JP2020083811A - Ampk activator containing 1,5-anhydro fructose derivative - Google Patents

Abstract

To provide an agent that can effectively activate AMP-activated protein kinase (AMPK).SOLUTION: An AMP-activated protein kinase (AMPK) activator contains a 1,5-anhydro fructose derivative.SELECTED DRAWING: Figure 2

Description

The present invention relates to an AMPK activator containing a 1,5-anhydrofructose derivative.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is activated with a decrease in intracellular ATP level. Recently, it was revealed that AMPK plays a central role in the cellular stress response. AMPK functions as a cell energy sensor against an increase in the AMP/ATP ratio (decrease in ATP and increase in AMP etc.) due to metabolic/physiological stress, etc., and is important as the highest regulator of cell energy homeostasis. Play a different role. AMPK positively regulates (enhances) ATP-producing signal pathways (catabolic pathways) such as glycolysis and fatty acid β-oxidation through phosphorylation of various downstream bioenzymes, while gluconeogenesis, lipids It is known to restore ATP levels by negatively regulating (suppressing) the ATP-consuming pathway (anabolic pathway) such as protein and protein biosynthesis.

Activation of AMPK has been reported to bring about various physiological effects. For example, cholesterol biosynthesis suppression, mitochondrial biosynthesis promotion, ischemia tolerance improvement, autophagy promotion, cell growth inhibition, gluconeogenesis inhibition, sugar It is known to be effective in promoting decomposition and increasing blood flow. There are various reports on the effect of AMPK activation to suppress cancer cell growth. AMPK activation is a treatment for many diseases and conditions such as hyperlipidemia, hypertension, metabolic syndrome such as obesity, type 2 diabetes, mitochondrial disease, autophagy-related diseases, aging-related symptoms, cancer and inflammatory diseases. It is considered to be effective in prevention, and the development of treatments/prevention methods targeting AMPK has received a great deal of attention worldwide.

Metformin, which is widely used as an antidiabetic drug in the world, has recently been proved to have an AMPK activating effect (Non-patent Document 1). Non-patent document 1 reports the results of examining AMPK phosphorylation after adding metformin to vascular endothelial cells at 1 mM and stimulating for 4 hours (see Fig. 2A of non-patent document 1).

1,5-Anhydrofructose (1,5-AF) is a monosaccharide showing structural similarity to glucose. Patent Document 1 discloses that 1,5-AF has a caspase I activation inhibitory action. Non-Patent Document 2 reports that 1,5-AF has AMPK phosphorylation activity.

However, the AMPK phosphorylation activity of 1,5-AF is not at a satisfactory level, and development of a more effective AMPK activator is still desired.

International Publication No. WO2015/016178

Shang F., et al., PLOS ONE, (2016) 11(3):e0151845 Kojima-Yuasa A., et al., Nat. Prod. Commun., (2012) 7(11): p.1501-1506

An object of the present invention is to provide a drug that can effectively activate AMP-activated protein kinase (AMPK).

As a result of intensive studies to solve the above problems, the present inventors have found that a given 1,5-AF derivative has a high AMPK activating ability, and have completed the present invention.

That is, the present invention includes the following.
[1] An AMP-activated protein kinase (AMPK) activator containing a 1,5-anhydrofructose derivative.
[2] The AMPK activator according to the above [1], wherein the 1,5-anhydrofructose derivative is a deoxy form or an enone form of 1,5-anhydrofructose.
[3] The 1,5-anhydrofructose derivative is 3-deoxy-1,5-anhydrofructose or 6-(hydroxymethyl)-2H-pyran-3-one, [1] or [2] above The AMPK activator according to 1.
[4] A medicament for treating or preventing a disease or condition for which AMPK activation is effective, which comprises the AMPK activator according to any of [1] to [3] above.

According to the present invention, AMPK can be effectively activated.

