3162
S. Sasmal et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3157–3162
7. Rossi, M.; Beak, S. A.; Choi, S.-J.; Small, C. J.; Morgan, D. G. A.; Ghatei, M. A.;
Smith, D. M.; Bloom, S. R. Brain Res. 1999, 846, 164.
22. Functional response of compounds was assessed using the IP3-SPA-YSI
method. Briefly, CHO-K1 cells stably expressing hMCHR1 were incubated
8. Gomori, A.; Ishihara, A.; Ito, M.; Mashiko, S.; Matsushita, H.; Yumoto, M.; Ito,
M.; Tanaka, T.; Tokita, S.; Moriya, M.; Iwaasa, H.; Kanatani, A. Am. J. Physiol.
Endocrinol. Metab. 2003, 284, E583–E588.
overnight with 0.5 lCi/well of 3H-myo-inositol to generate a pool of 3H-PIP2.
After aspiration of labeling medium, the cells were incubated with test
compounds followed by stimulation with 80 nM of agonist (MCH peptide,
corresponds to EC80). The resulting pool of 3H-IPs was extracted with formic
acid and the amount of 3H-IPs generated in the cells was detected using the
non derivatized yttrium silicate SPA beads.
9. Ludwig, D. S.; Tritos, N. A.; Mastaitis, J. W.; Kulkarni, R.; Kokkotou, E.; Elmquist,
J.; Lowell, B.; Flier, J. S.; Maratos-Flier, E. J. Clin. Invest. 2001, 107, 379.
10. (a) Qu, D. Q.; Ludwig, D. S.; Gammeltoft, S.; Piper, M.; Pelleymounter, M. A.;
Cullen, M. J.; Mathes, W. F.; Przypek, J.; Kanarek, R.; Maratos-Flier, E. Nature
1996, 380, 243; (b) Mizuno, T. M.; Kleopoulos, S. P.; Bergen, H. T.; Roberts, J. L.;
Priest, C. A.; Mobbs, C. V. Diabetes 1998, 47, 294; (c) Hanada, R.; Nakazato, M.;
Matsukura, S.; Murakami, N.; Yoshimatsu, H.; Sakata, T. Biochem. Biophys. Res.
Commun. 2000, 268, 88.
23. Protocol for solubility measurements: A solution of 25
lL of compound dissolved
in DMSO (10 mM) is added to a PBS solution to a final solution of 500
l
M in a
96-well PP Greiner deep-well plate. This mixture is allowed to precipitate
overnight at RT with the plate located on a plate-shaker. Next day solution (and
precipitate) is centrifuged and absorbance (260, 280 and 620 nm) of
11. Shimada, M.; Tritos, N. A.; Lowell, B. B.; Flier, J. S.; Maratos-Flier, E. Nature 1998,
396, 670.
supernatant is measured. Also a reference (100 and 500 lM compound)
dissolved in acetonitrile is measured and solubility (i.e., remaining compound
in PBS solution) is determined by dividing the absorbance measured in PBS
versus acetonitrile. A blind DMSO is also measured for all wave lengths as
reference and the absorbance of 620 nm is used to exclude possible
contaminations (i.e., visible) in samples.
12. (a) Chen, Y.; Hu, C.; Hsu, C.-K.; Zhang, Q.; Bi, C.; Asnicar, M.; Hsiung, H. M.; Fox,
N.; Slieker, L. J.; Yang, D. D.; Heiman, M. L.; Shi, Y. Endocrinology 2002, 143,
2469; (b) Marsh, D. J.; Weingarth, D. T.; Novi, D. E.; Chen, H. Y.; Trumbauer, M.
E.; Chen, A. S.; Guan, X.-M.; Jiang, M. M.; Feng, Y.; Camacho, R. E.; Shen, Z.;
Frazier, E. G.; Yu, H.; Metzger, J. M.; Kuca, S. J.; Shearman, L. P.; Gopal-Truter, S.;
MacNeil, D. J.; Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.; Qian, S.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3240.
13. (a) McBriar, M. D.; Kowalski, T. J. Ann. Rep. Med. Chem. 2005, 40, 119; (b) Dyke,
H.; Ray, N. C. Expert Opin. Ther. Pat. 2005, 15, 1303; (c) Shi, Y. Peptides 2004, 25,
1605; (d) Browning, A. Expert Opin. Ther. Pat. 2004, 14, 313; (e) Kowalski, T. J.;
McBriar, M. D. Expert Opin. Invest. Drugs 2004, 13, 1113.
14. (a) Mendez-Andino, J. L.; Wos, J. A. Drug Discovery Today 2007, 12, 972; (b)
McBriar, M. D.; Guzik, H.; Shapiro, S.; Paruchova, J.; Xu, R.; Palani, A.; Clader, J.
