Investigation of the bioactive conformation of histamine H3 receptor antagonists by the cyclopropylic strain-based conformational restriction strategy

Article abstract of DOI:10.1021/jm901848b

We previously identified the highly potent histamine H₃ receptor antagonists (1R,2S)-2-[2-(4-chlorobenzylamino)ethyl]-1-(1H-imidazol-4-yl) cyclopropane (1) and its enantiomer ent-1. Although the conformations of 1 and ent-1 are restricted by the central cyclopropane ring, the 2-(4- chlorobenzylamino)ethyl side chain essential for the H₃ receptor binding may somewhat freely rotate. To investigate the bioactive conformation, the 1′-ethyl-substituted derivatives 2a and 2b and their enantiomers ent-2a and ent-2b were designed as side chain conformation-restricted analogues of 1 and ent-1, based on the cyclopropylic strain. These compounds were synthesized, and their analysis by NMR and calculations suggested that the side chain moiety was effectively restricted in a syn-form or an anti-form by the cyclopropylic strain as expected. Pharmacological evaluation and docking simulation showed that the bioactive conformations of 1 and ent-1 appear to be the syn-form and the anti-form, respectively. Thus, the cyclopropylic strain can be effectively used for conformational restriction of the side chain moiety of cyclopropane compounds.

Full text of DOI:10.1021/jm901848b

J. Med. Chem. 2010, 53, 3585–3593 3585
DOI: 10.1021/jm901848b
Investigation of the Bioactive Conformation of Histamine H3 Receptor Antagonists by the
Cyclopropylic Strain-Based Conformational Restriction Strategy
Mizuki Watanabe,†,# Takatsugu Hirokawa,‡,# Takaaki Kobayashi,† Akira Yoshida,§ Yoshihiko Ito,§ Shizuo Yamada,§
Naoki Orimoto, Yasundo Yamasaki, Mitsuhiro Arisawa,† and Satoshi Shuto*,†
†Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan, ‡Computational
Biology Research Center, National Institute of Advanced Industrial Science and Technology, Aomi, Koutou-ku, Tokyo 135-0064, Japan,
§Department of Pharmacokinetics and Pharmacodynammics and Global Center of Excellence (COE), School of Pharmaceutical Sciences,
University of Shizuoka, Yada, Shizuoka 422-8526, Japan, and Hanno Research Center, Taiho Pharmaceutical Co. Ltd., Misugidai,
Hanno 357-8527, Japan. # M.W. and T.H. contributed equally to this work.
Received December 15, 2009
We previously identified the highly potent histamine H3 receptor antagonists (1R,2S)-2-[2-(4-chloro-
benzylamino)ethyl]-1-(1H-imidazol-4-yl)cyclopropane (1) and its enantiomer ent-1. Although the
conformations of 1 and ent-1 are restricted by the central cyclopropane ring, the 2-(4-chlorobenzyl-
amino)ethyl side chain essential for the H3 receptor binding may somewhat freely rotate. To investigate
the bioactive conformation, the 10-ethyl-substituted derivatives 2a and 2b and their enantiomers ent-2a
and ent-2b were designed as side chain conformation-restricted analogues of 1 and ent-1, based on the
cyclopropylic strain. These compounds were synthesized, and their analysis by NMR and calculations
suggested that the side chain moiety was effectively restricted in a syn-form or an anti-form by the
cyclopropylic strain as expected. Pharmacological evaluation and docking simulation showed that the
bioactive conformations of 1 and ent-1 appear to be the syn-form and the anti-form, respectively. Thus,
the cyclopropylic strain can be effectively used for conformational restriction of the side chain moiety of
cyclopropane compounds.
Introduction
Conformational restriction of neurotransmitters may im-
prove the specific binding to one of the receptor subtypes.4 In
conformationally restricted analogues highly selectively bound
to the target receptor, the functional groups essential for the
receptor binding must assume a special arrangement super-
imposed on the bioactive conformation, in which these func-
tional groups effectively interact with certain amino acid
residues in the binding pocket of the receptor. The major
problem in designing conformationally restricted analogues
specific for a receptor subtype is that the conformation of the
conformationally flexible lead compound that binds to the
target subtype, i.e., the bioactive conformation, is often un-
known. This is mainly because structural analysis of membrane-
bound proteins is tremendously difficult,2 compared with that
of proteins soluble in blood or cytosol. Thus, a method for
effectively identifying compounds targeting GPCRs, which do
not involve structural data, would be highly useful in drug
development. Consequently, we have devised a stereochemical
diversity-oriented conformational restriction strategy to deve-
lop compounds that bind selectively to target proteins of
unknown structure such as GPCRs.5 In this strategy, the
versatile chiral cyclopropane units with different stereochemi-
stries (Figure 2)5a are effectively used as the key tool for the
design and synthesis of a series of conformationally restricted
analogues with stereochemical diversity.5
Attention has been focused on the histamine H3 receptor,
which is a G-protein-coupled receptor (GPCRa) distributed
mainly in the central nervous system.1 Antagonists to the
H3 receptor are considered to be potential drugs for various
diseases, such as Alzheimer’s disease, attention-deficit/
hyperactivity disorder (ADHD), schizophrenia, depression,
dementia, obesity, and epilepsy.1b,d,e As a result, attempts to
develop H3 receptor antagonists have led to the identification
of potent H3 receptor ligands,1b-f some of which are shown in
Figure 1.
GPCRs are considered to be major targets for drug deve-
lopment.2 Indeed, it is estimated that over 50% of all modern
drugs are targeted at GPCRs.2a However, because of the
membranous nature of these proteins and their very low
natural abundance, structural analysis of GPCRs is difficult.
In fact, until the most recent resolution of the adrenergic β2
receptor structures,3 the only high-resolution structure of a
GPCR available had been that of bovine rhodopsin.2b One
obvious drawback in drug development targeting GPCRs is
therefore poor structural data on these proteins.
