Discovery of N-(2-hydroxy-2-aryl-cyclohexyl) substituted spiropiperidines as GlyT1 antagonists with improved pharmacological profile

Article abstract of DOI:10.1016/j.bmcl.2005.09.067

During SAR exploration of N-(2-aryl-cyclohexyl) substituted spiropiperidine as GlyT1 inhibitors, it was found that introduction of an hydroxy group in position 2 of the cyclohexyl residue considerably improves the pharmacological profile. In particular, r

Full text of DOI:10.1016/j.bmcl.2005.09.067

Bioorganic & Medicinal Chemistry Letters 16 (2006) 354–357
Discovery of N-(2-hydroxy-2-aryl-cyclohexyl) substituted
spiropiperidines as GlyT1 antagonists with
improved pharmacological profile
Simona M. Ceccarelli,* Emmanuel Pinard, Henri Stalder and Daniela Alberati
F. Hoffmann-La Roche Ltd, Pharmaceutical Division Basel, Discovery Chemistry, CH-4070 Basel, Switzerland
Received 7 September 2005; revised 23 September 2005; accepted 23 September 2005
Available online 21 October 2005
Abstract—During SAR exploration of N-(2-aryl-cyclohexyl) substituted spiropiperidine as GlyT1 inhibitors, it was found that
introduction of an hydroxy group in position 2 of the cyclohexyl residue considerably improves the pharmacological profile. In
particular, reduction of the binding affinity at the nociceptin/orphanin FQ peptide and the l opioid receptors was achieved.
Ó 2005 Elsevier Ltd. All rights reserved.
Enhancement of glutamate transmission, in particular
via N-methyl-D-aspartate (NMDA) receptor activation,
is postulated to produce both anti-psychotic and cogni-
tive enhancing effects, thus constituting a potential ther-
apeutic target for the treatment of schizophrenia,
psychoses and cognitive impairment.1 The amino acid
glycine is known to act as a positive allosteric modulator
and obligatory co-agonist with glutamate at the NMDA
receptor complex.2 Glycine transporters (GlyT) play an
important role in the termination of post-synaptic gly-
cinergic actions and maintenance of low extracellular
glycine concentration by re-uptake of glycine into pre-
synaptic nerve terminals or glial cells. In particular,
GlyT1 is the only sodium chloride dependent glycine
transporter in the forebrain, where it is co-expressed
with the NMDA receptor. At this site, GlyT1 is thought
to be responsible for control of extracellular level of gly-
cine at the synapse.3 Therefore, one strategy to enhance
NMDA receptor synaptic function is to elevate the gly-
cine concentration in the local microenvironment of
synaptic NMDA receptors by inhibition of GlyT1.4,5
GlyT1, showing good selectivity against the type 2
isoform.6
The spiropiperidine motif is a privileged structure in a
variety of CNS active agents.7 Indeed, compounds 1
and 2 and their derivatives showed consistently high
level of binding to the l opioid receptor (l), with a
resulting high risk of addiction and tolerance liabilities.
Moreover, the trans diasteroisomer 2, while showing
comparable activity at the GlyT1 transporter, was also
a potent inhibitor of the nociceptin/orphanin FQ recep-
tor (NOP) (Table 1).8 In the course of SAR exploration
of the N-(2-aryl-cyclohexyl) needle, we discovered that
the introduction of a hydroxy group in position 2 had
a noticeable influence on affinity for the l receptor.
While the trans isomer 3 retains considerable activity
at NOP, the cis isomer 4 shows only micromolar affinity
towards the l and the NOP receptors (Table 1). The
most active enantiomer of 4 (obtained via chiral HPLC
separation) shows an even improved pharmacological
profile with respect to l and NOP receptors.9
In the course of a program aimed at identifying new
glycine transporter inhibitors for the treatment of schizo-
phrenia, we discovered N-(2-aryl-cyclohexyl) substituted
spiropiperidines of type 1 to be very potent inhibitors of
Rapid exploration of the substitution patterns at the two
aryl rings of the cis isomer via parallel chemistry allowed
identification of a number of active derivatives (Table
2), all sharing good pharmacological profile with respect
to NOP and l receptors. A number of different substit-
uents in positions meta and para of the phenyl in posi-
tion 2 of the cyclohexane ring are tolerated, while
substitution in ortho, as in compound 10, seems to be
detrimental to selectivity vs the l receptor. Also hetero-
Keywords: GlyT1; GlyT2; NMDA; Schizophrenia; Transporter;
Spiropiperidine.
*
Corresponding author. Tel.: +0041 061 6884437; fax: +0041 061
6886459; e-mail: simona_m.ceccarelli_grenz@roche.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.067

