D-amino acid homopiperazine amides: Discovery of A-320436, a potent and selective non-imidazole histamine H3-receptor antagonist

Article abstract of DOI:10.1002/ardp.200300844

Structure-activity relationships of homopiperazine-containing alkoxybiaryl nitriles employing various D-amino acid moieties and their N-furanoyl analogues were undertaken. This led to A-320436, a potent and selective non- imidazole H₃-receptor

Full text of DOI:10.1002/ardp.200300844

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Selective Non-Imidazole Histamine H3-Receptor Antagonist 219
Michael P. Curtis,
Wesley Dwight,
John Pratt,
D-Amino Acid Homopiperazine Amides:
Discovery of A-320436, a Potent and Selective
Non-Imidazole Histamine H3-Receptor
Antagonist
Marlon Cowart,
Timothy A. Esbenshade,
Kathy M. Krueger,
Gerard B. Fox,
Structure-activity relationships of homopiperazine-containing alkoxybiaryl nitriles
employing various D-amino acid moieties and their N-furanoyl analogues were
undertaken. This led to A-320436, a potent and selective non-imidazole H3-re-
ceptor antagonist possessing balanced affinity for both rat and human H3-recep-
tors. This compound was shown to demonstrate in vitro and in vivo functional
antagonism and is non-neurotoxic at doses (i.p.) up to 163 mg/kg in a general
observation test.
Jia Bao Pan,
Thomas G. Pagano,
Arthur A. Hancock,
Ramin Faghih,
Youssef L. Bennani
Neuroscience Research,
Global Pharmaceutical
Research and Development,
Abbott Laboratories,
Keywords: Histamine; H3-Receptor; Non-imidazole H3 antagonist; 5-Trial
inhibitory Avoidance model, A-320436
Abbott Park, USA
Received: September 25, 2003; Accepted: December 16, 2003 [FP844]
DOI 10.1002/ardp.200300844
Introduction
derivative 19 due to its equipotent binding profile (Fig-
ure 1). In order to expand the structure-activity re-
To date there are four distinct histamine receptors be-
longing to the super-family of G protein-coupled recep-
tors, known as H1-4, respectively [1, 2]. The histamine
H3-receptor, which is found in the central nervous sys-
tem (CNS), was first identified in 1983. This pre-syn-
aptic auto-receptor modulates histamine levels in the
brain through a negative feedback mechanism. The
H3-receptor can also act as a hetero-receptor to modu-
late the release of CNS neurotransmitters such as
acetylcholine, dopamine, γ-aminobutyric acid, and
noradrenaline [3Ϫ5]. The H3-receptor has been pro-
posed as a potential therapeutic target for such cogni-
tive disorders as attention deficit hyperactivity disorder
and Alzheimer’s disease [6]. Several imidazole-based
H3-antagonists and inverse agonists have been shown
to block the effect of histamine at these receptors and
thereby increase CNS histamine and other neuro-
transmitter levels [7Ϫ10]. However, due to the poten-
tial for unfavorable drug-drug interactions involving
imidazole compounds, attention has recently focused
on the development of highly selective non-imidazole
containing H3-antagonists [11Ϫ15]. We have pre-
viously reported a series of non-imidazole aminoalkox-
ybiphenylnitrile compounds possessing good affinity
and selectivity for both the rat and human H3-receptors
(rH3 and hH3, respectively) [16]. Within this series, we
became particularly interested in the homopiperizine
lationships (SAR) of this biaryl series, several aza-de-
rived homopiperazine-analogues have been synthe-
sized and tested (vide infra).
Additionally, we have previously reported on the pro-
pensity of D-amino acid motifs and their N-furanoyl
analogues to enhance rH3 and hH3-receptor binding
potency, respectively, in a series of aminoalkoxycyclo-
propylketones [17, 18]. However, these compounds
lacked adequate hH3-receptor affinities (A-317920,
Table 1). It was therefore postulated that the incorpor-
ation of D-amino acid amides into an equipotent biaryl
motif, such as 19, could improve potency and concur-
rently maintain a balanced affinity profile of less than
5-fold difference between rH3 and hH3-receptors. We,
herein, report SAR studies of a series of D-amino acid
homopiperazine amides leading to A-320436 (30), a
selective non-imidazole histamine H3-receptor antag-
onist with an improved balance of potencies between
rat and human H3-receptors.
Chemistry
Synthesis of the aza-derived analogues of compound
19 (Table 1) began by reacting 1,4-diazepane-1-car-
boxylic acid tert-butyl ester (1) with (3-bromo-pro-
poxy)-tert-butyl-dimethyl-silane in the presence of KI
and K2CO3 to give 2 (91% yield). Intermediate 2 was
subsequently converted to compound 3 in 70% yield
by treatment with tetrabutylammonium fluoride (TBAF)
in tetrahydrofuran (THF) at 0°C (Scheme 1). Com-
pound 3 was next treated with 2,5-dibromopyridine
and NaH in methyl sulfoxide (DMSO) to afford inter-
Correspondence: Michael P. Curtis, Neuroscience Re-
search, Global Pharmaceutical Research and Development,
Abbott Laboratories, Abbott Park, IL 60064-6123, USA.
Phone: +1 847 937-4482, Fax: +1 847 937-4143, e-mail:
michael.curtis@abbott.com
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220 Curtis et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Figure 1. Biphenylnitrile-homopiperazine H3-receptor antagonists.
Table 1. Binding affinities† (pKi) at rat cortical H3 and human H3 cloned receptors.
Compound
6
Structure‡
hH3
rH3
Compound
19
Structure
hH3
rH3
7.61 7.46
7.44 7.58
8.00 7.95
7.22 9.22
7.18 8.19
6.91 9.10
9
Ciproxifan
12
6.99 6.78 Thioperamide
15
6.84 6.93
A-317920
†
Values were estimated from at least 3 separate competition experiments (SEM Յ 0.36).
R = (CH2)3-homopiperazine.
‡
mediate 4 (80% yield). A Suzuki cross-coupling reac-
This was accomplished by utilizing a Mitsunobu reac-
tion of with 2-chloro 5-hydroxypyridine, tri-
tion [19] of 4 with 4-cyanophenyl boronic acid was
used next to generate compound 5, followed by treat-
ment with trifluoroacetic acid (TFA) in dichloromethane
(DCM) which gave 4-[6-(3-[1,4] diazepan-1-yl-pro-
poxy)-pyridin-3-yl]-benzonitrile (6) in 90% yield.
3
phenylphosphine, and dibenzyl azodicarboxylate
(DBAD) in THF providing intermediate 7 in 66% yield.
4-Cyanophenyl boronic acid was next reacted with 7
in a cross-coupling reaction, followed by quantitative
N-Boc removal to generate 4-[5-(3-1,4- diazepan-1-yl-
propoxy)-pyridin-2-yl]-benzonitrile (9). The next aza-
derivative in this series was prepared by the conver-
The nitrogen atom of the aryl ring was next moved to
a meta-position to the oxygen of the aliphatic chain.
