Discovery of a Novel Class of State-Dependent Na_V1.7 Inhibitors for the Treatment of Neuropathic Pain

Abstract

The discovery of a novel class of state-dependent voltage-gated sodium channel (Na_V)1.7 inhibitors is described. By the modification of amide or urethane bond in Na_V1.7 blocker III, structure–activity relationship studies that led to the identification of novel Na_V1.7 inhibitor 2i (DS01171986) were performed. Compound 2i exhibited state-dependent inhibition of Na_V1.7 without Na_V1.1, Na_V1.5 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).

Introduction

Neuropathic pain (NP) is a debilitating disease that affects patients spontaneously and has a significant impact on their QOL. Diabetic peripheral neuropathy (DPN) is one of the most common types of NP. It is estimated that around half of diabetic patients suffer from DPN.¹⁾ The first-line treatments for DPN include calcium channel α₂δ ligands and tricyclic antidepressants.²⁾ Although these drugs are efficacious and tolerable, central nervous system (CNS) adverse effects, such as drowsiness lightheadedness, dizziness, and sedation, limit their use.²⁾ Consequently, novel potent analgesic drugs with less CNS adverse effects have a high demand.

Voltage-gated sodium channels (Na_V) are responsible for transmitting neuronal signals not only in CNS but also in the peripheral nervous system (PNS). In humans, nine Na_V subtypes (Na_V1.1–Na_V1.9) have been identified, and Na_V1.7 has been shown to play a crucial role in pain signaling.^3,4) Several Na_V blockers, such as lidocaine (I) and mexiletine (II) (Fig. 1), have been used to treat chronic pain in a clinical setting. Furthermore, a high safety index over CNS adverse effects is expected by selective Na_V1.7 inhibition, because the Na_V1.7 isoform is predominantly expressed in the PNS.⁵⁾ In the discovery of novel Na_V1.7 inhibitors, a high subtype selectivity is crucial, because multiple Na_V subtypes are differently expressed in various organs; non-selective inhibitors could cause adverse effects. For example, Na_V1.1 is expressed in brain, and Na_V1.1 inhibition is known to cause CNS adverse effects, whereas the inhibition of Na_V1.5 leads to cardiac arrhythmias.^6,7) In fact, Na_V blockers used in clinics are non-selective, and the lack of selectivity limits the usage of such drugs due to safety concerns. Therefore, the selective inhibition of Na_V1.7 is a promising analgesic target.^8–10)

Fig. 1. Structures of Lidocaine (I), Mexiletine (II), and Compound III, Reported by the Merck Group

During the course of a project directed at acquiring potent selective Na_V1.7 inhibitors,¹¹⁾ we focused on benzazepinone derivative III (Fig. 1), reported by the Merck group, owing to its high state dependency.^12–14) As two amide and one urethane bonds are found in this molecule, our derivatization strategy consists of the modifications of such bonds. Herein, we describe the conversion of these moieties to identify a novel class of state-dependent Na_V1.7 inhibitors with potent analgesic efficacy in a model of NP as well as an excellent CNS safety margin.

Results and Discussion

Human in Vitro Profile

The in vitro evaluation of the compounds was performed utilizing the IonWorks Quattro automated electrophysiology platform. The inhibitory activity of hNa_V1.7 was evaluated at a half-inactivated state (V_hold −59 mV). In addition, the inhibitory potency of hNa_V1.7 was evaluated at an elevated membrane voltage (V_hold −30 mV), because an elevated Na_V1.7 membrane voltage is reported in damaged dorsal root ganglion (DRG) neurons.^15–18) This screening process enabled us to provide an analgesic agent with high safety index owing to the ability to inhibit only abnormal hyperactive neurons with frequent action potentials without affecting normal firing activity. In this assay condition, we confirmed the inhibitory activity of the antiepileptic drug lacosamide, which is known to enhance slow inactivation of voltage-gated sodium channels selectively.¹⁹⁾ In the in vitro screening, hNa_V1.1 and hNa_V1.5 inhibitory activities were acquired to monitor adverse effects.

We initially focused on the conversion of the benzazepinone moiety, as shown in Table 1. Compound III showed good inhibitory potency against hNa_V1.7 at V_hold −30 mV (IC₅₀ = 2.5 µM) without hNa_V1.1 or hNa_V1.5 liability. Compound III was found to inhibit hNa_V1.7 in a high state-dependent manner, because III did not inhibit hNa_V1.7 at a half-inactivated state. As the removal of the trifluoromethyl group or the reduction of ring size (benzazepinone to dihydroquinolinone) did not affect hNa_V1.7 potency (data not shown), tetrahydroquinoline 1a was synthesized, which resulted in the retention of hNa_V1.7 potency. Compound 1a was prepared as a diastereomeric mixture (1 : 1). These results encouraged us to synthesize quinoline and isoquinoline derivatives. As expected, isoquinoline 1b and quinoline 1c exhibited potent hNa_V1.7 activity. Compounds 1b and 1c also showed no hNa_V1.1 or hNa_V1.5 liabilities. As other regioisomers of 1b and 1c did not show potent inhibitory activity (data not shown), alternative heteroaromatic rings were investigated. 2-Benzoazoles such as 1d, 1e and 1f retained hNa_V1.7 activity. In particular, 2-benzimidazole 1f exhibited the best inhibitory potency among this series of compounds with increased hNa_V1.1 or hNa_V1.5 potency. In contrast to quinolone derivatives, the regioisomer of 1d, 5-benzothiazole 1g retained hNa_V1.7 potency without hNa_V1.1 or hNa_V1.5 liabilities. Although the enhancement of hNa_V1.7 activity was expected by the introduction of a nitrogen in 1g, thiazolopyridine 1h showed attenuated hNa_V1.7 potency.

Table 1. In Vitro Profile of Na_V1.7 Inhibitors 1^a)

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) Values at V_hold −30 mV. d) Diastereomeric mixture (1 : 1). e) Values of a single experiment run in quadruplicate.

The replacement of acid labile Boc group of 1c was then investigated (Table 2). hNa_V1.7 activity was found to be correlated to the lipophilicity of molecules in the modification of t-butyl group. The inhibitory activity of isopropyl 2a and ethyl derivative 2b is 2.3 and 10 µM, respectively, whereas methyl analog 2c almost lost activity. Consequently, t-butyl group was optimal in this position. The introduction of methyl group in the urethane moiety led to the loss of potency in vitro, suggesting that a hydrogen bond may exist between this moiety and the ion channel (compound 2d). The importance of this hydrogen bond network was confirmed by amide derivatives, because β-alanine 2e and neopentyl amide 2g lost potency, whereas neopentyl amide 2f maintained the activity. As the hydrogen bond donor exists in this position, urea 2h maintained the activity. Compounds 2e and 2g were prepared as a racemic mixture. Reverse urethane 2i displayed decent hNa_V1.7 potency (IC₅₀ = 5.2 µM), and 2i did not exhibit hNa_V1.1 or hNa_V1.5 liabilities at concentrations up to 100 µM.

Table 2. In Vitro Profile of Na_V1.7 Inhibitors 2^a)

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_hold −30 mV. d) Racemic mixture. e) Values of a single experiment run in quadruplicate. f) Not tested.

