Discovery, structure-activity relationship study, and oral analgesic efficacy of cyproheptadine derivatives possessing N-type calcium channel inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2006.03.040

Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization, we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium channel, histamine (H₁), and serotonin (5-HT_2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat formalin-induced pain model.

Full text of DOI:10.1016/j.bmc.2006.03.040

Bioorganic & Medicinal Chemistry 14 (2006) 5333–5339
Discovery, structure–activity relationship study, and oral analgesic
efficacy of cyproheptadine derivatives possessing N-type calcium
channel inhibitory activity
Takashi Yamamoto, Seiji Niwa, Satoshi Iwayama, Hajime Koganei, Shin-ichi Fujita,
Tomoko Takeda, Morikazu Kito, Yukitsugu Ono, Yuki Saitou, Akira Takahara,
Seinosuke Iwata, Hiroshi Yamamoto and Masataka Shoji*
Pharmaceutical Research Laboratory, Ajinomoto company Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Japan
Received 14 February 2006; revised 21 March 2006; accepted 22 March 2006
Available online 17 April 2006
Abstract—Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to
histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was
optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization,
we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium
channel, histamine (H1), and serotonin (5-HT2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat forma-
lin-induced pain model.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
different types of small molecules with N-type calcium
channel inhibitory activity have been explored4 to ob-
tain orally available drugs.
Neuronal functions, such as neurotransmitter release
and neuronal cell excitation, are regulated by voltage-
dependent calcium channels (VDCCs), which are classi-
fied into at least five subtypes (L: CaV1, P/Q: CaV2.1, N:
CaV2.2, R: CaV2.3, and T: CaV3) based on their phar-
macological and biophysical properties.1 Among them,
the N-type channel has been shown to play a significant
role in the pathophysiological processes of stroke and
neuropathic pain2 in addition to physiological regula-
tion. Indeed, Ziconotide (25-residue peptide, molecular
weight 2639, SNX-111, Prialt), which is a synthetic ver-
sion of x-conotoxin MVIIA as a neuron-specific N-type
calcium channel blocker, has been proven to have non-
addictive analgesic efficacy in clinical trials via intrathe-
cal administration. Moreover, tolerance to Ziconotide is
not detected3 as observed by opiates. Now, Ziconotide is
on market for the treatment of severe chronic pain.
However, due to its restricted administrative route,
Recently, some of the classical L-type calcium channel
blockers were found to have a potent inhibitory activity
against the N-type as well.5 Flunarizine (1) is one of
such kind of non-specific calcium channel blockers.5a
Interestingly, some of the newly found N-type calcium
channel blockers, PD0176078 (2),4a emopamil derivative
(3)2c, and 4-aminopiperidine derivative (4),6 have the
similar pharmacophore as flunarizine, in which a nitro-
gen atom locates at four atoms distance from a diphe-
nylmethyl moiety. We presumed that this structure is a
key for N-type calcium channel inhibitory activity and
noticed that cyproheptadine (Cyp: 5), which is an orally
active antiallergic drug on market, has this pharmaco-
phore (Chart 1).
From this point of view, we assumed that Cyp could have
N-type calcium channel inhibitory activity with the po-
tential to show analgesic efficacy. Then, we started the re-
search to discover Cyp derived analgesics with potent
inhibitory activity for N-type calcium channels. Howev-
er, Cyp was also reported to have the potent antagonistic
activity against L-type calcium channel using whole-cell
Keywords: N-type calcium channel; Analgesic activity; Structure–
activity relationship study; Cyproheptadine.
*
Corresponding author. Tel.: +81 44 210 5826; fax: +81 44 210
5876; e-mail: masataka_shouji@ajinomoto.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.040

5334
T. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 5333–5339
influence on the cardiovascular systems. Furthermore,
Cyp was also reported to have strong antagonistic
activities for histamine (H1) and serotonin (5-HT2A
receptors, which were not necessary for a novel analgesic
drug because of the expected side effects.10
F
F
F
)
O
N
N
N
H
N
H
Ph
Here, we would like to report the structure–activity rela-
tionship studies of Cyp derived analgesics with N-type
calcium channel inhibitory activity which have reduced
inhibitory activities against L-type calcium channel, H1
and 5-HT2A receptors compared to Cyp.
