Investigation of the histamine H3 receptor binding site. Design and synthesis of hybrid agonists with a lipophilic side chain

Article abstract of DOI:10.1021/jm100643t

As a part of our search for novel histamine H3 receptor agonists, we designed and synthesized hybrid compounds in which the lipophilic (4′-alkylphenylthio)ethyl moiety of a novel H3 receptor agonist, 4-(2-(4′-tert-butylphenylthio)ethyl)-1H-imidazole (1), was incorporated into N^α-methylhistamine, immepip, and immethridine derivatives. These hybrid compounds were expected to interact concurrently with the histamine-binding site and a putative hydrophobic region in the H3 receptor. Among them, piperidine- and pyridine-type derivatives displayed partial agonist activity, and (S)-4-(1-(1H-imidazol-4-yl)-2-(4-(trifluoromethyl)phenylthio) ethyl)piperidine (36) was identified as a potent H3 agonist. We performed computational docking studies to examine the binding mode of the agonists. The results indicated that immepip interacts with the key residues, Asp114 and Glu206, in a different manner from histamine. The binding mode of 36 to these residues is similar to that of immepip, and the lipophilic tail of 36 has an additional interaction with a hydrophobic region in transmembrane helix 6 of the receptor. These results indicated that 36 served as a useful tool for studies on receptor-agonist interactions and drug design.

Full text of DOI:10.1021/jm100643t

J. Med. Chem. 2010, 53, 6445–6456 6445
DOI: 10.1021/jm100643t
Investigation of the Histamine H3 Receptor Binding Site. Design and Synthesis of
Hybrid Agonists with a Lipophilic Side Chain
Makoto Ishikawa,* Takashi Watanabe, Toshiaki Kudo, Fumikazu Yokoyama, Miki Yamauchi, Kazuhiko Kato,
Nobukazu Kakui, and Yasuo Sato
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
Received May 27, 2010
As a part of our search for novel histamine H3 receptor agonists, we designed and synthesized hybrid
compounds in which the lipophilic (40-alkylphenylthio)ethyl moiety of a novel H3 receptor agonist, 4-(2-(40-
tert-butylphenylthio)ethyl)-1H-imidazole (1), was incorporated into NR-methylhistamine, immepip, and
immethridine derivatives. These hybrid compounds were expected to interact concurrently with the
histamine-binding site and a putative hydrophobic region in the H3 receptor. Among them, piperidine- and
pyridine-type derivatives displayed partial agonist activity, and (S)-4-(1-(1H-imidazol-4-yl)-2-(4-(trifluoro-
methyl)phenylthio)ethyl)piperidine (36) was identified as a potent H3 agonist. We performed computational
docking studies to examine the binding mode of the agonists. The results indicated that immepip interacts with
the key residues, Asp114 and Glu206, in a different manner from histamine. The binding mode of 36 to these
residues is similar to that of immepip, and the lipophilic tail of 36 has an additional interaction with a
hydrophobic region in transmembrane helix 6 of the receptor. These results indicated that 36 served as a useful
tool for studies on receptor-agonist interactions and drug design.
Introduction
It has been suggested that the pharmacophore of general H3
agonists, such as histamine, NR-methylhistamine 2,1 immepip
3,11 and imetit 4,12 consists of an imidazole ring and a terminal
basic amine13 (Figure 1). The physiologically protonated imi-
dazole or the basic amine of these compounds is supposed to
interact with the aspartate residue (Asp114) of the H3 receptor
in transmembrane helix 3 (TM3), which is highly conserved in
G-protein-coupled receptors (GPCRs) for biogenic neurotrans-
mitter amines.14,15 Site-directed mutagenesis and a modeling
study of histamine indicated that the interaction between the
protonated N-atom of agonists and the glutamate residue
(Glu206) in TM5 plays a key role in agonist-induced activation
of the H3 receptor.15 Interestingly, H3 agonists of another type,
possessing a less basic side chain or highly lipophilic moiety in
place of the terminal basic nitrogen atom, have been discovered,
as represented by immethridine 5, UCL 1470 8, and our com-
pound 1.10,13,16-21 We have found that the introduction of an
alkylated benzene ring to the terminal nitrogen atom of hista-
mine, 4-(2-(N-(alkylphenyl)amino)ethyl)-1H-imidazole deriva-
tives, partially maintained the agonistic activity of histamine.10
Noticeably, para CF3 and para t-Bu substituents on the benzene
ring improved the H3 receptor affinity, and the alteration of its
aminoethyl spacer part to the thioether of 1 increased the ago-
nistic efficacy. On the contrary, the SAR study of 8 revealed that
the para CF3 or CN on the benzene ring of 4-(4-(alkylphenoxy)-
butyl)-1H-imidazole derivatives dramatically decreased the ago-
nistic efficacy and provided antagonists, though meta CF3
substitution yielded a full agonist.19 It seems that the subtle
structural divergence of the lipophilic moieties of these ligands
greatly impacts their agonistic activities. They presumably acti-
vate the H3 receptor through hydrophobic interaction between
their lipophilic moiety and an unidentified hydrophobic region
of the receptor. We considered that hybridizing the pharmaco-
phore components of these different types of H3 agonists would
Histamine receptors (H1-H4a) are involved in various biolo-
gical pathways in both central and peripheral systems. Among
these receptors, H3 receptor was discovered as an inhibitory
autoreceptor by Arrang et al. in 1983, using classic pharmaco-
logical approaches.1 This receptor regulates the synthesis and
release of histamine in the central nervous system (CNS).2,3 It
behaves as a presynaptic heteroreceptor, modulating the release
of several important neurotransmitters, including serotonin,4
noradrenaline,5 dopamine,6 and acetylcholine.7 Specific ligands
for the H3 receptor are expected to have potential for treating
various CNS disorders.8,9 We recently reported a novel H3
receptor agonist 1 that possesses a lipophilic (40-tert-butylphe-
nylthio)ethyl moiety at the 4 position of the imidazole ring
(Figure 1).10 Compound 1 exhibited high affinity and potent
agonistic activity for the H3 receptor and showed a good phar-
macokinetic profile following oral administration in rodents. It
also showed a remarkable in vivo efficacy in the resident-
intruder aggression model, an atypical antidepressant effective
animal model of anxiety.10 We expect that H3 agonists would
offer an effective therapeutic option for the treatment of some
forms of anxiety disorders in which commonly prescribed
selective serotonin reuptake inhibitors (SSRIs) have not exerted
satisfactory therapeutic efficacy.9
*To whom correspondence should be addressed. Phone: þ81-45-541-
2521. Fax: þ81-45-541-0667. E-mail: makoto_ishikawa@meiji.co.jp.
a Abbreviations: CADD, computer-assisted computational drug de-
sign; CHO, Chinese hamster ovary; CNS, central nervous system; CYP,
cytochrome P450; DIBAL-H, diisobutylaluminum hydride; ECL, ex-
tracellular loop; GPCRs, G-protein-coupled receptors; H1, histamine
type 1; H2, histamine type 2; H3, histamine type 3; H4, histamine type 4;
ia, intrinsic activity; TM, transmembrane helix; RAMH, (R)-R-methyl-
histamine; [35S]GTPγS, [35S]guanosine 50-[γ-thio]triphosphate; SSRIs,
selective serotonin reuptake inhibitors.
r
2010 American Chemical Society
Published on Web 08/06/2010
pubs.acs.org/jmc

6446 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ishikawa et al.
Figure 1. H3 receptor agonists.
which were treated with NsNHBoc under Mitsunobu condi-
tions27 to provide Boc-protected Ns-amides 13a,b. Selected
deprotection of Boc group and subsequent methylation gave
N-methyl Ns-amides 14a,b. Deprotection of Ns group using 4-t-
Bu-PhSH and Cs2CO3 provided the target compounds 15 and
16. 4-t-Bu-PhSH was used instead of unsubstituted thiophenol
because of its reduced odor.
The synthesis of piperidine derivatives 23 and 24 is summa-
rized in Scheme 2. Dehydrative condensation between 4-cya-
nomethylimidazole 17 and N-Boc-4-piperidone 18 under basic
conditions gave olefin 19, which was reduced by catalytic hydro-
genation. Protection of imidazole provided 20. Hydrolysis of cya-
nide and reduction of the generated carboxylic group with BH3
gave alcohol 22. This alcohol was converted to the mesylate, then
coupled with substituted sodium thiophenates (generated from
sodium hydride and substituted thiophenols) to give thioethers.
Exposure of the thioethers to acidic conditions gave 23 and 24.
The synthesis of pyridine derivatives 30-35 was started from
the reported alcohols 25a-c16 (Scheme 3). The alcohols 25a-c
were oxidized with MnO2 to give ketones, which were converted
to epoxides 26a-c under Corey-Chaykovsky epoxidation con-
ditions.28 Reduction of epoxides 26a-c by catalytic hydrogena-
tion provided alcohols 27a-c. These alcohols 27a-c were con-
verted to the thioethers as described above, and treatment with 5
N HCl afforded the desired pyridine derivatives 30-35.
Enantiomer Separation and Determination of Absolute Con-
figuration. The enantiomers of 23 were separated by preparative
liquid chromatography on a Chiralpak AD-H 20 mm ꢀ 250 mm
column with a solution of 0.1% diethylamine-MeOH. The
eluate was analyzed by means of HPLC on a AD-H 4.6 mm ꢀ
150 mm column with the same solution (flow rate, 1 mL/min).
The retention times of the obtained enantiomers (S)-36 and (R)-
37 were 4.07 and 6.14 min, respectively, under the conditions
used. Crystals of each enantiomer were obtained as the 1 TsOH
salt from isopropanol-Et2O using the vapor phase diffusion
technique.29 The absolute configuration of each enantiomer was
identified by X-ray crystallography (Supporting Information pp
S3 and S4).
Figure 2. Target hybrid molecules.
afford a novel H3 agonist and provide information about the
recognition of agonist structure by the H3 receptor.
Herein we report the design, synthesis, and evaluation of
novel H3 receptor agonists that interact with both the hista-
mine-binding site and a hydrophobic region of the H3 recep-
tor. We also performed computational docking of one of the
new agonists, 36, with the homology-modeled human H3
receptor in order to examine the binding mode.
Compound Design
H3 agonists, such as histamine, 2, and 3, which possess a basic
moiety in the side chain, are physiologically protonated and
thought to activate the receptor through interaction with both
the aspartate residue (Asp114) in TM 3 and glutamate (Glu206)
in TM 5.14,15 The less basic pyridine derivative 5(thepKa value of
the pyridine in 5 was 5.53, calculated with SPARC online
calculator22,23) activates the receptor equipotently with 3, though
the binding mode of 5 has not been established.16 We anticipated
that introduction of the lipophilic part of 1, ethylthio-(4-alkyl)-
phenyl, into these agonists would generate novel compounds
that would have an additional interaction with a hydrophobic
region of the H3 receptor. To test this idea, we planned to intro-
duce the liphophilic part of 1 into three types of agonist: 2, 3, and
immethridine derivatives 5, VUF4857 6, and VUF4886 7.16 The
lipophilic side chains were introduced to the R-position of the
side chain attached to imidazole ring of the agonists (Figure 2).
