Highly selective KCNQ4 potassium channel agonists, methods for their preparation and use

Abstract

The present invention provides a highly selective KCNQ4 potassium channel agonist according to formula I, its preparation method and use.The compounds of the present invention not only further enhance KCNQ4 agonist activity, but also lose KCNQ2 agonist activity, and still have good KCNQ4 / KCNQ2 selectivity.The novel selective KCNQ4 agonist provided by the present invention overcomes the shortcomings of poor selectivity existing in traditional potassium channel agonists, improves activity, obviously reduces toxicity, and has the advantages of simpler structure and lower production cost, so it has better development prospects. TIFF2025500696000027.tif29135

Description

[Technical Field]

[0001] The present invention relates to the technical field of agonist synthesis, and in particular to a novel, highly selective KCNQ4 potassium channel agonist, a method for preparing the same, and the use of such potassium channel agonist in the manufacture of a medicament for treating related diseases of smooth or skeletal muscles, such as visceral pain, dyspepsia, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary arterial hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, pre-labor, pruritus, sexual dysfunction, and hearing loss. [Background technology]

[0002] Voltage-gated potassium ion channels (Kv channels) are the most abundant family of ion channels discovered to date, with 12 members (Kv1.X-Kv12.X). KCNQ channels are the seventh subfamily of Kv channels (Kv7) and include five isoforms, KCNQ1-KCNQ5. KCNQ1 is primarily distributed in the cardiac system and, together with its auxiliary subunit KCNQ1, forms the IKs current responsible for repolarization of the cardiac action potential. KCNQ2-KCNQ5 are primarily distributed in the central and peripheral nervous systems and are also called neuronal KCNQ channels, regulating membrane potential levels and neuronal excitability (Brown et al., Br J Pharmacol. 2009, 156(8):1185-1195). KCNQ4 and KCNQ5 channels are also distributed in smooth and skeletal muscles, where they regulate muscle contraction and relaxation by influencing membrane potential levels (Stott et al., Drug Discov Today. 2014, 19(4):413-424). Human genetic studies have shown that mutations in KCNQ genes can cause arrhythmias, epilepsy, and congenital hearing loss (Jentsch, Nat Rev Neurosci. 2000, 1(1):21-30). KCNQ channels play a fundamental role in the electrical excitability of excitable cells because they have a low threshold for current activation (opening at subthreshold potentials of ~60 mV), a slow activation rate, and no inactivation after activation. Expression distribution, genetic, and pharmacological studies all support the idea that potent nonselective KCNQ2-KCNQ5 channel agonists could be used as medicines to treat, alleviate, or control somatic, visceral, inflammatory, and neuropathic pain (Du et al., Br J Pharmacol. 2018, 175(12):2158-2172). Flupirtine, a nonselective KCNQ channel agonist, has been approved in Europe for the treatment of acute and chronic pain since 1984 (Szelenyi, Inflamm Res. 2013, 62(3):251-258).

[0003] KCNQ4 channels are specifically and highly expressed in various visceral organs and tissues, but are rarely expressed in the central nervous system. This specific tissue distribution provides a structural basis for targeted modulation of KCNQ4 channels and, ultimately, the treatment of related diseases. Most visceral tissues examined show a tendency for high expression of KCNQ4 isoforms. For example, analysis of KCNQ channel expression in various types of murine arterial smooth muscle revealed that the expression levels of the five isoforms were in the order KCNQ4 > KCNQ5 > KCNQ1, with KCNQ2 and KCNQ3 expression levels being significantly lower (Yeung et al., Br J Pharmacol. 2007, 151(6):758-770). KCNQ4 expression was highest in different regions of gastrointestinal smooth muscle, including the colon, jejunum, gastric antrum, and fundus (Ipavec et al., Pharmacol Res. 2011, 64(4):397-409). KCNQ4 channels are abundantly expressed in rodent airway and uterine smooth muscles (Evseev et al., Front Physiol. 2013, 4:277). Concurrently, KCNQ distribution studies in human visceral tissues also demonstrated high KCNQ4 expression (Ng et al., Br J Pharmacol. 2011, 162(1):42-53). Specifically targeting KCNQ4 can avoid side effects of other isoforms, such as suppressing long QT syndrome caused by KCNQ1 channels and their associated IKs currents (Terrenoire et al., Circ Res. 2005, 96(5):e25-34), and effectively avoiding side effects such as sedation and drowsiness caused by enhancement of central nervous system KCNQ2 and KCNQ3 channels (Orhan et al., Expert Opin Pharmacother. 2012, 13(12):1807-16). Currently, KCNQ agonists generally suffer from poor selectivity, especially for KCNQ2, KCNQ4 and KCNQ5 channels.All discovered KCNQ2 agonists simultaneously act on the KCNQ4 channel, with RL648_81 (ethyl (2-amino-3-fluoro-4-)((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate) potentiating only the KCNQ2 channel and not the KCNQ4 channel (Kumar et al., Mol Pharmacol. 2016, 89(6):667-677). The selectivity of existing KCNQ4 agonists is not ideal. With the exception of the vasodilator fasudil, reported by Xuan Zhang et al. in November 2016, which showed a relatively selective agonist effect on the KCNQ4 / KCNQ5 channels, no other KCNQ4-selective agonists have been reported (Zhang et al., Br J Pharmacol. 2016, 173(24):3480-3491).

