Discovery of a 1-isopropyltetrahydroisoquinoline derivative as an orally active N-type calcium channel blocker for neuropathic pain

Article abstract of DOI:10.1016/j.bmc.2015.05.030

N-type calcium channel blockade is a promising therapeutic approach for the treatment of neuropathic pain. Starting from lead compound (S)-1, we focused our optimization efforts on potency for N-type calcium channel inhibition and improvement of CYP inhib

Full text of DOI:10.1016/j.bmc.2015.05.030

Bioorganic & Medicinal Chemistry 23 (2015) 4624–4637
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a 1-isopropyltetrahydroisoquinoline derivative as an
orally active N-type calcium channel blocker for neuropathic pain
a
a
a
a
Takashi Ogiyama a, , Koichi Yonezawa , Makoto Inoue , Toshihiro Watanabe , Yukihito Sugano ,
⇑
Takayasu Gotoh a, Tetsuo Kiso a, Akiko Koakutsu b, Shuichiro Kakimoto a, Jun-ichi Shishikura a
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Drug Discovery Research, Astellas Pharma Inc., 2-1-6 Kashima, Yodogawa-ku, Osaka 532-8514, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
N-type calcium channel blockade is a promising therapeutic approach for the treatment of neuropathic
pain. Starting from lead compound (S)-1, we focused our optimization efforts on potency for N-type calcium
channel inhibition and improvement of CYP inhibition profile. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-
(1R)-(1-isopropyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone oxalate ((R)-5r) was identified
as a novel orally active small-molecule N-type calcium channel inhibitor with reduced CYP inhibition
liability. Oral administration of (R)-5r improved mechanical allodynia in a spinal nerve ligation model of
neuropathic pain in rats with an ED50 value of 2.5 mg/kg.
Received 20 April 2015
Revised 19 May 2015
Accepted 20 May 2015
Available online 27 May 2015
Keywords:
N-type calcium channel
CaV 2.2
Ó 2015 Elsevier Ltd. All rights reserved.
Pain
CYP2D6
CYP3A4
1. Introduction
clinical efficacy of ziconotide, a potent and selective N-type cal-
cium channel blocker.3 Although the synthetic peptide ziconotide
N-type (Cav2.2) calcium channels are members of the voltage-
dependent calcium channel (VDCC) family, expressed on the
plasma membranes of excitable cells and control calcium influx
in response to membrane potential.1 The VDCC family regulates
various physiological functions, such as muscular contraction, neu-
ronal excitation, gene expression and hormone secretion. Calcium
channels are composed of multimeric protein complexes, named
has demonstrated marked clinical efficacy in treating severe and
chronic pain, the utilization of this drug has been limited, as it
requires intrathecal administration and has serious adverse effects
on the central nervous system (CNS).3 To overcome these draw-
backs, a large number of studies have been conducted to identify
small-molecule N-type calcium channel blockers as novel orally
active analgesics.4,5
We previously reported the novel tetrahydroisoquinoline (S)-1
as an orally active small-molecule N-type calcium channel
blocker.6 Notably, (S)-1 fully ameliorated mechanical allodynia in
a spinal nerve ligation (SNL) model of neuropathic pain in rats at
30 mg/kg po. However, compound (S)-1 also exhibited inhibitory
activity against both CYP2D6 and CYP3A4 (Table 1).
CYP2D6 and CYP3A4 play major roles in drug metabolism and
are involved in the metabolism of approximately 25% and over
50% of drugs in clinical use, respectively.7 CYP inhibition-mediated
drug–drug interactions (DDIs) might lead to serious adverse events
or a decrease in drug efficacy. Notably, the unwanted effects of
time-dependent CYP inhibition persist after discontinuation of
drug administration. Thus, these DDIs often pose a risk and are
extremely difficult to manage in clinical application.
a1,
a2d, b, and
c
subunits.1 The
a
1 subunit is believed to form
the ion-conducting pore and hence determine the fundamental
functions of the channel.1
N-type calcium channels are found abundantly in the presynap-
tic terminals of primary afferent nerves in the dorsal horn of the
spinal cord. They modulate excitatory neurotransmission and con-
trol the release of neurotransmitters, including pain-related amino
acids and neuropeptides, such as glutamate, calcitonin gene-
related peptide (CGRP), and substance P.1
Several groups have reported that mice deficient in the pore-
forming
a1B subunit of N-type calcium channels exhibit reduced
hyperalgesic and allodynic responses to thermal, chemical or
mechanical stimuli compared to wild type mice.2 The validity of
targeting N-type calcium channels is further established by the
Pregabalin and gabapentin, which are widely prescribed for the
treatment of neuropathic pain, have clinical advantages in terms of
DDIs compared to other first line drugs, such as tricyclic
⇑
Corresponding author. Tel.: +81 29 863 6683; fax: +81 29 852 5387.
E-mail address: takashi.ogiyama@astellas.com (T. Ogiyama).
http://dx.doi.org/10.1016/j.bmc.2015.05.030
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
4625
Table 1
In vitro pharmacological properties of (S)-1
b
Compound
N-type FLIPR IC50
(
lM)
CYP2D6 IC50
(l
M)
CYP3A4a preincubation time
0 min (reversible) 30 min (time-dependent)
cLogD7.4
OH
1
N
N
H
1.0
0.60
87%
67%
3.48
O
(S)-1
a
Residual activities (%) of HLM were evaluated using midazolam as a probe substrate. Details are described in Section 5.
cLogD values at pH 7.4 were calculated with ACD/PhysChem Batch (version 12.01).
b
antidepressants (TCAs) and serotonin and norepinephrine reuptake
inhibitors (SNRIs).8 To mitigate these risks in drug development
and improve convenience in clinical use, the minimization of inhi-
bitory activities against CYP2D6 and CYP3A4 in lead optimization
from (S)-1 is required.
Acylation of the tetrahydroisoquinoline 9, substitution with 1-
(aminomethyl)cyclohexanol and reductive removal of the pivaloyl
group yielded compound 5d.
3. Results and discussion
Here, we describe the design and synthesis of a novel series of
tetrahydroisoquinolines and the discovery of (R)-5r as an orally
active small-molecule N-type calcium channel blocker with
minimized liability of CYP inhibition.
Inhibitory activity against N-type calcium channels was evalu-
ated using the fluorometric imaging plate reader (FLIPR) calcium
imaging assay in IMR-32 human neuroblastoma cells.
Experiments were performed in the presence of nitrendipine to
block endogenous L-type calcium channels. Selected compounds
were assessed for inhibitory activity against CYP2D6 using a fluo-
rescence substrate. The reversible and time-dependent inhibitory
effects of selected compounds were also evaluated using midazo-
lam as a probe substrate to monitor changes in CYP3A4 activity.
The calculation of cLogD values at pH 7.4 was performed with
ACD/PhysChem Batch (version 12.01).12
Our prior investigation demonstrated that the 1-
(aminomethyl)hydroxylcyclohexyl group of compound (S)-1
played an important role in attenuating the blockade of hERG
channels while maintaining potency for N-type calcium channels
and good aqueous solubility.6 However, compound (S)-1 exhibited
inhibitory activity against both CYP2D6 and CYP3A4 (Table 1). The
tendency of highly lipophilic compounds to induce potent inhibi-
tory activities against CYP2D6 and CYP3A4 has also been
reported.13 To improve the CYP profile of lead compound (S)-1,
we focused on the 1-cyclohxyltetrahydroisoquinoline moiety,
which is the most lipophilic part of the molecule. We therefore
investigated the conversion of the cyclohexyl moiety, which is sub-
stituted at C-1 position of tetrahydroisoquinoline, to the less lipo-
philic groups (Table 2).
2. Chemistry
The preparation of the 1-substituted tetrahydroisoquinoline
derivatives 5a–5t is outlined in Scheme 1. Acylation of phenylethy-
lamines (2b, 2k–2n, 2p, and 2q) yielded the amides (3b, 3g, 3h,
3k–3n, 3p, and 3q). These amides (3b, 3g, 3h, 3k–3n, 3p, and 3q)
were cyclized utilizing traditional Bischler–Napieralski reaction
or a modified version9 and followed by reduction with sodium
borohydride to give tetrahydroisoquinolines (4b, 4g, 4h, 4k–4n,
4p, and 4q). Tetrahydroisoquinolines (4e, 4f, 4i, 4o, and 4r–4t)
were prepared as previously described.10,11 (S)-4j was obtained
from a commercial source. Acylation of tetrahydroisoquinolines
(4a, 4b, 4e–4m, 4o, 4p, and 4r–4t) with chloroacetyl chloride fol-
lowed by substitution with 1-(aminomethyl)cyclohexanol yielded
the tetrahydroisoquinoline derivatives (5a, 5b, 5e–5m, 5o, 5p,
and 5r–5t). Optically active (R)-5r was synthesized from tetrahy-
droisoquinoline (R)-4r, which was prepared via the diastereomeric
salt formation method using racemic 4r with (S)-mandelic acid.
(S)-5r was obtained from tetrahydroisoquinoline (S)-4r in a similar
manner. The absolute stereochemistry of (R)-4r was determined by
single-crystal X-ray crystallography as shown in Figure 1.
The 1-isopropyl-8-substituted tetrahydroisoquinolines (5n
and 5q) were also synthesized as shown in Scheme 1. To
selectively obtain the 8-substituted tetrahydroisoquinolines, 2-bro-
mophenylethylamines (2n and 2q) were employed as starting mate-
rials.11 2-Bromophenylethylamines (2n and 2q) were converted to
1-isopropyl-5-bromo-8-substituted tetrahydroisoquinolines (4n
and 4q) in a three-step sequence. Acylation of 1-isopropyl-5-
bromo-8-substituted tetrahydroisoquinolines (4n and 4q) with
chloroacetyl chloride followed by substitution and reductive
removal of the bromine gave the 1-isopropyl-8-substituted tetrahy-
droisoquinolines (5n and 5q).
Compounds 5c and 5d were synthesized as shown in Scheme 2.
A benzyl group was installed on tetrahydroisoquinoline 6 by reduc-
tive amination followed by nucleophilic addition with methyl
lithium to give the alcohol 7. Methylation of alcohol 7 and subse-
quent deprotection of N-benzyl group afforded the tetrahydrois-
pquinoline 4c. Tetrahydroisoquinoline 4c was converted to
compound 5c in a two-step sequence. Lithiation of N-Boc protected
tetrahydroispquinoline 8 followed by nucleophilic addition to ace-
tone generated the alcohol. In the presence of trifluoroacetic acid,
the alcohol was transformed to the tetrahydroispquinoline 9.10a
The 1-unsubstituted analogue 5a resulted in a significant loss of
N-type calcium channel inhibitory activity. Ether group substituted
analogues (5b and 5c) exhibited an approximately 5-fold decrease
in N-type calcium channel potency compared to that of compound
1. Incorporation of dimethylcarbinol (5d) led to a loss of potency
for N-type calcium channels. Given that hydrophilic substitution
at C-1 position of tetrahydroisoquinoline did not result in potent
inhibitory activity against N-type calcium channels, small
hydrophobic alkyl substituent, methyl group (5e) was introduced
but remained insufficient to exert potent inhibitory activity. In
contrast, hydrophobic acyclic alkyl substituents such as isopropyl
(5f), t-butyl (5g) and 2-ethylpropyl (5h) groups exhibited potent
inhibitory activities against
N-type calcium channels.
Hydrophobic cyclic analogues cyclopentyl (5i) and (S)-phenyl
((S)-5j) also provided high potency for N-type calcium channels.
A degree of lipophilicity at the C-1 position of tetrahydroisoquino-
line was therefore required to exert potent inhibitory activity
against N-type calcium channels. In this investigation, the 2-iso-
propyl group was the smallest hydrophobic alkyl substituent to
exhibit desirable potency for N-type calcium channels. In a fluoro-
metric assay for CYP2D6 inhibition, no compounds showed