S. M. Ceccarelli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 354–357
355
Table 1. In vitro inhibitory activity at the GlyT1 and the GlyT2
transporters and potency in inhibiting the NOP and l receptors for
compounds 1–4
cycles are tolerated, with a preference for the 4-pyridine
motif, as in 15. Attempts to substitute the aryl ring with
an alkyl chain (compounds 16–20) met with no success.
The SAR at the phenyl group in position 1 of the spiro
systems parallels that of the derivatives lacking the hy-
droxy group, with 4-fluorophenyl derivatives 21 and
23 and 4-chloro derivative 22 showing the best activity.
The selectivity profile of compound 21 was further
explored by assessment of its activity at other glycine
receptor binding sites present in brain, the glycine site
of the NMDA receptor and the strychnine-sensitive gly-
cine receptor, as well as towards a panel of 50 selected
binding sites, ligand gated ion channels and other neuro-
transmitter transporters. In all cases, no sign of signifi-
cant interaction was detected at concentrations below
10 lM, thus confirming that introduction of the hydroxy
group in position 2 of the 2-aryl-cyclohexyl needle
grants a clean pharmacological profile to this class of
GlyT1 inhibitors.
H
N
H
N
O
O
N
N
N
N
trans, rac
cis, rac
1
2
H
H
O
O
N
N
N
N
HO
HO
N
N
trans, rac
cis, rac
3
4
Compound GlyT1
GlyT2
NOP
l
(EC50, lM)a (EC50, lM)a (IC50, lM)b (IC50, lM)c
The cyclohexyl ring of the 2-hydroxy-2-phenyl-cyclo-
hexyl needle can also be substituted with a tetrahydro-
pyran ring, as in compounds 25 and 26 with no loss in
activity or selectivity (Table 3).
1
2
3
4
0.026
0.073
0.056
0.044
12
6
0.15
0.54
1.6d
2.7d
10.3
26
0.31
0.13
72
15
a Radiometric assay using [3H]glycine.10
Synthesis of the cis isomer of 8-(2-hydroxy-2-phenyl-
cyclohexyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one
derivatives was performed as detailed in Scheme 1.
Reaction of commercially available 1-phenyl-1,3,8-tri-
azaspiro[4.5]decan-4-one 27 with the epoxides 28 or 29
(derived from cyclohexene or dihydropyran) in refluxing
ethanol affords the corresponding secondary alcohol
b Displacement of [3H]NOP in membranes prepared from permanently
transfected HEK293 cells expressing hNOP receptors.11
c Displacement of [3H]naloxone in membranes prepared from BHK
cells transiently expressing hl receptors.11
d Full (3) and partial (4) agonists as assessed in GTPcS binding assay
in the same cell membranes, by comparison with DAMGO (full
agonist) and morphine (partial agonist).
Table 2. In vitro inhibitory activity at the GlyT1 and the GlyT2 transporters and potency in inhibiting the NOP and l receptors for compounds 5–24
H
O
N
N
R1
HO
N
R2
cis, rac
Compound
R1
R2
GlyT1 (EC50, lM)
GlyT2 (EC50, lM)
NOP (IC50, lM)
l (IC50, lM)
5
6
4-MeO-Ph
4-Me-Ph
4-Cl-Ph
3,4-Cl2-Ph
4-F-Ph
2-Me-Ph
3-Cl-Ph
3-MeO-Ph
2-Py
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
0.261
0.140
0.080
0.173
0.040
0.050
0.130
0.130
0.130
0.110
0.062
9
67
25
7
>10
1
2.5
3
7
16
3.6
3.7
4
8
6
57
10
1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
70
21
1
0.26
1.09
2.17
1.42
1.8
1
3.85
5.8
15
41
>100
—
>100
3-Py
4-Py
13.4
>10
Me
c-C6H11
Bn
t-Bu
17
4.4
29
>3
i-Pr
4-F-Ph
4-F-Ph
4-Cl-Ph
4-Cl-Ph
0.024
0.015
0.024
0.099
36
2.5
19
25
10
>10
10.4
>10
4
Cl
F
2.2
2.4
4.4
MeO

