Fluorescent non-imidazole histamine H3 receptor ligands with nanomolar affinities

Article abstract of DOI:10.1016/j.bmcl.2005.12.084

ω-Piperidinoalkanamine derivatives with fluorescent moieties (2-cyanoisoindol-1-yl, 7-nitrobenzofurazan-4-yl) have been synthesized starting from piperidine in three steps. The compounds display moderate to good histamine hH₃ receptor affinities with K_i values ranging from 178 to 11 nM. The new compounds may act as tools for identification and understanding of the binding site on the histamine H₃ receptor.

Full text of DOI:10.1016/j.bmcl.2005.12.084

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1938–1940
Fluorescent non-imidazole histamine H receptor ligands
3
with nanomolar affinities
b
a
b
a,
*
Michael Amon, Xavier Ligneau, Jean-Charles Schwartz and Holger Stark
a
Johann Wolfgang Goethe-Universit a¨ t, ZAFES, Biozentrum, Institut f u¨ r Pharmazeutische Chemie, 60431 Frankfurt, Germany
b
Bioprojet Biotech, 4 rue du Chesnay Beauregard, BP 96205, 35762 Saint Gr e´ goire Cedex, France
Received 2 December 2005; revised 21 December 2005; accepted 21 December 2005
Available online 24 January 2006
Abstract—x-Piperidinoalkanamine derivatives with fluorescent moieties (2-cyanoisoindol-1-yl, 7-nitrobenzofurazan-4-yl) have been
synthesized starting from piperidine in three steps. The compounds display moderate to good histamine hH receptor affinities with
3
K
on the histamine H receptor.
i
values ranging from 178 to 11 nM. The new compounds may act as tools for identification and understanding of the binding site
3
Ó 2006 Elsevier Ltd. All rights reserved.
Within the four known histamine receptors, the H
1
3
subtype is predominantly expressed in the brain. The
histamine H receptor has been shown to inhibit the
3
histamine synthesis in and release from histaminergic
neurons via a negative feedback loop, but also to mod-
2
ulate the release of other neurotransmitters. Actually
there is great effort in the development potential of high-
ly potent and selective antagonists due to their discussed
importance in the treatment of various diseases, e.g.,
schizophrenia, Alzheimer’s disease, obesity and atten-
1
tion-deficit hyperactivity disorder (ADHD).
Figure 1. Different structurally related lead structures for histamine H
3–5
receptor antagonists.
3
The development of H receptor antagonists opens a
3
wide range of compounds differentiated into two main
classes. Almost all potent antagonists published before
1
999 were imidazole derivatives monosubstituted in
7
3
mechanism of ligand binding, localization, movement
8
and internalisation of receptors in living cells. They
the 4(5)-position (Fig. 1; A). By exchange of the imidaz-
ole moiety to piperidine, pyrrolidine, piperazine and
related structures an additional class of non-imidazole
can give hints on the environment of receptor binding
sites, because some fluorophores show excitation and
emission wavelengths depending on the surrounding,
lipophilicity, pH, temperature, solvent, etc.
mercially available fluorescent histamine derivative
1
,2,4
antagonists has been described (Fig. 1; B).
9
,10
A com-
Fluorescent compounds for G protein-coupled receptors
may be useful research tools for non-radioactive binding
assays or for investigations on the structural properties
of these receptor–ligand interactions. Such ligands
may offer a multiplicity of information such as the
Ò
(
BODIPY FL histamine; Molecular Probes) is able to
1
1
6
show lysosomal localization, but to our knowledge
receptor binding properties have not been described.
Based on the recent results on structure–activity rela-
1
–4,12
tionships
el non-imidazole histamine H receptor ligands which
we have designed and prepared some nov-
3
Keywords: Histamine; Antagonist; Ligand; Fluorescence; H3; Medic-
inal chemistry.
*
possess a fluorescent chromophore in the lipophilic part
1
Corresponding author. Fax: +49 69 79829258; e-mail: h.stark@
pharmchem.uni-frankfurt.de
of a general blueprint for antagonist structures. Very
recently during the preparation of this project related
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.084

M. Amon et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1938–1940
1939
fluorescent histamine H receptor ligands of a benzofu-
3
5
ran series have been presented (Fig. 1; C).
