WO2025239319A1 - Transmucosal intake agent containing compound with phenylpyrazole skeleton that acts on circadian clock protein cry - Google Patents

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

The present invention relates to a transmucosal ingestible agent, etc.

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

KL001, a carbazole derivative, has been reported as the world's first synthetic compound acting on CRY. KL001 interacts with both CRY1 and CRY2, which are closely related isoforms, and activates CRY function by inhibiting CRY degradation via the ubiquitin ligase FBXL3, thereby extending the circadian rhythm at the cellular and tissue levels. Furthermore, it inhibits glucagon-stimulated activation of gluconeogenesis in primary cultured mouse hepatocytes. Furthermore, an orally administrable 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.

In addition, phenylpyrazole derivatives have been reported as compounds that selectively activate both CRY1 and CRY2 (Non-Patent Document 2).

International Publication No. 2013/170186 International Publication No. 2015/157182

Humphries, P. S. 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).

The inventors' objective is to provide a compound that can exert a circadian rhythm regulating effect through transmucosal administration, particularly oral administration, taking into consideration the ease of administration.

In light of the above-mentioned problems, the inventors have conducted extensive research and have found that phenylpyrazole derivatives with a specific structure can exert a circadian rhythm regulating effect when taken transmucosally, particularly orally. They have also found that they can exert CRY1 or CRY2 selectivity. Based on this finding, the inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

Item 1. General formula (1):

[wherein X represents -S(=O) _2- or -S(=O)-; ^R1 's may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom; p represents an integer of 1 to 5; ^{and R2} represents a group represented by the general formula (2):

(In the formula, n represents 0 or 1. One of ^R3 and ^R4 is an alkyl group and the other is a hydrogen atom, or they are linked together to form —(CH ₂ ) _m — (m represents 2 to 6). R ⁵ represents a hydrogen atom or an alkyl group. R ⁶ represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. R ⁷ represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom, excluding the cases where n is 0 and R ⁷ is a fluorine atom, and where R ⁶ and R ⁷ are both alkoxy groups. R ⁸ represents a hydrogen atom or an alkyl group.)
represents a group represented by the following formula:
A transmucosally ingestible agent comprising at least one compound selected from the group consisting of a compound represented by the formula (I):

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

Item 3. The transmucosally administrable agent according to Item 1, wherein p is 1 to 3 and one ^R1 is at the para position.

Item 4. The transmucosally administrable agent according to Item 1, wherein n is 0, and ^R6 and ^R7 are the same or different and are alkyl groups.

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

Item 6. n is 0, and R ⁶ and R ⁷ are the same or different and are alkyl groups, or n is 1, R ³ and R ⁴ are linked together to form —(CH ₂ ) _m — (m is 2 to 6), and R ⁷ is a halogen atom;
Item 1. The transmucosally administrable agent according to Item 1.

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

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

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

Item 10. The transmucosal administration agent according to any one of Items 1 to 7, which is used for circadian rhythm regulation.

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

Section 12. General formula (1AA):

[wherein X represents -S(=O) _2- or -S(=O)-; ^R1 's may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom; ^R5 represents a hydrogen atom or an alkyl group; ^R6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom; ^R7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom, except when R7 is a fluorine atom or when both ^{R6 and R7} are alkoxy groups; and ^R8 represents a hydrogen atom or an alkyl group.] (provided that the following compounds:

), its salts, and solvates thereof.

The present invention provides a compound that can exert a circadian rhythm regulating effect through transmucosal administration, particularly oral administration.

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

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

1. Active ingredient In one aspect, the present invention provides an active ingredient represented by the general formula (1):

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.

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):

It is expressed as:

In general formula (1), R ^1s may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom.

The alkoxy group represented by ^R1 includes both linear and branched ones. 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.

The alkyl group represented by ^R1 includes both linear and branched ones. 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.

Examples of the halogen atom represented by ^R1 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.

^R1 is preferably an alkoxy group or an alkyl group. In one embodiment, ^R1 is an alkoxy group.

In general formula (1), p is an integer from 1 to 5. p is preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2. In one embodiment, p is 1.

The position of ^R1 is not particularly limited, but preferably one ^R1 is at the para position. In this case, when p is 2 or more, preferably one ^R1 is at the para position and another ^R1 is at the ortho position. The compound of the present invention is preferably represented by the general formula (1D):

It is expressed as:

In general formula (1D), R ¹¹ has the same definition as R ¹ .

^R12 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. The definitions of the alkyl group, the alkoxy group, and the halogen atom are the same as those for ^R1 . ^R12 is preferably a hydrogen atom or an alkyl group. In one embodiment, ^R12 is a hydrogen atom.

