Asymmetric synthesis and biological evaluations of (+)- and (-)-6-dimethoxymethyl-1,4-dihydropyridine-3-carboxylic acid derivatives blocking N-type calcium channels

Article abstract of DOI:10.1016/j.bmcl.2011.04.007

An efficient asymmetric synthesis of 1,4-dihydropyridine derivatives is described. The key step is the stereoselective Michael addition using t-butyl ester of l-valine as a chiral auxiliary to achieve good ee (>95% for all the tested experiments) and moderate yield. With this method, (+)-4-(3-chlorophenyl) -6-dimethoxymethyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid cinnamyl ester was obtained and was characterized as a promising N-type calcium channel blocker with improved selectivity over L-type compared to its (-)- and racemic isomers.

Full text of DOI:10.1016/j.bmcl.2011.04.007

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3317–3319
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Asymmetric synthesis and biological evaluations
of (+)- and (ꢀ)-6-dimethoxymethyl-1,4-dihydropyridine-3-carboxylic acid
derivatives blocking N-type calcium channels
⇑
Takashi Yamamoto , Seiji Ohno, Seiji Niwa, Munetaka Tokumasu, 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:
An efficient asymmetric synthesis of 1,4-dihydropyridine derivatives is described. The key step is the
Received 26 January 2011
Revised 1 April 2011
Accepted 5 April 2011
Available online 9 April 2011
stereoselective Michael addition using t-butyl ester of L-valine as a chiral auxiliary to achieve good ee
(>95% for all the tested experiments) and moderate yield. With this method, (+)-4-(3-chlorophenyl)-6-
dimethoxymethyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid cinnamyl ester was obtained
and was characterized as a promising N-type calcium channel blocker with improved selectivity over
L-type compared to its (ꢀ)- and racemic isomers.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
N-type calcium channel blocker
Structure–activity relationships
1,4-Dihydropyridine derivative
Asymmetric synthesis
Table 1
1,4-Dihydropyridines (DHPs) have been considered as a privi-
leged pharmacophore to modulate calcium current at the volt-
age-dependent calcium channels (VDCCs), and widely used in
clinical.1 VDCCs mediate calcium influx into cells in response to
diverse physiological changes, such as triggering various physio-
logical events such as neuronal excitability, muscle contraction
and secretion of neurotransmitters, and they are generally classi-
fied into at least five subtypes based on their pharmacological
and functional properties: L- (CaV1.1–CaV1.4), P/Q- (CaV2.1),
N- (CaV2.2), R- (CaV2.3) and T-type (CaV3.1–CaV3.3).2
Cilnidipine (1), one of the marketed drugs possessing a DHP
pharmacophore, is a potent dual blocker for N/L-type VDCCs and
is currently used for the treatment of essential hypertension in
Japan and South Korea (Table 1). The N-type calcium channel dis-
tributes mainly on the endings of central and peripheral nerves to
control neuronal excitability and secretion of neurotransmittersis
(ex. glutamate, substance P or CGRP) in strong association with
the pathological process of neuropathic pain. Thus, blockade of
the N-type VDCC has been considered as a promising therapeutic
target to be a novel type of analgesic. Therefore, many efforts have
been made to discover efficacious small-molecule N-type VDCC
blockers to date.3
Activity table of dihydropyridine derivatives. In vitro inhibition against N-type
(calcium influx using IMR-32 cells) and L-type (magnus method) calcium channels
3'
Cl
O
NO2
O
O
O
O
4
*
3
HO
O
O
Ph
R
Ph
5
2
6
N
H
1 N
H
O
Cilnidipine ( 1); R = OCH2CH2OMe
APJ2708 ( 2) ; R = OH
3
Compd
N-type
IMR-32
L-type
Magnus
IC50, lM
eea (%)
IC50
,
lM
Cilnidipine (1)
AJP2708 (2)
3
(+)-3
(ꢀ)-3
1.6
3.5
3.0
2.6
2.6
0.0011
0.046
8.1
13.2
1.8
Racemic
Racemic
Racemic
95.6b
97.1c
a
b
c
Determined as 2-cyanoethyl ester (10) by chiral HPLC.
