Novel actinomycete-derived compound having naphthol structure having rho kinase inhibitory ability and use thereof

Abstract

The present invention relates to a novel actinomycete-derived compound having a naphthol structure and having Rho kinase inhibitory ability, and a use thereof. The present invention has an excellent Rho kinase (ROCK) inhibitory ability and an excellent effect of inhibiting shrinkage of human trabecular meshwork (HTM) cells, and thus can be variously used as a pharmaceutical composition, a quasi-drug composition, a food composition and the like for preventing, alleviating, or treating glaucoma. In addition, the compound according to the present invention is derived from natural substances and has almost no side effects, and thus can be safely and continuously used.

Description

Novel compound derived from actinomycetes having a naphthol structure having low-kinase inhibitory activity and uses thereof

[0001] The present application claims priority to Korean Patent Application No. 10-2025-0014687 filed on February 5, 2025, and the entire specification is a reference to the present application.

[0002] The present invention relates to a novel compound derived from actinomycetes having a naphthol structure having rho kinase inhibitory ability and the use thereof.

[0003]

[0004] Glaucoma is a chronic neurodegenerative disease characterized by progressive damage to retinal ganglion cells and is known as one of the leading causes of blindness worldwide (Tham et al., 2014). This disease is multifactorial, involving various risk factors such as elevated intraocular pressure, aging, and family history; however, the only clinically controllable risk factor currently is elevated intraocular pressure. Intraocular pressure is maintained by the balance between the production and outflow of aqueous humor, and if the function of the trabecular meshwork (TM), the primary pathway for aqueous humor outflow, deteriorates, intraocular pressure rises, which can accelerate optic nerve damage (Jayaram et al., 2023).

[0005] Rho kinase (Rho-associated protein kinase, ROCK) is a serine / threonine kinase that controls cell growth, migration, metabolism, and apoptosis by regulating cell contraction and actin cytoskeleton assembly. Activated ROCK plays a crucial role in regulating actin-myosin contractility in smooth muscle and non-muscle cells by directly phosphorylating the myosin light chain (MLC) (Totsukawa et al., 2000). Additionally, ROCK inhibits the activity of myosin light chain phosphatase (MLCP) by phosphorylating myosin phosphatase targeting subunit 1 (MYPT1), thereby preventing the dephosphorylation of the myosin light chain (Ilvarez-Santos et al., 2020). Furthermore, activated ROCK phosphorylates LIM (Lin11, Isl1, Mec3) domain kinase (LIMK) to phosphorylate cofilin, which increases the contraction of the trabecular meshwork, thereby raising the resistance to aqueous humor outflow and inducing an increase in intraocular pressure.

[0006] ROCK inhibitors are glaucoma treatments that lower intraocular pressure by inhibiting the contraction of the trabecular meshwork and increasing aqueous humor outflow through the inhibition of phosphorylation of myosin light chains and copillin in the trabecular meshwork (Buffault et al., 2022). Currently, netarsudil is the only ROCK inhibitor approved by the FDA as a glaucoma treatment (Tanna & Johnson, 2018). However, these drugs can be accompanied by side effects such as blepharitis, conjunctival hyperemia, and inflammation (Berrino & Supuran, 2019). Since glaucoma is a chronic disease and most patients must use various types of intraocular pressure-lowering agents for a long period, the development of drugs that are safe and effective in the long term is essential. Accordingly, the discovery of new pharmacophores as ROCK inhibitors is required.

[0007] The inventors completed the present invention by conducting research to discover a natural product-based ROCK inhibitor drug with few side effects as an intraocular pressure-lowering agent for the treatment of glaucoma, and experimentally confirming that a novel compound isolated from actinomycetes inhibits Rho kinase (ROCK) and is useful for lowering intraocular pressure in a DBA / 2J aging mouse model.

[0008]

[0009] The inventors conducted research to develop a natural product capable of effectively preventing or treating glaucoma and confirmed that a novel compound derived from actinomycetes inhibits rock kinase and can increase aqueous humor drainage by reducing resistance in the trabecular meshwork, which is the main drainage pathway of aqueous humor, and completed the present invention by confirming that it has excellent efficacy in lowering intraocular pressure in a DBA / 2J aging mouse model.

[0010] The object of the present invention is to provide a novel compound represented by Formula I or Formula II and a pharmaceutically acceptable salt thereof.

[0011] In addition, the objective of the present invention is to provide a composition for the prevention, improvement, or treatment of glaucoma comprising a novel compound represented by Formula I or Formula II and a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0013]

[0014] The present invention provides a compound represented by the following formula I or formula II or a pharmaceutically acceptable salt thereof.

[0015] [Chemical Formula I]

[0016]

[0017] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

[0018] [Chemical Formula II]

[0019]

[0020] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

[0021]

[0022] The compound represented by the above chemical formula I may be a compound represented by the following chemical formula 1 or chemical formula 3.

[0023] [Chemical Formula 1]

[0024]

[0025] [Chemical Formula 3]

[0026]

[0027]

[0028] The compound represented by the above chemical formula II may be a compound represented by the following chemical formula 2 or chemical formula 4.

[0029] [Chemical Formula 2]

[0030]

[0031] [Chemical Formula 4]

[0032]

[0033]

[0034] The above compound may be derived from actinomycetes.

[0035] The above actinomycete may be a strain of the genus Norcadiopsis sp.

[0036] The above actinomycete may be the strain deposited under accession number KCCM13539P.

[0037] The above compound or its pharmaceutically acceptable salt can lower intraocular pressure.

[0038] The above compound or a pharmaceutically acceptable salt thereof may have rho kinase inhibitory ability.

[0039] The above compound or a pharmaceutically acceptable salt thereof may have inhibitory ability against ROCK1 (rho-associated protein kinase 1) and ROCK2 (rho-associated protein kinase 2).

[0040] The above compound or a pharmaceutically acceptable salt thereof can inhibit the shrinkage of HTM (human trabecular meshwork) cells.

[0041] The above compound or a pharmaceutically acceptable salt thereof can increase the permeability of a monolayer cell layer of HTM (human trabecular meshwork).

[0042] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of glaucoma comprising a compound represented by the above formula I or formula II or a pharmaceutically acceptable salt thereof as an active ingredient.

[0043] In addition, the present invention provides a quasi-drug composition for preventing or improving glaucoma comprising a compound represented by Formula I or Formula II or a pharmaceutically acceptable salt thereof as an active ingredient.

[0044] In addition, the present invention provides a food composition for preventing or improving glaucoma comprising a compound represented by the above formula I or formula II or a pharmaceutically acceptable salt thereof as an active ingredient.

[0045] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

[0046] In addition, the present invention provides a quasi-drug composition for the prevention or improvement of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

[0047] In addition, the present invention provides a food composition for preventing or improving glaucoma or a health functional food composition comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

[0048] In addition, the present invention provides a method for preventing or treating glaucoma comprising the step of administering to an individual a composition comprising as an active ingredient a compound represented by Formula I or Formula II or a pharmaceutically acceptable salt thereof, or an actinomycete deposited under accession number KCCM13539P or a culture thereof.

