Indole inhibitors of human nonpancreatic secretory phospholipase A2. 1. Indole-3-acetamides

Article abstract of DOI:10.1021/jm960485v

Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A₂ (hnps-PLA₂) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA₂. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure-activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X- ray crystal structures of complexes between the inhibitors and hnps-PLA₂ which were of great value in directing the SAR.

Full text of DOI:10.1021/jm960485v

J . Med. Chem. 1996, 39, 5119-5136
5119
In d ole In h ibitor s of Hu m a n Non p a n cr ea tic Secr etor y P h osp h olip a se A2. 1.
In d ole-3-a ceta m id es
Robert D. Dillard, Nicholas J . Bach,* Susan E. Draheim, Dennis R. Berry, Donald G. Carlson,
Nickolay Y. Chirgadze, David K. Clawson, Lawrence W. Hartley, Lea M. J ohnson, Noel D. J ones,
Emma R. McKinney, Edward D. Mihelich, J ennifer L. Olkowski, Richard W. Schevitz, Amy C. Smith,
David W. Snyder, Cynthia D. Sommers, and J ean-Pierre Wery
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285
Received J uly 5, 1996X
Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of
biologically active lipids involved in inflammatory processes. Increased levels of human
nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathologi-
cal conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening
program was carried out to identify chemical structures which could inhibit hnps-PLA2. One
of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-
(phenylmethyl)-1H-indole-3-acetic acid (13a ). We describe the syntheses, structure-activity
relationships, and pharmacological activities of a series of indole-3-acetamides and related
compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal
structures of complexes between the inhibitors and hnps-PLA2 which were of great value in
directing the SAR.
In tr od u ction
availability of the enzyme allowed for the development
of a secondary in vitro assay employing lung pleural
tissue as the “natural substrate” for hnps-PLA2. In this
tissue assay system,9 guinea pig pleural lung strips were
challenged with hnps-PLA2, resulting in a contractile
response which could be quantitated. Any of the above
hits that inhibited the response were potential hnps-
PLA2 inhibitors. However, this was only an indirect
measure of hnps-PLA2 inhibitory activity since the
contractile response is mediated by the catalytic release
of arachidonic acid and the subsequent formation of
eicosanoids. Cyclooxygenase or lipoxygenase inhibitors,
which act downstream to PLA2 in the arachidonic acid
cascade, would also suppress the contractile response
although they are not hnps-PLA2 inhibitors. To elimi-
nate these false positives, tissues were challenged in a
separate experiment with exogenously administered
arachidonic acid, in the presence and absence of drug,
and inhibition of the contractile response was measured.
The presence of arachidonic acid circumvents the re-
quirement for its PLA2-mediated release, and any agent
that demonstrated suppression of the arachidonic acid-
induced response, as would a cyclooxygenase inhibitor,
such as indomethacin, could be ruled out as a specific
inhibitor of hnps-PLA2 and was of less interest.
Human nonpancreatic secretory phospholipase A2
(hnps-PLA2) has been isolated from human synovial
fluid as well as other human cells and identified1 to be
a 14 kDa stable protein whose properties have been well
characterized. This enzyme contains the site charac-
teristic of phospholipases which is capable of hydrolyz-
ing the sn-2 position of certain cellular phospholipids,
liberating arachidonic acid and leading to the biosyn-
thesis of eicosanoid products (prostaglandins, throm-
boxanes, leukotrienes). The exact physiological role of
this enzyme is not known; however, high levels of
enzyme have been observed in the synovial fluid of
arthritic joints2 and in the serum of patients with severe
acute pancreatitis,3 septic shock,4 and multiple injuries.5
A potent and selective inhibitor of hnps-PLA2 would be
a useful pharmacological tool for investigating its
biological role in these diseases and other diseases
where high levels of eicosanoids are thought to have a
role.6 Such an inhibitor would have the potential of
being developed as a useful drug in treating diseases
where elevated levels of the enzyme are found.
A program was initiated to find novel structures that
were inhibitors of hnps-PLA2 and could be investigated
chemically in structure-activity relationship (SAR)
studies. Initially, the inhibition of hnps-PLA2 activity
was determined by measuring the release of 14C oleate
from [14C]oleate-labeled autoclaved Escherichia coli
membranes.7 This system was easily automated, used
very small amounts of enzyme, and was extremely
sensitive. The “hit” rate (compounds with IC50’s less
than 50 µM) was quite high, and it was necessary to
develop a secondary test where the hits could be further
evaluated to verify that activity was true inhibition at
the enzyme active site and not a nonspecific perturba-
tion of the enyzme or its substrate.
Using this tissue assay system, most of the hits from
the E. coli assay were eliminated in that they did not
inhibit the contractile response induced by hnps-PLA2.
The contraction was inhibited by 5-methoxy-2-methyl-
1-(phenylmethyl)-1H-indole-3-acetic acid (13a ), identify-
ing this compound as one of the first viable inhibitors.
This indole-3-acetic acid derivative blocked the effect
of hnps-PLA2, giving an apparent KB of 2.89 ( 0.86 µM.
At higher drug concentrations, it also antagonized the
effects of arachidonic acid; however, concentration
response comparisons indicated a component that most
likely reflected the blocking of hnps-PLA2. A large
number of related indole-3-acetic acids were made and
evaluated as hnps-PLA2 inhibitors using the E. coli
substrate system, but none of these were much more
Using recombinant DNA methods,8 a large quantity
of hnps-PLA2 was supplied for this program. This ready
X Abstract published in Advance ACS Abstracts, December 1, 1996.
S0022-2623(96)00485-2 CCC: $12.00 © 1996 American Chemical Society

5120 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Ta ble 1. Inhibitory Activity of 3-Substituted 1H-Indoles
Dillard et al.
crystallization studies in order to obtain further 3-di-
mensional information to facilitate future drug design.
Ch em istr y
The indole-3-acetamides and related compounds were
synthesized by known chemical processes. The general
synthetic route involved an intermediary indole unsub-
stituted in the 1- and 3-positions. Some of these indoles
(4) were commerically available, and others were made
by reported methods (see the Experimental Section).
Indoles 4, with a 2-alkyl group and a 4-, 5-, or 6-methoxy
substituent, were synthesized using substituted 2-me-
thylanilines 1 as starting materials (see Scheme 1). This
conversion was accomplished by procedures described
by Clark et al.13 in which treatment of 2-methyl-N-BOC-
anilines 2 with 2 equiv of sec-butyllithium afforded the
stabilized dianions which were acylated by N-methyl-
N-methoxyamides to produce ketones 3. These ketones
could be cyclized to N-BOC-indoles with dilute trifluo-
roacetic acid in dichloromethane and then hydrolyzed
to indoles 4 with aqueous base or cyclized and depro-
tected by trifluoroacetic acid to indoles 4. To obtain
2-ethyl-6-isopropropyl-5-methoxy-1H-indole (4e), the
5-isopropropyl-4-methoxy-2-methylaniline precursor was
prepared in two steps from aminothymol.14 The ami-
nothymol was first reacted with di-tert-butyl dicarbon-
ate, and then the hydroxy intermediate, 2d , was treated
with sodium hydride and methyl iodide to give 2e.
Similarly, to obtain 2-ethyl-6-methyl-5-methoxy-1H-
indole (4f), the 2,5-dimethyl-4-methoxyaniline precur-
sor, 2g, was prepared from 2f.
chromogenic assay
compd
R8
IC50 (µM)
mole fractiona
13a
9a n
17c
20b
21b
10f
24
25
27
28a
28b
29a
29b
CH2CO2H
13.6 ( 4.2
115.8 (n ) 1)
0.84 ( 0.17
1.23 ( 0.17
20.06 (n ) 1)
0.86 ( 0.09
28.1 ( 6.7
74.1 (n ) 1)
b
84.7 (n ) 1)
64.7 ( 7.1
141.8 (n ) 1)
40 ( 8
1.1 × 10-2
9.4 × 10-2
6.8 × 10-4
1.0 × 10-3
1.6 × 10-2
7.0 × 10-4
2.3 × 10-2
6.0 × 10-2
6.9 × 10-2
5.3 × 10-2
1.2 × 10-1
3.3 × 10-2
CH2CO2CH3
CH2CONH2
COCONH2
CH(OH)CONH2
CH2CONHNH2
CH2CH2CONH2
CH2CSNH2
CH2CN
CH2CN4Hc
CH2CNHNH2
CH2CONHCH3
CH2CONHOCH3
a
Mole fraction is the IC50 concentration value divided by the
b
total lipid concentration, 1230 µM. 47% inhibition at 45 µg/mL
(highest dose tested), c CN4H ) 5-(1H-tetrazoyl).
potent than the original lead compound 13a , and all
showed considerable inhibition of the arachidonic acid-
induced contractile response.
The availability of large quantites of recombinant
hnps-PLA2 aided the resolution of the X-ray crystal
structure of the enzyme.10 The crystallization of the
enzyme complexed with 13a was also accomplished and
recently reported by Schevitz et al.11a,b The 3-dimen-
sional structure was solved and the compound was in
fact located in the active site. These X-ray crystal-
lography studies and pH dependency studies suggested
that the carboxylic acid functionality should be replaced
with an amide functionality. The syntheses and biologi-
cal evalution of a series of 1H-indole-3-acetamides and
related acetic acid hydrazides are the topic of this
publication.
The 2-chloroindole 4n (Scheme 2) was obtained by
reacting 5-methoxy-1H-indole with formaldehyde and
dimethylamine to give 1-[(dimethylamino)methyl]-5-
methoxy-1H-indole (8), and then the lithium salt15 was
treated with benzenesulfonyl chloride and the ortho-
directing (dimethylamino)methyl group was hydrolyzed
with aqueous hydrochloric acid.
An acetic acid ester group was introduced into the
indole 3-positon by reacting the zinc salt of 4 with
2-bromoalkanoic acid esters16 to give the indole acetic
acid esters 5 (Scheme 2). The use of zinc salts of indoles
is the preferred method for producing 3-alkylation or
acylation without complication by reaction at the 1-posi-
tion. When the indole-3-acetic acids were available,
such as 6a , their methyl esters could be obtained by
treatment with methanol and acid. In some cases,
4-substituted phenylhydrazines (7) were converted by
the classical Fischer-indole synthesis17 to give 5 directly.
The indole-3-acetic acid esters were alkylated on the
indole nitrogen by forming the sodium salt with sodium
hydride or the potassium salt with potassium tert-
butoxide and treating with an arylmethyl halide to give
9 (Scheme 3). Following the N-alkylation, the 5-bromo
substituent of 9g could be replaced by phenyl to give
9h using the palladium(0)-catalyzed coupling of aryl-
boronic acids and aryl halides as described by Miyura
et al.18 Catalytic hydrogenation of 9c gave the 5-hy-
droxyindole, 9a i. Similarly, the 1-[(3-hydroxyphenyl)-
methyl]indole-3-acetic acid ester (9a l) and the 1-[(3-
aminophenyl)methyl]indole-3-acetic acid ester (9a k )
were obtained by catalytic hydrogenation of their in-
termediate benzyl ether and nitro derivatives, respec-
tively. The 5-carboxyindole-3-acetic acid ester 9a j was
obtained after basic hydrolysis of the diester of 9a h on
Although the hnps-PLA2 assay using E. coli mem-
branes as substrate was indeed able to identify inhibi-
tors, it was far too sensitive to other factors and gave
many erroneous results. Other assay systems were
investigated, and the system that used the synthetic
substrate 1,2-bis(heptanoylthio)-1,2-dideoxy-rac-glycero-
3-phosphorylcholine described by Reynolds et al.12 was
selected. In this test, hnps-PLA2 hydrolyzes the sn-2
thioester to liberate a free thio that reacts with the thio-
sensitive 5,5-dithiobis(2-nitrobenzoic acid) to give the
chromogenic agent, 5-thio-2-nitrobenzoic acid. This was
measured spectrophotometrically at 405 nM and al-
lowed for easy automation of the system. The enzyme
is not nearly as efficient in hydrolyzing the synthetic
substrate as for the E. coli membranes, and much larger
quantities of hnps-PLA2 have to be used in the test.
When this system was used in the broad screening, very
few hits were obtained; however, practically all of the
hits were corroborated very well by the secondary
guinea pig lung tissue test.
This chromogenic test system was used to evaluate
all of the newly synthesized amides and related com-
pounds as hnps-PLA2 inhibitors, and selected com-
pounds were further evaluated in the guinea pig lung
tissue test. Compounds showing interesting activities
in both of these systems were candidates for enzyme

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5121
Sch em e 1. Preparation of 2-Alkylindolesa
a
Reagents: (a) (t-BuO2C)2O, THF, heat; (b) 2 equiv of s-BuLi, R2CON(OMe)Me, THF, -40 °C f room temperature; (c) CF3CO2H or (1)
CF3CO2H, CH2Cl2, (2) NaOH, EtOH, H2O, heat; (d) NaH, CH3I, THF.
Sch em e 2. Preparation of Indole-3-acetic Acid Estersa
a
Reagents: (a) (1) n-BuLi, ZnCl2, THF, 0 °C, (2) BrCH2CO2R4, toluene, room temperature; (b) CH3SO3H, CH3OH; (c) HO2CCH2CH2COCH3,
HCl, EtOH, heat; (d) HCHO, H2O, THF, Me2NH, heat; (e) (1) s-BuLi, benzenesulfonyl chloride, THF, -50 °C, (2) HCl, H2O.
attempted crystallization of the resulting dicarboxylic
acid from methanol.
of the 2-chloroindole (the preferred route to the 2-chloro
compounds is that of Scheme 2). The methylthio group
of 9a r was readily oxidized to methylsulfinyl with
m-chloroperbenzoic acid to give 9a s.
The 1-phenylindole-3-acetic acid ester 9a m was ob-
tained by indolization of the diphenylhydrazone, 11,
with phosphorous trichloride19 (Scheme 4). This in-
dolization procedure, developed by Baccolini et al., is
quite distinct in mechanism from the Fischer process.
The phosphorous trichloride-induced reaction tolerates
A bromine atom was introduced into the 2-position
of 9b with N-bromosuccinimide to give 9m (Scheme 3).
Similarly, 9a o was reacted with N-bromosuccinimide
to give the 2-bromoindole 9a q (Scheme 4). When 9a o
was reacted with sulfuryl chloride and boron trifluoride
etherate, or with methylsulfenyl chloride, the 2-chloro-
and 2-(methylthio)indoles 9a p and 9a r were obtained,
respectively. The chlorination reaction gave a poor yield

5122 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Dillard et al.
Sch em e 3. Preparation of 1-Substituted Indole-3-acetic
sponding 1-(arylmethyl)indole-3-acetic acid hydrazides
(10, 16) by heating with hydrazine in ethanol. These
hydrazides served, not only as intermediates, but as
interesting compounds for evaluating as PLA2 inhibi-
tors.
Acid Esters and Hydrazidesa
Heating the hydrazides with Raney nickel in ethanol
gave the corresponding 1-(arylmethyl)indole-3-aceta-
mides 17 (Scheme 6). Amides 17m -o were obtained
by reacting the corresponding precursor esters with
methylchloroaluminum amide20 since the presence of
halogen and sulfur would have complicated the ap-
proach involving reductive cleavage of the hydrazides
to produce the amides. Amides unsubstituted at the
2-position could be treated with methanesulfenyl chlo-
ride to give the 2-methylthio deriative, such as 17p .
Treatment of indole-3-acetic acids with methyl chloro-
formate and triethylamine, followed by reaction of the
intermediate mixed anhydride with ammonia, offered
another route to the indole-3-acetamides, such as 17q.
This sequence, followed by acylation of the indole
sodium salt, was utilized in the preparation of the
1-benzoylindole 17t.
Additional 1-(arylmethyl)indole-3-acetamides were
made by the route shown in Scheme 7. Indoles 4 were
first alkylated on nitrogen with the arylmethyl group
to give 19, and these were reacted with oxalyl chloride
and then ammonia to give the R-oxoacetamides21 20.
Stepwise reduction first with sodium borohydride and
then with triethylsilane/trifluoroacetic acid22 gave the
amides 17u -z. The 5-aminoindole 17a a was obtained
by catalytic hydrogenation of the corresponding nitro
compound 17w .
The higher homolog, indole-3-propionamide 24 (Scheme
8), was made by methods previously described for
the indole-3-acetamide series, derived from a Fischer-
indole product. The indole-3-thioacetamide 25 was
made by heating the acetamide 17c with Lawesson’s
reagent23 in toluene. The analogous amidine compound
28b was obtained by treating 5-methoxy-2-methyl-1-
(phenylmethyl)-1H-indole-3-acetonitrile (27) with me-
thylchloroaluminum amide24 in toluene. When the
same intermediate (27) was treated with triethylam-
monium azide in DMF, the 3-(tetrazoylmethyl)indole
28a was obtained. The N-methyl- and N-methoxya-
mides 29a ,b were formed by treatment of 13a with
methyl chloroformate and the appriopriate substituted
amine.
a
Reagents: (a) NaH or t-BuOK, arylmethyl halide, DMF; (b)
[(C6H5)3P]4Pd(0), phenylboronic acid, Na2CO3, H2O, benzene,
EtOH, heat; (c) H2, Pd/BaSO4, EtOH; (d) (1) NaOH, EtOH, H2O,
heat, (2) HCl, H2O; (e) H2, Pd/C, EtOH; (f) NBS, CCl4; (g)
hydrazine, EtOH, heat.
P h a r m a cology
The initial screening program utilized the hnps-PLA2-
induced release of [14C]oleate from E. coli membranes
to identify and evaluate inhibitors of recombinant
human secreted phospholipase A2. This E. coli assay
was later replaced with a chromogenic assay system
utilizing a synthetic substrate, 1,2-bis(heptanoylthio)-
1,2-dideoxy-rac-glycero-3-phosphorylcholine. A general
description of the method has been published.12 The
assay has been adapted for high-volume screening using
96-well microtiter plates. All compounds were tested
in triplicate. Typically, compounds were tested at a
final concentration of 5 µg/mL. Lack of color develop-
ment evidenced inhibition. Compounds initially found
to be active were reassayed to confirm their activity,
and if they were sufficiently active, IC50 values were
determined.
a variety of functional groups and is particularly effec-
tive for the preparation of 1-substituted indoles from
N-substituted hydrazine precursors.
Indole-3-acetic acid esters such as 9a n were prepared
from the precursor indole-3-acetic acid by heating with
methanesulfonic acid in methanol.
To obtain indoles where the R-carbon atom of the
acetic acid ester group was substituted (15a -c) (Scheme
5), the zinc salts of the appropriate starting indoles were
treated with R-bromoalkanoic acid esters, and the
resulting indoles were N-alkylated by the reaction
sequences previously described.
The 1-(arylmethyl)indole-3-acetic acid esters 9 and 15
of Schemes 3-5 were readily converted to the corre-