The compounds were synthesized in three steps accord-
ing to the scheme shown in Figure 2: 1. Alkylation of
1
2
piperidine with x-bromoalkannitrile (65 – 85% yields);
. Reduction of the nitrile group with Raney-nickel/hy-
2
drogen at room temperature (90–95% yields); 3. Reac-
1
3
tion of amine compound with phthaldialdehyde/
1
4
NaCN or 4-chloro-7-nitrobenzofurazan (4-chloro-7-
1
5
nitrobenzo[c][1,2,5]oxadiazole) resulting in the final
compounds (25–85% yields).
All excitation and emission data were measured with an
Amico-Bowman Series 2 Spectrometer. The fluorescent
compounds were measured at a concentration of
Figure 4. Excitation and emission spectra of the prominent penta-
methylene derivative 6 of 4-nitrobenzofuran series.
À5
1
0
M in absolute spectroscopic ethanol at room tem-
perature. Emission spectra were recorded at the excita-
tion max. wavelength (Figs. 3 and 4).
Table 1. Binding affinities of histamine H
isoindole (1–4), 4-nitrofurazan (5, 6) and reference compounds
3
receptor ligands (cf. Fig. 2):
3–5
a
b
Pharmacological data of histamine H receptor affinities
3
Compound
n
K
i
± SEM
ClogP
Excitation Emission
max (nm) max (nm)
1
25
were obtained by [ I]iodoproxyfan binding assay on
(
nM)
k
k
CHO-K1 cells stably expressing the human H receptor
3
1
2
1
2
3
4
2
3
351 ± 6
178 ± 41
31 ± 10
31 ± 5
3.50
3.70
3.50
4.75
3.84
4.37
1.89
3.82
5.76
343
350
344
342
503
467
359
366
366
357
526
526
1
6
(
Table 1).
3
4
5
170 ± 34
11 ± 1
6
c
A
B
C
125
12 ± 3
d
39
0.10
e
e
e
465
535
a
[
I]Iodoproxyfan binding on CHO-K1 cells stably expressing the
1
6
hH
3
receptor.
b
The n-octanol/water partition coefficient based on established
chemical interactions; calculated with ChemDraw Ultra 7.0.
17
c,d
3
i
K values obtained in experiments of [ H]histamine release from rat
3
,4
cerebral cortex synaptosomes see, respectively.
Value from Ref. 5.
e
All tested compounds could be easily obtained by the
method described. They showed moderate to good affin-
ities for histamine H receptors in the nanomolar con-
3
centration range (Table 1).
Figure 2. Synthesis of compounds 1–6. (i) K
2
CO
, Raney-nickel, H , 12 h, rt; (iii) NaCN,
4
, 18 h, rt; (iv) abs dioxane, 18 h, rt.
3
, KI, abs acetonitrile,
1
8 h, rt; (ii) MeOH/ NH
3
2
In the series of fluorescent isoindole compounds 1–4 the
highest binding affinities (e.g., ‘lowest K values’) were
MeOH, NaBH
i
obtained with compounds having a chain length of five
(
3) to six (4) methylene groups as spacer. Therefore, in
the 4-nitrobenzofurazan series the related spacer lengths
from basic amine to aromatic residue have only been
considered. The lipophilicities of all compounds are in
acceptable calculated range of ClogP values below
1
7
5
. All compounds showed an acceptable difference of
excitation to emission maximal wavelengths (Stokes
shift). Especially the most potent compound 6 showed
the highest Stokes shift of 59 nm. The wavelengths for
excitation vary from 342 to 503 nm, whereas those for
emission vary from 377 to 526 nm.
It was demonstrated that it is possible to obtain fluores-
cent histamine H receptor ligands by application of the
3
Figure 3. Excitation and emission spectra of the prominent penta-
methylene derivative 3 of isoindole series.
general blueprint and knowledge on structure–activity
relationships. The fluorescent compounds represent a

1940
M. Amon et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1938–1940
new series of potent compounds, which may be utilized
for further in vivo or cell studies using modern imaging
techniques and have the potential for useful pharmaco-
logical tools to investigate receptor–ligand interactions,
for example, in fluorimetric binding assays or functional
studies. The moderate to good affinities and maximal
emission wavelengths open a wide area for further opti-
mization on these fluorescent histamine H3 receptor
ligands.
J = 4.9 Hz, 2H), 2.94–3.04 (4H), 2.29 (t, J = 7.1 Hz, 2H),
.01 (dt, J = 7.2 Hz, 4H), 1.69 (t, J = 4.6 Hz, 2H).
Elemental analysis, calcd: C, 59.36; H, 6.82; N, 10.93.
