Table 1) using the
appropriate substituted benzyl indole-5-carboxylates.
*To whom correspondence should be addressed. Phone: þ49251 83
33331. Fax: þ49251 83 32144. E-mail: lehrm@uni-muenster.de.
a Abbreviations: cPLA2R, cytosolic phospholipase A2R; COX, cy-
clooxygenase; LO, lipoxygenase; PAF, platelet activating factor; po,
peroral; iv, intravenous; THF, tetrahydrofuran; DMSO, dimethylsulf-
oxide; DMF, dimethylformamide; SAPC, 1-stearoyl-2-arachidonoyl-
sn-glycero-3-phosphocholine; DOG, 1,2-dioleoyl-sn-glycerol; TPA, 12-
O-tetradecanoylphorbol 13-acetate; ETYA, 5,8,11,14-eicosatetraynoic
acid; NDGA, nordihydroguaiaretic acid, UV, ultraviolet; HPLC, high
performance liquid chromatography; MS, mass spectrometry; EI, elec-
tron beam ionization; ESI, electrospray ionization; log P, partition
coefficient; NADPH, dihydronicotinamide adenine dinucleotide phos-
phate; iPLA2, calcium-independent phospholipase A2; sPLA2, secretory
phospholipase A2.
Several of the test compounds were prepared starting from
the methyl ester of indole-5-carboxylic acid (24) as highlighted
r
pubs.acs.org/jmc
Published on Web 11/10/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8299
for the 5-carboxypentanoyl derivative 30 (Scheme 2). First,
position 3 of 24 was substituted with a 5-methoxycarbonyl-
pentanoyl group. Then the 2-oxopropyl substituent was intro-
duced in position 1 of indole 25, applying the reaction steps
described for the synthesis of 9. Next the ketone group of the
oxopropyl residue of intermediate 28 was acetalized with
orthoformic acid trimethyl ester in the presence of catalytic
amounts of H2SO4. The methyl ester groups of obtained
compound 29 were saponified with aqueous NaOH. Finally,
the acetal protecting group was removed by aqueous HCl in
THF to yield the indole-5-carboxylic acid derivative 30.
Similarly, the 3-isobutanoyl, 3-benzoyl-, 3-butyl-, and 3-(4-car-
boxybutanoyl)-substituted indoles 12, 14, 20, and 23 (Table 1)
were obtained. In the case of the 3-(3-carboxybenzoyl)- and
3-(4-carboxybenzoyl) derivatives 31 and 32 (Table 1) this
route was slightly modified. Here, the methyl ester protected
target compounds were not acetalyzed prior to ester hydro-
lysis but directly saponified with aqueous KOH in ethanol at
room temperature.
For the synthesis of the 3-ethyl-substituted indole 19, the
acetyl group of starting compound 1524 was reduced with
NaBH4/BF3 etherate (Scheme 3). The obtained tert-butyl
3-ethylindole-5-carboxylate (16) was directly reacted with
2-(4-octylphenoxymethyl)oxirane24 as described recently.
Oxidationwith acetic anhydride/DMSO and ester cleavage by
trifluoroacetic acid gave the desired test compound. Similarly
the 3-pivaloyl-substituted indole-5-carboxylic acid 13 (Table 1)
was synthesized.
The synthesisof the indolederivativewithoxadiazolegroup
in position 3 (40) was performed as outlined in Scheme 5.
Thus, tert-butyl 3-methoxycarbonylindole-5-carboxylate24
(35) was reacted with N0-hydroxyacetamidine29 to afford the
oxadiazole-substitued indole 36. Alkylation of indole 1 posi-
tion with epichlorohydrin followed by reaction with 4-octyl-
phenol and Dess-Martin oxidation yielded 39. Finally, the
tert-butyl ester group was cleaved with trifluororacetic acid to
afford target compound 40.
The 4-phenoxyphenyl-substituted target compounds 41
and 42 (Table 2) were synthesized in a similar way as the
corresponding 4-octylphenyl derivatives 30 and 40 (Schemes 2
and 5).
Results and Discussion
Previously we have shown that introduction of an acetyl
group in position 3 of indole-5-carboxylic acid derivative 2
increased cPLA2R inhibitory potency about 3-fold.24 Exam-
ining the effect of more bulky acyl groups, we found that
elongation of the 3-acetyl residue of 4 by two carbons did not
change inhibitory potency (Table 1). The 3-butanoyl-substituted
compound 10 was as active as 4. Introduction of one or two
methyl substituents in R-position of a propanoyl-residue also
did not affect enzyme inhibiton, as shown by the inhibition
data of the isobutanoyl- and pivaloyl-substituted derivatives
12 and 13. In contrast, replacement of the 3-butanoyl residue
by the longer 3-hexanoyl moiety (11) as well as by the more
voluminous benzoyl group (14) led to a 5- to 3-fold drop of
cPLA2R inhibition.
In contrast to small acyl residues, small alkyl groups like
ethyl and butyl in indole 3 position did not enhance inhibitory
activity. With IC50 values of about 0.030 μM, 19 and 20 were
about as active as the unsubstituted lead compound 2. Elon-
gation of the butyl residue was not well tolerated by the active
site of the enzyme, as shown by the about 20-fold decrease of
activity after introduction of a hexyl substituent (21).
Replacement of the terminal CH3 of the highly active
3-butanoyl derivative 10 by a polar carboxy group (22)
resulted in a similar loss of activity. Interestingly, in the case of
the 3-hexanoyl indole-5-carboxylic acid 11, such a struc-
tural modification led to a reversed effect. Here, a carboxy
substitutent increased inhibitory potency. This is reflected by
the IC50 of 0.022 μM of compound 30.
The 3-trifluoroacetyl substituted indole 34 was synthesized
from tert-butyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-
carboxylate24 (33) in one step by reaction with trifluoroacetic
anhydride in CH2Cl2 (Scheme 4).
Conformational restriction of the flexible alkyl spacer
in the 3-acyl residue of 30 by its replacement by a phenyl
residue did not change activity. The comparable activity of
the 3-carboxybenzoyl- and the 4-carboxybenzoyl-substituted
Figure 1
Scheme 1a
a (a) (1) ZnCl2, BuLi, CH2Cl2, 0 °C to room temp, (2) butanoyl chloride, AlCl3, 0 °C to room temp; (b) epichlorohydrin, KOH, Bu4NþBr-, room
temp; (c) 4-octyloxyphenol, 4-dimethylaminopyridine, 100 °C; (d) acetic anhydride, DMSO, room temp; (e) H2, Pd/C, THF, room temp.

8300 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Bovens et al.
Scheme 2a
a (a) (1) ZnCl2, BuLi, CH2Cl2, 0 °C to room temp, (2) adipic acid monomethyl ester chloride, AlCl3, 0 °C to room temp; (b) epichlorohydrin, KOH,
Bu4NþBr-, room temp; (c) 4-octylphenol, 4-dimethylaminopyridine, 100 °C; (d) acetic anhydride, DMSO, room temp; (e) trimethyl orthoformate,
methanol, H2SO4, reflux; (f) (1) aqueous NaOH, methanol, reflux, (2) aqueous HCl, THF, reflux.
Scheme 3a
a (a) NaBH4, BF3 etherate, THF; (b) 2-(4-octylphenoxymethyl)oxirane, NaH, DMF, 100 °C; (c) acetic anhydride, DMSO, room temp; (d)
trifluoroacetic acid, CH2Cl2, room temp.
Scheme 4a
compounds 31 and 32 indicates that there may be no specific
interactions between the carboxylic acid residues of these
groups and the enzyme.
Replacement of the 3-acetyl group of 4 by a 3-trifluoro-
acetyl residue (34) increased cPLA2R inhibition about 2-fold.
