Synthesis, biological evaluation, and structure-activity relationships of 3-acylindole-2-carboxylic acids as inhibitors of the cytosolic phospholipase A2

Article abstract of DOI:10.1021/jm960863w

3-Acylindole-2-carboxylic acid derivatives were prepared and evaluated for their ability to inhibit the cytosolic phospholipase A₂ of intact bovine platelets. To define the structural requirements for enzyme inhibition, the carboxylic acid group, the acyl residue, and the moiety in position 1 were systematically modified. Furthermore, different substituents were introduced into the phenyl part of the indole. Replacement of the carboxylic acid group in position 2 of the indole with an acetic or propionic acid substituent led to a decrease of inhibitory potency. Enzyme inhibition was optimal when the acyl residue in position 3 had a length of 12 or more carbons. Conformational restriction of the acyl residue did not influence activity. Introduction of alkyl chains at position 1 of the indole with 8 or more carbons resulted in a loss of activity. However, replacing the Ω-methyl group of such compounds with a carboxylic acid moiety was found to increase inhibitory potency significantly. Among the tested indole derivatives, 1-[2-(4- carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (29b) had the highest potency. With an IC₅₀ of 0.5 μM it was about 20-fold more active than the standard cPLA₂ inhibitor arachidonyl trifiuoromethyl ketane (IC₅₀: 11μM).

Full text of DOI:10.1021/jm960863w

2694
J . Med. Chem. 1997, 40, 2694-2705
Syn th esis, Biologica l Eva lu a tion , a n d Str u ctu r e-Activity Rela tion sh ip s of
3-Acylin d ole-2-ca r boxylic Acid s a s In h ibitor s of th e Cytosolic P h osp h olip a se A2
Matthias Lehr
Institute of Pharmacy and Food Chemistry, Ludwig-Maximilians-University, Sophienstr. 10, D-80333 Munich, Germany
Received December 23, 1996X
3-Acylindole-2-carboxylic acid derivatives were prepared and evaluated for their ability to inhibit
the cytosolic phospholipase A2 of intact bovine platelets. To define the structural requirements
for enzyme inhibition, the carboxylic acid group, the acyl residue, and the moiety in position
1 were systematically modified. Furthermore, different substituents were introduced into the
phenyl part of the indole. Replacement of the carboxylic acid group in position 2 of the indole
with an acetic or propionic acid substituent led to a decrease of inhibitory potency. Enzyme
inhibition was optimal when the acyl residue in position 3 had a length of 12 or more carbons.
Conformational restriction of the acyl residue did not influence activity. Introduction of alkyl
chains at position 1 of the indole with 8 or more carbons resulted in a loss of activity. However,
replacing the ω-methyl group of such compounds with a carboxylic acid moiety was found to
increase inhibitory potency significantly. Among the tested indole derivatives, 1-[2-(4-
carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (29b) had the highest potency.
With an IC50 of 0.5 µM it was about 20-fold more active than the standard cPLA2 inhibitor
arachidonyl trifluoromethyl ketone (IC50: 11 µM).
In tr od u ction
was reported; evidence has since accumulated that this
enzyme controls the biosynthesis of the lipid mediators
mentioned above.19-26 So, for example, this enzyme
selectively cleaves phospholipids containing arachidonic
acid in the sn-2 position contrary to the sPLA2s, which
do not show any degree of selectivity for the hydrolysis
of arachidonic acid at the scissile ester position of the
substrate.27 Moreover, inhibitors of cPLA2 proved to be
active in acute and chronic models of inflammation12
and suppressed arachidonic acid release in arthritic
tissue.13 Such inhibitors might therefore serve as useful
therapeutics for inflammatory diseases and asthma.
Possibly the antiinflammatory and antiasthmatic ef-
fectiveness of potent cPLA2 inhibitors is similar to that
of glucocorticoids, since the latter compounds exert their
therapeutic effect at least in part by preventing activa-
tion of cPLA2 by cytokines.28-31
Phospholipases A2 (PLA2) are a class of enzymes
which catalyze the hydrolysis of membrane glycerophos-
pholipids at the sn-2 position to release fatty acids and
lysophospholipids. When the liberated fatty acid is
arachidonic acid, subsequent metabolism by the cy-
clooxygenase and the 5-lipoxygenase enzymes leads to
the formation of prostaglandins, which play a major part
in the inflammatory response, and leukotrienes, which
play a main role in the pathogenesis of asthma.1,2 The
other products of PLA2 action are cytolytic lysophos-
pholipids. From these the 1-O-alkyl-substituted lyso-
phosphocholines can be further metabolized to the
platelet-activating factor (PAF). The lysophospholipids
and the PAF are also potent mediators of inflamma-
tion.3,4
One problem associated with the in vitro search for
antiinflammatory PLA2 inhibitors is the selection of the
appropriate enzyme, because structurally different PLA2
enzymes are present in the organism. In the synovial
fluid of arthritic joints5 and in the serum of patients
with severe acute pancreatitis,6 septic shock,7 and
multiple injuries,8 high levels of the human nonpancre-
atic secretory PLA2 (type II sPLA2)9 have been observed.
This enzyme was therefore long regarded as the key
control point of the arachidonic acid cascade. Many
potent inhibitors of type II sPLA2 have been developed,
e.g. the recently published indole-3-acetamides and
-glyoxamides10 and the thielocin B3 derivatives.11 How-
ever, inhibitors of this PLA2 were effective only in some
of the standard in vivo models of inflammation12 and
did not reduce arachidonic acid release in arthritic
tissue.13 Furthermore, transgenic mice which overex-
pressed the enzyme did not show signs of a systemic
inflammation.14
Despite the fact that several inhibitors of cPLA2 have
been discovered, e.g. arachidonyl trifluoromethyl ketone
(AACOCF3)32 (1) and (S)-N-hexadecylpyrrolidine-2-car-
boxamide (Wy-48,489)33-35 (2), no compound has been
reported to be undergoing clinical development. More-
over, only a few structure-activity relationship inves-
tigations relating to those enzyme inhibitors have been
published to date.
In 1990, the isolation of a higher-molecular-weight (85
kDa) cytosolic PLA2 (cPLA2)15-18 from several cell types
Recently we reported that 1-methyl-3-octadecanoylin-
dole-2-carboxylic acid (3) is an inhibitor of cPLA2.36 In
this paper we describe the synthesis, biological activity,
X Abstract published in Advance ACS Abstracts, J uly 1, 1997.
S0022-2623(96)00863-1 CCC: $14.00 © 1997 American Chemical Society

Inhibitors of the Cytosolic Phospholipase A2
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2695
Sch em e 1
a
(i) N,N-Dimethyloctadecanamide, POCl3, benzene; (ii) aqueous KOH, EtOH; (iii) methyl p-toluenesulfonate, (C4H9)4N+Br-, powdered
NaOH, Et2O, CH2Cl2.
Sch em e 2
dride and polyphosphoric acid. Hydrolysis of the ester
intermediates gave the desired test compounds 16a -
m .
Similarly, 1-methyl-3-dodecanoylindole-2-carboxylic
acids with different substituents in the phenyl part of
the indole were prepared starting from the appropri-
ately substituted ethyl indole-2-carboxylates 17a -d ,f,g
or ethyl 5-acetoxyindole-2-carboxylate (17e). Methyla-
tion in position 1 with methyl p-toluenesulfonate fol-
lowed by acylation in position 3 with dodecanoic acid
and saponification of the resulting ethyl 3-dodecanoyl-
1-methylindole-2-carboxylates yielded the target com-
pounds 18a -g (Scheme 3).
a
In order to synthesize the pyrrole-2-carboxylic acid
21, ethyl 4,5-dimethylpyrrole-2-carboxylate (19) was
acylated with dodecanoyl chloride by the Friedel-Crafts
reaction to produce 20 (Scheme 3). This compound was
N-methylated and hydrolyzed employing reaction condi-
tions similar those described for the synthesis of 9 and
13.
(i) NaBH4, BF3‚Et2O, THF, methyl acetate; (ii) aqueous KOH,
EtOH; (iii) carboxylic acid, trifluoroacetic anhydride, polyphos-
phoric acid, CH2Cl2.
and structure-activity relationships of a series of
compounds derived from this lead.
Ch em istr y
3-Octadecanoylindole-2-carboxylic acids with different
1-alkyl chains (23a -d ) and 1-(ω-carboxyalkyl) chains
(24a -g), respectively, were obtained starting from ethyl
indole-2-carboxylate (22) (Scheme 4). This was N-
alkylated with the appropriate 1-bromoalkanes or ethyl
ω-bromoalkanoates in DMSO in the presence of potas-
sium tert-butoxide as base. The ester intermediates
were acylated and hydrolyzed in a similar manner as
described for 16a -m .
The synthesis of compounds with m- and p-methyl
cinnamic acid or m- and p-methyl hydrocinnamic acid
substituents (27a ,b, 28a ,b) in position 1 of the indole
started from the common intermediates 26a and 26b,
respectively (Scheme 4). These were hydrolyzed or
hydrogenated and hydrolyzed to afford the desired test
compounds. The intermediates 26a and 26b were
prepared on different routes. While 26a was obtained
by treatment of ethyl indole-2-carboxylate (22) with
ethyl 4-(bromomethyl)cinnamate followed by acylation
with dodecanoic acid/trifluoroacetic anhydride/polyphos-
phoric acid, 26b was prepared the other way around by
first acylating 22 and then coupling the intermediate
acyl ester 25 with ethyl 3-(bromomethyl)cinnamate.
The synthesis of 3-octadecanoylindoles with acetic
acid and propionic acid side chains in position 2 started
from ethyl (indol-2-yl)acetate (6) and methyl 3-(indol-
3-yl)propionate (10), respectively (Scheme 1). The
introduction of the octadecanoyl group in position 3 was
accomplished by reaction with N,N-dimethylocta-
decanamide/POCl3 in benzene. Saponification of the
intermediates 7 and 11 with KOH in ethanol yielded
the desired test compounds 8 and 12. The analogous
N-methylindole derivatives 9 and 13 were afforded by
treating 7 and 11, respectively, with methyl p-toluene-
sulfonate in a phase transfer reaction prior to KOH
hydrolysis.
The 1-methylindole-2-carboxylic acid with an octade-
cyl residue in position 3 (14) was prepared by reducing
the carbonyl function of ethyl 1-methyl-3-octadecanoylin-
dole-2-carboxylate36 (4) upon treatment with NaBH4/
BF3‚Et2O followed by KOH hydrolysis of the resulting
intermediate (Scheme 2).
The 1-methylindole-2-carboxylic acids with varying
3-acyl chains (16a -m ) were synthesized following a
procedure described by Murakami et al. (Scheme 2).37
Ethyl 1-methylindole-2-carboxylate (15) was acylated in
position 3 by reaction with the appropriate carboxylic
acid in CH2Cl2 in the presence of trifluoroacetic anhy-
The indoledicarboxylic acid derivatives 29a ,b and
30a ,b were synthesized starting from 25 and the ethyl
esters of m- or p-(2-bromoethoxy)benzoate and m- or

