Discovery and structure-activity relationship of N-(ureidoalkyl)-benzyl-piperidines as potent small molecule CC chemokine receptor-3 (CCR3) antagonists

Article abstract of DOI:10.1021/jm0201767

Structure-activity relationship (SAR) studies of initial screening hits from our corporate library of compounds and a structurally related series of CCR1 receptor antagonists were used to determine that an N-(alkyl)benzylpiperidine is an essential pharmacophore for selective CCR3 antagonists. Further SAR studies that introduced N-(ureidoalkyl) substituents improved the binding potency of these compounds from the micromolar to the low nanomolar range. This new series of compounds also displays highly potent, in vitro functional CCR3-mediated antagonism of eotaxin-induced Ca²⁺ mobilization and chemotaxis of human eosinophils.

Full text of DOI:10.1021/jm0201767

3794
J . Med. Chem. 2002, 45, 3794-3804
Discover y a n d Str u ctu r e-Activity Rela tion sh ip of
N-(Ur eid oa lk yl)-Ben zyl-P ip er id in es As P oten t Sm a ll Molecu le CC Ch em ok in e
Recep tor -3 (CCR3) An ta gon ists
George V. De Lucca,* Ui T. Kim, Curt J ohnson, Brian J . Vargo, Patricia K. Welch, Maryanne Covington,
Paul Davies, Kimberly A. Solomon, Robert C. Newton, George L. Trainor, Carl P. Decicco, and Soo S. Ko
Bristol-Myers Squibb Company, Experimental Station, Wilmington, Delaware 19880-0336
Received April 24, 2002
Structure-activity relationship (SAR) studies of initial screening hits from our corporate library
of compounds and a structurally related series of CCR1 receptor antagonists were used to
determine that an N-(alkyl)benzylpiperidine is an essential pharmacophore for selective CCR3
antagonists. Further SAR studies that introduced N-(ureidoalkyl) substituents improved the
binding potency of these compounds from the micromolar to the low nanomolar range. This
new series of compounds also displays highly potent, in vitro functional CCR3-mediated
antagonism of eotaxin-induced Ca2+ mobilization and chemotaxis of human eosinophils.
In tr od u ction
molecules, currently termed eotaxin-22c and eotaxin-3,2d
have been described. While they share the same name,
the eotaxins are not closely related structurally (in fact,
eotaxin and eotaxin-2 share only 39% amino acid
identity) but appear to have a spectrum of activity
identical to those of eotaxin. Although all three eotaxin
molecules have a very similar range of biological activi-
ties, eotaxin-2 and eotaxin-3 are somewhat less potent
than eotaxin. While many CC chemokines bind to
several CC receptors, the eotaxins are exceptional in
this regard and bind only to the CCR3 receptor. Fur-
thermore, only a limited range of cells have been found
to express CCR3, predominantly eosinophils and baso-
phils. Thus, it appears that only the eotaxins can
chemoattract and activate these target cells specifically.
Eosinophils express at least three chemokine recep-
tors including CCR3, CCR1, and CXCR2. Of these,
CCR3 achieves by far the highest expression levels and
is thought to be the major eosinophil chemokine
receptor.1i Eosinophils express lower levels of the closely
related receptor CCR1, which binds and signals in
response to MIP-1R, MCP-3, and RANTES. CCR1 and
CCR3 share significant homology at the protein level
(54% identity), with the majority of conservation occur-
ring in the transmembrane helices.
The normal immunological response to infection,
tissue damage, or exposure to antigens is the recruit-
ment of inflammatory cells to fight infection, remove
damaged cells, and stimulate healing. While this is a
normal process, the excessive recruitment of inflamma-
tory cells is the hallmark of diseases such as asthma. A
characteristic feature of asthma is leukocyte infiltration
into the bronchial wall in which eosinophils predomi-
nate. Within the bronchial mucosa, eosinophils de-
granulate, releasing toxic proteins such as major basic
protein, eosinophil cationic protein, and eosinophil
peroxidase that cause tissue damage (e.g., epithelial
injury and shedding). Eosinophil activation is thought
to play a significant role in the underlying bronchial
hyperreactivity that is characteristic of human asthma.1
Chemokines are chemotactic cytokines that play an
important role in the directed migration of selected
populations of leukocytes from the circulation to sites
of inflammation, and thus, an integral part of the
immune response. These small secreted proteins are
classified into two main groups, CXC and CC, based on
the position of the first two of their conserved N-
terminal cysteine residues.2 In general, the hallmark
of the CC chemokines is their ability to chemoattract
and activate inflammatory leukocytes, particularly sub-
sets of lymphocytes, monocytes, eosinophils, and baso-
phils, as well as some stromal cells, such as endothelial
and smooth muscle cells. These activities of the CC
chemokines are mediated through a family of receptors
belonging to the seven transmembrane-spanning G
protein-coupled receptors (7TM GPCR) on the surface
of immune and inflammatory cells.1
The central role of CCR3 in allergic inflammation has
made this receptor a major target for drug development1d
with several reports of small molecule CCR3 antagonists
appearing in the literature.3,4 In this paper, we provide
details of our efforts in this area.5
Resu lts a n d Discu ssion
In our initial efforts to discover potential CCR3
antagonists, we screened our corporate library of com-
pounds and identified several hits in the low micromolar
range. Several of the compounds identified seemed to
be structurally related, including compounds 1-3 as
shown in Figure 1.
Eosinophils are proinflammatory granulocytes that
play a major role in allergic diseases, such as bronchial
asthma1f,g and allergic rhinitis.1h Eotaxin, a CC chemo-
kine2b with high selectivity for eosinophils, has recently
been identified. Since the discovery of eotaxin, two other
In addition to the screening hits, we synthesized
several examples (Table 1) from a Takeda patent
application,6 which described a series of CCR1 receptor
* To whom correspondence should be addressed. Tel: 302-467-6063.
Fax: 302-467-6913. E-mail: george.delucca@bms.com.
10.1021/jm0201767 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/11/2002

CC Chemokine Receptor-3 Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3795
Ta ble 2
CCR1
CCR3
compd
R
IC50 (µM)a
IC50 (µM)a
4
10
11
12
13
14
15
16
17
18
19
20
21
3-hydroxy-propyl
methyl
isopropyl
tert-butyl
2-(4-fluorophenyl)ethyl
benzyl
0.031
<0.78
<0.78
0.071
0.295
<0.78
0.125
<0.78
0.78
1.41
0.89
1.34
0.167
2.35
8.3
6.1
3.0
2.2
8.3
2.4
9.8
7.6
19.3
5.2
5.7
3.5
cyclohexylmethyl
phenyl
F igu r e 1. Initial hits identified from screening compound
library. Common structural features are highlighted with
bold bonds.
4-methoxy-phenyl
3-trifluoromethyl-phenyl
4-trifluoromethyl-phenyl
2,5-difluoro-phenyl
4-pyridyl
Ta ble 1
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
Ta ble 3
CCR1
CCR3
compd
R
IC50 (µM)a IC50 (µM)a
4
5
6
7
8
9
3-hydroxy-propyl
4-hydroxy-butyl
2-piperidin-1-yl-ethyl
1-benzyl-piperidin-4-yl
1-methoxycarbonyl-piperidin-4-yl
2-dimethylamino-ethyl
0.031
0.240
0.360
0.310
0.500
0.067
2.35
5.95
2.20
1.62
5.30
2.23
CCR3
CCR1
compd
R1
R2
R3
n
IC50 (µM)a
IC50 (µM)a
22
23
24
25
26
Ph Ph CH2NH2
Ph Ph CN
Ph
Ph
Ph
3
3
3
1
0
12.0
0.76
0.63
ntb
nt
9.0
H
H
H
H
H
H
14.5
19% at 10 µM
18.7
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
nt
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
antagonists, as standards for our CCR1 counterscreen
and the excellent potency of these compounds was
confirmed in our assay (compound 4, CCR1, IC50 ) 30
nM). Because the Takeda compounds had structural
features similar to the screening hits 1-3, they were
tested in our CCR3 binding assay and found to have
CCR3 binding affinity similar to the screening hits (4,
CCR3, IC50 ) 2 µM). All of these compounds shared
structural features that are highlighted with the bold
bonds in Figure 1. Each contains a core 1,4-disubstituted
piperidine with an N-phenethyl to N-phenbutyl group
as the 1-substituent and a more variably linked aro-
matic group as the 4-substituent. Our initial analoging
efforts concentrated on defining the essential pharma-
cophore features that were responsible for the CCR3
binding affinity of all of these compounds by trying to
incorporate the features of one compound into the
structure of the others. Because the Takeda compounds
had the additional urea functionality that the screening
hits lacked, we were especially interested in exploiting
this functionality in our structure-activity relationship
(SAR) studies.
b
shown. nt denotes not tested.
