CCR3 antagonists: a potential new therapy for the treatment of asthma. Discovery and structure-activity relationships.

Article abstract of DOI:10.1016/S0960-894X(02)00206-8

CCR3 antagonist leads with IC(50) values in the microM range were converted into low nM binding compounds that displayed in vitro inhibition of human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-1-yl-n-propylureas and erythro-3-(4-benzyl-2-(alpha-hydroxyalkyl)piperidin-1-yl)-n-propylureas (obtained via Beak reaction of N-BOC-4-benzylpiperidine) exhibited single digit nanomolar IC(50) values for CCR3.

Full text of DOI:10.1016/S0960-894X(02)00206-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1785–1789
CCR3 Antagonists: A Potential New Therapy for the Treatment
of Asthma. Discovery and Structure–Activity Relationships
Dean A. Wacker,* Joseph B. Santella, III, Daniel S. Gardner, Jeffrey G. Varnes,
Melissa Estrella, George V. DeLucca, Soo S. Ko, Keiichi Tanabe, Paul S. Watson,
Patricia K. Welch, Maryanne Covington, Nicole C. Stowell, Eric A. Wadman,
Paul Davies, Kimberly A. Solomon, Robert C. Newton, George L. Trainor,
Steven M. Friedman, Carl P. Decicco and John V. Duncia*
Bristol-Myers Squibb Company, Experimental Station, PO Box 80336, Wilmington, DE 19880-0336, USA
Received 18 September 2001; accepted 8 January 2002
Abstract—CCR3 antagonist leads with IC50 values in the mM range were converted into low nM binding compounds that displayed
in vitro inhibition of human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-1-yl-n-propylureas
and erythro-3-(4-benzyl-2-(a-hydroxyalkyl)piperidin-1-yl)-n-propylureas (obtained via Beak reaction of N-BOC-4-benzylpiperidine)
exhibited single digit nanomolar IC50 values for CCR3. # 2002 Published by Elsevier Science Ltd.
Asthma is an inflammatory disease of the lungs, which
is characterized by the restriction of airflow and bron-
chial hyperresponsiveness. A hallmark of the disease is
the selective localization of eosinophils to the lung tissue
of allergic asthmatics.1 Clinical studies have implicated
eotaxin as the primary chemokine associated with the
recruitment of eosinophils to the airways of asthmatic
patients.2,3 The eotaxin receptor has been identified as
CC chemokine receptor 3 (CCR3)4À6 and has been
demonstrated to be the dominant functional chemokine
receptor on eosinophils.7 This growing body of clinical
studies and animal models suggest that eotaxin and
CCR3 play a primary role in the recruitment of eosino-
phils in allergic asthma and therefore, small molecule
antagonists8À15 of CCR3 may provide a novel mechan-
ism of inhibiting this recruitment process.
we could change the binding potency, we began our
SAR investigation by replacing the carbonyl group with
the amide, sulfonamide, and urea moieties since (1) they
closely resembled a carbonyl group and (2) permitted
the rapid synthesis of both linker and phenyl analogues.
Unfortunately, we were not able to increase potency
over our lead compounds 1–4 (Table 2). However, we
did learn that there are a few SAR trends. A four car-
bon chain linker was the best for amides (8–10) and
sulfonamides (11 and 12). Adamantyl is better than
cyclohexyl, which in turn is better than a t-butyl amide
(15>14>13). Moving a methoxy group around the
phenyl ring did not lead to a preferred position for
enhanced binding (16–18). Keeping the optimal chain
length (6 atoms) the same as for the butylamides and
sulfonamides, the propylureas were investigated.
Although a complete systematic SAR study was not
done, the data suggested that unlike for amides 16–18,
there was preference for a H-bond acceptor in the
3-position on the phenylureas as evidenced by the con-
tribution to binding affinity of the 3-cyano group 21.
