A highly selective kcnq4 potassium channel agonist, methods of making and uses thereof

Abstract

This invention provides a highly selective KCNQ4 potassium channel agonist according to Formula I, its preparation method, and its uses. The compound of this invention not only further enhances KCNQ4 agonist activity but also eliminates KCNQ2 agonist activity, maintaining excellent KCNQ4 / KCNQ2 selectivity. This novel selective KCNQ4 agonist overcomes the shortcomings of poor selectivity in existing potassium channel agonists, exhibiting improved activity, significantly reduced toxicity, and a simpler structure with lower production costs, thus possessing better development prospects.

Description

Technical Field

[0001] This invention relates to the field of agonist synthesis technology, and in particular to a novel class of highly selective KCNQ4 potassium channel agonists, their preparation methods, and the use of such potassium channel agonists in the preparation of drugs for treating smooth muscle or skeletal muscle-related diseases such as visceral pain, indigestion, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, prenatal labor pains, pruritus, sexual dysfunction, and deafness. Background Technology

[0002] Voltage-gated potassium channels (Kv channels) are the most widely discovered ion channel family, with 12 members (Kv1.X-Kv12.X). KCNQ channels are the 7th subfamily of Kv channels (Kv7), including five subtypes KCNQ1-KCNQ5. KCNQ1 is mainly distributed in the cardiac system, forming the IKs current in the myocardial action potential with the auxiliary subunit KCNQ1, and is responsible for myocardial action potential repolarization. KCNQ2-KCNQ5 are mainly distributed in the central and peripheral nervous systems, also known as neural KCNQ channels, which regulate membrane potential levels and nerve excitability (Brown et al., Br J Pharmacol. 2009, 156(8):1185-1195); KCNQ4 and KCNQ5 channels are also distributed in smooth muscle and skeletal muscle, regulating muscle contraction and relaxation by affecting membrane potential levels (Stott et al., Drug Discov Today. 2014, 19(4):413-424). Human genetic studies have shown that KCNQ gene mutations can lead to arrhythmias, epilepsy, and congenital deafness (Jentsch, Nat Rev Neurosci. 2000, 1(1): 21-30). KCNQ channels have a low current activation threshold (opening at a subthreshold potential of approximately -60 mV), slow activation, and no inactivation after activation, thus playing a fundamental role in the electrical excitability of excitable cells. Expression distribution, genetic, and pharmacological studies all support the use of non-selective KCNQ2-KCNQ5 channel agonists for the treatment, relief, or control of somatic pain, visceral pain, inflammatory pain, and neuropathic pain (Du et al., Br J Pharmacol. 2018, 175(12): 2158-2172). The non-selective KCNQ channel agonist flupirtine has been approved in Europe since 1984 for the treatment of acute or chronic pain (Szelenyi, Inflamm Res. 2013, 62(3): 251-258).

