Benzenesulfonamide indole inhibitors of cytosolic phospholipase A2α: Optimization of in vitro potency and rat pharmacokinetics for oral efficacy

Article abstract of DOI:10.1016/j.bmc.2007.10.060

The synthesis and structure-activity relationship of a series of benzenesulfonamide indole inhibitors of cPLA₂α are described. Substitution of the benzenesulfonamide led to analogues with 50-fold improvement in potency versus the unsubstituted benzenesulfonamide lead compound. Rat pharmacokinetics in a minimal formulation was used to prioritize compounds, leading to the discovery of a potent inhibitor of cPLA₂α with oral efficacy in models of rat carrageenan paw edema and Ascaris suum airway challenge in naturally sensitized sheep.

Full text of DOI:10.1016/j.bmc.2007.10.060

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1345–1358
Benzenesulfonamide indole inhibitors of cytosolic
phospholipase A2a: Optimization of in vitro potency
and rat pharmacokinetics for oral efficacy
Katherine L. Lee,a,* Mark L. Behnke,a, Megan A. Foley,a Lihren Chen,a
Weiheng Wang,a Richard Vargas,a Jill Nunez,a,à Steve Tam,a Nevena Mollova,b,§
Xin Xu,b Marina W. H. Shen,c Manjunath K. Ramarao,c Debra G. Goodwin,c
Cheryl L. Nickerson-Nutter,c William M. Abraham,d Cara Williams,c
James D. Clarkc and John C. McKewa
aDepartment of Chemical and Screening Sciences, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
bDepartment of Drug Safety and Metabolism, Wyeth Research, One Burtt Road, Andover, MA 01810, USA
cDepartment of Inflammation, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA
dMount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140, USA
Received 22 August 2007; revised 15 October 2007; accepted 17 October 2007
Available online 22 October 2007
Abstract—The synthesis and structure–activity relationship of a series of benzenesulfonamide indole inhibitors of cPLA2a are
described. Substitution of the benzenesulfonamide led to analogues with 50-fold improvement in potency versus the unsubstituted
benzenesulfonamide lead compound. Rat pharmacokinetics in a minimal formulation was used to prioritize compounds, leading to
the discovery of a potent inhibitor of cPLA2a with oral efficacy in models of rat carrageenan paw edema and Ascaris suum airway
challenge in naturally sensitized sheep.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
inflammatory mediator. cPLA2a deficient mice are gen-
erally healthy with a defect in induction of labor that is
also seen with the cyclooxygenase-1 (COX-1) deficient
mice1,2 and have slightly reduced litter size, which was
more pronounced in the COX-2 deficient mice.3,4
cPLA2a deficient mice are resistant to disease in multiple
models including an ova-induced anaphylaxis model of
asthma,5 a bleomycin-induced model of idiopathic pul-
monary fibrosis,1 and lipopolysaccharide (LPS) induced
adult respiratory distress syndrome,6 a collagen-induced
arthritis model of rheumatoid arthritis,7 a 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Par-
kinson’s model,8 a model of colon cancer in the APC
mouse,9 an ischemia reperfusion model of stroke10 and
a model of multiple sclerosis.11
Cytosolic phospholipase A2a (cPLA2a, type IVA phos-
pholipase) catalyzes the selective release of arachidonic
acid from the sn-2 position of glycerophospholipids to
initiate the production of leukotrienes, prostaglandins
and thromboxanes, all of which are inflammatory
mediators. The 1-O-alkyl-2-OH-glycerophosphocholine
fragment of the glycerophospholipid can undergo acety-
lation to form platelet-activating factor (PAF), another
Keywords: Phospholipase; Inflammation; Inhibitor; Pharmacokinetics;
Formulation; Animal model; Carrageenan paw edema; Asthma.
*
Corresponding author. Tel.: +1 617 665 5664; fax: +1 617 665
5682; e-mail: klee@wyeth.com
Present address: Infinity Pharmaceuticals, Inc., 780 Memorial Drive,
Cambridge, MA 02139, USA.
cPLA2a inhibitors have been recently reviewed by Lehr12
and Kokotos.13 In a recent publication,14 Dennis and
Kokotos described their work on 2-oxoamide cPLA2a
inhibitors. At Wyeth we have disclosed cPLA2a inhibi-
tors bearing an indole core and three pharmacophores:
à
Present address: Novartis Institutes for Biomedical Research Inc.,
250 Massachusetts Avenue, Cambridge, MA 02139, USA.
Present address: CV Therapeutics, Inc., 3172 Porter Drive, Palo Alto,
CA 94304, USA.
§
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.060

1346
K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
HO2C
HO2C
HO2C
Cl
Cl
Cl
Cl
O
O
O
O
O
O
O
S
S
S
Cl
Cl
Cl
NH
NH
NH
N
N
N
Ph
Ph
Ph
Ph
Ph
Ph
WAY-196025
Ecopladib
Efipladib
3
1
2
Figure 1. Examples of Wyeth indole phenylmethane sulfonamide cPLA2a inhibitors.
a benzoic acid, a benzhydryl group and a sulfon-
amide.15,16 Phenylmethane sulfonamide indoles 1,15 216
and 316 (Fig. 1) all are potent inhibitors of cPLA2a in
our primary in vitro assays and demonstrated oral effi-
cacy in animal models of inflammation.
of these compounds are also representative: the rat iv
clearance of compounds with the C3 propyl linker is
typically lower than that of the corresponding ethoxy
analogue (2 vs 1, 5 vs 4). These compounds are highly
lipophilic and have poor aqueous solubility, and the oral
bioavailability (F) of these inhibitors is poor, particu-
larly in a minimal formulation such as 0.5% methyl cel-
lulose (MC)/2% Tween (TW) 80. Use of a lipid-based
formulation (55.5% Phosal 53 medium chain triglyceride
(MCT), 5.6% Tween 80, 16.7% Labrasol and 22.2% pro-
pylene carbonate, in which the compound was dissolved
at 37.5 mg compound per mL vehicle, diluted with water
where necessary, and dosed at 4 mL/kg) led to improved
oral bioavailability for the three lead compounds (1, 2,
and 3). This Phosal formulation was employed for
in vivo studies in which these phenylmethanesulfona-
mides displayed oral efficacy. However, because the
Phosal formulation is a non-conventional, lipid-based
formulation, there are some potential disadvantages.
Such a vehicle requires a gel capsule, increasing the com-
plexity and cost of production. Chronic dosing of lipid-
The behavior of the three lead compounds and unsubsti-
tuted phenylmethane sulfonamide analogues 4 and 5 in
our two primary assays and rat pharmacokinetic studies
is summarized in Table 1. In the GLU micelle assay,
purified human cPLA2a cleaves an artificial substrate,
7-hydroxycoumarinyl-c-linolenate (GLU), in a deter-
gent- and lipid-rich medium, while the rat whole blood
(RWB) assay utilizes pooled rat blood stimulated with
A23187, a calcium ionophore.15 These compounds dem-
onstrate structure–activity relationship trends that we
have observed earlier16 for this class of indole cPLA2a
inhibitors, for example, that at C3, the propyl linker is
preferred to the ethoxy linker, and that at C2, ortho sub-
stitution of the sulfonamide phenylmethane moiety
leads to increased potency. The pharmacokinetic data
Table 1. In vitro potency and rat pharmacokinetic data of phenylmethane- and benzenesulfonamides
HO2C
R
X
O
O
S
n
Cl
NH
N
Ph
Ph
iv Clb
(mL/min/kg)
Fc (%) MC/TW
formulation
Fc (%) Phosal
formulation
a
a
Compound
n
X
R
GLU IC50
(lM)
RWB IC50
(lM)
1
2
3
4
5
6
1
1
1
1
1
0
O
3,4-diCl
3,4-diCl
2,6-diMe
H
0.15
0.04
0.014
0.11
0.04
3.0
0.16
0.07
0.03
0.12
0.08
1.0
14
5.0
4.0
69
13
73
6.0
1.0
1.0
3.9
3.8d
—
12
CH2
CH2
O
6.0
3.5
—
7.2e
—
CH2
O
H
H
a Data are the average of two or more independent measurements.
b iv clearance (Cl) was determined at a dose of 2 mg/kg in 50% PEG-400, 50% DMSO formulation unless otherwise specified.
c Oral bioavailability (F) was determined by the dose normalized AUC ratio between oral and iv administration. A po dose of 25 mg/kg was used
unless otherwise specified.
d po dose 5 mg/kg.
e po dose 10 mg/kg.

K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
1347
based formulations may change the plasma lipoprotein
profile.17 Because of these concerns, we sought to avoid
the use of a lipid-based formulation such as Phosal, and
instead to use a minimal formulation in selection of a
new lead.
sequent hydrolysis of the methyl ester of intermediates
11 and 12 afforded the desired products (13 and 14).
3. Results
The focus of the work reported here is on benzenesul-
fonamide cPLA2a inhibitors. The starting point was
compound 6, which as a low lM inhibitor in both pri-
mary assays was significantly less potent than the corre-
sponding phenylmethane analogue (4). The goals of this
effort were twofold: to improve the potency of the ben-
zenesulfonamide class by exploring substitution of the
benzene moiety and demonstrate oral efficacy in a min-
imal formulation. Compounds were triaged based on rat
pharmacokinetics, that is, iv clearance (Cl) and oral
exposure in a conventional MC/TW formulation; for
all of these studies, the benchmark compounds were
the phenylmethane lead compounds exemplified by 1,
2 and 3.
3.1. In vitro and pharmacokinetic data
The in vitro data of the benzenesulfonamides 13a–k and
14a–k in the GLU micelle and RWB assays (Table 2)
indicate that as with the phenylmethane sulfonamides,
the propyl linker (X = CH2) generally is preferred over
the ethoxy linker (X = O). Substitution of the benzene-
sulfonamide led to increased potency compared to the
unsubstituted parent compound 6. The IC50 values of
these compounds in the RWB assay ranged from 0.06
to 0.60 lM, with the more potent compounds compar-
ing favorably to the lead compounds in the phenylmeth-
ane series (1, 2, and 3).
Rat pharmacokinetics of these compounds were then
determined. Again, in agreement with the phenylmeth-
ane sulfonamide series, compounds bearing the propyl
linker (X = CH2) displayed lower iv clearance than
their ethoxy linked (X = O) analogues. The majority
of the compounds had reasonable clearance, of less
than 20 mL/min/kg. Oral bioavailability (F) was calcu-
lated upon dosing the compounds orally using a mini-
mal formulation of MC/TW. Most compounds
showed better F than the more potent phenylmethane
derivatives did in the same formulation (2 and 3 dis-
played 1% F in rats). Only compounds 14d, 13d, 13h,
and 14k had >10% F, and of these, 14k offered the best
combination of in vitro potency and pharmacokinetic
parameters in rats: 13d displayed both a higher clear-
ance and was fivefold less potent, and 13d and 13h were
2- to 3-fold less potent in the RWB assay than 14k.
Thus, 14k was further studied by pharmacokinetic
determination in two formulations in rat and dog, iso-
thermal calorimetry, selectivity assays, a human whole
blood assay, and animal models of acute inflammation
and asthma.
2. Chemistry
There are hundreds of commercially available benzene-
sulfonyl chlorides, and for this structure–activity study,
we employed mono- or disubstituted benzenesulfonyl
chlorides, mainly with ortho substitution. One sulfonyl
chloride (8) was prepared by lithium–halogen exchange
of bromide 7, reaction with sulfur dioxide, and subse-
quent reaction with sulfuryl chloride (Scheme 1).
Scheme 2 shows the synthesis of our benzenesulfon-
amide cPLA2a inhibitors via a two-step procedure. Sulf-
onylation of amino esters 915 and 1016 with a sulfonyl
chloride under Schotten–Baumann conditions and sub-
F
F
SO2Cl
CF3
Br
a, b
CF3
Rat and dog pharmacokinetics of 14k and phenylmeth-
ane sulfonamide lead compound 3 were determined in
both the lipid-based Phosal formulation which had
been developed to increase the oral exposure of the
phenylmethane sulfonamides and the minimal MC/TW
7
8
Scheme 1. Reagents and conditions: (a) n-BuLi/hexanes, SO2, Et2O–
THF, ꢀ78 ꢁC–rt; (b) SO2Cl2/CH2Cl2, hexanes, 0 ꢁC–rt, 48% (two
steps).
MeO2C
HO2C
MeO2C
Cl
X
X
X
O
O
R
R
O
O
S
S
Cl
Cl
NH2
NH
NH
a
b
N
N
N
Ph
Ph
Ph
Ph
Ph
Ph
9: X = O
11: X = O
13: X = O
10: X = CH2
12: X = CH2
14: X = CH2
Scheme 2. Reagents and conditions: (a) ArSO2Cl, satd NaHCO3, CH2Cl2, 61–93%; (b) 1 N NaOH, MeOH, THF, 50 ꢁC, 36–100%.

