Development of novel NK3 receptor antagonists with reduced environmental impact

Article abstract of DOI:10.1016/j.bmc.2016.05.054

The neurokinin B (NKB)–neurokinin-3 receptor (NK3R) signaling positively regulates the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The NK3R-selective antagonists may suppress the reproductive functions of mammals. For developme

Full text of DOI:10.1016/j.bmc.2016.05.054

Accepted Manuscript
Development of novel NK3 receptor antagonists with reduced environmental
impact
Koki Yamamoto, Shiho Okazaki, Hiroaki Ohno, Fuko Matsuda, Satoshi Ohkura,
Kei-Ichiro Maeda, Nobutaka Fujii, Shinya Oishi
PII:
S0968-0896(16)30389-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.054
BMC 13038
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2016
26 May 2016
27 May 2016
Please cite this article as: Yamamoto, K., Okazaki, S., Ohno, H., Matsuda, F., Ohkura, S., Maeda, K-I., Fujii, N.,
Oishi, S., Development of novel NK3 receptor antagonists with reduced environmental impact, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.05.054
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Development of novel NK3 receptor antagonists with reduced environmental impact
Koki Yamamoto,a Shiho Okazaki,a Hiroaki Ohno,a Fuko Matsuda,b,c Satoshi Ohkura,b Kei-Ichiro
Maeda,c Nobutaka Fujii,a, Shinya Oishia,*
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
c
Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo
113-8657, 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
1

ABSTRACT
The neurokinin B (NKB)-neurokinin-3 receptor (NK3R) signaling positively regulates the release
of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The NK3R-selective
antagonists may suppress the reproductive functions of mammals. For development of novel NK3R
antagonists with reduced environmental toxicity, a structure–activity relationship study of an NK3R
antagonist, talnetant, was carried out. Among several talnetant derivatives with labile functional
groups in the natural environment, 3-mercaptoquinoline 2f exhibited a comparable biological
activity to that of the parent talnetant. Additionally, compound 2f was converted into the disulfide 3f
or isothiazolone 8 by air-oxidation, both of which showed no binding affinity to NK3R.
Keywords: environmental toxicity, GnRH, neurokinin B, NK3 receptor, talnetant
Abbreviations: GnRH, gonadotropin-releasing hormone; NKB, neurokinin B; NK3R, neurokinin-3
receptor; PCOS, polycystic ovary syndrome.
2

1. Introduction
Pharmaceuticals are exploited to cure diseases and/or to alleviate symptoms in humans or
veterinary therapy. For these purposes, the bioactive ingredient(s) are designed primarily for specific
binding to the target molecule(s) of target species. The high chemical stability of the ingredients is
crucial for the quality control of drugs during long-term storage before administration. However, in
terms of the impacts on the natural environment and ecosystems, the stable bioactive substances
excreted from the treated patients or animals may have unfavorable effects on non-target species [1].
Some representative examples of environmental contaminations are antibiotics and steroid hormones.
Environmental exposure to antibiotics promotes the emergence of drug-resistant microbial strains,
which threatens human and animal health [2-4]. Estradiol derivatives extensively used in veterinary
medicine disrupt the endocrine systems of non-target species, thus leading to disabling reproductive
functions and increasing developmental abnormalities [5,6]. For the appropriate control of drug
delivery and distribution in time and space, a number of technologies have been developed to
modify the bioactivities and properties of pharmaceutical agents [7].
Recent studies have revealed that neurokinin B (NKB)-neurokinin-3 receptor (NK3R) signaling
plays an important role in controlling mammalian reproduction via regulation of the hypothalamo-
pituitary-gonadal (HPG) axis [8,9]. NKB positively regulates the reproductive hormone cascade via
activation of the gonadotropin-releasing hormone (GnRH) neuron in the hypothalamus, leading to
the pulsatile secretion of luteinizing hormone (LH) from the pituitary gland [9]. Administration of
senktide, an NK3R-selective agonist, stimulated LH secretion in diestrous rats [10]. GnRH generator
activity was increased by intravenous administration of senktide into ovariectomized goats, which
was monitored by recording multiple-unit activity in the mediobasal hypothalamus [11]. Through
the structure-activity relationship studies of senktide, we identified novel NK3R selective agonists
with high potency and resistance to proteolytic degradation [12,13]. In contrast, NK3R antagonists
such as talnetant (1a) and AZD2624 (recently renamed AZD4901, 1b), which were originally
developed as antipsychotics, could be negative regulators for reproductive functions (Figure 1). For
3

