WO2016195194A2 - Novel tlr2 antagonists - Google Patents

Abstract

The present invention relates to TLR2 antagonists, which are small novel molecules, and, particularly, to: 19 novel TLR2 antagonists; a pharmaceutical composition, containing the antagonists, for preventing or treating inflammatory diseases; and a TLR4 modulator, containing the antagonists. The novel TLR2 antagonists according to the present invention can be effectively used as a preparation for oral administration by having low molecular weight and high oral bioavailability, and can be useful in pharmaceutical compositions for preventing or treating inflammatory diseases since the secretion of IL-8 is effectively inhibited and in vivo cytotoxicity is not induced. In addition, the novel TLR2 antagonists according to the present invention can be used as a TLR4 modulator.

Description

New TLR2 Antagonists

The present invention relates to a novel small molecule TLR2 antagonist, and in particular to 19 novel TLR2 antagonists, pharmaceutical compositions for the prophylaxis or treatment of inflammatory diseases comprising the antagonist and TLR4 modulators.

Virtual screening is an in silico that can rapidly predict the binding of molecules from the compound library to target receptors and effectively prioritize molecules with related biological activities for further identification experiments. ) Approach. Overcoming the high cost problem, which is a disadvantage of experimental high-throughput screening (HTS), requires the massive downsizing of a large amount of compound libraries into a small set that can contain specific target drugs. To this end, virtual screening has been increasingly applied in advance of experimental HTS in drug discovery projects. Application of various virtual screening techniques can significantly increase research efficiency in the field of drug discovery. One widely applied technique in virtual screening is to compare two-dimensional (2D) properties between experimentally determined ligands and molecules from compound libraries. In this approach, the structural characteristics of the molecules are expressed by the type of constituent atoms and their bond level. Another technique is to use the three-dimensional structural properties of molecules to compare similarities between molecules. Virtual screening approaches based on three-dimensional structure are classified into approaches based on ligand coordinates and approaches based on receptor coordinates. Like the two-dimensional (2D) method, the ligand-based approach searches for molecules similar to the active ligands determined experimentally by comparing three-dimensional structural properties between molecules. This approach is typically applied where only limited information about the target receptor is available. Ligand-based methods essentially involve a comparative analysis of structural properties, and therefore their application requires the acquisition of information of known active ligands. One of the valid programs, Rapid Overlay of Chemical Structures (ROCS), uses an overlapping method for large-scale form-based comparisons. ROCS adopts a method of comparing structural similarities between two molecules based on three-dimensional form. Three-dimensional forms of specifically parameterized molecules use Gaussian-based overlap to obtain an optimal alignment of the largest volume overlap between the two molecules. Since the similarity of the chemical properties as well as the morphological similarity of the molecules is a decisive factor in biological activity, the overlap between the functional groups possessed by the compound is further calculated using the color force field. By precomputing the conformers ensemble and comparing each of them sequentially, the conformational flexibility of the molecules can be considered. The excellence of the ROCS method has been reported in a number of studies. If high-resolution coordinates of the target receptor are available, molecular docking is the common method of choice in virtual screening. Docking is used to quantify the binding affinity between molecules and receptors from the compound library by computer operation to predict the likelihood of binding between them. In essence, this method does not necessarily require information about compounds that are active against the drug target, but can increase performance by incorporating the binding properties of known active substances into the docking process.

An antagonist, on the other hand, is a substance that binds to an agonist's receptor but does not exhibit physiological action through the receptor. Toll-like receptor 2 (TLR2) is present in the plasma membrane or endosome and belongs to the first line of defense of the host during an inflammatory response. Signaling associated with TLR2 has been reported to be associated with cancer, tuberculosis, anemia, atopic dermatitis and atherosclerosis. In particular, antagonists of TLR2 have become a major pharmaceutical target due to its inhibitory effect on inflammatory diseases. Therefore, there is a need for screening a new pharmaceutically available antagonist of TRL2, and in particular, research to screen for a small molecule antagonist is needed.

