A. V. Purandare et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2669–2672
2671
Table 4.
I.; Wells, T. N. C.; Power, C. A. J. Exp. Med. 2000, 19,
1755; (b) Wakugawa, M.; Nakamura, K.; Kakinuma, T.;
Tamaki, K. Drugs News Perspect. 2002, 15, 175, and
references cited therein.
Cl
N
R
O
N
HN
5. (a) Allen, S.; Newhouse, B.; Anderson, A.; Fauber, B.;
Allen, A. C.; Davis; Eberhardt, C.; Odingo, J.; Burgess, L.
Bioorg. Med. Chem. Lett. 2004, 14, 1619; (b) Habashita,
H.; Kokubo, M.; Shibayama, S.; Tada, H.; Sagawa, K.
WO2004007472, 2004; (c) Baxter, A.; Johnson, T.; Kin-
don, N.; Roberts, B.; Steele, J.; Stocks, M.; Tomkinson,
N. WO03051870, 2003.
6. Rotella, D. P.; Sun, Z.; Zhu, Y.; Krupinski, J.; Pongrace,
R.; Seliger, L.; Normandin, D.; Macor, J. E. J. Med.
Chem. 2000, 43, 1257, and references cited therein.
7. All compounds were characterized by LC–MS and NMR
analysis. In addition, the yields were based on weight of
pure product unless mentioned otherwise.
Cl
CCR4 IC50 (lM)11
Compd #
R
11a
11b
11c
11d
11e
11f
–(CH)Me-(CH2)3N(Et)2
–(CH2)3-N(Et)2
–(CH2)4-N(Et)2
1.5
8
3
–(CH2)2-N(Et)2
–(CH2)6-N(Me)2
–(CH)Me-(CH2)3CH(Me)2
9
2
>10
Also removal of the terminal nitrogen was found to be
detrimental for the activity (compound 11f) (Table 4).
8. Haviv, F.; DeNet, R. W.; Michaels, R. J.; Ratajczyk, J.
D.; Carter, G. W.; Young, P. R. J. Med. Chem. 1983, 26,
218.
9. Tsunoda, T.; Yamamiya, Y.; Ito, S. Tetrahedron Lett.
1993, 34, 1639.
Finally, replacement of oxygen as the linking atom with
nitrogen was found to be optimum and this modification
enhanced the activity by >5-fold. Compound 12 also
10. (a) Campbell, S. F.; Plews, R. M. J. Med. Chem. 1987, 30,
1794; (b) Acevedo, O. L.; Andrews, R. S.; Dunkel, M.;
Cook, P. D. J. Heterocycl. Chem. 1994, 31, 989.
showed
significant
inhibition
of
chemotaxis
(IC50 = 5 lM) without cytotoxicity (CC50 > 100 M).
Compound 12 also showed selectivity against CCR3
(IC50 = 2 lM vs eotaxin), CCR2 (IC50 > 10 lM vs
MCP-1) and CXCR3 (IC50 > 30 lM vs I-TAC) in radio-
ligand based binding assays.
11. Assay conditions: CCR4 binding assay. A whole cell
scintillation proximity assay (SPA) format was used for
binding assays. HEK293 cells that were stably transfected
with the human CCR4 receptor (accession # X85740),
were suspended in assay buffer and plated at 4 · 104 cells/
well into poly-L-lysine (100 lg/ml in PBS, 100 ll/well, O/
N, 4 ꢁC) treated solid white 96-well plates (Costar #3917).
Assay buffer was phenol-red free DMEM (GIBCO
#31053-028) supplemented with 10% FBS and 2 mM L-
glutamine. Cells were incubated overnight at 37 ꢁC, with
5% CO2. Following incubation, 100 ll of binding mix was
added to each well. Binding mix contains WGA-PVT SPA
beads to give a 0.1 mg/well final concn, and 125I-MDC
[50 lC/ml] to give a 0.1 nM final concn, in binding buffer.
Binding buffer is phenol-red free DMEM, 0.5% BSA
(Sigma #A7284), and 2 mM L-glutamine. Compounds
diluted in DMSO were added to the wells (1 ll/well, 1%)
and 100 nM unlabeled MDC was added to non-specific
control wells. The plate was sealed using Top Seal A
(Packard) and the plate was mixed with slight agitation for
15 min at room temperature (rt). Following 24 h of
incubation at rt without shaking, the plate was counted
on a TopCount (Packard). Comparisons of SPA format
results with direct receptor binding filter-based assays gave
comparable results. Hill values for compound 12 ranged
from 0.7 to 0.8 in five IC50 determinations. A typical
binding assay would have a non-specific binding value
from 100 cpm to 150 cpm, a signal to noise ratio between
8:1 and 16:1, and a signal window ranging from 5 to 20
(based on the number of standard deviations of the largest
signal). Substitution of TARC in the binding assay for
MDC gave IC50 values within 2-fold of the MDC based
values. The HEK293/CCR4 cell line was determined to
have 36,000 receptors per cell by Scatchard analysis.
Cytotoxicity assay: to test compounds for cytotoxicity,
compounds were diluted as above to 2 · concentrations in
phenol-red free RPMI supplemented with 10% FBS and
Pen./Strep. at a volume of 50 ll/well. CCR4 transfected
L1.2 cells (murine pre-B cell) grown in the same media,
were harvested by centrifugation, and suspended in media
at 2 · 106 cells/ml. Cells (50 ll) were then added to each
well containing 2 · compound. The plate was mixed and
incubated for 24 h at rt. For 0% viability controls, 6 ll/
well of a 5% saponin solution was added to control wells
Cl
N
N
N
N
N
H
H
Cl
12
CCR4IC50 0.27 µM
In conclusion, we have identified a potent non-cytotoxic
small molecule antagonist of CCR4 that showed inhibi-
tion of functional response mediated by binding of che-
mokine to the receptor. Further work related to the
optimization of this chemotype will be reported in due
course.
References and notes
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217; (b) Zlontik, A.; Yoshie, O. Immunity 2000, 12, 121; (c)
Owen, C. Pulm. Pharmacol. Ther. 2001, 14, 193; (d)
Power, C. A.; Proudfoot, A. E. Curr. Opin. Pharmacol.
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Editorial. Clin. Exp. Allergy 2001, 31, 1809; (c) Lukacs, N.
W. Nat. Rev. Immunol. 2001, 1, 108; (d) Mantovani, A.;
Gray, P. A.; Damme, J. V.; Sozzani, S. J. Leukocyte Biol.
2000, 68, 400, and references cited therin.
3. (a) Gonzalo, J. A.; Pan, Y.; Lloyd, C. M.; Jia, G. Q.; Yu,
G.; Dussault, B.; Powers, C. A.; Proudfoot, A. E.; Coyle,
A. J.; Gearing, D.; Gutierrzez-Ramos, J. C. J. Immunol.
1999, 163, 403; (b) Kawasaki, S.; Takizawa, H.; Yone-
yama, H.; Nakayama, T.; Fujisawa, R.; Izumizaki, M.;
Imai, T.; Yoshie, O.; Homma, I.; Yamamoto, K.; Matsu-
shima, K. J. Immunol. 2001, 166, 2055.
4. (a) Chvatchko, Y.; Hoogewerf, A. J.; Meyer, A.; Alouani,
S.; Juillard, P.; Buse, R.; Conquest, F.; Proudfoot, A. E.