Optimization of 2-aminothiazole derivatives as CCR4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.01.126

A series of 2-aminothiazole-derived antagonists of the CCR4 receptor has been synthesized and their affinity for the receptor evaluated using a [¹²⁵I]TARC (CCL17) displacement assay. Optimization of these compounds for potency and pharmacokinetic properties led to the discovery of potent, orally bioavailable antagonists.

Full text of DOI:10.1016/j.bmcl.2006.01.126

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2800–2803
Optimization of 2-aminothiazole derivatives as CCR4 antagonists
Xuemei Wang,a Feng Xu,a Qingge Xu,a Hossen Mahmud,a Jonathan Houze,a
Liusheng Zhu,a Michelle Akerman,a George Tonn,a Liang Tang,a Brian E. McMaster,b
Daniel J. Dairaghi,b Thomas J. Schall,b Tassie L. Collinsa and Julio C. Medinaa,*
aAmgen Inc., 1120 Veterans Boulevard, South San Francisco, CA 94080, USA
bChemoCentryx Inc., 850 Maude Ave., Mountain View, CA 94043, USA
Received 25 December 2005; revised 28 January 2006; accepted 31 January 2006
Available online 23 February 2006
Abstract—A series of 2-aminothiazole-derived antagonists of the CCR4 receptor has been synthesized and their affinity for the
receptor evaluated using a [125I]TARC (CCL17) displacement assay. Optimization of these compounds for potency and pharmaco-
kinetic properties led to the discovery of potent, orally bioavailable antagonists.
ꢀ 2006 Elsevier Ltd. All rights reserved.
CCR4 is a G-protein-coupled receptor from the CC
family of chemokine receptors that is involved in the
migration of inflammatory cells into inflamed tissue.1
CCR4 is predominantly expressed on T lymphocytes,
mainly on those of the Type-2 (Th2) phenotype.
CCR4 is also expressed on monocytes, macrophages,
dendritic cells, and platelets.2 The ligands for CCR4
are Thymus and Activation Regulated Chemokine
(TARC, CCL17) and Macrophage-derived Chemokine
(MDC, CCL22).3 CCR4 and its ligands have been impli-
cated in a number of inflammatory diseases including
asthma,4 psoriasis,5 atopic dermatitis,6 and solid organ
transplant rejection.7 Therefore, it has been postulated
that CCR4 antagonists could play a beneficial role in
the treatment of these diseases.
tion in proof-of-concept experiments in vivo. The clear-
ance of 1 in rats after a 0.5 mg/kg iv dose is high (4.2 L/
h/kg) and the half-life is short (0.4 h). The bioavailabil-
ity after oral gavage dosing at 2 mg/kg is 2%. In this
publication, we describe the design of potent CCR4
antagonists with good pharmacokinetic properties in
rats.
The thiazole-derived compounds were synthesized
according to Scheme 1. Anilines were converted to thio-
ureas by treatment with benzoylisothiocyanate followed
by saponification of the benzoyl group. Subsequently,
the N-arylthioureas were treated with a-chloroketones
in refluxing methanol to obtain 2-amino-4-alkyl-substi-
tuted thiazoles (1–8 and 15–26). Compounds 11–14 were
obtained by reacting the N-arylthioureas with 1,3-dichlo-
roacetone in acetone at room temperature, followed by
alkylation of the chloromethylthiazole intermediate with
an appropriate secondary amine.
A series of 2-aminothiazole derivatives (lead structure
shown in Fig. 1) was discovered as antagonists of
CCR4. Compound 1 inhibited binding of radiolabeled
TARC and MDC to CCR4 receptors on the surface of
CEM cells8 and also inhibited the in vitro migration of
CEM cells mediated by TARC (IC50 = 6.4 lM).9
A series of compounds was synthesized that contained
pyrimidine, isoxazole, and pyrazole as potential replace-
In addition to potency optimization, it was also critical
to improve the pharmacokinetic properties of the lead
series in order to obtain compounds suitable for evalua-
HN
N
S
Keywords: CCR4; Chemokine; Chemokine receptor; TARC; CCL17;
MDC; CCL22; GPCR.
1
Figure 1. Potency of lead compound 1: CCR4/[125I]TARC:
IC50 = 1.7 lM, CCR4/[125I]MDC: IC50 = 3 lM.
