Transmucosal intake agent containing compound with phenylpyrazole skeleton that acts on circadian clock protein cry

Abstract

Provided is a compound that exhibits a circadian rhythm regulating action by transmucosal intake, particularly oral intake. Also provided is a transmucosal intake agent comprising at least one selected from the group consisting of a compound represented by general formula (1), a salt thereof, and solvates of the same.

Description

Transmucosal administration agent containing a compound having a phenylpyrazole skeleton that acts on the circadian clock protein CRY

[0001] The present invention relates to a transmucosally ingestible agent and the like.

[0002] The clock protein CRY plays a central role in the oscillation of the circadian clock and is a causative gene for human sleep rhythm disorders. Furthermore, Cry gene knockout mice show abnormalities in glucose metabolism. Therefore, CRY is expected to be a drug discovery target for sleep rhythm disorders and glucose metabolism disorders.

[0003] The carbazole derivative KL001 was reported as the world's first synthetic compound acting on CRY. KL001 interacts with both CRY1 and CRY2, which are closely related isoforms, and inhibits CRY degradation via the ubiquitin ligase FBXL3, thereby activating CRY function and extending the circadian rhythm at the cellular and tissue levels. Furthermore, KL001 inhibits glucagon-stimulated gluconeogenesis in primary cultured mouse hepatocytes. Furthermore, an orally available KL001 derivative has been developed and has been reported to improve glucose intolerance in diet-induced and genetically obese mouse models (Non-Patent Document 1, Patent Document 1, Patent Document 2). This derivative (SHP656) exhibits selectivity for CRY2.

[0004] Furthermore, phenylpyrazole derivatives have been reported as compounds that selectively activate CRY1 and CRY2 (Non-Patent Document 2).

[0005] International Publication No. 2013 / 170186 International Publication No. 2015 / 157182

[0006] Humphries, PS et al. Carbazole-containing sulfonamides and sulfamides: Discovery of cryptochrome modulators as antidiabetic agents. Bioorg Med Chem Lett 26, 757-760 (2016). Miller, S. et al. Isoform-selective regulation of mammalian cryptochromes. Nat Chem Biol 16, 676-685 (2020).

[0007] The present inventors have taken into consideration the ease of ingestion and have set an object to provide a compound that can exert a circadian rhythm regulating effect by transmucosal ingestion, particularly oral ingestion.

[0008] In view of the above problems, the present inventors have conducted extensive research and found that phenylpyrazole derivatives with specific structures can exert a circadian rhythm regulating effect through transmucosal administration, particularly oral administration. They have also found that they can exert CRY1 selectivity or CRY2 selectivity. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0009] Item 1. General formula (1):

[0010] [Wherein: X represents -S(=O)2- or -S(=O)-. R 1 are the same or different and represent an alkoxy group, an alkyl group, or a halogen atom. p represents an integer of 1 to 5. R 2 is represented by the general formula (2):

[0011] (wherein n represents 0 or 1. R 3 and R 4 are each an alkyl group and a hydrogen atom, or are each linked to form -(CH2) m - (m represents 2 to 6). R 5 represents a hydrogen atom or an alkyl group. 6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom.7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom, provided that n is 0 and R 7 is a fluorine atom, and R 6 and R 7 The case where both R and R are alkoxy groups is excluded. 8 represents a hydrogen atom or an alkyl group.) represents a group represented by the following formula: ], a salt thereof, and a solvate thereof.

[0012] Item 2. R 1 Item 3. The transmucosally ingestible agent according to Item 1, wherein are the same or different and are an alkoxy group having 1 to 4 carbon atoms or an alkyl group having 1 to 4 carbon atoms.

[0013] Item 3. p is 1 to 3 and one R 1 Item 2. The transmucosally administrable agent according to Item 1, wherein is at the para position.

[0014] Item 4. n is 0 and R 6 and R 7 Item 2. The transmucosally administrable agent according to Item 1, wherein are the same or different and are alkyl groups.

[0015] Item 5. n is 1 and R 3 and R 4 are linked together to form -(CH2) m - (m is 2 to 6), and R 7 Item 2. The transmucosally administrable agent according to Item 1, wherein is a halogen atom.

[0016] Item 6. n is 0 and R 6 and R 7 are the same or different and are alkyl groups, or n is 1 and R 3 and R 4 are linked together to form -(CH2) m - (m is 2 to 6), and R 7 Item 2. The transmucosally ingestible agent according to Item 1, wherein is a halogen atom.

