Optimization of CCR4 antagonists: Side-chain exploration

Article abstract of DOI:10.1016/j.bmcl.2005.09.022

The design, synthesis, and activity of novel and selective small molecule antagonists of the CC chemokine receptor-4 (CCR4) are presented. Compound 8c was efficacious in a murine allergic inflammation model (ED₅₀ 30 mg/kg).

Full text of DOI:10.1016/j.bmcl.2005.09.022

Bioorganic & Medicinal Chemistry Letters 16 (2006) 204–207
Optimization of CCR4 antagonists: Side-chain exploration
Ashok V. Purandare,* Honghe Wan, Aiming Gao, John Somerville, Christine Burke,
Wayne Vaccaro, XiaoXia Yang, Kim W. McIntyre and Michael A. Poss
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
Received 22 July 2005; revised 5 September 2005; accepted 7 September 2005
Available online 19 October 2005
Abstract—The design, synthesis, and activity of novel and selective small molecule antagonists of the CC chemokine receptor-4
(CCR4) are presented. Compound 8c was efficacious in a murine allergic inflammation model (ED50 30 mg/kg).
ꢀ 2005 Elsevier Ltd. All rights reserved.
Chemokines and their receptors act as a communica-
tion and signaling network between cells, organs,
and regions of the body to facilitate immunological
responses.1 Chemokine receptor-4 (CCR4) together
with its ligands from the CC chemokine family, mac-
rophage derived chemokine (MDC; CCL22) and thy-
mus and activation regulated chemokine (TARC;
CCL17), are responsible for recruitment, homing,
and education of activated leukocytes (mainly CD4+
Th2 lymphocytes).2 Recently, CCR4 and its ligands
have attracted significant attention due to their
involvement in mediating various allergic inflammato-
ry conditions such as asthma, acute dermatitis, etc.3
Supported by studies using monoclonal antibodies
for the CCR4 receptor and its ligand TARC in
OVA-induced murine asthma models,4 we5 and oth-
ers6 have targeted antagonism of the CCR4 receptor
as a mechanism of inhibiting recruitment of activated
leukocytes to the lungs, thus supporting CCR4 antag-
onism as a potential treatment for diseases such as
asthma and atopic dermatitis.
Cl
N
N
O
N
H
N
Cl
1
Figure 1. CCR4 IC50 1.5 lM.
We first embarked on a systematic study of the diamine
side chain of compound (1). A small array of com-
pounds (4a–g) containing different diamine linkers and
terminal R1 groups was synthesized as shown in Scheme
1. Intermediates (I–IV) where R1 = O-t-butyl were syn-
thesized by coupling of pyrimidine derivative 35 with
the requisite mono-Boc-containing diamines. N-Boc
deprotection of intermediates (I–IV) and subsequent
amide bond formation with acids bearing basic R1
7
groups provided compounds (4a–g). Amongst the dif-
ferent linkers and terminal R1 groups, piperazine and
1-methylnipecotate; respectively; were preferred, (com-
pounds 4c and 4d, Table 1) over other nitrogen contain-
ing aromatic heterocycles or linkers (compounds 4e–g).
Recently, we reported5 the identification of a pyrimidine
analog (1 in Fig. 1) as a non-cytotoxic and moderately
potent antagonist of CCR4 (MDC binding). We herein
report further SAR exploration of the side chain that
led to the identification of a potent CCR4 antagonist
with in vivo activity.
Next we examined the effect of the linking atom (N vs O)
and regiochemistry of attachment to the central core.
The compounds were prepared from the previously
reported intermediates (5a and 5b)6 as shown in Scheme
2.7 A 2-fold enhancement of activity by changing the
linking atom from oxygen to nitrogen atom was ob-
served (compound 7a; Table 2).8 This trend was the same
as that observed in a previously reported series.6 The reg-
ioisomer 7b was found to be 2-fold more active than 7a.
Keywords: Chemokine receptor CCR4; Antagonist; MDC; In vivo
active; Asthma.
*
Encouraged by these findings, we embarked upon
further optimization of the terminal amino moiety.
Corresponding author. Tel.: +1 609 252 4320; fax: +1 609 252
7446; e-mail: ashok.purandare@bms.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.022

