Discovery of Novel Bicyclic Pyrazole Amides as Potent Neuronal K_V7 Potassium Channel Openers with Strong Preclinical Antiseizure Activity

Abstract

There has been considerable interest in developing openers of neuronal K_V7 potassium channels, particularly K_V7.2 (KCNQ2) and K_V7.3 (KCNQ3), as both genetic and clinical evidence strongly support their therapeutic potential in seizure disorders. Herein, we describe the discovery of a series of neuronal K_V7 potassium channel openers, optimized primarily via conformational rigidification and carbon to oxygen replacement. The combination of these structural modifications, together with careful investigation of the structure–activity relationship (SAR), culminated in the discovery of S-35, a potent and metabolically stable neuronal K_V7 opener that demonstrated favorable pharmacokinetics (PK) in preclinical species and robust antiseizure efficacy in rodent alternating-current maximal electroshock seizure (AC-MES) assays.

K_V7 channels (K_V7.1–K_V7.5) constitute a family of voltage-gated potassium channels encoded by the KCNQ1–KCNQ5 genes, that assemble as either homotetramers or heterotetramers, including K_V7.1, K_V7.4, K_V7.2/7.3, and K_V7.3/7.5. These channels have gained significant interest in drug discovery because of their critical roles in regulating cardiac excitability, neuronal signaling, and peripheral smooth muscle contractility. The cardiac K_V7.1 subunit is predominantly expressed in cardiomyocytes and mutations are associated with severe cardiac arrhythmias. , The four neuronal K_V7.2–7.5 subunits are largely expressed in the nervous system, with K_V7.4 and K_V7.5 subunits also being expressed in smooth and skeletal muscle tissues. , The heteromeric K_V7.2/7.3 channels represent the predominant form of K_V7.2-containing channel assemblies and generate a substantially larger neuronal M-current compared with either homomeric K_V7.2 or K_V7.3 channels. − The M-current is a noninactivating, hyperpolarizing potassium current that serves as a critical regulator of neuronal excitability by acting as a brake on hyperexcitability. Loss-of-function mutations in either KCNQ2 or KCNQ3, which reduce K_V7.2/7.3-mediated M-current, lead to neuronal hyperexcitability and are associated with a spectrum of seizure disorders (e.g., benign familial neonatal seizures and epileptic encephalopathy) in both humans − and mice. − Loss-of-function mutations in these channels are also related to various other neurological disorders including depression, − bipolar disorder, and amyotrophic lateral sclerosis. Moreover, dysfunction or downregulation of either K_V7.2 or K_V7.3 channel activity has been implicated in altered pain states. −

Conversely, enhancement of the K_V7.2-mediated M-current can promote membrane hyperpolarization, thereby decreasing neuronal excitability and suppressing rapid action potential firing (i.e., burst firing). This stabilization of neuronal excitability underlies the therapeutic potential of K_V7 channels in seizure disorders, as shown clinically by the K_V7.2/7.3 channel opener azetukalner (1, Figure ). Azetukalner is a K_V7.2/7.3 channel opener that has demonstrated significant antiseizure efficacy in clinical studies and is currently being evaluated in phase 3 clinical trials across multiple neurological and neuropsychiatric indications.

1.

Chemical structures of selected K_V7 openers.

Numerous compounds have been reported in the literature which target the neuronal K_V7 potassium channels, − several of which progressed to clinical development, for example, azetukalner (1), , opakalim (2) and PF-04895162 (3) (Figure ). Despite these advances, we sought to identify structurally distinct compounds that are novel and potent K_V7.2/7.3 channel openers. Here, we describe our efforts toward the discovery of compound S-35 (Table , presented later in this work), a potent neuronal K_V7 potassium channel opener exhibiting favorable pharmacokinetics (PK) in preclinical species and robust antiseizure efficacy in rodent alternating current maximal electroshock seizure (AC-MES) assays.

7. SAR in Discovery of Compound S-35 .

compd	R	KV7.2/7.3 EC50 (μM)	E max (%)	H/R LM Clhep , (mL/min/kg)	P app AB (× 10–6 cm/s)/ER ,	mLogD ,
32	Me	0.15 (0.067–0.35, n = 4)	119 (115–125, n = 4)	11/31	5.9/1.5	3.80
33	Et	0.10 (0.053–0.12, n = 5)	95 (89–103, n = 5)	16/38	nd	4.38
34	c-Pr	0.29 (0.074–0.92, n = 3)	94 (83–115, n = 3)	15/33	nd	4.70
rac-35	CF3	0.37 (0.27–0.49, n = 2)	112 (108–115, n = 2)	12/24	7.7/2.8	4.86
S-35	CF3	0.066 (0.026–0.14, n = 5)	92 (85–98, n = 5)	13/23	11/3.6	4.66
R-35	CF3	0.72 (0.32–1.58, n = 8)	93 (83–104, n = 8)	11/27	nd	4.57

^aAll compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95%, as determined by HPLC analysis.