[0017] Item 7. The transmucosally administrable agent according to Item 1, wherein X is —S(═O)2—.

[0018] Item 8. The transmucosally ingested agent according to any one of Items 1 to 7, which is an orally ingested agent.

[0019] Item 9. The transmucosally ingestible agent according to any one of Items 1 to 7, which is a pharmaceutical composition or a food composition.

[0020] Item 10. The transmucosally ingested agent according to any one of Items 1 to 7, which is used for regulating circadian rhythm.

[0021] Item 11. The transmucosally ingested preparation according to any one of Items 1 to 7, which is used for regulating sleep-wake rhythm or controlling glucose metabolism.

[0022] Item 12. General formula (1AA):

[0023] [Wherein: X represents -S(=O)2- or -S(=O)-. R 1 R may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom. 5 represents a hydrogen atom or an alkyl group. 6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 7 is a fluorine atom, and R 6 and R 7 The case where both R and R are alkoxy groups is excluded. 8 represents a hydrogen atom or an alkyl group.] (provided that the following compounds:

[0024] ), its salts, and solvates thereof.

[0025] According to the present invention, it is possible to provide a compound that can exert a circadian rhythm regulating effect by transmucosal administration, particularly oral administration.

[0026] Pharmacokinetics of KL101 and TH301 in mice (Test Example 1). The vertical axis indicates blood concentration, and the horizontal axis indicates time elapsed after administration. In the legend, "iv" indicates intravenous injection, "po" indicates oral administration, and the numbers indicate the dose. The structures of KL101, TH301, and TH139 are shown (Test Example 1). The effect of compounds on the circadian rhythm period of the Bmal1-dLuc reporter in human U2OS cells is shown (Test Example 1). The vertical axis indicates the time over which the circadian rhythm period is extended, and the horizontal axis indicates compound concentration. The compounds are shown in the legend. The effect of compounds on the half-life of the CRY1-LUC or CRY2-LUC reporter in human U2OS cells is shown (Test Example 1). The vertical axis indicates the relative half-life value, and the horizontal axis indicates compound concentration. The compounds are shown in the legend. The effect of compounds on the half-life of the CRY1-LUC or CRY2-LUC reporter in human HEK293 cells is shown (Test Example 1). The vertical axis indicates the relative half-life, and the horizontal axis indicates the compound concentration. The compound is indicated in the legend. The effect of compounds on Per2::Luc reporter intensity in wild-type or Cry1 and / or Cry2 knockout mouse fibroblasts is shown (Test Example 1). The vertical axis indicates the relative reporter intensity, and the horizontal axis indicates the compound concentration. The compound is indicated in the legend. The pharmacokinetics of TH139 and TH301 in mice is shown (Test Example 1). The vertical axis indicates blood concentration, and the horizontal axis indicates the time elapsed after administration. In the legend, "iv" indicates intravenous injection, "po" indicates oral administration, and the numbers indicate the dose. The concentrations in mouse tissues of TH139 and TH301 are shown (Test Example 1). The vertical axis indicates the concentration in the tissue indicated in the legend, and the horizontal axis indicates the time elapsed after administration and the dose. The effect of TH301 on the behavioral rhythm of wild-type mice is shown (Test Example 1). The vertical axis of the graph on the right indicates the period of the behavioral rhythm, and the horizontal axis indicates whether or not the compound was administered. On the right side of the graph, ZT7 / ZT19 indicates administration at the corresponding time, and "Free run" indicates free continuation after the end of compound administration. This shows the effect of TH139 on the behavioral rhythm of wild-type mice (Test Example 1). The vertical axis of the graph on the right shows the period of the behavioral rhythm, and the horizontal axis shows whether or not the compound was administered. On the right side of the graph, ZT7 / ZT19 indicates administration at the corresponding time, and "Free run" indicates free continuation after the end of compound administration.Figure 1 shows the effects of TH139 and TH301 on the behavioral rhythms of Cry1 KO mice and Cry2 KO mice (Test Example 1). The vertical axis indicates the period of the behavioral rhythm, and the horizontal axis indicates whether or not a compound was administered and the type of compound. Figure 2 shows the effects of four weeks of oral administration of TH139 and TH301 on glucose tolerance in obese model mice (Test Example 1). The vertical axis indicates blood glucose levels, and the horizontal axis indicates the time elapsed after glucose administration. In the legend, "Pre" indicates before compound administration, and "Post" indicates four weeks after compound administration. Figure 3 shows the effects of one week of oral administration of TH139 on glucose tolerance in obese model mice (Test Example 1). The vertical axis indicates blood glucose levels, and the horizontal axis indicates the time elapsed after glucose administration. In the legend, "Pre" indicates before compound administration, and "Post" indicates one week after compound administration. The structures of KL101, TH321, and TH320 are shown (Test Example 2). Figure 1 shows the effect of compounds on the circadian rhythm period of the Bmal1-dLuc reporter in human U2OS cells (Test Example 2). The vertical axis indicates the time the circadian rhythm period was extended, and the horizontal axis indicates compound concentration. Compounds are indicated in the legend. Figure 1 shows the effect of compounds on the half-life of the CRY1-LUC or CRY2-LUC reporter in human HEK293 cells (Test Example 2). The vertical axis indicates the relative half-life, and the horizontal axis indicates compound concentration. Compounds are indicated in the legend. Figure 1 shows the effect of compounds on the circadian rhythm period in Cry1 / Cry2 knockout mouse fibroblasts in which CRY1 or CRY2 was rescued (Test Example 2). The vertical axis indicates the time the circadian rhythm period was extended, and the horizontal axis indicates compound concentration. Compounds are indicated in the legend. Figure 1 shows the results of X-ray crystal structure analysis of CRY1 bound to TH320. Figure 1 shows the results of X-ray crystal structure analysis of CRY1 bound to TH321. Figure 1 shows the results of X-ray crystal structure analysis of CRY1 bound to KL101.

