GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists derived from 3-benzazepines: Synthesis and pharmacological evaluation of benzo[7]annulen-7-amines

Article abstract of DOI:10.1002/cmdc.201300547

Given their high neuroprotective potential, ligands that block GluN2B-containing N-methyl-D-aspartate (NMDA) receptors by interacting with the ifenprodil binding site located on the GluN2B subunit are of great interest for the treatment of various neurona

Full text of DOI:10.1002/cmdc.201300547

CHEMMEDCHEM
FULL PAPERS
DOI: 10.1002/cmdc.201300547
GluN2B-Selective N-Methyl-d-aspartate (NMDA) Receptor
Antagonists Derived from 3-Benzazepines: Synthesis and
Pharmacological Evaluation of Benzo[7]annulen-7-amines
Andre Benner,[a] Alessandro Bonifazi,[b] Chikako Shirataki,[c] Louisa Temme,[a]
Dirk Schepmann,[a] Wilma Quaglia,[b] Osami Shoji,[c] Yoshihito Watanabe,[d]
Constantin Daniliuc,[e] and Bernhard Wꢀnsch*[a]
Given their high neuroprotective potential, ligands that block
GluN2B-containing N-methyl-d-aspartate (NMDA) receptors by
interacting with the ifenprodil binding site located on the
GluN2B subunit are of great interest for the treatment of vari-
ous neuronal disorders. In this study, a novel class of GluN2B-
selective NMDA receptor antagonists with the benzo[7]annu-
lene scaffold was prepared and pharmacologically evaluated.
The key intermediate, N-(2-methoxy-5-oxo-6,7,8,9-tetrahydro-
5H-benzo[7]annulen-7-yl)acetamide (11), was obtained by
cyclization of 3-acetamido-5-(3-methoxyphenyl)pentanoic acid
(10b). The final reaction steps comprise hydrolysis of the
amide, reduction of the ketone, and reductive alkylation, lead-
ing to cis- and trans-configured 7-(w-phenylalkylamino)ben-
zo[7]annulen-5-ols. High GluN2B affinity was observed with cis-
configured g-amino alcohols substituted with a 3-phenylpropyl
moiety at the amino group. Removal of the benzylic hydroxy
moiety led to the most potent GluN2B antagonists of
this series: 2-methoxy-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-
5H-benzo[7]annulen-7-amine (20a, Ki =10 nm) and 2-methoxy-
N-methyl-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]an-
nulen-7-amine (23a, Ki =7.9 nm). The selectivity over related
receptors (phencyclidine binding site of the NMDA receptor, s1
and s2 receptors) was recorded. In a functional assay measur-
ing the cytoprotective activity of the benzo[7]annulenamines,
all tested compounds showed potent NMDA receptor antago-
nistic activity. Cytotoxicity induced via GluN2A subunit-contain-
ing NMDA receptors was not inhibited by the new ligands.
Introduction
The excitatory amino acid neurotransmitter (S)-glutamate me-
diates its effects by activation of metabotropic (mGlu1–8) and
ionotropic (N-methyl-d-aspartate [NMDA], 2-amino-3-(3-hy-
droxy-5-methylisoxazol-4-yl)propionic acid [AMPA], kainate)
glutamate receptors. In contrast to AMPA and kainate recep-
tors, the NMDA receptor allows penetration of Ca2+ ions into
the neuron, which has to be carefully controlled. The influx of
Ca2+ ions, the voltage-dependent Mg2+ blocking, and the re-
quirement of two agonists for activation, (S)-glutamate and
glycine, render the NMDA receptor a challenging target for
drugs.[1–3]
The NMDA receptor is formed by four subunits.[4] Today,
seven different NMDA receptor-forming subunits encoded by
different genes are known, which are classified into three
types: one GluN1 subunit with eight splice variants termed
GluN1a–h, four GluN2 subunits termed GluN2a–d, and two
GluN3 subunits termed GluN3a–b. A functional NMDA receptor
protein contains at least one GluN1 and one GluN2 subunit
bearing the binding sites for the agonists glycine and (S)-gluta-
mate, respectively.[5,6]
The heteromeric assembly of four proteins offers different
binding sites for ligands to modulate the opening state of the
ligand-gated ion channel. In addition to the binding sites for
the two agonists (S)-glutamate and glycine, there exist binding
sites for phencyclidine within the channel pore and NO, H+,
Zn2+, polyamines, and ifenprodil (1) at the amino terminus. We
are particularly interested in ligands interacting with the ifen-
prodil binding site of the NMDA receptor, which is only found
at the amino terminal end of the GluN2B subunit. Ligands
blocking the NMDA receptor by interaction with the ifenprodil
binding site address only NMDA receptors containing the
GluN2B subunit. Expression of the GluN2B subunit is restricted
to only some regions of the central nervous system (CNS), for
example, the cortex, hippocampus, and striatum. Therefore,
[a] Dr. A. Benner, L. Temme, Dr. D. Schepmann, Prof. Dr. B. Wꢀnsch
Institut fꢀr Pharmazeutische und Medizinische Chemie der Universitꢁt
Mꢀnster, Corrensstraße 48, 48149 Mꢀnster (Germany)
E-mail: wuensch@uni-muenster.de
[b] A. Bonifazi, Prof. W. Quaglia
Scuola di Scienze del Farmaco e dei Prodotti della Salute
Universitꢂ di Camerino, Via S. Agostino 1, 62032 Camerino (Italy)
[c] C. Shirataki, Prof. O. Shoji
Department of Chemistry, Graduate School of Science
Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
[d] Prof. Y. Watanabe
Research Center for Materials Science
Nagoya University, Furo-cho, Chikusa-ku, 464-8602 (Japan)
[e] Dr. C. Daniliuc
Organisch-chemisches Institut der Westfꢁlischen Wilhelms-Universitꢁt
Mꢀnster, Corrensstraße 40, 48149 Mꢀnster (Germany)
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 741

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
GluN2B-selective antagonists can only block NMDA
receptors in those regions of the CNS. NMDA recep-
tors in other parts of the CNS, for example, the cere-
bellum, which does not express the GluN2B subunit,
are not affected by this type of antagonist. This
mode of action leads to a decreased side effect pro-
file.[5–9]
Due to their high neuroprotective potential,[10]
compounds blocking GluN2B-containing NMDA re-
ceptors by selective interaction with the ifenprodil
binding site of the GluN2B subunit can be used for
the treatment of traumatic brain injury,[11] stroke (cer-
ebral ischemia),[12] neuropathic pain,[7] and Parkinson’s
disease.[13] Migraine,[14] depression,[10] and alcohol
withdrawal symptoms[15] represent further indications
of GluN2B-selective antagonists.
Figure 1. Development of GluN2B-selective NMDA receptor antagonists with a -
benzo[7]annulen-7-amine scaffold.
Very recently, X-ray crystal structures of an artificial dimer of
the amino terminal parts of the GluN1b and the GluN2B subu-
nits, together with ifenprodil (1) and Ro25-6981, have shown
that these GluN2B-selective antagonists bind at the interface
between the two subunits. However, the GluN2B subunit
forms crucial polar interactions with the antagonists, that is,
two hydrogen bonds between Gln110 and the protonated pi-
peridine and the aliphatic hydroxy moiety, as well as a hydro-
gen bond between Glu236 and the phenolic hydroxy
moiety.[16]
type 5. In the benzo[7]annulenamines 5, the basic amino
moiety of 3-benzazepines 2 and 3 was shifted to the side
chain. Thus, benzo[7]annulen-7-amines 5 represent regioisom-
ers of 3-benzazepines 2 and 3, with an increased distance of
four bond lengths between the left benzene ring and the
basic amino moiety. An analogously increased distance be-
tween the basic amino moiety and the benzene moiety of the
benzoxazolone system was also realized in the potent GluN2B
antagonist besonprodil (4).[8,9] Different phenylalkyl residues
(n=1, 2) were considered in this project, due to their similarity
to the nitrogen residues of 2–4.
The piperidinopropanol derivative ifenprodil (Vadilexꢂ,
1),[17,18] which was originally developed as an a1-receptor an-
tagonist, represents the first NMDA receptor antagonist ad-
dressing the ifenprodil binding site of the GluN2B subunit
(IC50 =13.3 nm).[19] (Figure 1) However, low bioavailability
caused by fast biotransformation[20] and undesired side effects
(e.g., psychotomimetic effects, memory deficits, hypertension)
resulting from low receptor selectivity (5-HT1A, 5-HT2, a1, s1,
and s2 receptors were affected as well) prevented further de-
velopment of ifenprodil.
Synthesis
The synthesis of the benzo[7]annulene scaffold started with
phenylpropionic acid 6a, which was converted with oxalyl
chloride into acid chloride 6b. Reaction of acid chloride 6b
with Meldrum’s acid and subsequent methanolysis led to b-
keto ester 7 in 71% yield (Scheme 1).
After transformation of b-keto ester 7 into enamido ester 9
upon condensation with NH4OAc and subsequent acetylation,
hydrogenation provided amido ester 10a. Hydrolysis of 10a
with KOH yielded acid 10b, which represented the key com-
pound for the construction of the benzo[7]annulene scaffold.
Several reaction conditions (e.g., PPA, P4O10 in CH2Cl2, SOCl2/
AlCl3, cyanuric chloride/AlCl3, or cyanuric chloride/SnCl4) were
Despite these unfavorable properties, ifenprodil served as
a lead compound for the development of potent, selective,
and bioavailable GluN2B antagonists. Formal opening of the
piperidine ring and reconnection of the free end of the chain
to the phenol of ifenprodil led to the tetrahydro-3-benzaze-
pines 2 and 3 with low nanomolar affinity toward GluN2B-con-
taining NMDA receptors (Ki(2)=
14 nm, Ki(3)=5.4 nm). Whereas
phenol 2 revealed lower GluN2B
affinity than methyl ether 3, its
NMDA receptor antagonistic ac-
tivity was considerably higher
(IC50 =18.4 nm) than that of 3
(IC50 =360 nm).[21,22]
Due to the promising GluN2B
affinity and NMDA receptor an-
tagonistic activity of 3-benzaze-
pines 2 and 3, we planned to
expand this type of GluN2B-se-
lective NMDA antagonistic activi-
Scheme 1. Synthesis of benzo[7]annulen-5-one 11. Reagents and conditions: a) 1. oxalyl chloride, CH2Cl2, DMF, RT,
21 h, 94%; b) Meldrum’s acid, CH2Cl2, pyridine, 08C, 30 min, RT, 1 h, then CH3OH, reflux, 3 h, 71%; c) NH4OAc,
CH3OH, 358C, 16 h, 100%; d) Ac2O, pyridine, THF, reflux, 48 h, 100%; e) H2 (5 bar), Pd/C, CH3OH, RT, 48 h, 76%;
ty to benzo[7]annulenamines of f) KOH, CH3OH, RT, 3 h, 93%; g) P4O10, CH3SO3H, CH2Cl2, ꢀ108C, 30 min, RT, 2 h, 65%.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 742

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
tested for the intramolecular Friedel–Crafts acylation of acid
10b. Finally, a solution of P4O10 in methanesulfonic acid[23]
gave the highest yield (65%) of cyclic ketone 11. During this
cyclization, small amounts (<15%) of the regioisomeric ben-
zo[7]annulene with the methoxy moiety in position 1 were
formed, which were separated by flash chromatography.
alcohols 16 was reacted with 3-phenylpropanal and
NaBH(OAc)3 to give a mixture of four products, which were
[24]
separated by flash chromatography. In addition to the desired
cis- and trans-configured phenylpropylamines (cis-17a and
trans-18a), tertiary amine 19a was isolated. During the reduc-
tive amination, an unexpected hydrogenolytic cleavage of the
benzylic hydroxy moiety also occurred, leading to phenylpro-
pylamine 20a. The homologous phenylbutylamines cis-17b
and trans-18b were obtained by reductive alkylation of amino
alcohol 16 with phenylbutanal. Alternatively, alkylation of 16
with 1-chloro-4-phenylbutane and tetrabutylammonium iodide
in acetonitrile at reflux led to a mixture of diastereomeric phe-
nylbutylamines cis-17b/trans-18b and N,N-bis(4-phenylbutyl)a-
mine 19b as the main product.
