Structure-activity study of L-cysteine-based N-type calcium channel blockers: optimization of N- and C-terminal substituents.

Article abstract of DOI:10.1016/S0960-894X(02)00032-X

Synthesis and structure-activity relationship (SAR) studies of L-cysteine-based N-type calcium channel blockers are described. In the course of exploring SAR of the N- and C-terminal substituents, the L-cysteine derivative was found to be a potent N-type calcium channel blocker with an IC(50) value of 0.14 microM on IMR-32 assay. Compound showed 12-fold selectivity for N-type over L-type calcium channels on AtT-20 assay.

Full text of DOI:10.1016/S0960-894X(02)00032-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 915–918
Structure–Activity Study of L-Cysteine-Based N-Type Calcium
Channel Blockers: Optimization of N- and C-Terminal
Substituents
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 3 December 2001; accepted 12 January 2002
Abstract—Synthesis and structure–activity relationship (SAR) studies of l-cysteine-based N-type calcium channel blockers are
described. In the course of exploring SAR of the N- and C-terminal substituents, the l-cysteine derivative 4b was found to be a
potent N-type calcium channel blocker with an IC50 value of 0.14 mM on IMR-32 assay. Compound 4b showed 12-fold selectivity
for N-type over L-type calcium channels on AtT-20 assay. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
most of these compounds also block the other calcium
channel subtypes, including L-type channels. Although
blocking of neuronal L-type calcium channels shows
neuroprotective effects,5 inhibition of cardiac L-type
calcium channels probably causes hypotensive side
effects. Previously, we reported the discovery of a novel
series of N-type calcium channel blockers.6 N-(t-
Butoxycarbonyl)-l-cysteine derivative 2 was identified
as a lead compound for l-cysteine-based N-type calcium
channel blockers, which inhibited calcium influx into
IMR-32 human neuroblastoma cells7 with an IC50 of 0.61
mM. Here, we report the synthesis and in vitro structure–
activity relationship (SAR) studies of modification of
N- and C-terminal substituents of l-cysteine.
Voltage-sensitive calcium channels play crucial roles in
various biological processes, and are classified into sev-
eral subtypes on the basis of pharmacological and elec-
trophysiological properties as L-, N-, P-, Q-, R- or T-
type calcium channels. Among these calcium channels,
the N-, P-, Q-, and R-type channels have all been shown
to play key roles in neurotransmitter release.1 N-Type
calcium channels are located at presynaptic terminals
throughout neurons and directly mediate spinal trans-
mission of pain signals from the peripheral to the cen-
tral nervous system.
o-Conotoxin MVIIA, a 25-amino acid peptide, is a
selective blocker of N-type calcium channels that 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 blockers of N-type calcium
channels have been reported,3,4 some of which have
been shown to be active in analgesic models.4 However,
The compounds synthesized were evaluated for inhibi-
tory activity against both N-type (IMR-32 assay7,9) and
L-type (AtT-20 assay8,9) calcium channels, focusing on
selectivity to reduce cardiovascular side effects due to
blocking of L-type calcium channels (Fig. 1).
Chemistry
The synthesis of l-cysteine-based N-type calcium chan-
nel blockers is outlined as shown in Scheme 1. Treat-
ment of l-cysteine with cyclohexylmethyl bromide in
the presence of aqueous sodium hydroxide in ethanol
*Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-
9314; e-mail: seko@ono.co.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00032-X

