A novel compound that exhibits a therapeutic effect on inflammatory diseases as a p38 MAP kinase inhibitor

Abstract

The present invention relates to novel compounds exhibiting anti-inflammatory activity. The compounds of the present invention have excellent inhibitory effects against p38 MAPK, which is known to play a crucial role in the production of inflammatory precursors and induce inflammatory diseases, and therefore can be usefully used as therapeutic agents for inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to a novel compound exhibiting anti-inflammatory activity. The novel compound of the present invention has an excellent inhibitory effect on p38 MAP kinase and is usefully used as a therapeutic agent for inflammatory diseases.

Background Art

[0002] p38 is a kind of serine / threonine phosphorylation enzyme belonging to the mitogen-activated protein kinases (MAPK) family. The MAPK pathway is activated by various extracellular stimuli and switches so that various intracellular reactions occur through phosphorylation of specific cellular substrates. Subtypes of MAPK include JNK, ERK, etc. in addition to p38. MAPK is a protein phosphorylation enzyme activated by insulin, mitogen, growth factor, activation stimuli of immune cells, etc., and occupies an important position in the signal transduction pathway that transmits the signal of receptors on the cell surface into the cell.

[0003] The p38 signal transduction system regulates processes such as metabolism and cell death and undergoes a series of consecutive phosphorylation processes in which higher-stage phosphorylation enzymes transfer phosphate groups to lower-stage phosphorylation enzymes. As p38 MAPK, p38-α, p38-β, p38-γ, p38-δ, etc. have been clarified and are activated by osmotic shock, inflammatory cytokines, LPS (lipopolysaccharides), ultraviolet rays (UV), growth factors (GF), etc.

[0004] Inflammation is known to be caused by various factors. According to various studies, MAPK is activated by various stress stimuli. In particular, p38 MAPK is known to play a decisive role in the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and to induce inflammatory diseases (NATURE REVIEWS, Drug discovery (2003), Vol.2, 717-726). Accordingly, several small molecule compounds have been developed as inhibitors of p38 MAPK (such as Republic of Korea Patent Registration No. 10-1879481). Various small molecule inhibitors of p38 act as inflammatory regulators by suppressing IL-1 and TNF synthesis in human monocytes at low concentrations, and it has been revealed that such suppression of cytokines can regulate, alleviate, or reduce the inflammatory response.

[0005] In connection with this, as a result of researching novel compounds having anti-inflammatory activity through p38 MAPK inhibition, the inventors of the present invention completed the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel compound having excellent anti-inflammatory efficacy by effectively suppressing p38 MAP kinase for the effective treatment of inflammatory diseases.

Means for Solving the Problems

[0007] To achieve the above object, the present invention provides a compound of Chemical Formula 1 below or a pharmaceutically acceptable salt thereof.

[0008]

Chemical Formula

[0009] In the above formula,

[0010] [Chemical]

[0011] R 2 is H, halogen, or -C 1 -C 6 is alkyl ,

[0012] R 3 is H or halogen.

[0013] Specifically, the compounds of the present invention may include the compounds in Table 1 below or pharmaceutically acceptable salts thereof.

[0014] [Table 1]

[0015] A composition for treating or preventing inflammatory diseases containing the compounds according to the present invention can be provided. [Advantages of the Invention]

[0016] The compounds of the present invention play a decisive role in the production of pro-inflammatory substances and have excellent inhibitory efficacy against p38 MAPK, which is known to induce inflammatory diseases, and thus are usefully used as therapeutic agents for inflammatory diseases. [Brief Description of the Drawings]

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0018] Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement them. However, the present invention can be embodied in various forms and is not limited to the embodiments and examples described herein.

[0019] Throughout the specification of the present invention, when a certain part is said to "include" a certain component, this means that, unless otherwise stated to the contrary, it does not exclude other components and can further include other components.

[0020] In this specification, "pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable and retains the desirable pharmacological activity of the parent compound. The salts are not particularly limited as long as they are pharmaceutically acceptable.

[0021] In this specification, "alkyl" is a hydrocarbon having primary, secondary, tertiary, or quaternary carbon atoms and includes saturated aliphatic groups that are linear, branched, or cyclic, or combinations thereof. For example, an alkyl group can have 1 to 20 carbon atoms (i.e., C 1 -C 20 alkyl), 1 to 10 carbon atoms (i.e., C 1 -C 10 alkyl), or 1 to 6 carbon atoms (i.e., C 1 -C 6 alkyl). Examples of suitable alkyl groups include methyl (Me, -CH 3 ), ethyl (Et, -CH2 CH 3 )), 1-propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 )), 2-propyl (i-Pr, i-propyl, -CH(CH 3 )) 2 )), 1-butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 )), 2-methyl-1-propyl (i-Bu, i-butyl, -CH 2 CH(CH 3 )) 2 )), 2-butyl (s-Bu, s-butyl, -CH(CH 3 ))CH 2 CH 3 )), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 )) 3 )), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 2 CH 3 )), 2-pentyl (-CH(CH 3 ))CH 2 CH 2 CH 3 )), 3-pentyl (-CH(CH 2 CH 3 )) 2 )), 2-methyl-2-butyl (-C(CH 3 )) 2 CH 2 CH 3 )), 3-methyl-2-butyl (-CH(CH 3 ))CH(CH 3 )) 2 )), 3-methyl-1-butyl (-CH 2 CH 2 CH(CH 3 )) 2 )), 2-methyl-1-butyl (-CH 2 CH(CH 3 ))CH 2 CH 3 )), 1-hexyl (-CH 2 CH 2 CH2 CH 2 CH 2 CH 3 )), 2-hexyl (-CH(CH 3 )CH 2 CH 2 CH 2 CH 3 ))), 3-hexyl (-CH(CH 2 CH 3 )(CH 2 CH 2 CH 3 ))), 2-methyl-2-pentyl (-C(CH 3 )) 2 CH 2 CH 2 CH 3 ))), 3-methyl-2-pentyl (-CH(CH 3 ))CH(CH 3 ))CH 2 CH 3 ))), 4-methyl-2-pentyl (-CH(CH 3 ))CH 2 CH(CH 3 )) 2 ))), 3-methyl-3-pentyl (-C(CH 3 )(CH 2 CH 3 )) 2 ))), 2-methyl-3-pentyl (-CH(CH 2 CH 3 ))CH(CH 3 )) 2 ))), 2,3-dimethyl-2-butyl (-C(CH 3 )) 2 CH(CH 3 )) 2 ))), 3,3-dimethyl-2-butyl (-CH(CH 3 ))C(CH 3 )) 3 ))), and octyl (-(CH 2 )) 7 CH 3 ))), among others, but not limited thereto.