FIG. 1 is an electrophoretic photograph of Western blot analysis showing the AMPK phosphorylation activity of 1,5-AF and its 3-deoxy form. A shows the results using 1,5-AF and B shows the results using 1,5-AF 3-deoxy. A band of phosphorylated AMPK (p-AMPK) is observed in the electrophoretic photograph. FIG. 2 is an electrophoretic photograph of Western blot analysis showing the AMPK phosphorylation activity of 1,5-AF 3-deoxy form and enone form. A band of phosphorylated AMPK (p-AMPK) is observed in the electrophoretic photograph. FIG. 3 shows changes in body weight (A) and fat mass (B) after administration of 1,5-AF. In A, the rhombus symbol indicates 1,5-AF(+) and the square symbol indicates 1,5-AF(-). FIG. 4 shows the insulin secretagogue activity of 1,5-AF in a cultured pancreatic cell line (Min6). Square symbols indicate 1,5-AF 10 μg/ml addition group, triangle symbols indicate 1,5-AF 1 μg/ml addition group, and diamond symbols indicate control group. FIG. 5 is a photograph showing ischemic tolerance caused by pre-administration of 1,5-AF. In the leftmost lane, as a result of the control brain in which cerebral vascular network ligation occlusion was performed without administration of 1,5-AF, the other 4 lanes on the right side were cerebral vascular network ligation occlusion after administration of 1,5-AF. The results of the test brains performed are shown. White areas indicate ischemic necrotic lesions, respectively, but ischemic necrosis is reduced by pre-administration of 1,5-AF as compared with the left end (control).

Hereinafter, the present invention will be described in detail.
The present invention relates to an AMP-activated protein kinase activator containing a 1,5-anhydrofructose derivative. AMP-activated protein kinase (AMPK) is a heterotrimer composed of three subunits of α, β, and γ, and AMPK kinase (AMPKK) is present at the 172nd threonine of the α subunit (α chain). ), it is activated by being phosphorylated. Activated AMPK activates or suppresses various downstream biological factors by phosphorylation, and induces or inhibits downstream signal transduction.

In the present invention, the 1,5-anhydrofructose derivative that can be used as the active ingredient of the AMPK activator is, for example, the following general formula (I) or (II):

It may be a compound represented by.

In the general formula (I), ^one of R ¹ and R ² represents a hydroxyl group or an acyloxy group, and the other represents a hydrogen atom or a fluorine atom. Preferably, one of R ¹ and R ² represents a hydroxyl group or an acyloxy group, and the other represents a hydrogen atom. More preferably, ^one of R ¹ and R ² represents a hydroxyl group and the other represents a hydrogen atom. As the acyl group constituting the acyloxy group, an optionally substituted alkylcarbonyl group, an optionally substituted arylcarbonyl group, or the like can be used. More specifically, for example, an acetyl group, an alkylcarbonyl group such as a trifluoroacetyl group, or a benzoyl group (a benzene ring is a halogen atom such as a fluorine atom or a chlorine atom, an alkoxy group such as a methoxy group, an alkyl group or an acyl group). Amino group which may be substituted, or one or more groups selected from alkyl groups such as methyl group) may be used.

R ³ and R ⁴ each independently represent a hydrogen atom or a fluorine atom. When one of R ³ and R ⁴ is a hydrogen atom and the other is a fluorine atom, it is preferable that both R ³ and R ⁴ are hydrogen atoms.

In the formula, ---- represents a single bond or a double bond, but when ---- represents a double bond, R ¹ and R ⁴ do not exist, and only R ² and R ³ exist Means that. When ---- represents a double bond, R ² is preferably a hydrogen atom, R ³ is a fluorine atom, or both R ² and R ³ are preferably hydrogen atoms.

R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group, or a fluorine atom, but it is preferable that both R ⁵ and R ⁶ are hydrogen atoms. In the present specification, as the alkyl group, for example, a C _1-6 alkyl group can be used, and preferably a C _1-4 alkyl group can be used. The alkyl group may be linear, branched, cyclic, or a combination thereof. The same applies to the alkyl moiety of other substituents having an alkyl moiety (for example, an alkylcarbonyl group).

