Progress in the proxifan class: heterocyclic congeners as novel potent and selective histamine H3-receptor antagonists

Article abstract of DOI:10.1016/S0928-0987(02)00024-6

Histamine H₃ receptors are critically involved in the pathophysiology of several disorders of the central nervous system (CNS). Among other families of H₃-receptor ligands, the proxifan class has recently been described to contain numerous potent histamine H₃-receptor antagonists, e.g. ciproxifan or imoproxifan. In the present study, we report on the design of novel heterocyclic proxifan analogues and their antagonist potencies at histamine H₃ receptors. The new compounds were tested for in vitro and in vivo H₃-receptor antagonist potencies in different species as well as for H₃-receptor selectivity vs. H₁ and H₂ receptors. In vitro, all compounds investigated proved to be potent H₃-receptor antagonists in the rat as well as in the guinea-pig. In addition, they showed good to high oral CNS potency in vivo in mice. Especially, oxadiazole derivatives 24-26 displayed nanomolar antagonist activity in vitro and high potency in vivo (ED₅₀=0.47-0.57 mg/kg). The results show that the additional heteroaromatic moieties might act as bioisosteres of the ketone or oxime moieties of ciproxifan or imoproxifan, respectively, and might cause divergent pharmacokinetic properties. Thus, these novel H₃-receptor antagonists are interesting leads for further development. Copyright

Full text of DOI:10.1016/S0928-0987(02)00024-6

European Journal of Pharmaceutical Sciences 15 (2002) 367–378
www.elsevier.nl/locate/ejps
Progress in the proxifan class: heterocyclic congeners as novel potent and
selective histamine H3-receptor antagonistsq
a
b
c
d
Sven Graßmanna , , Bassem Sadek , Xavier Ligneau , Sigurd Elz , C. Robin Ganellin ,
*
Jean-Michel Arrange, Jean-Charles Schwartze, Holger Starkf, Walter Schunacka
a
¨
¨
¨
Institut fur Pharmazie, Freie Universitat Berlin, Konigin-Luise-Strasse 214, 14195 Berlin, Germany
bLaboratoire Bioprojet, 30 rue des Francs-Bourgeois, 75003 Paris, France
c
¨
¨
Institut fur Pharmazie, Universitat Regensburg, 93040 Regensburg, Germany
dDepartment of Chemistry, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London WC1H 0AJ, UK
e
´
´
´
Unite de Neurobiologie et Pharmacologie Moleculaire (U.109), Centre Paul Broca de l’INSERM, 2ter rue d’Alesia, 75014 Paris, France
f
¨
¨
Biozentrum, Institut fur Pharmazeutische Chemie, Johann Wolfgang Goethe-Universitat, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany
Received 16 November 2001; received in revised form 22 February 2002; accepted 1 March 2002
Abstract
Histamine H3 receptors are critically involved in the pathophysiology of several disorders of the central nervous system (CNS). Among
other families of H3-receptor ligands, the proxifan class has recently been described to contain numerous potent histamine H3-receptor
antagonists, e.g. ciproxifan or imoproxifan. In the present study, we report on the design of novel heterocyclic proxifan analogues and
their antagonist potencies at histamine H3 receptors. The new compounds were tested for in vitro and in vivo H3-receptor antagonist
potencies in different species as well as for H3-receptor selectivity vs. H1 and H2 receptors. In vitro, all compounds investigated proved to
be potent H3-receptor antagonists in the rat as well as in the guinea-pig. In addition, they showed good to high oral CNS potency in vivo
in mice. Especially, oxadiazole derivatives 24–26 displayed nanomolar antagonist activity in vitro and high potency in vivo
(ED5050.47–0.57 mg/kg). The results show that the additional heteroaromatic moieties might act as bioisosteres of the ketone or oxime
moieties of ciproxifan or imoproxifan, respectively, and might cause divergent pharmacokinetic properties. Thus, these novel H3-receptor
antagonists are interesting leads for further development.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Histamine; H3 receptor; Proxifan; Antagonist; Ciproxifan; Imoproxifan
1. Introduction
in man and other species has been described (Lovenberg et
al., 1999, 2000; Tardivel-Lacombe et al., 2000; Drutel et
al., 2001). Following these findings another putative
heptahelical histamine receptor (H4) has been discovered
(for example, see Oda et al., 2000). In the H3-receptor
field recent molecular studies revealed the existence of
multiple mRNA isoforms from a single H3 gene (Tardivel-
Lacombe et al., 2000; Drutel et al., 2001) as well as
differential isoform distribution in the brain (Drutel et al.,
2001). This heterogeneity is suggested to also occur in
humans (Wellendorf et al., 2000). Among this third type of
histamine receptor, histamine H3 receptors were initially
characterized as presynaptic autoreceptors playing a key
role in the regulation of synthesis (Arrang et al., 1987b)
and release (Arrang et al., 1985) of histamine by means of
a negative feedback mechanism. On non-histaminergic
neurons H3 heteroreceptors possess the ability to influence
several monoaminergic and peptidergic neurotransmitter
systems (Schlicker et al., 1994; Hill et al., 1997). The
Histamine is a neurotransmitter in the brain and until a
short time ago, only three histamine receptors (H1, H2, and
H3) were known, all of them being G protein-coupled
molecules (for a recent review, see Brown et al., 2001).
Classic experimental pharmacology (Arrang et al., 1983)
demonstrated the existence of histamine H3 receptors
almost two decades before they were cloned. Recently, the
long awaited elucidation of the H3-receptor gene structure
qThis work was presented in part at the XVIth International Sym-
posium on Medicinal Chemistry, Bologna, Italy, September 18–22, 2000
and the XXXth Annual Meeting of the European Histamine Research
Society, Turku, Finland, May 9–12, 2001.
*
Corresponding author. Tel.: 149-30-8385-6587; fax: 149-30-8385-
3854.
E-mail address: sveng@schunet.pharmazie.fu-berlin.de (S. Graßmann).
0928-0987/02/$ – see front matter
PII: S0928-0987(02)00024-6
2002 Elsevier Science B.V. All rights reserved.

368
S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
predominant localization in the brain (Arrang et al.,
1987a), the mediation of important (patho)physiological
processes (Stark et al., 1996c), e.g. arousal (Schwartz et
al., 1991; Schwartz and Arrang, in press), and the inter-
action with a variety of different signal transduction
mechanisms make the H3 receptor considerably important.
Therefore, the need for highly potent and selective H3-
receptor ligands applicable both for subtype characteriza-
tion at the receptor level and for establishing new concepts
in pharmacotherapy has greatly increased. For antagonists
of histamine H3 receptors, several therapeutic targets have
been proposed. Since these ligands increase histaminergic
neuron activity by inhibiting the feedback mechanism,
their application as cognition enhancers seems to be most
promising, e.g. in Alzheimer’s disease (Morisset et al.,
1996; Panula et al., 1998), attention-deficit hyperactivity
disorder (Leurs et al., 1998), and schizophrenia (Schwartz
and Arrang, in press).