The exchange of the carboxylic ester group of compound 3 by
a bioisosteric 3-methyl-1,2,4-oxadiazol-5-yl moiety alters ac-
tivity in the same way. With an IC50 value of 0.0021 μM,
compound 40 is the most potent cPLA2R inhibitor we have
synthesized up to now.
An important structural part of the compounds investi-
gated is their activated electrophilic ketone moiety, which is
supposed to form covalent binding interactions with a serine
of the active site of cPLA2R.22,24 Because it is known that
a (a) Trifluoroacetic anhydride, CH2Cl2, room temp.
activated ketones can be metabolically unstable toward keto
reduction30-32 and because reduction of the ketone groups of
our inhibitors to alcohols leads to inactive compounds,32 we
also tested the metabolic stability of all newly synthesized
target compounds in a test system applying rat liver microsomes.
The investigations revealed that introduction of alkyl, acyl, or
oxadiazole substituents in indole 3-position of the lead 2 signifi-
cantly increased stability toward metabolic reduction of the
central ketone function.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8301
Table 1. cPLA2R Inhibitory Potency, Metabolic Stability, and Aqueous Solubility
compd
R
inhibition of cPLA2R IC50 (μM)a
metabolic stability (%)b
aqueous solubility (μg/mL)c
log P d
2
H
0.035
0.0049
0.012
0.010
0.071
0.010
0.013
0.042
0.030
0.028
0.59
65 ( 5
77
89
93
93
82
93
95
84
82
94
93
95
93
87
87
87
81
<1
<1
6.3
6.2
5.8
6.7
>7
6.6
>7
6.9
>7
>7
>7
5.1
5.2
5.3
5.7
5.6
>7
6.3
3
COOCH3
COCH3
COC3H7
COC5H11
4
<1
<1
10
11
12
13
14
19
20
21
22
23
30
31
32
34
40
<1
COCH(CH3)2
COC(CH3)3
COphenyl
80 ( 6
5 ( 2
9 ( 3
<1
C2H5
C4H9
C6H13
<1
<1
CO(CH2)2COOH
CO(CH2)3COOH
CO(CH2)4COOH
COphenyl(3-COOH)
COphenyl(4-COOH)
COCF3
0.21
354 ( 70
401 ( 39
194 ( 34
12 ( 1
472 ( 4
<1
0.030
0.022
0.027
0.028
0.0067
0.0021
3-methyl-1,2,4-oxadiazol-5-yl
<1
a Values are the mean of at least two independent determinations; errors are within (20%. IC50 value of reference inhibitor 1: 0.011 μM.22,24
b Percentage of parent compound remaining after metabolism by rat liver microsomes. Values are the mean of at least two independent determinations.
In the case of 2: mean ( standard deviation, n = 5. c Mean ( standard deviation, n = 3. In the case of values of <1 μg/mL, n = 2. d Partition coefficient
(log P) determined by reversed phase HPLC; log P of reference indomethacin, 2.9.
Scheme 5a
a (a) N0-Hydroxyacetamidine, NaH, THF, reflux; (b) epichlorohydrin, KOH, Bu4NþBr-, room temp; (c) 4-octylphenol, 4-dimethylaminopyridine,
120 °C; (d) acetic anhydride, DMSO, room temp; (e) trifluoroacetic acid, CH2Cl2, room temp.
Because of the two long hydrocarbon chains, phospholipid
substrates of cPLA2R possess a substantial lipophilicity.
Therefore, it can be expected that inhibitors, which will bind
competitively to the active site of the enzyme, must possess
similar properties. This assumption is confirmed by the fact that
most of the known cPLA2Rinhibitors with pronounced potency
bear larger lipophilic residues,14-28 which leads to a high total
lipophilicity of the compounds. Such a high lipophilicity can
cause a low water solubility, which may result in poor drug
absorption because the drug does not dissolve sufficiently in
the aqueous content of the gastrointestinal tract. Thus, we
also measured the aqueous solubility of all new compounds
under thermodynamic conditions33,34 by equilibrating the
solids in phosphate buffer (pH 7.4) for 20 h at room tempera-
ture, separating nonsoluble material by centrifugation and
measuring the soluble aqueous concentration by HPLC.
Under these conditions the lead compound 2 showed poor
water solubility (<1 μg/mL) (Table 1). Introduction of linear
alkyl or acyl residues in indole 3 position of 2 did not result in
compounds with dissolution values higher than 1 μg/mL.
However, replacement of the unbranched butanoyl (10) by
a branched isobutanoyl residue (12) significantly increased
solubility to a value of 80 μg/mL. Solubility guidelines for
drugs under development are given by Lipinski and co-
workers.35 According to those, compounds with mid-range
permeability and average potency should possess a minimum
thermodynamic solubility of 50 μg/mL. Thus, the isobutanoyl
derivative 12 clearly exceeds this limit. The pivaloyl and the
benzoyl substituted derivatives 13 and 14 were less soluble but
still revealed solubilities higher than 1 μg/mL (5 and 9 μg/mL,
respectively).
Introduction of a carboxylic acid group at the end of a
propanoyl, butanoyl, or pentanoyl side chain in position 3
of the indole scaffold increased solubility substantially.

8302 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Bovens et al.
Table 2. cPLA2R Inhibitory Potency, Metabolic Stability and Aqueous Solubility
compd
R
inhibition of cPLA2R IC50 (μM)a
metabolic stability (%)b
aqueous solubility (μg/mL)c
log P d
41
42
CO(CH2)4COOH
3-methyl-1,2,4-oxadiazol-5-yl
0.26
0.0065
24
8
246 ( 25
31 ( 2
2.1
3.1
a Values are the mean of at least two independent determinations; errors are within (20%. IC50 value of reference inhibitor 1: 0.011 μM.22,24
b Percentage of parent compound remaining after metabolism by rat liver microsomes. Values are the mean of at least two independent determinations.
c Mean ( standard deviation, n = 3. d Partition coefficient (log P) determined by reversed phase HPLC; log P of reference indomethacin, 2.9.
Figure 2
The dissolution values of obtained compounds 22, 23, and 30
lay between about 200 and 400 μg/mL. The 4-carboxy sub-
stituted benzoyl compound 32 even possessed a value of about
500 μg/mL. Surprisingly, shifting the carboylic acid group of
thebenzoyl residue of32from the para- into the meta-position
dramatically decreased aqueous solubility. From the synthe-
sized meta-isomer (31) only 12 μg/mL were dissolvable under
test conditions.
Recently, we have shown that replacement of the octyl
residue of the lead 2 by a phenoxy moiety does not change
cPLA2R inhibitory potency but increases aqueous solubility
and decreases metabolic stability. We now want to study the
effect of such a structural variation on these properties in the
cases of the selected compounds 30 and 40.
As could be expected, the metabolic stability of both
phenoxy derivatives (41 and 42) was significantly lower than
that of the corresponding octyl-substituted parent com-
pounds 30 and 40 (Tables 1 and 2). A substantial increase of
water solubility could be determined after replacement of the
octyl group of the oxadiazole 40 by a phenoxy residue (42).
In contrast, in the case of the carboxypentanoyl substituted
derivatives 30 and 41, the amount of dissolvable substance did
not change significantly after the same structural variation.
Inhibitory potency against cPLA2R dropped in both cases;
however, its extent was different. The oxadiazole42 was about
3-fold less active than its parent compound 40. In the case of
the 5-carboxypentanoyl indoles 30 and 41 the loss of activity
was even more than 10-fold. This may be explained by the
relativelylowlog P value (2.1) of 41. Since cPLA2Rinits active
status works at the lipophilic phospholipid membrane inter-
face, its inhibitors have to partition into the membrane before
they can bind to the enzyme. Because of its low lipophilicity,
the ability of 41 to concentrate in the interface could be
affected notably.