In one embodiment of the present invention, from the viewpoint of circadian rhythm regulating activity, it is preferred that in general formula (1D), X is —S(═O) ₂ —, and R ¹¹ and R ¹² are alkyl groups.

In general formula (1), ^R2 represents general formula (2):

represents a group represented by the formula:

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.

In general formula (2), one of ^R3 and ^R4 is an alkyl group and the other is a hydrogen atom, or R3 and R4 are linked together to form -( _CH2 ) _m- (m is 2 to 6). From the viewpoint of CRY2 selectivity, it is preferable that ^R3 and ^R4 are linked together to form -( _CH2 ) _m- (m is 2 to 6).

The alkyl group represented by ^R3 or ^R4 includes both linear and branched ones. 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.

The expression "R ³ and R ⁴ are linked together to form -(CH ₂ ) _m - (m is 2 to 6)" means that the compound represented by the general formula (2a):

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

This indicates that

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.

In general formula (2), ^R5 represents a hydrogen atom or an ^alkyl group, and is preferably a hydrogen atom from the viewpoint of circadian rhythm regulating activity.

The alkyl group represented by ^R5 includes both linear and branched ones. 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.

In general formula (2), ^R6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. From the viewpoint of circadian rhythm regulating activity, ^R6 is preferably a hydrogen atom or an alkyl group. From the viewpoint of CRY1 selectivity, ^R6 is preferably an alkyl group. From the viewpoint of CRY2 selectivity, ^R6 is preferably a hydrogen atom.

The alkyl group represented by ^R6 has the same definition as that of the alkyl group represented by ^R5 .

The alkoxy group represented by ^R6 is defined in the same manner as the alkoxy group represented by ^R1 .

From the viewpoint of circadian rhythm regulating activity, the halogen atom represented by ^R6 is preferably a chlorine atom or a bromine atom.

In general formula (2), ^R7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. From the viewpoint of CRY1 selectivity, ^R7 is preferably an alkyl group. From the viewpoint of CRY2 selectivity, ^R7 is preferably a halogen atom.

The alkyl group represented by ^R7 has the same definition as the alkyl group represented by ^R5 .

The alkoxy group represented by ^R7 is defined in the same manner as the alkoxy group represented by ^R1 .

From the viewpoint of circadian rhythm regulating activity, the halogen atom represented by ^R7 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.

In general formula (2), however, the cases where n is 0 and R ⁷ is a fluorine atom and where R ⁶ and R ⁷ are both alkoxy groups are excluded.

In general formula (2), ^R8 represents a hydrogen atom or an ^alkyl group, and is preferably a hydrogen atom from the viewpoint of circadian rhythm regulating activity.

The alkyl group represented by ^R8 is defined in the same manner as the alkyl group represented by ^R5 .

In view of the circadian rhythm regulating effect upon transmucosal ingestion, particularly oral ingestion, of the compounds of the present invention, the alkoxy group represented by R ¹ (particularly when R ¹¹ in general formula (1D) is an alkoxy group) has, for example, 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, even more preferably 1 to 2 carbon atoms, and still more preferably 1 carbon atom (Aspect 1).

In the case of embodiment 1 or other cases, in terms of CRY1 selectivity, the compound of the present invention is preferably such that n is 0, and ^R6 and ^R7 are the same or different and are alkyl groups (having, for example, 1 to 8 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, even more preferably 1 to 2 carbon atoms, and even more preferably 1 carbon atom). In this case, ^R5 and ^R8 are particularly preferably hydrogen atoms. In this case, the compound of the present invention is, in one embodiment, a compound represented by the general formula (1A):

It is expressed as:

In the case of embodiment 1 or other cases, from the viewpoint of CRY2 selectivity, the compound of the present invention is preferably one in which n is 1, ^R3 and ^R4 are linked together to form -( _CH2 ) _m- (m is 2 to 6 (preferably 3 to 4, more preferably 4)), and ^R7 is a halogen atom (preferably a chlorine atom or bromine atom, more preferably a chlorine atom). In this case, it is particularly preferable that ^R5 , ^R6 , and ^R8 are hydrogen atoms. In this case, the compound of the present invention, in one embodiment, is represented by the general formula (1B):

It is expressed as:

In one aspect, the present invention relates to a compound of general formula (1AA):

(wherein R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ , and R ⁸ are the same as defined in general formula (1)), a salt thereof, and a solvate thereof (provided that the following compound:

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

Salts of the compounds of the present invention are not particularly limited, so long as they are pharmaceutically acceptable. Both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamines, dialkylamines, trialkylamines, mono(hydroxyalkyl)amines, di(hydroxyalkyl)amines, and tri(hydroxyalkyl)amines.