4626
T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
R1
R1
R1
H
N
NH2
R3
a or b
3
4
R2
c or d or e, f
R2
NH
R2
O
5
R3
2b: R1, R2 = H
3b: R1, R2 = H, R3 = 4-tetrahydropyrany
3g: R1, R2 = H, R3 = tert-butyl
4b: R1, R2 = H, R3 = 4-tetrahydropyrany
4g: R1, R2 = H, R3 = tert-butyl
(for 3b, 3g, 3h)
2k: R1 = Me, R2 = H
2l: R1 = H, R2 = 3-Me
2m: R1 = H, R2 = 4-Me
2n: R1 = Br, R2 = 5-Me
2p: R1 = H, R2 = 4-F
2q: R1 = Br, R2 = 5-F
3h: R1, R2 = H, R3 = 1-ethylpropyl
3k: R1 = Me, R2 = H, R3 = isopropyl
3l: R1 = H, R2 = 3-Me, R3 = isopropyl
3m: R1 = H, R2 = 4-Me, R3 = isopropyl
3n: R1 = Br, R2 = 5-Me, R3 = isopropyl
3p: R1 = H, R2 = 4-F, R3 = isopropyl
3q: R1 = Br, R2 = 5-F, R3 = isopropyl
4h: R1, R2 = H, R3 = 1-ethylpropyl
4k: R1 = Me, R2 = H, R3 = isopropyl
4l: R1 = H, R2 = 6-Me, R3 = isopropyl
4m: R1 = H, R2 = 7-Me, R3 = isopropyl
4n: R1 = Br, R2 = 8-Me, R3 = isopropyl
4p: R1 = H, R2 = 7-F, R3 = isopropyl
4q: R1 = Br, R2 = 8-F, R3 = isopropyl
5
6
5
6
g, h
R1
R1
OH
NH
N
7
7
N
H
R2
R2
R3
R3
O
4a: R1, R2, R3 = H
5a: R1, R2, R3 = H
4b: R1, R2 = H, R3 = 4-tetrahydropyranyl
4e: R1, R2 = H, R3 = methyl
5b: R1, R2 = H, R3 = 4-tetrahydropyranyl
5e: R1, R2 = H, R3 = methyl
4f: R1, R2 = H, R3 = isopropyl
5f: R1, R2 = H, R3 = isopropyl
4g: R1, R2 = H, R3 = tert-butyl
5g: R1, R2 = H, R3 = tert-butyl
4h: R1, R2 = H, R3 = 1-ethylpropyl
4i: R1, R2 = H, R3 = cyclopentyl
5h: R1, R2 = H, R3 = 1-ethylpropyl
5i: R1, R2 = H, R3 = cyclopentyl
(S)-4j: R1, R2 = H, R3 = S-phenyl
4k: R1 = 5-Me, R2 = H, R3 = isopropyl
4l: R1 = 6-Me, R2 = H, R3 = isopropyl
4m: R1= 7-Me, R2 = H, R3 = isopropyl
4o: R1 = 6-F, R2 = H, R3 = isopropyl
4p: R1 = 7-F, R2 = H, R3 = isopropyl
4r: R1 = 6-MeO, R2 = H, R3 = isopropyl
4s: R1 = 7-MeO, R2 = H, R3 = isopropyl
4t: R1 = 8-MeO, R2 = H, R3 = isopropyl
(S)-5j: R1, R2 = H, R3 = S-phenyl
5k: R1 = 5-Me, R2 = H, R3 = isopropyl
5l: R1 = 6-Me, R2 = H, R3 = isopropyl
5m: R1= 7-Me, R2 = H, R3 = isopropyl
5o: R1 = 6-F, R2 = H, R3 = isopropyl
5p: R1 = 7-F, R2 = H, R3 = isopropyl
5r: R1 = 6-MeO, R2 = H, R3 = isopropyl
5s: R1 = 7-MeO, R2 = H, R3 = isopropyl
5t: R1 = 8-MeO, R2 = H, R3 = isopropyl
Br
OH
g, h, i
NH
N
N
8
8
H
O
R
R
4n: R = Me
4q: R = F
5n: R = Me
5q: R = F
Scheme 1. Reagents and conditions: (a) RCO2H, WSCD, HOBt, CH2Cl2, DMF, rt, 1 d; (b) RCOCl, satd NaHCO3, AcOEt, rt, 3 h; (c) polyphosphate ester, 120 °C, 2 h; (d) (i) FeCl3,
(COCl)2, 1,2-DCE, rt, 5 h (ii) H2SO4, MeOH, reflux, 12 h; (e) P2O5, POCl3, 120 °C, 8 h; (f) NaBH4, MeOH, rt, 1 h; (g) chloroacetyl chloride, K2CO3, H2O, AcOEt, rt, 1 h;
(h) 1-(aminomethyl)cyclohexanol, K2CO3, CH3CN, 70 °C, 3 d; (i) 10% Pd/C, H2, Et3N, EtOH, rt, 6 h.
improved inhibition liability over compound 1. In an assay for
CYP3A4 inhibition using midazolam, cyclopentyl and (S)-phenyl
substituted analogues (5i and (S)-5j) only exhibited an improved
time-dependent inhibition (TDI) profile over compound 1.
Reducing lipophilicity at the C-1 position of tetrahydroisoquino-
line, compound 5f exhibited notable improvements in both rever-
sible and time-dependent CYP3A4 inhibition profiles, whereas
methyl group substituted analogue 5e reduced CYP3A4 liability
despite having low lipophilicity. Notably, isopropyl (5f) main-
tained potent inhibitory activity against N-type calcium channels
with an improved CYP3A4 inhibition profile.
benzene ring of the tetrahydroisoquinoline moiety using 1-iso-
propyltetrahydroisoquinoline 5f (Table 3).
Compound 5l, which was substituted at the C-6 position of
tetrahydroisoquinoline, exhibited the most favorable potency for
N-type calcium channels of the compounds bearing a methyl group
(5k–5n). However, these transformations deteriorated reversible
inhibitory activity against CYP3A4 regardless of the position of
the methyl substituent (5k–5n). These results might be related
to an increase in lipophilicity of the compounds (clogD7.4 values
of 5k–5n: 2.70–2.94 vs 5f: 2.37). Substitution at the C-6 position
with a fluorine group (5o) was also more beneficial to N-type cal-
cium channel inhibition than substitution at other positions (5p
and 5q). Compound 5o exhibited potent inhibitory activity against
N-type calcium channels comparable to compound 5l.
Incorporation of a methoxy group maintained a satisfactory level
of inhibitory activity against N-type calcium channel inhibition
regardless of the position of substitution but did not enhance
potency (5r–5t). Regarding the TDI liability of CYP3A4, introduc-
tion of substituents at the C-6 position (5l, 5o, and 5r) maintained
a preferable profile, whereas introduction of substituents at the C-8
position (5n, 5q, and 5t) deteriorated the profile. Of the substituted
compounds, 5r afforded favorable overall CYP3A4 inhibition liabil-
ity with N-type calcium channel potency. Regarding CYP2D6
Conversion of the cyclohexyl moiety at C-1 position of tetrahy-
droisoquinoline in (S)-1 to the less lipophilic isopropyl moiety
improved the CYP3A4 inhibition profile while maintaining inhibi-
tory activity against N-type calcium channels. However, these con-
versions did not eliminate CYP2D6 inhibition liability. Many
CYP2D6 inhibitors are structurally characterized by the presence
of a basic nitrogen function and a planar aromatic ring.13 Several
studies regarding monoamine reuptake inhibitors, whose struc-
tures are typical CYP2D6 inhibitors as mentioned above, demon-
strated that small modifications of substituents on the benzene
moiety had
Therefore, we next investigated the effect of substituents on the
a
remarkable impact on inhibitory activity.14

T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
4627
the relative arrangement, induced by steric repulsion between
the isopropyl moiety and substituents introduced to the C-8 posi-
tion of tetrahydroisoquinoline, might not sufficiently reduce
CYP2D6 liability (e.g., compound 5n and 5t). In addition, the 6-
methoxy group of compound 5r appeared to have little effect on
the conformation of the compound. (ii) Our investigation demon-
strated that, whereas CYP3A4 inhibition liability exhibited partial
correlation with lipophilicity of a compound, CYP2D6 inhibition
liability had a relatively poor relationship. A more dramatic
decrease in lipophilicity might be required to improve the
CYP2D6 profile, as exemplified by research for CCR3 antago-
nists.13a,b However, this transformation would not allow the
compound to exert potent N-type calcium channel inhibition, as
demonstrated by the investigation of hydrophilic substitution of
the C-1 position of tetrahydroisoquinoline (5a–5e) and
introduction of substituents on the benzene ring of 1-cyclohexyl
tetrahydroisoquinoline.15 (iii) Improvement of compound 5r in
CYP2D6 liability appeared to be mainly due to the impairment of
specific interactions with the CYP2D6 enzyme with either or both
the methoxy group itself or the effect of this group on the
p bonds.
Given that introduction of the 6-methoxy moiety (5r) resulted in
moderate improvement and that introduction of other substituents
had minor effect on CYP2D6 inhibition liability, regardless of the
polarity or position of the substitution, the impairment effect of
substituents appears to be relatively limited.
Consequently, the possibility remains that conformational
changes, particularly those induced by a steric repulsion of two
more bulky substituents between the C-1 and C-8 positions of
tetrahydroisoquinoline, reducing CYP2D6 liability. However, com-
pounds with only bulky substituents at the C-1 position of tetrahy-
droisoquinoline (1, 5g–5i, and (S)-5j) exhibited potent inhibitory
activities against CYP2D6. Further, these compounds still exhibited
CYP3A4 inhibition liabilities probably due to their high lipophilicity.
We also prepared and characterized both enantiomers of com-
pound 5r. Interestingly, in vitro profiles were similar between
the enantiomers except for human liver microsomal (HLM) stabil-
ity (Table 4). Both (R)- and (S)-5r exhibited slight decreases in N-
type calcium channel inhibition compared to racemate 5r. This
result might be one of many exceptions to Pfeiffer’s rule.16 In addi-
tion, (R)-5r exhibited slightly weaker CYP2D6 inhibition than race-
mate 5r and (S)-5r.17 Regarding inhibitory activity against the
other four major cytochrome P450 isoforms (CYP1A2, 2C9, 2C19,
Figure 1. Molecular structure of (R)-4r, showing 60% probability displacement
elipsoids. Disorder present in the isopropyl group is removed for clarity.
inhibitory activity, only the 6-methoxy group-bearing compound
5r led to an improvement compared to 5f. Unexpectedly, introduc-
tion of substituents to the benzene ring of the tetrahydroisoquino-
line had only minor influence on CYP2D6 inhibition liability.
de Groot et al. investigated monoamine reuptake inhibitors and
rationalized their findings on the reduction of CYP2D6 liability via
small peripheral modifications with the following explanations: (i)
difference in structure and conformation (ii) difference in physico-
chemical properties (notably, lipophilicity) (iii) (impairment of)
specific interaction with residue of CYP2D6 in the active site.13c
Taken together, our results may be interpreted as follows. (i) As
the isopropyl moiety substituted at the C-1 position of tetrahy-
droisoquinoline is relatively small, the conformational changes in
c,d
a, b
N
NH
NH
Bn
HO
O
CO2Et
6
7
4c
OH
e, f
N
N
H
O
O
5c
OH
NH
O
g, h
e, f, i
N
N
O
N
H
O
O
OH
5d
8
9
Scheme 2. Reagents and conditions: (a) PhCHO, NaBH(OAc)3, AcOH, rt, 15 h; (b) MeLi, THF, ꢀ78 °C to 0 °C, 3 h; (c) NaH, MeI, THF, rt, 20 h; (d) 10% Pd/C, H2, MeOH, rt, 8 h; (e)
chloroacetyl chloride, satd NaHCO3, AcOEt, rt, 2 h; (f) 1-(aminomethyl)cyclohexanol, K2CO3, 1,4-dioxane, 50 °C, 2 h; (g) (i) t-BuLi, TMEDA, THF, ꢀ78 °C, 10 min (ii) acetone,
ꢀ78 °C, 1 h; (h) TFA, rt, 4 h; (i) DIBAH, CH2Cl2, ꢀ78 °C to 0 °C, 7 h.

