JP7185226B2 - AMPK Activator Containing 1,5-Anhydrofructose Derivative - Google Patents

Description

The present invention relates to AMPK activators comprising 1,5-anhydrofructose derivatives.

AMP-activated protein kinase (AMPK) is a serine-threonine kinase that is activated with decreased intracellular ATP levels. Recently, it has become clear that AMPK plays a central role in cellular stress responses. AMPK functions as a cellular energy sensor in response to increases in the AMP/ATP ratio (decrease in ATP and increase in AMP, etc.) caused by metabolic and physiological stress, and is important as the top regulator of cellular energy homeostasis. playing a role. AMPK positively regulates (enhances) ATP-producing signaling pathways (catabolic pathways) such as glycolysis and fatty acid β-oxidation via phosphorylation of various downstream biological enzymes. It is known that ATP levels are restored by negatively regulating (suppressing) ATP-consuming pathways (anabolic pathways) such as protein biosynthesis.

Activation of AMPK has been reported to bring about various physiological effects, such as suppression of cholesterol biosynthesis, promotion of mitochondrial biosynthesis, enhancement of ischemic tolerance, promotion of autophagy, suppression of cell proliferation, suppression of gluconeogenesis, glucose It is known to have effects such as promoting decomposition and increasing blood flow. There are also various reports on cancer cell growth inhibitory effects due to AMPK activation. AMPK activation is useful in the treatment and treatment of many diseases and conditions, including metabolic syndrome such as hyperlipidemia, hypertension, and obesity, type 2 diabetes, mitochondrial diseases, autophagy-related diseases, aging-related conditions, cancer, and inflammatory diseases. It is thought to be effective in prevention, and the development of treatment/prevention methods targeting AMPK is attracting a great deal of attention worldwide.

Metformin, which is widely used worldwide as an antidiabetic drug, has recently been proven to have an AMPK activating action (Non-Patent Document 1). Non-Patent Document 1 reports the results of adding 1 mM metformin to vascular endothelial cells and examining AMPK phosphorylation after stimulation for 4 hours (see Fig. 2A of Non-Patent Document 1).

1,5-anhydrofructose (1,5-AF) is a monosaccharide that exhibits structural similarity to glucose. Patent Document 1 discloses that 1,5-AF has caspase I activation inhibitory activity. Non-Patent Document 2 reports that 1,5-AF has AMPK phosphorylating 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 capable of effectively activating AMP-activated protein kinase (AMPK).

The present inventors have made intensive studies to solve the above problems, and as a result, have found that certain 1,5-AF derivatives have a high AMPK activation ability, and have completed the present invention.

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

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

FIG. 1 is an electrophoresis 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 the 3-deoxy form of 1,5-AF. A phosphorylated AMPK (p-AMPK) band is observed in the electrophoresis photograph. FIG. 2 is an electrophoresis photograph of Western blot analysis showing AMPK phosphorylation activity of 3-deoxy form and enone form of 1,5-AF. A phosphorylated AMPK (p-AMPK) band is observed in the electrophoresis photograph. FIG. 3 shows changes in body weight (A) and fat mass (B) after administration of 1,5-AF. In A, a diamond symbol indicates 1,5-AF(+) and a square symbol indicates 1,5-AF(-). Figure 4 shows the insulinotropic activity of 1,5-AF in a cultured pancreatic cell line (Min6). Square symbols indicate the 10 μg/ml 1,5-AF addition group, triangle symbols the 1 μg/ml 1,5-AF addition group, and diamond symbols the control group. FIG. 5 is a photograph showing ischemic tolerance induced by preadministration of 1,5-AF. In the leftmost lane, cerebral vascular ligation occlusion was performed without administering 1,5-AF, and in the other 4 lanes on the right side, cerebral vascular ligation occlusion was performed after administration of 1,5-AF. The results of the test brains performed are shown. Each white portion indicates an ischemic necrosis focus, and compared to the left end (control), the ischemic necrosis was reduced by preadministration of 1,5-AF.

