Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1

Article abstract of DOI:10.1021/jm1001919

The inhibition of LTB₄ binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B₄ (LTB_4<

Full text of DOI:10.1021/jm1001919

3502 J. Med. Chem. 2010, 53, 3502–3516
DOI: 10.1021/jm1001919
Discovery of Novel and Potent Leukotriene B4 Receptor Antagonists. Part 1
Robert A. Goodnow, Jr.,*,† Alexandra Hicks,‡ Achyutharao Sidduri,† Agnieszka Kowalczyk,† Romyr Dominique,† Qi Qiao,†
Jian Ping Lou,† Paul Gillespie,† Nader Fotouhi,† Jefferson Tilley,† Noal Cohen,† Satish Choudhry,§ Gary Cavallo,‡
Shahid A. Tannu,‡ Jessica D. Ventre,‡ Danielle Lavelle,‡ Nadine S. Tare,‡ Hyesun Oh, Martin Lamb, Grazyna Kurylko,^
Rachid Hamid,^ Matthew B. Wright,^ Anjula Pamidimukkala, Thomas Egan, Ueli Gubler,^ Ann F. Hoffman,^ Xin Wei,#
Ying L. Li,# John O’Neil,¥ Ruben Marcano,¥ Karen Pozzani,¥ Tina Molinaro,¥ Jennifer Santiago,¥ Laura Singer,¥
Maureen Hargaden,¥ David Moore, A. Robert Catala, Lisa C. F. Chao,ꢀ Gesine Hermann,b Radhika Venkat,]
Helena Mancebo,] and Louis M. Renzetti‡
Departments of †Discovery Chemistry, ‡RNA Therapeutics, §Chemical Synthesis, Non-Clinical Safety, ^Discovery Technologies, #In Silico
Sciences, ¥Laboratory Animal Resources, and ꢀPDR Lifecycle Management, Roche Research Center, 340 Kingsland Street, Nutley,
New Jersey 07110-1199, bChemOvation Ltd., Graylands, Langhurst Wood Road, Horsham, West Sussex RH12 4QD, U.K., and ]Multispan Inc,
26219 Eden Landing Road, Hayward, California 94545
Received November 17, 2009
The inhibition of LTB4 binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the
premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the
leukotriene B4 (LTB4) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found
to be highly potent inhibitors of LTB4 binding to BLT1 and BLT2 receptors, with varying levels of
selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good
in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB4
and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity
experiment provided the rationale for differentiation and selection of one compound (33) for clinical
development.
Introduction
inflammatory processes, including mast cell migration, den-
dritic cell trafficking, and allergic lung inflammation.1-3
Therefore, blockade of LTB4 receptors is a potentially useful
strategy for the treatment of several pulmonary inflammtory
diseases, including asthma, acute respiratory distress syn-
drome (ARDS), acute lung injury (ALI), and chronic ob-
structive pulmonary disease (COPD).4 In addition to
extensive pharmacological data characterizing the potential
role of LTB4/BLT pathways in pulmonary diseases, the
rationale for treatment of these diseases with LTB4 antago-
nists is based in part on the observation of elevated levels of
LTB4 in pulmonary tissues in patients having these diseases. A
large body of recent evidence also strongly suggests a role for
BLT1 and BLT2 receptors in the initiation and progression of
atherosclerosis.5 Extensive reviews of preclinical and clinical
development of small molecules for LTB4 inhibitory-based
therapies have been published.6,7
Leukotriene B4 (LTB4,a (6Z,8E,10E,14Z)-(5S,12R)-5,12-
dihydroxyeicosa-6,8,10,14-tetraenoic acid, 1, Figure 1) is
derived from the action of several enzymes acting sequentially
on arachidonic acid, largely in inflammatory cells. Its effect on
the recruitment of inflammatory cells is mediated by agonism
of two G-protein-coupled receptors, BLT1 and BLT2.1 The
physiological implications of selective BLT1, BLT2, or dual
BLT1 and BLT2 antagonism are not completely understood;
however, BLT1 is known to play a key role in the activation
and migration of several inflammatory cell types, including
neutrophils, macrophages, and lymphocytes.2 Several recent
studies have demonstrated a significant role of BLT2 in
*To whom correspondence should be addressed. Phone: 973-235-
6689. Fax: 973-235-6084. E-mail: robert.goodnow@roche.com.
a Abbreviations: ADME, absorption, distribution, metabolism,
excretion; ADMET, absorption, distribution, metabolism, excretion,
toxicity; AIBN, 2,20-azobisisobutyronitrile; ALI, acute lung injury;
ALT, alanine aminotransferase; ARDS, acute respiratory distress syn-
drome; AST, aspartate aminotransferase; BAL, bronchoalveolar
lavage; BSA, bovine serum albumin; COPD, chronic obstructive pul-
monary disease; cyp, cytochrome p450; PdCl2(dppf), dichloro[1,10-bis-
(diphenylphosphino)ferrocene]palladium(II); DME, dimethoxyethane;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; GLP,
good laboratory practice; GPCR, G-protein-coupled receptor; HBSS,
Hank’s balanced salt solution; HCl, hydrochloric acid; HEPES, 4-(2-
hydroxyethyl)-1-piperazinesulfonic acid; hERG, human ether-a-go-go-
related gene; IBD, inflammatory bowel disease; LTB4, leukotriene B4;
NaOH, sodium hydroxide; PBS, phosphate buffer saline; PK, pharma-
cokinetic; PMA, phosphomolybdic acid; RA, rheumatoid arthritis;
SAR, structure-activity relationship; TBDMS, tert-butyldimethylsilyl;
TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; THF, tetrahydrofuran.
Several compounds that inhibit the action of LTB4 on
BLT1 and/or BLT2 that have entered clinical trials for vari-
ous inflammatory and oncologic indications are shown in
Figure 2. For example, amelubant (BIIL-284) is a carbamate
prodrug of an antagonist of both BLT1 and BLT2 receptors;
its pharmacology8 and progress in clinical trials9 have been
thoroughly reported. Phase I studies for treatment of rheu-
matoid arthritis (RA) and inflammatory bowel disease (IBD)
have been disclosed for CP-195543, a dual antagonist of the
BLT1 and BLT2 receptors. The preclinical pharmacology has
been thoroughly described.10 The development of LY293111,
a selective BLT1 receptor antagonist, for treatment of asthma,
IBD, and RA was reported in 2002.9 Other studies of this
r
pubs.acs.org/jmc
Published on Web 04/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3503
Chemistry
compound for the treatment of some cancers have also been
reported.11,12 The structures of several other compounds that
have been studied clinically are also shown in Figure 2.13
In 1996, Roche scientists disclosed the structures of a series
of potent LTB4 receptor antagonists. Typical of these mole-
cules is structure 2 (Figure 3).14 On the basis of its efficacy in
an in vivo mechanistic model, this molecule was advanced to
canine PK studies.15 Variability in exposure levels was ob-
served as a function of the feeding state of the dog, rendering it
unsuitable for further development. In this paper, we disclose
the chemistry efforts to identify a new series of antagonists of
the LTB4 receptors, with the goal of improving the properties
of 2 with respect to potency and oral bioavailability and
developing new chemistry space around this unique chemical
structure.
The strategy for further optimization of this series of LTB4
inhibitors focused first on the replacement of the 4,6-diaryl-
pyridin-2-ol moiety while leaving the 4-[2-(2-carboxyethyl)-3-
hexylphenoxy]butyric acid side chain unchanged. The middle
carbon chain length and the position of the two carboxylic
acids had previously been optimized during the discovery of 2.
After the synthesis and assay of several small molecule
libraries, it became clear that the 3,5-diarylphenoxy system
provided the most promising results. In this work, we describe
our efforts to optimize this moiety that led to the selection
of the asymmetrically substituted derivative 33 for clinical
development.
The LTB4 antagonists were prepared according to the
chemistry shown in Schemes 2, 3, and 4.16 Compound 9 is
the common intermediate for the preparation of symmetric
and asymmetric analogues, and its synthesis is shown in
Scheme 1. The three functionalized alkyl chains of compound
9 were introduced from 2,3-dimethylphenol 3 in nine steps.
First, the 2,3-dimethylphenol was alkylated with 4-bromo-
butyric acid ethyl ester in the presence of lithium hydride in
DMSO, resulting in 4. The more reactive methyl group at the
2-position of 4 could be selectively oxidized with copper(II)
sulfate pentahydrate and potassium persulfate in water and
acetonitrile to the corresponding aldehyde which was treated
with triethyl phosphonoacetate in the presence of sodium
ethoxide usinga modificationofa reported Horner-Emmons
condensation condition.17 At this point, the benzylic bromi-
nation was effected with N-bromosuccinimide in the presence
of AIBN in chlorobenzene to afford compound 5. The desired
TBDMS-protected five-carbon aldehyde intermediate 7 was
prepared from monohydroxyl TBDMS-protected 1,5-penta-
nediol 6, by a TEMPO-mediated oxidation. The aldehyde was
then treated with a Wittig salt generated in situ from com-
pound 5 to provide intermediate 8 in a cis to trans ratio of
Figure 1. Structure of leukotriene B4 (1).
Figure 3. Chemical structure of LTB4 receptor antagonist (2).
Figure 2. Chemical structures of LTB4 receptor antagonists that have been reported to have entered clinical development.

3504 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Goodnow et al.
Scheme 1. Synthesis of 4-[3-(6-Bromohexyl)-2-(2-ethoxycarbonylethyl)phenoxy]butyric Acid Ethyl Ester Intermediatea
a Reagents and conditions: (a) 4-bromobutyric acid ethyl ester, LiH, DMSO, room temp; (b) CuSO4 5H2O, K2S2O8, H2O, CH3CN, reflux; (c)
3
(EtO)2P(O)CH2CO2Et, NaOEt, EtOH, room temp; (d) N-bromosuccinimide, AIBN, chlorobenzene, 85 ꢀC, 1 h; (e) n-Bu4NHSO4, KBr, TEMPO,
NaClO, CH2Cl2, H2O, 5-10 ꢀC; (f) PPh3, CH3CN, reflux, 1 h, then 5, epoxybutane, reflux; (g) Pd/C, H2, EtOAc, room temp; (h) n-Bu4NF, THF, 0 ꢀC to
room temp; (i) CBr4, PPh3, CH2Cl2, 5-10 ꢀC.
Scheme 2. Synthesis of Symmetric 3,5-Bis-phenol Ether LTB4 Receptor Antagonistsa
a Reagents and conditions: (a) 3,5-dibromophenol, K2CO3, DMF, acetone, reflux; (b) Ar-B(OH)2, Pd(PPh3)4/Na2CO3, EtOH or PdCl2(dppf)/
Cs2CO3, DME, 80 ꢀC; (c) aqueous NaOH, EtOH, room temp.
∼1:3. This mixture of cis and trans compounds was converted
to the corresponding alkyl bromide intermediate 9 in three
steps: hydrogenation of the double bonds, removal of the
protecting group, and finally, conversion of the alcohol to the
corresponding bromide.
A general synthesis of symmetric 3,5-diarylphenyl ethers is
shown in Scheme 2. The common 3,5-dibromo intermediate
10 was prepared from compound 9 and 3,5-dibromophenol in
the presence of potassium carbonate in DMF and acetone in
1:2 ratio at reflux temperature. The 3,5-dibromo intermediate
10 was then reacted with various arylboronic acids under
Suzuki coupling conditions,18 and the resulting diesters were
saponified to obtain the final symmetric 3,5-bis-phenol ether
diacids (11-18).
The asymmetrically substituted 3,5-diarylphenyl ether ana-
logues were synthesized using two approaches as shown in
Schemes 3 and 4. Several analogues were prepared using a
lengthy process starting with 3,5-dinitroanisole (19) as shown
in Scheme 3. This approach depends on a selective reduction
of a single nitro group in 3,5-dinitroanisole (19) following
a reported procedure using sodium sulfide and sodium
bicarbonate in water and methanol to give the 3-amino-5-
nitroanisole intermediate.19 At this point, a classical Sandmeyer
reaction of the amine intermediate with sodium nitrite and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3505
Scheme 3. Synthesis of Asymmetric 3,5-Bis-phenol Ether LTB4 Receptor Antagonistsa
a Reagents and conditions: (a) NaHCO3, Na2S, H2O, MeOH, reflux; (b) NaNO2, HCl, KI, H2O, 0-25 ꢀC ; (c) Ar-B(OH)2, Pd(PPh3)4, K2CO3, EtOH,
160 ꢀC, microwave; (d) Zn dust, NH4Cl, MeOH, H2O, room temp; (e) TMSCl, NaI, CH3CN, reflux; (f) 4-[3-(6-bromohexyl)-2-(2-ethoxycarbonyl-
ethyl)phenoxy]butyric acid ethyl ester, K2CO3, DMF, acetone, reflux; (g) Ar1-B(OH)2, PdCl2(dppf), Cs2CO3, DME, 80 ꢀC; (h) aqueous NaOH, EtOH,
room temp.