[0049] In addition, the present invention provides a composition for the prevention or treatment of glaucoma comprising, as an active ingredient, a compound represented by Formula I or Formula II or a pharmaceutically acceptable salt thereof, or an actinomycete or culture thereof deposited under accession number KCCM13539P.

[0050]

[0051] The present invention has excellent ROCK inhibitory ability and excellent effect in reducing resistance in the trabecular meshwork pathway and lowering intraocular pressure, and can be utilized in various ways, such as pharmaceutical compositions and food compositions for the prevention, improvement, or treatment of glaucoma. In addition, the compound of the present invention is derived from natural products and has almost no side effects, so it can be safely and continuously used.

[0052]

[0053] Figure 1 shows the major COSY, HMBC, and ROESY correlations of novel compounds 1-4 (nocarnaphthol AD) derived from the SCY18 strain.

[0054] Figure 2 shows the results of evaluating the ROCK activity inhibitory ability of novel compounds 1-4 derived from the SCY18 strain: (A and B) Luciferase-based ADP-Glo ​​kinase activity evaluations were performed to evaluate the inhibitory effect of SCY18 compounds 1-4 (30 μM) on ROCK1 and ROCK2 activities in the presence of 1 μg S6K substrate and 1 μM ATP. Y-27632 (1 μM) was used as a positive control as a ROCK inhibitor. Results are expressed as a percentage of the kinase activity of a dimethyl sulfoxide (DMSO)-treated control (vehicle). (C and D) Concentration-dependent inhibitory effects of compounds 1 and 3 on ROCK1 and ROCK2. Values ​​represent mean ± SD (n = 3). *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001. CPD, Compound (novel compound derived from SCY18 strain); Y, Y-27632.

[0055] Figure 3 shows the results of the molecular docking analysis of the ligand binding domain of ROCK with Compound 1 and Compound 3: (AF) The bonding modes of Compound 1 (indicated in orange), Compound 3 (light green), and Y-27632 (dark gray) within the binding sites of ROCK1 (PDB ID: 3V8S, light pink) and ROCK2 (PDB ID: 7JNT, light blue). The residues interacting with the compounds and their bonding modes are also indicated. Oxygen and nitrogen atoms are indicated in red and blue, respectively. Hydrogen bonds are indicated by yellow dashes. The bond free energy scores of the compounds were calculated using Schrödinger software and are expressed in kcal / mol. CPD, Compound; LBD, Ligand binding domain; Y, Y-27632.

[0056] Figure 4 shows the results of the analysis of the effects of Compound 1 and Compound 3 on human trabecular meshwork (HTM) cell viability: HTM cells were treated with Compound 1 and Compound 3 (10 or 30 μM) for 24 hours in serum-rich medium. DMSO was treated as a control under the same conditions. Y-27632 (30 μM) was used as a positive control as a ROCK inhibitor. Cell viability was determined using WST analysis. Results are expressed as a percentage of cell viability relative to the DMSO-treated control (vehicle). Values ​​represent mean ± SD (n = 3). CPD, Compound; ns, No statistical significance; Y, Y-27632.

[0057] Figure 5 shows the results of analyzing the effects of Compounds 1 and 3 on the activity of MYPT1 and MLC2, proteins involved in fiber contraction and acting as ROCK regulatory downstream mechanisms in HTM cells: Lysates were collected from HTM cells treated with Compounds 1 and 3 (10 or 30 μM) or Y-27632 (30 μM) for 8 hours in serum-rich medium. Lysates were collected under the same conditions with DMSO as a control. Western blots were performed to analyze changes in the expression of p-MYPT1, MYPT1, p-MLC2, MLC2, and ROCK1 and ROCK2 proteins. GAPDH was used as a loading control. Values ​​represent mean ± SD (n = 3). *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001. CPD, Compound; Y, Y-27632.

[0058] Figure 6 shows the results of analyzing the effects of Compounds 1 and 3 on the activity of LIMK and Cofilin, proteins involved in actin structure stabilization and ROCK regulatory downstream mechanisms in HTM cells: Lysates were collected from HTM cells treated with Compounds 1 and 3 (10 or 30 μM) or Y-27632 (30 μM) for 8 hours in serum-rich medium. Lysates were collected after treating with DMSO under the same conditions as a control. Western blots were performed to analyze changes in the expression of p-LIMK, LIMK p-Cofilin, and Cofilin proteins. GAPDH was used as a loading control. Values ​​represent mean ± SD (n = 3). *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001. CPD, Compound; Y, Y-27632.

[0059] Figure 7 shows the results of analyzing the effects of Compound 1 and Compound 3 on transendothelial electrical resistance (TEER) to measure the permeability of the HTM monolayer cell layer; HTM cells were treated with 5 ng / mL of transforming growth factor beta-2 (TGF-β2) (DMSO was used as a control instead of TGF-β2) and Compounds 1 and 3 (0, 10, or 30 μM) for 24 hours in serum-rich medium. Y-27632 (10 or 30 μM) was used as a ROCK inhibitor. Values ​​represent mean ± SD (n = 3). *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001. CPD, Compound; Y, Y-27632.

[0060] Figure 8 shows the results of evaluating the intraocular pressure-lowering efficacy of Compound 1 (nocarnaphthol A) in a DBA / 2J aging mouse model. Compound 1 (0.03 and 0.1%) was used as the test substance, and netarsudil 0.02% (Rhopressa) was used as the positive control. ® ) and latanoprost 0.005% (Xalatan ® CON, latanoprost, and NET, normal saline was used as a negative control. (A) Change in mean intraocular pressure between groups over time. (B) Mean baseline intraocular pressure between groups before instillation. (C) Mean intraocular pressure between groups at 1 week after instillation. Values ​​represent mean ± SEM (n= 8-13). CON, control; d, day; IOP, intraocular pressure; LAT, latanoprost; NET, netarsudil; w, weeks.

[0061] Figure 9 shows (A) the results of confirming changes in body weight between groups during the DBA / 2J aging mouse model experiment. (B), (C) the results of comparing liver and kidney indices compared to a negative control. Values ​​represent mean ± SEM (n= 8-13). CON, control; d, day; LAT, latanoprost; NET, netarsudil.

[0062]

[0063] The inventors completed the present invention by conducting research to discover a natural product-based ROCK inhibitor drug as an intraocular pressure-lowering agent for the treatment of glaucoma, and confirming that a novel compound isolated from actinomycetes inhibits ROCK kinase and has an intraocular pressure-lowering effect in a DBA / 2J aging mouse model.

[0064]

[0065] The present invention will be described in detail below.

[0066] The present invention provides a compound represented by the following formula I or formula II or a pharmaceutically acceptable salt thereof.

