Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties

Article abstract of DOI:10.1021/jm9902739

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of str

Full text of DOI:10.1021/jm9902739

J . Med. Chem. 1999, 42, 4239-4249
4239
Syn th esis of a Ser ies of 4-Ben zyloxya n ilin e An a logu es a s Neu r on a l N-Typ e
Ca lciu m Ch a n n el Block er s w ith Im p r oved An ticon vu lsa n t a n d An a lgesic
P r op er ties
Lain-Yen Hu,*,† Todd R. Ryder,† Michael F. Rafferty,† M. Rose Feng,§ Susan M. Lotarski,‡ David M. Rock,‡
Michael Sinz,§ Sally J . Stoehr,‡ Charles P. Taylor,‡ Mark L. Weber,‡ S. Scott Bowersox,∇ George P. Miljanich,∇
Elizabeth Millerman,∇ Yong-Xiang Wang,∇ and Balazs G. Szoke∇
Departments of Chemistry, Neuroscience Therapeutics, and Pharmacokinetics, Dynamics and Metabolism,
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor,
Michigan 48105, and Elan Pharmaceuticals, Inc., 3760 Haven Avenue, Menlo Park, California 94025
Received J une 1, 1999
In this article, the rationale for the design, synthesis, and biological evaluation of a series of
N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers,
such as ziconotide, have shown utility in several models of stroke and pain. Modification of
the previously reported lead, 1a , led to several 4-(4-benzyloxylphenyl)piperidine structures with
potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-
amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-piperidin-1-yl}-4-methyl-pentan-
1-one (11), blocked N-type calcium channels (IC50 ) 0.67 µM in the IMR32 assay) and was
efficacious in the audiogenic DBA/2 seizure mouse model (ED50 ) 6 mg/kg, iv) as well as the
antiwrithing model (ED50 ) 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments
demonstrated that compound 11 blocked N-type Ca2+ channels and Na+ channels in superior
cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in
vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic
agents in this series.
In tr od u ction
ziconitide3 has demonstrated efficacy in animal models
of traumatic brain injury, focal cerebral ischemia, and
pain.2 An NDA of ziconitide will be filed for its applica-
tion in pain.
Excessive calcium entry into depolarized neurons
contributes significantly to neuronal injury. Voltage-
sensitive calcium channels (VSCCs) regulate intracel-
lular calcium concentration, which affects various im-
portant neuronal functions such as cellular excitability,
neurotransmitter release, hormone secretion, intracel-
lular metabolism, neurosecretory activity, and gene
expression.1 Neuronal VSCCs are classified into L, N,
P, Q, R, and T subtypes based upon their physical and
pharmacological properties. These channels differ in
their protein structures, function, conductance, activa-
tion/inactivation voltage, and sensitivity to various
drugs and/or toxins.1 N-type channels are found mainly
in central and peripheral neurons, being primarily
located on presynaptic nerve terminals. These channels
regulate the calcium flux subserving depolarization-
evoked release of transmitter from synaptic endings.1
It has been suggested that N-type VSCCs would be
ideal targets for the development of new pharmacologi-
cal agents for clinical use.2 ω-Conotoxin MVIIA,3 a 25
amino acid residue containing peptide found in the
venom of the piscivorous marine snail (Conus magus),
is a potent and selective N-type voltage-sensitive cal-
cium channel blocker. The synthetic version of MVIIA,
Drug discovery efforts during the past decade have
focused on small molecule N-type calcium channel
blockers with non-peptide structures particularly for
neuroprotection or analgesia.4 Examples of known non-
peptidyl Ca2+ channel antagonists include flunarizine,5
fluspirilene,6 PD-157767,7 SB-201823,8 SB-206284,9 NNC-
09-0026,10 cilnipine (FRC-8653),11 NS-649,12 and (2R)-
1-((S)-6-cyano-7-methyl-6-phenyl-octanoyl)-pyrrolidine-
2-carboxylic acid benzhydryl-amide (1c).13 However,
most of these are nonselective and block multiple
subtypes of Ca2+ channels as well as Na+ and K+
channels. In the process of searching for small molecule
N-type calcium channel blockers, we have discovered
and reported on compound 1a as a potential chemical
lead.14,15 Compound 1a is a potent antagonist for N-type
calcium channels in the IMR32 human neuroblastoma
cells (IC50 ) 0.32 µM)14 and showed moderate in vivo
activities in the audiogenic DBA/2 seizure mouse model
(protection: 100% at 30 mg/kg and 0% at 10 mg/kg, iv),
as well as the antiwrithing model (protection: 75% at
30 mg and 11% at 10 mg/kg, iv). Unfortunately, due to
unfavorable physicochemical properties, such as an high
ClogP (6.5) and poor aqueous solubility (measured
aqueous solubility < 1 µg/mL at pH 7), compound 1a
has limited therapeutic potential.
* To whom correspondence should be addressed. Phone: (734)-622-
5625. Fax: (734)-622-5165. E-mail: Lain-Yen.Hu@wl.com.
† Department of Chemistry, Parke-Davis Pharmaceutical Research.
‡ Department of Neuroscience Therapeutics, Parke-Davis Pharma-
ceutical Research.
Several compounds were prepared with important
structural modifications to avoid the problem encoun-
tered with antagonist 1a .14,15 One of the major
§ Department of Pharmacokinetics, Dynamics and Metabolism,
Parke-Davis Pharmaceutical Research.
∇ Elan Pharmaceuticals, Inc.
10.1021/jm9902739 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/18/1999

4240 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Ch a r t 1. Selected N-Type Ca2+ Channel Blockers
Hu et al.
Sch em e 1. Rationale for the Design of N-Type Ca2+
Sch em e 2a
Channel Blockers
a
Reagents and conditions: (i) triphosgene, hexamethlenimine;
(ii) H2, Pd/C; (iii) HBTU (O-benzotriazol-1-yl-N,N,N′,N′-tetram-
ethyluronium hexafluorophosphate), DIEA, DMF.
tidic motifs and an amine functionality which allowed
rapid generation of analogues. In this paper, several
analogues with this novel template are described. Their
in vitro activities for N-type Ca2+ channel blockade as
well as in vivo efficacy in the audiogenic DBA/2 seizure
mouse model are discussed.
changes was to convert the tert-butyl ester to its tert-
butyl amide derivative (1b, IC50 ) 0.59 µM); this change
was well-tolerated for N-type Ca2+ channel blocking
activity (Chart 1).14,15 The previous results also sug-
gested that some flexibility was present in terms of the
electronic interaction and volume in the C-terminal
region of the molecule.14,15 Therefore, we envisioned
migrating the (4-O-benzyl)-phenyl group of 1b to the
C-terminal end and replacing the tert-butyl amide with
a 3-methyl-2-butenylamine to generate the structure
type A (Scheme 1) for exploring even more active N-type
Ca2+ channel blockers. The obvious advantages of this
approach were to provide new structures with nonpep-
Meth od s
Ch em istr y. The preparation of (S)-2-[(azepane-1-
carbonyl)-amino]-4-methyl-pentanoic acid (2), which is
a common intermediate for compounds 1b14,15 and 3-10,
was carried out in two steps (Scheme 2). Condensation
of triphosgene and hexamethylenimine with (S)-2-
amino-4-methyl-pentanoic acid benzyl ester provided 2a ,
which was subsequently hydrogenated to provide the
acid (2).

Synthesis of Neuronal N-Type Calcium Channel Blockers
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4241
Sch em e 3a
a
Reagents and conditions: (i) HBTU, DIEA, DMF; (ii) BH3/THF; (iii) 4-bromo-2-methyl-2-butene, DIEA, THF; (iv) TFA, CH2Cl2.
Sch em e 4a
a
Reagents and conditions: (i) NaBH(OAc)3, CH2Cl2; (ii) TFA, CH2Cl2; (iii) HBTU, DIEA, DMF, 2; (iv) for 5: 4-bromo-2-methyl-2-
butene, THF, DIEA; for 8-10: NaBH(OAc)3, CH2Cl2 and aldehydes.
The preparation of compound 1b was previously
reported.15 Synthesis of compound 3 was carried out by
coupling the acid (2) with (S)-N-(4-benzyloxy-phenyl)-
N-(3-methyl-but-2-enyl)-propane-1,2-diamine(3d)(Scheme
2). Compound 4 was prepared by the same method used
for compound 3, except that 4d was the starting
material. The 1,2-diamine (3d ) was prepared by the
amide formation of NR-Boc-alanine and 4-benzyloxya-
niline, reduction of the amide (3a ) with diborane to
generate the amine (3b), alkylation of the amine (3b)
to provide compound 3c, and deprotection of compound
3c to yield intermediate 3d . (Scheme 3). The prepara-
tion of intermediate 4d was similar to that of 3d , except
that NR-Boc-Aib acid was the starting material (Scheme
3).
Synthesis of compounds 5, 7-10 is outlined in Scheme
4. The key compound, 7, was prepared in three steps
by the reductive amination of 4-benzyloxy-aniline with
1-tert-butyl-carbonyl-4-piperidone to yield 7a , followed
by deprotection with TFA to generate 7b and the
coupling of the acid (2) with 7b to provide analogue 7
(Scheme 4). Further alkylating the aniline nitrogen of
compound 7 resulted in 5, 8-10. Compound 6 was
synthesized by reductive amination of 4-benzyloxy-
aniline with 1-benzyl-3-piperidone to yield 6a , alkylation
of 6a to generate 6b, debenzylation of compound 6b to
provide 6c, and coupling of 6c with the acid (2) to form
analogue 6 (Scheme 5).
