JP2008100954A - Neurotrophic factor and its utilizing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material having neurotrophic action and its applied products. <P>SOLUTION: The neurotrophic factor and its applied products include a 5-acyl-2-amino-1,3-selenazole analog represented by the structural formula of the figure. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention relates to a neurotrophic factor having a 5-acyl-2-amino-1,3-selenazole analog as an active ingredient, for example, as an anti-dementia agent, a therapeutic agent for peripheral neuropathy, a nerve cell protective agent, etc. Expected to be used as Furthermore, it is related with the pharmaceutical composition and edible composition which use the neurotrophic factor of this invention as an active ingredient.

を有する化合物であり、５−クロロアセチル−２−ジメチルアミノ−１，３−セレナゾール、５−クロロアセチル−２−ピペリジノ−１，３−セレナゾール、５−クロロアセチル−２−モルフォリノ−１，３−セレナゾールの構造式は、

である。
セレナゾール類縁体の工業的な利用法として、写真感光材料（特許文献１）、アポトーシスに起因する脳細胞死の抑制薬（特許文献２）、シクロオキシゲナーゼ阻害剤（特許文献３）、脳動脈中膜肥厚抑制剤（特許文献４）、喘息治療剤（特許文献５）、抗癌性組成物（特許文献６）、経皮的冠動脈形成術後の再狭窄抑制剤（特許文献７）、難聴を治療する方法（特許文献８）、局所コルチコステロイドを用いた包茎の治療のための医薬組成物（特許文献９）、チオレドキシン・レダクターゼ基質（特許文献１０）、アルツハイマー病予防・治療剤（特許文献１１）、後天性免疫不全症候群治療剤（特許文献１２）として有用であることが知られている。 The 5-acyl-2-amino-1,3-selenazole analog has the following structural formula

5-chloroacetyl-2-dimethylamino-1,3-selenazole, 5-chloroacetyl-2-piperidino-1,3-selenazole, 5-chloroacetyl-2-morpholino-1,3- The structural formula of selenazole is

It is.
Industrial uses of selenazole analogs include photographic materials (Patent Document 1), inhibitors of brain cell death caused by apoptosis (Patent Document 2), cyclooxygenase inhibitors (Patent Document 3), cerebral artery medial thickness Inhibitor (Patent Document 4), Asthma therapeutic agent (Patent Document 5), Anticancer composition (Patent Document 6), Restenosis inhibitor after percutaneous coronary angioplasty (Patent Document 7), Treating hearing loss Method (patent document 8), pharmaceutical composition for treatment of phimosis using topical corticosteroid (patent document 9), thioredoxin reductase substrate (patent document 10), Alzheimer's disease preventive and therapeutic agent (patent document 11) It is known to be useful as a therapeutic agent for acquired immune deficiency syndrome (Patent Document 12).

So far, as a method for preventing diseases such as depression, stress, Alzheimer's disease, etc., a technique using curcumin, cholic acid, and simnole (for example, Patent Document 13), a technique using arachidonic acid and medium chain fatty acid (for example, Patent Document 14), mugwort, Technology using Kumazasa (for example, Patent Document 15), Technology using theanine (for example, Patent Document 16), Technology using L-carnitine or alkanoyl (for example, Patent Document 17), Technology using docosahexaenoic acid (for example, Patent Documents 18 and 19) Techniques using isoflavonoids (for example, Patent Documents 20 and 21) are known.

