Novel compound having rho kinase inhibitory ability, and use thereof

Abstract

The present invention relates to a novel compound having a 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 of the present invention is derived from a natural product, and has almost no side effects, and thus can be safely and continuously used.

Description

Novel compound having RHO KINASE inhibitory ability and uses thereof

[0001] The present application claims priority to Korean Patent Application No. 10-2024-0129135 filed on September 24, 2024, and the entire specification is a reference to the present application.

[0002] The present invention relates to a novel compound having rho kinase inhibitory ability and its use.

[0003] Glaucoma is a progressive optic neuropathy characterized by the refraction of the optic disc and the loss of apoptosis in retinal ganglion cells, accompanied by consequent vision loss. While the underlying pathophysiological mechanism is multifactorial, intraocular pressure (IOP) is a persistent risk factor for the onset and progression of the disease (Lilian et al., 2023). Intraocular pressure refers to the pressure at which the aqueous humor, produced in the ciliary body and flowing posteriorly, balances with the aqueous humor draining through the trabecular meshwork and Schlemm's canal. If the aqueous humor is not properly drained, intraocular pressure rises, which can lead to optic nerve damage (Johnson and Erickson, 2000; Johnson, 2006). The only therapeutic intervention proven effective in slowing disease progression is lowering intraocular pressure.

[0004] Rho kinase (ROCK) inhibitors are a new class of glaucoma treatments with novel mechanisms of action and excellent safety profiles (Angelo and Mark, 2018). Rho-associated protein kinase (ROCK) belongs to the AGC family of serine-threonine protein kinases and regulates cell morphology and movement primarily by affecting the cytoskeleton. When ROCK kinase is activated, it activates the myosin light chain (MLC), which increases fiber contraction. This blocks the aqueous humor outflow pathway in human trabecular meshwork cells, leading to an increase in intraocular pressure (Rao et al., 2005). The use of ROCK inhibitors as eye drops can provide a potential therapeutic effect for glaucoma by reducing fiber contraction in human trabecular meshwork cells and relaxing the trabecular pathway, thereby increasing aqueous humor outflow and decreasing intraocular pressure (Rao et al., 2001; Honjo et al., 2001; Honjo et al., 2001). Currently, netarsudil and ripasudil are the only drugs approved for the treatment of glaucoma (Sturdivant et al., 2016; Garnock-Jones, 2014). Netarsudil is used in the United States, while ripasudil is used in Japan and China.

[0005] 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 experimentally confirming that a novel compound isolated from actinomycetes is useful for inhibiting Rho kinase (ROCK).

[0006] The inventors completed the present invention by confirming, through research efforts to develop a natural product capable of effectively preventing or treating glaucoma, that a novel compound derived from actinomycetes inhibits rho-associated protein kinase (ROCK, rho kinase) and can increase the drainage of aqueous humor by relaxing the fibers of the trabecular meshwork pathway, which is the main drainage route of aqueous humor.

[0007] The object of the present invention is to provide a novel compound represented by the formula A and a pharmaceutically acceptable salt thereof.

[0008] In addition, the object of the present invention is to provide a composition for the prevention, improvement, or treatment of glaucoma comprising a novel compound represented by Formula A and a pharmaceutically acceptable salt thereof as an active ingredient.

[0009] Furthermore, 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 KCCM13502P as an active ingredient.

[0010] 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.

[0011] The present invention provides a compound represented by the following chemical formula A or a pharmaceutically acceptable salt thereof.

[0012] [Chemical Formula A]

[0013]

[0014] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

[0015] The compound represented by the above chemical formula A may include one or more selected from the compounds represented by the following chemical formula 3 or chemical formula 4.

[0016] [Chemical Formula 3]

[0017]

[0018] [Chemical Formula 4]

[0019]

[0020]

[0021] The above compound may be derived from actinomycetes.

[0022] The above actinomycete may be a strain of the genus Nocardiopsis sp.

[0023] The above actinomycete may be the strain deposited under accession number KCCM13502P.

[0024] The above compound may have rho kinase inhibitory ability.

[0025] The above compound may have inhibitory ability against ROCK1 (rho-associated protein kinase 1) and ROCK2 (rho-associated protein kinase 2).

[0026] The above compound can inhibit the contraction of HTM (human trabecular meshwork) cells.

[0027] The above compound can increase the permeability of the HTM (human trabecular meshwork) monolayer cell layer.

[0028] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of glaucoma comprising a compound represented by the above formula A or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] In addition, the present invention provides a quasi-drug composition for the prevention or improvement of glaucoma comprising a compound represented by the above formula A or a pharmaceutically acceptable salt thereof as an active ingredient.

[0030] In addition, the present invention provides a food composition for preventing or improving glaucoma comprising a compound represented by the above formula A or a pharmaceutically acceptable salt thereof as an active ingredient.

[0031] 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 KCCM13502P as an active ingredient.

[0032] 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 KCCM13502P as an active ingredient.

[0033] 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 KCCM13502P as an active ingredient.

[0034] The present invention also 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 the formula A or a pharmaceutically acceptable salt thereof, or an actinomycete or a culture thereof deposited under accession number KCCM13502P.

[0035] In addition, the present invention provides a composition for the prevention or treatment of glaucoma comprising, as an active ingredient, a compound represented by the formula A or a pharmaceutically acceptable salt thereof, or an actinomycete or a culture thereof deposited under accession number KCCM13502P.

[0036] The present invention has excellent Rho kinase (ROCK) inhibitory ability and excellent effect in inhibiting the contraction of HTM (human trabecular meshwork) cells, and can be utilized in various ways, such as pharmaceutical compositions and food compositions for the prevention, improvement, or treatment of glaucoma. Furthermore, the compound of the present invention is derived from natural products and has almost no side effects, so it can be used safely and continuously.

[0037] Figures 1a to 1e show the results of confirming compounds 1-6 through LC / UV profiling.

[0038] Figure 2 shows the structures of compounds obtained from actinomycetes (Compound 1: Cittilin A, Compound 2: Cittilin C, Compound 3: Norcanickelamide A, Compound 4: Norcanickelamide B, Compound 5: Iturin D, Compound 6: Iturin A4).

[0039] Figure 3 shows the correlations of HMBC, COSY, and ROESY of compounds derived from actinomycetes.

