Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors - Part 2

Article abstract of DOI:10.1016/j.bmc.2016.05.038

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood-brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H³)methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.

Full text of DOI:10.1016/j.bmc.2016.05.038

Bioorganic & Medicinal Chemistry 24 (2016) 3207–3217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of a novel series of
peripheral-selective noradrenaline reuptake inhibitors—Part 2
⇑
Tomoya Yukawa , Ikuo Fujimori, Taku Kamei, Yoshihisa Nakada, Nobuki Sakauchi, Masami Yamada,
Yusuke Ohba, Hiroyuki Ueno, Maiko Takiguchi, Masako Kuno, Izumi Kamo, Hideyuki Nakagawa,
Yasushi Fujioka, Tomoko Igari, Yuji Ishichi, Tetsuya Tsukamoto
Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of
stress urinary incontinence to overcome adverse effects associated with central action. Herein, we
describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhi-
bitors to avert the risk of P-gp-mediated DDI at the blood–brain barrier. We observed that steric shielding
of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resis-
tance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative,
was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of
PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a
deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabo-
lized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium
Received 22 April 2016
Revised 19 May 2016
Accepted 20 May 2016
Available online 21 May 2016
Keywords:
Peripheral-selective noradrenaline reuptake
inhibitor
Stress urinary incontinence
MDR1 efflux ratio
replacement for
a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluo-
Genetic polymorphism
Deuterium replacement
rophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H3)methyloxy]acetamide hydrochloride, which exhibited
peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-depen-
dent manner.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Furthermore, in the case of peripheral-selective NRIs, adverse
effects on the central nervous system are not a concern.4 Target-
The noradrenaline transporter (norepinephrine transporter,
NET), which is a membrane-bound protein, is responsible for the
reuptake of extracellular noradrenaline. Selective inhibition of
NET, which increases the noradrenaline concentration at synaptic
cleft, has been shown to be an attractive approach for the treat-
ment of many diseases.1 NET is known to express in both central
and peripheral nervous systems and to exhibit its own functional
outputs.2 Among the various target indications for noradrenaline
reuptake inhibitor (NRI), stress urinary incontinence (SUI) seems
one of good indications for peripheral-selective NRI, as our previ-
ous publication suggests that the urethral resistance-increasing
effects of NRIs are mainly achieved by peripheral action.3
selective distribution could also minimize clinically effective doses.
Taken together, novel peripheral-selective NRIs have the potential
to be safer and more effective drugs compared to traditional cen-
tral-acting NRIs as anti-SUI agents.
In our previous papers, we described our approaches to design
and synthesis of small molecule peripheral-selective NRIs based
on 7-phenyl-6-substituted oxazepane series.3 Among compounds
in this series, compound 1 exhibits potent NET inhibitory activity,
high selectivity against serotonin transporter (SERT) and dopamine
transporter (DAT), and high peripheral NET selectivity in occu-
pancy tests in rat brain (Fig. 1). However, this compound exhibits
a high multidrug resistance protein 1 (MDR1) efflux ratio, and is
thus a potential P-glycoprotein (P-gp) substrate. P-gp belongs the
superfamily of ATP-binding cassette transporters and pumps
numerous xenobiotics out of cells.5 P-gp is also known to be the
major efflux transporter at the blood brain barrier and to be
responsible for the efflux of a number of xenobiotic substances
from the central nervous system. Although some approved drugs
Abbreviations: SUI, stress urinary incontinence; DAT, dopamine transporter;
SERT, serotonin transporter; NET, norepinephrine transporter; MDR1, multidrug
resistance 1; P-gp, P-glycoprotein; CYP, cytochrome P450; hERG, human ether-a-
go-go-related gene K+ channel; CNS, central nervous system.
⇑
Corresponding author. Tel.: +81 466 32 1105; fax: +81 466 29 4454.
E-mail address: tomoya.yukawa@takeda.com (T. Yukawa).
http://dx.doi.org/10.1016/j.bmc.2016.05.038
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

3208
T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
Cl
2. Experimental
essential for potency
NH
R2
O
O
Cl
NH
The preparation of 6-aminomethyl 7-phenyl-1,4-oxazepane
derivatives 1–15 is shown in Scheme 1. Morita–Baylis–Hillman
reaction of 3-fluoro-4-chlorobenzaldehyde 16a–b with methyl
acrylate afforded ester 17a–b. Michael addition of benzyl amine
gave the threo isomer of methyl ester rac-18a–b. After transforma-
tion to silyl ether rac-19a–b via a hydroxymethyl intermediate, N-
selective acylation with chloroacetyl chloride gave amide rac-20a–
b. Cyclization of rac-20a–b under basic conditions gave 6-substi-
tuted 1,4-oxazepan-3-one rac-21a–b. After reduction of the amide
group, the N-benzyl group was replaced with Boc along with de-
protection of the hydroxyl group to afford rac-23a–b. Following
optical resolution using preparative chiral HPLC, the hydroxyl
group was converted into amine 26a–b via the mesylate 24a–b
and azide 25a–b. After condensation of amine 26a–b with corre-
sponding acid chloride or carboxylic acid, treatment with hydrogen
chloride furnished amide 1–15 as the HCl salt. The absolute stere-
ochemistry of 6 was confirmed by single-crystal X-ray analysis
(Fig. 2).
NH
NH
O
O
Me
Shielding
R1
1
Reuptake inhibitions (NET/SERT/DAT):
IC50= 0.33 / 150 / 1500 nM
NET Occupancy in rat brain
(3 mg/kg, i.v.): -14%
Designed structure I
MDR1 efflux ratio: 5.0
Figure 1.
inhibit P-gp,6 drug–drug interaction (DDI) of the newly developed
peripheral-selective NRIs with existing P-gp inhibitors might
reduce the peripheral NET selectivity of the compounds, if they
are P-gp substrates. To avoid such DDI risks, we attempted to
decrease the MDR1 efflux ratio.
Removing or weakening the hydrogen-bond acceptor (HBA)
and/or donor (HBD) is one of the guiding principles for reduced
recognition as a P-gp substrate.7 These modifications also increase
passive diffusion of compounds, and if the passive diffusion is suf-
ficiently fast, the efflux pumping efficiency will be overcome.8 Our
lead compound 1 has two HBDs and one HBA. In the initial SAR
study, the HBD in the oxazepane ring was necessary for NET inhi-
bitory activity. With regard to the amide motif, we have already
observed that the amide is a good enhancer of the NET inhibitory
activity, though several other substituents are also tolerated for
NET inhibitory activity. On this basis, we decided to focus on R1
substituent optimization in designed structure I while maintaining
the amide motif. We expected that the modifications of the R1 sub-
stituent would sterically shield the HBA and/or HBD, leading to an
increase in passive diffusion and improvement of P-gp recognition
(Fig. 1).8 In addition, designed structure I might have a liability of
CYP2D6 inhibition, hERG inhibition and phospholipidosis (PLsis)
due to the cationic amphiphilic drug (CAD) structure. Aware of
these risks, we evaluated CYP2D6 inhibitory activity and priori-
tized compounds based on the potency.9 In this study, we per-
formed the design and synthesis of 7-aryl-6-substituted
oxazepine derivatives to reduce the MDR1 efflux ratio.
Figure 2. ORTEP drawing of compound 6, thermal ellipsoids are drawn at 30%
probability.
R1
R1
R1
R1
OH
c
a
b
OH
OH
CO2Me
Cl
Cl
O
Cl
CHO
Cl
NHBn
MeO2C
NHBn
OTBDMS
16a-b
17a-b
18a-b
19a-b
rac-
rac-
R1
R1
R1
OH
O
O
d
e
f
g
Cl
Cl
Cl
NBn
NBn
O
BnN
OTBDMS
Cl
OTBDMS
OTBDMS
20a-b
21a-b
rac-
22a-b
rac-
rac-
R1
R1
R1
O
O
l, m
h, i
O
Cl
Cl
Cl
NBoc
NBoc
NH
HCl
16a-26a: R1=Cl
16b-26b : R1=F
R
OH
rac-23a-b
NH
R2
O
1-15
R = OMs: 24a-b
N3: 25a-b
j
k
26a-b
NH2:
Scheme 1. Synthesis of compound 1–15. Reagents and conditions: (a) Methyl acrylate, DABCO, DBU, MeCN, rt; (b) BnNH2, Et3N, MeOH, rt; (c) (1) CaCl2, NaBH4, THF, EtOH,
0 °C to rt; (2) TBDMSCl, Et3N, imidazole, DMAP, THF, 0 °C to rt; (d) chloroacetyl chloride, Et3N, THF, 0 °C to rt; (e) 1 M NaOH aq, THF, 0 °C to rt; (f) LiAlH4, AlCl3, THF, 0 °C to rt;
(g) (1) 1-chloroethyl chloroformate, MeCN, rt, (2) 1 M HCl aq, MeOH, 80 °C, (3) Boc2O, Et3N, 0 °C to rt; (h) optical resolution by HPLC; (i) MsCl, Et3N, THF, 0 °C to rt; (j) NaN3,
DMF, 80 °C; (k) PPh3, THF, H2O, rt. (l) R2COCl, Et3N, THF, rt or WSC, HOBt, R2COOH, Et3N, THF, rt; (m) 11.7 M HCl in EtOH, rt. or 4 M HCl in EtOAc, rt.

