KR20170081783A - Novel benzothiazole derivatives for differentiation of neural stem cells and use thereof - Google Patents
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Abstract
The present invention relates to novel benzothiazole derivatives and their uses for the regulation of neural stem cell differentiation. In the present invention, it has been confirmed that a novel benzothiazole derivative prepared by an organic synthetic method can be treated to neural stem cells to promote differentiation into neural cells. As a result, it is useful as a therapeutic agent for neuronal cell damage diseases In the process of promoting differentiation into neurons, the secretion of cytokines such as LIF IL-6 and CNTF is remarkably increased, and it is expected to be used as a reagent for producing them.
Description
The present invention relates to novel benzothiazole derivatives and their uses for the regulation of neural stem cell differentiation.
Alzheimer's disease, Parkinson's disease, demyelinating disease, and spinal cord injury, known as intractable diseases, are disorders in which neuronal damage is caused by nerve cell damage. The number of patients suffering from the disease is rapidly increasing due to the increase in the average life span and the rapid entry into the aging society. .
On the other hand, the damaged nerve cells are not regenerated due to diseases caused by nerve cell damage, and therefore fundamental treatment is difficult. In addition, drug therapy and surgical operation methods generally used for treating the above diseases are not limited to damaged cells, Cells, which cause side effects, and they are having difficulty in treatment.
In recent years, cell replacement therapy, which supplies cells destructed or damaged by diseases, has been attracting attention as an effective treatment method. Among these cell therapy therapies, stem cells capable of proliferation and differentiation stem cells) are in the spotlight. In particular, since the cervical nervous system tissue has almost no immunity rejection unlike other tissues, when the neural stem cells are transplanted, long-term survival of the transplanted cells can be expected. Therefore, as a method of treating various neurological diseases caused by nerve cell damage, various studies on cell therapeutic agents using neural stem cells have been conducted.
Neural stem cells are subtypes of progenitor cells present in the nervous system and have the ability to differentiate into astrocytes, oligodendrocytes, neurons, and the central nervous system (CNS) And the peripheral nervous system (PNS) to form multicellular neurospheres, which are known to differentiate into glia and nervous systems under the respective conditions.
Since endogenous neural stem cells are present in the adult, neural stem cells are present in the hippocampus and other brain regions damaged by Alzheimer's disease. Therefore, development of a substance that regulates endogenous neural stem cell differentiation enhances the possibility of development of a therapeutic agent in the future.
However, in order to increase the usefulness of neural stem cells as a cell therapy agent, there is a need for a technique for efficiently differentiating neural stem cells into specific cells, and studies have been made actively (Korean Patent Laid-Open No. 10-2014-0130593) .
DISCLOSURE OF THE INVENTION The present invention has been conceived to solve the above-mentioned problems. The present inventors have made intensive studies to find a novel substance for controlling the differentiation of neural stem cells. As a result, they have found that benzothiazole And the present invention was completed on the basis thereof.
Accordingly, an object of the present invention is to provide a benzothiazole derivative represented by the following formula (1) or a salt thereof.
[Chemical Formula 1]
Another object of the present invention is to provide a composition for inducing differentiation of neural stem cells into neural stem cells comprising the compound or a salt thereof as an active ingredient and / or a pharmaceutical composition for preventing or treating neuronal cell damage diseases.
Yet another object of the present invention is to provide a method of differentiating neural stem cells into neural cells, comprising the step of treating the composition for inducing differentiation to neural stem cells.
Still another object of the present invention is to provide a method for producing cytokines, which comprises treating the compound or a salt thereof with neural stem cells.
However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.
In order to accomplish the above object, the present invention provides a benzothiazole derivative represented by the following formula (I) or a salt thereof.
[Chemical Formula 1]
The present invention also provides a composition for inducing differentiation of neural stem cells into neural stem cells comprising the compound or a salt thereof as an active ingredient and / or a pharmaceutical composition for preventing or treating neuronal cell damage diseases.
In one embodiment of the present invention, the nerve cell may be an astrocyte.
In another embodiment of the present invention, the composition may comprise a benzothiazole derivative or a salt thereof in a concentration of 5 to 9 [mu] M.
