Cyclopropane-based conformational restriction of histamine. (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane, a highly selective agonist for the histamine H3 receptor, having a cis-cyclopropane structure

Article abstract of DOI:10.1021/jm020415q

A series of cyclopropane-based conformationally restricted analogues of histamine, the "folded" cis-analogues, i.e., (1S,2R)-2-(aminomethyl)-1-(1H-imidazol-4-yl)cyclopropane (11), (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane (13), and their e

Full text of DOI:10.1021/jm020415q

1980
J . Med. Chem. 2003, 46, 1980-1988
Cyclop r op a n e-Ba sed Con for m a tion a l Restr iction of Hista m in e.
(1S,2S)-2-(2-Am in oeth yl)-1-(1H-im id a zol-4-yl)cyclop r op a n e, a High ly Selective
Agon ist for th e Hista m in e H3 Recep tor , Ha vin g a cis-Cyclop r op a n e Str u ctu r e
Yuji Kazuta,† Kazufumi Hirano,‡ Kentaro Natsume,‡ Shizuo Yamada,‡ Ryohei Kimura,‡ Shun-ichiro Matsumoto,§
Kiyoshi Furuichi,§ Akira Matsuda,† and Satoshi Shuto*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, J apan,
Department of Biopharmacy, School of Pharmaceutical Sciences, University of Shizuoka, Yata, Shizuoka 422-8526, J apan, and
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., 21 Miyukigaonka, Tsukuba 305-8585, J apan
Received September 24, 2002
A series of cyclopropane-based conformationally restricted analogues of histamine, the “folded”
cis-analogues, i.e., (1S,2R)-2-(aminomethyl)-1-(1H-imidazol-4-yl)cyclopropane (11), (1S,2S)-2-
(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane (13), and their enantiomers ent-11 and ent-
13, and the “extended” trans-analogues, i.e., (1R,2R)-2-(aminomethyl)-1-(1H-imidazol-4-
yl)cyclopropane (12) and its enantiomer ent-12, were designed as histamine H3 receptor agonists.
These target compounds were synthesized from the versatile chiral cyclopropane units, (1S,2R)-
and (1R,2R)-2-(tert-butyldiphenylsilyloxy)methyl-1-formylcyclopropane (14 and 15, respectively)
or their enantiomers ent-14 and ent-15. Among the conformationally restricted analogues, the
“folded” analogue 13 (AEIC) having the cis-cyclopropane structure was identified as a potent
H3 receptor agonist, which showed a significant binding affinity (Ki ) 1.31 ( 0.16 nM) and
had an agonist effect (EC50 value of 10 ( 3 nM) on the receptor. This compound owes its
importance to being the first highly selective H3 receptor agonist to have virtually no effect on
the H4 subtype receptor. These studies showed that the cis-cyclopropane structure is very
effective in the conformational restriction of histamine to improve the specific binding to the
histamine H3 receptor.
In tr od u ction
A variety of homeostatic processes, such as sleeping
and wakefulness, eating and drinking, learning and
memory, or neuroendocrine, are known to be related to
the neurotransmitter histamine (1, Figure 1).1 The
effects of histamine are mediated by at least four
receptor subtypes termed H1, H2, H3, and recently
discovered H4 receptors, and the agonists and antago-
nists specific to each one of these subtypes can be used
as drugs as well as pharmacological tools.1
Recently, much attention has been focused on the H3
receptor,2 a Gi protein-coupled receptor, which was
cloned by Lovenberg and co-workers in 1999.2c Homol-
ogy analysis of the H3 receptor showed it to be signifi-
cantly different from the previously cloned H1 and H2
receptors.2c,d The H3 receptors are located presynapti-
cally and inhibit synthesis and release of histamine.3
They are also known to play an important role as
heteroreceptors in the regulation of the release of
F igu r e 1. Histamine and H3 receptor ligands.
several other important neurotransmitters.4 It has been
recognized that the H3 receptor is a potential therapeu-
tic target.5 Agonists to the H3 receptor are considered
to be potential drugs for the treatment of sleep disor-
ders, migraines, asthma, inflammation, and ulcers.5a On
the other hand, the H3 receptor antagonists may be
useful therapeutically for Alzheimer’s disease, attention-
deficit hyperactive disorder (ADHD), schizophrenia,
depression, dementia, and epilepsy.5b
In 2000 and 2001, another histamine receptor sub-
type, i.e., the H4 receptor, was cloned by several groups
and identified as a G protein-coupled receptor.6 The H4
receptor has significant sequence homology (about 40%)
to the H3 receptor cDNA and seems to couple with Gi
protein like that of the H3 receptor.6f It is noteworthy
that the H3 and the H4 receptors share about 60%
sequence identity in their transmembrane regions,6d
which suggests that the two receptors possibly have a
* Corresponding author. Phone: +81-11-706-3229. Fax: +81-11-
706-4980. E-mail: shu@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ University of Shizuoka.
§ Yamanouchi Pharmaceutical Co. Ltd.
10.1021/jm020415q CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/20/2003

Highly H3-Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1981
F igu r e 2. The cyclopropane-based conformationally restricted analogues of histamine.
similar mode of ligand recognition. In fact, previous H3
agonists, such as (R)-R-methylhistamine (2), imetit (3),
and immepip (4), which had been considered to be highly
selective H3 receptor agonists before the discovery of the
H4 receptor, were disclosed to bind not only to the H3
receptor but also interestingly to the H4 receptor.6b-d,f,g
Consequently, development of highly selective H3 recep-
tor agonists, which are completely or virtually lacking
in affinity for the H4 receptor, is required. This poten-
tially new class of highly selective H3 selective agonists
would not only be very useful as tools for pharmacologi-
cal studies but would also be important as leads for the
development of new drugs.
To develop the new class of highly H3 selective
agonists mentioned above, we have designed and syn-
thesized the conformationally restricted analogues of
histamine, 9-13 and their enantiomers ent-9-13, hav-
ing a cis- or a trans-cyclopropane ring. Among the newly
synthesized compounds, (1S,2S)-2-(2-aminoethyl)-1-(1H-
imidazol-4-yl)cyclopropane (13) having the cis-cyclopro-
pane structure was identified as a highly H3-selective
agonist. In this report, we describe the design, synthesis,
and pharmacological effects of these conformationally
restricted analogues of histamine.
is common to all potent H3 receptor agonists known so
far;5 and (2) introduction of a chiral center into the
histamine side chain can bring about the selective and
potent agonism to the H3 subtype,5 which is typically
observed in the eutomers (R)-R-methylhistamine (2),8a
immepyr (5),8b and imifuramine (6).8c These findings
suggest that the spatial arrangement of the imidazole
ring and the basic amino nitrogen atom is a determining
factor in the potency of the H3 subtype agonists. Thus,
we speculated that suitable conformational restriction
of histamine may be effective in developing highly
selective H3 subtype agonists.