Major progress in the pharmacology of H3 receptors was
obtained with the development of the first selective and
orally active H3-receptor antagonist thioperamide (Fig. 1;
Arrang et al., 1987a). Another reference antagonist is
clobenpropit which proved to be a very potent antagonist
in vitro, but with only moderate potency in vivo (Fig. 1;
van der Goot et al., 1992). Considering potential benefit in
affinity, selectivity, pharmacokinetics, and/or toxicology
we reported on the successful design of a new series of
H3-receptor antagonists: the proxifan class (Ganellin et al.,
1996; Ligneau et al., 1998; Kathmann et al., 1998; Stark et
al., 2000a,b; Sasse et al., 2000). Acetoproxifan (FUB 372,
Fig. 1) provided the starting point with nanomolar in vitro
potency and high antagonist activity in vivo. The com-
pound contains an imidazole heterocycle connected by a
three-carbon alkyl chain to a para-substituted phenyl ether
moiety, features present in all ligands of this novel class.
On the basis of the structure–activity relationships in the
proxifan series we consequently developed the cyclopropyl
ketone derivative ciproxifan (Fig. 1) with even higher in
vitro and in vivo potency (Stark et al., 2000b). Continuing
efforts to refine and enhance the H3-receptor potency and
selectivity resulted in the recent identification of the oxime
derivative imoproxifan (Fig. 1), an antagonist of sub-
nanomolar affinity, high selectivity, and extremely high in
vivo potency (Sasse et al., 2000). In this context, another
related class of compounds, the proxyfan series, should be
]
mentioned to avoid a possible mixing up with the class
cited before (Fig. 1). Compared to the proxi]fan class,
proxyfans possess a similar chemical structure, but with a
]
benzyl ether instead of a phenyl ether group. Proxyfan is
the only neutral H3-receptor antagonist, i.e. a drug that
opposes both agonists and inverse agonists, described so
far (Morisset et al., 2000). However, the behaviour of this
compound is currently a matter of debate depending on
both response measured and histamine H3 receptor expres-
sion level (Morisset et al., 2000). Substituted proxyfans
possess different pharmacological behaviour depending on
the test system (cf. [125I]iodoproxyfan (Ligneau et al.,
1994; Stark et al., 1996a)).
Numerous structural modifications of the reference
antagonists ciproxifan and imoproxifan have been de-
scribed which clearly show strong preference for a sub-
stituent in the 4-position (Stark et al., 2000a,b; Sasse et al.,
2000). Likewise, when the cyclopropyl moiety of ciprox-
ifan was replaced by different aromatic ring systems no
further improvement in antagonist activity could be de-
tected (Sasse et al., 2001). However, one interesting
compound emerging from this study is the 2-thienyl
methanone derivative FUB 478 (Fig. 1) with good antago-
nist activity in vitro and high oral potency in vivo, one of
the few examples of a compound with an additional
aromatic heterocycle and promising pharmacological prop-
erties. Considering this heteroaromatic compound and the
ketone or oxime structure of ciproxifan or imoproxifan,
respectively, possible bioisosteric replacements will be
investigated.
In this report, we describe in vitro and in vivo H3-
receptor antagonist properties of novel members of the
proxifan family with various heteroaromatic moieties. In
an effort to reveal principles and applicability of this
heterocycle introduction we additionally investigate the
new compounds for H3-receptor selectivity. In order to
further characterize these structures we compare all new
pharmacological results with data obtained from FUB 478
and already established histamine H3-receptor antagonists,
i.e. thioperamide, clobenpropit, acetoproxifan, ciproxifan,
and imoproxifan, respectively.
2. Materials and methods
2.1. Chemistry
2.1.1. General procedures
Fig. 1. Different histamine H3-receptor antagonists.
Melting points (mp) were determined on an Electrother-

S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
369
1
¨
mal IA 9000 digital or a Buchi 512 apparatus. H NMR
Key intermediates were the para-substituted phenyl methyl
ethers 8–12.
spectra were recorded on a Bruker AC 300 (300 MHz) or
DPX 400 Avance (400 MHz) spectrometer in Me2SO-d6
as standard solvent, unless stated otherwise. 13C NMR
recording was performed on a Bruker DPX 400 Avance
(100 MHz). Chemical shifts are reported in ppm downfield
from internal tetramethylsilane as reference. 1H NMR
signals are reported in order: multiplicity (s, singlet; d,
doublet; t, triplet; m, multiplet; *, exchangeable by D2O),
number of protons, and approximate coupling constants in
Hertz. EI mass spectra were obtained on a Finnigan MAT
CH7A (70 eV, 170 8C) and FAB1 spectra were recorded on
a Finnigan MAT CH5DF (Xe, 80 eV, Me2SO as solvent,
glycerol as matrix). Elemental analyses (C, H, N) were
measured for final compounds on Perkin-Elmer 240 B or
Perkin-Elmer 240 C instruments and are within 60.4% of
the theoretical values. Column chromatography was car-
ried out using silica gel 63–200 mm (Merck). Thin layer
chromatography was performed on silica gel F254 plates
(Merck). The following abbreviations are used: Benz,
benzoxathiazolyl, EtOH, ethanol; Et2O, diethyl ether; Im,
imidazolyl; Mal; maleic acid; MeOH, methanol; Me2SO,
dimethyl sulfoxide; Ox, oxadiazolyl; Ph, phenyl; Tet,
tetrazolyl; Th, thiazolyl; THF, tetrahydrofuran.
The most common and most versatile procedure for the
formation of 1,3-thiazoles is the cyclocondensation of
a-haloketones with appropriate thioamide derivatives (Ver-
nim, 1979). In this study, the reaction of commercially
available a-bromoketone 4 with thioacetamide under basic
conditions resulted in the 4-substituted heterocyclic deriva-
tive 8. The amidoxime 5 provided the starting point for the
synthesis of the 3,5-disubstituted 1,2,4-oxadiazole 9. It was
generated by reaction of hydroxylamine, liberated from its
hydrochloride using potassium carbonate, with p-anisonit-
rile in refluxing ethanol. Dehydration, cyclization, and
aromatization by subsequent treatment with acetic acid
methyl ester in the presence of sodium hydride in tetrahy-
drofuran afforded the corresponding 5-methyl-1,2,4-ox-
adiazole compound 9 (Swain et al., 1991). The 3-methyl-
1,2,4-oxadiazole derivative 10 was prepared adapting the
procedure described by Lin et al. (1979). Reaction of the
carboxamide 3 with N,N-dimethylacetamide dimethyl acet-
al and cyclization of the intermediate acylamidine with
hydroxylamine provided 10 in good yield. According to
the procedure described by Ainsworth (1955) synthesis of
the 5-methyl-1,3,4-oxadiazole derivative 11 was achieved
by treatment of the ester 2 with hydrazine resulting in the
intermediary hydrazide 6 followed by cyclization with
triethyl orthoacetate. In a direct one-pot reaction primary
amide 3 was converted with triazidochlorosilane into the
5-substituted tetrazole 7 (El-Ahl et al., 1997). Adapting the
protocol of Begtrup and Larsen (1990), 7 was methylated
2.1.2. Synthesis
With the exception of 20 and 22, all novel heterocyclic
compounds were synthesized by ring-closing reactions,
each derivative via a different synthetic pathway (Fig. 2).
