Design, synthesis, and biological evaluation of 3-benzazepin-1-ols as NR2B-selective NMDA receptor antagonists

Article abstract of DOI:10.1002/cmdc.201000005

Cleavage and reconstitution of a bond in the piperidine ring of ifenprodil (1) leads to 7-methoxy-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ols, a novel class of NR2B-selective NMDA receptor antagonists. The secondary amine 7-methoxy-2,3,4,5-tetrahydro-1H-3-be

Full text of DOI:10.1002/cmdc.201000005

DOI: 10.1002/cmdc.201000005
Design, Synthesis, and Biological Evaluation of 3-
Benzazepin-1-ols as NR2B-Selective NMDA Receptor
Antagonists
Bastian Tewes,[a] Bastian Frehland,[a] Dirk Schepmann,[a] Kai-Uwe Schmidtke,[b]
Thomas Winckler,[b] and Bernhard Wꢀnsch*[a]
Cleavage and reconstitution of a bond in the piperidine ring of
ifenprodil (1) leads to 7-methoxy-2,3,4,5-tetrahydro-1H-3-benz-
azepin-1-ols, a novel class of NR2B-selective NMDA receptor
antagonists. The secondary amine 7-methoxy-2,3,4,5-tetrahy-
dro-1H-3-benzazepin-1-ol (12), which was synthesized in six
steps starting from 2-phenylethylamine 3, represents the cen-
tral building block for the introduction of several N-linked resi-
dues. A distance of four methylene units between the basic ni-
trogen atom and the phenyl residue in the side chain results in
high NR2B affinity. The 4-phenylbutyl derivative 13 (WMS-1405,
Ki =5.4 nm) and the conformationally restricted 4-phenylcyclo-
hexyl derivative 31 (Ki =10 nm) represent the most potent
NR2B ligands of this series. Whereas 13 shows excellent selec-
tivity, the 4-phenylcyclohexyl derivative 31 also interacts with
s1 (Ki =33 nm) and s2 receptors (Ki =82 nm). In the excitotoxici-
ty assay the phenylbutyl derivative 13 inhibits the glutamate-
induced cytotoxicity with an IC50 value of 360 nm, indicating
that 13 is an NMDA antagonist.
Introduction
The class of ionotropic glutamate receptors can be divided
into three subtypes: N-methyl-d-aspartate (NMDA), 2-amino-3-
(3-hydroxy-5-methylisooxazol-4-yl)propionate (AMPA), and kai-
nate receptors.[1] The NMDA receptor mediates a number of
important physiological and pathophysiological processes[2] in
the mammalian central nervous system (CNS). Its key role in
these processes renders the NMDA receptor a potential target
for the treatment of neurological disorders.[3]
ment of Parkinson’s disease,[11] traumatic brain injury,[12]
stroke,[13] migraine,[14] alcohol withdrawal,[15] and chronic and
neuropathic pain.[16]
In 1971 ifenprodil (1, Figure 1) was developed as an a1 adre-
noceptor antagonist.[17] However, 1 has been found to interact
with several other receptors and ion channels including 5-HT1A,
5-HT2, s, and NMDA receptors.[18] Subsequently, 1 was shown
The NMDA receptor is a heteromeric complex of four subu-
nits. Three types of subunits have been identified thus far: the
NR1 subunit with eight splice variants (NR1a–h), the NR2 subu-
nit, which has four distinct subtypes (NR2A–D) encoded by
four distinct genes, and the NR3 subunits NR3A and NR3B (two
genes).[4,5] A functional NMDA receptor comprises at least one
NR1 subunit bearing the glycine binding site and one NR2 sub-
unit responsible for glutamate binding.[6,7] Whereas the NR1
subunit is ubiquitously expressed in the CNS, the density of
the different NR2 subunits varies depending on the region of
the CNS. The NR2A subunit is found throughout the whole
CNS, but the NR2B subunit is highly expressed only in the
cortex and hippocampus with a rather low density in the cere-
bellum and hypothalamus. The NR2C subunit predominates in
the cerebellum and both the NR2C and NR2D subunits are
preferentially formed in the brain stem and the spinal cord.[8]
NR3 subunits are expressed predominantly in the developing
CNS and seem to be of no relevance in the adult brain.
Figure 1. Design of 3-benzazepines 2 as NR2B-selective NMDA receptor
antagonists from 1.
to be a selective NR2B antagonist.[19] At first it was generally
accepted that 1 interacts with the polyamine binding site of
the NMDA receptor.[20,21] However, site-directed mutagenesis
experiments have shown that a discrete binding site for 1
exists on the NR2B subunit.[22] However, 1 is a nonselective
[a] Dr. B. Tewes, B. Frehland, Dr. D. Schepmann, Prof. Dr. B. Wꢀnsch
Institut fꢀr Pharmazeutische und Medizinische Chemie der Westfꢁlischen
Wilhelms-Universitꢁt Mꢀnster, Hittorfstraße 58–62, 48149 Mꢀnster (Ger-
many)
Fax: (+49)251-8332144
E-mail: wuensch@uni-muenster.de
Compared with classical unselective NMDA receptor antago-
nists (such as (+)-MK-801,[9] dexoxadrol[10]), NR2B-selective an-
tagonists show decreased cognitive side effects due to the re-
duced expression of this subunit in the cerebellum. This results
in an improved safety profile of these antagonists. Thus NR2B-
selective NMDA antagonists have good potential for the treat-
[b] K.-U. Schmidtke, Prof. Dr. T. Winckler
Institut fꢀr Pharmazie der Friedrich-Schiller-Universitꢁt Jena,
Semmelweisstraße 10, 07743 Jena (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000005.
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687

B. Wꢀnsch et al.
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drug inducing many side effects such as hypotension, cogni-
tive problems, and neurotoxicity.[23] In addition to these side
effects, the bioavailability of 1 is rather low because of its fast
and extensive metabolism.[24,25]
The mechanism for the formation of tetrahydroisoquinoline
8 is outlined in the Supporting Information. An analogous
mechanism for the formation of 2-benzazepines has already
been described.[28] It is assumed that after coordination of the
Lewis acid aluminum trichloride with acid chloride 7, a fast
decarbonylation occurred instead of acylation of the benzene
ring. The resulting iminium ion then reacted with the benzene
ring to afford tetrahydroisoquinoline 8.
As all cyclization reactions with acid chloride 7 led exclusive-
ly to tetrahydroisoquinoline 8, the carboxylic acid 6 was used
directly for the intramolecular Friedel–Crafts acylation. The
reaction of 6 with phosphorus pentoxide provided 3-benzaze-
pin-1-one 9 as the major product, whilst the regioisomer 10
and the tetrahydroisoquinoline 8 were the main side products.
