Novel D-amino acid derivatives and pharmaceutical compositions containing the same

Abstract

The present invention provides a novel peptide derivative containing a specific D-amino acid and a pharmaceutical composition containing the same for preventing, improving or treating ophthalmic diseases. The peptide derivative of the present invention exhibits excellent anti-inflammatory activity and also exhibits excellent improving activity in xerophthalmia and corneal injury models, and therefore can be usefully used for preventing, improving and treating various ophthalmic diseases.

Description

[Technical field]

[0001] The present invention relates to a novel D-amino acid derivative and a pharmaceutical composition containing the same. More specifically, the present invention relates to a novel peptide derivative containing a specific D-amino acid and a pharmaceutical composition containing the same for preventing, improving or treating an ophthalmic disease. [Background technology]

[0002] Dry eye syndrome (DES) is one of the most common ophthalmic diseases and is a dry eye condition. Dry eye occurs when the eye cannot produce enough tears or when the tear film is unstable and tears evaporate too quickly. Dry eye occurs due to various causes such as the use of contact lenses, meibomian gland dysfunction, etc. Artificial tears are generally used to improve dry eye, and cyclosporine-containing preparations (e.g., Restasis (registered trademark)) are used as drugs to treat dry eye.

[0003] Ocular inflammation includes inflammation occurring in the eyeball, such as conjunctivitis, keratitis, keratoconjunctivitis, uveitis, and scleritis. Ocular inflammation is known to occur due to various causes. For example, xerophthalmia is known to cause keratoconjunctivitis, and cyclosporine-containing preparations (e.g., Restasis (registered trademark)) are also used to treat ocular inflammation associated with keratoconjunctivitis. Various treatments are used for ocular inflammation depending on the symptoms and its causes. For example, antibiotics are used to treat bacterial keratitis / conjunctivitis, antihistamines are used to treat allergic keratitis / conjunctivitis, and steroid preparations are used to treat uveitis.

[0004] Conjunctival and / or corneal injuries refer to damages or injuries of the conjunctiva and / or cornea caused by various causes including heat, chemical burns, etc. Such injuries cause corneal ulcers (or ulcerative keratitis). As described above, various treatments are used for ocular inflammation caused by conjunctival and / or corneal injuries depending on the symptoms and causes. However, there is a need to develop drugs that have satisfactory improving or treating activity against conjunctival / corneal injuries and corneal ulcers themselves.

[0005] Ophthalmic diseases such as xerophthalmia, ocular inflammation (e.g., keratitis, conjunctivitis, keratoconjunctivitis, uveitis, scleritis, etc.), conjunctival / corneal damage, and corneal ulcers require long-term treatment and have a high recurrence rate. Therefore, there is a need in the art for the development of new drugs with new mechanisms of action to maintain ocular homeostasis, such as effective tear film stabilization, inhibition of corneal and conjunctival inflammation, and promotion of ocular wound healing. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have conducted various studies to develop peptide derivatives capable of improving or treating ophthalmic diseases including xerophthalmia, ocular inflammation, conjunctival and / or corneal damage, etc. In particular, the present inventors have selected D-amino acid-containing peptide derivatives that exhibit excellent anti-inflammatory activity through tests using cell lines, and conducted various studies. As a result, the present inventors have discovered that a specific D-amino acid-containing peptide derivative exhibits excellent improving activity in xerophthalmia and corneal damage models.

[0007] Therefore, an object of the present invention is to provide the specific D-amino acid-containing peptide derivative.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing, ameliorating, or treating an ophthalmic disease, which contains the specific D-amino acid-containing peptide derivative.

[0009] Another object of the present invention is to provide therapeutic uses of the specific D-amino acid-containing peptide derivatives.

[0010] Another object of the present invention is to provide use of the specific D-amino acid-containing peptide derivative in a method for preventing or treating ophthalmic diseases.

[0011] Another object of the present invention is to provide a method for preventing or treating an ophthalmic disease in a subject in need thereof, the method comprising administering the specific D-amino acid-containing peptide derivative to the subject. [Means for solving the problem]

[0012] According to one aspect of the present invention, there is provided a compound of formula 1, or a pharma- ceutically acceptable salt thereof:

[0013] [ka]

[0014] In the formula, X is -OH or -NH2.

[0015] In the compounds of the present invention or pharma- ceutically acceptable salts thereof, preferably, X may be --NH2.

[0016] In another aspect of the present invention, there is provided a pharmaceutical composition for preventing, ameliorating or treating an ophthalmic disease, comprising the compound or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0017] In another aspect, the present invention provides a cosmetic composition or cosmetic additive for preventing or improving an ophthalmic disease, which comprises the compound or a pharma- ceutical acceptable salt thereof.

[0018] According to a further aspect of the invention there is provided the compound, or a pharma- ceutically acceptable salt thereof, for use in therapy.

[0019] A further aspect of the present invention provides the use of the compound or a pharma- ceutically acceptable salt thereof for use in a method for the prophylaxis or treatment of an ophthalmic disease.

