Circadian rhythm regulators

Abstract

To provide a novel circadian rhythm regulator. [Solution] A circadian rhythm regulator comprising a phthalide compound having at least one substituent R on a benzene ring as an active ingredient, wherein the at least one substituent R is independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, hydroxymethyl, and acetoxy.

Description

[Technical Field]

[0001] This disclosure relates to novel compounds that serve as circadian rhythm regulators and their active ingredients. [Background technology]

[0002] The biological clock is a mechanism present in various organisms, including humans, that regulates metabolism and physiological functions on an approximately 24-hour cycle. The fluctuation of biological events on an approximately 24-hour cycle based on the biological clock is called a circadian rhythm. The biological clock is known to be centered around a coordinated transcription-translation feedback loop, which includes transcriptional activation of the Per and Cry genes by the Bmal1 and Clock gene products, and suppression of this transcriptional activation by the translation products of Per and Cry. The factors and DNA elements involved in this transcriptional regulation are highly conserved in mammalian evolution.

[0003] In recent years, due to the diversification of lifestyles and shift work, people are more susceptible to conditions such as social jet lag and sleep deprivation. These conditions occur when the body clock and circadian rhythm are out of sync with the circadian rhythm of the external environment, or when the body clock and circadian rhythm are disrupted. Disruptions in the regulation of body clock genes, and consequently disruptions in the circadian rhythm, can be deeply involved in various diseases such as sleep disorders, lifestyle-related diseases, depression, and cardiovascular disorders. Sleep disorders that result from the inability to properly synchronize the period of the body clock with the circadian rhythm of the external environment are called circadian rhythm sleep disorders. It is believed that properly regulating the circadian rhythm is useful for maintaining healthy life activities. However, there are few specific substances that are understood to be effective in regulating the circadian rhythm.

[0004] Patent Document 1 and Non-Patent Document 1 describe that certain diterpenoid compounds (named cyclocircadines) isolated from mushrooms have circadian rhythm regulatory activity. These compounds primarily exerted the effect of delaying the phase of the circadian rhythm. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2022 / 224820 [Non-patent literature]

[0006] [Non-Patent Document 1] J Biosci Bioeng. 2023 Oct;136(4):278-286 [Overview of the project] [Problems that the invention aims to solve]

[0007] This disclosure provides novel circadian rhythm regulators and novel compounds that can serve as active ingredients thereof. [Means for solving the problem]

[0008] This disclosure includes at least the following embodiments. [1] A circadian rhythm regulator comprising a phthalide compound having at least one substituent R on a benzene ring as an active ingredient, wherein the at least one substituent R is independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, hydroxymethyl, and acetoxy. [2] The phthalide compound is given by the following formula (V): [ka] It is a compound represented by, and here, R 1 ~R 4 One or more of these are independently the substituent R, R 1 ~R 4Among them, the group that is not the substituent R is independently selected from the group consisting of a hydrogen atom, a halogen atom, and a hydrocarbon group having 1 to 6 carbon atoms, and two adjacent hydrocarbon groups may be linked to form a cyclic structure. The circadian rhythm regulator according to [1]. [3] R 1 and R 4 are independently the substituent R, and R 2 and R 3 are linked to form the following formula (VII):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0009] [Figure 1] Figure 1 shows an overview of the mass spectrometry results (a) and NMR spectrum (b) of novel compound 1, isolated from Cyclocybe mushrooms and exhibiting circadian rhythm regulatory activity. [Figure 2] Figure 2 shows the structure of compound 1 as determined by two-dimensional NMR measurement. [Figure 3] Figure 3 shows data indicating that administering compound 1 to cells advances the phase of the circadian rhythm. [Figure 4] Figure 4 shows the results of investigating the circadian rhythm phase alteration activity of different compounds structurally related to compound 1, using the same experimental procedure described for Figure 3. [Modes for carrying out the invention]

[0010] In one aspect, this disclosure provides a circadian rhythm regulator comprising a phthalide compound as an active ingredient. This phthalide compound is a phthalide compound having at least one substituent R on the benzene ring portion inherent to phthalide, wherein the at least one substituent R is independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, hydroxymethyl, and acetoxy. The term phthalide compound may also be replaced with phthalide derivative, where the term phthalide derivative does not necessarily mean that the phthalide compound was synthesized starting from phthalide.

[0011] A circadian rhythm regulator refers to a drug used to induce a change in the circadian rhythm. Changes in the circadian rhythm may include, for example, phase advancement, phase delay, period shortening, or period lengthening. Those skilled in the art know that the phase of the circadian rhythm and changes thereof can be observed, for example, by linking the promoter of a Per gene or Cry gene to a reporter gene (e.g., a luminescent enzyme gene such as luciferase) and measuring the activity of the reporter gene over time in a cell or tissue. Circadian rhythm regulators of embodiments of this disclosure can typically "advance" the phase of the circadian rhythm. Therefore, a circadian rhythm regulator in a particular embodiment may be provided as a composition for advancing the phase of the circadian rhythm. In some embodiments, a circadian rhythm regulator may be provided as a composition for the treatment or prevention of circadian rhythm sleep disorders. Circadian rhythm regulators may be used, for example, as a pharmaceutical composition or a supplement.

