WO2016047924A2 - Fluorescent-labeled novel dieckol derivative - Google Patents

Abstract

The present invention relates to a fluorescent-labeled novel dieckol derivative.

Description

Fluorescently Labeled Novel Diechol Derivatives

The present invention relates to fluorescently labeled novel diecol derivatives.

Endoplasmic Reticulum (hereinafter referred to as ER) is a major site involved in protein synthesis, protein folding and calcium ion storage.

ER stress refers to stress that cells receive due to accumulation and aggregation of misfolded proteins, abnormal overexpression of specific proteins, and inhibition of normal post-translational processes such as glycosylation reactions. Continued ER stress or ER dysfunction not only causes Alzheimer's and Parkinson's disease, but also causes chronic inflammatory metabolic diseases such as diabetes and obesity. The cause of ER stress is due to the occurrence of oxidative stress in the ER. Therefore, if the antioxidant activity can be properly acted in the ER, it will be able to treat various inflammatory diseases by effectively preventing the ER stress (Stress).

However, there is still no technology for selectively delivering drugs, particularly antioxidants, to specific areas of cells, particularly ER, and thus, there is a difficulty in developing a safe and effective inflammatory disease treatment.

Dietol is a natural polyphenolic antioxidant that is produced from brown algae of Ecklonia and Eisenia, such as Ecklonia cava and rhubarb, and its safety has been verified, and various pharmacological effects such as anti-inflammatory, anti-diabetic, anti-allergic, antiviral and anti-cancer effects Activity is known. Therefore, if dietol can have a selective action on ER while having such activity, a new level of medicine for treating the above-mentioned disease, that is, a drug development strategy targeting ER is possible.

It is therefore an object of the present invention to provide novel fluorescently labeled diecol derivatives capable of targeting ER.

In addition, an object of the present invention is to provide a composition for the prevention and treatment of ER-stress mediated disease comprising the fluorescently labeled diecol derivatives as an active ingredient.

It is also an object of the present invention to provide a method for preparing the novel fluorescently labeled diecol derivatives.

In order to achieve the above object,

The present invention provides fluorescently labeled diecol derivatives of the following general formula (1) or (7).

[Formula 1]

[Formula 7]

In addition, the present invention provides a Dieckol compound comprising a propargyl group represented by the following Chemical Formula 2.

[Formula 2]

In addition, the present invention is a compound of Formula 1 by reacting a Dieckol compound containing a propargyl group of Formula 2 with a rhodamine B derivative containing an azo group of Formula 3 It provides a method of manufacturing.

[Formula 3]

In addition, the present invention is a fluorescein isothiocyanate (FITC) comprising a dieth (Dieckol) compound containing a propargyl group of formula (2) comprising an azo group of the formula (8) The present invention provides a method for preparing the compound of Formula 7 by reacting with a derivative.

 [Formula 8]

In another aspect, the present invention provides a composition for the prevention and treatment of ER-stress (Endoplasmic reticulum stress) mediated disease comprising the fluorescently labeled diethol derivative of Formula 1 or Formula 7 as an active ingredient.

The present invention has the effect of selectively targeting ER, thus providing a novel fluorescently labeled diecol derivative useful for the prevention and treatment of ER-stress mediated diseases.

In addition, the compositions comprising the novel fluorescently labeled diecol derivatives of the present invention have an excellent effect in the prevention and treatment of ER-stress mediated diseases.

In addition, the present invention can provide a new technology that can be used for various chronic diseases, such as diabetes, which is an inflammatory disease caused by ER stress, by providing a novel anti-inflammatory material that selectively targets ER.

1 is a schematic diagram showing a method for preparing a fluorescently labeled novel diethol derivative of Formula 1 of the present invention.

Figure 2 is a schematic diagram showing a method for producing a fluorescently labeled novel diethol derivative of formula (7) of the present invention.

3 is a confocal microscope image of Experimental Example 1. FIG.

4 is a graph of NO measurement results measured in Comparative Example 2. FIG.

5 is a graph of comparative measurements of TNF-α measured in Experimental Example 2. FIG.

6 is a graph of IL-6 measurement results of comparative measurements of Experimental Example 2. FIG.

