KR101793254B1 - Acrylonitrile derivatives and pharmaceutical composition cpmprising the same - Google Patents
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Abstract
The present invention relates to an acrylonitrile compound and a pharmaceutical composition comprising the same. More particularly, the present invention relates to a method for preventing and treating a neurodegenerative disease by preventing degeneration such as oligomer formation of intracellular tau protein, And a pharmaceutical composition comprising the same.
Description
The present invention relates to an acrylonitrile compound for inhibiting oligomerization of tau protein, which is a major cause of neurodegenerative diseases, and a pharmaceutical composition containing the same.
The main function of tau is known to promote and stabilize microtubule assembly of neuronal axons. When pathologically transformed, the tau separates from the microtubules and becomes an insoluble aggregate called neurofibrillary tangles (NFTs). Abnormal aggregation of tau is an important pathological feature of many neurodegenerative diseases called tauopathies. Thus, many efforts have been made to study the tau aggregation process and to ascertain the pathological forms of the degenerate tau aggregates. Recent studies suggest that soluble tau oligomers instead of NFTs are responsible for neurodegeneration and cognitive impairment, and tau oligomers are an important therapeutic target for stopping or reversing tau pathology symptoms.
In contrast to the pathological nature of tau aggregates, tau is a naturally "unfolded" protein that exhibits very high solubility under physiological conditions (Guo JL, Lee VMY: Seeding of Normal Tau by Pathological Tau Conformers Drives Pathogenesis of Alzheimer-like Tangles, Journal of Biological Chemistry 2011, 286 (17): 15317-31). To be an intermediate for aggregation, the tau must undergo a series of post-translational modifications, including processes such as hyperphosphorylation and proteolytic truncation. Among the various modifications, the formation of taurosulfide bonds has received a lot of attention in recent years, because the disulfide bonds greatly affect the tau aggregation phenomenon. Human full-length tau (441 amino acids) includes two cysteine residues (C291 and C322) that can form intramolecular and intermolecular disulfide bonds (Sahara N et al . , Assembly of two distinct dimers and higher-order oligomers from full-length tau. Eur J Neurosci 2007, 25 (10): 3020-9), and reports that intermolecular disulfide bonds formed by these cysteine residues promote tau aggregation on in vitro (Daebel V et al . : beta-sheet core of tau paired helical filaments revealed by solid-state NMR. Journal of the American Chemical Society < RTI ID = 0.0 > 2012, < / RTI > 134 (34): 13982-9). During the tau flocculation process, disulfide bonded dimers are first formed and then higher order oligomers are formed in cascade. These oligomers function as 'nuclei' to form paired helical filaments (PHF). In addition, substitution of both cysteines with alanine significantly reduced the rate of conversion to dimers and prevented the formation of higher order oligos (Barghorn S et al . , Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments. Biochemistry 2002, 41 (50): 14885-96). The formation of tau disulfide bonds plays a key role in the initiation of tau agglutination and accelerates the formation of PHF. Therefore, it is required to develop a therapeutic agent for inhibiting the formation of tau sulfide bonds to treat various neurodegenerative diseases or cognitive disorders.
The present invention has been made in view of the above problems, and it is an object of the present invention to provide an acrylonitrile compound which inhibits deformation such as oligomer formation of tau protein.
It is another object of the present invention to provide a pharmaceutical composition for inhibiting oligomer formation of intracellular tau protein comprising acrylonitrile compound according to various embodiments of the present invention.
It is another object of the present invention to provide a composition for treating neurodegenerative diseases comprising acrylonitrile compounds according to various embodiments of the present invention.
An aspect of the present invention relates to an acrylonitrile compound represented by the following formula (I).
(I)
Wherein X is C or S and R < 1 > And R 2 are the same or different from each other and each independently represents a hydrogen, a halogen group, a C 1 -C 7 alkyl group, a halogenated C 1 -C 7 alkyl group, a C 1 -C 7 alkoxy group and a halogenated C 1 -C 7 alkoxy group And when X is C, n is 1 or 2.
Another aspect of the present invention relates to a pharmaceutical composition for inhibiting oligomer formation of intracellular tau protein comprising an acrylonitrile compound.
Another aspect of the present invention relates to a composition for treating neurodegenerative diseases comprising an acrylonitrile compound.