[0020] According to yet another aspect of the present invention, there is provided a method of preventing or treating an ophthalmic disease in a subject in need thereof, comprising administering to said subject said compound or a pharma- ceutically acceptable salt thereof.

[0021] In the present invention, the ophthalmological disease may be one or more diseases selected from the group consisting of xerophthalmia, ocular inflammation, conjunctival damage, corneal damage, and corneal ulcer. In one embodiment, the ophthalmological disease may be xerophthalmia. In another embodiment, the ophthalmological disease may be ocular inflammation. In yet another embodiment, the ophthalmological disease may be conjunctival damage, corneal damage, or corneal ulcer.

[0022] The pharmaceutical composition of the present invention may have the form of eye drops. The eye drops may be in the form of a solution or a suspension. Effect of the Invention

[0023] The present invention has revealed that the D-amino acid-containing peptide derivative (i.e., the compound of Chemical Formula 1) or a pharma- ceutically acceptable salt thereof exhibits excellent anti-inflammatory activity and also excellent improving activity in xerophthalmia and corneal injury models. Therefore, the compound according to the present invention or a pharma- ceutically acceptable salt thereof can be usefully applied to the prevention, improvement and treatment of various ophthalmic diseases, such as xerophthalmia, ocular inflammation, conjunctival injury, corneal injury, corneal ulcer, etc. [Brief description of the drawings]

[0024] [Figure 1]The results of in vitro anti-inflammatory activity evaluation of the compounds of the present invention are shown in FIG. 1A. FIG. 1B. FIG. 1C. shows the results of evaluating the inhibitory activity of the compounds of the present invention against the expression of inflammatory cytokines (TNF-α). FIG. 1D. shows the results of evaluating the inhibitory activity of the compounds of the present invention against the expression of inflammatory cytokines (IL-1β). In FIG. 1A and FIG. 1B, "Compound 1" represents the compound of formula 1a prepared in Example 1, and "Compound 2" represents the acetate salt of the compound of formula 1b prepared in Example 2. [Diagram 2] The results are shown below, in which the compound of the present invention was instilled into the eyes of mice with xerophthalmia induced, and then tear volume was measured. ***: significant difference with respect to G1, p<0.001; ### / ## / #: significant difference with respect to G2, p<0.001 / p<0.01 / p<0.05; $$$ / $$: significant difference with respect to G3, p<0.001 / p<0.01. [Diagram 3] The compound of the present invention was instilled into the eyes of mice with xerophthalmia induced, and the corneal fluorescent dye staining score was measured. ** / *: significant difference with respect to G1, p<0.01 / p<0.05, ### / ##: significant difference with respect to G2, p<0.001 / p<0.01, $$$ / $$: significant difference with respect to G3, p<0.001 / p<0.01. [Figure 4] FIG. 1 shows the results of instilling a compound of the present invention into the eyes of mice with xerophthalmia induced, followed by measuring the level of inflammation by histopathological examination. [Diagram 5] The compound of the present invention was instilled into the eyes of rats with corneal damage induced, and the corneal fluorescent dye staining score was measured. **: significant difference with respect to G1, p<0.01; ### / ## / #: significant difference with respect to G2, p<0.001 / p<0.01 / p<0.05; $$$ / $$: significant difference with respect to G3, p<0.001 / p<0.01. [Figure 6]The compound of the present invention was instilled into the eyes of rats with corneal injury, and the results of measuring the levels of inflammatory cell infiltration in the corneal stromal cells and anterior chamber by histopathological examination are shown below. **: significant difference with respect to G1, p<0.01; ##: significant difference with respect to G2, p<0.01; $$$ / $$ / $: significant difference with respect to G3, p<0.001 / p<0.01 / p<0.05. [Figure 7] 1 shows the results of 1H NMR analysis of the compound of the present invention produced by liquid phase peptide synthesis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention provides a specific D-amino acid-containing peptide derivative, namely, a compound of the following Chemical Formula 1 or a pharma- ceutically acceptable salt thereof:

[0026] [ka]

[0027] In the formula, X is -OH or -NH2.

[0028] In one embodiment, the compound of formula 1 or a pharma- ceutically acceptable salt thereof of the present invention may be in the form of a D-amino acid-containing peptide, i.e., in the compound of formula 1 or a pharma- ceutically acceptable salt thereof of the present invention, X may be -OH and may have the structure of formula 1a below.

[0029] [ka]

[0030] In another embodiment, the compound of formula 1 or a pharma- ceutically acceptable salt thereof of the present invention may be preferably in the form of a D-amino acid-containing peptide derivative, i.e., in the compound of formula 1 or a pharma- ceutically acceptable salt thereof of the present invention, X may be -NH2, and may have the structure of formula 1b below.

[0031] [ka]

[0032] The compound of formula 1 of the present invention may be in the form of a pharma- ceutically acceptable salt. The salts include conventional acid addition salts, such as salts derived from inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, and bromic acid, and salts derived from 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, embonic acid, aspartic acid, glutamic acid, and acetylsalicylic acid. The salts also include salts of sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid.