4628
T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
Table 2
Effect of tetrahydroisoquinoline 1-substitutions on N-type calcium channels and CYP2D6 and 3A4 inhibitory activities
OH
1
N
N
H
R
O
b
Compound no.
R=
H
N-type FLIPR IC50
(l
M)
CYP2D6 IC50
(lM)
CYP3A4a preincubation time
0 min (reversible) 30 min (time-dependent)
cLogD7.4
1
0.77
8.0
0.78
N.T.c
86%
61%
3.48
1.10
5a
N.T.c
N.T.c
5b
5c
4.1
3.7
N.T.c
N.T.c
N.T.c
N.T.c
N.T.c
N.T.c
1.51
1.68
O
MeO
5d
5e
6.1
2.9
N.T.c
N.T.c
N.T.c
65%
N.T.c
33%
1.07
1.50
HO
Me
5f
1.1
<0.78
<0.78
<0.78
<0.39
92%
N.T.c
77%
89%
98%
N.T.c
59%
84%
2.37
2.78
3.39
2.95
5g
5h
5i
0.80
0.73
0.99
(S)-5j
1.0
<0.39
89%
78%
2.75
a
b
c
Residual activities (%) of HLM were evaluated using midazolam as a probe substrate. Details are described in Section 5.
cLogD values at pH 7.4 were calculated with ACD/PhysChem Batch (version 12.01).
Not tested.
and 3A4), (R)-5r exhibited little or no inhibitory activity. The HLM
stability of (R)-5r was approximately 2-fold superior to that of (S)-
5r. In addition, as might have been expected,6 (R)- and (S)-5r had
Table 3
Effect of tetrahydroisoquinoline substitutions on N-type calcium channels and
CYP2D6 and 3A4 inhibitory activities
no effect on hERG inhibition (IC50 >100
lM) and good aqueous sol-
5
6
ubility (>100 g/mL) at both pH 6.8 (the Japanese Pharmacopoeia
l
2nd fluid for the dissolution test) and pH 1.2 (the Japanese
Pharmacopoeia 1st fluid for the dissolution test). These results
prompted us to investigate in vivo efficacy.
R
OH
N
N
H
7
8
O
The antinociceptive effect of (R)-5r was examined using a for-
malin test in mice. Subcutaneous injection of formalin into a hind
paw elicited biphasic pain behavior. The first phase results were
due to the direct stimulation of nociceptive nerve terminals, while
the late phase results were due to a combination of peripheral
input and spinal cord sensitization. Intrathecal administration of
(R)-5r dose-dependently decreased the nociceptive behavior in
both the first and second phase of a formalin test (Fig. 2). No abnor-
mal behavior was observed in the test animals. (R)-5r has a more
potent analgesic effect on the second phase than on the first
(ED50 = 25.4 vs >100 nmol/body), which is consistent with previous
findings regarding nociceptive responses using formalin test in
b
R
N-type
FLIPR IC50 IC50
M) M)
CYP2D6
CYP3A4a preincubation
time
cLogD7.4
(compound
no.)
(
l
(l
0 min
(reversible) (time-
dependent)
30 min
H (5f)
1.1
1.4
0.85
1.4
1.6
0.89
1.3
1.5
1.3
1.6
1.4
<0.78
<0.78
<0.78
<0.78
<0.78
0.39
92%
68%
75%
72%
73%
86%
89%
100%
94%
N.T.c
72%
98%
87%
2.37
2.88
2.78
2.94
2.70
2.55
2.24
1.92
2.55
2.82
2.90
5-Me (5k)
6-Me (5l)
7-Me (5m)
8-Me (5n)
6-F (5o)
7-F (5p)
8-F (5q)
6-MeO (5r)
7-MeO (5s)
8-MeO (5t)
105%
103%
82%
101%
81%
0.68
a
1B subunit deficient mice.2,18 Therefore, (R)-5r is considered to
<0.78
1.8
85%
95%
induce an antinociceptive effect via N-type calcium channel block-
ade at a spinal level. In addition, orally administered (R)-5r exerted
potent antinociceptive activity (64% inhibition) in the second
phase of formalin test in mice at 100 mg/kg. In contrast, orally
administered (S)-5r showed only moderate antinociceptive activity
(34% inhibition). Considering that the HLM stability of (R)-5r was
superior to that of (S)-5r, the difference in the pharmacokinetic
N.T.c
<0.78
N.T.c
81%
a
Residual activities (%) of HLM were evaluated using midazolam as a probe
substrate. Details are described in Section 5.
b
cLogD values at pH 7.4 were calculated with ACD/PhysChem Batch (version
12.01).
c
Not tested.

T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
4629
Table 4
In vitro pharmacological properties of (R)-5r and (S)-5r
Compound N-type FLIPR IC50
Major CYP isoforms
HLM stability CL (mL/
min/kg)
(lM)
1A2 IC50
M)
2C9 IC50
M)
2C19 IC50
M)
2D6 IC50
M)
CYP3A4a preincubation time
30 min (time-
(
l
(
l
(
l
(l
0 min
(reversible)
dependent)
(R)-5r
(S)-5r
1.9
2.1
>50
>50
>50
>50
>50
2.9
2.0
99%
96%
92%
99%
101.1
187.8
N.T.b
a
Residual activities (%) of HLM were evaluated using midazolam as a probe substrate. Details are described in Section 5.
Not tested.
b
Figure 2. Effect of intrathecal bolus injection of compound (R)-5r on (A) acute (phase 1: 0–10 min) and (B) persistent (phase 2: 15–25 min) pain behavior in the formalin test
in mice. The duration of nociceptive behaviors (lifting/licking time of the hindpaw) induced by subcutaneous injection of 2% formalin (20 lL) into the plantar surface of the
hindpaw was measured. Each bar represents the mean SEM of the duration of nociceptive behaviors (n = 6). *P<0.05 and ***P<0.001, statistically significant compared with
vehicle-treated group (Dunnett’s test).
properties of these enantiomers might be reflected in their
antinociceptive activity.
was also consistent with previous findings regarding the threshold
for mechanical stimuli in an SNL model using 1B subunit deficient
a
Encouraged by these results, the oral analgesic effect of (R)-5r
was evaluated in an L5/L6 spinal nerve ligation (SNL) model of
neuropathic pain in rats.19 (R)-5r dose-dependently reduced
mechanical allodynia with oral administration from 1 to 10 mg/kg.
Oral administration of (R)-5r improved mechanical allodynia in a
rat SNL model with an ED50 value of 2.5 mg/kg (Fig. 3). This result
mice.2a Given the results of our formalin test, (R)-5r probably
exerted its effect via suppression of the central sensitization
process in the SNL model. The effect of (R)-5r on motor
coordination was assessed using an accelerating rotarod test in
rats. (R)-5r did not significantly decrease the time on the rod at
30 mg/kg po. In contrast, ziconotide had only a narrow margin
between the ED50 value and TD50 value, which represents the
dose of agent causing moderate to severe behavioral toxicity in
the rat SNL model.20 This class of N-type calcium channel blockers
such as (R)-5r are expected to exhibit efficacy without adverse
effects on the CNS in clinical use, unlike ziconotide.
4. Conclusion
In lead optimization efforts starting from tetrahydoroisoquino-
line (S)-1, we identified 2-{[(1-Hydroxycyclohexyl)methyl]amino}-
(1R)-(1-isopropyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)
ethanone oxalate ((R)-5r) as a novel orally active small-molecule
N-type calcium channel blocker. Reducing the lipophilicity by con-
version from a cyclohexyl moiety (S)-1 to isopropyl moiety (5f),
with substitution at the C-1 position of the tetrahydroisoquinoline
moiety, led to improvements in both CYP3A4 reversible and time-
dependent inhibition profiles. Further introduction of a 6-methoxy
group to the tetrahydroisoquinoline moiety resulted in approxi-
mately 5-fold attenuation of CYP2D6 inhibitory activity ((S)-1 vs
(R)-5r). Oral administration of (R)-5r exhibited marked efficacy in
rat SNL model of neuropathic pain with an ED50 value of 2.5 mg/kg.
Figure 3. Effect of compound (R)-5r on mechanical allodynia in SNL model rats.
Each bar represents the mean SEM withdrawal threshold of the hindpaw (n = 8).
#P<0.05, statistically significant compared with non-operated side paw in vehicle-
treated group (Normal) (Student’s t-test). *P<0.05, statistically significant compared
with operated side paw in vehicle-treated group (Dunnett’s test). Closed columns,
operated side paw in drug-treated and vehicle-treated group. Open column, non-
operated side paw in vehicle-treated group.

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T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
5.1.6. 3-Methyl-N-[2-(2-methylphenyl)ethyl]propanamide (3l)
5. Experimental section
5.1. Chemistry
Compound 3l was synthesized in a manner similar to that for
compound 3g. Compound 3l was obtained at a yield of 90% as a
pale yellow oil. 1H NMR (CDCl3): d 7.20 (1H, t, J = 7.3 Hz), 6.95–
7.08 (3H, m), 5.42 (1H, br s), 3.50 (2H, q, J = 6.8 Hz), 2.78 (2H, t,
J = 6.8 Hz), 2.33 (3H, s), 2.28 (1H, sep., J = 6.8 Hz), 1.12 (6H, d,
J = 6.8 Hz); FAB MS m/z 206 [M+H]+.
5.1.1. General
All reactions were carried out using commercially available
reagents and solvents without further purification. Column chro-
matography was performed using Shoko Scientific SI series silica
gel cartridge on a Shoko Scientific Purif-a
2. 1H NMR spectra were
5.1.7. 4-Methyl-N-[2-(2-methylphenyl)ethyl]propanamide (3m)
Compound 3m was synthesized in a manner similar to that for
compound 3g. Compound 3m was obtained at a yield of 60% as a
pale yellow oil. 1H NMR (CDCl3): d 7.05–7.15 (4H, m), 5.40 (1H,
br s), 3.49 (2H, q, J = 6.8 Hz), 2.77 (2H, t, J = 6.8 Hz), 2.33 (3H, s),
2.27 (1H, sep., J = 6.8 Hz) 1.11 (6H, d, J = 7.1 Hz); FAB MS m/z 206
[M+H]+.
recorded on a JNM-EX400 spectrometer. Chemical shifts are
expressed in d units (ppm) using tetramethylsilane as an internal
standard. Abbreviations used for the signal patterns are as follows:
s, singlet; d, doublet; t, triplet; q, quartet; sep., septet; m, multi-
plet; and br, broad. Mass spectra were recorded on a JEOL LX-
2000 or Waters ZQ-2000 mass spectrometer. Elemental analysis
was conducted using a Yanaco MT-5 microanalyzer. HPLC analysis
was performed using a Daicel OD-H or AD-RH chiral column on a
Hitachi HPLC system (L-7000 series), equipped with an UV source
(210 or 230 nm). Specific rotations were measured using a HORIBA
SEPA-300 polarimeter.
5.1.8. N-[2-(2-Bromo-5-methylphenyl)ethyl]-2-methylpropan-
amide (3n)
Compound 3n was synthesized in a manner similar to that for
compound 3g using 2n21 instead of 2b. Compound 3n was
obtained 83% yield as a red solid. 1H NMR (DMSO-d6): d 7.73–
7.89 (1H, m), 7.43 (1H, d, J = 8.4 Hz), 7.09 (1H, br s), 6.92–
7.01(1H, m), 3.26 (2H, q, J = 7.3 Hz), 2.79 (2H, t, J = 7.1 Hz), 2.31
(1H, sep., J = 6.9 Hz), 2.23 (3H, s), 0.97 (6H, d, J = 6.9 Hz); CI MS
m/z 284 [M]+.
5.1.2. N-(2-Phenylethyl)tetrahydro-2H-pyran-4-carboxamide
(3b)
To a solution of 2-phenylethylamine (2.91 mL) and tetrahydro-
2H-pyran-4-carboxylic acid (3.0 g) in dichloromethane (30 mL)
and N,N-dimethylformamide (15 mL) were added 1-(3-dimethy-
laminopropyl)-3-ethylcarbodiimide hydrochloride (4.42 g) and 1-
hydroxybenzotriazole (3.11 g). The mixture was stirred at room
temperature for 1 day. The reaction mixture was diluted with
water, extracted with chloroform, washed with brine, and dried
over magnesium sulfate. The resulting mixture was concentrated
in vacuo to give compound 3b (4.85 g, 90%) as a colorless solid.
1H NMR (CDCl3): d 7.11–7.39 (5H, m), 5.46 (1H, br s), 3.92–4.04
(1H, m), 3.53 (2H, q, J = 6.7 Hz), 3.37 (2H, dt, J = 6.7, 12.8 Hz),
2.82 (2H, t, J = 6.8 Hz), 2.26 (sep. J = 5.0 Hz), 1.61–1.83 (m, 4H);
APCI MS m/z 234 [M+H]+.
5.1.9. 4-Fluoro-N-[2-(2-methylphenyl)ethyl]propanamide (3p)
Compound 3p was synthesized in a manner similar to that for
compound 3g. Compound 3p was obtained at a yield of 99% as a
pale yellow solid. 1H NMR (DMSO-d6): d 7.76 (1H, br s), 7.16–
7.28 (2H, m), 7.04–7.14 (2H, m), 3.24 (2H, q, J = 7.3 Hz), 2.69 (2H,
t, J = 7.1 Hz), 2.30 (1H, sep., J = 6.8 Hz), 0.96 (6H, d, J = 6.8 Hz);
FAB MS m/z 210 [M+H]+.
5.1.10. N-[2-(2-Bromo-5-fluorophenyl)ethyl]-2-methylpropan-
amide (3q)
Compound 3q was synthesized in a manner similar to that for
compound 3g using 2q21 instead of 2b. Compound 3q was obtained
98% yield as a colorless solid. 1H NMR (DMSO-d6): d 7.82 (1H, br s),
7.61 (1H, dd, J = 5.4, 8.8 Hz), 7.15 (1H, dd, J = 3.1, 9.8 Hz), 7.05 (1H,
dt, J = 3.0, 8.4 Hz), 3.31 (2H, q, J = 6.8 Hz), 2.83 (2H, t, J = 7.0 Hz),
2.30 (1H, sep., J = 6.8 Hz), 0.96 (6H, d, J = 6.8 Hz); FAB MS m/z 288
[M+H]+.
5.1.3. 2,2-Dimethyl-N-(2-phenylethyl)propanamide (3g)
To a solution of 2-phenylethylamine (17 mL) and potassium car-
bonate (37 g) in ethyl acetate (80 mL) and water (150 mL) was added
dropwise a solution of 2,2-dimethylpropanoyl chloride (16.3 g) in
ethyl acetate (70 mL) at 4 °C. After stirring at room temperature for
1.5 h, the mixture was extracted with ethyl acetate, washed with
1 M aqueous hydrochloric acid, and dried over magnesium sulfate.
The resulting mixture was concentrated in vacuo to give compound
3 g (24.6 g, 89%) as a pale yellow solid. 1H NMR (DMSO-d6): d 7.50
(1H, br s), 7.24–7.31 (2H, m), 7.14–7.22 (3H, m), 3.20–3.29 (2H, m),
2.71 (2H, t, J = 7.1 Hz), 1.05 (9H, s); FAB MS m/z 206 [M+H]+.
5.1.11. 1-(Tetrahydro-2H-pyran-4-yl)-1,2,3,4-tetrahydroisoqui-
noline (4b)
Compound 3b (3.0 g) was combined with polyphosphate ester
(15 mL) and stirred at 120 °C for 2 h. The reaction mixture was
diluted with water, basified with ammonia, extracted with ethyl
acetate, washed with water and brine, and dried over magnesium
sulfate. The resulting mixture was concentrated in vacuo to obtain
5.1.4. 2-Ethyl-N-(2-phenylethyl)butanamide (3h)
Compound 3h was synthesized in a manner similar to that for
compound 3b. Compound 3h was obtained at a yield of 76% as a
colorless solid. 1H NMR (CDCl3): d 7.27–7.36 (2H, m), 7.17–7.26
(3H, m), 5.42 (1H, br s), 3.56 (2H, q, J = 6.8 Hz), 2.83 (2H, t,
J = 6.9 Hz), 1.71–1.81 (1H, m), 1.51–1.65 (2H, m), 1.37–1.49 (2H,
m), 0.85 (6H, t, J = 7.4 Hz); ESI MS m/z 220 [M+H]+.
1-(tetrahydro-2H-pyran-4-yl)-3,4-dihydroisoquinoline
(2.61 g,
94%), which was used in the next step without further purification.
APCI MS m/z 216 [M+H]+. To a solution of 1-(tetrahydro-2H-pyran-
4-yl)-3,4-dihydroisoquinoline (2.61 g) in methanol (15 mL) was
added sodium borohydride (458 mg) at 4 °C. The reaction mixture
was stirred at room temperature for 1 h. The reaction was
quenched by the addition of water, extracted with ethyl acetate,
washed with water and brine, and dried over magnesium sulfate.
The resulting mixture was concentrated in vacuo to give com-
pound 4b (1.61 g, 61%) as a brown solid. 1H NMR (CDCl3): d
7.03–7.36 (4H, m), 3.82–4.19 (2H, m), 3.18–3.58 (2H, m), 2.56–
3.07 (2H, m), 1.39–1.85 (3H, m), 1.26–1.38 (1H, m); APCI MS m/z
218 [M+H]+.
5.1.5. 2-Methyl-N-[2-(2-methylphenyl)ethyl]propanamide (3k)
Compound 3k was synthesized in a manner similar to that for
compound 3g. Compound 3k was obtained at a quantitative yield
as a pale brown oil. 1H NMR (DMSO-d6): d 7.75–7.88 (1H, br),
7.05–7.17 (3H, m), 3.33 (3H, s), 3.20 (2H, q, J = 8.0 Hz), 2.69 (2H,
t, J = 8.0 Hz), 2.22–2.37 (4H, m), 0.98 (6H, d, J = 4.0 Hz); EI MS m/z
205 [M]+.