T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
3209
with a decrease in the lipophilicity.3 Similarly, compound 6
retained the potent inhibitory activity and high membrane perme-
ability, with remarkable improvement of CYP2D6 inhibitory activ-
ity. 2-Methoxypropionylamino derivative 7 exhibited a high MDR1
efflux ratio. The insertion of one carbon substantially increased the
MDR1 efflux ratio, and the length of the linker between the amide
moiety and the oxygen atom was critical. An extended methoxy
alkyl moiety might be recognized as a HBA, resulting in decreased
permeability.8 Having achieved low MDR1 efflux ratio, compound
6 was selected for further evaluation.
3. Results and discussion
The synthesized compounds were evaluated for their in vitro
reuptake inhibitory activity against human NET, SERT, and DAT
(expressed as IC50 values), PAMPA, MDR1 efflux ratio and CYP2D6
inhibitory activity at 10 lM. The results are shown in Table 1. First,
we introduced lipophilic substituents to the R1 position. n-
Propanoylamide derivative 2 exhibited slight improvement of
membrane permeability and MDR1 efflux ratio, and this result
encouraged us to introduce bulkier substituents. n-Butanoylamide
derivative 3 demonstrated a further decrease of the MDR1 efflux
ratio. Benzoylamide derivative 4 showed a low MDR1 efflux ratio.
Incorporation of lipophilic substituents retained the potent NET
inhibitory activities (2–4). As R1 groups become larger along with
the clogP value, membrane permeability was improved and/or P-
gp recognition of the amide moiety would be blocked.10 However,
CYP2D6 inhibitory activities were enhanced along with increase in
lipophilicity. 1-Methoxyacetamide derivative 5, which has lower
clogP value than 2–4, also exhibited potent NET inhibitory activity
with a low MDR1 efflux ratio; this result suggests that the steric
shielding would be critical for the improvement of MDR1 efflux.
Intramolecular hydrogen bonding of the oxygen atom with the
amide moiety might also contribute the improvement. This com-
pound demonstrated slight improvement of CYP2D6 inhibitory
activity compared with benzoylamino derivative 4. In the previous
SAR study, the replacement of a chloro atom with a fluoro atom at
the 3-position of the phenyl group was tolerated for the potency
Compound 6 showed potent NET inhibitory activity, high selec-
tivity against SERT and DAT inhibitions, and a low MDR1 efflux
ratio. However, a phenotyping study using eight CYP isoforms
revealed that compound
6 was dominantly metabolized by
CYP2D6 and CYP2C19 (contribution rate of CYP2D6/CYP2C19/
others: 74.3/25.7/0).11 These two CYP isoforms are known to have
genetic polymorphism in humans, which can influence clinical
pharmacokinetics and therapeutic effects/adverse effects.12 In poor
metabolizers, the compound is slowly eliminated, whereas the
compound is rapidly eliminated in ultrarapid metabolizers. To
minimize the unpredictable clinical exposure due to genetic poly-
morphism, we decided to focus on identifying the compounds
mainly metabolized by CYPs other than CYP2D6 and CYP2C19. A
metabolite profiling using CYP2D6-expressing microsomes and
human hepatocytes revealed that O-demethylated hydroxymethyl
derivative 8 was a major presumed metabolite (Fig. 3). Given the
metabolic mechanism, C–H bond hydroxylation of the methoxy
Table 1
Monoamine reuptake inh., MDR1 efflux ratio and CYP2D6 inh.a
R2
O
Cl
NH
HCl
NH
O
R1
b
Compd R1
R2
Reuptake inh. IC50 (nM), 95% CIc
PAMPA at
pH7.4
MDR1 ratio (B to A/A to B) CYP2D6 inhibitiond
(%)
ClogP
NET
SERT
DAT
1
Me
Cl 0.33 (0.16–
0.67)
150 (120–180)
>1000
146
5.0
26
0.71
2
3
4
Et
Pr
Ph
Cl 1.5 (1.0–2.1)
Cl 2.6 (2.1–3.3)
Cl 1.7 (1.4–2.1)
120 (98–160)
62 (45–85)
25 (23–28)
>1000
>1000
>1000
207
260
252
4.8
3.0
1.8
41
62
90
1.23
1.76
3.72
1600 (1200–
2100)
5
6
7
Cl 1.2 (1.1–1.4)
70 (57–86)
279
221
151
2.1
2.1
70
48
44
0.88
0.43
0.69
OMe
OMe
F
F
3.2 (2.5–4.0)
3.9 (2.7–5.5)
440 (370–520)
>1000
>1000
890 (640–
1200)
OMe
12
a
b
c
All compounds were isolated as HCl salt.
These values were calculated from the results of two experiments.
95% confidence interval for each IC50 value.
d
CYP2D6 inhibition expressed as its % inhibition value at the drug concentration of 10 lM.
F
F
F
O
de-methylated
by CYP2D6
O
O
Cl
Cl
Cl
NH
NH
NH
NH
NH
NH
OH
O
O
O
O
R4
OMe
R3
6
8
Designed structure II
Figure 3. Metabolite profiling and design.

3210
T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
Table 3
CYP phenotyping study
moiety by CYP2D6 would be a first step of the O-demethylation
reaction.13 We hypothesized that a modification of the methoxy-
methyl moiety would affect the metabolic pathway, resulting in
a decrease in the contribution rate of CYP2D6. On the basis of this
hypothesis, we focused on the modification of R3 and R4 sub-
stituents to lead potential development candidates.
Compd
Contribution ratio of
metabolisma (%)
Metabolic rate (%/min/pmol)
3A4b 2D6 2C19 Othersc 3A4
2D6
2C19
Othersc
11
12
13
6
57.4
75.6
81.3
0
41.6
22.6
9.8
1.0
1.8
5.9
0
0
3.1
0
0.026 0.365 0.005
0.064 0.370 0.017
0
0
The results of modifications of substituents R3 and R4 are shown
0.015 0.035 0.012 0.008
0.081 0.016
in Table 2. The introduction of a methyl group at
a-position (9)
74.3 25.7
0
0
gave a reduction in NET inhibitory activity. Dimethyl substitution
(10) further reduced NET inhibitory activity. Replacements of the
methoxy moiety with an ethoxy (11) or isopropoxy group (12)
provided potent compounds with low MDR1 efflux ratio. However,
CYP2D6 inhibitory activities were enhanced along with lipophilic-
ity. Deuterium methoxy compound 13 demonstrated almost the
same profiles as compound 6. Oxygen-containing five-membered
ring (14) or six-membered ring derivatives (15) also demonstrated
potent NET inhibitory activity, but showed a relatively high MDR1
efflux ratio.
Compounds 11, 12, and 13 were evaluated in CYP phenotyping
study (Table 3). As we expected, all methoxy-modified compounds
were mainly metabolized by CYP3A4 instead of CYP2D6 and
CYP2C19, indicating that these modifications affected the CYP-
mediated metabolism. Compared to ethoxy derivative 11, bulkier
and more lipophilic isopropoxy derivative 12 showed further
decrease in the contribution rate of CYP2D6, whereas the rate of
CYP2D6-mediated metabolism was not improved (6 vs 11, 12).
As the size and the lipophilicity of the substituent were increased,
CYP3A4-mediated metabolism would be accelerated (6: 0%/min/
pmol, 11: 0.026%/min/pmol 12: 0.064%/min/pmol), leading to a
change of the contribution rate. Interestingly, a drastic contribu-
tion rate reduction of CYP2D6 was observed in the case of deu-
terium methoxy compound 13. In general, the shapes and sizes
a
Contribution ratios were calculated by abundance of cytochromes P450 in
human liver.
b
Contribution ratio of CYP3A4 was corrected by CYP3A abundance in human
liver.
c
2B6, 2C9, 2E1, 2C8, 1A2.
of deuterated compounds are considerably similar to those of the
corresponding all-hydrogen compounds.14 Small differences in
physicochemical properties (e.g., decrease in lipophilicity) are
observed; however, in most cases, the replacement negligibly
affects on the biochemical activity. This result is consistent with
our results related to NET, SERT, and DAT inhibitory activities,
CYP2D6 inhibitory activity, and MDR1 efflux ratio (6 vs 13). How-
ever, the cleavage of a C–D bond requires greater energy than that
of the corresponding C–H bond, which results in differing cleavage
rate (i.e., the kinetic isotope effect) if the C–H bond cleavage step is
at least partially rate limiting.15 Therefore, the replacement of a
metabolic C–H bond with a C–D bond is assumed to directly affect
the metabolic reaction, resulting in the observed changes in the
contribution rate of CYPs. In this case, CYP2D6-mediated metabo-
lism was decreased, whereas CYP3A4-mediated metabolism was
accelerated. Importantly, identification of the principal metabolites
of compound 13 using human hepatocytes revealed that
Table 2
Monoamine reuptake inh., MDR1 efflux ratio and CYP2D6 inh.a
F
O
Cl
NH
NH
O
O
R4
R =
R3
Reuptake inh. IC50 (nM), 95% CIc
MDR1 ratio (B to A/A to B)
CYP2D6 inhibition (%)d
ClogP
b
Compd
R
NET
SERT
DAT
9
8.8 (7.3–11)
21 (14–30)
800 (600–1100)
520 (390–700)
>1000
1.9
2.4
33
42
0.74
1.05
OMe
Me
Me
Me
OMe
10
>1000
11
12
13
3.3 (2.3-4.9)
4.8 (4.0–5.8)
3.3 (2.4–4.6)
220 (190–260)
390 (290–500)
680 (540–860)
>1000
>1000
>1000
1.4
1.7
2.1
79
66
49
0.82
1.13
0.43
OEt
OiPr
OCD3
O
14
15
5.3 (3.6–7.6)
2.6 (2.1–3.3)
200 (170–240)
540 (410–700)
>1000
>1000
3.5
3.6
80
66
2.23
1.56
O
O
a
b
c
All compounds were isolated as HCl salt.
