Novel D-amino acid derivative and pharmaceutical composition comprising same

Abstract

The present invention provides a novel peptide derivative comprising a specific D-amino acid and a pharmaceutical composition for preventing, ameliorating or treating ophthalmic diseases comprising the same. The peptide derivative of the present invention can exhibit excellent anti-inflammatory activity and excellent ameliorating activity in a dry eye syndrome model and a corneal injury model, and thus can be effectively used for preventing, ameliorating, and treating various ophthalmic diseases.

Description

Technical Field

[0001] The present disclosure relates to novel D-amino acid derivatives and pharmaceutical compositions comprising the same. More specifically, the present disclosure relates to peptide derivatives comprising novel specific D-amino acids and pharmaceutical compositions comprising the same for preventing, improving or treating ophthalmic diseases. Background Art

[0002] Dry eye syndrome (DES) is one of the most common eye diseases and refers to the condition of dry eyes. DES occurs when the eyes do not produce enough tears or when tears evaporate too quickly due to instability of the tear film. DES is caused by a variety of reasons, such as contact lens use and meibomian gland dysfunction. Artificial tears are often used to improve DES, as well as preparations containing cyclosporine (such as Restasis). ® ) is used to treat dry eye syndrome.

[0003] Ocular inflammation includes inflammation of the eye, such as conjunctivitis, keratitis, keratoconjunctivitis, uveitis, scleritis, etc. Ocular inflammation is known to be caused by a variety of reasons. For example, dry eye is known to cause keratoconjunctivitis, etc., and preparations containing cyclosporine (such as Restasis) ® ) are also used to treat eye inflammation associated with keratoconjunctivitis. For eye inflammation, a variety of treatments are used depending on the symptoms and cause of the disease. For example, antibiotics are used to treat bacterial keratitis / conjunctivitis, antihistamines are used to treat allergic keratitis / conjunctivitis, and steroids are used to treat uveitis.

[0004] Conjunctival and / or corneal damage refers to damage or impairment of the conjunctiva and / or cornea caused by a variety of reasons, including thermal burns, chemical burns, etc. These injuries lead to corneal ulcers or ulcerative keratitis. For ocular inflammation caused by conjunctival and / or corneal damage, a variety of treatment methods are used according to the symptoms and causes of the disease. However, it is necessary to develop drugs with satisfactory activity to improve or treat conjunctival / corneal damage and corneal ulcers themselves.

[0005] For example, ophthalmic diseases such as dry eye syndrome, ocular inflammation (such as keratitis, conjunctivitis, keratoconjunctivitis, uveitis, scleritis, etc.), conjunctival / corneal damage, corneal ulcers, etc. require long-term treatment and have a high recurrence rate. Therefore, it is necessary to develop new mechanisms of action to maintain eye homeostasis, such as effectively stabilizing the tear film, inhibiting corneal and conjunctival inflammation, and promoting eye wound recovery. Public Content

[0006] Technical issues

[0007] The inventors of the present disclosure have conducted a number of studies to develop peptide derivatives that can improve or treat ophthalmic diseases, including dry eye syndrome, ocular inflammation, and conjunctival and / or corneal damage. Specifically, the inventors of the present disclosure selected peptide derivatives containing D-amino acids that exhibited excellent anti-inflammatory activity in experiments using cell lines, and conducted a number of studies. It was found that peptide derivatives containing specific D-amino acids exhibited excellent improvement activity in dry eye syndrome models and corneal damage models.

[0008] Thus, the present disclosure is directed to providing peptide derivatives comprising specific D-amino acids.

[0009] Furthermore, the present disclosure relates to providing a pharmaceutical composition for preventing, improving or treating ophthalmic diseases, which contains a peptide derivative comprising a specific D-amino acid.

[0010] Furthermore, the present disclosure relates to providing peptide derivatives comprising specific D-amino acids for use in therapy.

[0011] Furthermore, the present disclosure relates to providing uses of peptide derivatives containing specific D-amino acids for preventing or treating ophthalmic diseases.

[0012] Furthermore, the present disclosure is directed to providing a method for preventing or treating an ophthalmic disease in a subject in need thereof, comprising administering a peptide derivative comprising a specific D-amino acid to the subject.

[0013] Technical Solutions

[0014] According to one aspect of the present disclosure, there is provided a compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof:

[0015] <Chemical Formula 1>

[0016]

[0017] Wherein X is -OH or -NH2.

[0018] In the compounds of the present disclosure or pharmaceutically acceptable salts thereof, X can specifically be -NH2.

[0019] According to another aspect of the present disclosure, provided is a pharmaceutical composition for preventing, improving or treating an ophthalmic disease, comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0020] According to another aspect of the present disclosure, provided is a cosmetic composition or a cosmetic additive for preventing or improving ophthalmic diseases, comprising the compound or a pharmaceutically acceptable salt thereof.

[0021] According to another aspect of the present disclosure, there is provided use of the compound or a pharmaceutically acceptable salt thereof in therapy.

[0022] According to another aspect of the present disclosure, provided is a use of the compound or a pharmaceutically acceptable salt thereof for preventing or treating an ophthalmic disease.

[0023] According to another aspect of the present disclosure, provided is a method for preventing or treating an ophthalmic disease in a subject in need thereof, comprising administering the compound or a pharmaceutically acceptable salt thereof to the subject.

