KR102023845B1 - Histone deacetylases inhibitor and use thereof - Google Patents

Abstract

The present invention relates to a novel histone deacetylase (HDAC) inhibitor and a medical use thereof. More specifically, it is confirmed that a novel aspirin derivative inhibits the activity of HDAC by bonding to a substrate bonding pocket of HDAC and significantly increases the acetylation of intracellular α-tubulin and histone H3, thereby exhibiting an effect of inhibiting cancer cell proliferation. The novel aspirin derivative is provided as HDAC inhibitors, thereby being able to be provided as a therapeutic agent effective for HDAC-related cancer diseases and central nervous system diseases.

Description

Histone deacetylases inhibitor and use thereof

The present invention relates to novel histone deacetylase inhibitors and their medical use.

Histone deacetylases (HDACs) can be attached to gene promoters by corepressors or multi-protein transcriptional complexes, where they do not directly bind DNA to chromatin. (chromatin) modification regulates transcription.

These HDAC enzymes are involved in the regulation of many cellular processes.Histon acetylconvertase (HAT) and HDAC enzymes affect transcriptional activity by acetylating or deacetylating lysine residues at the N-terminus of histone proteins. It is known to regulate acetylation after transcription of at least 50 nonhistone proteins such as α-tubulin, Hsp90, p53, c-Myc, NF-κB and E2P.

There are 18 encrypted human HDACs, which are Class I (HDAC 1, 2, 3 and 8), Class II (HDAC 4, 5, 6, 7, 9 and 10), Class III (SIRT 1-7), and Class IV (HDAC11) enzymes.

Histone deacetylases (HDACs) have recently been reported to cause a wide range of diseases including cancer, Alzheimer's disease, depression and drug addiction, attracting attention as an important pharmaceutical target for various diseases. .

HDAC inhibitors are generally classified into four types, depending on their chemical structure: hydroxamic acids, benzamides, cyclic peptides and short chain fatty acids. To date, the US FDA has identified SAHA (vorinostat), Four HDAC inhibitors such as FK-228 (romidepsin), PXD101 (belinostat) and LBH589 (panobinostat) were approved as anticancer drugs, and the Chinese Food and Drug Administration approved HBI-8000 (chidamide) as a treatment for T-cell lymphoma.

Most of these HDAC inhibitors have been developed primarily for the treatment of hematologic malignancies, but studies on the use of HDAC inhibitors for the treatment of central nervous system (CNS) diseases, including Alzheimer's disease, depression and drug addiction (Kazantsev and Thompson, 2008) have been reported. According to the burden, there is a need for the development of HDAC inhibitors that can be effectively applied to a wider range of diseases in addition to chemotherapy.

Republic of Korea Patent Publication No. 10-2014-0053871 (published May 8, 2014)

The present invention is to develop a novel acetyl donating HDAC inhibitor, and to provide it as a treatment for various cancer diseases or central nervous system diseases including Alzheimer's disease, drug addiction and depression.

The present invention provides a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Chemical Formula 1,

The acetyl (AcO) is meta or para position.

The present invention provides a composition for inhibiting histone deacetylases (HDACs) containing a compound represented by Formula 1 as an active ingredient.

The present invention provides a composition for treating cancer diseases, which contains the compound represented by Formula 1 as an active ingredient.

In another aspect, the present invention provides a composition for treating central nervous system diseases containing the compound represented by the formula (1) as an active ingredient.

The novel aspirin derivative according to the present invention inhibits the activity of HDAC by binding to the substrate binding pocket of histone deacetylase (HDAC) and significantly increases the acetylation of α -tubulin and histone H3 in cells to increase cancer cell proliferation. As shown to have an inhibitory effect, the novel aspirin derivatives can be provided as HDAC inhibitors and as effective therapeutic agents for cancer diseases and central nervous system diseases associated with HDAC.

