New high affinity H3 receptor agonists without a basic side chain

Article abstract of DOI:10.1016/j.bmc.2005.09.002

In this study, we replaced the basic amine function of the known histamine H₃ receptor agonists imbutamine or immepip with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human H₃ receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed an increased selectivity for the human H₃ receptor over the human H₄ receptor, but none of the compounds in this series possess increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-imidazol-4-yl)-pentan-1-ol, is the most potent histamine H₃ receptor agonist (pK_i = 8.0 and pEC₅₀ = 8.1) with a 320-fold selectivity at the human H₃ receptor over the human H ₄ receptor.

Full text of DOI:10.1016/j.bmc.2005.09.002

Bioorganic & Medicinal Chemistry 13 (2005) 6309–6323
New high affinity H3 receptor agonists without a basic side chain
Ruengwit Kitbunnadaj,a,b Marcel Hoffmann,a Silvina A. Fratantoni,a Gerold Bongers,a
Remko A. Bakker,a Kerstin Wieland,a Ahmed el Jilali,a Iwan J. P. De Esch,a
Wiro M. P. B. Menge,a Henk Timmermana and Rob Leursa,*
aLeiden/Amsterdam Center of Drug Research (LACDR), Division of Medicinal Chemistry,
Department of Pharmacochemistry, Faculty of Chemistry, Vrije Universiteit Amsterdam,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
bDepartment of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences,
Naresuan University, Phitsanulok, Thailand
Received 24 November 2004; revised 2 May 2005; accepted 6 May 2005
Available online 5 October 2005
Abstract—In this study, we replaced the basic amine function of the known histamine H3 receptor agonists imbutamine or immepip
with non-basic alcohol or hydrocarbon moieties. All compounds in this study show a moderate to high affinity for the cloned human
H3 receptor and, unexpectedly, almost all of them act as potent agonists. Moreover, in the alcohol series, we consistently observed
an increased selectivity for the human H3 receptor over the human H4 receptor, but none of the compounds in this series possess
increased affinity and functional activity compared to their alkylamine congeners. In this new series of compounds VUF5657, 5-(1H-
imidazol-4-yl)-pentan-1-ol, is the most potent histamine H3 receptor agonist (pKi = 8.0 and pEC50 = 8.1) with a 320-fold selectivity
at the human H3 receptor over the human H4 receptor.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
whereas the alkyl side chain often contains a positive-
ly charged amine moiety (see Chart 1 for (R)-a-meth-
ylhistamine, immepip) or isothiourea moiety (imetit).
Initial molecular modelling studies from our laborato-
ry were able to explain H3 agonism on the basis of
the presumed interaction of the positively charged
side chain with a putative carboxylate group of the
receptor protein.10 However, it has been shown that
a basic group in the imidazole side chain can be
omitted.11
The histamine H3 receptor was initially discovered as a
presynaptic autoreceptor on histaminergic neurons reg-
ulating the release and synthesis of histamine in the rat
and human brain.1–3 Nowadays, it is generally accepted
that the histamine H3 receptor also acts as a presynaptic
heteroreceptor on several non-histaminergic nerve ter-
minals inhibiting the release of a variety of neurotrans-
mitters in both the central and peripheral nervous
system.4–6 As such, the H3 receptor is considered to be
an important target for the treatment of various CNS-
related disorders; ligands for the H3 receptor have been
proposed as potential drugs for the treatment of demen-
tia, attention deficit hyperactivity disorder, narcolepsy
and obesity.7,8
The cloning of the H3 receptor cDNA by Lovenberg
et al.12 in 1999 has revealed that the H3 receptor protein
shows all the expected hallmarks of the rhodopsin-class
of G-protein coupled receptors (GPCR). As for all
GPCRs for aminergic neurotransmitters, the highly con-
served aspartate in transmembrane domain three (TM3)
is found in the histamine H3 receptor.13–15 For various
GPCRs, including the histamine H1 and H2 recep-
tors,16–18 this conserved aspartate residue has been con-
vincingly implicated in the binding of protonated side
chains found in both agonists and antagonists.19–23
Moreover, recent computational studies with a 3D-ho-
mology model of the histamine H3 receptor also hypoth-
esized an interaction of the protonated amine of the
In order to exploit the interesting therapeutic poten-
tial, a wide variety of H3 receptor agonists and
antagonists have been developed in the last decade.9
All potent H3 agonists possess an imidazole ring,
Keywords: Histamine H3 receptor; Agonists; Selective.
*
Corresponding author. Tel.: +31 20 4447600; fax: +31 20 4447610;
e-mail: leurs@few.vu.nl
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.002

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R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
hH3 receptor
hH4 receptor
compound
Immepip40
structure
selectivity*
46
pKi
pEC50
pKi
pEC50
NH
9.3
9.9
7.7
6.6
7.2
N
NH
NH
N
Immethridine27
Imetit
9.1
9.7
6.0
288
2
N
NH
S
9.143
8.443
9.743
9.243
8.944
6.644
8.544
6.044
NH2
N
NH
NH2
(R)-α-Methylhistamine
55
H
N
NH
*Selectivity = Ki(H4)/Ki(H3).
Chart 1. Binding affinities and functional activities of ligands at the human histamine H3 and H4 receptors.
ethylamine side chain of histamine with the conserved
Asp114 in TM3.24–26
human histamine H3 and its closely related H4
receptors.
Recently, it was observed that replacement of the
piperidine ring of immepip by a weakly basic pyridine
ring does not affect the binding affinity at the human
histamine H3 receptor. Immethridine exhibits high
affinity and agonist activity at the H3 receptor with a
pKi value of 9.1 and a pEC50 value of 9.7 (Chart
1).27 Surprisingly, immethridine shows an increased
selectivity at the H3 receptor over its closely related
H4 receptor. Since the basicity of the nitrogen atoms
in the side chain of immepip and immethridine is differ-
ent, we questioned whether a basic amine at the side
chain is necessary for binding and activation of the hu-
man histamine H3 receptor. This observation opens po-
tential new avenues for selective H3 receptor agonists.
In this study, ligands with different physicochemical
properties at the side chain (Fig. 1) were therefore syn-
thesized and tested for their affinity and activity at the
2. Chemistry
The syntheses for all targeted compounds are outlined
in Schemes 1–8. The synthesis of 5 was modified from
the procedure described by Stark et al.28 (Scheme 1).
Reflux of urocanic acid in methanol saturated with
hydrochloric acid gave the methyl ester (27) in a
quantitative yield. After protection of the imidazole
ring of 27 with a triphenylmethyl group, the interme-
diate 28 was subsequently hydrogenated under a H2
atmosphere in the presence of a catalytic amount of
5% Pd/C to give 29 in a 75% yield. Reduction of 29
by lithium aluminium hydride in THF gave the
alcohol 30 in a 70% yield. Deprotection of the imidaz-
ole ring under acidic conditions finally yielded com-
pound 5.
CH3
NH2
OH
n
n
n
N
N
N
NH
NH
NH
N
NH
N
N
N
NH
NH2
NH2
N
NH
n
n
n
n
N
NH
NH
N
NH
O
OH
n
n
n
N
NH
N
NH
NH
Figure 1. Design of ligands with various physicochemical properties at the side chain.

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6311
O
O
O
O
O
O
OH
O
ii
i
iii
iv
N
NH
N
Tr
N
N
N
NH
N
Tr
N
N
27
28
29
OH
OH
v
Tr
N
NH
30
5
Scheme 1. Synthetic pathway for 5. Reagents and conditions: (i) MeOH, HCl (g), D, 5 h; (ii) triphenylmethylchloride, THF, TEA, rt, 16 h; (iii) 5%
Pd/C, MeOH, 1 atm H2, 4 h; (iv) LiAlH4, THF, rt, 6 h; (v) 1 N HCl, D, 5 h.
O
O
O
H
O
H
O
O
ii
iii
i
Tr
N
N
Tr
Tr
N
N
N
Tr
N
N
Tr
N
N
32
33
34
31
iv
v
OH
+
H N
N
Cl-
N
v
OH
35
OH
N
NH
6
Scheme 2. Synthetic pathway for 6. Reagents and conditions: (i) n-BuLi, diisopropylamine, THF, [2-(1,3-dioxolan-2-yl)ethyl]-triphenylphosphonium
bromide, ꢀ30 ꢁC ! reflux 6 h; (ii) 5% Pd/C, 1 atm H2, MeOH, 4 h; (iii) acetone, 1 N HCl, 0 ꢁC, 1 h; (iv) LiAlH4, THF, rt, 6 h; (v) 1 N HCl, D, 5 h.
O
O
O
O
O
H
OH
i
iii
ii
Tr
N
N
Tr
N
N
Tr
N
iv
N
Tr
N
N
36
37
38
30
OH
OH
v
Tr
N
N
N
NH
39
7
Scheme 3. Synthetic pathway for 7. Reagents and conditions: (i) oxalyl chloride, DMSO, DCM, TEA, ꢀ60 ꢁC to rt, 2 h; (ii) triethyl
phosphonoacetate, NaH, THF, 0 ꢁC to rt, 6 h; (iii) 1 atm H2, 5% Pd/C, MeOH, 4 h; (iv) LiAlH4, THF, rt, 4 h; (v) 1 N HBr, D, 3 h.
Compound 6 was synthesized according to Scheme 2;
the protected imidazole carboxaldehyde was reacted
with [2-(1,3-dioxolan-2-yl)ethyl]-triphenylphosphonium
bromide in the presence of LDA to give 31 in a
85% yield. Attempts to hydrolyse the dioxolane ring
under aqueous acidic conditions did not result in the
expected aldehyde, but we speculate that the cyclic
compound 35 was obtained instead. However, the
aldehyde 33 was obtained after hydrogenation of the
double bond of 31 under a H2 atmosphere in the pres-
ence of 5% Pd/C and subsequently hydrolysis of the
dioxolane ring with 1 N HCl in acetone at 0 ꢁC.
Reduction of the aldehyde using lithium aluminium
hydride yielded the intermediated alcohol 34 in a
75% yield. Deprotection of the imidazole ring gave 6
in a good yield.