2696 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Lehr
Sch em e 3
a
(i) Methyl p-toluenesulfonate, (C4H9)4N+Br-, powdered NaOH, Et2O, CH2Cl2; (ii) dodecanoic acid, trifluoroacetic anhydride,
polyphosphoric acid, CH2Cl2; (iii) aqueous KOH, EtOH; (iv) dodecanoyl chloride, AlCl3, nitrobenzene.
Sch em e 4
a
(i) 1-Bromoalkane (23a -d ), ethyl ω-bromoalkanoate (24a -g), ethyl 3- or 4-(bromomethyl)cinnamate (26a ,b), ethyl 3- or 4-(2-
bromoethoxy)benzoate (29a ,b) or ethyl 3- or 4-(2-bromoethoxy)phenylacetate (30a ,b), t-BuOK, DMSO; (ii) octadecanoic or dodecanoic
acid, trifluoroacetic anhydride, polyphosphoric acid, CH2Cl2; (iii) aqueous KOH, EtOH; (iv) H2, Pd/C, THF.
p-(2-bromoethoxy)phenylacetate, respectively, applying
reaction conditions described above (Scheme 4).
cium ionophore A23187-induced arachidonic acid release
from bovine platelets with HPLC/UV detection.38 In our
opinion, such a cellular assay better correlates with the
conditions prevailing in vivo than a test system using
the isolated enzyme. In cells the cPLA2 interacts, once
activated, with the substrate in its physiological form
Biologica l Eva lu a tion
The cPLA2 inhibitory potency biological activity of the
test compounds was evaluated by measuring the cal-

Inhibitors of the Cytosolic Phospholipase A2
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2697
Ta ble 1. In Vitro Inhibition of Platelet cPLA2
by cleaving phospholipids of the intact cell membranes.
On the other hand, in test systems using the isolated
enzyme, the cPLA2 hydrolyzes an artificial substrate
assembly whose physiological relevance cannot be judged.
The activity of cPLA2 in the cells can be regulated by
changes in the intracellular concentration of calcium
and by a protein kinase mediated phosphorylation.26,39
A23187 activates the cPLA2 by causing a rapid increase
of the intracellular calcium concentration.40 Protein
kinases are not involved in this mechanism since the
protein kinase inhibitor staurosporin did not inhibit
A23187-induced arachidonic release (concentration: 1
µM). When using A23187 as stimulant, however, it has
to be considered that the decrease of arachidonic acid
liberation caused by a test compound may not only be
the result of an affection of cPLA2, but can also depend
on a modification of the A23187-induced activation
mechanism.
To rule out this possibility of action, we measured
inhibition of arachidonic acid release by one character-
istic compound after stimulation with 12-O-tetra-
decanoylphorbol-13-acetate (TPA).41 TPA stimulates
the cPLA2 in another way than A23187 via phosphoryl-
ation of the enzyme as a consequence of an activation
of a protein kinase C (PKC) or a mitogen-activated
protein kinase (MAP kinase).42-44
cell lysis at
compd
R1
R2
33 µM (%) IC50 (µM)a
3
4
5
8
9
12
13
CH3 COOH
CH3 COOC2H5
H
H
CH3 CH2COOH
H
CH3 CH2CH2COOH
0
0
0
0
0
0
0
31
0
8
NAb
NAb
NAb
>33c
NAb
NAb
11
COOH
CH2COOH
CH2CH2COOH
AACOCF3 (1)
Wy-48,489 (2)
13
a
IC50 values are the means of at least two independent
b
determinations. Errors are within (20%. NA: not active at the
highest concentration tested (33 µM). c 28% inhibition at 33 µM.
Ta ble 2. In Vitro Inhibition of Platelet cPLA2
Recently we have shown that cPLA2 inhibition is
simulated when a substance leads to lysis of the
platelets.36 Therefore we also determined the cell lytic
potency of each test compound by turbidimetry.
Finally it has to be noted that platelets also contain
type II sPLA2.9 However, this PLA2 is not involved in
the liberation of arachidonic acid.20-22,41,45 So in com-
bination with the determination of the cell lytic potency
and the evaluation of the inhibition of the TPA-induced
arachidonic acid liberation, the cellular test system
applying A23187 as stimulant is specific for the evalu-
ation of cPLA2 inhibitors.
cell lysis at
33 µM (%)
compd
R3
IC50 (µM)a
3
14
C17H35
17H35
C13H27
11H23
C9H19
C7H15
0
0
0
0
0
8
C
NAb
8
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
C
8
18
0
>33c
d
C6H4(2-OC12H25
C6H4(3-OC12H25
C6H4(4-OC12H25
CH2C6H4(2-OC10H21
CH2C6H4(3-OC10H21
CH2C6H4(4-OC10H21
CH2CH2C6H4(2-OC10H21
CH2CH2C6H4(3-OC10H21
CH2CH2C6H4(4-OC10H21
)
)
)
60
26
19
37
21
38
34
14
0
-
7
7
7
10
7
8
)
)
)
Str u ctu r e-Activity Rela tion sh ip s
)
)
)
(A) Va r ia tion of th e Ca r boxylic Acid Gr ou p . In
order to define the structural features necessary for
inhibition of cPLA2, we determined the inhibitory
potency of the ethyl ester (4)36 of the lead compound 3
(IC50: 8 µM) initially. Since 4 was inactive even at the
highest concentration measured (33 µM) (Table 1), it can
be assumed that the carboxylic acid group is part of the
pharmacophore. Next we replaced the carboxylic acid
moiety of 3 and of its desmethyl derivative 5,36 respec-
tively, with the homologous acetic acid and propionic
acid functions. A significant potency reduction was
observed for the 1-methylindoleacetic acid 9 (IC50 >33
µM). The 1-methylindolepropionic acid 13 showed no
inhibition of cPLA2 at 33 µM. The 1-desmethylindole-
2-carboxylic acid 5 and the homologous acetic and
propionic acids 8 and 12 also proved to be inactive. The
1-methyl group is therefore essential for the inhibitory
activity of the indoles 3 and 9.
11
10
a
IC50 values are the means of at least two independent
b
determinations. Errors are within (20%. NA: not active at the
highest concentration tested (33 µM). c 30% inhibition at 33 µM.
d
IC50 could not be determined because of cell lysis even at 10 µM
(15%); 26% inhibition of arachidonic acid release at 3.3 µM.
ylene (14) led to a total loss of activity (Table 2). This
carbonyl function therefore seems to be part of the
pharmacophore.
Replacement of the octadecanoyl residue of 3 with a
tetradecanoyl and a dodecanoyl chain, respectively, did
not change activity: For the compounds 3, 16a , and 16b,
an IC50 of 8 µM was found in each case. However, a
further shortening of the acyl chain led to a decrease of
inhibitory potency: While the 3-decanoyl derivative 16c
was about 2-fold less active (IC50: 18 µM) compared to
3, the IC50 of the 3-octanoyl compound 16d ascended to
a value greater than 33 µM (Table 2). Thus the length
of the acyl chain in position 3 is of importance for
inhibitory activity.
In an attempt to increase potency by conformational
restriction, a phenoxy group was incorporated into the
acyl chain at various positions. Thus the octadecanoyl
moiety of 3 was replaced with benzoyl, phenylacetyl, and
3-phenylpropionyl residues substituted with dodecyloxy
The reference compounds AACOCF3 (1) and Wy-
48,489 (2) inhibited cPLA2 of bovine platelets with an
IC50 of 11 and 13 µM, respectively. With the exception
of the reference substance 1 (31% cell lysis at 33 µM),
none of the compounds shown in Table 1 caused cell
lysis even up to 33 µM.
(B) Va r ia tion of th e Acyl Resid u e. Reduction of
the carbonyl of the 3-octadecanoyl chain of 3 to meth-

2698 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Ta ble 3. In Vitro Inhibition of Platelet cPLA2
Lehr
On the contrary, substitution of the 1-methyl group
of 3 by a hexyl residue (23a ) retained inhibitory potency.
However, lengthening the 1-hexyl residue by one further
carbon atom (23b) decreased activity (IC50 > 10 µM, 34%
inhibition at 10 µM), and elongation by two carbons
afforded a compound (23c) which was inactive at 10 µM.
In our opinion, two explanations are possible for these
findings: First, the 1-alkyl residue of the indole could
be bound in a hydrophobic pocket of the enzyme which
can accommodate carbon chains with up to six atoms;
introduction of 1-alky substituents with seven or more
atoms causes a partial or total loss of activity because
of sterical interference. On the other hand, it could be
possible that lipophilic 1-alkyl residues with more than
six carbons come into close proximity with a hydrophilic
part of the enzyme resulting in inactivity because of
electronic incompatibility. To obtain evidence for one
of these two assumptions, the terminal methyl group
of the 1-octyl residue of the indole 23c should be
replaced with a polar moiety. For this exploration we
chose the carboxyl function because of the results on
structure-activity relationship investigations with 3-(4-
acylpyrrol-2-yl)propionic acids: As with the related
3-acylindole-2-carboxylic acids, such pyrroles lost their
activity when an alkyl substituent was introduced in
position 1 which exceeded a certain chain length (five
carbons).46 Introduction of a polar functional group at
the end of the chain, however, restored inhibitory
potency in this case. The most preferable of the
investigated polar terminal functions (CH2OH, CH2N-
(CH3)2, CON(CH3)2, CN, and COOH) was the carboxylic
acid moiety. Replacement of the ω-methyl group of the
indole 23c with a carboxylic acid function also gave back
activity: With an IC50 of 1.4 µM, the dicarboxylic acid
24a was about 5-fold more active than the lead 3. These
results support the second hypothesis, that substituents
in position 1 of the indole-2-carboxylic acids point to a
hydrophilic part of the enzyme.
Unfortunately, compound 24a was highly cytotoxic:
At 33 µM it caused 69% cell lysis. However, this cell
lytic potency disappeared when the chain length of the
3-acyl residue of 24a was reduced from 18 to 12 carbons.
Since the afforded compound 24e (IC50: 1.6 µM) was
about as active as 24a (Table 4), it can be assumed that
cell lysis, which may be a result of membrane perturba-
tion, and inhibition of calcium ionophore A23187-
induced arachidonic acid release are two distinct prop-
erties of the compounds.
Next we investigated the consequence of varying the
chain length of the octanoic acid substituent of 24e
(Table 4). Elongation of the 1-alkanoic acid up to 11
carbons did not significantly affect activity (24f,g),
whereas shortening the chain length successively re-
duced inhibitory potency: The IC50 of the 1-heptanoic
acid derivative 24d increased to 3 µM, the compound
with a 1-hexanoic acid 24c inhibited the cPLA2 only to
the same extent as 1-methyl derivative 16b (IC50: 8
µM), and the derivative with an acetic acid function in
position 1 was significantly less active (IC50 > 33 µM)
than 16b.
cell lysis at
33 mM (%)
compd
R4
IC50 (µM)a
16b
18a
18b
18c
18d
18e
18f
18g
21
H
5-Cl
5-F
5-NO2
5-OCH3
5-OH
4-Cl
0
14
0
10
0
8
8
11
10
8
10
9
9
0
50
26
0
6-Cl
11
a
IC50 values are the means of at least two independent
determinations. Errors are within (20%.
or decyloxy groups to achieve an overall chain length
of about 18 carbon atoms. The alkoxy groups were
introduced into the ortho, meta, or para position of the
phenyl substituent, respectively. Surprisingly, all com-
pounds (16e-m ) showed about the same activity as the
lead 3. With one exception (16m ) the introduction of a
phenoxy group led to the appearance of cell lysis at 33
µM. Compound 16e even destroyed the cells at 10 µM
(15% lysis); at the concentration of 3.3 µM, at which lysis
could no longer be observed, 16e inhibited the cPLA2
to about the same extent as the other compounds of this
series.
(C) In tr od u ction of Su bstitu en ts in to th e P h en yl
Rin g of th e In d ole. Next we wanted to study the
effect of substituents in the phenyl ring of the 1-methyl-
3-octadecanoylindole-2-carboxylic acid (3). Since some
of the synthesized compounds impaired the determina-
tion of the arachidonic acid in the biological assay by
overlapping with the arachidonic acid peak in the
reversed phase HPLC-chromatogram, we synthesized
analogously substituted derivatives of 3-dodecanoyl-1-
methylindole-2-carboxylic acid (16b). The latter com-
pound was as active as the lead 3, and the phenyl ring
substituted derivatives did not interfere with the arachi-
donic acid during HPLC analysis.
The following test results were obtained (Table 3):
Introduction of a chlorine, fluorine, nitro, methoxy, or
hydroxy substituent into position 5 of the indole ring
system (18a -e) did not change activity significantly.
Furthermore, chlorine substituents in position 4 or 6 of
the indole (18f,g) did not alter enzyme inhibition.
Therefore, the region of the C5 of the indole does not
seem to be essential for activity. This presumption was
supported by the fact that the fragment analogue
3-dodecanoyl-1,4,5-trimethylpyrrole-2-carboxylic acid (21)
was about as active as the indole 16b and its substituted
derivatives, respectively.
The introduction of a chlorine atom in position 4, 5,
or 6 and a nitro group in position 5, respectively, led to
compounds which caused cell lysis at 33 µM.
Finally the rigidity of the 1-alkanoic acid side chain
was increased by introduction of phenyl, ethenylphenyl,
and phenoxy groups, respectively (Table 4). The activity
of the cinnamic acid and hydrocinnamic acid type
derivatives (27a ,b, 28a ,b) (IC50: 4-5 µM) lay in be-
tween the activity of the 1-methyl derivative 16b (IC50:
8 µM) and of the 1-octanoic acid compound 24e (IC50:
(D) Va r ia tion of th e Su bstitu en t in P osition 1 of
th e In d ole. As shown in Table 4, replacement of the
1-methyl group of 3 with a dodecyl chain resulted in a
total loss of inhibitory activity. The 1-dodecyl derivative
23d even stimulated calcium ionophore A23187-induced
arachidonic acid liberation at 33 µM about 1.25-fold.

