JP2010083841A - Cell death inhibitory substance characterized by containing 2-(4-oxo-3-phenyl-selenazolidin-2-ylidene)-malononitrile analog - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance having cell death inhibitory activity similar to that of NGF, and having the cell death inhibitory effect, a cell differentiation-inducing effect, the neural spine-elongating effect of nerve cells and further the actions of preventing or curing traumatic injury, injury caused by a metabolic factor, injury caused by a β-amyloid protein or cerebral ischemic injury. <P>SOLUTION: This cell death inhibitory substance is characterized by containing the 2-(4-oxo-3-phenyl-selenazolidin-2-ylidene)-malononitrile analog prepared by a known method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention contains a 2- (4-oxo-3-phenyl-selenazolidin-2-ylidene) -malononitrile analog. The present invention relates to a cell death inhibitor characterized by the following. Further, the cell death inhibitor of the present invention has a cell differentiation-inducing action, a neuronal neurite outgrowth action, and further, a traumatic disorder of brain neurons, a disorder due to metabolic factors, a disorder due to β-amyloid protein, or a brain imagination. Has the effect of preventing or curing blood disorders. Furthermore, a pharmaceutical composition or an edible composition containing the cell death inhibitor of the present invention as an active ingredient can be prepared.

  Until now, as a method for preventing diseases that cause cell death such as Alzheimer's disease, a technique using curcumin, cholic acid, or simnole (for example, Patent Document 1), a technique using arachidonic acid or a medium chain fatty acid (for example, Patent Document 2), mugwort And technology using, for example, Patent Document 3, technology using theanine (for example, Patent Document 4), technology using L-carnitine or alkanoyl (for example, Patent Document 5), technology using docosahexaenoic acid (for example, Patent Documents 6 and 7). ) And techniques using isoflavonoids (for example, Patent Documents 8 and 9) are known.

On the other hand, the prior art regarding the cell death inhibitory action of 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analogue is not known.
JP 2003-113117 A JP 2003-48831 A JP-A-10-276719 JP 7-173059 A Special table 2002-527389 Special table 2002-527387 JP 2002-58450 A JP-A-11-318387 JP-A-9-16962

  However, the above-described methods for preventing diseases that cause cell death such as Alzheimer's disease are not effective enough, and their practical application is delayed. Moreover, the prior art which utilizes 2- (4-oxo-3-phenyl selenazolidin-2-idene) -malononitrile analog as a cell death inhibitor is not known.

  Therefore, the present invention has a cell death inhibitory action similar to that of NGF, a cell differentiation inducing action, a neuronal neurite outgrowth action, a traumatic disorder of brain neurons, a disorder due to metabolic factors, and a β-amyloid protein Provided is a material having an effect of preventing or curing a disorder or cerebral ischemic disorder.

The present inventors have found that an extract containing 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog suppresses cell death, and has completed the present invention. . That is, the present invention includes the following [1] to [6].
[1] A cell death inhibitor comprising a 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog.
[2] 2- (4-Oxo-3-phenylselenazolidine-2-idene) -malononitrile analog is 3- (2,6-dimethyl-phenyl) -2-selenoxo-thiazolid 2-{-3- ( 4-methoxyphenyl) -4-oxo-selenazolidine-2-lidene} malononitrile, 2-{-3- (2,6-dimethylphenyl) -4-oxo-selenazolidine-2-lidene} malononitrile], [2- { -3- (4-fluorophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile], [2-{-3- (4-chlorophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile], [ 2-{-3- (4-Bromophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile], which contains one or more of [1] Of cell death inhibitors.
[3] The cell death inhibitor according to [1] or [2], which has an action of promoting phosphorylation of Akt, which is a signal transduction protein.
[4] The cell death inhibitor according to [1] or [2], which has an action of promoting phosphorylation of mitogen-activated kinase, which is a signal transduction protein.
[5] A pharmaceutical composition comprising the cell death inhibitor according to [1] to [4] as an active ingredient.
[6] An edible composition comprising the cell death inhibitor according to [1] to [4] as an active ingredient.

  That is, the present invention relates to a cell death inhibitor comprising 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog and a method for using the same.

  According to the present invention, a cell death-inhibiting action, a cell differentiation-inducing action, a neuronal cell comprising 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog It is possible to provide a cell death inhibitory substance having an action of preventing or curing neurite outgrowth action of cerebral nerve cells, traumatic damage of brain neurons, damage caused by metabolic factors, damage caused by β-amyloid protein, or cerebral ischemic damage.

Hereinafter, the present invention will be described in detail. The 2- (4-oxo-3-phenyl-selenazolidin-2-ylidene) -malononitrile analog used in the present invention is: It can be obtained by stirring various malononitriles in the presence of a base (such as triethylamine), adding various isoselenocyanates, and further reacting with various halogenated compounds.
Further, as a purification method, it can be obtained by a method using conventional chromatography or recrystallization method.

