KR20150027703A - Novel compound having optical activity for differentiation of neural stem cells and medical use thereof - Google Patents

Abstract

The present invention relates to an optically active compound for regulating neural stem cell differentiation and its medical use. Since the optically active compound can differentiate neural stem cells into neurons, particularly astrocytes, superior to other similar compounds, Can be usefully used for the treatment or prevention of neuronal cell damage diseases including stroke, Alzheimer's disease, Parkinson's disease or spinal cord injury diseases and the like.

Description

TECHNICAL FIELD The present invention relates to optically active compounds for modulating neural stem cell differentiation and medicinal uses thereof,

The present invention relates to an optically active compound for modulating neural stem cell differentiation and its medical use.

Stroke, Alzheimer's disease, Parkinson's disease, demyelinating disease, and spinal cord injury are disorders in which neuronal dysfunction is caused by nerve cell damage, Is difficult to treat because it is not regenerated, and the drug or surgical operation is performed with the symptom therapy. However, these treatments are pointed out as a problem due to damage to transient or normal cells.

Recently, it has been suggested that a cell therapy agent that supplies cells destructed or damaged by diseases from outside is effective. Neural stem cells (NSCs) are in the spotlight as such cell therapy drugs.

Neural stem cells are subtypes of progenitor cells present in the nervous system and have the ability to differentiate into astrocytes, oligodendrocytes, and neurons. It can separate from the central nervous system (CNS) and the peripheral nervous system (PNS) to form multicellular neurospheres, which differentiate into glia and nervous systems at each condition (Sally Temple et al . 2001).

Since the cervical nervous system tissue has almost no immune rejection reaction unlike other tissues, long-term survival of the transplanted cells can be expected when the neural stem cells are transplanted, and as a cell therapy agent for various neurological diseases caused by nerve cell damage Much research has been done.

However, in order to increase the utility 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.

On the other hand, Korean Patent Publication No. 2010-0047901 discloses an oxadiazole derivative which inhibits the hedgehog pathway or a pharmaceutically acceptable salt thereof and uses thereof, but the oxadiazole derivative is different from the present invention Compounds as well as their use as modulators of neural stem cell differentiation.

The present inventors have completed the present invention by discovering that a novel optically active compound is a neural stem cell differentiation regulator that differentiates neural stem cells into specific cells, that is, astrocyte cells.

Accordingly, an object of the present invention is to provide an optically active compound having neural stem cell differentiation-regulating activity.

Another object of the present invention is to provide a composition for inducing the differentiation of neural stem cells comprising the optically active compound into neurons.

It is still another object of the present invention to provide a method of differentiating neural stem cells into neural cells, comprising culturing the composition with neural stem cells.

Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing a neuronal cell damage disease comprising the optically active compound.

In order to achieve the above object, the present invention provides an optically active compound represented by the following general formula (1) or a salt thereof:

[Chemical Formula 1]

(2)

The present invention also provides a composition for inducing the differentiation of neural stem cells into neurons, comprising an optically active compound represented by the formula (1) or (2) or a salt thereof.

The present invention also provides a method for differentiating neural stem cells into neural cells, comprising culturing the composition together with neural stem cells.

The present invention also provides a pharmaceutical composition for treating or preventing a neuronal cell damage disease comprising an optically active compound represented by the formula (1) or (2) or a salt thereof.

Since the optically active compound according to the present invention can differentiate neural stem cells into neurons, particularly astrocytes, the composition containing the optically active compound is useful for the treatment of nerve cell damage including stroke, Alzheimer's disease, Parkinson's disease, Can be usefully used for the treatment or prevention of diseases.

FIG. 1 shows immunofluorescence images of GFAP obtained by observing the induction of astrocyte-derived neural stem cells by an optically active compound according to the present invention. FIG.
FIG. 2 is a photograph showing the induction of neural stem cells derived from human induced pluripotent stem cells (iPSC) by the optically active compound according to the present invention into astrocytes and showing immunofluorescence images of GFAP.

 The terms, techniques, and the like described in the present invention are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified. In addition, all documents referred to in this specification are included in the present specification as a document for explaining the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides an optically active compound represented by the following formula (1) or a salt thereof:

[Chemical Formula 1]

(2)

The optically active compounds according to the present invention can be prepared as corresponding salts by known methods. Such salts are preferably water-soluble without toxicity. Preferred salts include salts of alkali metals such as potassium, sodium and the like; Salts of alkaline earth metals such as calcium, magnesium and the like; And amines such as tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) amine, lysine, arginine, And salts of pharmaceutically acceptable amines such as N-methyl-D-glucamine and the like.