1348
K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
Table 2. In vitro and rat pharmacokinetic data of benzene sulfonamides
HO2C
X
O
R
O
S
Cl
NH
N
iv Clb (mL/min/kg)
Fc (%) MC/TW formulation
a
a
Compound
R
X
GLU IC50 (lM)
RWB IC50 (lM)
6
H
2-Ph
O
3.0
1.0
73
—
13a
14a
13b
14b
13c
14c
13d
14d
13e
14e
13f
14f
13g
14g
13h
14h
13i
14i
13j
14j
13k
14k
O
0.11
0.04
0.57
0.18
0.60
0.19
0.40
0.10
0.52
0.10
0.34
0.09
0.18
0.10
0.35
0.10
0.26
0.06
0.10
0.047
0.18
0.065
0.15
0.06
0.60
0.22
0.15
0.22
0.55
0.20
0.32
0.12
0.16
0.095
0.06
0.20
0.36
0.20
0.21
0.10
0.095
0.075
0.15
0.10
4.5
2.3
<0.1
0.4
—
2-Ph
3-Ph
CH2
O
—
—
3-Ph
2-F
CH2
O
—
28.0
8.8
6.0
4.0
39.0
13.7
<0.2
1.9
—
2-F
2-Cl
CH2
O
31.0
9.4
2-Cl
2-Br
CH2
O
3.1
6.5
2-Br
2-Me
2-Me
CH2
O
61.0
10.3
18.6
10.7
12.4
5.2
CH2
O
8.6
<0.1
7.8
15.4
4.6
2.9
3.8
6.0
1.3
3.0
13.0
2-OMe
2-OMe
2-OCF3
2-OCF3
2,6-diCl
2,6-diCl
2,6-diMe
2,6-diMe
2-F, 6-CF3
2-F, 6-CF3
CH2
O
CH2
O
16.3
8.5
CH2
O
6.1
CH2
O
10.5
15.3
14.0
CH2
a Data are the average of two or more independent measurements.
b iv clearance (Cl) was determined at a dose of 2 mg/kg in 50% PEG-400, 50% DMSO formulation.
c Oral bioavailability (F) was determined by the dose normalized AUC ratio between oral and iv administration. A po dose of 25 mg/kg was used.
formulation which had been used to filter the benzene-
sulfonamides (Table 3). In both rat and dog, the phenyl-
methane derivative 3 had higher %F in the lipid-based
formulation than in the conventional MC/TW formula-
tion. Benzenesulfonamide 14k, however, had a slightly
higher %F in the MC/TW formulation than in the Pho-
sal formulation. Although 13–16%F is generally consid-
ered low, this represented an improvement compared to
all cPLA2a inhibitors of suitable activity in whole blood
assays, which are highly lipophilic and have high molec-
ular weight, and typically have <5% oral bioavailability.
Benzenesulfonamide 14k demonstrated >10-fold higher
%F than 3 in the MC/TW formulation in both rat and
dog, thus fulfilling one of the goals of this effort.
3.2. Isothermal calorimetry data
The affinity and stoichiometry of compound 14k for hu-
man cPLA2a were determined using isothermal calorim-
etry at 30 ꢁC using buffer conditions similar to that of
the GLU micelle assay. An exothermic reaction was ob-
served. The binding isotherm displayed a good fit
(CHI2 = 92793.8) with a single site model (N = 1.03),
indicating that one molecule of compound 14k bound
Table 3. Rat and dog pharmacokinetic parameters of 3 and 14k in two formulations
Compound
Species
iv Cla (mL/min/kg)
Fb (%) Phosal formulation
Fb (%) MC/TW formulation
3
Rat
Rat
Dog
Dog
3.5
14
3.5
5.0
1.5
13
1.0
13
14k
3
0.6
1.4
0.08
16
14k
a For rats, iv dose 2 mg/kg in 50% PEG-400, 50% DMSO formulation. For dogs, iv dose 2 mg/kg in 20% DMSO, 80% PEG-200 formulation.
b For rats, po dose 25 mg/kg. For dogs, a po dose of 50 mg/kg was used for compound 3 and 5 mg/kg was used for compound 14k.

K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
1349
to one molecule of cPLA2a. The Kd was 80 nM, in
agreement with that IC50 seen in the GLU micelle assay
(65 nM).
Naproxen, dosed in the Phosal formulation, and the
COX-2 selective inhibitor Celecoxib, for which the vehi-
cle was MC/TW. Inhibitor 14k was dosed orally at two
concentrations in both vehicles. While 14k when dosed
at 25 mg/kg in the Phosal vehicle led to a 16% decrease
in paw volume, 14k dosed at 25 mg/kg in the minimal
formulation of MC/TW was more efficacious, with a
44% decrease in paw volume. At 10 mg/kg orally in
MC/TW, 14k led to a 34% decrease in paw volume.
14k was more efficacious at the 25 mg/kg dose (44% de-
crease in paw volume) than the earlier lead compound,
phenylmethane sulfonamide 3, at the same dose (36%
decrease in paw volume) when both compounds were
administered in their preferred formulations (p-value
from Student’s t-test: 0.03). The ED50 (defined as the
dose giving 50% of the maximum response) of com-
pound 14k in MC/TW formulation is <10 mg/kg.20
3.3. Selectivity data
Compound 14k was also evaluated in assays for COX
inhibition. At a compound concentration of 100 lM,
14k led to 45.0% inhibition of COX-1 and 9.9% inhibi-
tion of COX-2, while its IC50 in the GLU micelle assay
measuring inhibition of cPLA2a is 0.065 lM. Thus, 14k
was shown to be greater than 1000-fold selective for
cPLA2a versus COX-1 and -2.
3.4. Human whole blood data
The human whole blood assay employed at Wyeth has
been previously described.15 Compound 14k was tested
using blood from 3 different donors. At 0.3 lM, 14k
blocked the production of TXB2, PGE2, PGF2a, and
LTB4, as indicated in Table 4. The activity of 14k in
these assays is consistent with its behavior as a cPLA2a
inhibitor in the GLU micelle and RWB assays and dem-
onstrates that 14k effectively inhibited the production all
of the arachidonate metabolites tested.
3.6. Ascaris suum airway challenge in naturally sensitized
sheep
This large animal model21 can be utilized to measure the
effect of inhibitors in decreasing allergen-induced bron-
choconstriction and associated airway hyperresponsive-
ness (AHR), both of which are cardinal features of
asthma. Sheep that are naturally sensitized to the para-
sitic nematode worm A. suum when challenged via the
airways with the A. suum antigen develop both an early
phase (EAR) and late phase (LAR) bronchoconstriction
and AHR to aerosolized carbachol. This model has
proved useful in helping to elucidate the pathogenesis
of asthma as well as in predicting corticosteroid utility
in the disease. Efficacy of 14k was demonstrated in this
model by iv dosing (10 mg/kg BID) and oral dosing
(3 mg/kg BID) the day prior to antigen challenge and
BID the day of challenge administration (Table 5).
Additionally, 14k was orally efficacious at a lower dose
(1 mg/kg) when dosed BID for 4 days prior to challenge
and BID on the day of challenge. With all three dosing
regimens, 14k had minimal impact on the early asth-
matic response (EAR) while demonstrating a significant
impact versus historical control on the late asthmatic re-
sponse (LAR); for example, when 14k was dosed 5 days
at 1 mg/kg, the mean maximum LAR was reduced from
3.5. Rat carrageenan paw edema (CPE) model
The CPE model is an acute inflammation model that is
primarily driven by prostaglandins.18,19 It has been
highly predictive of NSAID and COX-2 utility. In this
study, all of the inhibitors were dosed orally (Fig. 2).
The positive controls in this study were the NSAID
Table 4. Inhibition of inflammatory mediators in human whole blood
by 14k
a
Product
IC50 (lM)
Std. dev.
TXB2
LTB4
PGE2
PGF2a
0.12
0.048
0.009
0.017
0.03
0.082
0.060
0.11
a Compound 14k was tested using blood from three different donors.
70
60
50
40
30
20
10
0
Naproxen Celecoxib
10 mg/kg 25 mg/kg 25 mg/kg 10 mg/kg 25 mg/kg 10 mg/kg 25 mg/kg
(Phosal) (MC/TW) (Phosal) (Phosal) (MC/TW) (MC/TW) (Phosal)
Cpd 14k
Cpd 14k
Cpd 14k
Cpd 14k
Cpd 3
-10
Figure 2. Percent inhibition of paw swelling in the rat carrageenan paw edema model by Naproxen, Celecoxib, 14k and 3.

