Novel partial agonists for the histamine H3 receptor with high in vitro and in vivo activity

Article abstract of DOI:10.1021/jm991068w

Novel branched N-alkylcarbamates and aliphatic ethers derived from 3- (1H-imidazol-4-yl)-propanol were prepared. The compounds were investigated on two functional histamine H₃-receptor assays. Some compounds displayed partial agonist activity on synaptosomes of rat brain cortex but behaved as pure competitive antagonists on the guinea pig ileum. Under in vivo conditions after po application to mice, some of the compounds showed partial or full agonist activity. Highest in vivo potency was found for the 3,3- dimethylbutyl ether 10 (ED₅₀ = 0.29 mg/kg, full intrinsic activity). These novel agonists are structurally diverse from classical aminergic histamine H₃-receptor agonists (e.g., (R)-α-methylhistamine, imetit) as they lack a basic moiety in the side chain, which is supposed to be important for the activation of the receptor protein. The selectivity for the histamine H₃ receptor was proven by determination of H₁- and H₂-receptor activity on functional assays of the guinea pig.

Full text of DOI:10.1021/jm991068w

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J . Med. Chem. 1999, 42, 4269-4274
4269
Novel P a r tia l Agon ists for th e Hista m in e H3 Recep tor w ith High in Vitr o a n d in
Vivo Activity†
Astrid Sasse, Holger Stark, Sibylle Reidemeister, Annette Hu¨ls, Sigurd Elz, Xavier Ligneau,‡
C. Robin Ganellin,∇ J ean-Charles Schwartz,§ and Walter Schunack*,
Institut fu¨r Pharmazie, Freie Universita¨t Berlin, Ko¨nigin-Luise-Strasse 2+4, D-14195 Berlin, Germany, Laboratoire Bioprojet,
30 rue des Francs-Bourgeois, F-75003 Paris, France, Department of Chemistry, Christopher Ingold Laboratories, University
College London, 20 Gordon Street, London WC1H 0AJ , U.K., and Unite´ de Neurobiologie et Pharmacologie Mole´culaire (U.
109), Centre Paul Broca de l’INSERM, 2ter rue d’Ale´sia, F-75014 Paris, France
Received May 3, 1999
Novel branched N-alkylcarbamates and aliphatic ethers derived from 3-(1H-imidazol-4-yl)-
propanol were prepared. The compounds were investigated on two functional histamine H3-
receptor assays. Some compounds displayed partial agonist activity on synaptosomes of rat
brain cortex but behaved as pure competitive antagonists on the guinea pig ileum. Under in
vivo conditions after po application to mice, some of the compounds showed partial or full agonist
activity. Highest in vivo potency was found for the 3,3-dimethylbutyl ether 10 (ED50 ) 0.29
mg/kg, full intrinsic activity). These novel agonists are structurally diverse from classical
aminergic histamine H3-receptor agonists (e.g., (R)-R-methylhistamine, imetit) as they lack a
basic moiety in the side chain, which is supposed to be important for the activation of the
receptor protein. The selectivity for the histamine H3 receptor was proven by determination of
H1- and H2-receptor activity on functional assays of the guinea pig.
Ch a r t 1
In tr od u ction
In 1983, Arrang et al. discovered a third histamine
receptor subtype (H3), which is located presynaptically
and inhibits synthesis and release of histamine.2
Additionally, the histamine H3 receptor plays an im-
portant role as a heteroreceptor in the regulation of the
release of other neurotransmitters.3 Therapeutic targets
of histamine H3-receptor antagonists and agonists have
been reviewed extensively.4
Selected agonists for the histamine H3 receptor are
shown in Chart 1. These typical H3-receptor agonists,
(R)-R-methylhistamine,5 immepip,6 and imetit,7,8 are
structurally related to histamine inasmuch as they
consist of an imidazole ring, an alkyl spacer, and a
second basic moiety. Under physiological conditions the
isothiourea or the aliphatic amino group is protonated
and presumably interacts with a carboxylate group at
the receptor.7 This ionic interaction seemed to be
essential for activation of the receptor. All classical H3-
receptor agonists have this structural feature in com-
mon. One disadvantage of these highly hydrophilic
compounds is their poor penetration of the blood-brain
barrier. To overcome this problem azomethine prodrugs
of (R)-R-methylhistamine have been developed.9 Impen-
tamine, a homologue of histamine, was reported to
display agonist or antagonist properties on the hista-
mine H3 receptor depending on the test model.10 Io-
doproxyfan showed partial agonist activity on H3-
receptor functional models of guinea pig ileum11 and
mouse brain cortex,11a whereas purely antagonist be-
havior on rat synaptosomes was observed.12 Iodoproxy-
fan and analogoues were the first non-aminergic com-
pounds showing partial agonism at H3 receptors.11a
Recently, we have described novel carbamates as
antagonists at the histamine H3 receptor, e.g., 1 and 3
(Table 1).13 These carbamates are derivatives of 3-(1H-
imidazol-4-yl)propanol with alkyl- or methyl-branched
chains on the nitrogen of the carbamate functionality.