High-throughput screening of the corporate compound
library yielded several lead structures, including a series
of indolinopiperidines which are summarized in
Table 1.16 The screening data indicated that the potency
of the leads could be affected by increasing the alkyl
chain linker (1–3) or by incorporating a carbonyl into
the linker (4). With the identification of a site in which
During the synthesis of the tetracycles, it was discovered
in our group that a series of N-substituted 4-benzylpi-
peridines (not shown) also displayed micromolar bind-
ing affinities for the CCR3 receptor.17 We hypothesized
that the 4-(N-phenylamino)-piperidine portion of the
*Corresponding authors. Fax: +1-302-467-6913; e-mail: dean.
wacker@bms.com (D. A. Wacker); john.duncia@bms.com (J. V.
Duncia).
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00206-8

1786
D. A. Wacker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1785–1789
Table 1. Binding affinities of CCR3 leads obtained from screening of
the corporate compound library: all compounds are racemic
Table 3. Binding affinities 4-benzylpiperidinealkylureas and amides
Compd
R1
M
n
R2
IC50 (mM)
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
H
H
H
H
H
H
H
H
3
3
2
3
4
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
3
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
1
1
Ph
3-MeO-Ph
3-CN-Ph
3-CN-Ph
3-CN-Ph
3-CN-Ph
4-CF3-Ph
4-Me2N-Ph
Ph
-CH2Ph
-CH2CH2Ph
3-CN-Ph
4-CN-Ph
2-CF3-Ph
3-CF3-Ph
4-CF3-Ph
2-NO2-Ph
3-NO2-Ph
4-NO2-Ph
3-Ac-Ph
0.4Æ0.1
0.3Æ0.1
Compd
R
% Inh.a
IC50 (mM)
0.5Æ0.1
1
2
3
4
(CH2)1-Ph-4-F
(CH2)2-Ph-4-F
(CH2)3-Ph-4-F
15
50
100
—
—
0.2Æ0.1
0.5Æ0.1
0.5Æ0.8
1.2Æ0.4
(CH2)3-CO-Ph-4-F
1.1Æ0.3
1.2Æ0.2
aPercent inhibitions are at 10 mM substrate.
1.6Æ0.2
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
2-F
4-Cl
4-CF3
4-Me
H
0.03Æ0.02
0.4Æ0.02
0.3Æ0.1
Table 2. Binding affinities of indolinopiperidinylalkylureas and
amides: all compounds are racemic
0.02Æ0.01
0.02Æ0.004
0.2Æ0.1
0.07Æ0.01
0.06Æ0.002
0.089Æ0.002
0.009Æ0.002
0.007Æ0.002
0.01Æ0.004
0.2Æ0.1
Compd
R
% Inh.a
IC50 (mM)
4-Ac-Ph
5
6
7
8
(CH2)2-NH2
(CH2)3-NH2
(CH2)4-NH2
15
14
5
—
—
—
3-MeS-Ph
3-MeSO-Ph
3-MeSO2-Ph
4-MeS-Ph
4-MeSO-Ph
4-MeSO2-Ph
3-MeO-Ph
3-(Furan-2-yl)-Ph
3-(Thiophen-2-yl)-Ph
3-(Imidazol-2-yl)-Ph
0.02Æ0.01
0.05Æ0.0
0.02Æ0.0005
0.04Æ0.03
0.2Æ0.05
0.07Æ0.03
0.03Æ0.01
0.1Æ0.01
0.2Æ0.05
0.1Æ0.03
(CH2)2-NHCO-Ph-4-F
(CH2)3-NHCO-Ph-4-F
(CH2)4-NHCO-Ph-4-F
(CH2)3-NHSO2-Ph-4-F
(CH2)4-NHSO2-Ph-4-F
(CH2)4-NHCO-t-butyl
(CH2)4-NHCO-cyclohexyl
(CH2)4-NHCO-adamant-1-yl
(CH2)4-NHCO-Ph-2-OMe
(CH2)4-NHCO-Ph-3-OMe
(CH2)4-NHCO-Ph-4-OMe
(CH2)3-NHCONH-Ph-4-NMe2
(CH2)3-NHCONH-Ph-4-CF3
(CH2)3-NHCONH-Ph-3-CN
(CH2)3-NHCONH-Ph-3-I
(CH2)3-NHCONH-Ph-4-I
8.3Æ1.9
5.5Æ1.5
3.9Æ1.8
10.1Æ0.1
6.4Æ6.2
25.6Æ2.9
11.0Æ0.0
6.6Æ2.1
6.8Æ0.4
8.1Æ1.2
5.1Æ0.7
4.8Æ3.1
3.7Æ2.1
0.7Æ0.1
1.1Æ0.5
1.5Æ0.4
9
10
11
12
13
14
15
16
17
18
19
20
21
23
24
45
45
73
3-(1-Me-tetrazol-5-yl)-Ph 0.005Æ0.003
3-CN-Ph
3-CN-Ph
3-CN-Ph
3-CN-Ph
Ph
0.2Æ0.1
0.02Æ0.01
52% @ 5 mM
0.5Æ0.3
0.8Æ0.3
H
H
H
4-F
4-F
3-MeO-Ph
4-CN-Ph
4-F-Ph
Adamant-1-yl
Cyclohexyl
1.2Æ0.5
1.3Æ0.8
1.0Æ0.8
0.09Æ0.02
0.1Æ0.05
aPercent inhibitions are at 10 mM substrate.