*To whom correspondence should be addressed. Phone and fax: þ81-
11-706-3769. E-mail: shu@pharm.hokudai.ac.jp.
a Abbreviations: ADHD, attention-deficit/hyperactivity disorder;
EBNA, Epstein-Barr virus nuclear antigen-1; GPCR, G-protein-
coupled receptor; GPCRDB, G-protein-coupled receptors data bank;
IFD, induced fit docking; OPLS-AA, optimized potentials for liquid
simulations-all-atom; PGME, phenylglycine methyl ester; XP, extra-
precision.
On the basis of this stereochemical diversity-oriented strategy,
we recently designed and synthesized a series of conformation-
ally restricted analogues of histamine, as shown in Figure 2, with
different stereochemistries.5 In these analogues, the imidazole
r
2010 American Chemical Society
Published on Web 04/16/2010
pubs.acs.org/jmc

3586 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Watanabe et al.
and the amino groups are located in a variety of spatial
arrangements because of the conformational restriction. Some
of these analogues were shown to be potent H3 receptor ligands,
and a conformationally restricted analogue 1 with a (1R)-trans-
cyclopropane structure and its enantiomer ent-1 (Figure 3) were
identified as highly potent H3 receptor antagonists.5c
structure, cyclopropane is effective in restricting the confor-
mation of a molecule without changing the chemical and
physical properties of the lead compound.8 A characteristic
structural feature of cyclopropane is that cis-oriented adja-
cent substituents on the ring exert significant mutual steric
repulsion because they are fixed in the eclipsed orientation,
which we previously termed “cyclopropylic strain”.7 Conse-
quently, conformation of the substituents on a cyclopropane
can be restricted so that the steric repulsion due to the strain
is minimal, as indicated in Figure 4.
Considering that the basic amino function of the H3
receptor antagonist 1 is likely to be important for the H3
receptor binding, the conformation of the side chain would
significantly affect the activity of the compound. While the
cyclopropyl-C10 (C2-C10) bond may freely rotate some-
what, the rotation can be restricted by the cyclopropylic
strain. Therefore, the two conformers A (syn, the C-3 of
the cyclopropane “up”/the benzylaminomethyl “up”) and
C (anti, the C-3 of the cyclopropane “up”/the benzyl-
aminomethyl “down”) would be preferable to conformer
With these results in hand, we thought that, based on the
structures of 1 and ent-1, identification of the bioactive
conformation for the H3 receptor antagonists might be pos-
sible. If indeed this could be accomplished, the information
obtained would be useful in designing further effective com-
pounds. Although the conformation of 1 is restricted by the
(1R)-trans-cyclopropane structure, the spatial arrangement of
the basicnitrogen of the sidechain, which seems to be essential
for activity, would be somewhat flexible. Therefore, we
decided to restrict the side chain conformation by the cyclo-
propylic strain-based method,6,7 a detail of which is described
below, to identify the bioactive conformation. During our
study, X-ray crystallographic structures of adrenergic β2
receptors were reported,3 and as a consequence, the H3
receptor model using the structural data of a β2 receptor
was constructed and used for investigating the binding con-
formation of the conformationally restricted analogues.
In this report, we describe the design, synthesis, pharmaco-
logical effects, and receptor modeling studies of the cyclo-
propylic strain-based conformationally restricted analogues
2a and 2b and their enantiomers ent-2a and ent-2b (Figure 3)
for the identification of their bioactive conformations.
Results and Discussion
Cyclopropylic Strain-Based Design of the Conformation-
ally Restricted Analogues. Because of its small and rigid ring
Figure 3. Previously synthesized H3 receptor antagonists 1 and
ent-1 and their side chain conformationally restricted analogues
newly designed.
Figure 1. Histamine and representative H3 receptor ligands.
Figure 4. Cyclopropylic strain-based conformational restriction.
Figure 2. A series of conformationally restricted analogues of histamine with stereochemical diversity synthesized from the chiral
cyclopropane units.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3587
Scheme 1
Figure 5. Presumed stable conformations of 1 (a), 2a (b), and 2b (c).
We designed the 10-ethyl-substituted derivatives 2a and 2b
(Figure 3) as side chain conformation-restricted analogues
of 1. Introducing an ethyl group into the R-position of the
amino function of 1 would prevent the rotation of the side
chain moiety by restricting the conformation due to the
cyclopropylic strain, i.e., the steric repulsion for the adjacent
eclipsed proton. Accordingly, depending on the configura-
tion at the C10 position, the conformation of the compounds
can be restricted; the syn-conformer would be quite stable in
2a of the 10R-configuration (Figure 5b); conversely, the anti-
conformer would be stable in 2b of the 10S-configuration
(Figure 5c).
Thus, while cyclopropane is very effective for conforma-
tional restriction of conformationally flexible lead com-
pounds, the cyclopropylic strain-based conformational
restriction makes the more precise conformational restric-
tion of cyclopropane compounds possible, especially in the
side chain moiety.
Synthesis. Although much effort has been devoted to
developing practical methods for preparing chiral cyclopro-
panes, synthesis of cyclopropane derivatives with the desired
stereochemistry is often troublesome.9 The chiral cyclopro-
pane units (Figure 2), which are composed of four stereo-
isomeric cyclopropane derivatives bearing two adjacent
carbon substituents in a cis or a trans relationship, are useful
for the synthesis of various cyclopropane compounds, parti-
cularly for those with stereochemical diversity.5
As summarized in Scheme 1, the target compounds 2a and
2b were synthesized from an imidazolylcylcopropanecarbox-
aldehyde 5 with the (1R,2R)-structure, which was prepared
from the unit 4 by our previous method.5a Treatment of 5 with
EtMgBr in THF gave a diastereomeric mixture of the addition
products, the Dess-Martin oxidation of which afforded the
corresponding ketone 6. Wittig reaction of 6 with MeOCH2-
PPh3Cl/NaN(TMS)2 followed by acidic treatment gave the
Figure 6. Stable structures of the conformationally restricted ana-
logues in the gas phase obtained by the conformational search
program in MacroModel: (a) the syn-conformer and the anti-
conformer for 1; (b) the syn-conformer for 2a and the anti-con-
former for 2b.