The ratio of regioisomers 9 and 10 was controlled by the re-
action temperature with higher temperatures decreasing the
preference for the regioisomer 9. The reaction of 6 with phos-
phorus pentoxide at 08C yielded the best ratio of 9/10 (90:10;
Table 1). The regioisomers 9 and 10 were separated by recrys-
tallization with ethanol. Whereas the reaction temperature
strongly influenced the ratio of regioisomers 9/10, the amount
of tetrahydroisoquinoline 8 was almost constant at different
reaction temperatures.
With the aim of increasing the selectivity of 1 without losing
affinity toward NR2B subunit containing NMDA receptors (Ki =
10 nm), a new class of NR2B-selective NMDA receptor antago-
nists with reduced conformational flexibility was designed
(Figure 1). To decrease the conformational freedom of the eth-
ylene spacer between the phenol and the piperidine moiety,
the C3ÀC4 bond of the piperidine ring was formally cleaved
and the resulting open chain was connected to the phenol
leading to a 3-benzazepine scaffold (2). Herein we report the
synthesis, structural modification, and NR2B receptor affinity of
novel 3-benzazepines of type 2. After establishing structure–
affinity relationships, the receptor selectivities and intrinsic
activities of the most promising ligands were investigated.
Chemistry
The designed 3-benzazepin-1-ols 2 were synthesized from 2-(3-
methoxyphenyl)ethan-1-amine (3, Scheme 1). Primary amine 3
was protected with a toluenesulfonyl group according to pub-
lished procedures.[26] The N-tolylsulfonyl protecting group elim-
inates the basicity of the nitrogen atom in 4, thereby allowing
the planned ring closure to be performed via Friedel–Crafts
acylation. Sulfonamide 4 was then treated with excess ethyl
bromoacetate in the presence of potassium carbonate to give
ester 5, which was saponified with sodium hydroxide to afford
carboxylic acid 6.[27]
Table 1. Optimization of the intramolecular Friedel–Crafts acylation of 6
using P2O5.
T [8C]
Yield 9/10 [%]
Yield 8 [%]
Ratio 9:10
0
10
RT
49
46
47
19
20
28
90:10
80:20
70:30
An intramolecular Friedel–Crafts acylation of carboxylic acid
6 should provide the 3-benzazepine scaffold. To enhance the
electrophilicity of the carboxy group, carboxylic acid 6 was
converted with thionyl chloride into acid chloride 7, which was
directly treated with aluminum trichloride. Surprisingly, in con-
trast to reported observations,[27] tetrahydroisoquinoline 8 was
formed during this reaction instead of the described 3-benz-
azepine 9 (Scheme 1).
In the next step ketone 9 was reduced with sodium borohy-
dride to yield 3-benzazepinol 11.[27] The N-tolylsulfonyl protect-
ing group of 11 was removed with activated[29] magnesium in
boiling methanol[30] to get the secondary amine 12 (Scheme 2).
The secondary amine 12 represents the central building
block for the synthesis of 3-benzazepin-1-ols with various N-
linked residues. A large number
with differently substituted 3-
benzazepines is required for the
establishment of reliable struc-
ture–affinity relationships. In the
first series of compounds the
distance between the basic ni-
trogen atom of the 3-benzaze-
pine scaffold and phenyl residue
in the side chain was systemati-
cally increased from one to five
methylene units. As the 4-phe-
nylbutyl derivative 13, which has
the same NÀPh distance as the
lead compound 1 (see Figure 1),
showed the highest affinity in
this series (see Figure 2), sub-
Scheme 1. Reagents and conditions: a) p-toluenesulfonyl chloride, pyridine, RT, 1.5 h; b) ethyl bromoacetate, ace-
tone, K2CO3, reflux, 20 h; c) NaOH, 50% EtOH, reflux, 6 h; d) SOCl2, benzene, reflux, 8 h; e) AlCl3, 1,2-dichloro-
ethane, À658C, 20 h; f) P2O5, 1,2-dichloroethane, 08C, 24 h.
stituents with a spacer of 4–5
methylene groups (or heteroa-
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NR2B-Selective Antagonists
Table 2. Synthesis of 3-benzazepines by alkylation of 12 with RX.
Compd
R
Y
X
Yield [%]
45
13
CH2
Cl
(WMS-1405)
Scheme 2. Reagents and conditions: a) NaBH4, CH3OH, RT, 2 h; b) Mg0,
CH3OH, reflux, 16 h.
14
15
16
O
S
SO2
Br
Cl
Cl
79
95
68
17
18
19
CH2
S
SO2
Cl
Cl
Cl
93
96
92
20
21
22
tBu
OCH3
F
Cl
Cl
Cl
80
79
74
23
24
–
–
Cl
Br
34
61
25
26
–
–
Cl
Br
81
91
Figure 2. Correlation between NR2B affinity and the distance between the
nitrogen atom and phenyl residue in the side chain.
octyl
tom equivalents) were preferentially selected. 3-Benzazepines
13–27 were synthesized by nucleophilic substitution of secon-
dary amine 12 with various haloalkanes (Scheme 3 and
Table 2). The transformations were performed in acetonitrile at
reflux. In the case of chloroalkanes, tetra(n-butyl)ammonium
iodide was added to the reaction mixture to activate the chlor-
oalkanes through in situ generation of iodoalkanes (Finkelstein
reaction[31]).
27
–
Br
91
Table 3. Synthesis of 3-benzazepines by reductive alkylation of 12.
Compd
28
Yield [%]
3-Benzazepines 28–32 were synthesized by reductive alkyla-
tion of secondary amine 12 with different aldehydes and a
ketone in the presence of sodium triacetoxyborohydride[32]
(Scheme 3 and Table 3). For the reductive alkylation of 12 with
76
81
29
30
31
82
69
32
97
Scheme 3. Reagents and conditions: a) RX (see Table 2), CH3CN, K2CO3,
(nBu)4NI, reflux, 8–72 h; b) various aldehydes and a ketone (see Table 3),
NaBH(OAc)3, 1,2-dichloroethane, RT, 3 h; c) ClCO2Bn, CH2Cl2, NEt3, RT, 16 h;
d) 4-phenylbutyric acid, EDC·HCl, CH2Cl2, RT, 6 h.
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B. Wꢀnsch et al.
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4-phenylcyclohexanone, acetic acid was added to the reaction
mixture to increase the activity of the ketone. The 1H NMR
spectrum of the resulting N-(4-phenylcyclohexyl) derivative 31
shows the signals of two diastereomers, cis-31 and trans-31, at
a ratio of 70:30, which were used in the receptor binding stud-
ies without separation.
