1-(2-Carboxyindol-5-yloxy)propan-2-ones as inhibitors of human cytosolic phospholipase A2α: Synthesis, biological activity, metabolic stability, and solubility

Article abstract of DOI:10.1016/j.bmc.2008.02.019

Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent inhibitors of cytosolic phospholipase A₂α (cPLA₂α) isolated from human platelets. In continuation of our attempts to develop novel cPLA₂α inhibitors, a series of structurally related indole-2-carboxylic acids containing 3-aryloxy-2-oxopropoxy residues in position 5 were synthesized and tested for their cPLA₂α-inhibitory potency. Furthermore, the thermodynamic solubility of these compounds and their metabolic stability against rat liver microsomes were evaluated.

Full text of DOI:10.1016/j.bmc.2008.02.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3489–3500
1-(2-Carboxyindol-5-yloxy)propan-2-ones as inhibitors of
human cytosolic phospholipase A2a: Synthesis, biological
activity, metabolic stability, and solubility
Alexandra Fritsche, Alwine Schulze Elfringhoff, Jorg Fabian and Matthias Lehr*
¨
Institute of Pharmaceutical and Medicinal Chemistry, University of Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany
Received 31 October 2007; revised 29 January 2008; accepted 8 February 2008
Available online 13 February 2008
Abstract—Indole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent inhib-
itors of cytosolic phospholipase A2a (cPLA2a) isolated from human platelets. In continuation of our attempts to develop novel
cPLA2a inhibitors, a series of structurally related indole-2-carboxylic acids containing 3-aryloxy-2-oxopropoxy residues in position
5 were synthesized and tested for their cPLA2a-inhibitory potency. Furthermore, the thermodynamic solubility of these compounds
and their metabolic stability against rat liver microsomes were evaluated.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
to be very potent inhibitors of cPLA2a later on.6 An
important part of the pharmacophore of these sub-
stances is the activated electrophilic ketone moiety,
which is supposed to form covalent-binding interactions
with a serine of the active site of cPLA2a. Recently, we
have published effective cPLA2a inhibitors like com-
pound 3, structurally related to 2.7,8 In these derivatives,
a 5-carboxyindol-1-yl-substituent is tethered to an aryl-
oxypropan-2-one scaffold.
Cytosolic phospholipase A2a (cPLA2a) is an esterase
that selectively cleaves the sn-2 position of arachido-
noyl-glycerophospholipids of biomembranes to generate
free arachidonic acid and lysophospholipids.1 Arachi-
donic acid in turn is metabolized to a variety of
inflammatory mediators including prostaglandins and
leukotrienes. When the phospholipid substrate of
cPLA2a is a phosphatidylcholine with an alkyl ether
moiety at the sn-1 position, the lysophospoholipid pro-
duced is the immediate precursor of platelet activating
factor (PAF), another mediator of inflammation.2 Thus,
the inhibition of cPLA2a would lead to the blockade of
the cellular production of these proinflammatory lipid
mediators. Therefore, this enzyme is considered to be
an attractive target for the design of new anti-inflamma-
tory drugs.3–5
Here we describe the synthesis of novel cPLA2a inhibitors
with electrophilic activated ketone groups such as 11, in
which position 1 of 3-aryloxypropan-2-one is bound via
an oxygen to the 5 position of indole-2-carboxylic acid.
Because it is known that activated ketones can be unstable
towards keto-reduction,9–11 and because reduction of the
keto groups of 2 and 3 to alcohol functionalities leads to
inactive compounds,6,11,12 we also tested the metabolic
stability of all newly synthesized target compounds in a
test system applying rat liver microsomes. Furthermore,
we also measured the solubility of the test compounds,
in view of the fact that solubility is one important con-
straint for oral bioavailability.
First-generation cPLA2a inhibitors were analogues of
arachidonic acid with the COOH group replaced by
COCF3 (AACOCF3, 1) (Fig. 1) or CH2PO(OCH3)F
(MAFP).3 Besides other agents, several 1,3-diaryloxy-
propan-2-ones, such as compound 2, have been found
2. Chemistry
Keywords: Cytosolic phospholipase A2a; Inhibitors; Indole-2-carbox-
ylic acid; Activated ketone; Metabolic stability; Aqueous solubility.
*
Indole-2-carboxylic acid derivative 11 was synthesized
by the route outlined in Scheme 1. 5-Benzyloxyindole-
Corresponding author. Tel.: +49 251 83 33331; fax: +49 251 83
32144; e-mail: lehrm@uni-muenster.de
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.019

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A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
Figure 1.
Scheme 1. Reagents and conditions: (a) N,N-Dimethylformamide di-tert-butyl acetal, benzene, reflux; (b) di-tert-butyl dicarbonate, 4-dimethyl-
aminopyridine, THF, rt; (c) H2, Pd/C, triethylamine, THF, rt; (d) epichlorohydrin, tetrabutylammonium bromide, KHCO3 or KOH, acetonitrile, rt;
(e) 4-octylphenol, 4-dimethylaminopyridine, 40 ꢁC or 80–100 ꢁC; (f) acetic anhydride, DMSO, rt; (g) trifluoroacetic acid, CH2Cl2, rt.
2-carboxylic acid (4) was converted to its tert-butylester
(5) by N,N-dimethylformamide di-tert-butyl acetal.13
Treatment of 5 with di-tert-butyl dicarbonate in the
presence of 4-dimethylaminopyridine led to the indole-
1,2-dicarboxylic acid di-tert-butylester 6. Hydrogenolyt-
ic cleavage of the benzyl ether of 6 yielded the phenol 7.
This compound was further reacted with epichlorohy-
drin in the presence of tetrabutylammonium bromide
and KHCO3 to afford the epoxide intermediate 8. Ring
opening of 8 with 4-octylphenol was achieved without
solvent in the presence of catalytic amounts of 4-dimeth-
ylaminopyridine. Oxidation of the resulting alcohol 9 to

A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
3491
ketone 10 was carried out with acetic anhydride/DMSO.
Concomitant deprotection of the indole nitrogen and
cleavage of indole-2-carboxylic acid tert-butyl ester by
trifluoroacetic anhydride yielded target compound 11.
with 1-bromopropane in the presence of tert-BuOK in
DMSO provided 18. After hydrogenolytic debenzyla-
tion, solvent-free reaction of intermediate 19 with
2-(4-octylphenoxy)methyloxirane in the presence of
4-dimethylaminopyridine led to the hydroxy compound
20. Subsequent oxidation of the alcohol with acetic
anhydride/DMSO and cleavage of the tert-butyl ester
by trifluoroacetic acid gave the desired acid 22.
The synthesis of the N-methylated indole-2-carboxylic
acid 17 is also shown in Scheme 1. 5-Benzyloxyindole-
2-carboxylic acid tert-butylester (5) was methylated in
position 1 with methyl p-toluenesulfonate to give 12.
After deprotection by hydrogenolysis, the phenolic hy-
droxy group was reacted with epichlorohydrin applying
KOH as base. Ring opening of the epoxide, oxidation of
the resulting hydroxy group and final cleavage of the
tert-butyl ester were performed in the same way as
described for the synthesis of 11.
The 1-functionalized target compounds 31–36 were pre-
pared starting from the appropriately substituted 5-ben-
zyloxyindolecarboxylates 23–29 (Scheme 3). The latter
compounds were obtained by reaction of 5 with 1-bro-
mohexane, benzylbromide, x-bromoalkanoic acid tert-
butylesters, 4-bromomethylbenzoic acid tert-butylester
and 2-chloro-N,N-dimethylethylamine, respectively.
For synthesis of 30 and 31, the route used for the prep-
aration of 22 was applied. Compounds 32–36 were
The preparation of N-propyl indole derivative 22 also
started from 5 (Scheme 2). Alkylation of this compound
Scheme 2. Reagents and conditions: (a) 1-Bromopropane, tert-BuOK, DMSO, 110 ꢁC; (b) H2, Pd/C, triethylamine, THF, rt; (c) 2-(4-
octylphenoxymethyl)oxirane, 4-dimethylaminopyridine, 80–100 ꢁC; (d) acetic anhydride, DMSO, rt; (e) trifluoroacetic acid, CH2Cl2, rt.
Scheme 3. Reagent and condition: (a) R–Br or R–Cl, tert-BuOK, DMSO, 110 ꢁC.