A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 16 (2006) 204–207
205
H
N
Cl
N
N
Cl
a, b, c, d
Cl
X
N
X
O
a, b, c
Y
N
Cl
Y
N
Cl
3
Cl
A
A
R
Cl
Cl
HN
5a; X = N, Y = CH
5b, X = CH, Y = N
6a-b
N
N
Cl
O
N
4
N
N
O
d
Cl
NHCOR1
(I)
R =
NHCOR1
Y
N
Cl
(II)
X
A
NHCOR1
NCOR1
Cl
7a-b
N
Scheme 2. Reagents and conditions: (a) N-Boc-piperazine, N-methyl-
pyrrolidine, 90 ꢁC, 82–85%; (b) TFA–CH2Cl2 (1:3), 98%; (c) SCX
purification, 92%; (d) 1-methylnipecotoic acid,1,3-di-isopropylcarbo-
diimide,1-hydroxy-7-azabenzotriazole, DMF–CH2Cl2 (1:1), 89%.
(III)
(IV)
Scheme 1. Reagents and conditions: (a) I–IV where R1 = –OtBu, N-
methyl-pyrrolidone, 60 ꢁC, 70–82%; (b) TFA-CH2Cl2 (1:3), 90–95%;
(c) SCX purification, 93–98%; (d) R1CO2H, 1,3-di-isopropylcarbodi-
imide, 1-hydroxy-7-azabenzotriazole, DMF–CH2Cl2 (1:1), 60–70%.
Table 2. SAR for ring atoms
Compound
A
X
Y
CCR4 IC50 (lM)8
4d
7a
7b
O
N
CH
N
1.2
0.7
0.3
Table 1. SAR for linker
NH
NH
CH
N
Compound Linker group R1
CCR4 IC50
(lM)8
CH
N
analogs. Within the subset made from a-amino acids,
the R-enantiomer was about 15-fold more active than
the corresponding S-enantiomer. The same trend was ob-
served in general across different cyclic amino acids but
there was no significant difference in the activity for five-
versus six-membered rings (i.e., proline 8d vs homopro-
line 8c).
4a
I
>10
O
4b
4c
4d
II
Same
Same
Same
>10
5.6
1.2
III
IV
N
Compound 8c was not only active in the in vitro
CCR4 binding assay, but it also blocked chemotaxis
(IC50 0.38 lM) and Ca2+ mobilization (IC50
0.01 lM).8,9 It was >1000-fold selective against
related chemokine receptors (CCR3, CCR2, and
CXCR3) and GPCRs (5-HT1A, 5-HT6, and 5-
HT7).8
4e
4f
IV
IV
>10
>10
O
N
O
Compound 8c was administered sub-cutaneously twice a
day at 10, 30, and 100 mg/kg dose to mice previously
challenged with ovalbumin (Fig. 2).10 Dexamethasone
administered orally, 5 mg/kg was used as control to as-
sess the relative efficacy. Compound 8c reduced the
recruitment of eosinophils with an ED50 of 30 mg/kg.
Additionally at 100 mg/kg dose, 8c was as efficacious
as dexamethasone 5 mg/kg in reducing eosinophilic infil-
tration into mouse BAL. These results clearly demon-
strate the proof of principle of using a small molecule
CCR4 antagonist as a potential treatment for asthmatic
disease.
4g
IV
>10
O
N
N
The analogs were prepared from the intermediate 6b and
Boc-protected cyclic amino acids followed by the Boc
group deprotection as shown (Scheme 3).7
As shown in Table 3, a-amino acids at the N terminus
were found to be preferred over either b or c-amino acid

