KR20130108876A - Composition for specifically inhibiting shp-2 activity and method the same - Google Patents

Abstract

PURPOSE: A composition which specifically suppresses the activation of Src homology 2-containing protein tyrosine phosphatase (SHP-2) is provided to study SHP-2-related diseases such as LEOPARD syndrome type1, Noonan syndrome, or pigmented villondular synovitis and an SHP-2 mechanism. CONSTITUTION: A compound of chemical formula 1 or a pharmaceutically acceptable salt thereof is provided. A pharmaceutical composition for treating breast cancer, lung cancer, colon cancer, melanoma, neuroblastoma, acute myeloid leukemia, LEOPARD syndrome type1, Noonan syndrome, and pigmented villondular synovitis contains the compound of chemical formula 1 as an active ingredient. The pharmaceutical composition specifically suppresses SHP-2.

Description

Composition for specifically inhibiting the activity of SHP-2 and its preparation method {Composition for specifically inhibiting SHP-2 activity and method the same}

The present invention relates to a composition that specifically inhibits the activity of SHP-2 and a method of preparing the same, and more particularly to breast cancer, lung cancer, melanoma, neuroblastoma, acute myeloid leukemia The present invention relates to a pharmaceutical composition for treating Leopard syndrome type 1, Nuonan syndrome or pigmented chorio nodular synovial disease, and a preparation method thereof.

Protein phosphorylation and dephosphorylation are well known as important mechanisms utilized to induce signaling by cells among the various stages of cellular function. Signals by the cells are primarily transmitted through phosphorylation involving kinases and dephosphorylation involving protein phosphatase. In particular, protein tyrosine phosphatase (PTP), which acts on the dephosphorylation process, due to its unique activity in vivo, modulates and regulates the basic cellular signaling mechanisms involved in metabolism, growth, proliferation and differentiation. It is known to play an important role.

It has been shown that the regulation of protein tyrosine phosphatase activity is involved in various cellular biological functions and diseases. For example, mutations in the PTP1B gene in mice are responsible for insulin signaling and PTPRC is involved in many sclerosis. Thus, they are likely to be important targets for therapeutic intervention in many diseases, including cancer and diabetes.

SHP-2 is a protein tyrosine phosphatase encoded by the PTPN11 gene and has two domains, N-SH2 and C-SH2. SHP-2 modulates cell signaling mediated by cytokine and cellular signaling growth factors such as EGF, hepatocyte growth factor and interleukin-6. SHP-2 is also involved in Erk 1/2 MAP kinase activity by EGF. SHP-2 mutations cause child development disorders, Noonan syndrome and various malignancies in humans. Therefore, SHP-2 has become an important target as a therapeutic agent for Nuonan syndrome and cancer associated with SHP-2.

In this regard, there have been studies of inhibitors that modulate SHP-2 activity, but this has been shown to be involved in the activity of other protein tyrosine phosphatase, including SHP-2 (Liwei Chen et al., Mol Pharmacol 80: 562-). 570,2006)

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a therapeutic pharmaceutical composition and method for providing the same specifically inhibiting the activity of SHP-2 in protein tyrosine phosphatase.

In order to achieve the above object,

The present invention provides MLS-1 compounds.

In addition, the present invention is a breast cancer, lung cancer, melanoma (melanoma), neuroblastoma (neuroblastoma), acute myeloid leukemia (LEOPARD syndrome type 1), nunan comprising MLS-1 as an active ingredient Provided is a pharmaceutical composition for treating Noonan syndrome or pigmented chorionic nodular synoviitis disease.

Also. The present invention provides a therapeutic pharmaceutical composition, the therapeutic pharmaceutical composition specifically inhibits SHP-2 (SH2) -containing protein tyrosine phosphatase (SHP-2).

