Structure-activity relationships for a series of bis(phenylalkyl)amines: Potent subtype-selective inhibitors of N-methyl-D-aspartate receptors

Article abstract of DOI:10.1021/jm980235+

A series of bis(phenylalkyl)amines, structural analogues of ifenprodil and nylidrin, were synthesized and tested for antagonism of N-methyl-D- aspartate (NMDA) receptors. Potency and subunit selectivity were assayed by electrical recordings in Xenopus ooc

Full text of DOI:10.1021/jm980235+

J . Med. Chem. 1998, 41, 3499-3506
3499
Str u ctu r e-Activity Rela tion sh ip s for a Ser ies of Bis(p h en yla lk yl)a m in es:
P oten t Su btyp e-Selective In h ibitor s of N-Meth yl-D-a sp a r ta te Recep tor s
Amir P. Tamiz,† Edward R. Whittemore,‡ Zhang-Lin Zhou,‡ J in-Cheng Huang,‡ J ohn A. Drewe,‡
J ie-Cheng Chen,‡ Sui-Xiong Cai,‡ Eckard Weber,‡ Richard M. Woodward,*,‡ and J ohn F. W. Keana*,†
Department of Chemistry, University of Oregon, Eugene, Oregon 97403, and CoCensys, Inc., 201 Technology Drive,
Irvine, California 92618
Received April 16, 1998
A series of bis(phenylalkyl)amines, structural analogues of ifenprodil and nylidrin, were
synthesized and tested for antagonism of N-methyl-D-aspartate (NMDA) receptors. Potency
and subunit selectivity were assayed by electrical recordings in Xenopus oocytes expressing
three binary combinations of cloned rat NMDA receptor subunits: NR1A expressed in
combination with either NR2A, NR2B, or NR2C. The bis(phenylalkyl)amines were selective
antagonists of NR1A/2B receptors. Assayed under steady-state conditions, the most potent of
these, N-[2-(4-hydroxyphenyl)ethyl]-5-phenylpentylamine hydrochloride (20), has an IC50 value
of 8 nM and >1000-fold selectivity with respect to NR1A/2A and NR1A/2C receptors. The
structure-activity relationship of the bis(phenylalkyl)amine series indicates that the piperidine
ring and alkyl chain substitutions common to NR2B-selective antagonists such as ifenprodil,
CP 101,606, and Ro 25-6981 are not necessary to generate potent and selective ligands. The
primary determinants of potency are the phenolic OH group, acting as a hydrogen bond donor,
the distance between the two rings, and an electrostatic interaction between the receptor and
the basic nitrogen atom. This study provides a framework for designing structurally novel
NR2B-selective antagonists which may be useful for treatment of a variety of neurological
disorders.
In tr od u ction
N-Methyl-D-aspartate (NMDA) receptor antagonists
have therapeutic potential as neuroprotectants, anti-
convulsants, analgesics, and agents that augment the
effects of L-DOPA for the treatment of Parkinson’s
disease.1 Development of clinically useful drugs, how-
ever, has been hampered by a variety of dose-limiting
side effects which include neurotoxicity, psychotomi-
metic behaviors, and a narrow therapeutic index with
respect to sedation.2 Studies at the molecular level
suggest that native NMDA receptors are heterooligo-
meric assemblies of two different types of subunits. The
subunits are designated NMDA receptor 1 (NR1), of
which there are eight isoforms generated by alternate
RNA splicing, and NR2, of which there are four distinct
types each transcribed from a separate gene.3 Subunit
composition and distribution of native receptors in adult
mammalian brain differ significantly from region to
region.4 Characterization of recombinant receptors of
defined subunit composition indicates that NMDA
receptor subtypes have different pharmacological prop-
F igu r e 1. Potent antagonists of the NR2B subtype of the
NMDA receptor. Compounds 1, 2, and 5 were tested as
racemates; only one enantiomer of 1 and 5 is shown.
erties and thus represent discrete therapeutic targets.3,4
It follows that by designing subtype-selective antago-
nists it may be possible to find high-potency drugs that
also have more favorable side effect profiles when
compared to broad-spectrum inhibitors.
The first subtype-selective NMDA receptor antagonist
to be discovered was ifenprodil (1), which has pro-
nounced selectivity for receptors composed of NR1 and
NR2B subunits.5 Using ifenprodil as a starting point,
novel series of NR2B-selective antagonists have been
designed leading to compounds such as eliprodil (2),6
CP 101,606 (3),7 and Ro 25-6981 (4) (Figure 1).8 These
drugs are all reported to have neuroprotective effects
in animal models of focal cerebral ischemia without
themselves inducing neurotoxicity or showing behav-
ioral liability in drug discrimination studies.6-8 In
addition to ifenprodil analogues, recent studies indicate
* To whom correspondence should be addressed.
† University of Oregon.
‡ CoCensys, Inc.
S0022-2623(98)00235-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/01/1998

3500 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Tamiz et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (i) NaH, DMF; (ii) 10% Pd/C, H2, MeOH.
Sch em e 3a
a
Reagents: (i) K2CO3, CH3CN; (ii) 1 M HCl/MeOH; (iii) 10%
Pd/C, H2, EtOH.
that haloperidol and related molecules are also NR2B-
selective antagonists.9,10 Whether related to ifenprodil
or haloperidol, all these compounds are 1,4-disubstitut-
ed piperidines, and all inhibit NMDA receptor function
by a noncompetitive, allosteric mechanism at a site, or
sites, which are not located in the membrane-spanning
region of the channel pore.11
In the present study we have designed a series of bis-
(phenylalkyl)amines to investigate some of the basic
determinants of potency for NR2B-selective inhibitors.