206
A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 16 (2006) 204–207
H
N
O
R
N
N
N
a, b
N
N
Cl
N
N
Cl
N
H
N
Cl
H
Cl
6b
8
Scheme 3. Reagents: (a) R1CO2H, 1,3-di-isopropylcarbodiimide,1-hydroxy-7-azabenzotriazole, DMF–CH2Cl2 (1:1); (b) TFA–CH2Cl2 (1:3),
65–75%.
Table 3. SAR for terminal amine
Cl
N
Compound
R
CCR4 IC50 (lM)8
N
H
N
N
O
N
Cl
N
H
8a
>10
O
NH
8c
O
CCR4 IC50 0.05 µM
H
Cytotoxicity IC50 > 100 µM
Chemotaxis IC50 0.38µM
Ca2+ mobilization 0.01µM
8b
8c
8d
8e
8f
1
0.05
0.06
>10
0.8
HN
O
H
HN
H
In summary, we have identified a potent and selective
CCR4 antagonist (compound 8c) that showed in vivo
efficacy in a murine allergic lung inflammation model.
Further studies pertaining to optimization of this com-
pound will be reported in due course.
O
HN
O
O
NH
References and notes
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NH
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Figure 2. Dose-dependent inhibition by compound 8c of eosinophil
infiltration into allergic lung airways. Total leukocytes (solid bars),
total eosinophils (hatched bars), and percent eosinophils (open bars) in
bronchoalveolar lavage (BAL) fluid were determined as described.10
Data represent means SEM of seven mice treatment group. *p < .05
versus vehicle group, StudentÕs t test.

A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 16 (2006) 204–207
207
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7. All compounds were characterized by LC–MS and NMR
analysis. In addition, the yields were based on the weight
of pure product unless mentioned otherwise.
8. In vitro (MDC binding, chemotaxis, and cytoxicity) assays
were performed using conditions as described in Ref 5.
Substitution of TARC in the binding assay for MDC gave
IC50 values within 2-fold of the MDC based values.
9. Calcium mobilization—log-sphase CEMS529 cells were
washed in loading buffer that contained HanksÕ balanced
salt solution (GIBCO #14025-076) supplemented with
10 mM Hepes (pH 7.2) and 0.1% bovine serum albumin
(Sigma #A7284), and re-suspended in loading buffer at
2 · 106 cells/ml. Fluo-4 (Molecular Probes #F-14201) was
added to the cells at 4 lg/ml and the cells were incubated
at 37 ꢁC for 45 min. Following incubation, the cells were
centrifuged, the excess dye was removed, the cells were
washed once with loading buffer, and the cells are re-
suspended in loading buffer at 2 · 106 cells/ml. 1 · 105
cells were added to each well of a 96-well BIOCOAT plate
(#36-6640) and the cells adhered to the plate by centrifu-
gation. The cells were then pre-incubated with selected
compounds for 20 min at room temperature before the
calcium mobilization in response to an empirically deter-
mined amount of MDC ligand was measured by FLIPR.
10. OVA lung inflammation in mice: BALBnc female mice, 6–
8 weeks of age (Harlan, Indianapolis), were immunized
intraperitoneally with 100 lg of ovalbumin (OVA; Sigma)
in alum adjuvant (Pierce) and similarly boosted 10 days
later. Ten days after the booster, the mice were challenged
intranasally with 100 lg OVA in 50 ll pyrogen-free saline.
Seventy-two hours after challenge, mice were killed by
barbiturate overdose and lungs were lavaged via a
tracheostomy with 1 ml ice-cold HanksÕ balanced salt
solution (Ca2+- and Mg2+-free) containing 10% fetal
bovine serum. Total recovered leukocytes were enumerat-
ed by electronic cell counter (Scharfe System, Reutingen,
Germany). Cytocentrifuge smears (Shandon, Pittsburg)
were stained with Wright–Giemsa stain and 200 cells per
sample were classified by microscopic differential.