Mice in Vitro Profile, in Vitro ADME Properties, and Pharmacokinetics (PK) Parameters in Mice

Potent compounds without hNa_V1.1 or hNa_V1.5 liabilities were evaluated for mouse Na_V activities, in vitro ADME properties and PK parameters in mice as shown in Table 3. To evaluate the state dependency, mNa_V1.7 activity at V_hold −30 mV was measured, and all selected compounds exhibited comparable mouse in vitro profiles to that of human.²⁰⁾ Compounds 1c, 1g, and 2i exerted high membrane permeability in Parallel Artificial Membrane Permeation Assay (PAMPA) and were expected to exhibit high plasma exposure in the PK study. Compound 1b lacked adequate aqueous solubility, whereas better solubility was observed in other compounds. Additionally, all evaluated compounds exhibited acceptable plasma protein binding (PB) ability (free fraction >1.0%). PK parameters were acquired by orally administering the compounds to mice at 30 mg/kg. Compounds 1b and 1c showed poor plasma exposure of the test compound, whereas a slight improvement of the PK profile was observed in reverse urethane 2i. Further improvement of the plasma exposure was observed in benzothiazole 1g. Considering the mouse in vitro activity, plasma PB ability, and PK parameters, 1g was expected to exhibit potent efficacy. In the PK evaluation, all evaluated compounds exhibited T_max less than 1 h, which would be expected immediate onset of pharmacological efficacy in vivo.

Table 3. Mouse Na_V IC₅₀ Values, in Vitro ADME Properties and PK Parameters

Compound		1b	1c	1g	2i
mNaV1.1 IC50 (µM)a,b)		18h)	29 ± 8.4	>100h)	>100h)
mNaV1.5 IC50 (µM)a,b)		43h)	43 ± 12	>100h)	>100h)
mNaV1.7 IC50 (µM)a,b)		>100h)	>100h)	>100h)	>100h)
mNaV1.7 IC50 (µM)a,c)		4.3h)	2.0 ± 0.24	4.8 ± 0.70	11.8 ± 2.2
PAMPA Papp (10−6 cm/s)d)		NTi)	>50	>50	>50
Solubility (µg/mL)e)		1.4	23	28	17
PB free (%)f)		2.54	2.72	3.04	1.04
PK parameters in ddY mice at 30 mg/kgg)	Cmax (µg/mL)	0.010 ± 0.0036	0.032 ± 0.0077	1.45 ± 0.090	0.10 ± 0.019
	Tmax (h)	0.67 ± 0.29	0.67 ± 0.29	0.50 ± 0.00	0.50 ± 0.00
	AUC0–24 h (h·µg/mL)	0.031 ± 0.020	0.051 ± 0.011	2.84 ± 0.24	0.20 ± 0.047
	T1/2 (h)	1.4 ± 0.47	1.1 ± 0.53	6.0 ± 2.4	6.8 ± 3.3
	Kp Brain	NTi)	1.65	NTi)	0.69

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_hold −30 mV. 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 30 mg/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.

To unveil the brain penetration of the compound, the Kp brain value of 1c and 2i was assessed. After 0.5 h of the compound administration (30 mg/kg, p.o.), the brain concentration of the compounds was determined (n = 2). Compound 2i indicated less exposure in the brain than in the plasma, while higher brain concentration of 1c was observed.

In Vivo Efficacy

The assessment of in vivo efficacy was conducted on thermal hyperalgesia in mice evoked by partial sciatic nerve ligation (PSL, Seltzer), a model of NP²¹⁾ (Fig. 2). The model was prepared by partial ligation of 1/2 to 1/3 of the left sciatic nerve with silk suture. The paw withdrawal latency (PWL, second) was assessed after 30, 60, 120, and 180 min of oral administration of the test compound (Plantar test). Compared with the normal group, the pain threshold was significantly lowered in the operated group, indicating the development of hyperalgesia. The time course of the efficacy of compound 2i administered to PSL mice is shown in Fig. 2a. The administration of 2i at 3 mg/kg p.o. (red curve) reversed the thermal PWL from 30 to 120 min. This reversal of hyperalgesia was maximized significantly from 30 to 60 min (p < 0.01 at 30 min, and p < 0.05 at 60 min). The peak efficacy improved PWL to almost normal levels. The therapeutic efficacy of 2i at 3 mg/kg was maximized from 30 to 60 min, whereas the plasma concentration of 2i at 30 mg/kg peaked at 30 min in the PK study. The thermal hyperalgesia was suppressed in a dose dependent manner (3, 10, and 30 mg/kg), and the administration of 2i at 30 mg/kg significantly reversed hyperalgesia at all timepoints evaluated (purple curve, p < 0.01 from 30 to 120 min, and p < 0.05 at 180 min). Potent analgesic efficacy lasted until 180 min when 10 or 30 mg/kg of 2i was administered. The retention of potent efficacy at 30 mg/kg administered group correlates with the PK parameters, because T_1/2 of 2i at 30 mg/kg was 6.8 h (Table 3). AUC_0–3 h (area under curve for 0–3 h) for each curve was calculated to analyze the total anti-hyperalgesic effect (Fig. 2b). The anti-hyperalgesic effect occurred in a clear dose dependent manner, and ED₅₀ of 2i was estimated to be 3.4 mg/kg. Compound 2i at 30 mg/kg significantly improved the total hyperalgesic effect to the normal levels (p < 0.01).

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) AUC_0–3 h of each curve in 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 significance 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.)

Isoquinoline 1b failed to show potent efficacy on thermal hyperalgesia at 30 mg/kg (data not shown), owing to less plasma exposure of 1b, as suggested by the PK study. The total anti-hyperalgesic effect of 1c and 1g is shown in Figs. 2c and 2d. Compound 1c suppressed thermal hyperalgesia in a dose-dependent manner (3, 10, 30 mg/kg), and ED₅₀ was estimated to be 23 mg/kg. Although a remarkable improvement in PK parameters was observed in 5-benzothiazole 1g, the efficacy of 1g in PSL mice was comparable to that of 1c, with ED₅₀ = 16 mg/kg. There was a difference observed in AUC_0–3 h of control groups (vehicle-treated and normal groups) in Figs. 2b, 2c, and 2d. AUC_0–3 h of control groups in Fig. 2d is higher than those in Figs. 2b and 2c. Consequently, the pain threshold of the study with 1g is higher than those with 2i and 1c. As ED₅₀ value is calculated defined PWL of vehicle-treated group as 0%, and PWL of normal mice as 100%, the inter-assay variation in pain threshold has no significant influence on ED₅₀.

