Discovery of CC chemokine receptor-3 (CCR3) antagonists with picomolar potency

Article abstract of DOI:10.1021/jm049530m

Starting with our previously described²⁰ class of CC chemokine receptor-3 (CCR3) antagonist, we improved the potency by replacing the phenyl linker of 1 with a cyclohexyl linker and by replacing the 4-benzylpiperidine with a 3-benzylpiperidine. The resulting compound, 32, is a potent and selective antagonist of CCR3. SAR studies showed that the 3-acetylphenyl urea of 32 could be replaced with heterocyclic ureas or heterocyclic-substituted phenyl ureas and still maintain the potency (inhibition of eotaxin-induced chemotaxis) of this class of compounds in the low-picomolar range (IC₅₀ = 10-60 pM), representing some of the most potent CCR3 antagonists reported to date. The potency of 32 for mouse CCR3 (chemotaxis IC₅₀ = 41 nM) and its oral bioavailability in mice (20% F) were adequate to assess the efficacy in animal models of allergic airway inflammation. Oral administration of 32 reduced eosinophil recruitment into the lungs in a dose-dependent manner in these animal models. On the basis of its overall potency, selectivity, efficacy, and safety profile, the benzenesulfonate salt of 32, designated DPC168, entered phase I clinical trials.

Full text of DOI:10.1021/jm049530m

2194
J. Med. Chem. 2005, 48, 2194-2211
Discovery of CC Chemokine Receptor-3 (CCR3) Antagonists with Picomolar
Potency
George V. De Lucca,* Ui Tae Kim, Brian J. Vargo, John V. Duncia, Joseph B. Santella, III, Daniel S. Gardner,
Changsheng Zheng, Ann Liauw, Zhang Wang, George Emmett, Dean A. Wacker, Patricia K. Welch,
Maryanne Covington, Nicole C. Stowell, Eric A. Wadman, Anuk M. Das, Paul Davies, Swamy Yeleswaram,
Danielle M. Graden, Kimberly A. Solomon, Robert C. Newton, George L. Trainor, Carl P. Decicco, and Soo. S. Ko
Bristol-Myers Squibb Company, Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000
Received June 16, 2004
Starting with our previously described20 class of CC chemokine receptor-3 (CCR3) antagonist,
we improved the potency by replacing the phenyl linker of 1 with a cyclohexyl linker and by
replacing the 4-benzylpiperidine with a 3-benzylpiperidine. The resulting compound, 32, is a
potent and selective antagonist of CCR3. SAR studies showed that the 3-acetylphenyl urea of
32 could be replaced with heterocyclic ureas or heterocyclic-substituted phenyl ureas and still
maintain the potency (inhibition of eotaxin-induced chemotaxis) of this class of compounds in
the low-picomolar range (IC50 ) 10-60 pM), representing some of the most potent CCR3
antagonists reported to date. The potency of 32 for mouse CCR3 (chemotaxis IC50 ) 41 nM)
and its oral bioavailability in mice (20% F ) were adequate to assess the efficacy in animal
models of allergic airway inflammation. Oral administration of 32 reduced eosinophil
recruitment into the lungs in a dose-dependent manner in these animal models. On the basis
of its overall potency, selectivity, efficacy, and safety profile, the benzenesulfonate salt of 32,
designated DPC168, entered phase I clinical trials.
Introduction
Chemokines are small secreted proteins that chemoat-
tract and activate leukocytes of a variety of types,
sometimes under normal, homeostatic conditions, at
other times during inflammation. They are classified
into two main groups, CXC and CC, based on the
positions of the first two of their conserved N-terminal
cysteine residues. Both groups mediate their effects
through seven transmembrane (7TM) G-protein-coupled
receptors (GPCRs).
Since 1980, the frequency of asthma has almost
doubled. The disease now affects 8-10% of the adult
population of the U.S. and is the leading cause of
hospitalization among children less than 15 years of age.
While the principal clinical manifestation of asthma is
reversible airway obstruction, it is also recognized as
an inflammatory disease.1
In allergic asthma, the pulmonary airways are char-
acterized by an inflammatory infiltrate in which eosi-
nophils often comprise the major component. Within the
bronchial mucosa, eosinophils degranulate, releasing
toxic proteins that promote tissue damage and bron-
choreactivity.2 They also release fibrogenic factors such
as transforming growth factor-â (TGFâ) that are impli-
cated in chronic airway remodeling.3 Although correla-
tive evidence links eosinophils with asthma symptoma-
tology, a direct role for these cells in the disease is
controversial, largely as a result of the failed clinical
trials of two humanized anti-IL-5 antibodies. Although
the antibody regimens dramatically reduced the num-
bers of circulating eosinophils, airway function was not
significantly improved. Subsequent studies, however,
have shown that, despite near elimination from the
blood, eosinophils were reduced in the airway tissue by
only 55%. Airway remodeling, however, was signifi-
cantly reduced.4 On the basis of these results, the
original negative interpretation of the studies is under-
going reassessment.5
CC chemokine receptor-3 (CCR3) was originally cloned
from human eosinophils6 and is regarded as the prin-
cipal mediator of eosinophil chemotaxis. CCR3 is also
known to be expressed on a variety of inflammatory cells
associated with allergic responses and prominent in
allergic tissues such as the nose, airways, and skin.
These cells include basophils,7 mast cells,8 and Th2
lymphocytes.9 CCR3 is thought to mediate the migration
of cells from the blood into the tissue and their subse-
quent activation. In addition to its role in migratory
cells, CCR3 is also expressed on resident tissue cells
such as airway epithelium.10
CCR3 has 11 known ligands, but several, including
eotaxin (CC ligand 11, CCL11),11b eotaxin-2 (CCL24),11c
and eotaxin-3 (CCL26),11d are exclusive high-affinity
agonists of CCR3. Eotaxin has also been described as
an antagonist of CCR2,12 a partial agonist of CCR2b,13
and a full agonist of CCR5.12 Eotaxin-3 is an antagonist
of CCR2 and stimulates the negative chemotaxis (repul-
sion) of monocytes in vitro.14 The role of CCR3 in the
eosinophilia seen in asthmatic airways is suggested by
the fact that its ligands are expressed in asthma at high
levels that correlate with disease severity and poor
airway function.
IL-5 mobilizes eosinophils from the bone marrow into
the blood. Their recruitment into the pulmonary air-
ways, however, occurs through the action of chemokines.
CCR3 knock-out mice are fully functional and repro-
duce normally. In ovalbumin aerosol models of allergic
* Corresponding author. Phone: 609-252-3121. Fax: 609-252-3993.
E-mail: george.delucca@bms.com.
10.1021/jm049530m CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/14/2005

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2195
Table 1. SAR of Cyclohexyl Linker Stereochemistry in the
4-Benzylpiperidine Analogues
CCR3b 5HT2A/5HT2C
D2
Ki
IC50
Ki
compd stereochemistrya
X
R
(nM)
(nM)c
(nM)c
5
6
1,2 cis
H
H
F
F
F
F
3-OMe
3-OMe
3-OMe
3-Ac
3-OMe
3-Ac
423
67
12
4
78
41
NTd
NTd
NTd
271
103
NTd
NTd
1,2 trans
NTd
7
1,2 trans
1,2 trans
1,3 cis/trans mix
1,3 cis/trans mix
869/2315
601/>10 000
NTd
8
9
10
NTd
a All compounds in the table are racemic. b The value represents
an average of usually three determinations with a standard
deviation of (20-50% of the mean value shown. c The value
represents an average of usually two determinations with a
deviation of (30% of the mean value shown. d NT, not tested.
(compare 1 vs 2). We hypothesized that most of these
deficiencies could be attributed to the lower basicity of
the piperidine nitrogen in the benzyl linker series
compared to the phenethyl or propyl linker series.
Figure 1. Structure of compounds discussed in text.
To test this hypothesis, we synthesized a series of
compounds where the central phenyl ring of 1 was
replaced with a cyclohexyl ring. For this initial survey,
we started our synthesis with the commercially avail-
able racemic cis or trans 2-hydroxymethyl-1-cyclohexyl-
amine and 3-aminocyclohexane carboxylic acid (cis/trans
mixture) and the results are summarized in Table 1.
airway inflammation, they exhibit reduced lung eosi-
nophilia (>50%) relative to wild-type mice.15 A similar
reduction in eosinophilia is seen in eotaxin postallergen
challenge knock-out mice.16
The studies cited above suggest that CCR3 is a
prominent mediator of allergic responses in the airways
and that antagonizing the receptor will lead to a
reduction in airway inflammation and remodeling. The
potential importance of CCR3 in allergic inflammation
has made this receptor a target of drug development,17
and we18 and others19 have previously reported on small
molecule CCR3 antagonists. In this paper, we provide
further details of our efforts in this area.
The 1,2-cis isomer 5 has the same potency as the
corresponding benzyl linker20 (data not shown) and is
clearly less potent than the 1,2-trans isomer 6 (Table
1). The 1,3 isomers (9, 10, Table 1) are also less potent
than the 1,2-trans isomers. Introduction of the 4-fluoro
substituent on the benzylpiperidine group improves the
potency of 6 (IC50 ) 67 nM) about 5-fold to give 7 (IC50
) 12 nM). This is less than the 10-fold improvement
observed for the phenethyl20 or propyl21 series but better
than the 3-fold improvement seen with the benzyl20
series. With the use of a more optimal substituent on
the phenyl urea, the CCR3 binding potency is further
improved (to IC50 ) 4 nM) for the acetyl analogue 8,
which is a significant increase over the corresponding
benzyl analogue 1 (IC50 ) 125 nM, Figure 1). The
structure-activity relationship (SAR) for serotonin
selectivity of the cyclohexyl series mirrors the SAR
noted for the 4-fluorobenzylpiperidine effect, namely,
that its behavior is between that of the benzyl and
phenethyl/propyl linker series. Thus, the serotonin
selectivity of the trans 1,2-cyclohexyl linker analogue 8
(5HT2A Ki ) 600 nM) is not as good as the benzyl
analogue 4 (5HT2A Ki ) 5600 nM, Figure 1), but it is
better than the phenethyl 2 or the propyl 3 analogues
(Figure 1). While the serotonin selectivity could be
modulated, 8 showed potent affinity for the dopamine
D2 receptor (Ki ) 103 nM) as shown in Table 1.
Results and Discussion
Discovery of Cyclohexyl Linker. We have previ-
ously reported on the discovery of a potent class of CCR3
receptor antagonists.20 The important pharmacophore
of the class consists of a benzylpiperidine linked to a
phenyl urea through a benzyl group20 as in 1, a
phenethyl20 group as in 2, or a propyl21 chain as in 3
(Figure 1). These compounds are very selective for CCR3
over other chemokine receptors that were tested (CCR1,
CCR2, CCR5); however, they showed significant affinity
for other 7TM GPCRs that were examined. For example,
the phenethyl linker analogue 2 displayed potent affin-
ity for the serotonin receptors and in particular the
5HT2A (Ki ) 11 nM) and 5HT2C (Ki ) 284 nM) subtypes,
while the propyl linker analogue 3 had affinity for
5HT2A (Ki ) 193 nM) as shown in Figure 1. Although
the benzyl-linked analogues (e.g., 4) were more selective
(5HT2A Ki ) 5600 nM), this class of compounds had less
desirable physicochemical properties. They were much
less water soluble, much more lipophilic, and highly
protein bound (>99%) and were inherently less potent
To further improve selectivity, we examined replacing
the 4-benzylpiperidine of these compounds with 3-ben-

2196 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Table 2. SAR of 3-Benzylpiperidine Analogues
De Lucca et al.
to establish the preferred absolute configuration for this
series. The absolute configuration about the cyclohexyl
ring was initially established by resolving a synthetic
intermediate by chiral HPLC or by using the previously
described enzymatic procedure23 using Lipase PS to give
the (R,R) N-CBZ amino alcohol 18. In either case, the
resolved intermediate was converted to the 4-benzylpi-
peridine analogue 8. The (+)-isomer 8 (CCR3 IC50 ) 1.8
nM) was more potent than the (-)-isomer 8 (CCR3 IC50
) 43 nM). This established that the desired amino
alcohol 17 required the R,R configuration.
To determine the preferred configuration about the
3-benzylpiperidine portion, the BOC-protected 3-(4-
fluorobenzyl)piperidine 25 was resolved by chiral HPLC
and used to synthesize the analogues summarized in
Table 3. The data in Table 3 show that there is very
little difference in binding affinity between the (R)- and
(S)-3-(4-fluorobenzyl)piperidine analogues. This result
is in contrast to the 16-fold difference in potency that
is observed in the propyl linker series22 in which the
(S)-enantiomer is preferred. The selectivity against
other GPCRs is also not very different between the (R)-
and (S)-3-(4-fluorobenzyl)piperidine in the trans cyclo-
hexyl linker series as shown in Table 3. However, when
a functional assay of CCR3 receptor antagonism was
used, the (S)-3-(4-fluorobenzyl)piperidine analogue 32
(chemotaxis IC50 ) 0.034 nM) was clearly more potent
than the (R)-3-(4-fluorobenzyl)piperidine analogue 29
(chemotaxis IC50 ) 0.364 nM). This established the
(1R,2S)-2-(((S)-3-(4-fluorobenzyl)piperidin-1-yl)methyl)-
cyclohexanamine core as the preferred isomer.
The required starting material, the chiral (R,R) amino
alcohol 17, was obtained following the literature24
synthetic procedures. All subsequently described ana-
logues in this report were synthesized from 17 using
the synthetic sequence outlined in Scheme 1. Thus, the
amino alcohol 17 was protected as the benzylcarbamate
by treatment with benzyl chloroformate to give the
protected amine 18. The N-CBZ amino alcohol 18 was
subjected to Swern oxidation conditions to give the
aldehyde 19. Reductive amination of 19 with benzylpi-
peridine gave 20. The hydrogenation of 20 gave the free
amine 21, which was treated with isocyanates to give
the final urea analogues. Alternatively, the amine 21
was treated with phenyl carbamates to give the ureas.
CCR3b
IC50 (nM)
5HT2A/5HT2C
D2
compda
X
R
Ki (nM)c
Ki (nM)c
11
12
13
14
15
16
H
H
H
F
3-OMe
3-CN
3-Ac
3-OMe
3-CN
3-Ac
28
9
10 000/10 000
9000/10 000
6000/10 000
NTd
911
1010
550
3
14
12
3
NTd
NTd
1000
F
NTd
F
1333/10 000
a All compounds in the table are mixtures of diastereomers.
b The value represents an average of usually three determinations
with a standard deviation of (20-50% of the mean value shown.
c The value represents an average of usually two determinations
with a deviation of (30% of the mean value shown. d NT, not
tested.
zylpiperidine.18 Again, for our initial survey we used
racemic 3-benzylpiperidine and racemic 1,2-trans cy-
clohexyl components for the analogues synthesized, and
the data are summarized in Table 2. The potency of the
3-benzylpiperidine analogues for the CCR3 receptor is
slightly better than the 4-benzylpiperidines (compare
6 and 11). The 4-fluoro substituent on 3-benzylpiperi-
dine shows little (compare 11 and 14) to no improve-
ment (compare 13 and 16) in potency. However, there
is a more pronounced improvement in selectivity against
other GPCRs that were tested. The serotonin selectivity
generally improves for all the compounds tested. For
example, the 3-acetylphenyl urea analogue improves
2-fold in going from the 4-benzylpiperidine analogue 8
(5HT2A Ki ) 601 nM) to the 3-benzylpiperidine analogue
16 (5HT2A Ki ) 1333 nM). More significantly, there is a
substantial improvement in selectivity against dopa-
mine D2 of 16 (Ki ) 1000 nM) over analogue 8 (Ki )
103 nM).
Chemistry
Having established that the cyclohexyl linker ana-
logues could yield potent CCR3 antagonists, we needed
Table 3. SAR of 3-Benzylpiperidine Stereochemistry
piperidine
stereochemistry
CCR3b
IC50 nM
chemotaxis
IC50 (nM)
5HT2A/5HT2C
D2
compda
R
Ki (nM)c
Ki (nM)c
27
28
29
30
31
32
R
R
R
S
S
S
3-OMe
3-CN
3-Ac
3-OMe
3-CN
3-Ac
29
9
NTd
NTd
4300/3000
5000/2400
NTd
1800/2600
1000/6000
NTd
1000
702
NTd
2.5
14
9
0.364e
NTd
NTd
550
NTd
2.0
0.034 ( 0.019
1020
a All compounds in the table are homochiral with the absolute configuration of the cyclohexyl as shown. b The value represents an
average of usually three determinations with a standard deviation of (20-50% of the mean value shown. c The value represents an
average of usually two determinations with a deviation of (30% of the mean value shown. d NT, not tested. e Value was measured once.