T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
4631
5.1.12. 1-tert-Butyl-1,2,3,4-tetrahydroisoquinoline hydrochlo-
ride (4g)
compound 4k (4.19 g, 96%) as a colorless solid. 1H NMR (DMSO-
d6): d 9.85 (1H, br s), 8.77 (1H, br s), 7.02–7.26 (3H, m), 4.36–
4.45 (1H, m), 3.39–3.63 (1H, m), 3.08–3.21 (1H, m), 2.78–2.98
(2H, m), 2.34–2.55 (1H, m), 2.22 (3H, s), 1.09 (3H, d, J = 7.1 Hz),
0.84 (3H, d, J = 6.9 Hz); FAB MS m/z 190 [M+H]+.
To a solution of compound 3g (10.0 g) in toluene (100 mL) was
added phosphorus pentoxide (13.83 g) and stirred at 70 °C.
Phosphoryl chloride (7 mL) was then added to the mixture,
warmed to 120 °C and stirred for 8 h. After cooling to room tem-
perature, the supernatant was removed. 20% aqueous sodium
hydroxide was added to the residue, extracted with ethyl acetate,
washed with brine, and dried over magnesium sulfate. The mixture
was concentrated in vacuo to obtain 1-tert-butyl-3,4-dihydroiso-
quinoline (4.9 g, 53%), which was used in the next step without
further purification. EI MS: m/z: 186 [MꢀH]+. To a solution of
1-tert-butyl-3,4-dihydroisoquinoline (4.83 g) in ethanol (40 mL)
and toluene (5 mL) was added sodium borohydride (1.10 g) at
4 °C. After stirring at room temperature for 1 h, 1 M aqueous
hydrochloric acid (20 mL) was added to the mixture and stirred
at room temperature for 30 min. The resulting mixture was basi-
fied with 1 M aqueous sodium hydroxide (40 mL), extracted with
ethyl acetate, dried over magnesium sulfate, and concentrated in
vacuo. The residue was dissolved in ethyl acetate (20 mL) and ether
(60 mL) and 4 M hydrogen chloride in ethyl acetate (7 mL) was
then added to the mixture. The precipitate was filtered off to
give compound 4g (5.82 g, 23%) as a pale yellow solid. 1H NMR
(DMSO-d6): d 10.5 (1H, br s), 8.26 (1H, br s), 7.23–7.32 (4H, m),
4.31 (1H, br s), 3.48–3.56 (1H, m), 2.69–2.91 (3H, m), 1.00 (9H,
s); ESI MS m/z 190 [M+H]+.
5.1.15. 1-Isopropyl-6-methyl-1,2,3,4-tetrahydroisoquinoline
hydrochloride (4l)
Compound 4l was synthesized in a manner similar to that for
compound 4g. Compound 4l was obtained at a yield of 88% yield
as a colorless solid. 1H NMR (DMSO-d6): d 9.85 (1H, br s), 8.72
(1H, br s), 7.17 (1H, d, J = 8.1 Hz), 7.08 (1H, d, J = 8.1 Hz), 7.03
(1H, s), 4.37 (1H, br s), 3.38–3.47 (1H, m), 2.98–3.16 (2H, m),
2.80–2.91 (1H, m), 2.40–2.48 (1H, m), 2.27 (3H, s), 1.09 (3H, d,
J = 6.9 Hz), 0.84 (3H, d, J = 7.0 Hz); CI MS m/z 190 [M+H]+.
5.1.16. 1-Isopropyl-7-methyl-1,2,3,4-tetrahydroisoquinoline
hydrochloride (4m)
Compound 4m was synthesized in a manner similar to that for
compound 4g. Compound 4m was obtained at a yield of 74% as a
colorless solid.1H NMR (DMSO-d6) d 9.89 (1H, br s), 8.72 (1H, br
s), 6.82–7.26 (3H, m), 4.38 (1H, br s), 3.38–3.46 (1H, m), 2.97–
3.14 (2H, m), 2.79–2.90 (1H, m), 2.41–2.52 (1H, m), 2.28 (3H, s),
1.10 (3H, d, J = 7.1 Hz), 0.84 (3H, d, J = 7.0 Hz); CI MS m/z 190
[M+H]+.
5.1.17. 5-Bromo-1-isopropyl-8-methyl-1,2,3,4-tetrahydroiso-
quinoline (4n)
5.1.13. 1-(Pentan-3-yl)-1,2,3,4-tetrahydroisoquinoline hydro-
chloride (4h)
Compound 4n was synthesized in a manner similar to that for
compound 4g. Compound 4n was obtained at a yield of 8% as a
brown oil. 1H NMR (CDCl3): d 7.32 (1H, d, J = 8.1 Hz), 6.87 (1H, d,
J = 8.1 Hz), 4.00 (1H, d, J = 5.9 Hz), 3.26–3.39 (1H, m), 2.83–3.01
(2H, m), 2.56–2.69 (1H, m), 2.15–2.37 (4H, m), 2.02–2.13 (1H,
m), 0.98 (3H, d, J = 6.8 Hz), 0.83 (3H, d, J = 6.8 Hz); CI MS m/z 268
[M]+.
Compound 4h was synthesized in a manner similar to that for
compound 4g. Compound 4h was obtained at a yield of 80% as a
colorless solid. 1H NMR (DMSO-d6): d 9.66 (1H, br s), 8.42 (1H, br
s), 7.18–7.34 (4H, m), 4.61 (1H, br s), 3.36–3.46 (1H, m) 3.03–
3.25 (2H, m), 2.85–2.96 (1H, m), 1.95 (1H, br s), 1.22–1.60 (3H,
m), 1.07–1.19 (1H, m), 1.00 (3H, t, J = 7.6 Hz), 0.80 (3H, t,
J = 7.5 Hz); ESI MS m/z 204 [M+H]+.
5.1.18. 7-Fluoro-1-isopropyl-1,2,3,4-tetrahydroisoquinoline
hydrochloride (4p)
5.1.14. 1-Isopropyl-5-methyl-1,2,3,4-tetrahydroisoquinoline
hydrochloride (4k)
Compound 4p was synthesized in a manner similar to that for
compound 4g. Compound 4p was obtained at a yield of 12% as a
pale yellow solid.1H NMR (DMSO-d6): d 10.07 (1H, br s), 8.90
(1H, br s), 7.25–7.34 (1H, m), 7.07–7.24 (1H, m), 4.46 (1H, br s),
3.43 (1H, br s), 2.82–3.15 (4H, m), 1.12 (3H, d, J = 7.1 Hz), 0.83
(3H, d, J = 7.1 Hz); FAB MS m/z 194 [M+H]+.
To a solution of compound 3k (7.64 g) in dichloromethane
(50 mL) was added oxalyl chloride (5.0 mL) at 4 °C. After stirring
at room temperature for 1.5 h, ferric chloride (6.7 g) was added
to the mixture at 4 °C. The mixture was then stirred at room
temperature for 4.5 h. The reaction was quenched by the addition
of 1 M aqueous hydrochloric acid, extracted with chloroform,
dried over magnesium sulfate and concentrated in vacuo. The
residue was washed with ether, and the precipitate was filtered
off to obtain 10b-isopropyl-7-methyl-6,10b-dihydro-5H-[1,3]oxa-
zolo[2,3-a]isoquinoline-2,3-dione (5.25 g, 55%) as a colorless solid.
FAB MS m/z 260 [M+H]+. To a solution of concentrated sulfuric acid
(3.0 mL) in methanol (30 mL) was added 10b-isopropyl-7-methyl-
6,10b-dihydro-5H-[1,3]oxazolo[2,3-a]isoquinoline-2,3-dione (5.22 g)
and the mixture was heated under reflux for 4 days. The mixture
was concentrated in vacuo, diluted with water and washed with
ether. The aqueous phase was neutralized with aqueous ammonia,
extracted with toluene, dried over magnesium sulfate and concen-
trated in vacuo to obtain 1-isopropyl-5-methyl-3,4-dihydroiso-
quinoline (3.66 g, 97%) as a brown oil. FAB MS m/z 188 [M+H]+.
5.1.19. 5-Bromo-8-fluoro-1-isopropyl-1,2,3,4-
tetrahydroisoquinoline hydrochloride (4s)
Compound 4s was synthesized in a manner similar to that for
compound 4k. Compound 4s was obtained at a yield of 7% as a col-
orless solid. 1H NMR (DMSO-d6): d 9.95 (1H, br s), 9.09 (1H, br s),
7.71 (1H, dd, J = 5.1, 8.8 Hz), 7.20 (1H, t, J = 9.3 Hz), 4.52 (1H, d,
J = 6.9 Hz), 3.39–3.51 (1H, m), 3.22–3.38 (1H, m), 2.89–2.06 (2H,
m), 2.19–2.35 (1H, m), 1.00 (3H, d, J = 6.8 Hz), 0.94 (3H, dd,
J = 2.5, 6.8 Hz); ESI MS m/z: 272 [M+H]+.
5.1.20. 1-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclo-
hexyl)methyl]amino}ethanone oxalate (5a)
To
a solution of 1,2,3,4-tetrahydroisoquinoline (2.0 g) in
To
a
solution of 1-isopropyl-5-methyl-3,4-dihydroisoquinoline
toluene (10 mL) and water (10 mL) was added potassium carbon-
ate (3.2 g) and chloroacetyl chloride (1.4 mL) at 4 °C. The reaction
mixture was stirred at room temperature for 23 h and then
diluted with water, extracted with ethyl acetate, washed with
1 M aqueous hydrochloric acid and brine, dried over magnesium
sulfate, and concentrated in vacuo to give 2-chloro-1-(3,4-dihy-
droisoquinolin-2(1H)-yl)ethanone (2.7 g, 86%) as a pale yellow
solid. EI MS m/z 209 [M]+. To a solution of 2-chloro-1-(3,4-
(3.61 g) in ethanol (40 mL) and toluene (10 mL) was added sodium
borohydride (0.80 g) at 4 °C. After stirring at room temperature for
14 h, the reaction was quenched by the addition of 1 M aqueous
hydrochloric acid. The mixture was basified with 1 M aqueous
sodium hydroxide, extracted with toluene, and dried over magne-
sium sulfate. 4 M hydrogen chloride in ethyl acetate (6 mL) was
added to the mixture. The precipitate was filtered off to give

