Discovery and evaluation of selective N-type calcium channel blockers: 6-Unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.04.051

A structure-activity relationship study of 6-unsubstituted-1,4- dihydropyridine and 2,6-unsubstituted-1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5- dicarboxylic acid 3-cinnamyl ester was found to be an effective and selective N-type calcium channel blocker with oral analgesic potential.

Full text of DOI:10.1016/j.bmcl.2012.04.051

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3639–3642
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and evaluation of selective N-type calcium channel blockers:
6-Unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives
⇑
Takashi Yamamoto , Seiji Niwa, Munetaka Tokumasu, Tomoyuki Onishi, Seiji Ohno, Masako Hagihara,
Hajime Koganei, Shin-ichi Fujita, Tomoko Takeda, Yuki Saitou, Satoshi Iwayama, Akira Takahara,
Seinosuke Iwata, Masataka Shoji
Exploratory Research Laboratories, Ajinomoto Pharmaceuticals Co. Ltd, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 January 2012
Revised 6 April 2012
Accepted 10 April 2012
Available online 17 April 2012
A structure–activity relationship study of 6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-
1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers
that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-
dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-cinnamyl ester was found to
be an effective and selective N-type calcium channel blocker with oral analgesic potential.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
N-Type calcium channel blocker
Structure–activity relationships
1,4-Dihydropyridine derivative
The N-type calcium channel (Cav2.2) is a well-established and
characterized subtype of voltage-dependent calcium channels
(VDCCs) that are distributed among central and peripheral nerve
terminals and are known to control calcium influx into cells in
response to changes in membrane potential. Activation of this
channel triggers various physiological events, such as neuronal
excitability and secretion of neurotransmitters (e.g., glutamate,
substance P, or CGRP), in strong association with the pathological
process of neuropathic pain and cerebral ischemia. Thus, blockade
of the N-type VDCCs has been considered as a promising therapeu-
tic target for these pathological conditions.1
O
O
H
N
S
N
N
O
O
H
O
CF3
S
N
N
N
Cl
O
O
GlaxoSmithKline, 2010
Purdue Pharma,2009
Cl
H
N
O
O
N
CF3
N
N
O
Merck, 2011
NH
The clinical treatment of pain, especially prolonged and neuro-
pathic pain, is still a major challenge, and the efficacy of current
analgesic drugs, including opioids, is often determined by unde-
sired dose-limiting side effects, such as tolerance and physical
dependence. Therefore, there is a strong need for a novel analgesic
drug that does not cause these adverse effects.2 The therapeutic po-
tential of N-type VDCC blockers for neuropathic pain states has
been proven in clinical and preclinical studies of N-type selective
Neuromed,2010
Figure 1. Recently reported N-type VDCC blockers.
Ziconotide, which is a synthetic version of
x-conotoxin MVIIA
and is approved as an analgesic drug to treat intractable pain con-
ditions, can be administered only through the intrathecal route,
which has very low patient compliance.3 Therefore, many efforts
have been made to discover systemically efficacious small-mole-
cule N-type VDCC blockers to overcome the limitations of conopep-
tides (recently reported small-molecule N-type VDCC blockers are
shown in Fig. 1).4
peptidic blockers,
x x-
-conotoxins MVIIA, GVIA, and CVID.3 These
conotoxins clearly showed therapeutic benefits, with minimal
development of tolerance and addiction. However, their usage
had several drawbacks related to peptide blockers, such as limited
route of administration, challenging synthesis, and high costs.
As previously reported,5 we performed a structure–activity
relationship study of unique 1,4-dihydropyridine-5-carboxylate
compounds. Our goal was to discover orally available potent N-type
VDCC blockers with high selectivity over L-type channels to ensure
minimal effects on the cardiovascular system. As a consequence of
⇑
Corresponding author. Tel.: +81 44 210 5826; fax: +81 44 210 5876.