[0012] As is known to those skilled in the art, phthalides are compounds consisting of a benzene ring fused to a γ-lactone. The basic chemical structures and standard numbering of atomic positions of phthalides are shown below. [ka] In this disclosure, the positions of substituents are indicated according to the numbering in the above formula.

[0013] Circadian rhythm regulators according to embodiments of this disclosure can be described as compositions containing an effective amount of a phthalide compound as an active ingredient, wherein the phthalide compound has at least one substituent R on its benzene ring portion, and the at least one substituent R is independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, hydroxymethyl, and acetoxy. It has been found that circadian rhythm regulatory activity is generated in phthalide compounds by the presentation of a polar substituent on the benzene ring portion of the phthalide, and therefore all of these substituent Rs are polar groups. In this disclosure, substituent R is also referred to as an R group. The phthalide compound preferably has one, two, or three R groups. It is particularly preferable that the R groups are independently a hydroxyl group, a methoxy group, or a hydroxymethyl group. In this disclosure, "independently" means that if there are multiple groups (in this case, R groups), they may be identical or different from one another within the definition.

[0014] Phthalide compounds may have R groups at one or more of the following positions: 4, 5, and 7. Non-limiting examples include a phthalide compound having at least one R group including one at position 4, at least one R group including one at position 5, at least one R group including one at position 7, at least two R groups including R groups at positions 4 and 5, or having R groups at positions 4 and / or 5 and an additional R group at position 7. Preferably, a phthalide compound has at least one R group at position 4 or 5 of the phthalide. If there are multiple R groups, they may be different from each other or identical. Positions of the benzene ring portion of a phthalide compound (positions 4-7 of the phthalide compound) that are not substituted with R groups may independently be hydrogen or substituents other than R groups. Examples of substituents other than R groups include, but are not limited to, hydrocarbon groups with 6 or fewer carbon atoms (e.g., alkyl, alkenyl, or aryl) and halogen atoms (e.g., fluorine, chlorine, bromine, or iodine). The hydrocarbon group may be substituted or unsubstituted. In some embodiments, the number of carbon atoms in the hydrocarbon group may be 4 or less. Two adjacent hydrocarbon groups (i.e., located at adjacent carbon positions on the benzene ring) may be linked to form a cyclic structure. For example, in the compound of formula (I) below, it can be understood that a branched alkyl group with 4 carbon atoms at position 5 and an alkyl group with 1 carbon atom at position 6 are linked to form a cyclic structure. In various embodiments, the γ-lactone portion of the phthalide compound is unsubstituted.

[0015] Therefore, in some embodiments, the phthalide compound is of the following formula (V): [ka] It is a compound represented by R, where R 1 ~R 4 One or more of the substituents R (corresponding to positions 7, 6, 5, and 4 of the phthalide skeleton, respectively) are independently R, 1 ~R 4Among these, the groups other than the substituent R are independently selected from the group consisting of hydrogen atoms, halogen atoms, and hydrocarbon groups having 1 to 6 carbon atoms, and two adjacent hydrocarbon groups may be linked to form a cyclic structure.

[0016] In some preferred embodiments, R is used in a phthalide compound represented by formula (V). 1 and R 4 The substituents R are independently of each other, 2 and R 3 These are combined into the following equation (VII): [ka] It forms a cyclic structure represented by , where * and ** represent bonding points to the benzene ring portion, respectively.

[0017] Several specific examples of preferred phthalide compounds are shown below. The hydroxyl, methoxy, and hydroxymethyl groups may be substituted for each other, or for other polar groups included in the definition of an R group. The compound of formula (I) below is a particularly preferred example as an active ingredient in circadian rhythm regulators and is described as Compound 1 in the Examples section. [ka]

[0018] The compounds that constitute the active ingredients of the circadian rhythm regulators of this disclosure can be isolated from organisms, particularly mushrooms, more specifically Cyclocybe mushrooms, and even more specifically Cyclocybe erebia or Cyclocybe cf. erebia, or can be chemically synthesized. Synthetic routes for phthalides with various substituents are known to those skilled in the art and can be applied to the synthesis of the active compounds of this embodiment. An example of a specific procedure for isolating the compound of formula (I) from Cyclocybe mushrooms is described in the following examples. It will be understood to those skilled in the art that the compounds that constitute the active ingredients of the circadian rhythm regulators are provided in an isolated state and can be added to the composition of the circadian rhythm regulators. "Isolated state" as used herein means substantially separated from other organic compounds that were associated with the target compound in a source such as a mushroom extract or a chemically synthesized reaction product (including substrates and by-products), such that the proportion of associated organic compounds from such a source is less than 50% by mass (i.e., less than the same amount as the target compound). The active compound contained in the circadian rhythm regulator of this disclosure may be added to other components of the circadian rhythm regulator (e.g., excipients, carriers, etc.) in a state of purity of 50% or more, 70% or more, 90% or more, or 100% by mass.

