Scaffold hopping of fused piperidine-type NK3 receptor antagonists to reduce environmental impact

Article abstract of DOI:10.1016/j.bmc.2019.03.059

Neurokinin-3 receptor (NK3R) plays a pivotal role in the release of gonadotropin-releasing hormone in the hypothalamus–pituitary–gonadal (HPG) axis. To develop novel NK3R antagonists with less environmental toxicity, a series of heterocyclic scaffolds for

Full text of DOI:10.1016/j.bmc.2019.03.059

Accepted Manuscript
Scaffold hopping of fused piperidine-type NK3 receptor antagonists to reduce
environmental impact
Koki Yamamoto, Shinsuke Inuki, Hiroaki Ohno, Shinya Oishi
PII:
S0968-0896(19)30435-3
https://doi.org/10.1016/j.bmc.2019.03.059
BMC 14851
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
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29 March 2019
30 March 2019
Please cite this article as: Yamamoto, K., Inuki, S., Ohno, H., Oishi, S., Scaffold hopping of fused piperidine-type
NK3 receptor antagonists to reduce environmental impact, Bioorganic & Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.bmc.2019.03.059
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Scaffold hopping of fused piperidine-type NK3 receptor antagonists to reduce environmental
impact
Koki Yamamoto, Shinsuke Inuki, Hiroaki Ohno, Shinya Oishi*
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
*Corresponding Author:
Shinya Oishi, Ph.D.
Graduate School of Pharmaceutical Sciences
Kyoto University
Sakyo-ku, Kyoto, 606-8501, Japan
Tel.: +81-75-753-4561; Fax: +81-75-753-4570,
E-mail: soishi@pharm.kyoto-u.ac.jp

ABSTRACT
Neurokinin-3 receptor (NK3R) plays a pivotal role in the release of gonadotropin-releasing hormone
in the hypothalamus–pituitary–gonadal (HPG) axis. To develop novel NK3R antagonists with less
environmental toxicity, a series of heterocyclic scaffolds for the triazolopiperazine substructure in an
NK3R antagonist fezolinetant were designed and synthesized. An isoxazolo[3,4-c]piperidine
derivative exhibited moderate NK3R antagonistic activity and favorable properties that were
decomposable under environmental conditions.
Keywords: environmental impact; fezolinetant; GnRH; NK3 receptor; scaffold hopping
This paper is dedicated to the memory of Prof. Kei-ichiro Maeda of The University of Tokyo, Japan.
Abbreviations: DMB, 2,4-dimethoxybenzoyl; 4-FB, 4-fluorobenzoyl; GnRH, gonadotropin-releasing
hormone; NKB, neurokinin B; NK3R, neurokinin-3 receptor; PCOS, polycystic ovary syndrome.

1. Introduction
Pharmaceuticals include one or multiple bioactive ingredients that cure and/or alleviate
symptoms in human or veterinary diseases. Many of these ingredients are excreted from the body in
an unmetabolized form and/or as active metabolites via urine and/or feces. The fate of each
ingredient following excretion depends on its chemical properties as well as the methods used to
treat water it is excreted into.[1] Even after wastewater treatment, bioactive ingredients and their
metabolites often survive to become ground and surface water contaminants in downstream areas.[2]
High chemical stability of the ingredients is crucial for quality control of pharmaceuticals during
long-term storage before administration. However, in terms of the impacts on the natural
environment and ecosystems, stable bioactive substances with high bioactivity may have
unfavorable effects on non-target species after excretion from patients or treated animals. Indeed,
there have been cases of environmental contamination by bioactive ingredients derived from human
and animal drugs that have led to the emergence of drug-resistant strains,[3] effects on the sex
determination of aquatic fauna and flora,[4] and accumulation in organisms higher up in the food
chain.[5] To overcome these disadvantages, drug design to turn off bioactivity after release into the
environment is needed.
Neurokinin-3 receptor (NK3R) regulates pulsatile secretion of gonadotropin-releasing hormone
(GnRH) in the hypothalamus–pituitary–gonadal (HPG) axis.[6] Activation of NK3R promotes the
reproductive hormone cascade via activation of the GnRH neuron in the hypothalamus, leading to

the pulsatile secretion of luteinizing hormone (LH) from the pituitary gland.[7–9] In contrast, NK3R
antagonists, such as osanetant (1) and talnetant (2a), which were initially investigated for the
treatment of schizophrenia, negatively regulate reproductive functions in mammals (Figure
1).[10,11] For example, talnetant decreased the secretion of testosterone in Guinia pigs.[12]
Additionally, a recent clinical study revealed that oral administration of pavinetant (2b), which is
expected to be applicable to treatment of polycystic ovarian syndrome (PCOS), effectively
decreased the testosterone levels of healthy volunteers.[13,14] Alternatively, fezolinetant (3), which
has a unique triazolopiperazine scaffold,[15] effectively decreased LH levels in rats and
monkeys.[16,17] Clinical investigations of fezolinetant for the treatment of hot flashes are now
ongoing. Thus, NK3R antagonists are expected to be therapeutic agents for various disorders of
reproductive functions.
However, these NK3R antagonists and their bioactive metabolite(s) may affect the reproductive
functions of non-target species via water pollution and/or soil contamination after excretion from
treated humans and animals. To minimize the possibility of these adverse effects on non-target
species, structures with potent NK3R antagonistic activity need to be converted into inactive form(s)
by drug metabolizing systems in the body before excretion and/or by spontaneous degradation in the
environment soon after excretion. In our structure-activity relationship studies of talnetant, we
identified a talnetant analog, 2c, that can be converted into inactive disulfide and isothiazolone
forms by air oxidation.[18] Although the NK3R antagonism of thiol 2c was favorably convertible

under the conditions of the natural environment, its gradual inactivation in serum was also observed,
which would potentially prevent prolonged in vivo biological activity. We also attempted to append
the degradable properties onto fezolinetant by scaffold hopping, which is a process in which the core
structure(s) are substituted and/or modified with other motifs.[19] Substitution of the
triazolopiperazine scaffold in fezolinetant with nonplanar oxadiazolopiperazine synthesized by
gold(I)-catalyzed domino cyclization unexpectedly led to loss of the NK3R antagonistic activity.[20]
Based on these findings from our previous investigations, we expected that a series of aromatic
heterocycles mimicking the planar 1,2,4-triazole moiety in fezolinetant could be appropriate
scaffolds for novel NK3R antagonists with maintenance of NK3R antagonistic activity as well as
favorable degradation properties. Herein we conducted a structure-activity relationship study of the
triazolopiperazine part of fezolinetant by scaffold hopping for decomposable motif(s). The stability
characteristics of a series of derivatives under simulated-sunlight irradiation and in serum were also
investigated.
Figure 1. Structures of NK3R antagonists.