[0022] Moreover, the term "alkyl" as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups.

[0023] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo, and iodo.

[0024] The term "Cx-y" or "Cx-Cy", when used with a chemical residue such as alkyl, alkenyl or alkynyl, is considered to include a group containing from x to y carbon atoms in the chain. C 0 Alkyl represents hydrogen when the group is at a terminal position and a bond when it is internal. For example, C 1 -C 20 The alkyl group contains from 1 to 20 carbon atoms in the chain.

[0025] The compounds according to the present invention can be prepared using modifications to the following described specific synthetic protocols well known to those skilled in the art from readily available starting materials.

[0026] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0027] [Production Example]

[0028] Equipment and reagents used in the synthesis method of the compounds according to the present invention

[0029] All starting materials and reagents used in the synthesis of the compounds of the present invention were purchased from Aldrich, Alfa Aesar, TCI, etc. and used without further purification. The various glasswares used in the reaction were used in a state dried in an 80 °C oven, and argon gas or nitrogen gas was injected to block air and moisture to proceed with the reaction.

[0030] The flash column chromatography of the present invention was carried out by appropriately adjusting solvents such as hexane, ethyl acetate, dichloromethane, and methanol using silica gel 60 (230 - 400 mesh). For thin-layer chromatography for analysis, a 0.25 mm silica gel plate was used.

[0031] The 1 1H-NMR spectrum and 13 the 13C-NMR spectrum of the present invention were analyzed using a Bruker Avance 400 (400 MHz for 1H; 100 MHz for 13C) spectrometer or a VARIAN VNMRS 500 (500 MHz for 13C) spectrometer. 1 1H and 13 13C NMR chemical shift values were expressed in ppm units using TMS (tetramethylsilane) as an internal standard. NMR signals were denoted as m (multiplet), s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), bs (broad singlet), bd (broad doublet), dd (doublet of doublets), dt (doublet of triplets), dq (doublet of quartets), etc., and coupling constants were expressed in Hz units.

[0032] The low resolution mass spectra and high resolution mass spectra of the present invention were measured using a VG Trio-2 GC-MS or a JEOL JMS-700 instrument.

[0033] In one embodiment, intermediate compound 10 of the present invention can be produced according to the following Reaction Scheme 1.

[0034] [Reverse 1]

Chem.

[0035] Reagents and conditions: (a) N-Iodosuccinimide, sulfuric acid, H 2 O, r.t., 85%; (b) Cyclopropylamine, 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N-Diisopropylethylamine, 1-Hydroxybenzotriazole hydrate, DMF, r.t, 82%; (c) bis(pinacolato)diboron, PdCl 2 (dppf) 2 , KOAc, DMF, 90 °C, 50%.

[0036] Using commercially available 3-Fluoro-4-methylbenzoic acid (Compound 7) as the starting material, Compound 8 was synthesized through an iodination reaction, and then Compound 9 was synthesized through EDC coupling with cyclopropylamine. The main intermediate Compound 10 was synthesized through cross coupling with bis(pinacolato)diboron.

[0037] In one embodiment, Intermediate Compound 16 to Intermediate Compound 21 of the present invention can be produced according to the following Reaction Scheme 2.

[0038] [Reverse 2]

Chem.

[0039] Reagents and conditions: (a) thionyl chloride, reflux; (b) N,O-dimethylhydroxylamine, Et 3 N, CH 2 Cl 2 , r.t., 62%; (c) 4-methoxyphenylmagnesium bromide, THF, 0 °C, 87%; (d) BBr 3 , CH 2 Cl 2 , -78 °C to 0 °C, 79 - 87%; (e) (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate, K 2CO 3 , DMF, 70 °C, 54 - 83%; (f) indole, AlCl 3 , CH 2 Cl 2 , 0 °C to r.t., 42%; (g) BnBr, NaH, DMF, 0 °C, 95 - 97%; (h) 6-methoxyindole, AlCl 3 , ZnCl 2 , EtMgBr, CH 2 Cl 2 、r.t.、24%。

[0040] Compound 12, which was produced by refluxing commercially available 6-bromonicotinic acid (Compound 11) and thionyl chloride, was used in the subsequent reaction without a purification process. After making Weinreb amide and introducing a methoxyphenyl group, demethylation was carried out to obtain Compound 15, and solketal was introduced to obtain intermediate Compound 16 ((6-bromopyridin-3-yl)(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)methanone); 1 H NMR (400 MHz, Chloroform-d) δ 8.73 (dd, J = 2.4, 0.8 Hz, 1H), 8.05 (dd, J = 8.2, 2.5 Hz, 1H), 7.48 (dd, J = 8.3, 0.9 Hz, 1H), 7.39 - 7.32 (m, 2H), 7.06 - 6.96 (m, 2H), 4.52 (p, J = 5.7 Hz, 1H), 4.28 (tt, J = 6.0, 5.1 Hz, 2H), 3.77 (dd, J = 8.7, 5.1 Hz, 2H), 1.48 (s, 3H), 1.42 (s, 3H).