[0024] In the present disclosure, the ophthalmic disease may be one or more than one selected from dry eye syndrome, ocular inflammation, conjunctival injury, corneal injury and corneal ulcer. In an exemplary embodiment, the ophthalmic disease may be dry eye syndrome. In another exemplary embodiment, the ophthalmic disease may be ocular inflammation. In another exemplary embodiment, the ophthalmic disease may be conjunctival injury, corneal injury or corneal ulcer.

[0025] The pharmaceutical composition of the present disclosure may be an eye drop preparation. The eye drop preparation may be in the form of a solution or a suspension.

[0026] Beneficial Effects

[0027] The present disclosure has shown that peptide derivatives containing D-amino acids (i.e., compounds of Chemical Formula 1) or pharmaceutically acceptable salts thereof exhibit excellent anti-inflammatory activity in dry eye syndrome models and corneal injury models, and also exhibit excellent improvement activity. Therefore, the compounds according to the present disclosure or pharmaceutically acceptable salts thereof can be used to prevent, improve and treat various ophthalmic diseases, such as dry eye syndrome, ocular inflammation, conjunctival injury, corneal injury, corneal ulcer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1a and Figure 1b Shown are the results of in vitro evaluation of the anti-inflammatory activity of compounds of the present disclosure. Figure 1a Shown are the results of evaluating the inhibitory activity of the compounds of the present disclosure on the expression of inflammatory cytokines (TNF-α). Figure 1b The results of evaluating the inhibitory activity of the compounds of the present disclosure on the expression of inflammatory cytokines (IL-1β) are shown. Figure 1a and Figure 1b In the above, “Compound 1” represents the compound of Formula 1a prepared in Example 1, and “Compound 2” represents the acetate of the compound of Formula 1b prepared in Example 2.

[0029] Figure 2The results of measuring tear volume after dropping the compound of the present disclosure into the eyes of mice induced with dry eye syndrome are shown. ***: significant difference of p<0.001 compared with G1, ### / ## / ##: significant difference of p<0.001 / p<0.01 / p<0.05 compared with G2, $$$ / $$: significant difference of p<0.001 / p<0.01 compared with G3.

[0030] Figure 3 The results of measuring corneal fluorescein staining scores after dropping the compounds of the present disclosure into the eyes of mice induced with dry eye syndrome are shown. ** / *: significant difference with p<0.01 / p<0.05 compared with G1, ### / ##: significant difference with p<0.001 / p<0.01 compared with G2, $$$ / $$: significant difference with p<0.001 / p<0.01 compared with G3.

[0031] Figure 4 The results of detecting the degree of inflammation by histopathological examination after dropping the compound of the present disclosure into the eyes of mice induced with dry eye syndrome are shown.

[0032] Figure 5 The results of measuring corneal fluorescein staining scores after dropping the compounds of the present disclosure into the eyes of rats induced with corneal damage are shown. **: significant difference with p<0.01 compared with G1, ### / ## / #: significant difference with p<0.001 / p<0.01 / p<0.05 compared with G2, $$$ / $$: significant difference with p<0.001 / p<0.01 compared with G3.

[0033] Figure 6 The results of detecting the degree of inflammatory cell infiltration in the corneal keratocytes and anterior chamber of the cornea by histopathological examination after dropping the compound of the present disclosure into the eyes of rats induced with corneal damage are shown. **: There is a significant difference of p<0.01 compared with G1, ##: There is a significant difference of p<0.01 compared with G2, $$$ / $$ / $: There is a significant difference of p<0.001 / p<0.01 / p<0.05 compared with G3.

[0034] Figure 7 The compounds of the present disclosure prepared by liquid phase peptide synthesis are shown 1 H NMR analysis results. Best Mode of Invention

[0035] The present disclosure provides a peptide derivative containing a specific D-amino acid, namely, a compound of Chemical Formula 1, or a pharmaceutically acceptable salt thereof.

[0036] <Chemical Formula 1>

[0037]

[0038] Wherein X is -OH or -NH2.

[0039] In an exemplary embodiment, the compound of Chemical Formula 1 of the present disclosure or a pharmaceutically acceptable salt thereof may be in the form of a peptide containing a D-amino acid. In other words, in the compound of Chemical Formula 1 of the present disclosure or a pharmaceutically acceptable salt thereof, X may be -OH and the compound may have the structure of Chemical Formula 1a.

[0040] <Chemical Formula 1a>

[0041]

[0042] In another exemplary embodiment, the compound of Chemical Formula 1 of the present disclosure or a pharmaceutically acceptable salt thereof may be in the form of a peptide derivative containing a D-amino acid. That is, in the compound of Chemical Formula 1 of the present disclosure or a pharmaceutically acceptable salt thereof, X may be specifically -NH2 and the compound may have the structure of Chemical Formula 1b.

[0043] <Chemical Formula 1b>

[0044]

[0045] The compound of the chemical formula 1 of the present disclosure can be in the form of a pharmaceutically acceptable salt. The salt includes conventional acid addition salts, such as salts derived from inorganic acids and salts derived from organic acids, the inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, the organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, malic acid, tartaric acid, gluconic acid, lactic acid, gentisic acid, fumaric acid, lactobionic acid, salicylic acid, phthalic acid, pamoic acid, aspartic acid, glutamic acid and acetylsalicylic acid. In addition, the salt includes salts of sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid.