1 is the structure of an FDA approved HDAC inhibitor.
FIG. 2 shows the acetylation ability of aspirin (2a), meta-analog 2b and para-analog 2c to lysine residues. MDA-MB-231 cells were treated with 5 mM concentration for 24 hours, and total In the protein acetylation state, the results of Western blot confirming the effects of compounds 2a, 2b and 2c, DMSO (D) is a negative control.
3 shows the design of acetyl donor HDAC inhibitors, where ADG is an acetyl-donating group and ZBG is a zinc-binding group.
4 shows the time and dose dependent anti-proliferative effects of 4c in MDA-MB-231 cells.
Figure 5 compares the effects of aspirin, compounds 4c and 4b on the acetylation status of α-tubulin, histone H3, resulting in 75 μM or 150 μM of aspirin, compounds 4c and 4b in MDA-MB-231 cells. Western blot results of 24 hours of concentration and confirm the expression level of specific proteins, DMSO (D) is a negative control.
6 is a molecular binding model of compound 4c in the binding pocket of HDAC6 (PDB code: 5EF7).

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

Aspirin (acetylsalicylic acid) is a drug that has long been used extensively for the treatment of inflammation, fever, pain and cardiovascular disease. It has been shown that aspirin (acetylsalicylic acid) is a covalent modification of COX-2 through acetylation of Ser530 near the active site. It is a drug that works by preventing the active site from binding.

Recent studies have shown that aspirin reduces the risk for various types of cancer, which is reported to be due to the aspirin's endogenous protein acetylation ability. The present invention was completed by synthesizing an aspirin derivative and confirming that the aspirin derivative exhibited superior histone deacetylase inhibitory activity than conventional aspirin.

The present invention can provide a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, acetyl (AcO) may be meta or para position.

The compound represented by Chemical Formula 1 may inhibit histone deacetylases (HDACs) activity.

More specifically, the compound represented by Chemical Formula 1 is a novel aspirin derivative, which binds to the substrate binding bag of histone deacetylase (HDAC) to inhibit HDAC activity and acetylation of α -tubulin and histone H3 in cells. Was significantly increased to show an effect of inhibiting cell proliferation.

Therefore, the present invention can provide a composition for inhibiting histone deacetylases (HDACs) containing the compound represented by Formula 1 as an active ingredient.

[Formula 1]

In Formula 1, acetyl (AcO) may be meta or para position.

The histone deacetylase may be selected from the group consisting of HDAC1 and HDAC6.

The present invention can provide a pharmaceutical composition for treating cancer diseases containing the compound represented by the formula (1) as an active ingredient.

[Formula 1]

In Formula 1, acetyl (AcO) may be meta or para position.

The cancer diseases include breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, acute myeloid leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, brain cancer and blood cancer. It may be selected from the group consisting of.

The present invention can provide a pharmaceutical composition for treating central nervous system diseases containing the compound represented by the formula (1) as an active ingredient.

[Formula 1]

In Formula 1, acetyl (AcO) may be meta or para position.

The central nervous system disease may be selected from the group consisting of Alzheimer's, Lou Gehrig's disease, Huntington's disease, Parkinson's disease, drug addiction and depression.

In one embodiment of the present invention, the pharmaceutical composition containing the compound represented by Formula 1 as an active ingredient is an injection, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions according to conventional methods , Any one formulation selected from the group consisting of drops or solutions may be used.

In another embodiment of the present invention, the pharmaceutical composition containing the compound represented by the formula (1) as an active ingredient is suitable carriers, excipients, disintegrants, sweeteners, coatings, swelling agents, lubricants, lubricants commonly used in the manufacture of pharmaceutical compositions Agents, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants may further comprise one or more additives selected from the group.

Specifically, the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and such solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.

According to one embodiment of the invention the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.

The preferred dosage of the compound represented by Formula 1 may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route of administration and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Reference Example 1 Compound Synthesis Conditions

All reactions were carried out under argon atmosphere using dried glass tools, and all reagents and solutions purchased were used without further purification. Thin layer chromatography (TLC) was performed using Merck silica gel 60 F ₂₅₄ plates.