^bTested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); Emax represents the maximal response of test compound relative to 5 μM of a reference compound 3.

^cData derived from each single test.

^dPredicted hepatic clearance from liver microsomes; H/R = human/rat.

^eDetermined using a MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability.

^fMeasured octanol/water distribution coefficient (Log D) at pH 7.4.

Pyrazole-based derivative 4 was identified as a screening hit from a targeted library-based approach and utilized as a starting point for the discovery of novel small molecule K_V7.2/7.3 potentiators. Compound 4 is a K_V7.2/7.3 channel opener with an EC₅₀ value of 0.57 μM but suffered from poor metabolic stability in both human (HLM) and rat liver microsomes (RLM) (Table ). Therefore, we aimed to increase potency and metabolic stability and utilized a conformational rigidification approach. We cyclized between the N-methyl group and the propyl side chain of the pyrazole core of 4 to rigidify the molecule. The fused, partially saturated compound 5 showed an EC₅₀ value of 0.096 μM and an E _max value of 118% on K_V7.2/7.3, representing a >5-fold boost in potency, compared to 4, along with a moderate improvement in metabolic stability. Assessing the permeability of 5 in a MDRI-MDCKII assay revealed high passive permeability and no active transport (P _app AB = 25 × 10^–6 cm/s, ER = 1.2). However, compound 5 showed moderate brain penetration with a brain/plasma ratio (C_b/p) of 0.3 in mouse snapshot PK studies (10 mg/kg, po). When administered intravenously (iv) to rats at a dose of 3 mg/kg, compound 5 exhibited moderate systemic plasma clearance (CL_p = 22 mL/min/kg), moderate volume of distribution (V _dss = 1.4 L/kg), and a short terminal half-life (T _1/2 = 0.7 h). Since metabolic stability can be associated with lipophilicity, we monitored mLogD to assess potential correlations. In this case, mLogD did not significantly influence either HLM or RLM clearance, suggesting that the observed improvement may result from reduced conformational flexibility. In addition, reducing the ring size by one methylene, homologue 6, resulted in markedly diminished activity (EC₅₀ = 1.74 μM), leading us to focus subsequent optimization efforts on compound 5.

1. SAR of Conformational Rigidification.

compd	KV7.2/7.3 EC50 (μM)	E max (%)	H/R LM Clhep , (mL/min/kg)	P app AB (× 10–6 cm/s)/ER ,	mLogD ,
3	0.96 (0.23–3.68, n = 1180)	80 (59–123, n = 1180)	NA	NA	NA
4	0.57 (0.37–0.76, n = 3)	141 (137–146, n = 3)	19/45	nd	3.77
5	0.096 (0.043–0.28, n = 9)	118 (99–132, n = 9)	16/37	25/1.2	3.74
6	1.74 (1.15–2.63, n = 2)	114 (112–116, n = 2)	14/29	nd	3.29

^aAll compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95%, as determined by HPLC analysis.

^bTested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); Emax represents the maximal response of test compound relative to 5 μM of a reference compound 3.

^cData were derived from each single test.

^dPredicted hepatic clearance from liver microsomes: H/R = human/rat.

^eDetermined using an MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability.

^fMeasured octanol/water distribution coefficient (log D) at pH 7.4.

^gNA = not available.