In another embodiment of the present invention, the composition is administered to a mammal such as a leukemia inhibitory factor (LIF), interleukin-6 (IL-6), bone morphogenic protein (BMP2) The secretion of any cytokine selected from the group consisting of neurotrophic factor (CNTF) can be increased.
In another embodiment of the present invention, the neuronal cell damage disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, , Demyelinating disease, and spinal cord injury. ≪ Desc /
The present invention also provides a method for differentiating neural stem cells into neural cells, comprising the step of treating the composition for inducing differentiation to neural stem cells.
The present invention also provides a method for producing a cytokine, comprising the step of treating the compound or a salt thereof with a neural stem cell.
In one embodiment of the present invention, the differentiation factor is selected from the group consisting of Leukemia inhibitory factor (LIF), interleukin-6 (IL-6), Bone morphogenic protein 2 (BMP2), and Ciliary neurotrophic factor It can be one.
The present invention provides a method for treating a neuronal cell damage disease comprising administering the above pharmaceutical composition to a subject.
The present invention provides a therapeutic use of a neuronal cell damage disease of a composition comprising said compound or a salt thereof.
The present invention relates to a novel benzothiazole derivative having the activity of promoting the differentiation of neural stem cells, a composition for inducing differentiation into neurons comprising the same, and / or a pharmaceutical composition for preventing or treating neuronal damage diseases. In the present invention, a novel benzothiazole derivative was synthesized by an organic synthetic method, and it was confirmed that treatment of the derivative with neural stem cells could promote differentiation into neural cells. As a result, Can be usefully used.
In addition, it was confirmed that the secretion of cytokines such as LIF, IL-6, and CNTF was remarkably increased in the process of promoting differentiation into neurons by treatment of the derivatives, and they were also used as reagents for producing them It is expected to be possible.
FIG. 1 shows (a) results of fluorescence microscopy and (b) quantification of differentiation into astrocytes according to various benzothiazole derivative treatments (
FIG. 2 shows the results of (a) the number of differentiated astrocytes, (b) the number of differentiated astrocytes, (a) the number of differentiated astrocytes, (C) quantifying the number of differentiated neurons, and (d) quantifying the total number of cells.
FIG. 3 shows the result of western blot analysis of expression of GFAP protein, which is a marker of astrocytic cells, in neural stem cells treated with 7.5 μM of
Fig. 4 shows the results of quantitative comparison of GFAP expression in neural stem cells treated with
FIG. 5 shows the result of Western blotting the degree of phosphorylation of (a) STAT3 and (b) SMAD1 / 5/8 in neural stem cells treated with
6 is a result of quantifying the expression of (a) LIF, (b) IL-6, (c) BMP-2 and (d) CNTF mRNA in the compound 5-treated neural stem cells.
FIG. 7 shows the results of quantifying the expression of (a) miR-9, (b) miR-124, and (c) miR-29a in neural stem cells treated with
Fig. 8 shows the result of western blotting the degree of phosphorylation of ERK1 / 2 in neural stem cells treated with
FIG. 9 shows the results of quantifying the expression of (a) TGF-? 1, (b) FGF2, and (c) FGF8 mRNA in neural stem cells treated with
FIG. 10 is a schematic diagram showing a correlation between the related factors in the process of promoting the differentiation of neural stem cells into astrocytes according to the treatment of
11 is a schematic diagram showing a process of promoting the differentiation of neural stem cells into astrocytes according to the treatment of
The present inventors produced a benzothiazole derivative that promotes differentiation from neural stem cells into astrocytes, which are neuronal cells, by an organic synthetic method. Concentration of derivatives capable of achieving the optimum effect was derived through specific experiments. In addition, it was confirmed that the phosphorylation of STAT3, SMADs and ERK1 / 2 was promoted by treating the derivative, and the related differentiation factors were markedly increased. Based on this finding, the present invention was completed.
Hereinafter, the present invention will be described in detail.
The present invention provides a compound represented by the following formula (2-phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) acetamide) or a salt thereof.
