Application of a thioamide compound in regulating circadian rhythm

Abstract

The present invention relates to an application of a thioamide compound in regulating circadian rhythm. The thioamide compound of the present invention has been subjected to in vitro and in vivo experiments, showing obvious rapid regulation of circadian rhythm disorders, reduction of synchronization time of circadian rhythm phase shifts, and regulation of the expression of hypothalamic circadian clock proteins Per2, Bmal1 and related phosphorylated proteins P‑Per2, P‑CaMKII. Therefore, it can be used to treat jet lag, shift work, continuous multi-day and night work or circadian rhythm-related sleep disorders, and has the characteristics of high efficiency and low toxicity, can improve and guarantee work ability, and is of great significance for people with circadian rhythm disorders who work in night shifts, shift work, and long-distance cross-time zone flights.

Description

Technical Field

[0001] The invention relates to application of a thioamide compound, and in particular to application in regulating circadian rhythm. Background Art

[0002] The rotation of the earth causes periodic changes in light, temperature, humidity and available food in a nearly 24-hour circadian rhythm. Therefore, organisms on the earth must adjust their physiological behaviors accordingly to adapt to the environment. In this process, most organisms have formed nearly 24-hour biological rhythms at the behavioral and physiological and biochemical levels. For example, various physiological and metabolic activities such as human sleep, wakefulness, eating, excretion, hormone secretion, body temperature, blood pressure, etc. have a certain rhythmicity in order to adapt to the periodic changes in the external environment.

[0003] Studies have found that the mammalian circadian rhythm system is mainly concentrated in a specific brain area within the central nervous system, and the circadian rhythm process mainly occurs in the suprachiasmatic nucleus (SCN) and its adjacent structures in the anterior hypothalamus. The SCN is the most important circadian rhythm center in mammals, and it is involved in controlling a variety of rhythmic activities such as sleep and wake cycles. The ability of the SCN activity rhythm to self-generate and maintain depends on the spontaneous transcriptional feedback loop formed by a few genes, including Bmal1, Clock, Per, Cry, and CKIε. Mutations or deletions in any of the above clock genes will lead to an extension or shortening of the body's free-running cycle. The SCN's own rhythm is affected by both the external environment and collective endogenous factors. The rhythmic disorder of the core biological clock and the peripheral biological clock system in the body caused by the external environment is the key cause of biological rhythm disorders and other complications in the human body.

[0004] In response to the needs of people with circadian rhythm disorders who work night shifts, shift work, and long-distance flights across time zones, the current domestic and foreign methods mainly adjust sleep and work schedules in accordance with work task requirements, control labor intensity and time, and reduce "shift work" to improve and correct circadian rhythm disorders in complex working environments. However, the effects are relatively limited or passive, and there is a lack of active and effective measures, especially a lack of effective targeted drugs with obvious effects, few side effects, and easy application.

[0005] In recent years, various countries have conducted a series of studies on conventional sleep control drugs. Caffeine, modafinil, and armofinil can be used as wakefulness-promoting drugs to improve symptoms of sleepiness and promote alertness; melatonin, melatonin receptor agonists, and hypnotic sedatives can be used to promote sleep; caffeine, melatonin, cordycepin, dexamethasone and other hormone-like drugs can also be used to adjust jet lag. However, the above drugs all have problems such as low efficacy and high tolerance, and have certain adverse reactions. They are not suitable for long-term use and cannot meet the needs of regulating circadian rhythms, improving the working ability of personnel in special environments, and medical protection. Therefore, it is of great significance to develop highly efficient and low-toxic drugs to improve circadian rhythm disorders.

[0006] As the main permeable Ca 2+ The transient receptor potential (TRP) superfamily of ion channels plays an important role in regulating intracellular Ca 2+ The thioamide compounds disclosed in the present invention are TRPV1 channel specific inhibitors, have anti-allodynia effects, and are used in a variety of malignant tumors, inflammation, skin, cardiovascular, gastrointestinal and mental diseases. Currently, there is no report on the use of this compound in regulating circadian rhythm disorders. Summary of the invention

[0007] The object of the present invention is to provide an application of a thioamide compound, wherein the compound is a nitrogen-[2-(4-chlorophenyl)ethyl]-7,8-dihydroxy-1,3,4,5-tetrahydro-2-hydro-2-benzazepine-2-thioamide compound as shown in the following formula (I). The compound shown in formula (I) or a pharmaceutically acceptable salt thereof can be used to regulate circadian rhythm, and the compound specifically has the following structure.