example, oral administration of AZD4901 effectively decreased testosterone levels and is now
expected to be applicable as a treatment for polycystic ovary syndrome (PCOS) [14]. ESN364 (1c)
is also a potent NK3R antagonist with a unique triazolopiperazine scaffold [15,16]. Oral
administration of ESN364 in monkeys led to reduction of gonadotropin and ovarian hormone levels
throughout the menstrual cycle [17]. Clinical investigations also revealed that ESN364 was an
effective inhibitor of the activity of the HPG axis by decreasing gonadotropin secretion [18]. Thus,
both NK3R agonists and antagonists may be useful for the treatment of reproductive disorders.
However, the NK3 modulators and the bioactive metabolite(s) excreted from treated humans or
animals may cause environmental and health risks including reproductive disturbances of non-target
species via water pollution and/or soil contamination. To minimize the possibility of these adverse
effects on non-target species, the bioactive ingredient(s) need be converted into the inactive
substance(s), by drug metabolizing systems in the body before excretion or by spontaneous
degradation in the environment soon after excretion (Figure 2). Herein, we report the investigation
of the structure–activity relationship of a quinoline-based antagonist, talnetant (1a), in an effect to
develop novel NK3R antagonists with decreased environmental impact.
Figure 1. Structures of NK3 receptor antagonists.
4

Figure 2. Drug activities over time and space for conventional drugs or our concept drugs.
2. Results and Discussion
2.1. Design of NK3R antagonists with minimal environmental impact
We designed novel NK3R antagonists whose biological activities could be altered via three
possible conversion processes under the conditions of the natural environment. Compound 2a having
an o-hydroxyphenyl group at the quinoline 2-position was designed to be converted under oxidative
conditions to provide the quinone methide form 3a (Figure 3A). It was expected that compound 3a
with a planar conformation would show decreased potency compared with the parent 2a because
talnetant (1a) binds to NK3R in the twisted biaryl conformation A [19]. Compounds 2c–d could also
be converted into the similar planar quinone methide-type products under oxidative conditions
(Table 1). For the second class of NK3R antagonists, 3-azidoquinoline 2e was designed so that it
could be subjected to photoactivation to provide the ring-expanded benzodiazepine 3e via nitrene
formation. Because the quinoline ring is essential to the biological activity [20], we estimated that
ring expansion leads to inactivation (Figure 3B). Compound 2f with a 3-mercapto group was also
designed to be dimerized to form a disulfide 3f under oxidative conditions. We envisioned that the
dimerization could lead to a loss or decrease in the biological activity (Figure 3C).
5

Figure 3. Design of NK3 receptor antagonists with reduced environmental toxicity.
2.2. Synthesis of talnetant derivatives
We synthesized the derivatives with the potential to be inactivated via the above-mentioned three
pathways. The Pfitzinger reaction of isatins 4a,b with the appropriate aryl ketones 5a–g provided
quinoline-4-carboxylic acids 6a–g in 20–74% yield. For the preparation of amides 7a–g, carboxylic
acids 6a–g were coupled with (S)-1-phenylpropan-1-amine using carbonyldiimidazole (CDI) in
acetonitrile or O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
(HATU) in DMF (Scheme 1) [21]. The talnetant derivatives 2a–d with a hydroxy group were
obtained by treatment of the methoxy precursors 7a–d with BBr3 (Scheme 2A and 2B). For the
6

preparation of compound 2e with an azide group at the quinoline 3-position, compound 7e was
treated with TMSN3 and t-BuNO2 under Sandmeyer conditions (Scheme 2C). The derivative 2f with
a 3-mercapto group was prepared from 7f in three steps via deprotection of the trimethoxybenzyl
group, formation of a temporary dimer by air-oxidation, and subsequent reduction with L-cysteine
(Scheme 2D). Of note is that the deprotection of the p-methoxybenzyl group in 7g by treatment with
TFA, TfOH, and thioanisole for the synthesis of 2f was unsuccessful.
Scheme 1. Synthesis of talnetant derivative precursors 7a–g.
c
aMs: methanesulfonyl. bTMB: 2,4,6-trimethoxybenzyl. PMB: 4-methoxybenzyl.
7