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5123
Sch em e 4. Alternate Preparation of 1-Substituted Indole-3-acetic Acid Esters and Hydrazidesa
a
Reagents: (a) EtO2CCH2CH2COCH3, benzene, heat; (b) PCl3, dichloroethane, heat; (c) hydrazine, EtOH, heat; (d) NaH, benzyl bromide,
DMF; (e) (1) NaOH, H2O, EtOH, (2) HCl, H2O; (f) MeSO3H, MeOH, heat; (g) SO2Cl2, BF3‚OEt2, CH2Cl2; (h) NBS, CCl4; (i) MeSCl, CH2Cl2;
(j) m-chloroperbenzoic acid, CH2Cl2.
Ta ble 2. Inhibitory Activity of 1H-Indole-3-acetic Acid
Hydrazides
Sch em e 5. Preparation of R-Alkylindole-3-acetic Acid
Hydrazidesa
contraction of GP lung tissue
chromogenic assay
PLA2-induced AA-induced
IC50 (µM)
(n ) 3)
mole
fraction
apparent KB
(µM) (n ) 4)
ED50 (µM)
(n ) 4)
compd
10a
10d
10e
10f
10g
10h
10i
10j
10k
10n
10o
10p
10q
10r
10s
10t
4.9 ( 1.2
4.0 × 10-3 5.64 ( 0.79
>10
>30
2.36 ( 0.60 1.9 × 10-3
0.55 ( 0.27 4.5 × 10-4
0.85 ( 0.14 6.9 × 10-4 1.57 ( 0.23
1.02 ( 0.43 8.3 × 10-4
0.86 ( 0.20 7.0 × 10-4
0.42 ( 0.16 3.4 × 10-4 2.04 ( 0.25
>30
>30
70.5 ( 2.2
5.7 × 10-2 27.13 ( 7.04
0.39 ( 0.13 3.2 × 10-4
5.32 ( 1.36 4.3 × 10-3
40.2 ( 2.7
3.3 × 10-2
2.12 ( 0.27 1.7 × 10-3
0.61 ( 0.17 5.0 × 10-4 3.86 ( 0.35
15.22 ( 0.55 1.2 × 10-2
>30
>30
1.71 ( 0.18 1.4 × 10-3
0.23 ( 0.09 1.9 × 10-4
a
1.36 ( 0.34 1.1 × 10-3 1.13 ( 0.25
9.28 ( 1.55 7.5 × 10-3
Reagents: (a) (1) n-BuLi, ZnCl2, THF, 0 °C; (2) BrCHRCO2Et,
10u
10w
10x
toluene, room temperature; (b) NaH, benzyl halide, DMF; (c)
hydrazine, EtOH, heat.
9.39 ( 1.56 7.6 × 10-3
10y
0.64 ( 0.13 5.2 × 10-4
10a a
10a b
10a c
10a d
10a f
10a g
10a j
10a k
10a l
10a m
10a p
10a q
10a r
10a s
16a
0.26 ( 0.06 2.1 × 10-4 >10
2.35 (n ) 1) 1.9 × 10-3 30.10 ( 4.71
0.94 ( 0.27 7.6 × 10-4 22.62 ( 5.43
27.18 ( 6.06 2.2 × 10-2
>10
>30
A guinea pig lung tissue bath assay was developed
to serve as the secondary in vitro assay to evaluate
inhibitors of hnps-PLA2. The rationale for establishing
this assay was based on the knowledge that hnps-PLA2
requires mM calcium to become catalytically active, and
this requirement can be met by its secretion from a cell
into the external environment. Challenging isolated
guinea pig lung pleural strips with hnps-PLA2, with mM
concentrations of calcium in the bath fluid, would be
analogous to the cellular secretion of the protein into
the tissue environment. This tissue preparation is
sensitive to the contractile effects of eicosanoids which
are formed from the arachidonic acid released via the
catalytic action of hnps-PLA2 on the tissues.
9.43 ( 2.35 7.7 × 10-3
16.7 ( 2.2
1.4 × 10-2
0.36 ( 0.02 2.9 × 10-4 1.39 ( 0.21
>30
>30
4.32 ( 0.60 3.5 × 10-3
3.0 ( 0.93
2.4 × 10-3 5.96 ( 0.91
3.1 × 10-2
38.2 ( 0.70
0.39 ( 0.03 3.2 × 10-4 0.85 ( 0.26
0.43 ( 0.02 3.5 × 10-4 0.76 ( 0.18
0.62 ( 0.10 5.0 × 10-4
9.37 ( 2.03
7.32 ( 1.75
46.0 ( 18.0
3.7 × 10-2
2.0 × 10-2
32.64 (n ) 1) 2.7 × 10-2
16b
16c
24.2 ( 7.4
101.2 (n ) 1) 8.2 × 10-2
Discu ssion
for their ability to inhibit the action of this enzyme. This
screening effort identified 5-methoxy-2-methyl-1-(phe-
nylmethyl)-1H-indole-3-acetic acid (13a ) as a potential
inhibitor of hnps-PLA2. This hit was verified by the
An assay measuring the hnps-PLA2-induced release
of [14C]oleate from [14C]oleate-labeled E. coli membranes
was used to screen a wide variety of chemical structures

5124 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Sch em e 6. Preparation of Indole-3-acetamidesa
Dillard et al.
a
Reagents: (a) Raney Ni, EtOH, heat; (b) MeClAlNH2, benzene/toluene; (c) CH3SCl, CH2Cl2; (d) (1) NaOH, EtOH, H2O, (2) HCl, H2O;
(e) (1) MeO2CCl, Et3N, EtOAc, (2) ammonia; (f) NaH, C6H5COCl, DMF.
secondary guinea pig lung pleural strip tissue assay
system, and a SAR for this lead compound was under-
taken to optimize its inhibitory activity.
mogenic assay results and to demonstrate that the
inhibitors were free of cyclooxygenase and lipoxygenase
inhibiting activity.
The preparation of numerous indole-3-acetic acid
derivatives with various substituents at other positions
of the indole nucleus produced only moderate changes
in activity. X-ray crystallographic studies of the crys-
talline complex between 13a and hnps-PLA2 showed
that the indole was located in the active site of the
enzyme. However, no active site calcium could be seen
in the structure, and the Asp 49, which normally
provides two of the seven ligands for calcium, was
protonated and formed a bifurcated hydrogen bond to
both oxygen atoms of the carboxyl of 13a . A comparison
of this structure with that from a mutant porcine
enzyme and an amide substrate analogue27 (ASA) led
to the supposition that replacing the 3-acetic acid
substituent with a 3-acetamide could enhance activity
by mimicking some of the interactions observed with
the ASA.
At this time the E. coli assay, which had produced a
number of hits not verified by the tissue assay, was
replaced by a chromogenic assay utilizing a thioPC
substrate.12 All of the newly synthesized substituted
indoles were evaluated in the chromogenic assay, and
selected compounds were further evaluated in the tissue
assay. The tissue assay was used to verify the chro-
The acetamide 17c, which differs from the lead
compound 13a only by replacement of the 3-acetic acid
with a 3-acetamide, was synthesized and proved to be
a more potent inhibitor than 13a . The IC50 of 17c in
the chromogenic assay showed a 20-fold improvement
over that of 13a . An X-ray crystal structure obtained
for the complex between 17c and hnps-PLA2 showed the
amide carbonyl interacting with the calcium in the
active site and the amide NH binding to the active site
His 48.
When the 1-benzyl group of 17c was replaced with
large alkyl groups or was substituted at the 2- or
3-positions of the phenyl ring, activity was retained or
improved. Substitution at the 4-position of the phenyl
ring caused a loss of activity, as could be expected from
the X-ray structure of the enzyme complex with 17c,
which shows this position directed into the main chain
of the enzyme.
The indole 2-methyl substituent could be replaced by
halo, methylthio, ethyl, or cyclopropyl to give more
potent inhibitors, while 2-H or other 2-alkyl-substituted
compounds lost effectiveness. These changes in activity
that were seen with variation of the 2-substituent could
also be anticipated from the X-ray structure of the

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5125
Ta ble 3. Inhibitory Activity of 1H-Indole-3-acetamides
Exp er im en ta l Section
contraction of GP lung tissue
PLA2-induced AA-induced
Melting points were obtained on a Thomas-Hoover Mel
Temp and are uncorrected. The NMR data were recorded on
a QE300 instrument. Mass spectral data were obtained on a
VG Analytical 70-SE instrument. The following indoles were
commercially available: 5-methoxy-1H-indole (4k ), 5-(benzy-
loxy)-1H-indole (4m ), 5-methoxy-2-methyl-1H-indole (4o),
4-methoxy-1H-indole (4p ), 2-methyl-1H-indole-3-acetic acid
(6a ), 5-methoxy-2-methyl-1H-indole-3-acetic acid (6b), and
5-methoxy-1H-indole-3-acetic acid (6c). The following com-
pounds were made by the reported syntheses: 6-chloro-5-
methoxy-1H-indole (4l),28 2-methyl-5-nitro-1H-indole (4q),29
and 2-ethyl-4-nitro-1H-indole (4r ).30
N-(ter t-Bu t oxyca r b on yl)-3-m et h oxy-2-m et h yla n ilin e
(2a ). A solution of 3-methoxy-2-methylaniline (1a ) (13.7 g, 100
mmol) and 25 g (115 mmol) of di-tert-butyl dicarbonate in 125
mL of THF was heated slowly to reflux, and reflux was
maintained for 2 h. After cooling, the reaction mixture was
concentrated at reduced pressure and the residue dissolved
in EtOAc. The EtOAc solution was washed with a 1 N citric
acid solution, dried over Na2SO4, and concentrated at reduced
pressure. The residue was crystallized from hexane to give
17.25 g (yield 73%) of 2a : mp 80-82 °C; 1H NMR (DMSO-d6)
∂ 8.52 (br s, 1H), 7.08 (t, 1H), 6.92 (d, 1H), 6.75 (d, 1H), 3.76
(s, 3H), 2.00 (s, 3H), 1.46 (s, 9H); MS (FD) 237 (M+). Anal.
(C13H19NO3) H, N; C: calcd, 65.80; found, 63.32.
chromogenic assay
IC50 (µM)
(n ) 3)
mole
fraction
apparent KB
(µM) (n ) 4)
ED50 (µM)
(n ) 4)
compd
17a
17b
17c
17d
17e
17f
17g
17h
17l
17m
17n
17o
17p
17q
17r
17s
17t
3.7 ( 2.0
3.0 × 10-3
1.9 × 10-3
6.8 × 10-4
2.1 × 10-4
3.1 × 10-2
1.1 × 10-3
7.4 × 10-4
9.0 × 10-3
2.0 × 10-4
1.9 × 10-4
9.8 × 10-5
3.7 × 10-4
2.36 ( 0.15
0.84 ( 0.17
0.26 ( 0.11
38.1 ( 1.9
3.43 ( 0.88 >30
5.91 ( 0.97 >30
0.336 ( 0.023 2.7 × 10-4
1.38 ( 0.42
0.91 ( 0.19
11.13 (n)1)
0.25 ( 0.03
0.23 ( 0.04
0.12 ( 0.02
0.45 ( 0.01
2.05 ( 0.66
7.93 ( 3.52
1.7 × 10-3 22.54 ( 3.91 25.65 ( 5.75
21.36 ( 6.07 1.7 × 10-2
9.47 ( 4.86
7.21 ( 0.70
1.23 ( 0.23
1.51 ( 0.45
1.18 ( 0.39
7.7 × 10-3
5.9 × 10-3
1.0 × 10-3
1.2 × 10-3
9.6 × 10-4
1.3 × 10-3
1.4 × 10-3
4.5 × 10-4
3.1 × 10-2
2.1 × 10-3
9.8 × 10-2
4.3 × 10-2
17v
17w
17x
17a a 1.61 ( 0.32
1.98 ( 0.35 14.50 ( 3.00
20c
20e
20f
20i
21c
21e
1.77 ( 0.15
0.55 ( 0.24
37.8 ( 6.0
2.56 ( 0.55
120.9 ( 7.0
53.0 ( 2.7
Using this procedure, the following were synthesized from
4- and 5-methoxy-2-methylanilines (1b and 1c), respectively.
N-(ter t-Bu t oxyca r b on yl)-4-m et h oxy-2-m et h yla n ilin e
(2b) (crystallization, hexane): yield 73%; mp 80-82 °C; MS
(FD+) 237 (M+). Anal. (C13H19NO3) C, H, N.
N-(ter t-Bu toxycar bon yl)-5-m eth oxy-2-m eth ylan ilin e (2c)
(crude product): yield 80%; mp 76-80 °C; MS (FD+) 237 (M+).
Anal. (C13H19NO3) C, H, N.
enzyme complex with 17c, since there is little room
between the methyl group and the cavity wall. The
2-ethyl group of 17d fills this space, providing a 3-fold
increase in potency. The 2-propyl group of 17e is too
large, and this results in almost a 50-fold loss in activity.
Replacing the 5-methoxy group with various alkoxy,
alkyl, aryl, halo, and carboxyl functionalites produced
only moderate changes in activity. No significant
change in activity was observed when the various
5-substituents were moved to the 4- or 6-positions of
the indole.
Substitution on the nitrogen of the acetamide caused
a large loss of activity with the exception of the 3-ac-
etohydrazides which usually equaled the activity of the
3-acetamides. Alkyl substitution of the R-carbon of the
3-acetohydrazide resulted in decreased activity (16a -
c). The use of thioamide, amidine, tetrazole, nitrile, or
carboxylic acid groups in place of the carboxamide gave
derivatives with little or no ability to inhibit the enzyme,
as did lengthening the side chain as in the 3-propiona-
mide compound 24.
The R-oxo-3-acetamide intermediates (3-glyoxamides)
of the alternate indole-3-acetamide synthesis (Scheme
7) which were tested in the chromogenic assay showed
some interesting activity which was further explored
and is detailed in the third paper of this series.
N-(ter t-Bu toxyca r bon yl-5)-isop r op yl-4-m eth oxy-2-m e-
th yla n ilin e (2e). A solution of 34.5 g (210 mmol) of ami-
nothymol14 (1d ) and 45.8 g (210 mmol) of di-tert-butyl dicar-
bonate in 400 mL of THF was stirred for 4 h. The solvent
was evaporated at reduced pressure, and the residue was
crystallized from 20% EtOAc/hexane to give 53.8 g (yield 97%)
of 2d : mp 154-156 °C; 1H NMR (DMSO-d6) ∂ 9.00 (s, 1H),
8.18 (br s, 1H), 6.88 (s, 1H), 6.55 (s, 1H), 3.16-3.04 (m, 1H),
2.30 (s, 3H), 1.40 (s, 9H), 1.12 (d, 6H). To 27 g (100 mmol) of
this material in 200 mL of DMF was added a suspension of 4
g (100 mmol) of 60% NaH/mineral oil (previously washed with
hexanes), and the mixture was stirred for 1 h. Iodomethane
(6.2 mL, 100 mmol) was added, the reaction mixture was
stirred 17 h, water was added, and the product was extracted
with EtOAc, washed with brine, and dried (MgSO4). The
solvent was evaporated, and the residue was chromatographed
on silica gel, eluting with 20% EtOAc/hexane to give 20.6 g
(yield 74%) of 2e: mp 95-103 °C; 1H NMR (DMSO-d6) ∂ 8.32
(br s, 1H), 7.00 (s, 1H), 6.75 (s, 1H), 3.75 (s, 3H), 3.25-3.08
(m, 1H), 2.13 (s, 3H), 1.46 (s, 9H), 1.13 (d, 6H); MS (FD+) 279
(M+). Anal. (C16H25NO3) H, N; C: calcd, 68.79; found, 69.40.
Using the above procedure, 1f was converted to 2g.
N -(t er t -Bu t oxyca r b on yl)-2,5-d im e t h yl-4-m e t h oxya -
n ilin e (2g) (chromatography on silica gel, 10% EtOAc/hex-
ane): yield 84%; mp 110-113 °C; MS (FD) 251 (M+). Anal.
(C14H21NO3) C, H, N.
1-[2-[(ter t-Bu toxyca r bon yl)a m in o]-4-m eth oxyp h en yl]-
2-p r op a n on e (3b). A solution of 1.3 M sec-butyllithium/
cyclohexane (106 mL, 138 mmol) was added slowly to 16.4 g
(69 mmol) of 2c in 200 mL of THF while the temperature was
kept below -40 °C with a dry ice/ethanol bath. After 0.25 h,
7.1 g (69 mmol) of N-methoxy-N-methylacetamide in an equal
volume of THF was added dropwise. The reaction mixture was
stirred for 1 h, the cooling bath was removed, and the mixture
was stirred an additional 1 h. It was then poured into a
mixture of 500 mL of Et2O and 500 mL of 1 N HCl. The
organic layer was separated, washed with water, dried over
Na2SO4, and concentrated at reduced pressure to give after
chromatography on silica gel, eluting with 33% EtOAc/hexane,
13.8 g (72% yield) of the title compound: 1H NMR (CDCl3) ∂
7.75 (br s, 1H), 7.32 (d, 1H), 6.84 (d, 1H), 6.25 (s, 1H), 3.88 (s,
Su m m a r y
These studies provided compounds that were tighter
binding inhibitors of hnps-PLA2 than the screening hit
13a and approximately 100-fold more potent. In order
to make further improvements to the series, it was
necessary to introduce an additional functional group
to the indole structure whose purpose was to coordinate
to the catalytic calcium.11 The SAR describing these
activities is presented in the following papers.

