Structure-activity relationship study of novel NR2B-selective antagonists with arylamides to avoid reactive metabolites formation

Article abstract of DOI:10.1016/j.bmcl.2007.08.033

A novel potent NMDA-NR2B selective antagonist (5b) without the reactive metabolites formation issue was identified. Through this study, a close correlation between reactive metabolites formation and calculated HOMO energies of parent compounds was found.

Full text of DOI:10.1016/j.bmcl.2007.08.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5537–5542
Structure–activity relationship study of novel NR2B-selective
antagonists with arylamides to avoid reactive metabolites formation
Makoto Kawai,a,* Isao Sakurada,a Asato Morita,a Yuko Iwamuro,a Kazuo Ando,a
Hirofumi Omura,a Sachiko Sakakibara,a Tsutomu Masuda,a Hiroki Koike,a
Teruki Honma,a Kazunari Hattori,a Tadayuki Takashima,b Kunihiko Mizuno,b
Mayumi Mizutanib and Mitsuhiro Kawamuraa
aDiscovery Chemistry, Pfizer Global Research & Development, Nagoya Laboratories, 5-2, Taketoyo, Aichi 470-2393, Japan
bPhamacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Nagoya Laboratories,
5-2, Taketoyo, Aichi 470-2393, Japan
Received 14 June 2007; revised 9 August 2007; accepted 15 August 2007
Available online 19 August 2007
Abstract—A novel potent NMDA-NR2B selective antagonist (5b) without the reactive metabolites formation issue was identified.
Through this study, a close correlation between reactive metabolites formation and calculated HOMO energies of parent com-
pounds was found.
Ó 2007 Elsevier Ltd. All rights reserved.
Glutamate is an essential amino acid in the central ner-
vous system (CNS) and plays an important role as the
principal excitatory neurotransmitter. There are two
major classes of receptors, ionotropic and metabotropic.
Ionotropic receptors are classified into three major sub-
classes, N-methyl-aspratate (NMDA), 2-amino-3(methyl-
3-hydroxyisoxazol-4-yl)propionic acid (AMPA), and
kainate. Hyperalgesia and allodynia following periphe-
ral tissue or nerve injury were reported to depend on
NMDA receptor-mediated central changes in synaptic
excitability.1 In fact, non-selective NMDA receptor
antagonists such as ketamine2 and dextromethorphan3
have been found to decrease both pain perception and
sensitization in humans. However, many available
NMDA receptor antagonists are prone to cause
potentially serious CNS related side effects.4,5 NMDA
subunits are differentially distributed in the CNS. Espe-
cially, NR2B is believed to be restricted to the forebrain
and laminas I and II of the dorsal horn. This more
discrete distribution of NR2B subunit in the CNS may
support a reduced side-effect profile of agents that act
selectively at this site. For example, CP-101,606 (1,
Fig. 1) is a highly selective NR2B NMDA antagonist
with good in vivo potency in a variety of animal pain
models, and its effect to suppress pain intensity in
patients with spinal cord injury was confirmed without
serious side effects in an experimental clinical study.6
Therefore, there has been considerable interest in devel-
oping NR2B antagonists as analgesics.
Our efforts began with a high throughput screening
(HTS) of Pfizer compound collection by measurement
of Ca2+ influx in HEK (human embryonic kidney) 293
cells stably transfected with human NR1b/2B receptors.
The HTS provided 876 compounds with an IC50 less
than 10 lM. In our preceding paper, we reported that
removal of aliphatic basic nitrogen in the center of a
molecule is beneficial to reduce HERG current inhibi-
tory activity.7 Therefore, compound 2 without a basic
amine center was selected among them, which showed
moderate binding activity for rat NR2B receptor
Keywords: Reactive metabolite; Bioactivation; NR2B; NMDA; Met-
abolic stability.
*
Figure 1. Structure of CP-101,606 (1).
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.2007.08.033

