Design, synthesis, and preliminary pharmacological evaluation of 4-aminopiperidine derivatives as N-type calcium channel blockers active on pain and neuropathic pain

Article abstract of DOI:10.1021/jm049923l

Several compounds with a 4-aminopiperidine scaffold decorated on both nitrogen atoms by alkyl or acyl moieties containing the structural motifs of verapamil and of flunarizine, as well as those that are more frequent in known N-type calcium channel antago

Full text of DOI:10.1021/jm049923l

6070
J. Med. Chem. 2004, 47, 6070-6081
Design, Synthesis, and Preliminary Pharmacological Evaluation of
4-Aminopiperidine Derivatives as N-Type Calcium Channel Blockers Active on
Pain and Neuropathic Pain
Elisabetta Teodori,*,† Elisabetta Baldi,§ Silvia Dei,† Fulvio Gualtieri,† Maria Novella Romanelli,†
Serena Scapecchi,† Cristina Bellucci,† Carla Ghelardini, and Rosanna Matucci
Dipartimento di Scienze Farmaceutiche, Universita` di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (FI), Italy, and
Dipartimento di Fisiopatologia Clinica and Dipartimento di Farmacologia Preclinica e Clinica, Universita` di Firenze,
Viale G. Pieraccini 6, I-50139 Firenze, Italy
Received January 28, 2004
Several compounds with a 4-aminopiperidine scaffold decorated on both nitrogen atoms by
alkyl or acyl moieties containing the structural motifs of verapamil and of flunarizine, as well
as those that are more frequent in known N-type calcium channel antagonists, have been
synthesized. Antinociceptive activity on the mouse hot-plate test was used to select molecules
to be submitted to further studies. Active compounds were tested in vitro on a PC12 rat
pheochromocytoma clonal cell line, to evaluate their action on N-type calcium channels, and
on a rat model of neuropathic pain. Two compounds that show N-type calcium channel
antagonism and are endowed with potent action on pain and neuropathic pain (3 and 18) have
been selected for further studies.
Introduction
filled these expectations and have stimulated a large
synthetic effort to individuate small molecules able to
block N-type calcium channels, while showing improved
pharmacokinetic properties. In fact, ziconotide has
marked therapeutic benefits in both analgesia and in
neuroprotection, is in phase III clinical trial and has
been made available to patients for compassionate use.10
Unfortunately, the drug has to be administered intra-
thecally to allow direct access to the spinal cord. This
has prompted the design and the synthesis11-13,14 of
many small molecules that would block the N-type
calcium channel, giving the same therapeutic benefits
as ziconotide, with more acceptable pharmacokinetic
properties.
Excitable cells possess voltage-dependent calcium
channels (VDCC) that control ion flux in to and out of
the cell, by opening and closing in response to changes
in membrane potential.1 They modulate many impor-
tant physiological functions such as neurotrasmitter
release, gene expression and muscle contraction. Bio-
physical and pharmacological studies have identified
five different subtypes of high-voltage activated (HVA)
calcium channels: L, N, P, Q and R and one low-voltage
activated channel: the T-type. Functional calcium
channels are formed by a combination of subunits, the
largest of which, the R1 subunit, forms the voltage-
sensitive ion-conducting pore.
Most neurons express multiple calcium channel sub-
types.2 Apparently, they contribute to different extents
to the physiology of neuronal cells depending on the
types of cells and synapses formed by them. It is
generally accepted that L-type channels, unlike N-type
and P/Q-type, make only minor contributions to neu-
rotransmitter release in the brain. Indeed, N-type
calcium channels, which are located presynaptically and
are selectively blocked by ω-conotoxin MVIIA,3 a peptide
toxin derived from Conus magus, seem to play a
fundamental role in neuronal cell excitability and, as a
consequence, in neuroprotection4 and pain perception.5,6
This suggests that N-type calcium channel blockers
could be useful in pathological states such as stroke,
pain and, in particular, neuropathic pain.2,7 It is inter-
esting that very recently T-calcium channels have also
been found to play a role in neurophatic pain.8
For several years we have been involved in the design
and synthesis of L-type calcium channel antagonists
related to verapamil,15-18 and we decided to take
advantage of the experience accumulated during this
researche to design and study N-type calcium channel
antagonists. Indeed, during the past decade, it was
found that the classic L-type antagonists are not that
specific, as a few of them show some action also on other
types of calcium channels, namely the N-type. As an
example, Chart 1 reports some L-type classical antago-
nists that have also shown remarkable N-type antago-
nism: (S)-emopamil,19 cilnidipine,20 flunarizine.21 More
interestingly, structural motifs of L-type antagonists can
be found in the new molecules designed as N-type
antagonists such as compounds A22 and B23 shown in
Chart 1. These results seem to suggest that it is possible
to obtain N-type calcium channel antagonists by mo-
lecular manipulation of the structure of L-type antago-
nists and on this basis, we have designed the series of
compounds shown in Chart 2. The 4-aminopiperidine
scaffold, that has been already used as supporting
structure for N-type antagonists,13 has been decorated
on both nitrogen atoms by alkyl or acyl moieties
The promising properties of ziconotide (SNX-111), the
synthetic equivalent of ω-conotoxin MVIIA,9 have ful-
* Corresponding author. Tel. ++39 55 4573693. Fax ++39 55
4573671. E-mail elisabetta.teodori@unifi.it.
† Dipartimento di Scienze Farmaceutiche.
§ Dipartimento di Fisiopatologia Clinica.
Dipartimento di Farmacologia Preclinica e Clinica.
10.1021/jm049923l CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/22/2004

N-Type Calcium Channel Blockers
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6071
Chart 1
reductive alkylation of 1-benzyl-4-piperidone with the
suitable amine (commercially available 2-(3,4-dimeth-
oxyphenyl)ethylamine, 2-phenoxyethylamine and butyl-
amine), using titanium(IV) isopropoxide as Lewis acid
catalyst and NaBH3CN as reducing agent, according to
the Mattson procedure25 (Scheme 1).
The 1-alkyl-4-alkylaminopiperidines 2-12 were syn-
thesized from the corresponding 1-benzyl-4-alkylami-
nopiperidine (1, 21-23) by alkylation, with the suitable
alkyl halide, of compounds 24-27, obtained by deben-
zylation of 1, 21-23 (Scheme 2). The alkyl halides used
are commercially available (butyl iodide, cinnamyl
bromide) or synthesized according to the literature (1,1′-
(4-bromobutylidene)bis(4-fluorobenzene),26 1-bromo-4,4-
diphenylbutane,27 (2-bromoethoxy)benzene,28 4-(2-bro-
moethyl)-1,2-dimethoxybenzene29). 5-Bromo-2-phenyl-
2-(methylethyl)pentanenitrile (33), used to obtain com-
pound 4, was obtained with the same procedure de-
scribed by Teodori24 for the 3,4-dimethoxyphenyl de-
rivative, starting from 3-methyl-2-phenylbutyronitrile.30
The 1-piperidine amide derivatives (13-15) were
synthesized from the corresponding 4-alkylaminopip-
eridine (24-27) and 4-cyano-4-(3,4-dimethoxyphenyl)-
5-methylhexanoic acid (28) using diethylcyanophospho-
nate as dehydrating agent. Compound 28 was obtained
from the corresponding alcohol31 by oxidation with
KMnO4 and tetrabutylammonium bromide in benzene
(Scheme 2).
4,4′-Bis-(4-fluorophenyl)-N-piperidine-4-ylbutyr-
amide 16 was obtained from 4,4′-bis-(4-fluorophenyl)-
butyric acid26 by reaction with 1-benzyl-4-aminopiperi-
dine to give 29, which was then debenzylated (Scheme
3). Reaction of 16 with chloroethanol in the presence of
triethylamine gave 17. 1-(4-Aminopiperidin-1-yl)-4,4′-
bis-(4-fluorophenyl)butan-1-one 18 was synthesized by
cleavage with Me3SiI in MeOH of the t-BOC protected
compound 30. The latter was obtained from 4,4′-bis-(4-
fluorophenyl)butyric acid26 and 4-(tert-butyloxycarbo-
containing the structural motifs of verapamil and of
flunarizine as well as those that are more frequent in
known N-type calcium channel antagonists.14 It must
be stressed that we did not intend to synthesize all
possible isomers corresponding to the X and Y groups
indicated in Chart 2, as we rather wanted to explore
the possibility of obtaining efficient and possibly selec-
tive N-type antagonists, using the approach described
above.
Chemistry. The reaction pathways used to synthe-
sise the designed compounds (1-20) are described in
Schemes 1-4 and their chemical and physical charac-
teristics are reported in Table 1.
The key intermediates 1-benzyl-4-alkylaminopipe-
ridines (1, 21-23) were obtained by alkylation of
1-benzyl-4-aminopiperidine with 5-bromo-2-(3,4-di-
methoxyphenyl)-2-(methylethyl) pentanenitrile24 or by
Chart 2

6072 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Table 1. Chemical and Physical Characteristics of Compounds 1-20
Teodori et al.