[0041] Indole was introduced into Compound 12 to synthesize intermediate Compound 17, ((6-bromopyridin-3-yl)(1H-indol-3-yl)methanone); 11H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.79 (d, J = 3.1 Hz, 0H), 8.27 - 8.18 (m, 2H), 7.69 (s, 1H), 7.52 (s, 1H), 7.31 - 7.24 (m, 1H), 7.26 (s, 2H). Intermediate compound 18 ((1-benzyl-1H-indol-3-yl)(6-bromopyridin-3-yl)methanone) was obtained through its benzylation; 1 1H NMR (400 MHz, Chloroform-d) δ 8.81 (dd, J = 2.4, 0.8 Hz, 1H), 8.45 - 8.38 (m, 1H), 8.10 (dt, J = 8.4, 2.0 Hz, 1H), 7.60 (d, J = 1.5 Hz, 1H), 7.47 (dt, J = 8.2, 1.1 Hz, 1H), 7.36 (s, 2H), 7.42 - 7.29 (m, 5H), 7.26 (s, 1H), 7.21 - 7.12 (m, 2H), 5.38 (s, 2H).

[0042] The coupling reaction of 6-methoxyindole and compound 12 was carried out. After introducing a protection group as a benzyl group, demethylation was performed, and solketal was introduced to obtain intermediate compound 21 ((1-benzyl-6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-1H-indol-3-yl)(5-bromopyridin-2-yl)methanone); 1 1H NMR (400 MHz, Chloroform-d) δ 8.80 (d, J = 2.4 Hz, 1H), 8.30 (d, J = 8.7 Hz, 1H), 8.12 - 8.05 (m, 1H), 7.53 - 7.42 (m, 2H), 7.34 (dd, J = 7.3, 3.2 Hz, 2H), 7.15 (t, J = 9.3 Hz, 3H), 7.02 (dd, J = 8.8, 2.2 Hz, 1H), 6.83 (d, J = 2.3 Hz, 1H), 5.31 (d, J = 4.2 Hz, 3H), 4.48 (q, J = 6.0 Hz, 1H), 4.17 (dd, J = 8.4, 6.4 Hz, 1H), 4.08 (dd, J = 9.4, 5.4 Hz, 1H), 3.93 (ddd, J = 19.6, 8.9, 5.7 Hz, 1H), 1.46 (s, 3H), 1.41 (s, 3H).

[0043] [Example 1]

[0044] Preparation of N-Cyclopropyl-3-(5-(4-(2,3-dihydroxypropoxy)benzoyl)pyridin-2-yl)-5-fluoro-4-methylbenzamide (Compound 1)

[0045]

Chem.

[0046] Compound 1 of the present invention was prepared according to the following Production Formula 1.

[0047] [Production Formula 1]

Chem.

[0048] Reagents and conditions: (a) Pd(PPh 3 ) 4 , K 2 CO 3 , DMF, 90 °C, 36%; (b) p-TsOH, H 2 O, MeOH, 50 °C, 54%.

[0049] 1-1. Synthesis of Intermediate Compound 22

[0050] Intermediate Compound 10 (100.8 mg, 0.316 mmol), Intermediate Compound 16 (82.6 mg, 0.210 mmol), K 2 CO 3 (58.0 mg, 0.420 mmol), Pd(PPh 3 ) 4 (24.3 mg, 0.021 mmol) were dissolved in dry DMF, purged with argon, and then stirred at 90 °C for 1 hour. After confirming the completion of the reaction by TLC, the temperature of the reaction mixture was lowered to room temperature, and saturated aqueous NH 4The reaction was terminated using Cl solution. The product was extracted into the organic layer with EtOAc and H 2 O, then dehydrated using MgSO 4 , filtered, and the filtrate was distilled under reduced pressure. After purification using flash column chromatography, intermediate compound 22 was synthesized (37.8 mg, 36%); 1 H NMR (400 MHz, Chloroform-d) δ 9.04 (dd, J = 2.3, 1.0 Hz, 1H), 8.17 (dd, J = 8.1, 2.2 Hz, 1H), 7.88 (dt, J = 9.6, 2.8 Hz, 2H), 7.61 (d, J = 1.9 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.04 (dd, J = 9.2, 2.3 Hz, 2H), 6.40 (s, 1H), 4.52 (q, J = 4.6, 3.3 Hz, 1H), 4.25 - 4.10 (m, 2H), 4.10 - 4.02 (m, 1H), 3.98 - 3.90 (m, 1H), 2.94 - 2.87 (m, 1H), 2.35 (d, J = 2.4 Hz, 3H), 1.42 (m, 1H), 0.92 - 0.81 (m, 2H), 0.67 - 0.58 (m, 2H).

[0051] Synthesis of 1 - 2. Compound 1

[0052] To intermediate compound 22 (37.8 mg, 0.075 mmol) and p - TsOH (11.4 mg, 0.060 mmol), 500 μl of H 2 O and 2 mL of MeOH were added dropwise, and then stirred at room temperature. After confirming by TLC that the starting material had disappeared, it was diluted with CH 2 Cl 2 and H 2 O, and the organic layer was extracted and dehydrated using Na 2 SO 4 . The filtered filtrate was distilled under reduced pressure and then purified using flash column chromatography to finally obtain compound 1 (19 mg, 54%); 11H NMR (500 MHz, DMSO-d6) δ 8.92 (dd, J = 2.3, 0.9 Hz, 1H), 8.55 (d, J = 4.2 Hz, 1H), 8.17 (dd, J = 8.1, 2.2 Hz, 1H), 7.86 - 7.79 (m, 1H), 7.82 - 7.71 (m, 3H), 7.67 (dd, J = 10.4, 1.7 Hz, 1H), 7.16 - 7.05 (m, 2H), 5.02 (d, J = 5.2 Hz, 1H), 4.70 (t, J = 5.7 Hz, 1H), 4.16 - 4.04 (m, 1H), 4.03 - 3.93 (m, 1H), 3.85 - 3.75 (m, 1H), 3.50 - 3.40 (m, 2H), 2.88 - 2.77 (m, 1H), 2.28 (d, J = 2.3 Hz, 3H), 0.73 - 0.60 (m, 2H), 0.60 - 0.48 (m, 2H).

