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Table 5
hCXCR3 and muscarinic receptor antagonism: enantiomeric effect
R2
N
X
Br
Y
N
R1
b
Compound
Chirality
X
Y
R1
R2
hCXCR3 binding
IC50 (lM)
Muscarinic receptor binding IC50
M2
(lM)
a
M1
9.6
M3
1
1a
1b
18
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
2,4-F
2,4-F
2,4-F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Acetyl
Acetyl
Acetyl
0.78
0.61
1.33
0.12
0.06
0.33
0.14
0.11
2.31
0.08
0.81
0.06
0.06
0.03
6.31
À
>10
>10
<0.001
+
<0.001
<0.001
18a
18b
41
41a
41b
47
47a
47b
48
48a
48b
À
>10
>10
>10
<0.001
+
<0.001
<0.001
À
10
>10
10
>10
3
>10
+
a-Acetamide
a-Acetamide
a-Acetamide
À
5
>10
2
>10
1.1
>10
+
Phenylureum
Phenylureum
Phenylureum
À
>10
8.9
>10
>10
>10
>10
+
a
GTP
c
S binding assay.14
Ref. 16 for assay.
b
4. Tensen, C. P.; Flier, J.; Van Der Raaij-Helmer, E. M.; Sampat-Sardjoepersad, S.;
Van Der Schors, R. C.; Leurs, R.; Scheper, R. J.; Boorsma, D. M.; Willemze, R. J.
J. Invest. Dermatol. 1999, 112, 716.
5. (a) Lazzeri, E.; Romagnani, P. Curr. Drug Targets 2005, 5, 109; (b) Hancock, W.;
Lu, B.; Gao, W.; Csizmadia, V.; Faia, K.; King, J. A.; Smiley, S. T.; Ling, M.; Gerard,
N. P.; Gerard, C. J. Exp. Med. 2000, 192, 1515; (c) Belperio, J. A.; Keane, M. P.;
Burdick, M. D.; Lynch, J. P., III; Zisman, D. A.; Xue, Y. Y.; Li, K.; Ardehali, A.; Ross,
D. J.; Strieter, R. M. J. Immunol. 2003, 171, 4844.
6. (a) Sørensen, T. L.; Trebst, C.; Kivisäll, P.; Klaege, K. L.; Majmudar, A.;
Ravid, R.; Lassmann, H. J. Neuroimmunol. 2002, 127, 59; (b) Sindern, E.;
Patzhold, T.; Ossege, L. M.; Gisevius, A.; Malin, J.-P. J. Neuroimmunol. 2002,
131, 186.
with the a-acetamide (47) and the phenylureum (48) substituents,
but at the cost of less favorable PK properties in the case of 48 (data
not shown).
Table 5 lists the CXCR3 and muscarinic receptor binding activity
of our most potent racemic compounds and their enantiomers. The
enantiomers were separated by chiral HPLC or SFC and the enanti-
omers were distinguished by their optical rotations (levo or dextro
rotatory), without further determination of their absolute
configuration.
The muscarinic activity of the opposite enantiomer of the hit
1a lead us to select the enantiomeric pairs of those compounds
for screening on a panel of muscarinic receptors in a radioligand
binding assay.16 The isomers of compounds 1 and 18, which
share the same benzetimide (3) scaffold, display comparable
hCXCR3 antagonistic activities, while their anti-muscarinic activ-
ity is almost totally confined to the (+)-isomer form. However,
N-substitution of the 3-piperidine ring with carbonyl-containing
functions (compounds 41, 47, and 48) indicated that their optical
isomers produced weak or no anti-muscarinic activity and that
strong CXCR3 antagonistic activity was largely confined to a sin-
gle enantiomer 41a, 47b, and 48a. Of note, although not tested
on a regular basis, our compounds showed comparable potencies
against mouse CXCR3 (data not shown), excluding issues of spe-
ciation that are often encountered in the search for chemokine
receptor antagonists.
Taken together, we have identified a novel series of 3-piperidine
compounds that produce submicromolar antagonistic activity
against human and murine CXCR3. The compounds were opti-
mized from the HTS hit 1, a structural analog of the anti-choliner-
gic benzetimide. From enantiomeric separation of the most potent
3-piperidine compounds it was learnt that CXCR3 antagonism and
anti-cholinergic effects were not linked. In vivo experiments are
underway to assess the potential of these compounds as anti-
inflammatory agents.
7. (a) Olsen, D. B.; Strieter, R. M.; Ransohoff, R. M.; Sellebjerg, F. J. Neuroimmunol.
