Deuterated benzamide diazepine compounds, methods of making and uses thereof

Abstract

The application discloses a kind of deuterated benzamide diazepine compounds and preparation method and purposes thereof, and structure is as shown in general formula I.The compound is used as selective OX2R antagonist, and shows good activity, selectivity, brain exposure, brain blood ratio (the ratio of drug concentration in brain and drug concentration in blood, is called B / P), little toxic side effect, moderate half-life effect etc..It can be used for preventing, treating and / or reducing the disease related to orexin receptor.General formula I

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a deuterated benzamide diazacycloheptane compound, its preparation method, and its uses. Background Technology

[0002] Numerous basic and clinical studies have shown that the orexin system is not only a core hub in the central nervous system regulating the sleep-wake cycle, but also widely involved in various physiological processes such as eating behavior, energy homeostasis, reward processing, stress response, autonomic nervous function, and neuroendocrine regulation. OX1R signaling is mainly associated with reward behavior, stress-induced responses, and drug addiction, while OX2R plays a dominant role in maintaining wakefulness, promoting appetite, and regulating energy metabolism. Orexin neurons project to multiple regions throughout the brain, including the locus coeruleus, raphe nuclei, tuberomammary nuclei, ventral tegmental area, and cortex. Dysfunction of orexin neurons has been shown to be closely related to the pathogenesis of a range of neuropsychiatric disorders, metabolic disorders, and visceral dysfunctions.

[0003] Currently approved orexin receptor modulators are all dual orexin receptor antagonists, such as suvorexin, leboresen, dalesen, and vonoresen. Despite their excellent performance in treating insomnia, they have the following limitations: some are classified as psychotropic drugs due to their potential for abuse; they are relatively expensive and lack sufficient long-term safety data; and there is a lack of adequate clinical validation for indications other than insomnia. Selective OX2R antagonists and selective OX1R antagonists are currently in clinical trials, and no related compounds have been marketed.

[0004] OX2R antagonists also possess antidepressant effects and can effectively treat insomnia by antagonizing only a single OX2R receptor. Using selective OX2R antagonists can avoid many of the side effects caused by inhibiting OX1R. Currently, only Johnson & Johnson's OX2R antagonist, Seltorexant, is in the clinical stage, with its main indications including major depressive disorder (MDD), primary and secondary insomnia, etc. Selective OX2R antagonists have the potential to treat neurological disorders such as insomnia, depression, and anxiety, and there is a huge clinical demand. The synthesis of selective OX2R antagonists is currently a major research direction. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention proposes a deuterated benzamide diazacycloheptane compound, its preparation method, and its uses. As a selective OX2R antagonist, it exhibits good activity, selectivity, low brain exposure, low brain-to-blood ratio (B / P), low toxicity, and a moderate half-life. It can be used to prevent, treat, and / or alleviate orexin receptor-related diseases, and is preferably used to treat insomnia, depression, Alzheimer's disease, and sleep apnea.

[0006] The technical solution of the present invention is as follows: The first object of this invention is to provide a compound as shown in general formula I, or a pharmaceutically acceptable salt or stereoisomer thereof:

[0007] General Formula I Ring A is a five-membered nitrogen-containing heteroaromatic ring, selected from... , , , , , , , , , ; R 1 Selected from one or more R b Replaced or unreplaced R c ; R b Each is independently selected from halogens, -C1-C4 alkyl groups, -CF3, and -NH2; R c It is a six-membered heteroaromatic ring containing two nitrogen elements, selected from , , Or a heteroaromatic ring selected from the following groups: , , ; R 2 It is a C1-C4 deuterated alkyl group; R 3 It is independently selected from one of hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

[0008] In one embodiment of the present invention, in general formula I, at least one position is substituted with deuterium, the position being selected from: hydrogen in C1-C4 alkyl or C1-C4 hydroxyalkyl being substituted with deuterium; hydrogen directly attached to a benzene ring, heteroaromatic ring, or heterocycle being substituted with deuterium.

[0009] In one embodiment of the present invention, the specific structure of the compound is any one of the following structures: .

[0010] In one embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt; The inorganic acids are selected from hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, and nitric acid; The organic acids are selected from acetic acid, malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, or oxalic acid.

