Potassium channel activators and their use in the treatment of diseases associated with hyperexcitability

Abstract

The present invention is based on the repositioning of a clinically safe compound as a potassium ion channel activator for the treatment of diseases associated with hyperexcitability. The present invention also relates to a pharmaceutical composition comprising a compound of the present invention, as well as to methods of using the disclosed compounds or the pharmaceutical compositions of the present invention for the treatment of diseases associated with hyperexcitability.

Description

[0001] Invention Field This invention is based on repositioning a clinically safe compound as a potassium channel activator for the treatment of diseases associated with overexcitation. The invention also relates to a pharmaceutical composition comprising the compound of this invention, and a method of treating diseases associated with overexcitation using the disclosed compound or the pharmaceutical composition of this invention. Summary of the Invention

[0002] Epilepsy, characterized by a persistent predisposition to seizures, is one of the most common neurological disorders, with a lifetime prevalence estimated at 1% to 2%. Treatment typically relies on long-term use of antiepileptic drugs (ASMs) to prevent seizures. However, in more than 30% of patients, seizures are not adequately controlled with existing antiepileptic medications, leading to drug-resistant epilepsy (PRE). Specifically, PRE is defined as a patient whose seizures are not adequately controlled after treatment with two appropriately dosed antiepileptic drugs selected based on their medical history.

[0003] Long-term prognostic studies have shown that the success rate of seizure control in patients with PRE gradually decreases after adding other medications to the treatment regimen. Uncontrolled seizures are associated with a variety of serious complications, including an increased risk of sudden epileptic death (SUDEP); in children, it can also lead to failure to reach neurodevelopmental milestones, resulting in severe intellectual disability. While treatments different from antiepileptic drugs (ASMs), such as surgical resection and / or a ketogenic diet, may help control seizures in some patients with refractory epilepsy (PRE), not all patients are eligible for surgery, and long-term use of a ketogenic diet can be accompanied by serious side effects.

[0004] Currently, existing antiepileptic drugs can be classified according to their mechanisms of action, among which the most widely used are: 1. voltage-gated sodium channel blockers; 2. those enhancing gamma-aminobutyric acid (GABAergic) neurotransmission; and 3. voltage-gated calcium channel blockers. It is generally believed that treatment with antiepileptic drugs (ASMs) with different, newer, and differentiated mechanisms of action may help reduce preepileptic recurrence (PRE), thus necessitating the design of innovative pharmacological strategies to combat the hyperexcitatory processes underlying epilepsy.

[0005] Activation of potassium channels has recently been considered one of the most promising mechanisms for the anticonvulsant effects of novel antiepileptic drugs (ASMs). Among voltage-gated (Kv) potassium channels, those encoded by the KCNQ subfamily have received the most attention in recent decades. The KCNQ genes (KCNQ1-5) encode five members (Kv7.1-Kv7.5) of the Kv7 subunit, each with unique tissue distribution and pathophysiological functions. The Kv7.1 subunit is expressed in the heart and is responsible for the ventricular repolarization current (i.e., Ik). Ks The main molecular components of Kv7 are: Kv7.2 and Kv7.3 subunits are mainly expressed in the central nervous system (CNS), forming the basis of M currents, which are active at resting membrane potentials and limit the firing frequency of neuronal action potentials; Kv7.4 is mainly expressed in the smooth muscle of the inner ear, viscera, blood vessels and lungs, and participates in the regulation of smooth muscle tone; finally, Kv7.5 is mainly expressed in the central nervous system, vascular smooth muscle and skeletal muscle. 11 The crucial role of neuronal Kv7 channels (primarily Kv7.2 and Kv7.3, but Kv7.5 also contributes) in neuronal excitability has been demonstrated in humans through highly phenotypic seizures caused by specific mutations in the corresponding genes.

[0006] Given that Kv7 channels play an important role in regulating neuronal excitability, and that neuronal overexcitation is a common feature of neuropsychiatric diseases such as epilepsy, neuropathic pain, ischemic stroke, amyotrophic lateral sclerosis, and many other neurodegenerative diseases, pharmacological regulation of Kv7 channels seems to be a reasonable approach to treating these diseases.

[0007] In fact, two neuronal Kv7 activators, flupirtine and retigabine, have already been approved and are in clinical use. Flupirtine was the first compound identified as a Kv7 activator, although its development predates the discovery of these pathways. Flupirtine is a non-opioid analgesic approved in Europe in 1984, but its marketing authorization was revoked by the European Medicines Agency (EMA) in 2018 due to its severe hepatotoxicity.

[0008] Limited preclinical and clinical studies conducted in the 1980s showed that flupirtine also had anticonvulsant effects in animal models and patients with primary epilepsy (PRE). Therefore, by optimizing the flupirtine molecule to enhance its antiepileptic activity, the synthesis and preclinical characterization of retigabine (US trade name ezogabine) were finally obtained, and this drug is now considered a prototype of a Kv7 channel opener.

[0009] Retigabine's anticonvulsant efficacy has been demonstrated in numerous animal models of epilepsy because it inhibits neuronal hyperexcitability through a unique Kv7.2 / 7.3 channel activation mechanism, including shifting the activation threshold of the current to a higher polarized membrane potential and enhancing the maximum current of Kv7.2 / 7.3. 16 Its mechanism of action differs from classic antiepileptic drugs, making retigabine particularly effective in treating patients with prodromal symptoms of epilepsy (PRE). In 2011, retigabine was approved for adjunctive treatment of partial-onset epilepsy in adults. However, long-term use of retigabine can cause blue discoloration of mucous membranes, skin tissues, and the retina due to photoinduced formation and accumulation of retigabine dimers, especially in melanin-rich tissues. Primarily due to these adverse reactions, the clinical use of retigabine gradually decreased, and it was ultimately withdrawn from the market in 2017.

[0010] Therefore, although multiple studies have confirmed that pharmacological activation of neuronal Kv7 channels is an effective mechanism for treating epilepsy and other diseases characterized by neuronal overexcitation, there are currently no Kv7 activators available for clinical use.

[0011] Currently, multiple industrial and academic research teams are developing novel Kv7 activators. Over the past two decades, researchers have not only developed retigabine / flupirtine analogues with improved physicochemical properties, pharmacokinetic or pharmacodynamic characteristics, but have also explored novel Kv7 channel openers derived from pharmacodynamic structures different from the two parent molecules mentioned above.

[0012] Among the newly synthesized retigabine analogues, several exhibited higher efficacy in activating Kv7.2 and Kv7.3 channels in vitro than the parent compound, and some also demonstrated enhanced anticonvulsant efficacy and potency in animal models of epilepsy. Furthermore, to overcome the tissue discoloration problem associated with long-term retigabine use, researchers successfully developed retigabine analogues that do not produce molecular byproducts leading to this adverse reaction using different synthetic strategies. Some of these compounds also demonstrated potent anticonvulsant effects in animal models of epileptic seizures.

[0013] In addition to retigabine analogues, several Kv7 activators with different chemical structures from retigabine have been reported; these compounds belong to the acrylamide, benzamide, and fenamic acid classes, or have unique structures that do not belong to any of these classes. Acrylamide compounds bind to the same region within the Kv7 channel pore as retigabine, and after in vitro experiments demonstrated their effectiveness in reducing neuronal excitability, researchers have explored their potential therapeutic effects in migraines and neuropathic pain. Fenamic acid classes, such as the nonsteroidal anti-inflammatory drug diclofenac, can activate Kv7.2 and Kv7.3 channels and have shown anticonvulsant activity in animal models of epilepsy. A few diclofenac derivatives designed to eliminate COX inhibitory activity while retaining the Kv7 channel opening properties have effectively reduced neuronal activity in vitro, but have not yet been tested in vivo.

[0014] Benzamides have different binding sites and mechanisms of action than retigabine. Studies have found that many benzamides are more active than retigabine in vitro and have shown antiepileptic effects in various animal models of epilepsy.

[0015] Several other chemical compounds, including synthetic and natural compounds, have been studied in vitro as Kv7 activators. Some of these molecules have also shown anticonvulsant activity in animal models, and one of them (KB 3061 or BHV-7000) can reverse the effects of pathogenic KCNQ mutations in vitro.

