Selective KCNQ channel opener and application thereof in pharmacy

Abstract

The invention provides a selective KCNQ channel opener and application of the selective KCNQ channel opener in pharmacy. According to the invention, a series of KCNQ channel openers with high selectivity are obtained by carrying out structural modification and activity and selectivity evaluation on a KCNQ2 agonist 124-A10 (CAS number: 1173735-00-6) obtained by screening. Compared with common KCNQ channel openers, such as retigabine, ICA27243 and the like, the subtype selectivity of the compounds is enhanced, so that some toxic and side effects generated in the application of pharmaceutical preparations due to poor selectivity can be effectively avoided. The invention also provides applications of the compounds in preparation of anti-epileptic drugs, anti-schizophrenia drugs, anti-depression drugs and drugs for treating cerebral arterial thrombosis diseases, and provides more effective choices for treatment of related diseases.

Description

Technical Field

[0001] This invention relates to KCNQ potassium ion channel openers, specifically a selective KCNQ channel opener and its application in pharmaceuticals. Background Technology

[0002] KCNQ potassium channels are a class of voltage-gated potassium channels (Kv), with five known subtypes (Kv7.1–Kv7.5) encoded by the KCNQ1–KCNQ5 genes, respectively. Different KCNQ subtypes differ in tissue distribution, physiological function, and pathological function. KCNQ1 is mainly distributed in the myocardium, and is also expressed in the inner ear, kidneys, stomach, thyroid, and pancreas. KCNQ2 / 3 are mainly distributed in the nervous system; KCNQ4 is mainly expressed in the inner ear, the auditory pathway of the central nervous system, and visceral and vascular smooth muscle, and is closely related to hearing impairment and pain; KCNQ5 is mainly distributed in the central and peripheral nervous systems, skeletal muscle, and smooth muscle.

[0003] KCNQ2 channels are widely distributed in the central and peripheral nervous systems. In the central nervous system, KCNQ3 and KCNQ2 channels largely overlap and are often co-expressed, forming heteromeric channels with stronger currents than those of single-channel expression. Therefore, in studies of KCNQ2 channel openers, KCNQ2 channels can be replaced by KCNQ2 / 3 channels. The M-channel currents mediated by KCNQ2 and KCNQ2 / 3 channels can be regulated by acetylcholine M1 receptors, hence they are also called M-channels. M-channels play a crucial role in regulating neuronal excitability and nerve signal transmission, and are essential for the normal functioning of the nervous system. KCNQ2 and KCNQ2 / 3 channels have become a key target in M-channel research, potential therapeutic targets for various related ion channel diseases, and a hot topic in potassium ion channel research.

[0004] The KCNQ2 channel is considered an important target for antiepileptic drugs. Retigabine (RTG), a KCNQ2 agonist, was approved by the FDA for the treatment of partial seizures in adults, but was withdrawn from the market due to adverse reactions such as central nervous system side effects, pigment deposition, and urinary retention. Pinegabin (HN37), synthesized by the Shanghai Institute of Materia Medica, has been approved for clinical trials in China. This drug has undergone structural optimization, reducing the risk of pigment deposition and enhancing drug activity, but subtype selectivity remains significantly unchanged, still activating KCNQ2-KCNQ5 channels. In addition, dozens of other KCNQ2 agonists have been reported, such as ICA069673, ICA27243, acrylamide, (S)-1, ML213, PF-05020182, RL12, and SCR268, but only a handful have actually entered clinical trials. This is mainly because most of the reported KCNQ2 agonists have poor selectivity. Therefore, enhancing subtype selectivity and effectively reducing side effects caused by multiple targets is a key focus of current KCNQ2 agonist research. In selectivity studies, not all compounds possess absolute selectivity, meaning they do not act on only a single target. In fact, many compounds exhibit relative selectivity, which is usually reflected in their action on only some subtypes or differences in the intensity of their action on different subtypes. Summary of the Invention

[0005] The purpose of this invention is to provide a selective KCNQ channel opener and its application in pharmaceuticals, in order to solve the problem of poor selectivity of existing KCNQ2 agonists.

[0006] This invention is implemented as follows:

[0007] A selective KCNQ channel opener, the chemical structure of which is shown in formula (I) a As shown in the image:

[0008] (I) a )

[0009] Wherein, R1 is -CH2-, phenyl, or heteroaryl-substituted methyl;

[0010] When R1 is -CH2-, R2 is cyclopentyl, cyclohexyl, cyclohexylmethyl, thiazolylmethyl, thiazolyl, methylthiazolyl, aryl, haloaryl, trifluoromethylaryl, 4-trifluoromethylbenzyl or 3,4-dibromobenzonitrile-2-yl, and R3 is naphthyl, haloaryl, trifluoromethylaryl, cyclopentyl, trifluoromethylcyclohexyl or halopyridyl.

[0011] When R1 is phenyl, R2 is thiazolyl or thiazolylmethyl, and R3 is aryl or haloaryl;

[0012] When R1 is a heteroaryl-substituted methyl group, R2 is a thiazolyl, aryl, or haloaryl group, and R3 is an aryl or haloaryl group.

[0013] This invention also provides another selective KCNQ channel opener with the following chemical structure: (I) b As shown in the image:

[0014] (Ⅰ) b )

[0015] Wherein, R1 is aryl, R2 is thiazolyl, aryl, haloaryl or 4-trifluoromethylbenzyl, and R3 is aryl or haloaryl.

[0016] The beneficial effects of this invention are reflected in the above chemical structural formula (I) a ) and (Ⅰ b The compound shown has high selectivity for KCNQ2 / 3 channels, but no significant effect on KCNQ1 and KCNQ5 channels. Compared with poorly selective KCNQ openers such as RTG and ICA27243, its subtype selectivity is enhanced. Therefore, it can effectively avoid some of the toxic side effects caused by the poor selectivity of existing KCNQ2 channel openers in pharmaceutical formulation applications.