Scheme 2. Synthesis of talnetant derivatives 2a–f.
2.3. Biological activity of talnetant derivatives
We investigated the biological activities of talnetant derivatives 2a–f along with the synthetic
intermediates 7a–c and 7g (Table 1). The biological activities were evaluated by a competitive
binding assay using ([125I]-His3, MePhe7)-NKB for human NK3R. The o-hydroxy modification at the
8

2-phenyl group on the quinoline (2a and 2d) led to slightly more potent NK3R inhibition [IC50 (2a):
6.0 nM, IC50 (2d): 3.9 nM] compared with talnetant (IC50: 8.4 nM). The 3-mercapto modification (2f)
also inhibited the NKB binding to NK3R with high potency [IC50 (2f): 5.6 nM]. In contrast, the 7-
methoxy, 7-hydroxyl, 3-azido and 3-(p-methoxybenzyl)thio modifications of the quinoline ring (7c,
2c, 2e, and 7g) decreased NK3R inhibition [IC50 (7c): 44 nM, IC50 (2c): 245 nM, IC50 (2e): 24 nM,
IC50 (7g): 154 nM]. The o-methoxy (7a), p-methoxy (7b), or p-hydroxy (2b) modifications at the 2-
phenyl group were also not favorable.
Table 1. Biological evaluation of talnetant derivatives.
R1
H
R2
H
R3
H
R4
OH
compound
IC50 (nM)
8.4 ± 2.0
1.6 ± 0.7
1527 ± 234
965 ± 133
44 ± 8.0
Talnetant
AZD2624
H
H
H
NH-Msb
OH
H
H
OMe
H
7a
7b
7c
2a
2b
2c
2d
2e
2f
H
OMe
H
OH
OMe
H
H
OH
H
OH
H
OH
6.0 ± 2.0
390 ± 68
245 ± 45
3.9 ± 0.8
24 ± 12
H
OH
H
OH
OH
H
H
OH
H
OH
H
NH-Msb
N3
H
H
H
H
H
SH
S-PMBc
5.6 ± 1.8
154 ± 65
H
H
H
7g
aIC50 values are the concentrations required for 50% inhibition of the ([125I]His3, MePhe7)-NKB
binding to the NK3 receptor (n = 6). bMs: methanesulfonyl. cPMB: 4-methoxybenzyl.
2.4. Investigation of the stability of potent NK3 derivatives under oxidative conditions or in
serum
We examined the structural conversion or degradation from the highly potent diol 2a and thiol 2f
9

under oxidative conditions (Table 2 and Figure 4). When compound 2a was treated with mild
oxidizing agents including hydrogen peroxide (H2O2), no reaction occurred to give the possible
quinone methide form 3a with the expected planar conformation. Treatment of 2a with highly
reactive oxidizing agents such as pyridinium chlorochromate (PCC) led to degradation without
formation of 3a. These results that a severe oxidizing condition is needed for conversion from 2a to
the inactive form. In contrast, quinolinethiol 2f was easily converted to the disulfide form 3f in
MeCN under air for 24 h at 37 °C (Figure 4A). In a solution of 20 mM phosphate buffer (pH 7.4)-
MeCN, thiol 2f was converted to isothiazolone 8 by the same treatment for 12 h at 37 °C (Figure
4B). Both oxidation products (3f and 8) did not inhibit NK3R binding (IC50 >10 μM), suggesting that
the thiol 2f has an appropriate chemical property to offset the pharmacological effects under the
conditions of the natural environment.
Table 2. Conversion of diol 2a under oxidative conditions.
entry
1
oxidizing agent
Air (K2CO3)
Air (DBU)
H2O2/NaHCO3
FeCl3
solvent
acetone
MeOH
acetone
MeOH
MeOH
acetone
acetone
MeOH
MeCN
MeOH
MeOH
acetone
time
24 h
24 h
24 h
24 h
24 h
24 h
24 h
5 min
5 min
30 min
3 h
result
no reaction
no reaction
no reaction
no reaction
no reaction
no reaction
no reaction
decomp.
2
3
4
5
DDQ
6
MnO2
7
KMnO4
PCC
8
9
CAN
decomp.
10
11
12
O3
mixture
NBS
mixture
Ag2O
3 h
mixture
DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DDQ: 2,3-dichloro-5,6-dicyano-p-benzoquinone; CAN:
ammonium cerium (IV) nitrate; PCC: pyridinium chlorochromate, NBS: N-bromosuccinimide.
10