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

[0005] There are several main types of KCNQ potassium channel agonists that have been reported:

[0006] 1) Patent US5384330 discloses a series of compounds having the following structure: [ka]

[0007] Its structural feature is that it contains one benzene ring substituted with an o-diamino group.

[0008] 2) Patent WO2005 / 087754 describes a KCNQ potassium channel agonist having the following structure: [ka]

[0009] Its structural features include a benzene ring substituted with a p-diamino group, one of which is also in a saturated ring (which may be a heterocyclic ring when W=O), and another nitrogen atom in the adjacent position is R 1 , R 2 is being replaced.

[0010] 3) Patent WO2008024398 describes the following structure: [ka]

[0011] The structure of such compounds is similar to that of patent WO2005 / 087754, but an additional fused benzene ring structural unit is further introduced into the nitrogen heterocycle.

[0012] The most clinically representative KCNQ potassium channel agonist is the antiepileptic drug retigabine (abbreviated as RTG), which was developed by GSK (GlaxoSmithKline) and approved in 2011. It has the following structure: [ka]

[0013] Retigabine is the first systematically studied KCNQ potassium channel agonist that activates KCNQ2-5 and is primarily used to treat adult patients with partial epilepsy.

[0014] Retigabine's structure contains an electron-rich benzene ring substituted with three amino groups. This structural feature makes it particularly susceptible to oxidation and degradation during synthesis and storage. At the same time, retigabine has relatively common side effects in clinical use, including dizziness, drowsiness, fatigue, confusion, tremors, impaired coordination, diplopia, blurred vision, attention deficit, memory loss, ataxia, aphasia, difficulty speaking, imbalance, increased appetite, hallucinations, myoclonus, peripheral edema, motor function loss, dry mouth, and difficulty swallowing. Urinary abnormalities, including bladder swelling, bladder wall thickening, and urinary retention, are also common adverse reactions associated with retigabine. On April 26, 2013, the FDA Drug Safety Committee announced that retigabine may cause pigmentary reactions, such as blue skin color and changes in retinal pigment, during clinical use. However, the specific mechanism of action is unknown, and the committee recommended that all patients taking this drug undergo regular ophthalmic 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).

[0015] In a previous publication (WO2013060097), the inventors disclosed a KCNQ potassium channel agonist having the following structure: [ka]

[0016] Among them, R 3 When is an allyl group or a propargyl group, the compound not only has KCNQ potassium channel agonist activity equal to or superior to that of retigabine, but also has significant antiepileptic effects in the body and protective effects equivalent to those of retigabine. Preliminary pharmacokinetic studies in mice have shown that such compounds have better brain exposure than retigabine. However, further safety evaluation studies have revealed that the compounds disclosed in WO2013060097 have significant neurotoxicity.

[0017] In a subsequent publication (CN105017085A), the inventors disclosed KCNQ potassium channel agonists having the following structures: [ka]

[0018] Among them, R 4 , R 5 When the substituent introduced into is an alkyl group, typically a methyl group (e.g., HN37), the resulting compound not only has stable physical properties and good absorption in brain tissue, but also significantly improves KCNQ2 agonist activity. However, with the increase in KCNQ2 agonist activity, the KCNQ4 agonist activity also increases accordingly.

