Application of compound in preparation of medicine for treating insomnia and / or anxiety disorder

Abstract

The invention belongs to the technical field of nerve drugs, and particularly relates to application of an orexin receptor 1 antagonist and / or an orexin receptor 2 antagonist in preparation of drugs for treating insomnia or anxiety, and the compound is a compound and salt shown in the following structure. In in-vivo experiments, the traditional Chinese medicine composition shows a more excellent anti-insomnia effect and shows a good anti-anxiety effect. In addition, the structure also has better stability and pharmacokinetic characteristics, such as higher exposure and half-life period.

Description

Technical Field

[0001] This invention belongs to the field of neurological drug technology, specifically relating to the application of compounds in the preparation of drugs for treating insomnia and / or anxiety. Background Technology

[0002] The rate of sleep disturbance among people aged 18 and above reaches 48.5%. The prevalence of sleep disorders is approximately 35.9% in the elderly over 60 years old and 26% in adolescents. Long-term insomnia increases the risk of hypertension by 3.7 times and diabetes by 2.5 times. Furthermore, 70%-80% of chronic insomnia patients also experience anxiety or depression symptoms; the risk of developing new-onset depression in insomniacs is 3-4 times higher than in those without insomnia, and the risk of anxiety is nearly 6 times higher. Insomnia is both a core symptom of anxiety and depression (accounting for 53% of sleep disorder causes) and an independent risk factor for both; persistent insomnia increases the recurrence rate of anxiety and depression to 65%.

[0003] Lotus seed heart, known for its calming and soothing effects, also possesses effective anti-insomnia properties. A study (PMID:37111298) disclosed the in vitro inhibitory activity of the alkaloid methyl lotusinin [4-(((R)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline-1-yl)methyl)-2-(((R)-6-methoxy-1-(4-methoxybenzyl)-2-methyl-1,2,3-4-tetrahydroisoquinoline-7-yl)oxy)phenol] from lotus seed heart against orexin receptors, suggesting its potential anti-insomnia potential. Summary of the Invention

[0004] The purpose of this invention is to provide the use of the compound in the preparation of a medicament for treating insomnia and / or anxiety.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The use of the compound in the preparation of orexin receptor 1 antagonists and / or orexin receptor 2 antagonists, wherein the compound is a compound with the following structure or a pharmaceutical salt thereof:

[0007]

[0008] The use of the compound in the preparation of a medicament for treating insomnia, wherein the compound is a compound with the following structure or a pharmaceutical salt thereof:

[0009]

[0010] The use of a compound in the preparation of a medicament for treating anxiety disorders, wherein the compound is a compound with the following structure or a pharmaceutical salt thereof:

[0011]

[0012] In one preferred embodiment, the pharmaceutical salt is a salt formed by the compound and an acidic salt of an acid or inorganic base.

[0013] In one preferred embodiment, the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-benzenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, tribromoacetic acid, trichloroacetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid.

[0014] In one preferred embodiment, the acid is hydrobromic acid, sulfuric acid, acetic acid, trifluoroacetic acid, tribromoacetic acid, or trichloroacetic acid.

[0015] In one preferred embodiment, the acid salt of the inorganic base is a salt containing a basic metal cation, a salt containing an alkaline earth metal cation, or a salt containing an ammonium ion.

[0016] In one preferred embodiment, the compound significantly prolongs sleep time and shortens sleep latency.

[0017] In one preferred embodiment, the compound significantly reduces exercise, energy expenditure, and food intake.

[0018] In one preferred embodiment, the compound significantly improves sleep cycle disorders.

[0019] In one preferred embodiment, the compound significantly reduces autonomous activity distance.

[0020] In one preferred embodiment, the drug further includes pharmaceutically commonly used excipients.

[0021] In one preferred embodiment, the excipients include one or more of solubilizers, preservatives, antioxidants, flavoring agents, colorants, and fragrances.

[0022] In one preferred embodiment, the dosage form of the drug is any one of granules, capsules, tablets, injections, infusions, or suppositories.

[0023] The inventors' experimental data show that the structure of this invention has better orexin receptor inhibitory activity than (4-(((R)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline-1-yl)methyl)-2-(((R)-6-methoxy-1-(4-methoxybenzyl)-2-methyl-1,2,3-4-tetrahydroisoquinoline-7-yl)oxy)phenol), exhibiting superior anti-insomnia efficacy and good anti-anxiety effects in in vivo experiments. Furthermore, this structure has better stability and pharmacokinetics, such as higher exposure and half-life, making it more suitable for preparing drugs for the prevention or treatment of insomnia and / or anxiety disorders. It can more significantly shorten sleep latency, prolong sleep time, and alleviate anxiety symptoms. Attached Figure Description

[0024] Figure 1 The graph shows the changes in calcium ion concentration and the concentration inhibition curves after drug treatment of CHO cells under the stimulation of orexin B; among them, Figure 1 A represents the calcium ion concentration change curve after (R,S)-1 treatment of CHO cells. Figure 1 B represents the calcium ion concentration inhibition curve after (R,S)-1 treatment of CHO cells. Figure 1 C represents the curve showing the change in calcium ion concentration in CHO cells after treatment with methylnephrine. Figure 1 D is the calcium ion concentration inhibition curve after treatment of CHO cells with methyl lotusine. Figure 1 E is a graph showing the change in calcium ion concentration in CHO cells after treatment with suvorexin. Figure 1 F is the calcium ion concentration inhibition curve after treatment of CHO cells with svorex.