In addition, the present invention includes the following steps,

1) After dissolving benzimidazole and sodium hydride in THF (Tetrahydrofuran), methylbromoacetate is added to the reaction mixture, the reaction mixture is diluted with ethyl acetate, washed with water and brine, and dried over anhydrous magnesium sulfate. Preparing a methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate by column chromatography on a residue obtained by concentrating the filtrate under reduced pressure and drying the mixture;

2) After dissolving the methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate in alcohol and adding hydrazine hydrate dissolved in alcohol, the reaction mixture was refluxed and recrystallized after completion of the reaction to give 2- ( Preparing 1-benzo [d] imidazole-1) acetohydrazide;

3) After dissolving potassium hydroxide in alcohol and adding the 2- (1-H-benzo [d] imidazol-yl) acetohydrazide and carbon disulfide, the precipitates separated after dilution with ether are collected and washed once more with ether. After drying, it was dissolved in water, hydrazine hydrate was added to reflux, and the precipitate obtained under acidic condition was dried to give 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1, Preparing 2,4-triazole-3-thiol; And

4) the 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol, py-toluic acid and Phosphoryl chloride is added and refluxed to form a precipitate and dried to provide a step for preparing MLS-1.

The present invention specifically inhibits the activity of SHP-2 in protein tyrosine phosphatase, such as breast cancer, lung cancer, melanoma, neuroblastoma, acute myeloid leukemia, leopard syndrome type 1 (LEOPARD) It is expected to be used for the study of SHP-2-related diseases such as syndrome type1, Nuonan syndrome or pigmented chorio nodular synovial disease, and the mechanism of SHP-2.

1 is a result showing the inhibitory effect of SHP-2 according to the concentration of MLS-1.
2 shows the results of kinectic analysis of MLS-1 on SHP-2.
3 is a result showing the cell permeability of MLS-1 and the SHP-2 inhibitory effect expressed in cells.

The present inventors have endeavored to develop an inhibitor that specifically inhibits the activity of SHP-2 in protein tyrosine phosphatase. As a result, a novel compound of MLS-1 and a method for preparing the same have been completed.

The present invention provides a compound of formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

Specifically, breast cancer, lung cancer, melanoma, neuroblastoma, acute myeloid leukemia, LEOPARD syndrome type 1 and Nunan syndrome, including MLS-1 as an active ingredient syndrome) or pigment chorio nodular synovial disease, the present invention provides a therapeutic pharmaceutical composition, and specifically inhibits SHP-2 (SH2) -containing protein tyrosine phosphatase (SHP-2).

In the present invention, MLS-1 refers to the compound of Formula 1.

The therapeutic pharmaceutical compositions of the invention may comprise existing therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers. Examples of suitable carriers, excipients and diluents which may be included in such compositions include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When formulated, the composition may further include, but is not limited to, conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like.

The therapeutic pharmaceutical composition of the present invention may be used in the form of a general pharmaceutical formulation, and when formulated, a pharmaceutically acceptable diluent or excipient may be used. The composition of the present invention may be formulated into various forms such as powders, granules, tablets, capsules, oral formulations such as suspensions, emulsions, syrups and aerosols, and sterilized injection solutions according to conventional methods And can be administered via various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. 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. Base materials for injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

Administration in the present invention means providing the desired material to the patient by any suitable method, and the administration route of the composition of the present invention may be administered orally or parenterally through any general route as long as it can reach the target tissue . In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug, the drug. Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In addition, the dosage of the compound according to the present invention may be changed according to body absorption, weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, and the like. Administration may be administered once a day or may be divided several times.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example  One  : MLS -1 synthesis

One. Methyl  2- (1H- benzo [d] imidazol-1- yl ) acetate Synthesis of

Benzimidazole (300 mg, 2.54 mmol) and NaH (91 mg, 3.75 mmol) were dissolved in THF (5 ml) and stirred at room temperature. After 30 minutes, Methyl Bromo Acetate (388mg, 2.5mmol) was added to the reaction mixture at 0 ° C, and stirred for 3 hours at room temperature. The reaction mixture was diluted with EtOAc and washed with water and brine. After drying over anhydrous MgSO ₄ , the filtrate was concentrated under reduced pressure. The residue obtained by column chromatography (MC: MeOH = 10: 1) was purified by Methyl 2- (1H-benzo [d] imidazol-1-yl) acetate (83mg, 0.41mmol, 16). %) Was synthesized.

¹ H-NMR (DMSO-d ₆ , 300 MHz): δ 8.20 (s, 1H), 7.66 (dd, 1H, J = 6Hz, 1.5Hz), 7.55 (dd, 1H, J = 6Hz, 1.5Hz), 7.21 7.26 (m, 2H), 5.27 (s, 2H), 3.71 (s, 3H).

