Discovery of a novel class of state-dependent NaV1.7 inhibitors for the treatment of neuropathic pain

Article abstract of DOI:10.1248/cpb.c20-00126

The discovery of a novel class of state-dependent voltage-gated sodium channel (NaV)1.7 inhibitors is described. By the modification of amide or urethane bond in NaV1.7 blocker III, structure-activity relationship studies that led to the identification of novel NaV1.7 inhibitor 2i (DS01171986) were performed. Compound 2i exhibited state-dependent inhibition of NaV1.7 without NaV1.1, NaV1.5 or human ether-a-go-go related gene (hERG) liabilities at concentrations up to 100 μM. Further biological profiling successfully revealed that 2i possessed potent analgesic properties in a murine model of neuropathic pain (ED50: 3.4 mg/kg) with an excellent central nervous system (CNS) safety margin (> 600 fold).

Full text of DOI:10.1248/cpb.c20-00126

Vol. 68, No. 7
Chem. Pharm. Bull. 68, 653–663 (2020)
653
Regular Article
Discovery of a Novel Class of State-Dependent Na 1.7 Inhibitors
V
for the Treatment of Neuropathic Pain
a
a
a
a
a,†
Kyosuke Tanaka, Hiroyuki Kobayashi, Sayaka Suzuki, Satoshi Shibuya, Hiroko Kimoto,
a
a
a
a
a
Yuki Domon, Kazufumi Kubota, Yutaka Kitano, Tomihisa Yokoyama, Akiko Shimizugawa,
Ryuta Koishi, Chie Fujiwara, Daigo Asano, and Tsuyoshi Shinozuka*
b
a
a
,a
a
b
R&D Division, Daiichi Sankyo Co., Ltd.; 1–2–58 Hiromachi, Shinagawa-ku, Tokyo 140–8710, Japan: and Daiichi
Sankyo RD Novare Co., Ltd.; 1–16–13 Kitakasai, Edogawa-ku, Tokyo 134–8630, Japan.
Received February 12, 2020; accepted April 3, 2020
The discovery of a novel class of state-dependent voltage-gated sodium channel (Na )1.7 inhibitors is de-
V
scribed. By the modification of amide or urethane bond in Na 1.7 blocker III, structure–activity relationship
V
studies that led to the identification of novel Na 1.7 inhibitor 2i (DS01171986) were performed. Compound
V
2
i exhibited state-dependent inhibition of Na 1.7 without Na 1.1, Na 1.5 or human ether-a-go-go related gene
V V V
(
hERG) liabilities at concentrations up to 100μM. Further biological profiling successfully revealed that 2i
possessed potent analgesic properties in a murine model of neuropathic pain (ED : 3.4mg/kg) with an excel-
5
0
lent central nervous system (CNS) safety margin (>600fold).
Key words voltage-gated sodium channel; pain; central nervous system (CNS) side effect; quinolone; ure-
thane
11)
Introduction
selective Na 1.7 inhibitors, we focused on benzazepinone
V
Neuropathic pain (NP) is a debilitating disease that affects derivative III (Fig. 1), reported by the Merck group, owing to
12–14)
patients spontaneously and has a significant impact on their its high state dependency.
As two amide and one urethane
QOL. Diabetic peripheral neuropathy (DPN) is one of the bonds are found in this molecule, our derivatization strat-
most common types of NP. It is estimated that around half of egy consists of the modifications of such bonds. Herein, we
1)
diabetic patients suffer from DPN. The first-line treatments describe the conversion of these moieties to identify a novel
for DPN include calcium channel α δ ligands and tricyclic class of state-dependent Na 1.7 inhibitors with potent anal-
2
V
2)
antidepressants. Although these drugs are efficacious and gesic efficacy in a model of NP as well as an excellent CNS
tolerable, central nervous system (CNS) adverse effects, such safety margin.
as drowsiness lightheadedness, dizziness, and sedation, limit
2)
their use. Consequently, novel potent analgesic drugs with Results and Discussion
less CNS adverse effects have a high demand.
Human in Vitro Profile The in vitro evaluation of the
Voltage-gated sodium channels (Na ) are responsible for compounds was performed utilizing the IonWorks Quattro
V
transmitting neuronal signals not only in CNS but also in the automated electrophysiology platform. The inhibitory activ-
peripheral nervous system (PNS). In humans, nine Na sub- ity of hNa 1.7 was evaluated at a half-inactivated state (V
V
V
hold
types (Na 1.1–Na 1.9) have been identified, and Na 1.7 has −59mV). In addition, the inhibitory potency of hNa 1.7 was
V
V
V
V
3
,4)
been shown to play a crucial role in pain signaling. Several evaluated at an elevated membrane voltage (Vhold −30mV),
Na blockers, such as lidocaine (I) and mexiletine (II) (Fig. because an elevated Na 1.7 membrane voltage is reported
), have been used to treat chronic pain in a clinical setting. in damaged dorsal root ganglion (DRG) neurons.
V
V
1
5–18)
1
This
Furthermore, a high safety index over CNS adverse effects is screening process enabled us to provide an analgesic agent
expected by selective Na 1.7 inhibition, because the Na 1.7 with high safety index owing to the ability to inhibit only
V
V
5
)
isoform is predominantly expressed in the PNS. In the dis- abnormal hyperactive neurons with frequent action potentials
covery of novel Na 1.7 inhibitors, a high subtype selectivity without affecting normal firing activity. In this assay condi-
V
is crucial, because multiple Na subtypes are differently ex- tion, we confirmed the inhibitory activity of the antiepileptic
V
pressed in various organs; non-selective inhibitors could cause drug lacosamide, which is known to enhance slow inactivation
19)
adverse effects. For example, Na 1.1 is expressed in brain, of voltage-gated sodium channels selectively. In the in vitro
V
and Na 1.1 inhibition is known to cause CNS adverse effects,
V
whereas the inhibition of Na 1.5 leads to cardiac arrhyth-
V
6
,7)
mias. In fact, Na blockers used in clinics are non-selective,
V
and the lack of selectivity limits the usage of such drugs
due to safety concerns. Therefore, the selective inhibition of
8
–10)
Na 1.7 is a promising analgesic target.
V
During the course of a project directed at acquiring potent
Fig. 1. Structures of Lidocaine (I), Mexiletine (II), and Compound III,
Reported by the Merck Group
†
Present address: KAN Research Institute, Inc.; Kobe 650–0047, Japan.
*
To whom correspondence should be addressed. e-mail: sinozu.xf6@gmail.com; shinozuka.tsuyoshi.s5@daiichisankyo.co.jp
2020 The Pharmaceutical Society of Japan
©