tetracycle was mimicking a 4-benzylpiperidine as shown
below. We also hypothesized that perhaps the tetra-
cyclic indolinopiperidine was too rigid to allow for an
optimal binding conformation to the CCR3 receptor.
Therefore, we replaced the indolinopiperidine with a 4-
benzylpiperidine and these new analogues are summar-
ized in Table 3.
vs 27). Of all of the substituents investigated on the
benzylpiperidine’s phenyl, the 4-F and 4-Cl substituents
were the most potent (35 vs 56–59). On the right-hand
side of the molecule, the phenylureas were more potent
than the alkylureas (32 vs 33, 34, 64, 65). The acetyl
group on the phenylurea preferred the 3- position to the
4-position (43 vs 44). The methylsulfide, sulfoxide, and
sulfone also preferred the 3-position (45 vs 48, 46 vs 49,
and 47 vs 50), although they are less potent than the
acetyl group. The nitro and cyano groups showed a
preference for the 3- and 4-positions on the phenylurea
(35, 36, and 40–42). H-bond acceptor substituents such
as CN, NO2, Ac, and tetrazole at the 3- and 4-positions
were generally more potent than more lipophillic sub-
stituents such as CF3, methyl, methylthio, furan, and
thiophene. Finally, the ureas were more potent than the
corresponding amides (60 vs 24; and 61 vs 25).
From Table 3 we see that the benzylpiperidines were 3–
7 times more potent than the indolinopiperidines (10 vs
63, 17 vs 61, 19 vs 31, 20 vs 30, and 21 vs 27). A chain
length of 3 carbon atoms was optimal as exhibited by
the 3-cyanophenylureas 26–29. On the left-hand side of
the molecule, the addition of a fluorine to the benzyl-
piperidine increased potency by 10-fold (32 vs 24 and 35
Concurrent with the alkyl linker efforts, a series of
heterocyclic linkers was investigated (Table 4). We

D. A. Wacker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1785–1789
1787
hypothesized that a more rigidified linker would provide
compounds with improved potency for CCR3. The
SAR efforts utilized the 3-methoxyphenylurea for direct
comparison to the alkyl linker compounds. The
4-piperidine 66 and 4-methylpiperidine 67 were less
potent than the alkyl linker 25. However, the 3-methyl-
piperidine linker 68 was equipotent with 25. Further
optimization of the heterocyclic linker was carried out
with 4-fluorobenzylpiperidine because it proved to be
about 6-fold more potent than the non-fluorinated ana-
logue (69 vs 68). Optimization first consisted of short-
ening 70 and lengthening 71 the spacer of the
3-substituted piperidine linker, which reduced the
potency of the compounds by about 5- to 10-fold. The
linker was moved to the 2-postion of the piperidine 72
and again the potency was reduced by about 3-fold.
Contraction of the 3-piperidine ring to a pyrrolidine
ring 73 reduced the potency by 5-fold, making it clear
that the 3-substituted methylpiperidine ring was the
optimal arrangement for the linker. Incorporation of an
O or N heteroatom into the piperidine ring to form a
morpholine 74 or piperazine 75 and 76 increased the
potency only marginally over the piperidine analogue.