[0046] The compound of Chemical Formula 1 according to the present disclosure or a pharmaceutically acceptable salt thereof can be prepared by a variety of methods. For example, the compound of Chemical Formula 1 according to the present disclosure or a pharmaceutically acceptable salt thereof can be prepared by solid phase peptide synthesis or liquid phase peptide synthesis, using a D-amino acid [such as D-aspartic acid (dASP) or D-asparagine (dASN)] having an amino protecting group [such as Boc (tert-butyloxycarbonyl)] and a carboxylic acid protecting group [such as Bzl (benzyl)].

[0047] Since the compound of Chemical Formula 1 of the present disclosure or its pharmaceutically acceptable salt exhibits excellent anti-inflammatory activity and also exhibits excellent improvement activity in dry eye syndrome model and corneal injury model, it can be effectively used to prevent, improve and treat a variety of ophthalmic diseases. Accordingly, the present disclosure provides a pharmaceutical composition for preventing, improving or treating ophthalmic diseases, which comprises a therapeutically effective amount of the compound of Chemical Formula 1 or its pharmaceutically acceptable salt as an active ingredient.

[0048] The present disclosure also provides therapeutic uses of the compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof. In certain aspects, the therapeutic use is for preventing or treating ophthalmic diseases.

[0049] The present disclosure also provides a method for preventing or treating an ophthalmic disease in a subject in need thereof, comprising administering a pharmaceutical composition comprising the compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof to the subject.

[0050] In this specification, the term "subject" refers to any mammalian subject to which the compound described in this specification or its pharmaceutically acceptable salt or composition is administered. Non-limiting examples include humans, livestock (such as dogs, cats, etc.), farm animals (such as cattle, sheep, pigs, horses, etc.) and experimental animals (such as monkeys, rats, mice, rabbits, guinea pigs, etc.) that need diagnosis, management or treatment, especially humans. The methods described in this specification are applicable to the prevention or treatment of humans and veterinary uses.

[0051] As used in the present specification, the phrase "a subject in need thereof" includes subjects such as mammalian subjects, to whom administration of the compound described in the present specification or a pharmaceutically acceptable salt or composition thereof is beneficial.

[0052] In this specification, the term "ophthalmic disease" refers to a disease or condition that affects or involves the eye, a part of the eye, or a region of the eye. In some aspects, the ophthalmic disease may be a disease caused by dryness of the eyeball. In some aspects, the ophthalmic disease may be a disease of the cornea or conjunctiva. In some aspects, the ophthalmic disease may be a disease caused by corneal damage or conjunctival damage. In some aspects, the ophthalmic disease may be a disease caused by a defect or necrosis of the cornea or a defect or necrosis of the conjunctiva. In some aspects, the ophthalmic disease may be a disease caused by inflammation of the eyeball.

[0053] In some aspects, the ophthalmic disease can be one or more than one disease selected from dry eye syndrome, ocular inflammation, conjunctival damage, corneal damage and corneal ulcer. In some aspects, the ophthalmic disease can be dry eye syndrome. In some aspects, the ophthalmic disease can be ocular inflammation, and can be selected from the following diseases, such as conjunctivitis, keratitis (including neurotrophic keratitis), keratoconjunctivitis, uveitis and scleritis. In some aspects, the ophthalmic disease can be conjunctival damage, corneal damage or corneal ulcer.

[0054] The pharmaceutical composition of the present disclosure can be prepared into various preparations by using pharmaceutically acceptable additives or carriers. Specifically, the pharmaceutical composition of the present disclosure can have the form of eye drop preparation, eye ointment or spray, and the eye drop preparation can be in the form of solution or suspension.

[0055] For example, the eye drop preparation in the form of a solution may include a solubilizer such as polyethylene glycol 400, glycerol, a stabilizer such as EDTA, a buffer such as boric acid, and a pH regulator such as hydrochloric acid or sodium hydroxide in addition to the compound of Chemical Formula 1 or its salt in sterile water. In addition, the eye drop preparation in the form of a suspension may include a viscosity controller such as cross-linked polyvinyl pyrrolidone (e.g., polyvinylpyrrolidone K-25), an isotonic agent such as sodium chloride, a stabilizer such as EDTA, a buffer such as boric acid and borax, and a pH regulator such as hydrochloric acid or sodium hydroxide in addition to the compound of Chemical Formula 1 or its salt in sterile water. If necessary, the pharmaceutical composition in the form of an eye drop preparation may be sterilized according to conventional methods, or may include excipients such as preservatives, hydrating agents, emulsifiers, solubilizers, salts for osmotic adjustment, and / or buffers.

[0056] The compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof can be in the form of an eye drop preparation, for example, having a concentration of about 0.01% to about 10%, and can be administered to patients with ophthalmic diseases at a dose of 1 to 12 drops per eye in divided doses 1 to 6 times a day. Of course, the dose can be varied depending on the patient's age, sex, susceptibility, symptoms, or severity of the disease.