1350
K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
Table 5. Efficacy of compound 14k in Ascaris suum airway challenge in naturally sensitized sheepa
Route and
frequency of
administration
Duration
of dosing
(days)
Dose (mg/kg)
Mean Max.
EARb (%)
Mean Max.
LARb (%)
Mean Max.
AHRc
iv BID
po BID
po BID
1
2
5
Controla
10
405
346
136
29
14.5
23.5
Controla
3
526
379
136
29
13.0
24.0
Controla
1
396
431
124
68
11.7
26.0
a Control values are from historical data obtained from the same sheep.
b Mean maximum percentage increase in specific lung resistance.
c Mean cumulative carbachol concentration that increased specific lung resistance by 400% over the post saline value.
124% to 68%. Also, in all three dosing regimens, 14k had
a significant impact on AHR, that is, the mean cumula-
tive carbachol concentration required to increase the
specific lung resistance by 400% versus saline in sheep
treated with 14k was comparable to the amount needed
for untreated animals which had not been exposed to
antigen.
CHNS/O analyzer. CHN analyses were carried out by
Robertson-Microlit. Low resolution mass spectra were
obtained using a Micromass Platform Electrospray Ion-
ization Quadrupole mass spectrometer. High resolution
mass spectra were obtained using a Bruker (Billerica,
MA) APEXIII Fourier transform ion cyclotron reso-
nance (FT-ICR) mass spectrometer equipped with an
actively shielded 7 Tesla superconducting magnet
(Magnex Scientific Ltd, UK) and an external Bruker
APOLLO electrospray ionization (ESI) source. Flash
chromatography was performed using EM Science
230–400 mesh silica gel or Biotage flash columns packed
with KP-SIL 60 Angstrom silica gel. Thin-layer chroma-
tography (TLC) was performed using EMD 250 lm pre-
scored silica gel 60 F254 plates. Purity in two solvent
systems (H2O–CH3CN and H2O–MeOH) was deter-
mined using an Agilent 1100HPLC instrument.
4. Conclusion
Starting from a benzenesulfonamide indole cPLA2a
inhibitor with in vitro potency in the low lM range,
we synthesized substituted benzenesulfonamide ana-
logues with at least 10-fold improved potency in isolated
enzyme and rat whole blood assays. The SAR of these
inhibitors indicates that at C3, the propyl linker is pre-
ferred to the ethoxy linker, and that ortho substitution
of the benzene moiety of the sulfonamide is favorable.
Triage of these inhibitors by rat pharmacokinetics, by
first determining iv clearance, then measuring oral expo-
sure in a minimal formulation, led to the selection of
14k, which showed superior oral exposure in the MC/
TW formulation in rat and dog compared to the phen-
ylmethane sulfonamide lead compound 3. In the rat
CPE model, benzenesulfonamide 14k demonstrated oral
efficacy at 10 mg/kg when administered in the MC/TW
formulation. Furthermore, in the same in vivo model,
14k, when administered in MC/TW, the preferred, min-
imal formulation, showed superior oral efficacy to the
lead phenylmethane sulfonamide WAY-196025 (3)
administered in a lipid-based formulation. Indole 14k
also was orally efficacious at 1 mg/kg in the attenuation
of both the LAR and the associated AHR to aerosolized
carbachol in naturally sensitized sheep that had been
challenged via the airways with A. suum antigen.
5.1.1. 2-Fluoro-6-trifluoromethylbenzenesulfonyl chloride
(8). To a solution of 2-bromo-3-fluoro-benzotrifluoride
7
(15 g, 62 mmol) in THF (150 mL) and Et2O
(150 mL) cooled to ꢀ78 ꢁC was added n-BuLi (2.5 M
in hexanes, 25 mL, 62 mmol) dropwise. The mixture
was stirred at ꢀ78 ꢁC for 30 min. Sulfur dioxide
(200 mL, 3.9 mol) was condensed into a separate flask
at ꢀ78 ꢁC and Et2O (200 mL), precooled to ꢀ78 ꢁC,
was added via cannula. The n-BuLi mixture was added
via cannula to the sulfur dioxide mixture, and the result-
ing suspension was allowed to warm to room tempera-
ture overnight. The mixture was concentrated, diluted
with Et2O (200 mL), and filtered. The off-white solid
was washed with Et2O (200 mL), transferred to a flask,
and suspended in hexanes (500 mL), and cooled to
0 ꢁC. Sulfuryl chloride (1.0 M in CH2Cl2, 65 mL,
65 mmol) was added dropwise. The mixture was allowed
to warm to room temperature overnight and concen-
trated. The residue was triturated with hexanes
(500 mL) at 40 ꢁC for 30 min, filtered, and dried to af-
ford sulfonyl chloride 8 (7.8 g, 48% yield), an off-white
5. Experimental
1
powder. H NMR (400 MHz, DMSO-d6) d 7.39–7.50
(m, 1H), 7.50–7.57 (m, 2H).
5.1. General methods: chemistry
All solvents and reagents were used as obtained. Proton
NMR spectra were recorded at 300 MHz on a Varian
Gemini 2000 or 400 MHz Bruker AV-400 spectrometer
using TMS (d 0.0) as a reference. Combustion analyses
were obtained using a Perkin Elmer Series II 2400
5.2. General procedure 1: sulfonylation of amines via
Schotten–Baumann reaction
To a solution of the amine 915 or 1016 (1.0 mmol) in
CH2Cl2 (10 mL) were added sulfonyl chloride

K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
1351
(1.2 mmol) and sat. NaHCO3 (10 mL). The resulting
suspension was stirred until the amine was consumed
(TLC analysis in 10% MeOH–CH2Cl2). The mixture
was diluted with CH2Cl2 (20 mL), washed with H2O
(20 mL) and brine, dried (MgSO4), and concentrated.
Purification of the crude product by flash chromatogra-
phy (EtOAc–hexanes) afforded the sulfonamide.
J = 8.8 Hz, 1H), 6.73–6.85 (m, 3H), 6.89 (s, 1H),
6.99–7.10 (m, 4H), 7.21–7.39 (m, 9H), 7.51 (d,
J = 2.0 Hz, 1H), 7.65 (dd, J = 7.8, 1.3 Hz, 1H),
7.88–8.01 (m, 3H).
5.2.6. Methyl 4-{2-[1-benzhydryl-5-chloro-2-(2-{[(2-meth-
ylphenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}ben-
zoate (11f). Sulfonylation of
9
with 2-methyl-
5.2.1. Methyl 4-[2-(1-benzhydryl-2-{2-[(1,10-biphenyl-
2-ylsulfonyl)amino]ethyl}-5-chloro-1H-indol-3-yl)ethoxy]-
benzenesulfonyl chloride, white foam, 61% yield. 1H
NMR (400 MHz, CDCl3) d 1.87–2.00 (m, 2H), 2.48 (s,
3H), 2.71 (appar q, J = 7.8 Hz, 4H), 2.78–2.88 (m,
2H), 2.91–3.07 (m, 2H), 3.91 (s, 3H), 4.36 (t,
J = 6.3 Hz, 1H), 6.48 (d, J = 8.8 Hz, 1H), 6.79 (dd,
J = 8.8, 2.0 Hz, 1H), 6.84 (s, 1H), 6.97–7.08 (m, 4H),
7.12–7.20 (m, 1H), 7.20–7.34 (m, 9H), 7.36–7.45 (m,
3H), 7.74 (dd, J = 7.8, 1.3 Hz, 1H), 7.96 (d, J = 8.3 Hz,
2H).
benzoate (11a). Sulfonylation of
9 with 2-phen-
1
ylbenzenesulfonyl chloride, white foam, 93% yield. H
NMR (400 MHz, CDCl3) d 2.47–2.66 (m, 2H), 2.7–
2.96 (m, 2H), 3.09 (t, J = 6.7 Hz, 2H), 3.59 (t,
J = 6.4 Hz, 1H), 3.89 (s, 3H), 4.08 (d, 2H), 6.47 (d,
J = 8.8 Hz, 1H), 6.66–6.85 (m, 4H), 6.92–7.04 (m, 4H),
7.20–7.42 (m, 13H), 7.46–7.57 (m, 2H), 7.85–7.98 (m,
2H), 8.02 (dd, J = 8.0, 1.1 Hz, 1H).
5.2.7. Methyl
4-{2-[1-benzhydryl-5-chloro-2-(2-{[(2-
5.2.2. Methyl 4-[2-(1-benzhydryl-2-{2-[(1,10-biphenyl-
3-ylsulfonyl)amino]ethyl}-5-chloro-1H-indol-3-yl)ethoxy]-
methoxyphenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]eth-
oxy}benzoate (11g). Sulfonylation of 9 with 2-meth-
oxybenzenesulfonyl chloride, white foam, 61% yield.
1H NMR (400 MHz, CDCl3) d 3.01–3.15 (m, 2H), 3.25
(t, J = 7.6 Hz, 2H), 3.34 (t, J = 6.7 Hz, 2H), 3.87 (s,
3H), 4.05 (s, 3H), 4.33 (t, J = 6.3 Hz, 2H), 5.18 (t,
J = 6.1 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 6.97 (appar
d, J = 9.1 Hz, 3H), 7.03–7.15 (m, 3H), 7.18 (appar dd,
J = 7.3, 1.8 Hz, 4H), 7.34–7.53 (m, 6H), 7.61–7.69 (m,
1H), 7.70 (d, J = 2.0 Hz, 1H), 7.94 (dd, J = 7.7, 1.6 Hz,
1H), 8.11 (d, J = 8.8 Hz, 2H).
benzoate (11b). Sulfonylation of
9 with 3-phen-
1
ylbenzenesulfonyl chloride, white foam, 61% yield. H
NMR (400 MHz, CDCl3) d 2.98–3.10 (m, 2H), 3.14–
3.34 (m, 4H), 3.96 (s, 3H), 4.24 (t, J = 6.6 Hz, 2H),
4.57 (t, J = 6.3 Hz, 1H), 6.59 (d, J = 8.8 Hz, 1H), 6.75–
6.94 (m, 3H), 6.96 (s, 1H), 7.02–7.17 (m, 4H), 7.25–
7.40 (m, 6H), 7.43–7.55 (m, 4H), 7.56–7.64 (m, 3H),
7.64–7.73 (m, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.96–8.08
(m, 3H).
5.2.3. Methyl 4-{2-[1-benzhydryl-5-chloro-2-(2-{[(2-fluo-
rophenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}-
5.2.8. Methyl 4-(2-{5-chloro-1-(diphenylmethyl)-2-[2-({[2-
(trifluoromethoxy)phenyl]sulfonyl}amino)ethyl]-1H-indol-
3- yl}ethoxy)benzoate (11h). Sulfonylation of 9 with 2-
trifluoromethoxybenzenesulfonyl chloride, white foam,
61% yield. 1H NMR (400 MHz, CDCl3) d 2.81–2.95
(m, 2H), 3.03–3.13 (m, 2H), 3.16 (t, J = 6.6 Hz, 2H),
3.88 (s, 3H), 4.17 (t, J = 6.6 Hz, 2H), 4.71 (t,
J = 6.2 Hz, 1H), 6.53 (d, J = 8.8 Hz, 1H), 6.78 (d,
J = 8.8 Hz, 2H), 6.81 (dd, J = 8.8, 2.3 Hz, 1H), 6.90 (s,
1H), 6.99–7.09 (m, 4H), 7.15–7.39 (m, 8H), 7.45–7.62
(m, 2H), 7.82 (dd, J = 7.8, 1.8 Hz, 1H), 7.92 (d,
J = 8.8 Hz, 2H).
benzoate (11c). Sulfonylation of
9
with 2-
fluorobenzenesulfonyl chloride, white foam, 61% yield.
1H NMR (400 MHz, CDCl3) d 2.82–2.94 (m, 2H),
2.98–3.06 (m, 2H), 3.10 (t, J = 6.7 Hz, 2H), 3.82 (s,
3H), 4.12 (t, J = 6.6 Hz, 2H), 4.68 (t, J = 6.3 Hz, 1H),
6.46 (d, J = 8.6 Hz, 1H), 6.69–6.78 (m, 3H), 6.83 (s,
1H), 6.93–7.01 (m, 4H), 7.01–7.09 (m, 2H), 7.16–7.30
(m, 6H), 7.37–7.48 (m, 2H), 7.60–7.70 (m, 1H), 7.87
(d, J = 9.1 Hz, 2H).
5.2.4. Methyl 4-{2-[1-benzhydryl-5-chloro-2-(2-{[(2-chlo-
rophenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}-
5.2.9. Methyl 4-{2-[1-benzhydryl-5-chloro-2-(2-{[(2,6-
dichlorophenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}-
benzoate (11i). Sulfonylation of 9 with 2,6-dic-
hlorobenzenesulfonyl chloride, white foam, 77% yield.
1H NMR (400 MHz, CDCl3) d 2.83–3.03 (m, 2H),
3.07–3.17 (m, 2H), 3.16–3.31 (m, 2H), 3.89 (s, 3H),
4.20 (t, J = 6.6 Hz, 2H), 5.33 (t, J = 5.3 Hz, 1H), 6.54
(d, J = 8.8 Hz, 1H), 6.72–6.86 (m, 3H), 6.90 (s, 1H),
6.98–7.15 (m, 4H), 7.18–7.32 (m, 7H), 7.37 (d,
J = 8.3 Hz, 2H), 7.53 (s, 1H), 7.93 (d, J = 8.8 Hz, 2H).
benzoate (11d). Sulfonylation of
9
with 2-
chlorobenzenesulfonyl chloride, white foam, 86% yield.
1H NMR (400 MHz, DMSO-d6) d 2.86–2.98 (m, 2H),
2.98–3.07 (m, 2H), 3.07–3.21 (m, 2H), 3.81 (s, 3H),
4.19 (t, J = 6.7 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 6.80
(dd, J = 8.8, 2.0 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H),
6.99–7.14 (m, 5H), 7.28–7.36 (m, 6H), 7.37–7.44 (m,
1H), 7.53–7.61 (m, 1H), 7.60–7.69 (m, 2H), 7.80 (dd,
J = 7.8, 1.5 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 8.11 (t,
J = 6.1 Hz, 1H).
5.2.10. Methyl 4-{2-[1-benzhydryl-5-chloro-2-(2-{[(2,6-
dimethylphenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]eth-
oxy}benzoate (11j). Sulfonylation of 9 with 2,6-dim-
ethylbenzenesulfonyl chloride, white foam, 88% yield.
1H NMR (400 MHz, CDCl3) 2.41–2.55 (m, 6H), 2.81–
2.98 (m, 2H), 2.99–3.10 (m, 2H), 3.16 (t, J = 6.7 Hz,
2H), 3.89 (s, 3H), 4.17 (t, J = 6.7 Hz, 2H), 4.50 (t,
J = 5.9 Hz, 1H), 6.53 (d, J = 8.8 Hz, 1H), 6.74–6.86
5.2.5. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2-bromophe-
nyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic
acid (11e). Sulfonylation of 9 with 2-bromobenzene-
sulfonyl chloride, white foam, 90% yield. 1H NMR
(400 MHz, CDCl3) d 2.72–2.96 (m, 2H), 3.04–3.13 (m,
2H), 3.16 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 4.17 (t,
J = 6.6 Hz, 2H), 5.20 (t, J = 6.4 Hz, 1H), 6.52 (d,