[0053] [Example 2]

[0054] Production of 6-Chloro-N-cyclopropyl-4'-(1H-indole-3-carbonyl)-[1,1'-biphenyl]-3-carboxamide (Compound 2)

[0055] [Chemical formula]

[0056] Compound 2 of the present invention was produced according to the following Production Formula 2.

[0057] [Production Formula 2] [Chemical formula]

[0058] Reagents and conditions: (a) Bis(pinacolato)diboron, PdCl 2 (dppf) 2 , KOAc, DMF, 90 °C, 37%; (b) 17, PdCl 2 (PPh 3) 2 、 K 3 PO 4 、 1,2 - dioxane, H 2 O, reflux, 10%; (c) cyclopropylamine, ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide), N,N - diisopropylethylamine, 1 - hydroxybenzotriazole hydrate, CH 2 Cl 2 , r.t., 12%.

[0059] 2 - 1. Synthesis of Intermediate Compound 24

[0060] Commercially available 3 - bromo - 4 - chlorobenzoic acid (Compound 23, 1.5 g, 6.3 mmol), bis(pinacolato)diboron (2.6 g, 10.5 mmol), KOAc (3.1 g, 31.8 mmol), PdCl 2 (dppf) 2 were dissolved in 23 mL of dry DMF. After purging with argon, the reaction was carried out at 90 °C. After confirming the completion of the reaction by TLC, the temperature of the reaction mixture was decreased to room temperature. The reaction was quenched with saturated aqueous NH 4 Cl solution, and the product was extracted into the organic layer with EtOAc and H 2 O. Then it was dried over MgSO 4 . The filtered filtrate was distilled under reduced pressure and then purified by flash column chromatography to obtain Intermediate Compound 24 (664 mg, 37%).

[0061] 2 - 2. Synthesis of Intermediate Compound 25

[0062] Intermediate Compound 17 (500 mg, 1.67 mmol), Intermediate Compound 24 (659 mg, 2.33 mmol), PdCl 2 (PPh 3 ) 2 (117 mg, 0.17 mmol), K 3 PO 4 (1.8 g, 8.33 mmol) were mixed with 9 mL of 1,2 - dioxane and H 2It was dissolved in a solvent mixed with 3 mL of O, and after argon substitution, it was refluxed. After 18 hours, it was confirmed by TLC that the reaction had terminated, and the temperature of the reaction product was decreased at room temperature. The remaining filtrate was filtered through Celite, and the filtrate was diluted with H 2 O and CH 2 Cl 2 After dilution, the resulting solid was filtered, and the filtrate was vacuum-decompressed to remove the remaining solvent, and Intermediate Compound 25 was synthesized without a separate purification process (101 mg, 10%).

[0063] 2-3. Synthesis of Compound 2

[0064] Intermediate Compound 25 (50 mg, 0.133 mmol) and 1-hydroxybenzotriazole hydrate (29 mg, 0.213 mmol) were dissolved in 0.7 mL of dry DMF. After argon substitution, 35 μl of N,N-diisopropylethylamine and 47 μl of ethyl-3-(3-dimethylaminopropyl)carbodiimide were sequentially added dropwise. After confirming by TLC that the reaction had terminated, the reaction was terminated using H 2 O, and the product was extracted into the organic layer using EtOAc and H 2 O, and then dehydrated using MgSO 4 After the filtered filtrate was distilled under reduced pressure, it was purified using flash column chromatography to finally obtain Compound 2 (6 mg, 12%); 1 H NMR (500 MHz, DMSO-d6) δ 12.15 (d, J = 3.0 Hz, 1H), 8.63 (d, J = 4.2 Hz, 1H), 8.34 - 8.27 (m, 1H), 8.04 (d, J = 3.0 Hz, 1H), 7.97 - 7.91 (m, 3H), 7.90 (dd, J = 8.4, 2.2 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.71 - 7.64 (m, 2H), 7.59 - 7.53 (m, 1H), 7.34 - 7.24 (m, 2H), 2.93 - 2.84 (m, 1H), 0.76 - 0.66 (m, 2H), 0.66 - 0.56 (m, 2H).

[0065] [Example 3]

[0066] Preparation of N-Cyclopropyl-3-(5-(6-(2,3-dihydroxypropoxy)-1H-indole-3-carbonyl)pyridin-2-yl)-5-fluoro-4-methylbenzamide (Compound 3)

[0067] [Chemical formula]

[0068] Compound 3 of the present invention was prepared according to the following Production Formula 3.

[0069] [Production Formula 3] [Chemical formula]

[0070] Reagents and conditions: (a) Pd(PPh 3 ) 4 , K 2 CO 3 , DMF, 90 °C, 100%; (b) Potassium tert-butoxide, O 2 , DMSO, 0 °C, 12%; (c) p-TsOH, H 2 O, MeOH, 50 °C, 67%.