2. Results and discussion
2.1. Design of fezolinetant derivatives with decomposable scaffold
Initially, we designed potential NK3R antagonists, in which the scaffolds could be decomposed
via hydrolysis or photolysis under environmental conditions (Figure 2). We selected the quinoline
derivative 4a[21] as a lead compound for this structure-activity relationship study because of the
synthetic feasibility of a series of derivatives. Based on substitution of the 1,2,4-triazole moiety in
4a with five-membered aromatic heterocycles consisting of a combination of nitrogens and/or
oxygens, three fused piperidine derivatives 4b–d were possible. The arrangements of hydrogen bond
acceptors derived from two imino nitrogens of 1,2,4-triazole in 4a were reproduced in 1,2,3-triazole
4b and isoxazoles 4c and 4d. The [1,2,3]triazolo[4,5-c]piperidine scaffold in 4b was expected to
degrade in response to sunlight exposure because 1,2,3-triazole is decomposed by UV
irradiation.[22] Isoxazolo[3,4-c]piperidine 4c and isoxazolo[5,4-c]piperidine 4d were also designed

on the basis of possible degradation of the isoxazole moiety via hydrolysis or
photodegradation.[23,24]
Figure 2. Scaffold hopping from a fezolinetant derivative 4a for design of novel NK3R antagonists
with reduced environmental toxicity.
2.2. Synthesis of fezolinetant derivatives
We synthesized the fezolinetant derivatives 4a–d. Triazolopiperazine 6 was obtained by treatment
of piperazine 5 with quinoline-2-carbohydrazide. [1,2,4]Triazolo-[3,4-a]piperazine (R)-4a was
prepared from 6 in two steps via deprotection of the 2,4-dimethoxybenzyl (DMB) group followed by
acylation with 4-fluorobenzoyl chloride (Scheme 1A).[15] The fused piperidine derivatives 4b–d
were synthesized from a common substrate, 3-oxopiperidine derivative 7 (Scheme 1B). Reaction of
ketone 7 with 1-azido-4-nitrobenzene provided 1,2,3-triazole 8.[25] N1-arylation on triazole 8

proceeded by treatment of triazole 8 with 2-chloroquinoline in the presence of i-Pr2NEt to afford the
desired 1-(quinolin-2-yl)triazole 9 after separation from a mixture of regioisomers. Boc deprotection
of
9
followed by acylation with 4-fluorobenzoyl chloride gave the expected
[1,2,3]triazolo[4,5-c]piperidine 4b. The structure of 4b was determined by X-ray crystal structure
analysis. Synthesis of isoxazolopiperidines 4c and 4d began with treatment of ketone 7 with LDA
and quinaldoyl chloride. The resulting diketone 10 was reacted with hydroxylamine under basic
conditions to provide isoxazolo[3,4-c]piperidine 11c and isoxazolo[5,4-c]piperidine 11d after
separation by column chromatography. Isoxazoles 4c and 4d were obtained by the same
manipulations of 11c and 11d, respectively, as for the synthesis of 4b.
Scheme 1. Synthesis of fezolinetant derivatives 4a–d. Reagent and conditions: (a)
quinoline-2-carbohydrazide, EtOH, 70 °C; (b) TFA, CH2Cl2, 0 °C to rt; (c) 4-fluorobenzoyl chloride,
Et3N, CH2Cl2, rt; (d) 1-azido-4-nitrobenzene, NH4OAc, DMF, 80 °C; (e) 2-chloroquinoline, i-Pr2NEt,
120 °C; (f) HCl/dioxane, CH2Cl2, rt; (g) LDA, quinaldoyl chloride, THF, −78 °C to rt; (h)
NH2OH·HCl, NaOH, i-PrOH, reflux. Abbreviations: DMB, 2,4-dimethoxybenzyl; 4-FB,
4-fluorobenzoyl.

To identify the structures of compounds 4c and 4d, which cannot be distinguished by X-ray
analysis, the isoxazolo[3,4-c]piperidine 4c was synthesized via an alternative route from
N-Boc-D-alaninol 12 (Scheme 2). Swern oxidation of alcohol (12) followed by treatment with
hydroxylamine gave the oxime 13. After chlorination of 13 using N-chlorosuccinimide (NCS), the
nitrile oxide intermediate 15 was generated under basic conditions, then subjected to 1,3-dipolar
cycloaddition with 2-ethynylquinoline to provide the expected isoxazole 16. Treatment of isoxazole
16 with N-bromosuccinimide (NBS) in the presence of HBr afforded a 4-bromoisoxazole 17.
Subsequently, Suzuki-Miyaura coupling of 17 with vinyl boronic acid pinacol ester provided the
4-vinylisoxazole 18. 9-BBN-mediated hydroboration of 18 followed by oxidative treatment gave the

4-hydroxyethyl isoxazole 19. After the hydroxy group in 19 was activated with a mesyl group, Boc
group deprotection and acylation was conducted using 4-fluorobenzoyl chloride to afford the
isoxazolo[3,4-c]piperidine (R)-4c. The spectral data of (R)-4c coincided well with those of
isoxazolo[3,4-c]piperidine 4c.
Scheme 2. Synthesis of (R)-4c. Reagent and conditions: (a) (COCl)2, DMSO, i-Pr2NEt, CH2Cl2,
−78 °C to 0 °C; (b) NH2OH·HCl, NaOAc, EtOH, 80 °C; (c) NCS, HCl/dioxane, DMF, 40 °C; (d)
2-ethynylquinoline, NaHCO3, EtOAc, 40 °C; (e) NBS, HBr, DMF, 60 °C; (f) vinylboronic acid
pinacol ester, Pd(Ph3P)4, CsCO3, DMF, 100 °C; (g) 9-BBN, THF, 0 °C to rt, then H2O2, NaOH,
MeOH, THF, rt; (h) MsCl, Et3N, CH2Cl2, rt; (i) HCl/dioxane, CH2Cl2, rt; (j) 4-fluorobenzoyl
chloride, Et3N, CH2Cl2, rt.
2.3. Biological evaluation of fezolinetant derivatives
We investigated the biological activity of fused piperidine derivatives 4b–d by evaluating the
antagonism of NKB-induced activation for human NK3R (Table 1).[20] 1,2,3-Triazole 4b exhibited

15-fold less potent NK3R inhibition than 1,2,4-triazole (R)-4a [IC50 ((R)-4a): 0.26 µM, IC50 (4b):
4.1 µM]. Similarly, isoxazoles 4c and 4d showed moderate NK3R inhibition [IC50 (4c): 10.3 µM,
IC50 (4d): 8.2 µM]. These findings suggested that the presence of two nitrogens of piperazine in 4a
was favorable to NK3R inhibition.

