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10. Fryer, A. D.; Adamko, D. J.; Yost, B. L.; Jacoby, D. B. Life Sci. 1999, 64, 449.
11. Jin, J.; Budzik, B.; Wang, Y.; Shi, D.; Wang, F.; Xie, H.; Wan, Z.; Zhu, C.; Foley, J. J.;
Webb, E. F.; Berlanga, M.; Burman, M.; Sarau, H. M.; Morrow, D. M.; Moore, M.
L.; Rivero, R. A.; Palovich, M.; Salmon, M.; Belmonte, K. E.; Lainé, D. I. J. Med.
Chem. 2008, 51, 5915.
12i showed decreased potency compared to 12a–12g, suggesting
that an outlying heteroatom may be needed for maximum M3
potency.
Unlike the RHS diamine region, quaternization was well toler-
ated in the LHS diamine region (Table 6). Quaternization of the out-
er nitrogen of 12a was more favored than that of the inner nitrogen
(13a) and led to a compound 13b, which was equipotent to 12a.
Other quaternized piperazines (13c–13e) also gave excellent M3
potencies with pA2 values around 10. Quaternary ammonium salts
such as 13b indeed had extremely low membrane permeability
(<3 nm/s).22 Interestingly, the increased basicity of the piperidine
derivative 14 compared to 12a,23 correlated with an increased
affinity for the M3 receptor, suggesting that the terminal N of these
molecules may be interacting with an acidic residue. As previously
reported, compound 14 was found to exhibit sub-type selectivity
for M3 over M1 and M2 (pA2s for M3, M2, and M1 are 11.0, 8.3,
and 9.7, respectively).11 In addition, 14 also displayed excellent
inhibitory activity and long duration of action in a bronchocon-
striction in vivo model via intranasal administration.11
12. (a) Measuring inhibition of acetylcholine-mediated [Ca2+]i-mobilization in
Chinese hamster ovary (CHO) cells stably expressing human recombinant M3
receptor.; (b) For FLIPR assay details, see supporting information in Ref. 11.
13. The biological assay results in the paper are a mean of at least 2 determinations
with standard deviation of < 0.3, and data for compounds M3 pIC50 > 7.0 are
from 96-well FLIPR assay, <7.0 from 384 well FLIPR assay, unless otherwise
noted.
14. (a) Jin, J.; Graybill, T. L.; Wang, M. A.; Davis, L. D.; Moore, M. L. J. Comb. Chem.
2001, 3, 97; (b) Available from Polymer Laboratories, Part number: 1466-6689,
150–300 lm, 1.5 mmol/g loading.
15. (a) All new compounds in this paper were characterized via LC/MS and 1H
NMR.; (b) For representative experimental procedures, see supporting
information in Ref. 11.
16. Compound 5h was synthesized by using 3-(dihydroxyboranyl)benzoic acid
instead of 3-formylphenyl boronic acid in step (c) of Scheme 1, followed by DIC
mediated coupling with Boc-piperazine, followed by cleavage step (e).
17. Use of any unprotected, 2-substituted piperazine in step (d) of Scheme 1 gives
completely selective reductive amination at the less hindered nitrogen.
18. Quaternization of the inner piperazine nitrogen in 5n was affected by use of
Boc-piperazine in step (d) of Scheme 1, followed by reaction of the washed
resin with excess CH3I in CH3CN at rt for overnight, followed by cleavage step
(e).
19. Quaternization of the outer piperazine nitrogen in 5o was affected by use of N-
methyl-piperazine in step (d) of Scheme 1, followed by reaction of the washed
resin with excess CH3I in CH3CN at rt for overnight, followed by cleavage step
(e), quaternization being completely selective for the less sterically hindered
outer nitrogen.
In summary, the extensive SAR exploration of multiple regions
of the HTS hit 1 led to the identification of key structural motifs
necessary to achieve high M3 potency. The combination of these
features resulted in the discovery of the highly potent M3 antago-
nist 14 (pA2 = 11.0).
20. Compounds 8a and 8c were prepared by performing sulfonylation (using 1,3-
benzodioxole-5-sulfonyl chloride in presence of DMAP in pyridine/DCE), or
urea formation (using 5-isocyanato-1,3-benzodioxole in DCE), respectively,
instead of amide formation in step (b) of Scheme 1. Compound 8b was
prepared by using 3-bromoaniline instead of 3-bromobenzylamine in step (a)
of Scheme 1, and required more forcing amide coupling conditions in step (b):
DMAP, DIC, in DCE/DMF at 80 °C.
21. The pIC50 limit of the M3 FLIPR assay was about 9.0. pA2 was determined and
used to compare potency for compounds with pIC50 reaching the limit. See Ref.
11 for assay details of pA2 determination.
Acknowledgments
We thank Bing Wang for NMR, Qian Jin for LC/MS, Carl Bennett
for purification, and Christina Schulz-Pritchard, Jim Fornwald, and
Jeffrey Guss for assay support.
References and notes
22. The artificial phospholipid membrane technique is similar to the widely used
Caco-2 cell monolayer permeation technique. In short, egg phosphatidyl
choline (1.8%) and cholesterol (1%) are dissolved in n-decane. A small amount
of the volatile mixture is applied to the bottom of the microfiltration filter
inserts. Phosphate buffer (0.05 M, pH 7.05) is quickly added to the donors and
receivers, and the lipids are allowed to form self-assembled lipid bilayers
across the small holes in the filter. Permeation experiment is initiated by
spiking the compounds of interest to the donor sides, and the experiment is
stopped at a pre-determined elapsed time. The samples are withdrawn and
transferred to appropriate vials for analysis by HPLC with UV detection
(215 nm).
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piperidine 14 are given in Ref. 11.