Acylglycinamides as inhibitors of glycine transporter type 1

Article abstract of DOI:10.1016/j.bmcl.2011.07.096

A screening hit was used as the basis for the core structure of a new series of acylglycinamide GlyT-1 inhibitors. Investigation of the SAR around four areas of diversity used facile chemistry to prepare compounds quickly. By focussing on reducing the lipophilicity and improving the aqueous solubility in the series we were able to prepare a compound (17e) with a good level of activity at GlyT-1, selectivity over GlyT-2 and moderate oral bioavailability.

Full text of DOI:10.1016/j.bmcl.2011.07.096

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6176–6179
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acylglycinamides as inhibitors of glycine transporter type 1
⇑
,
Richard Blunt a, , Roderick Porter a, Amanda Johns a, David Nash a, Gemma Puckey a, Paul Wyman a,
Hugh Herdon a, Simon Teague a, Victoria Hadden a, Stefano Fontana b, Laurie Gordon c
a Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Harlow, UK
b Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, Medicines Research Centre, Verona, Italy
c Molecular Discovery Research, GlaxoSmithKline, New Frontiers Science Park, Harlow, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A screening hit was used as the basis for the core structure of a new series of acylglycinamide GlyT-1
inhibitors. Investigation of the SAR around four areas of diversity used facile chemistry to prepare com-
pounds quickly. By focussing on reducing the lipophilicity and improving the aqueous solubility in the
series we were able to prepare a compound (17e) with a good level of activity at GlyT-1, selectivity over
GlyT-2 and moderate oral bioavailability.
Received 13 June 2011
Revised 25 July 2011
Accepted 26 July 2011
Available online 6 August 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Glycine transporter type 1
GlyT-1
Schizophrenia
Acylglycinamides
SAR
Molecular cloning has revealed the existence in mammalian
brains of two classes of glycine transporters, termed GlyT-1 and
GlyT-2. GlyT-1 is widely distributed throughput the CNS and is ex-
pressed on both neurones and glia.1 Some expression of GlyT-1 is
closely associated with N-methyl
which possess strychnine-insensitive glycine binding sites. GlyT-2,
in contrast, is found predominantly in the brain stem and spinal
cord, and its distribution corresponds closely to that of strych-
nine-sensitive inhibitory glycine receptors.1 Another distinguish-
ing feature of glycine transport mediated by GlyT-2 is that it is
not inhibited by sarcosine, as is the case for glycine transport
mediated by GlyT-1. It is thought that GlyT-1 has an overall role
D
-aspartate (NMDA) receptors,2
O
F3C
F
O
SO2Me
N
N
OH
N
N
N
HO
O
O
O
CF3
HN
S
O
2
1
F
O
Cl
N
N
3
F
N
O
H
N
N
H
O
.2HCl
NH2
F
5
4
Figure 1. GlyT1 inhibitors.
⇑
Corresponding author.
E-mail address: richard.blunt@xention.com (R. Blunt).
Current address: Xention Ltd, Iconix Park, London Road, Pampisford, Cambridge CB22 3EG, UK.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.096