These values were calculated from the results of two experiments.
95% confidence interval for each IC50 value.
d
CYP2D6 inhibition expressed as its % inhibition value at the drug concentration of 10 lM.

T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
5. Experimental section
3211
5.1. Chemistry
Melting points were determined with a Yanagimoto melting
point apparatus or a Büchi melting point apparatus B-545 and
are uncorrected. 1H NMR spectra were obtained at 300 MHz on a
Varian Ultra-300, or a Bruker DPX-300 spectrometer. Chemical
shifts are given in d values (ppm) using tetramethylsilane as the
internal standard. Peak multiplicities are expressed as follows: s,
singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet;
ddd, doublet of doublet of doublets; dt, double triplet; td, triple
doublet; quin, quintet; sxt, sextet; br s, broad singlet; m, multiplet.
Elemental analyses were carried out by Takeda Analytical Labora-
tories Ltd. Reactions were followed by TLC on Silica gel 60 F 254
precoated TLC plates (E. Merck) or NH TLC plates (Fuji Silysia
Chemical Ltd). Chromatographic separations were carried out on
silica gel 60 (0.063–0.200 or 0.040–0.063 mm, E. Merck) or basic
silica gel (ChromatorexÒ NH, 100–200 mesh, Fuji Silysia Chemical
Ltd) using the indicated eluents. Yields are unoptimized. The HPLC
analyses were performed using a Shimadzu UFLC instrument. Elu-
tion was done with a gradient of 5–90% solvent B in solvent A (sol-
vent A was 0.1% TFA in water, and solvent B was 0.1% TFA in MeCN)
Figure 4. Time course study of the urethral resistance-increasing effects of
compound 13 in rats. Data are expressed as the mean SEM in 5–12 rats.
*P <0.05, **P <0.01 versus prevalue by paired t-test with Holm’s correction.
O-demethylated hydroxymethyl derivative 8 was a major metabo-
lite like compound 6. With this modification, no metabolic switch-
ing, which is a change from one metabolic site to a different site,
was observed. The metabolic rates of these compounds are also
similar (data not shown). Taken together, the aforementioned
results indicate that the deuterium replacement changed only
the contribution rate of metabolic enzymes. Compound 13, which
avoided DDI risks (MDR1 liability) and genetic polymorphism
(contribution of CYP2D6 and CYP2C19), showed low potential for
the liabilities often observed with CAD structures (hERG inhibition
through
a
L-column
2
ODS (3.0 ꢁ 50 mm,
2 lm) column at
1.2 mL minꢀ1. Area % purity was measured at 254 nm. Abbrevia-
tions for solvents are the following: Et2O, diethyl ether; MeOH,
methanol; EtOH, ethanol; EtOAc, ethyl acetate; THF, tetrahydrofu-
ran; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
MeCN, acetonitrile; CDCl3, chroloform-d; Et3N, triethylamine;
Boc2O, di-tert-butyl dicarbonate; WSCꢂHCl, water soluble carbodi-
imide hydrochloride (1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride); HOBtꢂH2O, 1-hydroxybenzotriazole hydrate.
at 10 l
M: 17%),16 favorable pharmacokinetic profiles (rat, bioavail-
ability = 68%), and high peripheral NET selectivity in rats (NET
occupancy = ꢀ3.8%, 3 mg/kg, iv); we therefore selected 13 as a can-
didate for in vivo evaluation.
5.1.1. Typical procedure of obtaining compounds 1–15 by
deprotection of Boc
Typical procedure A: To a solution of Boc intermediate in EtOH
(0.2 M) was added 14.7 M HCl in EtOH (10–100 eq.) at room tem-
perature. The mixture was stirred at room temperature for 1–
24 h and then concentrated in vacuo. The residue was crystallized
or triturated with EtOAc/hexane to give target compound.
Typical procedure B: To a solution of Boc intermediate was added
4 M HCl in EtOAc (10–100 equiv) at room temperature. The mix-
ture was stirred at room temperature for 1–24 h and then concen-
trated in vacuo. The residue was crystallized or triturated with
EtOAc/hexane to give target compound.
To assess the in vivo anti-SUI-related efficacy of compound 13,
we measured the effects on leak point pressure (LPP) in rats, which
indicates urethral resistance (Fig. 4). The LPP was measured from
15 min to 4 h after administration. Compound 13 was adminis-
tered orally at doses of 1, 3, and 10 mg/kg. Compared with the
vehicle group, the results showed that compound 13 increased
the LPP in a dose-dependent manner (Fig. 3). The maximum LPP
elevation was comparable to that induced by esreboxetine,3 which
is a centrally acting NRI that has been demonstrated to be effective
in a clinical phase IIa study.1b Therefore, compound 13 was
selected as a promising peripheral-selective NRI advanced into
further drug development.
5.1.2. Methyl 2-((4-chloro-3-fluorophenyl)(hydroxy)methyl)
acrylate (17b)
To
a
mixture of 4-chloro-3-fluorobenzaldehyde (50 g,
4. Conclusion
315 mmol) and methyl acrylate (42.6 mL, 473 mmol) in MeCN
(160 mL) was added 1,4-diazabicyclo[2.2.2]octane (10.6 g,
94.6 mmol) at room temperature. The mixture was stirred at room
temperature for 3 days, and then concentrated in vacuo. The resi-
due was purified by column chromatography (silica gel, hexane/
EtOAc) to give 17b (69.1 g, 90%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) d 3.19 (1H, d, J = 6.1 Hz), 3.75 (3H, s), 5.51 (1H,
d, J = 6.1 Hz), 5.84 (1H, s), 6.36 (1H, s), 7.11 (1H, d, J = 8.3 Hz),
7.20 (1H, dd, J = 9.8, 1.9 Hz), 7.32–7.42 (1H, m).
We demonstrated that introducing of substituents at the
a-
position of the amide moiety of (6S,7R)-1,4-oxazepane derivatives
1 effectively reduced MDR1 efflux ratio. Among the investigated
derivatives, we observed that the derivative with substituted
methoxy group reduced MDR1 efflux ratio, with low CYP2D6 inhi-
bitory activity; we also discovered compound 6, which was mainly
metabolized by CYP2D6 and CYP2C19. By chemical optimization of
the metabolic hot spot, we observed that replacement of hydrogen
atoms with deuterium atoms changed the major metabolic
enzymes by relatively decreasing the CYP2D6 contribution and
increasing the CYP3A4 contribution in the overall CYP-mediated
metabolism. Peripheral NET selective compound 13 exhibited a
significant increase in urethral resistance in rats in a dose-depen-
dent manner, with maximum effects similar to those of central-
acting and clinically effective esreboxetine.
5.1.3. (2SR,3RS)-Methyl 2-((benzylamino)methyl)-3-(4-chloro-
3-fluorophenyl)-3-hydroxypropanoate (rac-18b)
To a mixture of 17b (69.1 g, 283 mmol) and Et3N (47.3 mL,
339 mmol) in MeOH (710 mL) was added benzylamine (37.1 mL,
339 mmol), and the mixture was stirred at room temperature for
3 days. The mixture was concentrated in vacuo, and the residue

3212
T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
was purified by column chromatography (silica gel, hexane/EtOAc)
to give rac-18b (58.2 g, 59%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 1.57 (1H, br s), 2.66–2.76 (2H, m), 3.12–3.22 (1H, m),
3.65–3.79 (5H, m), 5.28 (1H, d, J = 4.9 Hz), 6.98 (1H, dd, J = 8.3,
1.9 Hz), 7.13 (1H, dd, J = 9.8, 1.9 Hz), 7.24–7.39 (6H, m).
d ꢀ0.02–0.02 (6H, m), 0.89 (9H, s), 2.14–2.28 (1H, m), 2.64–2.82
(2H, m), 2.94 (2H, d, J = 4.2 Hz), 3.46–3.74 (5H, m), 4.00–4.10
(1H, m), 4.48 (1H, d, J = 7.9 Hz), 7.12–7.46 (8H, m).