Inhibitors of the Cytosolic Phospholipase A2
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2699
Ta ble 4. In Vitro Inhibition of Platelet cPLA2
cell lysis at
33 µM (%)
compd
R1
R3
IC50 (µM)a
3
CH3
C6H13
C7H15
C8H17
C17H35
C17H35
C17H35
C17H35
C17H35
C17H35
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
C11H23
0
27
16
0
0
69
0
0
0
0
0
0
0
0
0
0
0
0
0
8
9
23a
23b
23c
23d
24a
24b
24c
24d
24e
24f
24g
27a
27b
28a
28b
29a
29b
30a
30b
>10b
NAc
Actd
1.4
>33e
8
C12H25
(CH2)7COOH
CH2COOH
(CH2)5COOH
(CH2)6COOH
(CH2)7COOH
(CH2)8COOH
(CH2)10COOH
CH2C6H4(3-CHdCHCOOH)(E)
CH2C6H4(4-CHdCHCOOH)(E)
CH2C6H4(3-CH2CH2COOH)
CH2C6H4(4-CH2CH2COOH)
CH2CH2OC6H4(3-COOH)
3.0
1.6
1.6
1.4
5.3
4.1
4.3
4.0
1.9
0.5
1.6
1.6
CH2CH2OC6H4(4-COOH)
CH2CH2OC6H4(3-CH2COOH)
CH2CH2OC6H4(4-CH2COOH)
0
a
b
IC50 values are the means of at least two independent determinations. Errors are within (20%. 34% inhibition at 10 µM. c NA: not
active at 10 µM. Act: 1.25-fold activation of arachidonic acid release. e 39% inhibition at 33 µM.
d
1.6 µM). Replacement of the octanoic acid side chain
in position 1 with a 2-(3-carboxyphenoxy)ethyl (29a ),
2-[3-(carboxymethyl)phenoxy]ethyl (30a ), and 2-[4-(car-
boxymethyl)phenoxy]ethylresidue (30b) maintained in-
hibitory potency at nearly the same level. However, the
introduction of a 2-(4-carboxyphenoxy)ethyl moiety in
position 1 of the indole (29b) resulted in a 3-fold increase
of activity. A reason for this may be that when “locked”
to the enzyme the conformational energy of 29b is
significantly lower than that of the other potent com-
pounds with a longer flexible (24e-g) or semirigid acid
substituent in position 1. With an IC50 of 0.5 µM
compound 29b was about 20-fold more active than the
cPLA2 standard reference inhibitor arachidonyl trifluo-
romethyl ketone (1) (IC50: 11 µM).
To exclude the possibility that the potent indoledi-
carboxylic acids act by modifying the calcium ionophore
A23187-induced activation mechanism of cPLA2 and not
by affecting the cPLA2 directly, we measured the inhibi-
tion of arachidonic acid release by compound 24e after
stimulation with 12-O-tetradecanoylphorbol-13-acetate
(TPA) (see Biological Evaluation). Like the lead 3,36
compound 24e reduced the arachidonic acid formation
also in this assay. Therefore, it can be safely supposed
that the active indoles are actually inhibitors of cPLA2.
The IC50 for 24e measured with TPA as stimulant (2.8
µM) was slightly higher than the IC50 obtained after
calcium ionophore A23187 stimulation of the platelets
(1.6 µM). The same effect was observed for the lead 336
and the reference compound 2.34
Mod el for In h ibitor Bin d in g
Although several inhibitors of cPLA2 have been
described in the literature,32-35 little is known about the
way these substances affect enzyme activity. For AA-
COCF3 (1) a direct interaction with the active site of
the enzyme was demonstrated by NMR experiments
and a crude model for the structure of an enzyme-
inhibitor complex was postulated.32 In this model, the
carbon chain of the inhibitor is bound in a hydrophobic
pocket and the carbonyl of the AACOCF3 forms a
covalent bond with a serine residue of the active site,
generating a charged hemiketal oxoanion which inter-
acts with a positively charged group of the enzyme. We
have proposed that our lead compound 3 could be bound
to the active site in a similar mode:36 The long carbon
chain could lie in the hydrophobic pocket, the carbonyl
residue could be bound via a hydrogen bond to the serine
residue of the active site, and the carboxylate group
could form a salt bridge like the hemiketal oxoanion
(Figure 1).
The results of the structure-activity relationship
investigation give some support to this model. It can
be suggested further on that the carboxylic acid function
in position 2 interacts with a positively charged group
of the enzyme, since its esterfication leads to a loss of
activity. The carbonyl moiety of the 3-octadecanoyl
residue also appears to be part of the pharmacophore,
because deoxygenation of the acyl residue destroys
activity. The fact that a certain length of the acyl chain
in position 3 (12 or more carbons) is necessary for
optimal activity of the 3-acyl-1-methylindole-2-carbox-
ylic acids provides evidence for the assumption that the
acyl residue is bound in a large hydrophobic pocket of
the enzyme. The surprising result that constraining the
3-acyl residue in different conformations does not lead
to a significant change in activity, however, allows
another hypothesis: Possibly there is no specific inter-
In the inflamed paws of rats, the concentrations of
the arachidonic acid metabolites prostaglandin E2 and
leukotriene B4 were reduced by compound 24e to nearly
the same extent.47 These findings indicate that 24e can
inhibit the cPLA2-mediated arachidonic acid release in
vivo as well.

2700 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Lehr
F igu r e 2. Modified model for the interaction of 3-acylindole-
2-carboxylic acids with the cPLA2 using 1-(7-carboxyheptyl)-
3-dodecanoylindole-2-carboxylic acid (24e) as example.
F igu r e 1. Proposed interaction of 1-methyl-3-octadecanoylin-
dole-2-carboxylic acid (3) with the cPLA2 according to the
model of Trimble et al.32
tionally constrained carboxylic acid side chain in posi-
tion 1 of the indole shall be synthesized.
action between the acyl residue and a hydrophobic
pocket of the enzyme. The longer acyl residue is
probably only needed for anchoring the indole inhibitor
in the cell membrane. A certain chain length of the acyl
residue (12 or more carbons) may be necessary to fix
the molecules in the plasma membrane optimally,
indoles with a shorter acyl chain may be enriched in
the lipophilic part of the cell membrane to a lesser
extent and therefore the activated enzyme may encoun-
ter locally lower concentrations of these compounds
resulting in a lower enzyme inhibition. This latter
hypothesis correlates with a model of Clark et al.26 for
the catalytic mechanism of the cPLA2. They suggest
that during hydrolysis by cPLA2, the lipophilic long acyl
or alkyl chain of the phospholipid substrates may
remain in the cell membrane (nuclear envelope or
endoplasmic reticulum48) and that only the region
between the scissile ester group and the phosphate
group interacts with the enzyme; in this case the
selectivity of the cPLA2 for phospholipids with arachi-
donate in position 2 could be explained with localized
packing differences of the different substrate molecules
in the cell membrane and would not be due to a specific
interaction of the cis double bonds of the arachidonate
with the active site of the enzyme.
Exp er im en ta l Section
Ch em istr y. All organic extracts were dried over Na2SO4.
Melting points were determined in open capillary tubes with
a Bu¨chi melting point apparatus and are uncorrected. 1H
nuclear magnetic resonance spectra were recorded on a J EOL
J NM-GX 400 spectrometer (400 MHz); chemical shifts (δ) are
expressed in ppm, relative to internal tetramethylsilane. Mass
spectra were obtained on a Varian CH7 apparatus; electron
beam ionization at 70 eV (EI) or chemical ionization with
methane (CI) was applied. Elemental analyses were deter-
mined on a Heraeus CHN Rapid instrument and were within
(0.4% of the theroetical value. Column chromatography was
performed with Kieselgel 60 (70-230 mesh) silica gel (Merck).
The starting materials were obtained from commercial
suppliers and used without further purification, or they were
synthesized in the same or a similar manner as described in
the literature cited. Reference compounds for the biological
assays: arachidonyl trifluoromethyl ketone was purchased
from Biomol (Hamburg); (S)-N-hexadecylpyrrolidine-2-car-
boxamide was synthesized by known procedures.33
Eth yl (3-Octa d eca n oylin d ol-2-yl)a ceta te (7). To the
refluxed solution of N,N-dimethyloctadecanamide (374 mg, 1.2
mmol) and POCl3 (153 mg, 1 mmol) in dry benzene (10 mL)
was added in one portion the solution of ethyl (indol-2-yl)-
acetate (6)49 (203 mg, 0.4 mmol) in dry benzene (3 mL). After
3 h an aqueous sodium acetate (1 g/4 mL) was added, and
refluxing was continued for an additional 15 min with vigorous
stirring. The mixture was cooled, diluted with water, and
extracted twice with Et2O. The organic phases were dried and
filtered and the filtrate was concentrated. The crude product
was purified using silica gel chromatography (elution with
petroleum ether-ethyl acetate (1) 9 + 1, (2) 8 + 2) and
precipitated from petroleum ether to give 7 as solid: yield 47%;
mp 86-87 °C; CI-MS m/e 470 (M + 1)+; 1H-NMR (CDCl3) δ
0.88 (t, 3H), 1.16-1.43 (m, 26H), 1.32 (t, 3H), 1.44 (quint, 2H),
1.78 (quint, 2H), 3.03 (t, 2H), 4.26 (q, 2H), 4.40 (s, 2H), 7.23-
7.29 (m, 2H), 7.42-7.44 (m, 1H), 7.88-7.90 (m, 1H), 10.09 (s,
1H).
Our results further indicate that the enzyme pos-
sesses a polar binding site, which can interact with the
ω-carboxylate group of a carboxylic acid substituents in
position 1 of the indole insofar as such a residue exceeds
a certain length (e.g. 24e).
On the basis of our present data and the published
models for inhibitor binding and substrate cleavage,26,32
for the interaction of the indolecarboxylic acids with the
enzyme we now propose the modified model shown in
Figure 2.
(3-Octa d eca n oylin d ol-2-yl)a cetic Acid (8). The mixture
of 7 (80 mg, 0.17 mmol), EtOH (15 mL), and 10% aqueous KOH
(5 mL) was refluxed for 15 min, cooled, diluted with water,
acidified with dilute HCl, and extracted with Et2O. The
organic phase was washed with dilute HCl, dried and filtered
and most of the Et2O was removed in vacuo. After addition
of petroleum ether the title compound precipitated: yield 79%;
mp 134-136 °C; 1H-NMR (CDCl3) δ 0.88 (t, 3H), 1.16-1.41
Further structure-activity relationship investigations
will be carried out in order to optimize the activity of
the compounds and to evaluate the distance between
the binding sites for the two carboxylic acid functions
of the indoledicarboxylic acids in particular. For the
latter purpose some more derivatives with conforma-