[0025] Figure 2 The fluorescence intensity changes of calcium ion release after treatment with drugs overexpressing orexin receptor 1 (OX1R) and receptor 2 (OX2R) in response to orexin A stimulation were measured using FLIPR assays. Figure 2 A represents the changes in calcium ion release fluorescence intensity and IC50 values ​​after treatment of CHO cells overexpressing OX1R with (R,S)-1, (R,S)-1 trifluoroacetate, (R,S)-1 hydrochloride, (R,S)-1 acetate, (R,S)-1 sulfate, and (R,S)-1 hydrobromide. Figure 2 B represents the changes in calcium ion release fluorescence intensity and IC50 values ​​after treatment of CHO cells overexpressing OX2R with (R,S)-1, (R,S)-1 trifluoroacetate, (R,S)-1 hydrochloride, (R,S)-1 acetate, (R,S)-1 sulfate, and (R,S)-1 hydrobromide.

[0026] Figure 3 A scheme for constructing a chronic sleep deprivation mouse model.

[0027] Figure 4 The time taken for mice in the open and closed arms during the EPMT test. Figure 4 A is a heatmap showing the movement trajectory of mice in the elevated cross maze (EPMT). Figure 4 B is a bar chart showing the percentage of time spent in the open arm after the EPMT test in the control group and insomnia mice. Figure 4 C is a bar chart showing the percentage of time spent in the closed arm in the control group and insomnia mice during the EPMT test.

[0028] Figure 5 The distance and time percentage of the mouse's central region during the open field (OFT) test are shown, where Figure 5 A is a heatmap of the mouse's activity trajectory in OFT. Figure 5 B is a bar chart showing the percentage of distance traveled in the central region by the control group and the insomniac mice. Figure 5 C is a bar chart showing the percentage of time spent in the central region for the control group and insomniac mice.

[0029] Figure 6 The effect of suprathreshold doses of sodium pentobarbital on the rate of sleep episodes in mice of different groups.

[0030] Figure 7 The sleep latency and duration in the pentobarbital-induced sleep experiment are given. Figure 7 A is a bar chart of sleep latency in the pentobarbital-induced sleep experiment; Figure 7 B is a bar chart showing the duration of sleep induced by pentobarbital.

[0031] Figure 8 For dynamic observation of oxygen consumption and carbon dioxide production.

[0032] Figure 9 To evaluate the hypnotic effect and anxiety behavior in insomnia mice treated with 40 mg / kg (R, S)-1; among which, Figure 9 A is a heatmap of the movement trajectory of mice in the elevated cross maze (EPMT) after treatment with 40 mg / kg (R, S)-1. Figure 9 B is a bar chart showing the percentage of time spent in the open arm after treating insomnia mice with 40 mg / kg (R, S)-1 in the EPMT test. Figure 9 C is a bar chart showing the percentage of time spent in arm closure in insomnia mice treated with 40 mg / kg (R, S)-1 in the EPMT test. Figure 9 D is a heatmap of the activity trajectory of mice in the open field (OFT) experiment after treatment with 40 mg / kg (R, S)⁻¹. Figure 9 E is a bar graph showing the percentage of distance moved in the central region in insomnia mice treated with 40 mg / kg (R, S)-1 in the OFT test. Figure 9F is a bar chart showing the percentage of time in the central region after treating insomnia mice with 40 mg / kg (R, S)-1 in the OFT test. Figure 9 G represents the effect of suprathreshold doses of sodium pentobarbital on the rate of sleep episodes in each group of mice. Figure 9 H is a bar chart of sleep latency in the pentobarbital-induced sleep experiment; Figure 9 I is a bar chart of the duration in the pentobarbital-induced sleep experiment.

[0033] Figure 10 The effect of 40 mg / kg (R, S)⁻¹ on insomnia in mice; Figure 10 A is a graph showing the effect of 40 mg / kg (R, S)⁻¹ on the activity level of insomniac mice. Figure 10 B is a graph showing the effect of 40 mg / kg (R, S)⁻¹ on energy expenditure in insomnia mice. Figure 10 C is a bar graph showing the effect of 40 mg / kg (R, S)⁻¹ on food and water intake in insomnia mice. Figure 10 A graph showing the effect of D = 40 mg / kg (R, S)⁻¹ on the respiratory exchange rate in insomnia mice.

[0034] Figure 11 The effects of treatments with (R,S)-1, (R,S)-1 trifluoroacetate, and methylnephrine on mice in a pentobarbital-induced sleep experiment were investigated. Figure 11 A is a bar chart of sleep latency in mice treated with (R,S)-1, (R,S)-1 trifluoroacetate and methylnephrine. Figure 11 B represents the sleep duration in mice treated with (R,S)-1, (R,S)-1 trifluoroacetate, and methylnephrine.

[0035] Figure 12 A bar graph showing the total distance moved by mice 25 minutes after administration of (R,S)-1, (R,S)-1 trifluoroacetate and methylnephrine. Detailed Implementation

[0036] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0037] In this invention, all data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.

[0038] In the following examples, (R,S)-1 is 4-(((R)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline-1-yl)methyl)-2-(((S)-6-methoxy-1-(4-methoxybenzyl)-2-methyl-1,2,3-4-tetrahydroisoquinoline-7-yl)oxy)phenol, which is the compound and pharmaceutical salt to be protected in this invention, and its structural formula is:

[0039]

[0040] Example 1

[0041] Preparation of compounds

[0042] 1. Preparation of (R,S)-1

[0043] 1.1 Preparation of compound S-11

[0044]

[0045] In a 50 mL reaction flask, 2.4 g of compound 10 and 51.3 mg of ruthenium catalyst RuCl [(R,R)-TsDPEN(P-cymene)] were added to 8.0 mL of N,N-dimethylformamide. Then, 1.0 mL of a formic acid-triethylamine (5:2) azeotrope was added at 0 °C, and the reaction was allowed to proceed overnight at room temperature. After the reaction was complete, 200 mL of dichloromethane was added for extraction. The mixture was washed with semi-saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then subjected to column chromatography to obtain 2.3 g of compound S-11 as a brown solid, with a yield of 96% and an ee value of 91%. (ESI-HRMS m / z calcd C) 18 H 22 NO3[M+H] + 300.1594, found 300.1611.[α] D 25 -12.5 (c 0.28, MeOH);