Compounds 30 and 40 were also tested for cPLA2R inhibi-
tion in a cellular situation. In intact human platelets cPLA2R
activity triggered by phorbol ester 12-O-tetradecanoylphor-
bol 13-acetate (TPA) was potently inhibited by these target
compounds too. The IC50 valuedeterminedfor30was 0.076(
0.019 μM (mean ( standard deviation, n = 3). Indole 40
inhibited the enzyme even with a subnanomolar IC50 of
0.0006 ( 0.0002 μM (mean ( standard deviation, n=3). For
comparison, the potent reference inhibitor 1 showed an IC50
of 0.0047 μM in the same assay.24
To obtain some information about specifity of 30 for
cPLA2R inhibition, we examined its inhibitory potency toward

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8303
Table 3. Plasma Levels of the cPLA2R Inhibitor 30 and Its Alcohol
Metabolite 45 after po Administration of 100 mg/kg 30 to Mice and
Plasma Levels of the Reference Indomethacin after po Dosage of 10 mg/kg
these substances.40 Therefore, we also tested the bioavailabil-
ity of 30, which possesses a good cPLA2R inhibitory potency,
substantial aqueous solubility, and high metabolic stability
against reduction by rat liver microsomes. However, the
obtained results were disappointing. The plasma levels mea-
sured for 30 and its alcohol metabolite 45 (Figure 2) after 1
and 2 h were even lower than those of 43 and 44 (Table 3).
The total amount of 30, 45, and their glucuronides found in
bile 2 h after dosage was also significantly lower than that of
43 and its corresponding metabolites. After treatment with
glucuronidase, the mean concentration of 30 and 45 in bile
togetherwas 66μg/mL (Table4)whilethe biliarconcentration
levels of 43 and 44 after enzymatic cleavage of the glucu-
ronides were raised to maximal values of 6450 μg/mL.28
In the intestine of the mice dosed with 30, besides only small
amounts of the alcohol metabolite 45 (14 μg/gut), high levels
of unmetabolized compound 30 could be found (870 μg/gut).
Considerable concentrations of glucuronides of these two
substances were not detected. In contrast, in the case of 43
the intestinal concentration of the inactive alcohol metabolite
44 in free and glucuronidated form exceeded the concentra-
tion of the free and bound parent compound 43 (875 μg/gut vs
404 μg/gut; for details see Supporting Information). The high
concentration of unmetabolized 30 in intestine after po ad-
ministration makes this compound interesting for in vivo
testing in animal models of inflammatory bowel diseases.
The results of such experiments as well as investigations about
topical activity of 30 will be reported in due course.
plasma level (μg/mL)a
30
45
compd
time (h)
30 (100 mg/kg)
1
2
1
2
<0.2
<0.2
<0.1
<0.1
indomethacin (10 mg/kg)
42 ( 7.2
34 ( 5.3
a Values are mean ( standard deviations. Compound 30: n = 3.
Indomethacin: n = 4.
Table 4. Amount of Parent Compound 30 and Its Alcohol Metabolite 45
Found in Bile and Intestine 2 h after po Administration of 30 (100 mg/kg)
amount in bile (μg/mL)a
before treatment with
glucuronidase
after treatment with
glucuronidase
30
45
6.0 ( 3.6
<1
33 ( 12
33 ( 13
amount in intestine (μg/gut)b
before treatment with
glucuronidase
after treatment with
glucuronidase
30
45
870 ( 139
14 ( 1.7
ndc
ndc
a Values are the mean ( standard deviations; n = 3. b Values are the
mean ( standard deviations; n = 3. The weights of the intestines of the
animals were 2.37, 2.22, and 2.12 g, respectively. c nd: not determined,
since no glucuronides of 30 and 45 could be detected in the intestinal
extracts by HPLC.
Experimental Section
other PLA2 enzymes, namely, calcium-independent PLA2
(iPLA2) from rat brain cytosol and group IB secretory PLA2
(sPLA2) from porcine pancreas.36,37 At the highest test con-
centration of 10 μM, 30 did not inhibit these enzymes, while
the applied reference inhibitors for iPLA2 (bromoenol
lactone) and sPLA2 ((S)-5-(4-benzyloxyphenyl)-4-[(7-phenyl-
heptanoyl)amino]pentanoic acid)38 showed about 50% and
90% enzyme inhibition, respectively, at this concentration.
Recently, we have investigated the bioavailability of the
5-carboxyindol-1-ylpropan-2-one derivative 43 in mice.28
Although the compound possessed a considerable aqueous
solubility, the median plasma levels of 43 and its main
metabolite 44 (Figure 2) determined 1 and 2 h after po
administration of 100 mg/kg of 43 were only low (less than
5 μg/mL after 1 h and less than 1 μg/mL after 2 h). For
comparison, the concentration of indomethacin in plasma in
mice perorally dosed in a concentration of 10 mg/kg was 42
and 34 μg/mL, respectively, after these times.28 A high plasma
clearance of 43 seemed to be the main reason for its impaired
po bioavailability. This efficient clearance obviously was due
to a pronounced glucuronidation of 43 and 44 in combination
with an excessive biliar excretion. One hint for this was the
high level of free and bound 43 and 44 found in the bile of the
animals. Furthermore, this assumption was supported by
the fact that 30 min after iv administration of 10 mg/kg 43
more than 73% of the dose applied could be found in the
intestine of the animals in form of 43, 44, and their glucuronides.
Because of these results, it could be expected that the
bioavailability of other 5-carboxyindol-1-ylpropan-2-ones
was also limited. On the other hand, possible exceptions could
be the dicarboxylic acid derivatives like 30, since for structu-
rally related indoledicarboxylic acids, such as compound 46
(Figure 2),39 high plasma levels (about 100 μg/mL) were
observed in rats 4 h after po administration of 100 mg/kg of
1. Chemistry. General. Column chromatography was per-
formed on Merck silica gel 60, 230-400 mesh or 70-230 mesh.
€
Melting points were determined on a Buchi B-540 apparatus and
1
are uncorrected. H NMR spectra (400 MHz) and 13C NMR
spectra (100 MHz) were recorded on a Varian Mercury Plus 400
spectrometer. Mass spectra were obtained on Finnigan GCQ
and LCQ apparatuses applying electron beam ionization (EI) or
electrospray ionization (ESI). Purity of the target compounds
was determined by normal phase HPLC at 254 nm. For the
dicarboxylic acids 22, 23, 30-32, and 41 and the trifluoromethyl
derivative 34, a diol phase (LiChrospher 100 DIOL, 5 μm, 3.0 mm
(i.d.) ꢀ 125 mm, Merck, Darmstadt, Germany) with a heptane/
THF gradient at a flow rate of 0.75 mL/min was applied. The
other compounds were chromatographed on an amino phase
(Spherisorb NH2, 5 μm, 4.0 mm (i.d.) ꢀ 250 mm, Latek,
Heidelberg, Germany) using an isohexane/THF gradient at a
flow rate of 0.75 mL/min. All target compounds showed purities
greater or equal 95%, with exception of 10 (purity 92%).