Chemistry
Pharmacology
The synthesis of 15 and 16 is outlined in Scheme 1. 2-(1-Tosyl-
1H-imidazol-4-yl)acetate methyl ester 924 was converted to a
Mannich intermediate using Eschenmoser’s salt.25 Then treat-
ment with MeI gave R-methylene ester 10 via elimination of an
ammonium salt.26 Michael addition of substituted thiophenol to
methylene product 10 under basic conditions gave thioether
derivatives 11a,b. The methyl ester of compounds 11a,b was
reduced with DIBAL-H and NaBH4 to give alcohols 12a,b,
Binding Affinity and Functional Activity at Human Hista-
mine H3 Receptor. Affinity of the synthesized compounds
for the human histamine H3 receptor was measured in terms
of displacement of [3H]-(R)-R-methylhistamine binding to mem-
branes of CHO cells expressing the human H3 receptor.30
Functional activities of these ligands were measured by means
of [35S]GTPγS binding assay to membranes of CHO cells ex-
pressing the human H3 receptor.31 Intrinsic activity (ia) is the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6447
Scheme 1. Synthetic Pathway for 15 and 16a
a Reagents and conditions: (i) LiHMDS, H2CdNþMe2I-, THF, 0 °C to room temp; ii) MeI, MeOH, room temp; (iii) 4-R1-PhSH, t-BuOK, THF,
room temp; (iv) DIBAL-H, CH2Cl2, -78 to 0 °C; (v) NaBH4, MeOH, room temp; (vi) NsNHBoc, DEAD, PPh3, THF, room temp; (vii) TFA-H2O,
room temp, evap, then K2CO3, MeI, DMF, room temp; (viii) 4-t-Bu-PhSH, Cs2CO3, CH3CN, room temp.
Scheme 2. Synthetic Pathway for 23 and 24a
a Reagents and conditions: (i) KOH, MeOH, 60 °C; (ii) Pd/C, H2, EtOAc, roomtemp;(iii) Ph3CCl, Et3N, CH2Cl2, room temp; (iv) KOH aq, 2-ethoxyethanol,
100 °C; (v) BH3 THF, THF, room temp; (vi) MeSO2Cl, Et3N, CH2Cl2, 0 °C; (vii) 4-R1-PhSH, NaH, DMF, 50 °C; (viii) 4 N HCl-dioxane, 90 °C.
3
ratio of the maximum response to each ligand to the maximum
response to (R)-R-methylhistamine (RAMH).
used this structure as the template for constructing activated
form of human H3 receptor. Receptor model construction and
docking simulation were performed with the Discovery Studio
2.5 from Accelrys, Inc. (San Diego, CA). Molecular mechanics
calculations were performed using DS CHARMm module and
using the CHARMm force field. The human H3 receptor model
was developed using homology modeling techniques in the DS
MODELLER module using the crystal structure of bovine
opsin as a template (Protein Data Bank code 3CAP).32 The
sequence of the human histamine H3 receptor was taken from
GenBank (accession code BAB17030). The sequence alignment
between human H3 receptor and bovine opsin was performed
based on the results published by Baldwin et al.33 In docking
simulations, ligands with the most basic moiety were manually
placed in the active site to interact with Glu206 in TM5 and/or
Cytochrome P450 (CYP) Inhibitory Activity. Inhibitory
activity of synthesized compounds toward CYPs was measured
as the concentration giving 50% inhibition (IC50) of the meta-
bolism of CYP-specific substrates (CYP1A2, 3-cyano-7-ethox-
ycoumarin; CYP2C19, dibenzylfluorescein; CYP2D6, 3-[2-(N,
N-diethylamino)ethyl]-7-methoxy-4-methylcoumarin; CYP3A4,
7-benzyloxy-4-(trifluoromethyl)coumarin) (Supporting Infor-
mation p S5).
Computational Chemistry
Modeling. The crystal structure of ligand-free GPCR opsin
obtained at low pH has been reported and is thought to
represent an activated form of the receptor.32 In this study, we

6448 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Scheme 3. Synthetic Pathway for 30-35a
Ishikawa et al.
a Reagents and conditions: (i) MnO2, 1,2-dichloroethane, 50 °C; (ii) Me3SþI-, NaH, DMSO, 50 °C; (iii) Pd/C, H2, EtOAc-AcOH, room temp;
(iv) MeSO2Cl, Et3N, CH2Cl2, 2 °C; (v) 4-R1-PhSH, NaH, DMF, room temp; (vi) 5 N HCl aq, 80 °C.
Table 1. Affinity and Functional Activity at Human H3 Receptor of the Synthesized Compounds
a Mean of two experiments. b Results are presented as the mean ( SEM of at least three independent experiments. c Intrinsic activity, being the ratio of
the maximum response to each ligand to the maximum response to RAMH.
Asp114 in TM3 and minimized in the receptor model together
with surrounding amino acid residues.
The original agonist 1, 3, and 5 exhibited high affinity and
agonistic activity (Ki =0.43 nM, EC50 =0.75 nM, and ia=
76%; Ki=0.30 nM, EC50=0.67 nM, and ia=82%; Ki=0.77
nM, EC50=1.1 nM, and ia=94%; respectively). N-Methyl
derivatives 15 and 16 showed no affinity for the receptor.
In contrast, piperidine and pyridine derivatives (23,24 and
Results and Discussion
The affinity and functional activity at human H3 receptor
of the newly synthesized compounds are presented in Table 1.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6449
Table 2. Affinity and Functional Activity of the Two Enantiomers of 23 at Human H3 Receptor
a Results are presented as the mean ( SEM of at least three independent experiments. b Intrinsic activity, being the ratio of the maximum response of
each ligand to the maximum response of RAMH.
30-35, respectively) showed moderate to high affinity and
agonistic activity. Of the piperidine derivatives, para-CF3-
substituted compound 23 had higher binding affinity and ago-
nistic activity (Ki=18 nM, EC50=38 nM, and ia=76%) than
the para t-Bu substituted compound 24 (Ki=530 nM, EC50=
540 nM, and ia=65%). As for the pyridine derivatives, the
bulky para t-Bu substituent was somewhat preferable to CF3.
The substituent effect in pyridine-type compounds seems tobe
different from that in piperidine-type compounds. Among
pyridines with the same substituent on the benzene ring,
the rank order of binding affinity and agonistic efficacy was
2-pyridyl derivatives (30 and 31)>3-pyridyl derivatives (32
and 33) > 4-pyridyl derivatives (34 and 35). Intriguingly, the
original agonist7, 6, and 5 showed the reverse order of binding
affinity and agonistic efficacy: 4-pyridyl (5)>3-pyridyl (6) >
2-pyridyl (7).16 The binding mode of the hybrid pyridine deri-
vatives in the receptor is therefore likely to be different from
that of the original 5 derivatives. Among the pyridine deriva-
tives, 31 showed the highest binding affinity and EC50 (Ki =
16 nM, EC50=15 nM). The 2-pyridyl derivatives 30 and 31
and 3-pyridyl derivative 33 possessed comparatively high
intrinsic activity (95%, 86%, and 91%, respectively).
Neither the H3 receptor affinity nor agonistic efficacy of the
hybridized compounds (23,24 and 30-35) was superior to those
of the original agonists 1, 3, and 5. This result indicates that
the newly introduced each substructure did not contribute to the
efficiency of the ligands.34 The branched structure of the hybrid
agonists is thought to cause steric hindrance in the H3 receptor
binding site, which might reduce the biding affinity and agonistic
potency. The piperidine and pyridine derivatives possess rigid
ring structure and might serve to keep the basic moiety and the
lipophilic moiety in appropriate locations to interact with the
human H3 receptor. The amino side chain of N-methyl deriva-
tives is shorter and more flexible than the piperidine and the
pyridine moiety. These structural features of N-methyl deriva-
tives would not be suitable to make optimal interaction with the
H3 receptor.
functional activity than the (R)-enantiomer 37. In particular,
the binding affinity of (S)-36 exceeded that of (R)-37 by a
factor of over 13-fold. This result indicates that the human H3
receptor strictly recognizes the enantiomeric structure of
ligands for interaction with the three-point binding site.
In order to examine further the binding mode of the ago-
nists, we performed docking studies of histamine, 3, (S)-36, and
1 into the active site of the homology-modeled human histamine
H3 receptor, which was built based on the crystal structure of
bovine opsin (Figures 3 and 4). On the basis of the previous
report,15 the extended histamine molecule showed interaction
between the imidazole moiety and Glu206 in TM 5 and between
the basic nitrogen and Asp114 in TM3 (Figure 3A). These
interactions are considered to be critical for receptor activation.
Additionally, the imidazole ring was located in a hydrophobic
pocket near Glu206, consisting of aromatic amino acid residues
(Tyr115 in TM3, Tyr189 in extracellular loop 2 (ECL2), Trp371
in TM6, and Tyr374 in TM6). Although there is space between
the imidazole ring and the hydrophobic pocket, this hydropho-
bic interaction should stabilize histamine binding in the active
site. In the case of 3, the same binding orientation, with imida-
zole proximate to Glu206 and the basic nitrogen proximate to
Asp114, results in steric repulsion between the piperidine ring of
3 and Leu401 in TM7 (Figure 3B). This steric repulsion pushes 3
toward Tyr115 (Figure 3D), destabilizing the binding. On the
other hand, 3 was more stably docked into the receptor in a
reverse binding mode (Figure 3C), in which imidazole interacts
with Asp114 and basic nitrogen interacts with Glu206. In this
case, the piperidine ring was tightly fitted into the hydrophobic
pocket formed by the aromatic amino acid residues, resulting in
a favorable hydrophobic interaction. Thus 3, which is confor-
mationally restricted by the piperidine ring, appears to bind to
the homology-modeled H3 receptor in the reverse orientation
compared with histamine. Although this result should be verified
by means of experimental studies, such as site-directed mutagen-
esis, the calculated binding modes of histamine and 3 in the
receptor appear plausible.
The binding affinity and functional activity at human H3
receptor of the enantiomers (S)-36 and (R)-37 are shown in
Table 2. The (S)-enantiomer 36 exhibited higher affinity and
(S)-36 bound to the H3 receptor through imidazole-
Asp114 interaction and basic nitrogen-Glu206 interaction
(Figure 4). The lipophilic side chain of (S)-36 extended toward

6450 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ishikawa et al.
Figure 3. Homology models of human histamine H3 receptor. (A) Modeled binding mode of histamine in the receptor binding site. The
imidazole ring interacts with Glu206, and the basic nitrogen interacts with Asp114. (B) Modeled binding mode of 3 in the receptor binding
site (unstable model). (C) Modeled binding mode of 3 in the receptor binding site (stable model). The imidazole ring interacts with Asp114,
and the basic nitrogen interacts with Glu206. (D) Overlay of the unstable immepip binding model (blue) and histamine binding model (red).