E-mail address: takashia_yamamoto@ajinomoto.com (T. Yamamoto).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.051

3640
T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3639–3642
the structural optimization, 2-dimethoxymethyl-4-(3-chloro-
phenyl)-6-methyl-1,4-dihydropyridine-5-carboxylic acid (1) was
identified as a promising N-type VDCC blocker that showed an effi-
cacious analgesic potency with no detectable influence on systemic
blood pressure at the effective dose.5d Therefore, further modifica-
tion of 1 to achieve improvement in the N-type VDCC inhibitory
activity with reduced blocking activity on the L-type channel is a po-
tential strategy to obtain an analgesic drug candidate with minimal
adverse effects.
activities at both the N- and L-type VDCCs. The derivatives possess-
ing chlorine atoms at the 40-position (2b) and 30,50-positions (2c)
showed further improvement in N/L selectivity (7.9% and 4.0% inhi-
bition for L-type VDCCs at 10
inhibitory activity at the N-type (IC50 = 1.1 and 1.0
tively). The 30,40-dichloro derivative (2d) had a submicromolar
lM, respectively), with effective
lM, respec-
IC50 value of 0.52 lM for N-type VDCCs. The compound with a
methoxy group at the 40-position (2e and 2f) and the 40-trifluoro-
methyl derivative (2g) resulted in improved inhibitory activity at
In this Letter, we focused on the introduction of a hydrogen
atom at the 2 and 6 positions of the 1,4-dihydropyridine structure
of 1 to improve the activity of the N-type VDCCs and to reduce
inhibitory activity at the L-type channels.
N-type VDCCs (IC50 = 1.5, 1.2, and 1.1
creased activity at L-type channels (26%, 20%, and 17% inhibition
at 10 M, respectively) compared with those of 1, but their N/L-
lM, respectively), with de-
l
selectivity profiles were not comparable with those of 2b and 2c.
Further introduction of a hydrogen atom was made at the 2-po-
sition of the 1,4-dihydropyridine ring in 2a to obtain the 2,6-
unsubstituted-1,4-dihydropyridine derivative 3a (Table 2), which
showed effective N-type activity with equivalent inhibitory activ-
ity at the L-type VDCCs compared with that of 1 (IC50 = 1.6 and
The synthetic methods for the synthesis of 6-unsubstituted-1,
4-dihydropyridine derivatives are shown in Scheme 1. 4,4-Dime-
thoxy-2-benzylidene-3-oxo-butyricacid cinnamyl ester (6) was
obtained using Knoevenagel condensation of 4,4-dimethoxy-3-oxo-
butyric acid cinnamyl ester (4) with corresponding benzaldehyde
(5). The 2-propanol solution of 6 was heated with propynoic acid 2-
cyanoethyl ester (7) in the presence of ammonium acetate to obtain
1,4-dihydropyridine (8), which was treated with 1 M NaOH to yield
a 2-dimethoxymethyl-6-unsubstituted-1,4-dihydropyridine-5-carb-
oxylicacid derivative (2). A four-component cyclizing reaction that
used the corresponding benzaldehyde (5), 7, propynoic acid cinnamyl
ester (9), and ammonium acetate resulted in the formation of 2,
6-unsubstituted-1,4-dihydropyridine-3,5-dicarboxylicacid3-cinn-
amylester5-(2-cyanoethylester) (10). Subsequent hydrolysis pro-
vided 2,6-unsubstituted-1,4-dihydropyridine-5-carboxylicacid (3).
Preparative HPLC equipped with a chiral column (Daicel Chiralcel
OD) followed by hydrolysis was used to separate and obtain the
optical isomer of the 30,50-dichloro-40-methoxy-phenyl derivative 3f
from 4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-
dicarboxylic acid 3-cinnamyl ester 5-(2-cyanoethyl) ester (10f).6
IMR-32 human neuroblastoma cells for N-type VDCCs7 and a rat
thoracic aorta ring for L-type VDCCs were used to perform in vitro
characterizations of the synthesized compounds.8 Our study was
initiated by the replacement of the 6-methyl group in compound
1 with a hydrogen atom to afford 2a (Table 1). Interestingly, 2a
9.1
type blocking activity and selectivity over those of L-type channels
(16% inhibition at 10 M). Another derivative with two chlorine
atoms at the 30- and 40-positions (3c) showed submicromolar activ-
ity for N-type VDCCs (IC50 = 0.72 M). The introduction of a trifluo-
l
M, respectively). Its 40-chloro derivative 3b had improved N-
l
l
romethyl group at the 30-position (3d) resulted in a 1.2
lM IC50
value for the N-type. The 30-chloro derivative with another elec-
tron-donating group at the 40-position (3e) had an IC50 value of
1.3 lM for N-type VDCCs, with reduced inhibitory activities for
the L-type compared with that of 3a (15% inhibition at 10 lM). Fi-
nally, the derivative possessing two chlorine atoms at the 30- and
50-positions with a 40-methoxy group (3f) had improved activity
for the N-type VDCCs (IC50 = 0.58
lM) and reduced activity for
the L-types (19% inhibition at 10 M) compared with those of 3a.
