4-Phenoxypiperidines: Potent, conformationally restricted, non-imidazole histamine H3 antagonists

Article abstract of DOI:10.1021/jm049212n

Two new series of 4-(1-alkyl-piperidin-4-yloxy)-benzonitriles and 4-(1-isopropyl-piperidin-4-yloxy)-benzylamines have been prepared. In vitro activity was determined at the recombinant human H₃ receptor and several members of these new series were found to be potent H₃ antagonists. The present compounds contain a 4-phenoxypiperidine core, which behaves as a conformationally restricted version of the 3-amino-1-propanol moiety common to the many previously described non-imidazole histamine H ₃ ligands. One selected member of the new series, 4-[4-(1-isopropyl-piperidin-4-yloxy)-benzyl]-morpholine (13g), was found to be a potent, highly selective H₃ receptor antagonist with in vivo efficacy in a rat EEG model of wakefulness at doses as low as 1 mg/kg sc.

Full text of DOI:10.1021/jm049212n

J. Med. Chem. 2005, 48, 2229-2238
2229
4-Phenoxypiperidines: Potent, Conformationally Restricted, Non-Imidazole
Histamine H3 Antagonists
Curt A. Dvorak,*,† Richard Apodaca,† Ann J. Barbier,† Craig W. Berridge,‡ Sandy J. Wilson,† Jamin D. Boggs,†
Wei Xiao,† Timothy W. Lovenberg,† and Nicholas I. Carruthers†
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, San Diego, California 92121,
and Department of Psychology, University of Wisconsin, 1202 W. Johnson Street, Madison, Wisconsin 53706
Received September 29, 2004
Two new series of 4-(1-alkyl-piperidin-4-yloxy)-benzonitriles and 4-(1-isopropyl-piperidin-4-
yloxy)-benzylamines have been prepared. In vitro activity was determined at the recombinant
human H3 receptor and several members of these new series were found to be potent H3
antagonists. The present compounds contain a 4-phenoxypiperidine core, which behaves as a
conformationally restricted version of the 3-amino-1-propanol moiety common to the many
previously described non-imidazole histamine H3 ligands. One selected member of the new
series, 4-[4-(1-isopropyl-piperidin-4-yloxy)-benzyl]-morpholine (13g), was found to be a potent,
highly selective H3 receptor antagonist with in vivo efficacy in a rat EEG model of wakefulness
at doses as low as 1 mg/kg sc.
Chart 1. Representative Non-Imidazole Antagonists
with Activities at the Cloned Recombinant Human
Histamine H3 Receptor or Rat Cortex
Introduction
Histamine1 directs a complex array of physiological
actions through four distinct G protein-coupled receptors
known simply as H1, H2, H3 and H4.2 The histamine H1
and H4 receptors are associated with inflammatory and
immune responses while the H2 receptor mediates
gastric acid secretion. The H3 receptor,3 which has been
cloned and subsequently found to be predominantly
expressed in the central nervous system (CNS),4 has
been determined to be a presynaptic autoreceptor
regulating the synthesis5 and release of histamine
through negative feedback. In addition, H3 receptors
were found to have a modulatory effect on the release
of other neurotransmitters in the CNS including dopa-
mine, noradrenalin, GABA, serotonin, and acetylcho-
line.6 These effects, in turn, have prompted suggestions
for therapeutic applications of H3 agonists and antago-
nists in the areas of Parkinson’s and Alzheimer’s
diseases, schizophrenia, memory, learning and sleep
disorders.7 There have also been reports that H3 an-
tagonists alone or in combination with a H1 antagonist
may be useful for the treatment of upper airway allergic
responses.8 Compared with the successes of marketed
drugs targeting H1 and H2 receptors, the potential of
histamine H3 receptor antagonists or agonists has yet
to be fully realized.9
Since the discovery of the H3 receptor in 1983, many
research groups have been interested in developing
potent and selective ligands. Through the late 1990s,
most efforts in the field were directed toward the
discovery of imidazole-based ligands, many of which
have found utility as pharmacological tools.10 The basic
structural motif common to these ligands was the 4(5)-
substituted imidazole ring, which is present in the
endogenous ligand histamine, linked to a polar group
and a lipophilic residue. However, the imidazole het-
erocycle is a potential metabolic liability11 and early
efforts to identify imidazole replacements were met with
only moderate success.12 The more recent identification
of imidazole-free ligands represents a significant ad-
vancement in the histamine H3 field.13 Examples of non-
imidazole H3 ligands include 4-(aminoalkoxy)benzyl-
amine 1,14 imidazopyridine 2,15 N-(5-phenoxypentyl)-
pyrrolidine 3,13a 1-(4-(phenoxymethyl)benzyl)piperidine
4,16 2-aminoethylbenzofuran 5,17 and 1-alkyl-4-acylpi-
perazine 618 (Chart 1).
* To whom correspondence should be addressed. Phone: 858-784-
3284. Fax: 858-450-2089. E-mail: cdvorak@prdus.jnj.com.
† Johnson & Johnson Pharmaceutical Research and Development,
L.L.C.
‡ University of Wisconsin.
10.1021/jm049212n CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/05/2005

2230 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Dvorak et al.
Scheme 1. Synthesis of Conformationally Restricted H3
Ligandsa
Figure 1. Design of 4-phenoxypiperidines (8).
As previously reported, our ligand design began with
an attempt to develop a pharmacophore model to
account for the activity of the structurally diverse non-
imidazole H3 ligands in the literature as well as those
found in our own compound collection. Three common
structural motifs were identified: (1) a basic nitrogen
atom; (2) an aromatic ring; and (3) an additional polar
functional group adjacent to the lipophilic core. A readily
accessible chemical series that incorporates these struc-
tural features is depicted by the generic structure 7,
seen in Figure 1, which ultimately led to the discovery
of the potent H3 antagonist 1, JNJ-5207852.19 Having
identified a general template for the design of H3
antagonists, we set out to further explore this model
by examining modifications to the ubiquitous propyloxy
piperidinyl side chain present in 1. Thus, we now report
the discovery of a conformationally restricted replace-
ment of the propyloxy piperidine moiety. We envisioned
that this could be accomplished by simply “tying back”
one of the alkyl residues present on the basic amine onto
the carbon chain of the propyloxy linker (Figure 1). This
would result in a cyclic structure for the tether linking
the basic amine to the central aromatic core with an
a Reagents and conditions: (a) 9 (R3 ) Boc): i. NaH, DMF,
4-chlorobenzonitrile, 65 °C; ii. HCl, MeOH, rt., 81% overall;
iii. carbonyl analogue, NaBH(OAc)3, AcOH, CH2Cl2, rt. (a) 10
(R3 ) i-Pr): NaH, DMF, 4-chlorobenzonitrile, 80 °C, 79% (b)
DIBAL-H, CH2Cl2, 0 °C, 95% (c) HNR1R2, NaBH(OAc)3, AcOH,
CH2Cl2, rt.
complished with diisobutylaluminum hydride (DIBAL-
H), providing the key aldehyde 12 directly from ben-
zonitrile 11e. Benzaldehyde 12 was then condensed with
various primary and secondary amines by reductive
amination to give the corresponding benzylamines.
Results and Discussion
The objectives of the SAR study were to: (1) quickly
assess the alkyl residue on the basic nitrogen atom of
the 4-phenoxypiperidine; (2) explore the second basic
polar group adjacent to the lipophilic core; and, (3)
ultimately determine if the 4-hydroxypiperidine moiety
is a suitable replacement for the propyloxypiperidine
unit in our ligand model. The histamine H3 receptor
binding affinities of 4-phenoxypiperidines 11a-k and
(1-isopropyl-piperidin-4-yloxy)-benzylamines 13a-r were
determined and are summarized in Tables 1 and 2. In
addition, the potency of (1-isopropyl-piperidin-4-yloxy)-
benzylamines 13a-r as antagonists of the human
recombinant H3 receptor were also obtained (Table 2).