[0057] The present disclosure also provides a cosmetic composition for preventing or improving ophthalmic diseases, comprising a compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof. In some aspects, the compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof can be provided as a cosmetic additive. In some aspects, the cosmetic composition can be a mist formulation or a spray formulation. In some aspects, for the purpose of preventing or improving ophthalmic diseases, the cosmetic additive can be included in eye cosmetics, such as eyebrow pencils, eyeliners, eye shadows, mascara or eye makeup removers.

[0058] Hereinafter, the present disclosure will be described in more detail through Examples and Test Examples. However, the following Examples and Test Examples are only for illustrating the present disclosure, and the present disclosure is not limited thereto.

[0059] Example 1. Preparation of the compound of formula 1a

[0060] <Chemical Formula 1a>

[0061]

[0062] The compound of formula 1a was prepared by Fmoc solid phase peptide synthesis using an automatic synthesizer (PeptrEx-R48, Peptron, Daejeon, South Korea) using D-aspartic acid (dASP) and D-asparagine (dASN) with Boc as an amino protecting group and Bzl as a carboxylic acid protecting group. The Fmoc protecting group was removed by reacting with piperidine in 20% dimethylformamide twice for 10 minutes each time. Then, Fmoc-amino acid (6 equivalents), hydroxybenzotriazole (HOBt) (6 equivalents), benzotriazole tetramethyluronium hexafluorophosphate (HBTU) (6 equivalents) and N, N-diisopropylethylamine (12 equivalents) were used for coupling. In each step, the resin was washed twice with dimethylformamide and methanol. The synthesized crude peptide was reacted with a mixture of trifluoroacetic acid (TFA) / 1,2-ethanedithiol (EDT) / thioanisole / triisopropylsilane (TIS) / distilled water (DW) (90 / 2.5 / 2.5 / 2.5 / 2.5 volume) for 2 hours to remove the resin and amino acid protecting groups. After adding ether to the resulting mixture solution, the peptide was recovered by centrifugation. The crude peptide was dissolved in distilled water and purified by reverse phase HPLC using a C18 reverse phase column. HPLC was performed using a water-acetonitrile linear gradient (acetonitrile concentration: 0% (v / v) to 50% (v / v)). The purified fraction was freeze-dried, redissolved in water containing 0.5% acetic acid, and then freeze-dried again. The molecular weight of the purified peptide was determined by LC / MS (Shimadzu LC / MS-2020 series, Japan) and freeze-dried using FDT-12012 (Operon, South Korea).

[0063] Example 2. Preparation of the compound of formula 1b

[0064] <Chemical Formula 1b>

[0065]

[0066] The acetate ester of the compound of Chemical Formula 1b was prepared by solid phase peptide synthesis using D-aspartic acid (dASP) and D-asparagine (dASN) with Boc as an amino protecting group and Bzl as a carboxylic acid protecting group.

[0067] Step 1:

[0068]

[0069] Di-tert-butyl dicarbonate ((Boc)2O) (43.8 g, 201 mmol, 46.1 mL, 1.30 eq), pyridine (Py) (12.2 g, 154 mmol, 12.4 mL, 1.00 eq) and NH4HCO3 (18.3 g, 231 mmol, 19.1 mL, 1.50 eq) were added to a solution of compound 1 (50.0 g, 154 mmol, 1.00 eq) in tetrahydrofuran (500 mL). The mixture was stirred at 20 ° C for 15 hours. The results of TLC analysis (petroleum ether: ethyl acetate = 1:3) confirmed that compound 1 (R f =0.52) is completely consumed and a new point (R f =0.45). The reaction mixture was diluted with H2O (500 mL) and vacuum filtered to obtain a residue. The crude product was triturated with H2O (300 mL) at 20°C for 0.5 h and then filtered to obtain a white solid, which was treated with petroleum ether (100 mL) at 20°C for 0.5 h. 1 H NMR and LCMS (R t = 1.08 min, MS theoretical value: 322.1, MS observed value: [M+H] + =323.0) to obtain compound 2 (47.0 g, 143 mmol, 92.6% yield, 98.3% purity) as a white solid. 1 The results of HNMR analysis are as follows:

[0070] .

[0071] Step 2:

[0072]

[0073] HCl / EtOAc (4M, 196 mL, 5.49 eq) was added to a solution of compound 2 (47.0 g, 143 mmol, 98.3% purity, 1.00 eq) in ethyl acetate (EtOAc) (100 mL). The mixture was stirred at 20 °C for 15 hours. The results of TLC analysis (petroleum ether: ethyl acetate = 1:3) confirmed that compound 2 (R f =0.45) is completely consumed and a new point (R f = 0.18). The reaction mixture was filtered to give a residue, which was treated with EtOAc (100 mL) at 20°C for 0.5 h. 1 HNMR and LCMS (R t= 0.14 min, MS theoretical value: 222.1, MS observed value: [M+H] + =223.1) and HPLC (R t =1.63 min, purity 98.7%), and compound 3 (37.0 g, 141 mmol, yield 98.4%, purity 98.7%, HCl salt) was obtained as a white solid. 1 The results of H NMR analysis are as follows:

[0074] .

