(E)-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2- pyridinyl]-2-propenoic acid: A high-affinity leukotriene B4 receptor antagonist with oral antiinflammatory activity

Article abstract of DOI:10.1021/jm960248s

An extensive structure-activity study based around the high-affinity leukotriene B₄ (LTB₄) receptor antagonist SB 201146 (1) led to the identification of (E)-3-[6-[[(2,6-dichlorophenyl)thio]methyl]-3-(2- phenylethoxy)-2-pyridinyl]-2-

Full text of DOI:10.1021/jm960248s

J . Med. Chem. 1996, 39, 3837-3841
3837
Notes
(E)-3-[6-[[(2,6-Dich lor op h en yl)th io]m eth yl]-3-(2-p h en yleth oxy)-2-p yr id in yl]-2-
p r op en oic Acid : A High -Affin ity Leu k otr ien e B4 Recep tor An ta gon ist w ith Or a l
An tiin fla m m a tor y Activity
Robert A. Daines,*,† Pamela A. Chambers,† J ames J . Foley,‡ Don E. Griswold,‡ William D. Kingsbury,†
Lenox D. Martin,‡ Dulcie B. Schmidt,‡ Kelvin K. C. Sham,† and Henry M. Sarau‡
Departments of Medicinal Chemistry and Pharmacology, SmithKline Beecham Pharmaceuticals, P.O. Box 1539,
King of Prussia, Pennsylvania 19406-0939
Received March 28, 1996X
An extensive structure-activity study based around the high-affinity leukotriene B4 (LTB4)
receptor antagonist SB 201146 (1) led to the identification of (E)-3-[6-[[(2,6-dichlorophenyl)-
thio]methyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic acid (3). This compound displays high
affinity for the human neutrophil LTB4 receptor (Ki ) 0.78 nM), blocks LTB4-induced Ca2+
mobilization with an IC50 of 6.6 ( 1.5 nM, and demonstrates potent oral and topical
antiinflammatory activity in a murine model of dermal inflammation.
Leukotriene B4 (LTB4), a product of 5-lipoxygenase-
catalyzed oxygenation of arachidonic acid (AA), has been
postulated to be a mediator of a variety of inflammatory
diseases.1 Elevated levels of LTB4 found in psoriatic
lesional skin,2 colonic mucosa associated with inflam-
matory bowel disease,3 synovial fluid from patients with
active rheumatoid arthritis,4 and gouty effusions5 sup-
port the involvement of this mediator in human inflam-
matory diseases. Known pathophysiological responses
of LTB4 include potent neutrophil chemotactic activity,
promotion of adherence of polymorphonuclear leuko-
cytes (PMNs) to the vascular endothelium, stimulation
of the release of lysosomal enzymes and superoxide
radicals by PMNs, and an increase in vascular perme-
ability.1 The pharmacological effects of LTB4 are medi-
ated through interaction with specific cell surface
receptors on inflammatory cells. Therefore, the avail-
ability of potent and selective LTB4 receptor antagonists
should prove useful in elucidating the role of LTB4 in
human inflammatory diseases.
the present study was to identify high-affinity LTB4
receptor antagonists that demonstrate potent oral an-
tiinflammatory activity. One compound that fully meets
these criteria has been identified, i.e., (E)-3-[6-[[(2,6-
dichlorophenyl)thio]methyl]-3-(2-phenylethoxy)-2-pyrid-
inyl]-2-propenoic acid, SB 209247 (3).
Previously, we reported the identification of the novel
high-affinity LTB4 receptor antagonists SB 201146 (1,
Ki ) 4.7 nM)6 and SB 201993 (2, Ki ) 7.1 nM).7 In
murine models, these compounds were potent antiin-
flammatory agents when applied topically and demon-
strated oral LTB4 receptor antagonist activity. How-
ever, the compounds did not show significant oral
activity in a murine model of inflammation. The in vivo
test system selected to evaluate antiinflammatory activ-
ity was the AA-induced mouse ear inflammation model.8
In this model, an inflammatory response (neutrophil
infiltration) is initiated by the application of 1 mg of
arachidonic acid to the mouse ear. Antiinflammatory
activity of the test compounds was determined by
monitoring the neutrophil marker enzyme myeloper-
oxidase (MPO). Both SB 201146 (1) and SB 201993 (2)
were potent topical antiinflammatory agents in this
model but lacked significant oral activity. The goal of
Ch em istr y
Compounds 8, 9, 13, and 14 were synthesized accord-
ing to procedures previously described.7 The phenyl-
ethyl-containing compounds of the present study were
synthesized according to Scheme 1 (illustrated for
compound 3). Reaction of the pyridyl 2-carboxaldehyde6
4 with methyl (triphenylphosphoranylidene)acetate fol-
lowed by a Mitsunobu-type coupling with phenethyl
alcohol provided methylpyridine 5. Oxidation to the
pyridine N-oxide, trifluoroacetic anhydride-induced re-
arrangement, and reaction of the resulting primary
alcohol with SOCl2 provided chloride 6. Conversion of
chloride 6 to sulfide 7 was achieved using 2,6-dichlo-
robenzenethiol and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU).9 Hydrolysis of the methyl ester and acidification
provided 3 as a crystalline solid.10
† Department of Medicinal Chemistry.
‡ Department of Pharmacology.
X Abstract published in Advance ACS Abstracts, August 15, 1996.