l
As a consequence of the structural optimization, all of the tested
6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-1,4-
dihydropyridine derivatives showed improved N-type inhibitory
activity and selectivity over those of the L-types compared with
1, which indicated the crucial role of the hydrogen atom at the
6-position for these bioactivities. Among the derivatives, 2b, 2c,
and 3f were found to be particularly selective and effective N-type
calcium channel blockers.
showed improved inhibitory activity for N-type VDCC (IC50
1.1 M) and reduced activity at the L-type channels (44% inhibition
at 10 M) compared with those of 1, indicating that a sterically
=
l
l
Further biological evaluations were performed on the two enan-
least hindered hydrogen atom at the 6-position is important for
the inhibitory activity at N-type VDCCs as well as for selectivity
over L-type channels. Thus, further modification was performed
on the substituent groups in the 4-phenyl moiety in 2a to optimize
tiomers of 3f. (ꢀ)-3f had an IC50 of 0.84
with 28% inhibition of the L-type channel at 10
-3f had equipotent inhibitory activity at the N-type VDCCs
l
M for the N-type channel,
l
M. In addition, (+)
(IC50 = 0.72
l
M) compared with that of 3f and showed excellent
R'
R'
R'
O
O
O
O
O
O
O
O
CN
a
b
O
O
O
O
RO
O
O
O
+
+
O
O
CHO
N
H
4
5
6
7
NC
8: R =
2: R = H
c
R'
O
O
R'
O
O
NC
RO
O
d
O
O
+
+
N
H
CHO
NC
7
5
9
10: R =
3: R = H
e
Scheme 1. Reagents and conditions: (a) Cat. piperidine, benzene, reflux; (b) AcONH4, i-PrOH, 80 °C; (c) 1 M NaOH, MeOH; (d) AcONH4, i-PrOH, 80 °C; (e) 1 M NaOH, MeOH.

T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3639–3642
3641
Table 1
Activity table of dihydropyridine derivatives
R'
3'
4'
5'
O6'
2'O
3
4
5
HO
O
O
Ph
6
2
1N
R
H
O
Compound
R
R0
N-type IMR-32 IC50
,
l
M
L-type Magnus IC50, lM or inh. % at 10 lM
1
Me
H
H
H
H
H
H
H
30-Cl
3.0
1.1
1.1
1.0
0.52
1.5
1.2
1.1
8.1
2a
2b
2c
2d
2e
2f
30-Cl
44%
7.9%
4.0%
31%
26%
20%
17%
40-Cl
30,50-Cl
30,40-Cl
30-Cl, 40-OMe
30,50-Cl, 40-OMe
30-CF3
2g
In vitro inhibition against N-type (calcium influx using IMR-32 cells) and L-type (Magnus method) calcium channels.
Table 2
Activity table of optically pure dihydropyridine derivatives
R'
O
O
*
HO
O
Ph
N
H
Compound
R0
N-type IMR-32 IC50
,
l
M
L-type Magnus IC50, lM or inh. % at 10 lM
3a
3b
3c
3d
3e
3f
30-Cl
1.6
1.2
0.75
1.2
1.3
0.58
0.84
0.72
9.1
40-Cl
16%
41%
40%
15%
19%
28%
30,40-Cl
30-CF3
30-Cl, 40-OMe
30,50-Cl, 40-OMe
30,50-Cl, 40-OMe
30,50-Cl, 40-OMe
(ꢀ)-3f
(+)-3f
6.5% (IC50 = 44 lM)
In vitro inhibition against N-type (calcium influx using IMR-32 cells) and L-type (Magnus method) calcium channels.
Table 3
Inhibitory activity of the test compound in phase II pain responses following footpad injection of formalin in rats
Compound
Rat formalin test
Dose
Inhibition%
(+)-3f
Gabapentin
3 mg/kg, po
100 mg/kg, po
35 11% (n = 5)
33 12% (n = 3)
Inhibition% versus that of the control.
selectivity over the L-types (IC50 = 44
l
M). As a result, (+)-3f was
N-type VDCC blockers with potent analgesic efficacy. These com-
pounds could be interesting research tools and possibly promising
drug candidates for the control of severe to moderate pain states
with negligible effects on blood pressure.
found to be a promising N-type VDCC blocker with submicromolar
activity and the best selectivity over the L-type VDCCs among the
tested compounds.