Receptor Affinity
Affinity toward NR2B-containing NMDA receptors
The NR2B receptor affinity of the synthesized 3-benzazepines
12–36 was determined in a competitive receptor binding
assay recently developed in our group.[38] In this assay tritium-
labeled [3H]ifenprodil was employed as the radioligand. Mem-
brane homogenates prepared upon ultrasonic irradiation of
L(tkÀ) cells stably expressing recombinant human NR1a/NR2B
receptors served as receptor material.[39] The high density of
NMDA receptors renders this system selective. The
expression of NMDA receptors at the cell surface was
induced by addition of dexamethasone to the
growth medium. During this period cell death was
inhibited by addition of the NMDA antagonist keta-
mine (phencyclidine binding site) to the growth
medium.
A further variation of the N-linked substituent was achieved
upon hydrolysis of the cyclic ketals 20 and 21 with 1m hydro-
chloric acid to obtain ketones 35 and 36, respectively
(Scheme 4). The direct reaction of the secondary amine 12
with the commercially available g-chloroketones 37–39 result-
The receptor affinities of 3-benzazepines are sum-
marized in Table 4. The secondary amine 12 does not
exhibit considerable affinity toward NR2B containing
NMDA receptors. However, substitution of 12 with
various arylalkyl moieties led to derivatives with high
NR2B affinity (for example, 13, 17, 21, 31, and 35). In
Figure 2 the NR2B affinity of compounds 13, 17, and
26–29 is correlated with the distance between the
basic nitrogen atom and the phenyl group in the
side chain. A distance of 4–5 methylene units proved
to be optimal and resulted in the potent NR2B an-
tagonists 13 (WMS-1405, Ki =5.4 nm) with a 4-phe-
Scheme 4. Reagents and conditions: a) 2,2-dimethylpropane-1,3-diol, toluene, p-toluene-
sulfonic acid, reflux, 4–8 h; b) 12, CH3CN, K2CO3, (nBu)4NI, reflux, 50–72 h; c) 1m HCl, Et2O,
RT, 16 h.
ed in very low yields of the desired ketones 23, 35, and 36 but
with a major side product. After reaction of the tert-butyl
ketone 37, the ketone 43 bearing a hydroxy moiety instead of
a chloro group was isolated and identified as the major prod-
uct. We assume that the intermediate formation of a five-mem-
bered oxonium ion is the driving force for the formation of hy-
droxy ketone side products such as 43. The electron-donating
tert-butyl moiety of 37 supports the formation of the oxonium
ion and ultimately the formation of 43. The electron-withdraw-
ing fluoro substituent of 39 inhibits oxonium ion formation
and explains the moderate yield of 23 (34%) after direct reac-
tion with the secondary amine 12. Thus, acetalization of ke-
tones 37–39 to get ketals 40–42[33,34] completely suppressed
the formation of hydroxy ketones such as 43 and provided the
substitution products 20–22 in high yields.
nylbutyl residue and 17 (Ki =29 nm) with a 5-phenylpentyl resi-
due. A simple octyl chain (compound 26) was not tolerated by
the NR2B receptor. Consequently, N-arylalkyl substituents with
four or five methylene groups (or heteroatom equivalents) in
the side chain were selected for the establishment of struc-
ture–affinity relationships.
The replacement of the methylene group at position 4 of
the 4-phenylbutyl side chain of 13 with an oxygen (in 14) or
sulfur atom (in 15) led to a drastic decrease in affinity for NR2B
by a factor of 41 (compound 14) or even 346 (compound 15).
The same effect was observed with the homologous 5-phenyl-
pentyl derivative 17. Reasons for the decreased NR2B affinity
of these derivatives might be the high electron density of the
phenyl group, the increased polarity of the spacer or an alter-
nate conformation of the spacer.
Potent and selective NR2B antagonists without a basic
amine have been described.[35,36] Therefore, carbamate 33 and
amide 34 were synthesized and their NR2B receptor affinities
were determined (Scheme 3). 3-Benzazepine 33 was prepared
by acylation of secondary amine 12 with benzyl chloroformate.
The acylation of secondary amine 12 with 4-phenylbutyric acid
was performed using the coupling reagent N-ethyl-N’-(3-di-
methylaminopropyl)carbodiimide hydrochloride (EDC·HCl).[37]
The butyrophenone derivatives 23, 35, and 36 together with
the corresponding ketals 20–22 were synthesized to investi-
gate the influence of different aromatic substituents (F, OCH3,
tBu) on NR2B affinity. Compound 35, with the bulky tert-butyl
substituent, displays the highest NR2B affinity in this group
(Ki =19 nm), demonstrating that the NR2B receptor can toler-
ate a sterically large group at the para position of the phenyl
group. The slightly lower affinities of the corresponding ketals
20–22 (Ki =32–83 nm) indicate that the bulky 5,5-dimethyl-1,3-
dioxane ring at position 4 of the side chain is also tolerated by
the NR2B binding site.
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NR2B-Selective Antagonists
the 4-phenylcyclohexyl deriva-
tive 31 (cis/trans=70:30) was
highly potent (Ki =10 nm). The
high affinity of the 4-phenylcy-
clohexyl derivative 31 indicates
that the conformationally re-
stricted side chain fits well with-
out adaptation to the ifenprodil
binding pocket of the NR2B
containing NMDA receptor.
Table 4. Affinities of 3-benzazepin-1-ols for the ifenprodil binding site of NR2B-containing NMDA receptors,
the PCP binding site of the NMDA receptor, and for s1 and s2 receptors.
Ki ÆSEM [nm]
Compd
R
NR2B
PCP
s1
s2
12
13
H
15%[a]
5.4Æ0.4
23%[b]
22%[b]
23%[a]
182Æ38
0%[a]
554Æ127
(CH2)4-C6H5
(WMS-1405)
14
15
16
17
18
19
(CH2)3-O-C6H5
(CH2)3-S-C6H5
(CH2)3-SO2-C6H5
(CH2)5-C6H5
(CH2)4-S-C6H5
(CH2)4-SO2-C6H5
222Æ49.0
1880
934
29Æ2.0
1090
29%[a]
35%[a]
0%[a]
35%[a]
33%[a]
32%[a]
178Æ14
155
21%[a]
65Æ7.1
95Æ29
249
376
1520
The carbamate 33 and the
amide 34 with an N-aryl dis-
tance of four carbon atoms
were prepared as it has been
shown that a basic amine is not
essential for high NR2B affini-
ty.[33,34] However, the negligible
affinity of 33 and 34 indicates
that in the 3-benzazepine class
of NR2B antagonists the basic
amine cannot be omitted.