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A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
Scheme 4. Reagent and condition: (a) Trifluoroacetic acid, CH2Cl2, rt.
obtained in the manner as described above for the syn-
thesis of 17.
the mixtures were cooled and centrifuged. Aliquots of
the supernatants were subjected to reversed phase
HPLC/UV for determination of the extent of metabo-
lism. The structures of the main metabolites were con-
firmed with reference substances and LC/MS
investigations, respectively.
The indole-2-carboxylic acids 37 and 38 bearing hydro-
xy groups in the side chain at position 5 of the indole
(Scheme 4) were prepared by cleavage of the corre-
sponding tert-butyl esters with trifluoroacetic acid.
3.3. Water solubility
3. Evaluation of the target compounds
Water solubility was determined experimentally accord-
ing to a published procedure.16 Sodium phosphate buf-
fer (pH 7.4) was added to a test compound, and the
suspension obtained was equilibrated by sonication
and shaking at room temperature, followed by centrifu-
gation. An aliquot of the supernatant was diluted with
acetonitrile, and the concentration of the test compound
was determined by reversed phase HPLC/UV using a
regression curve.
3.1. Inhibition of cPLA2a
All newly synthesized indole derivatives were evaluated
in an assay applying cPLA2a isolated from human plate-
lets.12 Like other lipases, cPLA2a has evolved to work
optimally at a lipid-water interface. For this reason,
sonicated covesicles consisting of 1-stearoyl-2-arachido-
noyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-
sn-glycerol were used as enzyme substrate. 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 merely
by 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
inhibitor in the interface has to be kept low.14 Thus,
the highest concentration of test compounds evaluated
was 10 lM, whereas the concentration of the vesicle-
forming lipids was 300 lM. The enzyme activity was
determined by measuring the enzyme product arachi-
donic acid formed after an incubation time of 60 min
with reversed phase HPLC and UV-detection at 200 nm.
4. Results and discussion
Our investigations started from the N-unsubstituted in-
dole-2-carboxylic acid 11. With an IC50 of 3.3 lM this
compound inhibited isolated cPLA2a with similar po-
tency as the known cPLA2a inhibitor AACOCF3 (1)
(Table 1). In comparison with the structurally related
indolylpropan-2-one 3, however, 11 was about 100-fold
less active. Introduction of small lipophilic substituents
at the indole 1 position slightly increased activity,
whereas bigger lipophilic groups were not tolerated well
by the enzyme. Thus, IC50-values evaluated for the
methyl (17) and propyl (22) derivatives were about
1–2 lM and for the hexyl (30) and benzyl (31)
compounds about 10 lM. One reason for the reduced
activity of 30 and 31 could be that their terminal lipo-
philic hydrocarbon groups come close to a hydrophilic
domain of the enzyme resulting in negative interactions.
To obtain evidence for this possibility and to increase
polarity and solubility of the compounds, several
x-carboxyalkyl-chains and a 4-carboxybenzyl-substitu-
ent, respectively, were introduced at the indole
nitrogen. With IC50-values of 0.3–0.6 lM the carboxy-
Several compounds were also tested in cellular situation.
In this assay cPLA2a of intact human platelets was
activated with the phorbol ester 12-O-tetradecanoyl-
phorbol-13-acetate (TPA).7 The cPLA2a-catalyzed
liberation of arachidonic acid from membrane phospho-
lipids was measured after 60 min with HPLC and UV-
detection at 200 nm. To avoid the metabolism of arachi-
donic acid via the cyclooxygenase-1 and the 12-lipoxyge-
nase pathways, the dual cyclooxygenase/12-lipoxygenase
inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) was
added to the platelets in these experiments.
methyl,
3-carboxypropyl-
and
5-carboxypentyl-
substituted indoles 32–34 showed significantly higher
cPLA2a inhibition than the starting compound 11,
probably because of additional interactions of the new
polar moieties with the enzyme. The derivative with a
more rigid 4-carboxybenzyl-substituent (35) (IC50:
1.5 lM) was about threefold less active than the x-car-
boxyalkyl-derivatives 32–34. Introduction of a basic
dimethylaminoethyl-moiety at position 1 (36) even led
to a compound with an IC50 greater than 10 lM.
3.2. Metabolic stability
Test compounds were incubated with rat liver micro-
somes under aerobic conditions in the absence and pres-
ence of the cofactor NADPH in a similar manner as
described previously.11,15 The metabolic reactions were
terminated by addition of acetonitrile after 30 min. Then

A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
3493
Table 1. cPLA2a-inhibitory potency, thermodynamic solubility and metabolic stability of indole-2-carboxylic acid derivatives
O
O
O
COOH
N
C8H17
R
Inhibition of cPLA2a IC50 (lM) Metabolic stabilityb (%) Thermodynamic solubilityc (lg/mL)
a
Compound
R
11
17
H
CH3
3.3
1.2
2.1
> 10d
10
<5
<5
<5
<5
<5
18
8.4 2.0
<1
<1
22
30
C3H7
C6H13
Benzyl
<1
<1
31
32
CH2COOH
(CH2)3COOH
(CH2)5COOH
0.3
0.4
0.6
8.1 2.7
30 2.5
17 0.6
13 3.2
46 9.1
n.t.f
33
34
<5
<5
15
35
36
CH2phenyl(4-COOH) 1.5
(CH2)2N(CH3)2
>10e
2.3
9
1 (AACOCF3)
3
n.t.f
65
0.035
5
<1
a Values are means of at least two independent determinations, errors are within 20%.
b Percentage of parent compound remaining after metabolism by rat liver microsomes; values are the mean of at least two independent determi-
nations; in case of 3: means standard deviation, n = 5.
c Mean standard deviation, n = 3, in case of values <1 lg/mL: n = 2.
d 42% inhibition at 10 lM.
e 32% inhibition at 10 lM.
f n.t., not tested.
Structure–activity relationship studies have revealed
that important pharmacophoric elements of the cPLA2a
inhibitors 2 and 3 are their electrophilic ketone moieties.
Reduction of the ketones to the corresponding alcohols
led to a drastic loss of activity.6,7,11 The same effect was
observed for the novel inhibitor class. Although the in-
doles 17 and 32, which were examined as representa-
tives, showed IC50 values against cPLA2a of 1.2 lM
and 0.3 lM, respectively, their alcohol derivatives 37
and 38 were inactive at the highest test concentration
of 10 lM (Table 2).
the phorbol ester TPA was inhibited by 17 and 32–34
to about the same extent as the activity of the isolated
enzyme (IC50 range 0.3–1.3 lM) (Table 2). As it could
be expected, the alcohols 37 and 38 were lacking activity
at 10 lM also in the cellular assay.
Because it is known that activated ketones may be met-
abolically unstable,9–11 the metabolism of the new com-
pounds by rat liver microsomes was also investigated.
Figure 2 shows typical chromatograms obtained after
incubation of the substances (here compound 32) with
microsomes in the absence and presence of NADPH.
Metabolism of the new inhibitors and reference com-
pound 3 was not measured after ether extraction with
normal phase HPLC as published previously for 3, but
The most active ketones (17, 32–34) and the inactive
alcohols 37 and 38 were also tested in cellular situation.
In intact human platelets, cPLA2a activity triggered by
Table 2. Inhibition of cPLA2a activity in the isolated enzyme assay and in the cellular assay
X
Y
O
O
COOH
N
C8H17
R
Compound
R
X,Y
Vesicle assay with the
a
isolated enzyme IC50 (lM)
Cellular assay with intact human
a
platelets (stimulant TPA) IC50 (lM)
17
32
33
34
37
38
3
CH3
@O
@O
@O
@O
H, OH
H, OH
1.2
0.3
0.6
1.3
CH2COOH
(CH2)3COOH
(CH2)5COOH
CH3
0.4
0.3
0.6
0.5
n.a.b
n.a.b
0.035
n.a.b
n.a.b
0.018
CH2COOH
a Values are the means of at least two independent determinations; errors are within 20%.
b n.a., not active at 10 lM.

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A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
have similar properties. This assumption is confirmed by
the fact that all known cPLA2a inhibitors with pro-
nounced potency bear larger lipophilic residues,6,7,17–21
which lead to a high total lipophilicity of the com-
pounds. 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 solubility of all new compounds in
phosphate buffer (pH 7.4) under thermodynamic condi-
tions. The amount of test compound dissolved was
determined by reversed phase HPLC. Like in the metab-
olism experiments (Fig. 2A), the ketone peaks of the
chromatograms were used for quantification.
