Benzimidazolone bioisosteres of potent GluN2B selective NMDA receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2016.03.065

Overactivation of the NMDA receptor is associated with excitotoxic events leading to neurodegenerative processes as observed during the development of Alzheimer's disease, ParFnson's disease, Chorea Huntington and epilepsy. Negative allosteric modulators

Full text of DOI:10.1016/j.ejmech.2016.03.065

European Journal of Medicinal Chemistry 116 (2016) 136e146
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Benzimidazolone bioisosteres of potent GluN2B selective NMDA
receptor antagonists
a
a
a, b, *
Ines Lütnant , Dirk Schepmann , Bernhard Wünsch
a
Institut für Pharmazeutische und Medizinische Chemie der Universit a€ t Münster, Corrensstraße 48, D-48149 Münster, Germany
Cells-in-Motion Cluster of Excellence (EXC 1003 e CiM), Westf a€ lische Wilhelms-Universit a€ t Münster, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2015
Received in revised form
Overactivation of the NMDA receptor is associated with excitotoxic events leading to neurodegenerative
processes as observed during the development of Alzheimer's disease, ParFnson's disease, Chorea
Huntington and epilepsy. Negative allosteric modulators addressing selectively the ifenprodil binding
site of GluN2B subunit containing NMDA receptors are of major interest due to their neuroprotective
potential accompanied by few side effects. Herein benzimidazolone bioisosteres of potent GluN2B an-
tagonists 1e5 were designed and synthesized. A seven step sequence provided the central intermediate
27 January 2016
Accepted 21 March 2016
Available online 28 March 2016
1
9 in 28% yield. Elimination of water, methylation, epoxidation, epoxide rearrangement and finally
Keywords:
NMDA antagonists
GluN2B selective
reductive amination afforded the [7]annulenobenzimidazolone 30 with a 3-phenylpropylamino sub-
stituent in 6-position. Although 30 fits nicely into the pharmacophore of potent GluN2B antagonists, the
Benzimidazolone bioisosteres
Tricyclic benzo[7]annulenamines
Receptor selectivity
gluN2B binding affinity of 30 was only moderate (K
over the receptor (K
¼ 549 nM). The moderate GluN2B affinity was explained by the rigid tricyclic
structure of the [7]annulenobenzimidazolone 30.
i
¼ 697 nM). Additionally, 30 shows low selectivity
s
2
i
©
2016 Elsevier Masson SAS. All rights reserved.
1
. Introduction
development of neuronal connections [8]. Therefore it is discussed
for a long time that the NMDA receptor is involved in synaptic
plasticity [8], which is connected with neurogenesis [9], learning
and memory [10].
The excitatory amino acid neurotransmitter (S)-glutamate me-
diates its effects by activation of metabotropic and ionotropic
glutamate receptors [1]. AMPA, kainate and NMDA receptors
belong to the class of ionotropic glutamate receptors, which
represent ligand gated ion channels [2]. The NMDA receptor reveals
unique properties including coagonism, i.e. activation requires two
agonists glutamate and glycine binding simultaneously at the re-
ceptor, a high permeability for Ca -ions (in addition to Na - and
K -ions) and voltage dependent blockade by Mg -ions, which has
to be removed before the ion channel can be opened [3e6]. Due to
the Mg -block, NMDA receptors are slower activated and deacti-
vated than AMPA and kainate receptors [2].
Certain conditions (e.g. cerebral ischemia, stroke, epilepsy)
change the glutamate homoeostasis and increase the release of
glutamate resulting in overstimulation of neurons and finally in
neuronal dysfunction and damage (excitotoxicity). The NMDA re-
ceptor plays a central role in this process of excitotoxicity, because
the excessive amount of glutamate leads to its overactivation fol-
2
þ
þ
þ
2þ
2þ
lowed by massive influx of Ca -ions into the neuron. The high
2
þ
concentration of Ca -ions leads to uncontrolled overactivation of
several enzymes inducing necrosis and apoptosis [11]. This process
of excitotoxicity is associated with a number of neurodegenerative
diseases including Alzheimer's disease, Parkinson's disease, Chorea
Huntington and epilepsy [7]. The involvement of the NMDA re-
ceptor in the process of excitotoxicity supports the hypothesis that
a blockade of the receptor results in neuroprotective effects [3].
Thus the NMDA receptor represents a promising target for the
development of novel neuroprotective drugs indicated for the
treatment of acute and chronic neurodamaging processes.
2
þ
2þ
The activation of the NMDA receptor causes an influx of Ca
-
ions into the neuron, which activates several secondary signaling
cascades leading to long lasting changes in synaptic activity [7]. The
extraordinary alliance between the coagonism of glutamate and
2
þ
glycine and the voltage-dependent Mg -blockade results in the
*
Corresponding author Institut für Pharmazeutische und Medizinische Chemie
Three types of subunits termed GluN1-GluN3 have been iden-
tified so far [12]. Four of these subunits build up the NMDA receptor
der Universit €a t Münster, Corrensstraße 48, D-48149 Münster, Germany.
E-mail address: wuensch@uni-muenster.de (B. Wünsch).
http://dx.doi.org/10.1016/j.ejmech.2016.03.065
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
137
in a dimer of dimers fashion (heterotetramer) [13e15]. A functional
NMDA receptor contains at least two GluN1 subunits [13] and,
moreover, the composition of the NMDA receptor entails its phar-
macology and its functional properties [16]. The GluN1 subunit
existing in eight splice variants GluN1a-GluN1h is distributed
ubiquitously in the central nervous system [17]. However, four
genes encoding four different GluN2 subunits termed GluN2A-
GluN2D have been identified. The expression of GluN2 subunits
differs considerably in different regions of the central nervous
system [18]. Due to this fact, it is supposed that the specific prop-
erties of the heterotetrameric ion channel are related to the type of
GluN2 subunit incorporated into the receptor [18e20].
In order to restrict the adaptability to other receptors and thus
increase selectivity, the conformational flexibility of the phenyl-
ethylamine side chain of
benzazepine ring of 2. The phenol 2a (K
methyl ether 2b (K
¼ 5.4 nM) show the same or slightly increased
1
was incorporated into the 3-
i
¼ 14 nM) and the
i
GluN2B affinity but considerably increased selectivity compared to
ifenprodil [28,29]. A similar idea was followed during the devel-
opment of the benzo[7]annulen-7-amines 3, comprising an
increased distance between the basic amino moiety and the “left”
aromatic ring, which is comparable to the distance in the lead
compound Ro 25-6981. The GluN2B affinity of 3a and 3b is in the
same range as the affinity of ifenprodil and 3-benzazepines 2 [30].
The phenolic OH group of 1, 2a and 4 was replaced by a methoxy-
group in 2b, 3a and 3b without losing GluN2B affinity [28e30].
In order to get rid of the phenolic OH moiety, which is conju-
gated with glucuronic acid very fast [26], but retaining the H-bond
donor property, the phenol of 1 was bioisosterically replaced by a
benzoxazolone system as realized in besonprodil (5) and in the
tricyclic compounds 6. Whereas besonprodil (5) shows GluN2B
Therefore the development of NMDA receptor antagonists and
allosteric modulators addressing selectively receptors containing a
particular GluN2 subunit is of major interest. In particular GluN2B-
selective NMDA receptor antagonists have been reported to show
neuroprotective potential with little or less severe side effects
®
[
21e23]. Ifenprodil (1, Vadilex , Fig. 1) was the first and is still one
of the best known GluN2B-selective NMDA receptor antagonists
[
24]. Ifenprodil binds with high affinity (IC50 ¼ 13.3 nM) [15] to
affinity in the low nanomolar range (K
i
¼ 30 nM) [31], the tricyclic
GluN2B subunit containing NMDA receptors resulting in a negative
allosteric modulation [25]. In addition to its high GluN2B affinity,
ifenprodil originally developed as
poor selectivity over related receptors (
which lead to undesirable side effects like impairment of reaction
and reduced blood pressure. Moreover, the bioavailability of ifen-
prodil is rather low due to fast metabolism (e.g. phenol glucur-
onidation) [26].
Despite these unfavorable properties, ifenprodil served as lead
compound for the development of different GluN2B antagonists
with comparable affinity but higher selectivity. Some promising
GluN2B antagonists derived from ifenprodil are displayed in Fig. 1
benzoxazolones 6 were considerably less potent, which was
attributed to the rather rigid structure holding the basic amino
moiety and the H-bond donor in a precise orientation to each other
[32]. Very recently, aminomethyl substituted benzimidazoles with
high GluN2B affinity and selectivity were reported [33].
In this manuscript we report on [7]annulenobenzimidazolone
derivatives 7, which are derived from the lead compounds 1-6. [7]
Annulenobenzimidazolones 7a with protons at N-atoms should be
able to form H-bonds with appropriate H-bond acceptors. The
corresponding methyl derivatives 7b correspond to the methyl
ethers 2b, 3a and 3b, which display high GluN2B affinity. However
the protonated basic amino group should be attached at the rigid
system in order to allow a free orientation to an H-bond accepting
group or an anionic functional group. Therefore various positions of
the amino moiety were considered. The affinity of benzimidazo-
lones 7 towards GluN2B subunit containing NMDA receptors and
a
1
receptor antagonist shows
, 5-HT, receptors),
a
1
1 2
s , s
[
8]. The basic amino functionality of ifenprodil (1) and Ro 25-
981 (4) is found in the 4-benzylpiperidine structure. However, 1
and 4 differ in the distance between the “left” benzene ring and the
basic amino moiety. The additional CH moiety in the flexible side
chain of 4 does not influence the GluN2B affinity, considerably [27].
6
2
some related receptors (PCP binding site of the NMDA receptor,
1
s ,
Fig. 1. Design of novel GluN2B selective NMDA receptor antagonists 7 with tricyclic benzimidazolone scaffold derived from lead compounds 1e6.

138
I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
s
2
receptors) will be recorded in radioligand receptor binding
Several methods for the cyclization of amino substituted car-
studies.
bamates to form cyclic ureas have been reported. Generally, simple
heating, acids (e.g. glacial acetic acid, p-toluenesulfonic acid) [34],
coupling agents (e.g. carbonyl diimidazole (CDI)) [35] and bases
2
. Results and discussion
2 3
(e.g. K CO , triazabicyclodecen, NaH) [36,37] can be used for this
cyclization. However, heating of the aminocarbamate 18 in meth-
anol, THF or toluene, addition of HOAc, p-toluenesulfonic acid or
CDI did not lead to the desired benzimidazolone 19. However, an
excess of the strong base NaH in boiling THF induced the cyclization
of 18 and the benzimidazolone 19 was isolated in 77% yield.
In conclusion this seven-step procedure allows for the first time
the synthesis of the linear tricyclic ring system 19 consisting of a
benzene ring, which is annulated with a five-membered imidazo-
lone ring and a seven-membered [7]annulene ring. The overall
yield of 19 prepared via the optimized seven-step sequence starting
with benzosuberone (8) was 28%.
2
.1. Synthesis
For the synthesis of GluN2B antagonists of type 7 the [7]annu-
lenobenzimidazolone ring system has to be prepared. The tricyclic
system should be obtained by annulation of the seven-membered
carbocyclic ring at the benzimidazolone system or alternatively
by establishment of the imidazolone ring at the benzo[7]annulene
system.
At first a three-step synthesis was planned consisting of a
FriedeleCrafts acylation of benzimidazolone with a glutaric acid
derivative, Wolff-Kishner reduction and a final intramolecular
FriedeleCrafts acylation. However all attempts to react benzimi-
dazolone or 1,3-dibenzylbenzimidazolone with glutaric anhydride
or glutaric acid mono methyl ester mono acid chloride in the
presence of a Lewis acid failed to give the acylated benzimidazolone
system.
The tricyclic ketone 20 was envisaged as valuable building block
for the introduction of amino substituents in a- and b-position (6-
and 7-position) as well as at the ketone position itself (5-position).
The ketone 20 was obtained in 75% yield upon oxidation of the
secondary alcohol 19 with Dess-Martin-Periodinane. The
nation of ketone 20 was performed in DMSO using NBS as Br
The -bromoketone 21 was obtained with high chemoselectivity in
5% yield. However, further reactions of the ketones 20 and 21 failed.
