Influence of bulky substituents on histamine H3 receptor agonist/antagonist properties

Article abstract of DOI:10.1021/jm020910m

Novel derivatives of 3-(1H-imidazol-4-yl)propanol were designed on the basis of lead compounds belonging to the carbamate or ether series possessing (partial) agonist properties on screening assays of the histamine H₃ receptor. One pair of enantiomers in the series of α-methyl-branched chiral carbamates was stereoselectively prepared in high optical yields. Enantiomeric purity was checked by Mosher amide derivatives of precursors and capillary electrophoresis of the final compounds with trimethyl-β-cyclodextrin as chiral selector, and was determined to be ≥95%. The novel compounds were investigated in various histamine H₃ receptor assays in vitro and in vivo. Some compounds displayed partial agonist activity on synaptosomes of rat brain cortex, whereas others exhibited antagonist properties only. Selected compounds were investigated in [¹²⁵I]iodoproxyfan binding studies on the human histamine H₃ receptor and showed high affinity in the nanomolar concentration range. Under in vivo conditions after oral administration to mice, some of the compounds exhibited partial or full agonist activity in the brain at low dosages. The (S)-enantiomer of one pair of chiral carbamates (9) proved to be the eutomer; thus, the (S)-enantiomer was selected for further pharmacological studies. In a peripheral in vivo test model in rats, measuring the level of inhibition of capsaicin-induced plasma extravasation, (S)-9 again proved its high oral agonist potency with full intrinsic activity (ED₅₀ values of 0.07-0.1 mg/kg depending on tissue).

Full text of DOI:10.1021/jm020910m

4000
J . Med. Chem. 2002, 45, 4000-4010
In flu en ce of Bu lk y Su bstitu en ts on Hista m in e H3 Recep tor Agon ist/An ta gon ist
P r op er ties†
Astrid Sasse,‡,§ Xavier Ligneau,| Agne`s Rouleau,§ Sigurd Elz, C. Robin Ganellin,@ J ean-Michel Arrang,§
J ean-Charles Schwartz,§ Walter Schunack,‡ and Holger Stark*,#
Institut fu¨r Pharmazie, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 2+4, 14195 Berlin, Germany, Laboratoire Bioprojet,
30 rue des Francs-Bourgeois, 75003 Paris, France, Institut fu¨r Pharmazie, Universita¨t Regensburg, Universita¨tsstrasse 31,
93040 Regensburg, Germany, Department of Chemistry, Christopher Ingold Laboratories, University College London,
20 Gordon Street, London WC1H 0AJ , U.K., Unite´ de Neurobiologie et Pharmacologie Mole´culaire (U. 109),
Centre Paul Broca de l’INSERM, 2ter rue d’Ale´sia, 75014 Paris, France, and Institut fu¨r Pharmazeutische Chemie,
Biozentrum, J ohann Wolfgang Goethe-Universita¨t, Marie-Curie-Strasse 9, 60439 Frankfurt/ Main, Germany
Received April 15, 2002
Novel derivatives of 3-(1H-imidazol-4-yl)propanol were designed on the basis of lead compounds
belonging to the carbamate or ether series possessing (partial) agonist properties on screening
assays of the histamine H3 receptor. One pair of enantiomers in the series of R-methyl-branched
chiral carbamates was stereoselectively prepared in high optical yields. Enantiomeric purity
was checked by Mosher amide derivatives of precursors and capillary electrophoresis of the
final compounds with trimethyl-â-cyclodextrin as chiral selector, and was determined to be
g95%. The novel compounds were investigated in various histamine H3 receptor assays in
vitro and in vivo. Some compounds displayed partial agonist activity on synaptosomes of rat
brain cortex, whereas others exhibited antagonist properties only. Selected compounds were
investigated in [125I]iodoproxyfan binding studies on the human histamine H3 receptor and
showed high affinity in the nanomolar concentration range. Under in vivo conditions after
oral administration to mice, some of the compounds exhibited partial or full agonist activity in
the brain at low dosages. The (S)-enantiomer of one pair of chiral carbamates (9) proved to be
the eutomer; thus, the (S)-enantiomer was selected for further pharmacological studies. In a
peripheral in vivo test model in rats, measuring the level of inhibition of capsaicin-induced
plasma extravasation, (S)-9 again proved its high oral agonist potency with full intrinsic activity
(ED50 values of 0.07-0.1 mg/kg depending on tissue).
Ch a r t 1
In tr od u ction
In 1983, the histamine H3 receptor was first described
pharmacologically as an inhibitory autoreceptor on
histaminergic neurons in the central nervous system.3
Many highly selective and potent histamine H3 receptor
agonists and antagonists have been developed for
pharmacological characterization, e.g., radioligands,4
but also for therapeutical implications, e.g., BP 2-94,5,6
GT-23317 (cipralisant, formerly Perceptin), and ciproxi-
fan.8,9 In 1999, the human histamine H3 receptor cDNA
was discovered by Lovenberg et al.10 Binding properties
and functional investigations with known histamine H3
receptor ligands were performed. In following experi-
ments, species differences between human and rat H3
receptors were observed11,12 as well as high constitutive
activity of both H3 receptors.13-15 Therapeutic targets
for histamine H3 receptor agonists have been proposed
in neurogenic inflammation (e.g., airways and urinary
bladder), migraine and sleep disorders.16 In the search
for novel histamine H3 receptor agonists, which lack a
basic moiety in the side chain of the molecule, thus
improving pharmacokinetic properties such as resorp-
tion and penetration of the blood-brain barrier, car-
bamate and ether derivatives of 3-(1H-imidazol-4-yl)-
propanol with high agonist in vivo potencies have been
described.17,18 Derived from FUB 31618 (carbamate),
FUB 40717 (ether), and FUB 47517 (carbamate) as lead
compounds (Chart 1), new aliphatic ethers and carbam-
ates were designed as potential histamine H3 receptor
agonists and pharmacologically described in this study.
As one of the novel carbamate racemates [(R/S)-9]
exhibited interesting pharmacological behavior in stand-
† Presented in part at the XXIXth Annual Meeting of the European
Histamine Research Society (EHRS), Nemi/Rome, Italy, May 17-21,
2000, the Annual Meeting of the Deutsche Pharmazeutische Gesell-
schaft (DPhG), Mu¨nster, Germany, October 5-7, 2000, the Inter-
national Sendai Histamine Symposium, Sendai, J apan, November 22-
25, 2000, and the XXXth Annual Meeting of the EHRS, Turku, Finland,
May 10-13, 2001.1 Part of Int. Pat. Appl. WO 96/29 315.2
* To whom correspondence should be addressed. Telephone:
+49-69-789 29302. Fax: +49-69-798 29258. E-mail: h.stark@
pharmchem.uni-frankfurt.de.
‡ Freie Universita¨t Berlin.
§ Centre Paul Broca de l’INSERM.
| Laboratoire Bioprojet.
Universita¨t Regensburg.
@ University College London.
#
J ohann Wolfgang Goethe-Universita¨t.
10.1021/jm020910m CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/30/2002

Histamine H3 Receptor Agonist/ Antagonist Properties
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4001
Sch em e 1. Synthesis of Carbamates 1-11a
corresponding isocyanate. The alcoholic component for
building up the carbamate, 3-(1H-imidazol-4-yl)propanol‚
HCl, was then added to this one-pot two-step reaction
(Scheme 1, route 2). Amines needed for carbamate
synthesis by route 1 (Scheme 1) were either com-
mercially available [4a , 8a , 9a , 11a , (R)-11a , and (S)-
11a ] or synthesized from appropriate ketones/oximes as
outlined in Scheme 2. Amines 1b-3b were synthesized
by reductive amination as shown for 1b (Scheme 2). In
the presence of ammonium acetate, as a buffer and
nitrogen donor, ketones preferably react to imines,
which are rapidly reduced by NaBH3CN as described
by Borch et al.23 Competing direct reduction of ketones
was avoided by properly controlling the pH with am-
monium acetate, as ketones are reduced at lower pH
than imines. The sterically highly hindered di-tert-butyl
ketoxime 5a was commercially available and reduced
to amine 5b with TiCl4/NaBH4.24
a
(a) ClCOOCCl3, charcoal (cat.), ethyl acetate, reflux for 4-5
h; (b) DPPA, Et3N, dioxane, reflux for 30 min; (c) 3-(1H-imidazol-
4-yl)propanol‚HCl,21 acetonitrile, reflux for 4-5 h.
Chiral amines (R)-9d and (S)-9d were synthesized by
a stereoselective three-step reaction procedure shown
in Scheme 2 for (S)-9d , first described by Charles et al.25
However, the optimized reaction protocol developed by
Moss et al.26 was applied, leading to the high enantio-
meric purity of the optically active products by working
at low temperature for the formation of the Schiff base
and mild reaction conditions for the reduction to the
secondary amine. The prochiral ketone pinacolone (9a )
is transformed to the corresponding (E)-Schiff base with
an enantiomerically pure amine, e.g., (S)-1-phenylethan-
1-amine, leading to (S)-9b. By reduction of (S)-9b under
mild conditions, a diastereomeric secondary amine (S,S)-
9c is formed, its chirality determined by the chiral
auxiliary amine used for the initial formation of the
Schiff base [e.g., (S)-1-phenylethan-1-amine, Scheme 2].