7 is a confocal microscope image of Experimental Example 3. FIG.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail. The following detailed description is for the description of one embodiment of the present invention, although not limited to the scope of the claims defined by the claims.

In the prior art, there is still no technology for selectively delivering drugs, particularly antioxidants, to specific sites in the cell, particularly ER, and thus, there is a difficulty in developing a safe and effective therapeutic agent for inflammatory diseases.

Accordingly, the present inventors synthesized novel dietol derivatives of the following Chemical Formula 1 or the following Chemical Formula 7 which introduced fluorescent substances capable of tracking the intracellular location while maintaining the antioxidant and anti-inflammatory ability of the dietol.

[Formula 1]

[Formula 7]

In addition, the present invention provides a diethol derivative comprising a propazyl group of the following formula (2) in which a propazyl group is introduced into diethol.

[Formula 2]

According to a preferred embodiment of the present invention, the diethol derivative including the propazyl group of Formula 2 may provide a novel diethol derivative that can be usefully used for effective binding to various azo compounds.

Meanwhile, in the present invention, in order to synthesize the compounds of Formula 1 or 7, synthesized a novel compound of Formula 2 wherein a propargyl group is introduced into Dieckol, an antioxidant.

In addition, a compound of the following Chemical Formulas 3 and 8, in which an azo group was introduced into Rhodamine B or FITC (Fluorescein isothiocyanate) as a fluorescent substance, was synthesized. Thereafter, the compounds of Formula 2 and the compounds of Formulas 3 and 8 are combined to synthesize each.

 [Formula 3]

[Formula 8]

In the compound of Formula 1 or Formula 7, dietol is a natural polyphenolic antioxidant produced from brown algae of Ecklonia and Eisenia, such as Ecklonia cava and rhubarb, and has been proven to be safe, and has anti-inflammatory, anti-diabetic, anti-allergic and antiviral properties. And various pharmacological activities such as anticancer effects are known. The compound of formula (1) or compound of formula (7) labeled with Rhodamine B or FITC, which is a fluorescent label on a diet, is easily tracked in the body by a fluorescent group, and particularly has a superior effect of targeting ER.

The present invention also provides a composition for the prevention and treatment of ER-stress (Endoplasmic reticulum stress) mediated disease comprising the compound of Formula 1 or Formula 7 as an active ingredient.

In the composition for the prevention and treatment of ER-stress (Endoplasmic reticulum stress) mediated diseases, the compound of Formula 1 is preferably included 1 to 50% by weight relative to the total weight percentage of the composition.

In addition, the compound of Formula 7 in the composition for the prevention and treatment of ER-stress (Endoplasmic reticulum stress) mediated disease is preferably included 1 to 50% by weight relative to the total weight percentage of the composition.

According to a preferred embodiment of the present invention, the ER-stress-mediated disease is diabetes type 1, type 2 diabetes, Alzheimer's disease, obesity, immunoglobulin light chain amyloidosis, Parkinson's disease, amyotrophic lateral sclerosis (ALS), haemodialysis-related amyloidosis, reactive amyloidosis, cystic fibrosis, sickle cell anemia cell anemia, Huntington's disease, Creutzfeldt-Jakob disease and related disorders (prion encephalopathies), familial hypercholesterolaemia, alpha1-antitrypsin deficiency (Alpha1) antitrypsin deficiency, cirrhosis, emphysema systemic, cerebral hereditary amyloidoses), Wolfcott-Rallison syndrome, Wolfram syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, breast cancer and prostate cancer Can be. Among them, the prevention and treatment of the compositions of the present invention are particularly effective against Alzheimer's disease, Parkinson's disease and obesity.

Hereinafter, a method for preparing a fluorescently labeled novel diethol derivative of Chemical Formula 1 according to the present invention will be described with reference to FIG. 1.

In order to introduce the rhodamine B compound represented by the following formula (6) into dietol, a rhodamine B derivative having a piperazine group represented by the following formula (4) was synthesized, and a rhodamine B derivative represented by the following formula (3) having an azo group introduced therefrom.

[Formula 6]

[Formula 4]

[Formula 3]

Apart from this, a compound of formula 2 having a propazyl group introduced into a diecol of formula 5 may be synthesized, and reacted with a rhodamine B derivative of formula 3 to synthesize a compound of formula 1 below.