4632
T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
dihydroisoquinolin-2(1H)-yl)ethanone (0.35 g) in acetonitrile
(5 mL) were added 1-(aminomethyl)cyclohexanol hydrochloride
(0.7 g) and potassium carbonate (0.7 g). The mixture was stirred
at 70 °C for 3 days and then diluted with water, extracted with
ethyl acetate, washed with brine, dried over magnesium sulfate,
and concentrated in vacuo. The residue was purified by column
chromatography, eluting with chloroform–methanol, and dis-
solved in 2-propanol (4 mL). A solution of oxalic acid (54 mg)
in ether (10 mL) was added to the solution. The precipitate was
filtered off to obtain compound 5a (194 mg, 30%) as a colorless
solid. 1H NMR (DMSO-d6): d 7.62 (1H, br s), 7.13–7.27 (4H, m),
4.66 (1H, s), 4.60 (1H, s), 4.03–4.14 (2H, m), 3.73 (1H, t,
J = 6.1 Hz), 3.60 (1H, t, J = 6.0 Hz), 2.86–2.95 (3H, m), 2.81 (1H,
t, J = 6.0 Hz), 1.18–1.64 (11H, m); FAB MS m/z 303 [M+H]+.
5.1.25. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-[1-(pentan-
3-yl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanone oxalate (5h)
Compound 5h was synthesized in a manner similar to that for
compound 5a. Compound 5h was obtained at a yield of 50% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.13–7.31 (4H, m), 5.32 (major
rotamer 1H, d, J = 9.4 Hz), 4.43 (minor rotamer 1H, d, J = 9.9 Hz),
3.84–4.19 (2H, m), 3.55–3.72 (2H, m), 3.38 (minor rotamer 1H, q,
J = 6.9 Hz), 2.78–3.05 (4H, m), 1.13–1.77 (14H, m), 0.72–0.93 (6H,
m); FAB MS m/z 373 [M+H]+.
5.1.26. 1-(1-Cyclopentyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-
{[(1-hydroxycyclohexyl)methyl]amino}ethanone oxalate (5i)
Compound 5i was synthesized in a manner similar to that for
compound 5a. Compound 5i was obtained at a yield of 58% as a col-
orless solid. 1H NMR (DMSO-d6): This compound exists as a pair of
rotamers at room temperature. d 7.63 (1H, br s), 7.09–7.26 (9H, m),
5.24 (major rotamer 1H, d, J = 10.1 Hz), 4.49 (minor rotamer 1H, d,
J = 9.8 Hz), 4.14 (major rotamer 1H, d, J = 10.1 Hz), 3.95–4.08 (1H,
m), 3.55–3.74 (major rotamer 2H, m), 3.27–3.40 (minor rotamer
1H, m), 2.78–3.05 (4H, m), 2.07–2.31 (1H, m), 1.15–1.79 (19H,
m); FAB MS m/z 371 [M+H]+.
5.1.21. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-[1-(tetra-
hydro-2H-pyran-4-yl)-3,4-dihydroisoquinolin-2(1H)-
yl]ethanone oxalate (5b)
Compound 5b was synthesized in a manner similar to that for
compound 5a. Compound 5b was obtained at a yield of 44% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a
pair of rotamers at room temperature. d 7.12–7.27 (4H, m),
5.14 (major rotamer 1H, t, J = 9.7 Hz), 4.39 (minor rotamer 1H,
d, J = 9.8 Hz), 4.21–4.30 (1H, m), 4.14 (major rotamer 1H, d,
J = 16.2 Hz), 3.30–4.07 (7H, m), 3.07–3.28 (2H, m), 2.82–3.03
(4H, m), 1.80–2.00 (1H, m), 1.19–1.65 (15H, m); FAB MS m/z
387 [M+H]+.
5.1.27. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-[(1S)-1-phe-
nyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone oxalate ((S)-5j)
Compound (S)-5j was synthesized in a manner similar to that
for compound 5a. Compound (S)-5j was obtained at a yield of
36% as a colorless solid. 1H NMR (DMSO-d6): This compound exists
as a pair of rotamers at room temperature. d 7.14–7.42 (9H, m),
6.66 (major rotamer 1H, s), 6.16 (minor rotamer 1H, s), 4.34 (minor
rotamer 1H, d, J = 16.2 Hz), 4.19 (major rotamer 1H, d, J = 16.2 Hz),
3.99–4.13 (1H, m), 3.84 (minor rotamer 1H, d, J = 16.2 Hz), 3.41–
3.70 (2H, m), 2.73–3.08 (5H, m), 1.18–1.65 (11H, m); ESI MS m/z
379 [M+H]+; Anal. Calcd for C24H30N2O2ꢁC2H2O4: C, 66.65; H,
5.1.22. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-methyl-
3,4-dihydroisoquinolin-2(1H)-yl)ethanone oxalate (5e)
Compound 5e was synthesized in a manner similar to that for
compound 5a. Compound 5e was obtained at a yield of 8% as a col-
orless solid. 1H NMR (DMSO-d6): This compound exists as a pair of
rotamers at room temperature. d 7.11–7.29 (4H, m), 5.46 (major
rotamer 1H, q, J = 6.8 Hz), 4.96 (minor rotamer 1H, q, J = 6.7 Hz),
4.38–4.47 (minor rotamer 1H, m), 3.97–4.27 (2H, m), 3.62–3.73
(major rotamer 1H, m), 3.44–3.55 (major rotamer 1H, m), 3.10–
3.21 (minor rotamer 1H, m), 2.69–3.01 (4H, m), 1.18–1.65 (13H,
m); FAB MS m/z 317 [M+H]+.
26
6.88; N, 5.98. Found: C, 66.63; H, 6.88; N, 5.94.
[a]
D
+129.6°(c 0.1, MeOH).
5.1.28. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-5-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5k)
5.1.23. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-3,4-dihydroisoquinolin-2(1H)-yl)ethanone oxalate (5f)
Compound 5f was synthesized in a manner similar to that for
compound 5a. Compound 5f was obtained at a yield of 20% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.12–7.27 (4H, m), 5.09 (major
rotamer 1H, d, J = 9.3 Hz), 4.31 (minor rotamer 1H, d, J = 9.4 Hz),
4.18–4.27 (minor rotamer 1H, m), 4.12 (major rotamer 1H, d,
J = 16.2 Hz), 3.94–4.04 (1H, m), 3.64–3.74 (major rotamer 1H, m),
3.53–3.63 (major rotamer 1H, m), 3.32–3.42 (minor rotamer 1H,
m), 2.80–3.02 (4H, m), 1.92–2.10 (1H, m), 1.17–1.65 (10H, m),
0.78–1.02 (6H, m); FAB MS m/z 345 [M+H]+; Anal. Calcd for
Compound 5k was synthesized in a manner similar to that for
compound 5a. Compound 5k was obtained at a yield of 68% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.03–7.12 (3H, m), 5.06 (major
rotamer 1H, d, J = 9.4 Hz), 4.37–4.47 (minor rotamer 1H, m), 4.27
(minor rotamer 1H, d, J = 9.4 Hz), 4.16 (major rotamer 1H, d,
J = 16.2 Hz), 3.91–4.08 (1H, m), 3.58–3.75 (2H, m), 3.25–3.36
(minor rotamer 1H, m), 2.83–2.98 (1H, m), 2.72–2.82 (3H, m),
2.21 (major rotamer 3H, s), 2.19 (minor rotamer 3H, s), 1.92–2.11
(1H, m), 1.16–1.67 (10H, m), 0.82–1.01 (6H, m); FAB MS m/z 359
[M+H]+.
C
21H32N2O2ꢁC2H2O4: C, 63.57; H, 7.89; N, 6.45. Found: C, 63.44;
5.1.29. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-6-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5l)
H, 7.92; N, 6.49.
5.1.24. 1-(1-tert-Butyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-
hydroxycyclohexyl)methyl]amino}ethanone oxalate (5g)
Compound 5g was synthesized in a manner similar to that for
compound 5a. Compound 5g was obtained at a yield of 20% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.12–7.31 (4H, m), 5.33 (major
rotamer 1H, s), 4.48–4.59 (minor rotamer 1H, m), 4.46 (minor rota-
mer 1H, s), 4.08 (2H, q, J = 11.7 Hz), 3.62–3.43 (2H, m), 3.30–3.43
(minor rotamer 1H, m), 2.76–3.08 (4H, m), 1.19–1.66 (11H, m),
0.98 (9H, s), 0.94 (9H, s); FAB MS m/z 359 [M+H]+.
Compound 5l was synthesized in a manner similar to that for
compound 5a. Compound 5l was obtained at a yield of 66% as a col-
orless solid. 1H NMR (DMSO-d6): This compound exists as a pair of
rotamers at room temperature. d 6.95–7.11 (3H, m), 5.04 (major
rotamer 1H, d, J = 9.2 Hz), 4.26 (minor rotamer 1H, d, J = 9.6 Hz),
4.17–4.24 (minor rotamer 1H, m), 4.14 (major rotamer 1H, d,
J = 16.2 Hz), 3.95–4.05 (1H, m), 3.51–3.71 (2H, m), 3.29–3.38
(minor rotamer 1H, m), 2.77–2.98 (4H, m), 2.26 (3H, s), 1.88–
2.08 (1H, m), 1.15–1.68 (10H, m), 0.82–1.00 (6H, m); FAB MS m/z
359 [M+H]+.