[0033] The compound of formula 1 according to the present invention or a pharma- ceutically acceptable salt thereof may be prepared by various methods. For example, the compound of formula 1 according to the present invention or a pharma- ceutically acceptable salt thereof may be prepared by solid-phase peptide synthesis or solution-phase peptide synthesis using a D-amino acid (e.g., D-aspartic acid (dASP), D-asparagine (dASN)) having an amino protecting group (e.g., Boc (tert-butoxycarbonyl)) and a carboxylic acid protecting group (e.g., Bzl (benzyl)).

[0034] The compound of formula 1 or a pharma- ceutical acceptable salt thereof of the present invention exhibits excellent anti-inflammatory activity and also exhibits excellent improving activity in xerophthalmia and corneal injury models, and therefore can be usefully used for the prevention, improvement and treatment of various ophthalmic diseases. Therefore, the present invention includes a pharmaceutical composition for preventing, improving or treating ophthalmic diseases, comprising a therapeutically effective amount of the compound of formula 1 or a pharma- ceutical acceptable salt thereof as an active ingredient.

[0035] The present invention also provides a therapeutic use of the compound of Formula 1 or a pharma- ceutically acceptable salt thereof. In some embodiments, the therapeutic use is for use in a method for the prophylaxis or treatment of an ophthalmic disease.

[0036] The present invention also provides a method for preventing or treating an ophthalmic disease in a subject in need thereof, comprising administering to the subject the compound of Formula 1 or a pharma- ceutically acceptable salt thereof or the pharmaceutical composition.

[0037] The term "subject" as used herein refers to any mammalian subject to which the compound or its pharma- ceutically acceptable salt or composition described herein is administered. Non-limiting examples include humans, pets (e.g., dogs, cats, etc.), livestock animals (e.g., cows, sheep, pigs, horses, etc.) and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), particularly humans, that require diagnosis, treatment or therapy. The methods described herein are applicable to both human prophylactic or therapeutic and veterinary applications.

[0038] As used herein, the phrase "subject in need" includes subjects such as mammalian subjects who would benefit from the administration of a compound, or a pharma- ceutically acceptable salt or composition thereof, described herein.

[0039] As used herein, the term "ophthalmic disease" refers to a disease or condition that affects or is associated with the eye or a portion or area of ​​the eye. In some embodiments, the ophthalmic disease may be a disease that causes dryness of the eye. In some embodiments, the ophthalmic disease may be a disease that occurs in the cornea or conjunctiva. In some embodiments, the ophthalmic disease may be a disease that causes damage to the cornea or conjunctiva. In some embodiments, the ophthalmic disease may be a disease that causes defects or necrosis of the cornea or conjunctiva. In some embodiments, the ophthalmic disease may be a disease that causes inflammation of the eye.

[0040] In some embodiments, the ophthalmic disease may be one or more diseases selected from the group consisting of xerophthalmia; ocular inflammation; conjunctival injury; corneal injury; and corneal ulcer. In some embodiments, the ophthalmic disease may be xerophthalmia. In some embodiments, the ophthalmic disease may be ocular inflammation, for example, selected from the group consisting of conjunctivitis, keratitis (including neurotrophic keratitis), keratoconjunctivitis, uveitis, and scleritis. In some embodiments, the ophthalmic disease may be conjunctival injury; corneal injury; or corneal ulcer.

[0041] The pharmaceutical composition of the present invention may be formulated into various dosage forms using pharma- ceutical acceptable excipients or carriers.Preferably, the pharmaceutical composition of the present invention may have the dosage form of eye drop, eye ointment, spray, and the eye drop may be in the form of a solution or suspension.

[0042] For example, an eye drop in the form of a solution may contain, in addition to the compound of formula 1 or a salt thereof, a solubilizing agent such as polyethylene glycol 400, glycerin, etc.; a stabilizer such as EDTA, etc.; a buffer such as boric acid, etc.; and a pH adjusting agent such as hydrochloric acid or sodium hydroxide, in sterile water. Also, an eye drop in the form of a suspension may contain, in addition to the compound of formula 1 or a salt thereof, a viscosity adjusting agent such as crosslinked polyvinylpyrrolidone (e.g., povidone K-25), etc.; an isotonicity adjusting agent such as sodium chloride, etc.; a stabilizer such as EDTA, etc.; a buffer such as boric acid, Borax, etc.; and a pH adjusting agent such as hydrochloric acid or sodium hydroxide, in sterile water. If necessary, the pharmaceutical composition in the form of eye drop may be sterilized by a conventional method, or may contain auxiliary agents such as preservatives, hydrating agents, emulsifiers, solubilizers, salts for adjusting osmotic pressure, and / or buffers.