[0017] Given that the compounds provided by this invention are highly active and selective KCNQ2 / 3 channel openers, this invention also proposes the following applications of them in pharmaceutical formulations:

[0018] The above chemical structural formula (I) a ) and (Ⅰ b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates or pharmaceutically acceptable salts in the preparation of antiepileptic drugs.

[0019] The above chemical structural formula (I) a ) and (Ⅰ b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antipsychotic drugs.

[0020] The above chemical structural formula (I) a ) and (Ⅰ b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates or pharmaceutically acceptable salts in the preparation of antidepressant drugs.

[0021] The above chemical structural formula (I) a ) and (Ⅰ bThe use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of medicaments for the treatment of ischemic stroke. Attached Figure Description

[0022] Figure 1 This is a graph showing the results of determining the activity of compound QO-A2 on the KCNQ2 channel based on ICR8000 technology.

[0023] Figure 2 This is a dose-response curve of compound QO-A2 on the KCNQ2 / 3 channel, determined using ICR8000 technology.

[0024] Figure 3 This is a dose-response curve of the lead compound 124-A10 on the KCNQ2 / 3 channel, determined using ICR8000 technology.

[0025] Figure 4 This is a graph showing the results of evaluating the activity of compound QO-A2 on the KCNQ2 channel based on patch-clamp technique. (A) shows the typical outward current induced by a step depolarization from a sustained potential of -80 mV to 0 mV, with a concentration of compound QO-A2 of 10 μM. (B) shows the activity results of compound QO-A2 at different concentrations on the KCNQ2 channel.

[0026] Figure 5 This is a graph showing the anticonvulsant activity of compound QO-A2 in a mouse MES model. (A) shows the protective rate of compound QO-A2 in the mouse MES model. (B) shows the dose-response curve of QO-A2 in the mouse MES model (n = 8).

[0027] Figure 6 This study investigated the effects of compound QO-A2 on different isoforms of KCNQ. n = 3, Mean ± SEM. A t-test was used to compare the effects with the control group. < 0.05, < 0.01. Detailed Implementation

[0028] This invention aims to develop a highly active and selective KCNQ opener by modifying the structure and evaluating the activity and selectivity of the screened KCNQ2 agonist 124-A10 (CAS No.: 1173735-00-6), providing a more effective option for the treatment of epilepsy and related diseases.

[0029] A selective KCNQ2 channel opener, the chemical structure of which is shown in formula (I):

[0030] (I)

[0031] When A1 is 0, and A2 and A3 are not of any structure, structure (Ⅰ) is represented as:

[0032] (Ⅰ) a )

[0033] When R1 is -CH2-, R2 is independently cyclopentyl, cyclohexyl, cyclohexylmethyl, thiazolylmethyl, thiazolyl, methylthiazolyl, aryl, haloaryl, trifluoromethylaryl, 4-trifluoromethylbenzyl or 3,4-dibromobenzonitrile-2-yl, and R3 is independently naphthyl, haloaryl, trifluoromethylaryl, cyclohexyl, cyclopentyl, trifluoromethylcyclohexyl or halopyridyl.

[0034] When R1 is phenyl, R2 is independently thiazolyl or thiazolylmethyl, and R3 is aryl or haloaryl.

[0035] When R1 is a heteroaryl-substituted methyl group, R2 is independently a thiazolyl, aryl, or haloaryl group, and R3 is independently an aryl or haloaryl group.

[0036] When A1, A2, and A3 are C, the structure (Ⅰ) is represented as:

[0037] (Ⅰ) b )

[0038] R1 is aryl, R2 is independently thiazolyl, aryl, haloaryl, or 4-trifluoromethylbenzyl. R3 is independently aryl or haloaryl.

[0039] The structures shown above, as well as their stereoisomers, hydrates, solvates, and pharmaceutically acceptable salts, can all be used to prepare antiepileptic drugs.

[0040] I. Synthesis of Compounds

[0041] 1.1 Synthesis of intermediates

[0042] 1) Synthesis of 4-aminomethylthiazole (intermediate I)

[0043]

[0044] In a 150 mL three-necked flask, 5 g (50 mmol) of 4-methylthiazole, 0.09 g (0.55 mmol) of azobisisobutyronitrile (AIBN), and 70 mL of dichloromethane (DCM) were added. The flask was placed in a constant-temperature oil bath with a stirrer and heated to reflux at 70 °C while stirring to dissolve the methylthiazole. N-bromosuccinimide (7.79 g, 55 mmol) was added in portions, and the mixture was refluxed for 7 h. Thin-layer chromatography (TLC) was used to determine if the 4-methylthiazole reaction was complete. The reaction solution was cooled to room temperature, and a yellow solid precipitated. The solid was filtered, and the filtrate was washed with 100 mL of water and 50 mL of saturated NaCl water, dried over anhydrous magnesium sulfate, washed with n-hexane, and the solvent was evaporated to obtain a yellow liquid, 4-bromomethylthiazole (5.15 g, 58.2%).

[0045] 4-Bromomethylthiazole (4.5 g, 25.4 mmol), potassium phthalimide (4.7 g, 25.5 mmol), and tetrabutylammonium bromide (0.8 g, 2.5 mmol) were added to a round-bottom flask, and 30 mL of N,N-dimethylformamide (DMF) was added. The mixture was stirred at room temperature and reacted for 24 h. The reaction was confirmed to be complete by TLC. Water was added to precipitate the solid, which was filtered and dried in an oven at 50 °C to obtain 1.04 g of a yellowish-white needle-like solid. The filtrate was extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and the solvent was removed to obtain 0.24 g of a yellow solid. The solids were combined to obtain 1.28 g of 2-(4-thiazolylmethyl)-1H-isoindole-1,3(2H)-dione (21%).

