Dibasic biphenyl H3 receptor antagonists: Steric tolerance for a lipophilic side chain

Article abstract of DOI:10.1016/j.ejmech.2011.12.019

Within a series of histamine H₃-antagonists characterized by a biphenyl core and two basic groups, we identified (S)-1-{[4′-((2- methylpyrrolidin-1-yl)methyl)biphenyl-4-yl]methyl}piperidine as a lead scaffold to introduce an additional lipophil

Full text of DOI:10.1016/j.ejmech.2011.12.019

European Journal of Medicinal Chemistry 48 (2012) 214e230
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Original article
Dibasic biphenyl H3 receptor antagonists: Steric tolerance for a lipophilic
side chain
,
a
a
a
a
Fabrizio Bordi a, Silvia Rivara a , Elisa Dallaturca , Caterina Carmi , Daniele Pala , Alessio Lodola ,
Federica Vacondio a, Lisa Flammini b, Simona Bertoni b, Vigilio Ballabeni b, Elisabetta Barocelli b,
Marco Mor a
*
a Dipartimento Farmaceutico, Università degli Studi di Parma, V.le G.P. Usberti 27/A, I-43124 Parma, Italy
b Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università degli Studi di Parma, V.le G.P. Usberti 27/A, I-43124 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Within a series of histamine H3-antagonists characterized by a biphenyl core and two basic groups, we
identified (S)-1-{[40-((2-methylpyrrolidin-1-yl)methyl)biphenyl-4-yl]methyl}piperidine as a lead scaffold
to introduce an additional lipophilic chain at the benzylic carbon close to the pyrrolidine ring. A series of
derivatives was synthesized and tested for their binding affinity at human and rat histamine H3 recep-
tors, and for their antagonist potency. For compounds with two chiral centers, the synthetic procedure
provided mixtures of diastereomeric couples, which were separated by flash chromatography. Combi-
nation of experimental NMR data and molecular dynamics simulation allowed the assignment of
absolute stereochemistry, based on characteristic differences detected within each diastereomeric
couple. The additional lipophilic group was tolerated by the receptor, supporting the hypothesis that the
two regions described within the H3 receptor binding site can be simultaneously occupied by antago-
nists. Diastereoisomers with opposite chirality at the benzylic carbon showed limited or no stereo-
selectivity at both human and rat receptors.
Received 5 August 2011
Received in revised form
7 November 2011
Accepted 10 December 2011
Available online 20 December 2011
Keywords:
Histamine H3 receptor
H3-antagonist
NMR analysis
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
neurotransmitters, including norepinephrine [7,8], dopamine [9],
serotonin [10], GABA [11], glutamate [12] and acetylcholine [13]. It
The histaminergic system is involved in the regulation of several
physiological processes. The natural ligand histamine is produced
from the amino acid L-histidine and exerts its actions through the
has been observed that administration of H3-antagonists in the CNS
induces increased availability of these neurotransmitters, causing
improvement in cognition and attention in animal models [14,15].
Thus, H3-antagonists have been proposed in the treatment of
cognitive disorders, such as attention deficit hyperactivity disorder
(ADHD), Parkinson’s and Alzheimer’s diseases, as well as of
narcolepsy, obesity, dementia, schizophrenia and epilepsy, and
a number of compounds are currently evaluated in clinical trials
[16e20].
First generation H3-antagonists were characterized by an
imidazole ring, in analogy with the natural ligand histamine
[16,21,22]. However, imidazole-based antagonists showed possible
drawbacks, such as affinity for CYP450, causing potential druge
drug interactions, poor brain penetration, due to the presence of
polar hydrogen-bonding groups, and significant data discrepancies
across species [23,24]. Thus, to overcome these drawbacks, many
research efforts have been focused on the discovery and develop-
ment of non-imidazole H3-antagonists. Despite significant differ-
ences in compounds molecular weight and polarity, a general
pharmacophore model has been developed for non-imidazole H3-
activation of four distinct G-protein-coupled receptors (GPCRs),
known as H1, H2, H3 and H4 [1]. While antagonists of the H1 receptor
are widely used in the treatment of allergic responses [2], H2
receptor antagonists inhibit gastric acid secretion, finding appli-
cation in the treatment of peptic ulcer and gastroesophageal reflux
disease [3]. H4-antagonists have immunomodulatory actions and
are under investigation as anti-inflammatory and anti-allergic
drugs [4].
Identified in 1983 by Arrang and co-workers [5], the histamine
H3 receptors are primarily located in the central nervous system
(CNS). They have a predominantly presynaptic localization and
they modulate, through a negative feed-back mechanism, the
biosynthesis and release of histamine [6]. In addition, H3 receptors
act as heteroreceptors, inhibiting the release of different
* Corresponding author. Tel.: þ39 0521 905062; fax: þ39 0521 905006.
E-mail address: silvia.rivara@unipr.it (S. Rivara).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.019

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
215
antagonists. They are usually characterized by a basic group, often
a tertiary cyclic amine, connected through an aliphatic spacer to
a second pharmacophoric fragment which includes, depending on
the particular series, a lipophilic substituent (e.g. 1 and 2, Fig. 1),
a second basic portion (e.g. 3 and 4, Fig. 1), or a hydrophilic
substituent (e.g. 5, Fig. 1) [18,24,25].
synthetic protocol led to couples of diastereomeric compounds,
which were separated and singularly tested, to evaluate possible
stereoselectivity. These couples resulted composed by two isomers
with characteristic differences in their NMR spectra, also shown by
their intermediates lacking the R1 piperidine ring. To study the
relationship between conformational equilibria and compound
stereochemistry, we performed molecular dynamics simulations
for the intermediates 29c,d (Scheme 2), comparing calculated and
experimental NMR data. As simulations for these two stereoiso-
mers were in good agreement with their spectra, the characteristic
differences observed within each diastereomeric couple were
applied to the assignment of absolute stereochemistry.
We have recently described a series of non-imidazole H3-
antagonists characterized by a central biphenyl scaffold connected,
via alkyl spacers, to two basic groups [26,27]. This series was the
result of a structural rigidification, obtained replacing the flexible
polimethylene linker of a previous series with the rigid biphenyl
scaffold [28,29]. The most potent derivative (6, Fig. 1) showed
subnanomolar affinity for human and rat H3 receptors (hH3
pKi ¼ 9.47, rH3 pKi ¼ 8.92). When docked within a model of rat H3
receptor, two possible accommodations were identified for 6. In the
“horizontal” arrangement, compound 6 is perpendicular to the
helix bundle and the piperidine nitrogens interact with Asp114 in
transmembrane helix (TM) 3 and Glu206 in TM5. These amino acids
are supposed to interact with the basic nitrogen and the imidazole
ring of histamine, respectively, on the basis of mutagenesis studies.
In the “vertical” arrangement, compound 6 lies parallel to the
transmembrane helices and its basic centers interact with Asp114
and with Asn404 in TM7, located at the end of a second pocket
perpendicular to that supposed to host the natural agonist [26].
Indeed, other docking studies on homology-based models of the H3
receptor provided either “horizontal” or “vertical” poses for H3-
antagonists [30e34]. The possibility that two perpendicular
pockets could simultaneously exist into the H3 receptor implies
that dibasic H3-antagonists with a perpendicular side chain could
undertake favorable interactions with the receptor binding site.
This hypothesis is supported by some examples of dibasic H3-
antagonists with a lipophilic side chain, recently reported in liter-
ature (Fig. 2) [35e37].
2. Chemistry
Compounds 7e8 were synthesized starting from commercially
available 4,40-bis(chloromethyl)-biphenyl 25, as reported in
Scheme 1. Monosubstitution of 25 with piperidine to the inter-
mediate 26, followed by reaction with optically pure pyrrolidines,
led to the desired products (Scheme 1). The synthetic route to
compounds 9e24 and 34e35 is described in Scheme 2. Derivatives
9e11 were obtained from 4-iodobenzaldehyde 27, that was reacted
with benzyl zinc bromide and appropriate amine to obtain the
iodophenyl intermediates 28aeb. These compounds were
submitted to Suzuki coupling giving the corresponding biphenyl
derivates 29aeb. Finally, mesylation of 29aeb followed by substi-
tution with the appropriate amine gave the final compounds 9e11.
Reaction of 27 with butyl magnesium bromide led to the secondary
alcohol 30, that was coupled with 4-hydroxymethylphenylboronic
acid to 31 and substituted with piperidine to obtain the final
product 12. Compounds 13e18 and 34 were synthesized from
alcohol 30, that was first oxidized to ketone 32a, then reacted with
the proper amine and reduced to give 28ceh and 28p. As already
described, these iodophenyl intermediates were submitted to
Suzuki coupling (to intermediates 29ceh), followed by mesylation
and amine substitution, to obtain products 13e18. Compound 35
was obtaine from 28p by Suzuki coupling reaction. Similar proce-
dures were employed to synthesize compounds 19e20 and 21e24.
Compounds 19 and 20 were obtained from 4-iodobenzonitrile 33,
that was reacted with 3-methoxypropyl magnesium chloride fol-
lowed by hydrolysis to 32b. Ketone 32b was converted to 28iej by
reductive amination with (S)-2-methylpyrrolidine. Suzuki coupling
of 28iej (to 29iej), followed by mesylation and substitution with
piperidine, gave the target compounds 19e20. Final products
21e24 were prepared from 27 and allyloxybenzene derivates to
obtain ketones 32ced, that were reductively aminated with (S)-2-
methylpyrrolidine to 28ken. Suzuki coupling of to 29ken and
substitution allowed to obtain products 21e24. Finally, compound
34 was obtained following the same synthetic route (i.e. Suzuki
Therefore, to explore and further extend the structure-activity
relationships (SARs) on our class of biphenyl-based H3-antago-
nists, new derivatives carrying
a lipophilic side chain were
prepared. In these compounds (7e24, Table 1) the biphenyl core is
linked to two basic groups (R1 and R2) through methylene spacers,
and the additional side chain (R3) is attached to a methylene linker.
The tolerance of the H3 receptor to the steric hindrance of the R3
substituent was first evaluated, with substituents of different size
and shape (9e12, Table 1). Then, the best tolerated n-butyl R3 group
was maintained, and the effect of 2-methylpiperidine or 2-
methylpyrrolidine as R2 substituent was investigated (12e18,
Table 1). Finally, the R2 group providing the best affinity values, i.e.
an (S)-2-methylpyrrolidine, was maintained and the lipophilic R3
substituent was replaced by bulkier groups (19e24, Table 1).
When the additional side chain was inserted close to a chiral 2-
methylpyrrolidine or 2-methylpiperidine (13e24, Table 1), the
Fig. 1. Representative non-imidazole H3-antagonists.

216
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
Fig. 2. Dibasic H3-antagonists carrying a lipophilic substituent.
Fig. 3. Diastereoisomers 29c and 29d with atom numbering used for NMR and molecular modeling discussion, and
s1 and s2 dihedral angle definition. The notation “a” or “b” refers
to the relative position of protons with respect to the pyrrolidine plane: protons “a” are below the ring plane, while protons “b” are above the plane.
coupling and substitution with amine) from commercially available
iodophenyl derivative 28o.
cortex membranes [39]. H3 Receptor antagonist potency was
evaluated on SK-N-MC cells expressing the human H3 receptor and
the reporter gene -galactosidase, by a colorimetric assay [40].
b
3. Pharmacology
Histamine H4 receptor affinity of the new molecules was
measured by displacement of [3H]-histamine from GPCR97-
transfected SK-N-MC cells stably expressing the human H4
receptor [38].
In terms of selectivity, the compounds under study were
investigated in the functional assays performed on guinea-pig
Histamine H3 receptor affinity of the newly synthesized
compounds was measured by displacement of [3H]-(R)-
a-methyl-
histamine ([3H]RAMHA) from GPCR97-transfected SK-N-MC cells
stably expressing the human H3 receptor [38], and from rat cerebral
Fig. 4. Time evolution of dihedral angles s1 (C2-N1-C6-C11, panels A and C) and s2 (N1-C6-C7-C8, panels B and D) for (R,S) isomer (panels A, B) and (R,R) isomer (panels C, D).

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
217
Fig. 5. Representative conformations of the (S,R) isomer with s1 z 300ꢁ and s2 z 180ꢁ (left), or s2 z 300ꢁ (right).
isolated ileum (histamine H1 receptor) and atria (histamine H2
receptor) pharmacologically stimulated [41].
of the (S)-methylpyrrolidine with a bulkier (S)-methylpiperidine
(17, 18) led to lower binding affinity. In all cases both human and rat
receptors showed limited and/or variable stereoselectivity for the
chiral center at the benzylic carbon. This lack of stereoselectivity,
probably related to chain flexibility, was further confirmed for all
the couples of diastereomeric derivatives of (S)-methylpyrrolidine
tested (vide infra).
4. Results and discussion
4.1. Structureeactivity relationships
Binding affinity and antagonist potency data for the newly
synthesized compounds are reported in Table 1, together with
those for the reference compound 6. Replacement of one piperidine
of the lead compound 6 with a 2-methylpyrrolidine provided better
binding affinities when the methyl substituent was in (S) configu-
ration (8). (R)-Methylpyrrolidine (7) was tolerated by human and
rat receptors, but led to a slight reduction of binding affinity
compared to the piperidine ring. Compound 8 showed sub-
nanomolar binding affinity at both human and rat H3 receptors,
and remarkable antagonist potency at the human H3 receptor. The
higher binding affinity displayed by (S)-8 was unexpected, as other
series of H3-antagonists, having a terminal methylpyrrolidine
group, had shown higher affinity for the (R) isomers [42,43].
The first results suggested that a benzyl substituent was detri-
mental for binding affinity, as can be seen from binding data of
compounds 9 and 10. The de-benzyl analog of 10, previously
synthesized by us, had shown hH3 pKi ¼ 8.66 and rH3 pKi ¼ 8.88
[27]. Similarly, compound 11, tested as a mixture of diastereoiso-
mers, showed lower binding affinity than 7, both carrying a (R)-
methylpyrrolidine group. On the other hand, a n-butyl substituent
introduced on the structure of 6 led to a limited reduction of
binding affinity (12), suggesting that lipophilic groups in the
proximity of the basic center could be tolerated, and leading us to
focus on the butyl chain to further explore the steric requirements
for this portion of the molecule.
R3 bulkiness and lipophilicity were further increased with
methoxypropyl (19 and 20), phenoxypropyl (21 and 22) and 4-
methoxyphenoxypropyl (23 and 24) side chains, while keeping
the R2 substituent
a
(S)-methylpyrrolidine. The resulting
compounds maintained binding affinities comparable to those of
butyl derivatives 15 and 16, reaching subnanomolar affinities for
human H3 receptor with compounds 20 and 21. Compound 20, with
a pKB ¼ 10.02 was the most potent H3-antagonist in the functional
assay on human receptors. These results confirmed the remarkable
steric tolerance of human and rat H3 receptors for lipophilic chains
stemming out from the rigid biphenyl scaffold, in an almost-
perpendicular direction with respect to the two basic groups.
Finally, compounds 34 and 35, lacking one of the cyclic amine
fragment either close to the lipophilic tail or at the opposite site,
were syntehsized to assess the role of the two basic groups. Both
these compounds showed remarkable drops in binding affinity
(pKi < 5), suggesting that the two basic groups are both important
for binding interactions within this series.
The compounds under study were selective for H3 receptor,
showing no activity on other histamine receptors, up to micromolar
concentration. In detail, compounds 7e24 did not display antago-
nism towards guinea-pig H1 receptors at concentrations lower than
100-fold those required to inhibit H3 receptors. On guinea-pig
histamine H2 receptors, compound 14 behaved as
competitive antagonist (pKB ¼ 5.13), while for other compounds no
significant activity was detected at concentration of 1 M. All the
a weak
Keeping fixed R1 piperidine and R3 butyl substituents, 2-
methylpyrrolidine with (R) or (S) chirality was evaluated as R2
substituent (13e16). The diastereomeric mixtures obtained during
the synthetic procedure were separated and each stereoisomer was
tested separately. Consistently with what observed for compounds
7 and 8, (S)-methylpyrrolidine provided compounds with slightly
higher binding affinity compared to the (R)-isomers. Replacement
m
substances showed no or negligible affinity for human H4 receptor
(pKi ꢀ 4.5). Compound 21, characterized by the highest hH3 binding
affinity, was also tested on bungarotoxin-sensitive nicotinic
receptors. It showed no affinity for human
and only 27% displacement of radiolabelled bungarotoxin from
human muscle-type nicotinic receptors at 1 M concentration.
a7 nicotinic receptors
m
Fig. 6. Representative conformations of the (R,R) isomer with s1 z 60ꢁ (left) or s1 z 180ꢁ (right) and s2 z 180ꢁ.