[0019] The present inventors summarized previous reports and found that conventional KCNQ agonists generally suffer from poor selectivity, particularly for KCNQ2, KCNQ4, and KCNQ5 channels. All discovered KCNQ2 agonists simultaneously affect the KCNQ4 channel, and among them, RL648_81 (ethyl (2-amino-3-fluoro-4-)((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate) enhances only the KCNQ2 channel but has no effect on the KCNQ4 channel (Kumar et al., Mol Pharmacol. 2016, 89(6):667-677). Currently, the selectivity of existing KCNQ4 agonists is not ideal. Apart from the vasodilator fasudil, reported by Xuan Zhang et al. in November 2016, which has relatively selective agonistic effects on the KCNQ4 / KCNQ5 channels, no other KCNQ4-selective agonists have been reported (Zhang et al., Br J Pharmacol. 2016, 173(24):3480-3491). Because of the drawback of these existing KCNQ potassium channel agonists, there is a need to develop novel, more selective KCNQ4 potassium channel agonists for use in novel medications for diseases such as visceral pain, dyspepsia, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary arterial hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, pre-labor pain, pruritus, sexual dysfunction, and hearing loss. Summary of the Invention

[0020] A first object of the present invention is to provide novel compounds that are highly selective KCNQ4 potassium channel agonists.

[0021] A second object of the present invention is to provide a method for producing the above compound.

[0022] A third object of the present invention is to provide a pharmaceutical composition comprising the above compound or a pharmacologically acceptable salt thereof as an active ingredient, and any pharmacologically acceptable additive.

[0023] A fourth object of the present invention is to provide use of the above compound, or a pharmacologically acceptable salt thereof, or a composition containing the same in the manufacture of a KCNQ4 potassium channel agonist.

[0024] A fifth object of the present invention is to provide use of the above-mentioned compound, or a pharmacologically acceptable salt thereof, or a composition containing the same in the manufacture of a medicament for treating diseases related to smooth muscle or skeletal muscle.

[0025] In one aspect of the present invention, there is provided a compound of Formula I, or a pharmaceutically acceptable salt thereof: [ka]

[0026] Among them, R 1 and R 2 are each independently selected from the group consisting of hydrogen, halogen, a C1-C6 alkoxy group, a haloC1-C6 alkoxy group, a C1-C6 alkylsulfanyl group, a C4-C6 tert-alkyl group, a haloC1-C6 alkyl group, a nitro group, or a cyano group; R 1 and R 2 are preferably each independently selected from the group consisting of a halogen, a tert-butyl group, a nitro group, a cyano group, or a trifluoromethyl group; R 3 and R 4 are each independently selected from the group consisting of hydrogen, halogen, and C1-C6 alkyl groups; R 3 and R 4 are preferably each independently hydrogen; R 5 C1-C6 alkyl group, C1-C3 alkoxy group, C4-C 14 R is selected from the group consisting of a -tert-alkoxy group, a phenoxy group, and a benzyloxy group; 5 is preferably a C1-C3 alkoxy group, a C4-C6 tert-alkoxy group, a phenoxy group, or a benzyloxy group; R 5More preferably, R is a methoxy group, an ethoxy group, an isopropoxy group, a tert-butoxy group, a phenoxy group, or a benzyloxy group; 5 However, most preferably, it is a tert-butoxy group.

[0027] In some embodiments, the compound of formula I is selected from the group consisting of compounds of formula II: [ka]

[0028] Among them, R 1 , R 2 , R 3 , R 4 and R 5 The definition of is the same as above.

[0029] In some embodiments, the compound of Formula I is selected from the group consisting of compounds of Formula III: [ka]

[0030] Among them, R 1 , R 2 The definition of is the same as above, R 6 is a C4-C6-tert-alkyl group; R 6 is preferably a tert-butyl group.

[0031] In some preferred embodiments, the compound of formula I is selected from the group consisting of compounds of formula c: [ka]

[0032] Among them, R 1 , R 2 The definition of is the same as above.