Benzyl 3-Butanoylindole-5-carboxylate (6). To a mixture of a
ZnCl2 solution in dry diethyl ether (2.2 M, 2.0 mL) and dry
CH2Cl2 (10 mL) a solution of butyllithium in dry hexane (1.6 M,
2.4 mL) was slowly added under nitrogen at 0 °C. The mixture
was allowed to warm to room temperature, stirred for 1 h at
room temperature, treated with a solution of benzyl indole-5-
carboxylate41 (5) (1.0 g, 4.0 mmol) in dry CH2Cl2 (10 mL), and
further stirred for 1 h. The reaction mixture was cooled to 0 °C,
butanoyl chloride (0.9 mL, 8.4 mmol) was added, and stirring
was continued for 1 h at room temperature. Then the mixture
was treated with AlCl3 (0.58 g, 4.4 mmol) and further stirred for
1 h. After addition of half-saturated brine and THF the mixture
was extracted exhaustively with ethyl acetate. The combined
organic layers were washed with brine, dried (Na2SO4), and
concentrated. The residue was recrystallized from ethyl acetate
to afford 6 as a solid (559 mg, 44%); mp 182-183 °C. 1H NMR
(DMSO-d6) δ 0.92 (t, 3H), 1.65 (sext, 2H), 2.83 (t, 2H), 5.36 (s,
2H), 7.35-7.41 (m, 3H), 7.46-7.49 (m, 2H), 7.55 (d, 1H), 7.85

8304 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Bovens et al.
(dd, 1H), 8.45 (d, 1H), 8.90-8.91 (m, 1H), 12.23 (sbroad, 1H). MS
(EI): m/z (%) 321 (84) [Mþ], 278 (100).
aqueous NaHCO3 and ice-water and extracted exhaustively
with diethyl ether. The combined organic phases were dried
(Na2SO4) and evaporated. Chromatography on silica gel
(hexane/ethyl acetate, 8:2) yielded 16 as an oil (236 mg, 89%).
1H NMR (CDCl3): δ 1.33 (t, 3H), 1.63 (s, 9H), 2.80 (q, 2H),
7.01 (s, 1H), 7.32 (d, 1H), 7.85 (dd, 1H), 8.17 (sbroad, 1H), 8.33
(s, 1H). MS (EI): m/z (%) 245 (47) [Mþ], 174 (100).
Benzyl 3-Butanoyl-1-oxiranylmethylindole-5-carboxylate (7).
To a mixture of 6 (400 mg, 1.25 mmol), powdered KOH (88%,
140 mg, 2.5 mmol), and tetrabutylammonium bromide (40 mg,
0.13 mmol) was added epichlorohydrin (4 mL). The reaction
mixture was stirred at room temperature until 6 had disappeared
(about 2 h). The mixture was purified by silica gel chromatog-
raphy (hexane/ethyl acetate, (a) 9:1, (b) 8:2, (c) 1:1) to give 7 as
tert-Butyl 3-Ethyl-1-[2-hydroxy-3-(4-octylphenoxy)propyl]indole-
5-carboxylate (17). Under a nitrogen atmosphere, a suspension
of NaH (60% in mineral oil, 40 mg, 1.01 mmol) in dry DMF
(10 mL) was stirred at room temperature for 10 min. After
addition of the solution of 16 (236 mg, 0.96 mmol) in dry DMF
(10 mL), the mixture was stirred for 1 h. A solution of 2-(4-
octylphenoxymethyl)oxirane24 (252 mg, 0.96 mmol) in dry
DMF (10 mL) was added dropwise at room temperature, and
the reaction mixture was heated at 60 °C for 4 h. After cooling,
the mixture was treated with half-saturated brine and extracted
exhaustively with diethyl ether. The combined organic phases
were washed with half-saturated brine, dried (Na2SO4), and the
solvent was evaporated. Chromatography on silica gel with
hexane/ethyl acetate (9:1) followed by chromatography on
reversed phase silica gel with acetonitrile yielded 17 as an oil
(198 mg, 41%). 1H NMR (CDCl3): δ 0.87 (t, 3H), 1.26-1.31 (m,
13H), 1.54-1.61 (m, 11H), 2.51-2.55 (m, 3H), 2.77 (quart, 2H),
3.83 (dd, 1H), 3.90 (dd, 1H), 4.25-4.38 (m, 3H), 6.79 (d, 2H),
6.95 (s, 1H), 7.08 (d, 2H), 7.31 (d, 1H), 7.83 (d, 1H), 8.29 (s, 1H).
MS (EI): m/z (%) 507 (53) [Mþ], 451 (100).
tert-Butyl 3-Ethyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-
5-carboxylate (18). Compound 17 (153 mg, 0.30 mmol) was
oxidized according to the procedure described above for the
preparation of 9. The crude product was purified by silica gel
chromatography (hexane/ethyl acetate, 9:1) to yield 18 as an oil
(77 mg, 66%). 1H NMR (CDCl3): δ 0.89 (t, 3H), 1.24-1.39 (m,
13H), 1.54-1.64 (m, 11H), 2.57 (t, 2H), 2.81 (quart, 2H), 4.59 (s,
2H), 5.13 (s, 2H), 6.80-6.86 (m, 3H), 7.04 (d, 1H), 7.14 (d, 2H),
7.86 (d, 1H), 8.32 (s, 1H). MS (EI): m/z (%) 505 (2) [Mþ], 202
(100).
1
an oil (411 mg, 87%). H NMR (CDCl3): δ 1.00 (t, 3H), 1.79
(sext, 2H), 2.43-2.45 (m, 1H), 2.80-2.85 (m, 3H), 3.30-3.32
(m, 1H), 4.08-4.10 (m, 1H), 4.56 (dd, 1H), 5.39 (s, 2H),
7.31-7.39 (m, 4H), 7.46-7.48 (m, 2H), 7.82 (s, 1H), 8.02 (dd,
1H), 9.13 (d, 1H). MS (EI): m/z (%) 377 (41) [Mþ], 334 (100).
Benzyl 3-Butanoyl-1-[2-hydroxy-3-(4-octylphenoxy)propyl]indole-
5-carboxylate (8). Under a nitrogen atmosphere a mixture of 7
(390 mg, 1.03 mmol), 4-octylphenol (213 mg, 1.03 mmol), and
4-dimethylaminopyridine (25 mg, 0.21 mmol) was stirred at 100
°C for 3 h. The reaction mixture was dissolved in toluene and
purified by silica gel chromatography (hexane/ethyl acetate, (a)
9:1, (b) 8:2, (c) 1:1) to afford 8 as a solid (192 mg, 32%); mp
1
74-75 °C. H NMR (DMSO-d6): δ 0.83 (t, 3H), 0.90 (t, 3H),
1.22-1.24 (m, 10H), 1.48-1.49 (m, 2H), 1.63 (sext, 2H),
2.48-2.49 (m, 2H), 2.74 (t, 2H), 3.86-3.89 (m, 2H), 4.19-4.21
(m, 1H), 4.26-4.28 (m, 1H), 4.51-4.53 (m, 1H), 5.36 (s, 2H),
6.84 (d, 2H), 7.07 (d, 2H), 7.39-7.42 (m, 3H), 7.46-7.48 (m,
2H), 7.69 (d, 1H), 7.85 (d, 1H), 8.42 (s, 1H), 8.90-8.91 (m, 1H).
MS (EI): m/z (%) 583 (18) [Mþ], 492 (100).
Benzyl 3-Butanoyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-
5-carboxylate (9). Acetic anhydride (1.1 mL, 12 mmol) was
added to dry DMSO (15 mL), and the mixture was stirred under
a nitrogen atmosphere at room temperature for 10 min. Then
this solution was added drop by drop to a solution of 8 (172 mg,
0.29 mmol) in dry DMSO (15 mL). The mixture was stirred for
18 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 exhaustively with diethyl ether.
The combined organic phases were washed three times with
brine, dried (Na2SO4), and concentrated. The residue was
purified by chromatography on silica gel (hexane/ethyl acetate,
(a) 9:1, (b) 8:2, (c) 1:1) to give 9 as an oil (79 mg, 48%). 1H NMR
(DMSO-d6): δ 0.84 (t, 3H), 0.93 (t, 3H), 1.22-1.24 (m, 10H),
1.48-1.49 (m, 2H), 1.65 (sext, 2H), 2.48-2.49 (m, 2H), 2.79 (t,
2H), 5.02 (s, 2H), 5.37 (s, 2H), 5.53 (s, 2H), 6.89 (d, 2H), 7.10 (d,
2H), 7.35-7.42 (m, 3H), 7.46-7.49 (m, 2H), 7.59 (d, 1H), 7.86
(dd, 1H), 8.38 (s, 1H), 8.90-8.91 (m, 1H). MS (EI): m/z (%) 581
(3) [Mþ], 377 (100).