In the design of conformationally restricted ana-
logues,9 it is essential that the conformationally re-
stricted analogues should be as similar as possible to
the parent compound in size, shape, and molecular
weight.9a Conformationally restricted analogues have
usually been designed and synthesized by introducing
often bulky cyclic moieties into the lead compounds.
Consequently, the chemical and physical properties of
these analogues can be quite different from those of the
original leads. Because of its characteristic structural
feature, a cyclopropane ring is likely to be effective in
restricting the conformation of a molecule without
changing the chemical and physical properties of the
lead compounds.10 In fact, cyclopropanes have already
been successfully used to restrict the bioactive confor-
mations of neurotransmitters, amino acids, peptides and
nucleosides.10 We also devised a new method for re-
stricting the conformation of cyclopropane derivatives
based on the fact that adjacent substituents on the ring
exert mutual steric repulsion because of their eclipsed
conformation to one another.11a This method has been
successfully used in the design of NMDA (N-methyl-D-
aspartic acid) receptor antagonists.11
De Esch and co-workers designed the four stereoiso-
mers of 2-amino-1-(1H-imidazol-4-yl)cyclopropane, i.e.,
the “folded” cis-isomer 9 and its enantiomer ent-9 and
the “extended” trans-isomer 10 and its enantiomer ent-
10 (Figure 2), in which the basic amino group was
directly attached to the cyclopropane ring.12 Although
they failed to synthesize the “folded” 9 and ent-9, the
synthesis of the “extended” trans-analogues 10 and ent-
10, via optical resolution of a racemic intermediate, was
accomplished, and ent-10 was identified as a potent H3
agonist.12 Furthermore, a very potent H3-receptor an-
tagonist GT-2331 (8, Figure 1), having an “extended”
Resu lts a n d Discu ssion
Design of th e Con for m a tion a lly Restr icted An a -
logu es of H ist a m in e. Histamine, like other neuro-
transmitters, is conformationally flexible due to the
“aromatic ring-C(sp3)-C(sp3)-N” backbone. Conse-
quently, histamine can assume a variety of conforma-
tions, which may make it possible for it to bind to
several receptor subtypes via different conformations.7
For example, the conformation binding to the H3 recep-
tor, i.e., the bioactive conformation for the H3 subtype,
may be different from those for the H1, H2, and/or H4
subtype receptors. Similarly the bioactive conformation
for the H4 subtype may be different from those for the
other receptor subtypes. Therefore, because conforma-
tional restriction of histamine may improve the specific
binding to one of the receptor subtypes, we planned to
develop novel H3 selective agonists by restricting the
conformation of histamine.
The previous structure-activity relationship studies
on the H3 receptor agonists showed that (1) the 4-alkyl-
imidazole moiety of the histamine molecule is an es-
sential partial structure for H3 receptor agonism, which

1982 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Kazuta et al.
trans-cyclopropylimidazole structure, was reported by
Ali and co-workers.13 These studies suggest that the
trans-cyclopropane ring is likely to be effective in
restricting the bioactive conformation of the histamine
H3 receptor. However, potency of the cis-cyclopropane
ring as a template for the conformational restriction of
histamine is unknown.
On the other hand, SKF91606 (7),14 having its two-
carbon-elongated structure (imidazole-C-C-C-C-N)
compared with that of histamine (imidazole-C-C-N),
is the strongest H3-receptor agonist reported to date.
Such carbon-elongation in SKF 91606 may relate to its
strong affinity for the H3 receptor subtype.
F igu r e 3. The versatile chiral cyclopropane units.
Sch em e 1a
On the basis of these findings and considerations, we
designed the histamine analogues 11, ent-11, 12, and
ent-12 (Figure 2), conformationally restricted by the cis-
or the trans-cyclopropane ring, which have the carbon-
elongated “imidazole-C(1)-C(2)-C(1′)-N” and/or the
“imidazole-C(1)-C(2)-C(3)-C(1′)-N” structure. In these
conformationally restricted analogues the spatial rela-
tionship between the imidazole ring and the basic
nitrogen of the cis-substituted 11 and ent-11 are re-
stricted in the “folded” form, while those of the trans-
isomers 12 and ent-12 are in the “extended” form. The
conformationally restricted analogues 9, ent-9, 10, and
ent-10 previously designed by De Esch and co-workers
described above were also included as synthetic targets
in this study to compare their biological activities with
those of the newly designed compounds. We were
especially interested in the pharmacological effect of the
unknown “folded” 9 and ent-9.
Since, as described below, the “folded” analogue 11
with its (1S,2R)-configuration proved to be a very potent
and selective H3 receptor agonist, the further carbon-
elongated “folded” analogue 13 with the same stereo-
chemistry as for 11 and its enantiomer ent-13 were also
designed as other synthetic targets.
Investigation of the effects of these compounds on the
histamine receptors may clarify the three-dimensional
structure-activity relationship of the conformationally
restricted analogues of histamine having a cyclopropane
ring.
a
Conditions: (a) (1) TsCH2NC, NaCN, EtOH, (2) NH3, EtOH,
(3) TrCl, Et3N, CH2Cl2, (4) TBAF, THF, (5) Swern ox. (ref 16); (b)
NaClO2, NaH2PO4, aq acetone, rt, 89% (17), 89% (21); (c)
(PhO)2PON3, Et3N, CH2Cl2, rt, 80% (18), 74% (22); (d) t-BuOH,
reflux, 96% (19), 48% (23); (e) 6 N HCl, reflux, 92%; (f) HCl, aq
MeOH, reflux, 98%.