B in compound 1 because of the significant steric repulsion
for the adjacent cis-proton in conformer B, as shown in
Figure 5a.
The conformation of 1 was analyzed by molecular me-
€
chanics calculations with MacroModel (Schrodinger, LLC).
As a result, as shown in Figure 6a, two significantly stable
structures were obtained, which correspond to the syn- and
the anti-conformers in Figure 5a, respectively. The two
conformers are nearly equally stable, while the anti-confor-
mer is only 0.31 kcal/mol more stable than the syn-confor-
mer. Thus, the results of calculations are in accord with the
above hypothesis on the conformation of 1, which suggests
that the bioactive conformation may be analogous to either
the syn-conformer or the anti-conformer.

3588 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Watanabe et al.
Table 1. Effects of Compounds on the Human H3 Receptor Subtypea
confor-
mation
inhibition
(%)b
compd
configuration
Ki (nM)
2a
2b
1
ent-2a
ent-2b
(1R)-trans-(10R)
(1R)-trans-(10S)
(1R)-trans
(1S)-trans-(10S)
(1S)-trans-(10R)
(1S)-trans
syn
anti
syn/anti
syn
anti
95
73
19.8 ( 2.8
129 ( 6.0
8.4 ( 1.5c
63.0 ( 6.1
6.7 ( 0.4
100
96
88
ent-1
thioperamide
syn/anti
100
98
3.6 ( 0.4c
51.1 ( 3.8c
Figure 7. Determination of the 10-configurations based on the Δδ
values (DMSO-d6, 500 MHz) of the (R)- and (S)-PGME amides.
a Assays were carried out with 293-EBNA cells or cell membranes
expressing the human H3 receptor subtype. b Inhibitory effect of com-
pound (10-4 M) on the agonistic activity of histamine (10-6 M). c Data
were taken from ref 5c.
The calculated energy barriers for the rotation of the C2-C10
bond between the syn-form and the anti-form are significant,
which are 5.22 kcal/mol for 2a and 5.55 kcal/mol for 2b,
respectively.
Thus, these conformational analyses suggested that the
cyclopropylic strain-based conformational restriction seems
to work effectively in 2a and 2b, and therefore, pharmaco-
logical evaluations of these compounds would help to iden-
tify the bioactive conformation.
Figure 8. NOE data of 2a and 2b in D2O.
Pharmacological Effects. Effects of compounds on the H3
receptor were investigated by luciferase reporter gene assay.
The human histamine receptor subtypes were individually
expressed in 293-Epstein-Barr virus nuclear antigen-1 (EB-
NA) cells according to the previously reported method,5b
and the function of the compounds on these receptors
expressed on the cells was evaluated. None of the newly
synthesized compounds 2a, 2b, ent-2a, and ent-2b showed
any agonistic activity to the H3 receptor at 10-5 M (data not
shown). On the other hand, all of these compounds inhibited
the agonistic effect of histamine to show that they are
antagonists of the H3 receptor as are the parent compounds
1 and ent-1, as shown in Table 1.
Binding affinities of compounds 2a, 2b, ent-2a, and ent-2b
for the human H3 receptor subtype using [3H]NR-methyl-
histamine5c were next investigated and were compared with
those of their parent compounds 1 and ent-1 (Table 1). In this
system, the well-known H3 receptor antagonist thioperamide
showed a Ki value of 51.1 nM, and compounds 1 and ent-1
displayed much higher binding affinity for the human H3
receptor as shown by the Ki values of 8.4 and 3.6 nM,
respectively.
Although compound 2a, which is restricted in the syn-
conformation, showed remarkable binding affinity for the
receptor with a Ki value of 19.8 nM, compound 2b, restricted
in the anti-conformation, showed more than 15-fold reduc-
tion of potency (Ki =129 nM), compared with the parent
compound 1. On the other hand, of the enantiomers ent-2a
and ent-2b, ent-2b (Ki =6.7 nM), which is restricted in the
anti-conformation, showed 10-fold higher binding affinity
for the human H3 receptor than the ent-2a (Ki = 63 nM),
which is restricted in the syn-conformation.
Docking Simulation by Homology Modeling. The above
conformational analysis and pharmacological results
showed that in the diastereomeric pair of 2a and 2b, the
syn-restricted 2a was more potent than the anti-restricted 2b,
while in their enantiomers ent-2a and ent-2b, the anti-
restricted ent-2b was more potent than the syn-restricted
ent-2a. In order to understand the discrepancy in the con-
formation-activity relationship between 2a/2b and ent-2a/
ent-2b, we planned to perform a docking simulation with a
aldehyde 7 as an inseparable diastereomeric mixture (dr,
1/1.1). Reductive amination of 7 with 4-chlorobenzylamine
and 2-picoline borane in AcOH/MeOH and subsequent acidic
removal of the trityl group of the product gave the desired
cyclopropylic strain-based conformationally restricted ana-
logues 2a and 2b as a diastereomeric mixture. Although the
diastereomers were inseparable at this stage, they were success-
fully separated by HPLC after protection of the imidazole and
amino nitrogens with Boc groups to give the diastereomerically
pure 8a and 8b, respectively. Acidic removal of the Boc groups
of 8a and 8b afforded the target (10R)-product 2a and the (10S)-
product 2b, respectively. The enantiomers ent-2a and ent-2b
were similarly synthesized from ent-5.