N
X
Y
Z
R
R1
R2
yield, %
salt/mp (°C)
analysisa
1
a
f
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
88
57
31
83
56
80
35
64
34
58
10
24
32
43
32
62
44
45
69
38
HCl/260-265b
HCl/234-235b
oxalate/217-218c
oxalate/233-234c
oxalate/198-200c
oxalate/252-253c
oxalate/234-235b
HCl/190-192b
HCl/130-132b
HCl/240-245b
HCl/210-215b
HCl/135-140b
oxalate/80-82c
oxalate/104-105c
HCl/90-92b
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
22H31ClN2O2
25H42ClN3O2
31H43N3O7
2
g
b
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CO
OCH3
-
3
g
d
OCH3
-
4
g
f
f
H
-
31H43N3O6
5
g
OCH3
-
33H47N3O8
27H38N2O4
6
h
h
h
b
b
-
H
H
F
-
7
f
f
-
31H41ClN2O2
31H39ClF2N2O2
25H42ClN3O2
30H46ClN3O3
30H42ClN3O2
31H45N3O4
8
-
9
g
OCH3
10
11
12
13
14
15
16
17
18
19
20
d
c
g
OCH3
-
g
OCH3
-
f
g
OCH3
-
g
f
OCH3
-
33H45N3O9
31H41N3O8
g
d
CO
OCH3
-
g
b
CO
OCH3
-
25H40ClN3O3
23H26F2N2O5
23H29ClF2N2O2
21H25ClF2N2O
27H36ClF2N3O2
28H38ClF2N3O2
none
e
h
CO
-
-
-
-
-
F
F
F
F
F
oxalate/100-102c
HCl/170-175b
HCl/138-141b
HCl/165-167b
HCl/85-87c
h
CO
h
h
h
none
CO
i
i
CO
H
CO
CH3
a All compounds have been analyzed for C, H, N after vacuum-drying at a temperature below the melting point; the results obtained
range within (0.4% of the theoretical values. b Recrystallization solvent: absolute ethanol/ anhydrous diethyl ether. c Recrystallization
solvent: ethyl acetate.
Scheme 1a
N-t-BOC-L-leucine (commercially available) and N-t-
BOC-N-methyl-L-leucine33 respectively, followed by cleav-
age of the protected intermediates 31 and 32 (Scheme
4).
Pharmacological Studies. Antinociceptive activity
of compounds 1-20 was evaluated on the mouse hot-
plate test according to O’Callaghan and Holtzman.34 All
compounds were administered icv at the maximal dose
soluble in isotonic (NaCl 0.9%) saline solution (saline)
or in a vehicle constituted by water and DMSO (2:1).
L-Type calcium channel binding affinity at the 1,4-
dihydropyridine and phenylalkylamine binding sites of
the compounds showing antinociceptive activity (2, 3,
6, 8-11, 16, 18-20) was evaluated on rat ventricle
membranes using [3H]D888 and [3H]PN200-110, respec-
tively. N-Type calcium channel blocking activity of
compounds 2, 3, 6, 8-11, 15, 16, 18-20 was determined
in vitro on a PC12 rat pheochromocytoma clonal cell
line, using a spectrophotofluorimetric method employing
a fluorescent probe sensitive to intracellular calcium
changes.35,36 Different concentrations of the tested
compounds (dissolved in DMSO) and ω-conotoxin
MVIIA, as the reference compound, were used.
a Reagents and conditions: (a) (iPrO)4Ti, 2-(3,4-dimethoxyphen-
yl)ethylamine or 2-phenoxyethylamine or butylamine. (b)
NaBH3CN. (c) Et3N.
Compounds 2, 3, 6, 10, 15, 18-20 were also tested
on a neuropathic pain model. Toward this end, a
peripheral mono neuropathy was produced in adult rats
by loosely placing constrictive ligatures around the
nylamino)piperidine32 using diethylcyanophosphonate
as dehydrating agent (Scheme 3). The leucine deriva-
tives, 19 and 20, were obtained from 18 by reaction with

N-Type Calcium Channel Blockers
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6073
Scheme 2a
a Reagents and conditions: (a) HCOONH4, MeOH, Pd/C. (b) XBr or XI, Et3N. (c) Diethylcyanophosphonate, DMF, Et3N. (d) KMnO4/
But4NBr, benzene. For the meaning of X and Y see Table 1.
Scheme 3a
a Reagents and conditions: (a) 4,4′-Bis(4-fluorophenyl)butyric acid,26 diethylcyanophosphonate, DMF, Et3N. (b) HCOONH4, MeOH,
Pd/C. (d) ClCH2CH2OH, Et3N. (d) t-BOC, THF. (e) Me3SiI, MeOH.
common sciatic nerve according to the method described
by Bennett.37 The nociceptive threshold in the rat was
determined with an analgesimeter according to the paw
pressure test.38
pressed as the % of the antinociception produced by
morphine (10 µg per mouse icv). It must be emphasized
that, due to the poor solubility of many of the com-
pounds of the series, their antinociceptive activity has
been evaluated at very low doses, and therefore their
analgesic activity may be underestimated and some of
the inactive compounds could indeed be active, at higher
doses.
With the exception of compound 9, all the active
compounds (2, 3, 6, 8, 10, 11, 16, 18-20) show an
analgesic efficacy that is more than 50% of that exerted
by morphine (Table 2). Three compounds are outstand-
ing in the series: compounds 11, 19 that possess 69%,
71% of morphine efficacy at the dose of 20, 40 µg/mouse
icv, respectively, and compound 18 which, at the dose
Results and Discussion
The mouse hot-plate test was used to identify, among
the compounds synthesized the molecules able to induce
analgesia and that, therefore, may possibly be endowed
with activity on N-type calcium channels.
As it can be seen from Table 2, intracerebroventricu-
lar injection of several compounds of the series (2, 3, 6,
8-11, 16, 18-20) induced a statistically significant
antinociception in the hot-plate test. The analgesic
efficacy of active compounds was evaluated and ex-

6074 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Teodori et al.
Scheme 4a
animals’ gross behavior nor modified the spontaneous
motility and inspection activity as revealed by the hole-
board test (data not shown). The zero percent value, that
appears in Table 2 in correspondence of some com-
pounds, means that those molecules are devoid of any
statistical significance versus the corresponding control
group treated with saline or vehicle.
Since significant antinociception can be produced also
by L-type calcium channel antagonists,39,40 binding
affinity at the 1,4-dihidropiridine and phenylalkylamine
binding sites of L-type calcium channel of the com-
pounds showing antinociceptive activity (2, 3, 6, 8-11,
16, 18-20) was evaluated. On rat ventricle membranes,
using [3H]D888 and [3H]PN200-110 as radioactive
ligands, such compounds have shown very low affinity
(KD ) 10-100 µM, data not shown) for the two binding
sites of L-type calcium channels.
Therefore, compounds (2, 3, 6, 8-11, 16, 18-20)
which induced a statistically significant antinociception
in the mouse hot-plate test and, as a control, compound
15 which is devoid of analgesic efficacy, were selected
for testing their ability to block N-type calcium chan-
nels.
A number of studies have demonstrated that PC12
cells express N- and L-type calcium channels.41,42 In
preliminary experiments, we have tested whether the
PC12 clone used in our studies (see Experimental
Section), which is characterized by the ability to grow
in suspension, expresses N-type calcium channels, by
evaluating the increase of intracellular calcium stimu-
lated by KCl (50 mM, final concentration) after complete
blockade of L-type calcium channels with 10 µM nife-
dipine. As can be observed in Figure 1, in these
conditions the residual Ca2+-flux (shown in Figure 2)
is abolished by ω-conotoxin MVIIA at concentrations
(0.1-1 µΜ) known to specifically inhibit N-type calcium
a Reagents and conditions: (a) Diethylcyanophosphonate, DMF,
Et3N. (b) 2 N HCl/ethyl acetate.