The new 1-unsubtituted indole-2-carboxylic acid 11 pos-
sesses a significant higher water solubility than reference
compound 3 (8.4 lg/mL vs. <1 lg/mL) (Table 1). Intro-
duction of lipophilic methyl, propyl, hexyl or benzyl
substituents at the indole-1-position (17, 22, 30, 31) de-
creased the amount of dissolvable substance to less than
1 lg/mL. In contrast, with a value of 8.1 lg/mL the
compound with carboxymethyl residue at position 1
(32) showed similar solubility as the unsubstituted deriv-
ative 11. Replacement of carboxymethyl by the larger
carboxylic acid residues carboxypropyl (33), carboxyl-
pentyl (34) and 4-carboxybenzyl (35) further increased
experimental solubility about 1.5- to 4-fold. Introduc-
tion of a basic dimethylaminoethyl moiety at indole-1-
position finally led to a compound (36), which was even
more soluble (46 lg/mL) than the best soluble dicarbox-
ylic acid of the synthesized series (33, 30 lg/mL).
Figure 2. Reversed phase HPLC chromatograms of 32 following
incubation with rat liver microsomes in the absence (A) and in the
presence (B) of NADPH. 32*: hydrate form of 32; 38: corresponding
alcohol of 32 (main metabolite). Column: Phenomenex Aqua 3 l C18
column (4.6 mm inside diameter · 75 mm); solvent: acetonitrile/water/
phosphoric acid (85%) (70:30:0.1, v/v/v); flow: 0.7 ml/min; UV-detec-
tion: 240 nm.
Solubility guidelines for drugs under development are
given by Lipinski and coworkers.22 According to those,
compounds with mid-range permeability and average
potency should possess a minimum thermodynamic sol-
ubility of 50 lg/mL. With solubilities of 30 lg/mL and
46 lg/mL, respectively, compounds 33 and 36 come
closest to this limit.
directly by reversed phase HPLC after protein precipita-
tion with acetonitrile. Chromatography under aqueous
conditions led to the formation of second peaks, which
were identified as the hydrate forms of the electrophilic
ketones by LC–MS.11 However, since the ketone peaks
of the compounds in the chromatograms increased line-
arly with the concentration of the substances in the con-
centration range evaluated, the metabolism could be
measured by comparing only the ketone peaks formed
in the presence and absence of NADPH. The ratio of
metabolism of reference compound 3 evaluated with this
reversed phase method (65%) was nearly identical with
the value obtained with the normal phase HPLC sys-
tem.11 All new substances were significantly less stable
against keto reduction than 3. After incubation with
rat liver microsomes in the presence of NADPH, only
about 10–20% of 32, 35 and 36 and less than 5% of
the other compounds of this series could be detected
in comparison with reference incubations in the absence
of NADPH (Table 1). In all cases the corresponding
inactive alcohols were the main metabolites as shown
by LC–MS experiments.
In conclusion, we have described a new series of cPLA2a
inhibitors with activated ketone groups. Although the
inhibitory activity against cPLA2a and the stability
against metabolic keto reduction of these compounds
were lower than that of the structurally related reference
compound 3 published by us recently, several of the new
compounds possessed a significantly increased aqueous
solubility in comparison with 3. The aim of further stud-
ies will be to develop derivatives of these compounds,
concomitantly possessing high cPLA2a-inhibitory po-
tency, adequate water solubility and good stability
against metabolic reduction of the pharmacophoric ke-
tone group.
5. Experimental
5.1. Chemistry
Due to the two long acyl chains, the phospholipid sub-
strates of cPLA2a possess a substantial lipophilicity.
Therefore it can be expected that inhibitors, which shall
bind competitively to the active site of the enzyme, must
5.1.1. General. Column chromatography was performed
on Merck silica gel 60, 230–400 mesh or 70–230 mesh.
Melting points were determined on a Buchi B-540 appa-
¨

A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
3495
1
ratus and are uncorrected. H NMR spectra were re-
1.63 (s, 9H), 6.91–6.93 (m, 2H), 6.96 (d, 1H), 7.86 (d,
corded on a Varian Mercury Plus 400 spectrometer
(400 MHz). Mass spectra were obtained on Finnigan
GCQ and LCQ apparatuses applying electron beam ion-
ization (EI). With the exception of 36, the purity of all
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 lm,
4.0 mm (ID) · 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 sep-
aration was performed using a cyano phase (LiChro-
spher 100 CN, 5 lm, 3.0 mm (ID) · 250 mm, Merck,
Darmstadt, Germany) with an isohexane/THF gradient
containing 0.1% trifluoroacetic acid at a flow rate of
0.5 mL/min. All these compounds showed purities great-
er than 95% in both HPLC systems. The purity of 36
was 97%, evaluated on a reversed phase (Symmetry
C18, 3.5 lm, 4.6 mm (ID) · 75 mm, Waters, Eschborn,
Germany) at 254 nm applying acetonitrile/water with
0.1% phosphoric acid (85%) as mobile phase at a flow
rate of 0.7 mL/min.
1H). MS (EI): m/z (%) 333 (8) [M+], 77 (100).
5.1.5. Di-tert-butyl 5-(oxiran-2-ylmethoxy)indole-1,2-
dicarboxylate (8). A solution of 7 (1.05 g, 3.2 mmol) in
dry acetonitrile (55 mL) was treated under a nitrogen
atmosphere with KHCO3 (593 mg, 6.3 mmol) and tetra-
butylammonium bromide (95 mg, 0.32 mmol). To the
refluxing mixture epichlorohydrin (1.92 mL, 25 mmol)
was added dropwise, and the resulting mixture was
heated under reflux for additional 24 h. Then the solvent
was distilled off and the residue was purified by silica gel
chromatography (hexane/chloroform/ethyl acetate,
2:1:0.1) to give 8 as an oil (1.12 g, 92%). 1H NMR
(CDCl3): d 1.59 (s, 9H), 1.63 (s, 9H), 2.78 (dd, 1H),
2.91 (d, 1H), 3.39–3.41 (m, 1H), 3.98 (dd, 1H), 4.25
(dd, 1H), 6.97 (s, 1H), 7.03–7.05 (m, 2H), 7.93 (d, 1H).
MS (EI): m/z (%) 289 (18) [M+], 233 (100).
5.1.6. Di-tert-butyl 5-[2-hydroxy-3-(4-octylphenoxy)pro-
poxy]indole-1,2-dicarboxylate (9).
A mixture of 8
(450 mg, 1.2 mmol), 4-octylphenol (238 mg, 1.2 mmol)
and 4-dimethylaminopyridine (14 mg, 0.12 mmol) was
stirred at 40 ꢁC for 48 h. The reaction mixture was sub-
jected to silica gel chromatography (hexane/ethyl ace-
5.1.2. tert-Butyl 5-benzyloxyindole-2-carboxylate (5). To
a boiling suspension of 5-benzyloxyindole-5-carboxylic
acid (5.0 g, 18.7 mmol) and dry benzene (100 mL) was
added dropwise under a nitrogen atmosphere over a per-
iod of 45 min a solution of N,N-dimethylformamide di-
tert-butyl acetal (15.2 g, 74.9 mmol) in dry benzene
(50 mL). After being refluxed for further 2 h, the solvent
was removed under reduced pressure and the residue
was purified by chromatography on silica gel (hexane/
ethyl acetate, 20:1) to afford 5 as solid (3.15 g, 52%);
1
tate, 9:1) to yield 9 as an oil (556 mg, 81%). H NMR
(CDCl3): d (ppm) 0.87 (t, 3H), 1.21–1.35 (m, 10H),
1.60 (s, 9H), 1.64 (s, 9H), 1.51–1.60 (m, 2H), 2.54 (t,
2H), 4.13–4.19 (m, 4H), 4.38–4.43 (m, 1H), 6.86 (d,
2H), 6.97 (s, 1H), 7.01–7.03 (m, 2H), 7.09 (d, 2H),
7.93 (d, 1H). MS (EI): m/z (%) 595 (5) [M+], 233 (100).