Novel compounds have been designed and prepared
with higher degrees of substitution on the N-alkyl chain
(Table 1, 2 and 4-7). These bulkier residues in some
cases led to a different and unexpected pharmacological
behavior on histamine H3 receptors, as some of these
compounds showed partial or full agonist action in vitro
and/or in vivo. Similar compounds with an ether moiety
instead of the carbamate group were also prepared and
tested (Table 1, 8-11).
Ch em istr y
3-(1H-Imidazol-4-yl)propanol12 was the key interme-
diate for the novel compounds. This synthon was
conveniently synthesized from urocanic acid and ob-
tained in its trityl-protected and deprotected form.12
Carbamates 1 and 3 have been described previously,13
with the synthetic route analogous to that of compound
† Part of Int. Pat. Appl. WO 96/29 315; see ref 1.
* To whom correspondence should be addressed.
Freie Universita¨t Berlin.
‡ Laboratoire Bioprojet.
∇ University College London.
§ Centre Paul Broca de l’INSERM.
10.1021/jm991068w CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/11/1999

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4270 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Brief Articles
Sch em e 1a
all compounds showed moderate to high antagonist
potency. In the series of carbamates, compounds with
short N-alkyl chains (1 and 2) were slightly less active
than the homologous compounds 3-6 and the multi-
branched compound 7. In the ether series the same
pattern of increasing antagonist potency on the guinea
pig ileum with increasing chain length was observed (8
f 9 f 10 f 11). Compound 11 showed the highest
antagonist potency (KB ) 13 nM) on this functional
model. No agonist activity was observed.
Surprisingly, some of the compounds (4, 9-11) dis-
played partial agonist activity on the rat synaptosome
model, and under in vivo conditions two compounds (9,
10) showed agonist action with full intrinsic activity at
very low doses. Partial agonism was observed in vivo
for 2, 4, 5, and 11, whereas only weak partial agonism
(4, 11) or antagonism (2, 5) in vitro was displayed. Due
to low intrinsic activities, the EC50 values could not be
calculated in every case (4, 11).
a
(a) ClCOOCCl3, charcoal (cat.), ethyl acetate, 4-5 h reflux;
(b) DPPA, Et3N, dioxane, 30 min reflux; (c) 3-(1H-imidazol-4-
yl)propanol‚HCl,12 acetonitrile, 4-5 h reflux.
Sch em e 2a
In general the intrinsic activities of compounds found
in vitro on the synaptosomal preparation were lower
than those of the same compounds evaluated from
modulation of Nτ-methylhistamine levels in brain in
vivo. Thus compound 9 with an intrinsic activity of 0.22
behaved as full agonist in vivo. This apparent difference
in receptor reserve may appear surprising inasmuch as
both tests evaluate the influence of H3-autoreceptor
stimulation upon endogenous histamine release. How-
ever, whereas the in vitro response is selectively medi-
ated by receptors on nerve terminals, in vivo response
may also involve somato-dentritic autoreceptors de-
tected autoradiographically19 controlling the firing rate
of histaminergic neurons. In addition, the character of
the response in vitro depends on the parameters of the
depolarizing stimulus.2 Here release from synaptosomes
was monitored after a continuous 30 mM K+ stimulus,
whereas physiological release in vivo results from trains
of impulses, i.e., intermittent depolarizations, which
may result in different receptor reserves in the two
models.
a
(a) 48% HBr, H2SO4 (concd), 1.5 h reflux; (b) i, Na 3-(1-
(triphenylmethyl)-1H-imidazol-4-yl)propanolate, 15-crown-5 (cat.),
(C4H9)4NI (cat.), toluene, 12 h reflux, ii, 2 N HCl/THF (20/30), 2 h
reflux.
