Development of a selective and potent radioactive ligand for histamine H3 receptors: A compound potentially useful for receptor occupancy studies

Article abstract of DOI:10.1016/j.bmcl.2009.06.025

Radioligands are powerful tools for examining the pharmacological profiles of chemical leads and thus facilitate drug discovery. In this study, we identified and characterized 3-([1,1,1-³H]methyl)-2-(4-{[3-(1-pyrrolidinyl)propyl]oxy} phenyl)-4(

Full text of DOI:10.1016/j.bmcl.2009.06.025

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4075–4078
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a selective and potent radioactive ligand for histamine H3
receptors: A compound potentially useful for receptor occupancy studies
*
Yuko Mitobe, Sayaka Ito, Takashi Mizutani, Tsuyoshi Nagase, Nagaaki Sato, Shigeru Tokita
Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co., Ltd, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Radioligands are powerful tools for examining the pharmacological profiles of chemical leads and thus
facilitate drug discovery. In this study, we identified and characterized 3-([1,1,1-3H]methyl)-2-(4-{[3-
(1-pyrrolidinyl)propyl]oxy} phenyl)-4(3H)-quinazolinone ([3H]1) as a potent and selective radioligand
for histamine H3 receptors. Radioligand [3H]1 exhibited appreciable specific signal in brain slices pre-
pared from wild-type mice but not from histamine H3 receptor-deficient mice, demonstrating the spec-
ificity and utility of [3H]1 as a selective histamine H3 receptor radioligand for ex-vivo receptor occupancy
assays.
Received 27 March 2009
Revised 2 June 2009
Accepted 3 June 2009
Available online 13 June 2009
Keywords:
Histamine H3 receptors
Inverse agonist
Radioligand
Ó 2009 Elsevier Ltd. All rights reserved.
Receptor occupancy
The histamine H3 receptor, one of the G protein-coupled recep-
tors (GPCRs) for histamine, is predominantly expressed in the cen-
tral nervous system (CNS) and is known to play important roles in
various physiological functions. Accumulating evidence has sug-
gested that histamine H3 receptor antagonists/inverse agonists
may be promising agents for the treatment of cognitive dysfunc-
tion, epilepsy, hypersomnia, and obesity.1 Several classes of imid-
azole- and non-imidazole H3 receptor antagonists/inverse
agonists have been developed to target CNS H3 receptors.2–4
Among them, BF2.649,5,6 ABT-2397, and GSK189254,8 which are
non-imidazole compounds, have entered clinical trials for treat-
ment of CNS disorders, for indications which include excessive
daytime sleepiness, schizophrenia, and cognitive dysfunction
(Fig. 1).
(e.g., loss of specific signals in receptor-deficient mice) remains an
essential but as yet unmet goal integral for the development of
useful and reliable radiotracers.14
In histamine H3 receptor biological and pharmacological stud-
a
ies, [3H] N -methylhistamine, a H3 receptor agonist, has often been
used as a standard radiotracer for ligand binding assays of tissues
a
and cells. However, the lack of selectivity of N -methylhistamine
a
for histamine H4 receptors limits the usefulness of [3H] N -methyl-
histamine for whole-tissue studies.15
Recently, we have reported the synthesis of a number of quinaz-
olinone analogues that exhibited high potency and selectivity for
histamine H3 receptors.16,17 In the present study, we report the
synthesis and pharmacological characterization of [3H]1 as a po-
tent and selective inverse agonist radioligand for histamine H3
receptors (Fig. 2).
We previously reported novel 2-[4-(alkoxy)phenyl]-4(3H)-qui-
nazolinone derivatives which exhibited potent human histamine
H3 receptor binding activity.9 In the course of structure–activity
relationship (SAR) studies, a highly potent compound, [3H]1 was
identified as a promising radioligand candidate with low hydrophi-
licity (Log D7.4 = 1.2). With respect to P-glycoprotein (P-gp) trans-
porter susceptibility, the transcellular transport ratios ((B-to-A)/
(A-to-B)) of compound 1 for human MDR1 and mouse mdrla pro-
teins were 1.9 and 2.1, respectively, indicating the low susceptibil-
ity of compound 1 to P-gp. Consistent with these observations,
compound 1 exhibited high brain penetrability 2 h after 10 mg/
kg oral administration to SD rats (brain = 2.22 nmol/g, plas-
In drug discovery, potent and selective radioligands for target
molecules are powerful tools and facilitate not only in vitro charac-
terization but also in vivo pharmacodynamic profiling (i.e., deter-
mination of relationships between efficacy and levels of target
engagement) of test compounds. Several radioligands including
positron emission tomography (PET) tracers have been developed
and characterized for histamine H3 receptors.9–12 However, low
brain penetrability and/or high background noise limit the applica-
tion of these radioligands as reliable and useful radiotracers.13,14
For example, despite its high performance in in vitro assays,
[
11C]JNJ-10181457 exhibited no discernable specific binding in
rodent brains, suggesting that demonstration of in vivo selectivity
ma = 1.36 lM, brain-to-plasma ratio = 1.6) (Table 1).
Tritium-labeled compound 1 was synthesized as outlined in
Scheme 1. The 2-aminobenzamide 2 was thermally condensed
* Corresponding author. Tel.: +81 29 877 2000; fax: +81 29 877 2027.
E-mail addresses: shigeru_tokita@merck.com, shigeru.tokita@po.rd.taisho.co.jp
(S. Tokita).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.025

