Patent Application
20250197363
The present disclosure relates to a thiadiazole compound, and a pharmaceutical composition for preventing or treating inflammatory disease including the same.
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor ligand-activated transcription factors. According to their ligand specificity, physiological role, and tissue distribution, PPARs are classified into three types of PPARα, PPARγ, and PPARδ. In particular, PPARδ (also known as PPARβ) is one of the PPAR transcription factors essential for several biological functions, and unlike PPARα and PPARγ, PPARδ is ubiquitously expressed. Therefore, its function and application in the field of drug development are the least known among PPARs. However, since the discovery of high affinity PPARδ agonists such as GW0742 and GW501516, several researchers have studied the role of PPARδ in diseases such as obesity and diabetes, as well as cancer, neurological disorders, inflammation, dyslipidemia, heart disease, and liver disease.
One embodiment is intended to provide a novel thiadiazole derivative compound.
Another embodiment is intended to provide a pharmaceutical composition including a thiadiazole derivative compound for preventing or treating inflammatory disease.
Another embodiment is intended to provide a health functional food composition for preventing or ameliorating inflammatory disease including a thiadiazole derivative compound.
Another embodiment is intended to provide a method for treating inflammatory disease, including administering the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof to an individual or subject in need thereof.
Another embodiment is intended to provide the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof for use in a treatment of inflammatory disease.
Another embodiment is intended to provide a use of the compound, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof for use in the manufacture of a drug for treating inflammatory disease.
One embodiment provides a compound represented by the following Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a salt thereof.
In the Chemical Formula 1,
Another embodiment provides a pharmaceutical composition for preventing or treating inflammatory disease, including the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
Another embodiment provides a health functional food composition for preventing or ameliorating inflammatory disease, including the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
Another embodiment provides a method for treating inflammatory disease, including administering the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof to an individual or subject in need thereof.
Another embodiment provides the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof for use in the treatment of inflammatory disease.
The present disclosure relates to a novel thiadiazole derivative compound and a pharmaceutical composition for preventing or treating inflammatory disease including the same as an active ingredient, the compound according to one embodiment is an effective PPARδ agonist that can effectively inhibit the production of inflammatory factors and nitric oxide, and is effective in preventing the infiltration of macrophages into the inflammatory site, so it can be usefully used as a pharmaceutical composition for preventing or treating inflammatory disease.
The embodiments described herein may be modified into various other forms, and the technology according to one embodiment is not limited to the embodiments described below. In addition, the embodiment of one embodiment is provided to more completely explain the present disclosure to those with average knowledge in the relevant technical field. Furthermore, throughout the specification, “comprising” a certain element means that other elements may be further included rather than excluding other elements, unless specifically stated to the contrary.
As used herein, numerical ranges include lower and upper limits and all values within that range, increments that are logically derived from the shape and width of the range being defined, all double restricted values, and all possible combinations of upper and lower limits of numerical ranges defined in different forms. As an example, if the content of the composition is limited to 10% to 80% or 20% to 50%, the numerical range of 10% to 50% or 50% to 80% should also be interpreted as described herein. Unless otherwise specified herein, values outside the numerical range that may occur due to experimental error or rounding of values are also included in the defined numerical range.
Hereinafter, unless otherwise specified herein, “about” may be considered a value within 30%, 25%, 20%, 15%, 10% or 5% of the specified value.
One embodiment provides a compound represented by the following Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a salt thereof.
In the Chemical Formula 1,
In one embodiment, the compound represented by the Chemical Formula 1 may be a compound represented by the following Chemical Formula 2.
In the Chemical Formula 2,
In one embodiment, the compound represented by the Chemical Formula 1 may be a compound represented by the following Chemical Formula 3.
In the Chemical Formula 3,
In one embodiment, the R1, R11, R12 and R13 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3, C1-4haloalkyl, C1-3haloalkyl, C1-2haloalkyl, C1haloalkyl, —CF3, C1-4haloalkoxy, C1-3haloalkoxy, C1-2haloalkoxy, C1haloalkoxy or —OCF3.
In one embodiment, the R2 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3.
In one embodiment, the R21 may be cyano C1-4alkyl, cyano C1-3alkyl, cyano C1-2alkyl, heteroaryl C1-4alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S, heteroaryl C1-3alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S, heteroaryl C1-2alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S, heteroaryl C1alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S,
In one embodiment, the R211 and R212 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3.
In one embodiment, the R214 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3.
In one embodiment, the x and y may each independently be integers of 1 to 4 or 1 to 3.
In one embodiment, the compound represented by Chemical Formula 1 may be any one selected from the following group of compounds.
In one embodiment, the compound represented by Chemical Formula 1 can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. Acid addition salts are obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., non-toxic organic acids such as aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. These types of pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate etc.
One embodiment includes not only the compound represented by Chemical Formula 1 and a pharmaceutically acceptable salt thereof, but also solvates, optical isomers, hydrates, etc., that can be prepared therefrom.
One embodiment provides a pharmaceutical composition for preventing or treating inflammatory disease, including a compound represented by the following Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
In the Chemical Formula 1,
In one embodiment, the compound represented by the Chemical Formula 1 may be a compound represented by the following Chemical Formula 2.
In the Chemical Formula 2,
In one embodiment, the compound represented by the Chemical Formula 1 may be a compound represented by the following Chemical Formula 3.
In the Chemical Formula 3,
In one embodiment, the R1, R11, R12 and R13 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3, C1-4haloalkyl, C1-3haloalkyl, C1-2haloalkyl, C1haloalkyl, —CF3, C1-4haloalkoxy, C1-3haloalkoxy, C1-2haloalkoxy, C1haloalkoxy or —OCF3.
In one embodiment, the R2 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3.
In one embodiment, the R21 may be cyano C1-4alkyl, cyano C1-3alkyl, cyano C1-2alkyl, heteroaryl C1-4alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S, heteroaryl C1-3alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S, heteroaryl C1-2alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S, heteroaryl C1alkyl of 5 to 6 atoms containing one or more heteroatoms selected from a group consisting of N, O and S,
In one embodiment, the R211 and R212 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3.
In one embodiment, the R214 may each independently be C1-4alkyl, C1-3alkyl, C1-2alkyl, —CH3.
In one embodiment, the x and y may each independently be integers of 1 to 4 or 1 to 3.
In one embodiment, the compound represented by Chemical Formula 1 may be any one selected from the following group of compounds.
In one embodiment, the inflammatory disease is not particularly limited as long as it is an inflammatory disease. Examples of inflammatory diseases include, but are not limited to, inflammatory bowel disease, osteoarthritis, bronchitis, rheumatoid arthritis, degenerative arthritis, asthma, atopy, diabetes, myocardial infarction, Crohn's disease, or psoriasis.
The compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof may be administered in various oral and parenteral formulations during clinical administration. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are conventionally used. Solid preparations for oral administration include tablets, pills, acids, granules, and capsules, etc., and such solid preparations may be prepared by mixing, in addition to one or more compounds, at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives, etc., may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, and emulsions. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable ester such as ethyl oleate may be used.
