Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors – Part 3

Article abstract of DOI:10.1016/j.bmc.2016.06.014

Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Here, we describe our medicinal chemistry approach to discover a no

Full text of DOI:10.1016/j.bmc.2016.06.014

Accepted Manuscript
Design, synthesis, and biological evaluation of a novel series of peripheral-se-
lective noradrenaline reuptake inhibitors: Part 3
Tomoya Yukawa, Yoshihisa Nakada, Nobuki Sakauchi, Taku Kamei, Masami
Yamada, Yusuke Ohba, Ikuo Fujimori, Hiroyuki Ueno, Maiko Takiguchi,
Masako Kuno, Izumi Kamo, Hideyuki Nakagawa, Yasushi Fujioka, Tomoko
Igari, Yuji Ishichi, Tetsuya Tsukamoto
PII:
S0968-0896(16)30438-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.014
BMC 13070
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 April 2016
4 June 2016
6 June 2016
Please cite this article as: Yukawa, T., Nakada, Y., Sakauchi, N., Kamei, T., Yamada, M., Ohba, Y., Fujimori, I.,
Ueno, H., Takiguchi, M., Kuno, M., Kamo, I., Nakagawa, H., Fujioka, Y., Igari, T., Ishichi, Y., Tsukamoto, T.,
Design, synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake
inhibitors: Part 3, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.014
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Design, synthesis, and biological evaluation of a novel series of peripheral-selective
noradrenaline reuptake inhibitors: Part 3
Tomoya Yukawa*, Yoshihisa Nakada, Nobuki Sakauchi, Taku Kamei, Masami Yamada, Yusuke Ohba,
Ikuo Fujimori, 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
Abstract
Peripheral-selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of
stress urinary incontinence to overcome adverse effects associated with central action. Here, we
describe our medicinal chemistry approach to discover a novel series of highly potent, peripheral-
selective, and orally available noradrenaline reuptake inhibitors with a low multidrug resistance protein
1 (MDR1) efflux ratio by cyclization of an amide moiety and introduction of an acidic group. We
observed that the MDR1 efflux ratio was correlated with the pKa value of the acidic moiety. The
resulting compound 9 exhibited favorable PK profiles, probably because of the effect of intramolecular
hydrogen bond, which was supported by a its single-crystal structure. The compound 9, 1-{[(6S,7R)-7-
(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-oxo-1,2-dihydropyridine-3-carboxylic
acid
hydrochloride, which exhibited peripheral NET-selective inhibition at tested doses in rats by oral
administration, increased urethral resistance in a dose-dependent manner.
1

KEYWORDS: Peripheral-selective noradrenaline reuptake inhibitor; 7-Aryl-1,4-oxazepane derivatives;
Stress urinary incontinence; MDR1 efflux ratio; Intramolecular hydrogen bond.
ABBREVIATIONS: SUI, Stress urinary incontinence; DAT, dopamine transporter; SERT, serotonin
transporter; NET, norepinephrine transporter; MDR1, multidrug resistance protein 1; P-gp, P-
glycoprotein.
*Corresponding author. Tel: +81-466-32-1105. Fax: +81-466-29-4454;
e-mail: tomoya.yukawa@takeda.com
1. Introduction
Peripheral-selective inhibition of noradrenaline reuptake has emerged as a new drug concept with the
potential for the treatment of stress urinary incontinence (SUI). Biological evaluation of highly
peripheral-selective noradrenaline reuptake inhibitors (NRIs) have been studied in our laboratory.1,2
Peripheral-selective NRIs are assumed to have several advantages with respect to safety and efficacy
over traditional central-acting NRIs as anti-SUI agents.3
We previously reported 7-phenyl-6-substituted oxazepane derivatives as peripheral-selective NRIs.1,2
In these studies, our chemical modification focused on enhancing peripheral norepinephrine transporter
(NET) selectivity and reducing the multidrug resistance protein 1 (MDR1) efflux ratio to mitigate drug–
drug interaction (DDI) risks. P-glycoprotein (P-gp) is the major efflux transporter at the blood–brain
barrier and is responsible for the efflux of a number of xenobiotic substances from the central nervous
system.4 DDI with P-gp inhibitor5 may affect the balance of distribution of our compound to the central
and peripheral nervous systems, resulting in a low peripheral NET selectivity. Derivatization of 7-
phenyl-6-substituted oxazepane derivative 1 gave trideuterium methoxy derivative 2 as a potent and
peripheral NET-selective inhibitor with a low MDR1 efflux ratio.2
2

In parallel with the aforementioned study, we investigated a new series of compounds for novel
peripheral-selective NRIs. We initially explored a new scaffold exhibiting strong NET inhibitory
activity. In our previous SAR study, we observed that a 3,4-disubstituted phenyl ring and an oxazepane
ring were necessary for NET inhibitory activity. We also observed that the terminal amide moiety is a
good enhancer for the NET inhibitory activity. For further study of the side chain containing the amide
moiety, we investigated cyclization of the terminal amide moiety, and observed that pyrrolidone
derivative 3 exhibited potent NET inhibitory activity compared with acyclic derivative 1. We selected
cyclic compound 3 as a lead compound for a new series of NRIs and investigated the peripheral NET
selectivity. However, we also observed that the N-methylated derivative 4 exhibited high NET
occupancy in the rat brain after intracavernous administration in our initial SAR. We speculated that the
decrease in peripheral NET selectivity was due to the removal of one hydrogen bond donor (HBD),6 and
that the introduction of a hydrophilic group resulted in the recovery of high peripheral NET selectivity
by increasing hydrogen bond aceptor (HBA) and/or HBD.7 However, in general, adding HBA and/or
HBD increases the MDR1 efflux ratio.8 To identify peripheral NET-selective candidates with a low
MDR1 efflux ratio, we selected an acidic group as a hydrophilic group. Acidic groups are known to
increase peripheral selectivity9 and acidic compounds commonly exhibit relatively low MDR1 efflux
ratios.10 In this study, we performed the synthesis of 7-aryl-6-substituted oxazepane derivatives with an
acidic group on the designed amide-containing ring on the basis of basic scaffold A.
[Figure 1]
Figure 1. Design concept
3

2. Chemistry
The preparation of cyclic ring derivatives 3, 5, and 6 is shown in Scheme 1. (6R,7R)-Amine
intermediate 17 was prepared according a previously reported procedure.2 Condensation of amine 17
with acid chloride or isocyanate afforded alkyl chlorides 18a–18c. Cyclization of 18a–18c under basic
conditions followed by treatment with hydrogen chloride gave 3, 5, and 6 as the HCl salts.
[Scheme 1]
Scheme 1. Synthesis of Compounds 3, 5, and 6 a
4