The preparation of compound 11 is illustrated in
Scheme 6. Alkylation of 4-(4-benzyloxy-phenylamino)-
piperidine-1-carboxylic acid tert-butyl ester (7a ) with
4-bromo-2-methyl-2-butene gave 11a . Deprotection of
the Boc group of 11a generated the amine (11b), which
was then coupled with NR-Boc-leucine to produce the
amide (11c). Further deprotection of the Boc group of
11c provided antagonist 11. Finally, alkylation of
compound 11 with various groups resulted in com-
pounds 12-14 (Scheme 6).
P h a r m a cology. N-type Ca2+ channel blocking poten-
cies of the compounds were determined using a fluo-
rescence-based Ca2+-flux assay in IMR32 human neu-
roblastoma cells. Compound 1a was run in parallel in
each assay as a standard to compare the relative
potency of experimental compounds with the initial lead.
The anticonvulsant effects of selected compounds were
evaluated in an audiogenic seizure model using DBA/2
mice (6.5-12 g, 20-23 days of age).16 The known N-type
Ca2+ channel blocker, ω-conotoxin GVIA, blocked sei-
zures in this model.17 Data are expressed as a percent-
age of animals showing no tonic seizure response to the
sound stimulation (Table 1).
Resu lts a n d Discu ssion
Initially, compound 3 was prepared to test the hy-
pothesis that structure type A could be a potent N-type
Ca2+ channel antagonist. Structure type A was derived

4242 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Hu et al.
Sch em e 5a
a
Reagents and conditions: (i) NaBH(OAc)3, CH2Cl2; (ii) 4-bromo-2-methyl-2-butene, DIEA; (iii) 1,2-dichloroethane, R-chloroethylchlo-
roformate; (iv) HBTU, DIEA, 2b, DMF.
Sch em e 6a
a
Reagents and conditions: (i) 4-bromo-2-methyl-2-butene, THF, DIEA; (ii) TFA, CH2Cl2, (iii) HBTU, DIEA, Boc-leucine, DMF; (iv)
TFA, CH2Cl2; (v) NaBH(OAc)3, CH2Cl2, aldehydes or ketones.
Ta ble 1. Activities of N-Type Calcium Channel Blockers
compd
IMR32 IC50 (µM)
0.32 ( 0.04 (n ) 10)a
DBA/2 activity, iv (n ) 5 mice/dose tested)
ClogP/solubility
6.5/0.8 µM (pH 7.4)
1a
100% @ 30 mg/kg
0% @ 10 mg/kg
1b
3
4
5
6
7
8
9
10
11
11b
12
13
0.59 (n ) 2)
1.0 (n ) 1)
0.26 (n ) 1)
0.34 ( 0.06 (n ) 4)
1.5 (n ) 1)
2.5 (n ) 1)
0.83 (n ) 1)
4.6 (n ) 1)
0.43 (n ) 1)
0.67 ( 0.07 (n ) 8)
4.7 (n ) 1)
0.75 (n ) 1)
0.48 (n ) 1)
5.5/11 µM (pH 7.4)
6.9
7.2
7.0
7.2
5.1
0% @ 30 mg/kg
60% @ 30 mg/kg
0% @ 30 mg/kg
60% @ 30 mg/kg
6.0
5.6/4 µM (pH 7.4)
7.1
5.5/87 µM (pH 7.4) and 100 mM (pH 4.0)
5.0
7.0
7.8
ED50 ) 6 mg/kg
80% @ 30 mg/kg
20% @ 10 mg/kg
14
0.51 (n ) 1)
8.1
from compound 1b by migrating the (4-O-benzyl)-phenyl
group of 1b to the C-terminal and replacing the tert-
butyl amide of 1b with a 3-methyl-2-butenylamine. In
the IMR32 assay, this non-peptidyl compound (3, IC50
) 1.0 µM) displayed reasonable potency: slightly less
potent than the parent compound (1b, IC50 ) 0.59 µM)
or 3-fold less active than compound 1a (IC50 ) 0.32 µM).
Further exploration of potent Ca2+ channel blockers led
us to modify the 1-methyl-ethylene-1,2-diamine linker
of 3 with a 1,1-dimethyl-ethylene-1,2-diamine linker
resulting in analogue 4 (IC50 ) 0.26 µM), which en-
hanced the activity by 4-fold relative to 3. For structure-
activity relationship studies, the 1-methyl-ethylene-1,2-
diamine linker of 3 was also substituted with a piperidin-
4-yl-amine linker obtaining compound 5 (IC50 ) 0.34
µM), which showed 3-fold improvement in potency

Synthesis of Neuronal N-Type Calcium Channel Blockers
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4243
compared to 3. Furthermore, the piperidin-3-yl-amine
analogue (6, IC50 ) 1.5 µM) was 5-fold less potent than
its piperidin-4-yl-amine counterpart (4) and slightly less
active than 3 for blocking N-type Ca2+ channels. From
the above four analogues, it was concluded that the N-
(2),N(3)-linker of structure type A was important for
activity. Both 1,1-dimethyl-ethylene-1,2-diamine and
piperidin-4-yl-amine moieties were advantageous for
IMR32 activity in this brief set of compounds; 1-methyl-
ethylene-1,2-diamine and piperidin-3-yl-amine linkers
are less preferred. Compounds 4 and 5 blocked N-type
Ca2+ channels as effectively as our screening lead, 1a .
4-Aminopiperidine has been shown to be a successful
scaffold for several known agents applied in central
nervous system,18 and this template is likely to be a good
motif for design of further drug candidates. Therefore,
further SAR studies in this series are all focused on the
4-piperidine analogues.
(such as solubility) in this series. Furthermore, (4-
benzyloxy-phenyl)-(3-methyl-but-2-enyl)-piperidin-4-yl-
amine (11b), a des-leucine analogue of compound 11,
was evaluated and found inactive. Therefore, it was
determined that the leucine moiety was essential for
blocking N-type Ca2+ channels in this series, even
though the N-substituted analogues, compounds 11-
14, displayed a flat SAR.
Compounds 11 and 13 were tested in the audiogenic
seizure model and displayed improved in vivo efficacy
compared to 1a . Remarkably, the free amine analogue
(11) demonstrated a potent anticonvulsant effect with
an ED50 of 6 mg/kg after iv administration. This is the
most potent anticonvulsant agent in this report. Com-
pound 11 was further characterized in electrophysiology
assays and found to block neuronal N-type Ca2+ chan-
nels in superior cervical ganglion neurons with an IC50
of 3.5 ( 0.62 µM (n ) 3) and blocked voltage-gated
sodium channels with an IC50 of 4.2 ( 0.9 µM (n ) 3).
These results suggested that compound 11 was a
balanced channel blocker and blocked audiogenic sei-
zures by activity at both voltage-gated N-type calcium
and sodium channels.19a,b Compound 11 was further
evaluated in an antiwrithing model and found to be
efficacious (ED50 ) 6 mg/kg iv). The assessment of its
cardiovascular side effects in rat indicated that 11 had
no significant effect on the mean blood pressure or heart
rate at 10 mg/kg iv.
The subsequent synthesis of several N(3)-substituted
analogues (5, 7-10) was directed toward optimizing
N-type Ca2+ channel activity. In the 1a /1b series, the
ester/amide substitution at the C-terminal of the dipep-
tides played a key role for blockade of N-type Ca2+
channels.14 Generally speaking, lipophilic groups were
advantageous for high IMR32 potency for analogues of
1a . Here, the SAR of N(3)-disubstituted analogues
(nitrogen of the aniline) of 5 were compared among a
set of antagonists. These were hydrogen (7), ethyl (8),
3-methyl-but-2-enyl (5), benzyl (10), and 3-hydroxy-
butyl (9). The potencies of these compounds for the
blockade of N-type Ca2+ channels ranged from 0.34 to
4.6 µM and suggested that affinity was strongly influ-
enced by substituent effects. A careful comparison
revealed that a graded preference for 3-methyl-but-2-
enyl over benzyl, ethyl, hydrogen, or 3-hydroxy-butyl
existed. A lipophilic substituent with certain size fa-
vored active blockade, while the hydrophilic substituent
was less preferred. This result was in agreement with
our previous observations in the compound 1a /1b series.
Compound 11 showed improved physicochemical prop-
erties compared to 1a (11, logP: measured logP ) 5.7
and ClogP ) 5.53, aqueous solubility: 90 µM at pH 7
and 100 mM at pH 4). Ligand 11 showed significantly
improved metabolic stability in a human liver mi-
crosome assay, with a half-life (t1/2) of 116 min, com-
pared to the dipeptidyl compounds (1a /1b with t1/2
<
10 min for both). This stability demonstrated that the
de novo designed nonpeptidyl Ca2+ channel antagonists
could be advantageous for in vivo application. Unfor-
tunately, pharmacokinetic (PK) studies of compound 11
indicated that plasma concentration declined very rap-
idly over 10 min after a single 5 mg/kg bolus intravenous
dose to rats. This appeared to be due to high clearance
(>90 mL/min/kg) and large volume of distribution (>20
L/kg). This undesirable PK profile hampered the further
development of compound 11 as a neuroprotection/pain
candidate. Further SAR of compounds 11 and 5 will be
presented elsewhere.19c
Further evaluation of compounds 5, 7-9 in the
audiogenic seizure model demonstrated that no anti-
convulsant effect was observed for 5 and 8 at an iv dose
of 30 mg/kg, while the less active compounds, 7 and 9
(IMR32 IC50 ) 2.5 and 4.6 µM, respectively), exhibited
moderate antiseizure activities (60% protection at 30
mg/kg, Table 1). The relatively low level of in vivo
efficacy of 5 was unusual, since 5 was a rather potent
N-type Ca2+ channel blocker in vitro (IMR32 IC50 ) 0.34
µM). It was speculated that this low central nervous
system activity was a consequence of the poor physico-
chemical properties of compound 5. Indeed, the antago-
nists 7 and 9 had lower ClogP values and demonstrated
improved in vivo efficacy. With the goal of enhancing
the in vivo potency of this series, the azepane-1-
carboxylic amide group of 5 was replaced with an NH2,
isopropylamine, 3-methylbutylamine, and cyclohexy-
lamine. Compounds 11-14 displayed good activity in
blocking N-type Ca2+ channels. Detailed analysis of
these ligands (11-14) suggested a relatively flat struc-
ture-activity relationship (IC50 values ranged from 0.48
to 0.75 µM) and the IMR32 activities were not sensitive
to the N-substitution. The free amine analogue (11) was
chosen for further evaluation, since antagonist 11
possessed the most favorable physicochemical properties
Con clu sion
In summary, a series of 4-(4-benzyloxylphenyl)pip-
eridine-based N-type calcium channel blockers has been
described. These compounds were derived from the high-
throughput screening hit (1a ) which was a dipeptide.