T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
4633
5.1.30. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-7-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5m)
3.50–3.70 (major rotamer 2H, m), 3.28–3.39 (minor rotamer 1H,
m), 2.79–3.00 (4H, m), 1.88–2.06 (1H, m), 1.18–1.66 (10H, m),
0.81–1.07 (6H, m); HPLC (Chiralpak OD-H [0.46 cm I.D. ꢂ 25 cm],
hexane/ethanol/diethylamine = 90:10:0.1 flow rate 0.5 mL/min,
column temp: 40 °C UV: 230 nm): retention time: tr = 14.96 min
(major), ts = 11.76 min (minor); 98.4% ee. FAB MS m/z 375
Compound 5m was synthesized in a manner similar to that for
compound 5a. Compound 5m was obtained at a yield of 44% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 6.97–7.12 (3H, m), 5.04 (major
rotamer 1H, d, J = 9.2 Hz), 4.25 (minor rotamer 1H, d, J = 9.5 Hz),
4.15–4.22 (minor rotamer 1H, m), 4.11 (major rotamer 1H, d,
J = 16.2 Hz), 3.92–4.07 (1H, m), 3.50–3.71 (major rotamer 2H, m),
3.29–3.40 (minor rotamer 1H, m), 2.77–2.97 (4H, m), 2.27 (major
rotamer 3H, s), 2.28 (minor rotamer 3H, s), 1.90–2.12 (1H, m),
1.18–1.65 (10H, m), 0.83–0.99 (6H, m); FAB MS m/z 359 [M+H]+.
[M+H]+; Anal. Calcd for C22H34N2O3ꢁC2H2O4: C, 62.05; H, 7.81; N,
26
6.03. Found: C, 61.80; H, 7.83; N, 5.96. [
a]
+31.8°(c 0.1, MeOH).
D
(R)-4r was prepared via the following procedure: To a solution of
4r (12.7 g) in 2-propanol (150 mL) was added (2S)-mandelic acid
(9.20 g) and a small portion of the seed crystal. After stirring for
30 min, the precipitate was filtered off to obtain rough crystal
(75% ee). Further recrystallization using 2-propanol (150 mL) was
repeated three times to furnish enantiomerically pure (1R)-iso-
propyl-6-methoxy-1,2,3,4-tetrahydroisoquinoline (2S)-mandelate
(6.11 g, 28%, >98% ee) as a colorless solid. 1H NMR (DMSO-d6): d
7.37 (2H, d, J = 7.4 Hz), 7.26 (2H, t, J = 7.7 Hz), 7.18 (1H, t,
J = 7.3 Hz), 7.13 (1H, d, J = 8.6 Hz), 6.78 (1H, dd, J = 2.7, 8.6 Hz),
6.72 (1H, d, J = 2.5 Hz), 4.68 (1H, s), 4.13 (1H, d, J = 4.0 Hz), 3.73
(3H, s), 3.23–3.35 (1H, m), 2.82–3.01 (2H, m), 2.67–2.79 (1H, m),
2.24–2.37 (1H, m), 0.99 (3H, d, J = 7.0 Hz), 0.73 (3H, d, J = 6.8 Hz);
HPLC (Chiralpak AD-RH [0.46 cm I.D. ꢂ 15 cm], acetonitrile:
20 mM borate buffer (pH 9.0) = 40/60 flow rate 0.5 mL/min, col-
umn temp: 40 °C UV: 210 nm): retention time: tr = 18.31 min
(major), ts = 14.30 min (minor); >98% ee. FAB MS m/z 206 [M+H]+.
5.1.31. 1-(6-Fluoro-1-isopropyl-3,4-dihydroisoquinolin-2(1H)-
yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone oxalate
(5o)
Compound 5o was synthesized in a manner similar to that for
compound 5a. Compound 5o was obtained at a yield of 62% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.25 (1H, dd, J = 5.9, 8.5 Hz),
6.98–7.12 (2H, m), 5.11 (major rotamer 1H, d, J = 9.3 Hz), 4.36
(minor rotamer 1H, d, J = 9.4 Hz), 4.18–4.26 (minor rotamer 1H,
m), 4.13 (major rotamer 1H, d, J = 16.3 Hz), 3.97–4.06 (1H, m),
3.55–3.71 (2H, m), 3.29–3.38 (minor rotamer 1H, m), 2.83–3.02
(4H, m), 1.90–2.09 (1H, m), 1.16–1.64 (10H, m), 0.80–0.98 (6H,
m); FAB MS m/z 363 [M+H]+.
5.1.35. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-(1S)-(1-isopr-
opyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate ((S)-5r)
5.1.32. 1-(7-Fluoro-1-isopropyl-3,4-dihydroisoquinolin-2(1H)-
yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone oxalate
(5t)
Compound (S)-5r was synthesized in a manner similar to that
for compound 5r, except that (S)-4r was used instead of 4r.
Compound (S)-5r was obtained at a yield of 70% as a colorless solid.
1H NMR (DMSO-d6): This compound exists as a pair of rotamers at
room temperature. d 7.04–7.14 (1H, m), 6.72–6.82 (2H, m), 5.03
(major rotamer 1H, d, J = 9.3 Hz), 4.25 (minor rotamer 1H, d,
J = 9.4 Hz), 4.06–4.23 (1H, m), 3.93–4.05 (1H, m), 3.73 (3H, s),
3.51–3.70 (major rotamer 2H, m), 3.28–3.38 (minor rotamer 1H,
m), 2.79–3.00 (4H, m), 1.88–2.07 (1H, m), 1.18–1.65 (10H, m),
0.82–1.07 (6H, m); HPLC (Chiralpak OD-H [0.46 cm I.D. ꢂ 25 cm],
hexane/ethanol/diethylamine = 90:10:0.1 flow rate 0.5 mL/min,
column temp: 40 °C UV: 230 nm): retention time: ts = 11.76 min
(major), tr = 14.96 min (minor). FAB MS m/z 375 [M+H]+; Anal.
Compound 5t was synthesized in a manner similar to that for
compound 5a. Compound 5t was obtained at a yield of 53% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.02–7.36 (3H, m), 5.14 (major
rotamer 1H, d, J = 9.2 Hz), 4.38 (minor rotamer 1H, d, J = 9.4 Hz),
4.09–4.26 (1H, m), 3.96–4.05 (1H, m), 3.54–3.71 (major rotamer
2H, m), 3.30–3.44 (minor rotamer 1H, m), 2.80–3.00 (4H, m),
1.94–2.12 (1H, m), 1.18–1.63 (10H, m), 0.81–0.99 (6H, m); FAB
MS m/z 363 [M+H]+.
5.1.33. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5r)
Calcd for C22H34N2O3ꢁC2H2O4: C, 62.05; H, 7.81; N, 6.03. Found: C,
23
61.83; H, 7.86; N, 6.00. [
a]
ꢀ24.3°(c 0.1, MeOH).
D
Compound 5r was synthesized in a manner similar to that for
compound 5a. Compound 5r was obtained at a yield of 60% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.07–7.15 (1H, m), 6.71–6.83
(2H, m), 5.03 (major rotamer 1H, d, J = 9.3 Hz), 4.54 (minor rotamer
1H, d, J = 9.7 Hz), 4.08–4.22 (1H, m), 3.95–4.05 (1H, m), 3.73 (3H, s),
3.51–3.70 (major rotamer 2H, m), 3.29–3.38 (minor rotamer 1H,
m), 2.82–2.98 (4H, m), 1.88–2.04 (1H, m), 1.19–1.63 (10H, m),
0.83–0.97 (6H, m); FAB MS m/z 375 [M+H]+; Anal. Calcd for
(S)-4r was prepared in a manner similar to that for (R)-4r,
except that (2R)-mandelic acid was used instead of (2S)-mandelic
acid. Compound (S)-4r was obtained at a yield of 29% as a colorless
solid. 1H NMR (DMSO-d6): d 7.37 (2H, d, J = 7.1 Hz), 7.24–7.28 (2H,
m), 7.17–7.21 (1H, m), 7.13 (1H, d, J = 8.6 Hz), 6.78 (1H, dd, J = 2.8,
8.7 Hz), 6.71 (1H, d, J = 2.6 Hz), 4.70 (1H, s), 4.08 (1H, d, J = 4.1 Hz),
3.72 (3H, s), 3.24–3.30 (1H, m), 2.83–2.97 (2H, m), 2.68–2.75 (1H,
m), 2.26–2.34 (1H, m), 1.00 (3H, d, J = 7.0 Hz), 0.72 (3H, d,
J = 6.9 Hz); HPLC (Chiralpak AD-RH [0.46 cm I.D. ꢂ 15 cm], acetoni-
trile: 20 mM borate buffer (pH 9.0) = 40/60 flow rate 0.5 mL/min,
column temp: 40 °C UV: 210 nm): retention time: ts = 14.30 min
(major), tr = 18.31 min (minor); >99% ee. ESI MS m/z 206 [M+H]+.
C22H34N2O3ꢁC2H2O4: C, 62.05; H, 7.81; N, 6.03. Found: C, 61.96;
H, 7.91; N, 6.05.
5.1.34. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-(1R)-(1-iso-
propyl-6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate ((R)-5r)
Compound (R)-5r was synthesized in a manner similar to that
for compound 5r, except that (R)-4r was used instead of 4r.
Compound (R)-5r was obtained at a yield of 64% as a colorless
solid. 1H NMR (DMSO-d6): This compound exists as a pair of rota-
mers at room temperature. d 7.07–7.14 (1H, m), 6.72–6.82 (2H, m),
5.03 (major rotamer 1H, d, J = 9.3 Hz), 4.25 (minor rotamer 1H, d,
J = 9.5 Hz), 4.07–4.23 (1H, m), 3.94–4.06 (1H, m), 3.73 (3H, s),
5.1.36. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-7-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5s)
Compound 5s was synthesized in a manner similar to that for
compound 5a. Compound 5s was obtained at a yield of 62% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.06–7.16 (1H, m), 6.74–6.86
(2H, m), 5.08 (major rotamer 1H, d, J = 9.2 Hz), 4.30 (minor rotamer
1H, d, J = 9.4 Hz), 4.07–4.22 (1H, m), 3.93–4.03 (1H, m), 3.74 (major