[0027] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0028] 1. Active ingredient In one aspect, the present invention provides a compound represented by the general formula (1):

[0029] The present invention relates to a transmucosally ingestible agent (sometimes referred to herein as "the agent of the present invention") containing at least one compound selected from the group consisting of a compound represented by the formula (sometimes referred to herein as "the compound of the present invention"), a salt thereof, and a solvate thereof (sometimes referred to herein as "the active ingredient of the present invention"). This will be explained below.

[0030] In general formula (1), X represents -S(=O)2- or -S(=O)-. From the viewpoint of circadian rhythm regulating activity, X is preferably -S(=O)2-. Therefore, the compound of the present invention is preferably a compound represented by general formula (1C):

[0031] It is expressed as:

[0032] In general formula (1), R 1 are the same or different and represent an alkoxy group, an alkyl group, or a halogen atom.

[0033] R 1 The alkoxy group represented by the formula (I) includes both straight-chain and branched-chain alkoxy groups. The number of carbon atoms in the alkoxy group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 2, and still more preferably 1. Specific examples of the alkoxy group include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, and a t-butoxy group.

[0034] R 1 The alkyl group represented by the formula (I) includes both straight-chain and branched-chain alkyl groups. The number of carbon atoms in the alkyl group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 2, and still more preferably 1. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0035] R 1Examples of the halogen atom represented by the formula (I) include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc. Among these, a fluorine atom and a chlorine atom are preferred.

[0036] R 1 is preferably an alkoxy group or an alkyl group. 1 is an alkoxy group.

[0037] In general formula (1), p is an integer of 1 to 5. p is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 or 2. In one embodiment, p is 1.

[0038] R 1 The position of is not particularly limited, but preferably one R 1 In this case, when p is 2 or more, preferably one R 1 is in para position, and another R 1 is in the ortho position. The compound of the present invention preferably has the general formula (1D):

[0039] It is expressed as:

[0040] In general formula (1D), R 11 The definition of R 1 is the same as

[0041] R 12 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. The definitions of an alkyl group, an alkoxy group, and a halogen atom are given by R 1 These definitions are the same as for R 12 is preferably a hydrogen atom or an alkyl group. 12 In one embodiment, is a hydrogen atom.

[0042] In one embodiment of the present invention, from the viewpoint of circadian rhythm regulating activity, X is -S(=O)2- in general formula (1D), and R 11 and R 12 is preferably an alkyl group.

[0043] In general formula (1), R 2 is represented by the general formula (2):

[0044] represents a group represented by the formula:

[0045] In general formula (2), n represents 0 or 1. From the viewpoint of CRY1 selectivity, n is particularly preferably 0. From the viewpoint of CRY2 selectivity, n is particularly preferably 1.