218
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
Table 1
Binding affinity at human and rat H3 receptor (pKi) and antagonist potency at human H3 receptor (pKB) for newly synthesized compounds.
R1
R2
R3
Stereochemical assignmenta
hH3 pKib
rH3 pKic
hH3 pKB
d
6
7
eH
9.4726
8.9226
8.7741
eH
eH
(R)
(S)
8.83 ꢂ 0.10
9.18 ꢂ 0.02
8.45 ꢂ 0.04
9.43 ꢂ 0.12
9.16 ꢂ 0.17
9.46 ꢂ 0.39
8
9
eCH2Ph
eCH2Ph
eCH2Ph
(ꢂ)
7.72 ꢂ 0.08
7.78 ꢂ 0.01
8.25 ꢂ 0.12
6.95 ꢂ 0.06
7.52 ꢂ 0.10
7.53 ꢂ 0.16
8.30 ꢂ 0.12
7.56 ꢂ 0.11
8.87 ꢂ 0.18
10
11
eN(CH3)2
(ꢂ)
(S,R)/(R,R)
12
13
e(CH2)3CH3
e(CH2)3CH3
(ꢂ)
8.89 ꢂ 0.02
8.63 ꢂ 0.06
8.37 ꢂ 0.05
7.79 ꢂ 0.03
8.81 ꢂ 0.16
8.58 ꢂ 0.21
(S,R)
14
15
16
17
18
19
20
21
22
23
e(CH2)3CH3
(R,R)
(R,S)
(S,S)
(R,S)
(S,S)
(R,S)
(S,S)
(R,S)
(S,S)
(R,S)
(S,S)
8.61 ꢂ 0.07
8.81 ꢂ 0.02
8.90 ꢂ 0.17
8.52 ꢂ 0.04
8.61 ꢂ 0.13
8.70 ꢂ 0.09
9.06 ꢂ 0.14
9.29 ꢂ 0.10
8.59 ꢂ 0.10
8.90 ꢂ 0.12
7.90 ꢂ 0.15
8.06 ꢂ 0.12
8.85 ꢂ 0.08
7.58 ꢂ 0.01
7.58 ꢂ 0.13
8.83 ꢂ 0.01
8.75 ꢂ 0.10
8.18 ꢂ 0.10
8.33 ꢂ 0.09
8.18 ꢂ 0.01
8.80 ꢂ 0.30
8.81 ꢂ 0.32
8.37 ꢂ 0.43
8.91 ꢂ 0.33
8.94 ꢂ 0.30
9.64 ꢂ 0.25
10.02 ꢂ 0.09
8.48 ꢂ 0.18
9.15 ꢂ 0.18
8.71 ꢂ 0.24
e(CH2)3CH3
e(CH2)3CH3
e(CH2)3CH3
e(CH2)3CH3
e(CH2)3OCH3
e(CH2)3OCH3
e(CH2)3OPh
e(CH2)3OPh
e(CH2)3O(4’OCH3Ph)
24
34
e(CH2)3O(4’OCH3Ph)
e(CH2)3CH3
8.69 ꢂ 0.10
4.89 ꢂ 0.09
8.68 ꢂ 0.10
4.22 ꢂ 0.05
8.71 ꢂ 0.25
eH
N.D.

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
219
Table 1 (continued )
R1
R2
R3
Stereochemical assignmenta
hH3 pKib
rH3 pKic
hH3 pKB
d
35
eH
e(CH2)3CH3
(ꢂ)
4.87 ꢂ 0.06
4.36 ꢂ 0.05
N.D.
a
For compounds 13e24 stereochemical assignment was based on NMR analysis. Absolute configuration is reported in the order (benzylic carbon, R2).
Inhibition of [3H]RAMHA binding to SK-N-MC cells stably expressing the human histamine H3 receptor.
Inhibition of [3H]RAMHA binding to rat brain membranes.
b
c
d
Antagonist potency at human histamine H3 receptors expressed in SK-N-MC cells. N.D. Not determined.
Starting from previous modeling work, which had proposed the
loops. The 4-methoxyphenoxypropyl chains of 23 and 24 extend
into the “vertical” pocket lined by TM3, TM6 and TM7, confirming
that the two pockets outlined in the receptor binding site can be
simultaneously occupied by the antagonists. The lack of stereo-
selectivity at the benzylic carbon observed throughout the series is
consistent with the similar docking poses for the two stereoiso-
mers represented in Fig. 7. Although this pose showed the highest
docking score, alternative orientations for these compounds
cannot be ruled out, and other docking solutions were obtained. In
one pose the pyrrolidine nitrogen is bound to Asp114 and the
piperidine ring is inserted into the “vertical” pocket, interacting
with polar amino acids, such as Asp80 in TM2, Ser121 in TM3 and
Ser405 in TM7. This pose is consistent with the “vertical” accom-
modation, previously found for the symmetrical derivative 6. The
two basic groups can also exchange their residue counterpart,
with the pyrrolidine nitrogen interacting with Glu206 and the
piperidine one with Asp114; in this case the lipophilic tail points
toward the extracellular portion of the receptor. No correlation
was found between hH3 binding affinities of compounds 6e24 and
the docking scores obtained from an induced-fit docking (see
Supplementary Material).
existence of two mutually perpendicular pockets into the H3
receptor [26], we tested the possibility that those two pockets could
be simultaneously fitted by our compounds. We thus built a new
model of the human H3 receptor by homology modeling, based on
the three-dimensional structure of human H1 receptor co-
crystallized with the antagonist doxepin [44].
H1 and H3 receptors are characterized by 43% homology and 21%
identity, considering their whole sequences. Although homology-
based models should be regarded as heuristic, previously devel-
oped models of H3 receptor showed acceptable performance in
discriminating active from inactive compounds [32] and repro-
ducing antagonist SARs [45], even if they had been built from less
similar templates. Transmembrane (TM) sequences of H1 and H3
receptors show 68% homology and 36% identity of amino acids,
sharing some key amino acids involved in ligand recognition. In
TM3, the conserved aspartic acid (Asp107) of the H1 crystal struc-
ture interacts with the basic nitrogen of doxepin. Asn198 in TM5 of
the H1 receptor is thought to bind the imidazole ring of the natural
ligand histamine [46], while it does not interact with doxepin in the
crystal structure. The corresponding amino acid in the H3 receptor
is Glu206, important for histamine and (R)-a-methylhistamine
binding, as highlighted by mutagenesis studies [47]. Some other
amino acids lining the doxepin binding site in the H1 receptor are
conserved in the H3 receptor, such as Tyr108 in TM3 close to the
conserved aspartic acid, Trp428 belonging to the CWXP motif in
TM6, Thr112, Phe199, Phe424, and Tyr431.
Human and rat H3 receptors are characterized by high
homology in their amino acid sequences (92% identity) and,
although some molecular models had been invoked to explain
different binding affinities for some H3-antagonists [30], we did
not try to rationalize the differences observed in this work given
the similarity between the SARs on the receptors from the two
species. Compounds 23 and 24, with the bulkiest lipophilic chain
in the series, were docked within the receptor binding site. In the
best docking solutions the piperidine nitrogen of both compounds
undertakes a hydrogen-bond with Glu206 in TM5 and the pyrro-
lidine interacts with Asp114 in TM3 (Fig. 7), similarly to what had
been reported for compound 6 in its “horizontal” pose. The
4.2. Diastereomeric characterization by NMR studies and molecular
dynamics
Several new compounds were synthesized as mixtures of two
diastereoisomers (13e14, 15e16, 17e18, 19e20, 21e22, and 23e24)
and then separated by silica gel chromatography. As only the
chirality of one stereocenter was known, assignment of their
absolute configuration was achieved combining high resolution
(600 MHz) 1D and 2D NMR data and computational studies. To
define a rule for stereochemical assignment, we focused on the
intermediates 29c,d (Fig. 3), as these compounds lack the piperi-
dine ring, which gave NMR signals highly overlapping those of the
pyrrolidine one. Compound 29c was the isomer that eluted first,
and 29d the latter one (see Experimental Section). Complete and
unambiguous assignment of all resonances was achieved by the
combined analysis of 1D 1H and 13C, 2D COSY, HSQC and NOESY
spectra (see Experimental Section and Supporting Information,
Figures S2eS5). Relevant features for stereochemical assignment
are here summarized.
aromatic ring of the benzylpiperidine portion forms a
p-stacking
interaction with Tyr115 in TM3 (not shown), similarly to what had
been described for another series of dibasic H3-antagonists [31].
No interactions are seen with amino acids of the extracellular
The 1H NMR spectrum of compound 29c showed a more shiel-
ded signal for proton H2a (Fig. 3), compared to the same proton of
Scheme 1. Reagents and conditions: (a) piperidine, Et3N, CH3CN, MW 150 ꢁC, 150 W, 120 psi, 5 min, 48%; (b) (R)- or (S)-2-methylpyrrolidine, CH3CN, MW 150 ꢁC, 150 W, 120 psi,
5 min, 78e87%.

220
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
Scheme 2. Reagents and conditions: (a) i. piperidine (to 28a) or dimethylamine (to 28b), anhydrous CH3CN, r.t.; ii. benzyl zinc bromide, anhydrous CH3CN, anhydrous THF, ꢃ5 ꢁC to
r.t., 73e87%; (b) butyl magnesium bromide, anhydrous Et2O, ꢃ5 ꢁC to r.t., 93%; (c) PCC, CH2Cl2, molecular sieve 4 Å, 93% (to 32a); (d) i. methoxypropyl magnesium chloride, I2,
anhydrous Et2O, 16 h; ii. 6 M HCl, 68% (to 32b); (e) allyloxybenzene derivate, 2-ammino-3-picoline, benzoic acid, aniline, [Rh(PPh3)3Cl], anhydrous toluene, 130 ꢁC, 1 h, 43e66%, (to
32ced); (f) i. (R)- or (S)-2-methylpyrrolidine (to 28cef,ien), (S)-2-methylpiperidine (to 28geh) or piperidine (to 28p), Ti(Oi-Pr)4, anhydrous THF, reflux, 16 h; ii. NaBH(OAc)3,
CH3OH, ꢃ48 ꢁC to r.t., 6 h, 32e85%; (g) 4-hydroxymethylphenylboronic acid (to 29aeo and 31) or p-tolylboronic acid (to 35), Cs2CO3, Pd(Ac)2, Acetone, CH3OH, H2O, 65 ꢁC, 1 h or
MW 100 ꢁC, 50 W, 120 psi, 10 min, 61e92%; (h) i. MsCl, Et3N, CH2Cl2, 0 ꢁC to r.t.; ii. piperidine (to 9e10, 12e24 and 34)or (R)-2-methylpyrrolidine (to 11), Et3N, CH3CN, MW 100 ꢁC,
80 W, 120 psi, 5 min, 55e89%.
29d (dH2a ¼ 2.37 and 2.97 ppm, respectively). On the other hand, 1H
NMR of 29c showed less shielded signals for protons H6 and CH3
(29c: dH6 ¼ 3.7 and dCH3 ¼ 1.1 ppm; 29d: dH6 ¼ 3.4 and
dCH3 ¼ 0.82 ppm).
and H5b. Pyrrolidine protons H2a and H5b showed broad signals at
2.97 and 2.76 ppm, respectively.
Molecular dynamics (MD) simulations were performed on the
(S,R) and (R,R) stereoisomers of {40-[1-(2-methylpyrrolidin-1-yl)
pentyl]biphenyl-4-yl}methanol (Fig. 3), simulating the solvent
effect of chloroform by a GB/SA model. 10,000 Snapshots were
In the 1H NMR spectrum of 29c butylic H7 protons were
3
overlapped (dH7a,H7b ¼ 1.79e1.89 ppm), with JH6H7 ¼ 9.6 and
6.0 Hz. Peaks corresponding to H2a and H5b were sharp, with
collected during 1 ms of simulation. The most relevant differences in
3
3
coupling constants JH2aH3a
and 3.0 Hz.
¼
3JH2aH3b ¼ 6.6 Hz, and JH5bH4 ¼ 8.4
conformational equilibria among the two stereoisomers regarded
butyl chain and pyrrolidine rotation. For the (S,R) stereoisomer, the
pyrrolidine adopted one preferred orientation (s1 z 300ꢁ in
Fig. 4A), with the methyl substituent close to the benzylic hydrogen
H6 (Fig. 5). For the (R,R) stereoisomer, the pyrrolidine exchanged
In the 1H NMR spectrum of compound 29d, butylic protons H7a
and H7b gave well-defined separated signals (dH7a ¼ 1.82 and
dH7b ¼ 1.74 ppm), with coupling constants toward H6 corre-
sponding to
a
gauche (3JH6H7a
¼
3.6 Hz) and an anti
between two equally populated orientations (s
1 z 60ꢁ and 180ꢁ in
(3JH6H7b ¼ 10.8 Hz) conformation, respectively. Only proton H7b
showed strong NOE signal with aromatic H12 protons, while H7a
showed cross peaks to pyrrolidine protons H2a, H3a, H3b, H4a, H5a
Fig. 4C). In the first (s
1 z 60ꢁ) the methyl substituent points toward
the biphenyl scaffold (Fig. 6, left), while in the second (s1 z 180ꢁ)
the methyl group is close to the benzylic hydrogen (Fig. 6, right).