5126 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Sch em e 7. Alternate Preparation of Indole-3-acetamidesa
Dillard et al.
a
Reagents: (a) NaH, arylmethyl halide, DMF; (b) (1) oxalyl chloride, CH2Cl2, (2) ammonia; (c) NaBH4, EtOH; (d) Et3SiH, TFA; (e) H2,
Pd/C, EtOH.
Sch em e 8. Preparation of Other 3-Substituted Indolesa
a
Reagents: (a) EtO2C(CH2)3COCH3, HCl, EtOH, heat; (b) t-BuOK, benzyl chloride, DMF; (c) (1) MeO2CCl, Et3N, THF, (2) RNH2; (d)
Lawesson’s reagent, toluene, heat; (e) (1) n-BuLi, ZnCl2, THF, 0 °C, (2) BrCH2CN, toluene; (f) Et3N‚HCl, NaN3, DMF, 95-125 °C; (g)
MeClAlNH2, toluene, heat.
3H), 2.54 (s, 3H), 1.69 (s, 9H); MS (FD+) 279 (M+). Anal.
(C15H21N2O4) H, N; C: calcd, 64.50; found, 63.80.
Using this procedure, the following conversions were made
using the appropriate N-methoxy-N-methylamide: 2b to 3d ,
3h and 3j; 2e to 3e.
1-[2-[(ter t-Bu toxyca r bon yl)a m in o]-5-m eth oxyp h en yl]-
2-bu ta n on e (3d ) (chromatography on silica gel, 5% EtOAc/
toluene): yield 74%; mp 80-81 °C; MS (FD+) 293 (M+). Anal.
(C16H23NO4) C, H, N.
1-[2-[(ter t-Bu toxyca r bon yl)a m in o]-4-isop r op yl-5-m eth -
oxyp h en yl]-2-bu ta n on e (3e) (crude product): yield 90%; oil;
1H NMR (DMSO-d6) ∂ 10.55 (br s, 1H), 8.35 (br s, 1H), 7.05 (s,
1H), 6.75 (s, 1H), 3.75 (s, 3H), 3.35 (s, 2H), 3.25-3.05 (m, 1H),
2.45 (q, 2H), 1.40 (s, 9H), 1.10 (d, 6H); MS (FD) 335 (M+). Anal.
(C19H29NO4) H; C: calcd, 68.03; found, 69.99; N: calcd, 4.18;
found, 5.09.
1-[2-[(ter t-Bu toxyca r bon yl)a m in o]-5-m eth oxyp h en yl]-
2-p en ta n on e (3h ) (chromatography on silica gel, 5% EtOAc/
toluene): yield 73%; mp 77-78 °C; MS (FD) 307 (M+). Anal.
(C17H25NO4) C, H, N.
sec-butyllithium/cyclohexane (280 mL, 0.36 mol) and then 18.5
g (0.18 mol) of N-methoxy-N-methylacetamide as described for
3b to give a mixture of 1-[2-[(tert-butoxycarbonyl)amino]-6-
methoxyphenyl]-2-propanone (3a ) and starting anilide, 2a
(NMR analysis). This mixture was dissolved in 100 mL of CH2-
Cl2 and 40 mL of trifluoroacetic acid, stirred for a total of 26
h, washed with water, dried (MgSO4), and concentrated at
reduced pressure. The residue was chromatographed on silica
gel, eluting with 20% EtOAc/hexane to give on crystallization
from CH2Cl2/hexane, 13.9 g (yield 48%, 2 steps) of 4a : mp 80-
86 °C; MS (FD) 161 (M+). Anal. (C10H11NO) C, H, N.
2-Eth yl-4-m eth oxy-1H-in d ole (4c). Using the procedure
described for 4a , 2a was converted to crude 1-[2-[(tert-butoxy-
carbonyl)amino]-6-methoxyphenyl]-2-butanone (3c), which was
reacted with trifluoroacetic acid to give 4c in 48% overall yield
(chromatography on silica gel, 20% EtOAc/hexane): oil; 1H
NMR (CDCl3) ∂ 7.90 (br s, 1H), 7.05 (t, 1H), 6.95 (d, 1H), 6.53
(d, 1H), 6.36 (s, 1H), 3.97 (s, 3H), 2.78 (q, 2H), 1.34 (t, 3H);
MS (FD) 175 (M+). Anal. (C11H13NO) H; C: calcd,75.40; found,
74.41; N: calcd, 7.99; found, 4.97.
[2-[(ter t-Bu toxyca r bon yl)a m in o]-5-m eth oxyp h en yl]m -
eth yl cyclop r op yl k eton e (3j) (crystallization, hexane):
2-Eth yl-5-m eth oxy-6-m eth yl-1H-in d ole (4f). To a solu-
tion of 27.3 g (109 mmol) of 2g in 400 mL of THF at -60 °C
was slowly added 168 mL (218 mmol) of a 1.3 M solution of
s-BuLi in cyclohexane. Following the addition, the cooling
bath was removed until the temperature reached 0 °C, the
bath was then replaced, and 12.8 g (109 mmol) of N-methoxy-
yield 77%; mp 96-97 °C; MS (FD+) 305 (M+). Anal. (C17H23
-
NO4) C, H, N.
4-Meth oxy-2-m eth yl-1H-in d ole (4a ). A solution of 43 g
(0.18 mol) of 2a in 280 mL of THF was treated with 1.3 M

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5127
N-methylpropionamide was added at -60 °C. The cooling bath
was removed, and the reaction mixture was stirred for 16 h
and then poured into Et2O/water. The organic layer was
washed with brine, dried (MgSO4), and evaporated at reduced
pressure to give 24.3 g (yield 73%) of 1-[2-[(tert-butoxycarbo-
nyl)amino]-5-methoxy-4-methylphenyl]-2-butanone (3f). The
crude product was stirred with 20 mL of trifluoroacetic acid
in 400 mL of CH2Cl2 for 5 h, diluted with water, and
neutralized with NaHCO3. The CH2Cl2 layer was washed with
brine, dried (MgSO4), evaporated at reduced pressure, and
then chromatographed on silica gel, eluting with 10% EtOAc/
hexane to give 8.21 g (28.4 mmol, yield 36%) of 1-(tert-
butoxycarbonyl)-2-ethyl-5-methoxy-6-methyl-1H-indole as an
oil. Anal. (C17H23NO3) C, H, N. A solution of this indole and
20 mL of 5 N NaOH in 100 mL of EtOH was heated at reflux
for 18 h. The solution was diluted with water, extracted with
EtOAc, washed with brine, dried (MgSO4), evaporated at
reduced pressure, and then chromatographed on silica gel,
eluting with 10% EtOAc/hexane to give 3.6 g (yield 67%) of 4f
as an oil: MS (FD+) 189 (M+). Anal. (C12H15NO) C, H, N.
Using this procedure, 2a was converted to 4g and 4i.
4-Meth oxy-2-p r op yl-1H-in d ole (4g) (chromatography on
silica gel, 20% EtOAc/hexane): yield 36%; oil; 1H NMR (CDCl3)
∂ 7.89 (br s, 1H), 7.05 (t, 1H), 6.94 (d, 1H), 6.51 (d, 1H), 6.34
(s, 1H), 3.95 (s, 3H), 2.72 (t, 2H), 1.74 (q, 2H), 1.00 (t, 3H).
Anal. (C12H15NO‚0.2EtOAc) C, H, N.
EtOAc/toluene): yield 64%; oil; MS (FD) 253 (M - 1, 100), 255
(M + 1, 40). Anal. (C12H12ClNO3) C, H, N.
5-(Ben zyloxy)-1H-in d ole-3-a cetic a cid eth yl ester (5c)
(chromatography on silica gel, gradient, toluene-5% EtOAc/
toluene): yield 27%; mp 57-59 °C; 1H NMR (DMSO-d6) ∂ 10.80
(br s, 1H), 7.50-7.21 (m, 7H), 7.11 (d, 1H), 6.83 (dd, 1H), 5.08
(s, 2H), 4.08 (q, 2H), 3.67 (s, 2H), 1.71 (t, 3H); MS (FD+) 309
(M+). Anal. (C19H19NO3) C, H; N: calcd, 5.43; found, 4.50.
4-Meth oxy-2-m eth yl-1H-in d ole-3-a cetic a cid eth yl es-
ter (5e) (chromatography on silica gel, 20% EtOAc/hexane):
yield 53%; mp 117-121 °C; MS (FD) 247 (M+). Anal. (C14H17
NO3) C, H, N.
-
2-Eth yl-5-m eth oxy-1H-in d ole-3-a cetic a cid m eth yl es-
ter (5i) (chromatography on silica gel, gradient, toluene-10%
EtOAc/toluene); yield 59%; oil; MS (FD) 247 (M+). Anal.
(C14H17NO3) C, H, N.
5-Meth oxy-2-p r op yl-1H-in d ole-3-a cetic a cid m eth yl es-
ter (5j) (chromatography on silica gel, 20% EtOAc/hexane):
yield 66%; oil; MS (FD+) 261 (M+). Anal. (C15H19NO3) C, H,
N.
2-Cyclopr opyl-5-m eth oxy-1H-in dole-3-acetic acid m eth -
yl ester (5k ) (chromatography on silica gel, gradient, 5-15%
1
EtOAc/toluene): yield 61%; oil; H NMR (DMSO-d6) ∂ 10.35
(br s, 1H), 7.11 (d, 1H), 6.88 (d, 1H), 6.65 (dd, 1H), 3.75 (s,
3H), 3.61 (s, 2H), 2.13-1.9 (m, 1H), 0.98-0.92 (m, 2H), 0.86-
0.77 (m, 2H); MS (FD+) 259 (M+). Anal. (C15H17NO3‚0.9H2O)
C, H, N.
2-Cyclop r op yl-4-m eth oxy-1H-in d ole (4i) (chromatogra-
phy on silica gel, 5% EtOAc/toluene): yield 24%; oil; MS (FD+)
187 (M+). Anal. (C12H13NO‚0.3EtOAc) C, H, N.
2-Meth yl-1H-in d ole-3-a cetic Acid Meth yl Ester (5d ). A
mixture of 25 g (0.32 mol) of 2-methyl-1H-indole-3-acetic acid
(6a ) and 10 mL of methanesulfonic acid in 200 mL of MeOH
was stirred for 24 h, poured into water, and extracted with
EtOAc. The EtOAc solution was washed with a NaHCO3
soution and water and dried (MgSO4). The solvent was
evaporated at reduced pressure to give 26.62 g (yield 97%) of
5d : MS (FD+) 203 (M+). Anal. (C12H13NO2) C, H, N.
5-Meth oxy-2-m eth yl-1H-in d ole-3-a cetic Acid Eth yl Es-
ter (5f). Dry hydrogen chloride was bubbled into a solution
of 27.95 g (0.16 mol) of (4-methoxyphenyl)hydrazine hydro-
chloride (7a ) and 19.72 g (0.17 mol) of levulinic acid in 500
mL of EtOH for 0.5 h while cooling with an ice/water bath.
The bath was removed, the reaction mixture was slowly heated
to reflux, and reflux was maintained for 20 h. After cooling,
the mixture was poured into water and extracted with EtOAc.
The EtOAc solution was washed with a NaHCO3 solution and
dried over Na2SO4. After the solvent was removed at reduced
pressure, the residue was chromatographed on silica gel,
eluting with 5% EtOAc/toluene to give 14.2 g (36% yield) of
the title compound: mp 38-40 °C; MS (FD+) 247 (M+). Anal.
(C14H17NO3) C, H, N.
6-Meth oxy-2-m eth yl-1H-in d ole (4b). A solution of 13.7
g (49 mmol) of 3b and 20 mL of trifluoroacetic acid in 250 mL
of CH2Cl2 was stirred for 72 h and washed with water. After
the solution was dried over MgSO4 and concentrated, the
residue was chromatographed on silica gel, eluting with 20%
EtOAc/hexane to give 4.8 g (61% yield) of 4b: 1H NMR (CDCl3)
∂ 7.70 (br s, 1H), 7.37 (d, 1H), 6.84-6.72 (m, 2H), 6.13 (br s,
1H), 3.83 (s, 3H), 2.43 (s, 3H).
Using a similar procedure, the following conversions were
made: 3d -e,h ,j to 4d -e,h ,j, respectively.
2-Eth yl-5-m eth oxy-1H-in d ole (4d ) (chromatography silica
gel, 20% EtOAc/hexane): yield 58%; mp 49-50 °C; MS (FD+)
175 (M+). Anal. (C11H13NO) C, H, N.
2-Eth yl-6-isop r op yl-5-m eth oxy-1H-in d ole (4e) (chroma-
tography on silica gel, 20% EtOAc/hexane): yield 62%; mp 65-
72 °C: MS (FD+) 217 (M+). Anal. (C14H19NO) C, H, N.
5-Met h oxy-2-p r op yl-1H -in d ole (4h ) (crystallization,
hexane): yield 58%; mp 49-50 °C; MS (FD+) 189 (M+). Anal.
(C12H15NO) C, H, N.
2-Cyclop r op yl-5-m eth oxy-1H-in d ole (4j) (chromatogra-
phy on silica gel, gradient, toluene-20% EtOAc/hexane): yield
The two following compounds were made by the above
procedure from (4-carbethoxyphenyl)hydrazine (7b ) and (4-
bromophenyl)hydrazine hydrochloride (7c), respectively.
5-(Eth oxyca r bon yl)-2-m eth yl-1H-in d ole-3-a cetic a cid
eth yl ester (5g) (chromatography on silica gel, gradient,
toluene-20% EtOAc/toluene): yield 8%; mp 74-76 °C; MS
(FD) 289 (M+). Anal. (C16H19NO4) C, H, N.
1
49%; oil; H NMR (DMSO-d6) ∂ 10.65 (br s, 1H), 7.17 (d, 1H),
6.88 (d, 1H), 6.59 (dd, 1H), 5.98 (s, 1H), 3.72 (s, 3H), 2.04-
1.96 (m, 1H), 0.96-0.90 (m, 2H), 0.79-0.70 (m, 2H); MS (FD+)
187 (M+). Anal. (C12H13NO) H, N; C: calcd, 76.98; found,
74.46.
5-Meth oxy-1H-in d ole-3-a cetic Acid Eth yl Ester (5a ).
To a cooled solution of 29.44 g (0.2 mol) of 5-methoxy-1H-indole
(4k ) in 400 mL of THF was added 125 mL (0.02 mol) of a 1.6
M solution of n-butyllithum in hexane, while the temperature
was kept below 10 °C with an ice/ethanol bath. After 0.25 h,
200 mL (0.2 mol) of a 1 M solution of ZnCl2 in Et2O was added.
The cooling bath was removed, and the mixture was stirred
for 2 h and then concentrated at reduced pressure to a give a
wax which was dissolved in 400 mL of toluene. To this
solution was added 22.2 mL (0.2 mol) of ethyl 2-bromoacetate,
and the mixture was stirred for 24 h and poured into 1 L of 1
N HCl and 1 L of EtOAc. The organic layer was washed twice
with water, dried (Na2SO4), and concentrated at reduced
pressure. The residue was chromatographed on silica gel and
eluted with 5% EtOAc/toluene to give 20 g (43%) of 5a , as an
oil: MS (FD) 233 (M+). Anal. (C13H15NO3) C, H, N.
Using the preceding method, the following conversions were
made using the appropriate 2-bromoacetic acid esters: 4l to
5b; 4m to 5c; 4a to 5e; 4d to 5i; 4h to 5j; and 4j to 5k .
6-Ch lor o-5-m et h oxy-1H -in d ole-3-a cet ic a cid m et h yl
est er (5b ) (chromatography on silica gel gradient, 5-10%
5-Br om o-2-m eth yl-1H-in d ole-3-a cetic a cid eth yl ester
(5h ) (chromatography on silica gel, 5% EtOAc/toluene): yield
83%; mp 65-68 °C; 1H NMR (DMSO-d6) ∂ 11.10 (br s, 1H),
7.56 (s, 1H), 7.25 (d, 1H), 7.11 (d, 1H), 4.06 (q, 2H), 3.65 (s,
2H), 2.34 (s, 3H), 1.17 (t, 3H); MS (FD+) 295 (M - 1), 297 (M
+ 1). Anal. (C13H14BrNO2) H, N; C: calcd, 52.72; found, 53.99.
1-[(Dim eth yla m in o)m eth yl]-5-m eth oxy-1H-in d ole (8).
A 37% aqueous solution of formaldehyde (11 g, 0.176 mol) was
added dropwise to 10 g (0.068 mol) of 5-methoxy-1H-indole (4k )
and 17 mL (0.176 mol) of 40% aqueous dimethylamine in 100
mL of THF and the mixture heated to maintain reflux for 3 h.
After cooling, water was added and the mixture extracted with
EtOAc. The EtOAc solution was washed twice with water,
dried (Na2SO4), and concentrated at reduced pressure. The
residue was chromatographed on silica gel, eluting with a
gradient of CH2Cl2-2% MeOH/CH2Cl2, to give 6.26 g (45%
yield) of 8 as an oil: MS (FD+) 204 (M+). Anal. (C12H16N2O)
C, H, N.
2-Ch lor o-5-m et h oxy-1H -in d ole-3-a cet ic Acid Met h yl
Ester (5l). Using a dry ice/ethanol bath for cooling, 20 mL