A detailed exploration of the structure–activity relationship (SAR) related to the bottom phenyl ring of 5 was undertaken (Table ). Replacement of the methyl group with a chlorine atom provided compound 7 with comparable potency relative to 5, while replacing the methyl group with a fluorine atom (8) or with a nitrile group (9) resulted in approximately 2- and 4-fold decreases in potency, respectively. Repositioning the fluorine from 4-position (see 5) to 3-position of the phenyl ring, as in compound 10, slightly improved potency and metabolic stability compared with 5. Subsequent replacement of the methyl group in 10 with a chlorine atom, yielding compound 11, further reduced HLM and RLM clearance (Cl_hep = 13 and 30 mL/min/kg, respectively). Compound 11 exhibited high passive permeability and no active transport (P _app AB = 15 × 10^–6 cm/s, ER = 2.2) with high brain penetration in mouse (C _b/p = 0.7, 10 mg/kg, po). Analogues with a monosubstituted aryl group, for example, trifluoromethoxy analogue (12) and trifluoromethyl analogue (13), showed comparable potency to compound 5. Replacing phenyl with a pyridyl ring, analogue 14 showed good HLM and RLM stability but exhibited a significant loss in activity (EC₅₀ = 3.29 μM), which may be due to a decrease in lipophilicity. In addition, we also explored the replacement of the phenyl ring with nonaromatic Csp³-rich components, for example, a 4,4-difluorocyclohexyl ring (15) or a 2,2,6,6-tetramethyl-4-tetrahydropyranyl ring (16). While both compounds showed improved metabolic stability, compound 15 was nearly 8-fold less active and compound 16 exhibited comparable activity (EC₅₀ = 0.11 μM) relative to compound 5. However, further characterization revealed that compound 16 had a high efflux ratio (ER = 12), as measured in the MDRI-MDCKII assay, resulting in low brain penetration in mice (C _b/p = 0.2, 3 mg/kg, po). Overall, this SAR data suggested that the 3-chloro-5-fluorophenyl (11) provided an advantageous balance of potency, metabolic stability, and brain penetration in the explored compounds.

2. SAR of Bottom Aryl Variation.

^a All compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95%, as determined by HPLC analysis. ^b Tested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); E _max represents the maximal response of test compound relative to 5 μM of a reference compound 3. ^c Data derived from each single test. ^d Predicted hepatic clearance from liver microsomes; H/R = human/rat. ^e Determined using a MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability. ^f Measured octanol/water distribution coefficient (Log D) at pH 7.4.

Concurrently, we replaced hydrogen with fluorine on the left-hand partially saturated ring of compound 5. We looked to explore whether this modification would enhance binding interactions, reduce local electron density, and/or protect a potential metabolic soft spot, thereby potentially improving potency and metabolic stability. For ease of access, this study began with the synthesis of difluoro analogues 17, 18, and 19 (Table ), wherein fluorine atoms were appended on C4, C5, and C6, respectively. This investigation identified the C5-difluoro analogue 18 as the most potent analogue in this series with an EC₅₀ value of 0.018 μM on K_V7.2/7.3. In addition, 18 achieved reasonable levels of HLM and RLM stability and exhibited high permeability and no active transport (P _app AB = 30 × 10^–6 cm/s, ER = 2.2) with significantly improved brain penetration in mouse (C _b/p = 0.5, 3 mg/kg, po) compared to 5. In comparison, the C4-difluoro analog 17 and the C6-difluoro analog 19 were less potent (7- and 5-fold, respectively) and less metabolically stable than 5. Compound 18 provides an example of how fluorination can have a positive effect on potency, metabolism, and brain penetration.

3. Effects of Fluorine Substituent Patterns on the Saturated Ring of the Core.

compd	KV7.2/7.3 EC50 (μM)	E max (%)	H/R LM Clhep , (mL/min/kg)	Papp AB (x 10–6 cm/s)/ER ,	mLogD ,
17	0.73 (0.61–0.86, n = 2)	135 (125–145, n = 2)	18/43	nd	3.83
18	0.018 (0.009–0.039, n = 9)	99 (85–123, n = 9)	14/34	30/2.2	3.64
19	0.53 (0.32–0.71, n = 7)	89 (82–95, n = 7)	16/36	24/2.2	3.47

^aAll compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95% as determined by HPLC analysis.

^bTested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); Emax represents the maximal response of test compound relative to 5 μM of a reference compound 3.

^cData derived from each single test.

^dPredicted hepatic clearance from liver microsomes; H/R = human/rat.

^eDetermined using a MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability.

^fMeasured octanol/water distribution coefficient (Log D) at pH 7.4.

Next, replacement of the 3-fluoro-4-methylphenyl with a 3-chloro-5-fluorophenyl group yielded analogue 20 (Table ), which displayed improved metabolic stability and comparable potency (EC₅₀ = 0.026 μM) relative to compound 18. The favorable potency and metabolic stability demonstrated by 20 prompted a more detailed pharmacological and metabolic characterization. Compound 20 exhibited good permeability and no active transport (P _app AB = 9.7 × 10^–6 cm/s, ER = 2.2 and 0.8 for P-gp and mBCRP, respectively). When administrated intravenously to rats (0.3 mg/kg), compound 20 demonstrated CL_p of 19 mL/min/kg, V _dss of 4.1 L/kg, and a T _1/2 of 2.6 h. However, when dosed orally, this compound exhibited flip-flop kinetics with a prolonged T _1/2 value of ∼38 h, indicating a markedly low absorption rate.