Potency and selectivity of ligands were assessed by
functional assays in Xenopus oocytes expressing recom-
binant NMDA receptors.9 The impetus for the series
came from the observation that nylidrin (5), a nonpip-
eridine analogue of ifenprodil, is a surprisingly potent
NR2B-selective NMDA receptor antagonist.12 The study
led us to the discovery of the amine 20, one of the most
potent and selective inhibitors of NR2B NMDA recep-
tors reported to date. Amine 20 shows low-nanomolar
potency for the NR1A/2B subunit combination and
>1000-fold selectivity with respect to NR1A/2A and
NR1A/2C.
a
Reagents: (i) PCC, CH2Cl2; (ii) 2-(4-hydroxyphenyl)ethyl-
amine, NaCNBH3, MeOH; (iii) HCl/ether.
Sch em e 4a
Ch em istr y
Piperidine 11 was prepared by N-alkylation of 4-ben-
zylpiperidine (6) with mesylate 8 followed by O-deben-
zylation (Scheme 1). N-Alkylation of 6 with mesylate
7 gave piperidine 9. Ether analogue 15 was prepared
by the reaction the sodium salt of alcohol 13 with
tosylate 12, followed by O-debenzylation (Scheme 2).
For the synthesis of amines 20 and 21, commercially
available alcohols 16 and 17 were first oxidized to the
corresponding aldehydes (18 and 19) with pyridinium
chlorochromate (PCC) in CH2Cl2 in high yield (Scheme
3). Reductive amination with 2-(4-hydroxyphenyl)-
ethylamine using NaCNBH3 in MeOH gave the free
amines which were isolated as their respective HCl salts
20 and 21.
a
Reagents: (i) DCC, HOBT, DMF, 60 °C; (ii) LiAlH4, THF,
reflux; (iii) HCl/MeOH; (iv) CH3CH2I, NaHCO3, CH3CN.
Open-chain secondary amines 29-34 were prepared
as depicted in Scheme 4. Condensation of acids 22-24
with the corresponding amines 25-28 in the presence
of 1,3-dicyclohexylcarbodiimide (DCC) and 1-hydroxy-
benzotriazole (HOBT) followed by LiAlH4 reduction of
the intermediate amide gave amines 29-34 in good
yields. Amines 29-33 were isolated as the hydrochloric
salts. N-Alkylation of 34 with ethyl iodide gave amine
35.

SAR for a Series of Bis(phenylalkyl)amines
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3501
Ta ble 1. Functional Antagonism of Substituted Amines at
NMDA Receptor Subtypes
results in a small but significant decrease in potency.
Removal of the phenolic hydroxyl group gave 9, the
unsubstituted analogue of ifenprodil. Potency of pip-
eridine 9 is 10-fold weaker than that of ifenprodil at
NR1A/2B receptors. Opening the piperidine ring of 11
to form the tertiary amine 35 causes only a slight
decrease in potency. The activity of this non-piperidine
amine was portended by the potent inhibitory effects
of nylidrin. Interestingly, removing the ethyl group
from the nitrogen atom to form the secondary amine
34 results in a 7-fold increase in potency. This simple
molecule has an apparent potency for NR1A/2B recep-
tors that is more than twice that of ifenprodil. The
increase in potency can be attributed to the less
hindered nature of the basic nitrogen atom providing a
more effective electrostatic interaction with the receptor
pocket. The potency and selectivity of 34 indicate that
conformational constraints conferred by the piperidine
ring are not important for generating high-potency
NR2B-selective antagonists. In the secondary amine
series, removing the hydroxyl group from the phenolic
ring of 34 gives 29 and results in an 80-fold drop in
potency as compared to a 5-fold drop in potency in going
from 11 to 9. The non-phenolic 29 is only ∼10-fold
selective for NR1A/2B as compared to the other two
subunit combinations. Shortening the chain between
the nitrogen atom and the non-phenolic ring in 34 by
one methylene gave 32 and results in a 2-fold reduction
in potency. Phenol 32 is an analogue of nylidrin with
all substitutions on the chain removed. These struc-
tural modifications result in a 2-fold increase in potency
with respect to the parent structure 5. It is also
noteworthy that 32 can be considered as the secondary
amine analogue of amine 3, by removing the CH3 and
OH substituents on the chain and the OH substitution
on the piperidine ring. In our assays amine 3 (synthe-
sized in house as a reference) has an IC50 value of ∼0.1
µM, a value similar to that of 32. Whether comparing
32 with nylidrin or amine 3, the results indicate that
the piperidine ring and substituents on the chain with
all the associated stereochemistry are superfluous to
generating potent NR2B-selective antagonists. In con-
trast, removing the phenolic hydroxyl group (33) causes
a substantial drop in activity.
IC50 (µM)a
compd no.