1352
K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
(m, 4H), 6.97–7.05 (m, 4H), 7.07 (s, 1H), 7.19–7.25 (m,
1H), 7.25–7.32 (m, 7H), 7.53 (d, J = 2.0 Hz, 1H), 7.93
(d, J = 8.8 Hz, 2H).
(dd, J = 7.7, 1.4 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 8.06
(t, J = 5.7Hz, 1H).
5.2.16. Methyl 4-{3-[1-benzhydryl-2-(2-{[(2-bromophe-
nyl)sulfonyl]amino}ethyl)-5-chloro-1H-indol-3-yl]propyl}-
benzoate (12e). Sulfonylation of 10 with 2-
bromobenzenesulfonyl chloride, white foam, 76% yield.
1H NMR (400 MHz, CDCl3) d 1.85–2.02 (m, 2H), 2.49–
2.86 (m, 6H), 2.88–3.06 (m, 2H), 3.91 (s, 3H), 5.10 (t,
J = 6.3 Hz, 1H), 6.48 (d, J = 8.8 Hz, 1H), 6.79 (dd,
J = 9.0, 2.1 Hz, 1H), 6.83 (s, 1H), 6.99–7.06 (m, 4H),
7.24 (d, J = 8.3 Hz, 2H), 7.25–7.43 (m, 10H), 7.62–7.69
(m, 1H), 7.89–8.01 (m, 3H).
5.2.11. Methyl 4-(2-{1-benzhydryl-5-chloro-2-[2-({[2-flu-
oro-6-(trifluoromethyl)phenyl]sulfonyl}amino)ethyl]-1H-
indol-3-yl}ethoxy)benzoate (11k). Sulfonylation of 9 with
2-fluoro-6-trifluoromethylbenzenesulfonyl chloride (8),
pale yellow foam, 89% yield. 1H NMR (400 MHz,
CDCl3) 3.00 (q, J = 6.8 Hz, 2H), 3.08–3.16 (m, 2H),
3.20 (t, J = 6.4 Hz, 2H), 3.88 (s, 3H), 4.20 (t,
J = 6.4 Hz, 2H), 4.99 (t, J = 6.2 Hz, 1H), 6.54 (d,
J = 8.8 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.84 (dd,
J = 9.0, 2.1 Hz, 1H), 6.88 (s, 1H), 7.01–7.07 (m, 4H),
7.22–7.37 (m, 7H), 7.54 (d, J = 2.0 Hz, 1H), 7.57–7.67
(m, 2H), 7.93 (d, J = 9.1 Hz, 2H).
5.2.17. Methyl 4-{3-[1-benzhydryl-5-chloro-2-(2-{[(2-
methylphenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}-
benzoate (12f). Sulfonylation of 10 with 2-meth-
1
5.2.12. Methyl 4-[3-(1-benzhydryl-2-{2-[(1,10-biphenyl-2-
ylsulfonyl)amino]ethyl}-5-chloro-1H-indol-3-yl)propyl]-
benzoate (12a). Sulfonylation of 10 with 2-phen-
ylbenzenesulfonyl chloride, white foam, 65% yield. H
NMR (400 MHz, CDCl3) d 2.42 (s, 3H), 2.76–2.94 (m,
2H), 2.95–3.05 (m, 2H), 3.08 (t, J = 6.7 Hz, 2H), 3.80
(s, 3H), 4.10 (t, J = 6.6 Hz, 2H), 4.41 (t, J = 6.3 Hz,
1H), 6.44 (d, J = 8.8 Hz, 1H), 6.66–6.78 (m, 3H), 6.82
(s, 1H), 6.92–7.00 (m, 4H), 7.05 (t, J = 8.3 Hz, 1H),
7.12–7.26 (m, 8H), 7.25–7.36 (m, 1H), 7.44 (d,
J = 2.0 Hz, 1H), 7.66 (dd, J = 8.0, 1.4 Hz, 1H), 7.85 (d,
J = 8.8 Hz, 2H).
1
ylbenzenesulfonyl chloride, white foam, 83% yield. H
NMR (400 MHz, CDCl3) d 1.78–1.93 (m, 2H), 2.44–
2.56 (m, 2H), 2.56–2.72 (m, 4H), 2.73–2.83 (m, 2H),
3.55 (t, J = 6.1 Hz, 1H), 3.91 (s, 3H), 6.44 (d,
J = 8.8 Hz, 1H,) 6.73 (s, 1H), 6.79 (dd, J = 8.8, 2.0 Hz,
1H), 6.90–7.04 (m, 4H), 7.10–7.33 (m, 14H), 7.35–7.46
(m, 2H), 7.48–7.63 (m, 1H), 7.90–7.99 (m, 2H), 8.02
(dd, J = 8.0, 1.1 Hz, 1H).
5.2.18. Methyl 4-{3-[1-benzhydryl-5-chloro-2-(2-{[(2-
methoxyphenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]pro-
pyl}benzoate (12g). Sulfonylation of 10 with 2-meth-
oxybenzenesulfonyl chloride, pale yellow foam, 65%
yield. 1H NMR (400 MHz, CDCl3) d 1.84–2.00 (m,
2H), 2.60–2.78 (m, 4H), 2.80–2.91 (m, 2H), 2.96 (t,
J = 7.2 Hz, 2H), 3.57 (s, 3H), 3.91 (s, 3H), 4.97 (t,
J = 6.1 Hz, 1H), 6.41 (d, J = 9.3 Hz, 1H), 6.77 (dd,
J = 8.8, 2.3 Hz, 1H), 6.80 (s, 1H), 6.89 (d, J = 8.3 Hz,
1H), 6.94–7.03 (m, 5H), 7.12–7.34 (m, 9H), 7.40 (d,
J = 2.0 Hz, 1H), 7.45–7.57 (m, 1H), 7.78 (dd, J = 7.8,
1.8 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H).
5.2.13. Methyl 4-[3-(1-benzhydryl-2-{2-[(1,10-biphenyl-3-
ylsulfonyl)amino]ethyl}-5-chloro-1H-indol-3-yl)propyl]-
benzoate (12b). Sulfonylation of 10 with 3-phen-
1
ylbenzenesulfonyl chloride, white foam, 65% yield. H
NMR (400 MHz, CDCl3) d 1.82–2.02 (m, 2H), 2.56–
2.84 (m, 4H), 2.84–2.97 (m, 2H), 2.97–3.13 (m, 2H),
3.92 (s, 3H), 4.64 (t, J = 6.3 Hz, 1H), 6.49 (d,
J = 8.8 Hz, 1H), 6.81 (dd, J = 8.8, 2.3 Hz, 1H), 6.86 (s,
1H), 6.96–7.10 (m, 4H), 7.15–7.35 (m, 8H), 7.37–7.51
(m, 5H), 7.51–7.58 (m, 2H), 7.63 (dd, J = 8.1, 1.5 Hz,
1H), 7.76 (dd, J = 6.4, 1.6 Hz, 1H), 7.87–8.05 (m, 3H).
5.2.19. Methyl 4-(3-{5-chloro-1-(diphenylmethyl)-2-[2-
({[2-(trifluoromethoxy)phenyl]sulfonyl}amino)ethyl]-1H-
indol-3-yl}propyl)benzoate (12h). Sulfonylation of 10
with 2-trifluoromethoxybenzenesulfonyl chloride, white
5.2.14. Methyl 4-{3-[1-benzhydryl-5-chloro-2-(2-{[(2-fluo-
rophenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}ben-
zoate (12c). Sulfonylation of 10 with 2-fluoro-
benzenesulfonyl chloride, white foam, 65% yield. 1H
NMR (400 MHz, CDCl3) d 1.82–2.00 (m, 2H), 2.72 (q,
J = 8.1 Hz, 4H), 2.82–2.91 (m, 2H), 2.92–3.02 (m, 2H),
3.91 (s, 3H), 4.63 (t, J = 6.1 Hz, 1H), 6.49 (d, J = 9.1 Hz,
1H), 6.80 (dd, J = 8.8, 2.3 Hz, 1H), 6.85 (s, 1H), 7.00–
7.07 (m, 3H), 7.08–7.19 (m, 2H), 7.21–7.35 (m, 9H), 7.40
(d, J = 1.8 Hz, 1H), 7.46–7.58 (m, 1H), 7.71 (dt, J = 7.6,
1.8 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H).
1
foam, 65% yield. H NMR (400 MHz, CDCl3) d 1.85–
2.00 (m, 2H), 2.65–2.76 (m, 4H), 2.76–2.86 (m, 2H),
2.91–3.15 (m, 2H), 3.91 (s, 3H), 4.63 (t, J = 6.2 Hz,
1H), 6.50 (d, J = 8.8 Hz, 1H), 6.80 (dd, J = 8.8, 2.3 Hz,
1H), 6.85 (s, 1H), 6.98–7.11 (m, 4H), 7.17–7.35 (m,
10H), 7.39 (d, J = 2.0 Hz, 1H), 7.48–7.66 (m, 1H), 7.82
(dd, J = 7.8, 1.5 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H).
5.2.20. Methyl 4-{3-[1-benzhydryl-5-chloro-2-(2-{[(2,6-
dichlorophenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}-
benzoate (12i). Sulfonylation of 10 with 2,6-dic-
hlorobenzenesulfonyl chloride, pale orange foam, 93%
yield. 1H NMR (400 MHz, CDCl3) d 1.84–2.01 (m,
2H), 2.66–2.78 (m, 4H), 2.82–2.91 (m, 2H), 2.94–3.06
(m, 2H), 3.91 (s, 3H), 5.25 (t, J = 6.2 Hz, 1H), 6.49 (d,
J = 8.8 Hz, 1H), 6.80 (dd, J = 8.8, 2.0 Hz, 1H), 6.84 (s,
1H), 7.00–7.08 (m, 4H), 7.20–7.32 (m, 9H), 7.35–7.43
(m, 3H), 7.95 (d, J = 8.3 Hz, 2H).
5.2.15. Methyl 4-{3-[1-benzhydryl-5-chloro-2-(2-{[(2-
chlorophenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}-
benzoate (12d). Sulfonylation of 10 with 2-chloro-
benzenesulfonyl chloride, white foam, 66% yield. 1H
NMR (400 MHz, DMSO-d6) d 1.72–1.91 (m, 2H),
2.59–2.74 (m, 4H), 2.79–3.04 (m, 4H), 3.84 (s, 3H),
6.45 (d, J = 8.8 Hz, 1H), 6.77 (dd, J = 9.0, 2.1 Hz, 1H),
6.99 (s, 1H), 7.04 (dd, J = 7.5, 1.9 Hz, 4H), 7.26–7.38
(m, 8H), 7.39–7.47 (m, 2H), 7.54–7.70 (m, 2H), 7.82