2. 2- (1H- benzo [d] imidazol-1- yl ) acetohydrazide Synthesis of

The Methyl 2- (1H-benzo [d] imidazol-1-yl) acetate (83mg, 0.41mmol) was dissolved in 10ml of EtOH and stirred while adding Hydrazine hydrate (40mg, 0.81mmol) dissolved in EtOH (1ml) drop by drop. It was. The reaction mixture was refluxed for 14 hours. After completion of the reaction, recrystallization was performed to synthesize 2- (1H-benzo [d] imidazol-1-yl) acetohydrazide (20mg, 0.36mmol, 89%).

1 H-NMR (DMSO-d 6,300 MHz): δ 9.55 (s, 1H), 8.15 (s, 1H), 7.65 (d, 1H, J = 8.1 Hz), 7.47 (d, 1H, J = 7.5 Hz), 7.18-7.27 (m, 2H), 4.88 (s, 2H), 4.45 (br, 2H).

3. Synthesis of 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol

Dissolve KOH (20mg, 0.36mmol) in Absolute ethanol (1ml) and 2- (1H-benzo [d] imidazol-1-yl) acetohydrazide (20mg, 0.36mmol) and carbon disulfide (27mg, 0.36mmol) at 0 ℃ Add and stir for 1 hour. After dilution with Ether collected the sediment that fell. Washed once more with ether and dried to proceed to the next reaction. The precipitate was dissolved in water (2ml) and Hydrazine hydrate (12mg, 0.36mmol) dissolved in water (1ml) was added dropwise to the reaction flask. The reaction mixture was refluxed for 12 hours. After completion of the reaction, the precipitate obtained under acidic condition was dried to give 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol (20mg, 0.08 mmol, 30%).

1 H-NMR (DMSO-d 6,300 MHz): δ 8.28 (s, 1H), 7.65 (d, 1H, J = 6 Hz), 7.57 (d, 1H, J = 6 Hz), 7.71-7.26 (m, 2H), 5.66 (s, 2 H), 5.59 (s, 2 H).

4. 3 - ((1H- benzo [ d] imidazol-1- yl) methyl) -6-p- tolyl - [1,2,4] triazolo [3,4-b] [1,3,4] t Synthesis of hiadiazole (MLS-001)

5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol (20mg, 0.08mmol), p-toluic acid in the reaction flask (16 mg, 0.12 mmol) and POCl 3 (2 ml) were added and refluxed. pH is adjusted to 8 to form a precipitate and dried to 3-((1H-benzo [d] imidazol-1-yl) methyl) -6-p-tolyl- [1,2,4] triazolo [3,4- b] [1,3,4] thiadiazole (MLS-001) (20 mg, 0.06 mmol, 72%) was obtained.

1 H-NMR (DMSO-d 6,300 MHz): δ 8.44 (s, 1H), 7.76 (d, 2H, J = 5.1 Hz), 7.65 (t, 2H, J = 4.5 Hz), 7.36 (d, 2H, J = 5.1 Hz), 7.19-7.25 (m, 2H), 6.04 (s, 2H), 2.39 (s, 3H).

[Reaction Scheme]

Example  2  : 6x His - tagged Phosphatase  Purification of proteins

PTP (protein tyrosine phosphatase) expression plasmid was used pET28a (+) (Novagen, Darmstadt) and transformed to BL21 (DE3) -RIL E. coli. The recombinant protein was induced by treatment of E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37, 30 or 22 ° C. for 3 to 16 hours. The cells were cultured and then sonicated in 50 mM Tris-HCl (pH 8), 300 mM NaCl, 1% Tergitol-type NP-40, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The lysate was treated at 10000 rpm at 4 ° C. for 30 minutes. The supernatant was then passed through a Ni-NTA resin (PEPTRON, Daejon) column. The resin was washed under 20 mM Tris-HCl (pH 8), 500 mM NaCl, 50 mM imidazole and separated under 20 mM Tris-HCl (pH 8), 500 mM NaCl, 200-300 mM imidazole. The isolated protein was dialyzed overnight under 20 mM Tris-HCl (pH 8), 100 mM NaCl, 30% glycerol, 0.5 mM PMSF before storage at -80 ° C.