[0046] 1.2 Preparation of compound S-11 hydrochloride

[0047]

[0048] 2.3 g of compound S-11 was dissolved in 5 mL of THF, followed by the addition of 2 mL of ether solution in 2.0 M HCl. After thorough mixing, the mixture was placed in a -20°C freezer and allowed to stand for 2 days. A solid precipitated at the bottom; after filtration, 1.33 g of S-11 hydrochloride was obtained as a light yellow solid powder, with a yield of 52%. ESI-HRMS m / z calcd C 18 H 22 NO3[M+H]+ 300.1594, found 300.1613. [α] D 25 -5.8 (c 0.45, CHCl3); 1 H NMR(500MHz, CDCl3)δ9.15(d,J=19.5Hz,2H),8.93(s,1H),7.27(d,J=8.6Hz,2H), 6.92(d,J=8.6Hz,2H),6.75(s,1H),6.55(s,1H),4.54(s,1H),3.75(s,6H),3.32– 3.28(m,1H),3.21(dd,J=14.3,6.0Hz,1H),3.15(dd,J=12.0,6.5Hz,1H),3.10(dd ,J=14.3,7.8Hz,1H),2.97(dt,J=16.4,6.0Hz,1H),2.85(dt,J=16.6,6.0Hz,1H).

[0049] 1.3 Preparation of compound S-12

[0050]

[0051] Stepwise method: In a 50 mL reaction flask, 1.06 g of compound S-11 hydrochloride was added to 7 mL of methanol, followed by 0.53 mL of formaldehyde. 200 mg of sodium borohydride was slowly added at 0 °C, and the reaction was allowed to proceed for 3 hours at room temperature. After the reaction was complete, 5 mL of water was added to terminate the reaction. The methanol was removed by concentration under reduced pressure, and the reaction was extracted with 100 mL of dichloromethane. The mixture was washed twice with water, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain 969 mg of compound S-12 as a white solid, with a yield of 98%. ESI-HRMSm / z calcd C 19 H 24 NO3 + [M+H] + 314.1751, found 314.1778. [α] D 25 +52.1 (c 1.0, CHCl3); 1H NMR (500MHz, CDCl3) δ7.04(d,J=8.6Hz,2H),6.81–6.75(m,2H),6.53(s,1H),6.40(s,1H),3.85(s,3H),3.78(s,3H),3.66(t,J=6.0Hz, 1H),3.19–3.12(m,1H),3.05(dd,J=14.2,6.1Hz,1H),2.84(dd,J=14.2,6.0Hz,1H),2.80–2.69(m,2H),2.60–2.51(m,1H),2.46(s,3H).

[0052] 1.4 Preparation of compound (R,S)-13

[0053]

[0054] In a 200 mL Schlenk flask, 891 mg of compound R-6, 960 mg of compound S-12, 83.9 mg of a mixture of cuprous dimethyl sulfide bromide, 100.6 mg of 2-pyridinecarboxylic acid, and 867.0 mg of potassium phosphate were added. Under anhydrous and anaerobic conditions, 41 mL of pyridine was added, followed by heating at 110 °C for 7 days. After the reaction was complete, the pyridine was removed by vacuum concentration, and the reaction was extracted with 200 mL of dichloromethane. The mixture was washed twice with water, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by column chromatography to give 1.2 g of compound (R,S)-13, a brown viscous liquid, with a yield of 88%. ESI-HRMS m / z calcd C 40 H 49 N₂O₇[M+H] + 669.3534, found 669.3559.[α] D 25 -63.3 (c 0.1, CHCl3); 1H NMR(500MHz, CDCl3)δ7.02(d,J=8.2Hz,1H),6.92(d,J=8.1Hz,2H),6.68–6.59(m,4H),6.56(s,1 H),6.52(s,1H),6.14(s,1H),5.76(s,1H),5.15–5.08(m,2H),3.98–3.88(m,2H),3.82(s,3H),3 .77(s,3H),3.69(s,3H),3.61–3.53(m,1H),3.48(s,3H),3.41(s,3H),3.38–3.29(m,2H),3.14- 3.07(m,1H),3.04–2.90(m,4H),2.89–2.79(m,4H),2.76–2.71(m,1H),2.68(s,3H),2.67(s,3H). 13 C NMR (126MHz, CDCl3) δ158.5,150.2,148.9,147.5,147.3,146.6,130.9,125.4,120.0,119.3,118.2,116.2,116.2,114.0,112.7 ,111.5,111.4,95.9,65.2,64.8,56.3,56.1,55.8,55.4,46.6,45.5,41.7,40.8,40.7,40.2,31.8,30.3,29.9,29.5,23.0,22.9.

[0055] 1.5 Preparation of the chiral isomer (R,S)-Nef of methylnepenone

[0056]

[0057] 1.2 g of compound (R,S)-13, 10 mL of methanol, and 5 mL of trifluoroacetic acid were added to a reaction flask and stirred at room temperature for 3 days. After the reaction was complete, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain 1.07 g of the chiral isomer of methylnepenthes (R,S)-Nef, a yellow solid powder, with a yield of 95%. The chromatographic data of (R,S)-Nef are as follows: ESI-HRMS m / z calcd C 38 H 45 N₂O₆[M+H] + 625.3272, found 625.3298.[α] D 25 -20.0 (c 1.55, CHCl3); [α] D 25 -21.5 (c 1.66, CHCl3); 1HNMR(400MHz, CDCl3) δ6.90(d,J=8.6Hz,2H),6.82(d,J=8.2Hz,1H),6.66(dd,J=12.6,5.2Hz,3H),6.6 2(s,1H),6.55(d,J=1.8Hz,1H),6.50(s,1H),6.37(s,1H),6.00(s,1H),3.80(s,3H),3.77(s,3H),3.70 (s,3H),3.59(dd,J=13.5,7.3Hz,2H),3.53(s,3H),3.17–3.06(m,2H),3.01(td,J=13.9,5.3Hz,2H),2 .85–2.73(m,3H).,2.73–2.67(m,2H),2.66–2.57(m,2H),2.57–2.49(m,1H),2.46(s,3H),2.43(s,3H). 13 C NMR (101MHz, CDCl3) δ158.0,149.2,147.5,146.6,145.6,144.9,143.0,132.2,131.7,131.2,130.7,130.6,129.4,125.9,125.5,12 0.2,119.4,115.7,113.7,112.6,111.4,111.2,65.0,64.7,56.1,55.9,55.7,55.3,47.3,47.0,42.9,42.7,40.9,40.2,26.3,25.5.