[0046] An ethanol solution of 2-(4-thiazolylmethyl)-1H-isoindole-1,3(2H)-dione (1 g, 4.1 mmol) and 35% hydrazine hydrate (0.64 g, 4.5 mmol) was refluxed in an oil bath at a constant temperature and stirred for 3 h. The starting material disappeared as detected by TLC. The mixture was cooled to room temperature, and a solid precipitated. The solid was filtered off. The filtrate was concentrated, and the concentrate was cooled to room temperature. A small amount of ethyl acetate was added, and the solid was filtered off. An ethyl acetate solution of HCl was added to the filtrate, and a yellow precipitate precipitated. The precipitate was filtered, washed with ethyl acetate, and dried in an oven at 50 °C to give the hydrochloride salt of intermediate I (0.25 g, 40.0%). ESI-MS (m / z): 115.1 [M + H] + .

[0047] 2) Synthesis of intermediate II

[0048]

[0049] Synthesis of intermediate IIa (N-(3-fluorobenzoyl)glycine):

[0050] Glycine (2.6 g, 34.7 mmol) and potassium carbonate (9.6 g, 69.4 mmol) were placed in a round-bottom flask, and 25 mL of DCM was added. The mixture was stirred and dissolved at room temperature. 15 mL of a DCM solution containing 3-fluorobenzoyl chloride (5 g, 31.5 mmol) was slowly added dropwise using a constant-pressure dropping funnel. The reaction was carried out at room temperature for 24 h. The solvent was evaporated, 50 mL of water was added, and the mixture was filtered. The pH of the filtrate was adjusted to 2–3 with 2 M hydrochloric acid, resulting in the precipitation of a yellow-green solid. This solid was filtered again, and the filter cake was dried in a 50°C oven to obtain intermediate IIa (2.3 g, 37%). ESI-MS (m / z): 196.1 [M–H]. - .

[0051] Synthesis of intermediate IIb (N-(3-fluorobenzoyl)histidine):

[0052] Following the method for synthesizing IIa, 3-fluorobenzoyl chloride (1 g, 6.3 mmol) was added to a 30 mL DCM mixture of histidine (1.1 g, 7.1 mmol) and potassium carbonate (1.7 g, 12.3 mmol), and reacted at room temperature to give intermediate IIb (0.94 g, 53.8%), a white solid, ESI-MS (m / z): 276.1 [M – H]. - .

[0053] Synthesis of intermediate IIc (3-[(3-fluorobenzoyl)amino]benzoic acid):

[0054] Following the method for synthesizing IIa, m-aminobenzoic acid (0.95 g, 7.8 mmol), potassium carbonate (1.7 g, 12.3 mmol), 3-fluorobenzoyl chloride (1 g, 6.3 mmol), and DCM were placed in a round-bottom flask and reacted at room temperature to give intermediate IIc (1.6 g, 98%), a white solid, ESI-MS (m / z): 258.1 [M – H]. - .

[0055] 3) Synthesis of intermediate III

[0056]

[0057] Synthesis of 2-amino-N-(thiazole-2)acetamide (IIIa):

[0058] In a 100 mL round-bottom flask, N-tert-butoxycarbonylglycine (2.0 g, 11.4 mmol), 2-aminothiazole (1.15 g, 11.5 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (2.82 g, 11.4 mmol) were added, followed by the addition of 40 mL of DCM. The mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC until complete. The solvent was evaporated, the mixture was washed with 2 M hydrochloric acid, filtered, the filter cake was washed with water, and dried in an oven at 50 °C to give 1,1-dimethylethyl N-[2-oxo-2-(2-thiazolylamino)ethyl]carbamate (2.6 g, 89%), a yellowish-white solid.

[0059] All of 1,1-dimethylethyl N-[2-oxo-2-(2-thiazolylamino)ethyl]carbamate was added to 40 mL of DCM, followed by 6 mL of HCl-methanol solution (4 M). The mixture was stirred at room temperature for 3 h, and the starting material disappeared as detected by TLC. The solvent was evaporated to obtain a thick paste, which was dissolved in 20 mL of water. The pH was adjusted to 9-10 with 6 M sodium hydroxide solution, resulting in the precipitation of a brown solid. The paste was filtered and dried in an oven at 50 °C. The filtrate was extracted with 60 mL of ethyl acetate (partial extraction until no product was observed in the aqueous layer). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated to obtain a brown solid. The solids were combined to give intermediate IIIa (0.64 g, 40%), ESI-MS (m / z): 158.1 [M + H]. + .

[0060] Synthesis of 2-amino-N-(5-methylthiazol-2)acetamide (Ⅲb):

[0061] N-tert-butoxycarbonylglycine (1.5 g, 8.56 mmol), 2-amino-4-methylthiazole (0.99 g, 8.7 mmol), and EEDQ (2.12 g, 8.56 mmol) were synthesized according to the method of IIIa to obtain 1,1-dimethylethyl N-[2-oxo-2-(4-methylthiazol-2-ylamino)ethyl]carbamate (2.2 g, 94.8%), a yellow solid. The product was dissolved in 40 mL of DCM, and 4 mL of HCl-methanol solution (4 M) was added. Following the method of IIIa, intermediate IIIb (0.673 g, 48.5%) was obtained as a yellowish-white solid. ESI-MS (m / z): 172.1 [M + H] + .