[0011] A second object of the present invention is to provide a pharmaceutical composition comprising the above-described compound or a pharmaceutically acceptable salt or stereoisomer thereof, and pharmaceutical excipients.

[0012] In one embodiment of the present invention, the pharmaceutical excipient includes a pharmaceutically acceptable carrier, excipient, or diluent; the pharmaceutically acceptable carrier includes microspheres, nanoparticles, and liposomes.

[0013] A third object of the present invention is to provide the use of the above-described compound or its pharmaceutically acceptable salt, stereoisomer, or the above-described pharmaceutical composition in the preparation of an orexin receptor antagonist.

[0014] In one embodiment of the present invention, the orexin receptor antagonist is orexin. 2-receptor antagonists.

[0015] In one embodiment of the present invention, the orexin receptor antagonist regulates the orexin system through a mechanism of action selected from at least one of the following: a) Regulate the activity of orexin receptors and promote the normalization of orexin receptor function; b) Antagonizes orexin receptors, exerting its biological effects by inhibiting orexin receptor-mediated signal transduction pathways; c) Selectively antagonize orexin receptor-2 (OX2R), thereby specifically inhibiting the physiological function of orexin receptor-2.

[0016] A fourth object of the present invention is to provide the use of the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating nervous system diseases, such as insomnia, depression, anxiety, or drug addiction.

[0017] In one embodiment of the present invention, the neurological disease is major depressive disorder, primary and secondary insomnia, or depression accompanied by insomnia.

[0018] Beneficial effects: The compounds of this invention exhibit good activity as selective OX2R antagonists, IC50 50 With a concentration as low as 13.8 nM, a selectivity of up to 141, and moderate efficacy in terms of brain exposure, brain-to-blood ratio (B / P), low toxicity, and moderate half-life, this drug can be used to prevent, treat, and / or alleviate orexin receptor-related diseases, and is preferred for the treatment of insomnia, depression, Alzheimer's disease, and sleep apnea. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific examples.

[0020] Example 1 Synthesis route:

[0021] Synthesis of Compound 1-1 Ethyl acetate (132 g, 1.5 mol) was added to a LiHMDS solution (3 L, 3 mol, 1 mol / L tetrahydrofuran solution), and stirred at -78°C for 1 h. Acetyl chloride-d3 (120 g, 1.48 mol) was then added dropwise to the reaction system, and the mixture was stirred at -78°C for 2 h. After the reaction was complete, 6N HCl was added to quench the reaction, and the pH was adjusted to neutral. The mixture was extracted with diethyl ether / water, and the organic phases were combined and concentrated under reduced pressure to give the crude compound 1-1. HRMS: m / z Calculation for C6H8D3O3[M+H] + : 134.0896, found 134.0892.

[0022] Synthesis of Compounds 1-2 Crude compound 1-1 was dissolved in 1 L of dichloromethane, then 500 g of 200-300 mesh silica gel was added, followed by N-Boc-ethylenediamine (240 g, 1.5 mol). The reaction was carried out at room temperature for 3 h. After the reaction was complete, the silica gel was removed by filtration. The crude compound 1-2 was obtained by concentration. HRMS: m / z calcd. for C 13 H 22 D3N2O4[M+H] + : 276.2002, found 276.1997.

[0023] Synthesis of compounds 1-3 The crude compounds 1-2 were dissolved in acetonitrile, and sodium triacetoxyborohydride (318 g, 1.5 mol) was slowly added under ice bath conditions. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the pH was adjusted to 8-10 with 10 N NaOH, and the mixture was extracted three times with dichloromethane / water. The organic phases were combined and concentrated. A large amount of petroleum ether was added, and the product precipitated. Filtration yielded 111 g of compounds 1-3. The overall yield of the three steps was 27%. HRMS: m / z calcd. for C 13 H 24 D3N2O4[M+H] + : 278.2159, found278.2154.

[0024] Synthesis of compounds 1-4 Compounds 1-3 were dissolved in 2 L of ethyl acetate / water (2:1) solution, and benzyl chloroformate (78 g, 462 mmol) was added. The pH was adjusted to 8-10 with 10 N NaOH, and the reaction was carried out at room temperature for 1 h. After the reaction was completed, the mixture was separated, the organic phase was washed with saturated sodium bicarbonate, and the solution was concentrated to obtain crude compounds 1-4. HRMS: m / z calcd. for C 21 H 30 D3N2O6[M+H] + : 415.2527, found 415.2523.