The acrylonitrile compound according to the present invention inhibits deformation such as disulfide bonding, oligomerization and aggregation of cellular tau protein to prevent the expression of tau pathology, which is an important pathological feature of neurodegenerative diseases, Can be widely used for treatment.
1 is a schematic diagram of a process in which BiFC is matured according to tau phosphorylation.
Fig. 2 is a photograph taken after incubation of Tau-BiFC cells for 24 hours with Okada acid (30 nM) and phoscholine (20 μM).
3A is a graph showing RFU of Tau-BiFC cells in various compound libraries after culturing in 6% FBS (Fetal Bovine Serum).
FIG. 3B is a graph showing the RFU of Tau-BiFC cells in various compound libraries after culturing in a medium containing 3% HS (horse serum) and 6% FBS (Fetal Bovine Serum).
Figure 4a is a photograph of a Tau-BiFC incubated with 6% FBS followed by incubation with DSMO, okadaic acid (30 nM), phoscholine (20 [mu] M) and an acrylonitrile compound according to one embodiment of the invention It is a picture afterwards.
Figure 4b shows a photograph of the cells treated with DSMO, okadaic acid (30 nM), phoscholin (20 [mu] M) and acrylonitrile compound according to one embodiment of the present invention, respectively, after the tau-BiFC was cultured in 3% HS and 6% ≪ / RTI >
Hereinafter, the present invention will be described in more detail with reference to the drawings.
An aspect of the present invention relates to an acrylonitrile compound represented by the following formula (I).
(I)
Wherein X is C or S, R 1 and R 2 are the same or different and each independently represents hydrogen, a halogen group, a C 1 -C 7 alkyl group, a halogenated C 1 -C 7 alkyl group, a C 1 -C 7 alkoxy group And a halogenated C 1 -C 7 alkoxy group, and when X is C, n is 1 or 2.
In one aspect, R 1 may be a C 1 -C 7 alkoxy group, and R 2 may be a halogen group or a halogenated C 1 -C 7 alkyl group.
In another aspect, R 1 may be OCH 3 and R 2 may be CF 3 or Cl.
The acrylonitrile compound represented by the above formula (I) may be selected from compounds represented by the following formulas (1) to (5).
[Chemical Formula 1]
(2)
(3)
[Chemical Formula 4]
[Chemical Formula 5]
Another aspect of the present invention relates to a pharmaceutical composition for inhibiting oligomer formation of intracellular tau protein comprising an acrylonitrile compound.
Meanwhile, the pharmaceutical composition according to the present invention can be used in addition to tablets, capsules, syrups, coatings and patches.
The tablet is not particularly limited in terms of the production method. As a preferred embodiment, the pharmaceutical composition containing the PAC-1 or a mixture thereof is sieved, and lactose, starch and pregelatinized corn starch The granules are mixed with magnesium stearate and compressed. The preferred components and contents of the tablet are 5.0 mg of the pharmaceutical composition according to the present invention, 150.0 mg of lactose BP, 30.0 mg of starch BP, 15.0 mg of pregelatinized corn starch BP and 1.0 mg of magnesium stearate.
In addition, there is no particular limitation in the preparation method of the capsules, and as a preferable example, the pharmaceutical composition comprising any one of the above-mentioned gallan or camper rolls or a mixture thereof is sieved and then mixed with the excipient The preferred components and contents of the capsules are 5.0 mg of the pharmaceutical composition according to the present invention, 100.0 mg of starch and 1.0 mg of magnesium stearate BP.
There is no particular limitation on the preparation method of the syrup agent. In one preferred embodiment, white sugar is dissolved in purified water (500 ml), carboxymethyl cellulose sodium is dissolved in 400 ml of purified water in a separate vessel, Purified water and purified water in which methyl cellulose sodium was dissolved were mixed and dissolved by adding methylparaben and propylparaben. After addition of ethanol, purified water was added so that the total volume of the solution became 1,000 ml, , Or a mixture thereof. The preferred ingredients and the content of the syrup are 5.0 g of the pharmaceutical composition according to the present invention, 637.5 g of white sugar, 2.0 g of carboxymethylcellulose sodium 2.0 g, 0.28 g of methylparaben, 0.12 g of propylparaben, 20 ml of ethanol, The.
Another aspect of the present invention relates to a composition for treating neurodegenerative diseases comprising an acrylonitrile compound.
Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.
