Benzyloxybenzyl aminyl amino acid derivatives that modulate TSLP / TSLPR signaling

Abstract

The present invention aims to provide a pharmaceutical composition for the prevention, treatment, or prevention and treatment of cancer, inflammatory diseases, itch, allergies, and / or immune diseases, which comprises, as an active ingredient, a novel benzyloxybenzyl aminyl amino acid derivative, a salt and / or a solvate thereof that inhibits intracellular signal transduction by the cytokine TSLP (thymic stromal lymphopoietin), TSLP receptor (TSLPR), and IL-7Rα.

Description

[Technical Field]

[0001] The present invention relates to benzyloxybenzylaminyl amino acid derivatives and pharmaceutical compositions containing them as active ingredients. Specifically, the present invention relates to anti-inflammatory pharmaceutical compositions that regulate intracellular signal transduction of TSLP. [Background technology]

[0002] Cytokines play an important role in the immune system, including cell-mediated immunity and allergic responses. Cytokines are divided into proinflammatory and anti-inflammatory cytokines. Cytokines are primarily produced by T lymphocytes, and antigen-specific receptors on the surface of T lymphocytes recognize foreign pathogens. T lymphocytes are classified into CD4 and CD8 types based on the types of cell surface molecules. CD4-expressing T lymphocytes produce the most cytokines and are known as helper T cells. Helper T cells are further divided into Th1, which produce Th1-type cytokines, and Th2, which produce Th2-type cytokines. Th1-type cytokines primarily initiate inflammatory responses that maintain the immune response to eliminate intracellular foreign pathogens. Interferon gamma is the primary Th1 cytokine. Th2-type cytokines include interleukin-4, interleukin-5, interleukin-13, and interleukin-10, which are associated with promoting IgE and eosinophilic responses in atopy.

[0003] Thymic stromal lymphopoietin (TSLP) is produced by skin keratinocytes or lung and intestinal epithelial cells in response to stimuli such as viruses, bacteria, or fungi, protease-active antigens, inflammatory cytokines, chemicals, and allergens. TSLP exerts its biological function by first binding to the TSLP receptor (TSLPR) and then forming a heterodimer with the IL-7 receptor (IL7Rα, interleukin-7 receptor subunit alpha), activating a signaling cascade. The heterodimer then binds to dendritic cells (DCs).

[0004] TSLP polarizes TSLPR cells, inducing a type 2 inflammatory response, and acts as a master regulator of Th2 immune responses at the surface of the skin, gut, and airways. TSLP complexes with TSLPR and IL-7Ra, resulting in the cross-phosphorylation of JAK1 and JAK2 and the subsequent induction of STAT5 (signal transducer and activator of transcription).

[0005] TSLP is secreted not only by epithelial and epidermal cells but also by mast cells, airway smooth muscle, fibroblasts, and dendritic cells, and is known to regulate immune responses. TSLP expression in the skin has been reported to be associated with various allergic diseases (Steven F. Ziegler et al., J Allergy Clin Immunol, 2012;130:845-52). TSLP has also been detected in bronchial lavage fluid from asthma patients, and a correlation has been reported between the sensitivity of the disease and increased TSLP expression in airway epithelial cells. TSLP secreted from skin cells has been shown to deepen the allergic inflammatory response upon antigen exposure, promoting the onset of asthma (Juan et al., Journal of Investigative Dermatology, 2012). The TSLP receptor (TSLPR) is primarily expressed in hematopoietic cells, most abundantly in dendritic cells (DCs).

[0006] TSLP is involved in the interaction of the innate and adaptive immune systems, including communication between tissue cells and immune cells. Therefore, TSLP and its signaling pathways are known to be associated with diseases such as atopic dermatitis (AD), psoriasis, asthma, allergic rhinitis (AR), and eosinophilic esophagitis (EoE). Furthermore, TSLP signaling has been reported to be associated with other immune-mediated diseases, such as solid tumors (e.g., breast, colon, and pancreatic cancer) and hematological cancers (e.g., B-cell acute lymphocytic leukemia (B-ALL)). TSLP is also an important pruritus inducer. Tezepelumab, a human monoclonal antibody targeting TSLP, is currently under development as a treatment for asthma and atopic dermatitis (TSLP: from allergy to cancer, Jonathan Corren & Steven F. Ziegler, Nature Immunology volume 20, 1603-1609 (2019)). However, biological drugs such as antibodies have limited tissue penetration, making them difficult to develop as oral agents and less economical.

[0007] Therefore, the present inventors have made efforts to develop drugs for the treatment of inflammation, Th2 immune diseases, cancer, and the like by regulating TSLP signaling, and as a result, have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a pharmaceutical composition for regulating TSLP / TSLPR signaling, which comprises a novel benzyloxybenzylaminyl amino acid derivative as an active ingredient.

[0009] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment, or prevention and treatment, of inflammation, immune diseases and / or cancer, which comprises a novel benzyloxybenzylaminyl amino acid derivative as an active ingredient.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating, or both preventing and treating itching, which comprises a novel benzyloxybenzyl aminyl amino acid derivative as an active ingredient.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating, or both preventing and treating, allergic diseases, which comprises a novel benzyloxybenzyl aminyl amino acid derivative as an active ingredient.

[0012] Another object of the present invention is to provide a method for treating cancer, inflammatory diseases, itch, allergies, and / or immune diseases by administering a pharmaceutical composition containing a novel benzyloxybenzylaminyl amino acid derivative as an active ingredient. [Means for solving the problem]

[0013] To achieve the above object, the present invention provides a benzyloxybenzylamine amino acid derivative represented by the following [Chemical Formula 1], its S-form, R-form or a racemic mixture thereof, or a pharmaceutically acceptable salt, tautomer, polymorph, prodrug, and / or solvate thereof. [ka]

[0014] In the above [Chemical Formula 1], R4, R5, R6, R7 and R8 are the same or different and each independently represents hydrogen, C 1-7 Alkyl, hydroxy, halogen, halogenated C 1-7 Alkyl, C 1-7Alkyloxy and halogenated C 1-7 alkyloxy, and Y is selected from the following [Chemical Formula 2] to [Chemical Formula 4]: [ka] [ka] [ka] R1 and R3 are the same or different and each independently represent hydrogen or C 1-7 is alkyl, R2 is hydrogen or C 1-7 It is alkyl.

[0015] In the present invention, the halogen can be selected from fluoro, chloro, bromo and iodo; Said C 1-6 The alkyl may be a straight chain, branched chain or cyclic alkyl and may be selected from among methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl and octyl.

[0016] Said C 1-7 The alkyloxy group may be selected from among methoxy, ethoxy, propoxy, butoxy, pentoxy, hexyloxy, heptyloxy and octyloxy.

[0017] The halogenated C 1-7 alkyl may be selected from among difluoromethyl, trifluoromethyl, difluoroethyl, trifluoroethyl, trifluoropropyl, trifluoropentyl, trifluorohexyl and trifluoroheptyl; The halogenated C 1-7Alkyloxy may be selected from among difluoromethyloxy, trifluoromethyloxy, difluoroethyloxy, trifluoroethyloxy, trifluoropropyloxy, trifluoropentyloxy, trifluorohexyloxy and trifluoroheptyloxy.

[0018] According to the present invention, in the above [Chemical Formula 1], any one of R4, R5, R6, R7 and R8 is C 1-7 Alkyl, hydroxy, halogen, halogenated C 1-7 Alkyl, C 1-7 Alkyloxy and halogenated C 1-7 When it is alkyloxy, the other four can be hydrogen.

[0019] Specific examples of the compound represented by Chemical Formula 1 include: (R) / (S)-1-((4-((2-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 17); (R) / (S)-1-((4-((3-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 18); (R) / (S)-1-((4-((4-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 19); (R) / (S)-1-oxo-1-((4-((2-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 20); (R) / (S)-1-oxo-1-((4-((3-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 21); (R) / (S)-1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 22); (R) / (S)-1-((4-((3-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 23); (R) / (S)-1-((4-((4-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 24); (R) / (S)-1-((4-((3-fluorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 25); (R) / (S)-1-((4-((4-(trifluorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 26); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 27); (R) / (S)-N-(4-((3-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (compound 28); (R) / (S)-N-(4-((4-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (compound 29); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 30); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 31); (R) / (S)-tert-butyl-2-((4-((3-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 34); (R) / (S)-tert-butyl-2-((4-((4-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 35); (R) / (S)-tert-butyl-2-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 36); (R) / (S)-tert-butyl-2-((4-((4-chlorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 37); (R) / (S)-N-(4-((3-fluorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (compound 38); (R) / (S)-N-(4-((4-fluorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (compound 39); (R) / (S)—N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (compound 40); (R) / (S)-N-(4-((4-chlorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (compound 41); (R) / (S)-1-methyl-N-(4-((3-fluorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide (compound 42); (R) / (S)-1-methyl-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pyrrolidine-2-carboxamide (compound 43); (R) / (S)-1-methyl-N-(4-((4-chlorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide (compound 44); (R) / (S)-tert-butyl-3-((4((3-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 46); (R) / (S)-tert-butyl-3-((4((4-(trifluoromethyl)benzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 47); (R) / (S)-tert-butyl-3-((4((4-chlorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (compound 48); (R) / (S)-2-((4-((3-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidin-1-nium chloride (compound 49); (R) / (S)-2-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)carbamoyl)pyrrolidin-1-nium chloride (compound 50); (R) / (S)-2-((4-((4-chlorobenzyl)oxy)benzyl)carbamoyl)pyrrolidin-1-nium chloride (compound 51); (R) / (S)-1-methyl-N-(4-((3-fluorobenzyl)oxy)benzyl)pyrrolidine-3-carboxamide (compound 52); (R) / (S)-1-methyl-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pyrrolidine-3-carboxamide (compound 53); (R) / (S)-1-methyl-N-(4-((4-chlorobenzyl)oxy)benzyl)pyrrolidine-3-carboxamide (compound 54); (R) / (S)-tert-butyl(1-((4-((3,4-chlorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (compound 55); (R) / (S)-tert-butyl(1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)pent-2-yl)carbamate (compound 56); (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 57); (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 58); (R) / (S)-2-(dimethylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 59); (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (compound 60); (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (compound 61); (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 62); (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)butanamide (compound 63); (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 64); (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)pentanamide (compound 65); (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 66); (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)butanamide (compound 67); (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 68); (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)pentanamide (Compound 69); and may be any of the R-form, S-form, or a racemic mixture thereof.

[0020] In the present invention, the pharmaceutically acceptable salt is not particularly limited as long as it is one commonly used in the art. Specific examples thereof include, but are not limited to, salts formed using inorganic acids such as hydrochloric acid, bromic acid, sulfonic acid, amidosulfuric acid, phosphoric acid, and nitric acid, and organic acids such as carboxylic acid and sulfonic acid.

[0021] In addition, the types of carboxylic acids include acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, succinic acid, propionic acid, glycolic acid, stearic acid, and lactic acid, and the types of sulfonic acids include methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, and naphthalenedisulfonic acid, but the types of carboxylic acids and sulfonic acids are not limited to the above compounds.

[0022] The benzyloxybenzyl aminyl amino acid derivative represented by [Chemical Formula 1] according to the present invention effectively inhibits or kills fungal growth. Therefore, a composition containing the benzyloxybenzyl aminyl amino acid derivative represented by [Chemical Formula 1] as an active ingredient can be useful as a pharmaceutical composition for preventing or treating inflammatory diseases, such as atopic dermatitis, psoriasis, asthma, inflammatory arthritis, inflammatory bowel disease, immune disorders, allergies, and cancer. In addition, it can be prepared and used as a pharmaceutical composition for co-administration with existing drugs.

[0023] The benzyloxybenzylaminyl amino acid derivative according to the present invention can be prepared by a method comprising the following steps, as described in the applicant's prior patent (Korean Patent Publication No. 10-2099980). 1) protecting the amine group of an amino acid selected from the following [Chemical Formula 5] to [Chemical Formula 7] with a protecting group; 2) reacting the amino acid having the protected amine group with a benzylamine derivative to prepare a benzyloxybenzylaminyl amino acid derivative in which the amine group of the amino acid is protected with a protecting group; and 3) Removal of the amine protecting group.

[0024] [ka] [ka] [ka] In the above [Chemical Formula 5] to [Chemical Formula 7], R1 is hydrogen or C 1-7 is alkyl, R2 is C 1-7 It is alkyl.

[0025] First, the amine group of an amino acid selected from the following [Chemical Formula 5] to [Chemical Formula 7] is protected with an amine-protecting group. Any amine-protecting group that is commonly used for amine protection can be used.

[0026] Next, the amino acid with the protected amine group is reacted with a benzylamine derivative to produce a benzyloxybenzylaminyl amino acid derivative in which the amine group of the amino acid is protected with a protecting group. The reaction can consist of one step or two steps. If the reaction is one step, it can be by reacting the amino acid with the protected amine group with a benzyloxybenzylamine derivative, or if the reaction is two steps, it can be by reacting the amino acid with the protected amine group with 4-hydroxybenzylamine to produce a 4-hydroxybenzylaminyl amino acid derivative in which the amine group of the amino acid is protected with a protecting group, followed by reaction with a benzyl bromide derivative.

[0027] The amine protecting group can be removed using standard amine removal methods, in this case by treatment with perhydrochloric acid.

[0028] In the benzyloxybenzylaminyl amino acid derivative according to the present invention, the amine of the amino acid residue may be primary, secondary, or tertiary. When the amine is secondary or tertiary, an alkyl group can be introduced by alkylation.

[0029] According to the present invention, the benzyloxybenzylaminyl amino acid derivative or a pharmaceutically acceptable salt thereof may be mixed with a conventional carrier, adjuvant, or diluent and formulated by a conventional formulation method to prepare a form suitable for oral or parenteral administration.

[0030] The carriers, excipients, adjuvants or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0031] When formulated, the formulation may further contain conventional fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., and may further contain lubricants such as magnesium stearate and talc.

[0032] In the case of oral administration, the compound may be prepared in the form of a tablet, capsule, solution, syrup, suspension, etc., and in the case of parenteral administration, the compound may be prepared in the form of an injection for peritoneal, subcutaneous, intramuscular, or transdermal administration, an inhalation preparation used in an inhaler or nebulizer, a patch preparation such as a microneedle, or an external preparation such as a skin application.