TLC plates were visualized using a mix of UV, p-anisaldehyde, ceric ammonium molybdate (CAM), ninhydrin and potassium permanganate stains.

Column chromatography was performed under medium pressure conditions on silica (Merck Silica Gel 40-63 νm) or MPLC (Biotage Isolera One instrument) conditions on silica columns (Biotage SNAP HP-Sil) or C18 columns (Biotage SNAP Ultra C18).

NMR analysis was performed using JNM-ECZ500R (500 MHz) manufactured by Jeol resonance, and the purity of all experimental compounds was dual pump Shimadzu LC-6AD equipped with VP-ODS C18 column (4.6 mmХ250 mm, 5 νm, Shimadzu) Analytical HPLC was performed with the system to confirm it was at least 95%.

Reference Example 2 Substance

DMEM (Dulbecco's Modified Eagle's medium) and L-glutamine were purchased from GenDEPOT (Barker, TX, USA) and fetal bovine serum (FBS) and penicillin / streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA).

α- tubulin, Ac- α- tubulin, histone H3, Ac-histone H3, β- actin and Ac-lysine specific antibodies were purchased from Cell Signaling Technology (Boston, MA, USA), and goth anti-rabbit IgG horseshoe Dish peroxidase conjugates were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell Titer 96 Aqueous One Solution cell proliferation assay kit was purchased from Promega (Madison, WI, USA) and Amersham ^™ ECL select ^™ Western Blotting Detection Reagent was purchased from GE Healthcare.

HDAC fluorogenic assay kits (HDAC1, HDAC3, HDAC6, and HDAC7) were purchased from BPS Bioscience (San Diego, CA, USA).

< Synthesis Example > Aspirin Analog Synthesis

1-3 Acetoxybenzoic  Acid [3- Acetoxybenzoic  acid; 2b]

Pyridine (10 mL) mixture with 3-hydroxybenzoic acid (1.0 g, 138.12 mmol) and acetic anhydride (5 mL) was added for 2 hours at 125 ° C. using a reflux condenser under argon. Was stirred.

After cooling to room temperature, the mixture was quenched with saturated aqueous NaHCO ₃ , acidified to pH 2 by addition of 12 N HCl, the solid obtained was filtered, washed with H ₂ O (50 mL) and dried under high vacuum to give compound 2b. Obtained in 94% yield.

¹ H NMR (500 MHz, CDCl ₃ ) δ 7.99 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.37-7.34 (m, 1H) , 2.34 (s, 3 H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ 171.1, 169.3, 150.7, 130.8, 129.7, 127.7, 127.3, 123.5, 21.1.

2. 4- ( Hydroxycarbamoyl ) Phenyl acetate [4- ( Hydroxycarbamoyl ) phenyl acetate; 2c]

Pyridine (10 mL) mixture with 4-hydroxybenzoic acid (1.0 g, 138.12 mmol) and acetic anhydride (5 mL) was stirred using a reflux condenser under argon for 2 hours at 125 ° C. After cooling to room temperature, the reaction was stopped with saturated aqueous NaHCO 3 and acidified to pH 2 by addition of 12 N HCl. The solid obtained was filtered, washed with H ₂ O (50 mL) and dried under high vacuum to give Compound 2c 66%. Obtained in yield.

¹ H NMR (500 MHz, (CDCl ₃ ) δ 8.14 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 2.34 (s, 3H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ 171.0, 169.0, 155.0, 131.9, 126.9, 121.9, 21.3.

3. 2-(( Benzyloxy ) Carbamoyl ) Phenyl acetate [2-((benzyloxy) carbamoyl) phenyl acetate; 3a]

Compound 2a (0.2 g, 1.11 mmol) , o - benzyl hydroxylamine hydrochloride (hydrochloride o -benzylhydroxylamine; 0.19 g, 1.22 mmol), EDC (0.43 g, 2.22 mmol) and DIPEA (0.39 mL, 2.22 mmol) is dissolved DCM (25 mL) mixture was stirred at rt for 4 h.