Reagents and conditions: (a) R1COCl, Et3N, THF, rt or R2CNO, Et3N, CH3CN, rt; (b) NaH, THF or
a
DMF, 0°C to rt; (c) 4 M HCl in EtOAc, rt.
The preparation of 2-pyridone or 3(2H)-pyridazinone derivatives 7–16 is illustrated in Scheme 2.
Compound 7 or 8 was synthesized by the reaction of 2-pyridone or pyridazinone with mesylate 202 and
subsequent de-protection of the Boc group.
The pyridone possessing the ester or cyano groups were coupled with mesylate 20 to give N-
substituted pyridones 21 or 22a–22d. Removal of the Boc group of 21 resulted in ester 13. The
carboxylic acid 9 was prepared by modification of 21 followed by de-protection reaction. The tetrazole
(10), 1,2,4-triazole (12), or 1,2,4-oxadiazolone (11, 14–16)-substituted derivatives were synthesized via
cyano-substituted derivatives 22a–22d.
[Scheme 2]
Scheme 2. Synthesis of Compounds 7–16 a
a Reagents and conditions: (a) corresponding pyridone, NaH, LiBr2, DME, DMF, 80°C or K2CO3, DME,
80°C or pyridazinone, K2CO3, DMF, 80°C; (b) 4 M HCl in EtOAc or 11.7 M HCl in EtOH, EtOH, rt; (c)
2 M NaOH, EtOH, 30°C; (d) azidotributyltin, toluene, 100°C; (e) (i) O,O'-diethyl dithiophosphate, 4 M
HCl in EtOAc, rt; (ii) NaHCO3, Boc2O, EtOAc, rt; (iii) MeI, K2CO3, acetone, rt; (iv) acetohydrazide,
toluene, 120°C; (f) (i) NH4Cl, NaHCO3, DMSO, 80°C; (ii) CDI, DBU, THF, 70°C.
5

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. In this study, we initially explored the terminal
ring system and then investigated the acidic groups on the selected ring to identify highly peripheral
NET selective compounds with a low MDR1 efflux ratio. As expected, all the cyclic amide derivatives
(3–8) showed marked enhancement of NET inhibitory activities compared with acyclic amide derivative
1 (Table 1). The six-membered cyclic amide 5 and urea 6 showed decreased selectivity against SERT.
In contrast, high selectivity against SERT and DAT was observed for the unsaturated amide series
(pyridone derivative 7 and pyridazinone derivative 8). Among the evaluated compounds, 2-pyridone
derivative 7 demonstrated potent NET inhibitory activity and high selectivity against SERT and DAT
inhibitions; we therefore selected pyridone (7) as the terminal ring system for further investigation.
[Table 1]
6

Next, we investigated the effects of introducing acidic groups to compound 7 (Table 2). Initially, we
introduced carboxylic acid and bioisosteres of acidic groups at the 2-position of the ring. All the
resulting acidic groups demonstrated potent NET inhibitory activities (9–12). Among them, carboxylic
acid derivative 9 showed excellent selectivity against SERT and DAT. As we expected, the MDR1
efflux ratio was correlated with the pKa value of the acidic parts; compounds 9–11, which have pKa
values of less than 6, exhibited low MDR1 efflux ratios.11 In addition, non-acidic methyl ester derivative
13 showed a high MDR1 efflux ratio, indicating that the introduction of acidic groups is suitable to our
strategy. With several potent acidic motifs in hand, we next optimized of the substituent position on the
pyridone ring using 1,2,4(4H)-oxadiazol-5-one (14–16). As a result, we observed that the ring position
7

was critical for NET inhibitory activity and that 2-substituted derivative 11 exhibited the most potent
NET inhibitory activity, and highest selectivity against SERT, and DAT among 2-, 3-, 4-, and 5-
substituted derivatives. Encouraged by these results, we next evaluated the pharmacokinetic (PK)
profiles of 9, 10 and 11, and performed NET occupancy tests in rat brain (Table 3). Among the
investigated derivatives, only carboxylic acid compound 9 exhibited favorable PK profiles, including
good oral bioavailability in rats. In the single-crystal structure of 9, intramolecular hydrogen bond
between the carboxylic acid and the carbonyl group of pyridone (2.53 Å) was observed (Figure 2). It
was suggested that compound 9 demonstrated good PK profiles as a consequence of this effect.12
Compound 9 also demonstrated high peripheral NET selectivity13 compared to that of 7, consistent with
our design hypothesis. Compound 9 was therefore selected as a candidate for in vivo evaluation.
[Table 2]
[Table 3]
[Figure 2]
8

9

Figure 2. ORTEP drawing of compound 9, thermal ellipsoids are drawn at 30% probability.
The leak point pressure of compound 9 was measured in rats to evaluate the urethral resistance-
increasing effect. The ability of a compound to increase the leak point pressure predicts the compound's
efficacy toward an anti-SUI effect. The leak point pressure was measured from 15 min to 4 h after
administration. Compound 9 was administered orally at doses of 0.3, 1, 3, and 10 mg/kg. The results
showed that, compared with the vehicle group, compound 9 increased the leak point pressure in a dose-
dependent manner (Figure 3). The maximum leak point pressure elevation was comparable to that by
esreboxetine, which is a centrally acting NRI.1 Therefore, compound 9 was selected as a promising
peripheral-selective NRI for further drug development.
[Figure 3]
10

ff
25
20
15
10
5
0
mg/kg, p.o.
**
0.3 mg/kg, p.o.
1
3
mg/kg, p.o.
mg/kg, p.o.
10 mg/kg, p.o.
0
-5
120
240
15 30
60
Time (minutes)
Figure 3. Time course study of the urethral resistance-increasing effects of compound 9 in rats. Data are
expressed as the mean ± SEM in 5–12 rats. * P < 0.05, ** P < 0.01 vs. prevalue by paired t-
test with Holm's correction.
4. Conclusion
Novel peripheral-selective NRIs were designed and synthesized, and a compound with high peripheral
NET selectivity was evaluated for its effects on urethral function. Cyclization of an amide moiety led to
promising lead compound 3 with extremely enhanced NET inhibitory activity, following identification
of pyridone as the best terminal ring system. Subsequently, investigation of the role of the acidic group
led to the identification of carboxylic acid derivative 9 as a peripheral-selective NET inhibitor with high
selectivity against SERT and DAT, low MDR1 efflux ratio and favorable PK profiles. Intramolecular
hydrogen bond between the carboxylic acid group and the carbonyl group of pyridone, which was
observed in the single-crystal structure, was assumed to contribute to the good PK profile. Compound 9
exhibited a significant increase in urethral resistance in rats in a dose dependent manner, with the
maximum effects similar to those of the central acting and clinically effective esreboxetine.
5. Experimental Section
5.1. Chemistry
11

Melting points were determined with a Yanagimoto melting point apparatus or a Büchi melting point
1
13
apparatus B-545 and are uncorrected. H NMR and C NMR spectra were obtained on a Varian Ultra-
300, or a Bruker DPX-300 spectrometer (300 MHz for 1H NMR, 75 MHz for 13C NMR). Chemical shifts
are given in δ 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; brs, broad singlet; m, multiplet.
Elemental analyses were carried out by Takeda Analytical Laboratories 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 (Chromatorex® 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. Elution was done with a gradient of 5–90% solvent B in solvent A (solvent
A was 0.1% TFA in water, and solvent B was 0.1% TFA in MeCN) through a L-column 2 ODS (3.0 ×
50 mm, 2 μm) column at 1.2 mL min-1. Area % purity was measured at 254 nm. Abbreviations for
solvents are the following: Et2O, diethyl ether; MeOH, methanol; EtOH, ethanol; EtOAc, ethyl acetate;
THF, tetrahydrofuran; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; MeCN, acetonitrile;
DME, 1,2-dimethoxyethane; CDCl3, chroloform-d; Et3N, triethylamine; Boc2O, di-tert-butyl
dicarbonate.
5.1.1. Typical procedure of obtaining compounds 3–16 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 temperature. 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 eq.)
at room temperature. 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.
12