6 and following route 1 in Scheme 1. Compounds 2 and
7 were prepared from commercially available isocyan-
ates (Scheme 1, route 1). Starting with the carboxylic
acids, 4 and 5 were synthesized via a modified Curtius
reaction using diphenyl phosphorazidate (DPPA) (Scheme
1, route 2).14
Ethers 8-11 were prepared by classical Williamson
synthesis15 from 3-(1-(triphenylmethyl)-1H-imidazol-4-
yl)propanolate and corresponding alkyl halides with
subsequent deprotection of the products (Scheme 2).
P h a r m a cology
When tested as antagonists on rat synaptosomes, the
compounds showed higher potency compared to the
antagonist action observed on guinea pig ileum.
Compounds were tested on three pharmacological
models for the histamine H3 receptor. The functional
in vitro assay on rat cerebral cortex synaptosomes with
K+-evoked depolarization-induced release of [3H]hista-
mine was performed according to Garbarg et al.7 An-
other functional in vitro assay was performed on guinea
pig ileum measuring the concentration-dependent in-
hibition of electrically evoked twitches by (R)-R-meth-
ylhistamine in the presence of the antagonist.16,17
Agonist measurements recorded concentration -res-
ponse curves of the new compounds instead of (R)-R-
methylhistamine. In vivo testing was performed after
po administration of the compounds to Swiss mice. The
increased (antagonist) or decreased (agonist) level of the
main histamine metabolite, Nτ-methylhistamine, in
cerebral cortex was measured.7 The compounds were
also screened for H1- and H2-receptor activity on func-
tional models on ileum and right atrium of guinea pigs.18
Small structural changes in the side chain of the new
compounds influence the pharmacological behavior to
a large extent. Changing from an isopropyl (1) to tert-
butyl (2) on the N-residue of the carbamates leads to a
transition from weak antagonism to partial agonism in
vivo. The same change is found for 3 and 4 which are
homologues of 1 and 2 with an isobutyl (3) and neopent-
yl (4) group. Compound 4 is a partial agonist with the
same range of intrinsic activity but with higher in vivo
potency than 2. Further elongation of the N-alkyl chain
(4 f 6) or introduction of more methyl groups (7) led to
a loss of agonist activity. Compound 6 showed pro-
nounced antagonist potency, whereas 7 was inactive
under in vivo conditions. A loss in intrinsic activity but
increase in potency was observed for 5.
Structure-activity relationships in the group of ethers
are comparable to those of the carbamates. Compound
8 possessing an isobutyl residue showed antagonist
potency in vitro as well as in vivo. Increasing chain
length, leading to the isopentyl derivative 9, converted
the antagonist mode of action of 8 to partial agonism
Resu lts a n d Discu ssion
The structures of the novel compounds and the results
of pharmacological testing on histamine H3-receptor
assays are presented in Table 1. On guinea pig ileum

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Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4271
Ta ble 1. Structures, Chemical Data, and Pharmacological Results of Screening for Histamine H3-Receptor Agonist/Antagonist
Activity in Vitro and in Vivo
a
b
Crystallization solvent: Et2O/EtOH. Functional H3-receptor assay on guinea pig ileum.16,17 c Functional H3-receptor assay on
synaptosomes of rat cerebral cortex.7 Central H3-receptor screening with po administration as a methylcellulose suspension to mice.7
d
e i.a., intrinsic activity. f Reference 13. n.c., not calculable due to low intrinsic activity. References 7, 8. EC50 value.17 Reference 7.
g
h
i
k
in vitro on rat synaptosomes and full agonism in vivo.
It should be noted that the antagonist potency of 8
under in vivo conditions exceeds that of the reference
antagonist thioperamide20 (ED50 ) 1 mg/kg). Terminal
addition of another methyl group, leading to the 3,3-
dimethylbutyl derivative 10, increased in vivo potency
and maintained full agonism. Of all the new compounds,
10 showed highest agonist affinity and highest intrinsic
activity in vitro on rat synaptosomes. Increasing the
chain length by one more methylene group (11) de-
creased intrinsic activity while maintaining high po-
tency in vivo. In vitro, only slight partial agonism could
be observed, not allowing exact quantification.