[0058] 2. Preparation of (R,S)-1 trifluoroacetate

[0059]

[0060] Dissolve (R,S)-1 (200 mg) in dichloromethane (4.0 mL), add 2.0 mL of trifluoroacetic acid, stir and react overnight at room temperature, then evaporate the solvent under reduced pressure to obtain a yellowish-brown solid powder, which is (R,S)-1 trifluoroacetate. 1H NMR (400MHz, CDCl3) δ6.92(d,J=8.2Hz,2H),6.85(d,J=8.2Hz,1H),6.74(s,1H),6.65(d,J=8.2 Hz,2H),6.61(d,J=5.2Hz,2H),6.51(d,J=7.9Hz,1H),6.01(s,1H),4.29(d,J=4.6Hz,1H),4.15( d,J=7.8Hz,1H),3.84(s,3H),3.80(s,3H),3.69(s,3H),3.66-3.51(m,4H),3.48(s,3H),3.38–3 .26(m,2H),3.14–2.98(m,4H),2.95–2.88(m,1H),2.86(s,3H),2.81(s,3H),2.81-2.76(m,1H). 13 C NMR (125MHz, CDCl3) δ158.55,150.06,148.82,147.17,146.67,144.94,143.57,131.02,126.30,120.67,120.61,116.27,114.02,112.6 5,111.38,111.22,65.22,64.41,56.22,56.19,55.56,55.44,46.13,45.38,41.34,40.66,40.58,31.83,29.90,29.87,22.89.ESI-HRMS m / z calcd C 38 H 45 N2O6[M-CF3COO]+625.3272,found625.3302

[0061] 3. Preparation of (R,S)-1 hydrochloride

[0062]

[0063] Dissolve (R,S)-1 (200 mg) in 2.0 mL of 0.48 M HCl in diethyl ether. Stir for 1 hour and then evaporate the solvent under reduced pressure. Repeat this process 3 times to obtain a yellowish-brown solid powder, which is (R,S)-1 hydrochloride. 1H NMR(500MHz, CDCl3)δ6.88(t,J=7.8Hz,3H),6.75–6.65(m,4H),6.59(s,1H ),6.53(s,1H),6.17(s,1H),5.72(s,1H),4.12–3.97(m,2H),3.87(s,3H), 3.83(s,3H),3.73(s,3H),3.69–3.60(m,1H),3.54–3.40(m,3H),3.37(s,3 H),3.22–3.16(m,1H),3.13–2.98(m,4H),2.95–2.83(m,3H),2.74(s,6H). 13 C NMR (125MHz, CDCl3) δ158.55,150.06,148.82,147.17,146.67,144.94,143.57,131.02,126.30,120.67,120.61,116.27,114.02,1 12.65,111.38,111.22,65.22,64.41,56.22,56.19,55.56,55.44,46.13,45.38,41.34,40.66,40.58,31.83,29.90,29.87,22.89.

[0064] 4. Preparation of (R,S)-1 acetate

[0065] (R,S)-1 (200 mg) was dissolved in a mixed solution of acetic acid (2.0 mL) and dichloromethane (2.0 mL). After stirring for 1 hour, the solvent was evaporated under reduced pressure. This process was repeated 3 times to obtain a yellowish-brown solid powder, which is (R,S)-1 acetate.

[0066] 5. Preparation of (R,S)-1 sulfate

[0067] Dissolve (R,S)-1 (200 mg) in methanol (1.0 mL), add 30% sulfuric acid aqueous solution (2.0 mL) dropwise at 0 °C, stir for 1 hour in an ice bath, and slowly add diethyl ether (5-10 mL) until a solid precipitates. Filter and dry the solid to obtain (R,S)-1 sulfate.

[0068] 6. Preparation of (R,S)-1 hydrobromide

[0069] Dissolve (R,S)-1 (200 mg) in methanol (1.0 mL), and add 48% HBr aqueous solution (2.0 mL) dropwise at 0 °C. Stir for 1 hour in an ice bath, then slowly add diethyl ether (5-10 mL) until a solid precipitates. Filter and dry the solid to obtain (R,S)-1 hydrobromide.

[0070] Example 2

[0071] Inhibitory activity of orexin receptors of (R,S)-1, (R,S)-1 trifluoroacetate and (R,S)-1 hydrochloride

[0072] 2.1 Experimental Methods: Intracellular Calcium Release Assay

[0073] 2.1.1 Flow cytometry for Ca 2+ Analysis: CHO cells were treated with (R,S)-1, methylcamocarpine, and the positive control drug sulforaphane (SUV), and collected after 24 hours. Cells were incubated with dye (S1061S, Beyotime, China) according to the manufacturer's instructions, and treated with Ca... 2+ Fluo-4 indicator was used for staining of isolated cells. Intracellular calcium levels were detected by flow cytometry after stimulation with Orexin B (a specific agonist of the orexin receptor). 2+ Increased concentration, flow cytometry monitoring of Ca 2+ Changes in the green fluorescence intensity of the bound Fluo-4.