6312
R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
ii iii
i
iv
O
O
O
O
O
O
O
O
HO
H
O
41
O
40
O
42
43
v
O
N
NH
13
Scheme 4. Synthetic pathway for 13. Reagents and conditions: (i) triethyl phosphonoacetate, NaH, THF, 0 ꢁC to rt, 6 h; (ii) 1 atm H2, 5% Pd/C,
MeOH, 4 h; (iii) LiAlH4, THF, rt, 4 h; (iv) oxalyl chloride, DMSO, DCM, TEA, ꢀ60 ꢁC to rt, 2 h; (v) 1—TosMIC, NaCN, EtOH; 2—satd NH3 in
EtOH, 90–110 ꢁC, 16 h.
O
O
OH
O
OH
O
O
O
O
i
ii
iii
iv
v
O
O
O
H
HO
O
O
O
O
O
O
44
45
46
47
48
O
O
OH
OH
O
O
vi
vii
viii
ix
x
N
NH
49
Boc
N
N
Boc
N
N
N
NH
N
NH
23
51
50
52
xi
HN Boc
x
NH2
Boc
N
N
N
NH
53a (cis)
53b (trans)
20 (cis)
21 (trans)
Scheme 5. Synthetic pathway for 20 and 21. Reagents and conditions: (i) 100 atm H2, 5% Rh/C, THF, 18 h; (ii) pyridinium chlorochromate (PCC),
DCM, Celite, sodium acetate, rt, 4 h; (iii) p-TSA, ethylene glycol, toluene, D, 18 h; (iv) LiAlH4, ether, rt, 3 h; (v) DMSO, DCM, TEA, ꢀ60 ꢁC to rt,
2 h; (vi) 1—TosMIC, NaCN, EtOH; 2—satd NH3 in EtOH, 90–110 ꢁC, 16 h; (vii) 1 N HCl, rt, 30 min; (viii) di-tert-butyldicarbonate, triethylamine,
CHCl3, rt, 16 h; (ix) NaBH4, MeOH, rt, 16 h; (x) 1 N HCl, rt, (xi) 1—ammonium acetate, NaBH4, MeOH, rt, 18 h; 2—di-tert-butyldicarbonate,
triethylamine, CHCl3, rt, 16 h.
The synthesis of compound 7 followed Scheme 3. The
intermediate aldehyde 36 was obtained from the alcohol
30 via a Swern oxidation29 in a 78% yield. Next, a Wittig
reaction of the aldehyde with triethyl phosphonoacetate
resulted in the compound 37. Following the aforemen-
tioned procedure for the synthesis for 5, the targeted
alcohol 7 was obtained.
Following the procedure outlined in Scheme 5, the com-
pounds 20, 21 and 23 were synthesized from commer-
cially available methyl-p-hydroxybenzoate. Attempts to
hydrogenate the benzene ring using 10% Pd/C as cata-
lyst in methanol resulted in hydrogenolysis. However,
hydrogenation performed under high pressure
(100 atm) of H2 in THF with a catalytic amount of 5%
Rh/C gave the desired product 44 in a quantitative yield.
Next, the alcohol 44 was oxidized by pyridinium chloro-
chromate (PCC) to give the intermediate ketone 45 in a
90% yield. The ketone was converted to the intermediate
dioxolane 46 by refluxing with propylene glycol in the
presence of p-toluenesulfonic acid. The ester 46 was re-
duced into the alcohol 47 using lithium aluminium hy-
dride, whereafter the alcohol was converted into 48 by
a Swern oxidation. Once the aldehyde was formed, it
was easily converted into an imidazole ring using Tos-
MIC chemistry to give 50 in a 94% yield. Next, the
Compound 13 was synthesized according to Scheme 4;
a
Wittig reaction of tetrahydropyran-4-one with
triethyl phosphonoacetate gave 40 in a 88% yield. Fol-
lowing hydrogenation and reduction as described for
the synthesis of 5, the intermediate alcohol 42 was
obtained and was subsequently transformed into the
aldehyde 43 via
a
Swern oxidation. Using
TosMIC chemistry,30 the aldehyde of 43 was convert-
ed to an imidazole ring to give compound 13 in a
75% yield.

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6313
O
O
O
O
O
O
i
ii
iii
O
O
O
HO
O
O
O
O
O
54
55
vi
56
O
iv
v
vii
O
H
O
O
O
N
NH
N
NH
57
58
61
59
viii
ix
O
OH
OH
Boc
Boc
N
N
N
Boc
N
N
N
NH
60
24
x
ix
NH2
NH2
N
N
NH
22
62
Scheme 6. Synthetic pathway for 22 and 24. Reagents and conditions: (i) triethyl phosphonoacetate, NaH, THF, 0 ꢁC to rt, 6 h; (ii) 1 atm H2, 5% Pd/
C, MeOH, 4 h; (iii) LiAlH4, THF, rt, 4 h; (iv) oxalyl chloride, DMSO, DCM, TEA, ꢀ60 ꢁC to rt, 2 h; (v) 1—TosMIC, NaCN, EtOH; 2—satd NH3 in
EtOH, 90–110 ꢁC, 16 h; (vi) 1 N HCl, rt, 30 min; (vii) di-tert-butyldicarbonate, triethylamine, CHCl3, rt, 16 h; (viii) NaBH4, MeOH, rt, 16 h; (ix) 1 N
HCl, rt; (x) ammonium acetate, NaBH4, MeOH, rt, 16 h.
NO2
NH2
carbonate in order to increase the lipophilicity of the
molecule to give 53. The cis-(20) and trans-(21) iso-
mers were successfully separated by column
chromatography.
i
ii
N
NH
63
N
NH
NH
25
N
Compounds 22 and 24 were synthesized according to
Scheme 6. Following the aforementioned procedure
for the synthesis of 13 but using 1,4-dioxa-spiro[4,5]dec-
an-8-one as starting material, the intermediate dioxolane
58 was obtained in a moderate yield. Hydrolysis of the
dioxolane ring under mildly acidic conditions provided
the ketone 59, which was subsequently treated with di-
tert-butyldicarbonate to give 60. Using the procedures
described above for the synthesis of 20, 21 and 23, the
ketone 60 was converted into the alcohol 61 and the
amine 62. After deprotection of 61 and 62 under mildly
acidic conditions, compounds 22 and 24 were obtained
as racemic mixtures and tested for their activities with-
out further separation.
Scheme 7. Synthetic pathway for 25. Reagents and conditions: (i)
fuming HNO3, H2SO4, 0 ꢁC, 2 h; (ii) 5% Pd/C, MeOH, 1 atm H2, rt,
4 h.
resulting imidazole was treated with di-tert-butyldicar-
bonate to obtain the protected-imidazole intermediate
51 whose ketone can be transformed into 1 the
alcohol by reduction using sodium borohydride or 2
the amine by reductive amination using ammonium
acetate (as ammonia generator) and sodium triacetoxy
borohydride. Since the reduction of the ketone to the
alcohol 52 resulted in more than 95% trans-isomer
(based on NMR spectra), the compound was treated
under mildly acid conditions to give 23, which was
tested for its activity without further isomeric
separation. The reductive amination of the ketone
gave a mixture of the cis- and trans-isomers (1:3);
the resulting mixture was treated with di-tert-butyldi-
Compound 25 was prepared following Scheme 7. Nitra-
tion of the commercially available 4-phenyl-imidazole,
using fuming nitric acid and concentrated sulfuric acid,
gave 63 in 45% yield. Next, the nitro group was reduced
under an atmosphere of H2 to give the product 25 in a
quantitative yield.
O
O
HO
NO2
NH2
I
NO2
i
ii
iii
Tr
N
N
N
NH
Tr
N
N
Tr
N
N
64
65
26
Scheme 8. Synthetic pathway for 26. Reagents and conditions: (i) EtMgBr, DCM, 4-nitrobenzaldehyde, rt, 16 h; (ii) DMAP, DCM, acetic anhydride,
D, 6 h; (iii) 1—10% Pd/C, MeOH, 1 atm H2, rt, 4 h; 2—1 N HBr, D, 5 h.

6314
R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
As shown in Scheme 8, compound 26 was prepared from
protected 4-iodo-imidazole. Treatment of the iodo com-
pound with ethyl magnesium bromide and 4-nitrobenz-
aldehyde gave the alcohol 64 in a 85% yield. Acetylation
of the alcohol using acetic anhydride resulted in the
intermediate 65, from which the acetoxy group was re-
moved by hydrogenation under an atmosphere of H2
in the presence of 10% Pd/C. In this step, the nitro group
was simultaneously reduced to an aniline. After depro-
tection in acidic conditions, derivative 26 was obtained
in a good yield.
idine ring leads to the highly potent H3 agonist imme-
thridine (16), which exhibits an improved (300-fold)
selectivity at the human H3 receptor over the H4 recep-
tor.27 A decreased basicity of the side chain is apparently
well tolerated for H3 receptor agonism, whereas the
requirements for H4 receptor activity seem to be more
stringent.27 As a result, increased selectivity at the
human histamine H3 receptor might be obtained by
alterations of the side chain.
In this study, we evaluated two new series of compounds,
in which the basic nitrogen function of histamine homo-
logues was replaced with a non-basic alcohol or methyl
moiety. As can be seen in Table 1, these compounds
(5–10) still retained moderate to high H3 receptor affini-
ties, which were not dramatically lower compared to
their respective histamine homologues. In the new series
of non-basic H3 receptor agonists VUF5657 (7), the
3. Results and discussion
Previously, it was observed that replacement of the
piperidine ring of the potent and moderately selective
H3 agonist immepip (11, Table 1) with a less basic pyr-
Table 1. Affinities and functional activities of various ligands on the human histamine H3/4 receptors
X
X
X
X
X
n
n
n
n
n
N
NH
N
NH
II
N
NH
N
NH
IV
N
NH
I
III
V
Compound
VUF
Structure
X
n
hH3
hH4
Selectivitye
pKi SEMa
pEC50 SEMb
ac
pKi SEMd
1
2
Histaminef
8326f
Imbutaminef
Impentaminef
5698
I
–NH2
–NH2
–NH2
–NH2
–OH
2
3
4
5
3
4
5
3
4
5
1
2
1
1
2
1
2
1
2
0
0
1
0
1
0
1
8.0 0.1
7.3 0.0
8.4 0.0
8.3 0.1
6.5 0.1
7.4 0.0
8.0 0.0
7.6 0.0
7.6 0.1
7.5 0.1
9.3 0.0
7.7 0.1
6.7 0.1
7.4 0.1
6.8 0.0
9.1 0.0
8.2 0.0
6.4 0.0
6.1 0.0
7.0 0.0
7.4 0.0
7.2 0.0
5.4 0.1
6.8 0.1
6.0 0.0
6.1 0.0
8.0 0.0
7.4 0.2
8.1 0.1
8.3 0.1
6.1 0.1
7.3 0.3
8.1 0.1
7.5 0.1
7.3 0.3
7.6 0.4
9.9 0.0
7.9 0.1
6.9 0.1
7.4 0.2
n.d.i
1.0
0.9
0.9
0.9
0.9
1.3
1.3
1.1
1.2
0.9
1.0
0.6
0.9
1.4
n.d.
0.9
0.9
n.d.
n.d.
1.3
1.1
7.8 0.0
7.3 0.1
7.8 0.1
6.4 0.1
5.6 0.1
6.0 0.3
5.5 0.0
6.2 0.1
6.7 0.1
6.8 0.0
7.7 0.0
6.4 0.0
5.2 0.1
6.2 0.1
6.4 0.0
6.6 0.0
5.9 0.0
6.7 0.1
6.1 0.2
7.7 0.3
6.5 0.3
6.4 0.0
<4
1.5
0.7
I
3
I
3.6
4
I
72.0
7.8
5
I
6
5793
5657
I
–OH
–OH
23.0
316.0
26.0
8.9
7
I
8
5523
5524
I
–CH3
–CH3
–CH3
–NH–
–NH–
–O–
9
I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
5466
I
3.9
Immepipf
4929f
5870
II
II
II
II
II
III
III
III
III
IV
IV
IV
IV
IV
V
V
46.0
19.0
34.0
17.0
2.2
5653
5654
Immethridineg
5889g
5511
–CH2–
–CH2–
–N–
9.7 0.1
8.2 0.0
n.d.
288.0
191.0
0.6
–N–
–CH–
–CH–
–NH2
–NH2
–NH2
–OH
5512
n.d.
1.6
0.2
5803 (cis)
5804 (trans)
5805
7.6 0.3
7.9 0.1
7.4 0.1
n.d.
7.9
6.6
h
0.5
5796
5798
n.d.
1.1
n.d.
38.0
1.5
–OH
–NH2
–NH2
6.3 0.2
n.d.
n.d.
5.2 0.1
5.8 0.1
5.7 0.1
5801
5802
n.d.
n.d.
2.3
a The pKi values were measured by [3H]-Na-methylhistamine binding to membranes of SK-N-MC cells expressing the human H3 receptor in the
presence of the ligand.
b The pEC50 values were determined by the inhibition of the cAMP-stimulated b-galactosidase transcription in SK-N-MC cells stably expressing the
human H3 receptor. Results are presented as mean SEM of at least three independent experiments.
c a = intrinsic activity, as the ratio of the maximum response of each ligand to the maximum response of histamine.
d The pKi values were measured by [3H]-histamine for H4 binding to membranes of SK-N-MC cells expressing the human H4 receptor in the presence
of the ligand. Results are presented as mean SEM of at least three independent experiments.
e Selectivity = Ki(H4)/Ki(H3).
f See Ref. 27.
g See Ref. 26.
h Inverse agonism.
i n.d. = not determined.