[0016] Despite encouraging results in preclinical models, few Kv7 activators have progressed to clinical trials. These include: - The retigabine analogue HN37 has been advanced to the clinical trial stage for the treatment of epilepsy in China; Compound XEN1101 is currently undergoing three different Phase II / III clinical trials: 1. for the treatment of focal seizures (ClinicalTrials.gov ID: NCT05614063); 2. as adjunctive therapy for primary generalized tonic-clonic seizures (ClinicalTrials.gov ID: NCT05667142); 3. for the treatment of major depressive disorder (ClinicalTrials.gov ID: NCT04827901).

[0017] The compound BHV-7000 has passed Phase I safety trials and announced that it will begin Phase II / III trials for focal epilepsy and bipolar disorder in 2023.

[0018] In addition, the clinical trial of XEN496, a novel pediatric formulation of retigabine developed by Xenon Pharmaceuticals, in patients with KCNQ-related diseases was terminated in 2023, and the results have not yet been published (ClinicalTrials.gov ID: NCT04639310).

[0019] The gap between the number of novel Kv7 channel openers in the preclinical research stage and the number of those undergoing clinical trials underscores the extremely high risk of failure in drug development based on novel chemical entities.

[0020] Ideally, if an alternative drug can be found that is in a more mature stage of development, usually already in clinical trials or approved, with comparable or even better efficacy, it can reduce the risk of failure, economic investment, and the time required to start treatment for patients.

[0021] Therefore, the object of this invention is to reposition a known antipsychotic drug as a potassium channel activator for the treatment of Kv7-related diseases.

[0022] Invention Summary In general, the main aspects of the present invention can be summarized as follows: In a first aspect, the present invention relates to a compound for treating a subject with a Kv7-related disease, wherein the compound is selected from compounds represented by formula (I) as defined below, or pharmaceutically acceptable salts, hydrates or solvates thereof, or stereoisomers thereof.

[0023] In a second aspect, the present invention relates to a method for treating a neurological disorder in a subject, the method comprising the step of administering a therapeutically effective amount of the compound of the present invention to the subject, wherein the subject suffers from a nonpsychiatric neurological disorder but has experienced or is experiencing seizures.

[0024] In a third aspect, the present invention relates to a pharmaceutical composition comprising the compounds of the present invention and a pharmaceutically acceptable carrier and / or excipients. Invention Details The elements of the invention will now be described. While specific embodiments are listed, it should be understood that these elements can be combined in any manner and in any number to form other embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to the explicitly described embodiments. It should be understood that this description is intended to support and cover embodiments combining two or more explicitly described embodiments, or combining one or more explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any permutation or combination of all the elements described in this application should be considered as disclosed by the description of this application.

[0026] In a first aspect, the technical problem is solved by providing a compound for treating a subject's Kv7-related disease, wherein the compound is selected from compounds of formula (I), or pharmaceutically acceptable salts, hydrates, or solvates thereof, or stereoisomers thereof. in R is H or C 1-6 alkyl; R 1 It is a phenyl group; a phenyl group substituted with 1, 2 or 3 substituents selected from the group consisting of: H, halogen, cyano, C. 1-4 Alkyl, C 1-4 Alkoxy, perfluorinated C 1-4 Alkyl, diC 1-4 Alkylamino; thiophene; thiophene group substituted with one or two substituents selected from the group consisting of halogens and C. 1-4 Alkyl; C 3-8 cycloalkyl; or C 5-7 Cycloalkenyl; R 2 For H or C 1-6 alkyl; R 3 Halogen, C 1-4 Alkyl or perfluorinated C 1-4 Alkyl; and R 4 and R 5 Each is independently selected from H or halogens.

[0027] In a preferred embodiment of the compound used in this invention, the R 3 It is trifluoromethyl; R, R 4 and R 5 All are H.

[0028] In another preferred embodiment of the compound used in this invention, R is H or methyl.

[0029] In another preferred embodiment of the compound used in this invention, the R 1 It is 4-fluorophenyl or 3,4-difluorophenyl.

[0030] In another preferred embodiment, the compound used in this invention is N -[1-[(3,4-difluorophenyl)methyl]-4-piperidinyl]-6-(trifluoromethyl)-3-pyridazinamine, or compounds having the following formula (II). .

[0031] In another preferred embodiment, the compound used in this invention is N-[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (III). .

[0032] In another preferred embodiment, the compound used in this invention is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (IV). .

[0033] In another preferred embodiment, the compound used in this invention is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (V). .

[0034] In another preferred embodiment, the compound used in this invention is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (VI). .

[0035] The term "alkyl" refers to a single group of a saturated straight-chain or branched hydrocarbon. Preferably, the alkyl group comprises 1 to 12 (e.g., 1 to 10) carbon atoms, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl (also known as 2-propyl or 1-methylethyl), butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, secondary pentyl, neopentyl, 1,2-dimethylpropyl, isopentyl, n-hexyl, isohexyl, secondary hexyl, n-heptyl, isoheptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, etc. "Substituted alkyl" means that one or more (e.g., 1 to the maximum number of hydrogen atoms contained in the alkyl group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, such as 1 to 5, 1 to 4, 1 to 3, or 1 or 2) of an alkyl group are replaced by substituents other than hydrogen (when more than one hydrogen atom is substituted, these substituents may be the same or different). Preferably, the non-hydrogen substituent is a primary, secondary, or tertiary substituent as specified in this specification, such as halogen, -OH, -NH2, -NHCH3, -N(CH3)2, -CN, -OCH3, -OCF3, or optionally substituted aryl groups. Examples of substituted alkyl groups include trifluoromethyl, 2,2,2-trichloroethyl, 2-hydroxyethyl, 2-aminoethyl, 2-(dimethylamino)ethyl, arylalkyl (also known as "aralkyl group", such as benzyl, chloro(phenyl)methyl, 4-methylphenylmethyl, (2,4-dimethylphenyl)methyl, o-fluorophenylmethyl, 2-phenylpropyl, 2-, 3- or 4-carboxyphenylalkyl), or heteroarylalkyl (also known as "heteroarylalkyl group").

[0036] C x1-x2 Alkyl refers to the alkyl group described above, whose chain length or number of carbon atoms is between X1 and X2 as indicated by the subscript. For example, the term "C 1-3 "Alkyl" refers to an alkyl group with a chain length of 1 to 3 carbon atoms.

[0037] The term "halogen" or "halogenated" refers to fluorine, chlorine, bromine, or iodine.

[0038] The term "cyano" refers to a carbon atom bonded to a nitrogen atom by three single bonds, with the nitrogen atom bonded to three carbon atoms.

[0039] The term "alkoxy" refers to an alkyl group as described above, which is bonded to an oxygen atom by a single bond.

[0040] The term "perfluorinated" refers to a chemical group in which all CH bonds are replaced by CF bonds.

[0041] The term "alkylamino" refers to an alkyl group as described above, which is attached to the rest of the molecule by a nitrogen atom.

[0042] The term "cycloalkyl" refers to the cyclic non-aromatic form of an alkyl group, preferably having 3 to 14 carbon atoms, for example 3 to 12 or 3 to 10, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl and adamantyl. The term "cycloalkyl" also includes its bicyclic and tricyclic forms. If a bicyclic ring is formed, it is preferred that the rings are connected to each other at two adjacent carbon atoms; however, as an alternative, the two rings may also be connected by the same carbon atom, i.e., forming a spirocyclic system or a "bridged" ring system. Preferred examples of cycloalkyl groups include C3-8 cycloalkyl groups, particularly cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, bicyclo[4.1,0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, and bicyclo[4.2.0]octyl. Cycloalkyl groups do not include fullerenes. "Substituted cycloalkyl" means that one or more hydrogen atoms (e.g., from 1 to the maximum number of hydrogen atoms in the cycloalkyl group, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as 1 to 5, 1 to 4, 1 to 3, or 1 or 2) are substituted with substituents other than hydrogen (when more than one hydrogen atom is substituted, the substituents can be the same or different). Preferably, the non-hydrogen substituent is a primary, secondary, or tertiary substituent as described herein, such as halogen, -CN, nitro, -OR11 (e.g., -OH), -SR11 (e.g., -SH), -N(R12)(R13) (e.g., -NH), =X (e.g., =O, =S, or =NH), alkyl (e.g., C... 1-6 Alkyl groups. Examples of substituted cycloalkyl groups include oxocyclohexyl, oxocyclopentyl, fluorocyclohexyl, and carbonylcyclohexenyl.

