Dibasic non-imidazole histamine H3 receptor antagonists with a rigid biphenyl scaffold

Article abstract of DOI:10.1016/j.bmcl.2006.04.092

A class of rigid, dibasic, non-imidazole H₃ antagonists was developed, starting from a series of previously described flexible compounds. The original polymethylene chain between two tertiary amine groups was replaced by a rigid scaffold, compo

Full text of DOI:10.1016/j.bmcl.2006.04.092

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4063–4067
Dibasic non-imidazole histamine H receptor antagonists
3
with a rigid biphenyl scaffold
a
a,
a
a
a
*
Giovanni Morini, Mara Comini, Mirko Rivara, Silvia Rivara, Simone Lorenzi,
a
a
b
b
b
Fabrizio Bordi, Marco Mor, Lisa Flammini, Simona Bertoni, Vigilio Ballabeni,
b
a
Elisabetta Barocelli and Pier Vincenzo Plazzi
a
Dipartimento Farmaceutico, Universit a` degli Studi di Parma, V. le G.P. Usberti 27/A, I-43100 Parma, Italy
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universit a` degli Studi di Parma,
V. le G.P. Usberti 27/A, I-43100 Parma, Italy
b
Received 12 April 2006; revised 28 April 2006; accepted 29 April 2006
Available online 18 May 2006
3
Abstract—A class of rigid, dibasic, non-imidazole H antagonists was developed, starting from a series of previously described flex-
ible compounds. The original polymethylene chain between two tertiary amine groups was replaced by a rigid scaffold, composed by
a phenyl ring or a biphenyl fragment. Modulation of the distance between the two amine groups, and of their alkyl substituents, was
0
0
driven by superposition of molecular models and docking into a receptor model, resulting in the identification of 1,1 -[biphenyl-4,4 -
diylbis(methylene)]bis-piperidine (5) as a subtype-selective H
histamine receptor.
3 i 3
antagonist with high binding affinity (pK = 9.47) at human H
Ó 2006 Elsevier Ltd. All rights reserved.
The biogenic amine histamine activates different G
1
Recently, the attention in the field of H antagonists
3
protein-coupled receptors (H , H , H , and H ) and
3
turned toward compounds lacking the imidazole ring
1
1
2
4
1
exerts multiple pharmacological effects, both in the
central nervous system (CNS) and in peripheral tissues.
The histamine H receptor was initially described as an
that characterized classical antagonists. In a previous
work, we described a series of non-imidazole H antag-
3
onists characterized by a basic piperidine ring connect-
ed through an alkyl spacer of variable length to
another piperidine group (symmetrical compounds) or
3
2
3
autoreceptor, mainly expressed in CNS, modulating
the biosynthesis and release of histamine from
histaminergic neurons. Indeed, the H receptor is also
1
2,13
to heterocyclic rings (asymmetrical compounds).
These compounds showed interesting H binding affin-
3
located presynaptically on non-histaminergic neurons,
modulating the release of other neurotransmitters, such
as dopamine, serotonin, norepinephrine, acetylcholine,
3
ity and antagonistic activity, as well as good selectivity
for the H receptor. Compounds carrying two piperi-
3
4–7
and neuropeptides.
Different potential therapeutic
dine rings were the most potent ones, with an optimal
length of the alkylene spacer comprised between 7 and
10 methylene groups. Despite the good biological data,
polymethylene chain flexibility hampered the interpre-
tation of structure–activity relationships (SARs) in
terms of spatial requirements. To further investigate
these requirements, we modulated the structure of the
applications have been proposed for H3 receptor
antagonists, mainly for the treatment of CNS disorders.