[0008]

[0009]

[0010] The pharmaceutically acceptable salts of the compound of formula (I) of the present invention include inorganic acid salts and organic acid salts, such as hydrochloride, phosphate, sulfate, acetate, maleate, methanesulfonate, fumarate, citrate, benzenesulfonate, toluenesulfonate or tartrate.

[0011] The dosage of the compound of formula (I) or a pharmaceutically acceptable salt thereof. Although the dosage varies depending on the subject, administration method, symptoms and other factors, it is generally about 0.1 mg / kg-1000 mg / kg body weight per dose for adults who are preparing to regulate circadian rhythms, preferably about 0.1 mg / kg-800 mg / kg body weight, more preferably 0.5 mg / kg-500 mg / kg body weight, and can be administered once or several times a day.

[0012] The present invention also discloses a pharmaceutical composition for regulating circadian rhythm, which comprises a nitrogen-[2-(4-chlorophenyl)ethyl]-7,8-dihydroxy-1,3,4,5-tetrahydro-2-hydro-2-benzazepine-2-thioamide compound or a pharmaceutically acceptable salt thereof as shown in formula (I) and a pharmaceutically acceptable excipient.

[0013] The compound of formula (I) of the present invention and its pharmaceutical salt can be safely administered orally or non-orally, or can be safely administered orally or non-orally in the form of a pharmaceutical composition such as tablets, sustained-release preparations, sugar-coated preparations, capsules, injections, solutions, etc. formed with pharmaceutically acceptable excipients such as carriers, excipients and other additives. For the preparation of oral pharmaceutical composition preparations, lactose or starch can be used as a carrier, and gelatin, sodium carboxymethyl cellulose, methylcellulose polyvinyl pyrrolidone, etc. are suitable binders or granules. Starch or microcrystalline cellulose can be selected as a disintegrant, and talcum powder, colloidal silica, glyceryl stearate, calcium or magnesium stearate, etc. are often used as suitable anti-adhesive agents and lubricants. For example, tablets can be prepared by pressing wet granules. The active ingredient is mixed with a carrier and optionally with a portion of a disintegrant additive, and the mixture is granulated with an aqueous solution, an alcoholic or aqueous alcoholic solution of a binder in a suitable device, and the dried granules are then added with other disintegrants, lubricants and anti-adhesives to tablet the mixture.

[0014] The compound of formula (I) of the present invention is not easily soluble in water. To increase solubility, the heterocyclic derivatives can be freed and prepared into pharmaceutically acceptable inorganic acids and organic acid salts, preferably maleic acid, methanesulfonic acid, fumaric acid and the like salts, which are advantageous for parenteral administration in the form of injections. When preparing the injection, the active ingredient compound of formula (I) or its pharmaceutical salt is dissolved in distilled water or various organic solvents, and a cosolvent such as sorbitol monolaurate, monostearate or monooleate can also be added. The injection can also contain various commonly used additives, preservatives, etc. Before filling the ampoule, the injection needs to be filtered and sterilized after filling.

[0015] In different dosage forms, the content of the compound of formula (I) or its pharmaceutically acceptable salt can be adjusted as needed, and in oral dosage forms such as tablets or capsules, the weight concentration can be 10%-30%. The effect of the compound of the present invention on regulating circadian rhythm has been verified by experiments.

[0016] The application of the compound of formula (I) of the present invention is verified by experiments. In vitro experiments have confirmed that the compound of formula (I) of the present invention can promote the phase shift of the biological rhythm of U2OS cells. In vivo experiments on mice have confirmed that the compound of formula (I) has a significant promoting effect on the synchronization of circadian rhythms after 8 hours of light delay, can significantly reduce the synchronization time of circadian rhythm phase shift, and can regulate the expression of hypothalamic clock proteins Per2, Bmal1 and related phosphorylated proteins P-Per2, P-CaMKII, which plays an important role in regulating circadian rhythms and improving circadian rhythm disorders. In summary, the compound of formula (I) of the present invention or its pharmaceutical salt shows a significant effect of improving circadian rhythm disorders, and has the characteristics of high efficiency and low toxicity, so it can be used to regulate circadian rhythms, regulate circadian rhythm disorders caused by jet lag, shift work, continuous multi-day and night work, and circadian rhythm-related sleep disorders.