2
Found: C, 59.27; H, 6.58; N, 10.90. Compound 2: 2-(4-
Piperidin-1-ylbutyl)-2H-isoindol-1-carbonitrile,
yield:
calcd: 281.40, found:
81.9; H NMR (DMSO): d (ppm) 7.69 (s, 1H), 7.66 (d,
9
2
1%, mp 139 °C; ESI-MS C18
23 3
H N
1
J = 10.6 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.24 (t,
J = 7.8 Hz, 1H), 7.09 (t, J = 6.9 Hz, 1H), 4.47 (t,
J = 6.9 Hz, 2H), 3.47 (t, J = 6.9 Hz, 2H), 3.13 (t,
J = 8.4 Hz, 2H), 2.81–2.92 (4H), 2.03 (t, J = 7.6 Hz, 2H),
1
.76–1.84 (4H), 1.49–1.52 (2H). Elemental analysis, calcd:
C, 62.40; H, 6.94; N, 10.92. Found: C, 62.75; H, 6.79; N,
0.54. Compound 3: 2-(5-Piperidin-1-ylpentyl)-2H-isoin-
dol-1-carbonitrile. Yield: 98%, mp 140 °C; ESI-MS
References and notes
1
1
2
. Stark, H. Expert Opin. Ther. Pat. 2003, 13, 851.
. Leurs, R.; Bakker, R. A.; Timmerman, H.; de Esch, J. P.
Nat. Rev. Drug Dis. 2005, 4, 107.
1
19 25 3
C H N calcd: 295.43, found, 295.8; H NMR (DMSO):
d (ppm) 7.84 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.57 (d,
J = 8.5 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 7.08 (t,
J = 6.1 Hz, 1H), 4.38 (t, J = 6.9 Hz, 2H), 2.95–3.01 (4H),
2.89 (t, J= 8.4 Hz, 4H), 1.68 (t, J = 7.4 Hz, 4H), 1.46–1.50
(4H), 1.27 (dd, J = 7.4 Hz, 2H). Elemental analysis, calcd:
C, 63.71; H, 6.85; N, 10.41. Found: C, 63.81; H, 6.92; N,
10.05. Compound 4: 2-(6-Piperidin-1-ylhexyl)-2H-isoin-
3
4
5
. Ganellin, C. R.; Fkyerat, A.; Bang-Andersen, B.;
Athmani, S.; Tertiuk, W.; Garbarg, M.; Ligneau, X.;
Schwartz, J.-C. J. Med. Chem. 1996, 39, 3806.
. Ganellin, C. R.; Leurquin, F.; Piripitsi, A.; Arrang, J.-M.;
Garbarg, M.; Ligneau, X.; Schunack, W.; Schwartz, J.-C.
Arch. Pharm. 1998, 331, 395.
. Cowart, M.; Gfesser, G. A.; Bhatia, K.; Esser, R.; Sun,
M.; Miller, T. R.; Krueger, K.; Witte, D.; Esbenshade, T.
A.; Hancock, A. A. Abstracts of Posters, Part 49, 34th
Meeting of European Histamine Research Society, Bled/
Slovenia, May 11–14, 2005.
dol-1-carbonitrile. Yield: 97%, mp 141 °C; ESI-MS
1
calcd: 309.46, found: 310.0; H NMR (DMSO):
C
H
20
N
27
3
d (ppm) 7.88 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.57 (d,
J = 8.6 Hz, 1H), 7.24 (t, J = 6.7 Hz, 1H), 7.08 (t,
J = 8.1 Hz, 1H), 4.36 (t,J = 6.9 Hz, 2H), 2.90–3.03 (4H)
2.81 (t, J = 7.9 Hz, 4H), 1.88 (t, J = 6.8 Hz, 2H), 1.66 (t,
J = 5.2 Hz, 4H), 1.56–1.60 (4H), 1.45–1.47 (4H), 1.26–1.28
(2H). Elemental analysis, calcd: C, 65.41; H, 7.36; N,
10.40. Found: C, 65.39; H, 7.33; N, 10.14.
6
7
8
. Garcia, M.; Floran, B.; Arias-Montano, J. A.; Young, J.
M.; Aceves, J. Neuroscience 1997, 80, 241.
. Madsen, B. W.; Beglan, C. L.; Spivak, C. E. J. Neurosci.
Methods 2000, 97, 123.
. Terillon, S.; Cheng, L. L.; Stoev, S.; Mouillac, B.;
Barberis, C.; Manning, M.; Durroux, T. J. Med. Chem.
