KR101641829B1 - New thienopyridine compound having inhibitory activity on Hsp90 and medical use thereof - Google Patents

Abstract

The present invention relates to a novel thienopyridine compound having Hsp90 inhibitory activity and a medicinal use thereof, and the thienopyridine compound according to the present invention is excellent in inhibiting effect of Hsp90 and is useful as a therapeutic agent for cancer diseases or neurodegenerative diseases And can be useful as a medicament or a health food for the treatment or prevention of Hsp90 mediated diseases such as cancer diseases or neurodegenerative diseases.

Description

TECHNICAL FIELD The present invention relates to novel thienopyridine compounds having Hsp90 inhibitory activity and medicinal uses thereof.

The present invention relates to novel thienopyridine compounds having Hsp90 inhibitory activity and their medical use.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that regulates the modification, activation and stabilization of the client protein, the substrate protein of Hsp90. Many types of Hsp90 client proteins are known to induce cancer, including tyrosine kinase (EGFR, Her2, Met), metastable signal transduction proteins (Akt, Raf-1, IKK) and mutated signal transduction proteins , v-Src), and the like.

Because cancer cells are more dependent on Hsp90 chaperone function than normal cells due to their genetic instability and stress environment, the expression of Hsp90 is 2- to 10-fold higher in cancer cells than in normal cells.

Accordingly, Hsp90 can be a target for effective cancer therapy, and the inhibitor of Hsp90 protein activity can simultaneously reduce various cancer-inducing proteins, and can be effective in the treatment of cancer with resistance.

In particular, the mutation and expression increase of the Hsp90 client proteins EGFR and Met causes resistance to non-small cell lung cancer drug, and the inhibitor of Hsp90 activity may be the most effective treatment for such non-small cell lung cancer.

Korean Patent Publication No. 10-2007-0038565 (2007.04.10)

In the present invention, a new structure of Hsp90 inhibitor is developed, and it is intended to provide a medical use for treating a cancer disease including non-small cell lung cancer or a therapeutic agent for a neurodegenerative disease including Alzheimer's disease.

In order to achieve the above object, the present invention provides a thienopyridine compound represented by the following formula (1).

[Chemical Formula 1]

The present invention also provides a pharmaceutical composition for preventing or treating Hsp90 (Heat Shock Protein 90: heat shock protein 90) mediated diseases containing a thienopyridine compound represented by Chemical Formula 1 as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or ameliorating Hsp90 (Heat Shock Protein 90: Heat Shock Protein 90) mediated diseases containing a thienopyridine compound represented by Chemical Formula 1 as an active ingredient.

The thienopyridine compound according to the present invention is excellent in the inhibitory effect of Hsp90 and induces degradation of Hsp90 client protein causing cancer disease or degenerative neuropathy through inhibition of Hsp90, And can be useful as medicines or health foods for treatment or prevention.

1 is a photograph showing the Hsp90 binding site of a thienopyridine compound according to the present invention.
FIG. 2 shows the results of confirming the H1975 cell proliferation inhibitory effect according to the concentration of the thienopyridine compound according to the present invention.
FIG. 3 is a Western blot result obtained by confirming the Hsp90 client protein inhibitory effect of a thienopyridine compound according to the present invention.
Fig. 4 is a Western blot result showing the inhibitory effect of the thiopyridine compound of the present invention on the apoptosis inducing protein associated with Hsp90.

The present invention provides a thienopyridine compound represented by the following formula (1).

[Chemical Formula 1]

The thienopyridine compound may be 6,7-dihydrothieno [3,2-c] pyridin-5 (4H) -yl) (2,4-dihydroxy-5-isopropylphenyl) methanone.

The thienopyridine compound may inhibit the activity of Hsp90 by binding to the N-terminal of Hsp90 (Heat Shock Protein 90: Thermal Shock Protein 90).

The thienopyridine compounds according to the present invention inhibit Hsp90 activity, decrease cancer-causing proteins such as Her2, Met, Akt and the like, increase Hsp70, and, through molecular modeling, such compounds inhibit the N-terminal ATP And confirmed to bind to the pocket.