The steric repulsion between the piperidine ring and Leu401 pushed 3 toward Tyr115. Only ligands, Leu401 and Try115 are depicted.
with the active GPCR state. Thus, hydrophobic interaction
between the liphophilic side chain of (S)-36 and the hydropho-
bic region in TM6 is speculated to participate in the recep-
tor activation. However, our model indicated that the bulky
branched (S)-36 developed some steric repulsion in the H3
receptor binding site. This might reduce the biding affinity and
agonistic potency of (S)-36, compared to 3. The(R)-enantiomer
37 is supposed to bind to the receptor as same way as (S)-36, but
the lipophilic tail of (R)-37 is thought to be more distant from
the hydrophobic region in TM6 compared with that of (S)-36,
and this unfavorable orientation should reduce the binding
affinity to the receptor. Both the lipophilic tail and the piper-
idine ring of (S)-36 have the same direction in the receptor, and
the lipophilic tail is pinched between the piperidine ring and the
hydrophobic region in TM6. In this constrained binding mode,
bulky substituents at the 4-position of the benzene ring is likely
to cause steric repulsion between the hydrophobic region in
Figure 4. Modeled binding mode of 36 (red) and 1 (green) in the
human H3 receptor binding site from the docking model with 36 is
TM6, and this is presumably the reason why the binding affinity
of t-Bu-substituted 24 is declined.
depicted. The terminal lipohilic moieties of ligands interact with the
hydrophobic region in TM6.
The newly synthesized pyridine derivatives are thought to bind
to the receptor similarly to (S)-36. In this case, the pyridine ring is
close to Glu206 in the binding site of the receptor. The nitrogen
atom of the pyridine ring has a weak negative charge because the
pyridine nitrogen is not likely to be protonated under physiolo-
gical conditions. (The calculated pKa values22,23 of the pyridine in
2-pyridyl 31, 3-pyridyl 33, and 4-pyridyl 35 were 3.15, 4.56, and
5.32, respectively.) In the case of the 4-pyridine derivatives, the
a hydrophobic region comprised Trp371, Tyr374, Thr375,
and Met378 in TM 6, which is one of the key segments for
GPCR activation. When bovine rhodopsin is compared with
active bovine opsin, the whole TM6 region of opsin is moved
slightly away from the center bundle and the cytoplasmic
segment in TM6 is tilted outward so that it runs almost
parallel to TM5.32 These structural changes are associated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6451
Table 3. Binding Affinity of Selected Compounds for Human Hista-
mine H1, H2, H3, and H4 Receptors
Table 4. CYP-Inhibitory Activity of Selected Compounds
CYP IC50 a (μM)
human histamine receptor
binding (Ki, nM)
compd
1A2
2C19
12
1.4
2D6
3A4
23
24
31
34
1
>94
>100
5.1
9.5
3.6
17
6.5
H1(Ki) /
H3(Ki)
H4(Ki) /
H3(Ki)
compd H3
H1a
H2a
H4a
<0.010
0.055
0.0085
0.82
4.3
0.27
0.35
0.47
31
33
34
16
64
>10000 >10000 840
>10000 >10000 360
>620
>150
2.7
53
8.6
0.68
5.6
0.085
93
0.68
200 540
280
(R)-37 180 48
>10000 17
>10000 1300
>10000 310
a Mean of two experiments.
(S)-36 14
20
0.27
1.7
high affinity for the H4 receptor (H1(Ki)/H3(Ki) = 2.7, H4(Ki)/
H3(Ki) = 0.085). Of the enantiomers (S)-36 and (R)-37, (S)-36
exhibited good selectivity for the H3 receptor (H1(Ki)/H3(Ki) =
20, H4(Ki)/H3(Ki) = 93). (R)-37 had moderate affinity for the
H1 and H4 receptors. The position of the basic N atom and the
enantiomeric configuration appear to be important determi-
nants of selectivity.
a Mean of two experiments.
pyridine nitrogen atom is located close to the deprotonated
carboxylic acid of Glu206. This may result in weak electrostatic
repulsion, which would disfavor binding. Consequently, the
binding affinity of 34 and 35 is inferior to that of other pyridine
derivatives. Compared to the piperidine derivatives, the pyridine
ring has a flat conformation, which is thought to alleviate steric
repulsion involving the lipophilic tail. This may be the reason why
t-Bu on the benzene ring interacts with the hydrophobic region in
TM6 more favorably than does CF3.
In the case of N-methyl derivatives 15 and 16, steric repulsion
would be generated between their lipophilic side chains and
TM3 if they take the similar binding mode of histamine, in
which the imidazoles and the N-methylamines are placed
proximate to Glu206 and Asp114, respectively. 15 and 16 were
able to bind to the receptor without generating such repulsion if
they took the binding mode of (S)-36. However, in this mode of
binding, the somewhat short N-methylamino moieties cannot
form effective three point interaction (Figure S3, see Supporting
Information p S7), interaction with Glu206, Asp114, and some
hydrophobic region in the receptor. Our model suggested that
this might be one of the fundamental reasons for the loss of
affinity of the N-methyl derivatives to the H3 receptor.
In the docking model, 1bound to the H3 receptor through the
interaction of imidazole with Asp114, and the peripheral lipo-
philic tail with the hydrophobic region in TM6 (Figure 4).
Although 1did not interact with Glu206, its molecular flexibility
may allow it to take a suitable conformation for strong hydro-
phobic interaction between the lipophilic tail and the hydro-
phobic region in TM6 without steric repulsion, resulting in high
binding affinity for the receptor. This two-point interaction
appears to be sufficient to induce receptor activation and is con-
sistent with the hypothesis of De Esch et al.21 On the basis of a
CADD (computer-assisted computational drug design) ap-
proach, they predicted that a conformationally constrained ago-
nist, which contained imidazole as the only basic moiety and
oxazoline as the lipophilic moiety, would interact with Asp114
through the imidazole moiety and with a putative hydrophobic
pocket via the peripheral lipophilic group. The results of our
docking study suggest that the hydrophobic region comprising
Trp371, Tyr374, Thr375, and Met378 in TM6 adjacent to the
histamine-binding site has a key role in the receptor activation
induced by agonists such as (S)-36 and 1.
The binding profiles of selected compounds with other hu-
man histamine receptor congeners, H1, H2, and H4, were next
examined (Table 3). None of the evaluated compounds showed
affinity for human H2 receptor. Among the pyridine derivatives,
2-pyridyl derivative 31 exhibited the highest selectivity for the
H3 receptor. The 3-pyridyl derivative 33 did not show affinity
for the H1 receptor but possessed moderate affinity for the H4
receptor, resulting in low selectivity for the H3 receptor (H4(Ki)/
H3(Ki) = 5.6). The 4-pyridyl derivative 34 showed relatively
Imidazole-based drug candidates may have the potential to
inhibit the function of cytochrome P450 (CYP) isoforms, owing
to imidazole nitrogen complexation to heme iron in the active
site of the enzyme.35 Since these enzymes play roles in the meta-
bolism of many medicines, inhibitors of CYPs may cause
drug-drug interactions by reducing or blocking the clearance
of coadministered medicines.36,37 Therefore, we tested selected
compounds for inhibitory activity toward important CYP iso-
forms (Table 4). The lead compound 1 had an unfavorable
profile of CYP inhibition. Compounds 31 and 34, which contain
a pyridine ring, also exhibited strong CYP-inhibitory ability,
especially toward CYP2C19 and CYP3A4. In contrast, the
piperidine derivatives 23 and 24 showed weaker inhibition.
Reducing the lipophilicity by introducing a piperidine side chain
has been reported to suppress CYP-inhibitory activity of H3
antagonists.38 The IC50 values of 23 for CYPs are in the range
from 9.5 to >94 μM, differing by >500-fold from the Ki value
for human H3 receptor. Compound 23 and its enantiomers (36
and 37) seem unlikely to be involved in CYP-mediated drug-
drug interactions. Unfortunately, 23 showed no oral bioavail-
ability at preliminary pharmacokinetic profile study in mice.
Parallel artificial membrane permeation assay39of 23 indicated
that it had very low membrane permeability (data not shown),
which is possibly due to the polar nature of piperidine moiety of
23. This should be considered in further compound design.
Conclusion
Novel hybrid compounds, possessing an imidazole ring, a
basic moiety, and a lipophilic moiety, were designed and synthe-
sized as candidate H3 receptor ligands. Among them, piperidine
and pyridine derivatives showed partial agonist activity at hu-
man H3 receptor, and (S)-36 was identified as a potent H3 ago-
nist. To examine the binding mode of the agonists, computa-
tional homology-modeled docking studies with the human H3
receptor were performed. The results suggested that 3and (S)-36
bind to the histamine H3 receptor through interactions between
imidazole and Asp114 in TM3 and between the basic nitrogen
and Glu206 in TM5, whereas histamine binds in a reverse orien-
tation through interactions between the imidazole and Glu206
in TM5 and between the basic nitrogen and Asp114 in TM3.
The lipophilic tail of (S)-36 may have a hydrophobic interaction
with the hydrophobic region in TM6, comprising Trp371, Tyr-
374, Thr375, and Met378. Hydrophobic interaction with TM6 is
considered to play an important role in receptor activation.
From the viewpoint of clinical potential, the piperidine
derivative (S)-36 exhibited high selectivity for H3 receptor

6452 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ishikawa et al.
over other histamine receptor congeners and weak CYP-
inhibitory activity (as examined with the racemate 23). These
characteristics suggest that (S)-36 is not only a useful tool for
probing the receptor active site but also a good lead com-
pound for developing human H3 receptor agonists with
therapeutic potential for treating anxiety disorders.
(4.2 mL) was added DIBAL-H (1.0 M in toluene, 1.30 mL, 1.30
mmol) at -78 °C. The reaction mixture was stirred at the same
temperature for 30 min, then warmed to 2 °C and stirred for 15
min. The reaction was quenched with 1 N aqueous HCl (20 mL),
and the mixture was diluted with EtOAc (50 mL) and stirred at
room temperature for 10 min. The residue was partitioned, and
the obtained organic layer was washed with 1 N aqueous HCl
(20 mL), saturated aqueous NaHCO3 (20 mL), and brine (20
mL), successively, and dried (Na2SO4). After concentration of
the mixture under reduced pressure, the residue was dissolved in
MeOH (4 mL)-THF (2 mL). To the mixture was added NaBH4
(16 mg, 0.42 mmol) at 0 °C, and then the mixture was stirred at
room temperature overnight. The reaction was quenched with
saturated aqueous NH4Cl (5 mL), and the mixture was poured
into brine (20 mL). The mixture was extracted with EtOAc (50
mL), and the organic layer was dried (Na2SO4), evaporated, and
purified with SiO2 column chromatography (EtOAc/hexane =
Experimental Section
NMR spectra were obtained on JEOL GX-400 FT-NMR
spectrometers. The following abbreviations were used: s=singlet,
d=doublet, t=triplet, q=quartet, m=multiplet, and br=broad.
Mass spectra were measured with Hitachi M-80B, Agilent HP-
5989A, and JEOL JMS-700 instruments. Elemental analysis data
were obtained on a VarioEL (Elementar). Compounds 1, 3, 5, 6,
and 7 were synthesized using procedures earlier described.10,11,16
Column chromatography was carried out using silica gel 60N 40-
50 μm (Kanto Chemical) or NH-chromatography silica gel 100-
200 mesh (Fulisilysia Chemical) or Sephadex LH-20 (Amersham
Biosciences). Thin layer chromatography was performed on silica
gel 60 F254 plates (Merck) or NH TLC plates (Fulisilysia
Chemical). All final compounds have a purity of >95% (see
Supporting Information for details). Profiling of binding affinity
for receptors other than H3R was done by MDS Pharma Services.