R. Blunt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6176–6179
6177
Table 2
SAR for amide replacements
O
H
N
12
14
N
Compound
GlyT-1 pIC50
GlyT-2 pIC50
N
O
8a
8b
8c
4.8
<4.6
5.6
<4.6
<4.6
4.9
O
Cl
6
The sarcosine derivative Org259357 (compound 1) is in Phase 2
clinical trials, and R16788 (2) recently reported a positive outcome
in a Phase 2 study on negative symptoms of schizophrenia. We
have previously reported a non-sarcosine series of diaminopropa-
nols (e.g., 39), and more recently compounds such as 410 and 511
have been reported (Fig. 1).
Figure 2. Example imidazolone.
H
N
H
N
N
N
N
Screening of the GSK compound collection12 gave a number of
hits containing an imidazolone moiety, for example, compound 6
(Fig. 2).
O
O
O
O
As part of the lead optimisation process we identified a set of
ring-opened analogues—the acylglycinamides—as a possible alter-
native series (Fig. 3). This series has structural similarities to
known bis-amides (e.g., 4) but is differentiated by having a fully-
substituted amide nitrogen. The facile chemistry required to con-
struct the molecules allowed a large number of compounds to be
prepared quickly and thus extensive structure–activity relation-
ship (SAR) data could be obtained rapidly. In addition, it was antic-
ipated that SAR data from this series might be used to inform
further work in the imidazolone series, where more difficult chem-
istry slowed the preparation of large numbers of compounds.
The first investigations in this series concerned the acylglycina-
mide core. We treated a set of analogous amines with 4-chloroben-
zoyl chloride13 to give compounds 7a–d (Fig. 4) and the results are
summarised in Table 1. The simple cyclohexyl-glycinamide (com-
pound 7a) appeared to be the most potent core with a pIC50 of 6.2.
Next we investigated the effect of replacing one of the amides in
the acylglycinamide, by treating the amine precursor of 7a with
4-chlorobenzenesulfonyl chloride, 4-chlorobenzyl chloride and
4-chlorophenyl isocyanate to give sulfonamide 8a, tertiary amine
8b and urea 8c, respectively (Fig. 5). All showed reduced activity
compared to the diamide 6b (Table 2).
Figure 3. ‘Ring-opened imidazolones’.
H
H
N
F
N
F
N
N
O
O
O
O
F
F
Cl
Cl
7b
7a
F
H
O
F
N
N
F
N
H
N
O
O
O
F
Cl
Cl
7c
7d
Figure 4. Acyglycinamide core variations.
Table 1
SAR for acylglycinamide variations
12
14
Compound
GlyT-1 pIC50
GlyT-2 pIC50
Having established the optimum core structure we investigated
the effect of replacing the 4-chlorophenyl moiety in compound 7a.
The synthetic route is shown in Scheme 1. By putting the diversity
step at the end we were able to prepare a large number of com-
pounds in parallel. R-groups were chosen, in part, for their poten-
tial to increase the polarity of the final compounds, as it was
anticipated that this would improve the aqueous solubility.
Forty compounds were prepared in the first iteration and se-
lected results are summarised in Table 3. From these results sev-
eral conclusions were drawn: selectivity for GlyT-1 over GlyT-2
is highly substituent-dependent (compounds 7a and 12a); aro-
matic rings are preferred over aliphatic (compounds 12a, 12b,
12c and 12d); small polar aromatic rings, though increasing solu-
bility, greatly reduce the activity (compounds 12e and 12f); para
substitution on phenyl rings is preferred over meta and ortho
7a
7b
7c
7d
6.2
5.4
5.0
5.5
5.0
<4.6
<4.6
<4.6
in regulating glycine levels at both NMDA receptors and inhibitory
glycine receptors,3,4 whereas GlyT-2 has a more specific role in
influencing glycine in glycinergic neurones.1
NMDA receptors are critically involved in memory and learning
and, furthermore, decreased function of NMDA-mediated neuro-
transmission appears to underlie, or contribute to, the symptoms
of schizophrenia.5 Thus, agents that inhibit GlyT-1 and thereby in-
crease glycine activation of NMDA receptors may be useful as novel
anti-psychotics and anti-dementia agents.6
H
N
H
H
N
F
F
N
F
N
S
O
N
N
O
O
O
O
O
NH
Cl
F
F
F
Cl
Cl
8a
8b
8c
Figure 5. Amide replacements.