4. SAR of Fluorine Substituent Patterns on Saturated Core Ring System.

compd	X	KV7.2/7.3 EC50 (μM)	E max (%)	H/R LM Clhep , (mL/min/kg)	P app AB (× 10–6 cm/s)/ER,	mLogD ,
20	F	0.026 (0.018–0.032, n = 3)	94 (93–95, n = 3)	13/32	9.7/2.2	3.36
21E1	H	0.071 (0.061–0.095, n = 3)	91 (88–93, n = 3)	13/29	12/2.4	3.67
21E2	H	0.29 (0.28–0.30, n = 3)	96 (91–103, n = 3)	10/23	12/1.7	3.71
22E1	Me	0.48 (0.36–0.94, n = 5)	102 (96–115, n = 5)	16/28	nd	4.20
22E2	Me	0.059 (0.053–0.065, n = 2)	106 (105–108, n = 2)	14/39	11/3.3	4.06

^aAll compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95%, as determined by HPLC analysis.

^bTested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); E _max represents the maximal response of test compound relative to 5 μM of a reference compound 3.

^cData derived from each single test.

^dPredicted hepatic clearance from liver microsomes; H/R = human/rat.

^eDetermined using a MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability.

^fMeasured octanol/water distribution coefficient (LogD) at pH 7.4.

We postulated that the low absorption rate of compound 20 in rats might be attributed to its low aqueous solubility (<1.6 μM at pH 7.4). To address this, we removed one F atom from the molecule and prepared optically pure enantiomers 21E1 (first-eluting enantiomer; [α]_D ²⁰ = −10.79, c 0.81, λ = 589 nm, methanol) and 21E2 (second first-eluting enantiomer; [α]_D ²⁰ = +11.18, c 0.81, λ = 589 nm, methanol), the absolute configuration was not determined. Both compounds 21E1 and 21E2 were less potent than compound 20 (∼3- and 11-fold, respectively). Compound 21E1 did show improved aqueous solubility (35.5 μM at pH 7.4). In rats (0.3 mg/kg, iv), 21E1 showed CL_p of 17 mL/min/kg, V _dss of 2.3 L/kg, and a T _1/2 of 1.6 h. In addition, we replaced one F atom with a methyl group and prepared enantiomers 22E1 (first-eluting enantiomer; [α]_D ²⁰ = +14.40, c 0.87, λ = 589 nm, methanol) and 22E2 (second-eluting enantiomer; [α]_D ²⁰ = −14.89, c 0.83, λ 589 nm, methanol), the absolute configuration was not determined. However, both enantiomers were characterized to be less potent and more metabolically labile than 20 (Table ). These data suggested that 21E1–22E2 did not offer any significant advantage over 20.

Subsequent optimization efforts focused on replacing the carbon atom in the left-hand partially saturated ring of compound 5 with heteroatoms. Such modification could alter the molecular physiochemical properties, potentially leading to improved potency, metabolic stability, and PK properties. The results are summarized in Table . Replacement of C4 and C5 with oxygen atoms yielded compounds 23 and 24. Both compounds exhibited markedly improved metabolic stability compared to 5, correlating with lower Log D values. However, this was accompanied by a greater than 6- and 16-fold reduction in K_V7.2/7.3 activity, respectively. We next replaced C5 with nitrogen-containing fragments and prepared compounds 25–28. SAR analysis revealed that the N-ethyl analogue 27 exhibited K_V7.2/7.3 potency comparable to compound 5; however, it showed a substantial reduction in both HLM and RLM stability, despite its lower lipophilicity. Other, relatively less lipophilic analoguessuch as when this nitrogen was unsubstituted (25), substituted with a small methyl group (26), or converted to an acetamide (28), exhibited significantly reduced potency. Overall, increasing polarity of this region did not provide any observable benefits and resulted in reduced K_V7.2/7.3 activity.