of 50 µg/mouse icv, shows an analgesic efficacy higher
than that of morphine (147%). At 25 µg/mouse icv,
efficacy of 18 was still very good (80%) while at 10 µg/
mouse icv the compound was ineffective. Its analgesic
activity reached the maximum peak 15 min after
administration, persisted almost unchanged up to 30
min, then progressively diminished, disappearing at 60
min (data not shown). The analgesic activity of 18 was
not inhibited by naloxone, showing that opioid receptors
are not involved in its action. Active compounds, at the
effective doses, neither produced any alteration of the
Table 2. Analgesic Activity and Efficacy of Compounds 1-20 Evaluated in the Mouse Hot-Plate Test
licking latency (s) after treatmentd
dose icv,
µg/mouse
% analgesic efficacy
treatment
before treatment
15 min
30 min
45 min
compared to morphinea
saline
-
-
14.4 ( 1.0
15.7 ( 1.2
15.3 ( 1.1
13.7 ( 1.5
15.3 ( 1.3
14.2 ( 0.6
13.8 ( 1.1
15.0 ( 0.9
14.0 ( 1.2
13.7 ( 0.9
14.7 ( 0.9
15.3 ( 1.5
14.7 ( 1.0
15.1 ( 1.6
13.5 ( 0.8
14.6 ( 0.8
14.9 ( 1.3
14.4 ( 1.0
13.3 ( 1.4
14.5 ( 1.0
14.5 ( 0.9
13.9 ( 1.0
14.3 ( 1.1
13.8 ( 1.5
14.1 ( 1.1
13.9 ( 1.6
14.8 ( 1.7
28.6 ( 2.5*
15.5 ( 1.5
23.1 ( 1.9*
22.3 ( 2.2*
16.6 ( 1.0
17.2 ( 1.9
22.6 ( 1.5*
17.7 ( 2.0
21.5 ( 1.5∧
20.5 ( 1.6∧
22.6 ( 2.1*
24.3 ( 1.9*
17.7 ( 1.2
16.5 ( 1.8
15.7 ( 1.7
18.7 ( 1.6
21.8 ( 1.1*
16.9 ( 1.7
34.1 ( 1.8*
24.6 ( 1.8*
15.7 ( 2.0
23.2 ( 1.5*
21.5 ( 1.8∧
14.8 ( 2.1
15.5 ( 1.6
27.7 ( 1.5*
16.7 ( 1.9
21.3 ( 1.8∧
20.8 ( 1.5∧
18.1 ( 1.7
18.3 ( 2.1
22.4 ( 2.3*
16.4 ( 1.6
16.3 ( 2.2
21.6 ( 1.9∧
16.5 ( 1.4
20.2 ( 1.5∧
18.3 ( 1.6
18.8 ( 2.2
18.5 ( 1.9
17.4 ( 2.1
16.7 ( 1.3
16.8 ( 1.5
31.7 ( 1.4*
23.9 ( 1.8*
16.1 ( 1.9
22.6 ( 1.6∧
20.7 ( 1.8∧
14.0 ( 1.9
16.0 ( 1.3
22.4 ( 1.9*
15.3 ( 1.8
18.7 ( 2.2
18.9 ( 1.3
17.5 ( 2.0
17.6 ( 2.0
15.8 ( 2.5
16.8 ( 1.9
16.6 ( 2.3
19.0 ( 2.0
15.2 ( 1.9
16.2 ( 1.6
15.4 ( 1.4
17.3 ( 1.5
13.8 ( 1.6
17.0 ( 1.7
17.1 ( 1.8
17.3 ( 2.0
29.5 ( 2.0*
20.5 ( 2.3∧
16.4 ( 1.8
16.6 ( 1.6
15.5 ( 1.9
0
0
vehicle
morphine
10
50
30
50
10
10
10
5
10
25
30
20
15
10
10
30
20
15
50
25
10
40
40
-
1
0b
59
61
0b
0b
64
0b
51
47
59
69
0b
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18c
0b
0b
0b
64
0b
147
80
0b
19
20
71
56
a The % analgesic efficacy was evaluated at the maximal soluble dose in saline or in vehicle H2O/DMSO (2:1) with respect to the
maximal analgesic effect of morphine (10 µg/mouse icv) taken as 100%. b Zero percent value means that the molecule is devoid of any
statistical significance versus the control group. c The analgesic effect was not blocked by naloxone at 1 mg/kg ip. d *P < 0.01; ∧P < 0.05
in comparison with controls treated with saline or vehicle.

N-Type Calcium Channel Blockers
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6075
Table 3. N-Type Calcium Channels Antagonism of Compounds
3, 10, 18, 20 Evaluated on PC12 Cellsa
IC50 (µM) in the
presence of nifedipine
N
IC50 (µM)
3
0.70 ( 0.30
3.80 ( 1.20
1.03 ( 0.10
3.00 ( 0.90
0.60 ( 0.30
0.40 ( 0.07
3.10 ( 0.50
2.40 ( 0.60
ntb
10
18
20
ω-conotoxin
0.50 ( 0.20
a Compounds 6 and 15 did not change calcium influx, while 2,
8, 9, 11, 16 and 19 were able to antagonize calcium influx, but
their potency was too low to calculate an IC50.
b nt ) not tested.
subtype of channels (KD ) 100 µM). Indeed, when 18 is
tested in the presence of the L-type calcium channels
blocker nifedipine (10 µM), Ca2+-flux was almost com-
pletely antagonized at lower doses (Figure 3B), sug-
gesting that compound 18 is an N-type calcium channel
antagonist.
Figure 1. Increase in intracellular [Ca2+]i in response to KCl
(50 mM) in basal conditions (control) and following addition
of nifedipine (10 µM) alone or in combination with ω-conotoxin
(Ctx) (0.1-1 µM).
As regards structure-activity relationships (even if
this was not the main goal of the present research), it
can be said that the position of the residue on the
4-aminopiperidine moiety is not crucial and analgesic
compounds are found in both series of regioisomers
(compare 2 and 9, 3 and 10, 16 and 18). Both 4,4′-bis-
(4-fluorophenyl)butyramide and 4,4′-bis(4-fluorophenyl)-
butylamine series possess analgesic activity (see 6, 8,
18-20), while this is not true for the corresponding (3,4-
dimethoxyphenyl)isopropylpentanenitrile derivatives,
where only the amines show analgesic activity (amides
13, 14, 15 are inactive, whereas amines 2, 3, 9, 10, 11
are active). Finally, our efforts to increase the solubility
of the most potent compound 18 were not fully satisfac-
tory, as compound 17 is inactive as analgesic, while 19
and 20 show lower, although remarkable, efficacy with
respect to the parent compound (71% and 56%, respec-
tively, at 40 µg/mouse icv).
On the basis of the well documented involvement of
N-type calcium channels in the neuropathic pain,2,7,8,44
compounds 2, 3, 6, 10, 15, 18-20 were tested on the
model described by Bennett37 (Table 4). Experiments
were performed on rats submitted to paw-pressure test
14 days after the operation since at this time a signifi-
cant reduction of the pain threshold of the injured paw
(dx) was observed. By contrast, in the controlateral paw
the pain perception remained unchanged. Compounds
2, 3, 6, 18 and 19 showed appreciable antihyperalgic
activity on this model when compared with the saline-
treated group 30, 45 and 60 min after administration.
All active compounds did not modify pain threshold in
controlateral, nonoperated paw and at the doses ad-
ministered were devoid of any antihyperalgic effect.
Rats treated with compounds 2, 3, 6, 18 and 19 at the
highest doses showed a normal behavior compared to
saline control rats.
Figure 2. Intracellular calcium waves in response to KCl (50
mM) in the presence of nifedipine (10 µM) and different
concentrations of compound 18 (0.1-10 µM).
channels.43 Although in most studies PC12 cells expres-
sion of N-type calcium channels is induced by treatment
with NGF,41 we argued from these experiments that the
PC12 clone used expresses a significant amount of
N-type calcium channels, even without treatment with
this expensive factor. Subsequent experiments were
performed evaluating the effect of our compounds on the
residual calcium fluxes obtained with KCl after nife-
dipine block.
All the compounds tested, except 6 and 15, which was
inactive as analgesic, were able to reduce, to a different
extent, the calcium flux in the presence of nifedipine,
showing that they are inhibiting N-type calcium chan-
nels. The result of a typical experiment, performed with
the compound 18 is reported in Figure 2.
The IC50 of the most potent compounds (3, 10, 18, 20)
was evaluated and compared to that of ω-conotoxin
MVIIA (Table 3). Compound 3, which is moderately
analgesic, shows an IC50 comparable with that of
ω-conotoxin MVIIA in this test while compound 18,
which is the most effective analgesic, shows an IC50
value 2.5 times greater. The discrepancy between the
analgesic activity and N-channel inhibition may be
explained, among other reasons, by different pharma-
cokinetics of the two drugs in the mouse.
It can be observed that the results obtained from
neuropathic pain model do not completely overlap those
obtained in the hot plate test. The discrepancy existing
between the results obtained in the two analgesic tests
can be due to the different stimulus applied (thermic
in the hot-plate and mechanical in the paw-pressure)
or to the different animal species. As a matter of fact,
the pharmacokinetic profile of the compounds could
differ greatly in mouse and rat species. Furthermore,
it has to be considered that in the Bennett model the
rat has a chronic pain caused by the operation and
Compound 18 is able to block the Ca2+-flux in a dose
dependent manner, and the Ca2+-flux is completely
blocked at 100 µM (Figure 3A). However, at this dose,
the compound is very likely blocking also the L-type
channels, according with its binding affinity for this

6076 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Teodori et al.
Figure 3. Increase in intracellular [Ca2+]i in response to KCl (50 mM) in basal conditions (control) and following addition of
increasing concentrations of compound 18 alone (panel A) or in combination with nifedipine (10 µM) (panel B).
