Potent and selective CC-chemokine receptor-2 (CCR2) antagonists as a potential treatment for asthma

Article abstract of DOI:10.1016/j.bmcl.2007.01.115

A number of compounds bearing a quaternary ammonium moiety were found to be antagonists with nanomolar binding affinity for the chemokine receptor-2. The structure-activity relationships in the series are described herein along with some detailed characte

Full text of DOI:10.1016/j.bmcl.2007.01.115

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4382–4386
Potent and selective CC-chemokine receptor-2 (CCR2) antagonists
as a potential treatment for asthma
Bharat Lagu,* Chrissy Gerchak, Meng Pan, Cuifen Hou, Monica Singer, Ravi Malaviya,
Michele Matheis, Gil Olini, Druie Cavender and Michael Wachter
Johnson & Johnson Pharmaceutical Research and Development, Cranbury, NJ 08512, USA
Received 5 December 2006; revised 24 January 2007; accepted 25 January 2007
Available online 8 February 2007
Abstract—A number of compounds bearing a quaternary ammonium moiety were found to be antagonists with nanomolar binding
affinity for the chemokine receptor-2. The structure–activity relationships in the series are described herein along with some detailed
characterization of the interesting compounds.
Ó 2007 Published by Elsevier Ltd.
Monocyte chemotactic protein-1 (MCP-1) is an impor-
tant member of a large family of chemokines that plays
a significant role in directing the influx of mononuclear
leukocytes into sites of inflammation (i.e., chemotaxis)
and tissue injury.1 MCP-1 is secreted by monocytes,
memory T cells, macrophages, fibroblasts, endothelial
cells, and mast cells, and stimulates the movement of
responsive leukocytes along a chemotactic gradient fol-
lowing binding to its cognate cell-surface receptor,
CC-chemokine receptor-2 (CCR2).2 The binding of
MCP-1 to the negatively charged extracellular loops of
CCR2 is believed to initiate a signaling cascade which
plays a critical role in both acute and chronic inflamma-
tory processes.3 Elevated expression of MCP-1 and
CCR2 has been observed in various diseases character-
ized by chronic inflammation and large numbers of infil-
trating monocytes including rheumatoid arthritis,
multiple sclerosis, inflammatory bowel diseases, athero-
sclerosis, asthma, and uveitis.2–5 There is strong evi-
dence, using both MCP-1 and CCR2 knockout mice,
that the MCP-1/CCR2 ligand/receptor pair plays a crit-
ical role in inflammatory processes.6,7 These observa-
tions have led to the hypothesis that an antagonist of
the G-protein coupled receptor CCR2 would be an effec-
tive treatment for inflammatory disorders.
Structures of several CCR2 antagonists have been pub-
lished by many research laboratories.4,5,8 In general
these compounds contain two lipophilic regions separat-
ed by a basic amine such as piperidine or pyrrolidine.9–14
However, our attention was caught by a paper from
Takeda Pharmaceuticals on a structurally unique
CCR5 antagonist, TAK-779, which was in Phase I clin-
ical trials as an anti-HIV treatment (Fig. 1).15 In addi-
tion to its high affinity for the CCR5 receptor, this
compound also showed affinity for the closely related
CCR2 receptor (IC50 for CCR5 = 1.4 nM, IC50 for
CCR2 = 27 nM). We were intrigued by the possibility
of analoging out the (undesired) CCR5 activity of these
types of compounds while maintaining or improving the
binding affinity for CCR2. In order to test the hypothe-
sis, the ‘A’, ‘B’, and ‘C’ segments of the molecule were
systematically modified (Fig. 1). The synthesis and
structure–activity of these CCR2 antagonists is
described in this letter.
B
C
A
H
N
O
N
O
Keywords: Chemokine receptor-2; CCR2 antagonist; Chemotaxis;
MCP-1; Asthma; Monocytes; Quaternary ammonium salt.
TAK-779
*
Corresponding author at present address: Novartis Institute for
Biomedical Research, Cambridge, MA 02139, USA. Tel.: +1 617 871
4257; fax: +1 617 871 4081; e-mail: bharat.lagu@novartis.com
Figure 1. Structure of TAK-779 and various segments targeted for
CCR2 affinity.
0960-894X/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.01.115