These new structures demonstrated potent in vitro
activity in the IMR32 assay and moderate in vivo
efficacy in the audiogenic seizure mouse model. From
this study, a new chemical lead (11) was discovered with
several advantages over 1a : good aqueous solubility,
potent in vivo efficacy, and increased metabolic stability.
(S)-2-Amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-
2-enyl)-amino]-piperidin-1-yl}-4-methyl-pentan-1-one (11)
was a potent N-type calcium channel antagonist (IC50
) 0.67 µM in the IMR32 assay), with anticonvulsant
(ED50 ) 6 mg/kg, iv) and analgesic activities (ED50 ) 6

4244 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Hu et al.
brine, dried over Na2SO4, and concentrated. The crude mate-
rial was chromatographed on silica gel eluting with 4% MeOH/
CH2Cl2 to give 6.54 g (67%) of the desired product as a white
solid: mp 183-184 °C; IR (KBr, cm-1) 3314, 2979, 1688, 1531,
mg/kg, iv). This compound was a balanced channel
blocker for both neuronal N-type calcium channels and
voltage-gated sodium channels, and it stands out as an
interesting lead for the future development of neuro-
protective and analgesic agents.
1
1511, 1232, 1168, 827; H NMR (CDCl3) δ 1.41 (s, 9 H), 1.52
(s, 6 H), 4.80 (br, 1 H), 5.01 (s, 2 H), 6.89 (d, 2 H, J ) 9.0 Hz),
7.26-7.39 (m, 7 H), 8.80 (br, 1 H); MS m/z 385.1 (M + 1). Anal.
(C22H28N2O4) C, H, N.
Exp er im en ta l Section
[2-(4-Ben zyloxy-p h en yla m in o)-1,1-d im eth yl-eth yl]-ca r -
ba m ic Acid ter t-Bu tyl Ester (4b). [1-(4-Benzyloxy-phenyl-
carbamoyl)-1-methyl-ethyl]-carbamic acid tert-butyl ester (4a ,
6.54 g, 17.0 mmol) was dissolved in THF (50 mL) and cooled
to 0 °C. A solution of diborane (51 mL, 1.0 M) in THF was
added, and the reaction mixture was kept at 0 °C for 10 min
and then heated to 65 °C for 18 h. The reaction was cooled to
room temperature and treated with 0.1 M HCl (100 mL) and
then neutralized with saturated sodium bicarbonate solution.
The aqueous layer was extracted three times with EtOAc,
dried over Na2SO4, and concentrated. The residue was chro-
matographed on silica gel eluting with 5:1 hexane/EtOAc to
give 1.32 g (22%) of the desired product as an oil: IR (KBr,
Ch em istr y. Melting points were determined in open capil-
lary tubes on a Thomas-Hoover apparatus and are uncorrected.
IR spectra were recorded as KBr disks on a Nicolet MX-1 F
spectrophotometer. Mass spectra were recorded on a Finnigan
4500 or VG Analytical 7070E/HF mass spectrometer. Thin-
layer chromatography was performed on E. Merck silica gel
F254 (0.25 mm) glass plates. Elemental analyses and poten-
tiometric logP measurement were performed by Robertson
Laboratories. Flash chromatography was performed with E.
Merck silica gel 60, 230-400 mesh ASTM. 1H NMR spectra
were recorded at 400 MHz on a Varian Unity 400, and the
chemical shifts were reported in ppm (δ) relative to trimeth-
ylsilane (0.0 ppm). The 1H NMR spectra of all compounds were
consistent with their assigned structures. All Ca2+ antagonists
were analyzed for C, H, and N elemental analyses. Compounds
analyzed for exact mass were further checked for homogeneity
by analytical reversed-phase HPLC on a Phenomenex C-18
column eluting with 1:1 CH3CN/H2O (with 0.1% TFA), detec-
tion at 254 nm.
(S)-2-[(Azep a n e-1-ca r bon yl)-a m in o]-4-m eth yl-p en ta n o-
ic Acid Ben zyl Ester (2a ). A solution of triphosgene (15.7 g,
52.9 mmol) in CH2Cl2 (600 mL) was cooled to -10 °C. The
solution was treated dropwise with a solution of leucine benzyl
ester (28.1 g, 127 mmol) and pyridine (26 mL, 320 mmol) in
CH2Cl2 (150 mL). The reaction was stirred at -10 °C for 90
min and then treated with a solution of hexamethyleneimine
(22 mL, 380 mmol) in CH2Cl2 (75 mL). The solution was stirred
for 48 h at room temperature. The reaction mixture was
concentrated, and the residue was dissolved in ether and
washed with 1 N HCl solution, water, and saturated aqueous
CuSO4 solution. The organic layer was dried over MgSO4,
1
cm-1) 3380, 2975, 1708, 1513, 1230, 1164, 819, 737; H NMR
(CDCl3) δ 1.31 (s, 6 H), 1.39 (s, 9 H), 3.17 (s, 2 H), 3.65 (br, 1
H), 4.60 (br, 1 H), 4.95 (s, 2 H), 6.57 (d, 2 H, J ) 8.8 Hz), 6.80
(d, 2 H, J ) 8.8 Hz), 7.27-7.39 (m, 5 H); MS m/z 371.2 (M +
1). Anal. (C22H30N2O3) C, H, N.
{2-[(4-Ben zyloxy-ph en yl)-(3-m eth yl-bu t-2-en yl)-am in o]-
1,1-d im eth yl-eth yl}-ca r ba m ic Acid ter t-Bu tyl Ester (4c).
[2-(4-Benzyloxy-phenylamino)-1,1-dimethyl-ethyl]-carbamic acid
tert-butyl ester (4b, 0.6 g, 1.62 mmol) was dissolved in THF
(32 mL) and treated with diisopropylethylamine (1.1 mL, 6.48
mmol) and 4-bromo-2-methyl-2-butene (0.37 mL, 3.24 mmol).
The solution was heated to 40 °C overnight. The solution was
cooled to room temperature, filtered, and concentrated. The
residue was chromatographed on silica gel eluting with 8:1
hexane/EtOAc to give 0.56 (81%) of the desired product as an
oil: IR (KBr, cm-1) 2973, 1714, 1511, 1239, 1164, 733; 1H NMR
(CDCl3) δ 1.27 (s, 6 H), 1.39 (s, 9 H), 1.62 (s, 3 H), 1.65 (s, 3
H), 3.38 (s, 2 H), 3.82 (d, 2 H, J ) 5.6 Hz), 4.59 (s, 1 H), 4.95
(s, 2 H), 5.10 (br, 1 H), 6.70 (d, 2 H, J ) 9.3 Hz), 6.82 (d, 2 H,
J ) 9.0 Hz), 7.25-7.39 (m, 5 H); MS m/z 439.1 (M + 1). Anal.
(C27H38N2O3) C, H, N.
treated with activated charcoal, and filtered. The filtrate was
1
concentrated to
/ volume and treated with hexane. The
2
resulting suspension was stored overnight at -10 °C. The solid
was collected by filtration, washed with hexane, and dried
under vacuum to give 38.6 g (88%) of the desired product as a
white solid: mp 87-88 °C; IR (KBr, cm-1) 3560, 3399, 3280,
3044, 2930, 1723, 1625, 1537, 1195; 1H NMR (DMSO-d6) δ 0.83
(d, 3 H, J ) 6.5 Hz), 0.89 (d, 3 H, J ) 6.3 Hz), 1.38-1.72 (m,
11 H), 3.22-3.32 (m, 4 H), 4.20-4.26 (m, 1 H), 5.09 (dd, 2 H,
J ) 19.0, 12.5 Hz), 6.35 (d, 1 H, J ) 8.0 Hz), 7.30-7.39 (m, 5
H); MS m/z 347 (M + 1 for C20H30N2O3).