Mean value of two independent experiments (96.0 and 95.2% ee).
Mean value of two independent experiments (98.7 and 95.5% ee).
As previously reported,4 we have performed a structure–activ-
ity relationship study on APJ2708 (2), a 3-carboxylate DHP deriva-
⇑
Corresponding author. Tel.: +81 44 210 5826.
E-mail address: takashia_yamamoto@ajinomoto.com (T. Yamamoto).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.007

3318
T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3317–3319
Cl
O
a
CN
O
CN
O
O
4
5
O
O
O
O
O
tBuO
O
O
O
NH
b
O
Ph
O
O
Ph
6
7
O
O
c
Cl
O
Cl
O
O
*
*
NC
NC
O
O
Ph
O
O
O
Ph
OH
O
HN
O
N
H
O
O
O
OtBu
9
8
Cl
Cl
O
O
O
O
*
NC
*
O
O
O
O
Ph
d
HO
Ph
O
N
N
H
H
O
O
(+)-10
(+)-3
Scheme 1. Reagents and conditions: (a) 3-chlorobenzaldehyde, cat. piperidine, cat. AcOH, i-PrOH, 66.3%; (b)
then ꢀ35 °C, 71.5%; (ii) 1 M HCl aq, rt; (iii) AcONH4, EtOH, 50 °C, then 120 °C; (d) 1 M NaOH, MeOH, 83.7%.
L-Val-O-tBu, benzene, reflux, 61.6%; (c) (i) 5, LDA, THF, ꢀ78 °C
tive of 1, to find an effective and selective N-type calcium channel
inhibitor with the least influence on cardiovascular system. As a re-
sult of the structural optimization, 4-(3-chlorophenyl)-6-dimethyl-
acetal DHP derivative 3 was identified as an promising N-type
VDCC blocker showing significant analgesic potency with more
than 170-fold lower activity for L-type channel compared to that
of 2.4d
easily available and its bulky isopropyl group plays an effective role
as a driving force for the highly enantiomeric purity (>95% ee in all
the tested experiments) of the obtained chiral DHPs.
The experiments were performed as follows.10 2-Benzylidene-3-
oxo-butyric acid 2-cyanoethyl ester (5) was prepared using Knoeve-
nagel condensation of 3-oxo-butyric acid 2-cyanoethyl ester (4)
with the 3-chlorobenzaldehyde. The 4,4-dimethoxy-3-oxo-butyric
Here, when DHP derivative has non-identical ester groups in the
3- and 5-positions, the molecule is chiral, with the carbon at 4 po-
sition as the stereogenic center, which sometimes has critical effect
on its biological activities; for example, S-configuration is crucial
for potent inhibitory activity for L-type VDCC.5 Thus, the separation
of two enantiomers of racemic DHP derivative 3 could be an effec-
tive strategy to identify a compound with the improved selectivity
for the N-type VDCC over the L-type channels.
acid cinnamyl ester (6) and L-valine t-butyl ester was refluxed in
benzene to afford enamine 7, which was treated with LDA at
ꢀ78 °C followed by slow addition of 5 to conduct key stereoselective
step. The obtained crude 8 was dissolved in ethanol, and then
ammonium acetate was added to yield the mixture of DHP diester
(+)-10 and its hydrated intermediate 9, which was heated to convert
into (+)-10 by a single spot. The obtained (+)-10 showed 96.0% ee
based on the HPLC analysis using Daicel chiralcel OD-H column.