Purification of the diastereomeric amine (S,S)-9c from
traces of the (R,S)-diastereomer was easily achieved by
column chromatography and repeated recrystallization.
Subsequent liberation of the enantiomerically pure
primary amine (S)-9d was performed by debenzylation
according to mild standard deprotection methods (Scheme
2). Racemic amines 7d and 10d were prepared by the
same procedure, using (RS)-1-phenylethan-1-amine and
benzylamine, respectively. Enantiomeric purity of the
stereoselectively synthesized amines (S)- and (R)-9d was
checked by preparing the corresponding Mosher amide
derivatives27 and determining the diastereomeric ratios
ard in vitro and in vivo screening assays, its enanti-
omers were synthesized stereoselectively. Enantiomeric
purity of the enantiomers was determined by capillary
electrophoresis (CE) by a method developed for struc-
turally similar optically active compounds.19,20 For
selected compounds, additional pharmacological inves-
tigations were performed such as [125I]iodoproxyfan
binding studies on the human H3 receptor, stably
transfected into CHO-K1 cells as well as peripheral in
vivo agonist activity based on a capsaicin-induced
plasma extravasation assay in rats after oral treatment,
and a receptor profile.
Ch em istr y
3-(1H-Imidazol-4-yl)propanol in its unprotected and
trityl-protected form was synthesized as described
previously from urocanic acid.21 Carbamates (1-5 and
7-11) were prepared from appropriate amines by reac-
tion of the amine with diphosgene, forming an inter-
mediate isocyanate, which was isolated from the reac-
tion mixture by careful distillation. The isocyanate that
was obtained was then added to 3-(1H-imidazol-4-yl)-
propanol‚HCl (Scheme 1, route 1). 2,2,3,3-Tetramethyl-
cyclopropyl carbamate 6 was synthesized from 2,2,3,3-
tetramethylcarboxylic acid by a modified Curtius reac-
tion using diphenyl phosphorazidate (DPPA) under
basic conditions,22 leading to an intermediate carboxylic
acid azide, which reacts by nitrogen liberation to the
1
by H NMR spectroscopy as well as HPLC (de g 95%).
Sch em e 2. Synthesis of Aminesa
a
(a) NaBH3CN, NH4CH3COO, MeOH, rt, 196 h; (b) NaBH4/TiCl4, 1,2-dimethoxyethane, 0 °C f rt, 12 h; (c) Et3N, TiCl4, CH2Cl2, 0 °C
f reflux for 2 h; (d) NaBH4, EtOH, -78 °C f -25 °C, 2 h; (e) Pd(OH)2, H2 (1 bar), MeOH, rt, 3 h.

4002 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Sasse et al.
Sch em e 3. Synthesis of Esters (12 and 13) and Ethers 14-17a
a
(a) R ) H for 12 and 13, R ) triphenylmethyl for 14a , 4-(dimethylamino)pyridine, pyridine, rt, 12 h; (b) Tebbe’s reagent,30 THF, 0 °C,
1 h; (c) Pd/C, H2 (1 bar), MeOH, 48 h; (d) SOCl2, THF, 0 °C f rt, 2 h; (e) 15-crown-5 (cat.), (C4H9)4NI (cat.), toluene, reflux for 12 h; (f)
2 N HCl/THF (20:30), 2 h, reflux; (g) 3-chloropropan-1-ol, KI (cat.), EtOH, reflux, 12 h; (h) NaH (60% in mineral oil), toluene, rt, 12 h.
triphenylmethyl-protected analogue of 13 was prepared
(14a ). Methylenation of ester 14a was carried out with
Tebbe’s reagent [(µ-chloro)(µ-methylene)bis(cyclopenta-
dienyl)(dimethylaluminum)titanium] under inert condi-
tions in THF.30 The triphenylmethyl-protected deriva-
tive of 12 did not react, which might be due to steric
hindrance of the tert-butyl group. Isolation of substi-
tuted vinyl ether 14a was achieved; however, deprotec-
tion of the imidazole ring by mild acid hydrolysis under
different conditions resulted in degradation to the
deprotected starting material, 3-(1H-imidazol-4-yl)pro-
panol. Upon hydrogenation under mild conditions, a
deprotection of the imidazole and hydrogenation of the
double bond was achieved which led to R-methyl-
branched ether 14 (Scheme 3).
Ethers 15-17 were synthesized by classical William-
son ether synthesis31 (Scheme 3). To achieve higher
reaction yields, 3-(1-triphenylmethyl-1H-imidazol-4-yl)-
F igu r e 1. Separation of enantiomers of 9 by CE. Background
electrolyte: 20 mM TM-â-CD, 150 mM H3PO4, pH 2.7 (tri-
ethylamine), 25 °C, sample concentration of 2.5 mg/mL, and
injection time of 2 s.
propanol was transformed into the corresponding alkyl
chloride to which freshly prepared sodium alkyl alco-
holate was added (Scheme 3). Alcoholate precursors of
15 and 16 were synthesized from 1-chloro-3,3-di-
methylbutane by elongation of the alkyl chain via the
corresponding nitrile and malonic acid ester, respec-
tively, according to standard methods. 3-Piperidino-
propan-1-ol was prepared from piperidine and 3-chloro-
propan-1-ol as described previously28,32 and transformed
into its sodium alcoholate to which 1-chloro-3,3-di-
methylbutane was added (Scheme 3).
Final carbamates (S)-9 and (R)-9 were then synthesized
as shown in Scheme 1, route 1, although, to increase
reaction yields, a solution of phosgene in toluene was
used for the formation of the isocyanate. This change
in protocol was necessary because the intermediate
isocyanate that was obtained had a low boiling point
which is close to that of diphosgene. Thus, problems
occurred in the fractional distillation of remaining traces
of diphosgene. Thus, advantage was taken of the low
boiling point of phosgene, which was removed from the
reaction vessel with nitrogen and securely inactivated
with aqueous KOH and NH3 solutions through which
it was passed. Piperidine analogue 18 was likewise
synthesized from (S)-9d and 3-piperidinopropan-1-ol.28
Enantiomeric purities of final carbamates (S)-9 and
(R)-9 were determined by capillary electrophoresis (CE).
The development of the CE method has been described
by Sasse et al. for comparable histamine H3 receptor
ligands.19,20 Trimethyl-â-cyclodextrin (TM-â-CD) as a
chiral selector was able to achieve baseline separation
with a resolution (Rs) of 2.2 for (R/ S)-9 (Figure 1). The
migration order of the enantiomers was determined by
spiking experiments. Enantiomeric purity of (S)-9 and
(R)-9 was determined to be as high as g95% (ee).
Esters 12 and 13 were prepared by standard methods
according to Einhorn29 from deprotected 3-(1H-imidazol-
4-yl)propanol‚HCl and 3,3-dimethylbutanoic acid chlo-
ride (Scheme 3). For subsequent reaction to 14, the
P h a r m a cologica l Resu lts a n d Discu ssion
F u n ction a l in Vitr o Assa ys. Many of the novel
compounds were tested on two functional in vitro models
for histamine H3 receptor activity in different species.
Modulated release of [3H]histamine from K+-evoked
depolarized synaptosomes of rat cerebral cortex33 and
concentration-dependent inhibition of electrically evoked
twitches of guinea pig ileum by (R)-R-methylhistamine
in the presence of an antagonist were used as screening
assays.34,35 All novel imidazole-containing carbamates
(1-11) are highly potent histamine H3 receptor antago-
nists in the guinea pig ileum assay (Table 1). Most of
the carbamates exhibit KB values between 10 and 25
nM, except for 1, which is less potent, and for dicyclo-
propylmethyl carbamate 3, which is more potent (1 nM),
but does not seem to be a pure competitive antagonist
as the slope of the Schild plot was observed to be
significantly less than 1 (not shown). Ester compounds
with a tert-butyl moiety (12 and 13) are far less potent
than the corresponding carbamates, which is in agree-

Histamine H3 Receptor Agonist/ Antagonist Properties
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4003
Ta ble 1. Structures, Chemical Data, and Pharmacological Results of Screening for Histamine H3 Receptor Agonist and Antagonist
Activity in Vitro and in Vivo
a
b
Crystallization solvent, Et2O/EtOH.
