M. W. Walter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5233–5238
5237
11. Alberati, D.; Hainzl, D.; Jolidon, S.; Krafft, E. A.; Kurt,
A.; Maier, A.; Pinard, E.; Thomas, A. W.; Zimmerli, D.
Bioorg. Med. Chem. Lett. 2006, 16, 4311.
12. Alberati, D.; Ceccarelli, S. M.; Jolidon, S.; Krafft, E. A.;
Kurt, A.; Maier, A.; Pinard, E.; Stalder, H.; Studer, D.;
Thomas, A. W.; Zimmerli, D. Bioorg. Med. Chem. Lett.
2006, 16, 4305.
ration of alkoxy and alkyl substituents into five or six-
membered aliphatic rings as in 45–47 and 49–51 was
found to be particularly beneficial for in vitro activity.
In order to assess the ability of our compounds to selec-
tively increase cortical glycine levels in an animal model,
we measured their effect on glycine levels in rat cerebro-
spinal fluid (CSF) after subcutaneous administration
(Table 5) at a dose of 30 mg/kg.22 Animals were eutha-
nized and 50–100 ml of CSF was sampled immediately
after death from the cisterna magna.23–25 The concentra-
tion of glycine and test compounds in the CSF was
determined using an LC/MS method in positive electro-
spray mode (Table 5).26
13. Ceccarelli, S. M.; Pinard, E.; Stalder, H.; Alberati, D.
Bioorg. Med. Chem. Lett. 2006, 16, 354.
14. Pinard, E., Alberati, D., Borroni, E., Ceccarelli, S.,
Fischer, H., Hainzl, D., Jolidon, S., Moreau, J. L.,
Stalder, H., Thomas, A. W. Abstracts of Papers, 231st
ACS National Meeting, Atlanta, GA, United States,
March 26–30, 2006; MEDI-407.
15. Dargazanli, G. et al. WO2838739.
16. Man, T. et al. WO2005100301.
17. Tsunoda, T.; Yamamiya, Y.; Ito, S. Tetrahedron Lett.
1993, 34, 1639.
18. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
19. The reactions were performed using Radley’s Greenhouse
Parallel Synthesizer.
Our initial hit molecule 5 showed no significant effect on
CSF glycine levels. 2-Chloro-phenyl substituted ana-
logue 32 had a weak effect on CSF glycine levels, but
2-thiophene substituted 30 proved to have a similar
effect on glycine levels as ALX-5407 1. Corresponding
to the increase in glycine levels compound levels of 30
measured in CSF were also significantly higher than
those measured for 5, 32, and 1.
20. Huang, Y.; Mahmood, K.; Mathis, C. A. J. Labelled
Compd. Radiopharm. 1999, 42, 949.
21. Compound IC50 values were determined using a whole
cell transport assay formatted as a solid-scintillant-
based assay using the Amersham 96-well Cytostar-T
plate technology. Sixteen to 20 h prior to initiation of
the assay, human medulloblastoma cells, BE(2)-C,
stably expressing a human GlyT1a cDNA were tryp-
sinized, counted, and plated at a cell density of 60,000
cells/well in culture medium (1:1 mixture of Eagle’s
minimum essential medium with non-essential amino
acids and Ham’s F12 medium, 90% and fetal bovine
serum, 10%). After washing the cell layer with assay
buffer (25 mM Hepes, 125 mM NaCl, 4.8 mM KCl,
1.2 mM KH2PO4, 1.2 mM MgSO4, 5.6 mM, glucose,
and 4 mM alanine), assay buffer and test compounds at
various concentrations (0.1 nM–30 lM) were added to
the cell layer. The uptake assay was initiated by
addition of 14C-glycine or 14C-sarcosine (7.5 lM final)
and allowed to proceed at 25 ꢁC for 4–5 h prior to
counting on a Wallac MicroBeta liquid scintillation
spectrometer. Non-specific uptake was defined using
10 mM sarcosine (for 14C-glycine) or 10 mM glycine
(for 14C-sarcosine). Each IC50 was obtained from at
least two-independent experiments. The assay had a
MSR of 2.5.
22. Compounds were dissolved in a 14% (2-hydroxypropyl)-
b-cyclodextrin vehicle solution and administered to male
Sprague–Dawley weighing 250–300 g in groups of 6–9
rats with an injection volume of 0.1 ml/100 g. CSF was
sampled 1–24 h after compound administration. Animals
were euthanized by CO2 asphyxiation and, very shortly
after death, a 27-gauge hypodermic needle was carefully
inserted approximately 1 mm into the rat cisterna magna
in order to withdraw 50–100 ll of CSF in a 1 ml
syringe. Samples with any sign of blood contamination
were discarded. CSF samples were immediately placed
on ice. Analysis of CSF levels of test compounds was
accomplished using an LC/MS in positive electrospray
mode and ChemStation data analysis software (1100
Series, Agilent Technologies, Palo Alto, CA, USA).
Three microliter injections of CSF samples were made
Major SAR trends in a new class of GlyT1 inhibitors
have been established. Optimization of in vitro potency
led to the discovery of highly potent and selective GlyT1
inhibitors. The in vivo activity of selected members of
this series was demonstrated by measurements of glycine
levels in CSF. Further optimization of our lead mole-
cules and activity after oral dosing will be disclosed in
due course.
Acknowledgment
´
The authors thank Veronique Dehlinger for helpful dis-
cussions and suggestions.
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onto
150 mm, Agilent Technologies) that was maintained at
mixture of
a
5
micron Zorbax C-18 column (4.6 mm ·
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30 ꢁC. The mobile phase used was
a
acetonitrile:water: 0.1% formic acid at a flow rate of
0.5 ml/min. The mixture of acetonitrile:water was varied
in order to have the analyte retention time stay within a