1 M sodium hydroxide solution was treated to the desired chiral
DHP dicarboxylic acid mono cinnamyl ester (+)-3 by 44.0% overall
yield from 6.11,12 The corresponding enantiomer (ꢀ)-3 was also pre-
Several methods for synthesis of chiral DHPs have been reported,
including asymmetric induction,6 resolution of racemic mixture,7 or
chemoenzymatic method.8 Asymmetric Michael addition using
a-
methylbenzylamine as chiral inducer was also reported by Ash-
pared with the same procedure using D-valine t-butyl ester.
worth et al.,9 but their procedure suffered from low d.e. (about
The obtained (+)- and (ꢀ)-3 were characterized using IMR-32
human neuroblastoma cells for N-type VDCC13 and rat thoracic
aorta ring for L-type VDCC.14,15 As found in Table 1, two enantio-
mers showed different inhibitory activities for L-type VDCC: (ꢀ)-
70%). We hypothesized that steric hindrance of the a-methylbenzyl-
amine was not enough to achieve sufficient stereoselectivity, and
applied novel approach to synthesis of highly enantioselective iso-
mer of compound 3. Our synthetic route was shown in the Scheme
3 was 7 times more potent than (+)-3 (1.8 and 13.2 lM, respec-
1. The key of this method is using t-butyl ester of
L
-valine, which is
tively). For the N-type VDCC, these enantiomers both showed

T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3317–3319
3319
2.26 (1H, m), 3.33 (6H, s), 4.25 (1H, dd, J = 4.5, 10.8 Hz), 4.61 (1H, s), 4.76 (2H,
m), 4.89 (1H, s), 6.32 (1H, m), 6.65 (1H, d, J = 15.9 Hz), 7.20–7.31 (5H, m), 8.48
(1H, d, J = 9.0 Hz). MS (ESI, m/z) 434 (M+H)+.
potent activities with no detectable difference (2.6 lM), implying
that the chirarity at the C4 position affects only on the binding at
the L-type VDCC, but not at the N-type channels.
Step 3. Synthesis of (+)-4-(3-chlorophenyl)-6-dimethoxymethyl-2-methyl-1,4-
dihydropyridine-3,5-dicarboxylic acid cinnamyl ester 5-(2-cyanoethyl) ester ((+)-
10): 567 mg of 7 (1.31 mmol) was dissolved in THF (50 mL) and 2 M solution of
LDA in THF (0.678 mL) was slowly added to the obtained solution at ꢀ78 °C,
and then stirred for 2 h 30 min. 364 mg of 5 (1.31 mmol) in THF (32 mL) was
added to the reaction mixture, stirred at ꢀ78 °C for 3 h and then at ꢀ35 °C for
overnight. 1 M HClaq (3 mL) was slowly added to the obtained mixture at
ꢀ35 °C followed by additional 1 M HCl aq (3 mL) at rt. After stirred for 6 h, the
reaction mixture was extracted three times with EtOAc, the organic layer was
dried over anhydrous sodium sulfate and the solvent was evaporated under
reduced pressure. The obtained residue was dissolved in ethanol (40 mL) and
ammonium acetate 537 mg (6.55 mmol) was added to the obtained solution.
After stirring for overnight at 50 °C, the solvent was evaporated under reduced
pressure. After extracting three times with EtOAc from water, the organic layer
was dried over anhydrous sodium sulfate and the solvent was evaporated
under reduced pressure. The residue was heated at 120 °C for 2 h 30 min, and
then purified by silica gel chromatography (hexane/EtOAc, 10:1) to obtain the
title compound (502 mg, 0.94 mmol, 71.5%). Optical purity: 96.0% ee
(analytical HPLC system: HITACHI L-6200 system; Column: Daicel chiralcel
OD-H, 25 cm ꢂ 0.46 cm ID, Daicel Chemical Industries Inc.; Solvent: n-hexane/
In summary, novel asymmetric Michael addition was per-
formed using L- or D-valine t-butyl ester as a chiral auxiliary for
the preparation of enantioenriched 6-dimethoxymethyl-1,4-dihy-
dropyridine-3-carboxylic acid DHP derivatives. Among the two
synthesized enantiomers, (+)-3 was found as an effective blocker
for N-type VDCC with the improved selectivity over L-type chan-
nel, compared to its (ꢀ)- and racemic isomers. Thus, (+)-3 could
be considered as an interesting research tool to seek for a promis-
ing drug candidate to control severe to moderate pain states.