R ⁷ is ^{_{-CH 2 -OR 8, -CHF-OR}} 8, -CF 2 -OR 8, a hydrogen atom, an alkyl group, or show trifluoromethyl group, R ⁷ is -CH ₂ -OR ⁸ Is preferred. R ⁸ represents a hydrogen atom, an alkyl group, or an acyl group, or R ⁸ is combined with R ⁶ to be —R ⁸ —R ⁶ — and represents a single bond. As the acyl group, an optionally substituted alkylcarbonyl group, an optionally substituted arylcarbonyl group, or the like can be used. More specifically, for example, an acetyl group, an alkylcarbonyl group such as a trifluoroacetyl group, or a benzoyl group (a benzene ring is a halogen atom such as a fluorine atom or a chlorine atom, an alkoxy group such as a methoxy group, an alkyl group or an acyl group). Amino group which may be substituted, or one or more groups selected from alkyl groups such as methyl group) may be used. Preferably, R ⁸ represents a hydrogen atom or an acyl group, or R ⁸ is bonded to R ⁶ to form —R ⁸ —R ⁶ — which represents a single bond.

A preferred combination is
(a) ^one of R ¹ and R ² is a hydroxyl group or an acyloxy group and the other is a hydrogen atom; R ³ and R ⁴ each independently represent a hydrogen atom or a fluorine atom; -Indicates a single bond or a double bond, and when ----indicates a double bond, R ¹ and R ⁴ are not present; R ⁵ and R ⁶ are hydrogen atoms; R ⁷ is -CH _2- OR ⁸ and R ⁸ represents a hydrogen atom, an alkyl group, or an acyl group, or R ⁸ is bonded to R ⁶ to form -R ⁸ -R ⁶ -, which is a single bond;
(b) R ² is a hydrogen atom or a fluorine atom; R ³ is a hydrogen atom or a fluorine atom; ---- in the formula represents a double bond; R ⁵ and R ⁶ are hydrogen atoms; R ⁷ is —CH ₂ —OR ⁸ and R ⁸ represents a hydrogen atom or an acyl group, or R ⁸ is bonded to R ⁶ to form —R ⁸ —R ⁶ — to represent a single bond. Can be mentioned.

A more preferable combination is
(c) R ² is a hydrogen atom; R ³ is a hydrogen atom or a fluorine atom; ----in the formula represents a double bond; R ⁵ and R ⁶ are hydrogen atoms; R ⁷ is -CH ₂ -OR ⁸ and R ⁸ represents a hydrogen atom or an acyl group, or R ⁸ is bonded to R ⁶ to form -R ⁸ -R ⁶ -, which is a single bond;
(d) R ² is a hydrogen atom; R ³ is a hydrogen atom or a fluorine atom; ---- represents a double bond in the formula; R ⁵ is a hydrogen atom; R ⁷ is -CH ₂ -OR ⁸ and R ⁸ is bonded to R ⁶ to form -R ⁸ -R ⁶ -, which represents a single bond. However, the preferable combination is not limited to these.

In the general formula (II), R ¹¹ and R ¹² are the same as R ¹ and R ² described above. R ¹⁴ represents an acyl group, and as the acyl group, those described as the acyl group constituting the acyloxy group in the description of R ¹ and R ² can be used. R ¹⁷ and R ¹⁸ are the same as R ⁷ and R ⁸ .

A preferred combination is
(e) one of R ¹¹ and R ¹² is a hydroxyl group or an acyloxy group, the other is a hydrogen atom; R ¹³ is a hydrogen atom or a fluorine atom; R ¹⁴ is an acyl group; R ¹⁵ and R ¹⁶ are each independently a hydrogen atom or a fluorine atom; R ¹⁷ is —CH ₂ —OR ¹⁸ , and R ¹⁸ is a hydrogen atom, an alkyl group, or an acyl group. Is
(f) one of R ¹¹ and R ¹² is an acyloxy group, the other is a hydrogen atom; R ¹³ is a hydrogen atom; R ¹⁴ is an acyl group; R ¹⁵ and R ¹⁶ are hydrogen atoms Yes; R ¹⁷ is —CH ₂ —OR ¹⁸ , and R ¹⁸ is a hydrogen atom, an alkyl group, or an acyl group. However, the preferable combination is not limited to these.

The compound represented by the general formula (I) or (II) may have one or more asymmetric centers, and stereoisomers such as optical antipodes or diastereoisomers based on such asymmetric centers May exist. A stereoisomer in a pure form, an arbitrary mixture of stereoisomers, a racemate and the like may be used as an active ingredient of the medicament of the present invention. Regarding two or more stereoisomers due to the cyclic structure, any stereoisomer in pure form or any mixture of stereoisomers can be used as an active ingredient of the medicament of the present invention.