Table 1. NK3R antagonism of fused piperidine derivatives.
compound
structure
IC50 (µM)a
(R)-4a
0.26 ± 0.02
4.1 ± 0.5
10.3 ± 1.5
11.8 ± 1.8
13.7 ± 1.8
8.2 ± 1.7
4b
4c
(R)-4c
(S)-4c
4d
aIC50 values are the concentrations required for 50% inhibition of the NKB-mediated activation of
NK3R.
To identify the stereoisomer of isoxazolo[3,4-c]piperidine 4c that predominantly contributed to
the bioactivity, two isoxazolo[3,4-c]piperidines (R)-4c and (S)-4c were evaluated for NK3R
antagonistic activity after separation by chiral chromatography using a COSMOSIL CHiRAL 5A
column. In contrast to the (R)-isomer of fezolinetant having more potent activity toward NK3R than
the (S)-isomer in the SAR study,[15a] the derivatives (R)-4c and (S)-4c exhibited essentially
identical NK3R antagonism [IC50 ((R)-4c): 11.8 ± 1.8 µM, IC50 ((S)-4c): 13.7 ± 1.8 µM]. These
findings suggest that the chirality of the 7-methyl group of the isoxazolo[3,4-c]piperidine in 4c had
little effect on the biological activity.
2.4. Investigation of photodegradation of fezolinetant derivatives

We investigated the spectral features and photodegradation profiles of 4a–d. It has been reported
that the absorption spectra of photodegradable compounds overlap with the actinic spectrum.[26]
Initially, the absorption spectra of all derivatives were evaluated to identify those with the potential
for photodegradation by sunlight exposure. The UV-vis spectrum of isoxazolo[3,4-c]piperidine 4c
showed strong absorption in the wavelength range of 300–350 nm (Figure 3). In contrast,
[1,2,4]triazolo[3,4-a]piperazine
4a,
[1,2,3]triazolo[4,5-c]piperidine
4b
and
isoxazolo[5,4-c]piperidine 4d exhibited low UV-visible absorption at above 300 nm.
Figure 3. Absorption spectra of fused piperidine-type NK3R antagonists in 50 mM phosphine buffer
(pH 7.4)–EtOH (7:3).
4
3
4a
4b
2
1
0
4c
4d
200
250
300
nm
350
400
The photochemical stability was assessed by irradiation via a mercury xenon lamp through a UV
cut-off filter (below 325 nm) in which the optical spectrum corresponds to that of UV-A (Figure
4A).[27] Among triazolopiperazine 4a and fused piperidines 4b–d, isoxazolo[3,4-c]piperidine 4c
was promptly decomposed, while other scaffolds (4a, 4b and 4d) were stable under irradiation with
a mercury xenon lamp. The simulated sunlight irradiation by the xenon lamp (>325 nm) also led to
decomposition of 4c to provide multiple products (Figure 4B and Supplementary Figure S1). After

UV-irradiation of 4c, the sample did not show NK3R inhibition (IC50 >100 µM). These findings
indicated that decomposition would depend on the excitation characteristics of the scaffold by
photoirradiation, and that isoxazole 4c could be inactivated by sunlight exposure.
Figure 4. Photodegradation of fused piperidine-type NK3R antagonists.
(A) UV light irradiation (>325 nm) by mercury xenon lamp (B) Simulated-sunlight (>325 nm) irradiation by xenon lamp
120
100
80
60
40
20
0
120
100
80
60
40
20
0
4a
4b
4c
4d
4a
4b
4c
4d
0
10
20
30
0
10
20
30
time (min)
time (min)
aThe ratios of compounds were calculated by HPLC analysis using calibration curves.
2.4. Investigation of the stability of fezolinetant derivatives under aqueous conditions
We also assessed the stability of fused piperidine derivatives in aqueous buffer by monitoring the
intact compounds by HPLC (Supplementary Figure 2A–C). No hydrolysate was observed from any
derivatives at any pH (pH 4.0, 7.4 and 10.0), suggesting that fused piperidine scaffolds were
unlikely to be decomposed via hydrolysis under environmental conditions. Additionally, we
investigated the stability of fused piperidine derivatives in rat serum (Supplementary Figure 3) and
found no degradation of any of the compounds. Thus, derivative 4c could be a novel NK3R
antagonist that is stable in serum and inactivated in the natural environment via solar light
irradiation after excretion.

3. Conclusion
We designed and synthesized a series of fezolinetant derivatives with fused piperidine scaffolds,
in which the 1,2,4-triazole moiety in fezolinetant was substituted with five-membered aromatic
heterocycles. The resulting derivatives showed moderate NK3R antagonistic activity. Among them,
isoxazolo[3,4-c]piperidine 4c was stable in aqueous buffer and rat serum and had a favorable
photodegradable property, being decomposed by UV irradiation. These results indicated
isoxazolo[3,4-c]piperidine 4c may be a candidate for the treatment of sex-hormone disorders with
less environmental impact.

4. Experimental Section
4.1. Synthesis
4.1.1. General synthesis
1H NMR spectra were recorded using a JEOL AL-400 or a JEOL ECA-500 spectrometer.
13
Chemical shifts are reported in δ (ppm) relative to Me4Si as an internal standard. C NMR spectra
were recorded using a JEOL AL-400 or a JEOL ECA-500 and referenced to the residual solvent
signal. Exact mass (HRMS) spectra were recorded on Shimadzu LC-ESI-IT-TOF-MS equipment. IR
spectra were obtained on a JASCO FT/IR-4100 spectrometer. Melting points were measured by a
hot stage melting points apparatus (uncorrected). Optical rotations were measured with a JASCO
P-1020 polarimeter. Melting points were measured by a hot stage melting points apparatus
(uncorrected). For chromatography, Wakogel C-300E (Wako) was employed.
(R)-1-(2,4-dimethoxylbenzyl)-5-ethoxy-6-methylpiperazine
5[19b] (158 mg, 1.00 mmol) in EtOH (2.00 mL) was added quinoline-2-carbohydrazide[28] (187
mg, 1.00 mmol). After being stirred for 17 h at 70 °C, the reaction mixture was concentrated. The
residue was dissolved with EtOAc, and the whole was washed with 1 M NaOH and brine, and dried
over Na2SO4. After the filtrate was concentrated, the residue was purified by column
chromatography on silica gel (n-hexane/EtOAc = 1/3) to give the title compound 6 (107 mg, 26%):
pale yellow amorphous solid; [α]24D +13.3 (c 1.06, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 1.77 (d, J

