366
X. Chen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 363–366
Table 5
monkey, so that this compounds could serve as a tool for further
Pharmacokinetic profiles of select analogs in rat
unraveling the physiological functions of the MCHR2 receptor
and its involvement in various diseases. In vivo pharmacological
evaluation of this compound will be published in due course.
Compound
CL (L/h/kg)a
MRT (h)a
F (%)b
16
23
19
35
8.7
1.2
2.2
0.8
2.8
18.2
14.2
9.2
5.9
7.5
9.0
49
References and notes
1. Kawauchi, H.; Kawazoe, I.; Tsubokawa, M.; Kishida, M.; Baker, B. I. Nature 1983,
305, 321.
2. Skofitsch, G.; Jacobowitz, D. M.; Zamir, N. Brain Res. Bull. 1985, 15, 635.
3. Bittencourt, J. C.; Presse, F.; Arias, C.; Peto, C.; Vaughan, J.; Nahon, J. L.; Vale, W.;
Sawchenko, P. E. J. Comp. Neurol. 1992, 319, 218.
a
Following i.v. dosing in rat at 0.5 mg/kg. MRT (Mean Residence Time).
Following p.o. dosing in rat at 2 mg/kg.
b
4. Saito, Y.; Maruyama, K. J. Exp. Zoolog. A. Comp. Exp. 2006, 305, 761.
5. Chung, S.; Parks, G. S.; Le, C.; Civelli, O. J. Mol. Neurosci. 2011, 43, 115.
6. Qu, D. S.; Ludwig, S.; Gammeltoft, M.; Piper, M. A.; Pelleymounter, M. J.; Cullen,
W. F.; Mathes, J.; Przypek, R.; Kanarek; Marathos-Flier, E. Nature 1996, 380, 243.
7. 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.
8. Shimada, M.; Tritos, N. A.; Lowell, B. B.; Flier, J. S. Nature 1998, 396, 670.
9. (a) Chambers, J.; Ames, R. S.; Bergsma, D.; Muir, A.; Fitzgerald, L. R.; Hervieu, G.;
Dytko, G. M.; Foley, J. J.; Martin, J.; Liu, W. S.; Park, J.; Ellis, C.; Ganguly, S.;
Konchar, S.; Cluderay, J.; Leslie, R.; Wilson, S.; Sarau, H. M. Nature 1999, 400,
261; (b) Saito, Y.; Nothacker, H. P.; Wang, Z.; Lin, S. H.; Leslie, F.; Civelli, O.
Nature 1999, 400, 265.
Table 6
Pharmacokinetic profiles of compound 38 in rat, dog and rhesus monkey
Species
Cl (L/h/Kg)a
MRT (h)a
F (%)b
Rat
1.0
0.6
0.6
18
11
6.1
37
73
47
Beagle dog
Rhesus monkey
a
Following i.v. dosing in rat at 0.5 mg/kg. MRT (Mean Residence Time).
Following p.o. dosing in rat, dog and rhesus monkey at 2 mg/kg.
b
10. 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.; Qian, S. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 3240.
be responsible for the high clearance. While the exact location of
the oxidation on the carbazole ring was not clearly revealed, we
were hoping that by reducing the overall electronic density of
the carbazole we would reduce its propensity for in vivo oxidation.
We synthesized several 4-substituted carbazole analogs with F, Cl,
Br and CF3 (Table 4). The Cl substitution of the carbazole compound
35 only lost 10-fold MCHR2 activity compared to the unsubstituted
compound 23. F, Br and CF3 substitution were not tolerated with a
loss of more than 100-fold MCHR2 activity over compound 23.
Compound 35 was dosed in rat to evaluate its PK profile. Com-
pound 35 had an i.v. clearance of 0.8 L/h/Kg and 49% oral bioavail-
ability in rat. This was a large improvement over the PK profile of
compound 23. However, when compound 35 was screened in the
dopamine receptor D2 and serotonin transporter binding assays,17
11. Méndez-Andino, J. L.; Wos, J. A. Drug Discovery Today 2007, 12, 972.
12. (a) An, S.; Cutler, G.; Zhao, J. J.; Huang, S. G.; Tian, H.; Li, W.; Liang, L.; Rich, M.;
Bakleh, A.; Du, J.; Chen, J. L.; Dai, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7576; (b)
Andreas, W.; Sailer, H. S.; Zeng, Z.; McDonald, T. P.; Pan, J.; Pong, S. S.; Feighner,
S. D.; Tan, C. P.; Fukami, T.; Iwaasa, H.; Hreniuk, D. L.; Morin, N. R.; Sadowski, S.
J.; Ito, M.; Ito, M.; Bansal, A.; Ky, B.; Figueroa, D. J.; Jiang, Q.; Austin, C. P.;
MacNeil, D. J.; Ishihara, A.; Ihara, M.; Kanatani, A.; Van der Ploeg, L. H. T.;
Howard, A. D.; Liu, Q. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7564; (c) Hill, J.;
Duckworth, M.; Murdock, P.; Rennie, G.; Sabido-David, C.; Ames, R. S.;
Szekeres, P.; Wilson, S.; Bergsma, D. J.; Gloger, I. S.; Levy, D. S.; Chambers, J.