[0046] In general formula (2), R 3 and R 4 are each an alkyl group and a hydrogen atom, or are each linked to form -(CH2) m - (m represents 2 to 6). R 3 and R 4 From the viewpoint of CRY2 selectivity, they are linked together to form -(CH2) m Preferably, m is 2 to 6.

[0047] R 3 or R 4 The alkyl group represented by the formula (I) includes both straight-chain and branched-chain alkyl groups. The number of carbon atoms in the alkyl group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, and more preferably 1 to 4. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0048] R 3 and R 4 are linked together to form -(CH2) m - (m represents 2 to 6) means a compound represented by the general formula (2a):

[0049] The partial structure represented by is one of the following partial structures:

[0050] This indicates that

[0051] From the viewpoint of CRY2 selectivity, m is preferably 3 to 4, and particularly preferably 4. From the viewpoint of circadian rhythm regulating activity, m is preferably 2 to 5, and particularly preferably 4 to 5.

[0052] In general formula (2), R 5represents a hydrogen atom or an alkyl group. 5 is preferably a hydrogen atom from the viewpoint of circadian rhythm regulating activity.

[0053] R 5 The alkyl group represented by the formula (I) includes both straight-chain and branched-chain alkyl groups. The number of carbon atoms in the alkyl group is not particularly limited, but is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 2, and still more preferably 1. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0054] In general formula (2), R 6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 6 From the viewpoint of the circadian rhythm regulating effect, R is preferably a hydrogen atom or an alkyl group. 6 From the viewpoint of CRY1 selectivity, R is preferably an alkyl group. 6 is preferably a hydrogen atom from the viewpoint of CRY2 selectivity.

[0055] R 6 For alkyl groups represented by R 5 The same definition applies to the alkyl group represented by the formula:

[0056] R 6 For the alkoxy group represented by R 1 The same definition applies to the alkoxy group represented by the formula:

[0057] R 6 From the viewpoint of circadian rhythm regulating activity, the halogen atom represented by the formula (I) is preferably a chlorine atom or a bromine atom.

[0058] In general formula (2), R 7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 7 From the viewpoint of CRY1 selectivity, R is preferably an alkyl group. 7 is preferably a halogen atom from the viewpoint of CRY2 selectivity.

[0059] R 7 For alkyl groups represented by R 5 The same definition applies to the alkyl group represented by the formula:

[0060] R 7 For the alkoxy group represented by R 1 The same definition applies to the alkoxy group represented by the formula:

[0061] R 7 From the viewpoint of circadian rhythm regulating activity, the halogen atom represented by the formula (I) is preferably a halogen atom other than a fluorine atom, such as a chlorine atom or a bromine atom. From the viewpoint of CRY2 selectivity, a chlorine atom is particularly preferred.

[0062] In the general formula (2), n is 0 and R 7 is a fluorine atom, and R 6 and R 7 The case where both are alkoxy groups is excluded.

[0063] In general formula (2), R 8 represents a hydrogen atom or an alkyl group. 8 is preferably a hydrogen atom from the viewpoint of circadian rhythm regulating activity.

[0064] R 8 For alkyl groups represented by R 5 The same definition applies to the alkyl group represented by the formula:

[0065] The compound of the present invention has the following circadian rhythm regulating effect when taken transmucosally, particularly orally: 1 (particularly, in the case of general formula (1D), R 11 is an alkoxy group), the number of carbon atoms in the alkoxy group is, for example, 1 to 8, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 or 2, and still more preferably 1 (Aspect 1).

[0066] In the case of embodiment 1 or other cases, the compound of the present invention is preferably such that n is 0 and R 6 and R 7are the same or different and are alkyl groups (having, for example, 1 to 8 carbon atoms, preferably 1 to 6, more preferably 1 to 4, even more preferably 1 to 2, and still more preferably 1 carbon atom). In this case, R 5 and R 8 is a hydrogen atom. In this case, the compound of the present invention is, in one embodiment, a compound represented by the general formula (1A):

[0067] It is expressed as:

[0068] In the case of embodiment 1 or otherwise, the compound of the present invention is preferably such that n is 1 and R 3 and R 4 are linked together to form -(CH2) m - (m is 2 to 6 (preferably 3 to 4, more preferably 4)), and R 7 is a halogen atom (preferably a chlorine atom or a bromine atom, more preferably a chlorine atom). In this case, particularly preferably R 5 , R 6 , and R 8 is a hydrogen atom. In this case, the compound of the present invention is, in one embodiment, a compound represented by the general formula (1B):

[0069] It is expressed as:

[0070] In one aspect, the present invention provides a compound represented by the general formula (1AA):

[0071] (In the formula, R 1 , R 2 , R 5 , R 6 , R 7 , and R 8 is the same as defined in general formula (1), a salt thereof, and a solvate thereof (provided that the following compounds:

[0072] The general formula (1AA) is preferably a preferred embodiment of the general formula (1) in terms of CRY1 selectivity.