The optically active compound used in the present invention can be prepared by a known method as the corresponding acid addition salt. These acid addition salts are preferably water-soluble without toxicity. Preferred acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate and nitrate, or organic acid salts such as acetate, lactate, tartrate, oxalate, fumarate, maleate, citrate, benzoate, methane Organic acid salts such as sulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate and gluconate.

The optically active compound or salt thereof used in the present invention can be prepared as a hydrate by a known method.

As shown in FIG. 1 and FIG. 2, the experimental group treated with the optically active compound according to the present invention induces the differentiation of neural stem cells into astrocytes, as compared with the control group not treated with any of the optically active compounds, The optically active compound or its salt according to the present invention promotes the differentiation of neurons into astrocytes, particularly by the stimulation of differentiation of neurons into astrocytes, Can be usefully used for the treatment or prevention of cell damage diseases.

The present invention also provides a composition for inducing differentiation of neural stem cells into neurons, comprising an optically active compound represented by the following formula (1) or (2) or a salt thereof as an active ingredient:

[Chemical Formula 1]

(2)

In particular, the composition according to the present invention preferably contains the optically active compound or its salt in an amount of 1 to 10 [mu] M.

The neural stem cells that can be differentiated according to the present invention are differentiated into neural cells through the steps of neural progenitor cells that produce specific neural cells. Therefore, it is not particularly limited as long as it is an undifferentiated cell and has a function of infinite proliferation and differentiation into neurons. For example, embryonic neural stem cells, adult neural stem cells, embryonic germinal neural stem cells, and embryonic tumor neural stem cells.

The neurons differentiated from the neural stem cells may be neurons, astrocyte, oligodendrocytes or microglia cells.

The present invention also provides a method for differentiating neural stem cells into neural cells, comprising culturing the composition together with neural stem cells.

The culture may be performed in a cell culture medium such as DMEM (Dulbecco's modified Eagle's medium, Hyclone) / F12 medium. The culture medium may include B27, fibroblast-derived growth factor (FGF) a growth factor such as an epidermal growth factor (EGF), and the like may be further added. The incubation period is preferably 3 to 10 days.

The present invention also provides a pharmaceutical composition for treating or preventing a neuronal cell damage disease, comprising an optically active compound represented by the following formula (1) or (2) or a salt thereof as an active ingredient:

[Chemical Formula 1]

(2)

The neuronal cell damage diseases include Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic brain disease, demyelinating disease, And spinal cord injury. ≪ Desc / Clms Page number 2 >

More specifically, when the neuron is administered to a damaged area of a subject in need of treatment of neuronal damage disease with a therapeutically effective amount, neuronal progenitor cells or neural stem cells of peripheral nervous system or peripheral nervous system are differentiated into neuronal cells To restore damaged nerve function and to treat nerve damage diseases. Wherein the subject may be a mammal, including a human.

The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable additives in addition to the active ingredient.

The pharmaceutical composition of the present invention can be formulated into a unit dosage form suitable for administration to a patient in the body according to a conventional method in the field of pharmacy. As formulations suitable for this purpose, injectable preparations or preparations for enteral administration are preferred as parenteral administration preparations. At this time, a commonly used diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient may be used together.

Then, the single dose of the nerve cells is 1 x 10 ⁵ cells / kg (body weight), and preferably may be administered once or several dividing circuit. However, the actual dosage of the active ingredient may be determined taking into account various relevant factors such as the amount of neuron to be differentiated and proliferated, the route of administration, the weight of the patient, age and sex, And are not intended to limit the scope of the invention.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Manufacturing example  1 > 2- ( Rat - Butoxycarbonylamino ) -2-phenylacetic acid (1) Synthesis

Compound 1 was synthesized according to a known method (J. Labelled Comp. Radiopharm. 2004, 47, 599-608).

< Manufacturing example  2> Rat - Butyl phenyl (5-phenyl-1,3,4-oxadiazol-2-yl) methylcarbamate (3) Synthesis

Compound 3 was synthesized according to a known method (Tetrahedron Lett. 2006, 47, 4827-4830) in the literature.