A pharmaceutical composition including the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
At this time, in order to formulate into a formulation for parenteral administration, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water along with a stabilizer or buffer to prepare a solution or suspension, which can be prepared in an ampoule or vial unit dosage form. The composition may be sterile and/or contain auxiliaries such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for adjusting osmotic pressure, and other therapeutically useful substances, and may be formulated according to conventional mixing, granulating or coating methods.
The formulations for oral administration include, for example, tablets, pills, hard/soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, etc., and in addition to the active ingredients, such formulations contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine) and lubricants (e.g., silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, etc., and depending on cases, may contain disintegrants such as starch, agar, alginic acid or sodium salts thereof, etc., or effervescent mixtures and/or absorbents, colorants, flavoring agents, and sweeteners.
One embodiment provides a health functional food composition for preventing or ameliorating inflammatory disease, including the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a salt thereof as an active ingredient.
The compound represented by Chemical Formula 1 according to one embodiment may be added to food as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention or amelioration). Generally, the amount of the above compound in health food may be 0.1 to 90 parts by weight of the total weight of the food. However, in the case of long-term intake for health and hygiene purposes or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in amounts exceeding the above range.
In addition, the health functional food composition according to one embodiment has no particular restrictions on other ingredients other than containing the above compound, and may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages.
Furthermore, it may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.
One embodiment provides a method for treating inflammatory disease, including administering the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof to an individual or subject in need thereof.
One embodiment provides the compound represented by Chemical Formula 1, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof for use in the treatment of inflammatory disease.
One embodiment provides a use of the compound, a stereoisomer thereof, a hydrate thereof, or a pharmaceutically acceptable salt thereof for use in the manufacture of a drug for treating inflammatory disease.
Hereinafter, examples and experimental examples will be described in detail below. However, the examples and experimental examples described below are only illustrative of some, and the technology described in this specification is not limited thereto.
Ethyl 4-(trifluoromethyl)benzoate (2.033 mL, 11.46 mmol) was dissolved in MeOH (10 mL), hydrazine hydrate (1.671 mL, 27.5 mmol) was added at 0° C. and stirred for 5 hours at 25° C., followed by stirring at 70° C. for 1 hour. After the reaction was completed, cold water was added to solidify to obtain compound b-1 (2.21 g, 10.83 mmol).
Compound b-1 (2.21 g, 10.83 mmol) was dissolved in dichloromethane (20 mL), TEA (1.521 mL, 10.83 mmol) was added dropwise at 0° C., and then ethyl 2-chloro-2-oxoacetate (1.210 mL, 10.83 mmol) was slowly added. The mixture was stirred at room temperature for 1 hour. After the reaction was completed, it was quenched with water, extracted with dichloromethane, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain Compound c-1 (2.8 g, 85% yield). It is used without further purification in the next step.
Compound c-1 (1 g, 3.29 mmol) was dissolved in THE (1 mL), Lawesson's Reagent (1.99 g, 4.93 mmol) was added, and the mixture was heated at 55° C. for 12 hours. After the reaction was completed, it was filtered through celite, washed with NaHCO3, and the organic layer was dried over anhydrous MgSO4. The obtained residue was concentrated under reduced pressure and purified by MPLC (silica gel, 0-25%, EtOAc/Hexane) to obtain Compound d-1 (0.88 g, 89% yield).
ESI LC/MS: m/z calcd. for C12H10F3N2O2S [M+H]+: 303.04; found 303.1. 1H NMR (CD3OD, 400 MHz) δ8.31 (d, J=7.2 Hz, 2H), 7.91 (d, J=7.44 Hz, 2H), 4.55 (q, J=6.64 Hz, 2H), 1.47 (t, J=6.1 Hz, 3H).
Compound d-1 (0.8 g, 2.64 mmol) was dissolved in methanol, and sodium borohydride (0.31 g, 7.94 mmol) was slowly added at 0° C., followed by stirring at room temperature for 1 hour. After the reaction is completed, it is quenched with NH4Cl aqueous solution, concentrated under reduced pressure, extracted with ethyl acetate, and dried over anhydrous Na2SO4. The obtained residue was concentrated under reduced pressure, purified by MPLC (silica gel, 5-30%, EtOAc/Hexane), and concentrated under reduced pressure to obtain Compound e-1 (0.61 g, 89% yield) as a light yellow solid.
ESI LC/MS: m/z calcd. for C10H8F3N2OS [M+H]+: 261.03; found 261.11. 1H NMR (CD3OD, 400 MHz) δ8.19 (d, J=8.12 Hz, 2H), 7.86 (d, J=8.24 Hz, 2H), 5.03 (s, 2H). 13C NMR (CD3OD, 100 MHz) 174.77, 167.79, 133.38, 132.17, 128.06, 127.63, 126.03, 125.91, 122.49, 58.61.
Compound e-1 (1 g, 3.84 mmol) was dissolved in CH2Cl2 (10 mL), then triphenylporphine (1.2 g, 4.61 mmol) and iothiocyanate (NCS) (0.616 g, 4.61 mmol) were added, followed by stirring at room temperature for 2 hours. After the reaction was completed, it was washed with water and saline solution and dried over anhydrous MgSO4. Volatile substances were removed by concentration under reduced pressure, and the obtained residue was purified by MPLC (silica gel, 0-20%, EtOAc/Hexane) to obtain Compound f-1 (0.203 g, 19% yield).
ESI LC/MS: m/z calcd. for C10H7ClF3N2S [M+H]+: 279.01; found 279.1. 1H NMR (CD3OD, 400 MHz) δ8.22 (d, J=8.12 Hz, 2H), 7.87 (d, J=8.24 Hz, 2H), 5.17 (s, 2H). 13C NMR (CD3OD, 100 MHz) δ169.38, 168.29, 133.05, 132.48, 128.20, 128.06, 125.98, 125.14, 122.45, 37.42.
4-mercapto-2-methylphenol (0.27 g, 1.97 mmol) was dissolved in acetonitrile (8 mL), then cesium carbonate (0.64 g, 1.97 mmol) and Compound f-1 (0.5 g, 1.97 mmol) were added dropwise, followed by stirring at room temperature for 2 hours. After concentrating under reduced pressure to remove the solvent, it was extracted with ethyl acetate and dried over anhydrous Na2SO4. The obtained residue was concentrated under reduced pressure, purified by MPLC (silica gel, 5-20%, EtOAc/Hexane), and concentrated under reduced pressure to obtain Compound g-1 (0.64 g, 85% yield).
ESI LC/MS: m/z calcd. for C17H14F3N2OS2 [M+H]+: 383.04; found 383.1. 1H NMR (DMSO-d6, 400 MHz) δ9.63 (s, 1H), 8.15 (d, J=8.12 Hz, 2H), 7.91 (d, J=8.28 Hz, 2H), 7.20 (s, 1H), 7.09 (d, J=8.24 Hz, 1H), 6.71 (d, J=8.28 Hz, 2H), 4.57 (s, 2H), 2.06 (s, 3H). 13C NMR (DMSO-d6, 100 MHz) δ171.03, 167.61, 156.28, 135.19, 133.72, 131.59, 128.85, 126.86, 126.83, 125.71, 121.19, 115.81, 34.02, 16.28.