5.1.7. Di-tert-butyl 5-[3-(4-octylphenoxy)-2-oxoprop-
oxy]indole-1,2-dicarboxylate (10). Acetic anhydride
(1.3 mL, 13 mmol) was added to dry DMSO (7 mL),
and the mixture was stirred under a nitrogen atmo-
sphere at room temperature for 10 min. Then this solu-
tion was added dropwise to a solution of 9 (200 mg,
0.34 mmol) in dry DMSO (7 mL). After being stirred
at room temperature for 12 h, the reaction mixture
was poured into a mixture of 5% aqueous NaHCO3
and brine (1:1), and extracted exhaustively with ethyl
acetate. The combined organic phases were washed with
brine, dried (Na2SO4) and concentrated. The residue
was purified by silica gel chromatography (hexane/ethyl
1
mp 124 ꢁC. H NMR (CDCl3): d 1.61 (s, 9H), 5.10 (s,
2H), 7.04–7.08 (m, 2H), 7.15 (d, 1H), 7.30–7.35 (m,
2H), 7.37–7.42 (m, 2H), 7.47 (d, 2H). MS (EI): m/z
(%) 323 (97) [M+], 267 (100).
5.1.3. Di-tert-butyl 5-benzyloxyindole-1,2-dicarboxylate
(6). To a solution of 5 (1.07 g, 3.3 mmol) in dry THF
(20 mL) was added di-tert-butyl dicarbonate (1.42 g,
6.6 mmol) and 4-dimethylaminopyridine (357 mg,
0.33 mmol). The mixture was stirred at room tempera-
ture for 2 h. Then the solvent was distilled off and the
residue was purified by silica gel chromatography (hex-
ane/ethyl acetate, 20:1) to give 6 as solid (1.31 g, 94%);
1
acetate, 20:1) to give 10 as an oil (141 mg, 71%). H
1
mp 72 ꢁC. H NMR (CDCl3): d 1.59 (s, 9H), 1.63 (s,
NMR (CDCl3): d 0.88 (t, 3H), 1.22–1.35 (m, 10H),
1.59 (s, 9H), 1.64 (s, 9H), 1.52–1.62 (m, 2H), 2.55 (t,
2H), 4.86 (s, 2H), 4.92 (s, 2H), 6.84 (d, 2H), 6.97 (s,
1H), 6.99 (d, 1H), 7.07 (dd, 1H), 7.11 (d, 2H), 7.96 (d,
1H). MS (EI): m/z (%) 593 (1) [M+], 437 (100).
9H), 5.10 (s, 2H), 6.97 (s, 1H), 7.07–7.11 (m, 2H), 7.33
(d, 1H), 7.37–7.41 (m, 2H), 7.45 (d, 2H), 7.93 (d, 1H).
MS (EI): m/z (%) 423 (8) [M+], 267 (100).
5.1.4. Di-tert-butyl 5-hydroxyindole-1,2-dicarboxylate
(7). A solution of 6 (1.69 g, 4.0 mmol) in dry THF
(60 mL) was treated with triethylamine (0.55 mL) and
Pd/C (10% Pd, 550 mg). Then a balloon filled with
hydrogen was attached. After stirring at room tempera-
ture for 1 h, the mixture was filtered and the filtrate
evaporated to dryness. The residue was dissolved in
ethyl acetate and filtered through a membrane filter
(0.2 lm diameter). The filtrate was concentrated and
the residue purified by chromatography on silica gel
(hexane/ethyl acetate, 9:1) to afford 7 as solid (1.12 g,
5.1.8. 5-[3-(4-Octylphenoxy)-2-oxopropoxy]indole-2-car-
boxylic acid (11). To a solution of 10 (112 mg,
0.19 mmol) in dry CH2Cl2 (26 mL) was added trifluoro-
acetic acid (1.28 mL, 15 mmol), and the mixture was
stirred at room temperature for 7 h. Then the reaction
mixture was concentrated to dryness. The residue was
treated twice with hexane and the solvent was evapo-
rated each time. The residue was purified by silica gel
chromatography (1. hexane/ethyl acetate, 1:3; 2. hex-
ane/ethyl acetate/acetic acid, 1:3:0.01) to give 11 as solid
1
1
84%); mp 165 ꢁC. H NMR (CDCl3): d 1.59 (s, 9H),
(69 mg, 84%); mp 167 ꢁC. H NMR (DMSO-d6): d 0.82

3496
A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
(t, 3H), 1.15–1.31 (m, 10H), 1.43–1.52 (m, 2H), 2.54 (m,
2H), 4.96 (s, 2H), 4.97 (s, 2H), 6.82 (d, 2H), 6.93–6.96
(m, 2H), 7.05–7.07 (m, 3H), 7.33 (d, 1H), 11.63 (s,
1H). MS (EI): m/z (%) 437 (51) [M+], 107 (100).
thesis of 10. The crude product was purified by silica gel
chromatography (hexane/ethyl acetate, 20:1) to yield 16
as solid; mp 89 ꢁC. H NMR (CDCl3): d 0.88 (t, 3H),
1.22–1.35 (m, 10H), 1.51–1.61 (m, 2H), 1.61 (s, 9H),
2.55 (t, 2H), 4.03 (s, 3H), 4.88 (s, 2H), 4.91 (s, 2H),
6.84 (d, 2H), 7.02 (d, 1H), 7.08 (dd, 1H), 7.10 (d, 2H),
7.11 (s, 1H), 7.30 (d, 1H). MS (EI): m/z (%) 507 (50)
[M+], 451 (100).
1
5.1.9. tert-Butyl 5-benzyloxy-1-methylindole-2-carboxyl-
ate (12). A mixture of 5 (1.5 g, 4.6 mmol), methyl-p-tol-
uenesulfonate (947 mg, 5.1 mmol), tetrabutylammonium
bromide (176 mg, 0.46 mmol), powdered NaOH (1.22 g,
30 mmol) and dry diethyl ether (100 mL) was stirred at
room temperature for 18 h. After addition of water,
the reaction mixture was extracted exhaustively with
ethyl acetate. The combined organic phases were washed
with 5% aqueous NaHCO3-solution and brine, dried
(Na2SO4) and concentrated. The residue was recrystal-
lized from hexane/ethyl acetate to give 12 (1.3 g, 83%);
5.1.14. 1-Methyl 5-[3-(4-octylphenoxy)-2-oxopropoxy]-
indole-2-carboxylic acid (17). tert-Butyl ester 16 was
hydrolyzed according to the procedure described above
for the preparation of 11. The crude product was puri-
fied by silica gel chromatography (hexane/ethyl acetate,
1
1:3) to yield 17 as solid; mp 148 ꢁC. H-NMR (DMSO-
d6): d 0.81 (t, 3H), 1.17–1.28 (m, 10H), 1.44–1.56 (m,
2H), 2.48 (m, 2H), 3.98 (s, 3H), 4.97 (s, 2H), 4.98 (s,
2H), 6.83 (d, 2H), 6.98–7.02 (m, 2H), 7.04–7.08 (m,
3H), 7.45 (d, 1H). MS (EI): m/z (%) 451 (30) [M+], 107
(100).
1
mp 112 ꢁC. H NMR (CDCl3): d 1.61 (s, 9H), 4.03 (s,
3H), 5.10 (s, 2H), 7.10 (dd, 1H), 7.11–7.13 (m, 2H),
7.28 (d, 1H), 7.33 (d, 1H), 7.38–7.41 (m, 2H), 7.47 (d,
2H). MS (EI): m/z (%) 337 (79) [M+], 190 (100).
5.1.10. tert-Butyl 5-hydroxy-1-methylindole-2-carboxyl-
ate (13). The benzyl ether group of 12 was cleaved
applying the procedure described for the synthesis of
7. Chromatography on silica gel (hexane/ethyl acetate,
4:1) gave 13 as solid (831 mg, 93%); mp 99 ꢁC. 1H
NMR (CDCl3): d 1.60 (s, 9H), 4.01 (s, 3H), 6.94 (dd,
1H), 7.03 (d, 1H), 7.08 (s, 1H), 7.24 (d, 1H). MS (EI):
m/z (%) 247 (38) [M+], 191 (100).