1A/2A
1A/2B
1A/2C
1
5b
9
20 ( 6
32 ( 2.0
>100
0.11 ( 0.01
0.18 ( 0.03
1.1 ( 0.2
>100
42 ( 1.1
>100
11
15
20
21
29
30
31
32
33
34
35
57 ( 8
>100
0.20 ( 0.02
3.7 ( 0.3
>100
>100
12 ( 2
17 ( 4
46 ( 7
18 ( 2
18 ( 3
26 ( 13
>100
0.008 ( 0.001
0.035 ( 0.008
3.5 ( 0.3
0.015 ( 0.002
0.045 ( 0.01
0.096 ( 0.01
7.3 ( 0.8
0.043 ( 0.004
0.30 ( 0.03
39 ( 4
30 ( 2
36 ( 4
48 ( 3
39 ( 4
>100
>100
65 ( 5
88 ( 13
15 ( 3
27 ( 5
a
IC50 values ((SEM) were determined by electrical assays in
Xenopus oocytes expressing the NMDA receptor combinations.
Values were obtained from g3 oocytes for NR1A/2B and g2 oocytes
b
for the other subunit combinations. See ref 12a.
F igu r e 2. Concentration-inhibition curves for amine 20:
effect of varying the NR2 subunit.
Biologica l Eva lu a tion
Potencies of antagonism at the three binary NMDA
receptor subunit combinations are given in Table 1.
Potency and subunit selectivity were assayed by electri-
cal recordings under steady-state conditions in Xenopus
oocytes expressing three binary combinations (NR1A
expressed in combination with either NR2A, NR2B, or
NR2C) of cloned rat NMDA receptor subunits. The IC50
values were determined by curve fitting to concentra-
tion-inhibition data pooled from 2-7 separate experi-
ments (see the Experimental Section for details). Sample
data are given in Figure 2. All compounds showed high
or moderate potency (IC50 e 7.5 µM) at the NR1A/2B
subunit combinations. In the following discussion we
consider the high-potency inhibition of NR1A/2B recep-
tors first, before briefly addressing the weak inhibition
of NR1A/2A and NR1A/2C receptors.
Extending the chain length of 34 by addition of one
methylene between the nitrogen atom and the unsub-
stituted ring gives 20 and results in a further 5-fold
increase in potency. With an IC50 of 8 nM, 20 is among
the most potent NR2B-selective antagonists yet re-
ported. Levels of selectivity for 20 are >1000-fold with
respect to either the NR2A- or NR2C-containing subunit
combinations. Similarly, extending the chain of 34 by
one methylene between the nitrogen atom and the
phenolic ring gives 30 and also increases potency. This
molecule can be considered the secondary amine ana-
logue of amine 4 with OH and CH3 substituents
removed from the chain. The two molecules again have
comparable IC50 values,8 further suggesting that the
substituents on the chain are not important determi-
nants of potency. It is noteworthy that the methylene
count between the two phenyl groups in compounds 20
and 30 is the same. Adding one methylene between the
piperidine and phenolic ring of ifenprodil results in
amine 4 and a ∼10-fold increase in potency on recom-
Str u ctu r e-Activity Rela tion sh ip
Structural relationships between ifenprodil, nylidrin,
and the related bis(phenylalkyl)amines are outlined in
Figure 3. In the current experiments ifenprodil inhib-
ited NR1A/2B receptors with an IC50 value of 0.11 µM.
This is a slightly higher potency than we had reported
in a previous study,10 but it is comparable with other
data from recombinant receptors5 and native receptors
in cultured rat neurons.10,11 Removal of the methyl and
hydroxyl groups from the ethylene linker gives 11 and

3502 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Tamiz et al.
F igu r e 3. Structure-activity relationship of the substituted amines at the NR1A/2B subtype.

SAR for a Series of Bis(phenylalkyl)amines
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3503
Ta ble 2. Intramolecular Distances (Å) between Atoms
Measured on the Fully Extended Conformer Calculated at the
Semiempirical (AM1) Level
compd no.
C-O
N-O
C-N
1/2B (µM)
1
4
5
20
30
13.9
15.0
13.8
17.5
17.0
7.7
8.8
7.8
7.8
8.8
6.6
6.6
7.1
9.9
8.9
0.11
0.009a
0.18
F igu r e 4. Receptor features presumed important for the
binding of inhibitors at the NR1A/2B subtype.
0.008
0.015
a
methylene linker toward the A-ring for most effective
binding. Extending the total length of the molecule to
19 Å in amine 21 renders the molecule less potent.
Inhibition of NR1A/2A and NR1A/2C receptors by this
series of compounds was consistently weaker when
compared to inhibition of NR1A/2B receptors, though
NR1A/2A receptors generally had higher sensitivity
than NR1A/2C. It is difficult to develop a structure-
activity relationship (SAR) for inhibition of NR1A/2A
because differences in potency between compounds are
not great: the most potent inhibitor 20 has an IC50 of
12 µM, and the cutoff for measurements was 100 µM.
Within this narrow range, there appears to be a trend
such that the most potent NR1A/2B antagonists also
have the higher potency for inhibition of NR1A/2A.
Exceptions include amine 29 that is more active at
NR1A/2A than would have been predicted by its NR1A/
2B potency. Even given this trend, the most potent
NR1A/2B antagonists still have the highest levels of
selectivity with respect to the other two subunit com-
binations. Developing a SAR for inhibition of NR1A/
2C receptors, where IC50 values only range from 30 to
100 µM, is even more fruitless than for the NR1A/2A
receptor. The most potent NR1A/2B antagonists also
appear to have higher potency at NR1A/2C. Again the
obvious exception is 29, which has an IC50 of 3.5 µM at
NR1A/2B but is still among the most potent inhibitors
at NR1A/2C.