Chemistry. Methyl 2-(1-Tosyl-1H-imidazol-4-yl)-3-(4-(trifluo-
romethyl)phenylthio)propanoate (11a). 11a was synthesized as a
colorless oil, using the procedure for 11b from 9 and 4-trifluor-
1
1:1, then 3:2) to give 12b as a colorless oil (134 mg, 71%). H
NMR (CDCL3) δ 7.90 (d, 1H, J = 1.2 Hz), 7.83 (d, 2H, J = 8.5
Hz), 7.35 (d, 2H, J = 8.5 Hz), 7.29-7.23 (m, 4H), 7.14 (m, 1H),
3.90 (m, 2H), 3.18 (dd, 1H, J = 7.3, 13.0 Hz), 3.15 (dd, 1H, J =
7.0, 13.0 Hz), 2.99-2.93 (m, 2H), 2.44 (s, 3H), 1.32 (s, 9H); MS
(FABþ) m/z 445 (M þ H)þ.
tert-Butyl (2-Nitrophenyl)sulfonyl(2-(1-tosyl-1H-imidazol-4-yl)-
3-(4-(trifluoromethyl)phenylthio)propyl)carbamate (13a). 13a
was synthesized as a colorless oil, using the procedure for 13b
from 12a (99%). 1H NMR (CDCL3) δ 8.25 (m, 1H), 7.92 (d, 1H,
J = 1.2 Hz), 7.82 (d, 2H, J = 8.5 Hz), 7.80-7.72 (m, 3H), 7.45
(d, 2H, J = 8.8 Hz), 7.36 (d, 2H, J = 8.0 Hz), 7.30 (d, 2H, J =
8.0 Hz), 7.18 (d, 1H, J = 1.2 Hz), 4.13 (m, 1H), 4.02 (dd, 1H, J =
6.1, 14.4 Hz), 3.42 (m, 1H), 3.37-3.32 (m, 2H), 2.43 (s, 3H), 1.24
(s, 9H); MS (FABþ) m/z 741 (M þ H)þ.
1
omethylbenzenethiol (25%). H NMR (CDCL3) δ 7.91 (d, 1H,
J = 1.2 Hz), 7.83 (ddd, 2H, J = 1.9, 1.9, 8.5 Hz), 7.48 (d, 2H, J =
8.3 Hz), 7.37 (d, 2H, J = 8.3 Hz), 7.35 (d, 2H, J = 8.5 Hz), 7.21 (m,
1H), 3.87 (dd, 1H, J = 6.6, 8.3 Hz), 3.69 (s, 3H), 3.52 (dd, 1H, J =
8.3, 13.6 Hz), 3.43 (dd, 1H, J = 6.6, 13.6 Hz), 2.45 (s, 3H); MS
(FABþ) m/z 485 (M þ H)þ.
tert-Butyl (3-(4-tert-Butylphenylthio)-2-(1-tosyl-1H-imidazol-
4-yl)propyl)-(2-nitrophenylsulfonyl)carbamate (13b). To a solu-
tion of 12b (133 mg, 0.30 mmol), PPh3 (118 mg, 0.45 mmol), and
tert-butyl 2-nitrophenylsulfonylcarbamate (109 mg, 0.36 mmol)
was added DEAD (2.2 M in toluene, 0.20 mL, 0.44 mmol), and
the mixture was stirred at room temperature for 10 min, then
50 °C for 20 min. After the evaporation of the solvent, the
resulting residue was purified with SiO2 column chromatogra-
phy (EtOAc/hexane = 2:3) to give 13b as a colorless oil (133 mg,
61%). 1H NMR (CDCL3) δ 8.22 (m, 1H), 7.89 (d, 1H, J = 1.0
Hz), 7.80 (d, 2H, J = 8.3 Hz), 7.74-7.69 (m, 3H), 7.34 (d, 2H,
J = 8.3 Hz), 7.26-7.18 (m, 5H), 4.09 (m, 1H), 4.06 (dd, 1H, J =
7.3, 14.6 Hz), 3.36-3.23 (m, 3H), 2.42 (s, 3H), 1.31 (s, 9H), 1.27
(s, 9H); MS (FABþ) m/z 729 (M þ H)þ.
Methyl 3-(4-tert-Butylphenylthio)-2-(1-tosyl-1H-imidazol-4-
yl)propanoate (11b). LiHMDS (1.6 M in THF, 1.30 mL, 2.08
mmol) was added to a solution of 2-(tosyl-1H-imidazol-4-yl)-
acetate methyl ester 9 (460 mg, 1.56 mmol) in THF (7.8 mL) at
0 °C, and the mixture was stirred at the same temperature for
30 min. To the mixture was added N,N-dimethylmethyleneam-
monium iodide (580 mg, 3.13 mmol) at 0 °C, and the mixture
was stirred for 20 min, then at room temperature for 10 min. The
reaction mixture was poured into brine (30 mL) and extracted
with CHCl3 (50 mL ꢀ 3). The organic layer was dried (Na2SO4)
and evaporated. The residue was dissolved in MeOH (6 mL),
and MeI (0.90 mL, 14.4 mmol) was added. After being stirred at
room temperature for 1 h, the reaction mixture was poured into
brine (30 mL) and extracted with EtOAc (50 mL). The organic
layer was dried (Na2SO4), evaporated, and the residue was
dissolved in THF (7 mL). To the mixture was added t-BuOK
(18 mg, 0.16 mmol) and 4-tert-butylbenzenethiol (0.26 mL, 1.56
mmol) successively, and the mixture was stirred at room tem-
perature for 10 min. The reaction mixture was diluted with
EtOAc (50 mL) and filtered by a short pad of Celite. The filtrate
was evaporated and purified with SiO2 thin layer chromatogra-
phy (EtOAc/hexane = 2:3) to give 11b as a colorless oil (199 mg,
27%). 1H NMR (CDCL3) δ 7.89 (d, 1H, J=1.2 Hz), 7.82 (d, 2H,
J=8.3 Hz), 7.37 (d, 2H, J=8.3 Hz), 7.25 (d, 4H, J=1.4 Hz),
7.19 (m, 1H), 3.84 (dd, 1H, J=6.2, 8.5 Hz), 3.67 (s, 3H), 3.42 (dd,
1H, J=8.5, 13.4 Hz), 3.42 (dd, 1H, J=6.2, 13.4 Hz), 2.43 (s, 3H),
1.30 (s, 9H); MS (FABþ) m/z 473 (M þ H)þ.
N-Methyl-N-3-(4-trifluoromethylphenylthio)-2-(1-tosyl-1H-imi-
dazol-4-yl)propyl-2-nitrobenzensulfonamide (14a). 14a was syn-
thesized as a colorless oil, using the procedure for 14b from 13a
1
(86%). H NMR (CDCL3) δ 7.94-7.80 (m, 2H), 7.80 (d, 2H,
J = 7.2 Hz), 7.69-7.63 (m, 2H), 7.57 (m, 1H), 7.44 (d, 2H, J =
7.8 Hz), 7.35 (d, 2H, J = 7.8 Hz), 7.29 (d, 2H, J = 7.3 Hz), 7.19
(s, 1H), 3.73 (dd, 1H, J = 5.1, 14.0 Hz), 3.43 (dd, 1H, J = 6.1,
14.0 Hz), 3.31 (m, 1H), 3.29-3.21 (m, 2H), 2.69 (s, 3H), 2.43 (s,
3H); MS (FABþ) m/z 655 (M þ H)þ.
N-3-(4-tert-Butylphenylthio)-2-(1-tosyl-1H-imidazol-4-yl)pro-
pyl-N-methyl-2-nitrobenzensulfonamide (14b). A solution of 13b
(132 mg, 0.18 mmol) in TFA (1.8 mL)-H2O (10 μL) was stirred
at room temperature for 2.5 h. After concentration of the
solvent, the residue was dissolved in DMF (1.8 mL). To the
solution was added K2CO3 (50 mg, 0.36 mmol) and MeI (17 μL,
0.27 mmol), successively, and the mixture was stirred at room
temperature overnight. Then the reaction mixture was poured
into H2O (20 mL) and extracted with EtOAc (50 mL ꢀ 2). The
organic layer was washed with brine, dried (Na2SO4), evapo-
rated, and purified with SiO2 thin layer chromatography
(EtOAc/hexane = 1:1) to give 14b as a colorless oil (88 mg,
77%). 1H NMR (CDCL3) δ 7.89 (dd, 1H, J = 1.7, 7.6 Hz), 7.89
(s, 1H), 7.88 (d, 2H, J = 8.8 Hz), 7.65 (dd, 1H, J = 1.7, 7.6 Hz),
7.62 (dd, 1H, J = 1.7, 7.4 Hz), 7.56 (dd, 1H, J = 1.7, 7.4 Hz),
2-(1-Tosyl-1H-imidazol-4-yl)-3-(4-trifluoromethylphenylthio)-
1-propanol (12a). 12a was synthesized as a colorless oil, using the
procedure for 12b from 11a (68%). 1H NMR (CDCL3) δ 7.92 (d,
1H, J=1.2 Hz), 7.83 (d, 2H, J=8.3 Hz), 7.45 (d, 2H, J=8.5 Hz),
7.35 (d, 2H, J=8.3), 7.27 (d, 2H, J=8.0), 7.14 (m, 1H), 3.90 (m,
2H), 3.34 (dd, 1H, J=7.1, 13.2 Hz), 3.28 (dd, 1H, J=7.3, 13.2
Hz), 3.19 (br s, 1H), 3.00 (m, 1H), 2.44 (s, 3H); MS (FABþ) m/z
457 (M þ H)þ.
3-(4-tert-Butylphenylthio)-2-(1-tosyl-1H-imidazol-4-yl)-1-pro-
panol (12b). To a solution of 11b (199 mg. 0.42 mmol) in CH2Cl2

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6453
7.34 (d, 2H, J = 8.8 Hz), 7.27 (ddd, 2H, J = 2.2, 2.2, 8.6 Hz),
7.19 (ddd, 2H, J = 2.2, 2.2, 8.6 Hz), 7.15 (d, 1H, J = 1.2 Hz),
3.66 (m, 1H), 3.43 (m, 1H), 3.19-3.13 (m, 3H), 2.64 (s, 3H), 2.43
(s, 3H), 1.24 (s, 9H); MS (FABþ) m/z 643 (M þ H)þ.
(m, 2H), 1.43 (s, 9H), 1.24 (m, 2H), 0.80 (m, 2H); MS (ESIþ) m/z
552 (M þ H)þ.
1-(tert-Butoxycarbonyl)-4-(2-hydroxy-(1-trityl-1H-imidazol-
4-yl)ethyl)piperidine (22). To a solution of 21 (4.14 g, 7.50 mmol)
in THF (75 mL) was added BH3 THF (1.2 M in THF, 25.0 mL, 30.0
2-(1H-Imidazol-4-yl)-N-methyl-3-(4-(trifluoromethyl)phenyl-
thio)propane-1-amine (15). 15 was synthesized as a colorless oil,
using the procedure for 16 from 14a (12%). 1H NMR (CDCL3) δ
7.58 (d, 1H, J = 1.0 Hz), 7.48 (d, 2H, J = 8.3 Hz), 7.34 (d, 2H, J =
8.3 Hz), 6.88 (s, 1H), 3.37 (dd, 1H, J = 7.0, 12.9 Hz), 3.28 (dd, 1H,
J = 6.6, 12.9 Hz), 3.16 (m, 1H), 2.98 (d, 2H, J = 6.3 Hz), 2.43 (s,
3H); MS (ESIþ) m/z 316 (M þ H)þ; HRMS (FABþ) calcd for
C14H17N3F3S (M þ H)þ 316.1095, found 316.1101.