[0062] 1.2 Synthesis of the target compound

[0063]

[0064] 3-Fluoro-N-[2-[(4-trifluoromethylphenyl)methylamino]-2-oxo-ethyl]benzamide (QO-A2):

[0065]

[0066] Intermediate IIa (50 mg, 0.25 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (58.7 mg, 0.31 mmol), and 4-dimethylaminopyridine (DMAP) (15.6 mg, 0.128 mmol) were placed in a 25 mL flask, and 10 mL of DCM was added. The mixture was stirred and dissolved at room temperature. 4-Trifluoromethylbenzylamine (49 mg, 0.28 mmol) was then added, and the reaction was allowed to proceed for 2 h. TLC was used to confirm the completeness of the reaction. The solvent was evaporated to obtain a thick paste, which was washed several times with 2 M hydrochloric acid, filtered, and the filter cake was washed with water and dried in an oven at 50 °C to give a yellow-green solid (71.7 mg, 81%). Melting point: 103.4 ~ 104.7 °C, ESI-MS (m / z): 355.6 [M + H] + . 1 H NMR (600 MHz, Chloroform-d) δ 7.61 (d, J = 8.0 Hz, 2H), 7.57-7.50(m, 2H), 7.49-7.44 (m, 2H), 7.42 (td, J = 8.0, 5.5 Hz, 1H), 7.22 (tdd, J =8.2, 2.6, 1.0 Hz, 1H), 6.53 (s, 1H), 4.70 (d, J = 5.9 Hz, 2H), 1.61 (s, 1H), 1.28 (d, J = 33.0 Hz, 1H), 0.94-0.74 (m, 1H). 13 C NMR (151 MHz, deuterated dimethyl sulfoxide-d6, DMSO-d6) δ 165.53, 144.76, 136.93, 131.06 (d, J = 7.6 Hz), 128.46 (d, J = 22.8 Hz), 125.70, 123.92, 118.78 (d, J = 20.2 Hz), 114.56 (d, J = 21.9 Hz), 42.89, 39.61.

[0067] 2-[(3-fluorobenzoyl)amino]-N-[4-fluorophenylmethyl]-3-(1H-imidazol-4-yl)propionamide (QO-A3):

[0068]

[0069] Intermediate IIb (100 mg, 0.36 mmol), EDCl (68.8 mg, 0.36 mmol), DMAP (22 mg, 0.18 mmol), and 4-fluorobenzylamine (50 mg, 0.4 mmol) were synthesized using the same procedure as QO-A2, yielding a pale yellow solid (105.1 mg, 76%). Melting point: 93.3 ~ 95.6 °C, ESI-MS (m / z): 385.3 [M + H] - , 1 H NMR (600 MHz, Chloroform-d)δ 7.68–7.46 (m, 3H), 7.40 (td, J = 8.0, 5.5 Hz, 1H), 7.35–7.28 (m, 2H), 7.20 (td, J = 8.3, 2.6 Hz, 1H), 7.11–6.88 (m, 3H), 6.45 (s, 1H), 4.60 (d, J = 5.7Hz, 3H), 3.20 (s, 1H), 1.26 (s, 1H), 0.86 (dt, J = 21.1, 6.5 Hz, 1H).

[0070] 3-Fluoro-N-[2-[(cyclohexylmethyl)amino]-2-oxo-ethyl]benzamide (QO-A4):

[0071]

[0072] Intermediate IIa (50 mg, 0.25 mmol), EDCl (58.7 mg, 0.31 mmol), and DMAP (15.6 mg, 0.128 mmol) were ground in a mortar at room temperature. Then, aminomethylcyclohexane (31.6 mg, 0.28 mmol) was added and the mixture was ground for 15 min. The mixture was washed several times with 2 M hydrochloric acid, filtered, and the solid was washed with water and dried in an oven at 50 °C to obtain the product (25 mg, 30.4%). Melting point: 123.3 ~ 124.6 °C, gray solid, ESI-MS (m / z): 291.4 [M – H] - . 1H NMR (600 MHz, CDCl3) δ 7.46 – 7.39 (m, 2H), 7.33 (q, J = 7.5 Hz, 1H), 7.12 (td, J = 8.3,2.6 Hz, 1H), 6.07 (s, 1H), 3.23 (t, J = 6.5 Hz, 2H), 1.74 – 1.65 (m, 4H), 1.61 (d, J = 12.5 Hz, 1H), 1.52 (tt, J = 7.5, 3.6 Hz, 1H), 1.23 – 1.08 (m,4H), 0.94 (tt, J = 12.8, 6.5 Hz, 2H).

[0073] 2-[(3-fluorobenzoyl)amino]-N-[4-thiazolylmethyl]-3-(1H-imidazol-4-yl)propionamide (QO-A9):

[0074]

[0075] Intermediate IIb (100 mg, 0.36 mmol), EDCl (68.8 mg, 0.36 mmol), DMAP (22 mg, 0.18 mmol), and 4-aminomethylthiazole (intermediate I) (45.6 mg, 0.4 mmol) were synthesized using the same procedure as QO-A2 to give a white solid (94.0 mg, 70%). Melting point: 98.6 ~ 100.3 °C, ESI-MS (m / z): 374.5 [M + H] + .

[0076] 3-[(3-fluorobenzoyl)amino]-N-4-thiazolylmethylbenzamide (QO-A10):

[0077]

[0078] Intermediate IIc (100 mg, 0.386 mmol), EDCl (114.6 mg, 0.6 mmol), DMAP (30.5 mg, 0.25 mmol), and 2-amino-5-methylthiazole (45.6 mg, 0.4 mmol) reacted in the same manner as QO-A4 to give a white solid (69.7 mg, 51%). Melting point: 198.4 ~ 199.5 °C, ESI-MS (m / z): 356.4 [M + H] - .

[0079] N-[2-[(2-thiazolyl)amino]-2-oxo-ethyl]naphthyl-1-carboxamide (QO-A11):

[0080]

[0081] α-Naphthoic acid (51.6 mg, 0.3 mmol) and EEDQ (78.7 mg, 0.32 mmol) were placed in a 25 mL flask, and 10 mL of DCM was added. The mixture was stirred for 10 min, and then intermediate IIIa (50 mg, 0.32 mmol) was added. The mixture was stirred at room temperature for 2 h, and the reaction was monitored by TLC until complete. The solvent was evaporated to obtain a thick paste, which was washed with 2 M hydrochloric acid, filtered, and a brown solid was obtained. The paste was washed with water and dried in an oven at 50 °C. The solid was redissolved in ethyl acetate and separated by a 200–300 mesh silica gel column (petroleum ether:ethyl acetate = 3:1) to obtain a white solid (76.5 mg, 82.3%). Melting point: 155.3–156.1 °C, ESI-MS (m / z): 312.4 [M + H] - .