Separate studies indicate that high-potency NR1A/
2B antagonism by this class of drug is largely unaffected
by voltage, whereas the low-potency inhibition of NR1A/
2A and NR1A/2C is voltage-dependent.5b,9,10 For the
low-potency antagonism this implies that the primary
site of inhibition is probably located in the channel pore.
Thus, there are no a priori reasons why there should
be parallels between SARs for the different subunit
combinations. To the extent that such relationships
exist, it would suggest that the distinct sites mediating
the high-potency and low-potency inhibition share com-
mon structural features in their ligand binding pockets.
See ref 8.
binant NR1A/2B receptors.8 In contrast, making the
same modification on 20 to give 31 does not produce an
increase in potency; instead, potency is reduced ∼5-fold.
One explanation for this difference is that it is not the
distance between the nitrogen atom and the phenolic
ring that is being optimized with amine 4 but instead
the total distance separating the aromatic rings (Table
2). Thus the increase in potency between 34 and 20
may occur for the same reason as the increase in potency
between ifenprodil and amine 4. Extending the length
of 20 by an additional methylene between the nitrogen
atom and the unsubstituted ring gives 21 and results
in a 5-fold loss of potency. Similarly, extending 30 by
one methylene to give 31 reduces potency 3-fold. There
is no significant difference in potency between 21 and
31. In both cases it would appear that the optimum
distance between the two phenyl groups has been
exceeded and that the additional chain length is now
becoming a steric liability. Finally, substituting the
nitrogen atom in 34 by oxygen gives 15 and results in
a 100-fold reduction in potency. Hence the existence of
effective electrostatic interaction between the nitrogen
atom and the receptor is very important for high
potency.
Intramolecular distances (Table 2) were measured (Å)
corresponding to the fully extended minimized con-
former calculated using AM1 semiempirical calcula-
tions. After geometry optimization, the fully elongated
conformer for amine 20 has an overall length of 17.5 Å
(distance measured from the para carbon of the A-ring
to the hydroxyl oxygen of the B-ring). This length is
approximately 3 Å longer than that calculated for
ifenprodil or nylidrin. The distance from the nitrogen
atom to the phenolic hydroxyl group 20 is ∼7.8 Å. This
distance is an important component leading to effective
binding and is close to the distance observed for nylidrin
and ifenprodil. Comparison of the distances between
the nitrogen atom and the A-ring (see Table 2) indicates
that a hydrophobic pocket presumably interacting at the
A-ring side of the inhibitor should be located within
9-10 Å from the nitrogen binding site. This distance
is about ∼2 Å longer when compared with the piperi-
dine-based molecules (having an out-of-plane equatorial
benzylic moiety). Yet amine 4 (a piperdine-based
molecule) has comparable potency when directly com-
pared to amine 30. Thus the bound secondary amine
inhibitors possibly adopt a bent conformation on the
Con clu sion
For the current series of subtype-selective NMDA
receptor antagonists, the primary determinants of
potency at the 1A/2B NMDA receptors are (1) the
phenolic OH group which presumably serves as a
H-bond donor; (2) the distance between the two aromatic
rings; (3) an electrostatic interaction between the recep-
tor and the nitrogen atom (see Figure 4). Our data

3504 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Tamiz et al.
The combined ether was dried over Na2SO4, and the solvent
was removed in vacuo. Column chromatography (CH2Cl2/
hexane) resulted in the title compound as a transparent oil,
0.59 g (93%): 1H NMR (CDCl3) δ 1.6-1.8 (m, 4H), 2.63 (t, J )
6.9 Hz, 2H), 2.83 (t, J ) 7.2 Hz, 2H), 3.46 (t, J ) 6.0 Hz, 2H),
3.60 (t, J ) 6.9 Hz, 2H), 5.04 (s, 2H), 6.93 (d, J ) 9.0 Hz, 2H),
7.0-7.5 (m, 12H).
2-(4-Hyd r oxyp h en yl)et h yl 4-P h en ylbu t yl E t h er (15).
Ether 14 (0.50 g, 1.4 mmol) and Pd/C (10%, 0.10 g) in MeOH
(30 mL) were shaken in a Parr apparatus under 45 psi of H2
for 12 h. The catalyst was removed by filtration and the
solvent removed in vacuo. Column chromatography (CH2Cl2/
hexane) resulted in the title compound as a transparent oil,
0.32 g (85%): 1H NMR (CDCl3) δ 1.6-1.8 (m, 4H), 2.62 (t, J )
6.9 Hz, 2H), 2.82 (t, J ) 7.2 Hz, 2H), 3.46 (t, J ) 6.0 Hz, 2H),
3.58 (t, J ) 6.9 Hz, 2H), 5.29 (s, 1H), 6.71 (d, J ) 8.9 Hz, 2H),
7.0-7.4 (m, 12H). Anal. (C18H22O2) C, H.
demonstrate that the piperidine ring and alkyl chain
substitutions common to many NR2B-selective antago-
nists such as ifenprodil are not necessary for high
potency and selectivity, while the presence of a basic
nitrogen atom, whether secondary or tertiary, in the
chain does contribute to more potent binding. The
potency and subtype selectivity of this series of bis-
(phenylalkyl)amines provide a framework for designing
other types of novel NR1A/2B-selective antagonists.