Fig. 2. Synthetic procedures for key intermediates 8–12. a H2NOH?HCl, K2CO3, EtOH, reflux, 18 h; b H2NNH2?H2O, 120 8C, 3 h; c SiCl4, NaN3,
e
H3CCN, reflux, 3 h; d H3CCSNH2, K2CO3, N,N-dimethylformamide, 100–120 8C, 2 h; (i) molecular sieves (4 A), THF, 30 min; (ii) NaH,
˚
H3CCOOCH3, THF, reflux, 6 h; f (i) N,N-dimethylacetamide dimethyl acetal, 120 8C, 2.5 h; (ii) H2NOH?HCl, 1 N NaOH, dioxane, AcOH, 90 8C, 3 h;
g (i) triethyl orthoacetate, 120 8C, 3 h; (ii) 140 8C, 4 h; h H3CI, KOH, MeOH, reflux, 1 h.

370
S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
with methyl iodide in methanolic potassium hydroxide.
The resulting mixture of the isomeric 1-methyl- and 2-
methyltetrazole derivatives was separated by column chro-
matography and showed a product ratio of 4:1 in favour of
12. The 1H and 13C NMR data for 12 were in full
agreement with literature data (Fraser and Haque, 1968;
Begtrup, 1973).
readily prepared from commercially available 6-hydroxy-
1,3-benzoxathiazol-2-one and 4-(1H-imidazol-1-yl)phenol
as mentioned above.
2.1.2.1. Methyl 4-(2-methyl-1,3-thiazol-4-yl)phenyl ether
(8)
In one-pot reactions with tosylmethyl isocyanide (Tos-
MIC) carbonyl compounds can efficiently be converted
into 5-substituted oxazoles (van Leusen et al., 1972). Here,
the oxazole compound 21 was obtained in 64% yield by
reaction of equimolar quantities of TosMIC and the
benzaldehyde derivative FUB 402 (Stark et al., 2000b)
with potassium carbonate in refluxing methanol (Fig. 3).
Proceeding from the different heterocyclic derivatives
8–12 methyl ether cleavage with BBr3 in dichloromethane
provided the corresponding phenols 13–17 in high yields.
All final compounds except 26 were synthesized by phenol
etherification with trityl-protected 3-(1H-imidazol-4-
yl)propanol (18) under Mitsunobu conditions (Mitsunobu,
1981) and acidic hydrolysis of the protecting group (Stark
et al., 1996a,b). When 26 was synthesized the lower
stability of the 1,3,4-oxadiazole system did not allow
acidolytic or hydrogenolytic deprotection of the trityl
group. Hence, 16 was etherified by analogy to Krause et al.
(1998) with Boc-protected 3-(1H-imidazol-4-yl)propanol
(19) followed by hydrazinolysis in methanol under mild
conditions. Benzoxathiazole 20 and imidazole 22 were
4-Methoxyphenacyl bromide (4, 1.49 g, 6.5 mmol),
thioacetamide (0.48 g, 6.5 mmol), and K2CO3 (1.20 g, 8.7
mmol) were dissolved in dry N,N-dimethylformamide (30
ml). The mixture was stirred for 2 h at 100–120 8C,
cooled, and the solvent was removed under reduced
pressure. The solid residue was recrystallized from EtOH:
1
Yield 79%; mp 59–60 8C (Das and Rout, 1957: 63 8C); H
NMR d 2.70 (s, 3H, Th–CH3), 3.79 (s, 3H, H3CO), 6.99
(d, J58.7 Hz, 2H, Ph-3,5H), 7.74 (s, 1H, Th-5H), 7.87 (d,
J58.7 Hz, 2H, Ph-2,6H); EI–MS m/z (%) 205 (M1, 100).
2.1.2.2. 4-Methoxyphenylcarboxamidoxime (5)
p-Anisonitrile (1, 6.66 g, 50 mmol), hydroxylamine
hydrochloride (6.95 g, 100 mmol), and K2CO3 (10.34 g,
75 mmol) in dry EtOH (80 ml) were heated under reflux
for 18 h. The mixture was cooled, filtered, and concen-
trated under reduced pressure. The crystalline residue was
suspended in MeOH, and the final product was isolated by
filtration: Yield 22%; mp 122–124 8C (Eloy and Lenaers,
1
1962: 122–123 8C); H NMR d 3.77 (s, 3H, H3CO), 5.71
Fig. 3. Synthesis of heterocyclic derivatives 20–27. Het, 2-methyl-1,3-thiazol-4-yl (8, 13, 23), 5-methyl-1,2,4-oxadiazol-3-yl (9, 14, 24), 3-methyl-1,2,4-
oxadiazol-5-yl (10, 15, 25), 5-methyl-1,3,4-oxadiazol-2-yl (11, 16, 26), or 2H-2-methyl-1,2,3,4-tetrazol-5-yl (12, 17, 27); Y, tert-butoxycarbonyl (Boc) (19,
synthesis of 26) or triphenylmethyl (18, all other compounds). a BBr3, CH2Cl2, 72 h, 280 8C; b (i) Ph3P, DEAD, THF, r.t., 12–72 h; (ii) H2NNH2?H2O,
MeOH, r.t., 30 min (synthesis of 26) or 2 N HCl, reflux, 2 h (all other compounds); c TosMIC, K2CO3, MeOH, reflux, 18 h.

S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
371
*
(s , 2H, NH2), 6.92 (d, J58.8 Hz, 2H, Ph-2,6H), 7.59–
2.1.2.6. Methyl 4-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl
ether (11)
*
7.62 (m, 2H, Ph-3,5H), 9.45 (s , 1H, OH); EI–MS m/z
(%) 166 (M1, 100).
Triethyl orthoacetate (10 ml) and 6 (1.66 g, 10 mmol)
were heated at 120 8C for 3 h. Excess triethyl orthoacetate
was evaporated, and the residue heated for further 2 h at
140 8C. The reaction was diluted with H2O (10 ml),
saturated with K2CO3, and extracted with Et2O. Evapora-
tion of the dried extracts afforded 11 as an oil: Yield 66%;
mp 88 8C (Huisgen et al., 1960: 91–92 8C); 1H NMR d
2.56 (s, 3H, Ox–CH3), 3.85 (s, 3H, H3CO), 7.13 (d,
J58.8 Hz, 2H, Ph-3,5H), 7.91 (d, J58.7 Hz, 2H, Ph-
2,6H); EI–MS m/z (%) 190 (M1, 93).