15. Compound
5:
7-Nitro-N-(4-(piperidin-1-yl)butyl)-
benzo[c][1,2,5]oxadiazol-4-amine was synthesized from
(4-aminobutyl)piperidine (20 mmol) and 4-chloro-7-nitro-
benzo[c][1,2,5]oxadiazole (10 mmol) in abs. dioxane
(20 mL) in the dark over 17 h at room temperature.
Purification was performed after evaporation in vacuum
2
002, 45, 2579.
. Neefjes, J.; Dantuma, N. P. Nat. Rev. Drug Dis. 2004, 3,
8.
9
5
1
1
0. Daly, C. J.; McGrath, J. C. Pharmacol. Ther. 2003, 100.
1. Molecular Probes (assessed on Dec. 2005 https://catalog.
invitrogen.com/index.cfm?fuseaction=viewCatalog.view-
ProductDetails&productDescription=16775&CMP= LEC-
GCMSSEARCH&HQS=histamine).
2. Meier, G.; Apelt, J.; Reichert, U.; Graßmann, S.; Ligneau,
X.; Elz, S.; Leurquin, F.; Ganellin, C. R.; Schwartz, J.-C.;
Schunack, W.; Stark, H. Eur. J. Pharm. Sci. 2001, 13, 249.
3. Albert, A.; Magrath, D. J. Chem. Soc. 1944, 240, 678.
4. Compound 1: 2-(3-Piperidin-1-ylpropyl)-2H-isoindol-1-
carbonitrile was synthesized from (3-aminopropyl)piperi-
dine with 1,2-phthaldialdehyde (10 mmol), NaCN
by column chromatography using CH
saturated (95:5). Yield: 65%, mp 157 °C;1 ESI-MS
calcd: 319.16, found; 319.8; H NMR
2 2 3
Cl -MeOH, NH
15 21 5 3
C H N O
(DMSO): d (ppm) 8.49 (d, J = 8.9 Hz, 1H), 6.42 (d,
J = 9.0 Hz, 1H), 3.94 (s, 1H, NH), 3.43 (t, J = 6.9 Hz, 2H),
2.81–3.15 (6H), 1.65–1.85 (8H), 1.48–1.54 (2H). Elemental
analysis, calcd: C, 48.38; H, 5.43; N, 15.94. Found: C,
48.55; H, 5.48; N, 15.63. Compound 6: 7-Nitro-N-(5-
(piperidin-1-yl)pentyl)benzo[c][1,2,5]oxadiazol-4-amine.
1
1
1
Yield: 62%, mp 186 °C; ESI-MS C16
H N O calcd:
23 5 3
1
333.39; found: 333.9; H NMR (DMSO): d (ppm) 8.50
(d, J = 8.9 Hz, 1H), 6.42 (d, J = 9.0 Hz, 1H), 3.96 (s, 1H,
NH), 3.42 (t, J = 7.0 Hz, 2H), 3.02–3.08 (2H), 1.69–1.75
(6H), 1.45–1.55 (8H), 1.27–1.29 (2H). Elemental analysis,
calcd: C, 49.15; H, 5.66; N, 15.24. Found: C, 49.26; H,
5.80; N, 15.00.
(
(
12 mmol) and sodium tetraborate (15 mmol) in methanol
20 mL). After stirring in the dark for over 18 h at room
temperature, the reaction mixture was added to ice/water.
Filtration, drying, and purification by column chroma-
tography using CH Cl -MeOH, NH saturated (95:5)
2 2 3
resulted in the final product. Yield: 65%, mp 141 °C;
16. Ligneau, X.; Morisset, S.; Tardivel-Lacombe, J.; Gba-
hou, F.; Ganellin, C. R.; Stark, H.; Schunack, W.;
Schwartz, J.-C.; Arrang, J.-M. Br. J. Pharmacol. 2000,
131, 1247.
1
ESI-MS C H N calcd: 267.38, found: 267.9; H NMR
1
7
21
3
(
7
(
DMSO): d (ppm) 7.91 (s, 1H), 7.74 (d, J = 13.2 Hz, 1H),
.65 (d, J = 7.5 Hz, 1H), 7.28 (dd, J = 7.0 Hz, 1H), 7.10
dd, J = 7.8 Hz, 1H), 4.46 (t, J = 7.8 Hz, 2H), 3.34 (t,
17. ChemOffice 2002, CambridgeSoft Corporation 2002.