Exp er im en ta l Section
Gen er a l. Compounds 1 and 5 were purchased from com-
mercial sources. Compound 3 was prepared according to the
literature procedures.7a Reagents and solvents were purchased
from commercial suppliers and used as received. All starting
materials were commercially available unless otherwise indi-
cated. Melting points were taken on a Mel-Temp melting point
apparatus and are uncorrected. Tetrahydrofuran (THF) was
distilled from blue sodium benzophenone ketyl solution.
Column chromatography was performed in the flash mode on
Davisil silica gel (200-425 mesh), unless otherwise stated.
Yields are of purified product and are not optimized. 1H NMR
spectra were recorded on a 300-MHz Varian spectrometer;
chemical shifts are reported in δ units referenced to residual
proton signals of the deuterated solvents (chloroform-d1, 7.26;
dimethyl-d6 sulfoxide, 2.49; methyl alcohol-d4, 3.31), and
coupling constants are reported in Hz.
5-P h en ylp en ta ld eh yd e (18). The title compound was
prepared from 5-phenyl-1-pentanol (16) by the method de-
scribed for 19: 1H NMR (CDCl3) δ 1.6-1.8 (4H, m), 2.4-2.8
(4H, m), 7.0-7.4 (5H, m), 9.76 (1H, s).
6-P h en ylh exa ld eh yd e (19). A solution of 6-phenyl-1-
hexanol (17; 1.0 g, 5.6 mmol) in CH2Cl2 (10 mL) was added to
a solution of PCC (1.8 g, 8.4 mmol) in CH2Cl2 (30 mL), and
the mixture was stirred at room temperature for 3 h. Ether
(30 mL) was added, and the solution was passed through a
short path of florosile. The solvent was removed in vacuo to
give an oil. Column chromatography (CH2Cl2/hexane) resulted
in the title compound as a transparent oil, 0.40 g (85%): 1H
NMR (CDCl3) δ 1.40 (p, J ) 6.3 Hz, 2H), 1.6-1.8 (m, 4H), 2.42
(dt, J ) 1.2 Hz, 7.8, 2H), 2.62 (t, J ) 7.5 Hz, 2H), 7.0-7.4 (m,
5H), 9.76 (s, 1H).
4-Ben zyl-1-(2-p h en yleth yl)p ip er id in e Hyd r och lor id e
(9). The title compound was prepared from 2-phenylethyl
methanesulfonate13a (7) by the method described for 10: mp
251-252 °C; 1H NMR (CDCl3) δ 1.3-1.4 (m, 2H), 1.4-1.6 (m,
1H), 1.6-1.7 (m, 3H), 1.9-2.0 (m, 2H), 2.5-2.6 (m, 3H), 2.7-
N-[2-(4-Hydr oxyph en yl)eth yl]-5-ph en ylpen tylam in e Hy-
d r och lor id e (20). The title compound was prepared from
aldehyde 18 by the method described for 21: mp 189-190 °C;
1H NMR (CDCl3) δ 1.30 (p, J ) 7.8 Hz, 2 H), 1.59 (m, 4H),
2.56 (t, J ) 7.8 Hz, 2H), 2.7-2.9 (m, 4H), 3.01 (t, J ) 7.2 Hz,
2H), 6.71 (d, J ) 8.7 Hz, 2H), 7.03 (d, J ) 8.4 Hz, 2H), 7.1-
7.3 (m, 5H), 8.71 (2H, s), 9.33 (s, 1H). Anal. (C19H26NClO) C,
H, N.
2.8 (m, 2H), 2.98 (m, 2H), 7.1-7.3 (m, 10H). Anal. (C20H26
NCl) C, H, N.
-
4-Ben zyl-1-[2-(4-(ben zyloxy)ph en yl)eth yl]piper idin e Hy-
d r och lor id e (10). A mixture of 2-[(methylsulfonyloxy)ethyl]-
4-(benzyloxy)benzene13a (8; 0.96 g, 3.5 mmol), 4-benzylpiperi-
dine (6; 0.53 g, 3.0 mmol), and potassium carbonate (1.0 g,
7.5 mmol) in acetonitrile (20 mL) was refluxed for 24 h. The
inorganic salts were removed through a short column of silica
gel and washed with EtOAc (3 × 25 mL). The combined
filtrate was evaporated in vacuo to give a crude mixture.
Column chromatography (20-50% EtOAc in hexanes then 20%
MeOH in EtOAc) resulted in the free base of the title
compound as an oil. A solution of the free base (0.47 g) in
methanolic HCl (5 mL, 5 M) was magnetically stirred for 20
min. The solvent was removed in vacuo to give a white solid
which was precipitated from EtOAc/MeOH to yield the title
compound as a colorless powder, 0.50 g (34%): mp 183-185
°C; 1H NMR (CDCl3) δ 1.72 (m, 1H), 1.80 (d, J ) 12.6 Hz, 2H),
2.11 (m, 2H), 2.57 (brs, 2H), 2.62 (d, J ) 6.9 Hz, 2H), 3.08 (m,
2H), 3.19 (m, 2H), 3.57 (m, 2H), 5.03 (s, 2H), 6.89 (d, J ) 8.4
Hz, 2H), 7.11 (m, 3H), 7.2-7.4 (m, 9H), 12.42 (brs, 1H).