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T. Seko et al. / Bioorg. Med. Chem. Lett. 12 (2002) 915–918
afforded sulfide 5. Subsequent reaction of compound 5
with di-tert-butyl dicarbonate gave N-Boc-protected
compound 6 from l-cysteine by one-pot procedure.
Reaction of carboxylic acid 6 with appropriately sub-
stituted benzylamines in the presence of EDC and
HOBt and subsequent deprotection gave intermediates
7 for N-terminal modification. Treatment of com-
pounds 7 with N-Boc-(R)-thiazolidine-4-carboxylic acid
in the presence of EDC and HOBt afforded compounds
4. Compounds shown in Tables 1 and 2 were synthe-
sized by the same method as described for compounds
4. This procedure can be easily applied to solution-
phase combinatorial synthesis using polymer-supported
condensation reagents.
lead for a novel series of N-type calcium channel
blockers.6 Compound 1 inhibited calcium influx into
IMR-32 cells with an IC50 of 3.4 mM. In the process of
exploring the SARs of this series of N-type calcium
channel blockers using l-amino acids as structural
motifs, compounds 2 and 3 were discovered as lead
compounds for further modification.6 These two com-
pounds have l-cysteine residues and showed improved
in vitro N-type calcium channel blocking potency (IC50
of 0.61 and 0.64 mM, respectively) compared to the
initial lead compound 1. In this study, we investigated
the SARs and further optimization of substituents at
N- and C-termini using l-cysteine as a central scaffold.
The SAR of l-cysteine-based N-type calcium channel
blockers was initially investigated for N-terminal sub-
stituent R1 (Table 1). While replacement of the tert-
butoxycarbonyl group with an iso-butoxycarbonyl
group resulted in slight loss of activity (9, IC50 0.72
mM), removal of the tert-butoxycarbonyl group resulted
in 10-fold loss of inhibitory activity for N-type calcium
Compounds shown in Table 3 were synthesized from
compound 4b by deprotection followed by condensation
with carboxylic acids or reductive amination with alde-
hydes.
Results and Discussion
Table 1. Modification of the N-terminal acyl group R1 of l-cysteine:
in vitro inhibition of calcium influx in IMR-32 and AtT-20 assays
The starting point in this study was an aspartic acid-
derived compound 1, which was identified as an initial
Compd
R1
IC50 (mM)a
N-type (IMR-32)
L-type (AtT-20)
2
0.61
1.7
7a
9
H
5.8
—
0.72
1.5
Figure 1.
10
11
1.2
2.7
2.3
0.92
12
4a
13
8a
14
1.3
1.9
0.68
1.9
3.5
2.7
0.39
0.88
2.7
1.9
Scheme 1. Reagents: (a) cyclohexylmethyl bromide, 2N NaOH, EtOH;
(b) Boc2O; (c) 4-methoxybenzylamine or 4-phenoxybenzylamine,
EDC, HOBt, dichloromethane; (d) 4N HCl/dioxane; (e) N-Boc-(R)-
thiazolidine-4-carboxylic acid, EDC, HOBt, dichloromethane.
aValues represent means of multiple determinations performed in
duplicate.