Figure 4. Investigation of the oxidation process of thiol 2f.
100
80
60
40
20
2f
3f
8
0
0
3
6
9
12 15 18 21 24
time (h)
100
80
60
40
20
2f
3f
8
0
0
3
6
9
12 15 18 21 24
time (h)
aThe ratios of monomer units were calculated by HPLC analysis using calibration curves.
We also investigated the stability of thiol 2f in pig serum by HPLC analysis. Compound 2f was
slowly converted to isothiazolone 8 via air-oxidation in pig serum (Figure 5A), while no change
from 8 was observed under the identical conditions over 24 h (Figure 5B). Thus, compound 2f could
be a novel NK3R antagonist, which is irreversibly converted into the inactive form 8 in the body
after administration as well as under aerobic conditions after excretion from the body [22]. The
inactive compound 8 would be the predominant form present if compound 2f-derived substances
were uptaken from the environment to other organisms.
11

Figure 5. Stability of thiol 2f and isothiazolone 8 in pig serum under air at 37 °C.
(A) Conversion of thiol 2f
100
2f
80
3f
8
60
40
20
0
0
3
6
9
12 15 18 21 24
time (h)
(B) Conversion of isothiazolone 8
100
80
60
40
20
0
2f
3f
8
0
3
6
9
12 15 18 21 24
time (h)
aThe ratios of monomer units were calculated by HPLC analysis using calibration curves.
3. Conclusions
In this study, we revealed that talnetant derivatives 2a,d and 2f with hydroxyl- or mercapto-group
modifications exhibited comparable NK3 receptor binding inhibition to that of the parent compound
talnetant. Among these potent derivatives, thiol 2f was spontaneously converted to the inactive
disulfide (3f) or isothiazolone (8) forms under aerobic conditions. Accordingly, the thiol 2f may be a
novel NK3 receptor antagonist for the treatment of sex-hormone disorders with decreased
environmental impact.
12

4. Experimental Section
4.1. Synthesis
4.1.1. General synthesis
1H NMR spectra were recorded using a JEOL ECA-500 spectrometer. Chemical shifts are
reported in δ (ppm) relative to Me4Si as an internal standard. 13C NMR spectra were referenced to the
residual solvent signal. Exact mass spectra (HRMS) were recorded on a JMS-HX/HX 110A mass
spectrometer or Shimadzu LC-ESI-IT-TOF-MS equipment. IR spectra were determined on a JASCO
FT/IR-4100 spectrometer. Optical rotations were measured with a JASCO P-1020 polarimeter. For
flash chromatography, Wakogel C-300E (Wako) was employed. The purity of the compounds was
determined as no less than 95% by combustion analysis or HPLC analysis.
4.1.2. 3-Hydroxy-2-(2-methoxyphenyl)quinoline-4-carboxylic acid (6a)
2-Acetoxy-2’-methoxyacetophenone (5a) (920 mg, 4.12 mmol) in EtOH (5.2 mL) was added to a
solution of isatin (4a) (500 mg, 3.40 mmol) in 10N NaOH (3.0 mL) and EtOH(800 μL) at 85 °C.
After the mixture was stirred at 85 °C for 1 h, H2O (9.0 mL) was added to the reaction mixture at 0
°C. The mixture was acidified to pH 1 with 1N HCl. The precipitate was collected by filtration and
washed with H2O and EtOH to give the title compound 6a as pale yellow solids (583 mg, 58%); mp
1
227-229 °C (recrystallized from EtOH); IR (neat) cm-1: 1654 (C=O); H NMR (500 MHz, DMSO-
d6): δ 3.77 (s, 3H, CH3), 7.13 (dd, J = 7.4, 7.4 Hz, 1H, Ar), 7.21 (d, J = 8.0 Hz, 1H, Ar), 7.48 (d, J =
6.9 Hz, 1H, Ar), 7.52-7.56 (m, 1H, Ar), 7.61-7.64 (m, 1H, Ar), 7.68-7.71 (m, 1H, Ar), 8.01 (d, J =
8.0 Hz, 1H, Ar), 8.91 (d, J = 6.3 Hz, 1H, Ar); 13C NMR (125 MHz, DMSO-d6): δ 55.7, 111.5 (2C),
120.3 (2C), 124.8, 126.0, 126.5 (2C), 128.6, 130.8 (2C), 131.1, 151.6, 157.3 (2C), 170.4; HRMS
(ESI): m/z calcd for C17H14NO4 [M+H]+ 296.0917, found: 296.0916.
4.1.3. (S)-3-Hydroxy-2-(2-methoxyphenyl)-N-(1-phenylpropyl)quinoline-4-carboxamide (7a)
13

