7384
H. Xiong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7381–7384
Neurol. Disord. 2002, 1, 261; (d) Marino, M. J.; Conn, P. J. Curr. Drug Targets CNS
Neurol. Disord. 2002, 1, 1; (e) Moghaddam, B. Psychopharmacology 2004, 174,
39.
intrinsic agonist activity at the mGluR5 receptor. The radio-tracer
3H-MPEP was used in a competitive binding experiment to mea-
sure the affinity of 2b for the allosteric binding site of mGluR5 in
rat cortical membranes. The Ki of 2b was determined to be
3. (a) Swanson, C. J.; Bures, M.; Johnson, M. P.; Linden, A.-M.; Monn, J. A.; Schoepp,
D. D. Nat. Rev. Drug Disc. 2005, 4, 131; (b) Sanacora, G.; Zarate, C. A.; Krystal, J.
H.; Manji, H. K. Nat. Rev. Drug Disc. 2008, 7, 426.
4. (a) Chen, Y.; Conn, P. J. Drugs Future 2008, 33, 355; (b) Rodriguez, A. L.; Williams,
R. Curr. Opin. Drug Disc. 2007, 10, 715; (c) Lindsley, C. W.; Shipe, W. D.;
Wolkenberg, S. E.; Theberge, C. R.; Williams, D. L.; Sur, C.; Kinney, G. G. Curr. Top.
Med. Chem. 2006, 6, 771; (d) Shipe, W. D.; Wolkenberg, S. E.; Williams, D. L.;
Lindsley, C. W. Curr. Opin. Drug Disc. 2005, 8, 449.
5. (a) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Nat. Rev. Drug Disc. 2009, 8, 41;
(b) Bridges, T. M.; Lindsley, C. W. ACS Chem. Biol. 2008, 3, 530; (c) May, L. T.;
Leach, K.; Sexton, P. M.; Christopoulos, A. Annu. Rev. Pharmacol. Toxicol. 2007,
47, 1; (d) Christopoulos, A. Nat. Rev. Drug Disc. 2002, 1, 198.
6. O’Brien, J. A.; Lemaire, W.; Chen, T. B.; Chang, R. S.; Jacobson, M. A.; Ha, S. N.;
Lindsley, C. W.; Schaffhauser, H. J.; Sur, C.; Pettibone, D. J.; Conn, P. J.; Wiliams,
D. L. Mol. Pharmacol. 2003, 64, 731.
7. Vanejevs, M.; Jatzke, C.; Renner, S.; Müller, S.; Hechenberger, M.; Bauer, T.;
Klochkova, A.; Pyatkin, I.; Kazyulkin, D.; Aksenova, E.; Shulepin, S.; Timonina,
O.; Haasis, A.; Gutcaits, A.; Parsons, C. G.; Kauss, V.; Weil, T. J. Med. Chem. 2008,
51, 634.
8. (a) Lindsley, C. W.; Wisnoski, D. D.; Leister, W. H.; O’Brien, J. A.; Lemiare, W.;
Williams, D. L. J.; Burno, M.; Sur, C.; Kinney, G. G.; Pettibone, D. J.; Tiller, P. R.;
Smith, S.; Duggan, M. E.; Hartman, G. D.; Conn, P. J.; Huff, J. R. J. Med. Chem.
2004, 47, 5825; (b) Kinney, G. G.; O’Brien, J. A.; Lemaire, W.; Burno, M.; Bickel,
D. J.; Clements, M. K.; Chen, T.-B.; Wisnoski, D. D.; Lindsley, C. W.; Tiller, P. R.;
Smith, S.; Jacobson, M. A.; Sur, C.; Duggan, M. E.; Pettibone, D. J.; Conn, P. J.;
Williams, D. L. J. Pharmacol. Exp. Ther. 2005, 313, 199.
9. (a) Bessis, A.-S.; Bonnet, B.; Le Poul, E.; Rocher, J.-P.; Epping-Jordan, M. P. PCT
Pat. Appl. WO 2005/044797, 2005.; (b) Epping-Jordan, M. P.; Nayak, S.; Derouet,
F.; Dominguez, H.; Bessis, A. S.; Le Poul, E.; Ludwig, B. L.; Mutel, V.; Poli, S. M.;
Rocher, J. P. Neuropharmacology 2005, 49, 231; (c) Engers, D. W.; Rodriguez, A.
L.; Williams, R.; Hammond, A. S.; Venable, D.; Oluwatola, O.; Sulikowski, G. A.;
Conn, P. J.; Lindsley, C. W. Chem. Med. Chem. 2009, 4, 505.
2.37 l
M and it completely displaced 3H-MPEP which indicated
that these compounds were competing for the same allosteric
binding site. As a comparison, CDPPB was found to be a much bet-
ter binder (Ki ꢀ0.25
lM) under our experimental conditions. PAM
2b was also screened for agonist, antagonist and modulator activ-
ities against all other mGluRs family members, and displayed
excellent selectivity, without showing any activity at concentra-
tions up to 25 lM.