[0043] The compound described in this invention was initially developed as a highly selective dopamine D2 receptor antagonist with a fast dissociation rate (see WO2007048779), which has antipsychotic effects and no exercise side effects.

[0044] The inventors conducted an extensive screening of a compound library containing a large number of compounds that have been proven to be clinically safe, and unexpectedly discovered that there was indeed a specific Kv7 activator (the compound of the present invention) that had been safely used in clinical practice (Example 1).

[0045] Furthermore, the compounds of this invention have been shown to be activators of neuronal Kv7 channels, particularly Kv7.2 / 7.3 channels, and through this mechanism of action, have effectively controlled epileptic seizures in two different animal models of epilepsy (Examples 2 and 4). The preferred Kv7 channel according to this invention is a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0046] An advantageous embodiment is that the compounds of the present invention have been shown to be safe for human use, have not caused any serious side effects in a 12-week clinical trial, and exhibit favorable pharmacodynamic characteristics such as good brain penetration.

[0047] Furthermore, docking simulation data revealed that the compound used in this invention interacts with W234 of Kv7.2 via its piperazine ring. This piperazine ring was found to form a hydrogen bond with the indole nitrogen atom of W236 in Kv7.2 (SEQ ID NO 1). This simulates the interaction between the carbamate group of retigabine and W236 at the same site in Kv7.2. Notably, the W235 binding residue is located in the pore region of Kv7.2, making it a plausible binding target for modulating Kv7.2 activity. Additionally, the two phenylalanine residues (F240 and F305) in Kv7.2, known to interact with the p-fluorophenyl fragment of retigabine, also interact with the difluorophenyl fragment in the compound of this invention. All amino acid positions are referenced based on the amino acid sequence shown in SEQ ID NO:1.

[0048] Therefore, another preferred embodiment of the compounds of the present invention is a compound comprising a piperazine or similar group, which can act as an acceptor for the hydrogen bonds provided by W236. References to amino acid positions are based on the amino acid sequence shown in SEQ ID NO: 1.

[0049] Through docking simulation, interaction sites can be modeled as follows: Figure 4 As shown. Another preferred embodiment is that, when the compound of the present invention binds to Kv7.2, it retains the ability to interact with or maintain contact with at least one (preferably all) of the residues F104, L242, F304, L312, I300, P308, S303, W236, L299, F305, and F240, which are the Kv7.2 binding sites of the compound of the present invention. Reference to amino acid positions is based on the amino acid sequence described in SEQ ID NO: 1.

[0050] In addition to the binding mechanism, the inventors further demonstrated that Kv7 channels are activated upon contact with the compounds of the present invention. Therefore, another important embodiment of the compounds of the present invention is the ability to modulate the activity of Kv7 channels (particularly Kv7.2 / Kv7.3) (Example 2).

[0051] In another preferred embodiment, the compounds of the present invention function as Kv7 agonists, preferably Kv7.2 and Kv7.3 agonists. As used herein, "agonist" should mean a compound capable of activating a biological target (particularly a protein receptor) to produce a biological effect.

[0052] In one related embodiment, the compounds of the present invention interact with Kv7 potassium ion channels, thereby increasing the ion flux through the Kv7 potassium ion channels, particularly the potassium ion flux. As used herein, the term "ion flux" refers to the movement of ions through, typically across, a membrane.

[0053] Therefore, in another preferred embodiment of the compound of the present invention, administration of the compound to a subject can reduce the incidence, prevalence, frequency, duration and / or severity of epileptic seizures, wherein the subject suffers from a disease that induces epilepsy.

[0054] As used herein, the term "Kv7 channel" refers to a member of a group of voltage-gated potassium ion channels encoded by the KCNG gene. The Kv7 family is a group of tetrameric voltage-gated potassium ion channels consisting of five members: Kv7.1, Kv7.2, Kv7.3, Kv7.4, and Kv7.5, whose subunits are encoded by the KCNG1-5 genes, respectively. Therefore, another important embodiment of the compounds of this invention is capable of inducing the opening of Kv7.2 and / or Kv7.3 channels.

[0055] Kv7 channels can be activated and allow potassium ions to pass through, for example, across the cell membrane; this induced potassium permeability is referred to here as the “opening” or “activation” of the corresponding channel protein.

[0056] In another embodiment, the compound used in this invention exists in the form of a salt, preferably a dihydrochloride.

[0057] In another embodiment, the compounds used in this invention are used as antiepileptic drugs (ASM). As used herein, antiepileptic drugs should be understood as drugs used to treat epileptic seizures.

[0058] Given that the activity of Kv7.2 / 7.3 is associated with neurological disorders, a second aspect of the invention relates to a method of treating a subject with a neurological disorder, the method comprising the step of administering a therapeutically effective amount of a compound of the invention to the subject, wherein the subject suffers from a nonpsychiatric neurological disorder but has experienced or is experiencing spastic epileptic seizures. Surprisingly, the compound as defined herein has been found to exhibit seizure-relieving activity in vivo.

[0059] Therefore, in a preferred embodiment of the method of the present invention, the neurological disease is selected from Kv7-related diseases. In particular, it can be selected from diseases involving neuronal overexcitation, such as epilepsy syndromes, neuropathic pain, amyotrophic lateral sclerosis (ALS), ischemic stroke, neurodegenerative diseases, etc. The neurological disease can be, for example, but not limited to, epilepsy, including generalized epilepsy, focal epilepsy, mixed generalized and focal epilepsy, epilepsy of unknown cause, early myoclonic encephalopathy, Ōtahara syndrome, West syndrome, Draway syndrome, non-progressive encephalopathy, status myoclonic, Lennox-Gasto syndrome, Landau-Cleffner syndrome, persistent spike-and-wave epilepsy during slow-wave sleep, migratory partial seizures in infancy, neuropathic pain, ischemic stroke, ALS, Alzheimer's disease, and Parkinson's disease.

[0060] In the context of this invention, the term “Kv7-related disease” should be understood as any disease or disorder in a particular subject that is primarily caused by dysfunction of the Kv7 (KCNQ) family voltage-gated potassium channels.

[0061] However, given that the purpose of this invention is to provide a novel treatment for neurological disorders related to potassium ion channels (e.g., disorders related to seizures), such neurological disorders should not encompass disorders related to dopamine D2 receptor dysfunction, particularly central nervous system disorders related to dopamine D2 receptor dysfunction. Specifically, such neurological disorders are not those unrelated to motor function.

[0062] In another embodiment of the invention, Kv7-related diseases may also be selected from hypertension, bladder dysfunction, intestinal diseases, and respiratory diseases. As used herein, the term "respiratory disease" refers to a disease or condition related to the respiratory system. Examples include, but are not limited to, airway inflammation, allergies, asthma, respiratory obstruction, cystic fibrosis (CF), allergic rhinitis (AR), acute respiratory distress syndrome (ARDS), pulmonary hypertension, lung inflammation, bronchitis, airway obstruction, bronchospasm, microbial infections, and viral infections, such as, but not limited to, severe acute respiratory syndrome (SARS) or severe acute respiratory syndrome coronavirus 2 (COVID). Intestinal diseases in this invention can be gastrointestinal diseases that cause, for example, infectious enterocolitis, diarrhea, irritable bowel syndrome (IBS), small intestinal bacterial overgrowth (SIBO), Crohn's disease (CD), pancreatic insufficiency, enteritis, fibromyalgia, etc.

[0063] The term "treatment" refers to the partial or complete suppression and / or relief of symptoms of a subject's specific disease (e.g., neurological disorders such as epilepsy); the term "prevention" refers to the prevention or delay of the onset of a clinically evident disease (e.g., neurological disorders such as epilepsy) in a subject. In the context of this invention, prevention and / or treatment should include preventative and / or actual treatment of symptoms of a disease (e.g., proliferative diseases such as cancer), which can be alleviated and / or even completely eliminated by said treatment.

