Synthesis and structure-activity relationships for biphenyl H3 receptor antagonists with moderate anti-cholinesterase activity

Article abstract of DOI:10.1016/j.bmc.2008.10.029

The combination of antagonism at histamine H₃ receptors and inhibition of acetylcholinesterase has been recently proposed as an approach to devise putative new therapeutic agents for cognitive diseases. The 4,4′-biphenyl fragment has been repor

Full text of DOI:10.1016/j.bmc.2008.10.029

Bioorganic & Medicinal Chemistry 16 (2008) 9911–9924
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships for biphenyl H3 receptor
antagonists with moderate anti-cholinesterase activity
a
a
Giovanni Morini a, Mara Comini a, Mirko Rivara a, Silvia Rivara a, , Fabrizio Bordi , Pier Vincenzo Plazzi ,
*
Lisa Flammini b, Francesca Saccani 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-43100 Parma, Italy
b Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università degli Studi di Parma, V. le G.P. Usberti 27/A, I-43100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2008
Revised 2 October 2008
Accepted 12 October 2008
Available online 17 October 2008
The combination of antagonism at histamine H3 receptors and inhibition of acetylcholinesterase has been
recently proposed as an approach to devise putative new therapeutic agents for cognitive diseases. The
4,40-biphenyl fragment has been reported by us as a rigid scaffold leading to potent and selective non-
imidazole H3-antagonists. Starting from these premises, the current work presents an expanded series
of histamine H3 receptor antagonists, characterized by a central 4,40-biphenyl scaffold, where the struc-
ture–activity profile of both mono-basic and di-basic compounds is further explored and their ability to
inhibit rat brain cholinesterase activity is determined. The steric properties and basicity of the terminal
groups were modulated in symmetrical compounds, carrying identical substituents, and in asymmetrical
compounds, having a piperidine ring at one end and different groups at the other. The length of the linker
connecting the biphenyl scaffold to the terminal groups was also modulated. Binding studies at rat and
human H3 receptors evidenced the highest binding affinities for di-basic compounds, in the order of nM
concentrations, and that the steric requirements for the two terminal groups are different. Many potent
compounds showed good selectivity profiles over the other histamine receptors. Interestingly, some
derivatives displayed a moderate ability to inhibit rat brain cholinesterase, for example compound 12
(1-[2-(40-piperidinomethyl-biphenyl-4-yl)ethyl]piperidine) has a pIC50 = 5.96 for cholinesterase inhibi-
tion and high H3 receptor binding affinity and antagonist potency (pKi = 8.70; pKB = 9.28). These com-
pounds can be considered as rigid analogs of a recently reported class of dual-acting compounds and
as a promising starting point for the design of new H3-antagonists with anti-cholinesterase activity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Histamine H3 receptor
H3-antagonist
Cholinesterase inhibition
Structure–activity relationship
1. Introduction
The high density of H3 receptors in different CNS areas and their
influence on the release of a large variety of neurotransmitters
Histamine is a biogenic amine that influences a wide range of
pathophysiological processes through the activation of different
G protein-coupled receptors (GPCRs). At present, four subtypes of
histamine GPCRs are known. H1 and H2 receptors are implicated
in allergic responses and gastric acid secretion, respectively. The
most recently discovered H4 receptor is mainly located on mast
cells, eosinophils and lymphoid tissues and seems to be involved
in inflammatory processes.1 The histamine H3 receptor was identi-
fied in 1983 and was initially described as an autoreceptor, mainly
expressed in the central nervous system (CNS), regulating hista-
mine biosynthesis and release from histaminergic neurons.2 Subse-
quently, H3 receptors have also been shown to act as
heteroreceptors on non-histaminergic neurons, where they inhibit
the release of other neurotransmitters such as acetylcholine, dopa-
mine, norepinephrine, serotonin and various neuropeptides.3–6
encouraged wide pharmacological investigation on their physio-
logical role and the quest for potential therapeutic applications of
H3-antagonists in the treatment of various CNS diseases. Among
them, the most promising ones include attention-deficit hyperac-
tivity disorders (ADHD), Alzheimer’s disease, epilepsy, schizophre-
nia, obesity and eating disorders.7,8
Since the discovery of the reference antagonist thioperamide,9
many classes of potent and selective H3-antagonists have been re-
ported. The earliest generation of H3-antagonists was derived from
the endogenous neurotransmitter histamine and the compounds
contained an imidazole ring in their structures.10,11 It is now well
established that the presence of the imidazole ring may lead to
low CNS penetration12,13 and potential metabolic liabilities due
to the interaction with cytochrome P450.14–18 Such liabilities seem
to be avoided by new classes of non-imidazole antagonists,19–22
comprising some interesting compounds that proved to block the
H3-receptor at nanomolar concentrations and to possess promising
efficacy in several experimental models of central disorders. This
* Corresponding author. Tel.: +39 0521 905062; fax: +39 0521 905006.
E-mail address: silvia.rivara@unipr.it (S. Rivara).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.029

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G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
approach led to the selection of some imidazole-free compounds
for clinical studies.21,23 Recently, some new classes of H3-antago-
nists have been described, endowed with additional pharmacolog-
ical properties that may synergistically potentiate their therapeutic
efficacy in the treatment of disorders related to neurotransmitter
deficits, such as Alzheimer’s disease. Accordingly, the development
of dual-acting compounds combining H3 receptor-blocking proper-
ties with cholinesterase inhibition has been put forward
(Fig. 1),24,25 as well as dual H3 and muscarinic M2 antagonists.26
Our researches in the field of H3-antagonists led us to the prep-
aration of a series of non-imidazole derivatives, characterized by a
basic piperidine ring connected through an alkyl spacer of variable
length to another piperidine or to heterocyclic rings (e.g. 1).27–29
This flexible class of H3-antagonists was subsequently rigidified
by the introduction of a biphenyl central core, leading to com-
pounds, such as 3a with two piperidines on the 4,40-bis-methyl-
biphenyl scaffold, displaying nM binding affinities at both human
one side a piperidine ring and on the other side a second group
chosen to modulate its size, shape and basicity (5a–5i). The length
of the linker connecting the biphenyl scaffold to the amine frag-
ments was varied by introducing an ethylene chain, instead of
the methylene spacers, in a symmetrical or asymmetrical way (
7b, 7c, 12, and 16). Finally, we evaluated the effect produced by
the presence of only one basic fragment in compounds with in-
creased spacer length (14, 15). The compounds were characterized
for their binding affinity at human H3 and H4 receptors and at rat
H3 receptors; their H3-antagonist behavior was assessed by func-
tional tests performed on human and guinea-pig receptors, while
H1 and H2 antagonism was measured on guinea-pig preparations.
Inhibition of cholinesterase activity was evaluated on rat brain
preparations and the effect of the compounds on cell viability
was also assessed. Moreover, the synthesis, the characterization
of H1 and H2 antagonist behavior, the binding affinity at H4 recep-
tor and the cholinesterase inhibitory activity of compounds 3a,
3d–3k, 5j–5l, and 7a are also reported for the first time. The results
obtained for the newly synthesized compounds, merged with the
data previously obtained for symmetric compounds, allow a better
characterization of the structural features required to combine po-
tent H3 antagonism with moderate anti-cholinesterase activity for
this class of biphenyl derivatives.
30
and rodent H3 receptors (Chart 1).
Within this class of biphenyl H3-antagonists, the reduction of
basicity of one or both the terminal fragments or the introduction
of primary or secondary amines led to a huge drop in binding affin-
ity.30 The most interesting derivative 3a was further characterized,
resulting devoid of any significant activity at the other histamine
receptors and endowed with weak anti-cholinesterase activity
(pIC50 = 5.23). Moreover, in ex-vivo binding experiments it dis-
played a prompt and long-lasting presence in rat CNS, and in the
passive avoidance test it resulted as effective as the reference
anti-Alzheimer drug donepezil in attenuating the scopolamine-in-
duced amnesia in rats.31
2. Chemistry
The biphenyl derivatives were prepared following the synthetic
routes outlined in Schemes 1 and 2.
With the aim to extend the structure–activity relationships
(SAR) exploration for this class of biphenyl H3-antagonists, we syn-
thesized a larger series of molecules, testing their ability to selec-
tively block histamine H3 receptors and to inhibit cholinesterase
activity in rat brain tissues. Accordingly, while conserving the
4,40-bis-methyl-biphenyl core scaffold, we prepared both symmet-
rical compounds (3b and 3c) and asymmetrical ones, carrying on
R2
R1
7a-7c
f,g
COOH
HOOC
6
N
N
O
N
d,e
N
H
FUB 833
Cl
Cl
2
O
OH
a
b
N
17
R1
N
Figure 1. Histamine H3 receptors antagonists-acetylcholinesterase inhibitors.
R2
Cl
3a-3k
4
N
N
c
1
rH3 pKi=7.80 hH3 pKi=8.35
N
R2
5a-5l
N
N
Scheme 1. Reagents and conditions: (a) primary or secondary amine, THF, MW; (b)
piperidine, Et3N, THF, MW; (c) amine (THF, MW), or sodium salt of 2-piperidone or
4-hydroxybenzonitrile (anhydrous toluene, reflux); (d) NaCN , H2O/CH3CN, MW; (e)
i—KOH , EtOH, MW; ii—HCl 2N; (f) i—CDI, THF, MW; ii—amine, THF, MW; (g)
RedAlÒ, anhydrous toluene.
3a rH3 pKi=8.92 hH3 pKi=8.47
Chart 1.