(S)-2-[(Azep a n e-1-ca r bon yl)-a m in o]-4-m eth yl-p en ta n o-
ic Acid (2). A solution of (S)-2-[(azepane-1-carbonyl)-amino]-
4-methyl-pentanoic acid benzyl ester (2a , 8.97 g, 25.9 mmol)
in THF (100 mL) was hydrogenated at 50 psi over Pd/C (0.25
g, 20% Pd) for 16 h. The reaction mixture was filtered through
Celite and concentrated to dryness. The residue was heated
in hexane (50 mL). The resulting suspension was cooled and
the solid collected by filtration and washed with hexane. The
solid was dried at room temperature under vacuum to give
6.1 g (92%) of the desired product 2 as a white solid: mp 88-
89 °C; IR (KBr, cm-1) 3366, 2930, 2869, 1727, 1609, 1544, 1210;
1H NMR (DMSO-d6) δ 0.79 (d, 3 H, J ) 6.3 Hz), 0.84 (d, 3 H,
J ) 6.3 Hz), 1.39-1.44 (m, 5 H), 1.55-1.62 (m, 6 H), 3.22-
3.33 (m, 4 H), 4.04-4.10 (m, 1 H), 6.10 (d, 1 H, J ) 8.0 Hz),
12.20 (s, 1 H); MS m/z 258 (M + 1). Anal. (C13H24N2O3) C, H,
N.
N-1-(4-Ben zyloxy-p h en yl)-2-m eth yl-N-1-(3-m eth yl-bu t-
2-en yl)-p r op a n e-1,2-d ia m in e (4d ). {2-[(4-Benzyloxy-phenyl)-
(3-methyl-but-2-enyl)-amino]-1,1-dimethyl-ethyl}-carbamic acid
tert-butyl ester (4c, 0.56 g, 1.32 mmol) was dissolved in CH2-
Cl2 (7 mL) and treated with TFA (3 mL). The solution was
stirred for 2.5 h and then concentrated. The residue was twice
redissolved in toluene and concentrated, then dissolved in
EtOAc, washed three times with saturated sodium bicarbonate
solution, once with brine, dried over Na2SO4, and concentrated
to give 0.43 g (100%) of the desired product as an oil: IR (KBr,
1
cm-1) 2963, 1511, 1237, 813, 736; H NMR (CDCl3) δ 1.18 (s,
6 H), 1.63 (s, 3 H), 1.66 (s, 3 H), 3.16 (s, 2 H), 3.88 (s, 2 H),
4.97 (s, 2 H), 5.13 (s, 1 H), 6.78-6.86 (m, 4 H), 7.23-7.41 (m,
5 H); MS m/z 339.2 (M + 1). Anal. (C22H30N2O1) C, H, N.
(S)-Azep a n e-1-ca r b oxylic Acid (1-{2-[(4-Ben zyloxy-
p h en yl)-(3-m eth yl-bu t-2-en yl)-a m in o]-1,1-d im eth yl-eth -
ylcar bam oyl}-3-m eth yl-bu tyl)-am ide (4). N-1-(4-Benzyloxy-
phenyl)-2-methyl-N-1-(3-methyl-but-2-enyl)-propane-1,2-
diamine (4d , 0.45 g, 1.32 mmol) was dissolved in DMF (7 mL)
and treated with diisopropylethylamine (1.4 mL, 7.9 mmol),
(S)-2-[(azepane-1-carbonyl)-amino]-4-methyl-pentanoic acid (2,
0.34 g, 1.32 mmol), and HBTU (0.5 g, 1.32 mmol). The reaction
was stirred for 1 h, then diluted with EtOAc, washed once with
saturated sodium bicarbonate solution, once with brine, dried
over Na2SO4, and concentrated. The residue was chromato-
graphed on silica gel eluting with 2:1 hexane/EtOAc to give
0.57 g (75%) of the desired product as a white foam: mp 104-
107 °C; IR (KBr, cm-1) 3283, 2928, 1650, 1619, 1542, 1511,
[1-(4-Ben zyloxy-ph en ylcar bam oyl)-1-m eth yl-eth yl]-car -
ba m ic Acid ter t-Bu tyl Ester (4a ). 4-Benzyloxyaniline (5.05
g, 25.4 mmol) was dissolved in acetonitrile (125 mL) and
treated with diisopropylethylamine (6.6 mL, 38.1 mmol), NR-
Boc-Aib acid (5.16 g, 25.4 mmol), and HBTU (9.63 g, 25.4
mmol). The solution was stirred for 3 h, then concentrated in
vacuo. The residue was dissolved in EtOAc and filtered, then
washed twice with saturated bicarbonate solution, once with
1
1239; H NMR (CDCl3) δ 0.87 (d, 3 H, J ) 4.2 Hz), 0.88 (d, 3
H, J ) 4.2 Hz), 1.31-1.64 (m, 22 H), 3.22-3.38 (m, 6 H), 3.77-
3.80 (m, 2 H), 4.16-4.21 (m, 1 H), 4.61 (d, 2 H, J ) 8.1 Hz),
4.94 (s, 2 H), 5.07-5.09 (m, 1 H), 6.31 (s, 1 H), 6.73 (d, 2 H, J

Synthesis of Neuronal N-Type Calcium Channel Blockers
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4245
) 9.3 Hz), 6.82 (d, 2 H, J ) 9.3 Hz), 7.25-7.39 (m, 5 H); MS
m/z 577.1 (M + 1). Anal. (C35H52N4O3) C, H, N.
4-(4-Ben zyloxy-p h en yla m in o)-p ip er id in e (7b). A solu-
tion of 4-(4-Benzyloxy-phenylamino)-piperidine-1-carboxylic
acid tert-butyl ester (7a , 3 g, 7.84 mmol) in CH2Cl2 (20 mL)
was treated with TFA (20 mL) and stirred for 30 min. The
reaction was concentrated, diluted with EtOAc, washed with
saturated sodium bicarbonate solution and brine, dried over
Na2SO4, and concentrated to give 1.56 g (70%) of the TFA salt
of the desired product as a white solid: mp 235-236 °C; IR
(KBr, cm-1) 3386, 2927, 1509, 1232, 1019, 698; 1H NMR
(DMSO-d6) δ 1.40 (dd, 2 H, J ) 21.5, 10.3 Hz), 1.91 (d, 2 H, J
) 13.7 Hz), 2.82 (t, 2 H, J ) 11.5 Hz), 3.13-3.30 (m, 4 H),
4.90 (s, 2 H), 5.17 (d, 1 H, J ) 8.1 Hz), 6.49 (d, 2 H, J ) 8.8
Hz), 6.73 (d, 2 H, J ) 8.5 Hz), 7.23-7.36 (m, 5 H); MS m/z
283.1 (M + 1); HRMS m/z 283.1810 (calcd for M + 1), 283.1815
(found); HPLC purity 100%. Anal. (C18H22N2O1‚0.5TFA‚
1.5H2O) C, H, N.
(S)-[1-(4-Ben zyloxy-p h en ylca r ba m oyl)-eth yl]-ca r ba m -
ic Acid ter t-Bu tyl Ester (3a ). N-Boc-L-alanine was subjected
to the same procedure as described for compound 4a , to give
the desired product as a solid: IR (KBr, cm-1) 3333, 1689,
1
1663, 1528, 1511, 1238, 1169, 827; H NMR (CDCl3) δ 1.28-
1.41 (m, 12 H), 2.71-2.80 (m, 1 H), 4.23 (br, 1 H), 4.94 (s, 2
H), 5.32 (br d, 1 H, J ) 6.1 Hz), 6.82 (d, 2 H, J ) 6.8 Hz),
7.22-7.41 (m, 7 H); MS m/z 371.2 (M + 1 for C21H26N2O4).
(S)-[2-(4-Ben zyloxy-p h en yla m in o)-1-m eth yl-eth yl]-ca r -
ba m ic Acid ter t-Bu tyl Ester (3b). Compound 3a was
subjected to the same procedure as described for compound
4b, to give the desired product as an oil: IR (KBr, cm-1) 3890,
1
2980, 1679, 1517, 1461, 1366, 1237, 1170, 1047, 812, 731; H
NMR (CDCl3) δ 1.17 (d, 3 H, J ) 6.8 Hz), 1.41 (s, 9 H), 3.00
(dd, 1 H, J ) 12.5, 7.3 Hz), 3.07 (dd, 1 H, J ) 12.5, 4.9 Hz),
3.88 (br, 2 H), 4.48 (br, 1 H), 4.95 (s, 2 H), 6.54 (d, 2 H, J ) 8.8
Hz), 6.81 (d, 2 H, J ) 9.0 Hz), 7.26-7.39 (m, 5 H); MS m/z
357.2 (M + 1 for C21H28N2O3).
(S)-Azep a n e-1-ca r boxylic Acid {1-[4-(4-Ben zyloxy-p h e-
n y la m in o )-p ip e r id in e -1-c a r b o n y l]-3-m e t h y l-b u t y l}-
a m id e (7). A solution of 7b was subjected to the same
procedure as described for compound 4, to give the desired
product as a white foam: mp 65-67 °C; IR (KBr, cm-1) 3351,
(S )-{2-[(4-Be n zyloxy-p h e n yl)-(3-m e t h yl-b u t -2-e n yl)-
a m in o]-1-m eth yl-eth yl}-ca r ba m ic Acid ter t-Bu tyl Ester
(3c). Compound 3b was subjected to the same procedure as
described for compound 4c, to give the desired product as an
oil: IR (KBr, cm-1) 3371, 2967, 1677, 1518, 1235, 1175, 1049,
802, 730; 1H NMR (CDCl3) δ 1.12 (d, 3 H, J ) 6.3 Hz), 1.39 (s,
9 H), 1.64 (s, 3 H), 1.66 (s, 3 H), 2.99 (dd, 1 H, J ) 13.4, 6.8
Hz), 3.26 (dd, 1 H, J ) 14.4, 6.3 Hz), 3.71-3.84 (m, 3 H), 4.41
(br, 1 H), 4.96 (s, 2 H), 5.12 (br, 1 H), 6.70 (d, 2 H, J ) 9.0 Hz),
6.84 (d, 2 H, J ) 9.0 Hz), 7.23-7.40 (m, 5 H); MS m/z 425.3
(M + 1 for C26H36N2O3).