[0067] [Chemical Formula I]

[0068]

[0069] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

[0070] [Chemical Formula II]

[0071]

[0072] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

[0073] In addition, the present invention provides a composition for the prevention, improvement, or treatment of glaucoma comprising a compound represented by Formula I or Formula II or a pharmaceutically acceptable salt thereof as an active ingredient.

[0074] In addition, the present invention provides a composition for the prevention, improvement, or treatment of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

[0075] The above composition may be a pharmaceutical composition, a quasi-drug composition, a food composition, or a health functional food composition.

[0076] The compound represented by the above chemical formula I may be a compound represented by the following chemical formula 1 or chemical formula 3.

[0077] [Chemical Formula 1]

[0078]

[0079] [Chemical Formula 3]

[0080]

[0081] The compound represented by the above chemical formula II may be a compound represented by the following chemical formula 2 or chemical formula 4.

[0082] [Chemical Formula 2]

[0083]

[0084] [Chemical Formula 4]

[0085]

[0086]

[0087] The above compound may be derived from actinomycetes.

[0088] The above actinomycete may be a strain of the genus Norcadiopsis sp.

[0089] The above actinomycete may be the strain deposited under accession number KCCM13539P.

[0090] The above compound or its pharmaceutically acceptable salt can lower intraocular pressure.

[0091] The above compound or a pharmaceutically acceptable salt thereof may have rho kinase inhibitory ability.

[0092] The above compound or a pharmaceutically acceptable salt thereof may have inhibitory ability against ROCK1 (rho-associated protein kinase 1) and ROCK2 (rho-associated protein kinase 2).

[0093] The above compound or a pharmaceutically acceptable salt thereof can inhibit the shrinkage of HTM (human trabecular meshwork) cells.

[0094] The above compound or a pharmaceutically acceptable salt thereof can increase the permeability of a monolayer cell layer of HTM (human trabecular meshwork).

[0095]

[0096] In this specification, "prevention" refers to any act of delaying the onset of glaucoma through the administration of a composition of the present invention, and "treatment" and "improvement" refer to any act of improving or beneficially altering the symptoms of glaucoma through the administration of a composition of the present invention.

[0097]

[0098] Pharmaceutical composition for the prevention or treatment of glaucoma

[0099] The composition of the present invention can be prepared as a pharmaceutical composition.

[0100] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0101] According to a preferred embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition comprising (a) a pharmaceutically effective amount of an active ingredient, such as the compound of the present invention or a pharmaceutically acceptable salt thereof described above; and (b) a pharmaceutically acceptable carrier. In this specification, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the active ingredient described above.

[0102] Pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0103] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0104] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The general dosage of the pharmaceutical composition of the present invention is within the range of 0.001 to 100 mg / kg for adults. Administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosages.

[0105] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0106]

[0107] quasi-drug composition for the prevention or improvement of glaucoma

[0108] The composition of the present invention may be provided as a quasi-drug composition.

[0109] The above active ingredient may be added as is, or used in combination with ingredients of other quasi-drugs, and may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0110]

[0111] Food composition for the prevention or improvement of glaucoma

[0112] The composition of the present invention may be provided as a food composition or a health functional food composition. When a composition for the prevention, improvement, or treatment of glaucoma containing the active ingredient of the present invention (the compound or a pharmaceutically acceptable salt thereof, etc.) is prepared as a food composition, it includes not only the active ingredient but also ingredients that are typically added during food preparation, such as, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of the carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc., which are conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavorings, natural flavorings [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavorings (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to the natural product extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0113] The formulation of the above food composition or health functional food composition can be in the form of powder, granule, pill, tablet, or capsule, as well as any form of general food or beverage.

[0114] There are no specific restrictions on the types of food mentioned above, and examples of food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all food in the conventional sense.

[0115] Generally, when manufacturing food or beverages, the above active ingredient may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range, and furthermore, since the present invention uses natural substances, there is no problem in terms of safety, so it may be used in an amount greater than the above range.

[0116]

[0117] The present invention will be explained in more detail below through examples. The purpose, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the examples described herein and may be embodied in other forms. The examples introduced herein are provided to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following examples.

[0118]

[0119] <Example>

[0120] Experimental method

[0121] General experimental procedures

[0122] Optical rotation was recorded using a Jasco P-2000 polarimeter with a 1.0 cm cell (Tokyo, Japan). IR spectra were measured using a PerkinElmer FT-IR spectrometer (MA, United States). UV spectra and low-resolution LC-MS data for electrospray ionization (ESI) were recorded using an Agilent G6125B MSD system coupled with an Agilent Technologies 1260 Series Infinity II LC system using a reversed-phase C18 column (Agilent column, 100 x 4.6 mm, 5 μm). Nuclear magnetic resonance spectroscopic data were collected at the Korea Basic Science Institute (KBSI) in Ochang, Korea, using a Bruker Advance and a 900 MHz spectrometer (Bruker, Billerica, MA, USA). High-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an Agilent Technologies 1290 series HPLC and an Agilent 6530 iFunnel LC-Q-TOF-MS system (Agilent Technologies). Compounds were purified using a Waters HPLC system (1525 binary pump and 996 photodiode array detector).

[0123]

[0124] bacterial material

[0125] Bacterial strain SCY18 was isolated from mud collected in Jeungdo, Korea. Strain SCY18 was identified as Nocardiopsis lucentensis based on 16sRNA sequencing analysis (99.41% similarity). The isolated strain was deposited under accession number KCCM13539P.

[0126]

[0127] Fermentation, extraction, and separation

[0128] Nocardiopsis lucentensisSCY18 was cultured for 2 days in a 150 mL flask containing 50 mL of YEME medium (yeast extract 3.0 g, malt extract 3.0 g, soytone 2.0 g, peptone 5.0 g, glucose 10.0 g, sea salt 33.0 g, distilled water 1.0 L). Then, 3.5 mL of the culture medium was transferred to a 500 mL flask containing 150 mL of R4 medium (yeast extract 1.0 g, MgCl2.6H2O 5.0 g, CaCl2.2H2O 2.0 g, glucose 5.0 g, proline 1.5 g, valine 1.2 g, TES 3.0 g, casamino acid 50 mg, K2SO4 100 mg, trace elements 1 mL) and cultured for 3 days. Then, 20 mL of culture medium was used to incubate in an ultra-high yield plastic flask containing 1.0 L of R4 medium. To isolate bacterial metabolites, 20 L of SCY18 culture medium was prepared in the same manner. All cultures were incubated at 30°C with shaking at 200 rpm. After 7 days of fermentation, the entire fermented SCY18 medium (20 L) was centrifuged and filtered to discard the cell pellet. Next, the supernatant was loaded onto C18 resin to extract metabolites. For elution, stepwise methanol-water gradients (2:8, 4:6, 6:4, 8:2, 10:0) were used to obtain five fractions (fractions 20%, 40%, 60%, 80%, and 100%). Finally, the 40% fraction was treated by HPLC using isosolvent elution (acetonitrile 35% (0.1% formic acid): water 65% (0.1% formic acid), detection wavelength 230 nm) on a reversed-phase column (YMC Pack ODS-A; 250 x 10.0 mm; 5 μM) to obtain Compound 1 (4.3 mg, t R = 12.7 min), Compound 2 (3.0 mg, t R = 30.2 min), Compound 3 (3.1 mg, t R = 15.3 min), Compound 4 (3.0 mg, t R = 40.0 minutes) obtained.