Example 3 : In vitro phosphatase assays and kinetic 분석

PTP activity was measured using 3-Omethylfluorescein phosphate (OMFP; Sigma, St. Louis, MO) in a 96-well microtiter plate assay. MLS-1 and OMFP were available in DMSO and final concentrations were performed in 1% DMSO for all reactions. The final culture mixture (100 μL) was optimized for enzymatic activity, 30 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.33% bovine serum albumin (BSA), 100 nM of PTP and Consist of inhibitors. The reaction started with the addition of OMFP and incubated at 37 ° C. for 30 minutes. Fluorescence emission from the product was measured with a multiwell plate reader (synergy H1 (BioTek); excitation filter, 485 nm; emission filter, 535 nm). The concentration of enzyme and substrate was directly proportional to the experiment time. Half-maximal inhibition constant (IC ₅₀ ) is the concentration that reduces PTP activity by 50%. The curve fit of the half-maximal inhibition constant (IC ₅₀ ) and Lineweaver-Burk plots was determined using the curve fitting program Prism 3.0 (GraphPad Software). All experiments were performed three times.

Example  4  : Inhibition studies

Inhibition constants (K _i ) of inhibitors against PTP were determined by measuring the respective fixed inhibitor concentrations and the initial rate of various OMFPs. The inhibition constant of the competitive inhibitor can be obtained by the following equation. The slope was expressed according to the inhibitor concentration. K _i values are obtained by slope.

1 / V = Km (1 + [I] / K _i ) V _max [S] + 1 / V _max

Example  5  : MLS -1 of Intracellular SHP -2 Phosphatase  Activity Verification Experiment

HEK293 cells were transformed using a plasmid expressing FLAG-SHP-2 phosphatase. 48 hours after conversion, MLS-1 (20 μΜ) was pretreated for 3 hours. Cells were washed twice with phosphate buffered saline (PBS) buffer and PTP lysis buffer (0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) for 30 minutes at 4 ° C. , 1 mM EDTA, 1% glycerol, 1 mM PMSF, and 1 μg / ml aprotinin). Cell lysates from the centrifuge were mixed with washed FLAG M2-agarose (Sigma-Aldrich, St. Louis, MO) and incubated using a rotator at 4 ° C. for 1 hour. After incubation, FLAG M2-agarose was washed three times with PTP lysis buffer, and phosphatase activity was measured.

Example 6 : Intracellular Experiment confirming the effect of MLS- 1 on ERK phosphorylation

BOSC cells cultured in serum-free medium were pretreated for 3 hours in MLS-1 (0, 20 μM) and stimulated with EGF (1 ng / ml, 5 min). The cells comprise 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 mM Na ₃ VO ₄ , and 1 mM NaF. It was dissolved in lysis buffer. These samples were separated by SDS-PAGE and western blotting was performed.

Example 7 : Western Blotting analysis

Samples were passed through SDS-12% polyacrylamide gel and delivered to nitrocellulose membrane. Membranes were blocked with 5% low fat skim milk, incubated with appropriate antibodies, and cultured by binding a secondary antibody to mustard peroxidase. Immunoreactive bands were observed using an ECL system (Younginfrontier).

Example  8  : SHP Identification of specific activity inhibitors of -2: MLS -One

1. Screening of PTPs Inhibited by MLS-1

Experiments have determined the inhibitory effects of MLS-1 on 12 recombinant proteins. Specifically, MLS-1 inhibition of PTPs including ACP-1, cdc25A, DUSP3, DUSP14, DUSP18, DUSP22, DUSP23, DUSP26, PTPN6, PTPn2, SSh3 and SHP-2 was measured. Each experiment was conducted three times. When PTPs were treated at various concentrations of MLS-1 (0-50 μM), only SHP-2 activity decreased dose dependently of MLS-1 (Table 1). Inhibition curves of SHP-2 were shown and IC ₅₀ values were calculated. IC ₅₀ values showed 4.49 ± 0.34 μM (n = 3) (FIG. 2).