[
125I]Iodoproxyfan binding assay on the human H3 receptor.12 c Functional H3 receptor assay
on guinea pig ileum.34,35 Functional H3 receptor assay on synaptosomes of rat cerebral cortex, compounds tested as antagonists.33
e Functional H3 receptor assay on synaptosomes of rat cerebral cortex, compounds tested as agonists.33 f Central H3 receptor screening
d
after oral administration as a methylcellulose suspension to mice.33 i.a. ) intrinsic activity (histamine i.a. ) 1.0). The 95% confidence
g
h
i
j
interval, 0.58-1.91 nM; slope of the Schild plot, 0.85 ( 0.05 (P < 0.01). At 30 mg/kg, toxicity signs appeared. n.c. ) cannot be calculated
due to a low i.a. From ref 17. From ref 33. EC50 value.35a
k
l
m

4004 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Ta ble 2. Piperidine Replacement of the Imidazole Heterocyclee
Sasse et al.
a
b
Crystallization solvent, Et2O/EtOH. Functional H3 receptor assay on guinea pig ileum.34,35 c Functional H3 receptor assay on
synaptosomes of rat cerebral cortex.33 Central H3 receptor screening after oral administration as a methylcellulose suspension to mice.33
d
e Structures, chemical data, and pharmacological results of screening for histamine H3 receptor antagonist activity in vitro and in vivo.
ment with previous results.21 Furthermore, imidazole
replacement of (S)-9 by piperidine (18) also led to an
enormous drop in histamine H3 receptor potency on the
guinea pig assay (from 13 to 5000 nM, Table 2). In some
other classes of compounds, a similar structural replace-
ment of the imidazole heterocycle maintained antago-
nist histamine H3 receptor potency.32 Aliphatic ethers
(14-16) structurally derived from lead FUB 40717
(Chart 1, Table 1, KB ) 42 nM) showed similar potencies
in the ileal preparation of guinea pig, with 14 being
slightly less potent, 15 being more potent, and 16 being
equipotent (cf. Table 1). In agreement with the observa-
tion on carbamates, 17, the piperidine analogue of FUB
407, did not retain H3 receptor potency (Table 2).
Some compounds were tested in a second functional
model on the modulated release of [3H]histamine from
K+-evoked depolarized synaptosomes of rat cerebral
cortex33 to assess histamine H3 receptor potency and to
investigate their potential agonist properties (Table 1).
In previous studies on structurally related antagonists,
partial agonism of some carbamates and ethers could
be observed using this functional model, e.g., FUB 316,18
FUB 407,17 and FUB 475,17 whereas the same com-
pounds did not induce a depression of contraction of
electrically evoked twitches of guinea pig ileum, but
shifted the concentration-response curve of (R)-R-
methylhistamine competitively to the right, thus dis-
playing histamine H3 receptor antagonist properties. A
similar behavior was found for some of the new com-
pounds described here. Whereas compounds 1-3 behave
as antagonists in the [3H]histamine release model on
rat synaptosomes, partial agonist behavior was observed
for racemate 9, but due to low intrinsic activity (R ≈
0.2), an exact EC50 value could not be calculated (Table
1). Structurally related to the tert-butyl derivative 9 is
the cyclopropylmethyl derivative 10, which also showed
partial agonist properties on rat synaptosomes. Thus,
the nature of the bulky alkyl group determines whether
antagonist or agonist properties are displayed. These
differences will be important in following theoretical
studies on models of receptor activation. For general
histamine H3 receptor potency, the imidazole moiety
seems to be very important within this class of com-
pounds, as the piperidine analogue of FUB 407 is clearly
less potent (Table 2, FUB 407 f 17).
for the human histamine H3 receptor, although some
differences from values obtained from functional studies
on guinea pig and rat were observed. However, this
effect was not surprising as binding studies with the
rat H3 receptor in comparison to the human counterpart
revealed distinct pharmacology for certain compounds,
with binding affinities being equipotent or in favor of
either hH3 or rH3 receptors.11,12 The most important
compounds in the study presented here, the enanti-
omers of 9, showed similar potencies both in in vitro
binding and in functional assays (Table 1).
In Vivo Assa y on Sw iss Mice. All novel compounds
were screened for modulating effects on Nτ-methyl-
histamine levels in the brain cortex of Swiss mice after
oral treatment (Tables 1 and 2). Compounds 1, 2, and
4-6 enhanced Nτ-methylhistamine levels with weak
antagonist potency only. However, dicyclopropylmethyl
derivative 3 showed antagonist behavior equipotent to
that of thioperamide (ED50 ) 1 mg/kg, po).36 Some
R-methyl-branched compounds revealed agonist behav-
ior with full or partial intrinsic activities (8-10),
whereas compounds with a cyclopropyl (7) or cyclohexyl
(11) moiety showed antagonist behavior and were only
moderately potent. Isopropyl (8), tert-butyl (9), and
cyclopropylmethyl derivatives (10) were able to decrease
Nτ-methylhistamine levels, thus revealing agonist action
with high potency (ED50 e 0.5 mg/kg, po). In this in vivo
assay, carbamates 9, (S)-9, and 10 displayed full ago-
nism.
In vivo, no effect was observed for esters 12 and 13.
Aliphatic ethers 14 and 16 showed antagonist potency,
but 15, the higher homologue of FUB 407, showed
extraordinarily high in vivo potency with partial intrin-
sic activity (ED50 ) 0.13 mg/kg, po, ia ) 0.6). Meanwhile,
FUB 407 has proven its high histamine H3 receptor
agonist activity in further functional in vitro experi-
ments regarding [35S]GTPγ[S] binding and electrically
evoked [3H]noradrenaline overflow from mouse cortex
membranes.37 Recent in vivo experiments in rats dem-
onstrated that FUB 407 possesses gastroprotective
properties on gastric mucosal lesions induced by HCl
by increasing the level of histamine H3 receptor-depend-
ent mucus secretion and intracellular mucus content.38
Taken together, these results verify the high interest
in this group of compounds. From a structural point of
view, it can be concluded that a bulky group within a
distinct distance and specific three-dimensional orienta-
tion for sterically rigidified structures such as carbam-
ates is an essential prerequisite in this class of imidazole
compounds for agonist activity. It must also be stressed
that the piperidine analogues 17 and 18 do not show
Bin din g Assay on Hu m an H3 Receptor s. [125I]Iodo-
proxyfan binding was performed for selected compounds
in CHO-K1 cells stably transfected with the full-length
coding sequence of the hH3 receptor, as described
previously.12 All compounds that were investigated (3,
5, 7, 9-11, and 14-16) showed good to high affinities

Histamine H3 Receptor Agonist/ Antagonist Properties
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4005
Ta ble 3. Capsaicin-Induced Plasma Extravasation in Rats
Ta ble 4. Receptor Profile Regarding Histamine Receptor
after Oral Treatment
Subtypes
BP 2-94a
ED50
(mg/kg)
(S)-9
H3
H2
H1
b
c
d
Emax
(%)
ED50
(mg/kg)
Emax
(%)
no.
pKia
pA2
pD′2
pA2
tissue
1
2
3
4
5
6
7
8
9
7.3
7.4
8.0
7.0
7.7
9.0
8.0
7.9
7.9
7.7
7.7
7.9
7.8
7.7
7.9
7.3
7.6
7.4
6.5
5.4
4.6
4.2
4.0
3.6
4.7
5.1
4.5
conjunctiva
trachea
esophagus
0.40 ( 0.20 65 ( 6 0.10 ( 0.05 67 ( 6
0.30 ( 0.10 55 ( 5 0.07 ( 0.05 67 ( 8
1.00 ( 0.40 65 ( 5 0.10 ( 0.04 68 ( 6
<4.3
4.0e
4.9
urinary bladder 0.20 ( 0.03 63 ( 2 0.07 ( 0.05 81 ( 10
4.1
3.6e
5.7
a
From ref 5.
<4.0
<4.0
3.7
<4.0
<4.5
3.8
4.3
<4.0
4.3
4.7
5.0
4.6
4.4
any agonist behavior. They are inactive or only weakly
active in vivo in mice as expected from in vitro studies.
En a n tiom er s (R)- a n d (S)-9. Stereoisomers of (R/
S)-9 were synthesized because of the interesting phar-
macological behavior of the racemate to investigate the
role of the steric demands of the bulky aliphatic group,
which seems to be responsible for activation of the
receptor protein within this class of compounds. Stereo-
chemical effects of H3 receptor agonists have been
observed previously in the series of compounds struc-
turally related to histamine.39
7.9
8.3
10
<4.0f
4.2
(R)-11
(S)-11
14
15
16
4.1
4.3
4.9
4.5
17
18
6.5
4.7
4.5f
H3 receptor assay on synaptosomes of rat cerebral cortex.33
a
H3 receptor assay on guinea pig ileum.34,35 c H2 receptor assay
b
on guinea pig atrium.41 H1 receptor assay on guinea pig ileum.41
d
e pD′2 value. f pA2 value.
Compounds (R)- and (S)-9 clearly show differences in
pharmacological behavior. In the in vitro assay on rat
synaptosomes, the (R)-distomer still retained weak
agonist properties, whereas the (S)-enantiomer proved
to be the eutomer with increased intrinsic activity and
a low EC50 value. When tested as antagonists, the
enantiomers differed only slightly. However, the (R)-
enantiomer displayed slightly higher antagonist potency
on the guinea pig ileum model, pointing out differences
within the two functional assays, as described previ-
ously.17,40 125I]Iodoproxyfan binding experiments with
[
the human histamine H3 receptor revealed an affinity
similar to that observed on the guinea pig ileum assay
(Table 1). Under in vivo conditions, the (S)-eutomer
showed higher agonist activity than the (R)-enantiomer
which, in addition, has lost full intrinsic activity. Thus,
(S)-9 seemed to be a promising candidate for further
development.
F igu r e 2. Receptor profile of racemic 9.