Ch em istr y. Chiral cyclopropanes are not only useful
for restricting the conformation of biologically active
compounds in order to improve the activity, but are also
important as key fragments in many natural products.15
Therefore, considerable effort has been devoted to
developing practical methods for preparing chiral cy-
clopropanes, including enantioselective cyclopropana-
tions, chemical or enzymatic optical resolutions, and
transformations from chiral synthons.10,11,15-17
and ent-12 were likewise synthesized from ent-14 and
ent-15, respectively (Scheme 1).16
As mentioned above, De Esch and co-workers suc-
cessfully synthesized the “extended” analogues 10 and
ent-10 via optical resolution of a racemic intermediate.12
They also tried various approaches to the synthesis of
the corresponding “folded” cis-isomer 9 and its enanti-
omer ent-9, but these were unsuccessful,12 which sug-
gests that the synthesis of the conformationally re-
stricted analogues of histamine having the cis-cyclo-
propane structure would be rather difficult. However,
we thought that the chiral imidazolylcyclopropanecar-
baldehydes 16 and 20 and their enantiomers ent-16 and
ent-20 should be effective intermediates not only for
synthesizing the trans-analogues 10 and ent-10 but also
synthesizing for the cis-analogues 9 and ent-9. Since the
basic amino group is directly attached to the cyclopro-
pane ring in these target compounds, a one-carbon
contraction was required for their synthesis, which
We most recently developed the four types of chiral
cyclopropane units bearing two adjacent substituents
in a cis or a trans relationship, namely 14 and 15, and
their enantiomers ent-14 and ent-15, as shown in Figure
3.16 These are versatile intermediates for synthesizing
various biologically important compounds having an
asymmetric cyclopropane structure, such as the target
conformationally restricted analogues in this study.
Thus, the cis and trans units 14 and 15 were converted
into the corresponding imidazole derivatives 16 and 20,
from which the conformationally restricted analogues
11 and 12 were synthesized. The enantiomers ent-11

Highly H3-Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1983
Sch em e 2a
a
Conditions: (a) (1) MeOCH2PPh3Cl, NaHMDS, THF, 0 °C, (2)
HCl, aq MeOH, rt, 76%; (b) BnONH2‚HCl, MS 4A, THF, rt, quant;
(c) LiAlH4, THF, rt, 97%; (d) HCl, aq MeOH, reflux, 99%.
F igu r e 4. Effects of compounds on specific binding of [3H]-
NR-methylhistamine.
seemed possible via the Curtius rearrangement. Oxida-
tion of the cis-imidazolylcyclopropanecarbaldehyde 16
with NaClO2 in aqueous acetone gave the cyclopro-
panecarboxylic acid 17, which was then treated with
(PhO)2PON3/Et3N in CH2Cl2 to produce the correspond-
ing acid azide 18. When the acid azide 18 was heated
in t-BuOH under reflux, the expected Curtius rear-
rangement and simultaneous removal of the trityl group
occurred to give the one-carbon-contracted isocyanate
19. Acidic hydrolysis of the isocyanate moiety of 19 by
heating it in 6 N HCl under reflux furnished the target
folded analogue 9 as a hydrochloride. From the trans-
imidazolylcyclopropanecarbaldehyde 20, the trans-acid
azide 22 was prepared according to a similar procedure
as that for the cis-series. Heating the trans-acid azide
22 in t-BuOH under the same conditions as those for
the cis-18 resulted in producing the Curtius-rearranged
Boc-amino derivative 23 without removing the N-trityl
group. Treatment of 23 with HCl in aqueous MeOH
gave the “extended” (1R,2R)-conformationally restricted
analogue 10. The spectral data of the synthesized 10
were in accord with those reported previously.12 The
enantiomers ent-9 and ent-10 were also effectively
synthesized from ent-16 and ent-20, respectively.
Synthesis of the one-carbon-elongated “folded” ana-
logues 13 and ent-13 was next investigated (Scheme 2).
The Wittig reaction of the cis-aldehyde 16 with MeOCH2-
PPh3Cl/NaHMDS in THF followed by acidic treatment
of the product gave the corresponding homologous
aldehyde 24. After conversion of 24 into the benzyloxime
25, reduction with LiAlH4 afforded the amine 26. The
N-trityl group was finally removed by the usual acidic
treatment with aqueous HCl to afford the one-carbon-
elongated analogue 13. Similarly, its enantiomer ent-
13 was synthesized from ent-16.
Ta ble 1. Affinity of Compounds for the Rat H3 Receptor
Subtypea
compd
Ki, nM
9
91.2 ( 6.5
1380 ( 140
42.2 ( 3.7
4.30 ( 0.63
2.50 ( 0.38
64.7 ( 6.8
22.6 ( 3.6
10.1 ( 0.6
1.31 ( 0.16
4.78 ( 0.02
2.65 ( 0.76
0.88 ( 0.06
ent-9
10
ent-10
11
ent-11
12
ent-12
13 (AEIC)
ent-13
thioperamide
(R)-R-methylhistamine (2)
a
Assay was carried out with rat brains using [3H]NR-methyl-
histamine (n ) 3).
rat brains in a concentration-dependent manner (0.1 nM
to 10 µM), as shown in Figure 4.19 In order of the binding
affinity, the compounds ranked as follows: 13 > 11 >
ent-10, ent-13 > ent-12 > 12 > 10 > ent-11 > 9 . ent-
9. Among the compounds, 13, 11, ent-10, and ent-13 had
a significant nM order Ki value. Compound 13 [(1S,2S)-
2-(2-aminoethyl)-1-(1H-imidazol-4-yl)cyclopropane, AEIC]
showed the strongest binding affinity (Ki ) 1.31 ( 0.16
nM) in this series of compounds, which is comparable
to that of (R)-R-methylhistamine8a (2, Ki ) 0.88 ( 0.06
nM) and is more potent than the well-known H3 receptor
antagonist thioperamide5b (Ki ) 2.65 ( 0.76 nM).
The binding affinities of the compounds 13 (AEIC),
which strongly bind to the rat H3 receptor, for human
H1, H2, and H3 receptor subtypes were next evaluated
using [3H]pyrilamine (H1), [3H]tiotidine (H2), and [3H]-
NR-methylhistamine (H3) as radioligands.6b AEIC showed
a significant binding affinity for the human H3 receptor
with the Ki values of 1.5 nM, which is similar to that
for the rat brain H3 receptor described above. On the
other hand, AEIC had any effect on the specific bindings
of [3H]pyrilamine to the human H1 receptor and [3H]-
tiotidine to the human H2 receptor at concentrations up
to 10 µM.
These results showed that the chiral cyclopropane
units 14 and 15, and their enantiomers ent-14 and ent-
15, are useful as versatile intermediates for synthesizing
various compounds having asymmetric a cis- or a trans-
cyclopropane structure.