The compound represented by the general formula (I) may be keto type. The compound represented by the general formula (II) may be a hydrate of the compound represented by the general formula (I).

The compound represented by the general formula (I) or (II) obviously does not include 1,5-anhydrofructose represented by the following formula (III).

As an example of the 1,5-AF derivative used as the active ingredient of the AMPK activator in the present invention, a deoxy form of 1,5-anhydrofructose (typically 1,5-D-anhydrofructose) (for example, , 3-deoxy form) or enone form, but not limited thereto.

As a specific example of the 1,5-AF derivative used as the active ingredient of the AMPK activator in the present invention, 3-deoxy-1,5-anhydrofructose represented by the following formula (IV), and the following formula (V): 6-(Hydroxymethyl)-2H-pyran-3-one represented.

The compound represented by the general formula (I) or (II) can be synthesized by, for example, the method specifically shown in Examples of the present specification, or a known method, or can be obtained as a commercial product. it can.

The 1,5-AF derivative according to the present invention can phosphorylate AMPK (specifically, phosphorylate the threonine at the 172nd position from the N terminus of the α subunit of AMPK), that is, the ability to activate AMPK. Have. The AMPK phosphorylation activity of 1,5-AF derivatives (that is, AMPK activation ability) is determined by adding 1,5-AF derivatives to vascular endothelial cells and reacting them for a certain period of time (for example, 1 to 2 hours). Phosphorylated AMPK in cells can be quantified and evaluated by comparison with the quantified value in a negative control tested under the same conditions except that the 1,5-AF derivative is not added. Such measurement of the AMPK phosphorylation activity of the 1,5-AF derivative using vascular endothelial cells can be carried out, for example, according to the description in Examples below. When phosphorylated AMPK is increased compared to the negative control when 1,5-AF derivative is added to vascular endothelial cells, the 1,5-AF derivative has AMPK phosphorylation activity (that is, AMPK activation ability) Is determined to have. For example, if the quantified value of phosphorylated AMPK in the 1,5-AF derivative-treated group is 20% or more higher than that of the negative control, it may be judged that the phosphorylated AMPK is increased compared to the negative control. it can.

The AMPK activator containing the 1,5-AF derivative according to the present invention may contain the 1,5-AF derivative in an effective amount. The AMPK activator according to the present invention may be a composition for activating AMPK containing the 1,5-AF derivative as an active ingredient. The AMPK activator according to the present invention may be used for cells, tissues or organs isolated in vitro, or may be used for cells, tissues or organs in the body in vivo. ..

The present invention also provides a medicament comprising the 1,5-AF derivative according to the present invention, or an AMPK activator containing the same. The drug according to the present invention can be used for AMPK activation based on the AMPK phosphorylation activity (AMPK activation ability) of the 1,5-AF derivative.

The AMPK activator or medicine according to the present invention is, in addition to the 1,5-AF derivative, a pharmaceutically acceptable additive such as a carrier (solid or liquid carrier), an excipient, a surfactant, Binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, coloring agents, flavoring agents, preservatives, buffers, pH adjusters, diluents, stabilizers, propellants, etc. It may further include.

The medicament according to the present invention may be in any dosage form. The drug according to the present invention includes, for example, tablets, capsules, powders, solid preparations such as granules, emulsions, liquid preparations such as syrups, solutions, suspensions, suppositories, inhalants, aerosols, ointments, It may be in the form of a cream, gel, patch, transdermal absorption agent, transmucosal absorption agent, etc., but is not limited thereto. The medicine according to the present invention may be oral or parenteral.

The compounding amount of the 1,5-AF derivative in the medicine according to the present invention can be appropriately set by those skilled in the art, and can be adjusted depending on the dosage form, additives, severity of target disease and the like. The drug according to the present invention may include one type of the above 1,5-AF derivative or two or more types thereof.