In addition to improving the CCR3 binding potency
of the initial hits, the dissection of the structure and
the synthetic studies also focused on determining the
structural features responsible for receptor selectivity
in order to reverse the selectivity found in 4 from CCR1
to CCR3.8 In an effort to improve potency and selectivity
for CCR3, we first examined changing the type of urea
substituent from the polar alkyl preferred in the Takeda
patent6 (examples in Table 1) to more lipophilic alkyls
or aryl substituents. As the data summarized in Table
2 show, changing the urea substituents of 4 to small
alkyl (10), branched alkyl (12), cycloalkyl (15), or phenyl
(16) and substituted phenyls (17-20) had only a small
effect on CCR1 selectivity or CCR3 potency.
To identify the essential pharmacophore responsible
for the CCR3 activity, the Takeda compounds were
systematically simplified by incorporating some of the
structural features found in the screening hits 1-3
(simple N-alkylphenyl substituents). Removal of the
urea functionality in Takeda compound 4 to give 22 (and
23, Table 3) had only a modest effect on CCR3 potency
or CCR1 selectivity. That the urea functionality did
contribute to the potency for CCR3 was evidenced by
the synthesis of several urea compounds, such as 27
(Figure 2), which although weak, still had some CCR3
binding affinity. As the data in Table 3 summarizes, the
benzhydrylpropyl motif of the Takeda compounds (22)
could be further simplified to the N-benzyl analogue 26
To streamline the discussion for this paper, we will
concentrate our comments in terms of Takeda com-
pounds such as 4. However, as results from concurrent
work7 in our labs starting with screening hit 3 indicate,
because all of these compounds (1-4) have common
structural features, all SAR studies beginning with any
one compound would eventually lead to a common
consensus structure as the key pharmacophore.

3796 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
De Lucca et al.
Ta ble 5
CCR3
CCR1
compd
R
m
n
IC50 (µM)a IC50 (µM)a
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
1-benzyl-piperidin-4-yl
2-piperidin-1-yl-ethyl
3-hydroxy-propyl
benzyl
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
1
3.9
7.6
2.6
3.4
>50
>50
>10
>10
>10
> 50
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
phenyl
0.90
1.75
0.60
0.75
0.75
0.45
0.45
0.14
0.18
0.41
0.081
0.049
2,5-difluoro-phenyl
3-methoxy-phenyl
4-methoxy-phenyl
3-cyano-phenyl
5-indazolyl
6-indazolyl
3-cyano-phenyl
3-methoxy-phenyl
2,5-difluoro-phenyl
3-methoxy-phenyl
3-cyano-phenyl
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
F igu r e 2. Structure of analogues discussed in the text.
by synthetic expediency to give the analogues sum-
marized in Table 5. The use of the polar alkyl urea
substituents (34-36, Table 5) originally found in the
Takeda compounds (of Table 1) did not improve the
CCR3 binding affinity. However, these compounds did
maintain selectivity for the CCR3 receptor as shown in
Table 5. On the other hand, phenyl and substituted
phenyl urea analogues provided compounds that were
more potent, with the IC50 improving to below the
micromolar range (38, Table 5). The substitution pattern
on the phenyl urea was important since analogues with
a 2-substituent (39 and 47) were less potent than phenyl
ureas with 3- or 4-substituents (40 and 41). The
heterocyclic ureas (43 and 44) showed a further im-
provement in potency.
Ta ble 4
CCR3
CCR1
compd
R1
R2
R3
X
n
IC50 (µM)a
IC50 (µM)a
28
29
30
31
32
33
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
CN
CN
H
H
H
C
N
C
N
C
C
3
3
3
3
1
0
4.6
14.1
3.8
8.4
7.1
>50
>50
>50
ntb
>50
>50
H
H
8.3
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
b
shown. nt denotes not tested.
and still maintain significant binding affinity for the
CCR3 receptor.
An increase in potency was also observed in changing
the length of the linker between the piperidine ring and
the urea functionality from the benzyl 42 to the phen-
ethyl 45 or the hydroxyethylphenyl 49 as shown in
Table 5. However, the acetylphenyl 50 or the oxime 51
(Figure 2) containing linkers were significantly less
potent than the other phenethyl linkers. These changes
affect not only the spacing but also the basicity of the
piperidine nitrogen and suggest a preference for a more
basic amine.9
Alternatives to the urea functionality were examined
briefly and were found less potent. A typical result is
exemplified by the amide 52, which is about 5-fold less
potent than the corresponding urea analogue 53, as
shown in Figure 2.
Further structure-activity studies were carried out
to investigate the regiochemical requirements of the
linker phenyl group to optimize the relative spatial
orientation between the 4-benzylpiperidine and the urea
substituents. The data in Table 6 show that analogues
with the para substitution pattern are clearly less
potent (compare 41 vs 55 or 42 vs 57). Ortho- or meta-
substituted analogues are in general of similar potency
(compare 34 vs 54). However, with the more potent
(phenethyl linked) analogues, there seems to be a
Next, an examination of a variety of other substituted
piperidines was undertaken to study the importance of
the 4-hydroxy-4-phenylpiperidine motif found in the
Takeda analogue 22 (Table 3). Several replacements
were synthesized, including the substituted piperidine
found in screening hit 3, and all were found to be less
potent (data not shown). However, the 4-benzylpiperi-
dine substituent, as in 28 (Table 4), provided compounds
that are more potent and selective for CCR3 over CCR1.
The corresponding piperazine analogues (29), while less
potent, also provided CCR3 selectivity. Similar to the
Takeda-like analogues (in Table 3), the benzhydryl-
propyl motif of 28 could be simplified to the phenethyl
(32) or benzyl analogue (33), while maintaining binding
affinity and selectivity for the CCR3 receptor as sum-
marized in Table 4.
Having established that an N-benzyl- or N-phenethyl-
substituted 4-benzylpiperdine was required for optimal
CCR3 binding affinity and selectivity, we then reincor-
porated the urea functionality to improve the potency.
The initial incorporation of the urea functionality into
the simplest N-benzyl analogue (33, Table 4) at the
ortho position of the N-benzyl ring was mainly driven

CC Chemokine Receptor-3 Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3797
Ta ble 6
Ta ble 8
CCR3
CCR3
IC50 (µM)a
compd
R
m
position
IC50 (µM)a
compd
R
m
n
75
76
77
78
79
80
81
82
83
H
0
0
0
1
1
1
0
0
0
1
1
1
1
1
1
2
2
2
0.361
0.310
0.125
0.024
0.021
0.011
0.014
0.011
0.0026
34
54
41
55
42
56
57
43
58
59
60
1-benzyl-piperidin-4-yl
1-benzyl-piperidin-4-yl
4-methoxy-phenyl
4-methoxy-phenyl
3-cyano-phenyl
3-cyano-phenyl
3-cyano-phenyl
5-indazolyl
0
0
0
0
0
0
0
0
0
1
1
ortho
meta
ortho
para
ortho
meta
para
ortho
meta
ortho
meta
3.9
3.7
0.75
48% at 5 µM
0.75
0.45
47% at 5 µM
0.45
0.62
0.249
cyano
acetyl
H
methoxy
cyano
cyano
methoxy
acetyl
5-indazolyl
phenyl
phenyl
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
71% at 5 µM
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
the binding potency of CCR3 antagonists. To examine
whether the effect was also observed in the phenyl
linker series, several compounds incorporating this
group were synthesized. As shown in Table 8, the
4-fluorobenzyl substituent has a significant effect on
binding potency. There is about a 3-fold improvement
in CCR3 binding potency with the benzyl linker (com-
pare 38, Table 5, vs 75, Table 8) and about a 10-fold
improvement with the phenethyl linker analogues
(compare 45, Table 7, vs 81, Table 8).
With the SAR on the piperidine and urea portions
established, the focus shifted to the phenyl ring in the
linker portion. The synthetic effort concentrated on the
more accessible benzyl linker analogues, and the results
are summarized in Table 9. Systematic substitution of
the four possible positions on the phenyl ring was
examined. Substitutions at the 5- and 6-positions were
either equivalent or less potent than the unsubstituted
analogues (compare 42, Table 5, vs 87, Table 9). Sub-
stituents at the 3- and 4-positions showed a dramatic
improvement in CCR3 binding potency. In particular,
the carboxylic acid derivatives (methyl esters and
amides) at the 3-position consistently showed single
digit nanomolar affinities (compare 77, Table 8, vs 107
and 115, Table 9).
Ta ble 7
CCR3
compd
R1
R2
R3
X
n
IC50 (µM)a
39
61
62
63
64
65
42
45
66
67
68
69
70
71
2,5-difluoro-phenyl
2,5-difluoro-phenyl
2,5-difluoro-phenyl
2,5-difluoro-phenyl
2,5-difluoro-phenyl
2,5-difluoro-phenyl
3-cyano-phenyl
3-cyano-phenyl
2-(4-fluorophenyl)ethyl
2-(4-fluorophenyl)ethyl
CH2Ph
C(O)Ph
Ph
4-Cl-Ph OH
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
H
H
H
C
C
C
C
N
C
C
C
N
C
C
C
C
C
1
1
1
1
1
0
1
2
1
1
1
1
1
1
1.75
4.65
11.0
21% at 5 µM
28% at 5 µM
9% at 5 µM
0.75
0.14
9.20
3.80
1.75
7.35
2.65
H
H
H
H
H
H
H
H
benzo[1,3]dioxol-5-ylmethyl CH2Ph
benzo[1,3]dioxol-5-ylmethyl C(O)Ph
2,2-diphenyl-ethyl
2,2-diphenyl-ethyl
CH2Ph
C(O)Ph
3.90
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
preference for the ortho substitution pattern (compare
59 vs 60).