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2197
Scheme 1. Synthesis of Urea Analoguesa
a Key: (a) benzyl chloroformate/Na2CO3(aq)/CH2Cl2; (b) Swern oxidation; (c) NaBH(OAc)3; (d) H2/10% Pd/C; (e) R-NdCdO/THF or
RHNCOOPh/THF.
Scheme 2. Synthesis of Phenyl Carbamates
BOC-protected 3-(4-fluorobenzyl)piperidine 25. This
compound was initially resolved by a preparative chiral
HPLC to give the required (S)-(+)-isomer. However, a
practical large-scale resolution method was developed,
which uses (R)-(-)-mandelic acid. Thus, 25 was depro-
tected to give the free amine 3-(4-fluorobenzyl)piperi-
dine, which was resolved by the formation of a diaster-
eomeric salt to selectively crystallize the (S)-(+)-3-(4-
fluorobenzyl)piperidine mandelic acid salt (98% ee after
two recrystallizations).26
In Vitro Structure-Activity Relationship (SAR)
The phenyl carbamates were readily available from
amines by treatment with phenyl chloroformate (Scheme
2). However, this procedure did not always work well
with some amines, especially the heterocyclic amines.
In these cases, the amine was first converted to the
sodium salt (NaH in DMF) and then treated with
diphenyl carbonate to give good yields of the desired
phenyl carbamate (Scheme 2). The phenyl carbamates
reacted readily with the amine 21 to give the urea
analogues.
Binding SAR. The evaluation of the in vitro phar-
macological properties of the compounds was performed
using the binding assay previously described.20 We
examined a series of substituents on the phenyl urea,
and the results are summarized in Table 4. The unsub-
stituted phenyl urea 33 itself is a potent CCR3 antago-
nist with a binding IC50 ) 2.5 nM. Substitution at the
2, 3, or 4 position of the phenyl ring with electron-
donating groups such as a methoxy (compounds 35, 30,
36, Table 4) or electron-withdrawing groups such as a
fluoride (37-39) was examined. The 2- or 4-isomers all
give similarly potent compounds. However, the 3-iso-
mers are less potent with the 3-methoxy analogue 30
decreasing to an IC50 ) 18 nM and the 3-fluoro analogue
38 decreasing to an IC50 ) 10 nM. The trend continued
with the larger halogens, with the 3-chloro (43) or
3-bromo (47) analogues (IC50 ) 31, 33 nM, respectively)
losing some potency. The disubstituted analogues are
The 3-benzylpiperidine fragment was prepared25 from
commercially available N-benzyl-3-piperidone (22) as
shown in Scheme 3. First, the benzyl group was con-
verted to N-BOC-3-piperidone (23) by a catalytic hy-
drogenolysis, followed by acylation with di-tert-butyl
dicarbonate. A Wittig reaction with 4-fluoro-benzylphos-
phonium chloride and n-butyllithium to give the olefin
24 was followed by a catalytic hydrogenation to afford

2198 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Scheme 3. Synthesis of (S)-3-(4-Fluorobenzyl)piperidinea
De Lucca et al.
a Key: (a) H2/10% Pd/C/MeOH; (b) di-tert-butyl-dicarbonate/NaHCO3(aq)/THF; (c) (4-F-benzyl)P(Ph)3Cl/BuLi/THF; (d) 1. 4N HCl/dioxane
2, Na2CO3; (e) (R)-(-)-mandelic acid/AcCN, recrystallize; (f) NaOH (aq)/ether.
Table 4. SAR of Phenyl Urea Substitution
Table 5. SAR of Heterocyclic Urea Analogues
CCR3
CCR3
CCR3
compd
R
IC50 (nM)a compd
R
IC50 (nM)a
compd
R
IC50 (nM)a
33
34
35
30
36
37
38
39
40
41
42
43
H
2.5
1.6
1.4
44
45
46
47
48
31
49
32
50
51
52
4-Cl
8
26
53
33
6
8.8
30
2.0
3.0
1.1
1.5
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
indol-6-yl
indol-5-yl
1.6
0.4
1.8
0.3
0.7
1.3
1.7
1.2
1.0
2.3
1.9
1.4
0.9
4.0
1.9
2.7
1.1
2.0
2.2
1.4
0.9
0.7
0.7
1.0
2-Me
3,5-Cl
3,4-Cl
3-Br
2-OMe
3-OMe
4-OMe
2-F
2,3-dimethyl-indol-6-yl
indazol-5-yl
18
2.0
1.8
10
5
12
4
4.6
31
4-Br
3-CN
3-CN-4-F
3-Ac
4-Ac
3,5-Ac
3-Ac-4-F
indazol-6-yl
3-Cl-indazol-5-yl
3-F
indolin-5-yl
4-F
benzo[d]thiazol-6-yl
3,4-F
benzimidazol-5-yl
3-F-4-Me
5-F-2-Me
3-Cl
1,3,4-thiadiazol-2-yl
thiazol-2-yl
4-methylthiazol-2-yl
5-acetyl-4-methylthiazol-2-yl
4-chlorobenzo[d]-thiazol-2-yl
1-methyl-pyrazol-3-yl
a The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
5-methylisoxazol-3-yl
pyridin-4-yl
3-cyano-4-(1H-pyrazol-1-yl)phenyl
4-(1H-1,2,4-triazol-1-yl)phenyl
3-(5-methyl-1H-tetrazol-1-yl)phenyl
2-(1-methyl-1H-tetrazol-5-yl)phenyl
3-(1-methyl-1H-tetrazol-5-yl)phenyl
4-(1-methyl-1H-tetrazol-5-yl)phenyl
3,5-bis-(1-methyl-1H-tetrazol-5-yl)phenyl
also less potent; for example, the 3,5-dichloro 45 (IC50
) 26 nM) and 3,4-dichloro analogues 46 (IC50 ) 53 nM)
both give weaker compounds. However, with electron-
withdrawing, hydrogen-bond-accepting substituents, all
isomers give potent compounds. For example, the 3-acetyl
isomer 32 (IC50 ) 2.0 nM) and the 4-acetyl isomer 50
(IC50 ) 3.0 nM) as well as the 3,5-diacetyl analogue 51
(IC50 ) 1.1 nM) and the 3-acetyl-4-fluoro analogue 52
(IC50 ) 1.5 nM) all provide potent compounds.
a The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
The apparent benefit of having hydrogen-bond-ac-
cepting groups prompted us to explore the use of
heterocycle-containing urea groups. Several benzo-fused
heterocycles, five- and six-membered ring heterocycles,
and heterocyclic-substituted phenyl ureas were pre-
pared, and the data for representative examples are
summarized in Table 5. As the data in Table 5 high-
lights, they were all uniformly potent. Thus, the 5- or
6-isomeric indoles (53-55) and indazoles (56-58), benz-
thiazole (60), and benzimidazole (61) all have bind-
ing potencies of about 1 nM or less. Five-membered
ring heterocycles such as 2-thiadiazole (62), 2-thiazoles
(63-66), pyrazole (67), and isoxazole (68) as well as the
six-membered ring pyridine urea (69) are all equally
potent. Phenyl ureas substituted with pyrazoles (70),
triazoles (71), and tetrazoles (72-76) are all potent
without regard to the position of the heterocycle on the
phenyl ring.
Functional Assays. Selected compounds were fur-
ther characterized in functional assays using freshly
isolated human eosinophils, as previously20 described.
The first assay used fluorescent imaging plate reader
(FLIPR) technology to monitor intracellular calcium

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2199
Table 6. SAR of Urea Analogues in Functional Assays
CCR3
Ca2+
chemotaxis
IC50 (nM)
compd
R
IC50 (nM)a
IC50 (nM)b
33
34
35
36
39
48
41
32
52
51
54
56
57
65
66
70
72
74
75
76
Ph
2.5
1.6
1.4
2.0
5
17
2-Me-Ph
2-OMe-Ph
4-OMe-Ph
4-F-Ph
>100c
115 ( 60
1.0c
10
50
50
35
8
4-Br-Ph
3-F-4-Me-Ph
3-Ac-Ph
3-Ac-4-F-Ph
3,5-Ac-Ph
indol-5-yl
indazol-5-yl
indazol-6-yl
5-acetyl-4-methylthiazol-2-yl
4-chlorobenzo[d]-thiazol-2-yl
3-cyano-4-(1H-pyrazol-1-yl)phenyl
3-(5-methyl-1H-tetrazol-1-yl)phenyl
3-(1-methyl-1H-tetrazol-5-yl)phenyl
4-(1-methyl-1H-tetrazol-5-yl)phenyl
3,5-bis-(1-methyl-1H-tetrazol-5-yl)phenyl
6
4
2.0
1.5
1.1
0.4
0.3
0.7
0.9
4.0
2.0
1.4
0.7
0.7
1.0
0.034 ( 0.019
0.015 ( 0.003
0.007 ( 0.007
0.016 ( 0.001
0.045 ( 0.015
0.060 ( 0.008
0.030 ( 0.020
0.090c
4
2
2
2
4
4
4
2
1
0.100c
0.010 ( 0.010
0.250c
2
0.042c
a The value represents an average of usually three determinations with a standard deviation of (20-50% of the mean value shown.
b The value represents an average of usually two determinations with a deviation of (50% of the mean value shown. c Value was measured
once.
mobilization stimulated by eotaxin (CCL11) at 10 nM.
The second assay determined eosinophil chemotaxis
across a membrane in response to eotaxin (CCL11) at
10 nM. The results are summarized in Table 6. These
compounds are functional antagonists since the com-
pounds themselves did not stimulate calcium mobiliza-
tion, chemotaxis, or random chemokinesis. While it is
hard to discriminate between different analogues based
on CCR3 receptor binding affinities (Tables 4 and 5),
the differences between them become apparent in the
functional assays.
Chemotaxis SAR. A more pronounced distinction
among the various analogues is observed in the chemo-
taxis assay. The 2-substituted phenyl urea analogues
34 and 35 are weak antagonists with IC50’s more than
100 nM, while the 4-substituted phenyl ureas, such as
the 4-methoxyphenyl 36 (IC50 ) 1 nM), are more potent.
However, substitution at the 3-position of the phenyl
urea with a hydrogen-bond-accepting group, such as an
acetyl (32, 52) or a heterocycle (72, 74, 75), gives
extremely potent compounds with chemotaxis IC50’s in
the 10-50 pM range. The nitrogen-linked tetrazole
analogue 72 (IC50 ) 0.100 nM), although very potent,
is 10-fold weaker than the corresponding carbon-linked
analogue 74 (IC50 ) 0.010 nM). The 3-position on the
phenyl urea seems to be the best for the heterocyclic
Calcium Mobilization SAR. In the calcium mobil-
ization assay the IC50 potency range is from about 1 to
50 nM and clusters around two features. Analogues with
simple phenyl ureas have IC50’s in the double-digit
range, and analogues with heterocycle-containing ureas
have IC50’s in the low single-digit range. Thus, the
parent phenyl urea 33 (IC50 ) 17 nM), the 2-methoxy-
phenyl 35 (IC50 ) 10 nM), and the 4-fluorophenyl 39
(IC50 ) 50 nM) are some of the weaker compounds listed
in Table 6 and are about 10-fold from their binding IC50
potency. The heterocyclic-substituted phenyl ureas show
a better correlation between binding and the calcium
mobilization assays. For example, the pyrazole-phenyl
70 (Ca2+ IC50 ) 4 nM) or tetrazole-phenyl 72 (Ca2+ IC50
) 2 nM) are within 2-fold of their binding potency (Table
6). The correlation between binding and calcium mobil-
ization assays for the heterocyclic ureas is again not as
good. Thus, while the binding of the indole 54, indazoles
56 and 57, and thiazole 65 are all below 1 nM, the
calcium mobilization IC50’s range from 2 to 4 nM, about
5-fold from their binding numbers.
substituent since the 4-tetrazole analogue 75 (IC50
)
0.250 nM) is more than 10-fold weaker than the
3-isomer 74. The 3,5-disubstitued phenyl analogues,
substituted with acetyl (51; IC50 ) 0.007 nM) or tetra-
zole (76; IC50 ) 0.042 nM), are also very potent. The
heterocyclic ureas, such as indole 54, indazoles 56 and
57, and thiazoles 65, also all give extremely potent
compounds as summarized in Table 6.
There seem to be several specific structural require-
ments in order to obtain compounds with extremely
potent inhibition of chemotaxis. One is the requirement
for a hydrogen-bond-accepting group on the urea, such
as an acetyl or a heterocycle, as discussed above.
However, other parts of the molecule, such as the benzyl
piperidine portion, are also important. For example, as
noted earlier, the (R)-3-(4-fluorobenzyl)piperidine ana-
logue 29 (chemotaxis IC50 ) 0.364 nM) is clearly less
potent than (S)-isomer 32 (chemotaxis IC50 ) 0.034 nM).

2200 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
De Lucca et al.
antagonists seem to be able to displace the natural
ligand, eotaxin, equally well from the receptor, but they
vary widely in their ability to interfere with the different
conformations of the receptor responsible for the various
functional activities observed. It would appear that the
conformation of the receptor required for signal trans-
duction leading to calcium mobilization is different from
the conformation required for chemotaxis.30
The picomolar potency of compounds in the chemo-
taxis assay relative to their nanomolar potency in the
binding assay suggests that the compounds inhibit
chemotaxis at fractional receptor occupancy. We postu-
late that such compounds stabilize a ternary complex
in which the receptor is coupled to inactive G (GDP)
proteins.31,32 The sequestration of G proteins in the
complex makes them unavailable for shuttling to recep-
tors that are free to bind natural ligand. If the chemo-
taxis-relevant G protein is present in limiting amounts
relative to receptor, functional inhibition would be
achieved even at low receptor occupancy.33 Intuitively,
this seems reasonable for a functionality such as chemo-
taxis which depends on the ability to sense a concentra-
tion gradient of the chemokine. Since compounds in the
calcium assay manifest nanomolar rather than picomo-
lar potency, however, we assume that calcium mobiliza-
tion signals through a different G protein or through
one that is more poorly coupled and thus requires a
higher receptor occupancy. Again, this seems reasonable
if calcium mobilization is associated with eosinophil
activation at the inflammation site where the maximum
concentration of chemokine would be present.
Recent reports34-36 suggest that the extracellular
signal-regulated kinase (ERK) signaling pathway is
important for eotaxin-induced chemotaxis of eosinophils
and is blocked by MEK inhibitors. In agreement with
this, we find that a selected group of compounds, with
a wide range of chemotaxis potencies, inhibited eotaxin-
dependent ERK phosphorylation with potencies similar
to those in the chemotaxis assay (data not shown).37
Since our objective is to develop a therapeutic agent
that inhibits the migration of eosinophils into tissues,
we concentrated our efforts on analogues that showed
the most potency in our chemotaxis assay, since that
measured the functional activity we wanted to optimize.
Selectivity. Many of the antagonists in this study
were examined against a panel of other 7TM GPCR to
determine their selectivity against other receptors. The
panel included other chemokine receptors (CCR1, CCR2,
CCR5, CXCR1, and CXCR2) as well as biogenic amine
and transporters, and the data for a few selected
analogues are summarized in Table 7. The selectivity
of the compounds highlighted in Table 7 is representa-
tive of the vast majority of this class of CCR3 antagonist.
They are more than 100-fold selective for CCR3 over
the other GPCRs using the CCR3 binding IC50 and
greater than 1000-fold selective using the chemotaxis
IC50 values. In general, they show some affinity for the
serotonin 5HT2A receptor subtype, dopamine D2, and
the dopamine and norepinephrine transporters as noted
in Table 7.
Figure 2. Chemotaxis SAR of compounds discussed in text.
An even more dramatic difference in potency is seen
between the 4-(4-fluorobenzyl)piperidine and the 3-(4-
fluorobenzyl)piperidine isomers. As shown in Figure 2,
the indole 78 (IC50 ) 21 nM) and the indazole 79 (IC50
) 23 nM) urea analogues in the 4-(4-fluorobenzyl)-
piperidine series are both about 1000-fold less potent
than the corresponding (S)-3-(4-fluorobenzyl)piperidine
series analogues 54 (IC50 ) 0.016 nM) and 56 (IC50
0.045 nM) in Table 6.
)
In addition, the conformational rigidity provided by
the trans cyclohexyl linker is also critical for the
potency. Again, there is large (about a 500-fold) differ-
ence in potency between the propyl linker series ana-
logue 77 (IC50 ) 25 nM in Figure 2) and the correspond-
ing cyclohexyl-linked analogue 32 (IC50 ) 0.034 nM in
Table 6).
Although the disconnection between the measured
binding affinity and the functional (calcium mobilization
and chemotaxis) assays is not understood, it is clear that
they are measuring different aspects of receptor phar-
macology. The fact that a Chinese hamster ovary (CHO)
cell line is used for the binding assay does not seem to
be an explanation. We have also measured the binding
affinities of several analogues using freshly isolated
human eosinophils and found only a small difference
(2- to 3-fold) in binding that does not account for the
large difference in functional activity using the same
cells (see Table 8).
Current models27 of GPCR activation, such as the
extended ternary complex model,28 propose different
conformational states of the receptor. Ligands having
different binding affinities for these various receptor
conformational states are able to produce a continuum
of receptor function.29 Viewed in this light, all our
We also examined the affinity of our compounds for
the CCR3 receptors of other species of interest. We
studied rat, mouse, and Cynomolgus monkey eosino-
phils in our CCR3 binding assay or in the chemotaxis