Bin d in g Affin ities for Hista m in e Recep tor Su b-
typ es. The binding affinity of the cyclopropane-based
conformationally restricted analogues of histamine for
the H3 subtype of rat brains was evaluated using [3H]-
NR-methylhistamine as a radio ligand.18 The results are
shown in Figure 4 and Table 1. All of the newly
synthesized analogues inhibited the specific binding of
[3H]NR-methylhistamine to the H3 subtype receptor of
F u n ction a l Effects on Hista m in e Recep tor Su b-
typ es. The functional potencies of the compounds on
the human H1, H2, H3, and H4 receptor subtypes were
assessed using luciferase reporter gene assay.6a The four
human histamine receptor subtypes were individually
expressed in 293-EBNA cells according to the previously
reported method,6a which were used in the evaluation

1984 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Kazuta et al.
co-workers were supported by this study.21 We synthe-
sized anew the corresponding cis-isomers 9 and ent-9
and clarified that their binding and functional effects
on the H3 receptor were relatively weak compared with
Ta ble 2. Functional Effects of Compounds on Human
Histamine Receptor Subtypes
functional activity (EC50 nM)a
compd
H1
H2
H3
H4
22
the trans-analogues 10 and ent-10.
9
>104
>104
>104
2330 ( 1520 >104
ent-9
10
ent-10
11
ent-11
12
ent-12
13 (AEIC)
ent-13
>104
>104
>104
>104
Pharmacological evaluation of the conformationally
restricted analogues 11, ent-11, 12, and ent-12, which
have the carbon-elongated “imidazole-C(1)-C(2)-C(1′)-
N” and/or the “imidazole-C(1)-C(2)-C(3)-C(1′)-N”
structure, showed that the cis-(1S,2R) analogue 11 was
a potent agonist of the H3 receptor. Although the
potency of 11 was somewhat weaker than ent-10 in the
functional assay (Table 2), it was much more selective
to the H3 subtype compared with ent-10. Thus, since the
one-carbon elongation resulted in improvement of the
H3-agonist potency in the cis-analogues, we synthesized
and evaluated the further carbon-elongated cis-analogue
13 (AEIC) having the same stereochemistry as that of
11 and its enantiomer ent-13. Consequently, AEIC (13)
was identified as the most potent and selective H3
receptor agonist in this series of compounds, which
showed that the cis-cyclopropane structure is very
effective in the conformational restriction of histamine
to improve the specific binding to the histamine H3
receptor. This compound owes its importance to being
the first highly selective H3 receptor agonist with
virtually no effect on the H4 subtype receptor, while the
previous H3 antagonists have been shown to clearly
affect the H4 subtype.6b-d,f,g AEIC (13) can be an efficient
lead for clinically useful H3 receptor agonists and would
also be useful as a pharmacological tool for various
studies on histaminergic systems.
These results of the cyclopropane-based conforma-
tionally restricted analogues of histamine as H3 subtype
receptor agonists showed that, in both the cis- and the
trans-series, the 1S-enantiomers 9, ent-10, 11, ent-12,
and 13 were more potent than the corresponding 1R-
enantiomers ent-9, 10, ent-11, 12, and ent-13, respec-
tively. The 1S-structure proved to be essential for the
strong binding to the H3 receptor.
Previous studies on GT2331 (8, Figure 1)13 as well as
on ent-1012 have demonstrated that the trans-cyclopro-
pane structure was very effective in the conformational
restriction of histamine for improving affinity for the
H3 receptor. This study proved that conformational
restriction of histamine by the cis-cyclopropane struc-
ture may be much more effective than the trans-one for
the development of potent and selective H3 receptor
ligands.
2500 ( 2190 724 ( 360
>104
864 ( 349 696 ( 218
42 ( 19
96 ( 54
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
>104
463 ( 25
-
>104
>104
>104
>104
>104
>104
10 ( 3
93 ( 5
77 ( 51
1.9 ( 0.4
>104
1410 ( 37
75 ( 21
51 ( 22
histamine (1) 13 ( 1
imetit (3)
-
a
Assay was carried out by luciferase reporter gene method with
the 293-EBNA cells expressing human H1, H2, H3, or H4 receptor
subtype (n ) 3).
of the compounds. The EC50 values of the conformation-
ally restricted analogues, as well as histamine, are
summarized in Table 2.
All the conformationally restricted analogues, except
for ent-10, were inactive or showed only a very weak
agonist activity (EC50 > 103 nM) to the H1 and the H2
subtype receptors, as expected from the results of the
above binding experiments, while ent-10 showed a
moderate agonist effect on the H1 (EC50 ) 864 ( 349
nM) and the H2 (EC50 ) 696 ( 218 nM) subtypes.
Although ent-9, ent-11, 12, and ent-12 were also inactive
to the H3 subtype (EC50 > 104 nM), the other confor-
mationally restricted analogues caused evident activa-
tion of the H3 receptor with the ranking of 13 > ent-10
> ent-13, 11 > 10 > 9, which was roughly in accord with
their binding affinities summarized in Table 1. In
particular, compound 13 (AEIC) showed an excellent
EC50 value of 10 ( 3 nM. On the other hand, none of
the compounds, except for ent-13 (EC50 ) 1410 ( 37
nM), showed any agonist effect on the H4 subtype (EC50
> 104 nM). None of the conformationally restricted
analogues showed an antagonist effect on any of the four
histamine receptor subtypes (data not shown).
These results demonstrated that en t-10, 11, 13, and
ent-13 were selective H3 receptor agonists. It is worth
noting that the conformationally restricted analogue 13
(AEIC) was an agonist of the H3 receptor with a
significant EC50 value of 10 ( 3 nM, lower than that of
endogenous ligand histamine (EC50 ) 76.7 ( 51 nM),
while it was inactive or almost inactive20 to the other
three histamine subtype receptors.
The results of 9, ent-9, 10, and ent-10 suggest that
the bioactive conformation of histamine for the H3
receptor subtype seems to be the “extended” form.
However, the highly selective H3 agonist potency of the
trans-analogues 11 and AEIC (13) contradicts the
“folded” bioactive conformation. The bioactive conforma-
tion may be between the “folded” and the “extended”
forms. Further studies are needed to confirm this
supposition.
Con clu sion . A series of cyclopropane-based confor-
mationally restricted analogues of histamine were
designed as novel H3 receptor agonists and were syn-
thesized from the versatile chiral cyclopropane units 14,
15, and their enantiomers ent-14 and ent-15. Among the
compounds, (1S,2S)-2-(2-aminoethyl)-1-(1H-imidazol-4-
Discu ssion . All of the target conformationally re-
stricted analogues having a cyclopropane ring were
efficiently synthesized from the chiral cyclopropane
units. These results proved that the chiral cyclopropane
units, 14 and 15, and their enantiomers ent-14 and ent-
15, are very useful synthetic intermediates for preparing
various conformationally restricted chiral cyclopropane
analogues of biologically active compounds.