32%[a]
305
308Æ67
1410
0%[a]
20
21
22
48Æ14
32Æ3.6
83Æ32
0%[a]
32%[a]
46%[a]
69
602
46
179
1260
103
Considering the affinity for
the NR2B receptor, phenylbutyl
compound 13 (Ki =5.2 nm), phe-
nylcyclohexyl compound 31
(Ki =10 nm),
phenyloxobutyl
compound 36 (Ki =19 nm), phe-
nylpentyl compound 17 (Ki =
29 nm), and dioxane derivative
21 (Ki =32 nm) represent the
five most potent compounds of
this series.
23
24
25
26
27
28
29
30
(CH2)3-CO-C6H4-4-F
CH2-C6H4-3-C6H5
CH2-CO-N(CH3)-CH2-C6H5
(CH2)7CH3
150Æ57
12%[a]
22%[a]
0%[a]
0%[a]
0%[a]
1100
6%[a]
1%[b]
34%[a]
1%*[a]
29%[a]
26%[a]
452
29%[a]
1140
14%[a]
108Æ52
497
6540
1450
1000
8%[a]
45Æ12
280
33Æ3.1
234
(CH2)3-C6H5
CH2-C6H5
(CH2)2-C6H5
3400
25%[a]
31%[a]
19%[a]
Receptor Selectivity
(CH2)3-O-CH2-C6H5
434Æ88
31
10Æ1.5
23%[a]
33Æ17
82Æ26
To investigate the selectivity of
this new class of NR2B ligands,
the compounds were tested
against the phencyclidine (PCP)
binding site of the NMDA recep-
tor[40] and both s receptor sub-
types (s1 and s2)[40,41] in receptor
binding studies with radio-
ligands. In Table 4 the PCP, s1,
and s2 receptor affinities of 3-
benzazepines 12–36 are com-
pared with their NR2B receptor
affinities. The 3-benzazepines do
not reveal significant interac-
tions with the PCP binding site
of the NMDA receptor, indicat-
ing high selectivity for the poly-
amine binding site over the
32
91Æ8.0
0%[a]
135Æ50
238
33
34
35
36
1
CO2-CH2-C6H5
CO-(CH2)3-C6H5
(CH2)3-CO-C6H4-4-C(CH3)3
(CH2)3-CO-C6H4-4-OCH3
ifenprodil
1180
19%[a]
19Æ6.0
341Æ64
10Æ0.7
13Æ2.5
–
28%[a]
0%[a]
43%[a]
44Æ8.1
12%[a]
125Æ24
–
0%[a]
0%[a]
267Æ39
2230
98.3Æ34
–
44%[a]
0%[a]
9%[b]
–
–
eliprodil
dexoxadrol
haloperidol
32Æ7.4
–
–
–
–
–
–
6.3Æ1.6
89Æ29
78.1Æ2.3
57.5Æ18
di-o-tolylguanidine (DTG)
[a] Due to low affinity, only the inhibition at a concentration of 1 mm is given. [b] Due to low affinity, only the
inhibition at a concentration of 10 mm is given.
Next, we investigated the NR2B affinity of 3-benzazepines
with conformationally constrained side chains. Whereas the bi-
phenylmethyl derivative 24 and compound 25 with an amide
in the side chain did not reveal significant NR2B interaction,
phencyclidine binding site of the NMDA receptor. The affinities
at the s2 receptor are also generally very low. In particular, the
most potent NR2B ligands show excellent selectivity against s2
receptors. Whereas the NR2B/s2 selectivity factors for 31 (8),
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17 (11), and 35 (14) are approximately 10, the corresponding
selectivity factors of 13 and 21 are 102 and 40, respectively.
The s1 receptor affinity of the 3-benzazepine compound
class has to be discussed separately. With NR2B/s1 selectivity
factors of 34 and 19, the very potent NR2B antagonists 13 and
21 reveal high selectivity for the NR2B receptor over the s1 re-
ceptor. However, the s1 affinities of phenylpentyl derivative 17,
phenylcyclohexyl derivative 31, and ketone 35 are in the same
range as their NR2B affinities [NR2B/s1: 2 (17), 3 (31), 2 (35)].
In addition to selective and unselective NR2B ligands, com-
pounds with a preference for the s1 receptor were uncovered
in the 3-benzazepine compound class. In particular, 15 and 18
with a sulfur atom in the side chain as well as 27, 28, and 29
with side chains shorter than butyl display a greater than ten-
fold preference for the s1 receptor over the NR2B receptor.
These results indicate that small structural modifications can
shift the receptor profile from an NR2B-selective ligand to a s1-
selective ligand. Therefore, investigation of both the NR2B and
s1 affinity is required during the development of novel selec-
tive NR2B and s1 receptor ligands.
Figure 3. Inhibition of NMDA receptor-mediated cell toxicity by compound
13. Different concentrations of the potent and selective ligand 13 were ap-
plied to L13-E6 cells, and NMDA receptors were activated by the addition of
(S)-glutamate and glycine. Excitotoxicity was determined by the amount of
lactate dehydrogenase (LDH) released. Compound 13 has an IC50 value of
360 nm.
The receptor binding profile of the phenylbutyl derivative
13 was investigated because of the promising NR2B affinity
(Ki =5.4 nm) and selectivity toward the s and the PCP recep-
tors. In competition experiments, the interaction of 13 with 15
relevant receptor systems including a1, D1, D2, NMDA (PCP
binding site), NMDA (glycine binding site), k-opioid, m-opioid,
d-opioid, 5-HT1A, 5-HT2, s1, and s2 receptors, with the noradre-
nalin and serotonin transporter, as well as with monoamine ox-
idase A was investigated. At a concentration of 100 nm 13 did
not compete significantly with the radioligands employed.
These results are of particular interest, as low receptor selec-
tivity is one of the major drawbacks of 1. Clearly, the decreased
conformational flexibility of the ethylene spacer in the 3-benz-
azepine 13 significantly reduces interaction with a1, 5-HT1A, 5-
HT2, s1, s2, and PCP receptors.
than 10%. Clearly, 13 does not interact with NMDA receptors
consisting of NR2A subunits.
Conclusions
A new compound class of NR2B-selective NMDA receptor an-
tagonists has been identified. The most promising compound
is 7-methoxy-3-(4-phenylbutyl)-2,3,4,5,-tetrahydro-1H-3-benza-
zepin-1-ol (13) showing high affinity (Ki =5.4 nm) toward NR2B
receptors. In contrast to the lead compound 1, benzazepine
13 does not interact with a1, NMDA (PCP binding site), 5-HT1A,
5-HT2, s1, and s2 receptors. In an assay using cells stably ex-
pressing only NR1a and NR2B subunits, 13 was able to inhibit
(IC50 =360 nm) the excitotoxicity caused by (S)-glutamate and
glycine, indicating the NMDA antagonistic activity of 13.