The present invention provides a pharmaceutical composition for preventing or treating Hsp90 (Heat Shock Protein 90: Heat Shock Protein 90) mediated diseases containing a thienopyridine compound represented by Chemical Formula 1 as an active ingredient.

The Hsp90 mediated disease may be a disease selected from the group consisting of cancer diseases, degenerative neurological diseases and viral infections.

The cancer disease may be a disease selected from the group consisting of non-small cell lung cancer, lung cancer, breast cancer, uterine cancer, pancreatic cancer, prostate cancer, gastric cancer, colon cancer, leukemia, brain tumor and head and neck cancer. But are not limited to, memory loss, memory impairment, dementia, amnesia, Parkinson's disease, Alzheimer's disease, Peak disease, Creutzfeldt-Jakob disease, Huntington's disease and Lou Gehrig's disease.

In one embodiment of the invention, the pharmaceutical composition may be formulated with suitable carriers, excipients, disintegrants, sweeteners, coatings, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, , A diluent, a dispersant, a surfactant, a binder, and a lubricant.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In another embodiment of the present invention, the pharmaceutical composition may be formulated into granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, Can be used.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, Can be administered to a subject in a conventional manner via the intradermal route.

The preferred dose of the thienopyridine compound may be varied depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

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

The present invention also provides a health food for preventing or ameliorating Hsp90 (Heat Shock Protein 90: Heat shock protein 90) -mediated disease containing a thienopyridine compound represented by the formula (1) as an active ingredient.

In one embodiment of the present invention, the health food comprises 0.01 to 90 parts by weight, 0.1 to 90 parts by weight, 1 to 90 parts by weight, or 10 to 90 parts by weight of a thienopyridine compound per 100 parts by weight of the whole health food But are not limited thereto.

In another embodiment of the present invention, the health food may further comprise at least one additive selected from the group consisting of organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar and sorbitol. The organic acid can be, but is not limited to, citric acid, fumaric acid, adipic acid, lactic acid or malic acid, and the phosphate can be sodium phosphate, potassium phosphate, acid pyrophosphate or polyphosphate (polymeric phosphate) But are not limited to, natural antioxidants such as polyphenols, catechins, alpha-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid or phytic acid.

In another embodiment of the present invention, the health food may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate, etc.) Organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition according to one embodiment of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink.

According to one embodiment of the invention, the formulation of a health food may be in the form of solid, powder, granule, tablet, capsule, liquid or drink, although not limited thereto.

In addition, the health food includes but is not limited to confectionery, saccharides, ice cream products, dairy products, meat products, fish meat products, tofu or glue, edible oils, noodles, Dried products of ginseng products, kimchi pickles, dried fruits, fruits, vegetables, fruits or vegetables, cutting products, fruit juices, vegetable juices, mixed juice thereof, nuts, noodles, processed livestock products, processed marine products, , Fermented milk food, bean curd food, cereal food, fermented microorganism food, confectionery bakery, condiments, meat processing, acidic beverage, licorice, herb.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Thienopyridine ( Thienopyridine ) Compound Synthesis

Except where otherwise noted, all reactions were carried out under an argon atmosphere and all products purchased were used without further purification. Thin layer chromatography (TLC) was performed using Merck silica gel 60 F ₂₅₄ plates. TLC plate visualization was performed using a combination of UV, p - anisaldehyde, ceric ammonium molybdate, ninhydrin, and potassium permanganate staining.

NMR spectra were measured using a Bruker 400 (400 MHz for ¹ H; 100 MHz for ¹³ C) spectrometer, and ¹ H and ¹³ C NMR chemical changes were shown per million (ppm) parts compared to TMS and the residual solvent peak It was used as an internal reference.

The signal may be a multiplet, a singlet, a doublet, a triplet, a quartet, a broad singlet, a broad doublet, a doublet of doublets, triplets) and dq (doublet of quartets); The connected integers are reported in hertz (Hz).

The synthesized final compound was purified by MPLC (Biotage Isolera One instrument) using a silica gel column (Biotage SNAP HP-Sil).