2.1.2.3. Methyl 4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl
ether (9)
Powdery molecular sieves (4 A, 4 g) were added to a
˚
solution of 5 (1.66 g, 10 mmol) in dry THF (60 ml). After
stirring for 30 min NaH (suspended in mineral oil, v5
60%, 0.22 g, 5.5 mmol) was added, and the mixture was
heated for 45 min at 60 8C. After cooling acetic acid
methyl ester (1.48 g, 20 mmol) dissolved in dry THF was
added dropwise. Subsequently, the mixture was refluxed
for 6 h under anhydrous conditions. Cooling, filtration, and
removal of the solvent under reduced pressure resulted in a
solid residue which was recrystallized from EtOH: Yield
2.1.2.7. Methyl 4-(2H-tetrazol-5-yl)phenyl ether (7)
A suspension of silicon(IV) chloride (9.35 g, 55 mmol)
and sodium azide (7.15 g, 110 mmol) in dry acetonitrile
(20 ml) was stirred for 1 h at ambient temperature. 4-
Methoxybenzamide (3, 4.00 g, 27 mmol) was added
dropwise. After stirring under reflux for 3 h the reaction
mixture was poured into an ice-cooled solution of K2CO3
in H2O, filtered, and acidified with aqueous HCl. The
precipitated product was filtered, washed with H2O, and
dried in vacuo: Yield 80%; mp 237–238 8C (Herbst and
Wilson, 1957: 238–239 8C); 1H NMR d 3.85 (s, 3H,
H3CO), 7.15–7.18 (m, 2H, Ph-3,5H), 7.97–8.00 (m, 2H,
Ph-2,6H); EI–MS m/z (%) 176 (M1, 43).
1
83%; mp 61–62 8C (Bianchi et al., 1974: 58–60 8C); H
NMR d 2.64 (s, 3H, Ox–CH3), 3.83 (s, 3H, H3CO), 7.11
(d, J58.8 Hz, 2H, Ph-3,5H), 7.92–7.95 (m, 2H, Ph-2,6H);
FAB1–MS m/z (%) 191 (M11H1, 100).
2.1.2.4. Methyl 4-(3-methyl-1,2,4-oxadiazol-5-yl)phenyl
ether (10)
A solution of 4-methoxybenzamide (3, 1.01 g, 6.7
mmol) in N,N-dimethylacetamide dimethyl acetal (15 ml)
was stirred for 2.5 h at 120 8C under N2. The solvent was
removed under reduced pressure and hydroxylamine hy-
drochloride (0.65 g, 9.4 mmol) dissolved in an aqueous
NaOH solution (9.14 ml, c51 mol/l) was added. After
addition of dioxane (10 ml) and acetic acid (12.5 ml) the
mixture was stirred for 1.5 h at room temperature. Sub-
sequently, the mixture was heated 3 h at 90 8C, cooled, and
a saturated solution of K2CO3 in H2O was added. Then,
the mixture was concentrated under vacuum and the
residual aqueous layer was extracted with CH2Cl2. The
organic extracts were combined, dried, and concentrated
under reduced pressure. The oily product was purified by
column chromatography (eluent: CH2Cl2) and crystallized
2.1.2.8. Methyl 4-(2-methyl-2H-tetrazol-5-yl)phenyl ether
(12)
A solution of 7 (1.86 g, 10.6 mmol) and KOH (2.81 g,
50 mmol) in dry MeOH (30 ml) was mixed at 225 8C
with iodomethane (4.56 g, 32.1 mmol) dissolved in dry
MeOH (5 ml). The temperature was raised to 20 8C during
1 h and the mixture refluxed for an additional hour. The
solvent was removed and the crude product purified by
column chromatography (eluent: CH2Cl2): Yield 60%; mp
1
56–57 8C (Butler et al., 1984: 85–86 8C); H NMR d 3.83
(s, 3H, H3CO), 4.40 (s, 3H, NCH3), 7.10–7.13 (m, 2H,
Ph-3,5H), 7.97–8.00 (m, 2H, Ph-2,6H); 13C NMR
(CDCl3) d 39.7 (NCH3), 55.8 (H3CO), 114.7 (Ph-2,6C),
120.4 (Ph-4C), 128.7 (Ph-3,5C), 161.7 (Ph-1C), 165.6
(Tet-5C); EI–MS m/z (%) 190 (M1, 21).
1
at 4 8C: Yield 66%; mp 59–61 8C; H NMR d 2.39 (s, 3H,
Ox–CH3), 3.87 (s, 3H, H3CO), 7.15–7.18 (m, 2H, Ph-
3,5H), 8.03 (d, J58.8 Hz, 2H, Ph-2,6H); EI–MS m/z (%)
190 (M1, 92).
2.1.2.9. General procedure for ether cleavage
A solution of the corresponding ether (4 mmol) in dry
CH2Cl2 (20 ml) was cooled to 280 8C under N2. BBr3 (4
mmol, c51.0 mol/l, solution in CH2Cl2), was added
dropwise with the temperature not exceeding 260 8C. The
reaction mixture was then allowed to warm to room
temperature and stirred for additional 72 h. Subsequently,
the mixture was cooled to 280 8C and MeOH (25 ml) was
added dropwise. The organic layer was removed from the
mixture under reduced pressure. After addition of a
saturated K2CO3 solution in H2O to the aqueous layer the
crude product precipitated. It was isolated by filtration and
recrystallized from EtOH.
2.1.2.5. 4-Methoxybenzoic acid hydrazide (6)
A mixture of 4-methoxybenzoic acid methyl ester (2,
4.99 g, 30 mmol) and hydrazine monohydrate (3.00 g, 60
mmol) was heated at 120 8C for 3 h. Cooling and dilution
with H2O (10 ml) resulted in a precipitate which was
filtered, washed with H2O, and dried in vacuo: Yield 89%;
1
mp 128–130 8C (Horner and Fernekess, 1961: 136 8C); H
*
NMR d 3.80 (s, 3H, H3CO), 4.42 (s , 2H, NH2), 6.96–
7.01 (m, 2H, Ph-3,5H), 7.79–7.84 (m, 2H, Ph-2,6H), 9.63
1
*
(s , 1H, NH); EI–MS m/z (%) 166 (M , 11).

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S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
2.1.2.9.1.
4-(2-Methyl-1,3-thiazol-4-yl)phenol
(13)
2.1.2.12. 6-[3-(1H-Imidazol-4-yl)propoxy]-1,3-benzox-
athiazol-2-one hydrogen maleate (20)
1
From 8; yield 70%; mp 151 8C; H NMR d 2.69 (s, 3H,
Th–CH3), 6.81 (d, J58.6 Hz, 2H, Ph-3,5H), 7.64 (s, 1H,
From 18 and 6-hydroxy-1,3-benzoxathiazol-2-one; 1H
NMR d 2.07 (m, 2H, Im–CH2CH2), 2.79 (t, J57.5 Hz,
2H, Im–CH2), 4.04 (t, J56.2 Hz, 2H, CH2O), 6.03 (s, 2H,
Mal), 6.93 (d, J58.8 Hz, 1H, Benz-5H), 7.16 (s, 1H,
Im-5H), 7.63 (d, J58.8 Hz, 2H, Benz-4H), 8.87 (s, 1H,
Im-2H); EI–MS m/z (%) 276 (M1, 9). Anal.