S0022-2623(96)00248-8 CCC: $12.00 © 1996 American Chemical Society

3838 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Notes
Sch em e 1a
a
Reagents: (a) Ph3PCHCO2Me, toluene, 50 °C, 85%; (b) PhCH2CH2OH, DEAD, Ph3P, 53%; (c) MCPBA, CH2Cl2; (d) TFAA, DMF (96%,
two steps); (e) SOCl2, toluene, 99%; (f) 2,6-dichlorobenzenethiol, DBU, MeCN, 50 °C, 87%; (g) (i) 1.0 N LiOH, THF, MeOH, (ii) HCO2H,
60%.
Ta ble 1. Inhibition of [3H]LTB4 Binding to Human
Neutrophils
Ta ble 2. Effect of LTB4 Receptor Antagonists on Arachidonic
Acid-Induced Mouse Ear Inflammationa
oral administration
topical administration
inhibition of MPO, % ED50
,
inhibition of MPO, % ED50,
compd
(dose, mg/kg)
mg/kg
(dose, µg/ear)
µg/ear
1
2
8
9
3
0 (50)
0 (50)
26 (50)
54 (50)
70 (25)
75 (25)
-
55 (500)
69 (500)
49 (1000)
82 (1000)
83 (50)
350
390
ND
ND
8
-
ND
57.6
14.8
14.1
compd
X
R
n
binding Ki, nMa
1
-
-
-
4
4
2
2
2
2
1
3
4.7 ( 0.4
2.8 ( 0.6
1.5 ( 0.4
1.2
0.78 ( 0.16
1.3 ( 0.4
1.5 ( 0.3
12
12
ND
ND
8b
H
4-MeO
4-MeO
4-MeO
H
a
9
Cl
Cl
Cl
Cl
Cl
Cl
Cl
ND: not determined. ED50 values are the doses required to
inhibit 50% of the arachidonic acid-induced inflammation obtained
from dose-response curves (4-5 concentrations of antagonist).
10b
3
11b
12
13b
14b
4-Cl
4-F
4-F
binding assay. Replacement of the (4-methoxyphenyl)-
ethyl group with phenylethyl provided 3. Compound 3
demonstrated slightly improved receptor affinity over
both 9 and 10; however, a 4-fold increase in oral activity
was obtained. In addition to having an ED50 of 14.8 mg/
kg for inhibiting neutrophil influx in the murine model,
3 inhibited the fluid phase (edema)8 of the inflammatory
response with an ED50 of 18.7 mg/kg.
H
1.4
a
The Ki values are stated as the mean of at least three
determinations ( standard error. All other values are the mean
b
Ki of two concentration-response curves. Compound tested as a
sodium salt.
Resu lts a n d Discu ssion
Additional modifications within this phenylethyl se-
ries resulted in compounds with slightly reduced recep-
tor affinities (i.e., 11 and 12, Table 1). Compound 12
also demonstrated potent oral antiinflammatory activity
exhibiting equivalent potency to that of 3 (Table 2).
However, in duration of action and pharmacokinetic
studies, compound 3 demonstrated a superior profile to
that of 12 (data not shown). Further reduction in the
tail length resulted in a compound with 1 order of
magnitude weaker affinity for the neutrophil LTB4
receptor (e.g., compare 12 and 13, Table 1). In contrast,
the phenylpropyl analog 14 was nearly as active in the
binding assay as 3.
In addition to being a high-affinity antagonist, com-
pound 3 demonstrated a high degree of selectivity for
the LTB4 receptor. Compound 3 competes with [3H]-
fMLP and [3H]LTD4 in respective receptor binding
assays with IC50 values of 3250 and 16 800 nM and with
another 14 ligands with IC50 values greater than 10 000
nM. Functional activity at the cellular level was
demonstrated in human PMNs by blocking the LTB4-
induced Ca2+ mobilization and LTB4-induced degranu-
lation with IC50 values of 6.6 ( 1.5 and 53 ( 7 nM,
respectively.
The search for a new class of orally active LTB4
receptor antagonists started with the aniline sulfoxide
1. Through an extensive structure-activity study, it
was discovered that the requirement for both the aniline
head group and the sulfoxide could be eliminated by
reducing the length of the lipid chain. Thus, compound
8 (Table 1) containing the (4-methoxyphenyl)butyl tail
and the unsubstituted phenyl head group had ap-
proximately 2-fold higher affinity for the LTB4 receptor
than 1. Evaluation of 8 in the AA-induced mouse ear
inflammation model revealed that the compound pos-
sessed significant, albeit modest, oral activity (Table 2).
Additional studies focused on aryl head group substitu-
tion revealed that further improvement in affinity could
be attained by incorporating a 2,6-dichlorophenyl moiety
leading to compound 9 (Table 1). This increase in
affinity for the LTB4 receptor resulted in a correspond-
ing increase in the oral antiinflammatory activity of the
compound (Table 2).
Having identified high-affinity compounds possessing
Ki values of approximately 1 nM, we focused on further
improving the oral antiinflammatory activity. It was
postulated that a further reduction in the lipophilic
character of these compounds may enhance the oral
activity provided the high receptor affinity was retained.