G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
9913
N
C
O
N
15
f
N
C
O
OH
13b
e
I
N
C
O
9b
a
a
I
HOOC
8
I
N
C
O
9a
b
e
COOH
N
C
O
N
N
C
OH
O
10
13a
14
c
f
N
O
C
O
N
N
C
O
11
12
g
d
N
N
N
N
16
Scheme 2. Reagents and conditions: (a) i—CDI, CH3CN, MW; ii—amine, CH3CN, MW; (b) i—4-carboxyphenylboronic acid, Pd(OAc)2, K2CO3, acetone, H2O, 65 °C; ii—HCl 1N; (c)
i—CDI, CH3CN, MW; ii—piperidine, MW; (d) RedAlÒ, anhydrous toluene; (e) 4-hydroxymethylphenylboronic acid, Pd(OAc)2, K2CO3, acetone, H2O, 65 °C; (f) i—MsCl, Et3N,
CH2Cl2; ii—amine, CH3CN; (g) RedAlÒ, anhydrous toluene.
As depicted in Scheme 1, the symmetrical compounds 3a–3k
were obtained by reacting the commercially available 4,40-
bis(chloromethyl)-biphenyl 2 with the appropriate amine.32 The
asymmetrical compounds 5a–5l were prepared reacting 2 with
piperidine to obtain the monosubstituted intermediate 4. After iso-
lation and purification, 4 was reacted with the suitable amine or
the sodium salt of 2-piperidone or of 4-hydroxybenzonitrile to ob-
tain the desired products. The synthesis of compounds 3b–3d and
3f, 3i, and 3j is described in Ref. 32.
reduced with RedAlÒ33 to obtain the target diamino compounds
7a–7c.
The synthesis of asymmetrical compounds 12, 14–16 is outlined
in Scheme 2. The commercially available 2-(4-iodophenyl)acetic
acid (8) was activated with CDI and reacted with piperidine or dipro-
pylamine to give the intermediates 9a and b. These intermediates
were submitted to Suzuki coupling with commercially available 4-
carboxyphenylboronicacid or with 4-hydroxymethylphenylboronic
acid to give the carboxylic acid 10 and the hydroxymethylbiphenyl
derivatives 13a and b, respectively. Compound 10 was activated
with CDI and reacted with piperidine to give the diamide 11 that
was reduced with RedAlÒ to obtain the final compound 12. Com-
pounds 13a and b were activated with mesyl chloride (MsCl) and re-
acted with the appropriate amine to obtain the final compounds 14
and 15. Reduction of compound 14 with RedAlÒ gave derivative 16.
Compounds 7a–7c were synthesized reacting 4,40-bis(chloro-
methyl)-biphenyl 2 with NaCN to obtain the dicyano derivative,
that was hydrolyzed in ethanolic KOH; acidification provided the
symmetrical dicarboxylic acid 6. Intermediate 6 was activated with
1,10-carbonyl-diimidazole (CDI) and reacted with the appropriate
amine to give the corresponding tertiary diamides that were finally

9914
G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
SK–N–MC cells were incubated with the compounds under study
(1–10 M) or with the vehicle for 6 h. At the end of the period of
incubation, 10 l of 5 mg/ml MTT solution were added to each well.
After 3 h the culture medium was removed, the cells washed with
phosphate buffer solution (PBS) and 200 l of formazan solubiliza-
tion solution (0.1 N HCl in anhydrous isopropanol) were added. Cul-
ture medium absorbance was spectrophotometrically read at
570 nm (Biorad microplate reader 550, Segrate, MI, Italy). Cell via-
bility was expressed as relative viability compared to control.
3. Pharmacology
3.1. Materials and methods
l
l
l
Drugs used were purchased from Sigma (St. Louis, MA, USA).
Cultured SK-N-MC cells stably expressing human histamine H3 or
H4 receptors and the reporter gene b-galactosidase (Johnson &
Johnson Pharmaceutical Research and Development, L.L.C.) were
used for binding and functional studies. Functional experiments
were also performed on isolated organs excised from guinea-pigs
(250–350 g) whereas binding and colorimetric assays were carried
out using brain preparations from male Wistar rats (150–200 g)
(Charles River, Italy). 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 com-
pliance with Italian regulations (DL 116/92) and with the local Eth-
ical Committee Guidelines for Animal Research.
3.6. Functional studies on isolated tissues
3.6.1. Field stimulated guinea-pig ileum
Portions of guinea-pig ileum were longitudinally mounted (1 g
load) in organ chambers, filled with Krebs–Henseleit solution
(mM composition: NaCl 118.9, KCl 4.6, CaCl2 2.5, KH2PO4 1.2, NaH-
CO3 25, MgSO4ꢁ7H2O 1.2, glucose 11.1) and gassed with 95% O2–5%
CO2 at 37 °C. The tissues were electrically stimulated (0.1 Hz, 1 ms,
submax voltage) (LACE, Ospedaletto PI, Italy). The H3-antagonist
3.2. Rat histamine H3 receptor binding assay
activity of the tested compounds (1 nM to 30
ally determined on twitch contractions inhibition induced by (R)-
-methylhistamine cumulatively administered (1 nM to 1 M) in
the presence of 1 M mepyramine.
lM), was function-
Rat brain membranes, prepared according to Kilpatrick’s meth-
od,34 were incubated for 45 min with [3H]-(R)-
a
-methylhistamine
0.5 nM ([3H]RAMHA) and the compounds under study (0.03 nM to
10 M), in Tris–HCl 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 bind-
ing was defined by 10 M thioperamide as competing ligand.
a
l
l
l
3.6.2. Guinea-pig isolated ileum and atria
l
Guinea-pig terminal ileum portions (H1 receptors) and atria (H2
receptors) were isometrically suspended in organ baths filled with
Krebs–Henseleit (37 °C) and Ringer–Locke solution (31 °C) (mM
composition: NaCl 154.0, KCl 5.6, CaCl2 1.08, NaHCO3 5.95, glucose
11.1), respectively, and aerated with 95% O2–5% CO2. Atrial re-
sponses were determined as changes in the rate of spontaneous
beating by means of a connected cardiotachograph. Cumulative
l
3.3. 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 or H4 receptors,
were used in radioligand displacement studies according to litera-
ture methods.35,36 Membranes were incubated for 60 min at room
dose–response curves of histamine in ileum (1 nM to 1
lM) or of
H2-agonist dimaprit in atria (0.1–100 M) were obtained in the ab-
l
temperature with 0.5 nM [3H]-(R)-
a-methylhistamine (47.0 Ci/
sence and in the presence of the compounds tested up to 10
lM
concentration.10
mmol, Amersham Bioscience) or with 10 nM [3H]histamine
(18.1 Ci/mmol, Perkin Elmer) in the absence or in the presence of
3.6.3. Rat brain cholinesterase inhibition
competing ligands (0.01 nM to 10 lM). Incubation was terminated
The inhibition of brain cholinesterase was determined spectro-
photometrically using acetylthiocholine as substrate according to
the method of Ellman et al.39 Aliquots of rat brain homogenates
were incubated in phosphate buffer 0.1 M (pH 8.0) with 5,50-dithi-
obis(2-nitrobenzoic acid) (DTNB) (5 mM) and test compounds at
by rapid filtration over Millipore AAWPO2500 filters followed by
two washes with ice-cold buffer (50 mM Tris–HCl/5 mM EDTA).
Nonspecific binding was defined by 10 or 100 lM histamine as
competing ligand for H3 and H4 receptors, respectively.
appropriate concentrations (1–100
lM). The reaction was started
3.4. Human histamine H3 receptor functional assay
at 37 °C by adding 20 l of acetylthiocholine (75 mM). The reaction
l
was stopped after 15 min by adding formalin (4%). The hydrolysis
of acetylthiocholine catalyzed by the enzyme was determined by
monitoring the formation of the yellow 5-thio-2-nitrobenzoate
anion at a wavelength of 412 nm (Biorad microplate reader 550,
Segrate, MI, Italy). The percent inhibition of cholinesterase was
calculated as follows: [(absorbancecontrol ꢂ absorbancesample)/
absorbancecontrol] ꢀ 100. Results are provided as pIC50 (ꢂLog con-
centration causing half-maximal inhibition of cholinesterase).
Compounds were added directly to the media containing SK-N-
MC cells expressing the human histamine H3 receptor as well as
the construct gene (b-galactosidase), followed, 5 min later, by addi-
tion of forskolin (5
added 10 min prior to (R)-
a 6 h incubation at 37 °C, the medium was aspirated and the cells
were lysed with 25
l of 0.1ꢀ assay buffer (mM composition:
NaH2PO4 10, Na2HPO4 10, pH 8, MgSO4 0.2, MnCl2 0.01) and after
10 min with 100
l of 1ꢀ assay buffer (NaH2PO4 100, Na2HPO4
100, pH 8, MgSO4 2, MnCl2 0.1) containing 0.5% Triton and
40 mM b-mercaptoethanol. Color was developed using 25 l of
1 mg/ml substrate solution (chlorophenol red b- -galactopyrano-
lM). The compounds (1 nM to 10
lM) were
a
-methylhistamine (0.1–100 nM). After
l
l
3.7. Data analysis
l
The data are presented as means SEM. In functional tests re-
sults are expressed as percentage of the maximum response in-
duced by the agonist. The pEC50 values (ꢂLog of the
concentration giving half-maximal effect) and slopes of regression
lines were calculated by analyzing agonist concentration–response
curves by the least-squares method. The antagonistic affinities
were estimated determining pKB (‘apparent pA2’) as described by
Furchgott’s equation.40 When non-surmountable antagonism was
D
side; Roche Molecular Biochemicals, Indianapolis, IN, USA) and
quantitated on a microplate reader by measuring the absorbance
at 570 nm (Biorad microplate reader 550, Segrate, MI, Italy).37
3.5. Cell viability
Cell viability was determined through colorimetric quantifica-
tion of formazan derived from 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) metabolic reduction.38
detected, the antagonist potency of the drugs was expressed by
0
pD2 values determined according to Van Rossum’s equation.41

