The first potent and selective inhibitors of the glycine transporter type 2 [1]

Full text of DOI:10.1021/jm0011272

© Copyright 2001 by the American Chemical Society
Volume 44, Number 17
August 16, 2001
Letters
provide skeletal muscle relaxation. A significant amount
of data has accumulated over recent years, indicating
that glycine also has an important role in the modula-
tion of nociceptive pathways.9 Thus, it was anticipated
that an increase in synaptic levels of endogenous glycine
by a selective inhibition of the GlyT-2 transporter in the
spinal cord may offer a unique approach for developing
a novel muscle relaxant, anesthetic, and/or analgesic
reagent, suitable for use during surgical anesthesia. Due
to the discrete localization of both ssGlyR and the
GlyT-2 transporter within the spinal cord and brain
stem, a glycine modulator might not be expected to lead
to serious CNS side effects that are characteristic for
currently used µ-opioid analgesics.
Since testing of this hypothesis has been hampered
by the lack of a suitable GlyT-2 inhibitor, we sought a
potent and selective inhibitor of the transporter that
would enable us to conduct proof-of-principle studies.
It is interesting to note that a tricyclic antidepressant,
amoxapine, has been recently shown to inhibit the GlyT-
2a transporter with a 10-fold selectivity over the GlyT-
1b transporter.10 It was suggested that the sedative and
psychomotor side effects associated with amoxapine
might be related to its inhibitory activity at the GlyT-2
transporter. Unfortunately, a relatively low GlyT-2
transporter activity (IC50 92 µM) and a high norepi-
nephrine transporter activity preclude use of this drug
in validation of our hypothesis.
Th e F ir st P oten t a n d Selective In h ibitor s
of th e Glycin e Tr a n sp or ter Typ e 2
Wilson L. Caulfield, Iain T. Collie, Rachel S. Dickins,
Ola Epemolu, Ross McGuire, David R. Hill,
Gillian McVey, J . Richard Morphy,
Zoran Rankovic,* and Hardy Sundaram
Lead Discovery Unit, Organon Laboratories Ltd.,
Newhouse ML1 5SH, Scotland, U.K.
Received November 30, 2000
In tr od u ction . Glycine is one of the major inhibitory
neurotransmitters in the spinal cord and brain stem of
vertebrates.1 The inhibitory actions of glycine are medi-
ated by the strychnine-sensitive glycine receptor (ssGlyR),
a ligand-gated chloride channel distributed throughout
the spinal cord and brain stem.2 Glycine is also known
to potentate the action of glutamate acting as an
essential co-agonist on postsynaptic N-methyl-D-aspar-
tate (NMDA) receptors.3 Synaptic levels of glycine are
believed to be controlled by high-affinity glycine trans-
porters. These transporters are members of a large
family of sodium/chloride-dependent transporters, which
are composed of single oligomeric proteins containing
12 hydrophobic membrane-spanning domains.4 Molec-
ular cloning has revealed the existence of two major
classes of glycine transporter subtypes: type 1 (GlyT-
1) and type 2 (GlyT-2).5 These have now been further
divided into three subtypes of GlyT-1 (a, b, and c) and
two splice variant versions of GlyT-2 (a and b).5 Recent
immunocytochemical studies showed that the GlyT-1
transporter has a wide distribution throughout the CNS
whereas the GlyT-2 transporter has a similar distribu-
tion to ssGlyR, being confined to the spinal cord and
brain stem.6
Screening of Organon’s compound collection, based on
measuring the ability of test compounds to inhibit [3H]-
glycine uptake into CHO cells stably expressing the
human GlyT-2 protein (hGlyT-2 assay),11 led to identi-
fication of 4-butyloxy-3,5-dimethoxy-N-[(1-dimethylami-
nocyclohexyl)methyl]benzamide (1), a compound with
an IC50 value of 214 nM at the hGlyT-2 and high
selectivity over the hGlyT-1 transporter12 (Table 1). The
There is evidence that glycine-mediated inhibition
produces muscle relaxation7 and blockade of this inhibi-
tion produces convulsions.8 Therefore, we postulated
that modulators of endogenous levels of glycine might
* To whom correspondence should be addressed. Tel: (+44) 1698
736157. Fax: (+44) 1698 736187. E-mail: z.rankovic@organon.nhe.
akzonobel.nl.