The present inventors screened about 7 million compounds based on drug-specific molecular group model, and selected the TLR2 antagonist, a novel small molecule that can be used for the prevention or treatment of inflammatory diseases. Was completed.

It is an object of the present invention to provide novel TLR2 antagonists.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease comprising the TLR2 antagonist, an oral dosage form, and a TLR4 modulator comprising the antagonist.

The novel TLR2 antagonist according to the present invention can be effectively used as an oral administration because of its low molecular weight and high oral bioavailability, and effectively inhibits IL-8 secretion and does not cause toxicity in vivo, thus preventing or treating inflammatory diseases. It can be usefully used in pharmaceutical compositions. The novel TLR2 antagonists according to the invention can also be used as modulators of TLR4.

1 is a simplified diagram of the overall steps for identifying a TLR2 antagonist.

2 is a diagram showing the results of identifying the major residues affecting the binding based on the free energy calculated as a result of in silico alanine scanning mutagenesis.

Figure 3 is a diagram showing the crystal structure of the TLR2-TLR1-Pam ₃ CSK ₄ complex used to build the receptor-ligand-based drug specific molecule model.

4 is a diagram showing all the drug-specific molecular cluster features obtained from Pam ₃ CSK ₄ bound to TLR2-TLR1.

FIG. 5 is a diagram showing the characteristics of five selected drug specific molecule groups in the receptor-ligand-based drug specific molecule model and the periphery of the residues labeled therein (asterisk: TLR1 residue, green sphere: HBA, purple sphere: HBD, blue spheres: HBY, gray spheres: space occupied by proteins).

6 is a diagram showing the main features shown by the receptor-ligand-based drug specific molecule model.

FIG. 7 is a diagram showing the results of constructing four sub-models of five features selected from a receptor-ligand-based drug specific molecule cluster model.

8 is a diagram showing the structure of compounds A, B, and C used to build a ligand-based drug specific molecule group model.

FIG. 9 is a diagram showing active molecules mapped from Ligand-based Drug Specific Molecule Group Model 1 constructed from Compound A.

FIG. 10 is a diagram showing active molecules mapped from ligand-based drug specific molecule group model 2 constructed from compounds B and C. FIG.

11 shows molecular docking using CDocker.

12 is a diagram showing a two-dimensional structure of the three screened compounds (S06690562, S01688300, S01382085).

13A is a diagram showing the docking result of screened compound S06690562.

13B is a view showing a docking result of the screened compound S01688300.

13C is a view showing the docking result of the screened compound S01382085.

14a is a diagram showing the docking result of screened compound S06690562.

14B is a view showing a docking result of the screened compound S01688300.

14C is a view showing the docking result of the screened compound S01382085.

15 is a diagram illustrating the secretion of IL-8 by treating 19 compounds in cells (* P <0.05, ** P <0.01).

FIG. 16 shows the results of inhibition of secretion of concentration-dependent IL-8 of three screened compounds (S06690562, S01688300, S01382085) (* P <0.05, ** P <0.01).

17 is a diagram confirming the cytotoxicity of the three screened compounds (S06690562, S01688300, S01382085).

Hereinafter, the present invention will be described in detail.

The present invention provides at least one TLR2 (Toll-like receptor 2) antagonist selected from the group consisting of the compounds of Table 1 below.

Compound name	Compound structure
S02546436
S02276077
S06696686
S06690562
S06713271
S02396152
S01525559
S06542401
S01739292
S01688300
S06570841
S06570001
S06568641
S06572801
S01577528
S01382085
S01442577
S01414289
S01292238

"TLR2 (Toll-like receptor 2)" of the present invention is located on the cell surface of monocytes, macrophages, neutrophils, etc., and bacterial cell components (fat polysaccharide, peptide glycan, adipocyte protein, anti-bacterial glycolipid, etc.) and heat shock protein (hsp) is a substance that functions as a receptor, and when TLR2 is stimulated, cells are activated to promote the production of inflammatory cytokines and inflammatory mediators (TNF, IL-1, IL-6, IL-8, NO, etc.). do.