G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
9915
In in vitro binding assays, pIC50 values were estimated from the
Generally, the compounds exhibited a good selectivity profile,
since some of them interacted with H1, H2, and H4 receptors only
at concentrations about 1000 or 10,000 times higher than those re-
quired to bind H3 receptors. Only compounds 5b and 7a interacted
at micromolar concentrations with H1 and H2 receptors, respec-
tively. Moreover, none of the compounds affected SK-N-MC cell
viability, since no alterations were observed in the MTT test at con-
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.42
4. Results and discussion
The binding and functional characterization of the newly syn-
thesized biphenyl derivatives 3b, 3c, 5a–5i, 7b, 7c, 12, and 14–16
are reported in Table 1, together with the previously described
data for compounds3a, 3d–3k, 5j–5l, and 7a.30,31 While all the pre-
viously reported biphenyl derivatives showed higher binding affin-
ities for human H3 receptors than for rat H3 receptors, some of the
newly synthesized compounds have similar affinity values (e.g. 3b
and 5d) or a slightly higher binding affinity for rat H3 receptors (3c,
5c, 5e, and 14). The new compounds proved to be antagonists at
human and guinea-pig H3 receptors, with highly correlated pKi
and pKB data at human H3 receptors (r = 0.83). The symmetric com-
pounds 3b and 3c, with diethylamine and dipropylamine substitu-
ents, respectively, confirmed the importance of tertiary amines for
high H3 binding affinity, since they roughly maintained the high
potency of the dimethylamino derivative 3d, compared to the
bis-secondary amines 3e and 3f. However, the best arrangement
for the lipophilic terminals of the basic groups remains that of
the piperidine rings of compound 3a, even if steric tolerance for
the acyclic tertiary amine 3c seems higher than for the more rigid
substituted piperidine 3g. As for the antagonist effect on human H3
receptors, in a cAMP-responsive element (CRE) mediated b-galac-
tosidase reporter gene assay and on guinea-pig native H3 receptors,
compound 3b was the most potent among the symmetrical com-
pounds (pKB = 8.96 and 9.34, respectively).
The asymmetric derivatives 5a–5l, maintaining a piperidine
ring on the 4,40-bis-methyl-biphenyl scaffold, generally exhibited
higher H3 affinity and antagonist potency than the corresponding
symmetric compounds (3c, 3d, and 3f–3k). A relevant increase in
binding affinity at both rat and human H3 receptors and in the
inhibitory potency at guinea-pig and human H3 receptors is evi-
dent for compounds5a, 5b, 5j, and 5k. Compound 5b, in particular,
was characterized by remarkable binding affinity, particularly at
human H3 receptors, showing that the binding sites for the two ba-
sic groups have different steric tolerance. On the other hand, inclu-
sion of electron-withdrawing fragments in close proximity to one
of the basic groups (5c and 5j) led to a significant decrease of
receptor affinity. This trend is confirmed by the lower affinity ob-
served for compounds 5d and 5l.
To further explore the SAR of the biphenyl series, we synthes-
ised a few symmetrical compounds with a longer spacer between
the central core and the basic fragments (compounds 7a–7c). As
previously observed for 7a, this modification is tolerated for recep-
tor binding, only producing a slight reduction of affinity and antag-
onist potency compared to those of the corresponding shorter
compounds, while maintaining the same rank order for the substi-
tuent effect (see compounds 3a, 3d, and 3h, respectively). The
introduction of one monomethylene and one two-carbon linker be-
tween the biphenyl scaffold and the nitrogen atoms led to asym-
metric compounds 12 and 14–16. Compound 12, having two
piperidine rings, showed a limited decrease in receptor affinity,
compared to its shorter and longer analogs, but it showed the high-
est potency on human H3 receptors in the CRE-mediated b-galacto-
sidase reporter gene assay. One of its piperidine rings can be
replaced by a dipropylamino group (16) with negligible reduction
of receptor affinity, while transformation of one of the two basic
amines into an amide fragment led to higher decreases of activity,
even if the N,N-dipropylamino-carbonyl group of 15 was better tol-
erated than other neutral groups on the piperidino-methyl-biphe-
nyl scaffold (see 5d and 5l).
centrations between 1 and 10 lM (data not shown).
Different series of compounds carrying two basic centers have
been described as cholinesterase inhibitors: they comprise poly-
amine derivatives,43–45 meptazinol derivatives,46 as well as H3-
antagonists.24 The interesting anti-cholinesterase activity already
described for symmetrical compounds 3a, 3f, 3h, 3k, and asym-
metrical compounds 5j and 5k31was maintained by asymmetrical
compounds 5a, 5b, 5g, 5h, and 5i and by compounds 12 and 14–
16. Comparing the inactivity of the symmetrical, longer com-
pounds 7a and 7c with the anti-cholinesterase property of their
shorter analogs 3a and 3h it can be speculated that the length of
the spacer connecting the basic substituents to the biphenyl scaf-
fold represents a critical feature for cholinesterase inhibition. In-
deed, compound 12, having an intermediate length, showed the
highest cholinesterase inhibitory potency coupled to good human
H3 binding affinity and antagonist potency. Even if its pIC50 value
on rat brain cholinesterase activity (5.96) is considerably lower
than that of donepezil in the same experimental conditions
(pIC50 = 7.45),31 this compound may represent a starting point to
develop new H3-antagonists also endowed with anti-cholinester-
ase activity. In fact, its antagonist potency on cells expressing hu-
man H3 receptors, its cholinesterase inhibitory potency and its
selectivity profile on other histamine receptors are comparable, if
not better, than those observed for compound 3a, that showed
promising efficacy in attenuating scopolamine-induced amnesia
in rats.31
Two different docking solutions for the most potent compound
3a, into a homology model of rat H3 receptor, have been previously
described by us.30 In the best scoring pose, compound 3a is ori-
ented ‘horizontally,’ in a direction perpendicular to the axes of
the transmembrane (TM) helices, and it forms two salt bridges be-
tween its protonated piperidines and the carboxylate groups of
Asp114 in TM3 and Glu206 in TM5. Glu206 is important for the
binding of the natural ligand histamine and is supposed to interact
with the imidazole ring,47 while Asp114, the conserved acidic res-
idue present in many class A G protein-coupled receptors, is
thought to bind the side chain amine of histamine. In the crystal
structure of the b2 adrenergic receptor Asp113, corresponding to
the H3 Asp114, binds the secondary amine group of the co-crystal-
lized inverse agonist carazolol.48 The docking protocol also pro-
vided an alternative, ‘vertical’ pose for 3a, roughly parallel to the
axes of the TM helices. In this pose, a piperidine nitrogen interacts
with Asp114 and the central scaffold occupies a region delimited
by TM3, 6, and 7, leading the second piperidine to form a hydrogen
bond with the side chain of Asn404 in TM7. Both orientations have
been proposed for other classes of non-imidazole H3 receptor
antagonists.49–51
Other representatives of this series of compounds (3g, 5b, 5d,
and 7a), docked into the rat H3 receptor model, gave alternative
solutions corresponding to horizontal and vertical poses. As the
distance between Glu206 and Asp114 is optimal for a double
acid–base interaction with the two piperidine rings of compound
3a, it was expected that the longer derivative 7a would hardly as-
sume the horizontal pose. Actually, it gave both vertical and hori-
zontal docking solutions (Fig. 2), with the first ones preferred,
according to Glide scoring function (see Section 6). On the other
hand, compound 3g, having two 3,5-dimethyl-piperidino-methyl
groups and significantly lower binding affinity, showed no docking
solution with horizontal poses and simultaneous interactions at

Table 1
aAffinity (pKi) and antagonist potency (pKB) of the compounds under study at human (hH3), rat (rH3), guinea-pig (gpH3) histamine H3 receptors, guinea-pig histamine H1 (gpH1) or H2 (gpH2) receptors, human histamine H4 receptors
(hH4).
R1 (CH2)n
(CH2)m R2
d
e
f
g
n
m
R1
R2
rH3 pKib
hH3 pKic
hH3 pKB
gpH3 pKB
gpH1 pKB
gpH2 pKB
hH4 pKih
Cholinesterase
Max Eff. (%)j
i
pIC50
5.23
#
#
3ak
1
1
8.92
9.47
8.77
8.58
pD2 = 4.84
4.60
82
0
N
N
#
#
#
#
#
3b
3c
1
1
1
1
1
1
–N(C2H5)2
–N(C3H7)2
–N(CH3)2
–N(C2H5)2
–N(C3H7)2
–N(CH3)2
8.62 0.10
8.50 0.14
8.18
8.55 0.14
8.03 0.00
8.73
8.96 0.19
7.80 0.09
8.17
9.34 0.27
8.78 0.18
8.46
5.54 0.12
pEC50 = 5.47 0.14
#
#
3dk
k
H
H
§
§
§
§
§
#
3e
1
1
5.48
6.12
6.51
5.19
(C5H11
)
(C5H11)
N
N
H
N
H
N
3fk
1
1
5.68
6.25
6.54
5.82
5.29
70
#
CH3
CH3
#
#
#
3gk
1
1
6.17
6.40
6.19
pD2 = 5.08
4.86
0
N
N
CH3
CH3
CH3
CH3
k
#
#
#
#
3h
3ik
1
1
1
1
7.82
7.09
8.21
7.91
7.68
7.67
7.30
ND
§
5.89
5.45
83
N
N
N
N
#
#
O
N
O
N
CH3
O
CH3
O
#
§
#
#
3jk
1
1
1
1
1
1
5.41
5.85
6.70
pD2 = 4.81
0
N
N
CH3
N
CH3
N
3kk
5a
6.67
7.43
7.27
7.45
pD2 = 5.24
5.19
5.78
57
93
0
CH3
N
#
#
8.13 0.13
8.56 0.01
9.19 0.15
8.46 0.18
5.56 0.02
6.15 0.09
5.86 0.11
5.62 0.09
N
N
CH3
CH3
&
#
5b
1
1
8.62 0.16
9.06 0.06
8.48 0.12
9.02 0.17
91
N

5c
1
1
1
1
7.81 0.08
7.15 0.23
7.71 0.10
7.12 0.06
7.77 0.08
7.27 0.09
9.07 0.13
7.27 0.03
#
#
#
§
#
#
#
#
N
N
N
O
5d
O
CN
#
#
&
#
#
#
#
#
#
#
#
#
5e
5f
1
1
1
1
1
1
1
1
–N(CH3)2
–N(C2H5)2
–N(C3H7)2
8.88 0.11
8.59 0.08
8.50 0.15
8.24 0.10
8.66 0.05
8.71 0.12
8.76 0.05
8.62 0.17
8.92 0.09
8.90 0.16
8.18 0.21
8.64 0.09
9.05 0.21
9.41 0.07
9.12 0.20
8.75 0.26
N
N
N
N
5g
5h
5.48 0.10
5.83 0.08
5.85 0.08
93
93
#
N
CH3
CH3
N
£
#
#
0
5i
1
1
1
1
7.85 0.03
8.17 0.03
7.98 0.13
7.34 0.14
pD2 = 4.75 0.11
5.50 0.11
79
N
N
CH3
O
#
5jk
7.33
8.20
7.86
7.79
pD2 = 4.46
4.94
79
73
0
N
N
H
N
#
#
§
#
#
5kk
5lk
1
1
1
1
8.04
6.94
8.77
7.33
8.03
7.46
7.59
7.57
5.24
N
N
O
N
#
#
#
#
#
#
#
#
7ak
7b
7c
2
2
2
2
2
2
8.40
8.93
8.50
8.43
5.32
6.38
N
N
#
#
–N(CH3)2
–N(CH3)2
7.71 0.11
7.26 0.09
8.29 0.05
7.78 0.14
8.06 0.13
8.17 0.23
8.04 0.17
7.43 0.16
£
5.85 0.01
N
CH3
N
CH3
(continued on next page)

Table 1 (continued)
d
e
f
g
n
m
R1
R2
rH3 pKib
hH3 pKic
hH3 pKB
gpH3 pKB
gpH1 pKB
gpH2 pKB
hH4 pKih
Cholinesterase
Max Eff. (%)j
i
pIC50
#
§
#
12
1
2
8.08 0.12
8.70 0.14
9.28 0.07
8.73 0.14
5.96 0.06
91
N
N
§
§
§
#
#
#
N
14
15
16
1
1
1
1
1
2
–N(C3H7)2
6.75 0.16
7.27 0.04
7.97 0.07
6.60 0.04
8.07 0.15
8.41 0.09
6.96 0.08
8.16 0.15
8.82 0.21
7.24 0.05
8.00 0.19
7.98 0.16
5.44 0.09
4.98 0.08
4.95 0.02
5.17 0.01
65
80
80
O
O
N(C3H7)2
5.44 0.13
N
#
–N(C3H7)2
N
(pIC50) of the compounds on rat brain cholinesterase.
pD20: ꢂlog reducing to 50% the maximal agonist effect.
ND, not determined.
a
Values are means SEM of 4–6 independent experiments.
b
Inhibition of [3H]RAMHA binding to rat brain membranes.
c
Inhibition of [3H]RAMHA binding to SK-N-MC cells stably expressing the human histamine H3-receptor.
Antagonist potency at human histamine H3-receptors expressed in SK-N-MC cells.
Antagonist potency at H3 receptors expressed in guinea-pig ileum.
Antagonist potency at H1 receptors expressed in guinea-pig ileum.
Antagonist potency at H2 receptors expressed in guinea-pig atrium.
Inhibition of [3H]RAMHA binding to SK-N-MC cells stably expressing the human histamine H4-receptor.
Rat brain cholinesterase inhibition.
d
e
f
g
h
i
j
Maximum percent inhibition of rat brain cholinesterase.
k
#
§
&
£
Ref. 30 and Ref. 31.
Inactive up to 10
l
M.
M.
M.
M.
Inactive up to 1
Inactive up to 3
Inactive up to 0.1
l
l
l