Although there are differences in the structure and physical properties of the substituents depending on the kind of the substituent, the reaction principle and conditions of the examples may be applied to the substituent-containing compounds not described in the examples of this specification, Therefore, it is obvious to those skilled in the art that these substituent-containing compounds can be easily carried out based on the disclosure of the examples and common sense in the art.
Example
Example 1: Synthesis of acrylonitrile compound represented by formula (1)
(E) -2- (4-methoxybenzoyl) -3- (4-methoxyphenyl) -3-oxopropanenitrile represented by the formula (1) (2- (trifluoromethyl) phenyl) acrylonitrile was synthesized.
[Reaction Scheme 1]
Propanol (2.0 M), and then 2- (trifluoromethyl) benzaldehyde (0.38 mL, 2.9 mmol) was added to a solution of 3- (4-methoxyphenyl) 2.9 mmol) and piperidine (0.04 mL, 0.4 mmol) were added thereto, followed by stirring at 60 ° C for 2 hours. Water is added dropwise to terminate the reaction. The reaction solution was diluted with ethyl acetate (EtOAc), washed with water and brine, and then the organic layer was dried with anhydrous Na 2 SO 4 and filtered. The solvent was distilled off under reduced pressure and the residue was purified by column chromatography (ethyl acetate / n-hexane 1: 9) to give (E) -2- (4- methoxybenzoyl) -3- (2- (trifluoromethyl ) Phenyl) acrylonitrile (2) (0.60 g) in a yield of 64%.
Yellow solid; Rf = 0.40 (n-hexane / EtOAc 3/1); mp: 72.0-74.0 DEG C; HPLC purity: 14.3 min, 99.5% (method A); 1 H NMR (400 MHz, CDCl 3) δ 8.30 (d, J = 1.8 Hz, C = C H), 8.15 (d, J = 7.8 Hz, Ar H), 7.96 (d, J = 9.0 Hz, 2 Ar H), 7.81 (d, J = 7.5 Hz, Ar H), 7.73 (t liked due to dd, J = 7.4 Hz, Ar H), 7.64 (t liked due to dd, J = 7.7 Hz, Ar H), 7.02 (d, J = 9.0 Hz , 2 Ar H), 3.91 (s, OCH 3); 13 C NMR (100 MHz, CDCl 3) δ 186.4 (C (O)), 164.5, 150.5, 132.6, 132.2, 131.5, 130.0,, 129.4 (q, J = 30.5 Hz), 127.6, 126.6 (q, J = 5.3 Hz), 123.7 (q, J = 272.3 Hz, C F 3), 116.0, 115.8, 114.3, 55.7 (O C H 3)
Examples 2 to 5: Synthesis of acrylonitrile compounds represented by Formulas 2 to 5
The acrylonitrile compounds represented by Formulas (2) to (5) were synthesized according to the following Reaction Scheme 2, and the compounds used were as shown in Table 1.
[Reaction Scheme 2]
Synthesis of Compound Represented by Formula 3a
(1.94 g, 25.7 mmol) and sodium carbonate (Na 2 CO 3 ; 3.54 g, 33.4 mmol) dissolved in acetone (2.0 M) were added to a solution of 2-methoxybenzenethiol mmol) are added and reacted at 50 ° C for 5 hours. After completion of the reaction, the reaction mixture was filtered under reduced pressure, and the residue was diluted with diethyl ether, washed with water and brine, and then the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The residue obtained by vacuum distillation was purified by column chromatography (ethyl acetate / n-hexane 1: 7) to obtain 2 - ((2-methoxyphenyl) thio) acetonitrile (3.5 g) .
Colorless oil; Rf = 0.45 (n-hexane / EtOAc 3/1); 1H NMR (400 MHz, CDCl 3 ) δ 7.52 (dd, J = 1.6, 7.6 Hz, ArH), 7.37 (td liked due to ddd, J = 1.6, 7.8 Hz, ArH), 6.97 (td liked due to ddd, J = 1.0, 7.6 Hz, ArH ), 6.94 (d, J = 8.2 Hz, ArH), 3.93 (s, OCH 3), 3.62 (s, SCH 2 CN); 13C NMR (100 MHz, CDCl 3 ) δ 158.5, 133.6, 130.5, 121.0, 119.2 (ArC), 116.6 (SCH 2 CN), 111.0 (ArC), 55.6 (OCH 3), 18.7 (SCH 2 CN)
Synthesis of Compound Represented by Formula 3b
(1.94 g, 25.7 mmol) and sodium carbonate (Na2CO3, 3.54 g, 33.4 mmol) were dissolved in acetone (2.0 M), followed by the addition of 4-methoxybenzenethiol (3.00 g, 21.4 mmol) And reacted at 50 ° C for 5 hours. After completion of the reaction, the reaction mixture was filtered under reduced pressure, and the residue was diluted with diethyl ether, washed with water and brine, and then the organic layer was dried over anhydrous Na 2 SO 4 and filtered. The residue obtained by vacuum distillation was purified by column chromatography (ethyl acetate / n-hexane 1: 5) to give 2 - ((4-methoxyphenyl) thio) acetonitrile (3.50 g) .
