The structure-activity relationship study on 2-, 5-, and 6-position of the water soluble 1,4-dihydropyridine derivatives blocking N-type calcium channels

Article abstract of DOI:10.1016/j.bmcl.2008.07.096

In order to find an injectable and selective N-type calcium channel blocker, we have performed the structure-activity relationship (SAR) study on the 2-, 5-, and 6-position of 1,4-dihydropyridine-3-carboxylate derivative APJ2708 (2), which is a derivative of Cilnidipine and has L/N-type calcium channel dual inhibitory activities. As a consequence of the optimization, 6-dimethylacetal derivative 7 was found to have an effective inhibitory activity against N-type calcium channels with more than 170-fold lower activity for L-type channel compared to that of APJ2708.

Full text of DOI:10.1016/j.bmcl.2008.07.096

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4813–4816
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The structure–activity relationship study on 2-, 5-, and 6-position of the
water soluble 1,4-dihydropyridine derivatives blocking N-type calcium channels
*
Takashi Yamamoto , Seiji Niwa, Seiji Ohno, Munetaka Tokumasu, Yoko Masuzawa, Chika Nakanishi,
Akira Nakajo, Tomoyuki Onishi, Hajime Koganei, Shin-ichi Fujita, Tomoko Takeda, Morikazu Kito,
Yukitsugu Ono, Yuki Saitou, Akira Takahara, Seinosuke Iwata, Masataka Shoji
Pharmaceutical Research Laboratory, Ajinomoto company Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to find an injectable and selective N-type calcium channel blocker, we have performed the struc-
ture–activity relationship (SAR) study on the 2-, 5-, and 6-position of 1,4-dihydropyridine-3-carboxylate
derivative APJ2708 (2), which is a derivative of Cilnidipine and has L/N-type calcium channel dual inhib-
itory activities. As a consequence of the optimization, 6-dimethylacetal derivative 7 was found to have an
effective inhibitory activity against N-type calcium channels with more than 170-fold lower activity for
L-type channel compared to that of APJ2708.
Received 23 April 2008
Revised 17 July 2008
Accepted 24 July 2008
Available online 27 July 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
N-type calcium channel blocker
Structure–activity relationships
1,4-dihydropyridine derivative
Calcium ions act as an intracellular signal and play a critical role
in variety of biological functions including muscle contraction, re-
lease of neurotransmitters, calcium-dependent gene transcription,
and the regulation of neuronal excitability. Several types of volt-
age-dependent calcium channels have been identified in native tis-
sues and classified into at least five subtypes based on their
pharmacological and functional properties: N-(CaV2.2), L-(CaV1.1-
CaV1.4), P/Q-(CaV2.1), R-(CaV2.3) and T-type (CaV3.1-CaV3.3).1
The N-type calcium channel is a well established and character-
ized subtype which is expressed on nerve endings to regulate neu-
ronal overexcitement and pain signals. Therefore, agents which
inhibit the N-type calcium channel have been shown to be effec-
tive therapies for several pathological states including ischemic
brain injury and neuropathic pain.2 In fact, Ziconotide, which is
the chemically synthesized version of 25-residue-peptide marine
in demand, especially for a patient who cannot take medicine oral-
ly (see Table 1).
3'
NO2
Cl
O
O
O
O
O
Ph
4
3
R
Ph
HO
O
Ph
5
2
6
1 N
N
CF3
H
H
Cilnidipine ( 1); R = OCH2CH2OMe
APJ2708 ( 2) ; R = OH
3
In order to find a selective N-type calcium channel blockers
with the least effects on cardiovascular system, we have performed
a structure–activity relationship study on APJ2708 (2), which is a
derivative of Cilnidipine (1) and has a unique 1,4-dihydropyri-
dine-3-carboxylate structure with dual blockage activities at the
L/N-type calcium channels. As a consequence of the optimization
on 4-, 5-, and 6-position of 1,4-dihydropyridine structure in 2, 3
was found as a promising N-type calcium channel blocker possess-
ing potent analgesic effect in vivo with a 40-fold lower activity
against L-type calcium channels than that of 2.4g In this study,
we made further optimization of 2-, 5-, and 6-position of 1,4-dihy-
dropyridine ring in 2 to find a selective N-type calcium channel
blocker with high aqueous solubility.