4634
T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
rotamer 3H, s), 3.73 (minor rotamer 3H, s), 3.61–3.70 (major rota-
mer 1H, m), 3.51–3.60 (major rotamer 1H, m), 3.29–3.12 (minor
rotamer 1H, m), 2.79–2.94 (4H, m), 1.92–2.06 (1H, m), 1.19–1.63
(10H, m), 0.83–0.98 (6H, m); FAB MS m/z 375 [M+H]+.
3.16 (4H, m), 1.95–2.18 (1H, m), 1.18–1.63 (10H, m), 0.99 (minor
rotamer 3H, d, J = 6.8 Hz), 0.96 (major rotamer 3H, d, J = 6.6 Hz),
0.88 (minor rotamer 3H, dd, J = 3.1, 6.6 Hz), 0.84 (major rotamer
3H, dd, J = 2.9, 6.7 Hz); ESI MS m/z 363 [M+H]+.
5.1.37. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-8-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5t)
5.1.40. 2-(2-Benzyl-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-
2-ol (7)
To a mixture of compound 6 (as a hydrochloride salt 4.98 g) and
benzaldehyde (2.72 g) in acetic acid (50 mL) was added sodium tri-
acetoxyborohydride (6.11 g) at 4 °C. After stirring at room temper-
ature for 15 h, the mixture was basified with 1 M aqueous sodium
hydroxide solution, extracted with chloroform, dried over magne-
sium sulfate and concentrated in vacuo. The residue was purified
by column chromatography, eluting with hexane-ethyl acetate
to obtain 2-benzyl-1,2,3,4-tetrahydroisoquinoline-1-carboxylate
(1.99 g, 33%) as a colorless oil. ESI MS m/z 297 [M+H]+. To a solution
of 2-benzyl-1,2,3,4-tetrahydroisoquinoline-1-carboxylate (1.99 g)
in tetrahydrofuran (20 mL) was added a solution of 1.04 M methyl
lithium in hexane (16.2 mL) at ꢀ78 °C under an argon atmosphere.
After stirring at ꢀ78 °C for 30 min, the mixture was warmed to 0 °C
and stirred for 1 h. The mixture was then cooled back to ꢀ78 °C,
and a solution of 1.04 M methyl lithium in hexane (3.24 mL) was
added to the mixture. After stirring at ꢀ78 °C for a further
30 min, the mixture was warmed to 0 °C and stirred for 1 h. The
reaction was then quenched by the addition of water, extracted
with ethyl acetate, washed with brine, dried over magnesium sul-
fate and then concentrated in vacuo. The residue was purified by
column chromatography, eluting with hexane-ethyl acetate to
obtain compound 6 (1.27 g, 67%) as a pale yellow oil. 1H NMR
(CDCl3): d 7.24–7.39 (5H, m), 7.11–7.23 (4H, m), 4.36 (1H, br s),
3.98 (1H, d, J = 13.2 Hz), 3.76 (1H, d, J = 13.2 Hz), 3.68 (1H, s),
3.26–3.35 (1H, m), 2.77–2.87 (1H, m), 2.66–2.75 (1H, m), 2.57–
2.65 (1H, m), 1.37 (3H, s), 0.97 (3H, s); ESI MS m/z 283 [M+H]+.
Compound 5t was synthesized in a manner similar to that for
compound 5a. Compound 5t was obtained at a yield of 56% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.16–7.24 (1H, m), 6.78–6.92
(2H, m), 5.58 (major rotamer 1H, d, J = 9.8 Hz), 4.30 (minor rotamer
1H, d, J = 9.6 Hz), 4.00–4.17 (1H, m), 3.95 (major rotamer 1H, d,
J = 16.3 Hz), 3.85 (minor rotamer 1H, d, J = 16.1 Hz), 3.81 (minor
rotamer 3H, s), 3.78 (major rotamer 3H, s), 3.68–3.79 (1H, m),
3.36–3.52 (1H, m), 2.98–3.13 (1H, m), 2.80–2.96 (3H, m), 2.05–
2.18 (minor rotamer 1H, m), 1.90–2.03 (1H, m), 1.17–1.65 (10H,
m), 0.98 (minor rotamer 3H, d, J = 6.8 Hz), 0.93 (major rotamer
3H, d, J = 6.8 Hz), 0.81 (minor rotamer 3H, d, J = 6.7 Hz), 0.78 (major
rotamer 1H, d, J = 6.6 Hz); FAB MS m/z 375 [M+H]+.
5.1.38. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-(1-isopro-
pyl-8-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethanone
oxalate (5n)
1-(5-Bromo-1-isopropyl-8-methyl-3,4-dihydroisoquinolin-2(1H)-
yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone was synthesized
in a manner similar to that for compound 5a. 1-(5-Bromo-1-iso-
propyl-8-methyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydrox-
ycyclohexyl)methyl]amino}ethanone was obtained at a yield of
63% as a brown oil. FAB MS m/z 437 [M]+.
To a mixture of 1-(5-Bromo-1-isopropyl-8-methyl-3,4-dihy-
droisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}
ethanone (116 mg) and triethylamine (0.05 mL) in ethanol (10 mL)
was added 10% palladium on charcoal (10 mg) under an argon
atmosphere. The mixture was stirred at room temperature under
a hydrogen atmosphere for 6 h. After the mixture was filtered
through a celite pad, the filtrate was concentrated in vacuo. The
residue was dissolved with chloroform, washed with 1 M aqueous
sodium hydroxide, dried over magnesium sulfate, and then con-
centrated in vacuo. The residue was purified by column chro-
matography, eluting with chloroform–methanol to give a pale
yellow oil (85 mg). The oil was dissolved in 2-propanol (0.8 mL).
Oxalic acid (23 mg) and ether (5 mL) were added to the solution.
The precipitate was filtered off to obtain compound 5n (66 mg,
56%) as a colorless solid. 1H NMR (DMSO-d6): This compound exists
as a pair of rotamers at room temperature. d 6.99–7.20 (3H, m),
5.46 (major rotamer 1H, d, J = 10.4 Hz), 4.41 (minor rotamer 1H,
d, J = 10.2 Hz), 4.19 (minor rotamer 1H, d, J = 15.8 Hz), 4.05 (major
rotamer 1H, d, J = 16.5 Hz), 3.90–4.00 (1H, m), 3.81 (1H, t,
J = 9.0 Hz), 3.48–3.59 (minor rotamer 1H, m), 3.32–3.43 (1H, m),
3.10–3.23 (1H, m), 2.82–2.97 (3H, m), 2.36 (minor rotamer 3H,
s), 2.29 (major rotamer 3H, s), 1.94–2.07 (1H, m), 1.20–1.64
(10H, m), 1.03 (minor rotamer 3H, d, J = 6.7 Hz), 0.99 (major rota-
mer 3H, d, J = 6.6 Hz), 0.74–0.84 (3H, m); ESI MS m/z 359 [M+H]+.
5.1.41. 1-(2-Methoxypropan-2-yl)-1,2,3,4-tetrahydroisoquino-
line (4c)
To a suspension of sodium hydride (60% dispersion in mineral
oil, 199 mg) in tetrahydrofuran (5 mL) was added dropwise a solu-
tion of 2-(2-benzyl-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-ol
(1.27 g) in THF (7 mL) at 4 °C and stirred at room temperature for
30 min. Methyl iodide (0.42 mL) was then added to the mixture
at 4 °C. After stirring at room temperature for 8 h, additional
sodium hydride (60% dispersion in mineral oil, 199 mg) and methyl
iodide (0.42 mL) were added to the mixture, followed by stirring at
room temperature for 12 h. The reaction was quenched by the
addition of water, extracted with ethyl acetate, washed with brine,
dried over magnesium sulfate, and then concentrated in vacuo. The
residue was purified by column chromatography, eluting with hex-
ane–ethyl acetate to obtain 2-benzyl-1-(2-methoxypropan-2-yl)-
1,2,3,4-tetrahydroisoquinoline (1.17 g, 88%) as a pale brown oil.
ESI MS m/z: 296 [M+H]+. To a mixture of 2-benzyl-1-(2-methoxy-
propan-2-yl)-1,2,3,4-tetrahydroisoquinoline (1.17 g) and in methanol
(12 mL) was added 10% palladium on charcoal (300 mg) under an
argon atmosphere. The mixture was stirred at room temperature
under a hydrogen atmosphere for 8 h. The mixture was filtered
through a celite pad, and the filtrate was concentrated in vacuo
to obtain compound 4c (770 mg, 95%) as a yellow gum. 1H NMR
(CDCl3): d 7.39–7.48 (1H, m), 7.03–7.20 (3H, m), 4.23 (1H, s),
3.25–3.38 (4H, m), 2.80–2.92 (2H, m), 2.61–2.70 (1H, m), 1.16
(3H, s), 1.12 (3H, s); ESI MS m/z 206 [M+H]+.
5.1.39. 1-(8-Fluoro-1-isopropyl-3,4-dihydroisoquinolin-2(1H)-
yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone oxalate
(5q)
Compound 5q was synthesized in a manner similar to that for
compound 5n. Compound 5q was obtained at a yield of 45% as a
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.26–7.32 (1H, m), 7.04–7.14
(2H, m), 5.44 (major rotamer 1H, d, J = 9.9 Hz), 4.49 (minor rotamer
1H, d, J = 10.0 Hz), 4.14–4.23 (minor rotamer 1H, m), 4.09 (1H, d,
J = 16.3 Hz), 3.97 (1H, d, J = 16.4 Hz), 3.34–3.92 (2H, m), 2.78–
5.1.42. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-[1-(2-
methoxypropan-2-yl)-3,4-dihydroisoquinolin-2(1H)-
yl]ethanone oxalate (5c)
Compound 5c was synthesized in a manner similar to that for
compound 5a. Compound 5c was obtained at a yield of 69% as a

T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
4635
colorless solid. 1H NMR (DMSO-d6): This compound exists as a pair
of rotamers at room temperature. d 7.29 (1H, d, J = 7.1 Hz), 7.11–
7.24 (3H, m), 5.44 (major rotamer 1H, s), 4.63 (minor rotamer
1H, s), 4.28–4.38 (minor rotamer 1H, m), 4.15 (1H, d, J = 16.2 Hz),
3.98–4.10 (1H, m), 3.89–3.78 (1H, m), 3.60–3.70 (1H, m), 3.50–
3.59 (1H, m), 3.07 (minor rotamer 3H, s), 3.04 (major rotamer
3H, s), 2.78–3.02 (4H, m), 1.30–1.64 (9H, m), 1.25 (major rotamer
3H, s), 1.21 (minor rotamer 3H, s), 1.17 (minor rotamer 3H, s),
1.12 (major rotamer 3H, s); FAB MS m/z 375 [M+H]+.
1H, s), 4.22–4.32 (minor rotamer 1H, m), 4.08–4.20 (1H, m),
3.91–4.07 (2H, m), 3.53–3.70 (1H, m), 3.07–3.19 (major rotamer
1H, m), 2.77–2.94 (minor rotamer 1H, m), 2.77–2.94 (3H, m),
1.07–1.66 (16H, m); FAB MS m/z 361 [M+H]+.
5.2. Pharmacology
5.2.1. Evaluation of inhibitory activity against N-type calcium
channels using an in vitro FLIPR assay in IMR-32 human
neuroblastoma cells
Details of experimental procedures were as previously
described.6 IC50 values were determined in duplicate in one exper-
iment. IC50 values and 95% confidence intervals were calculated
using Sigmoid-Emax nonlinear regression analysis with SAS soft-
ware (Cary, NC, USA).
5.1.43. 2-(1,2,3,4-Tetrahydroisoquinolin-1-yl)propan-2-yl
pivalate (9)
To a mixture of compound 8 (3.0 g) and tetramethylethylenedi-
amine (2.2 mL) in tetrahydrofuran (40 mL) was added to a solution
of 1.64 M tert-butyllithium in n-pentane (12 mL) at ꢀ78 °C under
an argon atmosphere. After stirring at ꢀ78 °C for 10 min, acetone
(1.8 mL) was added to the mixture and stirred at ꢀ78 °C for 1 h.
The reaction was quenched by the addition of acetic acid (2 mL),
warmed to room temperature, and then concentrated in vacuo.
The residue was dissolved with ethyl acetate and washed with
water, aqueous 5% citric acid solution, saturated aqueous sodium
bicarbonate, and brine. The resulting mixture was dried over mag-
nesium sulfate and concentrated in vacuo. The residue was purified
by column chromatography, eluting with hexane–ethyl acetate to
give 1-[1-(2-hydroxypropan-2-yl)-3,4-dihydroisoquinolin-2(1H)-
yl]-2,2-dimethylpropan-1-one (3.45 g, 68%) as a colorless solid.
FAB MS m/z: 276 [M+H]+.
Trifluoroacetic acid (2.9 mL) was combined with 1-[1-(2-hy-
droxypropan-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl]-2,2-dimethyl-
propan-1-one (300 mg) and stirred at room temperature for 4 h.
The mixture was concentrated in vacuo, basified with saturated
aqueous sodium bicarbonate, extracted with ethyl acetate, washed
with water, and dried over magnesium sulfate. The resulting solu-
tion was concentrated in vacuo to obtain compound 8 (294 mg
98%) as a yellow oil. 1H NMR (CDCl3): d 7.34 (1H, d, J = 7.3 Hz),
7.06–7.20 (3H, m), 4.60 (1H, br s), 3.25–3.35 (1H, m), 2.83–2.99
(2H, m), 2.63–2.74 (1H, m), 1.53 (3H, s), 1.45 (3H, s), 1.15 (9H, br
s); FAB MS m/z 276 [M+H]+.
5.2.2. Evaluation of inhibitory activity against hERG potassium
channels using an in vitro Rb Efflux assay in Chinese hamster
ovary (CHO) cells
The inhibitory activities of (R)-5r against hERG potassium chan-
nels were evaluated using an in vitro Rb efflux assay of Chinese
hamster ovary (CHO) cells. Details of experimental procedures
were as previously described.6
5.2.3. Measurement of CYP inhibition
The inhibitory activities of test compounds against CYP1A2,
2C9, 2C19, and 2D6 were determined using fluorescence sub-
strates. Details of experimental procedures were as previously
described.6 For the CYP3A4 inhibition assay, midazolam was used
as a probe substrate to monitor the changes in CYP3A4 activity
during the exposure to each test compound. Details of experimen-
tal procedures were also as previously described.6 Residual meta-
bolic activities for reversible (Eq. 1 and time-dependent (Eq. 2
inhibition were calculated using the following equations:
% Residual activity ¼ Activitycompound;0 min=Activityvehicle;0 min ꢂ 100
ð1Þ
5.1.44. 2-{[(1-Hydroxycyclohexyl)methyl]amino}-1-[1-(2-hydro-
xypropan-2-yl)-3,4-dihydroisoquinolin-2(1H)-yl]ethanone
oxalate (5d)
2-(2-{N-[(1-Hydroxycyclohexyl)methyl]glycyl}-1,2,3,4-tetrahy-
droisoquinolin-1-yl)propan-2-yl pivalate was synthesized in a
manner similar to that for compound 5a. 2-(2-{N-[(1-hydroxycy-
clohexyl)methyl]glycyl}-1,2,3,4-tetrahydroisoquinolin-1-yl)pro-
pan-2-yl pivalate was obtained at a yield of 81% as a colorless oil.
APCI MS m/z 445 [M+H]+.
À
Á
% Residual activity ¼ Activitycompound;30 min=Activityvehicle;30 min
À
Á
ꢂ Activitycompound;0 min=Activityvehicle;0 min
ꢂ 100
ð2Þ
where Activitycompound, 0 min denotes activity obtained in the pres-
ence of compound and without pre-incubation, Activityvehicle, 0 min
denotes activity obtained in the absence of compound and with-
out pre-incubation, Activitycompound,
denotes that obtained
30 min
To a solution of 2-(2-{N-[(1-hydroxycyclohexyl)methyl]glycyl}-
1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-yl pivalate (1.43 g) in
dichloromethane (15 mL) was added a solution of 1.01 M diisobu-
tyl aluminum hydride in hexane (9.55 mL) at ꢀ78 °C under an
argon atmosphere. After stirring at ꢀ78 °C for 5 h, the mixture
was warmed to 0 °C for 2 h. The reaction was then quenched by
the addition of saturated aqueous potassium sodium tartrate solu-
tion, stirred for 20 min and filtered through a celite pad. The filtrate
was extracted with chloroform, washed with brine, dried over
magnesium sulfate, and then concentrated in vacuo. The residue
was purified by column chromatography, eluting with chloro-
form-methanol to give a colorless oil (118 mg), which was dis-
solved in ethanol (4 mL). Oxalic acid (32 mg) was added to the
solution and concentrated in vacuo. The residue was washed with
acetonitrile and filtered off to obtain compound 5d (120 mg, 8%) as
a colorless solid. 1H NMR (DMSO-d6): This compound exists as a
pair of rotamers at room temperature. d 7.25–7.34 (1H, m), 7.24–
7.10 (2H, m), 5.30 (major rotamer 1H, s), 4.56 (minor rotamer
in the presence of compound and with pre-incubation, and
Activityvehicle, denotes that obtained in the absence of
compound and with pre-incubation.
30 min
5.2.4. In vitro DDI assay for CYP 2D6
Dextromethorphan was used as a probe substrate for CYP2D6.
Reaction mixtures containing 100 mM Na+–K+ phosphate buffer
(pH 7.4), 0.4 mg protein/mL human liver microsomes, 1 mM
NADPH, 0.1 mM EDTA, and 0.3–100
prepared and pre-incubated for 0 or 30 min at 37 °C. Reactions
were initiated by the addition of 7 M of dextromethorphan and
lM of test compounds were
l
incubated for an additional 20 min. Reactions were terminated
by the addition of 80% acetonitrile with internal standard. The
metabolite of dextromethorphan, dextrophan, was measured by
LC–MS/MS, and IC50 values were determined for 0 (reversible inhi-
bition) and 30 minutes (time-dependent inhibition) pre-
incubation.