[0040] Figure 4 shows the results of evaluating the inhibitory effect of MCY7 compounds 1-6 on ROCK activity: (A) A luciferase-based ADP-Glo ​​kinase assay was performed to evaluate the effect of MCY7 compounds 1-6 (30 μM) on ROCK1 and ROCK2 activity in the presence of 1 μg S6K substrate and 1 μM ATP. Rifasudil (1 μM) was used as a positive control for ROCK inhibition. Results are expressed as a percentage of kinase activity relative to a dimethyl sulfoxide (DMSO)-treated control (vehicle). (B and C) Concentration-dependent inhibitory effects of compounds 3 and 4 on ROCK1 and ROCK2. Values ​​represent mean ± SD (n = 3). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. CPD, compounds; RIP, ripasudil.

[0041] Figure 5 shows the results of the molecular docking analysis of the ligand binding domain of ROCK with compounds 3 and 4: (A) Docking poses of compounds 3 (indicated in green), 4 (orange), and ripasudyl (cyan) within the binding sites of ROCK1 (PDB ID: 3V8S, light blue) and ROCK2 (PDB ID: 7JNT, bright pink). Bars indicate residues within 4.5 Å of the ligand. Residues in close contact with the compounds are labeled. 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 Molecular Operating Environment software and are expressed in kcal / mol. CPD, Compound; LBD, Ligand Binding Domain; RIP, Ripasudyl.

[0042] Figure 6 shows the results of analyzing the effect of Compound 4 on the phosphorylation (activation form) of MYPT1 and MLC2 in HTM cells: (A) HTM cells were treated with Compound 4 (10, 30, or 60 μM) in serum-rich medium for 24 hours. Cell viability was determined using WST analysis. Rifasudil (10, 30, or 60 μM) was used as a reference ROCK inhibitor. Results are expressed as a percentage of cell viability relative to the DMSO-treated control (vehicle). Values ​​represent mean ± SD (n = 3). (B) Lysates were collected from HTM cells treated with Compound 4 (0, 10, or 30 μM) or Rifasudil (10 or 30 μM) in serum-rich medium for 6 hours. Western blot analysis was performed on phosphorylated MYPT1 (Thr696) and MLC2 (Thr18 / Ser19). GAPDH was used as a loading control. ns, not significant;; RIP, ripasudil.

[0043] Figure 7 shows the results of analyzing the effect of Compound 4 on transendothelial electrical resistance (TEER) to measure the permeability of the HTM monolayer. HTM cells were treated with 5 ng / mL of transforming growth factor beta-1 (TGF-β2) and Compound 4 (0, 3, 10, or 30 μM) in serum-rich medium for 72 hours. Rifasudil (30 μM) was used as a ROCK inhibitor. Values ​​represent mean ± SD (n = 3). **P ≤ 0.01 and ***P ≤ 0.001. RIP, Rifasudil.

[0044] 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 has the efficacy to inhibit Rho kinase (ROCK).

[0045]

[0046] Herbal extracts and plant secondary metabolites have been used to treat glaucoma for decades. Clinical studies have reported that Ginkgo biloba extract, composed primarily of flavonoids and terpene lactones, improves visual field indices after four weeks. Furthermore, the glaucoma damage-reducing effect of forskolin, a diterpene isolated from the roots of Coleus forskolin (Lamiaceae), has been confirmed through in vivo and in vitro evidence. To date, most naturally derived glaucoma treatments have originated from the plant kingdom. The inventors have focused on discovering bioactive metabolites from the microbiome to find novel glaucoma treatments in nature. Accordingly, LC / MS-based chemical analysis methods were used on microbial sources. Through this method, lead compounds such as actinoflavosides and cystagamides were isolated. In particular, the OSMAC (one strain many compound) strategy was employed to increase the chance of obtaining hidden metabolites by adding metal ions. Depending on the presence or absence of metal ions, the production of secondary metabolites can be affected or new metabolites can be induced. For example, anhydromevalonolactone in the culture of Streptomyces sp. SH-13125 Co 2+ and Zn 2+It was induced by the addition of a mixture. The metabolomic profile was influenced by the concentration and characteristics of metal ions. In particular, Streptomyces pratensis NA-ZhouS1 produced streptomycin A and B only when the nickel concentration was less than 100 µM. As the nickel concentration increased, the production of streptomycin AB changed dramatically, and streptomycin AB was not produced at all in Streptomyces pratensis NA-ZhouS1 treated with 800 µM nickel. Overall, the introduction of metal stress integrated with LC-MS analysis is a simple and effective technique for screening novel physiologically active natural products by activating silenced biosynthetic gene clusters.

[0047] In a preliminary study, the inventors cultured various types of Streptomyces strains under various types of metal ions. Then, they analyzed the LC / MS metabolite profiling data of the cultures to select the HIT strain and optimal culture conditions. As a result, 1 mM Ni 2+ or 1 mM Co 2+ It was confirmed that Norcadiopsis sp. MCY7 treated with [the substance] showed significant changes in the LC / MS metabolite profile. As a result of large-scale culture of Norcadiopsis sp. MCY7 under these conditions, a series of peptides (compounds 1-6) were isolated.

[0048]

[0049] The present invention will be described in detail below.

[0050] The present invention provides a compound represented by the following chemical formula A or a pharmaceutically acceptable salt thereof.

[0051] [Chemical Formula A]

[0052]

[0053] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

[0054] In addition, the present invention provides a composition for the prevention, improvement, or treatment of glaucoma comprising a compound represented by the above formula A or a pharmaceutically acceptable salt thereof as an active ingredient.

[0055] 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 KCCM13502P as an active ingredient.

[0056] The above composition may be a pharmaceutical composition, a quasi-drug composition, a food composition, or a health functional food composition.

[0057] The compound represented by the above chemical formula A may include one or more selected from the compounds represented by the following chemical formula 3 or chemical formula 4.

[0058] [Chemical Formula 3]

[0059]

[0060] [Chemical Formula 4]

[0061]

[0062]

[0063] The above compound may be derived from actinomycetes.

[0064] The above actinomycete may be a strain of the genus Nocardiopsis sp.

[0065] The above actinomycete may be the strain deposited under accession number KCCM13502P.

[0066] The above compound may have rho kinase inhibitory ability.

[0067] The above compound may have inhibitory ability against ROCK1 (rho-associated protein kinase 1) and ROCK2 (rho-associated protein kinase 2).

[0068] The above compound can inhibit the contraction of HTM (human trabecular meshwork) cells.

[0069] The above compound can increase the permeability of the HTM (human trabecular meshwork) monolayer cell layer.

[0070]

[0071] 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.