[0017] The present invention also discloses the use of a pharmaceutical composition containing a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a circadian rhythm regulating drug, wherein the circadian rhythm regulating drug includes adjusting circadian rhythm disorders caused by jet lag, shift work, continuous multi-day and night work, and circadian rhythm-related sleep disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the results of the Lumicycle experiment in a cell experiment, which evaluates the effect of drugs on the phase shift of the U2OS cell line.

[0019] Figure 2 To evaluate the effect of drugs on circadian phase adaptation of mice after an 8-hour delay in light time after two administrations at the original circadian time ZT15 (Zeitgeber Time, a time unit based on the jet lag cycle, ZT0 is the time when the lights come on and the activity starts, and ZT12 is the time when the lights go off).

[0020] Figure 3 To evaluate the effect of drugs on circadian phase synchronization in mice after 8-hour light shift after two administrations at the original circadian time ZT15.

[0021] Figure 4 To evaluate the effect of drugs on the expression of circadian clock protein levels in the mouse hypothalamus 12h and 24h after administration at the circadian time ZT12. DETAILED DESCRIPTION

[0022] In order to more fully explain the implementation of the present invention, formulation preparation examples 1 to 4 are provided. The examples provided by the present invention are only used to explain the present invention, rather than to limit the scope of the present invention.

[0023] Example 1 Preparation of tablets containing compound (I) of the present invention

[0024] composition Dosage / tablet (mg) Weight concentration (%) Compound of formula (I) 10 10.0 Microcrystalline Cellulose 35 35.0 starch 45 45.0 Polyvinylpyrrolidone 4 4.0 Sodium carboxymethyl starch 4.5 4.5 Magnesium Stearate 0.5 0.5 talc 1 1.0 total 100 100.0

[0025] The active ingredient, starch and cellulose are sieved and mixed thoroughly, the polyvinyl pyrrolidone solution is mixed with the above powder, sieved, and the wet granules are dried at 50-60°C, carboxymethyl starch sodium salt, magnesium stearate and talc are pre-sieved, and then added to the above granules for tableting. The compound of formula (I) is provided by Selleck, and the other reagents are commercially available, the same as in the following examples.

[0026] Example 2 Preparation of sustained-release tablets containing compound (I) of the present invention

[0027] composition Dosage / tablet (mg) Weight concentration (%) Compound of formula (I) 10 10.0 Eudragit RS-PO 10 10.0 lactose 40 40.0 Calcium Hydrogen Phosphate 20 20.0 total 100 100.0

[0028] The compound of formula (I), lactose and calcium hydrogen phosphate are sieved and fully mixed, Eudragit RS-PO is mixed with the above powders, and tablets are pressed using a wet granulation process.

[0029] Example 3 Preparation of capsules containing compound (I) of the present invention

[0030] Capsules containing 100 mg of active ingredient per capsule are prepared as follows:

[0031]

[0032] The compound of formula (I) and all the above ingredients are sieved and fully mixed, granulated by dry granulation method, dried at 50-60°C, the granule content is measured, the filling amount is calculated, and the granules are loaded into capsules.

[0033] Example 4 Preparation of injection containing compound (I) of the present invention

[0034] composition Dosage Compound of formula (I) 200mg Mannitol 700mg PEG3000 10mg Distilled water 100ml

[0035] The pH value is adjusted to 7.0-7.5, and the concentration of the filtered filtrate is 3 / ml. The filtrate is packaged at 2 ml per ampoule and freeze-dried to obtain the injection solution.

[0036] Example 5 Lumicycle experiment to evaluate the effect of drugs on phase shift of U2OS cell line

[0037] 1. Experimental Materials

[0038] Experimental cells: Per2-Luc U2OS cells, Bmal1-Luc U2OS cells

[0039] Experimental instruments: CO2 incubator, microscope, Lumicycle instrument (Actimetrics).

[0040] Experimental reagents: DMEM culture medium (GIBCO), Australian serum (GIBCO), PBS, DMEM powder, sterile water, HEPES, penicillin-streptomycin, sodium bicarbonate sulfate, gentamicin, DMSO, etc.