[0033] In some preferred embodiments, the compound of formula I is selected from the group consisting of the following compounds: [Table 1] TIFF0007818710000012.tif106167

[0034] The definitions of terms used in the present invention are as follows.

[0035] The "halogen" may be fluorine, chlorine, bromine, or iodine.

[0036] The "C1-C6 alkyl group" refers to a chain alkyl group having 1 to 6 carbon atoms; specific examples thereof may include, but are not limited to, a methyl group, an ethyl group, a propyl group, an n-propyl group, an isopropyl group, a butyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a 1-methylbutyl group, a 1-ethylbutyl group, a pentyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a tert-pentyl group, a hexyl group, a normal hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 4-methyl-2-pentyl group, a 3,3-dimethylbutyl group, a 2-ethylbutyl group, and similar groups.

[0037] The "halo C1-C6 alkyl group" refers to a C1-C6 heterochain alkyl group in which at least one hydrogen is substituted with a halogen; specific examples thereof include a trifluoromethyl group.

[0038] The "C4-C6-tert-alkyl group" refers to an alkyl group having two branched chains and having 4 to 6 carbon atoms; specific examples thereof may include a tert-butyl group, a tert-pentyl group, and the like.

[0039] The "C1-C3 alkoxy group" refers to an RO- group, where R is a C1-C14 alkyl group as defined above. Specific examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, and the like.

[0040] The term "C4-C6-tert-alkoxy group" refers to an alkoxy group having two branched chains and 4-6 carbon atoms; specific examples thereof may include a tert-butoxy group, a tert-pentyloxy group, etc. 14 The definition of the -tert-alkoxy group is the same.

[0041] The pharmacologically acceptable salt of the compound described in the present invention may be a salt of the compound with an acid, wherein the acid is selected from the group consisting of 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, ascorbic acid, gallic acid, almond acid, malic acid, sorbic acid, trifluoroacetic acid, taurine, hypotaurine, 2-hydroxyethanesulfonic acid, cinnamic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and perchloric acid.

[0042] The compounds and pharmaceutically acceptable salts of the present application may have isomers or racemates, such as, but not limited to, optical isomers (including diastereoisomers and enantiomers), atropisomers, geometric isomers (cis-trans isomers), conformational isomers, tautomers, and mixtures thereof, which are also included within the scope of the claims of the present invention.

[0043] In previous studies, the present inventors found that introducing different substituents into the benzene ring linked to the amino group, especially alkyl groups with two methyl groups (e.g., HN37, CN105017085A), significantly enhanced KCNQ2 agonist activity. In the present invention, the present inventors further found that, while maintaining the overall skeleton of HN37, substituting tert-butyl carboxylate for the substituent on the terminal nitrogen atom instead of methyl carboxylate significantly enhanced KCNQ4 agonist activity. Further research showed that removing the substituent on the benzene ring linked to the amino group, while retaining the tert-butyl carboxylate substituent on the terminal nitrogen atom, resulted in compound K31, which not only exhibited good KCNQ4 agonist activity but also lost its KCNQ2 agonist activity, demonstrating selectivity. Based on this, it was then found that when a different substituent was introduced into the benzene ring of the benzyl group bonded to the amino group, particularly when the introduced substituent was an electron-rich substituent such as a tert-butyl group, the resulting compound not only further increased KCNQ4 agonist activity, but also lost KCNQ2 agonist activity, while still maintaining very good KCNQ4 / KCNQ2 selectivity. In summary, the novel selective KCNQ4 agonists provided by the present invention overcome the drawback of the low selectivity of conventional potassium channel agonists and have the advantages of improved activity, significantly reduced toxicity, a simpler structure, and low production costs, making them more promising for development.

[0044] In another aspect of the present invention, there is provided a method for preparing a compound of formula c above, which is achieved by the following reaction scheme: [ka]

[0045] In the production method of the present invention, R 1 and R 2 The definition of is the same as above.

[0046] 1,4-Phenylenediamine and di-tert-butyl dicarboxylate react to produce N-(tert-butoxycarbonyl)-1,4-phenylenediamine, N-(tert-butoxycarbonyl)-1,4-phenylenediamine undergoes a substitution reaction with propargyl bromide to produce intermediate a, and intermediate a reacts with compound b to produce compound c.