5.1.8. (6RS,7SR)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-
(hydroxymethyl)-1,4-oxazepane-4-carboxylate (rac-23b)
Chloroformic acid 1-chloroethyl ester (7.35 mL, 68.2 mmol) was
added to a solution of rac-22b (26.4 g, 56.8 mmol) in MeCN
(300 mL). The mixture was stirred at room temperature for
30 min and then concentrated in vacuo. 1 M HCl aq (3.0 mL) was
added to the residue in MeOH (300 mL). The mixture was stirred
at 80 °C for 2 h. After cooling, the mixture was added Boc2O
(13.7 mL, 59.6 mmol) and Et3N (9.50 mL, 68.2 mmol) at 0 °C. The
mixture was stirred at room temperature overnight and then con-
centrated in vacuo. The residue was diluted with water, extracted
with EtOAc, washed with brine, dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by column chromatogra-
phy (silica gel, hexane/EtOAc) to give rac-23b (15.9 g, 78%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) d 1.51 (9H, s), 2.06–2.18
(1H, m), 3.04–3.34 (2H, m), 3.35–3.71 (3H, m), 4.01–4.29 (4H,
m), 4.34 (1H, d, J = 9.5 Hz), 7.08 (1H, d, J = 8.0 Hz), 7.18 (1H, dd,
J = 9.8, 1.9 Hz), 7.30–7.43 (1H, m).
5.1.4. (1RS,2RS)-3-(Benzylamino)-2-((tert-butyldimethylsilyloxy)-
methyl)-1-(4-chloro-3-fluorophenyl)propan-1-ol (rac-19b)
To a suspension of CaCl2 (27.6 g, 248 mmol) in THF (230 mL)
and EtOH (180 mL) was added NaBH4 (12.5 g, 331 mmol) at room
temperature. To the mixture was added dropwise rac-18b
(58.2 g, 166 mmol) in THF (200 mL) and EtOH (150 mL) at 0 °C
and then stirred for 5 h. The mixture was neutralized with 6 M
HCl aq and stirred at room temperature overnight. The mixture
was neutralized with 8 M NaOH aq, and then filtered through
Celite. The filtrate was concentrated in vacuo, extracted with
EtOAc, washed with brine, dried over Na2SO4 and concentrated in
vacuo to give an colorless oil. To a solution of the oil in THF
(420 mL) was added Et3N (21.1 mL, 151 mmol) at 0 °C and then
tert-butylchlorodimethylsilane (22.8 g, 151 mmol) in THF (40 mL)
was added at 0 °C. The mixture was stirred overnight, and then
poured into water and extracted with EtOAc. The organic layer
was separated, washed with brine, dried over MgSO4 and concen-
trated in vacuo. The residue was purified by column chromatogra-
phy (silica gel, hexane/EtOAc) to give rac-19b (29.3 g, 49%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) d 0.01 (6H, s), 0.89 (9H,
s), 1.71–1.81 (1H, m), 2.86–3.05 (2H, m), 3.44–3.53 (1H, m),
3.54–3.62 (1H, m), 3.79 (2H, s), 4.89 (1H, d, J = 6.8 Hz), 7.01–7.07
(1H, m), 7.16 (1H, dd, J = 10.2, 1.9 Hz), 7.29–7.40 (6H, m). MS m/
z: 438.4 [M+H]+.
5.1.9. tert-Butyl (6S,7R)-7-(3,4-dichlorophenyl)-6-(hydroxy-
methyl)-1,4-oxazepane-4-carboxylate
rac-23b (13.1 g) was separated by HPLC (CHIRALPAK AD,
50 mm ID ꢁ 500 mm L, manufactured by DAICEL CHEMICAL
INDUSTRIES, LTD, mobile phase: hexane/ethanol = 900/100) to give
(6S,7R)-isomer (6.4 g, >99.9% ee, recovery rate 97%) having a
shorter retention time and (6R,7S)-isomer (6.3 g, >99.6% ee, recov-
ery rate 97%) having a longer retention time. (6S,7R)-isomer: MS
m/z: 304.2 [M+Hꢀt-Bu]+, (6R,7S)-isomer: MS m/z: 304.2 [M+Hꢀt-
Bu]+.
5.1.5. N-Benzyl-N-((2RS,3RS)-2-((tert-butyldimethylsilyloxy)
methyl)-3-(4-chloro-3-fluorophenyl)-3-hydroxypropyl)-2-
chloroacetamide (rac-20b)
To a mixture of a mixture of rac-19b (29.3 g, 67.0 mmol) and
Et3N (11.2 mL, 80.4 mmol) in THF (340 mL) was added 2-chloroa-
cetyl chloride (5.37 mL, 67.0 mmol) at 0 °C. The mixture was stir-
red at 0 °C for 2 h and then concentrated in vacuo. The residue
was diluted with water, extracted with EtOAc, washed with water
and brine, dried over Na2SO4 and concentrated in vacuo to give
rac-20b (35.0 g, quant.) as a black oil. MS m/z: 514.1 [M+H]+.
5.1.10. (6S,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-
((methylsulfonyloxy)methyl)-1,4-oxazepane-4-carboxylate
(24b)
To a solution of tert-Butyl (6S,7R)-7-(3,4-dichlorophenyl)-6-
(hydroxymethyl)-1,4-oxazepane-4-carboxylate (2.0 g, 5.56 mmol)
in THF (20 mL) was added Et3N (1.55 mL, 11.1 mmol) and
methanesulfonyl chloride (0.86 mL, 11.1 mmol) at 0 °C. The mix-
ture was stirred at 0 °C for 1 h, concentrated in vacuo, diluted with
EtOAc, washed with water and saturated NaHCO3 aq, dried over
Na2SO4 and concentrated in vacuo to give 24b (2.68 g, quant.) as
a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 1.50 (9H, s), 2.30–
2.46 (1H, m), 3.41–4.38 (12H, m), 7.05 (1H, d, J = 7.9 Hz), 7.15
(1H, dd, J = 9.6, 1.7 Hz), 7.34–7.46 (1H, m).
5.1.6. (6SR,7RS)-4-Benzyl-6-((tert-butyldimethylsilyloxy)
methyl)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-3-one
(rac-21b)
To a solution of rac-20b (34.5 g, 67.0 mmol) in THF (1.3 L) was
added 1 M NaOH aq (80 mL, 80 mmol). The mixture was stirred
at room temperature overnight and then concentrated in vacuo.
The residue was diluted with water, extracted with EtOAc, washed
with water and brine, dried over Na2SO4 and concentrated in vacuo
to give rac-21b (31.5 g, 98%) as a pale yellow oil. MS m/z: 478.1 [M
+H]+.
5.1.11. (6S,7R)-tert-Butyl 6-(azidomethyl)-7-(4-chloro-3-fluoro-
phenyl)-1,4-oxazepane-4-carboxylate (25b)
To a solution of 24b (2.68 g, 6.12 mmol) in DMF (30 mL) was
added sodium azide (597 mg, 9.18 mmol) at room temperature
and stirred at 70 °C overnight. The mixture was poured into water
at room temperature, extracted with EtOAc, washed with satd
NH4Cl aq and water, dried over MgSO4 and concentrated in vacuo.
The residue was purified by column chromatography (silica gel,
hexane/EtOAc) to give 25b (1.50 g, 64%) as a colorless oil. 1H
NMR (300 MHz, CDCl3) d 1.51 (9H, s), 2.09–2.36 (1H, m), 3.13–
4.43 (9H, m), 7.00–7.23 (2H, m), 7.34–7.50 (1H, m). MS m/z:
285.2 [M+HꢀBoc]+.
5.1.7. (6SR,7RS)-4-Benzyl-6-((tert-butyldimethylsilyloxy)
methyl)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepane (rac-22b)
To a suspension of AlCl3 (5.28 g, 39.6 mmol) in THF (400 mL)
was added LiAlH4 (4.51 g, 119 mmol) at 0 °C and the mixture was
stirred at 0 °C for 1 h. A solution of rac-21b (31.5 g, 66.0 mmol)
in THF (260 mL) was added to the mixture at 0 °C. After being stir-
red at 0 °C for 2 h, a solution of potassium sodium (+)-tartrate
tetrahydrate (93 g, 330 mmol) was added to the reaction mixture
at 0 °C. The mixture was stirred overnight. The precipitate was
removed by filtration, and the filtrate was concentrated in vacuo.
The residue was diluted with water, extracted with EtOAc, washed
with brine, dried over Na2SO4 and concentrated in vacuo to give
rac-22b (94 g, 96%) as a pale orange oil. 1H NMR (300 MHz, CDCl3)
5.1.12. (6R,7R)-tert-Butyl 6-(aminomethyl)-7-(4-chloro-3-
fluorophenyl)-1,4-oxazepane-4-carboxylate (26b)
To a solution of 25b (8.15 g, 21.2 mmol) in THF (90 mL) and
water (20 mL) was added triphenylphosphine (6.67 g, 25.4 mmol)
and stirred at room temperature overnight. The mixture was

T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
3213
poured into water, extracted with EtOAc, washed with water and
5.1.18. N-(((6S,7R)-7-(3,4-Dichlorophenyl)-1,4-oxazepan-6-yl)
brine, dried over MgSO4 and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, EtOAc/MeOH)
to give 26b (7.18 g, 95%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 1.51 (9H, s), 1.83–2.12 (1H, m), 2.44–2.68 (2H, m), 3.24–
3.46 (1H, m), 3.47–4.24 (6H, m), 7.04 (1H, d, J = 7.5 Hz), 7.15 (1H,
d, J = 9.8 Hz), 7.31–7.43 (1H, m). MS m/z: 359.2 [M+H]+.
methyl)benzamide hydrochloride (4)
Typical procedure B, 94%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 2.68–2.86 (1H, m), 3.00–3.31 (5H, m), 3.34 (1H, s),
3.71–3.91 (1H, m), 3.98 (1H, br s), 4.48 (1H, d, J = 9.8 Hz),7.38–
7.57 (4H, m), 7.66 (1H, d, J = 8.3 Hz), 7.75–7.91 (3H, m), 8.69 (1H,
s), 9.01–9.82 (2H, m). MS m/z: 379.0 [M+H]+.