17, which is a weak histamine H3 receptor antagonist,
showed some H3 receptor preference over H1 or H2
receptors. For racemate 9, additional selectivity was
Since H3 receptors are not found exclusively in the
brain, but also control neurotransmission in the periph-
ery, (S)-9 was also investigated on a model of inflam-
matory processes. Peripheral in vivo potency was in-
vestigated by the inhibition of capsaicin-induced plasma
protein extravasation in rat tissues after oral adminis-
tration of (S)-9.5 The inhibitory effect of the agonist was
determined in various peripheral tissues, e.g., conjunc-
tiva, trachea, esophagus, and urinary bladder, by meas-
uring the amount of extravasated Evans Blue dye.
Compared to the promising prodrug BP 2-94, which is
already in clinical trials, (S)-9 induced at least the same
or even increased the maximal level of inhibition in all
tissues investigated with an ED50 up to 10-fold lower
than that of BP 2-94 (Table 3). Since BP 2-94 is a
prodrug, compound (S)-9 has the advantage of acting
directly without having to undergo the complex prodrug/
drug pharmacokinetics involved in drug liberation.
Recep tor P r ofile of Selected Com p ou n d s. The
receptor profile of selected compounds regarding his-
tamine H1 and H2 receptors was determined on func-
tional models of the guinea pig ileum41 (Table 4).
Compounds 1-16 showed high selectivity for the his-
tamine H3 receptor, with a 2-4 orders of magnitude
difference versus H1 and H2 receptors. Even compound
determined regarding various serotonergic42,43 (5-HT2A
,
5-HT3, and 5-HT4), adrenergic41,42 (R1D and â1/2), and
muscarinic42 (M3) receptors (Figure 2). Compound 9
proved to be highly selective, being at least 250 times
more potent on histamine H3 than on any other amin-
ergic receptor that was tested.
Con clu sion s
Novel derivatives of 3-(1H-imidazol-4-yl)propanol be-
longing to the carbamate, ester, or ether series were
prepared on the basis of lead compounds possessing
(partial) agonist properties on models for histamine H3
receptor activation. The compounds were investigated
on various histamine H3 receptor assays in vitro and in
vivo. Some compounds displayed partial agonist activity
on synaptosomes of rat brain cortex, but behaved as
pure competitive antagonists on the guinea pig ileum.
[
125I]Iodoproxyfan binding studies on the human his-
tamine H3 receptor provided high affinities in the
nanomolar concentration range. Under in vivo condi-
tions after oral administration to mice, some compounds
showed partial or full agonist activities with ED50 values
of <1 mg/kg. An exchange of the imidazole ring with a

4006 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Sasse et al.
3H, NH3x), 7.43-7.35 (m, 5H, 5Ph-H), 3.96 (d, J ) 8.4 Hz,
1H, CHNH3x), 1.93-0.74 (m, 11H, 11Cyhexl-H). Anal. (C13H19N‚
HCl) C, H, N. To a solution of trichloromethyl chloroformate
(0.6 g, 3 mmol) and a catalytic amount of activated charcoal
in 20 mL of dry ethyl acetate was rapidly added 1b hydro-
chloride (0.6 g, 2.5 mmol). The reaction mixture was heated
to reflux for 4 h, and then the black solution was cooled and
filtered and the solvent evaporated carefully under reduced
pressure. The freshly prepared isocyanate was dissolved in 20
mL of dry acetonitrile and added to 3-(1H-imidazol-4-yl)-
propanol hydrochloride21 (0.4 g, 2.5 mmol) in 10 mL of dry
acetonitrile. The solution was refluxed for 5 h and concentrated
in vacuo. The residue was purified by rotatory chromatography
[eluent, CH2Cl2/MeOH (gradient from 100:1 to 10:1), ammonia
atmosphere]. Separation was controlled by TLC. The product
(1) was obtained as a colorless oil and crystallized as a salt of
maleic acid from Et2O/EtOH: yield 27%; mp 136 °C; 1H NMR
(Me2SO-d6) δ 8.81 (s, 1H, Im-2-H), 7.64* (d, J ) 9.1 Hz, 1H,
CONH), 7.33-7.05 (m, 6H, 5Ph-H + Im-5-H), 6.03 (s, 2H, Mal),
4.19 (m, 1H, NHCH), 3.90 (t, J ) 6.8 Hz, 2H, CH2O), 2.64 (t,
piperidine moiety was not tolerated. Selectivity of the
novel compounds for the histamine H3 receptor was
established by determination of histamine H1 and H2
receptor activities in functional assays on guinea pigs.
Additionally, for carbamate 9 a receptor profile was
conducted regarding other neurotransmitter receptors.
The (S)-enantiomer of one pair of chiral carbamates (9)
proved to be the eutomer, with pharmacological agonist
activity superior to that of the (R)-distomer, which
displayed equal or slightly higher potency when tested
as an antagonist or on the human H3 receptor binding
assay. Thus, the stereochemical conformation of the
branched bulky side chain of the carbamate seems to
be highly important for induction of a change in receptor
conformation, which is necessary for activation of a
functional response in this receptor class. Thus, H3
receptors might be a good target for principal investiga-
tions on molecular studies for activation of G protein-
coupled receptors.
J ) 7.4 Hz, 2H, Im-CH2), 1.85-0.78 (m, 13H, Im-CH2CH2
+
11Cyhexl-H). Anal. (C20H27N3O2‚C4H4O4‚3/4H2O) C, H, N.
The (S)-enantiomer of 9 was selected for further
pharmacological studies. In a peripheral in vivo test
model in rats, measuring the level of inhibition of
capsaicin-induced plasma extravasation, (S)-9 showed
3-10-fold higher agonist activity than the prodrug
agonist BP 2-94. The promising data for (S)-9 shown in
this study give rise to the hope for a potential followup
candidate for clinical development.
3-(1H-Im id a zol-4-yl)p r op yl N-[3-(2,2,4,4-Tetr a m eth yl-
p en tyl)] Ca r ba m a te (5). To a solution of 2,2,4,4-tetramethyl-
pentanone oxime (5a ) (1.9 g, 12 mmol) in 15 mL of 1,2-
dimethoxyethane was added sodium boranate (1.9 g, 50 mmol).
TiCl4 (2.8 mL, 25 mmol) was added slowly under an argon
atmosphere at 0 °C. The mixture was warmed to room
temperature and stirred for 12 h. One hundred milliliters of
cold water was added, and the solution was basified with
ammonia and extracted with ethyl acetate. The organic layer
was washed with a saturated solution of NaCl, dried over
Na2SO4, and evaporated under reduced pressure. 2,2,4,4-
Tetramethylpentan-3-amine45 (5b) was crystallized as a hy-
drochloride salt from EtOH/Et2O: yield 58%; mp 234 °C dec;
1H NMR (Me2SO-d6) δ 7.72 (s, 3H, NH3x), 2.70 (d, J ) 5.2 Hz,
1H, CH), 1.08 {s, 18H, [C(CH3)3]2}. Anal. (C9H21N‚HCl‚1/8H2O)
C, H, N. Carbamate 5 was synthesized as described for 1 with
5b and crystallized as a salt of oxalic acid from Et2O/EtOH:
yield 24%; mp 179 °C; 1H NMR (Me2SO-d6) δ 8.32 (m, 1H, Im-
2-H), 7.11 (s, 1H, Im-5-H), 6.97* (d, J ) 10.7 Hz, 1H, CONH),
3.98 (t, J ) 6.6 Hz, 2H, CH2O), 3.19 (d, J ) 10.7 Hz, 1H, CH),
2.64 (t, J ) 7.5 Hz, 2H, Im-CH2), 1.89 (m, 2H, Im-CH2CH2),
0.97 {s, 18H, [C(CH3)3]2}. Anal. (C16H29N3O2‚C2H2O4) C, H, N.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Melting points were
determined on an Electrothermal IA 9000 digital or a Bu¨chi
512 melting point apparatus and are uncorrected. 1H NMR
spectra were recorded on a Bruker DPX 400 Avance (400 MHz)
spectrometer. Chemical shifts are expressed in parts per
million downfield from internal Me4Si as a reference. 1H NMR
data are reported in the following order: multiplicity (br,
broad; s, singlet; d, doublet; dd, doublet of a doublet; dt, doublet
of a triplet; t, triplet; and m, multiplet), approximate coupling
constants in hertz, and the number of protons (*) that can be
exchanged by D2O (Im, 4-imidazolyl; Ph, phenyl; Cyhexl,
cyclohexyl; Mal, maleic acid). Optical rotations were deter-
mined on a Perkin-Elmer 241 MC polarimeter. Elemental
analyses (C, H, N) were measured on a Perkin-Elmer 240 B
or Perkin-Elmer 240 C instrument and were within (0.4% of
theoretical values for all compounds. Preparative, centrifugally
accelerated, rotatory chromatography was performed using a
Chromatotron 7924T (Harrison Research) and glass rotors
with 4 mm layers of silica gel 60 PF254 containing gypsum
(Merck). Column chromatography was carried out using silica
gel 63-200 µm (Macherey, Nagel & Co.). “NH3-solution” means
an aqueous solution of ammonia (w ) 25%). Thin-layer
chromatography (TLC) was performed on silica gel PF254 plates
(Merck). Spectral data are shown only for parent compounds
which were obtained by different reactions or methods [1, 5,
6, (S)-9, 12, and 14-17].