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Chem. Pharm. Bull.
Vol. 68, No. 7 (2020)
a)
Table 1. In Vitro Profile of Na 1.7 Inhibitors 1
V
a) Values of at least two independent experiments run in quadruplicate, unless otherwise noted. Each
value represents the mean± standard error of the mean (S.E.M.) b) Values at a half-inactivated state. c) Val-
ues at V −30mV. d) Diastereomeric mixture (1:1). e) Values of a single experiment run in quadruplicate.
hold
screening, hNa 1.1 and hNa 1.5 inhibitory activities were ac- imidazole 1f exhibited the best inhibitory potency among this
V
V
quired to monitor adverse effects.
series of compounds with increased hNa 1.1 or hNa 1.5 poten-
V V
We initially focused on the conversion of the benzaz- cy. In contrast to quinolone derivatives, the regioisomer of 1d,
epinone moiety, as shown in Table 1. Compound III showed 5-benzothiazole 1g retained hNa 1.7 potency without hNa 1.1
V
V
good inhibitory potency against hNa 1.7 at V
−30mV or hNa 1.5 liabilities. Although the enhancement of hNa 1.7
V
hold
V
V
(
IC =2.5µM) without hNa 1.1 or hNa 1.5 liability. Com- activity was expected by the introduction of a nitrogen in 1g,
50 V V
pound III was found to inhibit hNa 1.7 in a high state- thiazolopyridine 1h showed attenuated hNa 1.7 potency.
V
V
dependent manner, because III did not inhibit hNa 1.7 at a
The replacement of acid labile Boc group of 1c was then
V
half-inactivated state. As the removal of the trifluoromethyl investigated (Table 2). hNa 1.7 activity was found to be cor-
V
group or the reduction of ring size (benzazepinone to dihydro- related to the lipophilicity of molecules in the modification
quinolinone) did not affect hNa 1.7 potency (data not shown), of t-butyl group. The inhibitory activity of isopropyl 2a and
V
tetrahydroquinoline 1a was synthesized, which resulted in the ethyl derivative 2b is 2.3 and 10µM, respectively, whereas
retention of hNa 1.7 potency. Compound 1a was prepared as a methyl analog 2c almost lost activity. Consequently, t-butyl
V
diastereomeric mixture (1:1). These results encouraged us to group was optimal in this position. The introduction of methyl
synthesize quinoline and isoquinoline derivatives. As expect- group in the urethane moiety led to the loss of potency in
ed, isoquinoline 1b and quinoline 1c exhibited potent hNa 1.7 vitro, suggesting that a hydrogen bond may exist between
V
activity. Compounds 1b and 1c also showed no hNa 1.1 or this moiety and the ion channel (compound 2d). The im-
V
hNa 1.5 liabilities. As other regioisomers of 1b and 1c did not portance of this hydrogen bond network was confirmed by
V
show potent inhibitory activity (data not shown), alternative amide derivatives, because β-alanine 2e and neopentyl amide
heteroaromatic rings were investigated. 2-Benzoazoles such as 2g lost potency, whereas neopentyl amide 2f maintained the
1
d, 1e and 1f retained hNa 1.7 activity. In particular, 2-benz- activity. As the hydrogen bond donor exists in this position,
V

Vol. 68, No. 7 (2020)
Chem. Pharm. Bull.
655
a)
Table 2. In Vitro Profile of Na 1.7 Inhibitors 2
V
a) Values of at least two independent experiments run in quadruplicate unless otherwise noted. Each value
represents the mean± S.E.M. b) Values at a half-inactivated state. c) Values at V
mixture. e) Values of a single experiment run in quadruplicate. f) Not tested.
−30mV. d) Racemic
hold
urea 2h maintained the activity. Compounds 2e and 2g were test compound, whereas a slight improvement of the PK pro-
prepared as a racemic mixture. Reverse urethane 2i displayed file was observed in reverse urethane 2i. Further improvement
decent hNa 1.7 potency (IC =5.2 µM), and 2i did not exhibit of the plasma exposure was observed in benzothiazole 1g.
V
50
hNa 1.1 or hNa 1.5 liabilities at concentrations up to 100µM.
Considering the mouse in vitro activity, plasma PB ability,
V
V
Mice in Vitro Profile, in Vitro ADME Properties, and and PK parameters, 1g was expected to exhibit potent efficacy.
Pharmacokinetics (PK) Parameters in Mice Potent com- In the PK evaluation, all evaluated compounds exhibited Tmax
pounds without hNa 1.1 or hNa 1.5 liabilities were evaluated less than 1h, which would be expected immediate onset of
V
V
for mouse Na activities, in vitro ADME properties and PK pharmacological efficacy in vivo.
V
parameters in mice as shown in Table 3. To evaluate the state
To unveil the brain penetration of the compound, the Kp
dependency, mNa 1.7 activity at Vhold −30mV was measured, brain value of 1c and 2i was assessed. After 0.5h of the com-
V
and all selected compounds exhibited comparable mouse in pound administration (30mg/kg, p.o.), the brain concentra-
2
0)
vitro profiles to that of human. Compounds 1c, 1g, and 2i tion of the compounds was determined (n=2). Compound 2i
exerted high membrane permeability in Parallel Artificial indicated less exposure in the brain than in the plasma, while
Membrane Permeation Assay (PAMPA) and were expected to higher brain concentration of 1c was observed.
exhibit high plasma exposure in the PK study. Compound 1b
In Vivo Efficacy The assessment of in vivo efficacy was
lacked adequate aqueous solubility, whereas better solubility conducted on thermal hyperalgesia in mice evoked by partial
21)
was observed in other compounds. Additionally, all evaluated sciatic nerve ligation (PSL, Seltzer), a model of NP (Fig.
compounds exhibited acceptable plasma protein binding (PB) 2). The model was prepared by partial ligation of 1/2 to 1/3
ability (free fraction >1.0%). PK parameters were acquired of the left sciatic nerve with silk suture. The paw withdrawal
by orally administering the compounds to mice at 30mg/kg. latency (PWL, second) was assessed after 30, 60, 120, and
Compounds 1b and 1c showed poor plasma exposure of the 180min of oral administration of the test compound (Plantar

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Chem. Pharm. Bull.
Vol. 68, No. 7 (2020)
Table 3. Mouse NaV IC50 Values, in Vitro ADME Properties and PK Parameters
Compound
1b
1c
1g
2i
a,b)
a,b)
a,b)
a,c)
h)
h)
h)
mNa 1.1 IC (µM)
18
29± 8.4
>100
>100
V
50
h)
h)
h)
h)
h)
mNa 1.5 IC (µM)
43
43± 12
>100
>100
>100
V
50
h)
h)
mNa 1.7 IC (µM)
>100
>100
>100
V
50
h)
mNa 1.7 IC (µM)
4.3
2.0± 0.24
>50
23
4.8± 0.70
>50
11.8± 2.2
>50
V
50
PAMPA Papp (10− cm/s)
6
d)
NT
1.4
i)
e)
Solubility (µg/mL)
28
17
f)
PB free (%)
2.54
2.72
3.04
1.04
g)
PK parameters in ddY mice at 30mg/kg
Cmax (µg/mL)
Tmax (h)
0.010± 0.0036
0.67± 0.29
0.031± 0.020
1.4± 0.47
0.032± 0.0077
0.67± 0.29
0.051± 0.011
1.1± 0.53
1.65
1.45± 0.090
0.50± 0.00
2.84± 0.24
6.0± 2.4
0.10± 0.019
0.50± 0.00
0.20± 0.047
6.8± 3.3
0.69
AUC0–24h (h·µg/mL)
T1/2 (h)
i)
i)
K Brain
NT
NT
p
a) Values of at least two independent experiments run in quadruplicate unless otherwise noted. Each value represents the mean± S.E.M. b) Values at a half-inactivated state.
c) Values at Vhold −30mV. d) PAMPA was performed at pH 7.4. Values of a single experiment run in duplicate. e) Aqueous thermodynamic solubility at pH 6.8. Values of a
single experiment run in duplicate. f) Unbound fractions (%) in mouse plasma. g) Average of three male ddY mice dosed at 30mg/kg per os (p.o.) in N,N-dimethylacetamide/
Tween80/saline: 10/10/80. h) Values of a single experiment run in quadruplicate. i) Not tested.
Fig. 2. Dose–Response Effect on Thermal Hyperalgesia (Plantar Test) in PSL Model of NP (Seltzer)
Compound 2i was administrated by po route in N,N-dimethylacetamide/Tween80/saline: 10/10/80 (n=5). (a) Time course of PWL (second). (b) AUC0 of each curve in
–3h
Fig. 2a (antihyperalgesic effect of 2i). (c) antihyperalgesic effect of 1c (n=6). (d) antihyperalgesic effect of 1g (n=5). Each value is the mean± S.E.M. Statistical signifi-
cance compared to vehicle treatment is denoted by * (p<0.05), and ** (p<0.01) as determined by the Dunnett multiple comparison test. (Color figure can be accessed in
the online version.)
test). Compared with the normal group, the pain threshold at 30min in the PK study. The thermal hyperalgesia was sup-
was significantly lowered in the operated group, indicating the pressed in a dose dependent manner (3, 10, and 30mg/kg), and
development of hyperalgesia. The time course of the efficacy the administration of 2i at 30mg/kg significantly reversed hy-
of compound 2i administered to PSL mice is shown in Fig. peralgesia at all timepoints evaluated (purple curve, p<0.01
2
a. The administration of 2i at 3mg/kg p.o. (red curve) re- from 30 to 120min, and p<0.05 at 180min). Potent analgesic
versed the thermal PWL from 30 to 120min. This reversal of efficacy lasted until 180min when 10 or 30mg/kg of 2i was
hyperalgesia was maximized significantly from 30 to 60min administered. The retention of potent efficacy at 30mg/kg ad-
(
p<0.01 at 30min, and p<0.05 at 60min). The peak efficacy ministered group correlates with the PK parameters, because
improved PWL to almost normal levels. The therapeutic ef- T1/2 of 2i at 30mg/kg was 6.8h (Table 3). AUC0–3h (area under
ficacy of 2i at 3mg/kg was maximized from 30 to 60min, curve for 0–3h) for each curve was calculated to analyze the
whereas the plasma concentration of 2i at 30mg/kg peaked total anti-hyperalgesic effect (Fig. 2b). The anti-hyperalgesic