[0129]

[0130] Physical and chemical data

[0131] Compound 1 (nocarnaphthol A): brown oil, [a] 25 D = +4.51 (c0.10, MeOH), UV (MeOH) λ max (log ε) 232 (2.92), 298 (1.63) nm; IR (neat) ν max 3409, 1627, 152 0, 1451, 1409, 1354, 1205, 1162, 1051 cm -1 . 1 H and 13 13C NMR data, Table 1. HR-ESI-MS [M+H] + m / z246.1130 (C 14 H 16 NO3), calculatedm / z246.1140 for [M+H] +

[0132]

[0133] Compound 2 (nocarnaphthol B): brown oil, [a] 25 D = +4.35 (c0.10, MeOH), UV (MeOH) λ max (log ε) 232 (2.61), 325 (1.88) nm; IR (neat) ν max 3421, 2989, 164 7, 1593, 1384, 1354, 1253, 1209, 1068 cm -1 . 1 H and 13 13C NMR data, Table 1. HR-ESI-MS [M+H] + m / z304.1193 (C 16 H 18 NO5), calculatedm / z304.1185 for [M+H] +

[0134]

[0135] Compound 3 (nocarnaphthol C): brown oil, [a]25 D = +2.25 (c0.10, MeOH), UV (MeOH) λ max (log ε) 234 (2.54), 325 (1.65) nm; IR (neat) ν max 3361, 2972, 169 3, 1627, 1563, 1454, 1219, 1160, 1050 cm -1 . 1 H and 13 13C NMR data, Table 2. HR-ESI-MS [MH] - m / z203.0708 (C 12 H 11 O3), calculatedm / z 203.0704 for [Ν-H] -

[0136]

[0137] Compound 4 (nocarnaphthol D): brown oil, [a] 25 D = +5.63 (c0.10, MeOH), UV (MeOH) λ max (log ε) 232 (2.75), 328 (1.92) nm; IR (neat) ν max 3390, 2962, 162 0, 1595, 1452, 1418, 1255, 1212, 1051 cm -1 . 1 H and 13 13C NMR data, Table 2. HR-ESI-MS [MH] - m / z261.0766 (C 14 H 13 O5), calculatedm / z261.0768 for [MH] -

[0138]

[0139]

[0140] [Structural formula of Compound 1-4]

[0141] Compound 1, nocarnaphthol A:

[0142] N-((6-hydroxy-3-methoxynaphthalen-1-yl)methyl)acetamide

[0143] Compound 2, nocarnaphthol B:

[0144] methyl-5-(acetamidomethyl)-2-hydroxy-7-methoxy-1-naphthoate

[0145] Compound 3, nocarnaphthol C:

[0146] 5-(hydroxymethyl)-7-methoxynaphthalen-2-ol

[0147] Compound 4, nocarnaphthol D:

[0148] methyl 2-hydroxy-5-(hydroxymethyl)-7-methoxy-1-naphthoate

[0149] DMSO-d 6 of Compound 1 and Compound 2 (nocarnaphthol A and B) 1 H and 13 C NMR data positioning nocarnaphthol Anocarnaphthol Bδ C , typeδ H , mult(Jin Hz)δ C , typeδ H, mult(Jin Hz)1136.6, C137.3, C2114.9, CH6.80, (2.5)114.7, CH6.89, (2.0)3157.2, C157.9, C4103.9, CH6.99, d (2.5)101.5, CH6.97, d (2.0)4a136.8, C133.2, C5109.2, CH7.06, d (2.5)112.9, C6156.1, C155.1, C7116.0, CH6.92, dd (9.0, 2.5)115.8, CH7.12, d (9.0)8124.97.77, d (9.0)127.7, CH7.92, d (9.0)8a120.9, C120.3, C939.6, CH24.57, d (5.5)39.6, CH24.60, d (5.5)10169.1, C169.1, C1122.7, CH31.85. s22.5, CH31.88, s1255.1, CH33.80, s55.0, CH33.80, s13168.8, C1452.1, CH33.90, s9-NH8.34, t (5.5)8.40, t (5.5)

[0150] 1 H and 13 CNMR data were recorded at 900 MHz and 225 MHz, respectively.

[0151] Compounds 3 and 4 (nocarnaphthol C and D) in DMSO-d6 1 H and 13 C NMR data position nocarnaphthol Cnocarnaphthol Dd C , typed H , mult(Jin Hz)d C , typed H, mult(Jin Hz)1139.7, C141.0, C2113.3, CH6.95, d (2.5)113.8, CH7.06*3157.2, C158.6, C4103.5, CH6.96, d (2.5)113.9, CH7.01, d (2.5)4a136.5, C133.3, C5108.9 C7.05, d (2.5)112.9 C6155.7, C155.4, C7115.6, CH6.91, dd (9.0, 2.5)115.8, CH7.05*8124.6, CH7.75, d (9.0)128.2, CH7.92, d (9.0)8a120.5, C120.6, C960.6, CH24.83, s61.4, CH24.86, s1055.2, CH33.82. s55.4, CH33.82. s11169.4, C1252.6, CH33.91, s

[0152] 1 H and 13 CNMR data were recorded at 900 MHz and 225 MHz, respectively.

[0153] * Redundant signal

[0154]

[0155] In vitro analysis of kinase (ROCK) inhibitory activity

[0156] ADP-Glo ​​according to the manufacturer's instructions TMROCK kinase (ROCK) activity was measured in vitro using an assay kit (Promega, Madison, WI, USA). This assay quantitatively measured the amount of ADP generated during the ROCK (ROCK1 and ROCK2) reaction. The measured luminescence signal was detected in proportion to the amount of ADP indicating ROCK activity. 10 ng ROCK1 (Promega) or 0.5 ng ROCK2 (Promega), an inhibitor (SCY18 Compound 1-4, Y-27632, or DMSO), 1 μg S6K peptide substrate (SignalChem), and 1 mM ATP (Promega) were diluted in kinase buffer and added to a 384-well plate with a total reaction volume of 20 μL. The components were added to the wells and incubated at 25 °C for 2 hours for ROCK1 and 1 hour for ROCK2. After incubation, 5 μL of ADP-Glo ​​reagent was added, and the mixture was incubated at 25 °C for 40 minutes. Subsequently, 10 μL of kinase detection reagent was added, and the mixture was incubated at 25 °C for 30 minutes. Luminescence was measured using a Biotek Synergy HT Multi-Mode Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). Relative luminescence units were obtained by setting the DMSO-treated group to 100% (control value) and the case without enzyme use to 0% (reference value). The half-maximal inhibitory concentration (IC10) was calculated. 50 The ) value was determined using logistic regression analysis. Y-27632 (ROCK inhibitor) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0157]