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Selective Non-Imidazole Histamine H3-Receptor Antagonist 221
Figure 2. A-320436, ciproxifan, thioperamide in a five-trial inhibitory avoidance model of cognition. Panels B + D
modified and reproduced with permission from Komater et al. [28].
sion of 3,6-dichloro-pyridazine and compound 3 with
NaH in DMSO to afford intermediate 10 in 62% yield.
This was followed by a cross coupling reaction with 4-
cyanophenylboronic acid providing 4-[3-(4’-cyano-
biphenyl-4-yloxy)-propyl]-1,4-diazepane-1-carboxylic
acid tert-butyl ester (11) in 55% yield and subsequent
N-Boc removal provided compound 12. The final aza-
derivative 15 was prepared in a manner similar to the
previously described analog starting with 5-bromo-2-
iodo-pyrimidine [20].
Synthesis of the biphenylnitrile motif began by treating
commercially available 4’-hydroxy-biphenyl-4-car-
bonitrile (16) with 1-bromo-3-chloropropane and
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222 Curtis et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Scheme 1. (a) (3-Bromo-propoxy)-tert-butyl-dimethyl-silane, KI/K2CO3, DMF, 85°C 16 h, 91%; (b) TBAF, THF,
0°C-rt. 16 h, 70%; (c) Aryl-halide , NaH, DMSO, 50°C 18 h, 62-80%; (d) 2-Chloro 5-hydroxypyridine, triphenyl-
phosphine, DBAD, THF, rt 48 h, 66%; (e) 4-Cyanophenyl boronic acid, Pd(PPh3)2Cl2, Na2CO3, DMF, 85°C 24 h,
45-65%; (f) TFA, DCM 0°C-rt 16 h, 82-92%.
Scheme 2. (a) Cl-(CH2)3-Br , K2CO3, 2-butanone, reflux 24 h, 91%; (b) 1,4- Diazepane-1-carboxylic acid tert-
butyl ester, KI, K2CO3, 2-butanone, reflux 72 h, 76%; (c) TFA, DCM 0°C- rt, 16 h, 86-92%; (d) N-Boc amino
acid, DMAP, Hunig’s base, EDCI, DMF rt. 16 h, 25Ϫ65%; (e) Furan-2-carbonyl chloride, triethylamine, DCM, rt.
18 h, 65-70%.
K2CO3 in 2-butanone (Scheme 2). The resulting inter-
mediate 17 was obtained in high yield and used with-
out further purification in the presence of 1,4-di-
azepane-1-carboxylic acid tert-butyl ester and KI/
K2CO3 to give 18. The N-Boc group was subsequently
removed to afford 4’-(3-1,4-diazepan-1-yl-propoxy)-bi-
phenyl-4-carbonitrile (19) in 91% yield.
Compounds 20Ϫ24 were prepared in a parallel fa-
shion (25Ϫ65% yield) by reacting 19 with N-Boc D-
amino acids in the presence of 4-dimethylaminopyrid-
ine (DMAP), Hunig’s base, and 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDCI)
(Scheme 2). After N-Boc removal, free amines 25Ϫ27
were further synthesized to their respective N-furanoyl
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Selective Non-Imidazole Histamine H3-Receptor Antagonist 223
Table 2. Binding affinities† (pKi) at rat cortical H3 and human H3 cloned receptors.
Compound
20
R =
hH3
rH3
Compound
24
R =
hH3
rH3
7.35
8.11
8.00
8.65
21
22
7.69
7.09
8.78
8.37
28
29
7.67
6.92
8.86
8.45
23
7.54
8.70
30
8.81
9.31
†
Values were estimated from at least 3 separate competition experiments (SEM Յ 0.26).
analogs using furan-2-carbonyl chloride and triethyl-
amine in DCM generating compounds 28Ϫ30 in
65Ϫ70% yield.
template for the introduction of a select group of D-
amino acids (available commercially or synthetically
derived as in the case of compound 24 [22]) and their
N-furanoyl analogues into the series. Rat cortex and
human cloned H3-receptor binding affinities for these
compounds are listed in Table 2. Data from this table
indicate enhanced rH3-receptor affinities, verses refer-
ence compound 19, for all D-amino acid derivatives
(20-24). This pattern is similar to what we witnessed
when D-amino acids were incorporated into the ami-
noalkoxycyclopropylketone series (vide supra). From
these results, the three most potent compounds (21,
23, and 24) were sequentially converted to their corre-
sponding N-furanoyl analogues. Here, a clear distinc-
tion in compound 30 (A-320436) was gained over all
other compounds, possessing high affinity (pKi >8.5)
for both rat and human H3-receptors while providing
the desired balance between species. This was the
Results and discussion
The homopiperazine derivatives 6, 9, 12, 15, and 19
were assayed in a binding experiment using rat cortex
and human cloned H3-receptors [21]. Biphenylnitrile
19 was the most potent with hH3 pKi = 8.00
0.26
and rH3 pKi = 7.95 0.10 (Table 1).
It is important to note that a balanced binding profile
was seen in each of the aza-derivatives, however, no
apparent improvement in binding affinity was gained
in comparison to 19. For this reason, coupled with the
increasing synthetic demands required to prepare
such structures, compound 19 was chosen as our
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224 Curtis et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Table 3. Binding affinities† (pKi) at rat and human biogenic amine receptors and transporters.
Compound
hH1
hH2
hH4
hM1 hM3 hD4.2 hα2C10 rUPTK-NE rUPTK-SER rUPTK-DA
5.00 6.49 5.92 4.96 5.45
A-320436
5.82 5.29 5.00 6.18 5.88
†
Values were estimated from at least 3 separate competition experiments (SEM Յ 0.30).
first compound of its class to possess such properties
and, therefore, A-320436 (30) was chosen for further
profiling in vitro and in vivo.
at 1 h. In comparison, thioperamide, a prototypical H3-
receptor antagonist, efficacious in this cognition model
(Figure 2, panels B and D), is also known to have poor
brain penetration [27]. However, thioperamide reaches
maximum brain concentrations that are 3.4-times
higher after a 10 mg/kg (i.p.) dose (48 ng/gm/mg drug)
than those measured for A-320436.
When tested against several biogenic amine receptors
and transporters such as those for norepinephrine,
serotonin and dopamine, A-320436 showed excellent
selectivity for the H3-receptors (Table 3) [21].