[0043] The compound of formula 1 or a pharma- ceutically acceptable salt thereof may be administered to a patient suffering from an ophthalmic disease in the form of eye drops having a concentration of, for example, about 0.01% to about 10%, in an amount of 1 to 12 drops per eye, divided and administered 1 to 6 times a day. Of course, the dose may vary depending on the age, sex, sensitivity, symptoms, or severity of the disease of the patient.

[0044] The present invention also provides a cosmetic composition for preventing or improving an ophthalmic disease, comprising a compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof. In some embodiments, the compound of Chemical Formula 1 or a pharma- ceutically acceptable salt thereof may be provided as a cosmetic additive. In some embodiments, the cosmetic composition may be in the form of a mist or spray. In some embodiments, the cosmetic additive may be included in an eye cosmetic product, such as an eyebrow pencil, an eyeliner, an eyeshadow, a mascara, or an eye makeup remover, for the purpose of preventing or improving the ophthalmic disease. EXAMPLES

[0045] The present invention will be described in more detail below using examples and test examples. However, the following examples and test examples are merely illustrative of the present invention, and the present invention is not limited thereto.

[0046] Example 1. Preparation of Compound of Formula 1a

[0047] [ka]

[0048] Compound 1a was prepared by FMOC solid-phase peptide synthesis using an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, Korea) with D-aspartic acid (dASP) and D-asparagine (dASN) with Boc as amino protecting group and Bzl as carboxylic acid protecting group. The Fmoc protecting group was removed by reacting twice with 20% piperidine in dimethylformamide for 10 min, and coupling was performed using Fmoc amino acid (6 equivalents), hydroxybenzotriazole (HOBt) (6 equivalents), hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) (6 equivalents), and N,N-diisopropylethylamine (DIPEA) (12 equivalents). At 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. The resulting mixture was centrifuged with ether to recover the peptide. The crude peptide was dissolved in distilled water and purified by reversed-phase HPLC using a C18 reversed-phase column. Separation was performed using a water-acetonitrile linear gradient (acetonitrile concentration: 0-50% (v / v)). The purified fraction was lyophilized, dissolved again in water containing 0.5% acetic acid, and then lyophilized again. The molecular weight of the purified peptide was confirmed using LC / MS (Shimadzu LC / MS-2020 series, Japan), and FDT-12012 (Operon, Korea) was used for lyophilization.

[0049] Example 2. Preparation of compound of formula 1b

[0050] [ka]

[0051] The acetate salt of compound of formula 1b was prepared by solution-phase peptide synthesis using D-aspartic acid (dASP) and D-asparagine (dASN) having Boc as the amino protecting group and Bzl as the carboxylate protecting group as follows.

[0052] Phase 1:

[0053] [ka]

[0054] To a solution of compound 1 (50.0 g, 154 mmol, 1.00 eq.) in tetrahydrofuran (500 mL) was added 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.). The mixture was stirred at 20 °C for 15 h. TLC (petroleum ether: ethyl acetate = 1:3) showed complete consumption of compound 1 (Rf = 0.52) and formation of one new spot (Rf = 0.45). The reaction mixture was diluted with H2O (500 mL) and vacuum filtered to obtain a residue. The crude product was triturated with HO (300 mL) for 0.5 h at 20 °C, then filtered to give a white solid, which was treated with petroleum ether (100 mL) for 0.5 h at 20 °C. Compound 2 (47.0 g, 143 mmol, 92.6% yield, 98.3% purity) was obtained as a white solid, which was 1 H NMR and LCMS (R t =1.08min, MS cal.:322.1, MS observed:[M+H] + =323.0). 1The H NMR analysis results are as follows:

[0055] 1 H NMR (400 MHz, DMSO-d6) δ 7.37 - 7.31 (m, 5H), 7.25 (s, 1H), 7.09 - 7.01 (m, 2H), 5.12 - 5.05 (m, 2H), 4.32 - 4.27 (m, 1H), 2.79 - 2.74 (m, 1H), 2.61 - 2.57 (m, 1H), 1.37 (s, 9H).

[0056] Phase 2:

[0057] [ka]

[0058] To a solution of compound 2 (47.0 g, 143 mmol, 98.3% purity, 1.00 eq.) in ethyl acetate (EtOAc) (100 mL) was added HCl / EtOAc (4 M, 196 mL, 5.49 eq.). The mixture was stirred at 20° C. for 15 h. TLC (petroleum ether:ethyl acetate=1:3) showed complete consumption of compound 2 (Rf=0.45) and formation of one new spot (Rf=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. Compound 3 (37.0 g, 141 mmol, 98.4% yield, 98.7% purity, HCl salt) was obtained as a white solid, which was 1 H NMR, LCMS(R t =0.14min, MS cal.:222.1, MS observed:[M+H] + =223.1), and HPLC (R t =1.63 min, 98.7% purity). 1 The H NMR analysis results are as follows:

[0059] 1H NMR (400 MHz, D2O) δ 7.41 (s, 5H), 5.20 (s, 2H), 4.37 -4.34 (m, 1H), 3.16 - 3.03 (m, 2H).