Toxicological Evaluation

Compounds with potent analgesic efficacy (1c, 1g, and 2i) were selected for CNS toxicological evaluation as indicated in Fig. 3. The in vivo CNS side effects were assessed using a mice rotarod test, a model of motor coordination at 30, 100, and 300 mg/kg (n = 10). TK parameters were acquired at 100 or 300 mg/kg from a satellite group (Table 4, n = 1 − 3). As the administration of compound 1g at 300 mg/kg was lethal (two mice died after 120 min), the results of rotarod test are shown in 30 and 100 mg/kg administered group (Fig. 2b). The administration of 1g at 30 or 100 mg/kg did not effect on rotarod activity in mice, and ID₅₀was calculated to be >100 mg/kg. Compared to anti-thermal hyperalgesia efficacy, plasma-exposure-based CNS safety margin of 1g was determined to be >8.6-fold (Table 4). On the contrary, two other compounds, 1c and 2i, demonstrated superior CNS safety properties. Although 300 mg/kg administration of compound 1c significantly affected the results of rotarod test at 30 min (p < 0.01), this side effect was attenuated at 120 min (ID₅₀ > 300 mg/kg). Consequently, the plasma exposure-based CNS safety margin of 1c was determined to be >100-fold. Furthermore, the administration of 2i at 300 mg/kg did not affect mice motor activity at all timepoints with ID₅₀ > 300 mg/kg. Consequently, the plasma exposure-based CNS safety margin of 2i was >600-fold.

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

Compound	TK parameters in ddY micea)				Thermal hyperalgesia ED50b) (mg/kg)	Rotarod ID50c) (mg/kg)	CNS safety margind)
	Dose (mg/kg)	Tmax (h)	Cmax (µg/mL)	AUCall (h·µg/mL)
1c	300e)	1.0e)	2.56e)	4.49e)	23	>300	>100
1g	100	0.50 ± 0.00f)	6.63 ± 0.87f)	15.2 ± 1.4f)	16	>100	>8.6
2i	300	1.0 (1.0, 1.0)g)	6.77 (7.65, 5.89)g)	15.0 (13.7, 16.3)g)	3.4	>300	>600

a) Average of ddY mice dosed at 100 or 300 mg/kg po in N,N-dimethylacetamide/Tween80/saline: 10/10/80 (n = 1–3). b) The ED₅₀ value was calculated from the suppression 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 C_max at rotarod ID₅₀ in TK study/calculated C_max at Plantar ED₅₀ in PK study. e) n = 1. f) n = 3. g) Individual data (n = 2).

With promising results, 2i was further evaluated. An in vitro assessment established that 2i did not inhibit other related ion channels, including human Na_V1.2, 1.3, 1.4, 1.6, or human ether-a-go-go related gene (hERG). Moreover, in vitro pharmacological activity of 2i on a total of 67 receptors, channels, and transporters was evaluated; 2i inhibited adenosine A1 and serotonin 5-HT2B by 50% at 10 µM.²²⁾ These data indicate that 2i is a selective Na_V1.7 inhibitor in vitro.

Conclusion

The discovery of quinoline amides as a novel class of state-dependent Na_V1.7 inhibitors has been described. The benzazepinone moiety of compound III was replaced with a heteroaromatic ring. Among a variety of heteroaromatic rings examined, quinoline ring was shown to be optimal regarding potency as well as Na_V1.1 or Na_V1.5 liabilities. The modification of acid-unstable Boc group led to the discovery of reverse urethane 2i (DS01171986). Compound 2i inhibited hNa_V1.7 at IC₅₀ = 5.2 µM in a state-dependent manner. Notably, 2i inhibited Na_V1.7 without affecting other receptors, channels, and transporters, including other Na_V subtypes and hERG. Compound 2i demonstrated potent analgesic efficacy in NP model with EC₅₀ = 3.4 mg/kg. A CNS adverse effect evaluation revealed that 2i possessed excellent CNS safety margin (>600 fold).

Experimental

General

Starting reagents were purchased from commercial suppliers and were used without further purification unless otherwise specified. Chromatographic elution was carried under continuous monitoring by TLC using silica gel 60F254 (Merck & Co., Inc., Germany) as the stationary phase; the mobile phase was the elution solvent used in column chromatography. A UV detector was used for detection. Silica gel SK-85 (230–400 mesh) or silica gel SK-34 (70–230 mesh), manufactured by Merck & Co., Inc., or Chromatorex NH (200–350 mesh), manufactured by Fuji Silysia Chemical Ltd. (Japan), was used as the column packing silica gel. ¹H-NMR spectra were obtained on Varian Unity 400- and 500-MHz spectrometers. Spectra were recorded in the indicated solvent at ambient temperature; chemical shifts are reported in ppm (δ) relative to the solvent peak. Resonance patterns are represented with the following notations: br (broad signal), s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). MS analysis was carried out by FAB, electron ionization (EI), or electrospray ionization (ESI). High resolution (HR)MS was carried out using an LC-MS system composed of a Waters Xevo Q-ToF MS and an Acquity UHPLC system. Elemental analyses were carried out on a Microcorder JM10 and a Dionex ICS-1500. The purity of compounds was confirmed as over 95% by diode array detector (DAD) signal area% performed on Agilent Infinity 1260 LCMS system. Conditions [column: Develosil Combi-RP-5 2.0 mmID × 50 mmL, gradient elution: 0.1% HCO₂H–H₂O/0.1% HCO₂H–MeCN = 98/2 to 0/100 (v/v), flow rate: 1.2 mL/min, UV detection: 254 nm, column temperature: 40°C, ionization: APCI/ESI].

Chemical Synthesis

Compounds 1a–h listed in Table 1 were synthesized by amidation of corresponding amine with N-Boc-D-phenylalanine under usual reaction conditions (Chart 1). The synthesis of thiazolopyridine 7 for 1h was accomplished by the annulation of chloropyridine 5 as a key step.²³⁾

Chart 1. Synthesis of Compounds 1a–h

Reagents and conditions: (a) WSC·HCl, HOBt, DIPEA, THF; (b) thioacetamide, 1,4-dioxane, 100°C, 27%; (c) Fe, HCl, EtOH, H₂O.

Chart 2 summaries the preparation of compounds 2a–i listed in Table 3. Compounds 2a–2c and 2f were synthesized from amine 8 derived from 1c by the reaction with carbonyl chlorides, whereas urea 2h was prepared from 8 reacted with tert-butyl isocyanate. N-Methyl urethane 2d was prepared by the condensation of 3-aminoquinoline with N-methyl-N-Boc-D-phenylalanine (structures not shown). The synthesis of β-alanine 2e was achieved from key intermediate 11, which was prepared from benzyl alcohol 10 and tert-butyl bromoacetate.²⁴⁾ Started from 15,²⁵⁾ neopentyl amide 2g was synthesized in a similar manner. Reverse urethane 2i was prepared from D-phenyllactic acid 18.

Chart 2. Synthesis of Compounds 2a–i

Reagents and conditions: (a) TFA, CH₂Cl₂; (b) RCOCl, Et₃N, CH₂Cl₂; (c) tert-butyl isocyanate, toluene, 77%; (d) BnOH, Et₃N, CH₂Cl₂, 91%; (e) tert-butyl bromoacetate, LDA, THF, −78°C, 56%; (f) tert-butylamine or neopentylamine, WSC·HCl, HOBt, CH₂Cl₂; (g) H₂, Pd/C, EtOH; (h) 3-aminoquinoline, WSC·HCl, HOBt, CH₂Cl₂; (i) tert-butyl isocyanate, Me₃SiCl, CH₂Cl₂; (j) 3-aminoquinoline, HATU, DMF, 45%.