[0025] Synthesis of compounds 1-5 Crude compounds 1-4 were dissolved in 1.5 L of ethyl acetate, and 200 mL of 4 N hydrochloric acid-ethyl acetate (800 mmol) was slowly added. The mixture was reacted overnight at room temperature. After the reaction was complete, the solution was concentrated under reduced pressure to give crude compounds 1-5. HRMS: m / z calcd.for C 16 H 22 D3N2O4[M+H] + : 312.2002, found 312.1997.

[0026] Synthesis of compounds 1-6 The crude compounds 1-5 were dissolved in 1.5 L of anhydrous methanol, and sodium methoxide (71 g, 1.3 mol) was slowly added under ice bath conditions, with stirring at room temperature for 3 h. After the reaction was complete, the solvent was removed by concentration, and the mixture was extracted with ethyl acetate / water. The combined organic phases were washed with saturated sodium bicarbonate, and the mixture was concentrated to give 90 g of compounds 1-6. The yield of the above three steps was 85%. HRMS: m / z calcd. for C 14 H 16 D3N2O3[M+H] + : 266.1584, found 266.1580.

[0027] Synthesis of compounds 1-7 Compounds 1-6 were dissolved in 100 mL of tetrahydrofuran. A tetrahydrofuran solution (400 mL, 800 mmol) of a 2 N borane-dimethyl sulfide complex was added at room temperature, and the mixture was heated to 65 °C for 3 h. After the reaction, 3 N HCl was added to adjust the pH to 1-2, and the mixture was refluxed at 75 °C overnight. The organic phase was extracted with ethyl acetate / water, discarded, and the pH was adjusted to 8-10 with 10 N NaOH. The mixture was extracted three times with ethyl acetate / water, and the organic phases were combined and concentrated to give compounds 1-7. HRMS: m / z calcd. forC 14 H 18 D3N2O2[M+H] + : 252.1791, found 252.1787.

[0028] Synthesis of compounds 1-8 Compounds 1-7 were dissolved in 1 L of dichloromethane, and Boc anhydride (81 g, 373 mmol) and triethylamine (69 g, 680 mmol) were added. The mixture was reacted at room temperature for 3 h. After the reaction was complete, the mixture was extracted three times with ethyl acetate / water. The organic phases were combined, concentrated, and purified by silica gel column chromatography. 57 g of compounds 1-8 were given, with a two-step yield of 47%. HRMS: m / z calcd. for C 19 H 26 D3N2O4[M+H] + :352.2315, found 352.2310.

[0029] Synthesis of compounds 1-9 Compounds 1-8 were dissolved in 1 L of methanol / ethyl acetate solution, 1% water was added, and 6 g of palladium / carbon was added. The reaction was carried out at room temperature for 24 h under a hydrogen atmosphere. After the reaction was completed, the palladium on carbon was removed by filtration, and 35 g of compounds 1-9 were obtained by silica gel column chromatography, with a reaction yield of 99%. HRMS: m / z calcd. for C 11 H 20 D3N2O2[M+H] + : 218.1948, found 218.1944.

[0030] Preparation of compounds 1-10 Supercritical fluid chromatography (SFC) was used for preparation. The sample was dissolved in methanol to prepare a sample solution with a concentration of 5.2 mg / mL. The chromatographic conditions were as follows: a YMC CHIRAL ART Amylose-C chiral preparative column (10 µm, 40 × 250 mm) was used at room temperature; the mobile phase consisted of supercritical carbon dioxide (food grade) and redistilled methanol in a volume ratio of 80:20; the flow rate was set at 140 mL / min; the injection volume was 2.0 mL; and the detection wavelengths were 214 nm and 254 nm. Under these conditions, the cycle time for a single preparation was 3.28 minutes, and compounds 1-10 were obtained by collecting the target components.