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2201
Table 7. GPCR Activities of Selected Analogues (nM)
pounds. Determining the binding potency was not
feasible for Cynomolgus monkey eosinophils, but in the
chemotaxis assay these compounds exhibit low picomo-
lar potency.
assay
32
2.0
65
0.9
74
0.7
a
b
b
b
CCR3 IC50
CCR1 IC50
CCR2 IC50
CCR5 IC50
>10 000 >10 000 >10 000
>10 000 >10 000 >10 000
In Vivo Pharmacology
>1000
>1000
>1000
b
CXCR1 IC50
CXCR2 IC50
>10 000 >10 000 >10 000
>10 000 >10 000 >10 000
Pharmacokinetics. Several analogues were exam-
ined for their oral bioavailability in species of interest,
and the data are summarized in Table 9. The perme-
ability for most analogues, as measured using the
Caco-2 assay, was reasonable. They were generally
highly protein bound (92-97%) and showed low to
moderate oral bioavailability, driven mainly by moder-
ate to high clearance in all species. The oral bioavail-
ability was best in the mouse and the chimp (15-22%
F) and less in the cyno (3-8% F), as summarized in
Table 9.
b
b
5HT2A IC50
920
1415
>1000
321
2350
718
611
3120
2600
487
3900
2260
1260
b
5HT2C IC50
>1000
1019
2938
492
b
D2 IC50
serotonin transporter, Ki
dopamine transporter, Ki
norepinephrine transporter, Ki 946
a The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown. b The value represents an average of usually two deter-
minations with a deviation of (30% of the mean value shown.
Table 8. Cross Reactivity of Selected Analogues (nM)
Animal Models of Eosinophil Migration. Having
established that these compounds were potent CCR3
antagonists in vitro, we next assessed their efficacy in
vivo. Since many of these compounds were active
against mouse CCR3 and had reasonable oral bioavail-
ability in mice, we examined these compounds in two
murine models of allergic airway inflammation.38 In the
first, mouse eotaxin, delivered intranasally to mice
previously sensitized to intraperitoneal (ip) injections
of ovalbumin (OVA), elicited a selective pulmonary
eosinophilia evident at 6 h in the bronchoalveolar lavage
(BAL) fluid. In the second model, an aerosolized OVA
challenge of OVA-sensitized mice stimulated pulmonary
inflammation in which eosinophils were a major com-
ponent in the BAL fluid at 24 h. The results are
summarized in Table 10.
assay
32
65
74
a
CCR3/CHO, IC50 2.0
0.9
0.7
0.5
Human eosb
0.5
0.034 ( 0.019 0.030 ( 0.020 0.010 ( 0.010
a
CCR3, IC50
0.8
chemotaxis, IC50
calcium, IC50
a
8
4
1
Mice eos
CCR3, IC50
chemotaxis, IC50
54 ( 21
41 ( 8
9 ( 4
10 ( 3
20 ( 8
20 ( 10
Rat eos
>1000
CCR3, IC50
>1000
>1000
Cyno eos
0.039 ( 0.018 0.007 ( 0.001 0.010 ( 0.010
chemotaxis, IC50
a The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown. b eos, eosinophils.
In the eotaxin challenge model, a dose dependent
inhibition of eosinophil recruitment into the lungs was
observed for all the compounds tested. The efficacy of
the compounds in this model was not uniform. While
both 32 and 65 showed efficacy consistent with their
mouse chemotaxis potency and pharmacokinetics (see
Tables 8 and 9), 74 was less efficacious than expected.
To test the efficacy in the OVA challenge model, the
compounds were administered orally at two doses,
corresponding to the EC50 (low dose) and 2-fold the EC90
(high dose) in the eotaxin challenge model. The ef-
fectiveness of 32 in the OVA challenge model was in
line with expectations of 86% reduction of eosinophil
infiltration observed at the high dose. In contrast, the
thiazole urea analogue 65 gave a similar response at
both doses with 60-69% reduction of eosinophils.
Clinical Candidate DPC168. Compounds were next
subjected to an extensive array of 117 selectivity assays
that included neurotransmitter, steroid, eicosanoid,
hormone, brain and gut peptide receptors, binding sites
on ion channels and transporters, and second messenger
and transport assays (performed by NovaScreen at 100
nM and 10 µM). This included all the 7TM GPCR assays
Table 9. PK Parameters of Selected Analogues
compounds
PK parameter
32
65
74
mouse F (%)
20
17
15
mouse t1/2 (h)
2.0
1.8
8
0.9
6.8
3
1.2
1.7
6
4.3
1.3
15
mouse CL ((L/h)/kg)
cyno F (%)
cyno t1/2 (h)
cyno CL ((L/h)/kg)
chimp F (%)
chimp t1/2 (h)
chimp CL ((L/h)/kg)
protein binding (hu) (%)
Caco-2, Papp (cm/s)
4.0
2.1
22
5.0
2.1
10
5.0
1.2
5.0
1.6
2.9
0.9
96.3
96.9
92.0
11 × 10-6
6 × 10-6
2 × 10-6
functional assay, and the data are summarized in Table
8. No compounds that were tested bind to rat CCR3.
Most did show affinity for mouse CCR3, with the acetyl-
phenyl urea analogue 32 (IC50 ) 54 nM) being less
potent than the thiazole urea 65 (IC50 ) 9 nM) or
tetrazole-containing phenyl urea analogue 74 (IC50
20 nM). The chemotaxis potency in the mouse correlates
well with the binding potency observed for these com-
)
Table 10. In Vivo Efficacy of Selected Analogues (po)
OVA challenge model (mice)
percent inhibition of eosinophils
eotaxin challenge model (mice)
compd
EC50 (mg/kg)
EC90 (mg/kg)
low dose
high dose
32
65
74
20
2.7
40
45
12.4
70
50% @ 30 mg/kg
60% @ 3 mg/kg
86% @ 100 mg/kg
69% @ 30 mg/kg

2202 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
De Lucca et al.
26587) available from Acros Organics. The 3-aminocyclohex-
anecarboxylic acid (catalog no. 26587) was obtained from TCI
America. The synthetic sequence used for the racemic com-
pounds is analogous to that described in detail below for the
chiral compounds using the same reaction conditions sum-
marized in Scheme 1.
1-((()-2-cis)-2-((4-Benzylpiperidin-1-yl)methyl)cyclo-
hexyl)-3-(3-methoxyphenyl)urea (5). This was synthesized
using the procedure outlined in Scheme 1, except using
commercially available 4-benzylpiperidine. 1H NMR (300 MHz,
CDCl3) δ TMS: 7.30-7.09 (m, 10 H), 6.93 (dd, J ) 1.5 Hz, J
) 8 Hz, 1 H), 6.61 (dd, J ) 2 Hz, J ) 8 Hz, 1 H), 3.87 (bs, 1
H), 3.81 (s, 3 H), 3.20 (bs, 1 H), 2.80 (bs, 1 H), 2.51 (m, 2 H),
2.08-1.20 (m, 18 H). MS ESI (M + H)+ ) 436.3. Anal. Calcd
for (C27H37N3O2‚(CHCl3)0.6): C, 65.36; H, 7.47; N, 8.28.
Found: C, 65.60; H, 7.48; N, 8.12.
available. In these assays, 32 exhibited greater than
100-fold selectivity in all assays (compared to the CCR3
binding IC50). Additionally, in vivo 5-day safety-assess-
ment studies were conducted in rats and dogs. In these
studies, 32 was well tolerated in both rats and dogs
without significant GI, CV, or CNS side effects up to
the maximum dose used (60 and 30 mg/kg, respectively).
Thus, while 32 was not ideal, with some affinity for
the dopamine transporter (Ki ) 492, Table 7), it is such
a potent inhibitor of eosinophil chemotaxis (IC50 ) 0.034
nM) that the potential therapeutic window was satisfac-
tory. This evaluation was also corroborated by the safety
assessment of 32 in rats and dogs. On the basis of its
overall potency, efficacy, and safety profile, 32, as the
benzenesulfonate salt, was advanced into phase I clini-
cal trials and designated as DPC168.
1-((()-2-trans-((4-Benzylpiperidin-1-yl)methyl)cyclo-
hexyl)-3-(3-methoxyphenyl)urea (6). This was synthesized
using the procedure outlined in Scheme 1, except using
commercially available 4-benzylpiperidine. 1H NMR (300 MHz,
CDCl3) δ TMS: 7.30-7.09 (m, 8 H), 6.78 (dd, J ) 1.5 Hz, J )
8 Hz, 1 H), 6.60 (dd, J ) 2 Hz, J ) 8 Hz, 1 H), 6.35 (bs, 1 H),
3.81 (s, 3 H), 3.28 (dt, J ) 3.3 Hz, J ) 10 Hz,1 H), 3.05 (bd, J
) 11 Hz, 1 H), 2.76 (bd, J ) 11 Hz, 1 H), 2.57-2.33 (m, 4 H),
Summary and Conclusions
Starting with our previously described20 class of CCR3
receptor antagonist, we improved the potency by replac-
ing the phenyl linker of 1 with a cyclohexyl linker and
by replacing the 4-benzylpiperidine with a 3-benzylpi-
peridine. The resulting compound, 32, is a potent and
selective antagonist of CCR3. SAR studies showed that
the 3-acetylphenyl urea of 32 could be replaced with
heterocyclic ureas or heterocyclic-substituted phenyl
ureas and still maintain the potency (inhibition of
eotaxin-induced chemotaxis) of this class of compounds
in the low picomolar range, representing some of the
most potent CCR3 antagonists reported to date. The
potency of 32 for mouse CCR3 (chemotaxis IC50 ) 41
nM) and its oral bioavailability in mice (20% F) were
adequate to assess the efficacy in animal models of
allergic airway inflammation. Oral administration of 32
reduced eosinophil recruitment into the lungs in a dose-
dependent manner in these animal models. The benzene-
sulfonate salt of 32, designated DPC168, entered phase
I clinical trials, and results from these studies will be
reported in due course. Our continuing efforts to further
improve this class of compounds will also be reported
in the near future.
2.08-1.89 (m, 2 H), 1.83-0.90 (m, 14 H). MS ESI (M + H)+
)
436.3. Anal. Calcd for (C27H37N3O2‚(H2O)0.66): C, 72.47; H, 8.63;
N, 9.39. Found: C, 72.46; H, 8.59; N, 9.18.
1-((()-2-trans-((4-(4-Fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexyl)-3-(3-methoxyphenyl)urea (7). This was
synthesized using the procedure outlined in Scheme 1, except
using commercially available 4-benzylpiperidine. 1H NMR (300
MHz, CDCl3) δ TMS: 7.66 (bs, 1 H), 7.20 (t, J ) 8 Hz, 1 H),
7.06-7.02 (m, 3 H), 6.98-6.92 (m, 2 H), 6.75 (bd, J ) 7 Hz, 1
H), 6.61 (dd, J ) 2 Hz, J ) 8 Hz, 1 H), 6.11 (bs, 1 H), 3.81 (s,
3 H), 3.26 (m, 1 H), 2.98 (bd, J ) 11 Hz, 1 H), 2.68 (bd, J ) 11
Hz, 1 H), 2.41-2.32 (m, 4 H), 2.02 (dd, J ) 2 Hz, J ) 13 Hz,
1 H), 1.89 (dt, J ) 2 Hz, J ) 11 Hz,1 H), 1.71-0.78 (m, 14 H).
MS ESI (M + H)+ ) 454.3. Anal. Calcd for (C27H36FN3O2‚
(H2O)0.33): C, 70.57; H, 8.04; N, 9.14. Found: C, 70.44; H, 7.92;
N, 9.04.
1-((()-2-trans-((4-(4-Fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexyl)-3-(3-acetylphenyl)urea (8). This was syn-
thesized using the procedure outlined in Scheme 1, except
using 4-(4-fluorobenzyl)piperidine. 1H NMR (300 MHz, CDCl3)
δ TMS: 7.95 (bs, 1 H), 7.62 (d, J ) 2 Hz, 1 H), 7.59 (d, J ) 2
Hz, 1 H), 7.56 (bs, 1 H), 7.36 (t, J ) 8 Hz, 1 H), 7.08-7.02 (m,
2 H), 6.99-6.93 (m, 2 H), 6.57 (bs, 1 H), 3.28 (m, 1 H), 3.07
(bd, J ) 10 Hz, 1 H), 2.79 (bd, J ) 10 Hz, 1 H), 2.61 (s, 3 H),
2.44 (d, J ) 7 Hz, 2 H), 2.50-2.33 (m, 2 H), 2.11-1.95 (m, 3
H), 1.84-0.98 (m, 13 H). MS ESI (M + H)+ ) 466.4. Anal.
Calcd for (C28H36FN3O2‚(H2O)0.66): C, 70.43; H, 7.88; N, 8.80.
Found: C, 70.46; H, 7.65; N, 8.72.
Experimental Section
General. All reactions were carried out under an atmo-
sphere of dry nitrogen. Commercial reagents were used
1
1-((1R,2S)-2-((4-(4-Fluorobenzyl)piperidin-1-yl)methyl-
)cyclohexyl)-3-(3-acetylphenyl)urea (+)-8. This was syn-
thesized using the procedure outlined for (()-8, except that
the tert-butyl 2-trans-((4-(4-fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexylcarbamate intermediate was resolved by chiral
HPLC (Chiralpak AD) and the first isomer obtained from the
without further purification. H NMR spectra were recorded
using tetramethylsilane (TMS) as an internal standard. TLC
was performed on E. Merck 15710 silica gel plates. Medium-
pressure liquid chromatography (MPLC) was carried out using
EM Science silica gel 60 (230-400 mesh). HPLC analysis was
carried out using Jasco PV-987 pumps, Jasco UV-975 detec-
tors, and DuPont Zorbax Sil or Zorbax NH2 1-in. preparative
columns. All final targets were obtained as noncrystalline
amorphous solids unless specified otherwise. Mass spectra
were measured with an HP5988A mass spectrometer with
particle beam interface using NH3 for chemical ionization or
a Finnigan MAT 8230 mass spectrometer with NH3-DCI or
VG TRIO 2000 for electrospray ionization (ESI). High-resolu-
tion mass spectra were measured on a VG 70-VSE instrument
with NH3 chemical ionization. Elemental analysis was per-
formed by Quantitative Technologies, Inc., Bound Brook, NJ.
For compounds where analysis was not obtained, HPLC
analysis was used, and purity was determined to be >95%.
Racemic analogues (5-16) in Tables 1 and 2 were synthe-
sized from the commercial (()-cis-2-hydroxymethyl-1-cyclo-
hexylamine hydrochloride (catalog no. 26586) and (()-trans-
2-hydroxymethyl-1-cyclohexylamine hydrochloride (catalog no.
column was used for the remainder of the synthesis to give
1
(+)-8. [R]25 ) +18.2° (MeOH, c ) 0.446 g/dL). H NMR (300
D
MHz, CDCl3) same as racemic above. MS ESI (M + H)+
)
466.3. Anal. Calcd for (C28H36FN3O2‚(H2O)0.50): C, 70.86; H,
7.86; N, 8.85. Found: C, 70.98; H, 7.72; N, 8.78.
1-((1S,2R)-2-((4-(4-Fluorobenzyl)piperidin-1-yl)methyl-
)cyclohexyl)-3-(3-acetylphenyl)urea (-)-8. This was syn-
thesized using the procedure outlined for (()-8, except that
the tert-butyl 2-trans-((4-(4-fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexylcarbamate intermediate was resolved by chiral
HPLC (Chiralpak AD) and the second isomer obtained from
the column was used to give (-)-8. [R]25 ) -20.5° (MeOH, c
D
) 0.434 g/dL). 1H NMR (300 MHz, CDCl3) same as racemic
above. MS ESI (M + H)+ ) 466.3. Anal. Calcd for (C28H36-
FN3O2‚(H2O)0.50): C, 70.86; H, 7.86; N, 8.85. Found: C, 71.00;
H, 7.66; N, 8.89.