The reaction mixture was diluted with DCM (100 mL) and then washed with 1N HCl (100 mL) and brine (100 mL) and dried over Na ₂ SO ₄ .

It was then concentrated under reduced pressure and purified by silica gel column chromatography to give compound 3a in 71% yield.

R _f = 0.32 (4: 6 ethyl acetate: hexane). ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.63 (s, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.50-7.44 (m, 3H), 7.39 (q, J = 7.3 Hz, 3H) , 7.29 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 5.03 (s, 2H), 2.10 (s, 3H). ¹³ C-NMR (125 MHz, CDCl ₃ ) δ 169.0, 164.1, 148.1, 135.4, 132.6, 130.0, 129.3, 128.9, 128.8, 126.4, 125.3, 123.3, 78.2, 20.94.

4. 3-(( Benzyloxy ) Carbamoyl ) Phenyl acetate [3-((benzyloxy) carbamoyl) phenyl acetate; 3b]

Compound 2b (0.2 g, 1.11 mmol) , o - benzyl hydroxylamine hydrochloride (hydrochloride o -benzylhydroxylamine; 0.19 g, 1.22 mmol) to a DCM (25 mL) mixture containing the DIPEA (0.39 mL) at room temperature containing the EDC (0.43 g, 2.22 mmol) was added with stirring.

The organic layer was washed twice with 1 N HCl (100 mL), brine (100 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure to give compound 3b in 68% yield.

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.82 (s, 1H), 7.49 (d, J = 7.4 Hz, 1H), 7.43 (m, 3H), 7.41-7.36 (m, 4H), 7.22 (d, J = 7.4 Hz, 1H), 5.01 (s, 2H), 2.29 (s, 3H). ¹³ C NMR (125 MHz, CDCl ₃ ) δ 169.4, 165.4, 150.7, 135.2, 133.5, 129.8, 129.4, 128.9, 128.7, 128.4, 127.9, 125.4, 124.5, 120.8, 78.4, 21.1.

5. 4-(( Benzyloxy ) Carbamoyl ) Phenyl acetate [4-((benzyloxy) carbamoyl) phenyl acetate; 3c]

Compound 2c (0.2 g, 1.11 mmol) , o - benzyl hydroxylamine hydrochloride (hydrochloride o -benzylhydroxylamine; 0.19 g, 1.22 mmol), DIPEA (0.39 mL) with DCM (25 mL) and the mixture EDC (0.43 g containing the , 2.22 mmol) and stirred at room temperature for 4 hours.

The mixture was diluted with DCM (100 mL). The organic layer was washed twice with 1 N HCl (100 mL), brine (100 mL), dried over Na ₂ SO ₄ , concentrated under reduced pressure to give compound 3c in 60% yield.

¹ H NMR (500 MHz, (CDCl ₃ ) δ 7.69 (d, J = 8.6 Hz, 2H), 7.41-7.40 (m, 2H), 7.37-7.33 (m, 3H), 7.08 (d, J = 8.6 Hz , 2H), 4.98 (s, 2H), 2.29 (s, 3H). 13 C NMR (125 MHz, CDCl 3) δ 169.1, 165.7, 153.5, 135.3, 129.6, 129.4, 128.9, 128.7, 128.4, 122.0, 121.6 , 78.4, 21.2.

6. 3- ( Hydroxycarbamoyl ) Phenyl acetate [3- ( hydroxycarbamoyl ) phenyl acetate; 4b]

MeOH (12.5 mL) mixture of compound 3b (0.203 g, 1.04 mmol) and Pd / C (0.05 g) was stirred under hydrogen at room temperature for 2 hours.

The reaction mixture was diluted with MeOH (50 mL), filtered, concentrated under reduced pressure and purified by MPLC (Biotage SNAP HP-Sil column) to give compound 4b in 45% yield.