Colorless oil; Rf = 0.31 (n-hexane / EtOAc 5/1); J = 8.7 Hz, 2 ArH), 4.04 (s, SCH 2 CN), 3.78 (s, OCH 3, )
Synthesis of Compound Represented by Formula 4a
Example 2 - (2-Methoxyphenyl) thio) acetonitrile (1.00 g, 5.6 mmol) was dissolved in methylene chloride (0.2 M) and meta- chloroperoxybenzoic acid (mCPBA; 1.25 g, 5.6 mmol) at 0 ° C, and the mixture was stirred at 0 ° C for 2 hours. After the reaction was terminated with sodium sulfite, the reaction mixture was diluted with ethyl acetate and washed with a saturated sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous Na 2 SO 4 and filtered. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (ethyl acetate / n-hexane 1: 1) to give 2 - ((2-methoxyphenyl) Yl) acetonitrile (1.0 g) in a yield of 93%.
White solid; Rf = 0.25 (n-hexane / EtOAc 1/1); mp: 147.0-153.0 [deg.] C; 1H NMR (400 MHz, CDCl 3 ) δ 7.88 (dd, J = 1.6, 7.6 Hz, ArH), 7.56 (td liked due to ddd, J = 1.6, 7.6 Hz, ArH), 7.27 (td liked due to ddd, J = 0.8, 7.6 Hz, ArH), 6.98 (d, J = 8.2 Hz, ArH), 3.80-3.97 (m, S (O) CH 2 CN, OCH 3 ); (100 MHz, CDCl 3 )? 155.0, 133.8, 128.7, 126.3, 122.3 (ArC), 111.5 (S (O) CH 2 CN), 111.0 (ArC), 56.1 (OCH 3 ), 41.7 ) CH 2 CN)
Synthesis of Compound Represented by Formula 4b
(2.4 g, 13.4 mmol) was dissolved in methylene chloride (0.2 M) and meta-chloroperoxybenzoic acid (mCPBA; 3.0 g, 13.4 mmol) was added to a solution of 2- ((4- methoxyphenyl) thio) acetonitrile At 0 < 0 > C, and then stirred at -20 [deg.] C for 2 hours. After the reaction was terminated with sodium sulfite, the reaction mixture was diluted with ethyl acetate and washed with a saturated sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous Na2SO4, filtered, and the solvent was distilled off under reduced pressure. The resulting residue was purified by column chromatography (ethyl acetate / n-hexane 2: 3) to give 2 - ((4-methoxyphenyl) Acetonitrile (2.6 g) was obtained in a yield of 99%.
White solid; Rf = 0.26 (n-hexane / EtOAc 3/2); mp: 64.8-66.1 캜; 1H NMR (400 MHz, CDCl 3 ) δ 7.70 (m, J = 8.8 Hz, 2 ArH), 7.10 (m, J = 8.8 Hz, ArH), 3.05 (s, OCH 3), 3.69 (q, J = 15.3 , 30.9 Hz, S (O) CH 2 CN); 13C NMR (75 MHz, CDCl 3 ) δ 163.3, 132.1, 126.2, 115.2 (ArC), 111.4 (CN), 55.6 (OCH 3), 44.8 (S (O) CH 2 CN)
Synthesis of Compound Represented by Formula 5a
(2.00 g, 11.5 mmol) was dissolved in methylene chloride (0.20 M) and meta-chloroperoxybenzoic acid (mCPBA; 5.71 g, 25.5 mmol) was added at room temperature And the mixture was stirred at room temperature for 2 hours. After the reaction was terminated with sodium sulfite, the reaction mixture was diluted with ethyl acetate and washed with a saturated sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and the solvent was distilled off under reduced pressure. The resulting residue was purified by column chromatography (ethyl acetate / n-hexane 1: 1) to give 2 - ((2-methoxyphenyl) (2.2 g) was obtained in a yield of 96%.