Strong indications, including active clinical trials,
support their use for cognitive and neurological
disorders, such as epilepsy, Alzheimer’s disease, memory
and learning deficits, sleep and wakefulness disorders,
8
–10
0
narcolepsy, and obesity.
most potent derivative 1-1 -nonamethylenedi-piperidine
synthesizing a new series of compounds (Table 1) in
which both the basic centers and the spacer were var-
ied. The piperidine moiety was replaced by primary,
secondary or tertiary amines or by a non-basic frag-
ment. The central portion of the polymethylene chain
was substituted by a rigid biphenyl scaffold, linked to
the two basic groups through one methylene spacer
3
Keywords: Histamine; H receptor antagonists; Non-imidazole; Biphe-
nyl; Dibasic.
*
Corresponding author. E-mail: giovanni.morini@unipr.it
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.092

4064
G. Morini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4063–4067
Table 1. Histamine H
3
receptor affinity (pK
i
) for human and rat H
3
receptor
R1
R
2
-11, 13-15
1
R
a
b
Compound
R
Human H
3
(pK
i
)
3 i
Rat H (pK )
1
7
2
3
4
5
6
–NH
2
–NH
2
5.35 (±0.01)
8.73 (±0.08)
6.12 (±0.06)
9.47 (±0.14)
8.21 (±0.03)
5.06 (±0.17)
8.18 (±0.06)
5.48 (±0.08)
8.92 (±0.03)
7.82 (±0.05)
CH3
CH3
CH3
CH3
N
N
H
N
H
N
C5H11
C5H11
24
N
N
N
CH3
CH3
CH3
CH3
7
8
9
0
1
3
4
5
7
8
N
N
N
N
6.40 (±0.09)
7.91 (±0.09)
5.85 (±0.20)
6.25 (±0.08)
7.43 (±0.01)
7.33 (±0.11)
8.20 (±0.03)
8.77 (±0.08)
8.93 (±0.17)
5.91 (±0.09)
8.59 (±0.05)
6.17 (±0.05)
7.09 (±0.05)
5.41 (±0.01)
5.68 (±0.06)
6.67 (±0.07)
6.94 (±0.08)
7.33 (±0.07)
8.04 (±0.15)
8.40 (±0.08)
5.39 (±0.09)
7.28 (±0.15)
CH3
CH3
1
1
1
5
5
5
O
O
O
O
N
N
N
N
H
N
H
N
1
1
1
1
1
1
1
N
N
CH3
CH3
N
O
N
N
N
O
N
N
H
N
N
N
18
N
N
c
c
Thioperamide
a
b
c
3
Inhibition of [ H]RAMHA binding to GPCR97-transfected SK-N-MC cells stably expressing the human histamine H
3
receptor.
3
Inhibition of [ H]RAMHA binding to rat brain membranes. Values are means of three experiments, standard error is given in parentheses.
Ref. 12.
at each position. This structure was selected on the
0
to those of the rigidified analogue 5. Similar structures
having a biphenyl scaffold have also been recently
reported in a patent on a series of H antagonists.
3
basis of its similarity to that of 1-1 -nonamethylenedi-
1
4
piperidine in its fully extended conformation, as can
be seen in Figure 1, where the protonated piperidine
NH groups of this flexible compound were superposed
To evaluate the influence of spacer length, we tested
the homologous derivative, having two methylene
0
groups each in 4 and 4 positions (17 in Table 1),
and the shorter analogue with only one phenyl ring
(
18 in Table 1).
Compounds 3–15 were synthesized as outlined in
Scheme 1. The symmetrical compounds 3–11 were
obtained by reacting the commercially available
0
4,4 -bis(chloromethyl)-biphenyl (1) with the appropriate
amine. The asymmetrical compounds 13–15 were
0
Figure 1. Superposition of 5 (yellow carbons) and 1-1 -nonamethy-
lenedi-piperidine (gray carbons). Only polar hydrogens are depicted.
1
5

G. Morini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4063–4067
4065
Cl
a
compounds was studied on RAMHA-induced inhibition
2
2
in electrically stimulated guinea pig isolated ileum.