6178
R. Blunt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6176–6179
H
H
F
N
F
NH
2
F
N
N
H
Br
a
b
O
O
F
F
F
11
9
10
H
N
F
N
c
O
O
R
F
12a-l
Scheme 1. Reagents and conditions: (a) Bromoacetyl bromide, NaHCO3, DCM, 2 h, yield 88%; (b) cyclohexylamine, Et3N, DCM, 4 h, yield 70%; (c) RCOCl, Et3N, DMAP, THF,
100 °C, 10 min, microwave, yields 62–72%; or RCO2H, HATU, Et3N, MeCN, 100 °C, 10 min, microwave, yields 25–61%.
Table 3
Table 4
SAR for substitution of the amide moiety
SAR of anilide replacements
Compound R2
GlyT-1 GlyT-2 CHI
Soln16
g mLÀ1
)
H
log D15
(
l
12
14
pIC50
pIC50
F
N
N
N
O
R
O
NH
16a
<4.6
<4.6
3.1
42
F
*
Compound
R
GlyT-1 GlyT-2 CHI
Soln16
O
N
log D15
(
l
g mLÀ1
)
12
14
pIC50
pIC50
<4.6
<4.6
16b
16c
16d
4.8
5.1
4.7
<4.6
4.8
4.1
4.6
2.9
6
2
*
O
12a
12b
6.3
3.7
7
*
S
N
<4.6
1.8
140
127
N.D.
160
*
N
*
H
N
12c
12d
12e
O
5.3
5.2
<4.6
<4.7
<4.6
3.0
*
<5.2
20
N
N
*
*
N.D.
2.7
*
*
N
N
16e
16f
16g
5.6
5.8
6.4
<4.6
<4.6
<4.6
3.5
5.0
4.2
28
<1
2
<4.6
N
*
N
Cl
12f
<4.6
5.8
<4.6
4.8
3.0
163
N
O
*
O
N
*
12g
N.D.
N.D.
*
Cl
*
12h
4.7
4.7
N.D.
N.D.
(compounds 7a, 12g and 12h); solubilising amine chain extensions
are not tolerated (compound 12i); 2-ring substituents (compounds
12j and 12m) offer the best compromise between Gly-T1 potency,
selectivity and lipophilicity.17
Cl
N
O
12i
12j
4.7
6.2
<4.6
<4.6
2.4
2.7
199
32
*
N
N
*
Compounds 12j and 12m were profiled further. They both
N
showed
high
microsomal
(rat);
intrinsic
clearance
(human)
(CLi):18
and
12k
12l
6.8
7.1
7.2
5.1
5.3
4.7
3.6
4.5
3.7
14
5
N
N
*
1.2 mL minÀ1 gÀ1
6.1 mL minÀ1 gÀ1
15.4 mL minÀ1 gÀ1 (rat) and 5.2 mL minÀ1 gÀ1 (human), respec-
tively. The solubilities in simulated gastric fluid (SGF; pH 1.2) were
*
N
also measured, giving encouraging results of >1000
l
g mLÀ1 and
12m
6
*
507
l
g mLÀ1, respectively.
In an attempt to reduce flexibility of the molecules we also
investigated replacing the anilide moiety with heterocycles. The
process used for the syntheses is described in Scheme 2. Commer-
cially-available heterocyclic aldehydes were subjected to a reduc-
tive amination step which was amenable to a parallel work-up
procedure using SCX (solid-supported sulfonic acid) cartridges
and the products required no further purification. Purification after
step b by mass-directed HPLC gave the final compounds in >95%
purity (by NMR). The results are shown in Table 4. 4-Methoxy-
phenyl was chosen as the amide substituent as it offered better
GlyT-1 selectivity than 4-chlorophenyl (compounds 7a and 12a).
From this array the 3,5-disubstituted isoxazole 16g was identi-
fied as having moderate potency at GlyT-1 but the lipophilicity was
O
a
b
R2
R2
R2
N
N
H
H
O
O
15a-g
14a-g
13a-g
Scheme 2. Reagents and conditions: (a) Cyclohexylamine, sodium triacetoxyboro-
hydride, DCM, 16 h, yields 70–100%; (b) 4-methoxybenzoyl chloride, Et3N, DMAP,
THF, 100 °C, 10 min, yields 52–75%.