The SAR of the alkyl group on the piperidine nitrogen
was initially examined (Table 1). The 4-phenoxypiperi-
dine 11a with the free NH lacked significant activity
at the H3 receptor with a pKi< 5. The addition of carbon
atoms onto the piperidine nitrogen in a linear succession
(11b-d) revealed a large gain in activity of more than
two log units with the N-ethyl substitution (11c) from
the initial unsubstituted parent compound (11a vs 11c).
The alpha branched N-isopropyl analogue 11e was
favored over the straight chain N-propyl compound 11d
(pKi 7.55 vs 6.97). The sec-butyl substituted phenoxypi-
peridine 11g gave a slight increase in activity over the
isopropyl analogue 11e. The addition of a hydroxyl
group (11f) on the isopropyl moiety resulted in over a
10-fold decrease in activity from 11e while the isobutyl
substituted phenoxypiperidine 11h, extending out the
isopropyl unit, gave only a slight decrease in binding
affinity over 11e. The ring substituted analogues 11i
overall reduction in the number of rotatable bonds.20
A
key design element was to conserve the number of atoms
between the central aromatic ring and the basic amine.
Thus, we chose to explore 4-hydroxypiperidine as a
potential replacement for the 3-amino-1-propanol func-
tionality. Another design element for the development
of this new ligand template was that the chemistry must
be concise, modular and driven by carbon-heteroatom
bond connections21 allowing for the rapid synthesis of
target compounds. This report describes the synthesis,
human H3 binding affinities and functional activities
of several members of this new series. Behavioral data
are also presented for a selected member of the series.
Chemistry. The syntheses of 4-(1-alkyl-piperidin-4-
yloxy)-benzonitriles 11a-k and 4-(1-isopropyl-piperidin-
4-yloxy)-benzylamines 13a-r were completed according
to the route shown in Scheme 1. To install the aryl ether
unit, the sodium salt of N-Boc-4-hydroxypiperidine or
N-isopropyl-4-hydroxypiperidine was prepared in DMF
with sodium hydride and then treated with 4-chloroben-
zonitrile thus affording 4-phenoxypiperidines 10 via
nucleophilic aromatic substitution. For compounds
11a-d and 11f-k, the tert-butyl carbamate (R3 ) Boc)
group was removed with HCl and the resulting second-
ary amine provided a flexible intermediate that was
alkylated via reductive amination.22 The installation of
the second basic polar group present in 4-(1-isopropyl-
piperidin-4-yloxy)-benzylamines 13a-r required a simple
functional group interconversion of the aromatic nitrile
moiety into the requisite benzaldehyde. This was ac-

4-Phenoxypiperidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2231
Table 1. Binding Affinities at the Human H3 Receptor
amine substitutions (13d-f) from pyrrolidine to azepine
provided excellent potency with the piperidine analogue
(11e) being the most active with pKi ) 9.20 ( 0.10 and
having a pA2 ) 9.90 ( 0.04. The introduction of a
heteroatom into the cyclic piperidine ring, either within
the ring by replacement of a carbon atom (13g and 13n)
or by simple attachment onto the ring (13h-i) was well
tolerated with only a minimal loss of functional activity.
The placement of larger groups on the ring (13j, o-p)
was also allowed. Secondary amines 13k-m are also
acceptable substrates with good affinity. Finally, the
piperazine 13n, homo-piperazine 13q and the ring
opened analogue 13r all exhibited comparable activity.
In general, the benzylamine portion of the molecule
confers potency on a wide array of substituents repre-
senting a key area where modifications could be made
to “fine-tune” the physical properties without jeopardiz-
ing potency.
The morpholine analogue 13g (JNJ-7737782), was
selected for further profiling over the more potent
piperidine, 13e, based on a preliminary pharmacokinetic
evaluation of both compounds in the rat. Thus 13e
exhibited a longer half-life and more extensive tissue
distribution compared to 13g, which we tentatively
attribute to differences in Log P of the compounds and
differences in pKa of the piperidine versus the morpho-
line fragments of the two molecules. Selectivity of 13g
was examined versus a panel of 50 monoamine and
hormone receptors, ion channels and neurotransmitter
uptake sites preformed by CEREP. A 1 µM screen
revealed no cross reactivity (<18% inhibition at all
targets). This promising selectivity profile was further
confirmed by in vitro autoradiography studies in rats.
As shown in Figure 2A, incubation of sagittal rat brain
slices with [3H]-13g showed preferential binding in the
cortex, striatum and substantia nigra, regions which are
known to be rich in H3 receptors.24 Most importantly,
the binding of [3H]-13g is likely localized specifically to
histamine receptors, since incubation with an excess of
histamine was able to completely block the binding of
[3H]-13g to the slices (Figure 2B). The potency of 13g
at the rat histamine H3 receptor was determined to be
7.81 ( 0.01 (pKi). There were slight differences observed
between the rat and human H3 binding affinities of 13g
(rH3 pKi ) 7.81 vs hH3 pKi ) 8.67). However, contrast-
ing ligand affinities between rat and human H3 recep-
tors have been well documented.25
Given the excellent selectivity and specificity of 13g,
its in vivo efficacy was examined in a model indicative
of H3 receptor antagonist activity. The role of the H3
receptor in the regulation of sleep/wake states has been
extensively studied. Histamine H3 antagonists have
been consistently shown to increase waking and sup-
press sleep in various animals such as rats26 and mice.27
We used a rat EEG model to evaluate the acute effects
of 13g on sleep-waking behavior.
Three doses of 13g (0.1, 1 and 10 mg/kg) or vehicle
were administered subcutaneously to rats. Time spent
in distinct sleep-wake states were monitored from EEG
and EMG recordings for 2 intervals of 30 min prior to,
and 3 intervals of 30 min after administration of vehicle
or test compound. As can be seen in Figure 3, admin-
istration of vehicle causes a small, transient increase
in waking in the first 30 min, probably as a consequence
a Displacement of [3H]-NR-methylhistamine from human H3
receptors expressed in SK-N-MC cells. Values reported as the
mean ( SEM of at least three independent determinations in
triplicate.
and 11j were also very potent H3 ligands with the
N-cyclopentyl substitution found to be the most potent
of this series. The N-benzyl analogue 11k resulted in a
more than 100-fold loss of activity. In comparison to the
propyloxypiperidine 14,23 the N-isopropyl phenoxypiper-
idine 11e has an identical molecular formula, fewer
rotatable bonds and comparable potency at the H3
receptor. Thus, it was determined to explore the addi-
tion of a second polar group for the fully assembled
ligand model (i.e. NR1R2, Figure 1) with the N-isopropyl
substituted 4-hydroxypiperidine as a possible replace-
ment tether from the central aromatic ring.
Table 2 lists the potency of compounds 13a-r at the
human H3 receptor. All compounds were found to be
antagonists with good correlation between the binding
affinities (pKi) and functional activities (pA2). The
replacement of the benzonitrile group in 11e with a
benzyldimethylamine moiety (13a) resulted in a 10-fold
increase in affinity at the H3 receptor (pKi 7.55 vs 8.52).
This improvement in activity with the addition of a
second basic amine was also observed in the chemical
series leading to 1.14 Increasing the size of the carbon
chain substituted on the benzyl nitrogen gave com-
pounds with improved activity (13a-c). The three cyclic

2232 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Dvorak et al.