[0047] In another aspect, the present invention provides a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, and any pharmaceutically acceptable additive.

[0048] Another aspect of the present invention provides use of a compound according to the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the present invention in the manufacture of a KCNQ4 potassium channel agonist.

[0049] In another aspect of the present invention, there is provided use of a compound according to the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing either of them, in the manufacture of a medicament for treating a disease associated with smooth muscle or skeletal muscle, etc.

[0050] In another aspect of the present invention, there is provided a method for treating a neurological disorder, comprising administering to a subject suffering from a related disorder such as smooth muscle or skeletal muscle a compound according to the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the present invention;

[0051] The smooth muscle or skeletal muscle-related diseases include smooth muscle or skeletal muscle-related diseases such as visceral pain, indigestion, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary arterial hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, pre-labor pain, pruritus, sexual dysfunction, hearing loss, etc.

[0052] The present invention has the following beneficial effects:

[0053] Compared with conventional KCNQ agonists, the compounds provided by the present invention have significantly improved selectivity for KCNQ4 over KCNQ2. For example, in in vitro electrophysiological experiments, compounds K31-K43 all have excellent selectivity for KCNQ4 over KCNQ2, and all have no effect on KCNO2, but only agonist activity for KCNQ4.

[0054] The compounds provided by the present invention have no free amino groups in their structure, and therefore are physically more stable and less susceptible to oxidative degradation than the existing drug retigabine, as evidenced by the fact that solutions of these compounds are less susceptible to oxidative discoloration when exposed to air.

[0055] In summary, the compounds provided by the present invention overcome the drawback of low selectivity of conventional agonists, and not only have stable physical properties and high activity, but also good selectivity and significantly reduced toxicity, and therefore have a larger therapeutic window and better therapeutic effect, showing good prospects for application.

[0056] Although the present invention has been described in detail above, the above embodiments are merely illustrative and are not intended to limit the present invention. Furthermore, the present application is not limited by the prior art, the summary of the invention, or the theory described in the following examples. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention will be further described below by means of examples, but it should be noted that the following examples are provided for illustrative purposes only and are not intended to limit the scope of protection claimed for the present invention.

[0058] Unless otherwise specified, the materials, reagents, methods and the like employed in the examples are conventional in the art.

[0059] Compound production examples In the following production examples, nuclear magnetic resonance (NMR) was measured using a Bruker NMR Spectrometer 400M device manufactured by Bruker, and the NMR calibration was δH 7.26 ppm (CDCl3), 2.50 ppm (DMSO-d6), and 3.15 ppm (CD3OD).

[0060] Reagents were mainly provided by Shanghai Bide PharmaTech Co.

[0061] The silica gel plate for thin layer chromatography (TLC) was produced by Shandong Yantai Huayou Silica Gel Development Co., Ltd., model number HSGF254.

[0062] The normal phase column chromatography silica gel used for compound purification was produced by the Shandong Qingdao Ocean Chemical Plant Branch, model number ZCX-11, 200-300 mesh. Manufacturing example: Preparation Example 1: Synthesis of tert-butyl 4-(Np-fluorobenzyl-N-propargyl-amino)-phenylaminocarboxylate (K31) [ka]

[0063] 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 propargyl bromide (0.71 g, 6.0 mmol) was added. The mixture was heated to 65 °C and reacted for 12 hours. 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), once with saturated brine (15 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5:1) to give product K31-b (1.21 g, 82% yield) as a white solid, which was used directly in the next step.

[0064] Compound K31-b (73.89 mg, 0.3 mmol) obtained in the above step 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 hours. 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), once with saturated brine (5 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20:1) to give product K31 as a white solid (0.95 g, 90% yield). 1 H NMR (400 MHz, CDCl3):δ 7.30-7.27(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).

[0065] The following compounds were prepared in the same manner as in Preparation Example 1. [Table 2] TIFF0007818710000016.tif195127TIFF0007818710000017.tif195125

[0066] Electrophysiological Experiment Examples Electrophysiological Experiment Example 1: The cell line used for electrophysiological experiments was the Chinese hamster ovary cell line (CHO-K1). KCNQ cDNA was transformed into Escherichia coli, expressed in Escherichia coli, and confirmed by plasmid extraction and sequencing.