1
2926, 2858, 1629, 1511, 1452, 1227, 697; H NMR (CDCl3) δ
0.88 (d, 3 H, J ) 6.8 Hz), 0.96 (d, 3 H, J ) 6.6 Hz), 1.20-1.68
(m, 13 H), 2.04-2.15 (m, 2 H), 2.76-2.89 (m, 1 H), 3.10-3.23
(m, 2 H), 3.36-3.41 (m, 5 H), 3.92 (t, 1 H, J ) 12.2 Hz), 4.40
(dd, 1 H, J ) 21.2, 14.4 Hz), 4.89-4.94 (m, 1 H), 4.95 (s, 2 H),
5.15-5.18, 1 H), 6.55 (d, 2 H, J ) 8.5 Hz), 6.81 (dd, 2 H, J )
8.8, 2.7 Hz), 7.25-7.39 (m, 5 H); MS m/z 521.2 (M + 1). Anal.
(C31H44N4O3) C, H, N.
(4-B e n z y lo x y -p h e n y l)-(1-b e n z y l-p i p e r i d i n -3-y l)-
a m in e (6a ). A solution of 1-benzyl-3-piperidone was subjected
to the same procedure as described for compound 7a , to give
the desired product as a solid: IR (KBr, cm-1) 3262, 2930,
(S)-N1-(4-Ben zyloxy-p h en yl)-N1-(3-m eth yl-bu t-2-en yl)-
p r op a n e-1,2-d ia m in e (3d ). Compound 3c was subjected to
the same procedure as described for compound 4d , to give the
desired product as a clear oil: IR (KBr, cm-1) 2967, 2908, 2871,
1
2807, 1506, 1452, 1295, 1217, 1045, 826, 732, 705; H NMR
(CDCl3) δ 1.49 (br, 2 H), 1.66 (br, 2 H), 2.24-2.35 (m, 3 H),
2.68 (br, 1 H), 3.47 (m, 3 H), 3.74 (br, 1 H), 4.94 (s, 2 H), 6.53
(d, 2 H, J ) 8.8 Hz), 6.79 (d, 2 H, J ) 8.5 Hz), 7.20-7.39 (m,
10 H); MS m/z 373.1 (M + 1). Anal. (C25H28N2O1‚0.2H2O) C,
H, N.
1
1510, 1237, 813, 735, 695; H NMR (CDCl3) δ 1.09 (d, 3 H, J
) 6.3 Hz), 1.63 (s, 3 H), 1.66 (s, 3 H), 2.61 (br, 2 H), 2.93 (dd,
1 H, J ) 14.2, 9.0 Hz), 3.09-3.22 (m, 2 H), 3.79 (s, 2 H), 4.95
(s, 2 H), 5.13 (br, 1 H), 6.71 (d, 2 H, J ) 9.3 Hz), 6.83 (d, 2 H,
J ) 6.8 Hz), 7.23-7.39 (m, 5 H); MS m/z 325.2 (M + 1 for
(4-Ben zyloxy-ph en yl)-(1-ben zyl-piper idin -3-yl)-(3-m eth -
yl-bu t-2-en yl)-a m in e (6b). A solution of (4-benzyloxy-phenyl)-
(1-benzyl-piperidin-3-yl)-amine (6a ) was subjected to the same
procedure as described for compound 10b, to give the desired
product as a gum: IR (KBr, cm-1) 2934, 2797, 1509, 1237,
C
21H28N2O1).
[S-(R*,R*)]-Azep a n e-1-ca r boxylic Acid (1-{2-[(4-Ben -
zyloxy-p h en yl)-(3-m et h yl-b u t -2-en yl)-a m in o]-1-m et h yl-
eth ylca r ba m oyl}-3-m eth yl-bu tyl)-a m id e (3). Compound 3d
was subjected to the same procedure as described for com-
pound 4, to give the desired product as a white solid: mp 130-
132 °C; IR (KBr, cm-1) 3262, 2927, 2865, 1619, 1536, 1512,
1229, 1026, 815, 734, 695; 1H NMR (CDCl3) δ 0.85 (dd, 6 H, J
) 6.6, 2.0 Hz), 1.13 (d, 3 H, J ) 6.6 Hz), 1.38-1.66 (m, 19 H),
2.96 (dd, 1 H, J ) 14.4, 7.3 Hz), 3.29-3.34 (m, 5 H), 3.73-
3.79 (m, 2 H), 4.07-4.23 (m, 2 H), 4.67 (d, 1 H, J ) 8.3 Hz),
4.94 (s, 2 H), 5.10 (br, 1 H), 6.24 (d, 1 H, J ) 7.1 Hz), 6.72 (d,
2 H, J ) 9.3 Hz), 6.84 (d, 2 H, J ) 9.0 Hz), 7.22-7.39 (m, 5
H); MS m/z 563.4 (M + 1). Anal. (C34H50N4O3) C, H, N.
4-(4-Ben zyloxy-p h en yla m in o)-p ip er id in e-1-ca r boxyl-
ic Acid ter t-Bu tyl Ester (7a ). 4-Benzyloxyaniline hydrochlo-
ride salt (10 g, 42.4 mmol) was suspended in EtOAc (500 mL)
and washed three times with saturated sodium bicarbonate
solution, once with brine, dried over Na2SO4, and concentrated.
The free base was dissolved in CH2Cl2 (250 mL), treated with
1-tert-butyl-carbonyl-4-piperidone (8.45 g, 42.4 mmol), stirred
for 30 min and then cooled to 0 °C. NaBH(OAc)3 (13.5 g, 63.6
mmol) was added, and the reaction was allowed to warm to
room temperature and stir for 18 h. The reaction was diluted
with CH2Cl2 (250 mL), washed with saturated sodium bicar-
bonate solution and brine, dried over Na2SO4, and concen-
trated to give 15.3 g (94%) the desired product as a white
solid: mp 101-102 °C; IR (KBr, cm-1) 3337, 2915, 1684, 1510,
1420, 1229, 1147, 1027, 835, 699; 1H NMR (CDCl3) δ 1.24-
1.27 (m, 2 H), 1.43 (s, 9 H), 1.98 (br, 2 H, J ) 12.0 Hz), 2.85 (t,
2 H, J ) 11.5 Hz), 3.29-3.31 (m, 2 H), 4.00 (br, 2 H), 4.95 (s,
2 H), 6.54 (d, 2 H, J ) 8.8 Hz), 6.81 (d, 2 H, J ) 8.8 Hz), 7.27-
7.39 (m, 5 H); MS m/z 383.1 (M + 1). Anal. (C23H30N2O3) C, H,
N.
1
1027, 811, 735, 697; H NMR (CDCl3) δ 1.28-1.36 (m, 1 H),
1.57-1.91 (m, 12 H), 2.78 (d, 1 H, J ) 10.7 Hz), 2.96 (d, 1 H,
J ) 10.3 Hz), 3.44-3.75 (m, 4 H), 4.95 (s, 2 H), 5.02 (s, 1 H),
6.66 (d, 2 H, J ) 8.3 Hz), 6.82 (d, 2 H, J ) 8.8 Hz), 7.20-7.40
(m, 10 H); MS m/z 441.2 (M + 1). Anal. (C30H36N2O1) C, H, N.
(4-Ben zyloxy-p h en yl)-(1-p ip er id in -3-yl)-(3-m eth yl-bu t-
2-en yl)-a m in e (6c). (4-Benzyloxy-phenyl)-(1-benzyl-piperidin-
3-yl)-(3-methyl-but-2-enyl)-amine (6b, 0.90 g, 2.04 mmol) was
dissolved in 1,2-dichloroethane (20 mL), cooled to 0 °C, and
treated with R-chloroethylchloroformate (0.24 mL, 2.04 mmol).
The reaction was stirred at 0 °C for 15 min and then heated
to 60 °C for 1 h. The reaction was cooled to room temperature,
concentrated in vacuo, redissolved in MeOH (20 mL), heated
to 50 °C for 1 h, and then concentrated in vacuo to give 0.86
g (>100%) of the crude material as an oil which was carried
on without further purification: IR (KBr, cm-1) 2928, 2803,
1511, 1452, 1220, 1039, 826, 740, 696; 1H NMR (CDCl3) δ 1.59
(s, 3 H), 1.65 (s, 3 H), 1.97-2.03 (m, 3 H), 2.70-2.76 (m, 2 H),
3.38-3.48 (m, 4 H), 3.73 (br, 2 H), 3.99 (br, 1 H), 4.98 (s, 2 H),
5.07 (s, 1 H), 6.84-6.89 (m, 2 H), 7.29-7.42 (m, 7 H); MS m/z
351.2 (M + 1 for C23H30N2O1).
(S,R/S)-Azep a n e-1-ca r boxylic Acid (1-{3-[(4-Ben zyloxy-
p h en yl)-(3-m eth yl-bu t-2-en yl)-a m in o]-p ip er id in e-1-ca r -
bon yl}-3-m eth yl-bu tyl)-am ide (6). A solution of (4-benzyloxy-
phenyl)-(1-piperidin-3-yl)-(3-methyl-but-2-enyl)-amine (6c) was
subjected to the same procedure as described for compound 4,
to give the desired product as a gum: IR (KBr, cm-1) 3337,
2928, 2863, 1734, 1632, 1509, 1453, 1406, 1375, 1284, 1240,
1212, 1025, 813, 738, 697; 1H NMR (two diastereomers)
(CDCl3) δ 0.87-0.98 (m, 6 H), 0.98 (s, 3 H), 1.00 (s, 3 H), 1.32-

4246 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Hu et al.