[0064] In a third aspect, the present invention relates to a pharmaceutical composition comprising the compounds of the present invention and a pharmaceutically acceptable carrier and / or excipients.

[0065] The compounds of the present invention are typically administered in the form of pharmaceutical compositions comprising at least one compound according to the invention, optionally, and an inert carrier (e.g., a pharmaceutically acceptable excipient), and, where appropriate, other compounds and / or pharmaceuticals.

[0066] Those skilled in the art are familiar with suitable dosage forms of pharmaceutical compositions, such as solid dosage forms like powders, granules, tablets (especially film-coated tablets), lozenges, sachets, sugar-coated tablets, capsules (such as hard capsules and soft capsules) or suppositories; semi-solid dosage forms like ointments, creams, hydrogels, pastes or patches; or liquid formulations like solutions, emulsions (especially oil-in-water emulsions), suspensions such as lotions, injectable formulations, and infusion formulations. Additionally, liposomes or microspheres may also be used.

[0067] In a particularly preferred embodiment, a pharmaceutical composition comprising at least one compound of the present invention, a pharmaceutically acceptable salt thereof, a solvate, or an optical isomer thereof is provided, said composition being in the form of a gel, ointment, cream, lotion, tablet, pill, capsule, tablet, sugar-coated tablet, powder, aerosol spray, nasal spray, suppository, and / or solution.

[0068] In preparing the pharmaceutical compositions of the present invention, at least one compound of the present invention may optionally be mixed or diluted with one or more carriers and / or excipients. The carriers and / or excipients of the present invention may be solid, semi-solid, or liquid materials, which may serve as solvents, carriers, or media for the active compound.

[0069] In another preferred embodiment, the pharmaceutical composition of the present invention comprises the compound of the present invention in an amount that can activate one or more Kv7 potassium ion channels in a subject.

[0070] In another preferred embodiment, the pharmaceutical composition of the present invention comprises the compounds of the present invention in an amount that can function as an antiepileptic drug (ASM).

[0071] In another preferred embodiment, the pharmaceutical composition of the present invention comprises the compound of the present invention in the form of a dihydrochloride salt.

[0072] In addition, the pharmaceutical compositions of the present invention may further comprise at least one acceptable excipient, such as a wetting agent, emulsifier, suspending agent, preservative, antioxidant, anti-irritant, chelating agent, coating agent, emulsion stabilizer, gelling agent, resin, solvent, solubilizer, neutralizing agent, pigment, silicone derivative, adhesive, filler, desiccant, thickener, wax, plasticizer, or similar substance.

[0073] As used herein, the terms “inventive,” “according to the invention,” “in accordance with the invention,” etc., are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0074] It should be noted that while the terms "compound of the present invention" and "according to the present invention" as used herein are intended to refer to compounds having all aspects and embodiments of the invention described and / or claimed herein, the term JNJ-37822681, depending on its context, refers to a compound containing all aspects and embodiments of the invention described and / or claimed herein, or a compound containing all aspects and embodiments of the invention described and / or claimed in patent application WO2007048779. Those skilled in the art should understand that although the two are structurally similar, they are not entirely identical.

[0075] As used herein, the term “comprising” should be interpreted to encompass both “including” and “consisting of”, both meanings being explicitly intended and therefore disclosed separately according to embodiments of the invention. As used herein, “and / or” should be considered as a specific disclosure of each of two particular features or components, regardless of whether the other is included. For example, “A and / or B” should be considered as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as listed separately herein. In the context of this invention, the terms “about” and “approximately” indicate a range of precision that, as those skilled in the art will understand, still ensures the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±20%, ±15%, ±10%, for example, ±5%. Those skilled in the art will understand that the specific range of deviation allowed for a numerical value of a particular technical effect depends on the nature of that technical effect. For example, natural or biotechnological effects typically have a larger range of deviation than man-made or engineered effects. When an indefinite or definite article is used to refer to a singular noun, such as “one,” “a,” or “the,” the plural form of the noun is also included unless otherwise explicitly stated.

[0076] It should be understood that applying the teachings of this invention to a particular problem or situation, as well as incorporating variations or additional features of this invention (e.g., further aspects and embodiments), is within the capabilities of those skilled in the art, taking into account the teachings contained herein.

[0077] Unless the context otherwise requires, the description and definition of the above features are not limited to any particular aspect or embodiment of the invention, and are equally applicable to all aspects and embodiments described.

[0078] All references, patents and publications cited in this article are incorporated herein by reference in their entirety.

[0079] In view of the foregoing, those skilled in the art should understand that the present invention also relates to the following listed embodiments: Item 1. A compound for treating a subject with a Kv7-related disease, wherein the Kv7-related disease is characterized by the occurrence of epileptic seizures and / or is an epileptic disorder; the compound is selected from compounds of formula (I), or pharmaceutically acceptable salts, hydrates or solvates thereof, or stereoisomers thereof, wherein R is H or C 1-6 Alkyl groups, especially methyl groups; R1 It is a phenyl group; a phenyl group substituted with 1, 2 or 3 substituents selected from the group consisting of: H, halogen, cyano, C. 1-4 Alkyl, C 1-4 Alkoxy, perfluorinated C 1-4 Alkyl, diC 1-4 Alkylamino, particularly 4-fluorophenyl or 3,4-difluorophenyl; thienyl; thienyl group substituted with one or two substituents selected from the group consisting of halogens and C. 1-4 Alkyl; C 3-8 cycloalkyl; or C 5-7 Cycloalkenyl; R 2 For H or C 1-6 alkyl; R 3 Halogen, C 1-4 Alkyl or perfluorinated C 1-4 Alkyl; and R 4 and R 5 Each is independently selected from H or halogens.

[0080] Item 2. The compound according to Item 1, wherein R... 3 It is trifluoromethyl; R, R 4 and R 5 All are H.

[0081] Item 3. The compound according to Item 1, wherein said compound is N -[1-[(3,4-difluorophenyl)methyl]-4-piperidinyl]-6-(trifluoromethyl)-3-pyridazinamine, or compounds having the following formula (II). .

[0082] Item 4. The compound according to Item 1, wherein said compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (III). .

[0083] Item 5. The compound according to Item 1, wherein said compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (IV). .

[0084] Item 6. The compound according to Item 1, wherein said compound is N-[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (V). .

[0085] Item 7. The compound according to Item 1, wherein said compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (VI). .

[0086] Item 8. The compound according to any of the preceding items, wherein the compound is in salt form, preferably, the salt is a dihydrochloride.

[0087] Item 9. A compound according to any of the preceding items, wherein the compound interacts with a Kv7 potassium ion channel, thereby increasing the ion flux through the Kv7 potassium ion channel.

[0088] Item 10. The compound according to any one of items 1 to 9, wherein the compound is a Kv7 agonist, preferably a Kv7.2 or Kv7.3 agonist.

[0089] Item 11. The compound according to any one of the preceding claims, wherein the compound, when bound to Kv7.2, has the ability to interact with or remain in contact with at least one (preferably all) of residues F104, L242, F304, L312, I300, P308, S303, W236, L299, F305, and F240, which are binding sites of Kv7.2 (SEQ ID NO: 1).

[0090] Item 12. The compound according to any of the preceding items, wherein the Kv7-related disease is characterized by the presence of epileptic seizures or epileptic disorders (such as seizures), and in particular, the Kv7-related disease is selected from the group consisting of: epilepsy, neonatal spasms, pain, migraine, neurotransmitter release disorders, smooth muscle contraction dysfunction, movement disorders, dystonia, mania, hearing impairment, neuropathic pain, inflammatory pain, persistent pain, cancer pain, postoperative pain, anxiety disorders, substance abuse, schizophrenia, bladder disorders, vascular disorders, tinnitus, frontotemporal dementia (FTD), familial frontotemporal dementia, or amyotrophic lateral sclerosis (ALS).

[0091] Item 13. The compound according to any of the preceding items, wherein the subject suffers from a disease that induces epilepsy, and administration of the compound to the subject reduces the incidence, prevalence, frequency, duration and / or severity of epileptic seizures.