Compound g-1 (0.3 g, 0.78 mmol) was dissolved in acetonitrile (5 mL), and cesium carbonate (0.38 g, 1.17 mmol) and methyl 2-bromoacetate (0.9 mL, 0.94 mmol) were added dropwise, followed by stirring at room temperature for 1 hour and 30 minutes. After the reaction was completed, it was concentrated under reduced pressure to remove acetonitrile, washed with water and saline solution, extracted with EtOAc, and dried over anhydrous Na2SO4. The obtained residue was concentrated under reduced pressure, purified by MPLC (silica gel, 5-20%, EtOAc/Hexane), and concentrated under reduced pressure to obtain Compound h-1 (0.32 g, 96% yield).
Compound h-1 (0.32 g, 0.7 mmol) was dissolved in tetrahydrofuran, then 2 M lithium hydroxide aqueous solution (1.5 mL) was added at 0° C., followed by stirring at room temperature for 1 hour. After the reaction was completed, it was quenched with water and acidified with 1N HCl. Afterwards, it was concentrated under reduced pressure to remove the solvent, extracted with EtOAc, and dried over anhydrous Na2SO4 to obtain Compound 1 (0.25 g, yield 93%).
ESI LC/MS: m/z calcd. for C19H16F3N2O3S2 [M+H]+: 441.06; found 441.1. 1H NMR (DMSO-d6, 400 MHz) δ8.14 (d, J=8.08 Hz, 2H), 7.88 (d, J=8.2 Hz, 2H), 7.28 (s, 1H), 7.21 (dd, J=8.44, 1.92 Hz, 1H), 6.77 (d, J=8.56 Hz, 1H), 4.67 (s, 2H), 4.64 (s, 2H), 2.13 (s, 3H). 13C NMR (DMSO-d6, 400 MHz) δ170.69, 170.29, 167.41, 156.01, 133.83, 133.44, 130.34, 128.61, 127.44, 126.59, 126.55, 123.78, 112.28, 64.88, 33.03, 16.12.
Compound i-1 (59 mg, 94% yield) was obtained in the same manner as in Step 7 of Example 1, except that ethyl 2-bromopropanoate was used instead of methyl 2-bromoacetate.
Compound 2 (46 mg, 83% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-1 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C20H18F3N2O3S2 [M+H]+: 455.07; found 455.1. 1H NMR (CD3OD, 400 MHz) δ8.08 (d, J=8.16 Hz, 2H), 7.80 (d, J=8.24 Hz, 2H), 7.25 (s, 1H), 7.19 (dd, J=8.44, 2.02 Hz, 1H), 6.89 (d, J=8.48 Hz, 1H), 4.78 (q, J=6.76 Hz, 1H), 4.46 (s, 2H), 2.19 (s, 3H), 1.59 (d, J=6.8 Hz, 3H), 13C NMR (CD3OD, 100 MHz) δ175.57, 172.53, 169.61, 157.85, 136.35, 132.69, 132.56, 129.89, 129.50, 127.46, 127.42, 125.23, 113.57, 73.65, 35.09, 19.01, 16.47.
Step 1: Preparation of ethyl 2-methyl-2-(2-methyl-4-(((5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)methyl)thio)phenoxy)propanoate (j-1)
Compound j-1 (18 mg, 28% yield) was obtained in the same manner as in Step 7 of Example 1, except that ethyl-2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 3 (9 mg, 64% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-1 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C21H20F3N2O3S2 [M+H]+: 469.09; found 469.12. 1H NMR (CD3OD, 400 MHz) δ8.11 (d, J=8.12 Hz, 2H), 7.82 (d, J=8.24 Hz, 2H), 7.26 (s, 1H), 7.16 (dd, J=8.48, 1.99 Hz, 1H), 6.69 (d, J=8.52 Hz, 1H), 4.51 (s, 2H), 2.16 (s, 3H), 1.56 (s, 6H), 13C NMR (CD3OD, 100 MHz) δ177.71, 172.53, 169.65, 155.73, 136.30, 131.93, 131.88, 129.51, 127.49, 127.45, 125.90, 118.15, 80.33, 34.91, 25.83, 16.87 [227]<Example 4> Preparation of 2-(2-methyl-4-(((5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)methyl)thio)phenoxy)acetonitrile
Compound 4 (0.163 g, 67% yield) was obtained in the same manner as in Step 7 of Example 1, except that 2-bromoacetonitrile was used instead of methyl 2-bromoacetate.
1H NMR (CDCl3, 400 MHz) δ8.04 (d, J=8.1 Hz, 2H), 7.72 (d, J=8.2 Hz, 2H), 7.27-7.24 (m, 2H), 6.82 (d, J=8.0 Hz, 1H), 4.76 (s, 2H), 4.45 (s, 2H), 2.20 (s, 3H).
Compound 4 (21 mg, 0.050 mmol), NaN3 (3.9 mg, 0.060 mmol), ZnCl2 (6.8 mg, 0.050 mmol) were dissolved in 0.4 mL of n-butanol and reacted at 120° C. for 12 hours. The reaction mixture was distilled under reduced pressure and separated using Prep-HPLC to obtain Compound 5 (9.5 mg, 41% yield). [235]1H NMR (CDCl3, 400 MHz) δ7.99 (d, J=8.1 Hz, 2H), 7.72 (d, J=8.2 Hz, 2H), 7.13-7.12 (m, 2H), 6.82 (d, J=8.0 Hz, 1H), 5.38 (s, 2H), 4.37 (s, 2H), 1.99 (s, 3H).
Compound 1 (3.7 mg, 0.008 mmol) was dissolved in 1 mL of THF, then 1,1′-carbonyldiimidazole (3.4 mg, 0.021 mmol) was added, followed by a reaction at room temperature for 1 hour. Hydroxylamine hydrochloride (1.5 mg, 0.021 mmol) was added to the reaction mixture, followed by a reaction at room temperature for another 16 hours. Potassium hydrogen sulfate (5.7 mg, 0.042 mmol) was added to the mixture, then 0.5 mL of distilled water was added, followed by a reaction at room temperature for another 1 hour. The solvent was removed through reduced pressure distillation and separated using Prep-HPLC to obtain Compound 6 (2.7 mg, 69% yield).
1H NMR (CD3OD, 400 MHz) δ8.14 (d, J=7.8 Hz, 2H), 7.85 (d, J=8.1 Hz, 2H), 7.30 (s, 1H), 7.26 (d, J=8.0 Hz, 1H), 6.83 (d, J=7.9 Hz, 1H), 4.56 (s, 2H), 4.53 (s, 2H), 2.24 (s, 3H).
Compound 7 (9.2 mg, yield 85%) was obtained in the same manner as in Example 6, except that O-methylhydroxylamine hydrochloride was used instead of hydroxylamine hydrochloride.
1H NMR (CD3OD, 400 MHz) δ8.12 (d, J=7.8 Hz, 2H), 7.84 (d, J=8.0 Hz, 2H), 7.30 (s, 1H), 7.26 (d, J=8.3 Hz, 1H), 6.82 (d, J=8.2 Hz, 1H), 4.56 (s, 2H), 4.52 (s, 2H), 3.71 (s, 3H), 2.24 (s, 3H).