Inparticular the synthesis of an -unsaturated ketone byoxidation
with IBX [37e40] or -elimination of HBr, the reductive amination of
0 with primary or secondary amines and the substitution of Br in
position of 21 were investigated. (Scheme 3)
a-bromi-
2
source.
Therefore, the second strategy, i.e. constructing the imidazolone
ring at the benzo[7]annulene system, was pursued. Benzosuberone
a
7
(
8) was nitrated regioselectively with fuming HNO
nitro derivative 9. After reduction with H and Pd/C the primary
amine 10 was acylated with methyl chloroformate. Treatment of
the carbamate 11 with fuming HNO in glacial acetic acid resulted
3
to afford the 3-
a,b
2
b
2
a-
3
in a mixture of 1- and 3-nitro derivatives 12 and 13. Although the 3-
nitro derivative 13 could be isolated in 33% yield, the separation of
the regioisomeric 1-nitro derivative 12 was very difficult. Moreover,
slight modifications of the reaction conditions led predominantly
to formation of 12. The unexpectedly facile nitration of carbamate
The failure of these transformations is explained by the very low
solubility of the ketones 20 and 21 in organic solvents. DMSO was
the only solvent, which allowed the preparation of solutions with
20 and 21. In addition to the low solubility, the carbonyl activity of
the ketones 20 and 21 is reduced due to the N-atom in p-position of
the benzene ring, which could explain the low reactivity during the
reductive amination experiments. Moreover, the ketones 20 and 21
represent phenylogous imides, which are deprotonated upon
treatment with base and thus are further deactivated for nucleo-
philic attack at the carbonyl moiety in 5-position.
In order to avoid deprotonation and increase the solubility, the
ketone 20 was methylated in a phase transfer-catalyzed trans-
formation [41,42]. However, despite improved solubility of the
dimethyl derivative 22, the reactivity of 22 was not increased
considerably.
In order to introduce amino moieties into the seven-membered
part of the tricyclic system, the alcohol 19 was dehydrated with p-
toluenesulfonic acid and the resulting alkene 23 was methylated in
a phase transfer catalyzed reaction [41,42] to obtain the dimethyl
derivative 24 in 71% yield. In contrast to the ketones 20 and 21 the
solubility of the methylated alkene 24 was sufficient to convert it
into the epoxide 25 upon treatment with m-chloroperbenzoic acid
1
1 between the keto and methoxycarbonylamino moieties was
explained by a neighbor group effect. Alternatively, the nitroketone
3 was obtained by oxidation of alcohol 17 with DMP in DMSO.
However, the nitroketone 13 was isolated in only 28% yield.
1
(Scheme 1)
In order to improve the reaction sequence the nitroketone 9 was
reduced first with NaBH
with H and Pd/C to afford the aminoalcohol 15. After conversion of
5 into the carbamate 16, nitration with fuming HNO took place
4
to yield the nitroalcohol 14 and secondly
2
1
3
with high regioselectivity providing the 3-nitro derivative 18 in 85%
yield. In contrast to the nitration of the ketone 11, nitration of the
alcohol 16 did not lead to the corresponding 1-nitro derivative. It is
assumed that the higher electron density and thus faster reaction of
the benzene moiety of 16 leads to the selective nitration of the less
hindered 3-position. The reduction of the nitro compound 18 was
2
performed with H (4 bar) in the presence of Pd/C affording the
primary amine in 77% yield. (Scheme 2)
(MCPBA) in CH
2 2
Cl . The epoxide 25 represents the central inter-
mediate for the introduction of amino moieties in various positions.
(see Scheme 4)
According to the structures of the lead compounds 1-6 the 3-
phenylpropylamino moiety was selected as preferred substituent.
Therefore the epoxide 25 was treated with phenylpropylamine
under various reaction conditions. These transformations led to
complex mixtures of products, which partly allowed the isolation of
some products. The isolated products are summarized in Scheme 4.
Treatment of 25 with 3-phenylpropylamine in the presence of NaH
and BF
and trans-27 could be isolated in 13% and 19% yield, respectively.
The opening of 25 with the Lewis acid BF .OEt seems to proceed
via a S 2-like mechanism. The reaction of 25 with CH MgBr in THF
resulted in the isolation of ketone 29 in a 36% yield which was
3 2
.OEt lead to an opening of the epoxide and the diols cis-26
Scheme 1. First approach for the synthesis of annulated benzimidazolones by nitra-
3
2
tion of carbamate 11. Reagents and reaction conditions: (a) HNO
3
fuming, ꢀ10
ꢁ
to ꢀ17 C, 1 h, 87%. (b) H
2
, 3 bar, Pd/C, CH
3
OH, rt, 44 h, 38%. (c) ClCO
2
CH , NEt , THF,
3
3
N
3
reflux, 24 h, 62%. (d) HNO
3
, fuming, HOAc, rt, 22 h, 16% (12), 33% (13).

I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
139
Scheme 2. Second approach for the synthesis of annulated benzimidazolones by nitration of carbamate 16. Reagents and reaction conditions: (a) NaBH
4
, CH
3
OH, rt, 0.5 h, 85%. (b)
H
2
, 4 bar, Pd/C, CH
3
OH, rt, 0.5e1 h, 91%. (c) ClCO
2
CH
3 3
, NEt , THF, rt, 0.5 h, 92%. (d) HNO
3
, fuming, HOAc, ꢀ10 ꢁC, 0.5 h, 85%. (e) H
2
, 4 bar, Pd/C, CH
3
OH, rt, 0.5 h, 69%. (f) NaH, THF,
reflux, 23 h, 77%.
ꢁ
Scheme 3. Oxidation of tricyclic alcohol 19. Reagents and reaction conditions: (a) Dess-Martin-Periodinane (DMP), DMSO, rt, 2 h, 75%. (b) N-bromosuccinimide (NBS), DMSO, 40 C,
2
þ ꢀ
ꢁ
3 2 4
4 h, 75%. (c) CH I, toluene, NaOH, H O, Bu N I , 30 C, 20 h, 26%.
ꢁ
þ ꢀ
Scheme 4. Synthesis of tricyclic phenylpropylamines 28 and 30. Reagents and reaction conditions: (a) p-TosOH, DMSO, 70 C, 40 min, 70%. (b) CH
3
I, toluene, NaOH, H
2
O, Bu
4
N I ,
NH
2
, rt, 45 min, 42%. The compounds in Scheme 4 represent racemic
ꢁ
30
C, 24 h, 71%. (c) MCPBA, CH
2
Cl
2
, Na
2
CO
3
, H
, CH
2
O, rt, 2 h, 51%. (d) Ph(CH
2
)
3
NH
2
, NaH, THF, rt, 10 d, 13% (cis-26). (e) Ph(CH
NH , NaBH(OAc) , CH Cl
2
)
3
NH
2
, BF
3
.OEt
2
, rt, 17 h, 19% (trans-27). (f) Ph(CH
2
)
3
2
,
ꢁ
LiHMDS, THF, rt, 11 d, 27%. (g) Ph(CH
2
)
3
NH
2
3
MgBr, THF, 35 C, 7.5 h, 36%. (h) Ph(CH
2
)
3
2
3
2
mixtures. In order to show the trans-configuration of compound 28 one enantiomer of the racemic mixture is displayed in Scheme 4.
converted by reductive amination into the amine 30 in 42% yield.
The aminoalcohol 28 was obtained by treatment of epoxide 25 with
the amines 28 and 30 towards GluN2B containing NMDA receptors
was investigated using the competitive receptor binding assay
recently developed in our group [43]. Membrane fragments pre-
pared by ultrasonic irradiation of L(tk-)-cells stably expressing re-
combinant human GluN1a and GluN2B subunits were employed as
receptor material in this assay. The synthesis of functional NMDA
receptors was induced by addition of dexamethasone to the growth
3
-phenylpropylamine and LiHMDS in THF in 27% yield.
2.2. Receptor affinity
The affinity of the diastereomeric diols cis-26 and trans-27 and

140
I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
medium of these L(tk-)-cells. In order to protect the cells against
cell death, ketamine was added to the medium, which blocks the
NMDA receptor by interaction with the phencyclidine binding site
suitable bioisostere of the phenol or the corresponding methyl
ether of potent GluN2B antagonists. Moreover, it is assumed that
the tricyclic [7]annulenobenzimidazolone system is too rigid to
induce a fit during approaching the ifenprodil binding site of the
NMDA receptor. Nevertheless, the tricyclic amine 30 opens the
possibility for several modifications at various ring positions.
Therefore, 30 represents a promising lead compound for the
development of novel types of GluN2B antagonists.
3
within the channel pore. Tritium labeled [ H]ifenprodil (1) served
as radioligand. Although the selectivity of the radioligand is rather
low, this assay is selective for the ifenprodil binding site of GluN2B
containing NMDA receptors due to the high amount of receptors in
this cell line.
In Table 1 the GluN2B affinity of the test compounds is sum-
marized. Generally the GluN2B affinity of the cis- and trans-
configured diols cis-26 and trans-27 is very low, since they are
lacking a basic amino moiety. The aminoalcohol 28 shows also a
very low GluN2B affinity, which is explained by the amino moiety
in benzylic position resulting in a short distance between the basic
amino moiety and the aromatic ring. Although the amine 30 fits
nicely into the pharmacophore model developed by Tamiz et al.
4. Experimental
4.1. Experimental, chemistry
4.1.1. General
Unless otherwise noted, moisture sensitive reactions were
conducted under dry nitrogen. Acetonitrile and dimethyl sulfoxide
[
27], it shows only moderate affinity (K
i
¼ 697 nM) to GluN2B
2 2 2
were dried over molecular sieves. CH Cl was distilled from CaH ,
subunit containing NMDA receptors. It can be speculated that the
methylated benzimidazolone is not an appropriate bioisostere of
the phenol of 1, 2a and 4 and the methyl phenyl ether of 2b and 3.
Moreover, it is possible that the tricyclic [7]annulenobenzimida-
zolone is too rigid to adapt to the complementary binding pocket of
the GluN2B binding site.
methanol was distilled from magnesium methanolate and tetra-
hydrofuran was distilled from sodium/benzophenone. Thin layer
chromatography (TLC): Silica gel 60 F254 plates (Merck). Flash
chromatography (FC): Silica gel 60 (40e63 mm, MachereyeNagel);
parentheses include: diameter of the column, length, fraction size,
Rf value, eluent. Automatic flash column chromatography: Isolera™
®
®
The diols cis-26 and trans-27 and the amine 28 do not interact
One (Biotage ). Biotage SNAP cartridge (10 g); stationary phase:
silica gel 60 (40e63 m, MachereyeNagel); column volume (CV):
considerably with
phenylpropylamine 30 shows weak
moderate affinity (K
¼ 549 nM). Thus the rigid tricyclic [7]
annulenobenzimidazolone 30 shows >5-fold selectivity for the
GluN2B receptor over the
the GluN2B receptor and
s
1
and
s
2
receptors [44e46]. However, the
m
s
1
affinity (K M) and
i
¼ 4
m
1CV ¼ 15 mL; flow rate: 12 mL/min; max. fraction volume: 18 mL;
detection at 254 nm (UV1) and 280 nm (UV2); baseline: on; start
threshold: 5 mAU; solvents: A: cyclohexane; B: ethyl acetate;
gradient elution: (A/B [%]): 2.0 CV: 20%, 10.0 CV: 20e100%, 6.0 CV:
100%. Melting point: Melting point apparatus SMP3 (Stuart Scien-
tific), uncorrected. MS: microTOF-Q II (Bruker Daltronics); APCI,
atmospheric pressure chemical ionization. IR: FT-IR MIRacle 10
s
2
i
s
1
receptor, but the same affinity towards
s
2
receptors.