[0060] Phase 3:

[0061] [ka]

[0062] To a solution of compound 4 (28.5 g, 122 mmol, 1.00 eq.) in acetone (500 mL) were added 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (25.8 g, 135 mmol, 1.10 eq.), hydroxybenzotriazole (HOBt) (16.5 g, 122 mmol, 1.00 eq.), and compound 3 (36.9 g, 141 mmol, 98.7% purity, 1.15 eq, HCl salt) at 20° C., followed by dropwise addition of N-methylmorpholine (NMM) (37.2 g, 368 mmol, 40.4 mL, 3.00 eq.) at 20° C. After 2 h, a solid precipitated. The resulting mixture was stirred at 20°C for 13 h. TLC (dichloromethane:methanol=7:1) showed the complete consumption of compound 3 (Rf=0.24) and the formation of 4 new spots (Rf=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. Compound 5 (40.6 g, 92.0 mmol, 74.9% yield, 98.9% purity) was obtained as a white solid, which was 1 H NMR, LCMS(R t =0.47min, MS cal.:436.2, MS observed:[M+H] + =437.0) and HPLC (Rt =1.77 min, 98.9% purity). 1 The H NMR analysis results are as follows:

[0063] 1 H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 8.4 Hz, 1H), 7.39 - 7.29 (m, 7H), 7.21 (s, 1H), 5.07 (s, 2H), 4.60 - 4.55 (m, 1H), 4.19 - 4.14 (m, 1H), 2.87 -2.81 (m, 1H), 2.68 - 2.62 (m, 1H), 2.55 - 2.53 (m, 1H), 2.42 - 2.36 (m, 1H), 1.36 (s, 9H).

[0064] Phase 4:

[0065] [ka]

[0066] 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 h. TLC (dichloromethane:methanol=7:1) showed the complete consumption of compound 5 (Rf=0.57) and the formation of one new spot (Rf=0.07). After diluting the reaction mixture with methyl tert-butyl ether (MTBE) (500 mL), the solid was precipitated and vacuum filtered to obtain a white solid. Compound 6 (35.4 g, crude product, HCl salt) was obtained as a white solid, which was 1 H NMR and LCMS (R t =0.12min, MS cal.:336.1, MS observed:[M+H] + =337.1). 1 The H NMR analysis results are as follows:

[0067] 1 H NMR (400 MHz, DMSO-d6) δ 7.32 - 7.25 (m, 5H), 5.04 (s, 2H), 4.59 - 4.55 (m, 1H), 4.04 -4.01 (t, J = 6.8 Hz, 1H), 2.80 -2.70 (m, 4H).

[0068] Phase 5:

[0069] [ka]

[0070] 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) was added N-methylmorpholine (NMM) (11.9 g, 118 mmol, 13.0 mL, 3.00 eq.). The mixture was stirred at 20° C. for 2.5 h. LCMS showed complete consumption of compound 7 and one major peak with the desired mass (R t =0.34min, MS cal.:675.2, MS observed:[M+H] + =676.1) was detected. After diluting the reaction mixture with H2O (500 mL) and ethyl acetate (150 mL), the solid was precipitated, stirred at 20 °C for 3 h, and vacuum filtered to obtain a yellow solid, which was treated with H2O (150 mL * 3) at 20 °C for 1 h. Compound 8 (22.0 g, crude product) was obtained as a yellow solid, which was identified by LCMS (R t =0.58min, MS cal.:675.2, MS observed:[M+H] + =676.3).

[0071] Phase 6:

[0072] [ka]

[0073] To a solution of compound 8 (24.0 g, 35.5 mmol, 1.00 eq.) in methanol (350 mL) and acetic acid (350 mL) was added Pd / C (10.0% purity, 4.80 g) under nitrogen atmosphere. The suspension was degassed and purged with hydrogen three times. The mixture was stirred under hydrogen (50 Psi) at 25° C. for 18 h. LCMS showed complete consumption of compound 8 and one major peak with the desired mass (R t =0.07min, MS cal.:361.1, MS observed:[M+H] + =362.1) was detected. The mixture was filtered, the filter cake was washed with H2O (50.0mL*2), and then the combined filtrate was concentrated to dryness to give a residue. The crude product was treated with ethanol (50.0mL) at 20°C for 0.6 h and vacuum filtered to give a yellow solid, which was treated with methanol (30.0mL) at 20°C for 0.6 h. The product (10.0g, crude) was obtained as a yellow solid, which was identified by LCMS (Rt=0.10min, MS cal.:361.1, MS observed:[M+H] + =361.9). The crude product was purified by prep-HPLC (0.5% HOAc) to give the product (5.00 g, 11.5 mmol, 97.4% purity, HOAc salt) as a white solid, which was analyzed by Special LCMS (R t =2.87min, MS cal.:361.1, MS observed:[M+H] + =362.1) and 1 Confirmed by 1 H NMR. 1 The H NMR analysis results (FIG. 7) are as follows:

[0074] 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 7.21 (s, 1H), 7.13 (s, 1H), 6.97 (s, 1H), 4.48 (s, 1H), 4.41 (dt, J1= 5.2 Hz, J2= 8.0 Hz, 1H), 3.81 (dd, J1= 5.2 Hz, J2=8.0 Hz, 1H), 2.69 - 2.52 (m, 4H), 2.46-2.37 (m, 2H).