5.1.15. tert-Butyl 5-benzyloxy-1-propylindole-2-carboxyl-
ate (18). A mixture of 5 (675 mg, 2.09 mmol), tert-BuOK
(264 mg, 2.3 mmol), and dry DMSO (7 mL) was heated
at 110 ꢁC for 15 min. After addition of a solution of 1-
bromopropane (257 mg, 2.1 mmol) in dry DMSO
(3 mL), heating was continued at the same temperature
for an additional 30 min. The reaction mixture was al-
lowed to cool, diluted with half-concentrated brine,
and extracted exhaustively with diethyl ether. The com-
bined organic layers were washed with water and brine,
dried (Na2SO4), and concentrated. The residue was puri-
fied by silica gel chromatography (hexane/ethyl acetate,
5.1.11. tert-Butyl 1-methyl-5-(oxiran-2-ylmethoxy)indole-
2-carboxylate (14). A solution of 13 (806 mg, 3.3 mmol)
in dry acetonitrile (57 mL) was treated under a nitrogen
atmosphere with KOH (183 mg, 3.3 mmol) and tetrabu-
tylammonium bromide (98 mg, 0.33 mmol). To the
refluxing mixture epichlorohydrin (1.98 mL, 26 mmol)
was added dropwise, and the resulting mixture was
heated under reflux for additional 2 h. Then the solvent
was distilled off and the residue was purified by silica gel
chromatography (hexane/ethyl acetate, 10:1) to give 14
1
20:1) to give 18 as solid (751 mg, 98%); mp 92 ꢁC. H
NMR (CDCl3): d 0.92 (t, 3H), 1.60 (s, 9H), 1.75–1.86
(m, 2H), 4.47 (t, 2H), 5.09 (s, 2H), 7.08 (dd, 1H),
7.11–7.13 (m, 2H), 7.29 (d, 1H), 7.35–7.41 (m, 3H),
7.47 (d, 2H). MS (EI): m/z (%) = 365 (74) [M+], 218
(100).
1
as solid (695 mg, 70%); mp 120 ꢁC. H NMR (CDCl3):
5.1.16. tert-Butyl 5-hydroxy-1-propylindole-2-carboxyl-
ate (19). The benzyl ether group of 18 (729 mg,
2.0 mmol) was cleaved applying the procedure described
for the synthesis of 7. Chromatography on silica gel
(hexane/ethyl acetate, 9:1) gave 19 as solid (474 mg,
d 1.60 (s, 9H), 2.79 (dd, 1H), 2.92 (d, 1H), 3.37–3.42
(m, 1H), 4.01 (dd, 1H), 4.02 (s, 3H), 4.24 (dd, 1H),
7.03–7.07 (m, 2H), 7.12 (s, 1H), 7.27 (d, 1H). MS (EI):
m/z (%) 303 (91) [M+], 247 (100).
1
87%); mp 101 ꢁC. H NMR (CDCl3): d 0.91 (t, 3H),
5.1.12. tert-Butyl 5-[2-hydroxy-3-(4-octylphenoxy)pro-
poxy]-1-methylindole-2-carboxylate (15). A mixture of
14 (180 mg, 0.59 mmol), 4-octylphenol (124 mg,
0.59 mmol) and 4-dimethylaminopyridine (7 mg,
0.06 mmol) was stirred at 100 ꢁC for 2.5 h. The reaction
mixture was subjected to silica gel chromatography
(hexane/ethyl acetate, 9:1) to yield 15 as solid (107 mg,
1.60 (s, 9H), 1.77–1.84 (m, 2H), 4.46 (t, 2H), 6.92 (dd,
1H), 7.03 (d, 1H), 7.08 (s, 1H), 7.25 (d, 1H). MS (EI):
m/z (%) 275 (42) [M+], 190 (100).
5.1.17. tert-Butyl 5-[2-hydroxy-3-(4-octylphenoxy)pro-
poxy]-1-propylindole-2-carboxylate (20). A mixture of
19 (424 mg, 1.5 mmol), 2-(4-octylphenoxymethyl)oxi-
rane7 (483 mg, 1.9 mmol) and 4-dimethylaminopyridine
(15 mg, 0.12 mmol) was stirred at 100 ꢁC for 12 h. The
reaction mixture was purified by silica gel chromatogra-
phy (hexane/ethyl acetate/methanol, 100:2:5) followed
by reversed phase chromatography on RP18 phase elut-
ing with acetonitrile to yield 20 as an oil (554 mg, 67%).
1H NMR (CDCl3): d 0.86–0.93 (m, 6H), 1.23–1.33 (m,
10H), 1.53–1.62 (m, 2H), 1.61 (s, 9H), 1.78–1.85 (m,
2H), 2.54 (t, 2H), 4.10–4.22 (m, 4H), 4.39–4.43 (m,
1H), 4.47 (t, 2H), 6.86 (d, 2H), 7.01 (dd, 1H), 7.08–
1
37%); mp 113 ꢁC. H NMR (CDCl3): d 0.88 (t, 3H),
1.22–1.36 (m, 10H), 1.53–1.62 (m, 2H), 1.61 (s, 9H),
2.54 (t, 2H), 2.62–2.67 (s, br), 4.03 (s, 3H), 4.12–4.22
(m, 4H), 4.38–4.45 (m, 1H), 6.86 (d, 2H), 7.03 (dd,
1H), 7.09–7.13 (m, 4H), 7.27 (d, 1H). MS (EI): m/z
(%) 509 (100) [M+].
5.1.13. tert-Butyl 1-methyl-5-[3-(4-octylphenoxy)-2-oxo-
propoxy]indole-2-carboxylate (16). Compound 15 was
oxidized in a similar way as described above for the syn-

A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
3497
7.14 (m, 4H), 7.28 (d, 1H). MS (EI): m/z (%) 537 (12)
9H), 1.60 (s, 9H), 2.03–2.12 (m, 2H), 2.23 (t, 2H), 4.56
(t, 2H), 5.09 (s, 2H), 7.08 (dd, 1H), 7.11 (d, 1H), 7.12
(s, 1H), 7.30–7.41 (m, 4H), 7.47 (d, 2H). MS (EI): m/z
(%) 465 (71) [M+], 176 (100).
[M+], 219 (100).
5.1.18. tert-Butyl 5-[3-(4-octylphenoxy)-2-oxopropoxy]-1-
propylindole-2-carboxylate (21). Compound 20 (513 mg,
0.96 mmol) was oxidized in a similar way as described
above for the synthesis of 10. The crude product was
purified by silica gel chromatography (hexane/ethyl ace-
tate/methanol, 200:1:5) to yield 20 as an oil (221 mg,
5.1.25. tert-Butyl 5-benzyloxy-1-(5-tert-butoxycarbonyl-
pentyl)indole-2-carboxylate (27). Purification by silica gel
chromatography (hexane/ethyl acetate, 50:1) gave 27 as
1
an oil; H NMR (CDCl3): d 1.30–1.39 (m, 2H), 1.41 (s,
1
43%). H NMR (CDCl3): d 0.86–0.95 (m, 6H), 1.22–
9H), 1.58–1.63 (m, 2H), 1.60 (s, 9H), 1.75–1.84 (m, 2H),
2.19 (t, 2H), 4.50 (t, 2H), 5.09 (s, 2H), 7.07 (dd, 1H),
7.11–7.13 (m, 2H), 7.27 (d, 1H), 7.31–7.42 (m, 3H),
7.47 (d, 2H). MS (EI): m/z (%) 493 (64) [M+], 381 (100).
1.35 (m, 10H), 1.54–1.62 (m, 2H), 1.60 (s, 9H), 1.77–
1.86 (m, 2H), 2.54 (t, 2H), 4.48 (t, 2H), 4.89 (s, 2H),
4.90 (s, 2H), 6.84 (d, 2H), 7.01 (d, 1H), 7.06 (dd, 1H),
7.08–7.13 (m, 3H), 7.31 (d, 1H). MS (EI): m/z (%) 535
(26) [M+], 479 (100).
5.1.26. tert-Butyl 5-benzyloxy-1-(4-tert-butoxycarbonyl-
benzyl)indole-2-carboxylate (28). Purification by silica
gel chromatography (hexane/ethyl acetate, 50:1) gave
5.1.19. 5-[3-(4-Octylphenoxy)-2-oxopropoxy]-1-propylin-
dole-2-carboxylic acid (22). tert-Butyl ester 21 was
hydrolyzed for 3 h according to the procedure described
above for the preparation of 11. The crude product was
purified by silica gel chromatography (chloroform/meth-
anol, 99:1) to yield 22 as solid (84 mg, 53%); mp 136 ꢁC.