To this end, the (4-methoxyphenyl)butyl tail was re-
placed with (4-methoxyphenyl)ethyl to provide 10 which
was determined to be equipotent with 9 in the receptor
Compound 3 was found to be a weak inhibitor of the
RBL-1 supernatant 5-lipoxygenase, inhibiting the en-
zyme with an IC50 of 8200 nM. When evaluated as an
inhibitor in several other enzyme assays, including CoA

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3839
1H, pyridyl), 4.26 (t, J ) 6.2 Hz, 2H, CH2O), 3.82 (s, 3H, OMe),
3.17 (t, J ) 6.8 Hz, 2H, benzylic), 2.45 (s, 3H, CH3).
independent transacylase, human phospholipase A2 (14
and 85 kDa), and protein kinase C (rat brain homoge-
nate, rhPKC-R), 3 was inactive at concentrations up to
10 000 nM.
Crude N-oxide (9.7 g, 31 mmol) was dissolved in anhydrous
DMF (95 mL) under argon, cooled to 0 °C, and treated with
TFAA (42 mL, 300 mmol) dropwise over 30 min. The cooling
bath was removed, and the reaction mixture was stirred for
18 h at room temperature. The solution was concentrated,
and the reaction was quenched slowly by addition to a mixture
of Na2CO3 (48 g) in H2O (100 mL) at 20-25 °C with rapid
stirring. After 1 h, the solution was poured into a mixture of
H2O (400 mL) and EtOAc (200 mL). The layers were sepa-
rated, and the aqueous phase was extracted with EtOAc (three
times). The combined organic extracts were washed with
brine, dried over MgSO4, filtered, and concentrated. The
alcohol was purified by column chromatography (silica gel, 33%
EtOAc in hexane) to provide 9.3 g (96%, two steps) of alcohol
as a waxy solid: 1H NMR (400 MHz, CDCl3) δ 8.05 (d, J )
15.9 Hz, 1H, vinyl), 7.25-7.33 (m, 5H, aryl), 7.17 (d, J ) 1.7
Hz, 2H, pyridyl), 6.99 (d, J ) 15.9 Hz, 1H, vinyl), 4.66 (s, 2H,
CH2O), 4.20 (t, J ) 6.9 Hz, 2H, CH2O), 3.82 (s, 3H, OMe), 3.74
(br s, 1H, OH), 3.15 (t, J ) 6.9 Hz, 2H, benzylic); MS (ES+)
314.0 (M + H).
The alcohol prepared above (9.0 g, 28.7 mmol) was dissolved
in anhydrous toluene (150 mL) under argon and cooled (0 °C).
Thionyl chloride (21 mL, 287 mmol) in toluene (20 mL) was
added over 15 min. The cooling bath was removed, and
stirring was continued for 1 h. The reaction solution was
concentrated to remove the excess SOCl2 and toluene; the
resulting tan solid was dried under vacuum to give hydrochlo-
ride 6 which was used without further purification.
(E)-3-[6-[[(2,6-Dich lor op h en yl)t h io]m et h yl]-3-(2-p h e-
n yleth oxy)-2-p yr id in yl]-2-p r op en oic Acid Meth yl Ester
(7). Hydrochloride 6 (∼28 mmol) was dissolved in anhydrous
MeCN (80 mL) and treated with 2,6-dichlorobenzenethiol (5.7
g, 31 mmol) and DBU (14.9 mL, 100 mmol). The reaction
mixture was heated at 50 °C for 1 h under argon and then
cooled to room temperature and concentrated in vacuo. The
residue was partitioned between CH2Cl2 and brine. The
organic layer was separated, washed with brine, dried over
MgSO4, filtered, and concentrated. The product was purified
by column chromatography (silica gel,20% EtOAc in hexane)
to give 11.7 g (87%) of 7 as an off-white solid: mp 53-56 °C;
1H NMR (400 MHz, CDCl3) δ 7.94 (d, J ) 15.6 Hz, 1H, vinyl),
7.26-7.34 (m, 7H, aryl), 7.11-7.15 (m, 3H, aryl, pyridyl), 7.06
(d, J ) 8.6 Hz, 1H, pyridyl), 6.70 (d, J ) 15.6 Hz, 1H, vinyl),
4.19 (t, J ) 7.0 Hz, 2H, CH2O), 4.14 (s, 2H, CH2S), 3.82 (s,
3H, OMe), 3.14 (t, J ) 7.0 Hz, 2H, benzylic); MS (ES+) 474
(M + H). Anal. (C24H21Cl2NO3S) C, H, N.
(E)-3-[6-[[(2,6-Dich lor op h en yl)t h io]m et h yl]-3-(2-p h e-
n ylet h oxy)-2-p yr id in yl]-2-p r op en oic Acid (3). Methyl
ester 7 (6.9 g, 14.5 mmol) was dissolved in THF (44 mL) and
methanol (22 mL) under argon and treated with 1.0 N LiOH
(22 mL, 22 mmol). The reaction mixture was stirred for 18 h
at room temperature. The solution was concentrated, and the
residue was stirred with distilled H2O (80 mL) for 5 h; the
resulting lithium salt was filtered off and washed with H2O.
