Piperazinyl CCR1 antagonists-optimization of human liver microsome stability

Article abstract of DOI:10.1016/j.bmcl.2007.03.037

The synthesis, biological activity, and pharmacokinetic profile of CCR1 antagonists are described.

Full text of DOI:10.1016/j.bmcl.2007.03.037

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3109–3112
Piperazinyl CCR1 antagonists—optimization of human
liver microsome stability
Matthew F. Brown,* Kevin B. Bahnck, Laura C. Blumberg, William H. Brissette,
Sara A. Burrell, James P. Driscoll, Flavia Fedeles, Michael B. Fisher, Robert S. Foti,
Ronald P. Gladue, Aikomari Guzman-Martinez, Matthew M. Hayward, Paul D. Lira,
Brett M. Lillie, Yi Lu, Greg D. Lundquist, Eric B. McElroy, Molly A. McGlynn,
Timothy J. Paradis, Christopher S. Poss, James H. Roache, Andrei Shavnya,
Richard M. Shepard, Kristen A. Trevena and Laurie A. Tylaska
Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
Received 28 February 2007; revised 9 March 2007; accepted 12 March 2007
Available online 15 March 2007
Abstract—The synthesis, biological activity, and pharmacokinetic profile of CCR1 antagonists are described.
ꢀ 2007 Elsevier Ltd. All rights reserved.
A number of published studies have demonstrated the
potential utility of CCR1 antagonists for the treatment
of human diseases including autoimmune disorders and
organ transplant rejection.1,2 We recently described
medicinal chemistry efforts that led to the identification
of the novel CCR1 antagonist CP-481,715 (1, see
human diseases. Herein, we describe efforts to identify
new CCR1 antagonists suitable for clinical evaluation.
A number of in-house and literature lead series were
evaluated for potential follow-up. The piperazine series
exemplified by BX471 (2), appeared to have the greatest
potential to produce compounds with the desired
attributes. BX471 (2) was reported to have excellent
intrinsic potency and was shown to be effective in a
variety of disease models while displaying little off-target
activity.8 Published pharmacokinetic studies conducted
with BX471 in dogs described good oral bioavailability
(ꢀ60%) and a moderate half-life of approximately
3 h.8 A human half-life of approximately 2.3 h has been
reported as well.9 We evaluated the in vitro metabolic
stability of BX471 (2) in liver microsome preparations
from a number of species, including rat, dog, monkey,
and human, to provide an understanding of metabolism
across species (see Table 1).10 Moderate to rapid metab-
olism was observed in all species, with the greatest
stability being observed in dog liver microsomes
(DLM). Metabolite identification studies conducted
with BX471 (2) following incubation in human liver
microsomes (HLM) suggested that N-debenzylation
contributed significantly to the moderate turnover
observed. The following describes our efforts to identify
potent, selective, piperazine-based CCR1 antagonists
with enhanced metabolic stability.
Scheme 1).3–5
A Phase I study conducted with
CP-481,715 (1) in rheumatoid arthritis (RA) patients
provided the first clinical evidence that blockade of
CCR1 signaling could be a viable treatment for RA
as a significant reduction of monocyte infiltration into
synovial tissue was observed following 2 weeks of dos-
ing (300 mg TID · 14 days).6 However, these positive
findings were tempered by a subsequent Phase II clini-
cal trial wherein CP-481,715 (1) failed to demonstrate
clinical efficacy in RA patients following 6 weeks of
treatment.1 In addition, efficacy was not observed in
a Phase II clinical trial wherein patients with relapsing
remitting multiple sclerosis (RRMS) were administered
the Berlex/Schering AG CCR1 antagonist BX471 (2)
(600 mg TID · 16 weeks).7 In spite of these clinical
setbacks, CCR1 antagonism remains to be an attractive
approach for the treatment of a number of other
Keywords: BX471; CP-481715; CCR1 antagonist.
*
Corresponding author. Tel.: +1 860 441 3522; fax: +1 860 686
1103; e-mail: matthew.f.brown@pfizer.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.037