(C13H12N2O3S?C4H4O4?1.75H2O) C, H, N.
*
Th-5H), 7.75 (d, J58.6 Hz, 2H, Ph-2,6H), 9.55 (s , 1H,
OH); EI–MS m/z (%) 191 (M1, 100).
2.1.2.9.2. 4-(5-Methyl-1,2,4-oxadiazol-3-yl)phenol (14)
From 9; yield 84%; mp 189–191 8C (Behr, 1962: 185 8C);
1H NMR d 2.62 (s, 3H, CH3), 6.89–6.92 (m, 2H, Ph-
3,5H), 7.81–7.83 (m, 2H, Ph-2,6H); EI–MS m/z (%) 176
(M1, 100).
2.1.2.9.3. 4-(3-Methyl-1,2,4-oxadiazol-5-yl)phenol (15)
From 10; yield 83%; mp 186 8C; H NMR d 2.37 (s, 3H,
CH3), 6.94–6.98 (m, 2H, Ph-3,5H), 7.92 (d, J58.7 Hz,
2.1.2.13. 3-(1H-Imidazol-4-yl)propyl
yl)phenyl ether hydrogen maleate (21)
4-(1,3-oxazol-5-
1
p-Toluenesulfonylmethyl isocyanide (0.27 g, 1.4 mmol)
and FUB 402 (0.32 g, 1.4 mmol), prepared according to
Stark et al. (2000b), were dissolved in dry MeOH (30 ml).
During 30 min K2CO3 (0.19 g, 1.4 mmol) was added
slowly. After refluxing for 18 h the solvent was removed.
The oily residue was purified by column chromatography
(eluent: CH2Cl2 /MeOH, NH3-sat.; 95:5, v/v) and crys-
*
2H, Ph-2,6H), 10.49 (s , 1H, OH); EI–MS m/z (%) 176
(M1, 69).
2.1.2.9.4. 4-(5-Methyl-1,3,4-oxadiazol-2-yl)phenol (16)
From 11; yield 55%; mp 236 8C (El Masri et al., 1958:
1
239 8C); H NMR d 2.56 (s, 3H, CH3), 6.98 (d, J58.6 Hz,
2H, Ph-3,5H), 7.85 (d, J58.6 Hz, 2H, Ph-2,6H), 10.26
1
1
*
(s , 1H, OH); EI–MS m/z (%) 176 (M , 100).
tallized with maleic acid from EtOH/Et2O. H NMR d
2.1.2.9.5.
4-(2-Methyl-2H-tetrazol-5-yl)phenol
(17)
2.04–2.09 (m, 2H, Im–CH2CH2), 2.80 (t, J57.4 Hz, 2H,
Im–CH2), 4.05 (t, J56.0 Hz, 2H, CH2O), 6.03 (s, 2H,
Mal), 7.02 (d, J58.6 Hz, 2H, Ph-3,5H), 7.40 (s, 1H,
Im-5H), 7.52 (s, 1H, Ox-4H), 7.64 (d, J58.6 Hz, 2H,
Ph-2,6H), 8.36 (s, 1H, Ox-2H), 8.83 (s, 1H, Im-2H);
EI–MS m/z (%) 269 (M1, 9). Anal. (C15H15N3O2?
C4H4O4?0.25H2O) C, H, N.
1
From 12; yield 95%; mp 210–212 8C; H NMR d 4.38 (s,
3H, NCH3), 6.92 (d, J58.6 Hz, 2H, Ph-3,5H), 7.87 (d,
*
J58.5 Hz, 2H, Ph-2,6H), 9.96 (s , 1H, OH); EI–MS m/z
(%) 176 (M1, 22).
2.1.2.10. General procedure for Mitsunobu-type etherifi-
cation (compounds 20–25, 27)
3-(1-Triphenylmethyl-1H-imidazol-4-yl)propanol (18,
1.84 g, 5 mmol) (Stark et al., 1996b), triphenylphosphine
(1.57 g, 6 mmol), and the corresponding phenol (6 mmol)
were dissolved in freshly distilled THF (15 ml) under Ar
atmosphere and ice-cooling. Then, diethyl azodicarboxy-
late (0.95 ml, 6 mmol) was slowly added followed by
additional stirring for 12–72 h at room temperature. After
removal of the solvent in vacuo the crude product was
purified by column chromatography (eluent: EtOAc). The
oily residue was dissolved in THF (40 ml), aqueous HCl
(c52 mol/l, 20 ml) was added and the mixture was
refluxed for 2 h. After removal of the organic solvent,
filtration, and extraction with Et2O the aqueous phase was
basified (K2CO3) and extracted with Et2O. The latter
organic extracts were combined, washed (H2O), dried
(Na2SO4), and concentrated under reduced pressure. The
residue was crystallized and recrystallized as free base,
hydrogen oxalate or hydrogen maleate from EtOH/Et2O.
2.1.2.14. 4-(1H-Imidazol-1-yl)phenyl 3-(1H-imidazol-4-
yl)propyl ether dihydrogen maleate (22)
From 18 and 4-(1H-imidazol-1-yl)phenol; 1H NMR d
2.08–2.13 (m, 2H, Im–CH2CH2), 2.83 (t, J57.6 Hz, 2H,
Im–CH2), 4.08 (t, J56.1 Hz, 2H, CH2O), 6.13 (s, 4H,
Mal), 7.11 (d, J59.0 Hz, 2H, Ph-3,5H), 7.47 (s, 1H,
Im-5H), 7.49 (m, 1H, Im-49H), 7.63 (d, J59.0 Hz, 2H,
Ph-2,6H), 7.90 (m, 1H, Im-59H), 8.81 (s, 1H, Im-2H), 9.00
(s, 1H, Im-29H); FAB1–MS m/z (%) 269 (M11H1,
100). Anal. (C15H16N4O?2C4H4O4?0.75H2O) C, H, N.
2.1.2.15. 3-(1H-Imidazol-4-yl)propyl
4-(2-methyl-1,3-
thiazol-4-yl)phenyl ether hydrogen maleate (23)
From 18 and 13; 1H NMR d 2.05–2.12 (m, 2H, Im–
CH2CH2), 2.70 (s, 3H, CH3), 2.84 (t, J58.1 Hz, 2H,
Im–CH2), 4.05 (t, J56.2 Hz, 2H, CH2O), 6.05 (s, 2H,
Mal), 6.97 (d, J58.7 Hz, 2H, Ph-3,5H), 7.43 (s, 1H,
Im-5H), 7.74 (s, 1H, Th-5H), 7.85 (d, J58.7 Hz, 2H,
Ph-2,6H), 8.87 (s, 1H, Im-2H); EI–MS m/z (%) 299 (M1,
3). Anal. (C16H17N3OS?C4H4O4) C, H, N.