[0033] According to one embodiment of the present invention, the effective daily dose of the benzyloxybenzyl aminyl amino acid derivative of [Chemical Formula 1] or its pharmaceutically acceptable salt in the pharmaceutical composition is 0.01 to 2,000 mg / day for an adult. However, the dose may vary depending on the age, weight, sex, dosage form, health condition, and severity of the disease of the patient, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist. [Effects of the Invention]

[0034] The pharmaceutical compositions of the present invention can be used in the development of drugs for the effective prevention and treatment of inflammatory diseases, immune diseases, allergies, itch, and / or cancer by inhibiting TSLP / TSLPR signaling. [Brief explanation of the drawings]

[0035] [Figure 1] The graph shows the inhibitory activity of the compounds of the present invention against IL-6, IL-4, IL-13 and TSLP. [Figure 2] The graph shows the inhibitory activity of the compounds of the present invention against IL-6, IL-4, IL-13 and TSLP. [Figure 3] The graph shows the inhibitory activity of the compounds of the present invention against IL-6, IL-4, IL-13 and TSLP. [Figure 4] This shows that the compounds of the present invention inhibit STAT5 phosphorylation in HEK293 cells. [Figure 5] This shows that the compounds of the present invention inhibit STAT5 phosphorylation in HEK293 cells. [Figure 6] SCOARD index determined from a DNCB-induced inflammation mouse model is shown in a graph. [Figure 7] This shows the results of H&E staining of skin tissue from DNCB-induced atopic dermatitis mice. [Figure 8] IgG and TSLP were confirmed in the serum of mice with DNCB-induced atopic dermatitis (NS, non-significant; *, P<0.05, **, P<0.01, ***, P<0.001). [Figure 9] This study confirmed the levels of TNF-α, IL-1β, CCL22 / MDC, IL-4, MMP-12, and S100A9 in the skin tissue of mice with DNCB-induced atopic dermatitis. [Figure 10] The itching-relieving effect of the compound of the present invention was confirmed in DNFB-induced atopic dermatitis mice. [Figure 11] This shows the results of observing inflammation scores in mice with house dust mite (HDM)-induced atopic dermatitis. [Figure 12] 1 is a graph showing the skin lesion scores of HDM atopic dermatitis mice. [Figure 13]IgE in the serum of HDM atopic dermatitis mice was analyzed. [Figure 14] These are photographs of changes in mouse skin tissue with HDM atopic dermatitis confirmed by H&E staining, and measurements of changes in eosinophils and skin thickness. [Figure 15] This shows the results of confirming skin-specific T cell fractions from skin tissue of HDM atopic dermatitis mice using FACS. [Figure 16] These are the results of confirming skin-specific T cell fractions using FACS from skin tissue isolated from patients with atopic dermatitis. DETAILED DESCRIPTION OF THE INVENTION

[0036] The reaction scheme for preparing the compounds of the present invention is as follows: In the following reaction scheme, numbers not used with an English letter indicate the compound numbers prepared in the present invention, i.e., 1 represents Compound 1. [ka] [ka] [ka] [ka] [ka]

[0037] Examples of syntheses for preparing compounds of the present invention are as follows: Synthesis Example 1: Amino Acid Amine Group Protection [ka] Amino acid (1.0 equivalent), Boc anhydride (1.5 equivalents), and sodium bicarbonate (1.5 equivalents) were dissolved in a 1:1 mixture of distilled water and methanol and reacted at room temperature for 36 to 48 hours. After the reaction was complete, the mixture was washed twice with diethyl ether, and the pH of the aqueous layer was adjusted to 2 with 1.0 M hydrochloric acid, followed by extraction with ethyl acetate (EA). The resulting organic layer was dehydrated using a drying agent (sodium sulfate) and dried under reduced pressure to obtain the desired compound, an amino acid whose amine group was protected with a protecting group (Boc).

[0038] Synthesis Example 2: 4-Hydroxybenzylamine bond [ka] The compound synthesized in Synthesis Example 1 (0.1 M) and N-methylmorpholine (NMM) (0.15 M, 1.5 equivalents) were added to anhydrous tetrahydrofuran at a low temperature of -78°C and stirred for 5 minutes. Then, isobutyl chloroformate (IBCF) (0.13 M, 1.3 equivalents) was added and stirred for 5 minutes. Next, 4-hydroxybenzylamine derivative (0.12 M, 1.2 equivalents) was added and stirred for 5 minutes. The reaction mixture was then warmed to room temperature and stirred for 30 to 60 minutes to check whether the reaction was progressing. After completion of the reaction, the mixture was filtered, dried under reduced pressure, and purified by silica gel column chromatography.

[0039] Synthesis Example 3: Benzyl bromide coupling reaction [ka] The compound from Synthesis Example 2 (1.0 equivalent), benzyl bromide (1.0-1.2 equivalents), and potassium carbonate (4.0 equivalents) were dissolved in acetone, and the reaction progress was monitored while refluxing at 50-60°C for 4-12 hours. After completion of the reaction, the reaction mixture was concentrated and extracted with methylene chloride, and the remaining water in the organic layer was removed with a desiccant (sodium sulfate). The resulting organic layer was concentrated and then purified by silica gel column chromatography.

[0040] Synthesis Example 4: Amine Protecting Group Removal The compound obtained in Synthesis Example 3 was dissolved in methylene chloride, and then an excess amount (6 to 10 equivalents) of 4.0 M hydrochloric acid was added and stirred at room temperature. After the reaction was completed, the resulting solid was washed with ethyl acetate and then dried under vacuum to remove the amine protecting group (boc), thereby obtaining the desired benzyloxybenzylaminyl amino acid derivative containing a primary amine structure. [ka]

[0041] Synthesis Example 5.1: Alkylation 1 [ka] A benzyloxybenzyl aminyl amino acid derivative in which the amino acid has a primary amine structure was alkylated to form a secondary amine structure. Specifically, the compound of Synthesis Example 4 (1.0 equivalent) and iodomethane (10 equivalents) were dissolved in tetrahydrofuran, and sodium hydride (10 equivalents) was slowly added dropwise at 0°C. The mixture was then reacted at room temperature for 24 hours. After the reaction was completed, the reaction mixture was extracted with methylene chloride, and the water was removed using a desiccant (sodium sulfate), followed by concentration under reduced pressure. The resulting concentrate was purified by silica gel column chromatography. The purified compound was dissolved in methylene chloride and stirred while adding 6.0 to 10.0 equivalents of 4.0 M hydrochloric acid. After the reaction was completed, the mixture was concentrated under reduced pressure, and the concentrate was purified by silica gel column chromatography to obtain a benzyloxybenzyl aminyl amino acid derivative containing a secondary amine structure.

[0042] Synthesis Example 5.2: Alkylation 2 [ka] A benzyloxybenzyl aminyl amino acid derivative, in which the amino acid has a primary amine structure, was alkylated to form a tertiary amine structure. Specifically, the compound of Synthesis Example 4 (1.0 equivalent) was dissolved in methanol, and then formaldehyde (37% by weight solution) and a 10% palladium catalyst were added sequentially. The reaction was carried out at room temperature for 18 hours. After the reaction, the catalyst was removed by filtration through Celite, and the filtrate was evaporated under reduced pressure. The reaction mixture was recrystallized from methanol / diethyl ether to obtain the purified target compound.

[0043] Synthesis Example 6: General Method for Ethylation and Diethylation of Amines [ka] The compound obtained in Synthesis Example 4 (1.0 equivalent) and iodoethane (0.9 equivalents) were dissolved in tetrahydrofuran solvent, and sodium hydride (10 equivalents) was added very slowly at 0°C. The reaction mixture was allowed to react at room temperature for 24 hours. After the reaction was completed, the mixture was diluted with dimethyl chloride solvent and washed with distilled water. The organic layer was dried over sodium sulfate and concentrated in vacuo. The resulting residue was separated and purified by silica gel chromatography. The purified product was dissolved in dimethyl chloride solvent, and 4.0 M hydrochloric acid (6.0-10.0 equivalents) was added. The mixture was stirred at room temperature and then concentrated in vacuo. The resulting residue was separated and purified by silica gel chromatography. The present invention will be described in more detail below with reference to preferred examples. However, it will be obvious to those skilled in the art that these examples are for the purpose of explaining the present invention in more detail and do not limit the scope of the present invention.

[0044] Manufacturing example Example 1: (R) / (S)-2-((tert-butoxycarbonyl)amino)butanoic acid (compound 3) Using the reaction scheme 6, compound 1 (3.00 g, 29.1 mmol), Boc anhyride (10.00 ml, 43.6 mmol), and NaHCO3 (3.67 g, 43.6 mmol) were reacted to synthesize compound 3 (5.89 g, 100%) as a white powder: f =0.50(DCM 19:Methanol 1 and few drops of acetic acid); 1 H NMR (DMSO-d6,400MHz) 12.40(C(O)OH), 7.02(d,J=7.9Hz,NH), 3.74-3.82(m,NHCHCH2), 1.50-1.73(m,CH2CH3), 1.38(s,Boc), 0.87(t,J=7.4Hz,CH2CH3).

[0045] Example 2: (R) / (S)-2-((tert-butoxycarbonyl)amino)pentanoic acid (compound 4) Using the reaction scheme 6, compound 2 (3.00 g, 25.6 mmol), Boc anhyride (8.82 ml, 38.4 mmol), and NaHCO3 (3.24 g, 38.4 mmol) were reacted to synthesize compound 4 (5.07 g, 91%) as a white powder: f =0.50(DCM 19:Methanol 1 and few drops of acetic acid); 1 H NMR (DMSO-d6,400MHz) 12.38(C(O)OH), 7.03(d,J=8.0Hz,NH), 3.81-3.89(m,NHCHCH2), 1.47-1.64(m,CH2CH3), 1.38(s,Boc), 0.85(t,J=7.4Hz,CH2CH3).

[0046] Example 3: (R) / (S)-tert-butyl (1-((4-hydroxybenzyl)amino)-1-oxobut-2-yl)carbamate (Compound 5) Using Reaction Scheme 7, compound 3 (1.05 g, 5.2 mmol), NMM (0.85 ml, 7.8 mmol), IBCF (0.85 ml, 6.6 mmol), and 4-hydroxybenzylamine (0.76 g, 6.2 mmol) were reacted to synthesize compound 5 (1.27 g, 80%) as a pale pink powder: R f =0.35(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz)9.27(s,Ar-OH), 8.15(t,J=5.7Hz,C(O)NH), 7.03(d,J=8.1Hz,ArH), 6.67(d,J=8.0Hz,ArH), 6.78(d,J=8. 0Hz,Boc-NH), 4.08-4.22(m,NHCH2), 3.79-3.87(m,NHCHCH2), 1.45-1.67(m,CH2CH3), 1.38(s,Boc), 0.82(t,J=7.3Hz,CH2CH3).

[0047] Example 4: (R) / (S)-tert-butyl(1-((4-hydroxybenzyl)amino)-1-oxopent-2-yl)carbamate (Compound 6) Using Reaction Scheme 7, compound 4 (4.50 g, 20.7 mmol), NMM (3.41 ml, 31.1 mmol), IBCF (3.41 ml, 26.3 mmol), and 4-hydroxybenzylamine (3.06 g, 24.9 mmol) were reacted to synthesize compound 6 (4.09 g, 61%) as a pale pink powder: R f =0.45(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz)9.27(s,Ar-OH), 8.15(t,J=5.7Hz,Boc-NH), 7.03(d,J=8.1Hz,ArH), 6.67(d,J=8.0Hz,ArH), 6.78(d,J=8. 0Hz,C(O)NH), 4.08-4.22(m,NHCH2), 3.79-3.87(m,NHCHCH2), 1.45-1.67(m,CH2CH3), 1.38(s,Boc), 0.82(t,J=7.3Hz,CH2CH3).

[0048] Example 5: (R) / (S)-tert-butyl(1-((4-((2-fluorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (Compound 7) Using the reaction scheme 8, compound 5 (0.10 g, 0.3 mmol), 2-fluorobenzyl bromide (0.07 g, 0.4 mmol), and K2CO3 (0.18 g, 1.3 mmol) were reacted to synthesize white powder 7 (0.11 g, 81%): R f =0.60(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz) 8.22(t,J=5.9Hz,C(O)NH), 7.50-7.57(m,ArH), 7.38-7.45(m,ArH), 7.15-7.28(m,ArH), 6.93-6.98(m,ArH), 6.78-6.83 (m,Boc-NH), 5.11(s,OCH2), 4.14-4.27(m,NHCH2), 3.80-3.87(m,NHCHCH2), 1.46-1.67(m,CH2CH3), 1.38(s,Boc), 0.82(t,J=7.4Hz,CH2CH3).

[0049] Example 6: (R) / (S)-tert-butyl(1-((4-((3-fluorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (Compound 8) Using the reaction scheme 8, compound 5 (0.20 g, 1.0 mmol), 3-fluorobenzyl bromide (0.27 g, 1.2 mmol), and K2CO3 (0.55 g, 4.0 mmol) were reacted to synthesize 8 (0.16 g, 39%) as a white powder: R f =0.50(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6, 400 MHz) 1H NMR (DMSO-d6, 400MHz) 8.21(t,J=5.8Hz,C(O)NH), 7.39-7.47(m,ArH), 7.23-7.29(m,ArH), 7.11-7.20(m,ArH), 6.91-6.97(m,ArH), 6.80(d,J=8 .0Hz,Boc-NH), 5.11(s,OCH2), 4.13-4.27(m,NHCH2), 3.80-3.87(m,NHCHCH2), 1.45-1.67(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.3Hz,CH2CH).

