Diamine-based human histamine H3 receptor antagonists: (4-Aminobutyn-1-yl)benzylamines

Article abstract of DOI:10.1016/j.ejmech.2009.04.049

A series of (4-aminobutyn-1-yl)benzylamines were prepared and the SAR around three key areas: (1) the amine attached to the butynyl linker (R³R⁴N-); (2) the benzylamine moiety (R¹R²N-); and (3) the point of attachment of the benzylamine group (R¹R²N- in the ortho, meta, or para positions) was examined. One compound, 4-[3-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-morpholine (9s) was chosen for further profiling and found to be a selective histamine H₃ antagonist with desirable drug-like properties. Ex vivo receptor occupancy studies established that 9s does occupy H₃ binding sites in the brain of rats after oral administration. Subcutaneous doses of 9s (10 mg/kg) given during the natural sleep phase demonstrated robust wake-promoting effects.

Full text of DOI:10.1016/j.ejmech.2009.04.049

European Journal of Medicinal Chemistry 44 (2009) 4098–4106
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Diamine-based human histamine H3 receptor antagonists:
(4-Aminobutyn-1-yl)benzylamines
*
Curt A. Dvorak , Richard Apodaca, Wei Xiao, Jill A. Jablonowski, Pascal Bonaventure,
Christine Dugovic, Jonathan Shelton, Brian Lord, Kirsten Miller, Lisa K. Dvorak,
Timothy W. Lovenberg, Nicholas I. Carruthers
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of (4-aminobutyn-1-yl)benzylamines were prepared and the SAR around three key areas: (1) the
amine attached to the butynyl linker (R3R4N–); (2) the benzylamine moiety (R1R2N–); and (3) the point
of attachment of the benzylamine group (R1R2N– in the ortho, meta, or para positions) was examined.
One compound, 4-[3-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-morpholine (9s) was chosen for further
profiling and found to be a selective histamine H3 antagonist with desirable drug-like properties. Ex vivo
receptor occupancy studies established that 9s does occupy H3 binding sites in the brain of rats after oral
administration. Subcutaneous doses of 9s (10 mg/kg) given during the natural sleep phase demonstrated
robust wake-promoting effects.
Received 16 January 2009
Received in revised form
10 April 2009
Accepted 30 April 2009
Available online 12 May 2009
Keywords:
Alkynes
Diamine H3 antagonists
Wake promoting
Pro-cognitive agents
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
capacity as a heteroreceptor [6], the H3 receptor also mediates the
release of several other neurotransmitters [7] including dopamine,
Since its initial discovery, the histamine H3 receptor [1] has
attracted the attention of numerous research groups exploring the
receptors’ pharmacology thus beginning the search for potent and
selective ligands. The successful cloning of the human H3 receptor
[2], and the design of selective ligands lacking the imidazole ring
found in the endogenous ligand histamine itself were significant
milestones in the H3 field [3]. Increasingly, medicinal chemistry
efforts are now focused on the refinement of these non-imidazole
based ligands thereby improving the physical–chemical properties
of the compounds [4]. This refinement is in part to address the next
key hurdle for histamine H3 ligands, human clinical trials, which
will ultimately determine their potential therapeutic value for the
treatment of human diseases.
noradrenaline, GABA, serotonin, and acetylcholine [8]. These effects
on neurotransmission have led to numerous suggestions for ther-
apeutic applications for H3 ligands including Parkinson’s and Alz-
heimer’s diseases, schizophrenia, obesity, cognition, learning and
sleep disorders [9]. Other clinical uses have been suggested due to
the presence of H3 receptors outside the CNS, for instance in the
heart, where they are involved in the regulation of noradrenaline
release. Thus, H3 agonists might be potentially useful in the treat-
ment of ischemic heart conditions [10]. Indeed, compared with the
marketed successes of compounds targeting histamine H1 and H2
receptors, the potential of histamine H3 receptor inverse agonists,
antagonists or agonists still remains to be determined [11].
We previously reported that several members of a series of
simple diamines 1 possessed high potency at the human histamine
H3 receptor (Chart 1) [12]. Structure–activity trends of these rela-
tively simple diamines 1 suggested a minimal H3 receptor phar-
macophore could be deduced consisting of a lipophilic core flanked
by two alkylamine groups. This conclusion is further reinforced by
the high potency of the marine natural product Aplysamine 4 [13]
and other more rigid diamines recently appearing in the literature
(e.g. 2 [14] and 3 [15]). In an effort to further improve our diamine
H3 pharmacophore model, and potentially enhancing the drug-like
properties of the molecules, we considered adding a conforma-
tionally restricted side-chain to replace the propyloxy group linking
The roles of histamine as a neurotransmitter and the H3 receptor
in the central nervous system (CNS) are now becoming quite clear.
The synthesis and release of histamine are regulated in the CNS by
H3 pre-synaptic autoreceptors through
a negative-feedback
mechanism [5]. Thus, H3 inverse agonists or antagonists would
increase the amount of histamine available in the synapse. In its
* Corresponding author. Tel.: þ1 858 784 3284; fax: þ1 858 784 3116.
E-mail address: cdvorak@its.jnj.com (C.A. Dvorak).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.049

C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
4099
R4
R4 R3
N
O
N
O
N
N
R4
N
R3
O
N
R3
O
N
N
R1
R1
N
R1
N
R2
N
N
R2
R2
1
2
3
7
8
9
pA2 = 9.84
pK = 9.7
pK = 9.47
i
i
Fig. 1. Design of (4-aminobutyn-1-yl)benzylamines.
N
O
N
utilized in the preparation of (4-aminobutyn-1-yl)benzylamines 9
and are shown in Fig. 2. Therefore, in order to explore the influence
of the amine attached to the butyne (R3R4N–), commercially
available 3-butynyl p-toluenesulfonate was treated with various
amines giving the corresponding 4-aminobutynes 10. The 3-
butynyl amines were then coupled to the appropriate appended
bromobenzyl amine fragments under Sonogashira conditions with
PdCl2(PPh3)2 and CuI to provide compounds 9a–g (Scheme 1).
The remaining compounds were synthesized by coupling 3-
butynyl p-toluenesulfonate to either o-, m- or p-bromobenzalde-
hyde with modified Sonogashira conditions developed by Fu et al.
[20] utilizing PdCl2(PhCN)2, CuI, and t-Bu3P at ambient tempera-
ture. The corresponding tosylates were then treated with piperi-
dine in DMF in the presence of Na2CO3 thus providing
benzaldehydes 15. The benzaldehydes 15 were then treated with
a diverse set of amines and sodium triacetoxyborohydride [21] to
install the benzylamine moieties to provide additional target
compounds 9h–w (Scheme 2).
O
N
Br
Br
N
N
N
N
N
4
5
6
pK = 7.52
pA2 = 8.68
pK = 8.2
i
i
Chart 1. Representative non-imidazole antagonists with activities at the cloned
recombinant human histamine H3 receptor.
the NR3R4 amine and the aryl ring found in 8. This resulted in
a series of aryloxypiperidines [16] (7) that utilized a hydroxy
piperidine attached to the core aryl ring thereby reducing the
number of rotatable bonds found in the structures [17]. Continued
refinement to our ligand design has led us to consider the use of
a butyne element as another conformationally restricted side-chain
replacement for the propyloxy moiety to give structures of type 9
providing a more rigid core (Fig. 1).
The use of alkynes as structural elements within non-imidazole
H3 receptor ligands has received relatively little attention. We
previously reported nearly identical human H3 receptor affinities of
imidazopyridines 5 and 6 [18], suggesting that the structural
changes in the manner we have envisioned may not adversely
affect the binding affinity. Subsequently, Stark et al. reported that
phenylalkyne 16 (Table 1) was a weak H3 receptor antagonist,
although some analogues showed improved potency [19]. Thus, we
set out to examine the structure–activity relationships (SAR) of
a series of diamines that utilize a 4-amino butynyl element as
a potential 3-amino propyloxy replacement.