[0092] Item 14. A pharmaceutical composition comprising a compound as described in any one of items 1 to 13, wherein the amount thereof is sufficient to effectively activate one or more Kv7 potassium ion channels in a subject, and a pharmaceutically acceptable carrier and / or excipient.

[0093] Item 15. The use of the pharmaceutical composition described in Item 14 is as described in any one of Items 1 to 13, preferably for treating a disease characterized by seizures, such as epilepsy, and more preferably, the pharmaceutical composition is used as an ASM.

[0094] Brief description of the accompanying figures and sequences The attached image shows: Figure 1 (A) Representative macroscopic current curves of CHO cells expressing the Kv7.3 A315T subunit before (left) and after (right) application of 1•M retigabine in response to a specified voltage protocol. Current bar, 500 pA; time bar, 0.2 s. (B) Representative macroscopic current curves of CHO cells expressing the Kv7.3 A315T subunit before (left) and after (right) application of 1•M JNJ-37822681 in response to a specified voltage protocol. Current bar, 500 pA; time bar, 0.2 s. (C) Dose-response curves of retigabine (RET, black dot) versus JNJ-37822681 (white dot) acting on the Kv7.3 A315T current. Solid lines represent the results of fitting the experimental data with a four-parameter logic equation to calculate EC. 50 value.

[0095] Figure 2 (A) Representative macroscopic current curves of CHO cells expressing the Kv7.2 / 7.3 subunit before (left) and after (right) application of 1•M retigabine in response to a specified voltage protocol. Current bar, 500 pA; time bar, 0.2 s. (B) Representative macroscopic current curves of CHO cells expressing the Kv7.2 / 7.3 subunit before (left) and after (right) application of 1•M JNJ-37822681 in response to a specified voltage protocol. Current bar, 500 pA; time bar, 0.2 s. (C) Dose-response curves of retigabine (RET, black dot) and JNJ-37822681 (white dot) acting on the Kv7.2 / 7.3 current. Solid lines represent the results of fitting the experimental data with a four-parameter logical equation to calculate EC. 50 value.

[0096] Figure 3 The image shows (A) the effect of 10 μM retigabine on the V channel in homologous Kv7.2 wild-type (black) or mutant W236L (white). 1 / 2 Offset (•V)1 / 2 The effect of 10 μM JNJ-37822681 on V in the homologous Kv7.2 wild-type (black) or mutant W236L (white) channels. 1 / 2 Offset (•V) 1 / 2 The effect of * indicates a significant difference compared to the corresponding control group (p<0.05). Figure 4 This demonstrates the binding sites identified using the PDB structure numbered 7cr2.

[0097] Figure 5 The effects of retigabin, JNJ-37822681, and sulpiride on the spontaneous firing activity of induced neurons are shown. (A) Representative curves of induced neurons recorded using the whole-cell current clamp method. The bar icons above each curve indicate drug administration. (BC) Quantitative analysis of Em (B) and spontaneous firing frequency (C) of induced neurons under no drug treatment (CTL, black bars) or with the specified drug treatment. Each drug was used at a concentration of 10•M. Each data point represents the mean ± standard error of 5–67 cells from at least 3 independent experiments. *p<0.05.

[0098] Figure 6 This demonstrates the effects of retigabin and JNJ-37822681 on induced neurons. Iso and induce neurons G290D Effects on individual neuron and synchronized population activity. As shown, retigabine (RET, blue dot) and JNJ-37822681 (JNJ, red dot) have effects on induced neuronal activity. Iso and induce neurons G290D Concentration-response curves for normalized active area (A), discharge rate (C), and cluster firing frequency (E) in the population. The effect of solvent (0.1% DMSO) was also evaluated (grey). In (A), the solid line represents the fitting of the experimental data with a four-parameter logistic equation to calculate EC. 50 Value. Induced neuron Iso and induce neurons G290D Quantitative analysis of active area (B), firing rate (D), and cluster firing frequency (F) after treatment with solvent (0.1% DMSO) or the following drugs: RET: 10 μM retigabine; JNJ: 10 μM JNJ-37822681; R+X: 10 μM retigabine + 20 μM XE991; J+X: 10 μM JNJ-37822681 + 20 μM XE991; SUL: 10*M sulpiride. Each data point represents the mean ± standard error of HD MEA recordings from 3–8 chips for each neuron. *p<0.05.

[0099] Sequence Listing: SEQ ID NO. 1 shows the amino acid sequence of human potassium voltage-gated channel subfamily KQT member 2: MVQKSRNGGVYPGPSGEKKLKVGFVGLDPGAPDSTRDGALLIAGSEAPKRGSILSKPRAGGAGAGKPPKRNAFYRKLQNFLYNVLERPRGWAFIYHAYVFLLVFSCLVLSVFSTIKEYEKSSEGALYILEIVTIVVFGVEYFVRIWAAGCCCRYRGWRGRLKFARKPFCVIDIMVLIASIAVLAAGSQGNVFATSALRSLRFLQILRMIRMDRRGGTWKLLGSVVYAHSKELVTAWYIGFLCLILASFLVYLAEKGENDHFDTYADALWWGLITLTTIGYGDKYPQTWNGRLLAATFTLIGVSFFALPAGILGSGFALKVQEQHRQKHFEKRRNPAAGLIQSAWRFYATNLSRTDLHSTWQYYERTVTVPMYSSQTQTYGASRLIPPLNQLELLRNLKSKSGLAFRKDPPPEPSPSKGSPCRGPLCGCCPGRSSQKVSLKDRVFSSPRGVAAKGKGSPQAQTVRRSPSADQSLEDSPSKVPKSWSFGDRSRARQAFRIKGAASRQNSEEASLPGEDIVDDKSCPCEFVTEDLTPGLKVSIRAVCVMRFLVSKRKFKESLRPYDVMDVIEQYSAGHLDMLSRIKSLQSRVDQIVGRGPAITDKDRTKGPAEAELPEDPSMMGRLGKVEKQVLSMEKKLDFLVNIYMQRMGIPPTETEAYFGAKEPEPAPPYHSPEDSREHVDRHGCIVKIVRSSSSTGQKNFSAPPAAPPVQCPPSTSWQPQSHPRQGHGTSPVGDHGSLVRIPPPPAHERSLSAYGGGNRASMEFLRQEDTPGCRPPEGNLRDSDTSISIPSVDHEELERSFSGFSISQSKENLDALNSCYAAVAPCAKVRPYIAEGESDTDSDLCTPCGPPPRSATGEGPFGDVGWAGPRK Examples Certain aspects and embodiments of the present invention will now be described with reference to the description, drawings, and tables herein, through examples. Such embodiments of the methods, uses, and other aspects of the invention are merely representative and should not be construed as limiting the scope of the invention to these representative embodiments.

[0100] Selected materials and methods: Human hiPSC-NGN neurons Originating from carrying pathogenic KCNQ2 G290D / + Human induced pluripotent stem cell neurons (hiPSCs) from mutant patients and their corresponding CRISPR / Cas9-repaired homologous cell lines ( Figure 6 (and Table 3), and KCNQ2 mutations originating from another carrier of DEE (KCNQ2). R2O1C / + ; Figure 5 CRISPR / Cas9-repaired homologous cell lines were established as part of an in vivo morphological study of KCNQ2-related diseases (paper in progress). In short, these hiPSCs were created by two individuals carrying KCNQ2. G290D / + and KCNQ2 R2O1C / + Peripheral blood mononuclear cells (PBMCs) from patients with mutated KCNQ2-DEE were reprogrammed. Sample collection was performed with informed consent and approval from the University Hospital of Antwerp and the University of Antwerp Medical Ethics Committee (No. 19 / 20 / 257). Subsequently, constructs containing human neuropoietin 1 and 2 (hNGN1 and hNGN2) under the control of doxycycline-inducible promoters were stably introduced into the CLYBR safe site of each hiPSC cell line. Neural induction was performed for three days using 2 μg / ml doxycycline to obtain predifferentiated neurons on day 3, which were frozen for subsequent differentiation and whole-cell patch-clamp or HD-MEA electrophysiological recording.

[0101] Anatomy of the mouse brain and isolation of astrocytes.