3-Benzazepine 13 represents a very promising new lead,
which will be further optimized by introduction of crucial
structural elements of the lead compound ifenprodil. At first
the methoxy group of 13 will be converted into a hydroxy
moiety, as a hydrogen bond donor at the aromatic system is
generally favorable for potent NR2B ligands. Secondly, the
methyl group of ifenprodil, which has been omitted in the first
series of NR2B ligands, will be added at position 8 of the 3-
benzazepine system. At the final stage, the most promising li-
gands will be prepared in enantiomerically pure form.
Functional Activity[42]
To verify the antagonistic activity of the potent NR2B-selective
ligand 13 the inhibition of excitotoxicity was investigated. In
this assay L(tkÀ)cells stably expressing NR1a/NR2B receptors
were employed. After addition of (S)-glutamate and glycine
the excitotoxicity was determined by the amount of lactate
dehydrogenase (LDH) released from the cells into the culture
supernatant. Different concentrations of the test compound 13
were added 30 min before addition of (S)-glutamate and gly-
cine and the release of LDH was measured.
In Figure 3 the excitotoxicity at different compound concen-
trations is shown. With an IC50 value of 360 nm, 13 inhibits the
cytotoxic effects of (S)-glutamate and glycine. This result
proves the NMDA receptor antagonistic activity of 13 and
shows that 13 not only binds at the polyamine binding site,
but also produces antagonistic effects.
Experimental Section
General synthesis protocols
Unless otherwise noted, moisture-sensitive reactions were conduct-
ed under dry N2. Flash chromatography (FC): silica gel 60, 40–
64 mm (Merck); parentheses include diameter of the column,
eluent, fraction size, Rf value. 1H NMR (400 MHz), 13C NMR
(100 MHz): Unity Mercury Plus 400 spectrometer (Varian); d in ppm
The same experiment was performed with L12-G10 cells
stably expressing NR1a/NR2A receptors. At a concentration of
10 mm, excitotoxicity inhibited by compound 13 was lower
692
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ChemMedChem 2010, 5, 687 – 695

NR2B-Selective Antagonists
relative to (CH3)4Si; coupling constants are given at a resolution of
0.5 Hz. According to HPLC analysis, the purity of all test com-
pounds is >95%.
1H, 4-H), 2.79 (d, J=12.2 Hz, 1H, 2-H), 3.12–3.27 (m, 3H, 2-H, 4-H,
5-H), 3.71 (s, 3H, OCH3), 4.52 (d, J=5.9 Hz, 1H, 1-H), 6.55–6.61 (m,
2H, 6-H and 8-H), 7.02–7.08 ppm (m, 1H, 9-H) signals for the NH
and OH protons are not visible; 13C NMR (CDCl3): d=39.5 (1C, C5),
49.7 (1C, C4), 54.0 (1C, C2), 55.4 (1C, OCH3), 74.4 (1C, C1), 110.3
(1C, C8), 117.1 (1C, C6), 130.2 (1C, C9), 135.9 (1C, C9a), 142.1 (1C,
C5a), 159.1 ppm (1C, C7); C11H15NO2 (193.2).
6-Methoxy-2-(4-methylphenylsulfonyl)-1,2,3,4-tetrahydroisoqui-
noline (8); 7-methoxy-3-(4-methylphenylsulfonyl)-2,3,4,5-tetra-
hydro-3-benzazepin-1-one (9); and 9-methoxy-3-(4-methylphe-
nylsulfonyl)-2,3,4,5-tetrahydro-3-benzazepin-1-one (10): Under
N2 the acid 6 (1.0 g, 2.76 mmol) was dissolved in CH2Cl2 (20 mL)
and cooled to 08C. Afterward P2O5 (1.96 g, 13.8 mmol) was added,
and the mixture was stirred for 24 h at 08C. Subsequently 3%
NaOH was added to the suspension to give pH 13–14. The mixture
was extracted with CH2Cl2 (3ꢂ40 mL); the CH2Cl2 layer was washed
with H2O (3ꢂ50 mL), dried (Na2SO4), and concentrated in vacuo.
The residue was purified by FC (4 cm, n-hexane/EtOAc 7:3 and 2%
N,N-dimethylethylamine, 50 mL, Rf (8)=0.57) and Rf (9 and 10)=
0.23). 9 and 10 were separated by recrystallization from EtOH.
7-Methoxy-3-(4-phenylbutyl)-2,3,4,5,-tetrahydro-1H-3-benzaze-
pin-1-ol (13=WMS-1405): A mixture of the secondary amine 12
(100.0 mg, 0.52 mmol), CH3CN (10 mL), (nBu)4NI (11.1 mg,
0.03 mmol), K2CO3 (575.0 mg, 4.16 mmol), and 1-chloro-4-phenyl-
butane (110.7 mL, 0.62 mmol) was heated at reflux for 48 h. It was
then filtered, and the solvent was evaporated in vacuo. The residue
was purified by FC (2 cm, n-hexane/EtOAc 8:2 and 1% N,N-dimeth-
ylethylamine, 10 mL, Rf =0.15) to afford 13 (76.1 mg, 45%) as color-
1
less solid, mp: 768C; H NMR (CDCl3): d=1.54–1.69 (m, 4H, N-CH2-
CH2-CH2-CH2-Ph), 2.39 (t, J=11.9 Hz, 1H, 4-H), 2.50 (d, J=12.1 Hz,
1H, 2-H), 2.58–2.66 (m, 5H, N-CH2-CH2-CH2-CH2-Ph and 5-H), 2.99
(dd, J=12.2/6.0 Hz, 1H, 4-H), 3.14–3.18 (m, 1H, 2-H), 3.26 (brt, J=
13.4 Hz, 1H, 5-H), 3.77 (s, 3H, OCH3), 4.57 (d, J=6.7 Hz, 1H, 1-H),
6.64–6.66 (m, 2H, 6-H and 8-H), 7.09 (d, J=7.8 Hz, 1H, 9-H), 7.17–
7.20 (m, 3H, 3-H phenyl, 4-H phenyl and 5-H phenyl), 7.27–
7.04 ppm (m, 2H, 2-H phenyl and 6-H phenyl) a signal for the OH
proton is not visible; 13C NMR (CDCl3): d=26.8 (1C, N-CH2-CH2-CH2-
CH2-Ph), 29.4 (1C, N-CH2-CH2-CH2-CH2-Ph), 36.0 (1C, N-CH2-CH2-CH2-
CH2-Ph), 37.1 (1C, C5), 55.4 (1C, OCH3), 56.3 (1C, C4), 59.9 (1C, N-
CH2-CH2-CH2-CH2-Ph), 61.0 (1C, C2), 72.6 (1C, C1), 110.4 (1C, C6),
116.8 (1C, C8), 126.0 (1C, C4 phenyl), 128.6 (2C, C3 phenyl and C5
phenyl), 128.6 (2C, C2 phenyl and C6 phenyl), 130.0 (1C, C9), 135.7
(1C, C9a), 141.4 (1C, C1 phenyl), 142.5 (1C, C5a), 159.1 ppm (1C,
C7); HRMS (ESI): calcd for C21H28NO2 326.2052, found 326.2115
[M+H]+; C21H27NO2 (325.2).