Inhibitors of the Cytosolic Phospholipase A2
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2701
(m, 26H), 1.45 (quint, 2H), 1.83 (quint, 2H), 3.15 (t, 2H), 4.12
(s, 2H), 7.28-7.35 (m, 2H), 7.45-7.47 (m, 1H), 7.81-7.83 (m,
1H), 10.17 (s, 1H). Anal. (C28H43NO3) C, H, N.
3-Dod eca n oyl-1-m eth ylin d ole-2-ca r boxylic Acid (16b):
yield 23%; mp 116-117 °C. Anal. (C22H31NO3) C, H, N.
3-Deca n oyl-1-m et h ylin d ole-2-ca r b oxylic Acid (16c):
yield 23%; mp 114-116 °C. Anal. (C20H27NO3) C, H, N.
1-Meth yl-3-octan oylin dole-2-car boxylic Acid (16d): yield
38%; mp 113-114 °C. Anal. (C18H23NO3) C, H, N.
(1-Meth yl-3-octadecan oylin dol-2-yl)acetic Acid (9). The
mixture of 7 (80 mg, 0.1 mmol), methyl p-toluenesulfonate (32
mg, 0.19 mmol), tetrabutylammonium bromide (22 mg, 0.07
mmol), Et2O (10 mL), CH2Cl2 (5 mL), and powdered NaOH
(105 mg, 2.6 mmol) was stirred at room temperature for 20 h.
The reaction mixture was filtered and the filter cake washed
twice with Et2O-CH2Cl2 (1 + 1). The filtrate was evaporated
and the residue chromatographed on silica gel with petroleum
ether-ethyl acetate (17 + 3) to give the methyl ester of 9,
which was saponified using the same method as for the
3-[2-(Dod ecyloxy)b en zoyl]-1-m et h ylin d ole-2-ca r b ox-
1
ylic Acid (16e): yield 28%; mp 63-65 °C; H-NMR (CDCl3)
δ 0.72 (quint, 2H), 0.88 (t, 3H), 0.92-1.04 (m, 4H), 1.04-1.15
(m, 2H), 1.15-1.32 (m, 12H), 3.75 (t, 2H), 4.28 (s, 3H), 6.65
(d, 1H), 6.98 (d, 1H), 7.05 (t, 1H), 7.12 (t, 1H), 7.36 (t, 1H),
7.47 (d, 1H), 7.50 (d, 1H), 7.57 (t, 1H), 16.06 (s, 1H). Anal.
(C29H37NO4) C, H, N.
1
synthesis of 8: yield 28%; mp 119-121 °C; H-NMR (CDCl3)
3-[3-(Dod ecyloxy)b en zoyl]-1-m et h ylin d ole-2-ca r b ox-
δ 0.88 (t, 3H), 1.09-1.39 (m, 26H), 1.45 (quint, 2H), 1.83 (quint,
2H), 3.15 (t, 2H), 3.91 (s, 3H), 4.09 (s, 2H), 7.33-7.44 (m, 3H),
7.82-7.85 (m, 1H), 13.05 (s, 1H). Anal. (C29H45NO3) C, H, N.
Met h yl 3-(3-Oct a d eca n oylin d ol-2-yl)p r op ion a t e (11).
Preparation started from methyl 3-(indol-2-yl)propionate (10)49
using the same method as for the synthesis of 7: yield 67%;
mp 91-93 °C; CI-MS m/e 470 (M + 1)+; 1H-NMR (CDCl3) δ
0.88 (t, 3H), 1.16-1.37 (m, 26H), 1.43 (quint, 2H), 1.78 (quint,
2H), 2.83 (t, 2H), 3.02 (t, 2H), 3.46 (t, 2H), 3.67 (s, 3H), 7.21-
7.25 (m, 2H), 7.37-7.40 (m, 1H), 7.86-7.88 (m, 1H), 9.22 (s,
1H).
ylic Acid (16f): yield 6%; mp 118-120 °C. Anal. (C29H37
-
NO4) C, H, N.
3-[4-(Dod ecyloxy)b en zoyl]-1-m et h ylin d ole-2-ca r b ox-
ylic Acid (16g): yield 34%; mp 90-92 °C. Anal. (C29H37NO4)
C, H, N.
3-[[2-(Decyloxy)p h en yl]a cet yl]-1-m et h ylin d ole-2-ca r -
boxylic Acid (16h ): yield 40%; mp 77-78 °C; 1H-NMR
(CDCl3) δ 0.86 (t, 3H), 0.98-1.32 (m, 14H), 1.58 (quint, 2H),
3.95 (t, 2H), 4.29 (s, 3H), 4.59 (s, 2H), 6.92 (d, 1H), 6.96 (t,
1H), 7.21 (d, 1H), 7.31 (t, 1H), 7.45 (t, 1H), 7.52 (t, 1H), 7.63
(d, 1H), 8.18 (d, 1H). Anal. (C28H35NO4) C, H, N.
3-[[3-(Decyloxy)p h en yl]a cet yl]-1-m et h ylin d ole-2-ca r -
boxylic Acid (16i): yield 9%; mp 80-81 °C; 1H-NMR (CDCl3)
δ 0.88 (t, 3H), 1.20-1.38 (m, 12H), 1.44 (quint, 2H), 1.77 (quint,
2H), 3.94 (t, 2H), 4.29 (s, 3H), 4.59 (s, 2H), 6.82-6.85 (m, 3H),
7.26-7.30 (m, 1H), 7.46 (t, 1H), 7.52 (t, 1H), 7.63 (d, 1H), 8.10
(d, 1H). Anal. (C28H35NO4) C, H, N.
3-[[4-(Decyloxy)p h en yl]a cet yl]-1-m et h ylin d ole-2-ca r -
boxylic Acid (16j): yield 15%; mp 98-99 °C; 1H-NMR
(CDCl3) δ 0.88 (t, 3H), 1.05-1.32 (m, 12H), 1.45 (quint, 2H),
1.78 (quint, 2H), 3.95 (t, 2H), 4.29 (s, 3H), 4.56 (s, 2H), 6.90
(d, 2H), 7.18 (d, 2H), 7.46 (t, 1H), 7.53 (t, 1H), 7.63 (d, 1H),
8.13 (d, 1H). Anal. (C28H35NO4) C, H, N.
3-(3-Octadecan oylin dol-2-yl)pr opion ic Acid (12). Prepa-
ration started from 11 using the same method as for the
1
synthesis of 8: yield 61%; mp 134-136 °C; H-NMR (CDCl3)
δ 0.88 (t, 3H), 1.21-1.37 (m, 26H), 1.44 (quint, 2H), 1.78 (quint,
2H), 2.90 (t, 2H), 3.03 (t, 2H), 3.44 (t, 2H), 7.21-7.28 (m, 2H),
7.37-7.39 (m, 1H), 7.86-7.88 (m, 1H). Anal. (C29H45NO3) C,
H, N.
3-(1-Meth yl-3-octa d eca n oylin d ol-2-yl)p r op ion ic Acid
(13). Preparation started from 11 in
a similar way as
described for 9: yield 47%; mp 106-108 °C; 1H-NMR (CDCl3)
δ 0.88 (t, 3H), 1.17-1.38 (m, 26H), 1.43 (quint, 2H), 1.79 (quint,
2H), 2.83 (t, 2H), 3.05 (t, 2H), 3.47 (t, 2H), 3.80 (s, 3H), 7.28-
7.32 (m, 2H), 7.36-7.40 (m, 1H), 7.86-7.91 (m, 1H). Anal.
(C30H47NO3) C, H, N.
3-[3-[2-(Decyloxy)p h en yl]p r op ion yl]-1-m eth ylin d ole-2-
1
ca r boxylic Acid (16k ): yield 14%; mp 84-85 °C; H-NMR
1-Meth yl-3-octadecylin dole-2-car boxylic Acid (14). The
mixture of ethyl 1-methyl-3-octadecanoylindole-2-carboxylate
(4)36 (94 mg, 0.2 mmol), dry THF (2 mL), dry methyl acetate
(3 mL), NaBH4 (40 mg), and BF3‚Et2O (0.2 mL) was stirred at
room temperature for 1 h. After addition of 50% MeOH (2
mL), the mixture was stirred for a further 15 min, diluted with
water, and extracted twice with Et2O. The organic phases
were dried and filtered, and the filtrate was concentrated to
give the ethyl ester of 14, which was saponified using a similar
method as for the synthesis of 8 (deviation: the reaction
mixture was refluxed for 1 h, the product was precipitated from
MeOH): yield 26%; mp 87-90 °C; 1H-NMR (CDCl3) δ 0.88 (t,
3H), 1.11-1.39 (m, 28H), 1.41 (quint, 2H), 1.68 (quint, 2H),
3.15 (t, 2H), 4.05 (s, 3H), 7.13-7.17 (m, 1H), 7.36-7.41 (m,
2H), 7.70 (d, 1H). Anal. (C30H49NO2) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-Acyl-1-
m eth ylin d ole-2-ca r boxylic Acid s (16a -m ). The mixture
of ethyl 1-methylindole-2-carboxylate50 (15) (122 mg, 0.6
mmol), the appropriate carboxylic acid (0.9 mmol),51-55 poly-
phosphoric acid (27 mg), dry CH2Cl2 (3 mL), and trifluoroacetic
anhydride (0.13 mL) was stirred at room temperature for 4 h.
The reaction mixture was diluted with Et2O, washed with
brine and a solution of sodium chloride in 1 M NaOH, dried,
and evaporated. The residue was chromatographed on silica
gel with petroleum ether-ethyl acetate (16a ,b 9 + 1; 16c-m ,
19 + 1) to give the ethyl ester of 16a -m , which was saponified
using a similar method as for the synthesis of 8 (deviation:
the reaction mixture was refluxed for 1 h).
(CDCl3) δ 0.86 (t, 3H), 1.08-1.34 (m, 14H), 1.76 (quint, 2H),
3.18 (t, 2H), 3.64 (t, 2H), 3.99 (t, 2H), 4.27 (s, 3H), 6.87 (d,
1H), 6.91 (t, 1H), 7.20-7.26 (m, 2H), 7.39 (t, 1H), 7.48 (t, 1H),
7.59 (t, 1H), 8.02 (d, 1H). Anal. (C29H37NO4) C, H, N.
3-[3-[3-(Decyloxy)p h en yl]p r op ion yl]-1-m eth ylin d ole-2-
ca r boxylic Acid (16l): yield 29%; mp 95-96 °C. Anal.
(C29H37NO4) C, H, N.
3-[3-[4-(Decyloxy)p h en yl]p r op ion yl]-1-m eth ylin d ole-2-
ca r boxylic Acid (16m ): yield 27%; mp 109-110 °C. Anal.
(C29H37NO4) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 4-, 5-, or
6-Su bstitu ted 3-Dod eca n oyl-1-m eth ylin d ole-2-ca r boxylic
Acid s (18a -g). The mixture of the appropriate substituted
ethyl indole-2-carboxylate (17a -g) (0.4 mmol),56-60 methyl
p-toluenesulfonate (82 mg, 0.44 mmol), tetrabutylammonium
bromide (13 mg, 0.04 mmol), Et2O (5 mL), and powdered
NaOH (20 mg, 0.5 mmol) was stirred at room temperature for
6 h. The reaction mixture was filtered, the filter cake washed
with Et2O-CH2Cl2 (1 + 1), and the filtrate concentrated in
vacuo. To the residue obtained were added dodecanoic acid
(120 mg, 0.6 mmol), polyphosphoric acid (27 mg), dry CH2Cl2
(3 mL), and trifluoroacetic anhydride (0.13 mL), and the
mixture was stirred at room temperature for 4-24 h. The
reaction mixture was diluted with Et2O, washed with brine
and a solution of sodium chloride in 1 M NaOH, dried, and
evaporated. The residue was chromatographed on silica gel
with petroleum ether-ethyl acetate (18a ,b,d ,f,g, 12 + 1, and
18c,e, 9 + 1).The substituted ethyl 3-dodecanoyl-1-methylin-
dolecarboxylate was saponified using a similar method as for
the synthesis of 8 (the reaction mixture was refluxed for 15
min to 1 h).
1-Meth yl-3-tetr adecan oylin dole-2-car boxylic Acid (16a).
The sodium tetradecanoate, which precipitated when the
organic phase was washed with the solution of sodium chloride
in 1 M NaOH, was filtered off from the organic phase after
1
addition of kieselguhr: yield 14%; mp 118-119 °C; H-NMR
5-Ch lor o-3-d od eca n oyl-1-m et h ylin d ole-2-ca r b oxylic
1
(CDCl3) δ 0.88 (t, 3H), 1.16-1.43 (m, 18H), 1.47 (quint, 2H),
1.86 (quint, 2H), 3.28 (t, 2H), 4.27 (s, 3H), 7.43-7.53 (m, 2H),
7.61 (d, 1H), 8.02 (d, 1H), 16.72 (s, 1H). Anal. (C24H35NO3)
C, H, N.
Acid (18a ): yield 29%; mp 105-106 °C; H-NMR (CDCl3) δ
0.88 (t, 3H), 1.19-1.56 (m, 16H), 1.86 (quint, 2H), 3.22 (t, 2H),
4.25 (s, 3H), 7.47 (dd, 1H), 7.54 (d, 1H), 7.98 (d, 1H). Anal.
(C22H30ClNO3) C, H, N.