[0041] Experimental drugs: Thioamide compounds of formula (I) were purchased from Selleck.

[0042] 2. Experimental Methods

[0043] Prepare 2×DMEM culture medium: add one bag of DMEM powder to 470 ml sterile water, 10 ml 1M HEPES, 10 ml penicillin-streptomycin, 5 ml 7.5% sodium bicarbonate solution and 500 mg gentamicin sulfate, stir thoroughly and adjust the pH value to between 7.2-7.4, and filter with a 0.22 μm filter.

[0044] Prepare XM medium: add 25 ml of 2×DMEM medium, 25 ml of sterile water, 1 ml of 100 mM Luciferin and 1 ml of B27.

[0045] Lumicycle monitoring of biological rhythms: Per2-Luc U2OS cells or Bmal1-Luc U2OS cells were cultured at 7.5×10 5 The cells were inoculated in a 3.5 cm culture dish. After 24 hours when the confluence reached 100%, XM medium containing 5 or 10 μM of the compound of formula (I) was prepared. The old medium in the culture dish was discarded, and the dish was washed twice with 1 ml of PBS and replaced with 2 ml of XM medium containing the compound of formula (I). The dish was sealed with high vacuum sealing grease and sealing film, and then placed in a Lumicycle instrument to monitor the fluorescence intensity in real time.

[0046] Data analysis: Real-time monitoring data were analyzed using Lumicycle Analysis software to analyze phase changes and compared with the control group.

[0047] 3. Experimental results

[0048] The phase shift diagram and phase shift statistics of Per2-Luc U2OS cells after administration of 5 and 10 μM compound of formula (I) are shown in the figure. Figure 1 As shown in A, compared with the control group, the phase shift was 0.96±0.16h (P=0.0032, statistically significant) after administration of 5μM compound (I), and 3.41±0.32h (P<0.0001, statistically significant) after administration of 10μM compound (I). Schematic diagram of phase shift and statistical results of phase shift after administration of 5 and 10μM compound (I) to Bmal1-Luc U2OS cells Figure 1As shown in B, after administration of 5 μM compound of formula (I), the phase shifted back by 1.31±0.35 h (P=0.0067, statistically significant), and after administration of 10 μM compound of formula (I), the phase shifted back by 3.76±0.68 h (P<0.0001, statistically significant).

[0049] Example 6: Evaluation of the effect of drugs on circadian rhythm phase synchronization in mice after 8 hours of illumination

[0050] 1. Experimental Materials

[0051] Experimental animals: C57BL / 6J male mice (Spefox Biotechnology Co., Ltd.), weighing (20±2) g.

[0052] Experimental instrument: CLOCKLAB rhythmic biology system (Wuhan Pubaike Technology Co., Ltd., model: ACT-556B)

[0053] Experimental drugs: Thioamide compounds of formula (I) were purchased from Selleck, and melatonin was purchased from MedChemExpress.

[0054] 2. Experimental Methods

[0055] Screening of spontaneous activity of mice: Purchased male C57BL / 6J mice were housed in cages equipped with running wheels, and each cage had a light intensity of about 200 lux. The running wheel activity of each animal was continuously recorded by using a digital system that records the number of wheel rotations, and stored every 6 minutes for further analysis. The mice lived in a normal 12:12 hour cycle lighting environment for about 14 days (CT8 is on, which means turning on the lights at 8 am; CT20 is off, which means turning off the lights at 8 pm, and so on in the following description, CT is a standard time unit); on the 14th day, the circadian rhythm phase was adjusted to delay the light for 8 hours (i.e., the light switching time was shifted back by 8 hours, CT16 was on, and CT4 was off). The Clocklab rhythm biology system was used to observe the animals' adaptation to the new light cycle, and mice with relatively uniform day and night activities were selected for subsequent experiments. Mice with too fast rhythm adjustment or irregular activities were eliminated. The 35 screened mice were randomly divided into a control group, a 5mg / kg melatonin group, a 2.5mg / kg compound of formula (I) group, a 5mg / kg compound of formula (I) group, and a 10mg / kg compound of formula (I) group, with 7 mice in each group. The drug was administered before the next light adjustment, and the administration time was 1 hour before turning on the lights. After administration, the light time was adjusted to 8 hours later. The drug was administered at CT15, and the light time was adjusted to CT0 to turn on the lights and CT12 to turn off the lights. The second administration was performed 24 hours after administration, and the drug was administered twice in total, and the administration method was intraperitoneal injection. After administration, the Clocklab rhythmic biology system was continued to record and observe the movement of the mice for ten days.