Scheme 4. Synthesis of Asymmetric 3,5-Bis-phenol Ether LTB4 Receptor Antagonistsa
a Reagents and conditions: (a) Ar-B(OH)2, Ar1-B(OH)2, PdCl2(dppf)/Cs2CO3, DME, 95 ꢀC; (b) 4-[3-(6-bromohexyl)-2-(2-ethoxycarbonylethyl)-
phenoxy]butyric acid ethyl ester, K2CO3, DMF, acetone, reflux; (c) aqueous NaOH, EtOH, room temp.
hydrochloric acid in the presence of potassium iodide furn-
ished the iodoanisole 20. The first aryl group was introduced
by reaction of 20 with arylboronic acids under Suzuki cou-
pling conditions with microwave irradiation at 160 ꢀC. The
second nitro group was reduced to the amine using zinc dust
and ammonium chloride under mild conditions. Again, the
amine was converted to the corresponding iodide using the
same Sandmeyer reaction conditions. The crucial 3-aryl-5-
iodophenol intermediate 22 was obtained by treatment of 21
with iodotrimethylsilane generated in situ from chlorotri-
methylsilane and sodium iodide in acetonitrile at reflux
temperature. The remaining steps were routine: alkylation
of 22 with the bromo intermediate 9 to provide 23, introduc-
tion of second aryl moiety using a Suzuki coupling with
arylboronic acids, and finally hydrolysis of the diester to
provide the desired asymmetrically substituted 3,5-diarylphe-
nyl ethers (25-28). Alternatively, the iodo intermediate (23)
was hydrolyzed first to the diacid (24) and then the second aryl
group was introduced using arylboronic acids under micro-
wave conditions to give compounds 29 and 30.
Scheme 4 illustrates a shorter synthetic approach for the
preparation of asymmetric 3,5-diarylphenol ethers in one pot
from 3,5-dibromophenol (31) and two different arylboronic
acids using PdCl2(dppf) and cesium carbonate in DME at

3506 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Goodnow et al.
Table 2. Antagonist Activity at BLT-1 Receptors Expressed in an
HL-60 Cell Line and LTB4-Evoked Chemotaxis of Human Neutrophils
by Compounds 2, 25-30, and 33-40
Figure 4. Chemical structure of LTB4 receptor antagonist
LY255283 (41).
Table 1. Antagonist Activity of Compounds 11-18 on BLT-1 Recep-
tors in a HL-60 Cell Line
95 ꢀC. The yield of the desired product in this reaction
(26-43%) depends on three factors: (1) the relative reactivity
of the two arylboronic acids, (2) control of the ratio of the
more reactive boronic acid to the less reactive boronic acid
(1:1.5, respectively), and (3) the feasibility of the separation of
the three possible products. The resulting asymmetric phenols
(32) were treated with the alkylbromo intermediate 9, and
hydrolysis of the diesters provided the desired diacids
(33-40).
toward a neutrophilic phenotype with retinoic acid. HL-60
cells express both BLT1 and BLT2 receptors; however, the
reportedly selective BLT2 antagonist LY25528320 (41,
Figure 4) had no antagonist activity in this assay in our hands,
supporting a role for BLT1 (data not shown). Compound 41
also exhibited no antagonist activity in a neutrophil chemo-
taxis assay, indicating this response is also mediated by BLT1
(data not shown). The potencies of symmetrical compounds
assayed in the HL-60 cell functional calcium flux assay are
Results and Discussion
The potency of these compounds as BLT1 receptor antago-
nists was first measured in a functional LTB4 stimulated
calcium flux assay in human HL-60 cells, differentiated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3507
shown in Table 1. It is interesting to note that substitution at
the para-position of both aryl rings caused a decrease in
inhibitor potency of these molecules as exemplified by the
results of compounds 11, 12, and 13. For example, a para
substituent as small as fluorine in 12 caused a 40-fold loss in
potency relative to the bis-phenyl analogue 11. The slightly
larger methoxy substitution in 13 resulted in an even larger
reduction in potency by 150-fold compared to the unsubsti-
tuted bis-aryl compound 11. The bis-4-pyridyl derivative (15)
was particularly potent, and other heteroaromatic moieties
such as the thiophenyl (14) and the 3,5-pyrimidinyl (16)
were well tolerated. Finally, the 26-fold drop in potency of
the bis-5-benzo[1,4]dioxolyl (18) compared to bis-5-benzo-
[1,3]dioxolyl (17) indicates that this area is surprisingly sensi-
tive to subtle differences in the size of the two aryl moieties.
In Table 2, the functional calcium flux assay results as well
as those for a neutrophil chemotaxis assay are shown for
selected unsymmetrically substituted compounds. The poten-
cies of the compounds shown in Table 2 in the two assays
correlate within a factor of 2-3 with exception the analogs 28
and 39; the reasons for this lack of correlation remain unclear.
Importantly, the pairing of 5-benzo[1,3]dioxolyl with thio-
phenyl, phenyl, 4-methoxyphenyl, pyridyl, pyrimidyl, 3-fluoro-
phenyl, or 2-fluorophenyl produced potent compounds
33-39. It is interesting to compare the results of compounds
13 and 35 in which one of the 4-methoxyphenyl moieties was
replaced with a benzo[1,3]dioxol-5-y-phenyl; this resulted in
an increase in the potency as measured by the calcium flux
assay (31 nM vs 3.3 nM). It seems that the nature of the
bicyclic ring is also crucial for maintaining good in vitro
potency. Comparison of the results of 38 and 30 in which
replacement of the 5-benzo[1,3]dioxolyl moiety with an indol-
5-yl moiety led to a 12-fold decrease in potency provides
further evidence for a unique effect of the five-member ring of
the 6-5 system rather than the ring size alone. The results
shown in the Tables 1 and 2 are consistent with a binding
model in which the one of the aryl moieties preferably has a
smallersubstitution ateither the ortho- ormeta-position when
the other aryl moiety has a small fused ring at the 3,4-position.
Inordertocharacterize thesecompounds in greater detail, a
small set of analogues was evaluated in a calcium mobilization
assay using cloned human BLT1 and BLT2 receptors stably
expressed in HEK293 cells to determine their relative selectiv-
ity for these receptors (Table 3). The potency of these com-
pounds was consistently less in this assay than their potency in
the HL-60and chemotaxis assays. Thehigh andlowaffinity of
LTB4 at cloned human BLT1 and BLT2 receptors, respec-
tively, as measured by the EC50 in the present studies, is
consistent with the well established pharmacology of these
receptors. Differences between the types and nature of
G-protein-receptor coupling or cell-specific translational
modification of the receptor protein may underlie the varia-
bility in the effects of LTB4 antagonists among these cellular
assays. The inhibitory effect of LTB4 antagonists on chemo-
taxis of primary human neutrophils may represent the most
relevant measure of compound efficacy to the in vivo setting.
As shown in Table 3, 2 and 11 have g15-fold selectivity for the
BLT1 receptor while compounds 33, 38, and 15 represent
potent dual BLT1 and BLT2 antagonists. Subsequent chemi-
stryeffortsalsoidentified additionalchemical seriesexhibiting
selective BLT2 antagonist activities; these compounds and
their results will be described in a future manuscript. The
potential clinical significance in human diseases of antagonist
activity at either or both of the BLT1 and BLT2 receptors
is not well understood at this time. However, the well-
established role of the BLT1 receptor in inflammatory pro-
cesses and the emerging evidence on the importance of BLT2
receptors in diverse inflammatory cell types suggest com-
pounds with selective or dual antagonist activity at these
receptors may be therapeutically useful in different disease
states with dependence on different cell types. This can only
accurately be assessed in the clinical setting in which differ-
ential effects of dual or selective antagonists may be observed
depending on the role of a particular cell type in a specific
human disease. Indeed, currently available animal models
do not reproduce the complexity of heterogeneous human
diseases and are unlikely to be able to address the importance
of dual versus selective BLT1 and BLT2 receptor antagonism.
Compounds with differing selectivities represent novel tools
with which to further investigate the pharmacology and roles
of the BLT1 and BLT2 receptors.
In Vitro Safety Characterization. As summarized in
Table 4, representative potent molecules in this series (2,
11, 15, 33, 38, and 39) were further characterized with in vitro
ADME and safety assays in order to select compounds for in
vivo evaluation. In general, despite high clogP,22 the log D 23
measurements were in an acceptable range. As one would
expect for a compounds having two carboxylic acid moieties,
these compounds had good solubility in a LYSA assay24 as
well as simulated intestinal fluid25 at either pH 5 (fed) or
6.5 (fasted) and a negligible level of hERG channel inhibition
at 3 μM.26 Metabolic clearance as estimated by incubation
with rat hepatocytes indicated the potential for good to
medium metabolic stability.
Table 3. Selectivity of Receptor Antagonists at Human BLT-1 and
BLT-221
IC50, nM
These compounds were also characterized initially in a
cytochrome p450 (cyp) high-throughput panel screen (1A2,
2D6, 2C9, 2C19, and 3A4) using fluorogenic sub-
strates based on published methods.27 All compounds had
receptor LTB4 EC50, nM (1)
2
11
15
33
38
BLT1
BLT2
1.1
25.1
205.0
3060.0 628.0 143.0 164.0 194.0
38.5
71.0 114.0 129.0
Table 4. Selected ADME and Safety Data for a Selection of BLT-1 and BLT-2 Antagonists
2
11
15
33
38
39
clogP
log D, pH 7.4
9.12
2.51
27.7
16.5
ND
9.51
0.7
6.65
1.78
61
8.75
2.34
32.9
34.6
16.2
>3
9.25
ND
ND
ND
ND
>3
9.25
2.44
38.1
27.1
21.2
ND
7.6
FEDSIF, pH 5.0 (μg/mL)
FASSIF, pH 6.5 (μg/mL)
hepatocytes clearance (rat), ((mL/min)/kg)
hERG binding IC50 (μM)
Cyp2C9 (μM) fluorescence method
Cyp2C9 (μM) LC/MS method
338
27.3
16.1
>3
22.7
14.9
ND
0.34
0.90
>3
>12.5
ND
>50
>12.5
3.9
22.5
>25
ND
ND

3508 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Table 5. Rat SDPK (n = 3)
Goodnow et al.
Table 6. Effects of 14-Day Treatment of Compounds 33 and 38 on
Clinical Chemistries in Male Ratsa
2
15
33
38
10
day of
sampling vehicle (mg/kg)/day) (mg/kg)/day) (mg/kg)/day) (mg/kg)/day)
33 (40
33 (400
38 (40
38 (400
dose (po, mg/kg)
T1/2 (h)
10
10
10
2.9
2.11
2.3
28
4.52
0.31
5
2.7
6.21
3
3.1
2.25
5
Cmax (μM)
Tmax (h)
Clinical Chemistry Parameter: ALT (IU/L)
5
15
45 ( 3.6 40 ( 3.4
45 ( 7.1 38 ( 1.7
58 ( 8.9*
125 ( 35.3*
40 ( 6.3 69 ( 13.2***
35 ( 1.8 121 ( 58.7*
a All data are the mean ( SD of four animals per group: (/) P < 0.05
and (///) P < 0.001 for compound-treated compared to vehicle-treated
animals.
F (%)
dose (iv, mg/kg)
10
5
70
5
88
5
2.5
2.1
0.3
7.53
T1/2 (h)
Vdss (L/kg)
CL ((mL/min)/kg)
4.52
1.55
28.14
2.1
0.22
5.47
2.4
0.98
11.6
Single Dose Pharmacokinetics in Rats. Several molecules
were characterized for oral bioavailability by means of rat
single dose pharmacokinetic studies (SDPK) (Table 5).
Compared to compound 2, 33 and 38 showed superior oral
bioavailability despite their high molecular weight (>600
Da), high clogP (∼9), and the presence of two carboxylic acid
groups. The exposure of bis-pyridyl 15 was inferior to that of
compound 2 which, coupled with its strong inhibition of
cyp2C9, disqualified this molecule from further experimen-
tal examination. These results with compound 15 limited any
interest in other compounds that also contained the pyridinyl
moiety. The volumes of distribution for these molecules are
generally low, as are their rates of hepatic clearance.