Table 4. Antihyperalgic Effect of Compounds 2, 3, 6, 10, 13, 18, 19, 20 in a Rat Model of Mononeuropathy dx Evaluated in the Paw
Pressure Testa
paw pressure in rats (g)b
treatment
dose icv, µg/rat
paw
before treatment
30 min
45 min
60 min
saline
saline
2
-
sn
dx
sn
dx
sn
dx
sn
dx
sn
dx
sn
dx
sn
dx
sn
dx
sn
dx
64.7 ( 4.6
27.3 ( 4.9
56.5 ( 3.7
28.7 ( 3.8
63.1 ( 5.0
29.4 ( 4.1
60.1 ( 3.7
25.8 ( 2.8
60.5 ( 4.8
27.8 ( 3.6
62.3 ( 4.1
30.5 ( 3.8
57.3 ( 3.9
30.1 ( 4.0
62.6 ( 5.2
32.2 ( 4.3
56.9 ( 4.2
25.7 ( 3.0
62.3 ( 5.7
24.2 ( 6.3
59.1 ( 7.1
47.3 ( 4.8∧
59.5 ( 5.8
61.7 ( 5.3*
57.5 ( 4.2
50.3 ( 4.4*
57.3 ( 5.4
33.4 ( 6.5
65.0 ( 7.2
28.4 ( 5.1
62.3 ( 7.3
55.1 ( 6.6*
55.9 ( 5.8
62.5 ( 8.7*
55.7 ( 4.9
28.1 ( 3.5
58.1 ( 4.5
25.1 ( 6.6
57.3 ( 6.4
43.5 ( 5.8∧
64.7 ( 6.3
58.3 ( 7.9*
61.2 ( 5.6
52.6 ( 5.0*
55.6 ( 6.3
38.6 ( 7.1
56.9 ( 6.6
26.7 ( 7.0
55.6 ( 6.7
54.8 ( 4.9*
61.3 ( 6.4
60.3 ( 5.3*
61.3 ( 6.5
33.4 ( 5.1
60.9 ( 6.1
25.4 ( 5.5
59.2 ( 5.1
38.6 ( 6.2
60.6 ( 7.5
54.8 ( 6.8*
63.0 ( 3.9
46.5 ( 6.7∧
59.4 ( 5.3
40.5 ( 5.9
59.3 ( 6.3
32.0 ( 3.7
54.7 ( 6.8
59.3 ( 8.1*
63.2 ( 7.0
55.8 ( 3.2*
66.4 ( 4.9
30.1 ( 4.2
-
30
30
50
50
10
10
30
30
30
30
50
50
50
50
40
40
2
3
3
6
6
10
10
15
15
18
18
19
19
20
20
a There were 9 to 10 rats per group. Each value represents the mean of two separate experiments. All compounds were administered
30 min before test. Vehicle: H2O/DMSO (2:1). b *P < 0.001; ∧P < 0.05 in comparison with controls.
a software for systematic names in organic chemistry. When
reactions were performed in anhydrous conditions, the mix-
tures were maintained under nitrogen.
therefore the experimental conditions are very different
from that observable in acute pain. However, it is
remarkable that compounds 3 and 18 are active in both
models and are antagonist of the N-type calcium chan-
nels with a potency close to that of ω-conotoxin MVIIA.
In conclusion we have identified a new series of
compounds that possess antinociceptive activity and
very likely work as N-type calcium channel antagonists.
Among them, compounds 3 and 18 are potent and
efficacious molecules that can be used as leads to
develop more potent and selective drugs.
(1-Benzylpiperidin-4-yl)-[2-(3,4-dimethoxyphenyl)ethyl]-
amine (1). A mixture of commercially available 1-benzyl-4-
piperidone (0.5 g, 2.64 mmol), 2-(3,4-dimethoxyphenyl)-
ethylamine (0.45 mL, 2.64 mmol) and titanium(IV) isopropoxide
(1 mL, 3.22 mmol) was stirred with a drying tube at room
temperature. After 4 h, the IR spectrum of the mixture showed
the absence of the carbonyl band, and the viscous solution was
diluted with absol ethanol (2 mL). Sodium cyanoborohydride
(0.3 g, 5.0 mmol) was added, and the solution was stirred for
20 h. Water (0.5 mL) was then added, the resulting inorganic
precipitate was filtered and washed with ethanol, and the
filtrate was concentrated in vacuo. The crude product was
dissolved in chloroform, filtered to remove the solids, and
washed with a solution of NaHCO3 and water; after drying
with Na2SO4, the solvent was removed under reduced pressure,
and the crude substance was purified by flash chromatography
using CHCl3/MeOH (90:10) as eluting system. Title compound
(620 mg, 88% yield) was obtained as an oil. IR (neat) ν cm-1
Experimental Section
Chemistry. All melting points were taken on a Bu¨chi
apparatus and are uncorrected. Infrared spectra were recorded
with a Perkin-Elmer 681 or a Perkin-Elmer Spectrum RX I
FT-IR spectrophotometer in Nujol mull for solids and neat for
liquids. NMR spectra were recorded on a Gemini 200 spec-
trometer. Chromatographic separations were performed on a
silica gel column by gravity chromatography (Kieselgel 40,
0.063-0.200 mm; Merck) or flash chromatography (Kieselgel
40, 0.040-0.063 mm; Merck). Yields are given after purifica-
tion, unless otherwise stated. Where analyses are indicated
by symbols, the analytical results are within (0.4% of the
theoretical values. Compounds were named following IUPAC
rules as applied by Beilstein-Institut AutoNom (version 2.1),
1
3300 (NH). H NMR (CDCl3) δ 1.21-1.59 (m, 3H, NH, CH2);
1.66-2.12 (m, 4H, 2CH2); 2.38-2.45 (m, 1H, CH); 2.65-2.88
(m, 6H, 3CH2); 3.45 (s, 2H, CH2); 3.84 (s, 6H, 2OCH3); 6.64-
6.76 (m, 3H, aromatics); 7.28 (s, 5H, aromatics) ppm.
The oily product was transformed into the hydrochloride,
treating the free base with HCl/absol EtOH and recrystallized

N-Type Calcium Channel Blockers
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6077
from absol EtOH/anhydrous ether. Mp: 260-265 °C. Anal.
1,2-dimetoxybenzene29). Their chemical and physical charac-
1
teristics are reported in Table 1 and IR and H NMR spectra
(C22H31ClN2O2) C, H, N.
in Table 5.
Compounds 21 and 22 were obtained in the same way using
4-Cyano-4-(3,4-dimethoxyphenyl)-5-methylhexanoic
Acid (28). To a solution of KMnO4 (310 mg, 1.96 mmol) in 3.2
mL of H2O at 0 °C, were added a solution of 2-(3,4-dimethoxy-
phenyl)-5-hydroxy-2-(methylethyl)pentanenitrile31 (200 mg,
0.72 mmol), dissolved in 2 mL of benzene, and 30 mg of
tetrabutylammonium bromide. The mixture was left at room
temperature for 12 h, and then a solution of NaHSO4 (3 mL)
was added. The mixture was then acidified with 6 N HCl and
extracted with diethyl ether. The organic layer was dried with
Na2SO4, and the solvent was removed under reduced pressure.
Title compound (190 mg, 90% yield) was obtained as a white
the suitable commercially available amine (2-phenoxyethyl-
1
amine and butylamine, respectively). Their IR and H NMR
spectra are consistent with the proposed structures.
5-(1-Benzylpiperidin-4-ylamino)-2-(3,4-dimethoxyphen-
yl)-2-(methylethyl)pentanenitrile (23). Commercially avail-
able 4-amino-1-benzylpiperidine (0.46 mL, 2.23 mmol), 5-bromo-
2-(3,4-dimethoxyphenyl)-2-(methylethyl)pentanenitrile24 (760
mg, 2.23 mmol) and 0.26 mL of dry triethylamine were heated
at 60 °C for 24h. The reaction mixture was cooled to room
temperature and treated with CH2Cl2, and the organic layer
was washed with 10% NaOH solution. After drying with Na2-
SO4, the solvent was removed under reduced pressure and the
residue purified by flash chromatography using CH2Cl2/Et2O/
absol EtOH/NH4OH (900:360:180:9.9) as eluting system. Title
compound (560 mg, 56% yield) was obtained as an oil. IR (neat)
ν cm-1 3300 (NH), 2240 (CN). 1H NMR (CDCl3) δ 0.78 (d, J )
6.6 Hz, 3H, CH3); 1.21 (d, J ) 6.6 Hz, 3H, CH3); 1.25-1.60
(m, 6H, 3CH2); 1.65-2.21 (m, 6H, NH, CH, 2CH2); 2.25-2.69
(m, 3H, CH, CH2); 2.75-2.89 (m, 2H, CH2); 3.47 (s, 2H, CH2);
3.89 (s, 6H, 2OCH3); 6.79-6.85 (m, 3H, aromatics); 7.26 (s,
5H, aromatics) ppm. Anal. (C28H39N3O2) C, H, N.
oil. IR (neat) ν 2500-3000 (OH), 2240 (CN), 1700 (CO) cm-1
.
1H NMR (CDCl3): δ 0.80 (d, J ) 6.5 Hz, 3H, CH3); 1.21 (d, J
) 6.2 Hz, 3H, CH3); 1.98-2.21 (m, 3H, CH, CH2); 2.36-2.58
(m, 2H, CH2); 3.89 (s, 6H, 2OCH3); 6.83-6.91 (m, 3H, aromat-
ics); 7.98 (bs, 1H, OH) ppm. Anal. (C16H21NO4) C, H, N.
2-(3,4-Dimethoxyphenyl)-5-(4-[2-(3,4-dimethoxyphenyl)-
ethylamino]piperidin-1-yl)-2-(methylethyl)-5-oxopen-
tanenitrile (13). Compound 24 (140 mg, 0.53 mmol), 5-cyano-
4-(3,4-dimethoxyphenyl)-5-methylhexanoic acid 28 (155 mg,
0.53 mmol), diethyl cyanophosphonate (0.1 mL, 0.66 mmol),
dry triethylamine (0.7 mL) and dry DMF (1.5 mL) were mixed
at 0 °C and then stirred at room temperature for 12 h. Brine
was added, the reaction mixture was extracted with ethyl
acetate, and the organic layer was washed first with brine and
then with a solution of NaHCO3. After drying with Na2SO4,
the solvent was removed under reduced pressure and the
residue purified by column chromatography using CHCl3/absol
EtOH/petroleum ether/NH4OH (340:65:60:8) as eluting system.