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
221
H2a (more shielded in 29c) and CH3 (more shielded in 29d)
protons. In fact, for the (S,R) isomer the majority of conformations
had H2a above the plane of the phenyl ring (Fig. 5) while, in the
case of the (R,R) isomer, one of the two equally preferred confor-
mation had the CH3 above the plane of the phenyl ring (Fig. 6).
For all couples of diastereoisomers 13e24, the isomer eluted
first from the chromatographic column (indicated as isomer A in
the Experimental Section) showed NMR features resembling those
of 29c (e.g. more shielded signals for proton H2a, closer coupling
constants between H6 and H7a and H7b protons), while the second
eluted (isomer B) showed features of the 29d spectrum (e.g. more
shielded signals for CH3 protons and H6, significantly different
coupling constants between H6 and H7a and H7b protons).
Configuration at the benzylic carbon was therefore assigned on the
basis of their NMR spectra, assuming that isomer A has the two
stereocenters with opposite absolute configuration. This resulted in
the assignments reported in Table 1.
5. Conclusions
Fig. 7. Superposition of the energy-minimized H3 receptor model-23 (green carbons)
and H3-24 (yellowcarbons) complexes. Asp114 and Glu206 are depicted with gray sticks.
TM6 and TM7 have been removed for clarity.
Starting from the structure of the lead compound 6, we devised
new H3-antagonists characterized by two basic centers and a lipo-
philic side chain at the benzylic carbon linking the biphenyl scaffold
to a chiral basic group. The high binding affinity and antagonist
behavior shown by these compounds, including those with a bulky
lipophilic chain, support the hypothesis that two previously
described regions within the H3 receptor binding site are simulta-
neously available for ligand binding. Stereoselectivity for diaste-
reomeric couples with opposite chirality at the benzylic carbon was
very limited or absent, probably due to the flexibility of the lipo-
philic substituent.
The butyl chain assumed two preferred orientations for the (S,R)
isomer (
in anti or gauche conformation (Fig. 5). On the contrary, for the (R,R)
isomer the butyl chain orientation was almost fixed with
2 z 180ꢁ
s
2 z 180ꢁ and 300ꢁ in Fig. 4B), with the first rotatable bond
s
(Figs. 4D and 6), with only a limited number of snapshots (w9%)
having s2 z 60ꢁ.
NMR data for 29c are in agreement with the conformational
behavior of the (S,R) isomer. The preferred orientation of the pyr-
rolidine ring is supported by NOE signals involving the ortho-
aromatic protons (H12): significant signals were observed with
H2a, H5a, H5b and CH3 protons, but not with H3a, H3b, H4a and
H4b (Supporting Information, Figure S3). Consistently, average
distances between H12 and H5a and H5b are 2.81 Å and 3.26 Å,
respectively; average minimum distance between H12 and CH3
protons is 3.07 Å. The two favored orientations of the butyl chain
are consistent with JH7a-H6 and JH7b-H6 values, deriving from
a combination of anti and gauche arrangements (Table 2).
NMR data for 29d are in agreement with MD simulation results
for the (R,R) isomer: the presence of one preferred conformation of
the butyl chain is consistent with the JH7a-H6 gauche and JH7b-H6 anti
values, as well as with the strong NOE signal between H12 and H7b,
and with the lack of any NOE signal between H12 and H7a. Rotation
of the pyrrolidine ring is also consistent with the high number of
weak NOE signals from both the aromatic ortho protons (H12) and
the butylic H7 protons to the pyrrolidine ones (Supporting Infor-
mation, Figure S5). Moreover, other 3J values calculated for the (S,R)
and (R,R) isomers were in good agreement with those observed for
29c and 29d, respectively (Table 2). Finally the assignment (S,R)-
6. Experimental section
6.1. General chemistry
Microwave reactions (MW) were conducted with
a CEM
Discover Synthesis Unit. Reactions were monitored by TLC, on
Kieselgel 60 F 254 (DC-Alufolien, Merck). Final compounds and
intermediates were purified by flash chromatography, using either
SiO2 column (Prepacked Cartridges, SiO2 60, 40e63 mm, BÜCHI) or
prepacked silica gel cartridges (Biotage). Melting points were
determined with a Gallenkamp melting point apparatus and are
uncorrected. Where melting points are not indicated compounds
were obtained as oils. NMR spectra were recorded on Bruker 300
Avance (300 MHz), Bruker 400 Avance (400 MHz) and Varian Inova
600 (600 MHz) spectrometers; chemical shifts (d scale) are reported
in parts per million (ppm) relative to the central peak of the solvent.
1H NMR spectra are reported in the following order: multiplicity,
approximate coupling constant (J value) in hertz (Hz), and number
of protons; signals were characterized as s (singlet), d (doublet), dd
(doublet of doublets), ddd (doublet of doublets of doublets), t
(triplet), td (triplet of doublets), q (quartet), quint (quintet), sest
(sestet), m (multiplet) br s (broad signal). The final compounds were
analyzed on a ThermoQuest (Italia) FlahEA 1112 Elemental Analyzer,
for C, H and N. The percentages we found were within ꢂ0.4% of the
theoretical values. Mass spectra were recorded using an API-150 EX
with APCI interface (Applied Biosystem, Mds Sciex, Foster City, CA).
When indicated, gaseous NH3 was added to the methanolic phase to
obtain a 5% w/w solution. Abbreviations are the following: EtOAc,
ethyl acetate; THF, tetrahydrofuran; MsCl, mesyl chloride. Couples
of diastereoisomers obtained as mixtures were separated by Flash
Chromatography: we indicated as isomer A (ds-A) the one that
eluted first, and as isomer B (ds-B) the latter one.
29c and (R,R)-29d is also consistent with the different
d values of
Table 2
1He1H vicinal coupling constants (3J, Hz) observed by NMR spectroscopy (600 MHz)
and calculated for (S,R) and (R,R) isomers 29c,d.
(S,R)-isomer
(R,R)-isomer
Coupling constant
Measured
Calculated
Measured
Calculated
H2eH3a
H2eH3b
6.6
6.6
3.0
8.4
6.0
9.6
7.1
7.5
3.2
8.3
6.8
7.3
e
e
e
e
H
H
H
H
5beH4a
5beH4b
7aeH6
7beH6
e
e
e
e
3.6
10.8
4.6
10.0

222
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
6.1.1. Preparation of 1-{[40-(chloromethyl)biphenyl-4-yl]methyl}
piperidine (26)
added carefully to the vigorously stirred solution. The ether solu-
tion was taken away and the ammonium salt was washed whit
diethyl ether (3 ꢄ 15 mL). The residue was dissolved in 5% NaOH
(35 mL) and the aqueous phase was extracted with CH2Cl2
(3 ꢄ 50 mL). The combined organic fractions were dried over
sodium sulfate and concentrated under vacuum to give the crude
products, that were purified by flash chromatography (SiO2,
CH2Cl2/CH3OH (NH3) 50:1).
A solution of 4,40-bis(chloromethyl)biphenyl 25 (1.0 mmol) in
CH3CN (3.5 mL) was reacted with piperidine (0.5 mmol) and Et3N
(0.5 mmol) (MW, 150 ꢁC, 150 W, 120 psi, 5 min). The solvent was
evaporated under reduced pressure and the residue was dissolved
in CH2Cl2 (50 mL). The organic phase was washed with brine and
dried over sodium sulfate. Removal of the solvent under reduced
pressure gave the crude product, which was purified by flash
chromatography (SiO2, CH2Cl2/CH3OH(NH3) 70:1). Yield: 48%; mp:
6.1.3.1. (ꢂ) 1-[1-(4-Iodophenyl)-2-phenylethyl]piperidine (28a). Yield:
174e176 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
1.52e1.40 (m, 2H),
87%; mp: 78e81 ꢁC. 1H NMR (CDCl3, 300 MHz)
d 1.41e1.36 (m, 2H),
1.64e1.57 (m, 4H), 2.43 (m, 4H), 3.56 (s, 2H), 4.63 (s, 2H), 7.41 (d,
J ¼ 8.2 Hz, 2H), 7.44 (d, J ¼ 8.2 Hz, 2H), 7.53 (d, J ¼ 8.2 Hz, 2H), 7.58
(d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 300.4 [Mþ1]þ.
1.58 (m, 4H), 2.43 (m, 4H), 3.94 (dd, J ¼ 13.3, 9.9 Hz, 1H), 3.32 (dd,
J ¼ 13.2, 4.6 Hz, 1H), 3.56 (dd, J ¼ 9.6, 4.6 Hz, 1H), 6.88 (d, J ¼ 8.3 Hz,
2H), 6.96 (d, J ¼ 7.8 Hz, 2H), 7.17e7.06 (m, 3H), 7.55 (d, J ¼ 8.2 Hz, 2H).
MS-(APCI): m/z 392.1 [Mþ1]þ.
6.1.2. Preparation of (R)- and (S)-1-{[40-((2-methylpyrrolidin-1-yl)
methyl)biphenyl-4-yl]methyl}piperidine (7e8)
6.1.3.2. (ꢂ) 1-(4-Iodophenyl)-N,N-dimethyl-2-phenylethanamine
A solution of 26 (0.33 mmol) in CH3CN (3.5 mL), was reacted
with (R)- or (S)-2-methylpyrrolidine (1.00 mmol) (MW, 150 ꢁC,
150 W, 120 psi, 5 min). CH2Cl2 (50 mL) was added to the reaction
mixture and the organic phase was extracted with 1 M HCl aqueous
solution (3 ꢄ 50 mL). The combined aqueous phases were made
alkaline with K2CO3 and were extracted with EtOAc (3 ꢄ 50 mL).
The organic layers were dried over sodium sulfate and the solvent
was evaporated under reduced pressure to afford the crude prod-
ucts which were purified by flash chromatography (SiO2, CH2Cl2/
CH3OH (NH3) 100:1 to 80:1).
(28b). Yield: 73%. 1H NMR (CDCl3, 300 MHz)
d 2.23 (s, 6H), 2.84
(dd, J ¼ 13.1, 9.8 Hz, 1H), 3.27 (dd, J ¼ 13.1, 4.8 Hz, 1H), 3.36 (dd,
J ¼ 9.7, 4.8 Hz, 1H), 6.84 (d, J ¼ 8.3 Hz, 2H), 6.90 (d, J ¼ 7.9 Hz, 2H),
7.15e7.05 (m, 3H), 7.25 (d, J ¼ 8.31 Hz, 2H). MS-(APCI): m/z 352.4
[Mþ1]þ.
6.1.4. Preparation of (ꢂ) 1-(4-iodophenyl)pentan-1-ol (30)
A solution of butyl bromide (10.5 mmol) in dry Et2O (10 mL) was
added dropwise to
a suspension of magnesium turnings
(10.5 mmol) in dry Et2O (2 mL) keeping reflux under argon. The
mixture was refluxed for 30 min. The solution of butyl magnesium
bromide thus obtained was cooled to ꢃ5 ꢁC and a solution of 4-
iodobenzaldehyde 27 (7.0 mmol) in dry Et2O (13 mL) was added.
The mixture was stirred for 1 h at room temperature, then was
poured onto ice and acidified with 6 M HCl. The organic layer was
separated and the aqueous phase was extracted with Et2O
(3 ꢄ 50 mL). The organic solutions were combined, washed with
10% NaHCO3 and brine, then dried over sodium sulfate and evap-
orated. The crude product was purified by flash chromatography
(SiO2, CH2Cl2/Hexane 1:1). Yield: 97%; mp: 38e40 ꢁC. 1H NMR
6.1.2.1. (R)-1-{[40-((2-methylpyrrolidin-1-yl)methyl)biphenyl-4-yl]
methyl}piperidine (7$2HCl·0.25H2O). Yield: 87%; crystallized from abs
EtOH/Et2O; mp: >300 ꢁC 1H NMR (DMSO-d6, 300 MHz)
d 1.28 (d,
J ¼ 6.5 Hz, 3H), 1.85e1.55 (m, 8H), 2.18e2.03 (m, 1H), 2.82e2.65 (m,
2H), 3.00 (sest, J ¼ 8.0 Hz, 1H), 3.22e3.09 (m, 4H), 3.34 (quint,
J ¼ 7.3 Hz, 1H), 4.06 (dd, J ¼ 12.7, 7.2 Hz, 1H), 4.17 (d, J ¼ 5.1 Hz, 2H),
4.52,(dd, J ¼ 13.0, 4.0 Hz, 1H), 7.69e7.60 (m, 8H), 10.76 (s, 1H). MS-
(APCI): m/z 349.6 [Mþ1]þ. Anal. Calcd for C24H32N2·2HCl·0.25H2O: C,
67.67; H, 8.16; N, 6.58. Found: C, 67.58; H, 8.12; N, 6.50.
(CDCl3, 300 MHz)
d
0.89 (t, J ¼ 6.9 Hz, 3H), 1.36e1.20 (m, 4H),
6.1.2.2. (S)-1-{[40-((2-methylpyrrolidin-1-yl)methyl)biphenyl-4-yl]
1.79e1.62 (m, 2H), 2.03 (s, 1H), 4.61 (t, J ¼ 6.5 Hz, 1H), 7.09 (d,
methyl}piperidine (8·2HCl·0.2H2O). Yield: 78%; crystallized from abs
J ¼ 8.2 Hz, 2H), 7.67 (d, J ¼ 8.3 Hz, 2H).
EtOH/Et2O; mp: >300 ꢁC 1H NMR (DMSO-d6, 300 MHz)
d 1.39 (d,
J ¼ 6.5 Hz, 3H), 1.95e1.65 (m, 8H), 2.28e2.13 (m, 1H), 2.90e2.78 (m,
2H), 3.11 (sest, J ¼ 8.6 Hz, 1H), 3.32e3.20 (m, 4H), 3.45 (quint,
J ¼ 7.5 Hz, 1H), 4.17 (dd, J ¼ 12.9, 7.1 Hz, 1H), 4.28 (d, J ¼ 5.3 Hz, 2H),
4.53 (dd, J ¼ 13.1, 3.7 Hz, 1H), 7.80e7.72 (m, 8H), 10.97 (s, 1H). MS-
(APCI): m/z 349.5 [Mþ1]þ. Anal. Calcd for C24H32N2·2HCl·0.2H2O: C,
67.82; H, 8.16; N, 6.59. Found: C, 67.79; H, 7.93; N, 6.50.
6.1.5. Preparation of 1-(4-iodophenyl)pentan-1-one (32a)
To a solution of 30 (2.2 mmol) in CH2Cl2 (25 mL) were added
molecular sieve 4 Å (5 g) and pyridinium chlorochromate (PCC)
(4.3 mmol) and the resulting mixture was stirred at room
temperature for 1 h. The suspension was then filtered over celite
and the solvent was evaporated under reduced pressure. The crude
product was purified by flash chromatography (SiO2, CH2Cl2). Yield:
6.1.3. General synthesis of 1-(4-iodophenyl)-2-phenylethylamine
(28aeb)
93%; mp: 62e64 ꢁC. 1H NMR (CDCl3, 300 MHz)
3H), 1.41 (sest, J ¼ 7.6 Hz, 2H), 1.72 (quint, J ¼ 7.3 Hz, 2H), 2.93
(t, J ¼ 7.3 Hz, 2H), 7.68 (dd, J ¼ 8.6, 2.0 Hz, 2H), 7.84 (dd, J ¼ 8.6,
1.8 Hz, 2H).
d
0.90 (t, J ¼ 7.3 Hz,
To a dried 100 mL flask kept under an atmosphere of argon
containing zinc dust (36.8 mmol) and zinc bromide (1.2 mmol) in
dry CH3CN (16 mL), phenyl bromide (1.2 mmol) and tri-
fluoromethansulfonic acid (0.1 mL) were added under vigorous
stirring. After 15 min, benzyl bromide (12.0 mmol) was added and
the reaction mixture was stirred at room temperature for 40 min.
Then the suspension was filtered over celite under nitrogen and the
solution was added dropwise to a mixture of 4-iodobenzaldehyde
27 (4.0 mmol) and the appropriate amine (4.0 mmol) in dry
CH3CN (8 mL). The mixture was stirred for additional 3 h at room
temperature. After completion of the reaction, 5% NaOH (50 mL)
was added and the mixture was extracted with CH2Cl2 (3 ꢄ 50 mL).
The combined organic fractions were dried over sodium sulfate and
the solvent was evaporated under reduced pressure. Et2O (50 mL)
was added to the residue and concentrated H2SO4 (0.25 mL) was
6.1.6. Preparation of 1-(4-iodophenyl)-4-methoxybutan-1-one
(32b)
To a flame-dried flask kept under argon, containing magnesium
turnings (2.66 mmol) and an iodine crystal, were added with stir-
ring dry Et2O (1 mL) and a solution of 1-chloro-3-methoxypropane
(2.62 mmol) in dry Et2O (2 mL), the latter at a rate to maintain
gentle refluxing. After the addition was completed, the mixture was
refluxed for 1 h then cooled to room temperature. A solution of 4-
iodobenzonitrile 33 (2.00 mmol) in dry Et2O (5 mL) was slowly
added and the resulting yellow mixture was refluxed overnight. A
mixture of 6 N H2SO4 and ice was then added and the biphasic
mixture was refluxed for 2 h. The aqueous phase of the cooled