[0075] Test Example 1: In vitro anti-inflammatory test Macrophages (RAW 264.7) treated with lipopolysaccharide (LPS) were treated with the compounds prepared in Examples 1 and 2 as test substances, and the changes in the production of inflammatory cytokines (TNF-α, IL-1β) were measured by ELISA and Western blotting, respectively.

[0076] Specifically, 1 × 10 cells were placed in each well of a 96-well plate. 4Each macrophage (RAW 264.7) was dispensed with 0.1 mL of DMEM (Dulbecco's Modified Eagle medium) containing 10% FBS (Gibco) and 1% penicillin / streptomycin. After culturing in a 5% CO2, 37°C incubator for 1 day, when the cell confluency reached 80% or more, LPS (100 ng / ml) and test substances were treated at concentrations of 1 μM and 10 μM, respectively, and incubated for 24 hours. The control group was treated with LPS (100 ng / ml) only and incubated for 24 hours. The supernatants of each test group were prepared as samples for ELISA analysis. TNF-α production was confirmed using an ELISA analysis kit (ELISA MAX Standard Set Human TNF-α; BioLegend, #430201) at 450 nm using a microplate reader according to the manufacturer's protocol. The results are shown in Figure 1A.

[0077] Also, 5 x 10 cells were added to each well of a 6-well plate. 6 Macrophages (RAW 264.7) were dispensed with 0.1 mL of DMEM (Dulbecco's Modified Eagle medium) containing 10% FBS (Gibco) and 1% penicillin / streptomycin. After culturing in a 5% CO2, 37°C incubator for 1 day, when the cell confluency reached 80% or more, LPS (100 ng / ml) and test substances were treated at concentrations of 1 μM and 10 μM, respectively, and incubated for 30 minutes. The control group was treated with LPS (100 ng / ml) only and incubated for 30 minutes. The cells were lysed using NP40 cell lysis buffer, quantified using the Bradford assay, and the expression level of IL-1β was measured by Western blotting. The results are shown in Figure 1B.

[0078] As can be seen from the results of Figures 1A and 1B, in the control group, the secretion and expression of inflammatory cytokines (TNF-α and IL-1β) increased due to LPS treatment. In contrast, in the test group treated with the test substance, the secretion and expression of inflammatory cytokines (TNF-α and IL-1β) was inhibited in a concentration-dependent manner. Therefore, the compounds of formula 1a and formula 1b obtained by the present invention exhibit excellent anti-inflammatory activity and can be usefully used to improve ocular inflammation such as conjunctivitis, keratitis, keratoconjunctivitis, uveitis, and scleritis.

[0079] Test Example 2: Activity evaluation in a dry eye syndrome model 1. Test Method (1) Administration of test substance Xerophthalmia was induced in specific pathogen-free (SPF) mice, C57BL / 6NHsd (male, 7 weeks old) [Coretech Co., Ltd., Korea]. Specifically, during the xerophthalmia induction period (Day -10 to Day 0), scopolamine hydrobromide was dissolved in physiological saline (PBS) and subcutaneously injected into the back skin of the mice at a dose of 0.5 mg / 0.2 mL, four times a day for 10 days. The tear volume of the mice in which xerophthalmia was induced was measured, and the mice were randomly divided into groups G2 to G6 according to Table 1 below so that the average tear volume of each group was uniformly distributed. The G1 group was a normal control group in which xerophthalmia was not induced.

[0080] From the day of induction of xerophthalmia (Day 0), the compound prepared in Example 2 (i.e., acetate salt of the compound of formula 1b, hereinafter referred to as "Compound 1b") was dissolved in saline (PBS) at concentrations of 0.1% and 1% as a test substance, and 2 μL / eye was instilled into the center of the right cornea of ​​G4 and G5 mice, three times a day for 10 days. The G2 group was an induced control group in which xerophthalmia was induced. The G3 group was instilled with saline (PBS) into the center of the right cornea of ​​mice in which xerophthalmia was induced. The G6 group (positive control group) was instilled with Restasis (registered trademark) eye drops (0.05%, containing cyclosporine) into the center of the right cornea of ​​mice in which xerophthalmia was induced.

[0081] [Table 1]

[0082] (2) Measurement of tear volume and corneal fluorescent dye staining score The tear volume was measured by the phenol red thread test (PRT test). Specifically, on the 5th and 10th days after the start of test substance administration, a phenol red thread was placed on the lateral canthus of the eye for 20 seconds, and the tear volume (mm) was measured.