= 6.9 Hz, 3H), 2.67-2.72 (m, 1H), 3.20-3.24 (m, 1H), 3.56 (d, J = 13.7 Hz, 1H), 3.78 (s, 6H), 3.94
(d, J = 13.7 Hz, 1H), 4.03-4.08 (m, 1H), 4.45-4.50 (m, 1H), 4.75-4.80 (m, 1H), 6.46-6.49 (m, 2H),
7.27-7.28 (m, 1H), 7.46-7.48 (m, 1H), 7.62-7.65 (m, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0
13
Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.36-8.37 (m, 1H); C-NMR (125 MHz, CDCl3) δ 17.3, 45.2,
45.6, 49.8, 53.6, 54.9, 55.0, 98.0, 103.7, 117.6, 119.6, 126.6, 127.1, 127.3, 128.9, 129.3, 130.6,
136.1, 146.7, 147.5, 150.5, 155.7, 158.4, 159.8; HRMS (ESI) calcd for C24H26N5O2 [M + H]+:
416.2081, found: 416.2086.
compound 6 (83.0 mg, 0.200 mmol) in CH2Cl2 (1.00
mL) was added TFA (115 µL, 1.50 mmol) at 0 °C. After being stirred for 4.5 h at room temperature,
the reaction mixture was extracted with EtOAc, and the whole was washed with H2O. The aqueous
layer was basified by 2 M NaOH and extracted with EtOAc. The organic layer was washed with
brine and dried over Na2SO4. After the filtrate was concentrated, the residue was dissolved in
CH2Cl2 (2.00 mL). To the solution were added Et3N (55.8 μL, 0.0642 mmol) and 4-fluorobenzoyl
chloride (3.88 μL, 0.0321 mmol). After being stirred for 15 min, the reaction mixture was diluted
with CH2Cl2, washed with 1 M HCl and brine, and dried over MgSO4. After the filtrate was
concentrated, the residue was purified by column chromatography on silica gel (n-hexane/EtOAc =
1/3) to give the title compound 4a (20.3 mg, 26%): colorless solid; mp 232-234 °C; [α]24D +57.3 (c
1
0.60, MeOH); IR (neat) 1641 (C=O) cm-1; H-NMR (500 MHz, DMSO-d6) δ 1.67 (d, J = 6.9 Hz,

3H), 3.66-3.71 (m, 1H), 4.19-4.22 (m, 1H), 4.42-4.46 (m, 1H), 5.08-5.11 (m, 1H), 5.67 (br s, 1H),
7.28-7.31 (m, 2H), 7.59-7.67 (m, 3H), 7.80-7.83 (m, 1H), 8.01-8.10 (m, 2H), 8.29-8.30 (m, 1H),
13
8.49 (d, J = 8.0 Hz, 1H); C-NMR (125 MHz, DMSO-d6) δ 18.6, 38.4, 45.3, 46.2, 115.0 (d, JC-F
=
=
21.6 Hz, 2C), 119.2, 126.9, 127.0, 128.6, 128.9 (d, JC-F = 9.6 Hz, 2C), 129.7 (2C), 131.5 (d, JC-F
3.6 Hz), 136.8, 146.3, 147.1, 150.1, 153.2, 163.4, 168.4; HRMS (ESI) calcd for C22H19FN5O [M +
H]+: 388.1568, found: 388.1566.
tert-butyl 2-methyl-3-oxopiperidine-1-carboxylate 7 (213 mg, 1.00 mmol) in DMF
(2.00 mL) were added 1-azido-4-nitrobenzene[29] (213 mg, 1.30 mmol) and NH4OAc (385 mg, 5.00
mmol). After being stirred for 11 h at 80 °C, the reaction mixture was extracted with EtOAc, washed
with 1 M HCl and brine, and dried over MgSO4. After the filtrate was concentrated, the residue was
purified by column chromatography on silica gel (n-hexane/EtOAc = 3/1) to give the title compound
1
8 (148 mg, 62%): colorless amorphous solid; IR (neat) 1693 (C=O) cm-1; H-NMR (500 MHz,
CDCl3) δ 1.47 (d, J = 6.3 Hz, 3H), 1.50 (s, 9H), 2.78-2.83 (m, 2H), 3.03-3.09 (m, 1H), 4.44 (br s,
13
1H), 5.40 (br s, 1H); C-NMR (125 MHz, CDCl3) δ 19.4, 22.4, 28.5 (3C), 37.4, 47.0, 80.4, 140.7,
145.3, 154.7; HRMS (ESI) calcd for C11H18N4O2 [M + H]+: 239.1503, found: 239.1501.

μ
After the filtrate was concentrated, the residue
was purified by column chromatography on silica gel (n-hexane/EtOAc = 6/1) to give the title
compound 9 (23.0 mg, 21%): colorless amorphous solid; IR (neat) 1695 (C=O) cm-1; 1H-NMR (500
MHz, CDCl3) δ 1.52 (s, 9H), 1.56 (d, J = 6.9 Hz, 3H), 3.08-3.13 (m, 1H), 3.27-3.34 (m, 1H),
3.55-3.59 (m, 1H), 4.51 (br s, 1H), 5.45 (br s, 1H), 7.57-7.60 (m, 1H), 7.74-7.77 (m, 1H), 7.87 (d, J
13
= 8.6 Hz, 1H), 8.01 (d, J = 8.6 Hz, 1H), 8.29-8.35 (m, 2H); C-NMR (125 MHz, CDCl3) δ 19.0,
25.0, 28.5 (3C), 36.5, 47.0, 80.2, 113.9, 127.0, 127.4, 127.7, 128.9, 130.5, 131.4, 139.3, 146.3,
146.9, 149.6, 154.5; HRMS (ESI) calcd for C20H24N5O2 [M + H]+: 366.1925, found: 366.1921.
(4-Fluorophenyl)[4-methyl-1-(quinolin-2-yl)-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo-
[4,5-c]pyridin-5-yl]methanone (4b).
(7.80 mg, 0.0214 mmol) in
CH2Cl2 (500 μL) was added HCl (4 M in dioxane; 250 μL, 1.00 mmol). After being stirred for 1 h,
the reaction mixture was basified by 2 M NaOH solution at 0 °C, and the whole was extracted with
CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue
was dissolved in CH2Cl2 (500 μL). To the solution were added Et3N (8.95 μL, 0.0642 mmol) and
4-fluorobenzoyl chloride (3.88 μL, 0.0321 mmol). After being stirred for 15 min, the reaction
mixture was diluted with CH2Cl2, washed with 1 M HCl and brine, and dried over MgSO4. After the
filtrate was concentrated, the residue was purified by column chromatography on silica gel
(n-hexane/EtOAc = 3/1) to give the title compound 4b (7.30 mg, 88%): colorless solid; mp 199-201