Vol. 68, No. 7 (2020)
Chem. Pharm. Bull.
657
Fig. 3. Dose–Response Effects of Compounds 1c (a), 1g (b), and 2i (c) on Motor Coordination in ddY Mice (Rotarod Test, n=10)
Each value is the mean± S.E.M. Statistical significance compared to vehicle treatment is denoted by ** (p<0.01) as determined by the Steel–Dwass test. (Color figure
can be accessed in the online version.)
Table 4. TK Parameters, Thermal Hyperalgesia ED50 Values, Rotarod ID50 Values and CNS Safety Margin
a)
TK parameters in ddY mice
c)
Thermal hyperalgesia Rotarod ID50
CNS safety
margin
Compound
b)
d)
Tmax
h)
Cmax
(µg/mL)
AUCall
(h·µg/mL)
ED50 (mg/kg)
(mg/kg)
Dose (mg/kg)
(
e)
e)
e)
e)
1c
1g
2i
300
1.0
0.50± 0.00
2.56
4.49
23
16
3.4
>300
>100
>300
>100
>8.6
>600
f)
g)
f)
f)
100
300
6.63± 0.87
6.77 (7.65, 5.89)
15.2± 1.4
15.0 (13.7, 16.3)
g)
g)
1.0 (1.0, 1.0)
a) Average of ddY mice dosed at 100 or 300mg/kg po in N,N-dimethylacetamide/Tween80/saline: 10/10/80 (n=1–3). b) The ED50 value was calculated from the suppres-
sion of thermal pain behavior in PSL mice after oral administration of compounds (Fig. 2). c) The ID50 value was calculated from the effect on motor coordination (Fig. 3). d)
CNS safety margin=Calculated Cmax at rotarod ID50 in TK study/calculated Cmax at Plantar ED50 in PK study. e) n=1. f) n=3. g) Individual data (n=2).
effect occurred in a clear dose dependent manner, and ED50 of As ED50 value is calculated defined PWL of vehicle-treated
2
i was estimated to be 3.4mg/kg. Compound 2i at 30mg/kg group as 0%, and PWL of normal mice as 100%, the inter-
significantly improved the total hyperalgesic effect to the nor- assay variation in pain threshold has no significant influence
mal levels (p<0.01).
Isoquinoline 1b failed to show potent efficacy on thermal
on ED .
50
Toxicological Evaluation Compounds with potent an-
hyperalgesia at 30mg/kg (data not shown), owing to less algesic efficacy (1c, 1g, and 2i) were selected for CNS toxi-
plasma exposure of 1b, as suggested by the PK study. The cological evaluation as indicated in Fig. 3. The in vivo CNS
total anti-hyperalgesic effect of 1c and 1g is shown in Figs. side effects were assessed using a mice rotarod test, a model
2
c and 2d. Compound 1c suppressed thermal hyperalgesia in of motor coordination at 30, 100, and 300mg/kg (n=10). TK
a dose-dependent manner (3, 10, 30mg/kg), and ED50 was es- parameters were acquired at 100 or 300mg/kg from a satellite
timated to be 23mg/kg. Although a remarkable improvement group (Table 4, n=1−3). As the administration of compound
in PK parameters was observed in 5-benzothiazole 1g, the 1g at 300mg/kg was lethal (two mice died after 120min),
efficacy of 1g in PSL mice was comparable to that of 1c, with the results of rotarod test are shown in 30 and 100mg/kg
ED =16mg/kg. There was a difference observed in AUC
administered group (Fig. 2b). The administration of 1g at 30
of control groups (vehicle-treated and normal groups) in Figs. or 100mg/kg did not effect on rotarod activity in mice, and
b, 2c, and 2d. AUC0–3h of control groups in Fig. 2d is higher ID was calculated to be >100mg/kg. Compared to anti-ther-
5
0
0–3h
2
50
than those in Figs. 2b and 2c. Consequently, the pain thresh- mal hyperalgesia efficacy, plasma-exposure-based CNS safety
old of the study with 1g is higher than those with 2i and 1c. margin of 1g was determined to be >8.6-fold (Table 4). On