[0071] 3-1. Synthesis of Intermediate Compound 26

[0072] Using Intermediate Compound 21 (127 mg, 0.244 mmol) as the starting material, Intermediate Compound 26 was synthesized in the same manner as Intermediate Compound 22 (158 mg, 100%); 11H NMR (400 MHz, Chloroform-d) δ 9.12 (s, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.22 (dd, J = 8.0, 2.2 Hz, 1H), 7.62 - 7.47 (m, 4H), 7.43 - 7.27 (m, 3H), 7.18 - 7.11 (m, 2H), 7.04 (dd, J = 8.8, 2.2 Hz, 1H), 6.84 (d, J = 2.2 Hz, 1H), 6.29 (s, 1H), 5.34 (s, 2H), 4.50 (p, J = 6.0 Hz, 1H), 4.18 (dd, J = 8.5, 6.4 Hz, 1H), 4.11 - 4.04 (m, 1H), 3.94 (ddd, J = 20.4, 8.9, 5.8 Hz, 2H), 2.91 (dt, J = 7.3, 3.8 Hz, 1H), 2.35 (d, J = 2.4 Hz, 3H), 2.28 - 2.22 (m, 1H), 2.05 (d, J = 0.8 Hz, 7H), 2.04 (s, 1H), 1.47 (s, 2H), 1.41 (s, 2H), 0.88 (q, J = 6.4 Hz, 3H), 0.62 (q, J = 6.0, 5.5 Hz, 2H).

[0073] 3 - 2. Synthesis of Intermediate Compound 27

[0074] A mixture of Intermediate Compound 26 (158 mg, 0.249 mmol) dissolved in 0.8 mL of DMSO was bubbled with O 2 and then potassium tert - butoxide (1.0 M in THF, 1.5 mL) was gradually added dropwise at 0 °C. After confirming the completion of the reaction by TLC, it was diluted with EtOAc and the reaction was terminated using saturated aqueous NH 4 Cl solution. After extracting the product into the organic layer with EtOAc and water, it was dehydrated using MgSO 4 filtered, and the filtrate was distilled under reduced pressure and then purified using flash column chromatography to synthesize Intermediate Compound 27 (18.1 mg, 12%).

[0075] 3 - 3. Synthesis of Compound 3

[0076] Using intermediate compound 27 (10.0 mg, 0.018 mmol) as the starting material, compound 3 was finally obtained through the same method as compound 1 (6 mg, 67%); 1 H NMR (400 MHz, Methanol-d4) δ 9.05 (d, J = 3.7 Hz, 1H), 8.35 (dt, J = 7.9, 2.7 Hz, 1H), 8.21 (dd, J = 8.9, 3.3 Hz, 1H), 7.87 (dd, J = 3.5, 1.7 Hz, 1H), 7.82 - 7.72 (m, 2H), 7.67 (d, J = 10.2 Hz, 1H), 7.09 (t, J = 2.8 Hz, 1H), 7.06 - 6.98 (m, 1H), 4.17 - 4.11 (m, 1H), 4.10 - 4.02 (m, 2H), 3.77 - 3.70 (m, 2H), 2.88 (d, J = 7.0 Hz, 1H), 2.36 (q, J = 2.5 Hz, 3H), 2.03 (s, 1H), 1.31 (s, 1H), 0.83 (d, J = 6.6 Hz, 2H), 0.66 (d, J = 5.7 Hz, 2H).

[0077] [Example 4]

[0078] Production of 3-(5-(1H-indole-3-carbonyl)pyridin-2-yl)-N-cyclopropyl-5-fluoro-4-methylbenzamide ((3-(5-(1H-indole-3-carbonyl)pyridin-2-yl)-N-cyclopropyl-5-fluoro-4-methylbenzamide, compound 4)

[0079] [Chemical formula]

[0080] Compound 4 of the present invention was produced according to the following production formula 4.

[0081] [Production formula 4] [Chemical formula]

[0082] Reagents and conditions: (a) Pd(PPh 3 )4 , K 2 CO 3 , dimethoxyethane, toluene, isopropyl alcohol, H 2 O, MW, 84%.

[0083] Intermediate compound 10 (48 mg, 0.15 mmol) and intermediate compound 17 (30 mg, 0.1 mmol), Pd(PPh 3 ), 4 (11.6 mg, 0.01 mmol), K 2 CO 3 (28 mg, 0.2 mmol) were placed in a reaction vessel, and 2 mL of a solvent mixed in (dimethoxyethane:toluene:isopropyl alcohol:H 2 O = 10:1:3:6) was added dropwise. After purging with argon, the reaction was carried out at 130 °C for 30 minutes in a microwave reactor. After the reaction was completed, the mixture was cooled to room temperature, the reaction was terminated with water, and the product was extracted into the organic layer with EtOAc and H 2 O, and then dehydrated using MgSO 4 . The filtered filtrate was distilled under reduced pressure and then purified using flash column chromatography to finally obtain compound 4 (34.8 mg, 84%); 1 1H NMR (400 MHz, DMSO-d6) δ 12.25 (s, 1H), 9.07 (s, 1H), 8.60 (d, J = 3.7 Hz, 1H), 8.31 (d, J = 7.5 Hz, 2H), 8.14 (s, 1H), 7.85 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 10.9 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.34 - 7.25 (m, 2H), 2.87 (dq, J = 7.6, 4.2, 3.3 Hz, 1H), 2.34 (s, 3H), 0.75 - 0.67 (m, 2H), 0.61 - 0.57 (m, 2H).

[0084] [Example 5]

[0085] Preparation of 3-(5-(1-Benzyl-1H-indole-3-carbonyl)pyridin-2-yl)-N-cyclopropyl-5-fluoro-4-methylbenzamide ((3-(5-(1-Benzyl-1H-indole-3-carbonyl)pyridin-2-yl)-N-cyclopropyl-5-fluoro-4-methylbenzamide, Compound 5))

[0086] [Chemical formula]

[0087] Compound 5 of the present invention was produced according to the following Production Formula 5.

[0088] [Production Formula 5] [Chemical formula]

[0089] Reagents and conditions: (a) Pd(PPh 3 ) 4 , K 2 CO 3 , DMF, 90 °C, 94%.