The salt was suspended in H2O (100 mL) and treated with
HCO2H to give a pH of 3.5. The free acid was extracted into
CH2Cl2, and the combined organic extracts were washed with
brine, dried over MgSO4, filtered, and concentrated. Recrys-
tallization from EtOAc provided 4.1 g (60%) of 3 as a white
solid: mp 124-125 °C (EtOAc); 1H NMR (400 MHz, CDCl3) δ
8.03 (d, J ) 15.7 Hz, 1H, vinyl), 7.28-7.36 (m, 7H, aryl), 7.18
(d, J ) 8.6 Hz, 1H, pyridyl), 7.13-7.17 (m, 1H, aryl), 7.08 (d,
J ) 8.6 Hz, 1H, pyridyl), 6.62 (d, J ) 15.7 Hz, 1H, vinyl), 4.19
(t, J ) 7.0 Hz, 2H, CH2O), 4.15 (s, 2H, CH2S), 3.14 (t, J ) 7.0
Hz, 2H, benzylic); MS (ES+) 460.0 (M + H); MS (ES-) 458.0
(M - H). Anal. (C23H19Cl2NO3S) C, H, N, S.
(E)-3-[3-[4-(4-Meth oxyp h en yl)bu toxy]-6-[(p h en ylth io)-
m eth yl]-2-p yr id in yl]-2-p r op en oic a cid sod iu m sa lt (8):
colorless amorphous solid; 1H NMR (250 MHz, MeOH-d4) δ
7.72 (d, J ) 15.7 Hz, 1H vinyl), 7.06 (d, J ) 8.6 Hz, 2H, aryl),
7.05-7.31 (m, 7H, aryl, pyridyl), 6.99 (d, J ) 15.7 Hz, 1H,
vinyl), 6.76 (d, J ) 8.6 Hz, 2H, aryl), 4.16 (s, 2H, CH2S), 3.97
(t, J ) 6.7 Hz, 2H, CH2O), 3.70 (s, 3H, OMe), 2.58 (t, J ) 6.7
Hz, 2H, benzylic), 1.78 (m, 4H, aliphatic); MS (ES+) 450.2 (M
In summary, (E)-3-[6-[[(2,6-dichlorophenyl)thio]m-
ethyl]-3-(2-phenylethoxy)-2-pyridinyl]-2-propenoic acid
(3) is a selective high-affinity antagonist of the LTB4
receptor. It selectively blocks the actions of LTB4 on
human PMNs in vitro, and in vivo it demonstrates
potent topical and oral antiinflammatory activity in a
murine model of dermal inflammation. Furthermore,
compound 3 represents a significant advantage over the
earlier aniline sulfoxide 1 in that it is structurally less
complex and lacks chiral centers. SB 209247 (3) has
been selected for further evaluation as a potential orally
active antiinflammatory agent.
Exp er im en ta l Section
Melting points were determined using a Thomas-Hoover
capillary melting point apparatus and are uncorrected. Nuclear
magnetic resonance (1H NMR) spectra were recorded on either
a Brucker AM-250 or AM-400 instrument with the solvents
indicated. All 1H NMR chemical shifts are reported in δ
relative to tetramethylsilane (TMS, δ 0.00) as the internal
standard. Elemental analyses were performed by the Analyti-
cal and Physical Chemistry Department of SmithKline Bee-
cham. Where analyses are indicated by symbols of the
elements, results obtained were within (0.40%. Mass spectra
were determined by the Physical and Structural Chemistry
Department of SmithKline Beecham.
(E)-3-[6-Meth yl-3-(2-p h en yleth oxy)-2-p yr id in yl]-2-p r o-
p en oic Acid Meth yl Ester (5). Aldehyde 47 (20 g, 146 mmol)
in anhydrous toluene (250 mL) was treated with methyl
(triphenylphosphoranylidene)acetate (48.8 g, 146 mmol) por-
tionwise, and the resulting mixture was heated for 0.5 h at
50 °C. The mixture was cooled to 0 °C and filtered, and the
cake was washed with toluene (40 mL) and dried to give 24 g
(85%) of (E)-3-(3-hydroxy-6-methyl-2-pyridinyl)-2-propenoic
acid methyl ester as a gold solid. This material was used
directly in the following step.
Hydroxypyridine prepared above (11.3 g, 58.5 mmol), triph-
enylphosphine (15.3 g, 58.5 mmol), and phenethyl alcohol (7.14
g, 58.5 mmol) were dissolved in anhydrous THF (350 mL)
under an argon atmosphere. The resulting solution was cooled
to 0 °C, and diethyl azodicarboxylate (10.2 g, 58.5 mmol) was
added dropwise over 15 min. The reaction mixture was stirred
at 0 °C for 15 min and then allowed to warm to room
temperature over 1 h. The reaction mixture was concentrated
and the residue triturated with hexane-EtOAc (3:1). The
resulting suspension was cooled (0 °C), and the solids were
filtered; concentration of the filtrate provided a dark amber
oil. Purification by column chromatography (silica gel, 20%
EtOAc in hexane) gave 9.2 g (53%) of 5 as a colorless oil that
gradually solidified: 1H NMR (250 MHz, CDCl3) δ 8.06 (d, J
) 15.2 Hz, 1H, vinyl), 7.25-7.34 (m, 5H, aryl), 7.07 (d, J )
1.7 Hz, 2H, pyridyl), 7.00 (d, J ) 15.2 Hz, 1H, vinyl), 4.18 (t,
J ) 7.0 Hz, 2H, CH2O), 3.82 (s, 3H, OMe), 3.14 (t, J ) 7.0 Hz,
2H, benzylic), 2.48 (s, 3H, CH3); MS (ES+) 297.2 (M + H).
