1-(4-Phenylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanones as novel CCR1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.01.005

A novel series of CCR1 antagonists based on the 1-(4-phenylpiperazin-1-yl)- 2-(1H-pyrazol-1-yl)ethanone scaffold was identified by screening a compound library utilizing CCR1-expressing human THP-1 cells. SAR studies led to the discovery of the highly pot

Full text of DOI:10.1016/j.bmcl.2013.01.005

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1228–1231
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
1-(4-Phenylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanones as novel
CCR1 antagonists
Andrew M. K. Pennell, James B. Aggen, Subhabrata Sen, Wei Chen, Yuan Xu, Edward Sullivan, Lianfa Li,
⇑
Kevin Greenman, Trevor Charvat, Derek Hansen, Daniel J. Dairaghi, J. J. Kim Wright, Penglie Zhang
ChemoCentryx, Inc., 850 Maude Avenue, Mountain View, CA 94043, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of CCR1 antagonists based on the 1-(4-phenylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanone
scaffold was identified by screening a compound library utilizing CCR1-expressing human THP-1 cells.
SAR studies led to the discovery of the highly potent and selective CCR1 antagonist 14 (CCR1 binding
IC50 = 4 nM using [125I]-CCL3 as the chemokine ligand). Compound 14 displayed promising pharmacoki-
netic and toxicological profiles in preclinical species.
Received 5 December 2012
Revised 31 December 2012
Accepted 2 January 2013
Available online 11 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
CCR1
Chemokines
Chemokine receptor antagonists
Pyrazoles
Rheumatoid arthritis
Multiple myeloma
Chemokines are a group of small proteins that are best known
for their ability to mediate leukocyte trafficking from blood into
tissues undergoing inflammation or other types of injury.1 Based
on the number and position of the invariant cysteines in their pro-
tein sequence, the more than 40 chemokines identified to date are
divided into four subfamilies of CC, CXC, CX3C, and C chemokines.
The first CC chemokine receptor cloned was designated as CCR1.2
The known CC chemokines that bind to CCR1 include CCL3 (MIP-
We began our effort with the high-throughput screening of a
small-molecule library using an optimized [125I]-CCL3/MIP-1
a
binding assay and utilizing THP-1 cells that endogenously express
CCR1. A novel series of CCR1 antagonists based on the 1-(4-phen-
ylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanone scaffold was iden-
tified. Preliminary characterization of representative compound 1
(Fig. 1, binding IC50 = 307 nM) confirmed its functional activity in
blocking CCR1-mediated THP-1 cell chemotaxis (IC50 = 93 nM)
and calcium mobilization (FLIPR, IC50 = 310 nM).11 Compound 1
displayed a promising PK profile in Sprague–Dawley rats with
the clearance and half-life values of 23 mL/min/kg and 1.9 h,
respectively, when dosed at 0.5 mg/kg intravenously. Compound
1 was also orally bioavailable (F = 10% at 5 mg/kg) in rats.
The SAR exploration began with modification of substituents on
the phenyl ring (Table 1). The para-fluoro moiety was replaced
with other functional groups in compounds 2–9. Among these
compounds, only the bromo- and chloro-containing analogues
demonstrated increased potency with compound 3 (R4 = Cl) being
the most potent. In the absence of a para-substituent, an electron
1a), CCL5 (RANTES), CCL15 (leukotactin 1) and CCL23 (CKb8).
CCR1 is expressed on a range of cell types, including monocytes,
immature dendritic cells, T-lymphocytes, basophils, eosinophils,
neutrophils, NK cells, mast cells, osteoclast precursors, and mature
osteoclasts. CCR1 and its chemokine ligands are implicated in the
pathology of autoimmune diseases such as rheumatoid arthritis.
Other CCR1-mediated indications such as multiple myeloma-asso-
ciated osteolytic bone disease may also benefit from disrupting the
interaction between CCR1 and its chemokine ligands.3,4 Many
small molecule CCR1 antagonists have been reported and a num-
ber of these have been evaluated in human trials.5–7 While the clin-
ical experience of earlier compounds has been mixed, positive
results from the recently completed CARAT-2 Phase 2 trial in rheu-
matoid arthritis using the small molecule CCR1 antagonist CCX354
provides the first clinical validation of the therapeutic potential of
CCR1 antagonists.8–10
O
CF3
F
N
N
N
N
Cl
H3C
1
⇑
Corresponding author. Tel.: +1 650 210 2955; fax: +1 650 625 8940.
E-mail address: pzhang@chemocentryx.com (P. Zhang).
Figure 1. Initial CCR1 antagonist screening hit.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.005