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
223
reaction mixture was separated and extracted with Et2O
(3 ꢄ 10 mL). The organic phases were combined and dried over
sodium sulfate. After evaporation of the solvent, the crude product
was purified by flash chromatography (SiO2, n-hexane/EtOAc 40:1).
6.1.8.2. (2S)-1-[1-(4-iodophenyl)pentyl]-2-methylpyrrolidine (28eef),
mixture of diastereoisomers. Starting from 1-(4-iodophenyl)pentan-
1-one (32a) and (S)-2-methylpyrrolidine, and after FC purification
(CH2Cl2/CH3OH(NH3) 75:1), compounds 28e and 28f were obtained
Yield: 68%; mp: 63e66 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
2.00 (quint,
as distereomeric mixture observed by NMR. Reaction yield: 77%,
1
J ¼ 6.0 Hz, 2H), 3.02 (t, J ¼ 7.1 Hz, 2H), 3.32 (s, 3H), 3.45 (t, J ¼ 6.0 Hz,
28e/28f 65:35. 28e: H NMR (CDCl3, 300 MHz)
d
0.80 (t, J ¼ 7.7 Hz,
2H), 7.68 (d, J ¼ 8.7 Hz, 2H), 7.82 (d, J ¼ 8.2 Hz, 2H).
3H), 1.08 (d, J ¼ 6.0 Hz, 3H), 1.42e0.95 (m, 4H), 1.58e1.45 (m, 1H),
1.95e1.60 (m, 5H), 2.50e2.28 (m, 2H), 2.84 (t, J ¼ 7.8 Hz, 1H), 3.64
(dd, J ¼ 9.2, 5.8 Hz,1H), 6.95 (d, J ¼ 7.7 Hz, 2H), 7.63 (d, J ¼ 7.4 Hz, 2H).
6.1.7. General synthesis of 1-(4-iodophenyl)-4-phenoxybutan-1-
one derivates (32ced)
A mixture of 27 (2.00 mmol), 2-amino-3-picoline (0.40 mmol),
benzoic acid (0.12 mmol), aniline (0.30 mmol), allyloxybenzene or
28f: 1H NMR (CDCl3, 300 MHz)
d
0.80 (t, J ¼ 7.7 Hz, 3H), 0.84 (d,
J ¼ 7.7 Hz, 3H), 1.42e0.95 (m, 4H), 1.58e1.45 (m, 1H), 1.95e1.60 (m,
5H), 2.50e2.28 (m, 1H), 2.80 (m, 1H), 3.00 (m, 1H), 3.36 (dd, J ¼ 10.5,
3.6 Hz,1H), 7.08 (d, J ¼ 7.7 Hz, 2H), 7.62 (d, J ¼ 7.6 Hz, 2H). MS-(APCI):
m/z 392.1 [Mþ1]þ.
1-allyloxy-4-methoxybenzene (10.0 mmol) and toluene (370 mL)
was degassed bubbling argon for 10 min, prior to the addition of
Rh(PPh3)3Cl (0.04 mmol). The mixture was stirred at 130 ꢁC for
1 h, then it was cooled, diluted with EtOAc (50 mL) and filtered
over celite. The solvent was evaporated and the crude products
were purified by flash chromatography (SiO2, n-hexane/EtOAc
30:1).
6.1.8.3. (2S)-1-[1-(4-iodophenyl)pentyl]-2-methylpiperidine (28g),
isomer A. Reaction of 32a and (S)-2-methylpiperidine, followed by
FC purification (CH2Cl2/CH3OH(NH3) 80:1) gave 28g as a single
diastereoisomer (ds-A). Reaction yield 32%, ds-A/ds-B 65:35. 1H
NMR (CDCl3, 300 MHz)
d
0.75 (t, J ¼ 7.3 Hz, 3H), 1.05e0.90 (m, 2H),
6.1.7.1. 1-(4-Iodophenyl)-4-phenoxybutan-1-one (32c). Yield: 66%;
0.99 (d, J ¼ 6.4 Hz, 3H), 1.35e1.10 (m, 6H), 1.72e1.40 (m, 4H), 2.07
(ddd, J ¼ 7.9, 6.9, 3.6 Hz, 1H), 2.24 (ddd, J ¼ 7.8, 7.7, 4.0 Hz, 1H),
2.95e2.80 (m, 1H), 3.55 (dd, J ¼ 10.2, 3.2 Hz, 1H), 6.99 (d, J ¼ 8.1 Hz,
2H), 7.51 (d, J ¼ 8.3 Hz, 2H). MS-(APCI): m/z 372.2 [Mþ1]þ.
mp: 74e77 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
2.22 (quint, J ¼ 6.0 Hz,
2H), 3.16 (t, J ¼ 7.1 Hz, 2H), 4.06 (t, J ¼ 6.0 Hz, 2H), 6.88 (d, J ¼ 7.7 Hz,
2H), 6.93 (dd, J ¼ 8.4, 7.4 Hz, 1H), 7.27 (dd, J ¼ 8.4, 7.5 Hz, 2H), 7.68
(d, J ¼ 8.6 Hz, 2H), 7.82 (d, J ¼ 8.3 Hz, 2H).
6.1.8.4. (2S)-1-[1-(4-Iodophenyl)pentyl]-2-methylpiperidine (28h),
isomer B. Reaction of 32a and (S)-2-methylpiperidine, followed
by FC purification (CH2Cl2/CH3OH(NH3) 80:1) gave 28h as
a single diastereoisomer. Reaction yield 32%, ds-A/ds-B 65:35. 1H
6.1.7.2. 1-(4-Iodophenyl)-4-(4-methoxyphenoxy)butan-1-one
(32d). Yield: 43%; mp: 136e139 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
2.10 (quint, J ¼ 6.2 Hz, 2H), 3.05 (t, J ¼ 7.1 Hz, 2H), 3.66 (s, 3H),
3.91 (t, J ¼ 6.0 Hz, 2H), 6.72 (s, 4H), 7.59 (d, J ¼ 8.5 Hz, 2H), 7.73 (d,
J ¼ 8.5 Hz, 2H).
NMR (CDCl3, 300 MHz)
d
0.74 (t, J ¼ 6.8 Hz, 3H), 1.05e0.92 (m,
2H), 1.04 (d, J ¼ 6.1 Hz, 3H), 1.25e1.10 (m, 4H), 1.52e1.30 (m, 4H),
1.71e1.58 (m, 2H), 1.80 (t, J ¼ 11.0, 10.4 Hz, 1H), 2.08e1.96 (m,
1H), 2.77 (d, J ¼ 11.2 Hz, 1H), 3.78 (dd, J ¼ 7.3, 6.5 Hz, 1H), 6.86 (d,
J ¼ 8.3 Hz, 2H), 7.52 (d, J ¼ 8.3 Hz, 2H). MS-(APCI): m/z 372.2
[Mþ1]þ.
6.1.8. General synthesis of (4-iodophenyl)methylen amines
(28cen,p)
A mixture of the proper ketone 32 (0.35 mmol) and the proper
secondary amine (0.42 mmol) in dry THF (3 mL) at room temper-
ature was treated with titanium tetra-isopropoxide (0.70 mmol)
under nitrogen and then stirred at 75 ꢁC for 16 h. The reaction was
cooled at ꢃ48 ꢁC and then treated with sodium triacetoxybor-
ohydride (0.70 mmol) followed by CH3OH (2 mL).The resulting
mixture was warmed to room temperature over 6 h. The reaction
mixture was diluted with THF (10 mL) and was neutralized with
10% NaOH. After filtration over celite, the solvent was evaporated
under reduced pressure. The residue was taken up in EtOAc
(50 mL), washed with brine, then the organic layer was dried over
sodium sulfate and concentrated under reduce pressure to give
crude products which were purified by flash chromatography
(SiO2). Compounds 28cen were obtained as couples of
diastereoisomers.
6.1.8.5. (2S)-1-[1-(4-Iodophenyl)-4-methoxybutyl]-2-methylpyr-
rolidine (28iej), mixture of diastereoisomers. Starting from
1-(4-iodophenyl)-4-methoxybutan-1-one 32b and (2S)-2-
methylpyrrolidine, compounds 28i and 28j were purified by FC
(CH2Cl2/CH3OH(NH3) 100:1) to obtain the distereomeric mixture
observed by NMR. Reaction yield: 64%, 28i/28j 65:35. 28i: 1H
NMR (CDCl3, 300 MHz)
d
0.97 (d, J ¼ 6.1 Hz, 3H), 1.35e1.10 (m,
2H), 1.48e1.35 (m, 1H), 1.72e1.50 (m, 3H), 1.90e1.72 (m, 2H), 2.30
(q, J ¼ 6.5 Hz, 1H), 2.36 (q, J ¼ 9.2 Hz, 1H), 2.72 (td, J ¼ 7.8, 3.33 Hz,
1H), 3.18 (s, 3H), 3.23 (t, J ¼ 6.5 Hz, 2H), 3.55 (dd, J ¼ 8.6, 6.3 Hz,
1H), 6.85 (d, J ¼ 8.2 Hz, 2H), 7.52 (d, J ¼ 8.2 Hz, 2H). 28j: 1H NMR
(CDCl3, 300 MHz)
d
0.73 (d, J ¼ 6.2 Hz, 3H), 1.35e1.10 (m, 2H),
1.48e1.35 (m, 1H), 1.72e1.50 (m, 3H), 1.90e1.72 (m, 2H), 2.20
(q, J ¼ 8.2 Hz, 1H), 2.66 (m, 1H), 2.89 (m, 1H), 3.10 (s, 3H), 3.18
(t, J ¼ 6.3 Hz, 2H), 3.28 (dd, J ¼ 10.5, 3.6 Hz, 1H), 6.96 (d, J ¼ 8.1 Hz,
2H), 7.51 (d, J ¼ 8.3 Hz, 2H). MS-(APCI): m/z 374.1 [Mþ1]þ.
6.1.8.1. (2R)-1-[1-(4-Iodophenyl)pentyl]-2-methylpyrrolidine (28ced),
mixture of diastereoisomers. Starting from 1-(4-iodophenyl)pentan-
1-one (32a) and (R)-2-methylpyrrolidine, compounds 28c and 28d
were obtained, after FC purification (CH2Cl2/CH3OH(NH3) 75:1), as
diastereomeric mixture observed by NMR. Reaction yield: 85%,
6.1.8.6. (2S)-1-[1-(4-Iodophenyl)-4-phenoxybutyl]-2-methylpyrrolidine
(28k), isomer A. The title compound was prepared from 1-(4-
iodophenyl)-4-phenoxybutan-1-one 32c and (S)-2-methylpyr-
rolidine. The diastereomeric mixture was separated and 28k iso-
lated as a single isomer by FC (CH2Cl2/CH3OH (NH3) 70:1). Reaction
28c/28d 65:35. 28c: 1H NMR (CDCl3, 300 MHz)
d
0.85 (t, J ¼ 8.8 Hz,
3H), 1.00 (m, 1H), 1.09 (d, J ¼ 6.1 Hz, 3H), 1.30 (m, 3H), 1.53 (m,1H),
1.75 (m, 3H), 1.88 (m, 2H), 2.33 (q, J ¼ 8.5 Hz, 1H), 2.40 (q, J ¼ 6.5 Hz,
1H), 2.85 (td, J ¼ 7.9, 3.3 Hz, 1H), 3.65 (dd, J ¼ 9.3, 5.6 Hz, 1H), 6.97
(d, J ¼ 8.2 Hz, 2H), 7.64 (d, J ¼ 10.5 Hz, 2H). MS-(APCI): m/z 392.1
yield 50%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 300 MHz)
d 1.10 (d,
J ¼ 6.2 Hz, 3H), 1.42e1.27 (m, 1H), 1.57e1.50 (m, 1H), 1.82e1.57 (m,
2H), 1.96e1.85 (m, 1H), 2.13e2.03 (m, 1H), 2.31 (q, J ¼ 8.2 Hz, 1H), 2.42
(sest, J ¼ 6.3 Hz, 1H), 2.86 (td, J ¼ 8.6, 8.0, 3.3 Hz, 1H), 3.74 (dd, J ¼ 8.3,
6.5 Hz, 1H), 3.94 (t, J ¼ 6.4 Hz, 2H), 6.86 (d, J ¼ 8.6 Hz, 2H), 6.93 (t,
J ¼ 7.7 Hz, 1H), 6.98 (d, J ¼ 8.3 Hz, 2H), 7.27 (dd, J ¼ 8.6, 7.2 Hz, 2H),
7.64 (d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 436.0 [Mþ1]þ.
[Mþ1]þ. 28d: 1H NMR (CDCl3, 300 MHz)
d
0.83 (t, J ¼ 7.4 Hz, 3H),
0.87 (d, J ¼ 5.9 Hz, 3H), 1.00 (m, 1H), 1.30 (m, 3H), 1.53 (m,1H),
1.75 (m, 3H), 1.88 (m, 2H), 2.47 (q, J ¼ 9.1 Hz, 1H), 2.80 (m, 1H),
3.00 (m, 1H), 3.37 (dd, J ¼ 10.5, 3.7 Hz, 1H), 7.09 (d, J ¼ 8.2 Hz, 2H),
7.63 (d, J ¼ 10.7 Hz, 2H). MS-(APCI): m/z 392.1 [Mþ1]þ.