Table 2. Binding Affinities and Functional Activity Values at the Human H3 Receptor
a pA2 values are reported as the mean ( SEM of at least three independent determinations in triplicate and are derived from Schild
regression analysis of the compound-induced rightward shifts in dose-response curves of histamine-induced inhibition of forskolin-
stimulated cAMP accumulation in SK-N-MC cells overexpressing the human histamine H3 receptor. b Displacement of [3H]- NR-
methylhistamine from human H3 receptors expressed in SK-N-MC cells. Values reported as the mean ( SEM of at least three independent
determinations in triplicate. c n ) 1.
of the manipulation during sc injection of the animals.
At 10 mg/kg, 13g elicited a clear and statistically
significant increase in total time spent awake in the first
half hour after dosing, as compared to vehicle (Figure
3A). This wake-promoting effect was maintained for the
next 30-minute observation interval. In the 1 mg/kg
group an increase in waking was observed in the second
30-min interval after dosing only, possibly reflecting a
longer time required to achieve sufficient brain levels.
At the lowest dose (0.1 mg/kg) there was no statistically
significant effect on waking or on any of the other
variables measured. The increase in waking was pre-
dominantly manifested as quiet waking, reaching sta-
tistical significance in the 10 mg/kg group (Figure 3B).
There appeared to be a trend toward an increase in time
spent in an active waking state, although the effect did
not reach statistical significance (Figure 3C). Slow-wave
sleep was significantly suppressed in the second 30 min
observation interval in both the 1 and 10 mg/kg groups,
mirroring the effect on total awake time (Figure 3E).

4-Phenoxypiperidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2233
3D). Thus, as expected, 13g induced a state of increased
arousal and wakefulness in rats, which was accompa-
nied by a decrease in time spent asleep. The wake-
promoting effect of 13g is consistent with previous
animal studies using H3 receptor antagonists, such as
thioperamide,28,27b carboperamide26b and ciproxifan.27a
In our hands, for instance, an intracerebroventricular
dose of thioperamide (80 µg, n ) 8) caused the animals
to spend an average time of 1619 ( 110 seconds awake,
with a suppression of non-REM sleep to a level of
181 ( 110 sec.
Although no human data are available, there is
considerable interest in the possible therapeutic ap-
plications of H3 receptor antagonists. Therapeutic in-
dications ranging from Alzheimer’s disease7a to obesity29
have been proposed. The wake-promoting effects dem-
onstrated in this paper with 13g could be beneficial for
conditions associated with excessive daytime sleepiness,
such as narcolepsy, sleep apnea, fibromyalgia and
multiple sclerosis. The mild stimulant effect could also
Figure 2. A. In vitro autoradiography of 13g in sagittal
sections from rat brain. Cortex (Cx), Substantia Nigra (SN),
and Striatum (Str) are indicated by arrows. Nonspecific
binding (B) was determined in the presence of 100 µM
histamine.
No statistically significant differences were seen in the
treated versus the vehicle group for REM sleep (Figure
Figure 3. Arousal response to 0.1, 1 and 10 mg/kg (sc) of 13g in the rat. The compound or vehicle was administered sc at t ) 0
min. Sleep-wake behavior was monitored for 2 epochs of 30 min prior to, and 4 epochs after dosing and expressed as average time
spent in each of the sleep/waking states. A, total time awake; B, quiet waking; C, active waking; D, REM sleep; E, slow-wave
sleep. Data are shown as average ( SEM of four animals. * p < 0.05, ** p <0.01, statistically significant from vehicle.

2234 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Dvorak et al.
aqueous sodium hydroxide (300 mL) and water (300 mL) were
added, and the mixture was extracted with CH2Cl2 (3 × 300
mL). The combined organic phases were dried over sodium
sulfate, and the solvent removed to reveal 12.23 g of an off-
white waxy solid (81%). 1H NMR (400 MHz) δ 7.57 (d, J ) 8.8
Hz, 2H), 6.94 (d, J ) 8.8 Hz, 2H), 4.48-4.41 (m, 1H), 3.18-
3.11 (m, 2H), 2.79-2.71 (m, 2H), 2.05-1.98 (m, 2H), 1.73-
1.65 (m, 2H); 13C NMR (125 MHz) δ 160.4, 133.9, 118.9, 116.0,
103.4, 71.5, 42.0, 29.9; MS m/z 203.1 (M + H+); Anal.
(C12H14N2O) H; C: calcd. 71.26; found 69.51; N: calcd. 13.85;
found 12.36.
General Procedure for Reductive Amination (Method
A). A solution of amine (1 equiv), the carbonyl analogue (1.2
equiv), and acetic acid (1 equiv) in CH2Cl2 (0.1 M) was treated
with NaBH(OAc)3 (1.6 equiv). After 16 h, the resulting mixture
was treated with 10% sodium hydroxide (5 mL), and the
mixture was extracted with CH2Cl2 (3 × 10 mL). The combined
organic phases were dried over sodium sulfate and evaporated.
Chromatography of the residue (1-7% 2M methanolic am-
monia/CH2Cl2) provided the desired product.
be beneficial for the treatment Attention Deficit Disor-
der with Hyperactivity, a disease for which low doses
of stimulants have been shown to improve attention.30
Conclusions
We have refined our previously described H3 antago-
nist template, replacing the piperidinyl propyloxy frag-
ment found in 1 with a conformationally restricted
4-hydroxypiperidine moiety. These two fragments were
determined to be freely interchangeable in our phar-
macophore design model. This has led to a new series
of H3 antagonists described herein. One selected mem-
ber of the new series, 4-[4-(1-isopropyl-piperidin-4-yl-
oxy)-benzyl]-morpholine 13g, was found to be a potent,
highly selective H3 receptor antagonist with in vivo
efficacy in a rat EEG model of wakefulness at doses as
low as 1 mg/kg sc.
General Procedure for Reductive Amination (Method
B). A solution of the aldehyde (1 equiv), the amine analogue
(1.2 equiv), and acetic acid (1.2 equiv) in CH2Cl2 or THF (0.1
M) was treated with NaBH(OAc)3 (1.6 equiv). After 16 h, the
resulting mixture was treated with 10% sodium hydroxide (5
mL), and the mixture was extracted with CH2Cl2 (3 × 10 mL).
The combined organic phases were dried over sodium sulfate
and evaporated. Chromatography of the residue (1-7% 2M
methanolic ammonia/CH2Cl2) provided the desired product.
4-(1-Methyl-piperidin-4-yloxy)-benzonitrile (11b). Pre-
pared according to Method A (40%). 1H NMR (400 MHz) δ 7.57
(d, J ) 9.1 Hz, 2H), 6.93 (d, J ) 9.1 Hz, 2H), 4.40 (m, 1H),
2.69 (br, 2H), 2.37-2.29 (br, 2H), 2.32 (s, 3H), 2.03 (m, 2H),
1.87 (m, 2H); 13C NMR (125 MHz) δ 160.8, 133.9, 119.1, 116.1,
103.6, 72.4, 52.3, 46.0, 30.5; MS m/z 217.1 (M + H+); Anal.
(C13H16N2O) C, H, N.
4-(1-Ethyl-piperidin-4-yloxy)-benzonitrile (11c). Pre-
pared according to Method A (26%). 1H NMR (400 MHz) δ 7.53
(d, J ) 8.8 Hz, 2H), 6.91 (d, J ) 8.8 Hz, 2H), 4.37 (m, 1H),
2.71 (m, 2H), 2.41 (q, J ) 7.1, 2H), 2.28 (m, 2H), 1.99 (m, 2H),
1.82 (m, 2H), 1.07 (t, J ) 7.1, 3H); 13C NMR (125 MHz) δ 160.9,
133.9, 119.2, 116.1, 103.6, 72.9, 52.2, 49.9, 30.5, 12.1; MS m/z
231.1 (M + H+); Anal. (C14H18N2O) C, H, N.