[Formula 5]

[Formula 2]

[Formula 1]

In addition, the method for preparing a fluorescently labeled novel diethol derivative of Chemical Formula 7 according to the present invention will be described with reference to FIG. 2.

In order to introduce fluorescein isothiocyanate (FITC) of formula (9) to dietol to react with 3-azidopropyl amine (3-azidopropyl amine), an azo group was introduced. FITC derivatives of were synthesized.

[Formula 9]

[Formula 8]

Separately, the compound of Formula 7 may be synthesized by reacting the compound of Formula 2 having a propazyl group in the diecol of Formula 5 with the FITC derivative of Formula 8.

[Formula 7]

Hereinafter, the present invention will be described in more detail with reference to Examples, but embodiments of the present invention disclosed below are exemplified to the last, and the scope of the present invention is not limited to these embodiments. The scope of the invention is indicated in the appended claims, and moreover contains all modifications within the meaning and range equivalent to the claims.

Synthesis Example 1 Synthesis of Compound of Formula 2

Dichloromethane (DCM) (5 mL), from which moisture was removed, was mixed with diecol (50 mg, 0.067 mmol) and anhydrous calcium carbonate (14 mg, 0.1 mmol) represented by the following formula (5). Propargyl bromide (0.04 ml, 0.08 mmol) was added slowly over 8 hours under normal temperature and stirring conditions.

After the reaction solvent was removed in vacuo, the resulting mixture was washed with distilled water (5 mL) and then centrifuged to remove the aqueous solution. The solid material thus obtained was washed with acetone to remove water and then vacuum dried.

The dried solid phase material was purified by HPLC (acetonitrile-water binary gradient solvent system) to separate the pure compound of formula 2 (20mg, 38% yield) as a brown solid.

To prepare a compound of formula 2 was confirmed by ¹ H-NMR, it is shown below.

¹ H-NMR (700 MHz, DMSO- d ₆ ): δ 9.14 -9.68 (ArOH, 10H, m), 6.21 (ArH, 1H, s), 6.19 (ArH, 1H, d, J = 2,7 Hz), 6.16 (ArH, 1H, s), 6.05 (ArH, 1H, d, J = 2.7 Hz), 6.04 (ArH, 1H, d, J = 2.8 Hz), 5.97 (ArH, 2H, s), 5.81 (ArH, 1H, t, J = 2.0 Hz), 5.80 (ArH, 1H, d, J = 2.9 Hz), 5.73 (ArH, 2H, d, J = 2.0 Hz), 4.68 (CH ₂ , 2H, d, J = 2.2 Hz), 3.57 (CH, 1H, t, J = 2.3 Hz); ¹³ C-NMR (176 MHz, DMSO- d ₆ ): δ 160.27, 158.79, 155.83, 154.17, 153.15, 151.19, 146.11, 146.06, 145.93, 145.81, 142.45, 142.37, 142.03, 141.86, 137.03, 136.81, 4.0. , 124.00, 123.09, 123.05, 122.25, 122.15, 98.49, 98.28, 98.18, 98.13, 96.32, 96.18, 94.42, 93.60, 93.39, 78.91, 78.83. 56.54; ESI-MS: m / z 781.07 [M + H] ⁺ (781.10, calcd for C ₃₉ H ₂₅ O ₁₈ ).

[Formula 5]

[Formula 2]

Synthesis Example 2 Synthesis of Compound of Formula 3

2-1. Synthesis of Compound of Formula 4 as Precursor of Compound of Formula 3

Piperazine (194 mg, 2.25 mmol) was added to Dichloromethane (DCM) (6 mL) and stirred under HATU (1- [Bis (dimethylamino) methylene] -1H-1,2,3- triazolo [4,5-b] pyridinium 3-oxid hexafluorophosphate, 198 mg, 0.52 mmol), N, N-diisopropylethylamine (DIPEA, N , N- Diisopropylethylamine, 0.17 mL, 1.0 mmol) and the following Chemical Formula 6 Compound Rhodamine B (200 mg, 0.45 mmol) was added. The reaction solution was stirred at room temperature for 3 hours and then stopped with dilute hydrochloric acid. The organic solution layer was washed thoroughly with dilute hydrochloric acid and sodium chloride solution, and then dried using sodium sulfate (Na ₂ SO ₄ ) to remove moisture, and then concentrated in vacuo. The obtained reaction product was purified using silica gel chromatography (CH ₂ Cl ₂ -MeOH, 30: 1) to obtain a compound of formula 4 (163 mg, 71% yield) as a purple purple solid.