[0072]

[0073] Pharmaceutical composition for the prevention or treatment of glaucoma

[0074] The composition of the present invention can be prepared as a pharmaceutical composition.

[0075] 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.

[0076] 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 the compound of the present invention or a pharmaceutically acceptable salt thereof described above; and (b) a pharmaceutically acceptable carrier. As specified herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the active ingredient described above.

[0077] 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).

[0078] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0079] 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.

[0080] 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.

[0081]

[0082] quasi-drug composition for the prevention or improvement of glaucoma

[0083] The composition of the present invention may be provided as a quasi-drug composition.

[0084] 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).

[0085]

[0086] Food composition for the prevention or improvement of glaucoma

[0087] 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 comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is prepared as a food composition, it includes not only the compound or a pharmaceutically acceptable salt thereof as an active ingredient, but also ingredients that are typically added during food preparation, such as 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 conventional sugars like dextrin, cyclodextrin, etc., 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092]

[0093] The compounds of the present invention were isolated from Norcadiopsis sp. MCY7 derived from tidal flats collected in Korea. As part of an effort to discover hidden metabolites in MCY7, the inventors applied metal stress by introducing various metal salts into the culture medium. Through LC-MS-based analysis, two novel linear peptides induced by metal stress, norcanickelamide A and B (Compounds 3 and 4), and two known cyclic lipopeptides, iturin D and A4 (Compounds 5 and 6), were isolated. The planar structures of the novel compounds were determined by comprehensively analyzing UV, mass, and nuclear magnetic resonance spectroscopy data. The absolute configurations of Compounds 3 and 4 were confirmed using advanced Marfey methods. Compounds 3 and 4 exhibited potent dual-inhibitory ROCK 1 / 2 kinase activity and bound to ATP binding sites in molecular simulations. Furthermore, in human trabecular meshwork cells, norcanickelamide B (Compound 4) inhibited the expression of ROCK-regulated fiber contraction markers p-MYPT1 and p-MLC. These findings suggest that Compound 4 is a novel dual ROCK1 / 2 inhibitor and a potential pharmacophore for designing new therapeutic agents for ocular hypertension and glaucoma.

[0094]

[0095] <Example>

[0096] Experimental method

[0097] General experimental procedures

[0098] Optical rotation was recorded using a 343 plus PerkinElmer polarimeter (MA, United States), and IR spectra were recorded using a PerkinElmer FT-IR spectrometer (MA, United States). Ultraviolet spectra and electrospray ionization (ESI) low-resolution LC / MS data were recorded using an inverted-phase C18 column (Agilent column, 100 × 4.6 mm, 5 μm) on an Agilent G6125B MSD system coupled with an Agilent Technologies 1260 series Infinity II LC system. 1 H, 13 C1 and 2D-NMR spectra were recorded using Bruker Avance 700 MHz and 900 MHz spectrometers (Bruker, Billerica, MA, USA) at the Korea Basic Science Institute (KBSI) in Ochang. High-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an Agilent Technologies 1290 series HPLC coupled to an Agilent 6530 iFunnel Q-TOF LC / MS system (Agilent Technologies). Purification was performed using a Waters HPLC system (1525 binary pump and 996 photodiode array detector). Optical rotation was obtained using a Jasco P-2000 polarimeter (Tokyo, Japan) with a 1.0 cm cell.

[0099]

[0100] Bacterial isolation and culture

[0101] The sample was a sediment collected from the Jeungdo salt field in South Korea in 2022. 2 g of the air-dried sample was diluted with 10 mL of sterile water and sonicated for 15 minutes. Then, 50 μL of the suspension was plated onto an agar plate of isolation medium (0.4 g casein, 1.0 g starch, 0.5 g KNO3, 0.2 g K2HPO4, 0.1 g MgSO4, 0.1 g CaCO3, 25 mg cylcoheximide, 10 mg nalidixic acid, 1 L seawater, and 18 g agar). The agar plates were incubated at 30 °C for 3 weeks, and colonies resembling actinomycetes were isolated and selected from the culture dishes. The MCY7 strain obtained from the subculture process was deposited as an international strain at the Korean Culture Collection (KCCC) (Accession No. KCCM13502P).

[0102] The seed culture of the actinobacterium (Norcadiopsis sp. MCY7) was prepared by inoculating the bacteria (MCY7) into 50 mL of YEME medium (3 g yeast, 10 g glucose, 3 g malt extract, 5 g peptone, 2 g soytone, 33 g sea salt, and 1 L water) and stirring at 180 rpm for 2 days at 30 ℃. Then, 5 mL of the seed culture was transferred to a 500 mL flask containing 150 mL of YEME medium and fermented under the same conditions as the seed culture. After 2 days, 20 mL of the broth culture was transferred to 1 L of YEME medium contained in a 2.5 L flask. For large-scale cultivation, 50 L of liquid culture was prepared for each culture condition. To stress the bacteria (MCY7), a metal ion solution was added to the medium until the metal ion concentration reached 1 mM.

[0103]

[0104] Extraction and purification

[0105] The entire liquid culture was centrifuged at 3,700 rpm for 1 hour at 4 ℃, then the supernatant was filtered and the packed C 18 Extraction was performed using a column. After extraction, the column was eluted with 200 mL of 20%, 40%, 60%, 80%, and 100% MeOH in water, respectively.

[0106] The 20% and 40% fractions of the native culture were combined and eluted with MeOH via a Sephadex lh-20 column to form subfractions, and Compound 1 and Compound 2 were detected in 100–130 subfractions by LC-MS. Then, Compound 1 and Compound 2 were purified using a reversed-phase semi-preparative HPLC column (YMC ODS column: 250 x 10 mm, 5 mm) with a gradient solvent system (25%–50% MeCN / H2O (0.1% formic acid) for 45 minutes, flow rate 2 mL / min, UV detection at 254 nm). Under these conditions, Compound 1 and Compound 2 (7.0 mg) were eluted at 22 minutes and 31 minutes, respectively.

[0107] Subsequently, Nickel-stress MCY7 (MCY7+1mM Ni 2+ The 40% and 60% fractions of ) were combined and loaded into Sephadex IH-20, and analyzed for 400 minutes using MeOH:H2O (1:1) as the solvent. The fractions at 300–330 minutes were analyzed by reverse-phase HPLC (YMC triart C 18 Using a column: 250 x 10 mm, 5 μm), further purified with 20% to 40% aqueous acetonitrile (0.1% trifluoroacetic acid) in a linear gradient for 30 minutes for compound (3) (3.5 mg, t R = 20 min) and compound (4) (4.2 mg, t R =21 minutes) obtained.