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2203
1-((()-3-((4-(4-Fluorobenzyl)piperidin-1-yl)methyl)cy-
clohexyl)-3-(3-methoxyphenyl)urea (9). This was synthe-
sized using the procedure outlined in Scheme 1, except using
racemic 3-hydroxymethyl-1-cyclohexylamine (cis/trans) as the
starting amine and 4-(4-fluorobenzyl)piperidine. 1H NMR (300
MHz, CDCl3) δ TMS: 7.20 (t, J ) 8 Hz, 1 H), 7.06-7.02 (m, 2
H), 6.98-6.92 (m, 3 H), 6.75 (bd, J ) 7 Hz, 1 H), 6.62 (dd, J )
2 Hz, J ) 8 Hz, 1 H), 6.30 (bs, 1 H), 4.67 (d, J ) 8 Hz, 1 H),
3.79 (s, 3 H), 3.66 (m, 1 H), 2.79 (bt, J ) 11 Hz, 2 H), 2.08 (d,
J ) 7 Hz, 2 H), 2.08-1.92 (m, 1 H), 1.85-0.68 (m, 15 H). MS
ESI (M + H)+ ) 454.3. Anal. Calcd for (C27H36FN3O2‚
(H2O)0.10): C, 71.21; H, 8.01; N, 9.23. Found: C, 71.06; H, 8.06;
N, 9.05.
1-((()-3-((4-(4-Fluorobenzyl)piperidin-1-yl)methyl)cy-
clohexyl)-3-(3-acetylphenyl)urea (10). This was synthe-
sized using the procedure outlined in Scheme 1, except using
racemic 3-hydroxymethyl-1-cyclohexylamine (cis/trans) as the
starting amine and 4-(4-fluorobenzyl)piperidine. 1H NMR (300
MHz, CDCl3) δ TMS: 7.82 (bs, 1 H), 7.78 (d, J ) 8 Hz, 1 H),
7.60 (d, J ) 8 Hz, 1 H), 7.38 (t, J ) 8 Hz, 1 H), 7.09-7.04 (m,
2 H), 6.98-6.92 (m, 3 H), 4.96 (d, J ) 8 Hz, 1 H), 3.64 (m, 1
H), 2.79 (bt, J ) 10 Hz, 2 H), 2.60 (s, 3 H), 2.47 (d, J ) 7 Hz,
2 H), 2.08-1.98 (m, 3 H), 1.85-0.68 (m, 15 H). MS ESI (M +
H)+ ) 466.3. Anal. Calcd for (C28H36FN3O2‚(H2O)0.50): C, 70.86;
H, 7.86; N, 8.85. Found: C, 70.91; H, 7.89; N, 8.70.
1-(((R/S-2-trans)-2-(((R/S)-3-Benzylpiperidin-1-yl)meth-
yl)cyclohexyl)-3-(3-methoxyphenyl)urea (11). This was
synthesized using the procedure outlined in Scheme 1, except
using racemic 3-benzylpiperidine. The NMR shows a mixture
of two diastereomers. 1H NMR (300 MHz, CDCl3) δ TMS: 7.60
(bs, 0.5 H), 7.40 (bs, 0.5 H), 7.27 (m, 1 H), 7.20-7.08 (m, 5 H),
7.03 (m, 1 H), 6.74 (m, 1 H), 6.59-6.51 (m, 1 H), 6.09 and 6.05
(bs, 1 H), 3.79 and 3.75 (s, 3 H), 3.22 (m, 1 H), 2.96 (bd, J ) 10
Hz, 1 H), 2.61 (m, 1 H), 2.42-2.30 (m, 4 H), 2.05-1.85 (m, 1
H), 1.80-0.85 (m, 15 H). MS ESI (M + H)+ ) 436.3. Anal.
Calcd for (C27H37N3O2): C, 74.45; H, 8.56; N, 9.65. Found: C,
74.23; H, 8.68; N, 9.45.
1-(((R/S-2-trans)-2-(((R/S)-3-Benzylpiperidin-1-yl)meth-
yl)cyclohexyl)-3-(3-cyanophenyl)urea (12). This was syn-
thesized using the procedure outlined in Scheme 1, except
using racemic 3-benzylpiperidine. The NMR shows a mixture
of two diastereomers. 1H NMR (300 MHz, CDCl3) δ TMS: 7.90
(bs, 0.5 H), 7.70 (bs, 0.5 H), 7.71 (m, 1 H), 7.55 (m, 1 H), 7.38-
7.20 (m, 3 H), 7.19-7.13 (m, 3 H), 7.00 (m, 1 H), 6.26 and 6.16
(bs, 1 H), 3.21 (m, 1 H), 3.00 (bd, J ) 9 Hz, 1 H), 2.69 (m, 1
H), 2.42-2.30 (m, 4 H), 2.10-1.95 (m, 1 H), 1.80-0.85 (m, 15
H). MS ESI (M + H)+ ) 431.3. Anal. Calcd for (C27H34N4O‚
(H2O)0.25): C, 74.53; H, 7.99; N, 12.88. Found: C, 74.52; H,
8.07; N, 12.85.
1-(((R/S)-2-trans)-2-(((R/S)-3-Benzylpiperidin-1-yl)meth-
yl)cyclohexyl)-3-(3-acetylphenyl)urea (13). This was syn-
thesized using the procedure outlined in Scheme 1, except
using racemic 3-benzylpiperidine. The NMR shows a mixture
of two diastereomers. 1H NMR (300 MHz, CDCl3) δ TMS: 7.92
and 7.87 (t, J ) 2 Hz, 1 H), 7.80 (bs, 0.5 H), 7.65 (bs, 0.5 H),
7.65-7.50 (m, 2 H), 7.41-7.20 (m, 2 H), 7.19-7.03 (m, 3 H),
7.00 - 6.97 (m, 1 H), 6.31 and 6.22 (bs, 1 H), 3.21 (m, 1 H),
2.97 (bd, J ) 9 Hz, 1 H), 2.69 (m, 1 H), 2.60 and 2.56 (s, 3 H),
2.42-2.30 (m, 4 H), 2.10-1.95 (m, 1 H), 1.85-0.85 (m, 15 H).
MS ESI (M + H)+ ) 448.3. Anal. Calcd for (C28H37N3O2‚
(H2O)0.25): C, 74.38; H, 8.36; N, 9.29. Found: C, 74.46; H, 8.28;
N, 9.29.
1-(((R/S)-2-trans)-2-(((R/S)-3-(4-Fluorobenzyl)piperidin-
1-yl)methyl)cyclohexyl)-3-(3-methoxyphenyl)urea (14).
This was synthesized using the procedure outlined in Scheme
1, except using racemic 3-(4-fluorobenzyl)piperidine. The NMR
shows a mixture of two diastereomers. 1H NMR (300 MHz,
CDCl3) δ TMS: 7.60 (bs, 0.5 H), 7.40 (bs, 0.5 H), 7.20-7.08
(m, 3 H), 7.03-6.90 (m, 2 H), 6.82 (m, 1 H), 6.76 (m, 1 H),
6.59-6.51 (m, 1 H), 6.10 and 6.06 (bs, 1 H), 3.79 and 3.75 (s,
3 H), 3.22 (m, 1 H), 2.96 (bd, J ) 10 Hz, 1 H), 2.61 (m, 1 H),
2.42-2.30 (m, 4 H), 2.05-1.85 (m, 1 H), 1.80-0.85 (m, 15 H).
MS ESI (M + H)+ ) 454.3. Anal. Calcd for (C27H36FN3O2): C,
71.49; H, 8.00; N, 9.26. Found: C, 71.19; H, 8.01; N, 9.20.
1-(((R/S)-2-trans)-2-(((R/S)-3-(4-Fluorobenzyl)piperidin-
1-yl)methyl)cyclohexyl)-3-(3-cyanophenyl)urea (15). This
was synthesized using the procedure outlined in Scheme 1,
except using racemic 3-(4-fluorobenzyl)piperidine. The NMR
shows a mixture of two diastereomers. 1H NMR (300 MHz,
CDCl3) δ TMS: 7.69 (m, 1 H), 7.55 (m, 1 H), 7.39-7.20 (m, 3
H), 7.19-7.13 (m, 1 H), 7.01-6.85 (m, 2 H), 6.82 (m, 1 H), 6.30
and 6.22 (bs, 1 H), 3.21 (m, 1 H), 3.00 (m, 1 H), 2.69 (m, 1 H),
2.49-2.30 (m, 4 H), 2.10-1.95 (m, 1 H), 1.80-0.85 (m, 15 H).
MS ESI (M + H)+ ) 449.3. Anal. Calcd for (C27H33FN4O‚
(H2O)0.25): C, 71.58; H, 7.45; N, 12.37. Found: C, 71.47; H,
7.48; N, 12.27.
1-(((R/S)-2-trans)-2-(((R/S)-3-(4-Fluorobenzyl)piperidin-
1-yl)methyl)cyclohexyl)-3-(3-acetylphenyl)urea (16). This
was synthesized using the procedure outlined in Scheme 1,
except using racemic 3-(4-fluorobenzyl)piperidine. The NMR
shows a mixture of two diastereomers. 1H NMR (300 MHz,
CDCl3) δ TMS: 7.85 (m, 1 H), 7.75 (bs, 0.5 H), 7.65-7.50 (m,
2 H), 7.41-7.20 (m, 1.5 H), 7.19-7.03 (m, 1 H), 7.00-6.87 (m,
2 H), 6.76 (m, 1 H), 6.31 and 6.22 (bs, 1 H), 3.21 (m, 1 H), 2.97
(m, 1 H), 2.69 (m, 1 H), 2.61 and 2.69 (s, 3 H), 2.42-2.28 (m,
4 H), 2.10-1.95 (m, 1 H), 1.85-0.80 (m, 15 H). MS ESI (M +
H)+ ) 466.3. Anal. Calcd for (C28H36FN3O2‚(H2O)0.25): C, 71.54;
H, 7.83; N, 8.94. Found: C, 71.49; H, 7.85; N, 8.99.
As examples of the synthetic procedures used in this work,
the synthesis of 32, DPC168, and 65, DPC-A37818, are
described in detail below. All other compounds were made
using essentially the same chemistry.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-acetylphenyl)urea (32). Synthe-
sis of (S)-3-(4-Fluorobenzyl)piperidine (26). To a stirring
solution of N-benzyl-3-piperidone hydrochloride hydrate (4.2
g, 18.6 mmol) and 10% palladium on carbon (0.8 g) in degassed
methanol (200 mL) was added hydrogen gas to 55 psi. The
reaction mixture was stirred for 16 h and then filtered through
a pad of Celite. The Celite was washed with methanol (200
mL). The filtrates were combined and concentrated in vacuo
to a colorless oil. The oil was dissolved in tetrahydrofuran
(THF, 200 mL) and then treated with di-tert-butyl-dicarbonate
(5.27 g, 24.1 mmol) and saturated aqueous sodium bicarbonate
(50 mL). The reaction mixture was stirred for 4 h and then
concentrated in vacuo to a white solid. The solid was partioned
between EtOAc and 1 N HCl. The organic layer was separated,
washed with 1 N NaOH and brine, dried over Na2SO4, and
evaporated in vacuo to a colorless oil. The oil was purified by
flash chromatography (silica gel, hexane/EtOAc 3:1) to yield
2.93 g of tert-butyl 3-oxopiperidine-1-carboxylate 23 as a
colorless oil. 1H NMR (300 MHz, CDCl3) δ TMS: 3.99 (s, 2 H),
3.58 (t, J ) 6.3 Hz, 2 H), 2.46 (t, J ) 6.3 Hz, 2 H), 1.97 (p, J
) 6.3 Hz, 2 H), 1.45 (s, 9 H).
To a stirring solution of (4-fluorophenylmethyl)triphenyl-
phosphonium chloride (17.68 g, 43.5 mmol) in dry THF (60
mL) at -78 °C was added 2.5 M n-butyllithium in hexane (14.6
mL, 36.5 mmol). The reaction mixture was warmed to 0 °C
for 1 h, and the piperidone 23 (3.46 g, 17.4 mmol) in THF (60
mL) was added. The mixture was stirred at room temperature
for 1 h and then heated to reflux for 16 h. The reaction mixture
was cooled to room temperature, and the reaction was quenched
by the addition of saturated aqueous NH4Cl. The reaction
mixture was extracted with EtOAc (3 × 100 mL). The organic
layers were combined, washed with brine, dried over MgSO4,
and evaporated in vacuo to a pale yellow oil. The oil was
purified by flash chromatography (silica gel, hexane/EtOAc 9:1)
to yield 3.82 g of a mixture of E and Z isomers of olefin 24 as
a colorless oil. 1H NMR (300 MHz, CDCl3) δ TMS: 7.22-7.14
(m, 2 H), 7.04-6.98 (m, 2 H), 6.36 (s, 0.33 H), 6.28 (s, 0.67 H),
4.14 (s, 1.34 H), 4.00 (s, 0.66 H) 3.50 (app t, J ) 5.5 Hz, 2 H),
2.47 (t, J ) 5.1 Hz, 0.66 H), 2.39 (t, J ) 5.1 Hz, 1.34 H), 1.75-
1.68 (m, 1.34 H), 1.65-1.57 (m, 0.66 H), 1.48 (s, 9 H).
To a stirring solution of the olefin 24 (3.82 g, 13.1 mmol)
and 10% palladium on carbon (0.76 g) in degassed methanol
(200 mL) was added hydrogen gas to 55 psi. The reaction
mixture was stirred for 16 h and then filtered through a pad
of Celite. The Celite was washed with methanol (200 mL). The