F
PD0176078 (2)
Flunarizine (1)
F
F
S
O
N
H
O
CN
N
H
N
H
2. Chemistry
NH2
3
4
The synthesis of Cyp derivatives 7–10 and 11a–c is
described in Scheme 1. Succinic acid derivative (7) and
N-tert-butoxycarbonyl-amino acid derivatives (8 and
9) were obtained via a coupling condition using des-
methylcyproheptadine 611 in the presence of EDC and
DMAP. The t-butoxycarbonyl group of 8 was removed
under acidic conditions to yield 10, and subsequent
condensation with appropriate acyl chloride or alkyl
chloroformate gave amide or carbamate compounds
11a-c. N-pivaloyl-b-alanine 12 was synthesized from
b-alanine and pivaloyl chloride. Subsequent coupling
with 6 yielded 13 by the same coupling method as
described for compounds 7–9.
N
Cyproheptadine (5)
Chart 1. Structures of flunarizine (1), PD0176078 (2), reported N-type
calcium channel blockers 3–4, and cyproheptadine (5).
patch-clamp technique.7 The strong inhibition on cardio-
vascular L-type channel invokes several side effects on
cardiovascular system including hypotensive action.
Moreover, although the inhibition of neuronal L-type
calcium channel is suggested to contribute analgesic
activities,8 we proved that the inhibitory activity for
N-type calcium channels is more essential than that for
L-type to show the analgesic efficacy.9 Therefore, the
lower activity against L-type channel might lead to lesser
3. Pharmacological studies
N-type calcium channel inhibitory activities of the
compounds were determined using fluorescence based
Ca2+-flux assay in IMR-32 human neuroblastoma cells.
O
O
b
a
N
N
NH
H
N
n
O
R1
O
O
6
8: n = 1
9: n = 2
7: R1 = NH-t-Bu
O
O
N
H
N
d
c
N
8
NH2
R3
O
11a: R3 = OEt
10
11b: R3 = O-i-Pr
11c: R3 = neopentyl
O
e
HO
O
f
β
-Alanine
O
N
N
O
H
N
H
13
12
Scheme 1. Reagents: (a) N-tert-butyl-succinamic acid, EDC, TEA, DMAP, CH2Cl2; (b) N-Boc-amino acids, EDC, TEA, DMAP, CH2Cl2; (c) 4N
HCl/1,4-dioxane, CH2Cl2; (d) acyl chloride or alkyl chloroformate, TEA, 1,4-dioxane, CH2Cl2; (e) pivaloyl chloride, 1N NaOH, Et2O; (f) 6, EDC,
TEA, DMAP, CH2Cl2.

T. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 5333–5339
Table 1. Activity table of cyproheptadine (5) and its derivatives
5335
N
R
Compound
R
Inhibitory activities against
calcium channelsa
Relative binding affinity
datab on human 5-HT2A
Relative binding affinity
datab on guinea-pig
cerebellum H1
N-type
(IC50, lM)
L-type
(IC50, lM)
Inhibition %
at 10 nM
Inhibition %
at 100 nM
Inhibition %
at 10 nM
Inhibition %
at 100 nM
Cyproheptadine (5)
Me
1.1
6.2
7.6
4.8
0.089
0.86
1.3
4.5 nM (IC50
)
10.5 nM (IC50
)
6
10
8
H
COCH2NH2
COCH2NHCO2-t-Bu
26%
<10%
<10%
80%
14%
13%
<10%
<10%
58%
<10%
<10%
0.98
<10%
a The inhibitory activities against N-type and L-type calcium channels were measured by calcium influx using IMR-32 cells and Magnus method,
respectively.
b The relative binding affinity (RBA) data were based on human 5-HT2A and guinea-pig cerebellum H1 receptors using [3H]ketanserin and
[3H]pyrilamine as radioligands.