356
S. M. Ceccarelli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 354–357
Table 3. In vitro inhibitory activity at the GlyT1 and the GlyT2
transporters and potency in inhibiting the NOP and l receptors for
compounds 25–26
H
N
O
H
N
O
O
a
OH
X
N
H
+
N
H
N
H
N
H
N
HN
O
O
X
Y
N
N
HO
HO
O
28 X = CH2
29 X = O
35a X = Y = CH2
35b X = O, Y = CH2
35c X = CH2, Y = O
N
N
34
O
R2
R2
cis, rac
cis, rac
H
H
N
O
N
O
25
26
c
b
OH
N
N
35a
HO
N
Compound GlyT1
GlyT2
NOP
l (IC50, lM)
N
(EC50, lM) (EC50, lM) (IC50, lM)
25
26
0.058
0.090
35
>100
>10
>10
5.3
2.5
36a
cis, rac
33
Scheme 2. Synthesis of N(1)-alkyl derivatives as, for example, 33.
Reagents and conditions: (a) EtOH, reflux, 40–55%; (b) propanal,
NaHB(OAc)3, 80%; (c) i—SO3–pyridine complex, DMSO, TEA,
DCM, 50%; ii—PhLi, THF, À78 °C–rt, 35%.
H
N
O
H
N
O
O
a
OH
N
+
N
N
HN
X
Y
of the hydroxy group on the parent 8-(2-phenyl-cyclo-
hexyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one
X
did
28 X = CH2
29 X = O
30a X = Y = CH2
27
not have any significant effect on metabolic rate mea-
sured in mouse and human microsomes (Table 4). The
corresponding N(1)-alkyl derivatives, on the contrary,
appear to be considerably stabilized by the introduction
of the hydroxy group in position 2. Microsomal clear-
ance data of compound 33, for example, predict a max-
imal achievable bioavailability (MAB) of up to 81% in
mouse and 84% in human microsomes (Table 4).13
30b X = O, Y = CH2
30c X = CH2, Y = O
H
H
N
O
N
O
R1
HO
c
b
O
X
N
N
N
N
Y
cis, rac
31a X = Y = CH2
31b X = O, Y = CH2
31c X = CH2, Y = O
The synthesis of N(1)-alkyl derivatives as 33 was
achieved in a way amenable to SAR exploration
through the key building blocks 35. These were obtained
by simple reaction of the cyclohexene or dihydropyran
epoxides 28 or 29 with 1,3,8-triazaspiro[4.5]decan-4-
one 34,14 yielding the secondary alcohols 35a–c. These
can perform reductive amination at the N(1) nitrogen,
as in the case of the reaction of 35a with propanal to
generate the N(1)-propyl derivative 36a. Oxidation fol-
lowed by reaction with phenyl lithium affords com-
pound 33. The two diversity vectors of such structures
can be varied very easily by employing different alde-
hydes in the reductive amination step and various aryl
5-20, 25-26
Scheme 1. Synthesis of compounds 5–20 and 25–26. Reagents and
conditions: (a) EtOH, reflux, 40–55%; (b) SO3–pyridine complex,
DMSO, TEA, DCM, 20–65%; (c) i—R1Br, BuLi, THF, À78 °C;
ii—31a–c, THF, À78 °C–rt, 20–70%.
30a,b, which can be easily oxidized to ketone 31a,b with
the SO3–pyridine complex. Reaction of 31 with a variety
of aryl-lithium reagents proceeded with very good
stereoselectivity and was amenable to parallel chemistry,
allowing easy access to compounds 5–20 and 25. Com-
pound 26 was obtained in an analogous way from the
minor isomer 30c deriving from epoxide opening of
dihydropyrane-3,4-epoxide Scheme 2.12
Table 4. Effects of the introduction of the 2-hydroxy group on
microsomal metabolic stability of N(1)-aryl and N(1)-alkyl derivatives
H
N
H
N
O
O
The synthesis of compounds 21–24, where the N(1)-
phenyl is substituted, required construction of the
N
N
HO
N
N
substituted
1-aryl-1,3,8-triazaspiro[4.5]decan-4-ones
starting from N-benzyl piperidin-4-one, in analogy to
what was described in the preceding publication.6a
These can then be employed in place of the unsubstitut-
ed analogue 27 as detailed in Scheme 1.
32
33
Compound
GlyT1
(EC50, lM)
Cl (mouse
microsomes)a
Cl (human
microsomes)a
Spiropiperidine systems of type 1 show suboptimal met-
abolic stability, which seems to be bound to the presence
of the 2-phenyl-cyclohexyl ring system. The effect of
2-hydroxy substitution on in vitro microsomal metabo-
lism was therefore of particular interest. Introduction
1
21
32
33
0.026
0.024
0.45
—
102
10
7
68
92
25
5
0.40
a Cl, clearance, lL/min/mg protein.