[0158] Molecular Docking Analysis

[0159] Molecular docking studies were performed using the Schrödinger program to dock compounds 1, 3, and the positive control (Y-27632) to the active sites of the ROCK1 and ROCK2 proteins. The protein structures used were obtained from the Protein Data Bank (PDB), specifically PDB IDs 3V8S (Li et al., 2012) and 7JNT (Hu et al., 2020), respectively. Protein preparation was performed according to the standard workflow of Maestro 2023-02 (Schrödinger LLC, NY). This process included the addition of hydrogen atoms, bond order assignment, removal of water molecules, hydrogen bond optimization, and energy minimization using the OPLS4 force field. PROPKA was used to predict the ionization state of the proteins, and ProtAssign was used to optimize the hydrogen bond network. Ligand preparation was performed at pH 7 ± 2.0 using LigPrep and Epik, taking into account protonation and tautomeric transformations, and applying an OPLS4 force field. Low-energy stereoisomers were generated, and those with stable 3D structures and correct chirality were selected. The ligands with these parameters were docked to the ROCK1 and ROCK2 active sites using the Glide module. Subsequently, a molecular docking grid box was set at the center of the co-crystallized ligands using the Glide grid generation function. Docking was performed in extra precision (XP) mode, and the ligands were evaluated based on the Glide XP scoring function (kcal / mol), which measures binding affinity.

[0160] XP Glide scores are defined as follows:

[0161] XP Glide score =E coul +E vdW +E bind +E penalty

[0162] Here E coul E is electrostatic potential energy. vdW is van der Waals potential energy, E bind represents the bond interaction energy, and E penalty It includes protein / ligand modification energy, entropy loss, and ligand / protein dehydration energy.

[0163]

[0164] Cell Culture

[0165] HTM cells were purchased from ScienCell (Carlsbad, CA, USA). The cells were poly- L -lysine (ploly- L -lysine, 2 μg / cm 2 Cells were cultured in Petri dishes pre-coated with ) and maintained at 37°C in a humid environment containing 5% CO2 in Trabecular Meshwork Cell Medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (10,000 U / mL; 10,000 μg / mL), and trabecular meshwork cell growth supplement. All culture-related products were purchased from ScienCell.

[0166]

[0167] Cell viability analysis

[0168] Cell viability was measured using the EZ-Cytox Kit (DoGenBio, Seoul, Korea). HTM cells were placed in a 96-well plate at a density of 1 × 10⁶ 4Cells were seeded at a cell / well density and cultured for 24 hours (at 37 °C, 5% CO2). Cells were treated with Compound 1, Compound 3 (10 and 30 μM), and Y-27632 (30 μM) in serum-rich medium for 24 hours. DMSO was used as a control under the same conditions. Subsequently, EZ-Cytox solution (DoGenBio, Seoul, Korea) was added, and the plates were incubated at 37 °C for 1–1.5 hours. Absorbance was then measured at a wavelength of 450 nm using a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA). The results were expressed as a percentage of cell viability relative to the DMSO-treated control (vehicle).

[0169]

[0170] Western blot analysis

[0171] 4.5 × 10⁶ HTM cells in a 6-well plate 5Cells were seeded at a cell / well density and cultured for 24 hours (at 37 °C, 5% CO2). Cells were treated with Compound 1, Compound 3 (10 and 30 μM), and Y-27632 (30 μM) in serum-rich medium for 8 hours. Cells were washed with cold phosphate-buffered saline and lysed on ice for 30 minutes with cold radioimmunoprecipitation lysis and extraction buffer (Thermo Fisher Scientific) containing phenylmethylsulfonyl fluoride and sodium orthovanadate, vortexing every 10 minutes. Subsequently, the lysate was centrifuged at 15,000 rpm for 15 minutes at 4 °C to obtain only the supernatant. After protein quantification, an equal amount of protein was separated from a sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% bovine serum albumin (GenDEPOT, Baker, TX, USA) and reacted with primary antibodies against ROCK1, ROCK2, p-MYPT1 (Thr696), MYPT1, p-MLC2 (Thr18 / Ser19), MLC2, Phospho-LIMK1 (Thr508) / LMK2 (Thr505), LIMK1, Phospho-Cofilin (Ser3), Cofilin, and GAPDH (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C for 16-20 hours or more. After washing three times with triple-buffered saline-tween 20 solution, the membrane was incubated with the corresponding anti-rabbit (1:3,000, Genetex Inc., Irvine, CA, USA) secondary antibody conjugated with HRP at 25°C for 1 hour.Finally, the membrane was washed three times with triple-buffered saline-Tween 20, and immunoreaction bands were developed using a chemiluminescence kit (Intron Biotechnology, Seoul, Korea) and visualized using a chemiluminescence kit (Thermo Fisher Scientific). The bands were imaged using an iBright CL1000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were quantified using Image J software (Rueden et al., 2017).

[0172]

[0173] Measurement of permeability of the trabecular monolayer (TEER, transendothelial electrical resistance)

[0174] 2×10⁴ HTM cells were placed in a 12-well Transwell insert (0.4 μm Polycarbonate membrane; Corning Costa, Corning, NY, USA). 4 Cells were seeded at a cell / well density and cultured for 24 hours (at 37 °C, 5% CO2). Cells were treated in serum-rich medium for 24 hours with 5 ng / mL of transforming growth factor beta-2 (TGF-β2) and compounds 1 and 3 (10 and 30 μM). Y-27632 (30 μM) was used as a positive control for ROCK inhibition. Permeability of the HTM cell line monolayer was quantified using an EVOM instrument (World Precision Instruments, Sarasota, FL, USA).

[0175]

[0176] Evaluation in the DBA / 2J aging mouse model

[0177] To confirm the preventive and therapeutic effects of Compound 1 (nocarnaphthol A) on glaucoma, Compound 1 and the positive control netarsudil 0.02% (Rhopressa) were tested in a DBA / 2J aging mouse model. ® ) and latanoprost 0.005% (Xalatan ® Changes in intraocular pressure due to ) treatment were confirmed. The animal experiment was conducted with the approval of the Animal Ethics Committee of the Korea Institute of Science and Technology, and the approval number is [KIST-IACUC-2025-035].