2002, 127, 59; (b) Sasaki, S.; Yoneyama, H.; Suzuki, K.; Suriki, H.; Aiba, T.;
Watanabe, S.; Kawachi, H.; Shimizu, F.; Matsuashima, K.; Asakurri, H.; Narumi,
S. Eur. J. Immunol. 2002, 32, 3197.
8. Frigerio, S.; Junt, T.; Lu, B.; Gerard, C.; Zumsted, U.; Holländer, G. A.; Piali, L. Nat.
Med. 2002, 8, 1414.
9. Saetta, M.; Mariani, M.; Panina-Bordignon, P.; Turato, G.; Buonsanti, C.; Baraldo,
S.; Bellettato, C. M.; Papi, A.; Corbetta, L.; Zuin, R.; Sinigaglia, F.; Fabbri, L. M.
Am. J. Respir. Crit. Care 2002, 165, 1404.
10. Flier, J.; Boorsma, D. M.; Bruynzeel, D. P.; van Beek, P.; Stoof, T. J.; Scheper, R. J.;
Willemze, R.; Tensen, C. P. J. Invest. Dematol. 1999, 113, 574.
11. Qin, S.; Rottman, J. B.; Myers, P.; Kassam, N.; Wienblatt, M.; Loetscher, M.;
Koch, A. E.; Moser, B.; Mackay, C. R. J. Clin. Invest. 1998, 101, 746.
12. (a) Storelli, S.; Verdijk, P.; Verzijl, D.; Timmerman, H.; van de Stolpe, A.; Tensen,
C. P.; Smit, M. J.; De Esch, I. J. P.; Leurs, R. Bioorg. Med. Chem. Lett. 2005, 15,
2910; (b) Allen, D. R.; Bolt, A.; Chapman, G. A.; Knight, R. L.; Meissner, J. W. G.;
Owen, D. A.; Watson, R. J. Bioorg. Med. Chem. Lett. 2007, 17, 697; (c) Johnson,
M.; Li, A.-R.; Liu, J.; Fu, Z.; Zhu, L.; Miao, S.; Wang, X.; Xu, Q.; Huang, A.; Marcus,
A.; Xu, F.; Ebsworth, K.; Sablan, E.; Danao, J.; Kumer, J.; Dairaghi, D.; Lawrence,
C.; Sullivan, T.; Tonn, G.; Schall, T.; Collins, T.; Medina, J. Bioorg. Med. Chem. Lett.
2007, 17, 3339; (d) Watson, R. J.; Allen, D. R.; Birch, H. L.; Chapman, G. A.;
Galvin, F. C.; Jopling, L. A.; Knight, R. L.; Meier, D.; Oliver, K.; Meissner, J. W. G.;
Owen, D. A.; Thomas, E. J.; Tremayne, N.; Williams, S. C. Bioorg. Med. Chem. Lett.
2008, 18, 147; (e) Du, X.; Chen, X.; Mihalic, J. T.; Deignan, J.; Duquette, J.; Li, A.-
R.; Lemon, B.; Ma, J.; Miao, S.; Ebsworth, K.; Sullivan, T. J.; Tonn, G.; Collins, T.
L.; Medina, J. C. Bioorg. Med. Chem. Lett. 2008, 18, 608; (f) Li, A. R.; Johnson, M.
G.; Liu, J.; Chen, X.; Du, X.; Mihalic, J. T.; Deignan, J.; Gustin, D. J.; Duquette, J.;
Fu, Z.; Zhu, L.; Marcus, A. P.; Bergeron, P.; McGee, L. R.; Danao, J.; Lemon, B.;
Carabeo, T.; Sullivan, T.; Ma, J.; Tang, L.; Tonn, G.; Collins, T. L.; Medina, J. C.
Bioorg. Med. Chem. Lett. 2008, 18, 688.
13. Stably transfected hCXCR3-expressing CHO cells were suspended at 106 cells/
well in DMEM/HAM’S F12 medium supplemented with 10% fetal bovine serum
and 1 mM isobutylmethylxanthine. After a 60-min pre-incubation at room
temperature with compound, forskolin (30 lM) and ITAC (0.3 lM, Peprotech)
were added. After 30 min, intracellular cAMP was measured using the cAMP
dynamic kit from CIS bio International.
References and notes
14. Membranes of hCXCR3-expressing CHO cells were diluted in incubation buffer
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H.; Oppenheimer, J. J.; Power, C. A. Pharmacol. Rev. 2000, 52, 145.
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(20 mM Hepes, 100 mM NaCl, 3
14 g/ml saponin) and pre-incubated with compound for 30 min at 30 °C in
96-well flashplates (Perkin-Elmer), before stimulation with human ITAC
(3 nM) or IP-10 (150 nM) (R&D Systems). [35 S]GTP
S (0.25 nM, ꢀ1119 Ci/
lM guanine diphosphate, 1 mM MgCl2, pH 7.4,
l
c
3. Medina, J.; Johnson, M. G.; Collins, T. L. Ann. Rep. Med. Chem. 2005, 40, 215.