[0067] 1. Cell Culture and Transfection Chinese hamster ovary (CHO) cells (Chinese Academy of Sciences Cell Bank, China) were used in a medium formulation of 50 / 50 DMEM / F-12 (Gibco), 10% fetal bovine serum (FBS) (Gibco, Australia), and 2 mM L-glutamine (Invitrogen). Transfection and KCNQ channel expression: 24 hours prior to transfection, cells were digested with trypsin (Sigma, China) and seeded into 6-well plates. Transfection was performed using Lipofectamine 2000™ reagent (Invitrogen) according to the accompanying protocol. 24 hours after transfection, cells were digested and re-spread onto poly-L-lysine (Sigma)-soaked slides. Co-transfection with GFP (green fluorescent protein) was used to confirm transfection 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 Liuhui Huada Co., Ltd. and constructed in the pcDNA3.1(+) vector plasmid. The KCNQ2 and KCNQ4 plasmids were transformed into DH5α E. coli and then confirmed by plasmid extraction and sequencing.

[0068] 2. Electrophysiological recordings on CHO cells: Whole-cell voltage clamp experiments were performed at room temperature (23–25°C) using an Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA) and a Digidata 1440A digital-to-analog converter (Molecular Devices). Signal acquisition was performed using pClamp 10.0 software (Molecular Devices, Sunnyvale, CA). Filtering was 2 kHz, the sampling frequency was 10 kHz, and series resistance compensation was 60%. Electrodes were fabricated from pulled borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). After filling the electrodes with intracellular solution, the electrical resistance was 3–5 MΩ. The perfusion system was a BPS-8 (ALA Scientific Instruments, Westburg, NY), and the rate was approximately 1 mL / min.

[0069] Intracellular solution formulation for electrophysiological experiments: 145 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 5 mM MgATP (adjusted to pH 7.3 with KOH).

[0070] Electrophysiology experiment extracellular solution formulation: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.5 mM MgCl2, 10 mM HEPES, 10 mM glucose (adjusted to pH 7.4 with NaOH).

[0071] Stimulation protocol for eliciting KCNQ2 and KCNQ4 channel currents: clamp voltage was -120 mV, followed by square wave stimulation at -10 mV for 1500 ms, with a stimulation frequency of 0.1 Hz.

[0072] 3. Dissolution and Preparation of Compounds A fixed mass of compound was dissolved in DMSO to prepare a 20 mM DMSO stock solution, which was then frozen and stored at -20°C. On the day of the test, the 20 mM compound stock solution was gradient diluted with extracellular solution to the required final concentration. The DMSO content in the test drug solution was kept below 0.5% to ensure that this concentration of DMSO would not affect the KCNQ channel currents tested. For example, to prepare 100 nM and 1 μM compound solutions, the gradient dilution method was as follows: 5 μL of DMSO stock solution was aspirated and added to 10 mL of extracellular solution for homogenous dissolution to obtain a 10 μM compound solution; 1 mL of the 10 μM compound was aspirated and added to 9 mL of extracellular solution for homogenous dissolution to obtain a 1 μM compound solution; and 1 mL of the 1 μM compound was aspirated and added to 9 mL of extracellular solution for homogenous dissolution to obtain a 100 nM compound solution.

[0073] 4. Experimental results and data analysis Data collection, analysis, and processing were performed using pClamp10 (Molecular Devices, Sunnyvale, CA), GraphPad Prism 5 (GraphPad Software, San Diego, CA), and Excel (Microsoft). All data are expressed as mean ± standard error (SEM). Significance analysis was performed using an unpaired student's t-test. Differences between groups were considered statistically significant if P < 0.05. The effect of compounds on current was calculated using the following formula: Enhancement factor = I Drug / I Control ;

[0074] where I Control is the steady-state maximum peak current value generated under a test voltage stimulus of -10 mV after administration of blank external solution to the cell, and I Drug is the steady-state maximum peak current value generated under a stimulation voltage of -10 mV after compound perfusion, and I Drug / I Control >1 indicates potentiation, I Drug / I Control<1 indicates inhibitory activity. n is the number of cells tested. [Table 3]

[0075] Results and Discussion: The results of the above electrophysiological tests showed that the compounds disclosed in this invention not only maintained good agonist activity against KCNQ4 potassium channels, but also showed good selectivity, being inactive against KCNQ2.