Mechanistic in Vivo Model of BLT-1/-2 Inhibition. After
elimination of compound 15, the attractive balance of BLT
receptor antagonist properieties and good rat PK measured
for compounds 33 and 38 prompted us to select them for in
vivo efficacy profiling. When guinea pigs are challenged with
an aerosol of LTB4, there is a significant influx of eosinophils
into the airway when compared to animals not treated
with LTB4. Oral pretreatment with 2, 33, or 38 (10 mg/kg,
formulated in 2% Klucel, 1% Tween) significantly attenu-
ated LTB4-evoked eosinophilic pulmonary inflammation as
shown in Figure 5. The effects of these compounds on
attenuation of eosinophil levels were not significantly differ-
ent from each other.
Figure 5. Effect of compounds 2, 33, and 38 and on LTB4 -evoked
pulmonary inflammation in guinea pigs when dosed orally (10 mg/
kg). Data are the mean ( SEM of 4-20 animals per group: (/)
P < 0.05 and (//) P < 0.01 for compound-treated animals
compared to vehicle-treated animals.
Allergen-Evoked Pulmonary Inflammation in Atopic Non-
human Primates. Having shown potent activity of com-
pounds 33 and 38 in a mechanistic in vivo model as well as
acceptable oral exposure in rat PK models, both compounds
were examined for their efficacy in a disease model in nonhu-
man primates. Allergen challenge with Ascaris suum antigen
in hypersensitive primates evoked significant increases in
percentages of neutrophils in bronchoalveolar lavage (BAL)
fluid compared to baseline cell counts (Figure 6). Pretreat-
ment with 2, 33, or 38 (10 mg/kg po, 1 h before allergen
challenge) inhibited allergen-evoked pulmonary inflamma-
tion, resulting in attenuation of allergen-evoked increases in
the percentages of neutrophils, with equal efficacy (Figure 6).
In similarity to the guinea pig, compounds 33 and 38 could
not be differentiated from each other in this nonhuman
primate efficacy model.
Differentiation by in Vivo Toxicity Studies. Given the
excellent in vivo effects as well as acceptable in vitro proper-
ties of both compounds 33 and 38, a non-GLP toxicity study
in rats was conducted as a means of differentiation. To this
end, male rats were treated with vehicle, 40 or 400 (mg/kg)/
day of compounds 33 and 38 (4 rats per group) for 14 days.
All rats survived until the end of the study, and there were
no abnormal in-life observations noted for either dose of
either compound. There were no effects at either dose on
body weight, food consumption, or hematological para-
meters (data not shown). Treatment-related findings for
Figure 6. Effect of compounds 2, 33, and 38 on percentages of
neutrophils in BAL fluid in allergen-challenged nonhuman pri-
mates. Baseline BAL data were obtained 24 h prior to allergen
challenge. Data are the mean ( SEM of 3-21 animals per group:
(###) P < 0.001 for vehicle compared to baseline; (/) P < 0.05, (//)
P < 0.01, and (///) P < 0.001 for analogue-treated animals
compared to vehicle-treated animals.
IC50 > 7.5 μΜ against these isoforms with the exception of
15 and 33 at 2C9. These two componds were further char-
acterized in a more rigorous follow-up assay in involving a
LC/MS detection metabolite detection protocol, again based
on published methods.28 A strong inhibition of cyp2C9
activity was noted for compound 15 (Table 4) presumably
through the interaction of the 4-pyridyl moieties with
cyp2C9. In contrast, compound 33 was only a weak cyp2C9
inhibitor using this protocol.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3509
Table 7. Metabolite Profiles of Compound 33 in Mouse, Rat, Dog, Monkey, and Human Cryopreserved Hepatocytesa
hepatocytes incubation (3 h)
P1 [M - H]-=805.3, %
P2 [M - H]-=805.3, T
[M - H]-=629.3, %
total % metabolism
recovery, %
gender pooled human
male CD1 mouse
male SD rat
15.4
16.5
10.7
25.0
5.2
1.9
0.4
9.7
2.8
2.5
ND
82.7
83.1
79.6
72.2
92.3
100
17.3
16.9
20.4
27.8
7.7
56.7
40.9
49.7
61.3
50.9
100.0
male beagle dog
male cyno monkey
cell-free
ND
ND
a Values in the table represent percent of each metabolite and parent remaining after 3 h of incubation based on relative UV absorbance peak area at
232-242 nm, assuming all peaks have same UV extinction coefficients. ND = not detectable.
both compounds consisted of a significant increase in alanine
aminotransferase (ALT) and total bilirubin at the higher
dose. As shown in Table 6, the increase in ALT was evident
after 5 days of treatment and was more pronounced for
compound 38 than for compound 33 (53% versus 29%,
respectively). After 14 days, the increase in ALT was almost
3-fold for both compounds. Neither compound had signifi-
cant effects on aspartate aminotransferase (AST) or alkaline
phosphatase levels at either dose tested (data not shown).
Furthermore, histological evaluation of the livers revealed
mild to moderate hepatocyte vacuolation in the group that
was treated with 400 (mg/kg)/day of compound 38, a finding
that was not observed in the livers of rats treated with
compound 33. After 14 days of dosing, the 40 (mg/kg)/day
dose level of compound 33 corresponded to AUC0-24h and
Conclusions
A newseriesof LTB4 antagonistswas successfullyidentified
and optimized, resulting in compounds with improved po-
tency and bioavailabiliy relative to the starting point (2).
Compounds having different selectivity profiles between
BLT1 and BLT2 receptors were identified. These molecules
may be useful as tools to better understand their individual
roles in mediating the effects of LTB4. In vitro ADMET
assays as well as rat PK studies guided the selection of the
best compounds among many attractive choices for in vivo
profiling in a guinea pig mechanistic model. With this assay, it
was possible to identify two particularly potent and bioavail-
able LTB4 receptor antagonists which were advanced into
nonhumanprimate efficacy models. Todifferentiatethe better
of two good compounds (33 and 38), a non-GLP 14-day rat
toxicity experiment was conducted. In this way, a rationale
was established to select compound 33 for further clinical
development as a dual BLT1 and BLT2 receptor antagonist
for the potential treatment of inflammatory diseases.
Cmax values of 90 400 ng h/mL and 11 300 ng/mL, respec-
3
tively. Greater than dose-proportional increase in exposure
was observed and the 400 (mg/kg)/day dose level of com-
pound 33 corresponded to AUC0-24h and Cmax values of
2 922 300 ng h/mL and 262 000 ng/mL, respectively. After
3
14 days of dosing, the 40 (mg/kg)/day dose level of com-
pound 38 corresponded to AUC0-24h and Cmax values of
Experimental Section
80 571 ng h/mL and 11 300 ng/mL, respectively. Greater
3
All reactions were carried out under a nitrogen or argon
atmosphere unless otherwise noted. Tetrahydrofuran was dis-
tilled over sodium and benzophenone. All other solvents and
reagents were purchased from Aldrich and used without further
purification unless otherwise noted. Melting points were taken
on a Thomas-Hoover apparatus and are uncorrected. 1H NMR
spectra were recorded with Mercury 300 and Unityplus
400 MHz spectrometers, using residual chloroform as internal
standard set to 7.26 ppm; for spectrum obtained for compounds
dissolved in DMSO-d6, residual proton DMSO-d5 signal was set
as 2.54 ppm. Electron impact (EI, 0 eV) and fast atom bombard-
ment (FAB) mass spectra were taken on VG Autospec or VG
70E-HF mass spectrometers, respectively. Biotage silica gel col-
umns or ISCO silica gel columns were used for flash chromato-
graphy; columns were run under a 0-5 psi head of nitrogen to
assist flow. Thin layer chromatograms were run on glass thin-
layer plates coated with silica gel as supplied by E. Merck
(E. Merck no. 1.05719) and were visualized by viewing under
254 nm UV light in a view box, by exposure to I2 vapor, or by
spraying with phosphomolybdic acid (PMA) in aqueous ethanol.
LC/MS (liquid chromatography/mass spectroscopy) chroma-
tograms were collected using a Waters ZQ mass detector/LC
system. Detectors include the Micromass ZQ spectrometer gen-
erally in ES ionization, positive ion mode (mass range, 150 -
1200 amu), a diode array detector, and a PL-ELS 2100 evapora-
tive light scattering detector. HPLC separations are achieved
using a reverse phase cartridge column (ES Industries Chrome-
gabond WR C-18 3 μm, 120 A, 3.2 mm ꢀ 30 mm) with a gradient
solvent method (mobile phase A, water (0.02% TFA); phase B,
acetonitrile (0.02% TFA); gradient 10% B to 90% B). The run
times were generally 3 min with a 1 min equilibration time. The
5 μL solution samples were injected with a pump flow rate of
2 mL/min. Other gradient conditions were utilized for samples
with extremely short or long retention times. The purity of all
than dose-proportional increase in exposure was observed
and the 400 (mg/kg)/day dose level of compound 38 corres-
ponded to AUC0-24h and Cmax values of 1 871 900 ng h/mL
3
and 124 000 ng/mL, respectively. The safety margins were
calculated to be 45- and 40-fold for compounds 33 and 38,
respectively, in the 40 (mg/kg)/day group and 1461- and
936-fold for compounds 33 and 38, respectively, in the
400 (mg/kg)/day group based on an efficacious AUC of
approximately 2000 ng h/mL in allergen-challenged nonhu-
3
man primates. The high safety margins indicated by these
results, in conjunction with the potent in vitro and in vivo
data set, prompted the selection of compound 33 for further
clinical development. A more detailed discussion of the
biological results of 33 has been published.29
Further clinical development studies included the identi-
fication of the metabolites of 33. Contrary to expectations
based on literature precedent,30,31 when compound 33 was
incubated with human, rat, monkey, mouse, and dog hepa-
tocytes, oxidative cleavage of the 5-benzo[1,3]dioxolyl moi-
ety was not observed to any measurable extent. Rather,
the formation of two glucuronide conjugates was observed
(P1 and P2) presumably corresponding to different conju-
gates of the two carboxyl groups (Table 7). These in vitro
observations were consistent with in vivo metabolic profiles
of compound 33 when dosed at 400 mg/kg in rats. Examina-
tion of rat plasma samples indicated that 33 was the major
component; the formation of acylglucuronide isomers was
observed to levels between 2% and 6% of that for compound
33. The product of oxidation of the 5-benzo[1,3]dioxolyl ring
amounted to less than 1% of the total.

3510 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Goodnow et al.
compounds was judged on the percentage of the integrated signal
at UV 214 nm. All final compounds submitted for bioassay were
at least 95% pure as judged by this method, unless indicated
otherwise.
mixture was cooled to room temperature, the charcoal was
filtered off and the filtrate was evaporated under vacuum
to give 4-[2-((E)-2-ethoxycarbonylvinyl)-3-methylphenoxy]-
butyric acid ethyl ester (13.25 g, 78%) as a colorless oil. 1H
NMR (300 MHz, CDCl3) δ 7.91 (d, J = 16.00 Hz, 1H), 7.18 (t,
J = 8.15 Hz, 1H), 6.83 (d, J = 8.15 Hz, 1H), 6.78 (d, J = 8.15
Hz, 1H), 6.62 (d, J = 16.00 Hz, 1H), 4.27 (q, J = 7.00 Hz, 2H),
4.15 (q, J = 7.00 Hz, 2H), 4.08 (t, J = 6.19 Hz, 2H), 2.54 (t, J =
6.90 Hz, 2H), 2.44 (s, 3H), 2.09-2.24 (m, 2H), 1.35 (t, J = 7.00
Hz, 3H), 1.26 (t, J = 7.00 Hz, 3H). EI(þ)-HRMS m/e calculated
for C18H24O5 (Mþ)þ 320.1624, found 320.1626.