Title compound (90 mg, 32% yield) was obtained as an oil. IR
(neat) ν cm-1 3300 (NH), 2240 (CN), 1650 (CO). 1H NMR
(CDCl3) δ 0.81 (d, J ) 6.6 Hz, 3H, CH3); 1.18-1.32 (m, 6H,
CH, CH2, CH3); 1.70-2.95 (m, 16H, 7CH2, NH, CH); 3.83 (s,
6H, 2OCH3); 3.92 (s, 6H, 2OCH3); 6.67-6.98 (m, 6H, aromatics)
ppm. The oily product was transformed into the oxalate that
was recrystallized from ethyl acetate. Mp: 80-82 °C. Anal.
(C33H45N3O9) C, H, N.
[2-(3,4-Dimethoxyphenyl)ethyl]piperidin-4-ylamine (24).
To 310 mg (0.87 mmol) of 1 dissolved in dry MeOH were added
Pd/C 10% (0.15 g) and HCOONH4 (276 mg, 4.37 mmol). The
reaction mixture was heated to reflux for 8 h; after cooling,
the solvent was filtered and evaporated. The residue was
dissolved in CH2Cl2 and washed with a solution of NaHCO3;
after drying with Na2SO4, the solvent was removed under
reduced pressure and the residue purified by column chroma-
tography using CHCl3/absol EtOH/petroleum ether/NH4OH
(340:65:60:8) as eluting system. Title compound (140 mg, 61%
1
yield) was obtained as an oil. IR (neat) ν cm-1 3300 (NH). H
NMR (CDCl3) δ 1.09-1.29 (m, 2H, CH2); 1.71-1.89 (m, 2H,
CH2); 2.09 (bs, 2H, 2NH); 2.42-3.09 (m, 9H, 4CH2, CH); 3.81
(s, 6H, 2OCH3); 6.68-6.81 (m, 3H, aromatics) ppm. Anal.
(C15H24N2O2) C, H, N.
Compounds 14 and 15 were obtained in the same way
starting from (2-ethoxyphenyl)piperidin-4-ylamine 25 and
butylpiperidin-4-ylamine 26, respectively. Their chemical and
physical characteristics are reported in Table 1 and IR and
1H NMR spectra in Table 5.
N-(1-Benzylpiperidin-4-yl)-4,4-bis(4-fluorophenyl)bu-
tyramide (29). Following the procedure described for com-
pound 13, starting from 1-benzyl-4-aminopiperidine (0.6 g, 3.16
mmol) and 4,4′-bis-(4-fluorophenyl)butyric acid26 (0.86 g, 3.16
mmol), compound 29 (430 mg, 30% yield) was obtained as a
colorless oil. IR (neat) ν cm-1 3300 (NH), 1650 (CO). 1H NMR
(CDCl3) δ 1.20-1.56 (m, 2H, CH2); 1.81-1.98 (m, 2H, CH2);
2.01-2.18 (m, 4H, 2CH2); 2.22-2.41 (m, 2H, CH2); 2.75-2.95
(m, 2H, CH2); 3.45 (s, 2H, CH2); 3.63-3.78 (m, 1H, CH); 3.83
(t, J ) 9.5 Hz, 1H, CH); 5.24 (bs, 1H, NH); 6.98-7.03 (m, 4H,
aromatics); 7.06-7.14 (m, 4H, aromatics); 7.25 (s, 5H, aromat-
ics) ppm. Anal. (C28H30F2N2O) C, H, N.
Compounds 25, 26 and 27 were obtained in the same way
from the corresponding benzyl derivative (21, 22 and 23,
respectively). Their IR and 1H NMR spectra are consistent with
the proposed structures.
2-(3,4-Dimethoxyphenyl)-5-(4-[2-(3,4-dimethoxyphenyl)-
ethylamino]piperidin-1-yl)-2-(methylethyl)pentaneni-
trile (5). Compound 24 (0.11 mg, 0.41 mmol), 5-bromo-2-(3,4-
dimethoxyphenyl)-2-(methylethyl)pentanenitrile24 (140 mg,
0.41 mmol), 0.1 mL of dry triethylamine and 1 mL of aceto-
nitrile were heated at 60 °C for 8 h. The reaction mixture was
cooled to room temperature and treated with CH2Cl2, and the
organic layer was washed with 10% NaOH solution. After
drying with Na2SO4, the solvent was removed under reduced
pressure and the residue purified by column chromatography
using CHCl3/absol EtOH/petroleum ether/NH4OH (340:65:60:
8) as eluting system. Title compound (120 mg, 56% yield) was
obtained as an oil. IR (neat) ν cm-1 3300 (NH), 2240 (CN). 1H
NMR (CDCl3) δ 0.81 (d, J ) 6.6 Hz, 3H, CH3); 1.18 (d, J ) 6.6
Hz, 3H, CH3); 1.10-1.37 (m, 2H, CH2); 1.43-1.67 (m, 2H, CH2);
1.71-1.96 (m, 6H, NH, 2CH2, CH); 1.99-2.13 (m, 2H, CH2);
2.16-2.29 (m, 2H, CH2); 2.32-2.49 (m, 1H, CH); 2.59-2.96
(m, 6H, 3CH2); 3.83 (s, 6H, 2OCH3); 3.90 (s, 6H, 2OCH3); 6.68-
6.98 (m, 6H, aromatics) ppm.
4,4-Bis(4-fluorophenyl)-N-piperidin-4-ylbutyramide
(16). Following the procedure described for compound 24,
starting from 29 (430 mg, 0.99 mmol), compound 16 (220 mg,
62% yield) was obtained. IR (neat) ν cm-1 3300 (NH), 1650
1
(CO). H NMR (CDCl3) δ 1.18-1.28 (m, 2H, CH2); 1.78-1.85
(m, 2H, CH2); 1.98-2.09 (m, 2H, CH2); 2.20-2.34 (m, 3H, CH2,
NH); 2.59 (t, J ) 13.0 Hz, 2H, CH2); 2.94-3.02 (m, 2H, CH2);
3.64-3.79 (m, 1H, CH); 3.83 (t, J ) 9.5 Hz, 1H, CH); 5.67 (bs,
1H, NH); 6.84-6.90 (m, 4H, aromatics); 7.08-7.23 (m, 4H,
aromatics) ppm.
The oily product was transformed into the oxalate that was
recrystallized from ethyl acetate. Mp: 198-200 °C. Anal.
(C33H47N3O8) C, H, N.
Compounds 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 were obtained in
the same way using the suitable halide (5-bromo-2-(3,4-
dimethoxyphenyl)-2-(methylethyl)pentanenitrile,24 5-bromo-2-
(methylethyl)-2-phenylpentanenitrile (33), 1-bromo-4,4-diphen-
ylbutane,27 1,1′-(4-bromobutylidene)bis(4-fluorobenzene),26 bu-
tyl iodide (commercially available), (2-bromoethoxy)benzene,28
cinnamyl bromide (commercially available), 4-(2-bromoethyl)-
The oily product was transformed into the oxalate that was
recrystallized from ethyl acetate. Mp: 100-102 °C. Anal.
(C23H26F2N2O5) C, H, N.
4,4-Bis(4-fluorophenyl)-N-[1-(2-hydroxyethyl)piperidin-
4-yl]butyramide (17). Following the procedure described for
compound 5, starting from 16 (240 mg, 0.67 mmol) and
chloroethanol (0.04 mL, 0.75 mmol), compound 17 (120 mg,

6078 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Table 5. IR and 1H NMR Spectra of Compounds 2-4, 6-15
Teodori et al.