G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
9919
Figure 2. Representation of compound 3a (orange carbons) and 7a (green carbons) docked into the rat H3 receptor model. Left: horizontal pose; right: vertical pose. Only
polar hydrogens are shown. The protein backbone is represented by a ribbon; for clarity, a portion of TM6 and TM7 are not shown.
both Glu206 and Asp114, while its asymmetric analog 5b, having
remarkably higher affinity, could accommodate the unsubstituted
piperidine ring close to Glu206 and take a second salt bridge be-
tween its 3,5-dimethyl-piperidine and Asp114. Moreover, com-
pound 5d, having only one basic group, can assume both
horizontal and vertical poses. While the relationship between the
limited decrease of binding affinity for the longer derivative 7a
and its docking preference is unclear, other solutions seem to sup-
port the horizontal pose as more consistent with affinity data. In
fact, if di-basic compounds must bind to their binding site at H3
receptors in a horizontal arrangement, the affinity loss for com-
pound 3g, compared to 5b, may be related to the lack of high-
scored horizontal poses, and that observed for the mono-basic
derivatives (e.g. 5d) can be explained by the lack of one salt bridge
with Glu206 or Asp114.
Compound 12 was docked into the catalytic cavity of mouse
acetylcholinesterase, whose molecular model was built refining a
crystal structure available at the PDB site.52 It gave a best scoring
Figure 3. Compound 12 (cyan carbons) and donepezil (orange carbons) docked into
the mouse acetylcholinesterase active site.
solution with the piperidino-ethyl group deeply inserted into the
active site gorge, similarly to the benzyl group in the docking solu-
tion obtained for donepezil, which was in turn similar to its accom-
modation into Torpedo californica acetylcholinesterase active site,
as observed for a crystal structure.53 As represented in Figure 3,
compound 12 occupies the acetylcholinesterase active site span-
ning from Trp86, near the catalytic triad, to Trp286 belonging to
the periferic anionic site, with the two protonated nitrogen atoms
pointing towards the tryptophan indole rings. This binding mode is
similar to the one reported for the dual-acting flexible H3-antago-
nist-acetylcholinesterase inhibitor 17 (Fig. 1),24 having comparable
inhibitory potency, and suggests that the more rigid and bulky
biphenyl scaffold is tolerated by the acetylcholinesterase active
site. The docking model represented in Figure 3, together with
homology models of the H3 receptor, can assist the design of
new H3-antagonists with anti-acetylcholinesterase activity.
decrease of potency. Some H3-antagonists showed an interesting,
although mild, anti-cholinesterase activity, providing the basis
for the development of dual-acting compounds. Moreover, the ver-
satility of the synthetic routes employed allowed to prepare com-
pounds with different combinations of substituents and of linkers
bound to the central biphenyl core with good yields.
6. Experimental
6.1. Chemistry
6.1.1. General methods
Melting points were not corrected and were determined with a
Gallenkamp melting point apparatus. The final compounds were
analyzed on a ThermoQuest (Italia) FlashEA 1112 Elemental Ana-
lyzer, for C, H, and N. The percentages we found were within
0.4% of the theoretical values. The 1H NMR spectra were recorded
on a Bruker 300 spectrometer (300 MHz); chemical shifts (d scale)
are reported in parts per million (ppm) relative to tetramethylsil-
ane as internal standard. 1H NMR spectra are reported in the fol-
lowing order: multiplicity, approximate coupling constants (J
value) in Hertz (Hz) and number of protons; signals were charac-
terized as s (singlet), d (doublet), dd (doublet of doublets), t (trip-
let), q (quartet), m (multiplet) br s (broad signal). Mass spectra
5. Conclusion
In summary, a series of rigid H3-antagonists characterized by
good receptor affinity and selectivity profile for human and rodent
histamine H3 receptors has been described. Analyzing the biologi-
cal data we can assert that, in di-basic compounds, the presence of
one piperidine connected to the central biphenyl scaffold provides
derivatives endowed with high H3 binding affinity and antagonist
potency. Introduction of non-basic moieties generally leads to a

9920
G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
were recorded using a API-150 EX with APCI interface (Applied Bio-
system, Mds Sciex, Toronto, Canada). Reactions were monitored by
TLC, on Kieselgel 60 F 254 (DC-Alufolien, Merck). Final compounds
and intermediates were purified by preparative flash chromatogra-
phy on BÜCHI SepacoreÒ, using SiO2 column (Prepacked Cartridges,
[M+1]+. Anal. calcd for C28H40N2: C, 83.10; H, 9.96; N, 6.92. Found:
C, 82.86; H, 9.87; N, 6.86.
1,10-[Biphenyl-4,40-diylbis(methylene)]bis-4-methylpiperidine
(3hꢁ0.25H2O). Crystallized from EtOH. Yield: 84%; mp: 98–100 °C.
1H NMR (CDCl3) d 0.92 (d, J = 5.7 Hz, 6H), 1.19–1.40 (m, 6H), 1.60
(d, J = 12.0 Hz, 4H), 1.96 (t, J = 11.7 Hz, 4H), 2.88 (d, J = 11.7 Hz,
4H), 3.51 (s, 4H), 7.38 (d, J = 8.1 Hz, 4H), 7.53 (d, J = 8.1 Hz, 4H).
MS (EI) 377 [M+1]+. Anal. calcd for C25e35N2ꢁ0.25H2O: C, 81.58; H,
9.58; N, 7.61. Found: C, 81.55; H, 9.52; N, 7.60.
SiO2 60, 40–63 lm, BÜCHI); the eluents were mixtures of CH2Cl2/
CH3OH at various volume ratios. When indicated, gaseous NH3
was added to the methanolic phase to obtain a 5% w/w solution.
Microwave reactions (MW) were conducted using a CEM Discover
Synthesis Unit. Abbreviations are the following: EtOAc, ethyl ace-
tate; THF, tetrahydrofuran; DMSO, dimethylsulfoxide; CDI, 1,10-
carbonyl-diimidazole; MsCl, mesyl chloride.
4,40-[Biphenyl-4,40-diylbis(methylene)]bis-morpholine (3i). Crys-
tallized from EtOH. Yield: 60%; mp: 123–125 °C. 1H NMR (CDCl3)
d 2.47 (t, J = 9.1 Hz, 8H), 3.53 (s, 4H), 3.73 (t, J = 9.2 Hz, 8H), 7.38
(d, J = 8.1 Hz, 4H), 7.54 (d, J = 8.1 Hz, 4H). MS (EI) 353 [M+1]+. Anal.
calcd for C22H28N2O2: C, 74.97; H, 8.01; N, 7.95. Found: C, 74.91; H,
8.06; N, 7.76.
6.1.1.1. General method of preparation of [biphenyl-4,40diyl-
bis(methylene)]bis-amine derivatives (3a–3k).
A solution of
4,40-bis(chloromethyl)-biphenyl 2 (1 mmol) was reacted with the
appropriate amine (4 mmol) in THF (2 mL) (MW, 100 °C, 150 W,
100 psi, 5 min). The solvent was removed in vacuo to give the
crude products, which were purified by flash chromatography
[SiO2, CH2Cl2/CH3OH (NH3) 97:3].
4,40-[Biphenyl-4,40-diylbis(methylene)]bis-(piperazin-1yl-ethan-2-
one) (3jꢁH2O). Crystallized from EtOH. Yield: 85%; mp: 188–190 °C.
1H NMR (CDCl3) d 2.07 ppm (s, 6H), 2.59 ppm (m, 8H), 2.37–
2.59 ppm (m, 8H), 3.46 ppm (t, 4H), 3.55 ppm (s, 4H), 3.63 ppm
(t, 4H), 7.37 ppm (d, J = 7.8 Hz, 4H), 7.54 ppm (d, J = 7.8 Hz, 4H).
MS (EI) 435 [M+1]+. Anal. calcd for C26H34N4O2ꢁH2O: C, 68.99; H,
7.57; N, 12.38. Found: C, 68.79; H, 8.00; N, 12.06.
1,10-[Biphenyl-4,40-diylbis(methylene)]bis-piperidine (3a). Crys-
tallized from EtOH. Yield: 64%; mp: 128–130 °C. 1H NMR (CDCl3)
d 1.41–1.51 ppm (m, 4H), 1.55–1.66 ppm (m, 8H), 2.37–2.48 ppm
(m, 8H), 3.50 ppm (s, 4H), 7.37 ppm (d, J = 8.1 Hz, 4H), 7.53 ppm
(d, J = 8.1 Hz, 4H). MS (EI) 349 [M+1]+. Anal. calcd for C24H32N2:
C, 82.71; H, 9.25; N, 8.04. Found: C, 82.51; H, 9.17; N, 7.84.
N,N0-[biphenyl-4,40diylbis(methylene)]bis-diethylamine
N,N0-[biphenyl-4,40diylbis(methylene)]bis-(N-methyl-N-cyclohex-
ylamine) (3k). Crystallized from EtOH. Yield: 69%; mp: 113–115 °C.
1H NMR (CDCl3) d 1.05–1.38 (m, 10H), 1.58–1.86 (m, 2H), 1.76–
1.94 (m, 8H), 2.22 (s, 6H), 2.40–2.51 (m, 2H), 3.59 (s, 4H), 7.37
(d, J = 8.1 Hz, 4H), 7.53 (d, J = 8.1 Hz, 4H). MS (EI) 405 [M+1]+. Anal.
calcd for C28H40N2: C, 83.11; H, 9.96; N, 6.92. Found: C, 83.24; H,
9.98; N, 6.83.
(3bꢁ2HClꢁH2O). Crystallized from abs EtOH/Et2O. Yield: 71%; mp:
262–264 °C. 1H NMR (D2O) d 1.38 ppm (t, J = 14.6 Hz, 12H), 3.21–
3.34 ppm (m, 8H), 4.41 ppm (s, 4H), 7.64 ppm (d, J = 8.2 Hz, 4H),
7.84 ppm (d, J = 8.3 Hz, 4H). MS (EI) 325 [M+1]+. Anal. calcd for
C22H32N2ꢁ2HClꢁ1H2O: C, 63.60; H, 8.73; N, 6.74. Found: C, 63.27;
H, 8.46; N, 6.43.
6.1.1.2. Preparation of 40-chloromethyl-biphenyl-4-ylmethylpi-
peridine (4).
A solution of 4,40-bis(chloromethyl)-biphenyl 2
(1 mmol) in THF (2 mL) was treated with piperidine (0.5 mmol)
and triethylamine (0.5 mmol) (MW, 100 °C, 150 W, 100 psi, 5
min). The solvent was evaporated under reduced pressure; the res-
idue was purified by flash chromatography [SiO2, CH2Cl2:CH3OH
(NH3) = 98:2]. Crystallized from EtOH/H2O. Yield: 63%; mp: 174–
176 °C. 1H NMR (CDCl3) d 1.39–1.49 (m, 2H), 1.53–1.63 (m, 4H),
2.38–2.44 (m, 4H), 3.51 (s, 2H), 4.63 (s, 2H), 7.39 (d, J = 8.1 Hz,
2H), 7.45 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H) 7.58 (d,
J = 8.1 Hz, 2H). MS (EI) 300 [M+1]+. Anal. calcd for C19H22NCl: C,
76.11; H, 7.39; N, 4.67. Found: C, 76.34; H, 7.67; N, 4.40.
N,N0-[biphenyl-4,40diylbis(methylene)]bis-dipropylamine
(3cꢁ2HClꢁ0.5H2O). Crystallized from abs EtOH/Et2O. Yield: 68%; mp:
274–276 °C. 1H NMR (D2O) d 0.95 ppm (t, J = 14.5 Hz, 12H), 1.78–
1.80 ppm (m, 12H), 3.10–3.15 ppm (m, 8H), 4.41 ppm (s, 4H),
7.62 ppm (d, J = 7.8 Hz, 4H), 7.83 ppm (d, J = 7.7 Hz, 4H). MS (EI)
381 [M+1]+. Anal. calcd for C26H40N2ꢁ2HCl ꢁ0.5H2O: C, 67.51; H,
9.37; N, 6.05. Found: C, 67.52; H, 9.00; N, 6.39.
N,N0-[biphenyl-4,40diylbis(methylene)]bis-dimethylamine
(3dꢁ2HClꢁ0.25H2O). Crystallized from abs EtOH/Et2O. Yield: 78%;
mp > 300 °C. 1H NMR (D2O) d 2.89 ppm (s, 12H), 4.38 ppm (s,
4H), 7.61 ppm (d, J = 8.3 Hz, 4H), 7.83 ppm (d, J = 8.4 Hz, 4H). MS
(EI) 269 [M+1]+. Anal. calcd for C18H24N2ꢁ2HClꢁ0.25H2O: C, 62.51;
H, 7.57; N, 8.10. Found: C, 62.36; H, 7.45; N, 7.60.
6.1.1.3. General method of preparation of (40-piperidin-1-
ylmethyl-biphenyl-4-ylmethyl)amine derivatives (5a–5c and
5e–k).
A solution of 40-chloromethyl-biphenyl-4-ylmethyl-
N,N0-[biphenyl-4,40diylbis(methylene)]bis-pentylamine (3eꢁ2HCl).
Crystallized from abs EtOH. Yield: 55%; mp > 300 °C. Free base
NMR: 1H NMR (DMSO) d 0.88 ppm (t, J = 13.15 Hz, 6H), 1.24–
1.32 ppm (m, 8H), 1.60–1.70 ppm (m, 4H), 2.90 ppm (t,
J = 14,12 Hz, 4H), 4.18 ppm (s, 4H), 7.63 ppm (d, J = 8.06 Hz, 4H),
7.79 ppm (d, J = 8.18 Hz, 4H). MS (EI) 353 [M+1]+. Anal. calcd for
C24H36N2ꢁ2HCl: C, 67.74; H, 9.00; N, 6.58. Found: C, 67.56; H,
8.90; N, 6.20.
piperidine 4 (1 mmol) in THF (2 mL) was treated with the appropri-
ate amine (2 mmol) (MW, 100 °C, 150 W, 100 psi, 5 min). Removal of
the solvent in vacuo gave the crude products, which were purified by
flash chromatography [SiO2, CH2Cl2/CH3OH (NH3) = 98:2].
Cyclohexyl-(40-piperidin-1-ylmethyl-biphenyl-4-ylmethyl)methyl-
amine (5aꢁ2HClꢁH2O). Crystallized from abs EtOH/Et2O. Yield: 65%;
mp: 264–266 °C. 1H NMR (D2O) d 1.15–2.07 (m, 16H), 2.75 (s, 3H),
2.91–3.07 (m, 2H), 3.25–3.35 (m, 1H), 3.44–3.54 (m, 2H), 4.21 (d,
J = 1.3 Hz, 1H), 4.33 (s, 2H), 4.49 (d, J = 1.3 Hz, 1H), 7.61 (dd,
J = 8.4 Hz, J = 1.6 Hz, 4H), 7.82 (dd, J = 8.4 Hz, 4H). MS (EI) 377
[M+1]+. Anal. calcd for C26H36N2ꢁ2HClꢁH2O: C, 66.79; H, 8.62; N,
5.99. Found: C, 66.83; H, 8.45; N, 5.90.
N,N0-[biphenyl-4,40diylbis(methylene)]bis-cyclohexylamine
(3f).
Crystallized from EtOH/H2O. Yield: 66%; mp: 98–100 °C. 1H NMR
(CDCl3) d 1.10–1.34 ppm (m, 10H), 1.58–1.67 ppm (m, 2H), 1.69–
1.82 ppm (m, 4H), 1.88–2.05 ppm (m, 4H), 2.45–2.63 ppm (m,
2H), 3.84 ppm (s, 4H), 7.38 ppm (d, J = 8.1 Hz, 4H), 7.54 ppm (d,
J = 8.1 Hz, 4H). MS (EI) 377 [M+1]+. Anal. calcd for C26H36N2: C,
82.92; H, 9.64; N, 7.44. Found: C, 82.84; H, 9.39; N, 7.21.
40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyl-3,5-dimethylpiperi-
dine (5bꢁ2HClꢁ0.25H2O). Crystallized from abs EtOH/Et2O. Yield:
82%; mp > 300 °C. 1H NMR (D2O) d 0.96 (d, J = 6.6 Hz, 6H), 1.41–
2.14 (m, 10H), 2.60 (t, J = 12 Hz, 2H), 2.93–3.15 (m, 2H), 3.35–
3.62 (m, 4H), 4.36 (s, 2H), 4.38 (s, 2H), 7.62 (d, J = 8.1 Hz, 4H),
7.84 (d, J = 8.1 Hz, 4H). MS (EI) 377 [M+1]+. Anal. calcd for
C26H36N2ꢁ2HClꢁ0.25H2O: C, 68.78; H, 8.55; N, 6.17. Found: C,
68.86; H, 8.46; N, 6.41.
1,10-[Biphenyl-4,40-diylbis(methylene)]bis(3,5-dimethylpiperidine)
(3g). Crystallized from EtOH. Yield: 73%; mp: 129–131 °C. 1H NMR
(CDCl3) d 0.45–0.60 (m, 2H), 0.83 (d, J = 6.3 Hz, 12H), 1.47 (t,
J = 11.1 Hz, 4H), 1.63–1.81 (m, 6H), 2.80–2.89 (m, 4H), 3.52 (s,
4H), 7.37 (d, J = 8.1 Hz, 4H), 7.56 (d, J = 8.1 Hz, 4H). MS (EI) 405