[0076] The above embodiments are intended to illustrate the technical means of the present invention, but are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical means described in the above embodiments may be modified, or some or all of the technical features may be replaced with equivalents, without departing from the spirit and scope of the present invention as defined by the claims, and such modifications or replacements still fall within the scope of the claims.

Claims

1. A compound of formula III or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 Among them, R １ and R ２ are each independently hydrogen, halogen, C ４ -C ６ -tert-alkyl group, haloC １ -C ６ selected from the group consisting of alkyl groups, nitro groups, and cyano groups; R 6 is a C 4 -C 6 -tert-alkyl group; The compound of formula III 【Chemistry 2】 isn't it, A compound or a pharmacologically acceptable salt thereof.

2. R １ and R ２ are each independently selected from the group consisting of a halogen, a tert-butyl group, a nitro group, a cyano group, and a trifluoromethyl group; 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The compound of formula III is selected from the group consisting of compounds of formula c: 【Transformation 3】 Among them, R １ , R ２ The definition of is the same as in claim 1.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

4. The compound of formula III is selected from the group consisting of the following compounds: Table 1 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

5. A method for producing the compound according to any one of claims 1 to 4, comprising the steps of: The compound is selected from the group consisting of compounds of formula c: 【Chemistry 4】 Among them, R １ , R ２ The definition of is the same as in the corresponding claim, The production method includes the steps of reacting 1,4-phenylenediamine with di-tert-butyl dicarboxylate to produce N-(tert-butoxycarbonyl)-1,4-phenylenediamine; subjecting N-(tert-butoxycarbonyl)-1,4-phenylenediamine to a substitution reaction with propargyl bromide to produce intermediate a; and reacting intermediate a with compound b to produce compound c. Manufacturing method.

6. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 or a pharmacologically acceptable salt thereof and any pharmacologically acceptable additive.

7. Use of a compound of formula III or a pharmacologically acceptable salt thereof, or a pharmaceutical composition comprising said compound or a pharmacologically acceptable salt thereof, in the manufacture of a KCNQ4 potassium channel agonist, comprising: 【Transformation 5】 wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, halogen, C 4 -C 6 -tert-alkyl, haloC 1 -C 6 alkyl, nitro, and cyano; R 6 is a C 4 -C 6 -tert-alkyl group; use.

8. The use according to claim 7, wherein R 1 and R 2 are each independently selected from the group consisting of halogen, a tert-butyl group, a nitro group, a cyano group, and a trifluoromethyl group.

9. The compound of formula III is selected from the group consisting of compounds of formula c: 【Transformation 6】 wherein R 1 and R 2 are defined as in claim 7; 8. The use according to claim 7.

10. The compound of formula III is selected from the group consisting of the following compounds: Table 2 8. The use according to claim 7.

11. Use of a compound of formula III or a pharmacologically acceptable salt thereof, or a pharmaceutical composition comprising said compound or a pharmacologically acceptable salt thereof, in the manufacture of a medicament for treating a smooth muscle or skeletal muscle-related disease, comprising: 【Transformation 7】 wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, halogen, C 4 -C 6 -tert-alkyl, haloC 1 -C 6 alkyl, nitro, and cyano; R 6 is a C 4 -C 6 -tert-alkyl group; use.

12. The use according to claim 11, wherein R 1 and R 2 are each independently selected from the group consisting of halogen, a tert-butyl group, a nitro group, a cyano group, and a trifluoromethyl group.

13. The compound of formula III is selected from the group consisting of compounds of formula c: 【Transformation 8】 wherein R 1 and R 2 are defined as in claim 11; 12. The use according to claim 11.

14. The compound of formula III is selected from the group consisting of the following compounds: Table 3 12. The use according to claim 11.

15. The smooth muscle or skeletal muscle-related diseases include visceral pain, indigestion, irritable bowel syndrome, overactive bladder syndrome, hypertension, pulmonary arterial hypertension, coronary artery disease, cerebral vasospasm, asthma, chronic obstructive pulmonary disease, pre-labor pain, pruritus, sexual dysfunction, and hearing loss.

12. The use according to claim 11.

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