[0075] Step 3:

[0076]

[0077] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (25.8 g, 135 mmol, 1.10 equiv), hydroxybenzotriazole (HOBt) (16.5 g, 122 mmol, 1.00 equiv) and compound 3 (36.9 g, 141 mmol, 98.7% purity, 1.15 equiv, HCl salt) were added to a solution of compound 4 (28.5 g, 122 mmol, 1.00 equiv) in acetone (500 mL) at 20°C, and N-methylmorpholine (NMM) (37.2 g, 368 mmol, 40.4 mL, 3.00 equiv) was added dropwise at 20°C. A solid precipitated after 2 hours. The resulting mixture was stirred at 20°C for 13 hours. The results of TLC analysis (dichloromethane: methanol = 7: 1) confirmed that compound 3 (R f =0.24) is completely consumed and a new point (R f = 0.11, 0.35, 0.41, 0.90). The reaction mixture was diluted with H2O (1000 mL) and filtered to obtain a residue. The crude product was treated with H2O (400 mL×3) at 20°C for 0.5 h and filtered to obtain a white solid, which was treated with petroleum ether (300 mL) at 20°C for 0.5 h. 1 H NMR and LCMS (R t = 0.47 min, MS theoretical value: 436.2, MS observed value: [M+H] + =437.0) and HPLC (R t = 1.77 min, purity 98.9%), and compound 5 (40.6 g, 92.0 mmol, 74.9% yield, 98.9% purity) was obtained as a white solid. 1 The results of HNMR analysis are as follows:

[0078] .

[0079] Step 4:

[0080]

[0081] A mixture of compound 5 (40.6 g, 92.0 mmol, 98.9% purity, 1.00 eq.) in HCl / dioxane (0.50 M, 552 mL, 3.00 eq.) was stirred at 20° C. for 15 hours. The results of TLC analysis (dichloromethane:methanol=7:1) confirmed that compound 5 (R f =0.57) is completely consumed and a new point (R f = 0.07). The reaction mixture was diluted with methyl tert-butyl ether (MTBE) (500 mL). After solid precipitation, vacuum filtration was performed to obtain a white solid. 1 H NMR and LCMS (R t = 0.12 min, MS theoretical value: 336.1, MS observed value: [M+H] + =337.1) to obtain compound 6 (35.4 g, crude product, HCl salt) as a white solid. 1 The results of H NMR analysis are as follows:

[0082] .

[0083] Step 5:

[0084]

[0085] N-methylmorpholine (NMM) (11.9 g, 118 mmol, 13.0 mL, 3.00 eq) was added to a solution of compound 7 (21.0 g, 39.4 mmol, 98.2% purity, 1.00 eq) and compound 6 (14.6 g, 39.4 mmol, 1.00 eq HCl salt) in dimethylformamide (100 mL). The mixture was stirred at 20 °C for 2.5 hours. The results of LCMS analysis confirmed that compound 7 was completely consumed, and a main peak (R t = 0.34 min, MS theoretical value: 675.2, MS observed value: [M+H] + =676.1). The reaction mixture was diluted with H2O (500 mL) and ethyl acetate (150 mL). After solid precipitation, it was stirred at 20°C for 3 hours and vacuum filtered to obtain a yellow solid, which was treated with H2O (150 mL×3) at 20°C for 1 hour. By LCMS (R t = 0.58 min, MS theoretical value: 675.2, MS observed value: [M+H] + = 676.3) to obtain compound 8 (22.0 g, crude product) as a yellow solid.

[0086] Step 6:

[0087]

[0088] Under nitrogen atmosphere, Pd / C (10.0% purity, 4.80 g) was added to a solution of compound 8 (24.0 g, 35.5 mmol, 1.00 eq) in methanol (350 mL) and acetic acid (350 mL). The resulting suspension was degassed and purged with hydrogen three times. The mixture was stirred under hydrogen (50 psi) at 25° C. for 18 hours. The results of LCMS analysis confirmed that compound 8 was completely consumed, and a main peak (R t = 0.07 min, MS theoretical value: 361.1, MS observed value: [M+H] + =362.1). The mixture was filtered and the filter cake was washed with H2O (50.0 mL×2). The filtrates were mixed and dried under vacuum to obtain a residue. The crude product was treated with ethanol (50.0 mL) at 20°C for 0.6 h. The residue was analyzed by LCMS (R t = 0.10 min, MS theoretical value: 361.1, MS observed value: [M+H] + =361.9) to obtain a yellow solid product (10.0 g, crude product). The crude product was purified by preparative HPLC (0.5% HOAc) and the specific LCMS (R t = 2.87 min, MS theoretical value: 361.1, MS observed value: [M+H] + =362.1) and 1 H NM confirmed that the product was obtained as a white solid (5.00 g, 11.5 mmol, 97.4% purity, HOAc salt). 1 H NMR analysis results ( Figure 7 )as follows:

[0089] .

[0090] Test Example 1: In vitro anti-inflammatory test

[0091] Macrophages (RAW 264.7) treated with lipopolysaccharide (LPS) were treated with the compounds prepared in Examples 1 and 2 as test substances, and changes in the production of inflammatory cytokines (TNF-α and IL-1β) were detected by ELISA and Western blotting, respectively.

