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 novel D-amino acid derivatives and pharmaceutical compositions containing the same. More specifically, the present invention relates to novel peptide derivatives containing specific D-amino acids and pharmaceutical compositions containing the same for preventing, ameliorating, or treating ophthalmic diseases. [Background technology]

[0002] Dry eye syndrome (DES) is a common ophthalmic disease and a condition characterized by dry eyes. DES occurs when the eyes do not produce enough tears or when the tear film is unstable, causing tears to evaporate too quickly. Dry eye syndrome can occur for a variety of reasons, including contact lens use and meibomian gland dysfunction. Artificial tears are commonly used to improve dry eye syndrome, and cyclosporine-containing preparations (e.g., Restasis®) are used as medications to treat dry eye syndrome.

[0003] Ocular inflammation includes inflammation occurring in the eyeball, such as conjunctivitis, keratitis, keratoconjunctivitis, uveitis, and scleritis. Ocular inflammation is known to occur for a variety of reasons. For example, xerophthalmia is known to cause keratoconjunctivitis, and cyclosporine-containing preparations (e.g., Restasis®) have also been used to treat ocular inflammation associated with keratoconjunctivitis. Various treatments for ocular inflammation are used depending on the symptoms and 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 damage or injuries to the conjunctiva and / or cornea caused by various factors, including heat and chemical burns. Such injuries can lead to corneal ulcers (also known as 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 for the development of drugs that have satisfactory improvement or therapeutic activity against conjunctival / corneal injuries and corneal ulcers themselves.

[0005] Ophthalmic diseases such as dry eye, ocular inflammation (e.g., keratitis, conjunctivitis, keratoconjunctivitis, uveitis, sclera, 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 that maintain ocular homeostasis, such as effective tear film stabilization, suppression of corneal and conjunctival inflammation, and the ability to promote ocular wound healing. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have conducted extensive research to develop peptide derivatives capable of ameliorating or treating ophthalmic diseases, including dry eye syndrome, ocular inflammation, and conjunctival and / or corneal damage. In particular, the present inventors screened D-amino acid-containing peptide derivatives that exhibited excellent anti-inflammatory activity through cell line tests and conducted extensive research. As a result, they discovered that specific D-amino acid-containing peptide derivatives exhibited excellent ameliorative activity in dry eye syndrome 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 ophthalmic diseases, which comprises 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 a use of the specific D-amino acid-containing peptide derivative for use 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, which comprises 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 below, or a pharmaceutically acceptable salt thereof:

[0013] [ka]

[0014] In the formula, X is —OH or —NH 2 .

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

[0016] In another aspect, the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating an ophthalmic disease, which comprises the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] Another aspect of the present invention provides a cosmetic composition or cosmetic additive for preventing or improving ophthalmic diseases, which comprises the compound or a pharmaceutically acceptable salt thereof.

[0018] A further aspect of the present invention provides the above compound or a pharmaceutically acceptable salt thereof for use in therapy.

[0019] Yet another aspect of the present invention provides use of the compound or a pharmaceutically acceptable salt thereof for use in a method for preventing or treating 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 the compound or a pharmaceutically 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 be in the form of eye drops, which may be in the form of a solution or a suspension. [Effects 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 pharmaceutically acceptable salt thereof exhibits excellent anti-inflammatory activity and also exhibits excellent ameliorative activity in xerophthalmia and corneal injury models. Therefore, the compound according to the present invention or a pharmaceutically acceptable salt thereof can be usefully applied to the prevention, amelioration, and treatment of various ophthalmic diseases, such as xerophthalmia, ocular inflammation, conjunctival injury, corneal injury, and corneal ulcer. [Brief explanation of the drawings]

[0024] [Figure 1]The results of evaluating the in vitro anti-inflammatory activity of the compounds of the present invention are shown below. FIG. 1A shows the results of evaluating the inhibitory activity of the compounds of the present invention against the expression of inflammatory cytokine (TNF-α). FIG. 1B shows the results of evaluating the inhibitory activity of the compounds of the present invention against the expression of inflammatory cytokine (IL-1β). In FIGS. 1A and 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. [Figure 2] The compounds of the present invention were instilled into the eyes of mice with xerophthalmia induced, and then tear volume was measured. ***: significant difference compared to G1 (p<0.001), ### / ## / #: significant difference compared to G2 (p<0.001 / p<0.01 / p<0.05), $$$ / $$: significant difference compared to G3 (p<0.001 / p<0.01). [Figure 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 then measured. ** / *: significant difference compared to G1 (p<0.01 / p<0.05), ### / ##: significant difference compared to G2 (p<0.001 / p<0.01), $$$ / $$: significant difference compared to G3 (p<0.001 / p<0.01). [Figure 4] The compounds of the present invention were instilled into the eyes of mice with xerophthalmia induced, and the inflammation level was measured by histopathological examination. [Figure 5] The compounds of the present invention were instilled into the eyes of rats with corneal injury induced, and the corneal fluorescent dye staining score was measured. **: significant difference compared to G1 (p<0.01), ### / ## / #: significant difference compared to G2 (p<0.001 / p<0.01 / p<0.05), $$$ / $$: significant difference compared 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 induced, and the levels of inflammatory cell infiltration in the keratocytes and anterior chamber were measured by histopathological examination. **: significant difference compared to G1 (p<0.01), ##: significant difference compared to G2 (p<0.01), $$$ / $$ / $: significant difference compared 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 INVENTION