General Procedure for Amidation (General Procedure A)

A solution of amine (3.47 mmol), carboxylic acid (3.47 mmol), N,N-diisopropylethylamine (DIPEA) (2.7 mL, 15.6 mmol), N-hydroxybenzotriazole (HOBt)·H₂O (0.84 g, 6.24 mmol) and WSC·HCl (1.20 g, 6.24 mmol) in tetrahydrofuran (THF) (23 mL) was stirred at room temperature overnight under N₂. Water was added to the reaction mixture, and the mixture was extracted with EtOAc several times. The combined organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel chromatography to obtain 1.

General Procedure for Amidation to Prepare 2 (General Procedure B)

A solution of amine 3 (70 mg, 0.24 mmol) and Et₃N (0.10 mL, 0.72 mmol) in CH₂Cl₂ (1.2 mL) was added chloroforamte or acid chloride (0.57 mmol) at room temperature under N₂. After being stirred at room temperature for several hours, the mixture was directly purified by silica gel chromatography to obtain 2a–2c or 2f.

General Procedure for Deprotection by Hydrogenolysis (General Procedure C)

A solution of benzyl ester (3.72 mmol) and Pd/C (10%, 300 mg) in EtOH (10 mL) was stirred at room temperature under H₂ for several hours. After the removal of the catalyst, the residue was concentrated to obtain carboxylic acid.

tert-Butyl[(2R)-1-oxo-3-phenyl-1-(1,2,3,4-tetrahydroquinolin-3-ylamino)propan-2-yl]carbamate (1a)

Prepared according to General Procedure A as a diastereomeric mixture. Yield: 99%. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₃H₃₀N₃O₃, 396.2293; found 396.2289.

tert-Butyl[(2R)-1-(isoquinolin-3-ylamino)-1-oxo-3-phenylpropan-2-yl]carbamate (1b)

Prepared according to General Procedure A. Yield: 63%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.42 (9H, s), 3.11–3.20 (1H, m), 3.27 (1H, dd, J = 6.3, 14.1 Hz), 4.64 (1H, s), 5.18 (1H, d, J = 7.0 Hz), 7.19–7.29 (5H, m), 7.48 (1H, t, J = 6.7 Hz), 7.64 (1H, t, J = 7.0 Hz), 7.83 (1H, d, J = 8.2 Hz), 7.87 (1H, d, J = 8.2 Hz), 8.58 (1H, s), 8.69 (1H, s), 8.94 (1H, s); MS (APCI/ESI) m/z: 392 [M + H]⁺; Anal. calcd for C₂₃H₂₅N₃O₃: C, 70.57; H, 6.44; N, 10.73. Found C, 70.49; H, 6.62; N, 10.91; [α]²⁵_D +6.85 (c = 0.974, CHCl₃).

tert-Butyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl]carbamate (1c)

Prepared according to General Procedure A. Yield: 45%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.43 (9H, s), 3.14 (1H, dd, J = 7.4, 14.1 Hz), 3.21 (1H, dd, J = 7.4, 14.1 Hz), 4.64 (1H, s), 5.39 (1H, d, J = 7.8 Hz), 7.21–7.30 (5H, m), 7.45 (1H, t, J = 7.2 Hz), 7.56 (1H, t, J = 7.8 Hz), 7.66 (1H, d, J = 7.4 Hz), 7.95 (1H, d, J = 8.6 Hz), 8.53 (1H, d, J = 2.3 Hz), 8.58 (1H, s), 8.78 (1H, s); MS (APCI/ESI) m/z: 392 [M + H]⁺; Anal. calcd for C₂₃H₂₅N₃O₃: C, 70.57; H, 6.44; N, 10.73. Found C, 70.40; H, 6.61; N, 10.75; [α]²⁵_D +5.92 (c = 0.980, CHCl₃).

N-1,3-Benzothiazol-2-yl-Nα-(tert-butoxycarbonyl)-D-phenylalaninamide (1d)

Prepared according to General Procedure A. Yield: 90%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.40 (9H, s), 3.08–3.16 (1H, m), 3.22 (1H, dd, J = 6.0, 14.4 Hz), 4.74 (1H, s), 5.19 (1H, s), 7.07–7.13 (2H, m), 7.18–7.25 (3H, m), 7.32 (1H, t, J = 6.8 Hz), 7.43 (1H, t, J = 8.4 Hz), 7.76 (1H, d, J = 8.0 Hz), 7.83 (1H, d, J = 8.0 Hz); MS (APCI/ESI) m/z: 398 [M + H]⁺; Anal. calcd for C₂₁H₂₃N₃O₃S·0.20H₂O: C, 62.89; H, 5.88; N, 10.48. Found C, 62.84; H, 5.83; N, 10.56; [α]²⁵_D +59.9 (c = 1.007, CHCl₃).

N-1,3-Benzoxazol-2-yl-Nα-(tert-butoxycarbonyl)-D-phenylalaninamide (1e)

Prepared according to General Procedure A. Yield: 50%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.41 (9H, s), 3.13 (1H, s), 3.28 (1H, dd, J = 5.9, 14.1 Hz), 4.69 (1H, s), 5.14 (1H, s), 7.20–7.32 (8H, m), 7.45 (1H, d, J = 8.2 Hz); MS (APCI/ESI) m/z: 382 [M + H]⁺; Anal. calcd for C₂₁H₂₃N₃O₄·0.10H₂O: C, 65.82; H, 6.10; N, 10.96. Found C, 65.69; H, 6.19; N, 10.82; [α]²⁵_D +14.0 (c = 1.007, CHCl₃).

Nα-(tert-Butoxycarbonyl)-N-(1-methyl-1H-benzimidazol-2-yl)-D-phenylalaninamide (1f)

Prepared according to General Procedure A. Yield: 30%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.45 (9H, s), 3.25 (2H, t, J = 5.3 Hz), 3.61 (3H, s), 4.62–4.67 (1H, m), 5.43 (1H, d, J = 7.0 Hz), 7.17–7.31 (9H, m); MS (APCI/ESI) m/z: 395 [M + H]⁺; Anal. calcd for C₂₂H₂₆N₄O₃·0.10H₂O: C, 66.68; H, 6.66; N, 14.14. Found C, 66.56; H, 6.58; N, 14.16; [α]²⁵_D +73.9 (c = 0.740, CHCl₃).

Nα-(tert-Butoxycarbonyl)-N-(2-methyl-1,3-benzothiazol-5-yl)-D-phenylalaninamide (1g)

Prepared according to General Procedure A. Yield: 88%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.44 (9H, s), 2.82 (3H, s), 3.12–3.20 (2H, m), 4.46 (1H, br), 5.14 (1H, br), 7.26–7.34 (5H, m), 7.45 (1H, dd, J = 2.0, 8.6 Hz), 7.71 (1H, d, J = 8.6 Hz), 7.80 (1H, br), 7.91 (1H, d, J = 2.0 Hz); HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₂H₂₆N₃O₃S, 412.1690; found 412.1709; Anal. calcd for C₂₂H₂₅N₃O₃S: C, 64.21; H, 6.12; N, 10.21. Found C,63.92; H, 6.36; N, 10.13; [α]²⁵_D +4.23 (c = 1.003, CHCl₃).