3. Pharmacological results and discussion
The human histamine H3 receptor binding affinities for a series
of (4-aminobutyn-1-yl)benzylamines and related compounds were
determined. The functional activities were also obtained for
selected analogues. Compounds were selected for their ability to
elucidate structure–activity relationships around three key struc-
tural areas of the molecules as mentioned above: (1) the butynyl-
amine group (R3R4N–); (2) the benzylamine group (R1R2N–); and
(3) the point of attachment of the benzylamine group (R1R2N– in
the ortho, meta, or para positions). In addition to determining these
structural modifications on potency and SAR trends, ultimately the
physical–chemical properties and any positive effects on the
pharmacokinetic profile were examined.
Initially holding the benzylamine group (R1R2N–) constant as
piperidinyl, compounds bearing cyclic and lipophilic –NR3R4
groups showed the highest binding affinity. Thus, binding affinity
increased slightly over the series of diethylamino, pyrrolidinyl and
piperidinyl (9a–c). Similarly, replacing a carbon atom in the
piperidine ring of the –NR3R4 group with a heteroatom led to a
2. Chemistry
The assembly of the compounds was designed to be modular,
flexible and adaptable for a parallel format whenever possible in
order to quickly elucidate the SAR. Two major disconnections were
R4
R4
N
R3
N
R3
Br
R3
i
N
R1
ii
R4
+
+
NH
R2
R1
R1
N
CHO
N
R2
R2
9
10
11
12
13
Fig. 2. Retrosynthesis of (4-aminobutyn-1-yl)benzylamines 9.

4100
C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
R3
allow the two basic amine moieties to obtain similar orientations.
N
Structure–activity trends of diamines 7, 8 and 9 further reinforce
the proposed minimal H3 receptor pharmacophore model con-
sisting of a lipophilic core flanked by two alkylamine groups.
Selected compounds were evaluated in a whole cell-based
model of receptor activation. All were found to be neutral antago-
nists with good correlation between the observed binding affinities
and functional activities. The compounds were triaged for possible
further profiling by receptor occupancy experiments in rodents (sc
or po). This useful and powerful tool provides plasma and brain
exposures and receptor occupancy over time as related to dose.
Given our experience with related structures, the importance in
shorter acting compounds to compliment 1 was desired for addi-
tional first-in-class CNS clinical candidates. As reported earlier in
a review of our H3 program, 9s (JNJ 10181457) is cleared from the
brain in rats within 24 h following a 10 mg/kg dose [22]. Full dose–
response ex vivo autoradiography of 9s was performed using [3H]-
R4
Br
R3
a
N
R4
+
N
N
9a-g
14
10
Scheme 1. Reagents and conditions: (a) PdCl2(PPh3)2, CuI, PPh3, Et2NH, DMF, 125 ꢁC.
10-fold decrease in binding affinity across the series piperidino-
(9c), thiomorpholino- (9d), morpholino- (9e) and methylpiper-
azinyl (9f). This drop in binding affinity correlates well with the
lipophilic nature (clog P) of the –NR3R4 fragments as well as the
corresponding pKa’s of the amines.
R-a-methylhistamine [23] as the radioligand. Dose-dependency of
To estimate the ligand binding contribution of the benzylamine
group (–CH2NR1R2) itself, the binding affinities of bispiperidine 9c
and monopiperidine 16 were compared. This analysis revealed
diamine 9c bound with higher affinity to the human histamine H3
receptor by over two orders of magnitude. Additional modifications
of the benzylamine group (–NR1R2) in the series of 4-(1-piper-
idinly)-butynes revealed a high degree of functional group toler-
ance. Most compounds displayed high H3 receptor affinity,
particularly piperidine and pyrrolidine 9c and 9j, N-methylpiper-
azine 9l, and carboxamide 9p. Functional groups tolerated include
secondary amines (9l and 9m), hydroxyl groups (9q) and primary
amides (9p). The marked increase in potency combined with
tolerance for a wide variety of functionality in this region (–NR1R2)
represents a key area allowing for the selection of optimized
physical properties (log P, amine pKa, TPSA, etc.)
the H3 receptor occupancy 1 h after oral administration of 9s (0.1,
0.3, 1, 3, 10, 30 mg/kg) is presented in Fig. 3. Maximal ex vivo
occupancy of the H3 receptor was observed at 30 mg/kg at 1 h post-
dose with an ED50 for occupancy being w6.5 mg/kg.
Compound 9s was selected for more detailed evaluation. The
potency of 9s was found to be slightly less at the rat H3 receptor vs
the human (pKi 8.15 rH3 vs 8.93 hH3). The selectivity of 9s over the
H1 and H4 receptors is very high with pKi’s of <5. Overall selectivity
against a panel of 50 neurotransmitter receptors, ion channels and
other drug targets indicated that 9s binds specifically to the H3
receptor (IC50’s of >1
effects on human hERG channel with an activity of 13.7% and 8.3%
at 10 and 3 M respectively and displayed high non-efflux limited
mM at all targets). In addition, 9s had minimal
m
absorption potential (Caco-2). Moreover, 9s has minimal drug–drug
interaction (DDI) potential having no effect on Cpy450’s 1A2, 2C9,
A series of compounds differing by virtue of the position of the
benzylamine substituent (–CH2NR1R2) on the aromatic ring were
also examined. Positional isomers of bispiperidine 9c exhibited
decreasing binding affinity in the order para (9c), meta (9r), then
ortho (9w), although the meta and para isomers were nearly equi-
potent. Additional meta substituted phenylalkynes were prepared
to further explore the binding affinities of meta substituted phe-
nylalkynes. Thus, both the meta and para carboxamides (9p and 9u)
bound with nearly equal affinity and the methylpiperazinyl
analogues (9t vs 9l) slightly favored the para orientation. The pyr-
rolidine 9v, piperidine 9r and morpholine 9s meta analogues all had
equivalent binding affinity with a preference over the para isomer.
In addition, a 10-fold loss in activity was observed in both the meta
and para analogues when the butynylamine group (R3R4N–) was
changed from piperidine to morpholine (9s and 9e). Overall, both
the para and meta benzylamine isomers of the 4-(1-piperidinly)-
butynes series were determined to be highly potent ligands at the
human H3 receptor. The SAR of the butyne linker on potency at the
human H3 receptor was comparable to both the propyloxy chain
linking the NR3R4 group and the central aryl ring as well as the 4-
aryloxypiperidines suggesting that the different scaffolds may
2C19, 2D6 and 3A4 at levels >40 mM. Plasma protein binding was
also determined and 9s displayed 45% binding to human plasma
proteins. The oral bioavailability (%F) of 9s was determined to be
11% in the rat. The potency of 9s at the rat receptor was considered
to be sufficient to explore animal models of efficacy (pA2
rH3 ¼ 8.33 ꢀ 0.08) (Table 2).
Histaminergic neurons, which originate in the tuber-
omammillary nuclei and project throughout the brain, play an
important role in arousal mechanisms, and histamine H3 receptors
N
N
a
R1
NH
+
R2
R1
OHC
N
R2
Fig. 3. Ex vivo occupancy of H3 receptor by 9s in rat striatum (coronal section): dose-
dependency after oral administration (1 h post-dose). Results are represented as mean
value of n ¼ 2.
15
13
9h-w
Scheme 2. Reagents and conditions: (a) NaBH(OAc)3, (R1)R2NH, CH2Cl2.

C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
4101
Table 1
Table 1 (continued)
In vitro human histamine H3 receptor binding affinities.