Table 2. The inhibitory activities of cyproheptadine derivativesa
Inhibitory activities against L-type calcium channel were
estimated from the effects on high K+-induced contrac-
tion in rat thoracic aorta ring by Magnus technique
(Tables 1,2).9,13 The radioligand-receptor binding assay
O
N
for human 5-HT2A and guinea-pig cerebellum H1 was
conducted at Cerep (www.cerep.com) using [3H]ketan-
serin and [3H]pyrilamine, respectively (Tables 1 and
3).14 The analgesic effect of the compound was evaluated
in a rat formalin-induced pain model.9,15 The test
compound was suspended in 0.5% tragacanth solution
and administered orally at a dosage of 3 mg/kg. The
cumulative flinching response in phase II (10–60 min
after injection of formalin) was calculated for each rat
(Table 3).
R
Compound
R
N-type (IC50, lM) L-type (IC50, lM)
H
N
8
4.8
0.98
O
O
O
N
9
4.6
4.5
0.24
2.6
H
O
H
N
4. Results and discussion
11a
O
O
We began our study by testing the potency of Cyp
against N-type calcium channel and found that Cyp
blocked the channel with the IC50 value of 1.1 lM (Ta-
ble 1). According to our previous results, this activity
seems to be enough to show the analgesic effect in vivo.9
However, its inhibitory activity against L-type calcium
channel was shown to be high enough to affect the car-
diovascular system (IC50 = 0.089 lM).7 Therefore, the
direction of our structure optimization on Cyp should
be to decrease its L-type activity maintaining the efficacy
against N-type calcium channel. Interestingly, des-
methylcyproheptadine (6) had micromolar IC50 value
against N-type calcium channel with a 10-fold lower
activity against L-type calcium channel compared to
Cyp (IC50 = 6.2 and 0.86 lM, respectively). This small
modification on the critical nitrogen atom in piperidine
ring led to the decreased antagonistic activities for both
H1 and 5-HT2A receptors than those of Cyp. Moreover,
the activities of N-glycyl Cyp 10, whose nitrogen atom in
piperidine ring was acylated, showed negligible activities
for both 5-HT2A and H1 receptors. On the other hand,
its activities against both N- and L-type calcium chan-
H
N
11b
11c
7.5
3.9
1.0
O
O
H
N
0.91
O
H
N
7
3.7
3.2
0.43
0.47
O
O
N
13
H
a The inhibitory activities against N-type and L-type calcium channels
were measured by calcium influx us ing IMR-32 cells and Magnus
method, respectively.
We have confirmed that the IMR model was about 1000
times less sensitive than the Magnus or in vivo model
for detecting each calcium channel blocking action.12

5336
T. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 5333–5339
Table 3. The biological activity table of 13
Compound
Relative binding affinity dataa on
human 5-HT2A
Relative binding affinity dataa on
guinea-pig cerebellum H1
Rat formalin test (3 mg/kg, po)b
Inhibition %
at 10 nM
Inhibition %
at 100 nM
Inhibition % Inhibition %
at 10 nM
<10%
Number of flinches
Inhibition %
at 100 nM
<10%
13
<10%
<10%
65 15 (n = 5)
44%
a The relative binding affinity (RBA) data were based on human 5-HT2A and guinea-pig cerebellum H1 receptors using [3H]ketanserin and
[3H]pyrilamine as radioligands.
b Inhibitory activity in rat formalin test is shown as the number of flinches in phase II pain responses following footpad injection of formalin in rat.