3-(1H -Im id a zol-4-yl)p r op yl
N-(2,2,3,3-Tet r a m et h yl-
cyclop r op yl) Ca r b a m a t e (6). A mixture of 2,2,3,3-tetra-
methylcyclopropylcarboxylic acid (1.0 g, 7.5 mmol), triethyl-
amine (1.4 mL, 10 mmol), diphenylphosphoryl azide (2.1 g, 7.5
mmol), and 3-(1H-imidazol-4-yl)propanol hydrochloride21 (0.8
g, 5 mmol) was refluxed for 16 h in 30 mL of dry dioxane. The
solvent was removed under reduced pressure, and the residue
was dissolved in ethyl acetate and extracted with a saturated
solution of K2CO3. After concentration of the combined organic
fractions, the residue was purified by column chromatography
[eluent, CH2Cl2/MeOH (10:1), ammonia atmosphere]. The
combined pure fractions were concentrated, dried, and crystal-
lized as a salt of maleic acid from Et2O/EtOH: yield 30%; mp
1
127 °C; H NMR (Me2SO-d6) δ 8.85 (s, 1H, Im-2-H), 7.38 (s,
1H, Im-5-H), 6.91* (s, 1H, CONH), 6.04 (s, 1H, Mal), 3.97 (t,
J ) 6.4 Hz, 2H, CH2O), 2.68 (m, 2H, Im-CH2), 1.91-1.85 (m,
3H, Im-CH2CH2 and CH), 1.02 [s, 6H, C(CH3)2], 0.88 [s, 6H,
C(CH3)2]. Anal. (C14H23N3O2‚C4H4O4) C, H, N.
(RS)-N-[(Cycloh exyl)p h en ylm eth yl] 3-(1H-Im id a zol-4-
yl)p r op yl Ca r ba m a te (1). Cyclohexylphenylmethanone (1a )
(2.8 g, 15 mmol), sodium cyanoboranate (0.63 g, 10 mmol), and
freshly sublimed ammonium acetate (11.6 g, 150 mmol) were
dissolved in 50 mL of dry MeOH and stirred under a nitrogen
atmosphere at room temperature for 1 week until no further
reaction was observed as monitored by TLC. The reaction was
quenched by addition of 20 mL of 6 N HCl; the white
precipitate was filtered, and the filtrate was extracted with
ethyl acetate. The aqueous phase was basified with 6 N NaOH
and extracted with ethyl acetate. The organic phase was dried
with Na2SO4, the solvent evaporated under reduced pressure,
and the yellow oily residue crystallized as a hydrochloride salt
from MeOH/Et2O to afford cyclohexylphenylmethanamine (1b):
(S)-3-(1H-Im id a zol-4-yl)p r op yl N-(1,2,2-Tr im eth ylp r o-
p yl) Ca r ba m a te [(S)-9]. (S)-1-Phenylethan-1-amine (6.1 g,
50 mmol) and triethylamine (25.3 g, 250 mmol) were dissolved
in dry CH2Cl2. The solution was cooled to 0 °C, and TiCl4 (4.0
g, 21 mmol) was added dropwise through a septum under a
nitrogen atmosphere. The reaction mixture was then heated
to reflux, and pinacolone (9a ) (4.2 g, 42 mmol) was added; the
mixture was refluxed for an additional 2 h and stirred at room
temperature overnight. Then, 50 mL of Et2O was added to the
ice-cooled mixture, and the white precipitate was filtered
repeatedly until a clear yellow filtrate was obtained, which
was evaporated. The dried yellow oil, (S)-1-phenylethyl-1-
44
1
yield 28%; mp 276 °C dec; H NMR (Me2SO-d6) δ 8.46* (s,

Histamine H3 Receptor Agonist/ Antagonist Properties
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4007
(1,2,2-trimethylpropane)imine25 [(S)-9b], was stored under
nitrogen at 4 °C until it was further needed: yield 90%; 1H
NMR (CDCl3) δ 7.41-7.17 (m, 5H, 5Ph-H, CDCl3), 4.57 (q, J
) 6.5 Hz, 0.84 × 1H, trans-CH), 4.28 (q, J ) 6.6 Hz, 0.16 ×
1H, cis-CH), 1.79 (s, 3H, NCCH3), 1.35 (d, J ) 6.6 Hz, 3H,
CHCH3), 1.12 [s, 9H, C(CH3)3]. Anal. (C14H21N). The imine (S)-
9b (6.1 g, 30 mmol) was dissolved in 50 mL of dry EtOH and
cooled to -78 °C. NaBH4 (0.56 g, 15 mmol) was added, and
the reaction mixture was stirred for 30 min at -78 °C. It was
then allowed to warm to -20 °C, and the reaction was
quenched by the dropwise addition of 30 mL of 6 N HCl. EtOH
was then evaporated under reduced pressure, and the aqueous
residue was alkalized with K2CO3 and extracted with ethyl
acetate. The product was purified by column chromatography
[eluent, ethyl acetate, NH3-solution (100:3)], and n(S,S)-N-
(1,2,2-trimethylpropyl) phenylethyl-1-amine R-methylbenzyl-
amine [(S,S)-9c] was crystallized as a hydrochloride salt from
14a } precursor was performed as described for 12 with 3,3-
dimethylbutyric acid chloride (0.67 g, 5 mmol) and 3-[1-
(triphenylmethyl)-1H-imidazol-4-yl]propanol (1.8 g, 5 mmol).
The crude product was purified by column chromatography
[eluent, ethyl acetate/NH3
solution (100:3)] and crystallized
at 4 °C: yield 95%; 1H NMR (Me2SO-d6) δ 7.42-7.35 (m, 10H,
10Ph-H), 7.26 (s, 1H, Im-2-H), 7.08 (m, 5H, 5Ph-H), 6.59 (s,
1H, Im-5-H), 3.97 (t, J ) 6.5 Hz, 2H, CH2O), 2.50 (t, J ) 7.2
Hz, 2H, Im-CH2 partially covered by DMSO), 2.14 (s, 2H,
COCH2), 1.83 (m, 2H, Im-CH2CH2), 0.94 [s, 9H, C(CH3)3]. Anal.
(C31H34N2O2) C, H, N. Ester 14a (0.9 g, 2 mmol) was dissolved
in 10 mL of freshly dried THF under an argon atmosphere.
Tebbe’s reagent46 (0.5 M solution in toluene, 4.4 mL, 2.2 mmol)
was added slowly through a septum. The mixture was stirred
for 1 h at 0 °C. Then 2 mL of 2 N NaOH was added carefully
to stop the reaction. The blue precipitate was filtered and the
orange filtrate concentrated under reduced pressure. The
residue was taken up in ethyl acetate/Et2O (1:1) and washed
with 0.2 N NaOH. The crude product was then purified by
column chromatography [eluent, ethyl acetate/NH3 solution
(100:3)], and 3-[1-(triphenylmethyl)-1H-imidazol-4-yl]propyl
2-(4,4-dimethylpent-1-enyl) ether 14b was obtained as a light
yellow oil: yield 60%; 1H NMR (Me2SO-d6) δ 7.37-7.13 (m,
16H, 15Ph-H + Im-2-H), 6.61 (s, 1H, Im-5-H), 3.86 (s, 1H, d
CHH), 3.75 (s, 1H, dCHH), 3.55 (t, J ) 6.1 Hz, 2H, CH2O),
Et2O/EtOH and recrystallized once: yield 76%; mp 222 °C;
1
[R]23 -8.45 (c 1.0, EtOH); H NMR (Me2SO-d6) δ 8.92* and
D
7.90* (two br s, 2H, NH2x), (br s, 1H, NHx), 7.72-7.40 (m, 5H,
5Ph-H), 4.39 (m, 1H, CHPh), 2.93 [m, 1H, CHC(CH3)3], 1.68
(d, J ) 6.8 Hz, 3H, CHCH3Ph), 1.08 [d, J ) 6.7 Hz, 3H,
CHCH3C(CH3)3], 0.98 [s, 9H, C(CH3)3]. Anal. (C14H23N‚HCl)
C, H, N. (S,S)-9c (3.6 g, 15 mmol) was dissolved in 50 mL of
MeOH, and 250 mg of Pd(OH)2/C was added. The mixture was
hydrogenated for 48 h at 1 bar until H2 consumation ceased.
The catalyst was filtered, methanolic HCl added, and the
solvent evaporated under reduced pressure. The residue was
taken up in MeOH, and (S)-1,2,2-trimethylpropylamine (S)-
9d was crystallized as a hydrochloride salt after addition of
Et2O: yield 83%. mp 256 °C; [R]23D 2.8 (c 4.0, MeOH); 1H NMR
(Me2SO-d6) δ 7.91* (s, 3H, NH3x), 2.94 (q, J ) 6.7 Hz, 1H,
CH), 1.13 (d, J ) 6.8 Hz, 3H, CH3), 0.92 [s, 9H, C(CH3)3]. Anal.