[0092] Specifically, 1×10 4Macrophages (RAW 264.7) were divided into each well of a 96-well plate together with 0.1 mL of Dulbecco's modified Eagle medium (DMEM) containing 10% FBS (GIBCO) and 1% penicillin / streptomycin. After culturing in a 5% CO2 incubator at 37°C for 1 day, when the cell confluence reached 80% or above, they were treated with LPS (100 ng / mL) and test substances at concentrations of 1 μM and 10 μM, respectively, and cultured for 24 hours. The control group was treated with LPS (100 ng / mL) only and cultured for 24 hours. The supernatant of each test group was prepared as a sample for ELISA analysis. According to the manufacturer's protocol, the production of TNF-α was detected at 450 nm using an ELISA detection kit (ELISA MAX Standard Kit Human TNF-α; BioLegend, #430201) and a microplate reader. The results are shown in Figure 2. Figure 1a shown.

[0093] In addition, 5×10 6 Macrophages (RAW 264.7) were divided into each well of a 96-well plate together with 0.1 mL of Dulbecco's modified Eagle medium (DMEM) containing 10% FBS (GIBCO) and 1% penicillin / streptomycin. After culturing in a 5% CO2 incubator at 37°C for 1 day, when the cell confluence reached 80% or above, they were treated with LPS (100 ng / mL) and test substances at concentrations of 1 μM and 10 μM, respectively, and cultured for 30 minutes. The control group was treated with LPS (100 ng / mL) only and cultured for 30 minutes. The cells were lysed using NP40 cell lysis buffer and quantified using the Bradford assay. The expression level of IL-1β was then detected by Western blotting. The results are shown in Figure 1b shown.

[0094] from Figure 1a and Figure 1b It can be seen that the secretion and expression of inflammatory cytokines (TNF-α and IL-1β) in the control group increased after treatment with LPS. On the other hand, it can be seen that in the test group treated with the test substance, the secretion and expression of inflammatory cytokines (TNF-α and IL-1β) were inhibited in a concentration-dependent manner. Therefore, since the compounds of Chemical Formula 1a and Chemical Formula 1b obtained according to the present disclosure show excellent anti-inflammatory activity, they can be effectively used to improve ocular inflammation, such as conjunctivitis, keratitis, keratoconjunctivitis, uveitis, scleritis, etc.

[0095] Experimental Example 2: Evaluation of activity in dry eye syndrome model

[0096] 1. Test methods

[0097] (1) Administration of test substances

[0098] Dry eye syndrome was induced in specific pathogen-free (SPF) C57BL / 6NHsd mice (male, 7 weeks old) [Koatech, South Korea]. Specifically, during the induction of dry eye syndrome (day -10 to day 0), scopolamine hydrobromide dissolved in physiological saline (PBS) was subcutaneously injected into the back skin of mice at a dose of 0.5 mg / 0.2 mL, 4 times a day, for 10 days. After measuring the tear volume of mice induced with dry eye syndrome, the mice were randomly divided into groups G2 to G6, as shown in Table 1, so that the average tear volume of each group was evenly distributed. G1 was a normal control group in which dry eye syndrome was not induced.

[0099] From the day of induction of dry eye syndrome (day 0), the compound prepared in Example 2 was dissolved in physiological saline (PBS) at a concentration of 0.1% and 1% as a test substance (i.e., the acetate of the compound of Chemical Formula 1b, hereinafter referred to as "Compound 1b") and dripped on the center of the right cornea of ​​G4 mice and G5 mice, respectively, 3 times a day for 10 days. G2 was the control group for induction of dry eye syndrome. For G3, physiological saline (PBS) was dripped on the center of the right cornea of ​​mice induced with dry eye syndrome. For G6 (positive control group), Restasis ® An ophthalmic solution (0.05%, containing cyclosporine) was dropped onto the center of the cornea of ​​the right eye of mice induced with dry eye syndrome.

[0100] [Table 1]

[0101]

[0102] G1: normal control, G2: induced control, G6: positive control.

[0103] (2) Measurement of tear volume and corneal fluorescein staining score

[0104] The tear volume was measured using the phenol red thread (PRT) test. Specifically, on the 5th and 10th days after the start of the test substance application, a phenol red thread was placed at the outer canthus of the eye for 20 seconds, and then the tear volume (mm) was measured.

[0105] After measuring the tear volume, the animals were anesthetized on the 5th and 10th days after the start of the test substance administration, and 1 μL of 1% sodium fluorescein was dropped into the inferior conjunctival sac of the right cornea of ​​the animals, and the degree of penetration in 5 corneal areas (superior, nasal, central, inferior, and temporal) was evaluated using cobalt blue light according to the following criteria of the National Eye Institute (NEI) grading system.

[0106] - Score: 0 to 3 (0: normal, 1: mild, 2: moderate, 3: severe).

[0107] -Total score: 15.

[0108] (3) Autopsy and histopathological examination

[0109] On the day of autopsy (day 10), the animals were euthanized, and the eyeballs including the conjunctiva were removed and fixed in 10% neutral buffered formalin solution. The fixed tissues were trimmed, dehydrated, paraffin-embedded, thin-sectioned, and then stained with hematoxylin and eosin (H&E) to prepare samples for histopathological examination. Then, the infiltration of inflammatory cells into the cornea, conjunctiva, and anterior chamber was evaluated by optical microscopy (Olympus BX53, Japan). Inflammation of the cornea and conjunctiva was evaluated according to known methods. The scoring criteria are as follows. Score: 0 (normal), 1 (mild), 2 (moderate), 3 (severe).