Number of flinches represents the mean SEM. Inhibition % is given by the control (116 8, n = 22) and test compound.
nels were slightly lower than those of 6 (IC50 = 7.6 and
1.3 lM, respectively). These results illustrated the fact
that the strength of positive charge on the piperidine
nitrogen had great influences on the activities for both
5-HT2A and H1 receptors, but fewer effects on those
for N- and L-type calcium channels. Finally, N-tert-bu-
toxycarbonyl-glycyl derivative 8, which had no charged
nitrogen atom at the terminal of the molecule as well as
in piperidine ring, had more potent activity against N-
type calcium channel (IC50 = 4.8 lM) than that of 10
with 11-fold lower activities for L-type channel
(IC50 = 0.98 lM) than that of Cyp. 8 also showed no
affinity for both 5-HT2A and H1 receptors. According
to this result, we chose 8 as the lead compound and
started the further structural optimizations on tert-bu-
toxycarbonyl-glycyl moiety without the charge on both
of the nitrogen atoms (Table 2).
analgesic activity of N-type to L-type channels was
previously reported, and the IC50 values coming from
IMR-32 assay were underestimated compared to those
of Magnus method.9,12
In summary, the optimization and structure–activity
relationship (SAR) studies on Cyp were performed to
find potent analgesics possessing N-type calcium chan-
nel inhibitory activity with reduced inhibitory activities
against L-type calcium channel, H1 and 5-HT2A recep-
tors. As a consequence, an N-pivaloyl-b-alanyl deriva-
tive (13) was discovered as the orally active analgesic
with effective N-type calcium channel inhibitory activity.
Because of the lower inhibitory activities for L-type cal-
cium channel, 5-HT2A and H1 receptors, 13 is expected
to have fewer side effects than Cyp is. Investigations into
the further optimizations and pharmacological profiles
are in progress and will be reported elsewhere.
First, the elongation of chain length by one carbon atom
(9) was tested. However, 9 showed no improvement for
N-type blocking potency (IC50 = 4.6 lM) with higher
activity against L-type (IC50 = 0.24 lM). The replace-
ment of the tert-butylcarbamate moiety of 8 with other
alkyl carbamates (11a or 11b) was performed without
changing their chain lengths. Interestingly, although eth-
ylcarbamate 11a had almost equipotent activities
against N-type calcium channel to 8, the activity of iso-
propylcarbamate 11b was decreased (IC50 = 7.5 lM).
Next, the urethane moiety of 8 was replaced by amides
(7, 11c, and 13). All of those derivatives had higher
activities for N-type calcium channel than that of 8
and the activity of 13 was the highest among all of the
derivatives (IC50 = 3.9, 3.7, and 3.2 lM, respectively).
Considering the analgesic action in vivo, higher activity
for N-type calcium channel is required. As for the activ-
ity against L-type, 13 had higher activity
(IC50 = 0.47 lM) than 8, but still more than 5 times low-
er than Cyp. Moreover, the radio binding assay proved
that 13 had no affinity for both 5-HT2A and H1 recep-
tors (Table 3). Therefore, even its N-type activity was
still less potent than that of Cyp, we concluded that 13
had the best potential to show an analgesic effect in vivo
with fewer side effects. In order to verify its analgesic
activity, 13 was tested in rat formalin-induced pain mod-
el. As expected, 13 showed promising analgesic activity
with 44% inhibition at 3 mg/kg po. (Table 3). In fact,
the inhibitory activities for both N-type and L-type cal-
cium channels could affect on the analgesic activity, we
might say that the effect of N-type calcium channel
was still important, because the superior influence on
5. Experimental
5.1. Chemistry
5.1.1. General. The reagents were purchased commer-
cially and used without further purification. Column
chromatography was performed using silica gel (Merck,
particle size 0.063–0.200 mm). NMR spectra were
recorded on a Varian EM-390 at 300 MHz. Mass spec-
tra (ESI) were measured on JEOL JMS-DX300 instru-
ments. The elemental analysis was carried out on an
Elemental Vario EL III analyzer. Where analyses are
indicated only by the symbols of the elements, the results
obtained were within 0.4% of the theoretical values.
Reagent abbreviations: EDC, 1-ethyl-3-(30-dimethyl-
aminopropyl)carbodiimide hydrochloride; TEA, trieth-
ylamine; DMAP, 4-dimethylaminopyridine.