Analog 2b was also profiled extensively in various in vitro and
in vivo ADME assays. It exhibited high plasma clearance in rat
(55 mL/min/kg), a moderate volume of distribution (4.8 L/kg), a
7.3 h half-life and oral bioavailability of 41%. Excellent in vivo
CNS exposure was observed with a brain/plasma ratio of 4.6. In
the dog, it exhibited high plasma clearance (38.5 mL/min/kg), a
moderate volume of distribution (3.2 L/kg), a 2.9 h half-life and
oral bioavailability of 37%.
Based on the favorable in vivo ADME profile, 2b was chosen for
study in several preclinical behavior models. It showed robust
activity in both rat conditioned avoidance response (CAR) assay
and mouse reversal of MK-801 induced locomotor activity assay,
which will be disclosed in a separate publication soon.
In this Letter, we have summarized our laboratory’s attempts to
broadly scope the key structural features of a series of mGluR5
PAMs containing either a piperazine or piperidine core. Linker
modifications have been proven to be not well tolerated. Small,
lipophilic groups such as halogen atoms at ortho- or para-positions
of LHS aryl groups are critical for good PAM activity. Exploration of
heterocyclic replacement at RHS provided several interesting leads
with balanced metabolic stability and PAM activity. In particular,
2b represents an opportunity to maintain good mGluR5 potency
10. Just before the submission, we became aware of this publication: Zhou, Y.;
Manka, J. T.; Rodriguez, A. L.; Weaver, C. D.; Days, E. L.; Vinson, P. N.; Jadhav, S.;
Hermann, E. J.; Jones, C. K.; Conn, P. J.; Lindsley, C. W.; Stauffer, S. R. ACS Med.
Chem. Lett. 2010, 1, 000.
11. All new compounds were characterized by 1H NMR and LCMS analysis.
12. Takahashif, A.; Shibasaki, M. J. Org. Chem. 1988, 53, 1227.
13. A Ca++ Flux FLIPR assay similar to literature14 was used to detect mGluR5
positive allosteric modulator (PAM) activity in an HEK293 cell line stably
expressing the d isoform of human MGluR5. PAM activity was assessed by
measuring potentiation of the EC20 response to
test compound.
L-glutamate in the presence of
(EC50 = 0.55 lM) with limited metabolic clearance (HLM 29 ll/
The day before the experiment 25,000 cells/well were plated in DMEM
containing 10% dialyzed FBS (Hyclone) in 384 well Poly- -Lysine coated plates
(Becton Dickinson). After removal of the plating medium the following day,
cells were labeled for 1 h at 37 °C in 4.3 M Fluo-4 AM (In Vitrogen) containing
min/mg). It was further characterized as a selective mGluR5 PAM
in several in vitro experiments, and was active in several preclini-
cal antipsychotic animal models.
D
l
10% Pluronic F-127 in assay buffer (HBSS(CellGro), 20 mM HEPES, 1 mM
Probenecid (Sigma) , pH 7.4). Cells were washed at rt to remove excess dye
prior to addition of test compounds serially diluted from 10 mM in 100% DMSO
into assay buffer.
Acknowledgements
The authors are grateful to James Hall for NMR support, Xia
Wang, Xincai Luo and Jenny Krumrine for computational chemistry
support, and My Linh Do for mouse locomotor assay.
Compounds were assayed for any underlying agonist activity by addition of
test compounds to the cells on the FLIPR instrument (first addition: 13
lL test
compound to 25 L assay buffer/well) and the response was recorded for
l
1 min. Eleven different concentrations were tested for each compound. After
(15 min) the 1st addition, positive modulator activity was assayed by challenge
with EC20 (200–300 nM)
final concentration 200–300 nM
L
-glutamate (second addition: 14
lL (740–1100 nM),
References and notes
L
-glutamate) and the response was recorded
for 1 min. Positive modulator activity was calculated from the fluorescence
max-min data normalized to yield responses for no modulation (EC20
1. (a) O’Hara, P. J.; Sheppard, P. O.; Thogerson, H.; Venezia, D.; Haldeman, B. A.;
McGrane, V.; Houamed, K. M.; Thomsen, C.; Gilbert, T. L.; Mulvihill, E. R. Neuron
1993, 11, 41; (b) Conn, P. J.; Pin, J.-P. Annu. Rev. Pharmacol. Toxicol. 1997, 37,
205; (c) Bockaert, J.; Pin, J.-P. EMBO J. 1999, 18, 1723; (d) Schoepp, D. D.; Jane, D.
E.; Monn, J. A. Neuropharmacology 1999, 38, 1431.
2. (a) Javitt, D. C.; Zukin, S. R. Am. J. Psychiatry 1991, 148, 1301; (b) Olney, J. W.;
Newcomer, J. W.; Farber, N. B. J. Psychiatry Res. 1999, 33, 523; (c) Chavez-
Noriega, L. E.; Schaffhauser, H. J. L.; Campbell, U. C. Curr. Drug Targets CNS
response) and full stimulation (10
l
M
L-glutamate) as 0% and 100%
modulation, respectively. Concentration–response data were fitted to the
four-parameter logistic equation to estimate compound potency (EC50) and
efficacy (Emax).
14. Ritzén, A.; Sindet, R.; Hentzer, M.; Svendsen, N.; Brodbeck, R. M.; Bundgaard, C.
Bioorg. Med. Chem. Lett. 2009, 19, 3275.