The present invention will be described in detail below.
The present invention relates to AMP-activated protein kinase activators containing 1,5-anhydrofructose derivatives. AMP-activated protein kinase (AMPK) is a heterotrimer composed of three subunits, α, β, and γ, and AMPK kinase (AMPKK ) is activated by being phosphorylated by 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 an active ingredient of the AMPK activator has, for example, the following general formula (I) or (II):

で表される化合物であってよい。

It may be a compound represented by

In 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 (the 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. may be substituted with one or more groups selected from an optionally substituted amino group, an alkyl group such as a methyl group, and the like).

R ³ and R ⁴ each independently represent a hydrogen atom or a fluorine atom. Preferably, one of R ³ and R ⁴ is a hydrogen atom and the other is a fluorine atom, or 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 are absent, and only R ² and R ³ are present means that When ---- represents a double bond, it is preferred that ^R2 is a hydrogen atom and ^R3 is a fluorine atom, or both ^R2 and ^R3 are hydrogen atoms.

R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkyl group, or a fluorine atom, but both R ⁵ and R ⁶ are preferably hydrogen atoms. In the present specification, for example, a C _1-6 alkyl group can be used as the alkyl group, and preferably a C _1-4 alkyl group can be used. Alkyl groups can be straight chain, branched chain, cyclic, or combinations thereof. The same applies to the alkyl moieties of other substituents having alkyl moieties (for example, alkylcarbonyl groups).

^R7 represents _-CH2 - ^OR8 , -CHF- ^OR8 , _-CF2 - ^OR8 , a hydrogen atom, an alkyl group, or a trifluoromethyl group, provided that ^R7 is _-CH2 - ^OR8 is preferred. R ⁸ represents a hydrogen atom, an alkyl group, or an acyl group, or R ⁸ combines with R ⁶ to form -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 (the 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. may be substituted with one or more groups selected from an optionally substituted amino group, an alkyl group such as a methyl group, and the like). Preferably, R ⁸ represents a hydrogen atom or an acyl group, or R ⁸ combines with R ⁶ to form -R ⁸ -R ⁶ - to represent a single bond.

A preferred combination is
(a) either 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; - represents a single bond or a double bond, and when ---- represents a double bond, R ¹ and R ⁴ are absent; 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 combines with R ⁶ to form -R ⁸ -R ⁶ - to represent a single bond; and
(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 combines with R ⁶ to become -R ⁸ -R ⁶ - and represents a single bond. can be mentioned.

A more preferred 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; -CH ₂ -OR ⁸ , and R ⁸ represents a hydrogen atom or an acyl group, or R ⁸ combines with R ⁶ to become -R ⁸ -R ⁶ - and represents a single bond;
⁽ d) 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 is _a hydrogen atom; -OR ⁸ and R ⁸ combines with R ⁶ to form -R ⁸ -R ⁶ - to form a single bond. However, preferred combinations are not limited to these.

In general formula (II), R ¹¹ and R ¹² are the same as R ¹ and R ² above. R ¹⁴ represents an acyl group, and as the acyl group, those described as the acyl groups constituting the acyloxy group in the description of R ¹ and R ² can be used. ^R17 and ^R18 are the same as ^R7 and ^R8 .

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

Compounds represented by 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 are May exist. Stereoisomers in pure form, any mixtures of stereoisomers, racemates, etc. may be used as active ingredients of the medicament of the present invention. Regarding two or more stereoisomers resulting from the cyclic structure, any stereoisomer in pure form or any mixture of stereoisomers can be used as the active ingredient of the medicament of the present invention.

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

The compound represented by general formula (I) or (II) clearly does not contain 1,5-anhydrofructose represented by formula (III) below.

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

Specific examples of the 1,5-AF derivative used as an active ingredient of the AMPK activator in the present invention include 3-deoxy-1,5-anhydrofructose represented by the following formula (IV) and and 6-(hydroxymethyl)-2H-pyran-3-one represented.