5.1.2. N-(tert-Butyl)-4-[4-(5H-dibenzo[a,d]cycloheptene-
5-ylidene)-1-piperidinyl]-4-oxobutanamide (7). 4-(5H-
Dibenzo
[a,d]cycloheptene-5-ylidene)-1-piperidine
(2.00 g, 7.32 mmol), 1.52 g (8.78 mmol) N-tert-butyl-
succinamic acid, 86 mg (0.70 mmol) DMAP, and
2.10 g (11.0 mmol) EDC were dissolved in 1 ml CH2Cl2.
TEA (1.48 g, 14.6 mmol) was added to the obtained
solution, and they were stirred for 2 h. One molar aque-
ous hydrochloride solution was added to the obtained
mixture. After extracting three times with CH2Cl2, the
organic layer was washed with brine. After drying over
anhydrous sodium sulfate, the solvent was evaporated

T. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 5333–5339
5337
under reduced pressure, and the residue was purified by
the silica gel chromatography (hexane/EtOAc, 2:1–3:7)
to obtain the title compound (2.96 g, 94%): H NMR
2.70 (4H, m), 3.81 (2H, m), 6.96 (2H, s), 7.20–7.44
(8H, m), 8.17(3H, bs): MS (ESI) 415 (MH+DMSO-
d6)+. Anal. C22H23ClN2OÆ0.9 H2O (C, H, N).
1
(CDCl3) d: 1.32 (9H, s), 2.08–2.36 (4H, m), 2.41 (2H,
m), 2.50–2.71 (2H, m), 2.24–2.96 (2H, m), 3.58 (1H,
m), 3.93 (1H, m), 5.77 (1H, br s), 6.92 (2H, s), 7.14–
7.38 (8H, m): MS (ESI) 429 (MH)+. Anal.
C28H32N2O2 (C, H, N).
5.1.6.
Ethyl
2-[4-(5H-dibenzo[a,d]cycloheptene-5-
ylidene)-1-piperidinyl]-2-oxoethylcarbamate (11a). Com-
pound 10 (375 mg) was suspended in 3 ml of CH2Cl2.
TEA (303 mg, 3.00 mmol) was added to the obtained
suspension. Then a solution of 130 mg (1.20 mmol) of
ethyl chloroformate in 3 ml CH2Cl2 was slowly added
to the reaction mixture. After stirring overnight, saturat-
ed aqueous sodium hydrogencarbonate solution was
added thereto. After extracting with EtOAc twice fol-
lowed by drying under anhydrous sodium sulfate, the
solvent was evaporated under reduced pressure. The res-
idue was roughly purified by the silica gel chromatogra-
phy (CH2Cl2/MeOH, 98:2) and then purified by the
silica gel chromatography (hexane/EtOAc, 1:2) to ob-
tain the title compound (213 mg, 53%): 1H NMR
(CDCl3) d: 1.24 (3H, t, J = 6.9 Hz), 2.12–2.36 (4H, m),
2.97–3.10 (2H, m), 3.44 (1H, m), 3.86–4.02 (3H, m),
4.13 (2H, q, J = 6.9Hz), 5.65 (1H, br s), 6.92 (2H, s),
7.14–7.20 (2H, m), 7.23–7.38 (6H, m): MS (ESI)
403(MH)+. Anal. C25H26N2O3 (C, H, N).
5.1.3. tert-Butyl 2-[4-(5H-dibenzo[a,d]cycloheptene-5-yli-
dene)-1-piperidinyl]-2-oxoethylcarbamate (8). 4-(5H-Dib-
enzo [a,d]cycloheptene-5-ylidene)-1-piperidine, (3.00 g,
10.9 mmol), 2.29 g (13.2 mmol) N-tert-butoxycarbonyl-
glycine, 3.14 g (16.4 mmol) EDC, and 122 mg
(1.00 mmol) DMAP were dissolved in 50 ml CH2Cl2.