5.1.13. (6R,7R)-tert-Butyl 7-(3,4-dichlorophenyl)-6-(propiona-
midomethyl)-1,4-oxazepane-4-carboxylate
To a solution of 26b (150 mg, 0.40 mmol) in THF (3 mL) was
added Et3N (84 lL, 0.60 mmol) and propionyl chloride (52 lL,
5.1.19. (6R,7R)-tert-Butyl 7-(3,4-dichlorophenyl)-6-((2-methoxy-
acetamido)methyl)-1,4-oxazepane-4-carboxylate
96%, colorless powder. 1H NMR (300 MHz, CDCl3) d 1.50 (11H,
s), 2.20–2.40 (1H, m), 2.91–3.09 (1H, m), 3.12–3.96 (9H, m), 4.03
(1H, d, J = 9.1 Hz), 4.07–4.17 (1H, m), 7.23 (1H, d, J = 8.3 Hz),
7.37–7.59 (3H, m).
0.60 mmol) and stirred at room temperature overnight. The mix-
ture was poured into water, extracted with EtOAc, washed with
water and brine, dried over MgSO4 and concentrated in vacuo.
The residue was purified by column chromatography (silica gel,
hexane/EtOAc) to give (6R,7R)-tert-butyl 7-(3,4-dichlorophenyl)-
6-(propionamidomethyl)-1,4-oxazepane-4-carboxylate (116 mg,
67%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 1.15 (3H, t,
J = 7.6 Hz), 1.51 (9H, s), 2.11–2.32 (3H, m), 2.92–3.09 (1H, m),
3.09–3.24 (1H, m), 3.24–3.42 (2H, m), 3.46–3.61 (1H, m), 3.92–
4.19 (4H, m), 7.12 (1H, br s), 7.24 (1H, d, J = 1.9 Hz), 7.42 (1H, d,
J = 8.3 Hz), 7.52 (1H, s). MS m/z: 431.0 [M+HꢀBoc]+.
5.1.20. N-(((6S,7R)-7-(3,4-Dichlorophenyl)-1,4-oxazepan-6-yl)
methyl)-2-methoxyacetamide hydrochloride (5)
Typical procedure A, 56%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 2.61 (1H, d, J = 8.7 Hz), 2.85 (1H, dt, J = 14.0, 4.7 Hz),
2.95–3.30 (7H, m), 3.71–3.88 (4H, m), 3.91–4.05 (1H, m), 4.39
(1H, d, J = 9.8 Hz), 7.45 (1H, dd, J = 8.3, 1.9 Hz), 7.63–7.71 (1H, m),
7.76 (1H, d, J = 2.3 Hz), 8.06 (1H, t, J = 6.0 Hz), 9.20 (2H, br s). MS
m/z: 347.3 [M+H]+.
5.1.14. N-(((6S,7R)-7-(3,4-Dichlorophenyl)-1,4-oxazepan-6-yl)
methyl)propionamide hydrochloride (2)
5.1.21. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((2-
methoxyacetamido)methyl)-1,4-oxazepane-4-carboxylate
71%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.50 (9H, s), 2.21–
2.37 (1H, m), 2.94–3.97 (12H, m), 4.01–4.18 (2H, m), 6.49–7.56
(4H, m). MS m/z: 431.2 [M+H]+.
4 M HCl–EtOAc (4 mL, 16.0 mmol) was added to (6R,7R)-tert-
butyl 7-(3,4-dichlorophenyl)-6-(propionamidomethyl)-1,4-oxaze-
pane-4-carboxylate (116 mg, 0.27 mmol) and stirred at room
temperature for 1 h. The mixture was concentrated in vacuo and
the residue was washed with Et2O to give 2 (38.0 mg, 38%) as
amorphous solid. 1H NMR (300 MHz, DMSO-d6) d 0.96 (3H, t,
J = 7.6 Hz), 1.96–2.12 (2H, m), 2.57 (1H, dd, J = 9.8, 6.4 Hz), 2.87
(2H, t, J = 5.9 Hz), 2.99–3.33 (4H, m), 3.71–3.90 (1H, m), 3.99 (1H,
dt, J = 13.7, 4.5 Hz), 4.39 (1H, d, J = 10.2 Hz), 7.45 (1H, dd, J = 8.3,
1.9 Hz), 7.67 (1H, d, J = 8.3 Hz), 7.76 (1H, d, J = 1.9 Hz), 8.01
(1H, t, J = 5.7 Hz), 9.17 (1H, br s), 9.65 (1H, br s). MS m/z: 331.3
[M+H]+.
5.1.22. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl)methyl)-2-methoxyacetamide hydrochloride (6)
Typical procedure A, 76%, colorless crystal. 1H NMR (300 MHz,
DMSO-d6) d 2.55–2.69 (1H, m), 2.80–2.90 (1H, m), 2.95–3.31 (8H,
m), 3.72–3.85 (3H, m), 3.92–4.01 (1H, m), 4.39 (1H, d,
J = 10.2 Hz), 7.33 (1H, dd, J = 8.3, 1.5 Hz), 7.55 (1H, dd, J = 10.6,
1.5 Hz), 7.58–7.66 (1H, m), 8.00–8.14 (1H, m), 8.92–9.58 (2H, m).
MS m/z: 331.1 [M+H]+. Mp: 178.0–178.7 °C. Anal. Calcd for C15H21
-
Compounds 3–6 were prepared in same manner using 26a or
26b.
Cl2N2O3F: C, 49.06; H, 5.76; N, 7.63. Found: C, 49.30; H, 5.60; N,
25
7.44. [
a
]
D
+5.2 (c 0.25, MeOH).
5.1.15. (6R,7R)-tert-Butyl 6-(butyramidomethyl)-7-(3,4-dichloro-
phenyl)-1,4-oxazepane-4-carboxylate
5.1.23. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((3-
methoxypropanamido)methyl)-1,4-oxazepane-4-carboxylate
To a mixture of 26b (100 mg, 0.28 mmol), 3-methoxypropionic
72%, colorless oil. 1H NMR (300 MHz, CDCl3) d 0.90–1.03 (3H,
m), 1.45–1.56 (9H, m), 1.60–1.67 (2H, m), 2.09–2.21 (2H, m),
2.21–2.33 (1H, m), 2.81–3.21 (2H, m), 3.21–3.41 (2H, m), 3.43–
3.60 (1H, m), 3.91–4.21 (4H, m), 7.04–7.19 (1H, m), 7.21–7.27
(1H, m), 7.42 (1H, d, J = 8.3 Hz), 7.51 (1H, s). MS m/z: 445.1 [M+H]+.
acid (31.4
Et3N (97
l
L, 0.33 mmol), HOBtꢂH2O (45.2 mg, 0.33 mmol) and
lL, 0.70 mmol) in THF (1.4 mL) was added WSCꢂHCl
(64.1 mg, 0.33 mmol). The mixture was stirred at room tempera-
ture for 3 h, and then poured into water and extracted with EtOAc.
The organic layer was separated, washed with water and brine,
dried over Na2SO4 and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, Hexane/EtOAc) to
give (6R,7R)-tert-butyl 7-(4-chloro-3-fluorophenyl)-6-((3-methox-
5.1.16. N-(((6S,7R)-7-(3,4-Dichlorophenyl)-1,4-oxazepan-6-yl)
methyl)butyramide hydrochloride (3)
Typical procedure B, 77%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 0.83 (3H, t, J = 7.4 Hz), 1.47 (2H, sxt, J = 7.4 Hz), 2.01
(2H, t, J = 7.4 Hz), 2.58 (1H, d, J = 2.6 Hz), 2.76–3.01 (2H, m),
3.02–3.36 (4H, m), 3.69–3.91 (1H, m), 4.00 (1H, dt, J = 13.9,
4.2 Hz), 4.39 (1H, d, J = 10.2 Hz), 7.35–7.52 (1H, m), 7.60–7.72
(1H, m), 7.76 (1H, d, J = 1.9 Hz), 8.04 (1H, t, J = 5.9 Hz), 9.22 (1H,
br s), 9.73 (1H, br s). MS m/z: 345.5 [M+H]+.
ypropanamido)methyl)-1,4-oxazepane-4-carboxylate
(81 mg,
65%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 1.50 (9H, s),
2.20–2.49 (3H, m), 2.94–3.40 (7H, m), 3.47–3.74 (3H, m), 3.91–
4.16 (4H, m), 6.13–7.47 (4H, m). MS m/z: 445.4 [M+H]+.