1
°C; IR (neat) 1637 (C=O) cm-1; H-NMR (500 MHz, DMSO-d6) δ 1.58 (d, J = 6.9 Hz, 3H),
3.30-3.54 (m, 3H), 4.17 (br s, 1H), 5.50 (br s, 1H), 7.26-7.29 (m, 2H), 7.53-7.55 (m, 2H), 7.65-7.67
(m, 1H), 7.83-7.84 (m, 1H), 8.01-8.07 (m, 2H), 8.22 (d, J = 8.6 Hz, 1H), 8.62 (d, J = 8.6 Hz, 1H);
13C-NMR (125 MHz, DMSO-d6) δ 18.5, 23.8, 38.0, 46.1, 113.2, 115.0 (d, JC-F = 21.6 Hz, 2C), 126.7
(2C), 127.5, 127.9, 128.6 (d, JC-F = 4.8 Hz, 2C), 130.3, 130.7, 132.4 (d, JC-F = 3.6 Hz), 139.6, 145.2,
145.3, 148.6, 162.3 (d, JC-F = 247.1 Hz), 168.7; HRMS (ESI) calcd for C22H19FN5O [M + H]+:
388.1568, found: 388.1566.
Crystallography of 4b. The data of the compound 4b (C22H18FN5O) was collected with a Rigaku
XtaLAB P200 diffractometer using multi-layer mirror monochromated Cu-Kα radiation at 93 K.
The substance was crystallized from CHCl3–n-hexane as clear colorless block crystals and solved in
primitive orthorhombic space group P21/c with Z = 4. The unit cell dimensions are a = 7.5117(3), b
= 35.6861(8), c = 7.4465(4), V = 1788.70(15) Å3, Dcalc = 1.439 g/cm3, Mw: 387.41. R = 0.0746,
GOF = 1.036. The CCDC deposition number: CCDC 1893155.
tert-Butyl 2-methyl-3-oxo-4-(quinoline-2-carbonyl)piperidine-1-carboxylate (10). To a
solution of i-Pr2NH (425 μL, 3.00 mmol) in THF (2.00 mL) was added n-BuLi (1.39 M in hexane;

2.45 mL, 3.40 mmol) at −78 °C under argon and the mixture was stirred for 20 min at 0 °C. A
solution of ketone 7 (426 mg, 2.00 mmol) in THF (2.00 mL) was added to the mixture at −78 °C and
the mixture was stirred for 30 min at this temperature. A solution of quinaldoyl chloride (497 mg,
2.60 mmol) in THF (4.00 mL) was added to the mixture. After being stirred for 6 h at room
temperature, the reaction mixture was quenched with 1 M HCl and the whole was extracted with
EtOAc. The organic layer was washed with saturated NaHCO3 and brine, dried over MgSO4 and
concentrated. The residue was purified by column chromatography on silica gel (n-hexane/EtOAc =
10/1) to give the title compound 10 (271 mg, 37%): yellow solid; mp 123-125 °C; IR (neat) 1687
1
(C=O) cm-1; H-NMR (400 MHz, CDCl3) δ 1.49-1.50 (m, 12H), 2.78-2.81 (m, 1H), 2.97-2.99 (m,
2H), 4.10-4.12 (m, 1H), 4.75 (br s, 1H), 7.62-7.66 (m, 1H), 7.76-7.80 (m, 1H), 7.87 (d, J = 8.6 Hz,
13
1H), 8.04 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 8.31 (d, J = 8.6 Hz, 1H); C-NMR (100
MHz, CDCl3) δ 17.5, 25.9, 28.5 (3C), 37.6, 52.8, 80.0, 106.8, 120.6, 127.7, 128.4, 128.6, 129.4,
130.4, 137.3, 137.5, 145.6, 153.9, 154.1, 186.6; HRMS (ESI) calcd for C21H25N2O4 [M + H]+:
369.1809, found: 369.1811.
tert-Butyl
7-methyl-3-(quinolin-2-yl)-4,5-dihydroisoxazolo[3,4-c]pyridine-6-(7H)-carboxylate (11c) and
tert-butyl 7-methyl-3-(quinolin-2-yl)-4,7-dihydroisoxazolo[5,4-c]pyridine-6-(5H)-carboxylate
(11d). To a solution of diketone 10 (159 mg, 0.432 mmol) in i-PrOH (2.00 mL) were added
NH2OH·HCl (60.0 mg, 0.864 mmol) and NaOH (52.0 mg, 1.30 mmol). After being stirred for 2 h

under reflux conditions, the reaction mixture was extracted with EtOAc. The organic layer was
washed with saturated NaHCO3 and brine, dried over Na2SO4, and concentrated. The residue was
purified by column chromatography on silica gel (n-hexane/EtOAc = 20/1) to give the title
compounds 11c (35.2 mg, 22%) and 11d (43.5 mg, 28%).
1
Compound 11c: colorless solid; mp 137-139 °C; IR (neat) 1698 (C=O) cm-1; H-NMR (400 MHz,
CDCl3) δ 1.52 (s, 9H), 1.56 (d, J = 7.0 Hz, 3H), 2.93-3.08 (m, 2H), 3.39-3.44 (m, 1H), 4.43 (br s,
1H), 5.55 (br s, 1H), 7.55-7.59 (m, 1H), 7.72-7.76 (m, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.97 (d, J = 8.7
13
Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 8.24 (d, J = 8.7 Hz, 1H); C-NMR (100 MHz, CDCl3) δ 19.7,
22.1, 28.4 (3C), 36.3, 46.4, 80.3, 113.1, 118.5, 127.3, 127.5, 127.6, 129.7, 130.0, 136.9, 147.6, 147.9,
154.4, 161.9, 163.9; HRMS (ESI) calcd for C21H24N3O3 [M + H]+: 366.1812, found: 366.1812.
1
Compound 11d: colorless solid; mp 127-129 °C; IR (neat) 1697 (C=O) cm-1; H-NMR (400 MHz,
CDCl3) δ 1.51-1.53 (m, 12H), 2.93-3.05 (m, 2H), 3.24-3.28 (m, 1H), 4.35-4.48 (br m, 1H), 5.29-5.45
(br m, 1H), 7.56-7.60 (m, 1H), 7.71-7.76 (m, 1H), 7.83-7.85 (m, 1H), 8.10-8.14 (m, 2H), 8.23 (d, J =
13
8.2 Hz, 1H); C-NMR (100 MHz, CDCl3) δ 17.9, 22.5, 28.4 (3C), 37.0, 47.6, 80.4, 111.7, 119.6,
127.2, 127.6, 127.9, 129.7, 129.8, 136.7, 147.9, 149.7, 154.3, 159.6, 169.3; HRMS (ESI) calcd for
C21H24N3O3 [M + H]+: 366.1812, found: 366.1822.
(4-Fluorophenyl)[7-methyl-3-(quinolin-2-yl)-4,5-dihydroisoxazolo[3,4-c]pyridin-6(7H)-yl]m
ethanone (4c). By use of a procedure similar to that described for the preparation of the compound
4b from 9, the compound
(43.5 mg, 0.0214 mmol) was converted to the title compound 4c (36.8