Selectivity against H and H histamine receptor sub-
Cl
1
1
2
b
types was studied on isolated ileal and atrial guinea
pig preparations; inhibition of cerebral rat AChE was
23
determined following Ellman’s method.
R
N
R
The compounds revealed an interesting binding profile,
with some derivatives characterized by remarkable H3
affinity (Table 1). All compounds showed a moderate
preference for human H3 receptor compared to rat
Cl
3
-11
12
c
N
receptor, with the highest pK difference (about one log
i
R1
unity) observed for compound 14. Compound 5, carry-
0
ing two piperidines on the central 4,4 -bis-methyl-biphe-
nyl moiety, was the most potent derivative at the
1
3-15
Scheme 1. Reagents and conditions: (a) primary or secondary amine
receptors of both species, showing subnanomolar affini-
0
2
1
equiv, anhydrous toluene, reflux, overnight, 60–80%; (b) piperidine
equiv, anhydrous toluene, reflux overnight, 55%; (c) 2-piperidone
ty for human H receptor. The 4,4 -bis-methyl-biphenyl
3
scaffold provided the optimum distance between the ba-
sic centers, as evidenced by the moderate, but significant
affinity reduction observed for the longest derivative 17
and by the huge affinity drop of the shortest derivative
(sodium salt), 1-acetylpiperazine or cyclohexylamine 2 equiv, anhy-
drous toluene, reflux, overnight, 60–70%.
1
8. Within this limited exploration the piperidine ring
prepared reacting 1 with one equivalent of piperidine to
obtain the monosubstituted intermediate 12. After
isolation and purification, 12 was reacted with 2-piperi-
done (sodium salt), 1-acetylpiperazine, and cyclohexyl-
amine to obtain the desired products. Compound 17
resulted in optimal receptor binding. Further steric hin-
drance on this ring was detrimental for binding affinity:
4-methyl-, 3,5-dimethyl-piperidine and 1-acetylpiper-
azine fragments (compounds 6, 7, and 9) led to a loss
of binding affinity in proportion to their increased bulk-
iness. The most potent compounds of the present series
are characterized by tertiary amine groups, with only a
modest loss in binding affinity when a dimethylamine
0
was synthesized as described in Scheme 2. 4,4 -Bis
(
chloromethyl)-biphenyl (1) was reacted with NaCN to
obtain the dicyano derivative, that was hydrolyzed in
ethanolic KOH. After acidification, the symmetrical
dicarboxylic acid (16) was obtained. Intermediate 16
was activated with CDI and reacted with piperidine to
(
3) replaced the piperidine ring. In contrast, the primary
amine derivative 2 showed a dramatic decrease of poten-
cy, and secondary amines (4, 10) were only moderately
active. The slightly higher potency of 11 compared to
give the corresponding tertiary amide that was finally
reduced with RedAl to the target diamino compound
7. Compound 2 was prepared according to Rosa
Ò16
1
0 is consistent with this observation, as well as the
binding affinity observed for the asymmetric derivative
5, being intermediate between those of the symmetric
1
et al. and compound 18 was purchased from Labo-
1
7
1
1
8 1
Test.
tures of all tested compounds.
H NMR and CHN data confirmed the struc-
compounds 5 and 10. Comparison of binding data for
compounds 3 and 11 suggests that the cyclohexyl substi-
tuent is hardly tolerated by the H receptor. The de-
creased basicity and higher hydrophilicity of 14, 8, and
13 led to reduced potency.
3
H receptor affinity was measured by displacement of
3
3
[
transfected SK-N-MC cells stably expressing the human
H](R)-a-methylhistamine (RAMHA) from GPCR97-
1
9
histamine H receptor, and from rat cerebral cortex
3
membranes.
The compounds were tested for their histamine receptor
subtype selectivity, showing no interaction with H and
2
0,21
The H3 antagonistic potency of the
1
H receptors up to a concentration of 10 lM, and all of
2
them behaved as competitive H antagonists on electri-
cally stimulated guinea pig isolated ileum (data not
shown).