The (1S,2S)-trans analogue ent-10, the amino function
of which directly bonded to the cyclopropane ring, was
previously shown to be a potent H3 agonist, which was
stronger than its enantiomer, the (1R,2R)-trans ana-
logue 10.12 However, ent-10 also had a moderate effect
on the H1 and the H2 subtypes.12 These previous results
on the trans-analogues 10 and ent-10 by De Esch and

Highly H3-Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1985
7.34 (9 H, m, aromatic); 13C NMR (125 MHz, CDCl3) δ 13.11
(C-3), 22.46 (C-2), 23.96 (C-1), 75.28 (CPh3), 120.43, 127.96,
128.04, 129.82, 136.10, 138.05, 142.34 (aromatic), 176.62 (C-
1′); LR-MS (FAB) m/z 420 [(M + H)+]. Anal. (C26H21N5O) C,
H, N.
yl)cyclopropane (13, AEIC) was identified as the first
highly selective H3 receptor agonist. These results
showed that the cis-cyclopropane structure is very
effective in the conformational restriction of histamine
to improve the specific binding to the histamine H3
receptor, and also that the cyclopropane-based confor-
mational restriction strategy is very effective in medici-
nal chemical studies for improving the potency of
compounds.
(1R,2S)-2-Azid oca r bon yl-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (en t-18). ent-18 (46 mg, 78%, white
solid) was prepared from ent-17 (55 mg, 0.14 mmol) as
described for preparing 18: [R]21 -51.2 (c 3.75, CHCl3); LR-
D
MS (FAB) m/z 420 [(M + H)+]. Anal. (C26H21N5O) C, H, N.
(1R,2R)-2-Azid oca r bon yl-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (22). Compound 22 (140 mg, 74%,
Exp er im en ta l Section
white solid) was prepared from 21 (178 mg, 0.45 mmol) as
Chemical shifts are reported in ppm downfield from TMS.
All of the H NMR assignments described were in agreement
1
described for preparing 18: [R]21 -205.3 (c 1.51, CHCl3); H
1
D
NMR (500 MHz, CDCl3) δ 1.58-1.61 (2 H, m, H-3), 2.05 (1 H,
m, H-1), 2.51 (1 H, m, H-2), 6.66 (1 H, d, CHd, J ) 1.3 Hz),
7.10-7.14 (6 H, m, aromatic), 7.29 (1 H, d, CHd, J ) 0.9 Hz),
7.33-7.34 (9 H, m, aromatic); 13C NMR (125 MHz, CDCl3) δ
15.68 (C-3), 22.72 (C-1), 25.59 (C-2), 75.21 (CPh3), 118.34,
128.00, 128.02, 129.67, 138.54, 138.75, 142.21 (aromatic),
179.79 (C-1′); LR-MS (FAB) m/z 420 [(M + H)+]. Anal.
(C26H21N5O) C, H, N.
with COSY spectra. Thin-layer chromatography was per-
formed on Merck coated plate 60F254. Silica gel chromatogra-
phy was performed on Merck silica gel 5715. Reactions were
carried out under an argon atmosphere.
(1S,2R)-1-(1-Tr ip h en ylm eth yl-1H-im id a zol-4-yl)cyclo-
p r op a n e-2-ca r boxylic Acid (17). A mixture of 1616 (151 mg,
0.40 mmol), NaClO2 (128 mg, 1.4 mmol), and NaH2PO4‚2H2O
(64 mg, 0.40 mmol) in acetone/CH2Cl2/H2O (4:2:1, 28 mL) was
stirred at room temperature for 12 h. The solvent was
evaporated, and the residue was partitioned between H2O and
CHCl3. The organic layer was washed with brine, dried (Na2-
SO4), evaporated, and purified by column chromatography
(1S,2S)-2-Azid oca r bon yl-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (en t-22). ent-22 (143 mg, 76%,
white solid) was prepared from ent-21 (178 mg, 0.45 mmol) as
described for preparing 18: [R]21D +203.5 (c 1.23, CHCl3); LR-
MS (FAB) m/z 420 [(M + H)+]. Anal. (C26H21N5O) C, H, N.
(silica gel; CHCl3/MeOH, 1:9) to give 17 as a white solid (141
1
mg, 89%, white solid): [R]21 +51.4 (c 1.59, CHCl3); H NMR
D
(1S,2R)-1-(1H-Im id a zol-4-yl)-2-a m in ocyclop r op a n e Di-
h yd r och lor id e (9). A solution of 18 (59 mg, 0.14 mmol) in
t-BuOH (2 mL) was heated under reflux for 2 h. The reaction
mixture was evaporated and purified by column chromatog-
raphy (silica gel; CHCl3/MeOH, 1:19) to give (1S,2R)-1-(1H-
imidazol-4-yl)cyclopropane-2-isocyanate (19) as a white solid
(20 mg, 96%): 1H NMR (500 MHz, CDCl3) δ 0.53 (1 H, m,
H-3a), 1.36 (1 H, m, H-3b), 2.41 (1 H, m, H-1), 3.23 (1 H, m,
H-2), 6.98 (1 H, s, CHd), 7.28 (1 H, s, CHd). Compound 19
was immediately used for the next reaction. A solution of 19
(15 mg, 100 µmol) in 6 N HCl (1 mL) was heated under reflux
for 5 h. The solvent was evaporated, and then the residue was
(500 MHz, CDCl3) δ 1.52 (1 H, m, H-3a), 1.56 (1 H, m, H-3b),
2.13 (1 H, m, H-1), 2.23 (1 H, m, H-2), 6.77 (1 H, s, CHd),
7.12-7.13 (6 H, m, aromatic), 7.35-7.37 (9 H, m, aromatic),
7.40 (1 H, s, CHd); 13C NMR (125 MHz, CDCl3) δ 15.04 (C-3),
23.53 (C-1), 29.36 (C-2), 75.89 (CPh3), 119.85, 128.20, 128.31,
129.69, 137.08, 137.32, 141.72 (aromatic), 174.78 (C-1′); LR-
MS (FAB) m/z 395 [(M + H)+]; HR-MS (FAB) calcd for
C
26H23N2O2 395.1759; found 395.1736 [(M + H)+]. Anal.
(C26H22N2O2‚2H2O) C, H, N.