R _f = 0.31 (7: 3 ethyl acetate: hexane) ¹ H NMR (500 MHz, acetone-d6) δ 7.71 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.50 (t, J = 7.7 Hz 1H), 7.30-7.28 (m, 1H), 2.28 (s, 3H). ¹³ C NMR (125 MHz, acetone-d ₆ ) δ 169.6, 164.8, 151.9, 134.6, 130.3, 130.1, 125.8, 124.8, 121.5, 20.9.

7. 4- ( Hydroxycarbamoyl ) Phenyl acetate [4- ( hydroxycarbamoyl ) phenyl acetate; 4c]

A MeOH (12.5 mL) mixture in which compound 3c (0.203 g, 1.04 mmol) and Pd / C (0.05 g) was dissolved was stirred under hydrogen at room temperature for 2 hours.

The reaction mixture was dissolved in MeOH (50 mL), filtered, concentrated under reduced pressure and purified by MPLC (Biotage SNAP HP-Sil column) to give compound 4c in 39% yield.

R _f = 0.24 (7: 3 ethyl acetate: hexane). ¹ H NMR (500 MHz, acetone-d ₆ ) δ 10.8 (s, 1H), 8.52 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H) , 2.28 (s, 3 H). ¹³ C NMR (125 MHz, acetone-d ₆ ) δ 168.6, 164.5, 153.5, 129.7, 128.4, 122.0, 20.2.

8. 4- Hydroxy -N-(( Tetrahydro -2H-pyran-2-yl) Oxy ) Benzamide  [4-hydroxy-N-((tetrahydro-2H-pyran-2-yl) oxy) benzamide; 5]

4-hydroxybenzoic acid (1.0 g, 7.24 mmol), o- (tetrahydro-2H-pyran-2-yl) hydroxylamine ( o- (Tetrahydro-2H-pyran-2-) in DMF (25 mL) yl) hydroxylamine; 0.93 g, 7.96 mmol) and a mixture of EDC (2.78 g, 14.48 mmol) were stirred at rt for 12 h and the reaction mixture was dissolved in DCM (100 mL).

The organic layer was washed with 1 N HCl (100 mL), washed with brine (100 mL), dried over Na ₂ SO ₄ , concentrated under reduced pressure and purified by silica gel column chromatography to give compound 5 in 22% yield.

R _f = 0.53 (7: 3 ethyl acetate: hexane). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.4 (s, 1H), 10.1 (s, 1H), 7.63 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 9.2 Hz, 2H) , 4.94 (s, 1H), 4.06-4.02 (m, 1H), 3.51-3.48 (m, 1H), 1.70-1.53 (m, 6H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 164.2, 160.2, 128.8, 122.8, 114.7, 100.8, 61.2, 27.7, 24.5, 18.1.

9. N, 4 - Dihydroxybenzamide  [ N, 4 - dihydroxybenzamide ; 6]

A mixture of compound 5 (0.075 g, 0.49 mmol) and 3 N HCl (10 mL) dissolved in acetonitrile (20 mL) was stirred at room temperature for 4 hours.

The mixture was concentrated under reduced pressure and purified by reverse phase MPLC (SNAP Ultra C18) to give compound 6 in 10% yield.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.77 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 9.2 Hz, 1H). ¹³ C NMR (125 MHz, DMSO-d ₆ ) δ 167.2, 161.6, 131.5, 121.5, 115.1

< Experimental Example  1> binding analysis

Using the AutoDock program downloaded from Scripps Research Institute's Molecular Graphics Laboratory, in silico binding of compound 4c was confirmed at three-dimensional coordinates of the X-ray crystal structure of HDAC6.

The AutoDock program was chosen because it uses genetic algorithms to generate the position of ligands in known or expected binding sites using Lamarckian versions of genetic algorithms. Used as a subsequent location.

In order to perform the coupling experiments, water was removed from the 3D X-ray coordinates, while Gasteiger charges were placed in the X-ray structure of HDAC6 as with 4c using the tools of the AutoDock product.

For ligand binding experiments, 60_60_60 points and 0.375 mm 3 spacing grid boxes were selected at the center of the substrate binding bag of the HDAC6 enzyme.