The 10-configurations of the cyclopropylic strain-based
conformationally restricted analogues synthesized were
determined by the phenylglycine methyl ester (PGME)
method10 by converting diastereomerically pure ent-7a and
ent-7b into the corresponding (R)-PGME amides ent-9a and
ent-9b and (S)-PGME amides ent-10a and ent-10b,11 respec-
tively, as shown in Figure 7.
Conformational Analysis by NMR and Calculations. Stable
structures of the conformationally restricted analogues 2a
and 2b were investigated by NOE experiments (Figure 8).
Irradiations of H-10 of 2a or 2b gave NOEs with both H-1
and H-3a oriented cis to H-1. Especially significant NOEs
were observed between H-10 and H-1 in both 2a and 2b,
which show that the side chain conformation of the two
compounds seems to be actually restricted by the cyclo-
propylic strain. When H-1 on the cyclopropane ring of the
(10R)-diastereomer 2a was irradiated, an NOE with the
terminal methyl protons of the ethyl group was observed
to suggest that it is restricted to the syn-form as expected. On
the other hand, during irradiation of H-1 of the (10S)-
diastereomer 2b, an NOE was observed with the methylene
proton H-20a adjacent to the basic nitrogen to demonstrate
that it is in the anti-form, as expected.
The conformations of 2a and 2b were also examined by
calculations with MacroModel. As shown in Figure 6b, the
most stable structures obtained by the calculations were
the syn-form for 2a and the anti-form for 2b, respectively.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3589
homology modeling of the H3 receptor to investigate the
binding mode of these cyclopropylic strain-based conforma-
tionally restricted analogues in the active site of the H3
receptor.
Previous studies showed that homology models of H3
receptor are useful for providing structural insight into the
ligand binding mechanism, QSAR analysis, and in silico
drug discovery.13 Furthermore, most recent resolutions of
the ligand-binding adrenergic β2 receptor structures3 give us
a chance to generate more accurate three-dimensional mod-
els for target GPCRs with a ligand using homology modeling
and docking simulation.14
Thus, in this study, a three-dimensional model of the H3
receptor was constructed on the basis of a structural template
from the crystal structure of the human β2-adrenergic GPCR
recently reported by Cherezov and co-workers,3a and dock-
ing simulations of the compounds into the H3 receptor model
were performed with ligand and receptor flexibility.
We constructed a homology model based on the confor-
mationally restricted analogue ent-2b, which is the most
potent H3 receptor ligand in this series of the cyclopropane-
based conformationally restricted analogues. Using this mod-
el, we performed docking simulation with a series of the
cyclopropane-based conformationally restricted H3-receptor
ligands having stereochemical diversity (16 compounds)
synthesized previously5c,12 and also with the four cyclopropylic
strain-based conformationally restricted analogues 2a, 2b, ent-
2a, and ent-2b synthesized in this study. Correlations between
the calculated binding score and the pKi were examined. As a
result, as shown in Figure 9, a reliable correlation (R2 =0.41)
between binding score and pKi was obtained. Consequently, the
ent-2b-bound H3 receptor model was also used for further studies.
In order to understand the binding modes of the newly
synthesized cyclopropylic strain-based conformationally re-
stricted analogues to the H3 receptor, docking simulations of
2a and 2b and their enantiomers ent-2b and ent-2b were
carried out by using the ent-2b-bound model. Figure 10
shows the proposed binding modes of the potent H3 antago-
nists 2a and ent-2b to the homology model of the H3 receptor
obtained by the simulation. These compounds are accom-
modated in the active site concavity formed by TM2, TM3,
TM5, TM6, and TM7. The H3 receptor-binding conforma-
tions of 2a and ent-2b are the syn-form and the anti-form,
respectively, as shown in Figures 10 and 11, which are in accord
with the stable forms proposed by their conformational analysis
by NMR and the calculations described above.
In the obtained binding models shown in Figure 10, the
NH of the imidazole ring of both compounds likewise serves
as a hydrogen donor and forms a hydrogen bond with an
oxygen atom of the side chain carboxyl group of Glu206.
Furthermore, the protonated amine in both 2a and ent-2b
forms a similar salt bridge with Asp114 in TM3, and the
4-chlorobenzyl group in 2a and ent-2b is observed to make a
π-π interaction with the indole ring of Trp110 in TM3.
Thus, the special positioning of the imidazole moiety and the
4-chlorobenzylamino moiety in 2a and ent-2b, which are
likely to be essential for their H3 receptor binding, is ana-
logous in the active site, and therefore 2a and ent-2b may
have a common pharmacophore. As shown in Figure 11a,
the imidazole and 4-chlorobenzylamino moieties of 2a and
ent-2b can be superimposed, where the two cyclopropane
rings orient oppositely, i.e., “up” in 2a and “down” in ent-2b,
respectively. This would explain why 2a and ent-2b have a
similar potent antagonistic effect on the H3 receptor, even
though the two compounds are conformationally restricted
Figure 9. Plot of binding score calculated by Glide extraprecision
(XP) based on ent-2b-bound H3 receptor model versus experimental
binding affinity pKi for 20 conformational restriction analogues.
The coefficient of determination, R2, between binding score and pKi
was 0.41 for 20 conformational restriction analogues.
Figure 10. Proposed models for 2a (a) and ent-2b (b) binding to the homology model of the H3 receptor from docking simulation. Receptor
˚
residues around the compounds within 4 A are shown in line representation. Carbon atoms of 2a and ent-2b are shown in magenta and green,
respectively. All nonpolar hydrogen atoms of receptor residues are omitted for clarify. Hydrogen bonding and salt bridge to side chain carboxyl
group of Glu206 and Asp114 are depicted by red dots.

3590 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Watanabe et al.