3
Cl
HOOC
a, b
Cl
COOH
1
16
c, d
Moreover, they showed no significant inhibition of
AChE activity up to a concentration of 10 lM, with
the exception of compounds 6 and 15 which caused
about 50% inhibition of rat brain AChE at 10 lM con-
centration. The most potent compound 5 was tested in a
functional assay performed on SK-N-MC cells express-
N
N
1
7
2
5
ing human H3 receptors. It showed an antagonist
behavior, with a pK value of 8.77 ± 0.17.
Scheme 2. Reagents and conditions: (a) NaCN 2 equiv, acetonitrile,
microwave irradiation (150 W, 150 °C, 10 bar, 5 min), 97%; (b) i—
KOH 2 equiv, ethanol, microwave irradiation (150 W, 150 °C, 10 bar,
B
The potent and rigid compound 5 was employed for
molecular modeling studies to investigate the putative
binding mode for this class of dibasic compounds. Com-
pound 5 was docked within a previously developed rat
5
min), ii—HCl 2 N, 92%; (c) i—CDI 2.2 equiv, THF, microwave
irradiation (250 W, 130 °C, 10 bar, 5 min); ii—piperidine 2 equiv,
THF, microwave irradiation (250 W, 130 °C, 10 bar, 5 min), 77%; (d)
RedAl 4 equiv, anhydrous toluene, 1 h rt, 3 h reflux, 85%.
Ò

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G. Morini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4063–4067
3
1
classical and non-imidazole H antagonists. Further
3
SAR studies to validate these docking hypotheses are
ongoing; at the moment, due to the nature of homolo-
gy-built receptor models, alternative binding mecha-
nisms cannot be excluded.
In conclusion, starting from a series of flexible ligands,
we developed a novel class of rigid dibasic H receptor
3
antagonists. The evaluation of different basic moieties
and the variation of spacer length led to compound 5 en-
dowed with subnanomolar potency at the human H3
receptor. The rigid scaffold also provided more informa-
tion for molecular modeling studies on the putative
interactions with H receptor.
3
3
Figure 2. Compound 5 (yellow carbons) within the H receptor model.
Only selected amino acids are represented, with gray carbons, and only
polar hydrogens are depicted. The protein backbone is represented by
a ribbon; for clarity, a portion of TM6 is not shown.
Acknowledgments
This work was supported by the Italian M.I.U.R.
della
(
Ministero dell’Istruzione, dell’Universita`
e
2
6
H receptor model by means of an automated docking
3
Ricerca). The Centro Interfacolt a` Misure and the
Centro di Calcolo Elettronico of the University of
Parma are gratefully acknowledged for providing
NMR instrumentation and software licenses. The
authors thank Dr. T. W. Lovenberg and Dr. S. Wilson,
Johnson & Johnson Pharmaceutical Research and
Development, San Diego, CA, for kindly providing
2
7,28
procedure,
and the resulting complexes were submit-
2
9
ted to molecular dynamics (MD) simulations to evalu-
ate their stability. The best solution is depicted in Figure 2.
The basic centers in compound 5 interact with the car-
boxyl groups of the highly conserved Asp114 in trans-
membrane (TM) 3 and of Glu206 in TM5, thought to
interact with the amine group and the imidazole ring
of histamine, respectively. One phenyl ring of 5 is sand-
wiched between Tyr115 and Tyr189, and this interaction
could stabilize the complex. The compound is near
Trp371, belonging to the CWXP motif in TM6 involved
in the process of receptor activation. This binding mode
is consistent with the docking results reported for other
the cells expressing the human histamine H receptor.
3
References and notes
1
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3
0
flexible dibasic non-imidazole H antagonists.