CDI (243 mg, 1.50 mmol) was added to a solution of compound 6a (400 mg, 1.36 mmol) and TEA
(247 μL, 1.77 mmol) in MeCN (1.60 mL). After the mixture was stirred at 50 °C for 5 h, (S)-1-
phenylpropylamine (218 μL, 1.50 mmol) was added to the reaction mixture at 50 °C. After being
stirred at 50 °C for 5 h, the reaction mixture was stirred at room temperature overnight. After the
reaction mixture was concentrated, the residue was dissolved in EtOAc. The extract was washed
with 10% NaHCO3, 1N HCl, H2O, and brine, and dried over MgSO4. The filtrate was concentrated,
and the residue was recrystallized from n-hexane–n-PrOAc to give the title compound 7a as
colorless crystals (137 mg, 25%); []28D –22.4 (c 0.93, CHCl3); mp 156-158 °C; IR (neat) cm-1: 3349-
3112 (OH), 1638 (C=O); 1H NMR (500 MHz, DMSO-d6): δ 0.94 (t, J = 7.4 Hz, 3H, CH ), 1.75-1.81
3
(m, 2H, CH2), 3.73 (s, 3H, CH3), 5.03 (dt, J = 7.6, 7.6 Hz, 1H, CH), 7.07 (ddd, J = 7.4, 7.4, 1.1 Hz,
1H, Ar), 7.13 (d, J = 8.0 Hz, 1H, Ar), 7.25-7.27 (m, 2H, Ar), 7.30 (dd, J = 7.4, 1.7 Hz, 1H, Ar), 7.35
(dd, J = 7.7, 7.7 Hz, 1H, Ar), 7.43-7.49 (m, 4H, Ar), 7.53-7.55 (m, 2H, Ar), 7.92 (d, J = 8.6 Hz, 1H,
13
Ar), 9.08 (d, J = 8.0 Hz, 1H, NH), 9.18 (s, 1H, OH); C NMR (125 MHz, DMSO-d6): δ 11.2, 29.3,
54.7, 55.4, 111.4, 120.2, 123.5, 125.7, 125.9, 126.3, 126.7 (3C), 126.8, 127.0, 128.2 (2C), 128.8,
130.1, 130.8, 141.9, 143.6, 145.1, 152.0, 157.3, 164.7; Anal. calcd for C26H24N2O3: C, 75.71; H, 5.86;
N, 6.79. found: C, 75.68; H, 5.91; N, 6.82.
4.1.4. (S)-2-Phenyl-N-(1-phenylpropyl)-3-[(2,4,6-trimethoxybenzyl)thio]quinoline-4-carboxam-
ide (7f)
HATU (6.34 g, 16.7 mmol), (S)-1-phenylpropylamine (3.20 mL, 22.2 mmol) and DIPEA (7.70 mL,
44.5 mmol) were added to a solution of 2-phenyl-3-[(2,4,6-trimethoxybenzyl)thio]quinoline-4-
carboxylic acid (6f) (5.10 g, 11.1 mmol) in DMF (16.7 mL). After being stirred at room temperature
for 4 h, the mixture was concentrated. After the residue was dissolved in EtOAc, the whole was
washed with 1N HCl, H2O, and brine, and dried over MgSO4. The filtrate was concentrated, and the
resulting residue was purified by flash chromatography over silica gel with n-hexane–EtOAc (2:1)
14