*
Corresponding author. Tel.: +1 6502442425; fax: +1 6502442015;
e-mail: medinaj@amgen.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.126

X. Wang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2800–2803
2801
O
OH
R1
N
R1
N
N
Cl
R5
HN
R2
a, b
R5 Br
N
S
R1 NH2
NH2
R5
HN
R6
H
H
CO2Et
R7
O
b, c
c
N
N
S
R2
R2 = alkyl
1-8, 15-26
a
O
OEt
R1
=
N
O
N
H
N
O
29
Cl
S
Cl
d
R5
HN
N
BOC
N
R5
HN
R6
R5
Br
N
R7
O
N
N
CO2Et
a, (d)
b, c
R1
R1
N
HN
OEt
N
N
HN
R3R4NH
e
N
S
N
N
N
H
H
30
R3
R4
e
N
N
R5
R5
R6
N
Cl
R7
O
HN
11-14
b, f, c
HN
N
N
N
OEt
N
31
Scheme 3. Reagents and conditions: (a) Na2CO3, CH2Cl2, rt, 24 h,
35%; (b) DIBAL, PhCH3, À78 ꢁC, 30 min, 42%; (c) R5R6NH,
NaHB(OAc)3, 1,2-DCE, rt, 24 h, 22%; (d) TFA, 1 h, 100%; (e) CH3I,
Cs2CO3, acetone, 0 ꢁC to rt, 24 h, 41%; (f) (COCl)2, DMSO, Et3N,
CH2Cl2, rt, 24 h, 98%.
Scheme 1. Reagents and conditions: (a) benzoylisothiocyanate, ace-
tone, rt, 1 h, 98%; (b) 10% NaOH, 98 ꢁC, 10 min.; 90%; (c) MeOH,
reflux, 3 h, 80–100%; (d) acetone, rt, overnight, 70–80%; (e) NaHCO3,
NaI, acetone, reflux, 2 h, 60–75%.
The compound optimization effort was primarily guided
by the [125I]TARC ligand displacement assay.8 The effect
of replacing the linker region between the naphthalene
and thiazole rings was explored first (Table 1). It was
ments to the thiazole moiety (Schemes 2 and 3). Pyrimi-
dine 27 was obtained by addition of t-BuLi to
2-chloropyrimidine followed by DDQ oxidation and
then reaction with a-naphthylamine. The pyridine com-
pound 32 was prepared by free-radical bromination of
2-bromo-6-picoline, and then displacement of the ali-
phatic and ring bromine atoms with appropriate amines
under thermal and Pd-catalyzed conditions, respectively.
Table 1. Thiazole derivatives as CCR4 antagonists
X
The isoxazole and pyrazole analogs (Scheme 3) were
prepared from a common aryl isocyanide intermediate.
Cyclization with the oxime or the BOC-protected hydra-
zone of ethyl bromopyruvate generated the isoxazole
and pyrazole carboxy esters, respectively. In cases where
the BOC group was not cleaved under the conditions of
the cyclization, a quick TFA treatment provided the
unprotected pyrazole. After conversion of the esters to
the corresponding aldehydes, reductive amination pro-
vided compounds 29–31.
N
R
S
Compound
X
R
[
125I]TARC
a
IC50 (lM)
1
2
3
4
5
6
7
–NH–
–N(CH3)–
–NH–
CH3
CH3
1.7
>25
0.17
>25
0.08
0.22
5.5
–CH(CH3)2
–CH(CH3)2
–C(CH3)3
–CH2C(CH3)3
–Ph
–CH2–
–NH–
–NH–
–NH–
8
–NH–
0.9
9
10
11
–NH–
–NH–
–NH–
–CH2NH2
–CH2NHCH2CH3
–CH2N(CH2CH3)2
>25
1.0
Cl
Br
N
N
HN
N
a,b
c-e
0.8
N
N
N
12
–NH–
0.045
27
R5
HN
N
R7
N
N
R6
13
–NH–
0.018
32
R7 =
CF3
F3C
N
R5 =
R6 =
14
–NH–
0.014
Scheme 2. Reagents and conditions: (a) t-BuLi, then DDQ, Et2O,
À30 ꢁC to 0 ꢁC, 1 h, 100%; (b) a-naphthylamine, ethylene glycol,
reflux, 54%; (c) NBS, AIBN, C6H6, 90 ꢁC, 24 h, 71%; (d) R6R7NH,
Cs2CO3, DMF, rt, 24 h, 9%; (e) R5NH2, Pd2(dba)3, dppp, t-BuOK,
PhCH3, 70 ꢁC, 24 h, 73%.
a Values are means of three experiments, standard deviation is 30%.