A binding model of compound 4c and HDAC6 was shown in FIG. 6, and PyMol (DeLanoScientific) was used for rendering the figure.

< Experimental Example  2> cell culture

MDA-MB-231 cells were cultured using DMEM medium containing L-glutamine, streptomycin (500 mg / mL), penicillin (100 units / mL) and 10% fetal bovine serum (FBS).

Cells were incubated until combined at 37 ° C., 5% CO ₂ conditions.

< Experimental Example  3> Cell Growth Assay

2000 cells were dispensed per well of a 96-well plate and the medium was added to a volume of 100 mL and cells were allowed to attach overnight.

The following day, compounds 2a-b, 4b-c and 6 were treated at various concentrations and 0.5% DMSO was added to the wells as a control.

Cell proliferation was confirmed using a Promega Cell Titer 96 Aqueous One Solution cell proliferation assay. After compound incubation, 20 mL of assay substrate solution was added to each well and further incubated at 37 ° C. for 1 hour.

Thereafter, 490 nm absorbance was measured in FLUO star ^® Omega (BMG LABTECH), and the value was expressed as a percentage of the absorbance of cells cultured with DMSO alone.

< Experimental Example  4> Weston Blot  analysis

50 nm in the culture dish dividing the MDA-MB-231 cells (1 × 10 ⁶ / dish), which was adhered for one night. Compounds 2a to 2c or compounds 4b and 4c were added to the cells and incubated for 24 hours, and the cells were cultured in 0.5% DMSO for comparison.

Collect cells with chilled lysis buffer (23 mM Tris-HCL pH 7.6, 139 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and isolate 30 μg lysate per lane on SDS-PAGE And transferred to PVDF membrane.

The membrane was blocked with TBS-T with 5% skim milk and incubated with a suitable antibody.

The appropriate horseradish peroxidase was then combined with the linked secondary antibody and the protein was visualized by ECL chemiluminescence according to the manufacturer's instructions.

< Example  1> Aspirin and Aspirin Derivatives Acetylation ability  Confirm

Scheme 1

(a) acetic anhydride, pyridine, rt, 2h, 94% for 2b, 66% for 2c

Aspirin derivatives 2b to 2c were synthesized in the same manner as in Scheme 1, in which the reported method (Zhang et al., 2014; Hrast et al., 2014) was modified. Briefly, compounds 1b to 1c were acetylated from pyridine to acetic anhydride to give compounds 2b to 2c in 66-94% yield.

The compounds 2b to 2c were identified as aspirin (2a), meta-analog 2b and para-analog 2c in MDA-MB-231 cells to determine whether the compounds can increase the acetylation level of the lysine ε-amino group.

Aspirin is known to covalently modify COX-2 through acetylation of Ser530 near the active site and to inhibit the active substrate from binding to the active site (Luei et al., 2015).

However, few studies on the acetylation ability of aspirin at lysine residues have been reported, confirming the acetylation ability of aspirin (2a), meta-analog 2b and para-analog 2c to lysine residues of the entire protein.

First, MDA-MB-231 cells were treated with aspirin (2a), meta-analog 2b and para-analog 2c for 24 hours and acetylation levels of lysine residues were confirmed by anti-Ac-lysine immunoblot.

As a result, as shown in FIG. 2, aspirin (2a) increased acetylation of lysine residues very effectively, and compounds 2b and 2c were also found to increase acetylation levels of lysine residues compared to DMSO control.

As the acetylation ability of the compounds 2a to 2c was found to be excellent from the above results, the scaffolds of the compounds 2a to 2c were used to develop a novel acetyl donating HDAC inhibitor.

< Example  2> Aspirin Analog Synthesis

Many HDAC inhibitors structurally mimic acetyl-lysine, an endogenous substrate, as shown in FIG. 3. The common characteristics of HDAC inhibitor drug-specific molecular groups, for example SAHA, are three elements: zinc binding group (ZBG), linker and cap group. Is determined.

Many HDAC inhibitors have been developed to treat a variety of human diseases, and most HDAC inhibitors, including clinically approved SAHA, PXD101 and LBH589, contain hydroxamic acid as a zinc binding group.