Nα-(tert-Butoxycarbonyl)-N-(2-methyl[1,3]thiazolo[5,4-b]pyridin-6-yl)-D-phenylalaninamide (1h)

Prepared according to General Procedure A. Yield: 46% (2 steps). ¹H-NMR (400 MHz, CDCl₃) δ: 1.45 (9H, s), 2.85 (3H, s), 3.13–3.25 (2H, m), 4.46–4.54 (1H, m), 5.13 (1H, br), 7.26–7.36 (5H, m), 7.99 (1H, br), 8.39 (1H, d, J = 2.3 Hz), 8.44 (1H, d, J = 2.0 Hz); HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₁H₂₅N₄O₃S, 413.1642; found 413.1648; [α]²⁵_D +4.4 (c = 0.302, CHCl₃).

Propan-2-yl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl]carbamate (2a)

Prepared as a mixture of conformers according to General Procedure B utilizing isopropyl chloroformate as an acylating reagent. Yield: 46%. HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₂H₂₄N₃O₃, 378.1823; found 378.1810; [α]²⁵_D +5.9 (c = 0.304, CHCl₃).

Ethyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl]carbamate (2b)

Prepared as a mixture of conformers according to General Procedure B utilizing ethyl chloroformate as an acylating reagent. Yield: 80%. MS (APCI/ESI) m/z: 364 [M + H]⁺; Anal. calcd for C₂₁H₂₁N₃O₃·0.50H₂O: C, 67.73; H, 5.95; N, 11.28. Found C, 67.77; H, 5.79; N, 11.26; [α]²⁵_D +2.6 (c = 1.002, CHCl₃).

Methyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl]carbamate (2c)

Prepared according to General Procedure B utilizing methyl chloroformate as an acylating reagent. Yield: 96%. ¹H-NMR (400 MHz, CDCl₃) δ: 3.16 (1H, dd, J = 7.4, 13.7 Hz), 3.26 (1H, dd, J = 6.7, 13.7 Hz), 3.73 (3H, s), 4.56–4.62 (1H, m), 5.37 (1H, s), 7.25–7.35 (5H, m), 7.52–7.55 (1H, m), 7.61–7.66 (1H, m), 7.79 (1H, d, J = 8.2 Hz), 8.02 (1H, d, J = 8.6 Hz), 8.51 (1H, d, J = 2.7 Hz), 8.64 (1H, d, J = 2.3 Hz); HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₀H₂₀N₃O₃, 350.1510; found 350.1504; [α]²⁵_D +6.2 (c = 0.306, CHCl₃).

tert-Butyl Methyl[(2R)-1-oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl]carbamate (2d)

Prepared according to General Procedure A utilizing N-(tert-butoxycarbonyl)-N-methyl-D-phenylalanine as a starting material. Yield: 99%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.44 (9H, s), 2.85 (3H, s), 3.09–3.16 (1H, m), 3.39–3.46 (1H, m), 5.01–5.06 (1H, m), 7.24–7.34 (5H, m), 7.53 (1H, t, J = 7.0 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.79 (1H, d, J = 8.2 Hz), 8.03 (1H, d, J = 8.2 Hz), 8.67 (1H, s), 8.73 (1H, s), 8.91 (1H, s); MS (APCI/ESI) m/z: 406 [M + H]⁺; Anal. calcd for C₂₄H₂₇N₃O₃·0.40H₂O: C, 69.85; H, 6.79; N, 10.18. Found C, 69.92; H, 6.74; N, 10.33; [α]²⁵_D +64.7 (c = 1.001, CHCl₃).

2-Benzyl-N4-tert-butyl-N1-(quinolin-3-yl)butanediamide (2e)

Prepared as a mixture of conformers according to General Procedure A utilizing CH₂Cl₂ as a solvent. Yield: 69%. MS (APCI/ESI) m/z: 390 [M + H]⁺; Anal. calcd for C₂₄H₂₇N₃O₂·0.20H₂O: C, 73.33; H, 7.03; N, 10.69. Found C, 73.33; H, 7.01; N, 10.59.

Nα-(3,3-Dimethylbutanoyl)-N-quinolin-3-yl-D-phenylalaninamide (2f)

Prepared as a mixture of conformers according to General Procedure B utilizing 3,3-dimethylbutanoyl chloride as an acylating reagent. Yield: 88%. MS (APCI/ESI) m/z: 390 [M + H]⁺; Anal. calcd for C₂₄H₂₇N₃O₂·0.30H₂O: C, 73.00; H, 7.04; N, 10.64. Found C, 73.11; H, 7.01; N, 10.64; [α]²⁵_D +12.7 (c = 1.008, CHCl₃).

2-Benzyl-N-(2,2-dimethylpropyl)-N′-(quinolin-3-yl)propanediamide (2g)

Prepared according to General Procedure A utilizing CH₂Cl₂ as a solvent. Yield: 63%. ¹H-NMR (400 MHz, CDCl₃) δ: 0.85 (9H, s), 3.02 (1H, dd, J = 6.1, 13.1 Hz), 3.09 (1H, dd, J = 6.7, 13.3 Hz), 3.27 (1H, dd, J = 8.6, 13.7 Hz), 3.36 (1H, dd, J = 7.0, 13.7 Hz), 3.64 (1H, t, J = 7.6 Hz), 6.89 (1H, t, J = 5.9 Hz), 7.06–7.27 (5H, m), 7.53 (1H, t, J = 7.2 Hz), 7.63 (1H, t, J = 7.0 Hz), 7.77 (1H, d, J = 7.8 Hz), 8.04 (1H, d, J = 8.6 Hz), 8.64 (1H, d, J = 2.3 Hz), 8.76 (1H, d, J = 2.7 Hz), 10.08 (1H, s); MS (APCI/ESI) m/z: 390 [M + H]⁺; Anal. calcd for C₂₄H₂₇N₃O₂·0.20H₂O: C, 73.33; H, 7.03; N, 10.69. Found C, 73.29; H, 7.02; N, 10.66.

α-(tert-Butylcarbamoyl)-N-quinolin-3-yl-D-phenylalaninamide (2h)

A solution of amine 3 (52 mg, 0.18 mmol) and tert-butylisocyanate (40 µL, 0.36 mmol) in toluene (2.3 mL) was stirred at room temperature for 18 h under N₂. The mixture was directly purified by silica gel chromatography to obtain 2h (54.1 mg, 77%) as a mixture of conformers. MS (APCI/ESI) m/z: 391 [M + H]⁺; Anal. calcd for C₂₃H₂₆N₄O₂·0.20H₂O: C, 70.10; H, 6.75; N, 14.22. Found C, 69.95; H, 6.73; N, 14.19; [α]²⁵_D +6.1 (c = 1.010, CHCl₃).