The medicament according to the present invention can be used particularly for treating or preventing a disease or condition for which AMPK activation is effective. Here, "the AMPK activation is effective" means that the AMPK activation has a therapeutic or prophylactic effect on the disease or condition. In the present invention, “treatment” includes complete cure, remission, delay or prevention of progression of disease state, improvement of prognosis, recovery, improvement or alleviation of symptoms, improvement of general condition and the like. In the present invention, “prevention” includes prevention or delay of onset, prevention or delay of recurrence, and the like. In one embodiment, the disease or condition for which AMPK activation is effective can be a disease or condition for which AMPK activation is known to be effective in treating or preventing that disease or condition.

AMPK activation is known to induce various physiological activities. The disease or condition for which activation of AMPK targeted by the present invention is effective, but is not limited to, metabolic syndrome (obesity, hypertension, abnormal glucose metabolism, hyperglycemia, hyperlipidemia, arteriosclerosis, etc.), type 2 Diabetes, aging-related diseases (frail, osteoporosis, cognitive impairment, etc.), mitochondrial disease (Parkinson's disease, neurodegenerative disease, Alzheimer's disease, age-related macular degeneration, etc.), autophagy-related diseases, malignant tumors (cancer), Examples thereof include, but are not limited to, inflammatory diseases (nephritis, hepatitis, NASH, fatty liver, etc.), ischemic diseases (ischemic diseases of the brain and cardiovascular system) and the like.

The present invention is based on administering the 1,5-AF derivative, the AMPK activator containing the 1,5-AF derivative, or the drug containing the AMPK activator to cells or subjects in vitro or in vivo. , AMPK activation method is also provided. The present invention is also effective for AMPK activation by administering to the subject a 1,5-AF derivative, an AMPK activator containing the 1,5-AF derivative, or a medicament containing an AMPK activator. Also provided are methods of treating or preventing the disease or condition. The “disease or condition for which AMPK activation is effective” and “treatment or prevention” are as described above.

The dose of the 1,5-AF derivative is not particularly limited, and it depends on various factors such as the body weight, age, and medical history of the patient, the purpose of prevention or treatment, the type and symptoms of the disease, and the administration route. It can be increased or decreased as appropriate. The 1,5-AF derivative, the AMPK activator containing the 1,5-AF derivative, or the medicine containing the AMPK activator may be administered once or plurally at intervals of several hours to several months. It may be administered once.

The 1,5-AF derivative according to the present invention, the AMPK activator containing the 1,5-AF derivative, or the medicament containing the AMPK activator may be administered parenterally or orally. May be administered. The 1,5-AF derivative according to the present invention, an AMPK activator containing the 1,5-AF derivative, or a medicament containing an AMPK activator may be directly administered to an affected area, or -The AF derivative may be administered for targeted delivery to the affected area.

The 1,5-AF derivative according to the present invention, an AMPK activator containing the 1,5-AF derivative, or a subject to which a drug containing the AMPK activator is administered, is not limited to , Any livestock (horse, cow, sheep, goat, pig, etc.), pet animals (dog, cat, rabbit, etc.), laboratory animals (mouse, rat, monkey, etc.), etc. Good. In one embodiment, the subject to be administered can be a subject suffering from or suspected of suffering from a disease or condition for which AMPK activation is effective. In one embodiment, the subject to be administered may be a subject that is predisposed to or in a susceptible environment for a disease or condition for which AMPK activation is effective.

The 1,5-AF derivative according to the present invention, the AMPK activator containing the 1,5-AF derivative, or the isolated cell, tissue or organ to which the drug containing the AMPK activator is administered is particularly limited. However, it may be, for example, a cell, tissue or organ derived from the subject. The cells, tissues or organs to be administered may be derived from an affected part of a disease or condition for which AMPK activation is effective (eg, tumor, inflammatory site, degenerated site, vascular endothelial cell, etc.).

According to the method of the present invention, AMPK can be strongly activated, whereby a disease or condition in which AMPK activation is effective can be treated or prevented.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Synthesis of 1,5-AF derivative
3-deoxy form of 1,5-anhydrofructose (1,5-AF) (3-deoxy-1,5-anhydrofructose) and enone form of 1,5-AF (6-(hydroxymethyl)- 2H-pyran-3-one) was synthesized.

(1) Process 1

Compound 1 (2.46 g, 9.45 mmol) and 1,1′-thiocarbonyldiimidazole (3.37 g, 18.9 mmol) were added to tetrahydrofuran (50 mL) in an argon atmosphere, and the mixture was stirred with heating under reflux overnight. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate=3:2) to obtain compound 2 (3.60 g, quant.). "quant." means "quantitative", that is, a yield of nearly 100%.