3-Butanoyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic
Acid (10). A mixture of 9 (75 mg, 0.13 mmol), palladium (10%)
on charcoal (19 mg), and THF (10 mL) was stirred under a
balloon filled with H2 at room temperature for 2.5 h. The
mixture was filtered through a cotton pad and evaporated.
The residue was recrystallized from hexane/ethyl acetate to give
10 as a solid (13 mg, 23%); mp 141-142 °C. 1H NMR (DMSO-
d6): δ 0.83 (t, 3H), 0.92 (t, 3H), 1.21-1.24 (m, 10H), 1.49-1.50
(m, 2H), 1.66 (sext, 2H), 2.49-2.50 (m, 2H), 2.79 (t, 2H), 5.01 (s,
2H), 5.51 (s, 2H), 6.89 (d, 2H), 7.10 (d, 2H), 7.54 (d, 1H), 7.81
(dd, 1H), 8.34 (s, 1H), 8.84-8.85 (m, 1H). 13C NMR (DMSO-
d6): δ 14.51, 14.63, 18.86, 22.76-31.95, 34.90, 41.62, 53.59, 71.55,
111.37, 115.08, 117.26, 124.65, 124.95, 125.30, 125.85, 129.91,
135.79, 139.74, 140.44, 156.38, 168.66, 195.80, 201.27. MS
(ESI-): m/z 490 [M - H]-.
3-Ethyl-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic
Acid (19). To the solution of 18 (77 mg, 0.15 mmol) in dry
CH2Cl2 (10 mL) was added trifluoroacetic acid (0.9 mL), and
the mixture was stirred at room temperature for 4 h. Then 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
1
(6:4) to give 19 as a solid (38 mg, 53%); mp 123-124 °C. H
NMR (DMSO-d6): δ 0.83 (t, 3H), 1.22-1.27 (m, 13H),
1.49-1.50 (m, 2H), 2.47-2.49 (m, 2H), 2.72 (q, 2H), 4.96 (s,
2H), 5.34 (s, 2H), 6.86 (d, 2H), 7.09 (d, 2H), 7.13 (s, 1H), 7.36 (d,
1H), 7.69 (dd, 1H), 8.32 (m, 1H), 12.44 (s, 1H). 13C NMR
(DMSO-d6): δ 14.63, 15.08, 18.24, 22.76-31.96, 34.90, 52.85,
71.37, 110.27, 115.02, 118.62, 121.74, 121.80, 123.08, 127.65,
128.21, 129.88, 135.69, 140.00, 156.40, 168.99, 202.10. MS
(ESI-): m/z 448.4 [M - H]-.
Methyl 3-(5-Methoxycarbonylpentanoyl)indole-5-carboxylate
(25). A suspension of AlCl3 (4.5 g, 34 mmol) in dry CH2Cl2 (40 mL)
was treated with adipic acid monomethyl ester chloride (2.5 mL,
15 mmol). After the mixture was stirred at room temperature for
1 h, methyl indole-5-carboxylate (24) (1.75 g, 10 mmol) was
added and stirring was continued for 30 min. Then water and
1 M HCl were added and the mixture was extracted exhaustively
with CH2Cl2. The combined organic layers were washed with
dilute aqueous Na2CO3 solution and water, dried (Na2SO4), and
concentrated. The residue was recrystallized from ethyl acetate
to afford 25 as a solid (2.3 g, 72%); mp 181-182 °C. 1H NMR
(DMSO-d6): δ 1.56-1.66 (m, 4H), 2.34 (t, 2H), 2.86 (t, 2H), 3.56
(s, 3H), 3.85 (s, 3H), 7.53 (d, 1H), 7.81 (dd, 1H), 8.42 (d, 1H),
8.87-8.88 (m, 1H), 12.21 (sbroad, 1H). MS (EI): m/z (%) 317 (13)
[Mþ], 217 (100).
tert-Butyl 3-Ethylindole-5-carboxylate (16). A solution of
tert-butyl 3-acetylindole-5-carboxylate24 (15) (280 mg, 1.1
mmol) in dry THF (30 mL) was treated with sodium borohy-
dride (81 mg, 2.2 mmol) under a nitrogen atmosphere at 30 °C.
Then borontrifluoride etherate (0.4 mL, 3.2 mmol) was
added dropwise at such a rate that the temperature did not
exceed 45 °C. After being stirred at room temperature for 1 h,
the reaction mixture was poured into a mixture of 5%
Methyl 3-(5-Methoxycarbonylpentanoyl)-1-oxiranylmethylin-
dole-5-carboxylate (26). To a mixture of 25 (1.3 g, 4.1 mmol),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8305
powdered KOH (88%, 460 mg, 8.2 mmol), and tetrabutylam-
monium bromide (132 mg, 0.41 mmol) was added epichlorohy-
drin (3 mL). The reaction mixture was stirred at room temperature
for 1.5 h. The mixture was purified by silica gel chromatography
(hexane/ethyl acetate, (a) 9:1, (b) 7:3, (c) 1:1) to give 26 as a solid
(939 mg, 61%); mp 140-141 °C. 1H NMR (DMSO-d6): δ
1.58-1.65 (m, 4H), 2.34 (t, 2H), 2.47-2.49 (m, 1H), 2.79-2.81
(m, 1H), 2.86 (t, 2H), 3.36-3.38 (m, 1H), 3.56 (s, 3H), 3.85 (s,
3H), 4.30 (dd, 1H), 4.66 (dd, 1H), 7.73 (d, 1H), 7.86 (dd, 1H),
8.48 (s, 1H), 8.87-8.88 (m, 1H). MS (EI): m/z (%) 373 (21) [Mþ],
273 (100).
Methyl 1-[2-Hydroxy-3-(4-octylphenoxy)propyl]-3-(5-meth-
oxycarbonylpentanoyl)indole-5-carboxylate (27). Under a nitro-
gen atmosphere a mixture of 26 (900 mg, 2.4 mmol), 4-octylphenol
(497 mg, 2.4 mmol), and 4-dimethylaminopyridine (59 mg, 0.48
mmol) was stirred at 100 °C for 30 min. The reaction mixture
was dissolved in toluene and purified by silica gel chromatog-
raphy (hexane/ethyl acetate, (a) 2:1, (b) 1:1) to afford 27 as a
solid (0.76 g, 54%); mp 103-104 °C. 1H NMR (CDCl3): δ 0.88
(t, 3H), 1.26-1.30 (m, 10H), 1.55-1.57 (m, 2H), 1.66-1.71 (m,
4H), 2.33 (t, 2H), 2.54 (t, 2H), 2.73 (t, 2H), 3.03 (m, 1H, OH),
3.64 (s, 3H), 3.91 (s, 3H), 3.95-3.99 (m, 2H), 4.31-4.39 (m, 2H),
4.42-4.49 (m, 1H), 6.83 (d, 2H), 7.11 (d, 2H), 7.41 (d, 1H), 7.89
(s, 1H), 7.95 (dd, 1H), 9.01-9.02 (m, 1H). MS (EI): m/z (%) 579
(100) [Mþ].