(2R)-1-Oxo-3-phenyl-1-(quinolin-3-ylamino)propan-2-yl-tert-butylcarbamate (2i)

A solution of 3-aminoquinoline (7.21 g, 50.0 mmol), 11 (13.3 g, 50.0 mmol) and HATU (19.01 g, 50.0 mmol) in N,N-dimethylformamide (DMF) (100 mL) was stirred at room temperature for 18 h under N₂. Water was added to the reaction mixture, and the mixture was extracted several times with EtOAc. The combined organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc, 50 : 50) to obtain 2i (8.70 g, 45%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.33 (9H, s), 3.25 (1H, dd, J = 6.8, 14.2 Hz), 3.35 (1H, dd, J = 5.6, 13.9 Hz), 4.85 (1H, s), 5.49 (1H, t, J = 5.6 Hz), 7.23–7.30 (5H, m), 7.54 (1H, t, J = 7.6 Hz), 7.64 (1H, t, J = 7.6 Hz), 7.81 (1H, d, J = 7.8 Hz), 7.98 (1H, s), 8.03 (1H, d, J = 8.3 Hz), 8.55 (1H, d, J = 2.4 Hz), 8.72 (1H, d, J = 2.4 Hz); HRMS (ESI) m/z: [M + H]⁺ calcd for C₂₃H₂₆N₃O₃, 392.1980; found 392.1983; Anal. calcd for C₂₃H₂₅N₃O₃: C, 70.57; H, 6.44; N, 10.73. Found C, 70.61; H, 6.28; N, 10.72. [α]²⁵_D +76.8 (c = 1.001, CHCl₃).

2-Methyl-6-nitro[1,3]thiazolo[5,4-b]pyridine (6)²²⁾

A solution of 2-chloro-3,5-dinitropyridine (5, 1.00 g, 4.91 mmol) and thioacetamide (1.48 g, 19.7 mmol) in 1,4-dioxane (10 mL) was stirred at 100°C for 1 h. After the addition of water the mixture was extracted with EtOAc twice. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc, 80 : 20) to obtain 6 (0.26 g, 27%) as a colorless solid. ¹H-NMR (400 MHz, DMSO-d₆) δ: 2.93 (3H, s), 9.06 (1H, d, J = 2.7 Hz), 9.38 (1H, d, J = 2.7 Hz).

2-Methyl[1,3]thiazolo[5,4-b]pyridin-6-amine HCl (7)

A solution of 6 (0.26 g, 1.33 mmol), iron powder (1.49 g, 26.6 mmol) and 12 M HCl (70 µL) in water (2.7 mL) and EtOH (13 mL) was stirred at room temperature for 2 h. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to obtain crude 7, which was used directly for the next reaction without further purification.

N-Quinolin-3-yl-D-phenylalaninamide (8)

A solution of 1c (1.20 g, 3.07 mmol) and TFA (6.0 mL) in CH₂Cl₂ (30 mL) was stirred at room temperature for 3 h. After the addition of saturated aqueous solution of NaHCO₃ (pH = 10), the mixture was extracted with CH₂Cl₂ twice. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to obtain 8 (0.89 g, 99%). ¹H-NMR (400 MHz, CDCl₃) δ: 2.88 (1H, dd, J = 9.4, 14.1 Hz), 3.41 (1H, dd, J = 4.1, 13.9 Hz), 3.83 (1H, dd, J = 3.9, 9.4 Hz), 7.26–7.30 (3H, m), 7.34–7.37 (2H, m), 7.52–7.56 (1H, m), 7.61–7.66 (1H, m), 7.83 (1H, dd, J = 1.4, 8.0 Hz), 8.05 (1H, d, J = 8.2 Hz), 8.76 (1H, d, J = 2.3 Hz), 8.87 (1H, d, J = 2.3 Hz), 9.80 (1H, s).

Benzyl 3-Phenylpropanoate (10)

A solution of benzyl 3-phenylpropanoyl chloride (4, 3.0 g, 17.8 mmol), Et₃N (3.0 mL, 21.5 mmol) and BnOH (2.0 g, 18.5 mmol) in CH₂Cl₂ (40 mL) was stirred at room temperature for 2 h. Water was added to the reaction mixture, and the mixture was extracted with EtOAc twice. The combined organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc, 90 : 10) to obtain 10 (3.90 g, 91%). ¹H-NMR (400 MHz, CDCl₃) δ: 2.69 (2H, t, J = 7.4 Hz), 2.97 (2H, t, J = 7.8 Hz), 5.11 (2H, s), 7.17–7.37 (10H, m).

1-Benzyl 4-tert-Butyl 2-Benzylbutanedioate (11)

n-BuLi (1.57 mmol/L in THF solution, 12.5 mL, 19.6 mmol) was added to a solution of iPr₂NH (2.75 mL, 19.5 mmol) in THF (50 mL) at 0°C under N₂. After being stirred for 20 min at 0°C, THF (20 mL) solution of 10 (3.90 g, 16.2 mmol) was added to the reaction mixture at −78°C, and the mixture was stirred at −78 0°C for 30 min. THF (10 mL) solution of tert-butyl bromoacetate (4.40 g, 22.6 mmol) was added to the reaction mixture at −78°C, and the mixture was stirred at 0°C for 2 h. Saturated aqueous solution of NH₄Cl was added to the reaction mixture at 0°C, followed by extraction with EtOAc twice. The combined organic layers were washed with water, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc, 90 : 10) to obtain 11 (3.29 g, 56%) as a colorless oil. ¹H-NMR (400 MHz, CDCl₃) δ: 1.39 (9H, s), 2.35 (1H, dd, J = 5.1, 16.4 Hz), 2.60 (1H, dd, J = 8.6, 16.4 Hz), 2.75–2.82 (1H, m), 3.02 (1H, dd, J = 6.7, 13.7 Hz), 3.12–3.14 (1H, m), 5.06 (1H, d, J = 12.5 Hz), 5.11 (1H, d, J = 12.5 Hz), 7.03–7.37 (10H, m).

3-Benzyl-4-(benzyloxy)-4-oxobutanoic Acid (12)²³⁾

A solution of 11 (1.60 g, 4.51 mmol) and TFA (15 mL) in CH₂Cl₂ (15 mL) was stirred at room temperature for 18 h. After concentration of the reaction mixture, the mixture was purified by silica gel chromatography (CH₂Cl₂/MeOH, 90 : 10) to obtain 12 (970 mg, 72%) as a colorless oil. ¹H-NMR (400 MHz, CDCl₃) δ: 2.46 (1H, dd, J = 5.1, 17.2 Hz), 2.73 (1H, dd, J = 9.8, 17.2 Hz), 2.79 (1H, dd, J = 8.2, 13.7 Hz), 3.07 (1H, dd, J = 6.3, 13.7 Hz), 3.13–3.20 (1H, m), 5.10 (2H, s), 7.11–7.36 (10H, m).

Benzyl 2-Benzyl-4-(tert-butylamino)-4-oxobutanoate (13)

Prepared according to General Procedure A utilizing CH₂Cl₂ as a solvent. Yield: 85%. ¹H-NMR (500 MHz, CDCl₃) δ: 1.28 (9H, s), 2.20 (1H, dd, J = 4.9, 14.2 Hz), 2.40 (1H, dd, J = 9.0, 14.9 Hz), 2.83 (1H, dd, J = 7.3, 12.7 Hz), 2.99 (1H, dd, J = 6.8, 13.7 Hz), 3.23–3.29 (1H, m), 5.06 (1H, d, J = 12.2 Hz), 5.11 (1H, d, J = 12.7 Hz), 5.26 (1H, s), 7.11–7.36 (10H, m).