[0082] 3-Fluoro-N-[2-[3,4-dibromobenzonitrile-2-amino]-2-oxo-ethyl]benzamide (QO-A12):

[0083]

[0084] Intermediate IIa (50 mg, 0.25 mmol), EDCl (58.7 mg, 0.31 mmol), DMAP (15.6 mg, 0.128 mmol), and 2-amino-3,4-dibromobenzonitrile (82.8 mg, 0.3 mmol) were synthesized using the same procedure as QO-A2, yielding an orange solid (104.2 mg, 92%). Melting point: 116.5 ~ 118.6 °C, ESI-MS (m / z): 453.6 [M + H] - .

[0085] N-[2-[(2-thiazolyl)amino]-2-oxo-ethyl]cyclopentylformamide (QO-A14):

[0086]

[0087] Intermediate IIIa (50 mg, 0.32 mmol), EEDQ (78.7 mg, 0.32 mmol), and cyclopentylcarboxylic acid (34.2 mg, 0.3 mmol) reacted in the same manner as QO-A11 to give a white solid (51 mg, 63.1%). Melting point: 188.1 ~ 191.2 °C, ESI-MS (m / z): 254.5 [M + H] - .

[0088] 4-Trifluoromethyl-N-[2-[(5-methyl-2-thiazolyl)amino]-2-oxo-ethyl]benzamide (QO-A16):

[0089]

[0090] Intermediate IIIb (50 mg, 0.29 mmol), EEDQ (71.7 mg, 0.29 mmol), and 4-trifluoromethylbenzoic acid (49.4 mg, 0.26 mmol) reacted in the same manner as QO-A11 to give a white solid (75.8 mg, 84%). Melting point: 183.2–186.5 °C, ESI-MS (m / z): 343.9 [M + H] + . 1 H NMR (600 MHz, DMSO) δ 8.11 (dd, J =24.3, 8.1 Hz, 2H), 7.89 (dd, J = 19.4, 8.2 Hz, 4H), 7.25 (d, J = 1.5 Hz, 1H), 4.00 (q, J = 7.1 Hz, 1H), 3.86 (d, J = 6.1 Hz, 1H), 2.38 (s, 3H).

[0091] N-[2-[(5-methyl-2-thiazolyl)amino]-2-oxo-ethyl]naphthyl-1-carboxamide (QO-A17):

[0092]

[0093] Intermediate IIIb (50 mg, 0.29 mmol), EEDQ (71.7 mg, 0.29 mmol), and α-naphthoic acid (44.7 mg, 0.26 mmol) reacted in the same manner as QO-A11 to give a white solid (65.2 mg, 77.2%). Melting point: 134.2 ~ 138.6 °C, ESI-MS (m / z): 324.6 [M – H] - .

[0094] 3-Trifluoromethyl-N-[2-[(5-methyl-2-thiazolyl)amino]-2-oxo-ethyl]-cyclohexylformamide (QO-A18):

[0095]

[0096] Intermediate IIIb (50 mg, 0.29 mmol), EEDQ (71.7 mg, 0.29 mmol), and 3-trifluoromethylcyclohexanecarboxylic acid (51 mg, 0.26 mmol) reacted in the same manner as QO-A11 to give a white solid (66.8 mg, 73.6%). Melting point: 155.6 ~ 156.4 °C, ESI-MS (m / z): 350.4 [M + H] + .

[0097] 3-Fluoro-N-[2-(3-fluoro-2-methylphenylamino)-2-oxo-ethyl]benzamide (QO-A20):

[0098]

[0099] Intermediate IIa (50 mg, 0.25 mmol), EDCl (58.7 mg, 0.31 mmol), DMAP (15.6 mg, 0.128 mmol), and 3-fluoro-2-methylaniline (35 mg, 0.28 mmol) were synthesized using the same procedure as QO-A2 to yield a yellow-green oil (49.7 mg, 65.4%), ESI-MS (m / z): 305.2 [M + H]. + .

[0100] 2,6-Dichloro-5-fluoro-N-[2-((5-methylthiazol-2-yl)amino)-2-oxoethyl]nicotinamide (QO-A21):

[0101]

[0102] Intermediate IIIb (40 mg, 0.23 mmol), EEDQ (56.9 mg, 0.23 mmol), and 2,6-dichloro-5-fluoronicotinic acid (42 mg, 0.2 mmol) reacted in the same manner as QO-A2 to give a white solid (57.6 mg, 79.6%). Melting point: 231.1 ~ 233.4 °C, ESI-MS (m / z): 363.1 [M + H] + .

[0103] N-(4-trifluoromethylbenzyl)-3-[2-(3-fluorophenyl)-1H-pyrrolo-1-yl]benzamide (QO-A19):

[0104]

[0105] Potassium hydroxide (0.784 g, 14 mmol) was added to an acetonitrile solution of pyrrole (5 g, 7.46 mmol) and 3-bromobenzoic acid (1.4 g, 7.1 mmol). The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC until complete. The solvent was evaporated, water was added and stirred to dissolve the solid, and the insoluble matter was filtered off. The pH of the filtrate was adjusted to 2-3 with 2 M hydrochloric acid, and a solid precipitated. The solid was filtered, and the filter cake was dried in an oven at 50 °C to give the product 3-(1H-pyrrole-1-yl)benzoic acid (1.32 g, 99.4%) as a gray solid. ESI-MS (m / z): 186.1 [M + H] - .