In addition to possessing a poor BBB penetration pro-
file, A-320436 also has shown low oral bioavailability
in the rat (F = 3.5%, T1/2 = 1.2 h). Although A-320436’s
cLogP of 3.63 is acceptable for oral dosing, the com-
pound’s other physico-chemical properties such as
molecular weight >500, number of rotatable bonds
In addition, A-320436 demonstrated in vitro functional
antagonism in an adenylate cyclase assay [21], with
pKb values of 7.06 0.06 and 7.84 0.34 for human
cloned and rat cloned H3-receptors, respectively. CNS
safety was determined based on observation of CNS
adverse effects in a general observation model [23]
and only minor adverse effects such as mild hypoactiv-
ity and ptosis were observed at doses up to 163 mg/
kg (i.p.). Seizure-like activity and lethality were ob-
served at 490 mg/kg. Next, in vivo functional antago-
nism was demonstrated in a mouse dipsogenia model
[24] whereby pronounced drinking, elicited acutely in
response to the potent and selective H3 agonist (R)-α-
methyl histamine, was attenuated by pre-adminis-
tration of A-320436. Agonist-induced dipsogenia was
dose-dependently attenuated over a range of 0.006-
1.75 mg/kg (i.p.), reaching statistical significance at
0.175, 0.58, and 1.75 mg/kg, with an approximate
ED50 of 0.175 mg/kg (data not shown).
>10, a total of 10 H-bond acceptors and donors, and a
2
˚
relatively high polar surface area of 112 A (calculated
according to methodology proposed by Ertl et al. [29])
certainly could help explain the low oral bioavailability
observed in the rat [30]. For these reasons, as well as
the less than optimal BBB penetration profile, further
examination of the compound at higher doses in cog-
nitive models was not merited.
In summary, we have demonstrated an improved po-
tency of H3-antagonist activity when D-amino acid mo-
tifs and their N-furanoyl analogues are incorporated
into a homopiperazine-alkoxybiphenylnitrile template
(19). A-320436 (30) is a selective non-imidazole hista-
mine H3-receptor antagonist in vitro and in vivo, with
balanced affinity at the rat and human H3-receptors.
Despite possessing a balanced affinity profile, weak in
vivo activity combined with poor pharmacokinetic
properties discouraged further behavioral evaluation in
this series of D-amino acid homopiperazine amides.
However this class of compounds might be of interest
as pharmacological tools for further studies of hista-
mine H3-receptors.
5-Trial inhibitory avoidance model [25] was employed
to test the efficacy of A-320436 to improve cognitive
and attentional behavior, using spontaneously hyper-
tensive rat (SHR) pups. A-320436 (10 mg/kg) was
compared to ciproxifan (a prototypical imidazole-con-
taining H3-receptor antagonist [26]), which served as
a positive control. Unfortunately, SHR pups treated
with A-320436 did not perform significantly better than
vehicleϪtreated controls on trials 2 through 5 (Figure
2, panels A and C).
Experimental
Chemistry
The lack of efficacy observed at this dose of A-320436
could in part be attributed to the compound’s poor
brain penetration. To examine this parameter, a rat in
vivo system was utilized for determination of the
blood-brainϪbarrier (BBB) penetration after a single
i.v. dose (5 mg/kg). A-320436 reached relatively low
brain concentrations of 0.07 µg/g (14 ng/gm/mg drug)
1H NMR spectra were recorded on a Varian Unity 300 (Palo
Alto, USA) spectrometer with chemical shifts in ppm down-
field from an internal TMS standard (300 MHz). The 13C NMR
spectrum was recorded on a Varian Unity 500 NMR spec-
trometer with chemical shifts in ppm relative to an internal
DMSO-d6 standard assigned as 39.5 ppm (125 MHz). Carbon
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Selective Non-Imidazole Histamine H3-Receptor Antagonist 225
signals in parentheses represent resonances due to the pres-
ence of rotamers and/or conformers. Desorption chemical
ionization (DCI/NH3) and electrospray ionization (ESI) were
recorded on a Finnigan SSQ 7000 (Bremen, Germany) and
Finnigan NewStar FT/MS T70, respectively. Elemental analy-
ses were performed by Robertson Microlit Laboratories, Ma-
dison, USA. Compounds tested in vivo were pure within
0.4%. All solvents were either distilled or of analytical re-
agent quality. All reactions were carried out under an inert
atmosphere of dry nitrogen using oven- or flame-dried glass-
ware. The progress of all reactions was monitored by TLC on
E. Merck (Gibbstown, USA) pre-coated silica gel (0.2 mm
layer) plates containing a fluorescent indicator. Detection was
first with UV (254 nm) and then by charring with a solution of
ammonium molybdate tetrahydrate (12.5 g) and cerium sul-
fate tetrahydrate (5.0 g) in 10% aqueous sulfuric acid (500
mL). Samples (<0.3 g) were purified by preparative HPLC on
a Waters Symmetry C8 column (Milford, USA, 40 mm X 100
mm, 7 mm particle size) using a gradient of 10%Ϫ100%
acetonitrile:0.1% aqueous TFA over 12 min (15 min run time)
at a flow rate of 70 mL/min. Flash chromatography was per-
formed with EM Science Silica Gel 60 (230Ϫ400 mesh,
Gibbstown, USA). Optical rotations were measured using a
Rudolph Autopol IV polarimeter (Flanders, USA).
an amber-colored oil. The crude product was purified by flash
chromatography (95:5 DCM:MeOH) to provide 4 (0.826 g) in
80% yield: 1H NMR (DMSO-D6) δ 8.26 (d, J = 3.0 Hz, 1H),
7.89 (dd, J = 3.0, 9.0 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H) 4.27
(t, J = 6.0 Hz, 2H), 3.34 (m, 4H), 2.58 (m, 6H), 1.85 (m, 2H),
1.72 (m, 2H), 1.38 (s, 9H); MS DCI m/z 414 (M+H)+.
4-[3-(6-Chloro-pyridazin-3-yloxy)-propyl]-1,4-diazepane-1-
carboxylic acid tert-butyl ester (10)
According to general procedure A, the reaction of 3 with 3,6-
dichloro-pyridazine afforded compound 10 in 62% yield after
chromatography: 1H NMR (DMSO-D6) δ 7.79 (d, J = 9.0 Hz,
1H), 7.33 (d, J = 9.0 Hz, 1H), 4.45 (t, J = 6.0 Hz, 2H), 3.35
(m, 4H), 2.60 (m, 6H), 1.91 (m, 2H), 1.72 (m, 2H), 1.38 (s,
9H); MS DCI m/z 371 (M+H)+ 271 (M+-Boc).
4-[3-(5-Bromo-pyrimidin-2-yloxy)-propyl]-1,4-diazepane-1-
carboxylic acid tert-butyl ester (13)
According to general procedure A, the reaction of 3 with 5-
bromo-2-iodo-pyrimidine afforded compound 13 in 65% yield
after chromatography: 1H NMR (CDCl3) δ 8.52 (s, 1H), 7.27
(s, 1H), 4.42 (t, J = 6.0 Hz, 2H), 3.49 (m, 4H), 2.69 (m, 6H),
1.99 (m, 2H), 1.82 (m, 2H), 1.46 (s, 9H); MS DCI m/z 415,
417 (M+), 315, 317 (M+-Boc).