As a result of the above MNR and HPLC (Shimadzu prominence, VP-ODS C18 column) analysis, the purity of all synthesized compounds was confirmed to be> 95%.

1. Synthesis of Compound 6

[Reaction Scheme 1]

Compound 6 was synthesized in the same manner as in Scheme 1.

2,4-Dihydroxyacetophenone (10.2 g, 66.0 mmol) and 5 mL of sulfuric acid were placed in 40 mL of methanol, and the mixture was stirred at 100 ° C for 12 hours in a reflux condenser under argon.

The mixture was cooled at room temperature, concentrated under reduced pressure, poured 40 mL of H ₂ O in an ice water bath and filtered to give a white solid.

The solids were dissolved with ethyl acetate and saturated NaHCO ₃ Solution. The organic layer was dried over Na ₂ SO ₄ and concentrated under reduced pressure to obtain Compound 6 in 86% yield.

^{1 H NMR (400 MHz, CDCl} 3) δ10.97 (s, 1H), 7.74 (d, J = 8.8 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H), 6.37 (dd, J = 8.8 Hz, 2.4 Hz, 1 H), 5.36 (s, 1 H), 3.91 (s, 1 H).

2. Synthesis of Compound 7

Compound 7 was synthesized in the same manner as in Reaction Scheme 1 above.

Compound 6 (3.9 g, 23.0 mmol) , 2- bromopropane (4.3 mL, 46.0 mmol) and aluminum chloride (6.1 g, 46.0 mmol) CH 2 Cl 2 And the mixture was stirred under a reflux condenser under argon at 50 &lt; 0 &gt; C for 24 hours.

2-Bromopropane (4.3 mL, 46.0 mmol) was added to the reaction mixture three times every 6 hours. The mixture was then neutralized by addition of 10% NaOH (pH 5), concentrated under reduced pressure and extracted with ethyl acetate.

The organic layer was washed with saturated NaHCO ₃ After the solution was washed three times, dried over Na ₂ SO ₄ and concentrated under reduced pressure. The concentrate was then purified by silica gel chromatography to give compound 7 in 45% yield. _Rf = 0.21 (1: 4 ethyl acetate: hexane). ¹ H NMR (400 MHz, CDCl ₃ )? 10.8 (s, IH), 7.64 (s, IH), 6.34 (m, 1 H), 1.25 (d, J = 10.8 Hz, 6H). ^{13 C NMR (100 MHz, CDCl} 3) δ170.7, 161.6, 159.6, 128.1, 127.1, 105.7, 103.2, 52.2, 26.7, 22.8.

3. Compound 8 Synthesis

[Reaction Scheme 2]

Compound 8 was synthesized in the same manner as in Scheme 2.

A mixture of compound 7 (2.1 g, 10.3 mmol), allyl bromide (2.3 mL, 26.8 mmol) and potassium carbonate (3.7 g, 26.8 mmol) in DMF was stirred for 18 hours and the mixture was dissolved in ethyl acetate.

The organic layer was washed with H ₂ O, dried over Na ₂ SO ₄ and concentrated under reduced pressure to give Compound 8 in 84% yield.

^{1 H NMR (400 MHz, CDCl} 3): δ7.71 (s, 1H), 6.41 (s, 1H), 6.10-5.97 (m, 2H), 5.50 (dd, J = 17.2 Hz, 1.6 Hz, 1H) , 5.41 (dd, J = 17.2 Hz, 1.2 Hz, 1H), 5.27 (d, J = 10.8 Hz, 2H), 4.56 (dd, J = 9.6 Hz, 4.8 Hz, 3.26-3.19 (m, 1H), 1.19 (d, J = 6.8 Hz, 6H).

4. Compound 9 Synthesis

Compound 9 was synthesized in the same manner as in Reaction Scheme 2 above.

Compound 8 (2.5 g, 8.6 mmol) and sodium (1.7 g, 43.1 mmol) with _{MeOH-H 2 O (30 mL} : 30 mL) hydroxide was stirred at room temperature for melting the mixture for 24 hours, and dissolving the mixture in ethyl acetate .