Methyl 3-(5-Methoxycarbonylpentanoyl)-1-[3-(4-octylphenoxy)-
2-oxopropyl]indole-5-carboxylate (28). A solution of 27 (0.75 g,
1.3 mmol) in dry CH2Cl2 (20 mL) was treated with Dess-
Martin periodinane reagent (1.2 g, 2.8 mmol) and stirred under
a nitrogen atmosphere at room temperature for 2 h. The
reaction mixture was poured into a solution of sodium thiosul-
fate (3.0 g) in saturated aqueous sodium bicarbonate solution
(60 mL) and extracted exhaustively with ethyl acetate. The
combined organic layers were dried (Na2SO4) and evaporated.
The residue was purified by chromatography on silica gel (hexane/
ethyl acetate, (a) 2:1, (b) 1.5:1) to yield 28 as a solid (0.69 g,
93%); mp 95-96 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H),
1.27-1.31 (m, 10H), 1.58-1.61 (m, 2H), 1.73-1.81 (m, 4H),
2.38 (t, 2H), 2.58 (t, 2H), 2.88 (t, 2H), 3.66 (s, 3H), 3.93 (s, 3H),
4.71 (s, 2H), 5.30 (s, 2H), 6.87 (d, 2H), 7.09 (d, 1H), 7.17 (d, 2H),
7.75 (s, 1H), 7.97 (d, 1H), 9.10 (s, 1H). MS (EI): m/z (%) 577 (33)
[Mþ], 373 (100).
Methyl 1-[2,2-Dimethoxy-3-(4-octylphenoxy)propyl]-3-(5-
methoxycarbonylpentanoyl)indole-5-carboxylate (29). Asolution
of 28 (0.69 g, 1.2 mmol) in dry methanol (25 mL) was treated
with trimethyl orthoformate (1.5 mL, 13 mmol) and three drops
of concentrated H2SO4. The mixture was heated under reflux for
3 h. After cooling, the reaction mixture was carefully poured
into a 5% aqueous NaHCO3 solution and extracted exhaus-
tively with ethyl acetate. The combined organic phases were
dried (Na2SO4) and concentrated. The residue was purified by
chromatography on silica gel (hexane/ethyl acetate, 8:2) to yield
29 as an oil (183 mg, 24%). 1HNMR(CDCl3):δ0.87 (t, 3H), 1.25-
1.28 (m, 10H), 1.52-1.55 (m, 2H), 1.63-1.72 (m, 4H), 2.32 (t, 2H),
2.50 (t, 2H), 2.64 (t, 2H), 3.41 (s, 6H), 3.66-3.68 (m, 5H), 3.89 (s,
3H), 4.47 (s, 2H), 6.72 (d, 2H), 7.04 (d, 2H), 7.45 (d, 1H), 7.77
(s, 2H), 7.85 (dd, 1H), 9.01-9.02 (m, 1H). MS (ESIþ): m/z 624
[M þ H]þ.
were dried (Na2SO4) and evaporated. The residue was chroma-
tographed on silica gel (hexane/ethyl acetate/formic acid, (a)
8:2:0.5, (b) 5:5:0.5) to give 30 as a solid (82 mg, 52%); mp
174-175 °C. H NMR (DMSO-d6): δ 0.82 (t, 3H), 1.21-1.24
1
(m, 10H), 1.48-1.65 (m, 6H), 2.24 (t, 2H), 2.49-2.50 (m, 2H),
2.82 (t, 2H), 5.02 (s, 2H), 5.51 (s, 2H), 6.89 (d, 2H), 7.10 (d, 2H),
7.54 (d, 1H), 7.82 (dd, 1H), 8.36 (s, 1H), 8.84-8.85 (m, 1H),
12.33 (sbroad, 2H). 13C NMR (DMSO-d6): δ 14.64, 22.76-31.94,
34.23, 34.90, 39.32, 53.61, 71.54, 111.40, 115.07, 117.16, 124.62,
124.67, 125.23, 125.86, 129.92, 135.80, 139.77, 140.46, 156.36,
168.64, 175.09, 195.67, 201.24. MS (ESIþ): m/z 550 [M þ H]þ.
1-[3-(4-Octylphenoxy)-2-oxopropyl]-3-(2,2,2-trifluoroacetyl)indole-
5-carboxylic Acid (34). To a solution of trifluoroacetic anhy-
dride (9.6 mL) in dry CH2Cl2 (140 mL) was added tert-butyl
1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylate (33)24
(0.69 g, 1.4 mmol) under a nitrogen atmosphere at 0 °C. After
being stirred at room temperature for 3 days, the solution was
concentrated to half of its volume and triturated with hexane
until precipitation occurred. The precipitate was separated by
filtration and purified by chromatography on silica gel (hexane/
ethyl acetate/formic acid, 3:1:0.1) to give 34 as a solid (0.35 g,
47%); mp 129 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H), 1.27-1.32
(m, 10H), 1.59-1.61 (m, 2H), 2.60 (t, 2H), 4.76 (s, 2H), 5.40 (s,
2H), 6.89 (d, 2H), 7.15-7.20 (m, 3H), 7.96 (s, 1H), 8.11 (dd, 1H),
9.19-9.20 (m, 1H). 13C NMR (CDCl3): δ 14.31, 22.90-32.11,
35.28, 53.99, 72.52, 109.96, 111.54, 114.38, 118.50, 125.37,
126.34, 126.58, 126.96, 130.18, 137.71, 139.00, 140.13, 155.22,
171.92, 175.00, 200.49. MS (ESIþ): m/z 518 [M þ H]þ.
tert-Butyl 3-(3-Methyl-1,2,4-oxadiazol-5-yl)indole-5-carboxylate
(36). Under a nitrogen atmosphere, a solution of N0-hydroxy-
acetamidine29 (135 mg, 1.82 mmol) in dry THF (30 mL) was
treated with NaH (60% dispersion in mineral oil) (73 mg,
1.8 mmol) and stirred at room temperature for 1 h. After
addition of 5-tert-butyl 3-methyl indole-3,5-dicarboxylate (35)24
(500 mg, 1.8 mmol), the mixture was heated under reflux for
24 h. Then water (150 mL) and ethyl acetate (150 mL) were
added and most of the remaining THF was evaporated in vacuo.
The concentrated mixture was extracted exhaustively with ethyl
acetate, and the combined organic layers were dried (Na2SO4)
and evaporated. The residue was chromatographed on silica gel
(hexane/ethyl acetate, 7:3) to afford 36 as a solid (310 mg, 57%); mp
205-206 °C. 1H NMR (CDCl3): δ 1.65 (s, 9H), 2.48 (s, 3H), 7.47
(d, 1H), 7.99 (dd, 1H), 8.10 (m, 1H), 8.97 (m, 1H), 9.20 (sbroad
,
1H). MS (EI): m/z (%) 299 (36) [Mþ], 243 (100).
tert-Butyl 3-(3-Methyl-1,2,4-oxadiazol-5-yl)-1-oxiranylmethy-
lindole-5-carboxylate (37). Compound 36 (305 mg, 1.0 mmol)
was reacted with epichlorohydrin as described for the prepara-
tion of compound 7. Chromatography on silica gel (hexane/
ethyl acetate, (a) 9:1, (b) 1:1) afforded 37 as a solid (311 mg,
86%); mp 138-139 °C. 1H NMR (CDCl3): δ 1.64 (s, 9H),
2.47-2.50 (m, 4H), 2.86-2.88 (m, 1H), 3.34-3.35 (m, 1H),
4.23 (dd, 1H), 4.57 (dd, 1H), 7.47 (d, 1H), 8.00-8.02 (m, 2H),
8.94 (m, 1H). MS (EI): m/z (%) 355 (48) [Mþ], 299 (100).
tert-Butyl 1-[2-Hydroxy-3-(4-octylphenoxy)propyl]-3-(3-methyl-
1,2,4-oxadiazol-5-yl)indole-5-carboxylate (38). Under a nitrogen
atmosphere a mixture of 37 (150 mg, 0.42 mmol), 4-octylphenol
(87 mg, 0.42 mmol), and 4-dimethylaminopyridine (10 mg, 0.08
mmol) was stirred at 120 °C for 1 h. The reaction mixture was
dissolved in toluene and purified by silica gel chromatography
(hexane/ethyl acetate, 7:3) to afford 38 as an oil (146 mg, 62%).