Ketone 11 was reduced with NaBH4 to yield amido alcohol
12 as a 6:4 mixture of diastereomers, which were not separat-
ed by flash chromatography (Scheme 2). However, all attempts
In addition to the secondary amines cis-17, trans-18, and
20a, we thought it interesting to test the corresponding terti-
ary amines as well. For this purpose, the secondary amines cis-
17a, trans-18a, and 20a were reductively methylated with
formaldehyde and NaBH(OAc)3 to yield tertiary amines cis-21a,
trans-22a, and 23a with a small third substituent.
Scheme 2. Elimination reactions of hydroxyamide 12. Reagents and condi-
tions: a) NaBH4, CH3OH, RT, 16 h, 56%; b) HCl, 5 m, DMF, 608C, 24 h, 12%
(13), 8% (14).
It was very difficult to unequivocally assign the relative con-
figuration of the amino and hydroxy moiety in the seven-mem-
bered ring of 17, 18, 21 and 22 by analyzing the coupling con-
1
stants in the corresponding H NMR spectra. Therefore, amino
to hydrolyze the amide to obtain the primary amine failed.
Either no reaction occurred, or undesired transformations took
place. For example, elimination products 13 and 14 were iso-
lated after treatment of amido alcohol 12 with 5m HCl at 608C
for 24 h. Therefore, the reaction sequence was changed.
alcohol cis-17a was recrystallized with ethyl acetate and diiso-
propyl ether (3:1) to yield crystals that were suitable for X-ray
crystal structure analysis. In Figure 2, only one of the two inde-
pendent enantiomers of cis-17a in the asymmetric unit is
shown. The figure shows that the hydroxy moiety and the phe-
nylpropylamino moiety are on the same side of the ben-
zo[7]annulene ring system, indicating cis-configuration. The rel-
ative configurations of cis-17b, trans-18a,b, cis-21a, and trans-
22a were defined by comparing their NMR spectra with the
NMR spectrum of cis-17a.
Hydrolysis of acetamide 11 with 2m HCl at 958C led to pri-
mary amine 15 in 85% yield after careful optimization
(Scheme 3). Reduction of amino ketone 15 with LiBH4 provided
amino alcohol 16, which was obtained in 85% yield as a 1:1
mixture of diastereomers. The mixture of diastereomeric amino
Scheme 3. Synthesis of various benzo[7]annulen-7-amines. Reagents and conditions: a) HCl (2m), 958C, 16 h, 85%; b) LiBH4, THF, ꢀ788C, 30 min, 85%;
c) PhCH2CH2CH=O, CH2CH2, RT, 20 min, then NaBH(OAc)3, RT, 4 h, 4% (cis-17a), 2%, (trans-18a), 13% (trans-19a), 5% (20a); d) Ph(CH2)3CH=O, MgSO4, CH2Cl2,
RT, 16 h, then NaBH(OAc)3, RT, 4 h, 2% (cis-17b: 2%), 2% (trans-18b); e) Ph(CH2)4Cl, Bu4NI, K2CO3, CH3CN, 858C, 24 h, 23% (cis-17b+trans-18b), 59% (19b);
f) CH2=O, H2O, CH2Cl2, NaBH(OAc)3, RT, 12–16 h, 71–75% (only phenylpropylamines of series a were reductively methylated).
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 743

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
1–4, traxoprodil, eliprodil, Ro25-6981). In conclusion, deoxy-
genated benzo[7]annulenamines 20a and 23a show the high-
est GluN2B affinity within this series of ligands.
Selectivity over related receptors
To prove the selectivity of the GluN2B ligands, the affinity
toward a number of related receptors was recorded in compet-
itive radioligand receptor binding studies. First, affinity toward
the phencyclidine (PCP, 1-(1-phenylcyclohexyl)piperidine) bind-
ing site within the channel pore was determined using the
radioligand [3H](+)-MK-801.[25,26] The benzo[7]annulenamines
did not show considerable interaction with the PCP binding
site, indicating high selectivity over this binding site. (Table 1)
As ifenprodil exhibits considerable affinity toward both the
s1 and s2 receptors, the affinity of the new ligands toward
these receptor systems was recorded employing the radioli-
Figure 2. X-ray crystal structure analysis of cis-17a. Only one of the two in-
dependent enantiomers in the asymmetric unit is shown. Thermal ellipsoids
are shown at 30% probability.
Biological Activity
Affinity toward the ifenprodil binding site of GluN2B--
containing NMDA receptors
The affinity of the diastereomeric benzo[7]annulen-7-amines
toward GluN2B-containing NMDA receptors was investigated
using the competitive receptor binding assay recently devel-
oped by our group.[19] Membrane preparations obtained by ul-
trasonic irradiation of L(tkꢀ) cells stably expressing recombi-
nant human GluN1a and GluN2B subunits were employed as
receptor materials in this assay. The synthesis of functional
NMDA receptors was induced by addition of dexamethasone
to the growth medium of these L(tkꢀ) cells. To protect against
cell death, ketamine was added to the medium, which blocks
the NMDA receptor by interaction with the phencyclidine
binding site within the channel pore. Tritium-labeled
[3H]ifenprodil (1) served as a radioligand. Although the selectiv-
ity of the radioligand was rather low, this assay is se-
gands
[3H](+)-pentazocine
and
[3H]di-o-tolylguanidine
([3H]DTG), respectively.[27–29] (Table 1). The Ki(s1) values for ben-
zo[7]annulenamines 17 and 18 are higher than 125 nm, indi-
cating considerable selectivity over the s1 receptor. However,
removal of the benzylic hydroxy moiety and methylation of
the secondary amines led to increased s1 affinity, with Ki(s1)
values in the range of 21–81 nm. Deoxygenated compounds
20a and 23a, which represent the GluN2B ligands with the
highest affinity, also show the highest s1 affinity, which leads
to a decreased GluN2B/s1 selectivity of 2.7. With respect to
GluN2B affinity and GluN2B/s1 selectivity, cis-17a represents
a promising ligand.
lective for the ifenprodil binding site of GluN2B-con-
Table 1. Affinities of benzo[7]annulenes toward the ifenprodil binding site of GluN2B-
containing NMDA receptors, the PCP binding site of the NMDA receptor, and s1 and
s2 receptors.
taining NMDA receptors, due to the high number of
receptors in this cell line.
In Table 1, the GluN2B affinity of the test com-
pounds is summarized. Generally, the GluN2B affinity
of cis-configured amino alcohols is higher than the
GluN2B affinity of the corresponding trans-configured
diastereomers (e.g., cis-17a/trans-18a). Elongation of
the distance between the basic amine and the
phenyl moiety in the side chain led to lower GluN2B
affinity (e.g., trans-18a/trans-18b). Introduction of an
additional methyl group to the basic amino moiety
did not affect the GluN2B affinity considerably. The
secondary amine (cis-17a, Ki =16 nm) and the tertiary
amine (cis-21a, Ki =15 nm) exhibit almost identical
GluN2B affinity. Reductive elimination of the hydroxy
moiety in the benzylic position resulted in ben-
zo[7]annulenamines with increased GluN2B affinity.
The GluN2B affinities of deoxygenated benzo[7]annu-
lenamines 20a (Ki =10 nm) and 23a (Ki =7.9 nm) are
slightly higher than the GluN2B affinities of corre-
sponding hydroxy derivatives cis-17a and cis-21a.
The high GluN2B affinity of deoxygenated ben-
zo[7]annulenamines 20a and 23a was unexpected,
as most of the lead compounds contain a benzylic
hydroxy moiety or a bioisosteric replacement (e.g.,
Compd
R
X
n
Ki [nm][a]
[b]
[b]
GluN2B[b]
PCP
s1
s2
2[22]
3[21]
H
CH3
H
H
H
H
H
CH3
CH3
CH3
–
–
–
–
3
4
3
4
3
3
3
3
14ꢁ1.5
5.4ꢁ0.4
16ꢁ3
24ꢁ10
48ꢁ11
127ꢁ16
10ꢁ4
15ꢁ2
39ꢁ3
7.9ꢁ4
10ꢁ0.7
13ꢁ2.5
–
35%
22%
12%
0%
4%
5%
0%
0%
32%
1%
–
194
182
132
489
167
197
27ꢁ7
81ꢁ11
42ꢁ1
21ꢁ7
125ꢁ24
–
>10 mm
554
9.2ꢁ2.1
209
cis-17a
cis-17b
trans-18a
trans-18b
20a
cis-21a
trans-22a
23a
ifenprodil (1)
eliprodil
dexoxadrol
haloperidol
OH
OH
OH
OH
H
OH
OH
H
1100
1400
15ꢁ5
33 ꢁ4
600
137
98.3ꢁ34
–
–
78.1ꢁ2.3
57.5ꢁ18
–
32ꢁ7.4
–
–
–
–
6.3ꢁ1.6
89ꢁ29
di-o-tolylguanidine
–
[a] For low-affinity compounds, only the inhibition of the radioligand binding at a test
compound concentration of 1 mm is given. [b] Ki values are the meanꢁSEM of three
independent experiments performed in triplicate.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 744

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Trans-configured benzo[7]annulenamines (trans-18a,b, trans-
22a) and phenylbutylamines (cis-17b, trans-18b) show rather
low s2 affinity, indicating high selectivity. The highest s2 affinity
was found for the cis-configured phenylpropylamine cis-17a.
Its s2 affinity (Ki =9.2 nm) even exceeds the GluN2B affinity
(Ki =16 nm). Removal of the benzylic hydroxy moiety (20a: Ki =
15 nm), N-methylation (cis-21a: Ki =33 nm), and both modifica-
tions (23a: Ki =137 nm) led to a progressive decrease in s2 af-
finity. With the exception of cis-17a, the highest affinity
GluN2B ligands (20a, cis-21a, and 23a) display a 1.5–17-fold
preference for the GluN2B receptor over the s2 receptor.
nulenamines interacting with high affinity with the ifenprodil
binding site of the GluN2B subunit did not inhibit (S)-gluta-
mate/glycine-induced damage of cells expressing GluN2A-con-
taining NMDA receptors. This result proves unequivocally the
high GluN2B selectivity of this novel class of benzo[7]annulena-
mines.
Conclusions
cis- and trans-Configured benzo[7]annulenes with a 5-hydroxy
moiety and various phenylalkylamino moieties in the 7-posi-
tion were prepared in 10–11 synthetic steps. High GluN2B af-
finity was observed for cis-configured 3-phenylpropylamines
cis-17a and cis-21a. Unexpectedly, the GluN2B affinity of cis-
17a and cis-21a was exceeded by that of deoxygenated ben-
zo[7]annulenamines 20a and 23a. With respect to selectivity
over the s receptors 20a, cis-21a, and 23a represent the most
promising GluN2B antagonists. In the functional GluN2B assay
recording cytoprotective effects, the cis-configured phenylpro-
pylamines cis-17a and cis-21a and the deoxygenated phenyl-
propylamines 20a and 23a show high antagonistic activity.
Due to their high selectivity over GluN2A-containing NMDA re-
ceptors, the benzo[7]annulenamines represent novel GluN2B-
selective antagonists with a promising therapeutic potential
and side effect profile.