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

[0026] [ka]

[0027] In the formula, X is —OH or —NH 2 .

[0028] In one embodiment, the compound of Formula 1 or a pharmaceutically 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 pharmaceutically 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 pharmaceutically 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 pharmaceutically acceptable salt thereof of the present invention, X may preferably be —NH 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 pharmaceutically 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 Chemical Formula 1 according to the present invention or a pharmaceutically acceptable salt thereof may be prepared by various methods. For example, the compound of Chemical Formula 1 according to the present invention or a pharmaceutically 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 pharmaceutically 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 useful for preventing, improving, and treating various ophthalmic diseases. Therefore, the present invention includes a pharmaceutical composition for preventing, improving, or treating ophthalmic diseases, which comprises a therapeutically effective amount of the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0035] The present invention also provides therapeutic uses of the compound of Formula 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutic use is for use in a method for preventing or treating 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 Chemical Formula 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0037] As used herein, the term "subject" refers to any mammalian subject to which a compound described herein or a pharmaceutically acceptable salt thereof or composition thereof 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, in need of diagnosis, treatment, or therapy. The methods described herein are applicable to both human prophylaxis or therapy and veterinary use.

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

[0039] As used herein, the term "ophthalmic disease" refers to a disease or condition that affects or is related to 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 pharmaceutically acceptable excipients or carriers. Preferably, the pharmaceutical composition of the present invention may have the dosage form of eye drops, eye ointment, or spray, and the eye drops may be in the form of a solution or suspension.

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

[0043] The compound of Formula 1 or a pharmaceutically 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%, at a dose of 1 to 12 drops per eye, 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 ameliorating ophthalmic diseases, comprising a compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Chemical Formula 1 or a pharmaceutically 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 eye cosmetic products such as eyebrow pencils, eyeliners, eyeshadows, mascaras, or eye makeup removers for the purpose of preventing or ameliorating the ophthalmic diseases. [Example]

[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 are not intended to limit the present invention.

[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) bearing Boc as the amino-protecting group and Bzl as the carboxylic acid-protecting group. The Fmoc protecting group was removed by two 10-minute reactions with 20% piperidine in dimethylformamide. Coupling was then carried out using the 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), and distilled water (DW) (90 / 2.5 / 2.5 / 2.5 / 2.5 volumes) for 2 hours to remove the resin and amino acid protecting groups. The resulting mixture was added with ether and centrifuged to recover the peptide. The crude peptide was dissolved in distilled water and purified by reverse-phase HPLC using a C18 reverse-phase column. Separation was performed using a water-acetonitrile linear gradient (acetonitrile concentration: 0–50% (v / v)). The purified fraction was lyophilized, redissolved 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 lyophilization was performed using an FDT-12012 (Operon, Korea).

[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) with Boc as the amino protecting group and Bzl as the carboxylic acid 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)O) (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 NHHCO (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 the complete consumption of compound 1 (Rf = 0.52) and the formation of one new spot (Rf = 0.45). The reaction mixture was diluted with HO (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 and 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 results of the H NMR analysis 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 the complete consumption of compound 2 (Rf = 0.45) and the formation of one new spot (Rf = 0.18). The reaction mixture was filtered to give a residue, which was treated with EtOAc (100 mL) for 0.5 h at 20 °C. 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 results of the H NMR analysis 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), 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) were added 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 four new spots (Rf = 0.11, 0.35, 0.41, 0.90). The reaction mixture was diluted with HO (1000 mL) and filtered to obtain a residue. The crude product was treated with HO (400 mL * 3) at 20 °C for 0.5 h, filtered, and obtained 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 results of the H NMR analysis 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] Stage 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 subsequently purified. 1 H NMR and LCMS (R t =0.12min, MS cal.:336.1, MS observed:[M+H] + =337.1). 1 The results of the H NMR analysis 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] Stage 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 and 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 analyzed by LCMS (R t =0.58min, MS cal.:675.2, MS observed:[M+H] + =676.3).

