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C. Zheng et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1442–1446
Table 5
Pharmacokinetic property of compound 19
Species
Dose (mg)
Cl (L/h/kg)
Vdss (L/kg)
t1/2 (iv) (h)
AUC (po)(
lMÁh)
F (%)
Rat
Cyno
5/10
2.5/10
3.8
2.2
17
6.2
5
3
3.0
5.3
84
57
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Owing to its superior in vitro profile, compound 19 was further
evaluated. It was active against mouse CCR2, rat CCR2, and cyno-
molgus monkey CCR2 with a binding IC50 of 2.8, 2.9, and 4.6 nM,
respectively, but inactive against canine CCR2, with an IC50 of
>10 lM. It was also active against mouse CCR5 and cynomolgus
monkey CCR5, with an IC50 of 71 and 4.0 nM, respectively. Count-
erscreen against a panel of >50 receptors, enzymes and ion chan-
nels proved that 19 is a selective dual CCR2 and CCR5 antagonist.
Compound 19 did not inhibit the tested major isoforms of human
CYP enzymes, with an IC50 of >25 lM against CYP3A4 and CYP2D6.
In hERG potassium channel activity, 19 displayed an IC50 of 1.7 lM
in a hERG patch clamp assay.
ADME profiling revealed that compound 19 exhibited a high
permeability across Caco-2 monolayers with a value of 5.3 cm/s,
and a high free fraction (fu = 56%) in human protein binding. When
treated with human liver microsomes in vitro, compound 19
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2003, 100, 7947.
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WO2005060625.
21. Yang, L.; Butora, G.; Jiao, R. X.; Pasternak, A.; Zhou, C.; Parsons, W. H.; Mills, S.
G.; Vicario, P. P.; Ayala, J. M.; Cascieri, M. A.; MacCoss, M. J. Med. Chem. 2007, 50,
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DeMartino, J. A.; Mills, S. G.; Yang, L. Bioorg. Med. Chem. Lett. 2008, 18, 994.
24. Pasternak, A.; Goble, S. D.; Struthers, M.; Vicario, P. P.; Ayala, J. M.; Di Salvo, J.;
Kilburn, R.; Wisniewski, T.; DeMartino, J. A.; Mills, S. G.; Yang, L. ACS Med. Chem.
Lett. 2010, 1, 14.
25. For CCR2 assay protocols, see: Xue, C.-B.; Wang, A.; Meloni, D.; Zhang, K.; Kong,
L.; Feng, H.; Glenn, J.; Huang, T.; Zhang, Y.; Cao, G.; Anand, R.; Zheng, C.; Xia, M.;
Han, Q.; Robinson, D. J.; Storace, L.; Shao, L.; Li, M.; Brodmerkel, C. M.;
Covington, M.; Scherle, P.; Diamond, S.; Yeleswaram, S.; Vaddi, K.; Newton, R.;
Hollis, G.; Friedman, S.; Metcalf, B. Bioorg. Med. Chem. Lett. 2010, 20, 7473.
26. For CCR5 assay protocols, see: Xue, C.-B.; Chen, L.; Cao, G.; Zhang, K.; Wang, A.;
Meloni, D.; Glenn, J.; Anand, R.; Xia, M.; Kong, L.; Huang, T.; Feng, H.; Zheng, C.;
Li, M.; Galya, L.; Zhou, J.; Shin, N.; Baribaud, F.; Solomon, K.; Scherle, P.; Zhao,
B.; Diamond, S.; Emm, T.; Keller, D.; Contel, N.; Yeleswaram, S.; Vaddi, K.;
Hollis, G.; Newton, R.; Friedman, S.; Metcalf, B. ACS Med. Chem. Lett. 2010, 1,
483.
exhibited good metabolic stability, with
a half-life (t1/2) of
93 min. In vivo, moderate to high systemic clearance was observed
in rats and cynomolgus monkeys, with Cl = 3.8 L/h/kg in rats and
2.2 L/h/kg in cynomolgus monkeys. However, the high volume of
distribution in both species (Table 5) resulted in a moderate to long
half-life for this compound, with t1/2 = 5 h (iv) in rats and 3 h (iv) in
cynomolgus monkeys. Compound 19 was orally well-absorbed,
with an oral bioavailability (F %) of 84% in rats and 57% in cynomol-
gus monkeys.
In conclusion, we identified piperazine as a suitable linker be-
tween the carbonyl in the center and the aryl on the right-hand
side using 3-aminocyclopentanecarboxamide as our core structure.
Modifications on both left-hand and right-hand sides led to the dis-
covery of a potent, selective and orally bioavailable dual CCR2 and
CCR5 antagonist 19 ((3S,4S)-N-[(1R,3S)-3-isopropyl-3-({4-[4-(tri-
fluoromethyl)pyridin-2-yl]piperazin-1-yl}carbonyl)cyclopentyl]-
3-methoxytetrahydro-2H-pyran-4-amine). The potency and PK
property of this compound predicted a low human dose of
20–50 mg twice-a-day dosing (BID).29 Compound 19 (INCB10820/
PF-4178903), after evaluation in 28-day toxicology studies, was
selected as
a clinical candidate. Further evaluation of this
compound will be reported in due course.
27. Turner, J. E.; Steinmetz, O. M.; Stahl, R. A.; Panzer, U. Mini-Rev. Med. Chem.
2007, 7, 1089.
28. A volume of 200 ll of whole blood collected in heparin coated vacutainer tubes
Acknowledgments
(BD Biosciences) from normal volunteers is preincubated with nothing or with
indicated amounts of a competitor (compound) for 5 min at rt. MCP-Alexa 488
at 30 nM final concentration together with an anti-CD14-allophycocyanin
(APC)-conjugated antibody (Caltag, CA) at 1/200 is then added to the samples
and further incubated for 30 min at rt, being protected from light. Washing of
the samples is done using 3 mL of cold FACS buffer (FB, 3% FBS in PBS) followed
by centrifugation (5 min/500 g/rt). The red blood cells are then lysed for 10 min
at rt in 2 mL of FACS™ lysing solution (BD Biosciences) followed by a 5 min/
We thank Lynn Leffet, Karen Gallagher, Patricia Feldman, Bitao
Zhao, Yanlong Li, Robert Collins, and Gengjie Yang for technical
assistance.
References and notes
500 g/rt centrifugation. The samples are then resuspended in 250 ll of FB
containing 1% paraformaldehyde and analyzed by flow cytometry (Facs
Calibur, BD Biosciences).
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29. The human dose projection was based upon the human trough concentration
required to cover the human whole blood (HWB) IC80. The human trough
concentration was obtained from the projected human PK by an allometric
scaling of the rat PK.