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References and Notes
1. Leroy, V.; Mauser, P.; Gao, Z.; Peet, N. P. Exp. Opin.
Invest. Drugs 2000, 9, 735.
2. Seward, E. M.; Swain, C. J. Exp. Opin. Ther. Pat. 1999, 9,
571.
3. Swain, C. J.; Rupniak, N. M. J. Ann. Rep. Med. Chem.
1999, 33, 51, and references cited therein
4. Cooper, L. C.; Chicchi, G. G.; Dinnell, K.; Elliott, J. M.;
Hollingworth, G. J.; Kurtz, M. M.; Locker, K. L.; Morrison,
D.; Shaw, D. E.; Tsao, K.-L.; Watt, A. P.; Williams, A. R.;
Swain, C. J. Bioorg. Med. Chem. Lett. 2001, 11, 1233.
5. Dinnell, K.; Chicchi, G. G.; Dhar, M. J.; Elliott, J. M.;
Hollingworth, G. J.; Kurtz, M. M.; Ridgill, M. P.; Rycroft,
W.; Tsao, K.-L.; Williams, A. R.; Swain, C. J. Bioorg. Med.
Chem. Lett. 2001, 11, 1237.
Scheme 4. (i) MeMgCl, THF (0 ꢀC to rt, 16 h); (ii) TFA, DCM (rt,
16 h).
6. Rupniak, N. M. J.; Tattersall, F. D.; Williams, A. R.;
Rycroft, W.; Carlson, E.; Cascieri, M. A.; Sadowski, S.; Ber,
E.; Hale, J. J.; Mills, S. G.; MacCoss, M.; Seward, E.;
Huscroft, I.; Owen, S.; Swain, C. J.; Hill, R. G.; Hargreaves,
R. J. Eur. J. Pharmacol. 1997, 326, 201.
7. Elliott, J. M.; Castro, J. L.; Chicchi, G. G.; Cooper, L.
C.; Dinnell, K.; Hollingworth, G. J.; Ridgill, M. P.;
Rycroft, W.; Kurtz, M. M.; Shaw, D. E.; Swain, C. J.;
Tsao, K.-L.; Yang, L. Bioorg. Med. Chem. Lett. 2002, 12,
1755.
8. Cross, P. E.; Arrowsmith, J. E.; Thomas, G. N.; Gwilt, M.;
Burges, R. A.; Higgins, A. J. J. Med. Chem. 1990, 33, 1151.
9. Cascieri, M. A.; Ber, E.; Fong, T. M.; Sadowski, S.; Bansal,
A.; Swain, C. J.; Seward, E. M.; Frances, B.; Burns, D.; Stra-
der, C. D. Mol. Pharmocol. 1992, 42, 458.
10. IKr affinity is measured using the MK-499 (L-706000)11
binding assay which is performed on membrane preparations
from human embryonic kidney (HEK) cells constitutively
expressing hERG. The membranes are prepared by homo-
genization of the HEK cells in Tris–EDTA buffer (50 mM
Tris, 1 mM EDTA, pH 7.5) and centrifugation at 45,000g for
20 min at 4 ꢀC. The pellet containing membrane is resuspended
in Tris–EDTA and stored at À70 ꢀC. In the binding assay,
hERG membranes are diluted into assay buffer (60 mM KCl,
71.5 mM NaCl, 1 mM CaCl2, 10 mM Hepes, pH 7.4) to a final
concentration of 4.16 mg per 400 mL well volume. Radio-
labelled MK-499 (35S-L-706000; specific activity ꢁ1000 Ci/
mmol) is then added to a final concentration of 50 pM and
400 mL of membrane-ligand mixture is added per well to 96-
well assay blocks containing 4 mL of 100Â stocks of tested
drugs or 100% DMSO (maximum binding control) or 100 mM
cold MK-499 (non-specific binding control). Membranes are
incubated at room temperature for 75 min, filtered through
GF/B Unifilters presoaked in 0.1% BSA and washed 5Â500 mL
with wash buffer at 4 ꢀC (10 mM Hepes, 131.5 mM NaCl, 1 mM
CaCl2, 2 mM MgCl2, pH 7.4). Filters are dried at room tem-
perature, 50 mL Microscint-20 is added to each well, and Uni-
filters are counted for 1 min in Topcount. Dose-inhibition
curves and inflection points are determined by curve-fitting the
equation: response=[(maxÀmin)/(1+([I]/IP)s)]+min, where I
is the concentration of inhibitor, max is the maximum response
(DMSO control), min is the minimum response (excess, cold
MK-499 control), IP is the inflection point, and s is the slope.
Generally, the inflection point is similar to the concentration of
drug that inhibits 50% of radiotracer binding.
The in vitro and in vivo results for compounds 5m and
5n are shown in Table 3. Both compounds retain excel-
lent NK1 receptor binding affinity and the improvement
already seen in IKr affinity is maintained in 5m and
increased in 5n. Both compounds showgood activity in
vivo following a 2 h pre-treatment and 5m maintains
this at 24 h, however duration of action is much reduced
in 5n which gave only 7.5% inhibition of foot-tapping
after a 24 h pre-treatment time. This is disappointing,
especially in light of its much reduced IKr affinity.
Table 3. NK1, IKr and gerbil activities for compounds 5m and 5n
Compd NR1R2
hNK1
IC50 (nM)a
hIKr
Ki (nM)b
Gerbil FT
ID50 (mg/kg iv)e
t=2 h
t=24 h
5m
5n
0.35Æ0.15
950Æ170c
1.0
72% @ 3
0.26Æ0.08 2200Æ1300d
1.1
7.5% @ 3
aDisplacement of [125I]-labelled substance P from the cloned hNK1
receptor expressed in CHO cells. Data are meanÆSD (n=3).9
bDisplacement of labelled MK-499 from the cloned channel expressed
in HEK cells. Data are meanÆSD.10
cn=4.
dn=3.
eInhibition of GR 73632-induced foot-tapping in gerbils.6
In conclusion, we have investigated substitution of the
amine portion of the lead compound 2 and found that a
wide range of large lipophilic amines is tolerated by the
NK1 receptor although the changes in IKr affinity are
unpredictable. Three compounds, 5i, 5j and 5m,
achieved the objectives of retaining NK1 receptor affi-
nity and in vivo activity while significantly reducing IKr
affinity.
11. Claremon, D. A.; Baldwin, J. J.; Elliott, J. M.; Remy, D.
C.; Ponticello, G. S.; Selnick, H. G.; Lynch, J. J., Jr.; Sangui-
netti, M. C. In Perspectives in Medicinal Chemistry; Testa, B.,
Kyburz, E., Fuhrer, W., Giger, R., Eds.; Helvetica Chimica
Acta: Basel, 1993; p 391.