This result motivated us to use a rat formalin-induced pain
model to evaluate (+)-3f in vivo using rat formalin-induced pain
model (Table 3). Our previously reported compounds were used
to confirm the applicability of this model for N-type calcium chan-
nel blockers.5a,b (+)-3f (3 mg/kg po) displayed significant analgesic
efficacy that was comparable to that of Gabapentin (100 mg/kg,
po), which was indicative of its effective pain-relieving potential.9
In summary, in a structure–activity relationship study at the 2-
and 6-positions of the 1,4-dihydropyridine ring of 1, the deriva-
tives 2b, 2c, and (+)-3f were found to be effective and selective
References and notes
1. Yaksh, T. L. J. Pain 2006, 7, S13.
2. (a) Yamamoto, T.; Nair, P.; Davis, P.; Ma, S. W.; Navratilova, E.; Moye, M.; Tumati,
S.; Vanderah, T. W.; Lai, J.; Porreca, F.; Yamamura, H. I.; Hruby, V. J. J. Med. Chem.
2007, 50, 2779; (b) Yamamoto, T.; Nair, P.; Vagner, J.; Davis, P.; Ma, S. W.;
Navratilova, E.; Moye, M.; Tumati, S.; Vanderah, T. W.; Lai, J.; Porreca, F.;
Yamamura, H. I.; Hruby, V. J. J. Med. Chem. 2008, 51, 1369; (c) Yamamoto, T.;
Nair, P.; Jacobsen, N. E.; Vagner, J.; Kulkarni, V.; Davis, P.; Ma, S. W.; Navratilova,
E.; Yamamura, H. I.; Vanderah, T. W.; Porreca, F.; Lai, J.; Hruby, V. J. J. Med. Chem.
2009, 52, 5164.

3642
T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3639–3642
3. (a) Miljanich, G. P. Curr. Med. Chem. 2004, 11, 3029; (b) Schroeder, C. I.; Lewis, R.
J. Mar. Drugs 2006, 4, 193.
7. Measurement of N-type calcium channel (CaV2.2) inhibitory activity was
performed as follows (previously reported in the Ref. 5): Human
neuroblastoma cells IMR-32 were obtained from the American Type Culture
Collection and cultured with a medium, which comprised the following: a
phenol red-free Eagle’s minimum essential medium containing Earle’s salts
(GIBCO) supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate
(pH 6.5) (GIBCO), antibiotic/antimycotic mixture (GIBCO), and 10% fetal calf
serum (Cell Culture Technologies). For measurement of intracellular calcium
concentrations, 3 mL of 1 ꢁ 105 cells/mL IMR-32 cells were spread on the glass
bottom of a dish (Iwaki Glass Co., Ltd) having a diameter of 35 mm that had been
treated with poly-L-lysine (SIGMA) and collagen (COLLAGEN VITROGEN 100;
4. (a) Yamamoto, T.; Takahara, A. Curr. Top. Med. Chem. 2009, 9, 377; (b) Barrow, J.
C.; Duffy, J. L. Ann. Rev. med. Chem. 2010, 45, 2; (c) Tyagarajan, S.; Chakravarty, P.
K.; Park, M.; Zhou, B.; Herrington, J. B.; Ratliff, K.; Bugianesi, R. M.; Williams, B.;
Haedo, R. J.; Swensen, A. M.; Warren, V. A.; Smith, M.; Garcia, M.; Kaczorowski,
G. J.; McManus, O. B.; Lyons, K. A.; Li, X.; Madeira, M.; Karanam, B.; Green, M.;
Forrest, M. J.; Abbadie, C.; McGowan, E.; Mistry, S.; Jochnowitz, N.; Duffy, J. L.
Bioorg. Med. Chem. Lett. 2011, 21, 869; (d) Pajouhesh, H.; Feng, Z. P.; Ding, Y.;
Zhang, L.; Pajouhesh, H.; Morrison, J. L.; Belardetti, F.; Tringham, E.; Simonson,
E.; Vanderah, T. W.; Porreca, F.; Zamponi, G. W.; Mitscher, L. A.; Snutch, T. P.
Bioorg. Med. Chem. Lett. 2010, 20, 1378; (e) Shao, B.; Yao, J. Patent Application
WO 2009/040659, 2009.; (f) Beswick, P. J.; Campbell, A.; Cridland, A.; Gleave, R.
J.; Page, L. W. Patent Application WO 2010/102663, 2010.