Compound 8 (14 mg, 42% yield) was obtained in the same manner as in Example 6, except that cyanamide was used instead of hydroxylamine hydrochloride.
1H NMR (CDCl3, 400 MHz) δ8.11 (d, J=8.0 Hz, 2H), 7.83 (d, J=8.3 Hz, 2H), 7.30-7.22 (m, 2H), 6.81-6.75 (m, 1H), 4.69 (s, 2H), 4.51 (s, 2H), 2.23 (s, 3H).
Compound b-2 (1.03 g, 67% yield) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-2 was used instead of Compound a-1.
Compound c-2 (0.236 g, 93% yield) was obtained in the same manner as in step 2 of Example 1, except that Compound b-2 was used instead of Compound b-1.
Compound d-2 (0.256 g, 78% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-2 was used instead of Compound c-1.
Compound e-2 (0.185 g, 88% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-2 was used instead of Compound d-1.
Compound f-2 (0.178 g, 78% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-2 was used instead of Compound e-1.
Compound g-2 (0.332 g, 97% yield) was obtained in the same manner as in Step 6 of Example 1, except that Compound f-2 was used instead of Compound f-1.
Compound h-2 (0.340 g, 84% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-2 was used instead of Compound g-1.
Compound 9 (0.363 g, 93% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-2 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C18H16FN2O3S2 [M+H]+: 391.06; found 391. 1H NMR (400 MHz, CDCl3) δ7.84 (dd, J=8.5, 5.3 Hz, 2H), 7.13-7.07 (m, 4H), 6.54 (d, J=8.4 Hz, 1H), 4.47 (s, 2H), 4.33 (s, 2H), 2.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ171.38, 169.49, 168.57, 164.44 (d, JCF=251.2 Hz), 156.14, 134.76, 130.75, 129.85, 128.60, 126.11, 124.51, 116.28, 112.21, 60.50, 34.28, 16.13, 14.20.
Compound i-2 (0.320 g, 74% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-2 was used instead of Compound g-1 and ethyl 2-bromopropanoate was used instead of methyl 2-bromoacetate.
Compound 10 (0.259 g, 64% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-2 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C19H18FN2O3S2 [M+H]+: 405.08; found 405. 1H NMR (DMSO-d6, 400 MHz) δ 8.01-7.97 (m, 2H), 7.38 (t, J=8.8 Hz, 2H), 7.28 (s, 1H), 7.20 (dd, J=8.5, 1.8 Hz, 1H), 6.72 (d, J=8.6 Hz, 1H), 4.77 (q, J=6.7 Hz, 1H), 4.61 (s, 2H), 2.13 (s, 3H), 1.49 (d, J=6.7 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz) δ 173.56, 169.86, 167.97, 164.19 (d, JCF=248.0 Hz), 156.09, 134.09, 130.45, 127.87, 126.64, 123.96, 116.91, 112.98, 72.42, 49.07, 33.30, 18.83, 16.41.
Compound j-2 (0.174 g, 39% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-2 was used instead of Compound g-1, and ethyl 2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 11 (0.167 g, 40% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-2 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C20H20FN2O3S2 [M+H]+: 419.09; found 419. 1H NMR (400 MHz, CDCl3) δ7.89 (dd, J=8.4, 5.2 Hz, 2H), 7.21 (s, 1H), 7.15 (t, J=8.5 Hz, 2H), 7.10 (d, J=8.0 Hz, 1H), 6.70 (d, J=8.5 Hz, 1H), 4.40 (s, 2H), 2.17 (s, 3H), 1.25 (s, 3H). 13C NMR (CDCl3, 100 MHz) δ171.34, 169.42, 168.50, 164.45 (d, JCF=250.9 Hz), 153.60, 134.55, 131.01, 129.87, 126.25, 125.33, 117.79, 116.28, 79.52, 60.49, 34.15, 25.27, 16.75.
Compound b-3 (0.34 g, 73.5% yield) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-3 was used instead of Compound a-1.
Compound c-3 (0.36 g, 67% yield) was obtained in the same manner as in Step 2 of Example 1, except that Compound b-3 was used instead of Compound b-1.
Compound d-3 (54 mg, 34% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-3 was used instead of Compound c-1.
Compound e-3 (45 mg, 99% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-3 was used instead of Compound d-1.
Compound f-3 (36 mg, 74% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-3 was used instead of Compound e-1.
Compound g-3 (50 mg, 98% yield) was obtained in the same manner as in Step 6 of Example 1, except that Compound f-3 was used instead of Compound f-1.
Compound h-3 (64 mg, 99% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-3 was used instead of Compound g-1.
Compound 12 (25 mg, 39.8% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-3 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C18H15F2N2O3S2 [M+H]+: 409.05; found 409. 1H NMR (DMSO, 400 MHz) δ 8.07 (s, 1H), 7.81 (s, 1H), 7.64 (d, J=7.28 Hz, 1H), 7.25 (s, 1H), 7.19 (s, 1H), 6.71 (s, 1H), 4.62 (s, 2H), 4.43 (s, 2H), 2.13 (d, J=7.3 Hz, 3H).
Compound i-3 (67 mg, 97% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-3 was used instead of Compound g-1, and 2-bromopropanoate was used instead of methyl 2-bromoacetate.
Compound 13 (3 mg, 5% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-3 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C19H18FN2O3S2 [M+H]h: 423.07; found 423. H NMR (CD3OD, 400 MHz) δ 7.93 (m, 1H), 7.74 (s, 1H), 7.47 (m, 1H), 7.27 (s, 1H), 7.22 (d, J=8.44 Hz, 1H), 6.73 (d, J=8.40 Hz, 1H), 4.47 (s, 2H), 2.21 (s, 3H), 1.61 (d, J=6.72 Hz, 3H).
Step 1: Preparation of ethyl 2-(4-(((5-(3,4-difluorophenyl)-1,3,4-thiadiazol-2-yl)methyl)thio)-2-methylphenoxy)-2-methylpropanoate (j-3)
Compound j-3 (33 mg, 50% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-3 was used instead of Compound g-1, and ethyl 2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 14 (12 mg, 38% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-3 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C19H18FN2O3S2 [M+H]+: 437.08; found 437.1. 1H NMR (CD3OD, 400 MHz) δ 7.93 (m, 1H), 7.74 (m, 1H), 7.47 (m, 1H), 7.27 (s, 1H), 7.18 (dd, J=2.12 Hz, 6.40 Hz, 1H), 6.72 (d, J=8.52 Hz, 1H), 4.49 (s, 2H), 2.17 (s, 3H), 1.58 (s, 6H). 13C NMR (CD3OD, 100 MHz) δ15.37, 24.33, 33.37, 78.81, 116.08, 116.28, 116.61, 118.07, 118.25, 124.39, 124.71, 130.38, 134.76, 154.20, 170.68, 176.20.
Compound b-4 (0.74 g, 39% yield) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-4 was used instead of Compound a-1.
Compound c-4 (0.209 g, 72% yield) was obtained in the same manner as in Step 2 of Example 1, except that Compound b-4 was used instead of Compound b-1.