224
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
6.1.8.7. (2S)-1-[1-(4-Iodophenyl)-4-phenoxybutyl]-2-methylpyrrolidine
(28l), isomer B. The title compound was prepared from 1-(4-
iodophenyl)-4-phenoxybutan-1-one 32c and (S)-2-methylpyrro-
lidine. The diastereomeric mixture was purified and 28l isolated as
a single isomer by FC (CH2Cl2/CH3OH (NH3) 70:1). Reaction yield 50%,
purification (CH2Cl2/CH3OH/CH3OH(NH3) 50:0.9:0.1) gave the
desired product. Yield: 84%. 1H NMR (CDCl3, 300 MHz)
1.43e1.35
d
(m, 2H), 1.68e1.56 (m, 4H), 2.32 (t, J ¼ 5.8 Hz, 1H), 2.52e2.48 (m,
4H), 3.06 (dd, J ¼ 13.3, 9.6 Hz, 1H), 3.36 (dd, J ¼ 13.3, 4.9 Hz, 1H),
3.67 (dd, J ¼ 9.5, 5.0 Hz, 1H), 4.73 (d, J ¼ 5.6, 2H), 7.03 (d, J ¼ 8.1 Hz,
2H), 7.18e7.06 (m, 3H), 7.21 (d, J ¼ 8.2 Hz, 2H), 7.43 (d, J ¼ 8.3 Hz,
2H), 7.48 (d, J ¼ 8.2 Hz, 2H), 7.58 (d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z
372.3 [Mþ1]þ.
ds-A/ds-B 65:35. 1H NMR (CDCl3, 300 MHz)
d
0.74 (t, J ¼ 6.3 Hz, 3H),
1.57e1.25 (m, 3H), 1.70e1.57 (m, 2H), 1.86e1.70 (m, 2H), 2.05e1.93
(m, 1H), 2.38 (q, J ¼ 7.8 Hz, 1H), 2.69 (ddd, J ¼ 8.8, 8.0, 4.2 Hz, 1H), 2.93
(m, 1H), 3.35 (dd, J ¼ 10.5, 3.8 Hz, 1H), 3.75 (t, J ¼ 6.4 Hz, 2H), 6.73 (d,
J ¼ 8.7 Hz, 2H), 6.81 (d, J ¼ 7.4 Hz, 1H), 7.00 (d, J ¼ 8.3 Hz, 2H), 7.16 (dd,
J ¼ 8.6, 8.5 Hz, 2H), 7.52 (d, J ¼ 8.3 Hz, 2H). MS-(APCI): m/z 436.2
[Mþ1]þ.
6.1.9.2. (ꢂ) [40-(1-dimethylamino-2-phenylethyl)biphenyl-4-yl]meth-
anol (29b). The title compound was synthesized from 28b. FC
purification (CH2Cl2/CH3OH/CH3OH(NH3) 50:0.9:0.1) gave the
desired product. Yield: 90%; mp: 100e102 ꢁC. 1H NMR (CDCl3,
6.1.8.8. (2S)-1-[1-(4-Iodophenyl)-4-(4-methoxyphenoxy)butyl]-2-
methylpyrrolidine (28m), isomer A. The title compound was
prepared from 1-(4-iodophenyl)-4-(4-methoxyphenoxy)butan-
1-one 32d and (S)-2-methylpyrrolidine. The diastereomeric
mixture was purified and 28m isolated as a single isomer by FC
(CH2Cl2/CH3OH (NH3) 70:1). Reaction yield 74%, ds-A/ds-B 65:35.
300 MHz)
d
2.30 (s, 6H), 3.00 (dd, J ¼ 13.3, 9.7 Hz, 1H), 3.35 (dd,
J ¼ 11.0, 4.9 Hz, 1H), 3.53 (dd, J ¼ 9.7, 4.9 Hz, 1H), 4.74 (s, 2H), 7.03 (d,
J ¼ 8.1 Hz, 2H), 7.19e7.10 (m, 3H), 7.21 (d, J ¼ 8.2 Hz, 2H), 7.43 (d,
J ¼ 8.3 Hz, 2H), 7.48 (d, J ¼ 8.2 Hz, 2H), 7.58 (d, J ¼ 8.2 Hz, 2H). MS-
(APCI): m/z 332.2 [Mþ1]þ.
1H NMR (CDCl3, 300 MHz)
d
1.09 (br s, 3H), 2.15e1.20 (m, 8H),
6.1.9.3. {40-[1-((R)-2-methylpyrrolidin-1-yl)pentyl]biphenyl-4-yl}
methanol (29c), isomer A. The title compound was synthesized
from 28ced. FC purification (CH2Cl2/Acetone/CH3OH (NH3)
15:2:0.1) gave the desired product as a single diastereoisomer.
Reaction yield 84%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 600 MHz)
2.52e2.20 (m, 2H), 2.95e2.70 (m, 1H), 3.80e3.70 (m, 1H), 3.76 (s,
3H), 3.87 (t, J ¼ 6.4 Hz, 2H), 6.82e6.76 (m, 4H), 6.97 (d, J ¼ 6.9 Hz,
2H), 7.65 (d, J ¼ 6.9 Hz, 2H). MS-(APCI): m/z 466.1 [Mþ1]þ.
6.1.8.9. (2S)-1-[1-(4-Iodophenyl)-4-(4-methoxyphenoxy)butyl]-2-
methylpyrrolidine (28n), isomer B. The title compound was
prepared from 1-(4-iodophenyl)-4-(4-methoxyphenoxy)butan-
1-one 32d and (S)-2-methylpyrrolidine. The diastereomeric
mixture was purified and 28n isolated as a single isomer by FC
(CH2Cl2/CH3OH (NH3) 70:1). Reaction yield 74%, ds-A/ds-B 65:35.
d
0.79 (t, J ¼ 7.2 Hz, 3H), 1.02e1.07 (m, 1H), 1.08 (d, J ¼ 6.0 Hz, 3H),
1.13e1.18 (m, 1H), 1.20e1.28 (m, 1H), 1.28e1.34 (m, 1H), 1.44e1.49
(m, 1H), 1.60e1.72 (m, 1H), 1.67e1.73 (m, 1H), 1.79e1.89 (m, 2H),
2.35 (ddd like a q, J ¼ 8.4 Hz, 1H), 2.37 (ddd, J ¼ 7.2, 6.6, 6.0 Hz, 1H),
2.84 (td, J ¼ 8.4, 3.0 Hz, 1H), 3.69 (dd, J ¼ 9.6, 6.0 Hz, 1H), 4.64 (s,
2H), 7.20 (d, J ¼ 7.8 Hz, 2H), 7.36 (d, J ¼ 9.0 Hz, 2H), 7.48 (d,
J ¼ 8.4 Hz, 2H), 7.53 (d, J ¼ 8.4 Hz, 2H). MS-(APCI): m/z 338.5
[Mþ1]þ.
1H NMR (CDCl3, 300 MHz)
d
0.85 (d, J ¼ 5.8 Hz, 3H), 1.60e1.35 (m,
4H), 2.15e1.60 (m, 4H), 2.58e2.42 (m, 1H), 2.90e2.70 (m, 1H),
3.15e2.95 (m, 1H), 3.45 (d, J ¼ 7.3 Hz, 1H), 3.75 (s, 3H), 3.81 (t,
J ¼ 6.4 Hz, 2H), 6.82e6.73 (m, 4H), 7.10 (d, J ¼ 7.1 Hz, 2H), 7.63 (d,
J ¼ 8.4 Hz, 2H). MS-(APCI): m/z 466.1 [Mþ1]þ.
6.1.9.4. {40-[1-((R)-2-methylpyrrolidin-1-yl)pentyl]biphenyl-4-yl}
methanol (29d), isomer B. The title compound was synthesized
from 28ced. FC purification (CH2Cl2/Acetone/CH3OH (NH3)
15:2:0.1) gave the desired product as a single diastereoisomer.
Reaction yield 84%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 600 MHz)
6.1.8.10. (ꢂ) 1-(1-(4-Iodophenyl)pentyl)piperidine (28p). Starting
from 1-(4-iodophenyl)pentan-1-one (32a) and piperidine, and
after FC purification (CH2Cl2/CH3OH(NH3) 80:1), compound 28p
was obtained as racemic mixture. Yield 60%.1H NMR (CDCl3,
d
0.75 (t, J ¼ 7.4 Hz, 3H), 0.82 (d, J ¼ 6.3 Hz, 3H), 0.90e1.02 (m, 2H),
1.15e1.28 (m, 2H), 1.31e1.36 (m, 1H), 1.55e1.62 (m, 1H), 1.63e1.70
(m, 1H), 1.71e1.77 (m, 1H), 1.81e1.87 (m, 2H), 2.11 (m, 1H), 2.44
(ddd like a q, J ¼ 8.4 Hz, 1H), 2.72e2.80 (m, 1H), 2.94e3.00 (m,
1H), 3.40 (dd, J ¼ 10.8, 3.6 Hz, 1H), 4.64 (s, 2H), 7.29 (d, J ¼ 7.8 Hz,
2H), 7.35 (d, J ¼ 7.8 Hz, 2H), 7.45 (d, J ¼ 7.8 Hz, 2H), 7.52 (d,
J ¼ 8.4 Hz, 2H). MS-(APCI): m/z 338.7 [Mþ1]þ.
300 MHz)
d
0.82 (t, J ¼ 7.1 Hz, 3H), 0.97e1.16 (m, 2H), 1.19e1.37 (m,
4H), 1.45e1.59 (m, 4H), 1.65e1.86 (m, 2H), 2.31 (t, J ¼ 5.2 Hz, 4H),
3.23 (dd, J ¼ 9.4, 4.9 Hz, 1H), 6.95 (d, J ¼ 8.3 Hz, 2H), 7.62 (d,
J ¼ 8.3 Hz, 2H).
6.1.9. General method for preparation of hydroxymethylbiphenyl
derivates (29aeo, 31) via Suzuki coupling
6.1.9.5. {40-[1-((S)-2-methylpyrrolidin-1-yl)pentyl]biphenyl-4-yl}
methanol (29e), isomer A. The title compound was synthesized
from 28eef. FC purification (CH2Cl2/Acetone/CH3OH (NH3)
15:2:0.1) gave the desired product as a single diastereoisomer.
Reaction yield 86%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 300 MHz)
A solution of the proper iodophenyl derivate 28aeo or 30
(0.50 mmol), 4-hydroxymethylphenylboronic acid (0.55 mmol),
cesium carbonate (1.50 mmol) in acetone (1.5 mL), CH3OH (2 mL)
and H2O (2 mL) was degassed bubbling nitrogen for 10 min before
the addition of Pd(OAc)2 (0.05 mmol). The mixture was heated at
100 ꢁC for 10 min under MW irradiation (50 W, 120 psi). After
filtration over celite and evaporation of the solvent, the residue was
d
0.85 (t, J ¼ 7.4 Hz, 3H), 1.18 (d, J ¼ 6.2 Hz, 3H), 1.20e1.15 (m, 1H),
1.45e1.25 (m, 4H), 1.62e1.48 (m, 1H), 1.85e1.65 (m, 2H),
1.93e1.86 (m, 2H), 2.45 (q, J ¼ 8.8 Hz, 1H), 2.46 (q, J ¼ 5.9 Hz, 1H),
2.93 (td, J ¼ 8.2, 3.1 Hz, 1H), 3.27 (m, 1H), 3.78 (dd, J ¼ 8.9, 6.0 Hz,
1H), 4.71 (s, 2H), 7.28 (d, J ¼ 8.2 Hz, 2H), 7.43 (d, J ¼ 8.1 Hz, 2H),
7.55 (d, J ¼ 8.2 Hz, 2H), 7.59 (d, J ¼ 8.3 Hz, 2H). MS-(APCI): m/z
338.3 [Mþ1]þ.
dissolved in CH2Cl2 (50 mL) and extracted with
1 M HCl
(3 ꢄ 50 mL). The combined aqueous layers were made alkaline with
K2CO3 and extracted with EtOAc (3 ꢄ 50 mL). The organic phases
were dried over sodium sulfate and concentrated under reduced
pressure to afford the crude product that was purified by flash
chromatography (SiO2). When products were obtained as mixture
of diastereoisomers, we named isomer A the one that eluted first,
and isomer B the latter one.
6.1.9.6. {40-[1-((S)-2-methylpyrrolidin-1-yl)pentyl]biphenyl-4-yl}
methanol (29f), isomer B. The title compound was synthesized
from 28eef. FC purification (CH2Cl2/Acetone/CH3OH (NH3)
15:2:0.1) gave the desired product as a single diastereoisomer.
Reaction yield 86%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 300 MHz)
6.1.9.1. (ꢂ)
methanol (29a). The title compound was synthesized from 28a. FC
{40-[1-(Piperidin-1-yl)-2-phenylethyl]biphenyl-4-yl}
d
0.82 (t, J ¼ 7.2 Hz, 3H), 0.96 (d, J ¼ 6.3 Hz, 3H), 1.10e0.95 (m, 2H),