On the other hand, nerve growth factor (hereinafter referred to as NGF) is known as a typical neurotrophic factor. NGF has an action of regulating neurite outgrowth and production of neurotransmitters, and has been demonstrated in vitro to have a regenerative action on nerve cells of old animals. However, NGF has no permeability to the cerebrovascular barrier (hereinafter referred to as BBB), so it cannot be transferred into the brain from the periphery or by oral administration, and it is practical for living organisms even though it is effective for cultured cells. It has become clear that it is not. Therefore, unlike NGF, a compound having a BBB permeation action is required as an anti-dementia agent, a peripheral neuropathy therapeutic agent, or a nerve cell protective agent.
JP 2006-119428 A JP 2000-344663 A JP 2000-16935 A JP 2001-261555 A JP-A-9-263533 JP-A-6-172330 JP-A-5-255084 Japanese translation of PCT publication No. 2005-516028 Special table 2004-519451 gazette WO00 / 58281 WO 98/08831 WO96 / 33712 WO96 / 33712 JP 2003-48831 A JP-A-10-276719 JP 7-173059 A Special table 2002-527389 Special table 2002-527387 Special table flat 8-511533 Special table 2001-511117 Special table 2001-500480 JP2003-2813A JP 2002-58450 A JP-A-11-318387 JP-A-9-16962 Biochemical pharmacology; Vol. 33, No. 20, 3235-3239 (1984) Biochemical pharmacology; Vol.33, No.20, 3241-3245 (1984) I. Gay et al. Biochim Biophys Acta 1684, 18-28, 2004 M. Koketsu et al. Synthesis, 31-36, 2006.

That is, the neurotrophic action of the known technique is insufficient in practical use and is delayed in practical use. Also, the neurotrophic action of 5-acyl-2-amino-1,3-selenazole analogs, ie, neurite outgrowth action, apoptosis inhibitory action, mitogen-activated protein kinase kinase (MEK1 / 2) and mitogen activation of neurons Prior art using the phosphorylation promoting action and the neurofilament expression enhancing action of protein kinase (ERK1 / 2) is not known.

Therefore, the present invention provides a material having a neurotrophic action.

The present inventors have found that 5-acyl-2-amino-1,3-selenazole analogs have a neurotrophic effect and have completed the present invention. That is, the present invention includes the following [1] to [9].
[1] A neurotrophic factor comprising a 5-acyl-2-amino-1,3-selenazole analog.
[2] 5-acyl-2-amino-1,3-selenazole analog is 5-chloroacetyl-2-dimethylamino-1,3-selenazole, 5-chloroacetyl-2-piperidino-1,3-selenazole, The neurotrophic factor according to the above [1], which is one or a mixture of two or more of 5-chloroacetyl-2-morpholino-1,3-selenazole.
[3] The neurotrophic factor according to the above [1] or [2], which has an action of promoting phosphorylation of a mitogen-activated protein kinase (ERK1 / 2) in a nerve cell.
[4] The neurotrophic factor according to [1] or [2] above, which has an action of promoting phosphorylation of mitogen-activated protein kinase kinase (MEK1 / 2) in nerve cells.
[5] The neurotrophic factor according to [1] or [2] above, which has an action of promoting expression of a protein (neurofilament) of a neuronal cell.
[6] The neurotrophic factor according to [1] or [2] above, which has an action of suppressing apoptosis of nerve cells.
[7] The neurotrophic factor according to [1] or [2] above, which has an action of promoting neurite outgrowth of nerve cells.
[8] A pharmaceutical composition comprising the neurotrophic factor according to [1] to [7] as an active ingredient.
[9] An edible composition comprising the neurotrophic factor according to [1] to [7] as an active ingredient.

That is, the present invention relates to a neurotrophic factor characterized by containing a 5-acyl-2-amino-1,3-selenazole analog and a method for using the same.

By taking the neurotrophic factor of the present invention as a food or a medicine, an effect as an anti-dementia agent, a peripheral neuropathy treatment agent, a nerve cell protective agent, or the like can be expected.

Hereinafter, the present invention will be described in detail. The 5-acyl-2-amino-1,3-selenazole analog used in the present invention is a fat-soluble organic selenium compound, and a 5-membered ring compound containing selenium element and nitrogen element at the 1-position and 3-position. It is. The 5-acyl-2-amino-1,3-selenazole analog used in the present invention can be obtained by chemical synthesis by a method described in the literature (Non-Patent Document 4).