[0083] After measuring the tear volume, the animals were anesthetized on the 5th and 10th days after the start of test substance administration, and 1 μL of 1% sodium fluorescein was instilled into the lower conjunctival sac of the cornea of ​​the right eye of the animals. The degree of penetration of the corneal dye into five corneal zones (superior, nasal, central, inferior, and temporal) was evaluated using cobalt blue light according to the following criteria of the National Eye Institute grading system (NEI).

[0084] - Score:0~3(0:normal, 1:mild, 2:moderate, 3:severe) - Total score:15 (3) Autopsy and histopathological examination On the day of necropsy (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, and thin-sectioned to prepare specimens for histopathological examination, and stained with hematoxylin and eosin (H&E). The inflammatory cell infiltration in the cornea, conjunctiva, and anterior chamber was evaluated using an optical microscope (Olympus BX53, Japan). Inflammation of the cornea and conjunctiva was evaluated by a known method, and the score scale was as follows: Score: 0 (normal), 1 (mild), 2 (moderate), 3 (severe).

[0085] (4) Statistical analysis Statistical analysis was performed using Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA), and a p value of less than 0.05 was considered statistically significant.

[0086] 2. Test Results (1) Tear volume measurement The results of tear volume measurement are shown in Figure 2. On the 5th and 10th days after the start of test substance administration, the tear volume levels of the induction control group (G2), PBS administration 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 that of the normal control group (G1) (p<0.001). The tear volume levels of G4, G5, and G6 were significantly higher than that of G2 (p<0.001, p<0.01, or p<0.05). On the 5th day after the start of test substance administration, the tear volume levels of G5 and G6 were statistically significantly higher than that of G3 (p<0.01), and on the 10th day after the start of test substance administration, the tear volume levels of G4, G5, and G6 were significantly higher than that of G3 (p<0.001).

[0087] (2) Corneal fluorescent dye staining score measurement The results of the corneal fluorescent dye staining score are shown in Figure 3. From the fifth to the tenth day after the start of the administration of the test substance, the corneal fluorescent dye staining score levels of G2, G3, G4, G5 and G6 were statistically significantly higher than that of G1 (p<0.01 or p<0.05). The corneal fluorescent dye staining score levels of G4, G5 and G6 were significantly lower than that of G2 (p<0.001 or p<0.01), and the corneal fluorescent dye staining score levels of G4, G5 and G6 were statistically significantly lower than that of G3 (p<0.001 or p<0.01).

[0088] (3) Histopathological examination The histopathological examination results are shown in Figure 4. The inflammation levels in G2 and G3 were statistically significantly higher than that in G1 (p<0.01), while the inflammation and fibrosis levels in G4, G5, and G6 were significantly lower than that in G2 and G3 (p<0.001 or p<0.05).

[0089] 3. Discussion The effects of the test substances on a C57BL / 6NHsd mouse model in which xerophthalmia was induced 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).

[0090] The tear volume was measured using a phenol red thread. From the 5th to 10th day after the start of test substance administration, the tear volume levels in the 0.1% and 1% test substance groups were statistically significantly higher than the induced control group, and the tear volume level in the 1% test substance group was significantly higher than the PBS group.

[0091] As a result of measuring the corneal fluorescent dye staining score, from 5 to 10 days after the start of test substance administration, the corneal fluorescent dye staining score levels in the 0.1% test substance administration group and the 1% test substance administration group were observed to be at statistically significantly lower levels than the induction control group and the PBS administration group.

[0092] As a result of histopathological examination, no inflammation was observed in the cornea or conjunctiva in the groups administered 0.1% or 1% of the test substance.

[0093] Therefore, repeated ocular instillation of the test substance may be useful in ameliorating xerophthalmia and ocular inflammation in a C57BL / 6 mouse model in which xerophthalmia is induced.

[0094] Test Example 3: Activity evaluation in corneal injury model 1. Test Method (1) Administration of test substance Corneal injury was induced in specific pathogen-free (SPF) Sprague Dawley rats (male, 7 weeks old) [Coretech Co., Ltd., Korea]. Specifically, the rats were anesthetized, and a filter paper soaked in 2 μL of 0.1 M NaOH solution was applied to the midline of the cornea of ​​the right eye for about 10 seconds, and then washed with saline. The rats in which corneal injury was induced were weighed, and randomly divided into groups G2 to G6 according to Table 2 below so that the average weight of each group was evenly distributed. Group G1 was a normal control group in which no corneal injury was induced.

[0095] From the day of corneal injury induction (Day 0), the compound prepared in Example 2 (i.e., acetate salt of the compound of formula 1b, hereinafter referred to as "Compound 1b") was dissolved in saline (PBS) at concentrations of 0.1% and 1% as a test substance, and 10 μL / eye was instilled into the eyes of G4 and G5 mice, respectively, three times a day for 7 days. The G2 group was a control group in which corneal injury was induced. The G3 group was rats in which corneal injury was induced and saline (PBS) was instilled into their eyes. The G6 group (positive control group) was rats in which corneal injury was induced and Restasis (registered trademark) eye drops (0.05%, containing cyclosporine) were instilled into their eyes.