[0031] 1 H NMR (600 MHz, Chloroform- d ) δ 3.66 (dt,J = 14.0, 3.5 Hz, 1H), 3.56(ddt, J = 17.3, 14.0, 4.6 Hz, 1H), 3.44 (dt, J = 12.7, 5.4 Hz, 1H), 3.36 (ddd, J = 13.9, 8.4, 4.4 Hz, 1H), 3.30 – 3.20 (m, 1H), 3.05 (ddt, J = 21.7, 13.8, 3.7Hz, 1H), 2.79 (dt, J = 14.1, 7.4 Hz, 1H), 2.70 (dq, J = 9.9, 5.2, 4.7 Hz, 1H),1.86 (dt, J = 26.5, 13.0 Hz, 1H), 1.44 (d, J = 2.4 Hz, 9H), 1.41 – 1.33 (m,1H), 1.09 (dq, J = 16.5, 5.5, 5.0 Hz, 1H). Example 2

[0032] Synthesis of compounds 1-11 1-10 (50 mg, 0.23 mmol), 2-(2H-1,2,3-triazol-2-yl)benzoic acid (38 mg, 0.2 mmol), HATU (87 mg, 0.23 mmol), and N,N-diisopropylethylamine (52 mg, 0.4 mmol) were dissolved in 1 mL of anhydrous N,N-dimethylformamide and reacted at room temperature for 3 h. After the reaction was completed, the mixture was quenched with saturated ammonium chloride, extracted with ethyl acetate / water, and the combined organic phases were concentrated to give crude compound 1-11.

[0033] Synthesis of compounds 1-12 Crude compound 1-11 was dissolved in 0.5 mL of ethyl acetate, and 1 mL of 4 N hydrochloric acid-ethyl acetate was added. The mixture was reacted at room temperature for 1 h. After the reaction was completed, the solution was concentrated under reduced pressure to obtain crude compound 1-12.

[0034] Synthesis of Compound 1 The crude compounds 1-12 were dissolved in 3 mL of anhydrous N,N-dimethylformamide, and 2,6-dimethyl-4,5-dichloropyrimidine (35 mg, 0.2 mmol) and triethylamine (202 mg, 2 mmol) were added. The reaction was carried out under nitrogen protection at 75 °C for 2 h. After the reaction was completed, the mixture was quenched with saturated ammonium chloride, extracted with ethyl acetate / water, and the combined organic phases were concentrated and purified by silica gel column chromatography. Compound 1 was given as 66 mg of white solid, with a three-step yield of 77%. HRMS: m / z calcd. for C 21 H 22 D3ClN7O[M+H] + : 428.1997, found 429.1993.

[0035] Example 3

[0036] Following the synthetic method for compound 1, compound 2 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 2-methyl-4,5-dichloropyrimidine. The product was 74 mg of white solid, with a three-step reaction yield of 86%. HRMS: m / z calcd. forC 21 H 22 D3ClN7O [M+H] + : 428.1997, found 429.1994.

[0037] Example 4

[0038] Following the synthetic method for compound 1, replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid yields compound 3. The product is 39 mg of a white solid, with a three-step reaction yield of 44%. HRMS: m / z calcd. for C 22 H 24 D3ClN7O [M+H] + : 443.2154, found 443.2150.

[0039] Example 5

[0040] Following the synthetic method for compound 1, replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-fluoro-2-(2H-1,2,3-triazol-2-yl)benzoic acid yields compound 4. The product is 74 mg of a white solid, with a three-step reaction yield of 86%. HRMS: m / z calcd. for C 21 H 21 D3ClFN7O [M+H] + : 447.1903, found 447.1897.

[0041] Example 6

[0042] Following the synthetic method for compound 1, compound 5 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 4,6-dimethyl-2-chloropyrimidine. The product was a white solid (37 mg), with a three-step reaction yield of 60%. HRMS: m / z calcd. forC 22 H 25 D3N7O [M+H] + : 408.2543, found 409.2540.

[0043] Example 7

[0044] Following the synthetic method for compound 1, compound 6 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 2,6-dimethyl-4-chloropyrimidine. The product was 27 mg of white solid, with a three-step reaction yield of 55%. HRMS: m / z calcd. forC 22 H 25 D3N7O [M+H] + : 408.2465543, found 409.2538.

[0045] Example 8

[0046] Following the synthetic method for compound 1, compound 7 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 2,5,6-trimethyl-4-chloropyrimidine. The product was a white solid (16 mg), with a three-step reaction yield of 31%. HRMS: m / z calcd. forC 23 H 27 D3N7O [M+H] + : 422.2700, found 423.2695.