Selected analogues were characterized in various
functional assays of human CCR3 receptor activity. Two
main assays were used; the first is an eotaxin-induced
intracellular calcium mobilization assay using FLIPR
(fluorometric imaging plate reader) technology and the
second, an eotaxin-induced eosinophil chemotaxis assay.
Both of these assays use freshly isolated human eosi-
nophils. The results for a few of the more potent
analogues are summarized in Table 10. These analogues
inhibited eotaxin-induced Ca2+ mobilization and eosi-
nophil chemotaxis. The compounds themselves did not
cause Ca2+ mobilization or chemotaxis at concentrations
up to 10 µM (results not shown) indicating that these
molecules are functional antagonists of the human
CCR3 receptor in human eosinophils with an IC50 in
the low nanomolar range in the calcium assay and
between 30 and 80 nM in the chemotaxis assay. These
compounds are not only potent receptor antagonists of
CCR3 but also selective for CCR3. All of the compounds
listed in Table 10, as well as all compounds in this study
With the SAR of the urea and linker portions estab-
lished, alternatives to the 4-benzylpiperidine were again
explored to confirm that it was still optimal. As shown
in Table 7, all replacements that were examined re-
sulted in reduced binding potency. This included 4-ben-
zoyl-, 4-phenyl-, and 4-hydroxy-4-phenyl-substituted
piperidines, as well as 4-benzylpiperazine, all of which
showed lower binding affinity (compare 39 to 61-64).
Having the 4-benzyl-piperidine directly attached to the
phenyl ring also caused a significant decrease in binding
potency (65, Table 7). Insertion of a methylene group
between the linker phenyl ring and the urea functional-
ity (as in 72-74, Figure 2) also decreased the binding
affinity. As noted earlier, an ethyl spacer between the
piperidine ring and the phenyl group (45, Table 7) gave
the most potent compounds.
Results from concurrent work7 in our labs with the
propyl linker CCR3 antagonist showed that a 4-fluoro-
benzyl piperidine substituent had a dramatic effect on

3798 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
De Lucca et al.
Ta ble 9
Ta ble 11
CCR3
compd
84 phenyl
85 phenyl
86 CO2Me
87 CO2Me
R
position
R1
X
IC50 (µM)a
CCR3
compd
R
position
R1
n
X
IC50 (µM)a
6-5 fused methoxy
6-5 fused cyano
5
5
5
5
F
F
H
H
H
H
H
H
H
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
0.423
0.529
0.777
72% at 5 µM
0.360
0.114
0.260
0.228
0.237
0.085
0.040
0.141
0.067
0.064
0.042
0.055
0.047
0.008
0.003
0.008
0.009
0.007
0.011
0.008
0.053
0.017
0.003
0.002
0.002
0.001
0.002
0.001
92
117
dioxymethylene 5-4 fused cyano
dioxymethylene 5-4 fused cyano
0
1
0
1
0
1
0
1
H
H
H
H
F
F
F
F
0.237
0.011
0.277
0.021
0.085
0.002
0.040
0.001
methoxy
cyano
methoxy
cyano
118
119
93
120
94
HO
HO
4
4
cyano
cyano
88 CH2OH
89 CH2OH
dioxymethylene 5-4 fused cyano
dioxymethylene 5-4 fused cyano
dioxymethylene 5-4 fused acetyl
dioxymethylene 5-4 fused acetyl
90 dioxymethylene 5-4 fused 5-indazolyl
91 dioxymethylene 5-4 fused acetyl
92 dioxymethylene 5-4 fused cyano
93 dioxymethylene 5-4 fused cyano
94 dioxymethylene 5-4 fused acetyl
95 dioxymethylene 5-4 fused methoxy
96 CO2Me
97 CO2Me
98 CO2Me
121
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
3
3
3
cyano
methoxy
acetyl
cyano
methoxy
cyano
acetyl
acetyl
H
cyano
methoxy
acetyl
H
they indeed demonstrated a 10-40-fold increase in
binding affinity as compared to the parent piperidine
derivatives as shown in Table 11.
99 CH2OH
100 CH2OH
101 CONH2
102 CONH2
103 CONHMe
104 CO2Me
105 CO2Me
106 CO2Me
107 CO2Me
108 CH2OH
109 COOH
110 CONH2
111 CONH2
112 CONHMe
113 CONHMe
114 CONHMe
115 CONHMe
Interestingly, the quaternary piperdinium salts listed
in Table 11 were found to be potent functional agonists
in the calcium and chemotaxis assays, while the corre-
sponding parent piperidine derivatives (in Table 11)
were themselves antagonists. For example, compound
121 (Table 11) caused Ca2+ mobilization at a concentra-
tion of 7 nM. The quaternary piperdinium salt phar-
macophore does not appear to be a prerequisite for
agonist activity, since, while several salts were synthe-
sized, only some were agonists. In addition, some parent
piperidine derivatives (nonpiperdinium salts) were found
to be weak agonists (data not shown). All of the agonists
displayed a rather flat SAR profile for agonist activity.
The structural requirements are limited mainly to the
oxygenated phenyl linker analogues listed in Table 11
and an ester urea derivative with much weaker activity.
Because the potential of quaternary salts is limited due
to pharmacokinetic liabilities (low oral bioavailability)
and the therapeutic value of a CCR3 agonist is ques-
tionable, these compounds were not pursued much
further. However, the data are presented in this paper
as an interesting observation that other workers might
find useful as a research tool.
H
cyano
acetyl
H
cyano
methoxy
acetyl
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
shown.
Ta ble 10
intracellular
calcium mobilization
(IC50, nM)
eotaxin-induced
chemotaxis
CCR3
compd
(IC50, nM)a
(IC50, nM)
83
102
103
107
111
112
113
114
115
2.6
3.0
8.0
8.0
2.0
2.0
1.0
2.0
1.0
ntb
71c
nt
9.5c
11.8c
nt
8.2c
76c
46c
nt
5.9c
Ch em istr y
4.3 ( 2.2
5.3 ( 2.2
4.2 ( 1.1
1.4 ( 0.8
nt
The syntheses of the compounds are summarized in
Schemes 1-4. Examples from the Takeda patent were
synthesized from the key intermediate 23 (Scheme 1),
which was obtained following the procedure described
in the patent.6 The cyano group was reduced to the
amine 22 by hydrogenaton over Raney Nickel. The
desired ureas were then obtained by reaction of the
amine 22 with commericially available isocyanates.
When the isocyanates were not available, 22 was first
converted to the phenylcarbamate 122 with phenyl
chloroformate (Scheme 1) and then treated with the
appropriate amine to give the ureas shown in Tables 1
and 2. Similar chemistry was used to synthesize the
compounds shown in Tables 3 and 4.
nt
30c
a
The value represents an average of usually three determina-
tions with a standard deviation of (20-50% of the mean value
b
shown. nt denotes not tested. c Value represents a single deter-
mination.
with CCR3 binding affinities less than 20 nM, were
examined against a panel of other chemokine receptors
and found to be selective for CCR3 by displaying low
binding affinity for CCR1, CCR2, or CCR5 (IC50 > 10
µM).
Finally, reports of the improved binding affinities of
quaternary ammonium salts in CCR112 and CCR33c
receptor antagonists prompted us, early in our program,
to explore the use of salts in our series. Several
quaternary piperidinium salts were synthesized, and
The benzyl and phenethyl linker analogues were
synthesized as shown in Schemes 2 and 3. Commercially
available 4-benzylpiperidine was alkylated with 2-ni-

CC Chemokine Receptor-3 Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3799
Sch em e 1a,b
Sch em e 3a,b
a
Conditions: (a) Raney Ni, H2 60 psi, NH3/EtOH, 80%. (b)
OCNR, THF, room temperature, 70-80%. (c) ClC(O)OPh, THF,
70%. (d) NH2R, THF, 70-80%. bSynthesized according to ref 6.
Sch em e 2a,b
a
Conditions: (a) K2CO3, DMF, room temperature, 80-90% or
(a) K2CO3, DMF, room temperature, 50 °C, 70-80%. (b) 10% Pd-
C, H2 50 psi, MeOH, 90%. (c) OCNR, THF, room temperature, 70-
80%. (d) NaBH4, MeOH, room temperature, 90%. bSame synthetic
scheme was used starting with the 3-NO2 isomers of compound
127.