To prepare a compound of formula 4 was confirmed by ¹ H-NMR, it is shown below.

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.66 (ArH, 2H, m); 7.54 (ArH, 1H, m), 7.32 (ArH, 1H, m), 7.27 (ArH, 1H, s), 7.23 (ArH, 1H, s), 6.93 (ArH, 2H, m), 6.76 (ArH, 2H , d, J = 4 Hz), 3.61 (CH ₂ , 8H, m), 3.35 (CH ₂ , 4H, m), 2.69 (CH ₂ , 4H, t, J = 8 Hz), 1.32 (CH ₃ , 12H , t, J = 8 Hz); ¹³ C-NMR (100 MHz, CDCl ₃ ): δ 167.63, 157.94, 156.28, 155.82, 135.91, 132.34, 130.75, 130.35, 130.28, 129.89, 127.87, 114.25, 113.93, 96.41, 49.05, 46.25, 43.10, 12.77.

[Formula 6]

[Formula 4]

2-2. Synthesis of Compound of Formula 3

To the DMF (5 mL) was added the compound of Formula 4 (150 mg, 0.294 mmol) prepared in Synthesis Example 2-1 and stirred while HATU (128 mg, 0.34 mmol), DIPEA (0.12 mL, 0.66 mmol) And 2-azidoacetic acid (33 mg, 0.32 mmol) were added. The reaction solution was stirred at room temperature overnight (overnight) and then the solvent was removed under vacuum. The obtained reaction product was purified using silica gel chromatography (CH ₂ Cl ₂ -MeOH, 35: 1) to obtain a compound of formula 3 (131 mg, 75% yield) as a purple purple solid.

To prepare a compound of formula 3 was confirmed by ¹ H-NMR, it is shown below.

¹ H-NMR (400 MHz, DMSO- d ₆ ): δ 7.70 (ArH, 3H, m), 7.50 (ArH, 1H, s), 7.10 (ArH, 4H, m), 6.91 (ArH, 2H, s) , 4.09 (CH ₂ , 2H, s), 3.62 (CH ₂ , 8H, m), 3.13 (CH ₂ , 4H, m), 2.65 (CH ₂ , 4H, m), 1.17 (CH ₃ , 12H, m) ; ¹³ C-NMR (100 MHz, DMSO- d ₆ ): δ 167.27, 166.69, 157.73, 156.24, 155.77, 135.78, 132.40, 131.39, 131.03, 130.54, 128.24, 128.04, 114.91, 113.70, 96.56, 50.32, 47.31, 46.06 , 38.94, 13.10; ESI-MS: m / z 594.50 (M ⁺ ) (594.32, calcd for C ₃₄ H ₄₀ N ₇ O ₃ ⁺ ).

 [Formula 3]

Synthesis Example 3 Synthesis of Compound of Formula 8

myriad Dichloromethane (DCM) (500 uL) was added to the compound of formula 9 (FITC, Fluorescein isothiocyanate, 100mg, 0.257 mmol) and stirred while adding TEA (Triethylamine, 400 ul). To this, 3-azidopropylamine (3-azidopropyl amine, 27 mg, 0.27 mmol) was dissolved in anhydrous DMF (200ul) and added under an ice bath. The reaction solution was stirred for 2 hours at room temperature and then the solvent was removed under vacuum. The obtained reaction product was purified using silica gel chromatography (CH ₂ Cl ₂ -MeOH, 95: 5) to obtain a compound of formula 8 (108 mg, 86% yield) as a light yellow solid.

To prepare a compound of formula 8 was confirmed by ¹ H-NMR, it is shown below.