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6315
hydroxyl analogue of impentamine (4) is the most potent
agonist with a pKi value of 8.0 and a pEC50 value of 8.1
at the human H3 receptor. Although the H3 receptor
affinity of 7 was not increased compared to impentamine
4 (Table 1), the H4 receptor affinity of 7 was considerably
lower (Table 1), resulting in an increased selectivity (320-
fold) at the human histamine H3 receptor over the H4
receptor compared to impentamine (Table 1). On the
other hand, a replacement of the side-chain amine with
a methyl group leads to ligands with both reasonable
H3 and H4 receptor affinity. For instance, compound
10 shows good affinity (pKi = 7.5) for the human H3
receptor and does not show a considerable decrease in
H4 receptor affinity as observed for the related hydroxy
compound 7. Consequently, compared to impentamine
(4) compound 10 shows a decreased selectivity for the
human H3 receptor over the H4 receptor (Table 1).
Previously, it has indeed been reported that high H3
receptor affinity could be obtained without charged or
basic groups in the side chain31–33 and the alkyl imidaz-
oles 8–10 previously proved to be interesting H3 receptor
antagonists, when tested on the guinea-pig intestine.34
Interestingly, compounds 5–10 all acted as full agonists
at the human H3 receptor in the recombinant test system
used in the present study (Table 1). This finding resembles
previous observations with other H3 receptor ligands.
With the cloning of the H3 receptor gene by Lovenberg
et al.12 and the availability of recombinant cellular test
systems we characterized the histamine homologues
imbutamine and impentamine and the thiourea burima-
mide as potent and effective agonists at the human H3
receptor35 (Table 1), although we previously had also
identified these compounds as potent H3 antagonists at
guinea-pig intestinal preparations.36 In view of the sur-
prising finding of H3 agonism of the alkyl imidazoles 8–
10, we also investigated in this study the pharmacological
characteristics of the acetylene-based H3 antagonist
(1S,2S)-4-(2-(5,5-dimethylhex-1-ynyl)cyclopropyl)imidaz-
ole (GT-2331, Fig. 2A).37,38 Previously, in vitro studies on
the guinea-pig ileum identified GT-2331 as a potent hista-
mine H3 antagonist. GT-2331 shows high affinity for the
cloned human H3 receptor stably expressed in SK-N-MC
cells (pKi = 8.1). Moreover, as found for the alkyl imidaz-
oles, GT-2331 also acts as an effective full agonist
(pEC50 = 7.7, a = 1.0) at the human H3 receptor
(Fig. 2B), confirming recent results by others.43 The ago-
nist actions of GT-2331 are competitively antagonized by
clobenpropit, which, as reported previously35, shows no
agonist effects at the human H3 receptor. Schild plot anal-
ysis of the obtained data yields the expected pA2 value of
9.5 for the H3 antagonist (Fig. 2B).
Figure 2. (A) Chemical structure of (1S,2S)-(+)-4-[2-(5,5-dimethylhex-
1-ynyl)-cyclopropyl]-1H-imidazole, GT-2331. (B) Agonistic action of
GT-2331 at the human H3 receptor and its competitive antagonism by
clobenpropit. The human H3 receptor was stably expressed in SK-N-
MC neuroblastoma cells, which were co-transfected with the b-
galactosidase gene under the control of cAMP-responsive elements. H3
agonism was measured as the inhibition of forskolin (10 lM)-induced
expression of b-galactosidase. The actions of GT-2331 were measured
in the absence (d) or presence of 10 (s), 100 (j) or 1000 nM (h) of
clobenpropit. The inset shows the transformation of the data by Schild
plot analysis, yielding a pA2 value of 9.5 for clobenpropit.
H4: pKi = 5.2) compared to immepip. Interestingly, the
cyclohexyl analogue 14 shows a significantly better H3
receptor affinity (pKi = 7.4) compared to the pyridine
compound 17 (Table 1). Like with the related impenta-
mine analogues, the non-basic immepip analogues 13
and 14 also act as full H3 receptor agonists (Table 1).
As shown in Table 1, the phenyl-based immethridine
analogues 18 and 19 possess a much lower affinity at
the human H3 receptor compared to immethridine
(16) or its longer homologue 17. Interestingly, no ma-
jor differences in affinity for the H4 receptor were ob-
served between 18 and immethridine (16) or 19 and
17 (Table 1). These data indicate that the H3 receptor
binding site is very efficiently targeted by the piperidine
and pyridine rings of immepip and immethridine,
respectively. These moieties contribute mainly to high
H3 receptor affinity and do not seem to be crucial for
H3 receptor activation.
In view of the fact that the amine functions of imbutamine
and impentamine are apparently not prerequisites for po-
tent H3 receptor agonism, we also examined the basic
function in the side chain of the potent H3 agonists imme-
pip (11) and immethridine (16). Replacement of the piper-
idine nitrogen of immepip 11 or its homologue VUF4929
(12) with an oxygen or carbon resulted in strong reduc-
tions in H3 and H4 receptor affinity (Table 1). The pyri-
dine analogue 17 displays a 400-fold lower affinity at
both the human H3 and H4 receptors (H3: pKi = 6.7;
To further examine the role of a basic amine in the side
chain, we incorporated cyclohexylamines (20–22), cyclo-

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R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
hexylalcohols (23 and 24) or anilines (25 and 26) and
tested these compounds for their affinities and effects
at the human H3 and H4 receptors. The cyclohexylam-
ines 20–22 can be seen as rigidified imbutamine (3) or
impentamine (4) analogues and lose approximately one
log unit affinity at the human H3 receptor. Interestingly,
the cis-(20) and trans-(21) isomers both act as agonists at
the H3 receptor and do not show any major difference in
their interaction with the human H3 receptor (Table 1
and Fig. 3A). In contrast, at the human H4 receptor
the cis-isomer (20) shows high affinity (pKi = 7.7), is as
effective as histamine or immepip at the H4 receptor
(pEC50 = 7.7, a = 1.1) and displays a 30-fold higher
affinity compared to the trans-isomer 21 (Fig. 3B,
pKi = 6.5, pEC50 = 6.0, a = 1). As such these two iso-
mers (20 and 21) can be regarded as interesting tools
for the human H4 receptor. An extension of the spacer
length between the imidazole ring and the cyclohexyl-
amine does not show improved affinity for the H3 or
H4 receptor (Tables 1 and 2), but interestingly, the long-
er homologue 22 behaves as a partial inverse agonist at
the human H3 receptor. Rigidification of the side chain
of impentamine 4 apparently not only causes a decrease
in affinity, but also causes 22 to prefer to bind to the
inactive form of the H3 receptor. Replacement of the
side-chain nitrogen of the cyclohexylamines (20–22) with
an alcohol (23 and 24) resulted in strongly decreased
affinity at both human histamine H3 and H4 receptors
(Table 1). Also the replacement of the cyclohexylamine
with an aniline resulted in strongly decreased affinities
at both the human histamine H3 and H4 receptors
(Table 1).
4. Conclusion
By changing the nature of the side chain of potent H3
receptor agonists like imbutamine and immepip a varie-
ty of interesting new tools for histamine H3 and H4
receptors have been identified. VUF5657 (7), a hydroxyl
analogue of impentamine (4), was identified as a potent
H3 receptor agonist with a 320-fold selectivity for the
human histamine H3 receptor over the H4 receptor. This
selectivity equals the selectivity of the recently reported
selective H3 agonist immethridine27 but VUF5657 shows
somewhat lower H3 receptor affinity.
VUF5803 and VUF5804, the cis- and trans-isomers of
the cyclohexylamine analogue of immepip, show inter-
esting differences in activities at the H4 receptor and
may become valuable tools in the H4 receptor field. Var-
ious alkylimidazoles and the related GT-2331 were effi-
cacious H3 receptor agonists, despite the fact that
these molecules lack any polar group in the side chain.
Most models for aminergic G-protein coupled receptors,
including the H3 receptor, assume that the amine moiety
of the agonists interacts with a conserved aspartate res-
idue in transmembrane domain three.19,25,26 Our results
give further evidence that replacement of the amine
group of H3 receptor agonists with non-basic groups
does not always lead to a significant loss in affinity
and agonist activity at the human H3 receptor.
5. Experimental section
5.1. Materials
Reagents were obtained from commercial suppliers
and used without further purification. Solvents used
were of either AR or HPLC grade. Dry THF and
DCM were obtained from distillation over lithium alu-
minium hydride and calcium hydride, respectively.
Compounds 1–4,36 8–1227,36 and 16–1927 were
obtained according to the literature procedures.
GT-2331 Perceptinꢂ, (1S,2S)-4-(2-(5,5-dimethylhex-1-
ynyl)cyclopropyl)imidazole was synthesized as de-
scribed earlier.42 Thin-layer chromatography was
carried out on Merck Kieselgel 60 F254 on aluminium
sheets and preparative flash chromatography was per-
formed on J. T. Baker Kieselgel 60 under pressure.
Melting points were determined with an Electrother-
mal IA9200 apparatus. 1H spectra were recorded on
a Bruker AC-200 spectrometer with the residual
undeuterated solvent peak as reference.
A
100
80
60
40
20
0
VUF5803 (20)
VUF5804 (21)
-11 -10 -9
-8
-7
-6
-5
log[compound]
B
100
80
60
40
20
0
VUF5803 (20)
VUF5804 (21)
6. Methods
6.1. 3-(1H-Imidazol-4-yl)-acrylic acid methyl ester
hydrochloride (27)
-11 -10 -9
-8
-7
-6
-5
log[compound]
Urocanic acid (1 g, 7.25 mmol) was dissolved in MeOH
(50 mL) and HCl gas was bubbled through the solution
for 10 min. The resulting solution was heated at reflux
temperature for 5 h and the solvent was evaporated to
obtain 27 as a white solid (1.35 g, 100%). 1H NMR
Figure 3. Comparison of the affinities between VUF5803 (20) and
VUF5804 (21) at the human histamine H3 (3A) and H4 receptors (3B)
indicates binding preference of the cis-isomer (VUF5803) for the
human H4 receptor.