The morpholine linker was synthesized enantiomerically
pure by the procedures of Brown et al.18 yielding 77 and
78 proving that the R-stereoisomer was the most potent.
All of the heterocyclic-linked compounds exhibited
similar, but not improved, potencies to the alkyl linkers.
With knowledge that piperidine ring conformation and
substitution pattern can affect binding of compounds to
7-transmembrane receptors, we began an investigation
using the Beak reaction to substitute at the piperidine
2-position.20 These compounds are summarized in
Table 5. Allyl bromide alkylation of N-Boc-4-benzyl-
piperidine led to both cis and trans compounds 79 and
80, both sharing the same binding affinity. Alkylation
using benzyl bromide led to only the cis compound 81,
also with moderate binding affinity. Quenching the N-
Boc-piperidinyl anion with aldehydes yielded only cis
alkylated products with a 1:1 erythro/threo mixture at
the alcohol carbon. The erythro diastereomer was
always more potent (85 vs 86). The presence of the
newly introduced OH group increased binding affinity
10-fold (85 vs 79). The erythro isomer can be separated
via chiral HPLC into its enantiomers, one being more
potent than the other, such as with 87a versus 87b and
93a versus 93b. The longer the alkyl chain, the more
potent the binding. Branching in the alkyl side chain
lowered binding affinity (89, 91, and 92). A pentanol
side chain yielded compounds with binding affinities in
the 1 nM range (93b and 94). Elimination of the chiral
center via oxidation21 of alcohol 82 to ketone 83 or via
gem disubstitution (84: via Beak reaction with acetone)
lowered affinity. A fluoro-substituent in this series (95)
did not increase affinity as previously observed.
Finally, a representative set of compounds was char-
acterized for their ability to block the in vitro functional
The heterocyclic linker compounds were utilized to
characterize our compounds for their functional
response at CCR3 by using an eotaxin induced Ca2+
mobilization assay.19 Several compounds (Table 4)
inhibited eotaxin induced Ca2+ mobilization with IC50s
between 139 and 822 nM. The compounds by them-
selves did not produce a functional response in the
Ca2+ mobilization assay at concentrations up to 10 mM
(results not shown). These experiments indicate that this
series of molecules is acting as functional antagonists of
CCR3.
Table 5. Binding affinities for the ‘Beak’ alkylated benzylpiperidine
analogues: compounds are racemic unless otherwise specified
Compd R1
R2
R3
OH or H
R4
IC50 (mM)a
79
80
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Et
Et (trans)
Ph
H
H
H
H
H
H
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
0.1Æ0.02
0.2Æ0.01
Table 4. Structure–activity relationships for p-fluorobenzylpiperidine
heterocyclic CCR3 antagonists
81
0.2Æ0.1
82c
83
Me
Me
Me
Et
H
Et
Et
i-Pr
H
n-Pr
i-Bu
H
n-Bu
n-Bu
n-Bu
n-Bu
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
0.02Æ0.01
0.05Æ0.02
0.2Æ0.05
OH
Me
H
Et
H
H
H
i-Pr
H
H
i-Bu
H
H
H
H
84
85
86
87ab
87bb
88
0.006Æ0.003
0.05Æ0.01
40%
Compd Ent.
R
X
m
n
y
CCR3
Ca2+
0.008Æ0.003
0.05Æ0.01
0.1Æ0.08
IC50 (mM)a IC50 (mM)a
89
66
67
H
H
H
F
F
F
F
F
F
F
F
F
F
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
0
0
1
1
1
1
2
0
1
1
1
1
1
2
2
1
1
1
1
0
1
1
1
1
1
1
0
1
1
1
0
2
1
1
1
1
1
1
1
2.3
1.6
0.33Æ0.05
90c
91
0.007Æ0.002
0.02Æ0.01
0.06Æ0.004
0.08Æ0.002
0.001Æ0.0005
92
68
69
70
71
72
73
74
75
76
77
78
(Æ)
(Æ)
(Æ)
(Æ)
(Æ)
(Æ)
(Æ)
(Æ)
(Æ)
R
0.05Æ0.01
0.53Æ0.04
0.25Æ0.01
0.17Æ0.03
0.24Æ0.03
0.05Æ0.01
0.04Æ0.01
0.03Æ0.01
0.03Æ0.01
0.26Æ0.06
0.82Æ0.08
93ab
93bb
94b
95
Tetd 0.001Æ0.0007
Ac
0.005Æ0.002
aPercent inhibitions are at 0.5 mM substrate.