(E)-3-[6-(Ch lor om eth yl)-3-(2-ph en yleth oxy)-2-pyr idin yl]-
2-p r op en oic Acid Meth yl Ester Hyd r och lor id e (6). Py-
ridyl ester 5 (9.2 g, 31.0 mmol) was dissolved in CH2Cl2 (100
mL) and cooled to 0 °C. MCPBA (11.7 g, 33.7 mmol; 50-60%)
was added portionwise over 15 min; the cooling bath was
removed, and stirring was continued at room temperature for
18 h. The reaction mixture was poured into H2O (100 mL),
and concentrated NH4OH was added to bring pH to 8.8. The
organic layer was separated, and the aqueous layer was
extracted with CH2Cl2. The combined organic extracts were
washed with brine, dried over MgSO4, filtered, and concen-
trated. The crude N-oxide (10.4 g) was obtained as a yellow
semisolid: 1H NMR (250 MHz, CDCl3) δ 8.2 (d, J ) 16.2 Hz,
1H, vinyl), 7.53 (d, J ) 16.2 Hz, 1H, vinyl), 7.26-7.34 (m, 5H,
aryl), 7.11 (d, J ) 8.8 Hz, 1H, pyridyl), 6.77 (d, J ) 8.8 Hz,

3840 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19
Notes
+ H, free acid); MS (ES-) 448.0 (M - H, free acid). Anal.
(C26H26NO4SNa‚0.75H2O) C, H, N.
nM [3H]LTB4 in a total volume of 500 µL. Total and nonspe-
cific binding of [3H]LTB4 were determined in the absence and
presence of 1 µM unlabeled LTB4, respectively. For radioli-
gand competition experiments, increasing concentrations of
LTB4 (0.05-10 nM) or test compound (0.1 nM-10 µM) was
included. Unbound radioligand and competing compounds
were separated from cell-bound ligand by vacuum filtration
through Whatman GF/C filters. Cell-bound radioactivity was
determined by liquid scintillation spectrometry. The percent
inhibition of specific [3H]LTB4 binding was determined for each
concentration, and the IC50 is defined as the concentration of
test compound required to inhibit 50% of the specific [3H]LTB4
binding.
(E)-3-[6-[[(2,6-Dich lor oph en yl)th io]m eth yl]-3-[4-(4-m eth -
oxyph en yl)bu toxy]-2-pyr idin yl]-2-pr open oic acid (9): col-
orless solid; mp 129-130 °C; 1H NMR (400 MHz, CDCl3) δ
8.03 (d, J ) 15.6 Hz, 1H, vinyl), 7.33 (d, J ) 8.1 Hz, 1H, aryl),
7.12 (d, J ) 8.6 Hz, 2H, aryl), 7.07-7.20 (m, 4H, aryl, pyridyl),
6.84 (d, J ) 8.6 Hz, 2H, aryl), 6.62 (d, J ) 15.6 Hz, 1H, vinyl),
4.15 (s, 2H, CH2S), 3.98 (t, J ) 6.0 Hz, 2H, CH2O), 3.78 (s,
3H, OMe), 2.64 (t, J ) 7.2 Hz, 2H, benzylic), 1.78-1.85 (m,
4H, aliphatic); MS (ES+) 518.0 (M + H). Anal. (C26H25Cl2-
NO4S‚2.75H2O) C, H, N.
(E)-3-[6-[[(2,6-Dich lor oph en yl)th io]m eth yl]-3-[2-(4-m eth -
oxyp h en yl)et h oxy]-2-p yr id in yl]-2-p r op en oic a cid so-
d iu m sa lt (10): colorless amorphous solid; 1H NMR (400 MHz,
MeOH-d4) δ 7.70 (d, J ) 15.6 Hz, 1H, vinyl), 7.36 (d, J ) 8.0
Hz, 2H, aryl), 7.22 (d, J ) 7.4 Hz, 2H, pyridyl), 7.15-7.20 (m,
2H, aryl), 7.00 (d, J ) 8.5 Hz, 1H, aryl), 6.84 (d, J ) 8.0 Hz,
2H, aryl), 6.83 (d, J ) 15.6 Hz, 1H, vinyl), 4.14 (t, J ) 6.7 Hz,
2H, CH2O), 4.12 (s, 2H, CH2S), 3.76 (s, 3H, OMe), 3.05 (t, J )
6.7 Hz, 2H, benzylic); MS (ES+) 490.0 (M + H, free acid); MS
(ES-) 487.6 (M - H, free acid). Anal. (C24H20Cl2NO4-
SNa‚2.5H2O) C, H, N.
(E)-3-[6-[[(2,6-Dich lor oph en yl)th io]m eth yl]-3-[2-(4-ch lo-
r op h en yl)eth oxy]-2-p yr id in yl]-2-p r op en oic a cid sod iu m
sa lt (11): colorless amorphous solid; 1H NMR (400 MHz,
MeOH-d4) δ 7.67 (d, J ) 15.9 Hz, 1H, vinyl), 7.36 (d, J ) 7.9
Hz, 2H, aryl), 7.30 (AB quartet, J ) 8.4 Hz, 2H, pyridyl), 7.23
(d, J ) 8.4 Hz, 1H, aryl), 7.17 (d, J ) 8.6 Hz, 2H, aryl), 7.01
(d, J ) 8.6 Hz, 2H, aryl), 6.81 (d, J ) 15.9 Hz, 1H, vinyl), 4.18
(t, J ) 6.7 Hz, 2H, CH2O), 4.12 (s, 2H, CH2S), 3.11 (t, J ) 6.7
Hz, 2H, benzylic); MS (ES+) 493.8 (M + H, free acid); MS
(ES-) 491.8 (M - H, free acid). Anal. (C23H17Cl3NO3-
SNa‚2.95H2O) C, H, N.