  The nerve cell in the present invention is a so-called neuron, which is roughly divided into cell body, dendrite, axon and synapse. The neurotrophic action in the present invention includes neuronal neurite outgrowth action, neuronal apoptosis inhibitory action, neuronal mitogen-activated protein kinase kinase (MEK1 / 2) and mitogen-activated protein kinase (ERK1 / 2) phosphorous. It refers to the action of promoting oxidation and enhancing neurofilament expression in neurons.

As a mechanism of action of the neurotrophic factor of the present invention, a 5-acyl-2-amino-1,3-selenazole analog activates a cell membrane receptor, and then a mitogen-activated kinase pathway which is a signal transduction pathway of cell survival. And a protein that induces neuronal differentiation such as neurofilament and MAP2 is expressed and finally induces neuronal differentiation.

The neurotrophic factor of the present invention contains 5-acyl-2-amino-1,3-selenazole analogs 0.01 to 50%, preferably 0.1 to 30%, more preferably 1 to 10%. When the content of 5-acyl-2-amino-1,3-selenazole analog is less than 0.01%, no neurotrophic action is observed. Also. Even if the 5-acyl-2-amino-1,3-selenazole analog content is more than 50%, no significant increase in activity is observed.

Next, a pharmaceutical composition and an edible composition comprising the neurotrophic factor of the present invention will be described. The preparation comprising the neurotrophic factor 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 conventional methods using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, and the like. . The compounding amount of the neurotrophic factor of the present invention in these preparations is 0.01 to 50%, preferably 0.1 to 30%, more preferably 1 to 10%. If the content of neurotrophic factor is less than 0.01%, no neurotrophic activity is observed. Also. Even if the content of neurotrophic factor is more than 50%, no significant increase in activity is observed. In addition to the neurotrophic factor of the present invention, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a coloring agent, a fragrance and the like may be appropriately used for this type of preparation. it can.

The pharmaceutical composition containing the above-mentioned neurotrophic factor can also be administered as a suspension, emulsion, syrup, or elixir. These various dosage forms contain a flavoring agent and a coloring agent. May be.

The neurotrophic factor of the present invention can also be used as an edible composition. That is, a neurotrophic factor comprising the 5-acyl-2-amino-1,3-selenazole analog obtained as described above as an active ingredient is used as it is in a liquid, gel or solid food such as juice, Soft drinks, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc. or dextrin as needed It can be processed into pellets, tablets, granules, etc. together with excipients such as lactose and starch, fragrances, pigments, etc., or coated with gelatin and formed into capsules for use as health foods, dietary supplements, and the like.

The compounding amount of the neurotrophic factor of the present invention in these foods or edible compositions is difficult to define uniformly depending on the type and condition of the food or composition, but is preferably about 0.01 to 50% by weight, more preferably Is 0.1 to 30% by weight. If the blending amount is less than 0.01% by weight, the desired effect by oral intake is small, and if it exceeds 50% by weight, the flavor may be impaired or the food may not be prepared depending on the type of food. In addition, if the raw material is a foodstuff, the neurotrophic factor of this invention can use this as it is for food.

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.

Production Examples and Properties of 5-Chloroacetyl-2-dimethylamino-1,3-selenazol N, N-Dimethyl-N '-(dimethylaminoselenocarbonyl) formamidine (63 mg, 0.3 mmol) ) In methanol solution (5 ml) and 1,3-dichloroacetone (197
mg, 1.5 mmol) was added and refluxed for 2 hours. The completion of the reaction was confirmed by TLC (ethyl acetate), and the mixed solution was extracted with dichloromethane, and then the solvent was distilled off. This was isolated and purified by silica gel column chromatography (ethyl acetate: hexane = 1: 5), and yellow. A white solid of 5-chloroacetyl-2-dimethylamino-1,3-selenazole (49 mg, 64%) was obtained.
Physical properties: Mp: 102-104 ° C, IR
(KBr): 1655 cm ^-1 , ¹ H NMR (500 MHz, CDCl _3, rt):
d
3.21 (br. S, 6H, N-CH ₃ ), 4.45 (s, 2H, CH ₂ ), 7.91 (s, 1H,
CH), ¹³ C NMR (125 MHz, CDCl _3, rt): d 44.2, 130.3,
150.6, 178.9, 183.7, ¹³ C NMR (125 MHz, CDCl _3, -40 ° C): d 39.3, 43.9,
44.5, 129.6, 150.6, 178.7, 183.5, ⁷⁷ Se NMR (95 MHz, CDCl ₃ ):
d 527.5,
MS (CI): m / z = 253 [M ⁺ +1], Anal. Calcd for C ₇ H ₉ ClN ₂ OSe:
C, 33.42; H, 3.61; N, 11.14.Found; C, 33.63; H, 3.86; N, 10.84.