5.1.2.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((4-chlorobutanamido)methyl)-1,4-
oxazepane-4-carboxylate (18a). A mixture of 17 (139 mg, 0.39 mmol), 4-chlorobutanoyl chloride (52
µL, 0.47 mmol) and Et3N (108 µL, 0.78 mmol) in THF (3 mL) was stirred overnight at rt, poured into
water and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4
and concentrated in vacuo. The residue was purified by column chromatography (silica gel,
hexane/EtOAc) to give 18a (173 mg, 96%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ1.52 (9H, s),
2.05–2.44 (5H, m), 2.96–3.21 (2H, m), 3.24–3.42 (2H, m), 3.47–3.66 (3H, m), 3.95–4.17 (4H, m), 7.09–
7.15 (1H, m), 7.19–7.25 (1H, m), 7.33–7.40 (1H, m). MS m/z: 363.1 [M+H-Boc]+.
5.1.3.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxopyrrolidin-1-yl)methyl)-1,4-
oxazepane-4-carboxylate (19a). To a solution of 18a (173 mg, 0.37 mmol) in THF (3 mL) was added
60% NaH in oil (35.9 mg, 0.75 mmol) at 0°C. The reaction mixture was stirred overnight at rt, poured
into water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc)
to give 19a (126 mg, 79%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ1.50 (9H, s), 1.54–1.76 (2H,
m), 1.76–2.03 (1H, m), 2.06–2.48 (3H, m), 2.82–3.53 (5H, m), 3.56–3.86 (3H, m), 4.01–4.21 (2H, m),
7.01–7.08 (1H, m), 7.13 (1H, dd, J = 9.8, 1.9 Hz), 7.36 (1H, t, J = 8.0 Hz). MS m/z: 427.3 [M+H]+.
5.1.4.
1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)pyrrolidin-2-one
1
hydrochloride (3). Typical procedure B, Quant., Colorless oil. H NMR (300 MHz, DMSO-d6) δ1.62–
1.89 (2H, m), 1.98–2.30 (2H, m), 2.62 (1H, dd, J = 14.0, 4.9 Hz), 2.80–3.04 (2H, m), 3.06–3.40 (4H,
m), 3.71–4.12 (4H, m), 4.42 (1H, d, J = 9.8 Hz), 7.29 (1H, dd, J = 8.1, 1.7 Hz), 7.50 (1H, dd, J = 10.4,
13
1.7 Hz), 7.63 (1H, t, J = 7.9 Hz), 9.19–9.48 (2H, m). C NMR (DMSO-d6): δ 17.31, 30.15, 40.45,
41.92, 45.15, 46.36, 46.56, 63.27, 82.35, 115.86 (d, J = 21.5 Hz), 119.15 (d, J = 17.6 Hz), 124.80 (d, J =
3.3 Hz), 130.65, 141.97 (d, J = 6.1 Hz), 157.06 (d, J = 247.0 Hz), 174.63. MS m/z: 327.2 [M+H] +.
HPLC purity 100%. Anal. Calcd for C16H21Cl2N2O2F•0.6 H2O: C,51.37; H,5.98; N,7.49. Found: C,51.26;
H,5.82; N,7.29.
13

5.1.5.
(6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((5-chloropentanamido)methyl)-1,4-
oxazepane-4-carboxylate (18b). A mixture of 17 (111 mg, 0.31 mmol), 5-chloropentanoyl chloride (48
µL, 0.37 mmol) and Et3N (86 µL, 0.62 mmol) in THF (3 mL) was stirred overnight at rt, poured into
water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc)
1
to give 18b (148 mg, 100%) as a colorless oil. H NMR (300 MHz, CDCl3) δ1.51 (9H, s), 1.73–1.86
(4H, m), 2.16–2.34 (3H, m), 2.92–3.22 (2H, m), 3.27–3.41 (2H, m), 3.47–3.60 (3H, m), 3.93–4.18 (4H,
m), 7.10–7.16 (1H, m), 7.18–7.25 (1H, m), 7.32–7.41 (1H, m). MS m/z: 477.3 [M+H]+.
5.1.6.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxopiperidin-1-yl)methyl)-1,4-
oxazepane-4-carboxylate (19b). To a solution of 18b (148 mg, 0.31 mmol) in THF (3 mL) was added
60% NaH in oil (29.7 mg, 0.62 mmol) at 0°C. The reaction mixture was stirred overnight at rt, poured
into water and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexane/EtOAc)
to give 19b (110 mg, 80%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ1.38–1.79 (13H, m), 2.07–
2.54 (3H, m), 2.75–3.83 (9H, m), 4.02–4.16 (1H, m), 4.21 (1H, d, J = 9.1 Hz), 7.02–7.08 (1H, m), 7.13
(1H, dd, J = 9.8, 1.9 Hz), 7.36 (1H, t, J = 7.8 Hz). MS m/z: 441.4 [M+H]+.
5.1.7.
1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)piperidin-2-one
1
hydrochloride (5). Typical procedure B, Quant., Colorless oil. H NMR (300 MHz, DMSO-d6) δ1.42–
1.68 (4H, m), 2.01–2.20 (2H, m), 2.65 (1H, dd, J = 13.8, 5.1 Hz), 2.78–3.33 (7H, m), 3.61 (1H, dd, J =
13.6, 9.4 Hz), 3.73–3.88 (1H, m), 3.98–4.11 (1H, m), 4.42 (1H, d, J = 10.2 Hz), 7.24–7.33 (1H, m), 7.51
13
(1H, dd, J = 10.6, 1.9 Hz), 7.63 (1H, t, J = 8.1 Hz), 9.18–9.63 (2H, m). C NMR (DMSO-d6): δ20.56,
22.39, 31.88, 40.20, 45.19, 45.87, 46.44, 47.36, 63.52, 82.53, 115.82 (d, J = 21.5 Hz), 119.10 (d, J =
17.6 Hz), 124.81 (d, J = 3.3 Hz), 130.61, 142.08 (d, J = 6.1 Hz), 157.06 (d, J = 247.0 Hz), 169.26. MS
m/z: 341.1 [M+H]+. HPLC purity 100%.
14

5.1.8. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((3-(3-chloropropyl)ureido)methyl)-1,4-
oxazepane-4-carboxylate (18c). The mixture of 17 (400 mg, 1.11 mmol) and Et3N (186 µL, 1.34
mmol) in CH3CN (3.7 mL) was added of 3-chloropropyl isocyanate (120 µL, 1.17 mmol). The mixture
was stirred at room temperature for 3 h, and concentrated in vacuo to give 18c as a colerless oil. This
product was subjected to the next reaction without further purification. MS m/z: 478.1 [M+H]+.
5.1.9.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxotetrahydropyrimidin-1(2H)-
yl)methyl)-1,4-oxazepane-4-carboxylate (19c). To a solution of 18c (531 mg, 1.11 mmol) in DMF (22
mL) was added 60% NaH in oil (48.8 mg, 1.22 mmol) at 0°C. The mixture was stirred at 0°C for 2 h
and then rt for 3 h. The mixture was quenched with 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 19c (563 mg, quant.) as a
1
colorless oil. H NMR (300 MHz, CDCl3) δ1.41–1.91 (11H, m), 2.24–2.49 (1H, m), 2.66–3.90 (11H,
m), 3.99–4.17 (1H, m), 4.23 (1H, d, J = 9.1 Hz), 4.55 (1H, brs), 7.07 (1H, d, J = 8.3 Hz), 7.16 (1H, dd, J
= 9.8, 1.9 Hz), 7.31–7.41 (1H, m). MS m/z: 442.3 [M+H]+.
5.1.10. 1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)tetrahydropyrimidin-
2(1H)-one hydrochloride (6). Typical procedure B, 22%, Colorless solid. 1H NMR (300 MHz, DMSO-
d6) δ1.59–1.77 (2H, m), 2.52–2.61 (2H, m), 2.77–2.88 (2H, m), 2.93–3.39 (6H, m), 3.54–3.65 (1H, m),
3.75–3.87 (1H, m), 3.98–4.10 (1H, m), 4.40 (1H, d, J = 9.1 Hz), 6.34 (1H, brs), 7.30 (1H, d, J = 8.3 Hz),
7.51 (1H, dd, J = 10.6, 1.9 Hz), 7.58–7.66 (1H, m), 9.14–9.47 (2H, m). MS m/z: 342.2 [M+H] +. HPLC
purity 100%. HPLC purity 100%.
5.1.11. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((2-oxopyridin-1(2H)-yl)methyl)-1,4-
oxazepane-4-carboxylate. 60% NaH in oil (45.3 mg, 1.13 mmol) was added to a solution of pyridin-
2(1H)-one (86 mg, 0.91 mmol) in DME (2 mL) and DMF (1 mL) at room temperature. After being
stirred for 5 min, lithium bromide (131 mg, 1.51 mmol) was added to the reaction mixture. After being
stirred at room temperature for 10 min, a solution of 20 (331 mg, 0.76 mmol) in DME (2 mL) was
15