[0003] KCNQ4 channels exhibit specific high expression in various visceral organs and tissues, while being largely absent in the central nervous system. This specific tissue distribution provides a structural basis for targeted regulation of KCNQ4 channels to treat related diseases. Most visceral tissues tested show a trend of high expression of KCNQ4 subtypes. For example, analysis of KCNQ channel expression in the smooth muscle of various mouse blood vessels revealed that the expression levels of the five subtypes were: KCNQ4>KCNQ5>>KCNQ1, with KCNQ2 and KCNQ3 showing extremely low expression levels (Yeung et al., Br J Pharmacol. 2007, 151(6):758-770). In different parts of the gastrointestinal smooth muscle, including the colon, jejunum, antrum, and fundus, KCNQ4 expression was highest (Ipavec et al., Pharmacol Res. 2011, 64(4):397-409). KCNQ4 channels are abundantly expressed in the tracheal smooth muscle and uterine smooth muscle of rodents (Evseev et al., Front Physiol. 2013, 4:277). Simultaneously, studies on the distribution of KCNQ4 in human visceral tissues also show high expression of KCNQ4 (Ng et al., Br J Pharmacol. 2011, 162(1):42-53). Specific targeting of KCNQ4 can avoid the side effects of stimulating other subtypes, such as inhibiting KCNQ1 channels and their mediated IKs currents leading to long QT syndrome (Terrenoire et al., Circ Res. 2005, 96(5):e25-34), and can also effectively avoid the sedative and drowsiness side effects caused by enhancing central nervous system KCNQ2 and KCNQ3 channels (Orhan et al., Expert Opin Pharmacother. 2012, 13(12):1807-16). Existing KCNQ agonists generally suffer from poor selectivity, particularly for KCNQ2, KCNQ4, and KCNQ5 channels. All known KCNQ2 agonists also affect KCNQ4 channels, with only RL648_81 (ethyl(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate) enhancing only KCNQ2 channels without affecting KCNQ4 channels (Kumar et al., Mol Pharmacol. 2016, 89(6):667-677). Currently available KCNQ4 agonists are not ideally selective. Apart from fasudil, a vasodilator reported by Xuan Zhang et al. in November 2016, which exhibits a relatively selective agonistic effect on both KCNQ4 and KCNQ5 channels, no other selective KCNQ4 agonists have been reported (Zhang et al., Br J Pharmacol. 2016, 173(24):3480-3491).

[0004] Given the important role of the KCNQ4 channel in the nervous and smooth muscle systems, KCNQ4 channel agonists can be used to treat, but are not limited to, the following conditions: visceral pain, dyspepsia, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, prenatal labor pains, pruritus, sexual dysfunction, and deafness (Haick et al., Pharmacol Ther. 2016, 165:14-25; Barrese et al., Annu RevPharmacol Toxicol. 2018, 58:625-648).

[0005] The reported KCNQ potassium channel agonists mainly include the following:

[0006] 1) Patent US5384330 discloses some compounds having the following structures:

[0007]

[0008] Its structural feature is that it contains a benzene ring that is substituted with an ortho-diamine group.

[0009] 2) Patent WO2005 / 087754 describes a KCNQ potassium channel agonist with the following structure:

[0010]

[0011] Its structural features include a benzene ring substituted with a diamine group, one of which is in a saturated ring (or possibly a heterocycle, in which case W=O), while the other nitrogen group has an R group adjacent to it. 1 R 2 replace.

[0012] 3) Patent WO2008024398 describes the following structure:

[0013]

[0014] The structure of these compounds is similar to that of patent WO2005 / 087754, except that an additional benzo[a]benzene ring structural unit is introduced on the nitrogen heterocyclic hydrocarbon.

[0015] Currently, the most representative KCNQ potassium channel agonist in clinical practice is retigabine (RTG), an antiepileptic drug developed by GSK (GlaxoSmithKline) and launched in 2011. Its structure is as follows:

[0016]

[0017] Retigabine is the first systematically studied KCNQ potassium channel agonist that activates KCNQ2-5 and is primarily used to treat adult patients with partial seizure-type epilepsy.

[0018] Retigabine contains an electron-rich benzene ring substituted with three amino groups, a structural feature that makes it particularly susceptible to oxidation and deterioration during synthesis and storage. Meanwhile, retigabine has numerous adverse reactions in clinical applications, including dizziness, drowsiness, fatigue, confusion, tremor, poor coordination, diplopia, blurred vision, attention deficit, memory loss, motor incoordination, aphasia, dysarthria, balance disorders, increased appetite, hallucinations, myoclonus, peripheral edema, decreased motor function, dry mouth, and dysphagia. Urinary abnormalities are also common adverse reactions of retigabine, including bladder swelling, bladder wall thickening, and urinary retention. On April 26, 2013, the FDA’s Drug Safety Committee disclosed that retigabine may cause some pigmentary reactions during clinical use, including skin turning blue and retinal pigment changes, but the specific mechanism of action is still unclear. The committee also recommended that all patients taking this drug have regular eye examinations (S. Jankovic et al., Expert Opinion on Drug Discovery, 2013, 8(11), 1-9; F. Rode et al., European Journal of Pharmacology, 2010, 638, 121-127).