[0106] 3-(1H-pyrrolo-1-yl)benzoic acid (0.3 g, 1.6 mmol) and N-bromosuccinimide (0.43 g, 2.4 mmol) were added to 20 mL of DMF and stirred overnight at room temperature. The reaction was monitored by TLC, and the starting material disappeared. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate until no product was observed in the aqueous layer. The ester layer was then washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain an orange viscous paste, namely 3-(2-bromo-1H-pyrrolo-1-yl)benzoic acid, which was used directly in the next step without further treatment. All the product from the previous step was added to 20 mL of ethanol, along with 3-fluorophenylboronic acid (0.224 g, 1.6 mmol) and 1,3-bis(diphenylphosphine propane) nickel dichloride (0.325 g, 0.6 mmol). The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC until complete. The reaction solution was filtered, the filtrate was concentrated, and separated by a 200-300 mesh silica gel column (petroleum ether: ethyl acetate = 3:1) to give the product 3-[2-(3-fluorophenyl)-1H-pyrrolo-1-yl]benzoic acid (0.29 g, 65.3%), a white solid, ESI-MS (m / z): 280.1 [M + H] - .

[0107] A solution of 3-[2-(3-fluorophenyl)-1H-pyrrolo-1-yl]benzoic acid (60 mg, 0.21 mmol), 4-trifluoromethylbenzylamine (43.8 mg, 0.25 mmol), and EEDQ (51.9 mg, 0.21 mmol) in DCM was reacted at room temperature, following the same procedure as QO-A11, to give product QO-A19 (80.3 mg, 73.3%), a white solid. Melting point: > 255℃, ESI-MS (m / z): 439.3 [M+ H] + . 1H NMR (600 MHz, DMSO) δ 10.53 (s, 1H), 8.42 (d, J = 2.1 Hz, 1H), 8.05 (dd, J = 8.0, 2.3 Hz, 1H), 7.97 – 7.77 (m, 5H), 7.70 (d, J = 7.7 Hz, 1H), 7.66 – 7.53 (m, 3H), 7.53 – 7.41 (m, 4H), 2.24 (t, J = 7.4 Hz, 1H).

[0108] II. Evaluation of Compound Activity and Selectivity

[0109] 2.1 The main reagents and equipment are shown in Table 1 below.

[0110] Table 1 Main Reagents and Equipment

[0111]

[0112] The synthesis method of compound QO-83 can be found in the master's thesis of Hebei Medical University, "Synthesis, Screening and Preliminary Establishment of Quality Standards for Novel KCNQ Potassium Channel Openers," Cui Fuchun, 2018 (Synthesis of Compound 2). The synthesis method of QO-58 can be found in "Qi Jinlong. Design, Synthesis and Screening of KCNQ2 / 3 and CaCCs Ion Channel Modulators and Optimization of Synthetic Process of Highly Active Compounds [D]" (Synthesis of Compound 58).

[0113] 2.2 Cells, Plasmids, and Animals

[0114] Cells: The human embryonic kidney cell line stably expressing the KCNQ1 / KCNE1 channel (KCNQ1 / KCNE1-HEK293 cell), the Chinese hamster ovary cell lines stably expressing the KCNQ2, KCNQ2 / 3 and KCNQ5 channels (KCNQ2-CHO cell, KCNQ2 / 3-CHO cell and KCNQ5-CHO cell), and the Chinese hamster ovary cells (CHO cell) used in this study were all derived from our laboratory's stable cell line bank.

[0115] Plasmids: The plasmid used in this study was human KCNQ4 (gene library code: AF105202), and the gene fragment was cloned into the pcDNA3.1(+) vector; the pcDNA3.1(+)-EGFP plasmid was provided by our laboratory's plasmid library.

[0116] Animals: Specific pathogen-free Kunming mice, half male and half female, with a body weight of about 20 g, provided by Beijing Huafukang Biotechnology Co., Ltd., license number SCXK(Beijing)2024-0003. Under 12 h light / dark conditions, environmental temperature of 22 - 25 °C, and fed separately in cages. They were raised in the animal house for three days before the experiment to adapt to the environment.

[0117] 2.3 Preparation of complete medium

[0118] The medium for the KCNQ1 / KCNE1-HEK293 stable cell line is Dulbecco's modified Eagle medium (DMEM): fetal bovine serum (FBS): penicillin and streptomycin = 90%:10%:1%.

[0119] The medium for the KCNQ2 / 3-CHO stable cell line is Ham’s F12 medium: FBS: penicillin and streptomycin: hygromycin B: G418 = 90%:10%:1%:600 μg / mL:600 μg / mL.

[0120] The medium for the KCNQ2-CHO stable cell line is DMEM-F12: FBS: penicillin and streptomycin: G418 = 90%:10%:1%:600 μg / mL.

[0121] The medium for the KCNQ5-CHO stable cell line is DMEM: FBS: penicillin and streptomycin: G418 = 90%:10%:1%:600 μg / mL.

[0122] The medium for the CHO cell line is DMEM: FBS: penicillin and streptomycin = 90%:10%:1%.

[0123] 2.4 Solutions related to ICR8000 high-throughput screening

[0124] 1) Loading Buffer: RbCl, Glucose, HEPES, NaCl, MgCl2, NaH2PO4·2H2O, CaCl2, with concentrations of 5.4 mM, 5 mM, 25 mM, 150 mM, 1 mM, 0.8 mM, 2 mM respectively.

[0125] 2) Washing Buffer: KCl, HEPES, NaCl, MgCl2, NaH2PO4·2H2O, CaCl2, with concentrations of 5.4 mM, 25 mM, 150 mM, 1 mM, 0.8 mM, 2 mM respectively.

[0126] 3) Open Buffer: KCl, HEPES, NaCl, MgCl2, NaH2PO4·2H2O, CaCl2, with concentrations of 20 mM, 25 mM, 130 mM, 1 mM, 0.8 mM, and 2 mM, respectively.