Sch em e 4a
a
Conditions: (a) For n ) 1: K2CO3, DMF, room temperature,
70-90%; or for n ) 2: THF, 3 days, room temperature, 75%. (b)
10% Pd-C, H2 50 psi, MeOH, 80-90%. (c) ClC(O)OPh, Et3N, THF,
70-80%. (d) NH2R1, DMF, 50 °C, 70-80%. (e) OCNR1, THF, room
temperature, 70-80%. bSame synthetic scheme was used starting
with the 3- and 4-NO2 isomers of compound 123.
trobenzyl bromide 123 in dimethyl formamide (DMF)
to give the N-alkylated product. Catalytic hydrogenation
of the nitro group gave the aniline 124, which was
converted to the desired ureas 125 by reaction with
isocyanates or via the carbamate 126. The alkylation
of 4-benzylpiperidine with 2-nitrophenethyl bromide
required tetrahydrofuran (THF) as the solvent to mini-
mize styrene formation, which was the main product
observed in DMF.
To preclude styrene formation, as well as to access
substituted phenethyl derivatives, 4-benzylpiperidine
was alkylated with the phenacyl bromide 127 (Scheme
3) to give the alkylated product. The nitro group was
then hydrogenated to give the aniline 128. The ketone
of 128 was reduced with NaBH4 to the aniline alcohol
130, and subsequent treatment with isocyanates yielded
the ureas 131. Alternately, the aniline alcohol 130 was
obtained from the reaction of nitro-epoxide 132 with
4-benzylpiperidine followed by catalytic hydrogenation.
The epoxide 132 was obtained from the phenacyl
bromide 127 by NaBH4 reduction and cyclizing the
resulting bromohydrin with NaOH.
a
Conditions: (a) K2CO3, DMF, room temperature, 80-90%. (b)
NaOH, MeOH, reflux - 2 h, 90%. (c) Oxalyl chloride, CH2Cl2, room
temperature. (d) RNH2, CH2Cl2, 80-90%, 2 steps. (e) 10% Pd-C,
H2 50 psi, MeOH, 90%. (f) ClC(O)OPh, Et3N, THF, 65-75%. (g)
NH2R1, DMF, 50 °C, 70-80%. (h) OCNR1, THF, room temperature,
70-80%.
Scheme 4. Alkylation of the benzyl bromide 133 (ob-
tained from NBS bromination of the commercially
available toluene) with 4-benzylpiperidine gave the ester
134. The ester was hydrolyzed, and the resulting acid
135 was converted to the acid chloride (oxalyl chloride)
and treated with the appropriate amine to give the
desired amide 136. The nitro group of 136 was hydro-
genated to give the amine 137. The amine was converted
to the desired urea by either direct treatment with the
appropriate isocyanate or via the carbamate intermedi-
ate 138.
Con clu sion
The synthesis of the carboxylic acid derivative-
substituted phenyl linker analogues is summarized in
In search of novel CCR3 antagonists, we identified
several hits with micromolar potency from our corporate

3800 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
De Lucca et al.
1-{5-[4-(4-Ch lor o-p h en yl)-4-h yd r oxy-p ip er id in -1-yl]-
2,2-d ip h en yl-p en tyl}-3-p yr id in -4-yl-u r ea (21). A solution
of amine 226 (200 mg, 0.44 mmol) in DMF was treated with
the carbamate, pyridin-4-yl-carbamic acid 4-nitro-phenyl ester
(obtained from the reaction of p-nitrophenyl chloroformate
with 4-amino pyridine) (110 mg, 0.44 mmol), and K2CO3 (300
mg, 2.2 mmol) and stirred overnight at room temperature. The
mixture was diluted with water, and the precipitated solid was
extracted into EtOAc and washed with water and brine. The
solvent was removed on a rotary evaporator, and the residue
was chromatographed on silica gel (30%MeOH/EtOAc) to give
150 mg (60%) of 21. 1H NMR (300 MHz, CDCl3) δ (TMS):
7.40-7.36 (m, 2 H), 7.30-7.16 (m, 13 H), 6.92-6.80 (m, 4 H),
3.81 (d, J ) 6 Hz, 2 H), 3.67 (bt, 1 H), 2.66 (m, 2 H), 2.36-
2.29 (m, 4 H), 2.08-1.95 (m, 4 H), 1.80 (bs, 1 H), 1.64 (m, 2
H), 1.27 (m, 2 H). MS ESI (M + H)+ ) 569.4. Anal. (C34H37N4O2-
Cl-(CDCl3)0.25) C, H, N.
5-(4-Ben zyl-p ip er id in -1-yl)-2,2-d ip h en yl-p en t a n en i-
tr ile (28). A solution of 5-bromo-2,2-diphenyl-pentanenitrile6
(200 mg, 0.64 mmol), 4-benzylpiperidine (113 mg, 0.64 mmol),
and K2CO3 in DMF was stirred at room temperature overnight.
The mixture was diluted with water, and the precipitated solid
was extracted into EtOAc and washed with water and brine.
The solvent was removed on a rotary evaporator, and the
residue was chromatographed on silica gel (70% EtOAc/
hexanes) to give 200 mg (77%) of 28 as a thick oil. 1H NMR
(300 MHz, CDCl3) δ (TMS): 7.40-7.06 (m, 15 H), 2.77 (m, 2
H), 2.50 (d, J ) 7 Hz, 2 H), 2.47-2.30 (m, 4 H), 1.78 (dt, J )
2 Hz, J ) 12 Hz, 2 H), 1.69-1.57 (m, 4 H), 1.45 (m, 1 H), 1.31-
1.18 (m, 4 H). MS ESI (M + H)+ ) 409.3.
1-[1-(P h en ylm et h yl)-4-p ip er id in yl]-3-[2-[[4-(p h en yl-
m eth yl)-1-p ip er id in yl]m eth yl]p h en yl]u r ea (34). A solu-
tion of 4-benzylpiperidine (1.75 g, 10 mmol) in 25 mL of DMF
was treated with 2-nitrobenzyl bromide (2.16 g, 10 mmol) and
K2CO3 (1.38 g, 10 mmol), and the reaction mixture was stirred
at room temperature for 2 h. The mixture was diluted with
water and extracted into ethyl acetate. The organic extracts
were washed successively with water and brine, and the
organic solvent was removed under vacuum on a rotary
evaporator to give 4-benzyl-1-(2-nitro-benzyl)piperidine (Scheme
2, R, Y ) H) as a yellow oil. Crude product was redissolved in
ethyl acetate (50 mL) and treated with 10% Pd/C and
hydrogenated at 50 psi at room temperature for 40 min. The
solution was filtered, the solvent was removed under vacuum,
and the residue was purified by chromatography (MPLC, 40%
ethyl acetate/hexane; silica gel) to give 2.0 g of 2-(4-benzyl-
piperidin-1-ylmethyl)phenylamine 124 (Scheme 2, R, Y ) H)
as a white solid.
A solution of aniline 124 (1.2 g, 4.3 mmol) in THF was
treated with Et3N (1.0 g, 10 mmol) and cooled in an ice bath
to °0 C. Phenyl chloroformate (0.71 g, 4.5 mmol) was added to
the mixture and stirred for 1 h. The mixture was diluted with
water and extracted into ethyl acetate. The extracts were
washed with water and brine, and the solvent was removed
under vacuum to give [2-(4-benzyl-piperidin-1-ylmethyl)phen-
yl]carbamic acid phenyl ester 126 as an off-white solid. The
crude product was used without further purification.
compound library. Using these hits and a structurally
related CCR1 antagonist reported in a Takeda patent6
as a starting point, we initiated a series of SAR studies
that determined that an essential pharmacophore for
CCR3 binding was an N-(alkyl)benzylpiperidine core.
Introduction of N-(ureidoalkyl) substituents improved
the binding potency of these compounds to sub-micro-
molar. Further SAR studies were carried out and led
to the identification of a potent series of CCR3 antago-
nists with single-digit nanomolar potency. These potent
N-(ureidoalkyl)benzylpiperidines displayed in vitro func-
tional antagonism of CCR3-mediated eotaxin-induced
calcium mobilization and chemotaxis of human eosino-
phils. Studies are continuing on this class of compounds
to improve their functional potency and will be reported
in due course.
Exp er im en ta l Section
Gen er a l. All reactions were carried out under an atmo-
sphere of dry nitrogen. Commercial reagents were used
without further purification. 1H NMR (300 MHz) spectra were
recorded using tetramethylsilane (TMS) as an internal stan-
dard. Thin-layer chromatography (TLC) was performed on E.
Merck 15710 silica gel plates. Medium-pressure liquid chro-
matography (MPLC) was carried out using EM Science silica
gel 60 (230-400 mesh). High-performance liquid chromatog-
raphy (HPLC) chromatography was carried out using J asco
PV-987 pumps, J asco UV-975 detectors, and DuPont Zorbax
Sil or Zorbax NH2 one inch preparative columns. All final
targets were obtained as noncrystalline amorphous solids
unless specified otherwise. Mass spectra were measured with
HP5988A Mass Spectrometer with particle beam interface
using NH3 for chemical ionization or Finnigan MAT 8230 Mass
Spectrometer with NH3-DCI or VG TRIO 2000 for ESI. High-
resolution mass spectra were measured on a VG 70-VSE
instrument with NH3 chemical ionization. Elemental analysis
was performed 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%.