1H NMR (CDCl3): d (t, 3H), 0.94 (t, 3H), 1.21–1.36 (m,
10H), 1.53–1.62 (m, 2H), 1.79–1.90 (m, 2H), 2.55 (t, 2H),
4.53 (t, 2H), 4.88 (s, 2H), 4.94 (s, 2H), 6.85 (d, 2H), 7.04
(d, 1H), 7.09–7.15 (m, 3H), 7.35 (d, 1H), 7.36 (s, 1H).
MS (EI): m/z (%) 479 (100) [M+].
1
28 as solid; mp 102 ꢁC. H NMR (CDCl3): d 1.53 (s,
9H), 1.55 (s, 9H), 5.08 (s, 2H), 5.83 (s, 2H), 7.00–7.05
(m, 3H), 7.11–7.17 (m, 2H), 7.21 (s, 1H), 7.29–7.41 (m,
3H), 7.45 (d, 2H), 7.86 (d, 2H). MS (EI): m/z (%) 513
(96) [M+], 457 (100).
5.1.27.
ethyl)indole-2-carboxylate (29).
tert-Butyl
5-benzyloxy-1-(2-dimethylamino-
mixture of
A
5
(880 mg, 2.72 mmol), tert-BuOK (343 mg, 3.0 mmol),
and dry DMF (9 mL) was heated at 110 ꢁC for
15 min. After addition of a solution of 2-chloro-N,N-
dimethylethylamine HCl (431 mg, 3.0 mmol) and tert-
BuOK (378 mg, 3.3 mmol) in dry DMF (5 mL), heating
was continued at the same temperature for 3 h. The
reaction mixture was allowed to cool, diluted with
half-concentrated brine, and extracted exhaustively with
diethyl ether. The combined organic layers were washed
with water and brine, dried (Na2SO4), and concentrated.
The residue was purified by silica gel chromatography
(hexane/ethyl acetate/triethylamine, 40:2:1) to yield 29
5.1.20. Synthesis of 23–28. Compounds 23–28 were pre-
pared from 5 in a manner similar to that described for
the synthesis of 18, utilizing 1-bromohexane, benzyl bro-
mide, the appropriate x-bromoalkanoic acid tert-buty-
lester and 4-bromomethylbenzoic acid tert-butylester,
respectively. Purification was performed by the method
indicated.
5.1.21. tert-Butyl 5-benzyloxy-1-hexylindole-2-carboxyl-
ate (23). Purification by silica gel chromatography (hex-
ane/ethyl acetate, 20:1) gave 23 as an oil; 1H NMR
(CDCl3): d 0.87 (t, 3H), 1.25–1.40 (m, 6H), 1.60 (s, 9H),
1.70–1.80 (m, 2H), 4.50 (t, 2H), 5.09 (s, 2H), 7.10 (dd,
1H), 7.11–7.13 (m, 2H), 7.28 (d, 1H), 7.33–7.42 (m, 3H),
7.47 (d, 2H). MS (EI): m/z (%) 407 (26) [M+], 260 (100).
1
as solid (724 mg, 68%); mp 96 ꢁC. H NMR (CDCl3):
d 1.60 (s, 9H), 2.34 (s, 6H), 2.64 (t, 2H), 4.64 (t, 2H),
5.09 (s, 2H), 7.06–7.12 (m, 3H), 7.30–7.42 (m, 4H),
7.46 (d, 2H). MS (EI): m/z (%) 394 (26) [M+], 58 (100).
5.1.28. Synthesis of compounds 30–36. Compounds 30
and 31 were prepared by the route used for the synthesis
of 22, starting from 23 and 24, respectively. Compounds
32–36 were synthesized from 25 to 29 in the manner as
described for the synthesis 17. The target compounds
were purified by the method indicated.
5.1.22. tert-Butyl 1-benzyl-5-benzyloxyindole-2-carboxyl-
ate (24). Purification by silica gel chromatography (hex-
1
ane/ethyl acetate, 50:1) gave 24 as solid; mp 141 ꢁC. H
NMR (CDCl3): d 1.53 (s, 9H), 5.09 (s, 2H), 5.80 (s, 2H),
7.01–7.08 (m, 3H), 7.16–7.26 (m, 6H), 7.30–7.41 (m, 3H),
7.46 (d, 2H). MS (EI): m/z (%) 413 (69) [M+], 357 (100).
5.1.29. 1-Hexyl-5-[3-(4-octylphenoxy)-2-oxopropoxy]indole-
2-carboxylic acid (30). Purification by silica gel chroma-
tography (chloroform/methanol, 99:1) and recrystalliza-
5.1.23. tert-Butyl 5-benzyloxy-1-(tert-butoxycarbonyl-
methyl)indole-2-carboxylate (25). Purification by silica
gel chromatography (hexane/ethyl acetate, 50:1) gave
1
tion from ethyl acetate gave 30; mp 127 ꢁC. H NMR
(CDCl3): d 0.83–0.91(m, 6H), 1.20–1.40 (m, 16H),
1.53–1.62 (m, 2H), 1.75–1.83 (m, 2H), 2.55 (t, 2H),
4.55 (t, 2H), 4.89 (s, 2H), 4.94 (s, 2H), 6.84 (d, 2H),
7.02 (d, 1H), 7.08–7.16 (m, 3H), 7.35 (d, 1H), 7.37
(s,1H). MS (EI): m/z (%) 521 (100) [M+].
1
25 as solid; mp 143 ꢁC. H NMR (CDCl3): d 1.43 (s,
9H), 1.58 (s, 9H), 5.09 (s, 2H), 5.15 (s, 2H), 7.09 (dd,
1H), 7.14 (d, 1H), 7.17 (s, 1H), 7.18 (d, 1H), 7.30–7.42
(m, 3H), 7.47 (d, 2H). MS (EI): m/z (%) 437 (100) [M+].
5.1.24. tert-Butyl 5-benzyloxy-1-(3-tert-butoxycarbonyl-
propyl)indole-2-carboxylate (26). Purification by silica
gel chromatography (hexane/ethyl acetate, 50:1) gave
26 as solid; mp 110 ꢁC. H NMR (CDCl3): d 1.45 (s,
5.1.30. 1-Benzyl-5-[3-(4-octylphenoxy)-2-oxopropoxy]-
indole-2-carboxylic acid (31). Purification by silica gel
chromatography (hexane/ethyl acetate, 4:1) yielded 31;
mp 137 ꢁC. 1H NMR (CDCl3): d 0.83–0.92 (m, 3H),
1

3498
A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
1.21–1.37 (m, 10H), 1.52–1.63 (m, 2H), 2.55 (t, 2H), 4.86
(s, 2H), 4.87 (s, 2H), 5.82 (s, 2H), 6.84 (d, 2H), 7.01–7.10
(m, 4H), 7.11 (d, 2H), 7.19–7.31 (m, 5H). MS (EI): m/z
(%) 527 (100) [M+].
5.1.37.
1-Carboxymethyl-5-[2-hydroxy-3-(4-octylphe-
noxy)propoxy]indole-2-carboxylic acid (38). Compound
38 was synthesized from the corresponding tert-butyl es-
ter in the manner described for the synthesis of 37. H
1
NMR (CDCl3): d 0.88 (t, 3H), 1.25–1.29 (m, 10H),
1.57 (quint, 2H), 2.54 (t, 2H), 4.13–4.19 (m, 4H), 4.37–
4.43 (m, 1H), 5.24 (s, 2H), 6.86 (d, 2H), 7.08–7.12 (m,
4H), 7.22 (d, 1H), 7.43 (s, 1H). MS (EI): m/z (%) 497
(75) [M+], 191 (100).
5.1.31. 1-(Carboxymethyl)-5-[3-(4-octylphenoxy)-2-oxo-
propoxy]indole-2-carboxylic acid (32). Recrystallization
from hexane/ethyl acetate gave 32; mp 171 ꢁC. 1H
NMR (DMSO-d6): d 0.83 (t, 3H), 1.18–1.31 (m, 10H),
1.45–1.55 (m, 2H), 2.47 (t, 2H), 4.98 (s, 2H), 4.99 (s,
2H), 5.25 (s, 2H), 6.84 (d, 2H), 7.02 (dd, 1H), 7.07 (d,
2H), 7.11 (d, 1H), 7.12 (s, 1H), 7.52 (d, 1H). MS (EI):
m/z (%) 495 (7) [M+], 107 (100).