2702 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Lehr
3-Dodecan oyl-5-flu or o-1-m eth ylin dole-2-car boxylic Acid
(18b): yield 29%; mp 87-88 °C; H-NMR (CDCl3) δ 0.88 (t,
3H), 1.05-1.39 (m, 16H), 1.85 (quint, 2H), 3.20 (t, 2H), 4.27
(s, 3H), 7.25-7.30 (m, 1H), 7.57 (dd, 1H), 7.66 (dd, 1H). Anal.
(C22H30FNO3) C, H, N.
stirred at room temperature for 4 h. The reaction mixture was
diluted with brine and extracted with Et2O. The extract was
washed with a solution of sodium chloride in 1 M NaOH and
the precipitated sodium octadecanoate filtered off after addi-
tion of kieselguhr. The organic phase was dried and evapo-
rated. The residue was chromatographed on silica gel with
petroleum ether-ethyl acetate (23a -c, 29 + 1; 23d , 200 + 1)
and the 1-alkylated ethyl 3-octadecanoylindole-2-carboxylate
obtained was saponified using a similar method as for the
synthesis of 8 (deviation: the reaction mixture was refluxed
for 1 h).
1-Hexyl-3-octa d eca n oylin d ole-2-ca r boxylic Acid (23a ):
yield 13%; mp 75-77 °C; 1H-NMR (CDCl3) δ 0.88 (t, 3H), 0.89
(t, 3H), 1.16-1.55 (m, 34H), 1.86 (quint, 4H), 3.27 (t, 2H), 4.79
(t, 2H), 7.44 (t, 1H), 7.49 (t, 1H), 7.59 (d, 1H), 8.01 (d, 1H),
16.67 (br, 1H). Anal. (C33H53NO3) C, H, N.
1
3-Dodecan oyl-1-m eth yl-5-n itr oin dole-2-car boxylic Acid
(18c): yield 7%; mp 97-99 °C; 1H-NMR (DMSO-d6) δ 0.88 (t,
3H), 1.18-1.45 (m, 16H), 1.89 (quint, 2H), 3.34 (t, 2H), 4.31
(s, 3H), 7.71 (d, 1H), 8.39 (d, 1H), 9.03 (s, 1H). Anal.
(C22H30N2O5) C, H, N.
3-Dod e ca n oyl-5-m e t h oxy-1-m e t h ylin d ole -2-ca r b ox-
ylic Acid (18d ): yield 14%; mp 111-113 °C; 1H-NMR (CDCl3)
δ 0.88 (t, 3H), 1.05-1.29 (m, 14H), 1.48 (quint, 2H), 1.87 (quint,
2H), 3.21 (t, 2H), 3.93 (s, 3H), 4.25 (s, 3H), 7.16 (dd, 1H), 7.35
(d, 1H), 7.51 (d, 1H). Anal. (C23H33NO4) C, H, N.
3-Dod e ca n oyl-5-h yd r oxy-1-m e t h ylin d ole -2-ca r b ox-
ylic Acid (18e). The synthesis started from ethyl 5-acetoxy-
indole-2-carboxylate57(17e). To dissolve 17e, CH2Cl2 (2.5 mL)
was added to the reaction mixture. The N-methyl derivative
of 17e was purified by silica gel chromatography (elution with
petroleum ether-ethyl acetate, (1) 9 + 1 and (2) 8 + 2). The
final product 18e was precipitated from Et2O: yield 15%; mp
169-170 °C; 1H-NMR (DMSO-d6) δ 0.85 (t, 3H), 1.19-1.41 (m,
16H), 1.60 (quint, 2H), 2.81 (t, 2H), 3.80 (s, 3H), 6.85 (dd, 1H),
7.35 (d, 1H), 7.42 (d, 1H), 9.22 (s, 1H). Anal. (C22H31NO4) C,
H, N.
1-Heptyl-3-octadecan oylin dole-2-car boxylic Acid (23b):
yield 14%; mp 61-62 °C. Anal. (C34H55NO3) C, H, N.
3-Octa d eca n oyl-1-octylin d ole-2-ca r boxylic Acid (23c):
yield 8%; mp 64-65 °C. Anal. (C35H57NO3) C, H, N.
1-Dod ecyl-3-oct a d eca n oylin d ole-2-ca r b oxylic
Acid
(23d ): yield 23%; mp 74-76 °C. Anal. (C39H65NO3) C, H, N.
The 3-acylindole-2-carboxylic acids with an 1-(ω-carboxy-
alkyl) substituent (24a -g) were synthesized similar to 23a -d
using the appropriate ethyl ω-bromoalkanoate62-64 instead of
the 1-bromoalkane. The crude ethyl 1-[ω-(ethoxycarbonyl)-
alkyl]indole-2-carboxylate intermediates were purified by
chromatography on silica gel (elution with petroleum ether-
ethyl acetate, 9 + 1) prior reaction with octadecanoic acid or
dodecanoic acid.
4-Ch lor o-3-d od eca n oyl-1-m et h ylin d ole-2-ca r b oxylic
Acid (18f): yield 22%; mp 136-137 °C; 1H-NMR (CDCl3) δ
0.87 (t, 3H), 1.11-1.44 (m, 16H), 1.80 (quint, 2H), 3.02 (t, 2H),
4.08 (s, 3H), 7.20 (t, 1H), 7.30-7.34 (m, 2H). Anal. (C22H30
ClNO3) C, H, N.
-
1-(7-Ca r b oxyh ep t yl)-3-oct a d eca n oylin d ole-2-ca r b ox-
ylic Acid (24a ): yield 12%; mp 99-101 °C. Anal. (C35H55
-
6-Ch lor o-3-d od eca n oyl-1-m et h ylin d ole-2-ca r b oxylic
Acid (18g): yield 17%; mp 113-114 °C; H-NMR (CDCl3) δ
0.88 (t, 3H), 1.05-1.37 (m, 16H), 1.85 (quint, 2H), 3.24 (t, 2H),
4.23 (s, 3H), 7.41 (dd, 1H), 7.60 (d, 1H), 7.93 (d, 1H). Anal.
(C22H30ClNO3) C, H, N.
1
NO5) C, H, N.
1-(Ca r b oxym et h yl)-3-d od eca n oylin d ole-2-ca r b oxylic
1
Acid (24b): yield 30%; mp 182-183 °C; H-NMR (CDCl3) δ
0.86 (t, 3H), 1.11-1.34 (m, 16H), 1.61 (quint, 2H), 2.90 (t, 2H),
5.19 (s, 3H), 7.22 (t, 1H), 7.33 (t, 1H), 7.61 (d, 1H), 7.85 (d,
1H). Anal. (C23H31NO5) C, H, N.
E t h yl 3-Dod eca n oyl-4,5-d im et h ylp yr r ole-2-ca r b oxyl-
a te (20). To a stirred solution of ethyl 4,5-dimethylpyrrole-
2-carboxylate (19)61 (1.67 g, 10 mmol) and dodecanoyl chloride
(4.38 g, 20 mmol) in dry nitrobenzene was added AlCl3 (2.67
g, 20 mmol). The mixture was allowed to stand for 3 days.
Then the mixture was poured into water and extracted twice
with CH2Cl2. The organic phases were washed with dilute
NaOH, dried, and evaporated. Chromatography on silica gel
(elution with (1) CH2Cl2-petroleum ether, 3 + 1, and (2)
petroleum ether-ethyl acetate, 8 + 2) and precipitation from
petroleum ether gave 20 as solid: yield 31%; mp 88-90 °C;
1H-NMR (CDCl3) δ 0.88 (t, 3H), 1.16-1.37 (m, 16H), 1.33 (t,
3H), 1.65 (quint, 2H), 1.97 (s, 3H), 2.20 (s, 3H), 2.86 (t, 2H),
4.30 (q, 2H), 8.73 (s, 1H).
1-(5-Ca r b oxyp e n t yl)-3-d od e ca n oylin d ole -2-ca r b ox-
ylic Acid (24c): yield 9%; mp 104-106 °C. Anal. (C27H39
NO5) C, H, N.
1-(6-Ca r b oxyh exyl)-3-d od eca n oylin d ole-2-ca r b oxylic
Acid (24d ): yield 8%; mp 104-105 °C. Anal. (C28H41NO5)
C, H, N.
1-(7-Ca r b oxyh e p t yl)-3-d od e ca n oylin d ole -2-ca r b ox-
ylic Acid (24e): yield 12%; mp 109-110 °C; 1H-NMR (CDCl3)
δ 0.88 (t, 3H), 1.18-1.53 (m, 22H), 1.64 (quint, 2H), 1.82-
1.88 (m, 4H), 2.35 (t, 2H), 3.28 (t, 2H), 4.79 (t, 2H), 7.46 (t,
-
1H), 7.49 (t, 1H), 7.58 (d, 1H), 8.01 (d, 1H). Anal. (C29H43
NO5) C, H, N.
-
1-(8-Ca r b oxyoct yl)-3-d od eca n oylin d ole-2-ca r b oxylic
Acid (24f): yield 9%; mp 110-111 °C. Anal. (C30H45NO5) C,
H, N.
3-Dodecan oyl-1,4,5-tr im eth ylpyr r ole-2-car boxylic Acid
(21). The mixture of 20 (175 mg, 0.5 mmol), methyl p-
toluenesulfonate (102 mg, 0.55 mmol), tetrabutylammonium
bromide (16 mg, 0.05 mmol), Et2O (10 mL), CH2Cl2 (6 mL),
and powdered NaOH (80 mg, 2 mmol) was stirred at room
temperature for 8 h. After addition of water the mixture was
extracted twice with Et2O. The organic phases were dried and
evaporated. Chromatography on silica gel (elution with
petroleum ether-ethyl acetate, 9 + 1) yielded the ethyl ester
of 21 which was saponified using a similar method as for the
synthesis of 8 (deviation: the reaction mixture was refluxed
for 1 h): yield 58%; mp 83-84 °C; 1H-NMR (CDCl3) δ 0.88 (t,
3H), 1.18-1.45 (m, 14H), 1.72 (quint, 2H), 2.23 (s, 3H), 2.29
(s, 3H), 2.91 (t, 2H), 3.95 (s, 3H). Anal. (C20H33NO3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 1-Alk yl-3-
octadecan oylin dole-2-car boxylic Acids (23a-d). The mix-
ture of ethyl indole-2-carboxylate (22) (189 mg, 1 mmol),
t-BuOK (135 mg, 1.2 mmol), and dry DMSO (4 mL) was heated
at 110-120 °C for 15 min. After addition of the appropriate
1-bromoalkane (1.2 mmol), the mixture was heated for an
additional 15 min at the same temperature. The mixture was
cooled, diluted with water, and extracted with Et2O. The
organic phase was dried and the solvent evaporated. To the
residue obtained were added octadecanoic acid (427 mg, 1.5
mmol), polyphosphoric acid (67 mg), dry CH2Cl2 (6 mL), and
trifluoroacetic anhydride (0.33 mL), and the mixture was
1-(10-Ca r b oxyd e cyl)-3-d od e ca n oylin d ole -2-ca r b ox-
ylic Acid (24g): yield 15%; mp 103-104 °C. Anal. (C32H49
NO5) C, H, N.
-
Eth yl 3-Dod eca n oylin dole-2-ca r boxyla te (25). The mix-
ture of ethyl indole-2-carboxylate (22) (3.8 g, 20 mmol),
dodecanoic acid (6.0 g, 30 mmol), polyphosphoric acid (1.0
mmol), dry CH2Cl2 (20 mL) and trifluoroacetic anhydride (4.4
mL) was stirred at room temperature for 4 h. The reaction
mixture was diluted with 1 M NaOH and extracted with Et2O.
The organic phase was dried and the solvent evaporated. After
addition of petroleum ether 25 precipitated: yield 58%; mp
75-76 °C; EI-MS m/e 371 (M+); 1H-NMR (CDCl3) δ 0.88 (t,
3H), 1.13-1.39 (m, 16H), 1.43 (t, 3H), 1.74 (quint, 2H), 3.06
(t, 2H), 4.45 (q, 2H), 7.24 (t, 1H), 7.36 (d, 1H), 7.41 (t, 1H),
7.92 (d, 1H), 9.02 (s, 1H).
Eth yl (E)-3-Dod eca n oyl-1-[3-[2-(eth oxyca r bon yl)eth en -
1-yl]ben zyl]in d ole-2-ca r boxyla te (26a ). The mixture of 25
(372 mg, 1 mmol), t-BuOK (124 mg, 1.1 mmol), and dry DMSO
(3 mL) was heated at 110-120 °C for 5 min. After addition of
ethyl (E)-3-(bromomethyl)cinnamate65 (296 mg, 1.1 mmol), the
mixture was heated for an additional 10 min at the same
temperature. The mixture was cooled, diluted with brine, and
extracted with Et2O. The organic phase was dried and the