TEA (2.20 g, 21.8 mmol) was added to the obtained
solution, and they were stirred overnight. Saturated
aqueous sodium hydrogencarbonate solution was added
to the obtained mixture. After extracting three times
with CH2Cl2, the organic layer was washed with brine.
After drying over anhydrous sodium sulfate, the solvent
was evaporated under reduced pressure, and the residue
was purified by the silica gel chromatography (hexane/
EtOAc, 4:1–2:1) to obtain the title compound (4.29 g,
94%): 1H NMR (CDCl3) d: 1.44 (9H, s), 2.15–2.35
(4H, m), 3.02 (2H, m), 3.42 (1H, m), 3.81–4.01 (3H,
m), 5.51 (1H, br s), 6.92 (2H, s), 7.15–7.38 (8H, m):
MS (ESI) 431 (MH)+. Anal. C27H30N2O3 (C, H, N).
5.1.7. Isopropyl 2-[4-(5H-dibenzo[a,d]cycloheptene-5-
ylidene)-1-piperidinyl]-2-oxoethylcarbamate (11b). The
title compound was prepared from 10 and isopropyl
1
chloroformate as described for 11a: H NMR (CDCl3)
5.1.4. tert-Butyl 3-[4-(5H-dibenzo[a,d]cycloheptene-5-yli-
dene)-1-piperidinyl]-3-oxopropylcarbamate (9). The title
compound was prepared from N-tert-butoxycarbonyl-
3-aminopropionic acid and 4-(5H-dibenzo[a,d]cyclohep-
tene-5-ylidene)-1-piperidine as described for 8: 1H NMR
(CDCl3) d: 1.49 (9H, s), 2.12–2.36 (4H, m), 2.86–3.36
(4H, m), 3.42 (1H, m), 3.97 (1H, m), 4.49 (1H, d), 4.76
(1H, m), 5.16 (1H, br s), 6.92 (2H, s), 7.13–7.22 (2H,
m), 7.22–7.39 (6H, m): MS (ESI) 445 (MH)+. Anal.
C28H32N2O3Æ0.5 H2O (C, H, N).
d: 1.23 (6H, d, J = 6.3 Hz), 2.12–2.48 (4H, m), 2.92–
3.11 (2H, m), 3.44 (1H, m), 3.83–4.09 (3H, m), 4.90
(1H, m), 5.59 (1H, br s), 6.92 (2H, s), 7.14–7.20 (2H,
m), 7.23–7.38 (6H, m): MS (ESI) 417 (MH)+. Anal.
C26H28N2O3Æ0.25 H2O (C, H, N).
5.1.8. N-{2-[4-(5H-Dibenzo[a,d]cycloheptene-5-ylidene)-
1-piperidinyl]-2-oxoethyl}-3,3-dimethylbutanamide (11c).
The title compound was prepared from 10 and 3,3-dim-
1
ethylbutanoyl chloride as described for 11a: H NMR
(CDCl3) d: 1.03 (9H, s), 2.12 (2H, s), 2.15–2.39 (4H,
m), 2.96–3.11 (2H, m), 3.47 (1H, m), 3.88–4.13 (3H,
m), 6.49 (1H, br s), 6.92 (2H, s), 7.14–7.21 (2H, m),
7.21–7.41 (6H, m): MS (ESI) 429 (MH)+. Anal.
C28H32N2O2Æ0.2 H2O (C, H, N).
5.1.5.
2-[4-(5H-Dibenzo[a,d]cycloheptene-5-ylidene)-
1-piperidinyl]-2-oxo-ethanamine hydrochloride (10). tert-
Butyl 2-[4-(5H-dibenzo[a,d]cycloheptene-5-ylidene)-1-
piperidinyl]-2-oxoethylcarbamate,
(8),
(1.40 g,
3.25 mmol) was dissolved in 20 ml of 1,4-dioxane.
