Discovery of a novel CCR3 selective antagonist

Article abstract of DOI:10.1016/S0960-894X(01)00176-7

In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide derivatives based on the structure of our potent antagonists for human CCR1 and CCR3 receptors, and screened the new compounds for inhibitory activity against ¹²⁵I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC₅₀ values of 750 and 1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human CCR1 receptors. We selected 1a as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over CCR1 receptors. Derivatization of 1a by incorporating substituents into each benzene ring of the benzothiazole and piperidine side chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC₅₀ = 2.3 nM) over CCR1 receptors (IC₅₀ = 1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin (IC₅₀ = 27 nM)- or RANTES (IC₅₀ = 13 nM)-induced Ca²⁺ increases in eosinophils.

Full text of DOI:10.1016/S0960-894X(01)00176-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1219–1223
Discovery of a Novel CCR3 Selective Antagonist
Akira Naya,* Kensuke Kobayashi, Makoto Ishikawa, Kenji Ohwaki,
Toshihiko Saeki, Kazuhito Noguchi and Norikazu Ohtake
Banyu Tsukuba Research Institute, Okubo-3, Tsukuba 300-2611, Ibaraki, Japan
Received 11 January 2001; accepted 10 March 2001
Abstract—In searching for a novel CCR3 receptor antagonist, we designed a library that included a variety of carboxamide deri-
vatives based on the structure of our potent antagonists for human CCR1and CCR3 receptors, and screened the new
compounds for inhibitory activity against 125I-Eotaxin binding to human CCR3 receptors expressed in CHO cells. Among
them, two 2-(benzothiazolethio)acetamide derivatives (1a and 2a) showed binding affinities with IC50 values of 750 and
1000 nM, respectively, for human CCR3 receptors. These compounds (1a and 2a) also possessed weak binding affinities for human
CCR1receptors. We selected 1a as a lead compound for derivatization to improve in vitro potency and selectivity for CCR3 over
CCR1receptors. Derivatization of 1a by incorporating substituents into each benzene ring of the benzothiazole and piperidine side
chain resulted in the discovery of a compound (1b) exhibiting 820-fold selectivity for CCR3 receptors (IC50=2.3 nM) over CCR1
receptors (IC50=1900 nM). This compound (1b) also showed potent functional antagonist activity for inhibiting Eotaxin
(IC50=27 nM)- or RANTES (IC50=13 nM)-induced Ca2+ increases in eosinophils. # 2001Elsevier Science Ltd. All rights
reserved.
Introduction
patients with atopic asthma.4 Thus, a CCR3 receptor
selective antagonist that suppresses the infiltration of
eosinophils to inflammatory sites may have clinical
potential in allergic diseases.
Chemokines, a large sub-family of chemoattractant cyto-
kines, are generally classified into four sub-families, CXC
chemokines, CC chemokines, C chemokine and CX3C
chemokine, based on the arrangement of the cysteines in
the N-terminal region.1 These chemokines elicit their
biological effects by activation of a subset of the seven
transmembrane G-protein-coupled receptors (GPCRs).
There have been 16 human chemokine receptors cloned
and characterized to date, which are referred to as
CCR1–11, CXCR1–5, XCR1 and CX3CR1receptors.
To identify an orally active, non-peptide antagonist
selective for CCR3 receptors, we initiated exploration of
a lead structure. Screening of a focused library includ-
ing 770 compounds to search for compounds exhibiting
greater than 50% inhibition at a concentration of 1 mM
to 125I-Eotaxin binding led to the selection of a 2-(ben-
zothiazolethio)acetamide derivative (1a). In this paper,
we describe the identification of a lead CCR3 receptor
antagonist from a focused library and the structure–
activity relationships of the 2-(benzothiazolethio)acet-
amides that were derived from the lead structure (1a).
Selective accumulation of eosinophils to inflammatory
sites is characteristic in allergic diseases such as asthma.2
Therefore, eosinophils are thought to play an important
role in the initiation and progression of these diseases.
There is a growing body of evidence that CC chemo-
kines such as Eotaxin, RANTES, MCP-3 and MCP-4,
which are ligands for the CCR3 receptor, are respon-
sible for the recruitment of eosinophils from the circu-
lation to allergic sites.3 It should also be noted that
enhanced expression of Eotaxin and CCR3 receptor
mRNA was observed in bronchial biopsies from
*Corresponding author. Tel.:+81-298-77-2000; fax: +81-298-77-
2029; e-mail: nayaak@banyu.co.jp
0960-894X/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00176-7