[0073] The salt of the compound of the present invention is not particularly limited as long as it is a pharmaceutically acceptable salt.The salt can be either an acidic salt or a basic salt.Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, paratoluenesulfonate, etc., and examples of basic salts include alkali metal salts such as sodium salt and potassium salt, and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, tri(hydroxyalkyl)amine, etc.

[0074] The compound of the present invention and its salts can be in the form of a solvate. Examples of the solvent include water and pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0075] The compound of the present invention and the active ingredient of the present invention can be produced according to or in accordance with a known method (for example, the method described in Non-Patent Document 2). In addition, the compound of the present invention and the active ingredient of the present invention can also be obtained as a commercially available product and used.

[0076] 2. Uses The active ingredient of the present invention can exert a circadian rhythm regulating effect (particularly, a period-lengthening effect) through transmucosal ingestion, particularly oral ingestion, and therefore can be used as an active ingredient in transmucosal ingestible agents.

[0077] The transmucosal intake is not particularly limited as long as it is an intake (e.g., administration) mode in which the active ingredient of the present invention is taken into the body via a mucous membrane. Specific examples of transmucosal intake agents include oral intake agents, nasal administration agents, inhalants, suppositories, etc. Among these, oral intake agents (e.g., tablets, capsules, granules, powders, fine granules, syrups, enteric-coated agents, sustained-release capsules, chewable tablets, drops, pills, oral liquids, confectionery tablets, sustained-release agents, sustained-release granules, etc.) are particularly preferred from the standpoint of ease of intake and the sense of burden on the user.

[0078] The agent of the present invention is not particularly limited as long as it contains the compound of the present invention, and may further contain other components as necessary. The other components are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0079] The agent of the present invention can be, for example, a pharmaceutical composition or a food composition.

[0080] The subject to which the agent of the present invention is applied is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

[0081] The content of the active ingredient of the present invention in the agent of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.

[0082] When the agent of the present invention is administered to an animal, the dosage is not particularly limited as long as it is an effective amount that produces the desired effect. Generally, when the agent is orally administered, the weight of the active ingredient of the present invention is 0.1 to 1000 mg / kg body weight per day, preferably 0.5 to 500 mg / kg body weight per day. The above-mentioned dosage can be administered once a day or in two or three divided doses, and can be increased or decreased as appropriate depending on the age, pathological condition, and symptoms.

[0083] The agent of the present invention can be used to regulate circadian rhythms. More specifically, the agent of the present invention can regulate circadian rhythms (expression cycles of clock genes (e.g., Bmal1, Clock, Per, and Cry)) controlled by CRY1 and / or CRY2. CRY1 (cryptochrome 1) and CRY2 (cryptochrome 2) are known in various biological species, with human CRY1 being the expression product of the gene identified by NCBI Gene ID: 1407 and human CRY2 being the expression product of the gene identified by NCBI Gene ID: 1408.

[0084] Based on the circadian rhythm regulating activity, the agent of the present invention can also be used for regulating sleep-wake rhythm or for regulating glucose metabolism. Sleep-wake rhythm regulation refers to adjusting the timing of sleep and wakefulness. Specifically, the agent can be used, for example, to improve, treat, or prevent sleep rhythm disorders. Furthermore, glucose metabolism regulation refers to the control of in vivo glucose metabolism, which is reflected in blood glucose concentrations. Specifically, the agent can be used, for example, to improve, treat, or prevent diabetes, suppress blood glucose levels, improve glucose tolerance, etc.

[0085] Based on its circadian rhythm regulating effect, the agent of the present invention can also be used to improve, treat, and prevent various diseases other than those mentioned above, such as metabolic diseases, cancer, mental diseases, and cardiovascular diseases.