3
. Conclusion
(Shimadzu) equipped with ATR technique. Nuclear magnetic reso-
nance (NMR) spectra were recorded on Agilent 600-MR (600 MHz
for H, 151 MHz for C) or Agilent 400-MR spectrometer (400 MHz
Starting with the structures of potent GluN2B antagonists, the
7]annulenobenzimidazolone system 7 was designed. A seven-step
1
13
[
1
13
for H, 101 MHz for C);
d in ppm related to tetramethylsilane and
sequence is described allowing the efficient synthesis of the linear
tricyclic ring system 19 consisting of a benzene ring, which is
annulated with a five-membered imidazolone ring and a seven-
membered [7]annulene ring. The overall yield of 19 prepared via
the optimized seven-step sequence starting with benzosuberone
1
measured referring to CDCl3 (
d
¼ 7.26 ppm ( H NMR) and
1
3
1
d
d
d
¼ 77.16 ppm ( C NMR)), CD
3
OD (
d
¼ 4.87 ppm ( H NMR) and
1
1
3
¼ 49.0 ppm ( C NMR)) and CD
3
OD (
d
¼ 2.50 ppm ( H NMR) and
1
3
¼ 39.52 ppm ( C NMR)); coupling constants are given with 0.5 Hz
resolution. Analytical HPLC: Merck Hitachi Equipment; UV detec-
tor: L-7400; autosampler: L-7200; pump: L-7100; interface: D-
(8) was 28%. Five more steps including elimination of water,
dimethylation to increase solubility, epoxidation followed by
epoxide rearrangement and, finally, reductive amination led to the
phenylpropylamine 30. Although the amine 30 fits nicely into the
postulated GluN2B pharmacophore, it shows only moderate
7
2
5
000; column: LiChrospher® 60 RP-select B (5 m); LiChroCART®
m
50-4 mm cartridge; flow rate: 1.0 mL/min; injection volume:
.0 L; detection at
m
l
¼ 210 nm; solvents: A: water with 0.05% (v/v)
trifluoroacetic acid; B: acetonitrile with 0.05% (v/v) trifluoroacetic
acid: gradient elution: (A %): 0e4 min: 90%, 4e29 min: 90 / 0%,
GluN2B affinity (K
receptors (K
selectivity. It is postulated that the benzimidazolone system is not a
i
¼ 697 nM). Additionally, 30 interacts with
s
2
i
¼ 549 nM) with similar affinity, indicating poor
29e31 min: 0%, 31e31.5 min: 0 / 90%, 31.5e40 min: 90%. The
purity of all compounds was determined by this method. The purity
of all test compounds is higher than 95%.
Table 1
Affinities of [7]annulenobenzimidazolones towards the ifenprodil binding site of
4
.1.2. 3-Nitro-6,7,8,9-tatrahydrobenzo[7]annulen-5-one (9)
Fuming nitric acid (29.5 mL, 703 mmol) was poured into a three
GluN2B containing NMDA receptors, as well as towards
s
1
and
s
2
receptors.
Compd.
K
i
± SEM (nM)a
necked flask. 1-Benzo-suberone (8) (9.3 mL, 62.42 mmol) was
added dropwise under vigorous stirring and cooling to ꢀ10
GluN2Bb
s
1
b
s
2
b
ꢁ
to ꢀ17 C by acetone and dry ice. After complete addition of 2 the
cis-26
trans-27
0%
18%
11%
0%
5%
18%
ꢁ
mixture was stirred for 1 h at < ꢀ10 C. The mixture was poured on
2
3
8
0
0%
19%
32%
ice and stirred till the ice was molten. The solid residue was filtered
off in vacuo and washed several times with ice cold water. The
crude product was purified by recrystallization from EtOH 96% to
697 ± 18
10 ± 0.7
13 ± 2.5
e
4
m
M
549 ± 84
98.3 ± 34
e
Ifenprodil (1)
Eliprodil
Haloperidol
125 ± 24
e
ꢁ
form a light yellow solid, mp 92e93 C, yield 11.2 g (87%).
C
6.3 ± 1.6
89 ± 29
78.1 ± 2.3
57.5 ± 18
Di-o-tolylguanidine
e
11
H
11NO
3
(205.2). TLC (petroleum ether: ethyl acetate ¼ 8:2):
1
a
Rf ¼ 0.29. H NMR (CDCl
.91e1.99 (m, 2H, 8-H), 2.83e2.87 (m, 2H, 6-H), 3.02e3.07 (m, 2H,
9-H), 7.40 (d, 3J ¼ 8.4Hz, 1H, 1-H), 8.26 (dd, 3J ¼ 8.3 Hz, 4J ¼ 2.5 Hz,
3
):
d
[ppm] ¼ 1.82e1.90 (m, 2H, 7-H),
For low-affinity compounds only the inhibition of the radioligand binding at a
M is given in %.
-values were recorded three times (n ¼ 3).
1
test compound concentration of 1
m
b
All K
i

I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
141
1
d
6
H, 2-H), 8.56 (d, 4J ¼ 2.5 Hz, 1H, 4-H). 13C NMR (CDCl
3
):
and fuming HNO3 (1 mL, 24.2 mmol) was dropwise added under
vigorous stirring and cooling to 0 C (water and ice). After complete
addition, the mixture was stirred at ambient temperature for 22 h.
ꢁ
[ppm] ¼ 20.8 (1C, C-7), 25.0 (1C, C-8), 32.7 (1C, C-9), 40.7 (1C, C-
), 124.1 (1C, C-4), 126.4 (1C, C-2), 131.2 (1C, C-1), 140.0 (1C, C-4a),
1
48.2 (1C, C-3), 152.9 (1C, C-9a), 203.6 (1C, C-5). Exact mass (APCI):
m/z ¼ 206.0829 (calcd. 206.0812 for C11 [MH]þ). IR (neat): ṽ
cm-1] ¼ 2951 (w, C-H), 1670 (s, C ¼ O aryl), 1604 (m, C]C
arom.), 1519 (s, N]O), 1342 (s, N]O konj.). Purity (HPLC):
tR ¼ 17.43 min, purity 97.4%.
The mixture was poured into H
0.5 h CH Cl was added and the layers were separated. The sepa-
rated organic layer was extracted with 2 M NaOH (3ꢂ), dried
(Na SO ), filtered and the solvent was removed in vacuo. The
products were separated by flash column chromatography
2
O and the mixture was stirred for
H12NO
3
2
2
[
n
n
n
n
n
2
4
(
Ø ¼ 2.3 cm, h ¼ 18.5 cm, petroleum ether: ethyl acetate ¼ 8:2,
4
.1.3. 3-Amino-6,7,8,9-tetrahydrobenzo[7]annulen-5-one (10)
Nitroketone 9 (0.30 g, 1.46 mmol) was dissolved in abs. CH
15 mL) and Pd/C (30 mg, 10% m/m) was added. The mixture was
stirred under H -atmosphere (3 bar) for 44 h. The crude product
was filtered over Celite 450 to remove the catalyst and Celite 450®
was washed with CH
V ¼ 10 mL). Two fractions were isolated containing compound 12
3
OH
(Rf ¼ 0.24) and compound 13 (Rf ¼ 0.29).
(
12: Pale yellow oil, yield 10 mg (16%). C13
H
14
N
2
O
5
(278.3). TLC
):
[ppm] ¼ 1.79e1.91 (m, 2H, 7-H), 1.90e2.01 (m, 2H, 6-H),
2.73e2.79 (m, 2H, 8-H), 2.83e2.87 (m, 2H, 5-H), 3.81 (s, 3H,
COOCH
), 7.34e7.37 (m, 1H, 4-H), 8.25 (d, 3J ¼ 8.6 Hz,1H, 3-H), 8.73
(s, 1H, NH). C NMR (CDCl
7), 32.5 (1C, C-8), 41.8 (1C, C-5), 53.1 (1C, COOCH
1
2
(petroleum ether: ethyl acetate ¼ 8:2): Rf ¼ 0.24. H NMR (CDCl
3
®
d
3
OH (3ꢂ). The solvent was removed in vacuo
and the residue was purified by fc (Ø ¼ 2.3 cm, h ¼ 19.5 cm,
3
13
cyclohexane:ethyl acetate ¼ 2:8, V ¼ 10 mL) to form an orange
3
):
d
[ppm] ¼ 24.5 (1C, C-6), 25.9 (1C, C–
), 122.5 (1C, C-3),
ꢁ
solid, mp 104e105 C, yield 0.1 g, (38%). C11
H
13NO (175.2). TLC
3
1
(
d
cyclohexane:ethyl acetate ¼ 7:3): Rf ¼ 0.21. H NMR (CDCl
3
):
131.8 (1C, C-9a), 132.8 (1C, C-2), 134.8 (1C, C-4), 138.8 (1C, C-4a),
146.0 (1C, C-1), 153.8 (1C, NCOO), 205.6 (1C, C-9). Exact mass
[ppm] ¼ 1.75e1.88 (m, 4H, 7-H, 8-H), 2.68e2.73 (m, 2H, 6-H),
.79e2.85 (m, 2H, 9-H), 6.75 (dd, 3J ¼ 8.0 Hz, 4J ¼ 2.6 Hz, 1H, 2-H),
.99 (d, 3J ¼ 8.0 Hz, 1H, 1-H), 7.06 (d, 4J ¼ 2.6 Hz, 1H, 4-H). Signals
2
6
(APCI): m/z ¼ 279.0871 (calcd. 279.0975 for C13
H
17
N
2
O
5
[MH]þ). IR
C-H), 1751 (m,
C]O Carbamate), 1612 (w, -C]C), 1578
N ¼ O), 1344 (m, N]O). Purity (HPLC):
tR ¼ 17.87 min, purity 93.4%.
13: Pale yellow solid, mp 166e167 C, yield 25 mg (33%).
(278.3). TLC (petroleum ether: ethyl acetate ¼ 8:2):
-
1
(neat): ṽ [cm ] ¼ 3356 (w,
C]O Ketone), 1701 (m,
(w,. -C]C), 1505 (s,
neCONHe), 2954 (w, n
13
for the protons of the amino group are not seen in the spectrum.
C
n
n
n
NMR (CDCl
1.0 (1C, C-6), 114.9 (1C, C-4), 119.2 (1C, C-2), 130.9 (1C, C-1), 132.0
1C, C-9a), 139.5 (1C, C-4a), 144.8 (1C, C-3), 206.4 (1C, C-5). Exact
mass (APCI): m/z ¼ 176.1063 (calcd. 176.1070 for C13 14NO [MH]þ).
IR (neat): ṽ [cm-1] ¼ 3429 (m, N-H), 3348 (m, N-H), 2928 (m, C-
H), 1639 (s, C]O aryl), 1625 (s,
C ¼ C arom). Purity (HPLC):
tR ¼ 8.76 min, purity 97.1%.
3
):
d
[ppm] ¼ 21.1 (1C, C-7), 25.6 (1C, C-8), 31.8 (1C, C-9),
n
n
n
4
(
ꢁ
H
13 14 2 5
C H N O
1
n
n
n
Rf ¼ 0.29. H NMR (CDCl
3
):
d
[ppm] ¼ 1.79e1.95 (m, 4H, 6-H, 7-H),
n
n
2.71e2.79 (m, 2H, 8-H), 2.88e2.96 (m, 2H, 5-H), 3.83 (s, 3H,
1
3
COOCH
NMR (CDCl
40.9 (1C, C-8), 53.1 (1C, COOCH
3
3
), 8.03 (s, 1H, 4-H), 8.73 (s, 1H, 1-H), 9.63 (s, 1H, NH).
):
[ppm] ¼ 21.3 (1C, C-6), 25.3 (1C, C-7), 31.8 (1C, C-5),
), 121.2 (1C, C-1), 126.6 (1H, C-4),
C
3
d
4.1.4. Methyl N-(9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-
yl)carbamate (11)
133.8 (1C, C-2), 134.3 (1C, C-3), 137.2 (1C, C4a), 145.5 (1C, C-9a),
Aminoketone 10 (1.5 g, 8.6 mmol) was dissolved in abs. THF
153.5 (1C, NCOO), 204.5 (1C, C-9). Exact mass (APCI): m/
(
70 mL). Triethylamine (3.23 mL, 21.4 mmol) and methyl chlor-
oformate (1.67 mL, 21.4 mmol) were added. The mixture was
heated to reflux under N -atmosphere for 24 h. The mixture was
cooled to ambient temperature and the solvent was removed in
vacuo. The residue was dissolved in CH Cl and extracted with
water and 2M HCl (4ꢂ). The separated organic layer was dried
Na SO ), filtered and the solvent was removed in vacuo. The crude
z ¼ 279.0952 (calcd. 279.0975 for C13
H
15
N
2
O
n
5
[MH]þ). IR (neat): ṽ
C-H), 1732 (m, C]O
C]O Carbamate),1612 (w, -C]C),1516 (s, N]
N]O), 1230 (s,
C-O). Purity (HPLC): tR ¼ 18.81 min,
-
1
[cm ] ¼ 3283 (w,
Ketone),1667 (m,
O), 1358 (w,
purity 95.7%.
n
eCONHe), 2959 (w,
n
2
n
n
n
n
n
2
2
(
2
4
Method 2: Synthesis of 13 by oxidation of nitroalcohol 17
product was purified by flash column chromatography (Ø ¼ 4.5 cm,
h ¼ 13.5 cm, cyclohexane:ethyl acetate ¼ 7:3, V ¼ 30 mL) to give a
A solution of nitroalcohol 17 (0.20 g, 0.71 mmol) in abs. DMSO
(3 mL) was added dropwise to a solution of Dess-Martin Period-
inane (0.36 g, 0.86 mmol) in abs. DMSO (10 mL) under vigorous
ꢁ
colorless solid, mp 98e99 C, yield 1.24 g (62%). C13
H
15NO
3
(232.3).