[0110] (4) Statistical analysis

[0111] Statistical analysis was performed using Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA), and p values ​​less than 0.05 were considered statistically significant.

[0112] 2. Test results

[0113] (1) Measurement of tear volume

[0114] The tear volume measurement results are as follows Figure 2 As shown. On the 5th and 10th days after the start of the test substance administration, the tear volume levels of the induced control group (G2), PBS group (G3), 0.1% test substance administration group (G4), 1% test substance administration group (G5) and positive control group (G6) were statistically significantly lower than those of the normal control group (G1) (p<0.001). The tear volume levels of G4, G5 and G6 were significantly higher than those of G2 (p<0.001, p<0.01 or p<0.05). On the 5th day after the start of the test substance administration, the tear volume levels of G5 and G6 were statistically significantly higher than those of G3 (p<0.01). On the 10th day after the start of the test substance administration, the tear volume levels of G4, G5 and G6 were significantly higher than those of G3 (p<0.001).

[0115] (2) Measurement of corneal fluorescein staining score

[0116] The corneal fluorescein staining score was measured as Figure 3As shown. On the 5th to 10th day after the start of the test substance administration, the corneal fluorescein staining score levels of G2, G3, G4, G5 and G6 were statistically significantly higher than those of G1 (p<0.01 or p<0.05). The corneal fluorescein staining score levels of G4, G5 and G6 were significantly lower than those of G2, and the corneal fluorescein staining score levels of G4, G5 and G6 were significantly lower than those of G3 (p<0.001 or p<0.01).

[0117] (3) Histopathological examination

[0118] The results of histopathological examination were Figure 4 Compared with G1, the inflammation levels of G2 and G3 were statistically significantly higher (p<0.01), and the inflammation and fibrosis levels of G4, G5, and G6 were significantly lower than those of G2 and G3 (p<0.001 or p<0.05).

[0119] 3. Summary

[0120] The effects of the test substances on the C57BL / 6NHsd mouse model induced with dry eye syndrome (G1: normal control group, G2: induced control group, G3: PBS administration group, G4: 0.1% test substance administration group, G5: 1% test substance administration group, G6: positive control group) were evaluated.

[0121] The results of measuring tear volume using phenol red cotton thread showed that from the 5th to the 10th day after the start of test substance administration, the tear volume levels of the 0.1% test substance administration group and the 1% test substance administration group were statistically significantly higher than those of the induced control group, and the tear volume level of the 1% test substance administration group was significantly higher than that of the PBS administration group.

[0122] The results of measurement of corneal fluorescein staining scores showed that on the 5th to 10th day after the start of test substance administration, significantly lower corneal fluorescein staining score levels were observed in the 0.1% test substance administration group and the 1% test substance administration group than in the induction control group and the PBS administration group.

[0123] As a result of histopathological examination, no inflammation was observed in the cornea and conjunctiva of both the 0.1% test substance administration group and the 1% test substance administration group.

[0124] Therefore, repeated instillation of the test substance in the C57BL / 6 mouse model of induced dry eye syndrome can help improve dry eye syndrome and ocular inflammation.

[0125] Test Example 3: Evaluation of activity in corneal injury model

[0126] 1. Test methods

[0127] (1) Administration of test substances

[0128] Corneal injury was induced in specific pathogen-free (SPF) Sprague-Dawley rats (male, 7 weeks old) [Koatech, South Korea]. Specifically, after anesthetizing the rats, filter paper soaked in 2 μL of 0.1 M NaOH solution was applied to the center of the right cornea for about 10 seconds and then washed with saline. The body weights of rats in which corneal injury was induced were measured and randomly divided into groups G2 to G6 as shown in Table 2 so that the average body weight of each group was evenly distributed. G1 was a normal control group in which corneal injury was not induced.

[0129] From the day of corneal injury (day 0), the compound prepared in Example 2 was used as a test substance (i.e., the acetate of the compound of Chemical Formula 1b, hereinafter referred to as "Compound 1b"), dissolved in physiological saline (PBS) at a concentration of 0.1% and 1%, and dripped into the eyes of G4 rats and G5 rats at 10 μL / eye, 3 times a day, for 7 days. G2 was an induced control group for inducing corneal injury. For G3, physiological saline (PBS) was dripped into the eyes of rats induced with corneal injury. For G6 (positive control), Restasis ® An ophthalmic solution (0.05%, containing cyclosporine) was instilled into the eyes of rats in which corneal damage was induced.

[0130] [Table 2]

[0131]

[0132] G1: normal control, G2: induced control, G6: positive control.

[0133] (2) Measurement of corneal fluorescein staining score

[0134] The animals were anesthetized 6 hours or on days 1, 2, 3, 5, and 7 after the induction of corneal injury, and 1 μL of 1% sodium fluorescein was dropped into the inferior conjunctival sac of the right cornea of ​​the animals, and the degree of penetration in five corneal areas (superior, nasal, central, inferior, and temporal) was evaluated using cobalt blue light according to the following criteria of the National Eye Institute (NEI) grading system.

[0135] - Score: 0 to 3 (0: normal, 1: mild, 2: moderate, 3: severe).