[0178] The DBA / 2J mouse model is a representative chronic glaucoma model characterized by genetically spontaneous elevation of intraocular pressure. It is utilized in spontaneous glaucoma models because intraocular pressure gradually increases after 6 months of age due to anterior chamber angle occlusion, iris atrophy, and pigment cell deposition, leading to progressive optic nerve degeneration and retinal ganglion cell (RGC) damage. Male DBA / 2J mice (10 weeks old) were purchased from Dooyul Biotech Co., Ltd. and aged until 6 months of age. All mice were provided with the same diet (5L79 diet), and water was allowed to be consumed freely. The Dark:Light cycle was maintained at 12 hours:12 hours, and intraocular pressure and body weight were measured weekly starting from 5 months of age. Intraocular pressure was measured using a tonometer (Icare, Vantaa, Finland) during a set time period (6:00 PM–9:00 PM) after local anesthesia with proparakine. In addition, accuracy was continuously verified through comparison with measurements from the Tono-Pen AVIA tonometer (Reichert, Depew, NY, USA). Groups were divided by random assignment based on intraocular pressure (n= 8-13) at the point when the overall mean intraocular pressure reached 20 mmHg (29 weeks of age). Compound 1 (0.03 and 0.1%) was used as the test substance, and netarsudil 0.02% (Rhopressa) was used as the positive control. ® ) and latanoprost 0.005% (Xalatan ®) was used, and physiological saline was used as a negative control. The test substance was instilled twice a day for 12 weeks, and intraocular pressure was measured and recorded weekly (every other week after 4 weeks of instillation).

[0179]

[0180] Statistical analysis

[0181] Experimental values ​​represent the mean ± standard deviation of at least three experiments. Statistical analysis was performed using GraphPad Prism version 10.4.1 (GraphPad Software Inc.; San Diego, CA, USA) with one-way and two-way analysis of variance (ANOVA), post-hoc Welch's t-tests, and Sidak's multiple comparisons test. Criteria for statistical significance were set to *P< 0.05, **P< 0.01, ***P< 0.001, and ****P< 0.0001.

[0182]

[0183] abbreviation

[0184] DMSO, dimethyl sulfoxide; HTM, human trabecular meshwork; IC 50, half-maximal inhibitory concentration; MLC, myosin light chain; MLCP, myosin light chain phosphatase; MYPT1, myosin phosphatase target subunit 1; PDB, protein data bank; ROCK, rho kinase (rho-associated protein kinase); TEER, transendothelial electrical resistance; TGF-β2, transforming growth factor-beta 2; TM, trabecular meshwork; XP, extra precision.

[0185]

[0186] Experimental results

[0187] Structure determination

[0188] Compound 1, Nocarnaphthol A, is a previously unreported compound identified through HRMS and NMR spectroscopic analysis. Compound 1 was isolated as a brown oil, and its molecular formula is C 14 H 15 NO3(obsd.m / z246.1130, calcd.m / z246.1140, [M + H] + It was determined to be ). The structure features a naphthalene core with characteristic signals for 10 aromatic carbons (δC 157.2, 156.1, 136.8, 136.6, 124.9, 120.9, 116.0, 114.9, 109.2, and 103.9) confirming the aromatic framework. The presence of acetylated amine groups indicates δ H 1.85 ppm (CH₃) singlet and δ CThis is evidenced by the carbonyl concentration of 169.1 ppm, which correlates with the C-10 methyl (CH₃) group, confirming its location at the C-1 position of the naphthalene ring. Further confirmation of the structure is provided by COSY and HMBC correlations. H-9(δ H 4.57, CH₂) is an amide proton (δ H It binds to (8.34) and is confirmed by HMBC correlations for C-1 and C-10. Additional O-methyl group (δ H 3.80; δ C 55.1) is located at C-3, and the HMBC correlation for C-3 (δ C This is evidenced by 157.2 ppm. These spectral data confirm the structure of the compound as a methoxylated, hydroxylated naphthalene ring connected to the acetamide moiety, along with major proton-proton (COSY) and proton-carbon (HMBC) correlations, showing a unique substitution pattern that enhances the structural diversity of naphthalene derivatives.

[0189]

[0190] Compound 2, Nocarnaphthol B, is also a previously unreported compound that is structurally similar to Compound 1 due to the addition of two carbons corresponding to an ester functional group. NMR data reveal a characteristic naphthalene core with a novel substituent at the C-5 position, which is evidence of the absence of an H-5 signal. Key spectroscopic features include δ C 168.8 ppm of carbonyl carbon and δ H 3.90(δ C 52.1) has an O-methyl group, which suggests an ester bond confirmed by the HMBC correlation between H-14 (3.90 ppm) and C-13 (168.8 ppm). Further HMBC correlations indicate that the acetamide moiety is methylene (δ at the C-5 position). H 4.60; δ CIt is attached via 39.6), and COSY and HMBC data match the methyl ester group at C-1, imparting a unique functional group pattern to the structure. Mass spectrometry data of molecular formula C 16 H 17 Supporting NO5, the observed mass is 304.1193 [M + H] + (Theoretical m / z = 304.1185) The minimum mass error is 2.63 ppm.

[0191]

[0192] Compound 3, Nocarnaphthol C, was isolated as a brown oil and is a compound structurally related to 1; the main difference is the absence of an acetyl group and an N-CH₂ group (δ in Compound 1). H 4.57, δ C Observed at 39.6) O-CH₂ group (δ H 4.86, δ C 61.4) is the fact that it was replaced. The structure consists of a naphthalene core and 13 This framework is supported by maintaining the C-carbon NMR signal. The O-CH₂ signal confirms the hydroxymethyl substitution at C-5, whereas the methoxy group is δ H It is found at 3.82 (δC 55.4). Also, according to the expected coupling pattern (dd and d), δ H The aromatic protons at 7.06 ppm, 6.96 ppm, 7.05 ppm, and 7.92 ppm correspond to positions around the naphthalene ring. Molecular formula C 12 H 12 Through O₃ and specific NMR correlations, the hydroxymethyl and methoxy functional groups supporting the chemical structure of compound 3, 5-(hydroxymethyl)-7-methoxynaphthalen-2-ol can be identified.

[0193]

[0194] Compound 4, nocarnaphthol D, was refined into brown oil, is structurally related to compound 3, and δ C 169.4 ppm of carbonyl and δH 3.92ppm(δ C The presence of two additional carbons containing an O-methyl group (52.6) confirms the ester bond. The absence of the H-5 signal and the appearance of this new signal indicate a substitution at the C-5 position. The HMBC correlation, particularly the correlation between H-12 (3.92 ppm) and C-11 (169.4 ppm), confirms that a methyl group is attached to the carbonyl of the ester group. δ around the ring H The retained aromatic signals with characteristic protons at 7.06, 6.96, 7.05, and 7.92 indicate that the naphthalene core is undamaged. Mass spectrometry results showing close agreement between theoretical and observed m / z values ​​(261.0768 and 261.076, respectively, with a mass error of -0.5362 ppm) correspond to molecular formula C 13 H 12 It supports O₄, and thus confirms that the structure of compound 4 is methyl 2-hydroxy-5-(hydroxymethyl)-7-methoxy-1-naphthoate, which is a methyl ester derivative.