Preparation of 4-[3-(6-Bromohexyl)-2-(2-ethoxycarbonylethyl)-
phenoxy]butyric Acid Ethyl Ester (9). a. Preparation of 4-(2,3-
Dimethylphenoxy)butyric Acid Ethyl Ester (4).32 To a solution of
2,3-dimethylphenol (25 g, 204 mmol) in DMSO (205 mL) was
added 4-bromobutyric acid ethyl ester (40.96 g, 210 mmol) and
lithium hydride (2.0 g, 250 mmol) at room temperature. The
resulting light-brown solution was stirred for 2 days. Then the
reaction mixture was cooled to 0 ꢀC and water (200 mL) was
added slowly. The organic compound was extracted with hexanes
(2 ꢀ 200 mL). The combined organic extracts were washed with
brine solution (150 mL), and the organic solution was dried over
anhydrous magnesium sulfate. Filtration of the drying agent and
removal of the solvent gave a light-brown oil. The crude mixture
was purified using a Biotage (40L) column, eluting with 5% ethyl
acetate in hexanes to isolate 4-(2,3-dimethylphenoxy)butyric acid
ethyl ester (45.32 g, 94%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) δ 6.99-7.11 (m, 1H), 6.77 (d, J = 7.55 Hz, 1H), 6.68 (d,
J = 8.45 Hz, 1H), 4.15 (q, J = 7.06 Hz, 2H), 3.98 (t, J = 5.89 Hz,
2H), 2.54 (t, J = 7.40 Hz, 1H), 2.27 (s, 3H), 2.14 (s, 3H),
2.08-2.18 (m, 2H), 1.26 (t, J = 7.06 Hz, 3H). ES(þ)-HRMS
m/e calculated for C14H20O3 (Mþ)þ 236.1412, found 236.1419.
b. Preparation of 4-(2-Formyl-3-methylphenoxy)butyric Acid
Ethyl Ester. A mixture of copper(II) sulfate pentahydrate
(21.98 g, 88.06 mmol) and potassium persulfate (71.42 g,
264 mmol) in water (396 mL) was heated to 63-65 ꢀC to give
a blue solution. Then a solution of 4-(2,3-dimethylphenoxy)-
butyric acid ethyl ester (20.81 g, 88.06 mmol) in acetonitrile
(220 mL) was added at the above temperature. The resulting
light-green solution was refluxed for 40 min. Then the reaction
mixture was cooled to ∼5 ꢀC in order to precipitate most of the
inorganic solids. The resulting solids were collected by filtration,
and the solid cake was washed with dichloromethane (1.0 L).
The two layers of filtrate were separated, and the aqueous layer
was extracted with dichloromethane (200 mL). The combined
organic extracts were washed with brine solution (150 mL), and
the organic solution was dried over anhydrous magnesium
sulfate. Filtration of the drying agent and removal of the solvent
gave a brown oil. The crude mixture was purified using a Biotage
(40L) column eluting with 5-10% ethyl acetate in hexanes to
give 4-(2-formyl-3-methylphenoxy)butyric acid ethyl ester
(20.70 g, 94%) as a colorless oil. 1H NMR (300 MHz, CDCl3)
δ 10.67 (s, 1H), 7.30-7.41 (m, 1H), 6.83 (d, J = 8.45 Hz, 1H),
6.81 (d, J = 7.55 Hz, 1H), 4.15 (q, J = 7.00 Hz, 2H), 4.11 (t, J =
6.30 Hz, 2H), 2.58 (s, 3H), 2.54 (t, J = 7.24 Hz, 2H), 2.08-2.26
(m, 2H), 1.27 (t, J = 7.00 Hz, 3H). EI(þ)-HRMS m/e calculated
for C14H18O4 (Mþ)þ 250.1205, found 250.1202.
d. Preparation of 4-[3-Bromomethyl-2-((E)-2-ethoxycarbonyl-
vinyl)phenoxy]butyric Acid Ethyl Ester (5). To a solution of
4-[2-((E)-2-ethoxycarbonylvinyl)-3-methylphenoxy]butyric acid
ethyl ester (8.0 g, 25.0 mmol) in chlorobenzene (190 mL) were
added N-bromosuccinimide (6.67 g, 37.5 mmol) and 2,20-azobi-
sisobutyronitrile (AIBN) (591 mg, 3.6 mmol) at room tempera-
ture. The resulting solution was heated to 85 ꢀC and stirred for
1 h. Then the reaction mixture was cooled to room temperature
and diluted with water (100 mL) and the mixture was extracted
with hexanes (3 ꢀ 100 mL). The combined organic extracts were
washed with brine solution (150 mL) and dried over anhydrous
magnesium sulfate. Filtration of the drying agent and removal of
the solvent gave a crude oil which was purified using a Biotage
(40L) column, eluting with 15-25% ethyl acetate in hexanes to
give 4-[3-bromomethyl-2-((E)-2-ethoxycarbonylvinyl)phenoxy]-
butyric acid ethyl ester (7.11 g, 71%) as a low melting solid. 1H
NMR (300 MHz, CDCl3) δ 7.94 (d, J = 16.15 Hz, 1H), 7.27 (t,
J = 8.15 Hz, 1H), 7.03 (dd, J = 0.91, 8.15 Hz, 1H), 6.90 (d, J =
8.15Hz, 1H), 6.73 (d, J =16.15 Hz, 1H), 4.59 (s, 2H), 4.30(q, J =
7.05 Hz, 2H), 4.15 (q, J = 7.05 Hz, 2H), 4.09 (t, J = 6.00 Hz, 2H),
2.54(t, J = 7.20 Hz, 2H), 2.11-2.24 (m, 2H), 1.36 (t, J = 7.05 Hz,
3H), 1.27 (t, J = 7.05 Hz, 3H). ES(þ)-HRMS m/e calculated for
C18H23BrO5 (M þ Na)þ 421.0621, found 421.0621.
e. Preparation of 5-(tert-Butyldimethylsilanyloxy)pentanal (7).
To
a solution of 5-(tert-butyldimethylsilanyloxy)pentanol
(3.66 g, 15.1 mmol) in dichloromethane (30 mL) were added
water (5.6 mL), potassium bromide (202 mg, 1.7 mmol),
n-tetrabutylammonium hydrogen sulfate (290 mg, 0.84 mmol),
and TEMPO (30 mg) at room temperature. The resulting light-
brown solution was cooled to ∼5 ꢀC, and a solution of sodium
hypochlorite (30 mL, 19.3 mmol, 5%) was added dropwise at
this temperature. After the addition of half of the sodium
hypochlorite solution, solid potassium carbonate (300 mg)
was added to keep the reaction mixture basic. The remaining
sodium hypochlorite solution was then added at 5-10 ꢀC. By
this point, a precipitate had formed and the reaction mixture
was stirred for another 1 h at ∼10-15 ꢀC. Then water (100 mL)
was added and the resulting solution was extracted with diethyl
ether (2 ꢀ 100 mL). The combined organic extracts were washed
with brine solution (150 mL), and the organic layer was dried
over anhydrous magnesium sulfate. Filtration of the drying
agent and removal of the solvent gave 5-(tert-butyldimethyl-
silanyloxy)pentanal (3.32 g, 99%) as a light-brown oil. 1H NMR
(300 MHz, CDCl3) δ 9.78 (s, 1H), 3.63 (t, J = 6.04 Hz, 2H), 2.47
(td, J = 1.81, 7.24 Hz, 2H), 1.64-1.78 (m, 2H), 1.48-1.62 (m,
2H), 0.90 (s, 9H), 0.05 (s, 6H). ES(þ)-HRMS m/e calculated for
C11H24O2Si (M þ H)þ 217.1619, found 217.1619.
c. Preparation of 4-[2-((E)-2-Ethoxycarbonylvinyl)-3-methyl-
phenoxy]butyric Acid Ethyl Ester. Sodium metal spheres (1.6 g,
69.6 mmol) were added to ethanol (100 mL) with stirring at
room temperature under a nitrogen atmosphere over 15 min. An
exothermic reaction occurred, and the mixture was stirred for
another 15 min. After the mixture was cooled to room tempera-
ture, triethyl phosphonoacetate (14.7 mL, 73.4 mmol) and 4-(2-
formyl-3-methylphenoxy)butyric acid ethyl ester (13.25 g, 52.9
mmol) were added sequentially. During the addition of 4-(2-
formyl-3-methylphenoxy)butyric acid ethyl ester, the solution
turned brown and the temperature increased to ∼55 ꢀC. The
resulting brown solution was stirred for 2 days at room
temperature. The reaction mixture was then diluted with water
(150 mL), stirred for 1 h, and extracted with hexanes (3 ꢀ
100 mL). The combined organic extracts were washed with brine
solution (150 mL), and the organic solution was dried over
anhydrous magnesium sulfate. Filtration of the drying agent
and removal of the solvent gave a light-yellow oil. The crude oil
was taken up in hexanes and ethyl acetate (3:1 ratio), treated
with charcoal, and heated gently with a heat gun. After the
f. Preparation of 4-[3-[6-(tert-Butyldimethylsilanyloxy)hex-1-
enyl]-2-((E)-2-ethoxycarbonylvinyl)phenoxy]butyric Acid Ethyl
Ester (8). A solution of 4-[3-bromomethyl-2-((E)-2-ethoxycar-
bonylvinyl)phenoxy]butyric acid ethyl ester (798 mg, 2.0 mmol)
and triphenylphosphine (577 mg, 2.2 mmol) in acetonitrile
(12 mL) was heated to reflux for 1 h under a nitrogen atmo-
sphere. Then it was cooled to room temperature and a solution
of 5-(tert-butyldimethylsilanyloxy)pentanal (606 mg, 2.8 mmol)
in 1,2-epoxybutane (22 mL) was added at room temperature
and the mixture was again heated to reflux for 15 h. During
this period, the mixture first turned to a brick-red color, and at
the end of the reaction it had become a pale-yellow solution.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3511
Then the reaction mixture was cooled to room temperature
and the solvent was removed under vacuum. The residue
was dissolved in a solution of ethyl acetate and hexanes (1:3,
150 mL), and the resulting cloudy solution was washed with a
mixture of methanol and water (2:1, 225 mL). The aqueous layer
was extracted one more time with ethyl acetate and hexanes (1:3,
50 mL). The combined organic extracts were washed with brine
solution (150 mL), and the organic solution was dried over
anhydrous magnesium sulfate. Filtration of the drying agent
and removal of the solvent gave light-brown oil. The crude
mixture was purified using a Biotage (40L) column, eluting with
5% and 15% ethyl acetate in hexanes to give the desired
4-[3-[6-(tert-butyldimethylsilanyloxy)hex-1-enyl]-2-((E)-2-ethoxy-
carbonylvinyl)phenoxy]butyric acid ethyl ester (760 mg, 74%)
as a trans/cis (∼2:1) mixture at six-carbon chain, and it was
obtained as a colorless oil. 1H NMR (300 MHz, CDCl3) δ
7.84-8.02 (m, 1H), 7.16-7.23 (m, 1H), 6.74-7.09 (m, 2H), 6.63 (d,
J = 16.00 Hz, 1H), 6.46 (d, J = 16.00 Hz, 1H), 5.68-6.15 (m, 1H),
4.01-4.34 (m, 8H), 3.49-3.70 (m, 2H), 2.45-2.64 (m, 2H),
2.01-2.34 (m, 4H), 1.19-1.40 (m, 8H), 0.82-0.94 (m, 9H),
0.04-0.12 (m, 6H). Note: Several peaks were broad and were
assigned as multiplets; thus, the ratio was assigned approximately
on the basis of olefinic proton multiplet integration. ES(þ)-HRMS
m/e calculated for C29H46O6Si (M þ Na)þ 541.2956, found
541.2953.
g. Preparation of 4-[3-[6-(tert-Butyldimethylsilanyloxy)hexyl]-
2-(2-ethoxycarbonylethyl)phenoxy]butyric Acid Ethyl Ester. To
a solution of 4-[3-[6-(tert-butyldimethylsilanyloxy)hex-1-enyl]-
2-((E)-2-ethoxycarbonylvinyl)phenoxy]butyric acid ethyl ester
(507 mg, 0.977 mmol) in ethyl acetate (10 mL) was added 10%
palladium on carbon (350 mg) at room temperature. The
resulting black mixture was stirred in the presence of atmo-
spheric hydrogen gas in a balloon for 36 h at room temperature.
Then the catalyst was removed by filtration using a filter paper
and the residue was washed with hot ethyl acetate (∼60 mL).