Displacement of [125I]TARC from the CCR4 receptor expressed on
CEM.8

2802
X. Wang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2800–2803
discovered that a secondary amino group is preferred
over a tertiary amine or a methylene linker (compare 1
to 2 and 4).
moiety by unsubstituted phenyl (15) resulted in a signif-
icant loss of activity. While a series of phenyl analogs
with substitutions at positions 2 and/or 3 (16–20)
showed improved activity compared to the unsubstitut-
ed analog, none of these compounds were as potent as
the naphthyl group. A series of bicyclic-heteroaromatic
replacements, which included quinoline (21, 23), iso-
quinoline (22), and indole (24–26) derivatives, was also
explored for potential replacements of the naphthyl moi-
ety. Among these replacements, indoles demonstrated
improved potency. Thus, N-methyl indole 25 is more
potent than the naphthyl-derived analog (5).
Exploration of the 4-position of the thiazole ring demon-
strated that bulky alkyl groups are optimal for activity
(5–6). Furthermore, alkyl substitutions are preferred
over aryl substitutions (7–8). In addition, while hydro-
phobic substituents appear to be preferred at this posi-
tion, the hydrophobic alkyl groups can be successfully
replaced by bulky secondary and tertiary amines (11–14).
Concurrently with the optimization of the thiazole sub-
stituents, a series of naphthalene replacements were
evaluated (Table 2). Replacement of the naphthalene
A systematic survey of thiazole ring replacements was
also performed. Replacement of the thiazole ring by a
pyrimidine (27) or unsubstituted pyrazole (30) resulted
in the substantial loss of activity, while N-methyl pyra-
zole (31) and pyridine (32) both showed significant activ-
ity (0.37 and 0.34 lM, respectively, against hCCR4/
Table 2. CCR4 antagonists with varying N-substituents
R
HN
N
Table 3. CCR4 antagonists with varying modifications at the thiazole
moiety
S
Compound
R
[
125I]TARC IC50 (lM)
a
X
15
4.5
HN
N
R
Y
S
16
17
18
19
20
21
22
23
24
25
26
0.9
Compound
X
Y
[
125I]TARC
a
IC50 (lM)
2.0
N
N
27
>25
N
0.44
0.26
0.21
0.3
28
29
0.08
12.1
O
O
HN
N
N
N
N
N
N
N
CF3
CF3
CF3
CF3
N
N
30
31
32
>25
0.37
0.34
1.5
>25
N
H
N
N
N
0.49
Me
N
0.037
0.21
Et
N
a Values are means of three experiments, standard deviation is 30%.
Displacement of [125I]-labeled TARC from the CCR4 receptor
expressed on CEM.8
a Values are means of three experiments, standard deviation is 30%.
Displacement of [125I]TARC from the CCR4 receptor expressed on
CEM.8

X. Wang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2800–2803
2803
[
125I]TARC). However, the most successful example in
3. Imai, T.; Chantry, D.; Raport, C. J.; Wood, C. L.;
Nishimura, M.; Godiska, R.; Yoshie, O.; Gray, P. W.
J. Biol. Chem. 1998, 273, 1764.
4. Hirata, H.; Arima, M.; Cheng, G.; Honda, K.; Fukushima,
F.; Yoshida, N.; Eda, F.; Fukuda, T. J. Clin. Immunol.
2003, 23, 34.
this area is the oxazole replacement (28). Compared with
the corresponding thiazole compound (5), the oxazole
possesses comparable potency against CCR4 Table 3.