[0127] 2.5 Patch clamp related solutions

[0128] Electrode internal solution: Composition: KCl:MgCl2:EDTA:HEPES = 145 mM: 1 mM: 5 mM: 10 mM, pH adjusted to 7.2 with KOH, sterilized by filtration through a 0.22 μm microporous membrane, aliquoted, and stored at -20℃. Extracellular solution: Composition: NaCl:KCl:HEPES:MgCl2:CaCl2:Glucose = 144 mM: 5 mM: 10 mM: 1 mM: 2 mM: 10 mM, pH adjusted to 7.4 with NaOH, and stored at 4℃.

[0129] The present invention will be further illustrated below through specific embodiments.

[0130] Example 1: Preliminary evaluation of the effect of compound QO-A2 on KCNQ2 channels based on ICR8000 technology.

[0131] Parameter settings: Average Current: 8 mA, Voltage: 300 V, Entrance slit wildth: 0.6 nm, automatic correction of monochromator wavelength to ensure the hollow cathode lamp energy is approximately 70%, 100 µL injection per sample, adjustment of robotic arm parameters before experiment to ensure accurate positioning of the plate under test, and acquisition of standard curves. Standard curve concentrations were set to 0 ppm, 1 ppm, 2 ppm, and 4 ppm. R 2 A value > 0.99 is required to measure the sample.

[0132] operate:

[0133] 1) Cells in logarithmic growth phase (KCNQ2-CHO cells) were subjected to a 2 × 10⁻⁶ thiocyanate infusion. 5 Inoculate the plates at a density of 100 cells / well into 96-well plates and incubate them in an incubator.

[0134] 2) After 24 hours, discard the culture medium, wash twice with 200 μL of Loading Buffer each time, and finally add 200 μL of Loading Buffer and incubate at 37°C for 3 hours.

[0135] 3) Remove the Loading Buffer and gently wash 3 times with Washing Buffer, 200 μL each time.

[0136] 4) Preliminary activity evaluation of the compound: Add 200 µL of Open Buffer containing the compound to each well and incubate the cells at 37°C for 15 min. Then transfer the supernatant to a new 96-well plate and measure the rubidium ion concentration in each well using an ICR8000.

[0137] Two concentrations of 1 µM and 10 µM were set for the positive control (RTG, QO-83), the lead compound (124-A10), and the test compound (the required solutions were prepared using Open Buffer). Three replicates were set for each concentration, and three replicates were set for the solvent control wells.

[0138] Data processing: Calculate the average value for each concentration group. The change in rubidium ion efflux caused by the depolarization buffer is represented by Fsupern, calculated using the formula Fsupern = Rb. + (supern / cpd) / Rb + (supern / d) × 100%, of which Rb + (supern / cpd) To determine the Rb content in the supernatant after the addition of the compound. + concentration of Rb + (supern / d) Rb in the supernatant of the solvent control group + The concentrations were determined. GraphPad Prism 8 was used for data analysis and graphing. Data are expressed as mean ± standard error of means (SEM), and significance was calculated using a t-test. < 0.05, < 0.01, A difference of less than 0.001 is considered statistically significant.

[0139] Compound QO-A2 exhibits excellent opening properties for KCNQ2 channels; see details. Figure 1 And Table 2.

[0140] Table 2. Preliminary activity evaluation of different compounds on KCNQ2 channels

[0141]

[0142] C in Table 2 compound C control The values ​​of Rb in the supernatant of the compound and the control group are respectively represented. + The concentration.

[0143] Example 2: Evaluation of the effect of compound QO-A2 on KCNQ2 / 3 channels based on ICR8000 technology.

[0144] Parameter settings, experimental procedures, and data processing were the same as in Example 1. KCNQ2 / 3-CHO cells were used. The dose-effect concentrations of lead compound 124-A10 were set at 0.01, 0.1, 0.3, 3, 10, and 30 µM; the dose-effect concentrations of compound QO-A2 were set at 0.1, 0.3, 1, 10, and 50 µM. The positive control was QO-83 at a concentration of 10 µM, and a solvent control was included. Each group had three replicates. The dose-effect equation was Y = Bottom + (Top - Bottom) / (1 + 10^((lg EC)) 50 - X) × HillSlope) ).

[0145] The dose-response curves of compound QO-A2 and lead compound 124-A10 on the KCNQ2 / 3 channel are shown in the figures below. Figure 2 and Figure 3 As shown in the figure, compound QO-A2 has a good opening effect on KCNQ2 / 3 channels.

[0146] Example 3: Evaluation of the effect of compound QO-A2 on KCNQ2 channels based on patch clamp technique.

[0147] Electrophysiological patch-clamp recording experiments were performed at room temperature. Cells were captured using the perforated whole-cell voltage-clamp method with a 2 kHz filter. Digital signals were filtered, acquired, and observed using Pclamp 10.7 software. KCNQ2-CHO cells were used.

[0148] RTG was prepared in extracellular fluid at a concentration of 10 μM as a control drug, and QO-A2 was prepared in extracellular fluid at concentrations of 0.1, 1, 3, 10, and 30 μM for electrophysiological patch-clamp experiments.

[0149] Data processing: The raw data were processed using Clampfit 10.7 software, followed by statistical analysis and plotting using OriginPro 2021 and GraphPad Prism 8. All data are expressed as mean ± SEM. Significance was calculated using a t-test. < 0.05, A difference of < 0.01 is considered statistically significant.

[0150] Compound QO-A2 exhibits excellent opening properties for KCNQ2 channels; see details. Figure 4 . Figure 4In the diagram, each point represents one cell per drug administration. Data were analyzed using a t-test, comparing the results with the control group. < 0.05, < 0.01. Compared with the control group, compound QO-A2 showed significant activity, with opening folds of 1.89 ± 0.308, 1.96 ± 0.374, and 2.51 ± 0.276 at 1 μM, 10 μM, and 30 μM, respectively.