Compound d-4 (0.21 g, 56% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-4 was used instead of Compound c-1.
Compound e-4 (0.24 g, 99% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-4 was used instead of Compound d-1.
Compound f-4 (0.18 g, 68% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-4 was used instead of Compound e-1.
Compound g-4 (0.368 g, 99% yield) was obtained in the same manner as in Step 6 of Example 1, except that Compound f-4 was used instead of Compound f-1.
Compound h-4 (0.247 g, 56% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-4 was used instead of Compound g-1.
Compound 15 (64 mg, 15% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-4 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C18H14F3N2O3S2 [M+H]+: 427.04; found 427. 1H NMR (CD3OD, 400 MHz) δ 7.67 (t, J=7.4 Hz, 2H), 7.15-7.09 (m, 2H), 6.63 (d, J=8.3 Hz, 1H), 4.37 (s, 2H), 3.55 (s, 2H), 2.10 (s, 3H).
Compound b-5 (0.60 g, 88% yield) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-5 was used instead of Compound a-1.
Compound c-5 (0.60 g, 88% yield) was obtained in the same manner as in Step 2 of Example 1, except that Compound b-5 was used instead of Compound b-1.
Compound d-5 (0.556 g, 60% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-5 was used instead of Compound c-1.
Compound e-5 (0.451 g, 87% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-5 was used instead of Compound d-1.
1H NMR (400 MHz, CDCl3) δ8.06 (dd, J=6.9, 2.2 Hz, 1H), 7.84 (ddd, J=8.6, 4.4, 2.2 Hz, 1H), 7.27 (t, J=8.6 Hz, 1H), 5.14 (s, 2H), 2.72 (brs, 1H)
Compound f-5 (0.259 g, 53% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-5 was used instead of Compound e-1.
1H NMR (400 MHz, CDCl3) δ8.07 (dd, J=6.8, 2.2 Hz, 1H), 7.84 (ddd, J=8.6, 4.4, 2.3 Hz, 1H), 7.28 (t, J=8.5 Hz, 1H), 4.99 (s, 2H)
Compound g-5 (69 mg, 97% yield) was obtained in the same manner as in Step 6 of Example 1, except that Compound f-5 was used instead of Compound f-1.
Compound h-5 (74 mg, 89% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-5 was used instead of Compound g-1.
1H NMR (400 MHz, CDCl3) δ8.01 (dd, J=6.9, 2.2 Hz, 1H), 7.79 (ddd, J=8.6, 4.5, 2.2 Hz, 1H), 7.26-7.21 (m, J=5.3 Hz, 2H), 7.18 (dd, J=8.4, 2.2 Hz, 1H), 6.61 (d, J=8.5 Hz, 1H), 4.63 (s, 2H), 4.41 (s, 2H), 3.79 (s, 3H), 2.25 (s, 3H).
Compound 16 (44 mg, 88% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-5 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C18H15ClFN2O3S2 [M+H]+: 425.02; found 425. 1H NMR (CD3OD, 400 MHz) δ8.12-8.04 (m, 1H), 7.91-7.82 (m, 1H), 7.41 (t, J=8.8 Hz, 1H), 7.27 (s, 1H), 7.23 (d, J=8.5 Hz, 1H), 6.76 (d, J=8.4 Hz, 1H), 4.60 (s, 2H), 4.48 (s, 2H), 2.23 (s, 3H). 13C NMR (CD3OD, 100 MHz) δ172.21, 170.76, 167.23, 159.62, 156.92, 134.75, 131.26, 129.36, 128.22, 128.16, 127.32, 123.52, 121.68, 117.35, 111.57, 65.45, 33.65, 14.94.
Compound i-5 (74.8 mg, 70.2% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-5 was used instead of Compound g-1, and the compound ethyl 2-bromopropanoate was used instead of methyl 2-bromoacetate.
1H NMR (CDCl3, 400 MHz) δ 8.01 (dd, J=6.9, 2.2 Hz, 1H), 7.79 (ddd, J=8.6, 4.5, 2.2 Hz, 1H), 7.25-7.21 (m, 2H), 7.15 (dd, J=8.5, 2.2 Hz, 1H), 6.58 (d, J=8.5 Hz, 1H), 4.69 (q, J=6.8 Hz, 1H), 4.40 (s, 2H), 4.19 (qd, J=7.1, 1.3 Hz, 2H), 2.23 (s, 3H), 1.62 (d, J=6.8 Hz, 3H), 1.22 (t, J=7.1 Hz, 3H).
Compound 17 (33 mg, 85% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-5 was used instead of Compound h-1.
1H NMR (CDCl3, 400 MHz) δ7.99 (dd, J=6.8, 1.9 Hz, 1H), 7.81-7.71 (m, 1H), 7.34 (brs, 1H), 7.23 (m, 2H), 7.15 (d, J=8.4 Hz, 1H), 6.63 (d, J=8.5 Hz, 1H), 4.73 (q, J=6.7 Hz, 1H), 4.41 (s, 2H), 2.21 (s, 3H), 1.65 (d, J=6.8 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ176.05, 170.12, 167.17, 159.73, 155.89, 134.86, 130.73, 130.06, 128.93, 127.84, 127.22, 124.40, 122.28, 117.44, 112.53, 72.43, 34.34, 18.49, 16.25.
Compound j-5 (12 mg, 13% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-5 was used instead of Compound g-1, and the compound ethyl 2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 18 (3 mg, 26% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-5 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C20H19ClFN2O3S2 [M+H]+: 453.05; found 453. 1H NMR (CD3OD, 400 MHz) δ8.15-8.03 (m, 1H), 7.93-7.83 (m, 1H), 7.42 (t, J=8.8 Hz, 1H), 7.28 (s, 1H), 7.18 (d, J=8.3 Hz, 1H), 6.73 (d, J=8.4 Hz, 1H), 4.49 (s, 2H), 2.18 (s, 3H), 1.58 (s, 6H). 13C NMR (CD3OD, 400 MHz) δ170.69, 170.62, 167.27, 159.65 (d, J=253.4 Hz), 154.39, 134.78, 130.45, 130.29, 129.40, 128.13, 127.32, 124.14, 121.69, 117.37, 116.67, 79.08, 33.44, 24.43, 15.37.
Compound b-6 (2.044 g, 92% yield) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-6 was used instead of Compound a-1.
Compound c-6 (0.139 g, 43% yield) was obtained in the same manner as in Step 2 of Example 1, except that Compound b-6 was used instead of Compound b-1.
Compound d-6 (0.371 g, 89% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-6 was used instead of Compound c-1.
Compound e-6 (0.253 g, 91% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-6 was used instead of Compound d-1.
Compound f-6 (0.234 g, 79% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-6 was used instead of Compound e-1.
Compound g-6 (0.401 g, 97% yield) was prepared in the same manner as in Step 6 of Example 1, except that Compound f-6 was used instead of Compound f-1.
Compound h-6 (0.421 g, 89% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-6 was used instead of Compound g-1.
Compound 19 (0.264 g, 60% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-6 was used instead of Compound h-1.