Figure 11. Superimposition of the structures of 2a (magenta) and ent-2b (green) binding to the homology model of the H3 receptor (a). Three-
feature pharmacophore model generated for 2a and ent-2b using MOE: hydrogen bond acceptor/donor (magenta feature), hydrogen bond
donor/cationic atom (blue feature), and aromatic ring center/hydrophobic region (green feature) (b). Known H3 receptor ligands are mapped
onto the pharmacophore model obtained from 2a and ent-2b (c).
simulations. As shown in Figure 12a, the bioactive confor-
mation of 1 is in accord with the syn-form of the two stable
syn- and anti-conformations. Figure 12b shows that the
stable syn-form of 2a is almost identical with the bioactive
conformation in 2a, which would make it highly potent. On
the other hand, in 2b, the bioactive conformation is the syn-
form similar to 1 and 2a, while 2b itself is stable in the anti-
form (Figure 12c). Thus, because of the entropic cost for the
conformational change from the stable anti-form into the
bioactive syn-form in its binding to the H3 receptor, the
binding affinity of 2b for the H3 receptor is significantly
decreased. In Figure 12d, the receptor-bound conformations
of 1 and 2a were superimposed, showing that their bio-
active conformations are the same. These results suggested
that the cyclopropylic strain-based conformational restric-
tion worked effectively as expected.
As described, we identified the potent H3 receptor antago-
nists 1 and ent-1 by the stereochemical diversity-oriented
conformational restriction method with chiral cyclopropane
units as shown in Figure 2. Their bioactive conformations
and a pharmacophore model were further elaborated by the
cyclopropylic strain-based conformational restriction method.
These results showed that the combinational use of the
Figure 12. Comparison of conformational changes between the
stable forms shown in Figure 6 and the bioactive forms proposed
stereochemical diversity-oriented and the cyclopropylic
strain-based conformational restriction methods seems to
be an effective strategy for developing significantly active
compounds and also for identifying their bioactive confor-
mations, especially in cases where the structural data of the
target biomolecule are lacking or poorly documented. In
these studies, simulations with homology modeling of the
target biomolecule can be effective. This is due to the fact
that a series of cyclopropane analogues and also cyclopro-
pane strain-based conformationally restricted analogues are
suitable for validating the homology models, since these
consist of compounds having diversity not only in their
conformation, i.e., three-dimensional structure, but also in
their binding affinity for the target.
by the H3 receptor-bound model. Shown are the two stable con-
formations and the bioactive conformations for 1 (a), the stable and
the bioactive conformations for 2a (b) and 2b (c), and superimposi-
tion of the bioactive conformations of 1 and 2a (d). Carbon atoms
of stable conformations for all compounds are shown in gray, and
carbon atoms of the bioactive conformations for 1, 2a, and 2b are
shown in blue, magenta, and yellow, respectively. All compounds
are aligned on the cyclopropane ring.
differently, i.e., the syn-form and the anti-form, respectively, by
the cyclopropylic strain. Figure 11b shows a common pharma-
cophore model for 2a and ent-2b, and the model effectively fitted
in known H3 receptor ligands,1 as shown in Figure 11c.
We next examined the possible conformational differences
between the stable form and the bioactive form of the
compounds, which could significantly affect the binding
affinity, based on the proposed receptor-bound model. In
Figure 12, the stable conformations of 1 and its conforma-
tionally restricted analogues 2a and 2b based on NMR and
calculation analysis are superimposed on their receptor-
bound (bioactive) conformations by the receptor modeling
Conclusion
In order to clarify the bioactive conformation of the pre-
viously developed H3 receptor antagonists 1 and ent-1, the
10-ethyl-substituted derivatives 2a and 2b and their enantiomers

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3591
ent-2a and ent-2b were designed as side chain conformation-
restricted analogues of 1 and ent-1, based on the cyclo-
propylic strain, and were synthesized from the versatile chiral
cyclopropane units. Conformational analysis, pharmacological
evaluation, and docking simulation of the compounds showed
that the bioactive conformations of 1 and ent-1 seem to be the
syn-form and the anti-form, respectively. On the basis of these
results, a common pharmacophore for the compounds was
obtained. These results suggest that the cyclopropylic strain-
based strategy can be effectively used for precise conformational
restriction of the side chain moiety and bioactive conformation
analysis of cyclopropane compounds.
The organic layer was washed with aqueous saturated NaHCO3,
brine, dried (Na2SO4), and evaporated. The residue was puri-
fied by silica gel column chromatography (25-40% AcOEt in
hexane) to give 7 (diastereomixture, 138 mg, 65%) as a yellow
amorphous solid: HRMS (EI) calcd for C29H28N2O 420.2202,
found 420.2200 (Mþ).
(1R,2S)-2-[1-Ethyl-2-(4-chlorobenzylamino)ethyl]-1-(1H-imi-
dazol-4-yl)cyclopropane (2a/2b). To a solution of 7 (112 mg,
0.266 mmol) and 4-chlorobenzylamine (35 μL, 0.28 mmol) in
MeOH/AcOH (10/1, 2.2 mL) was added 2-picolineborane (30
mg, 0.28 mmol) at room temperature, and the mixture was
stirred at the same temperature for 12 h. After evaporation of
the solvent, a solution of the residue in aqueous HCl (4 M,
4.0 mL) was stirred at 0 °C for 20 min, and then the mixture was
neutralized with Na2CO3. The mixture was partitioned between
CH2Cl2 and aqueous saturated NaHCO3, and the organic layer
was washed with brine, dried (Na2SO4), and evaporated. The
residue was purified by neutral silica gel column chromato-
graphy (0-10% MeOH in CHCl3) to give amine product
(diastereomixture, 108 mg) as a light-yellow amorphous solid.
To a solution of amine (108 mg) in EtOH (1.0 mL) was added
aqueous HCl (2 M, 1.0 mL), and the resulting solution was
stirred at 78 °C for 2 h, and then the solvent was evaporated. The
residue was partitioned between aqueous HCl (1 M) and CH2-
Cl2, and the aqueousueous layer was neutralized with aqueous
NaOH (2 M). The resulting solution was extracted with Et2O,
and the organic layer was washed with H2O, brine, dried
(Na2SO4), and evaporated. The residue was purified by NH
silica gel column chromatography (0-5% MeOH in CHCl3) to
give the diastereomixture of 2a and 2b (56 mg, 70%) as a
colorless amorphous solid: HRMS (EI) calcd for C17H22ClN3
303.1502, found 303.1500 (Mþ).