Collagen Co.). One day after the culture, 1 mM dibutyl-cAMP and 2.5 lM 5-
bromo-2-deoxyuridine (SIGMA) were added to express N-type calcium
channels. After culturing for an additional 10–14 days, the cells were
subjected to the assay.
5. (a) Yamamoto, T.; Niwa, S.; Ohno, S.; Onishi, T.; Matsueda, H.; Koganei, H.;
Uneyama, H.; Fujita, S.; Takeda, T.; Kito, M.; Ono, Y.; Saitou, Y.; Takahara, A.;
Iwata, S.; Yamamoto, H.; Shoji, M. Bioorg. Med. Chem. Lett. 2006, 16, 798; (b)
Yamamoto, T.; Niwa, S.; Iwayama, S.; Koganei, H.; Fujita, S.; Takeda, T.; Kito, M.;
Ono, Y.; Saitou, Y.; Takahara, A.; Iwata, S.; Yamamoto, H.; Shoji, M. Bioorg. Med.
Chem. 2006, 14, 5333; (c) Yamamoto, T.; Niwa, S.; Ohno, S.; Tokumasu, M.;
Masuzawa, Y.; Nakanishi, C.; Nakajo, A.; Onishi, T.; Koganei, H.; Fujita, S.;
Takeda, T.; Kito, M.; Ono, Y.; Saitou, Y.; Takahara, A.; Iwata, S.; Shoji, M. Bioorg.
Med. Chem. Lett. 2008, 18, 4813; (d) Yamamoto, T.; Niwa, S.; Ohno, S.; Tokumasu,
M.; Masuzawa, Y.; Nakanishi, C.; Nakajo, A.; Onishi, T.; Koganei, H.; Fujita, S.;
Takeda, T.; Kito, M.; Ono, Y.; Saitou, Y.; Takahara, A.; Iwata, S.; Shoji, M. Drugs
Future 2008, 33, 150; (e) Yamamoto, T.; Ohno, S.; Niwa, S.; Tokumasu, M.;
Hagihara, M.; Koganei, H.; Fujita, S.; Takeda, T.; Saitou, Y.; Iwayama, S.;
Takahara, A.; Iwata, S.; Shoji, M. Bioorg. Med. Chem. Lett. 2011, 21, 3317.
6. The synthesis of (+)-3f was performed as follows: Step 1. Synthesis of racemic 4-
(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-
cinnamyl ester 5-(2-cyanoethyl) ester (10f): 3,5-dichloro-4-methoxy-
benzaldehyde (5; 318 mg, 1.86 mmol), propynoic acid 2-cyanoethyl ester (7;
229 mg, 1.86 mmol), propynoic acid cinnamyl ester (9; 351 mg, 1.88 mmol), and
ammonium acetate (270 mg, 3.50 mmol) were dissolved in 2-propanol and
stirred overnight at 80 °C. The solvent was evaporated under reduced pressure,
and the residue was extracted three times with EtOAc from water. The title
compound (116 mg, 0.23 mmol, 12.2%) was obtained after purification by silica
gel chromatography (hexane/EtOAc, 3:1). 1H NMR (CDCl3): 2.56 (2H, t), 3.83 (3H,
s), 3.83 (3H, s), 4.18–4.40 (2H, m), 4.63–4.83 (2H, m), 4.87 (1H, s), 6.23 (1H, dt),
6.34 (1H, br d), 6.58 (1H, d), and 7.23-7.47 (9H, m). MS (ESI, m/z) 511 (MꢀH)ꢀ.
Step 2. Optical separation of 10f: the racemic mixture of 10f (3.50 g, 6.89 mmol;
ca 100 mg for each injection) was separated by HPLC using a chiral column
(semi-preparative HPLC system: HITACHI L-6200 system; Daicel Chiralcel OD,
Daicel Chemical Industries Inc.; 250 ꢁ 0.46 cm ID; n-hexane/ethanol = 85/15;
flow rate: 6 mL/min; detection: 254 nm). The fraction between 88 and 102 min
was collected to obtain (+)-10f (1.39 g, 2.70 mmol, 39.2%). Enantiomeric excess:
99.7%, (analytical HPLC system: HITACHI L-6200 system; column: Daicel
Chiralcel OD-H, 25 ꢁ 0.46 cm ID; Solvent: n-hexane/EtOH = 80:20; Flow rate:
The medium for the 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 performed at 37 °C for 30 min. Next, the medium was replaced with a
recording medium (20 mM HEPES–KOH, 115 mM NaCl, 5.4 mM KCl,
0.8 mM mgCl2, 1.8 mM CaCl2, and 13.8 mM D-glucose).