toxin
x-conotoxin MVIIA, has approved as an analgesic drug for se-
vere chronic pain treatment. However, although this drug has been
shown a non-addictive analgesic effect without opiates-like toler-
ance, its clinical use is limited because Ziconotide could be admin-
istered only via intrathecal route with least compliance for a
patient.3 Thus, systemically available N-type calcium channel
blocker is still strongly needed and many small molecules were re-
ported mostly for obtaining oral drug candidates.4 An injectable N-
type calcium channel blocker with high aqueous solubility is also
* 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 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.096

4814
T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4813–4816
Table 1
selective deprotection of 2-cyanoethyl ester group of 6 was per-
Activity table of dihydropyridine derivatives
formed by the treatment with 1 M sodium hydroxide solution to
give 7.4g The treatment of 6 under acidic condition yielded 6-alde-
hyde derivative 9 which was converted into cyanide 10 in the pres-
ence of acetic anhydride. Final selective hydrolysis with 1 M
sodium hydroxide gave 11. The rest of reported derivatives were
synthesized with same method as 7 from the corresponding substi-
tuted cinnamyl alcohol, b-ketoester, enamine and benzaldehyde as
starting materials.
Cl
O
O
HO
O
Ph
R1
N
R2
H
The biological evaluation of the synthesized 1,4-dihydropyri-
dine-3-carboxylate derivatives was performed using the procedure
as previously described.4g,h Their inhibitory activities at the N-type
Compound
R1
R2
N-type IMR-32
L-type Magnus
IC50
IC50
,
lM
, lM
Cilnidipine (1)
AJP2708 (2)
3
1.6
3.5
1.7
0.0011
0.046
1.8
calcium channel were determined using fluorescence based Ca2+
-
flux assay in IMR-32 human neuroblastoma cells.7,8 The L-type cal-
cium channel inhibitory activities were estimated from the effects
on high K+-induced contraction in rat thoracic aorta ring by Mag-
nus technique.9,10 In our previous in vivo study, 1 can inhibit both
the sympathetic N-type and vascular L-type calcium channels in
12a
12b
11
7
12c
Me
Me
Me
Me
Me
Et
CN
CH(OMe)2
Me
1.0
0.81
5.8
3.0
3.9
0.020
0.007
2.0
8.1
1.3
CH(OMe)2
antihypertensive doses (3 l
g/kg, i.v. administration),11 suggesting
In vitro inhibition against N-type (calcium influx using IMR-32 cells) and L-type
(magnus method) calcium channels.
that its ratio of L-type/N-type blocking activity is about 1. Based
on the information, the IMR model can be estimated to be about
1000 times less sensitive than the Magnus assay for detecting each
calcium channel blocking action. Therefore, the IC50 values of the
method against L-type calcium channel (Magnus method) could
not be simply compared with the IC50 values of the cell-based
IMR-32 method against N-type calcium channel.