  In the present invention, 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog is used as 2-{-3- (4-methoxyphenyl) -4-oxo-selenazolidine-2-lidene. } Malononitrile, 2-{-3- (2,6-dimethylphenyl) -4-oxo-selenazolidine-2-lidene} malononitrile], [2-{-3- (4-fluorophenyl) -4-oxo-selenazolidine -2-Lidene} malononitrile], [2-{-3- (4-Chlorophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile], [2-{-3- (4-bromophenyl) -4- One or more of oxo-selenazolidine-2-lidene} malononitrile] can be used. Of the above 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analogues, 2-{-3- (4-methoxyphenyl) -4-oxo-selenazolidine-2-lidene} Since the specific activity of malononitrile is the highest, it is most suitable for the use of the present invention.

  As the mechanism of action of the cell death inhibitor of the present invention, 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog activates a receptor on the cell membrane, and then signals cell survival. The mitogen-activated kinase pathway and the PI3K / Akt pathway, which are pathways, are activated, and finally cell death is suppressed through suppression of cytochrome C leakage from mitochondria, suppression of caspase activation, and the like.

  The cell death-suppressing substance of the present invention comprises 0.01 to 50%, preferably 0.1 to 30%, more preferably 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analog. Contains 1 to 10%. When the content of 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile is less than 0.01%, the cell death inhibitory effect is not observed. Also. Even if the content of 2- (4-oxo-3-phenylselenazolidine-2-idene) -malononitrile analogue is more than 50%, no significant increase in activity is observed.

  Next, a pharmaceutical composition and an edible composition comprising the cell death inhibitor of the present invention will be described. A preparation comprising the cell death inhibiting substance of the present invention can be administered to animals and humans as it is or as a pharmaceutical composition together with a conventional pharmaceutical preparation carrier. The dosage form of the pharmaceutical composition is not particularly limited, and may be appropriately selected according to need. For example, tablets, capsules, granules, fine granules, powders and other oral preparations, injections And parenterals such as suppositories and suppositories.

  In the present invention, oral preparations such as tablets, capsules, granules, fine granules, and powders are produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like. . The compounding amount of the cell death inhibitor of the present invention in these preparations is 0.01 to 50%, preferably 0.1 to 30%, more preferably 1 to 10%. When the content of the cell death inhibitor is less than 0.01%, the cell death inhibitory activity is not observed. Also. Even if the content of the cell death inhibitor is more than 50%, no significant increase in activity is observed. In addition to the cell death inhibitor of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance, and the like are appropriately used for this type of preparation. Can do.

  The above-mentioned pharmaceutical composition containing the cell death inhibitor can be administered as a suspension, emulsion, syrup, or elixir, and these various dosage forms contain a flavoring agent and a coloring agent. You may let them.

  If the pharmaceutical composition and edible composition of the present invention are used for the purpose of preventing or curing cell death, they are applied without any limitation on the use thereof.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely using an Example, this invention is not limited to this.

Experimental Example 1 2- (4-oxo-3-phenylselenazolidin-2-idene) -malononitrile (2- (4-Oxo) by reaction with malononitrile, phenyl isoselenocyanate and methyl 2-chloroacetate in the presence of triethylamine -3-phenyl-selenazolidin-2-ylidene) -malononitrile)
Triethylamine (0.15 mL, 1.1 mmol) was added to malononitrile (73 mg, 1.1 mmol) dissolved in DMF (10 mL), and the mixture was stirred at room temperature for 30 minutes. Phenyl isoselenocyanate (200.3 mg, 1.1 mmol) was added and stirred at room temperature for 1 hour. Thereafter, methyl 2-chloroacetate (119.4 mg, 1.1 mmol) was gradually added and stirring was continued for another 4 hours. After completion of the reaction, the residue was purified by silica gel column chromatography (eluent: hexane-ethyl acetate), and further recrystallized from ethyl acetate to obtain 235 mg of the target compound in a yield of 74%.

  PC-12 cells (distributed from Gifu Pharmaceutical University) are cultured at a concentration of 50,000 cells / mL in DMEM medium (Nissui Pharmaceutical) containing 10% horse serum and 5% fetal bovine serum, and serum free 24 hours later The DMEM medium was replaced. Further, after 24 hours, 2-{-3- (4-methoxyphenyl) -4-oxo-selenazolidine-2-lidene} malononitrile was added at 100 or 500 ng / mL, and the number of viable cells after 4 days was determined under an inverted microscope. did. As a positive target, 25 ng / mL of NGFβ (manufactured by Sigma) was added and the same operation was performed. The results are shown in Table 1. The symbols in the table represent the number of viable cells after 4 days of culture, and mean-: no live cells, +: some dead cells, ++ and ++: no dead cells.