[0047] Example 9

[0048] Following the synthetic method for compound 1, compound 8 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4-5-dichloropyrimidine with 2,6-methyl-4-chloro-5-fluoropyrimidine. The product was a white solid (30 mg), with a three-step reaction yield of 61%. HRMS: m / z calcd. forC 22 H 24 D3FN7O [M+H] + : 426.2449, found 427.2445.

[0049] Example 10

[0050] Following the synthetic method for compound 1, compound 9 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 2-methyl-4-chloro-5-fluoropyrimidine. The product was a white solid (34 mg), with a three-step reaction yield of 69%. HRMS: m / z calcd. forC 21 H 22 D3FN7O [M+H] + : 412.2293, found 413.2288.

[0051] Example 11

[0052] Following the synthetic method for compound 1, compound 10 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 6-methyl-4,5-dichloropyrimidine-2-amine. The product was a white solid (35 mg), with a three-step reaction yield of 66%. HRMS: m / z calcd.for C 21 H 23 D3ClN8O [M+H] + : 444.2106, found 444.2102.

[0053] Example 12

[0054] Following the synthetic method for compound 1, compound 11 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 2-methyl-6-chloropyrimidinyl-4-amine. The product was 4 mg of white solid, with a three-step reaction yield of 8%. HRMS: m / z calcd. forC 21 H 24 D3N8O [M+H] + : 410.2495, found 410.2491.

[0055] Example 13

[0056] Following the synthetic method for compound 1, compound 12 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 4-chloro-6-methyl-1H-pyrazolo[3,4-D]pyrimidine. The product was 44 mg of white solid, with a three-step reaction yield of 84%. HRMS: m / z calcd. for C 22 H 23 D3N9O [M+H] + : 435.2448, found 435.2441.

[0057] Example 14

[0058] Following the synthetic method for compound 1, compound 13 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 4-chloro-2-methyl-thiophene[3,2-D]pyrimidine. The product was a white solid, 45 mg in volume, with a three-step reaction yield of 83%. HRMS: m / z calcd. for C 23 H 23 D3N7OS [M+H] + : 451.2108, found 451.2103.

[0059] Example 15

[0060] Following the synthetic method for compound 1, compound 14 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and 2,6-dimethyl-4,5-dichloropyrimidine with 4-chloro-2-methyl-5H,6H,7H-cyclopentadien[d]pyrimidine. The product was 74 mg of white solid, with a three-step reaction yield of 86%. HRMS: m / z calcd. for C 24 H 27 D3N7O [M+H] + : 435.2700, found 435.2695.

[0061] Example 16

[0062] Following the synthetic method for compound 1, compound 15 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 6-fluoro-2-(2H-1,2,3-triazol-2-yl)benzoic acid. The product was a white solid (37 mg), with a three-step reaction yield of 43%. HRMS: m / z calcd. for C 21 H 21 D3ClFN7O [M+H] + : 447.1903, found 447.1897.

[0063] Example 17

[0064] Compound 16 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 4-fluoro-2-(2H-1,2,3-triazol-2-yl)benzoic acid, following the synthetic method for compound 1. The product was a 10 mg white solid, with a three-step reaction yield of 12%. HRMS: m / z calcd. for C 21 H 21 D3ClFN7O [M+H] + : 447.1903, found 447.1897.

[0065] Example 18

[0066] Compound 17 was obtained by replacing 2-(2H-1,2,3-triazol-2-yl)benzoic acid with 3-fluoro-2-(2H-1,2,3-triazol-2-yl)benzoic acid, following the synthetic method for compound 1. The product was a 14 mg white solid, with a three-step reaction yield of 16%. HRMS: m / z calcd. for C 21 H 21 D3ClFN7O [M+H] + : 447.1903, found 447.1897.