N
IR ν (cm-1
)
1H NMR (CDCl3) δ
2
3300 (NH),
0.77 (d, J ) 6.6 Hz, 3H, CH3); 0.91-0.94 (m, 3H, CH3); 1.17 (d, J ) 6.6 Hz, 3H, CH3); 1.22-1.67
(m, 9H, CH, 4CH2); 1.69-2.44 (m, 9H, NH, 4CH2); 2.54-2.61 (m, 5H, CH, 2CH2); 3.86 (s, 6H,
2OCH3); 6.82-6.88 (m, 3H, aromatics) ppm
0.74 (d, J ) 6.6 Hz, 3H, CH3); 1.28 (d, J ) 6.6 Hz, 3H, CH3); 1.27-1.66 (m, 7H, CH, 3CH2);
1.73-1.98 (m, 5H, NH, 2CH2); 1.98-2.14 (m, 2H, CH2); 2.20-2.34 (m, 2H, CH2); 2.36-2.57
(m, 1H, CH); 2.64-2.85 (m, 2H, CH2); 3.86 (s, 6H, 2OCH3); 4.05 (t, J ) 6.3 Hz, 2H, CH2O);
6.60-7.00 (m, 6H, aromatics); 7.19-7.33 (m, 2H, aromatics) ppm
0.76 (d, J ) 6.6 Hz, 3H, CH3); 1.18 (d, J ) 6.6 Hz, 3H, CH3); 1.25-1.63 (m, 4H, 2CH2); 1.65-1.98
(m, 6H, NH, CH, 2CH2); 2.05-2.31 (m, 4H, 2CH2); 2.36-2.57 (m, 1H, CH); 2.78-2.84 (m, 6H,
3CH2); 3.85 (s, 6H, 2OCH3); 6.72-6.76 (m, 3H, aromatics); 7.27-7.36 (m, 5H, aromatics) ppm
0.94 (t, J ) 6.3 Hz, 3H, CH3); 1.35-1.46 (m, 8H, 4CH2); 1.76-2.15 (m, 7H, NH, 3CH2); 2.50-2.60
2240 (CN)
3
3300 (NH),
2240 (CN)
4
6
3300 (NH),
2240 (CN)
3300 (NH)
(m, 3H, CH, CH2); 2.61 (t, J ) 8.6 Hz, 2H, CH2); 2.88-2.55 (m, 2H, CH2); 3.90 (t, J ) 9.5 Hz, 1H, CH);
7.25-7.28 (m, 10H, aromatics) ppm
1.12-1.53 (m, 5H, NH, 2CH2); 1.73-2.12 (m, 6H, 3CH2); 2.26-2.38 (m, 3H, CH, CH2); 2.66-2.93
(m, 6H, 3CH2); 3.85 (s, 7H, CH, 2OCH3); 6.69-6.85 (m, 3H, aromatics); 7.09-7.33 (m, 10H, aromatics) ppm
1.25-1.53 (m, 5H, NH, 2CH2); 2.73-2.06 (m, 6H, 3CH2); 2.19-2.53 (m, 3H, CH, CH2); 2.66-2.92
(m, 6H, 3CH2); 3.86 (s, 7H, CH, 2OCH3); 6.73-6.78 (m, 3H, aromatics); 6.91-7.11 (m, 4H,
aromatics); 7.12-7.18 (m, 4H, aromatics) ppm
0.76 (d, J ) 6.6 Hz, 3H, CH3); 0.87 (t, J ) 7.1 Hz, 3H, CH3); 1.16 (d, J ) 6.6 Hz, 3H, CH3);
1.19-2.60 (m, 8H, NH, CH, 3CH2); 1.69-2.15 (m, 8H, 4CH2); 2.17-2.42 (m, 4H, 2CH2); 2.44-2.69
(m, 1H, CH); 2.76-2.92 (m, 2H, CH2); 3.86 (s, 6H, 2OCH3); 6.82-6.87 (m, 3H, aromatics) ppm
0.79 (d, J ) 6.6 Hz, 3H, CH3); 1.19 (d, J ) 6.6 Hz, 3H, CH3); 1.28-1.69 (m, 3H, CH, CH2);
1.76-2.25 (m, 9H, NH, 4CH2); 2.34-2.69 (m, 3H, CH, CH2); 2.78 (t, J ) 5.9 Hz, 2H, CH2);
2.85-3.01 (m, 2H, CH2); 3.87 (s, 6H, 2OCH3); 4.06 (t, J ) 6.1 Hz, 2H, CH2O); 6.84-6.91 (m, 6H,
aromatics); 7.21-7.25 (m, 2H, aromatics) ppm
0.80 (d, J ) 6.6 Hz, 3H, CH3); 1.19 (d, J ) 6.6 Hz, 3H, CH3); 1.36-1.65 (m, 3H, CH, CH2);
1.79-2.30 (m, 8H, 4CH2); 2.41-2.73 (m, 4H, NH, CH, CH2); 2.92-3.09 (m, 2H, CH2); 3.23 (d, J ) 8.1 Hz,
2H, CH2); 3.89 (s, 6H, 2OCH3); 6.19-6.39 (m, 1H, CH); 6.54 (d, J ) 16.1 Hz, 1H, CH); 6.85-6.90
(m, 3H, aromatics); 7.27-7.36 (m, 5H, aromatics) ppm
0.78 (d, J ) 6.6 Hz, 3H, CH3); 1.18 (d, J ) 6.6 Hz, 3H, CH3); 1.28-1.63 (m, 3H, CH,CH2);
1.73-2.28 (m, 8H, 4CH2); 2.30-2.80 (m, 8H, NH, CH, 3CH2); 2.85-3.01 (m, 2H, CH2); 3.83
(m, 6H, 2OCH3); 3.89 (m, 6H, 2OCH3); 6.73-6.90 (m, 6H, aromatics) ppm
0.78 (d, J ) 6.6 Hz, 3H, CH3); 1.23 (d, J ) 6.6 Hz, 3H, CH3); 1.15 1.39 (m, 3H, CH, CH2);
1.76-2.15 (m, 5H, CH, 2CH2); 2.22-2.85 (m, 5H, CH, 2CH2); 2.90-3.03 (m, 2H, CH2);
3.50-3.69 (m, 1H, CH); 3.79 (s, 6H, 2OCH3); 4.06 (t, J ) 6.1 Hz, 2H, CH2); 4.37-4.50 (m, 1H, CH);
6.92-6.93 (m, 5H, aromatics); 7.22-7.27 (m, 3H, aromatics) ppm
0.77 (d, J ) 6.6 Hz, 3H, CH3); 0.89 (t, J ) 6.4 Hz, 3H, CH3); 1.18 (d, J ) 6.6 Hz, 3H, CH3);
1.10-1.53 (m, 5H, CH, 2CH2); 1.73-2.50 (m, 9H, CH, 4CH2); 2.53-2.73 (m, 4H, 2CH2);
2.80-3.03 (m, 1H, CH); 3.50-3.69 (m, 1H, CH); 3.87 (s, 6H, 2OCH3); 4.37-4.50 (m, 1H, CH);
6.82-6.84 (m, 3H, aromatics) ppm
7
8
3300 (NH)
3300 (NH)
9
3300 (NH),
2240 (CN)
10
3300 (NH),
2240 (CN)
11
3300 (NH)
12
14
3300 (NH),
2240 (CN)
3300 (NH),
2240 (CN),
1650 (CO)
15
3300 (NH),
2240 (CN),
1650 (CO)
44% yield) was obtained. IR (neat) ν cm-1 3500 (OH), 3300
(NH), 1650 (CO). 1H NMR (CDCl3) δ 1.17-1.46 (m, 2H, CH2);
1.89-1.99 (m, 2H, CH2); 2.02-2.24 (m, 4H, 2CH2); 2.26-2.28
(m, 2H, CH2); 2.50 (t, J ) 6.0 Hz, 2H, CH2); 2.60-2.84 (m,
3H, CH2, OH); 3.57 (t, J ) 5.0 Hz, 2H, CH2); 3.65-3.83 (m,
1H, CH); 3.90 (t, J ) 9.0 Hz, 1H, CH); 5.42 (bs, 1H, NH); 6.91-
7.00 (m, 4H, aromatics); 7.12-7.27 (m, 4H, aromatics) ppm.
The oily product was transformed into the hydrochloride by
treating the free base with HCl/absol EtOH and recrystallized
from absol EtOH/anhydrous ether Mp: 170-175 °C. Anal.
(C23H29ClF2N2O2) C, H, N.
by flash chromatography using CHCl3 as eluent. Title com-
pound (480 mg, 45% yield) was obtained as a green oil. IR
1
(neat) ν cm-1 3300-3400 (NH2) 1650 (CO). H NMR (CDCl3)
δ 0.92-1.19 (m, 2H, CH2); 1.54-1.65 (m, 4H, NH2, CH2); 2.02-
2.35 (m, 4H, 2CH2); 2.46 (t, J ) 12.8 Hz, 1H, CH); 2.62-2.85
(m, 2H, CH2); 3.37-3.59 (m, 1H, CH); 3.84 (t, J ) 9.0 Hz, 1H,
CH); 4.25-4.32 (m, 1H, CH); 6.75-6.98 (m, 4H, aromatics);
7.01-7.15 (m, 4H, aromatics) ppm.
The oily product was transformed into the hydrochloride,
treating the free base with HCl/absol EtOH and recrystallized
from absol EtOH/anhydrous ether. Mp: 138-141 °C. Anal.
(C21H25ClF2N2O) C, H, N.
(1-{1-[4,4-Bis(4-fluorophenyl)butyryl]piperidin-4-yl-
carbamoyl}-3-methylbutyl)carbamic Acid tert-Butyl Es-
ter (31). Following the procedure described for 13, starting
from 18 (100 mg, 0.28 mmol) and N-t-BOC-L-leucine (70 mg,
0.28 mmol), compound 31 (110 mg, 70% yield) was obtained
as an oil. IR (neat) ν cm-1 3300 (NH), 1650 (CO). 1H NMR
(CDCl3) δ 0.82-0.98 (m, 6H, 2CH3); 1.12-1.41 (m, 4H, 2CH2);
1.40 (s, 9H, 3CH3); 1.58-1.66 (m, 2H, CH2); 1.82-1.98 (m, 2H,
CH2); 2.18-2.39 (m, 3H, CH, CH2); 2.64-2.85 (m, 1H, CHH);
2.92-3.12 (m, 1H, CHH); 3.53-3.65 (m, 1H, CH); 3.83-4.10
(m, 3H, CH2, NH); 4.38-4.44 (m, 1H, CH); 4.84-4.98 (m, 1H,
CH); 6.25 (bs, 1H, NH); 6.90-7.04 (m, 4H, aromatics); 7.15-
7.22 (m, 4H, aromatics) ppm. Anal. (C32H43F2N3O4) C, H, N.