[0090] Using intermediate compound 18 (24.6 mg, 0.063 mmol) as the starting material, compound 5 was finally obtained through the same method as intermediate compound 22 (29.7 mg, 94%); 1 H NMR (400 MHz, Chloroform-d) δ 9.13 (dd, J = 2.2, 0.9 Hz, 1H), 8.48 (dd, J = 8.2, 1.7 Hz, 1H), 8.23 (dd, J = 8.0, 2.2 Hz, 1H), 7.69 (s, 1H), 7.62 - 7.52 (m, 3H), 7.43 - 7.25 (m, 6H), 7.17 (dd, J = 7.5, 2.0 Hz, 2H), 6.35 (s, 1H), 5.41 (s, 2H), 2.98 - 2.82 (m, 1H), 2.35 (d, J = 2.5 Hz, 3H), 0.96 - 0.81 (m, 2H), 0.67 - 0.55 (m, 2H).

[0091] [Example 6]

[0092] Preparation of N-Cyclopropyl-3-(5-(1-(2,3-dihydroxypropyl)-1H-indole-3-carbonyl)pyridin-2-yl)-5-fluoro-4-methylbenzamide (Compound 6)

[0093]

Chem.

[0094] Compound 6 of the present invention was prepared according to the following Production Formula 6.

[0095] [Production Formula 6]

Chem.

[0096] Reagents and conditions: (a) (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate, NaH, DMF, 90 °C, 19%; (b) p-TsOH, H 2 2O, MeOH, 50 °C, 62%.

[0097] 6-1. Synthesis of Intermediate Compound 28

[0098] After dissolving compound 4 (50 mg, 0.121 mmol) in 1.5 mL of dry DMF, NaH (60% dispersion in mineral oil, 7.24 mg, 0.181 mmol) was added at 0 °C. After purging with argon, the mixture was stirred. After 10 minutes, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 4-methylbenzenesulfonate (52.0 mg, 0.181 mmol) and 4-dimethylaminopyridine (catalytic amount) were added, and the temperature was gradually raised to 90 °C to proceed the reaction. When TLC confirmed that the starting material had disappeared, the temperature of the mixture was cooled to room temperature, and the reaction was terminated using saturated aqueous NH 4 Cl solution. The product was extracted into the organic layer with EtOAc and brine, and then dehydrated using MgSO 4 . The filtered filtrate was distilled under reduced pressure and then purified using flash column chromatography to synthesize intermediate compound 28 (13.5 mg, 19%).

[0099] 6-2. Synthesis of Compound 6

[0100] Using intermediate compound 28 (12 mg, 0.023 mmol) as the starting material, compound 6 was finally obtained in the same manner as compound 1 (7 mg, 62%); 1 1H NMR (500 MHz, Chloroform-d) δ 9.11 (s, 1H), 8.48 - 8.44 (m, 1H), 8.22 (dd, J = 8.1, 2.2 Hz, 1H), 7.76 (s, 1H), 7.59 (s, 1H), 7.56 (s, 1H), 7.54 (s, 1H), 7.48 - 7.43 (m, 1H), 7.38 (dd, J = 6.2, 3.1 Hz, 2H), 6.35 (s, 1H), 4.39 (dd, J = 14.5, 4.4 Hz, 1H), 4.26 (dd, J = 14.5, 7.3 Hz, 1H), 4.16 (s, 1H), 3.76 (d, J = 10.7 Hz, 1H), 3.58 (s, 1H), 2.89 (dd, J = 7.0, 3.5 Hz, 1H), 2.52 (s, 1H), 2.35 (d, J = 2.4 Hz, 3H), 1.40 (m, 1H), 0.87 (d, J = 6.0 Hz, 2H), 0.63 (d, J = 8.1 Hz, 1H).

[0101] [Experimental Example 1]

[0102] Evaluation of lipid affinity through calculated LogP (CLogP)

[0103] After writing the structural formulas of Compounds 1 to 6 using Chemdraw software, the CLogP values were confirmed, and the results are shown in Table 2 below.

[0104]

Table 2

[0105] As shown in Table 2 above, according to Lipinski's rule of five created by investigating conventional drugs, when CLogP is less than 5, it has the characteristics of a drug that is easy to administer orally. Therefore, it can be seen that the compounds of the present invention can be developed as orally administered drugs.

[0106] [Experimental Example 2]

[0107] Evaluation of the inhibitory ability of p38α MAPK (mitogen-activated protein kinase)

[0108] To confirm the ability to inhibit p38 MAPK, Compounds 1 to 5 were diluted to confirm the inhibitory effect depending on the concentration. Briefly, after diluting the compounds at concentrations of 1000, 333, 111, 37.0, 12.3, 4.12, 1.37, 0.457, 0.152, 0.0495 nM, peptide / kinase mixture (final 7.07 - 28.3 ng p38 alpha, 2 μM Serine / Threonine 15, 50 mM HEPES pH7.0, 0.01% NaN 3 ) and ATP solution were added. After reacting at room temperature for 1 hour, the development reagent was added and the reaction was carried out again at room temperature for 1 hour. The kinase inhibitory ability was confirmed at wavelengths of 445 nm and 520 nm, and the results are shown in Table 3 below.

[0109]

Table 3

[0110] As shown in Table 3 above, it was confirmed that the compound of the present invention is a potent inhibitor that inhibits p38 alpha by 50% at a concentration of 200 nM or less.

[0111] [Experimental Example 3]

[0112] Evaluation of the ability to inhibit p38 MAPK protein in cells

[0113] To confirm the inhibition of p-p38 MAPK, after treating each compound to Raw264.7 cells in which macrophages that induce inflammation by p-p38 MAPK activity were transformed into cancer cells, the inhibition of p38 MAPK was confirmed. Briefly, Raw264.7 cells were cultured in Dulbecco's modified Eagle Medium supplemented with 10% fetal bovine serum and penicillin / streptomycin (100 U / ml). Compounds 1 to 5 according to the present invention were each treated to Raw264.7 cells in a culture dish at 1 μM and cultured in a CO 2 After culturing in an incubator for 24 hours, each was treated with 100 ng / ml of LPS and cultured in a CO 2 Additional culture was performed in an incubator for 6 hours.