4-[3-(Tert-butyl-dimethyl-silanyloxy)-propyl]-1,4-diazepane-1-
carboxylic acid tert-butyl ester (2)
4-[3-(6-Chloro-pyridin-3-yloxy)-propyl]-1,4-diazepane-1-
carboxylic acid tert-butyl ester (7)
To a solution of 1 (5.0 g, 25.0 mmol) in 30 mL DMF was
added (3-bromo-propoxy)-tert-butyl-dimethyl-silane (6.96 g,
27.5 mmol), KI (5.0 g, 30 mmol), and K2CO3 (4.15 g, 30
mmol). The mixture was heated to 85°C for 16 h then par-
titioned between 500 mL ethylacetate and 300 mL H2O. The
organic phase was washed 3ϫ with 100 mL brine, dried with
Na2SO4, and filtered through a pad of silica. The filtrate was
concentrated under vacuum to provide 2 (8.5 g, 91%) as a
yellow oil: 1H NMR (DMSO-D6) δ 3.60 (t, J = 6.0 Hz, 2H),
3.32Ϫ3.27 (m, 4H), 2.57 (m, 6H), 1.68, (m, 2H), 1.60 (m, 2H),
1.37 (s, 9H), 0.84 (s, 6H); MS DCI m/z 373 (M+H)+.
To a solution of 3 (1.7 g, 6.6 mmol) in 50 mL THF, was added
6-chloro-pyridin-3-ol (0.85 g, 6.6 mmol), triphenylphosphine
(1.73 g, 6.6 mmol), and di-tert-butylazodicarboxylate (1.52 g,
6.6 mmol). The resulting mixture was stirred at room tem-
perature for 48 h and subsequently concentrated under vac-
uum. The residue was chromatographed using 95:5
DCM:MeOH to afford compound 7 (1.6 g, 66%) as a yellow
1
oil: H NMR (DMSO-D6) δ 8.11 (d, J = 3.0 Hz, 1H), 7.49 ( dd,
J = 3.0, 9.0 Hz, 1H ) 7.42 (d, J = 9.0 Hz, 1H), 4.10 (t, J = 6.0
Hz, 2H), 3.35 (m, 4H), 2.59 (m, 6H), 1.86 (m, 2H), 1.69 (m,
2H), 1.38 (s, 9H); MS ESI m/z 370 (M+H)+.
4-(3-Hydroxy-propyl)-1,4- diazepane-1-carboxylic acid tert-
butyl ester (3)
4-{3-[5-(4-Cyano-phenyl)-pyridin-2-yloxy]-propyl}-1,4-di-
azepane-1-carboxylic acid tert-butyl ester (5)
To a solution of 2 (8.5 g, 22.8 mmol) in tetrahydrofuran at 0°C
was added tetrabutylammonium- fluoride (TBAF) (34 mL, 34
mmol). The reaction mixture was allowed to gradually warm
to room temperature and stir for 18 h. The solution was next
concentrated and the residue was dissolved in 600 mL diethyl
ether, washed 4ϫ with 50 mL H2O, 2ϫ with 50 mL brine,
dried, concentrated under vacuum, and purified using flash
chromatography (100% DCM to 95:5 DCM:MeOH) to afford
3 (4.1 g, 70%): 1H NMR (DMSO-D6) δ 3.48 (t, J = 6.0 Hz,
2H), 3.39 (m, 4H), 2.62 (m, 6H), 1.75 (m, 2H), 1.62 (m, 2H),
1.43 (s, 9H); MS DCI m/z 259 (M+H)+.
General procedure B for compounds 8, 11, and 14 in
Scheme 1.
A solution of 4 (0.041 g, 0.1 mmol), 4-cyanophenyl boronic
acid (0.022 g, 0.15 mmol), Pd(PPh3)2Cl2 (0.007 g, 0.01
mmol), and 2M Na2CO3 (0.1 mL, 0.2 mmol) in 3 mL DMF
was heated to 85°C for 24 h. The reaction mixture was then
cooled, diluted to 50 mL with EtOAc, and filtered through ce-
lite. The filtrate was next washed 2ϫ with 20 mL H2O, 2ϫ
with 20 mL brine, dried, and chromatographed to afford 5
1
(0.019g, 45%) as a tan solid: H NMR (DMSO-D6) δ 8.58 (d,
4-[3-(5-Bromo-pyridin-2-yloxy)-propyl]-1,4- diazepane-1-car-
boxylic acid tert-butyl ester (4) General procedure A for com-
pounds 10 and 13 in Scheme 1.
J = 3.0 Hz, 1H), 8.12 (dd, J = 3.0, 9.0 Hz, 1H), 7.91 (m, 4H),
6.94 (d, J = 9.0 Hz, 1H), 4.36 (t, J = 6.0 Hz, 2H), 3.35 (m,
4H), 2.59 (m, 6H), 1.89 (m, 2H), 1.69 (m, 2H), 1.39 (s, 9H);
MS ESI m/z 437 (M+H)+.
To a suspension of 60% NaH in mineral oil (0.15 g, 3.75
mmol) in 2 mL DMSO was added drop wise a solution of 3
(0.645 g, 2.5 mmol) in 1 mL DMSO. Contents stirred at room
temperature for 1 h, then 20 min at 50°C, and cooled to room
temperature. Next, a solution of 2,5 dibromopyridine (0.621
g, 2.62 mmol) in 2 mL DMSO was added drop wise and the
resulting mixture was stirred for 18 h. The mixture was par-
titioned between ethylacetate (50 mL) and H2O (25 mL),
washed 2ϫwith 25 mL brine, dried and concentrated to give
4-{3-[6-(4-Cyano-phenyl)-pyridin-3-yloxy]-propyl}-1,4-di-
azepane-1-carboxylic acid tert-butyl ester (8)
According to general procedure B, the coupling of 7 with 4-
cyanophenyl boronic acid afforded compound 8 in 67% yield
after chromatography: 1H NMR (DMSO-D6) δ 8.42 (d, J = 3.0
Hz, 1H), 8.23 (d, J = 9.0 Hz, 2H) 8.06 (d, J = 9.0 Hz, 1H),
7.91 (d, J = 9.0 Hz, 2H), 7.54 (dd, J = 3.0, 9.0 Hz, 1H), 4.18
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226 Curtis et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
(t, J = 6.0 Hz, 2H), 3.38 (m, 4H), 2.62 (m, 6H), 1.90 (m, 2H),
1.86 (m, 2H), 1.39 (s, 9H); MS ESI m/z 437 (M+H)+.
Hz, 2H), 2.92 (m, 4H), 2.76 (m, 6H), 2.02 (m, 2H), 1.82 (m,
2H); MS DCI m/z 338 (M+H)+.