3
mmol) at 0 °C, and the mixture was then warmed to room tempera-
ture. After the mixture was stirred at room temperature for 2 h, the
reaction was carefully quenched with H2O (10 mL). The resulting
mixture was poured into brine (100 mL) and extracted with EtOAc
(200 mL ꢀ 3). The combined organic layer was dried (Na2SO4) and
evaporated. The residue was dissolved in THF (20 mL) and treated
with N,N-dimethylethylendiamine (2.5 mL, 23.2 mmol). The gener-
ated precipitate was filtered off, and the filtrate was evaporated. The
residue was purified with SiO2 column chromatography (EtOAc/
hexane=2:3 then 3:2) to give 22 as a white solid (2.65 g, 65%). 1H
NMR (CDCl3) δ 7.78 (d, 1H, J = 1.4 Hz), 7.40-7.33 (m, 9H),
7.11-7.08 (m, 6H), 6.61 (s, 1H), 4.14-4.04 (m, 2H), 3.86-3.83
(m, 2H), 3.21 (m, 1H), 2.63-2.54 (m, 2H), 1.80-1.77 (m, 2H), 1.45
(s, 9H), 1.36 (m, 1H), 1.18-1.15 (m, 2H); MS (ESIþ) m/z 538
(M þ H)þ.
3-(4-tert-Butylphenylthio)-2-(1H-imidazol-4-yl)-N-methylpro-
pane-1-amine (16). To a solution of 14b (88 mg, 0.14 mmol) in
CH3CN (1.4 mL) was added Cs2CO3 (67 mg, 0.21 mmol) and
4-tert-butylbenzenethiol (28 μL, 0.17 mmol). Then the mixture
was stirred at room temperature for 20 min. After additional
adding of Cs2CO3 (67 mg, 0.21 mmol) and 4-tert-butylbenze-
nethiol (28 μL, 0.17 mmol), the reaction mixture was stirred for
1.5 h. The solvent was evaporated and the resulting residue was
purified with NH thin layer chromatography (MeOH:CHCl3 =
1:19) to give 16 as a colorless oil (7 mg, 17%). 1H NMR
(CDCL3) δ 7.55 (s, 1H), 7.33-7.23 (m, 4H), 6.87 (s, 1H), 3.28
(dd, 1H, J = 6.1, 12.4 Hz), 3.17-3.07 (m, 2H), 2.96-2.91 (m,
2H), 2.40 (s, 3H), 1.29 (s, 9H); MS (FABþ) m/z 303 (M)þ;
HRMS (FABþ) calcd for C17H26N3S (M þ H)þ 304.1847,
found 304.1852.
tert-Butyl 4-(Cyano(1H-imidazol-4-yl)methylene)piperidine-
1-carboxylate (19). To a solution of KOH (1.06 g, 18.9 mmol)
in MeOH (6.3 mL) was added 1-(tert-butoxycarbonyl)-4-piperi-
done 18 (1.25 g, 6.27 mmol) and 4-cyanomethylimidazole 17 (0.67
g, 6.26 mmol), and the mixture was stirred at 60 °C for 3 h. The
reaction mixture was poured into H2O (50 mL) and extracted with
EtOAc (100 mL ꢀ 2). The combined organic layer was washed with
brine (50 mL), dried (Na2SO4), and evaporated. The resulting
residue was purified with SiO2 column chromatography (EtOAc/
hexane = 1:1, then EtOAc) to give 19 as a white solid (1.25 g, 68%).
1H NMR (CDCl3) δ 7.67 (s, 1H), 7.23 (s, 1H), 3.61-3.58 (m, 2H),
3.53-3.50 (m, 2H), 3.09 (m, 2H), 2.76 (t, 2H, J = 5.8 Hz), 1.48 (s,
9H); MS (FABþ) m/z 289 (M þ H)þ.
4-(1-(1H-Imidazol-4-yl)-2-(4-(trifluoromethyl)phenylthio)ethyl)-
piperidine Hydrochloride Salt (23). 23 was synthesized as a
colorless syrup, using the procedure for 24 from 22 and 4-tri-
1
fluoromethylbenzenethiol (57% in three steps, 2 HCl salt). H
NMR (CD3OD) δ 8.28 (d, 1H, J = 1.5 Hz), 7.37 (d, 2H, J = 8.3
Hz), 7.19 (d, 2H, J = 8.3 Hz), 7.07 (d, 1H, J = 1.5 Hz), 3.39 (m,
1H), 3.31 (m, 1H), 3.28-3.10 (m, 2H), 2.92 (m, 1H), 2.81-2.73
(m, 2H), 1.99-1.90 (m, 2H), 1.50 (m, 1H), 1.30 (m, 1H), 1.16 (m,
1H); MS (ESIþ) m/z 356 (M þ H)þ; HRMS (FABþ) calcd for
C17H21N3F3S (M þ H)þ 356.1408, found 356.1409.
4-(2-(4-tert-Butylphenylthio)-1-(1H-imidazol-4-yl)ethyl)piperi-
din Hydrochloride Salt (24). To a solution of 22 (153 mg, 0.28
mmol) in CH2Cl2 (2.8 mL) was added Et3N (0.12 mL, 0.86
mmol) and MsCl (54 μL, 0.70 mmol) at 0 °C, and the mixture
was stirred at the same temperature for 30 min. The reaction was
quenched with H2O (1 mL), and the resulting mixture was
poured into brine (20 mL). The mixture was extracted with
EtOAc (50 mL), and the organic layer was dried (Na2SO4) and
evaporated to give the crude mesylate.
To a solution of 4-tert-butylbenzenethiol (96 μL, 0.57 mmol) in
DMF (2 mL) was added NaH (60 wt %, 23 mg, 0.57 mmol), and the
mixture was stirred at room temperature for 20 min. To the mixture
was added the above mesylate in DMF (2 mL), and the mixture was
stirred at room temperature for 20 min, then 50 °C for 2 h. The reac-
tion mixture was poured into saturated aqueous NH4Cl (20 mL)
and extracted with CHCl3 (50 mL ꢀ 3 times). The combined
organic layer was dried (Na2SO4), evaporated, and purified with
short SiO2 column chromatography (EtOAc/hexane = 1:9, then
1:4) to give the thioether. This obtained thioether was used the next
step without further purification.
tert-Butyl 4-(Cyano(1-trityl-1H-imidazol-4-yl)methyl)piperi-
dine-1-carboxylate (20). A suspension of 19 (1.42 g, 4.92 mmol)
and 10% Pd/C (1.35 g) in EtOAc (40 mL) was stirred at room
temperature under H2 atmosphere for 20 h. The reaction mixture
was filtered with a pad of Celite, and the obtained filtrate was
concentrated under reduced pressure. The residue was dissolved in
CH2Cl2 (40 mL), and to the solution was added Et3N (1.4 mL, 10.0
mmol) and TrCl (1.80 g, 6.46 mmol). The mixture was stirred at
room temperature for 2 h, and the reaction was quenched with H2O
(5 mL). The resulting mixture was poured into brine (50 mL) and
extracted with EtOAc (100 mL ꢀ 2). The combined organic layer
was dried (Na2SO4), evaporated, and purified with SiO2 column
chromatography (EtOAc/hexane = 2:3, then 1:1) to give 20 as a
white solid (2.75 g, quant). 1H NMR (CDCl3) δ 7.42 (d, 1H, J =
1.4 Hz), 7.36-7.33 (m, 9H), 7.14-7.11 (m, 6H), 6.83 (s, 1H),
4.15-4.10 (m, 2H), 3.76 (m, 1H), 2.66 (m, 2H), 2.15 (m, 1H),
1.66-1.63 (m, 2H), 1.45 (s, 9H), 1.40-1.38 (m, 2H); MS (ESIþ) m/
z 533 (M þ H)þ.
A mixture of above thioether in 4 N HCl-dioxane solution (1.5
mL) was stirred at room temperature for 10 min, then at 60 °C for 1
h. After additional adding of 4 N HCl-dioxane solution (1.5 mL),
the reaction mixture was stirred at 90 °C for 1 h. The solvent was
evaporated, and the obtained residue was purified with LH-20
(MeOH/CHCl3 = 1:1) to give 24 as a colorless syrup (46 mg, 71%
in three steps, 2 HCl salt). 1H NMR (CD3OD) δ 8.21 (s, 1H), 7.19
(d, 2H, J = 8.2 Hz), 7.08 (d, 2H, J = 8.2 Hz), 7.07 (s, 1H), 3.30-
3.24 (m, 2H), 3.19 (m, 1H), 3.06 (dd, 1H, J = 10.4, 14.0 Hz), 2.89-
2.70 (m, 3H), 1.95-1.82 (m, 2H), 1.46 (m, 1H), 1.30-1.12 (m, 2H),
1.10 (s, 9H); MS (ESIþ) m/z 344 (M þ H)þ; HRMS (FABþ) calcd
for C20H30N3S (M þ H)þ 344.2160, found 344.2162.
2-(1-(tert-Butoxycarbonyl)piperidin-4-yl)-2-(1-trityl-1H-imi-
dazol-4-yl)acetic Acid (21). A solution of 20 (2.75 g, 5.17 mmol)
in 2-ethoxyethanol (5 mL) was added to the 40% KOH aqueous
solution (20 mL), and the mixture was stirred at 100 °C over-
night. The cooling reaction mixture was adjusted to pH 6.5 with
1 N aqueous HCl and then diluted with CHCl3 (50 mL). The
mixture was filtered with a pad of Celite, and the filtrate was
extracted with CHCl3 (50 mL ꢀ 3). The organic layer was dried
(Na2SO4), evaporated, and purified with SiO2 column chroma-
tography (MeOH/CHCl3 = 1:19 to 1:5) to give 21 as a white
solid (1.88 g, 66%). 1H NMR (CDCl3) δ 7.30 (m, 10H), 7.00 (m,
6H), 6.56 (s, 1H), 3.91 (m, 2H), 3.13 (m, 1H), 2.35 (m, 1H), 1.94
N,N-Dimethyl-4-(2-(pyridin-2-yl)oxirane-2-yl)-1H-imidazole-
1-sulfonamide (26a). 26a was synthesized as a white solid, using
the procedure for 26c from 4-(hydroxyl(pyridine-2-yl)methyl)-N,N-
dimethyl-1H-imidazole-1-sulfoneamide 25a (12% in two steps). 1H
NMR (CDCl3) δ 8.62 (m, 1H), 7.87 (d, 1H, J=1.2 Hz), 7.77 (ddd,
1H, J=1.2, 8.0, 8.0 Hz), 7.57 (d, 1H, J=8.0 Hz), 7.36 (d, 1H, J=1.2
Hz), 7.29 (m, 1H), 3.78 (d, 1H, J = 6.0 Hz), 3.27 (d, 1H, J = 6.0
Hz), 2.86 (s, 6H); MS (ESIþ) m/z 295 (M þ H)þ.

6454 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ishikawa et al.