4-(1-Propyl-piperidin-4-yloxy)-benzonitrile (11d). Pre-
pared according to Method A (48%). 1H NMR (400 MHz) δ 7.57
(d, J ) 9.1 Hz, 2H), 6.93 (d, J ) 9.1 Hz, 2H), 4.42 (br, 1H),
2.76 (br, 2H), 2.35 (br, 3H), 2.05 (br, 2H), 1.87 (br, 2H), 1.56
(br, 2H), 0.92 (t, J ) 7.3 Hz, 3H); 13C NMR (125 MHz) δ 160.9,
133.9, 119.2, 116.1, 103.5,73.2, 60.5, 50.4, 30.5, 20.2, 11.9; MS
m/z 245.1 (M + H); Anal. (C15H20N2O) C, H, N.
Experimental Section
General Procedures. Reagents were purchased from
commercial suppliers and were used without purification.
Anhydrous solvents were obtained from a GlassContour
Solvent Dispensing System. Reactions were performed at room
temperature (20-23 °C) under an atmosphere of N2 unless
otherwise noted. Chromatography was performed using pre-
packed ISCO RediSep silica cartridges utilizing gradient
1
elution. H and 13C NMR spectra were recorded on a Bruker
400 or 500 MHz spectrometers. Chemical shifts are reported
in parts per million downfield from an internal standard (Me4-
Si). All spectra were taken in CDCl3 unless otherwise noted.
Mass spectra were obtained on an Agilent series 1100 MSD
using electrospray ionization (ESI) in either positive or nega-
tive modes as indicated. Thin-layer chromatography was
performed using Merck silica gel 60 F254 2.5 cm × 7.5 cm 250
µm or 5.0 cm × 10.0 cm 250 µm precoated silica gel plates.
Preparative thin-layer chromatography was performed using
EM Science silica gel 60 F254 20 cm × 20 cm 0.5 mm precoated
plates with a 20 cm × 4 cm concentrating zone. Melting points
are uncorrected and were obtained on a MelTemp apparatus.
Analytical reverse phase HPLC was preformed on a Hewlett-
Packard Series 1100 instrument with an Agilent ZORBAX
Bonus RP, column utilizing an acetonitrile/water (0.05%TFA)
gradient. Combustion analyses were performed by Desert
Analytics or NuMega Resonance Labs.
1-Isopropyl-piperidin-4-ol (10). To a solution of 1-isopro-
pyl-piperidin-4-one (60.0 g, 425 mmol) in 600 mL of absolute
ethanol cooled to 0 °C (ice bath) was added NaBH4 (8.5 g, 225
mmol) in several portions. After the addition was complete the
mixture was stirred at room temperature for 24 h. The solvent
was then removed and the yellow paste was partitioned
between CH2Cl2 (300 mL) and 1.0 M NaOH (300 mL). This
mixture was stirred for 6 h (reaches clarity). The layers were
separated and the aqueous was extracted with CH2Cl2 (4 ×
100 mL) and the combined organics were dried over sodium
sulfate. The solvent was removed and the yellow oil distilled
bulb to bulb to reveal 39.3 g (bp 68 °C @ 1.5 mmHg, 65%) of a
4-(1-Isopropyl-piperidin-4-yloxy)-benzonitrile (11e). To
a suspension of 4-chlorobenzonitrile (38.4 g, 280 mmol) and
NaH (95%, 7.68 g) in DMF (200 mL) cooled to 0 °C was added
slowly a solution of 1-isopropyl-piperidin-4-ol 10 (39.3 g, 274
mmol) in DMF (100 mL). After the addition was complete, the
mixture was stirred at room temperature for 1 h then heated
to 80 °C for 12 h. The cooled reaction mixture was then poured
into water (1.5 L) and allowed to stand for 3 h. The product
was collected by suction filtration and the cake washed with
water and dried. The residual 4-chlorobenzonitrile was re-
moved by sublimation to reveal 52.9 g (79%) of a light brown
1
clear oil that solidified on standing to a waxy solid. H NMR
(400 MHz) δ 3.69-3.61 (m, 1H), 2.80-2.66 (m, 3H), 2.28-2.20
(m, 2H), 1.94-1.86 (m, 2H), 1.61-1.51 (m, 2H), 1.02 (d, J )
6.6 Hz, 6H); 13C NMR (100 MHz) δ 68.2, 54.2, 46.1, 34.7, 18.3;
MS m/z 144.1 (M + H+).
1
solid. H NMR (400 MHz) δ 7.56 (d, J ) 8.6 Hz, 2H), 6.93 (d,
J ) 8.6 Hz, 2H), 4.37 (m, 1H), 2.81-2.72 (m, 3H), 2.41 (m,
4H), 2.06-1.98 (m, 2H), 1.87-1.77 (m, 2H), 1.06 (d, J ) 6.6
Hz, 6H); 13C NMR (125 MHz) δ 161.0, 133.9, 119.2, 116.1,
103.5, 73.5, 54.3, 45.5, 30.9, 18.4; MS m/z 245.2 (M + H+); Anal.
(C15H20N2O) C, H, N.
4-(Piperidin-4-yloxy)-benzonitrile (11a). A solution of
4-hydroxy-piperidine-1-carboxylic acid tert-butyl ester (14.99
g, 74.5 mmol) and 4-chlorobenzonitrile (10.35 g, 75.2 mmol)
in DMF (100 mL) was treated with sodium hydride (60%, 3.8
g). The resulting dark mixture was then heated to 65 °C for
16 h. The cooled mixture was poured into water (1 L) and then
extracted with ether (3 × 400 mL). The combined organic
phases were evaporated, and the brown oil was dissolved in
methanol (500 mL) and treated with concentrated hydrochloric
acid (20 mL). After 24 h the methanol was removed, 5%
4-[1-(2-Hydroxy-1-methyl-ethyl)-piperidin-4-yloxy]-ben-
zonitrile (11f). Prepared according to Method A (61%). 1H
NMR (400 MHz) δ 7.57 (d, J ) 8.8 Hz, 2H), 6.93 (d, J ) 8.8
Hz, 2H), 4.42 (m, 1H), 3.42 (m, 1H), 3.32 (m, 1H), 2.88 (m,
2H), 2.64 (m, 2H), 2.33 (m, 1H), 2.03 (m, 2H), 1.84 (m, 2H),
0.92 (d, J ) 6.6 Hz, 3H); 13C NMR (125 MHz) δ 160.8, 134.0,

4-Phenoxypiperidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2235
119.1, 116.1, 103.7, 73.0, 62.3, 60.1, 31.1, 30.9; MS m/z 261.1
(M + H+); Anal. (C16H20N2O2) H, N; C: calcd. 69.20; found,
68.76.
4.26 (m, 1H), 3.49 (s, 2H), 2.82-2.70 (m, 4H), 2.50 (q, J ) 7.1
Hz, 4H), 2.37 (m, 2H), 2.04-1.96 (m, 2H), 1.85-1.76 (m, 2H),
1.60 (m, 9H), 1.07-1.00 (m, 12H); 13C NMR (100 MHz) δ 156.3,
130.0, 128.6, 115.7, 73.1, 56.7, 54.4, 46.4, 45.8, 31.2, 18.4, 11.6;
MS m/z 305.1 (M + H+); Anal. (C19H32N2O) C, H, N.
Butyl-[4-(1-isopropyl-piperidin-4-yloxy)-benzyl]-meth-
yl-amine (13c). Prepared according to Method B (73%). 1H
NMR (400 MHz) δ 7.19 (d, J ) 8.6 Hz, 2H), 6.85 (d, J ) 8.6
Hz, 2H), 4.27 (m, 1H), 3.40 (s, 2H), 2.82-2.71 (m, 3H), 2.39
(m, 2H), 2.34 (m, 2H), 2.16 (s, 3H), 2.00 (m, 2H), 1.85-1.76
(m, 2H), 1.52-1.45 (m, 2H), 1.38-1.27 (m, 2H) 1.06 (d, J )
6.6 Hz, 6H), 0.90 (t, J ) 7.3 Hz, 3H). 13C NMR (100 MHz) δ
156.4, 131.3, 130.1, 115.8, 73.2, 61.6, 57.1, 54.4, 45.7, 42.1, 31.2,
29.5, 20.6, 18.4, 14.0; MS m/z 319.2 (M + H+); Anal. (C20H34N2O)
C, H, N.