[0108] Compounds 5 and 6 were directly isolated from the 80% fraction of the 1 mM Cobalt-stress MCY7 (cobalt salt added) extract. Purification of compounds 5 and 6 was performed in a reverse-phase semi-preparative HPLC system (YMC ODS column: 250 x 10 mm, 5 mm), and the gradient used was 40%–65% aqueous acetonitrile (0.1% formic acid) for 40 minutes. Compounds 5 and 6 were then eluted at 28 minutes and 32 minutes, respectively.

[0109]

[0110] Acid hydrolysis and advanced Marfey's method

[0111] 1 mL of 6N HCl aqueous solution was added to Compound 3 (1 mg) and stirred at 90 °C overnight. After hydrolysis, the mixture was cooled and dried under vacuum, then resuspended in 3 mL of water and concentrated (three times) to remove residual acid. The hydrolysate was freeze-dried to completely remove moisture. Subsequently, the mixture was divided into two vials, and 100 mL of 1-fluoro-2,4-dinitrophenyl-5-D-leucine amide (D-FDLA) in 250 mL of 6N NaHCO3 and acetone was added to one vial. The same procedure was repeated for the other vial, but 1-fluoro-2,4-dinitrophenyl-5-L-leucine amide (L-FDLA) was used instead of D-FDLA. Subsequently, the mixture of the two vials was heated at 85°C for 10 minutes and neutralized with 125 mL of 2N HCl to obtain D-FDLA and L-FDLA derivatives. Finally, the L- / D-FDLA derivative was C 18Analysis was performed by LC / MS using a column (Agilent Eclipse Plus C18 column: 150 x 4.6 mm, 3.5 μm linear gradient (30%–65% MeCN / H2O, 0.1% formic acid, 50 min or more, flow rate 0.4 mL / min)). The retention time of FDLA-derived amino acid products was determined using UV values ​​and ion extraction. Standards for L-leucine, L-phenylalanine, cis-4-hydroxy-L-proline, and trans-4-hydroxy-L-proline were prepared using the same method as described above. The absolute composition of amino acid units was determined by comparing the retention times of the FDLA-derived products of standard amino acids with those of Compound 3.

[0112] cittilin C, Compound 2: White powder; [[α] D 25 -3 (c 0.2, MeOH);; UV (MeOH)λ max (logε) 195 (3.03), 230 (30.1), 288 (2.46) nm; IR (neat)vmax 3394, 3311 2968,1650, 1507, 1248, 1192, 1026 cm -1 ; 1 H and 13 For CNMR spectroscopic data, Table 2; HR-ESI-MSm / z673.2864 [M + H] + (calcd for C 36 H 41 N4O9,m / z673.2874).

[0113] nocarnickelamide A, Compound 3: White powder; ; [α] D 25 -5 (c 0.2, MeOH); UV (MeOH)λ max(logε) 193 (2.73) nm; IR (neat)vmax 3335, 2983, 1681, 1443, 1209, 1183, 1033 cm -1 ; H and 13 For CNMR spectroscopic data, Table 2; HR-ESI-MSm / z562.2878 [M + H] + (calcd for C 27 H 40 N5O8.2877).

[0114] nocarnickelamide B, compound 4: white powder; [α] D 25 -18 (c0.2, MeOH); UV (MeOH)λ max (logε) 194 (3.02) nm; IR (neat)vmax 3276, 1681, 1438, 1207, 1138, 1029 cm -1 ; H and 13 For CNMR spectroscopic data, Table 3; HRESIMSm / z619.3096 [M + H] + (calcd for C 29 H 42 N6O9,m / z619.3092).

[0115]

[0116] In vitro analysis of ROCK kinase inhibition

[0117] Kinase Enzyme Systems and ADP-Glo ​​according to the manufacturer's instructions TMROCK kinase activity was measured in vitro using an assay kit (Promega, Madison, WI, USA). This assay quantitatively measured the amount of ADP generated during the reaction of ROCK kinases (ROCK1 and ROCK2). The measured luminescence signal was detected in proportion to the amount of ADP indicating ROCK kinase activity. In a 384-well plate with a total reaction volume of 20 mL, 10 ng ROCK1 (Promega) or 0.5 ng ROCK2 enzyme (Promega), an inhibitor (MCY7 Compound 1-6, ripasudyl, or DMSO), 1 mg S6K peptide substrate (SignalChem), and 1 mM ATP (Promega) were diluted in kinase buffer and added. 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 (RLU) were obtained by setting the value to 100% (control value) when no inhibitor was used and 0% (reference value) when no enzyme was used. IC50 50 The values ​​were determined using logistic regression analysis. Rifasudil (a ROCK inhibitor) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0118]

[0119] Cell Culture

[0120] 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 in Trabecular Meshwork Cell Medium supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin (10,000 U / mL; 10,000 μg / mL), and growth supplement (serum-rich medium) at 37°C in a humid environment containing 5% CO2. All culture-related products were purchased from ScienCell.

[0121]

[0122] Cell viability analysis

[0123] 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 4 Cells were seeded at a cell / well density and cultured for 24 hours (at 37 °C, 5% CO2). Cells were treated with Compound 4 and Lipasudil at concentrations of 10, 30, or 60 μM in serum-rich medium for 24 hours. Then, EZ-Cytox solution was added and the cells were incubated for another 1.5 hours, after which absorbance was measured at a wavelength of 450 nm using a microplate reader (Multiskan SkyHigh Microplate Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA).

[0124]

[0125] Western blot analysis

[0126] 4 × 10⁶ HTM cells in a 6-well plate 5Cells were seeded at cell / well density and cultured for 24 hours (at 37 °C, 5% CO2). Cells were treated with Compound 4 and Lipasudil at concentrations of 10 or 30 μM for 6 hours in serum-rich medium. 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. Subsequently, the lysates were centrifuged (15,000 rpm, 15 min, 4 °C). Equal amounts of protein were separated on 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 incubated with primary antibodies against p-MYPT1 (Thr696), p-MLC2 (Thr18 / Ser19), and GAPDH (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA) at 4°C for at least 16 hours. After washing three times with triple-buffered saline-Tween 20 solution, the membrane was incubated with the corresponding anti-rabbit (1:4000, Genetex Inc., Irvine, CA, USA) secondary antibody conjugated with HRP at 25°C for 1 hour. Finally, the membrane was washed 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 the iBright CL1000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

[0127]

[0128] Measurement of transendothelial electrical resistance (TEER)

[0129] 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 with 5 ng / mL of transforming growth factor beta-1 (TGF-β2) and Compound 4 (0, 3, 10, or 30 μM) in serum-rich medium for 72 hours. Rifasudil (30 μM) was used as a positive control for ROCK inhibition. Permeability measurements of the HTM cell line monolayer were quantified using an EVOM instrument (World Precision Instruments, Sarasota, FL, USA).