0.22Æ0.03
0.20Æ0.03
0.14Æ0.02
0.19Æ0.03
bCompounds 87a and 87b, 93a and 93b are enantiomers of one
another and 94 is a single enantiomer, all of which have the above
assigned relative stereochemistry, but whose absolute configuration is
unknown.
NBOC
NH
O
S
O
cCompounds 82 and 90 are a mixture of erythro/threo diastereomers.
dTet=1-methyltetrazol-5-yl.
aValues are means of nꢀ2.

1788
D. A. Wacker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1785–1789
Table 6. Binding affinities and human eosinophil chemotaxis inhibi-
tion data
Compd
Binding IC50 mM
Chemotaxis IC50 mM
35
57
87b
0.02Æ0.01
0.02Æ0.01
0.08Æ0.05
0.2Æ0.15
0.02
0.008Æ0.003
activity of CCR3 by using an eotaxin induced human
eosinophil chemotaxis assay (Table 6).22 These com-
pounds proved to be potent inhibitors of chemotaxis
and the potency correlated to the binding assay results.
The compounds by themselves did not cause chemotaxis
and thus indicates these molecules are functional
antagonists of CCR3.
Scheme 1. (a) N-(3-bromopropyl)phthalimide, DBU, DMF, 100 ꢁC;
(b) NBS, AIBN, CCl4, reflux; (c) n-BuLi, THF, À78 ꢁC; (d) 90; (e) H2,
10% Pd/c, MeOH; (f) N2H42, EtOH, reflux; (g) ArNCO, THF, rt.
The synthesis of the piperidines, including the tetracyclic
indolino-piperidines, was accomplished by alkylating
with N-(bromoalkyl)-phthalimide in the presence of
K2CO3, KI and refluxing MEK. Subsequent hydrazino-
lysis followed by reaction of the amine with the appro-
priate isocyanate or phenylcarbamate yielded the desired
ureas. Tetracyclic indolino-piperidines were synthesized
via a Fisher–Indole reaction employing N-aminoindoline
and 4-piperidone hydrate hydrochloride in refluxing IPA.
Reduction of the indole double bond with sodium bor-
ohydride pellets (Caution! Use pellets) in TFA yielded the
tetracycle, which was alkylated as described above.
Scheme 2. (a) 10 equiv LDA, benzene, 0 ꢁC; (b) 10 equiv TMSCl,
THF, À70 ꢁC, 65%; (c) s-BuLi, TMEDA, À78 to À30 ꢁC; (d) RCHO,
warm to 0 ꢁC, 18%; (e) H2O; (f) NaOH, EtOH, reflux; (g) CsF, DMF,
H2O, 79%.
Substituted benzylpiperidines 56–59 were synthesized
via Wittig reaction with N-(phthalimidopropyl)-4-
piperidone 97 as shown in Scheme 1. Compounds 52
and 53 were made via Stille23 coupling of 2-tribu-
tyltinfuran/thiophene to 1-iodo-3-nitrobenzene. Imida-
zole 54 was made via reaction of 3-nitro-benzaldehyde
with glyoxal and ammonia.24 Tetrazole 55 was synthe-
sized via the method of Thomas25 (solvent–AcCN) from
N-methyl-3-nitrobenzamide, triflic anhydride and NaN3.
The synthesis of compound 95 (Scheme 2) exemplifies
the use of the Beak reaction which was employed to
make all of the compounds in Table 5. TMS protection
was necessitated in the case of 95 due to competing
metallation ortho to the fluorine.
