Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone-A potent NR2B-selective N-methyl d-aspartate (NMDA) antagonist for the treatment of pain

Article abstract of DOI:10.1016/j.bmcl.2007.08.014

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits c

Full text of DOI:10.1016/j.bmcl.2007.08.014

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5558–5562
Discovery of (ꢀ)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-
1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone—A potent
NR2B-selective N-methyl D-aspartate (NMDA) antagonist
for the treatment of pain
Makoto Kawai,a,* Kazuo Ando,a Yukari Matsumoto,a Isao Sakurada,a Masako Hirota,a
Hiroshi Nakamura,a Atsuko Ohta,a Masaki Sudo,a Kazunari Hattori,a
Tadashi Takashima,c Masanori Hizue,b Shuzo Watanabe,b Isami Fujita,b
Mayumi Mizutanic and Mitsuhiro Kawamuraa
aDiscovery Chemistry, Pfizer Global Research & Development, Nagoya laboratories, 5-2, Taketoyo, Aichi 470-2393, Japan
bDiscovery biology, Pfizer Global Research & Development, Nagoya laboratories, 5-2, Taketoyo, Aichi 470-2393, Japan
cPhamacokinetics Dynamics & Metabolism, Pfizer Global Research & Development, Nagoya laboratories, 5-2,
Taketoyo, Aichi 470-2393, Japan
Received 11 May 2007; revised 18 July 2007; accepted 9 August 2007
Available online 15 August 2007
Abstract—(ꢀ)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as
an orally active NR2B-subunit selective N-methyl-D-aspartate (NMDA) receptor antagonist. It has very high selectivity for
NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index = 4200 vs NR2B binding
IC50). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.
Ó 2007 Elsevier Ltd. All rights reserved.
The N-methyl-D-aspartate (NMDA) receptor is a gluta-
mate-gated ion channel playing important roles in a
variety of neuronal processes including learning, mem-
ory, and pain transmission.1 For treatment of pain,
there is considerable preclinical evidence that hyperalge-
sia and allodynia following peripheral tissue or nerve in-
jury are not only due to an increase in activity of
primary afferent nociceptors at the site of injury but also
depend on NMDA receptor-mediated central changes in
synaptic excitability.2–5 In humans, NMDA receptor
antagonists, such as ketamine and dextramethorphan,
have also been found to decrease both pain perception
and sensitization.6–13 However, while NMDA receptor
antagonists may have therapeutic utility for the treat-
ment of pain, there are significant hurdles to a wide-
spread clinical use. Notably, new approaches need to
be explored to overcome the potentially serious side ef-
fects such as psychotomimetic effects and cognitive
disruption.7,10,11
One approach to developing novel NMDA antagonists
is to target specific receptor subtypes. NMDA receptors
are heteromers consisting of NR1 and NR2A, NR2B,
NR2C, and NR2D subunits. Among them, the NMDA
receptors with NR2B subunits are distributed predomi-
nantly in forebrain and laminas I and II of the dorsal
horn, which are responsible for pain transmission. The
more discrete distribution of NR2B subunits in the cen-
tral nerve system (CNS) may support a reduced side-ef-
fect profile of agents that act selectively at this site. For
example, lack of expression in the cerebellum may sug-
gest a reduced propensity for causing ataxia. In fact,
Boyce et al. reported that CP-101,606 (1) (Fig. 1), an
NR2B-selective NMDA antagonist, demonstrated a
wider safety profile than existing nonselective NMDA
antagonists.14 More significantly, it was recently re-
ported that 1 significantly suppressed pain intensity in
patients with spinal cord injury and monoradiculopathy
compared to placebo with an acceptable tolerance.15
Keywords: NMDA; NR2B antagonist; PK variability; CYP2D6;
HERG.
*
Corresponding author. E-mail: makoto.kawai@pfizer.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.014