2-Benzyl-4-(tert-butylamino)-4-oxobutanoic Acid (14)

Prepared according to General Procedure C. Yield: 69%. ¹H-NMR (400 MHz, CDCl₃) δ: 1.33 (9H, s), 2.32–2.36 (2H, m), 2.77 (1H, dd, J = 9.2, 13.5 Hz), 3.09–3.23 (2H, m), 5.40 (1H, s), 7.18–7.33 (5H, m).

Benzyl 2-Benzyl-3-[(2,2-dimethylpropyl)amino]-3-oxopropanoate (16)

Prepared according to General Procedure A utilizing CH₂Cl₂ as a solvent. Yield: 89%. ¹H-NMR (400 MHz, CDCl₃) δ: 0.81 (9H, s), 2.97 (1H, dd, J = 5.9, 13.3 Hz), 3.06 (1H, dd, J = 6.7, 13.3 Hz), 3.19 (1H, dd, J = 8.6, 13.7 Hz), 3.29 (1H, dd, J = 6.3, 13.7 Hz), 3.56 (1H, dd, J = 6.3, 8.6 Hz), 5.07 (2H, s), 6.45 (1H, s), 7.10–7.37 (10H, m).

2-Benzyl-3-[(2,2-dimethylpropyl)amino]-3-oxopropanoic Acid (17)

Prepared according to General Procedure C. Yield: 99%. ¹H-NMR (400 MHz, CDCl₃) δ: 0.73 (9H, s), 2.83–2.94 (1H, m), 2.97–3.05 (1H, m), 3.12–3.20 (1H, m), 3.37–3.43 (2H, m), 5.56 (1H, s), 7.22–7.35 (5H, m).

(2R)-2-[(tert-Butylcarbamoyl)oxy]-3-phenylpropanoic Acid (20)

Me₃SiCl (15.8 mL, 125 mmol) was added to a solution of benzyl (2R)-2-hydroxy-3-phenylpropanoate (18, 14.50 g, 56.7 mmol) and tert-butylisocyanate (15.8 mL, 125 mmol) in CH₂Cl₂ (300 mL) at room temperature under N₂. After the reaction mixture was stirred at room temperature for 18 h, the mixture was concentrated to obtain crude 19. A solution of crude 19 and Pd/C (10%, 6.03 g) in EtOH (200 mL) was stirred at room temperature under H₂ for 2 h. After the removal of the catalyst, the residue was concentrated to obtain 20 (15.0 g, 99%, 2 steps) as a white powder. ¹H-NMR (500 MHz, CDCl₃) δ: 1.28 (9H, s), 3.07–3.13 (1H, m), 3.21 (1H, d, J = 15.1 Hz), 4.81 (1H, s), 5.22 (1H, s), 7.24–7.33 (5H, m).

High Throughput Electrophysiological Evaluation²⁶⁾

The IonWorks Quattro system (version 2.0, Molecular Devices) was used for electrophysiological recordings. A 384-well plate containing the compounds to be tested was placed in the plate-1 position. A PatchPlate was clamped into the PatchPlate station. Once the experiment was started, the fluidics-head (F-head) added 3.5 µL of Dulbecco’s Phosphate Buffered Saline (DPBS, with calcium and magnesium, Sigma-Aldrich Co. LLC) to each well of the PatchPlate and its underside was perfused with internal solution that had the following composition (in mM): 100.0 potassium D-gluconate, 40.0 KCl, 3.2 MgCl₂, 5.0 ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5.0 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.27 using 1 M KOH). After priming and debubbling, the electronics-head (E-head) moved around the PatchPlate to perform a whole test. The F-head then dispensed 3.5 µL of the cell suspension (hNa_V1.1/β1/β2/HEK293A, hNa_V1.5/β1/β2/HEK293A, hNa_V1.7/β1/β2/HEK293A, mNa_V1.1/β1/β2/HEK293A, mNa_V1.5/β1/β2/HEK293A, or mNa_V1.7/β1/β2/HEK293A) into each well of the PatchPlate, and the cells were given 400 s to reach and seal the hole in each well. After this, the E-head moved around the PatchPlate to determine the seal resistance obtained in each well. Then, the solution on the underside of the PatchPlate was changed to access solution that had the following composition (in mM): 100.0 potassium D-gluconate, 40.0 KCl, 3.2 MgCl₂, 5.0 EGTA, 5.0 HEPES (pH 7.27 using 1 M KOH) plus 100 µg/mL of amphotericin B (Sigma-Aldrich Co. LLC.). After 10 min of patch perforation was allowed to take place, the E-head moved around the PatchPlate to obtain Na_V current measurements.

After these pre-compound measurements, the F-head added 3.5 µL of solution from each well of the compound plate to each well on the PatchPlate. After about 5.5 min of incubation, the E-head moved around the PatchPlate to obtain post-compound Na_V current measurements. The pre- and post-compound Na_V current amplitude were measured from the peak current response subtracting the baseline current. The degree of Na_V current block was corrected by vehicle control currents as follows:

  

where, relative current (compound) is the value of the post-compound Na_V current amplitude divided by the respective pre-compound Na_V current amplitude and mean relative current (vehicle) is the mean value of the post-vehicle Na_V current amplitude divided by the pre-vehicle Na_V current amplitude.

Data was fitted with a four parameter logistic equation:

  

The voltage program for each Na_V subtype or species is summarized in Table 5.

Table 5. Voltage Program

	hNaV1.1a)	hNaV1.5a)	hNaV1.7a)	hNaV1.7b)	mNaV1.1a)	mNaV1.5a)	mNaV1.7a)	mNaV1.7b)
Holding pulse (for 5 s)	−100 mV	−120 mV	−100 mV	−100 mV	−100 mV	−120 mV	−100 mV	−100 mV
Second conditioning pulse (for 2 sa or 10 sb)	−43 mV	−68 mV	−59 mV	−30 mV	−44 mV	−69 mV	−56 mV	−30 mV
Holding pulse (for 50 ms)				−100 mV				−100 mV
Hyperpolarizing pulse (for 20 ms)				−140 mV				−140 mV
Test pulse (for analysis of inactivated-state channels, for 50 ms	0 mV	−20 mV	−10 mV	−10 mV	0 mV	−20 mV	−10 mV	−10 mV

a) Half-inactivated state. b) V_hold −30 mV.

PAMPA, Solubility, and Protein Binding Assay

Each assay was performed as previously described.^27),28)

Mouse PK Study

All experimental procedures were performed in accordance with the in-house guideline of the Institutional Animal Care and Use Committee of Daiichi 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-compartmental 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 Sankyo 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 180 min 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 20 s cut-off time was employed to avoid tissue damage. No animal reached the cutoff value. The measurement was conducted after the animal placed in the measuring cage became calm. The value indicated by the device was transcribed to the recording sheet, and this value was used as the pain threshold.