5.1.24. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl)methyl)-3-methoxypropanamide hydrochloride (7)
Typical procedure B, 81%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 2.28 (2H, t, J = 6.4 Hz), 2.53–2.64 (1H, m), 2.81–2.95
(2H, m), 3.05–3.39 (7H, m), 3.49 (2H, t, J = 6.4 Hz), 3.72–3.85 (1H,
m), 3.94–4.06 (1H, m), 4.38 (1H, d, J = 10.2 Hz), 7.33 (1H, d,
J = 8.3 Hz), 7.53 (1H, dd, J = 10.6, 1.5 Hz), 7.59–7.67 (1H, m), 8.01–
8.18 (1H, m), 8.88–9.77 (2H, m). MS m/z: 345.4 [M+H]+.
5.1.17. (6R,7R)-tert-Butyl 6-(benzamidomethyl)-7-(3,4-
dichlorophenyl)-1,4-oxazepane-4-carboxylate
81%, colorless powder. 1H NMR (300 MHz, CDCl3) d 1.49 (9H, s),
2.41 (1H, td, J = 7.7, 4.2 Hz), 3.03–3.26 (2H, m), 3.38 (1H, dd,
J = 14.7, 4.5 Hz), 3.45–3.81 (2H, m), 3.96–4.27 (4H, m), 7.29 (1H,
d, J = 2.3 Hz), 7.37–7.50 (4H, m), 7.50–7.62 (1H, m), 7.88 (2H, d,
J = 7.2 Hz), 8.02 (1H, br s). MS m/z: 479.3 [M+H]+.
Compounds 9–15 were prepared in same manner using 26b.

3214
T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
5.1.25. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((2-
methoxypropanamido)methyl)-1,4-oxazepane-4-carboxylate
76%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.24–1.75 (12H,
m), 2.19–2.37 (1H, m), 2.87–4.18 (13H, m), 6.59–7.67 (4H, m).
MS m/z: 445.4 [M+H]+.
5.1.33. tert-Butyl (6R,7R)-7-(4-chloro-3-fluorophenyl)-6-
[({[(2H3)methyloxy]acetyl}amino)methyl]-1,4-oxazepane-4-
carboxylate
84%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.46–1.55 (9H,
m), 2.19–2.38 (1H, m), 2.94–3.96 (9H, m), 4.01–4.18 (2H, m),
6.44–7.57 (4H, m). MS m/z: 434.2 [M+H]+.
5.1.26. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl)methyl)-2-methoxypropanamide hydrochloride (9)
Typical procedure B, 73%, colorless crystal. 1H NMR (300 MHz,
DMSO-d6) d 1.18 (3H, d, J = 6.8 Hz), 2.58–2.73 (1H, m), 2.76–2.90
(1H, m), 2.92–3.38 (8H, m), 3.63 (1H, q, J = 6.8 Hz), 3.72–3.86
(1H, m), 3.91–4.04 (1H, m), 4.34–4.43 (1H, m), 7.34 (1H, d,
J = 8.3 Hz), 7.51–7.68 (2H, m), 8.14 (1H, t, J = 5.9 Hz), 9.14–9.88
(2H, m). MS m/z: 345.2 [M+H]+.
5.1.34. N-{[(6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl]methyl}-2-[(2H3)methyloxy]acetamide hydrochloride (13)
Typical procedure B, 83%, colorless crystal. 1H NMR (300 MHz,
DMSO-d6) d 2.54–2.69 (1H, m), 2.79–2.90 (1H, m), 2.95–3.30 (5H,
m), 3.71–3.86 (3H, m), 3.91–4.01 (1H, m), 4.39 (1H, d, J = 9.8 Hz),
7.33 (1H, dd, J = 8.3, 1.9 Hz), 7.55 (1H, dd, J = 10.6, 1.9 Hz), 7.58–
7.66 (1H, m), 8.06 (1H, t, J = 6.0 Hz), 8.90–9.60 (2H, m). MS m/z:
334.1 [M+H]+. Anal. Calcd for C15H18Cl2N2O3FD3: C, 48.66; H,
25
4.90; N, 7.57. Found: C, 48.74; H, 4.88; N, 7.55. [
MeOH).
a]
+7.5 (c 0.25,
D
5.1.27. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((2-
methoxy-2-methylpropanamido)methyl)-1,4-oxazepane-4-
carboxylate
5.1.35. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-
((tetrahydrofuran-2-carboxamido)methyl)-1,4-oxazepane-4-
carboxylate
65%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.28–1.38 (6H,
m), 1.47–1.57 (9H, m), 2.19–2.36 (1H, m), 2.86–4.24 (12H, m),
6.72–7.71 (4H, m). MS m/z: 459.2 [M+H]+.
92%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.44–1.56 (9H,
m), 1.69–2.02 (3H, m), 2.16–2.34 (2H, m), 2.85–4.37 (12H, m),
6.58–7.73 (4H, m). MS m/z: 457.1 [M+H]+.
5.1.28. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-
yl)methyl)-2-methoxy-2-methylpropanamide hydrochloride (10)
Typical procedure B, 75%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 1.17–1.28 (6H, m), 2.55–2.67 (1H, m), 2.70–2.85
(1H, m), 2.92–3.26 (5H, m), 3.33 (3H, s), 3.70–3.87 (1H, m),
3.88–4.03 (1H, m), 4.39 (1H, d, J = 10.2 Hz), 7.34 (1H, dd,
J = 8.3, 1.5 Hz), 7.55 (1H, dd, J = 10.6, 1.9 Hz), 7.58–7.67 (1H,
m), 8.06 (1H, t, J = 6.0 Hz), 9.10 (1H, br s), 9.44 (1H, br s). MS
m/z: 359.2 [M+H]+.
5.1.36. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-
yl)methyl)tetrahydrofuran-2-carboxamide hydrochloride (14)
Typical procedure B, 39%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 1.70–1.86 (3H, m), 2.01–2.18 (1H, m), 2.57–2.70 (1H,
m), 2.73–2.86 (1H, m), 2.95–3.33 (5H, m), 3.69–3.88 (3H, m),
3.91–4.03 (1H, m), 4.12–4.20 (1H, m), 4.39 (1H, d, J = 9.8 Hz),
7.33 (1H, d, J = 8.3 Hz), 7.50–7.67 (2H, m), 8.02–8.12 (1H, m),
9.04–9.82 (2H, m). MS m/z: 357.4 [M+H]+.
5.1.29. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((2-
ethoxyacetamido)methyl)-1,4-oxazepane-4-carboxylate
67%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.14–1.31 (3H,
m), 1.46–1.55 (9H, m), 2.22–2.37 (1H, m), 2.85–4.00 (11H, m),
4.01–4.19 (2H, m), 6.49–7.76 (4H, m). MS m/z: 445.4 [M+H]+.
5.1.37. (6R,7R)-tert-Butyl 6-((1,4-dioxane-2-carboxamido)
methyl)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepane-4-
carboxylate
67%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.51 (9H, br s),
2.16–2.40 (1H, m), 2.93–4.27 (16H, m), 6.42–7.58 (4H, m). MS m/
z: 417.2 [M+HꢀBoc]+.
5.1.30. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl)methyl)-2-ethoxyacetamide hydrochloride (11)
Typical procedure B, 86%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 1.14 (3H, t, J = 7.0 Hz), 2.55–2.68 (1H, m), 2.78–
2.89 (1H, m), 2.98–3.29 (5H, m), 3.45 (2H, q, J = 6.8 Hz),
3.73–3.85 (3H, m), 3.90–4.02 (1H, m), 4.39 (1H, d,
J = 10.2 Hz), 7.32 (1H, d, J = 7.9 Hz), 7.54 (1H, d, J = 10.6 Hz),
7.59–7.67 (1H, m), 7.89–8.00 (1H, m), 8.77–9.45 (2H, m). MS
m/z: 345.2 [M+H]+.
5.1.38. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl)methyl)-1,4-dioxane-2-carboxamide hydrochloride (15)
Typical procedure B, 63%, colorless solid. 1H NMR (300 MHz,
DMSO-d6) d 2.57–2.70 (1H, m), 2.76–2.89 (1H, m), 2.90–3.52 (7H,
m), 3.56–3.72 (2H, m), 3.72–3.90 (3H, m), 3.90–4.03 (2H, m),
4.38 (1H, d, J = 9.8 Hz), 7.32 (1H, d, J = 8.3 Hz), 7.54 (1H, dd,
J = 10.6, 1.9 Hz), 7.58–7.68 (1H, m), 8.11 (1H, t, J = 5.1 Hz), 8.99–
9.80 (2H, m). MS m/z: 373.0 [M+H]+.
5.1.31. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((2-
isopropoxyacetamido)methyl)-1,4-oxazepane-4-carboxylate
100%, colorless oil. 1H NMR (300 MHz, CDCl3) d 1.09–1.31
(6H, m), 1.44–1.55 (9H, m), 2.22–2.40 (1H, m), 2.82–3.97 (9H,
m), 4.00–4.20 (3H, m), 6.49–7.78 (4H, m). MS m/z: 359.3 [M
+HꢀBoc]+.