2204 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
De Lucca et al.
filtrates were combined and concentrated in vacuo to yield 2.76
g of N-BOC-3-(4-fluorobenzyl)piperidine 25 as a colorless oil.
1H NMR (300 MHz, CDCl3) δ TMS: 7.12-7.07 (m, 2 H), 6.98-
6.93 (m, 2 H), 3.89 (dt, J ) 13.2 Hz, 4.0 Hz, 1 H), 3.84-3.74
(m, 1 H), 2.57-2.43 (m, 4 H), 1.75-1.60 (m, 4 H), 1.42 (s, 9
H), 1.15-1.09 (m, 1 H).
N-BOC-3-(4-fluorobenzyl)piperidine 25 (5 g) was dissolved
in 30 mL of 4 N HCl in dioxane. Some initial gassing occurred
which eventually subsided. After 1 h, the mixture was neutral-
ized with aqueous Na2CO3, and the dioxane was evaporated
off. The residue was extracted with ether, and the combined
ether extracts were dried over MgSO4 and evaporated off to
give 2.6 g of the free amine (()-26 as an oil.
water/1 N NaOH and stirred for 20 min. The organic layer
was separated and washed with water and brine. The solution
was dried over MgSO4; the organic solvent was removed under
vacuum, and the residue was chromatographed on basic
alumina (50% EtOAc/hexane) to give 32.1 g (73 mmol) of amine
1
20. H NMR (300 MHz, CDCl3) δ TMS: 7.79 (bs, 1 H), 7.38-
7.29 (m, 5 H), 6.95-6.84 (m, 4 H), 5.08 (m, 2 H), 3.06 (m, 1
H), 2.80 (m, 1 H), 2.55-2.36 (m, 2 H), 2.30 (dd, J ) 9 Hz, J )
13 Hz, 1 H), 2.05 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.81-0.90
(m, 16 H).
A solution of 20 (32 g, 73 mmol) in MeOH was treated with
8 g of 10% Pd/C and hydrogenated at 50 psi overnight in a
Parr apparatus. The mixture was filtered and the filtrate
concentrated on a rotary evaporator to give 20 g (65 mmol) of
the amine 21, which was used without further purification.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-acetylphenyl)urea (32). A solu-
tion of amine 21 (10 g, 32.8 mmol) in THF is treated with
3-acetylyphenyl isocyanate (5.3 g, 32.8 mmol), and the mixture
is stirred for 30 min. The solvent is removed on a rotary
evaporator, and the residue is chromatographed on silica gel
(0.5:4.5:95 NH4OH/MeOH/CH2Cl2) to give 11 g of urea 32 as
a solid. The urea 32 was further purified by a second chro-
matography on silica gel (40:60:1 EtAc/Hex/TEA) and final
recrystallization from ether to give a crystalline solid. Mp 115-
117 °C. 1H NMR (300 MHz, CDCl3) δ TMS: 7.86 (m, 1 H),
7.78 (bs, 1 H), 7.68-7.64 (m, 1 H), 7.62-7.59(m, 1 H), 7.38 (t,
J ) 8 Hz, 1 H), 6.95-6.90 (m, 2 H), 6.79-6.72 (m, 2 H), 6.25
(s, 1 H), 3.21 (dt, J ) 3 Hz, 11 Hz, 1 H), 3.00-2.97 (m, 1 H),
2.66-2.56 (m, 1 H), 2.61 (s, 3 H), 2.44-2.32 (m, 4 H), 2.06
(dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.86 (m, 15 H). MS ESI:
(M + H)+ ) 466.3 (100%). Anal. Calcd for C28H36N3O2F: C,
72.23; H, 7.70; N, 9.02. Found: C, 72.33; H, 7.91; N, 9.00.
Resolution of 3-(4-Fluorobenzyl)piperidine (26). Two
grams of the crude racemic 3-(4-fluorobenzyl)piperidine was
dissolved in 25 mL of acetonitrile and heated to reflux. The
solution was hazy. To this was added 1.56 g (1 equiv) of (R)-
(-)-mandelic acid dissolved in 15 mL of acetonitrile. Some
initial precipitation occurred when the cooler solution was
added, but it did redissolve when refluxing resumed. The heat
was turned off, and a small amount of enantiomerically pure
salt was added as the temperature dropped. At first, the seed
crystals dissolved, but when the temperature dropped to 75
°C, they remained suspended in the stirred solution. After a
few more degrees of cooling, crystal growth was obvious.
Cooling was continued at the rate of 1 deg/min. At 50 °C, the
solution was filtered to recover 0.9 g of salt, which melted at
164 °C. It was recrystallized from acetonitrile twice to give
(S)-(+)-3-(4-fluorobenzyl)piperidine mandelic acid salt in 98%
ee and melting at 168-171 °C. The salt was extracted into a
mixture of ether and 1 N NaOH. The ether extract was washed
with water and brine, dried over sodium sulfate, and evapo-
rated to give (S)-3-(4-fluorobenzyl)piperidine 26 as the free
base, which was used in subsequent reductive amination.
(1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexanamine (21). The preparation of ((1R,2R)-2-
aminocyclohexyl)methanol 17 was obtained following the
literature procedure24b except that we used LAH to reduce the
intermediate ester. To a solution of ((1R,2R)-2-aminocyclo-
hexyl)methanol 17 (9.5 g, 73.8 mmol) in CH2Cl2 (200 mL) is
added 200 mL of an aqueous solution of Na2CO3 (15 g, 141
mmol). While the mixture is stirred, benzyl chloroformate (12.6
g, 73.8 mmol) is added slowly, and the mixture is stirred at
room temperature for 1 h. The organic layer is separated and
washed with water and brine. The organic solvent is removed
on a rotary evaporator to give a white solid. The solid is
recrystallized from hexane to give 16.3 g (62 mmol) of the
alcohol 18 as a white solid. 1H NMR (300 MHz, CDCl3) δ
TMS: 7.40-7.29 (m, 5 H), 5.11 (s, 2 H), 4.71 (bd, 1 H), 3.76-
3.71 (m, 1 H), 3.53-3.28 (m, 3 H), 2.00-1.95 (m, 1 H), 1.90-
1.09 (m, 8 H). MS AP+ (M + H)+ ) 264.3 (100%).
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-acetylphenyl)urea‚Hydrochlo-
ride (32‚HCl). A solution of 32 (15 g, 32 mmol) in 300 mL of
THF was cooled in an ice bath and treated dropwise with 36
mL of a 1 M HCl/ether solution. The resulting solution was
stirred for 30 min and concentrated in vacuo. The resulting
solid was triturated with ether and the resulting white solid
dried under high vacuum overnight to give 16 g of the
hydrochloride salt designated as IQ657. Mp 58-60 °C. 1H
NMR (400 MHz, DMSO-d6) δ TMS: 9.61 (s, 1 H), 9.15 (s, 1
H), 8.00 (m, 1 H), 7.63-7.61 (m, 1 H), 7.51-7.49 (m, 1 H),
7.39-7.34 (m, 1 H), 7.22-7.17 (m, 2 H), 7.09-7.04 (m, 2 H),
6.86 (d, J ) 8 Hz, 1 H), 3.47-3.31 (m, 4 H), 3.11 (m, 1 H),
2.98-2.82 (m, 2 H), 2.67-2.62 (dd, J ) 5 Hz, J ) 13 Hz, 1 H),
2.58-2.50 (m, 2 H), 2.52 (s, 3 H), 2.39 (dd, J ) 8 Hz, J ) 13
Hz, 1 H), 2.16-2.06 (m, 2 H), 1.84-1.556 (m, 7 H), 1.30-1.00
(m, 4 H). Anal. Calcd for C28H37N3O2FCl‚H2O‚THF0.25
: C,
64.73; H, 7.68; N, 7.81. Found: C, 64.89; H, 7.41; N, 7.81.
A solution of DMSO (36 g, 430 mmol) in CH2Cl2 (200 mL)
is cooled to -78°C. To this solution is added dropwise oxalyl
chloride (27.41 g, 216 mmol), and the resulting solution is
stirred for an additional 10 min. A solution of alcohol 18 (38
g, 144 mmol) in CH2Cl2 (150 mL) is added via an addition
funnel and stirred for 10 min. Then, Et3N (58 g, 570 mmol) is
added and the solution is stirred for 20 min; the ice bath is
removed, and the solution is stirred for an additional 30 min.
The solution is diluted with water, and the organic layer is
separated and washed with water, 1 N HCl, and brine. The
organic layer is dried over Na2SO4, filtered, and concentrated
to give 38 g of aldehyde 19 as a white solid. The solid is
recrystallized from hexane to give 19.7 g of a first crop of
aldehyde 19 as white needles. A second crop gave an additional
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-acetylphenyl)urea-benzenesulfo-
nate (32‚Besylate): DPC168. Benzenesulfonic acid mono-
hydrate (1.06 g, 6 mmol) was dried by azeotroping off the water
of a benzene solution (twice) and adding the dried acid solution
to a solution of 32 (2.81 g, 6 mmol) in toluene (40 mL). The
solvents were removed in vacuo (twice) and the resulting
residue was recrystallized twice from toluene and dried under
high vacuum overnight to give 2.77 g of benzenesulfonic salt,
designated DPC168, as a white solid. Mp 157-159 °C. 1H NMR
(400 MHz, CDCl3) δ TMS: 9.46 (s, 1 H), 8.77 (s, 1 H), 8.29 (s,
1 H), 7.93 (m, 2 H), 7.55 (m, 2 H), 7.43 (m, 3 H), 7.31 (t, J )
8 Hz, 1 H), 6.95 (m, 2 H), 6.83 (m, 2 H), 6.64 (d, J ) 9 Hz, 1
H), 3.66 (m, 1 H), 3.56 (m, 1 H), 3.40 (m, 2 H), 2.57 (s, 3 H),
2.56-2.41 (m, 4 H), 2.37-2.29 (m, 2 H), 2.11-1.90 (m, 2 H),
1.86-1.70 (m, 6 H), 1.33-1.23 (m, 4 H), 1.00 (m, 1 H). Anal.
Calcd for C34H42N3O5FS: C, 65.47; H, 6.80; N, 6.75; S, 5.14.
Found: C, 65.48; H, 6.80; N, 6.70; S, 5.35.
1
11 g. H NMR (300 MHz, CDCl3) δ TMS: 9.59 (d, 3.6 Hz, 1
H), 7.38-7.28 (m, 5 H), 5.07 (m, 2 H), 4.69 (m, 1 H), 3.84 (m,
21 H), 2.19-2.11 (m,1 H), 2.09-2.01 (m, 1 H), 1.86-1.75 (m,
3 H), 1.54-1.17 (m, 4 H).
A solution of aldehyde 19 (19.6 g, 75 mmol) and (3S)-3-(4-
fluorophenylmethyl)piperidine 26 (14.5 g, 75 mmol) in dichlo-
roethane (400 mL) was treated with Na(OAc)3BH (32 g, 152
mmol) and stirred overnight at room temperature. The result-
ing solution was poured slowly into a stirred mixture of ice/
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(5-acetyl-4-methylthiazol-2-yl)urea
(65): DPC-A37818. Synthesis of Phenyl 5-Acetyl-4-me-
thylthiazol-2-ylcarbamate. In a round-bottom flask, NaH
60% dispersion in mineral oil (3.07 g, 77 mmol) was washed

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2205
2× with hexane and suspended in DMF. Then 2-amino-5-
acetyl-4-methyl-thiazole (10.0 g, 64 mmol) was added and
stirred while cooling in an ice bath. Stirring continued until
the NaH was consumed. Diphenyl carbonate (34 g, 160 mmol)
was added while cooling, and after the addition was complete,
the reaction mixture was stirred for an additional 30 min at
room temperature. The DMF was removed on a rotary
evaporator (high vacuum, 40 °C) to yield a brown residue. This
residue was dissolved in 1 L of CHCl3 and washed successively
with 2 L of 0.5 N HCl, 2 × 1 L of water, and finally with 1 L
of brine. The aqueous portions were back extracted twice with
300 mL of CHCl3. The combined organic fractions were dried
over anhydrous sodium sulfate, filtered, and concentrated on
a rotary evaporator to give a white solid. This was chromato-
graphed on silica (15-70% EtOAc/hexane) to give 15 g of the
desired carbamate as a white solid. Mp 180-182 °C. 1H NMR
(300 MHz, CDCl3) δ TMS: 11.42 (bs, 1 H), 7.47-7.40 (m, 2
H), 7.33-7.27 (m, 1 H), 7.22-7.18 (m, 2 H), 2.72 (s, 3 H), 2.50
(s, 3 H). ESI MS: (M + H)+ ) 277.1. Anal. Calcd for
C13H12N2O3S: C, 56.51; H, 4.39; N, 10.14; S, 11.60. Found: C,
56.42; H, 4.31; N, 10.11; S, 11.74.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(5-acetyl-4-methylthiazol-2-yl)urea
(65): DPC-A37818. A solution of (1R,2S)-2-[(3S)-3-(4-fluo-
robenzyl)piperidin-1-ylmethyl]cyclohexylamine (62.8 g, 206.3
mmol) and phenyl 5-acetyl-4-methylthiazol-2-ylcarbamate (57
g, 206.3 mmol) in a mixture of AcCN (6 L), CHCl3 (2 L), and
DMF (1 L) was stirred at room temperature overnight. The
solvents were removed under vacuum on a rotary evaporator,
and the residue was dissolved in methylene chloride, washed
with water and brine, and then dried over Na2SO4. The drying
agent was filtered off, and the filtrate was concentrated on a
rotary evaporator to give a solid. The solid was recrystallized
from acetonitrile (3 L) and EtOH (1 L) to give a first crop of
75 g of the desired urea. The mother liquor was concentrated,
and the solid was recrystallized from acetonitrile to give an
additional 17 g of urea 65 designated as DPC-A37818. Mp
174-175°C. [R]25D ) -40.4° (CHCl3, c ) 0.304 g/dL). 1H NMR
(500 MHz, DMSO-d6) δ TMS: 10.74 (bs, 1 H), 7.09-7.05 (m,
2 H), 6.98-6.930 (m, 2 H), 6.72 (bd, J ) 8 Hz, 1 H), 3.29-3.23
(m, 1 H), 2.69-2.67 (m, 1 H), 2.57 (bd, J ) 10 Hz, 1 H), 2.52
(s, 3 H), 2.43 (m, 1 H), 2.41 (s, 3 H), 2.33-2.26 (m, 2 H), 1.97-
1.93 (m, 1 H), 1.84-1.37 (m, 11 H), 1.24-1.10 (m, 3 H), 0.96-
0.79 (m, 2 H). ESI MS: (M + H)+ ) 487.3; HRMS: (M + H)+
) 487.256 1. Anal. Calcd for C26H35FN4O2S: C, 64.17; H, 7.26;
N, 11.51; S, 6.60. Found: C, 64.22; H, 7.34; N, 11.45; S, 6.67.
1-((1R,2S)-2-(((R)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-methoxyphenyl)urea (27). 1H
NMR (300 MHz, CDCl3) δ TMS: 7.27 (bs, 1 H), 7.16-7.01 (m,
4 H), 7.00-6.95 (m, 2 H), 6.74 (dd, J ) 1 Hz, J ) 8 Hz, 1 H),
6.54 (m, 1 H), 6.13 (bs, 1 H), 3.76 (s, 3 H), 3.26 (dt, J ) 10 Hz,
J ) 3 Hz, 1 H), 2.89 (bd, J ) 10 Hz, 1 H), 2.67 (bd, J ) 10 Hz,
1 H), 2.53 (m, 1 H), 2.41-2.37 (m, 4 H), 2.02 (dd, J ) 2 Hz, J
) 13 Hz, 1 H), 1.93 (bt, J ) 11 Hz, 1 H), 1.80-0.80 (m, 14 H).
MS ESI (M + H)+ ) 454.3. Anal. Calcd for (C27H36FN3O2): C,
71.49; H, 8.00; N, 9.26. Found: C, 71.23; H, 8.01; N, 9.15.
1-((1R,2S)-2-(((R)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-cyanophenyl)urea (28). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.71 (m, 1 H), 7.56 (bs, 1 H), 7.56-
7.52 (m, 1 H), 7.37 (m, 1 H), 7.35-7.29 (m, 1 H), 7.25 (m, 1
H), 7.13-7.08 (m, 2 H), 7.02-6.96 (m, 2 H), 6.31 (bs, 1 H),
3.23 (dt, J ) 10 Hz, J ) 4 Hz, 1 H), 2.96 (bd, J ) 10 Hz, 1 H),
2.72 (bd, J ) 10 Hz, 1 H), 2.58 (dd, J ) 5 Hz, J ) 13 Hz, 1 H),
2.45-2.32 (m, 4 H), 2.10-1.9 (m, 2 H), 1.83-0.85 (m, 14 H).
MS ESI (M + H)+ ) 449.3. Anal. Calcd for (C27H33FN4O‚
(H2O)0.33): C, 71.35; H, 7.46; N, 12.33. Found: C, 71.24; H,
7.29; N, 12.29.
H), 2.06 (dd, J ) 2 Hz, J ) 12 Hz, 1 H), 1.98 (m, 1 H), 1.78-
0.85 (m, 14 H). MS ESI: (M + H)+ ) 466.3.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-methoxyphenyl)urea (30). 1H
NMR (300 MHz, CDCl3) δ TMS: 7.60 (bs, 1 H), 7.17 (t, J ) 8
Hz, 1 H), 7.09 (t, J ) 2 Hz, 1 H), 6.85-6.76 (m, 2 H), 6.73 (dd,
J ) 1.5 Hz, J ) 8 Hz, 1 H), 6.59 (dd, J ) 2 Hz, J ) 8 Hz, 1 H),
6.04 (bs, 1 H), 3.80 (s, 3 H), 3.22 (m, 1 H), 2.96 (bd, J ) 10 Hz,
1 H), 2.61 (bd, J ) 10 Hz, 1 H), 2.39-2.30 (m, 4 H), 2.04 (dd,
J ) 2 Hz, J ) 13 Hz, 1 H), 1.83-0.85 (m, 15 H). MS ESI (M
+ H)+ ) 454.2. HRMS: calcd for C27H37FN3O2 (M + H)+,
454.287 0; found, 454.2883. Anal. Calcd for (C27H36FN3O2): C,
71.49; H, 8.00; N, 9.26. Found: C, 71.13; H, 8.00; N, 9.08.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-cyanophenyl)urea (31). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.86 (bs, 1 H), 7.68 (m, 1 H), 7.56-
7.52 (m, 1 H), 7.37 (m, 1 H), 7.30-7.27 (m, 1 H), 6.96-6.92
(m, 2 H), 6.85-6.78 (m, 2 H), 6.15 (bs, 1 H), 3.19 (m, 1 H),
2.99 (bd, J ) 10 Hz, 1 H), 2.65 (bd, J ) 10 Hz, 1 H), 2.44-2.32
(m, 4 H), 2.08 (dd, J ) 2 Hz, J ) 12 Hz, 1 H), 1.83-0.85 (m,
15 H). MS ESI (M + H)+ ) 449.2. HRMS: calcd for C27H34-
FN4O (M + H)+, 449.271 7; found, 449.2712. Anal. Calcd for
(C27H33FN4O‚(H2O)0.25): C, 71.58; H, 7.45; N, 12.37. Found: C,
71.72; H, 7.52; N, 12.27.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-phenylurea (33). 1H NMR (300 MHz,
CDCl3) δ TMS: 7.29-7.26 (m, 4 H), 7.05-6.99 (m, 1 H), 6.96-
6.91 (m, 2 H), 6.86-6.80 (m, 2 H), 6.04 (bs, 1 H), 3.22 (m, 1
H), 2.94 (bd, J ) 10 Hz, 1 H), 2.61 (bd, J ) 10 Hz, 1 H), 2.39-
2.30 (m, 4 H), 2.02 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.81
(m, 15 H). MS ESI (M + H)+ ) 424.2. HRMS: calcd for C26H35-
FN3O (M + H)+, 424.276 4; found, 424.275 5. Anal. Calcd for
(C26H34FN3O‚(H2O)0.25): C, 72.95; H, 8.12; N, 9.82. Found: C,
72.91; H, 8.10; N, 9.73.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-o-tolylurea (34). 1H NMR (300 MHz,
CDCl3) δ TMS: 7.57 (d, J ) 8 Hz, 1 H), 7.19-7.14 (m, 2 H),
7.08-6.85 (m, 6 H), 5.79 (bs, 1 H), 3.24 (m, 1 H), 2.85 (m, 1
H), 2.56 (bm, 1 H), 2.46-2.25 (m, 4 H), 2.22 (s, 3 H), 2.00 (dd,
J ) 12 Hz, J ) 2.5 Hz, 1 H), 1.70-0.83 (m, 15 H). MS ESI (M
+ H)+ ) 438.3. HRMS: calcd for C27H37FN3O (M + H)+,
439.292 1; found, 438.292 3. Anal. Calcd for (C27H36FN3O‚
(H2O)0.2): C, 73.50; H, 8.32; N, 9.52. Found: C, 73.59; H, 8.42;
N, 9.54.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(2-methoxyphenyl)urea (35). 1H
NMR (300 MHz, CDCl3) δ TMS: 8.18 (m, 1 H), 6.99-6.80 (m,
5 H), 6.94-6.88 (m, 2 H), 3.76 (s, 3 H), 3.20 (m, 1 H), 2.95 (m,
1 H), 2.58 (bm, 1 H), 2.46-2.31 (m, 4 H), 2.08 (dd, J ) 12 Hz,
J ) 2.5 Hz, 1 H), 1.80-0.88 (m, 15 H). MS ESI (M + H)+
)
454.3. HRMS: calcd for C27H37FN3O2 (M + H)+, 454.287 0;
found, 454.287 6. Anal. Calcd for (C27H36FN3O2): C, 71.49; H,
8.00; N, 9.26. Found: C, 71.18; H, 7.91; N, 9.13.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-methoxyphenyl)urea (36). 1H
NMR (400 MHz, CDCl3) δ TMS: 7.18 (m, 2 H), 7.09 (bs, 1 H),
7.00-6.96 (m, 2 H), 6.90-6.85 (m, 2 H), 6.81 (m, 2 H), 5.95
(bs, 1 H), 3.74 (s, 3 H), 3.27 (m, 1 H), 2.91 (m, 1 H), 2.64 (m,
1 H), 2.43-2.30 (m, 4 H), 2.04 (dd, J ) 13 Hz, J ) 3 Hz, 1 H),
1.80-0.88 (m, 15 H). MS ESI (M + H)+ ) 454.46.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(2-fluorophenyl)urea (37). 1H NMR
(400 MHz, CDCl3) δ TMS: 8.13 (m, 1 H), 8.00 (bs, 1 H), 7.12-
7.03 (m, 2 H), 6.97-6.91 (m, 3 H), 6.79-6.75 (m, 2 H), 6.21
(bs, 1 H), 3.19 (dt, J ) 3 Hz, J ) 10 Hz, 1 H), 2.99 (bd, J ) 10
Hz, 1 H), 2.61 (bd, J ) 10 Hz, 1 H), 2.40 (d, J ) 7 Hz, 2 H),
2.35-2.31 (m, 2 H), 2.07 (dd, J ) 2 Hz, J ) 12 Hz, 1 H), 1.83-
0.85 (m, 15 H). MS ESI (M + H)+ ) 442.47.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-fluorophenyl)urea (38). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.89 (bs, 1 H), 7.23-7.17 (m, 2 H),
6.97-6.91 (m, 3 H), 6.85-6.78 (m, 2 H), 6.71 (dt, J ) 2.2 Hz,
J ) 8 Hz, 1 H), 6.11 (bs, 1 H), 3.21 (dt, J ) 3 Hz, J ) 10 Hz,
1 H), 2.99 (bd, J ) 10 Hz, 1 H), 2.63 (bd, J ) 10 Hz, 1 H), 2.40
1-((1R,2S)-2-(((R)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-acetylphenyl)urea (29). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.86 (m, 1 H), 7.78 (bs, 1 H), 7.66-
7.62 (m, 1 H), 7.57-7.54 (m, 1 H), 7.35 (t, J ) 8 Hz, 1 H),
7.12-7.07 (m, 2 H), 7.02-6.96 (m, 2 H), 6.26 (s, 1 H), 3.28 (m,
1 H), 2.94 (bd, J ) 10 Hz, 1 H), 2.71 (bd, J ) 12 Hz, 1 H), 2.58
(dd, J ) 5 Hz, J ) 13 Hz, 1 H), 2.57 (s, 3 H), 2.44-2.33 (m, 3