[0019] In their previous work (WO2013060097), the inventors disclosed a KCNQ potassium channel agonist with the following structure:

[0020]

[0021] Where R 3 When the compound is allyl or propargyl, it not only maintains KCNQ potassium channel agonist activity comparable to or better than retigabine, but also exhibits significant in vivo antiepileptic effects and protective efficacy comparable to retigabine; preliminary pharmacokinetic studies in mice show that this class of compounds has superior brain exposure compared to retigabine. However, further safety evaluation studies have found that the compound disclosed in WO2013060097 has significant neurotoxicity.

[0022] In their subsequent work (CN105017085A), the inventors disclosed a KCNQ potassium channel agonist with the following structure:

[0023]

[0024] Where R 4 R 5When the introduced substituent is an alkyl group represented by methyl (such as HN37), the resulting compound not only has stable physical properties and good absorption by brain tissue, but also exhibits significantly enhanced KCNQ2 agonist activity. However, with the increase in KCNQ2 agonist activity, the corresponding KCNQ4 agonist activity also increases accordingly.

[0025] The inventors of this invention, in summarizing previously reported work, found that existing KCNQ agonists generally suffer from poor selectivity, particularly for KCNQ2 and KCNQ4 channels. All known KCNQ2 agonists simultaneously affect KCNQ4 channels, with only RL648_81 (ethyl(2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate) enhancing only KCNQ2 channels without affecting KCNQ4 channels (Kumar et al., Mol Pharmacol. 2016; 89(6):667-77). Currently available KCNQ4 agonists are not ideally selective. Apart from fasudil, a vasodilator reported by Xuan Zhang et al. in November 2016, which exhibits a relatively selective agonistic effect on KCNQ4 channels, no other selective KCNQ4 agonists have been reported (Zhang et al., Br J Pharmacol. 2016; 173(24):3480-3491). Given the shortcomings of existing KCNQ potassium channel agonists in terms of poor selectivity, it is necessary to develop novel potassium channel agonists of KCNQ4 with better selectivity for the treatment of visceral pain, indigestion, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, prenatal labor pains, pruritus, sexual dysfunction, deafness, and other diseases. Summary of the Invention

[0026] One of the objectives of this invention is to provide a novel class of compounds that can serve as highly selective KCNQ4 potassium channel agonists.

[0027] A second objective of this invention is to provide a method for preparing the above-mentioned compound.

[0028] A third objective of this invention is to provide a class of pharmaceutical compositions comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient and optionally pharmaceutically acceptable excipients.

[0029] A fourth objective of this invention is to provide the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof or a composition containing the thereof in the preparation of a KCNQ4 potassium channel agonist.

[0030] The fifth objective of this invention is to provide the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof or a composition containing the thereof in the preparation of a medicament for treating diseases of smooth muscle or skeletal muscle.

[0031] In one aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof.

[0032]

[0033] in:

[0034] R 1 and R 2 Each is independently selected from hydrogen, halogen, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C1-C6 alkylthio, C4-C6 tertiary alkyl, halogenated C1-C6 alkyl, nitro, or cyano; R 1 and R 2 Preferably, each is independently selected from halogen, tert-butyl, nitro, cyano, or trifluoromethyl;

[0035] R 3 and R 4 Each is independently selected from hydrogen, halogen, and C1-C6 alkyl; R 3 and R 4 Ideally, each should be hydrogen;

[0036] R 5 Selected from C1-C6 alkyl, C1-C3 alkoxy, C4-C 14 tertiary alkoxy, phenoxy, benzyloxy; R 5 Preferably, they are C1-C3 alkoxy, C4-C6 tertiary alkoxy, phenoxy, or benzyloxy; R 5 Further preferred are methoxy, ethoxy, isopropoxy, tert-butoxy, phenoxy, and benzyloxy; R 5 The preferred option is tert-butoxy.