In the present invention, an "antagonist" binds to a receptor of a drug or an agonist that acts to attenuate some or all of its action by combination with another drug, but itself binds the receptor. Means a substance that does not exhibit the physiological effect through. Thus, since the TLR2 antagonist has a strong binding force to TLR2, at the micromolecular level, the TLR2 antagonist may function to partially inhibit TLR2-related signaling but not completely abolish it.

The "TLR2 antagonist" of the present invention comprises 19 compounds of Table 1 and preferably 6 compounds (S02546436, S02276077, S06696686, S06690562, S01688300, S01382085), more preferably three compounds (S06690562, S01688300, S01382085). ). In addition, the TLR2 antagonist of the present invention may include, without limitation, the 19 compounds of Table 1 as well as analogs thereof having the same and similar activities.

Some of the compounds, such as S06690562, are tautomer molecules and may exist in enol or keto form by tautomerization depending on pH.

The TLR2 antagonist of the present invention is characterized by being a small molecule because it does not have a fatty acyl residue. Since such a decrease in molecular weight is advantageous in pharmacokinetics, the antagonists of the present invention and analogs thereof can be usefully used for designing a drug as an active ingredient of a pharmaceutical composition.

The "small molecule" means, but not limited to, an organic compound having a molecular weight of 900 Da or less.

The TLR2 antagonist of the present invention has a core structure different from the molecules previously presented as antagonists of TLR2. In addition, the nineteen compounds shown in Table 1 are characterized by having different structures.

The 19 TLR2 antagonists of the present invention include all materials purchased and used or synthesized through methods known in the art.

The present invention also provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising the TLR2 (Toll-like receptor 2) antagonist.

In the present invention, "inflammatory disease" is edema, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever , But are not limited to, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis.

As used herein, the term "prevention" means any action that inhibits or delays the progression of an inflammatory disease by administration of a composition of the present invention.

As used herein, the term "treatment" refers to any action in which an inflammatory disease is ameliorated or beneficially altered by administration of a composition of the present invention.

The composition of the present invention comprises a pharmaceutically acceptable carrier. Carriers included in the pharmaceutical composition of the present invention and acceptable carriers are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered saline (PBS) or Media and the like, but is not limited thereto.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

In addition, the pharmaceutical compositions of the present invention can be used together, simultaneously, and sequentially, including additional ingredients that can be used to prevent or treat inflammatory diseases associated with TLR2.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.

The present invention also provides an oral administration agent for the prevention or treatment of an inflammatory disease comprising the TLR2 (Toll-like receptor 2) antagonist.

Since the TLR2 antagonist of the present invention is characterized by being a small molecule, oral bioavailability is good. Bioavailability in the present invention means a portion of the amount of drug administered that falls into the subcategory of the absorption of the drug and reaches the systemic circulation. Therefore, when the oral administration of oral administration for the prevention or treatment of inflammatory diseases comprising the TLR2 antagonist of the present invention orally has the effect of reaching the systemic circulation at a high rate.

Oral dosage forms according to the present invention can be prepared in solid pharmaceutical preparations such as tablets, pills, capsules, powders and granules, or in pharmaceutically acceptable aqueous solutions, suspensions and emulsions, syrups, pharmaceutical preparations dissolved in use and liquid pharmaceutical preparations for elixirs oral. Characterized in that the dosage form is administered.

In addition, the present invention comprises the steps of (a) constructing a receptor-ligand-based pharmacophore model; (b) building a ligand-based pharmacophore model; (c) screening the products of (a) and (b); And (d) performing a biological experiment with the resultant of (c) for screening. The method of claim 1, further comprising a toll-like receptor 2 (TLR2) antagonist.

In the present invention, "pharmacophore" means a characteristic of a molecule necessary for molecular recognition of a ligand. The "pharmacophore model" describes how various ligands can bind to common receptor sites and can be used for virtual screening of novel ligands that bind to the same receptor.