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the contrary, two other compounds, 1c and 2i, demonstrated spectra were obtained on Varian Unity 400- and 500-MHz
superior CNS safety properties. Although 300mg/kg admin- spectrometers. Spectra were recorded in the indicated sol-
istration of compound 1c significantly affected the results of vent at ambient temperature; chemical shifts are reported in
rotarod test at 30min (p<0.01), this side effect was attenu- ppm (δ) relative to the solvent peak. Resonance patterns are
ated at 120min (ID >300mg/kg). Consequently, the plasma represented with the following notations: br (broad signal), s
5
0
exposure-based CNS safety margin of 1c was determined (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).
to be >100-fold. Furthermore, the administration of 2i at MS analysis was carried out by FAB, electron ionization (EI),
300mg/kg did not affect mice motor activity at all timepoints or electrospray ionization (ESI). High resolution (HR)MS was
with ID >300mg/kg. Consequently, the plasma exposure- carried out using an LC-MS system composed of a Waters
5
0
based CNS safety margin of 2i was >600-fold.
Xevo Q-ToF MS and an Acquity UHPLC system. Elemental
With promising results, 2i was further evaluated. An in analyses were carried out on a Microcorder JM10 and a Dion-
vitro assessment established that 2i did not inhibit other re- ex ICS-1500. The purity of compounds was confirmed as over
lated ion channels, including human Na 1.2, 1.3, 1.4, 1.6, or 95% by diode array detector (DAD) signal area% performed
V
human ether-a-go-go related gene (hERG). Moreover, in vitro on Agilent Infinity 1260 LCMS system. Conditions [column:
pharmacological activity of 2i on a total of 67 receptors, chan- Develosil Combi-RP-5 2.0 mmID×50mmL, gradient elution:
nels, and transporters was evaluated; 2i inhibited adenosine 0.1% HCO H–H O/0.1% HCO H–MeCN=98/2 to 0/100 (v/v),
2
2
2
2
2)
A1 and serotonin 5-HT2B by 50% at 10µM. These data flow rate: 1.2mL/min, UV detection: 254nm, column tem-
indicate that 2i is a selective Na 1.7 inhibitor in vitro.
perature: 40°C, ionization: APCI/ESI].
V
Chemical Synthesis Compounds 1a–h listed in Table 1
were synthesized by amidation of corresponding amine with
Conclusion
The discovery of quinoline amides as a novel class of N-Boc-D-phenylalanine under usual reaction conditions (Chart
state-dependent Na 1.7 inhibitors has been described. The 1). The synthesis of thiazolopyridine 7 for 1h was accom-
V
2
3)
benzazepinone moiety of compound III was replaced with a plished by the annulation of chloropyridine 5 as a key step.
heteroaromatic ring. Among a variety of heteroaromatic rings Chart 2 summaries the preparation of compounds 2a–i
examined, quinoline ring was shown to be optimal regarding listed in Table 3. Compounds 2a–2c and 2f were synthesized
potency as well as Na 1.1 or Na 1.5 liabilities. The modifica- from amine 8 derived from 1c by the reaction with carbonyl
V
V
tion of acid-unstable Boc group led to the discovery of reverse chlorides, whereas urea 2h was prepared from 8 reacted with
urethane 2i (DS01171986). Compound 2i inhibited hNa 1.7 tert-butyl isocyanate. N-Methyl urethane 2d was prepared
V
at IC =5.2 µM in a state-dependent manner. Notably, 2i by the condensation of 3-aminoquinoline with N-methyl-N-
5
0
inhibited Na 1.7 without affecting other receptors, channels, Boc-D-phenylalanine (structures not shown). The synthesis of
V
and transporters, including other NaV subtypes and hERG. β-alanine 2e was achieved from key intermediate 11, which
Compound 2i demonstrated potent analgesic efficacy in NP was prepared from benzyl alcohol 10 and tert-butyl bromoac-
2
4)
25)
model with EC =3.4mg/kg. A CNS adverse effect evaluation etate. Started from 15, neopentyl amide 2g was synthe-
5
0
revealed that 2i possessed excellent CNS safety margin (>600 sized in a similar manner. Reverse urethane 2i was prepared
fold).
from D-phenyllactic acid 18.
General Procedure for Amidation (General Proce-
dure A) A solution of amine (3.47mmol), carboxylic acid
Experimental
General Starting reagents were purchased from com- (3.47mmol), N,N-diisopropylethylamine (DIPEA) (2.7mL,
mercial suppliers and were used without further purification 15.6mmol), N-hydroxybenzotriazole (HOBt)·H O (0.84g,
2
unless otherwise specified. Chromatographic elution was 6.24mmol) and WSC·HCl (1.20g, 6.24mmol) in tetrahydro-
carried under continuous monitoring by TLC using silica gel furan (THF) (23mL) was stirred at room temperature over-
6
0F254 (Merck & Co., Inc., Germany) as the stationary phase; night under N . Water was added to the reaction mixture,
2
the mobile phase was the elution solvent used in column chro- and the mixture was extracted with EtOAc several times. The
matography. A UV detector was used for detection. Silica gel combined organic layer was dried over anhydrous Na SO and
2
4
SK-85 (230–400 mesh) or silica gel SK-34 (70–230 mesh), concentrated under reduced pressure. The residue was purified
manufactured by Merck & Co., Inc., or Chromatorex NH by silica gel chromatography to obtain 1.
(
(
200–350 mesh), manufactured by Fuji Silysia Chemical Ltd.
General Procedure for Amidation to Prepare 2 (General
Japan), was used as the column packing silica gel. H-NMR Procedure B) A solution of amine 3 (70mg, 0.24mmol) and
1
Reagents and conditions: (a) WSC·HCl, HOBt, DIPEA, THF; (b) thioacetamide, 1,4-dioxane, 100°C, 27%; (c) Fe, HCl, EtOH, H O.
2
Chart 1. Synthesis of Compounds 1a–h