5128 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Dillard et al.
(0.026 mol) of 1.3 M sec-butyllithium/cyclohexane was added
to 5.1 g (0.025 mol) of 8 in 100 mL of THF, while the
temperature was kept below -50 °C. The cooling bath was
removed and the temperature allowed to reach 0 °C. The bath
was then replaced. At -60 °C, 3.32 mL (0.026 mol) of
benzenesulfonyl chloride in 10 mL of THF was added, the
mixture stirred, 0.3 h, the bath removed, and the temperature
allowed to reach 20 °C over 1 h. To this mixture were added
100 mL of 1 N HCl and 50 mL of EtOAc, and the mixture was
stirred for 20 h. After the solution was made basic with 5 N
NaOH, the EtOAc layer was separated, washed with water,
dried (Na2SO4), and concentrated at reduced pressure. The
residue was chromatographed on silica, eluting with toluene
to give 1.37 g (29% yield) of crude 2-chloro-5-methoxy-1H-
indole (4n ). To this material (7.55 mmol) in 30 mL of THF
was added 4.7 mL (7.55 mmol) of 1.6 M n-butyllithium/hexane,
while the temperature was kept below 10 °C with an ethanol/
ice bath. After 0.25 h, 7.55 mL (7.55 mmol) of 1 M ZnCl2/
Et2O was added, the mixture stirred for 2 h and concentrated
at reduced pressure, and 40 mL of toluene added followed by
0.72 mL (7.55 mmol) of methyl 2-bromoacetate. The mixture
was stirred for 16 h, warmed at 76 °C for 4 h, and cooled, and
50 mL of 1 N HCl and 40 mL of EtOAc were added. After 0.5
h, the organic layer was separated, dried (Na2SO4), and
concentrated at reduced pressure. The residue was chromato-
graphed on silica gel, eluting with a gradient of toluene-20%
EtOAc/toluene to give 0.79 g (41% yield) of 5l as an oil: 1H
NMR (DMSO-d6) ∂ 11.60 (br s, 1H), 7.21 (d,1H), 6.98 (d, 1H),
6.77 (dd, 1H), 3.75 (s, 3H), 3.71 (s, 2H), 3.63 (s, 3H); MS (FD+)
253 (M - 1, 100), 255 (M + 1, 34). Anal. (C12H12ClNO3) C,
H; N: calcd, 5.52; found, 4.99.
2-Eth yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic a cid m eth yl ester (9i) (chromatography on silica gel,
gradient, toluene-10% EtOAc/toluene): yield 44%; oil; MS
(FD+) 337 (M+). Anal. (C21H23NO3) C, H, N.
4-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic Acid Eth yl Ester (9e). A suspension of 1.2 g (30 mmol)
of 60% NaH/mineral oil was washed with hexane and placed
in 50 mL of DMF. With ice-bath cooling, 7.4 g (30 mmol) of
5e was added, the mixture was stirred 1 h, 3.6 mL (30 mmol)
of benzyl bromide was then added, and stirring was main-
tained for 1.5 h. The mixture was diluted with water and
extracted with EtOAc, and the EtOAc solution was washed
with water and brine and dried (MgSO4). The solution was
concentrated at reduced pressure, and the product was chro-
matographed on silica gel, eluting with 25% EtOAc/hexane,
and crystallized from MeOH/hexane to give 6.16 g (61% yield)
of 9e: mp 75-80 °C; MS (FD) 337 (M+). Anal. (C21H23NO3)
C, H, N.
Using the above procedure, the following conversions were
made using the appropriately substituted alkyl halides: 5b
to 9b; 5e to 9t and 9z; 5h to 9g; 5j to 9j; 5k to 9k ; 5f to 9n -s,
9u , 9w -y, 9a a , 9a c, 9a d , and 9a g; 5i to 9v and 9a b.
6-Ch lor o-5-m eth oxy-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic a cid m eth yl ester (9b) (chromatography on silica gel,
gradient, 20-50% Et2O/hexane): yield 67%; mp 64-66 °C; MS
(FD+) 343 (M - 1, 100), 345 (M + 1, 37). Anal. (C19H18ClNO3)
C, H, N, Cl.
5-Br om o-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic a cid eth yl ester (9g) (chromatography on silica gel, 33%
EtOAc/hexane): yield 42%; mp 83-84 °C; MS (FD+) 385 (M
- 1) 387 (M + 1). Anal. (C20H20BrNO2) C, H, N.
5-Meth oxy-1-(p h en ylm eth yl)-2-p r op yl-1H-in d ole-3-a ce-
tic a cid m eth yl ester (9j) (chromatography on silica gel, 25%
EtOAc/hexane): yield 71%; oil; MS (FD) 352 (M+). Anal.
(C21H25N3O2) C, H, N.
2-Cyclop r op yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-
3-a cetic a cid m eth yl ester (9k ) (chromatography on silica
gel, gradient, 5-15% EtOAc/toluene): yield 40%; oil; MS (FD+)
349 (M+). Anal. (C22H23NO3) C, H, N.
2-Ch lor o-5-m eth oxy-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic a cid m eth yl ester (9l) (chromatography on silica gel,
gradient, 5-15% Et2O/hexane): yield 79%; oil; 1H NMR
(CDCl3) ∂ 7.35-7.25 (m, 4H), 7.15-7.05 (m, 4H), 6.85 (dd, 1H),
5.35 (s, 2H), 3.85 (s, 3H), 3.80 (s, 2H), 3.75 (s, 3H).
5-Meth oxy-2-m eth yl-1-(1-ph en yleth yl)-1H-in dole-3-ace-
tic a cid eth yl ester (9n ) (chromatography on silica gel, 25%
EtOAc/hexane): yield 22%; oil; MS (FD+) 337 (M+). Anal.
(C22H25NO3) C, H, N.
1-(Dip h en ylm eth yl)-5-m eth oxy-2-m eth yl-1H-in d ole-3-
a cetic a cid eth yl ester (9o) (chromatography on silica gel,
25% EtOAc/hexane): yield 54%; oil; MS (FD+) 413 (M+). Anal.
(C27H27NO3) C, H, N.
5-Meth oxy-1-[(3-m eth oxyp h en yl)m eth yl]-2-m eth yl-1H-
in d ole-3-a cetic a cid eth yl ester (9p ) (chromatography on
silica gel, 20% EtOAc/hexane, crystallization, MeOH): yield
58%; mp 88-90 °C; MS (FD+) 367 (M+). Anal. (C22H25NO4)
C, H, N.
1-[[3-(Ben zyloxy)p h en yl]m eth yl]-5-m eth oxy-2-m eth yl-
1H-in d ole-3-a cetic a cid eth yl ester (9q) (chromatography
on silica gel, 20% EtOAc/hexane, crystallization, MeOH): yield
42%; mp 60-70 °C. Anal. (C28H29NO4) C, H, N.
5-Meth oxy-2-m eth yl-1-[(3-n itr op h en yl)m eth yl]-1H-in -
d ole-3-a cetic a cid eth yl ester (9r ) (chromatography on silica
gel, 25% EtOAc/hexane, crystallization, MeOH): yield 8%; mp
105-106 °C; MS (FD+) 382 (M+). Anal. (C21H22N2O5) C, H,
N.
1-[(2-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H-
in d ole-3-a cetic a cid eth yl ester (9s) (chromatography on
silica gel, 30% EtOAc/hexane, crystallization, MeOH): yield
56%; mp 74-77 °C; MS (FD) 371 (M - 1, 100), 373 (M + 1,
40). Anal. (C21H22ClNO3) C, H, N.
5-Meth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a cetic Acid
Eth yl Ester (9a ). Potassium tert-butoxide (1.51 g, 13.5 mmol)
was added to 3.15 g (13.5 mmol) of 5a in 50 mL of DMF, the
mixture was stirred for 0.25 h, 1.55 mL (13.5 mmol) of benzyl
chloride was added, and the mixture was stirred for 72 h. After
being diluted with water, the mixture was extracted with
EtOAc, and the EtOAc solution was washed four times with
water and dried over Na2SO4. The solvent was removed at
reduced pressure and the residue chromatographed on silica
gel, eluting with a gradient of toluene-5% EtOAc/toluene, to
give 1.76 g (66% yield) of 9a as an oil: 1H NMR (DMSO-d6) ∂
7.42-7.13 (m, 7H), 7.00 (d, 1H), 6.77 (dd, 1H), 5.35 (s, 2H),
4.08 (q, 2H), 3.77 (s, 3H), 3.71 (s, 2H), 1.19 (t, 3H); MS (FD+)
323 (M+). Anal. (C20H21NO3) H, N; C: calcd, 74.28; found,
75.53.
Using the preceding chemical method, the following conver-
sions were made using the appropriately substituted alkyl
halides: 5c to 9c; 5d to 9d ; 5f to 9f, 9a e, and 9a f; 5g to 9a h ;
5i to 9i.
5-(Ben zyloxy)-1-(ph en ylm eth yl)-1H-in dole-3-acetic acid
eth yl ester (9c) (chromatography on silica gel, gradient,
toluene-10% EtOAc/toluene): yield 83%; mp 107-109 °C; MS
(FD+) 399.5 (M+). Anal. (C20H21NO3) C, H, N.
2-Met h yl-1-(p h en ylm et h yl)-1H -in d ole-3-a cet ic a cid
m eth yl ester (9d ) (chromatography on silica gel, gradient,
toluene-5% EtOAc/toluene): yield 68%; mp 71-73 °C; MS
(FD+) 293 (M+). Anal. (C19H19NO2) C, H, N.
5-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic a cid eth yl ester (9f) (chromatography on silica gel,
gradient, toluene-10% EtOAc/toluene): yield 68%; mp 63-
64 °C; MS (FD+) 338 (M+). Anal. (C21H23NO3) C, H, N.
1-(Cycloh exylm eth yl)-5-m eth oxy-2-m eth yl-1H-in d ole-
3-a cetic a cid eth yl ester (9a e) (chromatography on silica
gel, gradient, toluene-5% EtOAc/toluene): yield 48%; oil; MS
(FD+) 343 (M+). Anal. (C21H29NO3) C, H, N.
1-Decyl-5-m et h oxy-2-m et h yl-1H -in d ole-3-a cet ic a cid
eth yl ester (9a f) (chromatography on silica gel, gradient,
toluene-5% EtOAc/toluene): yield 56%; mp 40-42 °C; MS
(FD) 387 (M+). Anal. (C24H37NO3) C, H, N.
1-[(3-Ch lor oph en yl)m eth yl]-5-(eth oxycar bon yl)-2-m eth -
yl-1H-in d ole-3-a cetic a cid eth yl ester (9a h ) (chromatog-
raphy on silica gel, gradient, toluene-20% EtOAc/toluene):
yield 34%; mp 100-102 °C; MS (FD+) 413.5 (M - 1, 100), 415.5
(M + 1, 36). Anal. (C23H24ClNO4) C, H, N.
1-[(3-Ch lor op h en yl)m et h yl]-4-m et h oxy-2-m et h yl-1H -
in d ole-3-a cetic a cid eth yl ester (9t) (chromatography on
silica gel, 20% EtOAc/hexane): yield 70%; mp 113-115 °C;
1H NMR (CDCl3) ∂ 7.30-6.72 (m, 6H), 6.49 (d, 1H), 5.24 (s,
2H), 4.16 (q, 2H), 3.92 (s, 2H), 3.89 (s, 3H), 2.24 (s, 3H), 1.24

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5129
(t, 3H); MS (FD+) 371 (M - 1, 100), 373 (M + 1, 36). Anal.
(C21H22ClNO3) H, N; C: calcd, 67.83; found, 70.39.
Et2O/hexane-Et2O, to give 1.0 g (79% yield) of the title
compound that melted at 133-134 °C after crystallization from
CH2Cl2/Et2O: 1H NMR (CDCl3) ∂ 7.35-7.20 (m, 4H), 7.15-
700 (m, 3H), 5.25 (s, 2H), 3.90 (s, 3H), 3.75 (s, 2H), 3.70 (s,
3H); MS (FD) 421 (M - 1, 80%), 423 (M + 1, 100%). Anal.
(C19H17BrClNO3) H, N; C: calcd, 53.99; found, 54.70; Br: calcd,
18.90; found, 16.04; Cl: calcd, 8.40; found, 9.97.
1-[(3-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cetic a cid eth yl ester (9u ) (chromatography on
silica gel, 20% EtOAc/hexane): yield 70%; mp 113-115 °C;
MS (FD+) 371 (M - 1, 100), 373 (M + 1, 60). Anal. (C21H22
ClNO3) C, H, N.
-
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-5-m eth oxy-1H-in -
d ole-3-a cetic a cid m eth yl ester (9v) (chromatography on
silica gel, 25% EtOAc/hexane): yield 75%; oil; MS (FD+) 371
(M - 1, 100), 373 (M + 1, 38). Anal. (C22H24ClNO3) C, H, N.
1-[(4-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cetic a cid eth yl ester (9w ) (chromatography on
silica gel, 30% EtOAc/hexane, crystallization, MeOH): yield
47%; mp 98-100 °C; MS (FD+) 371 (M - 1, 100), 373 (M + 1,
43). Anal. (C21H22ClNO3) C, H, N.
1-[(2,5-Dich lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-
1H-in d ole-3-a cetic a cid eth yl ester (9x) (chromatography
on silica gel, 20% EtOAc/hexane, crystallization, MeOH): yield
29%; mp 146-148 °C; MS (FD+) 405 (M - 1, 100), 407 (M +
1, 75). Anal. (C21H21Cl2NO3) C, H, N.
5-Hyd r oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a cetic Acid
Eth yl Ester (9a i). A solution of 9c (8.1 g, 20.3 mmol) in 150
mL of EtOH and 25 mL THF was hydrogenated with 3 g of
10% Pd/C catalyst at approximately 40 psi of hydrogen. The
catalyst was filtered, and the filtrate was concentrated at
reduced pressure. The residue was chromatographed on silica
gel, eluting with a gradient of 30-50% Et2O/hexane to give
5.7 g (yield 90%) of 9a i: 1H NMR (CDCl3) ∂ 7.40-7.30 (m, 3H),
7.25-7.10 (m, 5H), 6.80 (dd, 1H), 6.00 (s, 1H), 5.25 (s, 2H),
4.25 (q, 2H), 3.75 (s, 3H), 1.30 (t, 3H).
1-[(3-Hyd r oxyp h en yl)m eth yl]-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cetic Acid Eth yl Ester (9a l). A solution of 357
mg (0.8 mmol) of 9q in 30 mL of 1:1 THF/EtOH was
hydrogenated at 60 psi of hydrogen for 16 h using 90 mg of
Pd/BaSO4. The catalyst was filtered and the filtrate concen-
trated at reduced pressure. The residue was dissolved in
EtOAc and washed with water and saturated NaCl solution.
After drying over MgSO4, the product was chromatographed
on silica gel, eluting with 50% EtOAc/hexane, and then EtOAc
to give, after crystallization from MeOH, 100 mg (35% yield)
1-[(2,6-Dich lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-
1H-in d ole-3-a cetic a cid eth yl ester (9y) (chromatography
on silica gel, 20% EtOAc/hexane): yield 68%; mp 131-131 °C;
MS (FD+) 405 (M - 1, 100), 407 (M + 1, 70). Anal. (C21H21
Cl2NO3) C, H, N.
-
1-([1,1′-Bip h en yl]-2-ylm eth yl)-4-m eth oxy-2-m eth yl-1H-
in d ole-3-a cetic a cid eth yl ester (9z) (chromatography on
silica gel, 20% EtOAc/hexane): yield 71%; oil; 1H NMR (CDCl3)
∂ 7.64-7.08 (m, 8H), 7.95 (t, 1H), 6.74 (d, 1H), 6.53-6.41 (m,
2H), 5.16 (s, 2H), 4.1 (q, 2H), 3.90 (s, 2H), 3.88 (s, 3H), 2.15 (s,
3H), 1.25 (t, 3H).
1-([1,1′-Bip h en yl]-2-ylm eth yl)-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cetic a cid eth yl ester (9a a ) (chromatography on
silica gel, 25% EtOAc/hexane): yield 69%; oil; MS (FD+) 413
(M+). Anal. (C27H27NO3) C, H, N.
1-([1,1′-Bip h en yl]-2-ylm et h yl)-2-et h yl-5-m et h oxy-1H -
in d ole-3-a cetic a cid m eth yl ester (9a b) (chromatography
on silica gel, 20% EtOAc/hexane): yield 44%; oil; MS (FD+)
413 (M+). Anal. (C27H27NO3) C, H, N.
1-([1,1′-Bip h en yl]-3-ylm eth yl)-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cetic a cid eth yl ester (9a c) (chromatography on
silica gel, 33% EtOAc/hexane): yield 62%; oil; 1H NMR (CDCl3)
∂ 7.54-7.26 (m, 8H), 7.15 (d, 1H), 7.08 (d, 1H), 6.88 (d, 1H),
6.79 (dd, 1H), 5.35 (s, 2H), 4.13 (q, 2H), 3.88 (s, 3H), 3.71 (s,
2H), 2.38 (s, 3H), 1.24 (t, 3H); MS (FD+) 413.5 (M+). Anal.
(C27H27NO3) H, N; C: calcd, 78.42; found, 80.59.
of 9a l: mp 114-116 °C; MS (FD+) 353 (M+). Anal. (C21H23
NO4) C, H, N.
-
1-[(3-Am in op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cetic Acid Eth yl Ester (9a k ). A solution of 500
mg (1.3 mmol) of 9r in 50 mL of EtOH was hydrogenated for
16 h at room temperature using 0.1 g of 5% Pd/C and 60 psi
of hydrogen. The catalyst was filtered and the filtrate
concentrated at reduced pressure. The residue was chromato-
graphed on silica gel, eluting with 25% EtOAc/hexane to give
234 mg (51% yield) of 9a k as an oil: MS (FD+) 352 (M+). Anal.
(C21H24N2O3) C, H, N.
5-Ca r boxy-1-[(3-ch lor op h en yl)m eth yl]-2-m eth yl-1H-in -
d ole-3-a cetic Acid Meth yl Ester (9a j). A solution of 0.27 g
(0.65 mmol) of 9a h and 2 mL of 5 N NaOH in 40 mL of EtOH
was heated to maintain reflux for 4 h. After cooling, the
mixture was diluted with water and extracted with EtOAc.
The EtOAc solution which contained some precipitate was
concentrated at reduced pressure, and the residue (containing
a dicarboxylic acid) was crystallized from MeOH to give 100
1
mg (43% yield) of 9a j: mp 216-217 °C; H NMR (DMSO-d6)
∂ 8.15 (s, 1H), 7.71 (d, 1H), 7.45 (d, 1H), 7.38-7.29 (m, 2H),
7.04 (s, 1H), 6.94 (d, 1H), 5.50 (s, 2H), 3.84 (s, 2H), 3.65 (s,
3H), 2.32 (s, 3H); MS (FD) 371 (M+). Anal. (C20H18ClNO4) C,
H; N: calcd, 3.77; found, 3.10.
5-Meth oxy-2-m eth yl-1-(2-p yr id ylm eth yl)-1H-in d ole-3-
a cetic a cid eth yl ester (9a d ) (chromatography on silica gel,
50% EtOAc/hexane): yield 75%; oil; MS (FD) 338 (M+). Anal.
(C20H22N2O3) C, H, N.
5-Met h oxy-2-m et h yl-1-(3-p h en ylp r op yl)-1H -in d ole-3-
a cetic a cid eth yl ester (9a g) (chromatography on silica gel,
25% EtOAc/hexane): yield 58%; oil; MS (FD+) 365 (M+). Anal.
(C23H27NO3) C, H, N.
2-Met h yl-1-p h en yl-1H-in d olea cet ic Acid E t h yl E st er
(9a m ). Diphenylhydrazine hydrochloride (11, 4.6 g, 21 mmol)
and 3 mL of ethyl levulinate in 250 mL of benzene was heated
at reflux with a water separator for 48 h, cooled, washed with
2-Meth yl-5-p h en yl-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic Acid Eth yl Ester (9h ). To 25 mL of EtOH were added
266 mg (0.7 mmol) of 9g, 194 mg (0.168 mmol) of Pd[P(Ph)3]4,
and 3.2 mL of a 2 M Na2CO3 solution. To this solution was
added 196 mg (1.6 mmol) of phenylboronic acid in 5 mL of
EtOH and the resulting mixture heated to maintain reflux for
16 h. After cooling, the mixture was diluted with EtOAc and
filtered through Celite. The filtrate was washed with water
and a saturated NaCl solution, dried (MgSO4), and concen-
trated at reduced pressure. The residue was chromatographed
on silica gel, eluting with 25% EtOAc/hexane, and then
recrystallized twice from MeOH to give 95 mg (35% yield) of
9h : mp 115-117 °C; MS (FD+) 383 (M+). Anal. (C26H25NO2)
C, H, N.
2-Br om o-6-ch lor o-5-m eth oxy-1-(p h en ylm eth yl)-1H-in -
d ole-3-a cetic Acid Meth yl Ester (9m ). A mixture of 1.0 g
(3.0 mmol) of 9b and 600 mg (3.3 mmol) of NBS in 100 mL of
CCl4 was stirred for 30 h. The mixture was washed with an
aqueous Na2S2O3 solution, washed with brine, dried (Na2SO4),
and concentrated at reduced pressure. The residue was
chromatographed on silica gel, eluted with a gradient of 20%
water, dried (Na2SO4), and concentrated at reduced pressure.
The residue was chromatographed on silica gel, eluting with
30% Et2O/hexane to give 4.5 g of the hydrazone intermediate,
12. A mixture of 2 g (6.45 mmol) of 12 and 3 mL of
phosphorous trichloride in 125 mL of 1,2-dichloroethane was
heated to maintain reflux for 17 h, cooled, and stirred with a
mixture of ice and concentrated ammonia. The organic layer
was separated and washed with brine, dried (Na2SO4), and
evaporated at reduced pressure. The residue was chromato-
graphed on silica gel, eluting with 10% Et2O/hexane to give
0.98 g (52% yield) of 9a m as an oil: 1H NMR (CDCl3) ∂ 7.75
(d, 1H), 7.65-7.45 (m, 3H), 7.40 (d, 2H), 7.25 -7.15 (m, 3H),
4.25 (q, 2H), 3.85 (s, 2H), 2.35 (s, 3H), 1.15 (t, 3H).
5-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic Acid (13a ). A solution of 438 mg (2.0 mmol) of 5-methoxy-
2-methyl-1H-indole-3-acetic acid (6b) in 5 mL of THF and 25
mL of DMSO was treated with 160 mg (4.0 mmol) of NaH (60%
in mineral oil) and then with 0.3 mL of benzyl bromide for 16
h. The solution which contained 5-methoxy-2-methyl-1-(phe-
nylmethyl)-1H-indole-3-acetic acid benzyl ester was diluted
with 2 N NaOH, stirred for 6 h, and washed with Et2O. The