Compds (9)
NR1R2
NR3R4
Isomer (o, m, p)
pKi ꢀ SEMa, b
8.72 ꢀ 0.04
R4
N
n
p
N
N
N
N
R3
N
o
p
p
p
8.46 ꢀ 0.22
9.12 ꢀ 0.02
N
N
R1
N
R2
16
9a-w
N
Compds (9)
NR1R2
NR3R4
Isomer (o, m, p)
pKi ꢀ SEMa, b
8.87 ꢀ 0.18
H2N
a
p
O
N
N
q
p
9.05 ꢀ 0.03
N
N
b
c
p
p
9.17 ꢀ 0.02
9.29 ꢀ 0.19
N
N
N
HO
N
N
r
s
t
m
m
m
m
9.11 ꢀ 0.06
8.93 ꢀ 0.16
9.14 ꢀ 0.09
9.26 ꢀ 0.09
N
N
N
N
N
d
e
f
p
8.42 ꢀ 0.23
8.30 ꢀ 0.05
8.28 ꢀ 0.05
7.84 ꢀ 0.23
N
N
N
O
S
p
N
N
N
N
N
O
u
p
N
H2N
N
N
O
g
m
v
m
o
7.09 ꢀ 0.09
7.08 ꢀ 0.09
N
N
N
O
h
i
p
p
p
p
p
p
9.04 ꢀ 0.01
8.96 ꢀ 0.01
9.21 ꢀ 0.06
8.76 ꢀ 0.03
9.01 ꢀ 0.10
9.06 ꢀ 0.03
w
N
N
N
N
N
N
N
N
N
16
–
–
–
7.25 ꢀ 0.06
7.50 ꢀ 0.10
N
Thioperamide
Displacement of N-[3H]-methylhistamine from human H3 receptors expressed
in SK-N-MC cells.
a
b
j
Values reported as the mean of at least three determinations.
N
N
k
l
Table 2
In vitro human histamine H3 receptor functional activities of selected compounds.
O
N
Compds
pA2 ꢀ SEMa, b
9c
9k
9r
9s
9s
9.83 ꢀ 0.06
8.49 ꢀ 0.05
10.09 ꢀ 0.07
9.31 ꢀ 0.08
8.33 ꢀ 0.08c
N
m
a
pA2 values are derived from Schild regression analysis of the compound-induced
rightward shifts in dose–response curves of histamine-induced inhibition of for-
skolin-stimulated cAMP accumulation in SK-N-MC cells overexpressing the human
histamine H3 receptor.
N
N
b
Values reported as the mean of at least three determinations.
Functional activity at the rat H3 receptor.
c

4102
C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
Wake Minutes
Wake Increment
100
75
50
25
0
300
200
100
0
**
Vehicle
9s
0
1
2
3
4
Time Interval (h)
5
6
7
8
Vehicle
9s
Wake Minutes
Wake Increment
100
75
50
25
0
***
300
200
100
0
Vehicle
Thioperamide
0
1
2
3
Time Interval (h)
4
5
6
7
8
Vehicle
Thioperamide
Fig. 4. Wake-promoting effects of 9s and thioperamide in rats. Male adult Sprague-Dawley rats chronically implanted with EEG/EMG electrodes received the compound (10 mg/kg
s.c.) or vehicle (saline) 2 h after the onset of the light phase. Polysomnographic traces were recorded for 8 h following the treatment and were visually scored by 10 s epoch as wake,
non-rapid eye movement (NREM) or rapid eye movement (REM) sleep. The increased time spent awake was analyzed for each rat per 1 h intervals and expressed as percentage of
recording time. In addition, the increment in wake was averaged over the 8-h recording period and expressed in minutes. Data are reported as mean ꢀ SEM (n ¼ 5 animals per
compound), and statistical significance was determined through comparison of vehicle vs compound treatment by paired t-test (**p < 0.01, ***p < 0.001).
are involved in this process by an inhibitory control of histamine
release and synthesis. Indeed, the pharmacological blockade of pre-
synaptic H3 receptors promotes wakefulness in different animal
models [22]. The development of specific H3 receptor antagonists
has therefore become a very attractive strategy for the treatment of
daytime sleepiness associated with various conditions (narcolepsy,
obstructive sleep apnea and shift work sleep disorders). Fig. 4
illustrates the wake-promoting effects of 9s in comparison with
thioperamide in rats after subcutaneous administration. Both
compounds significantly increased the time spent awake with
a similar duration of 7 h after injection. Concomitantly, the duration
of non-rapid eye movement (NREM) sleep was decreased whereas
REM sleep was not altered (data not shown).
extent beyond 24 h, with brain concentrations that roughly parallel
plasma concentrations.
5. Experimental protocols
5.1. Chemistry
Reagents were purchased from commercial suppliers and were
used without purification. Anhydrous solvents were obtained from
a Glass Contour Solvent Dispensing System. Reactions were per-
formed at room temperature (20–23 ꢁC) under an atmosphere of N2
unless otherwise noted. Chromatography was performed using
prepacked ISCO RediSepÔ silica cartridges utilizing gradient
elution. 1H and 13C NMR spectra were recorded on a Bruker 400 or
500 MHz spectrometer. Chemical shifts are reported in parts per
million downfield from an internal standard (Me4Si). All spectra
were taken in CDCl3 unless otherwise noted. Mass spectra were
obtained on an Agilent series 1100 MSD using electrospray ioni-
zation (ESI) in either positive or negative modes as indicated. Thin-
layer chromatography was performed using Merck silica gel 60 F254
4. Conclusion
A series of (4-aminobutyn-1-yl)benzylamines were prepared
and the SAR around three key areas: (1) the amine attached to the
butynyl linker (R3R4N–); (2) the benzylamine moiety (R1R2N–); and
(3) the point of attachment of the benzylamine group (R1R2N– in
the ortho, meta, or para positions) was examined. One compound,
4-[3-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-morpholine (9s) was
selected for further profiling and found to be a selective histamine
H3 antagonist that promotes wakefulness in rats as compared to
thioperamide. The compound distinguishes itself from previous
compounds mainly by its pharmacokinetic profile such that it has
a shorter plasma half-life (1.5 h) after single oral dose administra-
tion in rats. In addition, although 9s readily penetrates the blood–
brain barrier, it does not persist in the brain to an appreciable
2.5 cm ꢂ 7.5 cm, 250
mm or 5.0 cm ꢂ 10.0 cm, 250
mm pre-coated
silica gel plates. Preparative thin-layer chromatography was per-
formed using EM Science silica gel 60 F254 20 cm ꢂ 20 cm 0.5 mm
pre-coated plates with a 20 cm ꢂ 4 cm concentrating zone. Melting
points are uncorrected and were obtained on a MelTemp apparatus.
Analytical reverse phase HPLC was performed on a Hewlett Packard
Series 1100 instrument with an Agilent ZORBAXÒ Bonus RP, column
utilizing an acetonitrile/water (0.05%TFA) gradient. Combustion
analyses were performed by Desert Analytics or NuMega Reso-
nance Labs.

C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
4103
1
5.1.1. Method A. General procedure for the preparation
desired aldehyde. H NMR (500 MHz)
d
9.98 (s, 1H), 7.81–7.78 (m,
of 3-butynyl amines
2H), 7.54–7.51 (m, 2H), 2.69–2.61 (m, 4H), 2.51–2.43 (m, 4H), 1.63–
5.1.1.1. 4-But-3-ynyl-morpholine (10a). To a solution of 3-butynyl p-
toluenesulfonate (4.8 g, 22 mmol) in DMF (25 mL) were added
sodium carbonate (2.6 g, 24 mmol) and morpholine (2.1 mL,
24 mmol) and the reaction mixture was heated to 80 ꢁC. After 16 h,
the reaction was quenched with water and ether was added. The
organic portion was washed with brine, dried over Na2SO4 and
concentrated to provide 1.6 g (51%) of 4-but-3-ynyl-morpholine. 1H
1.57 (m, 4H), 1.48–1.42 (m, 2H); 13C NMR (125 MHz)
d
191.4, 135.0,
132.0,130.3,129.4, 93.4, 80.7, 57.7, 54.2, 25.9, 24.2,17.8; MS m/z 242.2
(M þ H).
5.1.1.9. Toluene-4-sulfonic acid 4-(3-formyl-phenyl)-but-3-ynyl ester.
To a solution of 3-bromobenzaldehyde (2.80 g, 15.1 mmol) in
dioxane (15 mL) were added 3-butynyl p-toluenesulfonate (3.1 mL,
17.1 mmol), iPr2NH (3.0 mL) and the flask was flushed with
nitrogen for ca. 10 min. The solid reagents, PdCl2(PhCN)2 (140.3 mg,
0.37 mmol), CuI (43.8 mg, 0.23 mmol), and P(t-Bu)3$HBF4
(210.0 mg, 0.72 mmol) were then added together in one portion.