(1R,2S)-1-(1-Tr ip h en ylm eth yl-1H-im id a zol-4-yl)cyclo-
p r op a n e-2-ca r boxylic Acid (en t-17). ent-17 (131 mg, 83%,
white solid) was prepared from ent-1616 (151 mg, 0.40 mmol)
as described for preparing 17: [R]22 -50.3 (c 1.11, CHCl3);
treated with Et2O/EtOH to give a white solid of 9 as dihydro-
D
1
chloride (18 mg, 92%): [R]20 +9.5 (c 0.33, CH3OH); H NMR
LR-MS (FAB) m/z 395 [(M + H)+]. Anal. (C26H22N2O2‚2H2O)
D
(500 MHz, CD3OD) δ 1.57 (1 H, m, H-3a), 1.77 (1 H, m, H-3b),
2.61 (1 H, m, H-1), 3.25 (1 H, m, H-2), 7.70 (1 H, s, CHd),
9.08 (1 H, s, CHd); 13C NMR (125 MHz, CDCl3) δ 10.27 (C-1),
10.99 (C-3), 29.06 (C-2) 119.73, 129.04, 136.28 (imidazole C);
HR-MS (EI) calcd for C6H10N3 124.0875; found 124.0880 [(M
+ H)+). Anal. (C6H11Cl2N3‚2H2O) C, H, N.
C, H, N.
(1R,2R)-1-(1-Tr ip h en ylm eth yl-1H-im id a zol-4-yl)cyclo-
p r op a n e-2-ca r boxylic Acid (21). Compound 21 (35 mg, 89%,
white crystals) was prepared from 2016 (38 mg, 0.10 mmol) as
described for preparing 17: mp (EtOH) 132-133 °C; [R]20
D
-75.1 (c 1.10, CHCl3); 1H NMR (500 MHz, CDCl3:CD3OD/1:2)
δ 1.25 (1 H, m, H-3a), 1.35 (1 H, m, H-3b), 1.79 (1 H, m, H-1),
2.28 (1 H, m, H-2), 6.63 (1 H, s, CHd), 7.03-7.04 (6 H, m,
aromatic), 7.25-7.28 (10 H, m, aromatic); 13C NMR (125 MHz,
CDCl3:CD3OD/1:2) δ 14.77 (C-3), 21.49 (C-1), 28.48 (C-2), 75.92
(CPh3), 117.15, 127.68, 127.75, 129.55, 136.88, 137.06, 140.39
(aromatic), 175.52 (C-1′); HR-MS (FAB) calcd for C26H23N2O2
395.1759; found 395.1734 ((M + H)+]; Anal. (C26H22N2O2‚2H2O)
C, H, N.
(1R,2S)-1-(1H-Im id a zol-4-yl)-2-a m in ocyclop r op a n e Di-
h yd r och lor id e (en t-9). ent-19 (20 mg, 95%), white solid) was
prepared from ent-18 (59 mg, 0.14 mmol) as described for
preparing 19 and was immediately used for the next reaction.
ent-9 (20 mg, 90%, white solid) was prepared from ent-19 (15
mg, 100 µmol) as described for preparing 9: [R]23 -10.2 (c
D
0.61, CH3OH); LR-MS (EI) m/z 124 [(M + H)+). Anal. (C6H11
Cl2N3‚2H2O) C, H, N.
-
(1S,2S)-1-(1-Tr ip h en ylm eth yl-1H-im id a zol-4-yl)cyclo-
p r op a n e-2-ca r boxylic Acid (en t-21). ent-21 (65 mg, 82%,
white crystals) was prepared from ent-2016 (76 mg, 0.20 mmol)
(1R,2R)-2-(t-Bu t yloxyca r b on yla m in o)-1-(1-t r ip h en yl-
m eth yl-1H-im id a zol-4-yl)cyclop r op a n e (23). A solution of
22 (138 mg, 0.33 mmol) in t-BuOH (2 mL) was heated under
reflux for 5 h. The reaction mixture was evaporated and
purified by column chromatography (silica gel; CHCl3/MeOH,
as described for preparing 17: mp (EtOH) 130-132 °C; [R]20
D
+73.2 (c 1.20, CHCl3); LR-MS (FAB) m/z 395 [(M + H)+]; Anal.
(C26H22N2O2‚2H2O) C, H, N.
1:19) to give 23 as a white solid (74 mg, 48%): [R]20 -54.3 (c
D
(1S,2R)-2-Azid oca r bon yl-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (18). A mixture of 17 (55 mg, 0.14
mmol), diphenylphosphoryl azide (120 mg, 0.56 mmol), and
Et3N (40 µL, 0.28 mmol) in CH2Cl2 (2 mL) was stirred at room
temperature for 5 h. The reaction mixture was partitioned
between H2O and CHCl3. The organic layer was washed with
brine, dried (Na2SO4), evaporated, and purified by column
chromatography (silica gel; AcOEt/hexane, 1:3 then 1:2) to give
18 as a white solid (47 mg, 80%): [R]21D +50.3 (c 2.40, CHCl3);
1H NMR (500 MHz, CDCl3) δ 1.38 (1 H, m, H-3a), 1.58 (1 H,
m, H-3b), 2.04 (1 H, m, H-1), 2.63 (1 H, m, H-2), 6.62 (1 H, s,
CHd), 7.11-7.15 (6 H, m, aromatic), 7.32 (1 H, s, CHd), 7.33-
1.31, CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.02 (1 H, m, H-3a),
1.20 (1 H, m, H-3b), 1.42 (9 H, s, (CH3)3C), 1.93 (1 H, m, H-1),
2.76 (1 H, m, H-2), 6.57 (1 H, s, CHd), 7.11-7.13 (6 H, m,
aromatic), 7.28 (1 H, s, CHd), 7.31-7.34 (9 H, m, aromatic);
13C NMR (125 MHz, CDCl3) δ 15.54 (C-3), 28.38 (CH3)3C), 31.41
(C-2), 75.14 (CPh3), 79.38 (C-1), 117.13, 127.95, 127.96, 129.76,
138.31, 140.67, 142.47 (aromatic), 156.32 (CdO); LR-MS (FAB)
m/z 466 [(M + H)+]. Anal. (C30H31N3O2) C, H, N.