Rotarod Performance in Mice

All experimental procedures were performed in accordance with the in-house guideline of the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd. ddY Mice able to walk on the rotarod (20 rpm, 3 cm in diameter; model MK-660C; Muromachi Kikai Co., Ltd., Tokyo, Japan) for 2 min 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 120 s in two trials. The rotarod test was conducted at 0, 30, 60, and 120 min after administration.

Conflict of Interest

Kyosuke Tanaka, Hiroyuki Kobayashi, Sayaka Suzuki, Satoshi Shibuya, Yuki Domon, 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.

Supplementary Materials

The online version of this article contains supplementary materials.

1. Eurofins Lead Profiling Data

2. Time course of antihyperalgesic effect of compounds 1c and 1d

References and Notes

• 1) Albers J. W., Pop-Busui R., Curr. Neurol. Neurosci. Rep., 14, 473 (2014).
• 2) Dworkin R. H., O’Connor A. B., Backonja M., Farrar J. T., Finnerup N. B., Jensen T. S., Kalso E. A., Loeser J. D., Miaskowski C., Nurmikko T. J., Portenoy R. K., Rice A. S., Stacey B. R., Treede R. D., Turk D. C., Wallace M. S., Pain, 132, 237–251 (2007).
• 3) Cox J. J., Reimann F., Nicholas A. K., Thornton G., Roberts E., Springell K., Karbani G., Jafri H., Mannan J., Raashid Y., Al-Gazali L., Hamamy H., Valente E. M., Gorman S., Williams R., McHale D. P., Wood J. N., Gribble F. M., Woods C. G., Nature (London), 444, 894–898 (2006).
• 4) Minett M. S., Nassar M. A., Clark A. K., Passmore G., Dickenson A. H., Wang F., Malcangio M., Wood J. N., Nat. Commun., 3, 791 (2012).
• 5) Eijkelkamp N., Linley J. E., Baker M. D., Minett M. S., Cregg R., Werdehausen R., Rugiero F., Wood J. N., Brain, 135, 2585–2612 (2012).
• 6) Schmunk G., Gargus J. J., Front. Genet., 4, 222 (2013).
• 7) Catterall W. A., Goldin A. L., Waxman S. G., Pharmacol. Rev., 57, 397–409 (2005).
• 8) Bagal S. K., Chapman M. L., Marron B. E., Prime R., Storer R. I., Swain N. A., Bioorg. Med. Chem. Lett., 24, 3690–3699 (2014).
• 9) Nardi A., Damann N., Hertrampf T., Kless A., ChemMedChem, 7, 1712–1740 (2012).
• 10) Bagal S. K., Brown A. D., Cox P. J., Omoto K., Owen R. M., Pryde D. C., Sidders B., Skerratt S. E., Stevens E. B., Storer R. I., Swain N. A., J. Med. Chem., 56, 593–624 (2013).
• 11) Suzuki S., Kuroda T., Kimoto H., Domon Y., Kubota K., Kitano Y., Yokoyama T., Shimizugawa A., Sugita R., Koishi R., Asano D., Tamaki K., Shinozuka T., Kobayashi H., Bioorg. Med. Chem. Lett., 25, 5419–5423 (2015).
• 12) Hoyt S. B., London C., Gorin D., Wyvratt M. J., Fisher M. H., Abbadie C., Felix J. P., Garcia M. L., Li X., Lyons K. A., McGowan E., MacIntyre D. E., Martin W. J., Priest B. T., Ritter A., Smith M. M., Warren V. A., Williams B. S., Kaczorowski G. J., Parsons W. H., Bioorg. Med. Chem. Lett., 17, 4630–4634 (2007).
• 13) Williams B. S., Felix J. P., Priest B. T., Brochu R. M., Dai K., Hoyt S. B., London C., Tang Y. S., Duffy J. L., Parsons W. H., Kaczorowski G. J., Garcia M. L., Biochemistry, 46, 14693–14703 (2007).
• 14) Schenkel L. B., DiMauro E. F., Nguyen H. N., Chakka N., Du B., Foti R. S., Guzman-Perez A., Jarosh M., La D. S., Ligutti J., Milgram B. C., Moyer B. D., Peterson E. A., Roberts J., Yu V. L., Weisset M. M., Bioorg. Med. Chem. Lett., 27, 3817–3824 (2017).
• 15) Bedi S. S., Yang Q., Crook R. J., Du J., Wu Z., Fishman H. M., Grill R. J., Carlton S. M., Walters E. T., J. Neurosci., 30, 14870–14882 (2010).
• 16) Ma C., LaMotte R. H. J., Neurosci., 27, 14059–14068 (2007).
• 17) Sapunar D., Ljubkovic M., Lirk P., McCallum J. B., Hogan Q. H., Anesthesiology, 103, 360–376 (2005).
• 18) Zhang X.-F., Zhu C. Z., Thimmapaya R., Choi W. S., Honore P., Scott V. E., Kroeger P. E., Sullivan J. P., Faltynek C. R., Gopalakrishnan M., Shieh C. C., Brain Res., 1009, 147–158 (2004).
• 19) Rogawski M. A., Tofighy A., White H. S., Matagne A., Wolff C., Epilepsy Res., 110, 189–205 (2015).
• 20) Compound 2i inhibited rat Na_V1.7 at IC₅₀ = 47.9 μM at V_hold −30 mV.
• 21) Seltzer Z., Dubnerb R., Shirc Y., Pain, 43, 205–218 (1990).
• 22) Eurofin’s Pan Labs lead profiling screen. This battery of radioligand binding assays consists of 67 common GPCRs, ion channels, and transporters, see Supporting Information for complete results.
• 23) Sahasrabudhe K. P., Estiarte M. A., Tan D., Zipfel S., Cox M., O’Mahony D. J. R., Edwards W. T., Duncton M. A. J., J. Heterocycl. Chem., 46, 1125–1131 (2009).
• 24) Plattner J. J., Marcotte P. A., Kleinert H. D., Stein H. H., Greer J., Bolis G., Fung A. K. L., Bopp B. A., Luly J. R., Sharn H. L., Kempf D. J., Rosenberg S. H., Dellaria J. F., Merits I., Perun T. J., J. Med. Chem., 31, 2277–2288 (1988).
• 25) Chorev M., Rubini E., Gilon C., Wormser U., Selinger Z., J. Med. Chem., 26, 129–135 (1983).
• 26) Finkel A., Wittel A., Yang N., Handran S., Hughes J., Costantin J., J. Biomol. Screen., 11, 488–496 (2006).
• 27) Shinozuka T., Tsukada T., Fujii K., Tokumaru E., Matsui Y., Wakimoto S., Ogata T., Araki K., Sawamura R., Watanabe N., Mori M., Tanaka J., ACS Med. Chem. Lett., 10, 358–362 (2019).
• 28) Nagata T., Yoshino T., Haginoya N., Yoshikawa K., Nagamochi M., Kobayashi S., Komoriya S., Yokomizo A., Muto R., Yamaguchi M., Osanai K., Suzuki M., Kanno H., Bioorg. Med. Chem., 17, 1193–1206 (2009).