In the present study, the structural optimization was started on
the 2,6-dimethyl-1,4-dihydropyridine-3-carboxylate derivative
12a, which had chlorine at 30-position in place of nitro group in
2, since 12a showed higher inhibitory activity for N-type calcium
The 1,4-dihydropyridine-3-carboxylate derivatives were syn-
thesized using Hantzsch 1,4-dihydropyridine synthesis reaction
as previously reported (Scheme 1).4g,5 The 4,4-dimethoxy-3-oxo-
butyric acid cinnamyl ester (5) was obtained by heating 4,4-dime-
thoxy-3-oxo-butyric acid ethyl ester (4)6 and cinnamyl alcohol to-
gether to conduct the transesterification. The 1,4-dihydropyridine
dicarboxylic acid 2-cyanoethyl ester 5-cinnamyl ester (6) was ob-
tained with two step reactions, Knoevenagel condensation with
3-chlorobenzaldehyde followed by 1,4-dihydropyridine ring con-
struction using 3-amino-2-butenoic acid 2-cyanoethyl ester. The
channels than that of 2 (IC50 = 1.0 and 0.020 lM for N- and L-type
calcium channels, respectively).4g The substitution of 6-methyl
group in 12a with ethyl group showed increased activities both
for the N- and L-type calcium channels (12b, IC50 = 0.81 and
0.007 lM for N- and L-type calcium channels, respectively). While
O
O
O
O
the introduction of cyano group (11) led to 5.8-times lower inhib-
itory activity at the N-type calcium channel than that of 12a, but its
blockage activity for L-type channel showed far larger decrease
(100-fold less activity than that of 12a), resulting in increased
a
O
O
O
Ph
O
O
O
4
5
selectivity for N-type channel (IC50 = 5.8 and 2.0 lM for N- and L-
type calcium channels, respectively). Furthermore, 7, possessing
dimethoxyacetal group at the 6-position, showed 400-fold de-
creased inhibitory activity for L-type calcium channel with only
3-times lower activity at N-type channel compared to those of
b
Cl
O
O
O
O
O
O
12a (IC50 = 3.0 and 8.1 lM for N- and L-type calcium channels,
O
Ph
c
R1O
O
O
Ph
respectively). This inhibitory activity for N-type calcium channel
of 7 was enough to show the analgesic effect in vivo based on
our previous result.4h However, the introduction of dimethoxyac-
etal group into 2-position (12c) was less effective than the substi-
O
Cl
N
H
8
6: R1 = CH2CH2CN
7: R1 = H
tution at the 6-position (IC50 = 3.9 and 1.3 lM for N- and L-type
d
calcium channels, respectively). This result indicated that the dim-
ethoxyacetal group at the 6-position strongly affected on the activ-
ity against L-type calcium channels but had fewer influence on the
N-type activities, leading to the better selectivity for N-type chan-
nel with the preserved potency. While the introduction of dim-
ethoxyacetal group at the 2-position of 1,4-dihydropyridine-3-
carboxylate structure resulted in a smaller decrease in the activity
for L-type calcium channel.
Cl
O
O
e
R1O
O
Ph
6
N
R2
H
Further optimization of 7 was made on the phenyl ring of cin-
namyl group (Table 2). The introduction of chlorine atom resulted
in the higher inhibitory activities for N-type calcium channel than
9: R1 = CH2CH2CN; R2 = CHO
10: R1 = CH2CH2CN; R2 = CN
f
g
2
11: R1 = H; R = CN
those of 7 (IC50 = 1.8, 1.4 and 1.7
tively). However, their activities for L-type channel showed larger
improvement than for N-type channel (IC50 = 0.7, 1.0 and 1.4
lM for 13a, 13b and 13c, respec-
Scheme 1. Reagents: (a) Cinnamyl alcohol, toluene; (b) 3-Chloro-benzaldehyde, i-
PrOH; (c) 3-Amino-2-butenoic acid 2-cyanoethyl ester, i-PrOH; (d) 1 M NaOH,
MeOH; (e) 6 M HCl, acetone; (f) NH2OH, AcONa, AcOH. Then Ac2O; (g) 1 M NaOH,
MeOH.
l
M
for 13a, 13b and 13c, respectively). The derivatives with fluorine
and trifluoromethyl group (13d and 13e) showed similar activity

T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4813–4816
4815
Table 2
References and notes
Activity table of dihydropyridine derivatives
1. (a) Bowersox, S. S.; Valentino, K. L.; Luther, R. R. Drug News Perspect. 1994, 7,
261; (b) Bean, B. P. Annu. Rev. Physiol. 1989, 51, 367; (c) Hess, P. Annu. Rev.