¹ H NMR (DMSOd ₆ , 400 MHz) δ 10.73 (COOH, 1H, s); 8.83 (1 H, s); 8.31 (ArH, 1 H, s); 7.72 (ArH, 1H, d, J = 8 Hz); 7.09 (ArH, 1H, d, J = 8 Hz); 6.66 (ArH, 2H, d, J = 8 Hz); 6.51 (ArH, 4H, m); 3.50 (CH ₂ , 2H, m); 3.37 (CH ₂ , 2H, t, J = 8 Hz); 1.75 (CH ₂ , 2H, m). ¹³ C-NMR (100 MHz, DMSO- d ₆ ): 181.27, 169.84, 165.62, 154.15, 141.95, 130.21, 127.84, 126.13, 119.11, 116.02, 111.34, 103.13, 102.98, 49.25, 49.20, 41.73, 28.39 ppm.

[Formula 9]

[Formula 8]

Example 1 Synthesis of Compound of Formula 1

After dissolving the compound of Formula 2 prepared in Synthesis Example 1 (52 mg, 0.067 mmol) and the compound of Formula 3 prepared in Synthesis Example 2 (40 mg, 0.067 mmol) in DMF (5 mL), the solution was The dissolved gas was removed by bubbling with argon for 1 hour.

Separately, copper perchlorate, (Cu (ClO ₄ ) (5.3 mg, 0.02 mmol) and sodium ascorbate (soduim L-ascorbate) (7.9 mg, 0.04 mmol) were dissolved in 3 mL of DMF, followed by argon as above. 1 hour of bubbling (bubbling) to remove the dissolved gas.

When the color of the copper solution changed from brown to light yellow, the copper (I) solution was injected into the prepared reaction solution by syringe. The reaction mixture thus obtained was stirred at room temperature for 48 hours, and then the reaction solution was poured into a large amount of diethyl ether to make a suspension, and then the solid was separated by centrifugation.

The obtained solid was washed with water (5 ml, 3 times), methanol (15 mL, 3 times), acetone (10 mL, 3 times) and dichloromethane, DCM (15 mL, 3 times). After washing with water and dried in vacuo to give the compound of formula 1 (32 mg, 35% yield) as a purple solid.

To prepare a compound of Formula 1 was confirmed by ¹ H-NMR, it is shown below.

¹ H-NMR (700 MHz, DMSO- d ₆ ): δ 9.12-9.7 (dieckol), 6.54-7.77 (rhodamine), 5.41-6.2 (dieckol), 1.1-4.36 (rhodamine); ¹³ C-NMR (176 MHz, DMSO- d ₆ ): δ 206.57, 204.58, 172.77, 166.66, 158.86, 158.74, 157.03, 155.46, 155.08, 146.02, 135.06, 131.75, 130.40, 129.83, 127.51, 126.40, 124.60, 124.60 , 123.83, 123.11, 122.10, 121.84, 121.28, 119.76, 115.59, 114.23, 112.99, 111.65, 105.39, 98.25, 95.87, 94.00, 93.55, 91.28, 87.86, 74.77, 73.22, 62.74, 45.36, 35.77, 31.26, 30.67 , 18.21, 12.41; MALDI-TOF-MS: m / z 1374.507 (M ⁺ ).

[Formula 1]

Example 2. Synthesis of Compound of Formula 7

The compound of Formula 2 prepared in Synthesis Example 1 (20 mg, 0.064 mmol) and the compound of Formula 8 prepared in Synthesis Example 3 (31 mg, 0.064 mmol) were diluted with dichloromethane (DCM) (5 mL). ), And the solution was bubbled with argon for 1 hour to remove dissolved gas.

Separately, copper perchlorate (Cu (ClO ₄ ) ₂ (5.3 mg, 0.02 mmol) and sodium ascorbate (7.9 mg, 0.04 mmol) were dissolved in 3 mL of DMF, followed by argon as above. 1 hour of bubbling (bubbling) to remove the dissolved gas.

When the color of the copper solution changed from brown to light yellow, the copper (I) solution was injected into the prepared reaction solution by syringe. The reaction mixture thus obtained was stirred at room temperature for 72 hours, the reaction solution was poured into a large amount of methanol (50 ml) to form a suspension, and the solids were separated by centrifugation.

The obtained solid was washed with water (5 ml, 3 times), methanol (15 mL, 3 times), acetone (10 mL, 3 times) and dichloromethane, DCM (15 mL, 3 times). It was washed with and then dried in vacuo to give the compound of formula 7 (20 mg, 24.5% yield) as a brown solid.