[Chemical Formula 1]
In addition, the compound represented by Formula 1 may be prepared through the preparation method of Example 2, but corresponds to an example of the method for producing a compound. For example, reaction conditions such as the reaction solvent and the amount of the base reactant to be used are not limited to those described above, and various known synthesis methods known to those skilled in the art can be used to produce the compound of
As used herein, the term "salt" is useful as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.
The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound represented by the formula (1) in an excess amount of an acid aqueous solution, and then mixing the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > It may also be prepared by evaporating a solvent or excess acid in this mixture and then drying or by suction filtration of the precipitated salt.
In addition, the base may be used to make a pharmaceutically acceptable metal salt. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (for example, silver nitrate).
In one embodiment of the present invention, a novel benzothiazole derivative was prepared through an organic synthetic method. When
Accordingly, the present invention provides a composition for inducing differentiation from neural stem cells into neurons, comprising the compound represented by the above-mentioned formula (1) or a salt thereof as an active ingredient.
As used herein, the term "neural stem cells" is a primitive, immature, self-renewing cell with the potential to occur in all differentiated neuronal cell types in all regions of the nerve axis, , Astrocyte, oligodendrocyte, and preferably stellate cells.
The composition for inducing differentiation according to the present invention has a technical feature in inducing differentiation into neurons from neural stem cells, and the compound represented by formula (1) as an active ingredient in the composition is preferably 5 to 9 μM, May be included at a concentration of 7.5 [mu] M.
In addition, the composition induces differentiation into neuronal cells, that is, astrocytes by increasing the expression of STAT3, SMADs, and ERK1 / 2 phosphorylation-related factors, and these factors are preferably selected from the group consisting of LIF (Leukemia inhibitory factor) but are not limited to, interleukin-6, bone morphogenic protein 2 (BMP2), and ciliary neurotrophic factor (CNTF).
In another aspect of the present invention, there is provided a method of differentiating a neural stem cell into a neural cell, comprising the step of treating the composition for inducing differentiation to neural stem cells.
According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a neuronal cell damage disease, comprising the compound represented by the above-mentioned formula (1) or a salt thereof as an active ingredient.
As used herein, the term "prophylactic " means the term used in the present invention, and" prophylactic "means any act that inhibits or delays neuronal cell damage disease by administration of the pharmaceutical composition according to the present invention .
As used herein, the term "treatment" refers to any action that improves or alters the symptoms of a neuronal cell damage disease by the administration of the pharmaceutical composition according to the present invention.
The neuronal cell damage disease to be prevented or treated according to the present invention is a disease in which neuronal damage is caused by nerve cell damage and includes, but is not limited to, Pick's disease, Huntington's disease, Amyotrophic lateral sclerosis, ischemic brain disease (stroke), demyelinating disease, and spinal cord injury.
The pharmaceutical composition of the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier. Herein, pharmaceutically acceptable carriers are those conventionally used at the time of formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.
The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may vary depending on the condition and the weight of the patient, The mode of administration, the route of administration, and the time, but may be appropriately selected by those skilled in the art.
The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, the degree of absorption of the active ingredient in the body, the rate of inactivation and excretion, the type of disease, 0.001 to 150 mg, preferably 0.01 to 100 mg, per 1 kg of body weight may be administered daily or every other day, or one to three divided doses per day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.
In another aspect of the present invention, the present invention provides a method for treating a neuronal cell damage disease comprising administering the pharmaceutical composition to a subject.
In the present invention, the term "individual" means a subject in need of treatment of a disease, and more specifically refers to a mammal such as a human or non-human primate, mouse, dog, cat, horse and cattle do.
In still another embodiment of the present invention, there is provided a method of producing a cytokine, which comprises treating a neural stem cell with the compound represented by the formula (1) or a salt thereof.
As used herein, the term "cytokine" is a signal substance that controls and stimulates the defense system of the body, preferably a substance that promotes differentiation into neurons, such as Leukemia inhibitory factor (LIF), interleukin -6), BMP2 (Bone morphogenic protein 2), and CNTF (Ciliary neurotrophic factor), but the present invention is not limited thereto.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
Example 1. Experimental Preparation and Experimental Method
1-1. Purification and analysis of compounds
Flash column chromatography was performed using Silica gel 60 (230-400 mesh; Millipore, Darmstadt, Germany). 1 H NMR spectra were recorded on a JNM-LA 300 model from JEOL Inc. and an Avance 400 MHz FT-NMR spectrometer from Bruker. The chemical shifts were expressed in ppm, based on tetramethylsilane . Mass spectrometry was performed using VG Trio-2 GC-MS and Aligent's 6460 Tiple Quad LC / MS.