[0056] 3. Experimental results

[0057] The phase adjustment of the control group, 5 mg / kg melatonin group, 2.5 mg / kg compound of formula (I) group, 5 mg / kg compound of formula (I) group, and 10 mg / kg compound of formula (I) group was as follows: Figure 2 As shown in A, Figure 2 B is the time of phase delay as the number of days of drug administration increases. Figure 3 The results of statistical analysis of the time required for the five groups to synchronize to the new rhythm showed that compared with the 7.57±1.72 days taken by the control group mice to adapt to the 8-hour light delay, the 5 mg / kg melatonin group required 5.29±0.95 days (P=0.0436, statistically significant), the 2.5 mg / kg compound of formula (I) group required 5.86±1.77 days (P=0.1998, no statistically significant), the 5 mg / kg compound of formula (I) group required 4.86±1.21 days (P=0.0114, statistically significant), and the 10 mg / kg compound of formula (I) group required 3.57±1.40 days (P=0.0001, statistically significant); compared with the 2.5 mg / kg compound of formula (I) group and the 10 mg / kg compound of formula (I) group, P=0.0436, there was a statistically significant difference; there was no statistically significant difference between the other groups.

[0058] 4. Conclusion

[0059] After the light on / off time was shifted back by 8 hours, the original circadian rhythm was disrupted. The effects of different doses of 2.5, 5 and 10 mg / kg of the compound of formula (I) were observed. 5 and 10 mg / kg of the compound of formula (I) could accelerate the adaptation of mice to light, while 2.5 mg / kg of the compound of formula (I) had no obvious effect on accelerating the adaptation. Figure 2 A. Figure 2 B. Figure 3 As shown. Under the action of 5mg / kg and 10mg / kg of the compound of formula (I), it can be clearly seen that the effect of 10mg / kg is more obvious than that of 5mg / kg. Therefore, the faster the mice adapt to the light changes as the dose of the compound of formula (I) increases, the faster the mice adapt to the light changes, which is dose-dependent. At the same time, it can be seen that the effect of 2.5mg / kg of the compound of formula (I) on accelerating the adaptation of mice to light is weaker than 5mg / kg of melatonin, but the effect of 5mg / kg and 10mg / kg of the compound of formula (I) on accelerating the adaptation of mice to light seems to be stronger than 5mg / kg of melatonin.

[0060] Example 7 Evaluation of the effect of drugs on the expression of circadian clock protein levels in the mouse hypothalamus 12 hours after administration at circadian rhythm time ZT12

[0061] 1. Experimental Materials

[0062] Experimental animals: C57 / B6J male mice (Spefox Biotechnology Co., Ltd.), weight (20±2) g

[0063] Experimental instruments: tissue grinder (Shanghai Jingxin Technology), ELISA reader, electrophoresis instrument (Bio-Rad, USA), Amersham Imager 680 (GE).

[0064] Experimental reagents: RIPA lysis buffer (Beyotime), protease inhibitor cocktail (Solybo), phosphatase inhibitor (Beyotime), BCA protein concentration assay kit (Beyotime), 5× protein loading buffer (Beyotime), skim milk powder (BD), 1.5M Tris-HCl buffer (Solybo), 1.0M Tris-HCl buffer (Solybo), 10% SDS (Solybo), AP (Thermo Scientific), Tris (Biotopped), glycine (Biotopped), ACTB antibody (SangonBiotech), Bmal1 antibody (Proteintech), P-Bmal1 antibody (CST), Per2 antibody (RabMAb), P-Per2 antibody (Immunoway), P-CaMKII antibody (mAb), ECL luminescent liquid (Mishu).

[0065] Experimental drugs: The compound of formula (I) was purchased from Selleck.