[0130]

[0131] Molecular Docking Analysis

[0132] Molecular docking studies were performed using MOE (Molecular Operating Environment) software version 2022.02 (Chemical Computing Group, Montreal, Canada). Atomic coordinates for target proteins ROCK1 and ROCK2 were obtained from the Protein Data Bank (PDB IDs: 3V8S and 7JNT, respectively). Protein structures were prepared using the QuickPrep module in MOE, which included steps such as hydrogen atom addition, partial charge assignment, and energy minimization. Compounds 3 and 4, along with the reference compound ripasudil, were constructed and optimized using MOE's Builder tool. Energy minimization of the ligands was performed using MMFF94 x force field to obtain the lowest energy form. The prepared ligands were docked to the active sites of ROCK1 and ROCK2. Docking was performed with specific settings for placement and refinement. The placement method used was Triangle Matcher, and the initial scoring function was London dG, which estimates the binding free energy of the ligand-protein complex. In the purification step, the Rigid Receptor method was used in conjunction with the GBVI / WSA dG scoring function, which is used to further evaluate binding affinity. Docking poses were ranked according to their scores, and the pose with the highest ranking was selected for further analysis. Binding modes and interactions were visually examined using MOE visualization tools. The reliability of the docking results was verified by re-docking the co-crystallized ligand and comparing the predicted binding modes with the experimental structure. This ensures the accuracy of the docking protocol and the relevance of the predicted binding interactions.

[0133]

[0134] Statistical analysis

[0135] Experimental values ​​represent the mean ± standard deviation of at least three experiments. Statistical analysis was performed using RStudio for Windows (RStudio Inc., Boston, MA, USA) with one-way analysis of variance (ANOVA) and post-hoc Welch's t-tests. Criteria for statistical significance were set to *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.01.

[0136]

[0137] abbreviation

[0138] DMSO, dimethyl sulfoxide; HTM, human trabecular meshwork; IOP, intraocular pressure; LBD, ligand binding domain; MLC, myosin light chain; MYPT, myosin phosphatase target subunit 1; PDB, protein data bank; ROCK, rho-associated protein kinase; TGF-β2, transforming growth factor-beta 1

[0139]

[0140] Marfey's advanced method (SI)Amino acid residue RL t RDm / zElution orderL-Phenylalanine35.543.3459L¨DPhenylalanine from 335.543.4459L¨DPhenylalanine from 435.343.9459L¨DL-Leucine34.146.2425L¨DLeucine from 334.446.4425L¨DLeucine from 434.846.7425L¨D4-cis-hydroxy-L-proline16.417.1425L¨D4-trans-hydroxy-L-proline14.915.4425L¨D4-hydroxyproline from 314.815.2425L¨D4-hydroxyproline from 415.115.6425L¨D

[0141]

[0142] Experimental results

[0143] Metal stress and LC-MS profile

[0144] MCY7 was cultured in YEME medium supplemented with various types of metal ions. As a result of analyzing the LC / UV / MS metabolite profile, Ni 2+ and Co 2+ It was found that the production of cittilin A (Compound 1) and cittilin C (Compound 2) decreased upon addition. MCY7 + 1mM Co 2+ In the LC / MS profile of (Cobalt-stressed MCY7), two new signals (Compound 5 and Compound 6) appeared at rt 15.9–16.0 min, which were less polar compounds. Compounds 3 and 4 were MCY7 + 1 mM Ni 2+ It was detected in the culture medium extract. 562 and 619 ions of Compound 3 and Compound 4 were extracted and observed by monitoring with an LC / MS profile (Fig. 1).

[0145]

[0146] Cittilin C (Compound 2) was purified as a white powder from the non-stressed culture medium of the MCY7 strain. The molecular formula of Compound 2 is C, having a degree of unsaturation of 19 degrees. 36 H 40 It was measured with N4O9 using high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). Compound 2 1 H-NMR and HSQC spectra show four exchangeable protons (δ H 7.06, 8.25, 8.58, and 8.75) and 4 α-amino protons (δ H It indicated the presence of 3.49, 4.21, 4.12, and 4.74. This initial analysis suggests that Compound 2 is a peptide-derived compound containing four amino acid residues. Additionally, nine aromatic protons (δ) in the downfield region H By closely analyzing 5.60, 6.45, 6.71, 6.88 (2H), 6.77, 7.27, 7.33, and 7.47), three aromatic rings were identified, accounting for 12 of the 19 saturation numbers. 1 Further H-NMR analysis revealed that compound 2 has four methyl groups (δ H There are (0.80, 0.83, 1.86, 3.75), one of which is a heteroatom (δ H It was found to be directly bound to 3.75) and contain 8 aliphatic methylene protons in the range of 1.10 to 3.50 ppm.

[0147] 13 The C-NMR spectrum showed 18 olefin carbons ranging from 110.0 to 165.0 ppm, and the structure of compound 2 had 3 aromatic rings and 4 alpha-amino carbons (δ c It was confirmed that 51.5, 52.0, 57.4, and 57.9) exist. Compound 2 may be a peptide with three aromatic rings.

[0148] Since Compound 2 is likely a peptide-derived compound, its peptide sequence was determined first before elucidating the structure of individual amino acid units. Interpretation of COSY, TOCSY, and HMBC spectroscopic data confirmed that Compound 2 possesses one isoleucine, one N-acetyl-O-methyltyrosine, and two tyrosine units.

[0149] First, characteristic 1 An aromatic ring with an ABX system was constructed by H-NMR signal: signal in the down-field region [(δ H 6.71(H), 6.88(2H)], H-8(δ H 6.88) and H-9(δ H COSY correlation between 6.88) and H-5(δ H 6.71) at C-7(δ c 155.3), C-9(δ c The HMBC correlation to 110.1) was confirmed. From H-10 (3.75) to C-7 (δ c Strong HMBC correlation up to 155.3) and C-sp 2 The weak down-field chemical shift determined the position of the O-methyl group connected to C-34. H-2(δ H 4.74) and H-3(δ H COSY correlation between (2.96) and H2-(δ H C-4(δ 4.74) c HMBC up to 127.8) clearly identified O-methyl tyrosine. The TOCSY spectrum showed C-2(δ c C-3(δ 52.0) c It showed good agreement with the spin system up to 36.6). In addition, N-acetyl-O-methyltyrosine has alpha-amino proton H-2(δ H At C-1'(δ 4.74) c 168.7) to, from proton NH-11(7.06) to C-2 (δ c 52.0), C-1' (δc It was successfully established through HMBC correlations (168.7), which established the bond between the acetyl group and the amino group. Other protein-producing amino acids (tyrosine, isoleucine) were determined by the combination analysis of COSY, TOCSY, and HMBC.