S. M. Ceccarelli et al. / Bioorg. Med. Chem. Lett. 16 (2006) 354–357
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lithium reagents in the final addition step. In this way,
the SAR of selective and metabolically stable GlyT1
inhibitors can be extensively explored.
In conclusion, introduction of a hydroxy group in posi-
tion 2 of the potent GlyT1 inhibitors 8-(2-aryl-cyclo-
hexyl)-1-aryl-1,3,8-triazaspiro[4.5]decan-4-ones had
a
considerable influence on the pharmacological profile
of such compounds, reducing, in particular, the affinity
towards the l and NOP receptors in the cis series. From
the stereochemical point of view, the relative 1,2 orienta-
tion showing the optimal profile is inverted with respect
to the non-hydroxy substituted derivatives. Synthetic
access to such compounds was amenable to parallel
synthesis, which allowed rapid exploration of the SAR
and modulation of the physicochemical properties. In
the N(1)-alkyl subset, introduction of the hydroxy group
brings about a notable metabolic stabilization, paving
the way for the identification of metabolically stable,
potent and selective GlyT1 antagonists.
8. Any data regarding the hNOP receptor mentioned in this
communication were generated in the years 2001 and
2002.
Acknowledgments
9. Unpublished results.
We thank Patrick Bourdeaux, Sylvia Meyer, Marianne
Rueher, Patrick Boissin, Serge Burner, and Daniel
Zimmerli for their dedicated technical assistance and
Robert Narquizian and Andrew Thomas for support
and advice.
10. Cells transfected with hGlyT-1b or hGlyT2 were seeded in
96-well culture plates. The cells were washed twice with
uptake buffer (UB) and then incubated for 30 min at 22 °C
with either (i) no potential competitor, (ii) 10 mM non-
radioactive glycine, or (iii) a test compound. A solution
was then immediately added containing [3H]glycine 60 nM
(11–16 Ci/mmol) and 25 lM non-radioactive glycine
(hGlyt1) or [3H]glycine 200 nM without cold glycine
(hGlyt2). The cells were then incubated with gentle
shaking for 30 min at 22–24 °C, after which the reaction
was stopped by aspiration of the mixture and washing
(three times) with ice-cold UB. The cells were lysed with
scintillation liquid, shaken for 3 h and the radioactivity in
the cells was counted using a scintillation counter.
11. Wichmann, J.; Adam, G.; Roever, S.; Hennig, M.;
Scalone, M.; Cesura, A. M.; Dautzenberg, F. M. Eur. J.
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