Structure-activity study and analgesic efficacy of amino acid derivatives as N-type calcium channel blockers

Article abstract of DOI:10.1016/S0960-894X(01)00414-0

The synthesis and structure-activity relationship (SAR) study of a novel series of N-type calcium channel blockers are described. L-Cysteine derivative 2a was found to be a potent and selective N-type calcium channel blocker with IC₅₀ 0.63 μM on IMR-32 assay. Compound 2a showed analgesic efficacy in the rat formalin-induced pain model by intrathecal and oral administration.

Full text of DOI:10.1016/S0960-894X(01)00414-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2067–2070
Structure–Activity Study and Analgesic Efficacy of Amino Acid
Derivatives as N-Type Calcium Channel Blockers
Takuya Seko,* Masashi Kato, Hiroshi Kohno, Shizuka Ono, Kazuya Hashimura,
Hideyuki Takimizu, Katsuhiko Nakai, Hitoshi Maegawa, Nobuo Katsube
and Masaaki Toda
Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
Received 26April 2001; accepted 13 June 2001
Abstract—The synthesis and structure–activity relationship (SAR) study of a novel series of N-type calcium channel blockers are
described. l-Cysteine derivative 2a was found to be a potent and selective N-type calcium channel blocker with IC50 0.63 mM on
IMR-32 assay. Compound 2a showed analgesic efficacy in the rat formalin-induced pain model by intrathecal and oral adminis-
tration. # 2001 Elsevier Science Ltd. All rights reserved.
In the course of screening of our compound library, N-
(t-butoxycarbonyl)-l-aspartic acid derivative 1a was
identified as an initial lead compound for a new series of
N-type calcium channel blockers, which inhibited cal-
cium influx into IMR-32 cells with an IC50 of 3.4 mM.6
The inhibitory activity of compound 1a for N-type cal-
cium channel was also confirmed by electro-
physiological study using IMR-32 cells (34% inhibition
at 10 mM, n=3).7 Here, we report the synthesis and
structure–activity study of N-type calcium channel
blockers that include the N-(t-butoxycarbonyl) l-amino
acid moiety as a structural motif.
Introduction
Six types of pharmacologically and/or biophysically
distinct voltage-dependent calcium channels (T, L, N, P,
Q, and R) have been described in neurons. Among these
calcium channels, the N, P, Q, and R-type channels
have all been shown to play key roles in neuro-
transmitter release.1 N-Type calcium channels are loca-
ted at presynaptic terminals throughout neurons and
directly mediate spinal transmission of pain signals from
the peripheral to the central nervous system. o-Con-
otoxin MVIIA, a 25-amino acid peptide, is a selective
blocker of N-type calcium channels which shows
analgesic activity when administered intrathecally.2
Over the last decade, synthetic efforts have focused on
small-molecule, non-peptide N-type calcium channel
blockers for analgesia or neuroprotection, since clinical
observations were reported for o-conotoxin MVIIA.2 A
number of small-molecule, non-peptide blockers of N-
type calcium channels have been reported, some of
which have been shown to be active in analgesic mod-
els.3,4 However, most of these compounds also block the
other calcium channel subtypes including L-type chan-
nels. Although blocking of neuronal L-type calcium
channels shows neuroprotective effects,5 inhibition of
cardiac L-type calcium channels probably causes a
decrease of blood pressure.
The compounds were evaluated for inhibitory activity
against both N-type and L-type calcium channels
focusing on selectivity to reduce cardiovascular side effects
due to blocking of L-type calcium channels (Fig. 1).
*Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-
9314; e-mail: seko@ono.co.jp
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00414-0

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T. Seko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2067–2070
Chemistry
Biological Results and Discussion
The synthesis of aspartic acid ester (1 and 3) was carried
out by sequential esterification as shown in Scheme 1.