(1S,2S)-2-(t-Bu t yloxyca r b on yla m in o)-1-(1-t r ip h en yl-
m eth yl-1H-im id a zol-4-yl)cyclop r op a n e (en t-23). ent-23 (77
mg, 50%, white solid) was prepared from ent-22 (138 mg, 0.33

1986 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10
Kazuta et al.
mmol) as described for preparing 23: [R]22 +53.5 (c 1.04,
saturated aqueous NH4Cl and brine, dried (Na2SO4), evapo-
rated, and purified by column chromatography (silica gel;
MeOH/CHCl3, 1:9 then 1:4) to give 26 as a white solid (38 mg,
97%): [R]24D +17.1 (c 0.28, MeOH); 1H NMR (500 MHz, CDCl3)
δ 0.26 (1 H, m, H-3a), 0.98 (1 H, m, H-3b), 1.07 (1 H, m, H-1′a),
1.23-1.32 (2 H, m, H-1′b and H-2), 2.03 (1 H, m, H-1), 3.16-
3.26 (2 H, m, H-2′), 6.62 (1 H, d, CHd), 7.09-7.11 (6 H, m,
aromatic), 7.34-7.37 (10 H, m, aromatic), 9.11 (2 H, br s,
-NH2); 13C NMR (125 MHz, CD3OD) δ 10.22 (C-3), 13.46 (C-
2), 13.62 (C-1), 26.10 (C-1′), 38.94 (C-2′), 75.56 (CPh3), 121.89,
128.15, 128.17, 129.67, 137.74, 139.49, 142.08 (aromatic); HR-
MS (FAB) calcd for C27H28N3 394.2283; found 394.2298 [(M +
H)+].
D
CHCl3); LR-MS (FAB) m/z 466 [(M + H)+]. Anal. (C30H31N3O2)
C, H, N.
(1R,2R)-1-(1H-Im id a zol-4-yl)-2-a m in ocyclop r op a n e Di-
h yd r och lor id e (10). A solution of 23 (23 mg, 50 µmol) in
MeOH (2 mL) and 3 N HCl (1 mL) was heated under reflux
for 3 h. The solvent was evaporated, and then residue was
treated with Et2O/EtOH to give a white solid of 10 as a
dihydrochloride (10 mg, 98%): 1H NMR (500 MHz, D2O) δ 1.46
(1 H, m, H-3a), 1.59 (1 H, m, H-3b), 2.54 (1 H, m, H-1), 3.07 (1
H, m, H-2), 7.29 (1 H, s, CHd), 8.61 (1 H, s, CHd); LR-MS
(EI) m/z 123 (M+). Anal. (C6H11Cl2N3) C, H, N. [R]25 +52.3
546
(dihydrobromide prepared by treating the dihydrochloride
successively with Diaion-PK-312 (OH-) and Diaion-WA-30
(1R,2R)-2-(2-Am in oeth yl)-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (en t-26). ent-26 (37 mg, 95%, white
solid) was prepared from ent-24 (39 mg, 0.10 mmol) as
(Br-) resins, c 0.68, H2O) [lit.12 [R]30 +54.4 (c 0.35, H2O)]
546
(1S,2S)-1-(1H-Im id a zol-4-yl)-2-a m in ocyclop r op a n e Di-
h yd r och lor id e (en t-10). ent-10 (10 mg, 98%, white solid) was
prepared from ent-23 (23 mg, 50 µmol) as described for
preparing 10: LR-MS (EI) m/z 123 (M+). Anal. (C6H11Cl2N3)
described for preparing 26: [R]24 -17.4 (c 0.48, MeOH); LR-
D
MS (FAB) m/z 394 [(M + H)+).
(1S,2S)-2-(2-Am in oeth yl)-1-(1H-im id a zol-4-yl)cyclop r o-
p a n e Dih yd r och lor id e (13). Compound 13 as a dihydrochlo-
ride (13 mg, 99%, white solid) was prepared from 26 (24 mg,
C, H, N. [R]25 -55.1 (dihydrobromide prepared by treating
546
the dihydrochloride successively with Diaion-PK-312 (OH-)
and Diaion-WA-30 (Br-) resins), c 1.28, H2O] [lit.12 [R]30
-54.2 (c 0.35, H2O)].
60 µmol) as described for preparing 10: [R]22 -35.1 (c 0.32,
546
D
1
MeOH); H NMR (500 MHz, CD3OD) δ 0.87 (1 H, m, H-3a),
1.23-1.39 (3 H, m, H-3b and H-1′), 1.67 (1 H, m, H-2), 2.16 (1
H, m, H-1), 2.92-3.03 (2 H, m, H-2′), 7.35 (1 H, s, CHd), 8.83
(1 H, d, CHd, J ) 1.1 Hz); 13C NMR (125 MHz, CD3OD) δ
10.91 (C-3), 11.27 (C-1), 16.78 (C-2), 27.75 (C-1′), 40.28 (C-2′),
118.06, 133.70, 135.01 (imidazole C); HR-MS (EI) calcd for
C8H13N3 151.1109; found 151.1101 (M+). Anal. (C8H15Cl2N3‚
2H2O) C, H, N.
(1S,2S)-2-F or m ylm eth yl-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (24). To a suspension of MeOCH2-
PPh3Cl (120 mg, 0.35 mmol) in THF (2 mL) was added
NaHMDS (1.0 M in THF, 0.30 mL, 0.30 mmol) dropwise at 0
°C, and the resulting mixture was stirred at the same
temperature for 5 min. To a solution of 16 (38 mg, 0.10 mmol)
in THF (2 mL) was added dropwise the prepared bright red
solution of the Wittig reagent, and the resulting mixture was
stirred at the same temperature for 30 min. The reaction was
quenched with saturated aqueous NH4Cl, and the solvent was
evaporated. The residue was partitioned between H2O and
CHCl3, and the organic layer was washed with brine, dried
(Na2SO4), evaporated, and purified by column chromatography
(silica gel; hexane/AcOEt, 1:1) to give the corresponding Wittig
reaction product as a white solid (32 mg). To a solution of the
product (32 mg) in acetone (2 mL) was added 12 N HCl (1 mL),
and the resulting solution was immediately added to a
saturated aqueous NaHCO3 solution. After concentration of
the mixture under reduced pressure (for removing acetone),
the resulting aqueous mixture was partitioned between H2O
and CHCl3. The organic layer was washed with brine, dried
(Na2SO4), evaporated, and purified by column chromatography
(silica gel; hexane/AcOEt, 1:1 then CHCl3/MeOH, 10:1) to give
(1R,2R)-2-(2-Am in oeth yl)-1-(1H-im ida zol-4-yl)cyclop r o-
p a n e Dih yd r och lor id e (en t-13). ent-13 was prepared from
ent-26 (24 mg, 60 µmol) as described for preparing 10: [R]17
D
+36.1 (c 0.22, MeOH); LR-MS (EI) m/z 151 (M+). Anal. (C8H15
-
Cl2N3‚2H2O) C, H, N.