[0074] 2.1.2 Cell Lines and Cell Culture: CHO cells were obtained from the National Identification Cell Culture Collection Center (China). Cells were cultured in F-12K medium supplemented with 10% fetal bovine serum (Gibco, Life Technologies, New York, NY, USA), penicillin (100 μg / mL), and streptomycin (100 μg / mL) in a humidified atmosphere of 5% CO2 at 37°C. Cells were passaged every 2 days or when they reached 90% confluence. CHO-K1 cells stably expressing human (h)OX1R or OX2R were established as follows: lentiviral vectors overexpressing OX1R or OX2R plasmids were purchased from GeneChem Co., Ltd. (Shanghai, China). Cells were infected with lentivirus according to the manufacturer's protocol. Briefly, cells were plated in 12-well plates at a rate of approximately 2 × 10^5 cells / well; lentivirus was added to the medium (multiple of infection (MOI) = 1:20), and the medium was refreshed after 6–8 hours. Stable cells were screened with puromycin at a concentration of 1 μg / mL 72 hours after lentiviral infection. Identification of stable cell lines by Western blotting (refer to existing techniques Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, ​​Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Anna RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell. 1998 Feb 20; 92(4):573-85. doi:10.1016 / s0092-8674(00)80949-6.PMID:9491897.).

[0075] 2.1.3 FLIPR high-throughput real-time fluorescence detection technology: FLIPR detection was performed using Chinese hamster ovary (CHO) cells expressing human orexin receptors (hOX1R or hOX2R). These cells were seeded into 96-well plates and incubated at 37°C in 5% CO2, followed by the addition of the fluorescent calcium indicator Fluo-4AM (molecular probe). After cell washing, intracellular calcium... 2+Changes in fluorescence intensity were measured using a FLIPR (Molecular Devices) system. Different concentrations of orexin antagonists were applied to plates before the addition of orexin A (a specific agonist of the orexin receptor), and the IC50 value (the concentration required to inhibit a 50% agonist response) of each antagonist was calculated.

[0076] 2.2 Experimental Results

[0077] Orexin is a neuropeptide synthesized and secreted by the lateral hypothalamus. It maintains wakefulness; activation of OX1R and OX2R induces wakefulness, while blocking these two receptors increases the duration of REM and NREM sleep, more closely resembling physiological sleep. Animal studies have shown that selective OX2 receptor antagonists or non-selective OX1 / OX2 receptor antagonists can promote sleep in animals. Currently, several orexin receptor antagonists are in clinical trials for the treatment of insomnia, such as Suvorexant, GSK649868, and MK-6096. These compounds have shown good activity in treating insomnia both in vivo and in vitro. Clinically, orexin receptor antagonists can improve sleep quality, prolong sleep duration, and shorten sleep onset time in insomnia patients without altering sleep structure, and without the effects of daytime residual effects or drug dependence.

[0078] 2.2.1 Comparison of the antagonistic activity of (R,S)-1 and methylnepenone against orexin receptors

[0079] This invention compares the antagonistic activities of (R,S)-1, methyllimonene, and the positive control drug svorexin (SUV) on orexin receptors. Flow cytometry was used to analyze Ca... 2+ The results of the analysis are as follows Figure 1 As shown, where, Figure 1 A represents the calcium ion concentration change curve after (R,S)-1 treatment of CHO cells. Figure 1 B represents the calcium ion concentration inhibition curve after (R,S)-1 treatment of CHO cells. Figure 1 C represents the curve showing the change in calcium ion concentration in CHO cells after treatment with methylnephrine. Figure 1 D is the calcium ion concentration inhibition curve after treatment of CHO cells with methyl lotusine. Figure 1 E is a graph showing the change in calcium ion concentration in CHO cells after treatment with suvorexin. Figure 1 F shows the calcium ion concentration inhibition curve after treatment with suvorexin in CHO cells. The results indicate that in the CHO cell line, 50 μg / mL of orexin B specifically activates orexin receptors, and the concentration induces intracellular calcium... 2+A transient increase in concentration was observed. Subsequently, the antagonistic activities of (R,S)-1, methylnevicine, and the positive control drug sulforaphane (SUV) against orexin B were tested at concentrations of 0.01 μM, 0.1 μM, 0.5 μM, 1.0 μM, 2.5 μM, and 5.0 μM, respectively, to evaluate the drug's antagonistic effect on orexin receptors. Figure 1 A- Figure 1 As shown in Figure F, after treating CHO cells with (R,S)-1, methylcaenine, and the positive control drug sulforaphane (SUV), the IC50 values ​​against the orexin receptor were 0.015 μM, 0.80 μM, and 0.48 μM, respectively, indicating that (R,S)-1 has better orexin receptor antagonistic activity than methylcaenine and sulforaphane.

[0080] 2.2.2 Antagonistic activity of (R,S)-1, (R,S)-1 trifluoroacetate, (R,S)-1 hydrochloride, (R,S)-1 acetate, (R,S)-1 sulfate, and (R,S)-1 hydrobromide

[0081] This invention utilizes FLIPR high-throughput real-time fluorescence detection technology to test the antagonistic activities of (R,S)-1, (R,S)-1 trifluoroacetate, and (R,S)-1 hydrochloride against orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R), respectively. The FLIPR measurement results are as follows: Figure 2 As shown, where Figure 2 A represents the change in calcium ion release fluorescence intensity after treatment with (R,S)-1, (R,S)-1 trifluoroacetate, and (R,S)-1 hydrochloride in CHO cells overexpressing OX1R (OX1R-CHO). Figure 2 B represents the change in calcium ion release fluorescence intensity after treatment of CHO cells overexpressing OX2R (OX2R-CHO) with (R,S)-1, (R,S)-1 trifluoroacetate, and (R,S)-1 hydrochloride. For example... Figure 2 As shown in A, in 10 -6 Under the stimulation of orexin A, the IC50 values ​​of (R,S)-1, (R,S)-1 trifluoroacetate, (R,S)-1 hydrochloride, (R,S)-1 acetate, (R,S)-1 sulfate and (R,S)-1 hydrobromide on OX1R-CHO cells were 31 nM, 13 nM, 106 nM, 12 nM, 11 nM and 10 nM, respectively. Figure 2 B shows that the IC50 values ​​against OX2R-CHO cells after treatment with (R,S)-1, (R,S)-1 trifluoroacetate, (R,S)-1 hydrochloride, (R,S)-1 acetate, (R,S)-1 sulfate, and (R,S)-1 hydrobromide were 60 nM, 43 nM, 180 nM, 45 nM, 34 nM, and 36 nM, respectively.