10.1021/jm0011272 CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/25/2001

2680 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Ch a r t 1. Building Blocks Used in Synthesis of the Librarya
Letters
a
Pn ) pentyl; cPn ) cyclopentyl.
IC50 value for glycine itself in this assay is approxi-
mately 100 µM.11
generated in situ by treating a suspension of LiAlH4 in
THF with H2SO4 at 0 °C.15 Attempts to reduce cy-
anoamine 2 with either LiAlH4 or BH3‚THF produced
complex mixtures containing only traces of the desired
product, mainly due to a retro-Strecker reaction induced
by these reagents. Acylation of diamine 3 with acid
chloride 5, obtained in three simple steps from a
commercially available ester 4, afforded 1 in 82% yield
(Scheme 1).
The compound’s physicochemical parameters ap-
peared to be consistent with CNS penetration:13 cLogP
3.93 (free base); MW 429.0; polar surface area14 41.4;
aqueous solubility 10 mg/mL. Thus, compound 1 was
selected as a starting point for a “hit to lead” optimiza-
tion program. In this communication we describe our
efforts to further improve activity while maintaining the
favorable selectivity profile and physicochemical proper-
ties of this hit.
To perform a rapid SAR exploration and optimization
program around 1, 16 diamines and 18 acid chlorides
were coupled in a matrix fashion to produce a 2D library
containing 288 amide analogues of compound 1 (Chart
1). In total 15 out of 34 building blocks were custom-
made following the synthetic routes depicted in the
Scheme 1. The presence of an internal tertiary amine
group conveniently eliminated the need for an external
base and, therefore, facilitated the reaction workup. The
synthesis of the library was performed in four 96-well
plates. After leaving the plates to stand overnight at
room temperature, the solvent was removed under
reduced pressure (GeneVac) to afford the desired prod-
ucts in quantities of 10-15 mg and of a purity sufficient
for testing in the hGlyT-2 assay (>80% by LC-MS). The
parent compound was resynthesized as an internal
standard within the library and was found to exhibit
similar activity (IC50 218 nM) as compared to 214 nM
for 1.
Sch em e 1a
a
(a) Me2H2N+Cl-, KCN(aq), 18 h; (b) H2SO4, LiAlH4, THF, 0-20
°C, 18 h; (c) BuBr, K2CO3, acetophenone, 135 °C, 18 h; (d) KOH,
MeOH, 65 °C; (e) SOCl2, toluene, 110 °C; (f) Et3N, THF.
Ch em istr y. Compound 1 was prepared following the
procedure outlined in Scheme 1.15 A standard Strecker
reaction, involving cyclohexanone and dimethylamine
hydrochloride, produced cyanoamine 2. This was fol-
lowed by a reduction of 2 with AlH3, to obtain the
corresponding diamine 3 in 57% overall yield. AlH3 was
Resu lts a n d Discu ssion . Initially, several closely
related analogues of 1 were selected from both internal
and external sources for testing in the hGlyT-2 assay.