added to the reaction mixture. The mixture was stirred at 80°C overnight. The mixture was diluted with
water and extracted with EtOAc. The organic layer was separated, washed with 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-((2-oxopyridin-1(2H)-
1
yl)methyl)-1,4-oxazepane-4-carboxylate (267 mg, 81%) as a colorless powder. H NMR (300 MHz,
CDCl3) δ1.33–1.58 (9H, m), 2.55–2.96 (1H, m), 3.11–3.35 (1H, m), 3.45–3.80 (3H, m), 3.82–4.01 (2H,
m), 4.02–4.26 (2H, m), 6.12 (1H, brs), 6.44 (1H, d, J = 9.1 Hz), 6.88–7.31 (4H, m), 7.37 (1H, t, J = 7.6
Hz), 7.78 (1H, brs). MS m/z: 437.2 [M+H]+.
5.1.12.
1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)pyridin-2(1H)-one
1
hydrochloride (7). Typical procedure A, Quant., Colorless solid. H NMR (300 MHz, DMSO-d6) δ
3.04–3.32 (5H, m), 3.64 (1H, dd, J = 13.2, 5.3 Hz), 3.77–3.88 (1H, m), 3.91–4.06 (2H, m), 4.54 (1H, d,
J = 9.4 Hz), 6.17 (1H, td, J = 6.7, 1.3 Hz), 6.27–6.33 (1H, m), 7.29–7.40 (2H, m), 7.46 (1H, dd, J = 6.6,
1.7 Hz), 7.50–7.61 (2H, m), 9.24 (1H, brs), 9.80 (1H, brs). 13C NMR (DMSO-d6): δ41.40, 44.68, 46.39,
48.61, 64.11, 82.35, 105.56, 115.95 (d, J = 21.5 Hz), 119.07 (d, J = 17.6 Hz), 119.62, 125.07 (d, J = 3.3
Hz), 130.44, 139.18, 140.10, 141.50 (d, J = 6.6 Hz), 156.94 (d, J = 246.5 Hz), 161.62. MS m/z: 337.1
[M+H] +. [α]25D +65.1 (c 0.25, MeOH). HPLC purity 100%. Anal. Calcd for C17H19Cl2N2O2F•1.2 H2O:
C,51.71; H,5.46; N,7.09. Found: C,51.67; H,5.39; N,6.98.
5.1.13. (6S,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((6-oxopyridazin-1(6H)-yl)methyl)-1,4-
oxazepane-4-carboxylate. Pyridazin-3(2H)-one (41.1 mg, 0.43 mmol) and K2CO3 (73.9 mg, 0.53
mmol) were added to a solution of 20 (156 mg, 0.36 mmol) in DMF (4 mL). The mixture was stirred at
80°C for 4 h. The residue was diluted with water and extracted with EtOAc. The organic layer was
separated, washed with 0.1 M HCl and brine, dried over Na2SO4 and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, hexane/EtOAc) to give (6S,7R)-tert-butyl 7-(4-
chloro-3-fluorophenyl)-6-((6-oxopyridazin-1(6H)-yl)methyl)-1,4-oxazepane-4-carboxylate (122 mg,
78%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ1.45 (9H, brs), 2.94 (1H, brs), 3.38–3.57 (1H, m),
16

3.64–3.80 (4H, m), 3.99–4.15 (3H, m), 4.28 (1H, d, J = 9.1 Hz), 6.72 (1H, dd, J = 9.4, 1.5 Hz), 6.98–
7.09 (3H, m), 7.24–7.31 (1H, m), 7.62 (1H, dd, J = 3.8, 1.5 Hz). MS m/z: 438.2 [M+H]+.
5.1.14. 2-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)pyridazin-3(2H)-one
1
hydrochloride (8). Typical procedure B, 86%, Colorless solid. H NMR (300 MHz, DMSO-d6) δ3.11
(2H, td, J = 8.8, 4.0 Hz), 3.18–3.37 (3H, m), 3.73–3.89 (2H, m), 3.95–4.12 (2H, m), 4.56 (1H, d, J =
10.2 Hz), 6.86 (1H, dd, J = 9.4, 1.9 Hz), 7.27–7.33 (1H, m), 7.36 (1H, dd, J = 9.4, 3.8 Hz), 7.52 (1H, dd,
J = 10.6, 1.9 Hz), 7.59 (1H, t, J = 8.1 Hz), 7.88 (1H, dd, J = 4.0, 1.7 Hz), 9.06–9.29 (1H, m), 9.65 (1H,
13
brs). C NMR (DMSO-d6): δ41.80, 44.74, 46.29, 50.37, 63.78, 82.10, 115.97 (d, J = 21.5 Hz), 119.22,
125.09, 129.36, 130.46, 132.15, 137.09, 141.53 (d, J = 6.6 Hz), 156.91 (d, J = 246.5 Hz), 159.84. MS
+
m/z: 338.2 [M+H] . HPLC purity 97%. Anal. Calcd for C16H18Cl2N3O2F: C,51.35; H,4.85; N,11.23.
Found: C,51.08; H,4.85; N,11.18.
5.1.15. (6R,7R)-tert-Butyl 7-(4-chloro-3-fluorophenyl)-6-((3-(methoxycarbonyl)-2-oxopyridin-
1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate (21). K2CO3 (2.23 g, 16.1 mmol) and methyl 2-oxo-
1,2-dihydropyridine-3-carboxylate (1.48 g, 9.67 mmol) was added to a solution of 20 (3.53 g, 8.06
mmol) in DME (70 mL). The mixture was stirred at 80°C overnight. The mixture was diluted with water
and concentrated in vacuo. The residue was diluted with water and extracted with EtOAc. The organic
layer was separated, washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, hexane/EtOAc) to give 21 (2.88 g, 72%) as a colorless
1
amorphous powder. H NMR (300 MHz, CDCl3) δ1.51 (9H, brs), 2.99 (1H, brs), 3.17 (2H, d, J = 13.6
Hz), 3.43–3.79 (3H, m), 3.87 (3H, s), 3.89–4.12 (4H, m), 6.23 (1H, brs), 7.06–7.24 (2H, m), 7.29–7.44
(1H, m), 7.97–8.16 (1H, m), 8.20 (1H, brs). MS m/z: 495.1 [M+H]+.
5.1.16. Methyl 1-(((6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-2-oxo-1,2-
1
dihydropyridine-3-carboxylate hydrochloride (13). Typical procedure B, 89%, Colorless solid. H
NMR (300 MHz, DMSO-d6) δ2.99–3.29 (5H, m), 3.63–3.87 (5H, m), 3.90–4.06 (2H, m), 4.51 (1H, d, J
= 9.8 Hz), 6.26 (1H, t, J = 7.0 Hz), 7.28 (1H, dd, J = 8.3, 1.5 Hz), 7.48 (1H, dd, J = 10.6, 1.9 Hz), 7.52–
17