Functional activity
The antagonistic activity of benzo[7]annulenamines cis-17a,
20a, cis-21a, and 23a was investigated by inhibition of the ex-
citotoxicity induced by activation of NMDA receptors with (S)-
glutamate and glycine. In the assays, L(tkꢀ) cells stably ex-
pressing the NMDA receptor subunits GluN1a and GluN2B
(GluN2B assay) or GluN1a and GluN2A (GluN2A assay) were
employed. Expression of the functional NMDA receptors was
induced by addition of dexamethasone. At the same time, (S)-
glutamate, glycine, and different concentrations of the test
compounds were added. After an incubation period of 12 h,
the amount of released lactate dehydrogenase (LDH) was re-
corded by measuring the amount of formed formazan dye.[30]
Table 2 displays the cytoprotective effect of benzo[7]annu-
lenamines. In the GluN2B assay, all tested compounds inhibited
(S)-glutamate/glycine-induced cytotoxicity in the low-to-
Experimental Section
Chemistry
General: Unless otherwise noted, moisture-sensitive reactions were
conducted under dry nitrogen. THF was dried with sodium/benzo-
phenone and was freshly distilled before use. Thin layer chroma-
tography (TLC): silica gel 60 F254 plates (Merck); flash chromatog-
raphy (FC): silica gel 60, 40–64 mm (Merck); parentheses include di-
ameter of the column, eluent, fraction size, and Rf value; melting
point. Melting points were taken using an SMP3 melting point ap-
paratus (Stuart Scientific) and are uncorrected. For microwave reac-
tions, a Discover microwave apparatus (CEM GmbH) was used.
Mass spectrometry (MS) measurements were taken using
a MAT GCQ (Thermo-Finnigan). IR spectrum were collected using
a 480Plus FT-ATR-IR spectrophotometer (Jasco); optical rotation
was determined using a Polarimeter 341 (PerkinElmer) at a wave-
length of 589 nm, path length (l): 1 dm, T+208C; unit
[deg mLꢀ1 dmꢀ1 gꢀ1] is omitted with concentration [mgmLꢀ1] and
solvent given in brackets. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were collected using a Unity Mercury Plus 400
spectrometer (Varian), with d in ppm related to tetramethylsilane;
coupling constants are given with 0.5 Hz resolution. HPLC was per-
formed using Merck Hitachi Equipment: l-7400 UV detector, L-
7200 autosampler, L-7100 pump, L-7614 degasser, column: LiChros-
pher60 RP-selectB (5 mm), LiChroCART250–4 mm cartridge, flow
rate: 1.0 mLminꢀ1, injection volume: 5.0 mL, detection at l=
210 nm, solvents: A) water with 0.05% (v/v) trifluoroacetic acid;
B) CH3CN with 0.05% (v/v) trifluoroacetic acid, with gradient elu-
tion (A%): 0–4 min: 90%, 4–29 min: 90 ! 0%, 29–31 min: 0%, 31–
31.5 min: 0 ! 90%, 31.5–40 min: 90%. Elemental analyses were
performed using a CHN-Rapid Analysator (Fons-Heraeus).
Table 2. Inhibition of cell death by GluN2B-selective NMDA receptor an-
tagonists.
Compd
R
X
N
Ki [nm][a]
IC50 [nm][a]
GluN2B
GluN2B
GluN2A
cis-17a
20a
cis-21a
23a
H
H
CH3
CH3
OH
H
OH
H
3
3
3
3
16ꢁ3
10ꢁ4
15ꢁ2
7.9ꢁ4
12ꢁ0.4
29ꢁ1
515ꢁ64
1700ꢁ400
882ꢁ109
450ꢁ163
55ꢁ24
9.7ꢁ3.9
[a] Ki and IC50 values are the meanꢁSEM of three independent experi-
ments performed in triplicate.
medium nanomolar range, which confirms that the new ben-
zo[7]annulene-based GluN2B-selective ligands represent
potent NMDA receptor antagonists. The inhibition of cytotoxic-
ity correlates nicely with the GluN2B affinity of these com-
pounds. Whereas cis-21a, with the lowest GluN2B affinity, has
the lowest cytoprotective effect (IC50 =55 nm), deoxygenated
benzo[7]annulenamine 23a shows the highest GluN2B binding
and cytoprotective activity (IC50 =9.7 nm). Thus, the ben-
zo[7]annulenamines reported herein show similar cytoprotec-
tive potential as channel blockers like phencyclidine and keta-
mine.
To show the selectivity over GluN2A-containing NMDA re-
ceptors, the same assay was conducted with GluN2A subunit-
producing cells. It can be seen in Table 2 that the benzo[7]an-
3-(3-Methoxyphenyl)propanoyl chloride (6b): Oxalyl chloride
(42.8 g, 337.2 mmol) was added under N2 to a solution of 3-(3-me-
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 745

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
thoxyphenyl)propanoic acid (6a, 30.5 g, 168.6 mmol) in CH2Cl2
(60 mL). After the mixture was stirred for 1 h at room temperature,
dimethylformamide (DMF; 0.5 mL) was added, and the mixture
was stirred for 20 h at room temperature. The solvent and excess
oxalyl chloride were removed by distillation under atmospheric
pressure, and the crude product was purified by distillation under
vacuum to yield 6b as a pale yellow oil (31.4 g, 94%): bp13 1458C;
1H NMR (CDCl3): d=2.99 (t, J=7.6 Hz, 2H, Ar-CH2-CH2), 3.21 (t, J=
7.6 Hz, 2H, -CH2-COCl), 3.80 (s, 3H, Ar-OCH3), 6.73 (t, J=2.0 Hz, 1H,
2-Harom), 6.78 (dd, J=7.5/2.0 Hz, 2H, 4-Harom, 6-Harom), and 7.23 ppm
(t, J=7.5 Hz, 1H, 5-Harom); C10H11ClO2, Mr =198.6 Da.
and 7.22 ppm (t, J=7.9 Hz, 1H, 5-Harom); 13C NMR (CDCl3): d=25.0
(1C, NHCO-CH3), 34.3 (1C, Ar-CH2-CH2), 35.1 (1C, Ar-CH2-CH2), 51.2
(1C, Ar-OCH3), 55.4 (1C, -CH-CO2CH3), 102.3 (1C, CHCO2CH3), 112.0
(1C, 4-Carom), 114.4 (1C, 2-Carom), 121.0 (1C, 6-Carom), 129.9 (1C, 5-
Carom), 142.6 (1C, 1-Harom), 152.3 (1C, -C-NHCOCH3), 160.1 (1C, 3-
C
arom), 168.6 (1C, -NHCO-CH3), and 169.2 ppm (1C, -CO2CH3); purity
by HPLC: 97.5% (tr =22.36 min); MS (ESI): m/z (%)=218
[MꢀC2H3O2, 100], 234 [MꢀC2H3O, 39], 278 [M+H, 15]; IR (neat): n˜ =
3267 (NꢀH), 3221 (NꢀH), 3028 (CꢀHarom), 2951 (CꢀHaliph), 1744 (C=
Oester), 1628 (C=Oamide), 779 cmꢀ1 (C=C1,3-disubstarom); C15H19NO4, Mr =
277.1 Da.
Methyl 5-(3-methoxyphenyl)-3-oxopentanoate (7): Meldrum’s
acid (20.5 g, 142.2 mmol) was dissolved in CH2Cl2 (34 mL) and
cooled to 08C. Pyridine (25.5 mL) was added dropwise, then a solu-
tion of 6b (31.4 g, 158.0 mmol) in CH2Cl2 (57 mL) was added at
08C over 60 min. The mixture was stirred for 30 min at 08C and
60 min at room temperature. The mixture was diluted with CH2Cl2
(100 mL), washed with 2m HCl (100 mL) and brine (100 mL), and
the organic layer was dried (Na2SO4) and concentrated in vacuum.
The residue (43.5 g) was dissolved in CH3OH (50 mL) and stirred at
reflux for 3 h. The solvent was evaporated under vacuum, and the
product was divided into three parts for FC purification (d=8 cm,
l=25 cm, V=65 mL, cyclohexane/EtOAc, 90:10, Rf =0.21) to yield 7
as a colorless oil (26.4 g, 71%): 1H NMR (CDCl3): d=2.84–2.93 (m,
4H, Ar-CH2-CH2), 3.43 (s, 2H, CO-CH2-CO2CH3), 3.71 (s, 3H, CO2CH3),
3.78 (s, 3H, Ar-OCH3), 6.71–6.80 (m, 3H, 2-Harom, 4-Harom, 6-Harom),
and 7.17–7.22 ppm (m, 1H, 5-Harom); purity by HPLC: 90% (tr =
19.82 min); MS (ESI): m/z (%)=254 [M+NH4+, 100], 495 [2M+Na,
95], 219 [MꢀH2O, 47]; FTIR: n˜ =3004 (s, n-CꢀHarom), 2953 (CꢀHaliph),
2837 (CꢀHOCH3), 1744 (C=Oester), 1714 (C=Oketone), and 776 cmꢀ1 (C=
C1,3-disubstarom); C13H16O4, Mr =236.3 Da;
Methyl 3-acetamido-5-(3-methoxyphenyl)pentanoate (10a): En-
amide 9 (8.85 g, 31.8 mmol) was dissolved in abs. MeOH (40 mL),
and 10% Pd/C (0.71 g) was added. The mixture was shaken for
48 h at room temperature under H2 atmosphere (5 bar). The cata-
lyst was removed by filtration, and the MeOH was removed under
reduced pressure. The residue was purified by FC (d=8 cm, l=
25 cm, V=65 mL, cyclohexane/EtOAc, 4:6, Rf =0.18) to yield 10a as
1
a colorless oil (6.7 g, 76%): H NMR (CDCl3): d=1.76–1.85 (m, 1H,
Ar-CH2-CH2), 1.86–1.94 (m, 1H, Ar-CH2-CH2), 1.96 (s, 3H, NHCO-CH3),
2.52 (dd, J=16.1/5.0 Hz, 1H, -CH2-CO2CH3) 2.59 (dd, J=16.1/5.0 Hz,
1H, -CH2-CO2CH3), 2.63 (t, J=8.2 Hz, 2H, Ar-CH2-), 3.67 (s, 3H,
CO2CH3), 3.78 (s, 3H, Ar-OCH3), 4.25–4.34 (m, 1H, -CH-NHCOCH3),
6.07 (s broad, 1H, NH), 6.72–6.77 (m, 3H, 2-Harom, 4-Harom, 6-Harom),
and 7.17–7.21 ppm (m, 1H, 5-Harom); purity by HPLC: 93.4% (tr =
21.33 min); MS (ESI): m/z (%)=279 [M, 10], 278 [MꢀH, 100]; IR
(neat): n˜ =3305 (NꢀH), 2916 (CꢀHaliph), 1728 (C=Oester), 1610 (C=
Oamide I), 1574 (C=Oamide II), 783 cmꢀ1 (C=C1,3-disubstarom); C15H21NO4,
Mr =279.1 Da.
3-Acetamido-5-(3-methoxyphenyl)pentanoic acid (10b): Ester
10a (8.5 g, 30.4 mmol) was dissolved in MeOH (20 mL) and cooled
to 08C. Next,1 m KOH (100 mL) was added, and the mixture was
stirred for 3 h at room temperature. The mixture was acidified with
5m HCl and extracted with EtOAc (4ꢃ). The organic layer was
washed with brine, dried (Na2SO4), filtered, and concentrated
under vacuum. Crude product 10b, a colorless oil, was used in the
next reaction step without purification (6.5 g, 93%): 1H NMR
(CDCl3): d=1.77–1.91 (m, 2H, Ar-CH2-CH2), 1.94 (s, 3H, NHCO-CH3),
2.48 (dd, J=15.9/5.4 Hz, 1H, -CH2-CO2H) 2.56 (dd, J=15.9/5.4 Hz,
1H, -CH2-CO2H), 2.61 (t, J=8.2 Hz, 2H, Ar-CH2-), 3.77 (s, 3H, Ar-
OCH3), 4.21–4.27 (m, 1H, -CH-NHCOCH3), 6.46–6.49 (m, 1H, NH),
6.70–6.75 (m, 3H, 2-Harom, 4-Harom, 6-Harom), and 7.14–7.18 ppm (m,
1H, 5-Harom), a signal for the CO2H proton is not observed in the
spectrum; purity by HPLC: 96.8% (tr =18.68 min); MS (ESI): m/z
(%)=265 [M, 80], 122 [MꢀC6H10NO3, 100]; IR (neat): n˜ =3267 (Nꢀ
H), 3221 (NꢀH), 3028 (CꢀHarom), 2951 (CꢀHaliph), 1744 (C=O), 1628
(C=Oamide), 779 cmꢀ1 (C=C1,3-disubstarom); C14H19NO4, Mr =265.3 Da.