This preliminary data indicated that, compared to the

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2681
Ta ble 1. In Vitro Activity of the Key Analogues
compd
R
R1
R2
R3
R4
R5
hGlyT-211 IC50 ( SEM (nM)a
hGlyT-112 IC50 (µM)b
1
6
7
8
9
10
11
12
13
14
15
16
Me2N
Me2N
Me2N
MeEtN
piperidine
Me2N
Me2N
Me2N
Me2N
Me2N
Me2N
Me2N
-CH2-(CH2)3-CH2-
-CH2-(CH2)3-CH2-
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OBu
OMe
OBu
OBu
OBu
OBu
OBu
OPr
OBn
OBu
OBu
OBn
OMe
OMe
OMe
OMe
OMe
H
214 ( 35.2
>10000
>100
ND
ND
ND
ND
ND
ND
ND
>100
ND
>100
>100
Ph
H
566 ( 178.5
1321 ( 507.0
1563 ( 637.5
2563 ( 170.5
2816 ( 297.2
2313 ( 397.5
84 ( 3.0
696 ( 205.1
77 ( 19.0
16 ( 1.9
-CH2-(CH2)3-CH2-
-CH2-(CH2)3-CH2-
-CH2-(CH2)3-CH2-
-CH2-(CH2)3-CH2-
-CH2-(CH2)3-CH2-
-CH2-(CH2)3-CH2-
H
OMe
OMe
OMe
OMe
OMe
Me
Me
-CH2-(CH2)2-CH2-
-CH2-(CH2)2-CH2-
a
Average of 3 determinations. b1 measurement.
compound’s aromatic region, the SAR around the
cyclohexyl group of 1 appears to be more flexible. For
example, replacement of the BuO by a MeO group in 6
abolished activity (IC50 >10 µM), whereas replacement
of the cyclohexyl by a phenyl group in 7 reduced activity
by 3-fold (IC50 566 nM), as shown in Table 1. This
preliminary SAR information was incorporated into the
design of the 2D library aimed not only to produce more
detailed SAR around 1 but also for purpose of hit
optimization and hit explosion. The library included a
range of “deletion” analogues (e.g. D7 and A13) provid-
ing information on the minimum structural require-
ments for activity. Furthermore, since the importance
of the BuO group has already been shown, a number of
analogues with various replacements at this position,
(e.g. A2 and A11) are also included (hit optimization).
Analogues designed to explore the bulk tolerance around
the basic nitrogen as well as more diverse derivatives,
containing the diamine moiety with varying degrees of
steric constraint and various substitutions at the acid
part of the molecule (hit explosion), were also included
within the library (Chart 1).
The percent inhibition for each of the 288 amides in
the hGlyT-2 assay was determined at 1 µM.16 A total of
39 compounds (13% of the collection) showed >60%
inhibition. These active compounds were derived pre-
dominately from 5 diamines (D1-D3, D10, and D13)
and 10 acid chlorides (A2, A3, A5, A7, A8, A10, A11,
and A13-A15). All “active” diamines contain at least
one substituent at the carbon adjacent (R) to the tertiary
amine functionality. Lack of R-substitution appeared to
be detrimental for activity in the GlyT-2 assay (e.g. D7).
Similarly, presence of â-substitution (e.g. D14) or a
secondary amino group (e.g. D12) abolished GlyT2
activity. Products derived from acid chlorides with a
para-alkyl or alkoxy group shorter than PrO (e.g. A16
and A18) showed no activity in the assay, indicating
the importance of hydrophobic interactions between this
region of the ligand and the transporter binding site.
IC50 values for the key analogues after purification
(prep-LC-MS) are presented in Table 1. The obtained
data indicated a very tight SAR around the Me2N group.