4-(1-sec-Butyl-piperidin-4-yloxy)-benzonitrile (11g). Pre-
pared according to Method A (34%). 1H NMR (400 MHz) δ 7.56
(d, J ) 9.1 Hz, 2H), 6.93 (d, J ) 9.1 Hz, 2H), 4.36 (br, 1H),
2.77 (br, 2H), 2.50 (br, 2H), 2.37 (br, 1H), 2.02 (br, 2H), 1.81
(br, 2H), 1.58 (br, 1H), 1.30 (m, 1H), 1.0 (br, d, J ) 6.1 Hz,
3H), 0.91 (t, J ) 7.3 Hz, 3H); 13C NMR (125 MHz) δ 161.0,
133.9, 119.2, 116.1, 103.5, 73.4, 60.8, 44.8, 31.1, 26.3, 13.8, 11.4;
MS m/z 259.1 (M + H+); Anal. (C16H22N2O) C, H, N.
4-(1-Isobutyl-piperidin-4-yloxy)-benzonitrile (11h). Pre-
pared according to Method A (72%). 1H NMR (400 MHz) δ 7.56
(d, J ) 9.1 Hz, 2H), 6.93 (d, J ) 9.1 Hz, 2H), 4.37 (br, 1H),
2.69 (br, 2H), 2.22 (br, 2H), 2.09 (br, 2H), 1.98 (br, 2H), 187-
1.73 (m, 3H), 0.90 (d, br, J ) 7.3 Hz, 6H); 13C NMR (125 MHz)
δ 161.0, 133.9, 119.2, 116.1, 103.4, 73.5, 66.7, 50.8, 30.6, 25.7,
20.8; MS m/z 259.1 (M + H+); Anal. (C16H22N2O) C, H, N.
4-(1-Cyclopentyl-piperidin-4-yloxy)-benzonitrile (11i).
1-Isopropyl-4-(4-pyrrolidin-1-ylmethyl-phenoxy)-pi-
1
peridine (13d). Prepared according to Method B (70%). H
NMR (400 MHz) δ 7.21 (d, J ) 8.8 Hz, 2H), 6.84 (d, J ) 8.8
Hz, 2H), 4.26 (m, 1H), 3.53 (s, 2H), 2.82-2.71 (m, 3H), 2.48
(m, 4H), 2.37 (m, 2H), 2.03-1.96 (m, 2H), 1.85-1.74 (m, 6H),
1.05 (d, J ) 6.6 Hz, 6H); 13C NMR (100 MHz) δ 156.4, 131.6,
130.0, 115.8, 73.2, 60.0, 54.0, 45.8, 31.2, 23.3, 18.4; MS m/z
303.1 (M + H+); Anal. (C19H30N2O) H, N; C: calcd. 75.45; found
75.03.
1
Prepared according to Method A (84%). H NMR (400 MHz,
CDCl3) δ 7.57 (d, J ) 8.8 Hz, 2H), 6.94 (d, J ) 8.9 Hz, 2H),
4.40 (br, 1H), 2.91-2.23 (br, m, 5H), 2.13-1.35 (m, 12H); 13C
NMR (125 MHz, CDCl3) δ 160.9, 133.9, 119.2, 116.1, 103.6,
73.1, 67.3, 49.3, 30.6, 30.5, 24.1; MS m/z 271.1 (M + H+); Anal.
(C17H22N2O) C, H, N.
1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-piperi-
1
dine (13e). Prepared according to Method B (84%). H NMR
4-(1-Cyclohexyl-piperidin-4-yloxy)-benzonitrile (11j).
Prepared according to Method A (79%). 1H NMR (400 MHz) δ
7.56 (d, J ) 8.8 Hz, 2H), 6.93 (d, J ) 8.8 Hz, 2H), 4.37 (m,
1H), 2.84 (m, 2H), 2.47 (m, 2H), 2.32 (br, 1H), 2.01 (br, 2H),
1.89-1.77 (m, 6H), 1.63 (m, 1H), 1.30-1.17 (m, 4H), 1.10 (m,
1H); 13C NMR (125 MHz) δ 161.0, 133.9, 119.2, 116.1, 103.4,
73.6, 63.6, 45.9, 31.1, 28.8, 26.2, 25.9; MS m/z 285.1 (M + H+);
Anal. (C18H24N2O) C, H, N.
(400 MHz) δ 7.19 (d, J ) 8.5 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H),
4.29-4.25 (m, 1H), 3.40 (s, 2H), 2.82-2.71 (m, 3H), 2.41-2.35
(m, 6H), 2.40-2.26 (m, 6H), 2.03-1.99 (m, 2H), 1.85-1.77 (m,
2H), 1.59-1.53 (m, 4H), 1.43-1.38 (m, 2H), 1.06 (d, J ) 6.6
Hz, 6H); 13C NMR (100 MHz) δ 156.4, 130.5, 130.4, 115.6, 73.0,
63.2, 54.3, 45.7, 31.2, 25.9, 24.3, 18.4; MS m/z 317.1 (M + H+);
Maleate salt Anal. (C24H36N2O5) H, N; C: calcd. 66.64; found
65.99.
4-(1-Benzyl-piperidin-4-yloxy)-benzonitrile (11k). Pre-
pared according to Method A (38%). 1H NMR (400 MHz) δ 7.56
(d, J ) 8.8 Hz, 2H), 7.33 (m, 3H), 7.27 (m, 2H), 6.93 (d, J )
8.8 Hz, 2H), 4.40 (br, 1H), 3.54 (br, 2H), 2.73 (br, 2H), 2.32
(br, 2H), 1.99 (br, 2H), 1.84 (br, 2H), 1.56 (br, 1H); 13C NMR
(100 MHz) δ 160.9, 138.2, 133.9, 129.0, 128.2, 127.0, 119.2,
116.1, 103.6, 73.2, 62.9, 50.3, 30.6; MS m/z 293.0 (M + H+);
Anal. (C18H20N2O) H, N; C: calcd. 78.05; found 77.54.
4-(1-Isopropyl-piperidin-4-yloxy)-benzaldehyde (12).
To a solution of 4-(1-isopropyl-piperidin-4-yloxy)-benzonitrile
11e (48.6 g, 199 mmol) in CH2Cl2 (500 mL) cooled to 0 °C was
added DIBAL-H (250 mL, 1.0 M CH2Cl2, 1.25 equiv) via
syringe. The reaction was stirred at 0 °C for 30 min then
quenched with ethyl acetate (30 mL). Aqueous Rochelle’s salt
(1.5 L) was added slowly. Caution! Gas Evolution! The result-
ing suspension was stirred for 12 h (reaches clarity). The layers
were separated and the aqueous was extracted with CH2Cl2
(4 × 400 mL). The solvent was removed to reveal a thick syrup
that was treated with HCl (300 mL, pH 1) and stirred for 2 h.
