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J. N. Burrows et al. / Bioorg. Med. Chem. Lett. 15 (2005) 25–28
chemokine receptors is of the order of 100-fold or better.
In order to survey the selectivity profile of the series rep-
resentative compounds were tested in a panel of assays
for binding to a range of GPCRs and ion channels (data
not shown).11 Binding to muscarinic and serotoninergic
receptors was seen at 1–10lM. On the basis of these re-
sults we established radioligand binding assays to meas-
ure activity at human M1 and 5-HT2A receptors,
compound 2ap showed IC50 values of 0.4 and 0.8lM,
respectively. In addition, compound 2aw showed micro-
molar activity at the hERG ion channel. These results
indicated to us that further optimisation was required
to increase the selectivity with respect to these other
activities.
the biological assays and Tom McInally for helpful ad-
vice on multiple parallel techniques.
References and notes
1. For a review of CCR5 as a target for HIV-1 and small
molecule CCR5 ligands, see: Kazmierski, W.; Bifulco, N.;
Yang, H.; Boone, L.; DeAnda, F.; Watson, C.; Kenakin,
T. Bioorg. Med. Chem. 2003, 11, 2663–2676.
2. Pipitone, N.; Pitzalis, C. Curr. Opin. Anti-inflammat.
Immunomodulat. Investigat. Drugs 2000, 2, 9.
3. Sellebjerg, F.; Madsen, H. O.; Jensen, C. V.; Jensen, J.;
Garred, P. J. Neuroimmunol. 2000, 102, 98–106.
4. Fischereder, M.; Luckow, B.; Wuthrich, R. P.; Rothenpi-
¨
eler, U.; Schneeberger, H.; Panzer, U.; Stahl, R. A. K.;
Hauser, I. A.; Budde, K.; Neumayer, H.-H.; Kra¨mer, B.
K.; Land, W.; Schlo¨ndorff, D. Lancet 2001, 357, 1758–
1761.
We then studied the effects of a representative com-
pound, 2aw, in functional systems. Compound 2aw
was tested for its ability to inhibit MIP-1b-stimulated
calcium transients in CHO cells stably expressing rec-
ombinant human CCR5 and was found to have an
IC50 of 78nM. Also 2aw was shown to have an IC50
of 38nM in an assay measuring the inhibition of the
chemotaxis of human AlloT cells in response to MIP-
1b. In the same system 2aw had no effect on the response
to MIP-1c at concentrations up to 10lM indicating that
the inhibition of chemotaxis is mediated through CCR5.
The similar potencies seen in these assays, in which
MIP-1b is used, to those previously observed in the
RANTES binding assay is evidence that the compound
is acting as an antagonist at the receptor since it is able
to block different CCR5 ligands.
5. Andres, P. G.; Beck, P. L.; Mizoguchi, E.; Mizoguchi, A.;
Bhan, A. K.; Dawson, T.; Kuziel, W. A.; Maeda, N.;
MacDermott, R. P.; Podolsky, D. K.; Reinecker, H. C. J.
Immunol. 2000, 164, 6303–6312.
6. For reviews, see: (a) Gao, Z.; Metz, W. A. Chem. Rev.
2003, 103, 3733; (b) Onuffer, J. J.; Horuk, R. Trends
Pharmacol. Sci. 2002, 23, 459–467.
7. Palani, A.; Shapiro, S.; Clader, J. W.; Greenlee, W. J.;
Blythin, D.; Cox, K.; Wagner, N. E.; Strizki, J.; Baroudy,
B. M.; Dan, N. Bioorg. Med. Chem. Lett. 2003, 13, 705–
708.
8. Compound 1 is an inhibitor of microsomal triglyceride
transfer protein, see: Jamil, H.; Gordon, D. A.; Eustice, D.
C.; Brooks, C. M.; Dickson, J. K., Jr.; Chen, Y.; Ricci, B.;
Chu, C.-H.; Harrity, T. W., et al. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 11991–11995.
In summary SAR studies around two weak screening
hits have led to the identification of a series of potent
and selective CCR5 functional antagonists (exemplified
by 2aw), which provide good leads for further optimisa-
tion. Our further development of this series to give po-
tent, orally bioavailable CCR5 antagonists will be the
subject of future publications.
9. Burrows, J.; Cooper, A.; Cumming, J.; McInally, T.;
Tucker, H. WO 01/087839, 2001; Chem. Abstr. 2002, 135,
371644.
10. SPA binding assays were conducted in 96-well Optiplates
[
125I]RANTES
(Packard 6005190). Displacement of
(Amersham IM-288) by compounds was measured using
membranes prepared from a h-CCR5 transfected CHO
cell line. Compounds were incubated overnight with
0.1nM
[
125I]RANTES, SPA beads (Amersham
RPNQ0001) and membranes expressing the human
CCR5 receptor. The signal was read on a Packard
Topcount.
Acknowledgements
The authors thank our colleagues Susan Mellor and
Lorraine D. Newboult for developing and carrying out
11. Work carried out on behalf of AstraZeneca by MDS
Pharma Services, Taipei, Taiwan.