B. Lagu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4382–4386
4383
The compounds were synthesized in a straightforward
manner using the synthetic routes described in Scheme
1.15,16 The flexible nature of synthesis made the explora-
tion of A, B, and C segments of the molecule in an effi-
cient manner. The compounds were initially screened for
the binding affinities to the CCR2 receptor using THP-1
ity (0.01 lM) to the CCR2 receptor. The compounds
devoid of the biphenyl group (1c–d) showed substantial-
ly decreased binding affinities for the receptor (>2 lM).
Interestingly, the 3,4-dichlorocinnamoyl moiety is also
present in the structures of CCR2 antagonists published
from Glaxo–Smith Kline.9,10
125
cell line and I-labeled MCP-1.17
However, when the cinnamoyl moiety in segment ‘A’
was replaced with a substituted 2H-chromene-3-carbox-
ylic acid moiety, the compounds (2a–e) displayed sub-
micromolar binding affinity (0.05–0.2 lM) for the
CCR2 as shown in Table 1. The presence of chlorine
at the 7-position on the 2H-chromene yielded com-
pounds with good binding affinities (0.05–0.2 lM).
When the segment ‘A’ was changed to a substituted
phenyl ring, an interesting SAR emerged for the result-
ing compounds.
It soon became obvious that the ‘B’ segment tolerated a
number of modifications without deleteriously affecting
the binding affinities for the CCR2 receptor as shown
in Scheme 1. The modifications of the segment ‘A’, on
the other hand, had a significant impact on the binding
affinities for the CCR2. Herein we describe the struc-
ture–activity relationship in a series of compounds that
differed from TAK-779 in the ‘A’ segment of the mole-
cule. The binding affinities for the compounds possess-
ing cinnamoyl, 2-H-chromenyl, and phenyl moieties in
the segment ‘A’ are summarized in Table 1.
In contrast to the compounds 1a and 1b, the biphenyl
moiety-containing compound 3h in this series of com-
pounds did not show good binding affinity
(IC50 = 3.4 lM). The presence of strong electron donat-
ing groups (in 3e and 3g) or a presence of electron with-
drawing groups (3d or 3f) at the 3-position of the phenyl
ring resulted in compounds with either the same or
diminished binding affinity for the CCR2 as compared
In general, the compounds lacking the quaternary nitro-
gen showed significantly reduced (>10 lM) binding
affinities for the CCR2 receptor as compared to the
compounds that incorporate the quaternary nitrogen.
Compounds 1a–b containing the biphenyl moiety at-
tached to a cinnamoyl group showed good binding affin-
O2N
H2N
H2N
c
O2N
X
N
( )
N
H
( )n
X
( )n
X
O
R
a, b
A
H
N
Cl
N
H
N
H
N
I
d
R
R
e
f
N
A
N
O
( )n
X
O
( )n
X
O
( )n
X
O
H
N
O2N
H2N
I
R
O2N
c-e
O
O
CHO
O
N
a, b
N
I
O
O
O
O2N
O2N
N
c-e
O
O
NH2
a, b
R
N
H
N
H
N
H2N
H
N
H
N
d, g
R
h, b
I
R
R
e
OH
O
O
N
O
N
CHO
O
O
Scheme 1. Reagents and conditions: (a) NaB(OAc)3H, cat AcOH, CH2Cl2, rt, 6 h; (b) 37% HCHO, NaB(OAc)3H, CH2Cl2, rt; (c) SnCl2Æ2H2O, THF;
(d) RCOOH, EDC, HOBt, Et3N, DMF or RCOCl, Et3N, THF; for the description of R groups, please see Table 1; (e) CH3I, CH2Cl2 or acetone; (f)
ion-exchange chromatography; (g) MnO2, CHCl3, rt, 12 h; (h) 4-aminotetrahydropyran, NaB(OAc)3H, cat AcOH, CH2Cl2, rt, 6 h.