1H NMR (CDCl3): δ 0.87 (t, 3H), 1.24-1.28 (m, 10H),
1.55-1.58 (m, 2H), 1.64 (s, 9H), 2.44 (s, 3H), 2.54 (t, 2H), 2.78
(sbroad, 1H), 3.93-3.99 (m, 2H), 4.38-4.42 (m, 2H), 4.49-4.52
(m, 1H), 6.80 (d, 2H), 7.09 (d, 2H), 7.46 (d, 1H), 7.96 (d, 1H),
8.09 (s, 1H), 8.91 (s, 1H). MS (EI): m/z (%) 561 (1) [Mþ], 264
(100).
3-(5-Carboxypentanoyl)-1-[3-(4-octylphenoxy)-2-oxopro-
pyl]indole-5-carboxylic Acid (30). To a solution of 29 (180 mg,
0.29 mmol) in methanol (15 mL) was added a solution of NaOH
(1.4 g) in water (15 mL). The mixture was heated under reflux for
2.5 h. Then most of the methanol was evaporated in vacuo. The
concentrated mixture was acidified with 6 M HCl (17 mL) and
extracted exhaustively with ethyl acetate. The combined organic
layers were dried (Na2SO4) and evaporated. The residue was
dissolved in THF (15 mL), treated with 6 M HCl (3 mL), and
heated under reflux for 3 h. After the mixture was cooled, water
was added (10 mL) and the reaction mixture was extracted
exhaustively with ethyl acetate. The combined organic layers
tert-Butyl 3-(3-Methyl-1,2,4-oxadiazol-5-yl)-1-[3-(4-octylphenoxy)-
2-oxopropyl]indole-5-carboxylate (39). Compound 38 (135 mg,
0.24 mmol) was oxidized according to the procedure described
above for the preparation of 9. The crude product was purified

8306 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Bovens et al.
by silica gel chromatography (hexane/ethyl acetate, 8:2) to yield
39 as an oil (48 mg, 38%). H NMR (CDCl3): δ 0.88 (t, 3H),
1.27-1.31 (m, 10H), 1.58-1.64 (m, 11H), 2.48 (s, 3H), 2.58 (t,
2H), 4.72 (s, 2H), 5.34 (s, 2H), 6.87 (d, 2H), 7.12 (d, 1H), 7.17 (d,
2H), 7.90 (s, 1H), 7.97 (d, 1H), 8.96 (s, 1H). MS (EI): m/z (%) 559
(1) [Mþ], 107 (100).
The microsomes were incubated with test substance in the
presence and absence of dihydronicotinamide adenine dinucleo-
tide phosphate (NADPH). Incubations were terminated after
30 min by addition of acetonitrile. After vigorous vortexing, the
samples were centrifuged and the supernatants were subjected to
reversed phase HPLC analysis with UV detection at 240 nm. The
metabolic stability was calculated by comparing the amount of the
ketone form of the target compound found in the presence and
absence of NADPH.
2.4. Solubility. Thermodynamic solubility was determined in
the way previously described.33 Briefly, to 1 mg of a test
compound was added phosphate buffered saline (0.1 M,
pH 7.4) (2 mL). The mixture was sonicated for 10 min in a bath
sonifier and then shaken for 20 h at room temperature. After
centrifugation, to an aliquot of the clear supernatant, acetoni-
trile was added, and the amount of the target compound present
in the sample was determined by reversed phase HPLC and UV
detection at 240 nm. With this method, for the reference
indomethacin an aqueous solubility of 246 ( 18 μg/mL
(mean ( standard deviation, n = 4) was measured.
1
3-(3-Methyl-1,2,4-oxadiazol-5-yl)-1-[3-(4-octylphenoxy)-2-
oxopropyl]indole-5-carboxylic Acid (40). To the solution of 39
(46 mg, 0.08 mmol) in dry CH2Cl2 (10 mL) was added trifluoro-
acetic acid (0.5 mL), and the mixture was stirred at room
temperature for 4 h. Then 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 ethyl acetate to give 40 as a solid (27 mg, 63%); mp
1
213-215 °C. H NMR (DMSO-d6): δ 0.83 (t, 3H), 1.22-1.25
(m, 10H), 1.50-1.52 (m, 2H), 2.43 (s, 3H), 2.49-2.50 (m, 2H),
5.03 (s, 2H), 5.60 (s, 2H), 6.91 (d, 2H), 7.11 (d, 2H), 7.66 (d, 1H,
3J = 8.6 Hz), 7.89 (d, 1H), 8.40 (s, 1H), 8.79 (s, 1H), 12.85 (sbroad
,
1H). 13C NMR (DMSO-d6): δ 11.96, 14.63, 22.75-31.93, 34.89,
53.74, 71.49, 101.45, 112.00, 115.07, 123.25, 124.75-124.82,
125.22, 129.92, 135.80, 136.35, 140.26, 156.34, 167.75, 168.42,
172.27, 201.14. MS (ESIþ): m/z 504 [M þ H]þ.
2.5. log P Values. Partition coefficients (log P) were determined
by reversed phase HPLC according to an OECD guideline,43
applying the specific procedure published recently.28 With this
method, for indomethacin and ibuprofen log P values of 2.9 and
3.2, respectively, were obtained. For determination of the log P
values of the target compounds, the retention times of the peak
of their ketone forms were used.
2. Evaluation of the Target Compounds. 2.1. General. The
HPLC system applied for measuring enzyme inhibition, meta-
bolic stability, solubility, partition coefficients (log P), and
blood plasma concentrations consisted of a Waters HPLC
pump model 515, a Waters autosampler model 717 plus, a
Waters column oven, and a Waters UV/vis detector model 2487.
Instrument control, data collection, and processing were
handled by Waters Millenium 32 chromatography software.
2.2. Inhibition of cPLA2r. 2.2.1. Assay with the Isolated
Enzyme. Inhibition of cPLA2R isolated from human platelets
was measured as previously published.42 Briefly, 1-stearoyl-2-
arachidonoyl-sn-glycero-3-phosphocholine (200 μM) sonicated
with 1,2-dioleoyl-sn-glycerol (100 μM) in a bath sonicator at
30-35 °C was used as substrate. Enzyme reaction was termi-
nated after 60 min by addition of a mixture of acetonitrile,
methanol, and 0.1 M aqueous EDTA-Na2 solution, which
contained 4-undecyloxybenzoic acid as internal standard and
nordihydroguaiaretic acid (NDGA) as oxygen scavenger. Re-
leased product arachidonic acid was determined with reversed
phase HPLC and UV detection at 200 nm after solid phase
extraction. Inhibition of cPLA2R activity was calculated by
comparing the arachidonic acid formed by the enzyme in the
absence and presence of a test compound.
2.6. Inhibition of Calcium-Independent Phospholipase A2
(iPLA2) and Secretory Phospholipase A2 from Porcine Pancreas
(sPLA2 Group IB). Inhibition of iPLA2 in rat brain cytosol and
sPLA2 from porcine pancreas (group IB) by 30 was assessed by the
methods described recently.28,36,37 Briefly, in the case of iPLA2,
1-palmitoyl-2-(10-pyren-1-yldecanoyl)-sn-glycero-3-phosphocho-
line was used as substrate. Enzyme activity in the presence and
absence of the test compound was determined by measuring the
release of 10-pyren-1-yldecanoic acid by reversed phase HPLC
and fluorescence detection. Inhibition of sPLA2 IB was evalu-
ated with the substrate 2-oleoyl-1-palmitoyl-sn-glycero-3-phos-
pho-rac-(1-glycerol) (ammonium salt) in the presence of sodium
deoxycholate. The release of the enzyme product oleic acid was
monitored by reversed phase HPLC and UV detection at 200 nm.