[0114] After culturing the cells according to the experimental conditions, they were washed with cold phosphate buffered saline (PBS). Subsequently, the cells were collected and centrifuged at 1200 rpm for 3 minutes. The precipitate was lysed with 2X sample buffer (1M Tris-HCl pH6.8, 50% Glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) and boiled at 100°C for 10 minutes. The prepared protein sample was subjected to Sodium Dodecyl sulfate poly acrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore). Blocking was performed with a 5% bovine serum albumin (BSA, RMbio) solution for the specific binding reaction between the protein on the PVDF membrane and the antibody. Subsequently, the BSA solution was discarded and the membrane was washed with PBS-T (0.5% tween 20 in PBS). The binding reaction between the protein on the membrane and the antibody was carried out overnight at 4°C and washed again with PBS-T. Finally, a secondary antibody (Cell signaling Technology) conjugated with Horse radish peroxidase (HRP) was diluted in PBS-T and reacted at room temperature for 2 hours. The amount of protein on the membrane was confirmed using Luminata Forte HRP substrate (Millipore), and the results are shown in Table 4 below.

[0115] [Table 4]

[0116] As shown in Table 4 above, as a result of immunoblotting, it was confirmed that the compound of the present invention effectively suppresses p38 phosphorylation in cells at a concentration of 1 μM.

[0117] [Experimental Example 4]

[0118] Evaluation of the ability to inhibit cytokine mRNA expression due to inflammatory response

[0119] To confirm the inhibition of p-p38 MAPK, after treating each compound to Raw264.7 cells in which macrophages that induce inflammation by p-p38 MAPK activity were transformed into cancer cells, the inhibition of p38 MAPK was confirmed. Briefly, Raw264.7 cells were cultured in Dulbecco’s modified Eagle Medium supplemented with 10% fetal bovine serum and penicillin / streptomycin (100 U / ml). The compounds 1 to 5 according to the present invention were each treated to Raw264.7 cells in a culture dish at 1 μM and then cultured in a CO 2 After culturing in an incubator for 24 hours, each was treated with 100 ng / ml of LPS and then cultured in a CO 2 Additional culturing was performed in an incubator for 24 hours.

[0120] After completion of cell culture according to the experimental conditions, the cells were washed with cold phosphate buffered saline (PBS). Thereafter, the cells were collected and centrifuged at 1200 rpm for 3 minutes. RNA was extracted by the method provided by the Hybrid-R total RNA purification kit (GeneAll (registered trademark), 305-101). After measuring the concentration and purity of RNA using Nanodrop, cDNA was synthesized by the method provided by TOPscript RT DryMIX (Enzynomics, RT200). The cDNA was reacted at 95°C for 3 minutes using SYBR Green Mix together with a primer, and then the reaction of 95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 30 seconds was repeated 30 times to confirm the RNA expression level. The results are shown in Table 5 and Table 6 below, and FIGS. 1 and 2.

[0121]

Table 5

[0122]

Table 6

[0123] As shown in Table 5 and Table 6, and FIGS. 1 and 2, it was confirmed that the compounds of the present invention suppress the mRNA expression of cytokines IL-1β and TNFα that cause inflammatory reactions, and it can be seen that the compounds of the present invention suppress the production of inflammatory cytokines through the suppression of p38.

[0124] [Experimental Example 5]

[0125] Evaluation of the ability to suppress TNFα by cell inflammatory response

[0126] To confirm the suppression of p-p38 MAPK, after treating each compound with Raw264.7 cells in which macrophages that induce inflammation by p-p38 MAPK activity were transformed into cancer cells, the suppression of p38 MAPK was confirmed. Briefly, Raw264.7 cells were cultured in Dulbecco's modified Eagle Medium supplemented with 10% fetal bovine serum and penicillin / streptomycin (100 U / ml). Compounds 1 to 5 according to the present invention were each treated with 1 μM on Raw264.7 cells in a culture dish and then cultured in a CO 2 After culturing in an incubator for 24 hours, each was treated with 100 ng / ml of LPS and then cultured in a CO 2 Additional culture was carried out in an incubator for 24 hours.

[0127] After the cell culture was completed according to the experimental conditions, the supernatant was obtained and centrifuged at 1200 rpm for 3 minutes. The centrifuged supernatant was dispensed into a plate coated with antibody, reacted at room temperature for 2 hours, and washed with PBS-T (0.5% tween 20 in PBS). After dispensing the detection antibody containing HRP, it was reacted at room temperature for 1 hour, and the protein amount was confirmed at a wavelength of 450 nm, and the results are shown in Table 7 below.

[0128]

Table 7

[0129] As shown in Table 7 above, it can be seen that the compound of the present invention suppresses the generation of the cytokine TNFα that causes an inflammatory reaction, and the compound of the present invention suppresses the inflammatory reaction through p38 inhibition.

[0130] [Experimental Example 6]

[0131] Evaluation of the ability to inhibit p38 MAPK protein in brain-derived cells

[0132] To confirm the inhibition of p-p38 MAPK in brain cells, after treating each compound to SH-SY5Y cells in which human neurons were cancerized, inflammation was induced to confirm the inhibition of p38 MAPK. Briefly, SH-SY5Y cells were cultured in Minimum essential medium / Eagle’s balanced salts supplemented with 10% fetal bovine serum and penicillin / streptomycin (100 U / ml). The compounds 4 and 6 according to the present invention were each treated at 1 μM to SH-SY5Y cells in a culture dish and cultured in a CO 2 After culturing in an incubator for 24 hours, each was treated with 1000 ng / ml of LPS and cultured in a CO 2 Additional culture was performed in an incubator for 6 hours.