7.60 (1H, m), 7.74 (1H, dd, J = 6.6, 2.1 Hz), 7.94 (1H, dd, J = 7.2, 1.9 Hz), 8.62–9.34 (2H, m). MS m/z:
395.3 [M+H] +. HPLC purity 96%. Anal. Calcd for C19H21Cl2N2O4F: C,52.91; H,4.91; N,6.50. Found:
C,52.93; H,4.98; N,6.36.
5.1.17.
1-(((6R,7R)-4-(tert-Butoxycarbonyl)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-
yl)methyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid. 2 M NaOH (44.9 mL, 89.8 mmol) was
added to a solution of 21 (22.2 g, 44.9 mmol) in EtOH (220 mL). The mixture was stirred at 30 °C
overnight. The mixture was concentrated in vacuo. The residue was diluted with Et2O and extracted with
water. The organic layer was removed, and the aqueous layer was acidified with 1 M HCl and extracted
with water. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo to
give
1-(((6R,7R)-4-(tert-butoxycarbonyl)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-2-
1
oxo-1,2-dihydropyridine-3-carboxylic acid (22.1 g, quant.) as a colorless solid. H NMR (300 MHz,
CDCl3) δ1.52 (9H, s), 2.85 (1H, brs), 3.06–3.27 (2H, m), 3.65 (1H, t, J = 11.0 Hz), 3.77–4.14 (6H, m),
6.54 (1H, t, J = 6.4 Hz), 7.18 (2H, dd, J = 19.6, 8.3 Hz), 7.42 (1H, t, J = 7.6 Hz), 8.45 (2H, d, J = 7.2
Hz), 14.13 (1H, brs). MS m/z: 481.1 [M+H]+.
5.1.18.
1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-2-oxo-1,2-
dihydropyridine-3-carboxylic acid hydrochloride (9). 4 M HCl in AcOEt (112 mL, 449 mmol) was
added to a solution of 1-(((6R,7R)-4-(tert-butoxycarbonyl)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-
yl)methyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid (21.6 g, 44.9 mmol) in EtOAc (20 mL). The
mixture was stirred at room temperature for 2 h, and then concentrated in vacuo. The residue was
crystallized from 10% water in MeCN to give 9 (15.9 g, 85%) as white crystals. 1H NMR (300 MHz,
CDCl3) δ3.10–3.30 (5H, m), 3.77–3.88 (1H, m), 3.89–3.98 (1H, m), 4.03 (1H, dt, J = 13.9, 4.4 Hz), 4.16
(1H, dd, J = 13.4, 7.7 Hz), 4.58 (1H, d, J = 9.1 Hz), 6.65 (1H, t, J = 6.8 Hz), 7.26 (1H, dd, J = 8.3, 1.5
Hz), 7.46 (1H, dd, J = 10.6, 1.9 Hz), 7.50–7.58 (1H, m), 8.07 (1H, dd, J = 6.8, 1.9 Hz), 8.26 (1H, dd, J =
7.4, 2.1 Hz), 9.45 (2H, brs), 14.19 (1H, brs). 13C NMR (DMSO-d6): δ40.60, 44.93, 46.55, 49.98, 63.92,
82.12, 108.44, 115.87, 116.16, 116.72, 119.30 (d, J = 17.1 Hz), 125.10 (d, J = 3.3 Hz), 130.45, 140.90
18

(d, J = 6.1 Hz), 145.15 (d, J = 14.9 Hz), 156.84 (d, J = 247.0 Hz), 163.64, 164.41. MS m/z: 381.1
[M+H]+. Anal. Calcd for C18H19Cl2N2O4F: C,51.81; H,4.59; N,6.71. Found: C,52.09; H,4.58; N,6.71.
[α]25 +96.2 (c 0.25, MeOH). HPLC purity 100%.
D
5.1.19.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((3-cyano-2-oxopyridin-1(2H)-
yl)methyl)-1,4-oxazepane-4-carboxylate (22a). 60% NaH in oil (274 mg, 6.85 mmol) was added to a
solution of 2-oxo-1,2-dihydropyridine-3-carbonitrile (658 mg, 5.48 mmol) in DME (10 mL) and DMF
(5 mL) at 0°C. After being stirred at 0°C for 20 min, lithium bromide (793 mg, 9.13 mmol) was added
to the reaction mixture. After being stirred at room temperature for 1.5 h, a solution of 20 (2.00 g, 4.57
mmol) in DME (10 mL) was added to the reaction mixture. The mixture was stirred at 60°C overnight.
The mixture was concentrated in vacuo. The residue was diluted with water and extracted with EtOAc.
The organic layer was separated, washed with brine, dried over Na2SO4 and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, hexane/EtOAc) to give 22a (1.76 g, 83%) as
a colorless powder. 1H NMR (300 MHz, CDCl3) δ1.52 (9H, s), 2.90 (1H, brs), 3.06–3.28 (2H, m), 3.55–
3.75 (2H, m), 3.81–4.12 (5H, m), 6.26 (1H, t, J = 6.2 Hz), 7.18 (2H, d, J = 7.9 Hz), 7.41 (1H, t, J = 7.9
Hz), 7.76 (1H, d, J = 6.4 Hz), 8.31 (1H, d, J = 5.3 Hz). MS m/z: 362.2 [M+H-Boc]+.
5.1.20
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxo-3-(1H-tetrazol-5-yl)pyridin-
1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate. Azidotributyltin (356 µL, 1.30 mmol) was added to a
solution of 22a (200 mg, 0.43 mmol) in toluene (5 mL). The mixture was stirred at 100°C overnight,
and then 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-((2-oxo-3-(1H-tetrazol-5-
1
yl)pyridin-1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate (87 mg, 40%) as a white solid. H NMR (300
MHz, CDCl3) δ1.56 (9H, s), 2.99 (1H, brs), 3.16 (1H, t, J = 11.5 Hz), 3.30 (1H, dd, J = 15.1, 4.9 Hz),
3.64 (1H, t, J = 11.3 Hz), 3.89–4.22 (5H, m), 4.45 (1H, d, J = 12.5 Hz), 6.62 (1H, t, J = 6.6 Hz), 6.75
(1H, t, J = 7.2 Hz), 6.94 (1H, d, J = 7.9 Hz), 7.38 (1H, d, J = 9.8 Hz), 8.68 (2H, d, J = 7.2 Hz), 14.45
(1H, brs). MS m/z: 505.3 [M+H]+.
19