2206 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
(d, J ) 7.3 Hz, 2 H), 2.35-2.31 (m, 2 H), 2.08 (dd, J ) 2 Hz,
De Lucca et al.
3 Hz, J ) 10 Hz, 1 H), 3.01 (bd, J ) 10 Hz, 1 H), 2.65 (bd, J
) 10 Hz, 1 H), 2.42 (d, J ) 7 Hz, 2 H), 2.39-2.31 (m, 2 H),
2.08 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.84-0.85 (m, 15 H). MS
ESI (M + H)+ ) 492.3. HRMS: calcd for C26H33Cl2FN3O (M +
H)+, 492.198 5; found, 492.198 4. Anal. Calcd for (C26H32Cl2-
FN3O): C, 63.41; H, 6.55; N, 8.53. Found: C, 63.13; H, 6.55;
N, 8.37.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3,4-dichlorophenyl)urea (46). 1H
NMR (300 MHz, CDCl3) δ TMS: 7.50 (d, J ) 2.5 Hz, 1 H),
7.32 (d, J ) 9 Hz, 1 H), 7.17 (dd, J ) 2.5 Hz, J ) 9 Hz, 1 H),
6.97-6.91 (m, 2 H), 6.88-6.80 (m, 2 H), 6.05 (bs, 1 H), 3.19
(m, 1 H), 2.99 (bd, J ) 10 Hz, 1 H), 2.64 (bd, J ) 10 Hz, 1 H),
2.43-2.31 (m, 4 H), 2.08 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.83-
0.81 (m, 15 H). MS ESI (M + H)+ ) 492.1. HRMS: calcd for
C26H33Cl2FN3O (M + H)+, 492.198 5; found, 492.197 5. Anal.
Calcd for (C26H32Cl2FN3O): C, 63.41; H, 6.55; N, 8.53. Found:
C, 63.44; H, 6.62; N, 8.37.
J ) 12 Hz, 1 H), 1.83-0.85 (m, 15 H). MS ESI (M + H)+
)
442.2. HRMS: calcd for C26H34F2N3O (M + H)+, 442.267 0;
found, 442.2693. Anal. Calcd for (C26H33F2N3O): C, 70.72; H,
7.53; N, 9.52. Found: C, 70.50; H, 7.53; N, 9.33.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-fluorophenyl)urea (39). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.26-7.20 (m, 2 H), 7.01-6.93 (m,
4 H), 6.88-6.80 (m, 2 H), 5.99 (bs, 1 H), 3.21 (m, 1 H), 2.94
(bd, J ) 10 Hz, 1 H), 2.61 (bd, J ) 10 Hz, 1 H), 2.41-2.30 (m,
4 H), 2.03 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.81 (m, 15
H). MS ESI (M + H)+ ) 442.2. HRMS: calcd for C26H34F2N3O
(M + H)+, 442.267 0; found, 442.269 6. Anal. Calcd for
(C26H33F2N3O): C, 70.72; H, 7.53; N, 9.52. Found: C, 70.54;
H, 7.58; N, 9.35.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3,4-difluorophenyl)urea (40). 1H
NMR (300 MHz, CDCl3) δ TMS): 7.65 (bs, 1 H), 7.38-7.30
(m, 1 H), 7.09-6.81 (m, 6 H), 6.14 (bs, 1 H), 3.20 (dt, J ) 3
Hz, J ) 10 Hz, 1 H), 2.96 (bd, J ) 10 Hz, 1 H), 2.63 (bd, J )
10 Hz, 1 H), 2.41 (d, J ) 7 Hz, 2 H), 2.38-2.31 (m, 2 H), 2.08
(dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.85 (m, 15 H). MS ESI
(M + H)+ ) 460.3. HRMS: calcd for C26H33F3N3O (M + H)+,
460.257 6; found, 460.258 7. Anal. Calcd for (C26H32F3N3O‚
(H2O)0.25): C, 67.29; H, 7.06; N, 9.05. Found: C, 67.19; H, 6.97;
N, 8.98.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-fluoro-4-methylphenyl)urea (41).
1H NMR (300 MHz, CDCl3) δ TMS: 7.60 (bs, 1 H), 7.12 (dd, J
) 1.8 Hz, J ) 12 Hz, 1 H), 7.08-6.81 (m, 6 H), 6.07 (bs, 1 H),
3.21 (dt, J ) 3 Hz, J ) 10 Hz, 1 H), 2.96 (bd, J ) 10 Hz, 1 H),
2.61 (bd, J ) 10 Hz, 1 H), 2.39-2.30 (m, 4 H), 2.19 (d, J ) 1.5
Hz, 3 H), 2.01 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.83 (m,
15 H). MS ESI (M + H)+ ) 456.3. HRMS: calcd for
C27H36F2N3O (M + H)+, 456.282 6; found, 456.282 4. Anal.
Calcd for (C27H35F2N3O): C, 71.18; H, 7.74; N, 9.22. Found:
C, 70.93; H, 7.65; N, 9.13.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(5-fluoro-2-methylphenyl)urea (42).
1H NMR (300 MHz, CDCl3) δ TMS: 7.62 (dd, J ) 11 Hz, J )
2.2 Hz, 1 H), 7.06 (m, 1 H), 6.98-6.82 (m, 5 H), 6.66 (dt, J )
2.5 Hz, J ) 8 Hz, 1 H), 5.83 (bs, 1 H), 3.22 (m, 1 H), 2.94 (m,
1 H), 2.60 (bm, 1 H), 2.45-2.30 (m, 4 H), 2.14 (s, 3 H), 2.05
(dd, J ) 12 Hz, J ) 2 Hz, 1 H), 1.80-0.83 (m, 15 H). MS ESI
(M + H)+ ) 456.3. HRMS: calcd for C27H36F2N3O (M + H)+,
456.282 6; found, 456.285 6. Anal. Calcd for (C27H35F2N3O‚
(H2O)0.2): C, 70.62; H, 7.77; N, 9.15. Found: C, 70.62; H, 7.63;
N, 8.96.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-chlorophenyl)urea (43). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.85 (bs, 1 H), 7.37 (s, 1 H), 7.21-
7.18 (m, 2 H), 7.01-6.81 (m, 5 H), 6.07 (bs, 1 H), 3.20 (dt, J )
3 Hz, J ) 10 Hz, 1 H), 3.00 (bd, J ) 10 Hz, 1 H), 2.64 (bd, J
) 10 Hz, 1 H), 2.40 (d, J ) 7 Hz, 2 H), 2.39-2.31 (m, 2 H),
2.08 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.86 (m, 15 H). MS
ESI (M + H)+ ) 458.3. HRMS: calcd for C26H34ClFN3O (M +
H)+, 458.237 4; found, 458.236 5. Anal. Calcd for (C26H33-
ClFN3O): C, 68.18; H, 7.26; N, 9.17. Found: C, 68.09; H, 7.27;
N, 8.99.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-chlorophenyl)urea (44). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.65 (bs, 1 H), 7.26 (bs, 2 H), 7.24
(bs, 2 H), 6.96-6.91 (m, 4 H), 6.87-6.80 (m, 2 H), 6.03 (bs, 1
H), 3.19 (dt, J ) 3 Hz, J ) 10 Hz, 1 H), 2.97 (bd, J ) 10 Hz,
1 H), 2.62 (bd, J ) 10 Hz, 1 H), 2.40 (d, J ) 7.3 Hz, 2 H),
2.38-2.31 (m, 2 H), 2.05 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.82-
0.82 (m, 15 H). MS ESI (M + H)+ ) 458.3. HRMS: calcd for
C26H34ClFN3O (M + H)+, 458.237 4; found, 458.237 7. Anal.
Calcd for (C26H33ClFN3O): C, 68.18; H, 7.26; N, 9.17. Found:
C, 68.56; H, 7.41; N, 9.04.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-bromophenyl)urea (47). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.75 (bs, 1 H), 7.52 (m, 1 H), 7.28-
7.22 (m, 1 H), 7.16-7.13 (m, 2 H), 6.97-6.92 (m, 4 H), 6.87-
6.81 (m, 2 H), 6.06 (bs, 1 H), 3.20 (dt, J ) 3 Hz, J ) 10 Hz, 1
H), 2.99 (bd, J ) 10 Hz, 1 H), 2.63 (bd, J ) 10 Hz, 1 H), 2.40
(d, J ) 7.3 Hz, 2 H), 2.35-2.31 (m, 2 H), 2.07 (dd, J ) 2 Hz,
J ) 13 Hz, 1 H), 1.82-0.82 (m, 15 H). MS ESI (M + H)+
)
502.3. HRMS: calcd for C26H34BrFN3O (M + H)+, 502.186 9;
found, 502.187 3. Anal. Calcd for (C26H33BrFN3O): C, 62.15;
H, 6.62; N, 8.36. Found: C, 62.17; H, 6.65; N, 8.24.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-bromophenyl)urea (48). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.65 (bs, 1 H), 7.48-7.36 (m, 2 H),
7.22-7.19 (m, 2 H), 6.96-6.91 (m, 4 H), 6.87-6.80 (m, 2 H),
6.03 (bs, 1 H), 3.19 (dt, J ) 3 Hz, J ) 10 Hz, 1 H), 2.97 (bd, J
) 10 Hz, 1 H), 2.61 (bd, J ) 10 Hz, 1 H), 2.40 (d, J ) 7.3 Hz,
2 H), 2.35-2.31 (m, 2 H), 2.05 (dd, J ) 2 Hz, J ) 13 Hz, 1 H),
1.82-0.82 (m, 15 H). MS ESI (M + H)+ ) 502.0. HRMS: calcd
for C26H34BrFN3O (M + H)+, 502.186 9; found, 502.186 8. Anal.
Calcd for (C26H33BrFN3O): C, 62.15; H, 6.62; N, 8.36. Found:
C, 62.11; H, 6.66; N, 8.21.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-cyano-4-fluorophenyl)urea (49).
1H NMR (300 MHz, CDCl3) δ TMS: 7.77 (bs, 1 H), 7.62 (dd, J
) 3 Hz, J ) 5 Hz, 1 H), 7.54-7.48 (m, 1 H), 7.12 (t, J ) 9 Hz,
1 H), 6.96-6.88 (m, 2 H), 6.87-6.80 (m, 2 H), 6.08 (bs, 1 H),
3.18 (dt, J ) 3 Hz, J ) 10 Hz, 1 H), 2.99 (bd, J ) 10 Hz, 1 H),
2.66 (bd, J ) 10 Hz, 1 H), 2.44 (d, J ) 7.3 Hz, 2 H), 2.39-2.31
(m, 2 H), 2.09 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.82-0.82 (m,
15 H). MS ESI (M + H)+ ) 467.3.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-acetylphenyl)urea (50). 1H NMR
(400 MHz, CDCl3) δ TMS: 7.86 (m, 3 H), 7.42 (m, 2 H), 6.93-
6.90 (m, 2 H), 6.80-6.76 (m, 2 H), 6.30 (bs, 1 H), 3.31 (dt, J )
3 Hz, 11 Hz, 1 H), 3.06 (m, 1 H), 2.66-2.56 (m, 1 H), 2.54 (s,
3 H), 2.46-2.32 (m, 4 H), 2.10 (m, 1 H), 1.90-0.86 (m, 15 H).
MS ESI: (M + H)+ ) 466.46.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3,5-diacetylphenyl)urea (51). 1H
NMR (300 MHz, CDCl3) δ TMS: 8.22 (d, J ) 1.5 Hz, 2 H),
8.19 (d, J ) 1.5 Hz, 1 H), 6.95-6.91 (m, 2 H), 6.73-6.67 (m, 2
H), 6.42 (bs, 1 H), 3.22 (m, 1 H), 3.05 (bd, J ) 12 Hz, 1 H),
2.68 (s, 6 H), 2.66 (bd, J ) 12 Hz, 1 H), 2.47-2.35 (m, 4 H),
2.08 (d, J ) 13 Hz, 1 H), 1.80-0.95 (m, 16 H). MS ESI (M +
H)+ ) 508.4. HRMS: calcd for C30H39FN3O3 (M + H)+,
508.297 5; found, 508.297 5. Anal. Calcd for (C30H38FN3O3‚
(H2O)0.5): C, 69.74; H, 7.61; N, 8.13. Found: C, 69.63; H, 7.51;
N, 8.04.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-acetyl-4-fluorophenyl)urea (52).
1H NMR (300 MHz, CDCl3) δ TMS: 7.87-7.83 (m, 1 H), 7.65
(bs, 1 H), 7.47 (dd, J ) 3 Hz, J ) 6 Hz, 1 H), 7.09 (dd, J ) 11
Hz, J ) 1 Hz, 1 H), 6.98-6.94 (m, 2 H), 6.84-6.78 (m, 2 H),
6.16 (bs, 1 H), 3.19 (dt, J ) 3 Hz, J ) 10 Hz, 1 H), 2.99 (bd, J
) 10 Hz, 1 H), 2.66 (d, J ) 5 Hz, 1 H), 2.66 (m, 1 H), 2.44-
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3,5-dichlorophenyl)urea (45). 1H
NMR (300 MHz, CDCl3) δ TMS: 7.85 (bs, 1 H), 7.30 (d, J )
1.9 Hz, 2 H), 7.01-6.84 (m, 5 H), 6.13 (bs, 1 H), 3.19 (dt, J )