OD):
[ppm] ¼ 1.74e1.90 (m, 4H, 7-H, 6-H), 2.69e2.75 (m, 2H, 8-H),
.89e2.95 (m, 2H, 5-H), 3.73 (s, 3H, COOCH
1
TLC (cyclohexane:ethyl acetate ¼ 7:3): Rf ¼ 0.23. H NMR (CD
3
d
2
stirring. The mixture was stirred under N -atmosphere at ambient
2
1
4
3
), 7.19 (d, 3J ¼ 8.2 Hz,
temperature for 2 h. Then, 1 M NaOH (20 mL), ethyl acetate (40 mL)
and brine (25 mL) were added. The separated aqueous layer was
extracted with ethyl acetate (6ꢂ). The combined organic layers
H, 4-H), 7.57 (dd, 3J ¼ 8.2 Hz, 4J ¼ 2.3 Hz, 1H, 3-H), 7.69 (d,
J ¼ 2.3 Hz, 1H, 1-H). Signal for the NH group is not seen in the
13
spectrum. C NMR (CD
6
3
OD):
d
[ppm] ¼ 22.0 (1C, C-7), 26.4 (1C, C-
2 4
were dried (Na SO ), filtered and the solvent was removed in
), 32.6 (1C, C-5), 41.6 (1C, C-8), 52.6 (1C, COOCH
3
), 119.4 (1C, C-1),
vacuo. The crude product was purified by flash column chroma-
tography (Ø ¼ 4.0 cm, h ¼ 19.5 cm, petroleum ether: ethyl
123.7 (1C, C-3), 131.5 (1C, C-4), 137.5 (1C, C-4a), 139.2 (1C, C-2),
1
40.2 (1C, C-9a), 156.5 (1C, COOCH
3
) 207.9 (1C, C-9). Exact mass
acetate ¼ 8:2, V ¼ 30 mL) to give a paella yellow solid, mp
ꢁ
(
APCI): m/z ¼ 234.1087 (calcd. 234.1026 for C13
H
16NO
3
[MH]þ). IR
C-H), 1724 (s, C]O
nC]O Carbamate), 1609 (m, n-C]C). Purity
166e167 C, yield 55.2 mg (28%). C13
H
14
N
2
O
5
(278.3). TLC (petro-
-1
(
neat): ṽ [cm ] ¼ 3321 (m,
n
eNHe), 2936 (m,
n
n
leum ether: ethyl acetate ¼ 8:2): Rf ¼ 0.29. Purity (HPLC):
Ketone), 1654 (s,
tR ¼ 19.97 min, purity 96.6%.
(
HPLC): tR ¼ 17.03 min, purity 99.9%.
4.1.6. 3-Nitro-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (14)
4.1.5. Methyl (1-nitro-9-oxo-6,7,8,9-tetrahydro-5H-benzo[7]
Nitroketone 9 (12.5 g, 61 mmol) was dissolved in abs. CH OH
3
annulen-2-yl)carbamate (12) and Methyl (3-nitro-9-oxo-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-2-yl)carbamate (13)
(525 mL) and NaBH
under N2-counter flow. The mixture was stirred under N
sphere at ambient temperature for 0.5 h. Water and 2 M HCl
(110 mL) were added. Then, CH Cl was added and the layers were
separated. The separated aqueous layer was extracted with CH Cl
(12ꢂ). The combined organic layers were dried (Na SO ) and the
4
(2.8 g, 73 mmol) were added portionwise
2
-atmo-
Method 1: Synthesis by nitration of carbamate 11
2
2
2
2
Carbamate 11 (0.5 g, 2.1 mmol) was dissolved in HOAc (10 mL)
2
4

142
I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
solvent was removed in vacuo. The crude product was purified by
recrystallization from EtOH 96% to form a fine colorless solid, mp
5), 38.1 (1C, C-8), 52.5 (1C, COOCH
118.1 (1C, C-3), 130.7 (1C, C-4), 136.7 (1C, C-4a), 138.0 (1C, C-2),
146.8 (1C, C-9a), 156.8 (1C, COOCH ). Exact mass (APCI): m/
z ¼ 218.1178 (calcd. 218.1176 for C13 [M e H2O þ H]þ). IR
eCONHe), 3275 (w, b, O-H), 2931 (w,
C]O Carbamate), 1240 (s, OeH), 1069 (s, -C-O).
Purity (HPLC): tR ¼ 16.39 min, purity 97.2%.
3
) 73.2 (1C, C-9), 117.0 (1C, C-1),
ꢁ
1
17e118 C, yield 10.7 g (85%). C11
H
13NO
0.29.
3
(207.2). TLC (cyclo-
3
1
hexane:ethyl acetate
[ppm] ¼ 1.33e1.46 (m, 1H, 8-H), 1.68e1.82 (m, 1H, 6-H), 1.80e1.93
m, 2H, 7-H, 8-H), 1.96 (d, 3J ¼ 4.1 Hz, 1H, OH), 2.01e2.11 (m, 2H, 6-
¼
7:3): Rf
¼
H
NMR (CDCl
3
):
H16NO
2
-
1
d
(
(neat): ṽ [cm ] ¼ 3406 (w,
C-H), 1694 (m,
n
n
n
n
n
n
H, 7-H), 2.74e2.83 (m, 1H, 9-H), 2.99 (dd, 2J ¼ 14.2 Hz, 3J ¼ 7.4 Hz,
1
3
(
2
H, 9-H), 4.99 (dd, 3J ¼ 9.0 Hz, 3J ¼ 3.6 Hz, 1H, 5-H), 7.23 (d,
J ¼ 8.2 Hz,1H,1-H), 8.00 (dd, 3J ¼ 8.3 Hz, 4J ¼ 2.5 Hz,1H, 2-H), 8.40
4.1.9. Methyl (9-hydroxy-3-nitro-6,7,8,9-tetrahydro-5H-benzo[7]
annulen-2-yl)carba-mate (17)
13
d, 4J ¼ 2.5 Hz, 1H, 4-H). C NMR (CDCl
3
):
d
[ppm] ¼ 26.9 (1C, C-8),
8.2 (1C, C-7), 35.8 (1C, C-9), 37.2 (1C, C-6), 72.9 (1C, C-5), 119.9 (1C,
Carbamate 16 (10.0 g, 42.5 mmol) was dissolved in glacial acetic
ꢁ
C-4),122.1 (1C, C-2),130.3 (C, C-1),146.0 (1C, C-4a),146.8 (1C, C-9a),
acid (100 mL). At ꢀ10 to 17 C (acetone and dry ice) fuming nitric
1
48.3 (1C, C-3). Exact mass (APCI): m/z ¼ 208.0979 (calcd. 208.0968
acid (19.8 mL, 479 mmol) was added dropwise under vigorous
stirring. After complete addition, the mixture was stirred
-
1
for C11
H14NO
3
[MH]þ). IR (neat): ṽ [cm ] ¼ 3499 (w,
O-H), 2928 (m, C-H), 1520 (m, N]O), 1330 (s,
konj.). Purity (HPLC): tR ¼ 17.29 min, purity 99.9%.
n
O-H), 3345
ꢁ
(
wbr,
n
n
n
nN]O
at < ꢀ10 C for 0.5 h. The mixture was poured on ice and stirred
until the ice was molten. The solid residue was filtered off in vacuo
and washed several times with ice cold water. The crude product
4
.1.7. 3-Amino-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-ol (15)
Nitroalcohol 14 (21.5 g, 103.7 mmol) was dissolved in abs.
was purified by recrystallization from ethyl acetate to give a light
ꢁ
yellow solid, mp 133e134 C, yield 10.1 g (85%). C13
H
16
N
2
O
5
(280.3).
1
CH
mixture was divided in six parts (5 ꢂ 4.0 g of 19, 19.3 mmol, and
ꢂ 1.5 g of 7, 7.2 mmol). The separated mixtures were shaken
under H atmosphere (4 bar) for 0.5e1 h. The crude product was
filtered over Celite 450 to remove the catalyst and Celite 450 was
washed with CH
3
OH (645 mL) and Pd/C (2.15 g, 10% m/m) was added. The
TLC (petroleum ether: ethyl acetate ¼ 8:2): Rf ¼ 0.18. H NMR
(CDCl ):
3
d
[ppm] ¼ 1.32e1.45 (m,1H, 6-H),1.68e1.91 (m, 3H, 6-H, 7-
1
H, 8-H), 1.99e2.09 (m, 2H, 7-H, 8-H),2.66e2.75 (m, 1H, 5-H),
2.85e2.94 (m, 1H, 5-H), 3.82 (s, 3H, COOCH3) 4.95 (d, 3J ¼ 9.3 Hz,
1H, 9-H), 7.93 (s,1H,1-H), 8.67 (s,1H, 4-H), 9.84 (1H, NH). The signal
2
®
®
13
3
OH (5ꢂ). The separated mixtures were combined
3
for the OH group is not seen in the spectrum. C NMR (CD OD):
and the solvent was removed in vacuo. The residue was purified by
flash column chromatography (Ø ¼ 8.0 cm, h ¼ 11.5 cm, cyclo-
d
[ppm] ¼ 28.6 (1C, C-6), 29.3 (1C, C-7), 35.6 (1C, C-8), 38.1 (1C, C-5),
3
53.3 (1C, COOCH ), 73.5 (1C, C-9), 119.2 (1C, C-4), 126.5 (1C, C-1),
hexane:ethyl acetate ¼ 6:4, V ¼ 100 mL) to give a light yellow solid,
133.6 (1C, C-2), 136.9 (1C, C-3), 137.3 (1C, C-4a), 155.3 (1C, C-9a),
ꢁ
mp 165e166 C, yield 17.0 g (91%). C11
H
0.15.
15NO (177.2). TLC (cyclo-
155.5 (1C, NCOO). Exact mass (APCI): m/z ¼ 281.1120 (calcd.
1
-1
hexane:ethyl acetate
d
2
2
2
¼
6:4): Rf
¼
H
NMR (CDCl
3
):
281.1132 for C13
17
H N
2
O
5
[MH]þ). IR (neat): ṽ [cm ] ¼ 3502 (w,
[ppm] ¼ 1.31e1.45 (m, 1H, 8-H), 1.57 (s br, 2H, NH2), 1.75 (tdd,
J ¼ 13.7 Hz, 3J ¼ 6.4 Hz, 3J ¼ 4.7 Hz, 3H, 6-H, 8-H), 1.91e2.07 (m,
H, 7-H), 2.61 (ddd, 2J ¼ 14.5 Hz, 3J ¼ 10.8 Hz, 3J ¼ 1.5 Hz, 1H, 9-H),
.77 (dd, 2J ¼ 14.7 Hz, 3J ¼ 7.5 Hz, 1H, 9-H), 3.59 (s br, 1H, OH), 4.84
n
OH), 3345 (w,
mate), 1578 (m,
O-H), 1072 (m,
n
eCONHe), 2932 (w,
-C]C), 1496 (s, N]O), 1327 (s,
C-O). Purity (HPLC): tR ¼ 18.40 min, purity 96.2%.
n
C-H), 1721 (s,
nC]O Carba-
n
n
nN]O), 1242 (s,
G
G
(
4
dd, 3J ¼ 8.3 Hz, 3J ¼ 1.5 Hz, 1H, 5-H), 6.47 (dd, 3J ¼ 7.8 Hz,
4.1.10. Methyl (3-amino-9-hydroxy-6,7,8,9- tetrahydro-5H-benzo
[7]annulen-2-yl)carba-mate (18)
Nitrocarbamate 17 (2.2 g, 7.8 mmol) was dissolved in abs.