The compounds of the present invention and their salts may also be solvated. Examples of solvents include water and pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

The compounds and active ingredients of the present invention can be manufactured according to or in accordance with known methods (for example, the method described in Non-Patent Document 2). The compounds and active ingredients of the present invention can also be obtained and used as commercially available products.

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.

There are no particular limitations on transmucosal intake, so 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 the mucous membrane. Specific examples of transmucosal intake agents include oral intake agents, nasal administration agents, inhalants, and suppositories. Of 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.

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 ingredients as necessary. The other ingredients are not particularly limited as long as they are pharmaceutically acceptable, and examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

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

The agents of the present invention may be used in any mammalian animal, including, but not limited to, humans, monkeys, mice, rats, dogs, cats, and rabbits.

The content of the active ingredient of the present invention in the agent of the present invention depends on factors such as the mode of use, the subject to which it is applied, and the condition of the subject to which it is applied, and is not limited to this amount, but can be, for example, 0.0001 to 95% by weight, and preferably 0.001 to 50% by weight.

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. Typically, when ingested orally, the weight of the active ingredient of the present invention is generally 0.1 to 1000 mg/kg of body weight per day, preferably 0.5 to 500 mg/kg of 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 animal's age, condition, and symptoms.

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, 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.

The agent of the present invention can also be used to regulate sleep-wake rhythms or control glucose metabolism based on its circadian rhythm regulating effect. Sleep-wake rhythm regulation refers to adjusting the timing of sleep and wakefulness. Specifically, it can be used, for example, to improve, treat, and 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, it can be used, for example, to improve, treat, and prevent diabetes, suppress blood sugar levels, and improve glucose tolerance.

The agent of the present invention, based on its circadian rhythm regulating effect, can also be used to improve, treat, and prevent a variety of other diseases in addition to those mentioned above, such as metabolic diseases, cancer, mental disorders, and cardiovascular diseases.

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

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.

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.

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

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

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

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.

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.

(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. However, KL101 exhibited very low blood concentrations, indicating that further modification was necessary for in vivo experiments.

Both KL101 and TH301 are phenylpyrazole derivatives with similar structures (Figure 2). Because the upper part 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 U S A 118, e2026191118 (2021)), we obtained TH139 (Figure 2), a molecule in which the lower part 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 expressing both CRY1 and CRY2 (Figure 6, top left), the repressive effect was attenuated in Cry1 KO cells lacking CRY1 (Figure 6, bottom left). On the other hand, the repressive effect was retained 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.

Next, we analyzed the pharmacokinetics of TH139 in individual 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. After oral administration of 100 mg/kg, both TH139 and TH301 maintained blood concentrations of 10 μM or higher for more than 4 hours. The brain is protected by the blood-brain barrier, preventing many compounds in the blood from reaching the brain. The suprachiasmatic nucleus in the brain controls sleep-wake behavioral rhythms, and compounds must reach the brain to control behavioral rhythms. Meanwhile, gluconeogenesis is primarily carried out in the liver. Measurement of TH139 and TH301 concentrations in the brain and liver after oral administration revealed that they were present in both tissues at concentrations equal to or higher than those in the blood (Figure 8). In other words, oral administration of TH139 and TH301 is expected to selectively activate the functions of CRY1 and CRY2, respectively, in various tissues, including the brain and liver.

We attempted to control behavioral rhythms in mice with the aim of applying this method to the treatment of sleep rhythm disorders. CRY protein activity exhibits a circadian rhythm, with activity in the suprachiasmatic nucleus increasing from ZT7 (7 hours after the onset of the light period) during the day and decreasing from ZT19 (7 hours after the onset of the dark period) in the middle of the night. Pharmacokinetic analysis showed that the blood 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 their effects 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 when administration was discontinued. Thus, by activating CRY2 using TH301, we 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, activation of CRY1 by oral administration of 50 mg/kg TH139 also altered the period of behavioral rhythms in a time-dependent and reversible manner (Figure 10). Only TH301 significantly extended the period in Cry1 KO mice, and only TH139 significantly extended the period in Cry2 KO mice (Figure 11), demonstrating that these compounds exhibit isoform selectivity even at the individual level.

Furthermore, with a view to applying this to diabetes treatment, we attempted to control blood glucose levels in mice. Mice fed a high-fat diet become obese and show impaired glucose tolerance. These obese model mice were orally administered 100 mg/kg of TH139 or TH301 once daily for four weeks, and the effect on glucose tolerance was analyzed (Figure 12). The administration time used was ZT1, at which CRY activity in the liver begins to decrease. While administration of the control DMSO worsened glucose tolerance within four weeks, administration of TH139 and TH301 prevented this deterioration. Furthermore, when 100 mg/kg of TH139 was orally administered for one week after glucose tolerance had progressed, an improving effect was observed (Figure 13).