N,N-Dimethyl-4-(2-(pyridin-3-yl)oxirane-2-yl)-1H-imidazole-
1-sulfonamide (26b). 26b was synthesized as a white solid, using
the procedure for 26c from 4-(hydroxyl(pyridine-3-yl)methyl)-N,N-
dimethyl-1H-imidazole-1-sulfoneamide 25b (12% in two steps). 1H
NMR (CDCl3) δ 8.78 (dd, 1H, J = 0.7, 2.2 Hz), 8.62 (dd, 1H, J =
1.7, 4.8 Hz), 7.89-7.85 (m, 2H), 7.35 (ddd, 1H, J= 1.0, 4.8, 8.0 Hz),
7.08 (d, 1H, J = 1.4 Hz), 3.78 (d, 1H, J = 5.8 Hz), 3.14 (d, 1H, J =
5.8 Hz), 2.86 (s, 6H); MS (ESIþ) m/z 295 (M þ H)þ.
as a colorless oil, using the procedure for 29b from 27b and 4-tri-
fluoromethylbenzenethiol (63% in two steps). 1H NMR (CDCl3)
δ 8.55 (s, 1H) 8.53 (dd, 1H, J = 1.5, 4.9 Hz), 7.85 (d, 1H, J = 1.2
Hz), 7.74 (m, 1H), 7.50 (d, 2H, J = 8.8), 7.35 (d, 2H, J = 8.8), 7.25
(m, 1H), 6.97 (d, 1H, J = 0.7 Hz), 4.17 (t, 1H, J = 7.6 Hz), 3.84
(dd, 1H, J = 7.6, 13.2 Hz), 3.47 (dd, 1H, J = 7.6, 13.2 Hz), 2.82 (s,
6H); MS (FABþ) m/z 457 (M þ H)þ.
N,N-Dimethyl-4-(1-(pyridine-4-yl)-2-(4-trifluoromethylphenyl-
thio)ethyl)-1H-imidazole-1-sulfonamide (28c). 28c was synthe-
sized as a colorless oil, using the procedure for 29b from 27c and
4-trifluoromethylbenzenethiol (84% in two steps). 1H NMR
(CDCl3) δ 8.57 (dd, 2H, J = 1.7, 4.6 Hz), 7.85 (d, 1H, J = 1.2
Hz), 7.51 (d, 2H, J = 8.2 Hz), 7.35 (d, 2H, J = 8.2 Hz), 7.27 (dd,
2H, J =1.7, 4.6 Hz), 6.99 (s, 1H), 4.13 (t, 1H, J = 7.6 Hz), 3.80
(dd, 1H, J = 7.6, 13.2 Hz), 3.48 (dd, 1H, J = 7.6, 13.2 Hz), 2.82
(s, 6H); MS (EIþ) m/z 456 (M)þ.
N,N-Dimethyl-4-(2-(pyridin-4-yl)oxirane-2-yl)-1H-imidazole-
1-sulfonamide (26c). To a solution of 4-(hydroxyl(pyridine-4-yl)-
methyl)-N,N-dimethyl-1H-imidazole-1-sulfoneamide 25c (351 mg,
1.24 mmol) in 1,2-dichloroethane (12 mL) was added MnO2 (650
mg, 7.47 mmol), and the mixture was stirred at 50 °Cfor1.5h. Tothe
mixture was added MnO2 (110 mg, 1.26 mmol) additionally, and the
mixture was stirred at the same temperature for 30 min. The reaction
mixture was filtrated by a pad of Celite, and the filtrate was concen-
trated under reduced pressure. The residue was purified with short
SiO2 column chromatography (EtOAc) to give the ketone. The
obtained ketone was used for the next reaction without further
purification.
4-(2-(4-tert-Butylphenylthio)-1-(pyridine-2-yl)ethyl)-N,N-di-
methyl-1H-imidazole-1-sulfonamide (29a). 29a was synthesized
as a colorless oil, using the procedure for 29b from 27a and
4-tert-butylbenzenethiol (64% in two steps). 1H NMR (CDCl3)
δ 8.55 (ddd, 1H, J = 0.7, 1.7, 4.6 Hz), 7.81 (d, 1H, J = 1.2 Hz),
7.63 (ddd, 1H, J = 1.9, 7.8, 7.8 Hz), 7.30-7.25 (m, 5H), 7.15
(ddd, 1H, J = 0.9, 4.8, 7.8 Hz), 7.07 (dd, 1H, J = 0.7, 1.2 Hz),
4.33 (t, 1H, J = 7.6 Hz), 3.68 (d, 2H, J = 7.6H), 2.51 (s, 6H),
1.28 (s, 9H); MS (FABþ) m/z 445 (M þ H)þ.
To a solution of trimethylsulfonium iodide (415 mg, 1.88
mmol) in DMSO (3.5 mL) was added NaH (60 wt % in mineral
oil, 73 mg, 1.85 mmol), and the mixture was stirred at room
temperature for 10 min. To the mixture was added above ketone
in DMSO (3 mL), and the mixture was stirred at room tem-
perature for 30 min, then at 50 °C for 20 min. The reaction
mixture was poured into brine (50 mL) and extracted with
EtOAc (100 mL). The organic layer was dried (Na2SO4),
evaporated, and purified with SiO2 column chromatography
(MeOH/CHCl3 = 1:19) to give 26c as a white solid (282 mg,
77% in two steps). 1H NMR (CDCl3) δ 8.64 (dd, 2H, J = 1.7, 4.6
Hz), 7.89 (d, 1H, J = 1.2 Hz), 7.46 (dd, 2H, J = 1.7, 4.6 Hz), 7.14
(d, 1H, J = 1.2 Hz), 3.78 (d, 1H, J = 5.8 Hz), 3.07 (d, 1H, J =
5.8 Hz), 2.87 (s, 6H); MS (FABþ) m/z 295 (M þ H)þ.
4-(2-(4-tert-Butylphenylthio)-1-(pyridine-3-yl)ethyl)-N,N-di-
methyl-1H-imidazole-1-sulfonamide (29b). To a solution of 27b
(74 mg, 0.25 mmol) in CH2Cl2 (2.5 mL) was added Et3N (87 μL,
0.62 mmol) and MsCl (23 μL, 0.30 mmol) at 0 °C, and the mixture
was stirred at the same temperature for 10 min. The reaction was
quenched with H2O (3 mL), and the resulting mixture was poured
into brine (20 mL). The mixture was extracted with CHCl3
(50 mL ꢀ 3), and the combined organic layer was dried (Na2SO4)
and evaporated to give the crud mesylate.
To a solution of 4-tert-butylbenzenethiol (0.12 mL, 0.71 mmol) in
DMF (2 mL) was added NaH (60 wt %, 30 mg, 0.75 mmol), and the
mixture was stirred at room temperature for 20 min. To the mixture
was added the above mesylate in DMF (2 mL), and the mixture was
stirred at room temperature overnight. The reaction mixture was
poured into saturated aqueous NH4Cl (20 mL) and extracted with
CHCl3 (50 mL ꢀ 3). The combined organic layer was dried (Na2-
SO4), evaporated, and purified with SiO2 thin layer chromatography
(MeOH/CHCl3 = 1:19) to give 29b as a colorless oil (93 mg, 84% in
two steps). 1H NMR (CDCl3) δ 8.52 (d, 1H, J = 1.9 Hz), 8.49 (dd,
1H, J = 1.6, 4.8 Hz), 7.82 (d, 1H, J = 1.2 Hz), 7.70 (ddd, 1H, J =
1.9,1.9, 8.0 Hz), 7.31-7.00 (m, 5H), 7.00 (s, 1H), 4.16 (t, 1H, J = 7.6
Hz), 3.72 (dd, 1H, J= 7.6, 13.2 Hz), 3.40 (dd, 1H, J= 7.6, 13.2 Hz),
2.83 (s, 6H), 1.27 (s, 9H); MS (FABþ) m/z 445 (M þ H)þ.
4-(2-(4-tert-Butylphenylthio)-1-(pyridine-4-yl)ethyl)-N,N-di-
methyl-1H-imidazole-1-sulfonamide (29c). 29c was synthesized
as a colorless oil, using the procedure for 29b from 27c and
4-tert-butylbenzenethiol (7% in two steps). 1H NMR (CDCl3) δ
8. 54 (m, 2H), 7.82 (d, 1H, J = 1.5 Hz), 7.32-7.23 (m, 6H), 7.01
(m, 1H), 4.10 (t, 1H, J = 7.6 Hz), 3.67 (dd, 1H, J = 7.8, 13.4 Hz),
3.50 (dd, 1H, J = 7.8, 13.4 Hz), 2.85 (s, 6H), 1.31 (s, 9H); MS
(EIþ) m/z 444 (M)þ.
4-(2-Hydroxy-1-(pyridine-2-yl)ethy)-N,N-dimethyl-1H-imi-
dazole-1-sulfonamide (27a). 27a was synthesized as a colorless
oil, using the procedure for 27c from 26a (52%). 1H NMR
(CDCl3) δ 8.54 (ddd, 1H, J = 0.7, 1.7, 4.8 Hz), 7.85 (d, 1H, J =
1.4 Hz), 7.67 (ddd, 1H, J = 1.7, 7.6, 7.6 Hz), 7.25 (d, 1H, J = 7.6
Hz), 7.21 (ddd, 1H, J = 1.2, 4.8, 7.6 Hz), 7.06 (s, 1H), 4.29-4.22
(m, 3H), 2.85 (s, 6H); MS (ESIþ) m/z 297 (M þ H)þ.
4-(2-Hydroxy-1-(pyridine-3-yl)ethy)-N,N-dimethyl-1H-imi-
dazole-1-sulfonamide (27b). 27b was synthesized as a colorless
oil, using the procedure for 27c from 26b (42%). 1H NMR
(CDCl3) δ 8.55 (d, 1H, J=2.2 Hz), 8.53 (dd, 1H, J=1.7, 4.8 Hz),
7.89 (d, 1H, J=1.4 Hz), 7.67 (ddd, 1H, J=1.9, 1.9, 7.8 Hz), 7.28
(dd, 1H, J = 4.8, 7.8 Hz), 6.89 (s, 1H), 4.16 (m, 1H), 4.05 (m,
1H), 3.54 (m, 1H), 2.85 (s, 6H); MS (EIþ) m/z 296 (M)þ.
4-(2-Hydroxy-1-(pyridine-4-yl)ethy)-N,N-dimethyl-1H-imi-
dazole-1-sulfonamide (27c). A suspension of 26c (47 mg, 0.16
mmol) and 10% Pd/C (568 mg) in EtOAc (1.6 mL)-AcOH (80
μL) was stirred at room temperature under H2 atmosphere for
45 min. The reaction mixture was filtered by a pad of Celite, and
the filtrate was evaporated. The residue was purified with SiO2
thin layer chromatography (MeOH/CHCl3 = 1:9) to give 27c as
a white solid (40 mg, 84%). 1H NMR (CDCl3) δ 8.56 (dd, 2H,
J = 1.7, 4.6 Hz), 7.90 (d, 1H, J = 1.2 Hz), 7.25 (dd, 2H, J = 1.7,
4.6 Hz), 6.94 (dd, 1H, J = 1.0, 1.0 Hz), 4.20-4.11 (m, 2H), 4.06
(m, 1H), 2.88 (s, 6H); MS (FABþ) m/z 297 (M þ H)þ.