[0019] The compounds that serve as the active ingredients of the circadian rhythm regulators disclosed herein, including the compound of formula (I), are advantageous because, compared to cyclocircadines, for example, in Non-Patent Document 1, they have a simpler structure and are phthalide-based compounds for which knowledge of chemical synthesis and modification has already been accumulated. They are also advantageous because they can exhibit activity even at low concentrations of less than 100 μM. Typically, activity can be confirmed even at concentrations diluted to 10 μM. These compounds are presumed to be highly safe because they are compounds found in or structurally similar to edible mushrooms.

[0020] It should be understood that the circadian rhythm regulators and compositions of this disclosure may contain, in addition to an effective amount of one or more of the above-described active ingredients, a suitable excipient or carrier, and may further contain one or more of various suitable additives known to those skilled in the art. The excipient or carrier may be a pharmaceutically acceptable excipient or carrier. The circadian rhythm regulators and compositions of this disclosure may be formulated in a variety of forms, including, but not limited to, tablets, capsules, powders, granules, liquids (e.g., aqueous solutions), and gels.

[0021] In another aspect, the Disclosure provides the use of the above-mentioned compounds or compositions for regulating circadian rhythms, advancing the phase of circadian rhythms, or for the treatment or prevention of circadian rhythm sleep disorders. In another aspect, the Disclosure provides methods for regulating circadian rhythms, advancing the phase of circadian rhythms, or for the treatment or prevention of circadian rhythm sleep disorders, comprising administering an effective amount of the above-mentioned compounds or compositions to a subject requiring such regulation. Circadian rhythm regulators, active compounds, and compositions can be systemically administered to subjects (particularly mammalian subjects, e.g., human subjects) via routes of administration known to those skilled in the art, including oral administration, intravenous administration, etc.

[0022] In another aspect, this disclosure provides novel compounds that may be active ingredients in circadian rhythm regulators or used in the manufacture of pharmaceuticals for circadian rhythm regulation. Compounds according to several embodiments are given by the following formula (V): [ka] A compound represented by the formula, where R 1 and R 4 R is independently the substituent R mentioned above, 2 and R 3 These are combined into the following equation (VII): [ka] It forms a cyclic group represented by , where * and ** represent bonding points to the benzene ring portion in formula (V), respectively.

[0023] More specifically, a compound represented by the following formula (I) is provided. [ka]

[0024] Further compounds are provided, including those represented by the following formula (II). [ka] [Examples]

[0025] The embodiments of this disclosure will be described in more detail below with reference to examples, but these examples are merely illustrative, and the present invention is not limited to these examples.

[0026] Here, Cyclocybe cf. erebia, which is generally available as TUFC strain catalog number 30219 from the Fungal Genetic Resources Center, Faculty of Agriculture, Tottori University (https: / / fungusdb.muses.tottori-u.ac.jp / distribution / ), was used as the extraction source. However, other Cyclocybe species, including other species of Pholiota nameko or closely related species, may also be used. The above mushrooms were cultured in liquid medium in malt, and the culture filtrate was extracted with ethyl acetate. The ethyl acetate extract was fractionated by silica gel column chromatography using acetone in hexane (0-100% (v / v)) as the mobile phase, and the 40% acetone fraction was recovered. The 40% acetone fraction was further fractionated by ODS column chromatography using methanol in water (20-100% (v / v)) as the mobile phase, and the 60% methanol fraction was recovered. This is a fraction containing circadian rhythm regulatory activity, which is different from the fraction containing cyclocircadin described in Patent Document 1 and Non-Patent Document 1. Compound 1 was obtained by purifying this 60% methanol fraction by preparative HPLC (ODS column, 29-50% gradient of acetonitrile in water).

[0027] Figures 1 and 2 show an overview of the mass spectrometry and nuclear magnetic resonance (NMR) analysis of compound 1. These analyses revealed that compound 1 is a novel compound represented by formula (I) above, 4-hydroxy-8-(hydroxymethyl)-6,6-dimethyl-3,5,6,7-tetrahydro-1H-indeno[5,6-c]furan-1-one.