[0067] Test example: 1. Determination of the effect of the compounds of the present invention on calcium flow in cells stably expressing OX1 and OX2 receptors. a. Experimental materials and instruments As shown in Table 1 below; Table 1

[0068] b. Cell information and culture conditions The two stably transfected CHO cell lines are involved, and the specific culture parameters are shown in Table 2 below: Table 2

[0069] Note: All incubation environments were 37°C in a 5% CO2 constant temperature incubator. 。

[0070] c. Experimental Procedure Collect CHO-OX1 or CHO-OX2 cells in logarithmic growth phase, discard the culture supernatant, wash with PBS, and digest with 0.25% trypsin-EDTA. Centrifuge to collect the cell pellet, resuspend in the corresponding complete culture medium, and agitate thoroughly. After cell counting, adjust to the appropriate concentration and seed at a density of 10,000 cells per well in 384-well cell culture plates. Incubate the plates at 37°C in a 5% CO2 incubator for 16-20 hours. On the second day, prepare the detection buffer according to the FLIPR Calcium 6 kit instructions, and dilute the fluorescent dye to 1× working concentration after allowing it to return to room temperature. Remove the cell culture plates from the incubator, discard the culture medium in the wells, add 35 μL of the prepared dye working solution to each well, briefly centrifuge to remove air bubbles, and incubate the cell culture plates at 37°C in the dark for 2 hours. During this period, prepare the compound loading plates: Prepare a stock solution of the test compound and the positive control drug Filorexant to a final concentration of 1000× using DMSO. Transfer 200 nL / well to a 384-well source plate using an ECHO 655 ultrasonic pipetting system, then add 20 μL of detection buffer to each well and mix well (this is a 10× intermediate dilution). Simultaneously, take another source plate and transfer 100 nL / well of Orexin A agonist using the ECHO system, then add 20 μL of detection buffer (note that the final concentration of Orexin A for OX1 receptor detection is 0.1 nM, and for OX2 receptor it is 1 nM). After incubation, place the cell plate on the stage of the FLIPR Penta instrument. First, the instrument aspirates 5 μL of the solution from the test compound plate and adds it to the cell plate, recording the fluorescence signal in real time for 5 minutes to read the baseline. After drug addition, incubate for 30 minutes, then the instrument aspirates 10 μL of the solution from the agonist plate and adds it to the cell plate, continuing to record the fluorescence signal in real time for 5 minutes. The optical parameters for the entire detection process were set to excitation wavelength 470-495 nm and emission wavelength 515-575 nm.

[0071] d. Data Analysis The validity of the experimental data was assessed using the Z' factor, where H was defined as the mean fluorescence signal value of the negative control group (DMSO only), and L was defined as the mean fluorescence signal value of the positive control group (reference inhibitor). The Z' factor was calculated as 1 - [3 × (SD_H + SD_L) / |Ave_L - Ave_H|]. A Z' factor greater than 0.5 indicated that the experimental system was stable and reliable, and the data could be used for subsequent IC50 analysis. 50The inhibition rate of each test compound was calculated using the formula [(sample signal value - H value) / (L value - H value)] × 100%. Finally, with the logarithm of the compound concentration as the X-axis and the corresponding inhibition percentage as the Y-axis, a nonlinear four-parameter regression equation was used: Y = Bottom + (Top - Bottom) / [1 + 10^((LogIC)] × 100%. 50 Curve fitting was performed using [-X)×HillSlope], and the data were processed using XLFit software to calculate the half-maximal inhibitory concentration (IC50) of each compound against the OX1 and OX2 receptors. 50 ).

[0072] e. Experimental Results The compounds of this invention showed good inhibitory effects on calcium flow in experiments on cells stably expressing OX2R, and the inhibitory effect on OX2R was significantly better than that on OX1R, demonstrating excellent selectivity. Their IC50 values... 50 The values ​​are shown in Table 3.

[0073] Table 3

[0074] 2. Pharmacokinetics of the compound in SD rats a. Experimental Procedure Six male SD rats, weighing between 210 and 240 g, were selected. The animals were housed in an SPF-grade barrier environment with free access to water and fed standard rodent feed. Environmental parameters in the animal housing were controlled as follows: temperature 18-29°C, relative humidity 30%-70%, and an electronic timed lighting system maintaining a 12-hour light / 12-hour dark cycle. Experimental animals underwent at least 3-5 days of acclimatization after arrival and were only enrolled in the experiment after passing a veterinary health check. On the day of the experiment, rat weight was measured, and the individual drug volume was calculated based on weight. All animals were fasted for 12 hours before drug administration (free access to water) and resumed feeding 2 hours after administration. The test compound was administered intravenously (IV) as a single dose of 2 mg / kg at a volume of 5 mL / kg. The solvent formulation was a DMSO / ethanol / PEG300 / 0.9% sodium chloride solution (volume ratio 5 / 5 / 40 / 50). The formulation was vortexed and sonicated until completely dissolved, resulting in a clear solution, and was freshly prepared immediately before use.