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A. Naya et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1219–1223
Results and Discussion
receptor; and (3) an aromatic group for a hydrophobic
interaction site, and that the quaternary ammonium
structure which was inappropriate for oral absorption
would be replaceable with a basic amine group. Fol-
lowing up on this hypothesis, we designed carboxamides
with aromatic group(s) in the acid part and with a ben-
zylamine in the amine part (vide infra). To construct a
library, we selected 70 kinds of commercially available
carboxylic acids6 from our in-house database after
clustering, based on the binary Tanimoto coefficient7a
calculated by the MDL keys7b as a structural descriptor,
and used 11 structurally diverse diamines. These car-
boxylic acids and diamines were coupled by a parallel
synthesis method using EDCI-HOBt as a coupling
reagent to produce a total of 770 carboxamides. The
structure of each compound was elucidated by its
molecular weight measured by LC–MS.
Chemistry
Our strategy to identify a lead structure for a potent
CCR3 selective antagonist involved the design of a
library constituted from carboxamide derivatives and
the derivatization of selected compounds.
Preparation of derivatives (1b–g, 2a–j) is summarized in
Scheme 1.8 Derivatives (1c–e) were prepared from sec-
ondary amine (4) by reductive alkylation with sub-
stituted benzaldehydes (5c–e) in >90% yields. Other
derivatives (1b and 1g) were synthesized from 4-aminopi-
peridine (6), which was reacted with bromoacetyl bromide
to produce bromoacetamide (7). Treatment of 7 with sub-
stituted benzothiazole (8b and 8g) yielded the desired
compounds in 55–65% yields (two steps). Compound 1f
was synthesized from 2-bromobenzothiazole (9f), which
was reacted with mercaptoacetate to produce 2-(ben-
zothiazolethio)acetate (10f) in 42% yields. Deprotection of
10f and coupling with 6 led to 1f in 98% yields (two steps).
Also, derivatives (2a–j) were prepared from an amine (11)
by reductive alkylation with appropriate aldehydes.
Figure 1.
Previously we reported that a xanthenecarboxamide (3)
showed potent inhibitory activity against both human
CCR1(IC
(IC50=0.58 nM).5 Based on the structure of 3, we
hypothesized that the following functional groups are
also required for CCR3 inhibitory activity: (1) a car-
bonyl group as a hydrogen bonding acceptor; (2) an
amine moiety for electrostatic interaction to the target
50=0.9 nM)
and
CCR3
receptors
Scheme 1.

A. Naya et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1219–1223
1221
Figure 2.
Biological properties
To investigate the role of substituent on the piperidine
nitrogen in the inhibitory activity against CCR3 recep-
tors, the phenyl ring of 2a was replaced with a variety of
substituents (Table 2). Evidently, only a benzyl group
was found to be effective in enhancing the activity.
Considering the potency of 1a and 2a for CCR3 recep-
tors and selectivity for CCR3 over CCR1receptors, we
prioritized to further derivatize 1a (Table 3).
The primary screening of compounds was conducted by
50% inhibition at 1 mM to 125I-Eotaxin binding to
CCR3 receptors expressed in CHO cells. Selected com-
pounds showing greater than 50% inhibition at 1 mM
were subsequently purified or re-synthesized, and tested
in the binding assay for human CCR3 receptors.
Among the compounds tested, only two 2-(benzothia-
zolethio)acetamide derivatives (1a and 2a) showed more
than 50% inhibition, and their IC50 values were 0.75
and 1.0 nM, respectively (Table 1). Unfortunately, these
compounds also showed potent inhibitory activity
against human CCR1receptors.
First, the effects of substituents on the benzene ring of
the piperidine nitrogen in 1a on inhibitory activity were
examined. Although substituents such as a methyl,
methoxy, or nitro group at the 4-position resulted in a
decrease in the potency for CCR3 receptors, introduction