to give the title compound 7f as brown amorphous solids (5.80 g, 91%); []28 +55.2 (c 0.53,
D
1
CHCl ); IR (neat) cm-1: 1647 (C=O); H NMR (500 MHz, DMSO-d6): δ 0.99 (t, J = 7.2 Hz, 3H,
3
CH3), 1.80-1.83 (m, 2H, CH2), 3.35 (s, 6H, CH3 × 2), 3.71-3.72 (m, 5H, CH2, CH3), 5.09 (dt, J = 7.6,
7.5 Hz, 1H, CH), 6.00 (s, 2H, Ar), 7.27 (dd, J = 7.4, 7.4 Hz, 1H, Ar), 7.35 (dd, J = 7.4, 7.4 Hz, 2H,
Ar), 7.39-7.41 (m, 3H, Ar), 7.46 (d, J = 6.9 Hz, 2H, Ar), 7.55-7.56 (m, 4H, Ar), 7.76-7.80 (m, 1H,
Ar), 8.03 (d, J = 8.6 Hz, 1H, Ar), 8.96 (d, J = 8.6 Hz, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ
11.2, 29.3, 29.7, 54.8, 55.2, 55.3 (2C), 90.5 (2C), 105.3, 124.1, 124.8, 124.9, 126.7 (2C), 126.8,
127.2 (3C), 127.9, 128.1 (2C), 129.0, 129.6 (2C), 130.3, 140.3, 143.2, 146.3, 150.3, 158.4 (2C),
160.3, 161.9, 165.3; HRMS (ESI): m/z calcd for C35H35N2O4S [M + H]+ 579.2312, found: 579.2309.
4.1.5. (S)-3-Hydroxy-2-(2-hydroxyphenyl)-N-(1-phenylpropyl)quinoline-4-carboxamide (2a)
BBr3 (1.21 mL, 2.17 mmol) was added to compound 7a (50.0 mg, 0.121 mmol) at 0 °C. After
being stirred at room temperature for 5 h, the mixture was quenched with H2O and extracted with
EtOAc. The extract was washed with brine and dried over MgSO4. After concentration, the residue
was purified by flash chromatography over silica gel with n-hexane–EtOAc (2:1) to give the title
compound 2a as colorless crystals (39.6 mg, 82%); []28D –21.7 (c 1.10, DMSO) mp 185–187 °C
(recrystallized from n-hexane–n-PrOAc); IR (neat)cm-1: 1636 (C=O); 1H NMR (500 MHz, DMSO-
d6): δ 0.96 (t, J = 7.2 Hz, 3H, CH3), 1.78-1.80 (m, 2H, CH2), 5.05 (dt, J = 7.6, 7.6 Hz, 1H, CH),
6.96-7.01 (m, 2H, Ar), 7.27 (dd, J = 7.4, 7.4 Hz, 1H, Ar) , 7.33-7.38 (m, 3H, Ar), 7.44 (d, J = 7.4
Hz, 2H, Ar), 7.49-7.52 (m, 1H, Ar), 7.55-7.61 (m, 2H, Ar), 7.83-7.85 (m, 1H, Ar), 7.97 (d, J = 8.0
13
Hz, 1H, Ar), 9.15 (d, J = 7.4 Hz, 1H, NH); C NMR (125 MHz, DMSO-d6): δ 11.2, 29.4, 54.8,
116.5, 119.0, 123.1, 123.6, 125.4, 126.7 (3C), 126.8, 127.0, 127.1, 128.2 (3C), 128.3, 130.7, 131.4,
143.5 (2C), 151.6, 156.4, 164.5; Anal. calcd for C25H22N2O3: C, 75.36; H, 5.57; N, 7.03. found: C,
75.27; H, 5.70; N, 7.00.
15