G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
9921
4-(40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyl)morpholine
(5cꢁ2HClꢁ0.5H2O). Crystallized from abs EtOH/Et2O. Yield: 76%;
mp > 300 °C. 1H NMR (D2O) d 1.39–2.08 (m, 6H), 2.88–3.10 (m,
2H), 3.16–3.60 (m, 6H), 3.66–4.21 (m, 4H), 4.33 (s, 2H), 4.43 (s,
2H), 7.61 (m, 4H), 7.82 (dd, J = 8.1 Hz, J = 3.3 Hz, 4H). MS (EI) 351
[M+1]+. Anal. calcd for C23H30N2ꢁ2HClꢁ0.5H2O: C, 63.88; H, 7.46;
N, 6.48. Found: C, 63.79; H, 7.40; N, 6.73.
NMR (CDCl3) d 1.44–1.47 (m, 2H), 1.62–1.65 (m, 4H), 2.45–2.48
(m, 4H), 3.57 (s, 2H), 5.15 (s, 2H), 7.04 (d, J = 8.8 Hz, 2H), 7.40–
7.64 (m, 10H). (EI) 385 [M+1]+. Anal. calcd for C26H26N2Oꢁ0.25H2O:
C, 80.69; H, 6.77; N, 7.23. Found: C, 80.89; H, 6.66; N, 7.18.
6.1.1.5. Preparation of 1-(40-piperidin-1-ylmethyl-biphenyl-4-
ylmethyl)piperidin-2-one (5l).
A solution of 2-piperidone
40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyldimethylamine
(5eꢁ0.25H2O). Crystallized from EtOH/H2O. Yield: 67%; mp: 78–
79 °C. 1H NMR (CDCl3) d 1.39–1.49 (m, 2H), 1.55–1.66 (m, 4H),
2.27 (s, 6H), 2.36–2.49 (m, 4H), 3.46 (s, 2H), 3.52 (s, 2H), 7.33–
7.42 (m, 4H), 7.50 (dd, J = 8.1 Hz, J = 2.1 Hz, 4H). MS (EI) 309
[M+1]+. Anal. calcd for C21H28N2ꢁ0.25H2O: C, 80.59; H, 9.02; N,
8.95. Found: C, 80.74; H, 8.99; N, 8.79.
(3.32 mmol) in anhydrous toluene (2 mL, under an atmosphere of
N2) was treated with NaH (0.14 g, 60% dispersion in mineral oil);
this mixture was added to a solution of 4 (1.67 mmol) in anhy-
drous toluene (3 mL). The reaction was stirred at 100 °C for 5 h
and the solid residue was filtered off. Removal of the solvent in va-
cuo gave the crude products, which was purified by flash chroma-
tography [SiO2, CH2Cl2:CH3OH (NH3) = 99:1]. Crystallized from
EtOH/H2O. Yield: 62%; mp: 108–110 °C. 1H NMR (CDCl3) d 1.39–
1.49 (m, 2H), 1.53–1.64 (m, 4H), 1.74–1.85 (m, 4H), 2.36–2.44
(m, 4H), 2.48 (t, J = 5.7 Hz, 2H), 3.23 (t, J = 5.7 Hz, 2H), 3.50 (s,
2H), 4.63 (s, 2H), 7.23–7.41 (m, 4H), 7.48–7.57 (m, 4H). MS (EI)
363 [M+1]+. Anal. calcd for C24H30N2O: C, 79.52; H, 8.34; N, 7.73.
Found: C, 79.31; H, 8.31; N, 7.53.
40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyldiethylamine
(5fꢁ2HClꢁ0.5H2O). Crystallized from abs EtOH/Et2O. Yield: 73%;
mp > 300 °C. 1H NMR (D2O) d 1.33 (t, J = 7.3 Hz, 6H), 1.42–2.01
(m, 6H), 2.88–3.06 (m, 2H), 3.24 (q, J = 7.2 Hz, 4H), 3.43–3.58 (m,
2H), 4.32 (s, 2H), 4.38 (s, 2H), 7.54–7.66 (m, 4H), 7.80 (dd,
J = 8.4 Hz, J = 2.1 Hz, 4H). MS (EI) 337 [M+1]+. Anal. calcd for
C23H32N2ꢁ2HClꢁ0.5H2O: C, 66.01; H, 8.19; N, 6.69. Found: C,
66.28; H, 8.08; N, 6.45.
6.1.1.6. Preparation of 2,20-biphenyl-4,40-diylbis-acetic acid
40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyldipropylamine
(5gꢁ2HClꢁH2O). Crystallized from abs EtOH/Et2O. Yield: 68%; mp:
240–242 °C. 1H NMR (D2O) d 0.95 (t, J = 7.2 Hz, 6H), 1.38–2.05
(m, 10H), 2.94–3.08 (m, 2H), 3.13 (t, J = 8.1 Hz, 4H), 3.43–3.63
(m, 2H), 4.34 (s, 2H), 4.42 (s, 2H), 7.61 (dd, J = 8.4 Hz, J = 2.7 Hz,
4H), 7.83 (dd, J = 8.1 Hz, J = 2.4 Hz, 4H). MS (EI) 365 [M+1]+. Anal.
calcd for C25H36N2ꢁ2HClꢁH2O: C, 65.92; H, 8.85; N, 6.15. Found: C,
66.17; H, 8.55; N, 6.33.
(6).
A solution of 4,40-bis(chloromethyl)-biphenyl 2 (1 mmol)
in CH3CN (2 mL) was reacted with NaCN (4 mmol) in H2O (1 mL) to
give [1,10-biphenyl]-4,40-diacetonitrile (MW, 100 °C, 150 W, 100
psi, 5 min). Yield: 94%. 1H NMR (DMSO) d 4.09 (s, 4H), 7.45 (d,
J = 8.2 Hz, 4H), 7.72 (d, J = 8.2 Hz, 4H). MS (EI) 233 [M+1]+.
A solution of [1,10-biphenyl]-4,40-diacetonitrile (1 mmol) in
EtOH (2 mL) was reacted with KOH (4 mmol) in EtOH (2 mL)
(MW, 150 °C, 200 W, 145 psi, 5 min). After cooling, the reaction
mixture was acidified with HCl (1 M) and the desired product
was obtained as precipitate. Yield: 91%. 1H NMR (DMSO) d 3.60
(s, 4H), 7.34 (d, J = 8.2 Hz, 4H), 7.59 (d, J = 8.2 Hz, 4H). MS (EI)
271 [M+1]+.
40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyl-4-methylpiperidine
(5hꢁ2HClꢁ0.5H2O). Crystallized from abs EtOH/Et2O. Yield: 82%;
mp > 300 °C. 1H NMR (D2O) d 0.99 (d, J = 6 Hz, 3H) 1.33–1.60 (m,
3H), 1.60–2.05 (m, 8H), 2.89–3.13 (m, 4H), 3.38–3.62 (m, 4H),
4.35 (s, 4H), 7.62 (d, J = 8.1 Hz, 4H), 7.83 (d, J = 8.1 Hz, 4H). MS
(EI) 363 [M+1]+. Anal. calcd for C25H34N2ꢁ2HClꢁ0.5H2O: C, 67.55;
H, 8.39; N, 6.30. Found: C, 67.39; H, 8.10; N, 6.39.
6.1.1.7. General method of preparation of [biphenyl-4,40diyl-
bis(ethylene)]bis-amine derivatives (7a–7c).
A solution of
Phenylmethyl-(40-piperidin-1-ylmethyl-biphenyl-4-ylmethyl)
methylamine (5iꢁ2HCl). Crystallized from abs EtOH. Yield: 64%; mp:
281–283 °C. 1H NMR (D2O) d 1.37–1.56 (m, 1H), 1.56–1.88 (m, 3H),
1.88–2.08 (m, 2H), 2.79 (s, 3H), 2.92–3.12 (m, 2H), 3.41–3.62 (m,
2H), 4.34 (s, 2H), 4.39 (s, 2H), 4.42 (s, 2H), 7.46–7.65 (m, 9H),
7.82 (d, J = 8.1 Hz, 4H). MS (EI) 385 [M+1]+. Anal. calcd for
C25H36N2ꢁ2HCl: C, 70.88; H, 7.49; N, 6.12. Found: C, 70.78; H,
7.43; N, 5.86.
2,20-(1,10-biphenyl)-4,40-diylbis-acetic acid 6 (0.3 mmol) in THF
(3 mL) were reacted with CDI (1.28 mmol) (MW, 150 °C, 200 W,
145 psi, 5 min). After this heating cycle, the suitable amine
(1.2 mmol) was added to the reaction and the mixtures were sub-
mitted to a second heating cycle as described above. After cooling,
THF was evaporated under reduced pressure; the residues were ta-
ken up in ethyl acetate, washed with HCl (1 N), NaOH (10% w/v),
and dried over Na2SO4. The organic layers were concentrated to
yield the desired amides which were characterized by 1H NMR
and mass spectra.
To the stirred solutions of the appropriate amide (1 mmol) in
anhydrous toluene (15 mL, under an atmosphere of N2), RedAlÒ
(8.0 mmol, 70% solution in toluene, 2.3 mL) was carefully added.
The resulting mixtures were stirred for 1 h at room temperature,
then heated to reflux for 3 h.33 After cooling, NaOH (10 mL, 3 N)
was added dropwise and the organic phases were evaporated under
reduced pressure to afford the crude products which were purified
by flash chromatography [SiO2, CH2Cl2/MeOH(NH3) = 15:1].
1,10-[Biphenyl-4,40-diylbis(ethylene)]bis-piperidine (7aꢁ2HClꢁH2O).
Crystallized from abs EtOH/Et2O. Yield: 70%; mp > 300 °C. 1H NMR
(CDCl3) d 1.46–1.50 (m, 4H), 1.60–1.69 (m, 8H), 2.54 (m, 8H), 2.56–
2.62 (m, 4H), 2.82–2.88 (m, 4H), 7.26 (d, J = 8.1 Hz, 4H), 7.49 (d,
J = 8.1 Hz, 4H). MS (EI) 377 [M+1]+. Anal. calcd for
C26H36N2ꢁ2HClꢁH2O: C, 66.79; H, 8.62; N, 5.99. Found: C, 66.68;
H, 8.07; N, 6.01.
1-[4-(40-Piperidin-1-ylmethyl-biphenyl-4-ylmethyl)-piperazin-
1yl]ethanone (5jꢁ2HClꢁ0.5H2O). Crystallized from abs EtOH/Et2O.
Yield: 65%; mp > 300 °C. 1H NMR (D2O) d 1.40–1.55 (m, 2H),
1.58–1.99 (m, 6H), 2.15 (s, 3H), 2.91–3.55 (m, 8H), 4.32 (s, 2H),
4.43 (s, 2H), 7.55–7.65 (m, 4H), 7.76–7.86 (m, 4H). MS (EI) 392
[M+1]+. Anal. calcd for C25H33N3Oꢁ2HClꢁ0.5H2O: C, 63.42; H, 7.45;
N, 8.87. Found: C, 63.68; H, 7.56; N, 8.68.
Cyclohexyl-(40-piperidin-1-ylmethyl-biphenyl-4-ylmethyl)amine
(5k). Crystallized from EtOH/H2O. Yield: 71%; mp: 76–78 °C. 1H
NMR (CDCl3) d 1.09–1.29 (m, 5H), 1.41–1.49 (m, 2H), 1.53–1.64
(m, 5H), 1.69–1.78 (m, 2H), 1.89–1.98 (m, 2H), 2.32–2.47 (m,
4H), 2.47–2.57 (m, 1H), 3.50 (s, 2H), 3.84 (s, 2H), 7.37 (d,
J = 7.2 Hz, 4H), 7.49–7.58 (m, 4H). MS (EI) 363 [M+1]+. Anal. calcd
for C25H34N2: C, 82.82; H, 9.45; N, 7.73. Found: C, 82.56; H, 9.48;
N, 7.65.
6.1.1.4. Preparation of 40-(4-cyanophenoxymethyl)-biphenyl-4-
ylmethylpiperidine (5dꢁ0.25H2O).
Starting from 4-hydrox-
N,N0-[biphenyl-4,40diylbis(ethylene)]bis-dimethylamine
(7bꢁ2HClꢁ0.5H2O). Crystallized from abs EtOH/Et2O. Yield: 81%;
mp > 300 °C. 1H NMR (D2O) d 2.93 (s, 12H), 3.12 (t, J = 15.7 Hz,
4H), 3.46 (t, J = 15.7 Hz, 4H), 7.43 (d, J = 8.1 Hz, 4H), 7.69 (d,
ybenzonitrile and the intermediate 4, the desired compound 5d
was obtained according to the procedure described for the synthe-
sis of 5l, Crystallized from EtOH. Yield: 63%; mp: 157–159 °C. 1H