(2) Process 2

Compound 2 (3.50 g, 9.45 mmol), tris(trimethylsilyl)silane (5.83 mL, 18.9 mmol) and azobisisobutyronitrile (307 mg, 1.89 mmol) were dissolved in toluene (50 mL), and the solution was degassed, The mixture was stirred at 100° C. for 2 hours in an argon atmosphere. The reaction solution was directly charged on silica gel column chromatography and purified (developing solvent n-hexane:ethyl acetate=4:1) to obtain a crude product of compound 3 (2.79 g).

(3) Process 3

A 0.1 M sulfuric acid aqueous solution (100 mL) was added to the crude product of compound 3 (2.79 g) obtained in Step 2, and the mixture was stirred at 60° C. overnight. The reaction solution was neutralized with sodium hydrogen carbonate (1.85 g), and the solvent was evaporated under reduced pressure. Pyridine (40 mL) and acetic anhydride (20 mL) were added to the obtained residue, and the mixture was stirred overnight at room temperature. After the reaction was stopped by adding methanol, the solvent was distilled off under reduced pressure, the obtained residue was dissolved in ethyl acetate, and the ethyl acetate phase was washed with water, 1M hydrochloric acid, a saturated sodium hydrogen carbonate aqueous solution, and a saturated saline solution. After that, it was dried over magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=3:1) to obtain compound 4 (2.96 g, 94% (3 steps)).

(4) Process 4

Compound 4 (1.52 g, 4.57 mmol) was dissolved in dichloromethane (6 mL), 25% hydrobromic acetic acid solution (9 mL) was added, and the mixture was stirred at room temperature for 2.5 hr. Dichloromethane was added to the reaction solution, and the organic phase was washed with ice water, saturated aqueous sodium hydrogen carbonate solution and saturated saline, and then dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, a crude product of compound 5 (1.53 g) was obtained.

(5) Process 5

The crude product of compound 5 (1.53 g) obtained in step 4 was dissolved in a mixed solvent of 1,4-dioxane (15 mL) and toluene (15 mL), and tris(trimethylsilyl)silane (4.23 mL, 13.7 mmol) was added. Triethylborane (1.0 M n-hexane solution, 1.37 mL, 1.37 mmol) was added, and the mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate=2:1) to give compound 6 (781 mg, 62% (2 steps)) ..

(6) Process 6

Compound 6 (774 mg, 2.82 mmol) was dissolved in methanol (15 mL), a catalytic amount of sodium methoxide (28% methanol solution) was added, and the mixture was reacted at room temperature for 4 hours. The reaction system was neutralized by adding an acidic ion exchange resin (AMBERLITE (registered trademark) IR120H). After the resin was filtered off, the solvent was distilled off under reduced pressure, the resulting residue (471 mg) was dissolved in acetonitrile (50 mL), and benzaldehyde dimethyl acetal (1.25 mL, 8.45 mmol) and (+)-10-camphor sulfone were added. Acid (100 mg, 0.43 mmol) was added and the mixture was stirred at room temperature overnight. The reaction was stopped by adding triethylamine (0.5 mL), and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate=1:1) to obtain compound 7 (523 mg, 78%).

(7) Process 7

Compound 7 (494 mg, 2.09 mmol) was dissolved in dichloromethane (20 mL), Dess-Martin periodinane (976 mg, 2.30 mmol) was added, and the mixture was stirred at room temperature under an argon atmosphere. After 2.5 hours, a saturated sodium hydrogen carbonate aqueous solution (10 mL) and a saturated sodium thiosulfate aqueous solution (10 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 20 minutes. Ethyl acetate was added to the reaction solution, and the obtained organic phase was washed with water, a saturated sodium hydrogen carbonate aqueous solution, a saturated sodium thiosulfate aqueous solution, and a saturated saline solution, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=10:1) to obtain compound 8 (477 mg, 97%).