Even a slight increase in the size of substituents at the
basic nitrogen in 8 and 9 resulted in a significant
decrease of activity: IC50 1321 and 1563 nM, respec-
tively. The substitution pattern on the aromatic ring
also appears to be important for activity. Deletion of one
of the two MeO groups at the aromatic ring in 10
resulted in a significant reduction of the binding affinity
(2563 nM), whereas only slight further reduction of
activity followed removal of the remaining MeO group
in compound 11 (2816 nM). This is probably due to both
MeO groups being required to suppress free rotation of
Ar-O-Bu bonds and therefore “freeze” the BuO group
in a bioactive conformation. The BuO group itself is
important for affinity perhaps due to hydrophobic
interactions with the transporter binding site. Replace-
ment of the BuO group by a PrO in 12 resulted in a
significant reduction of activity (2313 nM). Interestingly,
analogue 13 with the BuO replaced by a BnO group
showed 3-fold increase of activity (83 nM) which could
be attributed to the greater contact surface between the
BnO group and the hydrophobic pocket within the
transporter binding site. As the preliminary SAR indi-
cated, the region around the cyclohexyl group appears
to be more flexible to changes. Replacement of the
cyclohexyl either by a phenyl (7) or a gem-dimethyl
group (14) led to a ∼3-fold drop of activity (566 and 696
nM, respectively), whereas a cyclopentyl analogue 15
showed a 3-fold increase of activity (77 nM). Effects of
described structural changes on the GlyT-2 activity
appeared to be additive, so that analogue 16, containing
both cyclopentyl and BnO groups, was identified as the
most active compound in the library (IC50 16 nM).17 This
compound appeared to be highly selective not only over
the GlyT-1, where it showed no activity at concentra-
tions of up to 100 µM, but also over 56 other common
and relevant biological targets.18 As indicated by its
favorable physicochemical parameters (cLogP 3.97 (free
base); MW 429.0; polar surface area14 52.7; aqueous
solubility 10 mg/mL) compound 16 exhibits good blood-
brain barrier penetration (logBB 0.6).19 Since 16 also
shows good metabolic stability in plasma and mouse
hepatic microsomes (80% remaining after 30 min), the
compound should prove to be a valuable tool that might
help to define pharmacology of the GlyT-2 transporter.
Con clu sion . In summary, high-throughput screening
of Organon’s compound collection provided compound
1, an attractive drug-like GlyT-2 inhibitor suitable for
high-throughput synthesis. A detailed study of the SAR

2682 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Letters
transporters are differentially expressed among CNS cells. J .
Neurosci. 1995, 15, 3952-3969.
and rapid hit optimization were achieved through
synthesis of a solution-phase 2D library. This led to
identification of 4-benzyloxy-3,5-dimethoxy-N-[(1-di-
methylaminocyclopentyl)methyl]benzamide (16), the first
potent and selective GlyT-2 inhibitor. Further in vitro
and in vivo evaluations of 16 and related analogues will
be reported in due course.
(7) (a) Simpson, R. K.; Robertson, C. S.; Goodman, J . C. The role of
glycine in pain and spasticity. In Spinal Cord Monitoring, 2nd
ed.; Stalberg, E., Sharma, H. S., Ollson, Y., Eds.; Springer: New
York, 1998; pp 75-106. (b) Truong, D. D.; DeYebenes, J . G.;
Pezzoli, G.; J ackson-Lewis, V.; Fahn, S. Glycine involvement in
DDT induced myoclonus. Movement Dis. 1988, 3, 77-87.
(8) Becker, C. M. Convulsants acting at the Inhibitory Glycine
Receptor. Handbk. Exp. Pharmacol. 1992, 102, 539-575.
(9) (a) Simpson, R. K. J .; Gondo, M.; Robertson, C. S.; Goodman, J .
C. Reduction in the mechanociceptive response by intrathecal
administration of glycine and related compounds. Neurochem.
Res. 1996, 21, 1221-1226. (b) Sorkin, L. S.; Westlund, K. N.;
Sluka, K. A.; Dougherty, P. M.; Willis, W. D. Neural changes in
acute arthritis in monkeys. IV. Time-course of amino acid release
into the lumbar dorsal horn. Brain Res. Rev. 1992, 17, 39-50.
(10) Nunez, E.; Lopez-Corcuera, B.; Vazquez, J .; Gimenez, C.; Aragon,
C. Differential effects of the tricyclic antidepressant amoxapine
on glycine uptake mediated by the recombinant GlyT-1 and
GlyT2 glycine transporters. Br. J . Pharmacol. 2000, 129, 200-
206.
Su p p or tin g In for m a tion Ava ila ble: Detailed experi-
mental procedures for the synthesis of compound 1 and the
amide library, analytical data for all compounds presented in
Table 1, and percent inhibition in the hGlyT-2 assay for each
of the 288 compounds of the amide library. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Werman, R.; Davidoff, R. A.; Aprison, M. H. Evidence for glycine
as the principle transmitter mediating postsynaptic inhybition
in the spinal cord of the cat. J . Gen. Physiol. 1967, 50, 1093-
1094.