5130 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Dillard et al.
aqueous solution was acidified with 5 N HCl, extracted with
brine, dried (Na2SO4), and evaporated at reduced pressure to
give, after crystallization from EtOH, 540 mg (yield 87%) of
13a : mp 173-174 °C; MS (FD+) 309 (M+). Anal. (C19H19NO3)
C, H, N.
10 mL of hydrazine in 75 mL of EtOH was heated to maintain
reflux for 16 h. On cooling of the reaction mixture, a
precipitate formed that was filtered to give 1.33 g (93% yield)
of 10a : mp 143-144 °C; MS (FD+) 309 (M+). Anal.
(C18H19N3O2) C, H, N.
Using this procedure, 9d ,f,i,a f,a p -a s were converted to
5-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic Acid Meth yl Ester (9a n ). A solution of 0.2 g of 13a in
50 mL of MeOH and 1 mL of methanesulfonic acid was
refluxed for 3 h, diluted with water, and extracted with EtOAc.
The organic phase was washed with aqueous NaHCO3 and
water, dried over Na2SO4, and evaporated at reduced pressure
to give 100 mg (yield 48%) of 9a n as an oil which solidified on
standing, mp 72-75 °C. Anal. (C20H21NO3) C, H, N.
2-Ch lor o-5-m eth oxy-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic Acid P h en ylm eth yl Ester (9a p ). A solution of 2.0 g (10
mmol) of 5-methoxy-1H-indole-3-acetic acid (6c) in 100 mL of
DMF was treated in portions with 1.0 g (25 mmol) of 60% NaH/
mineral oil, and after 10 min, 3 mL of benzyl bromide was
added. After 22 h, the mixture was diluted with water and
extracted with EtOAc, and the EtOAc solution was washed
with water and saturated NaCl solution and dried over Na2-
SO4. After the mixture was concentrated at reduced pressure,
the residue was chromatographed on silica gel, eluting with
CH2Cl2, to give 3.7 g (96% yield) of 5-methoxy-1-(phenylm-
ethyl)-1H-indole-3-acetic acid phenylmethyl ester (9a o) as an
oil: 1H NMR (CDCl3) ∂ 7.35-7.15 (m, 9H), 7.10-7.00 (m, 4H),
6.75 (dd, 1H), 5.15 (s, 2H), 5.10 (s, 2H), 3.75 (s, 3H), 3.70 (s,
2H). While cooling at -5 °C, 0.6 mL (4.9 mmol) of boron
trifluoride etherate was added to 770 mg (2 mmol) of 9a o in
100 mL of CH2Cl2, followed by 0.24 mL (3 mmol) of SO2Cl2.
After 10 min, an aqueous NaHCO3 solution was added, and
the CH2Cl2 layer was separated, dried (Na2SO4), and concen-
trated at reduced pressure. The residue was chromatographed
on silica gel, eluting with a gradient of 15% Et2O/hexane-
Et2O, to give 100 mg (12% yield) of 9a p : 1H NMR (CDCl3) ∂
7.35-7.15 (m, 8H), 7.05-6.95 (m, 3H), 6.90 (d, 1H), 6.70 (dd,
1H), 5.25 (s, 2H), 5.10 (s, 2H), 3.75 (s, 3H), 3.70 (s, 2H).
2-Br om o-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic Acid P h en ylm eth yl Ester (9a q). With stirring, 450 mg
(2.5 mmol) of N-bromosuccinimide was added to 910 mg (2.4
mmol) of 9a o in 75 mL of CCl4. After 15 min, the reaction
mixture was washed with an aqueous Na2S2O3 solution, water,
and a saturated NaCl solution and dried over Na2SO4. After
the mixture was concentrated at reduced pressure, the residue
was chromatographed on silica gel, eluting with CH2Cl2, and
crystallized from Et2O/hexane to give 420 mg (69% yield) of
9a q: mp 89-90 °C; MS (FD) 463 (M - 1), 465 (M + 1). Anal.
(C25H22BrNO3) C, H, N.
5-Meth oxy-2-(m eth ylth io)-1-(ph en ylm eth yl)-1H-in dole-
3-a cetic Acid P h en ylm eth yl Ester (9a r ). A solution of 1.0
mL (11 mmol) of dimethyl disulfide in 25 mL of CH2Cl2 was
cooled to -25 °C, 0.8 mL (10 mmol) of SO2Cl2 was added, the
cooling bath was removed, and the mixture was stirred and
let warm to room temperature. Three milliliters of this
solution was added to 770 mg (2 mmol) of 9a o in 100 mL of
CH2Cl2. After 0.5 h, the reaction mixture was washed with
an aqueous Na2CO3 solution and a saturated NaCl solution
and dried over Na2SO4. After the mixture was concentrated
at reduced pressure, the residue was chromatographed on
silica gel, eluted with a gradient of 20-30% Et2O/hexane, and
crystallized from Et2O/hexane to give 600 mg (70% yield) of
9a r : mp 89-90 °C: MS (FD+) 431 (M+). Anal. (C26H25NO3S)
C, H, N, S.
5-Meth oxy-2-(m eth ylsu lfin yl)-1-(p h en ylm eth yl)-1H-in -
d ole-3-a cetic Acid P h en ylm eth yl Ester (9a s). To a solu-
tion of 460 mg (1 mmol) of 9a r in 50 mL of CH2Cl2 was added
200 mg (1.0 mmol) of m-chloroperbenzoic acid (80-85% pure),
and the mixture was stirred for 0.75 h. The reaction mixture
was washed with a Na2CO3 solution, dried (Na2SO4), and
concentrated at reduced pressure to give a residue that was
chromatographed on silica gel, eluted with CH2Cl2 and then
Et2O, and crystallized from EtOH to give 424 mg (95% yield)
of 9a s as a solid: MS (FD) 447 (M+). Anal. (C26H25NO4S) C,
H, N, S.
10d ,f,i,a f,a p -a s, respectively.
2-Meth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a cetic a cid h y-
d r a zid e (10d ) (crystallization, MeOH): yield 60%; mp 140-
143 °C; MS (FD+) 293 (M+). Anal. (C18H19N3O) C, H, N.
5-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic a cid h yd r a zid e (10f) (trituration with Et2O): yield 96%;
mp 161-162 °C; MS (FD) 323 (M+). Anal. (C19H21N3O2‚0.8H2O)
C, H, N.
2-Eth yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic a cid h yd r a zid e (10i) (crystallized from reaction mix-
ture): yield 77%; mp 138-140 °C; MS (FD+) 337 (M+). Anal.
(C20H23N3O2) C, H, N.
1-Decyl-5-m et h oxy-2-m et h yl-1H -in d ole-3-a cet ic a cid
h yd r a zid e (10a f) (crystallization, MeOH): yield 32%; mp
129-131 °C; MS (FD+) 373 (M+). Anal. (C22H35N3O2) C, H,
N.
2-Ch lor o-5-m eth oxy-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic a cid h yd r a zid e (10a p ) (trituration with Et2O): yield
100%; mp 186-187 °C; 1H NMR (DMSO-d6) ∂ 9.25 (br s, 1H),
7.40-7.10 (m, 7H), 6.90 (dd, 1H), 5.35 (s, 2H), 4.20 (br s, 2H),
3.70 (s, 3H), 3.45 (s, 2H); MS (FD) 343 (M - 1, 100%), 345 (M
+ 1, 60%). Anal. (C18H18ClN3O2) H, Cl; C: calcd, 62.88; found,
62.31; N: calcd, 12.22; found, 11.30.
2-Br om o-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic a cid h yd r a zid e (10a q) (chromatography on silica gel,
Et2O, then EtOAc): yield 71%; mp 178-180 °C; 1H NMR
(DMSO-d6) ∂ 9.30 (br s, 1H), 7.40-7.05 (m, 7H), 6.80 (dd, 1H),
5.45 (s, 2H), 4.30 (br s, 2H) 3.80 (s, 3H), 3.55 (s, 2H); MS (FD+)
387 (M - 1), 389 (M + 1). Anal. (C18H18BrN3O2) H, N; C:
calcd, 55.68; found, 54.02; Br: calcd, 20.58; found, 23.17.
5-Meth oxy-2-(m eth ylth io)-1-(ph en ylm eth yl)-1H-in dole-
3-a cetic a cid h yd r a zid e (10a r ) (crude product): yield 100%;
mp 181-182 °C; MS (FD) 355 (M+). Anal. (C19H21N3O2S) C,
H, N, S.
5-Met h oxy-2-(m et h ylsu lfin yl)-1-(p h en ylm et h yl)-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10a s) (crystallization,
EtOAc): yield 85%; mp 172-174 °C; MS (FD) 371 (M+). Anal.
(C19H21N3O3S) C, H, N, S.
4-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-ace-
tic a cid h yd r a zid e (10e). A solution of 2.8 g (8.3 mmol) of
9e and 10 mL of hydrazine in 40 mL of EtOH was heated to
maintain reflux for 16 h, diluted with water, and extracted
with EtOAc. The EtOAc solution was washed with brine, dried
(MgSO4), and concentrated at reduced pressure. The residue
was crystallized from MeOH to give 2.0 g (75% yield) of 10e:
mp 145-147 °C; MS (FD) 323 (M+). Anal. (C19H21N3O2) C,
H, N.
Using the above procedure, 9g,h ,j-k ,o-z,a a -a d ,a g,a j-a m
were converted to 10g,h ,j-k ,o-z,a a -a d ,a g,a j-a m , respec-
tively.
5-Br om o-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic a cid h yd r a zid e (10g) (crystallization, MeOH): yield 43%;
mp 181-182 °C; MS (FD
(C18H18BrN3O) C, H, N.
+) 371 (M - 1), 373 (M + 1). Anal.
2-Meth yl-5-p h en yl-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
tic a cid h yd r a zid e (10h ) (crystallization, MeOH): yield 35%;
mp 154-156 °C; MS (FD+) 369 (M+). Anal. (C24H23N3O) C,
H, N.
5-Meth oxy-1-(p h en ylm eth yl)-2-p r op yl-1H-in d ole-3-a ce-
tic a cid h yd r a zid e (10j) (crystallization, MeOH): yield 74%;
mp 140-141 °C; MS (FD+) 351 (M+). Anal. (C21H25N3O2) C,
H, N.
2-Cyclop r op yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-
3-a cetic a cid h yd r a zid e (10k ) (crystallization, EtOH): yield
74%; mp 173-174 °C; MS (FD+) 349 (M+). Anal. (C21H23N3O2)
C, H, N.
5-Meth oxy-2-m eth yl-1-(1-ph en yleth yl)-1H-in dole-3-ace-
tic a cid h yd r a zid e (10n ) (chromatography on silica gel,
EtOAc): yield 59%; foam; MS (FD+) 337 (M+). Anal.
(C20H23N3O2) C, H, N.
5-Meth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a cetic Acid
Hyd r a zid e (10a ). A solution of 1.4 g (4.33 mmol) of 9a and