3110
M. F. Brown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3109–3112
Me
Me
R'
O
O
O
F
Cl
F
N
N
a
N
N
Cl
R'
R
OH
R
3
Cl
5a: R, R' = H
5b: R = Me, R' = CO2Me
5c: R = Me, R' = CO2H
5d: R = Me, R' = CONH(CH2)2NH2
5e: R = Me, R' = NO2
5f: R = Me, R' = NHCONH(CH2)2NH2
5g: R = Me, R' = CH2CO2Et
5h: R = Me, R' = CH2CO2H
5i: R = Me, R' = CH2CONHSO2Me
5j: R = Me, R' = CO(CH2)2CO2Et
5k: R = Me, R' = CO(CH2)2CO2H
5l: R = Me, R' = CHO
4
F
d
e
b (4g)
or
c (4j)
O
O
N
N
f
N
NH2
H
OH
R"
d
g
OMe
CP-481,715 (1)
OH
Cl
d
h
O
6
Me
O
H2N NH
F
O
5m: R = Me, R' = CH2SO3H
N
N
Cl
BX471 (2)
Scheme 1. Reagents and conditions: (a) 2-butanone, K2CO3, KI, reflux; (b) R00 = CO2Et, (1) LAH, THF, 0 ꢁC ! reflux, (2) SOCl2, CH2Cl2,
(3) KCN, 18-C-6, CH3CN, rt, (4) KOH, H2O, reflux, (5) 48% HBr, reflux, (6) EtOH, HCl, rt; (c) R00 = H, (1) succinic anhydride, AlCl3, DCE, rt,
(2) EtOH, HCl, rt; (d) THF, methanol, H2O, LiOH hydrate, rt; (e) BOP, Hunig’s base, ethylenediamine, DMF; (f) (1) 5% PtO2 on carbon, 35 psi H2,
EtOH, rt, (2) 4-nitrophenylchloroformate, pyridine, CH2Cl2, rt, (3) ethylenediamine, MeOH, rt; (g) NH2SO2Me, EDCI, DMAP, NEt3, CH2Cl2; (h)
(1) NaBH4, MeOH, reflux, (2) SOCl2, CH2Cl2, (3) Na2SO3, EtOH, H2O, reflux.
Compounds were prepared in a straightforward fashion
as described in Scheme 1. Phenols (4) were either com-
mercially available or prepared as described, then cou-
pled with a-chloroacetamide 311 to provide analogs 5a,
5b, 5e, 5g, 5j, and 5l. Further elaboration provided ana-
logs 5c, 5d, 5f, 5h, 5i, 5k, and 5m.
Incorporation of 2-phenoxy groups containing basic
amines efficiently reduced ClogD values relative to
BX471 (2), which generally translated to improved
microsomal stability. For example, the amino analogs
5d and 5f, which were potent in binding, functional
and human whole blood assays16 were found to be rea-
sonably stable in liver microsomes across a number of
species. Unfortunately, the improved microsomal stabil-
ity did not translate in vivo as 5d and related analogs
were found to undergo rapid clearance in rats following
iv dosing. The reason for the rapid plasma clearance was
not discerned, however, it may have been the result of
non-CYP450 mediated metabolism, drug transporter-
mediated clearance, and/or partitioning into red blood
cells. Compound 5d was also evaluated for selectivity
against a panel of receptors. While BX471 (2) has been
reported to exhibit excellent selectivity for the CCR1
receptor,8 5d showed measurable activity against several
targets (adrenergic, dopaminergic, and calcium channel)
suggesting that the incorporation of an additional basic
moiety had led to a significant erosion of selectivity.15
Two general strategies were employed to improve meta-
bolic stability. One relied on incorporating structural
changes to block metabolism, the other focused on
reducing molecular lipophilicity, and the latter approach
is the focus of discussion for this letter. Our strategy was
as follows: (1) determine the minimum pharmacophore
for the series (lowest molecular weight analog which
maintains significant intrinsic potency), (2) determine
which position(s) of the molecule are most amenable
to incorporation of polar functionality to drive down
molecular ClogD,12 and (3) evaluate the impact of re-
duced ClogD on metabolic stability, both in vitro and
in vivo. Compound 5a was settled on as the minimum
pharmacophore as removal of either the 4-F, 4-Cl or
2-piperazinyl methyl groups led to a substantial loss of
potency in binding and/or functional chemotaxis as-
says.13,14 The 2-phenoxy vector was determined to be
an appropriate area to explore with new analog genera-
tion as BX471 (2) incorporates a polar urea in this
position. Indeed, a wide variety of neutral, basic and
acidic substituents could be incorporated at this position
without sacrificing intrinsic potency. While neutral polar
groups (e.g., amides, sulfonamides, ureas, carbamates,
etc.) maintained acceptable potency, a variety of
ADME issues plagued this set, including poor
microsome stability and/or intestinal efflux.15 Therefore,
analogs incorporating neutral 2-phenoxy substituents
were not extensively pursued.
Zwitterionic compounds derived from incorporation of
acidic groups at the 2-phenoxy position were explored
as well. Benzoic acid 5c displayed moderate potency in
the receptor binding and chemotaxis assays. However,
whole blood activity was poor (>10 lM), most likely
due to extensive binding to plasma proteins (<1% un-
bound). As was the case for the amines described above,
incorporation of an acidic moiety significantly lowered
the ClogD leading, in general, to improved microsomal
stability. However, unlike the amines, zwitterion 5c dis-
played much improved pharmacokinetic behavior
in vivo, prompting us to focus our efforts on identifying
zwitterions with acceptable whole blood activity.