Bromoenol lactone (Cayman, no. 70700) and (S)-5-(4-benzyl-
oxyphenyl)-4-[(7-phenylheptanoyl)amino]pentanoic acid (Sigma-
Aldrich, no. S3319), respectively, were tested as reference in-
hibitors.
2.2.2. Cellular Assay. The ability of compounds 30 and 40 to
inhibit cPLA2R activity in intact cells was determined by mea-
suring phorbolester-induced arachidonic acid release from hu-
man platelets with HPLC/UV detection according to a method
previously described.33 Briefly, platelets were isolated from
human buffy coat by differential centrifugation and washed
with phosphate buffered saline (pH 7.4). After addition of the
stimulant 12-O-tetradecanoylphorbol 13-acetate (TPA) (2 μM)
the cells were incubated for 60 min. Incubation was carried out
in the presence of a dual cyclooxygenase/12-lipoxygenase
inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) to avoid
metabolism of cPLA2R product arachidonic acid via the cy-
clooxygenase-1 and the 12-lipoxygenase pathways. Enzyme
reaction was terminated by addition of a mixture of acetonitrile,
methanol, and 0.1 M aqueous EDTA-Na2 solution, which
contained 3-(4-decyloxyphenyl)propanoic acid as internal stan-
dard and nordihydroguaiaretic acid (NDGA) as oxygen sca-
venger. Inhibition of cPLA2R activity was determined by
comparing the arachidonic acid released by the enzyme in the
absence and presence of a test compound with reversed phase
HPLC and UV detection at 200 nm after centrifugation of
the samples and cleaning up of the supernatant by solid phase
extraction.
2.7. Bioavailability Studies. 2.7.1. Determination of 30 and Its
Metabolite 45 in Mouse Plasma. The pharmacokinetic studies
with compound 30 were performed with C57Bl6 mice. Animals
(n = 3) were dosed perorally once via gavage with 100 mg/kg
30 (8 μL per gram of mouse of a suspension of 12.5 mg of 30 in
1000 μL of a 1% aqueous methylcellulose solution, Methocel
A4C, 408 mPas, Fa. Colorcon). Blood samples were withdrawn
from each animal after 1 and 2 h. All blood samples were
centrifuged at 3000g for 20 min, and the obtained plasma (about
100 μL each) was separated.
To 50 μL of each blood plasma were added 200 μL of
phosphate buffered saline (pH 7.4) and 250 μL of acetonitrile.
After vortexing, the mixtures were allowed to stand for 5 min at
room temperature. Then the samples were centrifuged at 2000g
for 15 min, and the supernatants were subjected to HPLC
analysis. Separation was achieved on a Phenomenex Aqua
C18 analytical column (4.6 mm inside diameter ꢀ 100 mm,
particle size 3 μm) protected with a Phenomenex C18 security
guard column (3 mm inside diameter ꢀ 4 mm). A 100 μL aliquot
of each sample was injected into the HPLC system. The mobile
phase consisted of acetonitrile/water/phosphoric acid (85%)
(70:30:0.1, v/v/v). The flow rate was 0.7 mL/min, and absorption
wavelength was set to 235 nm. The amounts of 30 and its alcohol
metabolite 45 were determined using standard curves of the
compounds.
2.3. Metabolic Stability. Metabolism of the target compounds
was studied as described previously using rat liver microsomes.33

Article
Control experiments with indomethacin had been performed
before in the same way dosing the mice (n = 4) perorally with
10 mg/kg (8 μL per gram of mouse of a suspension of 2.5 mg of
indomethacin in 2000 μL of a 1% aqueous methylcellulose
solution).28
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8307
samples of mice perorally dosed with 100 mg/kg 30; method for
determination of 43 and 44 in the intestine of mice treated with
43; HPLC-UV chromatograms of intestinal samples of a
mouse perorally dosed with 100 mg/kg of 43. This material is
available free of charge via the Internet at http://pubs.acs.org.
2.7.2. Determination of 30 and 45 and Their Glucuronides in the
Bile of the Mice. In the pharmacokinetic studies with 30, the
animals were killed after the final blood withdrawal (2 h after po
administration), and bile of each animal was collected.
2.7.2.1. Direct Measurement of 30 and 45 in Bile. To 2 μL of
the bile samples were added 98 μL of phosphate buffered saline
(pH 7.4) and 100 μL of acetonitrile. After being vortexed, the
mixtures were allowed to stand for 5 min at room temperature.
Then the samples were centrifuged at 2000g for 15 min and the
supernatants were subjected to HPLC analysis as described
above (see section 2.7.1.). The injection volume was 100 μL.
2.7.2.2. Measurement of 30 and 45 in Bile after Treatment with
Glucuronidase. To 2 μL of the bile samples were added 178 μL
of phosphate buffered saline (pH 5.5) and 20 μL of a solution of
β-glucuronidase type HP-2 from Helix pomatia (aqueous solu-
tion, g100000 units/mL, Sigma-Aldrich, no. G7017). The mix-
tures were heated in a water bath at 60 °C for 1 h. After addition
of 200 μL of acetonitrile, the samples were allowed to stand for
5 min at room temperature. Then they were centrifuged at 2000g
for 15 min and the supernatants were subjected to HPLC
analysis as described above (see section 2.7.1.). The injection
volume was 200 μL.
2.7.2.3. Measurement of the Glucuronides of 30 and 45 in Bile
with HPLC/MS. The obtained solutions were also investigated
by HPLC/high resolution mass spectrometry according to the
procedure described previously.28 The samples, which had not
been treated with glucuronidase, contained glucuronides of 30
and 45, as confirmed by their high resolution mass peaks of m/z
724.3004 and m/z 726.3146 ([M - H]-), respectively. In the
samples treated with glucuronidase increased concentrations of
30 (m/z 548.2685, [M - H]-) and 45 (m/z 550.2833, [M - H]þ)
occurred, while their glucuronides could not be detected any-
more.
2.7.3. Determination of 30 and 45 in the Intestine of the Mice.
After killing of the mice (2 h after po administration of 30), the
whole intestines of the animals were withdrawn. The weights of
the intestines were 2.37, 2.22, and 2.12 g. Each intestine was
crushed with a scalpel and transferred into a centrifuge tube with
phosphate buffered saline (pH 7.4) (2.5 mL). After homogeniza-
tion of the sample with an Ultra Turrax mixer at 0 °C, acetoni-
trile (3 mL) was added. The homogenate was vortexed and
centrifuged at 2000g and 4 °C for 5 min. The supernatant was
transferred into a 20 mL volumetric flask. The Ultra Turrax
mixer was washed with phosphate buffered saline (pH 7.4)/
acetonitrile, 1:1 (v/v) (4 mL). The washing liquid was given to
the centrifugation pellet. After being vortexed, the mixture was
centrifuged at 2000g and 4 °C for 5 min and the supernatant was
also transferred into a 20 mL volumetric flask. This washing
process was repeated twice. Then the volumetric flask was filled
up with phosphate buffered saline (pH 7.4)/acetonitrile, 1:1 (v/
v). An aliquot of the extract was centrifuged at 2000g and room
temperature for 5 min, filtered through a little cotton pad, and
subjected to HPLC analysis as described above (see section
2.7.1). The injection volume was 20 μL.
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Supporting Information Available: Synthesis and analytical
data of the target compounds 11-14, 20-23, 31, 32, 41, 42, and
their intermediates; synthesis and analytical data of the alcohol
metabolite 45; HPLC-UV chromatograms of bile and intestine

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