(8) Process 8

Compound 8 (475 mg, 2.03 mmol) was dissolved in dichloromethane (10 mL), 70% acetic acid aqueous solution (50 mL) was added, and the mixture was stirred at 70°C for 2.5 hr. The solvent was distilled off under reduced pressure, and the obtained residue was separated and purified by silica gel column chromatography (developing solvent chloroform:methanol:water=85:15:1) to give compound 9 and compound 10. 9) (251 mg, 85%) and a crude purified enone form (compound 10) (41 mg) were obtained. The crude purified enone form was dissolved in water, washed with ethyl acetate, the aqueous phase was concentrated, and the resulting residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate=1:2). Then, Compound 10 (25 mg, 10%) was obtained.
3-deoxy form (compound 9)
¹ H NMR (600 MHz, CDCl ₃ ): δ 2.50(dd,1H), 2.98(dd,1H), 3.54-3.59(m,1H), 3.85-3.97(m,2H), 3.99(d,1H), 4.15(d,1H), 4.19-4.25(m,1H).
Enone body (Compound 10)
¹ H NMR (600 MHz, CDCl ₃ ): δ 2.50(dd,1H), 3.78-3.82(m,1H), 3.85-3.88(m,1H), 4.18(dd,1H), 4.34(d,1H), 4.45-4.47(m,1H), 6.24(dd,1H), 7.01(d,1H).

[Example 2] Evaluation of AMPK phosphorylation activity of 1,5-AF derivatives-1
The AMPK phosphorylation activity of the 3-deoxy form of 1,5-AF was evaluated in comparison with 1,5-AF.
Specifically, cultured vascular endothelial cells (Lonza) were seeded at 1×10 ⁶ cells/well in a 6-well dish (Thermo Fisher Scientific). After seeding, it was left standing overnight. Next, change the medium to opti-MEM (Thermo Fisher Scientific), and after 2 hours, add 1,5-AF or the 3-deoxy form of the 1,5-AF derivative (final concentration 0 to 0.1 mg/ml). did. After 1-2 hours, the medium was removed and washed with cold phosphate buffered saline (PBS). After completely removing the cold PBS, 200 μL of protein lysing agent (containing a surfactant and 2-mercaptoethanol) was added. Phosphorylated AMPK was detected by Western blotting using 200 μL of the obtained cell lysate. An anti-human phosphorylated AMPK antibody (CST) was used as the primary antibody. Ez-Capture MG (Ato) was used for detection. ImageJ software was used for band quantification.

The results are shown in Figure 1. The 3-deoxy form of 1,5-AF showed about twice as much AMPK phosphorylation activity as 1,5-AF. Since AMPK is activated by phosphorylation, it was shown that the 3-deoxy form of 1,5-AF has a high AMPK activating ability.

[Example 3] Evaluation of AMPK phosphorylation activity of 1,5-AF derivatives-2
The AMPK phosphorylation activity of 1,5-AF 3-deoxy form and enone form was evaluated.
Specifically, cultured vascular endothelial cells (Lonza) were seeded at 1×10 ⁶ cells/well in a 6-well dish (Thermo Fisher Scientific). After seeding, it was left standing overnight. Next, the medium was replaced with opti-MEM (Thermo Fisher Scientific), and 2 hours later, the 1,5-AF derivative 3-deoxy form or enone form (final concentration 0 to 0.1 mg/ml) was added. After 1-2 hours, the medium was removed and washed with cold phosphate buffered saline (PBS). After completely removing the cold PBS, 200 μL of protein lysing agent (containing a surfactant and 2-mercaptoethanol) was added. Phosphorylated AMPK was detected by Western blotting using 200 μL of the obtained cell lysate. An anti-human phosphorylated AMPK antibody (CST) was used as the primary antibody. Ez-Capture MG (Ato) was used for detection. ImageJ software was used for band quantification. The solvent used in the preparation of the 1,5-AF derivative was used as a control.

The results are shown in Figure 2. The 3-deoxy form of 1,5-AF showed a high phosphorylation activity 1 hour after the initiation of the reaction, and a higher phosphorylation activity 2 hours after the start of the reaction. The 1,5-AF enone also showed phosphorylation activity 1 hour after the start of the reaction, and showed a sharp increase in phosphorylation activity 2 hours later. It was shown that not only the 3-deoxy form of 1,5-AF but also the enone form has a high AMPK activating ability.