4-{3-[6-(4-Cyano-phenyl)-pyridazin-3-yloxy]-propyl}-1,4-di-
azepane-1-carboxylic acid tert-butyl ester (11)
4Ј-(3-Chloro-propoxy)-biphenyl-4-carbonitrile (17)
To a solution of 4’-hydroxy-biphenyl-4-carbonitrile 16 (5.0 g,
25.6 mmol) in 2-butanone was added 1-bromo-3-chloro-pro-
pane (5.04 g, 32.0 mmol) and K2CO3 (5.0 g, 36.2 mmol). The
resulting mixture was heated to reflux for 24 h, cooled, and
filtered. The filtrate was dried and concentrated under vac-
uum to afford 17 (6.34 g, 91%) as a pale white solid; 1H NMR
(CDCl3) δ 7.70 (dd, J = 3.0 Hz, 1H), 8.12 (dd, J = 3.0, 9.0 Hz,
1H), 7.91 (m, 4H), 6.95 (d, J = 9.0 Hz, 1H), 4.35 (t, J = 6.0
Hz, 2H), 2.85 (m, 4H), 2.64 (m, 6H), 1.87 (m, 2H), 1.68 (m,
2H); MS ESI m/z 337 (M+H)+.
According to general procedure B, the coupling of 10 with 4-
cyanophenyl boronic acid afforded compound 11 in 58% yield
after chromatography: 1H NMR (DMSO-D6) δ 8.29 (d, J = 9.0
Hz, 2H), 8.01 (d, J = 9.0 Hz, 2H) 7.37 (d, J = 9.0 Hz, 2H),
4.56 (t, J = 6.0 Hz, 2H), 3.38 (m, 4H), 2.63 (m, 6H), 1.96 (m,
2H), 1.83 (m, 2H), 1.38 (s, 9H); MS ESI m/z 438 (M+H)+.
4-{3-[5-(4-Cyano-phenyl)-pyrimidin-2-yloxy]-propyl}-1,4-di-
azepane-1-carboxylic acid tert-butyl ester (14)
According to general procedure B, the coupling of 13 with 4-
cyanophenyl boronic acid afforded compound 14 in 70%
yield after chromatography: 1H NMR (CD3OD) δ 8.90 (s, 2H),
7.85 (s, 4H), 4.53 (t, J = 6.0 Hz, 2H), 3.48 (m, 4H), 2.74 (m,
6H), 2.02 (m, 2H), 1.89 (m, 2H), 1.46 (s, 9H); MS DCI m/z
438 (M+H)+ 338 (M+-Boc).
4-[3-(4Ј-Cyano-biphenyl-4-yloxy)-propyl]-1,4-diazepane-1-
carboxylic acid tert-butyl ester (18)
To a solution of 17 (5.0 g, 18.4 mmol) in 100 mL of 2-but-
anone was added 1,4-diazepane-1-carboxylic acid tert-butyl
ester (5.55 g, 27.8 mmol), K2CO3 (6.40 g, 46.3 mmol), and
KI (7.70 g, 46.3 mmol). The resulting mixture was heated to
reflux for 72 h, cooled, filtered and concentrated under vac-
uum. The residue was dissolved in 350 mL DCM and washed
2ϫ with 150 mL H2O, 2ϫ with 150 mL brine, dried (Na2SO4),
concentrated and purified using flash chromatography to af-
ford 18 (6.10 g, 76%) as a faint yellow oil; 1H NMR (CDCl3)
δ 7.69 (q, J = 9.0 Hz, 4H), 7.54 (d, J = 9.0 Hz, 2H), 7.01 (d,
J = 9.0 Hz, 2H), 4.09 (t, J = 6.0 Hz, 2H), 3.54 (m, 4H), 2.71
(m, 6H), 2.00 (m, 2H), 1.84 (m, 2H), 1.46 (s, 9H); MS ESI
m/z 436 (M+H)+.
4-[6-(3-1,4-Diazepan-1-yl-propoxy)-pyridin-3-yl]-benzonitrile
(6)
General procedure C for compounds 9, 12, 15, 19, 25, 26,
and 27 in Schemes 1 and 2.
To a solution of 5 (0.310 g, 0.71 mmol) in DCM at 0°C was
added 10 mL trifluoroacetic acid (TFA) at a fast drip. The re-
action mixture was then allowed to warm to room temperature
and stirred for an additional hour. After concentration under
vacuum, the residue was dissolved in 100 mL DCM washed
2ϫ with 20 mL 10% NaHCO3, 2ϫ with 20 mL brine, dried,
and concentrated to give 6 (0.206 g, 90%) as a viscous oil
which crystallized upon standing: 1H NMR (DMSO-D6) δ 8.58
(d, J = 3.0 Hz, 1H), 8.12 (dd, J = 3.0, 9.0 Hz, 1H), 7.91 (m,
4H), 6.95 (d, J = 9.0 Hz, 1H), 4.35 (t, J = 6.0 Hz, 2H), 2.85
(m, 4H), 2.64 (m, 6H), 1.87 (m, 2H), 1.68 (m, 2H); MS ESI
m/z 337 (M+H)+.
4Ј-(3-1,4-Diazepan-1-yl-propoxy)-biphenyl-4-carbonitrile (19)
According to general procedure C, TFA was added to 18 pro-
viding compound 19 in 95% yield after basic workup: 1H NMR
(CD3OD) δ 7.75 (s, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.05 (d, J =
9.0 Hz, 2H), 4.14 (t, J = 6.0 Hz, 2H), 3.32 (m, 4H), 2.95 (m,
6H), 2.01 (m, 4H); MS ESI m/z 336 (M+H)+.
2-(2-{4-[3-(4Ј-Cyano-biphenyl-4-yloxy)-propyl]-1,4-diazepan-
4-[5-(3-1,4-Diazepan-1-yl-propoxy)-pyridin-2-yl]-benzonitrile
(9)
1-yl}-2-oxo-ethyl)-pyrrolidine-1-carboxylic
acid
tert-butyl
ester (20)
According to general procedure C, TFA was added to 8 pro-
viding compound 9 in 92% yield after basic workup: 1H NMR
(DMSO-D6) δ 8.42 (d, J = 3.0 Hz, 1H), 8.23 (d, J = 9.0 Hz,
2H), 8.07 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 9.0 Hz, 2H), 7.54
(dd, J = 3.0, 9.0 Hz, 1H), 4.18 (t, J = 6.0 Hz, 2H), 2.78 (m,
4H), 2.66 (m, 6H), 1.90 (m, 2H), 1.68 (m, 2H); MS ESI m/z
337 (M+H)+.
General procedure D for compounds 21Ϫ24 in Scheme 2.