A fluorescence
microscope (Nikon Corporation) and an image analysis device ARGUS 50
(Hamamatsu Photonics) were used to analyze the intracellular calcium
concentrations. To prevent the activation of the L-type calcium channels in
the differentiated IMR-32 cells, a recording medium containing 1
selective L-type calcium channel blocker, nifedipine, was used throughout the
experiment. Then, stimulating medium containing 60 mM KCl (KCl was
lM of a
a
substituted for equimolar NaCl in the recording medium) was rapidly given by
the Y-tube method for 6 s. The change in the intracellular calcium concentration
was expressed as the N-type calcium channel activity (please see Takahara, A.;
Fujita, S.; Moki, K.; Ono, Y.; Koganei, H.; Iwayama, S.; Yamamoto, H. Hypertens
Res. 2003, 26, 743). Then, 60 mmol/L KCl solution was applied repeatedly at
5 min intervals, and 0.1, 1, or 10 lM of a test compound was applied 4–5 min
before the application of 60 mmol/L KCl. The antagonistic activity of each test
compound on the N-type calcium channel was expressed as a 50% inhibitory
concentration (IC50), which was calculated as previously reported (Dohmoto, H;
Takahara, A; Uneyama, H; Yoshimoto, R. J Pharmacol Sci. 2003, 91(2), 163) using
data from at least two independent experiments.
It should be noted that the tested compounds were evaluated at three
concentrations to prevent ‘‘run-down’’ of the cells used in the experiments, on
the basis of the reproducibility data of the KCl-induced responses in the absence
of drugs in our preliminary study.
8. Measurement of L-type calcium channel (CaV1.1–CaV1.4) inhibitory activity was
performed as follows (previously reported in the Ref. 5): 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 removed 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 a resting tension of 2 g for 1 h.
Next, the ring was incubated in a high K+ solution (containing 50 mM KCl; KCl
was substituted 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 attaining the maximum contraction reaction, the test
compound was cumulatively added at intervals of 90 min to attain
concentrations of 10ꢀ7, 10ꢀ6, and 10ꢀ5 M for the normal screening protocol
1.0 mL/min; Detection: 254 nm), ½
a
ꢂD25 = +12.05 (c = 0.95, MeOH).
Step 3. Synthesis of (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-
pyridine-3,5-dicarboxylic acid 3-cinnamyl ester (+)-3f): 1.37 g (2.66 mmol) of
(+)-10f was dissolved in MeOH (50 mL), and 1 M NaOH (8 mL) was added. After
stirring for 2 h, 1 M HCl (8 mL) was added, and the solvent was removed under
reduced pressure. After extracting three times with EtOAc, the organic layer was
dried over anhydrous sodium sulfate, and the solvent was evaporated under
reduced pressure. The residue was purified by silica gel chromatography (CHCl3/
MeOH = 500/1) to obtain the title compound (1.08 g, 2.34 mmol, 88.0%). purity:
97.1% (analytical HPLC system: HITACHI L-6200 system; C-18 reversed-phase
column: YMC-Pack ODS AM, 15 ꢁ 0.46 cm ID, YMC Co., Ltd; solvent: water/
acetonitrile = 50:50–100:0 in 30 min; flow rate: 1.0 mL/min; detection: 254 nm,
and of 10ꢀ7 10ꢀ6 10ꢀ5 10ꢀ4.5 and 10ꢀ4
, , , , M for compound (+)-3f. The
antagonistic activity of each test compound on the L-type calcium channel
was expressed as a 50% inhibitory concentration (IC50), which was calculated as
previously reported (Dohmoto, H; Takahara, A; Uneyama, H; Yoshimoto, R. J
Pharmacol Sci. 2003, 91(2), 163) using data from at least two independent
experiments.
½
a
ꢂD25 = +51.12 (c 1.00, MeOH). 1H NMR (DMSO-d6) d: 3.76(3H, s), 4.61–4.78(2H,
m), 4.73(1H, s), 6.33(1H, dt), 6.56(1H, d), 7.20–7.48(9H, m), and 9.27(1H, t). HR–
9. The experiments were performed as reported in the reference Koganei, H.; Shoji,
M.; Iwata, S. Biol. Pharm. Bull. 2009, 32, 1695.
MS (FAB, m/z) calcd 458.0562 (MꢀH)ꢀ observed 458.0565.