2.1.2.11. Modifications of Mitsunobu-type etherification
(compound 26)
4-(3-Hydroxypropyl)-1H-imidazol-1-yl carboxylic acid
tert-butyl ester (19, 1.13 g, 5 mmol) (Krause et al., 1998)
was used. Column chromatography was performed with a
different eluent (CH2Cl2 /MeOH, NH3-sat.; 95:5, v/v).
The Boc-protected product was deprotected by stirring in
methanolic hydrazine monohydrate solution (v510%, 25
ml) at ambient temperature for 30 min.
2.1.2.16. 3-(1H-Imidazol-4-yl)propyl 4-(5-methyl-1,2,4-
oxadiazol-3-yl)phenyl ether (24)
From 18 and 14; H NMR (CDCl3) d 2.12–2.21 (m, 2H,
Im–CH2CH2), 2.63 (s, 3H, CH3), 2.83 (t, J57.4 Hz, 2H,
Im–CH2), 4.05 (t, J56.2 Hz, 2H, CH2O), 6.81 (s, 1H,
Im-5H), 6.96 (d, J58.7 Hz, 2H, Ph-3,5H), 7.58 (s, 1H,
1

S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
373
Im-2H), 7.97 (d, J58.7 Hz, 2H, Ph-2,6H); EI–MS m/z
(%) 284 (M1, 6). Anal. (C15H16N4O2?H2O) C, H, N.
mean (S.E.M.) of experiments performed at least in
triplicate.
2.2.1.2. Histamine H3-receptor antagonist potency in vivo
in the mouse
2.1.2.17. 3-(1H-Imidazol-4-yl)propyl 4-(3-methyl-1,2,4-
oxadiazol-5-yl)phenyl ether hydrogen maleate (25)
From 18 and 15; 1H NMR d 2.08–2.15 (m, 2H, Im–
CH2CH2), 2.39 (s, 3H, CH3), 2.82 (t, J57.5 Hz, 2H,
Im–CH2), 4.14 (t, J56.2 Hz, 2H, CH2O), 6.05 (s, 2H,
Mal), 7.14 (d, J58.9 Hz, 2H, Ph-3,5H), 7.43 (s, 1H,
Im-5H), 8.03 (d, J58.8 Hz, 2H, Ph-2,6H), 8.88 (s, 1H,
Im-2H); EI–MS m/z (%) 284 (M1, 2). Anal.
(C15H16N4O2?C4H4O4?0.25H2O) C, H, N.
Compounds were examined for their antagonist poten-
cies in vivo after oral administration to male Swiss mice
(for details, see Garbarg et al., 1992). Brain histaminergic
neuronal activity was assessed by determination of the
main metabolite of histamine, Nt-methylhistamine. Mice
were fasted for 24 h before p.o. treatment. Animals were
decapitated 90 min after treatment. The brain was isolated
and homogenized in 10 vol of ice-cold perchloric acid (0.4
M). The Nt-methylhistamine level was measured by
radioimmunoassay (Garbarg et al., 1989). Treatment with
3 mg/kg ciproxifan gave the maximal increase in Nt-
methylhistamine level (Ligneau et al., 1998) which was
related to the level reached with the administered drug.
Each experiment was performed at least in triplicate. The
ED50 value is expressed as mean6S.E.M.
2.1.2.18. 3-(1H-imidazol-4-yl)propyl 4-(5-methyl-1,3,4-
oxadiazol-2-yl)phenyl ether hydrogen maleate (26)
From 19 and 16; 1H NMR d 2.08–2.11 (m, 2H, Im–
CH2CH2), 2.55 (s, 3H, CH3), 2.81 (t, J57.4 Hz, 2H,
Im–CH2), 4.10 (t, J56.1 Hz, 2H, CH2O), 6.03 (s, 2H,
Mal), 7.10 (d, J58.7 Hz, 2H, Ph-3,5H), 7.42 (s, 1H,
Im-5H), 7.89 (d, J58.7 Hz, 2H, Ph-2,6H), 8.85 (s, 1H,
Im-2H); EI–MS m/z (%) 284 (M1, 1). Anal.
(C15H16N4O2?C4H4O4?0.25H2O) C, H, N.
2.2.1.3. Histamine H3-receptor antagonist potency on
guinea-pig ileum
For selected compounds H3-receptor potency in vitro
was measured by inhibition of concentration-dependent
relaxation of field-stimulated isolated guinea-pig ileum
segments (longitudinal muscle with adhering plexus myen-
tericus) induced by (R)-a-methylhistamine according to
Ligneau et al. (1994). Four to seven experiments were
performed for each compound. Longitudinal muscle strips
were prepared from the small intestine, 20–50 cm proxim-
al to the ileocecal valve. The muscle strips were mounted
between two platinum electrodes (4 mm apart) in 20 ml of
Krebs buffer, containing 1 mM of mepyramine, connected
to an isometric transducer, continuously gassed with
oxygen containing 5% CO2 at 37 8C. After equilibration of
the muscle segments for 1 h accompanied by washing
every 10 min, they were stimulated continuously with
rectangular pulses of 15 V and 0.5 ms at a frequency of 0.1
Hz. After 30 min of stimulation, a cumulative concen-
tration–response curve to (R)-a-methylhistamine was re-
corded. Subsequently the preparations were washed three
times every 10 min without stimulation. The antagonist
was incubated 20–30 min before redetermination of the
concentration–response curve of (R)-a-methylhistamine
(Schlicker et al., 1994). The new antagonists were tested at
concentrations that did not block ileal muscarine M3
receptors (data not shown). In the absence of H3-receptor
antagonist, two successive concentration–relaxation curves
for (R)-a-methylhistamine were superimposable (data not
shown).
2.1.2.19. 3-(1H-Imidazol-4-yl)propyl
4-(2-methyl-2H-
tetrazol-5-yl)phenyl ether hydrogen oxalate (27)
From 18 and 17; 1H NMR d 2.08–2.11 (m, 2H, Im–
CH2CH2), 2.79 (t, J57.5 Hz, 2H, Im–CH2), 4.09 (t,
J56.2 Hz, 2H, CH2O), 4.40 (s, 3H, NCH3), 7.10 (d,
J58.8 Hz, 2H, Ph-3,5H), 7.27 (s, 1H, Im-5H), 7.98 (d,
J58.8 Hz, 2H, Ph-2,6H), 8.53 (s, 1H, Im-2H); EI–MS
m/z (%) 284 (M1, 2). Anal. (C14H16N6O?C2H2O4?
0.5H2O) C, H, N.