[0071] Stage 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), Pd / C (10.0% purity, 4.80 g) was added under a 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, and the filter cake was washed with H2O (50.0 mL*2), and then the combined filtrate was concentrated to dryness to give a residue. The crude product was treated with ethanol (50.0 mL) at 20 °C for 0.6 h and vacuum filtered to give a yellow solid, which was treated with methanol (30.0 mL) at 20 °C for 0.6 h. The product (10.0 g, crude) was obtained as a yellow solid, which was analyzed by LCMS (Rt = 0.10 min, 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 x 10 cells were placed in each well of a 96-well plate. 4Macrophages (RAW 264.7) were aliquoted with 0.1 mL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (Gibco) and 1% penicillin / streptomycin. After culturing for 1 day in a 5% CO2, 37°C incubator, when the cells reached 80% confluency, they were treated with LPS (100 ng / mL) and test substances at concentrations of 1 μM and 10 μM, respectively, and incubated for 24 hours. A control group was treated with LPS (100 ng / mL) alone and incubated for 24 hours. The supernatants from each test group were prepared for ELISA analysis. TNF-α production was determined using an ELISA kit (ELISA MAX Standard Set Human TNF-α; BioLegend, #430201) at 450 nm on 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 aliquoted 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 cells reached 80% confluency, they were treated with LPS (100 ng / mL) and the test compounds at 1 μM and 10 μM concentrations, respectively, and incubated for 30 minutes. A control group was treated with LPS (100 ng / mL) alone and incubated for 30 minutes. Cells were lysed using NP40 cell lysis buffer, quantified using a Bradford assay, and IL-1β expression was measured by Western blotting. The results are shown in Figure 1B.

[0078] As can be seen from the results in Figures 1A and 1B, the control group showed increased secretion and expression of inflammatory cytokines (TNF-α and IL-1β) upon treatment with LPS. In contrast, the test group treated with the test substance showed concentration-dependent suppression of secretion and expression of inflammatory cytokines (TNF-α and IL-1β). Therefore, the compounds of Formula 1a and Formula 1b obtained according to the present invention exhibit excellent anti-inflammatory activity and can be useful for ameliorating ocular inflammation such as conjunctivitis, keratitis, keratoconjunctivitis, uveitis, and sclera.

[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) C57BL / 6NHsd mice (male, 7 weeks old) [Coretech Co., Ltd., Korea]. Specifically, during the xerophthalmia induction period (Day -10 to Day 0), scopolamine hydrobromide (0.5 mg / 0.2 mL) was dissolved in physiological saline (PBS) and injected subcutaneously into the dorsal skin of the mice at a dose of 0.5 mg / 0.2 mL four times daily for 10 days. Tear volume was measured in xerophthalmia-induced mice, and they were randomly divided into groups G2 to G6 (Table 1 below) to ensure uniform distribution of mean tear volume in each group. Group G1 served as a normal control group without xerophthalmia induction.

[0080] Starting on Day 0, the compound prepared in Example 2 (i.e., the 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 the test substance. 2 μL / eye was instilled into the central cornea of ​​the right eyes of G4 and G5 mice, respectively, three times daily for 10 days. The G2 group was a control group in which xerophthalmia was induced. The G3 group received saline (PBS) instillation into the central cornea of ​​the right eyes of mice in which xerophthalmia was induced. The G6 group (positive control group) received Restasis® eye drops (0.05%, containing cyclosporine) instillation into the central cornea of ​​the right eyes of mice in which xerophthalmia was induced.

[0081] [Table 1]

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

[0083] After measuring 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 eyeball. 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 (NEI) grading system.

[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. The fixed tissues were trimmed, dehydrated, embedded in paraffin, and thin-sectioned to prepare specimens for histopathological examination. Hematoxylin and eosin (H&E) staining was performed, and inflammatory cell infiltration in the cornea, conjunctiva, and anterior chamber was evaluated using a light microscope (Olympus BX53, Japan). Corneal and conjunctival inflammation was evaluated using known methods and scored 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 tear volume measurement results are shown in Figure 2. On days 5 and 10 after the start of test substance administration, the tear volume levels of the induced control group (G2), PBS-administered 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 day 5 after the start of test substance administration, the tear volume levels of G5 and G6 were statistically significantly higher than those of G3 (p<0.01), and on day 10 after the start of test substance administration, the tear volume levels of G4, G5, and G6 were significantly higher than those of G3 (p<0.001).