5.2. Evaluation of the target compounds
5.2.1. General. The HPLC system applied for measuring
enzyme inhibition, metabolic stability and solubility
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.
5.1.32. 1-(Carboxypropyl)-5-[3-(4-octylphenoxy)-2-oxo-
propoxy]indole-2-carboxylic acid (33). Recrystallization
from hexane/ethyl acetate gave 33; mp 160 ꢁC. 1H
NMR (DMSO-d6): d 0.83 (t, 3H), 1.16–1.25 (m, 10H),
1.45–1.56 (m, 2H), 1.84–1.96 (m, 2H), 2.16 (t, 2H),
2.49 (t, 2H), 4.53 (t, 2H), 4.98 (s, 4H), 6.83 (d, 2H),
7.01–7.11 (m, 5H), 7.52 (d, 1H). MS (EI): m/z (%) 523
(93) [M+], 107 (100).
Acetonitrile (HPLC grade) and phosphoric acid (85%)
were obtained from Baker (Deventer, Netherlands).
Dimethylsulfoxide (DMSO) (p.a.) and magnesium chlo-
ride hexahydrate (ultra) were purchased from Fluka
(Buchs, Switzerland). Dihydronicotinamide adenine
dinucleotide phosphate tetrasodium salt (NADPH)
was acquired from Roth (Karlsruhe, Germany).
5,8,11,14-Eicosatetraynoic acid (ETYA) and arachidonyl
trifluoromethyl ketone (AACOCF3) were got from
Biomol (Hamburg, Germany). Phosphate-buffered
saline tablets for the preparation of phosphate-buffered
saline (0.1 M, pH 7.4), 12-O-tetradecanoylphorbol-13-
acetate (TPA) and nordihydroguaiaretic acid (NDGA)
were obtained from Sigma (Deisenhofen, Germany).
Methanol (HPLC grade) was purchased from Acros
Organics (Geel, Belgium). Calcium chloride, EDTA-
Na2 and (NH4)2HPO4 were acquired from Merck
(Darmstadt, Germany). Human buffy coat was a gift
5.1.33. 1-(Carboxypentyl)-5-[3-(4-octylphenoxy)-2-oxo-
propoxy]indole-2-carboxylic acid (34). Recrystallization
from hexane/ethyl acetate gave 34; mp 149 ꢁC. 1H
NMR (DMSO-d6): d 0.83 (t, 3H), 1.15–1.30 (m, 12H),
1.44–1.55 (m, 4H), 1.60–1.69 (m, 2H), 2.15 (t, 2H),
2.50 (t, 2H), 4.51 (t, 2H), 4.98 (s, 4H), 6.83 (d, 2H),
7.02 (dd, 1H), 7.04–7.09 (m, 4H), 7.49 (d, 1H). MS
(EI): m/z (%) 551 (33) [M+], 107 (100).
5.1.34. 1-(4-Carboxybenzyl)-5-[3-(4-octylphenoxy)-2-
oxopropoxy]indole-2-carboxylic acid (35). Recrystalliza-
1
tion from hexane/ethyl acetate gave 35; mp 212 ꢁC. H
NMR (DMSO-d6): d 0.78–0.81 (m, 3H), 1.12–1.25 (m,
10H), 1.40–1.51 (m, 2H), 2.45 (t, 2H), 4.95 (s, 2H),
4.97 (s, 2H), 5.86 (s, 2H), 6.80 (d, 2H), 6.97–7.06 (m,
5H), 7.11 (d, 1H), 7.20 (s, 1H), 7.39 (d, 1H), 7.79 (d,
2H). MS (EI): m/z (%) 571 (5) [M+], 107 (100).
from the German Red Cross (Munster, Germany).
¨
The internal standard 3-(4-decyloxyphenyl)propanoic
acid was synthesized according to
procedure.23
a
published
5.1.35. 1-(2-Dimethylaminoethyl)-5-[3-(4-octylphenoxy)-
2-oxopropoxy]indole-2-carboxylic acid (36). Recrystalli-
zation from hexane/ethyl acetate gave 36; mp 112 ꢁC.
1H NMR (DMSO-d6): d 0.80 (d, 3H), 1.17–1.23 (m,
10H), 1.41–1.51 (m, 2H), 2.46 (t, 2H) 2.85 (s, 6H),
3.37 (t, 2H), 4.81 (t, 2H), 4.96 (s, 2H), 4.99 (s, 2H),
6.81(d, 2H), 6.98–7.12 (m, 4H), 7.16 (s, 1H), 7.56 (d,
1H). MS (EI): m/z (%) 508 (1) [M+], 107 (100).
5.2.2. Inhibition of cPLA2a
5.2.2.1. Assay with the isolated enzyme. The ability of
test compounds to inhibit cPLA2a isolated from human
platelets was performed as previously described.12
Briefly, sonicated covesicles 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 sub-
strate. Enzyme reaction was terminated by addition of a
mixture of acetonitrile, methanol and 0.1 M aqueous
EDTA-Na2 solution, which contained 4-undecyloxyben-
zoic acid as internal standard and NDGA as oxygen
scavenger. cPLA2a activity was determined by measur-
ing 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 cleaning
up the samples by solid-phase extraction.
5.1.36. 5-[2-Hydroxy-3-(4-octylphenoxy)propoxy]-1-methyl-
indole-2-carboxylic acid (37). To a solution of 15 (30 mg,
0.06 mmol) in dry CH2Cl2 (3 mL) was added trifluoro-
acetic acid (0.4 mL, 4.7 mmol), and the mixture was stir-
red at room temperature for 6 h. Then the reaction
mixture was concentrated to dryness. The residue was
treated twice with hexane and the solvent was evapo-
rated each time. The product was precipitated from hex-
1
ane. H NMR (CDCl3): d 0.88 (t, 3H), 1.20–1.35 (m,
5.2.2.2. Cellular assay. Preparation of human blood
platelets: Buffy coat (about 50 mL) was centrifuged in
three 50 ml polypropylene tubes at 2000g for 2 min
and the platelet-rich supernatants were carefully sepa-
rated by aspiration. The obtained platelet-rich fraction
10H), 1.57 (quint, 2H), 2.55 (t, 2H), 4.07 (s, 3H), 4.16–
4.21 (m, 4H), 4.39–4.45 (m, 1H), 6.87 (d, 2H), 7.09–
7.12 (m, 4H), 7.30–7.35 (m. 2H). MS (EI): m/z (%) 453
(50) [M+], 191 (100).

A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
3499
was then centrifuged at 1000g for 15 min. The pellet was
resuspended in a mixture of phosphate-buffered saline
(0.1 M, pH 7.4) and 3.7% aqueous EDTA–Na2 (97:3,
v/v). The volume of this mixture was twice the volume
of the platelet-rich supernatant obtained before. The
suspension was centrifuged at 1000g for 15 min and
the platelets were resuspended in phosphate-buffered
saline (0.1 M, pH 7.4). The final cell concentration was
adjusted to about 108 cells/mL (0.1 mL of this cell sus-
pension diluted with 0.9 mL of phosphate buffered sal-
ine (0.1 M, pH 7.4) gave an absorbance of 0.12 at
800 nm). The platelets were stored at +4 ꢁC until used.
An aliquot of the microsome suspension (198 lL) was
added to a solution of test compound in DMSO (2 lL,
5 mM). The final concentration of the test compound
was 20 lM. After a 5 min preincubation at 37 ꢁC, the
metabolic reactions were started by addition of a solu-
tion of NADPH (12 mM) and MgCl2 (5 mM) in phos-
phate buffered saline (0.1 M, pH 7.4) (300 lL). The
incubation was terminated after 30 min by addition of
acetonitrile (500 lL) and cooling at 0 ꢁC. After further
15 min, the samples were centrifuged at 2000g for
5 min. The supernatants were stored in closed vials at
À20 ꢁC until subjection to HPLC. Reference incuba-
tions without NADPH were performed and analyzed
in the same way. Separation was achieved on a Waters
Symmetry C18 analytical column (3.5 lm, 4.6 mm
(ID) · 75 mm) or a Phenomenex Aqua C18 column
(3 lm, 4.6 mm (ID) · 75 mm). A 30 lL volume of each
sample was injected onto the HPLC system. The temper-
ature of the autosampler and the column oven were
maintained at 20 ꢁC. The mobile phase consisted of mix-
ture of solvent A (acetonitrile/water/phosphoric acid
(85%), 80:20:0.1, v/v/v) and solvent B (acetonitrile/
water/phosphoric acid (85%), 50:50:0.1, v/v/v). The ratio
of the two solvents applied depended on the lipophilicity
of the test compounds. The flow rate was 0.7 mL/min.