(1R,2S)-2-[(1R)-1-Ethyl-2-(4-chlorobenzylamino)ethyl]-1-(1H-
imidazol-4-yl)cyclopropane (2a) and (1R,2S)-2-[(1S)-1-Ethyl-
2-(4-chlorobenzylamino)ethyl]-1-(1H-imidazol-4-yl)cyclopro-
pane (2b). A solution of the diastereomixture of 2a and 2b
(56 mg, 0.15 mmol), Et3N (83 μL, 0.60 mmol), DMAP (1.8
mg, 0.015 mmol), and (Boc)2O (130 mg, 0.60 mmol) in MeOH
(1 mL) was stirred at room temperature for 16 h. After evapora-
tion of the solvent, the residue was partitioned between AcOEt
and H2O, and the organic layer was washed with brine, dried
(Na2SO4), and evaporated. The residue was separated by HPLC
(28% AcOEt in hexane, 13 mL/min, room temperature, 253 nm)
with Mightysil Si 60 (0.25 cm ꢀ 20 cm, Kanto Chemical Co.) to
give 8a (24 mg, a colorless amorphous solid) and 8b (26 mg, a
colorless amorphous solid). Each compound was dissolved in
EtOH (1.5 mL)/aqueous HCl (4 M, 0.5 mL), and the mixture
was stirred at 78 °C for 2 h. After the mixture was concentrated
and dried in vacuo, the residue was purified by NH silica gel
column chromatography (0-10% MeOH in CHCl3) to give 2a
(12 mg, 29% for three steps, a colorless amorphous solid) or 2b
(14 mg, 33% for three steps, a colorless amorphous solid) as a
free amine.
Experimental Section
Chemical shifts are reported in ppm downfield from tetra-
methylsilane. Thin-layer chromatography was done on Merck
coated plate 60F254. Silica gel chromatography was done on silica
gel 5715 (Merck) or NH silica gel (Chromatorex, Fuji Silysia
Chemical Ltd.). Reactions were carried out under an argon
atmosphere. Estimated purity of all of the final compounds by
combustional analysis was always at least 95%.
(1R,2R)-2-(1-Oxopropyl)-1-(1-triphenylmethyl-1H-imidazol-
4-yl)cyclopropane (6). To a solution of 55a (238 mg, 0.629 mmol)
in THF (5.0 mL) was added EtMgBr (0.91 M in THF, 830 μL,
0.755 mmol) at 0 °C, and the mixture was stirred at the same
temperature for 1 h. After addition of aqueous saturated
NH4Cl, the solvent was evaporated, and the residue was parti-
tioned between AcOEt and aqueous NH4Cl. The organic layer
was washed with brine, dried (Na2SO4), and evaporated. To a
solution of the residue in CH2Cl2 (5 mL) was added
Dess-Martin periodinane (320 mg, 0.755 mmol) at room
temperature, and the mixture was stirred at the same tempera-
ture for 1 h. To the reaction mixture was added a mixture of
aqueous saturated NaHCO3 and aqueous saturated Na2S2O3
(3/1, 12 mL) at room temperature, and the resulting mixture was
vigorously stirred at the same temperature for 10 min. The
resulting solution was extracted with AcOEt, and the organic
layer was washed with aqueous saturated NaHCO3, brine, dried
(Na2SO4), and evaporated. The residue was purified by silica gel
column chromatography (20-33% AcOEt in hexane) to give 6
(239 mg, 93%) as a white amorphous solid: [R]19 -217.3 (c
D
0.86, CHCl3); 1H NMR (400 MHz, CDCl3) δ 1.07 (3 H, t, J=7.3
Hz, CH3CH2-), 1.44 (1 H, m, H-3a), 1.50 (1 H, m, H-3b), 2.38 (2
H, m, H-1 and H-2), 2.61 (2 H, q, J=7.3 Hz, CH3CH2-), 6.64 (1
H, d, J=1.2 Hz, imidazolyl), 7.11-7.14 (6 H, m, aromatic), 7.28
(1 H, s, imidazolyl), 7.32-7.34 (9 H, m, aromatic); 13C NMR
(100 MHz, CDCl3) δ 7.96, 18.0, 22.5, 30.1, 37.0, 75.2, 118.0,
127.9, 129.7, 138.4, 138.4, 139.9, 142.2, 209.9; LRMS (EI) m/z
406 (Mþ); HRMS (EI) calcd for C28H26N2O 406.2045, found
406.2050 (Mþ). Anal. (C28H26N2O) C, H, N.
(1R,2R)-2-(1-Formylpropyl)-1-(1-triphenylmethyl-1H-imida-
zol-4-yl)cyclopropane (7). To a suspension of MeOCH2PPh3Cl
(608 mg, 1.77 mmol) in THF (5.0 mL) was added NaN(Si-
(CH3)3)2 (∼1.9 M in THF, 800 μL, 1.52 mmol) at 0 °C, and the
mixture was stirred at the same temperature for 15 min. To the
resulting solution was added a solution of 6 (206 mg, 0.507
mmol) in CH2Cl2 (2.0 mL) at 0 °C, and the reaction mixture was
stirred at the same temperature for 2 h. After addition of
aqueous saturated NH4Cl, the solvent was evaporated, and the
residue was partitioned between AcOEt and aqueous NH4Cl. The
organic layer was washed with brine, dried (Na2SO4), and evapo-
rated. The residue was purified by silica gel column chromato-
graphy (30% AcOEt in hexane) to give the enol ether product (151
mg) as a light-yellow solid. To a solution of the product in THF
(10 mL) was added aqueous HCl (12 M, 5.0 mL), and the mixture
was vigorously stirred at room temperature for 10 s. Immediately,
the mixture was poured into aqueous saturated NaHCO3 (100
mL). Then the resulting solution was extracted with AcOEt.