Half ester 4 was obtained from Boc-l-Asp anhydride
and esterification of 4 with the appropriate alcohol in
the presence of EDC and DMAP gave Boc-l-Asp di-
ester 1. Compound 3, an enantiomer of 1a, was synthe-
sized from Boc-d-Asp by the same method as described
for compound 1a.10
The initial lead compound 1a had moderate N-type
calcium channel blocking activity (IC50 of 3.4 mM;
IMR-32 assay6,9) and selectivity over L-type calcium
channels (IC50 of 15 mM; in AtT-20 assay,8,9 selectivity
ratio L/N=4.4). Compound 3, the enantiomer of 1a
derived from d-Asp, showed slightly higher activity in
IMR-32 assay (IC50=2.0 mM). However, inhibitory
activity for L-type calcium channels in AtT-20 assay
was increased by 5-fold (IC50=2.9 mM, L/N=1.5). Due
to lack of selectivity of the d-Asp derivative 3, all of the
following compounds were synthesized in optically
active forms, which were derived from the correspond-
ing l-amino acids.10
l-Serine ester 5 was synthesized from Boc-l-Ser by
reaction with 4-methoxybenzyl chloride and then with
acid chloride as shown in Scheme 2. l-Serine ether 7
was obtained from commercially available Boc-l-Ser
(OBzl) by catalytic hydrogenation of the benzene ring
using rhodium on alumina and subsequent condensa-
tion of carboxylic acid 6 with the appropriate alcohol or
amine (Scheme 2).
An initial structure–activity study was started with
replacement of the alkyl group on the side-chain ester
using the l-Asp skeleton as a structural motif (see Table
1: 1a–e). Methyl ester (1b: R=methyl) showed extre-
mely weak activity for N-type calcium channels
(IC50=26 mM) and low selectivity (L/N=0.5). In the
course of modification of ring size on the side-chain
ester (1a and 1c–e), compound 1c (R=cyclobutyl),
which has a cyclobutyl ring, showed 4-fold decreased N-
type inhibitory activity and selectivity. In contrast,
compounds 1d (R=cyclohexyl) and 1e (R=cycloheptyl)
showed similar potency to the initial lead compound 1a.
With respect to the selectivity for N-type calcium chan-
nels, cyclohexyl ester was better than cyclopentyl ester.
Modification of the substituents on the benzene ring
showed no significant influence on the potency (see
Table 1: compounds 1f–i). Thus, the compound 1d,
which has 4-methoxy benzyl ester at the a-carbonyl and
cyclohexyl ester at the side chain, was selected for
further SAR study.
A similar method was applied for the ether formation of
l-homoserine (l-Hse) ether 10 by reaction with cyclo-
hexene-3-bromide and Boc-l-Hse. Subsequent catalytic
reduction of compound 8 and condensation with 4-
methoxybenzylamine gave the Boc-l-Hse ether 10
(Scheme 3). Sulfide bond formation was carried out by
reaction of l-cysteine with halide or methanesulfonate
yielding compounds 11, which were easily derived to
compounds 2 by the usual method (Scheme 4).
Scheme 1. Reagents: (a) Ac2O; (b) 4-methoxybenzyl alcohol, DCHA,
THF–Et2O; (c) EDC, DMPA, CH2Cl2.
Scheme 3. (a) Cyclohexane-3-bromide, NaH; (b) H2, Pd/C, EtOH; (c)
4-methoxybenzylamine, EDC, DMAP.
Scheme 2. Reagents: (a) 4-methoxybenzyl chloride, K2CO3, NaI,
DMF; (b) cyclohexanecarbonyl chloride, Et3N, CH2Cl2; (c) Rh–Al2O3,
H2, i-PrOH; (d) 4-methoxybenzyl alcohol, EDC, HOBt, DMF; (e)
4-methoxybenzylamine, EDC, HOBt, DMF; (f) 4-methoxy-
benzylmethylamine, EDC, HOBt, DMF.
Scheme 4. Reagents: (a) cyclopentylmethyl methanesulfonate or
cyclohexylmethyl bromide, 2 N NaOH, EtOH; (b) Boc2O, 2 N NaOH,
EtOH; (c) 4-methoxybenzylamine, EDC, HOBt, DMF.