Vol. 68, No. 7 (2020)
Chem. Pharm. Bull.
659
Reagents and conditions: (a) TFA, CH Cl ; (b) RCOCl, Et N, CH Cl ; (c) tert-butyl isocyanate, toluene, 77%; (d) BnOH, Et N, CH Cl , 91%; (e) tert-butyl bromoacetate,
2
2
3
2
2
3
2
2
LDA, THF, −78°C, 56%; (f) tert-butylamine or neopentylamine, WSC·HCl, HOBt, CH Cl ; (g) H , Pd/C, EtOH; (h) 3-aminoquinoline, WSC·HCl, HOBt, CH Cl ; (i) tert-
2
2
2
2
2
butyl isocyanate, Me SiCl, CH Cl ; (j) 3-aminoquinoline, HATU, DMF, 45%.
3
2
2
Chart 2. Synthesis of Compounds 2a–i
Et N (0.10mL, 0.72mmol) in CH Cl (1.2mL) was added chlo- m), 7.45 (1H, t, J=7.2Hz), 7.56 (1H, t, J=7.8Hz), 7.66 (1H,
3
2
2
roforamte or acid chloride (0.57mmol) at room temperature d, J=7.4Hz), 7.95 (1H, d, J=8.6Hz), 8.53 (1H, d, J=2.3Hz),
+
under N . After being stirred at room temperature for several 8.58 (1H, s), 8.78 (1H, s); MS (APCI/ESI) m/z: 392 [M+H] ;
2
hours, the mixture was directly purified by silica gel chroma- Anal. calcd for C H N O : C, 70.57; H, 6.44; N, 10.73. Found
2
3
25
3
3
2
5
tography to obtain 2a–2c or 2f.
General Procedure for Deprotection by Hydroge-
C, 70.40; H, 6.61; N, 10.75; [α] +5.92 (c=0.980, CHCl ).
D
3
N-1,3-Benzothiazol-2-yl-Nα-(tert-butoxycarbonyl)-D-
nolysis (General Procedure C) A solution of benzyl ester phenylalaninamide (1d) Prepared according to General
1
(
3.72mmol) and Pd/C (10%, 300mg) in EtOH (10mL) was Procedure A. Yield: 90%. H-NMR (400MHz, CDCl ) δ: 1.40
3
stirred at room temperature under H for several hours. After (9H, s), 3.08–3.16 (1H, m), 3.22 (1H, dd, J=6.0, 14.4Hz), 4.74
2
the removal of the catalyst, the residue was concentrated to (1H, s), 5.19 (1H, s), 7.07–7.13 (2H, m), 7.18–7.25 (3H, m),
obtain carboxylic acid.
7.32 (1H, t, J=6.8Hz), 7.43 (1H, t, J=8.4Hz), 7.76 (1H, d,
tert-Butyl[(2R)-1-oxo-3-phenyl-1-(1,2,3,4-tetrahydroqui- J=8.0Hz), 7.83 (1H, d, J=8.0Hz); MS (APCI/ESI) m/z: 398
+
nolin-3-ylamino)propan-2-yl]carbamate (1a) Prepared ac- [M+H] ; Anal. calcd for C H N O S·0.20H O: C, 62.89; H,
21
23
3
3
2
2
5
cording to General Procedure A as a diastereomeric mixture. 5.88; N, 10.48. Found C, 62.84; H, 5.83; N, 10.56; [α] +59.9
D
+
Yield: 99%. HRMS (ESI) m/z: [M+H] calcd for C H N O , (c=1.007, CHCl3).
2
3
30
3
3
396.2293; found 396.2289.
N-1,3-Benzoxazol-2-yl-Nα-(tert-butoxycarbonyl)-D-
tert-Butyl[(2R)-1-(isoquinolin-3-ylamino)-1-oxo-3- phenylalaninamide (1e) Prepared according to General
1
phenylpropan-2-yl]carbamate (1b) Prepared according to Procedure A. Yield: 50%. H-NMR (400MHz, CDCl ) δ:
3
1
General Procedure A. Yield: 63%. H-NMR (400MHz, 1.41 (9H, s), 3.13 (1H, s), 3.28 (1H, dd, J=5.9, 14.1Hz),
CDCl ) δ: 1.42 (9H, s), 3.11–3.20 (1H, m), 3.27 (1H, dd, J=6.3, 4.69 (1H, s), 5.14 (1H, s), 7.20–7.32 (8H, m), 7.45 (1H, d,
3
+
1
4.1Hz), 4.64 (1H, s), 5.18 (1H, d, J=7.0Hz), 7.19–7.29 (5H, J=8.2Hz); MS (APCI/ESI) m/z: 382 [M+H] ; Anal. calcd
m), 7.48 (1H, t, J=6.7Hz), 7.64 (1H, t, J=7.0Hz), 7.83 (1H, d, for C H N O ·0.10H O: C, 65.82; H, 6.10; N, 10.96. Found C,
21
23
3
4
2
2
5
J=8.2Hz), 7.87 (1H, d, J=8.2Hz), 8.58 (1H, s), 8.69 (1H, s), 65.69; H, 6.19; N, 10.82; [α] +14.0 (c=1.007, CHCl ).
D
3
+
8
.94 (1H, s); MS (APCI/ESI) m/z: 392 [M+H] ; Anal. calcd
Nα-(tert-Butoxycarbonyl)-N-(1-methyl-1H-benzimidazol-
for C H N O : C, 70.57; H, 6.44; N, 10.73. Found C, 70.49; 2-yl)-D-phenylalaninamide (1f) Prepared according to
2
3
25
3
3
2
5
1
H, 6.62; N, 10.91; [α] +6.85 (c=0.974, CHCl ).
General Procedure A. Yield: 30%. H-NMR (400MHz,
D
3
tert-Butyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)- CDCl ) δ: 1.45 (9H, s), 3.25 (2H, t, J=5.3Hz), 3.61 (3H, s),
3
propan-2-yl]carbamate (1c) Prepared according to General 4.62–4.67 (1H, m), 5.43 (1H, d, J=7.0Hz), 7.17–7.31 (9H,
1
+
Procedure A. Yield: 45%. H-NMR (400MHz, CDCl ) δ: 1.43 m); MS (APCI/ESI) m/z: 395 [M+H] ; Anal. calcd for
3
(
9H, s), 3.14 (1H, dd, J=7.4, 14.1Hz), 3.21 (1H, dd, J=7.4, C H N O ·0.10H O: C, 66.68; H, 6.66; N, 14.14. Found C,
22 26 4 3 2
25
1
4.1Hz), 4.64 (1H, s), 5.39 (1H, d, J=7.8Hz), 7.21–7.30 (5H, 66.56; H, 6.58; N, 14.16; [α] +73.9 (c=0.740, CHCl ).
D
3