4.1.6. (S)-3-Azido-2-phenyl-N-(1-phenylpropyl)quinoline-4-carboxamide (2e)
Trimethylsilyl azide (39.0 μL, 0.296 mmol) and tert-butyl nitrite (35.4 μL, 0.296 mmol) were
added to a solution of (S)-3-amino-2-phenyl-N-(1-phenylpropyl)quinoline-4-carboxamide (7e) [23]
(50.0 mg, 0.197 mmol) at 0 °C. After being stirred at room temperature for 5h, the reaction mixture
was concentrated. Crude product was purified by flash chromatography over silica gel with EtOAc–
n-hexane (2:1) to give the title compound 2e as a pale yellow solid (35.0 mg, 44%.); []28 –20.3 (c
D
0.67, CHCl3); mp 135–137 °C (recrystallized from n-hexane–EtOAc); IR (neat) cm-1: 1632 (C=O);
1H NMR (500 MHz, DMSO-d6): δ 0.96 (t, J = 7.2 Hz, 3H, CH3), 1.78-1.83 (m, 2H, CH2), 5.05 (dt, J
= 7.6, 7.6 Hz, 1H, CH), 7.29 (dd, J = 7.4, 7.4 Hz, 1H, Ar), 7.38 (dd, J = 7.7, 7.7 Hz, 2H, Ar), 7.43
(d, J = 6.9 Hz, 2H, Ar), 7.53-7.65 (m, 5H, Ar), 7.77-7.82 (m, 3H, Ar), 8.08 (d, J = 8.6 Hz, 1H, Ar) ,
9.44 (d, J = 8.6 Hz, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 11.0, 29.3, 55.0, 124.1, 124.3,
126.6 (2C), 126.9, 127.0, 128.1, 128.3 (2C), 128.5 (2C), 129.2 (3C), 129.3, 129.8, 135.8, 137.3,
143.0, 144.9, 153.6, 163.2; Anal. calcd for C25H21N5O: C,73.69; H, 5.19; N, 17.19. found: C, 73.53;
H, 5.23; N, 17.25.
4.1.7. 3,3’-Dithiobis{2-phenyl-N-[(S)-1-phenylpropyl]quinoline-4-carboxamide} (3f)
Compound 7f (5.70 g, 9.90 mmol) was added to a solution of thioanisole (11.6 mL, 99.0 mmol)
in TFA (99.0 mL). After being stirred at room temperature for 2 h, the mixture was concentrated.
MeCN (49.5 mL) was added to the residue. After being stirred at room temperature under O2 for 4
days, the mixture was concentrated. Crude product was purified by flash chromatography over silica
gel with EtOAc–n-hexane (2:1) to give the title compound 3f as a pale yellow solid (2.0 g, 51%.);
[]28 –98.7 (c 1.12, MeOH); mp 222–224 °C (recrystallized from n-hexane–EtOAc); IR (neat) cm-1:
D
1630 (C=O); 1H NMR (500 MHz, DMSO-d6): δ 0.72 (t, J = 7.4 Hz, 6H, CH3 × 2), 1.46-1.60 (br m,
4H, CH2 × 2), 4.67 (dt, J = 7.3, 7.3 Hz, 2H, CH × 2), 7.12-7.16 (m, 8H, Ar), 7.19-7.27 (m, 8H, Ar),
7.35 (d, J = 6.9 Hz, 4H, Ar), 7.51-7.56 (m, 4H, Ar), 7.78-7.82 (m, 2H, Ar), 8.02 (d, J = 8.0 Hz, 2H,
16