Production Examples and Properties of 5-Chloroacetyl-2-piperidino-1,3-selenazol N, N-Dimethyl-N '-(piperidinoselenocarbonyl) formamidine (74 mg, 0.3 mmol) ) In methanol solution (3 ml) and 1,3-dichloroacetone (195
mg, 1.5 mmol) was added and refluxed for 3 hours. The completion of the reaction was confirmed by TLC (ethyl acetate), the mixed solution was extracted with dichloromethane, and then the solvent was distilled off. This was isolated and purified by silica gel column chromatography (ethyl acetate: hexane = 1: 5), and white. Solid 5-chloroacetyl-2-piperidino-1,3-selenazole (62 mg, 70%) was obtained.
Physical properties: Mp: 112-114 ° C, IR (KBr): 1648 cm ^-1 , ¹ H NMR
(500 MHz, CDCl _3, rt): d 1.72 (m, 6H, CH ₂ ), 3.60 (m, 4H, CH ₂ ),
4.44 (s, 2H, CH ₂ ), 7.87 (s, 1H, CH), ¹ H NMR (500 MHz,
CDCl _3, -40 ° C): d 1.74 (m, 6H, CH ₂ ), 3.37 (m, 2H, CH ₂ ), 3.87
(m, 2H, CH ₂ ), 4.55 (s, 2H, CH ₂ ), 7.87 (s, 1H, CH), ¹³ C
NMR (125 MHz, CDCl _3, rt): d 24.0, 25.3, 44.2, 129.4, 150.9, 178.6, 183.8,
¹³ C NMR (125 MHz, CDCl _3, -40 ° C): d 23.7, 25.1,
44.4, 47.7, 54.7, 128.6, 150.9, 178.3, 183.6, ⁷⁷ Se NMR (95 MHz, CDCl ₃ )
: d 526.4,
MS (FAB): m / z = 293 [M ⁺ +1], Anal. Calcd for C ₁₀ H ₁₃ ClN ₂ OSe:
C, 41.18; H, 4.49; N, 9.61. Found; C, 40.82; H, 4.64; N, 8.74.

Production Examples and Properties of 5-Chloroacetyl-2-dimethylamino-1,3-selenazol N, N-Dimethyl-N '-(morpholinoselenocarbonyl) formamidine (76 mg, 0.3 mmol) ) In methanol solution (4 ml) and 1,3-dichloroacetone (195
mg, 1.5 mmol) was added and refluxed for 1 hour. The completion of the reaction was confirmed by TLC (ethyl acetate), and the mixed solution was extracted with dichloromethane, and then the solvent was distilled off. This was isolated and purified by silica gel column chromatography (ethyl acetate: hexane = 1: 3), and white. Solid 5-chloroacetyl-2-dimethylamino-1,3-selenazole (62 mg, 70%) was obtained.
Physical properties: Mp: 138-140 ° C, IR (KBr): 1630 cm ^-1 , ¹ H NMR
(500 MHz, CDCl _3, rt): d 3.62 (t, J = 5.2 Hz, 4H, CH ₂ ),
3.82 (t, J = 5.2 Hz, 4H, CH ₂ ), 4.46 (s, 2H, CH ₂ ),
7.89 (s, 1H, CH), ¹³ C NMR (125 MHz, CDCl _3, rt): d 44.2, 49.7,
65.9, 130.5, 150.1, 179.0, 183.9, ⁷⁷ Se NMR (95 MHz, CDCl ₃ ):
d 536.1,
MS (CI): m / z = 295 [M ⁺ +1], Anal. Calcd for C ₉ H ₁₁ ClN ₂ O ₂ Se:
C, 36.82; H, 3.78; N, 9.54. Found; C, 37.44; H, 4.10; N, 9.29.