9922
G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
J = 8.1 Hz, 4H). MS (EI) 297 [M+1]+. Anal. calcd for
C20H28N2ꢁ2HClꢁ0.5H2O: C, 63.48; H, 8.26; N, 7.40. Found: C,
63.84; H, 7.80; N, 7.37.
6.1.1.12. General method of preparation of (40-hydroxymethyl-
biphenyl-4-yl)-1-aminoethanone derivatives (13a and
b). Using the same method described for the synthesis of
1,10-[Biphenyl-4,40-diylbis(ethylene)]bis-4-methylpiperidine
(7cꢁ2HClꢁH2O). Crystallized from abs EtOH/Et2O. Yield: 73%;
mp > 300 °C. 1H NMR (D2O) d 0.96 (d, J = 6.4 Hz, 6H), 1.42 (m,
J = 13.4 Hz, 4H), 1.71 (m, 2H), 1.94–1.99 (m, J = 14.45 Hz, 4H),
2.97–3.05 (m, J = 12.68 Hz, 4H), 3.11–3.16 (m, 4H), 3.37–3.47 (m,
4H), 3.61–3.65 (m, J = 12.32 Hz, 4H), 7.44 (d, J = 8.2 Hz, 4H), 7.70
(d, J = 8.2 Hz, 4H). MS (EI) 405 [M+1]+. Anal. calcd for
C28H40N2ꢁ2HClꢁH2O: C, 67.86; H, 8.94; N, 5.65. Found: C, 67.95;
H, 8.34; N, 5.68.
(40-carboxy-1,10-biphenyl-4-yl)-1-piperidin-1-yl-ethanone 10, the
title compounds were obtained starting from 4-iodophenyl-deriv-
atives 9a,b and 4-hydroxymethylphenylboronic acid. After filtra-
tion over celite and evaporation of the solvent, the crude
products were dissolved in H2O and extracted with CH2Cl2. The
combined organic layers were dried over Na2SO4 and the solvent
was removed under reduced pressure to give the colorless solids.
(40-Hydroxymethyl-biphenyl-4-yl)-1-piperidin-1-ylethanone
(13a). Yield: 70%. 1H NMR (CDCl3) d 1.37–1.44 (m, 2H), 1.54–1.60
(bs, 4H), 3.41 (t, 2H), 3.59 (t, 2H), 3.76 (s, 2H), 4.73 (s, 2H), 7.30
(d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 7.52–7.57 (m, 4H). MS
(EI) 310 [M+1]+.
6.1.1.8. General method of preparation of 4-iodophenyl-1-
aminoethanone derivatives (9a and b).
A solution of 2-(4-
iodophenyl)acetic acid 8 (1 mmol) in CH3CN (3 mL) was reacted
with CDI (1.2 mmol) (MW, 130 °C, 150 W, 145 psi, 5 min). After
this heating cycle, piperidine or dipropylamine (1.2 mmol) were
added to the reaction, and the mixtures were submitted to a sec-
ond heating cycle as described above. After cooling, H2O was added
and the crude products were obtained as precipitates.
4-Iodophenyl-1-piperidin-1-ylethanone (9a). Yield: 90%. 1H NMR
(DMSO) d 1.31–1.42 (m, 4H), 1.50–1.57 (m, 2H), 3.38–3.42 (m, 4H),
3.65 (s, 2H), 7.03 (d, J = 8.2 Hz, 4H), 7.63 (d, J = 8.2 Hz, 4H). MS (EI)
319 [M+1]+.
(40-Hydroxymethyl-biphenyl-4-yl)-1-dipropylaminoethanone
(13b). Yield: 73%. 1H NMR (CDCl3) d 0.89 (t, 6H), 1.52–1.62 (m, 4H),
3.22 (t, 2H), 3.30 (t, 2H), 3.72 (s, 2H), 4.69 (s, 2H), 7.29 (d, J = 8.3 Hz,
2H), 7.39 (d, J = 8.3 Hz, 2H), 7.50–7.54 (m, 4H). MS (EI) 326 [M+1]+.
6.1.1.13. General method of preparation of (40-aminomethyl-
biphenyl-4-yl)-1-aminoethanone
derivatives
(14
and
15). To a stirred solution of 13a and b (1.54 mmol) in CH2Cl2
(33 mL), MsCl (3.08 mmol) was added; after cooling to 0 °C, Et3N
(3.84 mmol) was added and the resulting mixture was stirred at
room temperature for 90 min. The solvent was evaporated to dry-
ness and the crude product, dissolved in CH3CN (2 mL), was re-
acted with the appropriate amine (1.7 mmol) (MW, 100 °C, 90
W, 100 psi, 5 min). After cooling, CH3CN was evaporated under re-
duced pressure and the crude product was purified by chromatog-
raphy [SiO2, CH2Cl2:MeOH(NH3) = 100:1].
4-Iodophenyl-1-dipropylaminoethanone (9b). Yield: 85%. 1H NMR
(CDCl3) d 0.85–0.92 (m, 6H), 1.49–1.61 (m, 4H), 3.18 (t, 2H), 3.28 (t,
2H), 3.63 (s, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H). MS
(EI) 346 [M+1]+.
6.1.1.9. Preparation of 40-(2-oxo-2-piperidino-ethyl)biphenyl-
4-carboxylic acid (10).
A solution of 9a (1mmol), 4-carbo-
2-(40-Dipropylaminomethyl-biphenyl-4-yl)-1-piperidin-1-yletha-
none (14ꢁHClꢁ0.5H2O). Crystallized from abs EtOH/Et2O. Yield: 80%;
mp: 174–176 °C. 1H NMR (D2O) d 0.82 (t, 6H), 1.32–1.55 (m, 10H),
2.30–2.31 (m, 4H), 3.42–3.45 (m, 4H), 2.99–3.04 (m, 2H), 3.53 (s,
2H), 3.72 (s, 2H), 7.29 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H),
7.59 (d, J = 8.2 Hz, 2H). MS (EI) 393 [M+1]+. Anal. calcd for
C26H36N2OꢁHClꢁ0.5H2O C, 71.29; H, 8.74; N, 6.39. Found: C, 71.45;
H, 8.71; N, 6.41.
xyphenylboronic acid (1.1 mmol) and K2CO3 (3 mmol) in acetone
(15 mL) and bidistilled H2O (15 mL) was degassed bubbling nitro-
gen for 10 min. To this solution Pd(OAc)2 (0.12 g) was added, and
the reaction mixture was stirred at 65 °C for 1 h.54 After filtration
of the catalyst, HCl (5 mL 1 N) was added and the product was ob-
tained as precipitate. Yield: 99%. 1H NMR (DMSO) d 1.36–1.41 (m,
4H), 1.52–1.54 (m, 2H), 3.41–3.45 (m, 4H), 3.74 (s, 2H), 7.33–8.07
(m, 8H), 12.99 (bs, 1H). MS (EI) 324 [M+1]+.
2-(40-Piperidin-1-ylmethyl-biphenyl-4-yl)-1-dipropylaminoetha-
none (15ꢁHCl). Crystallized from abs EtOH/Et2O. Yield: 85%; mp:
158–159 °C. 1H NMR (DMSO) d 0.76–0.88 (m, 6H), 1.30–1.78 (m,
10H), 2.83–2.87 (m, 2H), 3.17–3.30 (m, 6H), 4.27 (d, J = 4.9, 2H),
3.53 (s, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.62–7.68 (m, 4H), 7.75 (d,
J = 8.2 Hz, 2H), 10.40 (s, 1H). MS (EI) 393 [M+1]+. Anal. calcd for
C26H36N2OꢁHCl C, 72.78; H, 8.69; N, 6.53. Found: C, 73.03; H,
8.79; N, 6.54.
6.1.1.10. Preparation of 2-(40-piperidinocarbonyl-biphenyl-4-
yl)-1-piperidinoethanone (11).
A solution of 10 (1 mmol)
in 3 mL of CH3CN (3 mL) was reacted with CDI (1.2 mmol) (MW,
130 °C, 150 W, 145 psi, 5 min). After this heating cycle, piperidine
(1.2 mmol) was added to the reaction, and the mixture was sub-
mitted to a second heating cycle as described above. After comple-
tion of reaction, H2O was added and the solution was extracted
with ethyl acetate; the organic layer was dried over Na2SO4 and
evaporated under reduced pressure. The crude product was used
in the successive reaction without further purification. Yield:
97%. 1H NMR (CDCl3) d 1.40–1.66 (m, 12H), 3.40–3.69 (m, 8H),
3.74 (s, 2H), 7.29–7.62 (m, 8H). MS (EI) 391 [M+1]+.
6.1.1.14. Preparation of [40-(2-piperidin-1-yl-ethyl)-biphenyl-4-
ylmethyl]dipropylamine (16ꢁ2HClꢁ0.5H2O).
Reduction of
amide 14 was performed as described in the preparation of com-
pounds 7a–7c. The crude product 16 was purified by flash chroma-
tography [SiO2, CH2Cl2/MeOH(NH3) = 130:1]. Crystallized from abs
EtOH/Et2O. Yield: 60%; mp: 260–262 °C. 1H NMR (DMSO) d 0.86 (t,
6H), 1.36–1.83 (m, 10H), 2.90–2.94 (m, 6H), 3.08–3.15 (m, 2H),
3.20–3.27 (m, 2H), 3.46–3.57 (m, 2H), 4.32 (d, J = 4.9, 2H), 7.37–
7.40 (d, J = 8.2 Hz, 2H), 7.67–7.77 (m, 4H), 10.70 (s, 1H), 10.83 (s,
1H). MS (EI) 379 [M+1]+. Anal. calcd for C26H38N2ꢁ2HClꢁ0.5H2O C,
67.81; H, 8.97; N, 6.08. Found: C, 67.71; H, 9.05; N, 6.04.
6.1.1.11. Preparation of 1-[2-(40-piperidinomethyl-biphenyl-4-
yl)ethyl]piperidine (12ꢁ2HClꢁ0.5H2O).
Reduction of amide 11
was performed as described in the preparation of compounds 7a–
7c. The crude product 12 was purified by flash chromatography
[SiO2, CH2Cl2/MeOH(NH3) = 130:1]. Crystallized from abs EtOH/
Et2O. Yield: 90%; mp > 300 °C. 1H NMR (D2O) d 1.36–1.39 (m,
2H), 1.58–1.67 (m, 6H), 1.79–1.81 (m, 4H), 2.90 (t, J = 10.8 Hz,
4H), 2.99–3.04 (m, 2H), 3.20–3.23 (m, 2H), 3.28 (d, J = 12.1 Hz,
2H), 3.48 (d, J = 11.8 Hz, 2H), 4.22 (s, 2H), 7.39 (d, J = 8.2 Hz, 2H),
7.56 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.71 (d, J = 8.2 Hz,
2H). MS (EI) 363 [M+1]+. Anal. calcd for C25H34N2ꢁ2HClꢁ0.5H2O: C,
67.55; H, 8.39; N, 6.30. Found: C, 67.38; H, 8.28; N, 6.26.
6.2. Molecular modeling
Molecular modeling studies were performed with Schröedinger
software suite (Schröedinger L.L.C., NY). Ligand structures were
optimized with Macromodel 8.6, applying MMFF94s force field55
to an energy gradient of 0.05 kJ/mol Å.