R. Blunt et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6176–6179
6179
Table 5
GlyT-1 potency and selectivity. Further lead optimisation led to the
identification of compound 17e with improved lipophilic effi-
ciency, in vitro developability and good oral absorption, but it suf-
fered from high clearance in vivo. No further studies were
performed to identify potential metabolites or reduce clearance
in this series. However, four areas of diversity were explored, giv-
ing data which could be used to direct the work in the imidazolone
series. This will be reported in a future communication.
SAR of cyclohexyl replacements
H
R3
N
F
N
O
O
N
F
Compound R3
GlyT-1
pIC50
CHI
Soln16
g mLÀ1
CLi (rat/human)
log D15
(
l
)
(mL minÀ1 gÀ1
)
12
Acknowledgements
*
17a
7.0
3.3
12
4.9/1.3
We thank David Brown, Abir Khazragi, Kelly Locke, Meenal
Padhiar and Katya Helmich for providing screening data. We thank
Mark Healy for help in preparing the manuscript.
*
17b
6.7
7.2
3.1
3.4
25
14
5.0/<0.5
3.7/1.9
*
17c
*
References and notes
17d
5.9
3.0
42
1.8/<0.5
1. Eulenburg, V.; Armsen, W.; Betz, H.; Gomeza, J. Trends Biochem. Sci. 2005, 30,
325.
2. Cubelos, B.; Giménez, C.; Zafra, F. Cereb. Cortex 2005, 15, 448.
3. Sur, C.; Kinney, G. G. Curr. Drug Targets 2007, 8, 643.
4. Perry, K. W.; Falcone, J. F.; Fell, M. J.; Ryder, J. W.; Yu, H.; Love, P. L.; Katner, J.;
Gordon, K. D.; Wade, M. R.; Man, T.; Nomikos, G. G.; Phebus, L. A.; Cauvin, A. J.;
Johnson, K. W.; Jones, C. K.; Hoffmann, B. J.; Sandusky, G. E.; Walter, M. W.;
Porter, W. J.; Yang, L.; Merchant, K. M.; Shannon, H. E.; Svensson, K. A.
Neuropharmacology 2005, 55, 743.
5. Lisman, J. E.; Coyle, J. T.; Green, R. W.; Javitt, D. C.; Benes, F. B.; Heckers, S.;
Grace, A. A. Trends Neurosci. 2007, 31, 234.
6. Javitt, D. C. Curr. Opin. Drug Discov. Devel. 2009, 12, 468.
7. Gibson, S. G.; Jaap, D. R.; Thorn, S. N.; Gilfillan, R. WO 2000/07978.
8. (a) Jolidon, S.; Narquizian, R.; Nettekoven, M.; Norcross, R.; Pinard, E.; Stalder,
H. WO 2005/014563.; (b) NCT00616798.
O
17e
7.1
6.0
2.7
2.5
56
99
2.0/2.9
*
O
17f
17g
17h
17i
0.6/<0.5
*
*
6.4
6.9
5.9
3.3
3.5
3.0
29
7
3.8/4.3
4.8/2.0
4.2/5.8
*
*
58
O
O
*
*
17j
6.0
5.9
N.D.
3.3
N.D.
24
3.0/1.5
6.7/9.6
9. Rahman, S. S.; Coulton, S.; Herdon, H. J.; Joiner, G. F.; Jin, J.; Porter, R. A. Bioorg.
Med. Chem. Lett. 2007, 17, 1741.
*
17k
10. Jolidon, S.; Alberati, D.; Dowle, A.; Fischer, H.; Hainzl, D.; Narquizian, R.;
Norcross, R.; Pinard, E. Bioorg. Med. Chem. Lett. 2008, 18, 5533.
11. Lowe, J. A.; Hou, X.; Schmidt, C.; Tingley, F. D.; McHardy, S.; Kalman, M.;
DeNinno, S.; Sanner, M.; Ward, K.; Lebel, L.; Tunucci, D.; Valentine, J. Bioorg.
Med. Chem. Lett. 2009, 19, 2974.
O
O
17l
5.9
2.5
102
3.2/3.9
12. GlyT1 screening data was obtained using a [3H]-glycine uptake scintillation
proximity binding assay containing HEK293 cells expressing GlyT-1. Values are
still high and the compound had poor CLi (rat: 21.4 mL minÀ1 gÀ1
and human: 4.2 mL minÀ1 gÀ1).
expressed as pIC50 = Àlog IC50
, where IC50 is the half-maximal inhibitory
concentration of the substance, and are the means of at least two experiments.
Values given as ‘less than’ were below the sensitivity of the assay.
13. Work on the imidazolones had suggested that the 4-chlorophenyl and 3,5-
difluoroanilide fragments were likely to give the compounds high potency at
GlyT-1.
An alternative approach to improving the lipophilicity was to
replace the cyclohexyl group with more polar moieties. Twelve
compounds were prepared using the method of Scheme 1, with
the appropriate amines replacing cyclohexylamine and 4-(2-pyrid-
inyl)benzamide being retained as a consistent part of the mole-
cules. The results are summarised in Table 5. All the compounds
were inactive at GlyT-2.
Compounds 17e (racemic mixture) and 17f were judged to offer
the best compromise between GlyT-1 potency, lipophilicity and
in vitro clearance. Their pharmacokinetic properties were assessed
in vivo (rat): both had estimated clearances of 70 mL minÀ1 kgÀ1
(i.e., 75% liver blood flow) and brain: blood ratios of 0.2. However,
both compounds showed good oral absorption resulting in moder-
ate oral bioavailabilities of 15% and 25%, respectively.
14. GlyT2 screening data was obtained using a [3H]-glycine uptake scintillation
proximity binding assay containing HEK293 cells expressing GlyT-2. Values
given are means of at least two experiments. Values are expressed as
pIC50 = Àlog IC50, where IC50 is the half-maximal inhibitory concentration of
the substance. Values are expressed as pIC50 = Àlog IC50, where IC50 is the half-
maximal inhibitory concentration of the substance, and are the means of at
least two experiments. Values given as ‘less than’ were below the sensitivity of
the assay.
15. A chromatographic method of measuring lipophilicity. See Valko, K.; Du, C.
My.; Bevan, C.; Reynolds, D. P.; Abraham, M. H. Curr. Med. Chem. 2001, 8,
1137.
16. Aqueous solubility. Solubilities were determined from DMSO stock solutions
using chemiluminescent nitrogen detection. See Bhattachar, S. N.; Wesley, J. A.;
Seadeek, C. J. Pharm. Biomed. Anal. 2006, 41, 152.
17. A contributing factor to poor solubility and high clearance. See (a) Leeson, P. D.;
Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881; and (b) Gleeson, M. P. J. Med.
Chem. 2008, 51, 817.
In summary, rational design of compounds based on a screening
hit led to a new series of GlyT-1 inhibitors. Compounds 12j and
12m were identified as lipophilically efficient analogues with good
18. Values given are per gram of protein.