References and notes
1. (a) Safak, C.; Simsek, R. Mini-Rev. Med. Chem. 2006, 6, 747; (b) Furukawa, T.;
Nukada, T.; Namiki, Y.; Miyashita, Y.; Hatsuno, K.; Ueno, Y.; Yamakawa, T.;
Isshiki, T. Eur. J. Pharmacol. 2009, 613, 100.
2. Yaksh, T. L. J. Pain 2006, 7, S13.
EtOH = 92:8; Flow rate: 1.0 mL/min; Detect: 254 nm), ½a D25
ꢁ
= +43.59 (c 1.28,
3. (a) Yamamoto, T.; Takahara, A. Curr. Top. Med. Chem. 2009, 9, 377; (b) Barrow, J.
C.; Duffy, J. L. Annu. Rev. Med. Chem. 2010, 45, 2; (c) 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.
MeOH). 1H-NMR (CDCl3) d: 2.39 (3H, s), 2.61 (2H, t, J = 6.3 Hz), 3.44 (3H, s), 3.47
(3H, s), 4.19–4.32 (2H, m), 4.66–4.83 (2H, m), 5.05 (1H, s), 6.04 (1H, s), 6.24
(1H, dt, J = 9.3, 16.2 Hz), 6.56 (1H, d, J = 16.2 Hz), 6.83 (1H, br s), 7.10–7.39 (9H,
m). MS (ESI, m/z) 535 (MꢀH)ꢀ.
Step 4. Synthesis of (+)-4-(3-chlorophenyl)-6-dimethoxymethyl-2-methyl-1,4-
dihydropyridine-3,5-dicarboxylic acid cinnamyl ester ((+)-3): 64.6 mg of (+)-10
(0.121 mmol) was dissolved in MeOH (10 mL) and 1 M NaOH (0.13 mL) was
added to the obtained solution. After stirring for 4 h at rt, 1 M HCl aq (0.14 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 the silica gel chromatography (hexane/EtOAc, 10:1 to
2:1) to obtain the title compound (49.0 mg, 0.10 mmol, 83.7%). Purity: 98.9%
(analytical HPLC system: HITACHI L-6200 system; C-18 reversed-phase
column: YMC-Pack ODS AM, 15 cm ꢂ 0.46 cm ID, YMC Co., Ltd; Solvent:
water/acetonitrile = 50:50 to 80:20 in 30 min; Flow rate: 1.0 mL/min; Detect:
4. (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(Suppl. A), P150.
5. Arhancet, G. B.; Woodard, S. S.; Dietz, J. D.; Garland, D. J.; Wagner, G. M.; Iyanar,
K.; Collins, J. T.; Blinn, J. R.; Numann, R. E.; Hu, X.; Huang, H. C. J. Med. Chem.
2010, 53, 4300.
6. Martin, N.; Martinez-Grau, A.; Seoane, C.; Marco, J. L.; Albert, A.; Cano, F. H.
Tetrahedron: Asymmetry 1995, 6, 877.
254 nm), ½a 2D5
ꢁ
= +7.69 (c 0.858, MeOH). 1H-NMR (CDCl3) d: 2.38 (3H, s), 3.43
(3H, s), 3.47 (3H, s), 4.65–4.83 (2H, m), 5.06 (1H, s), 6.04 (1H, s), 6.23 (1H, dt,
J = 6.0, 15.9 Hz), 6.54 (1H, d, J = 15.9 Hz), 6.86 (1H, br s), 7.07–7.38 (9H, m). HR-
MS (FAB, m/z) calcd 482.1370 (MꢀH)ꢀ observed 482.1363.
11. The X-ray crystal structural study of a derivative of compound 3 (4-(3,4-
dichloro-5-methoxyphenyl)-6-dimethoxymethyl-2-methyl-1,4-
7. See the following reference and those cited within: Zhang, B. L.; He, W.; Shi, X.;
Huan, M. L.; Huang, Q. J.; Zhou, S. Y. Bioorg. Med. Chem. Lett. 2010, 20, 805. and
references herein.