2.2. Pharmacology
2.2.1. General methods
2.2.1.1. Histamine H3-receptor antagonist potency in vitro
on synaptosomes of rat cerebral cortex
Antagonist potency of the novel compounds was investi-
gated using an in vitro protocol where K1-evoked depolar-
ization induces [3H]histamine release from rat synapto-
somes (Garbarg et al., 1992). The synaptosomal fraction
was prepared according to Whittaker (1966), preincubated
with L-[3H]histidine (0.4 mM) at 37 8C for 30 min in a
modified Krebs–Ringer solution, washed extensively, and
transferred into a fresh Krebs–Ringer buffer containing 2
mM K1. Compounds and 1 mM histamine were added 5
min before the depolarization stimulus (30 mM K1 final
concentration). Incubation was terminated by rapid cen-
trifugation. [3H]Histamine levels were determined by
liquid scintillation spectrometry (Garbarg et al., 1992). Ki
values were calculated according to Cheng and Prusoff
(1973). Data are presented as mean6standard error of the
2.2.1.4. In vitro screening at other histamine receptors
Selected compounds were screened for histamine H2-
receptor activity on the isolated spontaneously beating
guinea-pig right atrium as well as for H1 receptor activity

374
S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
on the isolated guinea-pig ileum by standard methods
described by Hirschfeld et al. (1992). With the exception
of 20 (single experiment) H1-receptor potency was investi-
gated at least in triplicate for each compound, while for H2
receptors each experiment was performed at least in
duplicate.
compounds are the azole derivatives 21 and 22. Not
surprisingly, their physicochemical divergences reoccurred
in the in vitro pharmacology of the compounds, but the in
vivo data in both cases paralleled the result obtained with
20. With the introduction of a thiazole moiety (23) no
enhancement in in vitro potency, but still a favourable oral
in vivo potency could be detected. Major improvement in
biological activity was obtained with the next three
compounds, the methyloxadiazole derivatives 24–26.
Within this homogeneous series, the oxadiazoles differ
from each other solely in the position of the three
heteroatoms of the five-membered aromatic ring with
regard to the methyl or phenyl substituent. Whereas
potencies in vitro are significantly different from each
other, in vivo, the three compounds are equipotent at a
high level of oral potency (ED50 values of 0.47–0.57
mg/kg p.o.). Although the oxadiazoles showed reduced
potencies compared to those of ciproxifan and especially
imoproxifan, their high in vivo potencies are particularly
noteworthy as in vivo they exceed the antagonist activities
of the reference antagonists thioperamide and clobenpropit
(Table 1). The comparatively lower potencies of 24–26 in
vitro, but high potencies in vivo might be caused by
potential benefits in pharmacokinetics, e.g. metabolism or
distribution. Since a carboxylic acid group can bioisosteri-
cally be replaced by a tetrazole, the next compound (27)
can be interpreted as a potential bioisoster of the corre-
sponding benzoic acid methyl ester derivative FUB 567
(R54-H3CCOO–C6H4) (Sasse et al., 2000). The
equipotency of FUB 567 (ED50 54.1 mg/kg p.o.) with
tetrazole 27 (ED50 53.7 mg/kg p.o.) in the same assay is
in good accordance with the anticipation detailed above.
The discovery that all of the novel heterocyclic deriva-
tives described here possess significant antagonist poten-
cies at histamine H3 receptors in vitro and in vivo led us to
investigate selected compounds in another in vitro H3-
receptor assay (Table 2). The compounds were screened
on isolated segments of the guinea-pig ileum (Ligneau et
al., 1994; Schlicker and Marr, 1996). The results obtained
on rat cerebral cortex and those from the guinea-pig were
generally in good agreement. Minor inconsistencies for
some compounds may be attributed to species differences
between the rat and the guinea-pig amino acid sequences
(Tardivel-Lacombe et al., 2000) and/or assay conditions.
It is interesting to note that the affinities obtained from the
guinea-pig ileum as a rule are lower than those derived
from rat cerebral cortex by up to 1.5-orders of magnitude.
Similar results have repeatedly been reported (e.g. Sasse et
al., 2000), but the reasons thereof have yet to be clarified.
In summary, all compounds investigated proved to be
relatively potent histamine H3-receptor antagonists on
guinea-pig ileum (pA2 56.18–7.35).
3. Results and discussion
The novel heterocyclic compounds 20–26 were initially
evaluated for their abilities to effect the release of
[3H]histamine on synaptosomes of rat cerebral cortex
(Table 1), a functional assay for in vitro determination of
histamine H3-receptor potency (Garbarg et al., 1992).
Compounds 20–27 were further tested in an in vivo model
for their effect at histamine H3 receptors on mice brain
after oral administration measuring the increase in Nt-
methylhistamine level, the main histamine metabolite in
the brain (Garbarg et al., 1992). The results of the
pharmacological screening are summarized in Table 1.
From these data, it is obvious that all investigated com-
pounds clearly display antagonist properties at H3 re-
ceptors in vitro with affinities in the low to high nanomolar
concentration range (Ki 54.7–137 nM). Antagonist
potency can also be detected in vivo for all derivatives.
Depending on the structure of the heterocycle, some of the
novel compounds showed high antagonist potency in vivo,
while others were less potent.
The starting point for this study was the heterocyclic
derivative FUB 478, an antagonist of high in vitro and in
vivo potency (Sasse et al., 2001). Since this heteroaromatic
compound and the structurally related compounds ciprox-
ifan and imoproxifan display high H3-receptor affinity, we
tried to bioisosterically replace the ketone or oxime moiety
by different heteroaromatic structures. For this purpose, we
chose different heterocyclic moieties containing the corre-
sponding CO or NO element of the prototypes. To further
elaborate the structure-activity relationship, we extended
our investigation to derivatives with higher structural
diversity but still with substantial resemblance to ciprox-
ifan or imoproxifan. Since the derivatives selected differ
much in their chemical and/or physical properties, they are
ideal candidates for evaluating the scope and limitations of
bioisosteric replacement within this structural class.
Bicyclic derivative 20, containing a 1,3-benzoxathiazol-2-
one moiety, clearly showed antagonist activity in vitro and
in vivo, but at a lower level when compared to the parent
compound acetoproxifan (Stark et al., 2000b). This result
strengthens the importance of a para-substituted phenyl
ether functionality as seen in many other related potent
H3-receptor antagonists (e.g. Stark et al., 2000b; Sasse et
al., 2000). This led us to change the line and to move on to
compounds again possessing the para-substituted phenyl
spacer like FUB 478, but with various aromatic heterocy-
cles instead of the thienyl methanone group. The first two
In addition to the H3-receptor studies specified above
the selectivity of the compounds for the H3 receptor versus
the other two brain histamine receptors is of interest (Table
2). Determination of the H2-receptor potency was per-

S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
375
Table 1
Structures, physical data, and results of the pharmacological screening of aromatic 3-(1H-imidazol-4-yl)propyl ethers for histamine H3-receptor antagonist
potency in vitro and in vivo in rodents
a
b
No.