3
CH OH (100 mL) and Pd/C (0.22 g, 10% m/m) was added. The
J ¼ 2.5 Hz, 1H, 2-H), 6.84 (d, 4J ¼ 2.5 Hz, 1H, 4-H), 6.88 (d,
13
3
J ¼ 7.9 Hz,1H,1-H). C NMR (CDCl
3
):
d
[ppm] ¼ 28.2 (1C, C-8), 28.3
(
1
1
1C, C-7), 35.1 (1C, C-9), 37.1 (1C, C-6), 74.0 (1C, C-5), 112.0 (1C, C-4),
13.2 (1C, C-2), 130.5 (1C, C-1), 130.9 (1C, C-9a), 144.7 (1C, C-4a),
45.5 (1C, C-3). Exact mass (APCI): m/z ¼ 178.1233 (calcd. 178.1226
mixture was shaken under H2-atmosphere (4 bar) for 0.5 h. The
®
crude product was filtered over Celite 450 to remove the catalyst
-
1
and Celite 450® was washed with CH
for C11H16NO [MH]þ). IR (neat): ṽ [cm ] ¼ 3383 (w,
wbr, O-H), 2928 (m, C-H), 1612 (m, C]C arom), 1049 (s,
Purity (HPLC): tR ¼ 10.85 min, purity 98.6%.
nN-H), 3267
3
OH (3ꢂ). The solvent was
(
n
n
n
G
C-O).
removed in vacuo and the residue was purified by flash column
chromatography (Ø ¼ 4.0 cm, h ¼ 15.3 cm, cyclohexane:ethyl
ꢁ
acetate ¼ 2:8, V ¼ 25 mL) to give an orange solid, mp 117e118 C,
4.1.8. Methyl N-(9-hydroxy-6,7,8,9-tetrahydro-5H-benzo[7]
yield 1.5 g (69%).
annulen-2-yl)carbamate (16)
C
13
H
18
N
2
O
3
(250.3). TLC (cyclohexane:ethyl acetate ¼ 2:8):
1
Aminoalcohol 15 (2.8 g, 15.8 mmol) was dissolved in abs. THF
Rf ¼ 0.25. H NMR (CDCl
3
):
d
[ppm] ¼ 1.39e1.52 (m, 1H, 6-H),
(
120 mL). Triethylamine (3.29 mL, 23.7 mmol) and methyl chlor-
1.63e1.80 (m, 3H,, 6-H, 7-H, 8-H), 1.87 (ddt, 2J ¼ 10.6 Hz,
3J ¼ 4.9 Hz, 3J ¼ 2.3 Hz, 1H, 8-H), 1.98e2.09 (m, 1H, 7-H), 2.54e2.64
(m, 1H, 5-H), 2.83 (ddd, 2J ¼ 13.8 Hz, 3J ¼ 9.2 Hz, 3J ¼ 1.7 Hz, 1H, 5-
oformate (1.83 mL, 23.7 mmol) were added. The mixture was stir-
red under N atmosphere at ambient temperature for 0.5 h. The
solvent was removed in vacuo, the residue was dissolved in CH Cl
and the mixture was washed with water and 2 M HCl (6ꢂ). The
separated organic layer was dried (Na SO ), filtered and the solvent
2
2
2
H), 3.72 (s, 3H, COOCH
3
), 4.72 (d, 3J ¼ 8.7 Hz,1H, 9-H), 6.58 (s,1H, 4-
H), 7.13 (s, 1H, 1-H). The signals for the OH, NH
2
and NH groups are
[ppm] ¼ 28.7 (1C, C-
7), 29.3 (1C, C-6), 36.4 (1C, C-5), 38.0 (1C, C-8), 52.8 (1C, COOCH ),
74.4 (1C, C-9), 111.2 (1C, C-2), 119.7 (1C, C-4), 122.4 (1C, C-9a), 124.6
(1C, C-1), 136.3 (1C, C-4a),143.3 (1C, C-3), 213.1 (1C, COOCH ). Exact
mass (APCI): m/z ¼ 251.1392 (calcd. 251.1390 for C13
13
2
4
not seen in the spectrum. C NMR (CDCl3): d
was removed in vacuo. The crude product was purified by recrys-
tallization from ethyl acetate to give a fawn solid, mp 109e110 C,
3
ꢁ
yield 2.63 g (92%). C13
H
17NO
3
(235.3).
3
TLC (cyclohexane:ethyl acetate ¼ 7:3): Rf ¼ 0.13. 1H NMR
19 2 3
H N O
-
1
(
CD
3
OD):
d
[ppm] ¼ 1.32e1.44 (m, 1H, 6-H), 1.60e1.71 (m, 1H, 8-H),
[MH]þ). IR (neat): ṽ [cm ] ¼ 3372 (w,
2924 (w, C-H), 1728 (s,
C ¼ O Carbamate), 1593 (m,
(s, -C]C), 1238 (s, C-O), 1219 (s, O-H), 1045 (s,
(HPLC): tR ¼ 5.34 min, 5.73 min, purity 94.8%.
n
N-H), 3310 (w, b,
-C]C), 1524
C-O). Purity
n-O-H-),
1.72e1.85 (m, 2H, 6-H, 7-H), 1.90e1.98 (m, 1H, 8-H), 1.99e2.07 (m,
n
n
n
G
1
H, 7-H), 2.67 (ddd, 2J ¼ 14.3 Hz, 3J ¼ 10.8 Hz, 3J ¼ 1.7 Hz, 1H, 5-H),
n
n
G
2
.84 (dd, 2J ¼ 14.2 Hz, 3J ¼ 7.6 Hz, 1H, 5-H), 3.72 (s, 3H, COOCH
3
),
4
.81 (d, 3J ¼ 9.8 Hz, 1H, 9-H), 6.98 (d, 3J ¼ 8.0 Hz, 1H, 4-H), 7.22 (dd,
3
J ¼ 7.9 Hz, 4J ¼ 2.4 Hz, 1H, 3-H), 7.42 (d, 4J ¼ 2.3 Hz, 1H, 1-H).
4.1.11. 5-Hydroxy-3,5,6,7,8,9-hexahydro[7]annuleno[f]benz-
imidazol-2(1H)-one (19)
1
3
Signals for the OH and NH group are not seen in the spectrum.
C
NMR (CD
3
OD):
d
[ppm] ¼ 29.2 (1C, C-6), 30.7 (1C, C-7), 36.0 (1C, C-
2
Under N , sodium hydride (2.1 g, 60% dispersion in oil,

I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
143
6
3.1 mmol) was suspended in abs. THF (350 mL). Aminoalcohol 18
7.12 (s, 1H, 4-H), 10.74 (s, 1H, NH), 10.95 (s, 1H, NH). 13C NMR (D6-
DMSO):
(
4.7 g, 18.9 mmol) was dissolved in abs. THF (249 mL) and the so-
d
[ppm] ¼ 24.2 (1C, C-8), 32.08 (1C, C-7), 32.1 (1C, C-9),
lution was slowly added dropwise to the sodium hydride suspen-
sion under vigorous stirring. The mixture was heated to reflux for
55.7 (1C, C-6), 108.9 (1C, C-4), 109.3 (1C, C-10), 128.3 (1C, C-3a),
129.4 (1C, C-10a), 133.7 (1C, C-4a), 135.1 (1C, C-9a), 155.4 (1C, C-2),
197.4 (1C, C-5). Exact mass (APCI): m/z ¼ 295.0073 (calcd. 295.0077
2
3 h. After cooling to ambient temperature, saturated ammonium
chloride solution (100 mL), water (150 mL) and CH Cl (350 mL)
were added. The precipitate was collected and washed several
-
1
2
2
for C12
(m,
C ¼ O Ketone),1670(s,
(HPLC): tR ¼ 15.89 min, purity 54.8%.
H
13BrN
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 3928 (w,
n
C-H),1705
n
n
C ¼ O Urea), 714 (w,
nC-Br alkyl). Purity
times with CH
mp > 300 C (decomposition), yield 3.2 g (77%). C12
2
Cl
2
. The residue was dried in vacuo. Fawn solid,
ꢁ
H
14
1
N
2
O
2
(218.3).
TLC (cyclohexane:ethyl acetate ¼ 2:8): Rf ¼ 0.37. H NMR (D6-
4.1.14. 1,3-Dimethyl-1,3,6,7,8,9- hexahydro[7]annuleno[f]
benzimidazole-2,5-dione (22)
DMSO)
d
[ppm] ¼ 1.13e1.27 (m, 1H, 8-H), 1.37e1.52 (m, 1H, 6-H),
1
2
3
.61e1.79 (m, 2H, 7-H, 8-H), 1.82e1.94 (m, 2H, 6-H, 7-H), 2.63 (dd,
J ¼ 13.7 Hz, 3J ¼ 11.2 Hz, 1H, 9-H), 2.76 (dd, 2J ¼ 13.6 Hz,
J ¼ 6.5 Hz, 1H, 9-H), 4.69 (dd, 3J ¼ 9.5 Hz, 3J ¼ 4.1 Hz, 1H, 5-H), 5.11
Method 2 by methylation of ketone 20:
(
d, 3J ¼ 4.0 Hz, 1H, OH), 6.63 (s, 1H, 10-H), 7.05 (s, 1H, 4-H), 10.34 (s,
Ketone 20 (36 mg, 0.17 mmol) was suspended in toluene (3 mL)
and 50% aqueous NaOH (1 mL). Tetrabutylammonium iodide
13
1
H, NH), 10.37 (s, 1H, NH). C NMR (D6-DMSO):
d
[ppm] ¼ 28.5 (1C,
C-8), 28.7 (1C, C-7), 35.6 (1C, C-9), 38.5 (1C, C-6), 72.0 (1C, C-5),
06.0 (1C, C-4), 109.8 (1C, C-10), 110.0 (1C, C-10a), 128.1 (1C, C-3a),
33.0 (1C, C-4a), 139.2 (1C, C-9a), 156.3 (1C, C-2). Exact mass (APCI):
m/z ¼ 219.1116 (calcd. 219.1128 for C12 [MH]þ). IR (neat): ṽ
C-H), 1636 (s,
C ¼ O Urea),
C-O). Purity (HPLC): tR ¼ 12.03 min, purity
(9.2 mg, 0.025 mmol) and methyl iodide (63
added under vigorous stirring. The mixture was heated to 30 C for
20 h. Then, water was added and the separated aqueous layer was
m
L, 0.84 mmol) were
ꢁ
1
1
H
n
15
N
2
O
2
extracted with CH
was removed in vacuo. The crude product was purified by auto-
2
Cl
2
(4ꢂ), dried (Na
2 4
SO ), filtered and the solvent
-1
[
1
cm ] ¼ 3175 (w, b,
n
O-H), 2920 (m,
n
485 (m, O-H), 1018 (
G
n
matic flash column chromatography (h ¼ 5,5 cm) to form a color-
ꢁ
9
6.5%.
less solid, mp 152e153 C, yield 11 mg (26%). C14
H
16
N
2
O
2
(244.3).
TLC (cyclohexane:ethyl acetate ¼ 2:8): Rf ¼ 0.33. C14
16 2 2
H N O
1
4
.1.12. 1,3,6,7,8,9-Hexahydro[7]annuleno[f]benzimidazole-2,5-
(244.3). TLC (cyclohexane:ethyl acetate ¼ 2:8): Rf ¼ 0.33. H NMR
(CDCl ):
[ppm] ¼ 1.74e1.86 (m, 2H, 7-H), 1.84e1.96 (m, 2H, 8-H),
2.73e2.79 (m, 2H, 6-H), 2.96e3.04 (m, 2H, 9-H), 3.43 (s, 6H, NCH ),
[ppm] ¼ 20.6
), 27.52 (1C, NCH ), 32.9
dione (20)
A solution of alcohol 19 (0.4 g, 1.8 mmol) in abs. DMSO (6 mL)
was added dropwise to a solution of Dess-Martin Periodinane
0.93 g, 2.2 mmol) in abs. DMSO (10 mL) under vigorous stirring.