1
mg, 80%): colorless solid; mp 172-174 °C; IR (neat) 1640 (C=O) cm-1; H-NMR (500 MHz,
DMSO-d6) δ 1.60 (d, J = 6.9 Hz, 3H), 2.99-3.06 (m, 1H), 3.30-3.42 (m, 2H), 4.04-4.06 (m, 1H),
5.63-5.65 (m, 1H), 7.26-7.30 (m, 2H), 7.54-7.56 (m, 2H), 7.63-7.67 (m, 1H), 7.80-7.83 (m, 1H),
7.97-8.02 (m, 2H), 8.06 (d, J = 8.6 Hz, 1H), 8.51 (d, J = 8.6 Hz, 1H); 13C-NMR (125 MHz,
DMSO-d6) δ 18.9, 21.3, 38.4, 45.4, 112.0, 115.0 (d, JC-F = 21.6 Hz, 2C), 117.9, 127.0 (d, JC-F = 13.2
Hz, 2C), 127.5, 128.6, 128.7, 130.0, 132.1 (d, JC-F = 3.6 Hz), 137.1, 146.5, 147.0, 161.2, 161.3,
162.3 (d, JC-F = 248.3 Hz), 163.3, 168.7; HRMS (ESI) calcd for C23H19FN3O2 [M + H]+: 388.1456,
found: 388.1458.
(4-Fluorophenyl)[7-methyl-3-(quinolin-2-yl)-4,7-dihydroisoxazolo[5,4-c]pyridin-6(5H)-yl]m
ethanone (4d). By use of a procedure similar to that described for the preparation of the compound
4b from 9, the compound 11d (35.2 mg, 0.0961 mmol) was converted to the title compound 4d (25.2
1
mg, 68%): colorless solid; mp 161-163 °C; IR (neat) 1603 (C=O) cm-1; H-NMR (500 MHz,
DMSO-d6) δ 1.60 (d, J = 6.9 Hz, 3H), 2.99-3.06 (m, 1H), 3.30-3.42 (m, 2H), 4.04-4.06 (m, 1H),
5.63-5.65 (m, 1H), 7.26-7.30 (m, 2H), 7.54-7.56 (m, 2H), 7.63-7.67 (m, 1H), 7.80-7.83 (m, 1H),
7.97-8.02 (m, 2H), 8.06 (d, J = 8.6 Hz, 1H), 8.51 (d, J = 8.6 Hz, 1H); 13C-NMR (125 MHz,
DMSO-d6) δ 17.1, 21.8, 37.2, 46.3, 110.6, 115.0 (d, JC-F = 19.2 Hz, 2C), 118.7, 127.0, 127.4 (d, JC-F
= 18.0 Hz, 2C), 128.7 (2C), 129.6, 132.1 (d, JC-F = 3.6 Hz) , 136.8, 146.9, 148.6, 158.8, 161.3, 162.3
(d, JC-F = 247.1 Hz), 168.4, 168.6; HRMS (ESI) calcd for C23H19FN3O2 [M + H]+: 388.1456, found:
388.1464.

tert-Butyl (R)-{1-[5-(quinolin-2-yl)isoxazol-3-yl]ethyl}carbamate (16). DMSO (710 µL, 10.0
mmol) was added dropwise to a solution of oxalyl chloride (396 µL, 7.50 mmol) in CH2Cl2 (10.0
mL) under argon at −78 °C. After the reaction mixture was stirred for 15 min at same temperature, a
solution of N-(tert-butoxycarbonyl)-D-alaninol (12) (876 mg, 5.00 mmol) in CH2Cl2 (5.00 mL) was
added to the mixture dropwise. After the reaction mixture was stirred for 30 min at same
temperature, i-Pr2NEt (4.35 mL, 25.0 mmol) was added to the mixture dropwise. After being stirred
for 30 min at 0 °C, the reaction mixture was washed with 1 M HCl and brine, dried over MgSO4.
After the filtrate was concentrated, the residue was dissolved in EtOH (5.00 mL). To the mixture
were added
·
(417 mg, 6.00 mmol) and NaOAc (492 mg, 6.00 mmol) and the reaction
was continued for 30 min at 80 °C. The reaction mixture was diluted with EtOAc and washed with
H2O and brine, and dried over MgSO4. After the filtrate was concentrated, the residue was dissolved
in DMF (10.0 mL). To the mixture were added NCS (1.00 g, 7.50 mmol) and HCl (4 M in dioxane;
125 µL, 0.500 mmol and the reaction was continued for 1 h at 40 °C. The reaction mixture was
diluted with EtOAc, washed with H2O and brine, and dried over MgSO4. After the filtrate was
concentrated, the residue was dissolved in EtOAc (10.0 mL). To the mixture were added
2-ethynylquinoline[30] (459 mmol, 3.00 mmol) and NaHCO3 (1.01 g, 12.0 mmol). After being
stirred for 15 h at 40 °C, the whole was diluted with EtOAc and washed with H2O and brine, and
dried over MgSO4. After the filtrate was concentrated, the residue was purified by column
chromatography on silica gel (n-hexane/EtOAc = 20/1 to 5/1) to give the title compound 16 (575 mg,