[0037] In some embodiments, the compound of formula I is selected from the compounds of formula II:

[0038]

[0039] Among them, R 1 R 2 R 3 R 4 and R 5 The definition is the same as above.

[0040] In some embodiments, the compound of formula I is selected from the compounds of formula III:

[0041]

[0042] Among them, R1 R 2 The definition is the same as above;

[0043] R 6 It is a C4-C6 tertiary alkyl group; R 6 The preferred formulation is tert-butyl.

[0044] In some preferred embodiments, the compound of formula I is selected from the compounds of formula c:

[0045]

[0046] Among them, R 1 R 2 The definition is the same as above.

[0047] In some preferred embodiments, the compound of formula I is selected from the following compounds:

[0048]

[0049]

[0050] The terms used in this invention are defined as follows:

[0051] The "halogen" can be fluorine, chlorine, bromine or iodine.

[0052] The term "C1-C6 alkyl" refers to a chain alkyl group having 1-6 carbon atoms; specific examples may include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, and similar groups.

[0053] The term "halogenated C1-C6 alkyl" refers to a C1-C6 heterochain alkyl group in which at least one hydrogen atom is substituted by a halogen; specific examples include trifluoromethyl, etc.

[0054] The term "C4-C6 tertiary alkyl" refers to an alkyl group with two branches having 4-6 carbon atoms; specific examples may include tert-butyl, tert-pentyl, etc.

[0055] The term "C1-C3 alkoxy" refers to the RO- group, where R is a C1-C14 alkyl group as described above. Specific examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0056] The term "C4-C6 tertiary alkoxy" refers to an alkoxy group with two branches and 4-6 carbon atoms; specific examples may include tertiary butoxy, tertiary pentoxy, etc. C4-C14 The definition of tertiary alkoxy groups follows the same principle.

[0057] Pharmaceutically acceptable salts of the compounds described in this invention may be salts formed by the above compounds and acids selected from maleic acid, succinic acid, citric acid, tartaric acid, fumaric acid, formic acid, acetic acid, propionic acid, malonic acid, oxalic acid, benzoic acid, phthalic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, camphoric acid, camphorsulfonic acid, salicylic acid, acetylsalicylic acid, aspartic acid, glutamic acid, lactic acid, gluconic acid, retinoic acid, gallic acid, mandelic acid, malic acid, sorbic acid, trifluoroacetic acid, taurine, homotaurine, 2-hydroxyethanesulfonic acid, cinnamic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and perchloric acid.

[0058] The compounds and their pharmaceutically acceptable salts involved in this application may have isomers or racemates, such as optical isomers (including diastereomers and enantiomers), transisomers, geometric isomers (cis-trans isomers), conformational isomers, tautomers, and mixtures thereof, but are not limited thereto. These isomers are also included within the scope defined by the claims of this invention.

[0059] In their previous work, the inventors of this invention discovered that introducing different substituents onto the benzene ring linked to the amino group, particularly when the introduced substituents were alkyl groups represented by two methyl groups (such as HN37, CN105017085A), could significantly enhance the agonist activity of KCNQ2. In this invention, the inventors further discovered that replacing the substituent on the terminal nitrogen atom with tert-butyl formate while retaining the overall HN37 skeleton could significantly enhance the agonist activity of KCNQ4. Further research revealed that when the substituent on the terminal nitrogen atom was kept as tert-butyl formate while all substituents on the benzene ring linked to the amino group were removed, the resulting compound K31 not only retained good KCNQ4 agonist activity but also lost its KCNQ2 agonist activity, meaning it exhibited excellent selectivity. Furthermore, different substituents were introduced onto the benzyl ring linked to the amino group. Particularly when the introduced substituent was an electron-rich substituent, represented by tert-butyl, it was found that the resulting compound not only further enhanced KCNQ4 agonist activity but also still lost its KCNQ2 agonist activity, maintaining excellent KCNQ4 / KCNQ2 selectivity. In summary, the novel selective KCNQ4 agonist provided by this invention overcomes the shortcomings of poor selectivity in existing potassium channel agonists, offering improved activity, significantly reduced toxicity, and advantages such as a simpler structure and lower production cost, thus demonstrating better development prospects.