[0086] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0087] Test Example 1 (1-1) Materials and Methods Acquisition of compounds: KL101 was purchased from Life Chemicals (F0778-0202). TH301 was purchased from ChemDiv (K410-0714) and synthesized. TH139 was purchased from ChemDiv (K410-0679) and synthesized.

[0088] Pharmacokinetic analysis: KL101, TH301, or TH139 suspended in 1% Metolose was injected intravenously (1 mg / kg) or orally (10, 30, 50, or 100 mg / kg) into male C57BL / 6J or BALB / c mice, and blood samples were collected serially from the same mice. Additionally, brain and liver samples were collected 1 and 4 hours after oral administration. The concentrations of compounds in these samples were measured by LC-MS / MS.

[0089] Analysis of circadian rhythm in human U2OS cells: The method described in Non-Patent Document 2 was used.

[0090] Analysis of CRY degradation in human U2OS cells or human HEK293 cells: The method described in Non-Patent Document 2 was used.

[0091] Analysis of Per2::Luc reporter repression in mouse fibroblasts: The method described in Non-Patent Document 2 was used.

[0092] Analysis of mouse behavioral rhythms: Male C57BL / 6J mice were individually housed in a compartment and monitored using an infrared sensor. After entraining to a 12-hour light / 12-hour dark cycle, they were transferred to constant darkness and orally administered TH139, TH301, or control DMSO at 50 mg / kg once daily for 2 weeks at ZT7 (7 hours after the onset of the light phase) in 1% Metolose. After entraining to a light / dark cycle for 1 week, they were transferred to constant darkness and orally administered the same compounds at 50 mg / kg once daily at ZT19 (7 hours after the onset of the dark phase) for 2 weeks. After treatment, measurements were continued for another 2 weeks in constant darkness. Experiments using Cry1 KO and Cry2 KO mice were performed similarly, with treatment at ZT19 and subsequent measurements after treatment. Since the Cry1 KO mouse has a cycle approximately 70 minutes shorter than the wild-type mouse, the administration time was adjusted to be 70 minutes earlier each day. On the other hand, since the Cry2 KO mouse has a cycle approximately 50 minutes longer than the wild-type mouse, the administration time was adjusted to be 50 minutes later each day. The behavioral rhythm period was calculated using the analysis software ActogramJ.

[0093] Analysis of glucose tolerance in obese model mice: Male C57BL / 6J mice were fed a high-fat diet to create obese model mice. TH139, TH301, or control DMSO suspended in 1% Metolose was orally administered at 100 mg / kg once daily on ZT1 (1 hour after the onset of the light phase). Before and after the 1-week or 4-week treatment period, the mice were fasted for 16 hours and then subjected to glucose tolerance tests by intraperitoneal administration of a glucose solution.

[0094] (1-2) Test results We analyzed the pharmacokinetics of CRY1-selective KL101 and CRY2-selective TH301 in individual mice (Figure 1). TH301 maintained a blood concentration of 1 μM or higher for more than 4 hours after oral administration, suggesting that it could be used directly for in vivo experiments. On the other hand, KL101 exhibited very low blood concentrations, indicating that further modification was necessary for in vivo experiments.

[0095] KL101 and TH301 are both phenylpyrazole derivatives with similar structures (Figure 2). Because the upper portion of these molecules plays an important role in isoform selectivity (Miller, S. et al. Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation. Proc Natl Acad Sci USA 118, e2026191118 (2021)). Therefore, we obtained TH139 (Figure 2), a molecule in which the lower portion of KL101 was replaced with TH301, and analyzed its effects. In human U2OS cells, TH139 extended the circadian rhythm period of the Bmal1-dLuc reporter, similar to KL101 (Figure 3). Furthermore, TH139 inhibited the degradation of the CRY1-LUC reporter, increasing its half-life, while having little effect on the half-life of the CRY2-LUC reporter (U2OS cells: Figure 4; HEK293 cells: Figure 5). TH139 also exhibited a similar effect to KL101 in mouse fibroblasts (Figure 6). Because CRY is a transcriptional repressor of the Per2 gene, activation of CRY reduces the Per2::Luc reporter. While TH139 repressed the Per2::Luc reporter in wild-type cells containing both CRY1 and CRY2 (Figure 6, top left), the repression effect was attenuated in Cry1 KO cells lacking CRY1 (Figure 6, bottom left). On the other hand, the repression effect was maintained in Cry2 KO cells lacking CRY2 (Figure 6, bottom right), and was abolished in Cry1 / Cry2 KO cells lacking both CRY1 and CRY2 (Figure 6, top right). These results demonstrate that TH139 selectively activates CRY1.