5. Modification of the Saturated Ring of the Core.

compd	KV7.2/7.3 EC50 (μM)	E max (%)	H/R LM Clhep , (mL/min/kg)	Papp AB (× 10–6 cm/s)/ER ,	mLogD ,
23	0.64 (0.31–1.43, n = 12)	116 (92–143, n = 12)	11/25	18/1.9	3.17
24	1.60 (1.14–2.11, n = 3)	99 (96–102, n = 3)	12/30	20/1.5	2.87
25	16.9 (9.56–30, n = 2)	97 (97, n = 1)	11/31	nd	2.25
26	0.30 (0.21–0.45, n = 2)	144 (143–145, n = 2)	16/38	24/2.4	2.63
27	0.084 (0.034–0.14, n = 4)	135 (128–141, n = 4)	19/45	11/4.7	2.97
28	2.21 (2.02–2.42, n = 2)	105 (105–106, n = 2)	12/42	0.7/59	2.32

^aAll compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95%, as determined by HPLC analysis.

^bTested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); E _max represents the maximal response of test compound relative to 5 μM of a reference compound 3.

^cData derived from each single test.

^dPredicted hepatic clearance from liver microsomes; H/R = human/rat.

^eDetermined using a MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability.

^fMeasured octanol/water distribution coefficient (Log D) at pH 7.4.

Although these SAR trends did not improve the overall profile of the series, compound 23 displayed markedly low HLM and RLM clearance, with only a 6-fold decrease in potency, and we next investigated whether changes in the substituent position on the oxygen- containing ring could lead to enhanced biological activity. Preliminary SAR investigations, conducted on racemic material, evaluated the impact of a small methyl substituent at the C5-, C6-, and C7-positions of the left-hand partially saturated ring of 23, respectively (Table ). K_V7.2/7.3 potency was restored, compared to 23 upon installation of a methyl group at the C5-position (e.g., 29 was approximately 5-fold more potent than 23 with an EC₅₀ of 0.11 μM). In contrast, methylation at C6- and C7-positions was not well tolerated, as regio-isomers 30 and 31 were substantially less potent. In all cases, HLM and RLM stability decreased, which may be due to the increased lipophilicity.

6. Effects of Methyl Substituent Patterns on the Saturated Ring of the Core of 23 .

compd	KV7.2/7.3 EC50 (μM)	E max (%)	H/R LM Clhep , (mL/min/kg)	P app AB (× 10–6 cm/s)/ER ,	mLogD ,
29	0.11 (0.060–0.27, n = 5)	126 (120–129, n = 5)	15/37	8.1/3.1	3.41
30	1.01 (0.82–1.07, n = 4)	116 (114–124, n = 4)	14/37	16/1.4	3.54
31	0.86 (0.69–1.06, n = 3)	119 (113–123, n = 3)	15/38	nd	3.39

^aAll compounds exhibited spectral data consistent with their proposed structures and had analytical purities of ≥95%, as determined by HPLC analysis.

^bTested in a potassium influx assay; EC₅₀ and E _max data are reported as the geometric mean, with the range (minimum to maximum observed value) shown in parentheses, from n independent tests (n = number of replicates); Emax represents the maximal response of test compound relative to 5 μM of a reference compound 3.

^cData derived from each single test.

^dPredicted hepatic clearance from liver microsomes; H/R = human/rat.

^eDetermined using a MDRI-MDCKII assay; ER = efflux ratio calculated as the quotient of basolateral-to-apical (BA) permeability and apical-to-basolateral (AB) permeability.

^fMeasured octanol/water distribution coefficient (Log D) at pH 7.4.

Although the pronounced methyl effect on potency with compound 29 was encouraging, its increased HLM and RLM clearance was not satisfactory for the purposes of this exploration. Consistent with prior SAR in this series of compounds, replacing the 3-fluoro-4-methylphenyl with 3-chloro-5-fluorophenyl group yielded analogue 32, which showed improved metabolic stability and maintained K_V7.2/7.3 potency comparable to compound 29; however, its cellular permeability (P _app AB = 5.9 × 10^–6 cm/s) was only moderate. To address the limited permeability of 32, we replaced the methyl group with slightly more lipophilic substituents (Table ). While both the ethyl (33) and cyclopropyl (34) analogues showed high HLM and RLM clearance, the trifluoromethyl analogue rac-35 exhibited improved permeability, similar potency, and comparable metabolic stability relative to compound 32. This result prompted further evaluation of compound rac-35. Chiral separation of rac-35 by supercritical fluid chromatography (SFC) provided two optically pure enantiomers S-35 (first-eluting enantiomer; [α]_D ²⁰ = +35.20, c 0.87, λ = 589 nm, methanol) and R-35 (second-eluting enantiomer; [α]_D ²⁰ = −31.62, c 0.83, λ = 589 nm, methanol). The absolute configuration of S-35 was determined by small-molecule X-ray crystallographic analysis (CCDC Deposition No. 2577059). Compound S-35 was characterized as having an EC₅₀ value of 0.066 μM. In addition, S-35 was predicted to have good brain penetration, according to its permeability (P _app AB = 11 × 10^–6 cm/s) and transporter properties (ER = 3.6 and 2.6 for P-gp and mBCRP, respectively.).