4636
T. Ogiyama et al. / Bioorg. Med. Chem. 23 (2015) 4624–4637
Kim, S.; McEnery, M.; Chin, H.; Kim, H.; Park, J.; Kim, D.; Jung, S.; Kim, J.; Shin,
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Miyamoto, N.; Takahashi, E.; Yoshinaga, T.; Sawada, K.; Imoto, K.; Tanaka, I.;
Yoshizawa, T.; Nishizawa, Y.; Mori, Y.; Niidome, T.; Shoji, S. NeuroReport 2001,
12, 2423.
5.2.5. In vitro metabolic stability in human liver microsomes
Reaction mixtures containing 100 mM Na+–K+ phosphate buffer
(pH 7.4), 0.2 mg protein/mL human liver microsomes, 1 mM
NADPH and 0.1 mM EDTA were pre-incubated for 5 min at 37 °C.
The reactions were initiated by the addition of test compound solu-
3. Schmidtkco, A.; Lötsch, J.; Freynhagen, R.; Geisslinger, G. Lancet 2010, 375,
1569.
4. (a) Yamamoto, T.; Takahara, A. Curr. Top. Med. Chem. 2009, 9, 377; (b) Barrow, J.
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tion. The final concentration of test compound was 0.2 lM. After 0,
15, 30 and 45 min incubations at 37 °C, reactions were terminated
by the addition of acetonitrile with internal standard. The reaction
mixtures were centrifuged and supernatants injected into the LC–
MS/MS system to determine the residual ratio of the test compound.
5. (a) Beebe, X.; Yeung, C. M.; Darczak, D.; Shekhar, S.; Vortherms, T. A.; Miller, L.;
Milicic, I.; Swensen, A. M.; Zhu, C. Z.; Banfor, P.; Wetter, J. M.; Marsh, K. C.;
Jarvis, M. F.; Scott, V. E.; Schrimpf, M. R.; Lee, C.-H. Bioorg. Med. Chem. Lett. 2013,
23, 4857; (b) Shao, P. P. ACS Med. Chem. Lett. 2013, 1064, 4; (c) Winters, M. P.;
Subashinghe, N.; Wall, M.; Beck, E.; Brandt, M. R.; Finley, M. F. A.; Liu, Y.; Lubin,
M. L.; Neeper, M. P.; Qin, N.; Flores, C. M.; Sui, Z. Bioorg. Med. Chem. Lett. 2014,
24, 2053; (d) Winters, M. P.; Subashinghe, N.; Wall, M.; Beck, E.; Brandt, M. R.;
Finley, M. F. A.; Liu, Y.; Lubin, M. L.; Neeper, M. P.; Qin, N.; Flores, C. M.; Sui, Z.
Bioorg. Med. Chem. Lett. 2014, 24, 2057.
6. Ogiyama, T.; Inoue, M.; Honda, S.; Yamada, H.; Watanabe, T.; Kiso, T.; Goto, T.;
Koakutsu, A.; Kakimoto, S.; Shishikura, J. Bioorg. Med. Chem. 2014, 22, 6899.
7. (a) Zhou, S.-F.; Liu, J.-P.; Lai, X.-S. Curr. Med. Chem. 2009, 16, 2661; (b) Zhou, S.-F.
Curr. Pharm. Des. 2008, 14, 990.
8. (a) Bockbrader, H. N.; Wesche, D.; Miller, R.; Chapel, S.; Janiczek, N.; Burger, P.
Clin. Pharmacokinet. 2010, 49, 661; (b) Spina, E.; Trifiro, G.; Caraci, F. CNS Drugs
2012, 26, 39; (c) Wernicke, J. F.; Gahimer, J.; Yalcin, I.; Wulster-Radcliff, M.;
Viktrup, L. Expert Opin. Drug Saf. 2005, 4, 987; (d) Shin, J.-G.; Park, J.-Y.; Kim, M.-J.;
Shon, J.-H.; Yoon, Y.-R.; Cha, I.-J.; Lee, S.-S.; Oh, S.-W.; Kim, S.-W.; Flockhart, D. A.
Drug Metab. Dispos. 2002, 30, 1102.
5.2.6. Calculation of in vitro intrinsic clearance
In vitro intrinsic clearance (CLint, in vitro) was calculated using
Eq. 3, which is based on the time course of the residual ratio of the
test compounds, as determined using least-squares linear regres-
sion as follows:22
CLint; in vitro ð L=min=mg proteinÞ
¼ ke=microsomal protein concentration
l
ð3Þ
where ke denotes the disappearance rate constant.
5.2.7. Animal experiments
9. Larsen, R.; Reamer, R.; Corley, E.; Davis, P.; Grabowski, E.; Reider, P.; Shinkai, I. J.
Org. Chem. 1991, 56, 6034.
Male ddY mice or Sprague-Dawley rats (SLC, Hamamatsu, Japan)
were used for in vivo experiments. Animals were group-housed and
kept on a 12-hour light/dark cycle (lights on from 7:30 AM to 7:30
PM) with free access to food and water. All animal experimental
procedures were approved by the Committee for Animal
Experiments of Astellas Pharma Inc. and conformed to the
International Guiding Principles for Biomedical Research Involving
Animals (CIOMS) and Guidelines for Proper Conduct of Animal
Experiments (Science Council of Japan, 2006). All efforts were made
to minimize the number of animals used and their suffering.
We examined the antinociceptive effect of compound (R)-5r in
mice formalin test previously published by Hunskaar et al.23 with
10. The synthesis of compound 4e and 4f: (a) Seebach, D.; Lohmann, J.-J.; Syfrig, M.
A.; Yoshifuji, M. Tetrahedron 1983, 39, 1963; The synthesis of compound 4i: (b)
Kitabatake, M.; Nagai, J.; Abe, K.; Tsuchiya, Y.; Ogawa, K.; Yokoyama, T.; Mohri,
K.; Taguchi, K.; Horiguchi, Y. Eur. J. Med. Chem. 2009, 44, 4034.
11. The synthesis of compound 4o, 4r–4t: Watanuki, S.; Matsuura, K.; Tomura, Y.;
Okada, M.; Okazaki, T.; Ohta, M.; Tsukamoto, S. Chem. Pharm. Bull. 2011, 59,
1029.
12. The measured logD value at pH 7.4 of (R)-5r was 2.4, while the calculated one
was 2.55.
13. (a) Kumar, S.; Sharma, R.; Roychowdhury, A. Curr. Med. Chem. 2012, 19, 3605;
(b) Bourdonnec, B.; Leister, L. Curr. Med. Chem. 2009, 16, 3093; (c) de Groot, M.
J.; Wakenhut, F.; Whitlock, G.; Hyland, R. Drug Discovery Today 2009, 14, 964.
14. (a) Fray, M. J.; Bish, G.; Fish, P. V.; Stobie, A.; Wakenhut, F.; Whitlock, G. A.
Bioorg. Med. Chem. Lett. 2006, 16, 4349; (b) Fish, P. V.; Fray, M. J.; Stobie, A.;
Wakenhut, F.; Whitlock, G. A. Bioorg. Med. Chem. Lett. 2007, 17, 2022; (c)
Hudson, S.; Kiankarimi, M.; Eccles, W.; Dwight, W.; Mostofi, Y. S.; Genicot, M.
J.; Fleck, B. A.; Gogas, K.; Aparicio, A.; Wang, H.; Wen, J.; Wade, W. S. Bioorg.
Med. Chem. Lett. 2008, 18, 4491; (d) Pontillo, J.; Wu, D.; Ching, B.; Hudson, S.;
Genicot, M. J.; Gao, Y.; Ewing, T.; Fleck, B. A.; Gogas, K.; Aparicio, A.; Wang, H.;
Wen, J. Bioorg. Med. Chem. Lett. 2008, 18, 6151; (e) Ishichi, Y.; Kimura, E.;
Honda, E.; Yoshikawa, M.; Nakahata, T.; Terao, Y.; Suzuki, A.; Kawai, T.;
Arakawa, Y.; Ohta, H.; Kanzaki, N.; Nakagawa, H.; Terauchi, J. Bioorg. Med.
Chem. 2013, 21, 4600.
slight modifications. Briefly, 2% formalin (20 lL) was subcuta-
neously injected 15 min after intrathecal bolus administration of
compound (R)-5r. The summation of time spent in lifting/licking
of the paw that received injections was measured. The duration
of responses in the first 10 min and that from 15 to 25 min repre-
sent the first and second phases, respectively. In addition, the effi-
cacy of compound (R)-5r in neuropathic pain was evaluated in rat
SNL model reported by Chung et al.19 Effects of compound (R)-5r
on motor coordination was assessed via the rotarod test. Details
of the experimental procedures were as previously described.24
15. Ogiyama, T.; Yonezawa, K.; Inoue, M.; Katayama, N.; Watanabe, T.; Yoshimura,
S.; Gotoh, T.; Kiso, T.; Koakutsu, A.; Kakimoto, S.; Shishikura, J. Bioorg. Med.
Chem. 2015, 23, 4638.
16. Compound 1 exhibited a similar result among the racemate (IC50 = 0.77
and the optically pure compounds ((S)-1 IC50 = 1.0 M and (R)-1 IC50 = 1.5
l
M)
l
lM).
In addition, similar results were also reported by Zalicus scientists. See: Refs.
6,17a. Additional refferences are therein.
Acknowledgements
17. The reversible and time-dependent inhibitory effects of (R)-5r against CYP2D6
were scrutinized by the HLM-LC–MS assay using dextromethorphan as a probe
substrate (details of experimental procedures are described in Section 5).
We thank Drs. Seiji Yoshimura and Takahiro Ishii for their
advice and support regarding the determination of the absolute
stereochemistry of (R)-4r. We also thank Drs. Kenichi Onda,
Shinya Nagashima, and Susumu Watanuki for their advice and
careful reading of the manuscript. In addition, we thank the staff
of the Division of Analytical Science Laboratories for conducting
elemental analyses and spectral measurements.
Surprisingly, (R)-5r showed relatively weak inhibitory activity (IC50 = 37 lM)
although the reason for the discrepancy in results between the fluorometric
assay and HLM-LC–MS assay is not clear. In addition, (R)-5r had little effect on
TDI of CYP2D6 (IC50 = 41 lM), suggesting little concern of CYP2D6 inhibition-
mediated drug-drug interactions. Further evaluation is ongoing at present.
Discussion about the disparity in inhibitory activities between the fluorometric
assay and HLM-LC–MS assay reported by Wyeth (now part of Pfizer) scientists
might be informative. See: Di, L.; Kerns, E. H.; Li, S. Q.; Carter, G. T. Int. J. Pharm.
2007, 335, 1.
18. Zalicus scientists also reported similar results using their small-molecule N-
type calcium channel blockers. See: (a) Pajouhesh, H.; Feng, Z.-P.; Zhang, L.;
Pajouhesh, H.; Jiang, X.; Hendricson, A.; Dong, H.; Tringham, E.; Ding, Y.;
Vanderah, T. W.; Porreca, F.; Belardetti, F.; Zamponi, G. W.; Mitscher, L. A.;
Snutch, T. P. Bioorg. Med. Chem. Lett. 2012, 22, 4153; (b) Zamponi, G. W.; Feng,
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.05.030.
References and notes
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dd, J = 2.1, 8.2 Hz), 2.92–3.06 (4H, m), 2.27 (3H, s); FAB MS m/z 214 [M+H]+.
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4637
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