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6317
(D2O): d 8.87 (s, 1H), 7.84 (s, 1H), 7.66 (d, 1H,
J = 17.2 Hz), 6.62 (d, 1H, J = 17.2 Hz), 3.88 (s, 3H).
MS, m/z 127.7 [M+1]+. Anal. (C6H11N2OBrÆ1.5H2O)
C, H, N. Mp 83.1–84.0 ꢁC.
6.2. 3-(1-Trityl-1H-imidazol-4-yl)-acrylic acid methyl
ester (28)
6.6. 4-(3-[1,3]Dioxolan-2-yl-propenyl)-1-trityl-1H-imid-
azole (31)
Compound 27 (1.25 g, 6.63 mmol) was suspended in
THF (50 mL) and triethylamine (2.3 mL, 16.6 mmol)
was added to the suspension at 0 ꢁC (ice bath). Triphe-
nylmethyl chloride (2.1 g, 7.53 mmol) dissolved in
THF (50 mL) was added dropwise and the mixture
was stirred at 0 ꢁC for 1 h followed by 16 h at room tem-
perature. The resulting solution was concentrated under
reduced pressure and the residue was dissolved in DCM
(50 mL). The solution was washed with water
(3 · 50 mL) and the organic layer was dried over sodium
sulfate. After evaporation of the solvent under reduced
pressure, the product (28) was recrystallised from
Diisopropylamine (0.8 mL, 5.9 mmol) was added to dry
THF (50 mL) and the solution was cooled to ꢀ30 ꢁC. n-
BuLi (3.7 mL, 1.6 M in n-hexane) was added dropwise
and stirred at ꢀ30 ꢁC for 15 min. Finely powdered
[2-(1,3-dioxolan-2-yl)ethyl]-triphenylphosphonium bro-
mide (2.6 g, 5.9 mmol) was added in one portion
and the resulting mixture was stirred at 0 ꢁC for 1 h. A
solution of trityl-imidazol-4-yl carboxaldehyde (2 g,
5.9 mmol) in THF (50 mL) was added dropwise and
the mixture was heated at reflux for 6 h. Saturated sodi-
um carbonate (50 mL) was added, the organic solvent
was separated and evaporated under reduced pressure.
The residue was dissolved in DCM (50 mL) and washed
with water (3 · 50 mL). The organic layer was dried
over sodium sulfate and concentrated under reduced
pressure. The crude product was purified by flash chro-
matography (ether) to give 31 as a light yellow solid
1
EtOAc to give a white solid. (1.96 g, 75%). H NMR
(CDCl3): d 7.55 (d, 1H, J = 17.2 Hz), 7.48 (s, 1H),
7.15–7.36 (m, 15H), 7.09 (s, 1H), 6.60 (d, 1H,
J = 17.2 Hz), 3.80 (s, 3H).
1
6.3. 3-(1-Trityl-1H-imidazol-4-yl)-propionic acid methyl
ester (29)
(2.1 g, 85%). H NMR (CDCl3): d 7.39 (s, 1H), 7.15–
7.35 (m, 15H), 6.79 (s, 1H), 6.36 (d, 1H, J = 11.7 Hz),
5.57 (dt, 1H, J = 11.7 and 7.1 Hz), 4.96 (t, 1H,
J = 4.8 Hz), 3.79–3.92 (m, 4H), 2.87 (m, 2H).
Compound 28 (1.5 g, 3.81 mmol) and 5% Pd/C
(150 mg) were added to MeOH (25 mL) and the mix-
ture was hydrogenated under a hydrogen atmosphere
for 4 h. The suspension was filtered through a Celite-
packed column and the filtrate was evaporated under
reduced pressure to give 29 as a white solid (1.5 g,
6.7. 4-(3-[1,3]Dioxolan-2-yl-propyl)-1-trityl-1H-imidazole
(32)
Compound 31 (2.0 g, 4.7 mmol) and 25 mg of 5% Pd/C
were added to MeOH (25 mL). The mixture was hydro-
genated under a hydrogen atmosphere at room temper-
ature for 4 h and filtered through a Celite-packed
column. The filtrate was concentrated under reduced
pressure and recrystallised from EtOH to give 32
1
99%). H NMR (CDCl3): d 7.15–7.40 (m, 16H), 6.67
(s, 1H), 3.49 (s, 3H), 2.65 (t, 2H, J = 7.8 Hz), 2.10
(t, 2H, J = 7.8 Hz).
6.4. 3-(1-Trityl-1H-imidazol-4-yl)-propan-1-ol (30)
1
as a yellow solid (2.0 g, 100%). H NMR (CDCl3): d
Compound 29 (1.25 g, 3.16 mmol) was added to a sus-
pension of LiAlH4 (0.24 g, 6.32 mmol) in dry THF
(25 mL) and the resulting mixture was heated at reflux
temperature for 6 h. Slowly, saturated sodium carbonate
(5 mL) was added and stirred until all LiAlH4 was de-
stroyed. The mixture was filtered through a Celite-
packed column and the filtrate was concentrated under
reduced pressure. DCM (25 mL) was added and the
resulting solution was washed with saturated sodium
carbonate (3 · 50 mL) and dried over sodium sulfate.
Concentration in vacuo gave 30 as a white solid
(0.81 g, 70%). 1H NMR (CDCl3): d 7.15–7.40 (m,
16H), 6.59 (s, 1H), 3.74 (t, 2H, J = 7.5 Hz), 2.69 (t,
2H, J = 7.5 Hz), 1.87 (quint, 2H, J = 7.5 Hz).
7.52 (s, 1H), 7.15–7.35 (m, 15H), 6.55 (s, 1H), 4.82
(t, 1H, J = 4.5 Hz), 3.79–3.94 (m, 4H), 2.64 (t, 2H,
J = 7.5 Hz), 1.69 (m, 4H).
6.8. 4-(1-Trityl-1H-imidazol-4-yl)-butyraldehyde (33)
Compound 32 (1.8 g, 4.25 mmol) was dissolved in ace-
tone (25 mL) and the solution was cooled in an ice bath.
1 N HCl was slowly added until the solution was pH 3.
The resulting solution was stirred at 0 ꢁC for 1 h and sat-
urated sodium carbonate (25 mL) was added. The mix-
ture was concentrated under reduced pressure, DCM
(25 mL) was added to the residue and the organic layer
was washed with water (3 · 25 mL), dried over sodium
sulfate and concentrated under reduced pressure. The
crude product was purified by flash chromatography
(EtOAc) to give 33 as a light yellow solid (1.5 g, 95%).
1H NMR (CDCl3): d 9.70 (s, 1H), 7.15–7.35 (m, 16H),
6.51 (s, 1H), 2.56 (t, 2H, J = 7.4 Hz), 2.42 (t, 2H,
J = 7.4 Hz), 1.96 (quint, 2H, J = 7.4 Hz).
6.5. 3-(1H-Imidazol-4-yl)-propan-1-ol hydrobromide (5)
Compound 30 (0.5 g, 1.36 mmol) was dissolved in 1 N
HBr (5 mL) and heated at reflux for 5 h. The mixture
was washed with ether (3 · 25 mL) and the aqueous
layer was concentrated under reduced pressure. The
product was recrystallised from EtOH/ether to give a
light yellow solid (0.13 g, 45%). 1H NMR (D2O): d
8.54 (s, 1H), 7.21 (s, 1H), 3.62 (t, 2H, J = 7.2 Hz), 2.74
(t, 2H, J = 7.2 Hz), 1.92 (quint, 2H, J = 7.2 Hz). APCI/
6.9. 4-(1-Trityl-1H-imidazol-4-yl)-butan-1-ol (34)
Using the procedure described for the synthesis for 30.
The title compound (34) was obtained from 33 (1.25 g,