[0150] The arrangement of individual units was determined by comprehensively analyzing HMBC data. TNH-18(δ H 8.25) / C-1(δ c 169.8); NH-28(δ H 8.58) / C-12(δ c 171.0) HMBC correlation and H-20(δ H 3.49) / H-30 (δ H 4.21); H-2(δ H 4.74) / H-18(δ H 8.25) and H-28 (δ H 8.58) / H-13(δ H 4.12) The amino acid sequence N-acetyl-O-methyl tyrosine-isoleucine-tyrosine-1-tyrosine-2 was confirmed by ROESY correlation. Since five carbonyl groups and three aromatic rings occupy 17 of the 19 saturation numbers, Compound 2 must be a bicyclic compound. The first ring is C-26 (δ c 130.3) and C-6(δ c 127.2) It is composed of the linkage of N-acetyl-O-methyl and Tyr-1 through CC crosslinking between H-27(δ H 6.45) / C-6 (δ c 127.2) was shown as a correlation. The other link was C-35(δ c 162.4) and C-24 (δ c It is formed by O-crosslinking between 152.0), and ROESY correlation H-23 (δ H 5.60) / H-36 (δH 7.47); H-23 (δ H 5.60) / H-37 (δ H This can be explained by 7.33). This connectivity was established using ROESY because there is no two- or three-bond 1H-13C coupling. The structure of Compound 2 is reminiscent of Cittilins, particularly Cittilin A. Since the difference between Cittilin A and Compound 2 is the N-acetyl group, the absolute configurations of Compound 2 were determined by comparing their specific optical rotations, through which N-acetyl-O-methyl-L-tyrosine, L-isoleucine, and L-tyrosine were identified.

[0151]

[0152] Nocarnickelamide A (Compound 3) was isolated as a white powder, and based on HR-ESI-MS data, molecular formula C with an unsaturation number of 11 27 H 39 It was identified as a compound of N5O8. 1 Analysis of H-NMR and HSQC spectra revealed that Compound 3 contains two amide protons (δ H It was found to contain 8.00, 8.24), 2 hydroxyl protons, 5 aromatic protons, and 13 protons bonded to a carbon containing a heteroatom. In addition, 2 methyl groups (δ H 0.88, 0.89), 9 aliphatic methylene protons and 1 aliphatic proton were observed at 1.50–3.20 ppm.

[0153] 13 C-NMR shows five carbonyl carbons (δ c 167.2, 168.3, 170.4, 171.1, 173.1), 4 aromatic carbons (δ c 134.6, 129.9, 128.4, 127.2), 6 N / O-containing methines (δ c48.4, 52.3, 57.6, 58.9, 68.8, 68.9), 3 N / O-containing methylene carbons (δ c 41.7, 54.7, 54.9), 4 methylene carbons, 1 methine carbon (24.0) and 2 methyl groups (δ c It showed 21.6, 23.1).

[0154] The partial structure of Compound 3 was determined through the combined analysis of COSY, TOCSY, and HMBC. 1 The three H-NMR signals and the four signals of aromatic carbons are HMBC correlations H-27 / 31(δ H 7.36) / C-29(δ c It represents a single substituted aromatic ring identified by 127.2). Subsequently, H-24(δ H From 4.31) to C-23(δ c 167.2), C-25(δ c 36.0) and C-26(δ c Based on HMBC correlations up to 134.6), this ring was determined to be part of a phenylalanine residue. H-11(δ H 0.89) and H-12(δ H C-10(δ 0.88) c The HMBC correlation up to 24.0) (indicating terminal dimethyl) and the series of COSY correlations of H-8 / H-9 / H-10 / H-11 / H-12 represent the next amino acid unit, leucine. The glycine unit is H-15 (δ H 3.79) and H-16(δ H 8.24) COSY correlation between and H-15(δ H It was identified by HMBC correlations from C-14 (168.3) to C-3 (3.79). H-2 / H-3 / H-4 / H-5 (δ HThe consecutive COSY correlations between 4.25, 2.10, 4.60, and 3.62, and the HMBC correlations from H-2 (4.25) to C-4 (68.9) and C-5 (54.7) suggest the presence of γ-substituted proline. Two or three 1H-13C couplings from 4-OH (5.22) to C-3 (37.1), C-4 (38.9), and C-5 (54.7) indicate the remaining substructure, γ-hydroxyproline. In the same way, another γ-hydroxyproline was identified as the final amino acid unit.

[0155] The amino acid sequence of Compound 3 was established based on HMBC analysis. The HMBC correlation from H-2 (4.25) of Hpro-1 to C-7 (170.4) of Leu showed an association between OH-pro-1 and Leu. The HMBC correlation between H-13 (8.00) of Leu and C-14 (168.25) of Gly indicated that Leu is connected to Gly. The peptide sequence is elongated by OH-pro2 through an amide bond determined by the correlation between H-16 (8.24) of Gly and C-17 (171.1) of OH-pro-2. The Phe unit was positioned adjacent to OH-pro-2 by the ROESY correlation between H-21 (3.54) and H-24 (4.31). OH-pro-1 and Phe were identified as the C-terminus and N-terminus, respectively. The amino acid sequence was cross-confirmed through ROESY correlation analysis between α-protons.

[0156]

[0157] Norcanickelamide B (Compound 4) was obtained as a white powder, and according to HR-ESI-MS data, the molecular formula C of unsaturated water 11 29 H 42 It was identified as a compound of N6O9. Comparing the NMR-spectral data of compound 3 and compound 4, it was confirmed that compound 4 is structurally related to compound 3, which is a peptide compound. 1 H-NMR and 13C-NMR analysis revealed that Compound 4 has one more carbonyl group (166.4) and one more N / O-containing methylene group (3.47, 3.35; 40.0) compared to Compound 3. The presence of these two groups suggests that Compound 4 is Compound 3 extended by a single glycine unit, which is supported by the m / z 57 dalton difference between Compound 3 and Compound 4. An amide bond between OH-pro-1 and Gly-2 was confirmed by the 3JCH correlation from H-2' (3.47) to C-1 (173.2). Therefore, the structure of Compound 4 was determined to be a glycine extension analog of Compound 3.