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
225
1.37e1.21 (m, 3H), 1.55e1.42 (m, 1H), 2.05e1.68 (m, 5H),
2.70e2.50 (m, 1H), 3.28e2.85 (m, 2H), 3.57 (t, J ¼ 6.7 Hz, 1H), 4.74
(s, 2H), 7.43 (d, J ¼ 8.1 Hz, 4H), 7.56 (d, J ¼ 8.5 Hz, 2H), 7.61 (d,
J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 338.5 [Mþ1]þ.
from 28l. FC purification (CH2Cl2/CH3OH (NH3) 80:1) gave the desired
product. Yield: 63%. 1H NMR (CDCl3, 300 MHz)
0.80 (d, J ¼ 6.3 Hz,
d
3H), 1.40e1.26 (m, 1H), 1.52e1.40 (m, 2H), 1.75e1.52 (m, 2H),
1.95e1.75 (m, 2H), 2.13e1.98 (m, 1H), 2.41 (br s, 1H), 2.46 (ddd, J ¼ 9.1,
7.8, 7.7 Hz,1H), 2.76 (ddd, J ¼ 9.1, 7.7, 3.9 Hz, 1H), 2.99 (m, 1H), 3.46 (dd,
J ¼ 10.5, 3.6 Hz, 1H), 3.79 (t, J ¼ 6.5 Hz, 1H), 4.74 (s, 2H), 6.76 (d,
J ¼ 8.6 Hz, 2H), 6.82 (t, J ¼ 7.3 Hz, 1H), 7.16 (dd, J ¼ 8.4, 7.7 Hz, 2H), 7.31
(d, J ¼ 8.0 Hz, 2H), 7.33 (d, J ¼ 7.8 Hz, 2H), 7.44 (d, J ¼ 8.3 Hz, 2H), 7.50
(d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 416.3 [Mþ1]þ.
6.1.9.7. {40-[1-((S)-2-methylpiperidin-1-yl)pentyl]biphenyl-4-yl}
methanol (29g), isomer A. The title compound was synthesized
from 28g. FC purification (CH2Cl2/CH3OH (NH3) 70:1) gave the
desired product. Yield: 70%. 1H NMR (CDCl3, 300 MHz)
d 0.76 (t,
J ¼ 7.2 Hz, 3H), 1.10e0.90 (m, 2H), 1.05 (d, J ¼ 6.4 Hz, 3H), 1.38e1.15
(m, 6H), 1.55e1.45 (m, 1H), 1.80e1.50 (m, 3H), 2.16 (ddd, J ¼ 10.0,
6.5, 3.6 Hz, 1H), 2.36 (ddd, J ¼ 8.5, 7.3, 3.4 Hz, 1H), 2.52 (br s, 1H),
2.98e2.85 (m, 1H), 3.70 (dd, J ¼ 10.1, 3.5 Hz, 1H), 4.61 (s, 2H), 7.31
(d, J ¼ 8.2 Hz, 2H), 7.32 (d, J ¼ 8.3 Hz, 2H), 7.43 (d, J ¼ 8.3 Hz, 2H),
7.50 (d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 352.5 [Mþ1]þ.
6.1.9.13. {40-[4-(4-Methoxyphenoxy)-1-((S)-2-methylpyrrolidin-1-yl)
butyl]biphenyl-4-yl}methanol (29m), isomer A. The title compound
was synthesized from 28m. FC purification (CH2Cl2/CH3OH (NH3)
80:1) gave the desired product. Yield 72%; 1H NMR (CDCl3,
300 MHz)
d
1.07 (d, J ¼ 6.0 Hz, 3H), 1.34e1.22 (m, 1H), 1.55e1.38 (m,
1H), 1.74e1.53 (m, 4H), 2.10e1.85 (m, 2H), 2.27 (br s, 1H), 2.45e2.26
(m, 2H), 2.83 (td, J ¼ 8.0, 3.0 Hz, 1H), 3.65 (s, 3H), 3.75 (dd, J ¼ 8.7,
6.3 Hz, 1H), 3.80 (t, J ¼ 6.5 Hz, 2H), 4.63 (s, 2H), 6.71 (s, 4 H), 7.20 (d,
J ¼ 8.1 Hz, 2H), 7.33 (d, J ¼ 8.2 Hz, 2H), 7.46 (d, J ¼ 8.2 Hz, 2H), 7.50
(d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 446.1 [Mþ1]þ.
6.1.9.8. {40-[1-((S)-2-methylpiperidin-1-yl)pentyl]biphenyl-4-yl}
methanol (29h), isomer B. The title compound was synthesized
from 28h. FC purification (CH2Cl2/CH3OH (NH3) 70:1) gave the
desired product. Yield: 72%. 1H NMR (CDCl3, 300 MHz)
d 0.77 (t,
J ¼ 7.3 Hz, 3H), 1.12e0.95 (m, 2H), 1.12 (d, J ¼ 6.1 Hz, 3H),
1.28e1.12 (m, 4H), 1.55e1.32 (m, 4H), 1.76 (q, J ¼ 7.3 Hz, 2H), 1.90
(t, J ¼ 10.5 Hz, 1H), 2.09 (m, 1H), 2.09 (s, 1H), 2.88 (d, J ¼ 11.0 Hz,
1H), 3.91 (t, J ¼ 7.1 Hz, 1H), 4.64 (s, 2H), 7.19 (d, J ¼ 8.1 Hz, 2H),
7.33 (d, J ¼ 8.2 Hz, 2H), 7.44 (d, J ¼ 8.3 Hz, 2H), 7.51 (d, J ¼ 8.2 Hz,
2H). MS-(APCI): m/z 352.5 [Mþ1]þ.
6.1.9.14. {40-[4-(4-Methoxyphenoxy)-1-((S)-2-methylpyrrolidin-1-yl)
butyl]biphenyl-4-yl}methanol (29n), isomer B. The title compound
was synthesized from 28n. FC purification (CH2Cl2/CH3OH (NH3)
80:1) gave the desired product. Yield: 71%. 1H NMR (CDCl3,
300 MHz)
d
0.80 (d, J ¼ 6.2 Hz, 3H), 1.38e1.28 (m, 1H), 1.52e1.38 (m,
2H), 1.74e1.52 (m, 2H), 1.93e1.78 (m, 2H), 2.12e1.98 (m, 1H), 2.45
(q, J ¼ 7.8 Hz, 1H), 2.55 (br s, 1H), 2.80e2.70 (m, 1H), 3.03e2.93 (m,
1H), 3.44 (dd, J ¼ 10.6, 3.5 Hz, 1H), 3.65 (s, 3H), 3.74 (t, J ¼ 6.4 Hz,
2H), 4.62 (s, 2H), 6.70 (s, 4 H), 7.30 (d, J ¼ 7.8 Hz, 2H), 7.33 (d,
J ¼ 7.7 Hz, 2H), 7.43 (d, J ¼ 8.3 Hz, 2H), 7.59 (d, J ¼ 8.2 Hz, 2H). MS-
(APCI): m/z 446.3 [Mþ1]þ.
6.1.9.9. {40-[4-methoxy-1-((S)-2-methylpyrrolidin-1-yl)butyl]
biphenyl-4-yl}methanol (29i), isomer A. The title compound was
synthesized from 28iej. FC purification (CH2Cl2/CH3OH (NH3)
40:1) gave the desired product as single diasereoisomer. Reaction
yield 92%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 300 MHz)
d 1.05 (d,
J ¼ 6.1 Hz, 3H), 1.38e1.25 (m, 2H), 1.50e1.38 (m, 2H), 1.72e1.53 (m,
2H), 1.98e1.72 (m, 2H), 2.32 (q, J ¼ 7.8 Hz, 1H), 2.38 (q, J ¼ 6.3 Hz,
1H), 2.68 (br s, 1H), 2.81 (td, J ¼ 8.0, 3.2 Hz, 1H), 3.18 (s, 3H), 3.25 (t,
J ¼ 6.6 Hz, 1H), 3.68 (dd, J ¼ 9.0, 6.1 Hz, 1H), 4.67 (s, 2H), 7.17 (d,
J ¼ 8.0 Hz, 2H), 7.32 (d, J ¼ 8.2 Hz, 2H), 7.44 (d, J ¼ 8.2 Hz, 2H), 7.48
(d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 354.2 [Mþ1]þ.
6.1.9.15. (40-pentylbiphenyl-4-yl)methanol (29o). The title compound
was synthesized from commercially available 28o. FC purification
(CH2Cl2/CH3OH 30:1) gave the desired product. Yield: 41%; mp
(n-hexane): 103e104 ꢁC (lit.103.5 ꢁC)[48]. 1H NMR (CDCl3, 400 MHz)
d
0.91 (t, J ¼ 5.8 Hz, 3H),1.36 (m, 4H),1.65 (quint, J ¼ 6.4 Hz, 2H), 2.64
(t, J ¼ 7.9 Hz, 2H), 4.74 (s, 2H), 7.25 (d, J ¼ 7.8 Hz, 2H), 7.43 (d, J ¼ 7.8 Hz,
2H), 7.51 (d, J ¼ 7.9 Hz, 2H), 7.59 (d, J ¼ 8.2 Hz, 2H).
6.1.9.10. {40-[4-methoxy-1-((S)-2-methylpyrrolidin-1-yl)butyl]
biphenyl-4-yl}methanol (29j), isomer B. The title compound was
synthesized from 28iej. FC purification (CH2Cl2/CH3OH (NH3)
40:1) gave the desired product as single diasereoisomer. Reaction
6.1.11.16. (ꢂ)
1-[40-(Hydroxymethyl)biphenyl-4-yl]pentan-1-ol
(31). The title compound was synthesized from 30. FC purifica-
yield 92%, ds-A/ds-B 65:35. 1H NMR (CDCl3, 300 MHz)
d
0.78 (d,
tion (CH2Cl2/CH3OH/CH3OH(NH3) 30:1:0.2) gave the desired
J ¼ 6.2 Hz, 3H), 1.38e1.16 (m, 3H), 1.96e1.50 (m, 5H), 2.41 (q,
J ¼ 7.8 Hz, 1H), 2.71 (ddd, J ¼ 9.1, 7.9, 4.3 Hz, 1H), 3.00e2.90 (m,
1H), 3.03 (br s, 1H), 3.16 (s, H), 3.20 (t, J ¼ 6.6 Hz, 2H), 3.37 (dd,
J ¼ 10.6, 3.5 Hz, 1H), 4.58 (s, 2H), 7.26 (d, J ¼ 8.3 Hz, 2H), 7.30 (d,
J ¼ 7.8 Hz, 2H), 7.41 (d, J ¼ 8.3 Hz, 2H), 7.47 (d, J ¼ 8.2 Hz, 2H). MS-
(APCI): m/z 354.1 [Mþ1]þ.
product. Yield: 85%. 1H NMR (CDCl3, 300 MHz)
d
0.90 (t, J ¼ 6.9 Hz,
3H), 1.46e1.24 (m, 4H), 1.87e1.69 (m, 4H), 4.71 (t, J ¼ 7.0 Hz, 1H),
4.74 (s, 2H), 7.41 (d, J ¼ 8.0 Hz, 2H), 7.44 (d, J ¼ 8.1 Hz, 2H), 7.57 (d,
J ¼ 8.2 Hz, 2H), 7.59 (d, J ¼ 8.2 Hz, 2H).
6.1.10. General synthesis of aminomethylbiphenyl derivates (9e24,
and 34)
6.1.9.11. {40-[1-((S)-2-methylpyrrolidin-1-yl)-4-phenoxybutyl]
biphenyl-4-yl}methanol (29k), isomer A. The title compound was
synthesized from 28k. FC purification (CH2Cl2/CH3OH (NH3) 80:1)
gave the desired product. Yield: 61%. 1H NMR (CDCl3, 300 MHz)
To a stirred solution of hydroxymethylbiphenyl derivate 29aeo
or 31 (0.47 mmol) in CH2Cl2 (15 mL), MsCl (0.50 mmol for
compounds 29aeo, or 1.00 mmol for compound 31) was added.
After cooling to 0 ꢁC, Et3N (0.60 mmol for compounds 29aeo, or
1.20 mmol for compound 31) was added and the mixture was
stirred at room temperature for 90 min. The solvent was evapo-
rated to dryness and piperidine or 2-methylpyrrolidine(4.70 mmol)
was added to the crude product dissolved in CH3CN (3 mL). The
mixture was heated under MW irradiation at 100 ꢁC for 5 min
(80 W, 120 psi). After cooling, CH3CN was evaporated and the
residue was taken up in CH2Cl2 (40 mL) and was extracted with 1 M
HCl (3 ꢄ 50 mL). The aqueous phases were made alkaline with
K2CO3 and were extracted with EtOAc (3 ꢄ 50 mL). The organic
layers were dried over sodium sulfate and the solvent was
d
1.09 (d, J ¼ 6.1 Hz, 3H), 1.36e1.24 (m, 1H), 1.55e1.40 (m, 1H),
1.77e1.55 (m, 2H), 2.13e1.87 (m, 2H), 2.32 (q, J ¼ 8.3 Hz, 1H), 2.40
(q, J ¼ 6.4 Hz, 1H), 2.61 (br s, 1H), 2.84 (td, J ¼ 8.0, 3.1 Hz, 1H), 3.77
(dd, J ¼ 8.8, 6.2 Hz, 1H), 3.86 (t, J ¼ 6.5 Hz, 2H), 4.64 (s, 2H), 6.78 (d,
J ¼ 8.8 Hz, 2H), 6.84 (t, J ¼ 7.3 Hz, 1H), 7.18 (dd, J ¼ 8.5, 7.4 Hz, 2H),
7.21 (d, J ¼ 8.3 Hz, 2H), 7.36 (d, J ¼ 8.2 Hz, 2H), 7.47 (d, J ¼ 8.2 Hz,
2H), 7.51 (d, J ¼ 8.3 Hz, 2H). MS-(APCI): m/z 416.3 [Mþ1]þ.
6.1.9.12. {40-[1-((S)-2-methylpyrrolidin-1-yl)-4-phenoxybutyl]biphenyl-
4-yl}methanol (29l), isomer B. The title compound was synthesized