White solid; Rf = 0.35 (n-hexane / EtOAc 1/1); mp: 74.0-76.0 DEG C; 1H NMR (400 MHz, CDCl 3 ) δ 7.99 (dd, J = 1.7, 7.9 Hz, ArH), 7.69 (td liked due to ddd, J = 1.7, 7.6 Hz, ArH), 7.16 (td liked due to ddd, J = 0.9, 7.8 Hz, ArH ), 6.94 (d, J = 8.4 Hz, ArH), 4.37 (s, s (O) 2 CH 2 CN), 4.02 (s, OCH 3); 13C NMR (100 MHz, CDCl 3 ) δ 157.4, 137.4, 131.4, 124.9, 121.4, 112.8 (ArC), 110.5 (S (O) CH 2 CN), 56.7 (OCH3), 44.2 (S (O) 2 CH 2 CN)
Example 2: Acrylonitrile The compound ((E) -2 - ((2- Methoxyphenyl ) Sulfinyl ) -3- (2- ( Trifluoromethyl ) Phenyl ) Acrylonitrile ) Synthesis of
White solid; Yield: 79%; Rf = 0.35 (n-hexane / EtOAc 3/1); mp: 107.0-109.0 [deg.] C; HPLC purity: 13.4 min, 96.1% (method A); 1H NMR (400 MHz, CDCl 3 ) δ 8.24 (d, J = 1.6 Hz, C = CH), 8.02 (d, J = 7.8 Hz, ArH), 7.89 (dd, J = 1.6, 7.8 Hz, ArH), J = 7.6 Hz, ArH), 7.51-7.60 (m, 2 ArH), 7.26 (td corresponding to ddd, J = 0.8, 7.6 Hz, ArH), 6.97 ( d, J = 8.3 Hz, ArH), 3.90 (s, OCH 3); 13C NMR (100 MHz, CDCl 3 ) δ 156.4 (ArC), 143.3 (C = CH), 133.9, 132.6, 131.4, 130.1, 129.9 (ArC), 129.2 (q, JCF 3 = 30.5 Hz), 128.6 (ArC) , 126.6 (q, JCF 3 = 21.1 Hz), 125.8, 124.2 (ArC), 123.7 (q, JCF 3 = 272.4 Hz, CF 3), 122.1 (ArC), 111.9 (CN), 111.2 (ArC), 55.6 ( OCH 3 )
Example 3: Synthesis of acrylonitrile compound ((E) -2 - ((4-methoxyphenyl) sulfinyl) -3- (2- (trifluoromethyl) phenyl) acrylonitrile synthesis
White solid; Yield: 56%; Rf = 0.36 (n-hexane / EtOAc 5/1); mp: 103.1-105.2 [deg.] C; 1H NMR (400 MHz, CDCl 3 ) δ 8.29 (s, C = CH), 7.94 (d, J = 7.6 Hz, ArH), 7.78 (d, J = 7.6 Hz, ArH), 7.56-7.70 (m, J = 8.8, 18.0, 25.5 Hz, 4 ArH), 7.06 (m, J = 8.8 Hz, 2 ArH), 3.87 (s, OCH 3); 13C NMR (75 MHz, DMSO) δ 162.9 (ArC), 141.5 (C = CH), 133.3, 131.7, 131.6, 129.8, 129.64 (ArC), 127.1 (q, JCF 3 = 30.0 Hz), 126.6 (q, JCF 3 = 5.2 Hz), 124.8 ( CF 3), 123.7 (q, JCF3 = 272.2 Hz), 115.5 (ArC), 112.1 (CN), 55.7 (OCH 3)
Example 4: Synthesis of acrylonitrile compound ((E) -2 - ((4-methoxyphenyl) sulfinyl) -3- (2-chlorophenyl) acrylonitrile
White solid; Yield: 51%; Rf = 0.36 (n-hexane / EtOAc 5/1); mp: 118.9-121.7 [deg.] C; 1H NMR (400 MHz, CDCl 3 ) δ 8.34 (s, C = CH), 8.03 (d, J = 7.7 Hz, ArH), 7.70 (d, J = 8.6 Hz, 2 ArH), 7.50 (d, J = 7.9 Hz, ArH), 7.42 ( t, J = 7.3 Hz, ArH), 7.35 (t, J = 7.6 Hz, ArH), 7.06 (d, J = 8.6 Hz, 2 ArH), 3.87 (s, OCH 3) ; 13C NMR (75 MHz, CDCl 3 ) δ 163.3 (ArC), 140.5 (C = CH), 135.7, 132.9, 132.0, 130.43, 129.7, 129.2, 127.6, 127.5 (ArC), 122.1 (S (O) CCN), 115.3 (2 ArC), 112.8 ( CN), 55.7 (OCH 3)
Example 5: Synthesis of acrylonitrile compound ((E) -2 - ((2-methoxyphenyl) sulfonyl) -3- (2- (trifluoromethyl) methyl) acrylonitrile synthesis