The effluents were monitored at 240 nm. The degree of
metabolism of the parent compounds was calculated
from the ratio of the peak areas obtained in the presence
and absence of NADPH.
Incubation procedure: A DMSO solution of ETYA
(1.19 mg/mL) (5 lL; final concentration 10 lM), a
DMSO solution of the test compound (5 lL)-in the case
of the controls DMSO (5 lL) was taken instead-and
platelet suspension (1.59 lL) were preincubated in a
shaking water bath at 37 ꢁC. After 5 min, a CaCl2 solu-
tion (10 mM in 0.8% w/v saline) (0.4 lL) was added and
preincubation was carried on for further 5 min. Then the
cells were stimulated by adding a DMSO solution of 12-
O-tetradecanoylphorbol-13-acetate (TPA) (0.49 mg/mL)
(5 lL; final concentration 2 lM) and the incubation was
continued for 60 min at 37 ꢁC. The enzyme reaction was
terminated by the addition of a mixture of acetonitrile,
methanol and 0.1 M aqueous EDTA–Na2 solution
(16:15:1, v/v/v) (2 mL) containing NDGA as oxygen
scavenger (6 lg/2 mL) and 3-(4-decyloxyphenyl)propa-
noic acid as internal standard (0.8 lg/2 mL). After cool-
ing for 10 min in an ice bath, the samples were
centrifuged at 1000g for 10 min at +4 ꢁC. The superna-
tants were stored at À20 ꢁC.
LC–MS experiments were performed to elucidate the
structure of the respective main metabolites. A 75 lL
(100 lL at MSn investigations) volume of the assay solu-
tion was injected onto a Kromasil 100-5C18 column
(5 lm, 2 mm (ID) · 60 mm, inlet filter 2 lm) at a flow rate
of 0.4 mL/min. The mobile phase consisted of aqueous
ammonium formiate solution (20 mM)/acetonitrile
80:20 (v/v) (A) and aqueous ammonium formiate solution
(20 mM)/acetonitrile 15:85 (v/v) (B). The initial composi-
tion was 65 % A. The gradient was programmed linearly
to 0% A over 12 min and held for 3 min. Finally, the gra-
dient was linearly increased to 65% A over 1 min and held
for 4 min. The HPLC system consisting of a Waters 2690
separation module was coupled on-line to the LCQꢂ ion
trap mass spectrometer from Thermo-Finnigan via an
ESI-interface. The ESI-source voltage was set to 3 kV,
the sheath gas flow was adjusted to 80 arbitrary units
and the auxiliary gas flow-rate was set to 0 arbitrary units.
The temperature of the heated capillary was 200 ꢁC, the
tube lens offset was adjusted to 55 V and the capillary volt-
age set to À6 V. The mass spectra (m/z 250–1200) were re-
corded in negative mode. MSn-mode experiments were
accomplished with an isolation width of m/z 3 and nor-
malized collision energy of 35–40%. For the simultaneous
UV-detection a Waters UV/Vis-detector model 2487 was
used. The detection wavelength was set to 254 nm.
Sample preparation and HPLC-analysis: The superna-
tants were diluted with water (15 mL) and then applied
to a Bakerbond speTM octadecyl reversed phase solid
phase extraction column (500 mg, 19 mL wide mouth,
JT Baker, Deventer, Netherlands), which had succes-
sively been washed before with methanol (10 mL), water
(5 mL) and 0.1% aqueous EDTA-Na2 (5 mL) solution.
After washing the column with water (5 mL), the ad-
sorbed arachidonic acid was eluted with methanol
(3 · 1 mL). The eluate was diluted with water (5 mL)
and then subjected to HPLC. As stationary phase a
RP18 multospher 100 column (3 lm, 3.0 mm
(ID) · 125 mm) with a RP18 multospher 100 guard col-
umn (5 lm, 3.0 mm (ID) · 20 mm) (CS-chromatogra-
phie service, Langerwehe, Germany) was applied. The
mobile phase consisted of acetonitrile/aqueous
(NH4)2HPO4 (10 mM) adjusted to pH 7.4 with phos-
phoric acid (85%) (50:50, v/v). Oven temperature was
set to 30 ꢁC. The flow rate was 0.3 mL/min and the in-
jected sample volume was 0.6 mL. The detection wave-
length was 200 nm. After each run the column was
washed with 1.0 mL methanol. Control incubations in
the absence of the stimulant TPA were carried out in
parallel and used to calculate the specific hydrolysis.
In every mass chromatogram besides the expected
[MÀH]À ions for the ketones, hydrate ions of these sub-
stances with [M+18ÀH]À were observed (Fig. 2). For
the main metabolites, which eluted without exception
between hydrate and the ketone form of the test com-
pound (=parent peak), [M+2ÀH]À ions were detected.
5.2.3. Metabolic stability. Rat-liver microsomes were
prepared by differential centrifugation as described
previously11 and adjusted with phosphate-buffered sal-
ine (0.1 M, pH 7.4) to a protein content of 5.7 mg/mL.

3500
A. Fritsche et al. / Bioorg. Med. Chem. 16 (2008) 3489–3500
5. Lehr, M. Anti-Inflammatory Anti-Allergy Agents Med.
Chem. 2006, 5, 149.
Therefore, it can be assumed that the main metabolites
of the activated ketones are their corresponding alco-
hols. Additionally, in case of 17 and 32 the reference
alcohols 37 and 38 were synthesized and spiked to the
incubation probes leading to an increase of peak inten-
sity of the main metabolites. Furthermore, LC/MS3
investigations were performed for these two alcohols
by fragmenting the particular [M+2ÀH]À ions. After
isolation and collision of these ions, stable daughter ions
appeared at [M+2–44ÀH]À, which could be further frag-
mented to identical product ions in incubation and
spiked reference probes, respectively.
6. 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. J. Med. Chem. 2002, 45,
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´
9. Riendeau, D.; Guay, J.; Weech, P. K.; Laliberte, F.;
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5.2.4. Solubility. Thermodynamic solubility was deter-
mined in a similar way as previously described.16 To
1 mg of the test compound was added phosphate-buf-
fered saline (0.1 M, pH 7.4) (2 mL). The mixture was
sonicated for 10 min (Bandelin Sonorex TK52 sonica-
tion bath) and then shaken for 20 h at room temperature
(20–23 ꢁC). After centrifugation (4000g, 10 min) at room
temperature, about 1 mL of the supernatant was sepa-
rated. To 200 lL of this solution were added acetonitrile
(250 lL) and 0.1 M aqueous phosphoric acid (50 lL).
An aliquot of this solution (5–100 lL) was subjected
to HPLC analysis. Separation was achieved on a Waters
Symmetry C18 analytical column (3.5 lm, 4.6 mm
(ID) · 75 mm) or a Phenomenex Aqua C18 column
(3 lm, 4.6 mm (ID) · 75 mm). The mobile phase con-
sisted of mixture of solvent A (acetonitrile/water/phos-
phoric acid (85%), 80:20:0.1, v/v/v) and solvent B
(acetonitrile/water/phosphoric acid (85%), 50:50:0.1,
v/v/v). The ratio of the two solvents applied depended
on the lipophilicity of the test compounds. The flow rate
was 0.7 mL/min. The effluents were monitored at
240 nm. The amount of test compound dissolved was
calculated using a calibration curve. The calibration
solutions were prepared by mixing obtained standard
stock solutions of the test compounds in DMSO
(2 lL) with phosphate buffered saline (0.1 M, pH 7.4)
(198 lL), acetonitrile (250 lL) and 0.1 M aqueous phos-
phoric acid (50 lL). Before HPLC analysis, the solu-
tions were equilibrated for at least 30 min at 20 ꢁC.
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