[0050] Example 7: (R) / (S)-tert-butyl(1-((4-((4-fluorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (Compound 9) Using the reaction scheme 8, compound 5 (0.20 g, 0.7 mmol), 4-fluorobenzyl bromide (0.09 g, 0.7 mmol), and K2CO3 (0.36 g, 2.6 mmol) were reacted to synthesize white powder 9 (0.24 g, 89%): R f =0.63(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6, 400 MHz) 1 H NMR (DMSO-d6, 400MHz) 8.21(t,J=4.7Hz,C(O)NH), 7.44-7.53(m,ArH), 7.13-7.26(m,ArH), 6.90-6.97(m,ArH), 6.80(d,J=7.7Hz,Boc- NH), 5.06(s,OCH2), 4.13-4.28(m,NHCH2), 3.79-3.89(m,NHCHCH2), 1.45-1.69(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.0Hz,CH2CH3).

[0051] Example 8: (R) / (S)-tert-butyl(1-oxo-1-((4-((2-(trifluoromethyl)benzyl)oxy)benzyl)amino)but-2-yl)carbamate (Compound 10) Using the reaction scheme 8, compound 5 (0.10 g, 0.3 mmol), 2-(trifluoromethyl)benzyl bromide (0.08 g, 0.4 mmol), and K2CO3 (0.18 g, 1.3 mmol) were reacted to synthesize white powder 10 (0.13 g, 89%): R f =0.63(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz) 8.22(t,J=5.7Hz,C(O)NH), 7.68-7.83(m,ArH), 7.55-7.62(m,ArH), 7.16-7.23(m,ArH), 6.91-6.97(m,ArH), 6.80(d,J=7 .9Hz,Boc-NH), 5.21(s,OCH2), 4.14-4.29(m,NHCH2), 3.80-3.88(m,NHCHCH2), 1.47-1.69(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.2Hz,CH2CH3).

[0052] Example 9: (R) / (S)-tert-butyl(1-oxo-1-((4-((3-(trifluoromethyl)benzyl)oxy)benzyl)amino)but-2-yl)carbamate (Compound 11) Using the reaction scheme 8, compound 5 (0.30 g, 1.0 mmol), 3-(trifluoromethyl)benzyl bromide (0.17 ml, 1.1 mmol), and K2CO3 (0.54 g, 3.9 mmol) were reacted to synthesize 11 (0.20 g, 44%) as a white powder: R f =0.54(EtOAc 1: n-hexane 1) 1 H NMR (DMSO-d6,400MHz) 8.22(t,J=5.7Hz,C(O)NH), 7.57-7.82(m,ArH), 7.11-7.21(m,ArH), 6.90-6.99(m,ArH), 6.80(d,J=8.1Hz,Boc -NH), 5.20(s,OCH2), 4.14-4.28(m,NHCH2), 3.78-3.88(m,NHCHCH2), 1.44-1.68(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.2Hz,CH2CH3)

[0053] Example 10: (R) / (S)-tert-butyl (1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)propan-2-yl)carbamate (Compound 12) Using the reaction scheme 8, compound 5 (0.30 g, 1.0 mmol), 4-(trifluoromethyl)benzyl bromide (0.17 ml, 1.1 mmol), and K2CO3 (0.54 g, 3.9 mmol) were reacted to synthesize compound 12 (0.44 g, 96%) as a white powder: R f =0.24(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz) 8.21(t,J=5.8Hz,C(O)NH), 7.62-7.80(m,ArH), 7.14-7.21(m,ArH), 6.91-6.98(m,ArH), 6.80(d,J=7.9Hz,Boc- NH), 5.21(s,OCH2), 4.13-4.28(m,NHCH2), 3.80-3.87(m,NHCHCH2), 1.46-1.67(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.3Hz,CH2CH3).

[0054] Example 11: (R) / (S)-tert-butyl(1-((4-((3-chlorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (Compound 13) Using Reaction Scheme 8, compound 5 (0.30 g, 1.0 mmol), 3-chlorobenzyl bromide (0.22 g, 1.1 mmol), and K2CO3 (0.54 g, 3.9 mmol) were reacted to synthesize compound 13 (0.38 g, 89%) as a white powder: R f =0.54(EtOAc1:n-hexane1); 1H NMR (DMSO-d6,400MHz) 8.21(t,J=5.9Hz,C(O)NH), 7.48-7.51(m,ArH), 7.35-7.45(m,ArH), 7.15-7.19(m,ArH), 6.91-6.96(m,ArH), 6.80(d,J=8 .1Hz,Boc-NH), 5.21(s,OCH2), 4.13-4.27(m,NHCH2), 3.80-3.88(m,NHCHCH2), 1.45-1.67(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.4Hz,CH2CH3)

[0055] Example 12: (R) / (S)-tert-butyl(1-((4-((4-chlorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (Compound 14) Using Reaction Scheme 8, compound 5 (0.20 g, 0.7 mmol), 4-chlorobenzyl bromide (0.09 ml, 0.7 mmol), and K2CO3 (0.36 g, 2.6 mmol) were reacted to synthesize a white powdery compound (0.28 g, 100%): R f =0.50(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz) 8.21(t,J=5.9Hz,C(O)NH), 7.42-7.52(m,ArH), 7.13-7.20(m,ArH), 6.90-6.95(m,ArH), 6.80(d,J=8.0Hz,Boc -NH), 5.08(s,OCH2), 4.13-4.27(m,NHCH2), 3.79-3.87(m,NHCHCH2), 1.45-1.68(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.3Hz,CH2CH3)

[0056] Example 13: (R) / (S)-tert-butyl(1-((4-((3-fluorobenzyl)oxy)benzyl)amino)-1-oxopent-2-yl)carbamate (Compound 15) Using Reaction Scheme 8, compound 6 (0.50 g, 1.6 mmol), 3-fluorobenzyl bromide (0.31 g, 1.6 mmol), and K2CO3 (0.86 g, 6.2 mmol) were reacted to synthesize a white powdery compound (0.62 g, 92%): R f =0.33(EtOAc1:n-hexane3); 1 H NMR (DMSO-d6,400MHz) 8.20(t,J=5.8Hz,C(O)NH), 7.39-7.46(m,ArH), 7.23-7.2 9(m,ArH), 7.11-7.18(m,ArH), 6.91-6.96(m,ArH), 6.82(d,J=8.0Hz,Boc-NH), 5 .11(s,OCH2), 4.13-4.25(m,NHCH2), 3.86-3.94(m,NHCHCH2), 1.41-1.60(m,CH2 CH2CH3), 1.37(s,Boc), 1.20-1.32(m,CH2CH2CH3), 0.84(t,J=7.3Hz,CH2CH2CH3) 。

[0057] Example 14: (R) / (S)-tert-butyl(1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)pent-2-yl)carbamate (Compound 16) Using the reaction scheme 8, compound 6 (0.50 g, 1.6 mmol), 4-(trifluoromethyl)benzyl bromide (0.39 g, 1.6 mmol), and K2CO3 (0.86 g, 6.2 mmol) were reacted to synthesize a white powdery compound (0.70 g, 94%): R f =0.37(EtOAc1:n-hexane3); 1H NMR (DMSO-d6,400MHz) 8.20(t,J=5.9Hz,C(O)NH), 7.63-7.78(m,ArH), 7.13-7.19(m,ArH), 6.92-6.97(m,ArH), 6.82(d,J=8.2Hz,Boc-NH), 5.21(s,OCH) 2), 4.13-4.25(m,NHCH2), 3.86-3.94(m,NHCHCH2), 1.41-1.60(m,CH2CH2CH3), 1.37(s,Boc), 1.18-1.32(m,CH2CH2CH3), 0.84(t,J=7.3Hz,CH2CH2CH3).

[0058] Example 15: (R) / (S)-1-((4-((2-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (Compound 17) Using the reaction scheme 9, compound 7 (0.09 g, 0.22 mmol) was reacted with 4.0 M HCl in dioxane (0.38 ml, 10.8 mmol) to synthesize compound 17 (0.02 g, 22%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.85(t,J=5.3Hz,C(O)NH),8.06(broad,NH3 + Cl - ), 7.38-7.57(m,ArH), 7.20-7.29(m,ArH), 6.97-7.02(m,ArH), 5.13(s,OCH2), 4.22-4.34 (m,NHCH2), 3.66-3.72(m,NH3CHCH2), 1.70-1.80(m,CH2CH3), 0.87(t,J=7.5Hz,CH2CH3).

[0059] Example 16: (R) / (S)-1-((4-((3-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (Compound 18) Using the reaction scheme 9, compound 8 (0.14 g, 0.36 mmol) was reacted with 4.0 M HCl in dioxane (0.63 ml, 18.0 mmol) to synthesize compound 18 (0.10 g, 88%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.85(t,J=5.3Hz,C(O)NH),8.06(broad,NH3 + Cl - ), 7.38-7.57(m,ArH), 7.20-7.29(m,ArH), 6.97-7.02(m,ArH), 5.13(s,OCH2), 4.22-4.34 (m,NHCH2), 3.66-3.72(m,NH3CHCH2), 1.70-1.80(m,CH2CH3), 0.87(t,J=7.5Hz,CH2CH3).

[0060] Example 17: (R) / (S)-1-((4-((4-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (Compound 19) Using Reaction Scheme 9, compound 9 (0.14 g, 0.36 mmol) was reacted with 4.0 M HCl in dioxane (0.63 ml, 18.0 mmol) to synthesize compound 19 (0.10 g, 88%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.86(t,J=5.7Hz,C(O)NH),8.07(broad,NH3 + Cl - ), 7.63-7.82(m,ArH), 7.20-7.24(m,ArH), 6.97-7.03(m,ArH), 5.13(s,OCH2), 4.21-4.34 (m,NHCH2), 3.66-3.72(m,NH3CHCH2), 1.70-1.82(m,CH2CH3), 0.86(t,J=7.5Hz,CH2CH3).

[0061] Example 18: (R) / (S)-1-Oxo-1-((4-((2-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (Compound 20) Using Reaction Scheme 9, compound 10 (0.10 g, 0.21 mmol) was reacted with 4.0 M HCl in dioxane (0.37 ml, 10.7 mmol) to synthesize compound 20 (0.09 g, 99%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.88(t,J=5.6Hz,C(O)NH),8.15(broad,NH3 + Cl - ), 7.69-7.83(m,ArH), 7.56-7.62(m,ArH), 7.22-7.27(m,ArH), 6.96-7.02(m,ArH), 5.22(s,OCH2), 4.22-4.34(m,NHCH2), 3.68-3.74(m,NH3CHCH2), 1.72-1.81(m,CH2CH3), 0.87(t,J=7.4Hz,CH2CH3).

[0062] Example 19: (R) / (S)-1-Oxo-1-((4-((3-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (Compound 21) Using Reaction Scheme 9, compound 11 (0.09 g, 0.19 mmol) was reacted with 4.0 M HCl in dioxane (0.33 ml, 9.7 mmol) to synthesize compound 21 (0.07 g, 93%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.86(t,J=5.7Hz,C(O)NH),8.09(broad,NH3 + Cl -), 7.61-7.82(m,ArH), 7.20-7.25(m,ArH), 6.98-7.03(m,ArH), 5.21(s,OCH2), 4.22-4.34 (m,NHCH2), 3.67-3.72(m,NH3CHCH2), 1.70-1.80(m,CH2CH3), 0.86(t,J=7.5Hz,CH2CH3).

[0063] Example 20: (R) / (S)-1-Oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (Compound 22) Using Reaction Scheme 9, compound 12 (0.40 g, 0.86 mmol) was reacted with 4.0 M HCl in dioxane (1.49 ml, 42.8 mmol) to synthesize compound 22 (0.33 g, 97%) as a white powder; R f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.89(t,J=5.7Hz,C(O)NH),8.09(broad,NH3 + Cl - ), 7.61-7.82(m,ArH), 7.20-7.25(m,ArH), 6.98-7.03(m,ArH), 5.21(s,OCH2), 4.22-4.34 (m,NHCH2), 3.67-3.72(m,NH3CHCH2), 1.70-1.80(m,CH2CH3), 0.86(t,J=7.5Hz,CH2CH3).

[0064] Manufacturing Example 21 (R) / (S)-1-((4-((3-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (Compound 23) Using the reaction scheme 9, compound 13 (0.30 g, 0.7 mmol) was reacted with 4.0 M HCl in dioxane (1.20 ml, 34.7 mmol) to synthesize compound 23 (0.25 g, 97%) as a white powder; R f =0.00(EtOAc9:Acetone1); 1H NMR(DMSO-d6,400MHz)8.91(t,J=5.7Hz,C(O)NH),8.20(broad,NH3 + Cl - ), 7.48-7.51(m,ArH), 7.36-7.45(m,ArH), 7.19-7.24(m,ArH), 6.95-7.00(m,ArH), 5.12(s,OCH2), 4.21-4.33(m,NHCH2), 3.69-3.74(m,NH3CHCH2), 1.71-1.81(m,CH2CH3), 0.86(t,J=7.5Hz,CH2CH3).

[0065] Example 22: (R) / (S)-1-((4-((4-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (Compound 24) Using the reaction scheme 9, compound 14 (0.10 g, 0.2 mmol) was reacted with 4.0 M HCl in dioxane (0.40 ml, 11.5 mmol) to synthesize compound 24 (0.08 g, 98%) as a white powder; R f =0.00(EtOAc9:Acetone1); 1 H NMR (DMSO-d6,400MHz)8.86(t,J=5.8Hz,C(O)NH), 8.15(broad,NH3 + Cl - ), 7.44-7.46(m,ArH), 7.19-7.24(m,ArH), 6.95-7.00(m,ArH), 5.10(s,OCH2), 4.21-4.33 (m,NHCH2), 3.67-3.73(m,NH3CHCH2), 1.70-1.80(m,CH2CH3), 0.86(t,J=7.5Hz,CH2CH3).

[0066] Example 23: (R) / (S)-1-((4-((3-fluorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (Compound 25) Using Reaction Scheme 9, compound 15 (0.55 g, 1.3 mmol) was reacted with 4.0 M HCl in dioxane (2.22 ml, 64.0 mmol) to synthesize compound 25 (0.43 g, 92%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.90(t,J=5.7Hz,C(O)NH),8.16(broad,NH3 + Cl - ), 7.40-7.47(m,ArH), 7.11-7.30(m,ArH), 6.96-7.01(m,ArH), 5.13(s,OCH2), 4.24-4.30(m,NHCH2), 3.71 -3.77(m,NH3CHCH2), 1.65-1.73(m,CH2CH2CH3), 1.23-1.35(m,CH2CH2CH3), 0.86(t,J=7.3Hz,CH2CH2CH3).