[0133] After culturing the cells according to the experimental conditions, they were washed with cold phosphate buffered saline (PBS). Subsequently, the cells were collected and centrifuged at 1200 rpm for 3 minutes. The precipitate was lysed with 2X sample buffer (1M Tris-HCl pH6.8, 50% Glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) and boiled at 100°C for 10 minutes. The prepared protein sample was subjected to Sodium Dodecyl sulfate poly acrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore). For the specific binding reaction between the protein on the PVDF membrane and the antibody, blocking was performed with a 5% bovine serum albumin (BSA, RMbio) solution. Subsequently, the BSA solution was discarded and the membrane was washed with PBS-T (0.5% tween 20 in PBS). The binding reaction between the protein on the membrane and the antibody was carried out overnight at 4°C and washed again with PBS-T. Finally, a secondary antibody (Cell signaling Technology) conjugated with Horse radish peroxidase (HRP) was diluted in PBS-T and reacted at room temperature for 2 hours. The protein amount on the membrane was confirmed using Luminata Forte HRP substrate (Millipore), and the results are shown in Table 8 below.

[0134] [Table 8]

[0135] As shown in Table 8 above, as a result of immunoblotting, it was confirmed that Compound 4 and Compound 6 of the present invention effectively suppressed p38 phosphorylation in human-derived cells at a concentration of 1 μM.

[0136] [Experimental Example 7]

[0137] Evaluation of the ability to inhibit p38 MAPK protein in normal brain cells

[0138] To confirm the suppression of p-p38 MAPK in normal cells rather than cancer cells, each compound was treated with neuron cells collected from the brains of ICR mice, and inflammation was induced to confirm the suppression of p38 MAPK. Briefly, neurons were collected from the brains of fetuses of ICR mice that had been pregnant for about 17 - 18 days, and cultured for 7 days in Neurobasal Medium supplemented with 2% B27 supplements, 2 mM glutamine, and penicillin / streptomycin (100 U / ml). The primary neurons in the culture dish were treated with 1 μM each of Compound 4 and Compound 6 according to the present invention, and then CO 2 After culturing in an incubator for 24 hours, each was treated with 1000 ng / ml of LPS, and then CO 2 Additional culturing was performed in an incubator for 6 hours.

[0139] After culturing the cells according to the experimental conditions, they were washed with cold phosphate buffered saline (PBS). Subsequently, the cells were collected and centrifuged at 1200 rpm for 3 minutes. The precipitate was lysed with 2X sample buffer (1M Tris-HCl pH6.8, 50% Glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) and boiled at 100°C for 10 minutes. The prepared protein sample was subjected to Sodium Dodecyl sulfate poly acrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore). For the specific binding reaction between the protein on the PVDF membrane and the antibody, blocking was performed with a 5% bovine serum albumin (BSA, RMbio) solution. Subsequently, the BSA solution was discarded and the membrane was washed with PBS-T (0.5% tween 20 in PBS). The binding reaction between the protein on the membrane and the antibody was carried out overnight at 4°C and washed again with PBS-T. Finally, a secondary antibody (Cell signaling Technology) conjugated with Horse radish peroxidase (HRP) was diluted in PBS-T and reacted at room temperature for 2 hours. The protein amount on the membrane was confirmed using Luminata Forte HRP substrate (Millipore), and the results are shown in Table 9 below.

[0140]

Table 9

[0141] As shown in Table 9 above, as a result of immunoblotting, it was confirmed that Compound 4 and Compound 6 of the present invention effectively suppressed p38 phosphorylation due to inflammation not only in cancer cells but also in normal cells at a concentration of 1 μM.

[0142] [Experimental Example 8]

[0143] Evaluation of the ability to inhibit cytokine mRNA expression by inflammatory response

[0144] To confirm the inhibition of p-p38 MAPK, after treating each compound to BV2 cells in which microglia that induce inflammation by p-p38 MAPK activity in the brain were transformed into cancer cells, the inhibition of p38 MAPK was confirmed. Briefly, BV2 cells were cultured in Dulbecco’s modified Eagle Medium supplemented with 10% fetal bovine serum and penicillin / streptomycin (100 U / ml). BV2 cells in a culture dish were treated with 1 μM each of Compound 4 and Compound 6 according to the present invention and cultured in a CO 2 After culturing in an incubator for 24 hours, they were treated with 1000 ng / ml of LPS each and cultured in a CO 2 Additional culture was carried out in an incubator for 24 hours.

[0145] After the cell culture was completed according to the experimental conditions, it was washed with cold phosphate buffered saline (PBS). Thereafter, the cells were collected and centrifuged at 1200 rpm for 3 minutes. RNA was extracted by the method provided by the Hybrid-R total RNA purification kit (GeneAll (registered trademark), 305-101). After measuring the concentration and purity of RNA using Nanodrop, cDNA was synthesized by the method provided by TOPscript RT DryMIX (Enzynomics, RT200). cDNA was reacted with SYBR Green Mix together with a primer at 95°C for 3 minutes, and then the reaction of 95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 30 seconds was repeated 30 times to confirm the RNA expression level. The results are shown in Tables 10 to 12 below, and Figures 3 to 5.

[0146]

Table 10

[0147]

Table 11

[0148]

Table 12

[0149] As shown in Tables 10 to 12 and FIGS. 3 to 5, the compound of the present invention suppresses the generation of mRNAs of inflammatory cytokines IL-1β, TNFα and IL-6, and it can be seen that the compound of the present invention suppresses the expression of inflammatory cytokines through p38 inhibition.

Claims

Claim 1 A compound of the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof; 【Chemical 1】 In the above formula, 【Chemical Formula 2】 R ２ is H, halogen, or -C １ -C ６ is alkyl, R ３ is H or halogen. Claim 2 The compound according to claim 1, wherein the compound is a compound selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof: Claim 3 A pharmaceutical composition for treating or preventing an inflammatory disease, comprising the compound according to claim 1. Claim 4 A pharmaceutical composition for treating or preventing an inflammatory disease, comprising the compound according to claim 2.

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