Pyrazolone methylamino piperidine derivatives as novel CCR3 antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.05.035

The discovery and optimization of a novel class of potent CCR3 antagonists is described. Details of synthesis and SAR are given together with some ADME properties of selected compounds. An optimal balance between activities, physicochemical properties, and in vitro metabolic stability was reached by the proper choice of substituents.

Full text of DOI:10.1016/j.bmcl.2007.05.035

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4228–4231
Pyrazolone methylamino piperidine derivatives as novel
CCR3 antagonists
*
´
´
Cecile Pegurier, Philippe Collart, Pierre Danhaive, Sabine Defays, Michel Gillard,
´ ´
Frederic Gilson, Thierry Kogej, Patrick Pasau, Nathalie Van Houtvin,
Marc Van Thuyne and BerendJan van Keulen
UCB Pharma SA, R&D, Chemin du Foriest, B-1420 Braine-L’Alleud, Belgium
Received 30 March 2007; revised 9 May 2007; accepted 11 May 2007
Available online 17 May 2007
Abstract—The discovery and optimization of a novel class of potent CCR3 antagonists is described. Details of synthesis and SAR
are given together with some ADME properties of selected compounds. An optimal balance between activities, physicochemical
properties, and in vitro metabolic stability was reached by the proper choice of substituents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The prevalence of asthma is increasing in industrialized
countries and symptoms are not completely controlled
in many patients. There is a need for new therapies
against this chronic airway inflammatory disease. Pul-
monary eosinophil recruitment seems to be closely re-
lated to the symptoms of allergic asthma. Eosinophils
are attracted, via their CC chemokine receptor 3
(CCR3), in response to chemoattractants such as eotax-
in, Rantes, MCP-3, MCP-4 released in the airways of
asthmatics.1 Inhibiting pulmonary eosinophilia by
blocking the CCR3 receptor with small molecule antag-
onists may lead to a reduction in the inflammation and
the airway responsiveness seen in asthma. Several re-
search groups are investigating this approach.2 One
compound from GlaxoSmithKline (GW-766994)3 is cur-
rently undergoing Phase II clinical trials for the treat-
ment of asthma and allergic rhinitis.
aminopiperidine central core as a privileged structure to
explore the chemical space and find new ligands for the
CCR3 receptor. A large combinatorial library was de-
signed around three points of diversity. The selection of
the building blocks was based on different criteria like
commercial availability of the reagents, physicochemical
properties and diversity requirements of the generated vir-
tual library. A 2888-compound library was prepared by
solid-phase chemistry following Scheme 1. A Wang resin
was activated by reaction with p-nitrophenylchlorofor-
mate in order to allow the attachment of 4-piperidone.
The keto function was transformed after two consecutive
reductive amination reactions, first with primary amines
(R2NH2) then with aldehydes (R1CHO) to afford resin-
bound 4-dialkylamino piperidines. Cleavage from the re-
sin using trifluoroacetic acid yielded piperidines that were
finally reacted with acyl chlorides (R3COCl).
O
Cl
Cl
N
N
N
N+
Cl
H
H
O
O
N
H2N
O
S
N
N
H
Cl
N
H
Cl
O
O
S
GW - 766994
B
Cl
A
H2N
Among the first CCR3 antagonists described in the litera-
ture, two compounds with high affinities were disclosed by
Banyu Pharmaceuticals (A: IC50 = 0.58 nM and B:
IC50 = 2.3 nM).4 We initiated a library synthesis with an
Very few hits were identified from this large library,
compound 1 being the most potent for the human
CCR3 receptor (IC50 = 158 nM),5 all bearing a phenyl
pyrazolone moiety as R1 modulation. This subunit was
previously mentioned by Gong6 as providing some
Keywords: Chemokine; CCR3; Pyrazolone; Asthma.
*
Corresponding author. Fax: +32 2 386 27 04; e-mail: cecile.pegurier
@ucb-group.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.035