[0195]

[0196] Analysis of ROCK1 and ROCK2 activity inhibitory efficacy

[0197] To evaluate the ROCK activity inhibitory ability of compounds 1-4 derived from the SCY18 strain, an in vitro evaluation was performed using the luciferase-based ADP-Glo ​​assay (Fig. 2). Y-27632, used as a control, inhibits ROCK1 and ROCK2, thereby reducing the contraction of intracellular actin-myosin and inhibiting cell shrinkage. Additionally, this drug demonstrated high reproducibility of experimental results, making it suitable as a control, and was utilized as the control for this experiment. At 30 μM, compounds 1 and 3 significantly inhibited ROCK1 activity by 59.5% and 51.9%, respectively (Fig. 2A). Furthermore, compounds 1 and 3 significantly inhibited ROCK2 activity by 44.7% and 53.8%, respectively (Fig. 2B).

[0198] Next, the concentration-dependent inhibitory activity of these compounds was evaluated. IC50 of Compound 1 and Compound 3 for ROCK1 activity 50 The values ​​were 7.4 μM and 16.1 μM, respectively (Fig. 2C). For ROCK2, the ICs of Compound 1 and Compound 3 were 50 The values ​​were 12.1 μM and 9.6 μM, respectively (Fig. 2D). These results indicate that compounds 1 and 3 are potent dual inhibitors of ROCK1 and ROCK2.

[0199]

[0200] ROCK's ligand binding domain and molecular interaction model of Compound 1 and Compound 3

[0201] Molecular docking simulations were performed to identify the potential binding modes and affinities of Compound 1 and Compound 3 for the ligand binding domains of ROCK1 and ROCK2. The optimal docking modes of Compound 1 and Compound 3 were compared with the docking mode of Y-27632, which was used as a positive control (Fig. 3). An energy-minimizing optimal model for the ROCK1 ligand binding domain was constructed using the Schrdinger program, and the results showed that Compound 1 and Compound 3 bind to the hinge region of the ATP binding site. The free energy values ​​of these compounds were -6.5 kcal / mol (Compound 1), -7.5 kcal / mol (Compound 3), and -7.4 kcal / mol (Y-27632), respectively. In particular, all three compounds formed hydrogen bonds with the backbone NH of Met156 at the lip of the hinge binding site. These interactions are consistent with a previous study (Beroza et al., 2022) which found that all ligands bound to ROCK1 share essential hydrogen bond interactions with the NH of Met156 in the hinge region, and that several ligands form additional hydrogen bonds with DFG-1. Compound 1 and Y-27632 exhibited additional hydrogen bonds with Asp216 (Figs. 2A-2C). Hydrogen bonding with Asp216 residues is known to be a critical interaction in the active site of the ROCK1 enzyme, and when ROCK inhibitors form hydrogen bonds with Asp216, this can act as a mechanism to inhibit enzyme activity by blocking ATP binding sites or inducing structural changes. Therefore, inhibitors that form hydrogen bonds with Asp216 are highly likely to possess strong inhibitory activity or high selectivity toward ROCK1.

[0202] For ROCK2, the free energy values ​​of Compound 1, Compound 3, and Y-27632 were -7.0, -7.3, and -7.8 kcal / mol, respectively. Compound 1, Compound 3, and Y-27632 all formed hydrogen bonds with the backbone NH of Met172 (Figs. 2D-2F). Binding to Met172 enhances other electrical and hydrophobic interactions, helping the inhibitors bind more strongly to the protein. This binding stability can produce therapeutic effects, particularly in diseases requiring ROCK2 regulation such as cardiovascular or ophthalmic diseases, by inhibiting ROCK2 activity. Additionally, Compound 1 and Compound 3 form additional hydrogen bonds with Glu170, thereby enhancing the inhibitory effect on ROCK2.

[0203] In summary, Compounds 1 and 3 bind to the ATP binding sites of ROCK1 and ROCK2, and in particular, through interactions with residues important for the efficacy of each ROCK1 and ROCK2 inhibitor, they exhibit strong inhibitory activity and suggest the possibility of high selectivity.

[0204]

[0205] Analysis of expression of proteins involved in ROCK-regulated contraction in human trabecular meshwork cells

[0206] Compound 1 and Compound 3 significantly reduced the expression of fiber contraction protein, a ROCK regulatory downstream protein, in human fibroblast cells.

[0207] The trabecular meshwork is the primary drainage pathway for aqueous humor, and its dysfunction leads to elevated intraocular pressure, which is one of the major risk factors for glaucoma. The ROCK signaling pathway plays a crucial role in regulating cell contraction and relaxation. Activated ROCK phosphorylates the myosin light chain phosphatase target subunit 1 (MYPT1) and inhibits myosin light chain phosphatase (MLCP), thereby inducing cell contraction through the phosphorylation of the myosin light chain (MLC). ROCK also directly phosphorylates MLC, promoting the contraction of actin fibers. Consequently, the outflow resistance of aqueous humor in the trabecular meshwork pathway increases, leading to elevated intraocular pressure. Therefore, our research team evaluated the effects of Compound 1 and Compound 3, which exhibited ROCK inhibitory activity among SCY18 compounds, on the ROCK-regulated signaling pathway.

[0208] First, the effects of Compound 1 and Compound 3 on cell viability in human trabecular meshwork (HTM) cells were evaluated using WST analysis. HTM cells were treated with Compound 1 and Compound 3 at concentrations of 10 or 30 μM, respectively, for 24 hours, and Y-27632, used as a positive control, was also treated at a concentration of 30 μM for 24 hours for comparison. As a result, Compound 1, Compound 3, and Y-27632 all did not have a significant effect on cell viability at a maximum concentration of 30 μM (Fig. 4).

[0209] Under non-cytotoxic conditions, when compounds 1 and 3 (10 or 30 μM) were treated for 8 hours, it was confirmed that p-MYPT1 and p-MLC2, indicators of fiber shrinkage, were significantly reduced (Fig. 5; p- is an abbreviation for phosphorylated, meaning the phosphorylated form of the protein). In particular, compound 1 showed a superior inhibitory effect on p-MYPT1 and p-MLC2 compared to compound 3. On the other hand, compounds 1 and 3 did not affect the protein expression levels of ROCK1 and ROCK2 themselves (Fig. 5). Through this, it was confirmed that the compounds derived from the SCY18 strain regulate the activity of downstream proteins such as MYPT1 and MLC2 through the inhibition of kinase activity, rather than regulating the protein expression of ROCK1 / 2.

[0210]

[0211] Analysis of expression of ROCK-regulating cytoskeletal proteins in human trabecular meshwork cells

[0212] Compound 1 and Compound 3 significantly reduced the expression of cytoskeletal stabilization proteins, which are ROCK regulatory downstream proteins, in human filamentous fibroblasts.

[0213] Activated ROCK phosphorylates LIM (Lin11, Isl1, Mec3) domain kinase (LIMK), thereby phosphorylating cofilin. When cofilin is inactivated as a result, the disintegration of actin filaments is inhibited, and the cytoskeleton stabilizes, maintaining a contracted state. Conversely, when cofilin is dephosphorylated, it becomes activated, binds to actin filaments, and disintegrates the actin structure, changing the cytoskeleton to a dynamic state and contributing to relaxation.