5.1.21
1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-3-(1H-tetrazol-5-
1
yl)pyridin-2(1H)-one hydrochloride (10). Typical procedure B, 77%, Colorless solid. H NMR (300
MHz, DMSO-d6) δ3.10–3.42 (5H, m), 3.79–3.97 (2H, m), 4.00–4.15 (2H, m), 4.58 (1H, d, J = 9.8 Hz),
6.50 (1H, t, J = 7.0 Hz), 7.27–7.33 (1H, m), 7.44–7.54 (2H, m), 7.85 (1H, dd, J = 6.8, 1.9 Hz), 8.33 (1H,
13
dd, J = 7.2, 1.9 Hz), 9.46 (1H, brs). C NMR (DMSO-d6): δ41.19, 44.95, 46.49, 49.42, 64.20, 82.58,
106.19, 113.85, 115.97 (d, J = 21.5 Hz), 119.20 (d, J = 17.6 Hz), 125.01 (d, J = 3.3 Hz), 130.41, 140.59
(2 C), 141.22 (d, J = 6.1 Hz), 142.55, 152.74 (d, J = 374.1 Hz), 158.91. MS m/z: 405.1 [M+H] +. [α]25
D
+90.9 (c 0.25, MeOH). HPLC purity 100%. Anal. Calcd for C18H19Cl2N6O2F•2H2O: C,45.29; H,4.86;
N,17.61. Found: C,45.48; H,4.72; N,17.53.
5.1.22. (6R,7R)-tert-Butyl 6-((3-carbamothioyl-2-oxopyridin-1(2H)-yl)methyl)-7-(4-chloro-3-
fluorophenyl)-1,4-oxazepane-4-carboxylate. O,O'-Diethyl dithiophosphate (189 µL, 1.13 mmol) was
added to a solution of 22a (348 mg, 0.75 mmol) in 4 M HCl in AcOEt (5 mL, 20.0 mmol). The mixture
was stirred at room temperature overnight and then concentrated in vacuo. The residue was diluted with
sat. NaHCO3 aq. (10 mL) and EtOAc (10 mL). The mixture was added Boc2O (210 µL, 0.90 mmol). The
mixture was stirred at room temperature for 4 h. The mixture was diluted with water and extracted with
EtOAc. The organic layer was separated, washed with 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
6-((3-carbamothioyl-2-oxopyridin-1(2H)-yl)methyl)-7-(4-chloro-3-fluorophenyl)-1,4-
1
oxazepane-4-carboxylate (225 mg, 60%) as an yellow oil. H NMR (300 MHz, CDCl3) δ1.52 (9H, brs),
2.82 (1H, brs), 3.05–3.23 (2H, m), 3.57–3.82 (2H, m), 3.87–4.13 (5H, m), 6.44 (1H, t, J = 6.6 Hz),
7.07–7.28 (2H, m), 7.35–7.44 (1H, m), 7.90 (1H, brs), 8.35 (1H, brs), 9.12 (1H, d, J = 7.2 Hz), 11.39
(1H, brs). MS m/z: 496.1 [M+H]+.
5.1.23. (6R,7R)-tert-Butyl 6-((1,4-dioxane-2-carboxamido)methyl)-7-(4-chloro-3-fluorophenyl)-
1,4-oxazepane-4-carboxylate. K2CO3 (94 mg, 0.68 mmol) and MeI (142 µL, 2.27 mmol) was added to
a solution of (6R,7R)-tert-Butyl 6-((3-carbamothioyl-2-oxopyridin-1(2H)-yl)methyl)-7-(4-chloro-3-
20

fluorophenyl)-1,4-oxazepane-4-carboxylate (225 mg, 0.45 mmol) in acetone (4 mL). The mixture was
stirred at room temperature overnight and then concentrated in vacuo. The residue was diluted with
water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over Na2SO4
and concentrated in vacuo. The half amount of residue in toluene (5 mL) was added acetohydrazide
(33.3 mg, 0.45 mmol). The mixture was stirred at 120°C overnight and then diluted with water and
extracted with EtOAc. The organic layer was separated, washed with 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 6-((1,4-dioxane-2-carboxamido)methyl)-7-(4-chloro-3-fluorophenyl)-1,4-
1
oxazepane-4-carboxylate (92 mg, 79%) as a colorless gum. H NMR (300 MHz, CDCl3) δ 1.34–1.58
(9H, m), 2.45 (3H, s), 2.90 (1H, brs), 3.10–3.33 (2H, m), 3.55–4.15 (7H, m), 6.41 (1H, t, J = 5.3 Hz),
6.96–7.25 (2H, m), 7.30–7.43 (1H, m), 8.02–8.17 (1H, m), 8.37 (1H, d, J = 7.2 Hz), 12.42 (1H, brs). MS
m/z: 518.1 [M+H]+.
5.1.24. 1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-3-(5-methyl-4H-1,2,4-
1
triazol-3-yl)pyridin-2(1H)-one hydrochloride (12). Typical procedure B, 87%, Colorless solid. H
NMR (300 MHz, DMSO-d6) δ2.50 (3H, s), 3.08–3.39 (5H, m), 3.79–3.98 (2H, m), 4.02–4.20 (2H, m),
4.59 (1H, d, J = 9.4 Hz), 6.51 (1H, t, J = 7.0 Hz), 7.25–7.33 (1H, m), 7.44–7.55 (2H, m), 7.88 (1H, dd, J
= 6.4, 1.9 Hz), 8.30 (1H, dd, J = 7.2, 1.9 Hz), 9.44 (1H, brs), 9.96 (1H, brs). MS m/z: 418.1 [M+H]+.
HPLC purity 100%.
5.1.25.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxo-3-(5-oxo-4,5-dihydro-1,2,4-
oxadiazol-3-yl)pyridin-1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate. NaHCO3 (1.94 g, 23.1 mmol)
and hydroxylammonium chloride (1.60 g, 23.1 mmol) was added to a solution of 22a (1.33 g, 2.88
mmol) in DMSO (15 mL) and the mixture was stirred at 80°C for 8 h. The mixture was quenched with
water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over Na2SO4
and concentrated in vacuo to give a colorless oil. To the oil in THF (15 mL) was added 1,8-
diazabicyclo[5.4.0]undec-7-ene (431 µL, 2.88 mmol) and di(1H-imidazol-1-yl)methanone (700 mg,
21