M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5558–5562
5559
tion of 1 in human studies. For this reason, it is essential
to improve the metabolic profile of 1 for a chronic use.
QT prolongation induced by CP-101,606 increases the
likelihood of a polymorphous ventricular arrhythmia
(torsades de pointes, TdP), which may cause syncope
and degenerate into ventricular fibrillation and sudden
death.20 The QT prolongation is attributed to human
ether-a-go-go-related gene (HERG) K+ channel current
inhibitory activity of compound 1. Therefore, the
HERG current inhibitory activity needed to be mini-
mized to increase safety.
Figure 1. Structure of CP-101,606 (1).
Compound 1 is a highly selective NR2B NMDA antag-
16
onist discovered by Pfizer and is being developed for
stroke in the clinic. However, a chronic and safe use
of compound 1 for nonlife-threatening indications such
as pain is hampered by (1) its significant Pharmacoki-
netic (PK) variability and (2) QT prolongation issues.20
The cause of this PK variability is predominant metab-
olism by cytochrome P450 (CYP) 2D6.17–19 This enzyme
is a polymorphic member of the CYP superfamily and is
absent in 7% (poor metabolizers: PM) of the Caucasian
population.17–19 Thus, variation in expression of
CYP2D6 results in variability in the metabolic inactiva-
To identify a safer NR2B-selective NMDA antagonist
for the chronic pain therapy, further modification of
CP-101,606 was carried out with focus on alleviating
those two issues. First, improvement of the metabolic
profile of CP-101,606 by structural modification was
performed. Most of the drugs metabolized by CYP2D6
contain a basic nitrogen atom and a flat hydrophobic re-
gion coplanar to the oxidation site which is either 5 or 7
angstroms away from the basic nitrogen atom.21,22 In
Table 1. NR2B IC50, CYP2D6 contribution ( CYP2D6 ratio), and CYP2D6 inhibition of substituted phenols
Compounda
R
CYP2D6 t1/2 ratioc
>10
2.1
2D6 inhibition IC50 (lM)
b
NR2B IC50 (nM)
d
1
2
3
4
5
6
CP-101606
2-Me
3-Me
14
26
>10
<0.3
3.9
16
5.6
>4.5
1.6
NTe
NTe
2-Cl
3.5
2-tBu
2-Ph
>5000
570
NTe
8.4
a All compounds are racemate. The synthetic schemes for them are showed in Supplementary materials.
b Measured as the IC50 value for displacement of tritiated racemic CP-101,606 from the rat forebrain P2 membrane.
c
CYP2D6 t1/2 ratio = t1/2 (HHM0168 (1 mg) + control vector (0.3 mg))/t1/2 (HHM0168 (1 mg) + CYP2D6 (0.3 mg)): HHM0168 = PM-human liver
microsomes.
d Measured as the IC50 value for displacement of tritiated bufuralol from CYP2D6 supersomesÒ (Gentest, 0.1 mg/ml) added NADPH-regenerating
cofactor.
e NT, not tested.
Table 2. NR2B IC50, CYP2D6 contribution ( CYP2D6 t1/2 ratio), CYP2D6 inhibition, and HERG affinity of aniline derivatives
Compounda R1
R2
NR2B IC50 (nM)
41
CYP2D6 ratio 2D6 inhibition IC50 (lM) HERG IC50 (lM) HERG/NR2B ratio
b
7
O
1
1
1
1
1
1
11
>30
>30
>30
>30
>30
2.6
2.4
1.6
7.4
3.1
8.6
63
133
184
813
221
782
8
9
O
CH2 18
CH2
NH
O
CH2
8.7
9.1
10
11
12
NMe CH2 14
CH2 CH2 11
a All compounds are racemate. The synthetic scheme for them is showed in Supplementary materials.
b Measured as the IC50 value for displacement of tritiated dofetilide from human HERG channel expressed in HEK293 cells.