The filtrate was concentrated in vacuo and the resulting
residue was dried under high vacuum to give 4-[3-[6-(tert-
butyldimethylsilanyloxy)hexyl]-2-(2-ethoxycarbonylethyl)phe-
noxy]butyric acid ethyl ester (438 mg, 86%) as a colorless oil. 1H
NMR (300 MHz, CDCl3) δ 6.99-7.16 (m, 1H), 6.77 (dd, J =
0.91, 7.85 Hz, 1H), 6.69 (dd, J = 0.91, 8.15 Hz, 1H), 4.08-4.21
(m, 4H), 4.00 (t, J = 6.19 Hz, 2H), 3.60 (t, J = 6.64 Hz, 2H),
2.85-3.02 (m, 2H), 2.43-2.66 (m, 6H), 2.14 (quin, J = 6.72 Hz,
2H), 1.31-1.62 (m, 8H), 1.27 (t, J = 7.24 Hz, 3H), 1.26 (t, J =
7.24 Hz, 3H), 0.90 (s, 9H), 0.05 (s, 6H). ES(þ)-HRMS m/e cal-
culated for C29H50O6Si (M þ Na)þ 545.3269, found 545.3267.
h. Preparation of 4-[2-(2-Ethoxycarbonylethyl)-3-(6-hydroxy-
hexyl)phenoxy]butyric Acid Ethyl Ester. To a solution of
4-[3-[6-(tert-butyldimethylsilanyloxy)hexyl]-2-(2-ethoxycarbonyl-
ethyl)phenoxy]butyric acid ethyl ester (438 mg, 0.837 mmol) in
THF (12 mL) was added a solution of tetra-n-butylammonium
fluoride (1.25 mL, 1.25 mmol, 1.0 M in THF) at 0 ꢀC. Then the
resulting colorless solution was allowed to warm to room
temperature in 2 h and the mixture was stirred for another 2 h at
room temperature before being diluted with water (∼50 mL). The
mixture was extracted with ethyl acetate (2 ꢀ 50 mL), and the
combined extracts were washed with brine solution (100 mL). The
organic solution was dried over anhydrous magnesium sulfate,
and the solvent was removed under vacuum after filtration of the
drying agent. The crude residue was dried further under high
vacuum, and the desired 4-[2-(2-ethoxycarbonylethyl)-3-(6-hydroxy-
hexyl)phenoxy]butyric acid ethyl ester (342 mg, 99%) was isolated as
a colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.08 (t, J = 8.00
Hz, 1H), 6.75 (d, J = 8.00 Hz, 1H), 6.67 (d, J = 8.00 Hz, 1H),
4.07-4.20 (m, 4H), 3.98 (t, J = 6.04 Hz, 2H), 3.63 (t, J = 6.49
Hz, 2H), 2.88-3.01 (m, 2H), 2.41-2.65 (m, 6H), 2.01-2.17 (m,
2H), 1.35-1.63 (m, 9H), 1.16-1.30 (m, 6H). ES(þ)-HRMS m/e
calculated for C23H36O6 (M þ Na)þ 431.2404, found 431.2404.
i. Preparation of 4-[3-(6-Bromohexyl)-2-(2-ethoxycarbonyl-
ethyl)phenoxy]butyric Acid Ethyl Ester (9). To a solution of
4-[2-(2-ethoxycarbonylethyl)-3-(6-hydroxyhexyl)phenoxy]-
butyric acid ethyl ester (349 mg, 0.85 mmol) and carbon tetra-
bromide (423 mg, 1.26 mmol) in dichloromethane (10 mL) was
added triphenylphosphine (281 mg, 1.07 mmol) at ∼0 ꢀC. The
resulting colorless solution was stirred for 3 h at 5-10 ꢀC. Then
the solvent was removed under vacuum and a mixture of ethyl
acetate and hexanes (1:3, 50 mL) was added. As a result, a
cloudy solution containing some precipitate was formed, and
the cloudy solution was transferred into a separatory funnel and
was washed with a mixture of methanol and water (2:1, 150 mL).
The aqueous layer was extracted one more time with ethyl
acetate and hexanes (1:3, 50 mL). The combined organic
extracts were washed with brine solution (100 mL), and the
organic solution was dried over anhydrous magnesium sulfate.
Filtration of the drying agent and removal of the solvent gave a
colorless oil which was purified using a Biotage (40M) column,
eluting with 10% ethyl acetate in hexanes to give the desired
4-[3-(6-bromohexyl)-2-(2-ethoxycarbonylethyl)phenoxy]buty-
ric acid ethyl ester (350 mg, 87.5%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) δ 7.02-7.17 (m, 1H), 6.77 (d, J = 7.55 Hz,
1H), 6.69 (d, J = 8.15 Hz, 1H), 4.16 (q, J = 7.20 Hz, 2H), 4.15
(q, J = 7.20 Hz, 2H), 4.00 (t, J = 6.04 Hz, 2H), 3.42 (t, J = 6.79
Hz, 2H), 2.76-3.10 (m, 2H), 2.43-2.68 (m, 6H), 2.14 (quin, J =
6.70 Hz, 2H), 1.82-1.96 (m, 2H), 1.35-1.64 (m, 6H), 1.27 (t,
J=7.20Hz, 3H),1.26(t,J=7.20Hz, 3H).ES(þ)-HRMSm/ecal-
culated for C23H35BrO5 (M þ Na)þ 493.1560, found 493.1560.
4-{2-(2-Carboxyethyl)-3-[6-([1,10,3,100]terphenyl-50-yloxy)-
hexyl]phenoxy}butyric Acid (11). General Procedure A: Prepara-
tion of 4-{3-[6-(3,5-Dibromophenoxy)hexyl]-2-(2-ethoxycarbony-
lethyl)phenoxy}butyric Acid Ethyl Ester (10). To a mixture of
4-[3-(6-bromohexyl)-2-(2-ethoxycarbonylethyl)phenoxy]butyric
acid ethyl ester (14.54 g, 30.84 mmol), 3,5-dibromophenol (8.55 g,
33.92 mmol), and potassium carbonate (8.53 g, 61.68 mmol) were
added N,N-dimethylformamide (210 mL) and acetone (420 mL)
at room temperature. The resulting suspension was heated to
reflux for 2 days. Then the reaction mixture was cooled to room
temperature and diluted with water (200 mL). The mixture was
extracted with ethyl acetate (2 ꢀ 200 mL), and the combined
organic extracts were washed with brine solution (200 mL). The
organic layers were dried over anhydrous magnesium sulfate,
filtered, and concentrated in vacuo to give the crude product
which was purified using a Biotage column (40L), eluting with
10% ethyl acetate/hexanes to give 4-{3-[6-(3,5-dibromophe-
noxy)hexyl]-2-(2-ethoxycarbonylethyl)phenoxy}butyric acid ethyl
ester (19.61 g, 99%) as a colorless oil. 1H NMR (300 MHz, CDCl3)
δ 7.23 (t, J = 1.51 Hz, 1H), 7.10 (t, J = 7.85 Hz, 1H), 6.99 (d, J =
1.51 Hz, 2H), 6.77 (d, J = 7.85 Hz, 1H), 6.69 (d, J = 7.85 Hz, 1H),
4.16 (q, J = 7.24 Hz, 2H), 4.15 (q, J = 7.24 Hz, 2H), 4.00 (t, J =
6.04 Hz, 2H), 3.92 (t, J = 6.49 Hz, 2H), 2.91-3.04 (m, 2H),
2.59-2.68 (m, 2H), 2.43-2.58 (m, 4H), 2.14 (qd, J = 6.49, 6.69
Hz, 2H), 1.67-1.83 (m, 2H), 1.36-1.65 (m, 6H), 1.27 (t, J = 7.24
Hz, 6H). ES(þ)-HRMS m/e calcd for C29H38O6Br2 (M þ H)þ
641.1108, found 641.1101.
General Procedure B: Preparation of 4-{2-(2-Ethoxycarbony-
lethyl)-3-[6-([1,10,3,100]terphenyl-50-yloxy)hexyl]phenoxy}butyric
Acid Ethyl Ester. A solution of 4-{3-[6-(3,5-dibromophenoxy)-
hexyl]-2-(2-ethoxycarbonylethyl)phenoxy}butyric acid ethyl ester
(321 mg, 0.5 mmol) in dimethoxyethane (10 mL) was stirred for
5 min at room temperature under a nitrogen atmosphere. Then
tetrakis(triphenylphosphine)palladium(0) (115 mg, 0.1 mmol) was
added at room temperature and the resulting light-yellow solution
was heated to 80 ꢀC and stirred for 5 min. At this time, a solution of
phenylboronic acid (366 mg, 3.0 mmol) in ethanol (10 mL) was
added, followed by a solution of sodium carbonate (318 mg, 3.0
mmol) in water (1.0 mL). The resulting light-yellow suspension
was stirred for 15 h at reflux. Then the reaction mixture was cooled
to room temperature and diluted with water (20 mL) and ethyl
acetate (50 mL). The two layers were separated, and the aqueous
layer was extracted with ethyl acetate (50 mL). The combined
organic extracts were washed with water (100 mL), brine solution

3512 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Goodnow et al.
(100 mL) and dried over anhydrous magnesium sulfate. Filtration
and concentration of the solvent gave the crude residue which was
purified using an ISCO (40 g) column, eluting with 0-50% ethyl
acetate/hexanes to afford 4-{2-(2-ethoxycarbonylethyl)-3-[6-
([1,10,3,100]terphenyl-50-yloxy)hexyl]phenoxy}butyric acid ethyl
reaction mixture was cooled to room temperature and diluted
with water (20 mL) and ethyl acetate (20 mL). The two layers
were separated, and the ethyl acetate layer was discarded. Then
the aqueous layer was acidified with 1.0 N hydrochloric acid
and the organic compound was extracted with ethyl acetate (2 ꢀ
20 mL). The combined organic extracts were washed with brine
solution (20 mL), dried over anhydrous magnesium sulfate,
filtered, and concentrated to afford 4-{2-(2-carboxyethyl)-
3-[6-(3,5-dipyrimidin-5-ylphenoxy)hexyl]phenoxy}butyric acid
(85 mg, 57%) as a light-yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ 11.79 (br. s, 2H), 9.30 (s, 4H), 9.22 (s, 2H), 7.82 (s,
1H), 7.49 (d, J = 1.51 Hz, 2H), 6.91-7.18 (m, 1H), 6.67-6.79
(m, 2H), 4.17 (t, J = 6.34 Hz, 2H), 3.94 (t, J = 6.19 Hz, 2H),
2.75-2.87 (m, 2H), 2.54-2.64 (m, 2H), 2.24-2.43 (m, 4H),
1.88-1.97 (m, 2H), 1.68-1.85 (m, 2H), 1.35-1.59 (m, 6H).
ES(þ)-HRMS m/e calcd for C33H36N4O6 (M þ Na)þ 607.2527,
found 607.2527.
1
ester (147 mg, 46%) as a light-brown oil. H NMR (300 MHz,
CDCl3) δ 7.60-7.72 (m, 4H), 7.42-7.53 (m, 4H), 7.33-7.41 (m,
3H), 7.12 (d, J = 1.51 Hz, 2H), 7.03-7.12 (m, 1H), 6.78 (d, J =
7.85 Hz, 1H), 6.69 (d, J = 7.85 Hz, 1H), 4.10-4.20 (m, 4H),
4.06-4.12 (m, 2H), 4.00 (t, J = 6.04 Hz, 2H), 2.84-3.05 (m, 2H),
2.59-2.71 (m, 2H), 2.43-2.58 (m, 4H), 2.07-2.20 (m, 2H),
1.77-1.92 (m, 2H), 1.57 (s, 6H), 1.26 (t, J = 7.24 Hz, 3H), 1.26
(t, J = 7.24 Hz, 3H). ES(þ)-HRMS m/e calcd for C41H48O6 (M þ
Na)þ 659.3343, found 659.3343.
