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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
1. Chvatchko, Y.; Hoogewerf, A. J.; Meyer, A.; Alouani, S.;
Juillard, P.; Buser, R.; Conquet, F.; Proudfoot, A. E. I.;
Wells, T. N. C.; Power, C. A. J. Exp. Med. 2000, 191, 1755.
2. (a) Power, C. A.; Meyer, A.; Nemeth, K.; Bacon, K. B.;
Hoogewerf, A. J.; Proudfoot, A. E. I.; Wells, T. N. C. J.
Biol. Chem. 1997, 19495; (b) Imai, T.; Baba, M.; Nishi-
mura, M.; Kakizaki, M.; Takagi, S.; Yoshie, O. J. Biol.
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