Imetit has been described as a highly potent and
selective histamine H3-receptor agonist with full intrin-
sic activity in vitro as well as in vivo (Table 1).7,8 Imetit
and other classical H3-receptor agonists (Chart 1) exist
as monocations under physiological conditions due to
their second basic moiety. This monocationic moiety is
presumed to interact with acidic amino acids of the
receptor protein inducing a change in receptor confor-
mation, thus leading to agonist activity.21 The novel
compounds are devoid of a comparable structural fea-
ture. Most probably an interaction with a lipophilic
pocket with distinct steric demands could cause a
similar change in receptor conformation leading to
activation. The transition from antagonist to agonist
responses and vice versa is caused by only small
structural changes (1 f 2, 3 f 4). Activation of the
receptor only occurs when very distinct lipophilic and
steric demands are fulfilled. The extent of partial
agonism depends very much on the receptor tissue
system. The diversity of pharmacologic H3-receptor
action with different compounds needs further investi-
gation.
Selectivity of the novel compounds with regard to
other histamine receptors is presented in Table 2. Most
compounds are highly selective for the histamine H3

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4272 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Brief Articles
Ta ble 2. Antagonist Activity at Histamine Receptor Subtypes
Determined on Functional Models
2, 4, 6, and 8 which were obtained by different methods.
Elemental analyses (C, H, N) were measured on Perkin-Elmer
240 B or Perkin-Elmer 240 C instruments and were within
(0.4% of the theoretical values. 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.). Thin-layer chroma-
tography (TLC) was performed on silica gel PF254 plates
(Merck); the spots were visualized with Dragendorff’s reagent,
fast blue salt BB, or by UV absorption at 254 nm.
3-(1H-Im id a zol-4-yl)p r op yl N-ter t-Bu tylca r ba m a te (2).
tert-Butyl isocyanate (6 mmol, 0.6 g) was added to a solution
of 3-(1H-imidazol-4-yl)propanol‚HCl12 (5 mmol, 0.8 g) in 30 mL
of dry acetonitrile under nitrogen atmosphere and refluxed for
4 h. The solvent was removed under reduced pressure and the
reaction mixture purified by rotatory chromatography
[eluent: CHCl3/MeOH (gradient from 95/5 to 90/10), ammonia
atmosphere]. The combined fractions were concentrated, dried,
and crystallized as hydrogen maleate: 1H NMR (Me2SO-d6) δ
8.89 (s, 1H, Im-2-H), 7.40 (s, 1H, Im-5-H), 6.85 (s, 1H, CONH*),
6.05 (s, 2H, Mal), 3.93 (t, J ) 6.3 Hz, 2H, CH2-O), 2.68 (t, J
) 7.6 Hz, 2H, Im-CH2), 1.89 (m, 2H, Im-CH2-CH2), 1.21 (s,
9H, (CH3)3); MS m/z 225 (M•+, 12), 108 (85), 95 (100), 81 (77),
72 (29), 54 (30), 45 (25), 26 (42). Anal. (C11H19N3O2‚C4H4O4‚
0.25H2O) C, H, N.
3-(1H-Im id a zol-4-yl)p r op yl N-Neop en tylca r ba m a te (4).
A mixture of 3,3-dimethylbutanecarboxylic acid (5 mmol, 0.6
g), triethylamine (5 mmol, 0.5 g), and diphenyl phosphorazi-
date (5 mmol, 1.4 g) was stirred for 45 min in 30 mL of dry
acetonitrile at room temperature. The reaction mixture was
then heated to reflux for 30 min. Then 3-(1H-imidazol-4-yl)-
propanol‚HCl12 (5 mmol, 0.8 g) was added and the solution
again refluxed for 4-5 h. The solvent was removed under
reduced pressure and the residue was dissolved in Et2O and
extracted with a saturated solution of K2CO3 and NaHCO3.
After concentration of the combined organic fractions the
residue was purified by rotatory chromatography [eluent:
CHCl3/MeOH (gradient from 95/5 to 90/10), ammonia atmo-
sphere]. The combined fractions were concentrated, dried, and
crystallized as hydrogen oxalate: 1H NMR (Me2SO-d6) δ 8.63
(s, 1H, Im-2-H), 7.27 (s, 1H, Im-5-H), 7.12 (m, 1H, CONH*),
3.97 (t, J ) 6.4 Hz, 2H, CH2-O), 2.79 (d, 2H, J ) 6.3 Hz, NH-
CH2), 2.68 (t, J ) 7.5 Hz, 2H, Im-CH2), 1.89 (m, 2H, Im-CH2-
CH2), 0.82 (s, 9H, (CH3)3); MS m/z 239 (M•+, 17), 108 (100), 95
(62), 81 (78), 54 (11), 45 (63). Anal. (C12H21N3O2‚C2H2O4‚
0.5H2O) C, H, N.