The mixture was stirred at rt for 16 h. A solid ppt. was observed
after initial mild exotherm at ca. 5 min. The dark brown mixture
was then diluted with CH2Cl2 (400 mL) and washed with water. The
organic phase was dried over sodium sulfate and evaporated.
Chromatography of the residue (0–15% EtOAc/Hexanes) provided
NMR (CDCl3, 500 MHz)
d
3.69 (t, J ¼ 4.7 Hz, 4H), 2.56 (dd, J ¼ 7.8,
7.1 Hz, 2H), 2.46 (br t, J ¼ 4.4 Hz, 4H), 2.36 (dt, J ¼ 2.6, 7.5 Hz, 2H),
1.97 (t, J ¼ 2.6 Hz, 1H); MS m/z 140.2 (M þ H).
5.1.1.2. 1-But-3-ynyl-piperidine (10b). Prepared according to
method A (48%). 1H NMR (CDCl3, 500 MHz)
d
2.56 (dd, J ¼ 8.1,
7.4 Hz, 2H), 2.44–2.34 (m, 6H), 1.96 (t, J ¼ 2.6 Hz, 1H), 1.61–1.54 (m,
4H), 1.46–1.38 (m, 2H); MS m/z 138.2 (M þ H).
5.1.1.3. 4-But-3-ynyl-thiomorpholine (10c). Prepared according to
3.37 g (68%) of the desired tosylate. 1H NMR (500 MHz)
d 9.98 (s,
method A (74%). 1H NMR (CDCl3, 500 MHz)
d
2.79–2.73 (m, 4H),
1H), 7.85–7.79 (m, 4H), 4.60–7.57 (m, 1H), 7.46 (t, J ¼ 7.6 Hz, 1H),
2.68–2.65 (m, 4H), 2.63 (t, J ¼ 7.7 Hz, 2H), 2.35 (dt, J ¼ 2.2, 7.7 Hz,
7.33 (d, J ¼ 7.6 Hz, 2H), 4.20 (t, J ¼ 7.1 Hz, 2H), 2.81 (t, J ¼ 7.1 Hz, 2H),
2H), 2.00 (t, J ¼ 2.6 Hz, 1H); MS m/z 156.2 (M þ H).
2.42 (s, 3H); 13C NMR (125 MHz)
d 191.4, 144.9, 137.1, 136.3, 132.8,
129.8, 128.9, 127.9, 124.1, 85.7, 81.3, 67.4, 21.6, 20.3; MS m/z 329.2
(M þ H).
5.1.1.4. 1-But-3-ynyl-4-methyl-piperazine (10d). Prepared accord-
ing to method A (12%). 1H NMR (CDCl3, 500 MHz)
d 2.62 (t,
J ¼ 7.4 Hz, 3H), 2.59–2.43 (m, 6H), 2.39 (dt, J ¼ 2.6, 8.1 Hz, 3H), 2.30
5.1.1.10. 3-(4-Piperidin-1-yl-but-1-ynyl)-benzaldehyde (15a). To
a solution of toluene-4-sulfonic acid 4-(3-formyl-phenyl)-but-3-
ynyl ester (3.35 g, 10.2 mmol) in DMF (5 mL) were added piperidine
(2.2 mL, 22 mmol) and Na2CO3 (2.36 g, 22 mmol). The mixture then
placed into a preheated oil bath (80 ꢁC) and heated for ca. 2 h. The
mixture was then diluted with water (400 mL) and extracted with
Et2O (3 ꢂ 100 mL). The combined organic phases were dried over
magnesium sulfate and evaporated. Chromatography of the residue
(0–5% 2 M methanolic ammonia/CH2Cl2) provided 1.64 g (67%) of
(s, 3H), 1.99 (t, J ¼ 2.7 Hz, 1H); MS m/z 153.2 (M þ H).
5.1.1.5. But-3-ynyl-diethyl-amine (10e). Prepared according to
method A (14%). 1H NMR (CDCl3, 500 MHz)
d
2.70 (t, J ¼ 7.4 Hz, 2H),
2.54 (q, J ¼ 7.2 Hz, 4H), 2.31 (dt, J ¼ 2.7, 8.0 Hz, 2H), 1.96 (t,
J ¼ 2.6 Hz, 1H), 1.04 (t, J ¼ 7.1 Hz, 6H); MS m/z 126.2 (M þ H).
5.1.1.6. 1-But-3-ynyl-pyrrolidine (10f). Prepared according to
method A (28%). 1H NMR (CDCl3, 500 MHz)
2.55–2.48 (m, 4H), 2.39 (dt, J ¼ 2.7, 7.4 Hz, 2H), 1.97 (t, J ¼ 2.6 Hz,
1H), 1.81–1.74 (m, 4H); MS m/z 124.2 (M þ H).
d
2.65 (t, J ¼ 7.6 Hz, 2H),
the desired aldehyde. 1H NMR (500 MHz)
d 9.98 (s, 1H), 7.88 (s, 1H),
7.78 (d, J ¼ 7.7 Hz, 1H), 7.63 (d, J ¼ 7.7 Hz, 1H), 7.45 (t, J ¼ 7.7 Hz, 1H),
2.69–2.59 (m, 4H), 2.51–2.43 (m, 4H), 1.63–1.57 (m, 4H), 1.48–1.42
(m, 2H); 13C NMR (125 MHz)
d 191.6, 137.1, 136.3, 132.9, 128.9, 128.3,
5.1.1.7. Toluene-4-sulfonic acid 4-(4-formyl-phenyl)-but-3-ynyl ester.
To a solution of 4-bromobenzaldehyde (2.80 g, 15.1 mmol) in
dioxane (15 mL) were added 3-butynyl p-toluenesulfonate (3.1 mL,
17.1 mmol), iPr2NH (3.0 mL) and the flask was flushed with
nitrogen for ca. 10 min. The solid reagents, PdCl2(PhCN)2 (141.5 mg,
0.37 mmol), CuI (45.6 mg, 0.24 mmol), and P(t-Bu)3$HBF4
(211.1 mg, 0.73 mmol) were then added together in one portion.
The mixture was stirred at rt for 16 h. A solid ppt. was observed
after initial mild exotherm at ca. 5 min. The dark brown mixture
was then diluted with CH2Cl2 (400 mL) and washed with water. The
organic phase was dried over sodium sulfate and evaporated.
Chromatography of the residue (0–15% EtOAc/Hexanes) provided
125.0, 90.5, 79.9, 57.8, 54.2, 25.9, 24.2, 17.6; MS m/z 242.2 (M þ H).
5.1.1.11. Toluene-4-sulfonic acid 4-(2-formyl-phenyl)-but-3-ynyl ester.
To a solution of 2-bromobenzaldehyde (0.55 g, 2.9 mmol) in
dioxane (3 mL) were added 3-butynyl p-toluenesulfonate (0.6 mL,
3.4 mmol), iPr2NH (0.6 mL) and the flask was flushed with nitrogen
for ca. 10 min. The solid reagents, PdCl2(PhCN)2 (37.1 mg,
0.09 mmol), CuI (13.6 mg, 0.07 mmol), and P(t-Bu)3$HBF4 (57.1 mg,
0.19 mmol) were then added together in one portion. The mixture
was stirred at rt for 16 h. A solid ppt. was observed after initial mild
exotherm at ca. 5 min. The dark brown mixture was then diluted
with CH2Cl2 (400 mL) and washed with water. The organic phase
was dried over sodium sulfate and evaporated. Chromatography of
the residue (0–15% EtOAc/Hexanes) provided 0.52 g (54%) of the
3.65 g (73%) of the desired tosylate. 1H NMR (500 MHz)
d 10.0 (s,
1H), 7.84–7.79 (m, 4H), 7.48 (d, J ¼ 8.2 Hz, 2H), 7.32 (d, J ¼ 8.2 Hz,
2H), 4.20 (t, J ¼ 7.1 Hz, 2H), 2.82 (t, J ¼ 7.1 Hz, 2H), 2.43 (s, 3H); 13C
desired tosylate. 1H NMR (500 MHz)
d 10.36 (s, 1H), 7.88 (d,
NMR (125 MHz)
d
191.3, 144.9, 135.4, 132.9, 132.2, 129.8, 129.4,
J ¼ 6.6 Hz, 1H), 7.82 (d, J ¼ 8.2 Hz, 2H), 7.55–7.51 (m, 1H), 7.48–7.40
129.2, 127.9, 88.3, 81.9, 67.3, 21.6, 20.5; MS m/z 329.2 (M þ H).