R
Formula
Mr
Yield
(%)
mp
(8C)
Ki6S.E.M.
(nM)
ED506S.E.M.
(mg/kg)
20
C13H12N2O3S?C4H4O4?1.75H2O
423.9
70
64
61
60
56
57
78
80
147
1564
4.661.5
21
22
23
24
25
26
27
C15H15N3O2?C4H4O4?0.25H2O
C15H16N4O?2C4H4O4?0.75H2O
C16H17N3OS?C4H4O4
389.9
514.0
415.4
302.3
404.9
404.9
383.4
126–128
112
4.760.8
67615
137659
4467
6.362.0
6.061.5
149–151
145
7.962.2
C15H16N4O2?H2O
0.4760.16
0.5660.18
0.5760.21
3.761.5
C15H16N4O2?C4H4O4?0.25H2O
C15H16N4O2?C4H4O4?0.25H2O
C14H16N6O?C2H2O4?0.5H2O
160–162
161
118621
1363
c
194–196
n.d.
Acetoproxifand
Ciproxifand
4-H3CCO–C6H4 –
0.860.2
0.4960.09
0.2660.03
4.361.4
461
0.2460.06
0.1460.03
0.03460.009
1.060.6
1.060.5
2667
4-(cyclopropyl–CO)–C6H4 –
4-H3CC(NOH)–C6H4 –
4-(2-thienyl–CO)–C6H4 –
Imoproxifane
FUB 478f
Thioperamided
Clobenpropitd
0.660.1
a Functional H3-receptor assay in vitro on synaptosomes of rat cerebral cortex (Garbarg et al., 1992).
b Central H3-receptor screening in vivo after oral administration to mice (Garbarg et al., 1992).
c n.d., not determined.
d Data from Stark et al. (2000b) and literature therein.
e Data from Sasse et al. (2000).
f Data from Sasse et al. (2001).

376
S. Graßmann et al. / European Journal of Pharmaceutical Sciences 15 (2002) 367–378
Table 2
logical potencies in vitro and in vivo depending on the
type of the heterocycle.
Potency of selected compounds at histamine receptor subtypesa
No.
H3
H2
H1
In the course of this study, particularly the derivatives
possessing methyloxadiazole moieties (24–26) were iden-
tified as highly potent histamine H3-receptor antagonists.
In vivo, they proved to be more potent than the reference
antagonists clobenpropit or thioperamide with ED50 values
clearly below 1 mg/kg p.o. These results, combined with
potential benefits or at least differences in phar-
macokinetics make the oxadiazoles not only interesting
leads for the further optimization of the proxifan class but
also potentially interesting candidates for further improve-
ment in the development of H3-receptor antagonists for
CNS disorders.
pKib
pA26S.E.M.
pD26S.E.M.
pA26S.E.M.
c
d
e
9
20
21
23
24
25
26
27
7.8f
8.3f
6.9f
7.4f
6.9f
7.9f
7.3560.04
6.7760.05
,4.3
5.0g
4.3560.05e
4.5060.21
4.6160.02
4.6560.25
,3.8
4.4760.06
4.8160.23i
4.3260.14i
4.8060.08i
,4.5
,6.3h
6.4360.12
6.7960.14
6.7060.20
6.1860.10
j
n.d.
4.4460.05
4.3360.04i
a For structures of compounds, see Table 1.
b Functional H3-receptor assay in vitro on synaptosomes of rat cerebral
cortex (Garbarg et al., 1992).
c Functional H3-receptor assay on guinea-pig ileum (Ligneau et al.,
1994; Schlicker et al., 1994).
d Functional H2-receptor assay on guinea-pig atrium (Hirschfeld et al.,
1992).
Acknowledgements
e Functional H1-receptor assay on guinea-pig ileum (Hirschfeld et al.,
1992; Ligneau et al., 1994; Schlicker et al., 1994).
f For corresponding S.E.M., see Table 1.
The contribution of Inge Walther to some pharmaco-
logical experiments is greatly appreciated. This work was
supported by the Biomedical & Health Research Pro-
gramme (BIOMED) of the European Union and the Fonds
der Chemischen Industrie, Verband der Chemischen Indus-
trie, Frankfurt/Main, Germany.
g Single experiment.
h Exact value could not be determined with regard to the M3-receptor
blocking properties of 23 at higher concentrations.
i
9
pD2 value.
j n.d., not determined.
formed on the spontaneously beating guinea-pig atrium
and H1-receptor affinity was investigated on the isolated
guinea-pig ileum (Hirschfeld et al., 1992). All compounds
tested displayed no or low potency at H1 or H2 receptors
Appendix A
Results of elemental analysis (C, H, N) for final compounds
9
(pD2 or pA2 #5), thus proving their pronounced selectivity
No.
Formula
C
H
N
for the histamine H3 receptor. When data for the rat H3
receptor were compared with those for the other histamine
subreceptors investigated, H3 receptor affinities in all cases
were more than 100 times higher. Since potencies of the
ligands in the guinea-pig test model were generally slightly
lower, the differences were subsequently smaller, but still
differ by more than 1.5 log units.
20
C13H12N2O3S?C4H4O4?1.75H2O
Calc.
Found
48.2
48.0
4.64
4.48
6.61
6.38
21
22
23
24
25
26
27
C15H15N3O2?C4H4O4?0.25H2O
C15H16N4O?2C4H4O4?0.75H2O
C16H17N3OS?C4H4O4
Calc.
Found
58.5
58.3
5.04
4.95
10.8
10.5
Calc.
Found
53.8
53.6
5.00
5.05
10.9
10.7
Calc.
Found
57.8
57.7
5.10
5.18
10.1
9.91
C15H16N4O2?H2O
Calc.
Found
59.6
59.8
6.00
5.84
18.5
18.2
4. Conclusion
C15H16N4O2?C4H4O4?0.25H2O
C15H16N4O2?C4H4O4?0.25H2O
C14H16N6O?C2H2O4?0.5H2O
Calc.
Found
56.4
56.4
5.10
5.09
13.8
13.7
In this study we report on novel members of the
proxifan family. The new proxifans are non-symmetrical
aliphatic aromatic ethers containing a 3-(1H-imidazol-4-
yl)propyl moiety and a bicyclic structure (20) or a phenyl
group with different heteroaromatic substituents in para-
position (21–27). Except 20 and 22, each derivative was
prepared by ring formation via different multi-step syn-
thetic pathways. In vitro investigation in two different
species (rat and guinea-pig) proved their H3-receptor
antagonist potencies. Studies on histamine H1 and H2
receptors showed pronounced selectivity for H3 receptors.
Moreover, all of the new compounds displayed high CNS
activity when orally administered. The new compounds
presented here clearly differ in their corresponding bio-
Calc.
Found
56.4
56.4
5.10
5.05
13.8
13.6
Calc.
Found
50.1
49.9
5.00
4.68
21.9
21.5
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