Ch em istr y. The synthesis of illustrative examples are given
below. A complete listing of all of the compounds synthesized
for this work can be found in the Supporting Information.
1-{5-[4-(4-Ch lor o-p h en yl)-4-h yd r oxy-p ip er id in -1-yl]-
2,2-diph en yl-pen tyl}-3-[2-(4-flu or o-ph en yl)eth yl]u r ea (13).
A solution of carbamate 1226 (100 mg, 0.176 mmol) in DMF
was treated with 4-fluorophenylethylamine (48 mg, 0.35 mmol)
and K2CO3 (130 mg, 0.90 mmol) and stirred overnight at room
temperature. The mixture was diluted with water, and the
precipitated solid was filtered on a buchner funnel. The solid
was washed on the funnel with water and 1 N NaOH and
allowed to air-dry to give 90 mg (85%) of urea 13. 1H NMR
(300 MHz, CDCl3) δ (TMS): 7.44-7.42(m, 2 H), 7.39-7.16 (m,
12 H), 7.08-6.81 (m, 4 H), 4.16 (bt, 1 H), 3.91 (m, 3 H), 3.25
(m, 2 H), 2.76-2.60 (m, 3 H), 2.41-1.80 (m, 10 H), 1.64 (m, 2
H), 1.33-1.20 (m, 2 H). MS ESI (M + H)+ ) 614.2. Anal.
(C37H41N3O2ClF-(CDCl3)0.1) C, H, N.
1-{5-[4-(4-Ch lor o-p h en yl)-4-h yd r oxy-p ip er id in -1-yl]-
2,2-d ip h en yl-p en tyl}-3-(2,4-d iflu or o-p h en yl)u r ea (20). A
solution of amine 226 (200 mg, 0.44 mmol) in THF (5 mL) was
treated with 2,4-difluorophenyl isocyanate (69 mg, 0.44 mmol)
and stirred for 30 min at room temperature. The reaction
mixture was concentrated under vacuum, and the residue was
chromatographed on silica gel (EtOAc) to give 180 mg (70%)
of 20 as a white solid. 1H NMR (300 MHz, CDCl3) δ (TMS):
7.83 (m, 1 H), 7.42-7.39 (m, 2 H), 7.37-7.20 (m, 12 H), 6.92
(m, 1 H), 6.60 (m, 1 H), 6.34 (bs, 1 H), 4.38 (bs, 1 H), 4.04 (d,
J ) 6 Hz, 2 H), 2.67 (m, 2 H), 2.36-2.25 (m, 4 H), 2.20-2.15
(m, 2 H), 2.13-1.95 (m, 2 H), 1.63-1.45 (m, 3 H), 1.40-1.20
(m, 2 H). MS ESI (M + H)+ ) 604.4. Anal. (C35H36N3O2ClF2-
(H2O)0.5) C, H, N.
A solution of phenylcarbamate 126 (0.2 g, 0.5 mmol) in DMF
was treated with 4-amino-1-benzylpiperidine (95 mg, 0.5
mmol) and K2CO3 (138 mg, 1 mmol), and the mixture was
heated at 50 °C for 2 h. The mixture was diluted with water
and extracted into ethyl acetate. The extracts were washed
with water and brine, and the solvent was removed under
vacuum. The residue was purified by chromatography (MPLC,
0-25% MeOH/EtOAc; silica gel) to give 200 mg of 34 (83%)
as a white solid. 1H NMR (300 MHz, CDCl3) δ (TMS): 9.60
(bs, 1 H), 7.93 (d, J ) 8 Hz, 1 H), 7.33-7.21 (m, 9 H), 7.13 (d,
J ) 7 Hz, 2 H), 7.02 (d, J ) 6 Hz, 1 H), 6.89 (m, 1 H), 4.26 (bs,
1 H), 3.68 (m, 1 H), 3.52 (s, 2 H), 3.51 (s, 2 H), 2.83 (m, 4 H),
2.54 (d, J ) 7 Hz, 2 H), 2.19 (m, 2 H), 2.02-1.92 (m, 4 H),
1.88-1.45 (m, 5 H), 1.24-1.15 (m, 2 H). MS ESI (M + H)+
497. Anal. (C32H40N4O0-(CDCl3)0.12) C, H, N.
)
1-[2-(4-Ben zyl-p ip er id in -1-ylm eth yl)p h en yl]-3-(2,4-d i-
flu or o-p h en yl)u r ea (39). A solution of aniline 124 (as

CC Chemokine Receptor-3 Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3801
described above for the synthesis of 34) (140 mg, 0.5 mmol) in
THF was treated with 2,5-difluorophenyl isocyanate (80 mg,
0.5 mmol) at room temperature for 1 h. The solvent was
removed under vacuum, and the residue was purified by
chromatography (20% EtOAc/Hexane, silica gel) to give 150
mg (68%) of urea 39 as a white solid. 1H NMR (300 MHz,
CDCl3) δ (TMS): 11.0 (bs, 1 H), 8.06 (d, J ) 8 Hz, 1 H), 8.12-
8.02 (m, 1 H), 7.30-6.92 (m, 9 H), 6.67 (m, 1 H), 6.58 (bs, 1
H), 3.59 (s, 2 H), 2.88 (m, 2 H), 2.54 (d, J ) 7 Hz, 2 H), 1.95
(m, 2 H), 1.72 (m, 2 H), 1.60 (m, 1 H), 1.30-1.15 (m, 2 H). MS
ESI (M + H)+ ) 436.3. Anal. (C26H27N3OF2) C, H, N.
Hz, 1 H), 2.55 (d, J ) 7 Hz, 2 H), 2.51 (abx dd, J ) 4 Hz, J )
10 Hz, 1 H), 2.30 (m, 1 H), 2.02 (m, 1 H), 1.68 (m, 2 H), 1.57
(m, 1 H), 1.43-1.25 (m, 2 H). MS ESI (M + H)+ ) 466.4 Anal.
(C27H29N3O2F2-(H2O)0.5) C, H, N.
1-{2-[2-(4-Ben zyl-p ip er id in -1-yl)eth yl]p h en yl}-3-(3-cy-
a n o-p h en yl)u r ea (45). A solution of 4-benzylpiperidine (1.0
g, 5.7 mmol) in THF was treated with 2-nitrophenethyl
bromide (1.0 g, 4.3 mmol), and the reaction mixture was stirred
at room temperature for 3 days. The solvent was removed
under vacuum, and the residue was partitioned between 0.5
N NaOH and chloroform. The organic extract was washed
successively with water and brine. The solvent was removed
under vacuum on a rotary evaporator, and the residue was
chromatographed (MPLC, 50% ethyl acetate/hexane; silica gel)
to give 0.5 g (36%) of 4-benzyl-1-[2-(2-nitro-phenyl)ethyl]-
3-[4-(4-F lu or o-b e n zyl)p ip e r id in -1-ylm e t h yl]-4-[3-(3-
m eth oxy-p h en yl)u r eid o]ben zoic Acid Meth yl Ester (97).
The same procedure as for 39 was followed (see Scheme 2)
except starting with 3-bromomethyl-4-nitro-benzoic acid meth-
1
1
yl ester and 4-(4-fluorobenzyl)piperidine. H NMR (300 MHz,
piperidine (Scheme 2, n ) 2, R ) H) as a yellow oil. H NMR
CDCl3) δ (TMS): 10.63 (bs, 1 H), 8.24 (d, J ) 9 Hz, 1 H), 7.95
(dd, J ) 2 Hz, J ) 9 Hz, 1 H), 7.74 (d, J ) 2 Hz, 1 H), 7.26 (m,
1 H), 7.09-7.00 (m, 2 H), 6.98-6.93 (m, 2 H), 6.85 (dd, J )
1.5 Hz, J ) 8 Hz, 1 H), 6.71 (dd, J ) 2 Hz, J ) 8 Hz, 1 H),
6.26 (s, 1 H), 3.88 (s, 3 H), 3.83 (s, 3 H), 3.55 (s, 2 H), 2.71 (m,
2 H), 2.44 (d, J ) 7 Hz, 2 H), 1.87 (m, 2 H), 1.54 (m, 2 H), 1.46
(m, 1 H), 0.94 (m, 2 H). MS ESI (M + H)+ ) 506.3. Anal.
(C29H32N3O4F-(H2O)0.25) C, H, N.
1-{2-[4-(4-F lu or o-b en zyl)p ip er id in -1-ylm et h yl]-4-h y-
d r oxym eth yl-p h en yl}-3-(3-m eth oxy-p h en yl)u r ea (100).
Compound 100 was obtained from the LiBH4 reduction of 97.