3
3
4
. Ligneau, X.; Garbarg, M.; Vizuete, M. L.; Diaz, J.;
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ed another putative accommodation of 5 within the H3
receptor TM bundle (Fig. 3). According to this alterna-
tive binding scheme, a piperidine nitrogen interacts with
Asp114 side chain, while the compound is deeply insert-
ed in a lipophilic pocket, mainly delimited by TM helices
5
6
. Clapham, J.; Kilpatrick, G. J. Br. J. Pharmacol. 1992, 107,
9
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3
, 6, and 7, and close to Trp371. The other piperidine is
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Asn404 in TM7. A similar accommodation, parallel to
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955.
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3. Rivara, M.; Zuliani, V.; Cocconcelli, G.; Morini, G.;
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Figure 3. Alternative binding scheme for compound 5. For clarity, a
portion of TM 7 ribbon is not shown.

G. Morini et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4063–4067
4067
1
6. Binet, J.; Thomas, D.; Benmbarek, A.; De Fornel, D.;
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25. SK-N-MC cells expressing beta-galactosidase reporter
construct and the human H3 receptors were used. The
compounds (0.1 nM–10 lM) were added directly to the
cell media, followed 5 min later by addition of forskolin
(5 lM) and cumulative application of agonist (R)-a-
methylhistamine (0.1 nM–1 lM). After a 6 h incubation
at 37 °C, the media were aspirated and the cells were lysed
with 0.5% Triton and 40 mM beta-mercaptoethanol
Apodaca, R.; Dvorak, C. A.; Xiao, W.; Barbier, A. J.;
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1
1
1
2
2
8. Prof. Dr. J. Beger und Dipl.-Ing. U. Beger LaboTest
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1
1. Homogenates of SK-N-MC cells expressing human H
ꢀ
1
using 25 lL of 1 mg mL substrate solution (chlorophe-
nol red beta-D-galactopyranoside; Roche Molecular Bio-
chemicals, Indianapolis, IN, USA) and quantitated on a
microplate reader at absorbance 570 nm after overnight
incubation at 20 °C.
995, 30, 881.
3
histamine receptors or rat cerebral cortex membranes were
used for determining affinity values of the compounds
prepared in radioligand displacement studies. Membranes
26. 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.
were incubated for 60 min at room temperature with
0
3
.5 nM [ H](R)-a-methylhistamine (30.0 Ci/mmol, Amer-
sham Bioscience) in buffer solution (pH 7.4) (50 mM Tris–
HCl/5 mM EDTA for cells or 50 mM Tris–HCl/0.5 mM
EDTA/50 mM NaCl for rat) with or without competing
ligands. Incubation was terminated by rapid filtration over
Millipore AAWPO2500 filters followed by two washes
with ice-cold buffer (50 mM Tris–HCl/5 mM EDTA).
Retained radioactivity was determined by liquid scintilla-
tion counting. Non-specific binding was defined using
27. Glide 3.5, Schr o¨ dinger. L.L.C., NY Friesner, R. A.;
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Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S.
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28. Automated docking was performed on 5 in its diproto-
nated state, starting from a minimum energy conforma-
tion placed in an arbitrary position, within a box centered
on Asp114 and including the extracellular portion of the
receptor. Docking solutions were ranked according to
their Emodel values.
1
0 lM histamine or 10 lM thioperamide as competing
ligands in SK-N-MC cells or in rat cortex, respectively.
IC50 values were estimated from the displacement curves
3
of the tested compounds (0.01 nM–10 lM) versus [ H](R)-
29. MD simulations were performed with Macromodel 8.5
(Schr o¨ dinger. L.L.C., NY), applying MMFF94 force field,
with a time step of 1 fs, for 1 ns at 310 °K after 100 ps of
equilibration, with fixed protein backbone.
a-methylhistamine and converted to K values according
i
to Cheng and Prusoff’s equation Cheng, Y. C.; Prusoff, W.
H. Biochem. Pharmacol. 1973, 22, 3099.
2
2
2. Barocelli, E.; Ballabeni, V.; Caretta, A.; Bordi, F.; Silva,
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