(C6H15N‚HCl) C, H, N. (S)-9d (2.1 g, 15 mmol) was suspended
in 30 mL of dry toluene and the mixture cooled to 0 °C. Then,
phosgene (20% solution in toluene, 15 mL, 30 mmol) was added
rapidly through a septum and the mixture heated to 60 °C for
3 h. The solution was then cooled to room temperature, and
nitrogen was bubbled through for 1 h to remove the excess of
phosgene. This solution was then added to 3-(1H-imidazol-4-
yl)propanol hydrochloride21 (1.6 g, 10 mmol), dissolved in 2
mL of DMF, and heated to 80 °C for 12 h. MeOH was added,
the mixture concentrated on a rotatory evaporator, and the
residue purified by rotatory chromatography [eluent, CH2Cl2/
MeOH (gradient from 100:1 to 10:1), ammonia atmosphere].
Separation was controlled by TLC. The product (S)-9 was
obtained as a colorless oil and crystallized as a salt of oxalic
acid from Et2O/EtOH: yield 35%; [R]23D 9.66 (c 0.5, methanol);
2.63 (t, J ) 7.3 Hz, 2H, Im-CH2), 1.93 [m, 4H, Im-CH2CH2
+
CH2C(CH3)3], 0.88 [s, 9H, C(CH3)3]. Anal. (C32H36N2O) C, H,
N. Trityl-protected enyl ether 14b (0.6 g, 1.2 mmol) and Pd/C
(60 mg) were dissolved in 20 mL of methanol and hydrogenated
at 1 bar for 48 h. After filtration, the crude product was
purified by rotary chromatography [eluent, CH2Cl2/MeOH (10:
1), ammonia atmosphere] and then crystallized as a salt of
oxalic acid from Et2O/EtOH and recrystallized once: yield 95%;
1
mp 182 °C; H NMR (CD3OD) δ 8.50 (s, 1H, Im-2-H), 7.17 (s,
2
3
1H, Im-5-H), 3.57 (dt, J ) 9.2 Hz, J ) 6.1 Hz, 1H, CHHO),
3.51 (m, 1H, CH), 3.36 (dt, 2J ) 9.2 Hz, 3J ) 6.1 Hz, 1H,
CHHO), 2.77 (2t, J ) 7.5 Hz, 2H, Im-CH2), 1.90 (m, 2H, Im-
CH2CH2), 1.48 [dd, 2J ) 14.5 Hz, 3J ) 7.6 Hz, 1H, CHH(CH3)3],
2
3
1.23 [dd, J ) 14.5 Hz, J ) 3.2 Hz, 1H, CHH(CH3)3], 1.11 (d,
J ) 6.1 Hz, 3H, CH3), 0.93 [s, 9H, C(CH3)3]. Anal. (C13H24N2O‚
0.85C2H2O4) C, H, N.
3-(1H-Im id a zol-4-yl)p r op yl 4,4-Dim eth ylp en tyl Eth er
(15). 3,3-Dimethylbutyl chloride (15a ) (4.3 g, 36 mmol) was
added dropwise to a solution of KCN (2.9 g, 45 mmol) and a
catalytic amount of KI in 50 mL of dry DMSO and heated to
95 °C for 12 h. The solution was poured into 300 mL of H2O
after being cooled to room temperature and extracted with
Et2O. The organic phase was washed with a saturated solution
of NaCl, dried over Na2SO4, and concentrated. 3,3-Dimethyl-
butane nitrile47 (15b) was obtained as a colorless oil: yield
68%; 1H NMR (Me2SO-d6) δ 2.50 (t, J ) 8.0 Hz, 2H, CH2CN),
1.51 (t, J ) 7.9 Hz, 2H, CH2CH2CN), 0.86 [s, 9H, C(CH3)3].
Anal. (C7H13N) C, H, N. Nitrile 15b (2.7 g, 24 mmol) and KOH
(20 g) were refluxed for 12 h in EtOH/H2O (1:1). After the
solution was cooled to room temperature, EtOH was distilled
off, and the aqueous residue was washed with Et2O. The
aqueous solution was then carefully acidified with 2 N HCl
and extracted with Et2O. The organic phase was washed with
a saturated NaCl solution, dried over Na2SO4, and concen-
trated. 4,4-Dimethylpentanoic acid47 (15c) was obtained as a
colorless oil: yield 63%; 1H NMR (Me2SO-d6) δ 11.95* (s, 1H,
COOH), 2.15 (t, J ) 8.2 Hz, 2H, CH2COOH), 1.43 (t, J ) 8.3
Hz, 2H, CH2CH2COOH), 0.86 [s, 9H, C(CH3)3]. Anal. (C7H14O2)
C, H, N. The carboxylic acid 15c (2 g, 15 mmol), dissolved in
20 mL of Et2O, was carefully added dropwise to a suspension
of LiAlH4 (0.65 g, 17 mmol) in 50 mL of Et2O at 0 °C in an
argon atmosphere. The reaction mixture was heated to reflux
for 12 h. Excess LiAlH4 was destroyed by addition of EtOH
and hydrolyzed by addition of 2 N HCl. The organic solvents
were evaporated, and the aqueous residue was extracted with
Et2O, washed with saturated solutions of K2CO3 and NaCl,
dried over Na2SO4, and concentrated. 4,4-Dimethylpentan-1-
1
mp 122 °C; H NMR (Me2SO-d6) δ 8.48 (s, 1H, Im-2-H), 7.20
(s, 1H, Im-5-H), 6.88* (d, J ) 9.4 Hz, 1H, CONH), 3.95 (t, J )
6.5 Hz, 2H, CH2O), 3.36 (m, 1H, CH), 2.64 (t, J ) 7.5 Hz, 2H,
Im-CH2), 1.88 (m, 2H, Im-CH2CH2), 0.97 (d, J ) 6.9 Hz, 3H,
CH3), 0.82 [s, 9H, C(CH3)3]. Anal. (C13H23N3O2‚C2H2O4‚1/4H2O)
C, H, N.
3-(1H-Im idazol-4-yl)pr opyl 2,2-Dim eth ylpr opion ate (12).
3-(1H-Imidazol-4-yl)propanol hydrochloride21 (0.8 g, 5 mmol)
and a catalytic amount of 4-(dimethylamino)pyridine were
dissolved in 30 mL of pyridine. 2,2-Dimethylpropionic acid
chloride (0.6 g, 5 mmol) was added through a septum, and then
the mixture was stirred for 12 h at room temperature. The
solvent was evaporated under reduced pressure, and the
residue was taken up in ethyl acetate and washed with
saturated K2CO3 and NaCl solutions. The crude product was
then purified by column chromatography [eluent, CH2Cl2/
MeOH (10:1, ammonia atmosphere)], and the pure fractions
were evaporated, dried, and crystallized as a salt of oxalic acid
from Et2O/EtOH: yield 50%; mp 145 °C; 1H NMR (CD3OD) δ
8.74 (s, 1H, Im-2-H), 7.32 (s, 1H, Im-5-H), 4.12 (t, J ) 6.2 Hz,
2H, CH2O), 2.82 (t, J ) 7.8 Hz, 2H, Im-CH2), 2.04 (m, 2H, Im-
CH2CH2), 1.19 [s, 9H, C(CH3)3]. Anal. (C11H18N2O2‚C2H2O4) C,
H, N.
(RS)-3-(1H-Im id a zol-4-yl)p r op yl 1,3,3-Tr im eth ylbu tyl
Eth er (14). Synthesis of the trityl-protected ester {3-[1-(tri-
phenylmethyl)-1H-imidazol-4-yl]propyl 3,3-dimethylbutyrate,
1
ol (15d )48 was obtained as a colorless oil: yield 76%. H NMR
(Me2SO-d6) δ 4.35* (t, J ) 5.2 Hz, 1H, OH), 3.38 (m, 2H,

4008 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Sasse et al.
CH2OH), 1.37 (m, 2H, CH2CH2OH), 1.15 [m, 2H, CH2(CH3)3],
0.86 [s, 9H, C(CH3)3]. Anal. (C7H16O) C, H, N. A mixture of
4,4-dimethylpentan-1-ol (15d ) (0.7 g, 5 mmol) and NaH (60%)
(0.2 g, 5 mmol) was stirred in 10 mL of dry DMSO for 6 h at
room temperature under an argon atmosphere. Then 3-[1-
(triphenylmethyl)-1H-imidazol-4-yl]propyl chloride (0.6 g, 2
mmol), a catalytic amount of tetrabutylammonium iodide, and
15-crown-5 were added, and then the mixture was heated to
80 °C for 24 h. After the solution was cooled to room
temperature, 100 mL of H2O was added and the product
extracted with Et2O and purified by column chromatography
[eluent, ethyl acetate/NH3 solution (100:3)]. The pure fractions
were concentrated in vacuo, redissolved in 2 N HCl/THF (20:
30), and heated at 70 °C for 2 h. After removal of the organic
solvent under reduced pressure, the aqueous suspension was
filtered and extracted with Et2O. The aqueous solution was
basified (NaOH) and extracted with Et2O. The organic layer
was dried and concentrated in vacuo. The oily residue was
purified by column chromatography [eluent, CH2Cl2/MeOH
(10:1), ammonia atmosphere]. The colorless oil was crystallized
as a salt of oxalic acid from EtOH/Et2O and recrystallized
once: yield 10%; mp 180 °C; 1H NMR (Me2SO-d6) δ 8.39 (s,
1H, Im-2-H), 7.14 (s, 1H, Im-5-H), 3.35 (m, 4H, CH2OCH2),
2.62 (t, J ) 7.5 Hz, 2H, Im-CH2), 1.81 (br s, 2H, Im-CH2CH2),
1.44 [m, 2H, CH2CH2C(CH3)3], 1.16 [m, 2H, CH2(CH3)3], 0.86
[s, 9H, C(CH3)3]. Anal. (C13H24N2O‚0.8C2H2O4) C, H, N.