To a solution of 19 (1.0 g, 2.99 mmol) dissolved in 50 mL
DCM at 0°C was added 2-(R)-carboxymethyl-pyrrolidine-1-
carboxylic acid tert-butyl ester (0.752 g, 3.28 mmol), 4-di-
methylaminopyridine (0.13 g, 0.745 mmol), diisopropylethyl-
amine (1.3 mL, 7.45 mmol), and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (0.685 g, 3.58 mmol). The
resulting solution was allowed to warm to room temperature
and stir for 16 h. The solution was next diluted with 75 mL
DCM, washed 3ϫ with 25 mL saturated NaHCO3, 1ϫ with
25 mL 10% HCl, 1ϫ with 25 mL brine, dried, filtered, and
concentrated to afford crude 20 (0.70 g, 43%) which was
used directly without further purification; 1H NMR (CDCl3) δ
7.66 (q, J = 9.0 Hz, 4H), 7.53 (d, J = 9.0 Hz, 2H), 6.99 (d, J =
9.0 Hz, 2H), 5.02 (m, 1H), 4.06 (t, J = 6.0 Hz, 2H), 3.95 (m,
4H), 2.72 (m, 6H), 2.20 (m, 8H), 1.64 (m, 4H), 1.44 (s, 9H);
MS ESI m/z 547 (M+H)+.
4-[6-(3-1,4-Diazepan-1-yl-propoxy)-pyridazin-3-yl]-benzo-
nitrile (12)
According to general procedure C, TFA was added to 11 pro-
viding compound 12 in 89% yield after basic workup: 1H NMR
(DMSO-D6) δ 8.29 (d, J = 9.0 Hz, 2H), 8.02 (d, J = 9.0 Hz,
2H), 7.37 (d, J = 9.0 Hz, 1H), 4.57 (t, J = 6.0 Hz, 2H), 2.81
(m, 4H), 2.65 (m, 6H), 1.96 (m, 2H), 1.68 (m, 2H); MS ESI
m/z 338 (M+H)+.
4-[2-(3-1,4-Diazepan-1-yl-propoxy)-pyrimidin-5-yl]-benzo-
nitrile (15)
(2-{4-[3-(4Ј-Cyano-biphenyl-4-yloxy)-propyl]-1,4-diazepan-1-
yl}-1-methyl-2-oxo-ethyl)-carbamic acid tert-butyl ester (21)
According to general procedure C, TFA was added to 14 pro-
1
viding compound 15 in 82% yield after the basic workup: H
According to general procedure D, 2-(R)-tert-butoxycarbonyl-
amino-propionic acid was added to 20 providing compound
NMR (CD3OD) δ 8.89 (s, 2H), 7.85 (s, 4H), 4.53 (t, J = 6.0
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Selective Non-Imidazole Histamine H3-Receptor Antagonist 227
1
21 in 65% yield after workup; H NMR (CDCl3) δ 7.66 (q, J =
2H), 3.66 (m, 4H), 3.41 (m, 6H), 2.27 (m, 7H); MS ESI m/z
9.0 Hz, 4H), 7.53 (d, J = 9.0 Hz, 2H), 6.99 (d, J = 9.0 Hz,
2H), 5.50 (m, 1H), 4.60 (m, 1H) 4.05 (t, J = 6.0 Hz, 2H), 3.83
(m, 4H), 2.68 (m, 6H), 1.96 (m, 4H), 1.43 (s, 9H), 1.30 (dd,
J = 6.0 Hz, J = 3.0 Hz, 3H); MS ESI m/z 507 (M+H)+.
490 (M+H)+.
Furan-2-carboxylic acid (2-{4-[3-(4Ј-cyano-biphenyl-4-yloxy)-
propyl]-1,4- diazepan-1-yl}-1-methyl-2-oxo-ethyl)-amide (28)
General procedure E for compounds 29 and 30 in Scheme 2.
(1-{4-[3-(4Ј-Cyano-biphenyl-4-yloxy)-propyl]-1,4-diazepane-
1-carbonyl}-propyl)-carbamic acid tert-butyl ester (22)
To a stirred solution of 25 (1.0 g, 2.46 mmol) in 25 mL DCM
was added triethylamine (0.66 mL, 3.69 mmol) and furan-2-
carbonyl chloride (0.30 mL, 2.71 mmol). The reaction mixture
was allowed to stir at room temperature for 18 h. 100 mL
DCM was next added and the organic phase was washed 2ϫ
with 30 mL saturated NaHCO3, 2ϫ with 30 mL 0.5 M citric
acid, dried (Na2SO4), and concentrated under vacuum. The
crude product was chromatographed to afford 28 (0.84 g,
68%) as a viscous oil; 1H NMR (CDCl3) δ 7.70 (q, J = 9.0 Hz,
4H), 7.53 (d, J = 9.0 Hz, 2H), 7.45 (d, J = 0.5 Hz, 1H), 7.09
(d, J = 0.5 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 6.48 (m, 1H),
5.10 (m, 1H), 4.08 (t, J = 6.0 Hz, 2H), 3.83 (m, 4H), 2.68 (m,
6H), 2.00 (m, 4H), 1.65 (m, 1H), 1.45 (dd, J = 6.0 Hz, J = 3.0
Hz, 3H); MS ESI m/z 501 (M+H)+.
According to general procedure D, 2-(R)-tert-butoxycarbonyl-
amino-butyric acid was added to 20 providing compound 22
in 25% yield after workup; 1H NMR (CDCl3) δ 7.71 (q, J = 9.0
Hz, 4H), 7.54 (d, J = 9.0 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H),
5.38 (m, 1H), 4.58 (m, 1H) 4.10 (t, J = 6.0 Hz, 2H), 3.83 (m,
4H), 2.71 (m, 6H), 1.96 (m, 6H), 1.43 (s, 9H), 0.97 (m, 3H);
MS ESI m/z 521 (M+H)+.
(3-{4-[3-(4Ј-Cyano-biphenyl-4-yloxy)-propyl]-1,4-diazepan-1-
yl}-3-oxo-propyl)-carbamic acid tert-butyl ester (23)
According to general procedure D, 3-tert-butoxycarbonyl-
amino-propionic acid was added to 20 providing compound
1
23 in 65% yield after workup; H NMR (CDCl3) δ 7.66 (q, J =
Furan-2-carboxylic acid (3-{4-[3-(4Ј-cyano-biphenyl-4-yloxy)-
propyl]-1,4- diazepan-1-yl}-3-oxo-propyl)-amide (29)
9.0 Hz, 4H), 7.53 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz,
2H), 5.32 (m, 1H), 4.06 (t, J = 6.0 Hz, 2H), 3.62 (m, 4H), 2.65
(m, 6H), 2.51 (m, 2H), 1.96 (m, 2H), 1.42 (s, 9H), 0.97; MS
ESI m/z 507 (M+H)+.