T. Seko et al. / Bioorg. Med. Chem. Lett. 12 (2002) 915–918
917
channels in IMR-32 assay (7a, IC50 5.8 mM). This
observation suggested that the lipophilic substituents
enhance potency; however, substitution of the carba-
mate moiety with an acyl group showed decreased
potency by 2-fold (10, IC50 1.2 mM) compared to com-
pound 2a. Similarly, substitution with cycloalkyl group
(11 and 12) also reduced N-type blocking activity.
Screening of N-acyl substituents indicated that N-tert-
butoxycarbonyl-thiazolidine-4-carbonyl increased N-
type inhibitory potency (4a, IC50 0.39 mM). Interest-
ingly, compound 13, which has an l-proline residue
instead of a thiazolidine-4-carbonyl group on the N-
terminal, showed lower potency (IC50 0.88 mM). Com-
parison of 4a and 13 showed that the thiazolidine ring
led to a further enhancement of potency. Further
modification of the sulfur-containing ring structure was
considered, but compound 8a (IC50 2.7 mM), which did
not have a t-butoxycarbonyl group on the thiazolidine
ring, and compound 14, which had thiazole-4-carbonyl
moiety, showed very weak inhibitory activity for N-type
calcium channels.
Modification of C-terminal substituent R2 was next
investigated (Table 2). Replacement of substituents on
the benzene ring did not significantly affect the potency
for compounds 15 (R2=NMe2; IC50 0.61 mM) and 16
(R2=NO2; IC50 0.60 mM). Although both of these
compounds showed weaker activity than compound 4a,
there was no difference in the N-type inhibitory activity
between compounds 15 and 16. These results suggested
that there was no influence of the electronic effect of the
substituent on the benzene ring. On the other hand,
compound 17 with a free phenol moiety showed 5-fold
lower potency (R2=OH; IC50 1.8 mM). Compound 18,
which had benzyloxy moiety, was synthesized to esti-
mate steric effects. Compound 18 showed slightly lower
activity than compound 4a.
As a consequence of modification of substituents on the
benzene ring, compound 4b, which had a phenoxy resi-
due, was found to be the most potent N-type blocker
among this series of compounds. Compound 4b blocked
N-type calcium channels with an IC50 of 0.14 mM and
showed good selectivity over L-type calcium channels
(selectivity ratio IC50 L-type/N-type=12).
The inhibitory activity of compound 4a for N-type cal-
cium channels was also confirmed by electro-
physiological study using IMR-32 cells (48% inhibition
at 10 mM, n=3).10 Therefore, the following SAR study
was performed with compound 4a, which has an N-t-
butoxycarbonyl-thiazolidine-4-carbonyl at its N-terminus.
Finally, the SAR of modification of the substituents on
the nitrogen atom of thiazolidine was investigated
(Table 3). In contrast to the results with modification of
Table 3. Modification of substituents R3 on the nitrogen atom of the
thiazolidine ring: in vitro inhibition of calcium influx in IMR-32 and
AtT-20 assays
Table 2. Modification of the C-terminal amide group R2 of l-cysteine:
in vitro inhibition of calcium influx in IMR-32 and AtT-20 assays
Compd
R3
IC50 (mM)a
N-type (IMR-32)
Compd
R2
IC50 (mM)a
N-type (IMR-32)
L-type (AtT-20)
L-type (AtT-20)
4b
0.14
1.7
4a
15
16
17
18
4b
0.39
0.61
0.60
1.8
0.68
8b
19
H
0.29
0.12
2.0
0.87
0.95
0.50
2.8
20
21
22
23
24
0.36
0.33
0.12
0.20
0.35
1.2
0.44
0.93
0.60
1.1
0.50
0.14
1.5
1.7
aValues represent means of multiple determinations performed in
duplicate.
aValues represent means of multiple determinations performed in
duplicate.

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T. Seko et al. / Bioorg. Med. Chem. Lett. 12 (2002) 915–918
the N-terminal acyl group R1 (Table 1), no significant
influence of substituents was observed on the N-type
inhibitory activity. However, it should be noted that
most of these compounds showed increased inhibitory
activity and selectivity for N-type calcium channels
compared to the compounds shown in Table 1. These
results indicated that the phenoxy moiety played a crucial
role in inhibitory potency and selectivity. Among the
compounds shown in Table 3, compounds 19
(R3=methoxycarbonyl; IC50 0.12 mM) and 22 (R3=2-
methoxyacetyl; IC50 0.12 mM) were equipotent to
compound 4b.
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. 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, 2067.
7. IMR-32 cells were grown as described (Clementi, F.; Cab-
rini, D.; Gotti, C.; Sher, E. J. Neurochem. 1986, 47, 291. Car-
bone, E.; Sher, E.; Clementi, F. Pflugers Arch. 1990, 416, 170)
¨
In conclusion, the SAR study of a series of l-cysteine-
based compounds led to the discovery of novel neuronal
N-type calcium channel blockers. Compound 4b, which
had phenoxy residue on the C-terminal benzene ring,
was a potent N-type calcium channel blocker and was
12-fold more selective for N-type over L-type calcium
channels.
in Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100
mg/mL streptomycin.
8. (a) Xie, J.; Nagle, G. T.; Childs, G. V.; Ritchie, A. K.
Neuroendocrinology 1999, 70, 1. Mouse pituitary tumor-
derived cells (AtT-20/D16v-F2) were grown in Dulbecco’s
modified Eagle’s medium containing 10% FBS, penicillin–
streptomycin.
9. [Ca2+]I was measured in cell suspension. Cell suspensions
were 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
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Lotarski, S. M.; Malone, T.; Miljanich, G.; Millerman, E.;
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500 nm) was detected with a fluorometer. Cell suspensions
were 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 AtT-20/D16v-F2
cells were used for N-type and L-type calcium channels,
respectively.
10. The electrophysiological recordings were performed in the
conventional whole-cell configuration under voltage-clamp
conditions. Pipettes had a resistance of 3–6 Mꢀ. 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’.