Inhibitors of the Cytosolic Phospholipase A2
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2703
solvent evaporated. Chromatography on silica gel eluting with
petroleum ether-ethyl acetate (9 + 1) gave 26a as waxlike
substance: yield 70%; EI-MS m/e 559 (M+); 1H-NMR (CDCl3)
δ 0.88 (t, 3H), 1.18-1.48 (m, 22H), 1.77 (quint, 2H), 2.94 (t,
2H), 4.25 (q, 2H), 4.36 (q, 2H), 5.57 (s, 2H), 6.37 (d, 1H), 7.09
(d, 1H), 7.21-7.34 (m, 5H), 7.43 (d, 1H), 7.59 (d, 1H), 7.96 (d,
1H).
Eth yl (E)-3-Dod eca n oyl-1-[4-[2-(eth oxyca r bon yl)eth en -
1-yl]ben zyl]in d ole-2-ca r boxyla te (26b). The preparation
started from 22 and ethyl (E)-4-(bromomethyl)cinnamate65
using a similar method as for the synthesis of ester intermedi-
ates of 24a -g: yield 25%; mp 72-74 °C; EI-MS m/e 559 (M+);
1H-NMR (CDCl3) δ 0.88 (t, 3H), 1.17-1.45 (m, 16H), 1.28 (t,
3H), 1.32 (t, 3H), 1.76 (quint, 2H), 2.93 (t, 2H), 4.25 (q, 2H),
4.35 (q, 2H), 5.59 (s, 2H), 6.38 (d, 1H), 7.10 (d, 2H), 7.26-7.35
(m, 3H), 7.44 (d, 2H), 7.62 (d, 1H), 7.95 (d, 1H).
Compounds 27a ,b were synthesized by saponification of
26a ,b using a similar method as for the synthesis of 8
(deviation: the reaction mixture was refluxed for 1 h).
(E)-1-[3-(2-Ca r boxyeth en -1-yl)ben zyl]-3-d od eca n oylin -
d ole-2-ca r boxylic Acid (27a ): yield 42%; mp 186-187 °C;
1H-NMR (DMSO-d6) δ 0.85 (t, 3H), 1.12-1.37 (m, 16H), 1.63
(quint, 2H), 2.91 (t, 2H), 5.63 (s, 2H), 6.45 (d, 1H), 7.10 (d,
1H), 7.22-7.34 (m, 3H), 7.51 (d, 1H), 7.53-7.60 (m, 3H), 7.96
(d, 1H). Anal. (C31H37NO5) C, H, N.
(E)-1-[4-(2-Ca r boxyeth en -1-yl)ben zyl]-3-d od eca n oylin -
d ole-2-ca r boxylic Acid (27b): yield 30%; mp 224-230 °C;
1H-NMR (DMSO-d6) δ 0.85 (t, 3H), 1.12-1.36 (m, 16H), 1.63
(quint, 2H), 2.91 (t, 2H), 5.65 (s, 2H), 6.46 (d, 1H), 7.15 (d,
2H), 7.24 (t, 1H), 7.29 (t, 1H), 7.50-7.57 (m, 2H), 7.60 (d, 2H),
7.96 (d, 1H). Anal. (C31H37NO5) C, H, N.
1-[3-(2-Ca r b oxyet h yl)b en zyl]-3-d od eca n oylin d ole-2-
ca r boxylic Acid (28a ). A solution of 26a (56 mg, 0.1 mmol)
in THF (5 mL) was treated with a catalytic amount of 10%
Pd/C and the mixture hydrogenated at atmospheric pressure
for 4 h. After addition of kieselguhr the mixture was filtered
and the solvent evaporated. The residue was chromato-
graphed on silica gel with petroleum ether-ethyl acetate (9
+ 1), and the intermediate obtained was saponified using a
similar method as for the synthesis of 8 (deviation: the
reaction mixture was refluxed for 1 h and the product was
precipitated from Et2O): yield 22%; mp 131-132 °C; 1H-NMR
(DMSO-d6) δ 0.85 (t, 3H), 1.14-1.37 (m, 16H), 1.63 (quint, 2H),
2.48 (t, 2H), 2.76 (t, 2H), 2.90 (t, 2H), 5.57 (s, 2H), 6.86 (d,
1H), 7.10 (d, 1H), 7.15-7.31 (m, 4H), 7.55 (d, 1H), 7.96 (d, 1H).
Anal. (C31H39NO5) C, H, N.
1-[4-(2-Ca r b oxyet h yl)b en zyl]-3-d od eca n oylin d ole-2-
ca r boxylic Acid (28b). Preparation analogously 28a starting
from 26b. The product 28b was precipitated from petroleum
ether: yield 34%; mp 168-169 °C; 1H-NMR (CDCl3) δ 0.88 (t,
3H), 1.18-1.42 (m, 14H), 1.48 (quint, 2H), 1.88 (quint, 2H),
2.62 (t, 2H), 2.89 (t, 2H), 3.31 (t, 2H), 6.08 (s, 2H), 7.03 (d,
2H), 7.10 (d, 2H), 7.41-7.46 (m, 2H), 7.54-7.58 (m, 1H), 8.02-
8.05 (m, 2H). Anal. (C31H39NO5) C, H, N.
Compounds 29a ,b and 30a ,b were synthesized in a similar
manner as 26a starting from 25 and applying the appropriate
ethyl (2-bromoethoxy)benzoates66,67 and ethyl [(2-bromoethox-
y)phenyl]acetates.68
1-[2-(3-Ca r boxyp h en oxy)eth yl]-3-d od eca n oylin d ole-2-
ca r boxylic Acid (29a ). After the saponification the product
was extracted with CHCl3 and precipitated from Et2O: yield
43%; mp 195-196 °C; 1H-NMR (DMSO-d6) δ 0.85 (t, 3H),
1.12-1.34 (m, 16H), 1.61 (quint, 2H), 2.88 (t, 2H), 4.31 (t, 2H),
4.84 (t, 2H), 7.06 (dd, 1H), 7.25 (t, 1H), 7.33-7.39 (m, 3H),
7.48 (d, 1H), 7.76 (d, 1H), 7.92 (d, 1H). Anal. (C30H37NO6) C,
H, N.
NMR (DMSO-d6) δ 0.85 (t, 3H), 1.13-1.34 (m, 16H), 1.61
(quint, 2H), 2.88 (t, 2H), 3.47 (s, 2H), 4.23 (t, 2H), 4.80 (t, 2H),
6.71 (d, 1H), 6.72 (s, 1H), 6.79 (d, 1H), 7.16 (t, 1H), 7.25 (t,
1H), 7.36 (t, 1H), 7.74 (d, 1H), 7.93 (d, 1H). Anal. (C31H39
NO6) C, H, N.
-
1-[2-[4-(Car boxym eth yl)ph en oxy]eth yl]-3-dodecan oylin -
d ole-2-ca r boxylic Acid (30b). The product was precipitated
1
from petroleum ether: yield 20%; mp 130-131 °C; H-NMR
(DMSO-d6) δ 0.85 (t, 3H), 1.13-1.37 (m, 16H), 1.62 (quint, 2H),
2.88 (t, 2H), 3.43 (s, 2H), 4.23 (t, 2H), 4.80 (t, 2H), 6.76 (d,
2H), 7.10 (d, 2H), 7.24 (t, 1H), 7.36 (t, 1H), 7.72 (d, 1H, 7.92
(d, 1H). Anal. (C31H39NO6) C, H, N.
Bioch em istr y. cP LA2 In h ibition . Inhibition of cPLA2
was determined by measuring calcium ionophore A23187- or
TPA-induced arachidonic acid release from bovine platelets
with HPLC/UV detection as previously described.38,41 Briefly,
to a solution of 5,8,11,14-eicosatetraynoic acid (ETYA), which
inhibits formation of arachidonic acid metabolites in platelets,
was added the test compound solution or the solvent (in case
of the control test) followed by the platelet suspension and a
solution of calcium chloride at 37 °C. Then cPLA2 was
activated by calcium ionophore A23187 or TPA. After termi-
nation of the enzyme reaction the produced arachidonic acid
was cleaned up by solid-phase extraction and quantified with
HPLC/UV detection at 200 nm. Compounds 9, 13, and 23d
were dissolved in DMSO-0.05 M ethanolic NaOH (1 + 1), all
other test compounds were dissolved in DMSO, if necessary
with heating. When DMSO-0.05 M NaOH in EtOH was used
as solvent, the solution of the test compound (deviating from38
10 µL) was added after the platelet suspension to avoid
degradation of ETYA. The IC50 values (50% inhibitory con-
centration) were determined graphically and are the means
of at least two independent experiments. For AACOCF3 (1)
the IC50 was 11 ( 1.4 µM (mean ( SEM, n ) 3), for Wy-48,489
(2) it was 13 ( 1.0 µM (mean ( SEM, n ) 4), and for 16b it
was 8 ( 0.5 µM (mean ( SEM, n ) 5); the inhibition of
arachidonic acid release by 16b, which was included as
reference control in each experiment, ranged from 50% to 66%
(57 ( 1.3%, mean ( SEM; n ) 16) at a concentration of 10
µM. The enzyme reactions were performed within 36 h after
isolation of the platelets. The platelets were stored at +4 °C.
Cell Lysis. Cell lysis was measured by turbidimetry as
previously described.36 Briefly, to a solution of ETYA was
added the test compound solution or the solvent (in case of
the control test) followed by the platelet suspension and a
solution of calcium chloride at 37 °C. After dilution with
phosphate-buffered saline the absorbance of the cell suspen-
sions was measured at 800 nm. Cell lysis led to a decrease of
absorbance. Compounds 9, 13, and 23d were dissolved in
DMSO-0.05 M ethanolic NaOH (1 + 1); all other test
compounds were dissolved in DMSO, if necessary with heating.
When DMSO-0.05 M ethanolic NaOH was used as solvent,
the solution of the test compound (deviating from36 5 µL) was
added after the platelet suspension to avoid degradation of
ETYA. As reference substance (1-benzyl-3,5-dimethyl-4-octa-
decanoylpyrrol-2-yl)acetic acid69 was used. This compound
caused a cell lysis of 32 ( 2.7% (mean ( SEM, n ) 5, different
cell preparations were used). The cell lysis was determined
within 36 h after isolation of the platelets. The platelets were
stored at +4 °C.
Ack n ow led gm en t. I thank Prof. Dr. H.-D. Stachel
for supporting this study and Mrs. Monika Klimt for
technical assistance.
Refer en ces
(1) Moncada, S.; Higgs, E. A. Metabolism of arachidonic acid. Ann.
N.Y. Acad. Sci. 1988, 522, 454-463.
1-[2-(4-Ca r boxyp h en oxy)eth yl]-3-d od eca n oylin d ole-2-
ca r boxylic Acid (29b). After the saponification the product
was extracted with CHCl3 and precipitated from Et2O: yield
14%; mp 191-192 °C; 1H-NMR (DMSO-d6) δ 0.85 (t, 3H),
1.13-1.34 (m, 16H), 1.61 (quint, 2H), 2.87 (t, 2H), 4.34 (t, 2H),
4.85 (t, 2H), 6.90 (d, 2H), 7.26 (t, 1H), 7.37 (t, 1H), 7.75 (d,
1H), 7.82 (d, 2H), 7.91 (d, 1H). Anal. (C30H37NO6) C, H, N.
1-[2-[3-(Car boxym eth yl)ph en oxy]eth yl]-3-dodecan oylin -
d ole-2-ca r boxylic Acid (30a ). The product was precipitated
(2) Samuelsson, B.; Dahlen, S. E.; Lindgren, J . A.; Rouzer, C. A.;
Serhan, C. N. Leukotrienes and lipoxins: structures, biosyn-
thesis, and biological effects. Science 1991, 237, 1171-1176.
(3) Moreno, J . J .; Ferrer, X.; Ortega, E.; Carganico, G. PLA2-induced
edema in rat skin and histamine release in rat mast cells.
Evidence for involvement of lysophospholipids in the mechanism
of action. Agents Actions 1992, 36, 258-263.
(4) Venable, M. E.; Zimmerman, G. A.; McIntyre, T. M.; Prescott,
S. M. Platelet-activating factor: a phospholipid autacoid with
diverse actions. J . Lipid Res. 1993, 34, 691-702.
1
from Et2O-petroleum ether: yield 27%; mp 114-116 °C; H-