K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
1353
5.2.21. Methyl 4-{3-[1-benzhydryl-5-chloro-2-(2-{[(2,6-
dimethylphenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]pro-
pyl}benzoate (12j). Sulfonylation of 10 with 2,6-dim-
ethylbenzenesulfonyl chloride, white foam, 66% yield.
1H NMR (400 MHz, CDCl3) 1.87–1.99 (m, 2H), 2.50
(s, 6H), 2.64–2.74 (m, 4H), 2.78–2.87 (m, 2H), 2.88–
2.97 (m, 2H), 3.91 (s, 3H), 4.40 (t, J = 6.4 Hz, 1H),
6.47 (d, J = 8.8 Hz, 1H), 6.77 (s, 1H), 6.80 (dd, J = 8.8,
2.3 Hz, 1H), 6.95–7.03 (m, 4H), 7.07 (d, J = 7.6 Hz,
2H), 7.22 (d, J = 8.1 Hz, 2H), 7.24–7.32 (m, 7H), 7.40
(d, J = 2.0 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H).
741.21879. HPLC purity: CH3CN–H2O, 99.0%,
MeOH–H2O, 100%.
5.3.3. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2-fluorophenyl)-
sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic acid
(13c). Ester hydrolysis of 11c, white solid, 90% yield.
1H NMR (400 MHz, DMSO-d6) d 2.88–2.99 (m, 2H),
2.99–3.07 (m, J = 5.8 Hz, 2H), 3.10 (t, J = 6.6 Hz, 2H),
4.13 (t, J = 6.6 Hz, 2H), 6.49 (d, J = 9.1 Hz, 1H), 6.79
(appar dd, J = 8.8, 2.0 Hz, 3H), 7.04 (appar dd,
J = 7.7, 1.6 Hz, 5H), 7.20–7.42 (m, 8H), 7.55–7.68 (m,
3H), 7.80 (s, 2H), 8.19 (s, 1H); HRMS calcd for
[C38H33ClFN2O5S+H] 683.17773: found 683.17694.
Anal. Calcd for [C38H33ClFN2O5S]: C, H, N.
5.2.22. Methyl 4-(3-{1-benzhydryl-5-chloro-2-[2-({[2-flu-
oro-6-(trifluoromethyl)phenyl]sulfonyl}amino)ethyl]-1H-
indol-3-yl}propyl)benzoate (12k). Sulfonylation of 10
with 2-fluoro-6-trifluoromethylbenzenesulfonyl chloride
5.3.4. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2-chlorophenyl)-
sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic acid
(13d). Ester hydrolysis of 11d, white solid, 74% yield.
1H NMR (400 MHz, CDCl3) d 2.83–2.98 (m, 2H),
3.05–3.15 (m, 2H), 3.18 (t, J = 6.6 Hz, 2H), 4.20 (t,
J = 6.6 Hz, 2H), 5.09 (t, J = 6.3 Hz, 1H), 6.53 (d,
J = 8.8 Hz, 1H), 6.78–6.86 (m, 3H), 6.90 (s, 1H),
7.01–7.12 (m, 4H), 7.22–7.34 (m, 7H), 7.42–7.47
(m, 2H), 7.52 (d, J = 2.0 Hz, 1H), 7.85–7.94 (m,
1H), 8.00 (d, J = 8.8 Hz, 2H); HRMS calcd for
[C38H32Cl2N2O5S+H]: 699.14818: found 699.14786.
Anal. Calcd for [C38H32Cl2N2O5S]: C, H, N.
1
(8), pale yellow foam, 62% yield. H NMR (400 MHz,
CDCl3) d 1.84–2.02 (m, 2H), 2.56–2.82 (m, 4H), 2.84–
2.95 (m, 2H), 2.95–3.11 (m, 2H), 3.91 (s, 3H), 4.88 (t,
J = 6.3 Hz, 1H), 6.50 (d, J = 8.8 Hz, 1H), 6.72–6.92
(m, 2H), 7.02 (dd, J = 5.7, 3.4 Hz, 4H), 7.15–7.35 (m,
9H), 7.41 (d, J = 1.8 Hz, 1H), 7.52–7.73 (m, 2H), 7.95
(d, J = 8.1 Hz, 2H).
5.3. General procedure 2: ester hydrolysis
To a solution of the ester (1.0 mmol) in inhibitor-free
THF (20 mL) were added 1 N aq NaOH (3.0 mL,
3.0 mmol) and MeOH (10 mL). The mixture was heated
at 50 ꢁC until the ester starting material was consumed
(TLC analysis in 50% EtOAc–hexanes). The reaction
mixture was concentrated and the residue was diluted
with H2O (10 mL) and acidified to pH 1 using 1 N
HCl. The resulting mixture was extracted with EtOAc
(2· 20 mL). The organic extracts were washed with
H2O (20 mL) and brine (20 mL), dried, and concen-
trated. The residue was lyophilized to afford the carbox-
ylic acid.
5.3.5.
4-{2-[1-Benzhydryl-2-(2-{[(2-bromophenyl)sulfo-
nyl]amino}ethyl)-5-chloro-1H-indol-3-yl]ethoxy}benzoic
acid (13e). Ester hydrolysis of 11e, off-white powder,
91% yield. 1H NMR (400 MHz, DMSO-d6) d 2.87–
2.98 (m, J = 7.2, 3.9 Hz, 2H), 3.00–3.07 (m, 2H), 3.11
(t, J = 6.7 Hz, 2H), 4.18 (t, J = 6.6Hz, 2H), 6.49 (d,
J = 8.8 Hz, 1H), 6.80 (dd, J = 8.8, 2.3 Hz, 1H), 6.93 (d,
J = 9.1 Hz, 2H), 7.00–7.08 (m, 5H), 7.27–7.39 (m, 7H),
7.39–7.54 (m, 2H), 7.65 (d, J = 2.0 Hz, 1H), 7.76–7.89
(m, 4H), 8.08 (t, J = 5.8 z, 1H); HRMS calcd for
[C38H32BrClN2O5S+H] 743.09766: found 743.09697.
Anal. Calcd for [C38H32BrClN2O5S]: C, H, N.
5.3.1. 4-[2-(1-Benzhydryl-2-{2-[(1,10-biphenyl-2-ylsulfo-
nyl)amino]ethyl}-5-chloro-1H-indol-3-yl)ethoxy]benzoic
acid (13a). Ester hydrolysis of 11a, white solid, 94%
5.3.6. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2-methylphenyl)-
sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic acid
(13f). Ester hydrolysis of 11f, white solid, 90% yield.
1H NMR (400 MHz, DMSO-d6) d 1.66–1.91 (m, 2H),
2.52 (s, 3H), 2.65 (t, J = 6.7 Hz, 4H), 2.73–2.84 (m,
2H), 2.86–2.96 (m, 2H), 6.45 (d, J = 8.8 Hz, 1H), 6.77
(dd, J = 9.0, 2.1 Hz, 1H), 6.99 (s, 1H), 7.01–7.10 (m,
4H), 7.14–7.27 (m, 3H), 7.26–7.40 (m, 8H), 7.41–7.51
(m, 2H), 7.65 (dd, J = 7.8, 1.3 Hz, 1H), 7.83 (d,
J = 7.3 Hz, 2H), 7.94 (t, J = 5.8 Hz, 1H); HRMS calcd
for [C39H35ClN2O5S+H] 679.20280: found 679.20197.
HPLC purity: CH3CN–H2O, 100%, MeOH–H2O,
98.2%.
1
yield. H NMR (400 MHz, DMSO-d6) d 2.75–2.87 (m,
2H), 2.92–3.00 (m, 2H), 3.11 (t, J = 6.7 Hz, 2H), 4.17
(t, J = 6.7 Hz, 2H), 6.47 (d, J = 8.8 Hz, 1H), 6.80 (dd,
J = 8.8, 2.3 Hz, 1H), 6.88–6.94 (m, 2H), 6.95 (s, 1H),
6.96–7.03 (m, 4H), 7.21–7.36 (m, 12H), 7.40–7.53 (m,
2H), 7.56–7.63 (m, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.77–
7.90 (m, 3H); HRMS calcd for [C44H37ClN2O5S+H]:
741.21845: found 741.21709. Anal. Calcd for
[C44H37ClN2O5S]: C, H, N.
5.3.2. 4-[2-(1-Benzhydryl-2-{2-[(1,10-biphenyl-3-ylsulfo-
nyl)amino]ethyl}-5-chloro-1H-indol-3-yl)ethoxy]benzoic
acid (13b). Ester hydrolysis of 11b, white solid, 90%
1
yield. H NMR (400 MHz, DMSO-d6) d 2.81–2.96 (m,
5.3.7. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2-methoxyphenyl)-
sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic acid
(13g). Ester hydrolysis of 11g, white solid, 90% yield. 1H
NMR (400 MHz, DMSO-d6) d 2.83–3.03 (m, 4H), 3.02–
3.14 (m, 2H), 3.81 (s, 3H), 4.11 (t, J = 6.6 Hz, 2H), 6.46
(d, J = 8.8 Hz, 1H), 6.72–6.86 (m, 3H), 6.94 (t,
J = 7.8 Hz, 1H), 6.98–7.09 (m, 5H), 7.14 (d, J = 8.3
Hz, 1H), 7.25–7.43 (m, 6H), 7.45–7.56 (m, 2H),
2H), 2.96–3.06 (m, 2H), 3.11 (t, J = 5.8 Hz, 2H), 4.14
(t, J = 6.6 Hz, 2H), 6.49 (d, J = 8.6 Hz, 1H), 6.79 (dd,
J = 8.8, 2.3 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 6.91–
7.10 (m, 5H), 7.21–7.37 (m, 6H), 7.38–7.53 (m, 3H),
7.55–7.71 (m, 5H), 7.80 (d, J = 8.6 Hz, 2H), 7.88
(d, J = 7.6 Hz, 1H), 7.94–8.07 (m, 2H); HRMS
calcd for [C44H.37ClN2O5S+H] 741.21845: found