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A. Naya et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1219–1223
Table 1. Binding affinity of compounds 1a and 2a to CCR3 and
CCR1receptors
of a chlorine atom at the 4-position of the benzene ring
(1c) greatly enhanced the binding affinity for both
CCR1and CCR3 receptors. Therefore, the selectivity of
1c for CCR3 receptors was not improved. By contrast,
incorporation of a chlorine atom at the 3 position (1d)
led to approximately 3-fold improvement in the binding
affinity for CCR3 receptors, while reducing the affinity
for CCR1receptors. Although a 3,4-dichlorophenyl
derivative (1e) seemed most potent, its selectivity for
CCR3 over CCR1receptors also remained to be
improved.
IC50 (mM)
Next, we tried to introduce a substituent into the ben-
zothiazole ring of 1e. When an ethoxycarbonyl group
was incorporated into the 6 position, the resulting
derivative (1f) showed potent binding affinity for CCR3
receptors comparable to that of 1e. By contrast, the
affinity of 1f for CCR1receptors was much reduced as
compared to that of 1e. It should be noted that 1f, in
which a substituent was incorporated at the benzothia-
zole ring, showed 200-fold selectivity for CCR3 over
CCR1receptors. An ethoxy moiety ( 1g) resulted in
improvement in affinity for both CCR1and CCR3
receptors, while retaining the selectivity. Further deri-
vatization at this part revealed that the most effective
functional group seemed to be an amino moiety (1b),
which brought about 10-fold improvement in the bind-
ing affinity for CCR3 receptors as compared to that of
1e. The 6-aminobenzothiazole derivative (1b) showed
IC50 values of 2.3 and 1900 nM for human CCR3 and
CCR1receptors, respectively, and 820-fold selectivity
for CCR3 over CCR1receptors.
Compound
CCR3
CCR1
1a
2a
0.75
1.0
7.1
4.2
Table 2. Binding affinity of compounds 2a–j to CCR3 receptors
IC50 (nM)
CCR3
1000
Compound
R
2a
2b
2c
2d
2e
2f
2g
2h
2i
Ph
Cyclohexyl
Et
n-Butyl
2-Thiazolyl
3-Pyridyl
4-Pyridyl
2-Naphthyl
1-Naphthyl
CH2Ph
30%@
0%@
0%@
0%@
0%@
0%@
5%@
15%@
0%@
To determine whether 1b showed functional antagonism
of CCR3 receptors, we measured its ability to inhibit
Eotaxin- and RANTES-induced Ca2+ increases in
eosinophils. 1b inhibited the Ca2+ responses with IC50
values of 27 and 13 nM, respectively. These results indi-
cated that 1b is a potent antagonist selective for CCR3
receptors. Details on the biological properties of this
compound will be reported in the near future.
2j
@: inhibition% at 1 mM.
Conclusions
Table 3. Binding affinity of compounds 1a–g to CCR3 and CCR1
receptors
We discovered a novel CCR3 selective antagonist (1b)
by modifying the lead compound (1a), which was iden-
tified from the screening of a focused library (770 car-
boxamide derivatives). Since 1b possessed potent
functional antagonism against Eotaxin- and RANTES-
induced Ca2+ increases in eosinophils as well as high
potency in the binding assay, it may be a useful tool to
elucidate the role of CCR3 receptors in allergic diseases,
especially in the selective accumulation of eosinophils to
inflammatory sites.
IC50 (nM)
Compound
R1
R2
R3
CCR3
CCR1
1a
1c
1d
1e
1f
H
H
H
Cl
H
Cl
Cl
Cl
Cl
H
H
H
750
79
280
32
48
20
2.3
7200
260
>10,000
450
>10,000
3600
1900
Cl
Cl
Cl
Cl
Cl
Acknowledgements
H
COOEt
OEt
NH2
1g
1b
The authors are grateful to Ms. Anne Dobbins, Merck
& Co., Inc., for her critical reading of this manuscript.

A. Naya et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1219–1223
1223
References and Notes
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