At the same time, in the development of HDAC inhibitors, phenyl linkers of HDAC inhibitors with hydrophobicity have been widely used to fit the lipophilic channels of the HDAC substrate pockets.

Accordingly, by applying a hydroxyl zinc binding group and a phenyl linker to the structures of aspirin (2a), meta-analog 2b and para-analog 2c, a novel HDAC inhibitor was synthesized to induce an acetyl donor HDAC inhibitor as shown in FIG. It was.

Scheme 2

(a) NH 2 OBn, EDC, DIPEA, DCM, rt. 4h, 71% for 3a, 68% for 3b, 60% for 3c; (b) H ₂ , 10% Pd / C, MeOH, rt, 2h, 45% for 4b, 39% for 4c

First, acetyl donating HDAC inhibitors 4a to 4c were synthesized in the same manner as in Scheme 2.

The benzoyl-protected compounds 3a to 3c were synthesized in 60-71% yield by amine bonding of compounds 2a to 2c induced by EDC with NH ₂ OBn in the presence of DIPEA.

Next, the benzyl protecting group was removed in the presence of a 10% palladium catalyst and hydrogen atmosphere to yield compounds 4b and 4c in 39 and 45% yield.

On the other hand, the benzyl protecting group cleavage attempt of 3a under the same reaction conditions did not show an effective result. Compound 4a was not stable and, unlike the other analogs, provided degradation in the removal of protecting groups, thus providing a complex mixture.

Scheme 3

(a) NH ₂ OTHP, EDC, DIPEA, DMF, rt, 12h, 59%; (b) 3N HCl, MeCN, rt, 4h, 10%

To clarify the mode of activity of the acetyl donor HDAC inhibitor, Compound 6 lacking an acetyl donor group was synthesized in the same manner as in Scheme 3.

Compound 5 was obtained in 59% yield by combining 1c and NH ₂ OTHP in DMF in which EDC and DIPEA were dissolved, and acidic cleavage of the compound of THP protecting group 5 using 3N HCl in acetonitrile. 6 was obtained in 10% yield.

< Example  3> Confirmation of aspirin and aspirin analogues against cancer cell proliferation

The anti-proliferative activity of compounds 2a, 2b, 2c, 4b, 4c and 6 synthesized in the above process was confirmed in MDA-MB-231, a highly aggressive and invasive triple negative breast cancer (TNBC) cell.

As a result, as shown in Table 1, Compounds 2a (Aspirin), 2b and 2c showed very low anti-proliferative activity in MDA-MB-231 TNBC cells, and GI ₅₀ values of Compounds 2a and 2c were higher than 1,000 μM, The GI ₅₀ value of compound 2b was found to be 720 μM.

In contrast, compounds 4b and 4c showed very improved anticancer effects on MDA-MB-231 cells compared to compounds 2b and 2c, and the GI ₅₀ values were found to be 169 μM and 147 μM, respectively.

Interestingly, compound 6 lacking an acetyl donor group (ADG) (GI ₅₀ = 554 μM) was found to exhibit a relatively low anticancer effect compared to compound 4c (GI ₅₀ = 147 μM).

From these results, the acetyl donor group of compound 4c was found to play an important role in anticancer effects, thus demonstrating the molecular design efficiency of the acetyl donor HDAC inhibitor in the development of new HDAC inhibitors.

In addition, to confirm the time and dose dependent effects of compound 4c on MDA-MB-231 cell growth, compound 4c was administered to MDA-MB-231 cells at 1, 10, 100 and 300 μM concentrations in 1, 2 and 3 Days of treatment and cell proliferation was confirmed by MTS analysis.

As a result, as shown in FIG. 4, the cell-dependent inhibitory effect of MDA-MB-231 was confirmed depending on the dose of the compound 4c, and particularly, the cells treated with the compound 4c at the concentration of 300 μM showed an excellent anticancer effect, and 300 μM. Cells exposed to concentrations of compound 4c showed growth reduction effects of 37%, 21% and 22% for 1, 2 and 3 days, respectively.