White solid; Yield: 81%; R f = 0.30 (n-hexane / EtOAc 3/1); mp: 114.0-116.0 [deg.] C; HPLC purity: 13.7 min, 100.0% (method A); 1H NMR (400 MHz, CDCl 3 ) δ 8.68 (d, J = 1.6 Hz, C = CH), 8.08-8.13 (m, 2 ArH), 7.82 (d, J = 7.5 Hz, ArH), 7.65-7.73 ( m, 3 ArH), 7.18 ( td liked due to ddd, J = 0.9, 8.0 Hz, ArH), 7.06 (d, J = 8.4 Hz, ArH), 3.93 (s, OCH 3); 13C NMR (100 MHz, CDCl 3 ) δ 157.8 (ArC), 149.5 (C = CH), 137.3, 132.7, 132.4, 131.6 (4 ArC), 129.7 (q, JCF 3 = 31.6 Hz), 126.8 (q, JCF 3 = 5.2 Hz), 124.3 ( ArC), 123.5 (q, JCF 3 = 272.4 Hz, CF 3), 120.9, 119.7 (2 ArC), 112.6 (CN), 112.3 (ArC), 56.2 (OCH 3)
Test Example 1: Possibility as an indicator of the tau assembly
A tau-BiFC was used to verify that the acrylonitrile compound prepared in the examples could be used as an indicator of the tau assembly. BiFC (bimolecular fluorescence complementation) is a method for visualizing protein-protein interactions based on the technique of forming fluorescent protein complexes from non-transmissive components attached to the protein of interest (Kerppola TK 2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu. Rev. Biophys. 37: 465-487).
To fabricate the tau-BiFC sensor, a Venus based BiFC system was used. Venus protein is a type of yellow fluorescence protein (YFP) that is well suited for the spatio-temporal analysis of tau binding, which is either (i) fast and effective maturation of the Venus protein, (ii) (Iii) the fluorescence intensity of the Venus-based BiFC is 10 times higher than that of the EYFP-based BiFC (Shyu YJ, Liu H, Deng X, Hu CD (2006) Identification of new fluorescent protein fragments for bimolecular fluorescence complementation assay under physiological conditions. Biotechniques 40: 61-66 .; Kodama Y, Hu CD (2010) Biotechniques 49: 793-805).
To produce the Venus tau-BiFC sensor, full-length human tau (441 a.a.) was fused to the N-terminal fragment (1-172 a.a., VN173) and the C-terminal fragment (155-238 a.a, VC155) of Venus. Specifically, the mammalian expression vector pCMV6-hTau40-GFP was purchased from OriGene Technologies Inc. (Rockville, Md.). To replace GFP with BiFC moieties, pBiFC-VN173 and pBiFC-VC155 were purchased from Addgene (Cambridge, Mass.) And then amplified using PCR primers with XhoI / PmeI restriction enzyme sequences. Each forward and back primer sequence is as follows:
(VC155-F) 5'-AATTCGGTCG ACCGAGATCT CTCGAGGTAC-3 '
(VC155-R) 5'-CTAGTTGTGG TTTGTTTAAA CTCATCAATG TATC-3 '
(VN173-F) 5'-ATGACGACAA GCTCGAGGCC GCGAATTCAT CG-3 '
(VN173-R) 5'-CTAGTTGTGG TTTGTTTAAA CTCATCAATG TATC-3 '
pCMV6-hTau40-VN173 and pCMV6-hTau-VC155 were prepared by digesting and splicing pCMV6-hTau40-GFP and PCR amplified insights using XhoI / PmeI.