Methyl 3-amino-5-(3-methoxyphenyl)pent-2-enoate (8): b-Keto
ester 7 (8.0 g, 33.8 mmol) was dissolved in MeOH (50 mL) under
N2. Dry ammonium acetate (10.5 g, 172.0 mmol) was added, and
the mixture was stirred at 358C for 16 h. The MeOH was removed
under vacuum, and the residue was suspended in EtOAc and fil-
tered. The residue in the filter was washed with EtOAc (4ꢃ). The or-
ganic layer was dried (Na2SO4), filtered, and concentrated under
vacuum. The crude product, 8, was used in the next reaction with-
out purification as a colorless oil (8.12 g, 100%): 1H NMR (CDCl3):
d=2.42 (t, J=8.0 Hz, 2H, Ar-CH2-CH2), 2.84 (t, J=8.0 Hz, 2H, Ar-
CH2-CH2), 3.65 (s, 3H, -CO2CH3), 3.79 (s, 3H, Ar-OCH3), 4.59 (s, 1H, =
CH-CO2CH3), 6.73–6.80 (m, 3H, 2-Harom, 4-Harom, 6-Harom), and 7.20–
7.24 ppm (m, 1H, 5-Harom), signals for the NH2 group are not ob-
served in the spectrum; purity by HPLC: 88.5% (tr =17.49 min); MS
(ESI): m/z (%)=135 [MꢀC4H6NO, 100], 236 [M+H, 38]; IR (neat): n˜ =
3453 (NꢀH), 3333 (NꢀH), 3028 (CꢀHarom), 2947 (CꢀHaliph), 1740 (C=
O), 787 cmꢀ1 (C=C1,3-disubstarom); C13H17NO3, Mr =235.3 Da.
N-(2-Methoxy-5-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-
yl)acetamide (11): Acid 10b (6.5 g, 24.5 mmol) was dissolved in
abs. CH2Cl2 (100 mL) under N2, and the mixture was cooled to
ꢀ108C. A solution of P4O10 (11 g, 38.7 mmol) in methanesulfonic
acid (55 mL) was added slowly, and the mixture was stirred for
30 min at ꢀ108C and 2 h at room temperature. The mixture was
then cooled to 08C, and water (10 mL) and NaOH were added
(pH 10). The aqueous layer was extracted with CH2Cl2 (4ꢃ). The
combined organic layers were washed with brine and dried
(Na2SO4), the solvent was removed under reduced pressure, and
the residue was purified by crystallization (EtOAc/iPr2O/CH3OH,
8:2:1) to yield 11 as a colorless solid (3.9 g, 65%): mp: 158–1608C,
1H NMR (CDCl3): d=1.53 (dddd, J=14.7/9.3/6.7/5.5 Hz 1H, Ar-CH2-
CH2), 1.94 (s, 3H, NHCO-CH3), 2.48 (dddd, J=14.0/7.5/6.1/4.9 Hz 1H,
Ar-CH2-CH2), 2.78 (dd, J=13.7/5.6 Hz, 1H, Ar-CO-CH2), 2.95 (t, J=
Methyl 3-acetamido-5-(3-methoxyphenyl)pent-2-enoate (9): En-
amine 8 (7.5 g, 31.8 mmol) was dissolved in THF (40 mL) under N2.
Pyridine (5.3 g, 66.8 mmol) and acetic anhydride (19.9 g,
195.2 mmol) were added, and the reaction mixture was stirred at
reflux for 48 h. The THF was removed under vacuum, the residue
was dissolved in EtOAc, and the organic layer was washed with
H2O, 2m HCl, sat. NaHCO3, and brine. The organic layer was dried
(Na2SO4), filtered, and concentrated under vacuum. Crude product
9, a colorless oil, was used in the next reaction without purification
1
(yield: 8.95 g, 100%): H NMR (CDCl3): d=1.86 (s, 3H, NHCO-CH3),
2.87 (t, J=7.4 Hz, 2H, Ar-CH2-CH2), 3.00 (t, J=7.7 Hz, 2H, Ar-CH2-
CH2), 3.69 (s, 3H, CO2CH3), 3.79 (s, 3H, Ar-OCH3), 6.45 (s, 1H, NH),
6.76 (ddd, J=8.1/2.6/0.9 Hz, 1H, 4-Harom), 6.78–6.80 (m, 1H, 2-Harom),
6.80 (s, 1H, =CHCO2CH3), 6.84 (ddd, J=7.6/1.4/0.9 Hz, 1H, 6-Harom),
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 746

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
5.8 Hz, 2H, Ar-CH2-), 3.11 (dd, J=13.7 Hz/5.1, 1H, Ar-CO-CH2), 3.78
(s, 3H, Ar-OCH3), 4.42–4.51 (m, 1H, -CH-NHCOCH3), 5.67–5.68 (m,
1H, NH), 6.71 (d, J=2.5 Hz, 1H, 1-Harom), 6.80 (dd, J=8.7/2.5 Hz,
1H, 3-Harom), and 7.77 ppm (d, J=8.7 Hz, 1H, 4-Harom); 13C NMR
(CDCl3): d=23.6 (1C, -NC=O-CH3), 32.1 (1C, Ar -CH2-CH2), 33.5 (1C,
Ar-CH2-CH2-), 45.3 (1C, -CH-NHAc), 47.2 (1C, -CH=O-CH2-), 55.6 (1C,
Ar-OCH3), 112.2 (1C, 3-Carom), 115.5 (1C, 1-Carom), 131.4 (1C, 4-Carom),
131.5 (1Cq, 11-Carom), 145.7 (1Cq, 10-Carom), 162.8 (1Cq, 2-Carom), 169.7
(1 Cq, -NC=O-CH3), and 200.3 ppm (1Cq, -C=O-CH2-); purity by
HPLC: 95.5% (tr =13.93 min); MS (HRMS): m/z (%)=calcd for
C14H18NO3+: 248.1281, found: 248.1297; IR (neat): n˜ =3289 (NꢀH),
3060 (CꢀHarom), 2944 (CꢀHaliph), 1702 (C=Oketone), 1634 (C=Oamide I),
1599 (C=Oamide II), 819 cmꢀ1 (C=C1,3,4-trisubstarom); C14H17NO3, Mr =
247.3 Da.
perature, the mixture was washed with CH2Cl2 (2ꢃ). Next, NaOH
was added (pH>12), and the mixture was extracted with CH2Cl2
(4ꢃ). The organic layer was washed with brine, and dried (Na2SO4),
filtered, and concentrated under vacuum. The residue was purified
by FC (d=8 cm, l=18 cm, V=65 mL, CH2Cl2/CH3OH, 9:1, Rf =0.28)
1
to yield 15 as a pale yellow oil (1.1 g, 85%): H NMR (CDCl3): d=
1.61 (dtd, J=15.4/7.8/2.8 Hz, 1H, Ar-CH2-CH2), 2.24 (dddd, J=16.6/
9.6/6.7/2.8 Hz 1H, Ar-CH2-CH2), 2.77 (dd, J=13.4/7.6 Hz, 1H, Ar-CO-
CH2), 2.87 (ddd, J=16.0/8.0/2.5 Hz, 1H, Ar-CH2), 2.98 (dd, J=13.4/
4.5 Hz, 1H, Ar-CO-CH2), 2.87 (ddd, J=15.8/9.9/2.7 Hz, 1H, Ar-CH2),
3.42–3.48 (m, 1H, -CH-NHCOCH3), 3.84 (s, 3H, Ar-OCH3), 6.72 (d, J=
2.5 Hz, 1H, 1-Harom), 6.80 (dd, J=8.7/2.5 Hz, 1H, 3-Harom), and
7.80 ppm (d, J=8.7 Hz, 1H, 4-Harom), a signal for the NH2 group is
not observed in the spectrum; purity by HPLC: 94.7% (tr =
9.70 min); MS (HRMS): m/z (%)=calcd for C12H13O2+: 189.0910,
found: 189.0921 [MꢀNH2, 100]; C12H15NO2, Mr =205.1 Da.
cis- and trans-N-(5-Hydroxy-2-methoxy-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-7-yl)acetamide (cis-12 and trans-12): NaBH4
(14.0 mg, 0.4 mmol) was added to a solution of amido ketone 11
(92 mg, 0.4 mmol) in CH3OH (5 mL) at 08C. After stirring overnight
at room temperature, the solvent was removed under vacuum.
The residue was treated with 1m HCl (5 mL), and the aqueous
layer was extracted with EtOAc (3ꢃ). The combined organic layers
were washed with brine and dried (Na2SO4), the solvent was re-
moved under reduced pressure, and the residue (55 mg) was puri-
fied by FC (d=2 cm, l=18 cm, V=30 mL, cyclohexane/EtOAc, 1:9,
Rf =0.11) to yield the target compound mixture as a colorless oil
cis- and trans-7-Amino-2-methoxy-6,7,8,9-tetrahydro-5H-ben-
zo[7]annulen-5-ol (cis-16 and trans-16): LiBH4 (2 m in THF, 5.3 mL)
was added over 60 min to amino ketone 15 (1.1 g, 5.3 mmol) in
abs. THF (15 mL) at ꢀ788C under N2. After 30 min, the mixture was
warmed to room temperature, and H2O (1 mL) and HCl (2 m, 6 mL)
were added. The suspension was dissolved by addition of NaOH
(2 m) up to pH 8. The solvents were removed under vacuum, and
the residue was dissolved in EtOAc (10 mL) and H2O (10 mL). The
aqueous layer was separated and extracted with EtOAc (3ꢃ15 mL).
The combined organic layers were washed with brine and dried
(Na2SO4), and the solvent was removed under reduced pressure.