2-(1-(1H-Imidazole-4-yl)-2-(4-trifluoromeethylphenylthio)-
ethyl)pyridine Hydrochloride Salt (30). A solution of 28a (79 mg,
0.18 mmol) in 5 N aqueous HCl (1 mL) was stirred at 80 °C for 4
h. The solvent was evaporated, and the obtained residue was
purified with LH-20 (MeOH/CHCl3 = 1:1) to give 30 as a
colorless solid (53 mg, 70%, 2 HCl salt), mp 129 °C. 1H NMR
(CD3OD) δ 8.98 (s, 1H), 8.80 (d, 1H, J = 5.6 Hz), 8.5 (m, 1H),
8.06 (m, 1H), 7.95 (m, 1H), 7.81 (s, 1H), 7.56 (d, 2H, J = 8.5 Hz),
7.51 (d, 2H, J = 8.5 Hz), 5.03 (m, 1H), 4.06-4.03 (m, 2H); MS
(ESIþ) m/z 350 (M þ H)þ; HRMS (FABþ) calcd for
C17H15N3F3S (M þ H)þ 350.0939, found 350.0940.
N,N-Dimethyl-4-(1-(pyridine-2-yl)-2-(4-trifluoromethylphenyl-
thio)ethyl)-1H-imidazole-1-sulfonamide (28a). 28a was synthe-
sized as a colorless oil, using the procedure for 29b from 27a and
4-trifluoromethylbenzenethiol (77% in two steps). 1H NMR
(CDCl3) δ 8.58 (ddd, 1H, J = 0.7, 1.7, 4.6 Hz), 7.84 (d, 1H, J =
1.2 Hz), 7.63 (ddd, 1H, J = 1.7, 7.8, 7.8 Hz), 7.49 (d, 2H, J = 8.5
Hz), 7.37 (d, 2H, J = 8.5 Hz), 7.26 (m, 1H), 7.17 (m, 1H), 7.04 (s,
1H), 4.36 (t, 1H, J = 7.6 Hz), 3.78 (d, 2H, J = 7.6H), 2.80 (s,
6H); MS (FABþ) m/z 457 (M þ H)þ.
2-(2-(4-tert-Butylphenylthio)-1-(1H-imidazol-4-yl)ethyl)pyridine
(31). 31 was synthesized as a colorless oil, using the procedure
for 33 from 29a (80%). 1H NMR (CDCL3) δ 8.55(ddd, 1H, J =
N,N-Dimethyl-4-(1-(pyridine-3-yl)-2-(4-trifluoromethylphenyl-
thio)ethyl)-1H-imidazole-1-sulfonamide (28b). 28b was synthesized

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6455
0.7, 1.7, 4.8 Hz), 7.63 (ddd, 1H, J = 1.9, 7.8, 7.8 Hz), 7.55 (d, 1H,
J = 0.7 Hz), 7.30-7.23 (m, 5H), 7.14 (ddd, 1H, J = 1.2, 4.8, 7.3
Hz), 6.89 (s, 1H), 4.35 (t, 1H, J = 6.8 Hz), 3.65-3.48 (m, 2H), 1.29
(s, 9H);MS(FABþ) m/z 338 (M þ H)þ; HRMS (FABþ) calcd for
C20H24N3S (M þ H)þ 338.1691, found 338.1687.
[35S]GTPγS Binding Assay. Membranes prepared from CHO
cells stably expressing human recombinant histamine H3 recep-
tors were incubated with [35S]GTPγS (200 pM, GE Healthcare
Bio-Sciences) and test compounds alone or in the presence of
(R)-R-methylhistamine (10 nM, Sigma-Aldrich) in a buffer
containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 3 mM
MgCl2, 0.2 mM EDTA, 1 mM DTT, and 10 μM GDP at 25 °C
for 1 h. Radioligand binding was terminated by filtering through
GF/B filters, and the amount of bound radiolabel was deter-
mined by liquid scintillation counting.
3-(1-(1H-Imidazole-4-yl)-2-(4-trifluoromeethylphenylthio)ethyl)-
pyridine (32). 32 was synthesized as a colorless oil, using the
procedure for 33 from 28b (81%). 1H NMR (CDCL3) δ 8.53 (d,
1H, J = 2.2 Hz), 8.46 (dd, 1H, J = 1.7, 4.6 Hz), 7.70 (m, 1H),
7.60 (d, 1H, J = 1.2 Hz), 7.48 (d, 2H, J = 8.8 Hz), 7.33 (d, 2H,
J = 8.8 Hz), 7.23 (m, 1H), 6.78 (s, 1H), 4.23 (dd, 1H, J = 7.6, 7.6
Hz), 3.86 (dd, 1H, 7.0, 13.0 Hz), 3.48 (dd, 1H, 7.0, 13.0 Hz); MS
(FABþ) m/z 350 (M þ H)þ; HRMS (FABþ) calcd for
C17H15N3F3S (M þ H)þ 350.0939, found 350.0929.
Supporting Information Available: HPLC analytical data for
15, 16, 23, 24, and 30-37; crystal structure determination of
compounds 36 and 37; experimental details of in vitro human
CYP inhibition test; modeled binding mode of 16 in the human
H3 receptor binding site. This material is available free of charge
via the Internet at http://pubs.acs.org.
3-(2-(4-tert-Butylphenylthio)-1-(1H-imidazol-4-yl)ethyl)pyri-
dine (33). A solution of 29b (93 mg, 0.21 mmol) in 5 N aqueous
HCl (1.5 mL) was stirred at 80 °C for 20 h. The reaction mixture
was basified with 5 N aqueous NaOH and diluted with saturated
aqueous K2CO3 (20 mL) and brine (10 mL). The mixture was
extracted with CHCl3 (30 mL ꢀ 3), and the combined organic
layer was dried (Na2SO4), evaporated, and purified by NH thin
layer chromatography (MeOH/CHCl3 = 1:9) to give 33 as a
white solid (59 mg, 83%), mp 103 °C. 1H NMR (CDCl3) δ 8.47
(d, 1H, J = 1.7 Hz), 8.43 (dd, 1H, J = 1.7, 4.8 Hz), 7.66 (d, 1H,
J = 8.0 Hz), 7.56 (s, 1H), 7.30-7.19 (m, 6H), 6.78 (s, 1H), 4.19 (t,
1H, J = 7.6 Hz), 3.76-3.69 (m, 2H), 1.27 (s, 9H); MS (FABþ)
m/z 338 (M þ H)þ; HRMS (FABþ) calcd for C20H24N3S (M þ
H)þ 338.1691, found 338.1686.
4-(1-(1H-Imidazole-4-yl)-2-(4-trifluoromeethylphenylthio)ethyl)-
pyridine (34). 34 was synthesized as a colorless oil, using the
procedure for 33 from 28c (18%). 1H NMR (CDCL3) δ 8.52 (dd,
2H, J = 1.6, 3.0 Hz), 7.62 (s, 1H), 7.49 (d, 2H, J = 8.2 Hz), 7.35
(d, 2H, J = 8.2 Hz), 7.28 (dd, 2H, J = 1.6, 3.0 Hz), 6.79 (s, 1H),
4.20 (t, 1H, J = 7.6 Hz), 3.84 (dd, 1H, J = 7.6, 13.0 Hz), 3.50
(dd, 1H, J = 7.6, 13.0 Hz); MS (EIþ) m/z 349 (M)þ; HRMS
(FABþ) calcd for C17H15N3F3S (M þ H)þ 350.0939, found
350.0946.
References
(1) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Auto-inhibition of
brain histamine release mediated by a novel class (H3) of histamine
receptor. Nature 1983, 302, 832–837.
(2) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Autoregulation
of histamine release in brain by presynaptic H3-receptors. Neu-
roscience 1985, 15, 553–562.
(3) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Autoinhibition of
histamine synthesis mediated by presynaptic H3-receptors. Neu-
roscience 1987, 23, 149–157.
(4) Fink, K.; Schlicker, E.; Neise, A.; Gothert, M. Involvement of
presynaptic H3 receptors in the inhibitory effect of histamine on
serotonin release in the rat brain cortex. Naunyn Schmiedeberg’s
Arch. Pharmacol. 1990, 342, 513–519.
(5) Celuch, S. M. Possible participation of histamine H3 receptors in
the modulation of noradrenaline release from rat spinal cord slices.
Eur. J. Pharmacol. 1995, 287, 127–133.
(6) Garcia, M.; Floran, B.; Arias-Montano, J. A.; Young, J. M.;
Aceves, J. Histamine H3 receptor activation selectively inhibits
dopamine D1 receptor-dependent [3H]GABA release from depo-
larization-stimulated slices of rat substantia nigra pars reticulatta.
Neuroscience 1997, 80, 241–249.
4-(2-(4-tert-Butylphenylthio)-1-(1H-imidazol-4-yl)ethyl)pyri-
dine Hydrochloride Salt (35). 35 was synthesized as a colorless
syrup, using the procedure for 30 from 29c (52%, 2 HCl salt). 1H
NMR (CD3OD) δ 8.87 (d, 1H, J = 1.2 Hz), 8.77 (d, 2H, J = 6.6
Hz), 8.02 (d, 2H, J = 6.6 Hz), 7.68 (s, 1H), 7.32 (d, 2H, J = 6.0
Hz), 7.25 (d, 2H, J = 6.0 Hz), 4.73 (t, 1H, J = 7.6 Hz), 3.78 (dd,
1H, J = 7.6, 14.0 Hz), 3.71 (dd, 1H, J = 7.6, 14.0 Hz), 1.26 (s,
9H); MS (FABþ) m/z 338 (M þ H)þ; HRMS (FABþ) calcd for
C20H24N3S (M þ H)þ 338.1691, found 338.1687.
(7) Poli, E.; Stark, H.; Bertaccini, G. Histamine H3 receptor activation
inhibits acetylcholine release from the guinea pig myenteric plexus.
Agents Actions 1991, 33, 167–169.
(8) Sander, K.; Kottke, T.; Stark, H. Histamine H3 receptor antago-
nists go to clinics. Biol. Pharm. Bull. 2008, 31, 2163–2181.
(9) Yokoyama, F.; Yamauchi, M.; Oyama, M.; Okuma, K; Onozawa,
K.; Nagayama, T.; Shinei, R.; Ishikawa, M; Sato, Y.; Kakui, N.
Anxiolytic-like profiles of histamine H3 receptor agonists in animal
models of anxiety: a comparative study with antidepressants
and benzodiazepine anxiolytic. Psychopharmacology 2009, 205,
177–187.
(10) Ishikawa, M; Shinei, R; Yokoyama, F; Yamauchi, M; Oyama, M;
Okuma, K; Nagayama, T; Kato, K; Kakui, N; Sato, T. Role of
hydrophobic substituents on the terminal nitrogen of histamine in
receptor binding and agonist activity: development of an orally
active histamine type 3 receptor agonist and evaluation of its
antistress activity in mice. J. Med. Chem. 2010, 53, 3840–3844.
(11) Vollinga, R. C.; De Koning, J. P.; Jansen, F. P.; Leurs, R.; Menga,
W. M. P. B.; Timmerman, H. A new potent and selective histamine
H3 receptor agonist, 4-(1H-imidazole-4-ylmethyl)piperidine.
J. Med. Chem. 1994, 37, 332–333.
(12) Garbarg, M.; Arrang, J. M.; Rouleau, A.; Ligneau, X.; Tuong, M.
D. T; Schwartz, J. C.; Ganellin, C. R. S-[2-(4-Imidazo-
lyl)ethyl]isothiourea, a highly specific and potent histamine H3
receptor agonist. J. Pharmacol. Exp. Ther. 1992, 263, 304–310.