´
C. Pegurier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4228–4231
4229
O
O
N
a, b
OH
c
O
O
O
O
O
O
d
N
O
N
O
N
R2
R1
O
NH
R2
O
R3
N
f
e
HN
R1
N
R2
R1
N
R2
Scheme 1. Combinatorial chemistry procedure. Reagents and conditions: (a) p-nitrophenylchloroformate, pyridine, DCM; (b) 4-piperidone, DMF;
(c) R2NH2, NaBH(OAc)3, DCE, AcOH; (d) R1CHO, NaBH(OAc)3, DCE, AcOH; (e) TFA, DCM; (f) R3COCl, NEt3, DCE.
O
recognition for the CCR3 receptor. Further focused
H
d, e
combichem libraries were then generated in order to
optimize CCR3 binding affinity by modulating R2 and
R3 substituents. A total of 790 compounds were pre-
pared by the combichem procedure. The SARs revealed
that the para-substituted phenylethyl and the ortho,
ortho-disubstituted phenyl were good modulations for
R2 and R3 moieties, respectively. In particular, com-
a, b, c
N
R
NO2
R
N
R
NH2
O
N
h
O
O
f, g
O
N
N
R3
O
R2
O
O
pound
(IC50 = 32 nM).
2
displayed
a
good activity for CCR3
R2
N
N
O
i, j
R3
N
N
R
N
O
H
R3
.HCl
N
Cl
N
R
N
O
O
Scheme 2. Medicinal chemistry procedure. Reagents and conditions:
(a) SnCl2, conc. HCl, EtOH; (b) NaNO2, conc. HCl; (c) SnCl2, conc.
HCl; (d) methyl acetoacetate, CH3CN, reflux; (e) Me2SO4, CaO,
MeOH; (f) TFA, DCM; (g) R3COCl, NEt3, DCM; (h) R2NH2,
NaBH(OAc)3, DCE, AcOH, molecular sieves; (i) paraformaldehyde,
iPrOH, NH4Cl, 90 ꢁC; (j) methanolic HCl, ether.
O
F
O
N
N
N
N
N
N
N
F
N
2
1
IC50=32 nM
IC50=158 nM
We next decided to optimize the substitution pattern on
the phenyl ring attached to the pyrazolone. Compounds
were obtained following the route depicted in Scheme 2.
Diverse pyrazolones were prepared from commercially
available hydrazines by condensation with methyl aceto-
acetate followed by N-methylation with dimethylsulfate.
Some hydrazines were prepared from nitrobenzenes by
successive reduction into anilines, diazotation, and
reduction by SnCl2 of the diazo intermediates. The
aminopiperidine core was obtained in three steps from
N-Boc-piperidone following a Boc-deprotection, acyla-
tion, and then reductive amination sequence. The pyraz-
olone moiety is incorporated in the last step via a
Mannich reaction.
a fluorine atom around the phenyl ring indicated the
meta substitution is slightly disfavored. Most com-
pounds display an IC50 around 100 nM, regardless of
the presence of electron donating (OMe, NMe2) or with-
drawing (NO2, CN) substituents. A more dramatic de-
crease in affinity is observed with the para-carboxyl
analogue 14 (IC50 = 2500 nM) suggesting that the pres-
ence of a negative charge in this area is detrimental to
the binding with the receptor.
The most potent derivative was the para-fluoro phen-
ylpyrazolone analogue 5 (IC50 = 20 nM). Interestingly,
this compound exhibits comparable level of activity in
functional assays such as the inhibition of the in vitro
Ca2+ flux and the eotaxin-induced eosinophil shape
change (IC50 = 16 nM for both). Some physicochemical
properties and in vitro metabolic clearances of com-
pound 5 were measured and are reported in Table 2,
showing poor water solubility and metabolic stability.
The binding data on human CCR3 receptor5 of com-
pounds 2–18 are reported in Table 1. By comparison
with the unsubstituted phenyl analogue 2, the results
show an overall decrease in affinity, except for the
para-fluorophenyl and the 2-pyridyl derivatives. Moving