Measurement of apoptosis inhibitory action: Using a 96-well plate, 50,000 PC-12 cells (distributed from Gifu Pharmaceutical University) in DMEM medium (Nissui Pharmaceutical) containing 10% horse serum and 5% fetal calf serum The cells were cultured at a concentration of 48 cells / mL for 48 hours, and 5-chloroacetyl-2-piperidino-1,3-selenazole (prepared by the method of the above preparation example) was added to 100 μM, and after 48 or 120 hours After adding 6.3 μl of 8 mg / ml solution of MTT and incubating at 37 ° C. for 2 hours, Lysis Buffer (sodium dodecyl sulfate [Wako Pure Chemical Industries, Ltd.]: dimethylformamide [Wako Pure Chemical Industries, Ltd.]) = 20: 50 μl of 80 (w / w); pH 4.7) 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 at the start-OD562 (staining degree) at the time of culturing for a predetermined time was determined and used as an index of the number of living cells. The results are shown in Table 1.

Measurement of neurofilament expression level: Cerebral neurons were collected from an 18-day-old rat fetus by a conventional method, and cultured for 24 hours in DMEM medium (Nissui Pharmaceutical) containing 10% equine serum and 5% fetal calf serum. did. After 24 hours, the medium was replaced with serum-free DMEM medium, and further 24 hours later, 10 μM of 5-chloroacetyl-2-morpholino-1,3-selenazole (prepared by the method of the above production example) was added. After 48 or 120 hours, cerebral neuronal cytoplasmic proteins were harvested with lysis buffer. The collected proteins were separated by SDS-PAGE, and the proteins were blotted onto a nylon membrane. After blocking, it was reacted with a neurofilament mouse IgG antibody (Sigma) and then treated with an avidin-biotinylated horseradish peroxidase complex. The expression level of the neurofilament was measured by visualizing the neurofilament with peroxidase activity using diaminobenzidine-H 2 O 2. As a positive target, 25 ng / mL of NGFβ and EGF (both from Sigma) were added and the same operation was performed. The results are shown in Table 2. Symbols in the table mean-: no, +: yes, and ++: notably about neurofilament expression.

Measurement of phosphorylation ability of mitogen-activated protein kinase kinase (MEK1 / 2) and mitogen-activated protein kinase (ERK1 / 2): DMEM medium containing 10% horse serum and 5% fetal calf serum (Nissui Pharmaceutical) PC-12 cells (distributed from Gifu Pharmaceutical University) were cultured at a concentration of 2 million cells / mL for 48 hours, and maintained in DMEM medium (Nissui Pharmaceutical) containing 1% bovine serum albumin (Sigma) for 8 hours. Next, 5-chloroacetyl-2-dimethylamino-1,3-selenazole (prepared by the method of the above production example) was added to 100 μM, and after 10 minutes, the protein was collected with a lysis buffer. The collected proteins were separated by SDS-PAGE, and the proteins were blotted onto a nylon membrane. After blocking, reaction with mitogen-activated protein kinase kinase (MEK1 / 2) and mitogen-activated protein kinase (ERK1 / 2) rabbit IgG antibody (Cell Signal Technology), followed by avidin-biotinylated horseradish peroxidase complex (Santa Cruz) Processed. The peroxidase activity that correlates with the amount of protein was measured by the following method. Specifically, using BCL (GE Healthcare), the generated chemiluminescence was visualized by baking on an X-ray film (manufactured by GE Healthcare), and phosphorylated mitogen-activated protein kinase kinase (MEK1 / 2) and The amount of phosphorylated mitogen-activated protein kinase (ERK1 / 2) was quantified with image processing software. The numerical value is a ratio with respect to no addition. The results are shown in FIG.