The organic layer was washed with 3N HCl solution, dried over Na ₂ SO ₄ , concentrated under reduced pressure, and then purified by silica gel chromatography to obtain Compound 9 in 58% yield. _Rf = 0.18 (1: 4 ethyl acetate: hexane).

^{1 H NMR (400 MHz, CDCl} 3): δ 7.97 (s, 1H), 6.43 (s, 1H), 6.10-5.97 (m, 2H), 5.47-5.39 (m, 2H), 5.30 (d, J = 10.8 Hz, 2H), 4.73 ( d, J = 5.6 Hz, 2H), 4.57 (d, J = 4.8 Hz, 2H), 3.26-3.19 (m, 1H), 1.18 (d, J = 6.8 Hz, 6H) .

5. Compound 10 [6,7- Dihydrothieno [3,2-c] pyridine -5 (4H) -yl) (2,4- Di hardoxy -5- Small profile Neil) Methanone ] synthesis

[Reaction Scheme 3]

Compound 10 was synthesized in the same manner as in Scheme 3.

To a solution of compound 9 (0.23 g, 0.82 mmol), 4,5,6,7-tetrahydrothieno [3,2-c] pyridine hydrochloride (0.20 g, 1.24 mmol), EDC (0.32 g, , 1.65 mmol), HOBt (0.11 g, 0.82 mmol) and DIPEA (0.15 mL, 0.82 mmol) were stirred at 120 ° C for 3 hours while being irradiated with a microwave (Biotage Initiator) .

The organic layer was washed with 1N HCl solution, dried over Na ₂ SO ₄ and concentrated under reduced pressure.

The concentrate was purified using MPLC (Biotage SNAP HP-Sil column) to obtain an intermediate compound _Rf = 0.21 (1: 4 ethyl acetate: hexane).

4 mL of THF in which PdCl ₂ (PPh ₃ ) ₂ (40 mg) and ammonium formate (400 mg) were dissolved was mixed with the intermediate compound, and the mixture was stirred at 120 ° C. for 30 minutes while irradiating with microwave, The reaction mixture was diluted with ethyl acetate.

The organic layer was washed with water, dried over Na ₂ SO ₄ and concentrated under reduced pressure.

The concentrate was then purified using MPLC (Biotage SNAP HP-Sil column) to give compound 10 in 32% yield, R _f = 0.27 (3: 7 ethyl acetate: hexane).

^{1 H NMR (400 MHz, CDCl} 3) δ 7.15-7.14 (m, 2H), 6.76 (d, J = 5.2 Hz, 1H), 6.41 (s, 1H), 4.74 (s, 2H), 3.95 (t, J = 5.6 Hz, 5.6 Hz, 2H), 3.22-3.16 (m, 1H), 3.04 (t, J = 4.8 Hz, 5.6 Hz, 2H), 1.21 (d, J = 7.2 Hz, 6H)

< Example  2> Thienopyridine ( Thienopyridine ) Of compound Hsp  90 Identification of binding sites

The binding site of compound 10 was identified using the AutoDock program provided by the Molecular Graphics Laboratory of the Scripps Research Institute.

In The central square of the N-terminal domain of human Hsp90 (PDB code: 2XJX) magnified at 60_60_60 magnification in silico and 0.375 A spacing were selected for ligand docking experiments.

As a result, it can be confirmed that Compound 10 binds to the N-terminal ATP pocket of the human Hsp90 protein as shown in Fig.

< Example  3> Thienopyridine ( Thienopyridine ) Effect of derivatives

1. Substance

EGFR, Her2, Met, Akt, c-Raf, Hsp90, Hsp70, PARP, cleaved caspase8, cleaved caspase3, caspase3, Bax and β-actin specific antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Goat anti-rabbit IgG horseradish peroxidase and Goat-anti-mouse IgG horseradish peroxidase were purchased from Santa Cruz Biotechnology (Paso Robles, CA) and used.