[0060] Another aspect of the present invention provides a method for preparing the compound of formula c above, which is achieved through the following reaction route:

[0061]

[0062] In the preparation method of the present invention, R 1 and R 2 The definition is the same as above.

[0063] 1,4-Phenylenediamine reacts with di-tert-butyl dicarbonate to generate N-(tert-butyloxycarbonyl)-1,4-phenylenediamine; N-(tert-butyloxycarbonyl)-1,4-phenylenediamine undergoes a substitution reaction with bromopropyne to give intermediate a; intermediate a reacts with compound b to generate compound c.

[0064] In another aspect, the present invention provides a class of pharmaceutical compositions comprising, as an active ingredient, the compound of the present invention or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

[0065] Another aspect of the invention provides the use of the compound according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention for the preparation of a KCNQ4 potassium channel agonist.

[0066] Another aspect of the present invention is to provide the use of the compounds described herein or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing any of them, in the preparation of medicaments for treating diseases of smooth muscle or skeletal muscle.

[0067] Another aspect of the present invention provides a method for treating neurological diseases, wherein a subject suffering from a disease related to smooth muscle or skeletal muscle is administered a compound according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the invention.

[0068] The smooth muscle or skeletal muscle-related diseases include those related to visceral pain, indigestion, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, prenatal labor pains, pruritus, sexual dysfunction, and deafness.

[0069] The present invention has the following beneficial effects:

[0070] Compared with existing KCNQ agonists, the compounds provided in this invention exhibit significantly improved selectivity for KCNQ4 and KCNQ2. For example, in in vitro electrophysiological experiments, compounds K31-K43 all showed excellent selectivity for both KCNQ4 and KCNQ2, exhibiting only KCNQ4 agonist activity while having no effect on KCNO2.

[0071] Compared with the existing drug retigabine, the compounds provided by this invention have more stable physical properties and are less prone to oxidation and deterioration because they do not contain free amine groups in their structure. This is reflected in the fact that the solutions of these compounds are not easily oxidized and discolored even when exposed to air.

[0072] In summary, the compounds provided by this invention overcome the shortcomings of poor selectivity of existing agonists. They are not only physically stable and highly active, but also highly selective and have significantly reduced toxicity. Therefore, they have a larger therapeutic window and better therapeutic effects, showing promising application prospects.

[0073] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments. Detailed Implementation

[0074] The present invention will be further described below with reference to the embodiments. It should be noted that the following embodiments are provided for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0075] Unless otherwise specified, the raw materials, reagents, and methods used in the embodiments are all conventional raw materials, reagents, and methods in the art.

[0076] Preparation Examples of Compounds

[0077] In the following preparation examples, nuclear magnetic resonance (NMR) measurements were performed using a Bruker NMR Spectrometer 400M instrument manufactured by Bruker. NMR calibration: δH 7.26 ppm (CDCl3), 2.50 ppm (DMSO-d6), 3.15 ppm (CD3OD).

[0078] The reagents were mainly provided by Shanghai BIDE Pharmaceutical Technology Co., Ltd.

[0079] The thin-layer chromatography (TLC) silica gel plates were manufactured by Shandong Yantai Huiyou Silica Gel Development Co., Ltd., model HSGF254.

[0080] The silica gel used for normal-phase column chromatography in the compound purification was produced by the Qingdao Marine Chemical Plant Branch in Shandong Province, model ZCX-11, 200-300 mesh.

[0081] Preparation Example:

[0082] Preparation Example 1: Synthesis of tert-butyl 4-(N-p-fluorobenzyl-N-propargyl-amino)-phenylaminocarbamate (K31)

[0083]

[0084] Compound N-(tert-Butoxycarbonyl)-1,4-phenylenediamine K31-a (1.25 g, 6.0 mmol) was dissolved in DMF (20 mL) and diisopropylethylamine (1.16 g, 9.0 mmol), and bromopropyne (0.71 g, 6.0 mmol) was added. The mixture was heated to 65 °C and reacted for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and ethyl acetate (50 mL) was added. The organic layer was washed once with water (15 mL) and once with saturated brine (15 mL), dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5:1) to obtain product K31-b as a white solid (1.21 g, yield 82%), which was directly used in the next reaction.