5.1.39. X-ray structure analysis
Crystal data for compound 6: C15H21ClFN2O+3Å Clꢀ, MW = 367.25;
crystal size, 0.28 ꢁ 0.06 ꢁ 0.03 mm; colorless, needle; monoclinic,
space group P21, a = 8.10307(15) Å, b = 23.9821(4) Å, c = 9.94990
(18) Å,
a = c
= 90°, b = 115.8618(7)°, V = 1739.90(6) Å3, Z = 4,
Dx = 1.402 g/cm3, T = 100 K,
l
= 3.587 mmꢀ1
,
k = 1.54187 Å,
R1 = 0.046, wR2 = 0.110, Flack Parameter18 = ꢀ0.009(15).
All measurements were made on a Rigaku R-AXIS RAPID diffrac-
tometer using graphite monochromated Cu-Ka radiation. The
structure was solved by direct methods with SHELXS-9719 and
was refined using full-matrix least-squares on F2 with SHELXL-
97.19 All non-H atoms were refined with anisotropic displacement
parameters.
CCDC 1437357 for compounds 6 contains the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
5.1.32. N-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-
6-yl)methyl)-2-isopropoxyacetamide hydrochloride (12)
Typical procedure B, 46%, colorless crystal. 1H NMR
(300 MHz, DMSO-d6) d 1.11 (6H, d, J = 6.0 Hz), 2.56–2.71 (1H,
m), 2.78–2.91 (1H, m), 2.99–3.33 (7H, m), 3.48–3.62 (1H, m),
3.76–3.87 (1H, m), 3.90–4.05 (1H, m), 4.40 (1H, d, J = 9.8 Hz),
7.32 (1H, d, J = 8.3 Hz), 7.48–7.69 (2H, m), 7.83 (1H, t,
J = 6.0 Hz), 8.92–9.62 (2H, m). MS m/z: 359.2 [M+H]+. Anal.
Calcd for C17H25Cl2N2O3F: C, 51.65; H, 6.37; N, 7.09. Found: C,
51.70; H, 6.43; N, 7.08.

T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
3215
via
http://www.ccdc.cam.ac.uk/Community/Requestastructure/
(PerkinElmer) was dispensed to each well by 100
lL, and the mix-
Pages/DataRequest.aspx.
ture was stirred for about 30 min. The radioactivity was measured
by TopCount (PerkinElmer). The inhibitory activity of each com-
5.2. Monoamine reuptake inhibition
pound (10
lM) was calculated as a relative activity value based
on the inhibitory activity of 10
lM Paroxetine (serotonin trans-
5.2.1. Preparation of human monoamine expressing cell
Human serotonin transporter cDNA was amplified from human
brain cDNA library by PCR, and inserted into pCRII-TOPO vector
(manufactured by Invitrogen). The base sequence was confirmed
and modified, and subcloned to pcDNA3.1 vector (manufactured
by Invitrogen), whereby a human serotonin transporter expression
plasmid was constructed.
Human norepinephrine transporter cDNA was purchased from
Invitrogen, and the base sequence was confirm and modified, and
subcloned to pcDNA3.1 vector, whereby a human norepinephrine
transporter expression plasmid was constructed.
porter inhibitor) as 100%. IC50 and 95% confidence interval were
calculated by XLfit (n = 2).
5.2.3. Human norepinephrine transporter inhibitory activity
CHO cells stably expressing human norepinephrine transporter
were used for the measurement of human norepinephrine trans-
porter inhibitory activity. Unless otherwise indicated, these CHO
cells were cultured in Ham/F12 medium (Invitrogen) containing
10% fetal bovine serum (MOREGATE). The cultured cells that
reached almost confluent were rinsed with PBS (Invitrogen),
detached with Trypsin/EDTA (Invitrogen), and collected by a cen-
trifugal operation. The obtained cells were counted, and diluted
to 3 ꢁ 105 cells per 1 mL medium, and the mixture was dispensed
Human dopamine transporter expression plasmid was prepared
by following procedure. SR
a promoter contained in pTB1411
described in JP-A-H5-076385 was cleaved with restriction enzyme
HindIII (manufactured by TAKARA BIO INC.), blunt-ended, further
cleaved with restriction enzyme EcoRI (manufactured by TAKARA
BIO INC.), and fragmented. On the other hand, pCI vector was
cleaved with restriction enzyme BglII (manufactured by TAKARA
BIO INC.), blunt-ended with T4DNA polymerase, and further
cleaved with restriction enzyme EcoRI (manufactured by TAKARA
to a 96 well white plate (Corning) at 100 lL per well, and cultured
overnight in a CO2 incubator. Then, an assay buffer (126 mM NaCl,
4.95 mM KCl, 1.26 mM KH2PO4, 1.26 mM MgSO4, 10 mM HEPES,
2.32 mM CaCl2, 5.52 mM Glucose, 0.5% BSA) was prepared, the
medium in the cell plate was removed and the assay buffer was
added by 80 lL. A test compound was diluted with the assay buffer
to a 10-fold concentration of the final concentration, and the mix-
BIO INC.). Into this site was inserted a SR
a promoter fragment to
ture was dispensed to a 96 well polypropylene plate. The diluted
give pCI-SRa. Then, pCI-SRa was cleaved with restriction enzyme
ClaI (manufactured by TAKARA BIO INC.) and blunt–ended. Into
this site was inserted a 1.63 Kb fragment obtained by cleaving
pGFP-C1 (manufactured by TOYOBO) with restriction enzyme
Bsu36I (manufactured by Daiichi Pure Chemicals Co., Ltd) followed
test compound was dispensed to the cell plate by 10 l
L. [3H]-Nore-
pinephrine (GE Healthcare) was diluted with the assay buffer to
200 nM, and the mixture was dispensed to the cell plate by
10
fer was removed by suction, and the plate was washed twice with
150 L of PBS (Invitrogen) per well. Microscinti 20 (PerkinElmer)
was dispensed to each well by 100 L, and the mixture was stirred
for about 30 min. The radioactivity was measured by TopCount
(PerkinElmer). The inhibitory activity of each compound (10 M)
was calculated as a relative activity value based on the inhibitory
activity of 10 M DMI (norepinephrine transporter inhibitor) as
l
L. At 45 min from [3H]-norepinephrine addition, the assay buf-
by blunt-ending, whereby pMSR
a
neo was prepared. Human dopa-
l
mine transporter cDNA was amplified from human substantia
nigra cDNA library by PCR, and inserted into the pCRII vector (man-
ufactured by Invitrogen). The base sequence was confirmed, mod-
l
l
ified and subcloned to pMSRa neo, whereby a human dopamine
transporter expression plasmid was constructed.
l
The monoamine transporter expression plasmids thus prepared
were introduced into CHO-K1 cells using FuGENE6 (manufactured
by Roche Diagnostics) and according to the attached protocol,
whereby each expressing cell was established.
100%. IC50 and 95% confidence interval were calculated by XLfit
(n = 2).
5.2.4. Human dopamine transporter inhibitory activity
CHO cells stably expressing human dopamine transporter were
used for the measurement of human dopamine transporter inhibi-
tory activity. Unless otherwise indicated, these CHO cells were cul-
tured in Ham/F12 medium (Invitrogen) containing 10% fetal bovine
serum (MOREGATE). One day before the assay, the cultured cells
that reached almost confluent were rinsed with PBS (Invitrogen),
detached with Trypsin/EDTA (Invitrogen), and collected by a cen-
trifugal operation. The obtained cells were counted, and diluted
to 3 ꢁ 105 cells per 1 mL medium, and the mixture was dispensed
5.2.2. Human serotonin transporter inhibitory activity
CHO cells stably expressing a human serotonin transporter
were used for the measurement of human serotonin transporter
inhibitory activity. Unless particularly indicated, these CHO cells
were cultured in a Ham/F12 medium (Invitrogen) containing 10%
fetal bovine serum (MOREGATE). The cultured cells that reached
almost confluent were rinsed with PBS (Invitrogen), detached with
Trypsin/EDTA (Invitrogen), and collected by a centrifugal opera-
tion. The obtained cells were counted, and diluted to 3 ꢁ 105 cells
per 1 mL medium, the mixture was dispensed to a 96 well white
to a 96 well white plate (Corning) at 100 lL per well, and cultured
overnight in a CO2 incubator. On the day of the test, an assay buffer
(126 mM NaCl, 4.95 mM KCl, 1.26 mM KH2PO4, 1.26 mM MgSO4,
10 mM HEPES, 2.32 mM CaCl2, 5.52 mM Glucose, 0.5% BSA) was
prepared, the medium in the cell plate was removed and the assay
plate (Corning) at 100 lL per well, and cultured overnight in a
CO2 incubator. Then, an assay buffer (126 mM NaCl, 4.95 mM
KCl, 1.26 mM KH2PO4, 1.26 mM MgSO4, 10 mM HEPES, 2.32 mM
CaCl2, 5.52 mM Glucose, 0.5% BSA) was prepared, the medium in
the cell plate was removed and the assay buffer was added by
80 lL. A test compound was diluted with the assay buffer to a
10-fold concentration of the final concentration, and the mixture
buffer was added by 80 lL. A test compound was diluted with the
assay buffer to a 10-fold concentration of the final concentration,
and the mixture was dispensed to a 96 well polypropylene plate.