4.32 mmol). The mixture was stirred at 70°C for 5.5 h. The mixture was concentrated in vacuo. The
residue was diluted with 1 M HCl aq. 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-((2-oxo-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)pyridin-1(2H)-yl)methyl)-1,4-
1
oxazepane-4-carboxylate (1.46 g, 97%) as a colorless powder. H NMR (300 MHz, CDCl3) δ1.54 (9H,
s), 2.79–2.94 (1H, m), 3.06–3.20 (1H, m), 3.25 (1H, dd, J = 14.5, 4.3 Hz), 3.54–3.68 (1H, m), 3.76 (1H,
t, J = 11.7 Hz), 3.95–4.18 (4H, m), 4.45 (1H, d, J = 12.8 Hz), 6.48 (1H, t, J = 6.8 Hz), 7.08 (1H, d, J =
8.3 Hz), 7.17 (1H, t, J = 7.7 Hz), 7.34 (1H, d, J = 9.8 Hz), 8.27 (1H, d, J = 5.7 Hz), 8.66 (1H, d, J = 5.7
Hz), 10.44 (1H, brs). MS m/z: 421.1 [M+H-Boc]+.
5.1.26.
3-(1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-2-oxo-1,2-
dihydropyridin-3-yl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (11). Typical procedure A, 96%,
1
Colorless crystal. H NMR (300 MHz, DMSO-d6) δ3.05–3.33 (5H, m), 3.74–3.89 (2H, m), 3.96–4.11
(2H, m), 4.55 (1H, d, J = 9.8 Hz), 6.41 (1H, t, J = 7.0 Hz), 7.26–7.34 (1H, m), 7.49 (1H, dd, J = 10.6,
1.9 Hz), 7.52–7.60 (1H, m), 7.84 (1H, dd, J = 6.6, 2.1 Hz), 7.91 (1H, dd, J = 7.2, 1.9 Hz), 9.13 (1H,
13
brs), 9.61 (1H, brs), 12.22 (1H, brs). C NMR (DMSO-d6): δ40.95, 44.86, 46.55, 49.27, 64.11, 82.29,
105.62, 113.27, 116.03 (d, J = 21.5 Hz), 119.25 (d, J = 18.0 Hz), 125.05, 130.49, 142.26 (d, J = 198.6
Hz), 141.19 (d, J = 6.1 Hz), 155.15, 155.28, 158.44, 158.55, 159.33. MS m/z: 421.1 [M+H] +. Anal.
Calcd for C19H19Cl2N4O4F•0.2 H2O: C,49.51; H,4.24; N,12.16. Found: C,49.92; H,4.40; N,11.88. [α]25
D
+113.9 (c 0.25, MeOH). HPLC purity 100%.
Compounds 14–16 were prepared in same manner using 20.
5.1.27.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((4-cyano-2-oxopyridin-1(2H)-
yl)methyl)-1,4-oxazepane-4-carboxylate (22b). 26%, Colorless oil. 1H NMR (300 MHz, CDCl3) δ1.52
(9H, s), 2.80 (1H, brs), 3.07–3.26 (2H, m), 3.53–3.74 (2H, m), 3.80–3.95 (2H, m), 3.96–4.15 (3H, m),
22

6.27 (1H, d, J = 6.4 Hz), 6.75 (1H, s), 6.99–7.25 (2H, m), 7.41 (1H, t, J = 7.8 Hz), 8.16 (1H, d, J = 6.4
Hz). MS m/z: 362.3 [M+H-Boc]+.
5.1.28.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxo-4-(5-oxo-4,5-dihydro-1,2,4-
1
oxadiazol-3-yl)pyridin-1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate. 58%, Colorless solid. H
NMR (300 MHz, CDCl3) δ 1.55 (9H, s), 2.64 (1H, brs), 2.99–3.43 (2H, m), 3.45–4.10 (7H, m), 6.47
(1H, brs), 6.83 (1H, d, J = 7.2 Hz), 7.03 (1H, brs), 7.19 (2H, brs), 8.30 (1H, brs), 12.48 (1H, brs). MS
m/z: 421.3 [M+H-Boc]+.
5.1.29.
3-(1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-2-oxo-1,2-
dihydropyridin-4-yl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (14). Typical procedure B, 84%,
1
Colorless solid. H NMR (300 MHz, DMSO-d6) δ3.00–3.33 (5H, m), 3.66 (1H, dd, J = 13.4, 5.5 Hz),
3.76–3.88 (1H, m), 3.93–4.05 (2H, m), 4.53 (1H, d, J = 9.8 Hz), 6.51 (1H, dd, J = 6.8, 1.9 Hz), 6.77
(1H, d, J = 1.9 Hz), 7.27–7.33 (1H, m), 7.51 (1H, dd, J = 10.6, 1.9 Hz), 7.54–7.61 (1H, m), 7.64 (1H, d,
J = 7.2 Hz), 8.99 (1H, brs), 9.43 (1H, brs), 13.21 (1H, brs). MS m/z: 421.3 [M+H]+.
5.1.30.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((5-cyano-2-oxopyridin-1(2H)-
yl)methyl)-1,4-oxazepane-4-carboxylate (22c). 59%, Colorless oil. 1H NMR (300 MHz, CDCl3) δ1.52
(9H, s), 2.70–2.86 (1H, m), 3.04–3.26 (2H, m), 3.56–4.15 (7H, m), 6.45 (1H, d, J = 9.5 Hz), 7.09–7.25
(2H, m), 7.31–7.47 (2H, m), 8.59–8.71 (1H, m). MS m/z: 362.1 [M+H-Boc]+.
5.1.31.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxo-5-(5-oxo-4,5-dihydro-1,2,4-
1
oxadiazol-3-yl)pyridin-1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate. 79%, Colorless oil. H NMR
(300 MHz, CDCl3) δ1.58 (9H, s), 2.50–2.73 (1H, m), 3.36–3.92 (7H, m), 3.99–4.30 (2H, m), 6.59 (1H,
d, J = 9.8 Hz), 7.08–7.24 (2H, m), 7.36–7.48 (1H, m), 7.70–7.83 (1H, m), 8.41–8.58 (1H, m), 10.01–
10.47 (1H, m). MS m/z: 421.1 [M+H-Boc]+.
5.1.32.
3-(1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-6-oxo-1,6-
dihydropyridin-3-yl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (15). Typical procedure B, Quant.,
23

1
Colorless oil. H NMR (300 MHz, DMSO-d6) δ3.02–3.50 (5H, m), 3.56–3.67 (1H, m), 3.78–3.90 (1H,
m), 3.95–4.08 (2H, m), 4.60 (1H, d, J = 10.2 Hz), 6.42–6.50 (1H, m), 7.27 (1H, dd, J = 8.3, 1.5 Hz),
7.44–7.58 (2H, m), 7.63 (1H, dd, J = 9.4, 2.6 Hz), 8.06–8.23 (1H, m), 8.94–9.16 (1H, m), 9.45–9.62
(1H, m), 12.89–13.08 (1H, m). 13C NMR (DMSO-d6): δ45.17, 46.41, 49.61, 59.74, 63.88, 82.41, 102.24,
116.04 (d, J = 21.5 Hz), 119.29 (d, J = 17.6 Hz), 120.20, 125.09, 130.52, 135.62, 139.94, 141.15 (d, J =
6.6 Hz), 154.65, 156.92 (d, J = 247.0 Hz), 159.51, 161.09. MS m/z: 421.1 [M+H]+. HPLC purity 98%.
5.1.33.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((6-cyano-2-oxopyridin-1(2H)-
1
yl)methyl)-1,4-oxazepane-4-carboxylate (22d). 17%, Colorless oil. H NMR (300 MHz, CDCl3) δ
1.42–1.53 (9H, m), 2.88–3.17 (1H, m), 3.32–3.83 (5H, m), 3.96–4.38 (4H, m), 6.53 (1H, d, J = 6.8 Hz),
6.58–6.70 (1H, m), 7.00–7.10 (2H, m), 7.10–7.21 (1H, m), 7.27–7.36 (1H, m). MS m/z: 362.0 [M+H-
Boc]+.
5.1.34.
(6R,7R)-tert-Butyl
7-(4-chloro-3-fluorophenyl)-6-((2-oxo-6-(5-oxo-4,5-dihydro-1,2,4-
1
oxadiazol-3-yl)pyridin-1(2H)-yl)methyl)-1,4-oxazepane-4-carboxylate. 20%, Colorless oil. H NMR
(300 MHz, CDCl3) δ1.52 (10H, s), 2.22–2.42 (1H, m), 3.14–3.33 (1H, m), 3.38–3.51 (1H, m), 3.70–
4.01 (2H, m), 4.08–4.25 (3H, m), 4.27–4.37 (1H, m), 6.49 (1H, d, J = 6.8 Hz), 6.67–6.79 (1H, m), 6.95–
7.14 (2H, m), 7.33–7.46 (2H, m). MS m/z: 421.1 [M+H-Boc]+.
5.1.35.
3-(1-(((6S,7R)-7-(4-Chloro-3-fluorophenyl)-1,4-oxazepan-6-yl)methyl)-6-oxo-1,6-
dihydropyridin-2-yl)-1,2,4-oxadiazol-5(4H)-one hydrochloride (16). Typical procedure B, 96%,
1
Colorless oil. H NMR (300 MHz, DMSO-d6) δ 2.69–3.26 (5H, m), 3.55 (1H, dd, J = 14.2, 4.4 Hz),
3.77–3.91 (1H, m), 3.98–4.10 (1H, m), 4.29–4.43 (1H, m), 4.48 (1H, d, J = 9.8 Hz), 6.58–6.67 (2H, m),
7.16–7.24 (1H, m), 7.37–7.45 (1H, m), 7.48–7.59 (2H, m), 8.87–9.28 (2H, m). MS m/z: 421.1 [M+H] +.
HPLC purity 98%.
5.1.36.
N-(((6S,7R)-7-(3,4-dichlorophenyl)-1,4-oxazepan-6-yl)methyl)-N-methylacetamide
1
hydrochloride (4). Colorless solid. H NMR (300 MHz, DMSO-d6) δ 1.75–1.97 (3H, m), 2.15–2.30
24