[0075] At time points of 0 h before drug administration and at 0.05 h, 0.25 h, 0.75 h, 2 h, 4 h, 8 h, and 24 h after drug administration, approximately 200-300 μL of whole blood was collected via tail vein and placed in centrifuge tubes containing EDTA-K2 anticoagulant, and mixed by inversion. Within 30 minutes of collection, the whole blood sample was centrifuged at 3000-4000 rpm for 10 minutes at 4°C, the supernatant plasma was separated, transferred to clean cryovials, and stored at -80°C for analysis.

[0076] Plasma samples were pretreated using a protein precipitation method, with proteins precipitated in acetonitrile (containing an internal standard). The supernatant was collected after vortex centrifugation for analysis. The concentration of the test compounds in the plasma was determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The analytical method was validated, and the linear range and limit of quantitation met the detection requirements.

[0077] b. Experimental Results Based on blood drug concentration data at various time points, the main pharmacokinetic parameters were calculated using a non-compartmental (NCA) model in Phoenix WinNonlin software. Its T... 1 / 2 CL_ obs Vss_ obs The values ​​are shown in Table 4.

[0078] Table 4

[0079] Experimental results show that the pharmacokinetic properties of the compounds of this invention are significantly improved.

[0080] 3. hERG potassium channel current inhibition activity assay a. Experimental Procedure HEK-293 cell lines stably expressing hERG potassium channels were cultured in DMEM complete medium containing 10% fetal bovine serum and 0.8 mg / mL G418 at 37°C and 5% CO2. For cell passage, the old medium was discarded and the cells were washed once with PBS, followed by the addition of 1 mL TrypLE™ Express solution and incubation at 37°C for approximately 0.5 minutes. After the cells detached from the bottom of the dish, 5 mL of pre-warmed (37°C) complete medium was added to stop digestion, and the cells were gently pipetted to disperse them into a single-cell suspension. The cell suspension was transferred to sterile centrifuge tubes and centrifuged at 1000 rpm for 5 minutes to collect the cell pellet. Cells were then cultured at 2.5 × 10⁶ cells / mL. 5 Cells are seeded at a density of 1 cell per dish in 6 cm cell culture dishes for expansion or maintenance culture (final volume 5 mL). To maintain good electrophysiological activity, cell confluence should not exceed 80%.

[0081] Before patch clamp testing, use TrypLE TM Cells were digested and collected using Express culture, and centrifuged after adding complete culture medium to terminate the digestion. The supernatant was discarded, and the cell pellet was resuspended in an appropriate amount of extracellular fluid for counting. The cell density was adjusted to 2-3 × 10⁻⁶ cells / year. 6 cells / mL. Place the prepared cell suspension at room temperature and gently mix using a shaker for 15-20 minutes before analysis.

[0082] This experiment used the fully automated patch-clamp system QPatch 48X (Sophion) for electrophysiological assays. First, the prepared cell suspension was placed in the centrifuge station of the QPatch workbench. Cells were washed through multiple centrifugation / resuspending steps, replacing the cell culture medium with extracellular fluid. The MTP-96 compound plate was removed and placed in the MTP source position, and the QPlate chip was removed and placed in the QPlate source position. A robotic arm automatically scanned the barcodes on the MTP-96 plate and QPlate chip and picked them up, transporting them to the measurement station. The system aspirated intracellular and extracellular fluids from the liquid reservoir and added them to the corresponding liquid reservoirs on the QPlate chip. At the measurement station, all measurement sites on the QPlate underwent an initial quality control process, including aspirating the cell suspension from the centrifuge cell container, positioning the cells in the chip microwells using a pressure controller, establishing a high-resistance seal, and forming a whole-cell recording pattern.