[Reference Example 1] Promotion of GLP-1 secretion by ingestion of 1,5-AF
1,5-AF (240 mg) was orally administered to healthy human subjects (healthy volunteers). Blood GLP-1 (glucagon-like peptide-1) of the subject was measured before oral administration and 15 minutes after the oral administration. As an example of the measurement results, the blood GLP-1 concentration before oral administration was 2.7 pmol/L, and the blood GLP-1 concentration after oral administration was 3.8 pmol/L.
As a result of the measurement, GLP-1 secretion promoting activity of 1,5-AF was shown in a plurality of subjects.

[Reference Example 2] Effect of 1,5-AF administration
Normal diet with 1,5-AF (1,5-AF(+)) or normal diet without 1,5-AF (1,5-AF(-)) was fed to healthy rats and raised. , 1,5-AF intake, body weight, fat content, and blood component content were measured over time.
The average 1,5-AF intake (g/day/animal) (the average inter-individual 1,5-AF intake for 7 days of each week) is as follows.

The change in body weight with time is shown in FIG. 3A, and the amount of fat is shown in FIG. 3B. In the 1,5-AF administration group (1,5-AF(+)), the body weight increased while the fat mass decreased. It was considered that administration of 1,5-AF could promote the growth of animals in a healthier state by improving glucose metabolism and suppressing fat accumulation.
The blood analysis results are shown in Table 2.

The administration of 1.5-AF showed an increase in insulin secretion and a decrease in blood glucose level. A decrease in 1,5-AG indicated an improvement in glucose metabolism.

[Reference Example 3] Insulin secretion promoting activity of 1,5-AF administration
1,5-AF (320 mg) was orally administered to healthy human subjects. The fasting insulin level (μU/mL) and fasting blood glucose level (mg/dL) of the subject were measured before the oral administration and 2 hours after the oral administration. HOMA-β was calculated according to the following formula. HOMA-β <30% indicates reduced insulin secretion.
HOMA-β = (fasting insulin level x 360) / (fasting blood glucose level-63)
As an example of the measurement results, HOMA-β before oral administration was 60.7 and HOMA-β after oral administration was 91.4.
It was shown that administration of 1,5-AF promoted insulin secretion.

[Reference Example 4] Insulin secretion promoting activity of 1,5-AF on pancreatic β cells
1,5-AF was added to cultured mouse pancreatic β cells at 1 μg/mL or 10 μg/mL and cultured, and the insulin concentration in the supernatant was measured over time. As a control, the same test was conducted without adding 1,5-AF.
The results are shown in Fig. 4. Administration of 1,5-AF promoted insulin secretion. 1,5-AF was shown to directly stimulate pancreatic β cells and promote insulin secretion via AMPK activation.

[Reference Example 5] Ischemic tolerance imparted by 1,5-AF
1,5-AF (38.5 mg/day/kg body weight) was intraperitoneally administered to healthy rats for 3 consecutive days (pre-administration of 1,5-AF), and then cerebral vascular network ligation occlusion was performed. The result of observing the brain taken out from the rat after death is shown in FIG. The white part seen in the left half of the brain photograph in FIG. 5 is an ischemic cerebral nerve cell necrotic lesion.
As shown in FIG. 5, preadministration of 1,5-AF markedly reduced ischemic cerebral nerve cell necrotic lesions and improved ischemic tolerance.

INDUSTRIAL APPLICABILITY The present invention can provide a drug that brings about an effect of treating or preventing a disease or condition for which AMPK activation is effective, and an effect of promoting energy metabolism of cells based on AMPK activation.

Claims (4)

An AMP-activated protein kinase (AMPK) activator containing a 1,5-anhydrofructose derivative. The AMPK activator according to claim 1, wherein the 1,5-anhydrofructose derivative is a deoxy form or an enone form of 1,5-anhydrofructose. The AMPK activation according to claim 1 or 2, wherein the 1,5-anhydrofructose derivative is 3-deoxy-1,5-anhydrofructose or 6-(hydroxymethyl)-2H-pyran-3-one. Agent. A medicament for treating or preventing a disease or condition for which AMPK activation is effective, comprising the AMPK activator according to any one of claims 1 to 3.