Scheme 3. (a) (BOC)2O, CH2Cl2, rt, 1 h, 95%; (b) TPAP, NMO,
CH2Cl2, 3 A MS, 1 h, 70–90%; (c) NaBH(OAc)3, ClCH2CH2Cl, 50–
85%; (d) 4 M HCl, dioxane, 99%; (e) acyl chlorides, sulfonyl chlor-
ides, alkyl bromides, or isocyanates, TEA, CH2Cl2, 5 h, 70–90%.
Exploration of the heterocyclic benzylpiperidines was
facilitated by the synthesis outlined in Scheme 3. The
heterocyclic aldehydes were synthesized from their
commercially available precursor alcohols or, in the
case of morpholine analogues, according to literature
procedures.18 The alcohols were reacted with di-t-butyl
dicarbonate in CH2Cl2 and then oxidized with tetra-
propylammoniumperruthenate and N-methyl-morpho-
line oxide in CH2Cl2 to produce the carbonyl
compounds 106. The oxidation products 106 were
reductively alkylated with 4-benzylpiperidine and NaB-
H(OAc)3 in ClCH2CH2Cl to yield the amines 107.
Removal of the amine protecting group with HCl in
dioxane and then treatment with triethylamine and the
desired isocyanate afforded the final products.
Figure 1. Structural requirements for high affinity binding to CCR3.
atoms between the piperidine and the phenyl, (2) a H-
bond accepting substituent such as a tetrazole, acetyl,
cyano, etc. at the 3-position of the phenyl, (3) a fluorine
or chlorine at the 4-position on the benzyl group, or (4)
an optional erythro-pentanol side chain at the piperidine
2-position. Thus we have taken micromolar binding
Figure 1 summarizes what is required for high affinity
binding to the CCR3 receptor: (1) a chain length of 6

D. A. Wacker et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1785–1789
1789
leads and through structure–activity relationships man-
aged to increase affinity to the single digit nanomolar
range. These binding affinities also translate into a
pharmacological effect, namely the in vitro inhibition of
human eosinophil chemotaxis or eotaxin induced Ca2+
mobilization. It remains to be seen whether in human
clinical trials, compounds of this class will alleviate the
symptoms of asthma via the in vivo inhibition of eosi-
nophil chemotaxis.
10. Rodgers, D. H.; Saunders, J.; Williams, J. P. World Patent
Appl. WO 0031032.
11. Mori, N.; Kaneko, M.; Torii, Y.; Tanigawa, H. World
Patent Appl. WO 0034278.
12. Padia, J.; Hocker, M. D.; Ohashi, H.; Nishitoba, T.;
Sawa, E. World Patent Appl. WO 0109088.
13. Naya, A.; Kobayashi, K.; Ishikawa, M.; Ohwaki, K.;
Saeki, T.; Noguchi, K.; Ohtake, N. Bioorg. Med. Chem. Lett.
2001, 11, 1219.
14. Dhanak, D.; Christmann, L. T.; Darcy, M. G.; Jurewicz,
A. J.; Keenan, R. M.; Lee, J.; Sarau, H. M.; Widdowson,
K. L.; White, J. R. Bioorg. Med. Chem. Lett. 2001, 11, 1441.
15. Dhanak, D.; Christmann, L. T.; Darcy, M. G.; Keenan,
R. M.; Knight, S. D.; Lee, J.; Ridgers, L. H.; Sarau, H. M.;
Shah, D. H.; White, J. R.; Zhang, L. Bioorg. Med. Chem. Lett.
2001, 11, 1445.
16. The binding assay was carried out using 150 pM 125I
labeled human eotaxin, 5Â105 CHO cells, and 0.0001–1 mM
compound in 150 mLof binding buffer (0.5% bovine serum
albumen, 20 mM HEPES buffer and 5 mM magnesium chlo-
ride in RPMI 1640 media) in 96-well filtration plates (Milli-
pore) pretreated with 5 mg/mLprotamine in phosphate
buffered saline, pH 7.2. The assay was incubated at room
temperature for 30 min. The plates were vacuum filtered and
the remaining cells were washed three times with binding buf-
fer containing 0.5 M NaCl added. Radiolabel binding was
quantified upon filter removal via liquid scintillation counting.