4-Ben zyl-1-[2-(4-h yd r oxyp h en yl)eth yl]p ip er id in e Hy-
d r och lor id e (11). A mixture of piperidine 10 (0.20 g, 0.46
mmol) and Pd/C (10%, 50 mg) in EtOH (95%, 25 mL) was
shaken in a Parr flask under 30 psi of hydrogen for 2 h. The
catalyst was removed through a short column of Celite and
washed with MeOH (3 × 15 mL). The combined filtrate was
evaporated in vacuo to give an oil. This oil was titrated in
ether (30 mL) overnight. The white solid was collected by
filtration and dried in vacuo resulting in 0.16 g (98%) as the
N-[2-(4-Hydr oxyph en yl)eth yl]-6-ph en ylh exylam in e Hy-
d r och lor id e (21). Sodium cyanoborohydride (0.47 g, 7.4
mmol) was added to a stirring solution of aldehyde 19 (0.48 g,
2.7 mmol) and tyramine (0.34 g, 2.5 mmol) in MeOH (20 mL),
and the resulting solution was stirred for 24 h. The solvent
was removed in vacuo. The residual solid was dissolved in
EtOAc (20 mL) and washed with NaHCO3 (saturated in H2O,
2 × 20 mL) and water (20 mL). The solution was dried over
Na2SO4 and the solvent was removed in vacuo. Column
chromatography (EtOAc) resulted in a transparent oil. This
oil was dissolved in CH2Cl2 (5 mL) and treated dropwise with
a solution of HCl/ether (1.0 M, 10 mL). The solid formed was
collected and precipitated from EtOAc/MeOH to yield the title
compound as a white powder, 0.50 g (56%): mp 140-141 °C;
1H NMR (DMSO-d6) δ 1.29 (m, 4H), 1.55 (m, 4H), 2.55 (t, J )
7.5 Hz, 2H), 2.80 (m, 4H), 3.02 (t, J ) 8.4 Hz, 2H), 6.68 (d, J
) 8.7 Hz, 2H), 7.03 (d, J ) 8.1 Hz, 2H), 7.1-7.5 (m, 5H), 8.60
(s, 2H), 9.32 (s, 1H). Anal. (C20H28NClO) C, H, N.
N-[2-(4-Hydr oxyph en yl)eth yl]-4-ph en ylbu tylam in e (34).
A solution of 4-phenylbutyric acid (22; 1.0 g, 6.1 mmol), 1,3-
dicyclohexylcarbodiimide (1.3 g, 6.2 mmol), 1-hydroxybenzo-
triazole (0.84 g, 6.2 mmol), and tyramine (28; 0.85 mg, 6.2
mmol) in DMF (10 mL) was stirred at room temperature for 3
h and then at 60 °C for 24 h. The solid was removed by
filtration. The solution was diluted with H2O (150 mL), and
the yellow oil was extracted with CH2Cl2 (3 × 20 mL). The
combined CH2Cl2 portion was washed with H2O (2 × 50 mL)
and dried over Na2SO4, and the solvent was removed in vacuo
to give a yellow oil. Column chromatography (CH2Cl2/MeOH,
5:1) gave the intermediate N-[2-(4-hydroxyphenyl)ethyl]-4-
phenylbutyramide as a transparent oil, 1.6 g (92%): 1H NMR
(CDCl3) δ 1.93 (p, J ) 8.1 Hz, 2H), 2.13 (t, J ) 7.2 Hz, 2H),
2.60 (t, J ) 7.8 Hz, 2H), 2.71 (t, J ) 7.2 Hz, 2H), 3.48 (q, J )
1
title product: mp 222-224 °C; H NMR (CD3OD) δ 1.48 (m,
2H), 1.89 (m, 3H), 2.62 (d, J ) 6.6 Hz, 2H), 2.9-3.0 (m, 4H),
3.2-3.3 (m, 2H), 3.57 (m, 2H), 6.73 (d, J ) 8.4 Hz, 2H), 7.07
(d, J ) 8.4 Hz, 2H), 7.17 (m, 3H), 7.31 (m, 2H). Anal. (C20H26
NClO) C, H, N.
-
2-(4-(Ben zyloxy)ph en yl)eth yl 4-P h en ylbu tyl Eth er (14).
4-Phenylbutyl p-toluenesulfonate13b (12; 0.80 g, 2.6 mmol) was
added to a stirring solution of 2-(4-(benzyloxy)phenyl)ethanol13c
(13; 0.40 g, 1.8 mmol) and NaH (84 mg, 3.5 mmol) in DMF (5
mL), and the mixture was stirred for 12 h. H2O (30 mL) was
added, and the solution was extracted with ether (3 × 30 mL).

SAR for a Series of Bis(phenylalkyl)amines
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3505
were recorded 3-14 days after injection in a nominally Ca2+
free Ringer’s solution containing (in mM): NaCl, 115; KCl, 2;
BaCl2, 1.8; HEPES, 5 (pH ) 7.4).12 Levels of expression were
as reported previously.9b,10,12a In general, the aim was to limit
antagonist-evoked current responses to ∼100 nA in Ba2+
Ringer (-70 mV): i.e., sufficient current to do accurate
pharmacology without causing appreciable activation of sec-
ondary Ca2+-gated Cl- currents.5b
Potency of antagonism was estimated by measuring reduc-
tions in currents elicited by saturating, or near-saturating,
concentrations of agonists: 10 µM glycine plus 100 µM
glutamate for NR1A/2A; 1 µM glycine plus 100 µM glutamate
for NR1A/2B and NR1A/2C. In all cases the glutamate
concentration was sufficient to give an optimum measure of
potency.11b Inhibition was measured under conditions ap-
proximating steady state on desensitized receptors. Potent
antagonists had slower onset and washout kinetics than weak
antagonists, in some cases requiring >5 min to equilibrate.