General Procedure C: Preparation of 4-{2-(2-Carboxyethyl)-
3-[6-([1,10,3,100]terphenyl-50-yloxy)hexyl]phenoxy}butyric Acid
(11). To
a solution of 4-{2-(2-ethoxycarbonylethyl)-3-[6-
([1,10,3,100]terphenyl-50-yloxy)hexyl]phenoxy}butyric acid ethyl
ester (90 mg, 0.14 mmol) in ethanol (5 mL) was added 1.0 N
aqueous NaOH (5 mL) at room temperature. The mixture was
heated to 50-55 ꢀC, and the resulting solution was stirred for
3 h. Then the reaction mixture was concentrated and the residue
was diluted with water (20 mL) and extracted with diethyl ether
(50 mL) to remove any neutral impurities. The aqueous layer
was acidified with 1.0 N hydrochloric acid until the solution
became acidic. The resulting white solids were collected by
filtration and washed with water. After air-drying, 4-{2-(2-
carboxyethyl)-3-[6-([1,10,3,100]terphenyl-50-yloxy)hexyl]phenoxy}-
butyric acid (71 mg, 86%) was isolated as a white solid. 1H NMR
(300 MHz, DMSO-d6) δ 12.14 (br s, 2H), 7.76 (d, J = 7.55 Hz,
4H), 7.43-7.53 (m, 5H), 7.33-7.43 (m, 2H), 7.17 (d, J = 1.51 Hz,
2H), 7.01-7.09 (m, 1H), 6.70-6.80 (m, 2H), 4.12 (t, J = 6.34 Hz,
2H), 3.94 (t, J = 6.19 Hz, 2H), 2.73-2.90 (m, 2H), 2.59 (t, J= 7.24
Hz, 2H), 2.28-2.46 (m, 4H), 1.87-2.02 (m, 2H), 1.68-1.83 (m,
2H), 1.48 (d, J = 15.70 Hz, 6H). ES(þ)-HRMS m/e calcd for
C37H40O6 (M þ Na)þ 603.2717, found 603.2713.
4-{2-(2-Carboxyethyl)-3-[6-(5-thiophen-3-ylbiphenyl-3-yloxy)-
hexyl]phenoxy}butyric Acid (25). a. Preparation of 3-Methoxy-5-
nitrophenylamine. Sodium bicarbonate (5.62 g, 66.87 mmol) was
added to a solution of sodium sulfide (5.5 g, 70.58 mmol) in
deionized water (60 mL). When the sodium bicarbonate was
completely dissolved, methanol (50 mL) was added, and the
solution was cooled to 0 ꢀC. A precipitate formed, which was
removed by filtration through a Celite pad. Then the filtered
solution was added quickly to a solution of 3,5-dinitroanisole
(Apin Chemicals Ltd.) (7.36 g, 37.15 mmol) in methanol (50 mL).
The resulting suspension was heated to reflux for 30 min, and
then the solution was concentrated in vacuo to remove methanol.
The aqueous residue was poured into 200 mL of ice-water, and
the resulting orange precipitate was collected by filtration. After
air-drying, 3-methoxy-5-nitrophenylamine (5.82 g, 93%) was
1
obtained as light-brown solid. H NMR (300 MHz, CDCl3) δ
7.40 (s, 1H), 7.27 (br s, 1H), 6.61 (br s, 1H), 4.09 (br s, 2H), 3.97
(br s, 3H). ES(þ)-HRMS m/e calcd for C7H8N2O3 (M þ H)þ
169.0608, found 169.0608.
b. Preparation of 1-Iodo-3-methoxy-5-nitrobenzene (20). To a
solution of 3-methoxy-5-nitrophenylamine (7.5 g, 44.6 mmol) in
water (20 mL) was added concentrated hydrochloric acid (19.95
mL, 267.6 mmol, 36%) at 0 ꢀC. To this was added a chilled
solution of sodium nitrite (5.62 g, 81.5 mmol) in water (28.4 mL)
dropwise with a vigorous stirring. Then the resulting colored
mixture was stirred for 15 min at 0 ꢀC, and a cold solution of
potassium iodide (14.81 g, 89.2 mmol) in water (28.4 mL) was
added carefully. During this addition, a black-brown solid was
formed. After the addition the ice-cold bath was removed, and
the reaction mixture was heated to reflux. When the production
of purple vapor ceased, the mixture was cooled to room
temperature and the organic compound was extracted with
dichloromethane (3 ꢀ 200 mL). The combined organic extracts
were washed with brine solution (300 mL), dried over anhydrous
magnesium sulfate, filtered, and concentrated in vacuo. Then
the crude residue was purified using a LC 120 column, eluting
with 0-10% ethyl acetate in hexanes to give 1-iodo-3-methoxy-
5-nitrobenzene (10 g, 80%) as a white solid. 1H NMR (300 MHz,
CDCl3) δ 8.18 (s, 1H), 7.70 (s, 1H), 7.56 (s, 1H), 3.88 (s, 3H).
EI(þ)-HRMS m/e calcd for C7H6INO3 (Mþ)þ 278.9392, found
278.9393.
c. Preparation of 3-(3-Methoxy-5-nitrophenyl)thiophene. To a
solution of 1-iodo-3-methoxy-5-nitrobenzene (1.0 g, 3.59 mmol)
in ethanol (18 mL) in a microwave tube were added tetrakis-
(triphenylphosphine)palladium(0) (837 mg, 0.72 mmol), thio-
phen-3-ylboronic acid (748 mg, 5.85 mmol), and potassium
carbonate (496 mg, 3.58 mmol) at room temperature. Then
the mixture was heated to 160 ꢀC in a closed microwave tube for
30 min. After cooling to room temperature, the colored mixture
was filtered and the filter cake was washed with water. The
filtrate was diluted with 1.0 N HCl, and the mixture was
extracted with ethyl acetate (2 ꢀ 50 mL). The combined organic
extracts were washed with brine solution (100 mL) and dried
4-{2-(2-Carboxyethyl)-3-[6-(3,5-dipyrimidin-5-ylphenoxy)hexyl]-
phenoxy}butyric Acid (16). a. Preparation of 4-{2-(2-Carboxy-
ethyl)-3-[6-(3,5-dibromophenoxy)hexyl]phenoxy}butyric Acid. To a
solution of 4-{3-[6-(3,5-dibromophenoxy)hexyl]-2-(2-ethoxycarbo-
nylethyl)phenoxy}butyric acid ethyl ester (1.5 g, 2.33 mmol) in
ethanol (30 mL) was added aqueous 1.0 N sodium hydroxide
(25 mL) at room temperature. The resulting suspension was heated
to 50-55 ꢀC, and the mixture was stirred for 3 h. Then the reaction
mixture was concentrated and the residue was diluted with water
(20 mL) and extracted with diethyl ether (50 mL) to remove any
neutral impurities. The aqueous layer was acidified with 1.0 N
hydrochloric acid, and the organic compound was extracted with
ethyl acetate (2 ꢀ 50 mL). The combined ethyl acetate extracts were
washed with brine solution (50 mL), dried over anhydrous magne-
sium sulfate, filtered, and concentrated to give the crude product
which was purified using an ISCO 40 g column, eluting with
0-100% ethyl acetate/hexanes to give 4-{2-(2-carboxyethyl)-
3-[6-(3,5-dibromophenoxy)hexyl]phenoxy}butyric acid (1.26 g,
1
92%) as a white solid. H NMR (300 MHz, DMSO-d6) δ 12.10
(br s, 2H), 7.35 (t, J = 1.51 Hz, 1H), 7.18 (d, J = 1.51 Hz, 2H), 7.06
(t, J = 7.90 Hz, 1H), 6.76 (d, J = 7.90 Hz, 1H), 6.73 (d, J = 7.90
Hz, 1H), 3.88-4.02 (m, 4H), 2.76-2.86 (m, 2H), 2.53-2.62 (m,
2H), 2.29-2.46 (m, 4H), 1.94 (t, J = 6.34 Hz, 2H), 1.61-1.74 (m,
2H), 1.29-1.56 (m, 6H). ES(þ)-HRMSm/e calcd for C25H30Br2O6
(M þ Na)þ 607.0301, found 607.0298.
b. Preparation of 4-{2-(2-Carboxyethyl)-3-[6-(3,5-dipyrimi-
din-5-ylphenoxy)hexyl]phenoxy}butyric Acid (16). To a solution
of 4-{2-(2-carboxyethyl)-3-[6-(3,5-dibromophenoxy)hexyl]phe-
noxy}butyric acid (150 mg, 0.26 mmol) in ethanol (2 mL) in a
microwave tube were added tetrakis(triphenylphosphine)-
palladium(0) (29.5 mg, 0.03 mmol), pyrimidin-5-ylboronic acid
(371 mg, 3.0 mmol), and potassium carbonate (212 mg, 1.53
mmol) at room temperature. The microwave tube was sealed
and heated to 160 ꢀC in a closed microwave for 30 min. Then the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3513
over anhydrous magnesium sulfate, filtered, and concentrated
in vacuo. The crude mixture was purified using an ISCO
80 g column, eluting with 0-10% ethyl acetate in hexanes to
give 3-(3-methoxy-5-nitrophenyl)thiophene (776 mg, 92%) as a
light-yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.04-8.07 (m,
1H), 7.65 (t, J = 2.42 Hz, 1H), 7.57 (dt, J = 1.36, 2.42 Hz, 1H),
7.39-7.47 (m, 3H), 3.94 (s, 3H). EI(þ)-HRMS m/e calcd for
C11H9NO3S (Mþ)þ 235.0303, found 235.0298.
g. Preparation of 4-{2-(2-Ethoxycarbonylethyl)-3-[6-(3-iodo-
5-thiophen-3-ylphenoxy)hexyl]phenoxy}butyric Acid Ethyl Ester
(23a). The title compound was prepared in 99% yield from
4-[3-(6-bromohexyl)-2-(2-ethoxycarbonylethyl)phenoxy]butyric
acid ethyl ester (2.99 g, 6.35 mmol), 3-iodo-5-thiophen-3-ylphe-
nol (1.92 g, 6.35 mmol), and potassium carbonate (1.75 g, 12.7
mmol) using the general procedure A described for compound
11. 1H NMR (300 MHz, CDCl3) δ 7.52 (s, 1H), 7.44 (br s, 1H),
7.38 (br s, 1H), 7.30-7.36 (m, 1H), 7.17 (s, 1H), 7.04-7.15 (m,
2H), 6.78 (d, J=7.25 Hz, 1H), 6.69 (d, J = 8.45 Hz, 1H), 4.15 (q,
J = 6.87 Hz, 4H), 3.90-4.05 (m, 4H), 2.92-3.02 (m, 2H), 2.64
(t, J = 7.55 Hz, 2H), 2.45-2.59 (m, 4H), 2.07-2.21 (m, 2H),
1.70-1.88 (m, 2H), 1.34-1.67 (m, 6H), 1.26 (t, J = 6.87 Hz,
6H). ES(þ)-HRMS m/e calcd for C33H41IO6S (M þ Na)þ
715.1561, found 715.1561.
d. Preparation of 3-Methoxy-5-thiophen-3-ylphenylamine. To
a mixture of 3-(3-methoxy-5-nitrophenyl)thiophene (3.78 g,
16.07 mmol), zinc dust (10.72 g, 160.7 mmol), and ammonium
chloride (12.89 g, 241.1 mmol) were added methanol (50 mL)
and water (25 mL) at room temperature. An exothermic reac-
tion began on the addition of water. The suspension was stirred
for 1 h, and the reaction mixture was filtered through Celite. The
filter cake was washed with water and methanol. The filtrate was
concentrated to remove methanol, and the residue was extracted
with ethyl acetate (2 ꢀ 100 mL). The combined extracts were
washed with brine solution (100 mL), dried over anhydrous
magnesium sulfate, filtered, and concentrated in vacuo. The
residue was then purified using an ISCO 120 g column, eluting
with 0-20% ethyl acetate in hexanes to afford 3-methoxy-5-
thiophen-3-ylphenylamine (3.08 g, 93%) as a light-yellow solid.
1H NMR (300 MHz, CDCl3) δ 7.29-7.44 (m, 3H), 6.48-6.60 (m,
2H), 6.21 (br s, 1H), 3.82 (s, 3H), 3.74 (br s, 2H). ES(þ)-HRMS m/
e calcd for C11H11NOS (M þ H)þ 206.0634, found 206.0634.
e. Preparation of 3-(3-Iodo-5-methoxyphenyl)thiophene (21a).