2704 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17
Lehr
(5) Hara, S.; Kudo, I.; Chang, H. W.; Matsuta, K. Purification and
characterization of extracellular phospholipase A2 from human
synovial fluid in rheumatoid arthritis. J . Biochem. 1989, 105,
395-399.
(6) Gronroos, J . M.; Nevalainen, T. J . Increased concentrations of
synovial-type phospholipase A2 in serum and pulmonary and
renal complications in acute pancreatitis. Digestion 1992, 52,
232-236.
(7) Green, J . A.; Smith, G. M.; Buchta, R.; Lee, R.; Ho, K. Y.;
Rajkovic, I. A.; Scott, K. F. Circulating phospholipase A2 activity
associated with sepsis and septic shock is indistinguishable from
that associated with rheumatoid arthritis. Inflammation 1991,
15, 355-367.
(8) Uhl, W.; Buchler, M.; Nevalainen, T. J .; Deller, A.; Beger, H. G.
Serum phospholipase A2 in patients with multiple injuries. J .
Trauma 1990, 30, 1285-1290.
(9) Murakami, M.; Kudo, I.; Inoue, K. Secretory phospholipase A2.
J . Lipid Mediators Cell Signalling 1995, 12, 119-130.
(10) Dillard, R. D.; Bach N. J .; Draheim, S. E.; Berry, D. R.; Carlson,
D. G.; Chirgadze, N. Y.; Clawson, D. K.; Hartley, L. W.; J ohnson,
L. M.; J ones, N. D.; McKinney, E. R.; Mihelich, E. D.; Olkowski,
J . L.; Schevitz, R. W.; Smith, A. C.; Snyder, D. W.; Sommers, C.
D.; Wery, J .-P. Indole inhibitors of human nonpancreatic secre-
tory phospholipase A2. J . Med. Chem. 1996, 39, 5119-5175.
(11) Teshirogi, I.; Matsutani, S.; Shirahase, K.; Fujii, Y.; Yoshida T.;
Tanaka, K.; Ohtani, M. Synthesis and phospholipase A2 inhibi-
tory activity of thielocin B3 derivatives. J . Med. Chem. 1996,
39, 5183-5191.
(12) Tibes, U.; Vondran, A.; Rodewald, E.; Friebe, W.-G.; Scha¨fer, W.;
Scheuer, W. Inhibition of allergic and non-allergic inflammation
by phospholipase A2 inhibitors. Int. Arch. Allergy Immunol.
1995, 107, 432-434.
(13) Tibes, U.; Scheuer, W.; Burgermeister, E.; Thierse, H.-J .; Friebe,
W.-G. Cross talk between cPLA2 and iNOS in chronic inflam-
mation. Prostaglandins, Leukotrienes Essent. Fatty Acids 1996,
55 (Suppl. 1), 95.
(14) Fox, N.; Song, M.; Schrementi, J .; Sharp, J . D.; White, D. L.;
Snyder, D. W.; Hartley, L. W.; Carlson, D. G.; Bach, N. J .;
Dillard, R. D.; Draheim, S. E.; Bobbitt, J . L.; Fisher, L.; Mihelich,
E. D. Transgenic model for the discovery of novel human
secretory non-pancreatic phospholipase A2 inhibitors. Eur. J .
Pharmacol. 1996, 308, 195-203.
(15) Clark, J . D.; Milona, N.; Knopf, J . L. Purification of a 110-
kilodalton cytosolic phospholipase A2 from the human monocytic
cell line U937. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7708-
7712.
(16) Gronich, J . H.; Bonventre, J . V.; Nemonoff, R. A. Purification of
a high-molecular-mass form of phospholipase A2 from rat kidney
activated at physiological calcium concentrations. Biochem. J .
1990, 271, 37-43.
(17) Kramer, R. M.; Roberts, E. F.; Manetta, J .; Putnam, J . E. The
Ca2+-sensitive cytosolic phospholipase A2 is a 100-kDa protein
in human monoblast U937 cells. J . Biol. Chem. 1991, 266, 5268-
5272.
(18) Wijkander, J .; Sundler, R. An 100-kDa arachidonate-mobilizing
phospholipase A2 in mouse spleen and the macrophage cell line
J 774. Eur. J . Biochem. 1991, 202, 873-880.
(19) Lin, L. L.; Lin, A. Y.; Knopf, J . L. Cytosolic phospholipase A2 is
coupled to hormonally regulated release of arachidonic acid.
Proc. Nat. Acad. Sci. U.S.A. 1992, 89, 6147-51.
(20) Mounier, C.; Faili, A.; Vargaftig, B. B.; Bon, C.; Hatmi, M.
Secretory phospholipase A2 is not required for arachidonic acid
liberation during platelet activation. Eur. J . Biochem. 1993, 216,
169-175.
(21) Mounier, C.; Vargaftig, B. B.; Franken, P. A.; Verheij, H. M.;
Bon, C.; Touqui, L. Platelet secretory phospholipase A2 fails to
induce rabbit platelet activation and to release arachidonic acid
in contrast with venom phospholipases A2. Biochim. Biophys.
Acta 1994, 1214, 88-96.
(27) Mayer, R. J .; Marshall, L. A. New insights on mammalian
phospholipase A2(s); compari-son of arachidonyl-selective and
-nonselective enzymes. FASEB J . 1993, 7, 339-348.
(28) Gewert, K.; Sundler, R. Dexamethasone down-regulates the 85
kDa phospholipase A2 in mouse macrophages and suppresses
its activation. Biochem. J . 1995, 307, 499-504.
(29) Lin, L. L.; Lin, A. Y.; DeWitt, D. L. IL-1R induces the accumula-
tion of cPLA2 and the release of PGE2 in human fibroblasts. J .
Biol. Chem. 1992, 267, 23451-23454.
(30) Goppelt-Struebe, M.; Rehfeldt, W. Glucocorticoids inhibit TNF
R-induced cytosolic phospholipase A2 activity. Biochim. Biophys.
Acta 1992, 1127, 163-167.
(31) Schalkwijk, C. G.; Vervoordeldonk, M.; Pfeilschifter, J .; van den
Bosch, H. Interleukin-1â-induced cytosolic phospholipase A2
activity and protein synthesis is blocked by dexamethasone in
rat mesangial cells. FEBS Lett. 1993, 333, 339-343.
(32) Trimble, L. A.; Street, I. P.; Perrier, H.; Tremblay, N. M.; Weech,
P. K.; Bernstein, M. A. NMR structural studies of the tight
complex between a trifluoromethyl ketone inhibitor and the 85-
kDa human phospholipase A2. Biochemistry 1993, 32, 12560-
12565.
(33) McGregor, W. H.; Chang, J . Y. Preparation of R-aminoalkana-
mides as phospholipase A2 inhibitors. PCT Int. Appl. WO 88
06,885, 1988.
(34) Marshall, L. A.; Chang, J . Y. Pharmacological control of phos-
pholipase A2 activity in vitro and in vivo. In Advances in
Experimental Medicine and Biolology Phospholipase A2: Role
and Function in Inflammation; Wong, P. Y. K., Dennis, E. A.,
Eds.; Plenum Press: New York, 1990; Vol. 275, pp 169-182.
(35) Ma¨rki, F.; Breitenstein, W.; Beriger, E.; Bernasconi, R.; Cara-
vatti, G.; Francis, J . E.; Paioni, R.; Wehrli, H. U.; Wiederkehr,
R. Differential inhibition of human secretory and cytosolic
phospholipase A2. Agents Actions 1993, 38, 202-211.
(36) Lehr, M. 3-(Octadecanoylaminomethyl)indole-2-carboxylic acid
derivatives and 1-methyl-3-octadecanoylindole-2-carboxylic acid
as inhibitors of cytosolic phospholipase A2. Arch. Pharm. Pharm.
Med. Chem. 1996, 329, 386-392.
(37) Murakami, Y.; Masanobu, T.; Tanaka, K.; Yuusaku, Y. A simple
general method for the acylation of ethyl indole-2-carboxylates.
Heterocycles 1980, 14, 1939-1941.
(38) Lehr, M. In-vitro assay for the evaluation of phospholipase A2
inhibitors using bovine platelets and HPLC with UV-detection.
Pharm. Pharmacol. Lett. 1992, 2, 176-179.
(39) Wijkander, J .; Sundler, R. Regulation of arachidonate-mobilizing
phospholipase A2 by Ca2+ and by phosphorylation via protein
kinase C. J . Lipid Mediators Cell Signalling 1994, 10, 23-24.
(40) Urata, C.; Siraganian, R. P. Pharmacologic modulation of the
IgE or calcium ionophore A23187 mediated calcium influx,
phospholipase A2 activation, and histamine release in rat
basophilic leukemia cells. Arch. Allergy Appl. Immunol. 1985,
78, 92-100.
(41) Lehr, M. In vitro assay for the evaluation of inhibitors of 85kDa
cytosolic phospholipase A2 by measuring phorbol ester-induced
arachidonic acid release from bovine platelets with HPLC/UV-
detection. Pharm. Pharmacol. Lett. 1995, 5, 108-111.
(42) Qiu, Z. H.; Leslie, C. C. Protein kinase C-dependent and
-independent pathways of mitogen-activated protein kinase
activation in macrophages by stimuli that activate phospholipase
A2. J . Biol. Chem. 1994, 269, 19480- 19487.
(43) Visnjic, D.; Batinic, D.; Lasic, Z.; Knotek, M.; Marusic, M.; Banfic,
H. Phorbol 12-myristate 13-acetate-mediated signalling in mu-
rine bone marrow cells. Biochem. J . 1995, 310, 163-170.
(44) Kast, R.; Fu¨rstenberger, G.; Marks, F. Phorbol ester TPA- and
bradykinin-induced arachidonic acid release from keratinocytes
is catalyzed by a cytosolic phospholipase A2 (cPLA2). J . Invest.
Dermatol. 1993, 101, 567-572.
(45) Bartoli, F.; Lin, H.-K.; Ghomashchi, F.; Gelb, M. H.; J ain, M.
K.; Apitz-Castro, R. Tight binding inhibitors of 85-kDa phos-
pholipase A2 but not 14-kDa phospholipase A2 inhibit release of
free arachidonate in thrombin-stimulated human platelets. J .
Biol. Chem. 1994, 269, 15625-15630.
(22) Faili, A.; Emadi, S.; Vargaftig, B.; Hatmi, M. Dissociation
between the phospholipases C and A2 activities in stimulated
platelets and their involvement in the arachidonic acid libera-
tion. Br. J . Haematol. 1994, 88, 149-155.
(23) Angel, J .; Berenbaum, F.; Le Denmat, C.; Nevalainen, T.;
Masliah, J .; Fournier, C. Interleukin-1-induced prostaglandin
E2 biosynthesis in human synovial cells involves the activation
of cytosolic phospholipase A2 and cyclooxygenase-2. Eur. J .
Biochem. 1994, 226, 125-131.
(24) Hulkower, K. I.; Wertheimer, S. J .; Levin, W.; Coffey, J . W.;
Anderson, C.M.; Chen, T.; DeWitt, D. L.; Crowl, R. M.; Hope,
W. C.; Morgan, D. W. Interleukin-1â induces cytosolic phospho-
lipase A2 and prostaglandin H synthase in rheumatiod synovial
fibroblasts. Evidence for their roles in the production of pros-
taglandin E2. Arthritis Rheumatism 1994,37, 653-661.
(25) Connolly, S.; Robinson, D. H. A new phospholipase A2 comes to
the surface. Drug News Perspect. 1993, 6, 584-590.
(26) Clark, J . D.; Schievella, A. R.; Nalefski, E. A.; Lin, L. L. Cytosolic
phospholipase A2. J . Lipid Mediators Cell Signalling 1995, 12,
83-117.
(46) Lehr, M. Unpublished results.
(47) Laufer, S.; Lehr, M.; Tries, S. Effects of ML 3116, an inhibitor
of cPLA2, on zymosan- and carrageenan-induced paw edema.
Prostaglandins, Leukotrienes Essent. Fatty Acids 1996, 55
(Suppl. 1), 91.
(48) Schievella, A. R.; Regier, M. K.; Smith, W. L.; Lin, L. L. Calcium-
mediated translocation of cytosolic phospholipase A2 to the
nuclear envelope and endoplasmic reticulum. J . Biol. Chem.
1995, 270, 30749-30754.
(49) Capuano, L.; Ahlhelm, A.; Hartmann, H. New syntheses of
2-acylbenzofurans, 2-acylindoles, 2-indolylcarboxylates, and 2-qui-
nolones by intramolecular Wittig reaction. Chem. Ber. 1986, 119,
2069-2074.
(50) J ohnson, J . R.; Hasbrouck, R. B.; Dutcher, J . D.; Bruce, W. F.
Gliotoxin. V. The structure of certain indole derivatives related
to gliotoxin. J . Am. Chem. Soc. 1945, 67, 423-430.
(51) Pierce, J . S.; Salsbury, J . M.; Fredericksen, J . M. Local anesthe-
tics. I. â-Monoalkylaminoethyl esters of alkoxybenzoic acids. J .
Am. Chem. Soc. 1942, 64, 1691-1694.