Concentration-response curves (5-8 concentrations) for all
compounds were run in duplicate and tested in at least two
assays. Values presented are the mean Ki values which were
determined from the mean IC50 as described by Cheng and
Prusoff13 using the following equation:
IC50
Ki )
(1 + [L]/Kd)
where [L] is the concentration of added ligand and the Kd, as
determined from saturation studies, is 0.15 nM. Standard
errors are presented for all compounds where three or more
concentration-response curves were run and indicate the
precision of the assay method made.
LTB4-In d u ced Ca lciu m Mobiliza tion . The functional
assay used to assess antagonist activity of compound 3 was
LTB4-induced calcium mobilization in intact human PMNs.14
Cells were washed with 50 mM Tris, pH 7.4, containing 1 mM
EDTA. The [Ca2+]i was estimated with the calcium fluorescent
probe fura 2.15 Isolated PMNs were suspended in Krebs
Ringer Hensilet at 2 × 106 cells/mL containing 0.1% BSA, 1.1
mM MgCl2, and 5 mM HEPES, pH 7.4 (buffer A). The
diacetoxymethoxy ester of fura 2 (fura 2/AM) was added at a
concentration of 2 µM and incubated for 45 min at 37 °C. Cells
were centrifuged at 225g for 5 min, resuspended at 2 × 106
cells/mL in buffer A, and incubated an additional 20 min to
allow complete hydrolysis of the entrapped ester. Cells were
centrifuged as above and resuspended at 106 cells/mL in buffer
A containing 1 mM CaCl2. The cells were maintained at room
temperature until used in the fluorescent assay which was
performed within 3 h.
The fluorescence of fura 2-containing cells was measured
with a fluorometer designed by the J ohnson Foundation
Biomedical Instrumentation Group. The fluorometer was
equipped with a temperature control and a magnetic stirrer
under the cuvette holder. Wavelengths were set at 340 nm
(10 nm bandwidth) for excitation and 510 nm (20 nm band-
width) for emission. All experiments were performed at 37
°C with constant stirring. For compound studies, fura 2-loaded
cells were centrifuged and resuspended in buffer A containing
1 mM CaCl2 minus BSA at 106 cells/mL. For agonist activity,
a 2 mL aliquot of PMNs was added to a cuvette and warmed
in a water bath to 37 °C. The 1 cm2 cuvette was transferred
to the fluorometer, and fluorescence was recorded for 15 s to
ensure a stable base line before addition of compound. Fluo-
rescence was recorded continuously for up to 2 min after
addition of compound 3 to monitor for the presence of any
agonist activity; none was observed.
(E)-3-[6-[[(2,6-Dich lor op h en yl)th io]m eth yl]-3-[2-(4-flu -
or op h en yl)eth oxy]-2-p yr id in yl]-2-p r op en oic a cid (12):
1
colorless solid; mp 146-147 °C (EtOAc); H NMR (250 MHz,
MeOH-d4) δ 7.67 (d, J ) 15.8 Hz, 1H, vinyl), 7.35 (m, 4H, aryl),
7.25 (m, 2H, aryl), 7.05 (m, 3H, aryl), 6.82 (d, J ) 15.8 Hz,
1H, vinyl), 4.18 (t, J ) 6.7 Hz, 2H, CH2), 4.12 (s, 2H, pyr-CH2-
S), 3.10 (t, J ) 6.7 Hz, 2H, CH2); MS (ES+) 478 (M + H); MS
(ES-) 476 (M - H). Anal. (C23H18Cl2FNO3S) C, H, N, S.
(E)-3-[6-[[(2,6-Dich lor op h en yl)th io]m eth yl]-3-[(4-flu o-
r op h en yl)m et h oxy]-2-p yr id in yl]-2-p r op en oic a cid so-
d iu m sa lt (13): colorless amorphous solid; 1H NMR (400 MHz,
DMSO-d6) δ 7.50 (m, 4H, aryl), 7.35 (m, 3H, aryl, vinyl), 7.23
(dd, J ) 8.9 Hz, 2H, aryl), 7.07 (d, J ) 8.4 Hz, 1H, aryl), 6.59
(d, J ) 15 Hz, 1H, vinyl), 5.13 (s, 2H, OCH2), 4.14 (s, 2H, pyr-
CH2-S); MS (ES+) 464 (M + H). Anal. (C22H15Cl2FNO3-
SNa‚2.25H2O) C, H, N.