1.91 (m, 19 H), 2.58 (t, 1 H, J ) 12.7 Hz), 3.03-3.13 (m, 1 H),
3.37-3.41 (m, 3 H), 3.73-3.78 (m, 3 H), 4.04 (d, 1 H, J ) 13.7
Hz), 4.65-4.68 (m, 1 H), 4.92-4.95 (m, 1 H), 5.02 (s, 2 H),
5.25 (dd, 1 H, J ) 13.9, 5.6 Hz), 6.32-6.35 (m, 2 H), 7.07-
7.12 (m, 1 H), 7.27-7.42 (m, 4 H); MS m/z 589.3 (M + 1). Anal.
(C36H52N4O3) C, H, N.
then treated with N,N-diisopropylethylamine (9.1 mL, 52.4
mmol) and 4-bromo-2-methyl-2-butene (3.0 mL, 26.2 mmol).
The reaction was heated to 40 °C for 18 h and then concen-
trated in vacuo. The residue was chromatographed on silica
gel eluting with 5:1 hexane/EtOAc to give 5.15 g (87%) of the
desired compound as an oil: IR (KBr, cm-1) 2973, 2928, 2857,
1
1693, 1509, 1423, 1239, 1158, 1020; H NMR (CDCl3) δ 1.42
(S)-Azep a n e-1-ca r b oxylic Acid (1-{4-[(4-Ben zyloxy-
p h en yl)-(3-m eth yl-bu t-2-en yl)-a m in o]-p ip er id in e-1-ca r -
bon yl}-3-m eth yl-bu tyl)-a m id e (5). A solution of compound
7 (0.39 g, 0.75 mmol) in THF (15 mL) was treated with
diisopropylethylamine (0.4 g, 3 mmol) and 4-bromo-2-methyl-
2-butene (0.149 g, 1 mmol). The reaction was heated to 40 °C
for 18 h and then concentrated. The residue was chromato-
graphed on silica gel to give the desired product as a gum: IR
(KBr, cm-1) 3338, 2927, 2859, 1632, 1509, 1453, 1238; 1H NMR
(CDCl3) δ 0.87 (t, 3 H, J ) 7.1 Hz), 0.96 (d, 3 H, J ) 6.6 Hz),
1.32-1.67 (m, 20 H), 1.81-1.89 (m, 1 H), 2.59 (dd, 1 H, J )
22.7, 10.3 Hz), 3.01-3.08 (m, 1 H), 3.34-3.39 (m, 4 H), 3.49-
3.51 (m, 1 H), 3.65 (s, 2 H), 3.97-4.00 (m, 1 H), 4.61 (t, 1 H,
J ) 14.4 Hz), 4.87-5.06 (m, 4 H), 5.21 (dd, 1 H, J ) 22.0, 8.5
Hz), 6.70-6.76 (m, 2 H), 6.82-6.86 (m, 2 H), 7.26-7.40 (m, 5
H); MS m/z 589.2 (M + 1). Anal. (C36H52N4O3) C, H, N.
(s, 9 H), 1.43-1.56 (m, 3 H), 1.62 (s, 3 H), 1.63 (s, 3 H), 1.74
(d, 2 H, J ) 11.5 Hz), 2.65 (br, 2 H), 3.41-3.45 (m, 1 H), 3.66
(d, 2 H, J ) 5.4 Hz), 4.15 (br, 1 H), 4.96 (s, 2 H), 5.04 (br, 1 H),
6.71 (d, 2 H, J ) 9.0 Hz), 6.84 (d, 2 H, J ) 9.0 Hz), 7.27-7.40
(m, 5 H); MS m/z 451.4 (M + 1). Anal. (C28H38N2O3) C, H, N.
(4-Ben zyloxy-p h en yl)-(3-m eth yl-bu t-2-en yl)-p ip er id in -
4-yl-a m in e (11b). 4-[(4-Benzyloxy-phenyl)-(3-methyl-but-2-
enyl)-amino]-piperidine-1-carboxylic acid tert-butyl ester (11a ,
5.0 g, 11.1 mmol) was dissolved in CH2Cl2 (20 mL) and treated
with TFA (20 mL). The reaction was stirred for 10 min and
then concentrated in vacuo. The solution was washed with
saturated bicarbonate solution (2 × 400 mL) and brine (1 ×
400 mL), dried over Na2SO4, and concentrated in vacuo to give
3.9 g (99%) of the desired product as a oil: IR (KBr, cm-1
)
2927, 1678, 1509, 1237, 1203, 1177, 1131, 1024, 697; 1H NMR
(CDCl3) δ 1.60 (s, 3 H), 1.63 (s, 3 H), 1.65-1.75 (m, 2 H), 1.86
(d, 2 H, J ) 11.2 Hz), 2.75 (t, 2 H, J ) 12.5 Hz), 3.26 (d, 2 H,
J ) 12.7 Hz), 3.41-3.49 (m, 1 H), 3.67 (d, 2 H, J ) 5.4 Hz),
4.62 (br, 1 H), 4.97 (s, 2 H), 5.01-5.05 (m, 1 H), 6.73 (d, 2 H,
J ) 9.3 Hz), 6.84 (d, 2 H, J ) 9.0 Hz), 7.26-7.40 (m, 5 H); MS
m/z 351.3 (M + 1). HRMS 351.2436 (calcd M + 1), 351.2444
(found); HPLC purity 100%. Anal. (C23H30N2O1‚0.58TFA) C,
H, N.
(S)-Azep a n e-1-ca r b oxylic Acid (1-{4-[(4-Ben zyloxy-
p h en yl)-eth yl-a m in o]-p ip er id in e-1-ca r bon yl}-3-m eth yl-
bu t yl)-a m id e (8). (S)-Azepane-1-carboxylic acid {1-[4-(4-
benzyloxy-phenylamino)-piperidine-1-carbonyl]-3-methyl-butyl}-
amide (7, 0.30 g, 0.28 mmol) was dissolved in CH2Cl2 (5 mL)
and treated with acetaldehyde (32 µL, 0.58 mmol). The
reaction was stirred for 30 min at room temperature, then
cooled to 0 °C, treated with NaBH(OAc)3, allowed to warm to
room temperature as the ice melted and was stirred overnight.
The reaction was then diluted with EtOAc (100 mL), washed
with saturated bicarbonate solution and brine, dried over Na2-
SO4, and concentrated. The residue was chromatographed on
silica gel eluting with 3:2 hexane/EtOAc to give 128 mg (61%)
of the desired product as a gum: IR (KBr, cm-1) 3344, 2928,
(S)-(1-{4-[(4-Ben zyloxy-p h en yl)-(3-m eth yl-bu t-2-en yl)-
a m in o]-p ip er id in e-1-ca r bon yl}-3-m eth yl-bu tyl)-ca r ba m -
ic Acid ter t-Bu tyl Ester (11c). (4-Benzyloxy-phenyl)-(3-
methyl-but-2-enyl)-piperidin-4-yl-amine (11b, 1.03 g, 2.94
mmol) was dissolved DMF (15 mL) and treated with DIEA
(1.3 mL, 7.4 mmol), Boc-L-leucine hydrate (0.74 g, 2.94 mmol),
and HBTU (1.11 g, 2.94 mmol). The reaction was stirred for
4.5 h, then diluted with EtOAc (200 mL), washed with
saturated bicarbonate solution and brine, dried over Na2SO4,
and concentrated. The crude material was chromatographed
on silica gel eluting with 5% (MeOH/CH2Cl2) to give 1.17 g
1
2865, 1632, 1509, 1453, 1239, 1025, 752; H NMR (CDCl3) δ
0.88 (t, 3 H, J ) 6.8 Hz), 0.96-1.0 (m, 6 H), 1.32-1.91 (m, 15
H), 2.61 (dd, 1 H, J ) 23.9, 12.5 Hz), 3.00-3.10 (m, 3 H), 3.36-
3.44 (m, 5 H), 3.98 (t, 1 H, J ) 11.5 Hz), 4.56 (t, 1 H, J ) 14.6
Hz), 4.90 (br s, 1 H), 4.97 (s, 2 H), 5.21 (dd, 1 H, J ) 17.6, 8.3
Hz), 6.76-6.96 (m, 4 H), 7.28-7.41 (m, 5 H); MS m/z 549.3
(M + 1). Anal. (C33H48N4O3) C, H, N.
(71%) of the desired product as a white foam: IR (KBr, cm-1
)
3422, 2927, 2361, 1708, 1638, 1509, 1453, 1366, 1238, 1167,
1
1025, 697; H NMR (CDCl3) δ 0.88 (t, 3 H, J ) 6.3 Hz), 0.95
Azep a n e-1-ca r boxylic Acid (1-{4-[(4-Ben zyloxy-p h en -
yl)-(3-h yd r oxy-b u t yl)-a m in o]-p ip er id in e-1-ca r b on yl}-3-
m eth yl-bu tyl)-a m id e (9). A solution of 3-hydroxybutyralde-
hyde was subjected to the same procedure as described for
compound 8, to give the desired product as an oil: IR (KBr,
cm-1) 3347, 2928, 2865, 1627, 1509, 1454, 1371, 1239, 1027,
754; 1H NMR (CDCl3) δ 0.86 (d, 3 H, J ) 6.3 Hz), 0.94 (d, 3 H,
J ) 9.3 Hz), 1.08 (d, 3 H, J ) 5.9 Hz), 1.22-1.66 (m, 15 H),
1.82-1.87 (m, 2 H), 2.46-2.55 (m, 1 H), 2.91-3.35 (m, 8 H),
3.92-3.96 (m, 2 H), 4.40 (br, 1 H), 4.49-4.73 (m, 1 H), 4.84
(br, 1 H), 4.99 (s, 2H), 5.16 (t, 1 H, J ) 7.6 Hz), 6.87-6.98 (m,
4 H), 7.29-7.41 (m, 5 H); MS m/z 593.2 (M + 1). Anal.
(C35H52N4O4) C, H, N.