To a solution of 3-methoxy-5-thiophen-3-ylphenylamine
(2.45 g, 11.93 mmol) in water (7.2 mL) was added a concentrated
hydrochloric acid (5.34 mL, 65.2 mmol, 37%) at 0 ꢀC. To this
was added a chilled solution of sodium nitrite (1.5 g, 21.7 mmol)
in water (9.3 mL) dropwise with a vigorous stirring. Then the
resulting colored mixture was stirred for 15 min at 0 ꢀC, and a
cold solution of potassium iodide (3.96 g, 23.86 mmol) in water
(9.3 mL) was added carefully. During this addition, a black-
brown solid was formed. After addition the ice-cold bath was
removed, and the reaction mixture was heated to reflux. When
the production of purple vapor ceased, the mixture was cooled
to room temperature and the organic compound was extracted
with dichloromethane (3 ꢀ 100 mL). The combined organic
extracts were washed with brine solution (200 mL), dried
over anhydrous magnesium sulfate, filtered, and concentrated
in vacuo. Then the crude residue was purified using a LC
80 column, eluting with 0-10% ethyl acetate in hexanes to give
3-(3-iodo-5-methoxyphenyl)thiophene (2.19 g, 58%) as a white
solid. 1H NMR (300 MHz, CDCl3) δ 7.50-7.56 (m, 1H),
7.43-7.46 (m, 1H), 7.36-7.41 (m, 1H), 7.30-7.34 (m, 1H),
7.16-7.19 (m, 1H), 7.07 (dd, J = 1.36, 2.26 Hz, 1H), 3.83 (s,
3H). ES(þ)-HRMS m/e calcd for C11H9IOS (Mþ)þ 315.9419,
found 315.9418.
h. Preparation of 4-{2-(2-Ethoxycarbonylethyl)-3-[6-(5-thio-
phen-3-ylbiphenyl-3-yloxy)hexyl]phenoxy}butyric Acid Ethyl Es-
ter. The title compound was prepared in 55% yield from 4-{2-(2-
ethoxycarbonylethyl)-3-[6-(3-iodo-5-thiophen-3-ylphenoxy)hexyl]-
phenoxy}butyric acid ethyl ester (216 mg, 0.31 mmol) and phenyl-
boronic acid (152 mg, 1.25 mmol) using the general procedure B
1
described for compound 11. H NMR (300 MHz, CDCl3) δ
7.60-7.66 (m, 2H), 7.36-7.52 (m, 7H), 7.04-7.14 (m, 3H), 6.78
(d, J = 7.55 Hz, 1H), 6.69 (d, J = 8.15 Hz, 1H), 4.15 (q, J = 7.09
Hz, 4H), 4.07 (t, J = 6.49 Hz, 2H), 4.00 (t, J = 5.89 Hz, 2H),
2.88-3.04 (m, 2H), 2.59-2.70 (m, 2H), 2.45-2.58 (m, 4H), 2.14
(quin, J = 6.72 Hz, 2H), 1.78-1.91 (m, 2H), 1.41-1.68 (m, 6H),
1.26 (t, J = 7.09 Hz, 3H), 1.26 (t, J = 7.09 Hz, 3H). ES(þ)-
HRMS m/e calcd for C39H46O6S (M þ Na)þ 665.2907, found
665.2907.
i. Preparation of 4-{2-(2-Carboxyethyl)-3-[6-(5-thiophen-3-yl-
biphenyl-3-yloxy)hexyl]phenoxy}butyric Acid (25). The title
compound was prepared in 57% yield from the 4-{2-(2-
ethoxycarbonylethyl)-3-[6-(5-thiophen-3-ylbiphenyl-3-yloxy)hexyl]-
phenoxy}butyric acid ethyl ester (85 mg, 0.13 mmol) and 1.0 N
aqueous NaOH (8.0 mL) using the general procedure C described
for compound 11. 1H NMR (300 MHz, DMSO-d6) δ 12.14 (br s,
2H), 8.02 (s, 1H), 7.75 (d, J = 7.55 Hz, 2H), 7.62-7.70 (m, 2H),
7.54 (s, 1H), 7.42-7.51 (m, 2H), 7.32-7.43 (m, 1H), 7.25 (s, 1H),
7.10 (s, 1H), 7.05 (t, J = 7.85 Hz, 1H), 6.70-6.78 (m, 2H), 4.10 (t,
J = 6.34 Hz, 2H), 3.94 (t, J = 6.04 Hz, 2H), 2.80 (m, 2H), 2.58 (m,
2H), 2.28-2.44 (m, 4H), 1.86-2.02 (m, 2H), 1.75 (m, 2H),
1.30-1.60 (m, 6H). ES(þ)-HRMS m/e calcd for C35H39O6S
(M þ H)þ587.2467, found 587.246. The purity of this compound
as measured by HPLC was 80%.
4-{3-[6-(3-5-Benzo[1,3]dioxolyl-5-thiophen-3-ylphenoxy)hexyl]-
2-(2-carboxyethyl)phenoxy}butyric Acid (33). General Procedure
D: Unsymmetrical 3,5-Diarylphenyl Ethers. a. Preparation of 3-5-
Benzo[1,3]dioxolyl-5-thiophen-3-ylphenol (32a). To a mixture of
3,5-dibromophenol (7.55 g, 30 mmol), 3,4-methylenedioxyphe-
nylboronic acid (7.48 g, 45 mmol), 3-thiophenylboronic acid
(3.97 g, 31 mmol), dichloro[1,10-bis(diphenylphosphino)ferrocene]-
palladium(II) dichloromethane adduct (3.29 g, 4.0 mmol), and
cesium carbonate (48.87 g, 150 mmol) was added 1,2-dimethoxy-
ethane (300 mL) at room temperature under a nitrogen atmo-
sphere. The resulting brown suspension was heated to 95 ꢀC and
stirred for 36 h. Then the reaction mixture was cooled to room
temperature and diluted with water (200 mL) and ethyl acetate
(300 mL). The two layers were separated, and the aqueous layer was
extracted with ethyl acetate (2 ꢀ 100 mL). The combined organic
extracts were washed with water (500 mL) and brine solution
(500 mL). The organic layer was dried over anhydrous magnesium
sulfate, and filtration of the drying agent and removal of the solvent
in vacuo gave the colored residue which was purified using an ISCO
(330 g) column, eluting with 20-40% ethyl acetate in hexanes to
afford 3-5-benzo[1,3]dioxolyl-5-thiophen-3-ylphenol (3.26 g, 37%)
as a low melting white solid. 1HNMR(300MHz, DMSO-d6) δ9.60
(s, 1H), 7.90 (dd, J = 1.42, 2.95 Hz, 1H), 7.63 (dd, J = 2.95, 4.99
Hz, 1H), 7.57 (dd, J= 1.42, 4.99 Hz, 1H), 7.34 (t, J=1.66Hz, 1H),
7.27 (d, J = 1.81 Hz, 1H), 7.15 (dd, J = 1.81, 8.15 Hz, 1H),
f. Preparation of 3-Iodo-5-thiophen-3-ylphenol (22a). To a
suspension of 3-(3-iodo-5-methoxyphenyl)thiophene (2.08 g,
6.7 mmol) and sodium iodide (9.85 g, 65.73 mmol) in acetonitrile
(80 mL) was added trimethylsilyl chloride (4.16 mL, 32.86
mmol) at room temperature. Then the resulting light-yellow
suspension was heated to reflux for 48 h. Then it was cooled to
room temperature and diluted with water (50 mL). The mixture
was extracted with ethyl acetate (2 ꢀ 75 mL), and the combined
ethyl acetate extracts were washed with saturated sodium
thiosulfate solution (100 mL) to remove the iodine color and
then with brine solution (100 mL). The organic layer was dried
over anhydrous magnesium sulfate, filtered, and concentrated
in vacuo. The crude residue was purified using an ISCO 120 g
column, eluting with 0-20% ethyl acetate in hexanes to give
3-iodo-5-thiophen-3-ylphenol (1.92 g, 97%) as a light-brown oil.
1H NMR (300 MHz, CDCl3) δ 7.52 (dd, J = 1.5, 1.5 Hz, 1H), 7.43
(dd, J = 1.4, 3.0 Hz, 1H), 7.38 (dd, J = 3.0, 5.0 Hz, 1H), 7.31 (dd,
J = 1.4, 5.0 Hz, 1H), 7.14 (dd, J = 1.5, 2.4 Hz, 1H), 7.02 (dd, J =
1.5, 2.4 Hz, 1H), 4.83 (br s, 1H). ES(þ)-HRMS m/e calcd for
C10H7IOS (M - H)þ 300.9189, found 300.9189.

3514 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Goodnow et al.
7.01-7.04 (m, 1H), 6.99 (d, J = 8.15 Hz, 1H), 6.85-6.91 (m, 1H),
6.06 (s, 2H). ES(-)-HRMS m/e calcd for C17H12O3S (M - H)-
295.0434, found 295.0432.
Human Neutrophil Chemotaxis Assay. Human whole blood
was obtained from donors who had given written informed
consent, and the human whole blood was layered over an equal
volume of Lympholyte-poly (Cedarlane Laboratories Ltd.,
Ontario, Canada) in a 50 mL centrifuge tube. The tube was
centrifuged for 35 min, and the two cell bands at the interface
were harvested. The lower band containing polymorphonuclear
granulocytes was diluted with assay medium (HBSS with
calcium and magnesium, Invitrogen Corporation, CA, and
0.5% bovine serum albumin, Sigma, St. Louis, MO) and
centrifuged for 12 min. The pellet was resuspended and a
differential cell count performed on a Bayer ADVIA 120 hema-
tology system. Neutrophil purity was 95-97%. Neutrophils at
5 ꢀ 106 cells/mL were added to 96-well round-bottom plates and
incubated with the LTB4 antagonist analogue in question (3 ꢀ
10-12 to 1 ꢀ 10-6 M final assay concentration) for 30 min at
37 ꢀC. Quantification of neutrophil migration toward LTB4 was
determined using modifications to a polycarbonate membrane
migration assay kit (Cell Biolabs Inc., CA). LTB4 (100 ng/mL)
was added to the lower feeder tray, and the neutrophils were
added to the upper plate assembly. After 90 min at 37 ꢀC, the
migratory cell suspensions in the wells of the feeder tray were
transferred into a 96-well flat-bottom black plate. CyQuant GR
dye solution was added to the black plate to lyse the migrated
cells for fluorescent detection. After incubation at room tem-
perature for 30-45 min, fluorescence was read on a Tecan
Safire2 microplate reader at 480 nm/520 nm. Inhibition of
neutrophil migration was calculated by fitting the percent inhibi-
tion relative to the maximum migration minus background to a
sigmoidal dose-response equation using Microsoft Excel.
Human BLT1 and BLT2 Calcium Mobilization. Experiments
were performed at Multispan Inc. (CA) on cloned FLAG-
tagged human BLT1 (Genbank accession number BC004545)
and cloned FLAG-tagged human BLT2 (Genbank accession
number NM_019839.1) receptors stably expressed in HEK293
cells that were seeded into 96-well plates at 25 000 cells/well the
day before the experiment. To determine the EC50 of LTB4 for
each receptor, cell culture media were removed from the plate
and replaced with 100 μL of Hanks buffer. Then an amount of
100 μL of FLIPR Calcium 4 assay kit (Molecular Devices) was
added to the wells and incubated at 37 ꢀC at 5% CO2 for 60 min.
LTB4 was added (1 ꢀ 10-12 to 1 ꢀ 10-5 M final assay concen-
tration) to the compound plate and incubated for 1 h. The cell
plate was then transferred to FlexStation (Molecular Devices),
and within 90 s, LTB4 was injected automatically into the wells
and the calcium response measured. The EC50 of LTB4 was
determined using GraphPad Prism software (GraphPad Soft-
ware, CA) and used as the agonist concentration in the antago-
nist experiments. In these experiments, the compound plate was
incubated for 50 min and then the LTB4 antagonist analogue 6
added (1 ꢀ 10-8 to 1 ꢀ 10-5 final assay concentration) and
preincubated for 10 min. LTB4 was then injected into the wells
and the calcium response measured. Inhibition of LTB4-evoked
calcium mobilization was calculated by fitting the percentage
inhibition relative to the maximum response to a sigmoidal
dose-response equation using GraphPad Prism software. The
IC50 values of LTB4 antagonist analogues at BLT1 and BLT2
receptors were calculated and statistically compared using
Student’s unpaired t test using the same software.
b. Preparation of 4-{3-[6-(3-5-Benzo[1,3]dioxolyl-5-thiophen-
3-ylphenoxy)hexyl]-2-(2-ethoxycarbonylethyl)phenoxy}butyric
Acid Ethyl Ester. The title compound was prepared in 98% yield
from 3-5-benzo[1,3]dioxolyl-5-thiophen-3-ylphenol (5.05 g, 17.04
mmol), 4-[3-(6-bromo-hexyl)-2-(2-ethoxycarbonylethyl)phenoxy]-
butyric acid ethyl ester (8.44 g, 17.89 mmol), and potassium
carbonate (4.71 g, 34.08 mmol) using the general procedure A
described for compound 11. 1H NMR (300 MHz, CDCl3) δ
7.46-7.51 (m, 1H), 7.36-7.44 (m, 2H), 7.31 (d, J = 1.21 Hz,
1H), 7.04-7.13 (m, 4H), 6.97 (s, 1H), 6.89 (d, J = 8.75 Hz, 1H),
6.77 (d, J = 7.55 Hz, 1H), 6.68 (d, J = 7.85 Hz, 1H), 6.01 (s, 2H),
4.09-4.20 (m, 4H), 4.05 (t, J = 6.19 Hz, 2H), 3.99 (t, J = 6.19 Hz,
2H), 2.91-3.04 (m, 2H), 2.58-2.69 (m, 2H), 2.43-2.58 (m, 4H),
2.13 (dt, J = 6.75, 13.36 Hz, 2H), 1.75-1.91 (m, 2H), 1.39-1.68
(m, 6H), 1.20-1.30 (m, 6H). ES(þ)-HRMS m/e calcd for
C40H46O8S (M þ Na)þ 709.2805, found 709.2808.
c. Preparation of 4-{3-[6-(3-5-Benzo[1,3]dioxolyl-5-thiophen-
3-yl-phenoxy)hexyl]-2-(2-carboxyethyl)phenoxy}butyric Acid (33).