6
60
Chem. Pharm. Bull.
Vol. 68, No. 7 (2020)
Nα-(tert-Butoxycarbonyl)-N-(2-methyl-1,3-benzothiazol- formers according to General Procedure B utilizing 3,3-di-
-yl)-D-phenylalaninamide (1g) Prepared according to Gen- methylbutanoyl chloride as an acylating reagent. Yield:
5
1
+
eral Procedure A. Yield: 88%. H-NMR (400MHz, CDCl ) δ: 88%. MS (APCI/ESI) m/z: 390 [M+H] ; Anal. calcd for
3
1
5
7
.44 (9H, s), 2.82 (3H, s), 3.12–3.20 (2H, m), 4.46 (1H, br), C H N O ·0.30H O: C, 73.00; H, 7.04; N, 10.64. Found C,
24 27 3 2 2
2
5
.14 (1H, br), 7.26–7.34 (5H, m), 7.45 (1H, dd, J=2.0, 8.6Hz), 73.11; H, 7.01; N, 10.64; [α] +12.7 (c=1.008, CHCl ).
D
3
.71 (1H, d, J=8.6Hz), 7.80 (1H, br), 7.91 (1H, d, J=2.0Hz);
2-Benzyl-N-(2,2-dimethylpropyl)-N′-(quinolin-3-yl)pro-
+
HRMS (ESI) m/z: [M+H] calcd for C H N O S, 412.1690; panediamide (2g) Prepared according to General Proce-
2
2
26
3
3
1
found 412.1709; Anal. calcd for C H N O S: C, 64.21; H, dure A utilizing CH Cl as a solvent. Yield: 63%. H-NMR
2
2
25
3
3
2
2
2
5
6
(
.12; N, 10.21. Found C,63.92; H, 6.36; N, 10.13; [α] +4.23 (400MHz, CDCl ) δ: 0.85 (9H, s), 3.02 (1H, dd, J=6.1,
D
3
c=1.003, CHCl3).
13.1Hz), 3.09 (1H, dd, J=6.7, 13.3Hz), 3.27 (1H, dd, J=8.6,
Nα-(tert-Butoxycarbonyl)-N-(2-methyl[1,3]thiazolo[5,4- 13.7Hz), 3.36 (1H, dd, J=7.0, 13.7Hz), 3.64 (1H, t, J=7.6Hz),
b]pyridin-6-yl)-D-phenylalaninamide (1h) Prepared ac- 6.89 (1H, t, J=5.9Hz), 7.06–7.27 (5H, m), 7.53 (1H, t,
cording to General Procedure A. Yield: 46% (2 steps). J=7.2Hz), 7.63 (1H, t, J=7.0Hz), 7.77 (1H, d, J=7.8Hz),
1
H-NMR (400MHz, CDCl ) δ: 1.45 (9H, s), 2.85 (3H, s), 3.13– 8.04 (1H, d, J=8.6Hz), 8.64 (1H, d, J=2.3Hz), 8.76 (1H, d,
3
+
3
.25 (2H, m), 4.46–4.54 (1H, m), 5.13 (1H, br), 7.26–7.36 (5H, J=2.7Hz), 10.08 (1H, s); MS (APCI/ESI) m/z: 390 [M+H] ;
m), 7.99 (1H, br), 8.39 (1H, d, J=2.3Hz), 8.44 (1H, d, J= Anal. calcd for C H N O ·0.20H O: C, 73.33; H, 7.03; N,
2
4
2
4
27
3
2
2
+
.0Hz); HRMS (ESI) m/z: [M+H] calcd for C H N O S, 10.69. Found C, 73.29; H, 7.02; N, 10.66.
21 25 4 3
25
13.1642; found 413.1648; [α] +4.4 (c=0.302, CHCl ).
α - (t e r t - B ut y l c a r ba moy l ) - N - qu i no l i n - 3 -y l - D -
D
3
Propan-2-yl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)- phenylalaninamide (2h) A solution of amine 3 (52mg,
propan-2-yl]carbamate (2a) Prepared as a mixture of con- 0.18mmol) and tert-butylisocyanate (40µL, 0.36mmol) in
formers according to General Procedure B utilizing isopropyl toluene (2.3mL) was stirred at room temperature for 18h
chloroformate as an acylating reagent. Yield: 46%. HRMS under N . The mixture was directly purified by silica gel
2
+
(
3
ESI) m/z: [M+H] calcd for C H N O , 378.1823; found chromatography to obtain 2h (54.1mg, 77%) as a mixture of
2
2
24
3
3
2
5
+
78.1810; [α] +5.9 (c=0.304, CHCl ).
conformers. MS (APCI/ESI) m/z: 391 [M+H] ; Anal. calcd
D
3
Ethyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)propan- for C H N O ·0.20H O: C, 70.10; H, 6.75; N, 14.22. Found
2
3
26
4
2
2
2
5
2
-yl]carbamate (2b) Prepared as a mixture of conformers C, 69.95; H, 6.73; N, 14.19; [α] +6.1 (c=1.010, CHCl ).
D
3
according to General Procedure B utilizing ethyl chlorofor-
(2R)-1-Oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl-
mate as an acylating reagent. Yield: 80%. MS (APCI/ESI) tert-butylcarbamate (2i) A solution of 3-aminoquinoline
+
m/z: 364 [M+H] ; Anal. calcd for C H N O ·0.50H O: C, (7.21g, 50.0mmol), 11 (13.3g, 50.0mmol) and HATU (19.01g,
21
21
3
3
2
2
5
6
7.73; H, 5.95; N, 11.28. Found C, 67.77; H, 5.79; N, 11.26; [α]
50.0mmol) in N,N-dimethylformamide (DMF) (100mL) was
D
+
2.6 (c=1.002, CHCl3).
stirred at room temperature for 18h under N . Water was
Methyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)pro- added to the reaction mixture, and the mixture was extracted
2
pan-2-yl]carbamate (2c) Prepared according to General several times with EtOAc. The combined organic layer was
Procedure B utilizing methyl chloroformate as an acylating dried over anhydrous Na SO and concentrated under reduced
2
4
1
reagent. Yield: 96%. H-NMR (400MHz, CDCl ) δ: 3.16 pressure. The residue was purified by silica gel chroma-
3
(
1H, dd, J=7.4, 13.7Hz), 3.26 (1H, dd, J=6.7, 13.7Hz), 3.73 tography (hexane/EtOAc, 50:50) to obtain 2i (8.70g, 45%).
1
(3H, s), 4.56–4.62 (1H, m), 5.37 (1H, s), 7.25–7.35 (5H, m),
H-NMR (400MHz, CDCl ) δ: 1.33 (9H, s), 3.25 (1H, dd,
3
7
.52–7.55 (1H, m), 7.61–7.66 (1H, m), 7.79 (1H, d, J=8.2Hz), J=6.8, 14.2Hz), 3.35 (1H, dd, J=5.6, 13.9Hz), 4.85 (1H,
8
.02 (1H, d, J=8.6Hz), 8.51 (1H, d, J=2.7Hz), 8.64 (1H, d, s), 5.49 (1H, t, J=5.6Hz), 7.23–7.30 (5H, m), 7.54 (1H, t,
+
J=2.3Hz); HRMS (ESI) m/z: [M+H] calcd for C H N O , J=7.6Hz), 7.64 (1H, t, J=7.6Hz), 7.81 (1H, d, J=7.8Hz),
2
0
20
3
3
2
5
350.1510; found 350.1504; [α] +6.2 (c=0.306, CHCl ).
7.98 (1H, s), 8.03 (1H, d, J=8.3Hz), 8.55 (1H, d, J=2.4Hz),
D
3
+
tert-Butyl Methyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-yl- 8.72 (1H, d, J=2.4Hz); HRMS (ESI) m/z: [M+H] calcd
amino)propan-2-yl]carbamate (2d) Prepared according to for C H N O , 392.1980; found 392.1983; Anal. calcd for
2
3
26
3
3
General Procedure A utilizing N-(tert-butoxycarbonyl)-N- C H N O : C, 70.57; H, 6.44; N, 10.73. Found C, 70.61; H,
2
3
25
3
3
2
5
methyl-D-phenylalanine as a starting material. Yield: 99%. 6.28; N, 10.72. [α] +76.8 (c=1.001, CHCl ).
D
3
1
22)
H-NMR (400MHz, CDCl ) δ: 1.44 (9H, s), 2.85 (3H, s),
2-Methyl-6-nitro[1,3]thiazolo[5,4-b]pyridine (6)
.09–3.16 (1H, m), 3.39–3.46 (1H, m), 5.01–5.06 (1H, m), lution of 2-chloro-3,5-dinitropyridine (5, 1.00g, 4.91mmol)
.24–7.34 (5H, m), 7.53 (1H, t, J=7.0Hz), 7.62 (1H, t, and thioacetamide (1.48g, 19.7mmol) in 1,4-dioxane (10mL)
A so-
3
3
7
J=7.4Hz), 7.79 (1H, d, J=8.2Hz), 8.03 (1H, d, J=8.2Hz), was stirred at 100°C for 1h. After the addition of water the
8
4
.67 (1H, s), 8.73 (1H, s), 8.91 (1H, s); MS (APCI/ESI) m/z: mixture was extracted with EtOAc twice. The combined
+
06 [M+H] ; Anal. calcd for C H N O ·0.40H O: C, 69.85; organic layers were dried over anhydrous Na SO and con-
2
4
27
3
3
2
2
4
2
5
H, 6.79; N, 10.18. Found C, 69.92; H, 6.74; N, 10.33; [α]D centrated under reduced pressure. The residue was purified
+
64.7 (c=1.001, CHCl3).
by silica gel chromatography (hexane/EtOAc, 80:20) to ob-
-Benzyl-N4-tert-butyl-N1-(quinolin-3-yl)butanediamide tain 6 (0.26g, 27%) as a colorless solid. H-NMR (400MHz,
1
2
(
2e) Prepared as a mixture of conformers according to DMSO-d ) δ: 2.93 (3H, s), 9.06 (1H, d, J=2.7Hz), 9.38 (1H,
6
General Procedure A utilizing CH Cl2 as a solvent. Yield: d, J=2.7 Hz).
2
+
6
9%. MS (APCI/ESI) m/z: 390 [M+H] ; Anal. calcd for
2-Methyl[1,3]thiazolo[5,4-b]pyridin-6-amine HCl (7)
(0.26g, 1.33mmol), iron powder (1.49g,
26.6mmol) and 12M HCl (70µL) in water (2.7mL) and EtOH
Nα-(3, 3 -Dimethylbutanoyl)-N-quinolin-3 -yl-D - (13mL) was stirred at room temperature for 2h. The reaction
phenylalaninamide (2f) Prepared as a mixture of con- mixture was filtered through a pad of Celite, and the filtrate
A
C H N O ·0.20H O: C, 73.33; H, 7.03; N, 10.69. Found C, solution of
6
2
4
27
3
2
2
73.33; H, 7.01; N, 10.59.