57%): colorless needles; [α]24D +51.0 (c 1.35, CHCl3); mp 135-137 °C; IR (neat) 1684 (C=O) cm-1;
1H-NMR (500 MHz, CDCl3) δ 1.48 (s, 9H), 1.60 (d, J = 6.9 Hz, 3H), 5.07 (br s, 1H), 5.20 (br s, 1H),
7.06 (s, 1H), 7.57-7.60 (m, 1H), 7.74-7.78 (m, 1H), 7.83-7.85 (m, 1H), 7.98 (d, J = 8.6 Hz, 1H),
13
8.12-8.14 (m, 1H), 8.26 (d, J = 8.6 Hz, 1H); C-NMR (125 MHz, CDCl3) δ 20.8, 28.3 (3C), 44.0,
79.8, 101.4, 118.2, 127.5, 127.7, 128.0, 129.7, 130.3, 137.2, 146.2, 148.0, 155.0, 166.8, 169.7;
HRMS (ESI) calcd for C19H21N3O3Na [M + Na]+: 362.1475, found: 362.1476.
tert-Butyl (R)-{1-[4-bromo-5-(quinolin-2-yl)isoxazol-3-yl]ethyl}carbamate (17). To a solution
of isoxazole 16 (400 mg, 1.18 mmol) and NBS (630 mg, 3.54 mmol) in DMF (6.00 mL) was added
HBr (47% solution in H2O; 13.5 µL, 0.118 mmol). After being stirred for 12 h at 60 °C, the reaction
mixture was diluted with EtOAc, washed with H2O and brine, dried over MgSO4. After the filtrate
was concentrated, the residue was purified by column chromatography on silica gel
(n-hexane/EtOAc = 20/1 to 6/1) to give the title compound 17 (172 mg, 35%): colorless solid; [α]27
D
1
–7.81 (c 0.97, CHCl3); mp 117-119 °C; IR (neat) 1712 (C=O) cm-1; H-NMR (500 MHz, CDCl3) δ
1.47 (s, 9H), 1.60 (d, J = 7.4 Hz, 3H), 5.12-5.15 (m, 1H), 5.29 (d, J = 8.0 Hz, 1H), 7.61-7.64 (m,
1H), 7.77-7.81 (m, 1H), 7.86-7.88 (m, 1H), 8.05-8.07 (m, 1H), 8.23 (d, J = 8.6 Hz, 1H), 8.30 (d, J =
13
8.6 Hz, 1H); C-NMR (125 MHz, CDCl3) δ 20.5, 28.4 (3C), 43.7, 79.9, 91.7, 119.2, 127.6, 127.9,
128.0, 130.1, 130.4, 137.1, 145.9, 147.9, 154.8, 164.1, 165.2; HRMS (ESI) calcd for C19H21BrN3O3
[M + H]+: 418.0761, found: 418.0751.
tert-Butyl (R)-{1-[5-(quinolin-2-yl)-4-vinylisoxazol-3-yl]ethyl}carbamate (18). To a solution

of bromide 17 (313 mg, 0.750 mmol), vinylboronic acid pinacol ester (257 µL, 1.50 mmol), and
Pd(Ph3P)4 (86.7 mg, 0.0750 mmol) in DMF (3.75 mL) was added 2 M CsCO3 solution (3.75 mL).
After being stirred for 4 h at 100 °C, the reaction mixture was diluted with EtOAc, washed with H2O
and brine, dried over MgSO4. After the filtrate was concentrated, the residue was purified by column
chromatography on silica gel (n-hexane/EtOAc = 20/1 to 10/1) to give the title compound 18 (195
mg, 71%): colorless solid; [α]25D –54.5 (c 0.98, CHCl3); mp 122-124 °C; IR (neat) 1710 (C=O) cm-1;
1H-NMR (500 MHz, CDCl3) δ 1.47 (s, 9H), 1.61 (d, J = 6.3 Hz, 3H), 5.27-5.28 (m, 1H), 5.35-5.37
(m, 1H), 5.58 (d, J = 12.0 Hz, 1H), 5.80-5.84 (m, 1H), 7.55-7.62 (m, 2H), 7.75-7.78 (m, 1H),
7.85-7.86 (m, 1H), 7.99 (d, J = 8.6 Hz, 1H), 8.17 (d, J = 8.6 Hz, 1H), 8.27 (d, J = 8.6 Hz, 1H);
13C-NMR (125 MHz, CDCl3) δ 20.9, 28.4 (3C), 43.6, 79.8, 114.8, 119.2, 119.5, 125.3, 127.6 (3C),
129.9, 130.2, 136.9, 147.6, 147.8, 155.0, 163.6, 164.5; HRMS (ESI) calcd for C21H24N3O3 [M + H]+:
366.1812, found: 366.1819.
tert-Butyl (R)-{1-[4-(2-hydroxyethyl)-5-(quinolin-2-yl)isoxazol-3-yl]ethyl}carbamate (19). To
a solution of compound 18 (146 mg, 0.40 mmol) in THF (2.40 mL) was added 9-BBN (0.5 M in
THF; 2.40 mL, 1.20 mmol) at 0 °C and the mixture was stirred for 11 h at room temperature. To the
reaction mixture were added MeOH (400 µL), 2 M NaOH (400 µL), and H2O2 (30% solution in
H2O; 81.7 µL, 0.800 mmol). After being stirred for 5 h at room temperature, the reaction mixture
was diluted with EtOAc, washed with brine, dried over MgSO4. After the filtrate was concentrated,
the residue was purified by column chromatography on silica gel (n-hexane/EtOAc = 20/1 to 10/1)