(m, 2H), 7.31 (d, J ¼ 7.7 Hz, 2H), 4.23 (t, J ¼ 6.6 Hz, 2H), 2.87 (t,
J ¼ 6.6 Hz, 2H), 2.40 (s, 3H); 13C NMR (125 MHz)
d 191.4, 145.0,
5.1.1.8. 4-(4-Piperidin-1-yl-but-1-ynyl)-benzaldehyde (14). To a solu-
tion of toluene-4-sulfonic acid 4-(4-formyl-phenyl)-but-3-ynyl ester
(3.62 g, 11.0 mmol) in DMF (5 mL) were added piperidine (2.2 mL,
22 mmol) and Na2CO3 (2.41 g, 23 mmol). The mixture was placed
into a preheated oil bath (80 ꢁC) and heated for ca. 2 h. The mixture
was then diluted with water (400 mL) and extracted with Et2O
(3 ꢂ 100 mL). The combined organic phases were dried over sodium
sulfate and evaporated. Chromatography of the residue (0–5%
2 M methanolic ammonia/CH2Cl2) provided 1.35 g (51%) of the
136.0, 133.6, 133.4, 132.8, 129.9, 128.5, 127.9, 127.1, 126.5, 91.4, 78.4,
67.3, 21.6, 20.6; MS m/z 329.2 (M þ H).
5.1.1.12. 2-(4-Piperidin-1-yl-but-1-ynyl)-benzaldehyde (15b). To
a solution of toluene-4-sulfonic acid 4-(2-formyl-phenyl)-but-3-
ynyl ester (0.39 g, 1.2 mmol) in DMF (2 mL) were added piperidine
(0.5 mL, 5 mmol) and Na2CO3 (0.25 g, 2.4 mmol). The mixture then
placed into a preheated oil bath (80 ꢁC) and heated for ca. 12 h. The
mixture was then diluted with water (50 mL) and extracted with

4104
C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
Et2O (3 ꢂ 10 mL). The combined organic phases were dried over
magnesium sulfate and evaporated. Chromatography of the residue
(0–5% 2 M methanolic ammonia/CH2Cl2) provided 0.13 g (45%) of
(d, J ¼ 11.8 Hz, 2H), 3.20–3.06 (m, 7H), 3.06–2.97 (m, 2H), 2.63 (t,
J ¼ 12.6 Hz, 2H), 2.17–2.01 (m, 2H), 1.88–1.75 (m, 3H), 1.40–1.25 (m,
1H); 13C NMR (CD3OD, 125 MHz)
d 132.4, 131.6, 128.5, 124.6, 85.9,
the desired aldehyde. 1H NMR (500 MHz)
d
10.54 (s, 1H), 7.88 (d,
82.7, 63.7, 60.3, 55.8, 52.9, 22.7, 22.0, 15.3; MS m/z 313.3 (M þ H);
J ¼ 7.7 Hz, 1H), 7.53–7.47 (m, 2H), 7.40–7.36 (m, 1H), 2.67 (s, 4H),
Anal. (C20H30Cl2N2O) C, H, N.
2.51–2.42 (m, 4H), 1.64–1.57 (m, 4H), 1.47–1.41 (m, 2H); 13C NMR
(125 MHz)
d
192.2, 136.0, 133.6, 133.1, 127.9, 127.6, 126.9, 96.3, 57.6,
5.1.2.6. 1-Methyl-4-[4-(4-piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-
54.2, 25.9, 24.2, 17.9; MS m/z 242.2 (M þ H).
piperazine (9f). Prepared according to method B (63%). 1H NMR
(CD3OD, 500 MHz)
d
7.53 (d, J ¼ 8.3 Hz, 2H), 7.47 (d, J ¼ 8.3 Hz, 2H)
5.1.2. Method B. General procedure for the Sonogashira coupling
5.1.2.1. Diethyl-[4-(4-piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-amine
(9a). But-3-ynyl-diethyl-amine (300 mg, 2.4 mmol) was combined
in a sealed tube with 1-(4-bromo-benzyl)-piperidine (300 mg,
1.2 mmol), triphenylphosphine (56 mg, 0.21 mmol), copper (I)
iodide (11 mg, 0.06 mmol), dicholordiphenylphosphorylpalladium
(41 mg, 0.06 mmol) in a mixture of DMF (0.5 mL) and diethylamine
(2.5 mL). The reaction mixture was heated to 125 ꢁC for 1 h. After
cooling to room temperature, ether was added and all solids were
filtered off. The filtrate was concentrated and purified by RP-HPLC
to provide 221 mg (63%) of diethyl-[4-(4-piperidin-1-ylmethyl-
phenyl)-but-3-ynyl]-amine. The dihydrochloride salt was prepared
by addition of 2 mL of 1 M HCl in ether to the above compound in
methanol (7 mL). After 10 min a white precipitate was observed.
The solid was filtered off and rinsed with ether to provide 179 mg
(65%) of the dihydrochloride salt of diethyl-[4-(4-piperidin-1-
ylmethyl-phenyl)-but-3-ynyl]-amine 1H NMR (CD3OD, 500 MHz)
4.17–4.03 (m, 10H), 3.88–3.75 (m, 6H), 3.75–3.63 (m, 2H), 3.47 (dd,
J ¼ 7.2, 7.0 Hz, 2H), 3.43–3.36 (m, 2H), 3.08 (dd, J ¼ 7.2, 7.0, 2H), 2.95
(s, 3H), 2.74 (dt, J ¼ 2.0, 12.3 Hz, 2H), 2.09–1.94 (m, 2H), 1.91–1.79
(m, 3H), 1.46–1.33 (m, 1H); 13C NMR (CD3OD, 125 MHz)
d 132.4,
131.4, 128.5, 124.5, 85.2, 82.8, 60.1, 55.1, 52.7, 49.9, 48.4, 22.6, 21.7,
15.4; MS m/z 326.3 (M þ H); Anal. (C21H34Cl3N3) C, H, N.
5.1.2.7. 4-[4-(3-Piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-morpho-
line (9g). Prepared according to method B (51%). 1H NMR (CD3OD,
500 MHz)
d
7.78 (s, 1H), 7.53 (d, J ¼ 7.3 Hz, 1H), 7.48 (d, J ¼ 7.8 Hz,
1H), 7.40 (t, J ¼ 7.7 Hz, 1H), 4.22 (s, 2H), 4.12–3.97 (m, 4H), 3.59 (d,
J ¼ 12.3 Hz, 2H), 3.46–3.36 (m, 4H), 3.28–3.18 (m, 2H), 3.08 (t,
J ¼ 6.8 Hz, 2H), 2.96–2.85 (m, 2H), 2.01–1.81 (m, 5H), 1.55–1.40
(m, 1H); 13C NMR (CD3OD, 125 MHz)
d 134.5, 132.9, 131.2, 129.2,
129.1, 123.8, 123.8, 85.0, 85.5, 63.6, 60.0, 55.4, 52.8, 52.1, 22.7, 21.6,
15.0; MS m/z 313.3 (M þ H); Anal. (C20H30Cl2N2O) C, H, N.
d
7.55 (d, J ¼ 8.2 Hz, 2H), 7.45 (d, J ¼ 8.8 Hz, 2H), 4.19 (s, 2H), 3.44–
5.1.3. Method C. General procedure for reductive amination
A solution of the benzaldehyde (1 equiv) and amine (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 ꢂ10 mL). The combined organic phases were dried over sodium
sulfate and evaporated. Chromatography of the residue (0–5% 2 M
methanolic ammonia/CH2Cl2) provided the desired product. Ben-
zylamines were then dissolved in Et2O and treated with HCl
(2.5 equiv) and the resulting suspension stirred for 30 min. Solvent
removed and solids dried under high vacuum.