1H NMR (CD3OD, 400 MHz) δ 7.97 (d, J=11.2 Hz, 1H), 7.92-7.85 (m, 2H), 7.29 (s, 1H), 7.24 (d, J=8.5 Hz, 1H), 6.77 (d, J=8.4 Hz, 1H), 4.67 (s, 2H), 4.52 (s, 2H), 2.23 (s, 3H). 13C NMR (DMSO-d6, 100 MHz) δ 171.74, 170.62, 166.53, 159.52 (d, JCF=253.5 Hz), 156.42, 136.22, 134.06, 130.59, 129.04, 127.69, 124.63, 123.84, 122.76 (q, JCF=271.4 Hz), 116.32, 112.57, 65.46, 33.32, 16.38.
Compound i-6 (0.360 g, 72%) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-6 was used instead of Compound g-1, and the compound ethyl 2-bromopropanoate was used instead of the compound methyl 2-bromoacetate.
Compound 20 (0.300 g, 66% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-6 was used instead of Compound h-1.
1H NMR (CDCl3, 400 MHz) δ7.81-7.68 (m, 3H), 7.22 (s, 1H), 7.15 (dd, J=8.5, 1.9 Hz, 1H), 6.62 (d, J=8.5 Hz, 1H), 4.73 (q, J=6.8 Hz, 1H), 4.42 (s, 2H), 2.21 (s, 3H), 1.65 (d, J=6.8 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ176.22, 171.02, 166.72, 159.89 (d, JCF=254.5 Hz), 155.96, 135.47, 134.94, 130.83, 128.99, 128.16, 124.24, 123.43, 119.53 (q, JCF=246.2 Hz), 115.87, 112.50, 72.39, 34.40, 18.48, 16.23.
Compound j-6 (52 mg, 10% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-6 was used instead of Compound g-1, and ethyl 2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 21 (80 mg, 17% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-6 was used instead of Compound h-1.
1H NMR (CDCl3, 400 MHz) δ7.81-7.69 (m, 3H), 7.21 (s, 1H), 7.09 (d, J=8.2 Hz, 1H), 6.69 (d, J=8.6 Hz, 1H), 2.17 (s, 3H), 0.88 (s, 6H).
Compound b-7 (0.78 g, 85% yield) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-7 was used instead of Compound a-1.
Compound c-7 (1.25 g, 96% yield) was obtained in the same manner as in Step 2 of Example 1, except that Compound b-7 was used instead of Compound b-1.
Compound d-7 (0.840 g, 67.7% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-7 was used instead of Compound c-1.
Compound e-7 (0.690 g, 99% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-7 was used instead of Compound d-1.
Compound f-7 (0.359 g, 47.8% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-7 was used instead of Compound e-1.
Compound g-7 (0.560 g, 97% yield) was obtained in the same manner as in Step 6 of Example 1, except that Compound f-7 was used instead of Compound f-1.
Compound h-7 (70 mg, 97% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-7 was used instead of Compound g-1.
Compound 22 (34 mg, 96% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-7 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C19H19N2O3S2 [M+H]+: 387.09; found 386.8. 1H NMR (DMSO, 400 MHz) δ 7.80 (d, J=7.96 Hz, 2H), 7.34 (d, J=7.84 Hz, 2H), 7.24 (s, 1H), 7.19 (d, J=8.28 Hz, 1H), 6.73 (d, J=8.56 Hz, 1H), 4.57 (s, 2H), 4.47 (s, 2H), 2.36 (s, 3H), 2.12 (s, 3H).
Compound i-7 (0.100 g, 97% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-7 was used instead of Compound g-1, and ethyl 2-bromopropanoate was used instead of methyl 2-bromoacetate.
Compound 23 (34 mg, 35% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-7 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C20H21N2O3S2 [M+H]+: 401.10; found 401.1. 1H NMR (CD3OD, 400 MHz) δ 7.78 (d, J=7.84 Hz, 2H), 7.33 (d, J=7.76 Hz, 2H), 7.25 (s, 1H), 7.21 (d, J=8.44 Hz, 1H), 6.72 (d, J=8.40 Hz, 1H), 4.81 (m, 1H), 4.44 (s, 1H), 2.40 (s, 3H), 2.21 (s, 3H), 1.63 (d, J=6.76 Hz, 3H). 13C NMR (CD3OD, 100 MHz) δ15.00, 17.56, 20.07, 33.64, 60.71, 72.35, 112.03, 123.76, 126.84, 127.65 (2C), 128.29, 129.64 (2C), 131.19, 134.87, 141.99, 156.37, 169.57, 170.04.
Compound j-7 (94 mg, 97% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-7 was used instead of Compound g-1, and ethyl 2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 24 (2 mg, 3% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-7 was used instead of Compound h-1.
1H NMR (CD3OD, 400 MHz) δ 7.76 (d, J=8.20 Hz, 2H), 7.32 (d, J=8.04 Hz, 2H), 7.26 (s, 1H), 7.17 (dd, J=2.32 Hz, 6.20 Hz 1H), 6.72 (d, J=8.48 Hz, 1H), 4.45 (s, 2H), 2.39 (s, 3H), 2.16 (s, 3H), 1.57 (s, 6H). 13C NMR (CD3OD, 100 MHz) δ15.42, 20.08, 24.35 (2C), 78.82, 116.68, 124.53, 126.82, 127.31 (2C), 129.64 (2C), 130.34, 134.79, 142.01, 154.16, 169.46, 170.05, 176.24.
Compound b-8 (1.16 g, 53%) was obtained in the same manner as in Step 1 of Example 1, except that Compound a-8 was used instead of Compound a-1.
Compound c-8 (0.275 g, 86% yield) was obtained in the same manner as in Step 2 of Example 1, except that Compound b-8 was used instead of Compound b-1.
Compound d-8 (0.352 g, 85% yield) was obtained in the same manner as in Step 3 of Example 1, except that Compound c-8 was used instead of Compound c-1.
Compound e-8 (0.249 g, 90% yield) was obtained in the same manner as in Step 4 of Example 1, except that Compound d-8 was used instead of Compound d-1.
Compound f-8 (0.206 g, 70% yield) was obtained in the same manner as in Step 5 of Example 1, except that Compound e-8 was used instead of Compound e-1.
Compound g-8 (0.398 g, 97% yield) was obtained in the same manner as in Step 6 of Example 1, except that Compound f-8 was used instead of Compound f-1.
Compound h-8 (0.409 g, 87% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-8 was used instead of Compound g-1.
Compound 25 (0.393 g, 86% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound h-8 was used instead of Compound h-1,
1H NMR (DMSO-d6, 400 MHz) δ8.09-8.05 (m, 2H), 7.54 (d, J=8.1 Hz, 2H), 7.29 (d, J=1.8 Hz, 1H), 7.21 (dd, J=8.5, 2.3 Hz, 1H), 6.78 (d, J=8.6 Hz, 1H), 4.69 (s, 2H), 4.64 (s, 2H), 2.14 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) δ170.55, 170.39, 167.64, 156.26, 150.55, 134.09, 130.60, 130.21, 129.17, 127.68, 124.06, 122.90 (q, JCF=239.8 Hz), 122.30, 112.53, 65.17, 33.30, 16.38.