Neurosci. 1990, 13, 337; (d) Varadi, G.; Mori, Y.; Mikala, G.; Schwartz, A. Trends
Pharmacol. Sci. 1995, 16, 43; (e) Nooney, J. M.; Lambert, R. C.; Feltz, A. Trends
Pharmacol. Sci. 1997, 18, 363.
2. (a) Sheng, Z. H.; Rettig, J.; Cook, T.; Catterall, W. A. Nature 1996, 379, 451; (b)
Saegusa, H.; Kurihara, T.; Zong, S.; Kazuno, A.; Matsuda, Y.; Nonaka, T.; Han, W.;
Toriyama, H.; Tanabe, T. EMBO J. 2001, 20, 2349; (c) Cox, B.; Denyer, J. C. Exp.
Opin. Ther. Patents 1998, 8, 1237; (d) Miller, J. A.; Bialobok, P.; Gendron, P.;
Alnowske, J.; McCarthy, D.; Case, K.; Eisman, M.; Pierson, N. E.; Bostwick, J. R.
Society of Neuroscience Abstract 25 part 2, Rochester, NY and Worcester, Ma,
1999, pp. 896.3.; (e) Bowersox, S. S.; Singh, T.; Luther, R. R. Brain Res. 1997, 747,
343.
Cl
O
O
R
HO
O
O
N
H
O
Compound
R
N-type IMR-32 IC50
,
lM
L-type Magnus IC50, lM
7
H
3.0
1.8
1.4
1.7
1.8
2.0
2.5
3.0
2.1
8.1
0.7
1.0
1.4
2.0
3.2
5.9
4.4
0.54
3. Miljanich, G. P. Curr. Med. Chem. 2004, 11, 3029.
13a
13b
13c
13d
13e
13f
13g
13h
2-Cl
3-Cl
4-Cl
4-F
4. (a) Song, Y.; Bowersox, S. S.; Connor, D. T.; Dooley, D. J.; Lotarski, S. M.; Malone,
T.; Miljanich, G.; Millerman, E.; Rafferty, M. F.; Rock, D.; Roth, B. D.; Schmidt, J.;
Stoehr, S.; Szoke, B. G.; Taylor, C.; Vartanian, M.; Wang, Y.-X. J. Med. Chem. 2000,
43, 3474; (b) Seko, T.; Kato, M.; Kohno, H.; Ono, S.; Hashimura, K.; Takimizu, H.;
Nakai, K.; Maegawa, H.; Katsube, N.; Toda, M. Bioorg. Med. Chem. Lett. 2001, 11,
2065; (c) Seko, T.; Kato, M.; Kohno, H.; Ono, S.; Hashimura, K.; Takenobu, Y.;
Takimizu, H.; Nakai, K.; Maegawa, H.; Katsube, N.; Toda, M. Bioorg. Med. Chem.
Lett. 2002, 12, 2267; (d) Hu, L. Y.; Ryder, T. R.; Rafferty, M. F.; Taylor, C. P.; Feng,
M. R.; Kuo, B. S.; Lotarski, S. M.; Miljanich, G. P.; Millerman, E.; Siebers, K. M.;
Szoke, B. G. Bioorg. Med. Chem. 2000, 8, 1203; (e) Menzler, S.; Bikker, J. A.;
Suman-Chauhan, N.; Horwell, D. C. Bioorg. Med. Chem. Lett. 2000, 10, 345; (f)
Baell, J. B.; Duggan, P. J.; Forsyth, S. A.; Lewis, R. J.; Lok, Y. P.; Schroeder, C. I.
Bioorg. Med. Chem. 2004, 12, 4025; (g) 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; (h) 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.
5. (a) Hantzsch, A. Ann. Chem. 1882, 215, 72; (b) Mosher, H. S. Heterocycl.