To prepare a compound of formula 7 was confirmed by ¹ H-NMR, it is shown below.

¹ H NMR (DMSO-d ₆ , 500 MHz) δ 8.9 to 9.91 dieckol; 8.2-7.07 FITC; 6.38-66.6 FITC and dieckol; 1.1-3.5 FITC; ¹³ C-NMR (176 MHz, DMSO- d ₆ ): 168.89, 168.45, 159.45, 159.35, 158.87, 155.17, 155.14, 152.57 151.88, 151.85, 144.78, 142.20, 134.70, 129.82, 129.04, 126.93, 126.93, 126.93 , 124.54, 124.18, 124.13, 123.29, 121.82, 121.27, 116.85, 112.55, 112.49, 11.61, 109.95, 109.63, 102.21, 102.15, 95.02, 83.01, 82.84, 48.58, 48.47, 47.27, 41.43, 41.31, 41.19. , 31.28, 30.38, 28.96, 27.72, 27.48, 26.89 ppm.

[Formula 7]

Experimental Example 1. Confirmation of intracellular distribution of compounds of Formula 1 and Formula 3

In order to confirm the intracellular distribution of the compound of Chemical Formula 1, ER tracker (Endoplasmic Reticulum tracker, 1.0 μM), mitochondria tracker (0.2 μM) and a confocal laser microscopy in RAW 264.7 cells Lysosome tracker (2.0 μM) and co-localization experiments were performed.

That is, Confocal microscopy was performed by treating the compound of Formula 1 alone (2 μM) synthesized in Example 1 in each RAW 264.7 cell, treatment with each tracker alone, and simultaneous treatment (all cases at 37 ° C. for 20 minutes). Images (image size: 75 × 75 μm ² ) were obtained and compared (FIG. 3).

Excitaton frequency in the co-calibration experiment was 488nm, and the emission light was collected in the 560 ~ 620 nm region. In addition, each tracker alone has an excitation and emission frequency of Green, an ER Tracker of 488 nm and 500 to 540 nm, and a deep red Mito tracker. Mito Tracker is 488 nm and 660-750 nm. And Blue (Lyso tracker blue DND-22) was 740 nm and 360 ~ 520 nm.

 Meanwhile, a Confocal microscopy image of the compound of Formula 3, which is a phosphor of the compound of Formula 1, as the phosphor (2 μM) was obtained in the same manner as above, and compared with the compound of Formula 1 (FIG. 3).

According to the merged image in FIG. 3, the compound of Formula 1 of Example 1 is mostly present in ER, whereas the compound of Formula 3 of Synthesis Example 2 is present only in mitochondria as in the case of pure Rhodamine B. It can be seen.

Thus, it was found that a compound originally going to another region went to ER by linking the diecol.

Experimental Example 2 Activity Comparison Evaluation

LPS-stimulated RAW264.7 to confirm the activity of the compound of Formula 1 of Example 1, the compound of Formula 3 of Synthesis Example 2, Dieeckol of Formula 5 and Rhodamine B of Formula 6 The effects of NO, TNF-α and IL-6 inhibition on inflammation mediators in macrophages were evaluated.

Each of the compound of Formula 1 of Formula 1, Formula 3 of Synthesis Example 2, Dieeckol of Formula 5 and Rhodamine B of Formula 6 were treated in a cell culture solution after 24 hours after LPS treatment. The concentrations of NO, TNF-α and IL-6 were measured. The results are shown in FIGS. 4, 5, and 6, respectively.

Referring to Figure 4, it was found that when treated with the compound (Dieckol) of Formula 5 and the compound of Formula 1, both of them reduce the generation of NO by LPS.

But It can be seen that the compound of formula 1 (IC ₅₀ = 2.5 μM) exhibits 8 times stronger inhibitory power than that of diecol (IC ₅₀ = 20 μM).

Referring to FIG. 5, similarly to the result of NO, the compound of formula 1 also showed an effect superior to dietol in the ability to inhibit TNF-α generation by LPS.