In the present invention, a method for constructing "receptor-ligand-based pharmacophore models" is TLR2-TLR1-Pam ₃ CSK ₄ Residor at the binding site of Pam ₃ CSK ₄ and TLR2-TLR1 in the complex is an In Silico Alanine Scanning Mutagenesis technique, a receptor- ligand comprising mutation of alanine. Targeting binding sites of TLR2-TLR1-Pam ₃ CSK ₄ using the Pharmacophore Generation protocol includes techniques for identifying key residues that play an important role in binding. Using computer-based techniques saves time and costs.

"Pam ₃ CSK ₄ " of the present invention is an agonist of TLR2 and TLR1.

The method for constructing a "ligand-based pharmacophore model" in the present invention includes the process of characterizing a drug-specific molecular group model using an antagonist of TLR2, a known small molecule.

Screening the results identified from the drug-specific molecular cluster model constructed in the present invention may include fit value, molecular similarity, screening of drug-like compounds, molecular docking and scoring, and rescoring of docking complexes. rescoring). Through the screening process, it is possible to obtain a candidate compound of the antagonist of TLR2, which is a small molecule having excellent drug-like properties due to its binding to TLR2.

In the present invention, "biological experiment" includes IL-8 secretion, cell viability assay. Through the biological experiments, it is possible to determine whether candidate compounds of the selected antagonists of TLR2 act as antagonists of TLR2 and whether they exhibit toxicity.

The present invention also provides at least one TLR4 (Toll-like receptor 4) regulator selected from the group consisting of the compounds of Table 1 above.

In the present invention, "regulator" means a substance that increases or decreases the molecular level to a measurable level, and includes, but is not limited to, inhibitors, antagonists, agonists and the like.

TLR4 of the present invention is characterized by having structural and functional similarities to TLR2, the compound screened with an antagonist of TLR2 may have a modulator activity of TLR4. Thus, 19 compounds that are TLR2 antagonists of the invention can be utilized as modulators of TLR4.

Terms not defined otherwise in this specification are intended to have a meaning commonly used in the art to which the present invention pertains.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

The statistical analysis shown in the examples below is based on the data obtained from three independent experiments. Or less than or equal to 0.01).

Example  1-Inn Silico  Alanine Scanning Mutagenesis (In Silico Alanine  Scanning Mutagenesis)

Alanine scanning mutagenesis was used to analyze the key residues needed to build a pharmacophore model. TLR2-TLR1-Pam ₃ CSK ₄ The residues at the binding site of Pam ₃ CSK ₄ and TLR2-TLR1 in the complex were mutated to alanine, and the mutation energy was calculated based on the difference in binding free energy between mutation and wild-type. Calculate Mutation Energy (Binding) in Accelrys Discovery Studio ( DS ) 4.0 was calculated and determined that residues with mutation energy greater than 0.5 kcal / mol affect the binding capacity of the receptor-ligand complex because they make the binding unstable. Key residues that affect binding are shown in FIG. 2.

The values of the top ten destabilizing residues are shown in Table 2 and the values of all other residues are shown in Table 3.

* Residues of TLR1

* Residues of TLR1

The residues calculated in Table 2 and determined to affect the binding force were Phe325 of TLR2 and Gln316 of Phe349 and TLR1, and they were confirmed to be consistent with major residues found in biological experiments in previous studies.

Thus, the three residues are protein-ligand It was found to play an important role in the formation of the complex and was used to construct the following receptor-ligand-based drug specific molecule group model.

Example 2 Construction of a Receptor-ligand-Based Drug-Specific Molecule Group Model

To build a receptor-ligand-based drug specific molecule model, TLR2-TLR1-Pam ₃ CSK ₄ to identify the characteristics of ligands that enable binding to receptors The complex was used. The crystal structure of the composite is shown in FIG. 3.