Compounds 1950, 1, 462; (c) Richard, M.; Kellogg, T. J.; Bergen, V.; Van Doren,
H.; Hedstrand, D.; Kooi, J.; Kruizinga, W. H.; Troostwijk, C. B. J. Org. 1980, 45,
2854.
6. Claude, P.; Vilhjalmur, G. S.; Meyers, J. P.; Logan, J. I.; Lynous, H. J. Med. Chem.
1964, 7, 337.
7. The inhibitory activity against N-type calcium channel was measured by
calcium influx into IMR-32 cells as described in the Refs. 4g, 4h and 8.
8. Takahara, A.; Fujita, S.; Moki, K.; Ono, Y.; Koganei, H.; Iwayama, S.; Yamamoto,
H. Hypertens Res. 2003, 26, 743.
9. Inhibitory activity against L-type calcium channels was estimated from the
effects on high K+-induced contraction in rat thoracic aorta ring as described in
reference. Ref. 4g.
10. Feng, P. C.; Haynes, L. J.; Magnus, K. E. Nature 1961, 191, 1108.
11. Takahara, A.; Koganei, H.; Takeda, T.; Iwata, S. Eur. J. Pharmacol. 2002, 434, 43.
12. The sodium salt of selected compound was suspended in saline, which was
sonicated for 10 min followed by shaking for 2 h 30 min at 25 °C. The
suspension was kept for overnight and then, centrifuged at 10,000 rpm for
5 min. The supernatant liquid was diluted and analyzed by HPLC assay.
13. PALLAS for Windows 3.0, CompuDrug Chemistry Ltd., was employed: Tayar, N.
E.; Tsai, R. S.; Carrupt, P. A.; Testa, B. J. Chem. Soc. Perkin Trans. 1992, 2, 79.
14. The electrophysiological activity for N-type calcium channel of the test
compound was estimated as follows: (a) Isolation of superior cervical ganglia
(SCG) neurons: Male Wistar rats (2- to 4-week-old) were decapitated after an
injection of sodium pentobarbitone (50 mg/kg, i.p.). The SCG were dissected,
connective tissues were removed, and three to four cuts were made in each
ganglion in Ca2+-free Tyrode solution. The ganglia were incubated at 37 °C
under 100% O2 atmosphere for 30 min in 20 U/ml papain and then for 60 min
in a mixture of 600 U/ml collagenase and 16 mg/ml dispase. The ganglia were
rinsed with the Ca2+-free Tyrode solution and triturated gently through a
4-CF3
2-OMe
3-OMe
4-OMe
In vitro inhibition against N-type (calcium influx using IMR-32 cells) and L-type
(magnus method) calcium channels.
trends to 13a–c with improved inhibitory activities at L-type cal-
cium channel (IC50 = 2.0 and 3.2 lM for 13d and 13e, respectively).
Therefore, all of these derivatives with halogen atoms (13a–e)
showed larger improvement in the activities for L-type calcium
channel rather than for N-type channel, resulting in the decreased
selectivity for N-type calcium channel compared to those of 7. The
4-methoxy derivative (13h) showed similar result to those of hal-
ogenated derivatives (13a–e) with 15-fold improvement in the
inhibitory activity for L-type channel and 1.4-fold higher activity
for N-type channel compared to those of
0.54 M for N- and L-type calcium channels, respectively). How-
7 (IC50 = 2.1 and
l
ever, the blockage activity for L-type channel of 3-methoxy deriv-
ative (13g) was only two times higher than that of 7. Moreover,
this activity shift for the L-type calcium channel was 1.4-fold for
the 4-methoxy derivative (13f), whose inhibitory activity for
N-type channel showed 1.2-fold improvement with nearly equiva-
lent selectivity for N-type channel to that of 7 (IC50 = 2.1 and
5.9
l
M for N- and L-type calcium channels, respectively).