[0082] Example 3

[0083] The therapeutic effects of (R,S)-1 on insomnia and anxiety symptoms

[0084] 3.1 Construction of the insomnia model: The construction method is as follows Figure 3 As shown. A chronic sleep deprivation mouse model was constructed using a variety of unpredictable stimuli. Mice in the chronic sleep deprivation group received two random stimuli daily for 3 months, including 6 hours of restraint stimulation, 4 hours of ice stimulation, 12 hours of food and water deprivation, 12 hours of forced movement, and 12 hours of water stimulation. The intermittent occurrence of the same type of stimulation made it impossible for the mice to predict when the stimulation would occur, which was crucial for the success of the modeling. During the stimulation exposure, the CTRL group was placed in the same environment without stimulation. Regarding water stimulation, C57BL / 6J mice were placed in a box with four platforms, each 5 cm high and 3 cm in diameter, spaced 5 cm apart. The water temperature around the platforms was approximately 22°C, and the platforms were positioned 1 cm above the water surface. Mice were divided into five groups: control group (CTRL), insomnia group, and drug group (40 mg / kg). Except for the CTRL group, the other groups experienced a variety of unpredictable stimulation procedures. The drug was administered by gavage, dissolved in a mixture of DMSO (10%) and corn oil (90%).

[0085] 3.2 Experimental Evaluation Methods

[0086] 3.2.1 Sodium pentobarbital-induced sleep test: The sodium pentobarbital-induced sleep test is commonly used to assess whether a drug has sedative-hypnotic activity. A sodium pentobarbital-induced sleep test is performed 30 minutes after administration, in which mice are treated with 1% sodium pentobarbital (50 mg / kg, ip), which induces complete sleep, and then sleep signs are monitored. Sleep latency is defined as the time from sodium pentobarbital injection to the disappearance of the righting reflex. Sleep signs are observed when the righting reflex is lost for more than 1 minute. Sleep duration is defined as the time interval from the loss of the righting reflex to its recovery.

[0087] 3.2.2 Open Field Test (OFT): The open field test is used to assess anxiety-like behaviors. Mice were placed in the laboratory for 2 hours to acclimatize. Thirty minutes after drug administration, each group of mice underwent the OFT, and behavioral parameters were collected and counted within 5 minutes. Movement trajectories, total distance, and average speed were analyzed using a behavior analysis system (Smart version 3.0).

[0088] 3.2.3 Elevated Maze Test (EPM): The elevated maze (EPM) test was performed to assess anxiety-like behaviors. Mice were placed in the laboratory for 2 hours to acclimatize; EPM was performed on each group of mice 30 minutes after drug administration. The maze consisted of two open arms and two closed arms. To begin the test, mice were placed face-to-face in the central area of ​​the open arms, and behavioral parameters were collected and counted over 5 minutes. The percentage of time spent by the mouse on the open and closed arms was analyzed using a behavioral analysis system (Smart Version 3.0).

[0089] 3.2.4 LabMaster System: The circadian rhythms of movement, energy expenditure, respiratory exchange rate (RER), water intake, and food consumption were evaluated using the LabMaster system (TSE Systems, Germany). This system allows for long-term, continuous recording of these parameters without animal disturbance. The system consisted of 16 recording units, each comprising a test cage (42 cm × 6.5 cm × 15 cm, length × width × height), two external infrared frames, two weight sensors, and two probes for measuring oxygen and carbon dioxide concentrations. These devices were connected to a computer that used LabMaster software to collect and analyze data. The LabMaster software used a 48-minute sampling interval, with each unit recording 3 minutes of data. In an isolated environment, under a 12-hour light / dark cycle, the room temperature was maintained at 22.0 ± 1.0 °C. Mice were placed individually in the laboratory, and all parameters were analyzed according to the manufacturer's guidelines. The RER was estimated by calculating the VCO2 to VO2 ratio.

[0090] 3.3 Experimental Results

[0091] 3.3.1 Successful Construction of the Insomnia Model

[0092] Sleep-deprived mice are an ideal model for studying the efficacy of insomnia medications. To investigate the anti-insomnia efficacy of (R,S)-1, this invention used a 3-month chronic unpredictable stimulation to establish a sleep-deprivation mouse model. Figure 3 Following this period, the mouse model exhibited a depressive phenotype. The results of the pentobarbital-induced sleep experiment showed that, compared to the control group, the sleep onset rate in the insomnia model group after pentobarbital sodium induction was only 60% (compared to 100% in the control group). Figure 6 In a mouse model of insomnia, sleep latency was prolonged and sleep duration was shortened. Figure 7 A and Figure 7B). Exercise, energy expenditure, water intake, and food intake are activities influenced by sleep / wake rhythms. Therefore, this invention utilizes the LabMaster system (TSE system) to assess the effects of chronic, unpredictable stimuli on these diurnal behaviors. The LabMaster system can record long-term, undisturbed diurnal rhythms of activity and intake in animals. Results showed that, compared to the control group, diurnal exercise, energy expenditure, and respiratory exchange rate (RER) were significantly increased in the mouse model. Figure 4 A, Figure 4 B. Figure 4 C Figure 5 A, Figure 5 B. Figure 5 C Figure 8 The results indicate that sleep-deprived mice have more awake time than normal mice. These results demonstrate the successful establishment of an insomnia mouse model.