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M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5537–5542
Table 1. Biological data of lead compound 2 and advanced leads (3,
4a)
(Ki = 15 nM) and very weak HERG binding affinity
(IC50 > 30 lM). However, its in vivo activity was very
weak due to low CNS penetration. A preliminary struc-
ture–activity relationship (SAR) study indicated that
elimination of one of two amide groups was effective
to increase CNS penetration, leading to compounds 3
and 4a. (Table 1) Compound 4a especially showed better
in vivo activity to be selected as a lead compound. Here-
in, we wish to describe SAR around it.
Although efficacy of compound 4a was sufficient, meta-
bolic instability (t1/2 = 2.3 min) and formation of reac-
tive metabolites were major issues of this series. The
reactive metabolites resulting from bioactivation of
compounds by human liver microsomes (HLM) are elec-
trophilic presumably to cause toxicity by covalently
altering essential cellular macromolecules and can lead
to undesirable events such as cell death and carcinogen-
esis. Furthermore, adverse events in human caused by
reactive metabolites are not normally observed until
phase III or post-launch due to their sporadic nature.
The events generally lead to termination of development
or withdrawal of drugs from market. Therefore, forma-
tion of reactive metabolites has to be avoided to reduce
such risks.9,10 The formation of reactive metabolites was
Compound
NR2BaKi
(nM)
Rat haloperidol catalepsy,
po MEDb (mg/kg)c
2
15
>30
10
3
3
4a
5.6
6.8
a Measured as the Ki value for displacement of tritiated racemic CP-
101,606 from the rat forebrain P2 membrane.
b MED, Minimum Effective Dose.
c Determining the latency to remove both forepaws from the bar.8
Figure 2. Trapping reactive metabolites by glutathione.
Scheme 1. Reagents and conditions: (a) 1—concd H2SO4, MeOH, reflux, 21 h; 2—Ba(OH)2Æ8H2O, MeOH, H2O, rt, 24 h, 75%; (b) Bn2NH, EDCI,
HOBt, Et3N, DMF, rt, 72 h, 77%; (c) LiAlH4, THF, reflux, 2 h; 89%; (d) 1—MsCl, Et3N, CH2Cl2, 50 °C; 2—NaCN, 15-crown-5, DMSO, 60 °C,
19 h, 68%; (e) concd H2SO4, EtOH, reflux, 5 h, 63%; (f) LiAlH4, THF, 0 °C, 1 h, 73%; (g) DIAD, PhOH, PPh3, toluene, 0 °C-rt, 18 h, 93%, (h)
HCO2NH4, 10%Pd/C, MeOH, reflux, 1 h, 60%; (i) RCOOH, EDCI, HOBt, Et3N, DMF, rt.

M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5537–5542
Table 2. Biological data for phenol bioisosteres
5539
evaluated by trapping reactive metabolites using gluta-
thione (or glutathione ethyl ester) as a nucleophile.11
MS analysis of the conjugates with glutathione obtained
from derivatives around compound 4a revealed that a
major activation site was the phenol ring (Fig. 2). There-
fore optimization to avoid the formation of reactive
metabolites was initiated by exploring phenol bioisoster-
es. Improvement of metabolic stability was also
expected by this modification since the phenol moiety
was the major metabolic site.
Compound R
n
NR2B HLM Reactive
Ki (nM) t1/2 min metabolites
screeninga
Analogs of 4a with phenol bioisosteres were synthesized
as shown in Scheme 1. Dimethyl ester of 6 was mono-
hydrolyzed by barium hydroxide,12 followed by amida-
tion with dibenzylamine to afford compound 8. The
amide and ester of this compound were simultaneously
reduced by lithium aluminum hydride to afford alcohol
9. To extend the carbon chain, compound 9 was treated
with methansulfonyl chloride and sodium cyanide sub-
sequently to afford compound 10. After esterification
using concentrated sulfuric acid and ethanol, reduction
by lithium aluminum hydride led to the alcohol 12.
Mitsunobu reaction with the alcohols (9, 12) yielded
compounds 13 and 14, followed by deprotection to af-
ford the key intermediates (15, 16) which were converted
to amides (4, 5) by a parallel synthesis method.
4a
5a
1
2
6.8
20
2.3
Positive
4b
5b
1
2
14
44
44
Negative
Negative
4.2
4c
5c
1
2
>100
8.9
36
20
Positive
4d
5d
1
2
>100
5.6
Negative
The results of an SAR study for representative phenol
bioisosteres are summarized in Table 2. Most of the
analogs were found to retain potent NR2B antagonist
activity. The NR2B activity was generally improved by
one-carbon prolongation of the spacer (n = 2) between
the cyclohexane and the terminal benzene ring. Replace-
ments with pyrazole (4b, 5b), pyridazine 5d, and inda-
zole 5f suppressed reactive metabolite formation.
However, metabolic stabilities of compound, 5d and 5f
were insufficient. A regioisomer (5c) of 5b, other hetero-
cycles (5e, 5g), and a compound with phenol substituted
by a difluoro group (5h) were positive in reactive metab-
olites screening (RMS). Although 3,4-dihydro-2(1H)-
quinolinone derivatives (4i, 5i) were potent, they were
metabolized rapidly by HLM. Compounds 4b and 5b
were modified further with substituents on the pyrazole
moiety. However, all attempts resulted in loss of potency
(4j, 4k, 5k) or induction of reactive metabolites forma-
tion (5j).
4e
5e
1
2
>100
11
26
20
Positive
5f
2
15
Negative
4g
5g
1
2
84
6.8
33
Positive
Positive
4h
5h
1
2
12
13
25
8.5
A pharmacological profile of compound 5b,13 the most
favorable pyrazole derivative, is summarized in Table
3. Compound 5b exhibited potent analgesic activity in
the mouse partial sciatic nerve legation (PSL) model
4i
5i
1
2
18
16
5.9
8.4
Table 3. Pharmacological profile of compound 5b
4j
5j
1
2
>100
3.7
NR2B binding (Ki)
HERG binding (IC50
4.2 nM
>30 lM
36%
59
Positive
)
HERG current inhibition at 10 lM
t1/2 in human liver microsomes
Reactive metabolites screening
Bioavailability in rats
Mouse in vivo model (PSL)a
Minimum Effective Dose (sc)
44 min
Negative
43%
4k
5k
1
2
>100
>100
3 mg/kg
a This assay protocol is described in Ref. 11.
a The mouse partial sciatic nerve ligation (PSL) model.8