The mixture was stirred under N -atmosphere at ambient tem-
3
d
3
13
6.77 (s, 1H, 4-H), 7.46 (s, 1H, 10-H). C NMR (CDCl
3
): d
(
(1C, C-7), 25.5 (1C, C-8), 27.49 (1C, NCH
3
3
2
(1C, C-9), 40.9 (1C,C-6), 108.1 (1C, C-4), 108.2 (1C, C-10), 129.0 (1C,
C-4a), 132.3 (1C, C-3a), 133.6 (1C, C-9a), 136.9 (1C, C-10a), 155.1
(1C,C-2), 205.1 (1C, C-5). Exact mass (APCI): m/z ¼ 245.1298 (calcd.
perature for 2 h. Then, 1M NaOH (50 mL), ethyl acetate (100 mL)
and brine (60 mL) were added. The separated aqueous layer was
extracted with ethyl acetate (6ꢂ). The combined organic layers
-
1
245.1285 for C14
H), 1701 (s,
Purity (HPLC): tR ¼ 16.31 min, purity 90.6%.
H
17
N
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 2932(w,
nC-
were dried (Na
2
SO
4
), filtered and the solvent was removed in
nC]O Ketone), 1659(m, nC]O Urea), 1508 (m, nC]C).
vacuo. The crude product was purified by flash column chroma-
tography (Ø ¼ 4.0 cm, h ¼ 18.0 cm, cyclohexane:ethyl acetate ¼ 2:8,
ꢁ
V ¼ 20 mL) to give an orange solid, mp > 345 C (decomposition),
4.1.15. 3,5,6,7-Tetrahydro[7]annuleno[f]benzimidazol-2(1H)-one
(23)
12 2 2
yield 297 mg (75%). C12H N O (216.2). TLC (cyclohexane:ethyl
1
acetate ¼ 2:8): Rf ¼ 0.19. H NMR (D6-DMSO):
d
[ppm] ¼ 1.60
A solution of alcohol 19 (0.2 g, 0.92 mmol) and p-toluenesulfonic
acid (0.19 g, 1 mmol) in THF (28 mL) and DMSO (9 mL) was heated
(
2
quint, 3J ¼ 6.3 Hz, 2H, 7-H), 1.71 (quint, 3J ¼ 6.6 Hz, 2H, 8-H),
ꢁ
.57e2.63 (m, 2H, 6-H), 2.83e2.90 (m, 2H, 9-H), 6.77 (s, 1H, 10-H),
to 70 C for 40 min. Then, water (23 mL) was added and the
13
7
.16 (s, 1H, 4-H), 10.67 (s, 1H, NH), 10.86 (s, 1H, NH). C NMR (D6-
DMSO):
[ppm] ¼ 20.0 (1C, C-7), 24.9 (1C, C-8), 31.6 (1C, C-9),
0.2 (1C, C-6), 108.2 (1C, C-4), 109.3 (1C, C-10), 128.4 (1C, C-3a),
30.9 (1C, C-10a), 133.6 (1C, C-4a), 136.0 (1C, C-9a), 155.6 (1C, C-2),
separated aqueous layer was extracted with CH
2
Cl
2
(6ꢂ). The
d
combined organic layer was dried (Na SO ), filtered and the solvent
2
4
4
1
was removed in vacuo. The crude product was dried in vacuo
without further purification to give a pale yellow solid, mp > 335 C
ꢁ
2
03.6 (1C, C-5). Exact mass (APCI): m/z ¼ 217.0979 (calcd. 217.0972
(decomposition), yield 0.13 g (70%).C12
H
12
N
2
1
O (200.2). TLC (cyclo-
-
1
for C12
H
13
N
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 2928 (m,
n
C-H), 1721
hexane:ethyl acetate ¼ 2:8): Rf ¼ 0.17. H NMR (D6-DMSO):
(
s,
nC ¼ O Ketone), 1628 (s,
n
C ¼ O Urea). Purity (HPLC):
d
[ppm] ¼ 1.79e1.90 (m, 2H, 6-H), 2.28e2.37 (m, 2H, 7-H),
2.71e2.78 (m, 2H, 5-H), 5.73 (dt, 2J ¼ 12.1 Hz, 3J ¼ 4.1 Hz, 1H, 8-H),
.36 (dt, 3J ¼ 12.4 Hz, 3J ¼ 1.9 Hz,1H, 9-H), 6.68 (s,1H,10-H), 6.71 (s,
tR ¼ 12.91 min, purity 96.5%.
6
13
4.1.13. 6-Bromo-1,3,6,7,8,9-haxahydro [7]annuleno[f]
1H, 4-H), 10.42 (s, 1H, NH), 10.48 (s, 1H, NH). C NMR (D6-DMSO):
benzimidazole-2,5-dione (21)
d
[ppm] ¼ 27.2 (1C, C-6), 31.7 (1C, C-7), 35.4 (1C, C-5), 109.0 (1C, C-
Ketone 20 (0.05 g, 0.23 mmol) was dissolved in DMSO (10 mL)
and N-bromosuccinimide (80 mg, 0.46 mmol) was added. The
10), 110.5 (1C, C-4), 127.9 (1C, C-3a), 128.6 (2C, C-10a, C-9a), 129.5
(C-8), 130.0 (1C, C-9), 134.2 (1C, C-4a), 155.5 (1C, C-2). Exact mass
ꢁ
mixture was heated to 40 C for 24 h. Then water was added. The
(APCI): m/z ¼ 201.1020 (calcd. 201.1022 for C12
H
13
N
2
O [MH]þ). IR
C]O Urea), 1639 (w,
C]C konj). Purity (HPLC): tR ¼ 17.14 min, purity 96.4%.
-
1
separated aqueous layer was extracted witch CH
combined organic layers were dried (Na SO ), filtered and the
2
Cl
2
(4ꢂ). The
(neat): ṽ [cm ] ¼ 2929 (m,
nC-H), 1712(s, n
2
4
n
solvent was removed in vacuo. The crude product was purified by
flash column chromatography (Ø ¼ 2.0 cm, h ¼ 21.5 cm, cyclo-
4.1.16. 1,3-Dimethyl-3,5,6,7-tetrahydro[7]annuleno[f]benzimidazol-
2(1H)-one (24)
Tetrabutylammonium iodide (0.44 g, 1.2 mmol) and methyl io-
dide (3.0 mL, 47.9 mmol) were added to a suspension of alkene 23
(1.2 g, 6.0 mmol) in toluene (70 mL) and 50% aqueous NaOH
hexane:ethyl acetate ¼ 2:8, V ¼ 10 mL) to give a yellow solid,
ꢁ
mp > 260 C (decomposition), yield 51 mg (75%). C12
H
12BrN
2
O
2
1
(
(
1
295.1). TLC (cyclohexane:ethyl acetate ¼ 2:8): Rf ¼ 0.16. H NMR
D6-DMSO):
.98e2.15 (1H, 7-H), 2.25e2.39 (m, 1H, 7-H), 2.87e3.04 (m, 2H, 9-
H), 5.23 (dd, 3J ¼ 8.9 Hz, 3J ¼ 4.0 Hz, 1H, 6-H), 6.84 (s, 1H, 10-H),
d
[ppm] ¼ 1.62e1.79 (m, 1H, 8-H), 1.93e2.03 (1H, 8-H),
ꢁ
(23 mL). The mixture was stirred vigorously and heated to 30 C for
24 h. Then, water was added and the separated aqueous layer was

144
I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
extracted with CH
2
Cl
2
(15ꢂ), dried (Na
2
SO
4
), filtered and the sol-
6-OH), 4.74 (d, 3J ¼ 4.2 Hz, 1H, 5-H), 5.10 (d, 3J ¼ 4.1 Hz, 1H, 5-OH),
13
vent was removed in vacuo. The crude product was purified by flash
column chromatography (Ø ¼ 4.0 cm, h ¼ 17.0 cm, cyclo-
6.84 (s, 1H, 10-H), 7.07 (s, 1H, 4-H). C NMR (D6-DMSO):
), 34.4 (1C, C-9), 34.5
d
[ppm] ¼ 24.6 (1C, C-8), 27.0 (2C, 2 ꢂ NCH
3
hexane:ethyl acetate ¼ 2:8, V ¼ 30 mL) to give a solid, mp
(1C, C7), 72.7 (1C, C-6), 77.6 (1C, C-5), 108.0 (1C, C-10), 108.6 (1C, C-
4), 127.3 (1C, C-3a), 128.0 (1C, C-10a), 134.0 (1C, C-4a), 154.1 (1C, C-
9a), 206.7 (1C, C-2). Exact mass (APCI): m/z ¼ 263.1373 (calcd.
ꢁ
1
61e162 C, yield 0.965 g (71%). C14
H
16
N
2
O (228.3). TLC (cyclo-
1
hexane:ethyl acetate ¼ 2:8): Rf ¼ 0.44. H NMR (D6-DMSO):
-
1
d
[ppm] ¼ 1.83e1.91 (m, 2H, 6-H), 2.34 (tdd, 3 J ¼ 6.6 Hz,
J ¼ 4.5 Hz, 3 J ¼ 2.0 Hz, 2H, 7-H), 2.78e2.85 (m, 2H, 5-H), 3.29 (s,
263.1390 for C14
O-H), 2924(w,
(m,
C-O). Purity (HPLC): tR ¼ 12.63 min, purity 96.1%.
H
17
N
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 3345 (w, br,
3
3
8
6
2
n
n
C-H), 1670(s,
n
C]O Urea), 1508 (m,
n
C ¼ C), 1026
H, NCH3), 3.28 (s, 3H, NCH
3
), 5.79 (dt, 2J ¼ 12.2 Hz, 3J ¼ 4.5 Hz, 1H,
G
-H), 6.43 (dt, 2J ¼ 12.2 Hz, 3J ¼ 2.1 Hz, 1H, 9-H), 6.91 (s, 1H, 10-H),
.94 (s, 1H, 4-H). 13C NMR (D6-DMSO):
[ppm] ¼ 26.88 (1C, NCH ),
6.89 (1C,, NCH ), 27.3 (1C, C-6), 31.7 (1C, C-7), 35.4 (1C, C-5), 108.2
d
3
4.1.19. (5RS,6RS)-5,6-Dihydroxy-1,3-dimethyl-3,5,6,7,8,9-
hexahydro[7]annuleno[f]benz-imidazol-2(1H)-one (trans-27)
Epoxide 25 (40 mg, 0.16 mmol) was dissolved in abs. CH
3
(
(
(
C
1C, C-10), 109.6 (1C, C-4), 127.8 (1C, C-10a), 128.4 (1C, C-9a), 129.1
1C, C-3a), 129.8 (1C, C-8), 130.0 (1C, C-9), 134.7 (1C, C-4a), 154.0
1C, C-2). Exact mass (APCI): m/z ¼ 229.1369 (calcd. 229.1335 for
2
Cl
.OEt
2
(5 mL). 3-Phenylpropylamine (28 mL, 0.20 mmol) and BF
3
2
(catalytic amount) were added and the mixture was stirred at
ambient temperature for 17 h. Then, aqueous saturated sodium
hydrogen carbonate solution was added and the separated aqueous
-
1
H
14 17
N
2
O [MH]þ). IR (neat): ṽ [cm ] ¼ 2916(w,
nC-H), 1697 (s,
n
C]O Urea), 1512 (m,
nC]C). Purity (HPLC): tR ¼ 21.24 min, purity
6
8.3%.
layer was extracted with CH
were dried (Na SO ), filtered and the solvent was removed in
vacuo. The crude product was purified by flash column chroma-
2
Cl
2
(4ꢂ). The combined organic layers
2
4
4.1.17. (5RS, 6SR)- 1,3-dimethyl-5,6-epoxy-3,5,6,7,8,9-hexahydro[7]
annuleno[f]imidazol-2(1H)-one (25)
tography (Ø ¼ 2.0 cm, h ¼ 15.5 cm, CH
3
OH:CH
2
Cl
2
¼ 0.5:9.5 þ 0.75%
ꢁ
Alkene 24 (0.968 g, 4.2 mmol) was dissolved in CH
2
Cl
2
(117 mL).