[0093] 3.3.2 (R,S)-1 improves sleep and anxiety symptoms in insomnia mice

[0094] This invention utilizes an insomnia model to study the therapeutic effect of (R,S)-1 on sleep disorders and anxiety symptoms. Mice were randomly divided into a CTRL (control group), an insomnia group, and a (R,S)-1 treatment group (40 mg / kg). After 4 weeks of administration, the elevated maze test (EPMT) and open field test (OFT) were performed to assess whether (R,S)-1 improved the psychological behavior of insomniac mice. For example, the EPMT... Figure 9 A, Figure 9 B. Figure 9 As shown in C), the insomnia group spent less time on the open arm position and more time on the closed arm position, indicating a significant improvement compared to the (R,S)-1 treatment group. Furthermore, the insomnia group showed shorter walking distances and less time spent in the center, effects that were significantly reversed in the (R,S)-1 group. Figure 9 D、 Figure 9 E, Figure 9 F). These results indicate that (R,S)-1 can effectively improve anxiety symptoms in mice.

[0095] Subsequently, a pentobarbital-induced sleep experiment was conducted to assess the effects of (R,S)-1 on sleep latency and duration. Figure 9 As shown in G, after administration of a hypnotic dose of sodium pentobarbital (50 mg / kg), all mice except the insomnia group (60%) fell asleep. Compared with the insomnia group, (R,S)-1 significantly prolonged the sleep time and shortened the sleep latency of insomnia mice, making them closer to normal sleep. Figure 9 H, Figure 9 I). The above results indicate that (R,S)-1 can effectively improve sleep disorder symptoms in insomnia mice.

[0096] 3.3.3 Assessment of the effects of (R,S)-1 on diurnal behavior and respiratory exchange rate

[0097] This invention also investigated the effects of (R,S)-1 on diurnal behavior and respiratory exchange rate. These indicators are closely related to sleep / wake rhythms. Diurnal behavior recorded during the light phases of days 0, 1, and 2, and the dark eclipse phases of days 1 and 2, revealed characteristic diurnal rhythm temporal processes. Figure 10 A). As nocturnal animals, their activity levels during the dark feeding period are significantly higher than during the light feeding period. Notably, compared to the control group, the insomnia group showed a significant increase in daytime activity and energy expenditure. Figure 10 A, Figure 10 B). In contrast, treatment with (R,S)-1 significantly reduced exercise and energy expenditure in insomnia mice. Figure 10 A, Figure 10 B). Furthermore, the daily food and water intake in the insomnia group was significantly higher than that in the control group; (R, S)-1 administration significantly reduced the food intake of insomnia mice. Figure 10 C).

[0098] Mice exhibit different oxygen consumption and carbon dioxide production during sleep and wakefulness. In this study, carbon dioxide production versus oxygen consumption (RER, respiratory exchange ratio) was measured as an indicator of the sleep / wake cycle. RER was estimated by calculating the ratio of VCO2 to VO2. Values ​​were adjusted for mouse body weight. Figure 10 As shown in Figure D, the relative efficiency (RER) of insomnia mice was higher than that of the CTRL group in both dark and light conditions. Treatment with (R,S)-1 reduced the RER of insomnia mice, and the differences between the groups were statistically significant at all four time points. These results indicate that (R,S)-1 can significantly improve sleep / wake cycle disorders in insomnia mice.

[0099] Example 4

[0100] Comparative analysis of the anti-insomnia effects of (R,S)-1 and methylneferine.

[0101] This invention evaluated the sleep-promoting effects of (R,S)-1, (R,S)-1 trifluoroacetate, and methylneferine in a pentobarbital-induced sleep experiment in mice. The concentrations of (R,S)-1, (R,S)-1 trifluoroacetate, and methylneferine were all 40 mg / kg. Sleep latency was defined as the time from pentobarbital injection to the loss of the righting reflex. Sleep signs were observed when the righting reflex was lost for more than one minute. Sleep duration was defined as the time interval from the loss of the righting reflex to its recovery. Results are as follows: Figure 11As shown, after administration of (R,S)-1, (R,S)-1 trifluoroacetate, and methylneferine for 30 minutes, treatment with a hypnotic dose of sodium pentobarbital (50 mg / kg) resulted in all mice falling asleep. Compared with the insomnia group, the medication significantly shortened the sleep latency of the mice. Figure 11 A), and prolong sleep duration ( Figure 11 B). Compared with Neferine, (R,S)-1 and (R,S)-1 trifluoroacetate showed a more significant effect in shortening sleep latency and prolonging sleep time in insomniac mice. Among them, (R,S)-1 trifluoroacetate was more effective than (R,S)-1.

[0102] Example 5

[0103] Comparative analysis of the anxiolytic activity of (R,S)-1, (R,S)-1 trifluoroacetic acid, and methylneferine.

[0104] This invention used the open field test (OFT) to evaluate the therapeutic effects of neferine, (R,S)-1 trifluoroacetic acid, and (R,S)-1 on anxiety in mice. The concentrations of R,S)-1, (R,S)-1 trifluoroacetic acid, and neferine were all 40 mg / kg. The total distance of horizontal movement reflected the activity level of the mice, indicating the sedative and anxiolytic effects of the drugs. Results are as follows... Figure 12 As shown, during the 25-minute measurement period, spontaneous activity in all animals showed a decreasing trend. Compared with the control group, treatment with methylneferine, (R,S)-1 trifluoroacetic acid, and (R,S)-1 significantly reduced spontaneous activity in mice (p<0.05). Furthermore, compared with pre-administration levels, the distance of spontaneous activity in mice decreased significantly in a time-dependent manner after administration. Compared with methylneferine and daridorexant (commonly used positive control drugs for insomnia), (R,S)-1 and (R,S)-1 trifluoroacetic acid showed greater sedative effects. Figure 12 Furthermore, (R,S)-1 trifluoroacetate is more effective than (R,S)-1.