Vol. 68, No. 7 (2020)
Chem. Pharm. Bull.
661
was concentrated under reduced pressure to obtain crude 7,
2-Benzyl-4-(tert-butylamino)-4-oxobutanoic Acid (14)
which was used directly for the next reaction without further Prepared according to General Procedure C. Yield: 69%.
1
purification.
N-Quinolin-3-yl-D-phenylalaninamide (8) A solution of 2.77 (1H, dd, J=9.2, 13.5Hz), 3.09–3.23 (2H, m), 5.40 (1H, s),
c (1.20g, 3.07mmol) and TFA (6.0mL) in CH Cl (30mL) 7.18–7.33 (5H, m).
H-NMR (400MHz, CDCl ) δ: 1.33 (9H, s), 2.32–2.36 (2H, m),
3
1
2
2
was stirred at room temperature for 3h. After the addition
Benzyl 2-Benzyl-3-[(2,2-dimethylpropyl)amino]-3-oxo-
of saturated aqueous solution of NaHCO (pH=10), the mix- propanoate (16) Prepared according to General Procedure A
3
1
ture was extracted with CH Cl twice. The combined organic utilizing CH Cl as a solvent. Yield: 89%. H-NMR (400MHz,
2
2
2
2
layers were dried over anhydrous Na SO and concentrated CDCl ) δ: 0.81 (9H, s), 2.97 (1H, dd, J=5.9, 13.3Hz), 3.06
2
4
3
1
under reduced pressure to obtain 8 (0.89g, 99%). H-NMR (1H, dd, J=6.7, 13.3Hz), 3.19 (1H, dd, J=8.6, 13.7Hz), 3.29
400MHz, CDCl ) δ: 2.88 (1H, dd, J=9.4, 14.1Hz), 3.41 (1H, (1H, dd, J=6.3, 13.7Hz), 3.56 (1H, dd, J=6.3, 8.6Hz), 5.07
(
3
dd, J=4.1, 13.9Hz), 3.83 (1H, dd, J=3.9, 9.4Hz), 7.26–7.30 (2H, s), 6.45 (1H, s), 7.10–7.37 (10H, m).
(3H, m), 7.34–7.37 (2H, m), 7.52–7.56 (1H, m), 7.61–7.66 (1H,
2-Benzyl-3-[(2,2-dimethylpropyl)amino]-3-oxopropanoic
m), 7.83 (1H, dd, J=1.4, 8.0Hz), 8.05 (1H, d, J=8.2Hz), 8.76 Acid (17) Prepared according to General Procedure C.
1
(
1H, d, J=2.3Hz), 8.87 (1H, d, J=2.3Hz), 9.80 (1H, s).
Yield: 99%. H-NMR (400MHz, CDCl ) δ: 0.73 (9H, s),
3
Benzyl 3-Phenylpropanoate (10) A solution of benzyl 2.83–2.94 (1H, m), 2.97–3.05 (1H, m), 3.12–3.20 (1H, m),
3
-phenylpropanoyl chloride (4, 3.0g, 17.8mmol), Et N (3.0mL, 3.37–3.43 (2H, m), 5.56 (1H, s), 7.22–7.35 (5H, m).
3
2
1.5mmol) and BnOH (2.0g, 18.5mmol) in CH Cl (40mL)
(2R)-2-[(tert-Butylcarbamoyl)oxy]-3-phenylpropanoic
2
2
was stirred at room temperature for 2h. Water was added Acid (20) Me SiCl (15.8mL, 125mmol) was added to a solu-
3
to the reaction mixture, and the mixture was extracted with tion of benzyl (2R)-2-hydroxy-3-phenylpropanoate (18, 14.50g,
EtOAc twice. The combined organic layer was dried over 56.7mmol) and tert-butylisocyanate (15.8mL, 125mmol) in
anhydrous Na SO and concentrated under reduced pres- CH Cl2 (300mL) at room temperature under N . After the
2
4
2
2
sure. The residue was purified by silica gel chromatography reaction mixture was stirred at room temperature for 18h, the
1
(
hexane/EtOAc, 90:10) to obtain 10 (3.90g, 91%). H-NMR mixture was concentrated to obtain crude 19. A solution of
(
400MHz, CDCl ) δ: 2.69 (2H, t, J=7.4Hz), 2.97 (2H, t, crude 19 and Pd/C (10%, 6.03g) in EtOH (200mL) was stirred
3
J=7.8Hz), 5.11 (2H, s), 7.17–7.37 (10H, m).
at room temperature under H for 2h. After the removal of
2
1
-Benzyl 4-tert-Butyl 2-Benzylbutanedioate (11) n-BuLi the catalyst, the residue was concentrated to obtain 20 (15.0g,
1
(1.57mmol/L in THF solution, 12.5mL, 19.6mmol) was added 99%, 2 steps) as a white powder. H-NMR (500MHz, CDCl )
3
to a solution of iPr NH (2.75mL, 19.5mmol) in THF (50mL) δ: 1.28 (9H, s), 3.07–3.13 (1H, m), 3.21 (1H, d, J=15.1Hz),
2
at 0°C under N . After being stirred for 20min at 0°C, THF 4.81 (1H, s), 5.22 (1H, s), 7.24–7.33 (5H, m).
2
2
6)
(
20mL) solution of 10 (3.90g, 16.2mmol) was added to the re-
action mixture at −78°C, and the mixture was stirred at −78 The IonWorks Quattro system (version 2.0, Molecular Devic-
°C for 30min. THF (10mL) solution of tert-butyl bromoac- es) was used for electrophysiological recordings. A 384-well
etate (4.40g, 22.6mmol) was added to the reaction mixture at plate containing the compounds to be tested was placed in the
78°C, and the mixture was stirred at 0°C for 2h. Saturated plate-1 position. A PatchPlate was clamped into the PatchPlate
High Throughput Electrophysiological Evaluation
0
−
aqueous solution of NH Cl was added to the reaction mix- station. Once the experiment was started, the fluidics-head (F-
4
ture at 0°C, followed by extraction with EtOAc twice. The head) added 3.5µL of Dulbecco’s Phosphate Buffered Saline
combined organic layers were washed with water, dried over (DPBS, with calcium and magnesium, Sigma-Aldrich Co.
anhydrous Na SO , and concentrated under reduced pressure. LLC) to each well of the PatchPlate and its underside was
2
4
The residue was purified by silica gel chromatography (hex- perfused with internal solution that had the following com-
ane/EtOAc, 90:10) to obtain 11 (3.29g, 56%) as a colorless position (in mM): 100.0 potassium D-gluconate, 40.0 KCl, 3.2
1
oil. H-NMR (400MHz, CDCl ) δ: 1.39 (9H, s), 2.35 (1H, dd, MgCl , 5.0 ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-
3
2
J=5.1, 16.4Hz), 2.60 (1H, dd, J=8.6, 16.4Hz), 2.75–2.82 (1H, tetraacetic acid (EGTA), 5.0 N-(2-hydroxyethyl)piperazine-
m), 3.02 (1H, dd, J=6.7, 13.7Hz), 3.12–3.14 (1H, m), 5.06 (1H, N′-2-ethanesulfonic acid (HEPES) (pH 7.27 using 1M
d, J=12.5Hz), 5.11 (1H, d, J=12.5Hz), 7.03–7.37 (10H, m).
KOH). After priming and debubbling, the electronics-head
(E-head) moved around the PatchPlate to perform a whole
2
3)
3
-Benzyl-4-(benzyloxy)-4-oxobutanoic Acid (12)
A
solution of 11 (1.60g, 4.51mmol) and TFA (15mL) in CH Cl2 test. The F-head then dispensed 3.5µL of the cell suspension
2
(
15mL) was stirred at room temperature for 18h. After con- (hNa 1.1/β1/β2/HEK293A, hNa 1.5/β1/β2/HEK293A, hNaV
V
V
centration of the reaction mixture, the mixture was purified 1.7/β1/β2/HEK293A, mNa 1.1/β1/β2/HEK293A, mNa 1.5/β1/β2/
V
V
by silica gel chromatography (CH Cl /MeOH, 90:10) to ob- HEK293A, or mNa 1.7/β1/β2/HEK293A) into each well of the
2
2
V
1
tain 12 (970mg, 72%) as a colorless oil. H-NMR (400MHz, PatchPlate, and the cells were given 400s to reach and seal
CDCl ) δ: 2.46 (1H, dd, J=5.1, 17.2Hz), 2.73 (1H, dd, J=9.8, the hole in each well. After this, the E-head moved around the
3
1
7.2Hz), 2.79 (1H, dd, J=8.2, 13.7Hz), 3.07 (1H, dd, J=6.3, PatchPlate to determine the seal resistance obtained in each
3.7Hz), 3.13–3.20 (1H, m), 5.10 (2H, s), 7.11–7.36 (10H, m). well. Then, the solution on the underside of the PatchPlate
Benzyl 2-Benzyl-4-(tert-butylamino)-4-oxobutanoate (13) was changed to access solution that had the following com-
Prepared according to General Procedure A utilizing CH Cl position (in mM): 100.0 potassium D-gluconate, 40.0 KCl,
as a solvent. Yield: 85%. H-NMR (500MHz, CDCl ) δ: 1.28 3.2 MgCl , 5.0 EGTA, 5.0 HEPES (pH 7.27 using 1M KOH)
1
2
2
1
3
2
(
1
1
9H, s), 2.20 (1H, dd, J=4.9, 14.2Hz), 2.40 (1H, dd, J=9.0, plus 100µg/mL of amphotericin B (Sigma-Aldrich Co. LLC.).
4.9Hz), 2.83 (1H, dd, J=7.3, 12.7Hz), 2.99 (1H, dd, J=6.8, After 10min of patch perforation was allowed to take place,
3.7Hz), 3.23–3.29 (1H, m), 5.06 (1H, d, J=12.2Hz), 5.11 (1H, the E-head moved around the PatchPlate to obtain Na current
V
d, J=12.7Hz), 5.26 (1H, s), 7.11–7.36 (10H, m).
measurements.