(11) Morrow, J . A.; Collie, I. T.; Dunbar, D. R.; Walker, G. B.; Shahid,
M.; Hill D. R. Molecular Cloning and Functional Expression of
the Human Glycine Transporter GlyT-2 and Chromosomal
Localisation of the gene in the Human Genome. FEBS Lett.
1998, 439, 334-340.
(12) Glycine uptake assays were performed as described by Morrow
et al.11 using CHO cells stable transfected with hGlyT-1b. A full
protocol is described in the Supporting Information.
(13) Lipinski, C. A. Experimental and computational approaches to
estimated solubility and permeability in drug discovery and
development settings. Adv. Drug Deliv. Rev. 1997, 23, 3.
(14) Camenisch, G.; van de Waterbeemd, H. Estimation of Caco-2
cell permeability using calculated molecular descriptors. Quant.
Struct.-Act. Relat. 1996, 15, 480-490.
(15) Yang, D.; Bremont, B.; Shen, S.; Kefi, S.; Langlois, M. Sero-
toninergic properties of new conformationally restricted benz-
amides. Eur. J . Med. Chem. 1996, 31, 231-239.
(16) The percent inhibition for each of the 288 amides in the hGlyT2
assay is presented in Table 3 of the Supporting Information.
(17) Compounds with IC50 <100 nM were also tested for their
selectivity in the GlyT-1 assay and were found to be inactive at
10 µM concentration.
(18) Compound 16 was screened across 56 selected NovaScreen
targets at three concentrations: 0.1, 1, 10 µM. Limited affinity
was observed only in serotonin 5-HT2A binding assay (25%
inhibition at 100 nM). For more details, see Table 4 of the
Supporting Information.
(2) Langosch, D. M.; Becker, C.; Betz, H. The inhibitory glycine
receptor: a ligand gated chloride channel for the central nervous
system. Eur. J . Biochem. 1990, 194, 1-8.
(3) J ohnson, J . W.; Ascher, P. Glycine potentiates the NMDA
response in cultured mouse brain neurones. Nature 1987, 325,
529-531.
(4) Masson, J .; Sagne, C.; Hamon, M.; El Mestikawy, S. Neurotrans-
mitter transporters in the central nervous system. Pharmacol.
Rev. 1999, 51, 439-464.
(5) (a) Guastella, J .; Brecha, N.; Weigman, C.; Lester, H.; Davidson
N. Cloning, expression, and localization of a rat brain high
affinity glycine transporter. Proc. Natl. Acad. Sci. U.S.A. 1992,
89, 7189-7193. (b) Liu, Q.-R.; Nelson, H.; Mandiyan, S.; Lopez-
Corcuera, B.; Nelson, N. Cloning and expression of a glycine
transporter from mouse brain. FEBS Lett. 1992, 305, 110-114.
(c) Smith, K. E.; Borden, L. A.; Hartig, P. R.; Branchek, T.;
Weinshank, R. L. Cloning and expression of a glycine transporter
reveal colocalisation with NMDA receptors. Neuron 1992, 8,
927-937. (d) Ponce, J .; Poyatos, I.; Aragon, C.; Gimenez, C.;
Zafra, F. Characterisation of the 5′ region of the rat brain glycine
transporter GLYT2 gene: Identification of a novel isoform.
Neurosci. Lett. 1998 242, 25-28.
(6) (a) Zafra, F.; Aragon, C.; Gimenez, C. Molecular Biology of
Glycinergic Neurotransmission. Mol. Neurobiol. 1997, 14, 117-
142. (b) J ursky, F.; Nelson, N. Localisation of GlyT-2 reveals
correlation with the distribution of glycine receptor. J . Neuro-
chem. 1995, 64, 1026-1033. (c) Zafra, F.; Aragon, C.; Olivares,
L.; Danbolt, N. C.; Gimenez, C.; Storm-Mathisen, J . Glycine
(19) logBB ) log(concentration in brain/concentration in blood).
J M0011272