[0096] [Table 2]

[0097] (2) Corneal fluorescent dye staining score measurement Six hours, 1 day, 2 days, 3 days, 5 days, and 7 days after the induction of corneal injury, the animals were anesthetized and 1 μL of 1% sodium fluorescein was instilled into the lower conjunctival sac of the cornea of ​​the right eye of the animals. The degree of penetration of the corneal dye into five corneal zones (superior, nasal, central, inferior, temporal) was evaluated using cobalt blue light according to the following criteria of the NEI scoring system.

[0098] - Score:0~3(0:normal, 1:mild, 2:moderate, 3:severe) - Total score:15 (3) Autopsy and histopathological examination On the day of necropsy (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, and thin-sectioned to prepare specimens for histopathological examination, and stained with hematoxylin and eosin (H&E). The infiltration of inflammatory cells in the cornea, conjunctiva, and anterior chamber was evaluated using an optical microscope (Olympus BX53, Japan). Infiltration of inflammatory cells in the corneal stroma and in the anterior chamber was evaluated by a known method, and the score scale was as follows: Score: 0 (normal), 1 (mild), 2 (moderate), 3 (severe).

[0099] (4) Statistical analysis 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.

[0100] 2. Test Results (1) Corneal fluorescent dye staining score measurement The results of the corneal fluorescent dye staining score are shown in Figure 5. From 6 hours to 5 days after the start of test substance administration, the corneal fluorescent dye staining score levels of all corneal damage groups (G2 to G6) were statistically significantly higher than that of the normal control group (G1) (p<0.01). On the 6th day after the start of test substance administration, the corneal fluorescent dye staining score levels of the induced control group (G2) and the PBS-administered group (G3) were significantly higher than that of G1 (p<0.01). Six hours after the start of test substance administration and from the second to sixth days, the corneal fluorescent dye 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 that of G2 (p<0.001, p<0.01, or p<0.05), and from the third to sixth days after the start of test substance administration, the corneal fluorescent dye staining score levels of G4, G5, and G6 were significantly lower than that of G3 (p<0.001 or p<0.01).

[0101] (2) Histopathological examination The results of the histopathological examination are shown in Figure 6. The levels of inflammatory cell infiltration in the keratocytes and anterior chamber of G2 and G3 were statistically significantly higher than those in G1 (p<0.01 or p<0.05), while the levels of inflammatory cell infiltration in the keratocytes and anterior chamber of G4, G5, and G6 were statistically significantly lower than those in G2 and G3 (p<0.001, p<0.01, or p<0.01).

[0102] 3. Discussion The effects of the test substances were evaluated in a Sprague Dawley rat model in which corneal damage was induced (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).

[0103] As a result of measuring the corneal fluorescent dye staining score, the corneal fluorescent dye staining score levels of the 0.1% test substance administration group and the 1% test substance administration group were observed to be significantly lower than the induction control group throughout the entire test period except for the 1st day after the start of test substance administration, and the corneal fluorescent dye staining score levels of the 0.1% test substance administration group and the 1% test substance administration group were observed to be significantly lower than the PBS administration group from the 3rd day after the start of test substance administration to the end of the test period.

[0104] Histopathological examination showed that the inflammatory cell infiltration scores in the corneal stroma and anterior chamber of the 0.1% and 1% test substance groups were statistically significantly lower than the induction control and PBS groups. The histopathological examination results showed similar patterns to the corneal fluorescent dye staining scores.

[0105] Therefore, repeated instillation of a test substance in the Sprague-Dawley rat model in which corneal injury is induced may be useful for treating conjunctival injury, corneal injury, or corneal ulcers, and may be useful for improving ocular inflammation caused by the above diseases (e.g., conjunctivitis, keratitis, keratoconjunctivitis, neurotrophic keratitis, etc.).

Claims

1. A compound of formula 1 below, or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 In the formula, X is -OH or -NH ２ It is.

2. X is -NH ２ 2. The compound according to claim 1, wherein:

3. A pharmaceutical composition for preventing, ameliorating or treating an ophthalmological disease, comprising the compound according to claim 1 or 2 or a pharma- ceutically acceptable salt thereof as an active ingredient.

4. 4. The pharmaceutical composition according to claim 3, wherein the ophthalmological disease is one or more diseases selected from the group consisting of xerophthalmia, ocular inflammation, conjunctival injury, corneal injury, and corneal ulcer.

5. The pharmaceutical composition according to claim 3, wherein the ophthalmological disease is xerophthalmia.

6. 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 damage, corneal damage, or corneal ulcer.

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

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

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

11. 3. Therapeutic use of a compound according to claim 1 or 2 or a pharma- ceutically acceptable salt thereof.

12. 3. Use of the compound according to claim 1 or 2 or a pharma- ceutically acceptable salt thereof for use in a method for the prophylaxis or treatment of an ophthalmological disease.

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

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