[0067] Example 24: (R) / (S)-1-((4-((4-(trifluorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (Compound 26) Using Reaction Scheme 8, compound 16 (0.65 g, 1.4 mmol) was reacted with 4.0 M HCl in dioxane (2.34 ml, 67.5 mmol) to synthesize compound 26 (0.39 g, 70%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.89(t,J=5.7Hz,C(O)NH),8.15(broad,NH3 + Cl - ), 7.63-7.79(m,ArH), 7.20-7.25(m,ArH), 6.96-7.02(m,ArH), 5.23(s,OCH2), 4.25-4.29(m,NHCH2), 3.71 -3.77(m,NH3CHCH2), 1.65-1.72(m,CH2CH2CH3), 1.23-1.35(m,CH2CH2CH3), 0.86(t,J=7.3Hz,CH2CH2CH3).

[0068] Example 25: (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (Compound 27) Using Reaction Scheme 10, compound 12 (0.40 g, 0.86 mmol), NaH (10 equivalents), and CHCl (10 equivalents) were reacted to obtain a methylated compound, and the BoC group was removed using 4.0 M HCl in dioxane (1.49 ml, 42.8 mmol) to synthesize compound 27 (0.30 g); 1 H NMR(DMSO-d6,400MHz)8.89(t,J=5.7Hz,C(O)NH),8.09(broad,NH3 + Cl - ), 7.61-7.82(m,ArH), 7.20-7.25(m,ArH), 6.98-7.03(m,ArH), 5.18(s,OCH2), 4.22-4.34(m,NHC H2), 3.67-3.72(m,NHCHCH2), 2.39(s,NHCH3), 1.70-1.80(m,CH2CH3), 0.86(t,J=7.5Hz,CH2CH3).

[0069] Example 26: (R) / (S)-N-(4-((3-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (Compound 28) Using Reaction Scheme 10, compound 13 (1 equivalent), NaH (10 equivalents), and CHCl (10 equivalents) were reacted to obtain a methylated compound, and the BoC group was removed using 4.0 M HCl in dioxane (6 equivalents) to synthesize compound 28. 1 H NMR(DMSO-d6,400MHz)8.92(t,J=5.7Hz,C(O)NH), 8.19(broad,NH3 + Cl -), 7.40-7.49(m,ArH), 7.11-7.32(m,ArH), 6.96-7.03(m,ArH), 5.14(s,OCH2), 4.24-4.31(m,NHC H2), 3.72-3.77(m,NHCHCH2), 2.38(s,NHCH3), 1.68-1.79(m,CH2CH3), 0.87(t,J=7.3Hz,CH2CH3).

[0070] Example 27: (R) / (S)-N-(4-((4-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (Compound 29) Using Reaction Scheme 10, compound 14 (1 equivalent), NaH (10 equivalents), and CHCl (10 equivalents) were reacted to obtain a methylated compound, and the BoC group was removed using 4.0 M HCl in dioxane (6 equivalents) to synthesize compound 29. 1 H NMR(DMSO-d6,400MHz)8.88(t,J=5.7Hz,C(O)NH),8.07(broad,NH3 + Cl - ), 7.62-7.81(m,ArH), 7.21-7.26(m,ArH), 6.97-7.04(m,ArH), 5.13(s,OCH2), 4.22-4.31(m,NHC H2), 3.71-3.77(m,NHCHCH2), 2.40(s,NHCH3), 1.69-1.79(m,CH2CH3), 0.88(t,J=7.3Hz,CH2CH3).

[0071] Manufacturing Example 28 (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (Compound 30) Using Reaction Scheme 10, compound 12 (0.40 g, 0.86 mmol), NaH (10 equivalents), and CHCl (10 equivalents) were reacted to obtain a methylated compound, and the BoC group was removed using 4.0 M HCl in dioxane (1.49 ml, 42.8 mmol) to synthesize compound 27 (0.30 g). 1H NMR(DMSO-d6,400MHz)8.89(t,J=5.7Hz,C(O)NH),8.09(broad,NH3 + Cl - ), 7.61-7.82(m,ArH), 7.20-7.25(m,ArH), 6.98-7.03(m,ArH), 5.15(s,OCH2), 4.22-4.34(m,NHCH2), 3.67-3.72(m ,NHCHCH2), 2.39(s,NHCH3), 1.63-1.72(m,CH2CH2CH3), 1.22-1.35(m,CH2CH2CH3), 0.87(t,J=7.3Hz,CH2CH2CH3).

[0072] Example 29: (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (Compound 31) Using Reaction Scheme 11, compound 26 (0.31 g, 0.7 mmol), triethylamine (0.10 ml, 0.7 mmol), formaldehyde (0.15 ml), and a palladium catalyst (0.15 g) were reacted to synthesize compound 31 as a white powder. 1 H NMR (DMSO-d6,400MHz) 8.22(t,J=539Hz,C(O)NH), 7.63-7.79(m,ArH), 7.17-7.22(m,ArH), 6.94-6.99(m,ArH), 5.21(s,OCH2), 4.19-4.2 4(m,NHCH2), 2.70-2.76(NCH), 2.18(s,N(CH3)2), 1.65-1.73(m,CH2CH2CH3), 1.20-1.35(m,CH2CH2CH3), 0.86(t,J=7.4Hz,CH2CH2CH3).

[0073] Example 30: (R) / (S)-tert-Butyl-2-((4-((3-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 34) Using the reaction scheme 11, compound 33 (2.00 g, 9.3 mmol), 3-fluorobenzyl bromide (2.63 g, 11.2 mmol), and K2CO3 (5.14 g, 37.2 mmol) were reacted to synthesize compound 34 (3.91 g, 98%) as a white powder: R f =0.27(EtOH 1:n-hexane 5); 1 H NMR (DMSO-d6,400MHz) 8.27(t,J=5.8Hz,C(O)NH), 7.35-7.50(m,ArH), 7.20-7.30(m,ArH), 7.10-7.20(m,ArH), 6.93(d,J=7.8Hz) ,Boc-NH), 5.12(s,OCH2), 4.00-4.30(m,NCH,NHCH2), 3.02-3.45(NCHCH2CH2CH2), 1.70-2.15(m,NCHCH2CH2CH2), 1.30(s,Boc).

[0074] Preparation 31: (R) / (S)-tert-butyl-2-((4-((4-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 35) Using the above-mentioned Reaction Scheme 11, compound 33, 4-fluorobenzyl bromide, and K2CO3 were reacted to synthesize compound 35 as a white powder; 1 H NMR (DMSO-d6,400MHz) 8.27(t,J=5.8Hz,C(O)NH), 7.35-7.45(m,ArH), 7.15-7.25(m,ArH), 7.05-7.10(m,ArH), 6.85 6.95(m,ArH), 5.01(s,OCH2), 4.20-4.60(m,NCH,NHCH2), 2.10-2.50(NCHCH2CH2CH2), 1.80-200(m,NCHCH2CH2CH2), 1.25(s,Boc).

[0075] Preparation 32: (R) / (S)-tert-Butyl-2-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 36) Using the above-mentioned Reaction Scheme 11, compound 33, 4-(trifluoromethyl)benzyl bromide, and K2CO3 were reacted to synthesize compound 36 as a white powder; 1 H NMR (DMSO-d6,400MHz) 8.25(t,J=5.7Hz,C(O)NH), 7.70-7.60(m,ArH), 7.60-7.50(m,ArH), 7.15-7.25(m,ArH), 6.85-6.95(m ,ArH), 5.11(s,OCH2), 4.20-4.60(m,NCH,NHCH2), 2.10-2.40(NCHCH2CH2CH2), 1.80-1.95(m,NCHCH2CH2CH2), 1.25(s,Boc).

[0076] Preparation 33: (R) / (S)-tert-Butyl-2-((4-((4-chlorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 37) Using the above Reaction Scheme 11, compound 33, 4-chlorobenzyl bromide, and K2CO3 were reacted to synthesize compound 37 as a white powder; 1 H NMR (DMSO-d6,400MHz) 8.28(t,J=5.9Hz,C(O)NH), 7.70-7.60(m,ArH), 7.60-7.50(m,ArH), 7.15-7.25(m,ArH), 6.85-6.95(m ,ArH), 5.11(s,OCH2), 4.20-4.60(m,NCH,NHCH2), 2.10-2.40(NCHCH2CH2CH2), 1.80-1.95(m,NCHCH2CH2CH2), 1.27(s,Boc).

[0077] Preparation 34: (R) / (S)—N-(4-((3-fluorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (Compound 38) Using Reaction Scheme 9, compound 34 (1.00 g, 2.3 mmol) was reacted with 4.0 M HCl in dioxane (7.32 ml, 233.0 mmol) to synthesize compound 38 (0.77 g, 100%) as a white powder: f=0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.99(t,J=5.5Hz,C(O)NH),8.55(broad,NH2 + Cl - ), 7.39-7.47(m,ArH), 7.23-7.30(m,ArH), 7.18-7.23(m,ArH), 7.10-7.18(m,ArH), 6.92-7.01(m ,ArH), 5.13(s,OCH2), 4.23-4.30(m,NHCH2), 4.11-4.19(m,NHCH), 3.14-3.27(m,NHCHCH2CH2CH2) 、 2.24-2.34(m,NHCHCH2CHH'CH2), 1.77-1.93(m,NHCHCH2CHH'CH2).

[0078] Example 35: (R) / (S)—N-(4-((4-fluorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (Compound 39) Using Reaction Scheme 9, compound 35 (1.00 g, 2.3 mmol) was reacted with 4.0 M HCl in dioxane (4.05 ml, 117.0 mmol) to synthesize compound 39 (0.84 g, 99%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR (DMSO-d6,400MHz)9.04(t,J=5.7Hz,C(O)NH), 8.55(broad,NH2 + Cl - ), 7.46-7.53(m,ArH), 7.18-7.26(m,ArH), 6.95-7.01(m,ArH), 5.08(s,OCH2), 4.25-4.31(m,NHCH2), 4.12-4. 21(m,NHCH), 3.13-3.29(m,NHCHCH2CHHCH2), 2.25-2.35(m,NHCHCH2CHHCH2), 1.77-1.94(m,NHCHCH2CHHCH2).

[0079] Example 36: (R) / (S)—N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (Compound 40) Using Reaction Scheme 9, compound 36 (1.00 g, 2.09 mmol) was reacted with 4.0 M HCl in dioxane (3.63 ml, 104.0 mmol) to synthesize compound 40 (0.85 g, 98%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.98(t,J=5.8Hz,C(O)NH), 8.56(broad,NH2 + Cl - ), 7.63-7.79(m,ArH), 7.19-7.25(m,ArH), 6.97-7.03(m,ArH), 5.23(s,OCH2), 4.26-4.30(m,NHCH2), 4.12-4.1 8(m,NHCH), 3.13-3.27(m,NHCHCH2CH2CH2), 2.23-2.35(m,NHCHCH2CHH'CH2), 1.77-1.93(m,NHCHCH2CHH'CH2).

[0080] Preparation 37: (R) / (S)-N-(4-((4-chlorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide hydrochloride (Compound 41) Using Reaction Scheme 9, compound 37 (1.00 g, 2.09 mmol) was reacted with 4.0 M HCl in dioxane (3.63 ml, 104.0 mmol) to synthesize compound 41 (0.85 g, 98%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR (DMSO-d6,400MHz)8.97(t,J=5.4Hz,C(O)NH), 8.57(broad,NH2 + Cl -), 7.40-7.48(m,ArH), 7.20-7.29(m,ArH), 7.18-7.23(m,ArH), 7.08-7.18(m,ArH), 6.95-7.05(m,ArH), 5.11(s,OCH2), 4.20-4.31( m,NHCH2), 4.09-4.19(m,NHCH), 3.14-3.27(m,NHCHCH2CH2CH2), 2.25-2.36(m,NHCHCH2CHH'CH2), 1.70-1.83(m,NHCHCH2CHH'CH2).

[0081] Preparation 38: (R) / (S)-1-Methyl-N-(4-((3-fluorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide (Compound 42) Using the reaction scheme 11, compound 38 (0.31 g, 0.7 mmol), formaldehyde (0.15 ml), and a palladium catalyst (0.15 g) were reacted to synthesize compound 42 as a white powder. 1 H NMR (DMSO-d6, 400MHz)7.57(t,J=5.2Hz,C(O)NH), 7.36-7.43(m,ArH), 7.17-7.28(m, ArH), 7.01-7.08(m,ArH), 6.95-7.00(m,ArH), 5.05(s,OCH2), 4.33-4.37(m,NHCH2), 3.10-3.17(m,NCHCHHCH2CH2), 2.85-2.91(m,NCHCHHCH2CH2), 2.34(s,NCH3), 2.17-2 .32(m,NCHCH2CH2CH2), 1.82-1.92(m,NCHCH2CH2CH2), 1.67-1.82(m,NCHCH2CH2CH2).

[0082] Example 39: (R) / (S)-1-Methyl-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pyrrolidine-2-carboxamide (Compound 43) Using Reaction Scheme 11, compound 40 (0.31 g, 0.7 mmol), triethylamine (0.10 ml, 0.7 mmol), formaldehyde (0.15 ml), and a palladium catalyst (0.15 g) were reacted to synthesize compound 43 as a white powder. 1 H NMR (DMSO-d6,400MHz)7.62-7.67(m,ArH), 7.52-7.62(t,J=5.4Hz,C(O)NH,m,ArH), 7.18-7.23(m,ArH)), 6.90-6.96(m,ArH), 5.12(s,OCH2), 4.33-4.45 (m,NHCH2), 3.00-3.10(m,NCHCH2CH2CH2), 2.90-2.98(m,NCHCHHCH2CH2), 2.37(s,NCH3), 2.30-2.40(m,NCHCH2CH2CH2), 2.18-2.30(m,NCHCHHCH2CH2).