[0136] -Total score: 15.

[0137] (3) Autopsy and histopathological examination

[0138] On the day of autopsy (day 7), the animals were euthanized, and the eyeballs including the conjunctiva were removed and fixed in 10% neutral buffered formalin solution. The fixed tissues were trimmed, dehydrated, paraffin-embedded, thin-sectioned, and then stained with hematoxylin and eosin (H&E) to prepare samples for histopathological examination. Then, the infiltration of inflammatory cells into the cornea, conjunctiva, and anterior chamber was evaluated by optical microscopy (Olympus BX53, Japan). The infiltration of inflammatory cells into the cornea, conjunctiva, and anterior chamber was evaluated according to known methods. The scoring criteria are as follows. Score: 0 (normal), 1 (mild), 2 (moderate), 3 (severe).

[0139] (4) Statistical analysis

[0140] Statistical analysis was performed using Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA), and p values ​​less than 0.05 were considered statistically significant.

[0141] 2. Test results

[0142] (1) Measurement of corneal fluorescein staining score

[0143] The corneal fluorescein staining score was measured as Figure 5 As shown. From 6 hours to 5 days after the start of the test substance administration, the corneal fluorescein staining score levels of all corneal injury groups (G2 to G6) were statistically significantly higher than those of the normal control group (G1) (p<0.01). On the 6th day after the start of the test substance administration, the corneal fluorescein staining score levels of the induced control group (G2) and the PBS administration group (G3) were significantly higher than those of G1 (p<0.01). From 6 hours and 2 days to 6 days after the start of the test substance administration, the corneal fluorescein staining score levels of the 0.1% test substance administration group (G4), the 1% test substance administration group (G5) and the positive control group (G6) were significantly lower than those of G2 (p<0.001, p<0.01 or p<0.05). From the 3rd day to the 6th day after the start of the test substance administration, the corneal fluorescein staining score levels of G4, G5 and G6 were significantly lower than those of G3 (p<0.001 or p<0.01).

[0144] (2) Histopathological examination

[0145] The results of histopathological examination were Figure 6 The infiltration degree of inflammatory cells into corneal keratocytes and anterior chamber of the cornea in G2 and G3 was statistically significantly higher than that in G1 (p<0.01 or p<0.05), and the infiltration degree of inflammatory cells into corneal keratocytes and anterior chamber of the cornea in G4, G5 and G6 was statistically significantly lower than that in G2 and G3 (p<0.001, p<0.01 or p<0.01).

[0146] 3. Summary

[0147] The effects of the test substances on the Sprague-Dawley rat model inducing corneal damage were evaluated (G1: normal control group, G2: induced control group, G3: PBS administration group, G4: 0.1% test substance administration group, G5: 1% test substance administration group, G6: positive control group).

[0148] The measurement results of corneal fluorescein staining scores showed that, except for the first day after the start of the test substance administration, the corneal fluorescein staining score levels of the 0.1% test substance administration group and the 1% test substance administration group were significantly lower than those of the induced control group throughout the entire test period, and from the third day after the start of the test substance administration to the end of the test, it was observed that the corneal fluorescein staining score levels of the 0.1% test substance administration group and the 1% test substance administration group were significantly lower than those of the PBS administration group.

[0149] The results of the histopathological examination showed that the infiltration scores of inflammatory cells in the corneal stroma and anterior chamber of the eyes of the 0.1% test substance administration group and the 1% test substance administration group were statistically significantly lower than those of the induction control group and the PBS administration group. The results of the histopathological examination were consistent with the results of the measurement of the corneal fluorescein staining score.

[0150] Therefore, repeated instillation of the test substance in the Sprague-Dawley rat model of induced corneal damage can help conjunctival damage, corneal damage or corneal ulcer. In addition, repeated instillation of the test substance can help improve the ocular inflammation caused by the above-mentioned diseases (such as conjunctivitis, keratitis, keratoconjunctivitis, neurotrophic keratitis, etc.).

Claims

1. A compound of formula 1 or a pharmaceutically acceptable salt thereof: <Chemical Formula 1> Wherein X is -OH or -NH2.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -NH2.

3. A pharmaceutical composition for preventing, improving or treating ophthalmic diseases, comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

4. The pharmaceutical composition according to claim 3, wherein the ophthalmic disease is one or more than one disease selected from dry eye syndrome, ocular inflammation, conjunctival injury, corneal injury and corneal ulcer.

5. The pharmaceutical composition according to claim 3, wherein the ophthalmic disease is dry eye syndrome. The pharmaceutical composition according to claim 3 , wherein the ophthalmic disease is ocular inflammation.

7. The pharmaceutical composition according to claim 3, wherein the ophthalmic disease is conjunctival injury, corneal injury or corneal ulcer.

8. The pharmaceutical composition according to claim 3, which is in the form of an eye drop formulation, eye ointment or spray.

9. The pharmaceutical composition according to claim 8, wherein the eye drop preparation is in the form of a solution or a suspension.

10. A cosmetic composition for preventing or improving ophthalmic diseases, comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

11. The therapeutic use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

12. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof for preventing or treating ophthalmic diseases.

13. A method for preventing or treating an ophthalmic disease in a subject in need thereof, comprising administering the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof to the subject.

Images