T. Seko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2067–2070
Table 1. In vitro inhibition of calcium influx in IMR-32 and AtT-20 assays
2069
IC50 (mM)a
Compd
X
R1
Y
R2
Selectivity ratio (L/N)
N-type (IMR-32)
L-type (AtT-20)
1a
1b
1c
1d
1e
1f
1g
1h
1i
CO–O
CO–O
CO–O
CO–O
CO–O
CO–O
CO–O
CO–O
CO–O
O–CO
O–CH2
O–CH2
O–CH2
CH2–O
S–CH2
S–CH2
Cyclopentyl
Methyl
O
O
O
O
O
O
O
O
O
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
2-OMe
3-OMe
4-Cl
3.4
2614
15
15
0.5
28
167.0
4.6
15
4.4
1.9
Cyclobutyl
Cyclohexyl
Cycloheptyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclopentyl
Cyclohexyl
2.3
2.612
5.4
3.8
3.3
3.4
1.610
1.2
0.95
1.8
2.8
3.2
12
164.8
5.3
6
8.4
3.8
1.4
13
4-Br
1.6
5
O
O
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
.3
7a
7b
7c
10
2a
2b
7.0
4.0
0.8
3.3
6.2
2.8
NH
NMe
NH
NH
NH
4.0
0.63
0.61
3.9
1.7
side chain has a significant influence on the inhibitory
activity.
Table 2. Activities of selected compounds in electrophysiological
study and in vivo (rat) formalin-induced pain model
Compd
Electrophysiology
(IMR-32, 10 mM, n=3)
inhibition %
Formalin test
3 nmol/rat, it
Finally, we prepared some l-cysteine analogues, which
could be relatively stable against metabolic hydrolysis
compared with the initial lead. These compounds were
among the most potent analogues in this series (2a,
IC50=0.63 mM and 2b, IC50=0.61 mM). The inhibitory
activity for N-type calcium channels of compound 2a,
the most potent and selective compound, was evaluated
by electrophysiological assay. This compound blocked
N-type calcium channel current in IMR-32 cells (45% at
10 mM, n=3). This compound was also evaluated for
analgesic efficacy in the rat formalin-induced pain
model.11 Preliminary results showed that the compound
2a inhibited paw flinching during the persistent noci-
ceptive phase by intrathecal (47% inhibition at 3 nmol/
rat, it, n=3) and oral administration (43% inhibition at
100 mg/kg, po, n=3) without any significant effect on
blood pressure or neurological behavior. Activities of
selected compounds in electrophysiological study and in
the in vivo formalin test were summarized in Table 2.
Inhibition %
n
1a
7b
2a
2a
34
38
45
31
35
47
5
3
3
3
43
(100 mg/kg, po)
36
2b
66
3
Replacement of the amino acid skeleton was next
investigated. l-Ser analogue, the reverse type ester of
1a, showed increased N-type blocking activity in IMR-
32 assay (5, IC50=1.6 mM). Similar results were
observed in l-Ser ether (7a, IC50=1.2 mM), which had
3-fold improved N-type calcium channel blocking
potency than the lead compound 1a.
To evaluate in vivo analgesic efficacy, the ester was
converted to amide since the ester function seems to be
unstable against metabolic hydrolysis. Conversion to
amide was achieved without losing potency in IMR-32
assay (7b, IC50=0.95 mM) or selectivity (L/N=4.0). The
N-methyl analogue 7c (Y=NMe) was also synthesized
for the same purpose. However, this compound was not
selective (L/N=0.8).
In conclusion, the SAR study of a series of N-Boc-
amino acid derivatives led to the discovery of novel
neuronal N-type calcium channel blockers. Compound
2a was a potent N-type calcium channel blocker and
was 6-fold more selective over L-type calcium channels.
This compound also showed analgesic efficacy in the rat
formalin-induced pain model, and could be a potential
lead compound for further modification.