[0083] Example 40: (R) / (S)-1-Methyl-N-(4-((4-chlorobenzyl)oxy)benzyl)pyrrolidine-2-carboxamide (Compound 44) Using Reaction Scheme 11, compound 41 (0.31 g, 0.7 mmol), formaldehyde (0.15 ml), and a palladium catalyst (0.15 g) were reacted to synthesize compound 44 as a white powder. 1 H NMR (DMSO-d6, 400MHz) 7.75(t,J=5.4Hz,C(O)NH), 7.60-7.67(m,ArH), 7.52- 7.62(m,ArH), 7.18-7.23(m,ArH), 6.90-7.00(m,ArH), 5.11(s,OCH2), 4.34-4 .45(m,NHCH2), 3.00-3.09(m,NCHCH2CH2CH2), 2.90-2.99(m,NCHCHHCH2CH2), 2.35(s,NCH3), 2.28-2.40(m,NCHCH2CH2CH2), 2.18-2.30(m,NCHCHHCH2CH2).

[0084] Manufacturing Example 41 (R) / (S)-tert-Butyl-3-((4((3-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 46) Using Reaction Scheme 7, compound 45 (1.00 g, 4.6 mmol), NMM (0.77 ml, 7.0 mmol), IBCF (0.76 ml, 5.8 mmol), and 4-[(3-fluorophenyl)methoxy]-benzenemethanamine hydrochloride (1.47 g, 5.5 mmol) were reacted to synthesize compound 46 (1.97 g, 99%) as a white powder: R f =0.30(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6, 400MHz) 7.57(t,J=5.2Hz,C(O)NH), 7.32-7.39(m,ArH), 7.11-7.24(m,ArH), 6.89-6.94(m,ArH), 5.07(s,OCH2), 4 .33-4.37(m,NHCH2), 3.21-3.68(m,NCH2CHCH2CH2), 2.85-2.98(m,NCH2CHCH2CH2, 2.00-2.12(m,NCH2CHCH2CH2), 1.45(s,Boc).

[0085] Manufacturing Example 42 (R) / (S)-tert-Butyl-3-((4((4-(trifluoromethyl)benzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 47) Using Reaction Scheme 7, compound 45 (1.00 g, 4.6 mmol), NMM (0.77 ml, 7.0 mmol), IBCF (0.76 ml, 5.8 mmol), and 4-[(4-(trifluoromethyl)phenyl)methoxy]-benzenemethanamine hydrochloride (1.75 g, 5.5 mmol) were reacted to synthesize compound 47 (2.20 g, 99%) as a white powder: R f =0.33(EtOAc1:n-hexane1); 1 H NMR (DMSO-d 6、400MHz)7.78(t,J=5.3Hz,C(O)NH), 7.63-7.67(m,ArH), 7.52-7.57(m,ArH), 7.18-.23(m,ArH), 6.90-6.95(m,ArH), 5.12(s,OCH2), 4.37-4.42(m, NHCH2), 3.53-3.67(m,NCH2CHCH2CH2), 3.47-3.53(m,NCH2CHCH2CH2), 2.78-2.89(m,NCH2CHCH2CH2), 2.06-2.13(m,NCH2CHCH2CH2), 1.45(s,Boc).

[0086] Preparation 43: (R) / (S)-tert-Butyl-3-((4((4-chlorobenzyl)oxy)benzyl)carbamoyl)pyrrolidine-1-carboxylate (Compound 48) Using Reaction Scheme 7, compound 45 (1.00 g, 4.6 mmol), NMM (0.77 ml, 7.0 mmol), IBCF (0.76 ml, 5.8 mmol), and 4-[(4-(trifluoromethyl)phenyl)methoxy]-benzenemethanamine hydrochloride (1.75 g, 5.5 mmol) were reacted to synthesize compound 48 (2.20 g, 99%) as a white powder: R f =0.33(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz)7.80(t,J=5.4Hz,C(O)NH), 7.63-7.67(m,ArH), 7.52- 7.57(m,ArH), 7.18-7.23(m,ArH), 6.90-6.95(m,ArH), 5.12(s,OCH2), 4.37-4 .42(m,NHCH2), 3.53-3.67(m,NCH2CHCH2CH2), 3.47-3.53(m,NCH2CHCH2CH2), 2.78-2.89(m,NCH2CHCH2CH2), 2.06-2.13(m,NCH2CHCH2CH2), 1.45(s,Boc).

[0087] Preparation 44: (R) / (S)-2-((4-((3-fluorobenzyl)oxy)benzyl)carbamoyl)pyrrolidin-1-nium chloride (Compound 49) Using Reaction Scheme 9, compound 46 (1.00 g, 2.3 mmol) was reacted with 4.0 M HCl in dioxane (4.05 ml, 116.7 mmol) to synthesize compound 49 (0.84 g, 99%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)9.13(broad,NH2 + Cl - ), 8.65(t,J=5.6Hz,C(O)NH), 7.40-7.48(m,ArH), 7.23-7.30(m,ArH), 7.12-7.21(m,ArH), 6.94-7.00(m,ArH), 5.12(s,OCH2), 4.15-4.27(m,NHCH 2) , 3.04-3.32(m,NHCH2CHCH2CH2), 1.88-2.20(m,NHCH2CHCH2CH2).

[0088] Example 45: (R) / (S)-2-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)carbamoyl)pyrrolidin-1-nium chloride (Compound 50) Using Reaction Scheme 9, compound 47 (1.00 g, 2.09 mmol) was reacted with 4.0 M HCl in dioxane (3.63 ml, 104.5 mmol) to synthesize compound 50 (0.86 g, 99%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)9.13(broad,NH2 + Cl -), 8.65(t,J=5.6Hz,C(O)NH), 7.60-7.71(m,ArH), 7.20-7.27(m,ArH), 6.95-7.01(m,ArH), 5.20 (s,OCH2), 4.28-4.35(m,NHCH2), 3.36-3.56(m,NHCH2CHCH2CH2), 2.27-2.39(m,NHCH2CHCH2CH2) 、 2.10-2.20(m,NHCH2CHCH2CH2).

[0089] Example 46: (R) / (S)-2-((4-((4-chlorobenzyl)oxy)benzyl)carbamoyl)pyrrolidin-1-nium chloride (Compound 51) Using Reaction Scheme 9, compound 48 (1.00 g, 2.09 mmol) was reacted with 4.0 M HCl in dioxane (3.63 ml, 104.5 mmol) to synthesize compound 51 (0.86 g, 99%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)9.12(broad,NH2 + Cl - ), 8.63(t,J=5.6Hz,C(O)NH), 7.60-7.70(m,ArH), 7.20-7.29(m,ArH), 6.92-7.01(m,ArH), 5.18(s,OCH2), 4.27- 4.35(m,NHCH2), 3.38-3.56(m,NHCH2CHCH2CH2), 2.25-2.40(m,NHCH2CHCH2CH2), 2.10-2.20(m,NHCH2CHCH2CH2).

[0090] Preparation 47: (R) / (S)-1-Methyl-N-(4-((3-fluorobenzyl)oxy)benzyl)pyrrolidine-3-carboxamide (Compound 52) Using Reaction Scheme 11, compound 49 (0.14 g, 0.4 mmol), formaldehyde (0.03 ml), and a palladium catalyst (0.07 g) were reacted to synthesize compound 52 as a white powder. 1H NMR (DMSO-d6,400MHz)7.57(t,J=5.2Hz,C(O)NH), 7.36-7.43(m,ArH), 7.16-7. 28(m,ArH), 7.01-7.08(m,ArH), 6.94-6.99(m,ArH), 5.11(s,OCH2), 4.29-4.31 (m,NHCH2), 2.93-3.05(m,NHCH2CHCH2CH2), 2.78-2.85(m,NHCH2CHCH2CH2), 2.50-2.64(m,NHCH2CHCH2CH2), 2.39(s,NCH3), 2.02-2.15(m,m,NHCH2CHCH2CH2).

[0091] Example 48: (R) / (S)-1-Methyl-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pyrrolidine-3-carboxamide (Compound 53) Using the reaction scheme 11, compound 50 (0.14 g, 0.4 mmol), formaldehyde (0.03 ml), and a palladium catalyst (0.07 g) were reacted to synthesize compound 53 as a white powder. 1 H NMR (DMSO-d6,400MHz)7.62-7.69(m,ArH), 7.52-7.57(m,ArH), 7.17-7.23(m,ArH), 7.02(t,J=5.4Hz,C(O)NH), 6.89-6.95(m,ArH), 5.12(s ,OCH2), 4.30-4.45(m,NHCH2), 2.75-2.95(m,NCH2CH2CHCH2), 2.27-2.50(m,NCH2CH2CHCH2), 2.35(s,NCH3), 1.95-2.25(m,NCH2CHCH2CH2).

[0092] Example 49: (R) / (S)-1-Methyl-N-(4-((4-chlorobenzyl)oxy)benzyl)pyrrolidine-3-carboxamide (Compound 54) Using the reaction scheme 11, compound 51 (0.14 g, 0.4 mmol), formaldehyde (0.03 ml), and a palladium catalyst (0.07 g) were reacted to synthesize compound 54 as a white powder. 1H NMR (DMSO-d6,400MHz)7.60-7.69(m,ArH), 7.50-7.57(m,ArH), 7.17-7.25(m,ArH), 7.05(t,J=5.4Hz,C(O)NH), 6.89-6.95(m,ArH), 5.10(s ,OCH2), 4.32-4.43(m,NHCH2), 2.75-2.95(m,NCH2CH2CHCH2), 2.25-2.51(m,NCH2CH2CHCH2), 2.34(s,NCH3), 1.94-2.27(m,NCH2CHCH2CH2).

[0093] Example 50: (R) / (S)-tert-butyl(1-((4-((3,4-chlorobenzyl)oxy)benzyl)amino)-1-oxobut-2-yl)carbamate (Compound 55) Using Reaction Scheme 8, compound 5 (0.20 g, 0.7 mmol), 3,4-chlorobenzyl bromide (0.09 ml, 0.7 mmol), and K2CO3 (0.36 g, 2.6 mmol) were reacted to synthesize compound 55 (0.28 g, 100%) as a white powder: R f =0.50(EtOAc1:n-hexane1); 1 H NMR (DMSO-d6,400MHz) 8.21(t,J=5.9Hz,C(O)NH), 7.42-7.52(m,ArH), 7.13-7.20(m,ArH), 6.90-6.95(m,ArH), 6.80(d,J=8.0Hz,Boc -NH), 5.08(s,OCH2), 4.13-4.27(m,NHCH2), 3.79-3.87(m,NHCHCH2), 1.45-1.6(m,CH2CH3), 1.37(s,Boc), 0.82(t,J=7.3Hz,CH2CH3).

[0094] Preparation 51: (R) / (S)-tert-butyl (1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)pent-2-yl)carbamate (Compound 56) Using Reaction Scheme 8, compound 6 (1.00 g, 3.2 mmol), 3,4-dichlorobenzyl bromide (0.57 ml, 3.9 mmol), and K2CO3 (1.79 g, 13.0 mmol) were reacted to synthesize compound 56 (0.75 g, 50%) as a white powder: R f =0.24(EtOH1:n-hexane2); 1 H NMR (DMSO-d6,400MHz) 8.22(t,J=5.9Hz,C(O)NH), 7.60-7.75(m,ArH), 7.38-7.48(m,ArH), 7.10-7.25(m,ArH), 6.85-6.98(m,ArH), 6.82(d,J=7 .8Hz,Boc-NH), 5.12(s,OCH2), 4.10-4.30(m,NHCH2), 3.78-3.90(m,NHCHCH2), 1.41-1.70(m,CH2CH3), 1.38(s,Boc), 0.87(t,J=7.8Hz,CH2CH3).

[0095] Example 52: (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (Compound 57) Using Reaction Scheme 9, compound 55 (0.29 g, 0.6 mmol) was reacted with 4.0 M HCl in dioxane (1.05 ml, 30.1 mmol) to synthesize compound 57 (0.24 g, 95%) as a white powder: f =0.30(CH2Cl29:MeOH1); 1 H NMR(DMSO-d6,400MHz)8.85(t,J=5.6Hz,C(O)NH),8.10(broad,NH3 + Cl - ), 7.63-7.75(m,ArH), 7.40-7.50(m,ArH), 7.18-7.30(m,ArH), 6.93-7.03(m,ArH), 5.13(s,OCH2), 4.20-4.38(m,NHCH2), 3.65-3.78(m,NH3CHCH2), 1.70-1.80(m,CH2CH3), 0.87(t,J=7.4Hz,CH2CH3).

[0096] Example 53: (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (Compound 58) Using Reaction Scheme 9, compound 56 (0.65 g, 1.4 mmol) was reacted with 4.0 M HCl in dioxane (2.34 ml, 67.5 mmol) to synthesize compound 58 (0.39 g, 70%) as a white powder: f =0.00(EtOAc9:Acetone1); 1 H NMR(DMSO-d6,400MHz)8.99(t,J=7.3Hz,C(O)NH),8.26(broad,NH3 + Cl - ), 7.60-7.78(m,ArH), 7.38-7.48(m,ArH), 7.10-7.40(m,ArH), 6.90-7.10(m,ArH), 5.12(s,OCH2), 4.18-4.38(m,NHC H2), 3.70-3.83(m,NH3CHCH2), 1.60-1.80(m,CH2CH2CH3), 1.20-1.40(m,CH2CH2CH3), 0.86(t,J=9.6Hz,CH2CH2CH3).

[0097] Example 54: (R) / (S)-2-(Dimethylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (Compound 59) Using Reaction Scheme 11, compound 22 (0.10 g, 0.2 mmol), triethylamine (0.20 ml, 1.4 mmol), formaldehyde (0.03 ml), and palladium catalyst (0.10 g) were reacted to synthesize compound 59 as a white powder. 1H NMR (DMSO-d6, 400MHz) 8.23(t,J=5.9Hz,C(O)NH), 7.73-7.79(m,ArH), 7.62-7.70()M, ArH), 7.16-7.23(m,ArH), 6.93-6.99(m,Ar H), 5.21(s,OCH2), 4.17-4.24(m,NHCH2), 2.70-2.76(NCH), 2.18(s,N(CH3)2), 1.48-1.63(m,CH2CH3), 0.78(t,J=7.4Hz,CH2CH3).