G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
9923
6.2.1. Docking into the rat H3 receptor model
References and notes
Docking studies were performed with Glide 3.5,56 employing a
previously developed rat H3 receptor model.57 Protein structure
was subjected to the Protein Preparation procedure implemented
in Glide, applying the ‘Preparation Only’ procedure without neu-
tralization of amino acid residues. Docking experiments were per-
formed starting from minimum-energy conformations of the
ligands placed in arbitrary positions, within a region centered on
amino acid Asp114 in TM3, using enclosing and bounding boxes
of 46 and 14 Å on each side, respectively. Compounds 3g, 5b, and
7a were docked in their diprotonated state, compound 5d in the
protonated form. Van der Waals radii of the protein were not
scaled, whereas van der Waals radii of the ligand atoms with par-
tial atomic charge lower than |0.15| were scaled by 0.80. Standard
precision mode was applied and poses with a Coulomb–van der
Waals score greater than 0 kcal/mol were rejected. Docking solu-
tions were ranked according to their Emodel value.
1. Parsons, M. E.; Ganellin, C. R. Br. J. Pharmacol. 2006, 147, S127.
2. Arrang, J.-M.; Garbarg, M.; Schwartz, J.-C. Nature (London) 1983, 302, 832.
3. Clapham, J.; Kilpatrick, G. J. Br. J. Pharmacol. 1992, 107, 919.
4. Schlicker, E.; Fink, K.; Detzner, J.; Göthert, M. J. Neural. Transm. 1993, 93, 1.
5. Schlicker, E.; Betz, R.; Göthert, M. Arch. Pharmacol. 1988, 337, 588.
6. Burgaud, J. L.; Oudart, N. J. Pharm. Pharmacol. 1993, 45, 955.
7. Leurs, R.; Bakker, R. A.; Timmerman, H.; de Esch, I. J. P. Nat. Rev. Drug Discov.
2005, 4, 107.
8. Wijtmans, M.; Leurs, R.; de Esch, I. Expert Opin. Invest. Drugs 2007, 16, 967.
9. Arrang, J. M.; Garbag, M.; Lancelot, J. C.; Lecomte, J.-M.; Pollard, H.; Robba, M.;
Schunack, W.; Schwartz, J.-C. Nature 1987, 327, 117.
10. Barocelli, E.; Ballabeni, V.; Caretta, A.; Bordi, F.; Silva, C.; Morini, G.;
Impicciatore, M. Agents Actions 1993, 38, 158.
11. Leurs, R.; Vollinga, R. C.; Timmerman, H. Prog. Drug Res. 1995, 45, 107.
12. Ganellin, C. R.; Leurquin, F.; Piripitsi, A.; Arrang, J.-M.; Garbarg, M.; Ligneau, X.;
Schunack, W.; Schwartz, J.-C. Arch. Pharm. Weinheim 1998, 331, 395.
13. Liedtke, S.; Flau, K.; Kathmann, M.; Schlicker, E.; Stark, H.; Meier, G.; Schunack,
W. Naunyn-Schmiedeberg’s Arch. Pharmacol 2003, 367, 43.
14. La Bella, F. S.; Queen, G.; Glavin, G.; Durant, G.; Stein, D.; Brandes, L. J. Br. J.
Pharmacol. 1992, 107, 161.
The horizontal and vertical poses of compound 7a were merged
into the H3 receptor model. The complexes were energy-mini-
mized with MMFF94s force field to a gradient of 0.05 kJ/mol Å with
fixed protein backbone and they were submitted to molecular
dynamics (MD) simulation to evaluate their stability. MD was per-
formed with MMFF94 force field,58 with a time step of 1 fs, a
dielectric constant of 1 and a non-bonded cutoff of 8 Å, for 1 ns
at 310 K after equilibration for 100 ps, with fixed position of the
backbone atoms. The last conformation of each MD simulation
was energy-minimized as already described, and the resulting
structures are depicted in Figure 2.
15. Yang, R.; Hey, J. A.; Aslanian, R.; Rizzo, C. A. Pharmacology 2002, 66, 128.
16. Harper, E. A.; Shankley, N. P.; Black, J. W. Br. J. Pharmacol. 1999, 128, 881.
17. Alves-Rodrigues, A.; Leurs, R.; Wu, T.; Prell, G.; Foged, C.; Timmerman, H. Br. J.
Pharmacol. 1996, 118, 2045.
18. Ballabeni, V.; Impicciatore, M.; Bertoni, S.; Magnanini, F.; Zuliani, V.; Vacondio,
F.; Barocelli, E. Inflamm. Res. 2002, 51(Suppl. 1), ), S55.
19. Cowart, M.; Altenbach, R.; Black, L.; Faghih, R.; Zhao, C.; Hancock, A. A. Mini-
Rev. Med. Chem. 2004, 4, 979.
20. Stark, H. Expert Opin. Ther. Pat. 2003, 13, 851.
21. Celanire, S.; Wijtmans, M.; Talaga, P.; Leurs, R.; de Esch, I. J. P. Drug Discov.
Today 2005, 10, 1613.
22. Letavic, M. A.; Barbier, A. J.; Dvorak, C. A.; Carruthers, N. I. Prog. Med. Chem.
2006, 44, 181.
23. Hudkins, R. L.; Raddazz, R. Annu. Rev. Med. Chem. 2007, 42, 49.
24. Bembenek, S. D.; Keith, J. M.; Letavic, M. A.; Apodaca, R.; Barbier, A. J.; Dvorak,
L.; Aluisio, L.; Miller, K. L.; Lovenberg, T. W.; Carruthers, N. I. Bioorg. Med. Chem.
2008, 16, 2968.
6.2.2. Docking into the mouse acetylcholinesterase active site
Since the three-dimensional coordinates of rat acetylcholines-
terase are not available, the structure of the mouse acetylcholines-
terase was used for docking studies, given the high sequence
similarity and the identical composition of amino acids facing the
active site in the rat and mouse enzyme.59 The crystallographic
coordinates of acetylcholinesterase complex with succinylcholine
(PDB ID: 2HA2)52 were taken from the RCSB Protein Data Bank.60
This crystal structure has already been used to perform docking
studies for di-basic acetylcholinesterase inhibitors.46 The acetyl-
cholinesterase structure was prepared by deleting succinylcholine
and water molecules and by adding hydrogen atoms. Protein struc-
ture was subjected to the Protein Preparation procedure imple-
mented in Glide 3.5,56 applying the standard ‘Preparation and
Refinement’ protocol, without neutralization of amino acid resi-
dues. Docking experiments were performed starting from mini-
mum-energy conformations of the ligands placed in arbitrary
positions, within a region centered on the inhibitor succinylcho-
line, using enclosing and bounding boxes of 26 and 14 Å on each
side, respectively. Compound 12 was docked in the diprotonated
form and donepezil with protonated piperidine ring. Van der Waals
radii of the protein were not scaled, whereas van der Waals radii of
the ligand atoms with partial atomic charge lower than |0.15| were
scaled by 0.80. Standard precision mode was applied and poses
with a Coulomb–van der Waals score greater than 0 kcal/mol were
rejected. Docking solutions were ranked according to their Emodel
value. The best scoring solutions of compound 12 and donepezil
were merged into acetylcholinesterase and the complexes are
shown in Figure 3.
25. Petroianu, G.; Arafat, K.; Sasse, B. C.; Stark, H. Pharmazie 2006, 61, 179.
26. Hey, J.; Aslanian, R. G. WO02072093, 2002.
27. Barocelli, E.; Ballabeni, V.; Manenti, V.; Flammini, L.; Bertoni, S.; Morini, G.;
Comini, M.; Impicciatore, M. Pharmacol. Res. 2006, 53, 226.
28. Bertoni, S.; Flammini, L.; Manenti, V.; Ballabeni, V.; Morini, G.; Comini, M.;
Barocelli, E. Pharmacol. Res. 2007, 55, 111.
29. Rivara, M.; Zuliani, V.; Cocconcelli, G.; Morini, G.; Comini, M.; Rivara, S.; Mor,
M.; Bordi, F.; Barocelli, E.; Ballabeni, V.; Bertoni, S.; Plazzi, P. V. Bioorg. Med.
Chem. 2006, 14, 1413.
30. Morini, G.; Comini, M.; Rivara, M.; Rivara, S.; Lorenzi, S.; Bordi, F.; Mor, M.;
Flammini, L.; Bertoni, S.; Ballabeni, V.; Barocelli, E.; Plazzi, P. V. Bioorg. Med.
Chem. Lett. 2006, 16, 4063.
31. Bertoni, S.; Ballabeni, V.; Flammini, L.; Saccani, F.; Domenichini, G.; Morini, G.;
Comini, M.; Rivara, M.; Barocelli, E. Naunyn-Schmiedeberg’s Arch. Pharmacol.
2008, 378, 335.
32. Marquais-Bienewald, S.; Hoelzl, W.; Preuss, A.; Mehlin, A.; Brunner, F. PCT Int.
Appl. WO 04035521, 2004.
33. Binet, J.; Thomas, D.; Benmbarek, A.; De Fornel, D.; Reaut, P. Chem. Pharm. Bull.
2002, 50, 316.
34. Kilpatrick, G. J.; Michel, A. D. Agents Actions 1991, 33, 69.
35. Lovenberg, T. W.; Roland, B. L.; Wilson, S. J.; Jiang, X.; Pyati, J.; Huvar, A.;
Jackson, M. R.; Erlander, M. G. Mol. Pharmacol. 1999, 55, 1101.
36. Liu, C.; Ma, X.; Jiang, X.; Wilson, S. J.; Hofstra, C. L.; Blevitt, J.; Pyati, J.; Li,
X.; Chai, W.; Carruthers, N.; Lovenberg, T. W. Mol. Pharmacol. 2001, 59,
420.
37. Apodaca, R.; Dvorak, C. A.; Xiao, W.; Barbier, A. J.; Boggs, J. D.; Wilson, S. J.;
Lovenberg, T. W.; Carruthers, N. I. J. Med. Chem. 2003, 46, 3938.
38. Denizot, F.; Lang, R. J. Immunol. Methods 1986, 89, 271.
39. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
40. Furchgott, R. F.. In Handbook of Experimental Pharmacology; Blanschko, E.,
Muscholl, E., Eds.; Springer: Berlin, 1972; Vol. 33, pp 283–335.
41. Van Rossum, J. M. Arch. Int. Pharmacodyn. Ther. 1963, 143, 299.
42. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
43. Tumiatti, V.; Andrisano, V.; Banzi, R.; Bartolini, M.; Minarini, A.; Rosini, M.;
Melchiorre, C. J. Med. Chem. 2004, 47, 6490.
44. Bolognesi, M. L.; Banzi, R.; Bartolini, M.; Cavalli, A.; Tarozzi, A.; Andrisano, V.;
Minarini, A.; Rosini, M.; Tumiatti, V.; Bergamini, C.; Fato, R.; Lenaz, G.; Hrelia,
P.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. J. Med. Chem. 2007, 50, 4822.
45. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347.
Acknowledgments
The work was supported by the Italian Ministero dell’Istruzione,
dell’Università e della Ricerca. The Centro Interdipartimentale Mis-
ure and the Settore Innovazione e Tecnologie Informatiche of the
University of Parma are gratefully acknowledged for providing
NMR instrumentation and software licences.
46. Xie, Q.; Wang, H.; Xia, Z.; Lu, M.; Zhang, W.; Wang, X.; Fu, W.; Tang, Y.;
Sheng, W.; Li, W.; Zhou, W.; Zhu, X.; Qiu, Z.; Chen, H. J. Med. Chem. 2008,
51, 2027.
47. Uveges, A. J.; Kowal, D.; Zhang, Y.; Spangler, T. B.; Dunlop, J.; Semus, S.; Jones, P.
G. J. Pharmacol. Exp. Ther. 2002, 301, 451.