1
8 (Rf =0.57): Colorless solid, mp: 1138C, yield 0.12 g (14%); H NMR
(CDCl3): d=2.41 (s, 3H, Ph-CH3), 2.89 (t, J=5.9 Hz, 2H, 4-H), 3.32 (t,
J=5.9 Hz, 2H, 3-H), 3.74 (s, 3H, OCH3), 4.17 (s, 2H, 1-H), 6.59 (d, J=
2.3 Hz, 1H, 5-H), 6.71 (dd, J=8.5/2.6 Hz, 1H, 7-H), 6.92 (d, J=
8.5 Hz, 1H, 8-H), 7.31 (d, J=8.0 Hz, 2H, 3-H toluenesulfonyl and 5-
H toluenesulfonyl), 7.71 ppm (d, J=8.3 Hz, 2H, 2-H toluenesulfonyl
and 6-H toluenesulfonyl); C17H19NO3S (317.4).
9 (Rf =0.23): Colorless solid (EtOH), mp: 1688C, yield 0.37 g (39%);
1H NMR (CDCl3): d=2.37 (s, 3H, Ph-CH3), 2.95 (t, J=6.6 Hz, 2H, 5-
H), 3.67 (t, J=6.6 Hz, 2H, 4-H), 3.84 (s, 3H, OCH3), 4.17 (s, 2H, 2-H),
6.62 (d, J=2.7 Hz, 1H, 6-H), 6.75 (dd, J=8.6/2.7 Hz, 1H, 8-H), 7.12
(d, J=8.2 Hz, 2H, 3-H toluenesulfonyl and 5-H toluenesulfonyl),
7.45 (d, J=8.3 Hz, 2H, 2-H toluenesulfonyl and 6-H toluenesulfon-
yl), 7.48 ppm (d, J=8.8 Hz, 1H, 9-H); C18H19NO4S (345.1).
7-Methoxy-3-(4-methylphenylsulfonyl)-2,3,4,5-tetrahydro-1H-3-
benzazepin-1-ol (11): Ketone 9 (1.0 g, 2.90 mmol) was suspended
in abs CH3OH (15 mL) and NaBH4 (0.230 g, 6.1 mmol) was added in
several portions. After stirring for 2 h at room temperature, the sol-
vent was evaporated in vacuo. H2O (30 mL) was added to the resi-
due, and the mixture was extracted with CHCl3 (4ꢂ30 mL). The or-
ganic layer was washed with H2O (3ꢂ40 mL), dried (Na2SO4), and
concentrated in vacuo. The residue was purified by FC (6 cm,
CH2Cl2/Et2O 9.5:0.5, 50 mL, Rf =0.15) to afford 11 (0.98 g, 97%) as a
7-Methoxy-3-(5-phenylpentyl)-2,3,4,5-tetrahydro-1H-3-benzaze-
pin-1-ol (17): A mixture of 12 (103.9 mg, 0.54 mmol), CH3CN
(10 mL), K2CO3 (431.2 mg, 3.12 mmol), TBAI (144.1 mg, 0.39 mmol),
and 1-chloro-5-phenylpentane (104.5 mL, 0.58 mmol) was heated at
reflux for 39 h. It was then filtered, and the solvent was evaporated
in vacuo. The residue was purified by FC (3 cm, n-hexane/EtOAc
7:3 and 1% N,N-dimethylethylamine, 30 mL, Rf =0.28) to afford 17
1
(170.3 mg, 93%) as colorless oil; H NMR (CDCl3): d=1.31–1.38 (m,
1
2H, N-CH2-CH2-CH2-CH2-CH2-Ph), 1.48–1.56 (m, 2H, N-CH2-CH2-CH2-
CH2-CH2-Ph), 1.58–1.67 (m, 2H, N-CH2-CH2-CH2-CH2-CH2-Ph), 2.38 (t,
J=11.8 Hz, 1H, 4-H), 2.48 (d, J=12.0 Hz, 1H, 2-H), 2.53–2.65 (m,
5H, 5-H and N-CH2-CH2-CH2-CH2-CH2-Ph), 2.98 (ddt, J=12.3/6.1/
2.1 Hz, 1H, 4-H), 3.14 (ddd, J=12.0/6.8/1.9 Hz, 1H, 2-H), 3.24 (brt,
J=12.5 Hz, 1H, 5-H), 3.76 (s, 3H, OCH3), 4.55 (d, J=6.7 Hz, 1H, 1-
H), 6.62–6.65 (m, 2H, 6-H and 8-H), 7.10 (d, J=7.8 Hz, 1H, 9-H),
7.14–7.18 (m, 3H, 3-H phenyl, 4-H phenyl and 5-H phenyl), 7.25–
7.29 ppm (m, 2H, 2-H phenyl and 6-H phenyl) a signal for the OH
proton is not visible; C22H29NO2 (339.2).
colorless solid, mp: 988C; H NMR (CDCl3): d=2.39 (s, 3H, Ph-CH3),
2.83 (dd, J=15.1/7.2 Hz, 1H, 5-H), 3.08 (m, 1H, 4-H), 3.23 (d, J=
13.2 Hz, 1H, 2-H), 3.29 (m, 1H, 5-H), 3.61 (m, 1H, 4-H), 3.70 (dd, J=
12.9/7.0 Hz, 1H, 2-H), 3.76 (s, 3H, OCH3), 4.83 (d, J=6.3 Hz, 1H, 1-
H), 6.62 (d, J=2.4 Hz, 1H, 6-H), 6.69 (dd, J=8.4/2.5 Hz, 1H, 8-H),
7.21 (d, J=8.2 Hz, 1H, 9-H), 7.28 (d, J=8.2 Hz, 2H, 3-H toluenesul-
fonyl and 5-H toluenesulfonyl), 7.65 ppm (d, J=8.2 Hz, 2H, 2-H tol-
uenesulfonyl and 6-H toluenesulfonyl) a signal for the OH proton is
not visible; C18H21NO4S (347.1).