[0102] Primary mouse astrocytes were isolated from the cerebral cortex of C57BL / 6 mouse pups aged 0-3 days postnatally and cultured in T75 flasks in DMEM medium supplemented with 10% fetal bovine serum (FBS). After two passages, mycoplasma was detected in the astrocytes, and the cells were cryopreserved in FBS solution containing 10% dimethyl sulfoxide (DMSO). Animal experiments were approved by the Ethics Committee of the University of Antwerp (NAE 2020-076) and conducted in accordance with their established policies and guidelines.

[0103] Electrophysiological experiments of neurons derived from hiPSCs Whole-cell electrophysiological experiments of neurons derived from hiPSCs.Primary mouse astrocytes were seeded onto glass coverslips coated with 0.07% polyethyleneimine (PEI) and covered with 20 μg / ml laminin, and then cultured in Durbeco Modified Eagle Medium (MEM) containing 10% fetal bovine serum, 1% L-glutamine (100 mL), 1% penicillin (50 U / mL), and 1% streptomycin (50 μg / mL). Once a dense astrocyte layer had formed, 4 × 10⁶ cells were cultured. 4 Day 3 cultured neurons were seeded on top (DIV1). The co-culture system used neuronal-based (NB) complete medium supplemented with doxycycline (2 g / mL), human BDNF (10 ng / mL), human NT3 (10 ng / mL), B-27Plus supplement (50×), Rock inhibitor (10 M), and ARA-C (2 M). After 2 days (DIV3), the medium was replaced with NB supplemented with B27Plus (50×), FBS (50×), BDNF (10 ng / mL), and NT3 (10 ng / mL) to promote the final differentiation of iPSC-derived cortical excitatory neurons (induced neurons). Cells were cultured at 37°C and 5% CO2 humidified to 36 DIV, with half-volume medium changes twice weekly. The currents of induced neurons were recorded at room temperature (20-22°C) using a whole-cell patch-clamp technique with glass microelectrodes at a resistance of 5-7 MW. The preparation methods for the electrode solution and the oxygenated aCSF bath solution were as described above. The current signal was recorded using an Axopatch-200A amplifier, filtered at 5 kHz, and digitized using a DigiData 1440A (Molecular Devices). Data acquisition and analysis were performed using pCLAMP software (version 10.2) (Molecular Devices). Resting membrane potential (Em) and spontaneous action potentials per second (APs / sec) were measured in current-clamp mode. Basal neuronal activity was recorded for 30 seconds, followed by perfusion for 60 seconds with 10 μM retigabin, JNJ-37822681, or (-)-sulpiride, and then co-perfused with 10 μM XE-991 for 30–60 seconds according to the experimental protocol.

[0104] HD-MEA cell seeding.In the HD-MEA experiment, day 3 neurons from hiPSCs were co-cultured with primary mouse astrocytes in MaxTwo 6-well plates (MaxWell Biosystems). Mouse astrocytes were thawed and cultured for one week prior to inoculation to ensure cell viability. HD-MEA culture plates were pretreated sequentially with 1% tebuconazole (1 h) and then with enhanced neuronal basal medium supplemented with B-27 (48 h). Subsequently, the culture plates were coated with 0.14% PEI borate buffer (1 h) and dried overnight. On the day of cell inoculation (day 1 of in vitro culture; DIV1), the culture plates were coated with 20 g / ml CellAdhere Laminin-521 for 1 h prior to inoculation. Day 3 neurons (5e10^5) and mouse astrocytes (5e10^4) were seeded in 50 μl drops of enhanced neuronal basal culture medium supplemented with BDNF (10 ng / ml), NT3 (10 ng / ml), doxycycline (2 μg / ml) (Sigma, D9891), cytosine bD-arabinofuranoside (Ara-C), and CEPT complex reagent (HY-K1043, MCE). The medium was changed at half volume twice weekly until DIV26 to promote neuronal induction. G290D and induce neurons Iso The final differentiation.

[0105] HD-MEA record. All HD-MEA recordings were performed using MaxLab Live software and filtered with a 300 Hz high-pass filter and a peak detection threshold of 5.0 standard deviation. First, activity across the entire plate was measured using an activity scan assay with a pre-defined checkerboard electrode sampling pattern. Then, network-level cluster firing activity was recorded for 10 min using a network assay, selecting the 1,024 electrodes with the highest amplitude determined in the corresponding activity scan assay. On the day of drug treatment, untreated baseline recordings were obtained from all wells using both activity scan assays and network assays. 10 μL of drug was added to a 1.5 mL well. Two minutes after each drug administration, network assays were performed to detect changes in cluster firing parameters, and activity scan assays were performed to assess the overall effect on individual neurons. Recorded data were exported and analyzed in GraphPad Prism.

[0106] The example shows: Example 1: High-throughput screening of relocation libraries To identify repositioning compounds that could serve as novel and safer Kv7.2 / Kv7.3 channel openers, a cell-based high-throughput screening (HTS) library was used to screen the Fraunhofer repositioning library.

[0107] The Fraunhofer Repositioning Library, originating from the Bode Repositioning Center, contains approximately 5,600 bioactive molecules, the vast majority of which have been clinically approved for the treatment of neurological / psychiatric, infectious, cardiac, and endocrine disorders.

[0108] The Fraunhofer relocalization library was initially screened in a cell model consisting of Chinese hamster ovary (CHO) cells stably transfected with a Kv7.3 channel variant (carrying the A315T mutation to enhance current magnitude and improve signal-to-noise ratio). This Kv7.3 A315T cell line was used for fluorescence-based thallium flux assay (FluxOR, Thermo Fisher), an optimized readout indicator of Kv7 channel function, with retigabine as a positive control.

[0109] Compounds from the Fraunhofer relocalization library were tested in cells expressing Kv7.3 A315T at a concentration of 10-M, and their Kv7 channel-opening effects were compared with those observed with 10-M retigabine. Of the 5600 compounds, 59 were selected as candidates because they showed a relative percentage activation of ≥20% (relative to retigabine). Upon retesting in the same assay, 12 compounds showed reproducible activity, and their potency in the dose-response assay was subsequently analyzed using ten-point dose-response curves from 0.5 nM to 10 μM. Compound JNJ-37822681 was identified as the most active newly discovered Kv7 channel opener. v 7.3 A315T channel opener; in FluxOR assays, its potency and maximum titer are comparable to retigabine (retigabine's EC50). 50s The maximum activity of retigabine was 0.30 μM, while that of JNJ-37822681 was 0.14 μM; the maximum activity of retigabine was 100%, while that of JNJ-37822681 was 83.8%.

[0110] Example 2: Electrophysiological characteristics analysis of JNJ-37822681 as a Kv7 activator The effect of JNJ-37822681 on the opening of Kv7 channels was further investigated using whole-cell patch-clamp electrophysiology (the gold standard for studying ion channel activity and pharmacological regulation). This study was conducted on Kv7 channels transiently transfected with Kv7. v 7.3 A315T cDNA in CHO cells ( Figure 1 Voltage dependence of 1 μM retigabine-induced channel activation (•V) 1 / 2 The voltage shifted to the left by approximately 10 mV, and the maximum current density increased slightly; consistent with previous reports; similar effects were also observed when using 1 μM JNJ-37822681. Figure 1 ).

[0111] To better assess the quantitative difference between retigabin and JNJ-37822681 in Kv7 opening capacity, a dose-response experiment (0.01 • 30 • M) was performed, and the Kv7 opening capacity was measured using • V. 1 / 2 EC Functional parameter calculation 50 value( Figure 1 C). In cells transfected with Kv7.3 A315T, JNJ-37822681 was slightly less potent than retegabin; its EC50... 50 The values ​​were 2.0 ± 0.03 μM and 0.6 ± 0.1 μM, respectively, while the maximum •V 1 / 2 The values ​​were 37.0±5.4 mV and 60.9±6.1 mV, respectively (p<0.05, n=3-9). Figure 1 C).

[0112] Most importantly, when both compounds were tested in CHO cells expressing Kv7.2+Kv7.3 channels (representing a more physiological channel configuration), they both exhibited similar efficacy and potency; in fact, the calculated EC50 of JNJ-37822681 was similar. 50s The value was 1.2 ± 0.3 μM, and that of retigabine was 2.5 ± 1.8 μM (p > 0.05, n = 3-8); Figure 2 (A, B, C).