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

The 1,5-AF derivative according to the present invention can phosphorylate AMPK (specifically, phosphorylate the 172nd threonine from the N-terminal of the α-subunit of AMPK), that is, the ability to activate AMPK. have The AMPK phosphorylation activity (that is, AMPK activation ability) of 1,5-AF derivatives was evaluated by adding 1,5-AF derivatives to vascular endothelial cells and allowing them to react for a certain period of time (for example, 1 to 2 hours). It can be evaluated by quantifying phosphorylated AMPK in cells and comparing it with the quantified value in a negative control tested under the same conditions except that no 1,5-AF derivative was added. Such measurement of the AMPK phosphorylation activity of 1,5-AF derivatives using vascular endothelial cells can be performed, for example, as described in Examples below. If a 1,5-AF derivative increases phosphorylated AMPK compared to a negative control when added to vascular endothelial cells, the 1,5-AF derivative exhibits AMPK phosphorylation activity (i.e., ability to activate AMPK). is determined to have For example, if the quantitative value of phosphorylated AMPK is 20% or higher in the 1,5-AF derivative-treated group compared to the negative control, it can be determined that phosphorylated AMPK has increased compared to the negative control. can.

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

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

The AMPK activator or medicament according to the present invention contains, in addition to the above 1,5-AF derivative, pharmaceutically acceptable additives such as carriers (solid and liquid carriers, etc.), excipients, surfactants, Binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, coloring agents, flavoring agents, preservatives, buffers, pH adjusters, diluents, stabilizers, propellants, etc. It may contain further.

The medicament according to the present invention may be in any dosage form. The medicament according to the present invention includes, for example, solid formulations such as tablets, capsules, powders, and granules; liquid formulations such as emulsions, syrups, solutions, and suspensions; suppositories, inhalants, aerosols, ointments; It may be in the form of creams, gels, patches, transdermal absorbers, transmucosal absorbers, etc., but is not limited to these. The medicament according to the present invention may be for oral or parenteral use.

The amount of the 1,5-AF derivative to be incorporated in the medicament of the present invention can be appropriately determined by those skilled in the art, and can be adjusted according to the dosage form, additives, severity of target disease, and the like. The medicament according to the present invention may contain one type of the above 1,5-AF derivative, or may contain two or more types.

The medicament according to the present invention can be used particularly for the treatment or prevention of diseases or conditions for which AMPK activation is effective. Here, "AMPK activation is effective" means that AMPK activation has a therapeutic or prophylactic effect on the disease or condition. In the present invention, the term "treatment" includes complete cure, remission, delay or inhibition of progression of disease, 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, a disease or condition for which AMPK activation is beneficial can be a disease or condition for which AMPK activation is known to be effective in treating or preventing the disease or condition.

AMPK activation is known to induce various physiological activities. Diseases or conditions for which AMPK activation is effective in the present invention include, but are not limited to, metabolic syndrome (obesity, hypertension, glucose metabolism disorder, hyperglycemia, hyperlipidemia, arteriosclerosis, etc.), type 2 diabetes, aging-related diseases (frailty, osteoporosis, cognitive dysfunction, etc.), mitochondrial diseases (Parkinson's disease, neurodegenerative diseases, Alzheimer's disease, age-related macular degeneration, etc.), autophagy-related diseases, malignant tumors (cancer), Examples include, but are not limited to, inflammatory diseases (nephritis, hepatitis, NASH, fatty liver, etc.), ischemic diseases (cerebral and cardiovascular ischemic diseases), and the like.

The present invention provides the above-described 1,5-AF derivative, an AMPK activator containing the 1,5-AF derivative, or a medicament containing an AMPK activator to cells or subjects in vitro or in vivo. , also provides a method for activating AMPK. The present invention also provides a method in which AMPK activation is effective by administering to a subject the 1,5-AF derivative, an AMPK activator containing the 1,5-AF derivative, or a medicament containing the AMPK activator. Methods of treating or preventing diseases or conditions are also provided. "Disease or condition for which AMPK activation is effective" and "treatment or prevention" are as described above.

The dosage of the above 1,5-AF derivative is not particularly limited, and depends on various factors that should be considered normally, such as the patient's body weight, age, medical history, etc., the purpose of prevention or treatment, the type and symptoms of the disease, and the route of administration. , can be increased or decreased as appropriate. The 1,5-AF derivative, the AMPK activator containing the 1,5-AF derivative, or the drug containing the AMPK activator may be administered once or multiple times at intervals of several hours to several months. A single dose may be administered.