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2207
2.33 (m, 4 H), 2.07 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.81-0.82
(m, 15 H). MS ESI (M + H)+ ) 484.3.
12.2 Hz, J ) 2.2 Hz, 1 H), 1.80-0.86 (m, 15 H). MS ESI (M +
H)+ ) 481.4. HRMS: calcd for C27H34FN4OS (M + H)+,
481.243 7; found, 481.244 3. Anal. Calcd for (C27H33FN4OS‚
(H2O)0.25): C, 66.84; H, 6.96; N, 11.55. Found: C, 66.71; H,
6.88; N, 11.53.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3H-benzo[d]imidazol-5-yl)urea (61).
1H NMR (400 MHz, CDCl3) δ TMS: 7.86 (s, 1 H), 7.80 (m, 2
H), 7.51 (bs, 1 H), 7.01 (m, 1 H), 6.99-6.75 (m, 5 H), 3.30 (dt,
J ) 10 Hz, J ) 3 Hz, 1 H), 2.86 (m, 1 H), 2.63 (m, 1 H), 2.43-
2.25 (m, 4 H), 2.10 (dd, J ) 13 Hz, J ) 3 Hz, 1 H), 1.80-0.70
(m, 15 H). MS ESI (M + H)+ ) 464.4. Anal. Calcd for (C27H34-
FN5O‚H2O): C, 67.34; H, 7.53; N, 14.54. Found: C, 67.01; H,
7.52; N, 14.32.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(1,3,4-thiadiazol-2-yl)urea (62). 1H
NMR (300 MHz, CDCl3) δ TMS: 8.67 (s, 1 H), 7.06-6.91 (m,
2 H), 6.79-6.73 (m, 2 H), 3.36 (m, 1 H), 2.88 (bd, J ) 10 Hz,
1 H), 2.59 (bd, J ) 10 Hz, 1 H), 2.49-2.15 (m, 4 H), 2.09 (dd,
J ) 13 Hz, J ) 4.4 Hz, 1 H), 1.85-0.83 (m, 15 H). MS ESI (M
+ H)+ ) 432.3. HRMS: calcd for C22H31FN5OS (M + H)+,
432.223 3; found, 432.224 9. Anal. Calcd for (C22H30FN5OS‚
(H2O)0.2): C, 60.72; H, 7.04; N, 16.09. Found: C, 60.72; H, 7.03;
N, 15.82.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(thiazol-2-yl)urea (63). 1H NMR (300
MHz, CDCl3) δ TMS: 7.37 (d, J ) 3.6 Hz, 1 H), 7.01-6.97 (m,
2 H), 6.87 (d, J ) 3.6 Hz, 1 H), 6.75-6.69 (m, 2 H), 3.28 (m, 1
H), 2.93 (bd, J ) 10 Hz, 1 H), 2.53 (bd, J ) 10 Hz, 1 H), 2.49-
2.22 (m, 4 H), 2.32 (s, 3 H), 2.06 (dd, J ) 13 Hz, J ) 2.5 Hz,
1 H), 1.80-0.86 (m, 15 H). MS ESI (M + H)+ ) 431.4. HRMS:
calcd for C23H32FN4OS (M + H)+, 431.228 1; found, 431.228 4.
Anal. Calcd for (C23H31FN4OS): C, 64.16; H, 7.26; N, 13.01.
Found: C, 63.79; H, 7.24; N, 12.86.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(1H-indol-6-yl)urea (53). 1H NMR
(300 MHz, CDCl3) δ TMS: 8.51 (bs, 1 H), 7.58 (s, 1 H), 7.48
(d, J ) 8 Hz, 1 H), 7.09 (m, 1 H), 6.99-6.79 (m, 5 H), 6.45 (s,
1 H), 6.33 (bs, 1 H), 3.28 (dt, J ) 10 Hz, J ) 4 Hz, 1 H), 2.83
(m, 1 H), 2.57 (m, 1 H), 2.40-2.20 (m, 4 H), 2.01 (dd, J ) 13
Hz, J ) 3 Hz, 1 H), 1.80-0.70 (m, 15 H). MS ESI (M + H)+
)
463.3. Anal. Calcd for (C28H35FN4O‚(H2O)0.10): C, 71.64; H,
7.53; N, 11.89. Found: C, 71.82; H, 7.69; N, 11.51.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(1H-indol-5-yl)urea (54). 1H NMR
(300 MHz, CDCl3) δ TMS: 8.25 (bs, 1 H), 7.50 (m, 1 H), 7.29
(m, 1 H), 7.20 (m, 1 H), 7.07 (dd, J ) 2 Hz, J ) 8 Hz, 1 H),
6.96-6.86 (m, 4 H), 6.46 (m, 1 H), 6.09 (bs, 1 H), 3.26 (m, 1
H), 2.79 (bd, J ) 10 Hz, 1 H), 2.56 (bd, J ) 10 Hz, 1 H), 2.38-
2.00 (m, 4 H), 1.99 (dd, J ) 4 Hz, J ) 12 Hz, 1 H), 1.83-0.85
(m, 16 H). MS ESI (M + H)+ ) 463.3.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(2,3-dimethyl-1H-indol-6-yl)urea
1
(55). H NMR (300 MHz, CDCl3) δ TMS: 7.81 (bs, 1 H), 7.38
(s, 1 H), 7.14 (d, J ) 7 Hz, 1 H), 6.97-6.84 (m, 5 H), 6.10 (bs,
1 H), 3.11 (dt, J ) 11 Hz, J ) 4 Hz, 1 H), 2.75 (m, 1 H), 2.55
(m, 1 H), 2.40-2.20 (m, 4 H), 2.34 (s, 3 H), 2.17 (s, 3 H), 2.01
(dd, J ) 12 Hz, J ) 4 Hz, 1 H), 1.80-0.70 (m, 15 H). MS ESI
(M + H)+ ) 491.4. Anal. Calcd for (C30H39FN4O‚(H2O)0.50): C,
72.11; H, 8.07; N, 11.21. Found: C, 72.01; H, 7.99; N, 11.00.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(1H-indazol-5-yl)urea (56). 1H NMR
(300 MHz, CDCl3) δ TMS: 10.45 (bs, 1 H), 7.94 (bs, 1 H), 7.69
(bs, 1 H), 7.60 (bs, 1 H), 7.38 (m, 1 H), 7.25 9m, 1 H), 6.97-
6.81 (m, 4 H), 6.26 (bs, 1 H), 3.28 (dt, J ) 3 Hz, J ) 10 Hz, 1
H), 2.92 (bd, J ) 10 Hz, 1 H), 2.65 (bd, J ) 10 Hz, 1 H), 2.42-
2.28 (m, 4 H), 2.06 (dd, J ) 2 Hz, J ) 13 Hz, 1 H), 1.80-0.83
(m, 15 H). MS ESI (M + H)+ ) 464.4. HRMS: calcd for C27H35-
FN5O (M + H)+, 464.282 6; found, 464.282 8.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-methylthiazol-2-yl)urea (64). 1H
NMR (300 MHz, CDCl3) δ TMS: 8.16 (bs, 1 H), 7.07-6.97 (m,
2 H), 6.74-6.69 (m, 2 H), 6.41 (d, J ) 0.8 Hz, 1 H), 3.24 (m, 1
H), 2.93 (bd, J ) 10 Hz, 1 H), 2.53 (bd, J ) 10 Hz, 1 H), 2.49-
2.22 (m, 4 H), 2.32 (s, 3 H), 2.06 (dd, J ) 13 Hz, J ) 2.5 Hz,
1 H), 1.80-0.86 (m, 15 H). MS ESI (M + H)+ ) 445.4. HRMS:
calcd for C24H34FN4OS (M + H)+, 445.243 7; found, 445.243 5.
Anal. Calcd for (C24H33FN4OS‚(H2O)0.1): C, 64.57; H, 7.50; N,
12.55. Found: C, 64.42; H, 7.54; N, 12.44.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(1H-indazol-6-yl)urea (57). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.97 (s, 1 H), 7.95 (d, J ) 9 Hz, 1
H), 7.59 (d, J ) 9 Hz, 1 H), 7.52 (bs, 1 H), 6.93-6.89 (m, 2 H),
6.82-6.73 (m, 3 H), 3.30 (m, 1 H), 2.95 (bd, J ) 10 Hz, 1 H),
2.66 (bd, J ) 10 Hz, 1 H), 2.45-2.29 (m, 4 H), 2.06 (dd, J ) 3
Hz, J ) 13 Hz, 1 H), 1.81-1.42 (m, 7 H), 1.39-0.84 (m, 8 H).
MS ESI (M + H)+ ) 464.4. HRMS: calcd for C27H35FN5O (M
+ H)+, 464.282 6; found, 464.283 7. Anal. Calcd for (C27H34-
FN5O‚(H2O)0.75): C, 67.97; H, 7.50; N, 14.68. Found: C, 68.17;
H, 7.35; N, 14.64.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-chloro-1H-indazol-5-yl)urea (58).
1H NMR (300 MHz, CDCl3) δ TMS: 7.92 (bs, 1 H), 7.79 (m, 1
H), 7.30-7.23 (m, 3 H), 7.00 (bs, 1 H), 6.97-6.89 (m, 2 H),
6.85-6.81 (m, 2 H), 3.40 (m, 1 H), 3.30 (m, 1 H), 3.02 (bd, J )
8 Hz, 1 H), 2.85 (m, 1 H), 2.45-2.28 (m, 3 H), 2.19-1.95 (m,
4 H), 1.80-1.42 (m, 5 H), 1.39-0.84 (m, 7 H). MS ESI (M +
H)+ ) 498.3.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(indolin-5-yl)urea (59). 1H NMR
(300 MHz, CDCl3) δ TMS: 7.06-6.99 (m, 3 H), 6.94-6.88 (m,
2 H), 6.81 (d, J ) 8 Hz, 1 H), 6.53 (d, J ) 8 Hz, 1 H), 5.81 (bs,
1 H), 3.53 (m, 2 H), 3.25 (m, 1 H), 2.96 (m, 2 H), 2.80 (bd, J )
10 Hz, 1 H), 2.58 (bd, J ) 9 Hz, 1 H), 2.43-2.25 (m, 4 H), 2.00
(dd, J ) 12 Hz, J ) 2.5 Hz, 1 H), 1.80-0.86 (m, 15 H). MS
ESI (M + H)+ ) 465.4. HRMS: calcd for C28H38FN4O (M +
H)+, 465.303 0; found, 465.304 6. Anal. Calcd for (C28H37FN4O‚
(H2O)0.75): C, 70.34; H, 8.12; N, 11.72. Found: C, 70.36; H,
7.85; N, 11.49
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(benzo[d]thiazol-6-yl)urea (60). 1H
NMR (300 MHz, CDCl3) δ TMS: 8.87 (s, 1 H), 8.22 (d, J ) 2.2
Hz, 1 H), 8.01 (d, J ) 8.8 Hz, 1 H), 7.78 (bs, 1 H), 7.20 (dd, J
) 8.8 Hz, J ) 2.2 Hz, 1 H), 6.94-6.89 (m, 2 H), 6.82-6.76 (m,
2 H), 6.23 (bs, 1 H), 3.24 (m, 1 H), 2.96 (bd, J ) 10 Hz, 1 H),
2.63 (bd, J ) 10 Hz, 1 H), 2.41-2.32 (m, 4 H), 2.08 (dd, J )
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-chlorobenzo[d]thiazol-2-yl)-
1
urea (66). H NMR (300 MHz, CDCl3) δ TMS: 7.68 (dd, J )
1.1 Hz, J ) 8.0 Hz, 1 H), 7.44 ((dd, J ) 1.1 Hz, J ) 7.6 Hz, 1
H), 7.20 (t, J ) 8.0 Hz, 1 H), 7.04-6.99 (m, 2 H), 6.70-6.64
(m, 2 H), 3.27 (m, 1 H), 3.00 (bd, J ) 11 Hz, 1 H), 2.63 (bd, J
) 11 Hz, 1 H), 2.45-2.31 (m, 4 H), 2.12 (dd, J ) 11 Hz, J )
2.5 Hz, 1 H), 1.88-0.86 (m, 15 H). MS ESI (M + H)+ ) 515.4.
HRMS: calcd for C27H33ClFN4OS (M + H)+, 515.204 8; found,
515.206 1. Anal. Calcd for (C27H32ClFN4OS): C, 62.96; H, 6.26;
N, 10.88. Found: C, 62.84; H, 6.37; N, 10.56.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(1-methyl-1H-pyrazol-3-yl)urea (67).
1H NMR (300 MHz, CDCl3) δ TMS: 7.19 (d, J ) 2.2 Hz, 1 H),
7.17-7.02 (m, 2 H), 6.89-6.83 (m, 2 H), 6.56 (bs, 1 H), 6.01
(d, J ) 1.5 Hz, 1 H), 3.76 (s, 3 H), 3.36 (m, 1 H), 2.79 (bd, J )
10 Hz, 1 H), 2.68 (bd, J ) 8 Hz, 1 H), 2.51-2.28 (m, 3 H), 2.18
(bd, J ) 13 hz, 1 H), 2.08 (dd, J ) 13 Hz, J ) 2.5 Hz, 1 H),
1.98-0.82 (m, 15 H). MS ESI (M + H)+ ) 428.4. HRMS: calcd
for C24H35FN5O (M + H)+, 428.282 6; found, 428.283 1. Anal.
Calcd for (C24H34FN5O‚(H2O)0.33): C, 66.50; H, 8.06; N, 16.15.
Found: C, 66.41; H, 8.01; N, 16.26.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(5-methylisoxazol-3-yl)urea (68). 1H
NMR (300 MHz, CDCl3) δ TMS: 7.33 (bs, 1 H), 7.11-6.98 (m,
2 H), 6.89-6.83 (m, 2 H), 6.18 (s, 1 H), 3.31 (m, 1 H), 2.85 (bd,
J ) 10 Hz, 1 H), 2.65 (bd, J ) 8 Hz, 1 H), 2.49-2.28 (m, 3 H),
2.37 (s, 3 H), 2.18 (bd, J ) 13 Hz, 1 H), 2.08 (dd, J ) 13 Hz,
J ) 2.5 Hz, 1 H), 1.90-0.82 (m, 15 H). MS ESI (M + H)+
)
429.4. HRMS: calcd for C24H34FN4O2 (M + H)+, 429.266 6;