7-Methoxy-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ol (12): Before
use the surface of Mg turnings was activated by short treatment
with 0.01m HCl to remove MgO and subsequent washing with
H2O, dry CH3OH, and dry Et2O.[30] Mg turnings (2.83 g, 0.12 mol)
were added to a solution of 11 (1.85 g, 5.33 mmol) in abs CH3OH
(100 mL), and the mixture was heated at reflux for 5 h. Then, under
cooling with ice concd H2SO4 (6.55 mL) was added. The mixture
was filtered and adjusted with NaOH to pH 9–10. The aqueous
layer was extracted with CH2Cl2 (5ꢂ30 mL) and dried (Na2SO4). The
solvent was evaporated in vacuo, and the residue was purified by
FC (4.5 cm, CH2Cl2/CH3OH 19:1 and 2% NH3, 65 mL, Rf =0.11) to
afford 12 (0.80 g, 78%) as a colorless solid, mp: 1238C; 1H NMR
(CDCl3): d=2.58 (dd, J=15.3/5.9 Hz, 1H, 5-H), 2.72 (t, J=12.1 Hz,
3-Benzyl-7-methoxy-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ol
(28): A solution of 12 (100.0 mg, 0.52 mmol) in 1,2-dichloroethane
(1 mL) was treated with benzaldehyde (188.9 mL, 0.52 mmol) and
NaBH(OAc)3 (164.8 mg, 0.78 mmol). The mixture was stirred for 3 h
at room temperature. A saturated solution of NaHCO3 (10 mL) and
H2O (10 mL) were then added, the layers were separated, and the
aqueous layer was extracted with CH2Cl2 (3ꢂ10 mL). The combined
organic layers were dried (Na2SO4), the solvent was evaporated in
vacuo, and the residue was purified by FC (3 cm, n-hexane/EtOAc
8:2 and 1% N,N-dimethylethylamine, 20 mL, Rf =0.25) to afford 28
1
(111.9 mg, 76%) as a colorless oil; H NMR (CDCl3): d=2.43 (t, J=
11.9 Hz, 1H, 4-H), 2.57 (d, J=12.0 Hz, 1H, 2-H), 2.71 (dd, J=15.2/
ChemMedChem 2010, 5, 687 – 695
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693

B. Wꢀnsch et al.
MED
5.8 Hz, 1H, 5-H), 3.11 (dd, J=12.2/6.1 Hz, 1H, 4-H), 3.21–3.32 (m,
2H, 2-H and 5-H), 3.78 (s, 2H, N-CH2-Ph), 3.83 (s, 3H, OCH3), 4.64 (d,
J=6.7 Hz, 1H, 1-H), 6.69–6.72 (m, 2H, 6-H and 8-H), 7.15 (d, J=
8.1 Hz, 1H, 9-H), 7.27–7.29 (m, 1H, 4-H phenyl), 7.41–7.43 ppm (m,
4H, 2-H phenyl, 3-H phenyl, 5-H phenyl and 6-H phenyl) a signal
for the OH proton is not visible; C18H21NO2 (283.2).
For the binding assay, the cell pellet was resuspended in phos-
phate-buffered saline (PBS), and the number of cells was deter-
mined using an improved Neubauer’s counting chamber (VWR,
Darmstadt, Germany). The cells were subsequently lysed by sonica-
tion (48C, 6ꢂ10 s cycles with breaks of 10 s, device: Soniprep 150,
MSE, London, UK). The resulting cell fragments were centrifuged
with a high-performance cooled centrifuge (20000 g, 48C, Sorvall
RC-5 plus, Thermo Scientific). The supernatant was discarded, and
the pellet was resuspended in a defined volume of PBS yielding
cell fragments of ~500000 cells mLÀ1. The suspension of mem-
brane homogenates was sonicated again (48C, 2ꢂ10 s cycles with
a break of 10 min) and stored at À808C.
cis- and trans-7-Methoxy-3-(4-phenylcyclohexyl)-2,3,4,5-tetrahy-
dro-1H-3-benzazepin-1-ol
(cis-31/trans-31):
NaBH(OAc)3
(136.1 mg, 0.64 mmol) was added to a solution of 12 (82.7 mg,
0.43 mmol) in 1,2-dichloroethane (2 mL), 4-phenylcyclohexanone
(61.3 mL, 0.52 mmol), and CH3CO2H (36.6 mL, 0.64 mmol), and the
reaction mixture was stirred for 3 h at room temperature. A satu-
rated solution of NaHCO3 (10 mL) and H2O (10 mL) were added,
the layers were separated, and the aqueous layer was extracted
with CH2Cl2 (3ꢂ20 mL). The combined organic layers were dried
(Na2SO4), and the solvent was evaporated in vacuo. The residue
was purified by FC (2 cm, n-hexane/EtOAc 7:3 and 1% N,N-dimeth-
ylethylamine, 10 mL, Rf =0.15) to afford 31 (104.0 mg, 69%) as a
NR2B binding assay:[38] The competitive binding assay was per-
formed with the radioligand [3H]ifenprodil (60 CimmolÀ1; Perkin–
Elmer) using standard 96-well multiplates (Diagonal, Muenster, Ger-
many). The thawed cell membrane preparation (~20 mg protein)
was incubated with six different concentrations of test compounds,
5 nm [3H]ifenprodil, and Tris/EDTA buffer (5 mm/1 mm, pH 7.5) in a
total volume of 200 mL for 120 min at 378C. The incubation was
terminated by rapid filtration through the presoaked filter mats
using the cell harvester. After washing each well (5ꢂ300 mL H2O),
the filter mats were dried at 958C. Subsequently, the solid scintilla-
tor was placed on the filter mat and melted at 958C. After 5 min,
the solid scintillator was allowed to solidify at room temperature.