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R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
3.29 mmol) as a light yellow solid (0.94 g, 75%). 1H NMR
(CDCl3): d 7.15–7.35 (m, 16H), 6.52 (s, 1H), 3.75 (t, 2H,
J = 7.5 Hz), 2.70 (t, 2H, J = 7.5 Hz), 1.72 (m, 4H).
6.14. 5-(1-Trityl-1H-imidazol-4-yl)-pentanoic acid ethyl
ester (38)
Using the procedure described for the synthesis for 29
but 37 (1.25 g, 2.86 mmol) was used. The title compound
(38) was obtained as a light yellow solid (1.25 g, 100%).
1H NMR (CDCl3): d 7.15–7.35 (m, 16H), 6.54 (s, 1H),
4.08 (q, 2H, J = 7.5 Hz), 2.65 (t, 2H, J = 7.4 Hz), 2.28
(t, 2H, J = 7.4 Hz), 1.52–1.84 (m, 4H), 1.20 (t, 3H,
J = 7.5 Hz).
6.10. 5-Hydroxy-5,6-dihydro-2H-imidazo[1,5-a]pyridin-4-
ium (35)
NMR (D2O): d 8.82 (s, 1H), 7.30 (s, 1H), 6.65 (d,
J = 11.4 Hz, 1H), 6.20 (m, J = 11.4, 1H), 6.10 (m, 1H),
2.75–2.85 (m, 2H). MS (m/z): 138.11 [M+1]+.
6.11. 4-(1H-Imidazol-4-yl)-butan-1-ol hydrochloride (6)
6.15. 5-(1-Trityl-1H-imidazol-4-yl)-pentan-1-ol (39)
Compound 33 (0.75 g, 1.96 mmol) was dissolved in 1 N
HCl (5 mL) and heated under reflux for 5 h. The mixture
was washed with ether (3 · 25 mL) and the aqueous layer
was concentrated under reduced pressure. The product
was recrystallised from EtOH/ether to give a light yellow
solid (0.14 g, 40%). 1H NMR (D2O): d 8.52 (s, 1H), 7.23
(s, 1H), 3.71 (t, 2H, J = 7.6 Hz), 2.79 (t, 2H, J = 7.6 Hz),
1.55–1.89 (m, 4H). APCI/MS, m/z 141.2 [M+1]+. Anal.
(C7H13N2OClÆ0.7H2O) C, H, N. Mp 89.8–91.2 ꢁC.
Using the procedure described for the synthesis for 30
but 38 (1.25 g, 2.85 mmol) was used. The title compound
(39) was obtained as a light yellow solid (0.9 g, 80%). 1H
NMR (CDCl3): d 7.15–7.35 (m, 16H), 6.55 (s, 1H), 3.64
(t, 2H, J = 7.5 Hz), 2.62 (t, 2H, J = 7.5 Hz), 1.52–1.84
(m, 4H), 1.41 (m, 2H).
6.16. 5-(1H-Imidazol-4-yl)-pentan-1-ol hydrochloride (7)
Using the procedure described for the synthesis for 6 but
39 (0.9 g, 2.28 mmol) was used. The title compound (7)
was recrystallised from EtOH/ether to give a light yellow
solid (0.23 g, 52%). 1H NMR (D2O): d 8.49 (s, 1H), 7.15
(s, 1H), 3.56 (t, 2H, J = 7.8 Hz), 2.72 (t, 2H, J = 7.8 Hz),
1.52–1.84 (m, 4H), 1.38 (m, 2H). APCI/MS, m/z 155.2
[M+1]+. Anal. (C8H15N2OClÆ0.3H2O) C, H, N. Mp
94.5–95.8 ꢁC.
6.12. 3-(1-Trityl-1H-imidazol-4-yl)-propionaldehyde (36)
A solution of oxalyl chloride (0.6 mL, 6.8 mmol) in dry
DCM (50 mL) was cooled to ꢀ60 ꢁC under a nitrogen
atmosphere. A solution of dimethylsulfoxide (1.0 mL,
14.1 mmol) in DCM (50 mL) was added dropwise and
subsequently stirred for 15 min at ꢀ60 ꢁC. Next, a solu-
tion of 29 (2 g, 5.4 mmol) in dry DCM (50 mL) was add-
ed dropwise and stirred for 45 min at ꢀ60 ꢁC.
Subsequently, triethylamine (2.5 mL, 17.8 mmol) was
added and the mixture was warmed to room tempera-
ture. The reaction mixture was washed with water
(3 · 100 mL) and dried over sodium sulfate. The solvent
was evaporated under reduced pressure. After the puri-
fication by flash chromatography (EtOAc), the product
(36) was obtained as a light yellow solid (1.54 g, 78%).
1H NMR (CDCl3): d 9.7 (s, 1H), 7.1–7.3 (m, 16H), 6.5
(s, 1H), 2.8–2.9 (m, 4H).
6.17. (Tetrahydropyran-4-ylidene)-acetic acid ethyl ester
(40)
Using the procedure described for the synthesis for 37
but tetrahydropyran-4-one (1 g, 10 mmol) of was used.
The title compound (40) was obtained as a light yellow
oil (1.5 g, 88%). 1H NMR (CDCl3): d 5.66 (s, 1H),
4.12 (q, 2H, J = 7.1 Hz), 3.72 (m, 4H), 3.00 (m, 2H),
2.32 (m, 2H), 1.24 (t, 3H, J = 7.1 Hz).
6.18. (Tetrahydropyran-4-yl)-acetic acid ethyl ester (41)
6.13. 5-(1-Trityl-1H-imidazol-4-yl)-pent-2-enoic acid
ethyl ester (37)
Using the procedure described for the synthesis for 38
but 40 (1.5 g, 8.8 mmol) was used. The title compound
(41) was obtained as a light yellow oil (1.5 g, 100%).
1H NMR (CDCl3): d 4.14 (q, 2H, J = 7.1 Hz), 4.08 (m,
2H), 3.38 (m, 2H), 2.20 (d, 2H, J = 7.2 Hz), 1.99 (m,
1H), 1.55 (m, 2H), 1.24–1.41 (m, 5H).
NaH (60% dispersed in mineral oil, 0.18 g, 4.5 mmol)
was suspended in THF (25 mL) and cooled in an ice
bath. A solution of triethyl phosphonoacetate (0.9 mL,
4.5 mmol) in THF (25 mL) was added dropwise. The
mixture was stirred at room temperature for 30 min
and a solution of 36 (1.50 g, 4.1 mmol) in THF
(25 mL) was added dropwise. The resulting mixture
was stirred for additional 6 h at room temperature,
cooled to 0 ꢁC and water (100 mL) was added. The mix-
ture was extracted with ether (3 · 50 mL), the combined
ether layers were dried over sodium sulfate and concen-
trated under reduced pressure. Purification by flash
chromatography (EtOAc) yielded 37 as a light yellow
6.19. 2-(Tetrahydropyran-4-yl)-ethanol (42)
Using the procedure described for the synthesis for 39
but 41 (1.5 g, 8.8 mmol) was used. The title compound
(42) was obtained as a colourless oil (0.77 g, 67%). H
1
NMR (CDCl3): d 3.95 (m, 2H), 3.68 (t, 2H,
J = 7.1 Hz), 3.44 (m, 2H), 1.30–1.62 (m, 7H).
1
solid (1.35 g, 75%). H NMR (CDCl3): d 7.39 (s, 1H),
6.20. (Tetrahydropyran-4-yl)-acetaldehyde (43)
7.15–7.35 (m, 15H), 6.76 (dt, 1H, J = 14.5 and 7.6 Hz),
6.52 (s, 1H), 5.61 (d, 1H, J = 14.5 Hz), 4.15 (q, 2H,
J = 7.5 Hz), 2.62 (t, 2H, J = 7.6 Hz), 2.45 (m, 2H),
1.21 (t, 3H, J = 7.5 Hz).
Using the procedure described for the synthesis for 36
but 42 (0.75 g, 5.8 mmol) was used. The title compound
(43) was obtained as a light yellow oil (0.55 g, 75%). H
1