226
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
evaporated under reduced pressure to afford the crude products
which were purified by flash chromatography (SiO2, CH2Cl2/CH3OH
(NH3) 50:1).
1.35e1.20 (m, 2H), 1.50e1.38 (m, 3H), 1.66e1.56 (m, 4H),
1.96e1.58 (m, 5H), 2.48e2.38 (m, 4H), 2.54 (q, J ¼ 7.9 Hz, 1H), 2.88
(td, J ¼ 8.3, 3.5 Hz, 1H), 3.05 (q, J ¼ 6.2 Hz, 1H), 3.49 (dd, J ¼ 10.4,
3.8 Hz, 1H), 3.53 (s, 2H),7.38 (d, J ¼ 7.2 Hz, 4H), 7.56 (d, J ¼ 8.0 Hz,
2H), 7.58 (d, J ¼ 8.1 Hz, 2H). MS-(APCI): m/z 405.7 [Mþ1]þ. Anal.
Calcd for C28H40N2·2H2SO4·0.5H2O: C, 55.15; H, 7.44; N, 4.59.
Found: C, 55.00; H, 7.49; N, 4.75.
6.1.10.1. (ꢂ) 1-{2-phenyl-1-[40-(piperidin-1-ylmethyl)biphenyl-4-yl]
ethyl}piperidine (9·2HCl·H2O). The compound was synthesized
from 29a. Yield: 89%; crystallized from abs EtOH/Et2O; mp: 123 ꢁC
(dec). 1H NMR (D2O, 300 MHz)
d 1.46e1.27 (m, 2H), 1.95e1.57 (m,
10H), 2.94e2.74 (m, 4H), 3.50e3.37 (m, 4H), 3.60 (dd, J ¼ 13.2,
4.0 Hz, 1H), 3.72 (d, J ¼ 11.6 Hz, 1H), 4.24 (s, 2H), 4.62 (dd, J ¼ 11.8,
3.8 Hz, 1H), 7.13e7.05 (m, 5H), 7.50e7.42 (m, 8H). MS-(APCI): m/z
439.3 [Mþ1]þ. Anal. Calcd for C31H38N2·2HCl·H2O: C, 70.91; H, 7.99;
N, 5.29. Found: C, 70.13; H, 7.86; N, 5.22.
6.1.10.7. 1-{[40-(1-((S)-2-methylpyrrolidin-1-yl)pentyl)biphenyl-4-yl]
methyl}piperidine (15·1.65H2SO4), isomer A. The compound was
synthesized from 29e. Yield: 59%; crystallized from Et2O; mp:
160e164 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
d
0.80 (t, J ¼ 7.1 Hz, 3H),
1.40e0.85 (m, 6H), 1.78e1.40 (m, 5H), 1.95e1.85 (m, 2H),
2.20e2.00 (m, 3H), 3.10e2.80 (m, 4H), 3.60e3.10 (m, 6H),
4.22e4.05 (m, 2H), 4.40e4.20 (m, 1H), 7.54 (d, J ¼ 8.1 Hz, 2H), 7.50
(d, J ¼ 8.1 Hz, 2H), 7.78 (d, J ¼ 8.0 Hz, 2H), 7.79 (d, J ¼ 8.0 Hz, 2H),
9.54 (s, 1H). MS-(APCI): m/z 405.8 [Mþ1]þ. Anal. Calcd for
C28H40N2·1.65H2SO4: C, 59.37; H, 7.70; N, 4.94. Found: C, 59.25; H,
8.07; N, 4.84.
6.1.10.2. (ꢂ)
biphenyl-4-yl]ethanamine
synthesized from 29b. Yield: 60%; crystallized from Et2O; mp:
148e153 ꢁC. 1H NMR (CDCl3, 300 MHz)
1.46 (m, 2H), 1.63 (m, 4H),
N,N-dimethyl-2-phenyl-1-[40-(piperidin-1-ylmethyl)
(10·2H2SO4). The compound was
d
2.32 (s, 6H), 2.45 (m, 4H), 3.02 (dd, J ¼ 13.1, 9.8 Hz, 1H), 3.36 (dd,
J ¼ 13.3, 4.6 Hz, 1H), 3.55e3.51 (m, 1H), 3.55 (s, 2H), 7.00 (d,
J ¼ 6.7 Hz, 2H), 7.16e7.10 (m, 3H), 7.20 (d, J ¼ 8.1 Hz, 2H), 7.40 (d,
J ¼ 7.9 Hz, 2H), 7.50 (d, J ¼ 8.0 Hz, 2H), 7.56 (d, J ¼ 8.0 Hz, 2H). MS-
(APCI): m/z 399.4 [Mþ1]þ. Anal. Calcd for C28H34N2·2H2SO4: C,
56.55; H, 6.44; N, 4.71. Found: C, 56.60; H, 6.52; N, 4.71.
6.1.10.8. 1-{[40-(1-((S)-2-methylpyrrolidin-1-yl)pentyl)biphenyl-4-yl]
methyl}piperidine (16·1.8H2SO4), isomer B. The compound was
synthesized from 29f. Yield: 55%; crystallized from Et2O; mp:
92e98 ꢁC (dec). 1H NMR (DMSO-d6, 300 MHz)
d
0.78 (t, J ¼ 7.3 Hz,
3H), 1.02e0.85 (m, 1H), 1.40e1.04 (m, 6H), 1.80e1.40 (m, 5H),
2.18e1.90 (m, 4H), 3.07e2.65 (m, 3H), 3.70e3.10 (m, 7H), 4.20e3.90
(m, 2H), 4.50e4.20 (m, 1H), 7.57 (d, J ¼ 8.1 Hz, 2H), 7.66 (d,
J ¼ 8.2 Hz, 2H), 7.80 (d, J ¼ 8.0 Hz, 2H), 7.83 (d, J ¼ 7.9 Hz, 2H), 9.48
(s, 1H). MS-(APCI): m/z 405.6. [Mþ1]þ. Anal. Calcd for
C28H40N2·1.8H2SO4: C, 57.87; H, 7.56; N, 4.82. Found: C, 58.03; H,
7.73; N, 4.61.
6.1.10.3. 1-{1-[40-(((R)-20-methylpyrrolidin-1-yl)methyl)biphenyl-4-
yl]-2-phenylethyl}piperidine (11·2HCl·0.8H2O). The compound was
obtained from 29a as a 1:1 mixture of diastereoisomers. Yield: 67%;
crystallized from abs EtOH/Et2O; mp: 197 ꢁC (dec). 1H NMR (DMSO-
d6, 300 MHz)
d
1.36 (d, J ¼ 6.2 Hz, 3H), 1.95e1.60 (m, 6H), 2.28e2.03
(m, 2H), 2.65e2.55 (m, 2H), 3.15e3.04 (m, 1H), 3.30e3.18 (m, 1H),
3.61e3.40 (m, 5H), 3.78 (d, J ¼ 11.3 Hz, 1H), 3.86 (d, J ¼ 12.0 Hz, 1H),
4.17 (dd, J ¼ 12.9, 6.9 Hz, 1H), 4.52 (dd, J ¼ 12.6, 3.4 Hz, 1H), 4.72 (d,
J ¼ 11.8 Hz, 1H), 7.17e7.08 (m, 5H), 7.80e7.60 (m, 8H), 11.00 (s, 1H),
11.45 (s, 1H). MS-(APCI): m/z 439.4 [Mþ1]þ. Anal. Calcd for
C31H38N2·2HCl·0.8H2O: C, 70.79; H, 7.97; N, 5.33. Found: C, 70.62; H,
8.01; N, 5.16.
6.1.10.9. (2S)-2-methyl-1-{1-[40-(piperidin-1-ylmethyl)biphenyl-4-
yl]pentyl}piperidine (17·1.95H2SO4), isomer A. The compound was
synthesized from 29g. Yield: 63%; crystallized from Et2O; mp:
185e188 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
0.88 (t, J ¼ 6.7 Hz, 3H),
1.20e1.08 (m, 2H), 1.22 (d, J ¼ 6.1 Hz, 3H), 1.38e1.24 (m, 4H),
1.52e1.38 (m, 3H), 1.65e1.52 (m, 7H), 1.85 (q, J ¼ 7.7 Hz, 2H), 1.98
(td, J ¼ 10.8, 1.6 Hz, 2H), 2.28e2.15 (m, 1H), 2.50e2.30 (m, 4H),
2.97 (d, J ¼ 11.3 Hz, 1H), 3.51 (s, 2H), 4.01 (t, J ¼ 7.3 Hz, 1H), 7.28 (d,
J ¼ 8.0 Hz, 2H), 7.37 (d, J ¼ 8.0 Hz, 2H), 7.54 (d, J ¼ 7.9 Hz, 2H), 7.56
(d, J ¼ 8.0 Hz, 2H). MS-(APCI): m/z 419.5 [Mþ1]þ. Anal. Calcd for
C29H42N2·1.8H2SO4: C, 58.52; H, 7.72; N, 4.71. Found: C, 58.13; H,
7.74; N, 4.67.
6.1.10.4. (ꢂ) 1-{[40-(1-(Piperidin-1-yl)pentyl)biphenyl-4-yl]methyl}
piperidine (12·1.9H2SO4). The compound was synthesized from 31.
Yield: 85%; crystallized from Et2O; mp: 158 ꢁC 1H NMR (CDCl3,
300 MHz)
d
0.85 (t, J ¼ 7.0 Hz, 3H), 1.50e1.04 (m, 8H), 1.65e1.52 (m,
8H), 2.00e1.80 (m, 2H), 2.42 (m, 8H), 3.39 (dd, J ¼ 9.1, 4.9 Hz, 1H),
3.53 (s, 2H), 7.28 (d, J ¼ 8.1 Hz, 2H), 7.38 (d, J ¼ 8.1 Hz, 2H), 7.56 (d,
J ¼ 8.2 Hz, 2H), 7.57 (d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 405.7
[Mþ1]þ. Anal. Calcd for C28H40N2·1.9H2SO4: C, 56.90; H, 7.47; N,
4.74. Found: C, 56.99; H, 7.98; N, 4.29.
6.1.10.10. (2S)-2-methyl-1-{1-[40-(piperidin-1-ylmethyl)biphenyl-4-
yl]pentyl}piperidine (18·1.8H2SO4), isomer B. The compound was
synthesized from 29h. Yield: 73%; crystallized from Et2O; mp:
6.1.10.5. 1-{[40-(1-((R)-2-methylpyrrolidin-1-yl)pentyl)biphenyl-4-
yl]methyl}piperidine (13·1.7H2SO4), isomer A. The compound was
synthesized from 29c. Yield: 67%; crystallized from Et2O; mp:
175e180 ꢁC. 1H NMR (CDCl3, 300 MHz)
d
0.88 (t, J ¼ 7.2 Hz, 3H),
1.22e1.15 (m, 2H), 1.15 (d, J ¼ 6.3 Hz, 3H), 1.52e1.25 (m, 8H),
1.68e1.55 (m, 4H), 1.88e1.60 (m, 4H), 2.32e2.20 (m, 1H),
2.55e2.32 (m, 5H), 3.08e2.90 (m, 1H), 3.53 (s, 2H), 3.80 (dd,
J ¼ 9.8, 3.3 Hz, 1H), 7.38 (d, J ¼ 7.9 Hz, 2H), 7.41 (d, J ¼ 7.5 Hz, 2H),
7.55 (d, J ¼ 7.9 Hz, 2H), 7.57 (d, J ¼ 7.9 Hz, 2H). MS-(APCI): m/z 419.5
[Mþ1]þ. Anal. Calcd for C29H42N2·1.95H2SO4: C, 57.11; H, 7.59; N,
4.59. Found: C, 57.11; H, 7.62; N, 4.61.
187e191 ꢁC (dec). 1H NMR (CDCl3, 300 MHz)
d
0.87 (t, J ¼ 6.9 Hz,
3H), 1.16 (d, J ¼ 6.0 Hz, 3H), 1.23e1.12 (m, 2H), 1.53e1.25 (m, 4H),
1.65e1.53 (m, 6H), 1.80e1.65 (m, 2H), 1.95e1.87 (m, 2H), 2.46 (m,
6H), 2.94 (td, J ¼ 8.6, 2.9 Hz, 1H), 3.52 (s, 2H), 3.78 (dd, J ¼ 8.7,
6.3 Hz, 1H), 7.27 (d, J ¼ 8.1 Hz, 2H), 7.38 (d, J ¼ 8.3 Hz, 2H), 7.56 (d,
J ¼ 7.3 Hz, 4H). MS-(APCI): m/z 405.2 [Mþ1]þ. Anal. Calcd for
C28H40N2·1.7H2SO4: C, 58.86; H, 7.66; N, 4.90. Found: C, 58.98; H,
7.90; N, 4.66.
6.1.10.11. 1-{[40-(4-methoxy-1-((S)-2-methylpyrrolidin-1-yl)butyl)
biphenyl-4-yl]methyl}piperidine (19·1.65H2SO4), isomer A. The
compound was synthesized from 29i. Yield: 60%; crystallized
6.1.10.6. 1-{[40-(1-((R)-2-methylpyrrolidin-1-yl)pentyl)biphenyl-4-
from Et2O; mp: 98e102 ꢁC (dec). 1H NMR (CDCl3, 300 MHz)
d 1.06
yl]methyl}piperidine
(14·2H2SO4·0.5H2O),
isomer
B. The
(d, J ¼ 6.1 Hz, 3H), 1.55e1.23 (m, 10H), 1.75e1.55 (m, 2H),
1.95e1.75 (m, 2H), 2.38e2.25 (m, 5H), 2.40 (q, J ¼ 6.2 Hz, 1H), 2.83
(ddd, J ¼ 7.9, 7.2, 2.5 Hz, 1H), 3.19 (s, 3H), 3.26 (t, J ¼ 6.6 Hz, 2H),
3.41 (s, 2H), 3.69 (dd, J ¼ 8.8, 6.2 Hz, 1H), 7.17 (d, J ¼ 8.1 Hz, 2H),
compound was synthesized from 29d. Yield: 66%; crystallized
from Et2O; mp: 140e143 ꢁC (dec). 1H NMR (CDCl3, 300 MHz)
d
0.85 (t, J ¼ 7.2 Hz, 3H), 0.90 (d, J ¼ 6.2 Hz, 3H), 1.12e1.00 (m, 2H),