1H NMR (300 MHz, CDCl3) δ (TMS): 10.13 (bs, 1 H), 8.02 (d,
J ) 8 Hz, 1 H), 7.28-7.20 (m, 2 H), 7.08 (m, 4 H), 6.96 (m, 2
H), 6.85 (dd, J ) 1.5 Hz, J ) 8 Hz, 1 H), 6.71 (dd, J ) 2 Hz,
J ) 8 Hz, 1 H), 6.26 (s, 1 H), 4.61 (s, 2 H), 3.83 (s, 3 H), 3.49
(s, 2 H), 2.72 (m, 2 H), 2.43 (d, J ) 7 Hz, 2 H), 1.85 (m, 2 H),
1.62 (bs, 1 H), 1.54 (m, 2 H), 1.42 (m, 1 H), 0.94 (m, 2 H). MS
ESI (M + H)+ ) 478.3.
1-{2-[2-(4-Ben zyl-p ip er id in -1-yl)a cetyl]p h en yl}-3-(2,4-
diflu or o-ph en yl)u r ea (50). To an ice-cold solution of 2-bromo-
2′-nitro-acetophenone 127 (Scheme 3) (2.4 g, 10 mmol) in DMF
was added 4-benzylpiperidine (1.75 g, 10 mmol), and it was
stirred for 30 min. The solution was poured into a mixture of
K2CO3 (1.38 g, 10 mmol) in water/ice and extracted into ethyl
acetate. The ethyl acetate extract was washed several times
with water. The resultant ethyl acetate solution of crude nitro-
ketone was treated with 10% Pd/C and hydrogenated at 50
psi hydrogen at room temperature for 40 min. The solution
was then filtered, the solvent was removed under vacuum, and
the residue was purified by chromatography (MPLC, 30% ethyl
acetate/hexane; silica gel) to give 1.9 g (63%) of 1-(2-amino-
phenyl)-2-(4-benzyl-piperidin-1-yl)ethanone 128 (Scheme 3) as
a tan/brown solid.
A solution of aniline 128 (310 mg, 1.0 mmol) in THF was
treated with 2,5-difluoroisocyanate (160 mg, 1.0 mmol) at room
temperature for 1 h. The solvent was removed under vacuum,
and the residue was purified by chromatography (MPLC, 20%
EtOAc/Hexane, silica gel) to give 420 mg (91%) of the desired
urea-ketone 50 as a white solid. 1H NMR (300 MHz, CDCl3) δ
(TMS): 11.5 (bs, 1 H), 8.55 (d, J ) 8 Hz, 1 H), 8.09-8.02 (m,
1 H), 7.98 (dd, J ) 21 Hz, J ) 8 Hz, 1 H), 7.56 (m, 1 H), 7.30-
6.98 (m, 8 H), 6.89 (bs, 1 H), 6.67 (m, 1 H), 3.82 (s, 2 H), 2.95
(m, 2 H), 2.55 (d, J ) 7 Hz, 2 H), 2.09 (m, 2 H), 1.65 (m, 2 H),
1.55 (m, 1 H), 1.45-1.25 (m, 2 H). MS ESI (M + H)+ ) 464.3.
Anal. (C27H27N3O2F2-(H2O)0.25) C, H, N.
(300 MHz, CDCl3) δ (TMS): 7.88 (d, J ) 8 Hz, 1 H), 7.51 (m,
1 H), 7.37-7.13 (m, 7 H), 3.09 (m, 2 H), 2.94 (m, 2 H), 2.61
(m, 2 H), 2.54 (d, J ) 7 Hz, 2 H), 2.01 (m, 2 H), 1.65 (m, 2 H),
1.53 (m, 1 H), 1.38-1.24 (m, 2 H).
The oil (0.5 g, 1.5 mmol) was redissolved in ethyl acetate
(50 mL) and treated with 10% Pd/C and hydrogenated at 50
psi at room temperature for 45 min. The solution was filtered,
and the solvent was removed under vacuum to give 0.4 g (89%)
of 2-[2-(4-benzyl-piperidin-1-yl)ethyl]phenylamine 124 (Scheme
2, n ) 2, R ) H) as a white solid. The aniline was used without
further purification. A solution of 2-[2-(4-benzyl-piperidin-1-
yl)ethyl]phenylamine (240 mg, 0.81 mmol) in THF was treated
with 3-cynanophenyl isocyanate (144 mg, 1.0 mmol) and
stirred at room temperature for 2 h. The solvent was removed
under vacuum, and the residue was purified by chromatog-
raphy (MPLC, 50% EtOAc/Hexane to 100% EtOAc, silica gel)
to give 250 mg (71%) of the desired urea 45 as a white solid.
1H NMR (300 MHz, CDCl3) δ (TMS): 9.85 (bs, 1 H), 7.88 (bs,
1 H), 7.77 (d, J ) 8 Hz, 1 H), 7.65 (m, 1 H), 7.41-7.03 (m, 11
H), 3.11 (m, 2 H), 2.86 (m, 2 H), 2.65 (m, 2 H), 2.60 (d, J ) 7
Hz, 2 H), 2.13 (m, 2 H), 1.79 (m, 2 H), 1.63-1.45 (m, 3 H). MS
ESI (M + H)+ ) 439.3 Anal. (C28H30N4O-(H2O)0.5) C, H, N.
1-[2-(4-Ben zyl-p ip er id in -1-yl)p h en yl]-3-(2,4-d iflu or o-
p h en yl)u r ea (65). A solution of 4-benzyl piperidine (3.5 g,
20 mmol) in DMF was cooled in an ice bath and treated with
NaH (60% oil dispersion, 0.8 g, 20 mmol) and stirred for 30
min, removed from the ice bath, and stirred for an additional
30 min. The solution was again cooled in an ice bath and
treated with 2-fluoro-nitrobenzene (2.82, 20 mmol) and stirred
for 2 h. The mixture was removed from the bath and stirred
at room temperature overnight. The solution was diluted with
water and extracted into EtOAc. The organic extract was
washed with water and brine and concentrated, and the
residue was chromatographed (Hexane to 5% EtOAc/hexane,
silica gel) to give 5.8 g (98%) of 4-benzyl-1-(2-nitro-phenyl)-
piperidine as a yellow oil. The oil obtained was dissolved in
EtOAc, treated with 1.1 g of 10% Pd/C, and hydrogenated at
50 psi for 1 h. The solution was filtered, and the solvent was
removed under vacuum to give 5.2 g of 2-(4-benzyl-piperidin-
1-yl)phenylamine, which was used without further purifica-
tion. A solution of 2-(4-benzyl-piperidin-1-yl)phenylamine (133
mg, 0.50 mmol) in THF was treated with 2,4-difluorophenyl
isocyanate (80 mg, 0.52 mmol) at room temperature for 30 min.
The solvent was removed under vacuum, and the residue was
chromatographed (5-10% EtOAc/hexane, silica gel) to give 160
1
mg (76%) of 65 as a white solid. H NMR (300 MHz, CDCl3) δ
1-{2-[2-(4-Ben zyl-piper idin -1-yl)-1-h ydr oxy-eth yl]ph en -
yl}-3-(2,4-d iflu or o-p h en yl)u r ea (47). A solution of the urea-
ketone 50 (260 mg, 0.56 mmol) in MeOH was treated with
NaBH4 (400 mg, 11 mmol) at room temperature for 1 h. The
solvent was removed under vacuum, and the residue was
treated with 1 N NaOH and extracted into EtOAc. The extracts
were washed with water and brine, and the solvent was
removed under vacuum to give 0.25 g (96%) of the desired
alcohol 47 as a white solid. 1H NMR (300 MHz, CDCl3) δ
(TMS): 8.95 (bs, 1 H), 8.55 (d, J ) 8 Hz, 1 H), 8.06-7.99 (m,
1 H), 7.86 (d, J ) 8 Hz, 1 H), 7.33-6.98 (m, 9 H), 6.85 (bs, 1
H), 6.66 (m, 1 H), 4.81 (abx dd, J ) 10 Hz, J ) 4 Hz, 1 H),
3.02 (m, 1 H), 2.84 (m, 1 H), 2.78 (abx dd, J ) 10 Hz, J ) 17
(TMS): 8.01-7.92 (m, 2 H), 7.79 (bs, 1 H), 7.32-7.00 (m, 10
H), 6.71 (m, 1 H), 3.02 (m, 2 H), 2.64 (m, 2 H), 2.57 (d, J ) 7
Hz, 2 H), 1.76 (m, 2 H), 1.68 (m, 1 H), 1.59 (m, 2 H). MS ESI
(M + H)+ ) 422.3 Anal. (C25H25N3O2F2) C, H, N.