[0087] (2) Corneal fluorescent dye staining score measurement The results of the corneal fluorescent dye staining scores are shown in Figure 3. From day 5 to day 10 after the start of test substance administration, the corneal fluorescent dye staining scores 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 scores 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 scores 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 in G1 (p<0.01), while the inflammation and fibrosis levels in G4, G5, and G6 were significantly lower than in G2 and G3 (p<0.001 or p<0.05).

[0089] 3. Discussion The effects of the test substance on a C57BL / 6NHsd mouse model in which xerophthalmia was induced were evaluated (G1: normal control group, G2: induced control group, G3: PBS-administered group, G4: 0.1% test substance-administered group, G5: 1% test substance-administered group, G6: positive control group).

[0090] Tear volume was measured using a phenol red thread. From day 5 to day 10 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 those in the induced control group, and the tear volume level in the 1% test substance group was observed to be significantly higher than that of the PBS group.

[0091] As a result of measuring the corneal fluorescent dye staining score, from day 5 to day 10 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 statistically significantly lower than those in 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 0.1% and 1% test substance administration groups.

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

[0094] Test Example 3: Activity evaluation in a 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, after anesthetizing the rats, 2 μL of filter paper soaked in 0.1 M NaOH solution was applied to the midline of the cornea of ​​the right eye for approximately 10 seconds, followed by rinsing with saline. The rats with corneal injury were weighed and randomly divided into groups G2 to G6 (Table 2 below) to ensure uniform distribution of mean weight within each group. Group G1 served as a normal control group without corneal injury.

[0095] Starting from the day of corneal injury induction (Day 0), the compound prepared in Example 2 (i.e., the 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 instilled into the eyes of G4 and G5 mice at 10 μL / eye, three times daily 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® eye drops (0.05%, containing cyclosporine) was 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. 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 NEI scoring system, as follows:

[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. The fixed tissues were trimmed, dehydrated, embedded in paraffin, and thin-sectioned to prepare specimens for histopathological examination. Hematoxylin and eosin (H&E) staining was performed, and inflammatory cell infiltration in the cornea, conjunctiva, and anterior chamber was evaluated using a light microscope (Olympus BX53, Japan). Infiltration of inflammatory cells in the corneal stroma and anterior chamber was assessed using known methods and scored 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 a p-value of less than 0.05 was considered statistically significant.

[0100] 2. Test Results (1) Corneal fluorescent dye staining score measurement The results of the corneal fluorescent dye staining score measurement 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 injury groups (G2 to G6) were statistically significantly higher than that of the normal control group (G1) (p<0.01). On day 6 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 days 2 to 6, the corneal fluorescent dye staining score levels in the 0.1% test substance administration group (G4), 1% test substance administration group (G5), and positive control group (G6) were significantly lower than those in G2 (p<0.001, p<0.01, or p<0.05). From days 3 to 6 after the start of test substance administration, the corneal fluorescent dye staining score levels in G4, G5, and G6 were significantly lower than those in G3 (p<0.001 or p<0.01).

[0101] (2) Histopathological examination The histopathological examination results 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), whereas 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 substance on a Sprague Dawley rat model in which corneal injury was induced were evaluated (G1: normal control group, G2: induced control group, G3: PBS-administered group, G4: 0.1% test substance-administered group, G5: 1% test substance-administered group, G6: positive control group).

[0103] As a result of measuring the corneal fluorescent dye staining score, 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 significantly lower than the induced control group throughout the entire test period except for the first day after the start of test substance administration, and from the third day after the start of test substance administration to the end of the test, 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 significantly lower than the PBS administration group.

[0104] Histopathological examination revealed 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 control group and PBS group. The histopathological examination results showed similar patterns to the corneal fluorescent dye staining scores.

[0105] Therefore, repeated instillation of a test substance into the Sprague-Dawley rat model in which corneal injury is induced may be useful for treating conjunctival injury, corneal injury, or corneal ulcer, and may also be useful for ameliorating 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 pharmaceutically acceptable salt thereof: 【Chemical 1】 In the formula, X is —OH or —NH ２ is.

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

3. A pharmaceutical composition comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

4. A pharmaceutical composition according to claim 3 for the prevention, improvement or treatment of ophthalmic diseases.

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

6. The pharmaceutical composition according to claim 4, wherein the ophthalmological disease is xerophthalmia.

7. The pharmaceutical composition according to claim 4, wherein the ophthalmic disease is ocular inflammation.

8. The pharmaceutical composition according to claim 4, wherein the ophthalmic disease is conjunctival damage, corneal damage, or corneal ulcer.

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

10. 10. The pharmaceutical composition according to claim 9, wherein the eye drops are in the form of a solution or suspension.

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

12. The cosmetic composition of claim 11, in the form of a mist or spray.

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