Measurement of neurite outgrowth action: PC-12 cells (distributed from Gifu Pharmaceutical University) in DMEM medium (Nissui Pharmaceutical) containing 10% by volume of horse serum and 5% by volume of fetal bovine serum at a concentration of 50,000 cells / mL Incubate for hours. The medium was replaced with a medium containing 10 μM of 5-chloroacetyl-2-piperidino-1,3-selenazole, and the percentage of cells that had undergone neuronal differentiation at 1 mm 2 after 24 and 48 hours was visually measured under a microscope. The results are shown in Table 3.

Comparative Example 1

Mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), docosahexaenoic acid (Nippon Yushi Co., Ltd.) were each dissolved in 1 mg / ml in the medium shown in Example 1, OD562 at the start-OD562 (staining degree) at the time of culturing for a predetermined time was determined and used as an index of the number of living cells. The results are also shown in Table 1.

Comparative Example 2

Mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), docosahexaenoic acid (Nippon Yushi Co., Ltd.) were each dissolved in 1 mg / ml in the medium shown in Example 1, Regarding, the expression of neurofilament was measured by the method of Example 2. The results are shown in Table 2.

Comparative Example 3

Mitogen-activated protein kinase kinase (MEK1 / 2) according to the method of Example 3 for mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), docosahexaenoic acid (Nippon Yushi Co., Ltd.) And the phosphorylation ability of mitogen activated protein kinase (ERK1 / 2) was measured. The results are shown in FIG.

Comparative Example 4

The neurite elongation action was measured by the method of Example 4 with respect to mugwort extract (Ichimaru Falcos Co., Ltd.), theanine (Taiyo Kagaku Co., Ltd.), and docosahexaenoic acid (Nippon Yushi Co., Ltd.). The results are also shown in Table 3.

As shown in Tables 1 to 3 and FIG. 1, the neurotrophic factor of the present invention is far superior to mugwort extract, theanine, and docosahexaenoic acid, which have been conventionally said to have a nerve activating effect. The effect is recognized.

According to the present invention, a neurotrophic factor comprising a 5-acyl-2-amino-1,3-selenazole analog or a 5-acyl-2-amino-1,3-selenazole analog and The applied product can be provided.

It is explanatory drawing which compared the phosphorylation ability of the mitogen activated protein kinase kinase (MEK1 / 2) and mitogen activated protein kinase (ERK1 / 2) of this invention and a conventional product (Example 3, Comparative Example 3).

Claims (9)

A neurotrophic factor comprising a 5-acyl-2-amino-1,3-selenazole analog. 5-acyl-2-amino-1,3-selenazole analogs are 5-chloroacetyl-2-dimethylamino-1,3-selenazole, 5-chloroacetyl-2-piperidino-1,3-selenazole, 5-chloro The neurotrophic factor according to claim 1, which is one or a mixture of two or more of acetyl-2-morpholino-1,3-selenazole. The neurotrophic factor according to claim 1 or 2, which has an action of promoting phosphorylation of mitogen-activated protein kinase (ERK1 / 2) in nerve cells. The neurotrophic factor according to claim 1 or 2, which has an action of promoting phosphorylation of mitogen-activated protein kinase kinase (MEK1 / 2) in nerve cells. The neurotrophic factor according to claim 1 or 2, which has an action of promoting expression of a neuronal protein (neurofilament). The neurotrophic factor according to claim 1 or 2, which has an action of suppressing apoptosis of nerve cells. The neurotrophic factor according to claim 1 or 2, which has an action of promoting neurite outgrowth of nerve cells. The pharmaceutical composition formed by mix | blending the neurotrophic factor of Claims 1-7 as an active ingredient. The edible composition formed by mix | blending the neurotrophic factor of Claims 1-7 as an active ingredient.

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