The title compound was prepared in 95% yield from 4-{3-[6-(3-5-
benzo[1,3]dioxolyl-5-thiophen-3-ylphenoxy)hexyl]-2-(2-ethoxycar-
bonylethyl)phenoxy}butyric acid ethyl ester (21.34 g, 31.06 mmol)
and 1.0 N aqueous NaOH (180 mL) using the general procedure
C described for compound 11. Mp =114-116 ꢀC. 1H NMR
(300 MHz, DMSO-d6) δ 12.14 (br s, 2H), 8.01 (dd, J=1.51, 2.95
Hz, 1H), 7.67 (dd, J = 1.51, 5.13 Hz, 1H), 7.63 (dd, J = 2.95, 5.13
Hz, 1H), 7.48 (t, J = 1.36 Hz, 1H), 7.37 (d, J = 1.69 Hz, 1H), 7.25
(dd, J = 1.69, 8.08 Hz, 1H), 7.20 (t, J = 2.00 Hz, 1H), 7.02-7.10
(m, 2H), 7.00 (d, J = 8.08 Hz, 1H), 6.75 (dd, J = 2.87, 8.00 Hz,
2H), 6.07 (s, 2H), 4.09 (t, J = 6.19 Hz, 2H), 3.94 (t, J = 6.34 Hz,
2H), 2.69-2.92 (m, 2H), 2.55-2.65 (m, 2H), 2.28-2.44 (m, 4H),
1.87-2.02 (m, 2H), 1.67-1.82 (m, 2H), 1.36-1.61 (m, 6H). ES(þ)-
HRMS m/e calcd for C36H38O8S (M þ Na)þ 653.2179, found
653.2183. Elemental analysis (C36H38O8S): C = 68.49 (calcd,
68.55), H = 5.93 (6.07), S = 5.15 (5.08).
In Vitro Cellular Assays. HL-60 Calcium Mobilization. Hu-
man leukemia HL-60 cells endogenously expressing BLT1 and
BLT2 receptors were cultured in RPMI-1640 medium supple-
mented with 20% fetal bovine serum, 2 mM glutamine, 100 U/mL
penicillin, and 100 μg/mL streptomycin. Prior to the experiment,
cells were counted using ViaCount reagent, centrifuged, and
resuspended at 2.0 ꢀ 105 cells/ml (HL-60). Cells were plated in
growth media in 384-well plates containing 1 μM retinoic acid
(Sigma Aldrich, St. Louis, MO). On the day of the experiment,
media were removed and replaced with loading buffer (Calcium 3
assay kit, Molecular Devices, Sunnyvale, CA) which was prepared
by dissolving the contents of one vial (Express kit) into 500 mL of
Hank’s balanced salt solution (HBSS) containing 20 mM HEPES
and 5 mM probenecid and mixed with an equal volume of
replacement buffer (HBSS containing 20 mM HEPES, 0.05%
BSA, and 5 mM probenecid) and placed in a 37 ꢀC/5% CO2
incubator for an hour.
Following incubation, an amount 5 μL of the LTB4 antago-
nist analogue in question (5 ꢀ 10-11 to 3 ꢀ 10-6 M final assay
concentration) or vehicle was transferred to the cell plates using
an automated liquid handling system (fluorometric imaging
plate reader, Molecular Devices) and incubated for 30 min
(HL-60) at room temperature. During the assay, fluorescence
readings were taken every 1.5 s. Five readings were taken to
establish a stable baseline, and then 0.5 μM LTB4 was added.
The fluorescence was continuously monitored before, during,
and after sample addition for a total elapsed time of 100 s.
Responses (increase in peak fluorescence) following agonist
addition were determined. The initial fluorescence reading from
each well, prior to ligand stimulation, was used as a zero baseline
value for the data from that well. The responses are expressed as
percent inhibition of the neutral control (wells that received
buffer plus DMSO but no test compound).
Metabolite Characterization of Human, Mouse, Rat, Dog, and
Monkey Cryopreserved Hepatocytes. Incubations were carried
out with 1.5 million cells/mL hepatocytes in Liebovitz media
containing 1% fetal bovine serum (final concentrations). Sub-
strate concentration during incubation was 10 μM, and total
incubation volume was 500 μL. Incubations were carried out for
3 h in a 37 ꢀC water bath in glass flat-bottom tubes gently capped
while shaking at 40 strokes/min. Incubations were stopped with
equal volume methanol, samples were spun at 2000g, and
supernatants were collected for analysis. Cell-free incubations

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3515
were performed in the same manner except an aliquot of the
hepatocytes incubation media was used instead of the cells.
Human hepatocytes (male and female, N = 10, product lot
no. SX008055/CD1) were obtained from In Vitro. Mouse
hepatocytes (male, CD1, product lot no. Mc221) were obtained
from CellzDirect. Rat hepatocytes (male, Sprague-Dawley,
product lot no. R1000/0510160) were obtained from Xeno Tech
LLC. Monkey hepatocytes (male, cynomolgus, product lot no.
P2000/XHTcyno112002) were obtained from Xeno Tech LLC.
Dog hepatocytes (male, beagle, product lot no. Db139) were
obtained from CellzDirect.
In Vivo Assays. LTB4-Evoked Pulmonary Inflammation in
Guinea Pigs. Experiments were performed on male Hartley
guinea pigs (250-350 g; Charles River, MA). Animals were
dosed with vehicle (2% Klucel LF, 0.1% Tween-80 in water) or
10 mg/kg LTB4 antagonist analogue orally (po). One hour
postdose, animals were placed in a clear plastic chamber and
challenged with an aerosol solution of 10 μg/mL LTB4 for
20 min. Two hours post-LTB4 challenge, bronchoalveolar
lavage (BAL) was performed. Animals were anesthetized
(sodium pentobarbital, 40-60 mg/kg, ip), the trachea was
cannulated with a 15 gauge tubing adapter, and the lungs were
lavaged with 3 ꢀ 5 mL of sterile HBSS. The samples were
centrifuged at 200g for 10 min at 25 ꢀC, and red blood cells were
lysed from the resulting pellet with distilled water (1 mL for 30 s)
before restoring osmolarity with the addition of 10 mL of HBSS.
Samples were centrifuged a second time (200g, 10 min, 25 ꢀC),
and the resulting pellet was resuspended in 1 mL of HBSS. Total
cell number was determined using a Beckman-Coulter Z-1
particle counter. For differential cell counts, an aliquot of the
cell suspension was centrifuged in a Cytospin (5 min, 1300 rpm;
Shandon Southern Instruments, PA) and the slides were fixed
and stained with a modified Wright’s stain (Leukostat, Fisher
Scientific, PA). Standard morphological criteria were used to
classify at least 300 cells under light microscopy. Statistics were
performed using two-way ANOVA followed by Bonferroni
post-test.
Allergen-Evoked Pulmonary Inflammation in Atopic Nonhu-
man Primates. Experiments were performed on male cynomol-
gus monkeys (Macaca fascicularis, 4-10 kg; Charles River,
MA) exhibiting natural hypersensitivity to Ascaris suum anti-
gen. Baseline BAL was performed on all animals 24 h prior to
allergen challenge (see below for procedure; alternate sides of
lungs were used for pre- and post-BAL procedures). Animals
were dosed with vehicle (15% Labrasol in polyethylene glycol)
or 10 mg/kg LTB4 antagonist analogue po. One hour postdose,
animals were anesthetized with ketamine/xylazine (10 and
1 mg/kg, respectively, im) and challenged with an aerosol
of Ascaris suum antigen for 90 s via an endotracheal tube
(4-4.5 mm i.d.), at a concentration previously demonstrated
to evoke at least a 100% increase in lung resistance. Four hours
after allergen exposure, animals were reanesthetized and BAL
was performed. A pediatric bronchoscope was inserted transo-
rally into the trachea. The left or right diaphragmatic lobe was
lavaged twice with 5 mL of sterile PBS, which was placed on ice
immediately after collection and kept cold throughout proces-
sing. To determine leukocyte cell differentials, slides were pre-
pared by centrifuging 100 μL of the sample at 950 rpm for 2 min
at room temperature (Cytospin-3; Shandon-Lipshaw, PA). The
slides were stained using a LeukoStat stain set (Fisher Scientific,
PA), and 300 cells were counted. Total leukocyte cell count was
performed manually using a standard hemocytometer. Statistics
were performed using two-way ANOVA followed by Bonferroni
post-test.
each animal at least twice daily. Body weights were recorded for
each animal at least once prior to initiation of treatment and
on days 1, 8, and 14. Food consumption was recorded once
weekly. Blood samples were collected from toxicity animals for
evaluation of hematological and clinical chemistry parameters
on day 5 and at scheduled necropsy. At the end of the 14-day
study period, animals were euthanized by induction with iso-
flurane/O2 anesthesia followed by exsanguination. Organs and
tissues were removed and fixed in 10% neutral buffered for-
malin, embedded in paraffin, sectioned, mounted on glass slides,
and stained with hematoxylin and eosin (H & E). All data
were statistically analyzed using Bartlett’s nonparametric or
Bartlett’s parametric test as appropriate. Blood samples were
collected from rats under isoflurane/O2 anesthesia, from the
retro-orbital sinus. The plasma was separated after cold cen-
trifugation and stored frozen in polypropylene tubes in a -70 ꢀC
freezer until analysis.
Assessment of in Vitro Effect on hERG Channels. Compounds
were characterized for their effect on hERG current (IKr)
expressed in Chinese hamster ovary (CHO) cells by a non-
GLP in vitro assay of current inhibition using a cell-based
mammalian expression system. CHO cells were stably trans-
fected with hERG cDNA and selected by G418 resistance.
Selection is maintained by including G418 in the culture media.
The culture media consist of Ex-cell 301(JRH-14331), 10% FBS
(Sigma-Aldrich, catalog no. 8061799), and 0.25 mg/mL G418
(Sigma-Aldrich, catalog no. 8061799). Cells are cultured in 1 L
of polypropylene shaker flasks as a suspension with maximum
volume of 100 mL at 35-39 ꢀC. Cells are grown with 5% CO2
applied with a gas flow rate of 10-15 mL/min. Cells are cultured
in flasks on a shaker table at 85-100 rpm at a density less than
1.5 ꢀ 106 cells/mL. Compounds were were examined at three
concentrations (0.3, 3, and 30 μM) for the ability to reduce
current amplitude from the hERG potassium channel expressed
in CHO cells by automated electrophysiology using the PatchX-
press 7000A, with solubility as the limiting factor. If inhibition
of current were not represented in these concentrations, further
concentrations were selected in order to generate the percent
inhibition at low and high concentrations as well as an IC50 and
IC20 value. If hERG current inhibition was less than 50% at
the highest concentration tested, the IC50 will be reported as an
estimate. Compounds were initially dissolved in DMSO to
15 mM and stored at 4 ꢀC before further dilution in DMSO.
All testing solutions were made in the recording solution that
was composed of NaCl (150 mM), KCl (4 mM), MgCl2 (1 mM),
CaCl2 (1.2 mM), and HEPES (10 mM). The final DMSO
concentration was e0.3% DMSO.
Acknowledgment. Part of this chemistry was conducted
in a collaborative effort between Roche and ChemOvation.
Dr. Noal Cohen is acknowledged for expert consultation.
Dr. Liang Guo is gratefully acknowledged for his efforts
in characterizing the hERG binding potential of several
compounds.
Supporting Information Available: Experimental details and
analytical data for compounds 12-18, 26-30, and 34-40. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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