Placement of bulky hydrophobic tertiary amines at the 4-
position of the thiazole led to the discovery of potent com-
pounds with improved potency and pharmacokinetic
properties. Replacement of the methyl group on 1 by an
N,N-bis(cyclohexylmethyl)methyl amine moiety (13) led
to a significant improvement in potency. Compound 13
is approximately 100-fold more potent as an inhibitor of
TARC and MDC binding to CCR4 than 1 (13; CCR4/
5. Teraki, Y.; Miyake, A.; Takebayashi, R.; Shiohara, T. Clin.
Exp. Dermatol. 2004, 29, 658.
6. Kakinuma, T.; Nakamura, K.; Wakugawa, M.; Mitsui, H.;
Tada, Y.; Saeki, H.; Torii, H.; Asahina, A.; Onai, N.;
Matsushima, K.; Tamaki, K. J. Allergy Clin. Immunol.
2001, 107, 535.
7. Huser, N.; Tertilt, C.; Gerauer, K.; Maier, S.; Traeger, T.;
¨
Aßfalg, V.; Reiter, R.; Heidecke, C.; Pfeffer, K. Eur.
J. Immunol. 2005, 35, 128.
8. Assays for CCR4 were performed using the human T
lymphoblastic cell line CEM, obtained from the ATCC
(CCRF-CEM variant), which expresses high levels of
endogenous human CCR4. Compounds were diluted into
[
125I]TARC: IC50 = 0.018 lM; [125I]MDC IC50 = 0.031 lM).
This compound is also a functional antagonist of
CCR4, as demonstrated by its ability to inhibit TARC-
mediated cell migration (IC50 = 0.53 lM). Furthermore,
compound 13 has improved pharmacokinetic properties
in rats relative to compound 1. The clearance of 13 in rats
is approximately 4-fold lower than that of compound 1
(i.e., 1.07 L/h/kg vs 4.2 L/h/kg) after a 0.5 mg/kg iv dose.
The terminal half-life is also improved from 0.4 to 3.6 h.
The oral bioavailability of compound 13 is 63% following
a 2 mg/kg po dose. Thus, compound 13 can be used as a
reasonable tool to validate the CCR4 target in vivo.
DMSO and added to individual wells of
a 96-well
polypropylene assay plate. CEM cells were resuspended
to 5 · 106/mL in buffer (RPMI-1640 supplemented with
0.5% BSA); 90 lL was added to each well of the assay plate.
[
125I]TARC (Perkin-Elmer) was diluted to 100 pM in buffer;
100 lL was added to each well of the assay plate. The assay
plates were incubated at room temperature for 2 h. The
assay mixtures were filtered through GF/B filter plates
using a FilterMate Harvester (Perkin-Elmer). Retained
radioactivity was quantified by scintillation counting.
9. Migration assays for CCR4 were performed using 96-well
ChemoTX migration plates (NeuroProbe) with a 5 lm pore
size. Compounds were diluted into DMSO and added to
individual wells of two matched 96-well polypropylene
assay plates. TARC (R&D Systems) was diluted to 1 ng/mL
in buffer; 200 lL was added to each well of the second assay
plate to combine the TARC with compound and then
32 lL of this mixture was plated into the bottom chamber
of the migration plate. The filter membrane for the
migration plate was secured in place. CEM cells were
resuspended to 5 · 106/mL in buffer; 200 lL was added to
each well of one assay plate to combine the cells with
compound, then 50 lL of this mixture was plated onto the
filter membrane of the migration plate. The migration plate
was transferred to a 37 ꢁC incubator (5% CO2, 95%
humidity) and incubated for 2 h. At the end of this
incubation period, the cells were aspirated from the top
of the filter and the filter was removed. Alamar blue (5 lL)
was added to the bottom chamber, the plate was returned
to the incubator and incubated for another hour. At the end
of this incubation period, the plates were read on a
fluorescent plate reader (530 absorbance, 590 emission).
In summary, a novel series of 2-aminothiazole-derived
CCR4 antagonists was discovered and optimized for
potency and pharmacokinetic properties. It was found
that bulky hydrophobic substituents are preferred at
the 4-position of the thiazole ring. These observations
led to the discovery of compound 13, which displays
adequate potency and pharmacokinetic properties to
be used as a tool for further study of the CCR4
receptor.
References and notes
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Hoogewerf, A. J.; Proudfoot, A. E. I.; Wells, T. N. C. J.
Biol. Chem. 1997, 19495; (b) Imai, T.; Baba, M.; Nishi-
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