4384
B. Lagu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4382–4386
Table 1. Structure–activity relationship for CCR2 antagonists
Table 2. Structure–activity relationship for CCR2 antagonists
Compound R
IC50 (lM)a
R
H
N
H
N
R'
1a-d
R
Cl
N
R' =
3n
0.02 0.01
11.35 5.05b
0.20 0.04b
0.03 0.01
0.04 0.01b
0.29 0.17
0.09 0.04b
1.02 0.68b
>10
O
O
N
O
O
3a-k
R
Cl
O
Cl
2a-d
O
H
N
4
N
Compound
R
IC50 (lM)a
Cl
O
1a
1b
1c
1d
4-(p-toluyl)
0.01 0.01
0.01 0.01
1.81 1.04
2.55 0.95b
4-(p-CF3)-Ph
3-F
3,4-di-Cl
H
N
Cl
N
5
S
O
Cl
2a
2b
2c
2d
2e
2f
7-Br
7-Cl
0.18 0.10
0.12 0.07
0.17 0.12
0.22 0.13
0.05 0.03
0.20 0.10
0.16 0.03b
0.07 0.03
H
Cl
Cl
Cl
Cl
N
N
7-Me
9-Me
6
O
Cl
Cl
Cl
Cl
7-Cl-9-Me
6,8-di-Cl
7,9-di-Cl
4,5-benzo
H
N
2g
2h
N
N
N
7
O
3a
3b
3c
3d
3e
3f
H
3-Br
4.75 0.20
0.25 0.14
0.23 0.13
3.25 0.95b
9.55 1.85b
2.05 0.45b
4.25 2.15b
3.44 1.98
2.00 0.40b
>10
H
N
3-Cl
3-CN
8
O
3-OMe
3-CF3
3-Me
H
N
3g
3h
3i
3-(p-toluyl)
4-Cl
2-Cl
9
O
3j
3k
3l
2,3-di-Cl
2,4-di-Cl
3,5-di-Cl
3,4-di-Cl
3-Cl-4-F
>10
O
H
N
7.80 1.20b
0.71 0.35
0.02 0.01
0.39 0.23
Cl
N
10
11
12
3m
3n
3p
O
Cl
Cl
H
N
O
a SEM shown for n > 2.
b An average with SD is shown for n = 2 mean number for IC50 shown.
N
Cl
O
O
Cl
Cl
N
H
8.95 2.55b
to the compound, 3a. The presence of a chloro or a bro-
mo group at the 3-position (3b and 3c) yielded com-
pounds with significantly improved binding affinities
(IC50 = 0.2 lM). The position of the chlorine atom on
the phenyl ring was crucial for the observed binding
affinity (compounds 3i and 3j). Compound 3i, possess-
ing chloro group at the 4-position of the phenyl ring,
had reduced binding affinity (IC50 = 2 lM), while com-
pound 3j with 2-chloro group showed significantly
reduced binding affinity (IC50 > 10 lM) for CCR2. In
the case of di-substituted phenyl analogs, compound
3n bearing 3,4-di-chloro substituted phenyl ring showed
a superior binding affinity for the CCR2 receptor as
compared to the compounds 3l, 3m, and 3p containing
the other di-chloro substitutions.
N
O
a SEM shown for n > 2.
b An average with SD is shown for n = 2 mean number for IC50 shown.
ring in the segment ‘C’ by 5, 6 or 7-membered cycloalkyl
groups or norbornane (compounds 6–9) does not affect
the binding affinities (0.03–0.09 lM) in an adverse way.
The replacement of the tetrahydropyran ring by a tetra-
hydrothiopyranyl group results in compound 5 with 10-
fold lower binding affinity (0.2 lM vs 0.02 lM for 3n).
The presence of an additional methylene group on either
side of the phenyl ring (segment ‘B’) leads to compounds
11 and 12 with substantially reduced binding affinities
(>8 lM) for the CCR2. Similarly, compound 10 con-
taining an additional methylene group between the tetr-
ahydropyarn and the quaternary nitrogen resulted in the
loss of binding affinity (1.0 lM). Compound 3n was cho-
sen for more detailed evaluation.
Like segment ‘A’, modifications in the segments ‘B’ and
‘C’ also have some influence on the binding affinities of
the resulting compounds (Table 2). The meta substitu-
tion pattern in the di-substituted phenyl ring in the seg-
ment ‘B’ leads to compound 4 with significantly reduced
binding affinity (IC50 = 11 lM) as compared to the para-
substituted 3n. The replacement of the tetrahydropyran