K.; Muir, A. I. J. Biol. Chem. 2001, 276, 20125; (d) Logan, D. W.; Bryson-
Richardson, R. J.; Pagán, K. E.; Taylor, M. S.; Currie, P. D.; Jackson, I. J. Genomics
2003, 8, 184.
13. MCHR2 Ca2+ FLIPR assay: A FLIPR assay kit (Molecular Devices/MDS Analytical
Technologies) was used and the protocol provided by the manufacturer was
followed. Briefly, MCHR2 expressing cells were seeded in a 384 well FLIPR
assay plate and incubated overnight. The cells were washed with FLIPR buffer
and incubated with the buffer containing calcium sensitive dye for 1 h.
Dilutions of compounds were added to the cells and incubated for 10 min
it showed IC50 of 0.5 and 0.1 lM, respectively.
Since our earlier SAR indicated that the urea group could also
increase the MCHR2 activity compared to the unsubstituted carba-
zole (cf. 9 vs 7), compound 38 was synthesized. Synthesis of 38 was
achieved by reacting compound 23 with trichloroacetylisocyanate
in CH2Cl2 followed by stirring with aqueous NaOH.
before addition of MCH to
stimulated by MCH was recorded with a FLIPR system (Molecular Devices/MDS
Analytical Technologies). Data were analyzed for IC50 using nonlinear
a final concentration of 35 nM. Calcium flux
a
regression curve fitting program (GraphPad Prism, GraphPad Software, Inc.,
La Jolla, CA). Assays were repeated at least twice for IC50 determination.
14. Corey, E. J.; Helal, C. J. Angew. Chem., Int. Ed. 1998, 37, 1986.
15. Takemoto, T.; Nakajima, K.; Iio, Y.; Tamura, M.; Nishi, T. Tetrahedron:
Asymmetry 1999, 10, 1787.
Compound 38 had an IC50 of 1 nM in the MCHR2 Ca+2 FLIPR as-
say. Additionally, when compound 38 was tested in a MCHR2 bind-
ing assay,18 the measured Ki for binding was 13 nM. Compound 38
showed a very good selectivity against MCHR1 with no detectable
16. Staudinger, H.; Meyer, J. Helv. Chim. Acta 1919, 2, 635.
17. Membranes containing dopamine receptor D2 or serotonin transporter and the
corresponding radioligands were purchased from PerkinElmer. Assay protocols
and conditions provided by PerkinElmer were followed.
MCHR1 activity in the MCHR1 FLIPR assay up to 50
binding assay up to 10 M.
lM or a MCHR1
18. MCHR2 binding assays: Due to low specific binding signal of [125I]-MCH to the
membranes of MCHR2 stable cells, we developed binding assays using live cells
l
Compound 38 was evaluated in a panel of 23 receptors in MDS-
PanLabs and it showed greater than 500-fold selectivity for all
receptors tested. In particularly, the compound showed dopamine
transiently expressing MHCR2 and
compound. The compound used, 21, resulted from our lead optimization
a
[3H]-labeled MCHR2 antagonist
efforts. Briefly, HEK293 cells were transfected with
a MCHR2 expressing
plasmid.8 The transfected cells were detached with 2 ml of EDTA solution
(2 mM EDTA in PBS, pH7.4) and 8 ml of cold binding buffer (DMEM without
phenol red, 20 mM HEPES, pH7.6 and 0.2% BSA and proteinase inhibitors) was
D2 and serotonin transport binding IC50 of 1.5 and 10
a large improvement over compound 21. Compound 38 was tested
in rubidium (86Rb) efflux hERG assay.19 The IC50 was 2.0
lM. This was
lM
added to re-suspend the cells. The cells were counted and made
a
concentration of 2 Â 106/ml. 50,000 cells were used for each well of 96-well
plates in the final binding reaction. For displacement binding assay, final
concentration of [3H]-compound 21 used was 2 nM to determine Ki of the test
compounds. For saturation binding assay, different concentrations of [3H]-
compound 21 were added to determine Kd value. Non-specific binding in
indicated the compound may not have any issue against cardiac
K+ channel. The PK profile of compound 38 was evaluated in rat,
beagle dog and rhesus monkey. Compound 38 had modest to low
clearance in all animals (Table 6).
saturation binding assay was determined by using 10 lM cold compound 21.
In summary, we optimized a series of potent MCHR2 inhibitors
starting from a HTS hits. Among many potent MCHR2 inhibitors,
compound 38 showed good selectivity against MCHR1 and other
receptors in the MDS receptor screen and also demonstrated good
pharmacokinetic properties across rat, beagle dog and rhesus
The reactions were incubated at 4 °C with shaking for 2 h. The cells were then
harvested onto filter using a cell harvester and washed, and the radioactivity
counted as described previously.8
19. Cheng, C.; Alderman, D.; Kwash, J.; Dessaint, J.; Patel, R.; Kay, M.; Lescoe, M. K.;
Kinrade, M. B.; Yu, W. Informa 2002, 28, 177.