[0113] Example 3: Binding site of JNJ-37822681 in Kv7.2 To better elucidate the mechanism of action of JNJ-37822681 on the Kv7 channel, the molecular characterization of its binding site was investigated. It is known that Kv7 activators bind to two distinct sites in the Kv7 channel: one in the voltage-sensing domain, recognized by benzamide compounds, and the other in the pore domain, occupied by retigabine. In the latter, the hydrogen bond formed between the retigabine carbamate group and the indole nitrogen atom of a tryptophan residue (W236 in Kv7.2) within the Kv7.2-Kv7.5 channel pore domain is crucial for the opening effect of the Kv7.2-Kv7.5 channel. This tryptophan residue acts as a hydrogen bond donor (HBD), while the retigabine carbamate group acts as a hydrogen bond acceptor (HBA). In fact, the W236L mutation in Kv7.2 largely eliminates retigabine-induced Kv7.2 activation.

[0114] Computer simulations of molecular docking of JNJ-37822681 based on the cryo-electron microscopy structure of Kv7.2 revealed that the nitrogen atom in the piperazine ring of JNJ-37822681 can act as an acceptor for hydrogen bonds donated by W236, thereby generating a similar interaction with the carbamate group of retigabine. Furthermore, the two phenylalanine residues (F240 and F305) in Kv7.2, known to interact with the p-fluorophenyl group of retigabine, also interact with the difluorophenyl group in JNJ-37822681. These two observations suggest that the stabilization of JNJ-37822681 and retigabine in the Kv7.2 subunit involves similar structural determinants, thus supporting the view that the binding sites largely overlap.

[0115] Functional experiments fully supported the molecular docking simulation data and confirmed the hypothesis that JNJ-37822681 and retigabine have similar binding sites on the Kv7 channel; in fact, JNJ-37822681 (10 • M) failed to activate the retigabine-insensitive Kv7.2 channel carrying the W236L mutation. Figure 3 ).

[0116] Example 4: Anticonvulsant effect of JNJ-37822681 in two mouse epilepsy models Electrophysiological experiments showed that JNJ-37822681 is a novel Kv7.2 / 7.3 channel opener with a mechanism of action similar to retigabine. Given that activation of Kv7.2 / 7.3 channels is known to have antiepileptic effects in vivo, the potential anticonvulsant activity of JNJ-37822681 was investigated.

[0117] JNJ-37822681 was tested in two widely used animal models of acute epilepsy for evaluating novel ASMs: 1. A mouse model of generalized myoclonic epilepsy, for example, acute exposure to GABA. A 1. The receptor antagonist pentylenetetrazol (PTZ); 2. Auditory-induced seizures induced in genetically epilepsy-susceptible DBA / 2 mice. The efficacy of retigabine in reducing the severity / frequency of seizures was described in detail in both models; furthermore, other novel Kv7.2 / 7.3 activators also showed efficacy in antagonizing PTZ-induced seizures or sound-induced seizures in DBA / 2 mice, thus confirming the effectiveness of these two animal models in evaluating the anticonvulsant efficacy of Kv7 channel activators.

[0118] In both animal models, retegabine or JNJ-37822681 was administered via intraperitoneal injection 30 minutes before PTZ or sound-induced seizures.

[0119] In the PTZ model, retigabine (5-30 mg / kg) effectively reduced the severity of the clonic and tonic phases of epileptic seizures, and its ECV... 50S The values ​​were 13 and 9 mg / kg, respectively. JNJ-37822681 (10-60 mg / kg) was effective in controlling both the clonic and tonic phases of epileptic seizures, and its efficacy was comparable to that of retigabine (ECG value during the clonic phase). 50S The concentration was 15 mg / kg for the tonic phase and 9 mg / kg for the tetanic phase. When animals were pretreated with the Kv7 antagonist XE-991 (intraperitoneal injection, 3 mg / kg), the anticonvulsant effects of both retigabine and JNJ-37822681 were weakened (Table 1), thus demonstrating that the anticonvulsant effect of JNJ-37822681 is mediated by activation of the XE-991-sensitive Kv7 channel.

[0120] Table 1. Efficacy of retigabin and JNJ-37822681 in PTZ-induced seizures in C57BL / 6J mice * and # indicate p < 0.05; ** and ## indicate p < 0.01 (compared to the corresponding control group). In the DBA / 2 model, retigabin (3-20 mg / kg) reduced sound-induced seizures in a dose-dependent manner, showing a protective effect during the runaway, clonic, and tonic phases. The calculated ECGs... 50 The values ​​were 12, 7, and 4.6 mg / kg, respectively. Compared with retigabine, JNJ-37822681 (5-40 mg / kg) was slightly more effective in reducing the runaway, clonic, and tonic phases of seizures, with calculated EC50 values. 50 The values ​​were 11, 6, and 3 mg / kg, respectively. Pretreatment with the Kv7 antagonist XE-991 (intraperitoneal injection, 3 mg / kg) reversed the anticonvulsant effects of retigabine and JNJ-37822681, further demonstrating that the anticonvulsant effect observed in JNJ-37822681 is due to its Kv7 channel opening effect. The results are summarized in Table 2.

[0121] Table 2. Efficacy of retigabin and JNJ-37822681 in treating auditory-induced seizures in DBA / 2 mice * and # indicate p < 0.05; ** and ## indicate p < 0.01 (compared to the corresponding control group). Example 5: JNJ-37822681 reduces the spontaneous excitability of individual iPSC-derived cortical glutamatergic neurons. The Kv7 channel plays a major role in regulating neuronal firing. To investigate the effects of JNJ-37822681 on intrinsic neuronal excitability, the acute effects of this drug on spontaneous firing of mature cortical-like glutamatergic neurons (induced neurons) were examined. For this purpose, researchers used KCNQ2 mutants carrying DEE-induced KCNQ2 (KV7-induced K ... R2O1C / + Neurons differentiated from CRISPR / Cas9-corrected (homologous)-induced pluripotent stem cells (hiPSCs) in patients with KCNQ2. This cell line (and the cell line described below) was developed as part of an in vivo morphological study of KCNQ2-related diseases (paper in progress). Induced neurons differentiated after 21 to 36 days (DIV) of in vitro culture and were morphologically identified as neurite extension cells with a resting membrane potential (Em) of -41.9 ± 1.1 mV and generated spontaneous action potentials (APs) at a frequency of 3.14 ± 0.36 APs / second. After recording neuronal firing for approximately 30 seconds, the induced neurons were perfused with 10 μM retigabine or JNJ-37822681 for 60 seconds; both drugs hyperpolarized Em by approximately 8 mV and significantly reduced the firing frequency. Figure 5 (Table 2). Following perfusion with only 10 μM retigabine or JNJ-37822681, the Kv7 channel blocker XE-991 was added. 47 (10 μM) restored Em and AP firing frequencies to control values. Since JNJ-37822681 was developed as an antagonist of dopaminergic D2 receptor (D2R), and D2R is also expressed in induced neurons, as confirmed by RNAseq data (paper under writing), the effect of the selective D2R antagonist (-)-sulpiride was also investigated. 10 μM of (-)-sulpiride did not affect neuronal firing or Em ( Figure 5 This indicates that it is the activation of the Kv7.2-5 channel, rather than the blockade of D2R, that contributes to the inhibition of induced neuronal firing by JNJ-37822681.

[0122] Example 6: JNJ-37822681 inhibits the overall and synchronic activity of induced neuronal populations from KCNQ2-DEE patients. In patients with KCNQ2-DEE carrying recurrent Kv7.2 G290D LOF mutations, the induced neurons... G290D ) and homologous control cell lines corrected by CRISPR / Cas9 (induced neurons) IsoIn iPSC-derived induced neurons, the effect of JNJ-37822681 on neural network activity was investigated using HD MEA recording technology. Spontaneous extracellular electrical activity was monitored for up to 26 days using activity scanning and network detection techniques. Both cell lines showed peak activity around DIV10 and developed into strong synchrotron firing activity from DIV14 to DIV21. At DIV26, compared with induced neurons... Iso , induce neurons G290D They exhibited highly excitable characteristics, including a larger active area of ​​the electrode array, a higher spontaneous firing frequency, and a greater number of spikes per cluster firing, while the cluster firing frequency remained unchanged at this maturation stage (Table 3). Previously, increased firing rates and increased spikes per cluster firing have also been described in MEA records of iPSC-derived neurons carrying similar KCNQ2-DEE LOF mutants and other models of iPSC-derived neurons with hereditary cortical hyperexcitability.