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

Subjects to be administered the 1,5-AF derivative, the AMPK activator containing the 1,5-AF derivative, or the medicament containing the AMPK activator according to the present invention are not limited to, but are not limited to, human , domestic animals (horses, cows, sheep, goats, pigs, etc.), pet animals (dogs, cats, rabbits, etc.), laboratory animals (mice, rats, monkeys, etc.), etc. good. In one embodiment, the subject to be administered may be a subject suffering from or suspected of suffering from a disease or condition in which AMPK activation is beneficial. In one embodiment, the subject to whom AMPK activation is effective may be a subject that has a predisposition or is predisposed to a disease or condition in which AMPK activation is beneficial.

The isolated cell, tissue, or organ to which the 1,5-AF derivative, the AMPK activator containing the 1,5-AF derivative, or the pharmaceutical containing the AMPK activator according to the present invention is administered is particularly limited. It may be a cell, tissue or organ derived from, for example, the subject. Cells, tissues, or organs to be administered may be derived from diseased or condition-affected areas (eg, tumors, inflammatory sites, degenerative sites, vascular endothelial cells, etc.) in which AMPK activation is effective.

According to the method of the present invention, AMPK can be strongly activated, thereby treating or preventing diseases or conditions for which AMPK activation is effective.

EXAMPLES The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Synthesis of 1,5-AF derivatives
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 overnight under reflux. After distilling off the solvent under reduced pressure, the resulting 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", i.e. yield near 100%.

(2) Process 2

Compound 2 (3.50g, 9.45mmol), tris(trimethylsilyl)silane (5.83mL, 18.9mmol) and azobisisobutyronitrile (307mg, 1.89mmol) were dissolved in toluene (50mL). Stirred at 100° C. for 2 hours in an argon atmosphere. The reaction solution was directly charged to 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. After the reaction solution was neutralized with sodium hydrogencarbonate (1.85 g), the solvent was distilled off under reduced pressure. Pyridine (40 mL) and acetic anhydride (20 mL) were added to the resulting residue, and the mixture was stirred overnight at room temperature. After adding methanol to stop the reaction, the solvent was distilled off under reduced pressure, the resulting residue was dissolved in ethyl acetate, and the ethyl acetate phase was washed with water, 1M hydrochloric acid, saturated aqueous sodium hydrogencarbonate solution, and saturated brine. It was then dried over magnesium sulfate. After distilling off 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% hydrobromide in acetic acid (9 mL) was added, and the mixture was stirred at room temperature for 2.5 hours. Dichloromethane was added to the reaction solution, and the organic phase was washed with ice water, saturated aqueous sodium hydrogencarbonate solution and saturated brine, and 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), tris(trimethylsilyl)silane (4.23 mL, 13.7 mmol) and Triethylborane (1.0M n-hexane solution, 1.37mL, 1.37mmol) was added and stirred overnight at room temperature. The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate=2:1) to obtain 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. An acidic ion exchange resin (AMBERLITE® IR120 H) was added to neutralize the reaction. 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 treated with benzaldehyde dimethylacetal (1.25 mL, 8.45 mmol) and (+)-10-camphorsulfone. Acid (100 mg, 0.43 mmol) was added and stirred overnight at room temperature. Triethylamine (0.5 mL) was added to stop the reaction, and the solvent was distilled off under reduced pressure. The resulting 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, saturated aqueous sodium hydrogencarbonate solution (10 mL) and saturated aqueous sodium thiosulfate 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 resulting organic phase was washed with water, saturated aqueous sodium hydrogencarbonate solution, saturated aqueous sodium thiosulfate solution and saturated brine, and dried over magnesium sulfate. The solvent was distilled off 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% aqueous acetic acid solution (50 mL) was added, and the mixture was stirred at 70°C for 2.5 hours. The solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel column chromatography (developing solvent chloroform:methanol:water = 85:15:1) to separate and purify compound 9 and compound 10 to give the 3-deoxy form (compound 9) (251 mg, 85%) and a crude product (41 mg) of the enone form (compound 10) were obtained. Dissolve the crudely purified enone in water, wash with ethyl acetate, concentrate the aqueous phase, and purify the resulting residue by silica gel column chromatography (developing solvent n-hexane:ethyl acetate=1:2). to give compound 10 (25 mg, 10%).
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 form (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 in a 6-well dish (Thermo Fisher Scientific) at 1×10 ⁶ cells/well. After seeding, it was allowed to stand overnight. Next, the medium was replaced with opti-MEM (Thermo Fisher Scientific), and after 2 hours, 1,5-AF or 3-deoxy derivative of 1,5-AF (final concentration 0-0.1mg/ml) was added. did. Media was removed after 1-2 hours and washed with cold phosphate-buffered saline (PBS). After completely removing the cold PBS, 200 μL of protein dissolving agent (including surfactant and 2-mercaptoethanol) was added. Using 200 μL of the obtained cell lysate, phosphorylated AMPK was detected by Western blotting. An anti-human phosphorylated AMPK antibody (CST) was used as the primary antibody. Ez-Capture MG (ATTO) was used for detection. ImageJ software was used for band quantification.