Compounds were assayed at multiple concentrations over a
range that spanned the IC50 value. The number of separate
experiments for each compound was taken as the number of
different oocytes examined. Values from concentration-
inhibition experiments were then pooled to avoid issues of
weighting from unequal data sets and fit with logistic equa-
tions. For NR1A/2A and NR1A/2C, where inhibition was
complete and had a single component, data was fit with eq 1:
6.9 Hz, 2H), 5.57 (bt, 1H), 6.80 (d, J ) 6.3 Hz, 2H), 7.00 (d, J
) 8.4 Hz, 2H), 7.1-7.4 (m, 5H), 7.99 (s, 1H).
-
A solution of N-[2-(4-hydroxyphenyl)ethyl]-4-phenylbutyra-
mide (2.0 g, 3.7 mmol) in THF (10 mL) was slowly added to a
stirred suspension of LiAlH4 (1.0 g, 26 mmol) in THF (100 mL)
at room temperature. After addition, the mixture was stirred
at room temperature for 3 h and then at reflux for 24 h. H2O
(10 mL) was slowly added to the mixture. The resulting
precipitate was removed by filtration. The solvent was
removed in vacuo. The resulting yellow oil was partitioned
between H2O (25 mL) and CH2Cl2 (25 mL). The aqueous phase
was separated and extracted with CH2Cl2 (2 × 25 mL). The
combined CH2Cl2 extract was washed with H2O (50 mL), dried
over Na2SO4, and concentrated in vacuo to give a white
powder. Precipitation from EtOAc yielded the title compound
1
34 as a white solid, 0.95 g (50%): mp 113-114 °C; H NMR
(CDCl3) δ 1.56 (m, 4H), 2.61 (m, 4H), 2.73 (t, J ) 7.2 Hz, 2H),
2.85 (t, J ) 6.9 Hz, 2H), 6.25 (d, J ) 8.4 Hz, 2H), 7.02 (d, J )
8.4 Hz, 2H), 7.1-7.3 (m, 5H). Anal. (C18H23NO) C, H, N.
The following examples (29-33) were prepared by the
method described for 34 using the appropriate combination of
the carboxylic acid and the amine. All the compounds were
isolated as their respective HCl salts as described for 21.
N-(4-P h en ylbu tyl)-2-p h en yleth yla m in e h yd r och lor id e
(29): mp 192-194 °C; 1H NMR (CDCl3) δ 1.4-1.8 (m, 4H), 2.59
(t, J ) 6.3 Hz, 2H), 2.92 (m, 4H), 3.09 (t, J ) 6.3 Hz, 2H),
7.0-7.4 (m, 10H), 8.95 (s, 2H). Anal. (C18H24NClO) C, H, N.
N-[3-(4-Hydr oxyph en yl)pr opyl]-4-ph en ylbu tylam in e h y-
I/Icontrol ) 1/{1 + ([antagonist]/IC50)n}
(1)
1
d r och lor id e (30): mp 177-179 °C; H NMR (CDCl3) δ 1.81
(p, J ) 7.5 Hz, 2H), 2.54 (t, J ) 6.9 Hz, 2H), 2.66 (t, J ) 8.1
Hz, 2H), 2.86 (m, 4H), 6.63 (d, J ) 7.2 Hz, 2H), 6.92 (d, J )
8.4 Hz, 2H), 7.3 (m, 5H). Anal. (C19H26NClO) C, H, N.
N-[3-(4-Hyd r oxyp h en yl)p r op yl]-5-p h en ylp en tyla m in e
h yd r och lor id e (31): mp 115-116 °C; 1H NMR (DMSO) δ 1.29
(p, J ) 6.9 Hz, 2H), 1.4-1.6 (m, 4H), 1.81 (p, J ) 7.5 Hz, 2H),
2.4-2.6 (m, 4H), 2.8-3.0 (bm, 4 H), 6.65 (d, J ) 8.1 Hz, 2H),
6.96 (d, J ) 8.1 Hz, 2H), 7.0-7.4 (m, 5H), 8.59 (bs, 2H), 9.21
(s, 1H). Anal. (C20H28NClO) C, H, N.
where I is the measured current, Icontrol is the current in the
absence of antagonist, IC50 is the concentration of drug that
causes 50% inhibition of the control response, and n is the slope
factor of the inhibition curve. For NR1A/2B, where inhibition
had two components, data was fit with eq 2:
I/Icontrol ) min + {(1 - min)/(1 + ([antagonist]/IC50)n)} (2)
where min (minimum) is the residual fractional response at
concentrations of antagonist that are saturating for the first
component of inhibition and IC50 is the concentration that
causes half this level. For reasons that remain unclear,
residual fractional responses could vary from about 0.05 to 0.25
between different oocytes. Given the variability between
oocytes it was impractical to try to define individual min values
for each antagonist. Therefore, to make the analysis more
uniform, min was set at 0.15 for all experiments on NR1A/
2B. This value was chosen as the typical mean level of
residual current in the current study and is also consistent
with previous reports for this class of antagonist.5,10,12a Slope
values for the concentration-inhibition curves were varied
between -1.5 and -0.8. The degree to which differences in
potency measured between two antagonists were significant
was assessed by comparing mean IC50 values, <X1> and <X2>,
N-[2-(4-Hydr oxyph en yl)eth yl]-3-ph en ylpr opylam in e h y-
1
d r och lor id e (32): mp 161-162 °C; H NMR (CDCl3) δ 1.84
(p, J ) 7.4 Hz, 2H), 2.59 (t, J ) 7.8 Hz, 2H), 2.69 (t, J ) 7.2
Hz, 2H), 2.76 (t, J ) 6.3 Hz, 2H), 2.89 (t, J ) 7.2 Hz, 2H), 6.74
(d, J ) 8.4 Hz, 2H), 6.9-7.3 (m, 6H). Anal. (C17H22NClO) C,
H, N.