In addition, referring to Figure 6, 1.25 μM of the compound of formula (1) shows an excellent IL-6 inhibition inhibition compared to the case of 20 μM dietol. It can be seen that the compound of Formula 1 shows at least 16 times better effect than dietol.

On the other hand, in the case of Rhodamine B of Formula 6 and the compound of Formula 3 of Synthesis Example 1 used in the synthesis of the compound of Formula 1 showed no effect at all in NO, TNF-α and IL-6.

In conclusion, the activity of the compound of the formula (1) is due to the Dieckol (Dieckol), it was confirmed that entering the ER by the combination of Rhodamine B (Dihodol).

Therefore, it can be seen that the compound of Formula 1 is amplified anti-inflammatory activity by lowering the ER stress more effectively than dietol.

Experimental Example 3. Confirmation of intracellular distribution of compound of formula 7 and compound of formula 8

In order to confirm the intracellular distribution of the compound of Formula 7, ER tracker (Endoplasmic Reticulum tracker, 1.0 μM), Mitochondria tracker (0.2 μM) and the like using confocal laser microscopy in BV-2 cells. Lysosome tracker (2.0 μM) and co-localization experiments were performed. That is, the Confocal microscope was treated with the compound of Formula 7 (5 μM) synthesized in Example 2 alone, each tracker alone and concurrent treatment (20 minutes at 37 ° C.) in a RAW BV-2 cell. microscopy) images (image size: 75 × 75 μm ² ) were obtained and compared (FIG. 7).

Excitaton frequency in the co-calibration experiment was 514 nm, and the light was collected in the 530-600 nm region. In addition, each tracker alone has an excitation and emission frequency of Red, an ER tracker of 514 nm and 600-700 nm, and a deep red mito tracker. Mito Tracker is 633 nm and 650-750 nm. Blue Lyso trackers were 740 nm and 400-500 nm. Meanwhile, a Confocal microscopy image of the compound of Formula 8 (5 μM), which is a phosphor of the compound of Formula 7, was obtained and compared with the compound of Formula 7 (FIG. 7).

According to the merged image of FIG. 7, the compound of Formula 7 of Example 2 is present only in the ER, whereas the compound of Formula 8 of Synthesis Example 3 is interspersed without distinguishing between mitochondria and ER. have. Therefore, it was found that the compound of Formula 7 selectively enters ER through the combination with dietol as in the case of the compound of Formula 1.

Therefore, by chemically combining diethol, which has excellent antioxidant and anti-inflammatory properties, and rhodamine B or FITC, which have a fluorescence function, it is possible to provide a novel fluorescent diethyl derivative which selectively enters ER.

Claims (6)

Fluorescently labeled diecol derivatives of Formula 1 or Formula 7. [Formula 1]

[Formula 7] Dieckol derivative comprising a propargyl group of formula (2). [Formula 2]

The Dieckol derivative including the propargyl group of Formula 2 is reacted with Rhodamine B derivative including the azo group of Formula 3 below. Method for preparing fluorescently labeled diecol derivatives. [Formula 3]

The dieckol derivative including the propargyl group of Formula 2 is reacted with a FITC derivative including the azo group of Formula 8 below. Method for preparing kole derivatives. [Formula 8]

A composition for preventing and treating ER-stress (Endoplasmic reticulum stress) mediated diseases comprising the fluorescently labeled diecol derivative of Formula 1 or Formula 7 as an active ingredient. The method according to claim 5, The ER-stress mediated diseases include type 1 diabetes, type 2 diabetes, Alzheimer's disease, obesity, immunoglobulin light chain amyloidosis, Parkinson's disease, muscular dystrophy Amyotrophic lateral sclerosis (ALS), hemodialysis-related amyloidosis, active amyloidosis, cystic fibrosis, sickle cell anemia, Huntington's disease Creutzfeldt-Jakob disease and related disorders (prion encephalopathies), familial hypercholesterolaemia, Alpha1-antitrypsin deficiency, cirrhosis, Emphysema systemic, cerebral hereditary amyloidoses, and Wolcott-Rallison syndrome ER-stress (Endoplasmic reticulum stress), characterized in that it comprises any one or more selected from the group consisting of son syndrome, Wolfram syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, breast cancer and prostate cancer ) Composition for preventing and treating mediated diseases.

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