The diluted test compound was dispensed to the cell plate by
was dispensed to a 96 well polypropylene plate. The diluted test
10
buffer to 200 nM, cold dopamine was diluted to 10
mixture was dispensed to the cell plate by 10 L. At 60 min from
[3H]-dopamine addition, the assay buffer was removed by suction,
and the plate was washed twice with 150 L of PBS (Invitrogen)
per well. Microscinti 20 (PerkinElmer) was dispensed to each well
by 100 L, and the mixture was stirred for about 30 min. The
l
L. [3H]-Dopamine (GE Healthcare) was diluted with the assay
compound was dispensed to the cell plate by 10
l
L. [3H]-5-
lM, and the
Hydroxytryptamine (GE Healthcare) was diluted with the assay
buffer to 200 nM, and the mixture was dispensed to the cell plate
l
by 10
assay buffer was removed by suction, and the plate was washed
twice with 150 L of PBS (Invitrogen) per well. Microscinti 20
l
L. At 20 min from [3H]-5-hydroxytryptamine addition, the
l
l
l

3216
T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
radioactivity was measured by TopCount (PerkinElmer). The inhi-
bitory activity of each compound (10 M) was calculated as a rel-
ative activity value based on the inhibitory activity of 100
5.5. CYP2D6 inhibition
l
lM
Inhibition activity of the test compounds of CYP2D6 was evalu-
Nomifensine (dopamine transporter inhibitor) as 100%. IC50 and
ated by incubating 5
lmol/L bufuralol with 2 nmol/L CYP2D6
95% confidence interval were calculated by XLfit (n = 2).
derived from CYP2D6-expressing insect cells (BD Biosciences) in
the presence of 10 lmol/L test compound. The incubation mixture
5.3. NET ex vivo binding assay in rat brain cortex
was allowed to stand for 60 min at 27 °C. The concentration of 10-
hydroxybufuralol was measured by high performance liquid chro-
matography/tandem mass spectrometry (LC/MS/MS).
Adults female rats of Sprague–Dawley strain (CLEA Japan)
were studied using experimental protocols approved by Takeda’s
Experimental Animal Care and Use Committee. CNS penetrating
property was measured by investigating occupation rate of NET
in the brain cortex after drug administration. After rats were
anesthetized with intraperitoneal injection of urethane (Wako
Pure Chemical Industries, Ltd), the test compound was intra-
venously injected. Twenty minutes after the injection, the cortex
in the left brain was taken off and used for ex vivo binding study
with [3H]-nisoxetine (NEN). Fifteen times volume of assay buffer
[50 mmol/L Tris Cl (pH 7.5), 125 mmol/L NaCl, 5 mmol/L KCl] as
the taken cortex weight was added, homogenized, and plasma
membrane fraction was prepared so that protein concentration
5.6. Parallel artificial membrane permeability assay (PAMPA)
PAMPA Evolution system (pION) was applied for the assay. After
incubating the test compound (10 lmol/L) for 3 h at 25 °C, the per-
meation coefficient value was calculated with determining the
peak areas of test compound by high performance liquid chro-
matography or LC/MS/MS.
5.7. Permeability study across human MDR1 stably expressing
cells
of the fraction was 4 mg/mL. Six hundred
fraction, 200 L of the assay buffer and 100
solution (25 nmol/L) were mixed, and 100
(10 mol/L) or assay buffer was added, and the mixture was incu-
bated at room temperature for 1 hour. The mixture was suctioned
through GF/B glass filter (Whatman), and the glass filter was
washed with assay buffer. Radio activity with the glass filter
was measured using scintillation counter (Aloka, LSC6100). The
NET occupation rate was calculated with specific binding of the
vehicle group as 100%.
l
l
L of the membrane
L of [3H]-nisoxetine
L of desipramine
The transcellular transport was examined using human mul-
tidrug resistance 1 (MDR1) stably expressing LLC-PK1 cells. After
incubating the test compound (10 lmol/L) for 2 h at 37 °C, the api-
cal to basolateral permeation coefficient value and the basolateral
to apical permeation coefficient value as well as ratio of the perme-
ation coefficient (efflux ratio) were calculated with determining
the concentration of test compound by LC/MS/MS. The apparent
permeability (Papp) of test compounds in the receiver wells was
determined and the efflux ratio (ER) for MDR1 membrane perme-
ability test was calculated using the following equation:
l
l
l
Efflux ratio ¼ PappðBtoAÞ=PappðAtoBÞ;
ð1Þ
5.4. Evaluation of urethral resistance-increasing effects in rat
where Papp(AtoB) and Papp(BtoA) are ratio of permeation amount in
After rats were anesthetized with isoflurane (Abbott Japan)
inhalation, the spinal cord was transected at the T8–T9 level after
laminectomy. After closing the wound site on the back, the uri-
nary bladder was exposed through an abdominal incision. Two
polyethylene catheters (PE-100; Intramedic, Becton Dickson and
Company) with a fire-flared tip were inserted into the bladder
from the dome and secured with a ligature for bladder filling
and pressure recording, and then, the abdomen was closed. Rats
were placed in Bollman restraint cage (KN-326/3, Natsume Seisa-
kusho). A pressure transducer (DX-100; Nihon Koden) connected
to an amplifier (1257; NEC), an analog-to-digital converter (MP-
100A-CE; BIOPAK systems) and a computer equipped with a data
converting software (AcqKnowledge; BIOPAK systems) was used
to record the intravesical pressure. Data was acquired at the rate
of 100 Hz.
A to B and B to A, respectively.
5.8. Pharmacokinetic study in rats
The test compound was administered intravenously (0.1 mg/kg)
or orally (1 mg/kg) to 8 week old male Crl:CD(SD)(IGS) rats (cas-
sette dosing, n = 3 each). The blood samples were collected 5, 10,
15, 30 min, 1, 2, 4 and 8 h after intravenous administration and
15, 30 min, 1, 2, 4 and 8 h after oral administration. The blood sam-
ples were centrifuged to prepare the plasma samples. The plasma
samples were pretreated with protein precipitation method. The
concentrations of compounds were determined by LC/MS/MS.
5.9. Phenotyping study using CYP-expressing microsomes
The bladder was then filled continuously with saline (0.1 mL/
s) containing Evans blue dye (Wako Pure Chemical) via the
catheter using infusion pump (Kds100; KDScientific). The intrav-
esical pressure was raised by infusion until leakage of the Evans
blue solution was observed from the urethral orifice. The peak of
recorded intravesical pressure was recognized as the lowest
intravesical pressure inducing urinary leakage, and the value
was referred as the leak point pressure (LPP). As urine leakage
theoretically occurs when intravesical pressure exceeds total
urethral resistance, LPP is considered to show the total urethral
resistance. Measurements were repeated, and the mean of last
three LPPs was calculated. The vehicle and the test compound
were orally administered, and LPP was again measured 15, 30,
60, 120 and 240 min after oral administration. Data were ana-
lyzed with paired t-test, and P values <0.05 was considered to
be significant.
The in vitro metabolic stability in CYP-expressing microsomes
(CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1
and CYP3A4, BD Biosciences) was evaluated by incubating the test
compound (1 lmol/L) for 10 min at 37 °C with determining the
concentration of the test compound by ultra performance liquid
chromatography. The raw data was corrected with abundance of
cytochromes P450 in human liver obtained from literature20 to cal-
culate the contribution ratio of each CYP isoform.
5.10. Metabolite profiling study using cryopreserved human
hepatocytes
The in vitro metabolite profile were investigated by incubating
the test compound (10 lmol/L) for 6 h at 37 °C with cryopreserved
human hepatocytes (0.6 ꢁ 106 cells/mL). The structure elucidation
for metabolites were performed with LC/MS/MS.

T. Yukawa et al. / Bioorg. Med. Chem. 24 (2016) 3207–3217
3217
7. Cox, C. D.; Coleman, P. J.; Breslin, M. J.; Whitman, D. B.; Garbaccio, R. M.; Fraley,
M. E.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao,
W.; Davide, J. P.; Diehl, R. E.; Abrams, M. T.; South, V. J.; Huber, H. E.; Torrent,
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Software, Daylight Chemical Information Systems, Inc., Aliso Viejo, CA; http://
www.daylight.com).
11. Others: CYP3A4, 2E1, 2C9, 2C8, 2B6 and 1A2. Metabolic velocity (%/min/pmol):
0.081/0.016 (CYP2D6/2C19).
12. (a) Bertilsson, L.; Dahl, M. L.; Dalén, P.; Al-Shurbaji, A. Br. J. Clin. Pharmacol.
2002, 53, 111; (b) Varenhorst, C.; James, S.; Erlinge, D.; Brandt, J. T.; Braun, O.
O.; Man, M.; Siegbahn, A.; Walker, J.; Wallentin, L.; Winters, K. J.; Close, S. L.
Eur. Heart J. 2009, 30, 1744.
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Acknowledgments
We thank Dr. Satoshi Yamamoto and Dr. Teruaki Okuda for
helpful discussions; Mr. Mitsuyoshi Nishitani for the help in X-
ray crystallographic analysis.
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