1354
K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
7.58–7.68 (m, 2H), 7.84 (s, 2H); HRMS calcd for
[C39H35ClN2O6S+H] 695.19722: found 695.19701.
HPLC purity: CH3CN–H2O, 100%, MeOH–H2O, 100%.
1H), 6.99 (dd, J = 6.8, 2.5 Hz, 4H), 7.19–7.41 (m, 15H),
7.41–7.50 (m, 2H), 7.58–7.68 (m, 1H), 7.81 (dd, J = 8.0,
1.1 Hz, 1H), 7.83–7.89 (m, 2H); HRMS calcd for
[C45H39ClN2O4S+H] 739.23919: found 739.23825. HPLC
purity: CH3CN–H2O, 97.5%, MeOH–H2O, 97.0%.
5.3.8. 4-(2-{5-Chloro-1-(diphenylmethyl)-2-[2-({[2-(triflu-
oromethoxy)phenyl]sulfonyl}amino)ethyl]-1H-indol-3-yl}-
ethoxy)benzoic acid (13h). Ester hydrolysis of 11h, white
solid, 90% yield. 1H NMR (400 MHz, DMSO-d6) d 3.01
(dd, J = 19.6, 6.2 Hz, 4H), 3.11 (t, J = 6.4 Hz, 2H), 4.13
(t, J = 6.7 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 6.66–6.85
(m, 3H), 6.94–7.12 (m, 5H), 7.23–7.37 (m, 6H), 7.42 (t,
J = 7.5 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.65 (d,
J = 2.3 Hz, 1H), 7.67–7.73 (m, 1H), 7.74–7.90 (m, 3H),
8.22 (br s, 1H); HRMS calcd for [C39H35ClF3N2O6S+H]
749.16945: found 749.16813. HPLC purity: CH3CN–
H2O, 98.4%, MeOH–H2O, 97.9%.
5.3.13. 4-[3-(1-Benzhydryl-2-{2-[(1,10-biphenyl-3-ylsulfo-
nyl)amino]ethyl}-5-chloro-1H-indol-3-yl)propyl]benzoic
acid (14b). Ester hydrolysis of 12b, white solid, 100%
1
yield. H NMR (400 MHz, DMSO-d6) d 1.67–1.89 (m,
2H), 2.53–2.74 (m, 4H), 2.75–2.87 (m, 2H), 2.87–2.98
(m, 2H), 6.44 (d, J = 8.8 Hz, 1H), 6.75 (dd, J = 9.0,
2.1 Hz, 1H), 6.93–7.17 (m, 7H), 7.20–7.37 (m, 6H),
7.37–7.54 (m, 4H), 7.54–7.71 (m, 4H), 7.78 (d,
J = 7.6 Hz, 2H), 7.88 (d, J = 8.1 Hz, 1H), 7.98 (s, 1H),
8.05 (s, 1H); HRMS calcd for [C45H39ClN2O4S+H]
739.23919: found 739.23896. HPLC purity: CH3CN–
H2O, 97.6%, MeOH–H2O, 98.7%.
5.3.9. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2,6-dichloro-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic
acid (13i). Ester hydrolysis of 11i, white solid, 82% yield.
1H NMR (400 MHz, DMSO-d6) d 3.04 (br s, 4H), 3.14
(t, J = 6.6 Hz, 2H), 4.20 (t, J = 6.7 Hz, 2H), 6.49 (d,
J = 8.8 Hz, 1H), 6.81 (dd, J = 9.0, 2.1 Hz, 1H), 6.92 (d,
J = 8.8 Hz, 2H), 6.99–7.08 (m, 5H), 7.25–7.38 (m, 6H),
7.44–7.54 (m, 1H), 7.55–7.60 (m, 2H), 7.66 (d,
J = 2.3Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 8.30–8.41
(m, 1H), 12.59 (br s, 1H); HRMS calcd for
[C38H31Cl3N2O5S+H] 733.10921: found 733.10836.
Anal. Calcd for [C38H31Cl3N2O5S]: found C, H, N.
5.3.14.
4-{3-[1-Benzhydryl-5-chloro-2-(2-{[(2-fluoro-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}ben-
zoic acid (14c). Ester hydrolysis of 12c, white solid, 100%
1
yield. H NMR (400 MHz, DMSO-d6) d 1.72–1.90 (m,
2H), 2.57–2.73 (m, 4H), 2.92 (s, 4H), 6.45 (d, J =
8.8 Hz, 1H), 6.77 (dd, J = 8.8, 2.3 Hz, 1H), 7.01 (s,
1H), 7.02–7.09 (m, 4H), 7.15 (d, J = 8.3 Hz, 2H), 7.23–
7.43 (m, 8H), 7.45 (d, J = 2.3 Hz, 1H), 7.59–7.71 (m,
2H), 7.81 (d, J = 8.8 Hz, 2H), 8.16 (s, 1H); HRMS calcd
for [C39H34ClFN2O4S+H] 681.19846: found 681.19854.
HPLC purity: CH3CN–H2O, 99.1%, MeOH–H2O,
97.9%.
5.3.10. 4-{2-[1-Benzhydryl-5-chloro-2-(2-{[(2,6-dimethyl-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]ethoxy}benzoic
acid (13j). Ester hydrolysis of 11j, white solid, 79% yield.
1H NMR (400 MHz, DMF) d 3.07 (s, 6H), 3.37–3.50 (m,
2H), 3.49–3.60 (m, 2H), 3.67 (t, J = 6.7 Hz, 2H), 4.74 (t,
J = 6.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 1H), 7.38 (dd,
J = 8.8, 2.0 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.54 (s,
1H), 7.60 (dd, J = 7.2, 2.1 Hz, 4H), 7.73 (d, J = 7.6 Hz,
2H), 7.81–7.97 (m, 7H), 8.22 (d, J = 2.0 Hz, 1H), 8.32 (t,
J = 5.2 Hz, 1H), 8.41 (d, J = 8.8 Hz, 2H), 13.16 (br s, 1
H); HRMS calcd for [C40H37ClN2O5S+H] 693.21845:
found 693.21791. HPLC purity: CH3CN–H2O, 98.8%,
MeOH–H2O, 97.8%.
5.3.15.
4-{3-[1-Benzhydryl-5-chloro-2-(2-{[(2-chloro-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}ben-
zoic acid (14d). Ester hydrolysis of 12d, white foam, 84%
yield. 1H NMR (400 MHz, CDCl3) d 1.89–2.01 (m, 2H),
2.63–2.84 (m, 6H), 2.94–3.07 (m, 2H), 5.01 (t, J = 5.6 Hz,
1H), 6.48 (d, J = 8.8 Hz, 1H), 6.79 (dd, J = 8.8, 2.0 Hz,
1H), 6.84 (s, 1H), 7.04 (dd, J = 6.6, 2.8 Hz, 4H), 7.22–
7.33 (m, 9H), 7.40 (d, J = 1.8 Hz, 1H), 7.42–7.50 (m,
2H), 7.90 (dd, J = 7.7, 1.1 Hz, 1H), 8.02 (d, J = 8.3 Hz,
2H); HRMS calcd for [C39H34Cl2N2O4S–H] 695.15435:
found 695.15363. HPLC purity: CH3CN–H2O, 97.0%,
MeOH–H2O, 96.2%.
5.3.11.
4-(2-{1-Benzhydryl-5-chloro-2-[2-({[2-fluoro-6-
(trifluoromethyl)phenyl]sulfonyl}amino)ethyl]-1H-indol-3-
yl}ethoxy)benzoic acid (13k). Ester hydrolysis of 11k,
white solid, 36% yield. H NMR (400 MHz, CDCl3) d
5.3.16. 4-{3-[1-Benzhydryl-2-(2-{[(2-bromophenyl)sulfo-
nyl]amino}ethyl)-5-chloro-1H-indol-3-yl]propyl}benzoic
acid (14e). Ester hydrolysis of 12e, off-white powder,
95% yield. 1H NMR (400 MHz, DMSO-d6) d 1.72–
1.90 (m, 2H), 2.67 (q, J = 7.6 Hz, 4H), 2.78–2.99 (m,
4H), 6.45 (d, J = 8.8 Hz, 1H), 6.77 (dd, J = 8.8, 2.0 Hz,
1H), 6.99 (s, 1H), 7.05 (dd, J = 7.6, 1.8 Hz, 4H), 7.24–
7.40 (m, 9H), 7.42–7.55 (m, 3H), 7.78–7.91 (m, 4H),
8.03 (t, J = 5.8 Hz, 1H), 12.74 (br s, 1H); HRMS calcd
for [C39H34BrClN2O4S+H] 741.1184: found 741.11696.
Anal. Calcd for [C39H34BrClN2O4S]: C, H, N.
1
2.96–3.06 (m, 2H), 3.08–3.17 (m, 2H), 3.21 (t, J =
6.4 Hz, 2H), 4.22 (t, J = 6.4 Hz, 2H), 5.06 (t, J = 6.2 Hz,
1H), 6.55 (d, J = 9.1 Hz, 1H), 6.79–6.86 (m, 3H), 6.88 (s,
1H), 7.01–7.07 (m, 4H), 7.24–7.37 (m, 7H), 7.55 (d,
J = 2.3 Hz, 1H), 7.56–7.69 (m, 2H), 7.98 (d, J = 8.8 Hz,
2H); HRMS calcd for [C39H31ClF4N2O5S+H]
751.16511: found 751.16431. HPLC purity: CH3CN–
H2O, 98.8%, MeOH–H2O, 98.5%.
5.3.12. 4-[3-(1-Benzhydryl-2-{2-[(1,10-biphenyl-2-ylsulfo-
nyl)amino]ethyl}-5-chloro-1H-indol-3-yl)propyl]benzoic
acid (14a). Ester hydrolysis of 12a, white powder, 98%
yield. H NMR (400 MHz, DMSO-d6) d 1.69–1.88 (m,
2H), 2.59–2.78 (m, 6H), 2.80–2.94 (m, 2H), 6.44 (d,
J = 8.8 Hz, 1H), 6.77 (dd, J = 8.8, 2.3 Hz, 1H), 6.93 (s,
5.3.17. 4-{3-[1-Benzhydryl-5-chloro-2-(2-{[(2-methyl-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}ben-
zoic acid (14f). Ester hydrolysis of 12f, white solid, 100%
yield. H NMR (400 MHz, DMSO-d6) d 2.52 (s, 3H),
2.87 (br s, 2H), 2.93–3.03 (m, 2H), 3.09 (t, J = 7.3 Hz,
2H), 4.12 (t, J = 6.7 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H),
1
1