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5131
1-(Dip h en ylm eth yl)-5-m eth oxy-2-m eth yl-1H-in d ole-3-
a cetic a cid h yd r a zid e (10o) (chromatography on silica gel,
5% MeOH/EtOAc): yield 28%; foam; 1H NMR (CDCl3) ∂ 7.45-
6.80 (m, 12H), 6.58 (s, 1H), 3.84 (s, 3H), 3.71 (s, 2H), 2.26 (s,
3H); MS (FD+) 399 (M+). Anal. (C25H25N3O2) H; C: calcd,
75.16; found, 77.25; N: calcd, 10.52; found, 8.89.
2H), 2.28 (s, 3H), 2.16-2.00 (m, 2H); MS (FD) 336 (M+). Anal.
(C21H25N3O2) H, N; C: calcd, 74.97; found, 73.81.
5-Ca r boxy-1-[(3-ch lor op h en yl)m eth yl]-2-m eth yl-1H-in -
d ole-3-a cet ic a cid h yd r a zid e (10a j) (crystallization,
EtOH): yield 10%; mp 216-217 °C; MS (FD) 371 (M - 1, 100),
373 (M + 1, 50). Anal. (C19H18ClN3O3) C, H, N.
5-Meth oxy-1-[(3-m eth oxyp h en yl)m eth yl]-2-m eth yl-1H-
in d ole-3-a cet ic a cid h yd r a zid e (10p ) (crystallization,
MeOH): yield 62%; mp 161-163 °C. Anal. (C20H23N3O3) C,
H, N.
1-[(3-Am in op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10a k ) (crystallization,
MeOH): yield 40%; mp 154-156 °C; MS (FD) 338 (M+). Anal.
(C19H22N4O2) C, H, N.
1-[[3-(Ben zyloxy)p h en yl]m eth yl]-5-m eth oxy-2-m eth yl-
1H-in d ole-3-a cetic a cid h yd r a zid e (10q) (crystallization,
MeOH): yield 51%; mp 130-132 °C; MS (FD+) 353 (M+). Anal.
(C26H27N3O3) C, H, N.
1-[(3-Hyd r oxyp h en yl)m eth yl]-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cet ic a cid h yd r a zid e (10a l) (crystallization,
MeOH): yield 47%; mp 201-203 °C; MS (FD+) 339 (M+). Anal.
(C19H21N3O3) C, H, N.
5-Meth oxy-2-m eth yl-1-[(3-n itr op h en yl)m eth yl]-1H-in -
d ole-3-a cet ic a cid h yd r a zid e (10r ) (crystallization,
MeOH): yield 38%; mp 177-179 °C; H NMR (CDCl3) ∂ 8.14
2-Met h yl-1-p h en yl-1H -in d olea cet ic a cid h yd r a zid e
(10a m ) (crystallization, MeOH): yield 73%; mp 113-115 °C;
MS (FD+) 279 (M+). Anal. (C17H17N3O) C, H, N.
1
(d, 1H), 7.91 (s, 1H), 7.47 (t, 1H), 7.33-6.74 (m, 5H), 5.39 (s,
2H), 3.86 (s, 3H), 3.73 (s, 2H), 2.3 (s, 3H); MS (FD) 368 (M+).
Anal. (C19H20N4O4) H, N; C: calcd, 61.95; found, 62.53.
1-[(2-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10s) (crystallization,
MeOH): yield 53%; mp 99-100.5 °C; MS (FD) 357 (M - 1,
100), 359 (M + 1, 49). Anal. (C19H20ClN3O2) C, H, N.
1-[(3-Ch lor op h en yl)m et h yl]-4-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10t ) (crystallization,
MeOH): yield 98%; mp 160-162 °C; MS (FD+) 357 (M - 1,
100), 359 (M + 1, 36). Anal. (C19H20ClN3O2) C, H, N.
1-[(3-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10u ) (crystallization,
EtOAc): yield 98%; mp 160-162 °C; MS (FD) 357 (M - 1, 100),
359 (M + 1, 43). Anal. (C19H20ClN3O2) C, H, N.
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-5-m eth oxy-1H-in -
d ole-3-a cet ic a cid h yd r a zid e (10v) (crystallization,
MeOH): yield 62%; mp 115-117 °C. Anal. (C20H22ClN3O2)
C, H, N.
1-[(4-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10w ) (crystallization,
MeOH): yield 78%; mp 177-180 °C; MS (FD) 357 (M - 1, 100),
359 (M + 1, 33). Anal. (C19H20ClN3O2) C, H, N.
1-[(2,5-Dich lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-
1H-in d ole-3-a cetic a cid h yd r a zid e (10x) (crystallization,
MeOH): yield 64%; mp 168-170 °C; MS (FD+) 391 (M - 1,
100), 393 (M + 1, 66). Anal. (C19H19Cl2N3O2) C, H, N.
1-[(2,6-Dich lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-
1H-in d ole-3-a cetic a cid h yd r a zid e (10y) (crystallization,
MeOH): yield 61%; mp 194-196 °C; 1H NMR (CDCl3) ∂ 7.45-
7.20 (m, 3H), 6.50-6.40 (m, 2H), 6.38 (d, 1H), 6.21 (dd, 1H),
5.50 (s, 2H), 3.84 (s, 3H), 3.67 (s, 2H), 2.3 (s, 3H); MS (FD+)
391 (M - 1, 100), 393 (M + 1, 64). Anal. (C19H19Cl2N3O2) H,
N; C: calcd, 58.17; found, 58.65.
Using the procedure described for 5a , the following indoleal-
kanoic acid esters (14) were made by reacting 5-methoxy-2-
methyl-1H-indole (4o) with methyl 2-bromopropanoate, ethyl
2-bromobutyrate, and ethyl 2-bromooctanoate, respectively.
2-(5-Meth oxy-2-m eth yl-1H-in d ol-3-yl)p r op a n oic a cid
m et h yl est er (14a ) (chromatography on silica gel, 10%
EtOAc/hexane): yield 59%; oil; MS (FD
+) 247 (M+). Anal.
(C14H17NO3) C, H, N.
2-(5-Meth oxy-2-m eth yl-1H-in dol-3-yl)bu tan oic acid eth -
yl ester (14b) (chromatography on silica gel, gradient, toluene-
10% EtOAc/toluene): yield 56%; oil; MS (FD+) 275 (M+). Anal.
(C16H21NO3) C, H, N.
2-(5-Meth oxy-2-m eth yl-1H-in dol-3-yl)octan oic acid eth -
yl ester (14c) (additional 6 h at 80 °C, chromatography on
silica gel, gradient, toluene-10% EtOAc/toluene): yield 34%;
oil; MS (FD+) 331 (M+). Anal. (C20H29NO3) C, H, N.
Using the procedure described for 9e, the following indoles,
15a -c, were prepared by reacting 14a -c with benzyl bromide.
2-[5-Met h oxy-2-m et h yl-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]p r op a n oic a cid m eth yl ester (15a ) (chromatography on
silica gel, gradient, toluene-10% EtOAc/toluene): yield 61%;
oil; MS (FD) 338 (M+). Anal. (C21H23NO3) C, H, N.
2-[5-Met h oxy-2-m et h yl-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]bu ta n oic a cid eth yl ester (15b) (chromatography on
silica gel, 25% EtOAc/hexane): yield 79%; oil; MS (FD+) 365
(M+). Anal. (C23H27NO3) C, H, N.
2-[5-Met h oxy-2-m et h yl-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]octa n oic a cid eth yl ester (15c) (chromatography on silica
gel, 25% EtOAc/hexane): yield 86%; oil; 1H NMR (CDCl3) ∂
7.35-7.20 (m, 4H), 7.08 (d, 1H), 6.94 (d, 2H), 6.75 (dd, 1H),
5.27 (s, 2H), 4.20-4.00 (m, 2H), 3.88 (s, 3H), 3.79 (t, 1H), 2.34
(s, 3H), 2.30-2.15 (m, 1H), 2.05-1.90 (m, 1H), 1.40-1.15 (m,
11H), 0.84 (t, 3H); MS (FD+) 421 (M+). Anal. (C27H35NO3) C,
N; H: calcd, 8.37; found, 7.49.
1-([1,1′-Bip h en yl]-2-ylm eth yl)-4-m eth oxy-2-m eth yl-1H-
in d ole-3-a cetic a cid h yd r a zid e (10z) (chromatography on
silica gel, EtOAc, then 10% MeOH/EtOAc): yield 56%; mp
148-150 °C; MS (FD+) 399 (M+). Anal. (C25H25N3O2) C, H,
N.
1-([1,1′-Bip h en yl]-2-ylm eth yl)-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cetic a cid h yd r a zid e (10a a ) (chromatography on
silica gel, EtOAc): yield 28%: wax; MS (FD+) 399 (M+). Anal.
(C25H25N3O2) C, H, N.
1-([1,1′-Bip h en yl]-2-ylm et h yl)-2-et h yl-5-m et h oxy-1H -
in d ole-3-a cet ic a cid h yd r a zid e (10a b ) (crystallization,
MeOH): yield 49%; mp 67-73 °C; MS (FD+) 413 (M+). Anal.
(C26H27N3O2) C, H, N.
1-([1,1′-Bip h en yl]-3-ylm eth yl)-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cet ic a cid h yd r a zid e (10a c) (crystallization,
MeOH): yield 66%; mp 159-160 °C; MS (FD+) 399 (M+). Anal.
(C25H25N3O2) C, H, N.
Using the procedure for 10e, the following conversions of
esters to acid hydrazides were made, 15a -c to 16a -c.
2-[5-Met h oxy-2-m et h yl-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]p r op a n oic a cid h yd r a zid e (16a ) (chromatography on
silica gel, gradient, CH2Cl2-10% MeOH/CH2Cl2): yield 40%;
wax; MS (FD) 323 (M+). Anal. (C19H21N3O2) C, H, N.
2-[5-Met h oxy-2-m et h yl-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]bu ta n oic a cid h yd r a zid e (16b) (chromatography on silica
gel, 50% EtOAc/hexane, then EtOAc): yield 35%; oil; MS (FD)
337 (M+). Anal. (C20H23N3O2) C, H, N.
2-[5-Met h oxy-2-m et h yl-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]octa n oic a cid h yd r a zid e (16c) (chromatography on silica
gel, 50% EtOAc/hexane, then EtOAc): yield 56%; oil; MS (FD)
393 (M+). Anal. (C24H31N3O2) C, H, N.
5-Met h oxy-1-(p h en ylm et h yl)-1H -in d ole-3-a cet a m id e
(17a ). One gram of Raney nickel was added to 790 mg (2.4
mmol) of 10a in 120 mL of EtOH and the mixture heated at
reflux for 2 h. After the catalyst was filtered off, the filtrate
was concentrated at reduced pressure and the residue tritu-
rated with Et2O to give 675 mg (89% yield) of 17a : mp 156-
158 °C; MS (FD+) 294 (M+). Anal. (C18H18N2O2‚0.8H2O) C,
H, N.
5-Meth oxy-2-m eth yl-1-(2-p yr id ylm eth yl)-1H-in d ole-3-
a cetic a cid h yd r a zid e (10a d ) (crystallization, MeOH): yield
67%; mp 147-148 °C; MS (FD+) 324 (M+). Anal. (C18H20N4O2)
C, H, N.
5-Met h oxy-2-m et h yl-1-(3-p h en ylp r op yl)-1H -in d ole-3-
a cetic a cid h yd r a zid e (10a g) (crystallization, MeOH): yield
Using the above method, the following acetic acid hydrazides
were converted to acetamides: 10e,f,i,j,k ,t,u ,v,z,a a ,a g,a d ,a f
to 17b-l,17r -s, respectively:
1
31%; mp 133-135 °C; H NMR (CDCl3) ∂ 7.41-6.7 (m, 9H),
4.24 (t, 2H), 3.83 (s, 3H), 3.66 (s, 2H), 2.70 (t, 3H), 2.54 (br s,

5132 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Dillard et al.
4-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a c-
eta m id e (17b) (chromatography on silica gel, 5% MeOH/
EtOAc): yield 79%; mp 145-146 °C; 1H NMR (CDCl3) ∂ 7.34-
7.21 (m, 3H), 7.04 (t, 1H), 6.96 (d, 2H), 6.88 (d, 1H), 6.54 (d,
1H), 6.11 (br s, 1H), 5.29 (s, 2H), 5.19 (br s, 1H), 3.95 (s, 3H),
3.88 (s, 2H), 2.34 (s, 3H); MS (FD) 308 (M+). Anal.
(C19H20N2O2) H, N; C: calcd, 74.08; found, 75.09.
5-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a c-
eta m id e (17c) (filtered through silica gel, EtOAc, crystalliza-
tion, CH2Cl2/MeOH): yield 25%; mp 130-131 °C; MS (FD+)
308 (M+). Anal. (C19H20N2O2) C, H, N.
2-Eth yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
ta m id e (17d ) (chromatography on silica gel, 50% EtOAc/
hexane, EtOAc, and then 5% MeOH/EtOAc, crystallization,
MeOH): yield 21%; mp 161-166 °C; MS (FD+) 322 (M+). Anal.
(C20H22N2O2) C, H, N.
reduced pressure. Chromatography of the residue on silica
gel, eluted with a gradient of CH2Cl2-2% MeOH/CH2Cl2, gave
50 mg of starting material (9l) and 165 mg (50% yield) of
17m : mp 166-168 °C; MS (FD+) 328 (M - 1, 100), 330 (M +
1, 36). Anal. (C18H17ClN2O2) C, H, N, Cl.
Using the above procedure, 9a q and 9m were converted to
17n and 17o, respectively.
2-Br om o-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a c-
eta m id e (17n ) (heated an additional 20 h, chromatography
on silica gel, gradient, CH2Cl2-2% MeOH/CH2Cl2): yield 100;
mp 172-174 °C; MS (FD+) 372 (M - 1), 374 (M + 1). Anal.
(C18H17BrN2O2) C, H, N, Br.
2-Br om o-6-ch lor o-5-m eth oxy-1-(p h en ylm eth yl)-1H-in -
d ole-3-a ceta m id e (17o) (crystallization, EtOH/CH2Cl2): yield
1
65%; mp 205 °C dec; H NMR (DMSO-d6) ∂ 7.60 (s, 1H), 7.40
(br s, 1H), 7.30-7.20 (m, 4H), 7.00 (d, 2H), 6.90 (br s, 1H),
5.40 (s, 2H), 3.80 (s, 3H), 3.50 (s, 2H); MS (FD) 406 (M - 1,
75), 408 (M + 1, 100). Anal. (C18H16BrClN2O2) H, N, Br; C:
calcd, 53.03; found, 53.72; Cl: calcd, 8.70; found, 9.36.
5-Meth oxy-2-(m eth ylth io)-1-(ph en ylm eth yl)-1H-in dole-
3-a ceta m id e (17p ). Surfuryl chloride (0.8 mL, 10 mmol) was
added to an ice bath cooled solution of 1.0 mL of dimethyl
disulfide in 25 mL of CH2Cl2, the cooling bath was removed,
and the mixture was allowed to warm to room temperature.
Three milliliters of this solution (containing methanesulfenyl
chloride) was added to 320 mg (1.1 mmol) of 17a in 100 mL of
CH2Cl2, the mixture was stirred for 0.33 h, a saturated
NaHCO3 solution was added, the mixture was stirred well,
and the CH2Cl2 solution was separated, washed with brine,
dried (Na2SO4), and concentrated at reduced pressure. The
residue was chromatographed on silica gel, eluted with a
gradient of 40% EtOAc/hexane-EtOAc, to give 115 mg (31%
yield) of 17p : mp 195-197 °C; 1H NMR (DMSO-d6) ∂ 7.40 (br
s, 1H), 7.25-7.15 (m, 4H), 7.05 (d, 1H), 6.95 (d, 2H), 6.90 (br
s, 1H), 6.75 (dd, 1H), 3.70 (s, 3H), 3.60 (s, 2H), 2.10 (s, 3H);
MS (FD) 340 (M+). Anal. (C19H20N2O2S) H, N; C: calcd, 67.03;
found, 66.57; S: calcd, 9.42; found, 9.88.
5-Meth oxy-1-(p h en ylm eth yl)-2-p r op yl-1H-in d ole-3-a c-
eta m id e (17e) (chromatography on silica gel, EtOAc, crystal-
lization, MeOH): yield 36%; mp 154-156 °C. Anal.
(C21H24N2O2) C, H, N.
2-Cyclop r op yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-
3-a ceta m id e (17f) (crystallization, EtOH/water): yield 49%;
mp 156-158 °C; MS (FD+) 334 (M+). Anal. (C21H22N2O2) C,
H, N.
1-[(3-Ch lor op h en yl)m et h yl]-4-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet a m id e (17g) (chromatography on silica gel,
EtOAc): yield 77%; mp 171-173 °C; MS (FD+) 342 (M+). Anal.
(C19H19ClN2O2) C, H, N.
1-[(3-Ch lor op h en yl)m et h yl]-5-m et h oxy-2-m et h yl-1H -
in d ole-3-a cet a m id e (17h ) (chromatography on silica gel,
EtOAc): yield 77%; mp 171-173 °C; 1H NMR (CDCl3) ∂ 7.34-
6.76 (m, 7H), 5.63 (br s, 1H), 5.36-5.20 (m, 3H), 3.85 (s, 3H),
3.70 (s, 2H), 2.32 (s, 3H).
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-5-m eth oxy-1H-in -
d ole-3-a cet a m id e (17i) (chromatography on silica gel,
EtOAc): yield 74%; foam; MS (FD+) 357 (M+). Anal. (C19H20
ClN3O2) C, H, N.
-
1-([1,1′-Bip h en yl]-2-ylm eth yl)-4-m eth oxy-2-m eth yl-1H-
in d ole-3-a ceta m id e (17j) (crude product): yield 71%; mp
173-175 °C; 1H NMR (CDCl3) ∂ 7.62-7.08 (m, 8H), 7.04 (t,
1H), 6.78 (d, 1H), 6.5 (d, 1H), 6.48 (d, 1H), 6.07 (br s, 1H),
5.17 (s, 3H), 3.96 (s, 3H), 3.82 (s, 2H), 2.20 (s, 3H); MS (FD+)
384 (M+). Anal. (C25H24N2O2) H, N; C: calcd, 78.10; found,
78.94.
1-(Cycloh exylm eth yl)-5-m eth oxy-2-m eth yl-1H-in d ole-
3-a cetic Acid (13b). A solution of 3.1 g (9.0 mmol) of 9a e
and 5 mL of 5 N NaOH in 50 mL of EtOH was heated to
maintain reflux for 3 h, diluted with water, and made acidic
with 5 N HCl solution. The mixture was extracted with
EtOAc, and the EtOAc solution was dried over Na2SO4 and
concentrated at reduced pressure. The residue was crystal-
lized from toluene to give 2.1 g (74% yield) of 13b, melting at
173-175 °C after crystallization from toluene: MS (FD) 315
(M+). Anal. (C19H25NO3) C, H, N.
1-([1,1′-Bip h en yl]-2-ylm eth yl)-5-m eth oxy-2-m eth yl-1H-
in d ole-3-a cet a m id e (17k ) (chromatography on silica gel,
1
EtOAc): yield 67%; H NMR (CDCl3) ∂ 7.74-6.74 (m, 11H),
6.46 (d, 1H), 5.59 (br s, 1H), 5.31 (br s, 1H), 5.17 (s, 2H), 3.86
(s, 3H), 3.67 (s, 2H), 2.18 (s, 3H); MS (FD) 384 (M+).
5-Met h oxy-2-m et h yl-1-(3-p h en ylp r op yl)-1H -in d ole-3-
a ceta m id e (17l) (chromatography on silica gel, EtOAc, crys-
tallization, MeOH): yield 25%; mp 129-132 °C; 1H NMR
(CDCl3) ∂ 7.45-6.70 (m, 8H), 5.59 (br s, 1H), 5.25 (br s, 1H),
4.07 (t, 2H), 3.84 (s, 3H), 3.65 (s, 2H), 2.70 (t, 2H), 2.33 (s,
3H), 2.20-2.0 (m, 2H); MS (FD) 336 (M+). Anal. (C21H24N2O2)
H, N; C: calcd, 74.97; found, 73.81.
5-Meth oxy-2-m eth yl-1-(2-p yr id ylm eth yl)-1H-in d ole-3-
a ceta m id e (17r ) (chromatography on silica gel, EtOAc, then
5% MeOH/EtOAc): yield 28%; semisolid material; MS (FD+)
309 (M+). Anal. (C18H19N3O2) C, H, N.
1-Decyl-5-m et h oxy-2-m et h yl-1H -in d ole-3-a cet a m id e
(17s) (crystallization, EtOAc): yield 32%; mp 97-99 °C; 1H
NMR (DMSO-d6) ∂ 7.25 (d, 1H), 7.20 (br s, 1H), 7.04 (d, 1H),
6.77 (br s, 1H), 6.66 (dd, 1H), 4.04 (t, 2H), 3.80 (s, 3H), 3.42 (s,
2H), 2.34 (s, 3H), 1.70-1.50 (m, 2H), 1.35-1.17 (m, 14H), 0.88
(t, 3H); MS (FD+) 358 (M+). Anal. (C22H34N2O2) H, N; C:
calcd, 73.70; found, 76.80.
2-Ch lor o-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a c-
eta m id e (17m ). There was added 5 mL of 0.67 M methyl-
chloroaluminum amide in benzene to 344 mg (1 mmol) of 9l
in 20 mL of benzene, the mixture was heated to maintain
reflux for 2 h, an additional 5 mL of aluminum reagent was
added, and heating was continued for 1.5 h. After being cooled
with an ice bath, the mixture was decomposed with 1 N HCl
and extracted with EtOAc, and the EtOAc solution was washed
with saturated NaCl, dried (Na2SO4), and concentrated at
1-(Cycloh exylm eth yl)-5-m eth oxy-2-m eth yl-1H-in d ole-
3-a ceta m id e (17q). A solution of 0.63 g (2.0 mmol) of 13b in
25 mL of THF was cooled with an ice/water bath and 0.56 mL
(4.0 mmol) of triethylamine was added followed by 0.163 mL
(2.1 mmol) of methyl chloroformate. After 0.5 h, gaseous NH3
was bubbled into the reaction mixture for 0.5 h, the cooling
bath removed, and the mixture stirred for 2 h. It was then
poured into water and extracted with EtOAc, and the EtOAc
solution was washed with a Na2CO3 solution, dried (Na2SO4),
and concentrated at reduced pressure. The residue gave 0.3
g (48% yield) of 17q: mp 125-126 °C; MS (FD+) 314 (M+).
Anal. (C19H26N2O2) C, H, N.
5-Meth oxy-2-m eth yl-1H-in d ole-3-a ceta m id e (18). To a
solution of 6b (307 mg, 1.4 mmol) in 20 mL THF at 0 °C were
added 0.4 mL (2.8 mmol) of triethylamine and 0.17 mL (1.75
mmol) of ethyl chloroformate. The mixture was stirred for 30
min, ammonia gas was added for 30 min, and the mixture was
stirred an additional 3 h. Water was added, and the mixture
was extracted with EtOAc, washed with brine, dried (MgSO4),
concentrated at reduced pressure, and then crystallized from
CH2Cl2/MeOH to give 220 mg (yield 72%) of 18: mp 102-120
°C; MS (FD+) 218 (M+). Anal. (C12H14N2O2) C, H, N.
1-Be n zoyl-5-m e t h oxy-2-m e t h yl-1H -in d ole -3-a c e t a -
m id e (17t). To a suspension of 35 mg (0.87 mmol) of 60%
NaH/minerial oil (previously washed with hexane) in 5 mL of
DMF was added 190 mg (0.87 mmol) of 18. After 1 h, 0.1 mL
(0.87 mmol) of benzoyl chloride was added. After 4 h, the
mixture was diluted with water, extracted with EtOAc, washed
with brine, dried (MgSO4), and concentrated at reduced