NH
8.3
3
(25%), V ¼ 5 mL) to give a colorless solid, mp 214e215 C, yield
0
.5 M aqueous sodium carbonate solution (40 mL) was added and
the mixture was stirred vigorously. Then, m-chloroperbenzoic acid
1.46 g, 8.5 mmol) was added portionwise. The mixture was stirred
mg
OH:CH Cl
(D6-DMSO):
(19%).
C
14
H
18
N
2
O
3
(262.3).
TLC
1
(CH
3
2
2
d
¼ 0.5:9.5 þ 0.75% NH
3
(25%)): Rf ¼ 0.19. H NMR
(
[ppm] ¼ 1.20e1.34 (m, 1H, 8-H), 1.63 (tdd,
vigorously for 2 h. The layers were separated and the organic layer
2J ¼ 12.9 Hz, 3J ¼ 8.5 Hz, 3J ¼ 3.4 Hz, 1H, 7-H), 1.78e1.90 (m, 1H, 8-
was extracted with 1 M NaOH (10ꢂ) and water (10ꢂ). The organic
H), 1.92e2.02 (m, 1H, 7-H), 2.62e2.71 (m, 1H, 9-H), 2.82 (dd,
layer was dried (Na
2
SO
4
), filtered and the solvent was removed in
2J ¼ 14.5 Hz, 3J ¼ 6.2 Hz, 1H, 9-H), 3.28 (s, 3H, NCH
3
), 3.30 (s, 3H,
vacuo. The crude product was recrystallized from ethyl acetate to
NCH
3
), 4.54 (dd, 3J ¼ 8.2 Hz, 2J ¼ 3.3 Hz, 1H, 5-H), 4.77 (d,
ꢁ
give a yellow solid, mp 146e147 C, yield 527 mg, (51%). C14
H
16
N
2
O
2
3J ¼ 3.8 Hz, 1H, 6-OH), 5.26 (d, 2J ¼ 3.4 Hz, 1H, 5-OH), 6.88 (s, 1H,
1
(
244.3). TLC (cyclohexane:ethyl acetate ¼ 2:8): Rf ¼ 0.15. H NMR
10-H), 7.24 (s, 1H, 4-H). The signal by the CH of the OH group is not
13
(
CDCl ):
3
d
[ppm] ¼ 1.23e1.37 (m, 1H, 7-H), 1.66 (ddt, 3J ¼ 14.0 Hz,
seen in the spectrum. C NMR (D6-DMSO):
d
[ppm] ¼ 24.6 (1C, C-
2
3
3
3
3
J ¼ 11.7 Hz, 3J ¼ 5.8 Hz, 3J ¼ 4.2 Hz, 1H, 8-H), 1.90 (ddtd,
8), 26.9 (1C, NCH3), 27.0 (1C, NCH3), 34.1 (1C, C-9), 36.1 (1C, C-7),
72.0 (1C, C-6), 75.0 (1C, C-5), 105.4 (1C, C-4), 108.2 (1C, C-10), 127.7
(1C, C-10a), 128.0 (1C, C-3a), 133.5 (1C, C-9a), 134.6 (1C, C-4a), 154.1
(C-2). Exact mass (APCI): m/z ¼ 263.1378 (calcd. 263.1390 for
J ¼ 14.5 Hz, 2J ¼ 11.4 Hz, 3J ¼ 5.8 Hz, 3J ¼ 3.3 Hz, 1H, 8-H), 2.17 (dq,
J ¼ 14.1 Hz, 3J ¼ 4.4 Hz, 7-H), 2.72 (ddd, 2J ¼ 14.2 Hz, 3J ¼ 5.4 Hz,
J ¼ 3.2 Hz, 1H, 9-H), 3.04 (ddd, 2J ¼ 14.2 Hz, 3J ¼ 11.8 Hz,
J ¼ 5.9 Hz, 1H, 9-H), 3.37 (dt, 3J ¼ 7.5 Hz, 3J ¼ 4.4 Hz, 1H, 6-H), 3.39
-
1
C
14
H
19
N
2
O
3
[MH]þ). IR (neat): ṽ [cm ] ¼ 3352 (w, br,
n
O-H),
C ¼ C),1026 (m, C-
O). Purity (HPLC): tR ¼ 12.92 min, purity 98.4%.
(
(
s, 3H, NCH
s, 1H, 10-H), 7.11 (s, 1H, 4-H). CNMR (CDCl
), 27.4 (1C, NCH ), 27.4 (1C, C-7), 32.1 (1C, C-9),
5.0 (1C, C-6), 56.2 (1C, C-5), 108.0 (1C, Cd10), 109.5 (1C, C-4), 127.7
3
), 3.42 (s, 3H, NCH
3
), 4.10 (d, 3J ¼ 4.4 Hz, 1H, 5-H), 6.68
2916(w,
nC-H),1701(m,
n
C ¼ O Urea),1508 (m,
n
G
1
3
3
):
d
[ppm] ¼ 22.3 (1C,
C-8), 27.3 (1C, NCH
5
3
3
4.1.20. (5RS,6RS)-6-Hydroxy-1,3-dimethyl5-[(3-phenylpropyl)-
(
1C, C-3a), 129.0 (1C, C-10a), 130.1 (1C, C-9a), 132.1 (1C, C-4a), 154.9
amino]-3,5,6,7,8,9-hexahydro[7]annuleno[f]-benzimidazol-2(1H)-
(
C
n
1C, C-2). Exact mass (APCI): m/z ¼ 245.1286 (calcd. 245.1285 for
one (28)
-
1
H
14 17
N
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 2928 (w,
n
C-H), 1690 (s,
Under N
0.20 mmol) was added to a solution of 3-phenylpropan-1-amine
28 L, 0.20 mmol) in abs. THF (5 mL). The mixture was stirred at
2
, 1.0 M lithium bis(trimethylsilyl)amide (169 mL,
C ¼ O Uea), 1512 (m,
n
C ¼ C), 1265 (w,
nC-O Epoxid).
(
m
4
.1.18. (5RS,6SR)-5,6-Dihydroxy-1,3-dimethyl-3,5,6,7,8,9-
ambient temperature for 0.5 h. Epoxide 25 (40 mg, 0.16 mmol) was
added and the mixture was stirred at ambient temperature for
11.5 d. Then, water was added and the separated aqueous layer was
hexahydro[7]annuleno[f]benz-imidazol-2(1H)-one (cis-26)
Under N , sodium hydride (4.7 mg, 0.20 mmol) was added to a
solution of 3-phenylpropan-1-amine (28 L, 0.20 mmol) in abs. THF
5 mL). The mixture was stirred at ambient temperature for 1.5 h.
2
m
extracted with CH
dried (Na SO ), filtered and the solvent was removed in vacuo. The
crude product was purified by flash column chromatography
2
Cl
2
(4ꢂ). The combined organic layers were
(
2
4
Epoxide 25 (40 mg, 0.16 mmol) was added and the mixture was
stirred at ambient temperature for 10.5 d. Then, water and satu-
rated NH4Cl solution (20 mL) were added and the separated
(Ø ¼ 2.0 cm, h ¼ 15.3 cm, CH
3
OH:CH
2
Cl
2
¼ 0.5:9.5 þ 0.75% NH
3
ꢁ
(25%), V ¼ 5 mL) to give a pale yellow solid, mp 65e66 C, yield
aqueous layer was extracted with CH
organic layers were dried (Na SO ), filtered and the solvent was
removed in vacuo. The crude product was purified by flash column
OH:CH Cl
.5:9.5 þ 0.75% NH3 (25%), V ¼ 5 mL) to give a colorless solid, mp
2
Cl
2
(4ꢂ). The combined
16.9
mg
OH:CH Cl
(27%).
¼ 0.5:9.5 þ 0.75% NH
C
23
29
H N
3
O
2
(379.5).
TLC
1
2
4
(CH
3
2
2
3
(25%)): Rf ¼ 0.21. H NMR
(CDCl3):
d
[ppm] ¼ 1.37e1.50 (m, 1H, 8-H), 1.66e1.78 (m, 1H, 7-H),
chromatography (Ø ¼ 2.0 cm, h ¼ 15.6 cm, CH
3
2
2
]
1.96 (dtd, 2J ¼ 13.8 Hz, 3J ¼ 7.2 Hz, 3J ¼ 3.1 Hz, 3H, 8-H,
NHCH CH CH
Ph), 2.09e2.19 (m, 1H, 7-H), 2.57 (dt, 2J ¼ 11.7 Hz,
3J ¼ 7.1 Hz, 1H, 9-H), 2.72 (t, 3J ¼ 7.7 Hz, 2H, NHCH CH CH Ph),
2.80e2.91 (m, 3H, 9-H, NHCH CH CH Ph), 3.39 (s, 3H, NCH ), 3.41
(s, 3H, NCH
0
2
2
2
2
ꢁ
27e228 C, yield 5.7 mg (13%).
C
14
H
18
N
2
O
3
(262.3). TLC
2
2
2
1
(
CH
3
OH:CH
2
Cl
d
2
¼ 0.5:9.5 þ 0.75% NH3 (25%)): Rf ¼ 0.09. H NMR
2
2
2
3
(
D6-DMSO):
[ppm] ¼ 1.43 (q, 2J ¼ 10.2 Hz, 1H, 8-H), 1.60e1.79 (m,
3
), 3.87 (d, 3J ¼ 8.5 Hz, 5-H), 6.75 (s, 1H, 10-H), 6.91 (s,
2
3
3
H, 7-H, 8-H), 1.95e2.07 (m, 1H, 7-H), 2.59 (dd, 2J ¼ 13.8 Hz,
J ¼ 8.6 Hz, 1H, 9-H), 2.94e3.05 (m, 1H, 9-H), 3.28 (s, 3H, NCH ),
.29 (s, 3H, NCH
), 3.67e3.74 (m, 1H, 6-H), 4.34 (d, 3J ¼ 4.8 Hz, 1H,
1H, 4-H), 7.15e7.20 (m, 3H, AreH), 7.24e7.30 (m, 2H, AreH). Signals
3
for the protons of the 6-CH group and NH and OH groups are not
13
3
seen in the spectrum. C NMR (CDCl
3
):
d
[ppm] ¼ 24.9 (1C, C-8),

I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
145
2
7.4 (2C, 2 ꢂ NCH3), 31.5 (1C, NHCH
NHCH CH CH Ph), 34.8 (NHCH CH CH Ph), 37.2 (1C, C-7), 47.5 (1C,
C-9), 66.4 (1C, C-5), 71.3 (1C, C-6), 104.6 (1C, C-4), 109.0 (1C, C-10),
26.1 (1C, C-Arp), 128.5 (2C, C-Aro), 128.55 (2C, C-Arm), 128.6 (1C,
2
CH
2
CH
2
Ph), 33.5 (1C,
AreC), 128.47 (2C, AreC), 131.5 (1C, C-4a), 136.7 (1C, C-9a), 142.0
(1C, Ar-Cq). Exact mass (APCI): m/z ¼ 364.2370 (calcd. 364.2383 for
2
2
2
2
2
2
-
1
C
23
H
30
N
3O [MH]þ). IR (neat): ṽ [cm ] ¼ 3503 (w,
n
N-H), 2920(w,
C ¼ O Urea), 1508 (m, N-H), 1045
monosubst. arom), 698 (w, Gmonosubst. arom).
1
n
C-H), 1686(s,
(w, C-O), 741 (w,
Purity (HPLC): tR ¼ 17.73 min, purity 94.3%.
n
n
C ¼ C), 1458 (m,
G
C-10a), 128.8 (1C, C-3a), 131.1 (1C, C-4a), 135.7 (1C, C-9a), 141.7 (1C,
G
G
C-Arq), 155.0 (1C, C-2). Exact mass (APCI): m/z ¼ 380.2306 (calcd.
-
1
3
80.2333 for C14
H
n
17
N
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 3390 (w, br,
n
(
O-H), 3337 (w,
m,
N-H), 2924(w,
O-H), 1045 (w,
monosubst. arom). Purity (HPLC): tR ¼ 16.09 min,
n
C-H), 1682(s,
n
C ¼ O Urea), 1508
4
4
.2. Receptor binding studies
n
C ¼ C), 1396 (m,
G
G
C-O), 741 (w,
Gmonosubst.
arom), 698 (w,
purity 83.3%.