(1H, m), 2.63–2.96 (7H, m), 3.13–3.25 (2H, m), 3.42–3.58 (1H, m), 3.82–4.05 (1H, m), 4.22 (1H, dd, J
= 13.0, 8.9 Hz), 7.35 (1H, td, J = 7.9, 2.3 Hz), 7.55–7.67 (2H, m). MS m/z: 331.3 [M+H]+. HPLC purity
96%.
5.1.37. X-ray structure analysis
+
Crystal data for compound 9: C18H19ClFN2O4 ∙ Cl-, MW = 417.26; crystal size, 0.13 x 0.09 x 0.09
mm; colorless, block; orthorhombic, space group P212121, a = 6.93199(13) Å, b = 14.1848(3) Å, c =
18.7907(4) Å, α = β = γ = 90°, V = 1847.67(6) Å3, Z = 4, Dx = 1.500 g/cm3, T = 100 K, μ = 3.502 mm-1,
λ = 1.54187 Å, R1 = 0.035, wR2 = 0.071, Flack Parameter13 = 0.036(17).
All measurements were made on a Rigaku R-AXIS RAPID-191R diffractometer using graphite
monochromated Cu-K radiation. The structure was solved by direct methods with SIR200814 and was
refined using full-matrix least-squares on F2 with SHELXL-97.15 All non-H atoms were refined with
anisotropic displacement parameters.
CCDC 1456967 for compound 9 contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/Community/Requestastructure/Pages/DataRequest.aspx?.
5.2. Monoamine reuptake inhibition
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.
25

Human dopamine transporter expression plasmid was prepared by following procedure. SRαpromoter
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 BIO INC.).
Into this site was inserted a SRαpromoter fragment to 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 1.63Kb fragment obtained by cleaving pGFP-C1 (manufactured by TOYOBO) with
restriction enzyme Bsu36I (manufactured by Daiichi Pure Chemicals Co., Ltd.) followed by blunt-
ending, whereby pMSRα neo was prepared. Human dopamine transporter cDNA was amplified from
human substantia nigra cDNA library by PCR, and inserted into the pCRII vector (manufactured by
Invitrogen). The base sequence was confirmed, modified and subcloned to pMSRα neo, whereby a
human dopamine transporter expression plasmid was constructed.
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.
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 operation. The obtained cells were counted, and diluted to
3×105 cells per 1 mL medium, the mixture was dispensed to a 96 well white plate (Corning) at 100 μL
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%
26

BSA) was prepared, the medium in the cell plate was removed and the assay buffer was added by 80 μL.
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 10 μL. [3H]-5-Hydroxytryptamine (GE Healthcare) was diluted with the
assay buffer to 200 nM, and the mixture was dispensed to the cell plate by 10 μL. At 20 min from [3H]-
5-hydroxytryptamine addition, the assay buffer was removed by suction, and the plate was washed twice
with 150 μL of PBS (Invitrogen) per well. Microscinti 20 (PerkinElmer) was dispensed to each well by
100 μL, and the mixture was stirred for about 30 min. The radioactivity was measured by TopCount
(PerkinElmer). The inhibitory activity of each compound (10 μM) was calculated as a relative activity
value based on the inhibitory activity of 10 μM Paroxetine (serotonin transporter 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 transporter 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 centrifugal operation. The obtained cells were counted,
and diluted to 3×105 cells per 1 mL medium, and the mixture was dispensed to a 96 well white plate
(Corning) at 100 μL 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 μL. 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 10 μL. [3H]-Norepinephrine (GE Healthcare) was diluted
with the assay buffer to 200 nM, and the mixture was dispensed to the cell plate by 10 μL. At 45 min
from [3H]-norepinephrine addition, the assay buffer was removed by suction, and the plate was washed
27

twice with 150 μL of PBS (Invitrogen) per well. Microscinti 20 (PerkinElmer) was dispensed to each
well by 100 μL, and the mixture was stirred for about 30 min. The radioactivity was measured by
TopCount (PerkinElmer). The inhibitory activity of each compound (10 μM) was calculated as a relative
activity value based on the inhibitory activity of 10 μM DMI (norepinephrine transporter inhibitor) as
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 inhibitory activity. Unless otherwise indicated, these CHO cells were cultured 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 centrifugal operation. The obtained cells were counted,
and diluted to 3×105 cells per 1 mL medium, and the mixture was dispensed to a 96 well white plate
(Corning) at 100 μL 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 buffer was added by 80 μL. 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 10 μL. [3H]-Dopamine (GE
Healthcare) was diluted with the assay buffer to 200 nM, cold dopamine was diluted to 10 μM, and the
mixture was dispensed to the cell plate by 10 μL. At 60 min from [3H]-dopamine addition, the assay
buffer was removed by suction, and the plate was washed twice with 150 μL of PBS (Invitrogen) per
well. Microscinti 20 (PerkinElmer) was dispensed to each well by 100 μL, and the mixture was stirred
for about 30 min. The radioactivity was measured by TopCount (PerkinElmer). The inhibitory activity
of each compound (10 μM) was calculated as a relative activity value based on the inhibitory activity of
100 μM Nomifensine (dopamine transporter inhibitor) as 100%. IC50 and 95% confidence interval were
calculated by XLfit (n=2).
28

5.3. NET ex vivo binding assay in rat brain cortex
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 intravenously 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 of the fraction was 4 mg/mL. Six hundred μL of the membrane fraction, 200 μL of the
assay buffer and 100 μL of [3H]-nisoxetine solution (25 nmol/L) were mixed, and 100 μL of
desipramine (10 μmol/L) or assay buffer was added, and the mixture was incubated 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%.
5.4. Evaluation of urethral resistance-increasing effects in rat
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 urinary 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 Seisakusho). A pressure transducer (DX-100;
Nihon Koden) connected to an amplifier (1257; NEC), an analog-to-digital converter (MP-100 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.
29