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5133
pressure. The residue was stirred with EtOAc, and 17t was
filtered off and dried to give 77 mg (27% yield): mp 209-214
°C; MS (FD+) 322 (M+). Anal. (C19H18N2O3) C, H, N.
4-Meth oxy-1-(p h en ylm eth yl)-1H-in d ole (19a ). 4-Meth-
oxy-1H-indole (4p ) (1.5 g, 10 mmol) was dissolved in 20 mL of
DMF, and 400 mg (10 mmol) of 60% NaH/minerial oil was
added. After 1 h, 1.2 mL (10 mmol) of benzyl bromide was
added. After 3.5 h, the mixture was diluted with water and
extracted twice with EtOAc. The combined EtOAc was washed
with brine, dried (MgSO4), and concentrated at reduced
pressure. The residue was chromatographed on silica gel and
eluted with 20% EtOAc/hexane to give 1.77 g (75% yield) of
19a : MS (FD) 237 (M+). Anal. (C16H15NO) C, H, N.
Using the procedure above, the following conversions were
made: 4o to 19b; 4b to 19c; 2-methyl-5-nitro-1H-indole (4q)
to 19d ; 4c to 19e and 19h (alkylated with 3-chlorobenzyl
bromide); 2-ethyl-4-nitro-1H-indole (4r ) to 19f; 4e to 19g; 4d
to 19i; and 4f to 19j.
6-Met h oxy-2-m et h yl-1-r-oxo-1-(p h en ylm et h yl)-1H-in -
d ole-3-a ceta m id e (20c) (crystallization, EtOAc): yield 34%;
mp 230-234 °C; H NMR (DMSO-d6) ∂ 8.20 (br s, 1H), 7.95
1
(d, 1H), 7.75 (br s, 1H), 7.40-7.20 (m, 3H), 7.17 (d, 1H), 7.04
(d, 2H), 6.88 (dd, 1H), 5.55 (s, 2H), 3.75 (s, 3H), 2.61 (s, 3H);
MS (FD) 322 (M+). Anal. (C19H18N2O3) H, N; C: calcd, 70.79;
found, 70.11.
2-Meth yl-5-n itr o-r-oxo-1-(p h en ylm eth yl)-1H-in d ole-3-
a ceta m id e (20d ) (crystallization, MeOH): yield 67%; mp
204-206 °C; MS (FD+) 337 (M+). Anal. (C18H15N3O4) C, H,
N.
2-Eth yl-4-m eth oxy-r-oxo-1-(p h en ylm eth yl)-1H-in d ole-
3-a ceta m id e (20e) (crude product washed with H2O/EtOAc
and Et2O): yield 36%; mp 193-199 °C; 1H NMR (DMSO-d6) ∂
7.98 (br s, 1H), 7.80-6.90 (m, 8H), 6.67 (d, 1H), 5.52 (s, 2H),
3.77 (s, 3H), 2.89 (q, 2H), 1.06 (t, 3H); MS (FD+) 336 (M+).
Anal. (C20H20N2O3) H; C: calcd, 71.41; found, 66.22; N: calcd,
8.33; found, 10.42.
5-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole (19b)
(chromatography on silica gel, 25% EtOH/hexane, crystalliza-
tion, MeOH/CH2Cl2): yield 45%; mp 113-115 °C; MS (FD+)
251 (M+). Anal. (C17H17NO) C, H, N.
2-E t h yl-4-n it r o-r-oxo-1-(p h en ylm et h yl)-1H -in d ole-3-
a ceta m id e (20f) (chromatography on silica gel, 20% EtOAc/
hexane): yield 75%; mp 207-208 °C; MS (FD+) 351 (M+).
Anal. (C19H17N3O4) C, H, N.
6-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole (19c)
(crude product): yield 63%; oil; 1H NMR (CDCl3) ∂ 7.35-7.17
(m, 4H), 7.00 (d, 2H), 6.75 (d, 1H), 6.67 (s, 1H), 5.25 (s, 2H),
2-Eth yl-6-isopr opyl-5-m eth oxy-r-oxo-1-(ph en ylm eth yl)-
1H-in d ole-3-a ceta m id e (20g) (chromatography on silica gel,
50% EtOAc/hexane): yield 58%, wax; MS (FD) 378 (M+). Anal.
(C23H26N2O3) C, H, N.
3.77 (s, 3H), 2.34 (s, 3H); MS (FD+) 251 (M+). Anal. (C17H17
-
NO) H; C: calcd, 81.24; found, 80.52; N: calcd, 5.57; found,
4.78.
2-Meth yl-5-n itr o-1-(ph en ylm eth yl)-1H-in dole (19d) (crys-
tallization, EtOAc): yield 75%; mp 150-152 °C; MS (FD+) 266
(M+). Anal. (C16H14N2O2) C, H, N.
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-4-m eth oxy-r-oxo-
1H-in d ole-3-a ceta m id e (20h ) (chromatography-50% EtOAc/
hexane, then EtOAc, crystallization, MeOH): yield 75%; mp
186-189 °C; MS (FD) 370 (M - 1, 100), 372 (M + 1, 25). Anal.
(C20H19ClN2O3) C, H, N.
2-Eth yl-4-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole (19e)
(chromatography on silica gel, gradient, 5-10% Et2O/hex-
ane): yield 90%; oil; MS (FD+) 265 (M+). Anal. (C18H19NO)
C, H, N.
2-E t h y l-5-m e t h o x y -r-o x o -1-(p h e n y lm e t h y l)-1H -in -
d ole (20i) (chromatography on silica gel, gradient, CH2Cl2-
4% MeOH/CH2Cl2): yield 18%; MS (FD+) 336 (M+). Anal.
(C20H20N2O3‚0.8MeOH) C, H, N.
2-Eth yl-4-n itr o-1-(p h en ylm eth yl)-1H-in d ole (19f) (chro-
matography on silica gel, gradient, 25-50% EtOAc/hexane):
yield 84%; oil; MS (FD) 281 (M+). Anal. (C17H16N2O2) C, H,
N.
4-Met h oxy-r-h yd r oxy-1-(p h en ylm et h yl)-1H -in d ole-3-
a ceta m id e (21a ). A mixture of 1.4 g (4.5 mmol) of 20a and
213 mg (5.6 mmol) of NaBH4 and 50 mL of EtOH was stirred
for 20 h, 213 mg (5.6 mmol) of NaBH4 was added, and the
mixture was stirred for an additional 20 h, filtered, and
evaporated at reduced pressure. The residue was stirred with
EtOAc and water, and the insoluble material was filtered to
give 600 mg (43%) of 23a : mp 179-182 °C; MS (FD) 310 (M+).
Anal. (C18H18N2O3) C, H, N.
2-E t h yl-6-isop r op yl-5-m et h oxy-1-(p h en ylm et h yl)-1H -
in d ole (19g) (chromatography on silica gel, 20% EtOAc/
1
hexane): yield 47%; oil; H NMR (DMSO-d6) ∂ 7.34-6.76 (m,
7H), 6.17 (s, 1H), 5.36 (s, 2H), 3.77 (s, 3H), 3.35-3.15 (m, 1H),
2.78-2.60 (m, 2H), 1.30-1.00 (m, 9H); MS (FD+) 307 (M+).
Anal. (C21H25NO) H; C: calcd, 82.04; found, 80.95; N: calcd,
4.56; found, 4.11.
Using the above procedure, the following were converted:
20b-e,g,h to 21b-e,gm ,h .
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-4-m eth oxy-1H-in -
d ole (19h ) (chromatography on silica gel, gradient, 20-40%
CH2Cl2/hexane): yield 51%; oil; MS (FD) 299 (M - 1, 100),
301 (M + 1, 37). Anal. (C18H18ClNO) C, H, N, Cl.
2-E t h yl-5-m et h oxy-1-(p h en ylm et h yl)-1H-in d ole (19i)
(chromatography on silica gel, 20% EtOAc/hexane): yield 49%;
oil; MS (FD+) 265 (M+). Anal. (C18H19NO) C, H, N.
2-E t h yl-5-m et h oxy-6-m et h yl-1-(p h en ylm et h yl)-1H-in -
d ole (19j) (chromatography on silica gel, 10% EtOAc/hex-
ane): yield 57%; oil; 1H NMR (CDCl3) ∂ 7.49-6.90 (m, 7H),
6.26 (s, 1H), 5.25 (s, 2H), 3.87 (s, 3H), 2.65 (q, 2H), 2.27 (s,
3H), 1.29 (t, 3H); MS (FD+) 279 (M+). Anal. (C19H21NO) H,
N; C: calcd, 81.68; found, 83.01.
4-Meth oxy-r-oxo-1-(p h en ylm eth yl)-1H-in d ole-3-a ceta -
m id e (20a ). Oxalyl chloride (0.63 mL, 7.2 mmol) was added
to 1.7 g (7.2 mmol) of 19a in 20 mL of CH2Cl2, and the mixture
was stirred for 1 h and concentrated at reduced pressure. The
residue was redissolved in 25 mL of CH2Cl2, anhydrous
ammonia bubbled in for 0.25 h, and the mixture concentrated.
The residue was stirred with EtOAc and the insoluble material
filtered to give a mixture of 1.42 g of 20a and ammonium
chloride: 1H NMR (DMSO-d6) ∂ 8.48 (s, 1H), 8.05-708 (m, 9H),
6.72 (d, 1H), 5.50 (s, 2H), 3.80 (s, 3H); MS (FD) 308 (M+). Anal.
(C18H16N2O3‚1.8NH4Cl) C, H, N.
r-Hyd r oxy-5-m eth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-
in d ole-3-a ceta m id e (21b) (crystallization, MeOH): yield
1
83%; solid; H NMR (CDCl3) ∂ 7.37-6.70 (m, 9H), 6.08 (br s,
1H), 5.62 (br s, 1H), 5.39 (s, 1H), 5.26 (s, 2H), 3.83 (s, 3H),
2.40 (s, 3H); MS (FD+) 324 (M+). Anal. (C18H20N2O3) C, calcd
70.35, found 68.75; H, calcd 6.21, found 5.55; N, calcd 8.64,
found 8.14.
r-Hyd r oxy-6-m eth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-
in d ole-3-a ceta m id e (21c) (washed with EtOAc): yield 63%;
mp 196-198 °C; MS (FD) 324 (M+). Anal. (C19H20N2O3) C,
H, N.
r-H yd r oxy-2-m et h yl-5-n it r o-1-(p h en ylm et h yl)-1H -in -
d ole-3-a ceta m id e (21d ) (crude product): yield 100%; mp
120-124 °C; MS (FD+) 339 (M+). Anal. (C18H17N3O4) C, H,
N.
2-Eth yl-r-h yd r oxy-4-m eth oxy-1-(p h en ylm eth yl)-1H-in -
d ole-3-a ceta m id e (21e) (crude product washed with Et2O):
yield 88%; mp 160-162 °C; MS (FD+) 339 (M+). Anal.
(C20H22N2O3) C, H, N.
2-Eth yl-r-h yd r oxy-6-isop r op yl-5-m eth oxy-1-(p h en ylm -
eth yl)-1H-in d ole-3-a ceta m id e (21g) (triturated with Et2O/
hexane): yield 91%; mp 155-157 °C; MS (FD+) 380 (M+).
Anal. (C23H28N2O3) C, H, N.
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-r-h yd r oxy-4-m eth -
Using the above procedure, the following conversions were
made: 19b-h to 20b-h .
oxy-1H-in d ole (21h ) (crystallization, MeOH): yield 39%; mp
1
134-136 °C; H NMR (DMSO-d6) ∂ 7.34-6.84 (m, 8H), 6.55
5-Meth oxy-2-m eth yl-r-oxo-1-(ph en ylm eth yl)-1H-in dole-
3-a ceta m id e (20b) (chromatography on silica gel, EtOAc,
crystallization, MeOH): yield 23%; mp 205-207 °C; MS (FD+)
322 (M+). Anal. (C19H18N2O3) C, H, N.
(d, 1H), 5.54 (d, 1H), 5.40 (s, 2H), 5.33 (d, 1H), 3.85 (s, 3H),
2.86-2.64 (m, 2H), 1.03 (t, 3H); MS (FD) 372 (M+). Anal.
(C20H21ClN2O3) H; C: calcd, 64.43; found, 65.61; N: calcd, 7.51;
found, 11.24.