[0098] Example 55: (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (Compound 60) Using Reaction Scheme 11, compound 57 (0.20 g, 0.5 mmol), triethylamine (0.40 ml, 2.9 mmol), formaldehyde (0.11 ml), and palladium catalyst (0.04 g) were reacted to synthesize compound 60 as a white powder. 1 H NMR (DMSO-d6,400MHz) 8.23(t,J=5.8Hz,C(O)NH), 7.63-7.72(m,ArH), 7.30-7.47(m,ArH), 7.15-7.22(m,ArH), 6.93-6.99(m,ArH), 5.11(s,OCH2) , 4.10-4.30(m,NHCH2), 2.80-2.90(NCH), 2.19(s,N(CH3)2), 1.40-1.63(m,CH2CH2CH3), 1.25-1.30(m,CH2CH2CH3), 0.86(t,J=7.4Hz,CH2CH2CH3).

[0099] Example 56: (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (Compound 61) Using Reaction Scheme 11, compound 58 (0.31 g, 0.7 mmol), triethylamine (0.10 ml, 0.7 mmol), formaldehyde (0.15 ml), and palladium catalyst (0.15 g) were reacted to synthesize compound 61 as a white powder. 1H NMR (DMSO-d6,400MHz) 8.23(t,J=5.7Hz,C(O)NH), 7.60-7.72(m,ArH), 7.30-7.49(m,ArH), 7.10-7.25(m,ArH), 6.92-7.00(m,ArH), 5.15(s,OCH2) , 4.10-4.30(m,NHCH2), 2.79-2.89(NCH), 2.18(s,N(CH3)2), 1.40-1.60(m,CH2CH2CH3), 1.23-1.31(m,CH2CH2CH3), 0.87(t,J=7.4Hz,CH2CH2CH3).

[0100] Preparation 57: (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (Compound 62) Using Reaction Scheme 12, compound 22 (0.10 g, 0.3 mmol), triethylamine (0.21 ml, 1.5 mmol), and iodoethane (0.10 ml) were reacted to synthesize compound 62 as a white powder. 1 H NMR (DMSO-d6,400MHz)7.61-7.67(m,ArH), 7.51-7.60(m,ArH), 7.17-7.25(m,ArH), 6.89-6.95(m,ArH), 5.12(s,OCH) 2), 4.34-4.45(m,NHCH2), 3.03-3.09(NCH), 2.52-2.67(m,CH2CH2CH3), 1.75-1.87(m,CHHCH3), 1.55-1.68(m,CHHCH 3、 CH2CH2CH3, 1.06(t,J=7.1Hz,CH2CH3), 0.95(t,J=7.5Hz,CH2CH2CH3).

[0101] Example 58: (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)butanamide (Compound 63) Using Reaction Scheme 12, compound 57 (0.10 g, 0.7 mmol), triethylamine (0.21 ml, 1.5 mmol), and iodoethane (0.10 ml) were reacted to synthesize compound 63 as a white powder.1 H NMR (DMSO-d6,400MHz)7.61-7.70(m,ArH), 7.50-7.60(m,ArH), 7.25-7.38(m,ArH), 6.99-7.15(m,ArH), 5.10(s,OCH2), 4.30-4.45(m,NHCH2), 3.10-3.25 (NCH), 2.48-2.67(m,CH2CH2CH3), 1.75-1.88(m,CHHCH3), 1.53-1.69(m,CHHCH3, CH2CH2CH3), 1.06(t,J=7.1Hz,CH2CH3), 0.93(t,J=7.4Hz,CH2CH2CH3).

[0102] Example 59: (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (Compound 64) Using Reaction Scheme 12, compound 26 (0.13 g, 0.3 mmol), triethylamine (0.26 ml, 1.9 mmol), and iodoethane (0.02 ml) were reacted to synthesize compound 64 as a white powder. 1 H NMR (DMSO-d6,400MHz)7.63-7.80(m,ArH), 7.18-7.30(m,ArH), 6.90-7.00(m,ArH), 5.20(s,OCH2), 4.19-4.38(m,NHCH2), 3.35-3.42(m,CHCH2CH 2CH3), 1.63-1.73(m,NH), 1.33-1.48(m,CHHCH2CH3,CH2CH3), 1.15-1.19(m,CHHCH2CH3), 1.10-1.15(m,CH2CH3), 0.90(t,J=7.1Hz,CH2CH2CH3).

[0103] Example 60: (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)pentanamide (Compound 65) Using Reaction Scheme 12, compound 58 (0.20 g, 0.5 mmol), triethylamine (0.40 ml, 2.9 mmol), and iodoethane (0.04 ml, 0.4 mmol) were reacted to synthesize white powder 65. 1 H NMR (DMSO-d6,400MHz) 8.26(t,J=5.9Hz,C(O)NH), 7.68-7.72(m,ArH), 7.62-7.68(m,A rH), 67.40-7.46(m,ArH), 7.14-7.20(m,ArH), 6.91-6.99(m,ArH), 5.11(s,OCH2), 4.18 -4.25(m,NHCH2), 2.92-2.98(NCH), 2.31-2.48(m,CH2CH2CH3), 1.35-1.50(m,CH2CH3) , 1.20-1.35(m,CH2CH2CH3), 0.96(t,J=7.1Hz,CH2CH3), 0.84(t,J=7.2Hz,CH2CH2CH3).

[0104] Preparation 61: (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (Compound 66) Using Reaction Scheme 12, compound 22 (0.10 g, 0.3 mmol), triethylamine (0.21 ml, 1.5 mmol), and iodoethane (0.10 ml) were reacted to synthesize compound 66 as a white powder. 1 H NMR (DMSO-d6,400MHz) 8.20(t,J=5.4Hz,NHCH2), 7.57-7.70(m,ArH), 7.10-7.22(m,ArH), 6.88-7.02(m,ArH)), 5.25(s,OCH2), 4.08-4.23(m,NHCH2), 3.05 -3.13(m,CHCH2CH3), 2.52-2.63(m,CH2CH3), 2.35-2.4(m,CH2CH3), 1.42-1.67(m,CH2CH3), 0.93(t,J=7.0Hz,CH2CH3,CH2CH3), 0.84(t,J=7.4Hz,CH2CH3).

[0105] Example 62: (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)butanamide (Compound 67) Using Reaction Scheme 12, compound 57 (0.10 g, 0.7 mmol), triethylamine (0.21 ml, 1.5 mmol), and iodoethane (0.10 ml) were reacted to synthesize compound 67 as a white powder. 1 H NMR (DMSO-d6,400MHz)8.16(t,J=5.3Hz,NHCH2), 7.57-7.72(m,ArH), 7.10-7.25(m,ArH), 6.93-7.02(m,ArH), 5.21(s,OCH2), 4.10-4.26(m,NHCH2), 3.10- 3.18(m,CHCH2CH3), 2.50-2.63(m,CH2CH3), 2.35-2.48(m,CH2CH3), 1.35-1.55(m,CH2CH3), 0.92(t,J=7.0Hz,CH2CH3,CH2CH3), 0.84(t,J=7.4Hz,CH2CH3).

[0106] Preparation 63: (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (68) Using Reaction Scheme 12, compound 26 (0.13 g, 0.3 mmol), triethylamine (0.26 ml, 1.9 mmol), and iodoethane (0.02 ml) were reacted to synthesize white powder 68. 1 H NMR(DMSO-d6,400MHz)8.16(t,J=5.4Hz,NHCH2),7.60-7.80(m,ArH),7.15-7.25(m,Ar H), 6.90-7.00(m,ArH)), 5.21(s,OCH2), 4.12-4.26(m,NHCH2), 3.09-3.15(m,CHCH2CH 2CH3), 2.52-2.63(m,CH2CH3), 2.38-2.48(m,CH2CH3), 1.40-1.60(m,CH2CH2CH3), 1.2 0-1.31(m,CH2CH2CH3), 0.93(t,J=7.1Hz,CH2CH3,CH2CH3),0.86(t,J=7.3Hz,CH2CH3).

[0107] Example 64: (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)pentanamide (Compound 69) Using Reaction Scheme 12, compound 58 (0.20 g, 0.5 mmol), triethylamine (0.40 ml, 2.9 mmol), and iodoethane (0.04 ml, 0.4 mmol) were reacted to synthesize compound 69 as a white powder. 1 H NMR(DMSO-d6,400MHz)8.20(t,J=5.4Hz,NHCH2),7.60-7.80(m,ArH),7.13-7.22(m,Ar H), 6.90-7.02(m,ArH), 5.20(s,OCH2), 4.12-4.25(m,NHCH2), 3.10-3.15(m,CHCH2CH2 CH3), 2.50-2.63(m,CH2CH3), 2.37-2.49(m,CH2CH3), 1.40-1.60(m,CH2CH2CH3), 1.20 -1.33(m,CH2CH2CH3), 0.92(t,J=7.1Hz,CH2CH3,CH2CH3), 0.86(t,J=7.4Hz,CH2CH3). [Example]

[0108] Example 1: In vitro anti-inflammatory effect Example 1-1: Confirmation of anti-inflammatory effect using interleukin-6 (IL-6) Mouse macrophage RAW264.7 cells (obtained from the Korea Cell Line Bank) were cultured in a 12-well plate at an initial concentration of 5 × 10 5After seeding at 100 cells / well, the cells were cultured at 37°C for 16 to 24 hours. Next, the compounds of the present invention were dissolved in 100% DMSO to concentrations of 0, 1, 2, 4, and 8 μg / ml. The RAW264.7 cell culture medium was then treated with the compound samples at each concentration and incubated at 37°C for 1 hour. Next, lipopolysaccharide (LPS) (Sigma-Aldrich, USA) was added to a final concentration of 1 μg / ml, and the cells were cultured at 37°C for 24 hours to induce interleukin-6. The culture medium was then collected, and the amount of interleukin-6 induced in the RAW264.7 cells was determined using an ELISA kit (LABIS KOMA, Korea) according to the manufacturer's instructions. The compounds of the present invention exhibited IC values ​​within the treatment concentration range (up to 8 μg / ml) as shown in Tables 1 and 2 below. 50 In this example, the IC 50 The value is defined as the treatment dose of the compound of the present invention that can reduce the LPS-induced increase in IL-6 levels by 50%. Figure 1(a) shows the results for compound 27 of the present invention.

[0109] [Table 1-1] [Table 1-2]

[0110] [Table 2]

[0111] In addition, human skin keratinocytes, HaCaT cells (obtained from Korea Cell Line Bank), were cultured in a 12-well plate at an initial concentration of 2 × 10 5After seeding at 1000 cells / well, the cells were cultured at 37°C for 16 to 24 hours. Compound 27 of the present invention was added to the HaCaT cell culture medium at final concentrations of 0, 1, 2, 4, or 8 μg / ml and cultured at 37°C for 1 hour. To induce interleukin-6, the culture medium was treated with TNF-α (Sigma-Aldrich, USA) and IFN-γ (Sigma-Aldrich, USA) at final concentrations of 50 ng / ml each and cultured at 37°C for 24 hours. The culture medium was then harvested, and the amount of interleukin-6 induced in the HaCaT cells was determined using an ELISA kit (LABIS KOMA, Korea) according to the manufacturer's instructions. As shown in Figure 1(b), it was confirmed that interleukin-6 decreased with increasing concentrations of compound 27 of the present invention.

[0112] Example 1-2: Confirmation of anti-inflammatory effect using interleukin-4 (IL-4) RBL-2H3 cells, a type of rat basophilic leukemia cell line (obtained from the Korea Cell Line Bank), were cultured in a 48-well plate at an initial concentration of 2 × 10 5 After seeding at 1000 cells / well, the cells were cultured at 37°C for 16 to 24 hours. Compound 27 of the present invention was then added to the RBL-2H3 cell culture medium to final concentrations of 0, 1, 2, 4, and 8 μg / ml, and the cells were cultured at 37°C for 1 hour. To induce interleukin-4, the cells were treated with PMA (phosphomolybdic acid, Sigma-Aldrich, USA) and ionomycin (Sigma-Aldrich, USA) to final concentrations of 50 ng / ml and 0.5 μM, respectively, and cultured at 37°C for 24 hours. The culture medium was then collected, and the amount of interleukin-4 induced in the RBL-2H3 cells was determined using an ELISA kit (LABIS KOMA, Korea) according to the manufacturer's instructions. Figure 2(a) shows the results. As can be seen from Figure 2(a), the amount of interleukin-4 decreased in proportion to the treatment concentration of compound 27 of the present invention.

[0113] Example 1-3: Confirmation of anti-inflammatory effect using interleukin 13 (IL-13) RBL-2H3 cells were plated in a 24-well plate at an initial concentration of 2 × 105 Cells / well were seeded and cultured for 16 to 24 hours at 37°C. Compound 27 of the present invention was then added to the RBL-2H3 cell culture medium at final concentrations of 0, 1, 2, 4, or 8 μg / ml, followed by incubation at 37°C for 1 hour. To induce interleukin-13, PMA (phosphomolybdic acid, Sigma-Aldrich, USA) and ionomycin (Sigma-Aldrich, USA) were added to final concentrations of 50 ng / ml and 0.5 μM, respectively, and the cells were cultured at 37°C for 24 hours. The culture medium was then collected, and the amount of interleukin-13 induced in the RBL-2H3 cells was determined using an ELISA kit (R&D Systems, USA) according to the manufacturer's instructions. Figure 2(b) shows the results. As can be seen from Figure 2(b), the amount of interleukin-13 decreased with increasing concentrations of compound 27 of the present invention. This suggests that compound 27 of the present invention has an anti-inflammatory effect.

[0114] Example 1-4: Confirmation of anti-inflammatory effect after TSLP induction in HaCaT cells Human skin keratinocytes, HaCaT cells (obtained from the Korea Cell Line Bank), were cultured in a 12-well plate at an initial concentration of 2 × 10 5After seeding at 100 cells / well, the cells were cultured at 37°C for 16 to 24 hours. Compounds 27, 29, and FK506 (Sigma) of the present invention were added to the HaCaT cell culture medium at final concentrations of 0, 1, 2, 4, and 8 μg / ml, and the cells were cultured at 37°C for 1 hour. To induce TSLP, the culture medium was treated with TNF-α (Sigma-Aldrich, USA) at a final concentration of 100 ng / ml and then cultured at 37°C for 24 hours. The culture medium was then collected, and the amount of TSLP induced in the HaCaT cells was determined using an ELISA kit (R&D Systems, USA) according to the manufacturer's instructions. As shown in Figure 3(a), TSLP decreased with increasing concentrations of compound 27. To compare the TSLP reduction rates (RR) of compound 27, the positive control drug, and compound 29 under the same conditions, cells were pretreated with the same amounts of compounds 27, 29, and FK506 (Sigma-Aldrich, USA) for 1 hour. The amount of TSLP released from HaCat cells into the culture medium upon TNFα stimulation was quantitatively analyzed and is shown in Table 3 and Figure 3(c).