[0085] The compound K31-b obtained in the previous step (73.89 mg, 0.3 mmol) was dissolved in DMF (3 mL) and diisopropylethylamine (0.1 mL, 0.6 mmol), and p-fluorobenzyl bromide (68.05 mg, 0.36 mmol) was added. The mixture was heated to 65 °C and reacted for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and ethyl acetate (20 mL) was added. The organic layer was washed once with water (5 mL) and once with saturated brine (5 mL). The mixture was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20:1) to obtain product K31 as a white solid (0.95 g, yield 90%). 1 H NMR (400MHz, CDCl3): δ7.30-7.27(m,2H),7.24-7.22(d,J=8.0Hz,2H),7.01(t,J=8.0Hz,2H),6.86- 6.84(d,J=8.0Hz,2H),6.30(dr,1H),4.43(s,2H),3.93(s,2H),2.22(t,J=4.0Hz,1H),1.59(s,9H).

[0086] The following compounds were prepared using a similar procedure to that used in Example 1:

[0087]

[0088]

[0089]

[0090] Electrophysiological Experiment Examples

[0091] Electrophysiological Experiment Example 1:

[0092] The cell line used in the electrophysiological experiments was the Chinese hamster ovary cell line (CHO-K1); KCNQ cDNA was expressed in E. coli after transformation, and then confirmed by plasmid extraction and sequencing.

[0093] 1. Cell Culture and Transfection

[0094] Chinese hamster ovary cells (CHO) (Chinese Academy of Sciences Cell Bank), culture medium formulation: 50 / 50 DMEM / F-12 (Gibco), supplemented with 10% fetal bovine serum (FBS) (Gibco, Australia) and 2 mM L-glutamine (Invitrogen). KCNQ channel transfection and expression: 24 hours before transfection, cells were digested with trypsin (Sigma, China) and seeded into 6-well plates. Transfection was performed using Lipofectamine 2000™ reagent (Invitrogen), following the provided procedures. 24 hours after transfection, cells were digested and re-seeded onto poly-L-lysine (Sigma)-soaked slides. GFP (green fluorescent protein) was co-transfected for confirmation of transfected cells under a fluorescence microscope (Olympus). The human KCNQ2 plasmid (NCBI: NM_172107.4) and human KCNQ4 plasmid (NCBI: NM_004700.4) used were synthesized by Beijing Liuhe Huada Co., Ltd., and constructed in the pcDNA3.1(+) vector plasmid. The KCNQ2 and KCNQ4 plasmids were confirmed by plasmid extraction and sequencing after transformation into DH5α E. coli.

[0095] 2. Electrophysiological recordings on CHO cells:

[0096] Whole-cell voltage-clamp experiments were conducted at room temperature (23–25°C) using an Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA), a Digidata 1440A digital-to-analog converter (Molecular Devices), and pClamp 10.0 software (Molecular Devices, Sunnyvale, CA) with a 2kHz filter, a 10kHz sampling frequency, and 60% series resistance compensation. Borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) were used to fabricate electrodes, with a resistance of 3–5 MΩ after filling the electrodes with intracellular fluid. A BPS-8 perfusion drug delivery system (ALA Scientific Instruments, Westburg, NY) was used, with a flow rate of approximately 1 mL / min.

[0097] Intracellular solution formulation for electrophysiological experiments: 145mM KCl, 1mM MgCl2, 5mM EGTA, 10mM HEPES, 5mM MgATP (adjusted to pH 7.3 with KOH).

[0098] Extracellular solution formulation for electrophysiological experiments: 140mM NaCl, 5mM KCl, 2mM CaCl2, 1.5mM MgCl2, 10mM MEPES, 10mM glucose (adjusted to pH 7.4 with NaOH).