The PK profiles of compound S-35 was evaluated in Sprague–Dawley rats, beagle dogs, and cynomolgus monkeys (Table ). Compound S-35 showed low to moderate systemic plasma clearance (CL_p), moderate volume of distribution (V _dss), and a resulting moderate terminal half-life (T _1/2) of 1.8–6.0 h across species. Its oral bioavailability was moderate, ranging from 23% to 47%.

8. PK Profiles of S-35 in Rats, Dogs, and Cynomolgus Monkeys.

	CLp (mL/min/kg)	V dss (L/kg)	AUC (h·ng/mL)	T 1/2 (h) iv/po	F (%)
rat	4.0	2.1	5677	6.0/8.0	47
dog	14	2.2	361	2.0/4.9	36
cyno	18	2.6	197	1.8/5.6	23

^aIntravenous dose 0.3 mg/kg, formulation: DMSO/polyethylene glycol 400 (PEG400)/50% 2-hydroxypropyl)-β-cyclodextrin (2-HP-β-CD) in deionized water (5/40/55, v/v/v).

^bOral dose 3 mg/kg (Sprague–Dawley rats) or 1.0 mg/kg (beagle dogs and cynomolgus monkeys), formulation: 5% DMSO, 0.5% w/w methyl cellulose (400 cP), 0.2% v/v Tween 80 in deionized water for rat and dog, or Capryol 90, Maisine CC (20:80, v/v) for cyno.

We evaluated compound S-35 for its selectivity on neuronal isoforms K_V7.3/7.5 and K_V7.4 using a potassium ion flux assay (see the Supporting Information). It was found that S-35 was not selective over neuronal K_V7.3/7.5 and K_V7.4 subtypes (EC₅₀ = 0.069 μM and 0.11 μM for K_V7.3/7.5 and K_V7.4, respectively). Consistent with these findings, voltage-clamp electrophysiology (EP) assays (see the Supporting Information) demonstrated potent activation of K_V7.2/7.3, K_V7.3/7.5, and K_V7.4 by S-35 with EP EC₅₀ (E _max) values of 0.062 μM (−56 mV), 0.14 μM (−49 mV), and 0.11 μM (−54 mV), respectively (see Figure S1A–S1D in the Supporting Information). The EP EC₅₀ values were approximately 6.5–17-fold lower than that of a reference K_V7 opener 3 [K_V7.2/7.3, K_V7.3/7.5, and K_V7.4 EC₅₀ (E _max) values of 1.05 μM (−38 mV), 2.38 μM (−40 mV), and 0.74 μM (−29 mV), respectively] (see Figure S2A–S2D in the Supporting Information). Importantly, S-35 showed high selectivity over cardiac KCNQ1/mink channels with neither agonistic nor antagonistic activity (EC₅₀ > 10 μM, IC₅₀ > 10 μM). Compound S-35 was further characterized to have high selectivity (IC₅₀ > 10 μM) against key cardiac ion channels (hNa_V1.5, hERG, hKir2.1, hK_V4.3/ChIP2, and hCa_V1.2) in a panel of ion channel electrophysiological assays. Overall, S-35 had high selectivity over cardiac ion channels, but low/no selectivity over neuronal K_V7.3/7.5 and K_V7.4 subtypes. In addition, S-35 did not present genotoxicity or mitochondrial toxicity liabilities, as evident by negative findings in both the Ames and glucose/galactose assays, respectively. Furthermore, no significant inhibition was observed against cytochrome P450 enzymes with IC₅₀ values >10 μM for CYP1A2, 2C9, 2C19, 2D6, and 3A4. Collectively, these data demonstrate that compound S-35 is a selective neuronal K_V7 opener that exhibits sufficient selectivity over the evaluated off-targets.