9924
G. Morini et al. / Bioorg. Med. Chem. 16 (2008) 9911–9924
48. Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.;
Kobolka, T. S.; Choi, H.-J.; Yao, X.-J.; Weis, W. I.; Stevens, R. C.; Kobolka, B. K.
Science 2007, 318, 1266.
49. Axe, F. U.; Bembenek, S. D.; Szalma, S. J. Mol. Graph. Model. 2005, 24, 44.
50. Yao, B. B.; Hutchins, C. W.; Carr, T. L.; Cassar, S.; Masters, J. N.; Bennani, J. L.;
Esbenshade, T. A.; Hancock, A. A. Neuropharmacology 2003, 44, 773.
51. Schlegel, B.; Laggner, C.; Meier, R.; Langer, T.; Schnell, D.; Seifert, R.; Stark, H.;
Holtjie, H.-D.; Sippl, W. J. Comput. Aided Mol. Des. 2007, 21, 437.
52. Bourne, Y.; Radic, Z.; Sulzenbacher, G.; Kim, E.; Taylor, P.; Marchot, P. J. Biol.
Chem. 2006, 281, 29256.
55. Halgren, T. A. J. Comput. Chem. 1999, 20, 720–729.
56. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.;
Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.;
Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
57. Lorenzi, S.; Mor, M.; Bordi, F.; Rivara, S.; Rivara, M.; Morini, G.; Bertoni,
S.; Ballabeni, V.; Barocelli, E.; Plazzi, P. V. Bioorg. Med. Chem. 2005, 13,
5647.
58. Halgren, T. A. J. Comput. Chem. 1996, 17, 490–519.
59. Wiesner, J.; Kriz, Z.; Kuca, K.; Jun, D.; Koca, J. J. Enzyme Inhib. Med. Chem. 2007,
22, 417.
53. Kryger, G.; Silman, I.; Sussman, J. L. Struct. Fold Des. 1999, 7, 297.
54. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
60. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.