Twelve microliters of 4 M hydrochloric acid/1,4-dioxane
solution was added to the obtained solution, and they
were stirred overnight. After the neutralization with
4 M aqueous sodium hydroxide solution, the solvent
was evaporated under reduced pressure. Brine was add-
ed to the reaction mixture. After the extraction three
times with EtOAc, the extract was dried over anhydrous
sodium sulfate and then the solvent was evaporated un-
der reduced pressure. The residue was purified by the sil-
ica gel chromatography (hexane/EtOAc, 9:1 to EtOAc).
The obtained oil was dissolved in 10 ml of a solution of
EtOAc/hexane (1:2) and then 2 ml of 4 M hydrochloric
acid/1,4-dioxane solution was added to the residue.
The resultant precipitates were taken by the filtration,
washed with a solution of EtOAc/hexane (1:2), and
air-dried. After further drying under reduced pressure,
the title compound was obtained (1.15 g, 94%): 1H
NMR (DMSO-d6) d: 1.97 (2H, m), 2.29 (2H, m), 3.10–
5.1.9. N-(2,2-Dimethylpropanoyl)-b-alanine (12). b-Ala-
nine (4.35 g, 48.8 mmol) was dissolved in 49 ml of 1 M
aqueous sodium hydroxide solution and 2 ml Et2O.
Pivaloyl chloride (4.90 g, 40.6 mmol) in Et2O was added
to the vigorously stirred solution for 20 min. After stir-
ring for 1 h 15 min, the obtained solution was neutral-
ized with 1 M aqueous hydrochloride solution. The
solution was extracted with EtOAc three times. The ex-
tract was dried over anhydrous sodium sulfate and then
the solvent was evaporated under reduced pressure, the
1
title compound was obtained (5.40 g, 77%): H NMR
(CDCl3) d: 1.18 (9H, s), 2.60 (2H, t, J = 6.0Hz), 3.51
(2H, q, J = 6.0 Hz), 6.34 (1H, br s).
5.1.10. N-{3-[4-(5H-Dibenzo[a,d]cycloheptene-5-ylidene)-
1-piperidinyl]-3-oxopropyl}-2,2-dimethylpropanamide (13).
[4-(5H-Dibenzo[a,d]cycloheptene-5-ylidene)-1-piperidine
(275 mg, 1.01 mmol), 90.0 mg (0.480 mmol) N-(2,2-dim-

5338
T. Yamamoto et al. / Bioorg. Med. Chem. 14 (2006) 5333–5339
ethylpropanoyl)-b-alanine, 193 mg (1.01 mmol), EDC
and 6 mg (0.05 mmol) of DMAP were dissolved in 3 ml
CH2Cl2. TEA (152 mg, 1.50 mmol) was added to the ob-
tained solution. After stirring for 3 h, the obtained mix-
ture was purified by the silica gel chromatography
(ChromatorexTM NH, Fuji Silysia Chemical LTD., hex-
ane/EtOAc, 89:11–7:3) and then purified by the silica
gel chromatography (hexane/EtOAc, 2:3–1:4) to obtain
tic activity of each test compound on N-type calcium
channel was expressed as a 50% inhibitory concentra-
tion (IC50), as previously reported.7
5.2.4. L-type calcium channel inhibition. Male Sprague–
Dawley rats (7 weeks old) were used. The thoracic aorta
was isolated, cleared of adhering periadventitial fat, and
cut into rings of 3 mm width. The endothelium was re-
moved by gently rubbing the luminal surface. The ring
was mounted in an organ bath filled with warmed
(37 ꢁC), oxygenated (95% O2/5% CO2) Tyrode’s solution
(pH 7.4). The ring was equilibrated under resting ten-
sion of 2 g for 1 h. Then the ring was incubated in high
K+ solution (containing 50 mM KCl; KCl was substitut-
ed for equimolar NaCl in the Tyrode’s solution) and
then in Tyrode’s solution for 45 min each. The solution
was replaced with high K+ solution again. After attain-
ing the maximum contraction reaction, the test com-
pound was cumulatively added at intervals of 90 min
to attain concentrations of 10ꢀ9, 10ꢀ8, 10ꢀ7, and
10ꢀ6 M. The antagonistic activity of each test com-
pound on L-type calcium channel was expressed as a
50% inhibitory concentration (IC50), as previously
reported.7
1
the title compound (147 mg, 72%): H NMR (CDCl3)
d: 1.16 (9H, s), 2.11–2.36 (4H, m), 2.38–2.56 (2H, m),
2.94–3.12 (2H, m), 3.44–3.56 (3H, m), 3.92 (1H, m),
6.62 (1H, t), 6.92 (2H, s), 7.13–7.20 (2H, m), 7.22–7.38
(6H, m): MS (ESI) 429 (MH)+. Anal. C28H32N2O2 (C,
H, N).