8. Kosugi, Y.; Hori, M.; Tatsuo, N. Heterocycles 1994, 39, 591.
9. Ashworth, I.; Hopes, P.; Levin, D.; Patel, I.; Salloo, R. Tetrahedron Lett. 2002, 43,
4931.
dihydropyridine-3,5-dicarboxilic acid cinnamyl ester) showed that the
enantiomer synthesized using
data).
L-Val-O-tBu is as a (R)-isomer (unpublished
12. Due to peak broadening in chiral HPLC conditions, the optical purify of nether
(+)- nor (ꢀ)-3 could be directly determined. However, derivative of
compound (4-(3-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
a
3
10. The synthesis of (+)-3 was conducted as follows:
dicarboxylic acid cinnamyl ester) was obtained with 96.0% ee using same
synthetic procedure (unpublished data), implying that the hydrolysis step
induces negligible racemization at the C-4 position.
Step1. Synthesis of 2-Acetyl-3-(3-chlorophenyl)-acrylic acid (2-cyanoethyl) ester
(5): 3-Oxo-butenoic acid 2-cyanoethyl ester (4) 3.00 g (19.3 mmol), 3-
chlorobenzaldehyde 2.72 g (19.3 mmol), acetic adid 79 mg (0.97 mmol) and
piperidine 58 mg (0.97 mmol) was stirred in 2-propanol (50 mL) for overnight.
The solvent was evaporated under reduced pressure and the residue was
purified by the silica gel chromatography (hexane/EtOAc, 3:1) to obtain the
title compound as an yellow oil (3.55 g, 12.8 mmol, 66.3%). 1H NMR (CDCl3) d:
2.05 (3H, s), 2.63 (2H, t, J = 6.6 Hz), 3.92 (2H, t, J = 6.6 Hz), 7.00–7.88 (5H, m).
MS (ESI, m/z) 278 (M+H)+.
13. Takahara, A.; Fujita, S.; Moki, K.; Ono, Y.; Koganei, H.; Iwayama, S.; Yamamoto,
H. Hypertens. Res. 2003, 26, 743.
14. Feng, P. C.; Haynes, L. J.; Magnus, K. E. Nature 1961, 191, 1108.
15. As reported in the reference (Takahara, A.; Koganei, H.; Takeda, T.; Iwata, S. Eur.
J. Pharmacol. 2002, 434, 43.), cilnidipine (compound 1) can inhibit both the
sympathetic N-type and vascular L-type calcium channels in antihypertensive
doses (3 lg/kg, iv administration), suggesting that its ratio of L-type/N-type
Step 2. Synthesis of 3-(tert-butoxycarbonyl-2-methylpropylamino)-4,4-
dimethoxybutenoic acid cinnamyl ester (7): 3-Oxo-4,4-dimethoxybutenoic acid
cinnamyl ester (6) 16.24 g (58.4 mmol) and L-Val-O-tBu 10.12 g (58.4 mmol)
blocking activity is about 1. Based on the information, the IMR-32 assay system
for this series of compounds can be estimated to be about 1000 times less
sensitive than our assay in Magnus method using rat thoracic aorta ring for
detecting each calcium channel blocking action. Therefore, the IC50 values of
the method against L-type VDCC could not be simply compared with the IC50
values of the cell-based IMR-32 method against N-type channel.
was heated in benzene (60 mL) at reflux for 6 h while the water by-product
was collected in a Dean–Stark trap. The solvent was evaporated under reduced
pressure and the residue was purified by silica gel chromatography (hexane/
EtOAc, 9:1) to obtain the title compound (15.58 g, 35.9 mmol, 61.6%). ½a D25
ꢁ
=
+95.52 (c 1.00, MeOH). 1H NMR (CDCl3) d: 0.98–1.02 (6H, m), 1.46 (9H, s), 2.11–