1-2. Cultivation of neural stem cells
Neural stem cells (NSCs) were identified from the cortex of embryos in E14 rats (Sprague-Dawley rats, Orient Bio Inc., Gyeonggi-do, Korea). Then, the separated neural stem cells (200,000 cells / ml) were treated with 1% (v / v) PSA, 2% (v / v) B27 (Gibco, NY, USA), 20 ng / ml EGF, The cells were cultured in a neurosphere form in Dulbecoo's modified Eagle's medium / F12 supplemented with FGF2 (Chemicon, CA, USA) supplemented with 5% CO 2 at 37 ° C., Respectively. After 6 days, the neural nerves were treated with accutase (Chemicon), followed by incubation at 37 ° C for 10 minutes to obtain a suspension of single neural stem cells, and the cells were treated with 0.01% poly-D-lysine (Sigma-Aldrich ) And 10 [mu] m / ml laminin (Invitrogen, CA, USA). One day later, differentiation was induced without the addition of growth factors. During the experiment, compounds synthesized with 0.1% DMSO (Sigma-Aldrich) and 10 ng / ml CNTF or 20 μM Pd98059 (Mollipore, CA, USA) were treated with neural stem cells.
1-3. Immunocytochemical analysis
For immunocytochemical analysis, cell cultures were fixed in 4% paraformaldehyde for 30 minutes and washed with phosphate buffered saline (PBS). Immobilized cells were blocked with 5% normal goat serum (Milipore) and 0.2% Triton X-100 (Amresco, OH, USA) for 30 min. Subsequently, β-tubulin III (TuJ1, monoclonal; ; Sigma-Aldrich), glial fibrillary acidic protein (GFAP, polyclonal; 1: 1000; Dako) for 1 hour or more. After washing with PBS, the cells were incubated with Alex Fluor 488 (goat anti-mouse IgG, 1: 1000; Invitrogen) or Cy2 (goat anti-rabbit IgG, 1: 000; Jackson ImmunoResearch, And incubated with the secondary antibody for 30 minutes. (DAPI) (1: 10,000 in PBS; Sigma-Aldrich) was added for nuclear staining and the image was reconstructed using a reversed phase fluorescence microscope (DMIL; Leica, Hesse, Germany) . To reduce the error of the experiment, the images were randomly selected and the number of TuJ1-positive, GFAP-positive, or DAPI-positive cells was calculated. The number of TuJ1-positive or GFAP-positive cells was divided by the number of DAPI-positive cells and expressed as a percentage value. The% value in the group treated with the compound was divided by the percentage value of the control group and expressed as a Fold value.
1-4. Real-time RT-PCR
Total RNA was extracted from the cell culture using TRIzol reagent and 20 μL of the first strand complementary DNA (1 μg) was extracted from 1 μg of total RNA using QuantiTect Reverse Transcription Kit (Qiagen, Limburg, Nethelands) cDNA) was synthesized. Real-time RT PCR was performed using iQ SYBR green supermix (Bio-Rad, CA, USA) and PCR was performed under the following conditions: first, reaction was carried out at 95 ° C for 30 seconds and then 1) Denaturation of the strand DNA at 95 DEG C for 10 seconds; 2) annealing at 58 ° C for 15 seconds with template and primer binding; 3) The reaction was carried out at 72 ° C for 20 seconds with the synthesis of new strands. This step was repeated 30 times, and the housekeeping gene gapdh was used as an internal control. The ratio of gene expression between neural stem cells following treatment with DMSO or manufactured compound was calculated by ratio = 2 C (t) DMSO / Δ (t) compound . Where, C (t) DMSO means a value obtained by subtracting the C (t) gapdh in C (t) target gene in neural stem cells with DMSO to handle, and, C (t) compound is of the prepared compound was treated neural stem cells C (t) the target gene minus C (t) gapdh .