Water (1.4 L) was added and the pH adjusted with 50% NaOH
(pH ∼ 12). The produce was extracted with CH2Cl2 (5 × 300
mL) and washed with water and brine. The solvent was
removed to reveal 46.6 g (95%) of a thick oil that solidified on
standing to a light brown solid. 1H NMR (400 MHz) δ 9.86 (s,
1H), 7.81 (d, J ) 8.6 Hz, 2H), 6.99 (d, J ) 8.6 Hz, 2H), 4.43
(m, 1H), 2.82-2.72 (m, 3H), 2.42 (m, 4H), 2.08-2.00 (m, 2H),
1.89-1.80 (m, 2H), 1.06 (d, J ) 6.6 Hz, 6H); 13C NMR (100
MHz) δ 190.7, 162.8, 131.9, 129.6, 115.7, 73.2, 54.4, 45.5, 31.0,
18.4; MS m/z 248.3 (M + H+).
1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-azepane
1
(13f). Prepared according to Method B (73%). H NMR (400
MHz) δ 7.22 (d, J ) 8.6 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H), 4.26
(m, 1H), 3.56 (s, 2H), 2.85-2.71 (m, 3H), 2.60 (m, 4H), 2.38
(m, 2H), 2.04-1.96 (m, 2H), 1.85-1.76 (m, 2H), 1.60 (m, 9H),
1.06 (d, J ) 6.6 Hz, 6H); 13C NMR (100 MHz) δ 156.3, 132.0,
129.9, 115.7, 73.1, 62.0, 55.4, 54.4, 45.8, 31.2, 28.0, 27.0, 18.4;
MS m/z 331.2 (M + H+); Anal. (C21H34N2O) C, H, N.
4-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-morpho-
1
line (13g). Prepared according to Method B (96%). H NMR
(400 MHz) δ 7.20 (d, J ) 8.6 Hz, 2H), 6.82 (d, J ) 8.6 Hz, 2H),
4.27 (m, 1H), 3.69 (m, 4H), 3.42 (s, 2H), 2.82-2.70 (m, 3H),
2.41 (m, 4H), 2.36 (m, 2H), 2.04-1.96 (m, 2H), 1.84-1.75 (m,
2H), 1.05 (d, J ) 6.6 Hz, 6H); 13C NMR (100 MHz) δ 156.6,
130.3, 129.7, 115.8, 73.0, 66.9, 62.8, 54.4, 53.5, 45.7, 31.2, 18.4;
MS m/z 319.3 (M + H+). Maleate salt: Anal. (C23H34N2O6) C,
H, N.
1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-piperidin-
1
4-ol (13h). Prepared according to Method B (74%). H NMR
(400 MHz) δ 7.17 (d, J ) 8.6 Hz, 2H), 6.82 (d, J ) 8.6 Hz, 2H),
4.25 (m, 1H), 3.64 (m, 1H), 3.42 (s, 1H), 3.40 (s, 2H), 2.80-
2.69 (m, 5H), 2.36 (m, 2H), 2.08 (m, 2H), 2.02-1.94 (m, 2H),
1.87-1.74 (m, 2H), 1.55 (m, 2H), 1.04 (d, J ) 6.6 Hz, 6H); 13
C
NMR (100 MHz) δ 156.5, 130.2, 130.3, 115.6, 72.9, 67.8, 62.2,
54.3, 50.2, 45.6, 34.4, 31.0, 18.3; MS m/z 333.1 (M + H+); Anal.
(C20H32N2O2) C, H, N.
{1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-piperidin-
4-yl}-methanol (13i). Prepared according to Method B (75%).
1H NMR (400 MHz) δ 7.17 (d, J ) 8.6 Hz, 2H), 6.82 (d, J ) 8.6
Hz, 2H), 4.25 (m, 1H), 3.24 (d, J ) 6.6 Hz, 2H), 3.39 (s, 2H),
2.87 (m, 2H), 2.80-2.69 (m, 3H), 2.37 (m, 2H), 2.02-1.94 (m,
2H), 1.90 (m, 2H), 1.83-1.74 (m, 2H), 1.69 (m, 2H) 1.45 (m,
1H), 1.23 (m, 2H), 1.04 (d, J ) 6.6 Hz, 6H); 13C NMR (100
MHz) δ 156.4, 130.3, 130.4, 115.6, 72.8, 67.5, 62.7, 54.3, 53.2,
45.6, 38.5, 31.0, 28.7, 18.3; MS m/z 347.3 (M + H+); Anal.
(C21H34N2O2) H, N; calcd. 72.79; found 71.27.
{1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-piperidin-
4-}-methylbenzene (13j). Prepared according to Method B
(51%). 1H NMR (400 MHz) δ 7.29-7.23 (m, 3H), 7.19-7.10
(m, 4H), 6.83 (m, 2H), 4.26 (m, 1H), 3.39 (s, 2H), 2.87-2.70
[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-dimethyl-
amine (13a). Prepared according to Method B (86%). 1H NMR
(400 MHz) δ 7.18 (d, J ) 8.6 Hz, 2H), 6.85 (d, J ) 8.6 Hz, 2H),
4.27 (m, 1H), 3.34 (s, 2H), 3.37 (s, 3H), 2.81-2.70 (m, 3H), 2.38
(m, 2H), 2.21 (s, 6H), 2.05-1.96 (m, 2H), 1.85-1.75 (m, 2H),
1.05 (d, J ) 6.6 Hz, 6H); 13C NMR (100 MHz) δ 156.5, 130.9,
130.2, 115.8, 73.0, 63.7, 54.4, 45.7, 45.2, 31.2, 18.4; MS m/z
277.1 (M + H+); Anal. (C17H28N2O) H, N; C: calcd. 73.87; found
73.38.
Diethyl-[4-(1-isopropyl-piperidin-4-yloxy)-benzyl]-
amine (13b). Prepared according to Method B (37%). 1H NMR
(400 MHz) δ 7.21 (d, J ) 8.6 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H),

2236 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Dvorak et al.
(m, 5H), 2.52 (d, J ) 7.1 Hz, 2H), 2.38 (m, 2H), 2.03-1.96 (m,
2H), 1.59 (m, 2H), 1.50 (m, 1H), 1.29 (m, 2H), 1.06 (d, J ) 6.6
Hz, 6H); 13C NMR (100 MHz) δ 156.4, 140.7, 130.4, 130.3,
129.0, 128.0, 125.6, 115.6, 73.0, 62.8, 54.4, 53.6, 45.7, 43.2, 37.9,
32.1, 31.1, 18.4; MS m/z 407.3 (M + H+); Anal. (C27H38N2O) C,
H, N.
Cyclopropyl-[4-(1-isopropyl-piperidin-4-yloxy)-benzyl]-
amine (13k). Prepared according to Method B (30%). 1H NMR
(400 MHz) δ 7.19 (d, J ) 8.6 Hz, 2H), 6.84 (d, J ) 8.6 Hz, 2H),
4.25 (m, 1H), 3.74 (s, 2H), 2.81-2.69 (m, 3H), 2.37 (m, 2H),
2.12 (m, 1H), 2.03-1.95 (m, 2H), 1.83-1.74 (m, 2H), 1.04 (d,
J ) 6.6 Hz, 6H), 0.44-0.33 (m, 4H); 13C NMR (100 MHz) δ
156.3, 132.7, 129.2, 115.9, 73.0, 54.3, 53.0, 45.6, 31.1, 29.9, 18.3,
6.3; MS m/z 289.1 (M + H+); Anal. (C18H28N2O) C, H, N.
[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-phenyl-
amine (13l). A solution of benzaldehyde 12 (200 mg), aniline
(104 mg), and acetic acid (0.05 mL) in DCE (3 mL) was treated
with NaBH(OAc)3 (257 mg). After 16 h, the resulting mixture
was treated with 10% sodium hydroxide (1 mL), and the
mixture was extracted with CH2Cl2 (3 × 3 mL). The combined
organic phases were dried (magnesium sulfate) and evapo-
rated. Chromatography of the residue (0-8% 2 M methanolic
ammonia/CH2Cl2) gave 136 mg as a yellow amorphous solid.