The favorable overall profile of compound S-35 prompted evaluation of its antiseizure activity in the alternating current maximal electroshock seizure (AC-MES) assay, a preclinical model of generalized tonic-clonic seizures. Compound S-35 produced dose-dependent protection against AC-MES-induced tonic hindlimb extension in mice 30 min after oral dosing (1, 3, 10, and 30 mg/kg), with all vehicle-treated animals (n = 8) exhibiting seizures (see Figure S3A in the Supporting Information). A 50% reduction in the fraction of animals exhibiting seizures was achieved with an estimated brain EC₅₀ of 0.60 μM (95% CI: 0.49–0.74 μM) (Figure A) and a plasma EC₅₀ of 0.97 μM (95% CI: 0.74–1.35 μM) (Figure B). The brain EC₅₀ was approximately 2-fold lower than that of a reference K_V7 opener 3 (brain EC₅₀ of 1.22 μM) (see Figure S4A–S4C in the Supporting Information). Antiseizure activity was further confirmed in rats (n = 8) evaluated 60 min after oral dosing (1, 3, 10, and 30 mg/kg), where increasing doses were associated with progressive protection against AC-MES–induced seizures (see Figure S3B in the Supporting Information), yielding an estimated brain EC₅₀ of 0.24 μM (95% CI: 0.14–0.33 μM) (Figure C) and a plasma EC₅₀ of 0.33 μM (95% CI: 0.18–0.49) (Figure D). Logistic regression revealed a significant association between exposure and protection from MES-induced seizures in both mice and rats (likelihood ratio test, p < 0.0001). Notably, S-35 was well-tolerated, as there were no clinical observations over the course of these studies.

2.

Exposure-response relationships for compound S-35 in the mouse and rat AC-MES assays (n = 8 animals per dose group). Data points represent the (A, C) mean brain or (B, D) plasma exposure for each dose group following oral dosing, with error bars indicating the standard errors of the mean, plotted against the fraction of animals exhibiting seizures. Solid lines represent logistic regression fits to individual animal binary seizure outcome data as a function of exposure. EC₅₀ values were estimated from the fitted models. The statistical significance of the exposure–response relationship was assessed using a likelihood ratio test (p < 0.0001).

To assess whether compound S-35 displayed relevant GABAergic activity, its effect on GABA_A receptors were evaluated using a human ion-channel cell-based PAM IonFlux assay. Compound S-35 exhibited EC₅₀ values of 3.3 μM at GABA_A α1β2γ2 and 3.5 μM at α1β3γ2 receptors. These potencies were approximately 50-fold weaker than its activity at K_V7.2/7.3 channels, suggesting that GABA_A receptor engagement is unlikely to meaningfully contribute to the antiseizure effects of S-35.

The synthesis of S-35 (Scheme ) began with commercially available chemical 36, which could be prepared from 4,4,4-trifluorobutane-1,3-diol in 4 steps. Treatment of 36 with NBS provided intermediate 37. Miyaura–Suzuki coupling of 37 with (3-chloro-5-fluorophenyl)­boronic acid afforded biaryl ester 38, which was hydrolyzed to give acid 39. Upon treatment with DPPA under thermal conditions, 39 was readily converted to the amine 40. Amide formation of 40 with 3,3-dimethylbutanoyl chloride under basic conditions led to rac-35. Subsequent chiral SFC separation of rac-35 afforded optically pure enantiomers S-35 (first-eluting enantiomer) and R-35 (second-eluting enantiomer). The absolute configuration of S-35 was determined by small-molecule X-ray crystallographic analysis (CCDC Deposition No. 2577059).

1. Preparation of S-35 .

^a Reagents and conditions: (a) NBS, MeCN, 50 °C, 1 h, 81%. (b) (3-chloro-5-fluorophenyl)­boronic acid, Pd­(dppf)­Cl₂, CsCO₃, dioxane, H₂O, 100 °C, 2 h, 55%. (c) LiOH, THF, H₂O, rt, 3h, 98%. (d) DPPA, t-BuOH, DIPEA, toluene 105 °C, 12 h; then HCl, 1,4-dioxane, rt, 4 h, 68%. (e) 3,3-dimethylbutanoyl chloride, pyridine, MeCN, 0 °C to rt, 12 h, 68%. (f) chiral SFC, 20% for S -35, 16% for R -35.