1-5. Western blot
Neural stem cells were washed with PBS and resuspended in lysis buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 1% NP-40 (Amresco), Halt Phosphatase Inhibitor Cocktail (Thermo Scientific, IL , USA), 1 mM phenylmethylsulfonyl fluoride, 0.01 mg / ml leupeptin, and 0.01 mg / ml aprotinin (Sigma-Aldrich) were added to the solution and the mixture was centrifuged at 25200 xg for 30 minutes to remove debris. The proteins were incubated in SDS (sodium dodecyl sulfate) sample buffer (60 mM tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.1% bromophenol blue (Ameresco), 14.4 mM? -Mercaptoethanol , And distilled water) for 5 minutes. The membrane was separated by electrophoresis on a SDS polyacrylamide gel and transferred to a polyvinylidenedifluoride membrane. The membrane was washed with 5% non-fat dry milk in a 20 mM Tris-buffered saline supplemented with 0.03-0.1% Tween 20 (Amersco) Or bovine serum albumin (Millipore, IL, USA). Subsequently, anti-GFP (1: 500), TuJ1 (1: 2000) (Sigma-Aldrich), anti-STAT3 (1: 2000), anti-phospho-STAT3 (tyr705, 1: (Ser 463/465) / Smad 5 (Ser 463/465) / Smad 8 (Ser 426/428) (1: 1000),
1-6. Detection of miRNA expression
Expression of rno-miR-9, rno-miR-124 and rno-miR-29a was analyzed using the miScript PCR system (Qiagen) according to the manufacturer's instructions. Total RNA containing miRNA was extracted using TRIzol reagent (Invitrogen) and cDNA was synthesized from 2 μg of total RNA using miScript II RT kit. Real-time PCR was performed using miScript Primer Assays and miScript SYBR Green PCR kit of Rn_miR-9_1, Rn_miR-124_1, Rn_miR-29a_2 and Hs_RNU6-2_11. The PCR reaction was carried out at 95 ° C for 15 minutes, followed by 40 cycles of reaction at 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 70 ° C for 30 seconds. For quantification, U6 small nuclear RNA (RNU6) was used.
1-7. Statistical analysis
All the test results showed mean ± SD or standard error of mean, and statistical significance was tested using Student's t-test ( * P <0.05, ** P <0.01)
Example 2. 2-Phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) acetamide (2-Phenyl- N - (2-phenylbenzo [ d ] thiazol-7-yl) acetamide, Compound 5)
In this example, 2-phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) acetamide was obtained from N- (3-bromophenyl) benzothioamide as shown in
[Reaction Scheme 1]
2-1. 7-bromo-2-phenylbenzo [ d ] 7-Bromo-2-phenylbenzo [ d ] thiazole
Under conditions in which the nitrogen is supplied, CF 3 CH 2 OH (3mL ) to N - (3- bromophenyl) benzo thio amide (N - (3-Bromophenyl) benzothioamide) PIFA (Bis (trifluoroacetoxy) in a mixture solution of the iodo) benzene; Sigma-Aldrich). Thereafter, the mixed solution was stirred at room temperature for 2 hours, concentrated under reduced pressure, and purified by silica gel (100-200 mesh) column chromatography to obtain 7-bromo-2-phenylbenzo [ d ] thiazole .
2-2. 2-phenylbenzo [ d ] Thiazole l-7-amine (2-Phenylbenzo [ d ] thiazol-7-amine < / RTI >
To a mixed solution of 7-bromo-2-phenylbenzo [ d ] thiazole and benzophenone imine dissolved in 1,4-dioxane (10 mL) was added CS 2 CO 3 (357 mg, 1.1 mmol) was added and the argon gas was removed for 5 min. Pd 2 dba 3 (45 mg, 0.05 mmol) and Xantphos (29 mg, 0.05 mmol) were added and the gas was removed and then heated at 100 < 0 > C for 18 h. The reaction mixture was cooled at room temperature, passed through a celite pad, then washed with EtOAc (20 mL), brine (25 mL), dried over NaSO 4 and concentrated under reduced pressure. The residue was then diluted with 2M HCl (1 mL) dissolved in methanol (8 mL) with stirring for 30 minutes, then the volatiles were evaporated. It was diluted again with distilled water (10 mL), neutralized with NH 4 OH and extracted with EtOAc (2 x 15 mL). The EtOAc layer was separated, dried over Na 2 SO 4, and concentrated under reduced pressure. The residue was purified by silica gel (100-200 mesh) column chromatography using EtOAc / Pet ether (1: 4) as elution solvent to give phenylbenzo [ d ] thiazole l-7-amine.