5560
M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5558–5562
fact, MS analysis of the metabolites of CP-101,606 indi-
cated that the ortho position of the phenol ring and the
para position of the right benzene ring were mainly oxi-
dized by human liver microsomes (HLM).23 Thus, ef-
forts were put into introducing substituents at the sites
on the two aromatic rings, which are located around
5–7 angstroms away from the basic nitrogen on the
piperidine ring. Introduction of a metabolically stable
substituent at the ortho position of the phenol ring, how-
ever, unexpectedly resulted in increase of CYP2D6 inhi-
bition (Table 1), although this modification was effective
to reduce contribution of CYP2D6 metabolism.
Based on the result, attempts were made to replace the
phenol moiety with its isosteres to diminish the CYP2D6
metabolism and inhibition. Replacement with carboxani-
lides dramatically reduced contribution of CYP2D6
metabolism with weak CYP2D6 inhibition (Table 2).
Especially, 3,4-dihydroquinolinon-2(1H)-one deriva-
tives, represented by 12, were active in the haloperidol-in-
duced rat catalepsy model (MED = 10 mg/kg, po), with
which in vivo NR2B antagonism of compounds in the
CNS was evaluated. A 3,4-dihydroquinazolinon-2(1H)-
one derivative (10) was the best in terms of weak HERG
current inhibition, but not active in vivo probably due
Scheme 1. Synthetic route of 18. Reagents and conditions: (a) AlCl3, 2-chloropropionylchloride, CS2, 2 h, 50 °C, quant.; (b) 1,4-dioxa-8-
azaspiro[4.5]decane, Et3N, ethanol, reflux, 82%; (c) NaBH4, ethanol; 43%; (d) 6 N HCl aq, acetone, reflux, 57%; (e) ArBr, n-BuLi, THF (the
procedure was shown in Supplementary materials).
Table 3. NR2B IC50, HERG affinity, potency in rat haloperidol catalepsy model, and clogP of analogs
Compounda
R
NR2B
IC50 (nM)
HERG
IC50 (lM)
HERG/NR2B
ratio
Rat halodol catalepsyb,
po MEDc (mg/kg)
clogP
18a
18b
18c
18d
18e
18f
15
16
3.3
10
1067
323
>30
NTd
10
1.09
2.12
1.76
3.31
2.33
1.41
9.1
10
1000
<238
<151
1325
6.3
6.6
8.3
<1.5
<1
11
NTd
10
30
a All compounds are racemate.
b This assay is described in Supplementary materials.
c MED, minimum effective dose.
d NT, not tested.

M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5558–5562
5561
to its low CNS penetration. Therefore, further optimiza-
tion around the right aromatic portion for minimizing
the HERG current inhibitory activity was conducted with
a 3,4-dihydroquinolinon-2(1H)-one series.
A library of 4-aryl-4-hydroxypiperidines with physico-
chemical diversity was built in order to conduct an SAR
study of this series on HERG current inhibitory activity.
Instead of evaluating HERG current inhibitory activity,
a binding assay using [3H]dofetilide, a potent HERG cur-
rent inhibitor, was employed, because of its high assay
throughput andasufficient correlationbetweentheHERG
current inhibitory activity and the binding activity. Target
compounds were prepared according to Scheme 1.
Friedel–Crafts reaction24 of 3,4-dihydroquinolin-2(1H)-
one 13 with 2-chloropropionyl chloride yielded
a-chloroketone 14, which was subsequently aminated
to aminoketone 15. After reduction of 15 by sodium
borohydride, threo-alcohol 16 was isolated by crystalli-
zation. The key intermediate 17 was synthesized by
deprotection with aq HCl. Treatment with various aryl
lithiums (3 equiv) led to a compound library of 4-aryl-
4-hydroxypiperidines 18 (see Table 3).
Analysis of the HERG binding data of the compound li-
brary revealed that there was a good correlation between
HERG binding and lipophilicity as shown in Figure 2.
Especially, compounds with clogP of 1.5–2.5 are likely
to keep potent in vivo activity (MED 6 10 mg/kg, po)
with reduced HERG binding (<50% inhibition at
10 lM). The weak in vivo activity of less lipophilic
(clogP < 1.5) compounds was attributed to poor CNS
Figure 2. Correlation between HERG binding and clogP.
Table 4. NR2B IC50, HERG affinity, and clogP of analogs
Compounda
Linker
NR2B IC50 (nM)
10
HERG IC50 (lM)
HERG/NR2B ratio
1000
clogP
iHERGb IC20 (lM)
( )-18c
10
1.77
( )-19
(+)-19
(ꢀ)-19
(+)-1
4.5
6.8
5
13
9.8
21
10
2889
1441
4200
714
1.47
1.47
1.47
1.80
1.1
CP-101,606
14
0.15
a The synthetic scheme for them is showed in Supplementary materials.
b Measured as the degree of blockade by compounds on HERG potassium channels stably expressed in HEK-293 cells by an electrophysiological
method.