[0115] [Table 3]

[0116] Example 1-5: Confirmation of anti-inflammatory effect after interleukin-6 induction in HaCaT cells Human skin keratinocytes, HaCaT cells (obtained from the Korea Cell Line Bank), were cultured in a 12-well plate at an initial concentration of 2 × 10 5After seeding at 1000 cells / well, the cells were cultured at 37°C for 16 to 24 hours. Compound 27 of the present invention was added to the HaCaT cell culture medium at final concentrations of 0, 1, 2, 4, or 8 μg / ml and cultured at 37°C for 1 hour. To induce interleukin-6, the culture medium was treated with TNF-α (Sigma-Aldrich, USA) and IFN-γ (Sigma-Aldrich, USA) at final concentrations of 50 ng / ml each and cultured at 37°C for 24 hours. The culture medium was then harvested, and the amount of interleukin-6 induced in the HaCaT cells was determined using an ELISA kit (LABIS KOMA, Korea) according to the manufacturer's instructions. As shown in Figure 3(b), the amount of interleukin-6 decreased with increasing concentrations of compound 27.

[0117] Example 2: TSLP:TSLPR Interaction and Signaling Inhibition 2-1: Calcium imaging HEK293T cells (obtained from the Korean Cell Line Bank), a human embryonic kidney cell line, were transfected with mouse TSLPR (mTSLPR, NCBI Reference Sequence: NM_001164735 CDC sequence, Sequence Listing: 1) or human TSLPR (hTSLPR, NCBI Reference Sequence: NM_022148 CDC sequence, Sequence Listing: 2); mouse IL7Rα (mIL7R-alpha, NCBI Reference Sequence: NM_008372 CDC sequence, Sequence Listing: 3) or human IL-7Rα (hILIL-7Rα, NCBI Reference Sequence: NM_002185 CDC sequence, Sequence Listing: 4); and mouse TRPA1 (mTRPA1, NCBI Reference Sequence: NM_001348288 CDC sequence, Sequence Listing: 5) or human TRPA1 (hTRPA1, NCBI Reference Sequence: NM_007332 CDC sequence, Sequence Listing: 6). The cells were transfected with an expression vector (pcDNA3.1 plsamid, Invitrogen, USA) cloned to overexpress the TSLP protein (sequence 6, Sequence Listing). The cells were then pretreated with various concentrations of the compounds of the present invention (10 μM, 1 μM, 100 nM, 10 nM, 5 nM, 1 nM, and 100 pM) for 10 minutes. A calcium-specific staining reagent (Fluo-3, AM, Invitrogen, USA) was used to measure changes in intracellular calcium ions. Cells overexpressing the TSLP protein, either pretreated with or without the compounds of the present invention, were treated with 2.5 ng / ml of mouse recombinant TSLP protein (mTSLP, R&D Systems, USA) or 10 ng / ml of human recombinant TSLP protein (hTSLP, R&D Systems, USA), and the changes in intracellular calcium ions induced by the treatment were analyzed. Changes in intracellular calcium ions in cells not pretreated with the compound of the present invention were examined, and changes in intracellular calcium ions in the group treated with the compound of the present invention were examined.The signal (F) measured by the intracellular calcium-specific staining reagent in the group not treated with the compound of the present invention was compared with the signal (F) measured by the intracellular calcium-specific staining reagent in the group treated with the compound of the present invention. An F / F ratio of 1 indicates that intracellular calcium influx was completely suppressed by the compound of the present invention. In this example, it was confirmed that treatment with the compound of the present invention suppressed intracellular calcium influx, and IC was defined as 50% suppression. 50 The results are shown in Table 4 below. A:IC 50 Values ​​less than 500 nM, B:IC 50 Values ​​are 500 nM or greater.

[0118] [Table 4]

[0119] 2-2: Inhibition of STAT5 phosphorylation HEK293 cells were seeded into 12-well plates at 3 x 10^5 cells / well and cultured overnight (approximately 16 hours). TSLPR / IL-7Ra was transfected into the cells at 50 ng per well and allowed to express for 24 hours. Each well was treated with various concentrations of His-TSLP (0, 10, 50, and 100 ng / ml). After 0.5 hours, the supernatant was lysed using lysis buffer. The lysate and supernatant were mixed with sample buffer, boiled at 99°C for 5 minutes, and stored at -20°C until Western blotting (WB). The results are shown in Figures 4 and 5. For Western blotting, sections were blocked with 5% BSA (in TBST) for 2 hours at room temperature, followed by overnight incubation at 4°C using primary antibodies against phospho-STAT5, total STAT-5 (Abcam, USA), and gamma tubulin (Sigma-Aldrich, USA). The following day, sections were incubated with secondary antibodies against rabbit and mouse (SantaCruz, USA) for 2 hours at room temperature according to the procedure described above, followed by washing with TBST. Protein bands were detected using ECL reaction with HRP from the secondary antibody using an iBright (ThermoFisher Scientific) instrument. Bai is a known TSLP inhibitor (Baicalein, Sigma-Aldrich), and SD-1029 (CAS 118372-34-2, Sigma-Aldrich) is a JAK2 inhibitor.

[0120] Example 3: Efficacy experiment using a DNCB-induced atopic dermatitis mouse model Experimental Method ICR mice (Orient Bio Co., Ltd., male, 4 weeks old) were used, with four mice per group. Mice were acclimated for one week (23 ± 2°C, 55 ± 10% humidity, 12-hour light-dark cycle, Sungkyunkwan University Experimental Animal Center, Republic of Korea). Prior to drug treatment, hair was removed using clippers and depilatory cream. According to conventional methods, 1% DNCB (2,4-dinitrochlorobenzene, Sigma-Aldrich Co., St. Louis, USA) (200 μl) was applied to the back of the mice once every two days, four times a week, followed by 0.5% DNCB (200 μl) for eight applications at two-day intervals to induce atopic dermatitis (Ara, J., Mol Cell Toxicol 2019). One week after DNCB treatment, all mice were confirmed to have developed lesions. Compound 27 of the present invention, dissolved in sterilized water at a concentration of 1% (w / v), was then applied percutaneously in 200 μL doses daily for two weeks, starting from the time of the 0.5% DNCB application. Experimental animal powder feed (manufacturer: Purina, Switzerland) was provided, and feed and drinking water were available ad libitum throughout the experiment. Symptoms associated with atopic dermatitis, such as erythema, scaling, crusting, lichenified inflammation, bleeding, and dry skin, were observed and scored according to the Scoring Atopic Dermatitis (SCORAD) index shown in Table 2 below.

[0121] [Table 5]

[0122] Blood was collected from the orbital venous plexus using a capillary tube, and serum was separated. Skin tissue proteins were extracted with 0.2% phosphate buffered saline (PBS) using a homogenizer. IgE concentrations were measured using an IgE ELISA kit (Shibayagi, Japan) according to the manufacturer's instructions and analyzed with an ELISA reader. TNF-α, IL-4, CCL22 / MDC, MMP-12, and S100A9 concentrations were measured using Luminex RA Assays (R&D Systems, USA).

[0123] result 3-1:SCORAD indicators Erythema, scaling, crusting, lichenification, inflammation, bleeding, and dry skin were observed, and the Scoring Atopic Dermatitis (SCORAD) index (see Table 5) was recorded. The results showed that the DNCB-treated group showed progression of scarring, keratinization, and keratin loss, while the group treated with Compound 27 of the present invention showed a significant reduction in dermatitis symptoms. Figure 6 shows a graph of the SCOARD index.

[0124] 3-2: Histopathological analysis Skin tissue from the back of mice was isolated and fixed in 10% formalin (10% Neutral Buffered Formalin, Kangdong Trading Co., Ltd., Korea) for 72 hours. The fixed tissue was washed with water for approximately 1 hour and then treated with paraffin to obtain paraffin blocks. The paraffin blocks were cut into 8 μm sections using a microtome and stained with Harris's hematoxylin solution. The sections were then treated with 1% HCl-alcohol, neutralized with 0.5% ammonia water, and stained with eosin solution. The sections were then dehydrated with alcohol and xylene and mounted on slides in Canada balsam. Figures 7(a) and (b) show the results. As seen in Figures 6 and 7, the group treated with compound 27 of the present invention showed a significant decrease in inflammatory cell expression and a significant decrease in epidermal thickness compared to the DNCB-treated group.

[0125] 3-3: Serum analysis - IgE and TSLP expression To confirm the concentrations of IgE and TSLP in mouse blood, an IgE ELISA kit (Shibayagi, JP) and a TSLP ELISA kit (R&D Systems, USA) were used to measure serum IgE and TSLP concentrations from mouse serum according to the manufacturer's instructions. As shown in Figures 8(a) and (b), serum IgE and TSLP concentrations increased in the DNCB-treated group, whereas they decreased in the compound 27-treated group.

[0126] 3-4: Skin tissue analysis Proteins were extracted from the skin tissue of the backs of mice with atopic dermatitis, and TNF-α, IL-1β, CCL22 / MDC, IL-4, MMP-12, and S100A9 were measured using Luminex R Assays (R&D Systems, USA). As shown in Figures 9(a)-(f), the expression levels of these cytokines and chemokines were significantly increased in the DNCB-treated group, but significantly decreased in the compound-treated group.

[0127] Example 4: Efficacy experiment using a DNFB-induced atopic dermatitis mouse model Experimental Method ICR mice (obtained from Koatech, Gyeonggi, Korea, male, 9 weeks old) were used. They were divided into test drug and control groups and allowed to acclimate for one week, after which the hair from the drug application site (the back of the mouse) was removed. On day 0, 50 μl of 0.5% DNFB (99% DNFB diluted with acetone) was applied to the back of the mouse. Subsequently, 20 μl of 0.2% DNFB was applied on days 5, 8, 10, and 14 to induce atopic dermatitis. Seven days after DNFB treatment, all mice were confirmed to have developed lesions. Compound 27 of the present invention was then administered intraperitoneally (ip), orally, or transdermally (deep dermal) daily for one week.

[0128] result Itch scratching test The groups treated with Compound 27 (1-10 mg / kg) and the control group were separated on days 5, 8, 11, and 15, respectively, and allowed to acclimate for 1 hour. After this, the scratching behavior patterns of the mice were recorded and analyzed for 30 minutes. The results are shown in Figures 10(a) and (b). As shown in Figure 10, the number of scratching bouts in the group treated with Compound 27 was significantly reduced.

[0129] Example 5: Efficacy experiment using a mouse model of house dust mite (HDM)-induced atopic dermatitis Experimental Method Nc / Nga mice (obtained from Central Laboratory Animals, Republic of Korea, male, 8 weeks old) were used, with eight mice per group. The experimental animals were allowed free access to feed (Purina Nestle, Switzerland) and drinking water. The mice were depilated using clippers and depilatory cream the day before Df cream application (day 0). Df cream (dermatophagoides farina cream) (Biostir AD, Biostir Incorporation) was applied to the back of the mice twice a week for 5 weeks to induce atopic dermatitis. After confirming the development of lesions in all mice, compound 27 of the present invention was administered transdermally or orally for 3 weeks (21 days) starting 2 weeks after Df cream application. For transdermal administration, compound 27 of the present invention was mixed with 100% ethanol to a concentration of 0.3%, 1%, or 3%, and then applied once daily. 25 μl of the solution was applied to both ears and 70 μl to the back. 100% ethanol was used as the transdermal vehicle. For oral administration, Compound 27 of the present invention was administered at 25 mpk (once per day, QD), 50 mpk (QD), or 10 mpk (twice per day, BID). A mixture of DMSO (dimethyl sulfoxide):PEG300 (polyethylene glycol):HO = 1:4:5 was used as the oral administration vehicle. Dexamethasone (Sigma, Cat no. D1756) was used as a positive control and was orally administered at 25 mpk (once per day, QD). The positive control dexamethasone was prepared using sterile distilled water according to the dose and volume. The composition of the test groups is shown in Table 6 below.

[0130] [Table 6]

[0131] result 5-1: Dermatitis score Skin index values ​​were recorded by visual observation once a week (days 0, 7, 14, 21, 28, and 36). Evaluation items were recorded using the Modified SCORAD score. The items were recorded as erythema / hemorrhage, edema, excoriation / erosion, and scaling / dryness. Scores for each item were set as no symptoms (0 points), mild symptoms (1 point), normal symptoms (2 points), and severe symptoms (3 points), and the sum of all items was calculated. As can be seen from Figure 11, after 10 days of administration of the drug of the present invention, compound 27 of the present invention showed a superior effect on improving skin lesions compared to the control group.

[0132] Example 6: Skin-specific memory Tcell marker analysis Experimental Method Nc / Nga mice (obtained from Central Laboratory Animals, male, 6 weeks old) were used, with two mice per group. The experimental animals had free access to food and drinking water. Mice were depilated using clippers and depilatory cream before application of Df cream. Df cream (dermatophagoides farina cream) (Biostir AD, Biostir Incorporation) was applied to the backs of the mice three times a week for four weeks to induce atopic dermatitis. After confirming the development of lesions in all mice, compound 27 of the present invention was administered transdermally three times a week for three weeks starting four weeks after Df cream treatment. Unlike the other examples described above, this example did not include a period in which the test drug (compound 27 of the present invention) and the atopic dermatitis-inducing drug were simultaneously applied. Compounds of the present invention (8 μg / ml and 16 μg / ml) were applied to the skin in a volume of 100 μl. The control and test groups used in this example are listed in Table 7. As a positive control, 0.1% dexamethasone was applied to the mouse skin for comparison.