(1-{1-[4,4-Bis(4-fluorophenyl)butyryl]piperidin-4-yl-
carbamoyl}-3-methylbutyl)methylcarbamic Acid tert-
Butyl Ester (32). Following the procedure described for 13,
starting from 18 (110 mg, 0.30 mmol) and N-t-BOC-N-methyl-
L-leucine33 (120 mg, 0.48 mmol), compound 32 (130 mg, 40%
yield) was obtained as an oil. IR (neat) ν cm-1 3300 (NH), 1650
(CO). 1H NMR (CDCl3) δ 0.82-0.98 (m, 6H, 2CH3); 1.38-1.51
(1-[4,4-Bis(4-fluorophenyl)butyryl]piperidin-4-yl)car-
bamic Acid tert-Butyl Ester (30). Following the procedure
described for compound (13), starting from 4-(tert butyloxy-
carbonylamino)piperidine32 (600 mg, 3.03 mmol) and 4,4′-bis-
(4-fluorophenyl)butyric acid26 (824 mg, 3.03 mmol), compound
30 (1.36 g, 98% yield) was obtained as an oil. IR (neat) ν cm-1
1
3300 (NH), 1720 (CO). H NMR (CDCl3) δ 1.03-1.25 (m, 2H,
CH2); 1.33-1.41 (s, 9H, 3CH3); 1.83-1.98 (m, 2H, CH2); 2.12-
2.28 (m, 4H, 2 CH2); 2.68 (t, J ) 12.8 Hz, 1H, CH); 2.83-3.03
(m, 2H, CH2); 3.42-3.65 (m, 2H, CH2); 3.92 (t, J ) 9.0 Hz,
1H, CH); 4.45 (bs, 1H, NH); 6.86-6.99 (m, 4H, aromatics);
7.04-7.31 (m, 4H, aromatics) ppm. Anal. (C26H32F2N2O3) C,
H, N.
1-(4-Aminopiperidin-1-yl)-4,4-bis(fluorophenyl)butan-
1-one (18). To a solution of 30 (1.36 g, 2.97 mmol) dissolved
in CHCl3 was added Me3SiI (1.9 mL, 13.3 mmol). The reaction
mixture was left at room temperature for 6 h, the MeOH (8
mL) was added, the organic layer was removed under reduced
pressure and the residue was dissolved in CHCl3 and washed
with NaHCO3 solution. After drying with Na2SO4, the solvent
was removed under reduced pressure and the residue purified

N-Type Calcium Channel Blockers
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24 6079
(m, 2H, CH2); 1.45 (s, 9H, 3CH3); 1.58-1.98 (m, 4H, 2CH2);
2.18-2.39 (m, 5H, CH, 2CH2); 2.65 (s, 3H, CH3); 2.71-2.70
(m, 1H, CHH); 2.82-3.10 (m, 1H, CHH); 3.53-3.65 (m, 1H,
CH); 3.83-4.10 (m, 2H, CH2); 4.38-4.54 (m, 2H, CH2); 6.20-
6.33 (m, 1H, NH); 6.90-7.04 (m, 4H, aromatics); 7.15-7.22
(m, 4H, aromatics) ppm. Anal. (C33H45F2N3O4) C, H, N.
as a reference drug. Other chemicals were of the highest
quality commercially available. The percentage value inserted
in the table has been calculated as follows: A ) maximum
analgesic effect of morphine on the hot plate expressed as
latency in s (peak of analgesia) minus the latency of pretest
(about 15 s); B ) maximum analgesic effect of compound X on
the hot plate expressed as latency in s (peak of analgesia)
minus the latency of pretest (about 15 s); percentage ) B/A ×
100.
Animals. Male Swiss albino mice (23-30 g) from Morini
(San Polo d'Enza, Italy) and male rats from Harlan-Nossan
(Correzzana, Milano, Italy) breeding farm were used. Fifteen
mice or four rats were housed per cage. The cages were placed
in the experimental room 24 h before the test for acclimatiza-
tion. The animals were kept at 23 ( 1 °C with a 12 h light/
dark cycle, light at 7 a.m., with food and water ad libitum. All
experiments were carried out according to the guidelines of
the European Community Council.
Intracerebroventricular Injections. Icv administration
was performed during brief anaesthesia according to the
method described for mice by Haley and McCormick45 that we
have adapted also for rats. Briefly, during anaesthesia, mice
(22-25 g) or rats (150-200 g) were grasped firmly by the loose
skin behind the head. A 0.4 mm external diameter, hypodermic
needle attached to a 10 µL syringe was inserted perpendicu-
larly through the skull no more than 2 mm into the brain of
mouse and 4 mm into the brain of rat, where 5 µL (for mouse)
and 10 µL (for rat) of solution (H2O/DMSO 2:1) were injected.
The injection site was 1 mm (for mouse) or 2 mm (for rat) to
the right or left of the midline along a line drawn through the
anterior base of the ears. To ascertain that the drugs were
administered exactly into the cerebral ventricle, after the test
some assayed mice and rats were icv injected with 5-10 µL of
diluted 1:10 India ink and their brains examined macroscopi-
cally after sectioning.
Analgesic Action on Chronic Constriction Injury. A
peripheral mono neuropathy was produced in adult rats by
placing loosely constrictive ligatures around the common
sciatic nerve according to the method described by Bennett
and Xie.37 Rats were anaesthetised with chloral hydrate. The
common sciatic nerve was exposed at the level of the middle
of the thigh by blunt dissection through biceps femoris.
Proximal to sciatic nerve trifurcation, about 1 cm of the nerve
was freed of adhering tissue and four ligatures (3/0 silk tread)
were tied loosely around it with about 1 mm spacing. The
length of the nerve thus affected was 4-5 mm long. Great care
was taken to tie the ligatures such that the diameter of the
nerve was seen to be just barely constricted when viewed with
40× magnification. In every animal, an identical dissection
was performed on the opposite side except that the sciatic
nerve was not ligated. The left paw was untouched.
2-Amino-4-methylpentanoic Acid {1-[4,4-bis(4-fluo-
rophenyl)butyryl]piperidin-4-yl}amide (19). To a solution
of 31 (110 mg, 0.19 mmol) dissolved in ethyl acetate was added
2 N HCl (4 mL). The reaction mixture was left at room
temperature for 24 h, and then the solution was basified with
10% NaOH solution and extracted with CH2Cl2. After drying
with Na2SO4, the solvent was removed under reduced pressure
and the residue purified by column chromatography using
CHCl3/CH3OH (90:10) as eluting system. Title compound (60
mg, 69% yield) was obtained as a colorless oil. IR (neat) ν cm-1
3300-3400 (NH and NH2); 1630-1650 (2CO). 1H NMR
(CDCl3) 0.82-0.98 (m, 6H, 2CH3); 1.02-1.41 (m, 2H, CH2);
1.45-1.78 (m, 4H, NH2, CH2); 1.80-1.98 (m, 2H, CH2); 2.18-
2.41 (m, 5H, CH, 2CH2); 2.61-2.82 (m, 1H, CHH); 2.95-3.19
(m, 1H, CHH); 3.33-3.39 (m, 1H, CH); 3.55-3.65 (m, 1H, CH);
3.81-4.05 (m, 2H, CH2); 4.38-4.56 (m, 1H, CH); 6.86-7.04
(m, 4H, aromatics); 7.15-7.22 (m, 4H, aromatics); 7.25-7.38
(m, 1H, NH) ppm.
The oily product was transformed into the hydrochloride,
treating the free base with HCl/absol EtOH and recrystallized
from absol EtOH/anhydrous ether. Mp 165-167 °C. Anal.
(C27H36ClF2N3O2) C, H, N.
4-Methyl-2-methylaminopentanoic Acid {1-[4,4-bis(4-
fluorophenyl)butyryl]piperidin-4-yl}amide (20). Follow-
ing the procedure described for 19, starting from 32 (130 mg,
0.22 mmol), compound 20 (40 mg, 38% yield) was obtained as
an oil. IR (neat) ν cm-1 3300-3400 (NH and NH2); 1630-1650
1
(2CO). H NMR (CDCl3) 0.82-1.05 (m, 6H, 2CH3); 1.08-1.41
(m, 3H, CH,CH2); 1.45-1.78 (m, 3H, NH, CH2); 1.80-1.98 (m,
2H, CH2); 2.18-2.41 (m, 7H, CH3, 2CH2); 2.62-2.85 (m, 1H,
CHH); 2.91-3.12 (m, 2H, CH, CHH); 3.53-3.69 (m, 1H, CH);
3.85-4.05 (m, 2H, CH2); 4.41-4.58 (m, 1H, CH); 6.86-7.04
(m, 4H, aromatics); 7.15-7.22 (m, 4H, aromatics); 7.25-7.38
(m, 1H, NH) ppm.
The oily product was transformed into the hydrochloride,
treating the free base with HCl/absol EtOH and recrystallized
from absol EtOH/anhydrous ether. Mp 85-87 °C. Anal.
(C28H38ClF2N3O2) C, H, N.
5-Bromo-2-(methylethyl)-2-phenylpentanenitrile (33).