The crude residue, a 50:50 ratio of diastereomers, was used in the
next reaction step without purification (colorless oil, yield: 0.9 g,
1
(52 mg, 56%): H NMR (CD3OD): d=1.40–1.66 (m, 2H, Ar-CH2-CH2),
1.91 (s, 3ꢃ0.6H, NHCO-CH3), 1.92 (s, 3ꢃ0.4H, NHCO-CH3), 2.03–2.08
(m, 2ꢃ0.6H, -CHOH-CH2-), 2.17–2.20 (m, 2ꢃ0.4H, -CHOH-CH2-),
2.62–2.79 (m, 2H, Ar-CH2-), 3.75 (s, 3ꢃ0.4H, Ar-OCH3), 3.76 (s, 3ꢃ
0.6H, Ar-OCH3), 4.81 (d, J=10.1 Hz, 0.6H, -CHOH-), 4.07–4.16 (m,
0.6H, -CH-NH-), 4.39–4.47 (m, 0.4H, -CH-NH-), 4.86 (d, J=10.1 Hz,
0.4H, -CHOH-), 6.65–6.68 (m, 1H, 1-Harom), 6.73–6.76 (m, 1H, 3-
Harom), 7.12–7.13 (m, 0.4H, 4-Harom), and 7.39–7.41 ppm (m, 0.6H, 4-
Harom), signals for the NH and OH groups are not observed in the
spectrum; MS (ESI): m/z (%)=248 [MꢀH, 100; C14H19NO3, Mr =
249.3 Da.
1
85%): H NMR (CDCl3): d=1.02–1.24 (m, 1H, 8-CH2), 1.25–1.40 (m,
0.5H, 6-CH2), 1.41–1.54 (m, 0.5H, 8-CH2), 1.81–1.90 (m, 0.5H, 8-CH2),
1.99–2.18 (m, 1H, 6-CH2), 2.55–2.77 (m, 3ꢃ0.5H, 9-CH2, 6-CH2),
2.85–2.98 (m, 1H, 9-CH2), 3.10–3.25 (m, 0.5H, 7-CH), 3.31 (tt, J=8.9/
3.4 Hz, 0.5H, 7-CH), 3.75 (s, 3ꢃ0.5H, Ar-OCH3), 3.76 (s, 3ꢃ0.5H, Ar-
OCH3), 4.77 (d, J=8.0 Hz, 0.5H, cis-5-H), 4.99 (d, J=7.7 Hz, 0.5H,
trans-5-H), 6.62 (dd, J=8.2/2.8 Hz, 0.5H, trans-3-Harom), 6.66–6.69
(m, 1H, 1-Harom),6.72 (dd, J=8.4/2.8 Hz, 0.5H, cis-3-Harom), 7.15 (d,
J=8.3 Hz, 0.5H, trans-4-Harom), and 7.32 ppm (d, J=8.3 Hz, 0.5H,
cis-4-Harom), signals for the OH and NH2 protons are not observed in
the spectrum; C12H17NO2, Mr =207.3 Da.
N-(3-Methoxy-6,7,-dihydro-5H-benzo[7]annulen-7-yl)acetamide
(13) and 3-Methoxy-5H-benzo[7]annulene (14): Amido alcohol 12
(100 mg, 0.4 mmol) was dissolved in 5m HCl (8 mL) and N,N-dime-
thylformamide. The mixture was stirred at 608C for 24 h. After
cooling to room temperature, the mixture was washed with Et2O
(3ꢃ). Next, NaOH was added (pH>12), and the mixture was ex-
tracted with EtOAc (3ꢃ). The organic layer was washed with brine,
dried (Na2SO4), concentrated under vacuum, and the residue
(34 mg) was purified by FC (d=4 cm, l=6 cm, V=10 mL, EtOAc/cy-
clohexane, 8:2) to yield 13 and 14 as colorless oils. Compound 13
(yield 11 mg, 12%): Rf =0.15; 1H NMR (CDCl3): d=2.00 (s, 3H,
-NHCOCH3), 1.94–2.10 (m, 2H, 6-H), 2.77 (ddd, J=15.4/9.4/1.9 Hz,
1H, 5-H), 2.84 (ddd, J=15.4/8.6/2.06 Hz, 1H, 5-H), 3.80 (s, 3H, Ar-
OCH3), 4.78–4.85 (m, 1H, 7-H), 5.56 (dd, J=12.3/4.2 Hz, 1H, 8-H),
5.60–5.67 (m, 1H, NH), 6.41 (dd, J=12.3/1.9 Hz, 1H, 9-H), 6.68 (d,
J=2.7 Hz, 1H, 4-H), 6.70 (dd, J=8.3/2.7 Hz, 1H, 2-H), 7.10 ppm (d,
J=8.3 Hz, 1H, 1-H); C14H17NO2, Mr =231.3 Da. Compound 14 (yield:
cis-2-Methoxy-7-[N-(3-phenylpropyl)amino]-6,7,8,9-tetrahydro-
5H-benzo[7]annulen-5-ol (cis-17a), trans-2-methoxy-7-[N-(3-phe-
nylpropyl)amino]-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol
(trans-18a), trans-7-[N,N-Bis(3-phenylpropyl)amino]-2-methoxy-
6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (trans-19a), and 2-
methoxy-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]an-
nulen-7-amine (20a): Amino ketone 15 (915 mg, 4.5 mmol) was
reduced with LiBH4 as described above. After workup, the crude
amino alcohol 16 and 3-phenylpropionaldehyde (86) (510 mg,
3.8 mmol) were dissolved in abs. CH2Cl2 (20 mL). After the mixture
was stirred for 30 min at room temperature under N2, NaBH(OAc)3
(1.1 g, 5.3 mmol) was added. The reaction was stopped after 4 h by
addition of a sat. aqueous NaHCO3 solution (25 mL). The aqueous
layer was separated and extracted with CH2Cl2 (3ꢃ20 mL). The
combined organic layers were washed with brine and dried
(Na2SO4), the solvent was removed under reduced pressure, and
the residue (1.4 g) was purified by FC (d=3 cm, l=17 cm, V=
30 mL, CH2Cl2/CH3OH, 95:5).
1
5 mg, 8%): Rf =0.44; H NMR (CDCl3): d=2.97 (d, J=6.8 Hz, 2H, 5-
H), 3.75 (s, 3H, Ar-OCH3), 5.66 (dt, J=9.8/6.8 Hz 1H, 6-H), 5.99 (dd,
J=9.8/5.4 Hz, 1H, 7-H), 6.29 (dd, J=11.4/5.4 Hz, 1H, 8-H), 6.63 (d,
J=2.6 Hz, 1H, 4-H), 6.72 (dd, J=8.5/2.6 Hz, 1H, 2-H), 6.95 (d, J=
11.5 Hz, 1H, 9-H), and 7.17 ppm (d, J=8.6 Hz, 1H, 1-H); C12H12O,
Mr =172.2 Da.
cis-17a: The crude product (200 mg) was purified by recrystalliza-
tion (EtOAc/diisopropyl ether, 3:1) to yield a colorless solid (yield:
57 mg, 4%): Rf =0.25; 1H NMR (CDCl3): d=1.87–2.04 (m, 3H,
-CHOH-CH2-, Ar-CH2-CH2-), 1.99 (quint, J=7.5 Hz, 2H, -CH2-CH2-
7-Amino-2-methoxy-6,7,8,9-tetrahydrobenzo[7]annulen-5-one
(15): Amido ketone 11 (1.3 g, 5.3 mmol) was suspended in 2m HCl
(20 mL) and heated at 958C for 16 h. After cooling to room tem-
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 747

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
C6H5), 2.22–2.36 (m, 1H, -CHOH-CH2-), 2.49 (td, J=9.9/4.9 Hz, 1H,
Ar-CH2-), 2.68 (t, J=7.5 Hz, 2H, -CH2-CH2-C6H5), 2.78 (dt, J=11.6/
7.5 Hz, 1H, -NH-CH2-CH2-CH2-C6H5), 2.85 (dt, J=11.7/7.5 Hz, 1H,
-NH-CH2-CH2-CH2-C6H5), 3.19–3.37 (m, 2H, -CH-NH-, Ar-CH2-), 3.77 (s,
3H, Ar-OCH3), 4.84 (d, J=7.5 Hz, 1H, -CH-OH), 6.64 (d, J=2.4 Hz,
1H, 1-Harom), 6.67 (dd, J=8.2/2.4 Hz, 1H, 3-Harom), and 7.17–
7.31 ppm (m, 6H, -C6H5, 4-Harom), signals for the OH and NH groups
are not observed in the spectrum; 13C NMR (CDCl3): d=30.0 (1C,
Ar-CH2-), 30.3 (1C, -CH2-CH2-C6H5), 32.0 (1C, Ar-CH2-CH2-), 33.5 (1C,
-CH2-C6H5), 46.1 (1C, -NH-CH2-CH2-), 55.4 (1C, Ar-OCH3), 58.1 (1C,
-CH-NH-), 74.5 (1C, -CH-OH), 110.6 (1C, 3-Carom), 116.3 (1C, 1-Carom),
126.3 (1C, p-C6H5), 128.6 (2C, -C6H5), 128.7 (2C, -C6H5), 136.1 (1C, 4-
Carom), 141.0 (1Cq, 11-Carom), 142.0 (1Cq, -C6H5), 146.1 (1Cq, 10-Carom),
and 158.8 ppm (1Cq, 2-Carom), the signal for C-6 (CHOH-CH2) is not
observed in the spectrum; purity by HPLC: 99.3% (tr =16.43 min);
MS (HRMS): m/z (%)=calcd for C21H28NO2+: 326.2115, found:
326.2155; C21H27NO2, Mr =325.4 Da
Carom), 115.2 (1C, 1-Carom), 128.9 (1C, p-C6H5), 128.5 (2C, -C6H5), 128.6
(2C, -C6H5), 129.9 (1C, 4-Carom), 135.0 (1Cq, 11-Carom), 142.4 (1Cq,
-C6H5), 144.1 (1Cq, 10-Carom), and 159.1 ppm (1Cq, 2-Carom); purity by
HPLC: 97.1% (tr =18.37 min); MS (HRMS): m/z (%)=calcd for
C21H28NO+: 310.2165, found: 310.2131; C21H27NO, Mr =309.4 Da.
cis-2-Methoxy-7-[N-(4-phenylbutyl)amino]-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-5-ol (cis-17b) and trans-2-methoxy-7-[N-(4-phe-
nylbutyl)amino]-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol
(trans-18b): Amino ketone 15 (912 mg, 4.4 mmol) was reduced
with LiBH4 as described above. After workup, the crude amino al-
cohol 16, 4-phenylbutanal (204 mg, 1.38 mmol), and MgSO4
(159 mg, 1.32 mmol) were dissolved and suspended in abs. CH2Cl2
(25 mL), and the mixture was stirred at room temperature for 16 h.
NaBH(OAc)3 (426 mg, 2.01 mmol) was added, and the mixture was
stirred for 4 h at room temperature. The reaction was stopped by
addition of HCl (2 m, pH 1). The mixture was diluted with H2O
(10 mL) and CH2Cl2 (10 mL) and alkalized with NaOH (2 m, pH 8).
The aqueous layer was extracted with CH2Cl2 (2ꢃ15 mL). The com-
bined organic layers were washed with brine and dried (Na2SO4),
the solvent was removed under reduced pressure, and the residue
was purified by FC (d=2 cm, l=17 cm, V=10 mL, CH2Cl2/CH3OH,
18:1+0.84% NH3 (25% aq)).
trans-18a: The crude product (118 mg) was purified by additional
FC (d=2 cm, l=25 cm, V=10 mL, CH2Cl2/CH3OH, 18:1+0.84% NH3
(25% aq), Rf =0.50) to yield a colorless oil (yield: 21 mg, 2%);
1H NMR (CDCl3): d (ppm)=1.37–1.45 (m, 1H, Ar-CH2-CH2-), 1.61 (dd,
J=13.2/10.0 Hz, 1H, -CHOH-CH2-), 1.84 (quint, J=7.4 Hz, 2H, -CH2-
CH2-C6H5), 1.99–2.05 (m, 1H, Ar-CH2-CH2-), 2.20–2.29 (m, 1H,
-CHOH-CH2-), 2.67 (t, J=7.7 Hz, 1H, -CH2-CH2-C6H5), 2.68–2.77 (m,
3H, Ar-CH2-, -NH-CH2-CH2-CH2-C6H5), 2.99 (t, J=12.4 Hz, 1H, Ar-CH2-
), 3.18 (tt, J=9.5/3.3 Hz, 1H, -CH-NH-), 3.79 (s, 3H, Ar-OCH3), 4.99
(d, J=7.6 Hz, 1H, -CH-OH), 6.67 (dd, J=8.5/2.7 Hz, 1H, 3-Harom),
6.64 (d, J=2.4 Hz, 1H, 1-Harom), 7.15 (d, J=8.5 Hz, 1H, 4-Harom), and
7.17–7.32 (m, 5H, -C6H5), signals for the OH and NH groups are not
observed in the spectrum; 13C NMR (CDCl3): d=31.7 (1C, Ar-CH2-),
32.2 (1C, -CH2-CH2-C6H5), 34.0 (1C, -CH2-C6H5), 34.5 (1C, Ar-CH2-CH2-
), 41.5 (1C, -CHOH-CH2-), 47.0 (1C, -NH-CH2-CH2-), 55.1 (1C, Ar-
OCH3), 55.4 (1C, -CH-NH-), 72.3 (1C, -CH-OH), 110.3 (1C, 3-Carom),
116.3 (1C, 1-Carom), 126.0 (1C, p-C6H5), 128.5 (2C, -C6H5), 128.6 (2C,
-C6H5), 128.9 (1C, 4-Carom), 135.6 (1Cq, 11-Carom), 142.3 (1Cq, -C6H5),
143.4 (1Cq, 10-Carom), and 159.1 ppm (1Cq, 2-Carom); purity by HPLC:
98.0% (tr =16.88 min); C21H27NO2, Mr =325.4 Da.