< Example  4> Confirmation of anticancer activity of compound 4c

The relative effects of aspirin, compound 4c and compound 4b on the acetylation status of α -tubulin and histone H3, which are epigenetically removed by HDAC6 and HDAC1 enzymes, respectively, were confirmed.

As a result, acetylation of α -tubulin and histone H3 was significantly promoted in MDA-MB-231 cells treated with Compound 4c as shown in FIG. 5, whereas aspirin, a comparative drug, was used for acetylation of α -tubulin and histone H3. It could be confirmed that anger is not induced.

In addition, the acetylation effect of α -tubulin of compound 4b was less than that of compound 4c, and acetylation of histone H3 was not observed.

As expected, it was confirmed that the expression levels of α -tubulin and histone H3 were not affected by aspirin, compound 4c and compound 4b.

Meanwhile, the degree of total acetylation of all proteins in MDA-MD-231 cells was confirmed.

As a result of anti-Ac-lysine immunoblot, compound 4c acetylated the ε-amino group of the lysine residue more effectively than the comparator drug aspirin and other analogue 4b.

From these results, it was confirmed that para-analogue 4c inhibited the HDAC1 and HDAC6 enzymes very well and promoted the acetylation of different substrates, histone H3 and α -tubulin, respectively.

< Example  5> with compound 4c HDAC6  Check mating position

In order to confirm the exact binding position of Compound 4c with HDAC6, in silico binding simulation of Compound 4c was performed in the substrate binding bag (PDB code: 5EF7) of HDAC6.

As a result, it was confirmed that the compound 4c occupies the deep substrate binding pocket of HDAC6 through the binding simulation as shown in FIG. 6.

In addition, the para-acetyl group of the compound 4c is located at the edge of the substrate binding bag, and the carbonyl oxygen (C = O) of the acetyl group was confirmed to interact with the S531 residue to form a hydrogen bond.

The phenyl ring of 4c was injected into the lipophilic channel of HDAC6 to form a π-π interaction with the F583 and F643 residues, and the hydroxyl group OH and C═O functional groups of compound 4c have a double site of Zn ²⁺ ions as active sites. Coordination.

It was also confirmed that the 4c hydroxamate OH group participates in hydrogen bond interactions with the H573 residue.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A compound represented by formula (1) or a pharmaceutically acceptable salt thereof.
[Formula 1]

In Chemical Formula 1,
The acetyl (AcO) is meta or para position. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by Chemical Formula 1 inhibits histone deacetylases (HDACs) activity. Composition for inhibiting histone deacetylases (HDACs) containing a compound represented by the formula (1) as an active ingredient.
[Formula 1]

In Chemical Formula 1,
The acetyl (AcO) is meta or para position. The composition for inhibiting histone deacetylase according to claim 3, wherein the histone deacetylase is selected from the group consisting of HDAC1 and HDAC6. A pharmaceutical composition for treating cancer diseases containing the compound represented by the formula (1) as an active ingredient.
[Formula 1]

In Chemical Formula 1,
The acetyl (AcO) is meta or para position. The method of claim 5, wherein the cancer disease is breast cancer, stomach cancer, liver cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, acute myeloid leukemia, acute lymphocytic leukemia, uterine cancer, ovarian cancer, laryngeal cancer, prostate cancer, thyroid cancer, head or neck cancer, A pharmaceutical composition for the treatment of cancer diseases, characterized in that selected from the group consisting of brain cancer and blood cancer. A pharmaceutical composition for treating a central nervous system disease containing the compound represented by the formula (1) as an active ingredient.
[Formula 1]

In Chemical Formula 1,
The acetyl (AcO) is meta or para position. The pharmaceutical composition for treating central nervous system disease according to claim 7, wherein the central nervous system disease is selected from the group consisting of Alzheimer's, Lou Gehrig's disease, Huntington's disease, Parkinson's disease, drug addiction and depression.

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