2-[4-(4-F lu or o-ben zyl)p ip er id in -1-ylm eth yl]-3-(3-p h en -
yl-u r eid o)ben zoic Acid Meth yl Ester (104). A solution of
2-methyl-3-nitro-benzoic acid methyl ester (10.0 g, 51 mmol)
in CCl4 was heated to reflux and treated with NBS (9.1 g, 51
mmol) and a catalytic amount of benzoyl peroxide. The mixture
was heated to reflux for 24 h, cooled to room temperature, and
filtered. The filtrate was concentrated under vacuum, and the
residue was chromatographed (5-10% EtOAc/hexane, silica

3802 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
De Lucca et al.
gel) to give 10.3 g (73%) of the benzyl bromide 133 (see Scheme
4), 2-bromomethyl-3-nitro-benzoic acid methyl ester. 1H NMR
(300 MHz, CDCl3) δ (TMS): 8.11 (dd, J ) 1 Hz, J ) 8 Hz, 1
H), 7.97 (dd, J ) 1 Hz, J ) 8 Hz, 1 H), 7.53 (t, J ) 8 Hz, 1 H),
5.16 (s, 2 H), 4.00 (s, 3 H). A solution of the above benzyl
bromide 133 (4.9 g, 18 mmol) in DMF was treated with 4-(4-
fluorobenzyl)piperidine HCl (4.2 g, 18 mmol) and K2CO3 (2.76
g, 20 mmol), and the reaction mixture was stirred at room
temperature for 6 h. The mixture was diluted with water and
extracted into ethyl acetate. The organic extracts were washed
successively with water and brine, and the organic solvent was
removed under vacuum on a rotary evaporator to give 6.9 g
(100%) of 134 (Scheme 4), 2-[4-(4-fluoro-benzyl)piperidin-1-
ylmethyl]-3-nitro-benzoic acid methyl ester, as a yellow oil.
This was used without further purification. 1H NMR (300 MHz,
CDCl3) δ (TMS): 7.34-7.69 (m, 2 H), 7.40 (t, J ) 8 Hz, 1 H),
7.08-7.01 (m, 2 H), 6.97-6.90 (m, 2 H), 3.88 (s, 3 H), 3.77 (s,
2 H), 2.61 (m, 2 H), 2.45 (d, J ) 7 Hz, 2 H), 1.93 (dt, J ) 11
Hz, J ) 2 Hz, 2 H), 1.47 (m, 2 H), 1.40 (m, 1 H), 1.12 (m, 2 H).
vacuum, and the residue was purified by chromatography
(MPLC, EtOAc-10% MeOH/EtOAc, silica gel) to give 75 mg
1
(54%) of the desired urea 110 as a white solid. H NMR (300
MHz, CDCl3) δ (TMS): 8.01 (d, J ) 8 Hz, 1 H), 7.83 (s, 1 H),
7.62 (d, J ) 8 Hz, 1 H), 7.42-7.17 (m, 4 H), 7.06 (m, 2 H), 6.95
(m, 2 H), 6.70 (bs, 1 H), 5.75 (bs, 1 H), 3.74 (s, 2 H), 2.86 (m,
2 H), 2.50 (d, J ) 7 Hz, 2 H), 2.25 (m, 2 H), 1.68 (m, 2 H), 1.58
(m, 1 H), 1.16 (m, 2 H). MS ESI (M + H)+ ) 486.4 Anal.
(C28H28N5O2F-(CDCl3)0.12) C, H, N.
4-Ben zyl-1-{6-[3-(3-cya n o-p h en yl)u r eid o]b en zo[1,3]-
d ioxol-5-ylm eth yl}-1-m eth yl-p ip er id in iu m ; Iod id e (117).
A solution of 92 (200 mg, 0.42 mmol) in AcCN (10 mL) was
treated with MeI (0.052 mL, 0.84 mmol) and heated at 40 °C
overnight. The solvent was removed under vacuum, and the
residue was chromatographed (MPLC, EtOAc, silica gel) to
give 200 mg of solid. This was recrystallized from 80% EtOAc/
Hexane to give 120 mg (47%) of light yellow solid. 1H NMR
(300 MHz, DMSO-d6) δ (TMS): 9.16 (bs, 1 H), 8.32 (bs, 1 H),
7.96 (m, 1 H), 7.63 (m, 1 H), 7.46 (m, 1 H), 7.43 (m, 1 H), 7.29
(m, 2 H), 7.22-7.05 (m, 5 H), 6.12 (s, 2 H), 4.45 (s, 2 H), 3.30
(m, 4 H), 2.92 (s, 3 H), 2.58 (d, J ) 7 Hz, 2 H), 1.60-1.50 (m,
5 H). MS ESI (M - I)+ ) 483.4 Anal. (C29H31N4O3I) C, H, N.
1-(1,2-Dip h en yl-eth yl)-3-(3-h yd r oxy-p r op yl)u r ea (27).
This was obtained from the reaction of (1,2-diphenyl-ethyl)-
carbamic acid phenyl ester with 3-amino-propan-1-ol. 1H NMR
(300 MHz, DMSO-d6) δ (TMS): 7.29-7.12 (m, 10 H), 6.40 (d,
J ) 8 Hz, 1 H), 5.8 (m, 1 H), 4.86 (m, 1 H), 4.42 (t, J ) 5 Hz),
3.44 (m, 2 H), 2.96 (m, 2 H), 2.92 (m, 2 H), 1.43 (m, 2 H). MS
ESI (M + H)+ ) 299.2. HRMS Calcd for C18H23N2O2 (M + H)+,
299.1760; found, 299.1771. Anal. (C18H22N2O2) C, H, N.
A solution of the above crude nitrobenzene 134 (1.1 g, 2.8
mmol) in EtOAc (50 mL) was treated with 10% Pd/C (0.4 g)
and hydrogenated at 50 psi at room temperature for 45 min.
The solution was then filtered, and the solvent was removed
under vacuum to give the aniline as a thick oil. The aniline
was used without further purification. 1H NMR (300 MHz,
CDCl3) δ (TMS): 7.11-6.93 (m, 6 H), 6.74 (d, J ) 8 Hz, 1 H),
5.11 (bs, 2 H), 3.84 (s, 3 H), 3.71 (s, 2 H), 2.82 (m, 2 H), 2.48
(d, J ) 7 Hz, 2 H), 2.00 (dt, J ) 11 Hz, J ) 2 Hz, 2 H), 1.59
(m, 2 H), 1.51 (m, 1 H), 1.23 (m, 2 H). A solution of above
aniline (210 mg, 0.59 mmol) in THF was treated with phenyl
isocyanate (72 mg, 0.60 mmol) and stirred at room temperature
for 2 h. The solvent was removed under vacuum, and the
residue was purified by chromatography (MPLC, 50% EtOAc/
Hexane to 100% EtOAc, silica gel) to give 200 mg (74%) of the
1-[2-(4-Ben zyl-piper idin -1-ylm eth yl)ben zyl]-3-(3-cyan o-
p h en yl)u r ea (72). A similar procedure as described above for
39 was followed. Thus, 4-benzylpiperidine was alkylated with
2-bromomethyl-benzonitrile (in DMF) to give 2-(4-benzyl-
piperidin-1-ylmethyl)benzonitrile. The nitrile was reduced
using Raney Nickel in EtOH/NH3 (50 psi H2/room tempera-
ture/3 days) to give 2-(4-benzyl-piperidin-1-ylmethyl)benzyl-
amine. The resulting benzylamine was treated with 3-cy-
1
desired urea 104 as a white solid. H NMR (300 MHz, CDCl3)
δ (TMS): 10.5 8 (bs, 1 H), 8.15 (d, J ) 8 Hz, 1 H), 7.40-7.23
(m, 6 H), 7.15 (m, 1 H), 7.04 (m, 2 H), 6.95 (m, 2 H), 6.30 (bs,
1 H), 3.87 (s, 3 H), 3.78 (s, 2 H), 2.72 (m, 2 H), 2.43 (d, J ) 7
Hz, 2 H), 2.00 (m, 2 H), 1.52 (m, 2 H), 1.45 (m, 1 H), 0.92 (m,
2 H). MS ESI (M + H)+ ) 476.3 Anal. (C28H30N3O3F) C, H, N.
1
anophenyl isocyanate (in THF) to give the urea 72. H NMR
(300 MHz, CDCl3) δ (TMS): 7.73 (m, 1 H), 7.49-7.10 (m, 13
H), 5.99 (bs, 1 H), 4.42 (s, 2 H), 3.50 (s, 2 H), 2.92 (m, 2 H),
2.55 (d, J ) 7 Hz, 2 H), 2.03 (m, 2 H), 1.76 (m, 2 H), 1.66 (m,
1 H),1.20-1.04 (m, 2 H). MS ESI (M + H)+ ) 439.3. Anal.
(C28H30N4O) C, H, N.