As a result of the optimizations, 7 and 13f were found as the
selective and effective N-type calcium channel blockers. Moreover,
7 and 13f showed high aqueous solubility with low lipophilicity
and were proved to be good drug candidates which could be
administered via intravenous injection (Table 3).12,13
Finally, the N-type calcium channel inhibitory activity of com-
pound 7, which showed better solubility than 13f, was confirmed
by an electrophysiological study on rat superior cervical ganglion
(SCG) neuron (85% inhibition at 10ꢀ5 M).14
small-pore glass pipette. The isolated SCG neurones were plated on
a
In summary, we performed the structural optimization on 1,4-
polylysine coated recording chamber. (b) Electrical measurements:
Conventional whole-cell recordings of Ba2+ current through voltage-
dependent Ca2+ channels were made with voltage-clamped conditions. The
pipettes were pulled from 1.5 mm capillary glass (Narishige) in two stages by a
vertical pipette puller (Narishige, PB-7). The resistance between the recording
electrode filled with pipette solution and the reference electrode in external
dihydropyridine-3-carboxylate derivatives to find
a selective
blocker for the N-type calcium channel over the L-type channel.
The pharmacological characterizations of synthesized derivatives
were described and the 6-dimethoxymethyl derivatives 7 and
13f were found as the potent, selective, and aqueous soluble
N-type calcium channel blockers which could be novel candidates
for injectable neuroprotective and analgesic drugs.
solution was 2–5 MX. For whole-cell recording, an application of negative
pressure ruptures the captured membrane, providing continuity between the
pipette electrode and the cell’s cytoplasm. The dissociated SCG neuron was
held at a holding potential (VH) of ꢀ60 mV, and Na/K free Ba2+ solution was
applied from a tube within 200 lm from the SCG neuron by the ‘Y-tube’
method, as described elsewhere (Nakagawa, T.; Komune, S.; Vemura, T.;
Akaike, N. J. Neurophysiol., 1991, 65, 715., Uneyama, H.; Munakata, M.; Akaike,
N.; Brain Res., 1993, 604, 24., Uneyama, H.; Uneyama, C.; Akaike, N. J. Biol.
Chem., 1993, 268, 168.). The test compound was applied at 10ꢀ5 M. Neuron was
held at a VH of ꢀ60 mV and Ba2+ current was elicited by 200 ms depolarizing
pulses to 0 mV. Each concentration was applied to the neuron for 2 min before
subsequent five depolarizing pulses were applied every 10 s. The amplitude of
Ba2+ current was measured at the peak. The N-type current was defined as that
Table 3
Aquarius solubility and PrologD7.4 value of selected compounds
Compound
Solubility (mg/ml)
PrologD7.4
3
7
13f
3.5
>15
10.2
3.51
1.29
1.20
blocked by 10ꢀ6
M x-conotoxin-GVIA after the application of the test

4816
T. Yamamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4813–4816
compound. All the experiments were performed as
a
duplicate at room
HEPES 10 and glucose 10. The pH was adjusted to 7.4 with Tris-OH. The
conventional patch-pipette solution contained (in mM): CsCl 75, Cs-
methanesulfonate 75, ATP-Mg 2, EGTA 5 and HEPES 10. The pH was adjusted
to 7.2 with Tris-OH. (d) Positive control:(S)-4-methyl-2-(methylamino)
pentanoic acid [4,4-bis(4-fluorophenyl)butyl]amide hydrochloride (Ref. 4a)
was used as a positive control (83% inhibition at 10ꢀ5 M).
temperature. (c) Solutions: The ionic composition of Ca2+-free Tyrode solution
was (in mM): NaCl 150, KCl 5, MgCl2 1, N-2-hydroxye-thylpiperazine-N0-2-
ethanesulfonic acid (HEPES) 10 and glucose 10. The pH was adjusted to 7.4
with tris(hydroxymethyl)aminomethane (Tris)-OH. Na/K free Ba2+ solution
was (in mM): tetraethylammonium chloride 130, CsCl 4, MgCl2 1, BaCl2 10,