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M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5558–5562
4. Ren, K. Netherlands, 1994; p 157.
penetration according to PK studies. Several individual
data are shown in Table 2. Compounds with clogP of
>2.5 in this table showed potent HERG binding activity
(18d, HERG IC50 < 1.5 lM). On the other hand, com-
pounds with clogP of <1.5 showed weaker HERG bind-
ing (HERG IC50 = 18f: 11 lM, 18a: <16 lM) but weaker
in vivo activity (MED P 30 mg/kg, po). Among the syn-
thesized compounds, 18c had the best profile although the
safety index between NR2B activity and HERG activity
was still insufficient.
5. Sandkuhler, J.; Liu, X. Eur. J. Neurosci. 1998, 10, 2476.
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Max, M. B. Neurology 1997, 48, 1212.
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Wong, C. S. World J. Surg. 2000, 24, 512.
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G.; Rigby, M.; Sirinathsinghji, D.; Hill, R. G.; Rupniak,
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Neurosci. Abstr. 2003, 814.
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After thorough investigation of the SAR study around the
two aromatic rings, our attention was turned to the linker
moiety of 18c connecting the two aromatic rings (Table 4).
Removal of a methyl group from 18c afforded 19 with re-
duced clogP and HERG affinity (IC50 = 13 lM) as well
as a slight enhancement of NR2B affinity (IC50
=
4.5 nM). Compound (ꢀ)-19 turned out to exhibit the
better profile than the antipode (+)-1925 and exhibited
lower current inhibitory activity to HERG channel
compared to CP-101,606.
In summary, replacement of the phenol group by 3,4-
dihydroquinolinon-2(1H)-one solved the PK variability
issue of CP-101,606 (1). Finding a correlation between
HERG activity and clogP provided us with a clue to
reducing the HERG activity, leading to (ꢀ)-6-[2-[4-(3-flu-
orophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-
3, 4-dihydro-2(1H)-quinolinone (ꢀ)-1926 as a potential
development candidate for the pain therapy.
21. de Groot, M. J.; Vermeulen, N. P.; Kramer, J. D.; van, A.
´
F. A.; Donne-Op den Kelder, G. M. Chem. Res. Toxicol.
1996, 9, 1079.
Acknowledgments
22. Koymans, L.; Vermeulen, N. P.; van, A. S. A.; te, K. J. M.;
´
Heykants, J. J.; Lavrijsen, K.; Meuldermans, W.; Donne-Op
The authors thank Dr. Bertrand L. Chenard, Dr. Frank
S. Menniti, and Dr. Rodney W. Stevens for their valu-
able advice and discussions.
den Kelder, G. M. Chem. Res. Toxicol. 1992, 5, 211.
23. Investigator’s Brochure of CP-101,606, 2000. In-depth
data of the analysis are shown in Supplementary
materials.
24. Martinez, G. R.; Walker, K. A.; Hirschfeld, D. R.; Bruno,
J. J.; Yang, D. S.; Maloney, P. J. J. Med. Chem. 1992, 35,
620.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2007.
08.014.
25. Kawamura, M. (Pfizer Pharmaceuticals Inc., Japan; Pfizer
Inc.). WO2003091241; p 47.
1
26. Chemical data of (ꢀ)-19 (mesylate): H NMR (300 MHz,
DMSO-d6) d 10.14 (s, 1H), 9.23 (s, 1H) , 7.490–7.40 (m,
1H ), 7.32–7.18 (m, 4H), 7.15–7.07 (m, 1H), 6.87 (d,
J = 8.1 Hz, 1H), 6.22 (s, 1H), 5.63 (s 1H), 5.03 (d,
J = 10.4 Hz, 1H), 3.66–3.58 (m, 1H), 3.52–3.14 (m,7H),
2.93–2.84 (m, 2H), 2.32 (s, 3H), 2.53–2.21, (m, 2H), 1.92–
1.70 (m, 2H); MS (ESI) m/z 385.15 (M+H+), 383.20
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24
(MꢀH+); IR (KBr) 3261, 3050, 2737, 1655 cmꢀ1; ½aꢁD
ꢀ29.46 (c 0.1154, methanol); mp 180–182 °C; Anal. Calcd
for C22H25N2O3F, CH4O3S: C, 54.49; H, 6.08; N, 5.83.
Found: C, 57.32; H, 6.24; N, 5.74.