[0133] [Table 7]

[0134] result 6-1: Comparison of inflammatory responses The mice were visually observed for erythema hemorrhage, wound dryness, edema, and shedding, and the results were compared using the SCORAD scale. Erythema and hemorrhage were observed in the 0.1% dexamethasone-treated positive control group. Compared to the positive control group, the test group treated with compound 27 of the present invention showed significantly reduced erythema hemorrhage, wound dryness, edema, and shedding, indicating complete healing of the wound and erythema. Figure 12 graphically illustrates the changes in dermatitis score in a mouse model of atopic dermatitis following treatment with each test substance (Wilcoxon-signed rank test; P<0.05 with statistically significant data for three mice examined from each group). As can be seen from Figure 12, compound 27 of the present invention concentration-dependently improved atopic dermatitis symptoms (16 μg / ml, 8 μg / ml), demonstrating superiority over the positive control group, 0.1% dexamethasone-treated group.

[0135] 6-2: Serum analysis To measure IgE and mouse DF-specific IgE in mouse serum, a mouse IgE ELISA Kit (BioLegend, San Diego, CA, USA) and a mouse DF-specific IgE ELISA Kit (BioLegend, San Diego, CA, USA) were used according to the manufacturer's instructions. The changes in measured serum total IgE and Df cream-specific IgE are shown in Figures 13(a) and 13(b), respectively. Significant decreases in total IgE and Df-specific IgE were observed in the compound-treated group compared to the positive control group (0.1% dexamethasone).

[0136] 6-3: Organizational analysis Changes in skin tissue were confirmed by hematoxylin and eosin (H&E) staining (see Figure 15) (Data are shown as mean ± standard deviation (SD). **P<0.01, *P<0.05). In addition, changes in eosinophils and changes in mouse epidermal thickness were measured (see Figure 22). In the group treated with Compound 27 of the present invention, the number of eosinophils decreased in a concentration-dependent manner, and the thickness of the mouse epidermis became thinner as the drug concentration increased.

[0137] 6-4:FACS analysis (1) Atopic dermatitis mouse skin analysis The T cell fraction in the skin of a mouse model of atopic dermatitis was analyzed following treatment with Compound 27 of the present invention. The results are shown in Figures 16 and 17. (Data are shown as mean ± standard deviation (SD). **P<0.01, *P<0.05). In both Compound 27-treated groups (16 μg / ml and 8 μg / ml), the proportions of skin-specific T cells, including CD3+, CD4+, CD69+, and CD103+, were significantly reduced in a concentration-dependent manner. In particular, the numbers of CD69+ and CD103+ T cells were statistically significantly reduced, which was significantly greater than that of the positive control group (0.1% dexamethasone).

[0138] (2) Analysis of skin samples from patients with atopic dermatitis T cell fractions were compared in tissues isolated from the skin of patients with atopic dermatitis (Yonsei Severance Dermatology, Korea) treated with various drugs after treatment with Compound 27 of the present invention, and the results are shown in Figures 18 and 19. (Data are shown as mean ± standard deviation (SD). **P<0.01, *P<0.05) It was confirmed that the proportion of skin-specific T cells was significantly reduced in a concentration-dependent manner in both Compound 27-treated groups (16 μg / ml, 8 μg / ml).

[0139] Example 7: Confirmation of signal transduction regulation using bone marrow-derived dendritic cells (BMDCs) 7-1.Cell culture C57BL / 6J mice (JA BIO, DBL-0011, 6-12 weeks old, male) were cervically dissected, and the femur and tibia were isolated and stored in PBS (HyClone, Cat. No. SH30028.02). After maximum tissue removal from the femur and tibia, both epiphysis were vertically incised and connected to a 5ml syringe (Korea Baksin, 5cc syringe, 22g). The bone marrows were then rapidly collected in 5ml of PBS. After centrifugation at 1800 rpm for 3 minutes, the supernatant was discarded and the tissue was dissolved in 10ml of PBS. The tissue was then filtered through a 100µm cell strainer and centrifuged at 1800 rpm for 3 minutes. The supernatant was discarded, and the cells were cultured in a CO2 incubator with culture media [RPMI 1640 (Corning, Cat. No. 10-040-CV), 10% fetal bovine serum (FBS, Gibco, Cat. No. 26140-079), 1% penicillin-streptomycin (Gibco, Cat. No. 15140-122), 50 μM 2-mercaptoethanol (Gibco, Cat. No. 21985), and 20 μg / ml GM-CSF (Peprotech, Cat. No. 315-03)]. After 4 days, the same volume of culture media was added, and after 2 days of culture, bone marrow-derived dendritic cells (BMDCs) were harvested and placed in a 12-well plate at a density of 8 × 10 cells. 5 After seeding at 100 cells / ml, the cells were incubated overnight.

[0140] Drug and TSLP treatment The collected BMDCs were pretreated with compound 27, a Jak1 inhibitor (Filgotinib, Selleck, Cat. No. JS7605), and a Jak2 inhibitor (SD-1029, Sigma, Cat. No. 573098) for 30 minutes or 1 hour. After 5, 10, or 30 minutes of treatment with mTSLP (R&D Systems, Cat. No. 555-TS-010) or hTSLP, protein lysates were obtained using lysis buffer (Cell Signaling, 9806S). The lysates were centrifuged at 15,000 rpm for 20 minutes at 4°C, and the supernatant was collected and transferred to a new e-tube. After quantifying the protein using a BCA protein assay kit (Thermo, Cat No. 23225), 5X SDS-PAGE loading buffer (Biosesang, Cat No. SF2002-110-00) was added, the mixture was boiled at 103°C for 5 minutes, and then stored at -20°C.

[0141] 7-3: Western Blot SDS-PAGE gels were prepared with different compositions depending on the protein molecular weight. They were assembled using a BIO-RAD cassette, loaded with running buffer, and run at 60–100 V for a set time. The gels were then transferred to methanol-treated PVDF membranes (Amersham, Cat. No. 10600022) at 100 V for 2 hours. After blocking with 5% BSA (Bovogen, Cat. No. BSAS0.1) for 2 hours, the gels were incubated overnight at 4°C with primary antibodies: phospho-Stat5 antibody (Cell Signaling, Cat. No. 9351S), total Stat5 antibody (Cell Signaling, Cat. No. 94205S), γ-tubulin antibody (Invitrogen, Cat. No. T6557), and actin (Santa Cruz, Cat. No. sc-47778). The next day, after washing with TBS-T, the membrane was incubated with HRP-conjugated secondary antibodies [Anti-Rabbit IgG (BIO-RAD, Cat No. BR1706515) or Anti-Mouse IgG (BIO-RAD, Cat No. BR1706516)] for 2 hours at room temperature. After washing with TBS-T, the membrane was immersed in ECL solution (Thermo, Cat No. 32209) and then detected using an iBright imaging system (Invitrogen, Model No. FL1500). Band intensity was measured using ImageJ software (version 1.38; National Institutes of Health), and the pSTAT5 / STAT ratio was calculated and displayed graphically using GraphPad Prism software (version 5.01; GraphPad Software, Inc.). As shown in Figure 17, TSLP treatment increased p-STAT5 by 6.7-fold compared to Con, whereas treatment with compound 27 of the present invention significantly reduced p-STAT5.Compared to the TSLP-treated group, p-STAT5 was reduced by 40% when treated with 1 μM of the compound of the present invention, showing the same level as that of the JAK2 inhibitor, and was reduced by 51% and 59% when treated with 2 μM and 5 μM, respectively.

Claims

1. An anti-inflammatory pharmaceutical composition comprising, as a pharmacologically active ingredient, a benzyloxybenzylamine amino acid derivative represented by the following chemical formula 1, its R-form, S-form or racemic mixture, or a salt, tautomer, polymorph or solvate thereof, and a pharmaceutically acceptable excipient, which regulates intracellular signal transduction mediated by the cytokine TSLP (thymic stromal lymphopoietin), TSLP receptor (TSLPR) and IL-7Rα: 【Chemistry 1】 In the above Chemical Formula 1, R ４ , R ５ , R ６ , R ７ and R ８ are the same or different and each independently represent hydrogen, C １－７ Alkyl, hydroxy, halogen, halogenated C １－７ Alkyl, C １－７ Alkyloxy and halogenated C １－７ alkyloxy; Y is represented by the following chemical formula 2: 【Chemistry 2】 The R １ and R ３ are the same or different and each independently represent hydrogen or C １～７ is alkyl; R ２ is hydrogen or C １－７ It is alkyl.

2. Said C １－７ Alkyl is a straight chain, branched chain or cyclic C １－７ The anti-inflammatory pharmaceutical composition according to claim 1, wherein the alkyl group is alkyl.

3. The compound represented by Chemical Formula 1 is (R) / (S)-1-((4-((2-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 17); (R) / (S)-1-((4-((4-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 19); (R) / (S)-1-oxo-1-((4-((2-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 20); (R) / (S)-1-oxo-1-((4-((3-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 21); (R) / (S)-1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 22); (R) / (S)-1-((4-((3-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 23); (R) / (S)-1-((4-((4-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 24); (R) / (S)-1-((4-((4-(trifluorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 26); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 27); (R) / (S)—N-(4-((3-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (compound 28); (R) / (S)—N-(4-((4-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (compound 29); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 30); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 31); (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 57); (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 58); (R) / (S)-2-(dimethylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 59); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (compound 60); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (compound 61); (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 62); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)butanamide (compound 63); (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 64); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)pentanamide (compound 65); (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 66); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)butanamide (compound 67); (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 68); and The anti-inflammatory composition according to claim 1, which is any one selected from the group consisting of (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)pentanamide (compound 69).

4. The anti-inflammatory composition according to any one of claims 1 to 3, for the treatment of atopic dermatitis, psoriasis, asthma, rhinitis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, or allergic diseases.

5. An anti-cancer pharmaceutical composition comprising, as a pharmacologically active ingredient, a benzyloxybenzylamine amino acid derivative represented by the following chemical formula 1, its R-form, S-form or racemic mixture, or a salt, tautomer, polymorph or solvate thereof, and a pharmaceutically acceptable excipient, which regulates intracellular signal transduction mediated by the cytokine TSLP (thymic stromal lymphopoietin), TSLP receptor (TSLPR) and IL-7Rα: 【Transformation 3】 In the above Chemical Formula 1, R ４ , R ５ , R ６ , R ７ and R ８ are the same or different and each independently represent hydrogen, C １－７ Alkyl, hydroxy, halogen, halogenated C １－７ Alkyl, C １－７ Alkyloxy and halogenated C １－７ alkyloxy; Y is represented by the following chemical formula 2: 【Chemistry 4】 The R １ and R ３ are the same or different and each independently represent hydrogen or C １～７ is alkyl; R ２ is C １－７ alkyl (provided that R １ and R ３ is hydrogen, and R ２ Ga-CH ３ When R in Formula 1 is ５ or R ７ is not fluoro; R in Chemical 3 above １ is hydrogen, R in Formula 1 ４ ~R ８ is not hydrogen, and R ５ or R ７ is not fluoro).

6. Said C １－７ The alkyl may be a straight chain, branched chain or cyclic C １－７ The anticancer pharmaceutical composition according to claim 5, wherein the aryl group is alkyl.

7. The compound represented by Chemical Formula 1 is (R) / (S)-1-((4-((2-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 17); (R) / (S)-1-((4-((4-(fluorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 19); (R) / (S)-1-oxo-1-((4-((2-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 20); (R) / (S)-1-oxo-1-((4-((3-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 21); (R) / (S)-1-oxo-1-((4-((4-(trifluoromethyl)benzyl)oxy)benzyl)amino)butan-2-aminium chloride (compound 22); (R) / (S)-1-((4-((3-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 23); (R) / (S)-1-((4-((4-(chlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 24); (R) / (S)-1-((4-((4-(trifluorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 26); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 27); (R) / (S)—N-(4-((3-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (compound 28); (R) / (S)—N-(4-((4-chlorobenzyl)oxy)benzyl)-2-(methylamino)butanamide (compound 29); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 30); (R) / (S)-2-(methylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 31); (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxobutan-2-aminium chloride (compound 57); (R) / (S)-1-((4-((3,4-dichlorobenzyl)oxy)benzyl)amino)-1-oxopentan-2-aminium chloride (compound 58); (R) / (S)-2-(dimethylamino)-N-(4-((4-trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 59); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (compound 60); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl)-2-(dimethylamino)butanamide (compound 61); (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 62); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)butanamide (compound 63); (R) / (S)-2-(ethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 64); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(ethylamino)pentanamide (compound 65); (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)butanamide (compound 66); (R) / (S)—N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)butanamide (compound 67); (R) / (S)-2-(diethylamino)-N-(4-((4-(trifluoromethyl)benzyl)oxy)benzyl)pentanamide (compound 68); and The anticancer pharmaceutical composition according to claim 5, which is any one selected from the group consisting of (R) / (S)-N-(4-((3,4-dichlorobenzyl)oxy)benzyl-2-(diethylamino)pentanamide (compound 69).

8. The anti-cancer pharmaceutical composition according to any one of claims 5 to 7, which is for the prevention, treatment, or both prevention and treatment of pancreatic cancer, breast cancer, skin cancer, oropharyngeal cancer, or gastric cancer.

9. A pharmaceutical composition for preventing or treating allergic diseases, itch, or Th2 immune diseases, which comprises, as a pharmacologically active ingredient, a benzyloxybenzylamine amino acid derivative represented by the following chemical formula 1, its R-form, S-form, or racemic mixture, or a salt, tautomer, polymorph, or solvate thereof, and a pharmaceutically acceptable excipient, and which regulates intracellular signal transduction via cytokine TSLP (thymic stromal lymphopoietin), TSLP receptor (TSLPR), and IL-7Rα: 【Transformation 5】 In the above Chemical Formula 1, R ４ , R ５ , R ６ , R ７ and R ８ are the same or different and each independently represent hydrogen, C １－７ Alkyl, hydroxy, halogen, halogenated C １－７ Alkyl, C １－７ Alkyloxy and halogenated C １－７ alkyloxy; Y is represented by the following chemical formula 2: 【Transformation 6】 The R １ and R ３ are the same or different and each independently represent hydrogen or C １～７ is alkyl; R ２ is hydrogen or C １－７ It is alkyl.

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