W.; Cox, K.; Greenlee, W. J.; Hawes, B. E.; Kowalski, T. J.; O’Neill, K.; Spar, B. D.;
Weig, B.; Weston, D. J.; Farley, C.; Cook, J. J. Med. Chem. 2006, 49, 2294.
15. Andersen, D.; Storz, T.; Liu, P. L.; Wang, X.; Li, L. P.; Fan, P. C.; Chen, X. Q.;
Allgeier, A.; Burgos, A.; Tedrow, J.; Baum, J.; Chen, Y.; Crockett, R.; Huang, L.;
Syed, R.; Larsen, R. D.; Martinelli, M. J. Org. Chem. 2007, 72, 9648.
16. (a) Rokosz, L. L. Exp. Opin. Drug Discovery 2007, 2, 1301; (b) Liu, Y.; Sprenger, K.;
Maynard, G.; Friedman, H.; Anciro, L.; Rajachandran, L.; Changchit, A. J. Clin.
Pharmacol. 2009, 49, 1101.
24. Protocol for microsomal stability studies: Metabolic stability studies were
conducted by incubating the compounds at 10 lM with 1 mg/ml of mouse/
human liver microsomes diluted in phosphate buffer (0.1 M, pH 7.4) .The
reaction was initiated by the addition of NADPH and the samples were
incubated for 30 min at 37 °C. The reaction was terminated by the addition of
an organic solvent containing an appropriate internal standard. The samples
were vortexed, centrifuged and the clear supernatant was analysed via HPLC.
25. Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G.; Thian, F. S.;
Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C.
Science 2007, 318, 1258.
26. Generic numbering of amino acids in TM bundle according to Schwartz, T.W.
(e.g., Gln196 III:12), Curr. Opin. Biotechnol. 1994, 5, 434. A comparison with the
Ballesteros–Weinstein nomenclature (e.g., Gln196 3.36) is detailed in Frimurer,
T.M.; Högberg, T. Curr. Top. Med. Chem. 2011, 11, 1882.
27. A recent substructure search for compounds containing 4-hydroxypiperidines
of the ChEMBL (former Starlight) database containing more than 500000
bioactive ligands (predominantly small molecules) identified a total of 803
small molecules containing 4-hydroxypiperidine motifs (many more if
additional substituents in the piperidine ring are included). The compounds
distributed on 153 different chemotype clusters based on Daylight 2D
fingerprints and a Tannimoto similarity threshold of 0.7. The ligands were
associated with various different targets including several GPCR families.
28. Oral PK profile of compound 42 in Swiss Albino Mice (30 mg/kg); Plasma:
17. (a) Ulven, T.; Frimurer, T. M.; Receveur, J.-M.; Little, P. B.; Rist, Ø.; Nørregaard,
P. K.; Högberg, T. J. Med. Chem. 2005, 48, 5684; (b) Ulven, T.; Little, P. B.;
Receveur, J.-M.; Frimurer, T. M.; Rist, Ø.; Nørregaard, P. K.; Högberg, T. Bioorg.
Med. Chem. Lett. 2006, 16, 1070.
18. Clark, D. E.; Higgs, C.; Wren, S. P.; Dyke, H. J.; Wong, M.; Norman, D.; Lockey, P.
M.; Roach, A. G. J. Med. Chem. 2004, 47, 3962.
19. Arienzo, R.; Cramp, S.; Dyke, H. J.; Lockey, P. M.; Norman, D.; Roach, A. G.;
Smith, P.; Wong, M.; Wren, S. P. Bioorg. Med. Chem. Lett. 2007, 17, 1403.
20. Giraud, F.; Guillon, R.; Logé, C.; Pagniez, F.; Picot, C.; Borgne, M. L.; Pape, P. L.
Bioorg. Med. Chem. Lett. 2009, 19, 301.
AUC(0–8) 1.02
l
M.h, Cmax 0.43
lM, Tmax 1 h, t1/2 3.0 h; Brain: AUC(0–8)
6.21 M h, Cmax 2.66
l
lM, Tmax 1 h, t1/2 1.7 h.
29. b.i.d. stands for Latin word bis in die meaning twice a day.
30. C57BL/6J mice were fed a high fat diet (60% calories from fat, D12492 feed) for
nearly 3 months until they reached an average body weight of approximately
45 g. Animals were grouped based on initial body weight. Compounds to be
tested were then administered per orally twice daily for a period of 14 days.
Body weight and food intake of the animals was recorded on a daily basis
during the experimental duration.
21.
[
125I]-MCH binding was performed by incubating membranes from CHO-K1
cells stably expressing hMCHR1 with SPA beads and tracer in presence of
various test compounds for 2 h. Non specific binding was measured by
conducting the reaction in presence of 1 lM of cold MCH peptide. Readings
were taken in the top count scintillation counter. The extent of antagonism was
expressed as % displacement. The IC50 for the compound was calculated by
Assay Explorer from MDL software using the average CPM values.