M. F. Brown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3109–3112
Table 1. Potency data and pharmacokinetic study results
3111
Compound CCL3 binding CCL3
IC50 (lM)13
Human whole
blood IC50 (lM)16
aHLM RLM
ClogD at Species
%F
Clpb
Vdss T1/2
(L/kg) (h)
chemotaxis
IC50 (lM)14
DLM MLM pH 7.412
Cl1b0
1
0.048
0.066
0.22
0.062
NT
5.0
38
1.9
2.8
Dog 48
Monkey 10
13
17
1.2
1.1
1.5
0.9
11
20
16
62
28
42
14
63
27
40
2
0.031
0.086
0.059
0.030
0.008
0.038
0.044
0.013
0.023
0.004
0.086
0.068
0.003
0.001
0.002
0.002
<0.0005
0.003
Rat
Dog
2
NT
33
1.7
0.6
0.6
0.7
4.0
0.8
1.7
8.9
Monkey NT
5a
5c
5d
5f
5h
5i
3.7
NT
NT NT
NT
NT
> 10.00
0.007
0.011
>10.00
0.321
0.108
0.39
4.7
0.9
Rat
Dog
NT
NT
2.4
0.2
3.4
2.4
0.2
0.9
14
11
5.9
20
11
31
Monkey NT
5.9
1.0
Rat
NT
NT 184
7.7
1.0
22
25
36
4.9
1.4
NT NT
NT
NT
24
11
18
4.7
0.3
Rat
Dog
NT
NT
5.2
0.4
3.2
0.3
3.7
11
25
11
18
4.7
0.5
Rat
Dog
19
100
4.5 0.5
0.09 0.09
1.4
13
26
11
18
Monkey 24
1.2
0.2
1.2
5k
5m
4.7
0.9
Rat
Dog
9
100
42
9.7
4.0
4.2
2.3
5.8
31
11
18
4.7
ꢁ1.6
Rat
Dog
40
100
17
0.2
51
2.6
0.4
2.1
4.6
21
21
11
18
Monkey 19
1.1
NT, not tested.
a Human liver microsomes (HLM), rat liver microsomes (RLM), dog liver microsomes (DLM), monkey liver microsomes (MLM); units: mL/min/kg.
b Clp, plasma clearance (units: mL/min/kg).
Homologation of the benzoic acid 5c provided benzylic
acid 5h. While this analog displayed similar potency to
5c in the receptor binding assay, 5h was approximately
30-fold more potent in the functional chemotaxis assay.
Similarly, acylsulfonamide 5i, keto acid 5k, and sulfonic
acid 5m were all found to exhibit low nanomolar func-
tional activity, and importantly, all demonstrated
acceptable whole blood activity and HLM stability.
Having identified compounds with appropriate potency
and microsome stability, follow-on pharmacokinetic
studies were performed. As was the case with benzoic
acid 5c, benzylic acid 5h and acylsulfonamide 5i demon-
strated improved in vivo clearance as compared to
BX471 (2) in the species evaluated. However, keto acid
5k demonstrated similar rat clearance and somewhat
greater dog clearance as compared to BX471 (2). Sul-
fonic acid 5m demonstrated low plasma clearance fol-
lowing iv dosing in dogs and rats, and good exposure
following oral dosing. However, rapid clearance was ob-
served in monkeys for 5m. The greater than predicted
clearance observed for 5k in dogs and rats and, 5m in
monkeys may have been due to a number of factors
including phase II metabolism and/or drug transporter
mediated clearance. Additionally, while amine 5d
described above demonstrated poor broad selectivity,
sulfonic acid 5m displayed excellent selectivity for the
CCR1 receptor.15
In summary, medicinal chemistry efforts to identify
piperazine analogs with improved metabolic stability
were described. While analogs incorporating amino
groups at the 2-phenoxy position suffered a number of
issues, including poor in vivo pharmacokinetics and sub-
optimal broad selectivity, several zwitterionic analogs

3112
M. F. Brown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3109–3112
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