2a: 1H NMR (400 MHz, CDCl3) δ 0.76 (1 H, m, H-3a),
0.87-0.96 (6 H, m, H-3b and H-10 and H-2 and CH3CH2-), 1.45
(1 H, m, CH3CH2-), 1.54 (1 H, m, CH3CH2-), 1.64 (1 H, m, H-1),
2.65 (2 H, dd, J=5.0, 12.0 Hz, H-20), 3.77 (2 H, s, -CH2Ph), 6.63
(1 H, s, imidazolyl), 7.27 (4 H, dd, J=8.0, 8.6 Hz, aromatic),
7.48 (1 H, s, imidazolyl); 13C NMR (100 MHz, CDCl3) δ 12.0,
13.1, 14.6, 24.7, 26.3, 44.8, 53.8, 53.8, 128.7, 129.6, 132.8, 134.4,
139.2; HRMS (EI) calcd for C17H22ClN3 303.1502, found
303.1501 (Mþ).
The free amine 2a (12 mg) was dissolved in aqueous HCl
(4 M), and the solvent was then evaporated. The residue was
triturated with Et2O to give 2a dihydrochloride (15 mg, a white
amorphous solid): [R]21D -20.5 (c 0.81, MeOH); 1H NMR (400
MHz, D2O) δ 0.89 (3 H, t, J=7.5 Hz, CH3CH2-), 0.93 (1 H, m, H-
3a), 0.98-1.06 (2 H, m, H-2 and H-3b), 1.22 (1 H, m, H-10), 1.54 (2
H, m, CH3CH2-), 1.88 (1 H, m, H-1), 3.11 (2 H, dd, J=1.8, 7.1 Hz,
H-20), 4.20 (1 H, d, J=13.6 Hz, -CH2Ph), 4.26 (1 H, d, J=13.6 Hz,

3592 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Watanabe et al.
-CH2Ph), 7.10 (1 H, s, imidazolyl), 7.46 (4 H, dd, J=8.6, 8.6 Hz,
aromatic), 8.44 (1 H, d, J=1.4 Hz, imidazolyl); LRMS (EI) m/z
303 ((M - 2HCl)þ). Anal. (C17H24Cl3N3) C, H, N.
β2-adrenergic receptor. A three-dimensional model of H3 receptor
was constructed using a homology modeling approach incorpo-
rated in the program SegMod16 of GeneMine.17 Because it is very
long and predicted to be disordered, the third intracellular loop
was truncated by the five residues leading out of the helix V and
the five residues leading into helix VI. The second extracellular
loop was modeled without aligning them with those of the
β2-adrenergic receptor.
2b: 1H NMR (400 MHz, CDCl3) δ 0.72 (1 H, m, H-3a), 0.81
(1 H, m, H-3b), 0.93-0.99 (5 H, m, H-10 and H-2 and CH3CH2-),
1.47 (2 H, m, CH3CH2-), 1.64 (1 H, m, H-1), 2.64 (1 H, dd, J=
7.4, 11.4 Hz, H-20), 2.75 (1 H, dd, J=5.7, 11.4 Hz, H-20), 3.74 (1
H, d, J=13.3 Hz, -CH2Ph), 3.78 (1 H, d, J=13.3 Hz, -CH2Ph),
6.67 (1 H, s, imidazolyl), 7.22 (2 H, d, J=8.6 Hz, aromatic), 7.26
(2 H, d, J=8.6 Hz, aromatic), 7.44 (1 H, s, imidazolyl); 13C
NMR (100 MHz, CDCl3) δ 11.8, 12.5, 14.6, 24.9, 26.4, 44.8,
53.8, 54.4, 128.8, 129.7, 132.9, 134.5, 139.1; LRMS (EI) m/z 303
(Mþ); HRMS (EI) calcd for C17H22ClN3 303.1502, found
303.1509 (Mþ).
Docking Simulation. Initial coordinates of all compounds
were constructed using the Molecular Builder module in Maes-
€
tro (Schrodinger LLC.). Energy minimization of all compounds
was performed using the optimized potentials for liquid simula-
tions-all-atom (OPLS-AA) force field in the LigPrep in the
€
Maestro (Schrodinger LLC.). The homology model of H3
receptor was refined for docking simulations using the Protein
Preparation Wizard Script within Maestro. This protein pre-
paration procedure involves optimization of contacts by chan-
ging hydroxyl group orientations, flipping of Asn and Gln side
chains, and selecting His tautomeric states, followed by con-
strained energy refinement using the OPLS-AA force field.
Docking of the compounds into the H3 receptor model utilized
three main steps that take into account several levels of struc-
tural flexibility and scoring criteria: (1) molecular modeling of
compound bound H3 receptor model by docking the ent-2b
molecule, considering both ligand and receptor flexibility, (2)
rigid receptor docking of 20 cyclopropane-based conforma-
tional restriction analogues5c into the active site of ent-2b
compound bound H3 receptor model from the previous step,
(3) rescoring according to the calculated binding score by Glide
The free amine 2b (14 mg) was dissolved in aqueous HCl
(4 M), and the solvent was then evaporated. The residue was
triturated with Et2O to give 2b dihydrochloride (17 mg, a white
amorphous solid): [R]21D -64.5 (c 1.23, MeOH); 1H NMR (400
MHz, D2O) δ 0.92 (3 H, t, J=7.2 Hz, CH3CH2-), 1.05 (2 H, m,
H-3), 1.09 (1 H, m, H-2), 1.23 (1 H, m, H-10), 1.43-1.60 (2 H, m,
CH3CH2-), 1.74 (1 H, m, H-1), 3.12 (1 H, dd, J=7.4, 13.1 Hz,
H-2a0), 3.19 (1 H, dd, J=5.0, 13.1 Hz, H-2b0), 4.13 (1 H, d, J=
13.6 Hz, -CH2Ph), 4.26 (1 H, d, J=13.6 Hz, -CH2Ph), 6.97 (1 H,
s, imidazolyl), 7.39 (4 H, s, aromatic), 8.46 (1 H, d, J=1.4 Hz,
imidazolyl); LRMS (EI) m/z 303 ((M-2HCl)þ). Anal. (C17H24-
Cl3N3) C, H, N.