5134 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Dillard et al.
4-Met h oxy-1-(p h en ylm et h yl)-1H -in d ole-3-a cet a m id e
(17u ). A solution of 600 mg (1.9 mmol) of 21a and 0.32 mL (2
mmol) of triethylsilane in 5 mL of trifluoroacetic acid was
stirred for 16 h and concentrated under reduced pressure. The
residue was dissolved in EtOAc and water, and the EtOAc was
separated, washed with brine, and dried (MgSO4). The residue
was chromatographed on silica gel, eluted first with 50%
EtOAc/hexane and then EtOAc, to give, after crystallization
yphenyl)-1,3-dithia-2,4-diphosphetane 2,4-disulfide] in 20 mL
of toluene was refluxed 1 h, cooled, diluted with EtOAc, washed
with water, washed with brine, dried over Na2SO4, and
evaporated at reduced pressure. The residue was chromato-
graphed on silica gel, eluting with CH2Cl2, to give 25: 12 mg
(yield 10%); 1H NMR (CDCl3) ∂ 7.35-7.25 (m, 3H), 7.15 (d,
1H), 7.05 (d, 1H), 6.95 (d, 2H), 6.85 (dd, 1), 5.23 (s, 2H), 3.90
(s, 3H), 3.7 (s, 2H), 2.35 (s, 3H).
5-Meth oxy-2-m eth yl-1H-in d ole-3-a ceton itr ile (26). Us-
ing the procedure as described for 5a , 6.45 g (40 mmol) of 4o,
25 mL of 1.6 M n-BuLi/hexane (40 mmol), 40 mL of 1 M ZnCl2/
Et2O (40 mmol), and 2.70 mL (40 mmol) of bromoacetonitrile
were reacted to give after chromatography on silica gel, eluted
with 5% EtOAc/toluene, 5.2 g (yield 65%) of 26: mp 126-128
°C; MS (FD+) 200 (M+). Anal. (C12H12N2O) C, H, N.
5-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a c-
eton itr ile (27). Using the procedure described for 10a , 2.0 g
(10 mmol) of 26 was treated with 1.12 g (10 mmol) of t-BuOK
and 1.15 mL (10 mmol) of benzyl chloride to give 2.0 g of 27
(chromatography on silica gel, 5% EtOAc/toluene): yield 69%;
mp 126-128 °C; MS (FD+) 290 (M+). Anal. (C19H18N2O) C,
H, N.
5-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-3-[(1H-tetr a zol-
5-yl)m eth yl]-1H-in d ole (28a ). A mixture of 2.0 g (6.9 mmol)
of 27, 4.8 g (35 mmol) of triethylamine hydrochloride, 2.3 g
(30 mmol) of sodium azide, and 40 mL of DMF was heated at
95-105 °C for 20 h, cooled, concentrated at reduced pressure,
acidified with 6 N HCl, and extracted with EtOAc. The organic
phase was washed with water, dried over MgSO4, and evapo-
rated. The residue was purified by HPLC on an C-18 RP
column, eluted with 2% MeOH/CH2Cl2 to give 1.4 g (yield 60%)
of 28a : solid; MS (FD+) 333 (M+). Anal. (C19H19N5O) C, H,
N.
3-(Am id in om eth yl)-5-m eth oxy-2-m eth yl-1-(p h en ylm e-
th yl)-1H-in d ole (28b). To a solution of 3 mmol of methyl-
chloroaluminum amide24 in 15 mL of toluene was added 290
mg (1.0 mmol) of 27, and the resulting solution was refluxed
for 16 h, cooled, diluted with EtOAc, washed with aqueous
NaHCO3, washed with brine, dried over Na2SO4, and evapo-
rated at reduced pressure to give 28b: 50 mg (yield 20%); 1H
NMR (DMSO-d6) ∂ 9.00-8.60 (br s, 3H), 7.30-7.15 (m, 4H),
7.05 (s, 1H), 6.95 (d, 2H), 6.65 (d, 1H), 5.30 (s, 2H), 3.85 (s,
2H), 3.70 (s, 3H), 2.30 (s, 3H); MS (FD+) 307 (M+).
1
from MeOH, 262 mg (47% yield) of 17u : mp 184-187 °C; H
NMR (DMSO-d6) ∂ 7.34-6.96 (m, 9H), 6.77 (br s, 1H), 6.47 (t,
1H), 5.31 (s, 2H), 3.81 (s, 3H), 3.60 (s, 2H); MS (FD) 294 (M+).
Anal. (C18H18N2O2) C, calcd 73.45, found 77.20; H, calcd 6.16,
found 6.80; N, calcd 9.52, found 9.13.
By the above procedure, additional acetamides were pre-
pared from R-hydroxyacetamides: 21c to 17v, 21d to 17w , 21e
to 17x, 21g to 17y, and 21h to 17z:
6-Meth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-3-a c-
eta m id e (17v) (chromatography on silica gel, 33% EtOAc/
hexane, crystallization, CH2Cl2/MeOH): yield 24%; mp 136-
139 °C; MS (FD+) 308 (M+). Anal. (C19H20N2O2) C, H, N.
2-Meth yl-5-n itr o-1-(p h en ylm eth yl)-1H-in d ole-3-a ceta -
m id e (17w ) (chromatography on silica gel, EtOAc, crystal-
lization, MeOH/CH2Cl2): yield 52%; mp 189-192 °C; MS (FD+)
323 (M+). Anal. (C18H17N3O3) C, H, N.
2-Eth yl-4-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-3-a ce-
ta m id e (17x) (chromatography on silica gel, 50% EtOAc/
hexane, then EtOAc): yield 62%; mp 152-154 °C; MS (FD+)
322 (M+). Anal. (C20H22N2O2) C, H, N.
2-E t h yl-6-isop r op yl-5-m et h oxy-1-(p h en ylm et h yl)-1H -
in d ole-3-a ceta m id e (17y) (triturated with Et2O/MeOH):
yield 76%; mp 153-155 °C; MS (FD+) 364 (M+). Anal.
(C23H28N2O2) C, H, N.
1-[(3-Ch lor op h en yl)m eth yl]-2-eth yl-4-m eth oxy-1H-in -
d ole-3-a cet a m id e (17z) (chromatography on silica gel,
EtOAc): yield 48%; oil; 1H NMR (CDCl3) ∂ 7.35-6.74 (m, 6H),
6.58 (d, 1H), 6.25 (br s, 1H), 5.71 (br s, 1H), 5.29 (s, 2H), 3.97
(s, 3H), 3.86 (s, 2H), 2.75 (q, 2H), 1.13 (t, 3H); MS (FD+) 364
(M+).
5-Am in o-2-m et h yl-1-(p h en ylm et h yl)-1H-in d ole-3-a ce-
ta m id e (17a a ). A solution of 205 mg (0.634 mmol) of 17w in
30 mL of 2:1 THF/EtOH was hydrogenated at 60 psi of
hydrogen for 4 h using 0.1 g of Pd/C as catalyst. The catalyst
was filtered and the filtrate concentrated at reduced pressure.
The residue was chromatographed on silica gel, eluting
with EtOAc and then 5% MeOH/EtOAc to give 52 mg (28%
yield) of 17a a : mp 175-178 °C; MS (FD) 293 (M+). Anal.
(C18H19N3O) C, H, N.
3-[2-Met h yl-5-m et h oxy-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]p r op a n oic Acid Eth yl Ester (23). A solution 3.2 g (12.3
mmol) of 3-(2-methyl-5-methoxy-1H-indol-3-yl)propanoic acid
ethyl ester (22, crude product from 7a and 5-oxohexanoic acid
ethyl ester as described for 5f) in 40 mL of DMF was treated
with 1.37 g (12.3 mmol) of potassium tert-butoxide for 0.5 h
and then with 1.41 mL (12.3 mmol) of benzyl chloride for 24
h, poured into water, and extracted with EtOAc. The organic
phase was washed with water, dried over Na2SO4, and
evaporated at reduced pressure. The residue was chromato-
graphed on silica gel, eluting with a gradient of toluene-10%
EtOAc/toluene to give 1.66 g (yield 39%) of 23: mp 53-54 °C;
MS (FD+) 351 (M+). Anal. (C22H25NO3) C, H, N.
3-[2-Met h yl-5-m et h oxy-1-(p h en ylm et h yl)-1H -in d ol-3-
yl]p r op ion a m id e (24). A solution of 800 mg (2.3 mmol) of
23 and 2 mL of hydrazine in 20 mL of EtOH was refluxed for
18 h, cooled, diluted with water, and extracted with EtOAc.
The organic phase was washed with brine, dried over MgSO4,
and evaporated at reduced pressure to give 764 mg (yield 93%)
of the intermediate propanoic acid hydrazide as a solid. This
material and 0.3 g of Raney nickel catalyst (W-4) in 25 mL of
EtOH were refluxed for 2.5 h. The solution was decanted and
evaporated at reduced pressure and the residue chromato-
graphed on silica gel, eluting with EtOAc, to give 715 mg (yield
97%) of 24: mp 151-153 °C; MS (FD+) 322 (M+). Anal.
(C20H22N2O2) C, H, N.
N,5-Dim eth oxy-2-m eth yl-1-(p h en ylm eth yl)-1H-in d ole-
3-a ceta m id e (29b). A solution of 310 mg (1.0 mmol) of 13a
and 0.2 mL (1.4 mmol) of triethylamine in 50 mL of THF was
cooled to -5 °C and treated with 0.1 mL (1.3 mmol) of methyl
chloroformate for 10 min and then treated with a solution of
methoxylamine prepared by stirring 250 mg (1.3 mmol) of
methoxylamine hydrochloride and an excess of triethylamine
in THF. The mixture was left overnight at room temperature,
diluted with 1 N HCl, and extracted with EtOAc. The organic
phase was washed with brine, dried over Na2SO4, and evapo-
rated at reduced pressure. The residue was chromatographed
on silica gel, eluting with a gradient of 50% Et2O/hexane-
1
Et2O to give 45 mg of 29b (yield 13%): mp 123-125 °C; H
NMR (CDCl3) ∂ 8.40 (br s, 1H), 7.30-7.20 (m, 3H), 7.10 (d,
1H), 7.00-6.90 (m, 3H), 6.80 (dd, 1H), 5.25 (s, 2H), 3.80 (s,
3H), 3.65 (s, 3H), 2.30 (s, 3H); MS (FD) 338 (M+). Anal.
(C20H22N2O3) H; C: calcd, 70.99; found, 71.55; N: calcd, 8.28;
found, 7.84.
Also prepared by this procedure from 13a and methylamine
was the following.
N,2-Dim eth yl-5-m eth oxy-1-(p h en ylm eth yl)-1H-in d ole-
3-a ceta m id e (29a ) (crystallization, Et2O): yield 83%; mp
137-139 °C; 1H NMR (DMSO-d6) ∂ 7.70 (br s, 1H), 7.30-7.15
(m, 4H), 7.05 (d, 1H), 6.95 (d, 2H), 6.65 (dd, 1H), 5.30 (s, 2H),
3.75 (s, 3H), 3.45 (s, 2H), 2.55 (d, 3H), 2.25 (s, 3H); MS (FD+)
322 (M+). Anal. (C20H22N2O2) H, N; C: calcd, 74.51; found,
75.38.
(
14C)Olea te-La beled E. coli Assa y. A reaction mixture
(total volume 250 µL) composed of 2.5 × 108 autoclaved E. coli
labeled with [14C]oleate7 [a mix of labeled and unlabeled
bacteria to provide approximately 10 000 counts per minute
(cpm) of radioactivity], containing phospholipid, 40 mM of Tris-
HCl, and 10 mM of CaCl2, is incubated with enzyme (in
5-Meth oxy-2-m eth yl-1-(ph en ylm eth yl)-1H-in dole-3-th io-
a ceta m id e (25). A mixture of 100 mg (0.32 mmol) of 17c and
85 mg (0.2 mmol) of Lawesson’s reagent [2,4-bis(4-methox-

Indole Inhibitors of hnps-PLA2. 1
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5135
presence or absence of inhibitor) at 37 °C for 15 min, the
reaction stopped by the addition of ice-cold 0.5% BSA, and the
mixture promptly centrifuged at 10000g for 2 min at room
temperature. The sedimented bacteria contain the unde-
graded phospholipids, and the albumin-complexed products of
hydrolysis are in the supernatant. A measured sample of the
supernatant is counted by liquid scintillation for quantification
of hydrolysis.
in contact with the tissues.25 Agonist concentration was
increased after reaching the plateau of the contraction elicited
by the preceding concentration. One concentration-response
curve was obtained from each tissue. To minimize variability
between tissues obtained from different animals, contractile
responses were expressed as a percentage of the maximal
response obtained with the final KCl challenge. When study-
ing the effect of various drugs on the contractile effects of
sPLA2, the compounds and their respective vehicles were
added to the tissues 30 min prior to starting the sPLA2
concentration-response curves. This incubation period al-
lowed for observation of any intrinsic contractile activity
Ch r om ogen ic Assa y. The assay as described here has
been adapted for high-volume screening using 96-well micro-
titer plates.
Reagents:
(A) Rea ction Bu ffer : CaCl2‚2H2O, 1.47 g/L; KCl, 7.455 g/L;
bovine serum albumin (fatty acid free), 1 g/L; Tris‚HCl, 3.94
g/L; pH 7.5 (adjust with NaOH).
(B) En zym e Bu ffer : 0.05 N NaOAc‚3H2O; 0.2 N NaCl;
adjust pH to 4.5 with HOAc; DTNB, 5,5′-dithiobis(2-nitroben-
zoic acid); racemic diheptanoylthiopc; racemic 1,2-bis(hep-
tanoylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine; TRI-
TON X-100, prepared at 6.249 mg/mL in reaction buffer to
equal 10 µM.
Rea ction Mixtu r e. A measured volume of racemic dihep-
tanoylthio PC supplied in CHCl3 at a concentration of 100 mg/
mL is taken to dryness and redissolved in 10 mM Triton X-100
nonionic detergent aqueous solution. Reaction buffer is added
to the solution, then DTNB to give the reaction mixture which
contains 1 mM diheptanoylthio PC substrate, 0.29 mM Triton
X-100 detergent, and 0.12 mM DTNB in a buffered aqueous
solution at pH 7.5.
Assa y p r oced u r e: (1) add 0.2 mL of reaction mixture to
all wells; (2) add 10 µL of test compound (or solvent blank) to
appropriate wells, mix 20 s; (3) add 50 ng of sPLA2 (10 µL) to
appropriate wells; (4) incubate plate at 40 °C for 30 min; (5)
read absorbance of wells at 405 nm with an automatic plate
reader.
Typically, the IC50 values were determined by diluting test
compound serially 2-fold such that the final concentration in
the reaction ranged from 45 to 0.35 µg/mL. More potent
inhibitors required significantly greater dilution. In all cases,
the percent inhibition measured at 405 nm generated by
enzyme reactions containing inhibitors relative to the unin-
hibited control reactions was determined. Each sample was
titrated in triplicate, and result values were averaged for
plotting and calculation of IC50 values. The IC50 values were
determined by plotting log concentration versus inhibition
values in the range from 10 to 90% inhibition.
exhibited by the compounds, which was not
characteristic.
a desirable
Data from different experiments were pooled and presented
as a percentage of the maximal KCl responses (mean ( SEM).
To estimate the drug-induced rightward shifts in the concen-
tration-response curves, the curves were analyzed simulta-
neously using statistical nonlinear modeling methods similiar
to those described by Waud.26 The model includes four para-
meters: the maximum tissue response which was assumed
the same for each curve, the ED50 for the control curve, the
steepness of the curves, and the pA2 (apparent KB), the
concentration of antagonist that requires a 2-fold increase in
agonist to achieve an equivalent response. The Schild slope
was determined to be 1, using statistical nonlinear modeling
methods similiar to those described by Waud.26 The Schild
slope equal to 1 indicates the model is consistent with the
assumptions of a competitive antagonist; therefore the pA2 or
apparent KB could be interpreted as the dissociation constant
of the inhibitor.
To estimate the drug induced suppression of the maximal
responses, sPLA2 responses (10 µg/mL) were determined in
the absence and presence of drug, and percent suppression was
calculated for each pair of tissues.
Ack n ow led gm en t. We would like to acknowledge
our colleagues at the Shionogi Research Laboratories,
Osaka, J apan, who have collaborated with us during the
course of this research project. We would also like to
thank R. B. Hermann and R. E. Holmes for their SAR
suggestions.
Gu in ea P ig Lu n g Tissu e Ba th Assa y. Male Hartley
strain guinea pigs (500-700 g) were killed by cervical disloca-
tion and their heart and lungs removed intact and placed in
aerated (95% O2:5% CO2) Krebs’ buffer. The composition of
Krebs’ buffer was (mM) as follows: NaCl, 118.2; KCl, 4.6;
CaCl2‚2H2O, 2.5; MgSO4‚7H2O, 1.2; NaHCO3, 24.8; KH2PO4,
1.0; and dextrose, 10.0. Dorsal pleural strips (4 × 1 × 25 mm)
were dissected from intact parenchymal segments (8 × 4 ×
25 mm) cut parallel to the outer edge of the lower lung lobes.
Two adjacent pleural strips, obtained from a single lobe and
representing a single tissue sample, were tied at either end
and independently attached to a metal support rod. One rod
was attached to a Grass force-displacement transducer (FT03C).
Changes in isometric tension were displayed on a Modular
Instruments monitor and thermal recorder. All tissues were
placed in 10 mL jacketed tissue baths containing Krebs’ buffer
that was continuously aerated and maintained at 37 °C.
Pleural strips from the opposite lobes of the lung were used
for paired experiments. Preliminary data generated from
tension/response curves demonstrated that resting tension of
800 mg was optimal. The tissues were allowed to equilibrate
for 45 min as the bath fluid was changed periodically.
Initially tissues were challenged three times with KCl (40
mM) to test tissue viability and to obtain a consistent response.
After the maximal response to KCl was recorded, the tissues
were washed and allowed to return to baseline before the next
challenge. Cumulative concentration-response curves were
obtained from pleural strips by increasing the agonist concen-
tration (sPLA2 or arachidonic acid) in the tissue bath by half-
log10 increments while the previous concentration remained
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