Ar), 8.54 (d, J = 7.4 Hz, 2H, NH × 2); 13C NMR (125 MHz, DMSO-d6): δ 10.4 (2C), 28.4 (2C), 55.1
(2C), 124.0 (2C), 124.4 (2C), 125.3 (2C), 126.6 (2C), 126.7 (2C), 126.8 (2C), 127.1 (4C), 127.3
(2C), 127.7 (2C), 128.0 (2C), 128.1 (2C) 129.2 (2C), 129.4 (2C), 129.5 (2C), 130.9 (2C), 140.0
(2C), 142.1 (2C), 147.1 (2C), 149.5 (2C), 161.4 (2C), 164.5 (2C); Anal. calcd for
C50H42N4O2S2·1.5H2O: C, 73.05; H, 5.52; N, 6.82. found: C, 72.80; H, 5.31; N, 6.70.
4.1.8. (S)-3-Mercapto-2-phenyl-N-(1-phenylpropyl)quinoline-4-carboxamide (2f)
L-Cysteine hydrochloride (426 mg, 3.52 mmol) was added to a solution of compound 3f (1.0 g,
1.26 mmol) in MeOH(50.0 mL) and H2O(15.0 mL) at 0 °C. After being stirred at room temperature
for 30 min, the mixture was filtrated. The filtrate was concentrated and the precipitate was collected
by filtration to give the title compound 2f as a pink solid (746 mg, 74%); []28 –61.8 (c 0.75,
D
MeOH); mp 87-89 °C (recrystallized from H2O–MeOH); IR (neat) cm-1: 3330-3111 (SH), 1629
1
(C=O); H NMR (500 MHz, DMSO-d6): δ 0.98 (t, J = 7.2 Hz, 3H, CH3), 1.75-1.87 (m, 2H, CH2),
5.06 (dt, J = 7.6, 7.6 Hz, 1H, CH), 7.28-7.30 (m, 2H, Ar), 7.39 (dd, J = 7.2, 7.2 Hz, 2H, Ar), 7.44-
7.45 (m, 2H, Ar), 7.52-7.58 (m, 4H, Ar), 7.62-7.63 (m, 2H, Ar), 7.74 (dd, J = 7.4, 7.4 Hz, 1H, Ar),
8.02 (d, J = 8.6 Hz, 1H, Ar), 9.47 (d, J = 8.0 Hz, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 11.3,
29.2, 55.0, 123.9, 124.0, 126.7 (3C), 127.0, 127.8, 128.3 (4C), 128.9 (3C), 129.1, 129.4, 139.2,
142.3, 143.1, 144.5, 157.9, 165.4; Anal. calcd for C25H22N2OS: C, 75.35; H, 5.56; N, 7.03. found: C,
75.13; H, 5.72; N, 6.78.
4.2. Inhibitory activity of talnetant derivatives against NKB binding to NK3R (Table 1)
Binding inhibition assays were performed according to the procedure in our previous research
[12]. Membranes from NK3R-expressing CHO cells were incubated with 50 μL solution of talnetant
derivatives, 25 μL of radioactive ligand solution [([125I]His3, MePhe7)-NKB, 0.4 nM, PerkinElmer
17

Life Sciences], and 25 μL of NK3R membrane suspension (10 μg) in assay buffer [50 mM HEPES
(pH 7.4), 5 mM MgCl2, 1 mM CaCl2, 0.1% BSA]. Reaction mixtures were filtered through GF/B
filters, pretreated with 0.3% polyethyleneimine. Filters were washed with wash buffer [50 mM
HEPES (pH 7.4), 500 mM NaCl, 0.1% BSA] and dried at 55 °C. Bound radioactibity was measured
by TopCount (PerkinElmer Life Sciences) in the presence of MicroScint-O (30 μL) (PerkinElmer
Life Sciences).
4.3. Quantitative analysis of air oxidation process from thiol 2f
Compound 2f (1 mM) was incubated in MeCN or a mixture of 20 mM phosphate buffer (pH 7.4)
and MeCN [30:70 (v/v)] under air at 37 °C. An aliquot of the sample was analyzed by HPLC at the
indicated intervals and the peak area was recorded by UV detection at 254 nm. The ratios of the
resulting compounds were calculated from the calibration curves.
4.4. Quantitative analysis of the conversion of thiol 2f to isothiazolone 8 in pig serum
Compound 2f (0.1 mM) was incubated in pig serum (containing 0.5% DMSO) at 37 °C. A 20 μL
aliquot was sampled at the indicated intervals, and extracted with MeCN (80 μL). An aliquot of the
sample was analyzed by HPLC and the peak area was recorded by UV detection at 254 nm. The
ratios of the resulting compounds were calculated from the calibration curves.
Acknowledgements
This work was supported by Science and Technology Research Promotion Program for
Agriculture, Forestry, Fisheries and Food Industry from MAFF, Japan; Grants-in-Aid for Scientific
Research from JSPS, Japan; Platform for Drug Discovery, Informatics, and Structural Life Science
from MEXT, Japan. We are grateful to Dr. Akira Hirasawa (Kyoto University) for his generous
support of this research.
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Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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References and notes
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Graphical abstract
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