to give the title compound 19 (53.3 mg, 35%): orange solid; [α]25D –9.23 (c 0.81, CHCl3); mp
1
66-68 °C; IR (neat) 1678 (C=O) cm-1; H-NMR (500 MHz, CDCl3) δ 1.46 (s, 9H), 1.64 (d, J = 6.9
Hz, 3H), 3.12-3.27 (m, 2H), 4.04-4.06 (m, 2H), 5.08-5.09 (m, 1H), 5.24 (d, J = 8.6 Hz, 1H), 6.07 (br
s, 1H), 7.60-7.63 (m, 1H), 7.76-7.79 (m, 1H), 7.86-7.88 (m, 1H), 8.00 (d, J = 8.6 Hz, 1H), 8.14 (d, J
= 8.6 Hz, 1H), 8.33 (d, J = 8.6 Hz, 1H); 13C-NMR (125 MHz, CDCl3) δ 20.5, 25.4, 28.3 (3C), 42.4,
62.9, 80.0, 114.9, 119.3, 127.7, 127.8, 127.9, 128.8, 130.7, 137.9, 146.7 (2C), 154.9, 165.0, 165.9;
HRMS (ESI) calcd for C21H25N3O4Na [M + Na]+: 406.1737, found: 406.1736.
(R)-(4-Fluorophenyl)[7-methyl-3-(quinolin-2-yl)-4,5-dihydroisoxazolo[3,4-c]pyridin-6(7H)-y
l]methanone ((R)-4c). To a solution of compound 19 (38.3 mg, 0.100 mmol) in CH2Cl2 (500 µL)
were added Et3N (41.8 µL, 0.300 mmol) and MsCl (11.6 µL, 0.150 mmol). After being stirred for
2.5 h at room temperature, the reaction mixture was washed with brine, and dried over MgSO4.
After the filtrate was concentrated, the residue was dissolved in CH2Cl2 (1.00 mL) and HCl (4 M in
dioxane; 1.00 mL, 4.00 mmol). After being stirred for 2 h, the reaction mixture was basified by 2 M
NaOH solution, stirred for 1 h at room temperature, and the whole was extracted with CH2Cl2. The
organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was
dissolved in CH2Cl2 (1.00 mL). To the solution were added Et3N (40.9 μL, 0.300 mmol) and
4-fluorobenzoyl chloride (18.1 μL, 0.150 mmol). After being stirred for 30 min, the reaction mixture
was diluted with CH2Cl2, washed with 1 M HCl and brine, and dried over MgSO4. After the filtrate
was concentrated, the residue was purified by column chromatography on silica gel

(n-hexane/EtOAc = 3/1) to give the title compound (R)-4c (15.3 mg, 40%): pale yellow solid; [α]25
D
+132 (c 0.46, CHCl3); mp 181-183 °C; IR (neat) 1639 (C=O) cm-1; 1H-NMR (500 MHz, DMSO-d6)
δ 1.60 (d, J = 6.9 Hz, 3H), 3.02-3.07 (m, 1H), 3.31-3.41 (m, 2H), 4.03 (br s, 1H), 5.63 (br s, 1H),
7.26-7.29 (m, 2H), 7.53-7.56 (m, 2H), 7.64-7.67 (m, 1H), 7.81-7.84 (m, 1H), 7.99 (d, J = 8.6 Hz,
13
1H), 8.01-8.03 (m, 1H), 8.06 (d, J = 8.6 Hz, 1H), 8.53 (d, J = 8.6 Hz, 1H); C-NMR (125 MHz,
DMSO-d6) δ 18.9, 21.3, 38.3, 45.4, 112.0, 115.0 (d, JC-F = 20.4 Hz, 2C), 117.9, 127.0 (d, JC-F = 13.2
Hz, 2C), 127.5, 128.6, 128.7 (2C), 129.9, 132.1 (d, JC-F = 3.6 Hz), 137.1, 146.5, 147.1, 161.2, 162.3
(d, JC-F = 247.1 Hz), 162.7, 168.7; HRMS (ESI) calcd for C23H19FN3O2 [M + H]+: 388.1456, found:
388.1460.
(S)-(4-Fluorophenyl)[7-methyl-3-(quinolin-2-yl)-4,5-dihydroisoxazolo[3,4-c]pyridin-6(7H)-y
l]methanone ((S)-4c). The compound 4c (8.50 mg, 0.0220 mmol) was separated by COSMOSIL
CHiRAL 5A column (4.6 × 250 mm, Nacalai Tesque, Inc., 35% EtOH in n-hexane). After the
fractions were concentrated, the residue was purified by column chromatography on silica gel
(n-hexane/EtOAc = 3/1) to give the title compound (S)-4c (4.06 mg, 48%): pale yellow solid; [α]24
D
–98.0 (c 0.25, CHCl3); mp 174-176 °C; IR (neat) 1640 (C=O) cm-1; 1H-NMR (500 MHz, DMSO-d6)
δ 1.60 (d, J = 6.9 Hz, 3H), 3.02-3.06 (m, 1H), 3.30-3.41 (m, 2H), 4.03 (br s, 1H), 5.63 (br s, 1H),
7.26-7.29 (m, 2H), 7.53-7.56 (m, 2H), 7.64-7.67 (m, 1H), 7.80-7.84 (m, 1H), 7.99 (d, J = 8.6 Hz,
13
1H), 8.02 (d, J = 8.6 Hz, 1H), 8.06 (d, J = 8.6 Hz, 1H), 8.52 (d, J = 8.6 Hz, 1H); C-NMR (125
MHz, DMSO-d6) δ 18.9, 21.3, 38.4, 45.4, 112.0, 115.0 (d, JC-F = 20.4 Hz, 2C), 117.9, 127.0 (d, JC-F

= 14.4 Hz, 2C), 127.5, 128.6, 128.7 (2C), 129.9, 132.1, 137.1, 146.5, 147.1, 161.2, 162.4 (d, JC-F
=
260.3 Hz), 162.7, 168.7; HRMS (ESI) calcd for C23H19FN3O2 [M + H]+: 388.1456, found: 388.1453.
4.2. Inhibitory activity of the fezolinetant derivatives against NK3R
NK3R antagonistic activity of the fezolinetant derivatives was evaluated by [Ca2+]i flux assay.
NK3R expressing CHO cells (4.0  104 cells/50 μL/well) were inoculated in 10% FBS/Ham’s F-12
onto a 96-well black clear-bottom plate (Greiner), followed by incubation at 37 °C for 24 h in 5%
CO2. After the medium was removed, 50 μL of the pigment mixture (Calcium 4 assay kit, Molecular
Devices) and 50 μL of the compound solution at different concentrations in assay buffer
(HANKS/HEPES containing 2.5 mM probenecid, 0.2 % BSA, and 0.1% CHAPS) was dispensed
into each well of the plate, followed by incubation at 37 °C for 1 h. Separately, an NK3R agonist
solution (0.1 nM senktide) in assay buffer was prepared on a 96-well sample plate (V-bottom plate,
Coster). The cell and agonist solution plates were set in FDSS/μcell (Hamamatsu) and 25 μL of
agonist solution was automatically transferred to the cell plate.
4.3. UV-vis spectra
UV-vis spectra were recorded on Shimadzu UV-2450 UV-vis spectrophotometer. Compounds (30
µM) were dissolved in a mixture of 50 mM phosphate buffer (pH 7.4) and EtOH [70:30 (v/v)]
(containing 0.1% DMSO).
4.4. Investigation of photodegradation of fezolinetant derivatives