TLR2-TLR1-Pam ₃ CSK ₄ with default parameters Receptor- ligand by targeting lipopeptide binding sites of the complex Pharmacophore Generation protocol was used. As a result, TLR2-TLR1-Pam ₃ CSK ₄ All the features of the drug-specific molecular groups based on the interaction of the complex were confirmed, which is shown in FIG. 4. All of the above features were selected as five features and are shown in FIG. 5. The main features were found in the active site around the main residues identified in Example 1 (Phe325 and Phe349 of TLR2, and Gln316 of TLR1) and the results are shown in FIG.

As shown in FIG. 5 and FIG. 6, it was confirmed that the ligand that enables binding with the protein has two hydrogen bond receptors (HBA), one hydrogen bond provider (HBD), and two hydrophobic features (HBY).

The five features identified above were further classified into four sub-models, which are shown in FIG. 7.

Example 3 Construction of Ligand-Based Drug-Specific Molecule Group Model

In order to construct a ligand-based drug specific molecule group model, compounds A, B, and C, which are antagonists of the known small molecule TLR2, were used and their structure is shown in FIG. 8. Their two-dimensional structure was drawn using ChemBioDraw Ultra (CambridgeSoft). In addition, the HBD, HBA, HBY features identified in Example 2 were investigated using the Common Feature Pharmacophore Generation protocol.

Compound A was used as a query molecule in constructing the ligand-based drug specific molecule model 1. Ten drug specific molecule cluster models were constructed from various features of Compound A, of which three HBAs and two HBYs were constructed as ligand-based drug specific molecule cluster model 1. Based on this, active molecules were mapped and the results are shown in FIG. 9.

Compound B was used as a query molecule in constructing Ligand-based drug specific molecule group Model 2. The characteristics of the drug-specific molecule group identified from Compound B were confirmed to be one HBD, two HBAs, and two HBYs, and the mapping results thereof are shown in FIG. 10.

Also, because of the structural similarity between Compound C and Compound B, it was confirmed that the result of mapping the characteristic identified from Compound C is shown in FIG. 10.

Example 4-Screening with fit values

In order to select TLR2 antagonist candidates from a library of about 7 million molecules commercially available, four sub-receptor-ligand-based drug specific molecule models constructed in Example 2 were used. The Search 3D Database of DS 4.0 was used and each of the four models was used as input. Best search method was used to calculate the degree of fit of the ligand to the drug-specific molecular group model (fit value). Based on this value, 500 molecules (hit) were obtained which were ideally mapped to each model. As a result, 2000 hit molecules were selected from a total of four models.

Example 5 Screening Using Molecular Similarity

To select TLR2 antagonist candidates from a commercially available library of about 7 million molecules, from Ligand-based Drug Specific Molecule Group Model 1 with Compound A as input, according to the same method as Example 4 above. 2312604 hit molecules were selected from 1651005 hit molecules from Ligand-Based Drug Specific Molecule Group Model 2 with Compound B input.

To further select 3963609 hit molecules from two models, we used the scoring function of ROCS_tanimoto-Combo according to the similarity of the shape and atomic type of query and hit molecules. Compound A was used as a query molecule to compare 2312604 hit molecules obtained from Ligand-based drug specific molecule group Model 1, and compounds B and C to compare with 1651005 hit molecules obtained from ligand-based drug specific molecule group Model 2 Is the query molecule. As a result of the scoring, a total of 1500 hit molecules were selected from the ligand-based drug-specific molecular group model.

Example 6 Screening of Drug-Like Compounds

Lipinski and Veber rules for additional screening of drug-similar compounds required in the drug development phase out of the 2000 hit molecules obtained in Example 4 and 1500 hit molecules obtained in Example 5, that is, 3500 hit molecules in total And ADMET (absorption, distribution, metabolism, excretiom, toxicity) characteristics.

