WO2016195194A2 - Nouveaux antagonistes de tlr2 - Google Patents

Nouveaux antagonistes de tlr2 Download PDF

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WO2016195194A2
WO2016195194A2 PCT/KR2015/014202 KR2015014202W WO2016195194A2 WO 2016195194 A2 WO2016195194 A2 WO 2016195194A2 KR 2015014202 W KR2015014202 W KR 2015014202W WO 2016195194 A2 WO2016195194 A2 WO 2016195194A2
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tlr2
receptor
antagonist
compounds
ligand
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PCT/KR2015/014202
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Korean (ko)
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WO2016195194A3 (fr
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최상돈
듀라이프라산나벤카테시
아첵아스마
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아주대학교산학협력단
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Priority claimed from KR1020150175045A external-priority patent/KR101745524B1/ko
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Priority to US15/578,086 priority Critical patent/US10308655B2/en
Priority to EP15894366.2A priority patent/EP3305767B1/fr
Publication of WO2016195194A2 publication Critical patent/WO2016195194A2/fr
Publication of WO2016195194A3 publication Critical patent/WO2016195194A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings

Definitions

  • 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.
  • HTS experimental high-throughput screening
  • 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.
  • 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.
  • TLR2 Toll-like receptor 2
  • TLR2 Toll-like receptor 2
  • Signaling associated with TLR2 has been reported to be associated with cancer, tuberculosis, anemia, atopic dermatitis and atherosclerosis.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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).
  • FIG. 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.
  • FIG. 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. 10 is a diagram showing active molecules mapped from ligand-based drug specific molecule group model 2 constructed from compounds B and C.
  • FIG. 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).
  • 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.
  • TLR2 Toll-like receptor 2
  • TLR2 Toll-like receptor 2
  • bacterial cell components fat polysaccharide, peptide glycan, adipocyte protein, anti-bacterial glycolipid, etc.
  • heat shock protein hsp
  • TLR2 inflammatory cytokines and inflammatory mediators
  • 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.
  • the TLR2 antagonist 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).
  • 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.
  • 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.
  • TLR2 Toll-like receptor 2
  • 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.
  • prevention means any action that inhibits or delays the progression of an inflammatory disease by administration of a composition of the present invention.
  • 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.
  • PBS phosphate buffered saline
  • 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.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, a kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mann
  • 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.
  • TLR2 Toll-like receptor 2
  • 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.
  • 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.
  • TLR2 toll-like receptor 2
  • 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.
  • 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).
  • TLR2 the antagonist of TLR2
  • 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.
  • TLR4 Toll-like receptor 4
  • regulatory 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.
  • 19 compounds that are TLR2 antagonists of the invention can be utilized as modulators of TLR4.
  • 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.
  • TLR2-TLR1-Pam 3 CSK 4 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.
  • 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.
  • the ligand that enables binding with the protein has two hydrogen bond receptors (HBA), one hydrogen bond provider (HBD), and two hydrophobic features (HBY).
  • HBA hydrogen bond receptors
  • HBD hydrogen bond provider
  • HBY hydrophobic features
  • 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.
  • TLR2 antagonist candidates 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.
  • 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.
  • ADMET absorption, distribution, metabolism, excretiom, toxicity
  • 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.
  • TLR2-TLR1 dimers were 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.
  • two screening programs were performed: CDocker (using CD, CHARMM force field) and AutoDock (AD) Vina.
  • 1126 drug-like compounds obtained in Example 6 using four CD 4 Pam 3 CSK 4 Docked in position 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.
  • 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.
  • Equation 1 MM / PBSA binding free energy
  • 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)
  • G ligand is the binding free energy of ligand.
  • 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.
  • cell stimulation was done by treatment of 50 ⁇ M concentration of compound and 50 nM of Pam 3 CSK 4 (Invivogen, SanDiego, Calif., USA).
  • Pam 3 CSK 4 Invivogen, SanDiego, Calif., USA.
  • 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 .
  • 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.
  • HEK293-TLR2 and HEK293-Null cell lines were used for 10% heat-inactivated fetal bovine serum, Thermo Fisher Scientific Inc. (50 IU / mL penicillin, 50 ⁇ g / mL streptomycin). (Thermo Fisher Scientific Inc.) and Dulbecco's modified Eagle's medium (Thermo Fisher Scientific Inc., MA, USA) supplemented with Normocin TM 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.
  • dimethyl sulfoxide Sigma-Aldrich, St. Louis, Mo., USA
  • 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.
  • MTS assay 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 cultured cells were treated with three concentration conditions (12.5 ⁇ M, 25 ⁇ M, and 50 ⁇ M) of three screened compounds (S06690562, S01688300, S01382085).
  • 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.
  • 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.

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Abstract

La présente invention concerne des antagonistes de TLR2, qui sont de nouvelles petites molécules et, en particulier : 19 nouveaux antagonistes de TLR2 ; une composition pharmaceutique, contenant les antagonistes, pour prévenir ou traiter des maladies inflammatoires ; et un modulateur de TLR4 contenant les antagonistes. Les nouveaux antagonistes de TLR2 selon la présente invention peuvent être efficacement utilisés en tant que préparation pour administration orale car ils ont un faible poids moléculaire et une biodisponibilité orale élevée, et ils peuvent être utiles dans des compositions pharmaceutiques pour prévenir ou traiter des maladies inflammatoires du fait que la sécrétion d'IL-8 est efficacement inhibée et qu'aucune cytotoxicité in vivo n'est induite. De plus, les nouveaux antagonistes de TLR2 selon la présente invention peuvent être utilisés en tant que modulateur de TLR4.
PCT/KR2015/014202 2015-05-29 2015-12-23 Nouveaux antagonistes de tlr2 WO2016195194A2 (fr)

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US15/578,086 US10308655B2 (en) 2015-05-29 2015-12-23 Method of treating Crohn's disease comprising a TLR2 antagonist
EP15894366.2A EP3305767B1 (fr) 2015-05-29 2015-12-23 Nouveaux antagonistes de tlr2 pour le traitement des maladies inflammatoires

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020157620A1 (fr) * 2019-01-30 2020-08-06 Insilico Medicine Ip Limited Inhibiteurs de tlr
US11008303B2 (en) 2019-01-30 2021-05-18 Insilico Medicine Ip Limited TLR 9 inhibitors
US11807622B2 (en) 2019-01-30 2023-11-07 Insilico Medicine Ip Limited TLR 9 inhibitors

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US10988457B2 (en) 2019-01-30 2021-04-27 Insilico Medicine Ip Limited TLR inhibitors
US11008303B2 (en) 2019-01-30 2021-05-18 Insilico Medicine Ip Limited TLR 9 inhibitors
US11807622B2 (en) 2019-01-30 2023-11-07 Insilico Medicine Ip Limited TLR 9 inhibitors

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