cis-17b was isolated as a colorless oil (yield: 9 mg, 2%): Rf =0.43;
1H NMR (CDCl3): d=1.54–1.60 (m, 2H, -NH-CH2-CH2-), 1.70 (quint,
J=8.0 Hz, 2H, -CH2-CH2-C6H5), 1.72–1.81 (m, 1H, -CHOH-CH2-),
1.84–2.02 (m, 2H, Ar-CH2-CH2-), 2.25–2.38 (m, 1H, -CHOH-CH2-),
2.46–2.52 (m, 1H, Ar-CH2-), 2.65 (t, J=7.5 Hz, 2H, -CH2-CH2-C6H5),
2.68 (dt, J=11.3/7.1 Hz, 1H, N-CH2-CH2-), 2.83 (dt, J=11.2/7.1 Hz,
1H, N-CH2-CH2-), 3.21–3.27 (m, 1H, Ar-CH2-), 3.37–3.52 (m, 1H, -CH-
NH-), 3.78 (s, 3H, Ar-OCH3), 4.84 (d, J=6.5 Hz, 1H, -CH-OH), 6.65
(dd, J=8.1/2.6 Hz, 1H, 3-Harom), 6.68 (d, J=2.6 Hz, 1H, 1-Harom), 7.12
(d, J=8.2 Hz, 1H, 4-Harom), and 7.15–7.30 ppm (m, 5H, -C6H5), sig-
nals for the OH and NH groups are not observed in the spectrum;
13C NMR (CDCl3): d=29.5 (1C, -CH2-CH2-C6H5), 30.3 (1C, -NH-CH2-
CH2-), 31.7 (1C, Ar-CH2-), 34.5 (1C, Ar-CH2-CH2-), 36.1 (1C, -CH2-
C6H5), 41.4 (1C, -CHOH-CH2), 47.3 (1C, -NH-CH2-CH2-), 55.1 (1C, -CH-
NCH3-), 55.4 (1C, Ar-OCH3), 72.3 (1C, -CH-OH), 110.3 (1C, 3-Carom),
116.3 (1C, 1-Carom), 125.9 (1C, p-C6H5), 128.5 (2C, -C6H5), 128.6 (2C,
-C6H5), 128.9 (1C, 4-Carom), 135.6 (1Cq, 11-Carom), 142.7 (1Cq, -C6H5),
trans-19a: The crude product (255 mg) was purified by additional
FC (d=2.5 cm, l=15 cm, V=10 mL, EtOAc/cyclohexane, 8:2, Rf
1
0.28) to yield a colorless oil (yield: 173 mg, 13%); H NMR (CDCl3):
143.4 (1Cq, 10-Carom), and 159.1 ppm (1Cq, 2-Carom); purity by HPLC:
+
d=1.42 (q, J=11.9 Hz, 1H, Ar-CH2-CH2), 1.61 (m, 1H, Ar-CH2-CH2),
1.78 (quint, J=7.3 Hz, 4H, -CH2-CH2-C6H5), 2.06–2.14 (m, 1H,
-CHOH-CH2-), 2.29–2.36 (m, 1H, -CHOH-CH2-), 2.49 (t, J=7.2 Hz, 4H,
-CH2-CH2-C6H5), 2.62 (m, 1H, Ar-CH2), 2.63 (t, J=7.5 Hz, 4H, -CH2-
CH2-CH2-C6H5), 3.15 (t, J=12.8 Hz, 1H, Ar-CH2), 3.37–3.44 (m, 1H,
CH-N-), 3.80 (s, 3H, Ar-OCH3), 4.96 (d, J=6.0 Hz, 1H, -CH-OH), 6.66
(dd, J=8.1/2.6 Hz, 1H, 3-Harom), 6.71 (d, J=2.6 Hz, 1H, 1-Harom), 7.10
(d, J=8.2 Hz, 1H, 4-Harom), and 7.14–7.31 ppm (m, 10H, -C6H5),
a signal for the OH group is not observed in the spectrum;
C30H37NO2, Mr =443.6 Da.
98.9% (tr=17.86 min); MS (HRMS): m/z (%)=calcd for C22H30NO2
340.2271, found: 340.2245; C22H29NO2, Mr =339.5 Da.
:
trans-18b was isolated as a colorless oil (yield: 15 mg, 2%): Rf =
1
0.35; H NMR (CDCl3): d=1.36–1.44 (m, 1H, Ar-CH2-CH2-), 1.49–1.70
(m, 5H, -CHOH-CH2-, -CH2-CH2-CH2-C6H5), 1.99–2.04 (m, 1H, Ar-CH2-
CH2-), 2.21–2.27 (m, 1H, -CHOH-CH2-), 2.63 (t, J=7.5 Hz, 2H, -NH-
CH2-CH2-), 2.68 (dd, J=7.1/3.5 Hz, 1H, -CH2-CH2-C6H5), 2.70 (dd, J=
7.4/3.6 Hz, 1H, -CH2-CH2-C6H5), 2.71–2.77 (m, 1H, Ar-CH2-), 2.93–
3.04 (m, 1H, Ar-CH2-), 3.16 (tt, J=9.5/3.3 Hz, 1H, -CH-NH-), 3.78 (s,
3H, Ar-OCH3), 4.99 (d, J=7.5 Hz, 1H, -CH-OH), 6.65 (dd, J=7.7/
2.7 Hz, 1H, 3-Harom), 6.68 (d, J=2.7 Hz, 1H, 1-Harom), and 7.12–
7.30 ppm (m, 6H, 4-Harom, -C6H5), signals for the OH and NH groups
are not observed in the spectrum; 13C NMR (CDCl3): d=29.5 (1C,
-CH2-CH2-C6H5), 30.3 (1C, -NH-CH2-CH2-), 31.7 (1C, Ar-CH2-), 34.5
(1C, Ar-CH2-CH2-), 36.1 (1C, -CH2-C6H5), 41.4 (1C, -CHOH-CH2), 47.3
(1C, -NH-CH2-CH2-), 55.1 (1C, -CH-NCH3-), 55.4 (1C, Ar-OCH3), 72.3
(1C, -CH-OH), 110.3 (1C, 3-Carom), 116.3 (1C, 1-Carom), 125.9 (1C, p-
C6H5), 128.5 (2C, -C6H5), 128.6 (2C, -C6H5), 128.9 (1C, 4-Carom), 135.6
(1Cq, 11-Carom), 142.7 (1Cq, -C6H5), 143.4 (1Cq, 10-Carom), and
159.1 ppm (1Cq, 2-Carom); purity by HPLC: 95.1% (tr =17.81 min);
C22H29NO2, Mr =339.5 Da.
20a: The crude product (183 mg) was further purified by FC (d=
2.0 cm, l=17 cm, V=10 mL, EtOAc/cyclohexane, 9:1, Rf =0.29) to
yield a colorless oil (yield: 70 mg, 5%): 1H NMR (CDCl3): d=1.20–
1.41 (m, 4H, Ar-(CH2-CH2-)2CH-), 1.82 (quint, J=6.66 Hz, 2H, -CH2-
CH2-C6H5), 2.60–2.79 (m, 9H, CH-N-, 2ꢃAr-CH2, -NH-CH2-CH2-CH2-
C6H5), 3.78 (s, 3H, Ar-OCH3), 6.63 (dd, J=8.1/2.7 Hz, 1H, 3-Harom),
6.68 (d, J=2.7 Hz, 1H, 1-Harom), 7.01 (d, J=8.1 Hz, 1H, 4-Harom), and
7.19–7.31 ppm (m, 5H, -C6H5), a signal for the NH group is not ob-
served in the spectrum; 13C NMR (CDCl3): d=31.6 (1C, Ar-(CH2-)2),
32.4 (1C, -CH2-CH2-C6H5), 32.8 (1C, Ar-(CH2-)2), 34.0 (1C, -CH2-C6H5),
34.8 (1C, Ar-(CH2-CH2-)2), 35.2 (1C, Ar-(CH2-CH2-)2), 46.8 (1C, -NH-
CH2-CH2-), 55.4 (1C, Ar-OCH3), 61.6 (1C, -CH-NH-), 110.7 (1C, 3-
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 748

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
cis- and trans-2-Methoxy-7-[N-(4-phenylbutyl)amino]-6,7,8,9-tet-
rahydro-5H-benzo[7]annulen-5-ol (cis-17b/trans-18b) and cis-
trans-2-Methoxy-7-[N-methyl-N-(3-phenylpropyl)amino]-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-5-ol
(trans-22a):
trans-18a
and
trans-7-[N,N-Bis(4-phenylbutyl)amino]-2-methoxy-6,7,8,9-
suspension of
(46 mg, 0.14 mmol) and formaldehyde (37%, 228 mL, 2.8 mmol)
were dissolved in CH2Cl2 (5 mL). NaBH(OAc)3 (42 mg, 0.2 mmol)
was added, and the mixture was stirred for 12 h at room tempera-
ture. The reaction was stopped by the addition of H2O and HCl
(2 m, pH 1). The mixture was alkalized with NaOH (2 m, pH 8) and
extracted with CH2Cl2 (4ꢃ10 mL). The organic layers were washed
with brine, dried (Na2SO4), and concentrated under vacuum, and
the residue was purified by FC (d=2 cm, l=20 cm, V=5 mL,
CH2Cl2/CH3OH, 18:1+0.84% NH3 (25% aq), Rf =0.23) to yield a color-
tetrahydro-5H-benzo[7]annulen-5-ol (19b):
A
amino alcohol 16 (60 mg, 0.3 mmol), 1-chloro-4-phenylbutane
(108 mg, 0.6 mmol), Bu4NI (236 mg, 0.6 mmol) and K2CO3 (240 mg,
1.7 mmol) in CH3CN (6 mL) was stirred at 858C for 24 h. The mix-
ture was filtered, and the solvent was removed under reduced
pressure. The residue was purified by FC (d=2.5 cm, l=14 cm, V=
10 mL, EtOAc/cyclohexane, 95:5+2% N,N-dimethylethylamine).