Previous research has suggested that compounds selective for CRY2 are effective against impaired glucose tolerance. The above results indicate that not only CRY2-selective activation, but also CRY1-selective activation is effective against impaired glucose tolerance.

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 rhythm 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, we obtained and analyzed the activity of TH320, which has two oxygen atoms bound to it, and TH321, which has one oxygen atom bound to it (Figure 14). In human U2OS cells, TH321 was slightly more potent than KL101, and TH320 extended the circadian rhythm period of the Bmal1-dLuc reporter by approximately 10-fold (Figure 15). Furthermore, TH320 also enhanced the effect of inhibiting the degradation of the CRY1-LUC reporter and increasing its half-life (Figure 16). In Cry1/Cry2 knockout mouse fibroblasts rescued from CRY1 or CRY2, TH320 exhibited a stronger and more selective effect on CRY1 than KL101 (Figure 17). These results indicate that the activity of KL101 increases with the number of oxygen atoms bound to the sulfur atom. To clarify this molecular mechanism, we performed X-ray crystallography of CRY1 bound to TH320 and TH321. Three water molecules (W1, W2, and W3) were found near the S residue of the compounds. In TH320, two O molecules bound to the S residue interacted with W1 and W3, respectively (Figure 18). In TH321, one O molecule bound to the S residue interacted with W3 (Figure 19). In contrast, although W1 and W3 were present in the CRY1-KL101 complex described in Non-Patent Document 2, no interaction with KL101 was observed (Figure 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. It is thought that TH320 and TH321 interact with this network via O molecules, resulting in increased activity. These results suggest an effective means for increasing the activity of KL101, a compound selective for CRY1.

Claims (12)

General formula (1):
[wherein X represents -S(=O) _2- or -S(=O)-; ^R1 's may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom; p represents an integer of 1 to 5; ^{and R2} represents a group represented by the general formula (2):
(In the formula, n represents 0 or 1. One of ^R3 and ^R4 is an alkyl group and the other is a hydrogen atom, or they are linked together to form —(CH ₂ ) _m — (m represents 2 to 6). R ⁵ represents a hydrogen atom or an alkyl group. R ⁶ represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom. R ⁷ represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom, excluding the cases where n is 0 and R ⁷ is a fluorine atom, and where R ⁶ and R ⁷ are both alkoxy groups. R ⁸ represents a hydrogen atom or an alkyl group.)
represents a group represented by the following formula:
A transmucosally ingestible agent comprising at least one compound selected from the group consisting of a compound represented by the formula (I): The transmucosally ingestible agent according to claim 1, wherein R ¹ is the same or different and is an alkoxy group having 1 to 4 carbon atoms or an alkyl group having 1 to 4 carbon atoms. The transmucosally ingestible agent according to claim 1, wherein p is 1 to 3 and one ^R1 is at the para position. The transmucosally ingestible agent according to claim 1, wherein n is 0, and ^R6 and ^R7 are the same or different and each represent an alkyl group. 2. The transmucosally ingestible agent according to claim 1, wherein n is 1, ^R3 and ^R4 are linked together to form --( _CH2 ) _m-- (m is 2 to 6), and ^R7 is a halogen atom. n is 0, and R ⁶ and R ⁷ are the same or different and are alkyl groups, or n is 1, R ³ and R ⁴ are linked together to form —(CH ₂ ) _m — (m is 2 to 6), and R ⁷ is a halogen atom;
The transmucosal administration agent according to claim 1. The transmucosally ingestible agent according to claim 1, wherein X is —S(═O) ₂ —. The transmucosally ingested agent according to any one of claims 1 to 7, which is an orally ingested agent. The transmucosally ingestible agent according to any one of claims 1 to 7, which is a pharmaceutical composition or a food composition. The transmucosally ingested agent according to any one of claims 1 to 7, which is used for circadian rhythm regulation. The transmucosally ingested agent according to any one of claims 1 to 7, which is used for regulating sleep-wake rhythm or controlling glucose metabolism. General formula (1AA):
[wherein X represents -S(=O) _2- or -S(=O)-; ^R1 's may be the same or different and represent an alkoxy group, an alkyl group, or a halogen atom; ^R5 represents a hydrogen atom or an alkyl group; ^R6 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom; ^R7 represents a hydrogen atom, an alkyl group, an alkoxy group, or a halogen atom, except when R7 is a fluorine atom or when both ^{R6 and R7} are alkoxy groups; and ^R8 represents a hydrogen atom or an alkyl group.] (provided that the following compounds:
), its salts, and solvates thereof.