K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
1355
6.72–6.87 (m, 3H), 7.04 (d, J = 7.8 Hz, 5H), 7.20 (t,
J = 7.6 Hz, 1H), 7.26–7.38 (m, 7H), 7.40–7.50 (m, 1H),
7.58–7.69 (m, 2H), 7.74–7.87 (m, 2H), 7.95 (t,
J = 5.6 Hz, 2H); HRMS calcd for [C40H37ClN2O4S+H]
677.22354: found 677.22244. HPLC purity: CH3CN–
H2O, 98.6%, MeOH–H2O, 98.4%.
pale yellow powder, 86% yield. 1H NMR (400 MHz,
DMSO-d6) d 1.73–1.89 (m, 2H), 2.67 (t, J = 7.5 Hz,
4H), 2.94 (s, 4H), 6.44 (d, J = 8.8 Hz, 1H), 6.77 (dd,
J = 8.8, 2.0 Hz, 1H), 6.98 (s, 1H), 7.03 (d, J = 7.1 Hz,
4H), 7.20–7.40 (m, 8H), 7.45 (d, J = 2.0 Hz, 1H), 7.68–
7.99 (m, 5H), 8.40 (t, J = 5.1 Hz, 1H), 12.74 (br s, 1H);
HRMS calcd for [C40H33ClF4N2O4S+H] 749.18585:
found 749.18578. Anal. Calcd for [C40H33ClF4N2O4S]:
C, H, N.
5.3.18. 4-{3-[1-Benzhydryl-5-chloro-2-(2-{[(2-methoxy-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}benzoic
acid (14g). Ester hydrolysis of 12g, off-white solid, 100%
1
yield. H NMR (400 MHz, DMSO-d6) d 1.72–1.91 (m,
5.4. General procedure 3: GLU micelle assay conditions
2H), 2.59–2.75 (m, 4H), 2.87 (br s, 4H), 3.81 (s, 3H),
6.43 (d, J = 8.8 Hz, 1H), 6.76 (dd, J = 8.8, 2.3 Hz, 1H),
6.93–7.07 (m, 6H), 7.18 (d, J = 7.8 Hz, 1H), 7.27–7.39
(m, 9H), 7.38–7.48 (m, 2H), 7.53–7.60 (m, 1H), 7.64
(dd, J = 7.8, 1.8 Hz, 1H), 7.85 (d, J = 8.3 Hz, 2H),
12.80 (br s, 1H); HRMS calcd for [C40H37ClN2O5S+H]
693.2185: found 693.21852. Anal. Calcd for
[C40H37ClN2O5S]: C, H, N.
The assay was carried out in a 96-well format using a
fluorescent plate reader with a 355 nm excitation filter
and a 460 nm emission filter (Lab Systems Fluoroscan
II, Helsinki, Finland). The assay buffer contained
940 lM Triton X-100, 50 mM Hepes, pH 7.4, 0.3 mM
EDTA, 1 mM CaCl2, and 300 mM KCl. DTPC (1, 2-
O-tetradecyl-sn-glycero-3-phosphocholine, Avanti) at a
final concentration of 120 lM was added the day of
the experiment and GLU (7-hydroxycoumarinyl-c-linol-
enate, Biomol Research Lab, Inc.) at a final concentra-
tion of 90 lM was added immediately prior to each
assay.
5.3.19. 4-(3-{5-Chloro-1-(diphenylmethyl)-2-[2-({[2-(tri-
fluoromethoxy)phenyl]sulfonyl}amino)ethyl]-1H-indol-3-
yl}propyl)benzoic acid (14h). Ester hydrolysis of 12h,
1
white solid, 100% yield. H NMR (400 MHz, DMSO-
d6) d 1.69–1.90 (m, 2H), 2.54–2.77 (m, 4H), 2.93 (br s,
4H), 6.45 (d, J = 8.8 Hz, 1H), 6.77 (dd, J = 8.8, 2.0 Hz,
1H), 6.96–7.08 (m, 5H), 7.14 (d, J = 7.8 Hz, 2H), 7.23–
7.37 (m, 6H), 7.39–7.48 (m, 2H), 7.53 (d, J = 8.3 Hz,
1H), 7.72 (t, J = 8.0 Hz, 1H), 7.75–7.90 (m, 3H), 8.16
(br s, 1H); HRMS calcd for [C40H37ClF3N2O5S+H]
747.19019: found 747.18848. HPLC purity: CH3CN–
H2O, 98.5%, MeOH–H2O, 100%.
Compounds (10 lL) dissolved in DMSO were placed
in duplicate wells of a black 96-well plate. Wells cor-
responding to the positive and negative controls con-
tained DMSO without inhibitors. Just prior to the
experiment, 200 lL assay buffer containing 90 lM
GLU and 120 lM DTPC was added to all wells in
the assay plate. Assay buffer (50 lL) was added to
the negative, and 50 lL cPLA2a solution (5 mg/mL
in assay buffer) was added to all other wells to initiate
the reaction. The final concentration of enzyme was
1 lg/mL. The content of each well was mixed gently
during the addition of the enzyme, and the plate
was rapidly transferred to the fluorescent plate reader.
The increase in fluorescence was read every 4 min for
84 min. The slope of the resulting line was determined
and the inhibition was calculated.
5.3.20. 4-{3-[1-Benzhydryl-5-chloro-2-(2-{[(2,6-dichloro-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}benzoic
acid (14i). Ester hydrolysis of 12i, pale yellow solid, 71%
1
yield. H NMR (400 MHz, DMSO-d6) d 1.69–1.91 (m,
2H), 2.61–2.78 (m, 4H), 2.94 (br s, 4H), 6.44 (d,
J = 8.8 Hz, 1H), 6.78 (dd, J = 8.8, 2.0 Hz, 1H), 6.99
(s, 1H), 7.02–7.13 (m, 4H), 7.23–7.39 (m, 9H), 7.48–
7.56 (m, 1H), 7.57–7.68 (m, 2H), 7.84 (d, J = 8.3 Hz,
2H), 8.29 (t, J = 5.1 Hz, 1H), 12.74 (br s, 1H); HRMS
calcd for [C39H33Cl3N2O4S+H] 731.12994: found
731.13005. Anal. Calcd for [C39H33Cl3N2O4S]: C,
H, N.
5.5. General procedure 4: rat whole blood assay conditions
Fresh blood was collected in heparinized tubes by car-
diac puncture of male Sprague–Dawley rats. Aliquots
of blood (0.6 mL) were incubated with 6 lL vehicle
(DMSO) containing various concentrations of the test
compounds. After 15 min pre-incubation at 37 ꢁC,
blood was stimulated with 6 lL calcium ionophore,
A23187 (Sigma C-7522), in DMSO for 10 min at
37 ꢁC. The final concentration of A23187 was 5 lM.
DMSO (6 lL) was added in the unstimulated controls.
The reactions were stopped by mixing 60 lL cold
EDTA to give a final concentration of 20 mM. The
blood was centrifuged at 6500 rpm for 10 min on a
microcentrifuge to obtain plasma. A 70 lL aliquot
of plasma was mixed with 400 lL cold methanol for
protein precipitation. After incubation at ꢀ80 ꢁC for
30 min, the supernatant was obtained by centrifuging
at 6500 rpm for 10 min and was assayed for TXB2
according to the manufacturer’s procedure (Assay De-
signs, Inc.).
5.3.21. 4-{3-[1-Benzhydryl-5-chloro-2-(2-{[(2,6-dimethyl-
phenyl)sulfonyl]amino}ethyl)-1H-indol-3-yl]propyl}benzoic
acid (14j). Ester hydrolysis of 12j, white solid, 96% yield.
1H NMR (400 MHz, DMSO-d6) d 1.70–1.89 (m, 2H),
2.50 (s, 6H), 2.60–2.70 (m, 4H), 2.74–2.84 (m, 2H),
2.84–2.92 (m, 2H), 6.45 (d, J = 8.8 Hz, 1H), 6.77 (dd,
J = 8.8, 2.3 Hz, 1H), 6.94 (s, 1H), 7.03 (d, J = 2.3 Hz,
4H), 7.17 (d, J = 7.3 Hz, 2H), 7.24–7.38 (m, 9H), 7.43
(d, J = 2.3 Hz, 1H), 7.69 (t, J = 6.3 Hz, 1H), 7.85 (d,
J = 8.3 Hz, 2H), 12.74 (br s, 1H); HRMS calcd for
[C41H39ClN2O4S+H] 691.23919: found 691.23872.
HPLC purity: CH3CN–H2O, 97.1%, MeOH–H2O,
96.0%.
5.3.22.
4-(3-{1-Benzhydryl-5-chloro-2-[2-({[2-fluoro-6-
(trifluoromethyl)phenyl]sulfonyl}amino)ethyl]-1H-indol-3-
yl}propyl)benzoic acid (14k). Ester hydrolysis of 12k,