2208 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
De Lucca et al.
found, 429.264 8. Anal. Calcd for (C24H33FN4O2‚(H2O)0.1): C,
66.98; H, 7.78; N, 13.02. Found: C, 66.72; H, 7.80; N, 12.82.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(pyridin-4-yl)urea (69). 1H NMR
(300 MHz) (CDCl3) δ TMS: 8.38 (d, J ) 7 Hz, 2 H), 7.57 (s, J
) 7 Hz, 1 H), 7.31 (d, J ) 7 Hz, 2 H), 7.00-6.84 (m, 2 H),
6.84-6.70 (m, 2 H), 3.15 (m, 1 H), 3.07 (m, 1 H), 2.76 (m, 1
H), 2.60-2.46 (m, 1 H), 2.42 (d, J ) 7 Hz, 2 H), 2.20 (m, 2 H),
2.00-1.80 (m, 3 H), 1.80-1.40 (m, 7 H), 1.20-0.80 (m, 4 H).
MS ESI (M + H)+ ) 425.
dissolved in dichloromethane (1.4 L), and 2,6-lutidine (44.1 g,
411 mmol) was added. Phenyl chloroformate (21.2 g, 136 mmol)
was added in four portions over 15 min, and the mixture was
stirred for 1.5 h. The mixture was poured into 1 N aqueous
hydrochloric acid (200 mL), and the mixture was extracted
three times with dichloromethane (200 mL). The combined
organic layers were washed with saturated aqueous sodium
chloride, dried over sodium sulfate, and concentrated under
vacuum. The crude brown material was dissolved in hot
toluene, filtered, and allowed to precipitate at 0 °C to give a
white solid (34.1 g). The filtrate was concentrated and recrys-
tallized from toluene to give an additional crop of phenyl 3-(1-
methyl-1H-tetrazol-5-yl)phenylcarbamate as off-white crystals
(3.44 g, 93% total). 1H NMR (300 MHz, CDCl3), δ TMS: 10.51
(bs, 1 H), 8.01 (s, 1 H), 7.71 (dt, J ) 7, 2 Hz, 1 H), 7.55 (m, 2
H), 7.41 (m, 2 H), 7.24 (m, 2 H), 4.14 (s, 3 H).
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-(1-methyl-1H-tetrazol-5-yl)phenyl)-
urea (74). A solution of (1R,2S)-2-(((S)-3-(4-fluorobenzyl)-
piperidin-1-yl)methyl)cyclohexanamine (30.7 g, 101 mmol) and
phenyl 3-(1-methyl-1H-tetrazol-5-yl)phenylcarbamate (29.8 g,
101 mmol) in acetonitrile (800 mL) was stirred overnight at
room temperature. The mixture was concentrated in vacuo,
and the residue was purified over silica gel, eluting with 60:
40 CH2Cl2/EtOAc-95:5 CH2Cl2/MeOH-90:10 CH2Cl2/MeOH,
to yield 48.5 g of a white powder. This powder was dried
overnight under vacuum at 53 °C to remove residual solvents,
to yield 43.7 g of white powder as product. 1H NMR (300 MHz,
CDCl3) δ: 8.05-7.92 (m, 1 H), 7.80 (bs, 1 H), 7.56-7.36 (m, 3
H), 6.96 (dd, J ) 9 Hz, J ) 5 Hz, 2 H), 6.76 (t, J ) 9 Hz, 2 H),
6.39 (bs, 1 H), 4.22 (s, 3 H), 3.22 (dt, J ) 3 Hz, J ) 10 Hz, 1
H), 3.02 (d, J ) 11 Hz, 1 H), 2.69 (d, J ) 10 Hz, 1 H), 2.49-
2.26 (m, 4 H), 2.12 (dd, J ) 13 Hz, J ) 2 Hz, 1 H), 1.98-0.75
(m, 15 H). HRMS (M + H)+ ) 506.303 7. Anal. Calcd for
(C28H36FN7O): C, 66.51; H, 7.18; N, 19.39; F, 3.76. Found: C,
66.18; H, 7.14; N, 19.18; F, 4.06.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-cyano-4-(1H-pyrazol-1-yl)phen-
1
yl)urea (70). H NMR (300 MHz, CDCl3) δ TMS: 8.04 (m, 1
H), 7.79 (m, 2 H), 7.79 (bs, 1 H), 7.64 (m, 2 H), 6.99-6.86 (m,
2 H), 6.81-6.82 (m, 2 H), 6.53 (m, 1 H), 6.28 (bs, 1 H), 3.20
(m, 1 H), 3.02 (bd, J ) 11 Hz, 1 H), 2.68 (bd, J ) 11 Hz, 1 H),
2.45 (d, J ) 7 Hz, 2 H), 2.39-2.35 (m, 2 H), 2.08 (dd, J ) 13
Hz, J ) 2.5 Hz, 1 H), 1.90-0.82 (m, 15 H). MS ESI (M + H)+
) 515.3. Anal. Calcd for (C30H35FN6O‚(H2O)0.5): C, 68.81; H,
6.93; N, 16.05. Found: C, 68.80; H, 6.74; N, 15.98.
1-(4-(1H-1,2,4-Triazol-1-yl)phenyl)-3-((1R,2S)-2-(((S)-3-
(4-fluorobenzyl)piperidin-1-yl)methyl)cyclohexyl)urea
1
(71). H NMR (300 MHz, CDCl3) δ TMS: 8.47 (s, 1 H), 8.09
(s, 1 H), 7.72 (bs, 1 H), 7.57 (m, 2 H), 7.46 (m, 2 H), 6.99-6.93
(m, 2 H), 6.82 (m, 2 H), 6.30 (bs, 1 H), 3.22 (dt, J ) 10 Hz, J
) 3 Hz, 1 H), 3.00 (m, 1 H), 2.42-2.35 (m, 4 H), 2.10 (m, 1 H),
1.85-0.75 (m, 15 H). MS ESI (M + H)+ ) 491.3. Anal. Calcd
for (C28H35FN6O‚(H2O)0.25): C, 67.92; H, 7.23; N, 16.97.
Found: C, 67.86; H, 7.30; N, 16.81.
As an example of the tetrazole containing phenyl urea
analogues, a detailed procedure for 74 is given below. The
other analogues where synthesized using similar procedures.
Preparation of Phenyl 3-(1-Methyl-1H-tetrazol-5-yl)-
phenylcarbamate. 3-Nitrobenzoyl chloride (7.00 g, 37.7
mmol) was dissolved in tetrahydrofuran (300 mL), and meth-
ylamine (41.5 mL of a 2.0 M solution in tetrahydrofuran, 82.9
mmol) was added. The reaction mixture was stirred for 2 h.
The reaction mixture was diluted with ethyl acetate and
washed three times with water. The organic layer was dried
over sodium sulfate and concentrated to provide N-methyl-3-
nitro-benzamide as a solid (6.38 g, 94%) which was used
without further purification. 1H NMR (300 MHz, CDCl3), δ
TMS: 8.84 (bs, 1 H), 8.67 (m, 1 H), 8.37 (dd, J ) 8, 2 Hz, 1 H),
8.28 (d, J ) 7 Hz, 1 H), 7.78 (dd, J ) 8, 7 Hz, 1 H), 2.83 (m, 3
H). MS ESI (M + H)+ ) 181.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(3-(5-methyl-1H-tetrazol-1-yl)phenyl)-
urea (72). 1H NMR (300 MHz) (CDCl3) δ TMS: 7.75 (s, 1 H),
7.44 (t, J ) 7 Hz, 1 H), 7.30-7.20 (m, 2 H), 7.10 (d, J ) 7 Hz,
1 H), 7.00-6.85 (m, 2 H), 6.77 (t, J ) 7 Hz, 2 H), 6.36 (s, 1 H),
3.28-3.10 (m, 1 H), 3.05-2.90 (m, 1 H), 2.75-2.60 (m, 1 H),
2.60 (s, 3 H), 2.50-2.25 (m, 4 H), 2.15-2.00 (m, 1 H), 1.90-
1.55 (m, 9 H), 1.55-1.10 (m, 4 H), 1.10-0.85 (m, 2 H). MS
ESI (M + H)+ ) 506.
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(2-(1-methyl-1H-tetrazol-5-yl)phenyl)-
urea (73). 1H NMR (300 MHz) (CDCl3) δ TMS: 8.85 (s, 1 H),
8.40 (d, J ) 7 Hz, 1 H), 8.20 (bs, 1 H), 7.57 (t, J ) 7 Hz, 1 H),
7.38 (d, J ) 7 Hz, 1 H), 7.11 (t, J ) 7 Hz, 1 H), 6.95-6.60 (m,
2 H), 6.51 (t, J ) 7 Hz, 2 H), 4.13 (s, 3 H), 3.20-2.90 (m, 2 H),
2.65-2.20 (m, 5 H), 2.20-1.95 (m, 2 H), 1.95-1.10 (m, 13 H),
1.10-0.85 (m, 3 H). MS ESI (M + H)+ ) 506.
N-Methyl-3-nitro-benzamide (30.0 g, 167 mmol) was dis-
solved in acetonitrile (835 mL); sodium azide (10.9 g, 167
mmol) was added, and the mixture was cooled in an ice bath.
Trifluoromethanesulfonic anhydride (29 mL, 172 mmol) was
added dropwise at a rate which maintained the internal
temperature below 3 °C. The reaction mixture was stirred for
3.5 h at 0 °C. The solution was diluted with dichloromethane
and then was poured into 1 N aqueous sodium hydroxide (100
mL). The organic layer was separated, dried over sodium
sulfate, and concentrated under vacuum to 50 mL, and water
was added to precipitate a yellow solid (18.46 g, 54%). A second
crop of 1-methyl-5-(3-nitrophenyl)tetrazole crystals was ob-
tained by concentrating the filtrate in vacuo and adding it to
boiling ethyl acetate. When the mixture was cooled to 0 °C,
additional material (6.07 g, 18%) was isolated by filtration.
1H NMR (300 MHz, CDCl3), δ TMS: 8.67 (m, 1 H), 8.49 (dd, J
) 8, 2 Hz, 1 H), 8.31 (d, J ) 8 Hz, 1 H), 7.94 (dd, J ) 8, 8 Hz,
1 H), 4.22 (s, 3 H).
1-Methyl-5-(3-nitrophenyl)tetrazole (28.8 g, 140 mmol) was
dissolved in ethyl acetate (430 mL) and methanol (1270 mL).
Palladium on carbon (2.7 g, 10 wt %) was added, and the
mixture was shaken under a hydrogen atmosphere (60 psi)
for 1.5 h. The mixture was filtered, and the filtrate was
concentrated under vacuum to give 1-methyl-5-(3-aminophen-
yl)tetrazole as a white solid (24.0 g, 98%) which was used
without further purification. 1H NMR (300 MHz, CDCl3), δ
TMS: 7.21 (dd, J ) 8, 7 Hz, 1 H), 6.99 (s, 1 H), 6.90 (d, J ) 7
Hz, 1 H), 6.76 (d, J ) 8 Hz, 1 H), 5.44 (bs, 2 H), 4.10 (s, 3 H).
1-Methyl-5-(3-aminophenyl)tetrazole (24.0 g, 137 mmol) was
1-((1R,2S)-2-(((S)-3-(4-Fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexyl)-3-(4-(1-methyl-1H-tetrazol-5-yl)phenyl)-
1
urea (75). H NMR (300 MHz) (CDCl3) δ TMS: 7.65 (d, J )
7 Hz, 1 H), 7.54 (d, J ) 7 Hz, 1 H), 7.00-6.85 (m, 2 H), 6.80-
6.60 (m, 3 H), 4.12 (s, 3 H), 3.36-3.15 (m, 1 H), 3.10-2.90 (m,
1 H), 2.70 (d, J ) 7 Hz, 1 H), 2.50-2.20 (m, 4 H), 2.10 (d, J )
7 Hz, 1 H), 1.95-1.59 (m, 11 H), 1.59-0.85 (m, 4H). MS ESI
(M + H)+ ) 506.
1-(3,5-Bis(1-methyl-1H-tetrazol-5-yl)phenyl)-3-((1R,2S)-
2-(((S)-3-(4-fluorobenzyl)piperidin-1-yl)methyl)cyclohex-
1
yl)urea (76). H NMR (300 MHz, CDCl3) δ TMS: 8.23 (d, J
) 1.83 Hz, 2 H), 7.85 (t, J ) 1.65 Hz, 1 H), 7.19 (m, 1 H), 6.98
(m, 2 H), 6.70 (t, J ) 8.79 Hz, 2 H), 4.31 (s, 6 H), 3.25 (m, 1
H), 3.00 (d, J ) 11.35 Hz, 1 H), 2.70 (d, J ) 7.32 Hz, 1 H),
2.36 (m, 4 H), 2.11 (dd, J ) 12.82, 2.56 Hz, 1 H), 1.85-1.70
(m, 9 H), 1.65-0.80 (m, 6 H). MS ESI (M + H)+ ) 588.5
1-((1R,2S)-2-((4-(4-Fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexyl)-3-(1H-indol-5-yl)urea (78). 1H NMR (300
MHz, CDCl3) δ TMS: 8.27 (bs, 1 H), 7.52 (m, 1 H), 7.35 (d, J
) 11 Hz, 1 H), 7.23 (m, 1 H), 7.07 (dd, J ) 2 Hz, J ) 9 Hz, 1
h), 6.92 (d, J ) 7 Hz, 4 H), 6.54 (m, 1 H), 6.12 (bs, 1 H), 3.31

Discovery of CC Chemokine Receptor-3 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2209
(m, 1 H), 2.86 (bd, J ) 11 Hz, 1 H), 2.61 (bd, J ) 11 Hz, 1 H),
2.41-2.30 (m, 2 H), 1.99-1.90 (m, 3 H), 1.80-0.90 (m, 15 H).
MS ESI (M + H)+ ) 463.3.
changes in fluorescence in all wells simultaneously. To deter-
mine the IC50, compound or buffer alone was added and cells
were incubated for 5 min; eotaxin was then added to a final
concentration of 10 nM. The fluorescence shift was monitored,
and the base-to-peak excursion was computed automatically.
All conditions were tested in duplicate, and the mean shift
per condition was determined. The inhibition achieved by
graded concentrations of compound was calculated as a
percentage of the compound-free eotaxin control.
Animal Models of Eosinophil Migration. 1. Animals.
Female Balb/c (18-20 g) and male C57/B6 (18-20 g) mice were
obtained from Charles River Laboratories (Raleigh, NC).
Animals were housed at the Bristol Myers Squibb animal care
facility under a 12 h light-dark cycle and were allowed food
and water ad libitum. All animal protocols were reviewed and
approved by the Bristol Myers Squibb Wilmington Site Animal
Care and Use Committee.
1-((1R,2S)-2-((4-(4-Fluorobenzyl)piperidin-1-yl)meth-
yl)cyclohexyl)-3-(1H-indazol-5-yl)urea (79). 1H NMR (300
MHz, CDCl3) δ TMS: 10.42 (bs, 1 H), 8.04 (m, 1 H), 7.70 (m,
1 H), 7.45 (d, J ) 9 Hz, 1 H), 7.28 (m, 1 H), 6.98-6.89 (m, 4
H), 6.27 (bs, 1 H), 3.28 (m, 1 H), 2.93 (bd, J ) 12 Hz, 1 H),
2.66 (bd, J ) 12 Hz, 1 H), 2.44-2.33 (m, 2 H), 2.09 (m, 2 H),
1.99 (dd, J ) 13 Hz, J ) 2.5 Hz, 1 H), 1.82 (m, 1 H), 1.78-
0.80 (m, 14 H). MS ESI (M + H)+ ) 464.2.
Biological Assays. CCR3-Receptor Binding. Millipore
filter plates (no. MABVN1250) are treated with 5 µg/mL
protamine in phosphate buffered saline, pH 7.2, for 10 min at
room temperature. Plates are washed three times with phos-
phate buffered saline and incubated with phosphate buffered
saline for 30 min at room temperature. For binding, 50 µL of
binding buffer (0.5% bovine serum albumen, 20 mM HEPES
buffer, and 5 mM magnesium chloride in RPMI 1640 media)
with or without a test concentration of a compound present
at a known concentration is combined with 50 µL of 125I-labeled
human eotaxin (to give a final concentration of 150 pM
radioligand) and 50 µL of cell suspension in binding buffer
containing 5 × 105 total cells. Cells used for the binding assay
are CHO cell lines transfected with a gene expressing human
CCR3 as described by Daugherty.17i The mixture of compound,
cells, and radioligand is incubated at room temperature for
30 min. Plates are placed onto a vacuum manifold, vacuum is
applied, and the plates are washed three times with binding
buffer with 0.5 M NaCl added. The plastic skirt is removed
from the plate, and the plate is allowed to air-dry; the wells
are punched out, and the cpm are counted. The percent
inhibition of binding is calculated using the total count
obtained in the absence of any competing compound or
chemokine ligand and the background binding determined by
addition of 100 nM eotaxin in place of the test compound.
Human Eosinophil Chemotaxis Assay. Neuroprobe
MBA96 96-well chemotaxis chambers with Neuroprobe poly-
(vinylpyrrolidone)-free polycarbonate PFD5 5-µm filters in
place are warmed in a 37 °C incubator prior to the assay.
Freshly isolated human eosinophils39 are suspended in RPMI
1640 with 0.1% bovine serum albumin at 1 × 106 cells/mL and
warmed in a 37 °C incubator prior to the assay. A 20 nM
solution of human eotaxin in RPMI 1640 with 0.1% bovine
serum albumin is warmed in a 37 °C incubator prior to the
assay. The eosinophil suspension and the 20 nM eotaxin
solution are each mixed 1:1 with prewarmed RPMI 1640 with
0.1% bovine serum albumin with or without a dilution of a
test compound that is at 2-fold the desired final concentration.
The filter is separated, and the eotaxin/compound mixture is
placed into the bottom part of the chemotaxis chamber. The
filter and upper chamber are assembled, and 200 µL volume
of the cell suspension/compound mixture is added to the
appropriate wells of the upper chamber. The upper chamber
is covered with a plate sealer, and the assembled unit is placed
in a 37 °C incubator for 45 min. After incubation, the plate
sealer is removed and all remaining cell suspension is aspi-
rated off. The chamber is disassembled, and unmigrated cells
are washed away with phosphate buffered saline and then the
filter is wiped with a rubber tipped squeegee. The filter is
allowed to completely dry and stained with Wright Giemsa.
Migrated cells are enumerated by microscopy.
2. Sensitization of Mice to Ovalbumin. Mice received
an intraperitoneal (ip) injection of 10 µg of ovalbumin (OVA;
Sigma, St. Louis, MO) and 1 mg of aluminum hydroxide (in a
volume of 0.1 mL) on days 1 and 8. On days 15-17, mice were
subjected to either a single intranasal instillation of murine
eotaxin or an aerosolized OVA challenge.
3. Intranasal (IN) Administration of Eotaxin. OVA-
sensitized Balb/c mice were administered either vehicle (PBS
+ 0.1% low endotoxin BSA) or 10 µg of murine recombinant
eotaxin (carrier-free mr eotaxin; R & D Systems, Minneapolis,
MN) in 50 µL volume, under Metofane (Mallinckrodt Veteri-
nary Inc., Mundelein, IL) anesthesia. In some experiments,
oral gavage was used to administer vehicle (0.25% methylcel-
lulose) or test compound 30 min before the eotaxin challenge.
Six hours following eotaxin administration, bronchoalveolar
lavage (BAL) was performed with 1 mL of PBS (Ca2+/Mg2+
-
free) containing 10 mM EDTA (lavage buffer). BAL samples
were centrifuged (320g, 10 min) and resuspended in 200 µL
of lavage buffer containing 0.1% BSA (resuspension buffer).
An aliquot of the cell suspension was used for total counts,
performed using a Neubauer hemacytometer, and for cytospin
preparations used to count differentials after Wright’s Giemsa
staining (Sigma, St. Louis, MO).
4. Aerosol Antigen Challenge. OVA-sensitized C57/Bl 6
mice (11-12 per group) were placed in a plexiglass pie
chamber connected to a nebulizer (Pari Respiratory Equip-
ment, Midlothianm, VA) driven by compressed air. Mice were
exposed to an aerosol of either vehicle (saline containing 0.01%
Tween 20) or 1% OVA for 30 min. Mice were treated with
either vehicle (0.25% methycellulose) or test compound every
8 h, beginning at 24 h prior to aerosol challenge. The fourth
dose was administered 30 min prior to OVA challenge, and
the mice received two more doses at 8 h intervals following
OVA challenge during the course of the experiment. BAL was
performed 24 h following OVA challenge as described above.
The BAL samples were centrifuged (320g, 10 min) and
resuspended in 500 µL of resuspension buffer. Aliquots of the
cell suspension were used for total counts, performed using a
Technicon H1E automated hematology analyzer (Bayer Diag-
nostics, Tarryton, NY), and for differential counts on Wright’s
Giemsa-stained cytospin preparations.
Acknowledgment. We thank A. J. Mical, N. C.
Caputo, and K. A. Rathgeb of the Separations group for
the chiral separation of the 3-(4-fluorobenzyl)piperidine
and tert-butyl 2-trans-((4-(4-fluorobenzyl)piperidin-1-yl)-
methyl)cyclohexylcarbamate. We are grateful to G. A.
Cain and C. Teleha, of the Chemical Synthesis Group,
for providing all 4-fluorobenzylpiperidines used in this
work. We also thank all the members of the CCR3
working group, especially P. H. Carter for valuable
discussions.
Calcium Mobilization Assay. Intracellular calcium flux
was measured as the increase in fluorescence emitted by the
calcium-binding fluorophore, fluo-3, when preloaded cells were
stimulated with CCR3 ligand. Freshly isolated eosinophils
were loaded with fluorophore by resuspending them in a
HEPES-buffered PBS solution containing 5 µM fluo-3 and
o
incubating for 60 min at 37 C. After being washed twice to
remove excess fluorophore, cells were resuspended in binding
buffer (without phenol red) and plated into 96-well plates at
2 × 105/well. Plates were placed individually in a FLIPR-1
(Molecular Devices, CA) that uses an argon-ion laser to excite
the cells and robotically adds reagents while monitoring
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