Herein, we have described the discovery and optimization of a series of neuronal K_V7 potassium channel openers. Conformational rigidification of 4 led to compound 5, which exhibited promising K_V7.2/7.3 activity but was limited by poor metabolic stability and a short terminal half-life in rats. Initial optimization of 5 focused on fluorine substitutions with an aim to improve potency and metabolic stability, resulting in the identification of compound 20, which displayed significantly enhanced potency and metabolic stability in both human and rat liver microsomes. However, compound 20 retained suboptimal PK properties in rats. To further address these PK liabilities of 20, we replaced carbon with oxygen in 5, to yield compound 23, which showed markedly improved metabolic stability. Subsequent SAR optimization led to the identification of compound S-35 with substantially improved PK properties. Compound S -35 potently activated K_V7.2/7.3 channels, exhibited minimal activity on GABA_A receptors, and showed no detectable activity against a panel of cardiac ion channels. Importantly, compound S-35 was well-tolerated and demonstrated robust antiseizure efficacy in rodent alternating current maximal electroshock seizure (AC-MES) models.

Safety Statement

No unexpected or unusually high safety hazards were encountered in the synthesis of the compounds described in this manuscript.

Use of Animal Subjects

All animal use protocols were approved either by the Animal Care Committee of Xenon Pharmaceuticals (Burnaby, BC, Canada) and conducted in accordance with the guidelines of the CCAC (Canadian Council on Animal Care), or by the Institutional Animal Care and Use Committee of WuXi AppTec Co., Ltd. (China) and conducted in accordance with the guidelines of AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International).

Glossary

ABBREVIATIONS

SAR

structure–activity relationship

AC-MES

alternating current maximal electroshock seizure

HLM

human liver microsomes

RLM

rat liver microsomes

mLogD

measured distribution coefficient at pH 7.4

MDCK

Madin Darby Canine Kidney cell line

MDR1

multidrug resistance protein 1, also refer to P-glycoprotein (P-gp)

mBCRP

mouse breast cancer resistance protein

ER

efflux ratio

CYP

cytochrome P450

hERG

human Ether-à-go-go-Related Gene

PK

pharmacokinetics

AUC

area under curve

CL_p

systemic plasma clearance

V _dss

volume of distribution

T _1/2

terminal half-life

PEG400

polyethylene glycol 400

2-HP-β-CD

2-hydroxypropyl)-β-cyclodextrin

NBS

N-bromosuccinimide

DPPA

diphenylphosphoryl azide

SFC

supercritical fluid chromatography

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.6c00360.

• Analytical data of compounds 5–34; experimental procedure of compounds rac-35, S-35 and R-35; X-ray crystallographic analysis report of S-35; K_V7.X potassium channel influx assays; automated voltage clamp electrophysiology assays, electrophysiological characterization of compound S-35 on neuronal K_V7 channel subtypes (Figure S1A–S1D) and electrophysiological characterization of reference compound 3 on neuronal K_V7 channel subtypes. (Figure S2A–S2D); alternating current maximal electroshock seizure (AC-MES) assays; dose-dependent effects of compound S-35 in mouse (Figure S3A) and rat (Figure S3B) AC-MES assays; dose-dependent (Figure S4A) and concentration-dependent (Figure S4B and 4C) effects of reference compound 3 in mouse AC-MES assays; cardiac ion-channel protocols and GABA_A IonFlux HT PAM assays; microsomal stability, Log D, MDR1 transporter assays, CYP inhibition assays; PK study protocols (PDF)

The manuscript was written through the contributions of all authors, including conceptualization, compound design and synthesis, investigation, methodology, data curation and analysis, validation, visualization, project administration, supervision. All authors have given approval to the final version of the manuscript.

This work was fully funded by Xenon Pharmaceuticals, Inc.

The authors declare the following competing financial interest(s): Shaoyi Sun, Qi Jia, Girish Bankar, Kristen N. Burford, Jessica Christabel, Helen A. Clement, Gina de Boer, Zirui Feng, Stephen K. Jackson, Kuldip Khakh, Rainbow Kwan, Stephanie Lee, Jenny Li, Andrea Lindgren, Janette Mezeyova, Art Urrutia, Paul Charifson, Michael P. Clark, Steven S. Wesolowski, James P. Johnson, Jr., James .R. Empfield, Christoph M. Dehnhardt, and Richard Dean are employees of Xenon Pharmaceuticals, Inc.; Chido M. Hambira is no longer an employee of Xenon Pharmaceuticals, Inc.; Juliette Sabbatini is no longer an employee of X-Chem Canada (service company). They may hold equity or stocks in Xenon Pharmaceuticals, Inc.