3-Methyl-2-phenylbutyronitrile30 (0.5 g, 3.14 mmol) was dis-
solved in 10 mL of anhydrous THF and cooled to -78 °C; 2.7
mL (4.32 mmol) of n-butyllithium (1.6 M in hexane) was added,
and the mixture was left at -78 °C for 1.5 h. Then 0.64 mL
(6.37 mmol) of 1,3-dibromopropane was added, and the mixture
was allowed to warm to room temperature, treated with a
saturated solution of NH4Cl and extracted with diethyl ether.
The organic layer was dried over Na2SO4, the solvent removed
under reduced pressure, and the residue purified by column
chromatography using CH2Cl2 (100) as eluting system. Title
compound (520 mg, 59% yield) was obtained as an oil. IR (neat)
ν cm-1 2240 (CN). 1H NMR (CDCl3) δ 0.79 (d, J ) 5.5 Hz, 3H,
CH3); 1.23 (d, J ) 5.5 Hz, 3H, CH3); 1.40-1.59 (m, 1H, CH);
1.88-2.42 (m, 4H, 2CH2); 3.33 (t, J ) 3.6 Hz, 2H, CH2); 7.27-
7.40 (m, 5H, aromatics) ppm. Anal. (C14H18BrN) C, H, N.
Pharmacology. Analgesic Activity. Hot-Plate Test. The
method adopted was described by O’Callaghan and Holzman.34
Mice were placed inside a stainless steel container, thermo-
statically set at 52.5 ( 0.1 °C in a precision water-bath from
KW Mechanical Workshop, Siena, Italy. Reaction times (s)
were measured with a stop-watch before icv injections and at
regular intervals (15 min) up to a maximum of 60 min after
treatment (cutoff) in order to prevent tissue damage. The
endpoint used was the licking of the fore or hind paws.
Antinociception was seen as increased latencies to the re-
sponses evaluated, while increased nociception was seen by
shorter latencies. Those mice scoring below 12 and over 18 s
in the pretest were rejected (30%). An arbitrary cutoff time of
45 s was adopted. Morphine hydrochloride (SALARS) was used
Paw Pressure Test. The nociceptive threshold in the rat
was determined with an analgesimeter (Ugo Basile, Varese,
Italy), according to the method described by Leighton.38 The
instrument exerts a force which is applied at a costant rate
(32 g/s) with a cone-shaped pusher on the upper surface of the
rat hind paw. The force is continuously monitored by a pointer
moving along a linear scale. The pain threshold is given by
the force which induces the first struggling from the rat.
Pretested rats which scored below 40 g or over 75 g during
the test before drug administration (25%) were rejected. An
arbitrary cut-off value of 250 g was adopted.
Statistical Analysis. All experimental results are given
as the mean ( SEM. Analysis of variance (ANOVA), followed
by Fisher’s Protected Least Significant Difference (PLSD)
procedure for post-hoc comparison, was used to verify signifi-
cance between two means. Data were analyzed with the
StatView software for the Macintosh (1992). P values of less
than 0.05 were considered significant.
Binding Assays. Reversible, saturable and high affinity
binding sites were detected for [3H]D888 and [3H]PN200-110
in rat ventricle membranes: the KD amounted to 4.75 ( 1.05
nM for [3H]D888 and 98.10 ( 2.55 pM for [3H]PN200-110 and
the maximal binding capacities (Bmax) were 972.73 ( 223.73

6080 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 24
Teodori et al.
fmol/mg protein and 220.41 ( 34.08 fmol/mg protein for [3H]-
with 5% fetal bovine serum, 10% horse serum, 2 mM L-
glutamine. Before each experiment the cells were kept in
serum-free medium overnight.
D888 and [3H]PN200-110, respectively.
(-)-[N-Methyl-3H]desmethoxyverapamil
((-)-[3H]-
Measurement of [Ca2] Fluxes. PC12 cells, prepared as
described above, were loaded with 2 µM Fura-2/AM for 45 min
at 37 °C, washed, resuspended in Krebs Henseleit HEPES
(KHH) medium (140 mM Na+, 5.3 mM K+, 132.4 mM Cl-, 1
mM Ca++, 0.81 mM Mg2+, 5.5 mM glucose, 20.3 mM HEPES,
2.5 mM Na-pyruvate, 2 mM HEPES, 0.1% BSA, pH 7.5).
[Ca2+]i was measured as described previously using a spec-
trofluorimetric method35,36 employing a Perkin-Elmer LS50B
instrument equipped with a fast rotary filter shuttle for
alternate 340 and 380 nm excitation. KCl (50 mM) was added
directly in the cuvette either in the absence or presence of the
different concentrations of the tested compounds. In addition
the effect of the different compounds was tested also in the
presence of the L-type calcium channel inhibitor nifedipine at
the concentration of 10 µM. Each compound was initially
dissolved in DMSO (100%) at the concentration of 10 mM, and
further dilutions were performed in BSA-containing KHH
D-888) Binding Assays. Membrane Preparation. Hearts
were rapidly removed from male Sprague- Dawley rats (200-
250 g), the ventricles placed in ice-cold 20 mmol/L NaHCO3
supplemented with 0.1 mmol/L phenylmethylsulfonyl-fluoride
(PMSF), pH 7.4, and the wet weight-to-volume ratio was 1:10.
The membrane preparations were performed as described by
Goll et al.46 (with modification). Briefly, the tissue was
homogenized by 3 × 20 s with ultraturrax, and the crude
homogenate was centrifuged at 300g for 15 min. The resultant
pellet was resuspended 50 mmol/L Tris-HCl, 0.1 mmol/L PMSF
(pH 7.4) and centrifuged again. The supernatants of both runs
were collected and spun at 45 000g for 15 min. The pellets were
then suspended in the same buffer and sedimented twice at
45 000g for 15 min. The final crude membrane was stored at
-80 °C until use.
Binding Experiments. Binding assays for (-)-[3H]D-888
(86 Ci/mmol, Amersham Life Science) were performed in a
final volume of 0.5 mL; the frozen pellet was suspended in 50
mmol/L Tris-HCl supplemented with 0.1 mmol/L PMSF (pH
7.4) to a final concentration of 0.3-0.6 mg/mL and was
incubated at 25 °C for 60 min. In homologous competition
curves, the marker ligand was present at 0.5 nM in tubes
containing increasing concentrations of unlabeled D-888 (0.5
nM to 0.1 mM) and at 0.0625-0.5 nM in tubes without
unlabeled ligand. In heterologous competition curves fixed
concentrations of the tracer (0.5 nM) were displaced by
increasing concentrations of several unlabeled ligands (0.1 nM-
0.1 mM). The incubation was termined by filtration through
Whatman GF/C glass filter presoaked in buffer 20 mmol/L
Tris-HCl plus 10 mmol/L MgCl2 and 10% (poly(ethylene
glycol)) PEG 4000, pH 7.4, using a Brandel M-48R well cell
harvester. Filters were washed twice with 5 mL of 20 mmol/L
Tris-HCl. In all experiments, the radioactivity retained by
filters was measured in a liquid scintillation counter (TRI-
CARB 1900TR, Packard) after the addition of 4 mL of
scintillation fluid (Filter Count, Packard), and all measure-
ments were obtained in duplicate. Protein was measured by
the method of Bradford,47 using bovine serum albumine as
standard. The binding data were evaluated quantitatively with
nonlinear least-squares curve fittings using the computer
program LIGAND:48 this analysis provides optimal estimates
of “binding parameters” (such as affinity constants, binding
capacities and nonspecific binding).
(+)-[3H]-PN 200-110 Binding Assays. Membrane Prepa-
ration. Preparation of the membrane was carried out accord-
ing to Matucci49 with minor modification: hearts of male
Sprague-Dawley rats (200-250 g) were removed and placed
in ice cold solution containing 10 mmol/L Tris-HCl, 250 mmol/L
saccarose and 0.2 mmol/L PMSF, pH 7.4; the tissue was
homogenized by ultraturrax and filtered through a single layer
of cheesecloth and centrifuged at 1000g for 10 min. The
supernatant was centrifuged at 48 000g for 25 min. The final
pellet was stored at -80 °C until assayed.
Binding Experiments. For the binding experiments, the
defrozen membranes (0.3-0.6 mg/mL) were incubated in 50
mmol/L Tris-HCl plus 1.2 mmol/L MgCl2, pH 7.4, in a final
volume of 1 mL at 37 °C for 20 min. In homologous competition
curves, (+)-[3H]-PN 200-110 (85.1 Ci/mmol, Perkin-Elmer Life
Science) was present at 0.1 nM in tubes containing increasing
concentrations of unlabeled isradipine (0.1 nM to 0.1 mM) and
at 0.025-0.1 nM in tubes without unlabeled ligand. In
heterologous competition curves fixed concentrations of the
tracer (0.1 nM) were displaced by increasing concentrations
of the tested compounds that were used from 0.1 nM to 0.1
mM. At the end of incubation period, the samples were filtered
and processed as described above. All measurements were
obtained in duplicate.
medium. Fluorescence measurements were converted to [Ca2+
]
i
by determining maximal fluorescence (Fmax) with digitonin
(0.01% final concentration) followed by minimal fluorescence
(Fmin) with 10 mM EGTA (pH 10). [Ca2+]i was calculated
according to Grynkiewicz50 using the ratio 340/380 and as-
suming a dissociation constant of Fura-2 for calcium of 224
nM.
IC50 were evaluated, for each compound, using the computer
program ALLFIT,51 which allows the simultaneous analysis
of different families of dose-responses curves.
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