[0028] The Per gene family constitutes the core of the body clock, and its promoter activity changes periodically to reflect circadian rhythms. Here, we measured circadian rhythms using embryonic fibroblasts (PER2::LUC) derived from the embryos of knock-in mice in which the luciferase gene was fused in-frame to the 3' end of the promoter region of the mouse Per2 gene, following standard procedures. More specifically, we placed the cell line culture in a real-time luminescence measurement device and measured the luciferase emission value. We added samples of compounds from the extract, either in fractionation or after purification, to the culture medium and observed whether or not there was a change in the circadian rhythm. Contact between cells and the test compound was achieved by adding (administering) the sample to the culture medium.

[0029] In Figure 3a, time (in hours) is plotted on the horizontal axis, and the relative luminescence intensity of luciferase induced by the promoter of the circadian clock gene Per2 is plotted on the vertical axis. The timing of the addition of compound 1 to the culture medium containing cells with synchronized circadian rhythms is indicated by arrowheads. Figure 3b is a graph plotting the time shift from the addition of compound 1 to the culture medium until the circadian rhythm phase reaches its peak, against the concentration of compound 1 (μM). The time to reach the peak when the compound 1 concentration is 0 μM is set to 0.0, and the vertical axis shows how many hours (in hours) the time to reach the peak shifted. A shift represented by a negative value means that the phase has advanced.

[0030] In Figure 3a, the dashed square brackets indicate the position of the phase peak when compound 1 is not administered. It is clear that administration of purified compound 1 results in a phase advance compared to the circadian rhythm when compound 1 is not administered. As shown in Figure 3b, a concentration-dependent phase advance of the circadian rhythm was observed. At a concentration of 40 μM, an average phase advance of approximately 3 hours was observed. Phase advance was also observed when compound 1 was administered at different timings in the phase waveform (not shown).

[0031] Compound 1 has a phthalide skeleton and possesses methoxy and hydroxymethyl groups, which are polar groups capable of hydrogen bonding, on its benzene ring portion. Furthermore, it also has linked alkyl substituents forming a cyclic structure. Figure 4 shows an example of the results of the same circadian rhythm measurement performed using compounds that partially share structural commonalities with Compound 1. Of the compounds shown in Figure 4, Compound 1' (Compound 1 with the hydroxyl group replaced by a methoxy group) was prepared from Compound 1 using a known method, while the other compounds were purchased commercially. Phthalide itself did not significantly alter the circadian rhythm (Figure 4c). In contrast, compounds with polar groups such as hydroxy and methoxy on the benzene ring portion of phthalide, such as Compound 1' (Figure 4a), 4-methoxyphthalide, and 5-hydroxyphthalide (Figure 4b), showed the same effect as Compound 1 in advancing the phase of the circadian rhythm. Interestingly, gastrodigenin (4-hydroxybenzyl alcohol) showed activity in delaying the phase of the circadian rhythm in this assay (Figure 4c). Gastrodigenin is a compound known to be abundant in the plant Gastrodia elata, and extracts of Gastrodia elata have been reported to improve memory impairment caused by circadian rhythm disorders in mice (Life Sci Space Res. 2021 31:51-58). Therefore, circadian rhythm regulators containing gastrodigenin as an active ingredient are also being considered.

Claims

1. A circadian rhythm regulator comprising a phthalide compound having at least one substituent R on a benzene ring as an active ingredient, wherein the at least one substituent R is independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, hydroxymethyl, and acetoxy.

2. The phthalide compound is given by the following formula (V): 【Chemistry 1】 It is a compound represented by, and here, R １ ~R ４ One or more of these are independently the substituent R, R １ ~R ４ Among these, the group that is not substituent R is independently selected from the group consisting of a hydrogen atom, a halogen atom, and a hydrocarbon group having 1 to 6 carbon atoms, and two adjacent hydrocarbon groups may be linked to form a cyclic structure. A circadian rhythm regulator according to claim 1.

3. R １ and R ４ The substituents R are independently of each other, ２ and R ３ These are combined into the following equation (VII): 【Chemistry 2】 The circadian rhythm regulator according to claim 2, which forms a cyclic structure represented by , where * and ** each represent a bond point to the benzene ring portion.

4. Formula (I) below: 【Transformation 3】 A circadian rhythm regulator containing a compound represented by as an active ingredient.

5. The following equation (V): 【Chemistry 4】 A compound represented by the formula, in which, R １ and R ４ is a substituent R independently selected from the group consisting of hydroxy, methoxy, ethoxy, propoxy, hydroxymethyl, and acetoxy, R ２ and R ３ These are combined into the following equation (VII): 【Transformation 5】 A compound that forms a cyclic group represented by , where * and ** represent bonding sites to the benzene ring portion in formula (V), respectively.

6. Formula (I) below: 【Transformation 6】 The compound according to claim 5, which is a compound represented by .

7. Use of the compound according to claim 5 or 6 in the manufacture of a pharmaceutical product for regulating circadian rhythms.

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