A. M. K. Pennell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1228–1231
1229
Table 1
Table 2
Optimization of the phenyl ring substitution
Optimization of the pyrazole ring substitution
MeO
Cl
R3
O
O
R3a
R4a
CF3
Cl
R4
N
N
N
N
N
N
N
N
R6
R5a
H3C
14, 27-51
1-26
12
Compd
R3a
R4a
R5a
IC50 (nM)
R3
R4
R6
IC50 (nM)
12
Compd
14
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
Me
i-Pr
2-OH-i-Pr
Br
CN
SO2Me
NH2
CH2OH
CH2NH2
CO2H
Cl
H
F
Br
I
Me
Me
Me
Me
Me
Me
Me
H
CF3
i-Pr
Ph
CH2OH
CH2NH2
CH2NMe2
CH2NHAc
Me
Me
Me
Me
Me
Me
Me
4
19
5
8
7
6
500
18
740
1100
3400
10
50
47
68
120
40
8
16
20
10
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
F
307
5600
98
H
Cl
Br
Me
OMe
CF3
NO2
SO2Me
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
182
3650
32,000
9800
5650
6400
3300
2700
11,000
100
4
12
77
107
5
10
CN
NHAc
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Me
OMe
Cl
Me
OMe
OEt
O-i-Pr
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Cl
Br
CO2H
CONH2
CONHMe
CONMe2
CH2OH
14
420
52
210
220
12
10
340
97
120
5200
Me
Me
Me
Me
Cl
Cl
Cl
Cl
CH2NH2
16
Table 3
Modifications of the piperazine ring
donating group including methoxy and methyl at the meta-posi-
tion was preferred (10 and 11 vs 2 and 12). The combination of
R4 = Cl and R3 = OMe resulted in a significant potency boost (14
vs 3 and 11). The bulkier alkoxy groups in compounds 15 and 16
led to slightly decreased potency. Compounds 17–26 were pre-
pared to probe the R6 SAR. While a range of functional groups were
tolerated, only the fluoro analogue (18) was comparable to com-
pound 14 in CCR1 potency.
MeO
Cl
O
CF3
Cl
N
N
N
N
R2b
R3b
H3C
14, 52-56
12
Compd
R2b
R3b
IC50 (nM)
With 4-Cl, 3-OMe chosen as the substituents on the phenyl
group, we turned to modifying the pyrazole portion of the mole-
cule (Table 2). The decreased potency of compounds 27 and 32
compared to compounds 14 and 28–31 demonstrated that small
electron withdrawing groups were preferred over H and NHAc as
R4a. For R5a, a methyl was favored over a hydrogen atom, a bulkier
greasy group, or a trifluoromethyl moiety (14 vs 33–36). Attaching
a polar group to the R5a-methyl was allowed, but with moderately
decreased potency (37–40 vs 14). A variety of functional groups
were explored as R3a to replace the trifluoromethyl in compound
14. While most of them (except carboxylate) were allowed, the
resulting compounds (41–50) displayed decreased anti-CCR1
activity.
Further modification of compound 14 was investigated at the
piperazine template (Table 3). Methyl substitution at R2b or R3b
was tolerated, and in both cases the (S)-isomers were more potent
than the corresponding (R)-isomers. Compound 53, while more po-
tent, displayed supra-hepatic clearance in rats when dosed intrave-
nously at 1 mg/kg. In an attempt to address this problem by
introducing a polar moiety and increasing the aqueous solubility,
compound 54 incorporating a hydroxymethyl group was made.
However, the compound was found to be unstable under physio-
14
52
53
54
55
56
H
H
H
H
H
4
37
2
11
92
23
(R)-Me
(S)-Me
(S)-CH2OH
H
(R)-Me
(S)-Me
H
logically relevant conditions (37 °C and pH 7.4), likely due to an
intra-molecular acyl transfer as suggested by the LCMS analysis.
Compound 14 was evaluated in various assay formats on
different cell types for its potency on CCR1 (Table 4). In addition
to [125I]-CCL3/MIP-1 , it potently inhibited the binding of [125I]-
a
CCL15/Leukotactin to THP-1 cells. This high potency was also ob-
served in the calcium mobilization assay using either CCL3 or
CCL15 as the chemokine ligands. Additionally, compound 14
blocked the chemotaxis of THP-1 cells to all the known human
CCR1 ligands, including CCL3, CCL5, CCL15 and CCL23. Like many
CCR1 antagonists reported in the literature13, compound 14 dis-
played significantly lower activity against murine CCR1 in binding
as well as chemotaxis assays.