cis-17b/trans-18b was isolated as a colorless oil (yield: 23 mg,
23%): Rf =0.28; 1H NMR (CDCl3): d=1.43–1.55 (m, 2H, -NH-CH2-
CH2-), 1.56–1.74 (m, 3H, -CH2-CH2-C6H5, -CHOH-CH2-), 1.75–1.90 (m,
2H, Ar-CH2-CH2-), 2.13–2.26 (m, 1H, -CHOH-CH2-), 2.38–2.46 (m, 1H,
Ar-CH2-), 2.53–2.63 (m, 3H, -CH2-CH2-C6H5, N-CH2-CH2-), 2.71–2.78
(m, 1H, N-CH2-CH2-), 2.87–2.96 (m, 1H, Ar-CH2-), 3.13–3.18 (m, 1H,
-CH-NH-), 3.71 (s, 3H, Ar-OCH3), 4.74 (d, J=6.7 Hz, 0.7ꢃ1H, -CH-
OH), 4.92 (d, J=7.0 Hz, 0.3ꢃ1H, -CH-OH), 6.58 (dd, J=8.2/2.7 Hz,
1H, 3-Harom), 6.60 (d, J=2.7 Hz, 1H, 1-Harom), 7.05 (d, J=8.1 Hz, 1H,
4-Harom), and 7.08–7.23 ppm (m, 5H, -C6H5), signals for the OH and
NH groups are not observed in the spectrum; C22H29NO2, Mr =
339.5 Da.
1
less oil (25 mg, 74%): H NMR (CDCl3): d=1.45 (m, 1H, Ar-CH2-CH2-
), 1.65 (dd, J=11.9/1.4 Hz, 1H, -CHOH-CH2-), 1.81 (quint, J=7.5 Hz,
2H, -CH2-CH2-C6H5), 2.03–2.12 (m, 1H, Ar-CH2-CH2-), 2.23 (s, 3H,
-NCH3), 2.27–2.33 (m, 1H, -CHOH-CH2-), 2.47 (t, J=7.4 Hz, 2H,
-NCH3-CH2-CH2-), 2.63 (t, J=7.5 Hz, 2H, -CH2-CH2-C6H5), 2.63–2.69
(m, 1H, Ar-CH2-), 3.13 (t, J=12.8 Hz, 1H, Ar-CH2-), 3.31 (tt, J=11.7/
2.7 Hz, 1H, -CH-N-), 3.78 (s, 3H, Ar-OCH3), 4.97 (d, J=6.8 Hz, 1H,
-CH-OH), 6.64 (dd, J=8.2/2.6 Hz, 1H, 3-Harom), 6.69 (d, J=2.6 Hz,
1H, 1-Harom), 7.09 (d, J=8.4 Hz, 1H, 4-Harom), and 7.16–7.30 ppm (m,
5H, -C6H5), a signal for the OH group is not observed in the spec-
trum; 13C NMR (CDCl3): d=30.2 (1C, -CH2-CH2-C6H5), 30.5 (1C, Ar-
CH2-CH2-), 33.0 (1C, Ar-CH2-), 33.9 (1C, -CH2-C6H5), 36.1 (1C, -CHOH-
CH2-), 37.6 (1C, -NCH3), 53.4 (1C, -NCH3-CH2-CH2-), 55.4 (1C, Ar-
OCH3), 59.8 (1C, -CH-NCH3-), 74.1 (1C, -CH-OH), 110.3 (1C, 3-Carom),
116.6 (1C, 1-Carom), 125.9 (1C, p-C6H5), 128.5 (2C, -C6H5), 128.6 (2C,
-C6H5), 130.2 (1C, 4-Carom), 135.1 (1Cq, 11-Carom), 142.6 (1Cq, -C6H5),
19b was isolated as a colorless oil (yield: 81 mg, 59%): Rf =0.36;
1H NMR (CDCl3): d=1.28–1.42 (m, 5H, -NH-CH2-CH2-, Ar-CH2-CH2-),
1.46–1.58 (m, 5H, -CH2-CH2-C6H5, -CHOH-CH2-), 1.94–2.02 (m, 1H,
Ar-CH2-CH2-), 2.16–2.24 (m, 1H, -CHOH-CH2-), 2.32 (t, J=6.9 Hz, 4H,
N-CH2-CH2-), 2.52 (t, J=7.7 Hz, 4H, -CH2-CH2-C6H5), 2.52–2.59 (m,
1H, Ar-CH2-), 3.00–3.10 (m, 1H, Ar-CH2-), 3.20–3.28 (m, 1H, -CH-NH-
), 3.72 (s, 3H, Ar-OCH3), 4.69 (d, J=9.8 Hz, 0.4ꢃ1H, -CH-OH), 4.87
(d, J=6.3 Hz, 0.6ꢃ1H, -CH-OH), 6.57 (dd, J=8.2/2.6 Hz, 1H, 3-
Harom), 6.62 (d, J=2.6 Hz, 1H, 1-Harom), 7.01 (d, J=8.2 Hz, 1H, 4-
Harom), and 7.06–7.23 ppm (m, 10H, -C6H5), a signal for the OH
group is not observed in the spectrum; C32H41NO2, Mr =471.7 Da.
144.1 (1Cq, 10-Carom), and 159.4 ppm (1Cq, 2-Carom); purity by HPLC:
+
95.2% (tr =17.35 min); MS (HRMS): m/z (%)=calcd for C22H30NO2
340.2271, found: 340.2297; C22H29NO2, Mr =339.5 Da.
:
2-Methoxy-N-methyl-N-(3-phenylpropyl)-6,7,8,9-tetrahydro-5H-
benzo[7]annulen-7-amine (23a): Secondary amine 20a (31 mg,
0.1 mmol) and formaldehyde (37%, 162 mL, 2 mmol) were dissolved
in CH2Cl2 (3 mL). NaBH(OAc)3 (44 mg, 0.21 mmol) was added, and
the mixture was stirred for 12 h at room temperature. The reaction
was stopped by addition of H2O and HCl (2 m, pH 1), then the mix-
ture was alkalized with NaOH (2 m, pH 8) and extracted with
CH2Cl2 (4ꢃ10 mL). The organic layer was washed with brine, dried
(Na2SO4), and concentrated under vacuum, and the residue was
purified by FC (d=1.5 cm, l=15 cm, V=5 mL, CH2Cl2/CH3OH, 95:5,
cis-2-Methoxy-7-[N-methyl-N-(3-phenylpropyl)amino]-6,7,8,9-tet-
rahydro-5H-benzo[7]annulen-5-ol (cis-21a): cis-17a (35 mg,
0.1 mmol) and formaldehyde (37%, 179 mL, 2.2 mmol) were dis-
solved in CH2Cl2 (3 mL). NaBH(OAc)3 (46 mg, 0.22 mmol) was
added, and the mixture was stirred at room temperature for 16 h.
The reaction was stopped by addition of H2O and HCl (2 m, pH 1).
The mixture was alkalized with NaOH (2 m, pH 8) and extracted
with CH2Cl2 (4ꢃ10 mL). The organic layer was washed with brine,
dried (Na2SO4), and concentrated under vacuum, and the residue
was purified by FC (d=2 cm, l=14 cm, V=10 mL, CH2Cl2/CH3OH,
1
Rf =0.36) to yield a colorless oil (23 mg, 71%): H NMR (CDCl3): d=
1.30–1.43 (m, 2H, Ar-CH2-CH2-), 1.93 (quint, J=7.5 Hz, 2H, -CH2-
CH2-C6H5), 2.14–2.21 (m, 2H, Ar-CH2-CH2-), 2.33 (s, 3H, -N-CH3), 2.58
(t, J=7.6 Hz, 2H, -CH2-CH2-C6H5), 2.65 (t, J=7.5 Hz, 2H, -NH-CH2-
CH2-), 2.65–2.71 (m, 2H, Ar-CH2-), 2.73–2.80 (m, 2H, Ar-CH2-), 2.88–
2.93 (m, 1H, -CH-N-), 3.78 (s, 3H, Ar-OCH3), 6.65 (dd, J=8.1/2.7 Hz,
1H, 3-Harom), 6.69 (d, J=2.7 Hz, 1H, 1-Harom), 7.02 (d, J=8.1 Hz, 1H,
4-Harom), and 7.16–7.30 ppm (m, 5H, -C6H5); 13C NMR (CDCl3): d=
29.1 (1C, -CH2-CH2-C6H5), 29.5 (1C, Ar(-CH2-CH2-)2), 29.7 (1C, Ar(-
CH2-CH2-)2), 32.4 (1C, Ar(-CH2-)2), 33.6 (1C, Ar(-CH2-)2), 33.7 (1C,
-CH2-C6H5), 37.5 (1C, -NCH3), 53.3 (1C, -NCH3-CH2-CH2-), 55.5 (1C,
Ar-OCH3), 68.0 (1C, -CH-NH-), 111.0 (1C, 3-Carom), 115.2 (1C, 1-Carom),
126.2 (1C, p-C6H5), 128.5 (2C, -C6H5), 128.6 (2C, -C6H5), 130.1 (1C, 4-
Carom), 134.4 (1Cq, 11-Carom), 141.8 (1Cq, -C6H5), 143.5 (1Cq, 10-Carom),
and 158.3 ppm (1Cq, 2-Carom); purity by HPLC: 96.9% (tr =
19.14 min); MS (HRMS): m/z (%)=calcd for C22H30NO+: 324.2322,
found: 324.2298; C22H29NO, Mr =323.5 Da.
1
9:1, Rf =0.33) to yield a colorless oil (24 mg, 72%): H NMR (CDCl3):
d=1.50–1.66 (m, 1H, Ar-CH2-CH2-), 1.83 (quint, J=7.6 Hz, 2H, -CH2-
CH2-C6H5), 1.88–2.08 (m, 3H, Ar-CH2-CH2-, -CHOH-CH2-), 2.28 (s, 3H,
-N-CH3), 2.46–2.58 (m, 3H, -CH2-CH2-C6H5, Ar-CH2-), 2.63 (t, J=
7.6 Hz, 2H, -NH-CH2-CH2-), 2.86–2.98 (m, 2H, Ar-CH2-, -CH-N-), 3.79
(s, 3H, Ar-OCH3), 4.79 (d, J=10.4 Hz, 1H, -CH-OH), 6.67 (d, J=
2.7 Hz, 1H, 1-Harom), 6.73 (dd, J=8.4/2.7 Hz, 1H, 3-Harom), 7.16–7.30
(m, 5H, -C6H5), and 7.36 ppm (d, J=8.4 Hz, 1H, 4-Harom), a signal for
the OH group is not observed in the spectrum; 13C NMR (CDCl3):
d=29.3 (1C, -CH2-CH2-C6H5), 30.0 (1C, Ar-CH2-CH2-), 31.8 (1C, Ar-
CH2-), 33.8 (1C, -CH2-C6H5), 38.2 (1C, -NCH3), 53.5 (1C, -NCH3-CH2-
CH2-), 55.4 (1C, Ar-OCH3), 64.0 (1C, -CH-NCH3-), 72.1 (1C, -CH-OH),
110.6 (1C, 3-Carom), 115.8 (1C, 1-Carom), 126.0 (1C, p-C6H5), 128.5 (2C,
-C6H5), 128.6 (2C, -C6H5), 136.7 (1C, 4-Carom), 141.3 (1Cq, 11-Carom),
142.2 (1Cq, -C6H5), 146.1 (1Cq, 10-Carom), and 158.6 ppm (1Cq, 2-
Carom), the signal for C-6 (CHOH-CH2) is not observed in the spec-
trum; purity by HPLC: 99.2% (tr =16.93 min); MS (HRMS): m/z
(%)=calcd for C22H30NO2+: 340.2271, found: 340.2307; C22H29NO2,
Mr =339.5 Da.
X-ray crystal structure analysis of cis-17a
Data sets were collected with a Nonius KappaCCD diffractometer
using COLLECT (R. W. W. Hooft, Bruker AXS, 2008, Delft, The Neth-
erlands) for data collection, Denzo-SMN{31] for data reduction,
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 749

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
Denzo[32] for absorption correction, SHELXS-97[33] for structure solu-
tion, SHELXL-97[34] for structure refinement, and XP (BrukerAXS,
2000) for graphics. Thermals ellipsoids are shown with 30% proba-
bility, R-values are given for observed reflections, and wR2 values
are given for all reflections.
tions 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
presoaked filtermats using a cell harvester. After washing each well
five times with 300 mL of water, the filtermats were dried at 958C.