[0083] The voltage stimulation protocol for whole-cell patch-clamp recording of hERG currents was set as follows: the cell membrane clamp voltage was maintained at -80 mV, first depolarized from -80 mV to -50 mV and held for 0.5 seconds (for leakage current detection), then stepped to +30 mV and held for 2.5 seconds, and then rapidly restored to -50 mV and held for 4 seconds to elicit the characteristic tail current of the hERG channel. This stimulation protocol was repeated every 10 seconds to continuously observe the effect of the drug on the hERG tail current.

[0084] After the baseline control current stabilized, the test compound solutions were sequentially applied to the cell surface from the MTP-96 plate in ascending order of concentration. Each drug concentration was administered twice, with an application time of at least 5 minutes after each administration. The current value recorded for each cell in extracellular fluid without the compound before administration served as a self-control. Each test concentration was measured twice using at least two independent cells. All electrophysiological experiments were performed under isothermal conditions at 24°C.

[0085] b. Data Analysis First, the peak tail current after each drug concentration was normalized to the peak tail current of the blank solvent control group. The calculation formula is: Normalized current = Peak tail current after drug administration / Peak tail current of solvent control group. The inhibition rate at a given concentration is calculated as follows: Inhibition rate (%) = [1 - (peak tail current after administration / peak tail current of solvent control)] × 100%.

[0086] After calculating the mean and standard error of each concentration group, the dose-response relationship was fitted using the following four-parameter nonlinear regression equation: Y = Bottom + (Top - Bottom) / [1 + 10^((LogIC)] 50 -X)×HillSlope)], where X is the logarithm of the compound concentration and Y is the corresponding inhibition percentage. Curve fitting and IC 50 The value calculation was performed using GraphPad Prism software.

[0087] c. Experimental Results Table 5

[0088] Experimental results show that the compound has significantly lower cardiotoxicity than the positive control drug and has higher safety.

[0089] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A compound as shown in general formula I, or a pharmaceutically acceptable salt or stereoisomer thereof: General Formula I Ring A is a five-membered nitrogen-containing heteroaromatic ring, selected from... , , , , , , , , , ; R 1 Selected from one or more R b Replaced or unreplaced R c ; R b Each is independently selected from halogens, -C1-C4 alkyl groups, -CF3, and -NH2; R c It is a six-membered heteroaromatic ring containing two nitrogen elements, selected from , , Or a heteroaromatic ring selected from the following groups: , , ; R 2 It is a C1-C4 deuterated alkyl group; R 3 It is independently selected from one of hydrogen, C1-C4 alkyl, and C1-C4 hydroxyalkyl.

2. The compound according to claim 1, or its pharmaceutically acceptable salt or stereoisomer, characterized in that, In general formula I, at least one position is substituted with deuterium, the position being selected from: hydrogen in C1-C4 alkyl or C1-C4 hydroxyalkyl being substituted with deuterium; hydrogen directly attached to a benzene ring, heteroaromatic ring, or heterocycle being substituted with deuterium.

3. The compound according to claim 1, or its pharmaceutically acceptable salt or stereoisomer, characterized in that, The specific structure of the compound is any one of the following: 。 4. The compound according to claim 1, or its pharmaceutically acceptable salt or stereoisomer, characterized in that, Pharmaceutically acceptable salts are inorganic acid salts or organic acid salts; The inorganic acids are selected from hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid, and nitric acid; The organic acids are selected from acetic acid, malic acid, maleic acid, citric acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid, or oxalic acid.

5. A pharmaceutical composition, characterized in that, It contains the compound of any one of claims 1-4 or its pharmaceutically acceptable salt, stereoisomer, and pharmaceutical excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical excipients include pharmaceutically acceptable carriers, excipients, or diluents; the pharmaceutically acceptable carriers include microspheres, nanoparticles, and liposomes.

7. The use of a compound according to any one of claims 1-4 or a pharmaceutically acceptable salt, stereoisomer thereof, or the pharmaceutical composition according to claim 5 in the preparation of an orexin receptor antagonist.

8. The application according to claim 7, characterized in that, Orexin receptor antagonists are orexin 2-receptor antagonists.

9. The use of a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt or stereoisomer thereof, or the pharmaceutical composition according to claim 5, in the preparation of a medicament for treating nervous system diseases, characterized in that, Neurological disorders include insomnia, depression, anxiety, or drug addiction.

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