3-(1H-Im id a zol-4-yl)p r op yl N-(3,3-Dim eth ylbu tyl)ca r -
ba m a te (6). To a solution of trichloromethyl chloroformate (6
mmol, 1.2 g) and a catalytic amount of activated charcoal in
20 mL of dry ethyl acetate was added 3,3-dimethylbutylamine
(5 mmol, 0.5 g) rapidly. The reaction mixture was heated to
reflux for 4-5 h, the black solution was cooled and filtered,
and the solvent was evaporated carefully under reduced
pressure. The freshly prepared isocyanate was dissolved in 20
mL of dry acetonitrile and added to a mixture of 3-(1H-
imidazol-4-yl)propanol‚HCl12 (5 mmol, 0.8 g) in 10 mL of dry
acetonitrile. The solution was refluxed for 4-5 h and concen-
trated in vacuo. The residue was purified by rotatory chro-
matography [eluent: CH2Cl2/MeOH (gradient from 99/1 to 90/
10), ammonia atmosphere]. The product was obtained as a
colorless oil and crystallized as hydrogen maleate in Et2O/
EtOH: 1H NMR (Me2SO-d6) δ 8.83 (s, 1H, Im-2-H), 7.37 (s,
1H, Im-5-H), 6.98 (m, 1H, CONH*), 6.04 (s, 2H, Mal), 3.96 (t,
J ) 6.4 Hz, 2H, CH2-O), 2.97 (m, 2H, NH-CH2), 2.67 (t, J )
7.4 Hz, 2H, Im-CH2), 1.89 (m, 2H, Im-CH2-CH2), 1.32 (m, 2H,
NH-CH2-CH2), 0.88 (s, 9H, (CH3)3); MS m/z 253 (M•+, 14),
109 (42), 108 (100), 107 (33), 95 (72), 82 (30), 81 (58), 80 (11),
57 (11), 54 (11), 43 (12), 41 (21). Anal. (C13H23N3O2‚C4H4O4‚
0.5H2O) C, H, N.
a
H3-Receptor assay on synaptosomes of rat cerebral cortex;7 for
b
standard errors see Table 1. H3-Receptor assay on guinea pig
ileum;16,17 for SEM see Table 1. c H2-Receptor test on guinea pig
atrium;18 SEM ( 0.2. H1-Receptor test on guinea pig ileum;18
d
SEM < 0.1. e Reference 13. f n.d., not determined. pD′2 value.
g
receptor with pKB values below 4.5 at H1 and H2
receptors. Compounds 7 and 11 displayed moderate
activity for the H1 receptor. Except for 7, all tested
compounds showed higher histamine H3-receptor an-
tagonist activity by at least 2.5 log units compared to
H1 or H2 receptors, proving the high selectivity of the
new compounds for histamine H3 receptors.
Con clu sion s
Activation of histamine H3 receptors was achieved in
vitro and in vivo with some of the novel compounds of
the carbamate and the ether classes. These new agonists
are structurally diverse from classical histamine H3-
receptor agonists as they do not possess a basic moiety
in the side chain of the molecule. Hence they do not exist
as monocations under physiological conditions.
These compounds behave as partial agonists or an-
tagonists in two tests in vitro but as potent full agonists
in vivo (9, 10) on an index of histaminergic neuron
activity suggesting that they constitute not only inter-
esting pharmacological tools but also promising thera-
peutic agents with central activity.
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 Brucker DPX 400 Avance (400
MHz) spectrometer. Chemical shifts are expressed in ppm
downfield from internal Me4Si as reference. 1H NMR data are
reported in the following order: multiplicity (br, broad; s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; *,
exchangeable by D2O; Im, imidazole; Mal, maleic acid), number
of protons, and approximate coupling constants in hertz (Hz).
Mass spectra were obtained on an EI-MS Finnigan MAT
CH7A. Spectral data of parent compounds are shown only for
3-(1H-Im id a zol-4-yl)p r op yl 2-Meth ylp r op yl Eth er (8).
A mixture of 3-(1-(triphenylmethyl)-1H-imidazol-4-yl)pro-
panol12 (5 mmol, 1.84 g) and NaH (60%) (6 mmol, 0.24 g) was