Radio-immunoprecipitate-ion Assay (RIPA) buffer, fetal bovine serum (FBS), streptomycin / penicillin, Halt ^TM Protease and phosphatase inhibitor cocktail (100X) was purchased from Thermo Scientific (Waltham, Mass.) And used.

Cell Titer 96 Aqueous One Solution cell proliferation assay kit was purchased from Promega (Fithburg, WI).

2. Cell culture

The zetitinib-resistant non-small cell lung cancer cell line, H1975 cells (where purchased) was inoculated into a culture medium containing L-glutamine, streptomycin (500 mg / mL), penicillin (100 units / mL), and 10% fetal bovine serum The cells were cultured in RPMI 1640 medium at 37 ° C, 5% CO ₂ Cells were grown under culture conditions.

3. Thienopyridine ( Thienopyridine ) Derivatives for Cell Proliferation

3,000 H1975 cells per well of a 96-well plate were inoculated and the medium was dispensed to a final volume of 100 mL and incubated overnight.

The following day, 1% DMSO was added to each well at 0, 0.01, 0.1, 1, 5, 10, 20 and 30 μM concentration of compound 10 as a control and incubated at 37 ° C for 3 days.

Cell viability was analyzed using the Promega Cell Titer 96 Aqueous One Solution cell proliferation assay.

After compound incubation, 20 mL of the assay substrate solution was added to each well and incubated at 37 ° C for additional 1 hour. Absorbance was measured at 490 nm using a Tecan Infinite F200 Pro plate reader and the value was calculated as the absorbance of the DMSO control As a percentage.

As a result, it was confirmed that Compound 10 induced apoptosis of non-small cell lung cancer cells as shown in Fig.

4. Western Blot  Confirm

1x10 &lt; ⁶ &gt; cells of H1975 cells were inoculated in a 100 mm culture dish and adhered overnight. Compound 10 was then treated with 0, 0.05, 0.1, 0.5, 1 and 2 μM concentrations and further incubated for 24 hours. For comparison, cells were treated with 1% DMSO as negative control and 1 mM geladinamycin And incubated for 24 hours.

Each cell was then harvested using ice-cold lysis buffer (23 mM Tris-HCl pH 7.6, 130 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS) and 30 mg of SDS- Separated and transferred to a PVDF membrane (Bio-Rad).

The membrane was blocked with TBST in which 5% skim milk had been dissolved and then the membrane was blocked with EGFR, Her2, Met, Akt, c-Raf, Hsp90, Hsp70, PARP, caspase 3, truncated caspase 3, truncated caspase 8 , Bcl-2, Bax or &lt; RTI ID = 0.0 &gt; ss-Actin &lt; / RTI &gt;

Then, the appropriate secondary horseradish peroxidase conjugated secondary antibody was bound and the protein was visualized according to the manufacturer's instructions using ECL chemiluminescence (GE healthcare, USA).

As a result, as shown in FIG. 4, expression of Hsp90 was increased while expression of EGFR, Her2, Met, c-Raf and Akt, which are Hsp90 proteins, was decreased.

In addition, as shown in Fig. 5, it was confirmed that the expression of PARP, truncated caspase 8, truncated caspase 3, Bcl-2 and Bax was increased.

From the above results, it was confirmed that thienopyridine derivative (Compound 10) of the present invention inhibits the expression of Hsp90 client protein that induces cancer and expresses a protein that induces apoptosis, thereby effectively killing non-small cell lung cancer cells I could.

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)

A thienopyridine compound represented by the following formula (1).
[Chemical Formula 1]

The thienopyridine compound according to claim 1, wherein the thienopyridine compound inhibits the activity of Hsp90. 1. A pharmaceutical composition for the prevention or treatment of cancer diseases, which comprises a thienopyridine compound represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

delete The pharmaceutical composition according to claim 3, wherein the cancer is a disease selected from the group consisting of non-small cell lung cancer, lung cancer, breast cancer, uterine cancer, pancreatic cancer, prostate cancer, gastric cancer, colon cancer, leukemia, brain tumor and head and neck cancer. delete A health food for preventing or ameliorating cancer diseases, which comprises a thienopyridine compound represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

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