[0158]

[0159] Amino acid unit Position Cittilin C (Compound 2)d C ,typed H,mult (J in Hz)N-acetyl-O-methyl tyrosine1169.8, C11-NH7.06, d (8.0)252.0, CH4.74, s336.6, CH22.96, m4127.7, C5135,4, CH6.71, s6127.2, C7155.3, C8130.0, CH6.89,s9110.1, CH6.88,s1055.4, CH33.75, s1'168.7, C2'22.5, CH31.86, sIle12171,0, C188,25, d (7.5)1357,4, CH4,12, t (8.0)1435.6, CH1.60, m1524,8, CH21,16, m1,45, br s1610.8, CH30.80, t (7.5)1715.4, CH30.83, t (7.0)Tyr-219168.7, CNH8.58, d (8.5)2057.9, CH3.49, m2140.0, CH22.86, br s2.37, d (12.5)22134.6, C23118.5, CH5.60, s24151.9, C25143.6, C26130.3, C27125.2, C6.45, s25-OHNH8.75, sTyr-329171.0, C3051.5, CH4.21, br s3135.5, CH23.00, m3.46, m32133.0, C33134.4, CH7.27, d (8.0)34123.5, CH6.77, d (8.0)35162.4, C36126.0, CH7.47 (8.0)37129.6, CH7.33, br s

[0160] Regarding Cittilin C (Compound 2) 1 H and 13 C NMR spectroscopic data. 1 H and 13 C NMR data were recorded at 900 MHz and 225 MHz.

[0161]

[0162] Amino acid unit Position nocarnickelamide A (Compound 3) nocarnickelamide B (Compound 4) δ C,typeδ H ,mult(J in Hz)δ C ,typeδ H,mult(J in Hz)OH-pro-11173.1, C173.2, C257.6, CH4.25, t (8.0)57.6, CH4.24, t(8.0)337.1, CH22.10, m37.2 CH22.08, m1.90, m1.90, m468.9, CH4.36, m68.9, CH4.35, m4-OH-5.22, br s-5.20, br s554.7, CH23.62, dd(10.5, 4.0)54.9-, CH23.62, m3.56, d(10.5)3.56, m6-N----Leu7170.4, C-170.4, C-848.4, CH4.59, q (8.0)48.3, CH4.61, q (8.5)937.0, CH240.4, CH21.43, m1024.0, CH1.63, sept(7.0)23.9, CH1.63, sept(6.5)1121.6, CH30.88, d(7.0)21.6, CH30.86, dd(7.0, 2.0)1223.1, CH30.89, d(7.0)23.1, CH30.86, dd(7.0, 2.0)13-NH-8.00, d (8.5)-7.87, d (8.5)Gly-114168.3, C-168.3, C168.31541.7, CH23.79, dd(17.0, 6.0)41.7, CH23.72 (17.0, 6.0)3.70, dd(17.0, 6.0)3.70 (17.0, 6.0)16-NH-8.24, br s-8.22, br sOH-pro-217171.1, C-171.1, C1858.9, CH4.47, t(8.0)58.9, CH4.35, m1937.6, CH22.04, m37.6, CH21.91, m1.87, m1.99, m2068.8, CH4.28, br s68.6, CH4.35, m20-OH-5.20, br s-5.20, br s2154.9, CH23.54,d (10.5)55.0, CH23.55, t (9.0)3.12, br s3.61, m22-N----Phe23167.2, C-169.5, C-NH--8.72, br s2452.3, CH4.31, br s52.1, CH4.72, m2536.0, CH22.95, m36.0, CH22.74, dd(14.0, 9.0)2.08, m3.03 (14.0, 5.0)26134.6, CH-134.6, CH-27 / 31129.9, CH7.36, d (7.5)129.9, CH7.27, m28 / 30128.4, CH7.32, t (7.5)128.4, CH7.27, m29127.2, CH7.28, d (7.5)127.2, CH7.20, mGly 21'166.4, C2'40.0, CH23.473.35.

[0163] 1H and 13C NMR spectroscopic data of nocarnickelamide A (Compound 3) and nocarnickelamide B (Compound 4). 1 H and 13 C NMR data were recorded at 900 MHz and 225 MHz.

[0164]

[0165] Analysis of ROCK1 and ROCK2 kinase activity inhibitory efficacy

[0166] Compounds 3 and 4 significantly inhibited ROCK1 and ROCK2 kinase activity.

[0167] To evaluate the inhibitory effect of MCY7 compounds 1-6 on ROCK activity, a luciferase-based ADP-Glo ​​kinase assay (in vitro assay) was performed (Fig. 4). For the treatment of glaucoma and ocular hypertension, rifasudil (trade name Glanatec), a rho kinase inhibitor and derivative of rifasudil, was used as the reference compound. At 30 μM, compounds 3 and 4 significantly inhibited ROCK1 activity by 51.3% and 63.3%, respectively (Fig. 4A). Regarding ROCK2 activity, compounds 3 and 4 inhibited it by 53.0% and 60%, respectively.

[0168] Next, the concentration-dependent inhibitory activity of these compounds was evaluated. IC5 for ROCK1 inhibition 50The (half-maximal inhibitory concentration) values ​​were 29.8 μM for Compound 3 and 14.9 μM for Compound 4 (Fig. 4B). The IC50 values ​​of Compound 3 and Compound 4 for ROCK2 inhibition. 50 The values ​​were 27.0 and 21.9 μM, respectively (Fig. 4C). These results indicate that compounds 3 and 4 are potent dual inhibitors of ROCK1 and ROCK2.