In terms of oxygen position on the side chain, interest-
ing results were observed by comparison between l-Ser
ether 7b and l-Hse ether 10. The calcium channel inhi-
bitory activity of 10 was decreased 4-fold by this minor
modification. This observation was similar to the com-
parison between l-Asp ester 1a and l-Ser ester 5. These
results also showed that the position of oxygen on the
References and Notes
1. (a) Bean, B. P. Annu. Rev. Physiol. 1989, 51, 367. (b) Hess,
P. Annu. Rev. Neurosci. 1990, 13, 337.

2070
T. Seko et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2067–2070
2. (a) Jain, K. K. Expert Opin. Invest. Drugs 2000, 9, 2403. (b)
Bowersox, S.; Tich, N.; Mayo, M.; Luther, R. Drugs Future
1998, 23, 152.
3. Cox, B.; Denyer, J. C. Expert Opin. Ther. Pat. 1998, 8,
1237.
7. The electrophysiological recordings were performed in the
conventional whole-cell configuration under voltage-clamp
conditions. Pipettes had a resistance of 3–6M ꢀ. Membrane
currents were measured using a patch clamp amplifier (Axo-
patch 2B Axon Instruments). The test compounds were
applied using a rapid application method designated as the ‘Y-
tube method’.
8. (a) Xie, J.; Nagle, G. T.; Childs, G. V.; Ritchie, A. K.
Neuroendocrinology 1999, 70, 1. (b) Mouse pituitary tumor-
derived cells (AtT-20/D16v-F2), were grown in Dulbecco’s
Modified Eagle’s medium containing 10% FBS, penicillin-
streptomycin
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) 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. (c) Hu,
L.-Y.; Ryder, T. R.; Rafferty, M. F.; Siebers, K. M.; Malone,
T.; Chatterjee, A.; Feng, M. R.; Lotarski, S. M.; Rock, M.;
Stoehr, S. J.; Taylor, C. P.; Weber, M. L.; Miljanich, G. P.;
Millerman, E.; Wang, Y.-X.; Szoke, B. G. Drug Des. Discov.
2000, 17, 85. (d) Hu, L.-Y.; Ryder, T. R.; Rafferty, M. F.;
Feng, M. R.; Lotarski, S. M.; Rock, D. M.; Sinz, M.; Stoehr,
S. J.; Taylor, C. P.; Weber, M. L.; Bowersox, S. S.; Miljanich,
G. P.; Millerman, E.; Wang, Y.-X.; Szoke, B. G. J. Med.
Chem. 1999, 42, 4239.
5. Kobayashi, T.; Mori, Y. Eur. J. Pharmacol. 1998, 363, 1.
6. IMR-32 cells were grown as described (Clementi, F.; Cab-
rini, D.; Gotti, C.; Sher, E. Eur. J. Neurochem. 1986, 47, 291.
Carbone, E.; Sher, E.; Clementi, F. Pflugers Arch. 1990, 416,
170) in Dulbecco’s Modified Eagle’s medium (DMEM) con-
taining 10% fetal bovine serum (FBS), 100 IU/mL penicillin
and 100 mg/mL streptomycin
9. [Ca2+]I was measured in cell suspension. Cell suspension
was incubated with 5 mM fura-2/AM for 30 min at 37 ꢀC. Cells
were resuspended in Krebs–Ringer HEPES solution, and
adjusted to 1.0Â106 cells/mL (IMR-32), or 2.0Â106 cells/mL
(AtT20/D16v-F2). Fluorescence (lEx: 340 and 380 nm, lEm
:
500 nm) was detected with a fluorometer. Cell suspension was
incubated with test compound and 10 mM nifedipine (IMR-32)
or 3 mM o-conotoxin MVIIC (AtT-20/D16v-F2) for 360 s
before high-K+ stimulus. To evaluate the inhibitory activities
of test compounds, IMR-32 and AtT20/D16v-F2 cells were
used for N-type and L-type calcium channels, respectively.
10. No isomerized product was detected by chiral HPLC
analysis. 1a; [a]D=À16.0 ꢀ (c=1.15, MeOH). 3; [a]D=+15.1 ꢀ
(c 0.92, MeOH).
11. Dubuisson, D.; Dennis, S. G. Pain 1977, 4, 161.