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Y. Mitobe et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4075–4078
Figure 1. Structures of histamine H3 receptor (A) antagonists/inverse agonists and (B) agonists.
with 4-[3-(pyrrolidin-1-yl)propoxy]benzaldehyde
3
in the
presence of a catalytic amount of p-toluenesulfonic acid, followed
by oxidation with 2,3-dichloro-5,6-dicyanobenzoquinone to obtain
4. Methylation of 4 then furnished 1, establishing the route of syn-
thesis of [3H]1 from the corresponding precursor 4 using a
[3H]methyl iodide as a radiolabel donor. Subsequently, radioligand
[3H]1 was purified using radio-HPLC analysis, resulted in the pro-
duction of a high-specific radioligand with appreciable purity
(83.7 Ci/mmol, >95% purity) .
In the [35S]GTP
activity of ligands for G-protein coupled receptors,18 we observed
that compound 1 potently decreased basal [35S]GTP
S binding
cS binding assay, which measures the functional
Figure 2. Structure of compound 1.
c
activity in human embryonic kidney (HEK) 293 cell membranes
overexpressing recombinant mouse histamine H3 receptors, with
Table 1
Brain penetration and P-gp transport ratio of compound 1
Compd
Brian penetration in SD ratsa
P-gp transporter assayb
Transcellular transport ratio (B-to-A)/(A-to-B)
Plasma (
lM)
Brain (nmol/g)
2.22
CSF (
l
M)
MDR1
mdr1a
1
1.36
0.134
1.9
2.1
a
The values represent the means from three animals. The concentrations were determined at 2 h after 10 mg/kg po.
Transcellular transport ratios ((B-to-A)/(A-to-B)) for human and mouse P-glycoproteins were determined in the cellular transporter assay using porcine renal epithelial
b
cells expressing human MDR1 and murine mdr1a proteins.
Scheme 1. Reagents and conditions: (a) 4-[3-(pyrrolidin-1-yl)propoxy]benzaldehyde, p-toluenesulfonic acid, toluene, 100 °C, 15 h, then 2,3-dichloro-5,6-dicyanobenzoqui-
none, rt, 6 h, 67%; (b) NaH, CH3I or CT3I, DMF.