(S)-Azep a n e-1-ca r boxylic Acid (1-{4-[Ben zyl-(4-ben zy-
loxy-p h en yl)-a m in o]-p ip er id in e-1-ca r b on yl}-3-m et h yl-
bu tyl)-a m id e (10). A solution of benzaldehyde was subjected
to the same procedure as described for compound 8, to give
the desired product as a sticky solid: IR (KBr, cm-1) 3428,
2927, 1632, 1511, 1452, 1239, 697; 1H NMR (CDCl3) δ 0.87 (d,
3 H, J ) 6.1 Hz), 0.95 (t, 3 H, J ) 5.6 Hz), 1.28-1.55 (m, 8 H),
1.65-1.67 (m, 5 H), 1.85-1.98 (m, 2 H), 2.59 (dd, 1 H, J )
18.6, 7.3 Hz), 3.01-3.14 (m, 1 H), 3.35-3.38 (m, 4 H), 3.72
(br, 1 H), 4.00 (d, 1 H, J ) 12.7 Hz), 4.29 (s, 2 H), 4.61 (t, 1 H,
J ) 12.5 Hz), 4.86-4.89 (m, 1 H), 4.93 (s, 2 H), 5.18 (dd, 1 H,
J ) 15.6, 8.3 Hz), 6.70 (t, 2 H, J ) 7.8 Hz), 6.80 (d, 2 H, J )
8.3 Hz), 7.17-7.38 (m, 10 H); MS m/z 611.3 (M + 1). Anal.
(C38H50N4O3) C, H, N.
(dd, 3 H, J ) 6.3, 2.0 Hz), 1.28-1.67 (m, 20 H), 1.81-1.91 (m,
2 H), 2.59 (dd, 1 H, J ) 23.9, 12.9 Hz), 2.99-3.10 (m, 1 H),
3.49-3.53 (m, 1 H), 3.61-3.63 (m, 2 H), 3.94 (t, 1 H, J ) 15.1
Hz), 4.57-4.63 (m, 2 H), 4.97 (s, 2 H), 5.02-5.05 (m, 1 H),
5.25-5.28 (m, 1 H), 6.73 (dd, 2 H, J ) 11.5, 9.3 Hz), 6.84 (dd,
2 H, J ) 9.0, 3.2 Hz), 7.26-7.40 (m, 5 H); MS m/z 564.5 (M +
1). Anal. (C34H49N3O4) C, H, N.
(S)-2-Am in o-1-{4-[(4-ben zyloxy-p h en yl)-(3-m eth yl-bu t-
2-en yl)-am in o]-piper idin -1-yl}-4-m eth yl-pen tan -1-on e (11).
(S)-(1-{4-[(4-Benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-
piperidine-1-carbonyl}-3-methyl-butyl)-carbamic acid tert-butyl
ester (11c, 1.07 g, 1.90 mmol) was dissolved in CH2Cl2 (4.5
mL) and treated with TFA (4.5 mL). The reaction was stirred
for 25 min and then concentrated in vacuo. The residue was
dissolved in EtOAc (175 mL), and the solution was washed
twice with saturated bicarbonate solution and brine, dried over
Na2SO4, and concentrated in vacuo to give 0.87 g (99%) of the
desired product as an oil: IR (KBr, cm-1) 2953, 1640, 1509,
1
1453, 1237, 1191, 1025; H NMR (CDCl3) δ 0.90 (d, 6 H, J )
3.9 Hz), 1.21-1.31 (m, 1 H), 1.33-1.54 (m, 5 H), 1.60 (s, 3 H),
1.62 (s, 3 H), 1.79-1.85 (m, 3 H), 2.58 (dd, 1 H, J ) 20.0, 11.5
Hz), 3.01 (dd, 1 H, J ) 23.7, 11.2 Hz), 3.51 (t, 1 H, J ) 3.4
Hz), 3.62-3.70 (m, 3 H), 3.86 (d, 1 H, J ) 13.2 Hz), 4.66 (d, 1
H, J ) 12.5 Hz), 4.97 (s, 2 H), 5.03 (m, 1 H), 6.74 (t, 2 H, J )
8.8 Hz), 6.85 (d, 2 H, J ) 8.8 Hz), 7.26-7.40 (m, 5 H); MS m/z
464.3 (M + 1). Anal. (C29H41N3O2) C, H, N.
4-[(4-Ben zyloxy-p h en yl)-(3-m eth yl-bu t-2-en yl)-a m in o]-
p ip er id in e-1-ca r boxylic Acid ter t-Bu tyl Ester (11a ). 4-(4-
Benzyloxy-phenylamino)-piperidine-1-carboxylic acid tert-butyl
ester (7a , 5.0 g, 13.1 mmol) was dissolved in THF (65 mL) and
(S)-1-{4-[(4-Ben zyloxy-p h en yl)-(3-m et h yl-b u t -2-en yl)-
a m in o]-p ip er id in -1-yl}-2-isop r op yla m in o-4-m eth yl-p en -
ta n -1-on e (12). A solution of (S)-2-amino-1-{4-[(4-benzyloxy-
phenyl)-(3-methyl-but-2-enyl)-amino]-piperidin-1-yl}-4-methyl-

Synthesis of Neuronal N-Type Calcium Channel Blockers
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4247
pentan-1-one (10, 0.20 g, 0.43 mmol) and acetone (25 mg, 0.43
mmol) in CH2Cl2 (4 mL) was cooled to 0 °C, treated with
NaBH(OAc)3, allowed to warm to room temperature as ice
melted, and stirred overnight. The reaction was diluted with
EtOAc (100 mL), washed with saturated sodium bicarbonate
solution and brine, dried over Na2SO4, and concentrated. The
residue was chromatographed on silica gel eluting with 5%
MeOH/CH2Cl2 to give 176 mg (81%) of the desired product as
a gum: IR (KBr, cm-1) 3490, 2957, 1640, 1509, 1452, 1237,
eter with dual emission monochromators using excitation at
350 nm and emission at 400 and 490 nM. The ratio of the
emissions at the two wavelengths is a fraction of intracellular
Ca2+ concentration. Different concentrations of the test com-
pounds (dissolved and diluted in DMSO) were added to assay
buffer containing approximately 3 × 106 loaded cells with 5
µM nitrendipine added to block L-type Ca2+ channels. Samples
were incubated for 10 min for 30 °C, and then emission signals
at 400 and 490 nm were acquired from each cuvette at 30 °C
for 50 s. At 20 s after the start of reading, cells were
depolarized by the addition of a high K+ solution. Drug effects
were expressed as a percentage of the amplitude of the K+-
evoked change in intracellular Ca2+ in drug-treated compared
to control experiments. IC50 values in these studies were
calculated from five-point concentration response curves for
each compound and were estimated by fitting a four-parameter
logistic function to the data using the least-squares method.
1
1025, 738; H NMR (CDCl3) δ 0.86-0.89 (m, 6 H), 0.99 (m, 5
H), 1.41-1.64 (m, 13 H), 1.83-1.86 (m, 3 H), 2.55-2.65 (m, 2
H), 2.98-3.09 (m, 1 H), 3.49-3.65 (m, 3 H), 3.92-3.95 (m, 1
H), 4.68 (d, 1 H, J ) 13.4 Hz), 4.97 (s, 2 H), 5.05 (br s, 1 H),
6.74 (d, 2 H, J ) 9.0 Hz), 6.85 (d, 2 H, J ) 9.0 Hz), 7.26-7.40
(m, 5 H); MS m/z 506.4 (M + 1). Anal. (C32H47N3O2) C, H, N.
(S)-1-{4-[(4-Ben zyloxy-p h en yl)-(3-m et h yl-b u t -2-en yl)-
am in o]-piper idin -1-yl}-4-m eth yl-2-(3-m eth yl-bu tylam in o)-
p en ta n -1-on e (13). A solution of isovaleraldehyde was sub-
jected to the same procedure as described for compound 12,
to give the desired product as a gum:: IR (KBr, cm-1) 2953,
(2) Electr op h ysiology. The protocol for whole-cell voltage-
clamp experiments on superior cervical ganglion (SCG) neu-
rons evaluating drug actions on Ca2+ channels has been
previously described.21 Evaluation of Na+ channel currents in
from acutely isolated SCG neurons was done using similar
techniques except solutions and voltage protocols were altered
to isolate Na+ channel currents.
After obtaining a whole-cell recording, control Ca2+ or Na+
channel currents were elicited from holding potentials between
-65 mV and -55 mV to a depolarized test potential (+20 mV
for Ca2+ and 0 mV for Na+). After stable current amplitudes
were observed in control solution, the external solution was
rapidly exchanged with an external solution containing test
compound. Test compounds were applied until steady-state
block was achieved. Data were expressed as percent inhibition
of control amplitudes.