1H NMR (400 MHz) δ 7.27 (d, J ) 8.2 Hz, 1H), 7.19-7.15 (m,
2H), 6.89 (d, J ) 8.6 Hz, 2H), 6.73-6.69 (m, 1H), 6.65-6.62
(m, 2H), 4.31-4.23 (m, 3H), 3.96-3.90 (br s, 1H), 2.83-2.70
(m, 3H), 2.43-2.34 (m, 2H), 2.05-1.96 (m, 2H), 1.86-1.76 (m,
2H), 1.06 (d, J ) 6.7, 6H);13C NMR (100 MHz) δ 156.7, 148.1,
131.4, 129.2, 128.8, 117.4, 116.2, 112.7, 73.1, 54.4, 47.7, 45.7,
31.1, 18.4; MS m/z 325.5; (M + H+); Maleate salt: Anal.
(C25H32N2O5) C, H, N.
[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-pyridin-2-
yl-amine (13m). To a solution of benzaldehyde 12 (5.19 g, 20.9
mmol) in THF (100 mL) was added 2-aminopyridine (2.19 g,
23.3 mmol) followed by NaBH(OAc)3 (7.1 g, 33.5 mmol). This
mixture was stirred at room temperature for 16 h. The mixture
was quenched by addition of 1.0 M NaOH (40 mL) and poured
into water (700 mL). The pH was adjusted with 50% NaOH
(pH ∼ 10) and allowed to stand for 12 h. The resulting white
solid was collected by filtration and the filter cake washed with
water then dried (4.82 g, 71%). 1H NMR (400 MHz) δ 8.10 (m,
1H), 7.40 (m, 1H), 7.28-7.24 (m, 2H), 6.9-6.85 (m, 2H), 6.58
(m, 1H), 6.37 (m, 1H), 4.77 (m, 1H), 4.41 (d, J ) 5.8 Hz, 2H),
4.28 (m, 1H), 2.82-2.71 (m, 4H), 2.39 (m, 3H), 2.05-1.97 (m,
3H), 1.85-1.76 (m, 3H), 1.06 (d, J ) 6.6 Hz, 6H). 13C NMR
(100 MHz) δ 158.5, 156.7, 148.1, 137.3, 131.1, 128.7, 116.2,
113.0, 106.7, 72.9, 54.5, 45.8, 45.7, 31.0, 18.3; MS m/z 326.3
(M + H+); Maleate salt: Anal. (C24H31N3O5) C, H, N.
128.5, 127.3, 116.1, 73.5, 63.5, 62.8, 54.8, 53.4, 53.3, 46.2, 31.6,
18.9; MS m/z 408.3 (M + H+); Anal. (C26H37N3O) H, N; C:
calcd. 76.62; found 76.11.
1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-4-methyl-
[1,4]diazepane (13q). Prepared according to Method B (50%).
1H NMR (400 MHz) δ 7.22 (d, J ) 8.6 Hz, 2H), 6.84 (d, J ) 8.6
Hz, 2H), 4.26 (m, 1H), 3.55 (s, 2H), 2.82-2.72 (m, 3H), 2.71-
2.63 (m, 6H), 2.61-2.57 (m, 2H), 2.41-2.36 (m, 2H), 2.35 (s,
3H), 2.00 (m, 2H), 1.85-1.76 (m, 4H), 1.06 (d, J ) 6.6 Hz, 6H;
13C NMR (100 MHz) δ 156.4, 131.5, 129.9, 115.7, 72.8, 62.1,
57.9, 56.7, 54.5, 54.1, 46.9, 45.7, 31.0, 27.2, 18.3; MS m/z 346.2
(M + H+); Anal. (C21H35N3O) C, H, N.
N-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-N,N′,N′-
trimethyl-ethane-1,2-diamine (13r). Prepared according to
1
Method B (55%). H NMR (400 MHz) δ 7.19 (d, J ) 8.6 Hz,
2H), 6.84 (d, J ) 8.6 Hz, 2H), 4.26 (m, 1H), 3.44 (s, 2H), 2.82-
2.71 (m, 3H), 2.48-2.34 (m, 6H), 2.22 (s, 3H), 2.20 (s, 6H),
2.04-1.96 (m, 2H), 1.85-1.75 (m, 2H), 1.06 (d, J ) 6.6 Hz,
6H); 13C NMR (100 MHz) δ 156.5, 131.0, 130.2, 115.8, 73.0,
62.2, 57.4, 55.0, 54.4, 45.8, 45.7, 42.3, 31.1, 18.4; MS m/z 334.2
(M + H); Anal. (C20H35N3O) C, H, N.
In Vitro Pharmacology. Human and Rat Histamine H3
Binding Assays. Binding of compounds to the cloned human
H3 receptor, stably expressed in SK-N-MC cells, was performed
as described earlier.25d Briefly, cell pellets from SK-N-MC cells
expressing either the rat or human H3 receptor were homog-
enized in 50 mM Tris-HCl/5 mM EDTA and recentrifuged at
30 000g for 30 min. Pellets were rehomogenized in 50 mM
Tris/5 mM EDTA (pH 7.4). Membranes were incubated with
0.8 nM [3H]-NR-methylhistamine plus/minus test compounds
for 60 min at 25 °C and harvested by rapid filtration over GF/C
glass fiber filters (pretreated with 0.3% polyethylenimine)
followed by four washes with ice-cold buffer. Nonspecific
binding was defined in the presence of 10 µM histamine. IC50
values were determined by a single site curve-fitting program
(GraphPad, San Diego, CA) and converted to Ki values based
on a [3H]-NR-methylhistamine Kd of 800 pM and a ligand
concentration of 800 pM.31
CEREP Binding Panel. The compound was screened at a
concentration of 1 µM against a panel of 50 drug targets
(receptors, ion channels, transporters) by CEREP (15318 NE
95th Street, Redmond WA; www.cerep.com).
Human Histamine H3 Functional Assay. Sublines of SK-
N-MC cells were created that expressed a reporter construct
and the human H3 receptor. The reporter gene was â-galac-
tosidase under the control of multiple cyclic AMP responsive
elements. In 96-well plates, histamine was added directly to
the cell media followed 5 min later by an addition of forskolin
(5 µM final concentration). After a 6-h incubation at 37 °C,
the media was aspirated and the cells were washed with 200
µL of phosphate-buffered saline followed by a second aspira-
tion. Antagonists were added 10 min prior to the addition of
histamine. Cells were lysed with 25 µL 0.1 × assay buffer (10
mM Na-phosphate, pH 8, 0.2 mM MgSO4, 0.01 mM MnCl2)
and incubated at room temperature for 10 min. Cells were then
incubated for 10 min with 100 µL of 1 × assay buffer
containing 0.5% Triton and 40 mM â-mercaptoethanol. Color
was developed using 25 mL of 1 mg/mL substrate solution
(cholorphenolred â-D-galactopyranoside; Roche Molecular Bio-
chemicals, Indianapolis, IN). Color was quantitated on a
microplate reader at absorbance 570 nm. The pA2 values were
calculated by Schild regression analysis of the EC50 values.