2-3. 2-phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) acetamide (2-Phenyl- N - (2-phenylbenzo [ d ] thiazol-7-yl) acetamide)
To a mixed solution of phenylbenzo [ d ] thiazole l-7-amine (1.0 eq) dissolved in DCM (~ 10 mL) at 0 ° C was added phenylacetyl chloride (1.0 eq) and triethylamine (1.0 eq ). Thereafter, the mixed solution was diluted with distilled water (10 mL), the DCM layer was separated, dried over Na 2 SO 4, and concentrated under reduced pressure. Phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) acetamide was obtained by purifying by silica gel (100-200 mesh) column chromatography using hexane / EtOAc (1: 1) .
(M, 2H), 7.57-7.38 (m, 6H), 7.32-7.30 (m, 3H) ), 6.01 (d, J = 6.0 Hz, 1H), 3.90 (s, 2H); MS (ESI) m / z 345.1 [M + H]
Example 3 Confirmation of Effect of Promoting Differentiation into Astrocytes
Neural stem cells (NSCs) proliferate in the presence of EGF (epidermal growth factor) and / or FGF (Fibroblast growth factor), whereas in the absence of these factors, differentiation proceeds into neurons or glial cells. In this example, a benzothiazole derivative containing Compound 5 (2-phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) acetamide synthesized in Example 2) And the effect on the differentiation process. Specifically, the benzothiazole derivatives in the present Examples are shown in Tables 1 and 2, and as described in Example 1-3, immunocytochemical analysis revealed that the neuron stem cells were differentiated into Astrocyte Was evaluated.
[Table 1]
[Table 2]
As a result, as shown in Fig. 1, when
From the above results, it was found that the 2-phenyl- N- (2-phenylbenzo [d] thiazol-7-yl) acetamide according to the present invention promotes differentiation into astrocytes in neural stem cells.
Example 4. Derivation of optimal concentration for promoting differentiation into astrocytes
Based on the results of Example 3, it was tried to confirm the specific concentration of
As a result, as shown in FIG. 2, when the
On the other hand, the
As a result, as shown in Figs. 3 and 4, the expression of GFAP protein was increased according to the treatment of
From the above results, it was found that the 2-phenyl- N- (2-phenylbenzo [d] thiazol-7-yl) acetamide according to the present invention significantly promoted differentiation into astrocytes.
Example 5 Identification of Mechanism Related to Differentiation into Astrocytic Cells
In this Example, the specific mechanism of promoting the differentiation into the astrocytes from the neural stem cells according to the treatment of
In view of the fact that it took some time to phosphorylate the STATs and SMADs, it was hypothesized that other molecules could participate in the phosphorylation or activation according to the treatment of the
As a result, as shown in FIGS. 6 and 7, the expression of LIF, IL-6, BMP2, and CNTF mRNA was increased 50.20, 45.08, 6.52, or 1.28 times , The expression of miR-9, and miR-124, which decrease STAT3 activity, decreased with the treatment of
As shown in FIGS. 10 and 11, the 2-phenyl- N - (2-phenylbenzo [d] thiazol-7-yl) , And CNTF, indirectly induces phosphorylation of STAT3 and SMAD1 / 5/8, induces expression of FGFs and TGF-β1 to activate the ERK1 / 2 signaling mechanism, and activates related miRNAs (miR-9, miR-124), which promotes the differentiation of astrocytes.
It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
Claims (9)
[Chemical Formula 1]
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| KR102255066B1 (en) * | 2020-02-18 | 2021-05-24 | 중앙대학교 산학협력단 | Composition for Inducing Differentiation of Stem Cells into Astrocytes Comprising 3’-methoxyflavone as Active Ingredient |
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