Specifically, Lipinski and Veber rules were used as filters to screen for compounds with better oral bioavailability. ADMET (absorption, distribution, metabolism, excretiom, toxicity) features good absorption, adequate solubility, low blood-brain penetrability, cytochrome P450 2D6 non-inhibitory, non-hepatocytotoxic, and non-plasma It was used to identify protein binding capacity.

As a result, 1126 molecules selected as drug-like compounds were identified from a total of 3500 hit molecules. Drug-like characteristics of candidate compounds were calculated and screened to exclude molecules of low availability in drug development.

Example 7 Moleculatr Docking and Scoring

Molecular docking of TLR2-TLR1 dimers was used to select optimal docking poses for ligand-receptor binding and to screen ligands. Ligands were prepared with the Prepare Ligand module of DS 4.0. The binding position of the lipopeptide was selected as the docking position. For optimal screening, two screening programs were performed: CDocker (using CD, CHARMM force field) and AutoDock (AD) Vina.

More specifically, 1126 drug-like compounds obtained in Example 6 using four CD 4 Pam ₃ CSK ₄ Docked in position. At this time, as shown in Fig. 11, a sphere having a radius of 13 Å covering most of B and C positions was used. The CD interaction energy was calculated and the top 100 poses were screened for about 60 compounds.

The 100 poses were redocked using AD Vina. At this time, dock_runscreening protocol is applied. As a result, the top 26 poses with good docking were screened based on the AD affinity energy value.

Example 8-Rescoring of a Docking Composite

Docking complex rescoring using binding free energy was performed to determine the driving force of the protein-ligand interaction.

Specifically, in order to quantify the thermodynamic interaction of the ligand with the TRL2-TLR1 complex, the 26 docking complexes obtained in Example 7 were prepared by molecular calculation based on the calculation of Poisson-Boltzmann surface area (MM / PBSA) binding free energy. dynamics, MD). MD simulations were performed using the g_mmpbsa tool, and the average binding energy and its standard deviation / error were calculated with MnPbSaSatat.pyscript.

MM / PBSA binding free energy was calculated by Equation 1 below.

[Equation 1]

G _bind is the mean binding free energy, G _complex is the binding free energy of TRL2-TLR1 complex, G _protein is the binding free energy of protein (receptor), and G _ligand is the binding free energy of ligand.

Three compounds (S06690562, S01688300, S01382085) were selected based on the average MM / PBSA value obtained by the above calculation, the AD binding energy value and the CD binding energy value of Example 7. As a result, it was confirmed that two compounds were selected from the receptor-ligand-based model and one compound was selected from the ligand-based model and their two-dimensional structure is shown in FIG. 12. In addition, the molecular docking results are shown in FIGS. 13A to 13C and 14A to 14C.

Example 9-Confirmation of IL-8 Secretion

19 compounds out of 26 compounds screened in Example 7 (S02546436, S02276077, S06696686, S06690562, S06713271, S02396152, S01525559, S06542401, S01739292, S01688300, S06570841, S06570001, S06568641, S06572801, S01577528, 013,775, 026 , S01292238) was treated with the compound to confirm that it exhibits biological activity as an antagonist of TLR2, and the secretion changes of IL-8, a cytokine induced by TLR2, were confirmed.

HEK293-TLR2 (TLR1 is expressed at endogenous levels) and HEK293-Null cell line at 37 ° C., 95% air and 5% CO ₂ at a density of 1 × 10 ⁴ cells / well in 96-well tissue culture plates (BD Biosciences) Incubated for 24 hours at. To examine the activity of agonists, cell stimulation was done by treatment of 50 μM concentration of compound and 50 nM of Pam ₃ CSK ₄ (Invivogen, SanDiego, Calif., USA). To determine antagonist activity, cells were treated with various concentrations of compounds for 1 hour, followed by co-treatment with 50 nM of Pam ₃ CSK ₄ . The following day, IL-8 secretion was quantified in a human IL-8 ELISA Ready-SET-Go! ® (second-generation) kit (eBioscience, San Diego, Calif., USA) by the method according to the manufacturer's guide. 15 is shown.