B. Lagu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4382–4386
4385
such as CCR1, CCR3, CCR4, CCR6, CCR7, and
CCR8. Not surprisingly, the compound weakly inhibit-
ed binding of MIP-1a to the CCR5 receptor
(IC50 ꢀ 1 lM). This observation underscores the fact
that the original CCR5 antagonist (TAK-779) possess-
ing low-nanomolar affinity for CCR5 and lower CCR2
affinity has been transformed into a compound that
maintains a good binding affinity for the CCR2 receptor
but has significantly reduced binding affinity for the
CCR5 receptor. Such reversal of the chemokine receptor
selectivity can be attributed to the changes made in the
‘A’ region of TAK-779. Compound 3n did not show
any significant cross-reactivity when tested against a
panel of more than 30 receptors and ion channels
including some G-protein coupled receptors (e.g., 5-
HT, alpha adrenergic receptors, Ca2+ and ClÀ ion chan-
nels). The compound has excellent aqueous solubility
(>7 mg/mL) at pH 2 and 7. However, the compound
3n showed poor permeability through Caco-2 cell line
presumably due to its positive charge. The compound
exhibited excellent metabolic stability following
incubation with human liver microsomes for one hour
(the % recovery was 100%). The compound does not
show any oral bioavailability in rats (presumably due
to poor absorption), but has 74% and 100% bioavail-
ability upon intraperitoneal or subcutaneous adminis-
tration, respectively.
OVA
a
15.0
12.5
10.0
7.5
PBS
5.0
2.5
0.0
Vehicle
Vehicle
Cpd. 3n
OVA
b
250
200
150
100
50
PBS
0
Vehicle
Vehicle
Cpd. 3n
The anti-inflammatory property of the compound was
confirmed when compound 3n showed 79% inhibition
of the leukocyte recruitment in the zymosan-induced
peritonitis model in mice at a dose of 30 mg/kg (subcu-
taneously).18,19 Both CCR2 and MCP-1 have been pos-
tulated to play a role in the asthma.20–23 Therefore, the
compound was next evaluated in a mast cell-dependent
model of ovalbumin-induced asthma in BALB/c mice.24
The results are shown in Figure 2. A 95% inhibition of
the cell influx into the bronchoalveolar lavage fluid
was observed after dosing at 30 mpk (intraperitoneally).
In addition, the compound also inhibited production of
LTC4 and IL-4 by 90% and 85%, respectively. Together,
these beneficial effects on these critical markers of asth-
ma after the administration of 3n suggest that a potent
CCR2 antagonist could potentially serve as treatment
for asthma. The details of these experiments including
histological evaluation of the relevant tissues will be
published elsewhere.
OVA
c
75
50
25
0
PBS
Vehicle
Vehicle
Cpd. 3n
Figure 2. (a) Total cell count in bronchoalveolar lavage; (b) LTB4(pg/
mL); (c) IL-4 (pg/mL).
The compound 3n showed significantly reduced affinity
(2 lM) for disrupting the binding of radiolabeled mouse
MCP-1 to WEHI 265.1 cells (a mouse macrophage cell
line). These data are not completely surprising given
the fact that the human and mouse CCR2 proteins share
only 83% sequence homology, and the human and
mouse MCP-1 proteins share only 64% sequence identi-
ty. These species differences become critical when one
examines the results from in vivo studies in rodents. In
cell-based functional assays, the compound potently
inhibited MCP-1 induced chemotaxis (IC50 = 100 nM)
of THP-1 cells (a human monocyte-like cell line). The
compound also inhibited MCP-1 induced calcium mobi-
lization (IC50 = 100 nM) in both THP-1 cells and human
peripheral blood mononuclear cells. The compound 3n
did not inhibit binding of the relevant CC-chemokines
In summary, a number of CCR2-selective antagonists
were synthesized by modifications of the structure of a
known CCR5 antagonist. One of the compounds, 3n,
showed excellent binding affinity and selectivity for the
CCR2 receptor over a number of other GPCRs. When
dosed parenterally, the compound showed good activity
in two different models of inflammation. These observa-
tions suggest that CCR2 antagonists would be beneficial
for treatment of immunoinflammatory diseases.
Acknowledgment
The authors thank Dr. William Murray and Dr. Linda
Jolliffe for their constant support and encouragement.

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16. Hashimoto, H.; Ikemoto, T.; Itoh, T.; Maruyama, H.;
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