Compound i-8 (0.379 g, 76% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-8 was used instead of Compound g-1, and the compound ethyl 2-bromopropanoate was used instead of methyl 2-bromoacetate.
Compound 26 (0.409 g, 87% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound i-8 was used instead of Compound h-1.
1H NMR (DMSO-d6, 400 MHz) δ8.09-8.05 (m, 2H), 7.54 (d, J=8.1 Hz, 2H), 7.29 (d, J=1.8 Hz, 1H), 7.21 (dd, J=8.5, 2.3 Hz, 1H), 6.72 (d, J=8.6 Hz, 1H), 4.80 (q, J=6.7 Hz, 1H), 4.63 (s, 2H), 2.14 (s, 3H), 1.50 (d, J=6.8 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz) δ173.44, 170.38, 167.64, 156.03, 150.57, 134.13, 130.56, 130.21, 129.17, 127.92, 124.03, 122.30, 120.42 (q, JCF=255.7 Hz), 112.98, 72.26, 33.29, 18.79, 16.39.
Compound j-8 (0.179 g, 35% yield) was obtained in the same manner as in Step 7 of Example 1, except that Compound g-8 was used instead of Compound g-1, and the compound ethyl 2-bromo-2-methylpropanoate was used instead of methyl 2-bromoacetate.
Compound 27 (0.170 g, 35% yield) was obtained in the same manner as in Step 8 of Example 1, except that Compound j-8 was used instead of Compound h-1.
ESI LC/MS: m/z calcd. for C21H20F3N2O4S2 [M+H]+: 485.08; found 485. 1H NMR (CDCl3, 400 MHz) δ7.92 (d, J=8.2 Hz, 2H), 7.29 (d, J=8.2 Hz, 2H), 7.18 (s, 1H), 7.09 (s, 1H), 6.68 (s, 1H), 4.41 (s, 2H), 2.14 (s, 3H), 1.25 (s, 6H). 13C NMR (CDCl3, 100 MHz) δ169.86, 168.17, 160.90, 151.16, 142.81, 134.57, 134.52, 129.89, 129.47, 128.46, 121.60, 121.38, 119.03, 34.13, 29.72, 25.19, 16.74.
The compounds of Examples 1 to 27 are summarized in Tables 1 and 2 below.
Germ-free, 6-week-old immunocompetent C57BL/6 mice were obtained from SLC, Inc (Shizuoka, Japan). All animal experimental procedures were conducted in strict accordance with the appropriate institutional guidelines for animal research. The protocol was approved by the Animal Experiment Ethics Committee of Kyungpook National University (approval number: KNU 2012-43).
RAW 264.7 cells, which are macrophages, were purchased from the Korea Cell Line Bank and used, and were cultured in DMEM with 100 mg/mL inactivated fetal bovine serum and 10 mg/mL penicillin-streptomycin added as culture medium at 37° C. and 5% CO2 condition. All cells in the experimental process were subcultured when grown to a density of about 80% to 90%, and only cells that did not exceed 20 passages were used.
To evaluate the cytotoxicity of the sample, cell proliferation was tested using a cell counting kit [CCK-8; Dojindo Laboratories (Tokyo, Japan)]. RAW 264.7 cell 1×104 cells/mL were dispensed into a well plate, and after pre-culturing for 20 hours, 1 g/mL of LPS and example compounds were added at different concentrations and cultured for 24 hours in an incubator at 37° C. and 5% CO2. After culturing, 10 μl CCK-8 reagent was added and recultured for 1 to 2 hours, and the absorbance was measured. The absorbance was measured at 450 nm using a microplate reader [BMG Labtech (located in Offenburg, Germany)].
The concentration of NO was measured using the nitrite concentration in the culture medium with the Griess reagent system (Promega). Raw264.7 cells were adjusted to 2.5×105 cells/mL using DMEM medium, inoculated into 24-well plates, and pre-cultured for 20 hours in a 5% CO2 incubator. The cells were treated with 1 g/mL of LPS and various concentrations of the example compounds and recultured for 24 hours. After obtaining the supernatant of the culture medium, the NO concentration was measured using the Griess reagent system according to the protocol provided by the manufacturer. The absorbance was measured at 540 nm using a microplate reader [BMG Labtech (located in Offenburg, Germany)]. The concentration of NO in the cell culture medium was calculated by comparing it with a standard curve for each concentration of of sodium nitrite (NaNO2).
The supernatant was dispensed using the same method as used for measuring NO. High amounts of TNF-α and IL-6 in the supernatant were quantified using the multiplex bead-based immunoassay (BD™ Cytometric Bead Array) system and flow cytometry (BD Biosciences).
Raw264.7/NF-kb-luc cells were treated with 1 mg/mL LPS and various concentrations of compounds using the same method as used for measuring NO. 16 hours later, after adding the D-luciferin solution to the cells, luminescence images were obtained using the IVIS Lumina III. Quantification of the luminescence images was performed using Living Imaging software.
The cells were washed twice with cold PBS and lysed with RIPA buffer (Roche) containing a complete protease inhibitor cocktail. Protein concentration was quantified using the BCA protein assay kit (Pierce, IL, USA), and 30 μL of lysate was separated by 10% SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Biorad, CA, USA) at 70 mA for 1 hour and 30 minutes, then blocked with tris-buffered saline (TBST; pH 7.5) containing 50 mg/mL skim milk at room temperature for 2 hours. As antibodies to examine the expression levels of iNOS and pIKKαβ, anti-iNOS and pIKKαβ were used, diluted 1:1000 and reacted at room temperature for 2 hours, followed by washing three times with TBST. As secondary antibodies, horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were diluted 1:2000 and reacted at room temperature for 1 hour, followed by washing three times with TBST. Peroxidase activity was detected using ECL-Plus according to the manufacturer's protocol.
2 mL of thioglycollate medium (BD, USA) was injected into the abdominal cavity of a C57Bl/6 mouse, and three days later, the mouse was cervically dislocated, and 8 mL of Dulbecco's modified Eagle's medium (DMEM) was injected into the abdominal cavity to collect cells. After centrifugation, the cells were suspended in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and cultured in a 5% CO2 incubator at 37° C. for 2 hours. Non-adherent cells were removed, and the remaining cells were used for experiments.
The acute anti-inflammatory activity of the example compound was evaluated using a macrophage tracking technique with fluorescence imaging. Macrophages extracted from the mouse abdominal cavity were labeled with Vybrant™ DiD Cell-Labeling Solution and then administered to the mouse via intravenous injection. The next day, 50 mg/kg of the example compound was orally administered, and 1 hour later, a 0.1% Carrageenan (CG) solution or PBS was subcutaneously injected into the right or left sole of the mouse. Fluorescent images were obtained using IVIS Lumina III at the designated time.
All data were expressed as mean±standard deviation (SD) from at least three independent tests, and statistical significance of differences was determined by unpaired Student's test using GrahPad Prism 5. P values less than 0.05 were considered statistically significant.