(13) Stark, H.; Arrang, J. M.; Ligneau, X.; Garbarg, M.; Ganelline,
C. R.; Schwartz, J. C.; Schunack, W. The histamine H3 receptor
and its ligands. Prog. Med. Chem. 2001, 38, 279–308.
(14) De Esch, I. J. P.; Timmerman, H.; Merge, W. M. P. B.; Nederkoorm,
P. H. J. A qualitative model for the histamine H3 receptor explaining
agonist and antagonistic activity simultaneously. Arch. Pharm.
(Weinheim, Ger.) 2000, 333, 254–260.
(S)-4-(1-(1H-Imidazol-4-yl)-2-(4-(trifluoromethyl)phenylthio)-
ethyl)piperidine (36) and (R)-4-(1-(1H-Imidazol-4-yl)-2-(4-(tri-
fluoromethyl)phenylthio)ethyl)piperidine (37). The enantiomers
of 23 were separated by preparative liquid chromatography
using a Chiralpak AD-H (DAICEL) 20 mm ꢀ 250 mm (0.1%
diethylamine-MeOH). 36 (white foam) 99.5% ee; [R]D -22.5 (c
0.26, CHCl3); HRMS (FABþ) calcd for C17H21N3F3S (M þ
H)þ 356.1408, found 356.1406. Anal. Calcd for C17H20F3N3S
3
H2O: C, 54.68; H, 5.94; N, 11.25. Found: C, 54.67; H, 6.03; N,
11.17. 37 (white foam) 98.5% ee; [R]D þ20.4 (c 0.25, CHCl3);
HRMS (FABþ) calcd for C17H21N3F3S (M þ H)þ 356.1408,
found 356.1409. Anal. Calcd for C17H20F3N3S H2O: C, 54.68;
3
H, 5.94; N, 11.25. Found: C, 54.54; H, 5.85; N, 11.13.
Pharmacology. H3 Receptor Binding Assay. Membranes pre-
pared from CHO cells stably expressing human recombinant
histamine H3 receptors were incubated with [3H]-(R)-R-methyl-
histamine (3 nM, GE Healthcare Bio-Sciences) and test com-
pounds in a buffer containing 50 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, and 0.04% BSA at 25 °C for 1 h. Nonspecific binding
was assessed with (R)-R-methylhistamine (1 μM, Sigma-
Aldrich). Radioligand binding was terminated by filtering
through GF/B filters (PerkinElmer Life Sciences), and the
amount of bound radiolabel was determined by liquid scintilla-
tion counting.
(15) Uveges, A. J.; Kowal, D.; Zhang, Y.; Spangler, T. B.; Dunlop, J.;
Semus, S.; Jones, P. G. The role of transmembrane helix 5 in
agonist binding to the human H3 receptor. J. Pharmacol. Exp.
Ther. 2002, 301, 451–458.
(16) Kitbunnadaj, R.; Zuiderveld, O. P.; Christophe, B.; Hulscher, S.;
Menge, W. M. P. B.; Gelens, E.; Snip, E.; Bakker, R. A.; Celanire,

6456 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Ishikawa et al.
S.; Gillard, M.; Talaga, P.; Timmerman, H.; Leurs, R. Identifica-
tion of 4-(1H-imidazole-4(5)-ylmethyl)pyridine (immethridine) as
a novel, potent, and highly selective histamine H3 receptor agonist.
J. Med. Chem. 2004, 47, 2414–2417.
Formation and application to organic synthesis. J. Am. Chem. Soc.
1965, 87, 1353–1364.
(29) Abraham, D. J. X-ray Crystallography and Drug Design. Compu-
ter-Aided Drug Design; Methods and Applications; Marcel Dekker:
New York and Basel, Switzerland, 1989; pp 93-124.
(30) Denny, L. I.; Parihar, A. S.; Miller, T. R.; Kang, C. H.; Krueger,
K. M.; Esbenshade, T. A.; Hancock, A. A. Species-related phar-
macological heterogeneity of histamine H3 receptors. Eur. J.
Pharmacol. 2001, 433, 141–150.
(31) Coge, F.; Guenin, S. P.; Audinot, V.; Trenouard-try, A.; Beau-
verger, P.; Macia, C.; Ouvry, C.; Nagel, N.; Rique, H.; Boutin,
J. A.; Galizzi, J. P. Genomic organization and characterization of
splice variants of the human histamine H3 receptor. Biochem. J.
2001, 355, 279–288.
(32) Park, J. H.; Scheerer, P.; Hofmann, K. P.; Choe, H. W.; Ernst, O. P.
Crystal structure of the ligand-free G-protein-coupled receptor
opsin. Nature 2008, 454, 183–188.
(33) Baldwin, J. M; Schertler, G. F. X; Unger, V. M. An alpha-carbon
template for the transmembrane helices in the rhodopsin family of
G-protein-coupled receptors. J. Mol. Biol. 1997, 272, 144–164.
(34) Abad-Zapatero, C.; Metsz, J. T. Ligand efficiency indices as
guideposts for drug discovery. Drug Discovery Today 2005, 10,
464–469.
(17) Ishikawa, M.; Furuuchi, T.; Yamauchi, M.; Yokoyama, F.; Kakui,
N.; Sato, Y. Synthesis and structure-activity relationships of N-
aryl-piperidine derivatives as potent (partial) agonists for human
histamine H3 receptor. Bioorg. Med. Chem. 2010, 18, 5441–5448.
(18) Meier, G.; Krause, M.; Huls, A.; Ligneau, X.; Pertz, H. H.; Arrang,
J. M.; Ganellin, C. R.; Schwartz, J. C.; Schnack, W.; Stark, H.
4-(ω-(Alkyloxy)alkyl)-1H-imidazole derivatives as histamine H3
receptor antagonists/agonists. J. Med. Chem. 2004, 47, 2678–2687.
(19) Pelloux-Leon, N.; Fkyerat, A.; Piripitsi, A.; Tertiuk, W.; Schu-
nack, W.; Stark, H.; Garbarg, M.; Ligneau, X.; Arrang, J. M.;
Schwartz, J. C.; Ganellin, C. R. Meta-substituted aryl(thio)ethers
as potent partial agonists (or antagonists) for the histamine H3
receptor lacking a nitrogen atom in the side chain. J. Med. Chem.
2004, 47, 3264–3274.
(20) Kitbunnadaj, R.; Hoffmann, M.; Fratantoni, S. A.; Bongers, G.;
Bakker, R. A.; Wieland, K.; el Jilali, A.; De Esch, I. J. P.; Menge,
W. M. P. B.; Timmerman, H.; Leurs, R. New high affinity H3
receptor agonists without a basic side chain. Bioorg. Med. Chem.
2005, 13, 6309–6323.
(21) Wijtmans, M.; Celanire, S.; Snip, E.; Gillard, M. R.; Gelens, E.;
Collart, P. P.; Venhuis, B. J.; Christophe, B.; Hulscher, S.; van der
Goot, H.; Lebon, F.; Timmerman, H.; Bakker, R. A.; Lallemand,
B. I. L. F.; Leurs, R.; Talaga, P. E.; De Esch, I. J. P. 4-Benzyl-1H-
imidazoles with oxazoline termini as histamine H3 receptor ago-
nists. J. Med. Chem. 2008, 51, 2944–2953.
(35) Lin, J. H.; Liu, A. Y. H. Inhibition and induction of cytochrome
P450 and the clinical implications. Clin. Pharmacokinet. 1998, 35,
361–390.
(36) Newton, D. J.; Wang, R. W.; Lu, A. Y. Cytochrome P450
inhibitors. Evaluation of specificities in the in vitro metabolism
of therapeutic agents by human liver microsomes. Drug Metab.
Dispos. 1995, 23, 154–158.
(22) http://sparc.chem.uga.edu/sparc/.
(23) Hiral, S. H.; Karickhoff, S. W.; Carreira, L. A. A rigorous test for
SPARC’s chemical reactivity models: estimation of more than 4300
ionization pKas. Quant. Struct.-Act. Relat. 1995, 14, 348–355.
(24) Barrow, J. C.; Nantermet, P. G.; Stauffer, S. R.; Ngo, P. L.;
Steinbeiser, M. A.; Mao, S. S.; Carroll, S. S.; Bailey, C.; Colussi,
D.; Bosserman, M.; Burlein, C.; Cook, J. J.; Sitko, G.; Tiller, P. R.;
Miller-Stein, C. M.; Rose, M.; McMasters, D. R.; Vacca, J. P.;
Selnick, H. G. Synthesis and evaluation of imidazole acetic acid
inhibitors of activated thrombin-activatable fibrinolysis inhibitor
as novel antithrombotics. J. Med. Chem. 2003, 46, 5294–5297.
(25) Schreiber, J.; Maag, H.; Hashimoto, N.; Eschenmoser, A.
Dimethyl(methylene)ammonium iodide. Angew. Chem., Int. Ed.
1971, 10, 330–331.
(37) Bjornsson, T. D.; Callaghan, J. T.; Einolf, H. J.; Fischer, V.; Gan,
L.; Grimm, S.; Kao, J.; King, S. P.; Miwa, G.; Ni, L.; Kumar, G.;
McLeod, J.; Obach, S. R.; Roberts, S.; Roe, A.; Shah, A.; Snikeris,
F.; Sullivan, J. T.; Tweedie, D.; Vega, J. M.; Walsh, J.; Wrighton,
S. A. The conduct of in vitro and in vivo drug-drug interaction
studies: A PhRMA perspective. J. Clin. Pharmacol. 2003, 43, 443–
469.
(38) Berlin, M.; Ting, P. C.; Vaccaro, W. D.; Aslanian, R.; McCormick,
K. D.; Lee, J. F.; Albanese, M. M.; Mutahi, M. W.; Piwinski, J. J.;
Shih, N. Y.; Duguma, L.; Solomon, D. M.; Zhou, W.; Sher, R.;
Favreau, L.; Bryant, M.; Korfmacher, W. A.; Nardo, C.; West,
R. E., Jr.; Anthes, J. C.; Williams, S. M.; Wu, R. L.; She, H. S.;
Rivelli, M. A.; Corboz, M. R.; Hey, J. A. Reduction of CYP450
inhibition in the 4-[(1H-imidazol-4-yl)methyl]piperidine series of
histamine H3 receptor antagonists. Bioorg. Med. Chem. Lett. 2006,
16, 989–994.
(26) Ganem,B.;Ikota, N.;Muralidharan,V.B.;Wade,W.S.;Young,S. D.;
Yukimoto, Y. Shikimate-derived metabolites. 11. A total synthesis of
(()-chorismic acid. J. Am. Chem. Soc. 1982, 104, 6787–6788.
(27) Kan, T.; Fukuyama, T. Ns strategies: a highly versatile synthetic
method for amines. Chem. Commun. 2004, 353–359.
(39) Kansy, M.; Senner, F.; Gubernator, K. Physicochemical high
throughput screening: parallel artificial membrane permeation
assay in the description of passive absorption processes. J. Med.
Chem. 1998, 41, 1007–1010.
(28) Corey, E. J.; Chaykovsky, M. Dimethyloxosulfonium methylide
((CH3)2SOCH2) and dimethylsulfonium methylide ((CH3)2SCH2).