[0099] KCNQ2 and KCNQ4 channel current evoked stimulation protocol: clamp voltage of -120mV, followed by a square wave stimulation of -10mV for 1500ms, with a stimulation frequency of 0.1Hz.

[0100] 3. Compound dissolution and preparation

[0101] Weigh a certain mass of the compound and dissolve it in DMSO to prepare a 20 mM DMSO stock solution, which is then stored at -20°C for later use. On the day of testing, the 20 mM stock solution is serially diluted with extracellular fluid to the final concentration required for detection, ensuring that the DMSO content in the test drug solution does not exceed 0.5%, as this concentration of DMSO has no effect on the detected KCNQ channel current. For example, to prepare 100 nM and 1 μM compound solutions, the serial dilution method is as follows: First, add 5 μL of the DMSO stock solution to 10 mL of extracellular fluid and dissolve thoroughly to obtain a 10 μM compound solution; then, add 1 mL of the 10 μM compound solution to 9 mL of extracellular fluid and dissolve thoroughly to obtain a 1 μM compound solution; finally, add 1 mL of the 1 μM compound solution to 9 mL of extracellular fluid and dissolve thoroughly to obtain a 100 nM compound solution.

[0102] 4. Experimental Results and Data Analysis

[0103] Data acquisition, analysis, and processing were performed using pClamp10 (Molecular Devices, Sunnyvale, CA), GraphPadPrism 5 (GraphPad Software, San Diego, CA), and Excel (Microsoft). All data are expressed as mean ± standard error (Mean ± SEM). Significance analysis was performed using an unpaired student's-test, with P < 0.05 considered statistically significant. The effect of the compound on the current was calculated using the following formula:

[0104] Enhancement factor = I Drug / IControl ;

[0105] Among them, I Control I represents the steady-state maximum value of the peak current generated by cells after administration of blank exogenous solution under a test voltage of -10mV. Drug It is the steady-state maximum value of the peak current generated after perfusion of the compound at a stimulation voltage of -10mV, I Drug / I Control >1 indicates an enhancing effect, I Drug / I Control <1 indicates an inhibitory effect, I Drug / I Control =1 indicates no effect. n is the number of cells detected.

[0106]

[0107]

[0108] Results and Discussion: The electrophysiological test results above show that the compounds disclosed in this invention not only maintain the agonistic activity of the KCNQ4 potassium channel well, but also all compounds are inactive against KCNQ2, showing good selectivity.

[0109] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein, without departing from the spirit and substance defined by the claims of the present invention; and such modifications or substitutions are still within the scope defined by the claims of the present invention.

Claims

1. A compound of formula III or a pharmaceutically acceptable salt thereof, in: R 1 and R 2 Each is independently selected from hydrogen, halogen, C4-C6 tertiary alkyl, halogenated C1-C6 alkyl, nitro or cyano; R 6 C4-C6-cycloalkyl, C1-C6-haloalkyl, C1-C6-alkoxy, C1 wherein: the compound of Formula III is other than .

2. The compound or pharmaceutically acceptable salt thereof of claim 1, wherein, R 1 and R 2 each independently is selected from halogen, tert-butyl, nitro, cyano or trifluoromethyl.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein, Compound III is compound c of the following formula: wherein R 1 , R 2 are defined as in claim 1.

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, Compounds of Formula III are selected from the following compounds:

5. A method for preparing the compound of formula c as described in claim 3, the method comprising the following steps: 1,4-Phenylenediamine reacts with di-tert-butyl dicarbonate to generate N-(tert-butyloxycarbonyl)-1,4-phenylenediamine; N-(tert-butyloxycarbonyl)-1,4-phenylenediamine undergoes a substitution reaction with bromopropyne to give intermediate a; intermediate a reacts with compound b to generate compound c. wherein R 1 , R 2 are defined as in claim 1.

6. A pharmaceutical composition comprising the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

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