In Vitro Autoradiography. Tissue slices for autoradiog-
raphy were prepared as described by Bonaventure et al.32
Briefly, rats were asphyxiated by carbon dioxide and decapi-
tated. The brains were removed and frozen on dry ice. Sections
(20 µM thickness) were cut using a cryostat-microtome (Mi-
crom HM505E, Microm International GmbH, Walldorf, Ger-
many) and thaw-mounted on adhesive microscope slides
(Superfrost+ Plus, VWR, West Chester, PA). The sections were
kept at -70 °C until further use, when they were incubated
for 10 min at room temperature in 0.5 × PBS with the
radioligand. The concentration of [3H]-13g was 45 nM. The
slides were then washed three times for 20 s with agitation in
1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-4-methyl-
piperazine (13n). Prepared according to Method B (94%). 1H
NMR (400 MHz) δ 7.19 (d, J ) 8.6 Hz, 2H), 6.83 (d, J ) 8.6
Hz, 2H), 4.26 (m, 1H), 3.42 (s, 2H), 2.81-2.70 (m, 3H), 2.55-
2.33 (m, 8H), 2.26 (s, 3H), 2.03-1.97 (m, 2H), 1.84-1.74 (m,
2H), 1.04 (d, J ) 6.6 Hz, 6H); 13C NMR (100, MHz) δ 156.5,
130.3, 130.1, 115.7, 73.0, 62.4, 55.1, 54.3, 52.9, 46.0, 45.7, 31.2,
18.4; MS m/z 332.3 (M + H+); Anal. (C20H33N3O) H, N; C:
calcd. 72.46; found 71.72.
1-[4-(1-Isopropyl-piperidin-4-yloxy)-benzyl]-4-phenyl-
piperazine (13o). Prepared according to Method B (35%). 1H
NMR (400 MHz) δ 7.30-7.22 (m, 4H), 6.65-6.82 (m, 5H), 4.29
(m, 1H), 3.50 (s, 2H), 3.19 (m, 2H), 3.16-3.12 (m, 2H), 2.83-
2.71 (m, 3H), 2.59 (m, 2H), 2.39 (m, 2H), 2.06-1.99 (m, 2H),
1.87-1.78 (m, 2H), 1.07 (d, J ) 6.6 Hz, 6H); 13C NMR (100
MHz) δ 156.6, 151.7, 151.3, 130.3, 129.0, 119.4, 116.0, 115.7,
72.9, 62.3, 52.9, 50.3, 49.0, 46.0, 31.1, 18.3; MS m/z 394.2
(M + H+); Anal. (C25H35N3O) H, N; C: calcd. 76.29; found
74.42.
1-Benzyl-4-[4-(1-isopropyl-piperidin-4-yloxy)-benzyl]-
piperazine (13p). Prepared according to Method B (77%). 1H
NMR (400 MHz) δ 7.31-7.29 (m, 4H), 7.25-7.23 (m, 1H), 7.18
(d, J ) 8.6 Hz, 2H) 6.84 (d, J ) 8.6 Hz, 2H), 4.26 (m, 1H), 3.50
(s, 2H), 3.43 (s, 2H), 2.82-2.70 (m, 3H), 2.54-2.34 (m, 9H),
2.03-1.96 (m, 2H), 1.84-1.75 (m, 2H), 1.06 (d, J ) 6.6 Hz,
6H); 13C NMR (100 MHz) δ 157.0, 138.5, 130.8, 130.5, 129.6,

4-Phenoxypiperidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2237
ice-cold 0.5 × PBS. After dipping in water, the slides were
dried and put on Fujifilm BAS TR2025 for 5 days prior to
developing. Nonspecific binding was determined in the pres-
ence of 100 µM histamine.
[3H]-13g (26 Ci/mmol) was prepared through a contract with
Sibtech (Newington, CT). Radiochemical purity 99.6% by
HPLC analysis with radioactive flow detection. Analysis was
performed using a 4.6 × 250 mm Vydac TP C18 column
operated at a flow rate of 1 mL/min at 20 °C with a gradient
of acetonitrile and 50 mM pH 3 phosphate buffer with
radioactive flow detection using a 0.5 mL flow cell and In-
Flow ES cocktail at 2 mL/min.
Statistical Analysis. EEG/EMG effects were analyzed
using a one-way or two-way ANOVA with time as a repeated-
measures variable (five levels corresponding to five 0.5 h
epochs: the first two epochs corresponding to the preinjection
portion and the last three epochs corresponding to the postin-
jection portion of the experiment) and, when appropriate,
treatment as a between-subjects variable. When ANOVA
indicated a statistically significant effect of time, treatment
or a significant treatment × time interaction (P < 0.05),
posthoc analyses were conducted using Tukey’s HSD test
(between-subject analyses) or means-comparison contrasts
(within-subject analyses).
In Vivo EEG. All animal work reported in this paper was
performed in accordance with the Declaration of Helsinki.
Supporting Information Available: Results from com-
bustion analysis and 1H NMR of 11a, 11f, 11k, 13a, 13d, 13e,
13i, 13l, 13n, 13o and 13p. This material is available free of
charge via the Internet at http://pubs.acs.org.
Animals and Surgery. Male Sprague-Dawley rats (Charles
River, Wilmington, MA) weighing 280-350 g were used in all
studies. Stereotaxic surgery was performed using halothane
anesthesia administered through a nose cone, with the incisor
bar set at 11.5 mm below ear bar zero and body temperature
was maintained at approximately 36 °C. Animals were housed
in pairs prior to and following surgery. The postsurgical
recovery time was 5-7 days. All procedures conformed to
university, U.S. Department of Agriculture, and National
Institutes of Health guidelines for the care and use of labora-
tory animals. Electroencephalography and electromyography
(EEG and EMG) electrodes were implanted as described below.
EEG/EMG electrodes were cemented in position with acrylic
cement (Plastics One, Roanoke, VA).
EEG and EMG Recording and Analysis. EEG and EMG
were recorded as described previously.33 Briefly, a bipolar EEG
electrode was implanted into the frontal cortex (A + 3.0; L (
1.5), two EMG electrodes were implanted into the neck muscle,
and a ground screw electrode was placed over the cerebellum.
The free ends of the EEG, EMG and ground electrodes were
inserted into a five-pin plastic connector that was cemented
in place, along with the cannula, using acrylic cement (Plastics
One). EEG and EMG signals were amplified, filtered (0.3-
50.0 Hz band-pass), and continuously recorded on a polygraph
and on VCR recording tape using a four-channel headstage
FET amplifier connected to standard EEG amplifiers.
On the day prior to testing, pairs of animals were weighed
and transferred to testing chambers where they were housed
individually.33 The animals had free access to food and water.
On the day of testing, animals were connected to an EEG/EMG
recording FET headstage. Testing was conducted between the
hours of 10:00 AM and 3:00 PM. Following 60-min of collection
of baseline data, animals were injected subcutaneously with
0.1, 1.0, 10.0 mg/kg 13g or vehicle. Recording of behavior and
EEG/EMG data was continued for the subsequent 120-min.
Each animal was tested with both doses of 13g. Testing
sessions were separated by 3-4 days. All compounds were
dissolved in artificial extracellular fluid (147 mM NaCl, 1.3
mM CaCl2, 0.9 mM MgCl2, 2.5 mM KCl, 5.0 mM NaH2P04,
pH 7.4).
EEG and EMG were manually scored for the following
behavioral state categories: (1) slow-wave sleep (high-voltage
EEG, low-voltage EMG); (2) REM sleep (low-voltage EEG
combined with EMG activity of approximately 50% lower
amplitude than that observed in slow-wave sleep, with oc-
casional short-duration, large-amplitude deflections due to
muscle twitches); (3) quiet waking (low-voltage EEG with EMG
activity of an average amplitude twice that observed in slow-
wave sleep); active-waking (low-voltage EEG, sustained high-
voltage EMG of approximately twice that observed in quiet
waking, with occasional movement deflections). To be scored
as a distinct epoch, the appropriate EEG and EMG activity
patterns needed to persist for a minimum of 15 s. The time
spent in each state was scored and totaled for the five 30-min
epochs of the observation period. These included the two
segments immediately prior to injection (PRE1: 0-30 min;
PRE2: 30-60 min) and the three segments that followed the
injections (POST1 ) 60-90 min; POST2 ) 90-120 min;
POST3 ) 120-150 min). All measurements were conducted
by observers blind to experimental conditions.
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