[0096] Next, we analyzed the pharmacokinetics of TH139 in mice (Figure 7). After oral administration of 50 mg / kg, TH139 maintained a blood concentration of 1 μM or higher for more than 4 hours, suggesting its suitability for in vivo experiments. Following oral administration of 100 mg / kg, both TH139 and TH301 maintained blood concentrations of 10 μM or higher for more than 4 hours. Because the brain is protected by the blood-brain barrier, most compounds in the blood cannot reach the brain. The suprachiasmatic nucleus controls the behavioral rhythm of sleep and wakefulness, and compounds must reach the brain to control behavioral rhythms. Meanwhile, gluconeogenesis is primarily carried out in the liver. After oral administration, we measured the concentrations of TH139 and TH301 in the brain and liver and found that they were present in both tissues at concentrations equal to or higher than those in the blood (Figure 8). Thus, oral administration of TH139 and TH301 is expected to selectively activate CRY1 and CRY2 functions in various tissues, including the brain and liver.

[0097] To treat sleep rhythm disorders, we attempted to control behavioral rhythms in mice. CRY protein activity exhibits a circadian rhythm, increasing in daytime activity in the suprachiasmatic nucleus (SNU) beginning at ZT7 (7 hours after the onset of the light period) and decreasing in the middle of the night beginning at ZT19 (7 hours after the onset of the dark period). Pharmacokinetic analysis indicated that the plasma concentrations of both TH139 and TH301 significantly decreased approximately 8 hours after oral administration, suggesting that their effects could not be maintained for 24 hours. Therefore, we compared the effects of TH139 and TH301 at ZT7 and ZT19. Wild-type mice were orally administered 50 mg / kg of TH301 once daily under constant darkness for 2 weeks, and behavioral rhythms were measured (Figure 9). Administration at ZT7 did not affect the circadian period, but administration at ZT19 prolonged the period, and the period-prolonging effect disappeared upon discontinuation of administration. Thus, activation of CRY2 by TH301 successfully controlled behavioral rhythms in a time-of-day and reversible manner. The period-lengthening effect observed at ZT19, when CRY activity begins to decline, is consistent with the mechanism of action of TH301 activating CRY2. Similarly, CRY1 activation by oral administration of 50 mg / kg TH139 also altered the period of behavioral rhythms in a time-of-day-dependent and reversible manner (Fig. 10). Only TH301 significantly lengthened the period in Cry1 KO mice, and only TH139 significantly lengthened the period in Cry2 KO mice (Fig. 11), demonstrating that these compounds exhibit isoform selectivity even at the individual level.

[0098] Furthermore, we attempted to control blood glucose levels in mice with the aim of applying this to diabetes treatment. Mice fed a high-fat diet became obese and showed impaired glucose tolerance. 100 mg / kg of TH139 or TH301 was orally administered once daily for four weeks to these obese model mice, and the effects on glucose tolerance were analyzed (Figure 12). The administration time was ZT1, at which CRY activity in the liver begins to decrease. While administration of the control DMSO worsened glucose intolerance within four weeks, administration of TH139 and TH301 prevented this deterioration. Furthermore, oral administration of 100 mg / kg of TH139 for one week after glucose intolerance had progressed was effective in improving glucose intolerance (Figure 13).

[0099] Previous studies have suggested that compounds selective for CRY2 are effective in treating impaired glucose tolerance. The results above indicate that not only CRY2-selective activation but also CRY1-selective activation is effective in treating impaired glucose tolerance.