Inhibitors of the Cytosolic Phospholipase A2
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 17 2705
(52) J ones, B. The Halogenation of phenolic ethers and anilides. Part
XIV. m-Substituted phenyl ethers. J . Chem. Soc. 1943, 430-
432.
in the Fischer-Fink type synthesis. Monatsh. Chem. 1973, 104,
925-932.
(62) Salmon-Legagneur, F.; Neveu, C. R,R-Disubstituted diacids and
derivatives. VIII. R,R-Diphenylsuberic, R,R-diphenylazelaic, and
R,R-diphenylsebacic acids. Bull. Soc. Chim. Fr. 1956, 1345-1350.
(63) Tranchepain, I.; Le Berre, F.; Dure´ault, A.; Le Merrer, Y.;
Depezay, J . C. Total enantiospecific syntheses of 13(S)-hydroxy-
9Z,11E-octadecadienoic (coriolic) acid and 13(S)-N-tosylamino
analogue. Tetrahedron 1989, 45, 2057-2065.
(53) Gapinski, D. M.; Mallett, B. E.; Froelich, L. L.; J ackson, W. T.
Benzophenone dicarboxylic acid antagonists of leukotriene B4.
1. Structure-activity relationships of the benzophenone nucleus.
J . Med. Chem. 1990, 33, 2798-2807.
(54) Schubert, H.; Zaschke, H. Synthesis of crystalline liquid com-
pounds. III. n-Alkyl and n-alkoxy derivatives of 2,5-diphenyl-
pyrimidine. J . Prakt. Chem. 1970, 312, 494-506.
(64) Salmon-Legagneur, F.; Neveu, C.; Belot, A. R,R-Disubstituted
dioic acids and their derivatives. X. R,R-Diphenyl straight-chain
dioic acids from C11 to C14. Bull. Soc. Chim. Fr. 1957, 1463-
1469.
(65) Wang J .-Y, J en Y.-F. Tumor chemotherapy. XXI. Synthesis of
p-, m-, and o-bis(2-chloroethyl)aminomethylcinnamic acid hy-
drochloride and their esters. Yao Hsueh Hsueh Pao 1964, 11,
591-595; Chem. Abstr. 1965, 62:1592a.
(66) Caldwell, J . R.; J ackson, W. J . p-(Aminoethoxy)benzoic acids.
U.S. 2,790,825, 1957.
(55) Diczfalusy, E.; Ferno¨, O.; Fex, H.; Ho¨gberg, B. Long-acting
p-alkoxyhydrocinnamic acid esters of steroid hormones. Acta
Chem. Scand. 1963, 17, 2536-2547.
(56) Parmerter, S. M.; Cook, A. G.; Dixon, W. B. The synthesis of
4-nitro-, 5-nitro-, 6-nitro- and 7-nitroindole. J . Am. Chem. Soc.
1958, 80, 4621-4625.
(57) Suehiro T.; Niitsu, M. 4-Acetylation of 5-hydroxyindole deriva-
tives. Bull. Chem. Soc. J pn. 1971, 44, 550-553.
(58) Uhle, F. C. The synthesis of 5-keto-1,3,4,5-tetrahydrobenz[cd]-
indole. A synthesis of 4-substituted indoles. J . Am. Chem. Soc.
1949, 71, 761.
(67) Nippon Kayaku Co. 2-Bromoethyl aryl thio ethers. Fr. 1,403,337,
1965.
(59) Fox, S. W.; Bullock, M. W. Synthesis of indole-3-acetic acids and
2-carboxyindole-3-acetic acids with substituents in the benzene
ring. J . Am. Chem. Soc. 1951, 73, 2756-2759.
(68) Rosnati, V.; Pu¨schner, H. Synthetic curare compounds. IX. Ether-
esters of choline with p-hyroxyphenyl-substituted carboxylic
acids. Gazz. Chim. Ital. 1957, 586, 586-596.
(60) Pappalardo, G.; Vitali, T. Indoles. IV. Ultraviolet absorption
spectra of haloindoles and halo-2-indolecarboxylic acids and
esters. Gazz. Chim. Ital. 1958, 88, 1147-1169.
(69) Lehr, M. Preparation of acylpyrrolylalkanoates and related
compounds as inhibitors of phospholipase A2. Ger. Offen. DE
4,325,204.
(61) Falk, H.; Hofer, O.; Lehner, H. Concerning the selectivity
problem in the synthesis of pyrroles: isomers and homologues
J M960863W