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6319
NMR (CDCl3): d 9.77 (s, 1H), 3.94 (m, 2H), 3.40 (m,
2H), 2.40 (d, 2H, J = 7.0 Hz), 2.30 (m, 1H), 1.37–1.63
(m, 4H).
6.24. 1,4-Dioxa-spiro[4.5]decane-8-carboxylic acid meth-
yl ester (46)
Compound 45 (8.5 g, 54.4 mmol), ethylene glycol
(3.8 mL, 68.0 mmol) and p-toluenesulfonic acid monohy-
drate (1.1 g, 5.8 mmol) were dissolved in toluene
(250 mL) and heated at reflux in Dean–Stark apparatus
for 18 h. The reaction mixture was concentrated under re-
duced pressure and 50% NaHCO3 solution (200 mL) was
added. The resulting mixture was extracted with ether
(3 · 200 mL) and the combined organic layers were dried
over sodium sulfate and concentrated under reduced
pressure. The crude product was purified by bulb-to-bulb
distillation (138–145 ꢁC, 15 mmHg) to give the product
6.21. 4-(Tetrahydropyran-4-ylmethyl)-1H-imidazole
hydrochloride (13)
Finely powdered sodium cyanide (21.1 mg, 0.4 mmol)
was added in one portion to a stirred suspension of
tosylmethyl isocyanide (TosMIC) (0.92 g, 4.7 mmol)
and 43 (0.55 g, 4.3 mmol) in absolute ethanol (25 mL).
The reaction mixture became clear and the solution
was stirred for additional 2 h. The solvent was evaporat-
ed under reduced pressure and CHCl3 (25 mL) was add-
ed. The resulting mixture was filtered and the filtrate was
concentrated in vacuo to give a brown oil, which was
transferred to a stainless steel bomb. A saturated solu-
tion of ammonia in ethanol (50 mL) was added and
the mixture was heated at 90–110 ꢁC for 16 h. After
cooling to room temperature, the solvent was evaporat-
ed under reduced pressure. The residue was dissolved in
DCM (50 mL) and washed with 10% sodium carbonate
solution (50 mL) and water (2 · 50 mL). The organic
layer was dried over sodium sulfate and concentrated
under reduced pressure to give a dark brown oil which
was subsequently treated with activated charcoal. Filtra-
tion and purification using flash chromatography
(MeOH) gave the title compound (13) as a light-yellow
1
(46) as a colourless oil (9.8 g, 90%). H NMR (CDCl3):
d 3.95 (s, 4H), 3.68 (s, 3H), 2.29 (m, 1H), 1.40–2.0 (m, 8H).
6.25. (1,4-Dioxa-spiro[4.5]dec-8-yl)-methanol (47)
Using the procedure described for the synthesis for 30
but 46 (9.5 g, 47.5 mmol) was used. The title compound
(47) was obtained as a colourless oil (7.8 g, 95%). H
1
NMR (CDCl3): d 3.96 (s, 4H), 3.48 (d, 2H,
J = 7.5 Hz), 1.77 (m, 4H), 1.47 (m, 4H), 1.29 (m, 2H).
6.26. 1,4-Dioxa-spiro[4.5]decane-8-carbaldehyde (48)
Using the procedure described for the synthesis for 37
but 47 (7.5 g, 43.6 mmol) was used. The title compound
(48) was obtained as a light yellow oil (7.2 g, 97%). H
1
oil (0.54 g, 75%). H NMR (D2O): d 8.53 (s, 1H), 7.22
1
(s, 1H), 3.96 (m, 2H), 3.45 (m, 2H), 2.68 (d, 2H,
J = 7.0 Hz), 1.90 (m, 1H), 1.59 (m, 2H), 1.30 (m, 2H).
APCI/MS, m/z 167.2 [M+1]+. Anal. (C9H15N2OClÆ0.5-
H2O) C, H, N. Mp 142.9–144.2 ꢁC.
NMR (CDCl3): d 9.69 (s, 1H), 3.96 (s, 4H), 2.27 (m,
1H), 1.50–2.05 (m, 8H).
6.27. 4-(1,4-Dioxa-spiro[4.5]dec-8-yl)-1H-imidazole (49)
6.22. 4-Hydroxy-cyclohexanecarboxylic acid methyl ester
(44)
Using the procedure described for the synthesis for 13
but 48 (7.0 g, 41.1 mmol) was used. The title compound
(49) was obtained as a light yellow oil (6.4 g, 75%). H
NMR (CDCl3): d 7.60 (s, 1H), 6.79 (s, 1H), 3.96 (s,
4H), 2.72 (m, 1H), 2.09 (m, 2H), 1.55–1.90 (m, 6H).
1
In a stainless steel bomb, methyl p-hydroxybenzoate
(10 g, 65.8 mmol) and 5% Rh/C (1 g) were suspended
in THF (100 mL). The suspension was hydrogenated
under 100 atm hydrogen for 18 h. The mixture was fil-
tered through a Celite-packed column and the filtrate
was concentrated under reduced pressure. The crude
product was purified by bulb-to-bulb distillation (80–
100 ꢁC, 1 mmHg) to give the product (44) as a colourless
6.28. 4-(1H-Imidazol-4-yl)-cyclohexanone (50)
Compound 49 (6.2 g, 29.8 mmol) was dissolved in 1 N
HCl (150 mL) and stirred at room temperature for 1 h.
The resulting mixture was washed with CHCl3
(3 · 150 mL) and subsequently basified with sodium car-
bonate until pH ꢁ 12 and extracted with CHCl3
(3 · 150 mL). The combined CHCl3 layers were dried
over sodium sulfate and concentrated under reduced
pressure. Purification with flash chromatography
(MeOH) gave the product 50 as a light yellow oil
1
oil (10.2 g, 98%). H NMR (CDCl3): d 3.78 (m, 1H),
3.65 (s, 3H), 2.21–2.50 (m, 2H), 1.10–1.95 (m, 8H).
6.23. 4-Oxo-cyclohexanecarboxylic acid methyl ester (45)
Celite (10 g), sodium acetate (1.5 g, 19.0 mmol) and
pyridinium chlorochromate (20.5 g, 94.8 mmol) were
suspended in DCM (200 mL). Compound 44 (10 g,
63.2 mmol) was added dropwise and the mixture was
stirred at room temperature for 4 h. The resulting mix-
ture was filtered by suction through a column packed
with silica gel (75 g) and the silica gel was washed thor-
oughly with ether (250 mL). After concentration of the
filtrate under reduced pressure, the product (45) was
obtained as a colourless oil (8.9 g, 90%) and was used
1
(4.6 g, 94%). H NMR (CDCl3): d 7.61 (s, 1H), 6.82 (s,
1H), 3.12 (m, 1H), 2.20–2.55 (m, 6H), 1.80–1.95 (m, 2H).
6.29. 4-(4-Oxo-cyclohexyl)-imidazole-1-carboxylic acid
tert-butyl ester (51)
Compound 50 (4.5 g, 27.4 mmol) and triethylamine
(7.7 mL, 54.8 mmol) were dissolved in CHCl3 (100 mL).
The solution was cooled in an ice bath and a solution of
di-tert-butyldicarbonate (6.6 g, 30.1 mmol) in CHCl3
(100 mL) was added dropwise. The mixture was stirred
1
in the next step without further purification. H NMR
(CDCl3): d 3.71 (s, 3H), 2.78 (m, 1H), 1.89–2.55 (m,
8H).

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R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
at room temperature for 16 h and washed with saturated
sodium carbonate (3 · 200 mL). The organic layer was
dried over sodium sulfate and concentrated under re-
duced pressure. The product 51 was recrystallised from
ether as a light yellow solid (5.1 g, 70%). 1H NMR
(CDCl3): d 8.02 (s, 1H), 7.13 (s, 1H), 3.07 (m, 1H),
2.30–2.55 (m, 6H), 1.80–2.05 (m, 2H), 1.62 (s, 9H).
3.75 (m, 1H), 2.61 (m, 1H), 1.65–2.00 (m, 4H), 1.62 (s,
9H), 1.43 (s, 9H), 1.15–1.40 (m, 4H). Compound 53b
(trans) d 7.95 (s, 1H), 7.00 (s, 1H), 4.41 (br, 1H), 3.47
(m, 1H), 2.48 (m, 1H), 2.10 (m, 4H), 1.57 (s, 9H), 1.42
(s, 9H), 1.05–1.40 (m, 4H).
6.33. 4-(1H-Imidazol-4-yl)-cyclohexylamine dihydrobro-
mide (20 and 21)
6.30. 4-(4-Hydroxy-cyclohexyl)-imidazole-1-carboxylic
acid tert-butyl ester (52)
Using the procedure described for the synthesis for 23.
Compounds 53a or b (0.5 g, 1.4 mmol) of were used,
and the title products 20 and 21 were obtained, respec-
tively, as light yellow solids (0.20 and 0.25 g, respective-
Compound 51 (2.0 g, 7.5 mmol) was dissolved in MeOH
(50 mL) and sodium borohydride (0.3 g, 7.9 mmol) was
slowly added. The mixture was stirred at room temper-
ature for 16 h and concentrated under reduced pressure.
Saturated sodium carbonate (50 mL) was added and the
resulting mixture was washed with THF (3 · 50 mL).
The combined organic layers were dried over sodium
sulfate and concentrated. After flash chromatography
(MeOH), the product 52 was obtained as a light yellow
1
ly). H NMR (D2O): compound 20 (cis) d 8.56 (s, 1H),
7.26 (s, 1H), 3.43 (m, 1H), 3.09 (m, 1H), 1.96 (m, 6H),
1.68 (m, 2H). APCI/MS, m/z 166.3 [M+1]+. Anal.
(C9H17N3Br2Æ0.5H2O) C, H, N. Mp 241.2–242.4 ꢁC.
Compound 21 (trans) d 8.53 (s, 1H), 7.18 (s, 1H), 3.23
(m, 1H), 2.78 (m, 1H), 2.13 (m, 4H), 1.49 (m, 4H).
APCI/MS, m/z 166.6 [M+1]+. Anal. (C9H17N3Br2Æ0.3-
H2O) C, H, N. Mp > 300 ꢁC.
1
solid (1.3 g, 65%). H NMR (CDCl3): d 7.98 (s, 1H),
7.10 (s, 1H), 3.85 and 3.67 (m, 1H (cis and trans)),
2.65–2.85 (m, 1H), 2.00 (m, 4H), 1.65–1.85 (m, 2H),
1.62 (s, 9H), 1.40 (m, 2H).
6.34. (1,4-Dioxa-spiro[4.5]dec-8-ylidene)-acetic acid ethyl
ester (54)
6.31. 4-(1H-Imidazol-4-yl)-cyclohexanol hydrobromide
(23)
Using the procedure described for the synthesis for 40
but 1,4-Dioxa-spiro[4.5]decan-8-one (10.0 g, 64.0 mmol)
of was used. The title compound (54) was obtained as a
light yellow oil (13.8 g, 95%). H NMR (CDCl3): d 5.62
(s, 1H), 4.14 (q, 2H, J = 7.3 Hz), 3.98 (s, 4H), 2.95 (m,
2H), 2.33 (m, 2H), 1.72 (m, 4H), 1.21 (t, 3H, J = 7.3 Hz).
1
Compound 52 (1.0 g, 3.8 mmol) was added to 1 N HBr
(5 mL) and stirred at room temperature for 1 h. The sol-
vent was evaporated under reduced pressure and the
product 23 was recrystallised from EtOH/ether as a light
1
yellow solid (0.4 g, 45%). H NMR (D2O): d 8.47 and
6.35. (1,4-Dioxa-spiro[4.5]dec-8-yl)-acetic acid ethyl ester
(55)
8.50 (s, 1H (cis and trans)), 7.12 and 7.20 (s, 1H (cis
and trans)), 3.63 and 3.95 (m, 1H (cis and trans)),
2.68–2.85 (m, 1H), 2.00 (m, 2H), 1.65–1.85 (m, 2H),
1.40 (m, 4H). APCI/MS, m/z 167.6 [M+1]+. Anal.
(C9H15N2OBrÆ1.0H2O) C, H, N. Mp 167.2–169.0 ꢁC.
Using the procedure described for the synthesis for 41
but 54 (13.0 g, 57.4 mmol) was used. The title compound
(55) was obtained as a light yellow oil (13.2 g, 100%). 1H
NMR (CDCl3): d 4.14 (q, 2H, J = 7.5 Hz), 3.98 (s, 4H),
2.24 (d, 2H, J = 7.4 Hz), 1.45–1.95 (m, 9H), 1.25 (t, 3H,
J = 7.5 Hz).
6.32. 4-(4-tert-Butoxycarbonylamino-cyclohexyl)-imidaz-
ole-1-carboxylic acid tert-butyl ester (53a and b)
Compound 51 (2.0 g, 7.5 mmol), ammonium acetate
(5.8 g, 75.0 mmol) and sodium borohydride (0.3 g,
7.9 mmol) were added to MeOH (50 mL). The mixture
was stirred at room temperature for 16 h and concen-
trated under reduced pressure. Saturated sodium car-
bonate (50 mL) was added and the resulting mixture
was washed with THF (3 · 50 mL). The combined
organic layers were dried over sodium sulfate and con-
centrated. The residue was dissolved in CHCl3 (50 mL)
and triethylamine (2.1 mL, 15.0 mmol) was added. The
mixture was cooled in an ice bath and a solution of di-
tert-butyldicarbonate (1.6 g, 7.5 mmol) in CHCl3
(50 mL) was added dropwise. The mixture was stirred
at room temperature for 16 h and washed with saturated
sodium carbonate (3 · 500 mL). The organic layer was
dried over sodium sulfate and concentrated under re-
duced pressure. The products 53a and b were separated
by flash chromatography (n-hexane:DCM:EtOH (satu-
rated with NH3) 7.5:2:0.5) as a light yellow solid (0.5
6.36. 2-(1,4-Dioxa-spiro[4.5]dec-8-yl)-ethanol (56)
Using the procedure described for the synthesis for 42
but 55 (13.0 g, 57.0 mmol) was used. The title compound
(56) was obtained as a colourless oil (10.0 g, 95%). H
1
NMR (CDCl3):
d
J = 7.8 Hz), 1.20–1.95 (m, 11H).
3.98 (s, 4H), 3.67 (t, 2H,
6.37. (1,4-Dioxa-spiro[4.5]dec-8-yl)-acetaldehyde (57)
Using the procedure described for the synthesis for 43
but 56 (9.0 g, 48.4 mmol) was used. The title compound
(57) was obtained as a light yellow oil (8.0 g, 90%). H
NMR (CDCl3): d 9.75 (s, 1H), 3.98 (s, 4H), 2.45 (d,
2H, J = 7.8 Hz), 1.30–1.98 (m, 9H).
1
6.38. 4-(1,4-Dioxa-spiro[4.5]dec-8-ylmethyl)-1H-imidaz-
ole (58)
1
and 0.8 g, respectively). H NMR (CDCl3): compound
53a (cis) d 7.97 (s, 1H), 7.04 (s, 1H), 4.64 (br, 1H),
Using the procedure described for the synthesis for 44
but 57 (8.0 g, 43.3 mmol) was used. The title compound