[0105] Example 6

[0106] Pharmacokinetic characteristics and brain penetration evaluation of (R,S)-1

[0107] Experimental methods

[0108] Pharmacokinetic studies in rats

[0109] All animal experiments were approved by the Animal Care and Use Committee of Central South University. Fifteen female Sprague-Daw-ley rats (180-200g, 6 weeks old) were randomly divided into 5 groups, three of which received tail vein injections (control group: 5 mg / kg). -1 PBS), (R,S)-1 group (5 mg·kg -1 ), Methylnephrine group (5 mg·kg) -1 (R,S)-1 trifluoroacetate group (5 mg·kg) -1 (R,S)-1 were administered orally (10 mg / kg) -1 The (R,S)-1 solution was prepared by dissolving it in a mixture of DMSO / PEG400 / Tween 80 / physiological saline (10 / 40 / 5 / 45, V / V / V / V). Blood samples were collected before and after administration. Administration time was recorded, and 100 μL of blood was collected from the femoral vein of mice at nine time points (0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours). Plasma was separated from blood by centrifugation at 8000 rpm for 5 minutes and then immediately frozen at -80°C for further analysis. LC-MS / MS analysis was used to determine the concentration of (R,S)-1 in plasma. Briefly, 30 μL of plasma was added to 5 μL of internal standard (lemborexant, 1 μg / mL) and 200 μL of acetonitrile containing 0.1% formic acid in a 5 mL centrifuge tube, and centrifuged at 14000 g for 10 minutes. Collect the supernatant layer and inject 5 μL of the sample into it for LC-MS / MS analysis.

[0110] Brain Penetration Study of (R,S)-1 in SD Rats

[0111] Three female Sprague-Dawley rats (180–200 g, 6 weeks old) were orally administered the drug (10 mg·kg⁻¹). The (R,S)⁻¹ solution was prepared by dissolving the drug in a mixture of DMSO / PEG400 / Tween 80 / physiological saline (10 / 40 / 5 / 45, V / V / V / V). Blood and brain samples were collected at 2 hours to prepare plasma and brain homogenates, respectively. Similarly, 30 μL of plasma / brain homogenate was added to 5 μL of internal standard (lemborexant, 1 μg / mL) and 200 μL of acetonitrile containing 0.1% formic acid in a 5 mL centrifuge tube, and the mixture was centrifuged at 14000 g for 10 min. The supernatant was collected, and 5 μL aliquots were injected for LC-MS / MS analysis.

[0112] 6.2 Experimental Results

[0113] As shown in Table 1.

[0114] Table 1. Pharmacokinetic data in rats

[0115]

[0116] a PK parameters (mean ± standard deviation, n = 3), dosage: oral, 10 mg / kg; intravenous, 5 mg / kg. "-" indicates not measured. b. Drug concentrations in cardiac plasma and brain were measured at 2.0 hours after administration, and the ratio of brain drug concentration to cardiac plasma drug concentration was calculated.

[0117] The results showed that after oral administration of 10 mg / kg, the maximum concentration (Cmax) and area under the curve (AUC0-t) of (R,S)-1 were 770 ng / mL and 7639 h·ng / mL, respectively. After intravenous injection of 5 mg / kg, the half-life of (R,S)-1 was 9 hours, and the half-life of (R,S)-1 trifluoroacetate group was 7.2 hours; compared with the half-life of methylnephrine (6.1 hours, Biomed Chromatogr. 2021; 35(11):e5193.), the half-life was prolonged. This experiment also evaluated the blood-brain barrier permeability of (R,S)-1. By measuring the brain-to-blood plasma ratio (the ratio of drug concentration in the brain to drug concentration in cardiac plasma) at the maximum time (2 hours), the results showed that (R,S)-1 was detected in the brain with values ​​ranging from 0.28 to 1.91, indicating that it can cross the blood-brain barrier and has good brain distribution (B / P>0.3, Annu Rev Pharmacol Toxicol. 2000; 40:133-57.). These findings suggest that (R,S)-1 possesses the pharmacokinetic properties of a central nervous system drug.

[0118] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The use of the compound in the preparation of orexin receptor 1 antagonists and / or orexin receptor 2 antagonists, characterized in that, The compounds are compounds with the following structures and pharmaceutical salts:

2. The application of the compound in the preparation of a drug for treating insomnia, characterized in that, The compounds are compounds with the following structures and pharmaceutical salts:

3. The use of the compound in the preparation of a drug for treating anxiety disorders, characterized in that, The compounds are compounds with the following structures and pharmaceutical salts:

4. The application according to any one of claims 1-3, characterized in that, The medicinal salt is a salt formed by a compound and an acidic salt of an acid or inorganic base.

5. The application according to claim 4, characterized in that, The acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-benzenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, tribromoacetic acid, trichloroacetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid; the acidic salt of the inorganic base is a salt containing a basic metal cation, a salt containing an alkaline earth metal cation, or a salt containing ammonium ions.

6. The application according to any one of claims 1-3, characterized in that, The compound significantly prolongs sleep time, shortens sleep latency, and reduces exercise, energy expenditure, and food intake.

7. The application according to any one of claims 1-3, characterized in that, The compound significantly improves sleep cycle disorders.

8. The application according to any one of claims 1-3, characterized in that, The compound significantly reduces autonomous activity distance.

9. The application according to any one of claims 1-3, characterized in that, The drug also includes pharmaceutically commonly used excipients.

10. The application according to any one of claims 1-3, characterized in that, The dosage form of the drug is any one of granules, capsules, tablets, injections, infusions, or suppositories.

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