[0123] On day 26 of culture (DIV26), neurons were induced. lso and induce neurons G290D Exposure to escalating concentrations (0.03–10 μM) decreases overall neuronal activity in a concentration-dependent manner, reflected in a reduction in normalized active area, which measures the total amplitude and frequency of signals recorded at each electrode. This provides a reliable indicator of population drug sensitivity, even in cases of uncoordinated network cluster firing activity. Figure 6 A). Retigabine and JNJ-37822681 inhibited the induction of neurons lso and induce neurons G290D Both showed considerable effectiveness, and the calculated IC 50 The value was approximately 0.60 μM in homologous cell lines and approximately 1.2 μM in mutant strains (retigabine and JNJ-37822681 inducing neurons). lso The values ​​were 0.61±0.09 and 0.60±0.12, respectively, while in the induced neurons... G290D The mean values ​​were 1.14±0.22 and 1.21±0.28, respectively; n=6-8, p>0.05). It has been reported that retigabine has a similar IC50 value in reducing the spontaneous firing activity of hiPSC-derived sensory neurons. 50 Value. At a maximum concentration of 10 μM, retigabin and JNJ-37822681 silenced over 95% of active neurons; inducing neuronal... lso and induce neurons G290D In this study, incubation with 20 μM XE991 for 30 min could essentially reverse neuronal silencing induced by retigabine and JNJ-37822681. Figure 6B). After treatment with retigabin and JNJ-37822681, the discharge rate also showed a concentration-dependent decrease ( Figure 6 C), this effect was reversed by XE991, and no significant differences were observed between different drugs or different induced neuronal cell lines. Figure 6 D). To evaluate the effect of D2 receptor blockade on the inhibition of neurons induced by JNJ-37822681. lso and induce neurons G290D The potential role in electroactivity was also investigated by examining the effects of the selective D2 receptor antagonist (-)-sulpiride. 10 μM (-)-sulpiride induced neuronal activity at DIV26. lso and induce neurons G290D Total active area ( Figure 6 B) and discharge rate ( Figure 6 D) None of them have any effect.

[0124] Table 3. Induced neurons recorded by HD MEA at DIV26 lso and induce neurons G290D Electrophysiological characteristics of the population. Each data point is the mean ± standard error of 12-18 MEA chips recorded in 3 independent experiments. *p<0.05 indicates induced neurons. lso With induced neurons G290D compared to Retigabine and JNJ-37822681 inhibit network cluster firing in a concentration-dependent manner, and at concentrations of 3 μM or higher, induce neuronal firing. lso and induce neurons G290D All cluster releases were completely suppressed. Figure 6 E); however, after applying 20 μM XE991, the cluster firing frequency was fully restored ( Figure 6 F). In contrast to the cluster firing inhibition effects of retigabin and JNJ-37822681, sulpiride tended to increase cluster firing frequency in both cell lines, although this effect only occurred when neurons were induced. lso The significance level was statistically significant. Figure 6 F). In summary, the activity pattern of JNJ-37822681 is highly consistent with that of retigabine, which works by reducing the activity of induced neurons. lso and induce neurons G290D The excitability of individual neurons, in turn, inhibits the firing activity of synchronous clusters.

[0125] In induced neurons known to express D2R, 52Both JNJ-37822681 and retigabine induced XE991-sensitive hyperpolarization and inhibited spontaneous firing, while the selective D2R antagonist (-)-sulpiride was ineffective. JNJ-37822681 effectively reduced the activity of individual neurons and synchrotrons in induced neurons derived from KCNQ2-DEE patients and in homologous control cell lines, indicating its potency is comparable to retigabine. In these cell lines, unlike JNJ-37822681 and retigabine, sulpiride increased the frequency of synchrotron cluster firing, again demonstrating that in this in vitro model, JNJ-37822681 primarily inhibits intrinsic neuronal firing and synchrotron cluster firing by activating the Kv7.2-5 channel, rather than by antagonizing D2R.

Claims

1. A compound for treating a subject's Kv7-related disease, characterized in that, The Kv7-related disease is characterized by the occurrence of epileptic seizures and / or is an epileptic disorder; the compound is selected from compounds of formula (I), or pharmaceutically acceptable salts, hydrates or solvates thereof, or stereoisomers thereof. in, R is H or C 1-6 Alkyl groups, especially methyl groups; R 1 It is a phenyl group; a phenyl group substituted with 1, 2 or 3 substituents, each substituent being independently selected from the group consisting of: H, halogen, cyano, C. 1-4 Alkyl, C 1-4 Alkoxy, perfluorinated C 1-4 Alkyl, diC 1-4 Alkylamino, particularly 4-fluorophenyl or 3,4-difluorophenyl; thienyl; thienyl group substituted with one or two substituents selected from halogens and C. 1-4 Alkyl; C 3-8 cycloalkyl; or C 5-7 Cycloalkenyl; R 2 For H or C 1-6 alkyl; R 3 Halogen, C 1-4 Alkyl or perfluorinated C 1-4 Alkyl; and R 4 and R 5 Each is independently selected from H or halogens.

2. The compound according to claim 1, characterized in that, The R 3 It is trifluoromethyl; R, R 4 and R 5 All are H.

3. The compound according to claim 1, characterized in that, The compound is N -[1-[(3,4-difluorophenyl)methyl]-4-piperidinyl]-6-(trifluoromethyl)-3-pyridazinamine, or compounds having the following formula (II). 。 4. The compound according to claim 1, characterized in that, The compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (III). 。 5. The compound according to claim 1, characterized in that, The compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (IV). 。 6. The compound according to claim 1, characterized in that, The compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (V). 。 7. The compound according to claim 1, characterized in that, The compound is N -[1-(4-fluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazine-3-amine, or a compound conforming to formula (VI). 。 8. The compound according to any one of the preceding claims, characterized in that, The Kv7-related diseases are Kv7.2 and / or Kv7.3-related diseases.

9. The compound according to any one of the preceding claims, characterized in that, The compound interacts with the Kv7 potassium ion channel, thereby increasing the ion flux through the Kv7 potassium ion channel.

10. The compound according to any one of claims 1 to 9, characterized in that, The compound is a Kv7 agonist, preferably a Kv7.2 or Kv7.3 agonist.

11. The compound according to any one of the preceding claims, characterized in that, When the compound is bound to Kv7.2, it has the ability to interact with or maintain contact with at least one (preferably all) of the residues F104, L242, F304, L312, I300, P308, S303, W236, L299, F305, and F240, which are the binding sites of Kv7.2 (SEQ ID NO: 1).

12. The compound according to any one of the preceding claims, characterized in that, The Kv7-related diseases are selected from epilepsy, neonatal spasms, pain, migraine, neurotransmitter release disorders, smooth muscle contraction dysfunction, movement disorders, dystonia, mania, hearing impairment, neuropathic pain, inflammatory pain, persistent pain, cancer pain, postoperative pain, anxiety, substance abuse, schizophrenia, bladder disease, vascular disease, tinnitus, frontotemporal dementia (FTD), familial frontotemporal dementia, or amyotrophic lateral sclerosis (ALS).

13. The compound according to any one of the preceding claims, characterized in that, The subject suffers from a disease that triggers epileptic seizures, wherein administration of the compound to the subject reduces the incidence, prevalence, frequency, duration, and / or severity of epileptic seizures.

14. A pharmaceutical composition comprising the compound of any one of claims 1 to 13, wherein the amount thereof is sufficient to effectively activate one or more Kv7 potassium ion channels in a subject, and a pharmaceutically acceptable carrier and / or excipient.

15. The pharmaceutical composition according to claim 14, used for any one of claims 1 to 13, preferably for treating a disease characterized by seizures, such as epilepsy, and more preferably, the pharmaceutical composition as an ASM.

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