3,5-H), 1.52 (s, 2H, 2Pip-4-H), 1.43 [t, J ) 7.3 Hz, 2H, CH2C-
(CH3)3], 0.89 [s, 9H, C(CH3)3]. Anal. (C14H29NO‚1.1C2H2O4) C,
H, N.
Ca p illa r y Electr op h or esis (CE). CE separations were
carried out using an automated CE apparatus (Beckman
P/ACE 2100): Software Gold 7.11, capillary 57 cm × 50 µm
i.d. (Grohm), 50 cm to the detector according to Sasse et al.;20
UV detection at 200 nm; voltage of 25 kV; capillary temper-
ature thermostated at 25.0 °C; sample injection for 2 s with
pressure (5 bar). The composition of the background electrolyte
(BGE) was as follows: 150 mM phosphate buffer [aqueous
H3PO4 (85% w/w)] adjusted to pH 2.7 with triethanolamine
and 20 mM trimethyl-â-cyclodextrin (Fluka) and I ) 65 µA.
At the beginning of each working day, the capillary was rinsed
for 15 min with 0.1 N NaOH, for 5 min with H2O, and for 5
min with BGE. Between each run, the capillary was rinsed
for 1 min with 0.1 N NaOH and for 3 min with BGE. Each
experiment was performed at least in triplicate.
Gen er a l P h a r m a cology Meth od s
Hista m in e H3 Recep tor An ta gon ist Activity on Gu in ea
P ig Ileu m . For selected compounds, H3 receptor activity was
measured by concentration-dependent inhibition of electrically
evoked twitches of isolated guinea pig ileal segments induced
by (R)-R-methylhistamine in the absence and presence of the
antagonist according to Ligneau et al.35b In brief, longitudinal
muscle strips were prepared from the small intestine, 20-50
cm proximal to the ileocaecal valve. The muscle strips were
mounted between two platinum electrodes (4 mm apart) in 20
mL of Krebs buffer, containing 1 µM mepyramine, connected
to an isometric transducer, continuously gassed with oxygen
containing 5% CO2 at 37 °C. After equilibration of the muscle
segments for 1 h with washing every 10 min, they were
stimulated continuously with rectangular pulses of 15 V and
0.5 ms duration at a frequency of 0.1 Hz. After stimulation
for 30 min, a cumulative concentration-response curve was
recorded. Subsequently, the preparations were thoroughly
washed twice every 10 min without stimulation. The antago-
nist was incubated for 20-30 min before redetermination of
the (R)-R-methylhistamine concentration-response curve.34,35
After addition of the investigated compounds, no depression
of contraction, e.g., agonist activity, was observed. Each
experiment was performed at least in triplicate.
3-(1H -Im id a zol-4-yl)p r op yl 5,5-Dim et h ylh exyl E t h er
(16). To a solution of NaH (60% in mineral oil) (2.4 g, 60 mmol)
and a catalytic amount of KI in dry DMF was added malonic
acid diethyl ester (9.6 g, 60 mmol) dropwise at 0 °C under a
nitrogen atmosphere. After the reaction vessel had been
warmed to room temperature, 3,3-dimethylbutyl chloride (6.0
g, 50 mmol) was added and then heated to 80 °C for 12 h. The
cooled solution was poured into 300 mL of H2O and extracted
with ethyl acetate/Et2O (1:1). The organic phase was dried with
Na2SO4 and concentrated. The product was suspended in 200
mL of H2O/EtOH (1:1); 40 g of KOH was added, and the
mixture was refluxed for 12 h. EtOH was evaporated under
reduced pressure and the aqueous residue extracted with Et2O.
The organic phase was dried with Na2SO4 and concentrated.
3,3-Dimethylbutylmalonic acid (16b) was crystallized from
1
EtOH: yield 60% (two steps); mp 128 °C; H NMR (Me2SO-
d6) δ 12.60* (s, 2H, 2COOH), 3.11 (t, J ) 7.4 Hz, 1H, CH),
1.67 (m, 2H, CHCH2), 1.14 [m, 2H, CH2C(CH3)3], 0.86 [s, 9H,
C(CH3)3]. Anal. (C9H16O4) C, H, N. Compound 16b (5.5 g, 29
mmol) was heated to 150 °C for 3 h to decarboxylate it. The
cooled reaction product was dissolved in 2 N NaOH and
washed with Et2O. The aqueous phase was acidified with 2 N
HCl and extracted with ethyl acetate/Et2O (1:1). The organic
phase was dried with Na2SO4 and concentrated. 5,5-Di-
methylhexanoic acid (16c)49 crystallized at 4 °C: yield 36%;
Hista m in e H3 Recep tor Assa y on Syn a p tosom es of Ra t
Cer ebr a l Cor tex. Compounds were tested for their H3
receptor agonist and antagonist activity in an assay with K+-
evoked depolarization-induced release of [3H]histamine from
rat synaptosomes according to Garbarg et al.33 A synaptosomal
fraction from rat cerebral cortex prepared according to the
method of Whittaker50 was preincubated for 30 min with
L-[3H]histidine (0.4 µM) at 37 °C in a modified Krebs-Ringer
solution. The synaptosomes were washed extensively, resus-
pended in fresh 2 mM K+ Krebs-Ringer medium, and incu-
bated for 2 min with 2 or 30 mM K+ (final concentration).
Antagonists and 1 µM histamine or the agonist in increasing
concentrations were added 5 min before the depolarization
stimulus. Incubation was stopped by rapid centrifugation, and
[3H]histamine levels were determined after purification by
liquid scintillation spectrometry.33 Ki values were determined
according to the Cheng-Prussoff equation.51 The data that are
presented are given as mean values with the standard error
of the mean (SEM) each for a minimum of three separate
determinations.
1
mp 36 °C; H NMR (Me2SO-d6) δ 11.96* (s, 1H, COOH), 2.17
(t, J ) 7.3 Hz, 2H, CH2COOH), 1.45 (m, 2H, CH2CH2COOH),
1.15 [m, 2H, CH2C(CH3)3], 0.86 [s, 9H, C(CH3)3]. Anal. (C9H16O4)
C, H, N. Carboxylic acid (16c) was treated as described for
1
15c, to obtain 5,5-dimethylhexan-1-ol (16d ):48 yield 57%; H
NMR (Me2SO-d6) δ 4.31* (t, J ) 5.1 Hz, 1H, OH), 3.38 (m,
2H, CH2OH), 1.38 (m, 2H, CH2CH2OH), 1.28-1.12 [m, 4H,
(CH2)2(CH3)3], 0.85 [s, 9H, C(CH3)3]. Anal. (C8H18O) C, H, N.
Alcohol 16d was treated as described for 15d , to obtain 3-(1H-
imidazol-4-yl)propyl 5,5-dimethylhexyl ether (16) as a salt of
oxalic acid: yield 25%; mp 184 °C; 1H NMR (Me2SO-d6) δ 8.38
(s, 1H, Im-2-H), 7.13 (s, 1H, Im-5-H), 3.35 (m, 4H, CH2OCH2),
2.62 (t, J ) 7.6 Hz, 2H, Im-CH2), 1.81 (s, 2H, Im-CH2CH2),
1.46 [m, 2H, CH2(CH2)2C(CH3)3], 1.20 [m, 4H, CH2CH2(CH3)3],
0.85 [s, 9H, C(CH3)3]. Anal. (C14H26N2O‚0.8C2H2O4) C, H, N.
3,3-Dim eth ylbu tyl 3-P ip er id in op r op yl Eth er (17). 3-Pi-
peridinopropanol (1.43 g, 10 mmol) and 3,3-dimethylbutyl
chloride (0.6 g, 5 mmol) were treated as described for 15. The
colorless oil was crystallized as a salt of oxalic acid from EtOH/
Et2O and recrystallized once: yield 16%; mp 143 °C; 1H NMR
(Me2SO-d6) δ 3.40 (m, 4H, CH2OCH2), 3.00 (m, 6H, Pip-CH2
+ 4Pip-2,6-H), 1.82 (m, 2H, Pip-CH2CH2), 1.71 (m, 4H, 4Pip-
[
125I]Iod op r oxyfa n Bin d in g Assa y on Sta bly Tr a n s-
fected CHO-K1 Cells. CHO-K1 cells were transfected with
pCIneo-hH3 plasmids using SuperFect reagent (Quiagen).