1356
K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
5.6. General procedure 5: human whole blood assay
conditions
fasted rats (n = 3) were dosed via gavage at 25 mg/kg in
MC/TW vehicle (0.5% methylcellulose, Sigma and 2%
Tween 80, Spectrum) or Phosal formulation (55.5%
Phosal 53 MCT, American Lecithin Co.; 5.6% Tween
80, Spectrum; 16.7% Labrasol, Gattefosse; and 22.2%
propylene carbonate, Spectrum) with test compound
dissolved at 37.5 mg/mL vehicle, diluted with water
prior to use, and dosed at 4 mL/kg. Blood samples were
collected into EDTA/NaF tubes via the jugular vein
cannula at pre-selected time points over a 24-h period.
After a centrifugation at 4 ꢁC for 10 min, plasma sam-
ples (200 lL each) were harvested into a tube containing
10 lL of 2 N HCl (final pH of plasma samples approx-
imately 4). Plasma samples were kept at ꢀ70 ꢁC until
assay.
Fresh blood was collected in heparinized tubes by veni-
puncture of healthy male volunteers. Aliquots of blood
(0.9 mL) were incubated with 9 lL DMSO vehicle con-
taining various concentrations of test compound. After
15 min pre-incubation at 37 ꢁC, the blood was stimu-
lated with 9 lL of calcium ionophore A23187 (Sigma
C-7522) in DMSO for 15 min at 37 ꢁC. The final concen-
tration of A23187 was 30 lM. The unstimulated con-
trols received 9 lL DMSO. The reactions were stopped
by mixing 100 lL cold EDTA to give a final concentra-
tion of 20 mM and processed as described for the rat
whole blood assay. Supernatant was assayed for
TXB2, LTB4, PGE2, and PGF2a according to the man-
ufacturer’s procedures (Assay Designs, Inc.).
Male Beagle dogs (8.5–13.0 kg) were used in the study.
Dogs (n = 3 per treatment group) were fasted overnight
and received either iv administration at 2 mg/kg in 20%
DMSO/80% PEG-200 in a dosing volume of 0.2 mL/kg
or po administration at 5 mg/kg for compound 3 or
50 mg/kg for compound 14k in MC/TW or Phosal for-
mulation (for details of the formulations, see the rat
PK section). For compound 3, the po dosing volume
was 4 mL/kg and for compound 14k, the po dosing vol-
ume was 6 mL/kg. Blood samples were collected into
EDTA/NaF tubes via the saphenous vein at pre-selected
time points over a 24-h period. After a centrifugation at
4 ꢁC for 10 min, plasma samples (200 lL each) were har-
vested into tubes containing 10 lL of 2 N HCl (final pH
of plasma samples approximately 4). Plasma samples
were kept at ꢀ70 ꢁC until assay.
5.7. Isothermal calorimetry assay conditions
Purified human cPLA2a was dialyzed in 50 mM Hepes,
pH 7.4, 0.3 mM EDTA, 1 mM CaCl2, and 300 mM
KCl. DMSO and Triton X-100 were added to make a
2 mL protein solution consisting of 2.5 lM cPLA2a,
1% DMSO, and 940 lM Triton X-100. A stock solution
of compound (7.85 mM in 100% DMSO) was diluted
with the dialysis buffer in which cPLA2a was dialyzed
previously to make 2 mL working solution that con-
tained 78.5 lM compound 14k, 940 lM Triton X-100,
and 1% DMSO. A control solution containing the dial-
ysis buffer, 940 lM Triton X-100 and 1% DMSO was
made, and both solutions were degassed. The cPLA2a
protein solution was loaded carefully into the sample
cell of the calorimeter (VP-Isothermal Titration Calo-
rimeter, Microcal Inc., Northampton, MA), while the
syringe was filled with compound 14k working solution
(250 lL). The titration was carried out at 30 ꢁC. A total
of 20 injections (10 lL each) of the compound were
made and settings were as recommended by Microcal.
A separate control experiment was done in which com-
pound 14k was titrated against the buffer solution with-
out any protein to determine the background. The data
were analyzed using the Origin software supplied with
the VP-isothermal titration calorimeter and the binding
parameters were determined.
To determine plasma concentrations of cPLA2a inhibi-
tors, aliquots (50 lL) of plasma samples were used and
the plasma protein was precipitated with 100 lL of
ACN containing a structural analogue of the test article
as the internal standard. The supernatants (10 lL) were
injected directly onto the LC/MS/MS system for analy-
sis. The test article and its internal standard were sepa-
rated with
a Waters XTerra MS C18 column
(20 · 2.1 mm ID, 2.5 lm). The mobile phase consisted
of Solvent A with 0.1% TFA in H2O and Solvent B with
0.1% TFA in CH3CN. At a constant flow rate of 200 lL/
min, a linear mobile phase gradient from 100% Solvent
A to 100% Solvent B over a 4-min period was applied.
An API-3000-1 (Applied Biosystems, Concord, Ontario,
Canada) mass spectrometer fitted with an electrospray
ionization interface (ESI) was used for the mass detec-
tion. The positive MRM acquisition conditions were
optimized by infusing the standard solutions of the test
article and the internal standard. Under these experi-
mental conditions, the low quantitation limit was 5 ng/
mL for most cPLA2a inhibitors. The pharmacokinetic
parameters (AUC and clearance) were calculated with
non-compartment methods (WinNonlin, Version 4.0,
Pharsight Corp., Mountain View, CA).
5.8. Cyclooxygenase assay conditions
A colorimetric COX assay (Cayman, cat. # 760111) was
used to measure inhibition of both COX-1 and COX-2.
SC-560-7 (Cayman, cat. # 70340) was used as the refer-
ence compound for selective COX-1 inhibition and Cele-
coxib was used as the reference for COX-2 inhibition.
The assay was performed according to the manufac-
turer’s directions.
5.9. Pharmacokinetics in rats and dogs
Male Sprague–Dawley rats (200–300 g, Taconic, Ger-
mantown, NY) were used for PK assessment. For iv
administration, animals (n = 3) received a single bolus
dose of 2 mg/kg in vehicle (50% PEG-400, 50% DSMO)
via tail vein injection. For oral administration, overnight
5.10. Rat carrageenan paw edema (CPE) model
Male Sprague–Dawley rats (n = 10 per treatment group)
weighing 190–250 g from Taconic Farms were housed
for 1 week prior to experimentation and fed food and

K. L. Lee et al. / Bioorg. Med. Chem. 16 (2008) 1345–1358
1357
water ad libitum. The protocol for the CPE model was
adapted from procedures previously described.18,22 The
volume of the left hind footpad of the rat was measured
using an Ugo Basile plethysmometer prior to dosing.
The animal was then dosed orally with the test com-
pound in Phosal vehicle (55.5% Phosal 53 MCT, Amer-
ican Lecithin Co.; 5.6% Tween 80, Spectrum; 16.7%
Labrasol, Gattefosse; and 22.2% propylene carbonate,
Spectrum) or MC/TW vehicle (0.5% methylcellulose,
Sigma and 2% Tween 80, Spectrum), orally with
10 mg/kg of Naproxen in Phosal vehicle, orally with
25 mg/kg Celecoxib in MC/TW vehicle, or with vehicle
alone as described above. Two hours after dosing,
60 lL of 1% carrageenan in saline was injected subplan-
tar into the left hind footpad. The paw volume measure-
ment was repeated 3 h after the carrageenan injection.
Inhibition was calculated using the following formula:
long term oral administration, 14k was dosed BID for
4 d prior to challenge and on the day of challenge
(day 5) 2 h prior to challenge and 8 h post challenge.
For oral administration at 3 mg/kg, 14k was dissolved
in Phosal vehicle (150 mg/mL stock solution in 55.5%
Phosal 53 MCT, American Lecithin Co.; 5.6% Tween
80, Spectrum; 16.7% Labrasol, Gattefosse; and 22.2%
propylene carbonate, Spectrum). On the day of the
experiment the stock solution was diluted to 12 mg/mL
with water and dosed at 0.25 mL/kg. For evaluation at
1 mg/kg po the 150 mg/mL stock solution was diluted
to 4 mg/mL with water and dosed at 0.25 mL/kg.
Changes in airway resistance after administration of
14k were compared to historical control values for each
individual sheep.
Acknowledgments
Percent inhibition ¼ f1ꢀ½3 h paw volume
ꢀ0h paw volumeðtest groupÞꢁ
=½3h paw volume
The authors thank Nelson Huang, Ying Ge, Walter
Massefski, Ning Pan and Peter Tate for analytical sup-
port; and Weili Duan, Elizabeth Murphy and Yajuan
Zhao for assay support. We also thank Molly Hoke,
Wei Li, Jianchang Li and Dianne DeVincentis of Wyeth
Research; and Howard Sard, Anu Mahadevan, Mario
Gonzalez and Shanghao Liu of Organix, Inc. for the
preparation of chemical intermediates. We thank Mari-
anne Donnelly for reviewing this manuscript.
ꢀ0h paw volumeðvehicle groupÞꢁg
ꢂ100
5.11. Ascaris suum airway challenge in naturally sensi-
tized sheep
Allergic sheep (n = 2–3 per test group) weighing 27–
50 kg were used to assess efficacy in the A. suum asthma
model.21 All sheep had previously been shown to devel-
op both early and late bronchial responses to inhaled
A. suum antigen. The sheep were conscious and were re-
strained in a modified shopping cart in the prone posi-
tion with their heads immobilized. After topical
anesthesia of the nasal passages with 2% lidocaine, a
balloon catheter was advanced through one nostril into
the lower esophagus. The animals were intubated with a
cuffed endotracheal tube through the other nostril with a
flexible fiberoptic bronchoscope as a guide. Animals
were challenged via the airways with A. suum antigen
(Greer Diagnostics, Lenoir, NC) and breath-by breath
determination of mean pulmonary resistance (RL) was
measured with the esophageal balloon technique as de-
scribed previously.21 To assess bronchial responsiveness
(AHR), the day following allergen challenge, cumulative
concentration–response curves to carbachol were gener-
ated by measuring specific lung resistance immediately
after inhalation of buffer and after each consecutive
administration of 10 breaths of increasing concentra-
tions of carbachol. The provocation test was discontin-
ued when specific lung resistance increased by more
than 400% from the post saline value, or after the high-
est carbachol concentration had been administered.
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