5.2. In vitro pharmacology
5.2.1. N-type calcium channel inhibition
5.2.2. Cell culture. Human neuroblastoma cells IMR-32
were obtained from ATCC (American Type Culture
Collection) and cultured with the medium of the follow-
ing composition; a Phenol Red-free Eagle’s minimum
essential medium containing Earle’s salts (GIBCO) sup-
plemented with 2 mM of L-glutamine (GIBCO), 1 mM
of sodium pyruvate (pH 6.5) (GIBCO), antibiotic/anti-
mycotic mixture (GIBCO), and 10% fetal calf serum
(Cell Culture Technologies). For measurement of intra-
cellular calcium concentration, 3 ml of 1 · 105 cells/ml
IMR-32 cells was spread on the glass bottom of a dish
(Iwaki Glsss Co., Ltd.) having a diameter of 35 mm
which had been treated with poly-L-lysine (SIGMA)
and collagen (COLLAGEN VITROGEN 100; Collagen
Co.). One day after the culture, 1 mM dibutyl-cAMP
and 2.5 lM of 5-bromo-2-deoxyuridine (SIGMA) were
added to express N-type calcium channels. After the cul-
ture for additional 10–14 days, the cells were subjected
to the assay.
5.3. In vivo pharmacology
5.3.1. Rat formalin test. Male Sprague–Dawley rats were
used. One hundred microliters of formalin (2.5%) was
subcutaneously injected into the dorsal surface of the
left hind paw. Flinching behavior was detected as phase
I (0–9 min) and phase II (10–60 min). The cumulative
flinching response in phase II was observed for assessing
effect of each test compound on hyperalgesia. Test com-
pounds were suspended in 0.5% tragacanth solution and
orally administered 3 h before formalin injection.
Acknowledgments
5.2.3. Measurement of intracellular calcium concentra-
tion. The medium for IMR-32 cells thus prepared was
replaced with 1 ml of Phenol Red-free Eagle’s minimum
essential medium (GIBCO) containing 2.5 lM fura-2/
AM (Dojin Kagaku, Co.) and Earle’s salts supplement,
and the incubation was conducted at 37 ꢁC for 30 min.
Then, the medium was replaced with a recording medi-
um (20 mM Hepes–KOH, 115 mM NaCl, 5.4 mM KCl,
0.8 mM MgCl2, 1.8 mM CaCl2, and 13.8 mM D-glu-
cose). Intracellular calcium concentration was analyzed
using a fluorescence microscope (Nikon Corporation)
and an image analysis device ARGUS 50 (Hamamatsu
Photonics). To prevent the activation of the L-type cal-
cium channels in the differentiated IMR-32 cells, a
recording medium containing 1 lM of a selective L-type
calcium channel blocker Nifedipine was used through-
out the experiment. Then a stimulating medium contain-
ing 60 mM KCl (KCl was substituted for equimolar
NaCl in the recording medium) was rapidly given by
the Y-tube method for 6 s. The change in the intracellu-
lar calcium concentration was expressed as the N-type
calcium channel activity.12 Then stimulating medium
containing 60 mM KCl was reapplied in the presence
of 0.1, 1 or 10 lM of the test compound. The antagonis-
We thank members of the Analytical Chemistry Depart-
ment for mass spectral measurements.
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