The bound radioactivity trapped on the filters was counted in the
scintillation analyzer. The nonspecific binding was determined with
10 mm unlabeled 1. The Kd value of 1 is 7.6 nm.[38]
1
colorless oil; H NMR (CDCl3): d=1.39–1.54 (m, 4ꢂ0.3H, CH2 cyclo-
*
&
hexyl ), 1.56–1.67 (m, 3ꢂ0.7H, CH2 cyclohexyl ), 1.69–1.80 (m, 3ꢂ
0.7H, CH2 cyclohexyl*), 1.85–1.98 (m, 4ꢂ0.3H, CH2 cyclohexyl ),
&
2.16–2.22 (m, 2ꢂ0.7H, CH2 cyclohexyl*), 2.42 (brt, J=11.9 Hz, 0.7H,
*
*
&
4-H ), 2.46 (d, J=12.0 Hz, 0.7H, 2-H ), 2.45–2.50 (m, 0.3H, 4-H ),
&
&
2.56 (d, J=12.1 Hz, 0.3H, 2-H ), 2.60–2.68 (m, 2.3H, 1-H cyclohexyl ,
*
1-H cyclohexyl , 4-H cyclohexyl , 5-H and 5-H*), 2.92 (quint, J=
&
&
4.9 Hz, 0.7H, 4-H cyclohexyl*), 3.08 (ddt, J=12.4/5.8/2.2 Hz, 0.7H,
*
&
4-H ), 3.05–3.13 (m, 0.3H, 4-H ), 3.17–3.28 (m, 2H, 2-H and 5-H),
*
&
3.75 (s, 3ꢂ0.7H, OCH3 ), 3.76 (s, 3ꢂ0.3H, OCH3 ), 4.51 (d, J=
*
&
6.7 Hz, 0.7H, 1-H ), 4.54 (d, J=6.7 Hz, 0.3H, 1-H ), 6.59–6.60 (m,
Affinity toward s1 and s2 receptors and the PCP binding site of
the NMDA receptor: The receptor binding studies were performed
as previously described.[40,41]
*
1H, 6-H* and 6-H ), 6.61 (dd, J=7.8/2.5 Hz, 0.7H, 8-H ), 6.63 (dd,
&
*
&
J=9.0/2.5 Hz, 0.3H, 8-H ), 7.06 (d, J=7.8 Hz, 0.7H, 9-H ), 7.09 (d,
*
&
J=9.0 Hz, 0.3H, 9-H ), 7.10–7.19 (m, 1.6H, 4-H phenyl , 4-H
&
&
&
phenyl , 2-H phenyl and 6-H phenyl ), 7.25–7.28 (m, 0.6H, 3-H
Excitotoxicity assay:[42] Briefly, L(tkÀ) cells stably expressing either
NR1–1a/NR2A (L12-G10 cells) or NR1–1a/NR2B (L13-E6 cells) were
seeded in 96-well microtiter plates in MEM containing 10% FCS,
0.5 mm sodium pyruvate, penicillin/streptomycin (100 U/
100 mgmLÀ1), 100 mm ketamine, and 160 mm G418 in a humidified
incubator at 378C and 5% CO2. To perform excitotoxicity assays
ketamine was removed by washing the cells in MEM lacking
phenol red. Then compounds (volume: 200 mL; concentration
range: 1 nm–10 mm, dissolved in DMSO) diluted in MEM without
phenol red were added to each well (final DMSO content: 0.1%).
After 30 min a mixture of (S)-glutamate and glycine (10 mm each)
was added, and the cells were further incubated for 4 h. Excitotox-
icity was determined by detection of LDH release from the cells
into the culture supernatant using the Cytotoxicity Detection Kitꢃ
(Roche Diagnostics, Mannheim, Germany). Optical density was
measured at 492 nm using the HTS 7000 Bioassay Reader (Perkin–
Elmer) with background subtraction. Relative excitotoxicity values
were calculated relative to a negative control (0% excitotoxicity is
defined as LDH release in the absence of NMDA receptor agonist
and in the presence 100 mm ketamine) and a positive control
(100% excitotoxicity is defined as LDH release after addition of (S)-
glutamate/glycine in the absence of ketamine). Experiments were
repeated at least three times with six replicates per test concentra-
tion. IC50 values were calculated using GraphPad Prism v5.0. A
direct inhibitory effect of compound 13 on LDH activity was ex-
cluded by performing the excitotoxicity assay in the presence of
the test compound as described above; however, all cells were
lysed by addition of 1% Triton X-100 prior to quantification of LDH
activity. No effect of 13 was observed under this condition. Under
standard assay conditions 13 had no effect on L(tkÀ) untransfected
cells.
*
&
&
phenyl and 5-H phenyl ), 7.29–7.30 ppm (m, 2.8H, 2-H phenyl , 3-
H phenyl*, 5-H phenyl* and 6-H phenyl*) a signal for the OH
*
&
proton is not visible; the ratio of cis-31( )/trans-31( ) is 70:30;
C23H29NO2 (351.2).
Pharmacological studies
Materials and general procedures: Centrifuge: High-speed cooling
centrifuge model Sorvall RC-5C plus (Thermo Finnigan). Filter: Print-
ed Filtermat Type B (PerkinElmer), pre-soaked in 0.5% aqueous pol-
yethylenimine for 2 h at room temperature before use. The filtra-
tion was carried out with a MicroBeta FilterMate-96 Harvester (Per-
kinElmer). Scintillation analysis was performed using a Meltilex
(Type A) solid scintillator (PerkinElmer). The scintillation was mea-
sured using a MicroBeta Trilux scintillation analyzer (PerkinElmer).
The overall counting efficiency was 20%.
Cell culture and preparation of membrane homogenates for the
NR2B assay:[38] In the assay, mouse L(tkÀ) cells stably transfected
with the dexamethasone-inducible eukaryotic expression vectors
pMSG NR1a, pMSG NR2B at a 1:5 ratio were used. The transformed
L(tkÀ) cells were grown in modified Earl’s medium (MEM) contain-
ing 10% standardized fetal calf serum (FCS; Biochrom AG, Berlin,
Germany). The expression of the NMDA receptor at the cell surface
was induced after the adherent growing cells had reached a con-
fluency of ~90%. For induction, the original growth medium was
replaced by growth medium containing 4 mm dexamethasone and
4 mm ketamine (final concentrations). After 24 h the cells were har-
vested by scraping and centrifugation (10 min, 5000 g, Hettich
Rotina 35R centrifuge, Tuttlingen, Germany).
694
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 687 – 695

NR2B-Selective Antagonists
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Supporting Information available: Purity data for all test com-
pounds, physical and spectroscopic data for all new compounds,
mechanism of the formation of tetrahydroisoquinoline 8, general
chemistry methods, and details of the pharmacological assays.
Acknowledgements
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We thank Schwarz Pharma AG, Monheim for supporting this
work and for performing the receptor screening of compound 13.
We are also grateful to Prof. Dr. D. Steinhilber, Department of
Pharmacy, University of Frankfurt, for donating the L(tkÀ) cells
stably expressing NR1a/NR2B receptor proteins.
Keywords: 3-benzazepines · functional activity · medicinal
chemistry · NR2B-selective NMDA antagonists · structure–
affinity relationships
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Received: January 5, 2010
Revised: February 10, 2010
Published online on March 25, 2010
ChemMedChem 2010, 5, 687 – 695
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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