Bin d in g Assa y of Ra t Hista m in e H3 Recep tor . Male
Sprague-Dawley rats at 8 to 10 weeks of age (J apan SLC Inc.,
Shizuoka, J apan) were housed three or four per cage in the
laboratory with free access to food and water and maintained
on a 12 h dark/light cycle in a room with controlled temper-
ature (24 ( 1 °C) and humidity (55 ( 5%). Rats were killed
by taking the blood from the descending aorta under light
anesthesia with Et2O. The brain tissues (minus cerebellum)
were removed and used for the measurements of H3 receptor
subtype. The binding assay was performed by using [3H]NR-
methylhistamine (DuPont-NEN), as described previously.18
Briefly, the brain homogenates (800 µg) were incubated with
different concentrations of [3H]NR-methylhistamine (0.1-3.0
nM) for 1 h at 25 °C in 50 mM Na2HPO4/KH2PO4 buffer (pH
7.5). The reaction was terminated by rapid filtration (Cell
Harvester, Brandel Co., Gaithersburg, MD) through Whatman
GF/B glass fiber filters, and the filters were rinsed three times
with an ice-cold buffer (2 mL). Tissue-bound radioactivity was
extracted from filters overnight in scintillation fluid (toluene,
2 L; Triton X-100, 1 L; 2,5-diphenyloxazole, 15 g; 1,4-bis[2-(5-
phenyloxazolyl)]benzene, 0.3 g) and determined in a liquid
scintillation counter. The specific binding of each radioligand
was determined experimentally from the difference between
counts in the presence of 10 µM thioperamide. Analysis of the
binding data was performed as described previously.23,24 The
apparent dissociation constants (Kd) for [3H]NR-methylhista-
mine were estimated by Rosenthal analysis of the saturation
data. The ability of each compound to inhibit the specific
binding of [3H]NR-methylhistamine (0.25 nM) was estimated
by IC50 values, which are the molar concentrations of the
unlabeled drug necessary for displacing 50% of specific binding
(estimated by log probit analysis). The inhibition constant, K
value, was calculated from the equation, Ki ) IC50/(1 + L/Kd),
where L equals concentration of each radioligand. The data
were presented as mean ( SE.
24 as a yellow oil (30 mg, 76%): [R]23 +17.9 (c 1.02, CHCl3);
D
1H NMR (500 MHz, CDCl3) δ 0.79 (1 H, m, H-3a), 1.10 (1 H,
m, H-3b), 1.30 (1 H, m, H-2), 2.10 (1 H, m, H-1), 2.35 (2 H, m,
H-1′), 6.58 (1 H, s, CHd), 7.12-7.14 (6 H, m, aromatic), 7.32-
7.33 (10 H, m, aromatic), 9.66 (1 H, br s, H-2′); 13C NMR (125
MHz, CDCl3) δ 9.85 (C-3), 12.06 (C-1), 13.58 (C-2), 42.87 (C-
1′), 75.14 (CPh3), 119.52, 127.97, 127.99, 129.65, 138., 139.23,
142.41 (aromatic), 202.94 (C-2′); LR-MS (FAB) m/z 393 [(M +
H)+]; Anal. (C27H24N2O) C, H, N.
(1R,2R)-2-F or m ylm eth yl-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (en t-24). ent-24 (27 mg, 70%, yel-
low oil) was prepared from ent-16 (38 mg, 0.10 mmol) as
described for preparing 24: [R]23 -17.6 (c 1.32, CHCl3); LR-
D
MS (FAB) m/z 393 [(M + H)+]. Anal. (C27H24N2O) C, H, N.
(1S,2S)-2-(2-Am in oeth yl)-1-(1-tr ip h en ylm eth yl-1H-im i-
d a zol-4-yl)cyclop r op a n e (26). A solution of 24 (39 mg, 0.10
mmol), O-benzylhydroxylamine hydrochloride (32 mg, 0.20
mmol), and molecular sieves 4A (10 mg) in THF (3 mL) was
stirred at room temperature for 5 h. After filtration of the
resulting mixture with Florisil, the filtrate was evaporated and
purified by column chromatography (silica gel; AcOEt/hexane,
1:1) to give the corresponding benzyloxime 25 as a white solid
(E/ Z mixture, 49 mg, quant). To a solution of 25 (49 mg) in
THF (2 mL) was added LiAlH4 (1.0 M in THF, 0.30 mL, 0.30
mmol), and the mixture was stirred at room temperature for
1 h. The reaction was quenched with MeOH, and then the
solvent was evaporated. The residue was partitioned between
CHCl3 and H2O, and the organic layer was washed with
Bin d in g Assa y of Hu m a n Hista m in e Recep tor s. The
stable CHO cell lines expressing H1, H2, and H3 receptors are
established by transfection of the plasmids harboring human
H3 and H4 cDNA as described previously.6b The binding of the

Highly H3-Selective Agonist
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 10 1987
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compounds to the membrane fractions were evaluated using
[3H]pyrilamine (H1), [3H]tiotidine (H2), and [3H]NR-methyl-
histamine (H3) (Perkin-Elmer) as radioligands, respectively.
Lu cifer a se Rep or ter Assa y. Reporter gene assay was
performed according to the method described previously.6a
Briefly, 3 × 104 cells of 293-EBNA (Invitrogen) were harvested
on collagen-coated 48-well plates for 24 h. An H1-expression
plasmid and a pSRE-Luc (Stratagene) or an H2-expression
plasmid and pCRE-Luc (Stratagene) were cotransfected using
Fugene 6 (Roche Diagnostics) according to the manufacturer’s
recommendations. An expression plasmid for GRq/i, chimera GR
protein of GRq and GRi, was constructed25 and cotransfected
with an H3 or H4-expression plasmid and a pSRE-Luc. The
following day, the cells were treated with compounds for 5 h
and laid on ice. Intracellular luciferase activity in aliquots from
each lysate was measured using a model ML3000 luminometer
(Dynatech laboratories).
Ack n ow led gm en t . This investigation was sup-
ported by a Grant-in-Aid for Creative Scientific Re-
search (13NP0401) from the J apan Society for Promo-
tion of Science. We are grateful to Daiso Co., Ltd. for
the gift of chiral epichlorohydrins and to Ms. H. Mat-
sumoto, A. Maeda, and S. Oka (Center for Instrumental
Analysis, Hokkaido University) for technical assistance
with NMR, MS, and elemental analyses.
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