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
227
7.28 (d, J ¼ 8.2 Hz, 2H), 7.43 (d, J ¼ 8.2 Hz, 2H), 7.44 (d, J ¼ 8.2 Hz,
2H). MS-(APCI): m/z 421.4 [Mþ1]þ. Anal. Calcd for
C28H40N2O·1.65H2SO4: C, 57.74; H, 7.49; N, 4.81. Found: C, 57.77;
H, 7.72; N, 4.59.
J ¼ 8.3 Hz, 2H), 7.46 (d, J ¼ 8.1 Hz, 2H). MS-(APCI): m/z 513.3
[Mþ1]þ. Anal. Calcd for C33H44N2O2·2H2SO4: C, 57.61; H, 6.83; N,
3.95. Found: C, 57.53; H, 6.73; N, 3.74.
6.1.10.17. 1-((40-pentylbiphenyl-4-yl)methyl)piperidine (34·H2SO4). The
compound was synthesized from 29o. Yield: 90%; precipitated from
6.1.10.12. 1-{[40-(4-methoxy-1-((S)-2-methylpyrrolidin-1-yl)butyl)
biphenyl-4-yl]methyl}piperidine (20·1.6H2SO4), isomer B. The
compound was synthesized from 29j. Yield: 67%. Crystallized
Et2O. 1H NMR (CDCl3, 300 MHz)
d
0.92 (t, J ¼ 6.8 Hz, 3H), 1.34e1.39 (m,
4H), 1.46 (q, J ¼ 5.6 Hz, 2H), 1.57e1.72 (m, 6H), 2.42 (m, 4H), 2.65 (t,
J ¼ 7.5 Hz, 2H), 3.52 (s, 2H), 7.25 (d, J ¼ 8.3 Hz, 2H), 7.38 (d, J ¼ 8.3 Hz,
2H), 7.52 (d, J ¼ 8.2 Hz, 2H), 7.54 (d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z
322.6 [Mþ1]þ. Anal. Calcd for C23H31N·H2SO4: C, 65.84; H, 7.93; N,
3.34. Found: C, 65.60; H, 8.04; N, 3.11.
from Et2O; mp: 80e85 ꢁC (dec). 1H NMR (CDCl3, 300 MHz)
d 0.78
(d, J ¼ 6.2 Hz, 3H), 1.38e1.15 (m, 5H), 1.54e1.42 (m, 5H), 1.95e1.54
(m, 4H), 2.37e2.25 (m, 4H), 2.43 (q, J ¼ 8.9 Hz, 1H), 2.75 (td,
J ¼ 8.8, 4.1 Hz, 1H), 2.95 (m, 1H), 3.17 (s, 3H), 3.21 (t, J ¼ 6.5 Hz,
2H), 3.39 (dd, J ¼ 9.7, 3.8 Hz, 1H), 3.40 (s, 2H), 7.26 (d, J ¼ 8.1 Hz,
4H), 7.43 (d, J ¼ 8.3 Hz, 2H), 7.44 (d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/
z 421.4 [Mþ1]þ. Anal. Calcd for C28H40N2O·1.6H2SO4: C, 58.23; H,
7.54; N, 4.85. Found: C, 58.23; H, 7.92; N, 4.62.
6.1.11. Preparation of (ꢂ)1-(1-(40-methylbiphenyl-4-yl)pentyl)
piperidine (35·H2SO4)
The compound was synthesized via Suzuki coupling from 28p
and p-tolylboronic acid, following the procedure described for
compounds 29aen and 31. FC purification (CH2Cl2/CH3OH(NH3) 80:1)
gave the desired product. Yield: 76%; crystallized from Et2O; mp:
6.1.10.13. 1-{[40-(1-((S)-2-methylpyrrolidin-1-yl)-4-phenoxybutyl)
biphenyl-4-yl]methyl}piperidine (21·1.65H2SO4), isomer A. The
compound was synthesized from 29k. Yield: 86%; crystallized
120e122 ꢁC. NMR (CDCl3, 300 MHz)
d
0.86 (t, J ¼ 7.0 Hz, 3H), 1.10e1.18
from Et2O; mp: 110e115 ꢁC (dec). 1H NMR (CDCl3, 300 MHz)
d
1.16
(m, 2H),1.28e1.39 (m, 4H),1.53e1.63 (m, 4H),1.82e1.95 (m, 2H), 2.39
(m, 4H), 2.41(s, 3H), 3.37(dd, J¼ 9.2,5.1 Hz,1H), 7.26(d, J¼ 7.9Hz,2H),
7.27 (d, J ¼ 8.1 Hz, 2H), 7.53 (d, J ¼ 8.0 Hz, 2H), 7.55 (d, J ¼ 8.2 Hz, 2H).
MS-(APCI): m/z 322.4 [Mþ1]þ. Anal. Calcd for C23H31N·H2SO4: C,
65.84; H, 7.93; N, 3.34. Found: C, 65.84; H, 7.92; N, 3.33.
(d, J ¼ 6.0 Hz, 3H), 1.90e1.25 (m, 12H), 2.20e1.95 (m, 2H),
2.50e2.35 (m, 6H), 2.93 (td, J ¼ 8.6, 3.0 Hz, 1H), 3.51 (s, 2H), 3.85
(dd, J ¼ 8.4, 6.6 Hz, 1H), 3.96 (t, J ¼ 6.5 Hz, 2H), 6.87 (d, J ¼ 8.8 Hz,
2H), 6.92 (t, J ¼ 7.7 Hz, 1H), 7.26 (dd, J ¼ 8.5, 5.4 Hz, 2H), 7.27 (d,
J ¼ 8.9 Hz, 2H), 7.38 (d, J ¼ 8.2 Hz, 2H), 7.55 (d, J ¼ 8.2 Hz, 2H), 7.56
(d, J ¼ 8.2 Hz, 2H). MS-(APCI): m/z 483.2 [Mþ1]þ. Anal. Calcd for
C33H42N2O·1.65H2SO4: C, 61.49; H, 7.08; N, 4.35. Found: C, 61.48;
H, 6.92; N, 4.13.
6.2. Pharmacology
Drugs used were purchased from Sigma (St. Louis, MA, USA).
Cultured SK-N-MC cells stably expressing human histamine H3
receptors and the reporter gene b-galactosidase (Johnson & John-
6.1.10.14. 1-{[40-(1-((S)-2-methylpyrrolidin-1-yl)-4-phenoxybutyl)
biphenyl-4-yl]methyl}piperidine (22·1.7H2SO4), isomer B. The
compound was synthesized from 29l. Yield: 75%; crystallized
from Et2O; mp: 140e145 ꢁC (dec). 1H NMR (CDCl3, 300 MHz)
son Pharmaceutical Research and Development, L.L.C.) were used
for binding and functional studies. Functional experiments were
also performed on isolated ileal and atrial tissues excised from
guinea-pigs (250e350 g). Animals were housed, handled and cared
for according to the European Community Council Directive 86
(609) EEC and the experimental protocols were carried out in
compliance with Italian regulations (DL 116/92) and with the local
Ethical Committee Guidelines for Animal Research.
d
0.90 (d, J ¼ 5.9 Hz, 3H), 1.52e1.28 (m, 4H), 1.85e1.56 (m, 7H),
2.08e1.85 (m, 2H), 2.25e2.17 (m, 1H), 2.50e2.30 (m, 4H), 2.60 (q,
J ¼ 7.9 Hz, 1H), 2.95e2.83 (m, 1H), 3.15e3.03 (m, 1H), 3.52 (s, 2H),
3.57 (dd, J ¼ 10.5, 3.7 Hz, 1H), 3.89 (t, J ¼ 6.3 Hz, 2H), 6.84 (d,
J ¼ 8.6 Hz, 2H), 6.92 (t, J ¼ 6.8 Hz, 1H), 7.25 (t, J ¼ 7.8 Hz, 2H), 7.38
(d, J ¼ 8.2 Hz, 2H), 7.41 (d, J ¼ 8.4 Hz, 2H), 7.56 (d, J ¼ 7.9 Hz,
2H). MS-(APCI): m/z 483.4 [Mþ1]þ. Anal. Calcd for
C33H42N2O·1.7H2SO4: C, 61.03; H, 7.05; N, 4.31. Found: C, 60.98; H,
7.18; N, 4.04.
6.2.1. Human histamine H3 and H4 receptor binding assay
Homogenates of SK-N-MC cells, a human neuroblastoma cell line
stably expressing the human histamine H3 and H4 receptors, were
used in radioligand displacement studies according to literature
methods [38,49]. Membranes were incubated for 60 min at room
6.1.10.15. 1-{[40-(4-(4-Methoxyphenoxy)-1-((S)-2-methylpyrrolidin-
1-yl)-butyl)biphenyl-4-yl]methyl}piperidine (23·1.7H2SO4), isomer
A. The compound was synthesized from 29m. Yield: 62%; crys-
tallized from Et2O; mp: 153e161 ꢁC (dec). 1H NMR (CDCl3,
temperature with 0.5 nM [3H]-(R)-
a-methylhistamine (47.0 Ci/
mmol, Amersham Bioscience) or with 10 nM [3H]-histamine (18.1 Ci/
mmol, Perkin Elmer) in the absence or in the presence of competing
300 MHz)
d
1.06 (d, J ¼ 6.0 Hz, 3H), 1.40e1.26 (m, 3H), 1.53e1.40
ligands (0.01 nMe10 mM). Incubation was terminated by rapid
filtration over Millipore AAWPO2500 filters followed by two washes
with ice-cold buffer (50 mM TriseHCl/5 mM EDTA). Nonspecific
(m, 5H), 1.75e1.58 (m, 4H), 2.10e1.85 (m, 2H), 2.41e2.27 (m, 6H),
2.83 (td, J ¼ 8.6, 3.0 Hz, 1H), 3.42 (s, 2H), 3.66 (s, 3H), 3.74 (dd,
J ¼ 8.3, 6.7 Hz, 1H), 3.81 (t, J ¼ 6.5 Hz, 2H), 6.72 (s, 4H), 7.18 (d,
J ¼ 8.2 Hz, 2H), 7.28 (d, J ¼ 8.2 Hz, 2H), 7.45 (d, J ¼ 8.2 Hz, 4H). MS-
(APCI): m/z 513.3 [Mþ1]þ. Anal. Calcd for C33H44N2O2·1.7H2SO4: C,
60.10; H, 7.03; N, 4.12. Found: C, 60.05; H, 7.10; N, 3.89.
binding was defined by 10 mM histamine as competing ligand.
6.2.2. Rat histamine H3 receptor binding assay
Rat brain membranes, prepared according to Kilpatrick’s
method [50], were incubated for 45 min with [3H]-(R)-
a
-methyl-
histamine 0.5 nM and the compounds under study
(0.03 nMe10 M), in TriseHCl 50 mM, pH 7.4, NaCl 50 mM, EDTA
0.5 mM, then rapidly filtered (AAWP Millipore filters 0.8 m) under
vacuum and rinsed twice with the same ice-cold buffer. Nonspecific
binding was defined by 10 M thioperamide as competing ligand.
6.1.10.16. 1-{[40-(4-(4-Methoxyphenoxy)-1-((S)-2-methylpyrrolidin-
1-yl)-butyl)biphenyl-4-yl]methyl}piperidine (24·2H2SO4), isomer
B. The compound was synthesized from 29n. Yield: 78%; crystal-
m
m
lized from Et2O; mp: 131e138 ꢁC. 1H NMR (CDCl3, 300 MHz)
d 0.80
(d, J ¼ 6.3 Hz, 3H), 1.40e1.26 (m, 3H), 1.72e1.40 (m, 8H), 1.90e1.74
(m, 2H), 2.10e1.98 (m, 1H), 2.37e2.25 (m, 4H), 2.44 (q, J ¼ 7.8 Hz,
1H), 2.88 (ddd, J ¼ 8.9, 7.7, 4.1 Hz, 1H), 2.96 (m, 1H), 3.42 (s, 2H),
3.44 (dd, J ¼ 10.6, 3.7 Hz, 1H), 3.65 (s, 3H), 3.74 (t, J ¼ 6.4 Hz, 2H),
6.70 (s, 4H), 7.28 (d, J ¼ 8.1 Hz, 2H), 7.29 (d, J ¼ 8.2 Hz, 2H), 7.45 (d,
m
6.2.3. Human histamine H3 receptor functional assay
Compounds were added directly to the media containing SK-N-
MC cells expressing the human histamine H3 receptor as well as the

228
F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
construct gene (
b
-galactosidase), followed, 5 min later, by addition
M). The compounds (1 nMe10 M) were added
10 min prior to (R)- -methylhistamine (0.1e100 nM). After a 6 h
incubation at 37 ꢁC, the medium was aspirated and the cells were
lysed with 25
L of 0.1 ꢄ assay buffer (mM composition: NaH2PO4
10, Na2HPO4 10, pH 8, MgSO4 0.2, MnCl2 0.01) and after 10 min with
100
L of 1 ꢄ assay buffer (NaH2PO4 100, Na2HPO4 100, pH 8, MgSO4
2, MnCl2 0.1) containing 0.5% Triton and 40 mM -mercaptoetha-
nol. Color was developed using 25 L of 1 mg/mL substrate solution
(chlorophenol red -galactopyranoside; Roche Molecular
0.05 nM [125I]
a-bungarotoxin in the absence or presence of the test
compound in a buffer containing 50 mM K2HPO4/KH2PO4 (pH 7.4),
10 mM MgCl2 and 0.1% BSA [56]. Nonspecific binding was deter-
mined in the presence of 1 mM a-bungarotoxin. Following incuba-
tion, the samples were filtered rapidly under vacuum through glass
fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed
several times with ice-cold 50 mM TriseHCl using a 96-sample cell
harvester (Unifilter, Packard). The filters were dried then counted
for radioactivity in a scintillation counter (Topcount, Packard) using
a scintillation cocktail (Microscint 0, Packard). The results are
of forskolin (5
m
m
a
m
m
b
m
b-D
Biochemicals, Indianapolis, IN, USA) and quantitated on a micro-
plate reader by measuring the absorbance at 570 nm (Biorad
microplate reader 550, Segrate, MI, Italy) [40].
expressed as a percent inhibition of the control radioligand (
a
-
-
bungarotoxin) specific binding. The percent inhibition of [125I]
a
bungarotoxin binding exerted by compound 21 was evaluated at
100 and 1 M, in experiments performed in duplicate.
m
6.2.4. Guinea-pig histamine H1 and H2 receptor functional assay
Portions of guinea-pig ileum were longitudinally mounted (1 g
load) in organ chambers, filled with KrebseHenseleit solution (mM
composition: NaCl 118.9, KCl 4.6, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25,
MgSO4 1.2, glucose 11.1) and gassed with 95% O2-5% CO2 at 37 ꢁC.
Spontaneously beating isolated atria were isometrically mounted
(0.5 g load) in organ chambers filled with Ringer-Locke solution
(mM composition: NaCl 154.0, KCl 5.6, CaCl2 1.08, NaHCO3 5.95,
glucose 11.1). Atrial responses were determined as changes in the
rate of spontaneous beating by means of a connected car-
diotachograph. Cumulative doseeresponse curves of histamine in
6.3. Molecular modeling
6.3.1. Molecular dynamics simulations
Molecular modeling studies were performed with Macromodel
9.8 [57]. Starting structures of (S,R) and (R,R) isomers of {40-[1-
(2-methylpyrrolidin-1-yl)pentyl]biphenyl-4-yl}methanol (29c,d)
were optimized with the MM3 force field [58], using a convergence
criterion of 0.05 kJ molꢃ1 Åꢃ1. Stochastic dynamics (SD) simulations
were performed employing the MM3 force field, applying the GB/
SA [59] CHCl3 solvation treatment. SD was conducted for 1 ms to
the ileum (1 nMe1
m
M) or of the H2-agonist dimaprit in the atria
a temperature of 298 K, after 1000 ps of equilibration, applying
a time step of 1 fs. 10,000 snapshots were collected for data anal-
ysis. SD has already proved to be able to reproduce the conforma-
tional equilibrium of small molecules in CHCl3 applying an implicit
treatment of solvent molecules [60e66]. Protoneproton coupling
constants were calculated with the software Maestro 9.1 [67],
employing an implemented version of the Karplus equation [68].
(0.1e100 M) were obtained in the absence and presence of the
m
compounds tested, up to a concentration of 10
their H1 and H2 antagonistic activity [41].
mM, to ascertain
6.2.5. Data analysis
The data are presented as means ꢂ SEM. In functional tests
results are expressed as percentage of the maximum response
induced by the agonist. The antagonistic affinities were estimated
determining pKB as described by Furchgott’s equation [51]. When
non-surmountable antagonism was detected, the antagonist
6.3.2. H3 receptor homology modeling and docking
The X-ray crystal structure of the histamine H1 receptor (PDB ID:
3RZE) [44] was used as template for the H3 receptor modeling. The
amino acid sequence of the human H3 receptor was retrieved from
the Universal Protein Resource (UniProt ID: Q9Y5N1) [69,70]. Initial
alignment was carried out with ClustalW2 [71] using default
parameters and subsequently optimized considering conserved
sequence motifs among class A GPCRs [72e74]. Highly conserved
residues within transmembrane (TM) domains [72], as well as the
conserved disulfide bridge connecting the extracellular loop (ECL) 2
and TM3 have been taken into account during alignment refine-
ment. The disulfide bond connecting ECL3 and the top of TM6 in the
H1 histamine receptor crystal structure was also considered during
the optimization of the sequence alignment (Figure S6). Since T4-
lysozyme replaced intracellular loop 3 (ICL3) in the H1 receptor
crystal structure, the corresponding sequence of the H3 receptor was
excluded from homology modeling procedure and Thr229 at the end
of TM5 was linked to Phe348 belonging to the last portion of ICL3.
This should not impact the ligand docking experiments, since the
binding pocket of H3 receptor is located near the extracellular region.
Comparative modeling was carried out with Modeller 9v7 [75]
and thirty H3 receptor models were initially generated. The
stereochemical quality of the models was evaluated using Procheck
[76], as well as the Protein Report tool implemented in Maestro 9.1.
The best model was selected on the basis of geometrical parameters
quality and on Modeller objective function. Since the ECL2 portion
is known to play an important role in shaping the binding site and
could be directly involved in ligand stabilization, the selected
receptor model was submitted to an additional refinement proce-
dure, in which the terminal segment of ECL2 (from Tyr189 to
Asn195) was optimized using Modeller 9v7. The final H3 receptor
model was then refined with the Protein Preparation Wizard
0
potency of the drugs was expressed by pD2 values determined
according to Van Rossum’s equation [52].
In binding assays, pIC50 values were estimated from the
displacement curves of the tested compounds versus [3H]-(R)-
a-
methylhistamine or [3H]-histamine and converted to pKi values
according to Cheng and Prusoff’s equation [53].
6.2.6. Human muscle-type nicotinic receptor binding assay
The test was performed by Cerep [54]. Cell membrane homoge-
nates (60
m
g protein) were incubated for 120 min at 22 ꢁC with 0.5 nM
[
125I]
a-bungarotoxin in the absence or presenceof thetestcompound
in a buffer containing 20 mM Hepes/NaOH (pH 7.3), 118 mM NaCl,
4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4 and 0.1% BSA [55].
Nonspecific binding was determined in the presence of 5 mM a-
bungarotoxin. Following incubation, the samples were filtered
rapidly under vacuum through glass fiber filters (GF/B, Packard)
presoaked with 0.3% PEI and rinsed several times with an ice-cold
buffer containing 50 mM TriseHCl, 150 mM NaCl and 0.1% BSA
using a 96-sample cell harvester (Unifilter, Packard). The filters were
dried then counted for radioactivity in a scintillation counter (Top-
count, Packard) using a scintillation cocktail (Microscint 0, Packard).
The results are expressed as a percent inhibition of the control radi-
oligand (
125I]
-bungarotoxin binding exerted by compound 21 was evaluated
at 100 and 1 M, in experiments performed in duplicate.
a-bungarotoxin) specific binding. The percent inhibition of
[
a
m
6.2.7. Human a7 neuronal nicotinic receptor binding assay
The test was performed by Cerep [54]. Cell membrane homog-
enates (20 m
g protein) were incubated for 120 min at 37 ꢁC with

F. Bordi et al. / European Journal of Medicinal Chemistry 48 (2012) 214e230
229
workflow of Maestro 9.1. Hydrogen atoms were added and
References
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receptor model was subjected to restrained molecular mechanics
refinement to a RMSD of 0.3 Å with the OPLS2005 force field [77].
The Ramachandran plot for the final refined structure is reported in
Figure S7.
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Appendix. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.12.019

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