According to general procedure E, furan-2-carbonyl chloride
was added to 26 providing compound 29 in 65% yield after
chromatography; 1H NMR (CDCl3) δ 7.70 (q, J = 9.0 Hz, 4H),
7.54 (d, J = 9.0 Hz, 2H), 7.44 (d, J = 0.5 Hz, 1H), 7.10 (m,
2H), 6.96 (d, J = 9.0 Hz, 2H), 6.47 (m, 1H), 4.11 (t, J = 6.0
Hz, 2H), 3.75 (m, 2H), 3.62 (m, 2H), 3.48 (s, 2H) 3.33 (m,
2H), 2.94 (m, 6H), 1.96 (m, 4H); MS ESI m/z 501 (M+H)+.
(2-{4-[3-(4Ј-Cyano-biphenyl-4-yloxy)-propyl]-1,4-diazepan-1-
yl}-2-oxo-1-thiazol-4-ylmethyl-ethyl)-carbamic acid tert-butyl
ester (24)
According to general procedure D, 2-(R)-tert-butoxycarbonyl-
amino-3-thiazol-4-yl-propionic acid was added to 20 providing
compound 24 in 43% yield after workup; 1H NMR (CDCl3) δ
8.74 (d, J = 3.0 Hz, 1H), 7.66 (q, J = 9.0 Hz, 4H), 7.53 (d, J =
9.0 Hz, 2H), 7.11 (d, J = 3.0 Hz, 1H), 6.99 (d, J = 9.0 Hz,
2H), 5.65 (m, 1H), 5.10 (m, 1H), 4.06 (t, J = 6.0 Hz, 2H), 3.66
(m, 4H), 3.28 (m, 1H), 3.20 (m, 1H), 2.78 (m, 6H), 1.95 (m,
4H), 1.38 (s, 9H); MS ESI m/z 590 (M+H)+.
Furan-2-carboxylic acid (2-{4-[3-(4Ј-cyano-biphenyl-4-yloxy)-
propyl]-1,4- diazepan-1-yl}-2-oxo-1-thiazol-4-ylmethyl-ethyl)-
amide (30)
According to general procedure E, furan-2-carbonyl chloride
was added to 27 providing compound 30 in 70% yield after
chromatography; 1H NMR (CDCl3) δ 8.75 (d, J = 3.0 Hz, 1H),
7.70 (q, J = 9.0 Hz, 4H), 7.53 (d, J = 9.0 Hz, 2H), 7.44 (d, J =
0.5 Hz, 1H), 7.21 (d, J = 3.0 Hz, 1H), 7.10 (m, 2H), 6.96 (d,
J = 9.0 Hz, 2H), 6.47 (m, 1H), 5.51 (m, 1H), 4.06 (t, J = 6.0
Hz, 2H), 3.69 (m, 4H), 3.35 (m, 2H), 2.65 (m, 6H), 1.96 (m,
4H); 13C NMR (DMSO-D6) δ 169.8, 159.0, 156.8, 152.9,
152.6, 147.1, 144.6, 144.0, 132.2, 130.2, 127.8, 126.4, 118.3,
115.1, 115.0, 113.3, 111.2, 108.9, 65.7, 54.7 (53.9), 53.5
(53.0), 52.8, 48.3, 46.4 (45.4), 44.4 (44.0), 33.0, 26.9 (25.9),
26.3; HRMS ESI, calcd for C32H34N5O4S (M+H)+: 584.2326.
Found: 584.2322. [α]2D4° = Ϫ17° (c 0.5 in CHCl3).
4Ј-{3-[4-(2-Amino-propionyl)-1,4-diazepan-1-yl]-propoxy}-
biphenyl-4-carbonitrile (25)
According to general procedure C, TFA was added to 21 pro-
viding compound 25 in 92% yield after basic workup; 1H NMR
(CDCl3) δ 7.71 (q, J = 9.0 Hz, 4H), 7.54 (d, J = 9.0 Hz, 2H),
7.00 (d, J = 9.0 Hz, 2H), 6.04 (bs, 2H), 4.08 (t, J = 6.0 Hz,
2H), 3.83 (m, 4H), 2.68 (m, 6H), 2.00 (m, 4H), 1.30 (dd, J =
6.0 Hz, J = 3.0 Hz, 3H); MS ESI m/z 407 (M+H)+.
4Ј-{3-[4-(3-Amino-propionyl)-1,4-diazepan-1-yl]-propoxy}-
biphenyl-4-carbonitrile (26)
Pharmacology
Animals
According to general procedure C, TFA was added to 23 pro-
viding compound 26 in 90% yield after basic workup; 1H NMR
(CDCl3) δ 7.71 (q, J = 9.0 Hz, 4H), 7.54 (d, J = 9.0 Hz, 2H),
7.00 (d, J = 9.0 Hz, 2H), 4.08 (t, J = 6.0 Hz, 2H), 3.77 (bs,
2H), 3.62 (m, 4H), 3.06 (m, 2H), 2.74 (m, 6H), 2.55 (m, 2H),
1.96 (m, 4H); MS ESI m/z 407 (M+H)+.
Animals for experiments conducted in-house were housed in
AAALAC-approved facilities at Abbott Laboratories in a tem-
perature-regulated environment with lights on between 6:00
and 18:00 h. All mice were supplied through Charles River
Breeding Laboratories (Portage, Canada) and SHR pups
were obtained from Harlan (Indianapolis, USA). The animals
were acclimated to laboratory conditions for at least one week
before testing. All in-house testing was conducted according
to protocols approved by Abbott’s Institutional Animal Care
and Use Committee.
4Ј-{3-[4-(2-Amino-3-thiazol-4-yl-propionyl)-1,4-diazepan-1-
yl]-propoxy}-biphenyl-4-carbonitrile (27)
According to general procedure C, TFA was added to 24 pro-
viding compound 27 in 86% yield after basic workup; 1H NMR
(CDCl3) δ 8.85 (d, J = 3.0 Hz, 1H), 7.70 (q, J = 9.0 Hz, 4H),
7.53 (d, J = 9.0 Hz, 2H), 7.21 (d, J = 3.0 Hz, 1H), 6.96 (d, J =
9.0 Hz, 2H), 4.78 (m, 1H), 4.40 (m, 1H), 4.09 (t, J = 6.0 Hz,
H3-receptor (H3R) cloning and cell membrane preparation
The human histamine H3R gene was cloned using human
thalamus poly-A RNA (Clontech, Palo Alto, USA) with RT-
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228 Curtis et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
PCR methods using primers designed according to the pub-
lished human H3R gene sequences [31]; (GenBank ac-
cession number AF140538). The full-length (H3L) human his-
tamine H3R cDNA encoding 445 amino acids was cloned into
the pCIneo expression vector. A partial rat histamine H3R
gene was identified by homology searching using the InCyte
Pharmaceutical (Palo Alto, USA) database. This unique clone
shared significant homology to the published human hista-
mine H3LR sequence. RACE (rapid amplification of cDNA
ends) was performed with thalamus RNA from Long Evans
rat (Charles River Laboratories, Wilmington, USA) using pri-
mers designed according to the InCyte clone and the PCR
product was cloned into the pCIneo expression vector.
incubated with H3R antagonists for 2 min prior to the addition
of 30 nM (R)-α-MeHA. After additional 5 min incubation, 10
mM forskolin was added to provide a final volume of 150 mL.