G
.2.1. Materials and general procedures
Centrifuge: High-speed cooling centrifuge model Sorvall RC-5C
plus (Thermo Finnigan). Filter: Printed Filtermat Type B (Per-
kineElmer), presoaked in 0.5% aqueous polyethylenimine for 2 h at
rt before use. The filtration was carried out with a MicroBeta
FilterMate-96 Harvester (PerkineElmer). The scintillation analysis
was performed using Meltilex (Type A) solid scintillator (Per-
kineElmer). The scintillation was measured using a MicroBeta
Trilux scintillation analyzer (PerkineElmer). The overall counting
efficiency was 20%.
4
.1.21. 1,3-Dimethyl-5,7,8,9-tetrahydro[7]annuleno[f]
benzimidazole-2,6(1H,5H)-dione (29)
Under N , a solution of 3-phenylpropan-1-amine (28
.20 mmol) in abs. THF (4 mL) was slowly added dropwise to a 1 M
2
mL,
0
solution of methylmagnesium bromide (200
mL, 0.20 mmol). The
ꢁ
mixture was heated to 35 C for 1 h. The mixture was cooled to
ambient temperature. A solution of epoxide 25 (40 mg, 0.16 mmol)
in abs THF (3 mL) was slowly added and the reaction mixture was
ꢁ
heated to 35 C for 7.5 h. Then, water (5 mL), saturated aqueous
NH
6 mL) was added and the separated aqueous layer was extracted
with CH Cl
(2ꢂ). The separated aqueous layer was alkalized by
addition of 10% aqueous NaOH (10 mL) and extracted with CH Cl
4 2 2
Cl solution (20 mL) and CH Cl (10 mL) were added. 2 M HCl
4
.2.2. Cell culture and preparation of membrane homogenates for
(
the GluN2B assay [43]
2
2
In the assay mouse L(tk-)-cells stably transfected with the
dexamethasone inducible eukaryotic expression vectors pMSG
NR1a, pMSG NR2B in a 1:5 ratio were used. The transformed L(tk-
2
2
(
2ꢂ). The combined organic layers were dried (Na2SO4), filtered
and the solvent was removed in vacuo. The crude product was
)-cells were grown in Modified Earl's Medium (MEM) containing
purified by flash column chromatography (Ø ¼ 2.0 cm, h ¼ 15 cm,
10% of standardized FCS (Biochrom AG, Berlin, Germany). The
CH
3
OH:CH
2
Cl
2
¼ 0.5:9.5 þ 0.75% NH
3
(25%), V ¼ 5 mL) to give a pale
expression of the NMDA receptor at the cell surface was induced
after the cell density of the adherent growing cells had reached
approximately 90% of confluency. For the induction, the original
growth medium was replaced by growth medium containing 4
dexamethasone and 4 M ketamine (final concentration). After
ꢁ
yellow solid, mp 162e163 C, yield 14.5 mg (36%). C14
H
16
N
2
O
2
(
244.3). TLC (CH
3
OH:CH
2
Cl
d
2
¼ 0.5:9.5 þ 0.75% NH
[ppm] ¼ 1.96e2.04 (m, 2H, 8-H), 2.56
),
), 3.75 (s, 2H, 5-H), 6.78 (s, 2H, 4-H, 10-H). C NMR
[ppm] ¼ 27.2 (2C, 2 ꢂ NCH ), 27.4 (1C, C-8), 33.3 (1C, C-9),
3.9 (1C, C-7), 50.2 (1C, C-5), 108.4 (1C, C-10), 108.8 (1C, C-4), 126.6
3
(25%)):
1
Rf ¼ 0.33. H NMR (CDCl
3
):
mM
(
3
t, 3J ¼ 6.9 Hz, 2H, 7-H), 2.97e3.01 (m, 2H, 9-H), 3.39 (s, 3H, NCH
3
m
13
.40 (s, 3H, NCH
):
3
2
5
4 h the cells were harvested by scraping and pelleted (10 min,
000 ꢂ g, Hettich Rotina 35R centrifuge, Tuttlingen, Germany).
For the binding assay, the cell pellet was resuspended in phos-
(
CDCl
3
d
3
4
(
1C, C-3a), 129.0 (1C, C-10a), 129.3 (1C, C-9a), 133.9 (1C, C-4a), 154.8
phate buffer saline (PBS, pH 7.4) buffer and the number of cells was
determined using an improved Neubauer's counting chamber
(
2
1C, C-2), 208.9 (1C, C-6). Exact mass (APCI): m/z ¼ 245.1345 (calcd.
-
1
45.1285 for C14
H
17
N
2
O
2
[MH]þ). IR (neat): ṽ [cm ] ¼ 2920(w,
nC-
(VWR, Darmstadt, Germany). Subsequently, the cells were lysed by
H), 1686(m,
tR ¼ 15.73 min, purity 78.2%.
n
C ¼ O Urea), 1508 (m,
n
C ¼ C). Purity (HPLC):
ꢁ
sonication (4 C, 6 ꢂ 10 s cycles with breaks of 10 s, device: Soni-
prep 150, MSE, London, UK). The resulting cell fragments were
centrifuged with a high performance cool centrifuge (20,000 ꢂ g,
4
.1.22. 1,3-Dimethyl-6-[(3-phenylpropyl)amino]-3,5,6,7,8,9-
hexahydro[7]annuleno[f]-benzimidazol-2(1H)-one (30)
-Phenylpropan-1-amine (24 L, 0.168 mmol) and NaBH(OAc)3
35.6 mg, 0.168 mmol) were added to a solution of ketone 29
20.5 mg, 0.084 mmol) in CH Cl (5 mL). The mixture was stirred at
so-
lution (10 mL) was added and the separated aqueous layer was
extracted with CH Cl
(4ꢂ). The combined organic layers were
dried (Na SO ), filtered and the solvent was removed in vacuo. The
crude product was purified by flash column chromatography
ꢁ
4
C, Sorvall RC-5 plus, Thermo Scientific). The supernatant was
discarded and the pellet resuspended in a defined volume of PBS
3
m
yielding cell fragments of approximately 500,000 cells/mL. The
(
(
ꢁ
suspension of membrane homogenates was sonicated again (4 C,
2
2
ꢁ
2
ꢂ 10 s cycles with a break of 10 min) and stored at ꢀ80 C.
ambient temperature for 45 min. Saturated aqueous NaHCO
3
2
2
4.2.3. Performing of the GluN2B binding assay [43]
2
4
The competitive binding assay was performed with the radio-
ligand [3H]ifenprodil (60 Ci/mmol; Perkin Elmer) using standard
96-well-multiplates (Diagonal, Münster, Germany). The thawed
cell membrane preparation (about 20 mg protein) was incubated
with 6 different concentrations of test compounds, 5 nM [3H]-
(
Ø ¼ 2.0 cm, h ¼ 16 cm, CH
3
OH:CH
2
Cl
2
¼ 0.5:9.5 þ 0.75% NH
3
H
(25%),
V ¼ 5 mL) to give a yellow oil, yield 12.9 mg (42%). C23
29
N
3
O
(
363.5). TLC (CH
3
OH:CH
2
Cl
):
2
¼ 0.5:9.5 þ 0.75% NH3 (25%)):
1
Rf ¼ 0.16. H NMR (CDCl
3
d
[ppm] ¼ 1.43e1.57 (m, 1H, 8-H),
ifenprodil, and TRIS/EDTA-buffer (5 mM/1 mM, pH 7.5) in a total
ꢁ
1.70e1.79 (m, 1H, 7-H), 1.79e1.90 (m, 3H, 8-H, NHCH2CH2CH2Ph),
volume of 200
m
L for 120 min at 37 C. The incubation was termi-
1
.95e2.05 (m, 1H, 7-H), 2.61e2.85 (m, 7H, 9-H, 6-H,
NHCH2CH2CH2Ph), 2.93 (d, 3J ¼ 7.8 Hz, 2H, 5-H), 3.38 (s, 6H,
ꢂ NCH3), 6.71 (s, 1H, 10-H), 6.74 (1H, 4-H), 7.13e7.21 (3H, AreH),
nated by rapid filtration through the presoaked filtermats by using
the cell harvester. After washing each well five times with 300
m
L of
ꢁ
2
water, the filtermats were dried at 95 C. Subsequently, the solid
13
ꢁ
7
.22e7.30 (2H, AreH). C NMR (CDCl
3
):
), 27.33 (1C, NCH3), 31.6 (1C, NHCH
CH CH Ph), 36.1 (1C, C-9), 38.1 (1C, C-7), 42.0 (1C,
CH CH Ph), 56.7 (1C, C-6), 108.2 (1C, C-10),
09.4 (1C, C-4), 126.0 (1C, AreC), 128.3 (2C, C-3a, C-10a),128.45 (2C,
d
[ppm] ¼ 25.8 (1C, C-8),
scintillator was placed on the filtermat and melted at 95 C. After
2
3
7.29 (1C, NCH
3.8 (1C, NHCH
3
2
CH CH Ph),
2
2
5 min, the solid scintillator was allowed to solidify at rt. The bound
radioactivity trapped on the filters was counted in the scintillation
analyzer. The non-specific binding was determined with 10 mM
unlabeled ifenprodil. The Kd-value of ifenprodil is 10 nM [43].
2
2
2
C-5), 46.5 (1C, NHCH
1
2
2
2

146
I. Lütnant et al. / European Journal of Medicinal Chemistry 116 (2016) 136e146
4
.2.4. Affinity towards
The affinity towards
s
1 and
1 and
s
2 receptors
[19] P. Paoletti, C. Bellone, Q. Zhou, Nat. Rev. Neurosci. 14 (2013) 383e400.
[
[
[
20] K.B. Hansen, K.K. Ogden, H. Yuan, S.F. Traynelis, Neuron 81 (2014) 1084e1096.
21] J.A. McCauley, Expert Opin. Ther. Patents 15 (2005) 389e407.
22] R.M. Santangelo, T.M. Acker, S.S. Zimmerman, B.M. Katzman, K.J. Strong,
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23] S. Zhu, P. Paoletti, Curr. Opin. Pharmacol. 20 (2015) 14e23.
24] I. Reynolds, Mol. Pharmacol. 36 (1989) 758.
s
s2 receptors [44e46] was recorded
as previously described.
[
[
[
Acknowledgment
25] E. Karakas, N. Simorowski, H. Furukawa, Nat. 475 (2011) 249e253.
We are grateful to Prof. Dr. D. Steinhilber, Department of Phar-
macy, University of Frankfurt, for donating us the L(tk-)-cells stably
expressing GluN1a/GluN2A and GluN1a/GluN2B receptor proteins,
respectively. Financial support by the Deutsche For-
schungsgemeinschaft is gratefully acknowledged.
[26] E. Falck, F. Begrow, E. Verspohl, B. Wünsch, J. Pharm, Biomed. Anal. 88 (2014)
6e105.
9
[27] A.P. Tamiz, E.R. Whittemore, Z. Zhou, J. Huang, J.A. Drewe, J. Chen, S. Cai,
E. Weber, R.M. Woodward, J.F.W. Keana, J. Med. Chem. 41 (1998) 3499e3506.
[28] B. Tewes, B. Frehland, D. Schepmann, K.-U. Schmidtke, T. Winckler, B. Wünsch,
Chem. Med. Chem. 5 (2010) 687e695.
[29] B. Tewes, B. Frehland, D. Schepmann, K. Schmidtke, T. Winckler, B. Wünsch,
Bioorg. Med. Chem. 18 (2010) 8005e8015.
[30] A. Benner, A. Bonifazi, C. Shirataki, L. Temme, D. Schepmann, W. Quaglia,
O. Shoji, Y. Watanabe, C. Daniliuc, B. Wünsch, Chem. Med. Chem. 9 (2014)
Appendix A. Supplementary data
7
41e751.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.03.065.
[
[
31] A.H. Tahar, L. Gr eꢀ goire, A. Darr eꢀ , N. B eꢀ langer, L. Meltzer, P.J. B eꢀ dard, Neurobiol.
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