[0151] Example 4: Maximal electroshock seizure (MES) test of compound QO-A2 in mice.

[0152] The parameters of the S48 pharmacological and physiological experimental multi-purpose instrument are set as follows: stimulation type: continuous wave output, wave width: 20.00 ms, interval: 10.00 ms, trigger delay: 0.00 ms, wave number: 60, voltage: 160 V, current limit: 4.00 mA.

[0153] Half male and half female Kunming mice, weighing approximately 20 g, were selected 24 hours before the experiment. Alligator clips were moistened with physiological saline, and the mice's ears were clamped with the clips. The output button was pressed, and hind limb rigidity was used as a positive indicator to select qualified mice. Qualified mice were then randomly divided into 7 groups of 8 mice each, based on their weight. The experiment must be completed between 9:00 AM and 3:00 PM, with the room temperature controlled at 20-25°C. The time interval between drug administration and measurement should be as consistent as possible for each animal. The control group received 4 mg / kg of QO-83, the QO-A2 groups received 12.5 mg / kg, 20 mg / kg, 25 mg / kg, 40 mg / kg, and 50 mg / kg, and the control group received 25% β-cyclodextrin. The drug was administered intraperitoneally at a volume of 0.2 mL / 10 g. 15–20 min after administration, the animals were stimulated using an S48 pharmacological and physiological experimental multi-purpose instrument. The number of mice that did not induce convulsions during the experiment was recorded, and the percentage of convulsions induced by the drug was calculated.

[0154] Data processing: GraphPad Prism 8 was used for data analysis, graphing, and calculation of ED. 50 Value, by using χ 2 The significance is calculated using a test. < 0.05, < 0.01, A difference of less than 0.001 is considered statistically significant.

[0155] The results showed that compound QO-A2 has anticonvulsant activity. (See details...) Figure 5 . Figure 5In (A), the control group was given 25% β-cyclodextrin, and the positive control group was given QO-83 (4 mg / kg). n=8, one experiment, χ² 2 test, < 0.001.

[0156] Example 5: Evaluation of the subtype selectivity of compound QO-A2 based on ICR8000 technology.

[0157] KCNQ1 / KCNE1-HEK293 cells, KCNQ5-CHO cells, and KCNQ4-CHO transient cells were seeded at a density of 2 × 10⁵ cells / well in 96-well plates for selectivity evaluation. The parameter settings, experimental procedures, and data processing were the same as in Example 1.

[0158] For the activity assay of KCNQ1 / KCNE1 channels, QO-58 was used as the positive control. For the activity assays of KCNQ4 and KCNQ5 channels, QO-83 was used as the positive control. Both the positive control and the test compound were set at concentrations of 10 and 30 μM, with three replicates for each concentration. The solvent control was also set at three replicates. For the KCNQ2 / 3 channel assay, the compound was set at concentrations of 1 and 10 μM.

[0159] For detailed test results, please see Figure 6 See Table 3. The test results show that compound QO-A2 exhibits good selectivity, with no significant opening effect on KCNQ1, KCNQ4, and KCNQ5 channels. The selectivity of the other tested compounds is acceptable.

[0160] Table 3. Subtype selectivity of synthesized compounds for KCNQ channels

[0161]

[0162] When the opening multiple is less than 1.2, it is “-”; when 1.2 ≤ opening multiple < 1.4, it is “+”; when 1.4 ≤ opening multiple < 1.6, it is “++”; when 1.6 ≤ opening multiple, it is “+++”.

[0163] In Table 3, blank spaces indicate that no tests were performed. This is because compounds QO-A10 and QO-A20 showed poor activity towards KCNQ2 / 3, so KCNQ4 and KCNQ5 channel tests were not performed.

Claims

1. A selective KCNQ channel opener, characterized in that, Its chemical structural formula is as shown in formula (I) a As shown in the image: （I a ） Wherein, R1 is -CH2-, phenyl, or heteroaryl-substituted methyl; When R1 is -CH2-, R2 is cyclopentyl, cyclohexyl, cyclohexylmethyl, thiazolylmethyl, thiazolyl, methylthiazolyl, aryl, haloaryl, trifluoromethylaryl, 4-trifluoromethylbenzyl or 3,4-dibromobenzonitrile-2-yl, and R3 is naphthyl, haloaryl, trifluoromethylaryl, cyclopentyl, trifluoromethylcyclohexyl or halopyridyl. When R1 is phenyl, R2 is thiazolyl or thiazolylmethyl, and R3 is aryl or haloaryl; When R1 is a heteroaryl-substituted methyl group, R2 is a thiazolyl, aryl, or haloaryl group, and R3 is an aryl or haloaryl group.

2. The formula (I) in claim 1 a The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antiepileptic drugs.

3. The formula (I) in claim 1 a The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antipsychotic drugs.

4. The formula (I) in claim 1 a The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antidepressant drugs.

5. The formula (I) in claim 1 a The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of medicaments for the treatment of ischemic stroke.

6. A selective KCNQ channel opener, characterized in that, Its chemical structural formula is as shown in formula (I) b As shown in the image: （Ⅰ b ） Wherein, R1 is aryl, R2 is thiazolyl, aryl, haloaryl or 4-trifluoromethylbenzyl, and R3 is aryl or haloaryl.

7. The formula (I) in claim 6 b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antiepileptic drugs.

8. The formula (I) in claim 6 b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antipsychotic drugs.

9. The formula (I) in claim 6 b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of antidepressant drugs.

10. The formula (I) in claim 6 b The use of the compound shown in the diagram, its stereoisomers, hydrates, solvates, or pharmaceutically acceptable salts in the preparation of medicaments for the treatment of ischemic stroke.

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