17. DeLucca, G. V.; Kim, U. T.; Johnson, C.; Vargo, B. J.;
Welch, P. K.; Covington, M.; Davies, P.; Solomon, K. A.;
Newton, R. C.; Trainor, G. L.; Decicco, C. P., Ko, S. S. J.
Med. Chem., manuscript in preparation.
Acknowledgements
We gratefully acknowledge the help of A. J. Mical, K.
A. Rathgeb, D.-R. Wu, and N. C. Caputo, of the
Separations Group; M. X. Xia, G. A. Cain, and C.
Teleha, of the Chemical Synthesis Group; and T. H.
Scholz and G. A. Nemeth of the Spectroscopy Group.
We also thank Prof. Victor Snieckus for providing con-
ditions for the bis-TMS protection on the 4-fluoro-
benzylpiperidine to circumvent the competing ortho-
directed metallation reaction.
References and Notes
1. Bousquet, J.; Chanez, P.; Lacoste, J. Y.; Barneon, G.;
Ghavanian, N.; Enander, I.; Venge, P.; Ahlstedt, S.; Simony-
Lafontaine, J.; Godard, P. N. Engl. J. Med. 1990, 323, 1033.
2. Ying, S.; Robinson, D. S.; Meng, Q.; Rottman, J.; Ken-
nedy, R.; Ringler, D. J.; Mackay, C. R.; Daugherty, B. L.;
Springer, M. S.; Durham, S. R.; Williams, T. J.; Kay, B. A.
Eur. J. Immunol. 1997, 27, 3507.
3. Brown, J. R.; Kleimberg, J.; Marini, M.; Sun, G.; Bellini,
A.; Mattoli, S. Clin. Exp. Immunol. 1998, 114, 137.
4. Combadiere, C.; Ahuja, S. K.; Murphy, P. M. J. Biol.
Chem. 1995, 270, 16491.
18. Brown, G. R.; Forster, G.; Foubister, A. J.; Stribling, D. J.
Pharm. Pharmacol. 1990, 42, 797.
19. The Ca2+ mobilization assay was carried out using 10 nM
eotaxin, 2Â105 eosinophils preloaded with Fluo-3AM (Mole-
cular Probes) and and 0.0001–1 mM compound in 200 mLof
buffer (0.1% bovine serum albumen, 20 mM HEPES buffer
and 2.5 mM Probenecid in RPMI 1640 media) in 96-well
plates in a fluorescent imaging plate reader. Data was gener-
ated as arbitrary fluorescence units and compound dependent
inhibition was calculated as a percentage of the response of
eotaxin alone.
5. Ponath, P. D.; Qin, S.; Post, T. W.; Wang, J.; Gerard, N. P.;
Newman, W.; Gerard, C.; Mackay, C. R. J. Exp. Med. 1996,
183, 2437.
6. Daugherty, B. L.; Siciliano, S. J.; DeMartino, J. A.; Mal-
kowitz, L.; Sirotina, A.; Springer, M. S. J. Exp. Med. 1996,
183, 2349.
20. Beak, P.; Lee, W. K. J. Org. Chem. 1990, 55, 2578.
21. Griffith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A. D.
Chem. Comm. 1987, 1625.
7. Heath, H.; Qin, S.; Rao, P.; Wu, L.; LaRosa, G.; Kassam,
N.; Ponath, P. D.; Mackay, C. R. J. Clin. Invest. 1997, 99, 178.
8. Naya, A.; Owada, Y.; Saeki, T. O; Ohwaki, K.; Iwasawa, Y.
World Patent Appl. WO 9804554.
9. Gong, L.; Kertesz, D. J.; Smith, D. B.; Talamas, F. X.;
Wilhelm, R. S. European Patent Appl. EP 0 903 349.
22. Ko, S. S.; Delucca, G. V.; Duncia, J. V.; Santella, J B. III;
Wacker, D. A. U.S. Patent 6,331,545, 2001.
23. Bailey, T. R. Tetrahedron Lett. 1986, 27, 4407.
24. Rothenberg, A. S.; Duplaise, D. L.; Panzer, H. P. Angew.
Chem. Intl. Ed. 1983, 45, 573.
25. Thomas, E. W. Synthesis 1993, 767.