[0214] Under non-cytotoxic conditions, when Compound 1 and Compound 3 (10 or 30 μM) were treated for 8 hours, it was confirmed that p-LIMK1 / 2 and p-Cofilin, indicators of cytoskeletal stabilization proteins, were significantly reduced (Fig. 6; p- is an abbreviation for phosphorylated, meaning the phosphorylated form of the protein). In particular, Compound 3 showed a superior inhibitory effect on p-LIMK1 / 2 and p-Cofilin compared to Compound 1. Through this, it was confirmed that SCY18 compounds can inhibit the contraction of fibroblast cells by significantly regulating the expression of cytoskeletal stabilization proteins such as LIMK1 / 2 and Cofilin through the inhibition of kinase activity.

[0215]

[0216] Permeability analysis of the monolayer cell layer of the trabecular meshwork

[0217] Compounds 1 and 3 significantly reduced the electrical resistance of the monolayer cell layer increased by transforming growth factor beta (TGF-β2) in human fibroblast cells.

[0218] Previous ex vivo and in vitro studies have reported that TGF-β plays a significant role in the pathogenesis of elevated intraocular pressure (Bhattacharya et al., 2005). Changes in the extracellular matrix environment induced by TGF-β can cause an increase in intraocular pressure, and patients with high levels of TGF-β in the aqueous humor are at high risk of optic nerve damage and vision loss due to elevated intraocular pressure (Vittitow et al., 2004).

[0219] Therefore, in this study, conditions were established to reduce permeability by inducing fiber contraction in HTM cells through treatment with TGF-β2, and the effects of Compound 1 and Compound 3 were evaluated (Fig. 7). HTM cells were treated with TGF-β2 (5 ng / mL) and Compounds 1 and 3 (10 or 30 μM) for 24 hours, and the ROCK inhibitor Y-27632 (10 or 30 μM) was used as a positive control. Upon treatment with TGF-β2 (5 ng / mL), the penetration resistance (TEER) of the monolayer increased by 32% compared to the control group (Fig. 7), and when Compounds 1 and 3 were treated together, a significant decrease in penetration resistance was observed at both concentrations (decreases of 26.4%, 24.0%, and 24.2% at the concentrations of Compound 1, Compound 3, Y-27632, and 30 μM, respectively). This suggests that compounds 1 and 3, which are ROCK 1 / 2 inhibitors, may increase permeability by inhibiting TGF-β2-induced fiber contraction in HTM cells and contribute to the outflow of aqueous humor.

[0220]

[0221] Evaluation in the DBA / 2J aging mouse model

[0222] To confirm the preventive and therapeutic effects of Compound 1 (nocarnaphthol A) on glaucoma, Compound 1 and the positive control netarsudil 0.02% (Rhopressa) were tested in a DBA / 2J aging mouse model. ® ) and latanoprost 0.005% (Xalatan ®Changes in intraocular pressure induced by treatment were confirmed. Changes in intraocular pressure and body weight were observed in DBA / 2J mice starting from 24 weeks of age (Figs. 8A and 9A). Instillation of the test substance was initiated when the average intraocular pressure of the individuals reached 20 mmHg at 29 weeks of age. Prior to instillation, individuals were distributed equally among the groups to ensure similar average baseline intraocular pressures (Fig. 8B). At the first week after instillation of the test substance, the average intraocular pressure of the negative control group was 22.2 mgHg, an increase of approximately 11.7% compared to the previous week (Figs. 8B, 8C). The Compound 1 treatment group showed an intraocular pressure-lowering effect at both concentrations (0.03%: 25.7% decrease, 0.1%: 29.9% decrease). The positive control groups, netarsudil 0.02% and latanoprost 0.005%, reduced intraocular pressure by 17.8% and 7.9%, respectively, compared to the negative control group (Fig. 8C). The intraocular pressure-lowering effect of compound 1 was sustained, and a decrease in intraocular pressure was observed in the negative control group starting from 35 weeks of age in mice, suggesting that this was due to reduced aqueous humor production caused by impaired ciliary function and structural changes in the terminal stage (Fig. 8A).

[0223] There was no significant change in body weight between groups during the experimental period (Fig. 9A). In addition, no significant changes in liver and kidney indicators were observed (Figs. 9B, 9C).

[0224] [Consignment Number]

[0225] Depository Name: Korean Culture Collection Center (KCCM)

[0226] Trustee Number: KCCM13539P

[0227] Date of Trust: 20250109

[0228]

Claims

1. A compound represented by the following chemical formula I or chemical formula II, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl. [Chemical Formula II] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

2. In Claim 1, A compound represented by the above chemical formula I is characterized as being a compound represented by the following chemical formula 1 or chemical formula 3, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 3] 3. In Claim 1, A compound represented by the above Chemical Formula II is characterized as being a compound represented by the following Chemical Formula 2 or Chemical Formula 4, or a pharmaceutically acceptable salt thereof: [Chemical Formula 2] [Chemical Formula 4] 4. In Claim 1, The above compound is a compound or a pharmaceutically acceptable salt thereof characterized by being derived from actinomycetes.

5. In Claim 4, A compound or a pharmaceutically acceptable salt thereof characterized in that the above-mentioned actinomycete is a strain of the genus Nocardiopsis sp.

6. In Claim 4, A compound or a pharmaceutically acceptable salt thereof characterized in that the above-mentioned actinomycete is a strain deposited under accession number KCCM13539P.

7. In Claim 1, The above compound or pharmaceutically acceptable salt thereof is a compound or pharmaceutically acceptable salt thereof characterized by lowering intraocular pressure.

8. In Claim 1, The above compound or pharmaceutically acceptable salt thereof is characterized by having rho kinase inhibitory ability.

9. In Claim 1, The above compound or pharmaceutically acceptable salt thereof is characterized by having inhibitory ability against ROCK1 (rho-associated protein kinase 1) and ROCK2 (rho-associated protein kinase 2).

10. In Claim 1, The above compound or pharmaceutically acceptable salt thereof is characterized by inhibiting the shrinkage of HTM (human trabecular meshwork) cells or increasing the permeability of the HTM (human trabecular meshwork) monolayer cell layer.

11. A pharmaceutical composition for the prevention or treatment of glaucoma comprising, as an active ingredient, a compound represented by the following chemical formula I or chemical formula II or a pharmaceutically acceptable salt thereof: [Chemical Formula I] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl. [Chemical Formula II] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

12. A quasi-drug composition for the prevention or improvement of glaucoma comprising, as an active ingredient, a compound represented by the following chemical formula I or chemical formula II or a pharmaceutically acceptable salt thereof: [Chemical Formula I] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl. [Chemical Formula II] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

13. A food composition for the prevention or improvement of glaucoma comprising, as an active ingredient, a compound represented by the following chemical formula I or chemical formula II or a pharmaceutically acceptable salt thereof: [Chemical Formula I] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl. [Chemical Formula II] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

14. A pharmaceutical composition for the prevention or treatment of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

15. A quasi-drug composition for the prevention or improvement of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

16. A food composition for the prevention or improvement of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13539P as an active ingredient.

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