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C. Pegurier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4228–4231
4230
Table 1. CCR3 binding activities for compounds 2–18
In order to increase solubility and metabolic stability,
some less lipophilic analogues were prepared by the
introduction of ionizable groups. Data from selected
compounds with improved profiles are reported in Table
2. Several structural modifications generated more solu-
ble compounds like the substitution of the chlorine atom
in R4 moiety by a fluorine (19), a nitro (20), a sulfon-
amido (25) or an amino (26) group (100- to 900-fold).
The increase was even more significant with the replace-
ment of one phenyl ring by a pyridine (21–24) or a pyr-
idine N-oxide 27 (1200- to 2000-fold). Not surprisingly,
the metabolic stability was concomitantly improved
with those compounds, reaching acceptable levels on
rat microsomes for analogues 24–27 (Clint < 70 ll/min/
mg protein). Those results were confirmed on rat hepa-
tocytes for compounds 24, 26, and 27. On the contrary,
by lowering the lipophilicity, the intestinal permeability
of some analogues (22, 25–27) could be limited, as pre-
dicted in the Caco-2 assay (Papps < 2 · 10À6 cm/s).
Regarding CCR3 binding affinities, structural modula-
tions in the R4 moiety generally lead to less active ana-
logues, whereas modifications in the R3 part provided
potent compounds (23, 24, 27). Two candidate com-
pounds 24 and 27 will be proposed for in vivo DMPK
evaluation. They both have interesting CCR3 affinities
and solubility profile, 24 having good predicted intesti-
nal permeability and moderate metabolic stability and
27 showing a better metabolic profile but a lower pre-
dicted intestinal permeability.
Cl
O
O
F
N
N
R
N
N
F
2-18
a
Binding IC50 (nM)
Compound
R
2
3
Ph
2-Pyridyl
32
25
4
Cyclohexyl
4-F-Ph
3-F-Ph
100
20
5
6
100
40
7
2-F-Ph
4-Cl-Ph
8
63
9
4-OH-Ph
4-NO2-Ph
4-CN-Ph
4-OMe-Ph
4-NH2-Ph
100
100
158
100
79
10
11
12
13
14
15
16
17
18
4-COOH-Ph
4-NHCOMe-Ph
4-NMe2-Ph
2,500
400
100
100
250
4-Cl-(3-pyridyl)
4-OMe-(3-pyridyl)
a Competition binding experiments with
eosinophils.
[
125I]eotaxin on human
In conclusion, we described the discovery of a novel
class of CCR3 antagonists: the pyrazolone methylamino
piperidine derivatives. Lead optimization allowed the
identification of potent analogues characterized by im-
proved water solubility and metabolic stability.
Nevertheless, it is predicted to have good intestinal per-
meability according to the Caco-2 experimental model.
Table 2. Physicochemical properties and in vitro metabolic stability of selected compounds
R4
F
O
O
N
N
R3
N
N
Compound R4
R3
Binding IC50 Water
solubilityb microsomesc consumption on consumption on
Clint on rat
% parent drug
% parent drug
Pappsd
a
rat hepatocytes
after 2 h
rat hepatocytes
after 19 h
5
19
20
21
22
23
24
25
26
27
4-Cl-Ph
4-F-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
2,6-F2-Ph
20
200
0.001
0.1
0.1
472
303
235
113
82
52
nt
95
nt
8.1
nt
4-NO2-Ph
3-Pyridyl
2-Pyridyl
4-NO2-Ph
4-NO2-Ph
6.3
2,500
1,000
35
nt
95
nt
nt
>2
1.5
nt
35
38
29
43
23
15
78
94
82
86
71
55
1.4
nt
2,6-(Me)2-pyrid-3-yl 12
12
1.2
79
62
2-Pyridyl
1.26
0.4
7.8
<1
<1
1.3
4-SO2NH2-Ph 2,6-F2-Ph
4-NH2Ph
4-Cl-Ph
>10,000
2,500
200
<50
<50
<50
2,6-F2-Ph
2-Pyridyl-N-oxide
0.9
>2
a Competition binding experiments with [125I]eotaxin on human eosinophils in nM.
b In mg/ml at pH 7.4.
c In ll/min/mg protein.
d Apical to basolateral flux through a Caco-2 monolayer in 10À6 cm/s. nt, not tested.

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C. Pegurier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4228–4231
4231
RPMI 1640 containing 25 mM Hepes (pH 7.4), 0.1% BSA,
0.1 nM
125I]eotaxin (Amersham Biosciences, Belgium;
References and notes
[
2000 Ci/mmol), and a test compound at the desired
concentration. Final DMSO concentration in each sample
is 2%. After incubation, bound and free radioligand are
separated by rapid vacuum filtration through glass fiber
filters (type GF/B or GF/C) presoaked for 2 h in PEI 0.3%
in order to reduce nonspecific binding. Samples and filters
are washed with 4 · 2 ml of an ice-cold 50 mM Tris HCl
(pH 7.4) buffer containing 500 mM NaCl. Radioactivity
trapped onto the filters is measured by liquid scintillation.
Nonspecific binding is determined in the presence of 1 lM
of compound A.
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eosinophils were incubated for 60 min at 25 ꢁC in 200 ll of