3.37 (m, 2H), 3.36–3.31 (m, 3H), 3.30–3.22 (m, 4H), 3.03 (t,
J ¼ 7.2 Hz, 2H), 2.87–2.73 (m, 2H), 2.07–1.93 (m, 2H), 1.92–1.77 (m,
3H), 1.41 (t, J ¼ 7.3 Hz, 6H); 13C NMR (CD3OD, 125 MHz)
d 132.6,
131.8, 128.9, 124.9, 85.9, 82.7, 60.5, 53.1, 50.2, 47.7, 23.0, 22.0, 15.7,
8.8; MS m/z 299.3 (M þ H); Anal. (C20H32Cl2N2) C, H, N.
5.1.2.2. 1-[4-(4-Pyrrolidin-1-yl-but-1-ynyl)-benzyl]-piperidine (9b).
Prepared according to method B (24%). 1H NMR (CD3OD, 500 MHz)
d
7.52 (d, J ¼ 8.3 Hz, 2H), 7.43 (d, J ¼ 8.3 Hz, 2H), 4.18 (s, 2H), 3.96–
3.73 (m, 1H), 3.49–3.32 (m, 5H), 3.18–2.95 (m, 4H), 2.79–2.63 (m,
2H), 2.27–1.99 (m, 6H), 1.93–1.78 (m, 3H), 1.51–1.31 (m, 1H); 13C
NMR (CD3OD, 125 MHz)
d
132.6, 131.6, 128.5, 124.9, 86.0, 82.7, 60.4,
5.1.3.1. Dimethyl-[4-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-amine
54.3, 53.6, 53.0, 23.4, 22.8, 22.0, 17.2; MS m/z 297.3 (M þ H); Anal.
(9h). Prepared according to method C (75%). 1H NMR (500 MHz)
(C20H30Cl2N2) C, H, N.
d
7.35–7.32 (m, 2H), 7.23–7.20 (m, 2H), 3.38 (s, 2H), 2.68–2.63 (m,
2H), 2.61–2.57 (m, 2H), 2.49–2.44 (m, 4H), 2.21 (s, 6H), 1.62–1.57
(m, 4H), 1.47–1.41 (m, 2H); 13C NMR (125 MHz)
138.5, 131.4, 128.9,
5.1.2.3. 4-[4-(4-Piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-piperidine
d
(9c). Prepared according to method B (42%). 1H NMR (CD3OD,
122.4, 88.3, 81.1, 64.0, 58.0, 54.2, 45.3, 25.9, 24.2, 17.5; MS m/z 271.3
(M þ H); Anal. (C18H26N2) C, H, N.
500 MHz)
d
7.53 (d, J ¼ 8.2 Hz, 2H), 7.41 (d, J ¼ 8.1 Hz, 2H), 4.15 (s,
2H), 3.60 (d, J ¼ 11.7 Hz, 2H), 3.43–3.35 (m, 2H), 3.25 (t, J ¼ 7.36 Hz,
2H), 3.07 (t, J ¼ 7.1 Hz, 2H), 2.88–2.79 (m, 2H), 2.73–2.64 (m, 2H),
2.18–1.98 (m, 4H), 1.93–1.77 (m, 6H), 1.53–1.28 (m, 2H); 13C NMR
5.1.3.2. Diethyl-[4-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-amine (9i).
Prepared according to method C (74%). 1H NMR (400 MHz)
d 7.33 (d,
(CD3OD, 125 MHz)
d
132.5, 131.6, 128.5, 124.8, 86.2, 82.6, 60.4, 55.5,
J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 8.2 Hz, 2H), 3.53 (s, 2H), 2.68–2.57 (m,
53.6, 53.0, 22.8, 22.7, 22.0, 21.9, 15.6; MS m/z 311.3 (M þ H); Anal.
4H), 2.52–2.45 (m, 8H), 1.63–1.57 (m, 4H), 1.47–1.41 (m, 2H), 1.02 (t,
(C21H32Cl2N2) C, H, N.
J ¼ 7.1 Hz, 6H); 13C NMR (125 MHz)
d 139.7, 131.3, 128.6, 122.0, 88.1,
81.2, 58.1, 57.3, 54.2, 46.7, 25.9, 24.3, 17.6, 11.7; MS m/z 299.3
(M þ H); Anal. (C20H30N2) C, H, N.
5.1.2.4. 4-[4-(4-Piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-thiomorpho-
line (9d). Prepared according to method B (36%). 1H NMR (CD3OD,
500 MHz)
d
7.54 (d, J ¼ 8.1 Hz, 2H), 7.48 (d, J ¼ 8.1 Hz, 2H), 4.24 (s, 2H),
5.1.3.3. 1-[4-(4-Pyrrolidin-1-ylmethyl-phenyl)-but-3-ynyl]-piperi-
3.88 (br d, J ¼ 11.9 Hz, 2H), 3.44–3.33 (m, 6H), 3.30–3.24 (m, 2H), 3.09
(dd, J ¼ 7.3, 7.2 Hz, 2H), 2.95–2.85 (m, 2H), 2.84–2.76 (m, 2H), 1.97–
dine (9j). Prepared according to method
(400 MHz)
7.34 (d, J ¼ 8.0 Hz, 2H), 7.24 (d, J ¼ 8.0 Hz, 2H), 3.58 (s,
2H), 2.68–2.57 (m, 4H), 2.50–2.45 (m, 8H), 1.79–1.76 (m, 4H), 1.63–
1.57 (m, 4H), 1.47–1.41 (m, 2H); 13C NMR (125 MHz)
139.0, 131.3,
C
(96%). 1H NMR
d
1.81 (m, 5H), 1.56–1.40 (m, 1H); 13C NMR (CD3OD, 125 MHz)
d 132.1,
131.3, 128.7, 124.6, 85.6, 82.4, 60.1, 55.8, 54.1, 52.7, 24.6, 22.7, 21.5,
d
14.9; MS m/z 329.3 (M þ H); Anal. (C20H30Cl2N2S) C, H, N.
128.7, 122.2, 88.2, 81.1, 60.4, 58.1, 54.2, 54.1, 25.9, 24.2, 23.4, 17.6; MS
m/z 297.3 (M þ H); Anal. (C20H28N2) C, H, N.
5.1.2.5. 4-[4-(4-Piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-morpho-
line (9e). Prepared according to method B (68%). 1H NMR (CD3OD,
5.1.3.4. 4-[4-(4-Piperidin-1-yl-but-1-ynyl)-benzyl]-morpholine (9k).
500 MHz)
d
7.32 (d, J ¼ 8.1 Hz, 2H), 7.23 (d, J ¼ 8.0 Hz, 2H), 4.20–
Prepared according to method C (88%). 1H NMR (400 MHz)
d
7.34 (d,
4.06 (m, 4H), 4.02–3.92 (m, 2H), 3.54 (d, J ¼ 12 Hz, 2H), 3.37
J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 8.2 Hz, 2H), 3.70 (t, J ¼ 4.6 Hz, 4H), 3.47

C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
4105
(s, 2H), 2.68–2.57 (m, 4H), 2.50–2.41 (m, 8H), 1.63–1.57 (m, 4H),
1.48–1.42 (m, 2H); 13C NMR (125 MHz)
137.4, 131.4, 128.9, 122.6,
5.1.3.12. 4-[3-(4-Piperidin-1-yl-but-1-ynyl)-benzyl]-morpholine (9s).
Prepared according to method C (90%). 1H NMR (400 MHz)
7.36
d
d
88.4, 81.0, 66.9, 63.1, 58.0, 54.2, 53.5, 25.9, 24.2, 17.5; MS m/z 313.3
(M þ H); Anal. (C20H28N2O) C, H, N.