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6321
1
(58) was obtained as a light yellow oil (6.8 g, 70%). H
NMR (CDCl3): d 7.52 (s, 1H), 6.70 (s, 1H), 3.89 (s,
4H), 2.56 (d, 2H, J = 7.2 Hz), 1.73 (m, 4H), 1.25–1.49
(m, 5H).
6.44. 4-(1H-Imidazol-4-ylmethyl)-cyclohexylamine
dihydrobromide (22)
Using the procedure described for the synthesis for 23
but 62 (1.0 g, 3.6 mmol) was used. The title compound
(22) was obtained as a light yellow oil (0.5 g, 41%). H
1
6.39. 4-(1H-Imidazol-4-ylmethyl)-cyclohexanone (59)
NMR (D2O): d 8.50 (s, 1H), 7.17 (s, 1H), 3.09 and
3.38 (m, 1H (cis and trans)), 2.60 (d, 2H, J = 7.0 Hz),
1.96 (m, 2H), 1.74 (m, 2H), 1.60 (m, 1H), 1.38 (m,
2H), 1.11 (m, 2H). APCI/MS, m/z 180.6 [M+1]+. Anal.
(C10H19N3Br2) C, H, N. Mp > 300 ꢁC.
Using the procedure described for the synthesis for 50
but 58 (6.8 g, 30.4 mmol) was used. The title compound
(59) was obtained as a light yellow oil (5.2 g, 95%). H
NMR (CDCl3): d 7.57 (s, 1H), 6.79 (s, 1H), 2.54 (d,
2H, J = 7.5 Hz), 1.95–2.35 (m, 7H), 1.30–1.45 (m, 2H).
1
6.45. 4-(4-Nitro-phenyl)-1H-imidazole (63)
6.40. 4-(4-Oxo-cyclohexylmethyl)-imidazole-1-carboxylic
acid tert-butyl ester (60)
4-Phenyl-imidazole (0.5 g, 3.47 mmol) was dissolved in
concentrated sulfuric acid (5 mL) at 0 ꢁC and fuming ni-
tric acid (0.15 mL) was added. The mixture was stirred
for 2 h and ice cubes (ca. 25 g) were added. The resulting
mixture was stirred for 10 min and filtered. The residue
was washed with water (25 mL). The filtrate was cooled
in an ice bath and basified with 10% sodium hydroxide
solution until the pH was basic. The yellow suspension
was filtered and the remained solid was washed with
water (50 mL). The product (63) was purified by flash
chromatography (EtOAc) as a yellow solid (0.3 g,
Using the procedure described for the synthesis for 51
but 59 (5.0 g, 28.0 mmol) was used. The title compound
(60) was obtained as a light yellow oil (5.8 g, 75%). H
1
NMR (CDCl3): d 8.05 (s, 1H), 7.13 (s, 1H), 2.57 (d,
2H, J = 7.5 Hz), 2.39 (m, 4H), 2.10 (m, 3H), 1.67 (s,
9H), 1.40–1.65 (m, 2H).
6.41. 4-(4-Hydroxy-cyclohexylmethyl)-imidazole-1-
carboxylic acid tert-butyl ester (61)
1
45%). H NMR (CDCl3): d 8.00 (d, 2H, J = 7.8 Hz),
7.72 (d, 2H, J = 7.8 Hz), 7.47 (s, 1H), 7.29 (s, 1H).
Using the procedure described for the synthesis for 53
but 60 (2.9 g, 10.4 mmol) was used. The title compound
(61) was obtained as a light yellow oil (2.1 g, 70%). H
6.46. 4-(1H-Imidazol-4-yl)-phenylamine dihydrobromide
(25)
1
NMR (CDCl3): d 8.05 (s, 1H), 7.10 (s, 1H), 3.65 and
4.01 (m, 1H (cis and trans)), 2.45 and 2.50 (d, 2H (cis
and trans), J = 7.5 Hz), 1.89 (m, 2H), 1.70 (m, 2H),
1.62 (s, 9H), 0.95–1.60 (m, 5H).
Compound 63 (0.25 g, 1.32 mmol), 5% Pd/C (25 mg)
and 1 N HBr (5 mL) were added to MeOH (25 mL).
The mixture was hydrogenated under hydrogen atmo-
sphere at room temperature for 4 h and filtered through
a Celite-packed column. The filtrate was concentrated
under reduced pressure and recrystallised from EtOH
6.42. 4-(1H-Imidazol-4-ylmethyl)-cyclohexanol hydrobro-
mide (24)
1
to give 25 as a yellow solid (0.27 g, 65%). H NMR
Using the procedure described for the synthesis for 23
but 61 (2.0 g, 7.1 mmol) was used. The title compound
(24) was obtained as a light yellow solid (0.6 g, 35%).
1H NMR (D2O): d 8.52 (s, 1H), 7.19 (s, 1H), 3.57 and
3.92 (m, 1H (cis and trans)), 2.60 (d, 2H, J = 7.5 Hz),
1.97 (m, 2H), 1.78 (m, 2H), 1.62 (m, 1H), 1.00–1.45
(m, 4H). APCI/MS, m/z 181.2 [M+1]+. Anal.
(C10H17N2OBrÆ0.5H2O) C, H, N. Mp 167.2–168.9 ꢁC.
(D2O): d 8.79 (s, 1H), 7.82 (m, 3H), 7.53 (d, 2H,
J = 7.8 Hz). APCI/MS, m/z 160.3 [M+1]+. Anal.
(C9H11N3Br2) C, H, N. Mp > 300 ꢁC.
6.47. (4-Nitro-phenyl)-(1-trityl-1H-imidazol-4-yl)-metha-
nol (64)
1-Trityl-4-iodo-imidazole (2 g, 4.58 mmol) was dissolved
in DCM (50 mL), ethylmagnesium bromide (3.0 M in
THF, 1.6 mL) was added and the resulting mixture
was stirred at room temperature for 45 min. 4-Nitro-
benzaldehyde (0.76 g, 5.04 mmol) dissolved in DCM
(50 mL) was added dropwise and stirred at room tem-
perature for 16 h. The mixture was washed with saturat-
ed sodium carbonate solution (3 · 50 mL) and dried
over anhydrous sodium sulfate. Evaporation of the sol-
vent and recrystallisation from EtOAc yielded 64 as a
pale yellow solid (1.8 g, 85%). 1H NMR (CDCl3): d
8.14 (d, 2H, J = 7.8 Hz), 7.56 (d, 2H, J = 7.8 Hz), 7.41
(s, 1H), 7.04–7.32 (m, 15H), 6.60 (s, 1H), 5.83 (s, 1H).
6.43. 4-(4-Amino-cyclohexylmethyl)-imidazole-1-carbox-
ylic acid tert-butyl ester (62)
Compound 61 (2.9 g, 10.4 mmol), ammonium acetate
(8.0 g, 104.0 mmol) and sodium borohydride (0.4 g,
10.6 mmol) were added to MeOH (50 mL). The mixture
was stirred at room temperature for 16 h and concen-
trated under reduced pressure. Saturated sodium car-
bonate (50 mL) was added and the resulting mixture
was washed with THF (3 · 50 mL). The combined
organic layers were dried over sodium sulfate and con-
centrated. After flash chromatography (MeOH), the
product (62) was obtained as a light yellow oil (2.0 g,
6.48. Acetic acid (4-nitro-phenyl)-(1-trityl-1H-imidazol-4-
yl)-methyl ester (65)
1
70%). H NMR (CDCl3): d 8.12 (s, 1H), 7.15 (s, 1H),
2.76 and 3.08 (m, 1H (cis and trans)), 2.52 (m, 2H),
1.68–1.87 (m, 2H), 1.60 (s, 9H), 1.45 (m, 3H), 0.90–
1.25 (m, 4H).
Compound 64 (1.5 g, 3.25 mmol) and 4-dimethylami-
nopyridine (DMAP, 0.04 g, 0.33 mmol) were dissolved