1230
A. M. K. Pennell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1228–1231
Table 4
Potency of compound 14 on CCR1 in various assay formats and on a variety of cell types
Cells
Assay
Chemokine ligand
IC50 (nM)
THP-1 (human)
Binding
[
[
125I]-CCL3/MIP-1
a
4
12
125I]-CCL15/Leukotactin 1
Calcium flux
Chemotaxis
CCL3/MIP-1
CCL15/Leukotactin 1
CCL3/MIP-1
CCL15/Leukotactin 1
CCL5/RANTES
a
5.5
4.8
3.9
1.5
0.8
2.5
a
CCL23/CKb8.1
Monocytes (human)
WEHI-274.1 (mouse)
Binding
[
[
125I]-CCL3/MIP-1
a
6
13
15
125I]-CCL15/Leukotactin 1
Chemotaxis
CCL15/Leukotactin 1
Mouse [125I]-CCL3/MIP-1
Mouse CCL3/MIP-1a
Binding
Chemotaxis
a
3000
900
Compound 14 was tested against a wide panel of chemoattrac-
tant receptors, including CCR2 through CCR12, CXCR1 through
CXCR7, CX3CR1, Duffy, CMV US28, and HHV8 ORF74. No apprecia-
ble activity was observed in the radioligand binding, calcium flux,
Cl
MeO
N
NBoc
b
a
Cl
MeO
Br
58
57
or chemotaxis assays at a concentration of 10 lM. Additionally, no
significant activity was noted when the compound was screened
against a MDS Pharma panel of 146 targets at relevant concentra-
O
tions (up to 10
pound 14 showed IC50 values of >10
and 3A4, while it inhibited CYP2C19 moderately, with an
IC50 = 6.0 M. The compound displayed low affinity to the hERG
ion channel with an IC50 of 16 M in the patch clamp assay. Com-
lM). In competitive CYP inhibition assays com-
.
Cl
MeO
N
NH
c
2 HCl
Cl
MeO
N
N
lM for CYP1A2, 2C9, 2D6,
Cl
60
59
l
l
pound 14 was also shown to be negative in the Ames mutagenicity
test ( S9).
O
CF3
Cl
Cl
MeO
N
N
d
N
Dosed at 1 mg/kg intravenously, compound 14 displayed a plas-
ma clearance of 20 mL/min/kg in Sprague–Dawley rats and 9.6 mL/
min/kg in beagle dogs. The half-life was 2.2 h and 13.4 h in rats and
dogs, respectively. The bioavailability was 52% in rats and 20% in
dogs at a 5.0 mg/kg oral dose. No acute toxicity was observed at
a single dose of up to 1000 mg/kg in rats. In a rat 7-day toxicolog-
ical evaluation, compound 14 was dosed orally at 70, 210 and
630 mg/kg daily. There were no dose-related findings in body
weight, necropsy, blood chemistry, or complete blood cell counts.
Synthesis of this series of CCR1 antagonists as exemplified by
compound 14 is illustrated in Scheme 1. Palladium-catalyzed cou-
pling between 5-bromo-2-chloroanisole (57) and Boc-protected
piperazine gave compound 58, which was treated with concen-
trated aqueous hydrochloric acid to yield compound 59. Treatment
of 59 with chloroacetyl chloride gave the intermediate 60 which
was treated with 3-trifluoromethyl-4-chloro-5-methylpyrazole in
the presence of potassium carbonate as the base to provide com-
pound 14.
N
H3C
14
Scheme 1. Reagents and conditions: (a) Boc-piperazine (1 equiv), Pd2(dba)3
(0.005 equiv), rac-BINAP (0.03 equiv), NaO-t-Bu (1.4 equiv), toluene, 60 °C, 95%;
(b) concd HCl (10 equiv), MeOH, rt, quantitative; (c) ClCH2COCl (1.1 equiv), K2CO3
(2 equiv), CH2Cl2, H2O, 5–25 °C, 92%; (d) 3-CF3-4-Cl-5-Me-pyrazole (1 equiv), K2CO3
(2 equiv), DMF, 60 °C, 90%.
potential therapeutic agents will be reported in subsequent
publications.
Acknowledgment
The authors thank Dr. Juan C. Jaen and Dr. Jay P. Powers for
encouraging us to publish the results and proof-reading the
manuscript.
In summary, a novel series of CCR1 antagonists based on the 1-
(4-phenylpiperazin-1-yl)-2-(1H-pyrazol-1-yl)ethanone
scaffold
References and notes
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using hCCR1-expressing THP-1 cells or freshly isolated human
monocytes. Compound 14 was shown to be highly selective for
CCR1 over other biological targets including chemoattractant
receptors. Minimal interactions with CYP isozymes and the hERG
ion channel were observed for compound 14, and it was negative
in the Ames mutagenicity assay. In rats and dogs, compound 14
displayed good oral bioavailability and other promising pharmaco-
kinetic features. In vivo evaluation in rats indicated a clean toxico-
logical profile. Further studies on this series of CCR1 antagonists as
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12. All IC50 values in Tables 1–3 were from the binding assays employing THP-1
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a was used in the binding assay. CCL3/MIP-1a
was used as the chemokine ligand in the chemotaxis and calcium mobilization
assays. All IC50 values were the average of triplicates.