Stable transfectants were selected with 2 mg/mL G418 and
tested for [125I]iodoproxyfan binding.35b Positive clones were
maintained in the presence of 1 mg/mL G418. For [125I]iodo-
proxyfan binding experiments, cells were harvested, washed,
and homogenized (Polytron) in ice-cold binding buffer [50 mM
Na2HPO4/KH2PO4 (pH 6.8)]. The binding assay was performed
as described previously.12,35b Briefly, aliquots of membrane
suspensions (5-15 µg of protein) were incubated at 25 °C for

Histamine H3 Receptor Agonist/ Antagonist Properties
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 4009
60 min with 22-25 pM [125I]iodoproxyfan alone or together
with competing drugs (final volume of 200 µL). The level of
nonspecific binding was determined using imetit (1 µM). Each
experiment was performed at least in triplicate.
of the Deutsche Pharmazeutische Gesellschaft (DPhG), Mu¨nster,
Germany, October 5-7, 2000, Abstract D1.21; Arch. Pharm.
Pharm. Med. Chem. 2000, 333 (Suppl. 2), 9. (c) Schunack, W.;
Sasse, A.; Stark, H.; Elz, S.; Ligneau, X.; Ganellin, C. R.;
Schwartz, J .-C. Partial Agonists for the Histamine H3 Receptor
with High in Vivo Activity. International Sendai Histamine
Symposium, Sendai, J apan, November 22-25, 2000, Abstract
51 O-10; in Histamine Research in the New Millenium; Wa-
tanabe, T., Timmerman, H., Yanai, K., Eds.; Elsevier: Amster-
dam, 2001; pp 77-82. (d) Schunack, W.; Sasse, A.; Stark, H.;
Elz, S.; Ligneau, X.; Ganellin, C. R.; Schwartz, J .-C. Partial
Agonists for the Histamine H3 Receptor with High in Vivo
Activity. XXXth Annual Meeting of the European Histamine
Research Society (EHRS), Turku, Finland, May 10-13, 2001,
Abstract O-7, p 31.
Hista m in e H3 Recep tor An ta gon ist P oten cy in Vivo
in Mice. In vivo testing was performed after peroral admin-
istration of the antagonist to Swiss mice as described by
Garbarg et al.33 Brain histamine turnover was assessed by
measuring the level of the main metabolite of histamine, Nτ-
methylhistamine. Mice were fasted for 24 h before po treat-
ment. Animals were decapitated 90 min after treatment, and
the brain was dissected out and homogenized in 10 volumes
of ice-cold perchloric acid (0.4 M). The Nτ-methylhistamine
level was measured with a radioimmunoassay.52 By treatment
with 3 mg of ciproxifan/kg or 10 mg of imetit/kg, the maximum
and minimum Nτ-methylhistamine levels were obtained and
related to the level reached with the administered drug. The
ED50 value was calculated as the mean with the SEM.53
Ca p sa icin -In d u ced P la sm a Extr a va sa tion . Male Wistar
rats were orally administered BP 2-94 or (S)-9 in increasing
doses or vehicle. After 90 min, they were anesthetized with
pentobarbital (6 mg/kg, ip) and after an additional 30 min were
treated with capsaicin (90 µg/kg, iv) and Evans Blue dye (30
mg/kg, iv) or vehicle. Five minutes after capsaicin treatment,
rats were perfused with saline to remove the intravascular
dye. Tissues were dissected out and analyzed for extravasated
Evans Blue dye by measuring absorption at 630 nm after dye
extraction.5
In Vitr o Scr een in g a t Oth er Hista m in e Recep tor s.
Selected compounds were screened for histamine H2 receptor
potency on the isolated spontaneously beating guinea pig right
atrium as well as for H1 receptor potency on the isolated guinea
pig ileum by standard methods described by Hirschfeld et al.41
Each pharmacological test was performed at least in triplicate.
The values that are given represent means.
Mu sca r in ic M3 Recep tor Assa y on Gu in ea P ig Ileu m .
The procedure that was used was that described by Pertz and
Elz.42
(2) Schwartz, J .-C.; Arrang, J .-M.; Garbarg, M.; Quemener, A.;
Lecomte, J .-M.; Ligneau, X.; Schunack, W.; Stark, H.; Purand,
K.; Hu¨ls, A.; Reidemeister, S.; Athmani, S.; Ganellin, C. R.;
Pelloux-Le´on, N.; Tertiuk, W.; Krause, M.; Sadek, B. Imidazole
Derivatives as Histamine Receptor H3 (Ant)Agonists. Int. Pat.
Appl. WO 96/29 315, Sep 26, 1996.
(3) 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.
(4) (a) J ansen, F. P.; Leurs, R.; Timmerman, H. Radioligands for
the Histamine H3 Receptor and Their Use in Pharmacology. In
The Histamine H3 Receptor; Leurs, R., Timmerman, H., Eds.;
Elsevier: Amsterdam, 1998; pp 127-144. (b) Stark, H.; Arrang,
J .-M.; Ligneau, X.; Garbarg, M.; Ganellin, C. R.; Schwartz, J .-
C.; Schunack, W. The Histamine H3 Receptor and its Ligands.
Prog. Med. Chem. 2001, 38, 279-308.
(5) Rouleau, A.; Garbarg, M.; Ligneau, X.; Mantion, C.; Lavie, P.;
Advenier, C.; Lecomte, J .-M.; Krause, M.; Stark, H.; Schunack,
W.; Schwartz, J .-C. Bioavailability, Antinociceptive and Anti-
inflammatory Properties of BP 2-94, a Histamine H3 Receptor
Agonist Prodrug. J . Pharmacol. Exp. Ther. 1997, 281, 1085-
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(6) Rouleau, A.; Stark, H.; Schunack, W.; Schwartz, J .-C. Anti-
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Histamine H3-Receptor Agonist Prodrug. J . Pharmacol. Exp.
Ther. 2000, 295, 219-225.
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L.; Ganellin, C. R.; Stark, H.; Elz, S.; Schunack, W.; Schwartz,
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Ther. 1998, 287, 658-666.
(9) Stark, H.; Sadek, B.; Krause, M.; Hu¨ls, A.; Ligneau, X.; Ganellin,
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Histamine H3-Receptor Antagonists with Carbonyl-Substituted
4-(3-(Phenoxy)propyl)-1H-imidazole Structures like Ciproxifan
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Ad r en er gic r1D Recep tor Assa y on Ra t Aor ta . The
procedure that was used was that described by Hirschfeld et
al.41
Ad r en er gic â1/2 Recep tor Assa y on Gu in ea P ig Righ t
Atr iu m . The procedure that was used was that described by
Pertz and Elz.43
Ser ot on er gic 5-H T2A R ecep t or Assa y on R a t Ta il
Ar ter y. The procedure that was used was that described by
Pertz and Elz.42
Ser ot on er gic 5-H T3 R ecep t or Assa y on Gu in ea P ig
Ileu m . The procedure that was used was that described by
Elz and Keller.42
(11) Lovenberg, T. W.; Pyati, J .; Chang, H.; Wilson, S. J .; Erlander,
M. G. Cloning of Rat Histamine H3 Receptor Reveals Distinct
Species Pharmacological Profiles. J . Pharmacol. Exp. Ther. 2000,
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(12) (a) Ligneau, X.; Morisset, S.; Tardivel-Lacombe, J .; Gbahou, F.;
Ganellin, C. R.; Stark, H.; Schunack, W.; Schwartz, J .-C.; Arrang,
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Receptors: Role of Two Amino Acids in the Third Trans-
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Ser oton er gic 5-HT4 Recep tor Assa y on Ra t Esop h a -
gu s. The procedure that was used was that described by Elz
and Keller.43
Ack n ow led gm en t. We gratefully thank J . Gysler
and G. Blaschke for supporting help with CE analysis.
This work was supported by the Biomedical & Health
Research Programme (BIOMED) of the European Union,
the Fonds der Chemischen Industrie, Verband der
Chemischen Industrie, Frankfurt/Main, Germany, and
the Deutsche Pharmazeutische Gesellschaft, DPhG-
Stiftung zur Fo¨rderung des wissenschaftlichen Nach-
wuchses, Germany.
(13) Morisset, S.; Rouleau, A.; Ligneau, X.; Gbahou, F.; Tardivel-
Lacombe, J .; Stark, H.; Schunack, W.; Ganellin, C. R.; Schwartz,
J .-C.; Arrang, J .-M. High Constitutive Activity of Native H3
Receptors Regulates Histamine Neurons in Brain. Nature 2000,
408, 860-864.
(14) Wieland, K.; Bongers, G.; Yamamoto, Y.; Hashimoto, T.; Yama-
todani, A.; Menge, W. M. B. P.; Timmerman, H.; Lovenberg, T.
W.; Leurs, R. Constitutive Activity of Histamine H3 Receptors
Stably Expressed in SK-N-MC Cells: Display of Agonism and
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(15) Rouleau, A.; Ligneau, X.; Tardivel-Lacombe, J .; Morisset, S.;
Gbahou, F.; Schwartz, J .-C.; Arrang, J .-M. Histamine H3-
Receptor-Mediated [35S]GTPγ[S] Binding: Evidence for Consti-
tutive Activity of the Recombinant and Native Rat and Human
H3 Receptors. Br. J . Pharmacol. 2002, 135, 383-392.
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