The results are shown in FIG. The 3-deoxy form of 1,5-AF showed about twice the AMPK phosphorylation activity as compared to 1,5-AF. Since AMPK is activated by phosphorylation, the 3-deoxy form of 1,5-AF was shown to have a high ability to activate AMPK.

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

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

[Reference Example 1] Promotion of GLP-1 secretion by oral intake of 1,5-AF
1,5-AF (240 mg) was administered orally to healthy human subjects (healthy volunteers). Blood GLP-1 (glucagon-like peptide-1) in the subjects was measured before oral administration and 15 minutes after 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.
Measurements showed GLP-1 secretagogue activity of 1,5-AF in multiple subjects.

[Reference Example 2] Effects of administration of 1,5-AF
Normal diet with 1,5-AF (1,5-AF(+)) or normal diet without 1,5-AF (1,5-AF(-)) was fed to healthy rats. , 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) (average 1,5-AF intake among individuals for 7 days each week) is as follows.

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

Administration of 1.5-AF was shown to increase insulin secretion and lower blood glucose levels. A decrease in 1,5-AG indicated an improvement in glucose metabolism.

[Reference Example 3] Insulin secretagogue activity of 1,5-AF administration
1,5-AF (320 mg) was administered orally to healthy human subjects. Fasting insulin levels (μU/mL) and fasting blood glucose levels (mg/dL) of the subjects were measured before oral administration and 2 hours after oral administration. HOMA-β was calculated according to the following formula. HOMA-β <30% indicates decreased insulin secretion.
HOMA-β = (fasting insulin level x 360) / (fasting blood sugar 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 secretagogue 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, cultured, and the insulin concentration in the supernatant was measured over time. A similar test was performed without adding 1,5-AF as a control.
The results are shown in FIG. Administration of 1,5-AF stimulated insulin secretion. 1,5-AF was shown to directly stimulate pancreatic β-cells and promote insulin secretion via AMPK activation.

[Reference Example 5] Conferring ischemic resistance by 1,5-AF
1,5-AF (38.5 mg/day/kg body weight) was intraperitoneally administered to healthy rats for 3 consecutive days (preadministration of 1,5-AF), followed by cerebral vascular ligation and occlusion. FIG. 5 shows the results of observation of brains removed from rats after death. The white portion seen in the left half of the photograph of the brain in FIG. 5 is the ischemic cerebral nerve necrosis focus.
As shown in FIG. 5, pre-administration of 1,5-AF significantly reduced ischemic cerebral necrotic foci and improved ischemic tolerance.

INDUSTRIAL APPLICABILITY The present invention can provide therapeutic or prophylactic effects for diseases or conditions for which AMPK activation is effective, and drugs that provide cellular energy metabolism promoting effects based on AMPK activation.

Claims (1)

AMP-activated protein kinase (AMPK) activators, including 3-deoxy-1,5-anhydrofructose or 6-(hydroxymethyl)-2H-pyran-3-one .

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