(E)-3-[6-[[(2,6-Dich lor op h en yl)t h io]m et h yl]-3-(3-p h e-
n ylp r op oxy)-2-p yr id in yl]-2-p r op en oic a cid sod iu m sa lt
1
(14): colorless amorphous solid; H NMR (400 MHz, MeOH-
d4) δ 7.74 (d, J ) 15.6 Hz, 1H, vinyl), 7.36 (d, J ) 7.9 Hz, 2H,
aryl), 7.19-7.25 (m, 5H, aryl, pyridyl), 7.11-7.16 (m, 2H, aryl,
pyridyl), 6.99 (d, J ) 8.4 Hz, 1H, aryl), 6.86 (d, J ) 15.6 Hz,
1H, vinyl), 4.13 (s, 2H, CH2S), 3.97 (t, J ) 6.2 Hz, 2H, CH2O),
2.83 (t, J ) 7.5 Hz, 2H, benzylic), 2.12 (quintet, J ) 7.2, 6.6
Hz, 2H, aliphatic); MS (ES+) 474.0 (M + H,free acid); MS
(ES-) 472.0 (M - H, free acid). Anal. (C24H20Cl2NO3-
SNa‚2.25H2O) C, H, N.
Biologica l Eva lu a tion . [3H]LTB4 Bin d in g Assa ys. [3H]-
LTB4, with specific activity of 140-210 Ci/mmol, was obtained
from New England Nuclear (Boston, MA). Unlabeled LTB4
was synthesized by the Medicinal Chemistry Prep Group at
SmithKline Beecham.
Human peripheral blood from healthy aspirin-free donors
was phlebotomized into sterile heparinized syringes. PMNs
were isolated by the standard Ficoll-Hypaque centrifugation,
dextran 70 sedimentation, and hypotonic lysis procedure.11 Cell
preparations were >90% neutrophils and >95% viable.
Test compounds were evaluated for the ability to compete
with [3H]LTB4 for receptors on intact human PMNs utilizing
methods described previously.12 Equilibrium binding for
washed PMNs (106 cells) was performed at 0 °C for 20 min in
Hanks balanced salt solution with 0.1% ovalbumin and 0.2
For antagonist studies, varying concentrations of compound
3 or vehicle were added to the fura 2-loaded PMNs and
monitored for 1 min to ensure that there was no change in
base-line fluorescence followed by the addition of 1 nM LTB4.
The maximal [Ca2+]/fura 2 fluorescence was then determined
for each sample. The [Ca2+]i was calculated using the following
formula:
F - Fmin
[Ca2+]i ) 224 (nM)
Fmax - F
The percent of maximal LTB4 (1 nM)-induced [Ca2+]i was
determined for each concentration of compound, and the IC50
is defined as the concentration of test compound that inhibits

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 19 3841
(2) Thorsen, S.; Fogh, K.; Broby-J ohansen, U.; Sondergaard, J .
Leukotriene B4 in Atopic Dermatitis: Increased Skin Levels and
Alterred Sensitivity of Peripheral Blood T-Cells. Allergy 1990,
45, 457.
(3) Fretland, D. J .; Widomski, D. L.; Levin, S.; Gaginella, T. S.
Colonic Inflammation in the Rabbit Induced by Phorbol-12-
Myristate-13-Acetate. Inflammation 1990, 14, 143.
(4) Klickstein, L. B.; Shapleigh, C.; Goetzl, E. J . Lipoxygenation of
Arachidonic Acid as a Source of Polymorphonuclear Leukocyte
Chemotactic Factors in Synovial Fluid and Tissue in Rheumatoid
Arthritis and Spondyloarthritis. J . Clin. Invest. 1980, 66, 1166.
(5) Rae, S. A.; Davidson, E. M.; Smith, M. J . H. An Inflammatory
Mediator in Gout. Lancet 1982, 2, 1122.
(6) Daines, R. A.; Chambers, P. A.; Pendrak, I.; J akas, D. R.; Sarau,
H. M.; Foley, J . J .; Schmidt, D. B.; Kingsbury, W. D. Trisubsti-
tuted Pyridine Leukotriene B4 Receptor Antagonists. Synthesis
and Structure-Activity Relationships. J . Med. Chem. 1993, 36,
3321-3332.
(7) (a) Daines, R. A.; Chambers, P. A.; Pendrak, I.; J akas, D. R.;
Sarau, H. M.; Foley, J . J .; Schmidt, D. B.; Griswold, D. E.;
Martin, L. D.; Tzimas, M. N.; Kingsbury, W. D. (E)-3-[[[[6-(2-
Carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl]oxy]-2-pyridinyl]-
methyl]thio]methyl]benzoic Acid: A Novel High Affinity Leu-
kotriene B4 Receptor Antagonist. J . Med. Chem. 1993, 36, 2703-
2705. (b) Daines, R. A.; Chambers, P. A.; Eggleston, D. S.; Foley,
J . J .; Griswold, D. E.; Haltiwanger, R. C.; J akas, D. R.; Kings-
bury, W. D.; Martin, L. D.; Pendrak, I.; Schmidt, D. B.; Tzimas,
M. N.; Sarau, H. M. (E)-3-[[[[6-(2-Carboxyethenyl)-5-[[8-(4-
methoxyphenyl)octyl]oxy]-2-pyridinyl]methyl]thio]methyl]ben-
zoic Acid and Related Compounds: High Affinity Leukotriene
B4 Receptor Antagonists. J . Med. Chem. 1994, 37, 3327-3336.
(8) Griswold, D. E.; Webb, E.; Schwartz, L.; Hanna, N. Arachidonic
Acid-Induced Inflammation: Inhibition by Dual Inhibitor of
Arachidonic Acid Metabolism, SK&F 86002. Inflammation 1987,
11, 189-199.