N-(2-P h en ylet h yl)-3-p h en ylp r op yla m in e h yd r och lo-
r id e (33): mp 270-272 °C; 1H NMR (CDCl3) δ 1.32-1.40 (m,
2H), 1.46 (bs, 1H), 1.6-1.8 (m, 2H), 2.6-2.6 (m, 4H), 2.7-2.9
(m, 2H), 7.1-7.3 (m, 10H). Anal. (C17H22NCl) C, N; H: calcd,
8.04; found, 7.53.
N-Eth yl-N-[2-(4-h yd r oxyp h en yl)eth yl]-4-p h en ylbu tyl-
a m in e (35). A mixture of amine 34 (0.20 g, 0.74 mol),
iodoethane (0.13 g, 0.81 mmol), and NaHCO3 (68 mg, mmol)
in CH3CN was refluxed for 4 h. The solvent was removed in
vacuo, and the resulting oil was dissolved in H2O/CH2Cl2
mixture (1:1, 20 mL). The CH2Cl2 portion was separated, and
the water layer was extracted with CH2Cl2 (2 × 20 mL). The
combined CH2Cl2 portions was dried over Na2SO4, and the
solvent was removed in vacuo to give a yellow oil. Column
chromatography (EtOAc) resulted in the title compound as a
transparent oil, 70 mg (32%): 1H NMR (CDCl3) δ 1.08 (t, J )
7.2 Hz, 2H), 1.60 (m, 4H), 2.5-2.8 (m, 10H), 5.5 (s, 1H), 6.73
(d, J ) 8.4 Hz, 2H), 7.01 (d, J ) 8.7 Hz, 2H), 7.1-7.4 (m, 5H).
Anal. (C20H27NO) C, H, N.
P h a r m a cology. cDNAs encoding the rat NR1A, NR2A,
NR2B, and NR2C subunits were provided cloned into the
pRSSP6 vector (a modified version of the pSP64T vector
created by Dr. Rolf Schoepfer) in which the coding region for
each subunit is attached to a 150-base poly-A tail. The cDNA
template was linearized with MluI and cRNAs transcribed
using the SP6 mMessage mMachine system (Ambion, Austin,
TX). Xenopus oocytes were prepared as described previously.9b
Oocytes were injected with 1:1 mixtures of NR1- and NR2-
encoding cRNA: 0.5-1.5 pg of NR1A/2A; 50-150 pg of NR1A/
2B; 100-300 pg of NR1A/2C. Membrane current responses
2
each with an associated variance, S1 and S22, respectively.
First, the population variance, Sp2, was calculated using eq
3:14
2
2
Sp ) {(n1 - 1)S1 + (n2 - 1)S22}/(n1 + n2 - 2)
(3)
where n1 and n2 are the corresponding numbers of sample
points and n1 + n2 - 2 stands for the degrees of freedom. The
analysis assumes that both means come from populations with
normal distributions and equal variances. To test the two-
tailed hypothesis a t value was then calculated using eq 4:
t ) (<X1> - <X2>)/{Sp2(1/n1 + 1/n2)}1/2
(4)
Values for p were estimated using a built-in function in
Microsoft Excel Software (v. 5.0) to calculate the corresponding
critical values of t. A p value e0.05 was taken as significant.
Ack n ow led gm en t. The cDNAs encoding the rat
NR1A, NR2A, NR2B, and NR2C subunits were a

3506 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Tamiz et al.
(7) (a) Chenard, B. L.; Bordner, J .; Butler, T. W.; Chambers, L. K.;
Collins, M. A.; De Costa, D. L.; Ducat, M. F.; Dumont, M. L.;
Fox, C. B.; Mena, E. E.; Meniti, F. S.; Nielsen, J .; Pagnozzi, M.
J .; Richter, K. E. G.; Ronau, R. T.; Shalaby, I. A.; Stemple, J . Z.;
White, W. F. (1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phen-
ylpiperidino)-1-propanol: a Potent New Neuroprotectant which
Blocks N-methyl-D-aspartate Responses. J . Med. Chem. 1995,
38, 3138-3145. (b) Mott, D.; Zhang, D. S.; Washburn, M. S.;
Fendley, M.; Dingledine, R. CP-101,606 Antagonizes NMDA
Receptors by Interacting with the Polyamine Modulatory Site.
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generous gift from Dr. P. H. Seeburg (University of
Heidelberg, Heidelberg, Germany). We thank Drs. V. I.
Ilyin (CoCensys, Inc.) and J . E. Huettner (Washington
University Medical School) for help with statistical
methodology and Dr. L. Zhang for the preparation of
cRNA. Financial support to the University of Oregon
was provided by CoCensys, Inc.
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