1
1640, 1509, 1451, 1237, 1158, 1025, 738; H NMR (CDCl3) δ
0.82-0.89 (m, 11 H), 1.25-1.62 (m, 15 H), 1.73-1.86 (m, 3
H), 2.30-2.35 (m, 1 H), 2.44-2.50 (m, 1 H), 2.55-2.62 (m, 1
H), 3.03 (dd, 1 H, J ) 29.1, 11.7 Hz), 3.50-3.55 (m, 2 H), 3.65
(s, 2 H), 3.97 (d, 1 H, J ) 13.2 Hz), 4.69 (m, 1 H), 4.97 (s, 2 H),
5.05 (d, 1 H, J ) 4.9 Hz), 6.74 (dd, 2 H, J ) 9.0, 2.2 Hz), 6.85
(dd, 2 H, J ) 9.0, 2.0 Hz), 7.26-7.40 (m, 5 H); MS m/z 534.5
(M + 1). Anal. (C34H51N3O2) C, H, N.
(S)-1-{4-[(4-Ben zyloxy-p h en yl)-(3-m et h yl-b u t -2-en yl)-
a m in o]-p ip er id in -1-yl}-2-cycloh exyla m in o-4-m eth yl-p en -
ta n -1-on e (14). A solution of cyclohexanone was subjected to
the same procedure as described for compound 12, to give the
desired product as a gum: IR (KBr, cm-1) 2925, 2853, 1640,
1509, 1449, 1237, 1025, 811, 738; 1H NMR (CDCl3) δ 0.86-
0.89 (m, 6 H), 1.10-1.17 (m, 5 H), 1.38-1.85 (m, 20 H), 2.16
(m, 1 H), 2.54-2.66 (m, 1 H), 3.07 (dd, 1 H, J ) 28.1, 13.4
Hz), 3.52 (t, 1 H, J ) 11.2 Hz), 3.66 (d, 2 H, J ) 5.6 Hz), 3.94
(d, 1 H, J ) 12.5 Hz), 4.68 (br t, 1 H), 4.97 (d, 2 H, J ) 2.7
Hz), 5.05 (s, 1 H), 6.74 (d, 2 H, J ) 9.0 Hz), 6.85 (d, 2 H, J )
9.0 Hz), 7.26-7.40 (m, 5 H); MS m/z 546.5 (M + 1). Anal.
(C35H51N3O2) C, H, N.
(3) Meta bolic Sta bility in Hu m a n Liver Micr osom es.
The compounds were individually incubated (5 µM) with
human liver microsomes (0.5 mg/mL protein) at 37 °C in
duplicate or triplicate. At 0, 10, 20, and 40 min, 50 µL aliquots
were removed and added to 100 µL of acetonitrile and 25 µL
internal standard, PD 181210 (1 µg/mL) in acetonitrile.
Standard curves were run in a similar manner with each
compound at a concentration range of 1.25-5 µM. The samples
were analyzed for parent concentration by LC/MS/MS to
estimate in vitro metabolic half-life. These in vitro data
represent the rate of oxidative and hydrolytic reaction (CYP450,
FMO, esterases/amidases) but may not represent all possible
metabolic routes of elimination (e.g., conjugative reactions).
logP Deter m in a tion . The protocol (a potentiometric titra-
tion method) for this assay has been described earlier.20
ClogP Deter m in a tion . ClogP was calculated by Pallas
software (version 1.2, released for Windows CompuDrug
Chemistry Ltd., 1995).
Solu bility Deter m in a tion . Concentrated solution (10 mL)
of individual compounds in DMSO were diluted 20-fold with
PBS containing 25 mM potassium phosphate (pH 7.4) so that
precipitation was observed. The suspension was stirred for 1
h at 22 °C followed by centrifugation at 20000g for 5 min. An
aliquot of the supernatant was analyzed by HPLC; peak area
and retention time were compared to those of a standard
solution of the same compound in DMSO. The concentration
of the supernatant (which equals solubility in 5% DMSO/95%
25 mM potassium phosphate in PBS pH 7.4) was used as a
measure of aqueous solubility at neutral pH.
In Vitr o P h a r m a cology. (1) IMR32 Assa y. The IMR32
cell line was obtained from the American Type Culture
Collection (Rockville, MD). Cells were grown in Eagle’s Mini-
mum Essential Medium with Earle’s salts supplemented with
10% fetal bovine serum and 2 mM L-Gln and antibiotic/
antimycotic mixture (Gibco). At approximately 80% confluency,
differentiation was induced by the addition of 1 mM dibutyryl
cAMP and 2.5 µM bromodeoxyuridine to the medium. After
7-13 days of differentiation, cells were detached using 0.5 mM
EDTA and subsequently loaded with 5 µM Indo-1 acetoxym-
ethyl ester (Molecular Probe, Eugene, OR) at 30 °C for 45 min.
Dye-loaded cells at a concentration of ∼107 cells/mL were
resuspended in assay buffer (Hank’s balanced salt solution
with 10 mM HEPES/Tris pH 7.4 and 0.5% bovine serum
albumin) and kept on ice until use. Fluorescence measure-
ments were carried out in a Photon Technology International
(PTI, South Brunswick, NJ ) model RF-F3004 spectrofluorom-
In Vivo Exp er im en ts. (1) Au d iogen ic Seizu r e Mod el
in DBA/2 Mice. For in vivo testing, compounds were dissolved
in water using 1% (weight/volume) Emulphor/D5W (GAF
Corp., Wayne, NJ ) surfactant. Substances were administered
by intravenous injection into the retro-orbital venous sinus.
All testing was performed 15 min or 45 min after drug
injection. All the male mice, 3-4 weeks old were obtained from
J ackson Laboratories, Bar Harbor, ME. Mice were placed into
an enclosed acrylic plastic chamber (21 cm height, approxi-
mately 30 cm diameter) with a high-frequency speaker (4 cm
diameter) in the center of the top lid. An audio signal generator
(Protek model B-8 10) was used to produce a continuous
sinusoidal tone that was swept linearly in frequency between
8 and 16 kHz once each 10 ms. The average sound pressure
level (SPL) during stimulation was approximately 100 dB at
the floor of the chamber. Mice were placed within the chamber
and allowed to acclimatize for 1 min. DBA/2 mice in the
vehicle-treated group responded to the sound stimulus (applied
until tonic extension occurred, or for a maximum of 60 s) with
a characteristic seizure sequence consisting of wild running
followed by clonic seizures, and later by tonic extension, and
finally by respiratory arrest and death in 80% or more of the
mice. In vehicle-treated mice, the entire sequence of seizures
to respiratory arrest lasts approximately 15-20 s.
The incidence of all the seizure phases in the drug-treated
and vehicle-treated mice was recorded, and the occurrence of
tonic seizures was used for calculating anticonvulsant ED50

4248 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Hu et al.
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at each time and dose point.
(2) Acetic Acid Wr ith in g Test. The mouse acetic acid
writhing test measures the acute nociceptive response elicited
by injection of dilute acetic acid into the peritoneal cavity.22
Nociceptive behavior is quantified by counting the incidence
of abdominal constrictions in a fixed observation interval.
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of N-type voltage-sensitive calcium channels protects against
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Male, CF-1 mice (26 and 30 g) were given
a single,
intraperitoneal injection of 0.6% acetic acid. This injection-
evoked abdominal constrictions, defined as discrete episodes
of torso and hind limb stretching with or without neck arching,
were counted and recorded for 5 min, beginning 7 min after
acetic acid injection. The mice are individually housed in
Nalgene cages and allowed to move freely during the experi-
mental period (12 min). Animals are sacrificed by CO2 as-
phyxiation immediately after the 5 min observation period.
Test compounds were administered by intravenous or oral
routes approximately 10 min prior to administering the acetic
acid. The dose-response relationship for antinociceptive effects
during the acetic acid writhing test is assessed by plotting the
incidence of abdominal constrictions against dose of the test
compound. ED50 values are calculated using a four-parameter
logistic function.
(3) Ar ter ia l Blood P r essu r e a n d Hea r t Ra te Recor d in g
P r oced u r es. Male Sprague-Dawley rats weighing between
220 and 280 g (Simonson Laboratories, Gilroy, CA) were used.
Animals were acclimated to the laboratory enviroment for 5-7
days before entering the study. Approximately 2 h after
recovery from isoflurane anesthesia and surgery stress, arte-
rial blood pressure and heart rate (pulse rate) were recorded
using a pressure transducer (model BP-100; CB Sciences, Inc.,
Dover, NH) connected to a computerized data collection system
(MacLab, ADIstruments, Milford, MA). Rats were confined to
their home cages during recording but were otherwise unre-
strained. Animals were given intraarterial injection of 0.5-1
mL heparinized (50 IU/mL) saline, and their arterial lines were
connected to the recording system. Arterial blood pressure and
heart rate were recorded for at least 20 min until stable
baselines were obtained prior to test article administration.
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min. Arterial blood pressure and heart rate were continuously
monitored during and approximately 1 h post administration.
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and reduces the effects of focal ischemia in rats and mice. Stroke
(Dallas) 1995, 26, 1683-1689.
(4) P h a r m a cok in etics Stu d y. Three Wistar rats received
a 5 mg/kg bolus intravenous dose of compound 11 as a solution,
and serial plasma samples were collected at various times up
to 24 h postdose. Plasma samples were analyzed using direct
protein precipitation with acetonitrile, and compound 11 was
quantitated by Sciex LC/MS/MS system. A Betasil phenyl
column (2.1 mm × 12 cm) was used with a mobile phase of
acetonitrile:0.1% acetic acid (70:30, v/v).
Ack n ow led gm en t. The authors thank the Parke-
Davis Analytical Research for spectral and microanaly-
sis data. We acknowledge Dr. Thomas Malone for his
helpful discussion and Mr. Gregory Campbell for pre-
paring the cell culture for electrophysiology. We also
appreciate Dr. J ing Belfield for the literature search.
We thank Don J ohnson and Norman Colbry for running
catalytic hydrogenation experiments. Mr. Anthony Black-
burn is acknowledged for ClogP calculations.
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