(br s, 1H), 7.30–7.22 (m, 3H), 3.70 (t, J ¼ 4.6 Hz, 4H), 3.45 (s, 2H),
2.68–2.57 (m, 4H), 2.51–2.40 (m, 8H), 1.64–1.57 (m, 4H), 1.48–1.41
(m, 2H); 13C NMR (125 MHz)
d 137.9, 132.2, 130.3, 128.5, 128.1, 123.7,
88.5, 81.1, 66.9, 63.0, 58.0, 54.2, 53.5, 25.9, 24.2, 17.5; MS m/z 313.3
(M þ H); Anal. (C20H28N2O) C, H, N.
5.1.3.5. 1-Methyl-4-[4-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-pipera-
zine (9l). Prepared according to method
(400 MHz)
7.33 (d, J ¼ 8.2 Hz, 2H), 7.23 (d, J ¼ 8.2 Hz, 2H), 3.47 (s,
2H), 2.68–2.57 (m, 4H), 2.47 (m, 12H), 2.28 (s, 3H), 1.62–1.57
(m, 4H), 1.47–1.41 (m, 2H); 13C NMR (125 MHz)
137.9, 131.3, 128.9,
C
(84%). 1H NMR
d
5.1.3.13. 1-Methyl-4-[3-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-piper-
azine (9t). Prepared according to method
(500 MHz) 7.36 (s, 1H), 7.29–7.20 (m, 3H), 3.45 (s, 2H), 2.68–2.57
(m, 4H), 2.56–2.30 (m, 11H), 2.28 (s, 3H), 1.87 (br s, 1H), 1.63–1.57
(m, 4H), 1.48–1.41 (m, 2H); 13C NMR (125 MHz)
138.3, 132.2, 130.2,
C
(95%). 1H NMR
d
d
122.4, 88.3, 81.1, 62.7, 58.1, 55.1, 54.2, 53.1, 46.0, 25.9, 24.2, 17.6; MS
m/z 326.4 (M þ H); Anal. (C21H31N3) C, H, N.
d
128.5, 128.0, 123.6, 88.4, 81.2, 62.6, 58.1, 55.1, 54.2, 53.0, 46.0, 25.9,
24.2, 17.6; MS m/z 326.4 (M þ H); Anal. (C21H31N3) C, H, N.
5.1.3.6. 1-Isopropyl-4-[4-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-pipera-
zine (9m). Prepared according to method
(400 MHz)
7.33 (d, J ¼ 8.2 Hz, 2H), 7.23 (d, J ¼ 8.2 Hz, 2H), 3.48 (s,
C
(97%). 1H NMR
d
5.1.3.14. 1-[3-(4-Piperidin-1-yl-but-1-ynyl)-benzyl]-piperidine-4-car-
2H), 2.68–2.40 (m, 17H), 1.63–1.57 (m, 4H), 1.48–1.41 (m, 2H), 1.04
boxylic acid amide (9u). Prepared according to method C (84%). 1H
(d, J ¼ 6.6 Hz, 6H); 13C NMR (125 MHz)
d 137.8, 131.3, 129.0, 122.4,
88.3, 81.1, 62.8, 58.1, 54.4, 54.2, 53.4, 48.6, 25.9, 24.2, 18.6, 17.6; MS
NMR (500 MHz)
d 7.35 (s, 1H), 7.29–7.21 (m, 3H), 5.45 (br s, 1H),
5.36 (br s, 1H), 3.44 (s, 2H) 2.93–2.88 (m, 2H), 2.68–2.57 (m, 4H),
2.51–2.42 (m, 4H), 2.18–2.11 (m, 1H), 2.02–1.96 (m, 2H), 1.89–1.82
(m, 2H), 1.80–1.68 (m, 3H), 1.63–1.57 (m, 4H), 1.48–1.41 (m, 2H); 13C
m/z 354.4 (M þ H); Anal. (C23H35N3) C, H, N.
5.1.3.7. 1-Benzyl-4-[4-(4-piperidin-1-yl-but-1-ynyl)-benzyl]-pipera-
NMR (125 MHz) d 177.2, 138.5, 132.0, 130.1, 128.3, 128.0, 123.6, 88.5,
zine (9n). Prepared according to method
(400 MHz) 7.38–7.21 (m, 9H), 3.51 (s, 2H), 3.48 (s, 2H), 2.68–2.56
(m, 4H), 2.46 (m, 10H), 1.62–1.56 (m, 6H), 1.74–1.42 (m, 2H); 13C
NMR (125 MHz) 138.1, 137.8, 131.3, 129.1, 128.9, 128.1, 126.9, 122.4,
C
(38%). 1H NMR
81.2, 62.7, 58.1, 54.2, 53.0, 42.7, 28.9, 25.9, 24.2, 17.6; MS m/z 354.3
(M þ H); Anal. (C22H31N3O) C, H, N.
d
d
5.1.3.15. 1-[4-(3-Pyrrolidin-1-ylmethyl-phenyl)-but-3-ynyl]-piperidine
88.3, 81.1, 63.0, 62.7, 58.0, 54.2, 53.1, 53.0, 25.9, 24.2, 17.6; MS m/z
402.4 (M þ H); Anal. (C27H35N3) C, H, N.
(9v). Prepared according to method C (90%). 1H NMR (400 MHz)
d
7.37 (s, 1H), 7.28–7.22 (m, 3H), 3.56 (s, 2H), 2.68–2.57 (m, 4H),
2.51–2.42 (m, 8H), 1.82–1.74 (m, 4H), 1.63–1.57 (m, 4H) 1.48–1.41
(m, 2H); 13C NMR (125 MHz)
139.5, 131.9, 130.0, 128.2, 128.0,
5.1.3.8. 4-{4-[4-(4-Benzyl-piperidin-1-ylmethyl)-phenyl]-but-3-ynyl}-
d
piperidine (9o). Prepared according to method C (19%). 1H NMR
123.6, 88.3, 81.2, 60.3, 58.1, 54.2, 54.1, 25.9, 24.2, 23.4, 17.5; MS m/z
297.3 (M þ H); Anal. (C20H28N2) C, H, N.
(400 MHz)
d
7.32 (d, J ¼ 8.0 Hz, 2H), 7.28–7.15 (m, 5H), 7.12 (d,
J ¼ 7.1 Hz, 2H), 3.43 (s, 2H), 2.83–2.80 (m, 2H), 2.68–2.56 (m, 4H),
2.52 (d, 7.0 Hz, 2H), 2.46 (m, 4H), 1.87 (t, J ¼ 9.9 Hz, 2H), 1.62–1.57
(m, 6H), 1.53–1.41 (m, 3H), 1.34–1.24 (m, 2H); 13C NMR (125 MHz)
5.1.3.16. 4-[4-(2-Piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-piperidine
(9w). Prepared according to method C (85%). 1H NMR (400 MHz)
d
140.7, 131.3, 129.1, 128.9, 128.1, 125.7, 63.1, 58.1, 54.2, 53.8, 43.2,
d
7.43 (d, J ¼ 7.1 Hz, 1H), 7.36 (d, J ¼ 7.7 Hz, 1H), 7.26–7.22 (m, 1H),
7.16–7.12 (m, 1H), 3.63 (s, 2H), 2.70–2.61 (m, 4H), 2.53–2.37 (m, 8H),
1.64–1.55 (m, 8H), 1.49–1.39 (m, 4H); 13C NMR (125 MHz)
140.3,
37.8, 32.1, 28.5, 25.9, 24.2, 17.6; MS 401.4 m/z (M þ H); Anal.
(C28H36N2) C, H, N.
d
131.9, 129.0, 127.5, 126.3, 123.6, 92.8, 79.7, 61.0, 58.1, 54.5, 54.2, 26.0,
25.9, 24.3, 24.2, 17.8; MS m/z (M þ H); Anal. (C21H30N2) C, H, N.