50% of the maximal LTB4 response. Concentration-response
curves (5-7 concentrations) were run in four separate assays,
and results are reported as mean IC50 ( SEM.
LTB4-In d u ced Degr a n u la tion . Compound 3 was tested
in a second functional assay that requires higher concentra-
tions of the agonist, that is, LTB4-induced degranulation of
human PMNs.16 Degranulation was assessed using 106 washed
neutrophils, isolated as described above, in 1 mL of Earls
balanced salt solution with 20 mM HEPES and 0.1% ovalbu-
min. Cells were preincubated with 5 µg/mL cytochalasin B
for 10 min and compound 3 or vehicle for 5 min at 37 °C
followed by 100 nM LTB4 for 3 min. Incubation was termi-
nated by placing assay tubes on ice followed by centrifugation
(400g for 2 min). MPO activity in the supernatant fractions
was determined kinetically as described by Bradley et al.17 An
aliquot (50 µL) of the supernatant was incubated with 0.95
mL of assay reagent containing 0.167 mg/mL o-dianisidine
dihydrochloride, 0.0005% hydrogen peroxide, and 50 mM
potassium phosphate buffer (pH 6.0). Product formation was
linear for >2 min and measured spectrophotometrically every
15 s for 2 min at 460 nm. A unit of MPO activity is defined
as that degrading 1 µmol of peroxide/min at 25 °C. The percent
of LTB4-induced (100 nM) degranulation was determined for
each concentration of compound, and the IC50 is defined as
the concentration of test compound that inhibits 50% of the
100 nM LTB4 response. Four concentration-response curves
were run for compound 3 using 5-7 concentrations, and the
mean IC50 ( SEM is presented.
Ar a ch id on ic Acid -In d u ced Mou se Ea r In fla m m a tion
Mod el. For oral studies the test compounds were dissolved
in distilled water and administered directly into the stomach.
Balb/c mice, weighing 20 g, N ) 6-10 mice/group for vehicle
and typically N ) 5-6 mice/group for drug treatment, were
used for the study. Thirty minutes following vehicle or drug
administration, 1.0 mg of arachidonic acid in 20 µL of cold
acetone was applied topically to the left ear of the mouse. One
hour later, the left treated ear and right untreated ear were
measured with a thickness gauge. The difference represents
the degree of swelling (edema). The mice were then eutha-
nized with carbon dioxide gas and the left treated ears
harvested and placed in dry ice to await MPO analysis.
For topical studies, 1.0 mg/ear AA in 20 µL of cold acetone
was applied topically to the left ear for each vehicle and drug
group. The number of mice/group used was the same as for
the oral study. Immediately following AA application, 1.0 mg/
ear of test compound in 25 µL of methanol or acetone was
applied topically to the left ear. One hour later, the left and
right ears of each group were measured for ear thickness and
the mice euthanized. The left ears were harvested for MPO
analysis as a measure of neutrophil influx.
(9) Ono, N.; Miyake, H.; Saito, T.; Kaji, A. A Convenient Synthesis
of Sulfides, Formaldehydes, and Chloromethyl Sulfides. Syn-
thesis 1980, 952-953.
(10) Free acid; mp 124-125 °C from ethyl acetate.
(11) Boyum, A. Isolation of Mononuclear Cells and Granulocytes from
Human Blood. Scan. J . Clin. Lab. Invest. 1968, 21 (Suppl. 97),
77-89.
(12) Goldman, D. W.; Goetzl, E. J . Specific Binding of Leukotriene
B4 to Receptors on Human Polymorphonuclear Leukocytes. J .
Immunol. 1982, 129, 1600-1604.
(13) Cheng, Y.-C.; Prusoff, W. H. Relationship between the Inhibition
Constant (Ki) and the Concentration of Inhibitor which cause
50 Percent Inhibition (IC50) of an Enzymatic Reaction. Biochem.
Pharmacol. 1973, 22, 3099-3108.
(14) Goldman, D. W.; Gifford, L. A.; Olson, D. M.; Goetzl, E. J .
Transduction by Leukotriene B4 Receptors of Increases in
Cytosolic Calcium in Human Polymorphonuclear Leukocytes. J .
Immunol. 1985, 135, 525-530.
(15) Grynkiewicz, G.; Poenie, M.; Tsein, R. Y. A New Generation of
Ca2+ Indicators with Greatly Improved Fluorescence Properties.
J . Biol. Chem. 1985, 260, 3440-3450.
(16) Prescott, S. M.; Zimmerman, G. A.; Seeger, A. R. Leukotriene
B4 is an Incomplete Agonist for the Activation of Human
Neutrophils. Biochem. Biol. Res. Commun. 1984, 122, 535-541.
(17) Bradley, P. P.; Priebat, D. A.; Christensen, R. D.; Rothstern, G.
Measurement of Cutaneous Inflammation: Estimation of Neu-
trophil Content with an Enzyme Marker. J . Invest. Dermatol.
1982, 78, 206-209.
MPO was extracted from the ears, and the activity was
assayed spectrophotometrically by the method of Bradley17 (see
above). ED50 values were derived from multiple dose-
response studies and calculated using linear regression.
Refer en ces
(1) Bray, M. A. Leukotrienes in Inflammation. Agents Actions 1986,
19, 87-99. Brain, S. D.; Williams, T. J . Leukotrienes and
Inflammation. Pharmacol. Ther. 1990, 46, 57-66.
J M960248S