6
62
Chem. Pharm. Bull.
Vol. 68, No. 7 (2020)
Table 5. Voltage Program
a)
a)
a)
b)
a)
a)
a)
b)
hNa 1.1
hNa 1.5
hNa 1.7
hNa 1.7
mNa 1.1
mNa 1.5
mNa 1.7
mNa 1.7
V
V
V
V
V
V
V
V
Holding pulse (for 5s)
−100mV −120mV −100mV −100mV −100mV −120mV −100mV −100mV
a
b
Second conditioning pulse (for 2s or 10s )
Holding pulse (for 50ms)
−43mV
−68mV
−59mV
−30mV
−100mV
−140mV
−10mV
−44mV
−69mV
−56mV
−30mV
−100mV
−140mV
−10mV
Hyperpolarizing pulse (for 20ms)
Test pulse (for analysis of inactivated-state channels,
for 50ms
0mV
−20mV
−10mV
0mV
−20mV
−10mV
a) Half-inactivated state. b) Vhold −30mV.
After these pre-compound measurements, the F-head after the animal placed in the measuring cage became calm.
added 3.5µL of solution from each well of the compound The value indicated by the device was transcribed to the re-
plate to each well on the PatchPlate. After about 5.5min of cording sheet, and this value was used as the pain threshold.
incubation, the E-head moved around the PatchPlate to obtain
Rotarod Performance in Mice All experimental pro-
post-compound Na current measurements. The pre- and post- cedures were performed in accordance with the in-house
V
compound NaV current amplitude were measured from the guideline of the Institutional Animal Care and Use Committee
peak current response subtracting the baseline current. The of Daiichi Sankyo Co., Ltd. ddY Mice able to walk on the ro-
degree of Na current block was corrected by vehicle control tarod (20rpm, 3cm in diameter; model MK-660C; Muromachi
V
currents as follows:
Kikai Co., Ltd., Tokyo, Japan) for 2min were selected. After
oral administration of the test compound or vehicle (control),
each mouse was placed on the rotarod, and motor coordination
was considered to be impaired if the rat fell off the rotarod
within 120s in two trials. The rotarod test was conducted at 0,
%
inhibition

1
−
relative current(compound)

=100×


mean relative current(vehicle)


where, relative current (compound) is the value of the post- 30, 60, and 120min after administration.
compound NaV current amplitude divided by the respective
pre-compound Na current amplitude and mean relative cur-
Conflict of Interest Kyosuke Tanaka, Hiroyuki
V
rent (vehicle) is the mean value of the post-vehicle Na current Kobayashi, Sayaka Suzuki, Satoshi Shibuya, Yuki Domon,
V
amplitude divided by the pre-vehicle Na current amplitude.
Data was fitted with a four parameter logistic equation:
Kazufumi Kubota, Yutaka Kitano, Chie Fujiwara, Daigo
Asano, and Tsuyoshi Shinozuka are currently employees of
Daiichi Sankyo Co., Ltd. Ryuta Koishi is currently employees
of Daiichi Sankyo RD Novare Co., Ltd.
V
[(Log IC50
−X )×HillSlope])
Y =Bottom+(Top−Bottom) / (1+10
The voltage program for each NaV subtype or species is
summarized in Table 5.
Supplementary Materials The online version of this ar-
ticle contains supplementary materials.
PAMPA, Solubility, and Protein Binding Assay E a c h
assay was performed as previously described.
1.
2.
Eurofins Lead Profiling Data
Time course of antihyperalgesic effect of compounds
2
7),28)
Mouse PK Study All experimental procedures were 1c and 1d
performed in accordance with the in-house guideline of the
Institutional Animal Care and Use Committee of Daiichi References and Notes
Sankyo Co., Ltd. For the determination of test compound
exposures in male ddY mice, blood samples were collected at
several timepoints post-dose. The plasma was separated from
the blood by centrifugation, and stored at −70°C until use for
the measurement of plasma concentration. The determination
of the plasma concentration of the compound was performed
by LC-MS/MS using API 4000 (Applied Biosystems/MDS
SCIEX). PK parameters were calculated using a non-compart-
mental analysis techniques.
PSL Model in Mice All experimental procedures were
performed in accordance with the in-house guideline of the
Institutional Animal Care and Use Committee of Daiichi San-
kyo Co., Ltd. Four-week-old PSL mice were purchased from
Japan SLC, Inc., and were fed until 5 weeks old. The paw
withdrawal latency (second) in each animal was measured
at 0 (before administration) and at 30, 60, 120 and 180min
after administration in a blinded fashion. A planta thermal-
stimulation device (PAW THERMAL STIMULATOR, UCSD,
San Diego, CA, U.S.A.) was used for measurement. A 20s
cut-off time was employed to avoid tissue damage. No animal
reached the cutoff value. The measurement was conducted
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