5.1.3.9. 1-[4-(4-Piperidin-1-yl-but-1-ynyl)-benzyl]-piperidine-4-car-
boxylic acid amide (9p). Prepared according to method C (49%). 1H
NMR (400 MHz)
d
7.33 (d, J ¼ 8.0 Hz, 2H), 7.23 (d, J ¼ 8.0 Hz, 2H),
5.2. Biological evaluation
3.94 (s, 2H), 3.49 (s, 2H), 2.67–2.57 (m, 4H), 2.51–2.45 (m, 8H),1.77–
1.71 (m, 5H), 1.63–1.57 (m, 4H), 1.47–1.42 (m, 2H); 13C NMR
5.2.1. In vitro pharmacology
(125 MHz)
d 177.3, 138.1, 131.4, 128.8, 122.4, 88.4, 81.1, 62.8, 58.1,
5.2.1.1. 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 [24]. Briefly, cell
pellets from SK-N-MC cells expressing either the rat or human H3
receptor were homogenized in 50 mM Tris–HCl/5 mM EDTA and
recentrifuged at 30,000 g for 30 min. Pellets were rehomogenized
in 50 mM Tris/5 mM EDTA (pH 7.4). Membranes were incubated
with 0.8 nM [3H]-Na-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
54.2, 53.0, 42.7, 28.9, 25.9, 24.3, 17.6; MS m/z 354.3 (M þ H); Anal.
(C22H31N3O) C, H, N.
5.1.3.10. 1-[4-(4-Piperidin-1-yl-but-1-ynyl)-benzyl]-piperidin-4-ol
(9q). Prepared according to method C (84%). 1H NMR (400 MHz)
d
7.34 (d, J ¼ 8.0 Hz, 2H), 7.22 (d, J ¼ 8.0 Hz, 2H), 3.72–3.65 (m, 1H),
3.47 (s, 2H), 2.75–2.57 (m, 6H); 2.47 (m, 4H), 2.13 (t, J ¼ 9.6 Hz, 2H),
1.90–1.84 (m, 2H), 1.63–1.53 (m, 5H), 1.47–1.41 (m, 3H); 13C NMR
(125 MHz)
d 138.2, 131.3, 128.8, 122.4, 88.3, 81.1, 68.0, 62.6, 58.1,
54.2, 50.9, 34.5, 25.9, 24.2, 17.5; MS m/z 327.3 (M þ H); Anal.
(C21H30N2O) C, H, N.
in the presence of 10 mM 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]-Na-methylhistamine Kd of
800 pM and a ligand concentration of 800 pM [25].
5.1.3.11. 4-[4-(3-Piperidin-1-ylmethyl-phenyl)-but-3-ynyl]-piperidine
(9r). Prepared according to method C (92%). 1H NMR (400 MHz)
d
7.35 (br s, 1H), 7.28–7.21 (m, 3H), 3.42 (s, 2H), 2.67–2.57 (m, 4H),
2.50–2.43 (m, 4H), 2.39–2.31 (m, 4H), 1.63–1.53 (m, 8H), 1.48–1.38
(m, 3H); 13C NMR (125 MHz)
138.7,132.2,130.0,128.5,127.9,123.5,
5.2.1.2. Additional in vitro data. The compound was screened at
a concentration of 1 mM against a panel of 50 drug targets (recep-
d
88.3, 81.3, 63.5, 58.1, 54.4, 54.2, 26.0, 25.9, 24.3, 24.2, 17.6; MS m/z
(M þ H); Anal. (C21H30N2) C, H, N.
tors, ion channels, transporters) by CEREP (15318 NE 95th Street,
Redmond WA; www.cerep.com). Human plasma protein binding

4106
C.A. Dvorak et al. / European Journal of Medicinal Chemistry 44 (2009) 4098–4106
Table A1 (continued)
and Caco-2 permeability were determined by Absorbtion Systems,
Exton, PA.
Compd.
Formula
20H30Cl2N2
Anal. Calcd.
Anal. Found
9v
C
C, 65.03;
C, 65.02;
5.2.1.3. Human histamine H3 functional assay. Sublines of SK-N-MC
H, 8.19; N, 7.58
C, 61.05;
H, 8.61; N, 6.78
H, 7.95; N, 7.47
C, 61.12;
H, 8.29; N, 6.54
9w
C21H32Cl2N2 ꢂ 1.65 H2O
cells were created that expressed a reporter construct and the
human H3 receptor. The reporter gene was b-galactosidase 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
After a 6-h incubation at 37 ꢁC, the media were aspirated and the
cells washed with 200 L of phosphate-buffered saline followed by
a second aspiration. Antagonists were added 10 min prior to the
addition of histamine. Cells were lysed with 25
mM final concentration).
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m
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Table A1
Combustion analysis data for all biologically-assayed compounds.
Compd.
Formula
20H32Cl2N2
Anal. Calcd.
Anal. Found
(b) A. Rouleau, M. Garbarg, X. Ligneau, C. Mantion, P. Lavie, C. Advenier,
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9a
C
C, 64.68; H,
8.68; N, 7.54
C, 64.41; H,
8.22; N, 7.51
C, 65.48; H,
8.43; N, 7.27
C, 59.31;
H, 7.57; N, 6.92
C, 62.19;
H, 7.85; N, 7.25
C, 58.00;
H, 7.88; N, 9.66
C, 62.33;
H, 7.85; N, 7.27
C, 62.97;
H, 8.22; N, 8.16
C, 64.68;
H, 8.68; N, 7.54
C, 65.03;
H, 8.19; N, 7.58
C, 62.33;
H, 7.85; N, 7.27
C, 56.82;
H, 7.95; N, 9.47
C, 57.87;
H, 8.36; N, 8.80
C, 80.06;
H, 8.72; N, 10.35
C, 83.22;
H, 8.99; N, 6.92
C, 74.75;
H, 8.84; N, 11.89
C, 60.95;
H, 8.18; N, 6.77
C, 65.79;
C, 64.69;
H, 8.43; N, 7.37
C, 64.11;
H, 7.83; N, 7.54
C, 65.17;
H, 8.04; N, 7.11
C, 59.05;
H, 7.19; N, 7.08
C, 61.70;
H, 7.30; N, 7.19
C, 57.72;
H, 7.79; N, 9.51
C, 61.66;
H, 7.59; N, 7.18
C, 62.83;
H, 7.99; N, 8.00
C, 64.97;
H, 8.28; N, 7.46
C, 64.78;
H, 7.82; N, 7.36
C, 62.48;
H, 7.71; N, 7.05
C, 56.88;
H, 7.91; N, 9.40
C, 58.08;
H, 8.10; N, 8.59
C, 80.24;
H, 8.72; N, 10.19
C, 83.30;
H, 9.05; N, 6.86
C, 74.89;
9b
9c
9d
9e
9f
C20H30Cl2N2 ꢂ 0.2 H2O
C21H32Cl2N2 ꢂ 0.01 H2O
C20H30Cl2N2S ꢂ 0.2 H2O
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C
20H30Cl2N2O ꢂ 0.05 H2O
C20H34Cl3N3
9g
9h
9i
C20H30Cl2N2O ꢂ 0.15 H2O
C18H28Cl2N2
C
20H32Cl2N2
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9j
C20H30Cl2N2
9k
9l
C20H30Cl2N2O
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C
21H34Cl3N3 ꢂ 0.5 H2O
9m
9n
9o
9p
9q
9r
9s
9t
C23H38Cl3N3 ꢂ 0.8 H2O
C27H35N3 ꢂ 0.05 CH2Cl2
C28H36N2 ꢂ 0.05 CH2Cl2
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C
22H31N3O
H, 8.93; N, 11.59
C, 61.04;
H, 8.17; N, 6.61
C, 65.44;
H, 8.28; N, 6.97
C, 62.11;
H, 7.90; N, 7.10
C, 58.02;
C21H32Cl2N2O ꢂ 0.8 H2O
C21H32Cl2N2
H, 8.41; N, 7.31
C, 62.33;
H, 7.85; N, 7.27
C, 58.00;
H, 7.88; N, 9.66
C, 74.75;
H, 8.84; N, 11.89
C20H30Cl2N2O
C
21H34Cl3N3
H, 7.78; N, 9.28
C, 74.59;
H, 8.76; N, 11.80
9u
C22H31N3O