Cells were hydrolyzed after 10 min by the addition of 20 µL
of 1 M HCl and subsequent shaking for 10 min. After the
addition of 20 µl of 1 M NaOH, the level of cAMP was deter-
mined by scintillation proximity assay (Amersham Biosci-
ences, Piscataway, USA). Data were normalized to the
amount of cAMP produced in control wells and are expressed
as the% forskolin-stimulated cAMP response. Experiments
were run in triplicate and data were analyzed using GraphPad
Prism (San Diego, USA) to obtain IC50 values and Hill slopes.
pKb values were determined by the generalized Cheng-Pru-
soff equation and are presented as the mean S.E.M.
C6 cells were grown in DMEM media containing high glucose
that was supplemented with 10% fetal bovine serum and 20
mM L-glutamine. Transfection of C6 cells was performed with
Lipofectamine according to the protocol provided by the ven-
dor (Invitrogen, Carlsbad, USA) and cell lines were selected
using Geneticin. Cells from stable clonal lines were harvested
and homogenized in TE buffer (50 mM Tris-HCl, 5 mM EDTA,
pH 7.4) using a polytron at 20,000 rpm for 2 x 10 sec bursts
in the presence of protease inhibitors (1 mM benzamidine,
2 µg/ mL aprotinin, 1 µg/mL leupeptin, and 1 µg/mL pepstatin,
Sigma, St. Louis, USA), followed by centrifugation at
40,000 x g. The membrane pellets were further purified by
repeating the homogenization and centrifugation steps as de-
scribed above. Final membrane preparations were obtained
by re-homogenizing the pellets in 6.25 vol (w/v) of TE buffer
and were frozen at Ϫ70°C until used.
Dipsogenia model
Mice were tested in a dipsogenia model as previously de-
scribed [24]. Briefly, each mouse was injected i.p. with saline
or A-320436 (0.006, 0.018, 0.058, 0.175, 0.583, 1.75 mg/kg).
After 5 min, saline or (R)-α-MeHA (30 mg/kg), was injected
i.p. on the opposite side of the abdomen, and the animals
returned to their home cages. Following a period of 30 min,
mice were then placed separately into smaller cages with ac-
cess to water via a modified sipper. The mice were left alone
for a further 30 min after which they were removed to their
home cages once more and the amount of water consumed
was recorded to the nearest 0.01 mL. Sixteen animals were
treated at a time for a total of 12-16 mice per group. Statistical
analysis was performed with one-way ANOVA, followed by
Fisher’s PLSD.
Rat (Pelfreez, Rogers, USA) cortices expressing H3R were
homogenized in cold TE buffer containing protease inhibitors.
The homogenate was centrifuged at 40,000 x g for 20 min
at 4°C. This step was repeated and the resulting pellet was
resuspended in TE buffer in a final volume of three times
the wet weight of the tissue. Aliquots were frozen at Ϫ70°C
until needed.
5-Trial inhibitory avoidance model
SHR pups were trained in an inhibitory avoidance response
with 5 acquisition trials as previously described [25]. Briefly,
the animals were trained to avoid a mild footshock (0.1 mA,
1 s duration) delivered when the pup transferred from a
brightly lit to a darkened compartment of a computer-con-
trolled Gemini inhibitory avoidance system (San Diego Instru-
ments, San Diego, USA). After the first trial, the pup was re-
moved and returned to its home cage and littermates and the
transfer latency noted. One minute later, the same pup was
once again placed in the brightly lit compartment and the
training process repeated for a total of 5 trials. A criterion time
of 1 min applied for the first trial and 3 min for each of the 4
subsequent trials. Ciproxifan (3, 10 mg/kg Ϫ internal positive
control), A-320436 (10 mg/kg), thioperamide (10, 30 mg/kg)
or saline vehicle was injected s.c. 30 min prior to the first trial.
An oscilloscope (Hitachi V-212, Tokyo, Japan, 20 MHz) and
a 100 kOhm resistor were used frequently to ensure correct
calibration of the equipment in producing this relatively mild
footshock. Pups were not habituated to the avoidance
apparatus before the first trial to avoid potentially confounding
latent inhibitory effects. Each drug treatment was equally rep-
resented among each litter (n=12 per group) and the investi-
gator was normally blinded to treatment. Statistical analysis
was performed with non-parametric Kruskal-Wallis, followed
by individual Mann-Whitney U-tests.
Radioligand binding assays
For H3R-binding, membrane preparations were incubated
with [3H]-N-α-methylhistamine ([3H]-NAMH) (0.5Ϫ1.0 nM) in
the presence or absence of increasing concentrations of li-
gands for competition binding as previously described [21].
The binding reactions were carried out for 30 min at 25°C in
a final volume of 0.5 mL TE buffer. Nonspecific binding was
defined with 30 µM thioperamide. Radioligand binding assays
for cloned human histamine H1R and H2R were performed
using [3H]-mepyramine and [3H]-tiotidine, respectively.
For all of the radioligand competition binding assays, IC50 val-
ues and Hill slopes were determined by Hill transformation of
the data as previously described [21] and pKi values were
determined by the generalized Cheng-Prusoff equation [32].
Data are presented as the mean pKi standard error of the
mean.
Adenylate cyclase assay
C6 cells or HEK cells stably expressing the full-length human
or rat H3LR were plated the day before the assay at 75,000-
100,000 cells per well in a 96-well plate coated with either
collagen IV or polyethylenimine (PEI) as previously described
[21]. Medium was removed and cells were incubated with
Dulbecco’s phosphate buffered saline with calcium (DPBS,
Invitrogen, Carlsbad, USA) for 10 min followed by DPBS con-
taining 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 min
at 37°C with 5% CO2. Upon removal of buffer, cells were
General observation test
Adult mice were separated into groups of 3 and placed into
observation cages (23 x 21 x 20 cm) as previously described
[23]. Baseline core (rectal) body temperature was recorded
with a rapid read digital thermometer (Model BAT-12, Physi-
temp Instruments Inc., Clifton, USA). In separate experi-
ments, mice were then injected with vehicle or A-320436 (4.6,
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 219−229
Selective Non-Imidazole Histamine H3-Receptor Antagonist 229
16.3, 48.9, 163, 490 mg/kg i.p.). All mice were continuously
observed for adverse behavior such as general changes in
activity levels, piloerection, ptosis, loss of righting reflex and
seizure activity (including Straub tail, wild running, clonus,
tonus) for the first hour and then intermittently at 2, 3, 6 and
24 h following drug administration. All subjective observations
(e.g. activity) in drug-treated mice were made with constant
reference to a cage of vehicle-treated control mice.
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