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M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5537–5542
Table 4. Results of RMS and calculated HOMO energies for phenol bioisosteres
Compound
R
Amine (n)
Reactive metabolites screening
Positive
Calculated HOMO energy (eV)
4a
1
À9.37
5b
2
Negative
À9.43
5c
5d
2
2
Positive
À9.35
À9.66
Negative
5f
2
2
2
Negative
Positive
Positive
À9.17
5g
5h
À9.20
À9.72 (À3.84)b
5j
5l
2
2
Positive
À8.68
À9.70
Negative
5m
5n
2
2
Negative
Negative
À9.58
À9.63
5o
2
Positive
À8.92
5p
2
Negative
Positive
À9.95
À9.33
17a
A

M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5537–5542
5541
Table 4 (continued)
Compound
R
Amine (n)
Reactive metabolites screening
Positive
Calculated HOMO energy (eV)
18a
19a
B
À9.17
À9.27
C
Positive
a The syntheses of these compounds are described in Ref. 8.
b An anion form.
À9.73 eV) Introduction of an amino group to the pyra-
zole (5j) enhanced its HOMO energy, which turned out
to be positive in RMS. Despite an exception (5f), the
results of RMS are significantly correlated with calcu-
lated HOMO energies. Specifically compounds with
high HOMO energy are prone to formation of reactive
metabolites. The tendency would be applicable to other
chemotypes.
5h
-4.0
≈
-8.6
5j
-8.8
-9.0
5f
-9.2
In conclusion, we have explored bioisosteres for phenol
of the lead series to avoid reactive metabolite formation
and to improve metabolic stability. Replacement of phe-
nol with pyrazole was effective to prevent formation of
reactive metabolites, and pyrazolecarboxiamide 5b pos-
sessing analgesic activity and a good pharmacokinetic
profile was identified. The good correlation between
the formation of reactive metabolites and HOMO ener-
gies of parent compounds found in this study may be
applicable to other chemotypes.
-9.4
5b
-9.6
-9.8
-10
Negative
Positive
Reactive Metabolite Screening
Figure 3. Correlation between HOMO energy and RMS.
Acknowledgment
The authors thank Dr. Rodney W. Stevens for his valu-
able suggestion.
(Minimum Effective Dose = 3 mg/kg, sc) and showed
lower HERG current inhibitory activity (36% at
10 lM). In addition, this compound possesses a good
pharmacokinetic profile, high metabolic stability
(44 min), and bioavailability (43%).
References and notes
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The SAR for formation of reactive metabolites was ana-
lyzed using 16 different phenol bioisosteres (aromatic
rings) summarized in Table 4. From the result of the
structure of glutathione conjugate of reactive metabolite
(Fig. 2), a reaction between the heme oxygen in meta-
bolic enzyme(s) and HOMO (Highest Occupied Molec-
ular Orbital) of the compound is speculated to be the
first step for production of the reactive metabolite.14
The correlation between reactive metabolite formation
and calculated HOMO values is shown in Figure 3.
Compounds with low HOMO energy (<À9.4 eV) tend
to be negative in RMS presumably due to low reactivity
to the oxygen of HEME. In fact, all compounds were
negative. On the other hand, 9 out of 10 compounds
with high HOMO energy (>À9.4 eV) were positive. As
for compound 5h, the proton of the phenol was pre-
dicted to an anion form in this assay condition
(pH = 7.4), because its calculated pKa value is about
6.05. Therefore, its HOMO value (À3.84 eV) is calcu-
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M. Kawai et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5537–5542
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DMSO-d6) d 13.08 (br s, 1H), 8.12–7.91 (m, 3H),