(1S,2S)-2-(1-Oxopropyl)-1-(1-triphenylmethyl-1H-imidazol-
4-yl)cyclopropane (ent-6). Compound ent-6 (173 mg, 85%, a
white amorphous solid) was prepared from ent-5 (190 mg, 0.50
mmol) as described for the preparation of 6: [R]20 þ232.1 (c
€
extraprecision (XP) score (Schrodinger LLC).
D
Following are the details of each step. In order to account for
both compound and receptor flexibility in the first step, the
1.03, CHCl3); HRMS (EI) calcd for C28H26N2O 406.2045,
found 406.2044 (Mþ). Anal. (C28H26N2O) C, H, N.
€
Glide “Induced Fit Docking (IFD)” protocol (Schrodinger
(1S,2S)-2-(1-Formylpropyl)-1-(1-triphenylmethyl-1H-imida-
zol-4-yl)cyclopropane (ent-7). Compound ent-7 (94 mg, 60%, a
white amorphous solid) was prepared from ent-6 (152 mg, 0.37
mmol) as described for the preparation of 7: HRMS (EI) calcd
for C29H28N2O 420.2202, found 420.2220 (Mþ).
LLC.) was utilized, followed by iteratively combining rigid
receptor docking (Glide) and protein remodeling by side chain
searching and minimization (Prime) techniques. Hydrogen-
bonding constraints between the side chain COO- group of
Asp114 and Glu206 were introduced because this hydrogen-
bonding formation is highly conserved in almost all known
complexes of histamine receptor subfamily bound to histamine
and to a wide variety of inhibitors. In the protein remodeling
(1S,2R)-2-[(1S)-1-Ethyl-2-(4-chlorobenzylamino)ethyl]-1-(1H-
imidazol-4-yl)cyclopropane (ent-2a). Compound ent-2a (10 mg,
30%, a white amorphous solid) was prepared from ent-8 (35 mg,
0.11 mmol) as described for the preparation of 2a: HRMS (EI)
calcd for C17H22ClN3 303.1502, found 303.1500 (Mþ). The free
amine ent-2a (10 mg) was dissolved in aqueous HCl (4 M), and
the solvent was then evaporated. The residue was triturated with
Et2O to give ent-2a dihydrochloride (13 mg, a white amorphous
˚
stage, all residues within a 14.0 A radius of each initial docked
compound were refined using Prime. Compound was then
redocked into the refined receptor structure using Glide in the
standard precision (SP) mode. All of the docked structures were
then ranked according to GlideScore. After modeling of the
compound-H3 receptor complex using the IFD protocol, grid
generation and rigid receptor docking of the 20 cyclopropane-
based conformational restriction analogues using Glide (SP
mode) were carried out, using the hydrogen bonding constraint
to connect the side chain COO- group of Asp114 and Glu206.
The best orientation for each docked compound was rescored
according to its binding score, which was calculated using the
solid): [R]21 þ19.9 (c 0.62, MeOH); LRMS (EI) m/z 303
D
((M-2HCl)þ). Anal. (C17H24Cl3N3) C, H, N.
(1S,2R)-2-[(1R)-1-Ethyl-2-(4-chlorobenzylamino)ethyl]-1-(1H-
imidazol-4-yl)cyclopropane (ent-2b). Compound ent-2b (10 mg,
30%, a white amorphous solid) was prepared from ent-8 (35 mg,
0.11 mmol) as described for the preparation of 2b: LRMS (EI)
m/z 303 (Mþ); HRMS (EI) calcd for C17H22ClN3 303.1502,
found 303.1500 (Mþ). The free amine ent-2b (10 mg) was
dissolved in aqueous HCl (4 M), and the solvent was then
evaporated. The residue was triturated with Et2O to give ent-
2b dihydrochloride (13 mg, a white amorphous solid): [R]21
þ63.2 (c 1.11, MeOH); LRMS (EI) m/z 303 ((M-2HCl)þD).
Anal. (C17H24Cl3N3) C, H, N.
€
Glide XP Score (Schrodinger LLC).
Acknowledgment. This investigation was supported by a
Grant-in-Aids for Scientific Research (Grant 21390028) from
the Japan Society for the Promotion of Science. We are
grateful to Sanyo Fine Co., Ltd. for the gift of the chiral
epichlorohydrins.
Binding Assay with Human Histamine Receptors. The assay
was performed according to the method described previously.5c
Luciferase Reporter Gene Assay. The assay was performed
according to the method described previously.5b
Homology Modeling of the H3 Receptor. The histamine H3
receptor sequence was aligned with the human β2-adrenergic
receptor sequence3a and 40 representative sequences of class A
rhodopsin-like amine families (G-protein-coupled receptors data
bank (GPCRDB): http://www.gpcr.org/7tm/) using the CLUS-
TAL W (version 1.8) multiple alignment program.15 The align-
ment was refined manually on the basis of the compatibility of the
amino acid position with the corresponding structure of the
Supporting Information Available: Synthesis of PGME
amides ent-9a, ent-9b, ent-10a, and ent-10b; the experimental
details by which the determination of the 10-configurations of
ent-2a and ent-2b by the PGME method was effected; the
structures and pKi values of the H3 receptor antagonists used
for the docking simulations; and elemental analysis data of the
final compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3593
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