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available as starting materials, thiophenes 12 were well
suited to explore the role of alkyl substituents on this
part of the molecules (Table 3). Whereas the unsubsti-
tuted core structure 12a was as active (Ki: 28 nM) as
the comparable phenyl derivative 8b, small or larger
substituents R1 led to compounds weaker by a factor
of 10 or more. Compounds 12d and 12e, both with sub-
stituents R1 and R2, had similar acitivity as the cyclo-
propyl derivative 12b. In conclusion, substitution on
the aryl ring seems to be detrimental for CCR3 receptor
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Further variations of aryl ring substituents led to com-
pounds 13–15 (Table 4). Whereas the furane 13 retained
activity (compared to 12d), the pyridine 14 and the imid-
azole 15 were considerably less active than the compara-
ble substances 8b and 12a. Compounds with electron
rich aryl rings seem to be more active than those with
basic or electron poor ones. In summary, the SAR of
the discussed structures is very narrow. Although the
space to introduce further substituents is very limited,
it is possible that polar substituents are beneficial for
activity.
Compound 8b stimulates freshly isolated human periph-
eral blood eosinophils via the CCR3 receptor as mea-
sured by the transient increase the of intracellular
calcium concentration19 (Fig. 1b). The same increase is
seen with the natural agonist eotaxin-1 (Fig. 1a).
14. Spa¨th, E.; Platzer, N. Ber. 1936, 69, 255.
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´
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Furthermore, the CCR3 selective antagonist8 RO320-
2947/001 had no effect on intracellular calcium concen-
tration. However, it completely inhibited eosinophil
stimulation of compound 8b (Fig. 1b) as well as that
of eotaxin-1 (Fig. 1a).
4,240,820, 1980.
18. The CCR3 receptor binding test is based on a K562 cell
line (leukemia myelogenic blast cells) transfected with the
human chemokine receptor CCR3 (hCCR3-C1). The cell
membranes were prepared by disrupting the hCCR3
transfected K562 cells by nitrogen decomposition and
centrifugation at 40,000g, 4 ꢁC for 1 h. The membranes
were resuspended in the SPA incubation buffer (see below)
without bovine serum albumin for storage in aliquots at
ꢀ80 ꢁC.
Other compounds of this series (the imines 8a,c,f and
12a) showed essentially the same stimulation pattern
depending on sufficient affinity to the CCR3 receptor
(data not shown).
The CCR3 receptor binding assay with the radioligand
125Jodine-eotaxin-1 was performed in
a Scintillation
These findings clearly prove the agonist property of 8b
and strongly support that compound 8b interacts via
CCR3, that is, the same receptor as that used by eotax-
in-1.
Proximity Assay (SPA) design. Cell membranes of hCCR3
C1 cells were diluted in suitable concentrations (0.5–5 lg
protein/well) in 96-well microtiter plates (1450–401, Wal-
lac).
The test incubation mixture comprising 60 lL of the
membrane suspension, 80 lL of the Wheat Germ Agglu-
tinin coated PVT beads (organic scintillator, Amersham
Pharmacia biotech) in a concentration of 0.4 mg and
40 lL of radiolabelled 125J rhEotaxin (Amersham, IM290)
were incubated with 20 lL of the test compound (in
DMSO dilutions) for 2 h. The SPA incubation buffer
contained 25 mM HEPES, 25 mM MgCl2 6 · H2O, 1 mM
CaCl2 2 · H2O and 0.1% bovine serum albumin. Included
were controls for specific binding (no displacer added) and
nonspecific binding by adding unlabelled rhEotaxin (R&D
Systems) or a test compound. Bound radioactivity was
determined by scintillation counter (ÔMicro Beta TriluxÕ,
Wallac).
Acknowledgements
We thank Drs. H. Dollinger, C. Heine and P. Pouzet for
helpful discussions, and Dr. D. Martyres for proof
reading.
References and notes
1. (a) Combadiere, C.; Ahuja, S. K.; Murphy, P. M. J. Biol.
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Ringler, D. J.; Clark-Lewis, I.; Wang, J.; Kassam, N.;
Smith, H.; Shi, X.; Gonzalo, J.-A.; Newman, W.; Gut-
ierrez-Ramos, J.-C.; Mackay, C. R. J. Clin. Invest. 1996,
97, 604.
Determination of affinity of test compounds (dissociation
constant Ki) was calculated by iterative fitting of exper-
imental data using the law of mass action based program
Ôeasy sysÕ Schittkowski Num. Math. 1994, 68, 129–142.