[0066] 2. Experimental Methods

[0067] Hypothalamic protein extraction: Intraperitoneally inject 5 mg / kg of drugs (control solvent, melatonin, compound of formula (I)) at ZT12 of normal circadian rhythm of mice, take the mouse hypothalamus 12 hours later, put it into liquid nitrogen for rapid freezing, then add 100 μl protein lysis buffer (RIPA: phosphatase inhibitor: protease inhibitor = 970:29:10), grind it with a tissue grinder, the condition is 60 Hz, 20 s. After grinding, all samples are centrifuged in a centrifuge at 4°C, the speed is 12000r / min, 10min, and the supernatant liquid is aspirated into a clean ep tube. Use BCA protein concentration determination kit combined with microplate reader to determine the protein concentration, unify the protein concentration to 10μg / μl, add 5× protein loading buffer, mix well and boil at 100°C for 10 minutes, and then cool with ice water.

[0068] Western Blot experiment: The loading volume is 10μl, that is, the loading amount is 100μg, the electrophoresis conditions are 100V, 80 minutes, and the transfer conditions are 200mA, 90 minutes. After the transfer, the PVDF membrane is blocked with TBST containing 5% skim milk and shaken slowly at room temperature for 1h. After the blocking is completed, the PVDF membrane is transferred to the primary antibody and shaken slowly with the primary antibody at 4°C overnight. The next day, the PVDF membrane was washed 3 times with TBST, 10min each time. The PVDF membrane was then transferred to the HRP-conjugated secondary antibody and shaken at room temperature for 1h. After the secondary antibody was shaken at room temperature, the PVDF membrane was washed three times with TBST, 10min each time. Prepare the ECL luminescent solution in a 1:1 ratio, evenly add it to the PVDF membrane, and image it on the AMI680 system, with ACTB as the internal reference protein.

[0069] 3. Experimental results

[0070] Figure 4 A is a schematic diagram showing the effect of drugs on the expression of Bmal1, Per2, P-Bmal1, P-Per2 and P-CaMKII proteins after intraperitoneal injection of 5 mg / kg of drugs (control solvent, melatonin, compound of formula I) at circadian time ZT12 and extraction of proteins from the hypothalamus 12 hours later to verify the effect of drugs on the expression of Bmal1, Per2, P-Bmal1, P-Per2 and P-CaMKII proteins. Figure 4 BF shows the changes in the expression of Per2, P-Per2, Bmal1, P-Bmal1 and P-CaMKII proteins in the 5 mg / kg melatonin group and the 5 mg / kg compound of formula I group 12 h after administration, compared with the solvent control group. Figure 4 As shown in BF, compared with the solvent control group, the compound of formula (I) significantly reduced the expression of Bmal1, Per2, P-Per2, and P-CaMKII proteins in the hypothalamus 12 hours after administration.

[0071] 4. Conclusion

[0072] Twelve hours after administration at circadian time ZT12, the compound of formula (I) significantly reduced the expression of Bmal1, Per2, P-Per2, and P-CaMKII proteins in the hypothalamus at the protein level.

Claims

1. Use of a thioamide compound or a pharmaceutically acceptable salt thereof in the preparation of a circadian rhythm regulating drug, wherein the thioamide compound is a nitrogen-[2-(4-chlorophenyl)ethyl]-7,8-dihydroxy-1,3,4,5-tetrahydro-2-hydro-2-benzazepine-2-thioamide compound as shown in the following formula (I): Regulating circadian rhythm includes regulating circadian rhythm disorders or circadian rhythm-related sleep disorders caused by jet lag, shift work, and continuous multi-day and night work.

2. The use according to claim 1, wherein the pharmaceutically acceptable salt of the compound of formula (I) is a pharmaceutically acceptable inorganic acid salt or organic acid salt.

3. The use according to claim 1 or 2, wherein the pharmaceutically acceptable salt of the compound of formula (I) is hydrochloride, phosphate, sulfate, acetate, maleate, methanesulfonate, fumarate, citrate, benzenesulfonate, toluenesulfonate or tartrate.

4. The use according to claim 1 or 2, wherein the effective dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 0.1 mg / kg-1000 mg / kg body weight per dose, administered once or several times a day.

5. The use according to claim 4, wherein the effective dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 0.1 mg / kg-800 mg / kg body weight per dose, administered once or several times a day.

6. The use according to claim 5, wherein the effective dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is 0.5 mg / kg-500 mg / kg body weight per dose, administered once or several times a day.

7. Use of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for regulating circadian rhythm, wherein regulating circadian rhythm includes regulating circadian rhythm disorders caused by jet lag, shift work, continuous multi-day and night work, or circadian rhythm-related sleep disorders.

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