[0169]

[0170] ROCK's ligand binding domain and molecular interaction model of Compounds 3 and 4

[0171] Molecular docking simulations were performed to identify the potential binding modes and affinities of compounds 3 and 4 for the ligand binding domains (LBDs) of ROCK1 and ROCK2, thereby providing insights into the molecular basis of the interactions. The optimal docking modes of compounds 3 and 4 were compared with the docking mode of ripasudil (Fig. 5). For ROCK1, the energy-minimizing optimal model generated using Molecular Operating Environment (MOE) software showed that compounds 3 and 4 bind to the hinge region of the ATP binding site. The free energy values ​​of compounds 3, 4, and ripasudil were -7.3, -9.1, and -7.4 kcal / mol, respectively. Notably, all three compounds formed hydrogen bonds with the backbone NH of Met156 at the lip of the hinge binding site. Compounds 3 and 4 exhibited additional hydrogen bonding with Lys200 (Figs. 5A-5C). These interactions are consistent with a previous study (Beroza et al., 2022) that found 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 the loop amino acid Asp216. For ROCK2, the free energy values ​​of compounds 3, 4, and ripasudil were -8.4, -9.8, and -7.4 kcal / mol, respectively. Both compounds 4 and ripasudil formed hydrogen bonds with the backbone NH of Met172 and Asp218 (Figs. 5D-5F). Compound 4 exhibited more interactions than compounds 3 and ripasudil. These findings suggest that compounds 3 and 4 effectively bind to the ATP binding sites of ROCK1 and ROCK2, and specifically indicate that compound 4 exhibits superior binding affinity and interactions compared to compounds 3 and ripasudil. These results indicate that compound 4 is an effective inhibitor of ROCK1 and ROCK2.

[0172]

[0173] Analysis of ROCK-regulated fiber contraction marker expression

[0174] Compound 4 reduced the expression of the ROCK-regulated fiber contraction marker.

[0175] The human trabecular meshwork (HTM) is the primary site for the drainage of the aqueous humor, and dysfunction of this structure 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 the cytoskeleton and synthesizing extracellular matrix proteins in HTM cells. ROCK1 and ROCK2 activate the myosin light chain phosphatase complex (MYPT1) and inhibit myosin light chain phosphatase (MLCP), thereby activating MLC through phosphorylation. Phosphorylated MLC induces actin fiber contraction. Therefore, our research team evaluated the effects of Compound 4, one of the most potent ROCK inhibitors in the MCY7 series, on the ROCK-regulated signaling pathway in HTM cells. First, the viability of HTM cells treated with Compound 4 was assessed using the WST assay. HTM cells were treated with Compound 4 at various concentrations (10, 30, or 60 μM) for 24 hours. The data showed that Compound 4 did not exhibit significant cytotoxicity at concentrations up to 60 μM, similar to the ROCK inhibitor rifasudil (Fig. 6A). Treatment with 10 or 30 μM of Compound 4 for 6 hours (confirming that this condition does not affect cell viability) inhibited the phosphorylation of MYPT1 and MLC2, indicators of fiber shrinkage (Fig. 6B). On the other hand, it did not affect the total protein expression levels of MYPT1 and MLC2. Furthermore, Compound 4 did not affect the protein expression levels of ROCK1 and ROCK2 themselves, confirming that Compound 4 regulates downstream proteins (MYPT1, MLC2) through the inhibition of kinase activity rather than ROCK1 / 2 protein expression. These results suggest that Compound 4, a dual ROCK1 / 2 inhibitor, can inhibit the shrinkage of HTM cells and prevent obstruction of aqueous humor outflow.

[0176]

[0177] Permeability analysis of the monolayer cell layer of the trabecular meshwork

[0178] Compound 4 increased the permeability of the fibrous filament (HTM) monolayer cell layer reduced by converting growth factor beta (TGF-β2).

[0179] Ex vivo and in vitro studies have reported that TGF-β is involved in the pathogenesis of elevated intraocular pressure (Bhattacharya et al., 2005). TGF-β2 can cause changes in the extracellular matrix (ECM) environment associated with elevated intraocular pressure, and patients with high levels of TGF-β in the aqueous humor have an increased risk of elevated intraocular pressure, leading to a high risk of optic nerve damage and vision loss (Vittitow et al., 2004). Therefore, our research team treated HTM cells with TGF-β2 to induce fiber contraction and reduce permeability, and evaluated the effect of Compound 4. TGF-β2 (5 ng / mL) and Compound 4 (0, 3, 10, or 30 μM) were co-treated for 72 hours, and rifasudil (30 μM) was used as a positive control for ROCK inhibition. The permeability of the monolayer cell layer was significantly increased by 32% compared to the untreated control group by TGF-β2 (5 ng / mL) (Fig. 7). When compound 4 was co-treated, it was confirmed that the permeability decreased in a concentration-dependent manner, and at 30 μM, the effect was reduced to a level comparable to that of ripasudil (30 μM). This suggests that compound 4, a ROCK1 / 2 inhibitor, can increase permeability and facilitate the outflow of aqueous humor by inhibiting fiber contraction even under conditions where fiber contraction is induced by TGF-β2 in HTM cells.

[0180] [Consignment Number]

[0181] Depository Name: Korean Culture Collection Center (KCCM)

[0182] Trustee Number: KCCM13502P

[0183] Date of Trust: 20240801

[0184]

Claims

1. A compound represented by the following chemical formula A or a pharmaceutically acceptable salt thereof: [Chemical Formula A] 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 A is characterized by comprising one or more selected from compounds represented by the following chemical formula 3 or chemical formula 4, or a pharmaceutically acceptable salt thereof: [Chemical Formula 3] [Chemical Formula 4] 3. In Claim 1, The above compound is a compound or a pharmaceutically acceptable salt thereof characterized by being derived from actinomycetes.

4. In Claim 3, A compound or a pharmaceutically acceptable salt thereof characterized in that the above-mentioned actinomycete is a strain of the genus Nocardiopsis sp.

5. In Claim 3, A compound or a pharmaceutically acceptable salt thereof characterized in that the above-mentioned actinomycete is a strain deposited under accession number KCCM13502P.

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

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

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

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

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

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

12. A pharmaceutical composition for the prevention or treatment of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13502P as an active ingredient.

13. A quasi-drug composition for the prevention or improvement of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13502P as an active ingredient.

14. A food composition for the prevention or improvement of glaucoma comprising an actinomycete or a culture thereof deposited under accession number KCCM13502P as an active ingredient.

15. A method for the prevention or treatment of glaucoma comprising the step of administering to an individual a composition comprising as an active ingredient a compound represented by the following chemical formula A or a pharmaceutically acceptable salt thereof, or an actinomycete or culture thereof deposited under accession number KCCM13502P. [Chemical Formula A] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

16. Use for the prevention or treatment of glaucoma of a composition comprising, as an active ingredient, a compound represented by the following chemical formula A or a pharmaceutically acceptable salt thereof, or an actinomycete or culture thereof deposited under accession number KCCM13502P. [Chemical Formula A] R is -OH, -H, or C 1-10 It is a straight-chain or branched-chain alkyl.

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