Subsequently, the solid scintillator was placed on the filtermat 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. Nonspecific
binding was determined with 10 mm unlabeled ifenprodil. The Kd
value of ifenprodil is 10 nm.[19]
Formula C21H27NO2, M=325.44, colorless crystal, 0.30ꢃ0.13ꢃ
0.10 mm, a=9.9223(2), b=35.8022(7), c=10.7061(2) ꢄ, b=
105.745(1)8, V=3660.5(1) ꢄ3, 1calc =1.181 gcmꢀ3, m=0.587 mmꢀ1
,
empirical absorption correction (0.843ꢂTꢂ0.943), Z=8, monoclin-
ic, space group P21/n (No. 14), l=1.54178 ꢄ, T=223(2) K, w and f
scans, 23476 reflections collected (ꢁh, ꢁk, ꢁl), [(sinq)/l]=
0.60 ꢄꢀ1, 6030 independent (Rint =0.054) and 5093 observed reflec-
tions [I>2s(I)], 443 refined parameters, R=0.058, wR2 =0.170, max.
(min.) residual electron density 0.24 (ꢀ0.25) eꢄꢀ3, the hydrogens at
nitrogen atoms were refined freely, but with fixed U-values; others
were calculated and refined as riding atoms. CCDC 976582 con-
tains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Protocol for functional GluN2A and GluN2B assay: The test com-
pound solutions were prepared by dissolving ~10 mmol (usually 2–
4 mg) of test compound in DMSO so that a 10 mm stock solution
was obtained. To obtain the required test solutions for the assay,
the DMSO stock solution was diluted with phosphate buffered
saline solution (PBS).
Mouse L(tkꢀ) cells stably transfected with the dexamethasone-in-
ducible eukaryotic expression vectors pMSG NR1-1a and pMSG
NR2A (1:5 ratio) for the GluN2A assay or pMSG and NR2B (1:5
ratio) for the GluN2B assay, grown in sterile 96-well plates in Dul-
becco Modified Earl’s Medium (DMEM) containing 10% of standar-
dized fetal calf serum (FCS, Biochrom AG, Berlin, Germany). When
the monolayer of the adherent growing cells almost covered the
bottom area of each well, the medium was removed, and the cells
in each well were rinsed with 200 mL PBS.
Pharmacology
Materials and general procedures: A high-speed cooling Sorvall
RC-5C plus centrifuge (Thermo Finnigan) was used, with a printed
Filtermat Type B filter (PerkinElmer), presoaked in 0.5% aqueous
polyethylenimine for 2 h at room temperature before use. Filtration
was carried out with a MicroBeta FilterMate-96 Harvester (Perki-
nElmer). 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%.
Subsequently, the cells were incubated with 100 mL dexametha-
sone solution (8 mm in growth medium), 100 mL CSS reagent
(120 mm NaCl, 25 mm HEPES, 50 mm KCl, 4 mm EDTA, 1.8 mm
CaCl2, 15 mm glucose, 100 mm glycine and 100 mm Na-glutamate),
and 50 mL of the respective test compound in six different concen-
trations (40 mm, 4 mm, 400 nm, 40 nm, 4 nm and 0.4 nm). Each con-
centration of the test compound was incubated at least in tripli-
cate for 12 h at 378C.
Cell culture and preparation of membrane homogenates for the
GluN2B assay:[19] In the assay, mouse L(tkꢀ) cells stably transfected
with the dexamethasone-inducible eukaryotic expression vectors
pMSG NR1a and pMSG NR2B were used in a 1:5 ratio. The trans-
formed L(tkꢀ) cells were grown in Modified Earl’s Medium (MEM)
containing 10% of standardized FCS (Biochrom AG, Berlin, Germa-
ny). The expression of the NMDA receptor at the cell surface was
induced after the cell density of the adherent growing cells
reached ~90% confluency. For induction, the original growth
medium was replaced by growth medium containing 4 mm dexa-
methasone and 4 mm ketamine (final concentration). After 24 h, the
cells were harvested by scraping and pelleted (10 min, 5000ꢃg,
Hettich Rotina 35R centrifuge, Tuttlingen, Germany).
Fifty microliters of the supernatant were transferred into a second
96-well plate and incubated with 50 mL of lactate dehydrogenase
(LDH) assay mixture (18 U/mL diaphorase, 1% Na-lactate, 0.1%
NAD+, 0.08% BSA, 0.4% iodonitrotetrazolium chloride in 75 mm
PBS) at room temperature. Previously, the 96-well plate was
blocked with 1% BSA solution for 12 h. The reaction was terminat-
ed after 30 min by the addition of 100 mL of acetic acid (1 m), and
UV absorption was measured at l=490 nm in a plate reader
(TECAN, Crailsheim, Germany). Total LDH activity was determined
with buffer instead of ligand solution. A solution of 0.1% Triton-X
served as a positive control. Data were analyzed using Sigma Plot
by nonlinear regression analysis (sigmoidal dose response).
For the binding assay, the cell pellet was resuspended in phos-
phate buffered saline (PBS, pH 7.4), and the number of cells was
determined using an improved Neubauer’s counting chamber
(VWR, Darmstadt, Germany). Subsequently, the cells were lysed by
sonication (48C, 6ꢃ10 s cycles with breaks of 10 s, using a Soni-
prep 150, MSE, London, UK). The resulting cell fragments were cen-
trifuged with a high performance centrifuge (20000ꢃg, 48C, Sor-
vall RC-5 plus, Thermo Scientific). The supernatant was discarded,
and the pellet was resuspended in a defined volume of PBS yield-
ing cell fragments of ~500000 cells per mL. The suspension of
membrane homogenates was sonicated again (48C, 2ꢃ10 s cycles
with a break of 10 min) and stored at ꢀ808C.
Performing of the GluN2B binding assay:[19] The competitive
binding assay was performed with the radioligand [3H]ifenprodil
(60 Cimmolꢀ1; PerkinElmer) using standard 96-well multiplates (Di-
agonal, Mꢀnster, Germany). The thawed cell membrane prepara-
tion (~20 mg protein) was incubated with six different concentra-
Affinity toward s1 and s2 receptors and the PCP binding site of
the NMDA receptor: The affinity toward the PCP binding site of
the NMDA receptor[25,26] and the affinity toward s1 and s2 recep-
tors[27–29] were recorded as previously described.
Abbreviations
2-Amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA);
bovine serum albumin (BSA); central nervous system (CNS); di-o-
tolylguanidine (DTG); 1-(1-phenylcyclohexyl)piperidine (PCP); N-
methyl-d-aspartate (NMDA).
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 750

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[14] S. F. Menniti, J. M. Pagnozzi, P. Butler, B. L. Chenard, S. S. Jaw-Tasi, W. F.
White, Neuropharmacology 2000, 39, 1147–1155.
Acknowledgements
[15] J. Nagy, C. Horvath, S. Farkas, S. Kolok, Z. Szombathelyi, Neurochem. Int.
2004, 44, 17–23.
[16] E. Karakas, N. Simorowski, H. Furukawa, Nature 2011, 475, 249–253.
[17] I. J. Reynolds, R. J. Miller, Mol. Pharmacol. 1989, 36, 758–765.
[18] K. Williams, Curr. Drug Targets 2001, 2, 285–298.
The authors are grateful to Prof. Dr. D. Steinhilber (Department
of Pharmacy, University of Frankfurt) for donating L(tkꢀ) cells
stably expressing GluN1a/GluN2A and GluN1a/GluN2B receptor
proteins. Funding by the Deutsche Forschungsgemeinschaft is
gratefully acknowledged.
[19] D. Schepmann, B. Frehland, K. Lehmkuhl, B. Tewes, B. Wꢀnsch, J. Pharm.
Biomed. Anal. 2010, 53, 603–608.
[20] E. Falck, F. Begrow, E. Verspohl, B. Wꢀnsch, J. Pharm. Biomed. Anal.
2014, 88, 96–105.
[21] B. Tewes, B. Frehland, D. Schepmann, K.-U. Schmidtke, T. Winckler, B.
Wꢀnsch, ChemMedChem 2010, 5, 687–695.
[22] B. Tewes, B. Frehland, D. Schepmann, K.-U. Schmidtke, T. Winckler, B.
Wꢀnsch, Bioorg. Med. Chem. 2010, 18, 8005–8015.
Keywords: benzo[7]annulenamines · cytoprotective activity ·
GluN2B · NDMA receptors · selectivity · structure–affinity
relationships
[23] P. E. Eaton, G. R. Carlson, J. T. Lee, J. Org. Chem. 1973, 38, 4071–4073.
[24] A. F. Abdel-Magid, S. J. Mehrmann, Org. Process Res. Dev. 2006, 10, 971–
1031.
[25] T. Utech, J. Kçhler, B. Wꢀnsch, Eur. J. Med. Chem. 2011, 46, 2157–2169.
[26] U. Wirt, D. Schepmann, B. Wꢀnsch, Eur. J. Org. Chem. 2007, 462–475.
[27] C. A. Maier, B. Wꢀnsch, J. Med. Chem. 2002, 45, 438–448.
[28] C. A. Maier, B. Wꢀnsch, J. Med. Chem. 2002, 45, 4923–4930.
[29] C. Meyer, B. Neue, D. Schepmann, S. Yanagisawa, J. Yamaguchi, E.-U.
Wꢀrthwein, K. Itami, B. Wꢀnsch, Bioorg. Med. Chem. 2013, 21, 1844–
1856.
[30] Y. Rook, K.-U. Schmidtke, F. Gaube, D. Schepmann, B. Wꢀnsch, J. Heil-
mann, J. Lehmann, T. Winckler, J. Med. Chem. 2010, 53, 3611–3617.
[31] Z. Otwinowski, W. Minor, Methods in Enzymology, Vol. 276: Macromolec-
ular Crystallography, Part A (Eds.: C. W. Carter, Jr., R. M. Sweet), Academ-
ic Press, New York, 1997, pp. 307–326.
[1] M. B. Kennedy, Science 2000, 290, 750–754.
[2] H. Brꢅuner-Osborne, J. Egebjerg, E. O. Nielsen, U. Madsen, P. Krogs-
gaard-Larsen, J. Med. Chem. 2000, 43, 2609–2644.
[3] H. Stark, S. Graßmann, U. Reichert, Pharm. Unserer Zeit 2000, 29, 228–
236.
[4] H. Furukawa, S. K. Singh, R. Mancusco, E. Gouaux, Nature 2005, 438,
185–192.
[5] R. Paoletti, J. Neyton, Curr. Opin. Pharmacol. 2007, 7, 39–47.
[6] D. J. Goebel, M. S. Poosch, Mol. Brain Res. 1999, 69, 164–170.
[7] S. Boyce, A. Wyatt, J. K. Webb, R. O’Donnell, G. Mason, M. Rigby, D. Siri-
nathsinghji, R. G. Hill, N. M. J. Rupniak, Neuropharmacology 1999, 38,
611–623.
[8] M. E. Layton, M. J. Kelly III, K. J. Rodzinak, Curr. Top. Med. Chem. 2006, 6,
697–709.
[9] I. Borza, G. Domꢆny, Curr. Top. Med. Chem. 2006, 6, 687–695.
[10] L. Mony, J. N. C. Kew, M. J. Gunthorpe, P. Paoletti, Br. J. Pharmacol. 2009,
157, 1301–1317.
[32] Z. Otwinowski, D. Borek, W. Majewski, W. Minor, Acta Crystallogr. Sect. A
2003, 59, 228–234.
[33] G. M. Sheldrick, Acta Crystallogr. Sect. A 1990, 46, 467–473.
[34] G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112–122.
[11] L. Yurkewicz, J. Weaver, M. R. Bullock, L. F. Marshall, J. Neurotrauma
2005, 22, 1428–1443.
[12] X. Di, R. Bullock, J. Watson, P. Fatouros, B. Chenard, F. White, F. Corwin,
Stroke 1997, 28, 2244–2251.
Received: December 20, 2013
[13] R. H. Wessell, S. M. Ahmed, F. S. Menniti, G. L. Dunbar, T. N. Chase, J. D.
Oh, Neuropharmacology 2004, 47, 184–194.
Published online on February 23, 2014
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 741 – 751 751