In order to confirm the PPARδ activation effect of the example compounds, the following experiment was performed. First, the example compounds were diluted in DMSO to 100× based on the final concentration, then 1/50 was diluted in assay buffer, and 10 μL of each was added to a 384 well plate. Afterwards, GST-conjugated PPARδ ligand-binding domain (LBD) was added to a final concentration of 5 nM, and fluorescien-conjugated C33 coactivator peptide and Tb-α-GST antibody were added to a final concentration of 100 nM and 10 nM, respectively. The final volume of each test system was set to 20 μL, and the test for each concentration was repeated twice, and the binding activity after reaction was measured by TR-FRET method. In other words, after excitation at 340 nm and measurement of emission values at 495 nm and 520 nm, the results were analyzed using the measured value at 490 nm/measured at 520 nm, and the analysis program Prism 6 was used to calculate the EC50 value. The results are summarized in Table 2 below.
The principles of this test method are as follows. GST antibody binds to GST fused to the PPARδ LBD, thereby maintaining its inactive state, and binding of the agonist induces a structural change in the LBD protein. At this time, fluorescence energy is emitted as the fluorescence energy-donor bound to the GST antibody transfers energy to the fluorescence energy-acceptor (fluorescine) bound to the PPARδ substrate (C33 peptide), and by measuring this value, the degree of activation of PPARδ can be measured.
Meanwhile, the previously known PPARδ agonist compound GW501516 was used as a test material, and as a result of the experiment, the EC50 value in the case of GW501516 was 15.5 nM.
In order to evaluate the cytotoxicity of the compound of Example 1, Raw264.7 cells, a macrophage cell line, were treated with the compound of Example 1 at concentrations of 0, 12 μM, 25 μM, and 50 μM, respectively, and then CCK8 assay was performed according to <Experiment Method> 3 above. CCK8 assay results showed no cytotoxicity at all concentrations (
NO is produced from L-arginine by nitric oxide synthase (NOS), which exists in various tissues. There are three types of NOS, which are neuronal NOS, endothelial NOS, and inducible NOS (iNOS), and among these, iNOS, also called NOS-2, is expressed in various cells, including macrophages, by inflammatory stimuli such as bacterial endotoxin and various inflammatory cytokines during bacterial infection, and once iNOS is expressed, a large amount of NO is produced for a long period of time and participates in cytotoxicity during inflammation. In order to analyze the NO production amount by the compound of Example 1, the following experiment was performed.
1 mg/mL lipopolysaccharide (LPS) and the compounds of Example 1 at concentrations of 0, 12 μM, 25 μM, and 50 μM were added to Raw264.7 cells, cultured for 24 hours, and changes in NO concentration in the upper layer of the culture were analyzed.
As a result of the analysis, the concentration of NO secreted from LPS-stimulated Raw264.7 decreased in a concentration-dependent manner of Example Compound 1, and specifically, the NO concentrations produced in LPS-stimulated Raw26.7+0 μM, LPS-stimulated Raw26.7+12.5 μM, LPS-stimulated Raw26.7+25 μM and LPS-stimulated Raw26.7+50 μM, respectively, were 22.8±1.3 μM, 18.9±0.3 μM, 14.8±0.54 μM and 2.41±0.24 μM (
In order to confirm whether the NO production inhibitory effect of the compound of Example 1 is due to inhibition of iNOS protein expression, 1 mg/mL LPS and the compound of Example 1 at concentrations of 0, 12 μM, 25 μM, and 50 μM were added to Raw264.7 cells, and the effect on the expression of iNOS protein was evaluated by the method in <Experiment Method>7 above. As a result, Raw264.7 cells to which LPS was not added did not express iNOS protein, and Raw264.7 cells to which 1 mg/mL LPS was added showed high iNOS protein expression. In addition, when treated with the compound of Example 1, it was confirmed that iNOS protein expression in LPS-stimulated Raw264.7 was significantly suppressed (
LPS, well known as an endotoxin present in the outer cell membrane of Gram-negative bacteria, stimulates macrophages or monocytes to promote the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1l (IL-1), and interleukin-6 (IL-6). Accordingly, in order to analyze the effect of the compound of Example 1 on inhibiting cytokine secretion, RAW264.7 cells were cultured for 24 hours by adding 1 mg/mL of LPS and the compound of Example 1 at concentrations of 0, 12 μM, 25 μM, and 50 μM, and the concentrations of IL-6 and TNFα in the culture supernatant were measured. As a result, the compound of Example 1 effectively inhibited the production of TNFα and IL-6 in LPS-stimulated Raw264.7 cells in a concentration-dependent manner, and statistical significance was confirmed compared to the vehicle group (
Nuclear factor-kappa B (NF-κB), which plays an important role in inflammatory responses, is a transcription factor that regulates the synthesis of various cytokines, chemokines, and growth factors. NFκB is composed of p50 and p65, enters the nucleus, acts as a transcription factor, and synthesizes iNOS, COX-2, and inflammation-related cytokines, and generally binds to the inhibitor NF-κBα (IκBα) in the cytoplasm, thereby inhibiting the action of NF-κB. In order to confirm whether NF-κb-related cell signaling induced in Raw264.7 cells was affected by the compound of Example 1, NF-κb promoter-luciferase analysis and pIKKαβ expression change analysis were performed in Raw264.7 cells using 1 mg/mL LPS and the compound of Example 1 at concentrations of 0, 12 μM, 25 μM, and 50 μM. As a result, when 1 mg/ml LPS was treated with Raw264.7/NF-κb-luciferase cells, strong luminescent signals occurred, but when treated with the compound of Example 1, the luminescent signals decreased in a concentration-dependent manner, and statistical significance was confirmed when compared to the vehicle group (
Through this, it can be seen that the compound of Example 1 regulates NF-κb cell signaling in macrophages as a PPARδ agonist and is effective in controlling inflammatory cytokines.
The inflammation model induced by λ-carrageenan in the mouse foot region is an acute arthritis model and is useful when checking the effect of suppressing edema due to osteoarthritis. In order to confirm the ability of the compound of Example 1 to suppress acute inflammation, an experiment was performed according to the macrophage tracking technique (
As a result, no fluorescent signal could be confirmed in the soles of all experimental groups injected with 0.1% CG before inducing inflammation. On the other hand, 6 hours after inducing inflammation, a strong fluorescent signal was confirmed in the sole of the foot injected with 0.1% CG in the vehicle group. Meanwhile, in the sole of the foot injected with 0.1% CG in the group treated with 50 mg/kg compound of Example 1, a lower fluorescent signal was confirmed than that in the vehicle group, and statistical significance was also detected (
Through this, it could be confirmed that the compound of Example 1, which is a PPARδ agonist, has no cytotoxicity and is effective in suppressing the production of inflammatory cytokines and NO by regulating NF-κb cell signaling in macrophages. It was also confirmed to be effective in preventing macrophage infiltration into acute inflammatory areas.
Although the present disclosure has been described in detail through preferred embodiments and experimental examples above, the scope of the present disclosure is not limited to the specific embodiments and should be construed in accordance with the appended claims. In addition, those skilled in the art should understand that many modifications and variations are possible without departing from the scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0032759 | Mar 2022 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2023/003210 | 3/9/2023 | WO |