Tau-BiFC cells were treated with okadaic acid and forskolin, respectively, to confirm that the acrylonitrile compounds prepared in the examples could be used as indicators of the tau assembly. The above-mentioned okada acid and phoscholine are known to induce hyperphosphorylation of tau. The battle tau has 79 putative serine and threonine residues. Phosphorylation of these residues is precisely controlled by protein kinases and phosphatases to maintain the microtubule mechanics required for nerve flexibility. Among these regulatory enzymes, the predominant tau phosphatase in the human brain is the protein phosphatase 2A (PP2A), which regulates the activity of some protein kinases that phosphorylate tau. Okada acid is known to induce Alzheimer-like tau phosphorylation in rat brain with low PP2A (Arias C, Sharma N, Davies P, Shafit-Zagardo B (1993) 2 and tau phosphorylation prior to neurodegeneration in cultured cortical neurons. J. Neurochem. 61: 673-682 .; Zhang Z, Simpkins JW (2010) Okadaic acid induces tau phosphorylation in SH-SY5Y cells in an estrogen- 1345: 176-181). Activation of protein kinase A (PKA) by phokolin is known to induce hyperphosphorylation and memory decline of tau (Liu SJ, Zhang JY, Li HL, Fang ZY, Wang Q, et al. a more favorable substrate for GSK-3 when it is prephosphorylated by PKA in rat brain. 279: 50078-50088 .; Tian Q, Zhang JX, Zhang Y, Wu F, Tang Q, et al. 2009) Biphasic effects of forskolin on tau phosphorylation and spatial memory in rats J. Alzheimers Dis. 17: 631-642).
Fig. 1 is a schematic diagram of the process of maturation of BiFC according to tau phosphorylation, and Fig. 2 is a photograph taken after incubation of tau-BiFC cells with Okada acid (30 nM) and phoscholine (20 uM) for 24 hours.
Referring to FIG. 2, when the tau-BiFC cells were incubated with okadaic acid and phocolin for 24 hours, the BiFC fluorescence increased 2.2-fold and 1.9-fold, respectively. From this, it can be seen that the increase in BiFC fluorescence increased the level of Ta-tau interaction in the cells, resulting in oligomer formation of tau.
Test Example 2: Inhibitory effect of acrylonitrile derivatives on tau protein oligomer formation
Compound library was used to screen for the inhibitory effect of tau protein oligomer formation. In Tau-BiFC cells under two culture conditions with different kinds of culture media, various compounds contained in the above-mentioned compound library, the compound represented by the formula 1 prepared in Example 1 and the compound represented by the formula 2 prepared in Example 2 Were co-cultured for the inhibition of tau protein aggregation. When the DMSO, the okadaic acid, and the phoscholine were co-cultured together with the compound represented by the formula 1 prepared in Example 1 and the compound represented by the formula 2 prepared in Example 2, Tau- The relative fluorescence units (RFU) of BiFC cells were compared.
FIG. 3A is a graph showing the RFU of Tau-BiFC cells after culturing for 18h and 29h in DMEM medium containing 6% FBS (Fetal Bovine Serum). The results are shown in Table 1 below. It was confirmed that the compound represented by the formula (2) prepared in (1) significantly inhibited the aggregation of the tau protein by oligomer formation.
FIG. 3B is a graph showing RFU of Tau-BiFC cells after culturing for 18 h and 29 h in a medium containing 3% HS (horse serum) and 6% FBS (Fetal Bovine Serum) It was confirmed that the compound displayed markedly inhibited aggregation due to oligomer formation of tau protein.
The Tau-BiFC cell line was treated with the compound represented by the formula 2 prepared in Example 2, which showed excellent oligomer formation inhibitory effect on both of the two different culture conditions, and the degree of oligomer formation of tau was observed by fluorescence imaging Are shown in Figs. 4A and 4B.
FIGS. 4A and 4B are photographs showing cell distribution of Tau-BiFC fluorescence. Compared with other compounds, the compound represented by the formula 2 prepared in Example 2 is superior in the oligomer formation inhibitory effect of tau protein.
It will be apparent to those skilled in the art that the present invention is not limited to the embodiments described above and that various changes and modifications may be made without departing from the spirit and scope of the present invention as defined by the appended claims. As shown in FIG.
It will be understood by those skilled in the art that the present invention may be embodied in many other specific forms without departing from the spirit or scope of the invention as defined by the appended claims and their equivalents. .
Claims (7)
[Chemical Formula 1]
(2)
(3)
[Chemical Formula 4]
[Chemical Formula 5]
Wherein Me is a CH 3.
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