Y. Mitobe et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4075–4078
4077
a
Table 2
compound 1, thioperamide, clobenpropit, N -methylhistamine,
histamine, and imetit are 2.4, 4.8, 0.60, 19, 68, and 2.0 nM, respec-
tively. These findings suggest that radioligand [3H]1 will be useful
for studies of histamine H3 receptor ligands regardless of their
functional classes (Fig. 4).
EC50 values and maximum inhibition (% basal control) of H3 receptor ligands for
mouse histamine H3 receptor in the [35S]-GTP
c
S binding assay
Compound
Potency EC50 (nM)
Efficacy max. inhibition (%)
Compound 1
Thioperamide
Clobenpropit
9.6
11
1.7
24
27
14
Finally, we examined the profiles of binding of radioligand [3H]1
to brain membranes and slices prepared from wild-type and hista-
mine H3 receptor knockout mice.20 Consistent with the findings
obtained with recombinant mouse histamine H3 receptors de-
scribed above, radioligand [3H]1 displayed high binding affinity
to the brain membranes from wild-type mice (Kd = 3.1 nM) while
no significant specific binding to brain membranes was observed
in the receptor knockout mice (data not shown) .
The values represent the means from two independent experiments.
an EC50 of 9.6 nM, indicating that compound 1 is a potent inverse
agonist for mouse histamine H3 receptors (Table 2). The maximum
efficacy (i.e., inverse agonism) of compound 1 was comparable to
that of thioperamide. It has been reported that thioperamide is a
potent inverse agonist of rodent histamine H3 receptors while
exhibiting significantly lower activity for human and monkey
histamine H3 receptors,19 limiting the utility of thioperamide in
higher species. In contrast, compound 1 exhibited high intrinsic
potencies for rodents and human histamine H3 receptors in com-
petitive ligand binding assays (human Ki = 4.2 nM, rat Ki = 9.6 nM,
mouse Ki = 12 nM). In addition, compound 1 displayed high de-
grees of selectivity over other histamine receptor subtypes (IC50
In the receptor occupancy (RO) assay, the striatum was used as a
target area, since histamine H3 receptors are widely and abundantly
expressed in this region.21–23 Coronal brain sections (20
lm) includ-
ing striatum were incubated with 3 nM of radioligand [3H]1 in RO
assay buffer (pH 7.4, 150 mM NaH2PO4, 2 mM MgCl2, Tris–HCl) for
>10 lM for histamine H1, H2, and H4 receptors) and a panel of 86
GPCRs (data not shown). Furthermore, compound 1 exhibited
low lipophilicity as described above (Log D7.4 = 1.2), which might
be advantageous in terms of decreasing the background noise that
is often associated with non-specific binding.
With these promising biochemical and physicochemical proper-
ties, the potential of [3H]1 as a radioligand for histamine H3 recep-
tors was examined in in vitro membrane binding and ex vivo brain
slice binding assays.
Scatchard plot analysis of saturation binding of [3H]1 against re-
combinant mouse histamine H3 receptors revealed that radioli-
gand [3H]1 bound to
2.6 0.36 nM, consistent with its high potency in the GTP
a
single site with
a
Kd value of
S func-
c
Figure 4. Displacement curves for radioligand [3H]1 in competition with H3 ligand
in membranes expressing recombinant mouse H3 receptors. The membranes from
HEK293 cells expressing mouse H3 receptors were incubated with 2 nM [3H]1 for
competition binding assays in the presence of various compound concentrations in
buffer (50 mM Tris–HCl, pH 7.4) at 25 °C for 1 h. Following three washes,
membrane-bound radioactivity was measured by a liquid scintillation counter.
tional assay (Fig. 3). To further examine the receptor binding pro-
file of compound 1, we conducted a competitive binding assay
using [3H]1 as a radioligand. Binding of compound 1 to mouse his-
tamine H3 receptors was completely replaced by a series of known
histamine H3 receptor ligands including agonists (N -methylhista-
mine, histamine, and imetit) and the antagonists/inverse agonists
a
Non-specific binding was determined in the presence of excessive cold (R)-a-
methylhistamine (10
lM). The result shown is one of two independent
thioperamide, clobenpropit, and compound 1. The Ki values of
experiments.
Figure 3. Saturation binding of radioligand [3H]1 binding in membranes expressing recombinant mouse H3 receptors. Membranes from HEK293 cells expressing mouse H3
receptors were incubated with several concentrations of [3H]1 in the presence of DMSO (total binding) or 1
M compound 1 in assay buffer (pH 7.4, 50 mM Tris–HCl) at 25 °C
l
for 1 h. The membranes were filtered with GF/C filter and washed with washing buffer (pH 7.4, 50 mM Tris–HCl) three times. The residual radioactivities on the dried filter
were measured by microplate scintillation counter. The results shown are from a typical experiment from among three independent experiments. (A) Saturation binding
isotherm. (B) Scatchard plot analysis of binding of [3H]1 resulted in a Kd of 3.9 0.49 nM and a Bmax of 6.2 0.12 pmol/mg protein.

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Y. Mitobe et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4075–4078
Figure 5. Ex vivo autoradiography of radioligand [3H]1 binding to the striatum in mouse brains. (A-1) Ex vivo autoradiography with b-imager detection of radioligand [3H]1
binding to brain sections including striatum. Total binding to wild-type (H3R+/+), histamine H3 receptor hetero knockout (H3R+/À), and homo knockout (H3RÀ/À) mouse brain
sections. (A-2) In this schematic, the striatal region examined in this study is surrounded by a circle. (B) Quantitative analysis of signal potency of [3H]1 binding to the
striatum in the mouse brains. The signal potency of the striatum in the each section was determined in cpm/mm2 using the b-imager.
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nal in the wild-type, hetero, and homo knockout brains, suggesting
that radioligand [3H]1 can be used to quantitate levels of expression
of histamine H3 receptors in the brain.
In summary, we developed a highly potent and selective radio-
ligand [3H]1, which displayed competitive binding to histamine H3
receptorxs (vs known histamine H3 receptor ligands) and a specific
signal in brain sections. The high selectivity of and appreciable
brain penetration by compound 1 make it promising as a radio-
tracer for in vitro and in vivo studies. Given the importance of
pharmacodynamic findings for compounds in drug discovery, radi-
oligand [3H]1 will be a useful tool for the development of hista-
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