3-[3-(3-Cya n o-p h en yl)u r eid o]-2-[4-(4-flu or o-ben zyl)p i-
p er id in -1-ylm eth yl]ben za m id e (110). A solution of 2-[4-(4-
fluoro-benzyl)piperidin-1-ylmethyl]-3-nitro-benzoic acid methyl
ester 134 (described above for the synthesis of 104) (2.6 g, 6.7
mmol) in 75 mL of MeOH was treated with 21 mL of 1 N
NaOH, and the mixture was heated to reflux for 2 h. The
solution was concentrated under vacuum, and the residue was
diluted with water and neutralized with 20 mL of 1 N HCl (to
a pH of about 6) to give a precipitate. The solid was extracted
into EtOAc, washed with water and brine, and dried over
MgSO4. The solvent was removed under vacuum to give 2.5 g
(100%) of the acid 135 (Scheme 4) as a yellow foam. A solution
of the acid 135 (1.0 g, 2.68 mmol) in dichloromethane was
treated dropwise with oxalyl chloride (0.55 g, 4.3 mmol) at
room temperature [immediate bubbling and precipitate forma-
tions were observed] and stirred for 1.5 h. The solvent and
excess oxalyl chloride were removed under vacuum to give the
acid chloride as a yellow foam. The crude acid chloride was
treated with a saturated solution of ammonia in dioxane at
room temperature for 20 min. The solution was concentrated
under vacuum, and the residue was partitioned between water
and EtOAc. The organic extract was washed with water and
brine and dried over MgSO4. The solvent was removed under
vacuum to give 0.9 g (90%) of the corresponding amide 136
(Scheme 4) as a yellow foam. This was used without further
purification. A solution of the above crude amide 136 (0.9 g,
2.4 mmol) in THF (50 mL) was treated with 10% Pd/C (0.3 g)
and hydrogenated at 50 psi at room temperature for 30 min.
The solution was then filtered, and the solvent was removed
under vacuum to give the aniline 137 (Scheme 4) as a white
solid. The aniline was used without further purification. A
solution of aniline 137 (Scheme 4) (100 mg, 0.29 mmol) in THF
was treated with 3-cyanophenyl isocyanate (42 mg, 0.35 mmol)
at room temperature for 1 h. The solvent was removed under
1-{2-[2-(4-Ben zyl-p ip er id in -1-yl)-1-h yd r oxyim in o-eth -
yl]p h en yl}-3-p h en yl-u r ea (51). Following the procedure
detailed above for 50 except using phenyl isocyanate provided
1-{2-[2-(4-benzyl-piperidin-1-yl)acetyl]phenyl}-3-phenyl-urea
129 (Scheme 3). A solution of 129 (310 mg, 0.73 mmol) in EtOH
was treated with hydroxylamine hydrochloride (300 mg, 4.3
mmol) and heated to reflux for 2 h. The solution was diluted
with water and extracted into EtOAc. The extract was washed
with water and brine and concentrated under vacuum, and
the residue was chromatographed (MPLC, 50%-70% EtOAc/
hexane, silica gel) to give 200 mg (63%) of the oxime 51 (Figure
1
2) as a solid. H NMR (300 MHz, CDCl3) δ (TMS): 7.89 (d, J
) 8 Hz, 1 H), 7.75 (bs, 1 H), 7.59(s, 1 H), 7.46-6.99 (m, 12 H),
6.89 (m, 2 H), 3.43 (s, 2 H), 2.93 (m, 2 H), 2.26 (d, J ) 7 Hz,
2 H), 1.93 (m, 2 H), 1.47 (m, 2 H), 1.36 (m, 1 H), 1.25-1.10
(m, 2 H). MS ESI (M + H)+ ) 443.4. Anal. (C27H30N4O2-
(H2O)0.25) C, H, N.
1-(2-{2-[4-(4-F lu o r o -b e n zy l)-p ip e r id in -1-y l]e t h y l}-
p h en yl)-3-(4-flu or o-p h en yl)u r ea (53). This compound was
obtained from the reaction of 2-{2-[4-(4-fluoro-benzyl)piperidin-
1-yl]ethyl}phenylamine (detailed above for the synthesis of 81/
45) with 4-fluorophenyl isocyanate. 1H NMR (300 MHz, CDCl3)
δ (TMS): 9.34 (bs, 1 H), 7.77 (d, J ) 8 Hz, 1 H), 7.39-7.20 (m,
4 H), 7.15-6.92 (m, 8 H), 3.03 (m, 2 H), 2.81 (m, 2 H), 2.58
(m, 2 H), 2.50 (d, J ) 7 Hz, 2 H), 2.02 (m, 2 H), 1.68 (m, 2 H),
1.56-1.25 (m, 3 H). MS ESI (M + H)+ ) 450.3 Anal. (C27H29N3-
OF2-(H2O)0.5) C, H, N.
N-(2-{2-[4-(4-Flu or o-ben zyl)piper idin -1-yl]eth yl}ph en yl)-
2-(4-flu or o-p h en yl)a ceta m id e (52). This compound was
obtained from the reaction of 2-{2-[4-(4-fluoro-benzyl)piperidin-

CC Chemokine Receptor-3 Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3803
1-yl]ethyl}phenylamine (detailed above for the synthesis of 81/
45) with (4-fluoro-phenyl)acetyl chloride. H NMR (300 MHz,
filter was allowed to completely dry and was stained
with Wright Giemsa. Migrated cells were enumerated
by microscopy.
1
CDCl3) δ (TMS): 10.15 (bs, 1 H), 7.91 (d, J ) 8 Hz, 1 H), 7.39-
6.92 (m, 11 H), 3.67 (s, 2 H), 2.88 (m, 2 H), 2.65 (m, 2 H), 2.54
(m, 2 H), 2.45 (d, J ) 7 Hz, 2 H), 2.00 (m, 2 H), 1.60 (m, 2 H),
1.50 (m, 1 H), 1.25 (m, 2 H). MS ESI (M + H)+ ) 449.3. Anal.
(C28H30N2OF2) C, H, N.
Ca lciu m Mobiliza tion Assa y. 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 fluoro-
phore by resuspending them in a HEPES-buffered
phosphate-buffered saline containing 5 µM fluo-3 and
incubating for 60 min at 37 °C. After they were washed
twice to remove excess fluorophore, cells were resus-
pended 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 changes in
fluorescence in all wells simultaneously. To determine
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 com-
puted automatically. All conditions were tested in
duplicate, and the mean shift per condition was deter-
mined. The inhibition achieved by graded concentrations
of compound was calculated as a percentage of the
compound-free eotaxin control.
Biologica l Assa ys
CCR3 Recep tor Bin d in g. Millipore filter plates (no.
MABVN1250) were treated with 5 µg/mL protamine in
phosphate-buffered saline, pH 7.2, for 10 min at room
temperature. Plates were washed three times with
phosphate-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
albumin, 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 was combined with 50 µL of 125-I-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 were a Chinese hamster ovary cell line
transfected with a gene expressing human CCR3 as
described by Daugherty.13 The mixture of compound,
cells, and radioligand was incubated at room tempera-
ture for 30 min. Plates were placed onto a vacuum
manifold, the vacuum was applied, and plates were
washed three times with binding buffer with 0.5 M NaCl
added. The plastic skirt was removed from the plate,
the plate was allowed to air-dry, the wells were punched
out, and the CPM was counted. The percent inhibition
of binding was calculated using the total count obtained
in the absence of any competing compound or chemokine
ligand, and the background binding was determined by
addition of 100 nM eotaxin in place of the test com-
pound.
Ack n ow led gm en t. We are grateful to the chemical
synthesis group for providing the 4-F-benzylpiperidine
used in this study. We also thank all of the members of
the CCR3 working group, especially Drs. J . V. Duncia
and D. A. Wacker, and Mr. J . B. Santella for valuable
discussions.
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
mentals including analytical data (NMR, MS) for all of the
compounds in this study. This material is available free of
charge via the Internet at http://pubs.acs.org.
Hu m a n Eosin op h il Ch em ota xis Assa y. Neuro-
probe MBA96 96 well chemotaxis chambers with Neu-
roprobe poly(vinylpyrrolidone)-free polycarbonate PFD5
5 µm filters in place were warmed in a 37 °C incubator
prior to assay. Freshly isolated human eosinophils14
were suspended in RPMI 1640 with 0.1% bovine serum
albumin at 1 × 106 cells/mL and warmed in a 37 °C
incubator prior to assay. A 20 nM solution of human
eotaxin in RPMI 1640 with 0.1% bovine serum albumin
was warmed in a 37 °C incubator prior to assay. The
eosinophil suspension and the 20 nM eotaxin solution
were each mixed 1:1 with prewarmed RPMI 1640 with
0.1% bovine serum albumin with or without a dilution
of a test compound that was at least 2-fold the desired
final concentration. The filter was separated, and the
eotaxin/compound mixture was placed into the bottom
part of the chemotaxis chamber. The filter and upper
chamber were assembled, and a 200 µL volume of the
cell suspension/compound mixture was added to the
appropriate wells of the upper chamber. The upper
chamber was covered with a plate sealer, and the
assembled unit was placed in a 37 °C incubator for 45
min. After incubation, the plate sealer was removed and
all of the remaining cell suspension was aspirated off.
The chamber was disassembled, unmigrated cells were
washed away with phosphate-buffered saline, and then,
the filter was wiped with a rubber-tipped squeegee. The
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