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R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
in DCM (50 mL). The solution was cooled in an ice
bath and acetic anhydride (0.5 mL, 5 mmol) was add-
ed dropwise and stirred for 1 h and heated under re-
flux for 6 h. After cooling to the room temperature,
saturated sodium carbonate (50 mL) was added. The
organic layer was separated, washed with water
(3 · 50 mL), dried over sodium sulfate and evaporat-
ed under reduced pressure. The product (65) was
recrystallised from EtOAc as a light yellow solid
(1.55 g, 95%). 1H NMR (CDCl3): d 8.12 (d, 2H,
J = 7.8 Hz), 7.56 (d, 2H, J = 7.8 Hz), 7.43 (s, 1H),
7.04–7.35 (m, 15H), 6.77 (s, 1H), 6.72 (s, 1H), 2.14
(s, 3H).
7. Pharmacology
Radioligand displacement studies were performed as
follows; SK-N-MC cells, a human neuroblastoma cell
line, stably expressing the human histamine H3(445aa)
or H4 receptor12,39 were grown at 37 ꢁC in a humidified
atmosphere with 5% CO2 in minimum essential medi-
um eagle (EMEM), supplemented with foetal calf ser-
um (10%, v/v), penicillin (50 IU/ml), non-essential
amino acids, L-glutamine (2 mM), streptomycin
(50 lg/ml) and sodium pyruvate (50 lg/ml) in the pres-
ence of G418 (600 lg/ml). H3 and H4 receptor affinity
was determined by [3H]-Na-methylhistamine (82.0 Ci/
mmol, NEN Dupont, Boston MA, USA) and [3H]-
histamine (23.2 Ci/mmol, NEN Dupont, Boston MA,
USA) binding as described.40 Binding data were
analyzed using Graphpad Prism 3.0; Ki values were
calculated from the IC50 according to the Cheng and
Prusoff equation.41
6.49. 4-(1H-Imidazol-4-ylmethyl)-phenylamine dihydro-
bromide (26)
Compound 65 (1.25 g, 2.48 mmol) and 10% Pd/C
(0.15 g) were added to MeOH (25 mL). The mixture
was stirred under a hydrogen atmosphere at room tem-
perature for 4 h and filtered through a Celite-packed col-
umn. HBr (5 mL) was added and the solution was
concentrated under reduced pressure. The title com-
pound (26) was recrystallised from EtOH/ether as a light
The functional experiments were performed with SK-N-
MC cells, stably co-expressing the human H3 or H4
receptor and the b-galactosidase gene under the control
of cAMP-responsive elements. In these experiments,
cells were stimulated for 6 h with forskolin and hista-
mine receptor ligands, whereafter the cells were lysed
and b-galactosidase activity was measured colourimetri-
cally as described previously.27
1
yellow solid (0.45 g, 55%). H NMR (D2O): d 8.61 (s,
1H), 7.42 (m, 4H), 7.25 (s, 1H), 4.17 (s, 2H). APCI/
MS, m/z 174.2 [M+1]+. Anal. (C10H13N3Br2) C, H, N.
Mp > 300 ꢁC.
Appendix A
Compound VUF Mp (ꢁC)
APCI/MS [M+1]+ Formula
Elemental analysis (C, H, N)
5
5698
5793
5657
5870
5803
83.1–84.0
89.8–91.2
94.5–95.8
127.7
141.2
155.2
C6H11N2OBrÆ1.5H2O
C7H13N2OClÆ0.7H2O
C8H15N2OClÆ0.3H2O
C9H15N2OClÆ0.5H2O
C9H17N3Br2Æ0.5H2O
C9H17N3Br2Æ0.3H2O
C10H19N3Br2
Calcd (30.8, C; 6.0, H; 12.0, N)
Anal. (30.94, C; 5.85, H; 12.2, N)
Calcd (44.4, C; 7.7, H; 14.8, N)
Anal. (44.26, C; 7.83, H; 14.6, N)
Calcd (48.8, C; 8.0, H; 14.2, N)
Anal. (48.87, C; 8.06, H; 14.2, N)
Calcd (51.1, C; 7.6, H; 13.2, N)
Anal. (50.97, C; 7.48, H; 13.2, N)
Calcd (32.2, C; 5.4, H; 12.5, N)
Anal. (32.28, C; 5.35, H; 12.6, N)
Calcd (32.5, C; 5.3, H; 12.6, N)
Anal. (32.47, C; 5.44, H; 12.5, N)
Calcd (35.2, C; 5.6, H; 12.3, N)
Anal. (35.02, C; 5.58, H; 12.3, N)
Calcd (40.8, C; 6.5, H; 10.6, N)
Anal. (40.67, C; 6.43, H; 10.5, N)
6
7
13
20
21
22
23
24
25
26
142.9–144.2 167.2
241.2–242.4 166.3
5804 >300
5805 >300
166.6
180.6
5796
5798
167.2–169.0 167.2
167.2–168.9 181.2
C9H15N2OBrÆ1.0H2O
C10H17N2OBrÆ0.5H2O Calcd (44.5, C; 6.7, H; 10.4, N)
Anal. (44.45, C; 6.63, H; 10.4, N)
C9H11N3Br2
C10H13N3Br2
5801 >300
5802 >300
160.3
174.2
Calcd (33.7, C; 3.5, H; 13.1, N)
Anal. (33.76, C; 3.32, H; 13.0, N)
Calcd (35.8, C; 3.9, H; 12.5, N)
Anal. (36.02, C; 3.78, H; 12.5, N)

R. Kitbunnadaj et al. / Bioorg. Med. Chem. 13 (2005) 6309–6323
6323
23. Gantz, I.; DelValle, J.; Wang, L. D.; Tashiro, T.; Munzert,
G., et al. J. Biol. Chem. 1992, 267, 20840–20843.
24. Stark, H.; Sippl, W.; Ligneau, X.; Arrang, J. M.; Ganellin,
C. R., et al. Bioorg. Med. Chem. Lett. 2001, 11, 951–
954.
25. Uveges, A. J.; Kowal, D.; Zhang, Y.; Spangler, T. B.;
Dunlop, J., et al. J. Pharmacol. Exp. Ther. 2002, 301, 451–
458.
References and notes
1. Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Nature 1983,
302, 832–837.
2. Arrang, J. M.; Schwartz, J. C.; Schunack, W. Eur. J.
Pharmacol. 1985, 117, 109–114.
3. Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Neuroscience
1987, 23, 149–157.
26. Yao, B. B.; Hutchins, C. W.; Carr, T. L.; Cassar, S.;
Masters, J. N., et al. Neuropharmacology 2003, 44, 773–
786.
27. Kitbunnadaj, R.; Zuiderveld, O. P.; Christophe, B.;
Hulscher, S.; Menge, W. M. J. Med. Chem. 2004, 47,
2414–2417.
28. Stark, H.; Purand, K.; Huls, A.; Ligneau, X.; Garbarg,
M., et al. J. Med. Chem. 1996, 39, 1220–1226.
29. Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651–1660.
30. Horne, D. A.; Yakushijin, K.; Buchi, G. Heterocycles
1994, 391, 39–153.
31. Sasse, A.; Sadek, B.; Ligneau, X.; Elz, S.; Pertz, H. H.,
et al. J. Med. Chem. 2000, 43, 3335–3343.
32. Sasse, A.; Stark, H.; Ligneau, X.; Elz, S.; Reidemeister, S.,
et al. Bioorg. Med. Chem. 2000, 8, 1139–1149.
33. Pelloux-Leon, N.; Fkyerat, A.; Piripitsi, A.; Tertiuk, W.;
Schunack, W., et al. J. Med. Chem. 2004, 47, 3264–
3274.
4. Schwartz, J. C.; Arrang, J. M.; Garbarg, M.; Pollard, H.;
Ruat, M. Physiol. Rev. 1991, 71, 1–51.
5. Schlicker, E.; Malinowska, B.; Kathmann, M.; Gothert,
M. Fundam. Clin. Pharmacol. 1994, 8, 128–137.
6. Haas, H.; Panula, P. Nat. Rev. Neurosci. 2003, 4, 121–130.
7. Onodera, K.; Watanabe, T. Nihon Shinkei Seishin Yak-
urigaku Zasshi 1995, 15, 87–102.
8. Leurs, R.; Blandina, P.; Tedford, C.; Timmerman, H.
Trends Pharmacol. Sci. 1998, 19, 177–183.
9. Stark, H.; Arrang, J. M.; Ligneau, X.; Garbarg, M.;
Ganellin, C. R., et al. Prog. Med. Chem. 2001, 38, 279–
308.
10. De Esch, I. J.; Mills, J. E.; Perkins, T. D.; Romeo, G.;
Hoffmann, M., et al. J. Med. Chem. 2001, 44, 1666–1674.
11. Sasse, A.; Stark, H.; Reidemeister, S.; Huls, A.; Elz, S.,
et al. J. Med. Chem. 1999, 42, 4269–4274.
12. Lovenberg, T. W.; Roland, B. L.; Wilson, S. J.; Jiang, X.;
Pyati, J., et al. Mol. Pharmacol. 1999, 55, 1101–1107.
13. Lovenberg, T. W.; Pyati, J.; Chang, H.; Wilson, S. J.;
Erlander, M. G. J. Pharmacol. Exp. Ther. 2000, 293, 771–
778.
14. Drutel, G.; Peitsaro, N.; Karlstedt, K.; Wieland, K.; Smit,
M. J., et al. Mol. Pharmacol. 2001, 59, 1–8.
15. Tardivel-Lacombe, J.; Rouleau, A.; Heron, A.; Morisset,
S.; Pillot, C., et al. Neuroreport 2000, 11, 755–759.
16. Carson, M. R.; Shasby, S. S.; Shasby, D. M. Am. J.
Physiol. 1989, 257, L259–L264.
17. Gantz, I.; Schaffer, M.; DelValle, J.; Logsdon, C.; Camp-
bell, V., et al. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 429–
433.
34. De Esch, I. J.; Gaffar, A.; Menge, W. M.; Timmerman, H.
Bioorg. Med. Chem. 1999, 7, 3003–3009.
35. Wieland, K.; Bongers, G.; Yamamoto, Y.; Hashimoto, T.;
Yamatodani, A., et al. J. Pharmacol. Exp. Ther. 2001,
299, 908–914.
36. Vollinga, R. C.; Menge, W. M.; Leurs, R. J. Med. Chem.
1995, 38, 266–271.
37. Tedford, C. E.; Hoffmann, M.; Seyedi, N.; Maruyama, R.;
Levi, R., et al. Eur. J. Pharmacol. 1998, 351, 307–311.
38. Tedford, C. E.; Phillips, J. G.; Gregory, R.; Pawlowski, G.
P.; Fadnis, L., et al. J. Pharmacol. Exp. Ther. 1999, 289,
1160–1168.
39. Liu, C.; Ma, X.; Jiang, X.; Wilson, S. J.; Hofstra, C. L.,
et al. Mol. Pharmacol. 2001, 59, 420–426.
18. Karchin, R.; Karplus, K.; Haussler, D. Bioinformatics
2002, 18, 147–159.
40. Kitbunnadaj, R.; Zuiderveld, O. P.; De Esch, I. J.;
Vollinga, R. C.; Bakker, R., et al. J. Med. Chem. 2003,
46, 5445–5457.
41. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099–3108.
42. Liu, H.; Kerdesky, F. A.; Black, L. A.; Fitzgerald, M.;
Henry, R., et al. J. Org. Chem. 2004, 69, 192–194.
43. Wulff, B. S.; Hastrup, S.; Rimvall, K. Eur. J. Pharmacol.
2002, 453, 33–41.
19. Trumpp-Kallmeyer, S.; Hoflack, J.; Bruinvels, A.; Hibert,
M. J. Med. Chem. 1992, 35, 3448–3462.
20. Prasad Ratnala, V. R.; Hulsbbergen, F. B.; de Groot, H.
J.; de Grip, W. J. Inflamm. Res. 2003, 52, 417–423.
21. ter Laak, A. M.; Venhorst, J.; Donne-Op den Kelder, G.
M.; Timmerman, H. J. Med. Chem. 1995, 38, 3351–3360.
22. ter Laak, A. M.; Timmerman, H.; Leurs, R.; Nederkoorn,
P. H.; Smit, M. J., et al. J. Comput. Aided Mol. Des. 1995,
9, 319–330.