< Manufacturing example  3> Phenyl (5-phenyl-1,3,4-oxadiazol-2-yl) methanamine (5) Synthesis

TFA (0.93 mL, 12.1 mmol) was added to a solution of compound 3 (533 mg, 1.517 mmol) dissolved in dry CH ₂ Cl ₂ (7.0 mL) at room temperature under an argon atmosphere. After monitoring the completion of the reaction with TLC for 4 h, the reaction mixture was quenched with water (7.0 mL), extracted with CH ₂ Cl ₂ , saturated NaHCO ₃ Aqueous solution and brine. The organic layer was dried over MgSO _4, filtered and concentrated. Unrefined residue was purified via flash column chromatography on silica gel (EtOAc: n -hexane = 1: 1) to give compound 5 (348 mg, 91%

¹ H-NMR (CDCl ₃ , 300 MHz) 隆 7.93 (dd, 2H, J = 7.7, 1.4 Hz), 7.33 (m, 9H), 5.41 (s, 1H).

< Example  1 > (R) - N- ( Phenyl (5-phenyl-1,3,4-oxadiazol-2-yl) methyl ) -2- (4- ( Trifluoromethyl ) Phenyl ) Acetamide (7) Synthesis

The compound 7 was synthesized by the following method.

(DMAP) (1.5 eq.) And EDCI (1.2 eq.) In a solution of optically active compound 5 (1.0 eq.) Dissolved in dry CH ₂ Cl ₂ (0.1 M) and phenylacetic acid . The reaction mixture was stirred at room temperature until the completion of the reaction was confirmed by monitoring via TLC (2 hours to overnight), followed by quenching with aqueous 2N HCl solution and extraction with EtOAc. The obtained organic layer was saturated NaHCO ₃ Washed with aqueous solution and brine, dried over anhydrous MgSO ₄ , filtered under reduced pressure and concentrated. Unrefined residue was purified via flash column chromatography on silica gel (EtOAc: n -hexane = 1: 4) to give compound 7 (18 mg, 56%):

_{^{1 H NMR (300MHz, CDCl 3}} ): δ 3.71 (s, 2H), 6.50 (d, J = 7.95Hz, 1H), 6.95 (m, 1H), 7.30-7.61 (m, 12H), 7.92-8.01 ( m, 2H).

< Example  2 > (S) - N- ( Phenyl (5-phenyl-1,3,4-oxadiazol-2-yl) methyl ) Benzamide (10) Synthesis

The compound 10 was synthesized as follows.

Phenylsulfonyl which was dissolved in a solution of an optically active compound 5 (1.0 eq.) In 0 ℃ dry CH ₂ Cl ₂ (0.1M) in dissolved in dry CH ₂ Cl ₂ (0.05M) and triethylamine (1.7 eq.) At room temperature Chloride (1.0 eq.) Was added. The reaction mixture was stirred at 0 &lt; 0 &gt; C until monitoring was complete by monitoring through TLC, then the reaction was quenched with aqueous 2N HCl solution and extracted with EtOAc. The obtained organic layer was saturated NaHCO ₃ Washed with aqueous solution and brine, dried over anhydrous MgSO ₄ , filtered under reduced pressure and concentrated. Unrefined residue was purified via flash column chromatography on silica gel (EtOAc: n -hexane = 1: 3) to give pale yellow Compound 10 (13 mg, 29%

_{^{1 H NMR (300MHz, CDCl 3}} ): δ 6.83 (d, J = 7.95Hz, 1H), 7.35-7.65 (m, 11H), 7.99-8.04 (m, 4H), 8.81 (d, J = 7.8Hz, 1H).

< Comparative Example  1 > 2- Phenyl - N- ( Phenyl (5-phenyl-1,3,4-oxadiazol-2-yl) methyl ) Acetamide (11) Synthesis

4-Dimethylaminopyridine (DMAP) (1.5 eq.) And EDCI (1.2 eq.) Were added to a solution of compound 5 (1.0 eq.) Dissolved in dry CH ₂ Cl ₂ (0.1 M) and phenylacetic acid . The reaction mixture was stirred at room temperature until the completion of the reaction was confirmed by monitoring via TLC (2 hours to overnight), followed by quenching with aqueous 2N HCl solution and extraction with EtOAc. The obtained organic layer was saturated NaHCO ₃ Washed with aqueous solution and brine, dried over anhydrous MgSO ₄ , filtered under reduced pressure and concentrated. Unrefined residue was purified via flash column chromatography on silica gel (EtOAc: n -hexane = 1: 4) to afford 11 (18 mg, 56%):

_{^{1 H-NMR (CDCl 3,}} 500MHz) δ 7.93 (d, 2H, J = 7.2Hz), 7.46 (m, 3H), 7.31 (m, 10H), 6.63 (d, 1H, J = 7.8Hz), 6.46 (d, IH, J = 8.0 Hz), 3.66 (s, 2H).