As shown in FIG. 15, all 19 compounds were found to reduce the secretion of IL-8, and in particular, when treated with 6 compounds (S02546436, S02276077, S06696686, S06690562, S01688300, S01382085), Pam ₃ CSK ₄ -induced. It was confirmed that the amount of secreted IL-8 was greatly reduced. Therefore, it was confirmed that the six compounds act as an antagonist of TLR2 and significantly reduce the secretion of IL-8.

For concentration-dependent analysis of the top three of the six compounds (S06690562, S01688300, S01382085), the concentrations of the three compounds were varied at 12.5 μM, 25 μM, 50 μM and the method described above. As described above, the secretion amount of IL-8 was analyzed and the results are shown in FIG. 16.

As shown in FIG. 16, all three compounds were found to significantly reduce the secretion of IL-8 when the compounds were treated at a high concentration of 50 μM compared to the treatment at low concentrations. Therefore, the three compounds were confirmed to be a substance that reduces the secretion of IL-8 in a concentration-dependent manner.

Example 10 Cell Culture and Cell Viability Assay

HEK293-TLR2 and HEK293-Null cell lines (Invivogen, San Diego, Calif., USA) were used for 10% heat-inactivated fetal bovine serum, Thermo Fisher Scientific Inc. (50 IU / mL penicillin, 50 μg / mL streptomycin). (Thermo Fisher Scientific Inc.) and Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc., MA, USA) supplemented with Normocin ™ 100 mg / mL (Invivogen., San Diego, Calif., USA). The compound was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, Mo., USA) in a brown tube and stored at a concentration of 10 mM.

In order to confirm the cytotoxicity of the three compounds screened in Example 8 (S06690562, S01688300, S01382085), MTS technique was performed in HEK293-TLR2, and the results are shown in FIG.

Specifically, cell activity was measured using a CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS assay; Promega, Madison, WI, USA) according to the manufacturer's guidelines. Cells were incubated in 96-well plates at a concentration of 5 × 10 ³ cells / mL and kept overnight at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . The following day, the cultured cells were treated with three concentration conditions (12.5 μM, 25 μM, and 50 μM) of three screened compounds (S06690562, S01688300, S01382085). After 24 hours, the MTS solution was treated in the wells, and the absorbance was measured at 490 nm with a microplate spectrophotometer system (Molecular Devices Inc.), and the results are shown in FIG. 17.

As shown in FIG. 17, all three compounds did not show cytotoxicity at concentrations of 12.5 μM, 25 μM, and 50 μM, and thus were found to be safe substances and used as drugs.

Since the novel TLR2 antagonist according to the present invention effectively inhibits IL-8 secretion and does not cause toxicity in vivo, it may be usefully used in the pharmaceutical composition for preventing or treating inflammatory diseases. Since the TLR2 antagonist has a low molecular weight and high oral bioavailability, the TLR2 antagonist may be effectively used as an oral administration agent, and may also be used as a modulator of TLR4.

Claims (6)

1. At least one TLR2 (Toll-like receptor 2) antagonist selected from the group consisting of the compounds of Table 1.

   TABLE 1

   

   

   

   

   
2. The TLR2 (Toll-like receptor 2) antagonist according to claim 1, wherein the TLR2 antagonist is at least one selected from the group consisting of S06690562, S01688300 and S01382085 in Table 1.
3. A pharmaceutical composition for preventing or treating an inflammatory disease comprising the toll-like receptor 2 (TLR2) antagonist of claim 1.
4. According to claim 3, The inflammatory disease is edema, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, At least one member selected from the group consisting of rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis Characterized in that for the prevention or treatment of inflammatory diseases.
5. An oral dosage form for the prevention or treatment of an inflammatory disease comprising the toll-like receptor 2 (TLL2) antagonist of claim 1.
6. At least one TLR4 (Toll-like receptor 4) regulator selected from the group consisting of the compounds of Table 1.

   TABLE 1

   

   

   

   

   

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