PTPs Inhibited by MLS-1 Protein tyrosine phosphatase IC50 ACP1 No inhibition cdc25A No inhibition DUSP 3 No inhibition DUSP 14 No inhibition DUSP 18 No inhibition DUSP 22 No inhibition DUSP 23 No inhibition DUSP 26 No inhibition PTPN6 No inhibition PTPN2 No inhibition SSH3 No inhibition SHP-2 4.49 ± 0.34 (n = 3)

2. Kinetic assay

To determine the binding mechanism of SHP-2 and MLS-1, kinetic assay of SHP-2 and MLS-1 was performed. The kinetic assay revealed that SHP-2 and MLS-1 are competitive inhibitions. The K _M value of SHP-2 phsophatase of 3-Omethylfluorescein phosphate (OMFP) was 3.41 μM. Lineweaver-Burk plot showed that K _i was 3.477 μM (FIG. 3). These results suggest that MLS-1 competitively inhibits SHP-2 and that MLS-1 binds to the active site of SHP-2.

3. Inhibitory Effect of MLS-1 on SHP-2 Phosphatase Activity in Cells

Experiments were conducted to show whether MLS-1 enters animal cells and inhibits SHP-2 action. In the next experiment, we found that MLS-1 penetrated the cells. HEK 293 cells were transformed with plasmids expressing FLAG-SHP-2, MLS-1 was pretreated at 20 μM for 3 hours, HepG2 cells were lysed, and immunoprecipitation was performed using anti FLAG M2-agarose. Was performed. Phosphatase activity of SHP-2 immunoprecipitation was measured using OMFP (FIG. 4). Specifically, immunoprecipitated SHP-2 was incubated with OMFP at 37 ° C. for 30 minutes. MLS-1 inhibited phosphatase activity in SHP-2 cells. These results indicate that MLS-1 inhibits the activity of SHP-2 intracellularly.

4. Effect of MLS-1 on Intracellular ERK Phosphorylation

Intracellular SHP-2 activity of MLS-1 was determined by measuring the phosphorylation status of endogenous extracellular signal regulated kinase (ERK). BOSC cells were pretreated with 20 μM for 3 hours and stimulated with EGF (1ng / ml, 5min). The level of phospho-ERK was determined by Western blotting. EGF-mediated phosphorylation of ERK was markedly inhibited by MLS-1 (FIG. 9). These results indicate that as MLS-1 inhibits SHP-2 activity, SHP-2 mediated ERK phosphorylation process is inhibited.

Claims (4)

A compound of Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]

Breast cancer, lung cancer, rectal cancer, melanoma, neuroblastoma, neuroblastoma, acute myeloid leukemia, Leopard syndrome type 1, Nuonan syndrome (Noonan) syndrome) or pigmented chorionic nodular synoviitis disease, pharmaceutical composition for treatment. The therapeutic pharmaceutical composition of claim 2, wherein the therapeutic pharmaceutical composition specifically inhibits Src homology 2 (SH2) -containing protein tyrosine phosphatase (SHP-2). Including the following steps, the preparation method of Chemical Formula 1.
1) After dissolving benzimidazole and sodium hydride in THF (Tetrahydrofuran), methylbromoacetate was added to the reaction mixture, the reaction mixture was diluted with ethyl acetate, washed with water and brine, and dried over anhydrous magnesium sulfate. Preparing a methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate by column chromatography on a residue obtained by concentrating the filtrate under reduced pressure and drying the mixture;
2) After dissolving the methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate in alcohol and adding hydrazine hydrate dissolved in alcohol, the reaction mixture was refluxed and recrystallized after completion of the reaction to give 2- ( Preparing 1-benzo [d] imidazole-1) acetohydrazide;
3) After dissolving potassium hydroxide in alcohol and adding the 2- (1-H-benzo [d] imidazol-yl) acetohydrazide and carbon disulfide, the precipitates separated after dilution with ether are collected and washed once more with ether. After drying, it was dissolved in water, hydrazine hydrate was added to reflux, and the precipitate obtained under acidic condition was dried to give 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1, Preparing 2,4-triazole-3-thiol; And
4) the 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol, py-toluic acid and Phosphoryl chloride was added to reflux to form a precipitate and dried to prepare the formula (1).

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