< Experimental Example  1> Immuno-staining analysis

1. Rat fetal derived neural stem cells ( NSC ) Culture

Rat fetal neural stem cells (NSC) were purchased from invitrogen to confirm whether the compounds according to the examples induced normal cell differentiation.

At this time, the NSC (200,000 / ml) was cultured in DMEM / F12 medium (Gibco) supplemented with 2% B27 (Invitrogen), 20 ng / ml EGF (Chemicon, CA) and 20 ng / ml FGF- , Carlsbad, Calif.) For 7 days. At this time, the medium was changed once every two days. For differentiation, neurospheres were dispersed in a single cell suspension containing accutase for 10 min at 37 ° C and dispensed onto 0.01% poly-L-lysine and 10 μg / ml laminin (Sigma-Aldrich, MO). At this time, no growth factors were included at the time of dosing but it was reinforced with 2% B27. Cells treated with DMSO were cultured in a 5% CO ₂ incubator and cell differentiation was determined for 4 days thereafter.

2. Human induced pluripotent stem cell (iPSC) derived neural stem cells (NSC) culture

Using human induced pluripotent stem cell (iPSC) derived neural stem cells (NSC), we confirmed whether the compounds according to the Examples induce normal cell differentiation.

At this time, the NSC (200,000 / ml) was cultured in DMEM / F12 medium (Gibco) supplemented with 2% B27 (Invitrogen), 20 ng / ml EGF (Chemicon, CA) and 20 ng / ml FGF- , Carlsbad, Calif.) For 7 days. At this time, the medium was changed once every two days. For differentiation, neurospheres were dispersed in a single cell suspension containing accutase for 10 min at 37 ° C and dispensed onto 0.01% poly-L-lysine and 10 μg / ml laminin (Sigma-Aldrich, MO). At this time, no growth factors were included at the time of dosing but it was reinforced with 2% B27. Cells treated with DMSO were cultured in a 5% CO ₂ incubator and cell differentiation was determined for 4 days thereafter.

3. Immunostaining analysis

For immunostaining analysis, the cell culture obtained above was fixed in 4% paraformaldehyde for 30 minutes and washed with phosphate buffer (PBS). The fixed cells were broke with 5% normal goat serum and 0.2% Triton X-100 dissolved in PBS and incubated with β-tubulin III (TuJ1) (monoclonal; 1: 1,000; Sigma-Aldrich, MO) And cultured with primary antibody against glial fibrillary acidic protein (GFAP) (polyclonal; 1: 1,000; Dako, Carpinteria, CA). After washing with PBS, the cells were incubated with a second antibody conjugated to Cy3 (1: 1000, Molecular Probes, CA) for 30 minutes. After completion of incubation of the second antibody with nuclear staining, 4,6-diamidino-2-phenylindole (DAPI) (1: 10,000 in PBS) was added for 5 minutes. Images were obtained using reversed phase fluorescence microscopy (DMIL; Leica, Wetzlar).

As a result, as shown in FIG. 1, in the immunostaining using the neural stem cells of the rat fetal origin, the compounds 7 and 10 having optical activity as compared with the compound 11 synthesized in the comparative example are differentiated from neural stem cells into astrocytes As well as to promote excellence. As shown in FIG. 2, it was confirmed that Compound 7 and Compound 10, which have optical activity in immunostaining using neural stem cells derived from human induced pluripotential stem cell (iPSC), excellently promote the differentiation from neural stem cells into astrocytes .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (7)

An optically active compound represented by the following formula (1) or a salt thereof:
[Chemical Formula 1]

(2)

A composition for inducing differentiation of neural stem cells into neurons, comprising an optically active compound represented by the following formula (1) or (2) or a salt thereof as an active ingredient:
[Chemical Formula 1]

(2)

The composition according to claim 2, wherein the optically active compound or its salt is contained in an amount of 1 to 10 [mu] M to induce differentiation of neural stem cells into neurons. The composition according to claim 2, wherein the neural stem cells are selected from the group consisting of embryonic stem cell, adult neural stem cell, embryonic germinal stem cell, and embryonic tumor neural stem cell. The composition according to claim 2, wherein the nerve cell is a neuron, an astrocyte, an oligodendrocyte, or a microglia cell. . A pharmaceutical composition for treating or preventing a neuronal cell damage disease, comprising an optically active compound represented by the following formula (1) or (2) or a salt thereof as an active ingredient:
[Chemical Formula 1]

(2)

7. The method of claim 6, wherein 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, ischemic brain disease, demyelinating disease, and spinal cord injury. The present invention also provides a pharmaceutical composition for treating or preventing a neuronal cell injury disease.

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