  Cerebral neurons were collected from 18-day-old rat fetuses by a conventional method, and cultured for 24 hours in DMEM medium (Nissui Pharmaceutical) containing 10% horse serum and 5% fetal bovine serum. After 24 hours, the serum-free DMEM medium was replaced, and after 24 hours, 100 or 500 ng / mL of 2-{-3- (2,6-dimethylphenyl) -4-oxo-selenazolidine-2-lidene} malononitrile] was added. did. After 30 seconds, cytoplasmic proteins of cerebral neurons were collected with a lysis buffer. The collected proteins were separated by SDS-PAGE, and the proteins were blotted onto a nylon membrane. After blocking, the reaction with an anti-phosphorylated Akt rabbit IgG antibody was followed by treatment with an avidin-biotinylated horseradish peroxidase complex. The phosphorylation of Akt was measured by visualizing the phosphorylated Akt with peroxidase activity using diaminobenzidine-H2O2. As a positive target, 25 ng / mL of NGFβ (manufactured by Sigma) was added and the same operation was performed. The results are shown in Table 2. The symbols in the table mean-: none, +: yes, ++ and ++: notably about phosphorylation of Akt.

A 96-well plate was seeded with 50 μl, that is, 2 × 10 4 cells / well, of 4 × 10 5 cells / ml PC-12 cells (distributed from Gifu Pharmaceutical University). After culturing at 37 ° C. in the presence of 5% carbon dioxide gas for 2 days, the culture solution was removed, and the cells adhering to the bottom of the well were washed with PBS. 2-{-3- (4-methoxyphenyl) -4-oxo-selenazolidine- 50 μl of serum-free medium supplemented with 100 or 500 ng / mL of 2-redene} malononitrile was added. After culturing for a predetermined time (0, 24, 48 hours), 6.3 μl of an 8 mg / ml solution of MTT was added, incubated at 37 ° C. for 2 hours, and then 50 μl of lysis buffer was added to lyse the cells. After overnight incubation at 37 ° C., the absorbance at 562 nm was measured using a microplate reader (Amersham).
OD562 (degree of staining) was determined and used as an index of the number of viable cells. The results are shown in FIG.

(Comparative example 1) Cell death inhibitory effect was measured by the method of Example 1 with respect to mugwort extract (manufactured by Ichimaru Falcos), theanine (manufactured by Taiyo Kagaku), and docosahexaenoic acid (manufactured by NOF Corporation). did. The results are shown in Table 1.

(Comparative Example 2) Cell death inhibitory action was measured by the method of Example 2 with respect to mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), and docosahexaenoic acid (Nippon Yushi Co., Ltd.). did. The results are shown in Table 2.

(Comparative Example 3) The cell death inhibitory effect was measured by the method of Example 3 without additives. The results are shown in FIG.

  As shown in Tables 1 and 2 and FIG. 1, the cell death inhibitor of the present invention is a cell far superior to mugwort extract, theanine, and docosahexaenoic acid, which has been conventionally said to have a cell death inhibitory effect. Death-inhibiting action is observed. Moreover, it had the cell death inhibitory effect equivalent to NGF made into the positive object.

  The present invention can be used not only for humans but also for animals such as livestock.

It is explanatory drawing which compared the cell death inhibitory substance of this invention, the positive control, and the additive-free cell death inhibitory effect. (Example 3, Comparative Example 3)

Claims (6)

  Characterized by containing 2- (4-oxo-3-phenyl-selenazolidin-2-ylidene) -malononitrile analogue. Cell death inhibitor.   The 2- (4-oxo-3-phenylselenazolidin-2-ylidene) -malononitrile analog is 2-{-3- (4-Methoxyphenyl) -4-oxo-selenazolidine-2-lidene} malononitrile 2- [3- (4-Methoxy-phenyl) -4-oxo-selenazolidin-2-ylidene] -malononitrile, 2-{-3- (2,6-Dimethylphenyl) -4-oxo-selenazolidin-2-lidene} malononitrile] 2- [3- (2,6-Dimethyl-phenyl) -4-oxo-selenazolidin-2-ylidene] -malononitrile, [ 2-{-3- (4-fluorophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile] 2- [3- (4-Fluoro-phenyl) -4-oxo-selenazolidin-2-ylidene] -malononitrile , [2-{-3- (4-Chlorophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile] 2- [3- (4-Chloro-phenyl) -4-oxo-s elenazolidin-2-ylidene] -malononitrile, [2-{-3- (4-bromophenyl) -4-oxo-selenazolidine-2-lidene} malononitrile] 2- [3- (4-Bromo-phenyl) -4- The cell death inhibitor according to claim 1, comprising at least one of oxo-selenazolidin-2-ylidene] -malononitrile.   The cell death inhibitor according to claim 1, which has an action of promoting phosphorylation of Akt, which is a signal transduction protein.   The cell death inhibitor according to claim 1, which has an action of promoting phosphorylation of a mitogen-activated kinase that is a signal transduction protein.   The pharmaceutical composition formed by mix | blending the cell death inhibitor of Claims 1-4 as an active ingredient.   An edible composition comprising the cell death inhibitor according to claim 1 as an active ingredient.

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