[0100] Test Example 2 (2-1) Materials and Methods Acquisition of Compounds: TH320 was purchased from ChemDiv (K410-0861). TH321 was purchased from Life Chemicals (F0561-0620). Analysis of Circadian Rhythms in Human U2OS Cells: The method described in Non-Patent Document 2 was used. Analysis of CRY Degradation in Human HEK293 Cells: The method described in Non-Patent Document 2 was used. Analysis of Circadian Rhythms in Cry1 / Cry2 Knockout Mouse Fibroblasts Rescued from Cry: The method described in Non-Patent Document 2 was used. Crystal Structure Analysis of CRY1: The method described in Non-Patent Document 2 was used. (2-2) Test Results TH301 has two oxygen atoms bound to the sulfur atom, whereas KL101 has no oxygen atom bound to the sulfur atom (Figure 2). To clarify the effect of these oxygen atoms on the activity of the compounds, TH320 with two oxygen atoms bound and TH321 with one oxygen atom bound (Figure 14) were obtained and analyzed. In human U2OS cells, TH321 extended the circadian rhythm of the Bmal1-dLuc reporter slightly more strongly than KL101, whereas TH320 extended it approximately 10-fold more strongly (Fig. 15). Furthermore, TH320 also enhanced the inhibition of CRY1-LUC reporter degradation and increased its half-life (Fig. 16). In Cry1 / Cry2 knockout mouse fibroblasts in which CRY1 or CRY2 was rescued, TH320 exhibited a more potent and selective effect on CRY1 than KL101 (Fig. 17). These results suggest that the activity of KL101 increases depending on the number of oxygen atoms bound to the sulfur atom. To clarify the molecular mechanism, we performed X-ray crystallography of CRY1 bound to TH320 and TH321. As a result, three water molecules (W1, W2, and W3) were found near S in the compounds, and in TH320, two O atoms bound to S interacted with W1 and W3, respectively (Fig. 18), while in TH321, one O atom bound to S interacted with W3 (Fig. 19). On the other hand, although W1-W3 were present in the CRY1-KL101 complex described in Non-Patent Document 2, no interaction with KL101 was observed (Fig. 20).W1, W2, and W3 form a strong hydrogen-bond network with serine 252 (S252), arginine 293 (R293), aspartic acid 387 (D387), and aspartic acid 389 (D389) of CRY1, and it is thought that TH320 and TH321 increase the activity by interacting with this network via O. These results suggest an effective means to increase the activity of KL101, a compound selective for CRY1.

Claims

1. General formula (1): [Wherein: X represents -S(=O)2- or -S(=O)-. R 1 are the same or different and represent an alkoxy group, an alkyl group, or a halogen atom. p represents an integer of 1 to 5. R 2 is represented by the general formula (2): (wherein n represents 0 or 1. R 3 and R 4 are each an alkyl group and a hydrogen atom, or are each linked to form -(CH2) m - (m represents 2 to 6). R 5 represents a hydrogen atom or an alkyl group. 6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom, provided that n is 0 and R 7 is a fluorine atom, and R 6 and R 7 The case where both R and R are alkoxy groups is excluded. 8 represents a hydrogen atom or an alkyl group.) represents a group represented by the following formula: , a salt thereof, and a solvate thereof.

2. R 1 The transmucosally ingestible agent according to claim 1, wherein are the same or different and are an alkoxy group having 1 to 4 carbon atoms or an alkyl group having 1 to 4 carbon atoms.

3. p is 1 to 3 and one R 1 The transmucosally ingestible agent according to claim 1, wherein is at the para position.

4. n is 0 and R 6 and R 7 The transmucosally ingestible agent according to claim 1, wherein are the same or different and are alkyl groups.

5. n is 1 and R 3 and R 4 are linked together to form -(CH2) m - (m is 2 to 6), and R 7 The transmucosally ingestible agent according to claim 1, wherein is a halogen atom.

6. n is 0 and R 6 and R 7 are the same or different and are alkyl groups, or n is 1 and R 3 and R 4 are linked together to form -(CH2) m - (m is 2 to 6), and R 7 The transmucosally ingestible agent according to claim 1, wherein is a halogen atom.

7. The transmucosally ingestible agent according to claim 1, wherein X is -S(=O)2-.

8. The transmucosally ingested agent according to any one of claims 1 to 7, which is an orally ingested agent.

9. The transmucosally ingestible agent according to any one of claims 1 to 7, which is a pharmaceutical composition or a food composition.

10. The transmucosally ingested agent according to any one of claims 1 to 7, which is used for regulating circadian rhythm.

11. The transmucosally ingested preparation according to any one of claims 1 to 7, which is used for regulating sleep-wake rhythm or for controlling glucose metabolism.

12. General formula (1AA): [Wherein: X represents -S(=O)2- or -S(=O)-. R 1 R may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom. 5 represents a hydrogen atom or an alkyl group. 6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. 7 is a fluorine atom, and R 6 and R 7 The case where both R and R are alkoxy groups is excluded. 8 represents a hydrogen atom or an alkyl group.] (provided that the following compounds: ), its salts, and solvates thereof.

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